@article {pmid38609262,
year = {2024},
author = {Su, Z and Wang, X and Chen, X and Ding, L and Zeng, X and Xu, J and Peng, C},
title = {Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342519},
doi = {10.1016/j.aca.2024.342519},
pmid = {38609262},
issn = {1873-4324},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Biological Assay ; Biotechnology ; RNA ; },
abstract = {The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.},
}
@article {pmid38609257,
year = {2024},
author = {He, W and Li, X and Li, X and Guo, M and Zhang, M and Hu, R and Li, M and Ding, S and Yan, Y},
title = {Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342477},
doi = {10.1016/j.aca.2024.342477},
pmid = {38609257},
issn = {1873-4324},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Nucleic Acids ; DNA, Single-Stranded/genetics ; },
abstract = {CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.},
}
@article {pmid38609223,
year = {2024},
author = {Ma, J and Qian, C and Hu, Q and Zhang, J and Gu, G and Liang, X and Zhang, L},
title = {The bacteriome-coupled phage communities continuously contract and shift to orchestrate the traditional rice vinegar fermentation.},
journal = {Food research international (Ottawa, Ont.)},
volume = {184},
number = {},
pages = {114244},
doi = {10.1016/j.foodres.2024.114244},
pmid = {38609223},
issn = {1873-7145},
mesh = {Humans ; *Oryza ; Acetic Acid ; Fermentation ; *Bacteriophages/genetics ; *Microbiota/genetics ; },
abstract = {Amounts of microbiome studies have uncovered the microbial communities of traditional food fermentations, while in which the phageome development with time is poorly understood. Here, we conducted a study to decipher both phageome and bacteriome of the traditional rice vinegar fermentation. The vinegar phageomes showed significant differences in the alpha diversity, network density and clustering coefficient over time. Peduoviridae had the highest relative abundance. Moreover, the phageome negatively correlated to the cognate bacteriome in alpha diversity, and undergone constantly contracting and shifting across the temporal scale. Nevertheless, 257 core virial clusters (VCs) persistently occurred with time whatever the significant impacts imposed by the varied physiochemical properties. Glycoside hydrolase (GH) and glycosyltransferase (GT) families genes displayed the higher abundances across all samples. Intriguingly, diversely structuring of toxin-antitoxin systems (TAs) and CRISPR-Cas arrays were frequently harbored by phage genomes. Their divergent organization and encoding attributes underlie the multiple biological roles in modulation of network and/or contest of phage community as well as bacterial host community. This phageome-wide mapping will fuel the current insights of phage community ecology in other traditional fermented ecosystems that are challenging to decipher.},
}
@article {pmid38607027,
year = {2024},
author = {Chua, R and Wang, L and Singaraja, R and Ghosh, S},
title = {Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/cells13070588},
pmid = {38607027},
issn = {2073-4409},
support = {2U54GM104940, R01HL146462-01/NH/NIH HHS/United States ; },
mesh = {Humans ; Animals ; Mice ; *Monocytes/metabolism ; *Furin/genetics/metabolism ; U937 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Multiomics ; RNA, Guide, CRISPR-Cas Systems ; Cytokines/genetics ; },
abstract = {The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.},
}
@article {pmid38607007,
year = {2024},
author = {Wang, S and Zhu, Y and Du, S and Zheng, Y},
title = {Preclinical Advances in LNP-CRISPR Therapeutics for Solid Tumor Treatment.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
pmid = {38607007},
issn = {2073-4409},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; *Neoplasms/genetics/therapy ; *Nanostructures ; },
abstract = {Solid tumors, with their intricate cellular architecture and genetic heterogeneity, have long posed therapeutic challenges. The advent of the CRISPR genome editing system offers a promising, precise genetic intervention. However, the journey from bench to bedside is fraught with hurdles, chief among them being the efficient delivery of CRISPR components to tumor cells. Lipid nanoparticles (LNPs) have emerged as a potential solution. This biocompatible nanomaterial can encapsulate the CRISPR/Cas9 system, ensuring targeted delivery while mitigating off-target effects. Pre-clinical investigations underscore the efficacy of LNP-mediated CRISPR delivery, with marked disruption of oncogenic pathways and subsequent tumor regression. Overall, CRISPR/Cas9 technology, when combined with LNPs, presents a groundbreaking approach to cancer therapy, offering precision, efficacy, and potential solutions to current limitations. While further research and clinical testing are required, the future of personalized cancer treatment based on CRISPR/Cas9 holds immense promise.},
}
@article {pmid38605715,
year = {2024},
author = {Shen, Q and Ruan, H and Zhang, H and Wu, T and Zhu, K and Han, W and Dong, R and Ming, T and Qi, H and Zhang, Y},
title = {Utilization of CRISPR-Cas genome editing technology in filamentous fungi: function and advancement potentiality.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1375120},
pmid = {38605715},
issn = {1664-302X},
abstract = {Filamentous fungi play a crucial role in environmental pollution control, protein secretion, and the production of active secondary metabolites. The evolution of gene editing technology has significantly improved the study of filamentous fungi, which in the past was laborious and time-consuming. But recently, CRISPR-Cas systems, which utilize small guide RNA (sgRNA) to mediate clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), have demonstrated considerable promise in research and application for filamentous fungi. The principle, function, and classification of CRISPR-Cas, along with its application strategies and research progress in filamentous fungi, will all be covered in the review. Additionally, we will go over general matters to take into account when editing a genome with the CRISPR-Cas system, including the creation of vectors, different transformation methodologies, multiple editing approaches, CRISPR-mediated transcriptional activation (CRISPRa) or interference (CRISPRi), base editors (BEs), and Prime editors (PEs).},
}
@article {pmid38578074,
year = {2024},
author = {Meng, JN and Xu, ZK and Li, PR and Zeng, X and Liu, Y and Xu, ZL and Wang, J and Ding, Y and Shen, X},
title = {Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8823-8830},
doi = {10.1021/acs.jafc.3c06744},
pmid = {38578074},
issn = {1520-5118},
mesh = {*Food Microbiology ; *Emetics ; Recombinases/genetics ; Bacillus cereus/genetics ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleotidyltransferases/genetics ; },
abstract = {Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10[-2] ng μL[-1] and 10[2] CFU mL[-1], respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.},
}
@article {pmid38575365,
year = {2024},
author = {Wang, Y and Du, P and Shao, Y and Wang, W and Liu, Y and Ma, Y and Hu, P and Cao, J and Wang, X and Abd El-Aty, AM},
title = {An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8831-8839},
doi = {10.1021/acs.jafc.3c08829},
pmid = {38575365},
issn = {1520-5118},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; Coloring Agents ; DNA, Single-Stranded ; Recombinases ; Salmonella/genetics ; Polymerase Chain Reaction ; *Biosensing Techniques ; },
abstract = {Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 10[1]-2 × 10[9] CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.},
}
@article {pmid38564120,
year = {2024},
author = {Sarkar, P and Santiago Vazquez, J and Zhou, M and Levy, A and Mou, Z and Orbović, V},
title = {Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.},
journal = {Transgenic research},
volume = {33},
number = {1-2},
pages = {59-66},
pmid = {38564120},
issn = {1573-9368},
support = {2018-70016-27392//USDA/NIFA Emergency Citrus Disease Research and Extension Program/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Introns ; *Citrus/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Transfer/genetics ; },
abstract = {Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.},
}
@article {pmid38504013,
year = {2024},
author = {Abkallo, HM and Arbuthnot, P and Auer, TO and Berger, DK and Burger, J and Chakauya, E and Concordet, JP and Diabate, A and Di Donato, V and Groenewald, JH and Guindo, A and Koekemoer, LL and Nazare, F and Nolan, T and Okumu, F and Orefuwa, E and Paemka, L and Prieto-Godino, L and Runo, S and Sadler, M and Tesfaye, K and Tripathi, L and Wondji, C},
title = {Making genome editing a success story in Africa.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {551-554},
pmid = {38504013},
issn = {1546-1696},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Africa ; Genome ; },
}
@article {pmid38497640,
year = {2024},
author = {Wang, P and Du, X and Zhao, Y and Wang, W and Cai, T and Tang, K and Wang, X},
title = {Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.},
journal = {Applied and environmental microbiology},
volume = {90},
number = {4},
pages = {e0009524},
pmid = {38497640},
issn = {1098-5336},
support = {32070175//MOST | National Natural Science Foundation of China (NSFC)/ ; 42188102//MOST | National Natural Science Foundation of China (NSFC)/ ; 91951203//MOST | National Natural Science Foundation of China (NSFC)/ ; 2022YFC3103600//MOST | National Key Research and Development Program of China (NKPs)/ ; 2019BT02Y262//Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program/ ; 2021345//Youth Innovation Promotion Association of the Chinese Academy of Sciences (CAS YIPA)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Bacteria/genetics ; Genomic Islands ; Gene Transfer, Horizontal ; Plasmids/genetics ; Interspersed Repetitive Sequences ; },
abstract = {Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.},
}
@article {pmid38323580,
year = {2024},
author = {Romano, A and Mortellaro, A},
title = {The New Frontiers of Gene Therapy and Gene Editing in Inflammatory Diseases.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {219-231},
doi = {10.1089/hum.2023.210},
pmid = {38323580},
issn = {1557-7422},
mesh = {Animals ; Humans ; *Gene Editing/methods ; Quality of Life ; Genetic Therapy/methods ; Genetic Engineering ; *Severe Combined Immunodeficiency ; CRISPR-Cas Systems ; },
abstract = {Inflammatory diseases are conditions characterized by abnormal and often excessive immune responses, leading to tissue and organ inflammation. The complexity of these disorders arises from the intricate interplay of genetic factors and immune responses, which challenges conventional therapeutic approaches. However, the field of genetic manipulation has sparked unprecedented optimism in addressing these complex disorders. This review aims to comprehensively explore the application of gene therapy and gene editing in the context of inflammatory diseases, offering solutions that range from correcting genetic defects to precise immune modulation. These therapies have exhibited remarkable potential in ameliorating symptoms, improving quality of life, and even achieving disease remission. As we delve into recent breakthroughs and therapeutic applications, we illustrate how these advancements offer novel and transformative solutions for conditions that have traditionally eluded conventional treatments. By examining successful case studies and preclinical research, we emphasize the favorable results and substantial transformative impacts that gene-based interventions have demonstrated in patients and animal models of inflammatory diseases such as chronic granulomatous disease, cryopyrin-associated syndromes, and adenosine deaminase 2 deficiency, as well as those of multifactorial origins such as arthropathies (osteoarthritis, rheumatoid arthritis) and inflammatory bowel disease. In conclusion, gene therapy and gene editing offer transformative opportunities to address the underlying causes of inflammatory diseases, ushering in a new era of precision medicine and providing hope for personalized, targeted treatments.},
}
@article {pmid38251667,
year = {2024},
author = {Rai, R and Steinberg, Z and Romito, M and Zinghirino, F and Hu, YT and White, N and Naseem, A and Thrasher, AJ and Turchiano, G and Cavazza, A},
title = {CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {269-283},
doi = {10.1089/hum.2023.100},
pmid = {38251667},
issn = {1557-7422},
mesh = {Infant, Newborn ; Humans ; *Severe Combined Immunodeficiency/genetics/therapy ; T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; Gene Editing/methods ; Receptors, Interleukin-7/genetics/metabolism ; },
abstract = {Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.},
}
@article {pmid38159103,
year = {2024},
author = {Wang, R and Li, Y and Xu, S and Huang, Q and Tu, M and Zhu, Y and Cen, H and Dong, J and Jiang, L and Yao, X},
title = {Genome-wide association study reveals the genetic basis for petal-size formation in rapeseed (Brassica napus) and CRISPR-Cas9-mediated mutagenesis of BnFHY3 for petal-size reduction.},
journal = {The Plant journal : for cell and molecular biology},
volume = {118},
number = {2},
pages = {373-387},
doi = {10.1111/tpj.16609},
pmid = {38159103},
issn = {1365-313X},
support = {2022YFD1200404//National Key Research and Development Program of China/ ; },
mesh = {*Brassica napus/genetics ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Plant Breeding ; *Brassica rapa/genetics ; Mutagenesis ; },
abstract = {Petals in rapeseed (Brassica napus) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal-less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal-size regulation is limited, and the lack of markers and pre-breeding materials hinders targeted petal-size breeding. Here, we conducted a genome-wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal-size variation. Through a cross-analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal-size features can be developed. Leveraging CRISPR-Cas9 technology, we successfully generated a quadruple mutant of Far-Red Elongated Hypocotyl 3 (q-bnfhy3), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal-size regulation in rapeseed and offers abundant potential molecular markers for breeding. The q-bnfhy3 mutant unveiled a novel role of FHY3 orthologues in regulating petal size in addition to previously reported functions.},
}
@article {pmid38069547,
year = {2024},
author = {Zhang, M and Feng, J and Li, Y and Qin, PZ and Chai, Y},
title = {Generation of tamoxifen-inducible Tfap2b-CreER[T2] mice using CRISPR-Cas9.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {62},
number = {1},
pages = {e23582},
pmid = {38069547},
issn = {1526-968X},
support = {R01 DE012711/NH/NIH HHS/United States ; R01 DE022503/NH/NIH HHS/United States ; R01 DE012711/DE/NIDCR NIH HHS/United States ; T90 DE021982/DE/NIDCR NIH HHS/United States ; R01 DE022503/DE/NIDCR NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Tamoxifen/pharmacology ; Mice, Transgenic ; Red Fluorescent Protein ; Integrases/genetics/metabolism ; },
abstract = {Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreER[T2] knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreER[T2] transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.},
}
@article {pmid38062734,
year = {2024},
author = {Whittaker, TE and Moula, SE and Bahal, S and Bakri, FG and Hayajneh, WA and Daoud, AK and Naseem, A and Cavazza, A and Thrasher, AJ and Santilli, G},
title = {Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67[phox]-Deficient Chronic Granulomatous Disease.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {298-312},
pmid = {38062734},
issn = {1557-7422},
support = {/WT_/Wellcome Trust/United Kingdom ; 104807/WT_/Wellcome Trust/United Kingdom ; MR/S021930/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Granulomatous Disease, Chronic/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; Antigens, CD34/genetics ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34[+] hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34[+] cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34[+] cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67[phox]-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67[phox] expression and functional correction of CD34[+]-derived neutrophils from a CGD patient.},
}
@article {pmid37997697,
year = {2024},
author = {Zhang, D and Boch, J},
title = {Development of TALE-adenine base editors in plants.},
journal = {Plant biotechnology journal},
volume = {22},
number = {5},
pages = {1067-1077},
pmid = {37997697},
issn = {1467-7652},
mesh = {Humans ; *Cytosine Deaminase/genetics ; *Adenine ; Gene Editing/methods ; DNA/genetics ; CRISPR-Cas Systems ; *Cytidine Deaminase ; *Proteins ; },
abstract = {Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.},
}
@article {pmid37400521,
year = {2024},
author = {Wessels, HH and Stirn, A and Méndez-Mancilla, A and Kim, EJ and Hart, SK and Knowles, DA and Sanjana, NE},
title = {Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {628-637},
pmid = {37400521},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Deep Learning ; RNA ; Gene Editing ; },
abstract = {Transcriptome engineering applications in living cells with RNA-targeting CRISPR effectors depend on accurate prediction of on-target activity and off-target avoidance. Here we design and test ~200,000 RfxCas13d guide RNAs targeting essential genes in human cells with systematically designed mismatches and insertions and deletions (indels). We find that mismatches and indels have a position- and context-dependent impact on Cas13d activity, and mismatches that result in G-U wobble pairings are better tolerated than other single-base mismatches. Using this large-scale dataset, we train a convolutional neural network that we term targeted inhibition of gene expression via gRNA design (TIGER) to predict efficacy from guide sequence and context. TIGER outperforms the existing models at predicting on-target and off-target activity on our dataset and published datasets. We show that TIGER scoring combined with specific mismatches yields the first general framework to modulate transcript expression, enabling the use of RNA-targeting CRISPRs to precisely control gene dosage.},
}
@article {pmid37365261,
year = {2024},
author = {},
title = {Efficient A-to-C base editing with high specificity.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {578-579},
pmid = {37365261},
issn = {1546-1696},
support = {32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid37322276,
year = {2024},
author = {Chen, L and Hong, M and Luan, C and Gao, H and Ru, G and Guo, X and Zhang, D and Zhang, S and Li, C and Wu, J and Randolph, PB and Sousa, AA and Qu, C and Zhu, Y and Guan, Y and Wang, L and Liu, M and Feng, B and Song, G and Liu, DR and Li, D},
title = {Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {638-650},
pmid = {37322276},
issn = {1546-1696},
support = {82100773//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; U01AI142756//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing ; *Adenine/metabolism ; Mutation ; Cytosine/metabolism ; Adenosine ; CRISPR-Cas Systems/genetics ; Mammals/genetics ; },
abstract = {Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.},
}
@article {pmid38605260,
year = {2024},
author = {Rafiq, MS and Shabbir, MA and Raza, A and Irshad, S and Asghar, A and Maan, MK and Gondal, MA and Hao, H},
title = {CRISPR-Cas System: A New Dawn to Combat Antibiotic Resistance.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38605260},
issn = {1179-190X},
support = {32172914//the National Natural Science Foundation of China/ ; 2021YFD1800600//Key Technology Research and Development Program of Shandong Province/ ; . 2662022DKYJC005//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Antimicrobial resistance (AMR) can potentially harm global public health. Horizontal gene transfer (HGT), which speeds up the emergence of AMR and increases the burden of drug resistance in mobile genetic elements (MGEs), is the primary method by which AMR genes are transferred across bacterial pathogens. New approaches are urgently needed to halt the spread of bacterial diseases and antibiotic resistance. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an RNA-guided adaptive immune system, protects prokaryotes from foreign DNA like plasmids and phages. This approach may be essential in limiting horizontal gene transfer and halting the spread of antibiotic resistance. The CRISPR-Cas system has been crucial in identifying and understanding resistance mechanisms and developing novel therapeutic approaches. This review article investigates the CRISPR-Cas system's potential as a tool to combat bacterial AMR. Antibiotic-resistant bacteria can be targeted and eliminated by the CRISPR-Cas system. It has been proven to be an efficient method for removing carbapenem-resistant plasmids and regaining antibiotic susceptibility. The CRISPR-Cas system has enormous potential as a weapon against bacterial AMR. It precisely targets and eliminates antibiotic-resistant bacteria, facilitates resistance mechanism identification, and offers new possibilities in diagnostics and therapeutics.},
}
@article {pmid38605095,
year = {2024},
author = {de Boer, EN and Vroom, V and Scheper, AJ and Johansson, LF and Bosscher, L and Rietema, N and Commandeur-Jan, SZ and Knoers, NVAM and Sikkema-Raddatz, B and van den Berg, E and van Diemen, CC},
title = {Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8508},
pmid = {38605095},
issn = {2045-2322},
mesh = {Humans ; Reproducibility of Results ; *CRISPR-Cas Systems ; Karyotyping ; *Leukemia ; Chromosome Mapping ; },
abstract = {Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.},
}
@article {pmid38603815,
year = {2024},
author = {Bai, H and He, LY and Gao, FZ and Yao, KS and Zhang, M and Qiao, LK and Chen, ZY and He, LX and Liu, YS and Zhao, JL and Ying, GG},
title = {Airborne antibiotic resistome and microbiome in pharmaceutical factories.},
journal = {Environment international},
volume = {186},
number = {},
pages = {108639},
doi = {10.1016/j.envint.2024.108639},
pmid = {38603815},
issn = {1873-6750},
abstract = {Antimicrobial resistance is considered to be one of the biggest public health problems, and airborne transmission is an important but under-appreciated pathway for the spread of antibiotic resistance genes (ARGs) in the environment. Previous research has shown pharmaceutical factories to be a major source of ARGs and antibiotic resistant bacteria (ARB) in the surrounding receiving water and soil environments. Pharmaceutical factories are hotspots of antibiotic resistance, but the atmospheric transmission and its environmental risk remain more concerns. Here, we conducted a metagenomic investigation into the airborne microbiome and resistome in three pharmaceutical factories in China. Soil (average: 38.45%) and wastewater (average: 28.53%) were major contributors of airborne resistome. ARGs (vanR/vanS, blaOXA, and CfxA) conferring resistance to critically important clinically used antibiotics were identified in the air samples. The wastewater treatment area had significantly higher relative abundances of ARGs (average: 0.64 copies/16S rRNA). Approximately 28.2% of the detected airborne ARGs were found to be associated with plasmids, and this increased to about 50% in the wastewater treatment area. We have compiled a list of high-risk airborne ARGs found in pharmaceutical factories. Moreover, A total of 1,043 viral operational taxonomic units were identified and linked to 47 family-group taxa. Different CRISPR-Cas immune systems have been identified in bacterial hosts in response to phage infection. Similarly, higher phage abundance (average: 2451.70 PPM) was found in the air of the wastewater treatment area. Our data provide insights into the antibiotic resistance gene profiles and microbiome (bacterial and non-bacterial) in pharmaceutical factories and reveal the potential role of horizontal transfer in the spread of airborne ARGs, with implications for human and animal health.},
}
@article {pmid38602884,
year = {2024},
author = {van Beljouw, SPB and Haagsma, AC and Kalogeropoulos, K and Pabst, M and Brouns, SJJ},
title = {Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.},
journal = {ACS chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acschembio.3c00788},
pmid = {38602884},
issn = {1554-8937},
abstract = {The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.},
}
@article {pmid38600465,
year = {2024},
author = {Ci, Y and Zhang, Y and Zhang, X},
title = {Methylated lncRNAs suppress apoptosis of gastric cancer stem cells via the lncRNA-miRNA/protein axis.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {51},
pmid = {38600465},
issn = {1689-1392},
support = {DY135-B-04//China Ocean Mineral Resources Research and Development Association/ ; },
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems ; Carcinogenesis/genetics ; Apoptosis/genetics ; Neoplastic Stem Cells/metabolism ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; Methyltransferases/genetics ; },
abstract = {BACKGROUND: Long noncoding RNAs (lncRNAs) play essential roles in the tumorigenesis of gastric cancer. However, the influence of lncRNA methylation on gastric cancer stem cells (GCSCs) remains unclear.<br><br>METHODS: The N6-methyladenosine (m6A) levels of lncRNAs in gastric cancer stem cells were detected by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the results were validated by MeRIP-quantitative polymerase chain reaction (qPCR). Specific sites of m6A modification on lncRNAs were detected by single-base elongation- and ligation-based qPCR amplification (SELECT). By constructing and transfecting the plasmid expressing methyltransferase-like 3 (METTL3) fused with catalytically inactivated Cas13 (dCas13b) and guide RNA targeting specific methylation sites of lncRNAs, we obtained gastric cancer stem cells with site-specific methylation of lncRNAs. Reverse transcription (RT)-qPCR and Western blot were used for detecting the stemness of treated gastric cancer stem cells.<br><br>RESULTS: The site-specific methylation of PSMA3-AS1 and MIR22HG suppressed apoptosis and promoted stemness of GCSCs. LncRNA methylation enhanced the stability of PSMA3-AS1 and MIR22HG to suppress apoptosis of GCSCs via the PSMA3-AS1-miR-411-3p- or MIR22HG-miR-24-3p-SERTAD1 axis. Simultaneously, the methylated lncRNAs promoted the interaction between PSMA3-AS1 and the EEF1A1 protein or MIR22HG and the LRPPRC protein, stabilizing the proteins and leading to the suppression of apoptosis. The in vivo data revealed that the methylated PSMA3-AS1 and MIR22HG triggered tumorigenesis of GCSCs.<br><br>CONCLUSIONS: Our study revealed the requirement for site-specific methylation of lncRNAs in the tumorigenesis of GCSCs, contributing novel insights into cancer development.},
}
@article {pmid38600182,
year = {2024},
author = {Moncaut, N},
title = {Streamlining mouse genome editing by integrating AAV repair template delivery and CRISPR-Cas electroporation.},
journal = {Lab animal},
volume = {},
number = {},
pages = {},
pmid = {38600182},
issn = {1548-4475},
}
@article {pmid38599852,
year = {2024},
author = {Liu, S and Zhang, Y and Luo, Y and Liu, J},
title = {Traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine.},
journal = {Journal of diabetes},
volume = {16},
number = {4},
pages = {e13545},
pmid = {38599852},
issn = {1753-0407},
support = {20202ACBL206008//Natural Science Foundation of Jiangxi Provincial/ ; 81760150//National Natural Science Foundation of China/ ; 82160162//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Regenerative Medicine ; Pancreas ; *Diabetes Mellitus, Type 1 ; Hepatocytes ; },
abstract = {Although pancreas and islet cell transplantation are the only ways to prevent the late complications of insulin-dependent diabetes, a shortage of donors is a major obstacle to tissue and organ transplantation. Stem cell therapy is an effective treatment for diabetes and other pancreatic-related diseases, which can be achieved by inducing their differentiation into insulin-secreting cells. The liver is considered an ideal source of pancreatic cells due to its similar developmental origin and strong regenerative ability as the pancreas. This article reviews the traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine and evaluates their advantages and challenges. Gene reprogramming and chemical reprogramming technologies are traditional strategies with potential to improve the efficiency and specificity of cell reprogramming and promote the transformation of hepatocytes into islet cells. At the same time, organoid technology, as an emerging strategy, has received extensive attention. Biomaterials provide a three-dimensional culture microenvironment for cells, which helps improve cell survival and differentiation efficiency. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has brought new opportunities and challenges to the development of organoid technology.},
}
@article {pmid38599684,
year = {2024},
author = {Bukhari, H and Nithianadam, V and Battaglia, RA and Cicalo, A and Sarkar, S and Comjean, A and Hu, Y and Leventhal, MJ and Dong, X and Feany, MB},
title = {Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.},
journal = {Genome research},
volume = {},
number = {},
pages = {},
doi = {10.1101/gr.278576.123},
pmid = {38599684},
issn = {1549-5469},
abstract = {Missense mutations in the gene encoding the microtubule-associated protein tau cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human tau in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease, but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas mediated gene editing to model frontotemporal dementia caused by the tau P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for tau P251L display age-dependent neurodegeneration, metabolic defects, and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies we performed single-cell RNA sequencing on approximately 130,000 cells from brains of tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified. Gene expression was also altered in glial cells, suggestive of non-cell autonomous regulation. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell type-specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy, which faithfully recapitulates the genetic context and phenotypic features of the human disease and use the results of comprehensive single cell sequencing analysis to outline pathways of neurotoxicity and highlight the potential role of non-cell autonomous changes in glia.},
}
@article {pmid38598861,
year = {2024},
author = {Burbano, DA and Kiattisewee, C and Karanjia, AV and Cardiff, RAL and Faulkner, ID and Sugianto, W and Carothers, JM},
title = {CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.},
journal = {Annual review of chemical and biomolecular engineering},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-chembioeng-100522-114706},
pmid = {38598861},
issn = {1947-5446},
abstract = {In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.},
}
@article {pmid38521283,
year = {2024},
author = {Hwang, J and Ye, DY and Jung, GY and Jang, S},
title = {Mobile genetic element-based gene editing and genome engineering: Recent advances and applications.},
journal = {Biotechnology advances},
volume = {72},
number = {},
pages = {108343},
doi = {10.1016/j.biotechadv.2024.108343},
pmid = {38521283},
issn = {1873-1899},
mesh = {*Gene Editing ; *Genetic Engineering ; Mutagenesis ; Interspersed Repetitive Sequences ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.},
}
@article {pmid38490381,
year = {2024},
author = {Sun, Z and Wang, M and Wang, W and Li, D and Wang, J and Sui, G},
title = {Getah virus capsid protein undergoes co-condensation with viral genomic RNA to facilitate virion assembly.},
journal = {International journal of biological macromolecules},
volume = {265},
number = {Pt 1},
pages = {130847},
doi = {10.1016/j.ijbiomac.2024.130847},
pmid = {38490381},
issn = {1879-0003},
mesh = {Animals ; Humans ; *Alphavirus/genetics/metabolism ; Capsid Proteins/genetics/metabolism ; RNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; Virion/genetics ; },
abstract = {Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.},
}
@article {pmid38235591,
year = {2024},
author = {Ghahremani, S and Kanwal, A and Pettinato, A and Ladha, F and Legere, N and Thakar, K and Zhu, Y and Tjong, H and Wilderman, A and Stump, WT and Greenberg, L and Greenberg, MJ and Cotney, J and Wei, CL and Hinson, JT},
title = {CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.},
journal = {Circulation},
volume = {149},
number = {16},
pages = {1285-1297},
doi = {10.1161/CIRCULATIONAHA.123.063972},
pmid = {38235591},
issn = {1524-4539},
support = {R01 HL141086/HL/NHLBI NIH HHS/United States ; R01 HL165220/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Cardiomyopathy, Dilated/genetics/therapy/pathology ; Connectin/genetics ; Haploinsufficiency/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; Myocytes, Cardiac/metabolism ; },
abstract = {BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity.<br><br>METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation.<br><br>RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts.<br><br>CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.},
}
@article {pmid38598855,
year = {2024},
author = {Hsieh, SC and Peters, JE},
title = {Natural and Engineered Guide RNA-directed Transposition with CRISPR-Associated Tn7-like Transposons.},
journal = {Annual review of biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-biochem-030122-041908},
pmid = {38598855},
issn = {1545-4509},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.},
}
@article {pmid38597925,
year = {2024},
author = {Li, J and Yan, K and Wang, S and Wang, P and Jiao, J and Dong, Y},
title = {Alteration of the intestinal microbiota and serum metabolites in a mouse model of Pon1 gene ablation.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {38},
number = {7},
pages = {e23611},
doi = {10.1096/fj.202302344R},
pmid = {38597925},
issn = {1530-6860},
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; RNA, Guide, CRISPR-Cas Systems ; Disease Models, Animal ; *Microbiota ; Aryldialkylphosphatase/genetics ; Mice, Knockout ; },
abstract = {Mutations in the Paraoxonase 1 (Pon1) gene underlie aging, cardiovascular disease, and impairments of the nervous and gastrointestinal systems and are linked to the intestinal microbiome. The potential role of Pon1 in modulating the intestinal microbiota and serum metabolites is poorly understood. The present study demonstrated that mice with genomic excision of Pon1 by a multiplexed guide RNA CRISPR/Cas9 approach exhibited disrupted gut microbiota, such as significantly depressed alpha-diversity and distinctly separated beta diversity, accompanied by varied profiles of circulating metabolites. Furthermore, genomic knock in of Pon1 exerted a distinct effect on the intestinal microbiome and serum metabolome, including dramatically enriched Aerococcus, linoleic acid and depleted Bacillus, indolelactic acid. Specifically, a strong correlation was established between bacterial alterations and metabolites in Pon1 knockout mice. In addition, we identified metabolites related to gut bacteria in response to Pon1 knock in. Thus, the deletion of Pon1 affects the gut microbiome and functionally modifies serum metabolism, which can lead to dysbiosis, metabolic dysfunction, and infection risk. Together, these findings put forth a role for Pon1 in microbial alterations that contribute to metabolism variations. The function of Pon1 in diseases might at least partially depend on the microbiome.},
}
@article {pmid38597776,
year = {2024},
author = {Xie, H and Su, F and Niu, Q and Geng, L and Cao, X and Song, M and Dong, J and Zheng, Z and Guo, R and Zhang, Y and Deng, Y and Ji, Z and Pang, K and Zhu, JK and Zhu, J},
title = {Knockout of miR396 genes increases seed size and yield in soybean.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13660},
pmid = {38597776},
issn = {1744-7909},
support = {2021LZGC003//Seed-Industrialized Development Program in Shandong Province, China/ ; 2022AH010056//the Innovative research team of Anhui education/ ; 2021LZGC012//Key Research &amp; Development Program of Shandong Province, China/ ; 32270367//National Natural Science Foundation of China/ ; HSPHDSRF-2023-11-007//the PhD Scientific Research and Innovation Foundation of Sanya Yazhou Bay Science and Technology City/ ; NK2022010301//National Key Research and Development Program of China/ ; },
abstract = {Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.},
}
@article {pmid38414349,
year = {2024},
author = {Zeng, J and Liang, X and Duan, L and Tan, F and Chen, L and Qu, J and Li, J and Li, K and Luo, D and Hu, Z},
title = {Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.},
journal = {Acta biochimica et biophysica Sinica},
volume = {56},
number = {4},
pages = {525-537},
doi = {10.3724/abbs.2023280},
pmid = {38414349},
issn = {1745-7270},
mesh = {Humans ; Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems ; Fusion Proteins, bcr-abl/genetics/metabolism ; Genes, abl ; Proto-Oncogene Proteins c-bcr/genetics/metabolism ; CRISPR-Cas Systems ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism/therapy ; Apoptosis/genetics ; Cell Proliferation/genetics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein (BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase (ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.},
}
@article {pmid38365083,
year = {2024},
author = {Duan, X and Li, H and Tan, X and Liu, N and Wang, X and Zhang, W and Liu, Y and Ma, W and Wu, Y and Ma, L and Fan, Y},
title = {Polygonum cillinerve polysaccharide inhibits transmissible gastroenteritis virus by regulating microRNA-181.},
journal = {Veterinary journal (London, England : 1997)},
volume = {304},
number = {},
pages = {106083},
doi = {10.1016/j.tvjl.2024.106083},
pmid = {38365083},
issn = {1532-2971},
mesh = {Animals ; Swine ; *Transmissible gastroenteritis virus/genetics ; *Polygonum/genetics ; RNA, Guide, CRISPR-Cas Systems ; Signal Transduction ; *MicroRNAs/genetics ; },
abstract = {Transmissible gastroenteritis virus (TGEV) is an important pathogen capable of altering the expression profile of cellular miRNA. In this study, the potential of Polygonum cillinerve polysaccharide (PCP) to treat TGEV-infected piglets was evaluated through in vivo experiments. High-throughput sequencing technology was employed to identify 9 up-regulated and 17 down-regulated miRNAs during PCP-mediated inhibition of TGEV infection in PK15 cells. Additionally, miR-181 was found to be associated with target genes of key proteins in the apoptosis pathway. PK15 cells were treated with various concentrations of PCP following transfection with miR-181 mimic or inhibitor. Real-time PCR assessed the impact on TGEV replication, while electron microscopy (TEM) and Hoechst fluorescence staining evaluated cellular functionality. Western blot analysis was utilized to assess the expression of key signaling factors-cytochrome C (cyt C), caspase 9, and P53-in the apoptotic signaling pathway. The results showed that compared with the control group, 250 μg/mL PCP significantly inhibited TGEV gRNA replication and gene N expression (P < 0.01). Microscopic examination revealed uniform cell morphology and fewer floating cells in PCP-treated groups (250 and 125 μg/mL). TEM analysis showed no typical virus structure in the 250 μg/mL PCP group, and apoptosis staining indicated a significant reduction in apoptotic cells at this concentration. Furthermore, PCP may inhibit TGEV-induced apoptosis via the Caspase-dependent mitochondrial pathway following miR-181 transfection. These findings provide a theoretical basis for further exploration into the mechanism of PCP's anti-TGEV properties.},
}
@article {pmid38284432,
year = {2024},
author = {Bagheri, N and Chamorro, A and Idili, A and Porchetta, A},
title = {PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {63},
number = {17},
pages = {e202319677},
doi = {10.1002/anie.202319677},
pmid = {38284432},
issn = {1521-3773},
support = {MFAG 2022 - ID. 27151 project//Associazione Italiana per la Ricerca sul Cancro/ ; 2022FPYZ2N//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; 101086341//H2020 Marie Sk&#x142;odowska-Curie Actions/ ; MSCA_0000010//European Union NextGenerationEU/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; DNA/metabolism ; Nucleic Acid Conformation ; },
abstract = {The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.},
}
@article {pmid38589794,
year = {2024},
author = {Wang, Y and Zhai, Y and Zhang, M and Song, C and Zhang, Y and Zhang, G},
title = {Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.},
journal = {Cellular &amp; molecular biology letters},
volume = {29},
number = {1},
pages = {48},
pmid = {38589794},
issn = {1689-1392},
support = {31970666//the National Natural Science Foundation of China/ ; tsqn201812054//Taishan Scholar Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.},
}
@article {pmid38587910,
year = {2024},
author = {Sakamoto, N and Watanabe, K and Awazu, A and Yamamoto, T},
title = {CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.},
journal = {Zoological science},
volume = {41},
number = {2},
pages = {159-166},
doi = {10.2108/zs230052},
pmid = {38587910},
issn = {0289-0003},
mesh = {Animals ; *Anthocidaris/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Knockout Techniques ; Sea Urchins/genetics ; Gene Editing/methods ; },
abstract = {Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.},
}
@article {pmid38585710,
year = {2024},
author = {Xiao, Y and Qin, T and He, S and Chen, Y and Li, H and He, Q and Wang, X and Yang, S},
title = {Systematic investigation of TetR-family transcriptional regulators and their roles on lignocellulosic inhibitor acetate tolerance in Zymomonas mobilis.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {12},
number = {},
pages = {1385519},
pmid = {38585710},
issn = {2296-4185},
abstract = {TetR-family transcriptional regulators are widely distributed among bacteria and involved in various cellular processes such as multidrug and inhibitor resistance. Zymomonas mobilis is a industrial bacterium for lignocellulosic ethanol production. Although TetR-family regulators and their associated RND-family efflux pumps in Z. mobilis have been identified to be differentially expressed under various inhibitors and stressful conditions, there are no systematic investigation yet. In this study, bioinformatic analyses indicated that there are three TetR-family transcriptional regulators (ZMO0281, ZMO0963, ZMO1547) and two RND-family efflux pumps (ZMO0282-0285, ZMO0964-0966) adjacent to corresponding TetR-family regulators of ZMO0281 and ZMO0963 in Z. mobilis. Genetics studies were then carried out with various mutants of TetR-family regulators constructed, and ZMO0281 was characterized to be related to acetate tolerance. Combining transcriptomics and dual-reporter gene system, this study demonstrated that three TetR-family regulators repressed their adjacent genes specifically. Moreover, TetR-family regulator ZMO0281 might also be involved in other cellular processes in the presence of acetate. In addition, the upregulation of RND-family efflux pumps due to ZMO0281 deletion might lead to an energy imbalance and decreased cell growth in Z. mobilis under acetate stress. The systematic investigation of all three TetR-family regulators and their roles on a major lignocellulosic inhibitor acetate tolerance in Z. mobilis thus not only unravels the molecular mechanisms of TetR-family regulators and their potential cross-talks on regulating RND-family efflux pumps and other genes in Z. mobilis, but also provides guidance on understanding the roles of multiple regulators of same family in Z. mobilis and other microorganisms for efficient lignocellulosic biochemical production.},
}
@article {pmid38585687,
year = {2024},
author = {Fischer, J and Fedotova, A and Jaki, L and Sallard, E and Erhardt, A and Fuchs, J and Ruzsics, Z},
title = {Combining CRISPR-Cas-mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {32},
number = {2},
pages = {101241},
pmid = {38585687},
issn = {2329-0501},
abstract = {While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 10[6] unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 10[4] unique rAds per cm[2] of transfected cell culture.},
}
@article {pmid38584940,
year = {2024},
author = {Carhuaricra-Huaman, D and Gonzalez, IHL and Ramos, PL and da Silva, AM and Setubal, JC},
title = {Analysis of twelve genomes of the bacterium Kerstersia gyiorum from brown-throated sloths (Bradypus variegatus), the first from a non-human host.},
journal = {PeerJ},
volume = {12},
number = {},
pages = {e17206},
pmid = {38584940},
issn = {2167-8359},
mesh = {Animals ; *Sloths/genetics ; Phylogeny ; Brazil ; *Alcaligenaceae/genetics ; },
abstract = {Kerstersia gyiorum is a Gram-negative bacterium found in various animals, including humans, where it has been associated with various infections. Knowledge of the basic biology of K. gyiorum is essential to understand the evolutionary strategies of niche adaptation and how this organism contributes to infectious diseases; however, genomic data about K. gyiorum is very limited, especially from non-human hosts. In this work, we sequenced 12 K. gyiorum genomes isolated from healthy free-living brown-throated sloths (Bradypus variegatus) in the Parque Estadual das Fontes do Ipiranga (São Paulo, Brazil), and compared them with genomes from isolates of human origin, in order to gain insights into genomic diversity, phylogeny, and host specialization of this species. Phylogenetic analysis revealed that these K. gyiorum strains are structured according to host. Despite the fact that sloth isolates were sampled from a single geographic location, the intra-sloth K. gyiorum diversity was divided into three clusters, with differences of more than 1,000 single nucleotide polymorphisms between them, suggesting the circulation of various K. gyiorum lineages in sloths. Genes involved in mobilome and defense mechanisms against mobile genetic elements were the main source of gene content variation between isolates from different hosts. Sloth-specific K. gyiorum genome features include an IncN2 plasmid, a phage sequence, and a CRISPR-Cas system. The broad diversity of defense elements in K. gyiorum (14 systems) may prevent further mobile element flow and explain the low amount of mobile genetic elements in K. gyiorum genomes. Gene content variation may be important for the adaptation of K. gyiorum to different host niches. This study furthers our understanding of diversity, host adaptation, and evolution of K. gyiorum, by presenting and analyzing the first genomes of non-human isolates.},
}
@article {pmid38583547,
year = {2024},
author = {Saunier, M and Fortier, LC and Soutourina, O},
title = {RNA-based regulation in bacteria-phage interactions.},
journal = {Anaerobe},
volume = {87},
number = {},
pages = {102851},
doi = {10.1016/j.anaerobe.2024.102851},
pmid = {38583547},
issn = {1095-8274},
abstract = {Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.},
}
@article {pmid38582932,
year = {2024},
author = {Wang, Y and Peng, Y and Zi, G and Chen, J and Peng, B},
title = {Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8095},
pmid = {38582932},
issn = {2045-2322},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics/metabolism ; *Breast Neoplasms/genetics ; Gene Editing/methods ; },
abstract = {Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.},
}
@article {pmid38582885,
year = {2024},
author = {Chan, YT and Wu, J and Lu, Y and Li, Q and Feng, Z and Xu, L and Yuan, H and Xing, T and Zhang, C and Tan, HY and Feng, Y and Wang, N},
title = {Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC.},
journal = {Molecular cancer},
volume = {23},
number = {1},
pages = {74},
pmid = {38582885},
issn = {1476-4598},
support = {104006600//University Research Committee of The University of Hong Kong/ ; 17119621//Research Grant Committee of Hong Kong/ ; 19201591//The Health and Medical Research Fund/ ; PRP/028/22FX//Innovation and Technology Fund/ ; },
mesh = {Humans ; Mice ; Animals ; Sorafenib/pharmacology/therapeutic use ; *Carcinoma, Hepatocellular/drug therapy/genetics/metabolism ; *RNA, Long Noncoding/genetics ; *Liver Neoplasms/drug therapy/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; PPAR alpha/genetics/metabolism/therapeutic use ; Proteomics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND AND AIMS: Sorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC.<br><br>MATERIALS AND METHODS: A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array.<br><br>RESULTS: CRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPAR&#x3b1; is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPAR&#x3b1; inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC.<br><br>CONCLUSION: Our study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.},
}
@article {pmid38565025,
year = {2024},
author = {Zhu, Y and Lin, Y and Gong, B and Zhang, Y and Su, G and Yu, Y},
title = {Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping.},
journal = {Biosensors &amp; bioelectronics},
volume = {255},
number = {},
pages = {116255},
doi = {10.1016/j.bios.2024.116255},
pmid = {38565025},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Genotype ; Polymorphism, Single Nucleotide/genetics ; *Biosensing Techniques ; Apolipoproteins E ; Uracil-DNA Glycosidase ; },
abstract = {Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.},
}
@article {pmid38552526,
year = {2024},
author = {Yang, Y and Sun, L and Zhao, J and Jiao, Y and Han, T and Zhou, X},
title = {Improving trans-cleavage activity of CRISPR-Cas13a using engineered crRNA with a uridinylate-rich 5'-overhang.},
journal = {Biosensors &amp; bioelectronics},
volume = {255},
number = {},
pages = {116239},
doi = {10.1016/j.bios.2024.116239},
pmid = {38552526},
issn = {1873-4235},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Biosensing Techniques ; RNA ; Recombinases ; SARS-CoV-2 ; CRISPR-Cas Systems/genetics ; },
abstract = {The engieering of Cas13a crRNA to enhance its binding affinity with the Cas enzyme or target is a promising method of improving the collateral cleavage efficiency of CRISPR-Cas13a systems, thereby amplifying the sensitivity of nucleic acid detection. An examination of the top-performing engineered crRNA (24 nt 5'7U LbuCas13a crRNA, where the 5'-end was extended using 7-mer uridinylates) and optimized conditions revealed an increased rate of LbuCas13a-mediated collateral cleavage activity that was up to seven-fold higher than that of the original crRNA. Particularly, the 7-mer uridinylates extension to crRNA was determined to be spacer-independent for enhancing the LbuCas13a-mediacted collateral cleavage activity, and also benefited the LwaCas13a system. The improved trans-cleavage activity was explained by the interactions between crRNA and LbuCas13a at the molecular level, i.e. the 5'-overhangs were anchored in the cleft formed between the Helical-1 and HEPN2 domains with the consequence of more stable complex, and experimentally verified. Consequently, the improved CRISPR-Cas13a system detected the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a sensitivity of 2.36 fM that was 160-times higher than that of the original system. Using isothermal amplification via reverse transcription-recombinase polymerase amplification (RT-RPA), the system was capable to detect SARS-CoV-2 with attomolar sensitivity and accurately identified the SARS-CoV-2 Omicron variant (20/21 agreement) in clinical samples within 40 min.},
}
@article {pmid38504114,
year = {2024},
author = {Yao, D and Tycko, J and Oh, JW and Bounds, LR and Gosai, SJ and Lataniotis, L and Mackay-Smith, A and Doughty, BR and Gabdank, I and Schmidt, H and Guerrero-Altamirano, T and Siklenka, K and Guo, K and White, AD and Youngworth, I and Andreeva, K and Ren, X and Barrera, A and Luo, Y and Yardımcı, GG and Tewhey, R and Kundaje, A and Greenleaf, WJ and Sabeti, PC and Leslie, C and Pritykin, Y and Moore, JE and Beer, MA and Gersbach, CA and Reddy, TE and Shen, Y and Engreitz, JM and Bassik, MC and Reilly, SK},
title = {Multicenter integrated analysis of noncoding CRISPRi screens.},
journal = {Nature methods},
volume = {21},
number = {4},
pages = {723-734},
pmid = {38504114},
issn = {1548-7105},
support = {UM1 HG012053/HG/NHGRI NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; U01 HG012103/HG/NHGRI NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; R01 HG010741/HG/NHGRI NIH HHS/United States ; R00 HG010669/HG/NHGRI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; U24 HG009397/HG/NHGRI NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; U01 HG009380/HG/NHGRI NIH HHS/United States ; U01 HG009395/HG/NHGRI NIH HHS/United States ; U01 HG009431/HG/NHGRI NIH HHS/United States ; K00 DK126120/DK/NIDDK NIH HHS/United States ; UM1 HG009435/HG/NHGRI NIH HHS/United States ; R01 HG012872/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; U24 HG009446/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genome ; K562 Cells ; },
abstract = {The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.},
}
@article {pmid38499809,
year = {2024},
author = {Manjunath, LE and Singh, A and Devi Kumar, S and Vasu, K and Kar, D and Sellamuthu, K and Eswarappa, SM},
title = {Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.},
journal = {EMBO reports},
volume = {25},
number = {4},
pages = {2118-2143},
pmid = {38499809},
issn = {1469-3178},
support = {BT/PR38405/GET/119/309/2020//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; SB/SJF/2020-21/18//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; STARS/APR2019/BS/328/FS//Ministry of Education, Government of India/ ; ID-2021-13048//Indian Council of Medical Research (ICMR)/ ; },
mesh = {Animals ; Codon, Terminator/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Codon, Nonsense/genetics ; RNA, Messenger/genetics/metabolism ; Protein Biosynthesis ; Mammals/genetics/metabolism ; },
abstract = {Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.},
}
@article {pmid38490564,
year = {2024},
author = {Pennati, A and Jakobi, M and Zeng, F and Ciampa, L and Rothbächer, U},
title = {Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.},
journal = {Developmental biology},
volume = {510},
number = {},
pages = {31-39},
doi = {10.1016/j.ydbio.2024.03.003},
pmid = {38490564},
issn = {1095-564X},
mesh = {Animals ; *Ciona intestinalis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Ciona/genetics ; Electroporation ; Gene Editing/methods ; },
abstract = {CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.},
}
@article {pmid38412306,
year = {2024},
author = {Capin, J and Harrison, A and Raele, RA and Yadav, SKN and Baiwir, D and Mazzucchelli, G and Quinton, L and Satchwell, TJ and Toye, AM and Schaffitzel, C and Berger, I and Aulicino, F},
title = {An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.},
journal = {Nucleic acids research},
volume = {52},
number = {6},
pages = {3450-3468},
pmid = {38412306},
issn = {1362-4962},
support = {834631/ERC_/European Research Council/International ; MR/V010506/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Mammals/genetics ; Baculoviridae/genetics ; DNA/genetics ; },
abstract = {CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.},
}
@article {pmid38409225,
year = {2024},
author = {Montero, JJ and Trozzo, R and Sugden, M and Öllinger, R and Belka, A and Zhigalova, E and Waetzig, P and Engleitner, T and Schmidt-Supprian, M and Saur, D and Rad, R},
title = {Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx.},
journal = {Nature methods},
volume = {21},
number = {4},
pages = {584-596},
pmid = {38409225},
issn = {1548-7105},
support = {DFG RA1629/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB1321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB1371//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; (70114314)//Deutsche Krebshilfe (German Cancer Aid)/ ; Consolidator grant PACA-MET (819642)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; MSCA-ITNETN (861196//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; Long-Term Fellowship (ALFT 655-2019).//European Molecular Biology Organization (EMBO)/ ; },
mesh = {Humans ; Gene Expression Profiling ; *RNA, Long Noncoding/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Transcriptome ; *Neoplasms/genetics/metabolism ; },
abstract = {Although long noncoding RNAs (lncRNAs) dominate the transcriptome, their functions are largely unexplored. The extensive overlap of lncRNAs with coding and regulatory sequences restricts their systematic interrogation by DNA-directed perturbation. Here we developed genome-scale lncRNA transcriptome screening using Cas13d/CasRx. We show that RNA targeting overcomes limitations inherent to other screening methods, thereby considerably expanding the explorable space of the lncRNAome. By evolving the screening system toward pan-cancer applicability, it supports molecular and phenotypic data integration to contextualize screening hits or infer lncRNA function. We thereby addressed challenges posed by the enormous transcriptome size and tissue specificity through a size-reduced multiplexed gRNA library termed Albarossa, targeting 24,171 lncRNA genes. Its rational design incorporates target prioritization based on expression, evolutionary conservation and tissue specificity, thereby reconciling high discovery power and pan-cancer representation with scalable experimental throughput. Applied across entities, the screening platform identified numerous context-specific and common essential lncRNAs. Our work sets the stage for systematic exploration of lncRNA biology in health and disease.},
}
@article {pmid38379122,
year = {2024},
author = {Li, S and Mei, L and He, C and Cai, X and Wu, H and Wu, X and Liu, Y and Feng, Y and Song, J},
title = {Identification of a family with van der Hoeve's syndrome harboring a novel COL1A1 mutation and generation of patient-derived iPSC lines and CRISPR/Cas9-corrected isogenic iPSCs.},
journal = {Human cell},
volume = {37},
number = {3},
pages = {817-831},
pmid = {38379122},
issn = {1749-0774},
support = {82071065//National Natural Science Foundation of China/ ; 2014CB541702//Major State Basic Research Development Program of China/ ; 2020YFC2005204//Key Technologies Research and Development Program/ ; 2020SK2106//Key Research and Development Program of Hunan Province of China/ ; 2021zzts0345//Fundamental Research Funds for Central Universities of the Central South University/ ; CX20210355//Fundamental Research Funds for Central Universities of the Central South University/ ; 2021M693566//Postdoctoral Research Foundation of China/ ; 2021T140751//Postdoctoral Research Foundation of China/ ; 2020RC2013//Science and Technology Program of Hunan Province/ ; 2021JJ41017//Natural Science Foundation of&#xa0;Hunan Province/ ; 2021JJ31084//Natural Science Foundation of Hunan Province/ ; 202107010047//Health Commission of Hunan Province/ ; },
mesh = {Humans ; *Osteogenesis Imperfecta/genetics/therapy ; Collagen Type I/genetics ; *Induced Pluripotent Stem Cells ; Leukocytes, Mononuclear ; CRISPR-Cas Systems/genetics ; Collagen Type I, alpha 1 Chain ; Mutation ; },
abstract = {Van der Hoeve's syndrome, also known as osteogenesis imperfecta (OI), is a genetic connective tissue disorder characterized by fragile, fracture-prone bone and hearing loss. The disease is caused by a gene mutation in one of the two type I collagen genes COL1A1 or COL1A2. In this study, we identified a novel frameshift mutation of the COL1A1 gene (c.1607delG) in a family with OI using whole-exome sequencing, bioinformatics analysis and Sanger sequencing. This mutation may lead to the deletion of a portion of exon 23 and the generation of a premature stop codon in the COL1A1 gene. To further investigate the impact of this mutation, we established two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells of OI patients carrying a novel mutation in the COL1A1 gene. Osteoblasts (OB) derived from OI-iPSCs exhibited reduced production of type I collagen and diminished ability to differentiate into osteoblasts. Using a CRISPR-based homology-directed repair strategy, we corrected the OI disease-causing COL1A1 novel mutations in iPSCs generated from an affected individual. Our results demonstrated that the diminished expression of type I collagen and osteogenic potential were enhanced in OB induced from corrected OI-iPSCs compared to those from OI-iPSCs. Overall, our results provide new insights into the genetic basis of Van der Hoeve's syndrome and highlight the potential of iPSC technology for disease modeling and therapeutic development.},
}
@article {pmid38347223,
year = {2024},
author = {Krink, N and Nikel, PI and Beisel, CL},
title = {A Hitchhiker's guide to CRISPR editing tools in bacteria : CRISPR can help unlock the bacterial world, but technical and regulatory barriers persist.},
journal = {EMBO reports},
volume = {25},
number = {4},
pages = {1694-1699},
pmid = {38347223},
issn = {1469-3178},
support = {NNF20OC0065068//Novo Nordisk Fonden (NNF)/ ; NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF18OC0034818//Novo Nordisk Fonden (NNF)/ ; NNF21OC0067996//Novo Nordisk Fonden (NNF)/ ; 814418//EC | Horizon 2020 Framework Programme (H2020)/ ; 865973//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Bacteria/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Join us on a journey through the complex and ever-expanding universe of CRISPR approaches for genome editing in bacteria. Discover what is available, current technical challenges, successful implementation of these tools and the regulatory framework around their use. [Image: see text]},
}
@article {pmid38293815,
year = {2024},
author = {Wang, H and Tan, HY and Lian, J and Zhou, K},
title = {Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries.},
journal = {Biotechnology and bioengineering},
volume = {121},
number = {5},
pages = {1543-1553},
doi = {10.1002/bit.28664},
pmid = {38293815},
issn = {1097-0290},
support = {LR20B060003//Natural Science Foundation of Zhejiang Province/ ; 22278361//Natural Science Foundation of China/ ; 226-2023-00015//Fundamental Research Funds for the Central Universities/ ; 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; 226-2023-00085//Fundamental Research Funds for the Central Universities/ ; //National Research Foundation of Singapore/ ; //Ministry of Education of Singapore/ ; //China Scholarship Council (CSC)/ ; //National University of Singapore/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Saccharomyces cerevisiae/genetics/metabolism ; Transcriptional Activation ; *Nanopore Sequencing ; Cloning, Molecular ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing-based workflow demonstrated here should provide a cost-effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.},
}
@article {pmid38199243,
year = {2024},
author = {Song, N and Fan, X and Guo, X and Tang, J and Li, H and Tao, R and Li, F and Li, J and Yang, D and Yao, C and Liu, P},
title = {A DNA/Upconversion Nanoparticle Complex Enables Controlled Co-Delivery of CRISPR-Cas9 and Photodynamic Agents for Synergistic Cancer Therapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {36},
number = {15},
pages = {e2309534},
doi = {10.1002/adma.202309534},
pmid = {38199243},
issn = {1521-4095},
support = {22225505//National Natural Science Foundation of China/ ; 22322407//National Natural Science Foundation of China/ ; 32371468//National Natural Science Foundation of China/ ; //Innovative Research Team of High-level Local Universities in Shanghai/ ; },
mesh = {Mice ; Animals ; *Photochemotherapy ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; Antioxidants ; Hemin ; Hydrogen Peroxide ; RNA, Guide, CRISPR-Cas Systems ; *Nanoparticles/therapeutic use ; Photosensitizing Agents/pharmacology/therapeutic use ; Hypoxia ; Cell Line, Tumor ; *Neoplasms/drug therapy ; },
abstract = {Photodynamic therapy (PDT) depends on the light-irradiated exciting of photosensitizer (PS) to generate reactive oxygen species (ROS), which faces challenges and limitations in hypoxia and antioxidant response of cancer cells, and limited tissue-penetration of light. Herein, a multifunctional DNA/upconversion nanoparticles (UCNPs) complex is developed which enables controlled co-delivery of CRISPR-Cas9, hemin, and protoporphyrin (PP) for synergistic PDT. An ultralong single-stranded DNA (ssDNA) is prepared via rolling circle amplification (RCA), which contains recognition sequences of single guide RNA (sgRNA) for loading Cas9 ribonucleoprotein (RNP), G-quadruplex sequences for loading hemin and PP, and linker sequences for combining UCNP. Cas9 RNP cleaves the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), improving the sensitivity of cancer cells to ROS, and enhancing the synergistic PDT effect. The G-quadruplex/hemin DNAzyme mimicks horseradish peroxidase (HRP) to catalyze the endogenous H2O2 to O2, overcoming hypoxia condition in tumors. The introduced UCNP converts NIR irradiation with deep tissue penetration to light with shorter wavelength, exciting PP to transform the abundant O2 to [1]O2. The integration of gene editing and PDT allows substantial accumulation of [1]O2 in cancer cells for enhanced cell apoptosis, and this synergistic PDT has shown remarkable therapeutic efficacy in a breast cancer mouse model.},
}
@article {pmid38164916,
year = {2024},
author = {Yaylacıoğlu Tuncay, F and Talim, B and Dinçer, PR},
title = {Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea.},
journal = {Current eye research},
volume = {49},
number = {5},
pages = {458-466},
doi = {10.1080/02713683.2023.2298904},
pmid = {38164916},
issn = {1460-2202},
mesh = {Mice ; Animals ; Humans ; Transforming Growth Factor beta/genetics/metabolism ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; Cornea/metabolism ; *Corneal Dystrophies, Hereditary/genetics ; Mutation ; *Corneal Opacity/metabolism ; Extracellular Matrix Proteins/genetics/metabolism ; DNA Mutational Analysis ; Pedigree ; },
abstract = {PURPOSE: Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs.<br><br>METHODS: The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3&#x2009;months and 1&#x2009;year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits.<br><br>RESULTS: We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L&#x2009;+&#x2009;del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3&#x2009;months and 1&#x2009;year of age.<br><br>CONCLUSION: This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1&#x2009;year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.},
}
@article {pmid38582601,
year = {2024},
author = {Koo, J and Zhu, GH and Palli, SR},
title = {CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.},
journal = {Pesticide biochemistry and physiology},
volume = {200},
number = {},
pages = {105839},
doi = {10.1016/j.pestbp.2024.105839},
pmid = {38582601},
issn = {1095-9939},
mesh = {Animals ; RNA Interference ; Spodoptera/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA, Double-Stranded/genetics/metabolism ; Insecta/genetics ; Larva/genetics/metabolism ; },
abstract = {Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.},
}
@article {pmid38582582,
year = {2024},
author = {Li, JL and Li, SS and Luo, ZJ and Lu, J and Cai, XM and Luo, ZX and Bian, L and Xiu, CL and Fu, NX and Liu, NY and Li, ZQ},
title = {CRISPR/Cas9-mediated ebony knockout causes melanin pigmentation and prevents moth Eclosion in Ectropis grisescens.},
journal = {Pesticide biochemistry and physiology},
volume = {200},
number = {},
pages = {105810},
doi = {10.1016/j.pestbp.2024.105810},
pmid = {38582582},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics ; Melanins/genetics ; CRISPR-Cas Systems ; Larva/genetics ; Pigmentation/genetics ; },
abstract = {Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.},
}
@article {pmid38581602,
year = {2024},
author = {Zhang, H and Liang, M and Chen, J and Wang, H and Ma, L},
title = {Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.},
journal = {Planta},
volume = {259},
number = {5},
pages = {112},
pmid = {38581602},
issn = {1432-2048},
support = {LY24C130004//Natural Science Foundation of Zhejiang Province/ ; LY22C135104//Natural Science Foundation of Zhejiang Province/ ; LY23C130002//Natural Science Foundation of Zhejiang Province/ ; 31501288//National Natural Science Foundation of China/ ; 20210207//Open Project Program of State Key Laboratory of Rice Biology/ ; CPSIBRF-CNRRI-202306//Central Public-interest Scientific Institution Basal Research Fund/ ; },
mesh = {*CRISPR-Cas Systems ; *Oryza/genetics ; Disease Resistance/genetics ; Odorants ; Temperature ; Plant Breeding ; },
abstract = {The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.},
}
@article {pmid38580329,
year = {2024},
author = {Lo Presti, V and Meringa, A and Dunnebach, E and van Velzen, A and Moreira, AV and Stam, RW and Kotecha, RS and Krippner-Heidenreich, A and Heidenreich, OT and Plantinga, M and Cornel, A and Sebestyen, Z and Kuball, J and van Til, NP and Nierkens, S},
title = {Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML.},
journal = {Journal for immunotherapy of cancer},
volume = {12},
number = {4},
pages = {},
pmid = {38580329},
issn = {2051-1426},
mesh = {Humans ; Child ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Fetal Blood ; Receptors, Antigen, T-Cell/genetics ; *Leukemia, Myeloid, Acute/genetics/therapy ; Cell Line, Tumor ; *Antineoplastic Agents ; Recurrence ; },
abstract = {BACKGROUND: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft.<br><br>METHODS: We produced CB-CD8[+] T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCR&#x3b1; (TRAC) and TCR&#x3b2; (TRBC) chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts.<br><br>RESULTS: The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR[-/-] WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-&#x3b3; (IFN&#x3b3;), and tumor necrosis factor-&#x3b1; (TNF&#x3b1;), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR[+/+] WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR[-/-] WT1-TCR CD8[+] CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells.<br><br>CONCLUSIONS: In summary, we show the feasibility of developing a potent CB-derived CD8[+] T&#x2009;cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.},
}
@article {pmid38579680,
year = {2024},
author = {Cavazzana, M and Miccio, A},
title = {The difficult translational pathway from animal models to patients.},
journal = {Cell stem cell},
volume = {31},
number = {4},
pages = {435-436},
doi = {10.1016/j.stem.2024.03.010},
pmid = {38579680},
issn = {1875-9777},
mesh = {Animals ; Humans ; *Gene Editing ; Hematopoietic Stem Cells/metabolism ; Models, Animal ; CRISPR-Cas Systems/genetics ; *Hematopoietic Stem Cell Transplantation ; },
abstract = {Lee et al.[1] analyzed the impacts of lentiviral vector transduction and CRISPR-Cas9/homology-directed repair editing on hematopoietic stem and progenitor cell (HSPC) engraftment and clonal dynamics. The study suggests that relative to lentiviral-vector-mediated gene addition, homology-directed repair editing is inefficient in vivo and might impair the engraftment and differentiation of HSPCs.},
}
@article {pmid38579669,
year = {2024},
author = {Jalil, S and Keskinen, T and Juutila, J and Sartori Maldonado, R and Euro, L and Suomalainen, A and Lapatto, R and Kuuluvainen, E and Hietakangas, V and Otonkoski, T and Hyvönen, ME and Wartiovaara, K},
title = {Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.},
journal = {American journal of human genetics},
volume = {111},
number = {4},
pages = {714-728},
doi = {10.1016/j.ajhg.2024.03.004},
pmid = {38579669},
issn = {1537-6605},
mesh = {Humans ; *Argininosuccinic Aciduria/genetics ; Argininosuccinate Lyase/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; Urea ; },
abstract = {Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.},
}
@article {pmid38576085,
year = {2024},
author = {Song, J and Luo, N and Dong, L and Peng, J and Yi, C},
title = {RNA base editors: The emerging approach of RNA therapeutics.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {15},
number = {2},
pages = {e1844},
doi = {10.1002/wrna.1844},
pmid = {38576085},
issn = {1757-7012},
support = {2023YFC3402200//Ministry of Science and Technology of the People's Republic of China/ ; 2019YFA0802200//Ministry of Science and Technology of the People's Republic of China/ ; 92153303//National Natural Science Foundation of China/ ; 21825701//National Natural Science Foundation of China/ ; Z231100002723005//Beijing Municipal Science and Technology Commission/ ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Oligonucleotides, Antisense ; RNA Interference ; },
abstract = {RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m[6]A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.},
}
@article {pmid38575718,
year = {2024},
author = {Ren, K and Zhou, F and Zhang, F and Yin, M and Zhu, Y and Wang, S and Chen, Y and Huang, T and Wu, Z and He, J and Zhang, A and Guo, C and Huang, Z},
title = {Discovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
pmid = {38575718},
issn = {1748-7838},
support = {31825008//National Natural Science Foundation of China (National Science Foundation of China)/ ; U21A20276//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.},
}
@article {pmid37461250,
year = {2024},
author = {Chen, F and Guo, H and Lan, W and Zhou, M and Geng, W and Shen, G and Lin, P and Xia, Q and Zhao, P and Li, Z},
title = {Targeted DNA N[6]-methyladenine editing by dCas9 fused to METTL4 in the lepidopteran model insect Bombyx mori.},
journal = {Insect science},
volume = {31},
number = {2},
pages = {646-650},
doi = {10.1111/1744-7917.13222},
pmid = {37461250},
issn = {1744-7917},
support = {cstc2020jcyj-cxttX0001//Natural Science Foundation of Chongqing/ ; 2022YFD1201600//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Bombyx/genetics ; DNA Methylation ; DNA/genetics ; CRISPR-Cas Systems ; *Adenine ; },
abstract = {We have established a novel CRISPR-dCas9-METTL4 epigenome editing tool that can methylate target regions to achieve site-specific DNA 6mA methylation in both hypermethylated and hypomethylated genes. Targeted methylation on genes by dCas9-METTL4 results in misexpression, allowing for the functional investigation of target genes of interest in silkworm.},
}
@article {pmid38575625,
year = {2024},
author = {Flores, VS and Amgarten, DE and Iha, BKV and Ryon, KA and Danko, D and Tierney, BT and Mason, C and da Silva, AM and Setubal, JC},
title = {Discovery and description of novel phage genomes from urban microbiomes sampled by the MetaSUB consortium.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {7913},
pmid = {38575625},
issn = {2045-2322},
mesh = {*Bacteriophages/genetics ; *Railroads ; *Microbiota/genetics ; Metagenome/genetics ; Bacteria/genetics ; },
abstract = {Bacteriophages are recognized as the most abundant members of microbiomes and have therefore a profound impact on microbial communities through the interactions with their bacterial hosts. The International Metagenomics and Metadesign of Subways and Urban Biomes Consortium (MetaSUB) has sampled mass-transit systems in 60 cities over 3 years using metagenomics, throwing light into these hitherto largely unexplored urban environments. MetaSUB focused primarily on the bacterial community. In this work, we explored MetaSUB metagenomic data in order to recover and analyze bacteriophage genomes. We recovered and analyzed 1714 phage genomes with size at least 40 kbp, from the class Caudoviricetes, the vast majority of which (80%) are novel. The recovered genomes were predicted to belong to temperate (69%) and lytic (31%) phages. Thirty-three of these genomes have more than 200 kbp, and one of them reaches 572 kbp, placing it among the largest phage genomes ever found. In general, the phages tended to be site-specific or nearly so, but 194 genomes could be identified in every city from which phage genomes were retrieved. We predicted hosts for 48% of the phages and observed general agreement between phage abundance and the respective bacterial host abundance, which include the most common nosocomial multidrug-resistant pathogens. A small fraction of the phage genomes are carriers of antibiotic resistance genes, and such genomes tended to be particularly abundant in the sites where they were found. We also detected CRISPR-Cas systems in five phage genomes. This study expands the previously reported MetaSUB results and is a contribution to the knowledge about phage diversity, global distribution, and phage genome content.},
}
@article {pmid38575571,
year = {2024},
author = {Zhao, H and Sheng, Y and Zhang, T and Zhou, S and Zhu, Y and Qian, F and Liu, M and Xu, W and Zhang, D and Hu, J},
title = {The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2901},
pmid = {38575571},
issn = {2041-1723},
support = {2022A1515010370//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; 2022A1515011721//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; },
mesh = {Animals ; Humans ; RNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; *Epstein-Barr Virus Infections/genetics ; Herpesvirus 4, Human/genetics ; *MicroRNAs ; Mammals/genetics ; },
abstract = {Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.},
}
@article {pmid38573978,
year = {2024},
author = {Zheng, C and Liang, H and Dai, L and Yu, J and Long, C},
title = {Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.},
journal = {The journal of physical chemistry. B},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jpcb.3c07320},
pmid = {38573978},
issn = {1520-5207},
abstract = {Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&amp;Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&amp;Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.},
}
@article {pmid38573530,
year = {2024},
author = {Zhao, Z and Cao, S and Sun, M and Yang, Q and Huang, T and Yang, X and Li, J and Zhang, X and Li, X and Wang, X and Jiang, W and Gong, P},
title = {Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.},
journal = {Parasitology research},
volume = {123},
number = {4},
pages = {176},
pmid = {38573530},
issn = {1432-1955},
support = {20220508101RC//Jilin Scientific and Technological Development Program/ ; 21ZY24//Changchun Science and Technology Program/ ; CARS-39//China Wool-sheep &amp; Cashmere-goat Research System/ ; },
mesh = {Humans ; Animals ; Cattle ; *Giardia lamblia/genetics ; CRISPR-Cas Systems ; *Giardiasis/diagnosis/veterinary ; Giardia/genetics ; Biological Assay ; },
abstract = {Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10[-1] copies/μL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.},
}
@article {pmid38573428,
year = {2024},
author = {Calbry, J and Goudounet, G and Charlot, F and Guyon-Debast, A and Perroud, PF and Nogué, F},
title = {The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens.},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {38573428},
issn = {1573-9368},
support = {ANR-20-CE20-0027-02-TOR-DYRKcontrol//Agence Nationale de la Recherche/ ; ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR-20-CE20-0027-02-TOR-DYRKcontrol//Agence Nationale de la Recherche/ ; },
abstract = {Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.},
}
@article {pmid38556749,
year = {2024},
author = {You, S and Yao, S and Chen, X and Hou, Q and Liu, Z and Lei, G and Xie, X and Liang, Z and Yuchi, Z and You, M and Liu, Y and Xiong, L},
title = {CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {14},
pages = {8180-8188},
doi = {10.1021/acs.jafc.3c08721},
pmid = {38556749},
issn = {1520-5118},
mesh = {Animals ; Female ; *Moths/genetics/metabolism ; Larva/metabolism ; *Bacillus thuringiensis/genetics/metabolism ; Longevity ; CRISPR-Cas Systems ; Endotoxins/genetics/metabolism ; Bacillus thuringiensis Toxins/metabolism ; Bacterial Proteins/genetics/metabolism ; Hemolysin Proteins/genetics/metabolism ; Insecticide Resistance/genetics ; },
abstract = {Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.},
}
@article {pmid38490212,
year = {2024},
author = {Lin, CP and Levy, PL and Alflen, A and Apriamashvili, G and Ligtenberg, MA and Vredevoogd, DW and Bleijerveld, OB and Alkan, F and Malka, Y and Hoekman, L and Markovits, E and George, A and Traets, JJH and Krijgsman, O and van Vliet, A and Poźniak, J and Pulido-Vicuña, CA and de Bruijn, B and van Hal-van Veen, SE and Boshuizen, J and van der Helm, PW and Díaz-Gómez, J and Warda, H and Behrens, LM and Mardesic, P and Dehni, B and Visser, NL and Marine, JC and Markel, G and Faller, WJ and Altelaar, M and Agami, R and Besser, MJ and Peeper, DS},
title = {Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness.},
journal = {Cancer cell},
volume = {42},
number = {4},
pages = {623-645.e10},
pmid = {38490212},
issn = {1878-3686},
mesh = {Humans ; *CRISPR-Cas Systems ; CD8-Positive T-Lymphocytes ; *Neoplasms/genetics ; },
abstract = {Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.},
}
@article {pmid38462771,
year = {2024},
author = {Li, M and Zhang, CL and Zhou, DS and Chan, SH and Liu, XQ and Chen, SN and Yang, ZY and Ju, FE and Sang, XY and Liu, ZX and Zhang, QX and Pan, YM and Deng, SS and Wang, XM and Zhong, L and Zhang, XD and Du, X},
title = {Identification of COQ2 as a regulator of proliferation and lipid peroxidation through genome-scale CRISPR-Cas9 screening in myeloma cells.},
journal = {British journal of haematology},
volume = {204},
number = {4},
pages = {1307-1324},
doi = {10.1111/bjh.19375},
pmid = {38462771},
issn = {1365-2141},
support = {32100564//National Nature Science Foundation of China/ ; 81970191//National Nature Science Foundation of China/ ; 82002899//National Nature Science Foundation of China/ ; 82373026//National Nature Science Foundation of China/ ; JCYJ20190807160209294//Shenzhen Science and Technology Program/ ; JCYJ20220530144607017//Shenzhen Science and Technology Program/ ; 2022A1515220110//Guangdong Basic and Applied Basic Research Foundation/ ; 2023A1515012662//Guangdong Basic and Applied Basic Research Foundation/ ; A2303011//Shenzhen Medical Research Funds (SMART)/ ; 2021B1212040006//Science and Technology Planning Project of Guangdong Province/ ; SZXK008//Shenzhen Key Medical Discipline Construction Fund/ ; },
mesh = {Humans ; Animals ; Mice ; *Multiple Myeloma/drug therapy ; CRISPR-Cas Systems ; Lipid Peroxidation ; Mice, Inbred CBA ; Mice, Knockout ; Neoplasm Recurrence, Local ; Cell Proliferation ; Disease Models, Animal ; Mitogen-Activated Protein Kinase Kinases/therapeutic use ; Cell Line, Tumor ; },
abstract = {Multiple myeloma (MM) is the second most common malignant haematological disease with a poor prognosis. The limit therapeutic progress has been made in MM patients with cancer relapse, necessitating deeper research into the molecular mechanisms underlying its occurrence and development. A genome-wide CRISPR-Cas9 loss-of-function screening was utilized to identify potential therapeutic targets in our research. We revealed that COQ2 plays a crucial role in regulating MM cell proliferation and lipid peroxidation (LPO). Knockout of COQ2 inhibited cell proliferation, induced cell cycle arrest and reduced tumour growth in vivo. Mechanistically, COQ2 promoted the activation of the MEK/ERK cascade, which in turn stabilized and activated MYC protein. Moreover, we found that COQ2-deficient MM cells increased sensitivity to the LPO activator, RSL3. Using an inhibitor targeting COQ2 by 4-CBA enhanced the sensitivity to RSL3 in primary CD138[+] myeloma cells and in a xenograft mouse model. Nevertheless, co-treatment of 4-CBA and RSL3 induced cell death in bortezomib-resistant MM cells. Together, our findings suggest that COQ2 promotes cell proliferation and tumour growth through the activation of the MEK/ERK/MYC axis and targeting COQ2 could enhance the sensitivity to ferroptosis in MM cells, which may be a promising therapeutic strategy for the treatment of MM patients.},
}
@article {pmid38418802,
year = {2024},
author = {Stojak, J and Rocha, D and Mörke, C and Kühn, C and Blanquet, V and Taniguchi, H},
title = {Establishment of a cloning-free CRISPR/Cas9 protocol to generate large deletions in the bovine MDBK cell line.},
journal = {Journal of applied genetics},
volume = {65},
number = {2},
pages = {399-402},
pmid = {38418802},
issn = {2190-3883},
support = {815668//H2020 European Research Council/ ; },
mesh = {Cattle ; Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Base Sequence ; Cell Line ; },
abstract = {The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.},
}
@article {pmid38411080,
year = {2024},
author = {Rathore, U and Haas, P and Easwar Kumar, V and Hiatt, J and Haas, KM and Bouhaddou, M and Swaney, DL and Stevenson, E and Zuliani-Alvarez, L and McGregor, MJ and Turner-Groth, A and Ochieng' Olwal, C and Bediako, Y and Braberg, H and Soucheray, M and Ott, M and Eckhardt, M and Hultquist, JF and Marson, A and Kaake, RM and Krogan, NJ},
title = {CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.},
journal = {mBio},
volume = {15},
number = {4},
pages = {e0222223},
pmid = {38411080},
issn = {2150-7511},
support = {P50 AI150476, R01 AI120694, P01 AI063302//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1110189-69-RKRL//amfAR, The Foundation for AIDS Research (amfAR)/ ; DP2 DA042423-01//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; H22BD4486//California HIV/AIDS Research Program (CHRP)/ ; },
mesh = {Humans ; *HIV Infections ; TNF Receptor-Associated Factor 2/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; Virus Latency ; CRISPR-Cas Systems ; Virus Replication ; CD4-Positive T-Lymphocytes ; Ubiquitins/metabolism ; CCAAT-Enhancer-Binding Proteins/genetics/metabolism ; },
abstract = {UNLABELLED: During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.<br><br>IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.},
}
@article {pmid38351739,
year = {2024},
author = {Zhao, S and Han, X and Zhu, Y and Han, Y and Liu, H and Chen, Z and Li, H and Wang, D and Tian, C and Yuan, Y and Guo, Y and Si, X and Wang, D and Ji, X},
title = {CRISPR/CasΦ2-mediated gene editing in wheat and rye.},
journal = {Journal of integrative plant biology},
volume = {66},
number = {4},
pages = {638-641},
doi = {10.1111/jipb.13624},
pmid = {38351739},
issn = {1744-7909},
support = {32000286//National Natural Science Foundation of China/ ; 32370432//National Natural Science Foundation of China/ ; 2021YFF1000203//National Key Research and Development Program of China/ ; },
mesh = {*Gene Editing ; *Triticum/genetics ; CRISPR-Cas Systems/genetics ; Secale/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The compact CRISPR/CasΦ2 system provides a complementary genome engineering tool for efficient gene editing including cytosine and adenosine base editing in wheat and rye with high specificity, efficient use of the protospacer-adjacent motif TTN, and an alternative base-editing window.},
}
@article {pmid38320883,
year = {2024},
author = {Yong, J and Wu, M and Carroll, BJ and Xu, ZP and Zhang, R},
title = {Enhancing plant biotechnology by nanoparticle delivery of nucleic acids.},
journal = {Trends in genetics : TIG},
volume = {40},
number = {4},
pages = {352-363},
doi = {10.1016/j.tig.2024.01.005},
pmid = {38320883},
issn = {0168-9525},
mesh = {*Nucleic Acids ; CRISPR-Cas Systems ; Genome, Plant ; Plants/genetics ; Biotechnology ; Gene Editing ; *Nanoparticles ; Plants, Genetically Modified/genetics ; },
abstract = {Plant biotechnology plays a crucial role in developing modern agriculture and plant science research. However, the delivery of exogenous genetic material into plants has been a long-standing obstacle. Nanoparticle-based delivery systems are being established to address this limitation and are proving to be a feasible, versatile, and efficient approach to facilitate the internalization of functional RNA and DNA by plants. The nanoparticle-based delivery systems can also be designed for subcellular delivery and controlled release of the biomolecular cargo. In this review, we provide a concise overview of the recent advances in nanocarriers for the delivery of biomolecules into plants, with a specific focus on applications to enhance RNA interference, foreign gene transfer, and genome editing in plants.},
}
@article {pmid38217286,
year = {2024},
author = {Ma, G and Yan, F and Ren, B and Lu, Z and Xu, H and Wu, F and Li, S and Wang, D and Zhou, X and Zhou, H},
title = {LbCas12a-nuclease-mediated tiling deletion for large-scale targeted editing of non-coding regions in rice.},
journal = {Plant communications},
volume = {5},
number = {4},
pages = {100815},
doi = {10.1016/j.xplc.2024.100815},
pmid = {38217286},
issn = {2590-3462},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid38571947,
year = {2024},
author = {Yang, Y and Xu, Z and Tao, Q and Xu, L and Gu, S and Huang, Y and Liu, Z and Zhang, Y and Wen, J and Lai, S and Zhu, L},
title = {Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1339387},
pmid = {38571947},
issn = {1664-3224},
mesh = {Swine ; Animals ; Mice ; *Herpesvirus 1, Suid/genetics ; *Pseudorabies/prevention &amp; control ; Interleukin-4/genetics ; *Circovirus/genetics ; Vaccines, Synthetic ; },
abstract = {BACKGROUND: Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China.<br><br>METHODS: In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice.<br><br>RESULTS: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators.<br><br>CONCLUSION: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.},
}
@article {pmid38570691,
year = {2024},
author = {Yan, J and Oyler-Castrillo, P and Ravisankar, P and Ward, CC and Levesque, S and Jing, Y and Simpson, D and Zhao, A and Li, H and Yan, W and Goudy, L and Schmidt, R and Solley, SC and Gilbert, LA and Chan, MM and Bauer, DE and Marson, A and Parsons, LR and Adamson, B},
title = {Improving prime editing with an endogenous small RNA-binding protein.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {38570691},
issn = {1476-4687},
abstract = {Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs[1]. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts[2]. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.},
}
@article {pmid38570576,
year = {2024},
author = {Sutipatanasomboon, A and Wongsantichon, J and Sakdee, S and Naksith, P and Watthanadirek, A and Anuracpreeda, P and Blacksell, SD and Saisawang, C},
title = {RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {7820},
pmid = {38570576},
issn = {2045-2322},
support = {/WT_/Wellcome Trust/United Kingdom ; 220211/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Cattle ; Animals ; *Anaplasma marginale/genetics ; *Anaplasmosis/diagnosis/genetics ; CRISPR-Cas Systems ; *Cattle Diseases/genetics ; *Tick-Borne Diseases/genetics ; },
abstract = {Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.},
}
@article {pmid38567981,
year = {2024},
author = {Safarkhani, M and Farasati Far, B and Kim, SH and Makvandi, P and Park, MK and Huh, Y and Rabiee, N},
title = {Advances and Challenges of Sensing in Water Using CRISPR-Cas Technology.},
journal = {ACS biomaterials science &amp; engineering},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsbiomaterials.3c01689},
pmid = {38567981},
issn = {2373-9878},
abstract = {The groundbreaking gene-editing mechanism, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), paired with the protein Cas9, has significantly advanced the realms of biology, medicine, and agriculture. Through its precision in modifying genetic sequences, CRISPR holds the potential to alter the trajectory of genetic disorders and accelerate advancements in agriculture. While its therapeutic potential is profound, the technology also invites ethical debates centered on responsible use and equity in access. Parallelly, in the environmental monitoring sphere and sensing in water, especially biosensors have been instrumental in evaluating natural water sources' quality. These biosensors, integrating biological components with detection techniques, have the potential to revolutionize healthcare by providing rapid and minimally invasive diagnostic methods. However, the design and application of these sensors bring forth challenges, especially in ensuring sensitivity, selectivity, and ethical data handling. This article delves into the prospective use of CRISPR-Cas technology for sensing in water, exploring its capabilities in detecting diverse biomarkers, hazardous substances, and varied reactions in water and wastewater systems.},
}
@article {pmid38567969,
year = {2024},
author = {Maes, A and Botzki, A and Mathys, J and Impens, F and Saelens, X},
title = {Systematic review and meta-analysis of genome-wide pooled CRISPR screens to identify host factors involved in influenza A virus infection.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0185723},
doi = {10.1128/jvi.01857-23},
pmid = {38567969},
issn = {1098-5514},
abstract = {UNLABELLED: The host-virus interactome is increasingly recognized as an important research field to discover new therapeutic targets to treat influenza. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify new pro- and antiviral host factors of the influenza A virus. However, at present, a comprehensive summary of the results is lacking. We performed a systematic review of all reported CRISPR studies in this field in combination with a meta-analysis using the algorithm of meta-analysis by information content (MAIC). Two ranked gene lists were generated based on evidence in 15 proviral and 4 antiviral screens. Enriched pathways in the proviral MAIC results were compared to those of a prior array-based RNA interference (RNAi) meta-analysis. The top 50 proviral MAIC list contained genes whose role requires further elucidation, such as the endosomal ion channel TPCN1 and the kinase WEE1. Moreover, MAIC indicated that ALYREF, a component of the transcription export complex, has antiviral properties, whereas former knockdown experiments attributed a proviral role to this host factor. CRISPR-Cas-pooled screens displayed a bias toward early-replication events, whereas the prior RNAi meta-analysis covered early and late-stage events. RNAi screens led to the identification of a larger fraction of essential genes than CRISPR screens. In summary, the MAIC algorithm points toward the importance of several less well-known pathways in host-influenza virus interactions that merit further investigation. The results from this meta-analysis of CRISPR screens in influenza A virus infection may help guide future research efforts to develop host-directed anti-influenza drugs.<br><br>IMPORTANCE: Viruses rely on host factors for their replication, whereas the host cell has evolved virus restriction factors. These factors represent potential targets for host-oriented antiviral therapies. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify pro- and antiviral host factors in the context of influenza virus infection. We performed a comprehensive analysis of the outcome of these screens based on the publicly available gene lists, using the recently developed algorithm meta-analysis by information content (MAIC). MAIC allows the systematic integration of ranked and unranked gene lists into a final ranked gene list. This approach highlighted poorly characterized host factors and pathways with evidence from multiple screens, such as the vesicle docking and lipid metabolism pathways, which merit further exploration.},
}
@article {pmid38567606,
year = {2024},
author = {Saifullah, M and Laghzaoui, O and Ozyahyalar, H and Irfan, A},
title = {The CRISPR-Cas9 induced CCR5 Δ32 mutation as a potent gene therapy methodology for resistance to HIV-1 variant: a review.},
journal = {European review for medical and pharmacological sciences},
volume = {28},
number = {6},
pages = {2430-2463},
doi = {10.26355/eurrev_202403_35751},
pmid = {38567606},
issn = {2284-0729},
mesh = {Humans ; *HIV-1/genetics ; *Acquired Immunodeficiency Syndrome/genetics/therapy ; *HIV Infections/genetics/therapy ; CRISPR-Cas Systems/genetics ; Receptors, CCR5/genetics/metabolism ; Mutation ; Genetic Therapy ; Polymorphism, Single Nucleotide ; Gene Frequency ; },
abstract = {Human Immunodeficiency Virus (HIV) has continuously been the greatest epidemic for humanity over a period spanning almost five decades. With no specific cure or treatment available to date despite extensive research, the C-C Chemokine Receptor 5, Delta 32 (CCR5 Δ32) allele genetic point mutation plays an imperative role in the prevention of acquired immunodeficiency syndrome (AIDS). This comprehensive study aims to review the induction of the homozygous recessive deletion genotype using the Clustered Regularly Interspaced Short Palindromic Repeats, Cas 9 Enzyme (CRISPR-Cas9), and hematopoietic stem cell transplantation under positive selection pressure for active immunity in seropositive patients' populations as the phenotype. A methodology is proposed to trigger a significant increase in the expression of Delta 32 beneficial mutant alleles within controlled modern healthcare facilities utilizing totipotent stem cells through somatic gene therapy. It acts upon two dysfunctional CCR5 genes, translating mutant G protein-coupled co-receptors, whose primary function is similar to that of C-X-C Motif Chemokine receptor 4 (CXCR4), by blocking the entry of viral RNA into the CD4+ T helper lymphocytes, halting infection and seizing viral life cycle. This modification is endemic in Northern Europe, where it naturally pertains to the Caucasian descent population samples in the form of polymorphism, p (X=0.01), where X is the probability of frequency of complete immunity against HIV-1 in population samples. The epigenetics of the single nucleotide polymorphism (SNP) are analyzed as they play a significant role in immunity distribution. Furthermore, a comparative analysis within the ethical boundaries of CRISPR-Cas9 is conducted to discuss the practical aspects and challenges of the presented methodologies and treatment alternatives. Additionally, the study assembles all available data and summarizes preexisting research while providing a promising solution to this ethical dilemma. Finally, a methodology is devised to answer the question of whether the variant-specific epidemic of AIDS caused by HIV-1 can be cured via artificially inducing immunity by CRISPR-Cas9.},
}
@article {pmid38509377,
year = {2024},
author = {Lee, HG and Rone, JM and Li, Z and Akl, CF and Shin, SW and Lee, JH and Flausino, LE and Pernin, F and Chao, CC and Kleemann, KL and Srun, L and Illouz, T and Giovannoni, F and Charabati, M and Sanmarco, LM and Kenison, JE and Piester, G and Zandee, SEJ and Antel, JP and Rothhammer, V and Wheeler, MA and Prat, A and Clark, IC and Quintana, FJ},
title = {Disease-associated astrocyte epigenetic memory promotes CNS pathology.},
journal = {Nature},
volume = {627},
number = {8005},
pages = {865-872},
pmid = {38509377},
issn = {1476-4687},
support = {R00 NS114111/NS/NINDS NIH HHS/United States ; R01 MH130458/MH/NIMH NIH HHS/United States ; R01 MH132632/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Acetyl Coenzyme A/metabolism ; *Astrocytes/enzymology/metabolism/pathology ; ATP Citrate (pro-S)-Lyase/metabolism ; Chromatin/genetics/metabolism ; Chromatin Assembly and Disassembly ; Chromatin Immunoprecipitation Sequencing ; CRISPR-Cas Systems ; *Encephalomyelitis, Autoimmune, Experimental/enzymology/genetics/metabolism/pathology ; *Epigenetic Memory ; Inflammation/enzymology/genetics/metabolism/pathology ; *Multiple Sclerosis/enzymology/genetics/metabolism/pathology ; Single-Cell Gene Expression Analysis ; Transposases/metabolism ; },
abstract = {Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis[1-8] (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY[+]p300[+] memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY[+]p300[+] astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.},
}
@article {pmid38401849,
year = {2024},
author = {Wang, X and Li, Y and A, S and Lyu, J and Wang, X and He, Z and Lara-Sáez, I and Li, M and Wang, W},
title = {Cyclization-enhanced poly(β-amino ester)s vectors for efficient CRISPR gene editing therapy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {368},
number = {},
pages = {444-452},
doi = {10.1016/j.jconrel.2024.02.032},
pmid = {38401849},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing/methods ; Cyclization ; *Esters ; RNA, Guide, CRISPR-Cas Systems ; DNA/metabolism ; CRISPR-Cas Systems/genetics ; Collagen Type VII/genetics ; *Polymers ; },
abstract = {Among non-viral gene delivery vectors, poly(β-amino ester)s (PAEs) are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. Over two decades, PAEs have evolved from linear to highly branched structures, significantly enhancing gene delivery efficacy. Building on the proven efficient sets of monomers in highly branched PAEs (HPAEs), this work introduced a new class of cyclic PAEs (CPAEs) constructed via an A2 + B4 + C2 cyclization synthesis strategy and identified their markedly improved gene transfection capabilities in gene delivery applications. Two sets of cyclic PAEs (CPAEs) with rings of different sizes and topologies were obtained. Their chemical structures were confirmed via two-dimensional nuclear magnetic resonance and the photoluminescence phenomena, and their DNA delivery behaviours were investigated and compared with the HPAE counterparts. In vitro assessments demonstrated that the CPAEs with a macrocyclic architecture (MCPAEs), significantly enhanced DNA intracellular uptake and facilitated efficient gene expression while maintaining perfect biocompatibility. The top-performance MCPAEs have been further employed to deliver a plasmid coding dual single guide RNA-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. In recessive dystrophic epidermolysis bullosa (RDEB) patient-derived epidermal keratinocytes, MCPAEs facilitated the CRISPR plasmid delivery and achieved efficient targeted gene editing in multiple colonies.},
}
@article {pmid38199433,
year = {2024},
author = {Chen, J and Qin, H and Hao, J and Wang, Q and Chen, S and Yang, G and Li, M and Zhu, X and Wang, D and Chen, H and Cui, C and Chen, M},
title = {Cardiac-specific overexpression of CREM-IbΔC-X via CRISPR/Cas9 in mice presents a new model of atrial cardiomyopathy with spontaneous atrial fibrillation.},
journal = {Translational research : the journal of laboratory and clinical medicine},
volume = {267},
number = {},
pages = {54-66},
doi = {10.1016/j.trsl.2024.01.001},
pmid = {38199433},
issn = {1878-1810},
mesh = {Mice ; Humans ; Animals ; *Atrial Fibrillation ; CRISPR-Cas Systems/genetics ; Mice, Transgenic ; Heart Atria/pathology ; *Cardiomyopathies/genetics ; Cyclic AMP Response Element Modulator/genetics/metabolism ; },
abstract = {Atrial cardiomyopathy (ACM) forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. However, generating stable animal models that accurately replicate the entire progression of atrial lesions, particularly the onset of AF, presents significant challenges. In the present study, we found that the isoform of CRE-binding protein modulator (CREM-IbΔC-X), which is involved in the regulation of cardiac development and atrial rhythm, was highly expressed in atrial biopsies from patients with AF. Building upon this finding, we employed CRISPR/Cas9 technology to create a mouse model with cardiac-specific overexpression of CREM-IbΔC-X (referred to as CS-CREM mice). This animal model effectively illustrated the development of ACM through electrophysiological and structural remodelings over time. Proteomics and Chip-qPCR analysis of atrial samples revealed significant upregulation of cell-matrix adhesion and extracellular matrix structural components, alongside significant downregulation of genes related to atrial functions in the CS-CREM mice. Furthermore, the corresponding responses to anti-arrhythmia drugs, i.e., amiodarone and propafenone, suggested that CS-CREM mice could serve as an ideal in vivo model for drug testing. Our study introduced a novel ACM model with spontaneous AF by cardiac-specifically overexpressing CREM-IbΔC-X in mice, providing valuable insights into the mechanisms and therapeutic targets of ACM.},
}
@article {pmid38521024,
year = {2024},
author = {Zhu, L and Zhang, X and Yang, L and Qiu, S and Liu, G and Xiong, X and Xiao, T and Huang, K and Zhu, L},
title = {Label-free electrochemical sensing platform for sensitive detection of ampicillin by combining nucleic acid isothermal enzyme-free amplification circuits with CRISPR/Cas12a.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125950},
doi = {10.1016/j.talanta.2024.125950},
pmid = {38521024},
issn = {1873-3573},
mesh = {Humans ; *Nucleic Acids ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; Ampicillin ; *Biosensing Techniques/methods ; },
abstract = {The residue of ampicillin (AMP) in food and ecological environment poses a potential harm to human health. Therefore, a reliable system for detecting AMP is in great demand. Herein, a label-free and sensitive electrochemical sensor utilizing NH2-Co-MOF as an electrocatalytic active material for methylene blue (MB) was developed for rapid and facile AMP detection by combining hybridization chain reaction (HCR), catalytic hairpin assembly (CHA) with CRISPR/Cas12a. The surface of glassy carbon electrode modified with NH2-Co-MOF was able to undergo HCR independent of the AMP, forming long dsDNA complexes to load MB, resulting in strong original electrochemical signal. The presence of AMP could trigger upstream CHA circuit to activate the CRISPR/Cas12a system, thereby achieving rapid non-specific cleavage of the trigger ssDNA of HCR on the electrode surface, hindering the occurrence of HCR and reducing the load of MB. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the AMP with a detection limit as low as 1.60 pM (S/N = 3). The proposed sensor exhibited good stability, selectivity, and stability, and achieved reliable detection of AMP in milk and livestock wastewater samples, demonstrating its promising application prospects in food safety and environmental monitoring.},
}
@article {pmid38503125,
year = {2024},
author = {Peng, J and Liu, T and Guan, L and Xu, Z and Xiong, T and Zhang, Y and Song, J and Liu, X and Yang, Y and Hao, X},
title = {A highly sensitive Lock-Cas12a biosensor for detection and imaging of miRNA-21 in breast cancer cells.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125938},
doi = {10.1016/j.talanta.2024.125938},
pmid = {38503125},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems ; Diagnostic Imaging ; Limit of Detection ; *Nucleic Acids ; *Biosensing Techniques ; *Neoplasms ; },
abstract = {The expression levels of microRNA (miRNA) vary significantly in correlation with the occurrence and progression of cancer, making them valuable biomarkers for cancer diagnosis. However, their quantitative detection faces challenges due to the high sequence homology, low abundance and small size. In this work, we established a strand displacement amplification (SDA) approach based on miRNA-triggered structural "Lock" nucleic acid ("Lock" DNA), coupled with the CRISPR/Cas12a system, for detecting miRNA-21 in breast cancer cells. The "Lock" DNA freed the CRISPR-derived RNA (crRNA) from the dependence on the target sequence and greatly facilitated the extended detection of different miRNAs. Moreover, the CRISPR/Cas12a system provided excellent amplification ability and specificity. The designed biosensor achieved high sensitivity detection of miRNA-21 with a limit of detection (LOD) of 28.8 aM. In particular, the biosensor could distinguish breast cancer cells from other cancer cells through intracellular imaging. With its straightforward sequence design and ease of use, the Lock-Cas12a biosensor offers significant advantages for cell imaging and early clinical diagnosis.},
}
@article {pmid38503121,
year = {2024},
author = {Li, T and Wang, J and Fang, J and Chen, F and Wu, X and Wang, L and Gao, M and Zhang, L and Li, S},
title = {A universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification with multiple signal readout.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125922},
doi = {10.1016/j.talanta.2024.125922},
pmid = {38503121},
issn = {1873-3573},
mesh = {Male ; Humans ; CRISPR-Cas Systems ; Semen ; Biological Assay ; *Nucleic Acids ; DNA ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; },
abstract = {Rapid and sensitive detection of nucleic acids has become crucial in various fields. However, most current nucleic acid detection methods can only be used in specific scenarios, such as RT-qPCR, which relies on fluorometer for signal readout, limiting its application at home or in the field due to its high price. In this paper, a universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification (CRISPR-SDA) with multiple signal readout was established to adapt to different application scenarios. Nucleocapsid protein gene of SARS-CoV-2 (N gene) and hepatitis B virus (HBV) DNA were selected as model targets. The proposed strategy achieved the sensitivity of 53.1 fM, 0.15 pM, and 1 pM for N gene in fluorescence mode, personal glucose meter (PGM) mode and lateral flow assay (LFA) mode, respectively. It possessed the ability to differentiate single-base mismatch and the presence of salmon sperm DNA with a mass up to 10[5]-fold of the targets did not significantly interfere with the assay signal. The general and modular design idea made CRISPR-SDA as simple as building blocks to construct nucleic acid sensing methods to meet different requirements by simply changing the SDA template and selecting suitable signal report probes, which was expected to find a breadth of applications in nucleic acids detection.},
}
@article {pmid38432581,
year = {2024},
author = {Rodrigo-Faus, M and Vincelle-Nieto, A and Vidal, N and Puente, J and Saiz-Pardo, M and Lopez-Garcia, A and Mendiburu-Eliçabe, M and Palao, N and Baquero, C and Linzoain-Agos, P and Cuesta, AM and Qu, HQ and Hakonarson, H and Musteanu, M and Reyes-Palomares, A and Porras, A and Bragado, P and Gutierrez-Uzquiza, A},
title = {CRISPR/Cas9 screenings unearth protein arginine methyltransferase 7 as a novel essential gene in prostate cancer metastasis.},
journal = {Cancer letters},
volume = {588},
number = {},
pages = {216776},
doi = {10.1016/j.canlet.2024.216776},
pmid = {38432581},
issn = {1872-7980},
mesh = {Male ; Animals ; Mice ; Humans ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Prostatic Neoplasms, Castration-Resistant/pathology ; CRISPR-Cas Systems ; Genes, Essential ; Early Detection of Cancer ; },
abstract = {Due to the limited effectiveness of current treatments, the survival rate of patients with metastatic castration-resistant prostate cancer (mCRPC) is significantly reduced. Consequently, it is imperative to identify novel therapeutic targets for managing these patients. Since the invasive ability of cells is crucial for establishing and maintaining metastasis, the aim of this study was to identify the essential regulators of invasive abilities of mCRPC cells by conducting two independent high-throughput CRISPR/Cas9 screenings. Furthermore, some of the top hits were validated using siRNA technology, with protein arginine methyltransferase 7 (PRMT7) emerging as the most promising candidate. We demonstrated that its inhibition or depletion via genetic or pharmacological approaches significantly reduces invasive, migratory and proliferative abilities of mCRPC cells in vitro. Moreover, we confirmed that PRMT7 ablation reduces cell dissemination in chicken chorioallantoic membrane and mouse xenograft assays. Molecularly, PRMT7 reprograms the expression of several adhesion molecules by methylating various transcription factors, such as FoxK1, resulting in the loss of adhesion from the primary tumor and increased motility of mCRPC cells. Furthermore, PRMT7 higher expression correlates with tumor aggressivity and poor overall survival in prostate cancer patients. Thus, this study demonstrates that PRMT7 is a potential therapeutic target and potential biomarker for mPCa.},
}
@article {pmid38331220,
year = {2024},
author = {Rong, F and Wang, H and Tang, X and Xing, J and Sheng, X and Chi, H and Zhan, W},
title = {The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV).},
journal = {Journal of virological methods},
volume = {326},
number = {},
pages = {114892},
doi = {10.1016/j.jviromet.2024.114892},
pmid = {38331220},
issn = {1879-0984},
mesh = {Animals ; *Infectious hematopoietic necrosis virus/genetics ; CRISPR-Cas Systems ; Reverse Transcription ; Recombinases/genetics ; },
abstract = {Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.},
}
@article {pmid38565780,
year = {2024},
author = {Ly, LK and Ho, TM and Bui, TP and Nguyen, LT and Phan, Q and Le, NT and Khuat, LTM and Le, LH and Chu, HH and Pham, NB and Do, PT},
title = {CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice.},
journal = {Functional &amp; integrative genomics},
volume = {24},
number = {2},
pages = {70},
pmid = {38565780},
issn = {1438-7948},
mesh = {*Salt Tolerance/genetics ; CRISPR-Cas Systems ; *Oryza/metabolism ; Salt Stress ; Mutation ; },
abstract = {Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.},
}
@article {pmid38565160,
year = {2024},
author = {Morimoto, K and Suzuki, H and Kuno, A and Daitoku, Y and Tanimoto, Y and Kato, K and Murata, K and Sugiyama, F and Mizuno, S},
title = {Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.},
journal = {Open biology},
volume = {14},
number = {4},
pages = {240007},
pmid = {38565160},
issn = {2046-2441},
mesh = {Animals ; Mice ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Mutagenesis ; *MicroRNAs/genetics ; },
abstract = {Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.},
}
@article {pmid38564697,
year = {2024},
author = {Han, X and Lu, M and Zhang, Y and Liu, X and Zhang, Q and Bai, X and Man, S and Zhao, L and Ma, L},
title = {A Thermostable Cas12b-Powered Bioassay Coupled with Loop-Mediated Isothermal Amplification in a Customized "One-Pot" Vessel for Visual, Rapid, Sensitive, and On-Site Detection of Genetically Modified Crops.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.4c01028},
pmid = {38564697},
issn = {1520-5118},
abstract = {Genetically modified crops (GMCs) have been discussed due to unknown safety, and thus, it is imperative to develop an effective detection technology. CRISPR/Cas is deemed a burgeoning technology for nucleic acid detection. Herein, we developed a novel detection method for the first time, which combined thermostable Cas12b with loop-mediated isothermal amplification (LAMP), to detect genetically modified (GM) soybeans in a customized one-pot vessel. In our method, LAMP-specific primers were used to amplify the cauliflower mosaic virus 35S promoter (CaMV35S) of the GM soybean samples. The corresponding amplicons activated the trans-cleavage activity of Cas12b, which resulted in the change of fluorescence intensity. The proposed bioassay was capable of detecting synthetic plasmid DNA samples down to 10 copies/μL, and as few as 0.05% transgenic contents could be detected in less than 40 min. This work presented an original detection method for GMCs, which performed rapid, on-site, and deployable detection.},
}
@article {pmid38563791,
year = {2024},
author = {Zheng, Z and Xu, L and Dou, H and Zhou, Y and Feng, X and He, X and Tian, Z and Song, L and Gao, Y and Mo, G and Hu, J and Zhao, H and Wei, H and Church, GM and Yang, L},
title = {Testing multiplexed anti-ASFV CRISPR-Cas9 in reducing African swine fever virus.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0216423},
doi = {10.1128/spectrum.02164-23},
pmid = {38563791},
issn = {2165-0497},
abstract = {UNLABELLED: African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies.<br><br>IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.},
}
@article {pmid38563119,
year = {2024},
author = {Jia, HY and Zhang, XY and Ye, BC and Yin, BC},
title = {An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c05975},
pmid = {38563119},
issn = {1520-6882},
abstract = {CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.},
}
@article {pmid38561518,
year = {2024},
author = {Özcan, A and Yıbar, A and Kiraz, D and Ilıkkan, &#xd6;K},
title = {Comprehensive analysis of the CRISPR-Cas systems in Streptococcus thermophilus strains isolated from traditional yogurts.},
journal = {Antonie van Leeuwenhoek},
volume = {117},
number = {1},
pages = {63},
pmid = {38561518},
issn = {1572-9699},
mesh = {*Streptococcus thermophilus/genetics ; CRISPR-Cas Systems ; Yogurt ; *Bacteriophages/genetics ; Plasmids/genetics ; },
abstract = {Phage resistance is crucial for lactic acid bacteria in the dairy industry. However, identifying all phages affecting these bacteria is challenging. CRISPR-Cas systems offer a resistance mechanism developed by bacteria and archaea against phages and plasmids. In this study, 11 S. thermophilus strains from traditional yogurts underwent analysis using next-generation sequencing (NGS) and bioinformatics tools. Initial characterization involved molecular ribotyping. Bioinformatics analysis of the NGS raw data revealed that all 11 strains possessed at least one CRISPR type. A total of 21 CRISPR loci were identified, belonging to CRISPR types II-A, II-C, and III-A, including 13 Type II-A, 1 Type III-C, and 7 Type III-A CRISPR types. By analyzing spacer sequences in S. thermophilus bacterial genomes and matching them with phage/plasmid genomes, notable strains emerged. SY9 showed prominence with 132 phage matches and 30 plasmid matches, followed by SY12 with 35 phage matches and 25 plasmid matches, and SY18 with 49 phage matches and 13 plasmid matches. These findings indicate the potential of S. thermophilus strains in phage/plasmid resistance for selecting starter cultures, ultimately improving the quality and quantity of dairy products. Nevertheless, further research is required to validate these results and explore the practical applications of this approach.},
}
@article {pmid38559425,
year = {2024},
author = {Wang, X and Li, D and Qin, Z and Chen, J and Zhou, J},
title = {CRISPR/Cpf1-FOKI-induced gene editing in Gluconobacter oxydans.},
journal = {Synthetic and systems biotechnology},
volume = {9},
number = {2},
pages = {369-379},
pmid = {38559425},
issn = {2405-805X},
abstract = {Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system. Its incomplete oxidation system has many crucial industrial applications. However, it also leads to slow growth and low biomass, requiring further metabolic modification for balancing the cell growth and incomplete oxidation process. As a non-model strain, G. oxydans lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network regulation. In the last 15 years, our laboratory attempted to deploy multiple CRISPR/Cas systems in different G. oxydans strains and found none of them as functional. In this study, Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1, while Cpf1-FokI was deployed to study its nuclease activity. A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in G. oxydans but lacked the nuclease cleavage activity. Therefore, the CRISPR/Cpf1-FokI system based on FokI nuclease was constructed. Single-gene knockout with efficiency up to 100% and double-gene iterative editing were achieved in G. oxydans. Using this system, AcrVA6, the anti-CRISPR protein of G. oxydans was discovered for the first time, and efficient genome editing was realized.},
}
@article {pmid38557903,
year = {2024},
author = {Valencia-Lozano, E and Cabrera-Ponce, JL and Barraza, A and López-Calleja, AC and García-Vázquez, E and Rivera-Toro, DM and de Folter, S and Alvarez-Venegas, R},
title = {Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.},
journal = {PloS one},
volume = {19},
number = {4},
pages = {e0301169},
pmid = {38557903},
issn = {1932-6203},
mesh = {*Solanum lycopersicum/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Breeding ; Embryonic Development ; Regeneration ; Gene Editing ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.},
}
@article {pmid38557757,
year = {2024},
author = {Rodríguez-Román, E and Manuel, JA and Goldberg, D and Levin, BR},
title = {The contribution of abortive infection to preventing populations of Lactococcus lactis from succumbing to infections with bacteriophage.},
journal = {PloS one},
volume = {19},
number = {4},
pages = {e0298680},
pmid = {38557757},
issn = {1932-6203},
mesh = {*Bacteriophages/genetics ; *Lactococcus lactis/genetics ; Computer Simulation ; Bacterial Proteins ; Bacteria ; },
abstract = {In the dairy industry bacteriophage (phage) contamination significantly impairs the production and quality of products like yogurt and cheese. To combat this issue, the strains of bacteria used as starter cultures possess mechanisms that make them resistant to phage infection, such as envelope resistance, or processes that render them immune to phage infection, such as restriction-modification and CRISPR-Cas. Lactococcus lactis, used to manufacture cheese and other dairy products, can also block the reproduction of infecting phages by abortive infection (Abi), a process in which phage-infected cells die before the phage replicate. We employ mathematical-computer simulation models and experiments with two Lactococcus lactis strains and two lytic phages to investigate the conditions under which Abi can limit the proliferation of phages in L. lactis populations and prevent the extinction of their populations by these viruses. According to our model, if Abi is almost perfect and there are no other populations of bacteria capable of supporting the replication of the L. lactis phages, Abi can protect bacterial populations from succumbing to infections with these viruses. This prediction is supported by the results of our experiment, which indicate that Abi can help protect L. lactis populations from extinction by lytic phage infections. However, our results also predict abortive infection is only one element of L. lactis defenses against phage infection. Mutant phages that can circumvent the Abi systems of these bacteria emerge. The survival of L. lactis populations then depends on the evolution of envelope mutants that are resistant to the evolved host-range phage.},
}
@article {pmid38557598,
year = {2024},
author = {J Davis, D and McNew, JF and Walls, JN and Bethune, CE and Oswalt, PS and Bryda, EC},
title = {CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {205},
pages = {},
doi = {10.3791/66069},
pmid = {38557598},
issn = {1940-087X},
mesh = {Rats ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Electroporation/methods ; Zygote ; },
abstract = {Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.},
}
@article {pmid38556550,
year = {2024},
author = {Kim, HS and Kweon, J and Kim, Y},
title = {Recent advances in CRISPR-based functional genomics for the study of disease-associated genetic variants.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {38556550},
issn = {2092-6413},
support = {2021R1C1C1007162//National Research Foundation of Korea (NRF)/ ; RS-2023-00243993//National Research Foundation of Korea (NRF)/ ; },
abstract = {Advances in sequencing technology have greatly increased our ability to gather genomic data, yet understanding the impact of genetic mutations, particularly variants of uncertain significance (VUSs), remains a challenge in precision medicine. The CRISPR‒Cas system has emerged as a pivotal tool for genome engineering, enabling the precise incorporation of specific genetic variations, including VUSs, into DNA to facilitate their functional characterization. Additionally, the integration of CRISPR‒Cas technology with sequencing tools allows the high-throughput evaluation of mutations, transforming uncertain genetic data into actionable insights. This allows researchers to comprehensively study the functional consequences of point mutations, paving the way for enhanced understanding and increasing application to precision medicine. This review summarizes the current genome editing tools utilizing CRISPR‒Cas systems and their combination with sequencing tools for functional genomics, with a focus on point mutations.},
}
@article {pmid38556532,
year = {2024},
author = {Ishibashi, R and Maki, R and Toyoshima, F},
title = {Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {7615},
pmid = {38556532},
issn = {2045-2322},
support = {22K15029//Japan Society for the Promotion of Science/ ; JPMJCR2023//Core Research for Evolutional Science and Technology/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plasmids/genetics ; Gene Targeting/methods ; DNA ; },
abstract = {The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.},
}
@article {pmid38555443,
year = {2024},
author = {Huyen, DT and Reboud, J and Quyen, DT and Cooper, JM and Velavan, TP and Trung, NT and Song, LH},
title = {An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {23},
number = {1},
pages = {28},
pmid = {38555443},
issn = {1476-0711},
support = {364/2020/HD-NCKHCN//Vietnamese Ministry of National Defence/ ; 364/2020/HD-NCKHCN//Vietnamese Ministry of National Defence/ ; 364/2020/HD-NCKHCN//Vietnamese Ministry of National Defence/ ; 364/2020/HD-NCKHCN//Vietnamese Ministry of National Defence/ ; DAAD-57592343//PAN-ASEAN Coalition for Epidemic and Outbreak Preparedness (PACE-UP)/ ; },
mesh = {Humans ; *Neisseria meningitidis/genetics ; *Meningitis, Meningococcal/diagnosis/microbiology ; *Meningococcal Infections/diagnosis/microbiology ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; *Sepsis ; },
abstract = {BACKGROUND: Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.<br><br>METHODS: A total of n&#x2009;=&#x2009;139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.<br><br>RESULTS: Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4&#xa0;M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.<br><br>CONCLUSION: This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.},
}
@article {pmid38554739,
year = {2024},
author = {Panthi, VK and Fairfull-Smith, KE and Islam, N},
title = {Liposomal drug delivery strategies to eradicate bacterial biofilms: Challenges, recent advances, and future perspectives.},
journal = {International journal of pharmaceutics},
volume = {655},
number = {},
pages = {124046},
doi = {10.1016/j.ijpharm.2024.124046},
pmid = {38554739},
issn = {1873-3476},
abstract = {Typical antibiotic treatments are often ineffectual against biofilm-related infections since bacteria residing within biofilms have developed various mechanisms to resist antibiotics. To overcome these limitations, antimicrobial-loaded liposomal nanoparticles are a promising anti-biofilm strategy as they have demonstrated improved antibiotic delivery and eradication of bacteria residing in biofilms. Antibiotic-loaded liposomal nanoparticles revealed remarkably higher antibacterial and anti-biofilm activities than free drugs in experimental settings. Moreover, liposomal nanoparticles can be used efficaciously for the combinational delivery of antibiotics and other antimicrobial compounds/peptide which facilitate, for instance, significant breakdown of the biofilm matrix, increased bacterial elimination from biofilms and depletion of metabolic activity of various pathogens. Drug-loaded liposomes have mitigated recurrent infections and are considered a promising tool to address challenges associated to antibiotic resistance. Furthermore, it has been demonstrated that surface charge and polyethylene glycol modification of liposomes have a notable impact on their antibacterial biofilm activity. Future investigations should tackle the persistent hurdles associated with development of safe and effective liposomes for clinical application and investigate novel antibacterial treatments, including CRISPR-Cas gene editing, natural compounds, phages, and nano-mediated approaches. Herein, we emphasize the significance of liposomes in inhibition and eradication of various bacterial biofilms, their challenges, recent advances, and future perspectives.},
}
@article {pmid38521032,
year = {2024},
author = {Han, X and Song, D and Xu, W and Lu, L and Zhu, A and Long, F},
title = {CRISPR/Cas12a powered air-displacement enhanced evanescent wave fluorescence fiber-embedded microfluidic biochip for nucleic acid amplification-free detection of Escherichia coli O157:H7.},
journal = {Journal of hazardous materials},
volume = {469},
number = {},
pages = {134037},
doi = {10.1016/j.jhazmat.2024.134037},
pmid = {38521032},
issn = {1873-3336},
mesh = {*Escherichia coli O157/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; Microfluidics ; *Biosensing Techniques ; *Nucleic Acids ; },
abstract = {Simple yet ultrasensitive and contamination-free quantification of environmental pathogenic bacteria is in high demand. In this study, we present a portable clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) powered Air-displacement enhanced Evanescent wave fluorescence Fiber-embedded microfluidic Biochip (AEFB) for the high-frequency and nucleic acid amplification-free ultrasensitive detection of Escherichia coli O157:H7. The performance of AEFB was dramatically enhanced upon employing a simple air-solution displacement process. Theoretical assays demonstrated that air-solution displacement significantly enhances evanescent wave field intensity on the fiber biosensor surface and increases the V-number in tapered fiber biosensors. Consequently, light-matter interaction is strengthened, and fluorescence coupling and collection efficiency are improved, considerably enhancing sensitivity. By integrating the CRISPR biosensing mechanism, AEFB facilitated rapid, accurate, nucleic acid amplification-free detection of E.coli O157:H7 with polymerase chain reaction (PCR)-level sensitivity (176 cfu/mL). To validate its practicality, AEFB was used to detect E.coli O157:H7 in surface water and wastewater. Comparison with RT-PCR showed a strong linear relationship (R[2] = 0.9871), indicating the excellent accuracy and reliability of this technology in real applications. AEFB is highly versatile and can be easily extended to detect other pathogenic bacteria, which will significantly promote the high-frequency assessment and early-warning of bacterial contamination in aquatic environments.},
}
@article {pmid38503294,
year = {2024},
author = {Oh, Y and Lee, SH},
title = {Tiny CRISPR molecular switch tool opens a new world.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {4},
pages = {870-872},
doi = {10.1016/j.ymthe.2024.03.010},
pmid = {38503294},
issn = {1525-0024},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Gene Editing ; },
}
@article {pmid38491159,
year = {2024},
author = {Tavridou, A and Rogers, D and Farinelli, G and Gravanis, I and Jekerle, V},
title = {Genome-editing medicinal products: the EMA perspective.},
journal = {Nature reviews. Drug discovery},
volume = {23},
number = {4},
pages = {242-243},
doi = {10.1038/d41573-024-00050-2},
pmid = {38491159},
issn = {1474-1784},
mesh = {Humans ; *Genome ; *Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid38403745,
year = {2024},
author = {Barker, E and Morgan, A and Barclay, JW},
title = {Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14.},
journal = {Cell and tissue research},
volume = {396},
number = {1},
pages = {41-55},
pmid = {38403745},
issn = {1432-0878},
support = {/WT_/Wellcome Trust/United Kingdom ; BB/R01390X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 102172/B/13/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Male ; Animals ; Mice ; Humans ; *Caenorhabditis elegans/genetics/metabolism ; Tissue Distribution ; *CRISPR-Cas Systems/genetics ; Membrane Proteins/metabolism ; Mammals/metabolism ; *HSP40 Heat-Shock Proteins ; },
abstract = {Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPβ and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.},
}
@article {pmid38377993,
year = {2024},
author = {He, Y and Zhou, X and Chang, C and Chen, G and Liu, W and Li, G and Fan, X and Sun, M and Miao, C and Huang, Q and Ma, Y and Yuan, F and Chang, X},
title = {Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing.},
journal = {Molecular cell},
volume = {84},
number = {7},
pages = {1257-1270.e6},
doi = {10.1016/j.molcel.2024.01.021},
pmid = {38377993},
issn = {1097-4164},
mesh = {Animals ; Mice ; *Gene Editing ; *Uracil-DNA Glycosidase/genetics/metabolism ; Mutation ; Uracil ; CRISPR-Cas Systems ; *Alkanesulfonic Acids ; },
abstract = {Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.},
}
@article {pmid38351611,
year = {2024},
author = {Wang, X and Li, L and Guo, L and Feng, Y and Du, Z and Jiang, W and Wu, X and Zheng, J and Xiao, X and Zheng, H and Sun, Y and Ma, H},
title = {Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {4},
pages = {910-919},
doi = {10.1016/j.ymthe.2024.02.013},
pmid = {38351611},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Transcriptional Activation ; Genetic Therapy ; },
abstract = {The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1,319-fold increase in the activation of the ASCL1 gene. The activity can be further increased by tethering DNA-binding protein Sso7d to miniCRa and generating SminiCRa, which reached a 5,628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression of ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi used to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In summary, miniCRa, SminiCRa, miniCRi, and SminiCRi are robust miniature transcriptional modulators with high specificity that expand the toolbox for biomedical research and therapeutic applications.},
}
@article {pmid38342473,
year = {2024},
author = {Quintero-Ruiz, N and Oliveira, WL and Esteca, MV and Granato, DC and Simabuco, FM},
title = {Uncovering the bookshelves of CRISPR-based libraries: Advances and applications in cancer studies.},
journal = {Critical reviews in oncology/hematology},
volume = {196},
number = {},
pages = {104287},
doi = {10.1016/j.critrevonc.2024.104287},
pmid = {38342473},
issn = {1879-0461},
mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Gene Library ; *Neoplasms/genetics/therapy ; },
abstract = {The advent of CRISPR/Cas9 technology has revolutionized the genome editing field. CRISPR-based libraries have become powerful tools for high-throughput functional genomics and genetic screening. CRISPR-based libraries can represent a powerful approach to uncovering genes related to chemoresistance and therapy efficacy and to studying cancer cells' fitness. In this review, we conducted an extensive literature search and summarized multiple studies that utilized these libraries in both in vitro and in vivo research, emphasizing their key findings. We provide an overview of the design, construction, and applications of CRISPR-based libraries in different cancer-focused studies and discuss the different types of CRISPR-based libraries. We finally point out the challenges associated with library design, including guide RNA selection, off-target effects, and library complexity. This review provides an overview of the work conducted with CRISPR libraries in the search for new targets that could potentially assist in cancer therapy by contributing to functional approaches.},
}
@article {pmid38341611,
year = {2024},
author = {Zhang, W and Wang, H and Luo, Z and Jian, Y and Gong, C and Wang, H and Lin, X and Liu, M and Wang, Y and Shao, H},
title = {Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {4},
pages = {920-934},
doi = {10.1016/j.ymthe.2024.02.008},
pmid = {38341611},
issn = {1525-0024},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Nuclear Localization Signals/genetics ; },
abstract = {CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.},
}
@article {pmid38041781,
year = {2024},
author = {Wang, J and Wu, S and Ye, K},
title = {Complicated target recognition by archaeal box C/D guide RNAs.},
journal = {Science China. Life sciences},
volume = {67},
number = {4},
pages = {631-644},
pmid = {38041781},
issn = {1869-1889},
mesh = {Base Sequence ; *RNA, Archaeal/genetics ; Nucleic Acid Conformation ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Ribosomal/genetics ; },
abstract = {Box C/D RNAs guide the site-specific formation of 2'-O-methylated nucleotides (Nm) of RNAs in eukaryotes and archaea. Although C/D RNAs have been profiled in several archaea, their targets have not been experimentally determined. Here, we mapped Nm in rRNAs, tRNAs, and abundant small RNAs (sRNAs) and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus. The targets of C/D RNAs were assigned by analysis of base-pairing interactions, in vitro modification assays, and gene deletion experiments, revealing a complicated landscape of C/D RNA-target interactions. C/D RNAs widely use dual antisense elements to target adjacent sites in rRNAs, enhancing modification at weakly bound sites. Two consecutive sites can be guided with the same antisense element upstream of box D or D', a phenomenon known as double-specificity that is exclusive to internal box D' in eukaryotic C/D RNAs. Several C/D RNAs guide modification at a single non-canonical site. This study reveals the global landscape of RNA-guided 2'-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.},
}
@article {pmid38554115,
year = {2024},
author = {Enustun, E and Armbruster, EG and Lee, J and Zhang, S and Yee, BA and Malukhina, K and Gu, Y and Deep, A and Naritomi, JT and Liang, Q and Aigner, S and Adler, BA and Cress, BF and Doudna, JA and Chaikeeratisak, V and Cleveland, DW and Ghassemian, M and Bintu, B and Yeo, GW and Pogliano, J and Corbett, KD},
title = {A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae216},
pmid = {38554115},
issn = {1362-4962},
support = {R01 GM129245/NH/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
abstract = {Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.},
}
@article {pmid38552157,
year = {2024},
author = {Yang, S and Im, SH and Chung, JY and Lee, J and Lee, KH and Kang, YK and Chung, HJ},
title = {An Antibody-CRISPR/Cas Conjugate Platform for Target-Specific Delivery and Gene Editing in Cancer.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2308763},
doi = {10.1002/advs.202308763},
pmid = {38552157},
issn = {2198-3844},
support = {2021R1A2C2011763//National Research Foundation of Korea/ ; 2022R1A4A5028131//National Research Foundation of Korea/ ; HI22C2010//Ministry of Health and Welfare/ ; },
abstract = {The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody-CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2-positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody-conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2-positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody-CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2-positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody-mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases.},
}
@article {pmid38552011,
year = {2024},
author = {Liu, F and Xin, M and Feng, H and Zhang, W and Liao, Z and Sheng, T and Wen, P and Wu, Q and Liang, T and Shi, J and Zhou, R and He, K and Gu, Z and Li, H},
title = {Cryo-shocked tumor cells deliver CRISPR-Cas9 for lung cancer regression by synthetic lethality.},
journal = {Science advances},
volume = {10},
number = {13},
pages = {eadk8264},
pmid = {38552011},
issn = {2375-2548},
mesh = {Mice ; Animals ; *Lung Neoplasms/genetics/therapy ; CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Gene Transfer Techniques ; Synthetic Lethal Mutations ; Endothelial Cells ; Proto-Oncogene Proteins p21(ras)/genetics ; Cell Line, Tumor ; Gene Editing ; Lung ; },
abstract = {Although CRISPR-mediated genome editing holds promise for cancer therapy, inadequate tumor targeting and potential off-target side effects hamper its outcomes. In this study, we present a strategy using cryo-shocked lung tumor cells as a CRISPR-Cas9 delivery system for cyclin-dependent kinase 4 (CDK4) gene editing, which initiates synthetic lethal in KRAS-mutant non-small cell lung cancer (NSCLC). By rapidly liquid nitrogen shocking, we effectively eliminate the pathogenicity of tumor cells while preserving their structure and surface receptor activity. This delivery system enables the loaded CRISPR-Cas9 to efficiently target to lung through the capture in pulmonary capillaries and interactions with endothelial cells. In a NSCLC-bearing mouse model, the drug accumulation is increased nearly fourfold in lung, and intratumoral CDK4 expression is substantially down-regulated compared to CRISPR-Cas9 lipofectamine nanoparticles administration. Furthermore, CRISPR-Cas9 editing-mediated CDK4 ablation triggers synthetic lethal in KRAS-mutant NSCLC and prolongs the survival of mice.},
}
@article {pmid38551977,
year = {2024},
author = {Yang, L and Chen, G and Wu, J and Wei, W and Peng, C and Ding, L and Chen, X and Xu, X and Wang, X and Xu, J},
title = {A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c05545},
pmid = {38551977},
issn = {1520-6882},
abstract = {Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/μL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.},
}
@article {pmid38551745,
year = {2024},
author = {Shi, L and Gu, R and Long, J and Duan, G and Yang, H},
title = {Application of CRISPR-cas-based technology for the identification of tuberculosis, drug discovery and vaccine development.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {466},
pmid = {38551745},
issn = {1573-4978},
support = {No. 82273696//National Natural Science Foundation of China/ ; },
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Tuberculosis/prevention &amp; control/microbiology ; *Mycobacterium tuberculosis/genetics ; Drug Discovery ; Vaccine Development ; },
abstract = {Tuberculosis (TB), which caused by Mycobacterium tuberculosis, is the leading cause of death from a single infectious agent and continues to be a major public health burden for the global community. Despite being the only globally licenced prophylactic vaccine, Bacillus Calmette-Guérin (BCG) has multiple deficiencies, and effective diagnostic and therapeutic options are limited. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is an adaptive immune system that is found in bacteria and has great potential for the development of novel antituberculosis drugs and vaccines. In addition, CRISPR-Cas is currently recognized as a prospective tool for the development of therapies for TB infection with potential diagnostic and therapeutic value, and CRISPR-Cas may become a viable tool for eliminating TB in the future. Herein, we systematically summarize the current applications of CRISPR-Cas-based technology for TB detection and its potential roles in drug discovery and vaccine development.},
}
@article {pmid38551156,
year = {2024},
author = {Zhang, X and Wang, Y and Tang, Y and Yang, L and Zhao, C and Yang, G and Wang, P and Gao, S},
title = {A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for Vibrio vulnificus Detection.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {},
doi = {10.1089/fpd.2023.0119},
pmid = {38551156},
issn = {1556-7125},
abstract = {Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40-60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.},
}
@article {pmid38550570,
year = {2024},
author = {Bohle, F and Schneider, R and Mundorf, J and Zühl, L and Simon, S and Engelhard, M},
title = {Where does the EU-path on new genomic techniques lead us?.},
journal = {Frontiers in genome editing},
volume = {6},
number = {},
pages = {1377117},
pmid = {38550570},
issn = {2673-3439},
abstract = {Recently, the European Commission (EC) published a regulatory proposal on plants generated with new genomic techniques (NGTs) (5 July 2023). According to this proposal, NGT plant applications are categorized into category 1 NGT (NGT1) and category 2 NGT (NGT2) based on their molecular characteristics, which diverges from the current legislation centered around Directive 2001/18/EC. To demonstrate where the path of the proposal leads to in practice, we applied the proposed criteria for categorization to a list of NGT plant applications currently in the commercialization pipeline. Combining literature research and a descriptive statistical approach, we can show that 94% of the plant applications affected by the EC proposal, would be classified as NGT1 and thus would receive market approval without risk assessment, monitoring, and sufficient labeling provisions. The remaining 6% of applications would be classified as NGT2 plants, for which, in deviation from the current regulation, an adapted risk assessment is proposed. Screening of the intended traits in the pipeline highlights that certain NGT1 plants can pose similar environmental risks (e.g., invasiveness) to other genetically modified organisms (GMOs), as defined in Directive 2001/18/EC. For example, NGT1 applications based on RNA interference technology can exhibit insecticidal effects with potential side effects on non-target organisms (i.e., other insects). Our quantitative and case-specific elaboration of how the current EC regulatory proposal would affect the environment, health, and consumer protection will be informative for decision-makers and politicians.},
}
@article {pmid38549983,
year = {2024},
author = {International, BR},
title = {Retracted: CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.},
journal = {BioMed research international},
volume = {2024},
number = {},
pages = {9849515},
pmid = {38549983},
issn = {2314-6141},
abstract = {[This retracts the article DOI: 10.1155/2022/9080396.].},
}
@article {pmid38549686,
year = {2023},
author = {Wang, X and Xu, G and Johnson, WA and Qu, Y and Yin, D and Ramkissoon, N and Xiang, H and Cong, L},
title = {Long sequence insertion via CRISPR/Cas gene-editing with transposase, recombinase, and integrase.},
journal = {Current opinion in biomedical engineering},
volume = {28},
number = {},
pages = {},
pmid = {38549686},
issn = {2468-4511},
support = {R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; },
abstract = {CRISPR/Cas-based gene-editing technologies have emerged as one of the most transformative tools in genome science over the past decade, providing unprecedented possibilities for both fundamental and translational research. Following the initial wave of innovations for gene knock-out, epigenetic/RNA modulation, and nickase-mediated base-editing, recent efforts have pivoted towards long-sequence gene editing- specifically, the insertion of large fragments (>1 kb) into the endogenous genome. In this review, we survey the development of these CRISPR/Cas-based sequence insertion methodologies in conjunction with the emergence of novel families of editing enzymes, such as transposases, single-stranded DNA-annealing proteins, recombinases, and integrases. Despite facing a number of challenges, this field continues to evolve rapidly and holds the potential to catalyze a new wave of revolutionary biomedical applications.},
}
@article {pmid38548901,
year = {2024},
author = {Kim, H and Han, JH and Kim, H and Kim, M and Jo, SI and Lee, N and Cha, S and Oh, MJ and Choi, G and Kim, HS},
title = {CRISPR/Cas9 targeting of passenger single nucleotide variants in haploinsufficient or essential genes expands cancer therapy prospects.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {7436},
pmid = {38548901},
issn = {2045-2322},
support = {2019H1A2A1075632//National Research Foundation of Korea/ ; 2023R1A2C2003586//National Research Foundation of Korea/ ; HI14C1324//Korea Health Industry Development Institute/ ; 6-2021-0194//Yonsei University College of Medicine/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Genes, Essential ; Mutation ; Nucleotides ; Gene Editing ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR/Cas9 technology has effectively targeted cancer-specific oncogenic hotspot mutations or insertion-deletions. However, their limited prevalence in tumors restricts their application. We propose a novel approach targeting passenger single nucleotide variants (SNVs) in haploinsufficient or essential genes to broaden therapeutic options. By disrupting haploinsufficient or essential genes through the cleavage of DNA in the SNV region using CRISPR/Cas9, we achieved the selective elimination of cancer cells without affecting normal cells. We found that, on average, 44.8% of solid cancer patients are eligible for our approach, a substantial increase compared to the 14.4% of patients with CRISPR/Cas9-applicable oncogenic hotspot mutations. Through in vitro and in vivo experiments, we validated our strategy by targeting a passenger mutation in the essential ribosomal gene RRP9 and haploinsufficient gene SMG6. This demonstrates the potential of our strategy to selectively eliminate cancer cells and expand therapeutic opportunities.},
}
@article {pmid38548070,
year = {2024},
author = {Akter, S and Kamal, E and Schwarz, C and Lewin, A},
title = {Gene knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR/Cas.},
journal = {Journal of microbiological methods},
volume = {220},
number = {},
pages = {106924},
doi = {10.1016/j.mimet.2024.106924},
pmid = {38548070},
issn = {1872-8359},
abstract = {The CRISPRi system using dCas9Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity.},
}
@article {pmid38512977,
year = {2024},
author = {Peng, G and Liu, T and Qi, X and Wang, Y and Ren, J and Peng, J and Du, X and Hu, S and Wu, S and Zhao, Y and Li, D and Zheng, H},
title = {A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways.},
journal = {PLoS pathogens},
volume = {20},
number = {3},
pages = {e1012104},
pmid = {38512977},
issn = {1553-7374},
mesh = {Mice ; Swine ; Animals ; *Foot-and-Mouth Disease Virus/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Expression Regulation ; ErbB Receptors/metabolism ; *Foot-and-Mouth Disease/genetics ; },
abstract = {The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs.},
}
@article {pmid38410866,
year = {2024},
author = {Wang, L and Wang, J and Feng, D and Wang, B and Jahan-Mihan, Y and Wang, Y and Bi, Y and Lim, D and Ji, B},
title = {A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.},
journal = {American journal of physiology. Gastrointestinal and liver physiology},
volume = {326},
number = {4},
pages = {G473-G481},
doi = {10.1152/ajpgi.00013.2024},
pmid = {38410866},
issn = {1522-1547},
support = {DK117910//HHS | NIH | NIDDK | Division of Diabetes, Endocrinology, and Metabolic Diseases (DEM)/ ; CA255068//HHS | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Genotype ; Trypsinogen ; Workflow ; },
abstract = {Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW &amp; NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.},
}
@article {pmid38547405,
year = {2024},
author = {Li, J and Wang, Z and Fan, X and Yao, R and Zhang, G and Fan, R and Wang, Z},
title = {Rapid multiple protein sequence search by parallel and heterogeneous computation.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btae151},
pmid = {38547405},
issn = {1367-4811},
abstract = {MOTIVATION: Protein sequence database search and multiple sequence alignment (MSA) generation is a fundamental task in many bioinformatics analyses. As the data volume of sequences continues to grow rapidly, there is an increasing need for efficient and scalable multiple sequence query algorithms for super-large databases without expensive time and computational costs.<br><br>RESULTS: We introduce Chorus, a novel protein sequence query system that leverages parallel model and heterogeneous computation architecture to enable users to query thousands of protein sequences concurrently against large protein databases on a desktop workstation. Chorus achieves over 100x speedup over BLASTP without sacrificing sensitivity. We demonstrate the utility of Chorus through a case study of analyzing a &#x223c;1.5TB large-scale metagenomic datasets for novel CRISPR-Cas protein discovery within 30&#x2009;minutes.<br><br>AVAILABILITY: Chorus is open-source and its code repository is available at https://github.com/Bio-Acc/Chorus.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid38547339,
year = {2024},
author = {Berman, RE and Dampier, W and Nonnemacher, MR and Wigdahl, B},
title = {What's in a cure: designing a broad-spectrum HIV gene therapy.},
journal = {Current opinion in HIV and AIDS},
volume = {19},
number = {3},
pages = {150-156},
doi = {10.1097/COH.0000000000000846},
pmid = {38547339},
issn = {1746-6318},
abstract = {PURPOSE OF REVIEW: The leading gene editing strategy for a human immunodeficiency virus type 1 (HIV-1) cure involves the delivery of SaCas9 and two guide RNAs (gRNAs) in an adeno-associated viral (AAV) vector. As a dual-component system, CRISPR is targeted to a genetic locus through the choice of a Cas effector and gRNA protospacer design pair. As CRISPR research has expanded in recent years, these components have been investigated for utilization in cure strategies, which will be discussed in this article.<br><br>RECENT FINDINGS: Type II SpCas9 and SaCas9 have been the leading Cas effectors across gene editing therapeutics to date. Additionally, extensive research has expanded the potential to multiplex gRNAs and target them effectively to the highly genetically diverse HIV-1 provirus. More recently, the Type V family of Cas12 effectors opens a new opportunity to use a smaller Cas protein for packaging into an AAV vector with multiplexed gRNAs.<br><br>SUMMARY: In understanding the individual components of a CRISPR/Cas therapeutic cure for HIV-1, it is important to know that the currently used strategies can be improved upon. Future areas will include alternative smaller Cas effectors, multiplexed gRNAs designs, and/or alternative delivery modalities.},
}
@article {pmid38546638,
year = {2024},
author = {Neri, P and Boise, LH},
title = {TP53 function over forms in multiple myeloma.},
journal = {Blood},
volume = {143},
number = {13},
pages = {1202-1204},
doi = {10.1182/blood.2023023487},
pmid = {38546638},
issn = {1528-0020},
mesh = {Humans ; *Multiple Myeloma/genetics ; CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics ; bcl-2-Associated X Protein ; },
}
@article {pmid38545983,
year = {2024},
author = {Xie, H and Huang, L and Luo, Y and Zhang, G},
title = {[Prediction of CRISPR/Cas9 off-target activity using multi-scale convolutional neural network].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {40},
number = {3},
pages = {858-876},
doi = {10.13345/j.cjb.230382},
pmid = {38545983},
issn = {1872-2075},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Neural Networks, Computer ; Genome ; },
abstract = {Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) is a new generation of gene editing technology, which relies on single guide RNA to identify specific gene sites and guide Cas9 nuclease to edit specific location in the genome. However, the off-target effect of this technology hampers its development. In recent years, several deep learning models have been developed for predicting the CRISPR/Cas9 off-target activity, which contributes to more efficient and safe gene editing and gene therapy. However, the prediction accuracy remains to be improved. In this paper, we proposed a multi-scale convolutional neural network-based method, designated as CnnCRISPR, for CRISPR/Cas9 off-target prediction. First, we used one-hot encoding method to encode the sgRNA-DNA sequence pair, followed by a bitwise or operation on the two binary matrices. Second, the encoded sequence was fed into the Inception-based network for training and evaluating. Third, the well-trained model was applied to evaluate the off-target situation of the sgRNA-DNA sequence pair. Experiments on public datasets showed CnnCRISPR outperforms existing deep learning-based methods, which provides an effective and feasible method for addressing the off-target problems.},
}
@article {pmid38500378,
year = {2024},
author = {Zhu, D and Su, T and Sun, T and Qin, X and Su, S and Bai, Y and Li, F and Zhao, D and Shao, G and Chao, J and Feng, Z and Wang, L},
title = {Enhancing Point-of-Care Diagnosis of African Swine Fever Virus (ASFV) DNA with the CRISPR-Cas12a-Assisted Triplex Amplified Assay.},
journal = {Analytical chemistry},
volume = {96},
number = {13},
pages = {5178-5187},
doi = {10.1021/acs.analchem.3c05364},
pmid = {38500378},
issn = {1520-6882},
mesh = {Animals ; Swine ; *African Swine Fever Virus/genetics ; CRISPR-Cas Systems/genetics ; Gold ; *Metal Nanoparticles ; Point-of-Care Systems ; Hydrolases ; Recombinases ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques ; },
abstract = {Accurate, ultrasensitive, and point-of-care (POC) diagnosis of the African swine fever virus (ASFV) remains imperative to prevent its spread and limit the losses incurred. Herein, we propose a CRISPR-Cas12a-assisted triplex amplified colorimetric assay for ASFV DNA detection with ultrahigh sensitivity and specificity. The specific recognition of recombinase aided amplification (RAA)-amplified ASFV DNA could activate the Cas12a/crRNA/ASFV DNA complex, leading to the digestion of the linker DNA (bio-L1) on magnetic beads (MBs), thereby preventing its binding of gold nanoparticles (AuNPs) network. After magnetic separation, the release of AuNPs network comprising a substantial quantity of AuNPs could lead to a discernible alteration in color and significantly amplify the plasmonic signal, which could be read by spectrophotometers or smartphones. By combining the RAA, CRISPR/Cas12a-assisted cleavage, and AuNPs network-mediated colorimetric amplification together, the assay could detect as low as 0.1 copies/μL ASFV DNA within 1 h. The assay showed an accuracy of 100% for the detection of ASFV DNA in 16 swine tissue fluid samples, demonstrating its potential for on-site diagnosis of ASFV.},
}
@article {pmid38545958,
year = {2024},
author = {Dorner, L and Stratmann, B and Bader, L and Podobnik, M and Irion, U},
title = {Efficient genome editing using modified Cas9 proteins in zebrafish.},
journal = {Biology open},
volume = {13},
number = {4},
pages = {},
doi = {10.1242/bio.060401},
pmid = {38545958},
issn = {2046-6390},
support = {//Max-Planck-Gesellschaft/ ; 2816ERA06G//Bundesministerium f&#xfc;r Ern&#xe4;hrung und Landwirtschaft/ ; //Max Planck Institute for Biology T&#xfc;bingen/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Zebrafish/genetics/metabolism ; DNA Breaks, Double-Stranded ; },
abstract = {The zebrafish (Danio rerio) is an important model organism for basic as well as applied bio-medical research. One main advantage is its genetic tractability, which was greatly enhanced by the introduction of the CRISPR/Cas method a decade ago. The generation of loss-of-function alleles via the production of small insertions or deletions in the coding sequences of genes with CRISPR/Cas systems is now routinely achieved with high efficiency. The method is based on the error prone repair of precisely targeted DNA double strand breaks by non-homologous end joining (NHEJ) in the cell nucleus. However, editing the genome with base pair precision, by homology-directed repair (HDR), is by far less efficient and therefore often requires large-scale screening of potential carriers by labour intensive genotyping. Here we confirm that the Cas9 protein variant SpRY, with relaxed PAM requirement, can be used to target some sites in the zebrafish genome. In addition, we demonstrate that the incorporation of an artificial nuclear localisation signal (aNLS) into the Cas9 protein variants not only enhances the efficiency of gene knockout but also the frequency of HDR, thereby facilitating the efficient modification of single base pairs in the genome. Our protocols provide a guide for a cost-effective generation of versatile and potent Cas9 protein variants and efficient gene editing in zebrafish.},
}
@article {pmid38543787,
year = {2024},
author = {Liu, Y and Liang, Z and Yu, S and Ye, Y and Lin, Z},
title = {CRISPR RNA-Guided Transposases Facilitate Dispensable Gene Study in Phage.},
journal = {Viruses},
volume = {16},
number = {3},
pages = {},
pmid = {38543787},
issn = {1999-4915},
support = {2018YFA0901000, 2022YFC2104800//National Key R&amp;D Program of China/ ; },
mesh = {*Bacteriophages/genetics ; RNA ; Transposases/genetics ; CRISPR-Cas Systems ; Genes, Viral ; Bacteria/genetics ; },
abstract = {Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR-Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.},
}
@article {pmid38543774,
year = {2024},
author = {Hassanien, RT and Thieulent, CJ and Carossino, M and Li, G and Balasuriya, UBR},
title = {Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing.},
journal = {Viruses},
volume = {16},
number = {3},
pages = {},
pmid = {38543774},
issn = {1999-4915},
mesh = {Animals ; Horses ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; *Herpesvirus 1, Equid/genetics ; Genome, Viral ; },
abstract = {(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.},
}
@article {pmid38542493,
year = {2024},
author = {Sasaki, S and Ogawa, H and Katoh, H and Honda, T},
title = {Suppression of Borna Disease Virus Replication during Its Persistent Infection Using the CRISPR/Cas13b System.},
journal = {International journal of molecular sciences},
volume = {25},
number = {6},
pages = {},
pmid = {38542493},
issn = {1422-0067},
support = {JP18H02664, JP18K19449, JP21H02738, JP22K19436, JP22K06027//Japan Society for the Promotion of Science/ ; None//Takeda Science Foundation/ ; None//Kobayashi International Scholarship Foundation/ ; None//Naito Foundation/ ; },
mesh = {Animals ; Humans ; *Borna disease virus/genetics ; Persistent Infection ; RNA, Guide, CRISPR-Cas Systems ; *RNA Viruses/genetics ; Genome ; CRISPR-Cas Systems/genetics ; *Borna Disease/genetics ; Virus Replication/genetics ; },
abstract = {Borna disease virus (BoDV-1) is a bornavirus that infects the central nervous systems of various animal species, including humans, and causes fatal encephalitis. BoDV-1 also establishes persistent infection in neuronal cells and causes neurobehavioral abnormalities. Once neuronal cells or normal neural networks are lost by BoDV-1 infection, it is difficult to regenerate damaged neural networks. Therefore, the development of efficient anti-BoDV-1 treatments is important to improve the outcomes of the infection. Recently, one of the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems, CRISPR/Cas13, has been utilized as antiviral tools. However, it is still unrevealed whether the CRISPR/Cas13 system can suppress RNA viruses in persistently infected cells. In this study, we addressed this question using persistently BoDV-1-infected cells. The CRISPR/Cas13 system targeting viral mRNAs efficiently decreased the levels of target viral mRNAs and genomic RNA (gRNA) in persistently infected cells. Furthermore, the CRISPR/Cas13 system targeting viral mRNAs also suppressed BoDV-1 infection if the system was introduced prior to the infection. Collectively, we demonstrated that the CRISPR/Cas13 system can suppress BoDV-1 in both acute and persistent infections. Our findings will open the avenue to treat prolonged infection with RNA viruses using the CRISPR/Cas13 system.},
}
@article {pmid38542449,
year = {2024},
author = {Yang, Y and Kong, X and Yang, J and Xue, J and Niu, B and Chen, Q},
title = {Rapid Nucleic Acid Detection of Listeria monocytogenes Based on RAA-CRISPR Cas12a System.},
journal = {International journal of molecular sciences},
volume = {25},
number = {6},
pages = {},
pmid = {38542449},
issn = {1422-0067},
mesh = {Animals ; Cattle ; Humans ; *Listeria monocytogenes/genetics ; CRISPR-Cas Systems ; Food Microbiology ; Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics ; DNA ; },
abstract = {Listeria monocytogenes (L. monocytogenes) is a food-borne pathogenic bacteria that frequently contaminates animal-derived food and low-temperature preserved food. Listeriosis caused by its infection has a high mortality rate and poses a serious threat to human health. Therefore, it is crucial to establish a sensitive, rapid and easy-to-operate technique. In this study, a Recombinase Aided Amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform was established for highly sensitive nucleic acid detection of L. monocytogenes. The established RAA-CRISPR/Cas12a showed high sensitivity and high specificity, with the sensitivity of 350 CFU/mL and 5.4 × 10[-3] ng/μL for pure bacterial solution and genomic DNA, and good specificity for 5 strains of Listeria spp. and 14 strains of other common pathogenic bacteria. L. monocytogenes could be detected at an initial concentration of 2.3 CFU/25g within 2 h of enriching the beef in the food matrix, and this method could be applied to food samples that were easily contaminated with L. monocytogenes The results of RAA-CRISPR/Cas12a could be observed in 5 min, while the amplification was completed in 20-30 min. The speed and sensitivity of RAA-CRISPR/Cas12a were significantly higher than that of the national standard method. In conclusion, the RAA-CRISPR/Cas12a system established in this study has new application potential in the diagnosis of food-borne pathogens.},
}
@article {pmid38542245,
year = {2024},
author = {Devillars, A and Magon, G and Pirrello, C and Palumbo, F and Farinati, S and Barcaccia, G and Lucchin, M and Vannozzi, A},
title = {Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals.},
journal = {International journal of molecular sciences},
volume = {25},
number = {6},
pages = {},
pmid = {38542245},
issn = {1422-0067},
support = {CN00000022//European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Juniperus ; Gene Editing/methods ; Plants/genetics ; Genomics ; DNA ; },
abstract = {The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.},
}
@article {pmid38542067,
year = {2024},
author = {Zhou, J and Feng, Z and Lv, D and Wang, D and Sang, K and Liu, Z and Guo, D and Shen, Y and Chen, Q},
title = {Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.},
journal = {International journal of molecular sciences},
volume = {25},
number = {6},
pages = {},
pmid = {38542067},
issn = {1422-0067},
support = {2021J01202; 2023J011275; 2023J011568//Natural Science Foundation of Fujian province, China/ ; },
mesh = {Animals ; Humans ; Swine ; Chlorocebus aethiops ; *Porcine epidemic diarrhea virus/genetics ; Protein Kinase C-theta/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems ; Vero Cells ; *Swine Diseases/genetics ; Virus Replication/genetics ; },
abstract = {Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.},
}
@article {pmid38539119,
year = {2024},
author = {Hourigan, D and Stefanovic, E and Hill, C and Ross, RP},
title = {Promiscuous, persistent and problematic: insights into current enterococcal genomics to guide therapeutic strategy.},
journal = {BMC microbiology},
volume = {24},
number = {1},
pages = {103},
pmid = {38539119},
issn = {1471-2180},
support = {SFI/12/RC/2273_P2//Science Foundation Ireland (SFI)/ ; SFI/12/RC/2273_P2//Science Foundation Ireland (SFI)/ ; SFI/12/RC/2273_P2//Science Foundation Ireland (SFI)/ ; SFI/12/RC/2273_P2//Science Foundation Ireland (SFI)/ ; ERC, BACtheWINNER, Project No. 101054719//HORIZON EUROPE European Research Council/ ; },
mesh = {Humans ; Anti-Bacterial Agents/pharmacology/therapeutic use ; *Vancomycin-Resistant Enterococci/genetics ; *Enterococcus faecium/genetics ; *Gastrointestinal Microbiome/genetics ; Genomics ; *Gram-Positive Bacterial Infections/drug therapy/microbiology ; Microbial Sensitivity Tests ; },
abstract = {Vancomycin-resistant enterococci (VRE) are major opportunistic pathogens and the causative agents of serious diseases, such as urinary tract infections and endocarditis. VRE strains mainly include species of Enterococcus faecium and E. faecalis which can colonise the gastrointestinal tract (GIT) of patients and, following growth and persistence in the gut, can transfer to blood resulting in systemic dissemination in the body. Advancements in genomics have revealed that hospital-associated VRE strains are characterised by increased numbers of mobile genetic elements, higher numbers of antibiotic resistance genes and often lack active CRISPR-Cas systems. Additionally, comparative genomics have increased our understanding of dissemination routes among patients and healthcare workers. Since the efficiency of currently available antibiotics is rapidly declining, new measures to control infection and dissemination of these persistent pathogens are urgently needed. These approaches include combinatory administration of antibiotics, strengthening colonisation resistance of the gut microbiota to reduce VRE proliferation through commensals or probiotic bacteria, or switching to non-antibiotic bacterial killers, such as bacteriophages or bacteriocins. In this review, we discuss the current knowledge of the genomics of VRE isolates and state-of-the-art therapeutic advances against VRE infections.},
}
@article {pmid38538988,
year = {2024},
author = {Liu, Y and Balaji, R and de Toledo, MAS and Ernst, S and Hautvast, P and Kesdoğan, AB and Körner, J and Zenke, M and Neureiter, A and Lampert, A},
title = {The pain target NaV1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging.},
journal = {Pflugers Archiv : European journal of physiology},
volume = {},
number = {},
pages = {},
pmid = {38538988},
issn = {1432-2013},
support = {99999.001703/2014-05//Alexander von Humboldt-Stiftung/ ; IZKF TN1-1/IA 532001//Medizinische Fakult&#xe4;t, RWTH Aachen University/ ; 363055819/GRK2415//Deutsche Forschungsgemeinschaft/ ; 13GW0334B//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
abstract = {Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.},
}
@article {pmid38492217,
year = {2024},
author = {Yang, Z and Johnson, BA and Meliopoulos, VA and Ju, X and Zhang, P and Hughes, MP and Wu, J and Koreski, KP and Clary, JE and Chang, TC and Wu, G and Hixon, J and Duffner, J and Wong, K and Lemieux, R and Lokugamage, KG and Alvarado, RE and Crocquet-Valdes, PA and Walker, DH and Plante, KS and Plante, JA and Weaver, SC and Kim, HJ and Meyers, R and Schultz-Cherry, S and Ding, Q and Menachery, VD and Taylor, JP},
title = {Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.},
journal = {Cell reports},
volume = {43},
number = {3},
pages = {113965},
doi = {10.1016/j.celrep.2024.113965},
pmid = {38492217},
issn = {2211-1247},
mesh = {Humans ; *SARS-CoV-2/genetics ; DNA Helicases/metabolism ; RNA Helicases/metabolism ; RNA Recognition Motif Proteins/metabolism ; Poly-ADP-Ribose Binding Proteins/metabolism ; Virulence ; *COVID-19 ; RNA, Guide, CRISPR-Cas Systems ; Nucleocapsid Proteins ; Virus Replication ; RNA, Viral/genetics ; },
abstract = {G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.},
}
@article {pmid38431844,
year = {2024},
author = {Ren, CY and Liu, YS and He, YS and Zhang, LP and Rao, JH and Rao, Y and Chen, JH},
title = {Engineered CBEs based on Macaca fascicularis A3A with improved properties for precise genome editing.},
journal = {Cell reports},
volume = {43},
number = {3},
pages = {113878},
doi = {10.1016/j.celrep.2024.113878},
pmid = {38431844},
issn = {2211-1247},
mesh = {Rats ; Animals ; Humans ; *Gene Editing ; Macaca fascicularis ; *Cytosine ; Cytidine Deaminase/genetics ; RNA/genetics ; DNA/genetics ; CRISPR-Cas Systems ; *Proteins ; },
abstract = {Cytidine deaminase defines the properties of cytosine base editors (CBEs) for C-to-T conversion. Replacing the cytidine deaminase rat APOBEC1 (rA1) in CBEs with a human APOBEC3A (hA3A) improves CBE properties. However, the potential CBE application of macaque A3A orthologs remains undetermined. Our current study develops and evaluates engineered CBEs based on Macaca fascicularis A3A (mA3A). Here, we demonstrate that BE4-mA3A and its RNA-editing-derived variants exhibit improved CBE properties, except for DNA off-target activity, compared to BE3-rA1 and BE4-rA1. Unexpectedly, deleting Ser-Val-Arg (SVR) in BE4-mA3A dramatically reduces DNA and RNA off-target activities and improves editing accuracy, with on-target efficiency unaffected. In contrast, a chimeric BE4-hA3A-SVR[+] shows editing efficiency increased by about 50%, with other properties unaffected. Our findings demonstrate that mA3A-based CBEs could provide prototype options with advantages over rA1- and hA3A-based CBEs for further optimization, highlighting the importance of the SVR motif in defining CBE intrinsic properties.},
}
@article {pmid38431235,
year = {2024},
author = {Hajizadeh, Y and Badmasti, F and Oloomi, M},
title = {Inhibition of the blaOXA-48 gene expression in Klebsiella pneumoniae by a plasmid carrying CRISPRi-Cas9 system.},
journal = {Gene},
volume = {910},
number = {},
pages = {148332},
doi = {10.1016/j.gene.2024.148332},
pmid = {38431235},
issn = {1879-0038},
mesh = {Humans ; *Klebsiella pneumoniae/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; beta-Lactamases/genetics/metabolism ; Plasmids/genetics ; Gene Expression ; *Klebsiella Infections/genetics/microbiology ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; },
abstract = {Antibiotic resistance is an increasing concern that threatens the effectiveness of treating bacterial infections. The spread of carbapenem resistant Klebsiella pneumoniae poses a significant threat to global public health. To combat this issue, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system is being developed. This system includes a single guide RNA (sgRNA) and a nuclease dead Cas9 (dCas9), which work together to downregulate gene expression. Our project involved the use of the CRISPRi system to reduce gene expression of the beta-lactamase oxacillin-48 (blaOXA-48) gene in K. pneumoniae. We designed a sgRNA and cloned it into pJMP1363 plasmid harboring the CRISPRi system. The pJMP1363-sgRNA construct was transformed in K. pneumoniae harboring the blaOXA-48 gene. The MIC test was used to evaluate the antimicrobial resistance, and quantitative real-time RT-PCR was used to confirm the inhibition of the OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA construct expression. The Galleria mellonella larvae model was also utilized for in vivo assay. Following the transformation, the MIC test indicated a 4-fold reduction in meropenem resistance, and qRT-PCR analysis revealed a 60-fold decrease in the mRNA OXA-48 harboring the pJMP1363-sgRNA construct expression. Additionally, G. mellonella larvae infected with OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA showed higher survival rates. Based on the findings, it can be concluded that the CRISPR interference technique has successfully reduced antibiotic resistance and virulence in the K. pneumoniae harboring the blaOXA-48 gene.},
}
@article {pmid38401832,
year = {2024},
author = {Su, H and Zhi, D and Song, Y and Yang, Y and Wang, D and Li, X and Cao, G},
title = {Exploring the formation mechanism of short-tailed phenotypes in animals using mutant mice with the TBXT gene c.G334T developed by CRISPR/Cas9.},
journal = {Gene},
volume = {910},
number = {},
pages = {148310},
doi = {10.1016/j.gene.2024.148310},
pmid = {38401832},
issn = {1879-0038},
mesh = {Pregnancy ; Female ; Mice ; Animals ; Sheep/genetics ; *CRISPR-Cas Systems ; *Tail ; Embryonic Development/genetics ; Phenotype ; Gene Editing/methods ; },
abstract = {With the change in diet structure, individuals prefer to consume mutton with less fat. However, sheep tail has a lot of fat. We identified a breed of low-fat short-tailed sheep (i.e., Hulunbuir short-tailed sheep). It is necessary to develop an animal model that can promote research on the potential mechanisms of the short-tail phenotype in sheep, which results from the TBXT gene c.G334T mutation. To create animal models, we selected mice as experimental animals. Mouse embryos lacking the TBXT protein, which crucially regulates mouse embryonic development, cannot develop normally. We utilized CRISPR/Cas9 gene editing technology to generate site-specific mutation (c.G334T) in the TBXT gene of mice, and found that the mouse TBXT mutation (c.G334T) leads to a short-tail phenotype. Furthermore, we investigated the interaction between TBXT and Wnt signaling pathways. The expressions of TBXT, Axin2, Dkk1, Wnt3, Wnt3a, and Wnt5a were discovered to be significantly different between mutant embryos and wild embryos by obtaining mouse embryos at various developmental stages and examining the expression relationship between the TBXT and Wnt signaling pathway-related components in all of these embryos. Therefore, as a transcription factor, TBXT regulates the expression of the aforementioned Wnt signaling pathway components by forming a regulatory network for the normal development of mouse embryos. This study enriches the research on the functional role of the TBXT in the development of mouse embryos and the mechanism by which the short-tailed phenotype in sheep develops.},
}
@article {pmid38401312,
year = {2024},
author = {Zhang, X and Li, Z and Wang, X and Hong, L and Yin, X and Zhang, Y and Hu, B and Zheng, Q and Cao, J},
title = {CRISPR/Cas12a integrated electrochemiluminescence biosensor for pufferfish authenticity detection based on NiCo2O4 NCs@Au as a coreaction accelerator.},
journal = {Food chemistry},
volume = {445},
number = {},
pages = {138781},
doi = {10.1016/j.foodchem.2024.138781},
pmid = {38401312},
issn = {1873-7072},
mesh = {Animals ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; DNA Primers ; DNA, Single-Stranded ; *Tetraodontiformes/genetics ; *Biosensing Techniques ; },
abstract = {Meat adulteration has brought economic losses, health risks, and religious concerns, making it a pressing global issue. Herein, combining the high amplification efficiency of polymerase chain reaction (PCR) and the accurate recognition of CRISPR/Cas12, a sensitive and reliable electrochemiluminescence (ECL) biosensor was developed for the detection of pufferfish authenticity using NiCo2O4 NCs@Au-ABEI as nanoemitters. In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a is activated upon specific recognition by crRNA, and then it cleaves dopamine-modified single stranded DNA (ssDNA-DA), triggering the ECL signal from the "off" to "on" state. However, without target DNA, the trans-cleavage activity of CRISPR/Cas12a is silenced. By rationally designing corresponding primers and crRNA, the biosensor was applied to specific identification of four species of pufferfish. Furthermore, as low as 0.1 % (w/w) adulterate pufferfish in mixture samples could be detected. Overall, this work provides a simple, low-cost and sensitive approach to trace pufferfish adulteration.},
}
@article {pmid38332200,
year = {2024},
author = {Ma, Y and Su, S and Fu, Z and Zhou, C and Qiao, B and Wu, Y and Yuan, YJ},
title = {Convenient synthesis and delivery of a megabase-scale designer accessory chromosome empower biosynthetic capacity.},
journal = {Cell research},
volume = {34},
number = {4},
pages = {309-322},
pmid = {38332200},
issn = {1748-7838},
support = {31971351//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Chromosomes ; DNA/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic biology confers new functions to hosts by introducing exogenous genetic elements, yet rebuilding complex traits that are based on large-scale genetic information remains challenging. Here, we developed a CRISPR/Cas9-mediated haploidization method that bypasses the natural process of meiosis. Based on the programmed haploidization in yeast, we further developed an easy-to-use method designated HAnDy (Haploidization-based DNA Assembly and Delivery in yeast) that enables efficient assembly and delivery of large DNA, with no need for any fussy in vitro manipulations. Using HAnDy, a de novo designed 1.024 Mb synthetic accessory chromosome (synAC) encoding 542 exogenous genes was parallelly assembled and then directly transferred to six phylogenetically diverse yeasts. The synAC significantly promotes hosts' adaptations and increases the scope of the metabolic network, which allows the emergence of valuable compounds. Our approach should facilitate the assembly and delivery of large-scale DNA for expanding and deciphering complex biological functions.},
}
@article {pmid38243587,
year = {2024},
author = {Liu, D and Myers, EA and Xuan, S and Prichard, LE and Donahue, LI and Ellison, EE and Starker, CG and Voytas, DF},
title = {Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs.},
journal = {Plant physiology},
volume = {194},
number = {4},
pages = {2229-2239},
doi = {10.1093/plphys/kiae015},
pmid = {38243587},
issn = {1532-2548},
support = {DE-SC-0018277//U.S. Department of Energy/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Mutagenesis ; },
abstract = {CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.},
}
@article {pmid38128544,
year = {2024},
author = {Zhou, C and Okuno, M and Nakazato, I and Tsutsumi, N and Arimura, SI},
title = {Targeted A-to-G base editing in the organellar genomes of Arabidopsis with monomeric programmable deaminases.},
journal = {Plant physiology},
volume = {194},
number = {4},
pages = {2278-2287},
pmid = {38128544},
issn = {1532-2548},
support = {19KK0391//Japan Society for the Promotion of Science/ ; JPJSCCA20230008//Core-to-Core program/ ; JPMJTR22UG//Japan Science and Technology Agency/ ; },
mesh = {*Gene Editing ; *Arabidopsis/genetics ; RNA, Ribosomal, 16S ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Plastids ; Plants/genetics ; DNA ; CRISPR-Cas Systems ; },
abstract = {Plastids and mitochondria are 2 intracellular organelles containing DNA-encoding partial but essential components for their roles, photosynthesis, and respiration. Precise base editing in both plastid and mitochondrial genomes would benefit their gene functional analysis and crop breeding. Targeted base editing in organellar genomes relies on a protein-based genome-editing system that uses the TALE-DNA recognition motif with deaminases. This is because the efficient delivery of guide RNA for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems into organelles is currently impossible. Since TALE-based base editors used in organellar genomes are usually dimeric types, in this study, we used targeted A-to-G base editing in Arabidopsis (Arabidopsis thaliana) plastid and mitochondrial genomes with monomeric TALE-based deaminase for easier assembling of vectors. As a result, inheritable targeted A-to-G base editing of adenosine triphosphatase subunit 6-2 (atp6-2) in plant mitochondrial genomes and of 16S ribosomal RNA (16S rRNA) in plastid genomes of Arabidopsis was successfully induced by monomeric TALE-based adenine deaminase (AD) without off-target mutations. The monomeric TALE-based adenine deaminases also demonstrated a preference for editing the 8th T on the same strand from the recognition end. Phenotypic analysis showed that A-to-G conversion at 1139A of plastid 16S rRNA conferred substantial spectinomycin resistance in Arabidopsis, but not the other 2 potential-resistant mutations at 1131T and 1137T, predicted from the previous bacterial data. Our study demonstrated the feasibility of monomeric TALE-based ADs in plant organelles and their potential contribution to the functional analyses of plant organelles with easier assembling.},
}
@article {pmid38113345,
year = {2024},
author = {Capdeville, N and Schindele, P and Puchta, H},
title = {Increasing deletion sizes and the efficiency of CRISPR/Cas9-mediated mutagenesis by SunTag-mediated TREX1 recruitment.},
journal = {The Plant journal : for cell and molecular biology},
volume = {118},
number = {1},
pages = {277-287},
doi = {10.1111/tpj.16586},
pmid = {38113345},
issn = {1365-313X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; Mutagenesis ; Mutation ; Deoxyribonuclease I/genetics/metabolism ; Gene Editing ; },
abstract = {Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana. Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3'-5' exonucleases, namely the human TREX1 and E. coli ExoI. It turns out that SunTag-mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25 bp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag-dependent, indicating efficient target-specific recruitment. Using SunTag-mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40 bp around the cut site. Such sizes are often required for the manipulation of cis-regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively.},
}
@article {pmid37644142,
year = {2024},
author = {Reichel, LS and Traeger, A},
title = {Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.},
journal = {Handbook of experimental pharmacology},
volume = {284},
number = {},
pages = {27-43},
pmid = {37644142},
issn = {0171-2004},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; Gene Transfer Techniques ; Genetic Therapy ; *Nanoparticles ; RNA, Messenger ; },
abstract = {Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.},
}
@article {pmid38534351,
year = {2024},
author = {Gallego Villarejo, L and Gerding, WM and Bachmann, L and Hardt, LHI and Bormann, S and Nguyen, HP and Müller, T},
title = {Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research.},
journal = {Cells},
volume = {13},
number = {6},
pages = {},
pmid = {38534351},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/metabolism ; Genomics ; Brain ; Chromosome Mapping ; },
abstract = {Genome editing, notably CRISPR (cluster regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), has revolutionized genetic engineering allowing for precise targeted modifications. This technique's combination with human induced pluripotent stem cells (hiPSCs) is a particularly valuable tool in cerebral organoid (CO) research. In this study, CRISPR/Cas9-generated fluorescently labeled hiPSCs exhibited no significant morphological or growth rate differences compared with unedited controls. However, genomic aberrations during gene editing necessitate efficient genome integrity assessment methods. Optical genome mapping, a high-resolution genome-wide technique, revealed genomic alterations, including chromosomal copy number gain and losses affecting numerous genes. Despite these genomic alterations, hiPSCs retain their pluripotency and capacity to generate COs without major phenotypic changes but one edited cell line showed potential neuroectodermal differentiation impairment. Thus, this study highlights optical genome mapping in assessing genome integrity in CRISPR/Cas9-edited hiPSCs emphasizing the need for comprehensive integration of genomic and morphological analysis to ensure the robustness of hiPSC-based models in cerebral organoid research.},
}
@article {pmid38534252,
year = {2024},
author = {Li, X and Dang, Z and Tang, W and Zhang, H and Shao, J and Jiang, R and Zhang, X and Huang, F},
title = {Detection of Parasites in the Field: The Ever-Innovating CRISPR/Cas12a.},
journal = {Biosensors},
volume = {14},
number = {3},
pages = {},
pmid = {38534252},
issn = {2079-6374},
support = {XZNKY-CZ-2022-016-08//Open Project Program of State Key Laboratory of Hulless Barley and Yak Germplasm Resources and Genetic Improvement/ ; },
mesh = {Animals ; *Parasites ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Biological Assay ; *Body Fluids ; Nucleic Acid Amplification Techniques ; },
abstract = {The rapid and accurate identification of parasites is crucial for prompt therapeutic intervention in parasitosis and effective epidemiological surveillance. For accurate and effective clinical diagnosis, it is imperative to develop a nucleic-acid-based diagnostic tool that combines the sensitivity and specificity of nucleic acid amplification tests (NAATs) with the speed, cost-effectiveness, and convenience of isothermal amplification methods. A new nucleic acid detection method, utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, holds promise in point-of-care testing (POCT). CRISPR/Cas12a is presently employed for the detection of Plasmodium falciparum, Toxoplasma gondii, Schistosoma haematobium, and other parasites in blood, urine, or feces. Compared to traditional assays, the CRISPR assay has demonstrated notable advantages, including comparable sensitivity and specificity, simple observation of reaction results, easy and stable transportation conditions, and low equipment dependence. However, a common issue arises as both amplification and cis-cleavage compete in one-pot assays, leading to an extended reaction time. The use of suboptimal crRNA, light-activated crRNA, and spatial separation can potentially weaken or entirely eliminate the competition between amplification and cis-cleavage. This could lead to enhanced sensitivity and reduced reaction times in one-pot assays. Nevertheless, higher costs and complex pre-test genome extraction have hindered the popularization of CRISPR/Cas12a in POCT.},
}
@article {pmid38532479,
year = {2024},
author = {Hermantara, R and Richmond, L and Taqi, AF and Chilaka, S and Jeantet, V and Guerrini, I and West, K and West, A},
title = {Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.},
journal = {Journal of biomedical science},
volume = {31},
number = {1},
pages = {32},
pmid = {38532479},
issn = {1423-0127},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; Transgenes ; DNA ; },
abstract = {BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.<br><br>METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies.<br><br>RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.<br><br>CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.},
}
@article {pmid38530904,
year = {2024},
author = {Zhao, L and Wang, H and Chen, X and Wang, L and Abulaizi, W and Yang, Y and Li, B and Wang, C and Bai, X},
title = {Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.4c00204},
pmid = {38530904},
issn = {1520-5118},
abstract = {Rapid and accurate detection of the zoonotic nematode Anisakis is poised to control its epidemic. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated assay shows great potential in the detection of pathogenic microorganisms. The one-tube method integrated the CRISPR system with the recombinase polymerase amplification (RPA) system to avoid the risk of aerosol pollution; however, it suffers from low sensitivity due to the incompatibility of the two systems and additional manual operations. Therefore, in the present study, the agarose hydrogel boosted one-tube RPA-CRISPR/Cas12a assay was constructed by adding the CRISPR system to the agarose hydrogel, which avoided the initially low amplification efficiency of RPA caused by the cleavage of Cas12a and achieved reaction continuity. The sensitivity was 10-fold higher than that of the one-tube RPA-CRISPR/Cas12a system. This method was used for Anisakis detection within 80 min from the sample to result, achieving point-of-care testing (POCT) through a smartphone and a portable device. This study provided a novel toolbox for POCT with significant application value in preventing Anisakis infection.},
}
@article {pmid38530114,
year = {2024},
author = {Tanga, S and Hota, A and Karmakar, A and Banerjee, P and Maji, B},
title = {Cysteine-independent CRISPR-associated protein labeling for presentation and co-delivery of molecules toward genetic and epigenetic regulations.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202400149},
doi = {10.1002/cbic.202400149},
pmid = {38530114},
issn = {1439-7633},
abstract = {Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) is a huge challenge for their genome engineering applications. Cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. Introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for in vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.},
}
@article {pmid38529860,
year = {2024},
author = {Davydova, YD and Kazantseva, AV and Khusnutdinova, EK},
title = {[A perspective on the application of CRISPR/CAS9 genome editing system to study of molecular-genetic basis of mental disorders].},
journal = {Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova},
volume = {124},
number = {3},
pages = {27-33},
doi = {10.17116/jnevro202412403127},
pmid = {38529860},
issn = {1997-7298},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; *Mental Disorders/genetics/therapy ; },
abstract = {Schizophrenia, depression, bipolar disorder and autism spectrum disorders are common mental disorders that are among the leading causes of disability worldwide. The major complication to effective therapies for mental disorders is the poor understanding of their pathogenic mechanisms. Currently, an increasing number of research groups are focusing on uncovering the molecular mechanisms of mental disorders and developing novel therapies using the CRISPR/Cas9 (Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) - CRISPR-associated system 9 (Cas9)) system to determine the molecular mechanisms of developing mental disorders and novel therapy. The CRISPR/Cas9 system is the most promising among genome editing tools. Numerous advantages of the CRISPR/Cas9 system and its successful application in some studies provide wide opportunities for genome therapy and regeneration medicine. In this review we shortly describe structure and function of the CRISPR/Cas9 system and its application to study the molecular-genetic basis of mental disorders in human.},
}
@article {pmid38528986,
year = {2024},
author = {Kumar, S and Venkat, S},
title = {Editorial: Advances and challenges of genome engineering tools in healthcare: molecular insights into CRISPR/Cas technology.},
journal = {Frontiers in molecular biosciences},
volume = {11},
number = {},
pages = {1376509},
pmid = {38528986},
issn = {2296-889X},
}
@article {pmid38528620,
year = {2024},
author = {Ruta, GV and Ciciani, M and Kheir, E and Gentile, MD and Amistadi, S and Casini, A and Cereseto, A},
title = {Eukaryotic-driven directed evolution of Cas9 nucleases.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {79},
pmid = {38528620},
issn = {1474-760X},
support = {825825//Horizon 2020/ ; 01071041//HORIZON EUROPE European Innovation Council/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genome ; Mammals/genetics ; },
abstract = {BACKGROUND: Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity.<br><br>RESULTS: Here, we develop a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity.<br><br>CONCLUSIONS: We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.},
}
@article {pmid38528259,
year = {2024},
author = {Iori, S and D'Onofrio, C and Laham-Karam, N and Mushimiyimana, I and Lucatello, L and Lopparelli, RM and Gelain, ME and Capolongo, F and Pauletto, M and Dacasto, M and Giantin, M},
title = {Establishment and characterization of cytochrome P450 1A1 CRISPR/Cas9 Knockout Bovine Foetal Hepatocyte Cell Line (BFH12).},
journal = {Cell biology and toxicology},
volume = {40},
number = {1},
pages = {18},
pmid = {38528259},
issn = {1573-6822},
support = {PhD fellowship//Universit&#xe0; degli Studi di Padova/ ; SID 2020-Prot. BIRD 207109//Universit&#xe0; degli Studi di Padova/ ; },
mesh = {Cattle ; Animals ; *Cytochrome P-450 CYP1A1/genetics/metabolism ; *Xenobiotics ; CRISPR-Cas Systems/genetics ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Hepatocytes/metabolism ; Cell Line ; },
abstract = {The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.},
}
@article {pmid38527001,
year = {2024},
author = {Abugattas-Núñez Del Prado, J and Quintana Reyes, A and Leon, J and Blume La Torre, J and Gutiérrez Loli, R and Pinzón Olejua, A and Chamorro Chirinos, ER and Loza Mauricio, FA and Maguiña, JL and Rodriguez-Aliaga, P and Málaga-Trillo, E},
title = {Clinical validation of RCSMS: A rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva.},
journal = {PloS one},
volume = {19},
number = {3},
pages = {e0290466},
pmid = {38527001},
issn = {1932-6203},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis/genetics ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; Saliva/chemistry ; Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics/analysis ; Sensitivity and Specificity ; },
abstract = {Peru's holds the highest COVID death rate per capita worldwide. Key to this outcome is the lack of robust, rapid, and accurate molecular tests to circumvent the elevated costs and logistics of SARS-CoV-2 detection via RT-qPCR. To facilitate massive and timely COVID-19 testing in rural and socioeconomically deprived contexts, we implemented and validated RCSMS, a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. RCSMS uses the power of CRISPR-Cas technology and lateral flow strips to easily visualize the presence of SARS-CoV-2 even in laboratories with limited equipment. We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Notably, RCSMS performed outstandingly in a clinical validation done with 352 patients from two hospitals in Lima, detecting as low as 50 viral copies per 10 μl reaction in 40 min, with sensitivity and specificity of 96.5% and 99.0%, respectively, relative to RT-qPCR. The negative and positive predicted values obtained from this field validation indicate that RCSMS can be confidently deployed in both high and low prevalence settings. Like other CRISPR-Cas-based biosensors, RCSMS can be easily reprogrammed for the detection of new SARS-CoV-2 variants. We conclude that RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in Peru and other low- and middle-income countries with precarious healthcare systems.},
}
@article {pmid38526846,
year = {2024},
author = {Liu, J and Yang, F and Shang, L and Cai, S and Wu, Y and Liu, Y and Zhang, L and Fei, C and Wang, M and Gu, F},
title = {Recapitulating familial hypercholesterolemia in a mouse model by knock-in patient-specific LDLR mutation.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {38},
number = {6},
pages = {e23573},
doi = {10.1096/fj.202301216RRR},
pmid = {38526846},
issn = {1530-6860},
support = {32071443//MOST | National Natural Science Foundation of China (NSFC)/ ; 31871247//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Humans ; Mice ; Animals ; RNA, Guide, CRISPR-Cas Systems ; *Hyperlipoproteinemia Type II/genetics/therapy ; Mutation ; *Hypercholesterolemia/genetics ; Cholesterol ; Receptors, LDL/genetics/metabolism ; },
abstract = {Familial hypercholesterolemia (FH) is one of the most prevalent monogenetic disorders leading to cardiovascular disease (CVD) worldwide. Mutations in Ldlr, encoding a membrane-spanning protein, account for the majority of FH cases. No effective and safe clinical treatments are available for FH. Adenine base editor (ABE)-mediated molecular therapy is a promising therapeutic strategy to treat genetic diseases caused by point mutations, with evidence of successful treatment in mouse disease models. However, due to the differences in the genomes between mice and humans, ABE with specific sgRNA, a key gene correction component, cannot be directly used to treat FH patients. Thus, we generated a knock-in mouse model harboring the partial patient-specific fragment and including the Ldlr W490X mutation. Ldlr[W490X/W490X] mice recapitulated cholesterol metabolic disorder and clinical manifestations of atherosclerosis associated with FH patients, including high plasma low-density lipoprotein cholesterol levels and lipid deposition in aortic vessels. Additionally, we showed that the mutant Ldlr gene could be repaired using ABE with the cellular model. Taken together, these results pave the way for ABE-mediated molecular therapy for FH.},
}
@article {pmid38526790,
year = {2024},
author = {Hawley, RG and Hawley, TS},
title = {CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2779},
number = {},
pages = {273-286},
pmid = {38526790},
issn = {1940-6029},
mesh = {Humans ; *Paired Box Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; *Rhabdomyosarcoma/genetics ; Peptides/metabolism ; Oncogene Proteins, Fusion/genetics/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Red Fluorescent Protein ; },
abstract = {Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.},
}
@article {pmid38526727,
year = {2024},
author = {Schroven, K and Voet, M and Lavigne, R and Hendrix, H},
title = {Targeted Genome Editing of Virulent Pseudomonas Phages Using CRISPR-Cas3.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2793},
number = {},
pages = {113-128},
pmid = {38526727},
issn = {1940-6029},
mesh = {Gene Editing/methods ; *Pseudomonas Phages/genetics ; CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Homologous Recombination ; },
abstract = {The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.},
}
@article {pmid38526321,
year = {2024},
author = {Feng, W and Peng, H and Zhang, H and Weinfeld, M and Le, XC},
title = {A Sensitive Technique Unravels the Kinetics of Activation and Trans-Cleavage of CRISPR-Cas Systems.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202404069},
doi = {10.1002/anie.202404069},
pmid = {38526321},
issn = {1521-3773},
abstract = {Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).},
}
@article {pmid38525557,
year = {2024},
author = {Saha, S and Kroos, L},
title = {Regulation of late-acting operons by three transcription factors and a CRISPR-Cas component during Myxococcus xanthus development.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.15252},
pmid = {38525557},
issn = {1365-2958},
support = {IOS-1951025//National Science Foundation/ ; MCB-1411272//National Science Foundation/ ; //AgBioResearch, Michigan State University/ ; },
abstract = {Upon starvation, rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I, exoL-P, and nfsA-H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose produces spores suited to withstand starvation and environmental insults.},
}
@article {pmid38524180,
year = {2024},
author = {, },
title = {Erratum: Advances in application of CRISPR-Cas13a system.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1393044},
doi = {10.3389/fcimb.2024.1393044},
pmid = {38524180},
issn = {2235-2988},
abstract = {[This corrects the article DOI: 10.3389/fcimb.2024.1291557.].},
}
@article {pmid38524179,
year = {2024},
author = {Zhang, Y and Li, S and Li, R and Qiu, X and Fan, T and Wang, B and Zhang, B and Zhang, L},
title = {Advances in application of CRISPR-Cas13a system.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1291557},
pmid = {38524179},
issn = {2235-2988},
mesh = {Humans ; *Bacteria ; Archaea/genetics ; RNA ; CRISPR-Cas Systems ; *Viruses/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria contain various CRISPR-Cas systems. CRISPR-Cas systems depend on CRISPR RNAs (crRNAs). They act as a navigation system to specifically cut and destroy foreign nucleic acids by recognizing invading foreign nucleic acids and binding Cas proteins. In this review, we provide a brief overview of the evolution and classification of the CRISPR-Cas system, focusing on the functions and applications of the CRISPR-Cas13a system. We describe the CRISPR-Cas13a system and discuss its RNA-directed ribonuclease function. Meanwhile, we briefly introduce the mechanism of action of the CRISPR-Cas13a system and summarize the applications of the CRISPR-Cas13a system in pathogen detection, eukaryotes, agriculture, biosensors, and human gene therapy. We are right understanding of CRISPR-Cas13a has been broadened, and the CRISPR-Cas13a system will be useful for developing new RNA targeting tools. Therefore, understanding the basic details of the structure, function, and biological characterization of CRISPR-Cas13a effector proteins is critical for optimizing RNA targeting tools.},
}
@article {pmid38523559,
year = {2024},
author = {Haslam, TM and Herrfurth, C and Feussner, I},
title = {Diverse INOSITOL PHOSPHORYLCERAMIDE SYNTHASE mutant alleles of Physcomitrium patens offer new insight into complex sphingolipid metabolism.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.19667},
pmid = {38523559},
issn = {1469-8137},
support = {INST 186/1167-1//Deutsche Forschungsgemeinschaft/ ; INST 186/822-1//Deutsche Forschungsgemeinschaft/ ; CAN 1210075 HFST-P//Alexander von Humboldt-Stiftung/ ; MSCA-IF-EF-ST:892532-SMFP//HORIZON EUROPE Marie Sklodowska-Curie Actions/ ; },
abstract = {Sphingolipids are widespread, abundant, and essential lipids in plants and in other eukaryotes. Glycosyl inositol phosphorylceramides (GIPCs) are the most abundant class of plant sphingolipids, and are enriched in the plasma membrane of plant cells. They have been difficult to study due to lethal or pleiotropic mutant phenotypes. To overcome this, we developed a CRISPR/Cas9-based method for generating multiple and varied knockdown and knockout populations of mutants in a given gene of interest in the model moss Physcomitrium patens. This system is uniquely convenient due to the predominantly haploid state of the Physcomitrium life cycle, and totipotency of Physcomitrium protoplasts used for transformation. We used this approach to target the INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS) gene family, which catalyzes the first, committed step in the synthesis of GIPCs. We isolated knockout single mutants and knockdown higher-order mutants showing a spectrum of deficiencies in GIPC content. Remarkably, we also identified two mutant alleles accumulating inositol phosphorylceramides, the direct products of IPCS activity, and provide our best explanation for this unexpected phenotype. Our approach is broadly applicable for studying essential genes and gene families, and for obtaining unusual lesions within a gene of interest.},
}
@article {pmid38471390,
year = {2024},
author = {Briski, O and La Motta, GE and Ratner, LD and Allegroni, FA and Pillado, S and Álvarez, G and Gutierrez, B and Tarragona, L and Zaccagnini, A and Acerbo, M and Ciampi, C and Fernández-Martin, R and Salamone, DF},
title = {Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.},
journal = {Theriogenology},
volume = {220},
number = {},
pages = {43-55},
doi = {10.1016/j.theriogenology.2024.02.028},
pmid = {38471390},
issn = {1879-3231},
mesh = {Animals ; Swine/genetics ; Humans ; Male ; Animals, Genetically Modified ; *Gene Editing/veterinary ; *CRISPR-Cas Systems ; Transplantation, Heterologous/veterinary ; Sperm Injections, Intracytoplasmic/veterinary ; Semen ; Fertilization in Vitro/veterinary ; },
abstract = {Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and β4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.},
}
@article {pmid38442045,
year = {2024},
author = {Sun, J and Jiao, Y and Pan, F and Cheng, SH and Sun, D},
title = {A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9 System.},
journal = {IEEE transactions on nanobioscience},
volume = {23},
number = {2},
pages = {378-388},
doi = {10.1109/TNB.2024.3373597},
pmid = {38442045},
issn = {1558-2639},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Zebrafish/genetics ; Transfection ; DNA ; },
abstract = {The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.},
}
@article {pmid38096363,
year = {2024},
author = {Durand, R and Descamps, G and Bellanger, C and Dousset, C and Maïga, S and Alberge, JB and Derrien, J and Cruard, J and Minvielle, S and Lilli, NL and Godon, C and Le Bris, Y and Tessoulin, B and Amiot, M and Gomez-Bougie, P and Touzeau, C and Moreau, P and Chiron, D and Moreau-Aubry, A and Pellat-Deceunynck, C},
title = {A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics.},
journal = {Blood},
volume = {143},
number = {13},
pages = {1242-1258},
doi = {10.1182/blood.2023021581},
pmid = {38096363},
issn = {1528-0020},
mesh = {Humans ; *Multiple Myeloma/drug therapy/genetics/metabolism ; bcl-2-Associated X Protein/genetics/metabolism ; Myeloid Cell Leukemia Sequence 1 Protein/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Cell Line, Tumor ; Apoptosis ; *Antineoplastic Agents/therapeutic use ; *Pyrimidines ; *Thiophenes ; },
abstract = {To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.},
}
@article {pmid37437039,
year = {2024},
author = {Yi, K and Kong, H and Lao, YH and Li, D and Mintz, RL and Fang, T and Chen, G and Tao, Y and Li, M and Ding, J},
title = {Engineered Nanomaterials to Potentiate CRISPR/Cas9 Gene Editing for Cancer Therapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {36},
number = {13},
pages = {e2300665},
doi = {10.1002/adma.202300665},
pmid = {37437039},
issn = {1521-4095},
support = {2021YFC2400600//National Key Research and Development Program of China/ ; 2019YFA0111300//National Key Research and Development Program of China/ ; 52373166//National Natural Science Foundation of China/ ; 22277155//National Natural Science Foundation of China/ ; 52022095//National Natural Science Foundation of China/ ; 52073280//National Natural Science Foundation of China/ ; 51903256//National Natural Science Foundation of China/ ; 21907113//National Natural Science Foundation of China/ ; 51873207//National Natural Science Foundation of China/ ; 202102010225//Science and Technology Program of Guangzhou/ ; 2019QN01Y131//Guangdong Provincial Pearl River Talents Program/ ; 2019230//Youth Innovation Promotion Association of Chinese Academy of Sciences/ ; 2022-003//China Primary Health Care Foundation/ ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Genetic Therapy ; Genetic Vectors ; *Neoplasms/genetics/therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) gene-editing technology shows promise for manipulating single or multiple tumor-associated genes and engineering immune cells to treat cancers. Currently, most gene-editing strategies rely on viral delivery; yet, while being efficient, many limitations, mainly from safety and packaging capacity considerations, hinder the use of viral CRISPR vectors in cancer therapy. In contrast, recent advances in non-viral CRISPR/Cas9 nanoformulations have paved the way for better cancer gene editing, as these nanoformulations can be engineered to improve safety, efficiency, and specificity through optimizing the packaging capacity, pharmacokinetics, and targetability. In this review, the advance in non-viral CRISPR delivery is highlighted, and there is a discussion on how these approaches can be potentially used to treat cancers in addressing the aforementioned limitations, followed by the perspectives in designing a proper CRISPR/Cas9-based cancer nanomedicine system with translational potential.},
}
@article {pmid38523431,
year = {2024},
author = {Lanza, DG and Mao, J and Lorenzo, I and Liao, L and Seavitt, JR and Ljungberg, MC and Simpson, EM and DeMayo, FJ and Heaney, JD},
title = {An oocyte-specific Cas9-expressing mouse for germline CRISPR/Cas9-mediated genome editing.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {62},
number = {2},
pages = {e23589},
doi = {10.1002/dvg.23589},
pmid = {38523431},
issn = {1526-968X},
support = {P30CA12512/NH/NIH HHS/United States ; S10 OD016167/NH/NIH HHS/United States ; UM1 HG006348/NH/NIH HHS/United States ; U54 OD030165/NH/NIH HHS/United States ; },
mesh = {Female ; Male ; Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Mutation ; Zygote/metabolism ; Animals, Genetically Modified ; Oocytes ; },
abstract = {Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.},
}
@article {pmid38523187,
year = {2024},
author = {Rasheed, AAB and Birling, MC and Lauria, G and Gaveriaux-Ruff, C and Herault, Y},
title = {The COL6A5-p.Glu2272* mutation induces chronic itch in mice.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {},
number = {},
pages = {},
pmid = {38523187},
issn = {1432-1777},
support = {Marie Sk&#x142;odowska-Curie grant agreement No. 721841-Pain-Net//European Commission/ ; ANR-10-IDEX-0002-02//Agence Nationale de la Recherche/ ; ANR-10-LABX-0030-INRT//Agence Nationale de la Recherche/ ; ANR-10-INBS-07 PHENOMIN.//Agence Nationale de la Recherche/ ; },
abstract = {Pruritus is a common irritating sensation that provokes the desire to scratch. Environmental and genetic factors contribute to the onset of pruritus. Moreover, itch can become a major burden when it becomes chronic. Interestingly, the rare Collagen VI alpha 5 (COL6A5) gene variant p.Glu2272* has been identified in two families and an independent patient with chronic neuropathic itch. These patients showed reduced COL6A5 expression in skin and normal skin morphology. However, little progress has been made until now toward understanding the relationships between this mutation and chronic itch. Therefore, we developed the first mouse model that recapitulates COL6A5-p.Glu2272* mutation using the CRISPR-Cas technology and characterized this new mouse model. The mutant mRNA, measured by RT-ddPCR, was expressed at normal levels in dorsal root ganglia and was decreased in skin. The functional exploration showed effects of the mutation with some sex dysmorphology. Mutant mice had increased skin permeability. Elevated spontaneous scratching and grooming was detected in male and female mutants, with increased anxiety-like behavior in female mutants. These results suggest that the COL6A5-p.Glu2272* mutation found in patients contributes to chronic itch and induces in mice additional behavioral changes. The COL6A5-p.Glu2272* mouse model could elucidate the pathophysiological mechanisms underlying COL6A5 role in itch and help identify potential new therapeutic targets.},
}
@article {pmid38521856,
year = {2024},
author = {Patil, SV and Kaipa, BR and Ranshing, S and Sundaresan, Y and Millar, JC and Nagarajan, B and Kiehlbauch, C and Zhang, Q and Jain, A and Searby, CC and Scheetz, TE and Clark, AF and Sheffield, VC and Zode, GS},
title = {Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {6958},
pmid = {38521856},
issn = {2045-2322},
support = {EY026177/EY/NEI NIH HHS/United States ; EY030366/EY/NEI NIH HHS/United States ; EY025580/EY/NEI NIH HHS/United States ; EY034070/EY/NEI NIH HHS/United States ; },
mesh = {Mice ; Animals ; Intraocular Pressure/genetics ; CRISPR-Cas Systems/genetics ; *Glaucoma, Open-Angle/genetics/therapy/metabolism ; Lentivirus/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Glaucoma/metabolism ; Eye Proteins/genetics/metabolism ; Trabecular Meshwork/metabolism ; Disease Models, Animal ; *Cytoskeletal Proteins ; *Glycoproteins ; },
abstract = {Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOC[Y437H] mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.},
}
@article {pmid38521791,
year = {2024},
author = {Li, Z and You, L and Hermann, A and Bier, E},
title = {Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation classifier.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2629},
pmid = {38521791},
issn = {2041-1723},
support = {R01 AI162911/AI/NIAID NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; R01 GM144608/GM/NIGMS NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; },
mesh = {*DNA Breaks, Double-Stranded ; Alleles ; *DNA Repair/genetics ; DNA/metabolism ; DNA End-Joining Repair ; Mutation ; Recombinational DNA Repair ; CRISPR-Cas Systems/genetics ; },
abstract = {DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Factors influencing the choice of particular repair pathways, however remain poorly characterized. Here we develop an Integrated Classification Pipeline (ICP) to decompose and categorize CRISPR/Cas9 generated mutations on genomic target sites in complex multicellular insects. The ICP outputs graphic rank ordered classifications of mutant alleles to visualize discriminating DSB repair fingerprints generated from different target sites and alternative inheritance patterns of CRISPR components. We uncover highly reproducible lineage-specific mutation fingerprints in individual organisms and a developmental progression wherein Microhomology-Mediated End-Joining (MMEJ) or Insertion events predominate during early rapid mitotic cell cycles, switching to distinct subsets of Non-Homologous End-Joining (NHEJ) alleles, and then to Homology-Directed Repair (HDR)-based gene conversion. These repair signatures enable marker-free tracking of specific mutations in dynamic populations, including NHEJ and HDR events within the same samples, for in-depth analysis of diverse gene editing events.},
}
@article {pmid38521763,
year = {2024},
author = {Perez-Bermejo, JA and Efagene, O and Matern, WM and Holden, JK and Kabir, S and Chew, GM and Andreoletti, G and Catton, E and Ennis, CL and Garcia, A and Gerstenberg, TL and Hill, KA and Jain, A and Krassovsky, K and Lalisan, CD and Lord, D and Quejarro, BJ and Sales-Lee, J and Shah, M and Silva, BJ and Skowronski, J and Strukov, YG and Thomas, J and Veraz, M and Vijay, T and Wallace, KA and Yuan, Y and Grogan, JL and Wienert, B and Lahiri, P and Treusch, S and Dever, DP and Soros, VB and Partridge, JR and Seim, KL},
title = {Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2625},
pmid = {38521763},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Gene Editing/methods ; DNA Repair ; DNA End-Joining Repair ; },
abstract = {Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.},
}
@article {pmid38520741,
year = {2024},
author = {Martínez-Pizarro, A and Picó, S and López-Márquez, A and Rodriguez-López, C and Montalvo, E and Alvarez, M and Castro, M and Ramón-Maiques, S and Pérez, B and Lucas, JJ and Richard, E and Desviat, LR},
title = {PAH deficient pathology in humanized c.1066-11G&gt;A phenylketonuria mice.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddae051},
pmid = {38520741},
issn = {1460-2083},
support = {PID PID2022-137238OB-I00/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation and European Regional Development Fund/ ; },
abstract = {We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.},
}
@article {pmid38520090,
year = {2024},
author = {Schreiber, T and Prange, A and Schäfer, P and Iwen, T and Grützner, R and Marillonnet, S and Lepage, A and Javelle, M and Paul, W and Tissier, A},
title = {Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR/Cas endonucleases.},
journal = {Molecular plant},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molp.2024.03.013},
pmid = {38520090},
issn = {1752-9867},
abstract = {In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.},
}
@article {pmid38518680,
year = {2024},
author = {Leta, S and Chibssa, TR and Paeshuyse, J},
title = {CRISPR-Cas12/Cas13: Bibliometric analysis and systematic review of its application in infectious disease detection.},
journal = {Journal of infection and public health},
volume = {17},
number = {5},
pages = {741-747},
doi = {10.1016/j.jiph.2024.03.003},
pmid = {38518680},
issn = {1876-035X},
abstract = {BACKGROUND: Infectious diseases impose a significant burden on the global public health and economy, resulting in an estimated 15 million deaths out of 57 million annually worldwide. This study examines the current state of CRISPR-Cas12/Cas13 research, focusing on its applications in infectious disease detection and its evolutionary trajectory.<br><br>METHODS: A bibliometric analysis and systematic review were conducted by retrieving CRISPR-Cas12/Cas13-related articles published between January 1, 2015 to December 31, 2022, from the Web of Science database. The research protocol was registered with International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY202380062).<br><br>RESULTS: Our search identified 1987 articles, of which, 1856 were included in the bibliometric analysis and 445 were used in qualitative analysis. The study reveals a substantial increase in scientific production on CRISPR-Cas12/Cas13, with an annual growth rate of 104.5%. The United States leads in the number of published articles. The systematic review identified 580 different diagnostic assays targeting 170 pathogens, with&#xa0;SARS-CoV-2 dominating with 158 assays. Recombinase polymerase amplification (RPA)/reverse transcription-RPA (RT-RPA) emerged as the predominant amplification method, while lateral flow assay was the most common readout method. Approximately 72% of the diagnostic assays developed are suitable for point-of-care testing.<br><br>CONCLUSION: The rapid increase in research on CRISPR-Cas12/Cas13 between 2015 and 2022 suggests promising potential for advancements in infectious disease diagnosis. Given the numerous advantages of CRISPR-Cas technology for disease detection over other methods, and the dedicated efforts of scientists from around the world, it is reasonable to anticipate that CRISPR-Cas technology may emerge as a formidable alternative, offering the possibility of expedited point-of-care testing in the not-too-distant future.},
}
@article {pmid38517206,
year = {2024},
author = {Wakasa, K and Tamura, R and Osaka, S and Takei, H and Asai, A and Nittono, H and Kusuhara, H and Hayashi, H},
title = {Rapid in vivo evaluation system for cholestasis-related genes in mice with humanized bile acid profiles.},
journal = {Hepatology communications},
volume = {8},
number = {4},
pages = {},
pmid = {38517206},
issn = {2471-254X},
mesh = {Humans ; Mice ; Child ; Animals ; *Bile Acids and Salts/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Cholestasis/metabolism ; Liver/metabolism ; Phenotype ; },
abstract = {BACKGROUND: Pediatric cholestatic liver diseases (Ped-CLD) comprise many ultrarare disorders with a genetic basis. Pharmacologic therapy for severe cases of Ped-CLD has not been established. Species differences in bile acid (BA) metabolism between humans and rodents contribute to the lack of phenocopy of patients with Ped-CLD in rodents and hinder the development of therapeutic strategies. We aimed to establish an efficient in vivo system to understand BA-related pathogenesis, such as Ped-CLD.<br><br>METHODS: We generated mice that express spCas9 specifically in the liver (L-Cas9Tg/Tg [liver-specific Cas9Tg/Tg] mice) and designed recombinant adeno-associated virus serotype 8 encoding small-guide RNA (AAV8 sgRNA) targeting Abcc2, Abcb11, and Cyp2c70. In humans, ABCC2 and ABCB11 deficiencies cause constitutional hyperbilirubinemia and most severe Ped-CLD, respectively. Cyp2c70 encodes an enzyme responsible for the rodent-specific BA profile. Six-week-old L-Cas9Tg/Tg mice were injected with this AAV8 sgRNA and subjected to biochemical and histological analysis.<br><br>RESULTS: Fourteen days after the injection with AAV8 sgRNA targeting Abcc2, L-Cas9Tg/Tg mice exhibited jaundice and phenocopied patients with ABCC2 deficiency. L-Cas9Tg/Tg mice injected with AAV8 sgRNA targeting Abcb11 showed hepatomegaly and cholestasis without histological evidence of liver injury. Compared to Abcb11 alone, simultaneous injection of AAV8 sgRNA for Abcb11 and Cyp2c70 humanized the BA profile and caused higher transaminase levels and parenchymal necrosis, resembling phenotypes with ABCB11 deficiency.<br><br>CONCLUSIONS: This study provides proof of concept for efficient in vivo assessment of cholestasis-related genes in humanized bile acid profiles. Our platform offers a more time- and cost-effective alternative to conventional genetically engineered mice, increasing our understanding of BA-related pathogenesis such as Ped-CLD and expanding the potential for translational research.},
}
@article {pmid38516796,
year = {2024},
author = {Zhang, J and Li, Z and Guo, C and Guan, X and Avery, L and Banach, D and Liu, C},
title = {Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202403123},
doi = {10.1002/anie.202403123},
pmid = {38516796},
issn = {1521-3773},
abstract = {The CRISPR-Cas12a system has emerged as a powerful tool for nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by fully complementary RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a exhibits higher specificity compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay, demonstrating random target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.},
}
@article {pmid38512561,
year = {2024},
author = {Lu, S and Zeng, H and Xiong, F and Yao, M and He, S},
title = {Advances in environmental DNA monitoring: standardization, automation, and emerging technologies in aquatic ecosystems.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {38512561},
issn = {1869-1889},
abstract = {Environmental DNA (eDNA) monitoring, a rapidly advancing technique for assessing biodiversity and ecosystem health, offers a noninvasive approach for detecting and quantifying species from various environmental samples. In this review, a comprehensive overview of current eDNA collection and detection technologies is provided, emphasizing the necessity for standardization and automation in aquatic ecological monitoring. Furthermore, the intricacies of water bodies, from streams to the deep sea, and the associated challenges they pose for eDNA capture and analysis are explored. The paper delineates three primary eDNA survey methods, namely, bringing back water, bringing back filters, and bringing back data, each with specific advantages and constraints in terms of labor, transport, and data acquisition. Additionally, innovations in eDNA sampling equipment, including autonomous drones, subsurface samplers, and in-situ filtration devices, and their applications in monitoring diverse taxa are discussed. Moreover, recent advancements in species-specific detection and eDNA metabarcoding are addressed, highlighting the integration of novel techniques such as CRISPR-Cas and nanopore sequencing that enable precise and rapid detection of biodiversity. The implications of environmental RNA and epigenetic modifications are considered for future applications in providing nuanced ecological data. Lastly, the review stresses the critical role of standardization and automation in enhancing data consistency and comparability for robust long-term biomonitoring. We propose that the amalgamation of these technologies represents a paradigm shift in ecological monitoring, aligning with the urgent call for biodiversity conservation and sustainable management of aquatic ecosystems.},
}
@article {pmid38069501,
year = {2024},
author = {Djennane, S and Gersch, S and Le-Bohec, F and Piron, MC and Baltenweck, R and Lemaire, O and Merdinoglu, D and Hugueney, P and Nogué, F and Mestre, P},
title = {CRISPR/Cas9 editing of Downy mildew resistant 6 (DMR6-1) in grapevine leads to reduced susceptibility to Plasmopara viticola.},
journal = {Journal of experimental botany},
volume = {75},
number = {7},
pages = {2100-2112},
doi = {10.1093/jxb/erad487},
pmid = {38069501},
issn = {1460-2431},
support = {ANR-17-EUR-0007//Saclay Plant Sciences-SPS/ ; },
mesh = {Disease Resistance/genetics ; CRISPR-Cas Systems ; Plant Breeding ; *Oomycetes ; *Vitis/genetics ; Plant Diseases ; },
abstract = {Downy mildew of grapevine (Vitis vinifera), caused by the oomycete Plasmopara viticola, is an important disease that is present in cultivation areas worldwide, and using resistant varieties provides an environmentally friendly alternative to fungicides. DOWNY MILDEW RESISTANT 6 (DMR6) from Arabidopsis is a negative regulator of plant immunity and its loss of function confers resistance to downy mildew. In grapevine, DMR6 is present in two copies, named VvDMR6-1 and VvDMR6-2. Here, we describe the editing of VvDMR6-1 in embryogenic calli using CRISPR/Cas9 and the regeneration of the edited plants. All edited plants were found to be biallelic and chimeric, and whilst they all showed reduced growth compared with non-transformed control plants, they also had reduced susceptibility to P. viticola. Comparison between mock-inoculated genotypes showed that all edited lines presented higher levels of salicylic acid than controls, and lines subjected to transformation presented higher levels of cis-resveratrol than controls. Our results identify VvDMR6-1 as a promising target for breeding grapevine cultivars with improved resistance to downy mildew.},
}
@article {pmid37321345,
year = {2024},
author = {Han, H and Xu, M and Wang, J and Li, MD and Yang, Z},
title = {CRISPR/Cas9 based gene editing of Frizzled class receptor 6 (FZD6) reveals its role in depressive symptoms through disrupting Wnt/β-catenin signaling pathway.},
journal = {Journal of advanced research},
volume = {58},
number = {},
pages = {129-138},
doi = {10.1016/j.jare.2023.06.001},
pmid = {37321345},
issn = {2090-1224},
mesh = {Humans ; Mice ; Animals ; *Wnt Signaling Pathway ; *beta Catenin/genetics/metabolism ; Depression/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Glycogen Synthase Kinase 3 beta/genetics/metabolism ; Sucrose ; Frizzled Receptors/genetics/metabolism ; },
abstract = {INTRODUCTION: As one of the common psychiatric diseases, depression poses serious threats to human health. Although many genes have been nominated for depression, few of them were investigated in details at the molecular level.<br><br>OBJECTIVES: To demonstrate Frizzled class receptor 6 (FZD6) functions in depression through disrupting Wnt/&#x3b2;-catenin signal pathway.<br><br>METHODS: The FZD6 edited cell line and mouse model were generated by using CRISPR/Cas9 technique. The expression of key genes and proteins in Wnt/&#x3b2;-catenin pathway was determined by qRT-PCR and Western blotting, respectively. Animal behavioral tests, including open field test (OFT), elevated plus maze test (EPM), forced swimming test (FST), tail suspension test (TST), and sucrose preference test (SPT), were employed to determine anxiety- and depressive-like behaviors. Immunofluorescent staining was used to assess cell proliferation in the hippocampus of mouse brain.<br><br>RESULTS: Among patients with depression, FZD6, one of the receptors of Wnt ligand, was significantly decreased. In CRISPR/Cas9-based FZD6 knockdown cells, we showed that FZD6 plays a significant role in regulating expression of genes involved in Wnt/&#x3b2;-catenin pathway. Subsequently behavioral studies on Fzd6 knockdown mice (with a 5-nucleotide deletion; Fzd6-&#x394;5) revealed significant changes in depressive symptoms, including increased immobility duration in FST, less preference of sucrose in SPT, reduction of distance traveled in OFT, and decreased time spent in open arms in EPM. Immunofluorescent staining showed decreased cell proliferation in the hippocampus of Fzd6-&#x394;5 mice with reduced number of Ki67[+] and PCNA[+] cells. Moreover, decreased Gsk3&#x3b2; mRNA expression, phosphorylated GSK3&#x3b2;, and cytoplasmic &#x3b2;-catenin in the hippocampus of Fzd6-&#x394;5 mice provided further evidence supporting the role of Fzd6 in depression.<br><br>CONCLUSION: Together, above findings proved the significant role of FZD6 in depression through its effect on hippocampal cell proliferation and its ability to regulate canonical Wnt/&#x3b2;-catenin pathway.},
}
@article {pmid38512221,
year = {2024},
author = {Krueger, CJ and Dai, Z and Zhu, C and Zhang, B},
title = {Heritable CRISPR Mutagenesis of Essential Maternal Effect Genes as a Simple Tool for Sustained Population Suppression of Invasive Species in a Zebrafish Model.},
journal = {Zebrafish},
volume = {},
number = {},
pages = {},
doi = {10.1089/zeb.2023.0108},
pmid = {38512221},
issn = {1557-8542},
abstract = {Invasive species control is important for ecological and agricultural management. Genetic methods can provide species specificity for population control. We developed heritable maternal effect embryo lethality (HMEL), a novel strategy allowing negative population pressure from HMEL individuals to be transmitted within a population across generations. We demonstrate the HMEL technique in zebrafish through genome-integrated CRISPR/Cas targeted mutagenic disruption of nucleoplasmin 2b (npm2b), a female-specific essential maternal effect gene, causing heritable sex-limited disruption of reproduction. HMEL-induced high-efficiency mutation of npm2b in females suppresses population, while males transmit the HMEL allele across generations. HMEL could be easily modified to target other genes causing sex-specific sterility, or generalized to control invasive fish or other vertebrate species for environmental conservation or agricultural protection.},
}
@article {pmid38508195,
year = {2024},
author = {Lee, BC and Gin, A and Wu, C and Singh, K and Grice, M and Mortlock, R and Abraham, D and Fan, X and Zhou, Y and AlJanahi, A and Choi, U and DeRavin, SS and Shin, T and Hong, S and Dunbar, CE},
title = {Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.},
journal = {Cell stem cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.stem.2024.02.010},
pmid = {38508195},
issn = {1875-9777},
abstract = {For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.},
}
@article {pmid38507738,
year = {2024},
author = {Estupiñan Velasquez, HY and Bouderlique, T and He, C and Berglöf, A and Cappelleri, A and Frengen, N and Zain, R and Karlsson, MCI and Månsson, R and Smith, CIE},
title = {In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function.},
journal = {Blood advances},
volume = {},
number = {},
pages = {},
doi = {10.1182/bloodadvances.2024012706},
pmid = {38507738},
issn = {2473-9537},
abstract = {Bruton's tyrosine kinase (BTK) is an enzyme needed for B-cell survival and inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has for more than two decades generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using non-lymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas mediated knock-in mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness, while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and morphology of lymphoid organs as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control, and mounted similar humoral immune responses as Y223F and wildtype animals. Collectively our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase C(?)2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this post-translational modification is dispensable for the function of BTK.},
}
@article {pmid38507682,
year = {2024},
author = {Pudžiuvelytė, I and Olechnovič, K and Godliauskaite, E and Sermokas, K and Urbaitis, T and Gasiunas, G and Kazlauskas, D},
title = {TemStaPro: protein thermostability prediction using sequence representations from protein language models.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btae157},
pmid = {38507682},
issn = {1367-4811},
abstract = {MOTIVATION: Reliable prediction of protein thermostability from its sequence is valuable for both academic and industrial research. This prediction problem can be tackled using machine learning and by taking advantage of the recent blossoming of deep learning methods for sequence analysis. These methods can facilitate training on more data and, possibly, enable development of more versatile thermostability predictors for multiple ranges of temperatures.<br><br>RESULTS: We applied the principle of transfer learning to predict protein thermostability using embeddings generated by protein language models (pLMs) from an input protein sequence. We used large pLMs that were pre-trained on hundreds of millions of known sequences. The embeddings from such models allowed us to efficiently train and validate a high-performing prediction method using over one million sequences that we collected from organisms with annotated growth temperatures. Our method, TemStaPro (Temperatures of Stability for Proteins), was used to predict thermostability of CRISPR-Cas Class II effector proteins (C2EPs). Predictions indicated sharp differences among groups of C2EPs in terms of thermostability and were largely in tune with previously published and our newly obtained experimental data.<br><br>TemStaPro software and the related data are freely available from https://github.com/ievapudz/TemStaPro and https://doi.org/10.5281/zenodo.7743637.},
}
@article {pmid38507394,
year = {2024},
author = {Xu, S and Shiomi, H and Yamashita, Y and Koyama, S and Horie, T and Baba, O and Kimura, M and Nakashima, Y and Sowa, N and Hasegawa, K and Suzuki, A and Suzuki, Y and Kimura, T and Ono, K},
title = {CRISPR-Cas9-guided amplification-free genomic diagnosis for familial hypercholesterolemia using nanopore sequencing.},
journal = {PloS one},
volume = {19},
number = {3},
pages = {e0297231},
pmid = {38507394},
issn = {1932-6203},
mesh = {Humans ; Proprotein Convertase 9/genetics ; *Nanopore Sequencing ; CRISPR-Cas Systems/genetics ; Receptors, LDL/genetics/metabolism ; *Hyperlipoproteinemia Type II/diagnosis/genetics ; Mutation ; Genomics ; DNA ; },
abstract = {Familial hypercholesterolemia is an inherited disorder that remains underdiagnosed. Conventional genetic testing methods such as next-generation sequencing (NGS) or target PCR are based on the amplification process. Due to the efficiency limits of polymerase and ligase enzymes, these methods usually target short regions and do not detect large mutations straightforwardly. This study combined the long-read nanopore sequencing and CRISPR-Cas9 system to sequence the target DNA molecules without amplification. We originally designed and optimized the CRISPR-RNA panel to target the low-density lipoprotein receptor gene (LDLR) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9) from human genomic DNA followed by nanopore sequencing. The average coverages for LDLR and PCSK9 were 106× and 420×, versus 1.2× for the background genome. Among them, continuous reads were 52x and 307x, respectively, and spanned the entire length of LDLR and PCSK9. We identified pathogenic mutations in both coding and splicing donor regions in LDLR. We also detected an 11,029 bp large deletion in another case. Furthermore, using continuous long reads generated from the benchmark experiment, we demonstrated how a false-positive 670 bp deletion caused by PCR amplification errors was easily eliminated.},
}
@article {pmid38483980,
year = {2024},
author = {Sun, J and Guo, J and Liu, J},
title = {CRISPR-M: Predicting sgRNA off-target effect using a multi-view deep learning network.},
journal = {PLoS computational biology},
volume = {20},
number = {3},
pages = {e1011972},
pmid = {38483980},
issn = {1553-7358},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Deep Learning ; Gene Editing/methods ; Neural Networks, Computer ; },
abstract = {Using the CRISPR-Cas9 system to perform base substitutions at the target site is a typical technique for genome editing with the potential for applications in gene therapy and agricultural productivity. When the CRISPR-Cas9 system uses guide RNA to direct the Cas9 endonuclease to the target site, it may misdirect it to a potential off-target site, resulting in an unintended genome editing. Although several computational methods have been proposed to predict off-target effects, there is still room for improvement in the off-target effect prediction capability. In this paper, we present an effective approach called CRISPR-M with a new encoding scheme and a novel multi-view deep learning model to predict the sgRNA off-target effects for target sites containing indels and mismatches. CRISPR-M takes advantage of convolutional neural networks and bidirectional long short-term memory recurrent neural networks to construct a three-branch network towards multi-views. Compared with existing methods, CRISPR-M demonstrates significant performance advantages running on real-world datasets. Furthermore, experimental analysis of CRISPR-M under multiple metrics reveals its capability to extract features and validates its superiority on sgRNA off-target effect predictions.},
}
@article {pmid38466962,
year = {2024},
author = {Moitra, P and Skrodzki, D and Molinaro, M and Gunaseelan, N and Sar, D and Aditya, T and Dahal, D and Ray, P and Pan, D},
title = {Context-Responsive Nanoparticle Derived from Synthetic Zwitterionic Ionizable Phospholipids in Targeted CRISPR/Cas9 Therapy for Basal-like Breast Cancer.},
journal = {ACS nano},
volume = {18},
number = {12},
pages = {9199-9220},
doi = {10.1021/acsnano.4c01400},
pmid = {38466962},
issn = {1936-086X},
mesh = {Humans ; CRISPR-Cas Systems ; Phospholipids ; Tissue Distribution ; *Triple Negative Breast Neoplasms/drug therapy/genetics ; *Nanoparticles ; },
abstract = {The majority of triple negative breast cancers (TNBCs) are basal-like breast cancers (BLBCs), which tend to be more aggressive, proliferate rapidly, and have poor clinical outcomes. A key prognostic biomarker and regulator of BLBC is the Forkhead box C1 (FOXC1) transcription factor. However, because of its functional placement inside the cell nucleus and its structural similarity with other related proteins, targeting FOXC1 for therapeutic benefit, particularly for BLBC, continues to be difficult. We envision targeted nonviral delivery of CRISPR/Cas9 plasmid toward the efficacious knockdown of FOXC1. Keeping in mind the challenges associated with the use of CRISPR/Cas9 in vivo, including off-targeting modifications, and effective release of the cargo, a nanoparticle with context responsive properties can be designed for efficient targeted delivery of CRISPR/Cas9 plasmid. Consequently, we have designed, synthesized, and characterized a zwitterionic amino phospholipid-derived transfecting nanoparticle for delivery of CRISPR/Cas9. The construct becomes positively charged only at low pH, which encourages membrane instability and makes it easier for nanoparticles to exit endosomes. This has enabled effective in vitro and in vivo downregulation of protein expression and genome editing. Following this, we have used EpCAM aptamer to make the system targeted toward BLBC cell lines and to reduce its off-target toxicity. The in vivo efficacy, biodistribution, preliminary pharmacokinetics, and biosafety of the optimized targeted CRISPR nanoplatform is then validated in a rodent xenograft model. Overall, we have attempted to knockout the proto-oncogenic FOXC1 expression in BLBC cases by efficient delivery of CRISPR effectors via a context-responsive nanoparticle delivery system derived from a designer lipid derivative. We believe that the nonviral approach for in vitro and in vivo delivery of CRISPR/Cas9 targeted toward FOXC1, studied herein, will greatly emphasize the therapeutic regimen for BLBC.},
}
@article {pmid38412717,
year = {2024},
author = {Yang, M and Li, Q and Yang, H and Li, Y and Lu, L and Wu, X and Liu, Y and Li, W and Shen, J and Xiao, Z and Zhao, Y and Du, F and Chen, Y and Deng, S and Cho, CH and Li, X and Li, M},
title = {Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {173},
number = {},
pages = {116336},
doi = {10.1016/j.biopha.2024.116336},
pmid = {38412717},
issn = {1950-6007},
mesh = {Animals ; Mice ; Humans ; *Stomach Neoplasms/pathology ; Down-Regulation/genetics ; Protein Disulfide-Isomerases/genetics/metabolism ; Mice, Nude ; Cyclin G1/genetics ; RNA, Guide, CRISPR-Cas Systems ; Cell Proliferation/genetics ; Cell Line, Tumor ; Cell Cycle/genetics ; RNA, Small Interfering/genetics ; Cell Transformation, Neoplastic/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics ; *Benzothiazoles ; },
abstract = {OBJECTIVE: Protein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms.<br><br>METHODS: GC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression.<br><br>RESULTS: The expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin&#xa0;G1 regulation.<br><br>CONCLUSION: PDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.},
}
@article {pmid38411120,
year = {2024},
author = {Zhang, C and Yang, S and Quansah, E and Zhang, Z and Da, W and Wang, B},
title = {The dCas9-based genome editing in Plasmodium yoelii.},
journal = {mSphere},
volume = {9},
number = {3},
pages = {e0009524},
pmid = {38411120},
issn = {2379-5042},
support = {XJ201807//Anhui Medical University (AHMU)/ ; KJ2021A0213//Scientific Research Foundation of Education Department of Anhui Province of China (Research Foundation of Education Department of Anhui Province of China)/ ; },
mesh = {Humans ; Gene Editing ; *Plasmodium yoelii/genetics ; CRISPR-Cas Systems ; *Malaria ; DNA ; },
abstract = {Genetic editing is a powerful tool for functional characterization of genes in various organisms. With its simplicity and specificity, the CRISPR-Cas9 technology has become a popular editing tool, which introduces site-specific DNA double-strand breaks (DSBs), and then leverages the endogenous repair pathway for DSB repair via homology-directed repair (HDR) or the more error-prone non-homologous end joining (NHEJ) pathways. However, in the Plasmodium parasites, the lack of a typical NHEJ pathway selects for DSB repair through the HDR pathway when a homologous DNA template is available. The AT-rich nature of the Plasmodium genome exacerbates this drawback by making it difficult to clone longer homologous repair DNA templates. To circumvent these challenges, we adopted the hybrid catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP) editor to the Plasmodium genome. In Plasmodium yoelii, we demonstrated the use of the dCas9-SSAP, as the cleavage-free gene editor, by targeted gene deletion and gene tagging, even using shorter homologous DNA templates. This dCas9-SSAP method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, would be a great addition to the existing genetic toolbox and could be deployed for the functional characterization of genes in Plasmodium, contributing to improving the ability of the malaria research community in characterizing more than half of genes with unknown functions.IMPORTANCEMalaria caused by Plasmodium parasites infection remains a serious threat to human health, with an estimated 249 million malaria cases and 608,000 deaths worldwide in 2022, according to the latest report from the World Health Organization (WHO). Here, we demonstrated the use of dCas9-single-stranded annealing protein, as the cleavage-free gene editor in Plasmodium yoelii, by targeted deletion and gene tagging, even using shorter homologous DNA templates. This method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, showing the potential significance in greatly improving our ability to elucidate gene functions, would contribute to assisting the malaria research community in deciphering more than half of genes with unknown functions to identify new drug and vaccine targets.},
}
@article {pmid38408576,
year = {2024},
author = {Banazadeh, M and Abiri, A and Poortaheri, MM and Asnaashari, L and Langarizadeh, MA and Forootanfar, H},
title = {Unexplored power of CRISPR-Cas9 in neuroscience, a multi-OMICs review.},
journal = {International journal of biological macromolecules},
volume = {263},
number = {Pt 2},
pages = {130413},
doi = {10.1016/j.ijbiomac.2024.130413},
pmid = {38408576},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Multiomics ; Gene Editing/methods ; *Neurodegenerative Diseases/genetics ; DNA ; },
abstract = {The neuroscience and neurobiology of gene editing to enhance learning and memory is of paramount interest to the scientific community. The advancements of CRISPR system have created avenues to treat neurological disorders by means of versatile modalities varying from expression to suppression of genes and proteins. Neurodegenerative disorders have also been attributed to non-canonical DNA secondary structures by affecting neuron activity through controlling gene expression, nucleosome shape, transcription, translation, replication, and recombination. Changing DNA regulatory elements which could contribute to the fate and function of neurons are thoroughly discussed in this review. This study presents the ability of CRISPR system to boost learning power and memory, treat or cure genetically-based neurological disorders, and alleviate psychiatric diseases by altering the activity and the irritability of the neurons at the synaptic cleft through DNA manipulation, and also, epigenetic modifications using Cas9. We explore and examine how each different OMIC techniques can come useful when altering DNA sequences. Such insight into the underlying relationship between OMICs and cellular behaviors leads us to better neurological and psychiatric therapeutics by intelligently designing and utilizing the CRISPR/Cas9 technology.},
}
@article {pmid38372397,
year = {2024},
author = {Guo, B and Hu, C and Yang, Z and Tang, C and Zhang, C and Wang, F},
title = {Test strip coupled Cas12a-assisted signal amplification strategy for sensitive detection of uracil-DNA glycosylase.},
journal = {Lab on a chip},
volume = {24},
number = {7},
pages = {1987-1995},
doi = {10.1039/d4lc00096j},
pmid = {38372397},
issn = {1473-0189},
mesh = {*Uracil-DNA Glycosidase/chemistry/metabolism ; *CRISPR-Cas Systems ; Limit of Detection ; DNA/chemistry ; Uracil/chemistry ; },
abstract = {Uracil-DNA glycosylase (UDG) is a base excision repair (BER) enzyme, which catalyzes the hydrolysis of uracil bases in DNA chains that contain uracil and N-glycosidic bonds of the sugar phosphate backbone. The expression of UDG enzyme is associated with a variety of genetic diseases including cancers. Hence, the identification of UDG activity in cellular processes holds immense importance for clinical investigation and diagnosis. In this study, we employed Cas12a protein and enzyme-assisted cycle amplification technology with a test strip to establish a precise platform for the detection of UDG enzyme. The designed platform enabled amplifying and releasing the target probe by reacting with the UDG enzyme. The amplified target probe can subsequently fuse with crRNA and Cas12a protein, stimulating the activation of the Cas12a protein to cleave the signal probe, ultimately generating a fluorescent signal. This technique showed the ability for evaluating UDG enzyme activity in different cell lysates. In addition, we have designed a detection probe to convert the fluorescence signal into test strip bands that can then be observed with the naked eye. Hence, our tool presented potential in both biomedical research and clinical diagnosis related to DNA repair enzymes.},
}
@article {pmid38354935,
year = {2024},
author = {Ma, L and Xie, L and Wu, Q and Yang, L and Zhou, Y and Cui, Y and Zhang, Y and Jiao, B and Wang, C and He, Y},
title = {Integrating CRISPR-Cas12a and rolling circle-amplified G-quadruplex for naked-eye fluorescent "off-on" detection of citrus Alternaria.},
journal = {International journal of biological macromolecules},
volume = {262},
number = {Pt 2},
pages = {129983},
doi = {10.1016/j.ijbiomac.2024.129983},
pmid = {38354935},
issn = {1879-0003},
mesh = {*Alternaria/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; *Citrus ; Fluorescent Dyes ; },
abstract = {Alternaria is a plant pathogen that spreads globally and is prone to causing citrus brown spot disease and metabolizing mycotoxins, thus seriously hindering the development of this economic crop industry. Herein, a "label-free" and "turn on" visual fluorescent assay for citrus Alternaria based on CRISPR-Cas12a and rolling circle amplification (RCA) was described. Using ssDNA complementary to RCA primer as a trans-cleavage substrate for CRISPR-Cas12a, the two systems of CRISPR-Cas12a and RCA-amplified G-quadruplex were skillfully integrated. By using a portable light source for excitation, the positive sample produced obvious red fluorescence, while the negative sample remained almost colorless, making them easy to differentiate with the naked eye. In addition, the specificity was demonstrated by distinguishing Alternaria from other citrus disease related pathogens. Moreover, the practicality was verified by analyzing cultured Alternaria and Alternaria in actual citrus leaf and fruit samples. Therefore, this method may contribute to the on-site diagnosis of Alternaria.},
}
@article {pmid38344762,
year = {2024},
author = {Huang, Z and Yang, T and Yu, J and Gao, Y and Weng, Y and Huang, Y and Li, S},
title = {Ultra-efficient delivery of CRISPR/Cas9 using ionic liquid conjugated polymers for genome editing-based tumor therapy.},
journal = {Biomaterials science},
volume = {12},
number = {7},
pages = {1716-1725},
doi = {10.1039/d3bm01981k},
pmid = {38344762},
issn = {2047-4849},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Ionic Liquids ; Polymers ; Plasmids/genetics ; },
abstract = {Emerging CRISPR-Cas9 systems can rebuild DNA sequences in the genome in a spatiotemporal manner, offering a magic tool for biological research, drug discovery, and gene therapy. However, low delivery efficiency remains a major roadblock hampering the wide application of CRISPR-Cas9 gene editing talent. Herein, ionic liquid-conjugated polymers (IL-CPs) are explored as efficient platforms for CRISPR-Cas9 plasmid delivery and in vivo genome editing-based tumor therapy. Via molecular screening of IL-CPs, IL-CPs integrated with fluorination monomers (PBF) can encapsulate plasmids into hybrid nanoparticles and achieve over 90% delivery efficiency in various cells regardless of serum interference. In vitro and in vivo experiments demonstrate that PBF can mediate Cas9/PLK1 plasmids for intracellular delivery and therapeutic genome editing in tumor, achieving efficient tumor suppression. This work provides a new tool for safe and efficient CRISPR-Cas9 delivery and therapeutic genome editing, thus opening a new avenue for the development of ionic liquid polymeric vectors for genome editing and therapy.},
}
@article {pmid38230816,
year = {2024},
author = {Cho, T and Wierk, A and Gertsenstein, M and Rodgers, CE and Uetrecht, J and Henderson, JT},
title = {The development and characterization of a CRISPR/Cas9-mediated PD-1 functional knockout rat as a tool to study idiosyncratic drug reactions.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {198},
number = {2},
pages = {233-245},
pmid = {38230816},
issn = {1096-0929},
support = {/CAPMC/CIHR/Canada ; },
mesh = {Humans ; Rats ; Mice ; Animals ; *Nevirapine/toxicity/metabolism ; Programmed Cell Death 1 Receptor/metabolism ; CRISPR-Cas Systems ; Models, Animal ; Liver/metabolism ; *Drug-Related Side Effects and Adverse Reactions ; Mammals/metabolism ; },
abstract = {Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.},
}
@article {pmid38135764,
year = {2024},
author = {Guan, W},
title = {Probing when dCas9 tolerates DNA mismatches.},
journal = {Nature biomedical engineering},
volume = {8},
number = {3},
pages = {207-208},
pmid = {38135764},
issn = {2157-846X},
mesh = {*DNA/genetics ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid37550424,
year = {2024},
author = {Sandler, SE and Weckman, NE and Yorke, S and Das, A and Chen, K and Gutierrez, R and Keyser, UF},
title = {Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores.},
journal = {Nature biomedical engineering},
volume = {8},
number = {3},
pages = {325-334},
pmid = {37550424},
issn = {2157-846X},
support = {ERC-2019-POC PoreDetect 899538//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: 'Ideas ' Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; EP/S022953/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; EP/L015889/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; },
mesh = {*Nanopores ; CRISPR-Cas Systems ; DNA/chemistry ; Nanotechnology ; Proteins ; },
abstract = {Single-molecule quantification of the strength and sequence specificity of interactions between proteins and nucleic acids would facilitate the probing of protein-DNA binding. Here we show that binding events between the catalytically inactive Cas9 ribonucleoprotein and any pre-defined short sequence of double-stranded DNA can be identified by sensing changes in ionic current as suitably designed barcoded linear DNA nanostructures with Cas9-binding double-stranded DNA overhangs translocate through solid-state nanopores. We designed barcoded DNA nanostructures to study the relationships between DNA sequence and the DNA-binding specificity, DNA-binding efficiency and DNA-mismatch tolerance of Cas9 at the single-nucleotide level. Nanopore-based sensing of DNA-barcoded nanostructures may help to improve the design of efficient and specific ribonucleoproteins for biomedical applications, and could be developed into sensitive protein-sensing assays.},
}
@article {pmid38502511,
year = {2024},
author = {Giustini, C and Angulo, J and Courtois, F and Allorent, G},
title = {Targeted Gene Editing of Nuclear-Encoded Plastid Proteins in Phaeodactylum tricornutum via CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2776},
number = {},
pages = {269-287},
pmid = {38502511},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Nuclear Proteins/metabolism ; *Diatoms/genetics/metabolism ; Chloroplast Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields.},
}
@article {pmid38502493,
year = {2024},
author = {Heath, NG and Segal, DJ},
title = {CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2784},
number = {},
pages = {285-299},
pmid = {38502493},
issn = {1940-6029},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Base Sequence ; DNA/genetics/metabolism ; *Biosensing Techniques ; Luciferases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.},
}
@article {pmid38502446,
year = {2024},
author = {Pérez-García, I and Pérez-García, V},
title = {CRISPR Activation in Mouse Trophoblast Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2781},
number = {},
pages = {93-103},
pmid = {38502446},
issn = {1940-6029},
mesh = {Pregnancy ; Female ; Animals ; Mice ; *Placenta ; *RNA, Guide, CRISPR-Cas Systems ; Trophoblasts ; Placentation/physiology ; Cell Differentiation/genetics ; Stem Cells ; },
abstract = {The placenta is a vital organ that regulates nutrient supply to the developing embryo during gestation. In mice, the placenta is composed of trophoblast lineage and mesodermal derivatives, which merge through the chorioallantoic fusion process in a critical event for the progression of placenta development. The trophoblast lineage is derived from self-renewing, multipotent cells known as mouse trophoblast stem cells (mTSCs). These cells are a valuable tool that allows scientists to comprehend the signals regulating major placental cell types' self-renewal and differentiation capacity. Recent advances in CRISPR-Cas9 genome editing applied in mTSCs have provided novel insights into the molecular networks involved in placentation. Here, we present a comprehensive CRISPR activation (CRISPRa) protocol based on the CRISPR/gRNA-directed synergistic activation mediator (SAM) method to overexpress specific target genes in mTSCs.},
}
@article {pmid38502445,
year = {2024},
author = {Doria-Borrell, P and Moya-Navamuel, M and Hemberger, M and Pérez-García, V},
title = {Generation of Knockout Mouse Trophoblast Stem Cells by CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2781},
number = {},
pages = {81-91},
pmid = {38502445},
issn = {1940-6029},
mesh = {Pregnancy ; Female ; Animals ; Mice ; *Placenta ; *CRISPR-Cas Systems ; Mice, Knockout ; Trophoblasts ; Cell Differentiation/genetics ; Stem Cells ; Mammals ; },
abstract = {The placenta is the organ that dictates the reproductive outcome of mammalian pregnancy by supplying nutrients and oxygen to the developing fetus to sustain its normal growth. During early mammalian development, trophoblast cells are the earliest cell type to differentiate with multipotent capacity to generate the trophoblast components of the placenta. The isolation and use of mouse trophoblast stem cells (mTSCs) to model in vitro trophoblast differentiation, in combination with CRISPR/Cas9 genome editing technology, has provided tremendous insight into the molecular mechanisms governing early mouse placentation. By knocking out a specific gene of interest in mTSCs, researchers are shedding light onto the molecular pathways involved in normal placental development and pregnancy disorders associated with abnormal placentation. In this chapter, we provide a detailed protocol for the genetic modification of mTSCs by using CRISPR/Cas9 genome editing system.},
}
@article {pmid38363032,
year = {2024},
author = {Park, HJ and Kim, M and Lee, D and Kim, HJ and Jung, HW},
title = {CRISPR-Cas9 and beyond: identifying target genes for developing disease-resistant plants.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {26},
number = {3},
pages = {369-377},
doi = {10.1111/plb.13625},
pmid = {38363032},
issn = {1438-8677},
support = {//Dong-A University/ ; //Green Fusion Technology Graduate School Program of the Ministry of Environment/ ; 2020R1A6A1A03047729//National Research Foundation of Korea/ ; 2022R1A2C1010324//National Research Foundation of Korea/ ; 2022R1F1A1067928//National Research Foundation of Korea/ ; RS-2022-RD009977//New Breeding Technologies Development Program of the Rural Development Administration/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Gene Editing ; Crops, Agricultural/genetics ; Agriculture ; },
abstract = {Throughout the history of crop domestication, desirable traits have been selected in agricultural products. However, such selection often leads to crops and vegetables with weaker vitality and viability than their wild ancestors when exposed to adverse environmental conditions. Considering the increasing human population and climate change challenges, it is crucial to enhance crop quality and quantity. Accordingly, the identification and utilization of diverse genetic resources are imperative for developing disease-resistant plants that can withstand unexpected epidemics of plant diseases. In this review, we provide a brief overview of recent progress in genome-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) technologies. In particular, we classify disease-resistant mutants of Arabidopsis thaliana and several crop plants based on the roles or functions of the mutated genes in plant immunity and suggest potential target genes for molecular breeding of genome-edited disease-resistant plants. Genome-editing technologies are resilient tools for sustainable development and promising solutions for coping with climate change and population increases.},
}
@article {pmid38500478,
year = {2024},
author = {Lee, S and Ahn, SJ},
title = {CRISPR/Cas9-mediated knockout of scarlet gene produces eye color mutants in the soybean looper, Chrysodeixis includens.},
journal = {Archives of insect biochemistry and physiology},
volume = {115},
number = {3},
pages = {e22100},
doi = {10.1002/arch.22100},
pmid = {38500478},
issn = {1520-6327},
support = {#311360//MAFES Strategic Research Initiative/ ; #32-2023//Mississippi Soybean Promotion Board/ ; },
mesh = {Female ; Male ; Animals ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Glycine max/genetics ; Eye Color ; *Moths/genetics ; },
abstract = {The CRISPR/Cas9 technology has greatly progressed research on non-model organisms, demonstrating successful applications in genome editing for various insects. However, its utilization in the case of the soybean looper, Chrysodeixis includens, a notable pest affecting soybean crops, has not been explored due to constraints such as limited genomic information and the embryonic microinjection technique. This study presents successful outcomes in generating heritable knockout mutants for a pigment transporter gene, scarlet, in C. includens through CRISPR/Cas9-mediated mutagenesis. The scarlet locus identified in the genome assembly of C. includens consists of 14 exons, with a coding sequence extending for 1,986 bp. Two single guide RNAs (sgRNAs) were designed to target the first exon of scarlet. Microinjection of these two sgRNAs along with the Cas9 protein into fresh embryos resulted in the successful production of variable phenotypes, particularly mutant eyes. The observed mutation rate accounted for about 16%. Genotype analysis revealed diverse indel mutations at the target site, presumably originating from double-strand breaks followed by the nonhomologous end joining repair, leading to a premature stop codon due to frame shift. Single-pair mating of the mutant moths produced G1 offspring, and the establishment of a homozygous mutant strain occurred in G2. The mutant moths exhibited lightly greenish or yellowish compound eyes in both sexes, confirming the involvement of scarlet in pigmentation in C. includens. Notably, the CRISPR/Cas9-mediated genome editing technique serves as a visible phenotypic marker, demonstrating its proof-of-concept applicability in C. includens, as other pigment transporter genes have been utilized as visible markers to establish genetic control for various insects. These results provide the first successful case that the CRISPR/Cas9 method effectively induces mutations in C. includes, an economically important soybean insect pest.},
}
@article {pmid38498220,
year = {2024},
author = {Zhang, Y and Lin, XF and Li, L and Piao, RH and Wu, S and Song, A and Gao, M and Jin, YM},
title = {CRISPR/Cas9-mediated knockout of Bsr-d1 enhances the blast resistance of rice in Northeast China.},
journal = {Plant cell reports},
volume = {43},
number = {4},
pages = {100},
pmid = {38498220},
issn = {1432-203X},
support = {20210101026JC//Natural Science Foundation of Jilin Province/ ; CXGC2023RCY050//Jilin Provincial Agricultural Science and Technology Independent Innovation Fund/ ; },
mesh = {Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Plant Breeding ; Alleles ; Transcription Factors/genetics ; *Oryza/genetics/metabolism ; Disease Resistance/genetics ; Plant Diseases/genetics ; *Magnaporthe ; },
abstract = {The blast resistance allele of OsBsr-d1 does not exist in most japonica rice varieties of Jilin Province in China. The development of Bsr-d1 knockout mutants via CRISPR/Cas9 enhances broad-spectrum resistance to rice blast in Northeast China. Rice blast is a global disease that has a significant negative impact on rice yield and quality. Due to the complexity and variability of the physiological races of rice blast, controlling rice blast is challenging in agricultural production. Bsr-d1, a negative transcription factor that confers broad-spectrum resistance to rice blast, was identified in the indica rice cultivar Digu; however, its biological function in japonica rice varieties is still unclear. In this study, we analyzed the blast resistance allele of Bsr-d1 in a total of 256 japonica rice varieties from Jilin Province in Northeast China and found that this allele was not present in these varieties. Therefore, we generated Bsr-d1 knockout mutants via the CRISPR/Cas9 system using the japonica rice variety Jigeng88 (JG88) as a recipient variety. Compared with those of the wild-type JG88, the homozygous Bsr-d1 mutant lines KO#1 and KO#2 showed enhanced leaf blast resistance at the seedling stage to several Magnaporthe oryzae (M. oryzae) races collected from Jilin Province in Northeast China. Physiological and biochemical indices revealed that the homozygous mutant lines produced more hydrogen peroxide than did JG88 plants when infected with M. oryzae. Comparative RNA-seq revealed that the DEGs were mainly involved in the synthesis of amide compounds, zinc finger proteins, transmembrane transporters, etc. In summary, our results indicate that the development of Bsr-d1 knockout mutants through CRISPR/Cas9 can enhance the broad-spectrum resistance of rice in Northeast China to rice blast. This study not only provides a theoretical basis for disease resistance breeding involving the Bsr-d1 gene in Northeast China, but also provides new germplasm resources for disease-resistance rice breeding.},
}
@article {pmid38497929,
year = {2024},
author = {Kader, M and Sun, W and Ren, BG and Yu, YP and Tao, J and Foley, LM and Liu, S and Monga, SP and Luo, JH},
title = {Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.},
journal = {Hepatology communications},
volume = {8},
number = {4},
pages = {},
pmid = {38497929},
issn = {2471-254X},
mesh = {Humans ; Animals ; Mice ; Thymidine Kinase/genetics ; CRISPR-Cas Systems/genetics ; *Herpesvirus 1, Human/genetics ; *Liver Neoplasms/genetics/therapy ; Catenins ; Mutation/genetics ; },
abstract = {BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease.<br><br>METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers.<br><br>RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups.<br><br>CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.},
}
@article {pmid38494539,
year = {2024},
author = {Atia, M and Jiang, W and Sedeek, K and Butt, H and Mahfouz, M},
title = {Crop bioengineering via gene editing: reshaping the future of agriculture.},
journal = {Plant cell reports},
volume = {43},
number = {4},
pages = {98},
pmid = {38494539},
issn = {1432-203X},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; Bioengineering ; Agriculture ; },
abstract = {Genome-editing technologies have revolutionized research in plant biology, with major implications for agriculture and worldwide food security, particularly in the face of challenges such as climate change and increasing human populations. Among these technologies, clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated protein [Cas] systems are now widely used for editing crop plant genomes. In this review, we provide an overview of CRISPR-Cas technology and its most significant applications for improving crop sustainability. We also review current and potential technological advances that will aid in the future breeding of crops to enhance food security worldwide. Finally, we discuss the obstacles and challenges that must be overcome to realize the maximum potential of genome-editing technologies for future crop and food production.},
}
@article {pmid38493479,
year = {2024},
author = {Kath, J and Franke, C and Drosdek, V and Du, W and Glaser, V and Fuster-Garcia, C and Stein, M and Zittel, T and Schulenberg, S and Porter, CE and Andersch, L and Künkele, A and Alcaniz, J and Hoffmann, J and Abken, H and Abou-El-Enein, M and Pruß, A and Suzuki, M and Cathomen, T and Stripecke, R and Volk, HD and Reinke, P and Schmueck-Henneresse, M and Wagner, DL},
title = {Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.},
journal = {Blood},
volume = {},
number = {},
pages = {},
doi = {10.1182/blood.2023020973},
pmid = {38493479},
issn = {1528-0020},
abstract = {Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T-cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and redirection of various immune cell types, including conventional T-cells, TCRγ/δ T-cells, regulatory T-cells, and NK-cells. In T-cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T-cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T-cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK-cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.},
}
@article {pmid38493286,
year = {2024},
author = {Jiang, F and Liu, Y and Yang, X and Li, Y and Huang, J},
title = {Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay.},
journal = {BMC veterinary research},
volume = {20},
number = {1},
pages = {106},
pmid = {38493286},
issn = {1746-6148},
support = {ZYN2023046//The Fundamental Research Funds for the Central Universities, Southwest Minzu University/ ; },
mesh = {Cats ; Animals ; *Calicivirus, Feline ; *Herpesviridae ; Recombinases/genetics ; CRISPR-Cas Systems ; *Varicellovirus ; },
abstract = {BACKGROUND: Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay.<br><br>RESULTS: The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4&#x2009;&#xd7;&#x2009;10[-&#x2009;1] copies/&#x3bc;L for the FHV-1 TK gene and 5.5 copies/&#x3bc;L for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings.<br><br>CONCLUSIONS: The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.},
}
@article {pmid38493086,
year = {2024},
author = {Ravn, JL and Manfrão-Netto, JHC and Schaubeder, JB and Torello Pianale, L and Spirk, S and Ciklic, IF and Geijer, C},
title = {Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing.},
journal = {Microbial cell factories},
volume = {23},
number = {1},
pages = {85},
pmid = {38493086},
issn = {1475-2859},
mesh = {*Saccharomyces cerevisiae/metabolism ; Fermentation ; *Gene Editing ; Hydrolysis ; CRISPR-Cas Systems ; Ethanol/metabolism ; Polymers/metabolism ; Glucuronidase ; Xylose/metabolism ; *Xylans ; },
abstract = {BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.<br><br>RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-&#x3b2;-xylanase and &#x3b2;-xylosidase activities, &#x3b1;-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA &#x3b2;-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 &#x3b1;-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33&#xa0;g L[-&#x2009;1] after 48&#xa0;h under oxygen limited condition in bioreactor fermentations.<br><br>CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.},
}
@article {pmid38493013,
year = {2024},
author = {Zimmermann, A and Prieto-Vivas, JE and Voordeckers, K and Bi, C and Verstrepen, KJ},
title = {Mutagenesis techniques for evolutionary engineering of microbes - exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases.},
journal = {Trends in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tim.2024.02.006},
pmid = {38493013},
issn = {1878-4380},
abstract = {The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, evolutionary engineering is often used to generate variants that show increased yields or resistance to stressful industrial environments, thus obtaining superior microbial cell factories. However, even in large populations, the natural supply of beneficial mutations is typically low, which implies that obtaining improved microbes is often time-consuming and inefficient. To overcome this limitation, different techniques have been developed that boost mutation rates. While some of these methods simply increase the overall mutation rate across a genome, others use recent developments in DNA synthesis, synthetic biology, and CRISPR-Cas techniques to control the type and location of mutations. This review summarizes the most important recent developments and methods in the field of evolutionary engineering in model microorganisms. It discusses how both in vitro and in vivo approaches can increase the genetic diversity of the host, with a special emphasis on in vivo techniques for the optimization of metabolic pathways for precision fermentation.},
}
@article {pmid38490023,
year = {2024},
author = {Shi, J and Lei, C and Fan, W and Sun, Y and Liu, C},
title = {Ultrasensitive protein and exosome analysis based on a rolling circle amplification assisted-CRISPR/Cas12a strategy.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125906},
doi = {10.1016/j.talanta.2024.125906},
pmid = {38490023},
issn = {1873-3573},
abstract = {CRISPR/Cas12a system has attracted extensive concern in biosensing due to its high specificity and programmability. Nevertheless, existing Cas12a-based assays mainly focus on nucleic acid detection and have limitations in non-nucleic acid biomarker analysis. To broaden the application prospect of the CRISPR/Cas technology, a cascade Cas12a biosensing platform is reported by combining dual-functionalized gold nanoparticles (FGNPs)-assisted rolling circle amplification (RCA) and Cas12a trans-cleavage activity (GAR-Cas) for ultrasensitive protein and exosome analysis. FGNPs serve as a critical component in the transduction of protein or exosome recognition information into nucleic acid amplification events to produce Cas12a activators. In the GAR-Cas assay, by integrating the triple cascade amplification of FGNPs-assisted transduction, RCA, and Cas12a signal amplification, ultralow abundance of target molecules can arouse numerous concatemers to activate Cas12a trans-cleavage activity to release intense fluorescence, allowing the ultrasensitive detection of as low as 1 fg/mL (∼41 aM) cTnI and 5 exosomes per μL. Furthermore, the presented strategy can be applied to detect exosome levels from clinical samples, showing excellent performance in distinguishing cancer patients from healthy individuals. The GAR-Cas sensing platform exhibits great potential in clinical diagnosis and enlarges biosensing toolboxes based on CRISPR/Cas technology for non-nucleic acid target analysis.},
}
@article {pmid38489357,
year = {2024},
author = {Li, J and Zhao, D and Zhang, T and Xiong, H and Hu, M and Liu, H and Zhao, F and Sun, X and Fan, P and Qian, Y and Wang, D and Lai, L and Sui, T and Li, Z},
title = {Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3.},
journal = {Science advances},
volume = {10},
number = {11},
pages = {eadk8052},
pmid = {38489357},
issn = {2375-2548},
mesh = {Mice ; Humans ; Animals ; *CRISPR-Cas Systems ; Gene Editing ; Y Chromosome ; *CRISPR-Associated Proteins/genetics ; DNA/genetics ; Mammals/genetics ; },
abstract = {Currently, the Cas9 and Cas12a systems are widely used for genome editing, but their ability to precisely generate large chromosome fragment deletions is limited. Type I-E CRISPR mediates broad and unidirectional DNA degradation, but controlling the size of Cas3-mediated DNA deletions has proven elusive thus far. Here, we demonstrate that the endonuclease deactivation of Cas9 (dCas9) can precisely control Cas3-mediated large-fragment deletions in mammalian cells. In addition, we report the elimination of the Y chromosome and precise retention of the Sry gene in mice using CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3, respectively. In conclusion, dCas9-controlled CRISPR/Cas3-mediated precise large-fragment deletion provides an approach for establishing animal models by chromosome elimination. This method also holds promise as a potential therapeutic strategy for treating fragment mutations or human aneuploidy diseases that involve additional chromosomes.},
}
@article {pmid38489100,
year = {2024},
author = {Eskandari, A and Leow, TC and Rahman, MBA and Oslan, SN},
title = {Recent insight into the advances and prospects of microbial lipases and their potential applications in industry.},
journal = {International microbiology : the official journal of the Spanish Society for Microbiology},
volume = {},
number = {},
pages = {},
pmid = {38489100},
issn = {1618-1905},
abstract = {Enzymes play a crucial role in various industrial sectors. These biocatalysts not only ensure sustainability and safety but also enhance process efficiency through their unique specificity. Lipases possess versatility as biocatalysts and find utilization in diverse bioconversion reactions. Presently, microbial lipases are gaining significant focus owing to the rapid progress in enzyme technology and their widespread implementation in multiple industrial procedures. This updated review presents new knowledge about various origins of microbial lipases, such as fungi, bacteria, and yeast. It highlights both the traditional and modern purification methods, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, the aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF), moreover, delves into the diverse applications of microbial lipases across several industries, such as food, vitamin esters, textile, detergent, biodiesel, and bioremediation. Furthermore, the present research unveils the obstacles encountered in employing lipase, the patterns observed in lipase engineering, and the application of CRISPR/Cas genome editing technology for altering the genes responsible for lipase production. Additionally, the immobilization of microorganisms' lipases onto various carriers also contributes to enhancing the effectiveness and efficiencies of lipases in terms of their catalytic activities. This is achieved by boosting their resilience to heat and ionic conditions (such as inorganic solvents, high-level pH, and temperature). The process also facilitates the ease of recycling them and enables a more concentrated deposition of the enzyme onto the supporting material. Consequently, these characteristics have demonstrated their suitability for application as biocatalysts in diverse industries.},
}
@article {pmid38485709,
year = {2024},
author = {Fiflis, DN and Rey, NA and Venugopal-Lavanya, H and Sewell, B and Mitchell-Dick, A and Clements, KN and Milo, S and Benkert, AR and Rosales, A and Fergione, S and Asokan, A},
title = {Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2325},
pmid = {38485709},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics ; Trans-Splicing ; RNA/genetics ; Mammals/genetics ; },
abstract = {Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5' and 3' segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.},
}
@article {pmid38472686,
year = {2023},
author = {Kruglova, NA and Komkov, DS and Mazurov, DV and Shepelev, MV},
title = {The RRE-Rev Module Has No Effect on the Packaging Efficiency of Cas9 and Gag Proteins into NanoMEDIC Virus-like Particles.},
journal = {Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections},
volume = {513},
number = {Suppl 1},
pages = {S45-S50},
pmid = {38472686},
issn = {1608-3105},
mesh = {Humans ; *HIV-1/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Products, gag/genetics ; Response Elements ; },
abstract = {Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE-Rev module to enhance the packaging of Cas9 nuclease into VLPs.},
}
@article {pmid38460306,
year = {2024},
author = {Ludwik, KA and Hellwig, L and Fisch, T and Contzen, J and Schaar, C and Mergenthaler, P and Stachelscheid, H},
title = {Generation of two human induced pluripotent stem cell lines with BAX and BAK1 double knock-out using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103377},
doi = {10.1016/j.scr.2024.103377},
pmid = {38460306},
issn = {1876-7753},
mesh = {Humans ; bcl-2-Associated X Protein/genetics/metabolism ; *Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; bcl-2 Homologous Antagonist-Killer Protein/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Apoptosis/genetics ; },
abstract = {Bcl-2-associated X protein (BAX) and Blc-2 homologous antagonist killer 1 (BAK) are two pro-apoptotic members of BCL2 family. Here, two BAX/BAK double knock-out human induced pluripotent stem cell lines (iPSC) we generated using CRISPR-Cas9 to generate apoptosis incompetent cell lines. The resulting cell lines were karyotypically normal, had typical morphology and expressed typical markers for the undifferentiated state.},
}
@article {pmid38458031,
year = {2024},
author = {Ferreira, LGA and Cabral-da-Silva, MC and Pachernegg, S and van den Bergen, JA and Robevska, G and Vlahos, K and Howden, SE and Ng, ES and Dias-da-Silva, MR and Sinclair, AH and Ayers, KL},
title = {Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103374},
doi = {10.1016/j.scr.2024.103374},
pmid = {38458031},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; Heart ; Heterozygote ; Homozygote ; Phenotype ; CRISPR-Cas Systems/genetics ; COUP Transcription Factor II/genetics/metabolism ; },
abstract = {The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.},
}
@article {pmid38452707,
year = {2024},
author = {Agrawal, T and Maddileti, S and Mariappan, I},
title = {Generation and characterization of three CRISPR/Cas9 edited RB1 null hiPSC lines for retinoblastoma disease modelling.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103373},
doi = {10.1016/j.scr.2024.103373},
pmid = {38452707},
issn = {1876-7753},
mesh = {Humans ; *Retinoblastoma/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; *Retinal Neoplasms/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; Retinoblastoma Binding Proteins/genetics ; },
abstract = {Complete loss of RB1 causes retinoblastoma. Here, we report the generation of three RB1[-/-] iPSC lines using CRISPR/Cas9 based editing at exon 18 of RB1 in a healthy control hiPSC line. The edited cells were clonally expanded, genotyped and characterized to establish the mutant lines. Two of the mutant lines are compound heterozygous, with different in-del mutations in each of their alleles, while the third mutant line is homozygous, with identical edits in both alleles. All lines maintained their stemness, pluripotency, formed embryoid bodies with cell types of all three lineages, displayed a normal karyotype and lost RB1 expression.},
}
@article {pmid38452706,
year = {2024},
author = {Hou, X and Fan, W and Zeng, J and Gao, Z and Wan, J and Liao, B},
title = {Generation of a ISL1 homozygous knockout stem cell line (WAe009-A-1G) by episomal vector-based CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103376},
doi = {10.1016/j.scr.2024.103376},
pmid = {38452706},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Transcription Factors/genetics/metabolism ; Cell Line ; Heart ; Embryonic Stem Cells/metabolism ; *Human Embryonic Stem Cells/metabolism ; LIM-Homeodomain Proteins/genetics ; },
abstract = {The ISL LIM homeobox 1 (ISL1) gene belongs to the LIM/homeodomain transcription factor family and plays a pivotal role in conveying multipotent and proliferative properties of cardiac precursor cells. Mutations in ISL1 are linked to congenital heart disease. To further explore ISL1's role in the human heart, we have created a homozygous ISL1 knockout (ISL1-KO) human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this ISL1-KO cell line retains normal morphology, pluripotency, and karyotype. This resource serves as a valuable tool for investigating ISL1's function in cardiomyocyte differentiation.},
}
@article {pmid38451610,
year = {2024},
author = {Luo, L and Dong, F and Li, D and Li, X and Li, X and Fan, Y and Qi, C and Luo, J and Li, L and Shen, B},
title = {Enhancing 3D DNA Walker-Induced CRISPR/Cas12a Technology for Highly Sensitive Detection of ExomicroRNA Associated with Osteoporosis.},
journal = {ACS sensors},
volume = {9},
number = {3},
pages = {1438-1446},
doi = {10.1021/acssensors.3c02533},
pmid = {38451610},
issn = {2379-3694},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *MicroRNAs ; DNA/genetics ; *Osteoporosis/diagnosis/genetics ; Technology ; },
abstract = {Exosomal microRNAs (exomiRNAs) have emerged as promising biomarkers for the early clinical diagnosis of osteoporosis. However, their limited abundance and short length in peripheral blood present significant challenges for the accurate detection of exomiRNAs. Herein, we have designed and implemented an efficacious fluorescence-based biosensor for the highly sensitive detection of exomiRNA associated with osteoporosis, leveraging the enhancing 3D DNA walker-induced CRISPR/Cas12a technology. The engineered DNA walker is capable of efficiently transforming target exomiRNA into amplifying DNA strands, thereby enhancing the sensitivity of the developed biosensor. Concurrently, the liberated DNA strands serve as activators to trigger Cas12a trans-cleavage activity, culminating in a significantly amplified fluorescent signal for the highly sensitive detection of exomiRNA-214. Under optimal conditions, the devised technology demonstrated the capacity to detect target exomiRNA-214 at concentrations as low as 20.42 fM, encompassing a wide linear range extending from 50.0 fM to 10.0 nM. Moreover, the fluorescence-based biosensor could accurately differentiate between healthy individuals and osteoporosis patients via the detection of exomiRNA-214, which was in agreement with RT-qPCR results. As such, this biosensing technology offers promise as a valuable tool for the early diagnosis of osteoporosis.},
}
@article {pmid38442486,
year = {2024},
author = {Ci, Q and He, Y and Chen, J},
title = {Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA).},
journal = {ACS sensors},
volume = {9},
number = {3},
pages = {1162-1167},
doi = {10.1021/acssensors.4c00201},
pmid = {38442486},
issn = {2379-3694},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; CRISPR-Cas Systems/genetics ; DNA/genetics ; *Biosensing Techniques ; },
abstract = {Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.},
}
@article {pmid38430734,
year = {2024},
author = {Ratnavadivel, S and Dammeier, J and Gaertner, A and de Toledo, MAS and Zenke, M and Gummert, J and Bloch Rasmussen, T and Klinke, N and Jürgens, K and Meyer, H and Paululat, A and Milting, H},
title = {Generation of a TMEM43 knockout human induced pluripotent stem cell line (HDZi003-A-1) using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103354},
doi = {10.1016/j.scr.2024.103354},
pmid = {38430734},
issn = {1876-7753},
mesh = {Animals ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Drosophila melanogaster/metabolism ; *Arrhythmogenic Right Ventricular Dysplasia/genetics ; Mutation ; Membrane Proteins/genetics/metabolism ; },
abstract = {TMEM43 (LUMA) is a ubiquitously expressed protein with unknown function. The protein is phylogenetically highly conserved and also found in Drosophila melanogaster (Klinke et al., 2022). TMEM43-p.S358L is a rare, fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). To understand the function of the ARVC5-associated mutation it is first important to understand the function of the TMEM43 protein. Therefore, a TMEM43 knockout induced pluripotent stem cell (iPSC) line was generated using the CRISPR/Cas9 genome editing system. The resulting cell line had a deficiency of TMEM43 and showed normal morphology and a stable karyotype. The colonies were positive for pluripotency markers and could be differentiated into the three germ layers.},
}
@article {pmid38428348,
year = {2024},
author = {Jin, H and Fu, H and Wang, J and Wang, Z and Liu, J and Han, F and Zheng, H and Jiang, Y},
title = {Generation of a DMD loss-of-function mutant human embryonic stem cell lines by CRISPR base editing.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103343},
doi = {10.1016/j.scr.2024.103343},
pmid = {38428348},
issn = {1876-7753},
mesh = {Humans ; Dystrophin/genetics/metabolism ; *Muscular Dystrophy, Duchenne/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Human Embryonic Stem Cells/metabolism ; Exons/genetics ; Cell Line ; Mutation/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder, which is caused mostly by frame-disrupting, out-of-frame variation in the dystrophin (DMD) gene. Loss-of- function mutations are the most common type of mutation in DMD, accounting for approximately 60-90% of all DMD variations. In this study, we used adenine base editing to generate a human embryonic stem cell line with splice-site mutations to mimic exon deletion variants in clinical Duchenne muscular dystrophy patients. This cell line has differentiation potential and a normal karyotypic.},
}
@article {pmid38382214,
year = {2024},
author = {Pierre, B and Laëtitia, DB and Camille, B and Claire, P and Elise, B and Estelle, G and Vincent, F and Eric, V},
title = {Generation of CRISPR/Cas9 edited human induced pluripotent stem cell line carrying the heterozygous p.H695VfsX5 frameshift mutation in the exon 10 of the PKP2 gene.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103341},
doi = {10.1016/j.scr.2024.103341},
pmid = {38382214},
issn = {1876-7753},
mesh = {Humans ; *Frameshift Mutation ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; Exons/genetics ; Plakophilins/genetics/metabolism ; },
abstract = {Loss-of-function mutations in the PKP2 gene are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), a rare cardiac disease associated with a poor prognosis. The search for therapeutics and a better understanding of the molecular mechanisms of the disease require the development of cellular modelling. Using CRISPR/Cas9, we generated a hiPSC line with heterozygous 7-bp deletion in exon 10 of PKP2 (p.H695VfsX5). We demonstrated that hiPSCs were fully pluripotent and showed a high rate of differentiation into cardiomyocytes (iPS-CM). We also showed that PKP2 protein was expressed at the plasma membrane, with an overall decreased expression in iPS-CM indicating haploinsufficiency.},
}
@article {pmid38364506,
year = {2024},
author = {Hua, M and Williams, L and Burns, K and Liu, S and Ellis, J and Innes, AM and McPherson, M and Yang, G},
title = {Generation and characterization of a human iPSC line and gene-corrected isogenic line derived from a patient with a CELF2 gene mutation.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103344},
doi = {10.1016/j.scr.2024.103344},
pmid = {38364506},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Gene Editing ; Mutation, Missense ; Cell Differentiation ; CRISPR-Cas Systems/genetics ; CELF Proteins/genetics/metabolism ; Nerve Tissue Proteins/metabolism ; },
abstract = {The identification of neurodevelopmental defects in a patient harboring a heterozygous de novo missense variant (NM_006561.4, c.1517G > A, p.Arg506His) within the CELF2 gene. Here, we describe the establishment of a patient-derived induced pluripotent stem cell (iPSC) line, alongside an isogenic gene-corrected iPSC line, achieved through CRISPR/Cas9 genome editing. These lines exhibit the expression of pluripotency markers, demonstrate differentiation potential into all three germ layers, and maintain a normal karyotype. These iPSC lines serve as valuable tools for investigating the consequences of CELF2 related neurodevelopmental disorders.},
}
@article {pmid38324931,
year = {2024},
author = {Przymuszała, M and Martyniak, A and Kwiatkowska, J and Meyer-Szary, J and Śledzińska, K and Wierzba, J and Dulak, J and Florczyk-Soluch, U and Stępniewski, J},
title = {Generation of human induced pluripotent stem cell line derived from Becker muscular dystrophy patient with CRISPR/Cas9-mediated correction of DMD gene mutation.},
journal = {Stem cell research},
volume = {76},
number = {},
pages = {103327},
doi = {10.1016/j.scr.2024.103327},
pmid = {38324931},
issn = {1876-7753},
mesh = {Humans ; *Muscular Dystrophy, Duchenne/pathology ; Dystrophin/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; },
abstract = {Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by in-frame deletions in the dystrophin gene (DMD), leading to progressive muscle degeneration and weakness. We generated a human induced pluripotent stem cell (hiPSC) line from a BMD patient. BMD hiPSCs were then engineered by CRISPR/Cas9-mediated knock-in of missing exons 3-9 of DMD gene. Obtained hiPSC line may be a valuable tool for investigating the mechanisms underlying BMD pathogenesis.},
}
@article {pmid38317057,
year = {2024},
author = {Li, B and Li, Q and Qi, Z and Li, Z and Yan, X and Chen, Y and Xu, X and Pan, Q and Chen, Y and Huang, F and Ping, Y},
title = {Supramolecular Genome Editing: Targeted Delivery and Endogenous Activation of CRISPR/Cas9 by Dynamic Host-Guest Recognition.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {63},
number = {14},
pages = {e202316323},
doi = {10.1002/anie.202316323},
pmid = {38317057},
issn = {1521-3773},
support = {2021YFA0910100//National Key Research and Development Program of China/ ; 2021YFA0909903//National Key Research and Development Program of China/ ; 32261143727//National Natural Science Foundation of China/ ; 32000992//National Natural Science Foundation of China/ ; 82073779//National Natural Science Foundation of China/ ; LR21H300002//Natural Science Foundation of Zhejiang Province/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plasmids ; DNA ; Polyamines ; },
abstract = {We synthesize supramolecular poly(disulfide) (CPS) containing covalently attached cucurbit[7]uril (CB[7]), which is exploited not only as a carrier to deliver plasmid DNA encoding destabilized Cas9 (dsCas9), but also as a host to include trimethoprim (TMP) by CB[7] moieties through the supramolecular complexation to form TMP@CPS/dsCas9. Once the plasmid is transfected into tumor cells by CPS, the presence of polyamines can competitively trigger the decomplexation of TMP@CPS, thereby displacing and releasing TMP from CB[7] to stabilize dsCas9 that can target and edit the genomic locus of PLK1 to inhibit the growth of tumor cells. Following the systemic administration of TMP@CPS/dsCas9 decorated with hyaluronic acid (HA), tumor-specific editing of PLK1 is detected due to the elevated polyamines in tumor microenvironment, greatly minimizing off-target editing in healthy tissues and non-targeted organs. As the metabolism of polyamines is dysregulated in a wide range of disorders, this study offers a supramolecular approach to precisely control CRISPR/Cas9 functions under particular pathological contexts.},
}
@article {pmid38190037,
year = {2023},
author = {Golubev, DS and Komkov, DS and Shepelev, MV and Mazurov, DV and Kruglova, NA},
title = {Efficient Editing of the CXCR4 Locus Using Cas9 Ribonucleoprotein Complexes Stabilized with Polyglutamic Acid.},
journal = {Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections},
volume = {513},
number = {Suppl 1},
pages = {S28-S32},
pmid = {38190037},
issn = {1608-3105},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Polyglutamic Acid/genetics/metabolism ; *HIV Infections ; RNA, Guide, CRISPR-Cas Systems ; Ribonucleoproteins/genetics/chemistry/metabolism ; Receptors, CXCR4/genetics/metabolism ; },
abstract = {Gene editing using the CRISPR/Cas9 system provides new opportunities to treat human diseases. Approaches aimed at increasing the efficiency of genome editing are therefore important to develop. To increase the level of editing of the CXCR4 locus, which is a target for gene therapy of HIV infection, the Cas9 protein was modified by introducing additional NLS signals and ribonucleoprotein complexes of Cas9 and guide RNA were stabilized with poly-L-glutamic acid. The approach allowed a 1.8-fold increase in the level of CXCR4 knockout in the CEM/R5 T cell line and a 2-fold increase in the level of knock-in of the HIV-1 fusion peptide inhibitor MT-C34 in primary CD4[+] T lymphocytes.},
}
@article {pmid38485293,
year = {2024},
author = {Hu, YQ and Zhang, YH and Han, W and Hu, T and Du, Y and Zeng, YX},
title = {Complete genome sequence of the novel Antarctic Oceanisphaera sp. IT1-181 that carried five plasmids.},
journal = {Marine genomics},
volume = {74},
number = {},
pages = {101083},
doi = {10.1016/j.margen.2024.101083},
pmid = {38485293},
issn = {1876-7478},
mesh = {*Seawater/microbiology ; Fatty Acids/analysis ; Antarctic Regions ; DNA, Bacterial/genetics ; Phylogeny ; RNA, Ribosomal, 16S ; Bacterial Typing Techniques ; Plasmids/genetics ; Bacteria/genetics ; *Aeromonadaceae/genetics ; Sequence Analysis, DNA ; },
abstract = {Bacteria of the genus Oceanisphaera in the class Gammaproteobacteria are widely distributed in marine environments. Oceanisphaera sp. IT1-181 was isolated from intertidal sediment in the coastal region of the Chinese Great Wall Station on the Fildes Peninsula, King George Island, Antarctica. Here, we sequenced the complete genome of strain IT1-181, which contained a single chromosome of 3,572,184 bp (G + C content of 49.89 mol%) with five plasmids. A total of 3229 protein-coding genes, 88 tRNA genes, and 25 rRNA genes were obtained. Genome sequence analysis revealed that strain IT1-181 was not only a potentially novel species of the genus Oceanisphaera, but also harbored genes involved in biosynthesizing ectoine as well as poly-β-hydroxybutyric acid (PHB). In addition, genes of a complete type I-E CRISPR-Cas system were found in the bacterium. The results indicate the potential of strain Oceanisphaera sp. IT1-181 in biotechnology and are helpful for us understanding its ecological roles in the changing Antarctic intertidal zone environment.},
}
@article {pmid38484704,
year = {2024},
author = {McGee, AV and Liu, YV and Griffith, AL and Szegletes, ZM and Wen, B and Kraus, C and Miller, NW and Steger, RJ and Escude Velasco, B and Bosch, JA and Zirin, JD and Viswanatha, R and Sontheimer, EJ and Goodale, A and Greene, MA and Green, TM and Doench, JG},
title = {Modular vector assembly enables rapid assessment of emerging CRISPR technologies.},
journal = {Cell genomics},
volume = {4},
number = {3},
pages = {100519},
pmid = {38484704},
issn = {2666-979X},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; U01 CA250565/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Vectors/genetics ; },
abstract = {The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.},
}
@article {pmid38483978,
year = {2024},
author = {Chen, P and Zhou, J and Liu, H and Zhou, E and He, B and Wu, Y and Wang, H and Sun, Z and Paek, C and Lei, J and Chen, Y and Zhang, X and Yin, L},
title = {Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.},
journal = {PLoS biology},
volume = {22},
number = {3},
pages = {e3002514},
doi = {10.1371/journal.pbio.3002514},
pmid = {38483978},
issn = {1545-7885},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.},
}
@article {pmid38418885,
year = {2024},
author = {Ollivier, M and Soto, JS and Linker, KE and Moye, SL and Jami-Alahmadi, Y and Jones, AE and Divakaruni, AS and Kawaguchi, R and Wohlschlegel, JA and Khakh, BS},
title = {Crym-positive striatal astrocytes gate perseverative behaviour.},
journal = {Nature},
volume = {627},
number = {8003},
pages = {358-366},
pmid = {38418885},
issn = {1476-4687},
support = {F32 MH125598/MH/NIMH NIH HHS/United States ; R01 DA047444/DA/NIDA NIH HHS/United States ; R35 NS111583/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Humans ; Mice ; *Astrocytes/metabolism ; *Corpus Striatum/cytology/physiology ; Gene Editing ; Gene Knockout Techniques ; *mu-Crystallins/deficiency/genetics/metabolism ; *Rumination, Cognitive/physiology ; Synaptic Transmission ; CRISPR-Cas Systems ; Medium Spiny Neurons/metabolism ; Synapses/metabolism ; Prefrontal Cortex/cytology/metabolism ; Presynaptic Terminals/metabolism ; Neural Inhibition ; },
abstract = {Astrocytes are heterogeneous glial cells of the central nervous system[1-3]. However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses μ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders[4-7]. In adult mice, reducing the levels of μ-crystallin in striatal astrocytes through CRISPR-Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory-inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex-striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics[8], and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders[9-12]. Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte-neuron interaction mechanisms that could be targeted in treatments for perseveration.},
}
@article {pmid38483963,
year = {2024},
author = {Hossain, MJ and O'Connor, TJ},
title = {An efficient and cost-effective method for disrupting genes in RAW264.7 macrophages using CRISPR-Cas9.},
journal = {PloS one},
volume = {19},
number = {3},
pages = {e0299513},
pmid = {38483963},
issn = {1932-6203},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Cost-Benefit Analysis ; Gene Editing/methods ; Cells, Cultured ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are widely used for genome editing in cultured cell lines. However, the implementation of genome editing is still challenging due to the complex and often costly multi-step process associated with this technique. Moreover, the efficiency of genome editing varies across cell types, often limiting utility. Herein, we describe pCRISPR-EASY, a vector for simplified cloning of single guide RNAs (sgRNAs) and its simultaneous introduction with CRISPR-Cas9 into cultured cells using a non-viral delivery system. We outline a comprehensive, step-by-step protocol for genome editing in RAW264.7 macrophages, a mouse macrophage cell line widely used in biomedical research for which genome editing using CRISPR-Cas9 has been restricted to lentiviral or expensive commercial reagents. This provides an economical, highly efficient and reliable method for genome editing that can easily be adapted for use in other systems.},
}
@article {pmid38481662,
year = {2024},
author = {Ma, C and Zou, M and Xu, N and Liu, Y and Wang, Y},
title = {Portable, and ultrasensitive HR-HPV tests based on nucleic acid biosensors.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1357090},
pmid = {38481662},
issn = {2235-2988},
mesh = {Female ; Humans ; *Papillomavirus Infections/diagnosis ; *Uterine Cervical Neoplasms/diagnosis ; Reproducibility of Results ; Mass Screening ; Nucleic Acid Hybridization ; },
abstract = {Cervical cancer is the third most common cancer threatening women's health globally, and high-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer worldwide. Given the recurrent nature of HR-HPV infection, accurate screening is essential for its control. Since the commonly used polymerase chain reaction (PCR) technique is limited by professional equipment and personnel, convenient and ultrasensitive detection methods for HR-HPV are still highly needed. As new molecular detection methods, nucleic acid amplification-based biosensors have the advantages of high sensitivity, rapid operation, and portability, which are helpful for point-of-care testing in rural and remote areas. This review summarized nucleic acid biosensors for HR-HPV screening based on a variety of nucleic acid amplification strategies involved in improved PCR, loop-mediated isothermal amplification, recombinase polymerase amplification, hybridization chain reaction, catalyzed hairpin assembly, and CRISPR/Cas systems. In combination with microfluidic technology, lateral flow assays, electrochemical analysis and other sensing technologies, HR-HPV nucleic acid biosensors have the advantages of high throughput, short response time, high sensitivity and easy operation in the field. Although there are still shortcomings, such as high cost and poor reproducibility, this approach will be suitable for on-site screening of HR-HPV infection or cervical cancer and for auxiliary clinical diagnosis in complex environments and poor areas in the future.},
}
@article {pmid38457464,
year = {2024},
author = {McCabe, CV and Price, PD and Codner, GF and Allan, AJ and Caulder, A and Christou, S and Loeffler, J and Mackenzie, M and Malzer, E and Mianné, J and Nowicki, KJ and O'Neill, EJ and Pike, FJ and Hutchison, M and Petit-Demoulière, B and Stewart, ME and Gates, H and Wells, S and Sanderson, ND and Teboul, L},
title = {Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.},
journal = {PLoS genetics},
volume = {20},
number = {3},
pages = {e1011187},
pmid = {38457464},
issn = {1553-7404},
support = {U42 OD011174/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Alleles ; *Gene Editing/methods ; Recombinational DNA Repair ; Polymerase Chain Reaction ; },
abstract = {BACKGROUND: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders.<br><br>In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles.<br><br>CONCLUSION: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.},
}
@article {pmid38417717,
year = {2024},
author = {Dai, XY and Lan, HJ and Chen, Y and Liu, TY and Zhao, YT and Liu, JZ},
title = {Knocking out NtSARD1a/1b/1c/1d by CRISPR/CAS9 technology reduces the biosynthesis of salicylic acid (SA) and compromises immunity in tetraploid Nicotiana tabacum.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {342},
number = {},
pages = {112051},
doi = {10.1016/j.plantsci.2024.112051},
pmid = {38417717},
issn = {1873-2259},
mesh = {Salicylic Acid/metabolism ; Nicotiana/genetics ; CRISPR-Cas Systems ; Tetraploidy ; *Arabidopsis/genetics ; *Arabidopsis Proteins/metabolism ; Plant Diseases ; Pseudomonas syringae/physiology ; Gene Expression Regulation, Plant ; },
abstract = {Salicylic acid (SA) is a key phyto-hormone that is essential for plant immunity. SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1), a member of the CBP60 (CALMODULIN-BINDING PROTEIN60) gene family, is one of the major transcription factors regulating the expression of the genes in SA biosynthesis. SARD1 has been extensively studied in model plant Arabidopsis. However, the function of SARD1 homologues in SA biosynthesis and immune responses have rarely been investigated in other plant species. In this study, the CRISPR/CAS9 (Clustered Regularly Interspersed Short Palindromic Repeats/CAS9) technology was used in creating transgenic tobacco mutant lines with 6-8 alleles of four NtSARD1 homologous genes (NtSARD1a/1b/1c/1d) knocked out. No significant difference in morphological phenotype was observed between the transgenic knockout lines and the wild type tobacco plants, indicating that knocking out NtSARD1s does not affect the growth and development in tobacco. However, knocking out or partially knocking out of NtSARD1a/b/c/d resulted in a significantly reduced expression of NtICS1, the key gene in SA biosynthesis pathway, and thus the subsequently decreased SA/SAG accumulations in response to Pst DC3000 (Pseudomonas syrangae pv.tomato DC3000) infection, indicating a key role of NtSARD1 genes in SA biosynthesis in tobacco. As a consequence of reduced SA/SAG accumulation, the Pst DC3000-induced expression of NtPR genes as well as the resistance to Pst DC3000 were both significantly reduced in these knockout lines compared with the wild type tobacco plants. Interestingly, the reductions in the SA/SAG level, NtPR gene induction and Pst DC3000 resistance were positively correlated with the number of alleles being knocked out. Furthermore, LUC reporter gene driven by the promoter of NtICS1 containing two G(A/T)AATT(T/G) motifs could be activated by NtSARD1a, suggesting that NtSARD1a could bind to the core G(A/T)AATT(T/G) motifs and thus activate the expression of LUC reporter. Taken together, our results demonstrated that the NtSARD1 proteins play essential roles in SA biosynthesis and immune responses in tobacco. Our results also demonstrated that the CRISPR/CAS9 technology can overcome gene redundancy and is a powerful tool to study gene functions in polyploid plant species.},
}
@article {pmid38354755,
year = {2024},
author = {Sun, S and Han, X and Jin, R and Jiao, J and Wang, J and Niu, S and Yang, Z and Wu, D and Wang, Y},
title = {Generation of CRISPR-edited birch plants without DNA integration using Agrobacterium-mediated transformation technology.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {342},
number = {},
pages = {112029},
doi = {10.1016/j.plantsci.2024.112029},
pmid = {38354755},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems ; *Agrobacterium/genetics ; RNA, Guide, CRISPR-Cas Systems ; Betula/genetics ; Gene Editing/methods ; DNA/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR/Cas9 system has emerged as a powerful tool in genome editing; however, generation of CRISPR-edited DNA-free plants is still challenging. In this study, Betula platyphylla (birch) was used to build a method to generate CRISPR-edited plant without foreign DNA integration using Agrobacterium-mediated transformation (CPDAT method). This technique utilizes transient genetic transformation to introduce T-DNA coding gRNA and Cas9 into birch cells, and T-DNA will express to synthesize gRNA and Cas9 protein, which will form a complex to cleave the target DNA site. The genome may be mutated due to DNA repair, and these mutations will be preserved and accumulated not dependent on whether T-DNA is integrated into the genome or not. After transient transformation, birch plants were cut into explants to induce adventitious buds without antibiotic selection pressure. Each adventitious bud can be considered as an independent potentially CRISPR-edited line for mutation detection. CRISPR-edited birch plants without foreign DNA integration are further selected by screening CRISPR-edited lines without T-DNA integration. Among 65 randomly chosen independent lines, the mutation rate was 80.00% including 40.00% of lines with both alleles mutated. In addition, 5 lines out of 65 studied lines (7.69%) were CRISPR-edited birch plants without DNA integration. In conclusion, this innovative method presents a novel strategy for generating CRISPR-edited birch plants, thereby significantly enhancing the efficiency of generating common CRISPR-edited plants. These findings offer considerable potential to develop plant genome editing techniques further.},
}
@article {pmid38353529,
year = {2024},
author = {Almási, &#xc9;dH and Knischewski, N and Osbelt, L and Muthukumarasamy, U and El Mouali, Y and Vialetto, E and Beisel, CL and Strowig, T},
title = {An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition between Klebsiella species.},
journal = {Journal of bacteriology},
volume = {206},
number = {3},
pages = {e0031723},
pmid = {38353529},
issn = {1098-5530},
support = {01KI1824//Bundesministerium f&#xfc;r Bildung und Forschung (BMBF)/ ; 01KI2131//Bundesministerium f&#xfc;r Bildung und Forschung (BMBF)/ ; },
mesh = {*CRISPR-Cas Systems ; *Klebsiella/genetics ; Klebsiella pneumoniae/genetics ; },
abstract = {UNLABELLED: Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. Firstly, the system allowed efficient and precise full-length gene deletion via enhanced lambda Red expression. Furthermore, we tested the efficiency of two independent, functionally validated complementation strategies. Ultimately, the insertion of universal "bookmark" targets during gene deletion subsequently allows the most optimal genetic complementation in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. This approach offers a significant advantage by enabling the use of a single high-efficiency "bookmark" for complementing other loci or strains, eliminating the need for site-specific design. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition.<br><br>IMPORTANCE: Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We then adapted a recently developed protocol for functional complementation based on universal "bookmark" targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.},
}
@article {pmid38321963,
year = {2024},
author = {Wang, Q and Capelletti, S and Liu, J and Janssen, JM and Gonçalves, MAFV},
title = {Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells.},
journal = {Nucleic acids research},
volume = {52},
number = {5},
pages = {2740-2757},
doi = {10.1093/nar/gkae057},
pmid = {38321963},
issn = {1362-4962},
support = {W.OR21-01//Prinses Beatrix Spierfonds/ ; //China Scholarship Council/ ; 101072427//European Union's Horizon/ ; },
mesh = {Humans ; *Dystrophin/genetics/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Myoblasts/metabolism ; Myocytes, Cardiac/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
abstract = {Prime editors have high potential for disease modelling and regenerative medicine efforts including those directed at the muscle-wasting disorder Duchenne muscular dystrophy (DMD). However, the large size and multicomponent nature of prime editing systems pose substantial production and delivery issues. Here, we report that packaging optimized full-length prime editing constructs in adenovector particles (AdVPs) permits installing precise DMD edits in human myogenic cells, namely, myoblasts and mesenchymal stem cells (up to 80% and 64%, respectively). AdVP transductions identified optimized prime-editing reagents capable of correcting DMD reading frames of ∼14% of patient genotypes and restoring dystrophin synthesis and dystrophin-β-dystroglycan linkages in unselected DMD muscle cell populations. AdVPs were equally suitable for correcting DMD iPSC-derived cardiomyocytes and delivering dual prime editors tailored for DMD repair through targeted exon 51 deletion. Moreover, by exploiting the cell cycle-independent AdVP transduction process, we report that 2- and 3-component prime-editing modalities are both most active in cycling than in post-mitotic cells. Finally, we establish that combining AdVP transduction with seamless prime editing allows for stacking chromosomal edits through successive delivery rounds. In conclusion, AdVPs permit versatile investigation of advanced prime editing systems independently of their size and component numbers, which should facilitate their screening and application.},
}
@article {pmid38319218,
year = {2024},
author = {Neo, DM and Clatworthy, AE and Hung, DT},
title = {A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus.},
journal = {Journal of bacteriology},
volume = {206},
number = {3},
pages = {e0033523},
pmid = {38319218},
issn = {1098-5530},
support = {//Broad Institute Tuberculosis Donor Group/ ; //Pershing Square Foundation (PSF)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Mycobacterium abscessus/genetics ; Gene Editing/methods ; Plasmids/genetics ; },
abstract = {UNLABELLED: Mycobacterium abscessus is increasingly recognized for causing infections that are notoriously difficult to treat, owing to its large arsenal of intrinsic antibiotic resistance mechanisms. Tools for the genetic manipulation of the pathogen are critical for enabling a better understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods are largely recombination-based, which are relatively inefficient. Meanwhile, CRISPR/Cas9 has revolutionized the field of genome editing including its recent adaptation for use in mycobacteria. In this study, we report a streamlined and efficient method for rapid genetic disruptions in M. abscessus. Harnessing the CRISPR1 loci from Streptococcus thermophilus, we have developed a dual-plasmid workflow that introduces Cas9 and sgRNA cassettes in separate steps but requires no other additional factors to engineer mutations in single genes or multiple genes simultaneously or sequentially using multiple targeting sgRNAs. Importantly, the efficiency of mutant generation is several orders of magnitude higher than reported for homologous recombination-based methods. This work, thus, reports the first application of CRISPR/Cas9 for gene editing in M. abscessus and is an important tool in the arsenal for the genetic manipulation of this human pathogen.<br><br>IMPORTANCE: Mycobacterium abscessus is an opportunistic pathogen of increasing clinical importance due to its poor clinical outcomes and limited treatment options. Drug discovery and development in this highly antibiotic-resistant species will require further understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods for facile genetic engineering are relatively inefficient. This study reports on the first application of CRISPR/Cas9 for gene editing in M. abscessus using a dual-plasmid workflow. We establish that our method is easily programmable, efficient, and versatile for genetic disruptions in M. abscessus. This is a critical advancement to facilitating targeted gene function studies in this emerging pathogen.},
}
@article {pmid38197228,
year = {2024},
author = {Manakova, E and Golovinas, E and Pocevičiūtė, R and Sasnauskas, G and Silanskas, A and Rutkauskas, D and Jankunec, M and Zagorskaitė, E and Jurgelaitis, E and Grybauskas, A and Venclovas, &#x10c; and Zaremba, M},
title = {The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.},
journal = {Nucleic acids research},
volume = {52},
number = {5},
pages = {2530-2545},
pmid = {38197228},
issn = {1362-4962},
support = {S-MIP-23-131//Research Council of Lithuania/ ; 653706//Horizon 2020/ ; //Lithuanian Research Library Consortium LMBA/ ; 01.2.2-CPVA-V-716-01-0001//Cryo-EM/ ; },
mesh = {*Archaeoglobus fulgidus/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Protein Domains ; Prokaryotic Cells/metabolism ; Bacteria/genetics ; Eukaryota/genetics ; Argonaute Proteins/metabolism ; },
abstract = {Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.},
}
@article {pmid38147372,
year = {2024},
author = {Li, H and Gong, P and Xu, X and Zhou, X and Li, F},
title = {Knockout of the virus replication-related genes UbEF1B and CCR4/NOT3 by CRISPR/Cas9 confers high-efficiency and broad-spectrum resistance to geminiviruses in Nicotiana benthamiana.},
journal = {Plant biotechnology journal},
volume = {22},
number = {4},
pages = {793-795},
doi = {10.1111/pbi.14265},
pmid = {38147372},
issn = {1467-7652},
support = {2021YFD1400400//National Key Research and Development Program of China/ ; 32172385//National Natural Science Foundation of China/ ; 31930089//National Natural Science Foundation of China/ ; },
mesh = {*Geminiviridae/genetics ; CRISPR-Cas Systems/genetics ; Nicotiana/genetics ; Gene Editing ; Virus Replication/genetics ; },
}
@article {pmid38085691,
year = {2024},
author = {Zobrist, JD and Lee, K and Wang, K},
title = {Application of CRISPR/Cas9 for targeted mutagenesis in teosinte Zea mays ssp. parviglumis.},
journal = {Plant biotechnology journal},
volume = {22},
number = {4},
pages = {796-798},
doi = {10.1111/pbi.14269},
pmid = {38085691},
issn = {1467-7652},
support = {1917138//National Science Foundation/ ; 2121410//National Science Foundation/ ; },
mesh = {*Zea mays/genetics ; *CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Mutagenesis/genetics ; },
}
@article {pmid38480006,
year = {2024},
author = {Kaur, N and Pati, PK},
title = {Retron Library Recombineering: Next Powerful Tool for Genome Editing after CRISPR/Cas.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00667},
pmid = {38480006},
issn = {2161-5063},
abstract = {Retron library recombineering (RLR) is a powerful tool in the field of genome editing that exceeds the scope and specificity of the CRISPR/Cas technique. In RLR, single-stranded DNA produced in vivo by harnessing the in-built potential of bacterial retrons is used for replication-dependent genome editing. RLR introduces several genomic variations at once, resulting in pooled and barcoded variant libraries, thus permitting multiplexed applications. Retron-generated RT-DNA has already shown promise for use in genome editing. Thus, this new tool will result in fresh, intriguing, and surprising developments in molecular biology and its juncture with other disciplines of research, including medicine, agriculture, and microbiology. In this review, we discuss the current state of this brand-new tool that could eventually boost genome editing research.},
}
@article {pmid38479094,
year = {2024},
author = {Chhipa, AS and Radadiya, E and Patel, S},
title = {CRISPR-Cas based diagnostic tools: Bringing diagnosis out of labs.},
journal = {Diagnostic microbiology and infectious disease},
volume = {109},
number = {2},
pages = {116252},
doi = {10.1016/j.diagmicrobio.2024.116252},
pmid = {38479094},
issn = {1879-0070},
abstract = {Timely detection is important for the effective management of infectious diseases. Reverse Transcription Polymerase Chain Reaction (RT-PCR) stands as the prime nucleic acid based test that is employed for the detection of infectious diseases. The method ensures sensitivity and specificity. However, RT-PCR is a relatively expensive technique due to the requirement of costly equipment and reagents. Further, it requires skilled personnel and established laboratories that are usually inaccessible in underdeveloped areas. On the other hand, rapid antigen based techniques are cost effective and easily accessible, but are less effective in terms of sensitivity and specificity. CRISPR-Cas systems are advanced diagnostic tools that combine the advantages of both PCR and antigen based detection techniques, and allows the rapid detection with high sensitivity/specificity. The present review aims to discuss the applicability of CRISPR-Cas based diagnostic tools for the infectious disease detection. The review further attempts to highlight the current limitations and future research directions to improve the CRISPR based diagnostic tools for rapid and effective disease detection.},
}
@article {pmid38478115,
year = {2024},
author = {Prasad, K and Gadeela, H and Bommineni, PR and Reddy, PS and Tyagi, W and Yogendra, K},
title = {CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.},
journal = {Functional &amp; integrative genomics},
volume = {24},
number = {2},
pages = {57},
pmid = {38478115},
issn = {1438-7948},
support = {SRG/2021/000422//Science and Engineering Research Board/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; Mutagenesis ; *Fabaceae/genetics ; Plants, Genetically Modified/genetics ; *Oxidoreductases ; },
abstract = {The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.},
}
@article {pmid38477342,
year = {2024},
author = {Sun, K and Pu, L and Chen, C and Chen, M and Li, K and Li, X and Li, H and Geng, J},
title = {An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae176},
pmid = {38477342},
issn = {1362-4962},
support = {2022YFB3205600//National Key Research and Development Program of China/ ; 2020YFS0579//Science &amp; Technology Department of Sichuan Province/ ; ZYYC23015//West China Hospital, Sichuan University/ ; 2023TQ0230//China Postdoctoral Science Foundation/ ; },
abstract = {CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.},
}
@article {pmid38474238,
year = {2024},
author = {Locubiche, S and Ordóñez, V and Abad, E and Scotto di Mase, M and Di Donato, V and De Santis, F},
title = {A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0.},
journal = {International journal of molecular sciences},
volume = {25},
number = {5},
pages = {},
pmid = {38474238},
issn = {1422-0067},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; Drug Evaluation, Preclinical ; *Epilepsy/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {The zebrafish model has emerged as a reference tool for phenotypic drug screening. An increasing number of molecules have been brought from bench to bedside thanks to zebrafish-based assays over the last decade. The high homology between the zebrafish and the human genomes facilitates the generation of zebrafish lines carrying loss-of-function mutations in disease-relevant genes; nonetheless, even using this alternative model, the establishment of isogenic mutant lines requires a long generation time and an elevated number of animals. In this study, we developed a zebrafish-based high-throughput platform for the generation of F0 knock-out (KO) models and the screening of neuroactive compounds. We show that the simultaneous inactivation of a reporter gene (tyrosinase) and a second gene of interest allows the phenotypic selection of F0 somatic mutants (crispants) carrying the highest rates of mutations in both loci. As a proof of principle, we targeted genes associated with neurodevelopmental disorders and we efficiently generated de facto F0 mutants in seven genes involved in childhood epilepsy. We employed a high-throughput multiparametric behavioral analysis to characterize the response of these KO models to an epileptogenic stimulus, making it possible to employ kinematic parameters to identify seizure-like events. The combination of these co-injection, screening and phenotyping methods allowed us to generate crispants recapitulating epilepsy features and to test the efficacy of compounds already during the first days post fertilization. Since the strategy can be applied to a wide range of indications, this study paves the ground for high-throughput drug discovery and promotes the use of zebrafish in personalized medicine and neurotoxicity assessment.},
}
@article {pmid38473916,
year = {2024},
author = {Lee, HJ and Kim, HJ and Cho, IS and Jeong, RD},
title = {Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.},
journal = {International journal of molecular sciences},
volume = {25},
number = {5},
pages = {},
pmid = {38473916},
issn = {1422-0067},
support = {PJ014947032023//Rural Development Administration/ ; },
mesh = {*Nanopore Sequencing ; CRISPR-Cas Systems ; *Amaryllidaceae ; Cross Reactions ; *Latent Infection ; *Orchidaceae ; Recombinases ; },
abstract = {Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/μL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.},
}
@article {pmid38473859,
year = {2024},
author = {Larriba, E and Yaroshko, O and Pérez-Pérez, JM},
title = {Recent Advances in Tomato Gene Editing.},
journal = {International journal of molecular sciences},
volume = {25},
number = {5},
pages = {},
pmid = {38473859},
issn = {1422-0067},
support = {TED2021-132256B-C22//Ministerio de Ciencia e Innovaci&#xf3;n/ ; AGROALNEXT/2022/036//Conselleria de Innovaci&#xf3;n, Universidades, Ciencia y Sociedad Digital/ ; },
mesh = {*Gene Editing/methods ; *Solanum lycopersicum ; Genome, Plant ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Plant Breeding ; },
abstract = {The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification of physiological, morphological, and other characteristics in a wide range of crops to mitigate the negative effects of stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have the potential to improve crop resilience and increase yields in response to challenging environmental conditions. This review provides an overview of gene-editing techniques used in plants, focusing on the cultivated tomatoes. Several dozen genes that have been successfully edited with the CRISPR/Cas system were selected for inclusion to illustrate the possibilities of this technology in improving fruit yield and quality, tolerance to pathogens, or responses to drought and soil salinity, among other factors. Examples are also given of how the domestication of wild species can be accelerated using CRISPR/Cas to generate new crops that are better adapted to the new climatic situation or suited to use in indoor agriculture.},
}
@article {pmid38473704,
year = {2024},
author = {Leal, AF and Herreno-Pachón, AM and Benincore-Flórez, E and Karunathilaka, A and Tomatsu, S},
title = {Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.},
journal = {International journal of molecular sciences},
volume = {25},
number = {5},
pages = {},
pmid = {38473704},
issn = {1422-0067},
support = {R01 HD102545/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; DNA End-Joining Repair ; Gene Editing ; Genetic Therapy ; Mammals/genetics ; },
abstract = {Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.},
}
@article {pmid38472540,
year = {2024},
author = {Kahraman-Ilıkkan, &#xd6;},
title = {Comparative genomics of four lactic acid bacteria identified with Vitek MS (MALDI-TOF) and whole-genome sequencing.},
journal = {Molecular genetics and genomics : MGG},
volume = {299},
number = {1},
pages = {31},
pmid = {38472540},
issn = {1617-4623},
mesh = {*Lactobacillales ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Bacteria ; Whole Genome Sequencing ; Genomics ; *Lactobacillus ; },
abstract = {Lactic acid bacteria (LAB) can be used as a probiotic or starter culture in dairy, meat, and vegetable fermentation. Therefore, their isolation and identification are essential. Recent advances in omics technologies and high-throughput sequencing have made the identification and characterization of bacteria. This study firstly aimed to demonstrate the sensitivity of the Vitek MS (MALDI-TOF) system in the identification of lactic acid bacteria and, secondly, to characterize bacteria using various bioinformatics approaches. Probiotic potency-related genes and secondary metabolite biosynthesis gene clusters were examined. The Vitek MS (MALDI-TOF) system was able to identify all of the bacteria at the genus level. According to whole genome sequencing, the bacteria were confirmed to be Lentilactobacillus buchneri, Levilactobacillus brevis, Lactiplantibacillus plantarum, Levilactobacillus namurensis. Bacteria had most of the probiotic potency-related genes, and different toxin-antitoxin systems such as PemIK/MazEF, Hig A/B, YdcE/YdcD, YefM/YoeB. Also, some of the secondary metabolite biosynthesis gene clusters, some toxic metabolite-related genes, and antibiotic resistance-related genes were detected. In addition, Lentilactobacillus buchneri Egmn17 had a type II-A CRISPR/Cas system. Lactiplantibacillus plantarum Gmze16 had a bacteriocin, plantaricin E/F.},
}
@article {pmid38472198,
year = {2024},
author = {Spisak, S and Chen, D and Likasitwatanakul, P and Doan, P and Li, Z and Bala, P and Vizkeleti, L and Tisza, V and De Silva, P and Giannakis, M and Wolpin, B and Qi, J and Sethi, NS},
title = {Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2230},
pmid = {38472198},
issn = {2041-1723},
support = {P30 CA006516/CA/NCI NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; Cell Differentiation/genetics ; Stem Cells/metabolism ; *Colorectal Neoplasms/genetics ; },
abstract = {Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators of these key cellular programs, we engineer a dual endogenous reporter system by genome-editing the SOX9 and KRT20 loci of human CRC cell lines to express fluorescent reporters, broadcasting aberrant stem cell-like and differentiation activity, respectively. By applying a CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, we identify factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominate SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency and required for in vivo growth of human CRC models. These studies highlight the utility of biologically designed endogenous reporter platforms to uncover regulators with therapeutic potential.},
}
@article {pmid38472096,
year = {2024},
author = {Tian, Z and Yan, H and Zeng, Y},
title = {Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a.},
journal = {ACS applied materials &amp; interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.3c17039},
pmid = {38472096},
issn = {1944-8252},
abstract = {Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans-cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.},
}
@article {pmid38437635,
year = {2024},
author = {Kong, W and Li, X and Guo, X and Sun, Y and Chai, W and Chang, Y and Huang, Q and Wang, P and Wang, X},
title = {Ultrasound-Assisted CRISPRi-Exosome for Epigenetic Modification of α-Synuclein Gene in a Mouse Model of Parkinson's Disease.},
journal = {ACS nano},
volume = {18},
number = {11},
pages = {7837-7851},
doi = {10.1021/acsnano.3c05864},
pmid = {38437635},
issn = {1936-086X},
mesh = {Humans ; Mice ; Animals ; *Parkinson Disease/diagnostic imaging/genetics/metabolism ; alpha-Synuclein/genetics/metabolism ; Epigenesis, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Exosomes/metabolism ; },
abstract = {Currently, there is a lack of effective treatment for Parkinson's disease (PD). In PD patients, aberrant methylation of SNCA (α-synuclein gene) has been reported and may be a potential therapeutic target. In this study, we established an epigenetic regulation platform based on an exosomal CRISPR intervention system. With the assist of focused ultrasound (FUS) opening the blood-brain barrier, engineered exosomes carrying RVG (rabies viral glycoprotein) targeting peptide, sgRNA (single guide RNA), and dCas9-DNMT3A (named RVG-CRISPRi-Exo) were efficiently delivered into the brain lesions and induced specific methylation of SNCA. In vivo, FUS combined with RVG-CRISPRi-Exo significantly improved motor performance, balance coordination, and neurosensitivity in PD mice, greatly down-regulated the elevation of α-synuclein (α-syn) caused by modeling, rescued cell apoptosis, and alleviated the progression of PD in mice. [[18]F]-FP-DTBZ imaging suggested that the synaptic function of the nigrostriatal pathway could be restored, which was conducive to the control of motor behavior in PD mice. Pyrosequencing results showed that RVG-CRISPRi-Exo could methylate CpG at specific sites of SNCA, and this fine-tuned editing achieved good therapeutic effects in PD model mice. In vitro, RVG-CRISPRi-Exo down-regulated SNCA transcripts and α-syn expression and relieved neuronal cell damage. Collectively, our findings provide a proof-of-principle for the development of targeted brain nanodelivery based on engineered exosomes and provide insights into epigenetic regulation of brain diseases.},
}
@article {pmid38401123,
year = {2024},
author = {Navarro-Guerrero, E and Baronio, R and Tay, C and Knight, JC and Ebner, DV},
title = {Optimized protocol for CRISPR knockout of human iPSC-derived macrophages.},
journal = {STAR protocols},
volume = {5},
number = {1},
pages = {102903},
pmid = {38401123},
issn = {2666-1667},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; *Neurodegenerative Diseases ; Gene Editing/methods ; Macrophages ; },
abstract = {Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression. For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al.[1].},
}
@article {pmid38354082,
year = {2024},
author = {Ittiprasert, W and Moescheid, MM and Mann, VH and Brindley, PJ},
title = {Multiplexed CRISPR-Cas9 protocol for large transgene integration into the Schistosoma mansoni genome.},
journal = {STAR protocols},
volume = {5},
number = {1},
pages = {102886},
pmid = {38354082},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Schistosoma mansoni/genetics ; Gene Editing/methods ; Genome ; Transgenes/genetics ; },
abstract = {Precise, on-target CRISPR-Cas9 genome editing has been shown in Schistosoma mansoni, involving both non-homology end joining and homology-directed repair pathways. Here, we present a multiplexed CRISPR-Cas9 protocol for large transgene integration into the S. mansoni genome. We describe steps for deploying multiplexed ribonucleoprotein complexes (RNPs) and donor DNA preparation. We then detail procedures for introducing RNPs into schistosome eggs by square-wave electroporation in the presence of a 5' phosphorothioate-modified double-stranded donor transgene. For complete details on the use and execution of this protocol, please refer to Ittiprasert et al. (2023).[1].},
}
@article {pmid38471818,
year = {2024},
author = {Grüschow, S and McQuarrie, S and Ackermann, K and McMahon, S and Bode, BE and Gloster, TM and White, MF},
title = {CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae167},
pmid = {38471818},
issn = {1362-4962},
support = {BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.},
}
@article {pmid38454603,
year = {2024},
author = {Böck, D and Revers, IM and Bomhof, ASJ and Hillen, AEJ and Boeijink, C and Kissling, L and Egli, S and Moreno-Mateos, MA and van der Knaap, MS and van Til, NP and Schwank, G},
title = {In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2024.03.009},
pmid = {38454603},
issn = {1525-0024},
abstract = {Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5[R191H] variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.},
}
@article {pmid38452570,
year = {2024},
author = {Li, X and Ma, Y and He, M and Tan, B and Wang, G and Zhu, G},
title = {A novel fluorescent aptasensor for sensitive and selective detection of environmental toxins fumonisin B1 based on enzyme-assisted dual recycling amplification and 2D δ-FeOOH-NH2 nanosheets.},
journal = {Biosensors &amp; bioelectronics},
volume = {253},
number = {},
pages = {116183},
doi = {10.1016/j.bios.2024.116183},
pmid = {38452570},
issn = {1873-4235},
mesh = {Humans ; *Fumonisins ; *Biosensing Techniques ; DNA, Single-Stranded ; Fluorescent Dyes ; CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Fumonisin (FB) is a pervasive hazardous substance in the environment, presenting significant threats to human health and ecological systems. Thus, the selective and sensitive detection of fumonisin B1 (FB1) is crucial due to its high toxicity and wide distribution in corn, oats, and related products. In this work, we developed a novel and versatile fluorescent aptasensor by combining enzyme-assisted dual recycling amplification with 2D δ-FeOOH-NH2 nanosheets for the determination of FB1. The established CRISPR/Cas12a system was activated by using activator DNA (aDNA), which was released via a T7 exonuclease-assisted recycling reaction. Additionally, the activated Cas12a protein was utilized for non-specifically cleavage of the FAM-labeled single-stranded DNA (ssDNA-FAM) anchored on δ-FeOOH-NH2 nanosheets. The pre-quenched fluorescence signal was restored due to the desorption of the cleaved ssDNA-FAM. Due to the utilization of this T7 exonuclease-Cas12a-δ-FeOOH-NH2 aptasensor for signal amplification, the detection range of FB1 was expanded from 1 pg/mL to 100 ng/mL, with a limit of detection (LOD) as low as 0.45 pg/mL. This study not only provides novel insights into the development of fluorescence biosensors based on 2D nanomaterials combined with CRISPR/Cas12a, but also exhibits remarkable applicability in detecting other significant targets.},
}
@article {pmid38325641,
year = {2024},
author = {Torres, M and Mcconnaughie, D and Akhtar, S and Gaffney, CE and Fievet, B and Ingham, C and Stockdale, M and Dickson, AJ},
title = {Engineering mammalian cell growth dynamics for biomanufacturing.},
journal = {Metabolic engineering},
volume = {82},
number = {},
pages = {89-99},
doi = {10.1016/j.ymben.2024.01.006},
pmid = {38325641},
issn = {1096-7184},
mesh = {Cell Division ; *Gene Regulatory Networks ; *Biological Products ; CRISPR-Cas Systems ; Genetic Engineering/methods ; Cell Engineering ; },
abstract = {Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a "gas pedal", based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a "brake pedal". A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the "gas and brake pedals", enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.},
}
@article {pmid38471546,
year = {2024},
author = {Gandon, S and Guillemet, M and Gatchitch, F and Nicot, A and Renaud, AC and Tremblay, DM and Moineau, S},
title = {Building pyramids against the evolutionary emergence of pathogens.},
journal = {Proceedings. Biological sciences},
volume = {291},
number = {2018},
pages = {20231529},
pmid = {38471546},
issn = {1471-2954},
mesh = {Humans ; Bacteria/genetics ; *Bacteriophages ; *Communicable Diseases ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Mutations allowing pathogens to escape host immunity promote the spread of infectious diseases in heterogeneous host populations and can lead to major epidemics. Understanding the conditions that slow down this evolution is key for the development of durable control strategies against pathogens. Here, we use theory and experiments to compare the efficacy of three strategies for the deployment of resistance: (i) a mixing strategy where the host population contains two single-resistant genotypes, (ii) a pyramiding strategy where the host carries a double-resistant genotype, (iii) a combining strategy where the host population is a mix of a single-resistant genotype and a double-resistant genotype. First, we use evolutionary epidemiology theory to clarify the interplay between demographic stochasticity and evolutionary dynamics to show that the pyramiding strategy always yields lower probability of evolutionary emergence. Second, we test experimentally these predictions with the introduction of bacteriophages into bacterial populations where we manipulated the diversity and the depth of immunity using a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) system. These biological assays confirm that pyramiding multiple defences into the same host genotype and avoiding combination with single-defence genotypes is a robust way to reduce pathogen evolutionary emergence. The experimental validation of these theoretical recommendations has practical implications in various areas, including for the optimal deployment of resistance varieties in agriculture and for the design of durable vaccination strategies.},
}
@article {pmid38469793,
year = {2024},
author = {Ebstein, F and Latypova, X and Hung, KYS and Prado, MA and Lee, BH and Möller, S and Wendlandt, M and Zieba, BA and Florenceau, L and Vignard, V and Poirier, L and Toutain, B and Moroni, I and Dubucs, C and Chassaing, N and Horvath, J and Prokisch, H and Küry, S and Bézieau, S and Paulo, JA and Finley, D and Krüger, E and Ghezzi, D and Isidor, B},
title = {Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.},
journal = {Genetics in medicine : official journal of the American College of Medical Genetics},
volume = {},
number = {},
pages = {101120},
doi = {10.1016/j.gim.2024.101120},
pmid = {38469793},
issn = {1530-0366},
abstract = {PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy.<br><br>METHODS: Here, we identified biallelic USP14 variants in four individuals from three unrelated families: one fetus, a newborn with a syndromic NDD, and two siblings affected by a progressive neurological disease. Specifically, the two siblings from the latter family carried two compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330*), while the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs*24) variant and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs*11) variant. Functional studies were conducted using SDS-PAGE, Western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas-generated cells.<br><br>RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities.<br><br>CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.},
}
@article {pmid38468277,
year = {2024},
author = {Ponta, S and Bonato, A and Neidenbach, P and Bruhin, VF and Laurent, A and Applegate, LA and Zenobi-Wong, M and Barreto, G},
title = {Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations.},
journal = {Arthritis research &amp; therapy},
volume = {26},
number = {1},
pages = {66},
pmid = {38468277},
issn = {1478-6362},
support = {00220103//Suomen Kulttuurirahasto/ ; },
mesh = {Humans ; Animals ; Cattle ; Chondrocytes/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Interleukin-1beta/metabolism ; *Cartilage Diseases/metabolism ; *Polydactyly/metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources.<br><br>METHODS: We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-&#x3ba;B), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1&#x3b2; (IL-1&#x3b2;).<br><br>RESULTS: We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of&#xa0;~90%, confirming lower inflammatory pathways activation upon IL-1&#x3b2; stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions.<br><br>CONCLUSIONS: We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.},
}
@article {pmid38468094,
year = {2024},
author = {Ravagnan, G and Meliawati, M and Schmid, J},
title = {CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2760},
number = {},
pages = {267-280},
pmid = {38468094},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Paenibacillus polymyxa/genetics ; Genome ; },
abstract = {In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.},
}
@article {pmid38468089,
year = {2024},
author = {Ramesh, A and Lee, S and Wheeldon, I},
title = {Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2760},
number = {},
pages = {169-198},
pmid = {38468089},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Yarrowia/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Endonucleases/genetics ; Genetic Testing ; },
abstract = {Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.},
}
@article {pmid38468088,
year = {2024},
author = {Gao, J and Cheng, J and Lian, J},
title = {Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2760},
number = {},
pages = {157-167},
pmid = {38468088},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Methanol/metabolism ; Pichia/genetics/metabolism ; *Saccharomycetales/metabolism ; Butylene Glycols/metabolism ; },
abstract = {Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.},
}
@article {pmid38447574,
year = {2024},
author = {Noguchi, Y and Onodera, Y and Miyamoto, T and Maruoka, M and Kosako, H and Suzuki, J},
title = {In vivo CRISPR screening directly targeting testicular cells.},
journal = {Cell genomics},
volume = {4},
number = {3},
pages = {100510},
pmid = {38447574},
issn = {2666-979X},
mesh = {Male ; Humans ; *RNA, Guide, CRISPR-Cas Systems ; *Semen ; Testis ; Spermatids ; Spermatogenesis/genetics ; },
abstract = {CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.},
}
@article {pmid38445989,
year = {2024},
author = {Arévalo, S and Pérez Rico, D and Abarca, D and Dijkhuizen, LW and Sarasa-Buisan, C and Lindblad, P and Flores, E and Nierzwicki-Bauer, S and Schluepmann, H},
title = {Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.},
journal = {ACS synthetic biology},
volume = {13},
number = {3},
pages = {901-912},
doi = {10.1021/acssynbio.3c00583},
pmid = {38445989},
issn = {2161-5063},
mesh = {Humans ; RNA, Guide, CRISPR-Cas Systems ; RNA ; Plasmids/genetics ; *Anabaena/genetics ; *Cyanobacteria/genetics ; DNA ; Escherichia coli/genetics ; DNA Transposable Elements/genetics ; },
abstract = {In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.},
}
@article {pmid38428132,
year = {2024},
author = {Shi, J and Li, S and Shao, R and Jiang, Y and Qiao, Y and Liu, J and Zhou, Y and Li, Y},
title = {Electrochemiluminescence aptasensing method for ultrasensitive determination of lipopolysaccharide based on CRISPR-Cas12a accessory cleavage activity.},
journal = {Talanta},
volume = {272},
number = {},
pages = {125828},
doi = {10.1016/j.talanta.2024.125828},
pmid = {38428132},
issn = {1873-3573},
mesh = {Humans ; Lipopolysaccharides ; DNA, Single-Stranded ; Gold ; *Metal Nanoparticles ; CRISPR-Cas Systems ; Luminescent Measurements/methods ; Sulfhydryl Compounds ; *Biosensing Techniques/methods ; Electrochemical Techniques/methods ; *Fluorocarbon Polymers ; },
abstract = {In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)3[2+]) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)3[2+]/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)3[2+]/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)3[2+]/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)3[2+] due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.},
}
@article {pmid38374729,
year = {2024},
author = {De Marchi, D and Shaposhnikov, R and Gobaa, S and Pastorelli, D and Batt, G and Magni, P and Pasotti, L},
title = {Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria.},
journal = {ACS synthetic biology},
volume = {13},
number = {3},
pages = {763-780},
doi = {10.1021/acssynbio.3c00541},
pmid = {38374729},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Staphylococcus aureus/genetics ; Escherichia coli/genetics ; Promoter Regions, Genetic ; },
abstract = {Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up circuit complexity, the availability of parts toolkits is central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) is widely adopted for implementing programmable regulations in synthetic circuits, and alternative CRISPRi systems will further expand our toolkits of orthogonal components. Here, we showcase the potential of CRISPRi using the engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used in bacterial circuits, that is attractive for its low size and high specificity. We designed a collection of ∼20 increasingly complex circuits and variants in Escherichia coli, including circuits with static function like one-/two-input logic gates (NOT, NAND), circuits with dynamic behavior like incoherent feedforward loops (iFFLs), and applied sadCas9 to fix a T7 polymerase-based cascade. Data demonstrated specific and efficient target repression (100-fold) and qualitatively successful functioning for all circuits. Other advantageous features included low sadCas9-borne cell load and orthogonality with spdCas9. However, different circuit variants showed quantitatively unexpected and previously unreported steady-state responses: the dynamic range, switch point, and slope of NOT/NAND gates changed for different output promoters, and a multiphasic behavior was observed in iFFLs, differing from the expected bell-shaped or sigmoidal curves. Model analysis explained the observed curves by complex interplays among components, due to reporter gene-borne cell load and regulator competition. Overall, CRISPRi/sadCas9 successfully expanded the available toolkit for bacterial engineering. Analysis of our circuit collection depicted the impact of generally neglected effects modulating the shape of component dose-response curves, to avoid drawing wrong conclusions on circuit functioning.},
}
@article {pmid38350870,
year = {2024},
author = {Wang, Z and Pan, H and Ni, S and Li, Z and Lian, J},
title = {Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator.},
journal = {ACS synthetic biology},
volume = {13},
number = {3},
pages = {851-861},
doi = {10.1021/acssynbio.3c00664},
pmid = {38350870},
issn = {2161-5063},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Cupriavidus necator/genetics/metabolism ; Gene Expression ; Carbon/metabolism ; Evolution, Molecular ; },
abstract = {Cupriavidus necator H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.},
}
@article {pmid38330875,
year = {2024},
author = {Zeng, H and Daniel, TC and Lingineni, A and Chee, K and Talloo, K and Gao, X},
title = {Recent advances in prime editing technologies and their promises for therapeutic applications.},
journal = {Current opinion in biotechnology},
volume = {86},
number = {},
pages = {103071},
pmid = {38330875},
issn = {1879-0429},
support = {R01 HL157714/HL/NHLBI NIH HHS/United States ; R35 GM138207/GM/NIGMS NIH HHS/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Technology ; Epigenomics ; Gene Editing ; Protein Engineering ; CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) is a groundbreaking genome editing technology offering unparalleled precision in targeted genome modifications and has great potential for therapeutic applications. This review delves into the core principles of PE and emphasizes its advancements, applications, and prospects. We begin with a brief introduction to PE principles, followed by a detailed examination of recent improvements in efficiency, precision, and the scale of feasible edits. These improvements have been made to the PE systems through guide RNA engineering, protein engineering, DNA repair pathway screening, chromosomal or epigenomic modification, and in silico design and optimization tools. Furthermore, we highlight in vivo studies showcasing the therapeutic potential of PE to model and treat genetic diseases. Moreover, we discuss PE's versatile applications in saturation genome editing and its applicability to nonhuman organisms. In conclusion, we address the challenges and opportunities linked with PE, emphasizing its profound impact on biological research and therapeutics.},
}
@article {pmid38310724,
year = {2024},
author = {Kaul, T and Thangaraj, A and Jain, R and Bharti, J and Kaul, R and Verma, R and Sony, SK and Abdel Motelb, KF and Yadav, P and Agrawal, PK},
title = {CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.).},
journal = {Plant physiology and biochemistry : PPB},
volume = {207},
number = {},
pages = {108374},
doi = {10.1016/j.plaphy.2024.108374},
pmid = {38310724},
issn = {1873-2690},
mesh = {*Zea mays/genetics ; Gene Editing ; CRISPR-Cas Systems ; Herbicide Resistance/genetics ; Glyphosate ; *Herbicides/pharmacology ; Amino Acids, Aromatic/genetics ; },
abstract = {Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-mZmEPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.},
}
@article {pmid38104868,
year = {2024},
author = {Ran, X and Wu, BX and Shi, M and Song, L and Nixon, K and Philip, V and He, HH and Tsao, MS and Lok, BH},
title = {CRISPR Screen of Druggable Targets in Small Cell Lung Cancer Identified ATM Inhibitor (AZD1390) as a Radiosensitizer.},
journal = {International journal of radiation oncology, biology, physics},
volume = {118},
number = {5},
pages = {1308-1314},
doi = {10.1016/j.ijrobp.2023.12.011},
pmid = {38104868},
issn = {1879-355X},
mesh = {Humans ; Animals ; Mice ; *Small Cell Lung Carcinoma/genetics/radiotherapy/drug therapy ; *Lung Neoplasms/genetics/radiotherapy/drug therapy ; RNA, Guide, CRISPR-Cas Systems ; *Radiation-Sensitizing Agents/pharmacology/therapeutic use ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; Ataxia Telangiectasia Mutated Proteins/metabolism ; *Pyridines ; *Quinolones ; },
abstract = {PURPOSE: Small cell lung cancer (SCLC) is an aggressive and lethal form of lung cancer and the overall 5-year survival (OS) for patients is a dismal 7%. Radiation therapy (RT) provides some benefit for selected patients with SCLC but could be improved with radiosensitizing agents. In this study, we identified novel radiosensitizers for SCLC by a CRISPR-Cas9 screen and evaluated the efficacy of ATM inhibitor AZD1390 as a radiosensitizer of SCLC.<br><br>METHODS AND MATERIALS: We transduced the SCLC cell line SBC5 with a custom CRISPR sgRNA library focused on druggable gene targets and treated cells with RT. Cells collected at multiple timepoints were subjected to next-generation sequencing. We determined radiosensitization both in vitro with cell lines assessed by short-term viability and clonogenic assays, and in vivo mouse models by tumor growth delay. Pharmacodynamic effects of AZD1390 were quantified by ATM-Ser1981 phosphorylation, and RT-induced DNA damage by comet assay.<br><br>RESULTS: Using a CRISPR dropout screen, we identified multiple radiosensitizing genes for SCLC at various timepoints with ATM as a top determinant gene for radiosensitivity. Validation by ATM knockout (KO) demonstrated increased radiosensitivity by short-term viability assay (dose modification factor [DMF]50 = 3.25-3.73 in SBC5 ATM-KO) and clonogenic assays (DMF37 1.25-1.65 in SBC5 ATM-KO). ATM inhibition by AZD1390 effectively abrogated ATM Ser1981 phosphorylation in SCLC cell lines and increased RT-induced DNA damage. AZD1390 synergistically increased the radiosensitivity of SCLC cell lines (cell viability assay: SBC5 DMF37 = 2.19, SHP77 DMF37 = 1.56, H446 DMF37 = 3.27, KP1 DMF37 = 1.65 at 100nM; clonogenic assay: SBC5 DMF37 = 4.23, H1048 DMF37 = 1.91), and in vivo murine syngeneic, KP1, and patient-derived xenograft (PDX) models, JHU-LX108 and JHU-LX33.<br><br>CONCLUSIONS: In this study, we demonstrated that genetically and pharmacologically (AZD1390) inhibiting ATM markedly enhanced RT against SCLC, providing a novel pharmacologically tractable radiosensitizing strategy for patients with SCLC.},
}
@article {pmid38055160,
year = {2024},
author = {Vlashi, R and Zhang, X and Li, H and Chen, G},
title = {Potential therapeutic strategies for osteoarthritis via CRISPR/Cas9 mediated gene editing.},
journal = {Reviews in endocrine &amp; metabolic disorders},
volume = {25},
number = {2},
pages = {339-367},
pmid = {38055160},
issn = {1573-2606},
support = {2023AY11045, 2020AY10001//Jiaxing Science Technology Foundation/ ; 202002N3150, 2022S035//Social Development Public Welfare Foundation of Ningbo/ ; LZ23H140001//Zhejiang Provincial Natural Science Foundation of China/ ; 21040040-E//Zhejiang Qianjiang Talent Program/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; DNA ; },
abstract = {Osteoarthritis (OA) is an incapacitating and one of the most common physically degenerative conditions with an assorted etiology and a highly complicated molecular mechanism that to date lacks an efficient treatment. The capacity to design biological networks and accurately modify existing genomic sites holds an apt potential for applications across medical and biotechnological sciences. One of these highly specific genomes editing technologies is the CRISPR/Cas9 mechanism, referred to as the clustered regularly interspaced short palindromic repeats, which is a defense mechanism constituted by CRISPR associated protein 9 (Cas9) directed by small non-coding RNAs (sncRNA) that bind to target DNA through Watson-Crick base pairing rules where subsequent repair of the target DNA is initiated. Up-to-date research has established the effectiveness of the CRISPR/Cas9 mechanism in targeting the genetic and epigenetic alterations in OA by suppressing or deleting gene expressions and eventually distributing distinctive anti-arthritic properties in both in vitro and in vivo osteoarthritic models. This review aims to epitomize the role of this high-throughput and multiplexed gene editing method as an analogous therapeutic strategy that could greatly facilitate the clinical development of OA-related treatments since it's reportedly an easy, minimally invasive technique, and a comparatively less painful method for osteoarthritic patients.},
}
@article {pmid37696982,
year = {2024},
author = {Nouraein, S and Lee, S and Saenz, VA and Del Mundo, HC and Yiu, J and Szablowski, JO},
title = {Acoustically targeted noninvasive gene therapy in large brain volumes.},
journal = {Gene therapy},
volume = {31},
number = {3-4},
pages = {85-94},
pmid = {37696982},
issn = {1476-5462},
support = {C-2048-20200401//Welch Foundation/ ; },
mesh = {Animals ; *Genetic Therapy ; *Gene Editing ; Gene Transfer Techniques ; Blood-Brain Barrier ; Brain ; CRISPR-Cas Systems ; },
abstract = {Focused Ultrasound Blood-Brain Barrier Opening (FUS-BBBO) can deliver adeno-associated viral vectors (AAVs) to treat genetic disorders of the brain. However, such disorders often affect large brain regions. Moreover, the applicability of FUS-BBBO in the treatment of brain-wide genetic disorders has not yet been evaluated. Herein, we evaluated the transduction efficiency and safety of opening up to 105 sites simultaneously. Increasing the number of targeted sites increased gene delivery efficiency at each site. We achieved transduction of up to 60% of brain cells with comparable efficiency in the majority of the brain regions. Furthermore, gene delivery with FUS-BBBO was safe even when all 105 sites were targeted simultaneously without negative effects on animal weight or neuronal loss. To evaluate the application of multi-site FUS-BBBO for gene therapy, we used it for gene editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system and found effective gene editing, but also a loss of neurons at the targeted sites. Overall, this study provides a brain-wide map of transduction efficiency, shows the synergistic effect of multi-site targeting on transduction efficiency, and is the first example of large brain volume gene editing after noninvasive gene delivery with FUS-BBBO.},
}
@article {pmid37563300,
year = {2024},
author = {Katti, A and Vega-Pérez, A and Foronda, M and Zimmerman, J and Zafra, MP and Granowsky, E and Goswami, S and Gardner, EE and Diaz, BJ and Simon, JM and Wuest, A and Luan, W and Fernandez, MTC and Kadina, AP and Walker, JA and Holden, K and Lowe, SW and Sánchez Rivera, FJ and Dow, LE},
title = {Generation of precision preclinical cancer models using regulated in vivo base editing.},
journal = {Nature biotechnology},
volume = {42},
number = {3},
pages = {437-447},
pmid = {37563300},
issn = {1546-1696},
support = {R01 CA233944/CA/NCI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Gene Editing ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Neoplasms/genetics/therapy ; Lung ; },
abstract = {Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.},
}
@article {pmid37558852,
year = {2024},
author = {Klinnert, S and Schenkel, CD and Freitag, PC and Günthard, HF and Plückthun, A and Metzner, KJ},
title = {Targeted shock-and-kill HIV-1 gene therapy approach combining CRISPR activation, suicide gene tBid and retargeted adenovirus delivery.},
journal = {Gene therapy},
volume = {31},
number = {3-4},
pages = {74-84},
pmid = {37558852},
issn = {1476-5462},
support = {00408//Gilead Sciences (Gilead)/ ; FN20-0000000206//Novartis Foundation/ ; },
mesh = {Humans ; *HIV-1/genetics ; *HIV Infections/genetics ; Virus Activation/genetics ; Virus Latency/genetics ; Adenoviridae/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; Proviruses/genetics ; Genetic Therapy ; CD4-Positive T-Lymphocytes/metabolism ; },
abstract = {Infections with the human immunodeficiency virus type 1 (HIV-1) are incurable due the long-lasting, latent viral reservoir. The shock-and-kill cure approach aims to activate latent proviruses in HIV-1 infected cells and subsequently kill these cells with strategies such as therapeutic vaccines or immune enhancement. Here, we combined the dCas9-VPR CRISPR activation (CRISPRa) system with gRNA-V, the truncated Bid (tBid)-based suicide gene strategy and CD3-retargeted adenovirus (Ad) delivery vectors, in an all-in-one targeted shock-and-kill gene therapy approach to achieve specific elimination of latently HIV-1 infected cells. Simultaneous transduction of latently HIV-1 infected J-Lat 10.6 cells with a CD3-retargeted Ad-CRISPRa-V and Ad-tBid led to a 57.7 ± 17.0% reduction of productively HIV-1 infected cells and 2.4-fold ± 0.25 increase in cell death. The effective activation of latent HIV-1 provirus by Ad-CRISPRa-V was similar to the activation control TNF-α. The strictly HIV-1 dependent and non-leaky killing by tBid could be demonstrated. Furthermore, the high transduction efficiencies of up to 70.8 ± 0.4% by the CD3-retargeting technology in HIV-1 latently infected cell lines was the basis of successful shock-and-kill. This novel targeted shock-and-kill all-in-one gene therapy approach has the potential to safely and effectively eliminate HIV-1 infected cells in a highly HIV-1 and T cell specific manner.},
}
@article {pmid37217753,
year = {2024},
author = {},
title = {CRISPR-free, strand-selective mitochondrial DNA base editing using a nickase.},
journal = {Nature biotechnology},
volume = {42},
number = {3},
pages = {392-393},
pmid = {37217753},
issn = {1546-1696},
mesh = {*Gene Editing ; *DNA, Mitochondrial/genetics ; Deoxyribonuclease I/genetics ; CRISPR-Cas Systems/genetics ; Mitochondria/genetics ; },
}
@article {pmid37217751,
year = {2024},
author = {Yi, Z and Zhang, X and Tang, W and Yu, Y and Wei, X and Zhang, X and Wei, W},
title = {Strand-selective base editing of human mitochondrial DNA using mitoBEs.},
journal = {Nature biotechnology},
volume = {42},
number = {3},
pages = {498-509},
pmid = {37217751},
issn = {1546-1696},
mesh = {Humans ; *Gene Editing/methods ; DNA, Mitochondrial/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Mitochondria/genetics ; *Mitochondrial Diseases/genetics/therapy ; Deoxyribonuclease I/genetics ; Cytosine ; },
abstract = {A number of mitochondrial diseases in humans are caused by point mutations that could be corrected by base editors, but delivery of CRISPR guide RNAs into the mitochondria is difficult. In this study, we present mitochondrial DNA base editors (mitoBEs), which combine a transcription activator-like effector (TALE)-fused nickase and a deaminase for precise base editing in mitochondrial DNA. Combining mitochondria-localized, programmable TALE binding proteins with the nickase MutH or Nt.BspD6I(C) and either the single-stranded DNA-specific adenine deaminase TadA8e or the cytosine deaminase ABOBEC1 and UGI, we achieve A-to-G or C-to-T base editing with up to 77% efficiency and high specificity. We find that mitoBEs are DNA strand-selective mitochondrial base editors, with editing results more likely to be retained on the nonnicked DNA strand. Furthermore, we correct pathogenic mitochondrial DNA mutations in patient-derived cells by delivering mitoBEs encoded in circular RNAs. mitoBEs offer a precise, efficient DNA editing tool with broad applicability for therapy in mitochondrial genetic diseases.},
}
@article {pmid37188916,
year = {2024},
author = {Kim, N and Choi, S and Kim, S and Song, M and Seo, JH and Min, S and Park, J and Cho, SR and Kim, HH},
title = {Deep learning models to predict the editing efficiencies and outcomes of diverse base editors.},
journal = {Nature biotechnology},
volume = {42},
number = {3},
pages = {484-497},
pmid = {37188916},
issn = {1546-1696},
support = {2022R1A3B1078084//National Research Foundation of Korea (NRF)/ ; 2018R1A5A2025079//National Research Foundation of Korea (NRF)/ ; 2022M3A9E4017127//National Research Foundation of Korea (NRF)/ ; 2022M3A9F3017506//National Research Foundation of Korea (NRF)/ ; 2022R1C1C2004229//National Research Foundation of Korea (NRF)/ ; HN21C0917 (H.H.K.))//Ministry of Trade, Industry and Energy (Ministry of Trade, Industry and Energy, Korea)/ ; HI21C1314//Ministry of Health and Welfare (Ministry of Health, Welfare and Family Affairs)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Deep Learning ; *Alkanesulfonic Acids ; },
abstract = {Applications of base editing are frequently restricted by the requirement for a protospacer adjacent motif (PAM), and selecting the optimal base editor (BE) and single-guide RNA pair (sgRNA) for a given target can be difficult. To select for BEs and sgRNAs without extensive experimental work, we systematically compared the editing windows, outcomes and preferred motifs for seven BEs, including two cytosine BEs, two adenine BEs and three C•G to G•C BEs at thousands of target sequences. We also evaluated nine Cas9 variants that recognize different PAM sequences and developed a deep learning model, DeepCas9variants, for predicting which variants function most efficiently at sites with a given target sequence. We then develop a computational model, DeepBE, that predicts editing efficiencies and outcomes of 63 BEs that were generated by incorporating nine Cas9 variants as nickase domains into the seven BE variants. The predicted median efficiencies of BEs with DeepBE-based design were 2.9- to 20-fold higher than those of rationally designed SpCas9-containing BEs.},
}
@article {pmid37169967,
year = {2024},
author = {Ely, ZA and Mathey-Andrews, N and Naranjo, S and Gould, SI and Mercer, KL and Newby, GA and Cabana, CM and Rideout, WM and Jaramillo, GC and Khirallah, JM and Holland, K and Randolph, PB and Freed-Pastor, WA and Davis, JR and Kulstad, Z and Westcott, PMK and Lin, L and Anzalone, AV and Horton, BL and Pattada, NB and Shanahan, SL and Ye, Z and Spranger, S and Xu, Q and Sánchez-Rivera, FJ and Liu, DR and Jacks, T},
title = {A prime editor mouse to model a broad spectrum of somatic mutations in vivo.},
journal = {Nature biotechnology},
volume = {42},
number = {3},
pages = {424-436},
pmid = {37169967},
issn = {1546-1696},
support = {K99 HL163805/HL/NHLBI NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Mice ; Humans ; Animals ; Mice, Transgenic ; *RNA, Guide, CRISPR-Cas Systems ; Mutation/genetics ; *Pancreatic Neoplasms/genetics ; Cell Line ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
abstract = {Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR-Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models.},
}
@article {pmid37127662,
year = {2024},
author = {Allen, AG and Khan, SQ and Margulies, CM and Viswanathan, R and Lele, S and Blaha, L and Scott, SN and Izzo, KM and Gerew, A and Pattali, R and Cochran, NR and Holland, CS and Zhao, AH and Sherman, SE and Jaskolka, MC and Wu, M and Wilson, AC and Sun, X and Ciulla, DM and Zhang, D and Nelson, JD and Zhang, P and Mazzucato, P and Huang, Y and Giannoukos, G and Marco, E and Nehil, M and Follit, JA and Chang, KH and Shearman, MS and Wilson, CJ and Zuris, JA},
title = {A highly efficient transgene knock-in technology in clinically relevant cell types.},
journal = {Nature biotechnology},
volume = {42},
number = {3},
pages = {458-469},
pmid = {37127662},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques ; Transgenes/genetics ; *Gene Editing ; },
abstract = {Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.},
}
@article {pmid38468087,
year = {2024},
author = {Lee, HJ and Lee, SJ},
title = {Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2760},
number = {},
pages = {147-155},
pmid = {38468087},
issn = {1940-6029},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Nucleotides ; Escherichia coli/genetics ; Genome, Microbial ; DNA ; },
abstract = {Microbial genome editing can be achieved by donor DNA-directed mutagenesis and CRISPR-Cas12a-mediated negative selection. Single-nucleotide-level genome editing enables the manipulation of microbial cells exactly as designed. Here, we describe single-nucleotide substitutions/indels in the target DNA of E. coli genome using a mutagenic DNA oligonucleotide donor and truncated crRNA/Cas12a system. The maximal truncation of nucleotides at the 3'-end of the crRNA enables Cas12a-mediated single-nucleotide-level precise editing at galK targets in the genome of E. coli.},
}
@article {pmid38468085,
year = {2024},
author = {Kang, M and Kim, K and Cho, BK},
title = {CRISPRi-Driven Genetic Screening for Designing Novel Microbial Phenotypes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2760},
number = {},
pages = {117-132},
pmid = {38468085},
issn = {1940-6029},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Gene Expression Regulation ; Phenotype ; Genetic Testing ; CRISPR-Cas Systems ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has enabled rapid advances in genomic engineering and transcriptional regulation. Specifically, CRISPR interference (CRISPRi) system has been used to systematically investigate the gene functions of microbial strains in a high-throughput manner. This method involves growth profiling using cells that have been transformed with the deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) libraries that target individual genes. The fitness scores of each gene are calculated by measuring the abundance of individual sgRNAs during cell growth and represent gene essentiality. In this chapter, a process is described for functional genetic screening using CRISPRi at the whole-genome scale, starting from the synthesis of sgRNA libraries, construction of CRISPRi libraries, and identification of essential genes through growth profiling. The commensal bacterium Bacteroides thetaiotaomicron was used to implement the protocol. This method is expected to be applicable to a broader range of microorganisms to explore the novel phenotypic characteristics of microorganisms.},
}
@article {pmid38468084,
year = {2024},
author = {Yu, L and Marchisio, MA},
title = {dCas12a:Pre-crRNA: A New Tool to Induce mRNA Degradation in Saccharomyces cerevisiae Synthetic Gene Circuits.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2760},
number = {},
pages = {95-114},
pmid = {38468084},
issn = {1940-6029},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Saccharomyces cerevisiae/genetics/metabolism ; Genes, Synthetic ; Deoxyribonucleases/metabolism ; RNA Stability ; CRISPR-Cas Systems ; },
abstract = {We describe a new way to trigger mRNA degradation in Saccharomyces cerevisiae synthetic gene circuits. Our method demands to modify either the 5'- or the 3'-UTR that flanks a target gene with elements from the pre-crRNA of type V Cas12a proteins and expresses a DNase-deficient Cas12a (dCas12a). dCas12a recognizes and cleaves the pre-crRNA motifs on mRNA sequences. Our tool does not require complex engineering operations and permits an efficient control of protein expression via mRNA degradation.},
}
@article {pmid38467777,
year = {2024},
author = {O'Leary, K},
title = {MEGA CRISPR rejuvenates exhausted CAR T cells.},
journal = {Nature medicine},
volume = {},
number = {},
pages = {},
doi = {10.1038/d41591-024-00014-4},
pmid = {38467777},
issn = {1546-170X},
}
@article {pmid38467687,
year = {2024},
author = {Kogenaru, V and Isalan, M and Kogenaru, M},
title = {A drug stabilizable GAL80[ds] for conditional control of gene expression via GAL4-UAS and CRISPR-Cas9 systems in Drosophila.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {5893},
pmid = {38467687},
issn = {2045-2322},
mesh = {Animals ; *Drosophila/genetics/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Transcription Factors/genetics/metabolism ; Gene Expression ; *Drosophila Proteins/genetics/metabolism ; },
abstract = {The binary GAL4-UAS expression system has been widely used in Drosophila to achieve tissue-specific expression of genes. To further allow for simultaneous spatial and conditional control of gene expression in existing GAL4 expression lines backgrounds, temperature and chemical controllable GAL80 variants have been engineered. Here we add a new drug stabilizable GAL80[ds] variant, by fusing it to a low-background DHFR-22-DD. We first quantify both single (DD-GAL80) and double (DD-GAL80-DD) architectures and show varied background and activation levels. Next, we demonstrate the utility of GAL80[ds] Drosophila line to regulate a cell death gene ectopically, in a drug-dependent manner, by utilizing an existing tissue-specific GAL4 driver that regulates the expression of a cell death gene under a UAS. Finally, we showcase the usefulness of GAL80[ds] in tight drug-mediated regulation of a target gene, from an endogenous locus, by utilizing an existing tissue-specific GAL4 to drive the expression of a dead Cas9 variant fused to the transcriptional coactivator nejire, under a UAS and in gRNA lines. Overall, these new GAL80[ds] lines expand the use of the wide variety of existing tissue-specific GAL4 and gene-specific gRNA lines. This enables conditional control of genes, both ectopically and endogenously, for a broad array of gene expression control applications.},
}
@article {pmid38467613,
year = {2024},
author = {Apostolopoulos, A and Kawamoto, N and Chow, SYA and Tsuiji, H and Ikeuchi, Y and Shichino, Y and Iwasaki, S},
title = {dCas13-mediated translational repression for accurate gene silencing in mammalian cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2205},
pmid = {38467613},
issn = {2041-1723},
support = {JP20H05784//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP21H05278//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP21H05734//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP23H04268//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP20H05786//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP23H02415//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20K07016//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP23K05648//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21K15023//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP23KJ2175//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20gm1410001//Japan Agency for Medical Research and Development (AMED)/ ; JP20gm1410001//Japan Agency for Medical Research and Development (AMED)/ ; JP23gm6910005h0001//Japan Agency for Medical Research and Development (AMED)/ ; JP23gm6910005//Japan Agency for Medical Research and Development (AMED)/ ; JP20gm1410001//Japan Agency for Medical Research and Development (AMED)/ ; Pioneering Projects//MEXT | RIKEN/ ; Pioneering Projects//MEXT | RIKEN/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems ; Codon, Initiator/metabolism ; *Ribosomes/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Gene Silencing ; Protein Biosynthesis/genetics ; Peptide Chain Initiation, Translational ; Mammals/genetics ; },
abstract = {Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.},
}
@article {pmid38326036,
year = {2024},
author = {Saikia, BB and Bhowmick, S and Malat, A and Preetha Rani, MR and Thaha, A and Abdul-Muneer, PM},
title = {ICAM-1 Deletion Using CRISPR/Cas9 Protects the Brain from Traumatic Brain Injury-Induced Inflammatory Leukocyte Adhesion and Transmigration Cascades by Attenuating the Paxillin/FAK-Dependent Rho GTPase Pathway.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {44},
number = {11},
pages = {},
doi = {10.1523/JNEUROSCI.1742-23.2024},
pmid = {38326036},
issn = {1529-2401},
mesh = {Male ; Female ; Humans ; Mice ; Animals ; *Intercellular Adhesion Molecule-1 ; Paxillin ; Focal Adhesion Protein-Tyrosine Kinases/metabolism ; Endothelial Cells/metabolism ; rho GTP-Binding Proteins/metabolism ; CRISPR-Cas Systems ; Brain/metabolism ; *Brain Injuries, Traumatic/metabolism ; Leukocytes ; },
abstract = {Intercellular adhesion molecule-1 (ICAM-1) is identified as an initiator of neuroinflammatory responses that lead to neurodegeneration and cognitive and sensory-motor deficits in several pathophysiological conditions including traumatic brain injury (TBI). However, the underlying mechanisms of ICAM-1-mediated leukocyte adhesion and transmigration and its link with neuroinflammation and functional deficits following TBI remain elusive. Here, we hypothesize that blocking of ICAM-1 attenuates the transmigration of leukocytes to the brain and promotes functional recovery after TBI. The experimental TBI was induced in vivo by fluid percussion injury (25 psi) in male and female wild-type and ICAM-1[-/-] mice and in vitro by stretch injury (3 psi) in human brain microvascular endothelial cells (hBMVECs). We treated hBMVECs and animals with ICAM-1 CRISPR/Cas9 and conducted several biochemical analyses and demonstrated that CRISPR/Cas9-mediated ICAM-1 deletion mitigates blood-brain barrier (BBB) damage and leukocyte transmigration to the brain by attenuating the paxillin/focal adhesion kinase (FAK)-dependent Rho GTPase pathway. For analyzing functional outcomes, we used a cohort of behavioral tests that included sensorimotor functions, psychological stress analyses, and spatial memory and learning following TBI. In conclusion, this study could establish the significance of deletion or blocking of ICAM-1 in transforming into a novel preventive approach against the pathophysiology of TBI.},
}
@article {pmid38467609,
year = {2024},
author = {Wilson, EL and Yu, Y and Leal, NS and Woodward, JA and Patikas, N and Morris, JL and Field, SF and Plumbly, W and Paupe, V and Chowdhury, SR and Antrobus, R and Lindop, GE and Adia, YM and Loh, SHY and Prudent, J and Martins, LM and Metzakopian, E},
title = {Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.},
journal = {Cell death &amp; disease},
volume = {15},
number = {3},
pages = {203},
pmid = {38467609},
issn = {2041-4889},
mesh = {*CRISPR-Cas Systems/genetics ; *Mitochondria Associated Membranes ; Mitochondria/genetics/metabolism ; Mitochondrial Membranes/metabolism ; Endoplasmic Reticulum/genetics/metabolism ; Calcium/metabolism ; },
abstract = {Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca[2+] release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.},
}
@article {pmid38464062,
year = {2024},
author = {Ligunas, GD and Paniagua, G and LaBelle, J and Ramos-Martinez, A and Shen, K and Gerlt, EH and Aguilar, K and Nguyen, A and Materna, SC and Woo, S},
title = {Tissue-specific and endogenous protein labeling with split fluorescent proteins.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {38464062},
support = {R15 HD102829/HD/NICHD NIH HHS/United States ; T32 GM141862/GM/NIGMS NIH HHS/United States ; },
abstract = {The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein function. Our approach should be broadly useful for a wide range of applications.},
}
@article {pmid38461237,
year = {2024},
author = {Boudna, M and Campos, AD and Vychytilova-Faltejskova, P and Machackova, T and Slaby, O and Souckova, K},
title = {Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies.},
journal = {Cell communication and signaling : CCS},
volume = {22},
number = {1},
pages = {171},
pmid = {38461237},
issn = {1478-811X},
support = {20-18889S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; 20-18889S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; 20-18889S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; 20-18889S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; 20-18889S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; 20-18889S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; },
mesh = {*Extracellular Vesicles ; },
abstract = {This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.},
}
@article {pmid38460133,
year = {2024},
author = {Biase, FH and Schettini, G},
title = {Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.},
journal = {STAR protocols},
volume = {5},
number = {1},
pages = {102940},
doi = {10.1016/j.xpro.2024.102940},
pmid = {38460133},
issn = {2666-1667},
abstract = {The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation. The first electroporation introduces ribonucleoproteins formed by Cas9D10A with two guide RNAs to target DNA, and the second introduces the same ribonucleoprotein complex to target DNA plus Cas13a with one guide RNA to target RNAs. For complete details on the use and execution of this protocol, please refer to Nix et al.[1].},
}
@article {pmid38459694,
year = {2024},
author = {Klermund, J and Rhiel, M and Kocher, T and Chmielewski, KO and Bischof, J and Andrieux, G and Gaz, ME and Hainzl, S and Boerries, M and Cornu, TI and Koller, U and Cathomen, T},
title = {On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2024.03.006},
pmid = {38459694},
issn = {1525-0024},
abstract = {Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired nickase editing. While the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal aberrations at on-target sites.},
}
@article {pmid38459454,
year = {2024},
author = {Liu, Y and Chao, Z and Ding, W and Fang, T and Gu, X and Xue, M and Wang, W and Han, R and Sun, W},
title = {A multiplex RPA-CRISPR/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay.},
journal = {Cellular &amp; molecular biology letters},
volume = {29},
number = {1},
pages = {34},
pmid = {38459454},
issn = {1689-1392},
support = {2022SS18//Science and Technology Planning Project of Su Zhou/ ; },
mesh = {Humans ; *Recombinases ; CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis ; Reproducibility of Results ; Point-of-Care Testing ; Human Papillomavirus Viruses ; },
abstract = {Persistent infection with high-risk human papillomavirus (HR-HPV) is the primary and initiating factor for cervical cancer. With over 200 identified HPV types, including 14 high-risk types that integrate into the host cervical epithelial cell DNA, early determination of HPV infection type is crucial for effective risk stratification and management. Presently, on-site immediate testing during the HPV screening stage, known as Point of Care Testing (POCT), remains immature, severely limiting the scope and scenarios of HPV screening. This study, guided by the genomic sequence patterns of HPV, established a multiplex recombinase polymerase amplification (RPA) technology based on the concept of "universal primers." This approach achieved the multiple amplification of RPA, coupled with the CRISPR/Cas12a system serving as a medium for signal amplification and conversion. The study successfully constructed a POCT combined detection system, denoted as H-MRC12a (HPV-Multiple RPA-CRISPR/Cas12a), and applied it to high-risk HPV typing detection. The system accomplished the typing detection of six high-risk HPV types (16, 18, 31, 33, 35, and 45) can be completed within 40 min, and the entire process, from sample loading to result interpretation, can be accomplished within 45 min, with a detection depth reaching 1 copy/μL for each high-risk type. Validation of the H-MRC12a detection system's reproducibility and specificity was further conducted through QPCR on 34 clinical samples. Additionally, this study explored and optimized the multiplex RPA amplification system and CRISPR system at the molecular mechanism level. Furthermore, the primer design strategy developed in this study offers the potential to enhance the throughput of H-MRC12a detection while ensuring sensitivity, providing a novel research avenue for high-throughput detection in Point-of-Care molecular pathogen studies.},
}
@article {pmid38146164,
year = {2024},
author = {Han, Y and Liu, G and Wu, Y and Bao, Y and Zhang, Y and Zhang, T},
title = {CrisprStitch: Fast evaluation of the efficiency of CRISPR editing systems.},
journal = {Plant communications},
volume = {5},
number = {3},
pages = {100783},
doi = {10.1016/j.xplc.2023.100783},
pmid = {38146164},
issn = {2590-3462},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Gene Editing ; },
}
@article {pmid37946411,
year = {2024},
author = {Wang, H and Zhang, D and Chen, M and Meng, X and Bai, S and Xin, P and Yan, J and Chu, J and Li, J and Yu, H},
title = {Genome editing of 3' UTR-embedded inhibitory region enables generation of gene knock-up alleles in plants.},
journal = {Plant communications},
volume = {5},
number = {3},
pages = {100745},
doi = {10.1016/j.xplc.2023.100745},
pmid = {37946411},
issn = {2590-3462},
mesh = {*Gene Editing ; 3' Untranslated Regions ; Alleles ; *Plants/genetics ; CRISPR-Cas Systems ; },
}
@article {pmid38459056,
year = {2024},
author = {Beavogui, A and Lacroix, A and Wiart, N and Poulain, J and Delmont, TO and Paoli, L and Wincker, P and Oliveira, PH},
title = {The defensome of complex bacterial communities.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2146},
pmid = {38459056},
issn = {2041-1723},
mesh = {Humans ; *Bacteria/genetics ; *Genome, Bacterial ; Metagenomics ; Genome Size ; CRISPR-Cas Systems ; },
abstract = {Bacteria have developed various defense mechanisms to avoid infection and killing in response to the fast evolution and turnover of viruses and other genetic parasites. Such pan-immune system (defensome) encompasses a growing number of defense lines that include well-studied innate and adaptive systems such as restriction-modification, CRISPR-Cas and abortive infection, but also newly found ones whose mechanisms are still poorly understood. While the abundance and distribution of defense systems is well-known in complete and culturable genomes, there is a void in our understanding of their diversity and richness in complex microbial communities. Here we performed a large-scale in-depth analysis of the defensomes of 7759 high-quality bacterial population genomes reconstructed from soil, marine, and human gut environments. We observed a wide variation in the frequency and nature of the defensome among large phyla, which correlated with lifestyle, genome size, habitat, and geographic background. The defensome's genetic mobility, its clustering in defense islands, and genetic variability was found to be system-specific and shaped by the bacterial environment. Hence, our results provide a detailed picture of the multiple immune barriers present in environmentally distinct bacterial communities and set the stage for subsequent identification of novel and ingenious strategies of diversification among uncultivated microbes.},
}
@article {pmid38458740,
year = {2024},
author = {Martinsen, E and Jinnurine, T and Subramani, S and Rogne, M},
title = {Advances in RNA therapeutics for modulation of 'undruggable' targets.},
journal = {Progress in molecular biology and translational science},
volume = {204},
number = {},
pages = {249-294},
doi = {10.1016/bs.pmbts.2023.12.003},
pmid = {38458740},
issn = {1878-0814},
mesh = {Humans ; *RNA ; *Gene Editing ; RNA Interference ; RNA, Messenger ; Proteins ; },
abstract = {Over the past decades, drug discovery utilizing small pharmacological compounds, fragment-based therapeutics, and antibody therapy have significantly advanced treatment options for many human diseases. However, a major bottleneck has been that>70% of human proteins/genomic regions are 'undruggable' by the above-mentioned approaches. Many of these proteins constitute essential drug targets against complex multifactorial diseases like cancer, immunological disorders, and neurological diseases. Therefore, alternative approaches are required to target these proteins or genomic regions in human cells. RNA therapeutics is a promising approach for many of the traditionally 'undruggable' targets by utilizing methods such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based genome editing, aptamers, and the development of mRNA therapeutics. In the following chapter, we will put emphasis on recent advancements utilizing these approaches against challenging drug targets, such as intranuclear proteins, intrinsically disordered proteins, untranslated genomic regions, and targets expressed in inaccessible tissues.},
}
@article {pmid38456346,
year = {2024},
author = {Li, Y and Zhou, S and Wu, Q and Gong, C},
title = {CRISPR/Cas gene editing and delivery systems for cancer therapy.},
journal = {Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology},
volume = {16},
number = {1},
pages = {e1938},
doi = {10.1002/wnan.1938},
pmid = {38456346},
issn = {1939-0041},
support = {2023YFS0153//Key Research and Development Program of Sichuan Province/ ; 82172094//National Natural Science Foundation of China/ ; 2021JDJQ0037//Science Fund for Distinguished Young Scholars of Sichuan Province/ ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Gene Transfer Techniques ; Genetic Vectors ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR/Cas systems stand out because of simplicity, efficiency, and other superiorities, thus becoming attractive and brilliant gene-editing tools in biomedical field including cancer therapy. CRISPR/Cas systems bring promises for cancer therapy through manipulating and engineering on tumor cells or immune cells. However, there have been concerns about how to overcome the numerous physiological barriers and deliver CRISPR components to target cells efficiently and accurately. In this review, we introduced the mechanisms of CRISPR/Cas systems, summarized the current delivery strategies of CRISPR/Cas systems by physical methods, viral vectors, and nonviral vectors, and presented the current application of CRISPR/Cas systems in cancer clinical treatment. Furthermore, we discussed prospects related to delivery approaches of CRISPR/Cas systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.},
}
@article {pmid38453924,
year = {2024},
author = {Cai, H and Zhang, B and Ahrenfeldt, J and Joseph, JV and Riedel, M and Gao, Z and Thomsen, SK and Christensen, DS and Bak, RO and Hager, H and Vendelbo, MH and Gao, X and Birkbak, N and Thomsen, MK},
title = {CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2088},
pmid = {38453924},
issn = {2041-1723},
support = {R146-A9394//Kr&#xe6;ftens Bek&#xe6;mpelse (Danish Cancer Society)/ ; R204-A12490//Kr&#xe6;ftens Bek&#xe6;mpelse (Danish Cancer Society)/ ; AUFF-E-2015-FLS-9-8//Aarhus Universitets Forskningsfond (Aarhus University Research Foundation)/ ; AUFF-E-2018-7-14//Aarhus Universitets Forskningsfond (Aarhus University Research Foundation)/ ; R272-2017-4040//Lundbeckfonden (Lundbeck Foundation)/ ; NNF21OC0071483//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {Male ; Humans ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Prostatic Neoplasms/genetics/pathology ; Transcriptome ; Multigene Family ; },
abstract = {Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.},
}
@article {pmid38453913,
year = {2024},
author = {Hayashi, N and Lai, Y and Fuerte-Stone, J and Mimee, M and Lu, TK},
title = {Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2096},
pmid = {38453913},
issn = {2041-1723},
support = {R25 GM109439/GM/NIGMS NIH HHS/United States ; R35 GM147478/GM/NIGMS NIH HHS/United States ; T32 GM007183/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Containment of Biohazards ; Genetic Engineering ; Bacteria/genetics ; Thymidine ; },
abstract = {Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.},
}
@article {pmid38453904,
year = {2024},
author = {Weber, Y and Böck, D and Ivașcu, A and Mathis, N and Rothgangl, T and Ioannidi, EI and Blaudt, AC and Tidecks, L and Vadovics, M and Muramatsu, H and Reichmuth, A and Marquart, KF and Kissling, L and Pardi, N and Jinek, M and Schwank, G},
title = {Enhancing prime editor activity by directed protein evolution in yeast.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2092},
pmid = {38453904},
issn = {2041-1723},
support = {R01 AI153064/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Saccharomyces cerevisiae/genetics ; Amino Acid Substitution ; *Biological Assay ; Brain ; Cell Line ; CRISPR-Cas Systems/genetics ; Mammals ; },
abstract = {Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations - the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT - results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.},
}
@article {pmid38453634,
year = {2024},
author = {Wang, X and Leptihn, S},
title = {Defense and anti-defense mechanisms of bacteria and bacteriophages.},
journal = {Journal of Zhejiang University. Science. B},
volume = {25},
number = {3},
pages = {181-196},
pmid = {38453634},
issn = {1862-1783},
support = {2024KY592//the Western Medicine Program of the Zhejiang Provincial Health Commission/ ; 2-2050205-19-361//the Fundamental Research Funds for Central Universities of the Central South University/ ; },
mesh = {*Bacteriophages ; Bacteria ; CRISPR-Cas Systems ; },
abstract = {In the post-antibiotic era, the overuse of antimicrobials has led to a massive increase in antimicrobial resistance, leaving medical doctors few or no treatment options to fight infections caused by superbugs. The use of bacteriophages is a promising alternative to treat infections, supplementing or possibly even replacing antibiotics. Using phages for therapy is possible, since these bacterial viruses can kill bacteria specifically, causing no harm to the normal flora. However, bacteria have developed a multitude of sophisticated and complex ways to resist infection by phages, including abortive infection and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. Phages also can evolve and acquire new anti-defense strategies to continue predation. An in-depth exploration of both defense and anti-defense mechanisms would contribute to optimizing phage therapy, while we would also gain novel insights into the microbial world. In this paper, we summarize recent research on bacterial phage resistance and phage anti-defense mechanisms, as well as collaborative win-win systems involving both virus and host.},
}
@article {pmid38450402,
year = {2024},
author = {Das, S and Kwon, M and Kim, JY},
title = {Enhancement of specialized metabolites using CRISPR/Cas gene editing technology in medicinal plants.},
journal = {Frontiers in plant science},
volume = {15},
number = {},
pages = {1279738},
pmid = {38450402},
issn = {1664-462X},
abstract = {Plants are the richest source of specialized metabolites. The specialized metabolites offer a variety of physiological benefits and many adaptive evolutionary advantages and frequently linked to plant defense mechanisms. Medicinal plants are a vital source of nutrition and active pharmaceutical agents. The production of valuable specialized metabolites and bioactive compounds has increased with the improvement of transgenic techniques like gene silencing and gene overexpression. These techniques are beneficial for decreasing production costs and increasing nutritional value. Utilizing biotechnological applications to enhance specialized metabolites in medicinal plants needs characterization and identification of genes within an elucidated pathway. The breakthrough and advancement of CRISPR/Cas-based gene editing in improving the production of specific metabolites in medicinal plants have gained significant importance in contemporary times. This article imparts a comprehensive recapitulation of the latest advancements made in the implementation of CRISPR-gene editing techniques for the purpose of augmenting specific metabolites in medicinal plants. We also provide further insights and perspectives for improving metabolic engineering scenarios in medicinal plants.},
}
@article {pmid38449862,
year = {2024},
author = {Tao, R and Han, X and Bai, X and Yu, J and Ma, Y and Chen, W and Zhang, D and Li, Z},
title = {Revolutionizing cancer treatment: enhancing CAR-T cell therapy with CRISPR/Cas9 gene editing technology.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1354825},
pmid = {38449862},
issn = {1664-3224},
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics ; *Leukemia, Lymphocytic, Chronic, B-Cell ; Technology ; Cell- and Tissue-Based Therapy ; },
abstract = {CAR-T cell therapy, a novel immunotherapy, has made significant breakthroughs in clinical practice, particularly in treating B-cell-associated leukemia and lymphoma. However, it still faces challenges such as poor persistence, limited proliferation capacity, high manufacturing costs, and suboptimal efficacy. CRISPR/Cas system, an efficient and simple method for precise gene editing, offers new possibilities for optimizing CAR-T cells. It can increase the function of CAR-T cells and reduce manufacturing costs. The combination of CRISPR/Cas9 technology and CAR-T cell therapy may promote the development of this therapy and provide more effective and personalized treatment for cancer patients. Meanwhile, the safety issues surrounding the application of this technology in CAR-T cells require further research and evaluation. Future research should focus on improving the accuracy and safety of CRISPR/Cas9 technology to facilitate the better development and application of CAR-T cell therapy. This review focuses on the application of CRISPR/Cas9 technology in CAR-T cell therapy, including eliminating the inhibitory effect of immune checkpoints, enhancing the ability of CAR-T cells to resist exhaustion, assisting in the construction of universal CAR-T cells, reducing the manufacturing costs of CAR-T cells, and the security problems faced. The objective is to show the revolutionary role of CRISPR/Cas9 technology in CAR-T cell therapy for researchers.},
}
@article {pmid38448635,
year = {2024},
author = {Mori, K and Tanase, K and Sasaki, K},
title = {Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.},
journal = {Planta},
volume = {259},
number = {4},
pages = {84},
pmid = {38448635},
issn = {1432-2048},
mesh = {Humans ; *Dianthus ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Electroporation ; Ribonucleoproteins ; *Oxygenases ; },
abstract = {A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.},
}
@article {pmid38448490,
year = {2024},
author = {Uppuluri, L and Shi, CH and Varapula, D and Young, E and Ehrlich, RL and Wang, Y and Piazza, D and Mell, JC and Yip, KY and Xiao, M},
title = {A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {5583},
pmid = {38448490},
issn = {2045-2322},
support = {R01 HG005946/HG/NHGRI NIH HHS/United States ; R56 HG005946/HG/NHGRI NIH HHS/United States ; HG005946/NH/NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; Sequence Analysis, DNA ; Genomics ; *Bacteriophages ; CRISPR-Associated Protein 9 ; DNA/genetics ; },
abstract = {In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.},
}
@article {pmid38448164,
year = {2024},
author = {Wong, CH and Wingett, SW and Qian, C and Hunter, MR and Taliaferro, JM and Ross-Thriepland, D and Bullock, SL},
title = {Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.},
journal = {The Journal of cell biology},
volume = {223},
number = {5},
pages = {},
pmid = {38448164},
issn = {1540-8140},
support = {MC_U105178790/MRC_/Medical Research Council/United Kingdom ; R35 GM133385/GM/NIGMS NIH HHS/United States ; R35-GM133385/NH/NIH HHS/United States ; },
mesh = {Humans ; *Dyneins/genetics ; *Microtubules/genetics ; Peroxisomes/genetics ; CRISPR-Cas Systems ; Genetic Techniques ; },
abstract = {The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.},
}
@article {pmid38448140,
year = {2024},
author = {Mathur, S and Singh, D and Ranjan, R},
title = {Recent advances in plant translational genomics for crop improvement.},
journal = {Advances in protein chemistry and structural biology},
volume = {139},
number = {},
pages = {335-382},
doi = {10.1016/bs.apcsb.2023.11.009},
pmid = {38448140},
issn = {1876-1631},
mesh = {*Genomics ; *Gene Expression Profiling ; Phenotype ; Polymorphism, Single Nucleotide ; },
abstract = {The growing population, climate change, and limited agricultural resources put enormous pressure on agricultural systems. A plateau in crop yields is occurring and extreme weather events and urbanization threaten the livelihood of farmers. It is imperative that immediate attention is paid to addressing the increasing food demand, ensuring resilience against emerging threats, and meeting the demand for more nutritious, safer food. Under uncertain conditions, it is essential to expand genetic diversity and discover novel crop varieties or variations to develop higher and more stable yields. Genomics plays a significant role in developing abundant and nutrient-dense food crops. An alternative to traditional breeding approach, translational genomics is able to improve breeding programs in a more efficient and precise manner by translating genomic concepts into practical tools. Crop breeding based on genomics offers potential solutions to overcome the limitations of conventional breeding methods, including improved crop varieties that provide more nutritional value and are protected from biotic and abiotic stresses. Genetic markers, such as SNPs and ESTs, contribute to the discovery of QTLs controlling agronomic traits and stress tolerance. In order to meet the growing demand for food, there is a need to incorporate QTLs into breeding programs using marker-assisted selection/breeding and transgenic technologies. This chapter primarily focuses on the recent advances that are made in translational genomics for crop improvement and various omics techniques including transcriptomics, metagenomics, pangenomics, single cell omics etc. Numerous genome editing techniques including CRISPR Cas technology and their applications in crop improvement had been discussed.},
}
@article {pmid38447547,
year = {2024},
author = {Pasch, T and Bäumer, N and Bäumer, S and Buchholz, F and Mootz, HD},
title = {Towards targeted Cas9 (CRISPR-Cas) delivery: Preparation of IgG antibody-Cas9 conjugates using a split intein.},
journal = {Journal of peptide science : an official publication of the European Peptide Society},
volume = {},
number = {},
pages = {},
doi = {10.1002/psc.3592},
pmid = {38447547},
issn = {1099-1387},
support = {111418//University of M&#xfc;nster Medical School/ ; 111501//University of M&#xfc;nster Medical School/ ; 121802//University of M&#xfc;nster Medical School/ ; 211502//University of M&#xfc;nster Medical School/ ; 2014.054.1//Wilhelm Sander-Stiftung/ ; 2017.071.1//Wilhelm Sander-Stiftung/ ; CRC1450-431460824//Deutsche Forschungsgemeinschaft/ ; },
abstract = {The CRISPR-Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease-protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9-RNP. We opted for protein trans-splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine-less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG-Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double-strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N-terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG-Cas9 provides the first step towards a novel CRISPR-Cas9 targeting approach involving the preformed RNP complex.},
}
@article {pmid38422550,
year = {2024},
author = {Zhou, M and Li, C and Wei, R and Wang, H and Jia, H and Yan, C and Huang, L},
title = {Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA.},
journal = {Analytical chemistry},
volume = {96},
number = {10},
pages = {4322-4329},
doi = {10.1021/acs.analchem.4c00313},
pmid = {38422550},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; DNA/genetics ; *Neoplasms/diagnosis/genetics ; },
abstract = {As an important component of highly heterogeneous exosomes, exosomal microRNAs (miRNAs) have great potential as noninvasive biomarkers for cancer diagnosis. Therefore, a sensitive and simple sensor is the key for its clinical application. Herein, we designed an exponential amplification reaction (EXPAR) to induce the reactivation of the CRISPR-associated protein 9/small guide RNA (Cas9/sgRNA) complex, thus achieving sensitive and visual exosomal miRNAs-21 (miR-21) fluorescence sensing. In this design, we inactivated the sgRNA by hybridizing sgRNA and blocker DNA. Then, we used a trigger DNA to hybridize with miR-21 and produced a lot of activated DNA by EXPAR. Those activated DNA further hybridized with blocker DNA and released the free sgRNA to form the activated Cas9/sgRNA complex. Based on the quick cleavage of activated Cas9/sgRNA complex, the reporter DNA labeled by SYBR Green I was released from the surface of the magnetic nanoparticles (MNPs) into the supernatant, and thus was used to sensitively quantify the miRNAs concentration with a limit of detection of 3 × 10[3] particles/mL. In addition, this fluorescence sensor has also been successfully employed to distinguish healthy people and cancer patients by naked-eye observation of the fluorescence, thus demonstrating its great potential for accurate and point-of-care cancer diagnosis.},
}
@article {pmid38404229,
year = {2024},
author = {Wang, ZY and Teng, SQ and Zhao, NN and Han, Y and Li, DL and Zhang, CY},
title = {Ligase detection reaction amplification-activated CRISPR-Cas12a for single-molecule counting of FEN1 in breast cancer tissues.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {22},
pages = {3075-3078},
doi = {10.1039/d4cc00408f},
pmid = {38404229},
issn = {1364-548X},
mesh = {Humans ; Flap Endonucleases ; CRISPR-Cas Systems/genetics ; Coloring Agents ; Drug Discovery ; *Biosensing Techniques ; *Neoplasms ; },
abstract = {We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10[-8] U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.},
}
@article {pmid38302659,
year = {2024},
author = {Truong, DJ and Geilenkeuser, J and Wendel, SV and Wilming, JCH and Armbrust, N and Binder, EMH and Santl, TH and Siebenhaar, A and Gruber, C and Phlairaharn, T and Živanić, M and Westmeyer, GG},
title = {Exonuclease-enhanced prime editors.},
journal = {Nature methods},
volume = {21},
number = {3},
pages = {455-464},
pmid = {38302659},
issn = {1548-7105},
mesh = {*Exonucleases ; *RNA, Guide, CRISPR-Cas Systems ; Cell Line ; DNA, Single-Stranded/genetics ; Flow Cytometry ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.},
}
@article {pmid38301346,
year = {2024},
author = {Qu, Z and Gong, Z and Olajide, JS and Wang, J and Cai, J},
title = {CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella.},
journal = {Veterinary parasitology},
volume = {327},
number = {},
pages = {110131},
doi = {10.1016/j.vetpar.2024.110131},
pmid = {38301346},
issn = {1873-2550},
mesh = {Female ; Male ; Animals ; *Eimeria tenella/genetics ; CRISPR-Cas Systems ; *Coccidiosis/genetics/veterinary ; Life Cycle Stages ; Chickens ; *Poultry Diseases/parasitology ; *Red Fluorescent Protein ; },
abstract = {Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.},
}
@article {pmid38232511,
year = {2024},
author = {Wu, M and Wu, H and Chen, X and Wu, F and Ma, G and Du, A and Yang, Y},
title = {RPA-CRISPR/Cas9-based method for the detection of Toxoplasma gondii: A proof of concept.},
journal = {Veterinary parasitology},
volume = {327},
number = {},
pages = {110115},
doi = {10.1016/j.vetpar.2024.110115},
pmid = {38232511},
issn = {1873-2550},
mesh = {Animals ; Cats ; *Toxoplasma/genetics ; *Cryptosporidium ; *Cryptosporidiosis ; CRISPR-Cas Systems ; *Toxoplasmosis ; Oocysts/genetics ; Feces ; *Cat Diseases/diagnosis ; *Toxoplasmosis, Animal/epidemiology ; },
abstract = {Toxoplasma gondii is a widespread and specialized intracellular protozoan pathogen that affects one third of the world' s population, posing a great threat to public health. As the definitive host, cats excrete oocysts and play a crucial role in the transmission of toxoplasmosis. The current diagnostic tools usually require bulky equipment and expertize, which hinders the efficient diagnosis and intervention of Toxoplasma infection in cats. In this study, we combined (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to establish an easier method for the detection of T. gondii oocysts in cat fecal samples. The sensitivity, specificity, and practicability of the established RPA-CRISPR/Cas9 method were evaluated using a lateral flow strip, with the limitation of detection determined at 10 plasmid copies/μL (corresponding to about one oocyst), cross reactivity to none of Giardia lamblia, Cryptosporidium sp., Microsporidium biberi and Blastocystis hominis that also commonly found in cats, and comparable performance in detecting T. gondii in clinical samples to conventional PCR amplification. This RPA-CRISPR/Cas9 method provides an alternative to conventional molecular tools used in the clinical diagnosis of Toxoplasma infection in cats and other animals.},
}
@article {pmid38446895,
year = {2024},
author = {Skeens, E and Sinha, S and Ahsan, M and D'Ordine, AM and Jogl, G and Palermo, G and Lisi, GP},
title = {High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.},
journal = {Science advances},
volume = {10},
number = {10},
pages = {eadl1045},
pmid = {38446895},
issn = {2375-2548},
support = {P30 GM133893/GM/NIGMS NIH HHS/United States ; R01 GM136815/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Communication ; CRISPR-Associated Protein 9 ; Catalysis ; DNA/genetics ; },
abstract = {The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.},
}
@article {pmid38445556,
year = {2024},
author = {Maurya, VK and Sanjeevi, M and Rahul, CN and Mohan, A and Ramachandran, D and Siddalingappa, R and Rauniyar, R and Kanagaraj, S},
title = {Finding identical sequence repeats in multiple protein sequences: An algorithm.},
journal = {Journal of biosciences},
volume = {49},
number = {},
pages = {},
pmid = {38445556},
issn = {0973-7138},
mesh = {Amino Acid Sequence ; *Algorithms ; *Amino Acids ; Databases, Factual ; },
abstract = {In recent years, several experimental evidences suggest that amino acid repeats are closely linked to many disease conditions, as they have a significant role in evolution of disordered regions of the polypeptide segments. Even though many algorithms and databases were developed for such analysis, each algorithm has some caveats, like limitation on the number of amino acids within the repeat patterns and number of query protein sequences. To this end, in the present work, a new method called the internal sequence repeats across multiple protein sequences (ISRMPS) is proposed for the first time to identify identical repeats across multiple protein sequences. It also identifies distantly located repeat patterns in various protein sequences. Our method can be applied to study evolutionary relationships, epitope mapping, CRISPR-Cas sequencing methods, and other comparative analytical assessments of protein sequences.},
}
@article {pmid38443409,
year = {2024},
author = {Tomasi, F and Pozzi, M and Lauria, M},
title = {Investigating the mechanisms underlying resistance to chemoterapy and to CRISPR-Cas9 in cancer cell lines.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {5402},
pmid = {38443409},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; *Lung Neoplasms ; Cell Line ; Gene Knockout Techniques ; Gene Regulatory Networks ; Ubiquitin Thiolesterase ; Intracellular Signaling Peptides and Proteins ; },
abstract = {Cancer is one of the major causes of death worldwide and the development of multidrug resistance (MDR) in cancer cells is the principal cause of chemotherapy failure. To gain insights into the specific mechanisms of MDR in cancer cell lines, we developed a novel method for the combined analysis of recently published datasets on drug sensitivity and CRISPR loss-of-function screens for the same set of cancer cell lines. For our analysis, we first selected cell lines that consistently exhibit drug resistance across several classes of compounds. We then identified putative resistance genes for each class of compound and used inferred gene regulatory networks (GRNs) to study possible mechanisms underlying the development of MDR in the identified cancer cell lines. We show that the same method of analysis can also be used to identify cell lines that consistently exhibit resistance to the gene knockout effect of the CRISPR-Cas9 technique and to study the possible underlying mechanisms. In the GRN associated to the drug resistant cell lines, we identify genes previously associated with resistance (UHMK1, RALYL, MGST3, USP9X, and ESRG), genes for which an indirect association can be identified (SPINK13, LINC00664, MRPL38, and EMILIN3), and genes that are found to be overexpressed in non-resistant cancer cell lines (MRPL38, EMILIN3 and RALYL). In the GRNs associated to the CRISPR-Cas9 resistance mechanism, none of the identified genes has been previously reported in the admittedly sparse literature on the subject. However, some of these genes have a common role: APBB2, RUNX1T1, ZBTB7C, and ISX regulate transcription, while APBB2, BTG3, ZBTB7C, SZRD1 and LEF1 have a function in regulating proliferation, suggesting a role for these two pathways. While our results are specific for the lung cancer cell lines we selected for this work, our method of analysis can be applied to cell lines from other tissues and for which the required data is available.},
}
@article {pmid38443394,
year = {2024},
author = {Deng, F and Li, Y and Yang, B and Sang, R and Deng, W and Kansara, M and Lin, F and Thavaneswaran, S and Thomas, DM and Goldys, EM},
title = {Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1818},
pmid = {38443394},
issn = {2041-1723},
mesh = {DNA, Circular/genetics ; RNA/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; DNA/genetics ; *Helicobacter pylori ; *Nanostructures ; Ribonucleoproteins ; },
abstract = {Control of CRISPR/Cas12a trans-cleavage is crucial for biosensor development. Here, we show that small circular DNA nanostructures which partially match guide RNA sequences only minimally activate Cas12a ribonucleoproteins. However, linearizing these structures restores activation. Building on this finding, an Autocatalytic Cas12a Circular DNA Amplification Reaction (AutoCAR) system is established which allows a single nucleic acid target to activate multiple ribonucleoproteins, and greatly increases the achievable reporter cleavage rates per target. A rate-equation-based model explains the observed near-exponential rate trends. Autocatalysis is also sustained with DNA nanostructures modified with fluorophore-quencher pairs achieving 1 aM level (<1 copy/μL) DNA detection (10[6] times improvement), without additional amplification, within 15 min, at room temperature. The detection range is tuneable, spanning 3 to 11 orders of magnitude. We demonstrate 1 aM level detection of SNP mutations in circulating tumor DNA from blood plasma, genomic DNA (H. Pylori) and RNA (SARS-CoV-2) without reverse transcription as well as colorimetric lateral flow tests of cancer mutations with ~100 aM sensitivity.},
}
@article {pmid38443218,
year = {2024},
author = {Chang, CW and Truong, VA and Pham, NN and Hu, YC},
title = {RNA-guided genome engineering: paradigm shift towards transposons.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2024.02.006},
pmid = {38443218},
issn = {1879-3096},
abstract = {CRISPR-Cas systems revolutionized the genome engineering field but need to induce double-strand breaks (DSBs) and may be difficult to deliver due to their large protein size. Tn7-like transposons such as CRISPR-associated transposons (CASTs) can be repurposed for RNA-guided DSB-free integration, and obligate mobile element guided activity (OMEGA) proteins of the IS200/IS605 transposon family have been developed as hypercompact RNA-guided genome editing tools. CASTs and OMEGA are exciting, innovative genome engineering tools that can improve the precision and efficiency of editing. This review explores the recent developments and uses of CASTs and OMEGA in genome editing across prokaryotic and eukaryotic cells. The pros and cons of these transposon-based systems are deliberated in comparison to other CRISPR systems.},
}
@article {pmid38441816,
year = {2024},
author = {Hwarari, D and Radani, Y and Ke, Y and Chen, J and Yang, L},
title = {CRISPR/Cas genome editing in plants: mechanisms, applications, and overcoming bottlenecks.},
journal = {Functional &amp; integrative genomics},
volume = {24},
number = {2},
pages = {50},
pmid = {38441816},
issn = {1438-7948},
support = {31971682, 32071784//National Natural Science Foundation of China/ ; 31971682, 32071784//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Disease Resistance ; Gene Knockout Techniques ; Genome, Plant ; },
abstract = {The CRISPR/Cas systems have emerged as transformative tools for precisely manipulating plant genomes and enhancement. It has provided unparalleled applications from modifying the plant genomes to resistant enhancement. This review manuscript summarises the mechanism, application, and current challenges in the CRISPR/Cas genome editing technology. It addresses the molecular mechanisms of different Cas genes, elucidating their applications in various plants through crop improvement, disease resistance, and trait improvement. The advent of the CRISPR/Cas systems has enabled researchers to precisely modify plant genomes through gene knockouts, knock-ins, and gene expression modulation. Despite these successes, the CRISPR/Cas technology faces challenges, including off-target effects, Cas toxicity, and efficiency. In this manuscript, we also discuss these challenges and outline ongoing strategies employed to overcome these challenges, including the development of novel CRISPR/Cas variants with improved specificity and specific delivery methods for different plant species. The manuscript will conclude by addressing the future perspectives of the CRISPR/Cas technology in plants. Although this review manuscript is not conclusive, it aims to provide immense insights into the current state and future potential of CRISPR/Cas in sustainable and secure plant production.},
}
@article {pmid38394831,
year = {2024},
author = {Chrzanowski, S and Batra, R},
title = {CRISPR-Based Gene Editing Techniques in Pediatric Neurological Disorders.},
journal = {Pediatric neurology},
volume = {153},
number = {},
pages = {166-174},
doi = {10.1016/j.pediatrneurol.2024.01.021},
pmid = {38394831},
issn = {1873-5150},
mesh = {Humans ; Child ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genetic Therapy ; *Muscular Dystrophy, Duchenne ; *Nervous System Diseases/genetics/therapy ; },
abstract = {The emergence of gene editing technologies offers a unique opportunity to develop mutation-specific treatments for pediatric neurological disorders. Gene editing systems can potentially alter disease trajectory by correcting dysfunctional mutations or therapeutically altering gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches are attractive gene therapy platforms to personalize treatments because of their specificity, ease of design, versatility, and cost. However, many such approaches remain in the early stages of development, with ongoing efforts to optimize editing efficiency, minimize unintended off-target effects, and mitigate pathologic immune responses. Given the rapid evolution of CRISPR-based therapies, it is prudent for the clinically based child neurologist to have a conceptual understanding of what such therapies may entail, including both benefits and risks and how such therapies may be clinically applied. In this review, we describe the fundamentals of CRISPR-based therapies, discuss the opportunities and challenges that have arisen, and highlight preclinical work in several pediatric neurological diseases.},
}
@article {pmid38285472,
year = {2024},
author = {Salomonsson, SE and Clelland, CD},
title = {Building CRISPR Gene Therapies for the Central Nervous System: A Review.},
journal = {JAMA neurology},
volume = {81},
number = {3},
pages = {283-290},
doi = {10.1001/jamaneurol.2023.4983},
pmid = {38285472},
issn = {2168-6157},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Therapy ; Gene Editing ; Central Nervous System ; },
abstract = {IMPORTANCE: Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) holds the promise to arrest or cure monogenic disease if it can be determined which genetic change to create without inducing unintended cellular dysfunction and how to deliver this technology to the target organ reliably and safely. Clinical trials for blood and liver disorders, for which delivery of CRISPR is not limiting, show promise, yet no trials have begun for central nervous system (CNS) indications.<br><br>OBSERVATIONS: The CNS is arguably the most challenging target given its innate exclusion of large molecules and its defenses against bacterial invasion (from which CRISPR originates). Herein, the types of CRISPR editing (DNA cutting, base editing, and templated repair) and how these are applied to different genetic variants are summarized. The challenges of delivering genome editors to the CNS, including the viral and nonviral delivery vehicles that may ultimately circumvent these challenges, are discussed. Also, ways to minimize the potential in vivo genotoxic effects of genome editors through delivery vehicle design and preclinical off-target testing are considered. The ethical considerations of germline editing, a potential off-target outcome of any gene editing therapy, are explored. The unique regulatory challenges of a human-specific therapy that cannot be derisked solely in animal models are also discussed.<br><br>CONCLUSIONS AND RELEVANCE: An understanding of both the potential benefits and challenges of CRISPR gene therapy better informs the scientific, clinical, regulatory, and timeline considerations of developing CRISPR gene therapy for neurologic diseases.},
}
@article {pmid38441767,
year = {2024},
author = {Gebhardt, CM and Niopek, D},
title = {Anti-CRISPR Proteins and Their Application to Control CRISPR Effectors in Mammalian Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2774},
number = {},
pages = {205-231},
pmid = {38441767},
issn = {1940-6029},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems ; *Bacteriophages ; Mammals ; RNA ; Transcriptome ; },
abstract = {CRISPR-Cas effectors are powerful tools for genome and transcriptome targeting and editing. Naturally, these protein-RNA complexes are part of the microbial innate immune system, which emerged from the evolutionary arms race between microbes and phages. This coevolution has also given rise to so-called anti-CRISPR (Acr) proteins that counteract the CRISPR-Cas adaptive immunity. Acrs constitutively block cognate CRISPR-Cas effectors, e.g., by interfering with guide RNA binding, target DNA/RNA recognition, or target cleavage. In addition to their important role in microbiology and evolution, Acrs have recently gained particular attention for being useful tools and switches to regulate or fine-tune the activity of CRISPR-Cas effectors. Due to their commonly small size, high inhibition potency, and structural and mechanistic versatility, Acrs offer a wide range of potential applications for controlling CRISPR effectors in heterologous systems, including mammalian cells.Here, we review the diverse applications of Acrs in mammalian cells and organisms and discuss the underlying engineering strategies. These applications include (i) persistent blockage of CRISPR-Cas function to create write-protected cells, (ii) reduction of CRISPR-Cas off-target editing, (iii) focusing CRISPR-Cas activity to specific cell types and tissues, (iv) spatiotemporal control of CRISPR effectors based on engineered, opto-, or chemogenetic Acrs, and (v) the use of Acrs for selective binding and detection of CRISPR-Cas effectors in complex samples. We will also highlight potential future applications of Acrs in a biomedical context and point out present challenges that need to be overcome on the way.},
}
@article {pmid38441765,
year = {2024},
author = {Hirosawa, M and Saito, H},
title = {RNA Switches Using Cas Proteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2774},
number = {},
pages = {177-192},
pmid = {38441765},
issn = {1940-6029},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics ; 5' Untranslated Regions ; *CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics ; Mammals ; },
abstract = {Expanding the number of available RNA-binding proteins (RBPs) is vital to establishing posttranscriptional circuits in mammalian cells. We focused on CRISPR-Cas systems and exploited Cas proteins for their versatility as RBPs. The translation of genes encoded in an mRNA becomes regulatable by a Cas protein by inserting a crRNA/sgRNA sequence recognizable by the specific Cas protein into its 5'UTR. These Cas protein-responsive switches vastly expand the available tools in synthetic biology because of the wide range of Cas protein orthologs that can be used as trigger proteins.Here, we describe the design principle of Cas protein-responsive switches, both plasmid and RNA versions, using Streptococcus pyogenes Cas9 (SpCas9) as an example and show an example of its use in mammalian cells, HEK293FT cells.},
}
@article {pmid38441656,
year = {2024},
author = {Yarra, R and Krysan, PJ},
title = {An SpG-Cas9-based cytosine base editor expands the scope of genome editing in carrot plants.},
journal = {Plant cell reports},
volume = {43},
number = {3},
pages = {82},
pmid = {38441656},
issn = {1432-203X},
support = {2022-67013-37077//National Institute of Food and Agriculture/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Daucus carota/genetics ; Gene Editing ; Cytosine ; },
abstract = {SpG Cas9 significantly expands the genome editing scope in carrot with NGN PAM recognition.},
}
@article {pmid38441497,
year = {2024},
author = {Liu, M and Ding, Y and Ye, Q and Wu, S and Gu, Q and Chen, L and Zhang, Y and Wei, X and Deng, M and Zhang, J and Wu, Q and Wang, J},
title = {Cold-tolerance mechanisms in foodborne pathogens: Escherichia coli and Listeria monocytogenes as examples.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/10408398.2024.2322141},
pmid = {38441497},
issn = {1549-7852},
abstract = {The cold chain is an integral part of the modern food industry. Low temperatures can effectively alleviate food loss and the transmission of foodborne diseases caused by microbial reproduction. However, recent reports have highlighted shortcomings in the current cold chain technology's ability to prevent and control cold-tolerant foodborne pathogens. Furthermore, it has been observed that certain cold-chain foods have emerged as new sources of infection for foodborne disease outbreaks. Consequently, there is a pressing need to enhance control measures targeting cold-tolerant pathogens within the existing cold chain system. This paper aims to review the recent advancements in understanding the cold tolerance mechanisms of key model organisms, identify key issues in current research, and explore the potential of utilizing big data and omics technology in future studies.},
}
@article {pmid38440971,
year = {2024},
author = {Li, Y and Ma, R and Gao, C and Li, Z and Zheng, Y and Fang, F and Wang, C and Li, G and Du, X and Xu, C and Xu, M and Liu, R and Deng, X and Zheng, Z},
title = {Integrated bacterial transcriptome and host metabolome analysis reveals insights into "Candidatus Liberibacter asiaticus" population dynamics in the fruit pith of three citrus cultivars with different tolerance.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0405223},
doi = {10.1128/spectrum.04052-23},
pmid = {38440971},
issn = {2165-0497},
abstract = {"Candidatus Liberibacter asiaticus" (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars ("Shatangju" mandarin, "Guanxi" pomelo, and "Shatian" pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth.IMPORTANCECitrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, "Candidatus Liberibacter asiaticus" (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.},
}
@article {pmid38440193,
year = {2024},
author = {Nadeem, S and Riaz Ahmed, S and Luqman, T and Tan, DKY and Maryum, Z and Akhtar, KP and Muhy Ud Din Khan, S and Tariq, MS and Muhammad, N and Khan, MKR and Liu, Y},
title = {A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus.},
journal = {Frontiers in genetics},
volume = {15},
number = {},
pages = {1306469},
pmid = {38440193},
issn = {1664-8021},
abstract = {Cotton (Gossypium hirsutum L.) is a significant fiber crop. Being a major contributor to the textile industry requires continuous care and attention. Cotton is subjected to various biotic and abiotic constraints. Among these, biotic factors including cotton leaf curl virus (CLCuV) are dominant. CLCuV is a notorious disease of cotton and is acquired, carried, and transmitted by the whitefly (Bemisia tabaci). A cotton plant affected with CLCuV may show a wide range of symptoms such as yellowing of leaves, thickening of veins, upward or downward curling, formation of enations, and stunted growth. Though there are many efforts to protect the crop from CLCuV, long-term results are not yet obtained as CLCuV strains are capable of mutating and overcoming plant resistance. However, systemic-induced resistance using a gene-based approach remained effective until new virulent strains of CLCuV (like Cotton Leaf Curl Burewala Virus and others) came into existence. Disease control by biological means and the development of CLCuV-resistant cotton varieties are in progress. In this review, we first discussed in detail the evolution of cotton and CLCuV strains, the transmission mechanism of CLCuV, the genetic architecture of CLCuV vectors, and the use of pathogen and nonpathogen-based approaches to control CLCuD. Next, we delineate the uses of cutting-edge technologies like genome editing (with a special focus on CRISPR-Cas), next-generation technologies, and their application in cotton genomics and speed breeding to develop CLCuD resistant cotton germplasm in a short time. Finally, we delve into the current obstacles related to cotton genome editing and explore forthcoming pathways for enhancing precision in genome editing through the utilization of advanced genome editing technologies. These endeavors aim to enhance cotton's resilience against CLCuD.},
}
@article {pmid38262570,
year = {2024},
author = {Alhazza, A and Mahdipoor, P and Hall, R and Manda, A and Lohan, S and Parang, K and Aliabadi, HM},
title = {Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {195},
number = {},
pages = {106708},
doi = {10.1016/j.ejps.2024.106708},
pmid = {38262570},
issn = {1879-0720},
mesh = {*CRISPR-Cas Systems ; Ribonucleoproteins/genetics ; *Peptide Nucleic Acids ; RNA, Small Interfering ; Polyethylene Glycols ; Lipids ; Peptides ; },
abstract = {With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.},
}
@article {pmid38216671,
year = {2024},
author = {Gelsinger, DR and Vo, PLH and Klompe, SE and Ronda, C and Wang, HH and Sternberg, SH},
title = {Bacterial genome engineering using CRISPR-associated transposases.},
journal = {Nature protocols},
volume = {19},
number = {3},
pages = {752-790},
pmid = {38216671},
issn = {1750-2799},
support = {EB031935//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; 1R01EB031935//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; AI168976//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 2R01AI132403//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; HG011650//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 00034095//Pew Charitable Trusts/ ; FG-2020-12881//Alfred P. Sloan Foundation/ ; Career Scientist Award//Irma T. Hirschl Trust (Irma T. Hirschl Charitable Trust)/ ; Postdoctoral Diversity Enrichment Program//Burroughs Wellcome Fund (BWF)/ ; 1016691//Burroughs Wellcome Fund (BWF)/ ; 1R21AI146817//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; 1R01DK118044//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; MCB-2025515//National Science Foundation (NSF)/ ; W911NF-22-2-0210//U.S. Department of Defense (United States Department of Defense)/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Transposases/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genome, Bacterial ; DNA ; Escherichia coli/genetics ; CRISPR-Cas Systems/genetics ; Genetic Engineering/methods ; Gene Editing ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in Escherichia coli at efficiencies approaching ~100%. Moreover, they generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1-2 weeks using available plasmid constructs and standard molecular biology techniques.},
}
@article {pmid38158001,
year = {2024},
author = {Salem, AR and Bryant, WB and Doja, J and Griffin, SH and Shi, X and Han, W and Su, Y and Verin, AD and Miano, JM},
title = {Prime editing in mice with an engineered pegRNA.},
journal = {Vascular pharmacology},
volume = {154},
number = {},
pages = {107269},
doi = {10.1016/j.vph.2023.107269},
pmid = {38158001},
issn = {1879-3649},
support = {K99 HL169827/HL/NHLBI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; DNA/genetics ; Nucleotides ; },
abstract = {CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3'-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells. Here, we show the inability of three-component CRISPR or conventional PE to incorporate a nonsynonymous substitution in the Capn2 gene, expected to disrupt a phosphorylation site (S50A) in CAPN2. In contrast, an epegRNA with the same protospacer correctly installed the desired edit in two founder mice, as evidenced by robust genotyping assays for the detection of subtle nucleotide substitutions. Long-read sequencing demonstrated sequence fidelity around the edited site as well as top-ranked distal off-target sites. Western blotting and histological analysis of lipopolysaccharide-treated lung tissue revealed a decrease in phosphorylation of CAPN2 and notable alleviation of inflammation, respectively. These results demonstrate the first successful use of an epegRNA for germline transmission in an animal model and provide a solution to targeting essential developmental genes that otherwise may be challenging to edit.},
}
@article {pmid38103196,
year = {2023},
author = {Zhu, Y and Tahara, SM and Tsukamoto, H and Machida, K},
title = {Protocol for generation of humanized HCC mouse model and cancer-driver mutations using CRISPR-Cas9.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102389},
pmid = {38103196},
issn = {2666-1667},
support = {I01 BX001991/BX/BLRD VA/United States ; R24 AA012885/AA/NIAAA NIH HHS/United States ; R01 AA018857/AA/NIAAA NIH HHS/United States ; P50 AA011999/AA/NIAAA NIH HHS/United States ; P30 CA014089/CA/NCI NIH HHS/United States ; R01 AA025204/AA/NIAAA NIH HHS/United States ; P30 DK048522/DK/NIDDK NIH HHS/United States ; U01 AA027681/AA/NIAAA NIH HHS/United States ; IK6 BX004205/BX/BLRD VA/United States ; R21 AA025470/AA/NIAAA NIH HHS/United States ; },
mesh = {Mice ; Animals ; Humans ; *Carcinoma, Hepatocellular/genetics/pathology ; *Liver Neoplasms/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; Disease Models, Animal ; },
abstract = {We detail procedures for generating a humanized mouse model of hepatocellular carcinoma (HCC) recapitulating genetic mutations associated with metabolic liver diseases (MLD). We humanized liver parenchymal, non-parenchymal, and hematopoietic cells. We employed CRISPR-Cas9-based ARID1A knockout and constitutively active CTNNB1 knockin combined with an alcohol Western diet to generate cancer-driver mutations commonly found in MLD-HCC patients. This HCC model facilitates the study of tumor-promoting gene-environment interactions. For complete details on the use and execution of this protocol, please refer to Yeh et al.[1].},
}
@article {pmid38048220,
year = {2023},
author = {Yang, X and Duan, S and Li, Z and Wang, Z and Kon, N and Zhang, Z and Gu, W},
title = {Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102762},
pmid = {38048220},
issn = {2666-1667},
support = {R01 CA254970/CA/NCI NIH HHS/United States ; R01 CA258390/CA/NCI NIH HHS/United States ; R35 CA253059/CA/NCI NIH HHS/United States ; R35 GM118015/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Ferroptosis/genetics ; Genome ; Gene Library ; Computational Biology/methods ; },
abstract = {Ferroptosis, an iron-dependent programmed cell death triggered by excessive lipid peroxidation, has shown promising therapeutic potentials in human diseases. Here, we describe a protocol of a CRISPR-Cas9 loss-of-function screen to identify regulators in response to different inducers of ferroptosis. We emphasize the steps of library amplification, drug treatment, high-throughput sequencing preparation, and bioinformatics analysis using model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK). We also present a method to discover the regulators of ferroptosis and verify the potential targets efficiently. For complete details on use and execution of this protocol, please refer to Yang et al. (2023).[1].},
}
@article {pmid38041822,
year = {2023},
author = {Luo, J and Lu, C and Wang, M and Yang, X},
title = {Protocol for generating mutant zebrafish using CRISPR-Cas9 followed by quantitative evaluation of vascular formation.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102753},
pmid = {38041822},
issn = {2666-1667},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems ; *CRISPR-Cas Systems/genetics ; Zebrafish/genetics ; Animals, Genetically Modified/genetics ; Mutagenesis ; },
abstract = {The use of vascular-specific transgenic zebrafish provides advantages for identifying new mutations affecting angiogenesis and vascular development. Here, we present a protocol for establishing, screening, and phenotyping CRISPR-Cas9-based mutagenesis in fluorescently labeled transgenic zebrafish. We describe steps for designing single-guide RNA (sgRNA) oligos, synthesizing sgRNA and Cas9 mRNA, and microinjection and generation of mutant lines. We then detail procedures for visualizing dynamic vasculature and quantitatively evaluating vascular formation in transgenic zebrafish. For complete details on the use and execution of this protocol, please refer to Luo et al.[1].},
}
@article {pmid37967017,
year = {2023},
author = {Wang, P and Cao, Z and Wang, Q and Ma, X and Wang, N and Chen, L and Zhao, Y and Miao, L},
title = {Protocol for CRISPR-Cas9-mediated genome editing to study spermatogenesis in Caenorhabditis elegans.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102720},
pmid = {37967017},
issn = {2666-1667},
mesh = {Animals ; Male ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics ; Semen ; Spermatogenesis/genetics ; },
abstract = {Gene silencing by P-element-induced wimpy testis-interacting RNAs is a mechanism to maintain genome integrity in germ cells. Here, we present a protocol for knockin or knockout editing of male germline genome mediated by CRISPR-Cas9 technology in Caenorhabditis elegans. We describe steps for constructing edited plasmids, microinjecting worms with these plasmids, and screening edited worms. We then detail procedures for dissecting released sperm and their observation with fluorescence microscopy. Engineered worms provide a model for studying hermaphrodite/male fertility or protein localization in vivo. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).[1].},
}
@article {pmid37956708,
year = {2024},
author = {Wu, W and Yin, Y and Huang, J and Yang, R and Li, Q and Pan, J and Zhang, J},
title = {CRISPR/Cas9-meditated gene knockout in pigs proves that LGALS12 deficiency suppresses the proliferation and differentiation of porcine adipocytes.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1869},
number = {3},
pages = {159424},
doi = {10.1016/j.bbalip.2023.159424},
pmid = {37956708},
issn = {1879-2618},
mesh = {Swine ; Animals ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Adipocytes/metabolism ; Adipogenesis/genetics ; Cell Proliferation ; },
abstract = {LGALS12, also known as galectin12, belongs to the galectin family with β-galactoside-binding activity. We previously reported that LGALS12 is an important regulator of adipogenesis in porcine adipocytes in vitro, but its value in pig breeding needed to be explored in vivo. In this study, we used CRISPR/Cas9 to construct porcine fetal fibroblasts (PFFs) with a 43 bp deletion in LGALS12 exon 2. Using these PFFs as donor cells, a LGALS12 knockout pig model was generated via somatic cell nuclear transfer. Primary cultures of porcine intramuscular (IM) and subcutaneous (SC) adipocytes were established using cells from LGALS12 knockout pigs and wild-type pigs. A comparison of these cells proved that LGALS12 deficiency suppresses cell proliferation via the RAS-p38MAPK pathway and promotes lipolysis via the PKA pathway in both IM and SC adipocytes. In addition, we observed AKT activation only in IM adipocytes and suppression of the Wnt/β-catenin only in SC adipocytes. Our findings suggest that LGALS12 deficiency affects the adipogenesis of IM and SC adipocytes through different mechanisms.},
}
@article {pmid37948185,
year = {2023},
author = {Sahu, S and Sullivan, T and Southon, E and Caylor, D and Geh, J and Sharan, SK},
title = {Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102702},
pmid = {37948185},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; Genomics ; DNA ; },
abstract = {Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.[1].},
}
@article {pmid37922314,
year = {2023},
author = {Ravi, NS and George, A and Mohankumar, KM},
title = {Protocol for arrayed gRNA screening by base editors in mammalian cell lines using lentiviral system.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102668},
pmid = {37922314},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; Lentivirus/genetics ; Mammals ; Cell Line ; },
abstract = {Base editing, a CRISPR-based genome engineering technique, enables precise single-nucleotide modifications while minimizing double-strand breaks. Here, we present a protocol for arrayed mutagenesis using base editors to identify regulatory elements within the gamma-globin locus. We describe steps for guide RNA (gRNA) cloning into lentiviral vectors, establishing stable cell lines with base editor expression, transducing gRNAs, and assessing editing efficiency. This protocol can be applied to diverse genomic regions and cell lines for arrayed screening, facilitating genetic research, and target discovery. For complete details on the use and execution of this protocol, please refer to Ravi et al. (2022)[1].},
}
@article {pmid37889758,
year = {2023},
author = {Li, Z and Reint, G and Haapaniemi, EM},
title = {Protocol for editing fibroblasts with in vitro transcribed Cas9 mRNA and profile off-target editing by optimized GUIDE-seq.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102662},
pmid = {37889758},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics ; Gene Editing/methods ; DNA/genetics ; },
abstract = {CRISPR-Cas9 gene editing is an efficient technique to modify specific sites/regions of DNA. Delivery of the Cas9 by mRNA is particularly promising in pre-clinical genome editing applications for its transient, nonintegrating feature. However, the off-target of Cas9-gRNA still remains a concern and needs a specific monitor. Here, we present a revised protocol to edit fibroblasts by in vitro transcribed Cas9 mRNA and profile its off-target effect by the optimized GUIDE-seq method. This protocol can also be applied to other cell lines. For complete details on the use and execution of this protocol, please refer to Ganna Reint et al. (2021).[1].},
}
@article {pmid37773752,
year = {2023},
author = {Bril, O and Schwarzmueller, LJ and Moreno, LF and Vermeulen, L and Léveillé, N},
title = {Identifying essential long non-coding RNAs in cancer using CRISPRi-based dropout screens.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102588},
pmid = {37773752},
issn = {2666-1667},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics ; *Neoplasms/diagnosis/genetics ; Genome, Human ; },
abstract = {Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens.},
}
@article {pmid37738119,
year = {2023},
author = {Wu, Y and Sidharta, M and Zhong, A and Persily, B and Li, M and Zhou, T},
title = {Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102583},
pmid = {37738119},
issn = {2666-1667},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Pluripotent Stem Cells ; Mutation ; Genome ; Gene Editing/methods ; },
abstract = {Prime editing introduces single-nucleotide polymorphism changes, small deletions, or insertions at a specific genome site without double-stranded DNA breaks or the need for the donor template. Here, we present a protocol to design, conduct, and evaluate prime editing in human pluripotent stem cells. We describe steps for pegRNA and nicking sgRNA design and cloning, the prime editing tool electroporation, and the efficiency evaluation using Miseq. We elaborate the process of GBA (N370S) mutation induction and correction as an example. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).[1].},
}
@article {pmid37729058,
year = {2023},
author = {Ten Hacken, E and Gruber, M and Hernández-Sánchez, M and Hoffmann, GB and Baranowski, K and Redd, RA and Clement, K and Livak, K and Wu, CJ},
title = {Generation of mouse models carrying B cell restricted single or multiplexed loss-of-function mutations through CRISPR-Cas9 gene editing.},
journal = {STAR protocols},
volume = {4},
number = {4},
pages = {102165},
pmid = {37729058},
issn = {2666-1667},
support = {P01 CA206978/CA/NCI NIH HHS/United States ; R01 CA216273/CA/NCI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Disease Models, Animal ; Mutation ; *Lymphoproliferative Disorders/genetics ; },
abstract = {Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitro transduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders. For complete details on the use and execution of this protocol, please refer to ten Hacken et al.,[1] ten Hacken et al.,[2] and ten Hacken et al.[3].},
}
@article {pmid38440123,
year = {2024},
author = {Jung, WJ and Park, SJ and Cha, S and Kim, K},
title = {Factors affecting the cleavage efficiency of the CRISPR-Cas9 system.},
journal = {Animal cells and systems},
volume = {28},
number = {1},
pages = {75-83},
pmid = {38440123},
issn = {1976-8354},
abstract = {The CRISPR-Cas system stands out as a promising genome editing tool due to its cost-effectiveness and time efficiency compared to other methods. This system has tremendous potential for treating various diseases, including genetic disorders and cancer, and promotes therapeutic research for a wide range of genetic diseases. Additionally, the CRISPR-Cas system simplifies the generation of animal models, offering a more accessible alternative to traditional methods. The CRISPR-Cas9 system can be used to cleave target DNA strands that need to be corrected, causing double-strand breaks (DSBs). DNA with DSBs can then be recovered by the DNA repair pathway that the CRISPR-Cas9 system uses to edit target gene sequences. High cleavage efficiency of the CRISPR-Cas9 system is thus imperative for effective gene editing. Herein, we explore several factors affecting the cleavage efficiency of the CRISPR-Cas9 system. These factors include the GC content of the protospacer-adjacent motif (PAM) proximal and distal regions, single-guide RNA (sgRNA) properties, and chromatin state. These considerations contribute to the efficiency of genome editing.},
}
@article {pmid38439699,
year = {2024},
author = {Lee, Y and Hwang, S and Kim, W and Kim, JH and Palsson, BO and Cho, BK},
title = {CRISPR-aided genome engineering for secondary metabolite biosynthesis in Streptomyces.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jimb/kuae009},
pmid = {38439699},
issn = {1476-5535},
abstract = {The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent secondary metabolite biosynthetic gene clusters for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications.},
}
@article {pmid38437567,
year = {2024},
author = {Tsuchida, CA and Wasko, KM and Hamilton, JR and Doudna, JA},
title = {Targeted nonviral delivery of genome editors in vivo.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {11},
pages = {e2307796121},
doi = {10.1073/pnas.2307796121},
pmid = {38437567},
issn = {1091-6490},
mesh = {*Commerce ; *Gene Editing ; Genetic Therapy ; RNA, Messenger ; Ribonucleoproteins ; },
abstract = {Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.},
}
@article {pmid38437470,
year = {2024},
author = {Wang, T and Zeng, H and Liu, Q and Qian, W and Li, Y and Liu, J and Xu, R},
title = {Establishment of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus.},
journal = {Polish journal of microbiology},
volume = {73},
number = {1},
pages = {39-48},
pmid = {38437470},
issn = {2544-4646},
mesh = {Cats ; Animals ; Female ; *Recombinases ; *Feline Panleukopenia Virus ; CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/μl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV.},
}
@article {pmid38437462,
year = {2024},
author = {Hu, X and He, P and Jiang, T and Shen, J},
title = {Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a.},
journal = {Polish journal of microbiology},
volume = {73},
number = {1},
pages = {89-97},
pmid = {38437462},
issn = {2544-4646},
mesh = {Humans ; *CRISPR-Cas Systems ; China ; Diarrhea/diagnosis ; Disease Outbreaks ; *Norovirus/genetics ; },
abstract = {Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 10[2] copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.},
}
@article {pmid38433235,
year = {2024},
author = {Terrones, M and Deben, C and Rodrigues-Fortes, F and Schepers, A and de Beeck, KO and Van Camp, G and Vandeweyer, G},
title = {CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles.},
journal = {Journal of translational medicine},
volume = {22},
number = {1},
pages = {234},
pmid = {38433235},
issn = {1479-5876},
support = {G094820N//Fonds Wetenschappelijk Onderzoek/ ; },
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung ; Protein-Tyrosine Kinases/genetics ; Crizotinib ; CRISPR-Cas Systems/genetics ; *Lung Neoplasms/drug therapy/genetics ; Proto-Oncogene Proteins ; Drug Resistance ; *Pyrazoles ; *Sulfones ; *Benzamides ; *Aminopyridines ; *Indazoles ; *Lactams ; *Pyrimidines ; },
abstract = {INTRODUCTION: The study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting.<br><br>METHODS: In the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose-response curves was the variable used to quantify the cellular response towards TKIs.<br><br>RESULTS: Spheroids derived from ROS1[G2032R] cells were significantly more resistant to repotrectinib (AOC fold change&#x2009;=&#x2009;-&#xa0;7.33), lorlatinib (AOC fold change&#x2009;=&#x2009;-&#xa0;6.17), ceritinib (AOC fold change&#x2009;=&#x2009;-&#xa0;2.8) and entrectinib (AOC fold change&#x2009;=&#x2009;-&#xa0;2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change&#x2009;=&#x2009;-&#xa0;2.35), entrectinib (AOC fold change&#x2009;=&#x2009;-&#xa0;2.44) and ceritinib (AOC fold change&#x2009;=&#x2009;-&#xa0;2.12) in targeting the ROS1 G2032R mutation. ROS1[L2026M] cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change&#x2009;=&#x2009;-&#xa0;2.19) and entrectinib (spheroid AOC fold change&#x2009;=&#x2009;-&#xa0;1.98). ROS1[S1986Y] cells were resistant only towards crizotinib in 2D (AOC fold change&#x2009;=&#x2009;-&#xa0;1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose-response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells.<br><br>CONCLUSION: In this study we knock-in for the first time in a ROS1&#x2009;+&#x2009;patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.},
}
@article {pmid38431858,
year = {2024},
author = {Galun, E},
title = {[GENE THERAPY: FROM TECHNOLOGY TO REALITY].},
journal = {Harefuah},
volume = {163},
number = {2},
pages = {97-101},
pmid = {38431858},
issn = {0017-7768},
mesh = {Humans ; *Pandemics ; *COVID-19/therapy ; Europe ; Genetic Therapy ; Technology ; },
abstract = {Gene therapy has made major achievements in the last few decades. These were in numerous medical disciplines, including metabolic, oncologic, infectious and regenerative. As of today, regulatory agencies, both in the USA and Europe, approved for clinical usage numerous gene therapy treatments. However, we are still facing a number of significant obstacles including: 1. Efficient delivery systems, 2. Immunological responses, and 3. recently we have learned that many gene therapy approaches are very expensive. The COVID-19 pandemic was a period in which genetic vaccination had proved its efficacy, by which RNA in a synthetic delivery system was very effective. This review will focus on three fast developing technologies in gene therapy: 1. CAR-T cells, 2. CRISPR-Cas and 3. RNAi.},
}
@article {pmid38428389,
year = {2024},
author = {Pacesa, M and Pelea, O and Jinek, M},
title = {Past, present, and future of CRISPR genome editing technologies.},
journal = {Cell},
volume = {187},
number = {5},
pages = {1076-1100},
doi = {10.1016/j.cell.2024.01.042},
pmid = {38428389},
issn = {1097-4172},
mesh = {Humans ; Biological Science Disciplines ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Technology ; },
abstract = {Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.},
}
@article {pmid38427670,
year = {2024},
author = {Chariou, PL and Minnar, CM and Tandon, M and Guest, MR and Chari, R and Schlom, J and Gameiro, SR},
title = {Generation of murine tumor models refractory to αPD-1/-L1 therapies due to defects in antigen processing/presentation or IFNγ signaling using CRISPR/Cas9.},
journal = {PloS one},
volume = {19},
number = {3},
pages = {e0287733},
pmid = {38427670},
issn = {1932-6203},
mesh = {Animals ; Mice ; *Antigen Presentation ; B7-H1 Antigen ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *Programmed Cell Death 1 Receptor/genetics ; RNA, Guide, CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.},
}
@article {pmid38427560,
year = {2024},
author = {Workman, RE and Stoltzfus, MJ and Keith, NC and Euler, CW and Bondy-Denomy, J and Modell, JW},
title = {Anti-CRISPR proteins trigger a burst of CRISPR-Cas9 expression that enhances phage defense.},
journal = {Cell reports},
volume = {43},
number = {3},
pages = {113849},
doi = {10.1016/j.celrep.2024.113849},
pmid = {38427560},
issn = {2211-1247},
abstract = {CRISPR-Cas immune systems provide bacteria with adaptive immunity against bacteriophages, but they are often transcriptionally repressed to mitigate auto-immunity. In some cases, CRISPR-Cas expression increases in response to a phage infection, but the mechanisms of induction are largely unknown, and it is unclear whether induction occurs strongly and quickly enough to benefit the bacterial host. In S. pyogenes, Cas9 is both an immune effector and auto-repressor of CRISPR-Cas expression. Here, we show that phage-encoded anti-CRISPR proteins relieve Cas9 auto-repression and trigger a rapid increase in CRISPR-Cas levels during a single phage infective cycle. As a result, fewer cells succumb to lysis, leading to a striking survival benefit after multiple rounds of infection. CRISPR-Cas induction also reduces lysogeny, thereby limiting a route for horizontal gene transfer. Altogether, we show that Cas9 is not only a CRISPR-Cas effector and repressor but also a phage sensor that can mount an anti-anti-CRISPR transcriptional response.},
}
@article {pmid37590151,
year = {2023},
author = {Merenciano, M and Aguilera, L and González, J},
title = {Two-step CRISPR-Cas9 protocol for transposable element deletion in D. melanogaster natural populations.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102501},
pmid = {37590151},
issn = {2666-1667},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *DNA Transposable Elements/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Recombination, Genetic ; },
abstract = {We present a protocol for generating a precise deletion, without altering the genetic background of the strain, of a transposable element (TE) in a natural population of Drosophila melanogaster using two steps of CRISPR-Cas9 homology-directed repair. We describe steps for replacing the TE by a fluorescent marker and for subsequent marker removal using single-guide RNAs, repair plasmids, and microinjection. We also detail steps for screening the deletion of the TE and generating a homozygous mutant strain. For complete details on the use and execution of this protocol, please refer to Merenciano and Gonzalez.[1].},
}
@article {pmid37585297,
year = {2023},
author = {Zhang, Z},
title = {Generation of epitope tag knock-in mice with CRISPR-Cas9 to study the function of endogenous proteins.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102518},
pmid = {37585297},
issn = {2666-1667},
support = {R00 DK115766/DK/NIDDK NIH HHS/United States ; R01 DK130959/DK/NIDDK NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Epitopes/genetics ; *Gene Editing/methods ; },
abstract = {Studying endogenous proteins in mice has provided numerous insights into the physiological and pathological roles of these proteins. However, the availability and specificity of protein-specific antibodies often limit such studies. Here we present a protocol for generating epitope tag knock-in mice with CRISPR-Cas9-mediated gene editing. We discuss key considerations for tag selection and knock-in location and provide insights into CRISPR design. Subsequently, we outline the sequential steps involved in knock-in mouse generation, genotyping, and validation and explore potential applications. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).[1].},
}
@article {pmid37481731,
year = {2023},
author = {Ludwik, KA and Telugu, N and Schommer, S and Stachelscheid, H and Diecke, S},
title = {ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102406},
pmid = {37481731},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening.},
}
@article {pmid37432856,
year = {2023},
author = {Wu, L and Tan, JC and Gascoigne, NRJ},
title = {Generation of gene-of-interest double allele knockout clones in primary human T cells by CRISPR.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102445},
pmid = {37432856},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Alleles ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; T-Lymphocytes ; Clone Cells ; },
abstract = {Gene-of-interest (GOI) knockout is an important technique to study the genetic mechanisms of T cells. Here, we present a protocol to generate GOI double allele gene knockouts in primary human T cells by CRISPR, thus depleting proteins of interest expressed intracellularly or extracellularly in primary T cells. We describe steps for gRNA selection and efficiency validation, homology-directed repair (HDR) DNA template design and cloning, and genome editing and HDR gene insertion. We then detail clone isolation and GOI knockout validation. For complete details on the use and execution of this protocol, please refer to Wu et al.[1].},
}
@article {pmid37432855,
year = {2023},
author = {du Halgouet, A and Darbois, A and Alphonse, A and Yvorra, T and Colombeau, L and Rodriguez, R and Lantz, O and Salou, M},
title = {Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102419},
pmid = {37432855},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Mucosal-Associated Invariant T Cells ; CRISPR-Cas Systems/genetics ; Genomics ; Mice, Knockout ; Promoter Regions, Genetic ; },
abstract = {Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε[-/-] mice and characterizing them in the skin. For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).[1].},
}
@article {pmid37432853,
year = {2023},
author = {Zeng, X and Wang, S and Liang, M and Wang, W and Jiang, Y and Xu, F and Liu, L and Yan, H and Tong, Y and Zhang, L and Tan, GY},
title = {An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102435},
pmid = {37432853},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA ; Cloning, Molecular ; Genomics ; },
abstract = {Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al.[1].},
}
@article {pmid37421616,
year = {2023},
author = {Geyer, F and Geyer, M and Klapproth, S and Wolff, KD and Nieberler, M},
title = {Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102366},
pmid = {37421616},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Squamous Cell Carcinoma of Head and Neck/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Head and Neck Neoplasms/genetics ; Cell Line ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.},
}
@article {pmid37389994,
year = {2023},
author = {Shibata, Y and Okumura, A and Mochii, M and Suzuki, KT},
title = {Protocols for transgenesis at a safe harbor site in the Xenopus laevis genome using CRISPR-Cas9.},
journal = {STAR protocols},
volume = {4},
number = {3},
pages = {102382},
pmid = {37389994},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Xenopus laevis/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Transfer Techniques ; Transgenes ; },
abstract = {We have established a new transgenesis protocol based on CRISPR-Cas9, "New and Easy XenopusTransgenesis (NEXTrans)," and identified a novel safe harbor site in African clawed frogs, Xenopus laevis. We describe steps in detail for the construction of NEXTrans plasmid and guide RNA, CRISPR-Cas9-mediated NEXTrans plasmid integration into the locus, and its validation by genomic PCR. This improved strategy allows us to simply generate transgenic animals that stably express the transgene. For complete details on the use and execution of this protocol, please refer to Shibata et al. (2022).[1].},
}
@article {pmid38427231,
year = {2024},
author = {Song, MK and Kim, YS},
title = {Targeted Modification of Epigenetic Marks Using CRISPR/dCas9-SunTag-Based Modular Epigenetic Toolkit.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2761},
number = {},
pages = {81-91},
pmid = {38427231},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Neuroblastoma/genetics ; Epigenesis, Genetic ; },
abstract = {The epigenome, consisting of chemical modifications to DNA and histone proteins, can alter gene expression. Clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) systems enable precise target gene-specific gene modulation by attaching different "effector" domains to the dCas9 protein to activate or repress specific genes. CRISPR/dCas9-SunTag is an improved system version, allowing more efficient and precise gene activation or repression by recruiting multiple copies of the protein of interest. A CRISPR/dCas9-SunTag-based modular epigenetic toolkit was developed, enabling gene-specific epigenetic architecture modulation. This protocol generated a stable SH-SY5Y cell line expressing the CRISPR/dCas9-SunTag-JARID1A system to study H3K4Me3-mediated promoter regulation at a 200-400 bp of fine resolution. The procedure involved designing sgRNAs, subcloning dCas9-5XGCN4 into pLvx-DsRed, validating epigenetic mark changes with ChIP, and validating gene expression changes with RT-qPCR. This epigenetic toolkit is valuable for researchers to understand the relationship between gene-specific epigenetic modifications and gene expression.},
}
@article {pmid38427033,
year = {2024},
author = {Hua, HM and Xu, JF and Huang, XS and Zimin, AA and Wang, WF and Lu, YH},
title = {Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c09101},
pmid = {38427033},
issn = {1520-5118},
abstract = {Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.},
}
@article {pmid38426328,
year = {2024},
author = {Yang, Z and Zhang, Z and Li, J and Chen, W and Liu, C},
title = {CRISPRlnc: a machine learning method for lncRNA-specific single-guide RNA design of CRISPR/Cas9 system.},
journal = {Briefings in bioinformatics},
volume = {25},
number = {2},
pages = {},
pmid = {38426328},
issn = {1477-4054},
support = {XDA26030301//Chinese Academy of Sciences/ ; 31970609//National Natural Science Foundation of China/ ; //Top Talents Program in Science and Technology' from Yunnan Province/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics ; Gene Editing ; Machine Learning ; },
abstract = {CRISPR/Cas9 is a promising RNA-guided genome editing technology, which consists of a Cas9 nuclease and a single-guide RNA (sgRNA). So far, a number of sgRNA prediction softwares have been developed. However, they were usually designed for protein-coding genes without considering that long non-coding RNA (lncRNA) genes may have different characteristics. In this study, we first evaluated the performances of a series of known sgRNA-designing tools in the context of both coding and non-coding datasets. Meanwhile, we analyzed the underpinnings of their varied performances on the sgRNA's specificity for lncRNA including nucleic acid sequence, genome location and editing mechanism preference. Furthermore, we introduce a support vector machine-based machine learning algorithm named CRISPRlnc, which aims to model both CRISPR knock-out (CRISPRko) and CRISPR inhibition (CRISPRi) mechanisms to predict the on-target activity of targets. CRISPRlnc combined the paired-sgRNA design and off-target analysis to achieve one-stop design of CRISPR/Cas9 sgRNAs for non-coding genes. Performance comparison on multiple datasets showed that CRISPRlnc was far superior to existing methods for both CRISPRko and CRISPRi mechanisms during the lncRNA-specific sgRNA design. To maximize the availability of CRISPRlnc, we developed a web server (http://predict.crisprlnc.cc) and made it available for download on GitHub.},
}
@article {pmid38425279,
year = {2024},
author = {Parums, DV},
title = {Editorial: First Regulatory Approvals for CRISPR-Cas9 Therapeutic Gene Editing for Sickle Cell Disease and Transfusion-Dependent β-Thalassemia.},
journal = {Medical science monitor : international medical journal of experimental and clinical research},
volume = {30},
number = {},
pages = {e944204},
pmid = {38425279},
issn = {1643-3750},
mesh = {United States ; Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *beta-Thalassemia/genetics/therapy ; *Anemia, Sickle Cell/genetics/therapy ; DNA ; },
abstract = {In 2020, Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their research on the endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (CRISPR-Cas9) method for DNA editing. On 16 November 2023, the UK Medicines and Healthcare Products Regulatory Agency (MHRA) was the first to approve the CRISPR-Cas9 gene editing therapy, Casgevy (exagamglogene autotemcel), for the treatment of patients with transfusion-dependent b-thalassemia and the treatment of sickle cell disease in patients aged ≥12 years with recurrent vaso-occlusive crises. On 8 December 2023, the US Food and Drug Administration (FDA) approved both Casgevy and Lyfgenia (lovotibeglogene autotemcel) for patients with sickle cell disease. On 15 December 2023, the European Medicines Agency (EMA) approved Casgevy for sickle cell disease and transfusion-dependent ß-thalassemia. This Editorial aims to present an update on the landmark first regulatory approvals of CRISPR-Cas9 for patients with sickle cell disease and transfusion-dependent b-thalassemia and the potential challenges for therapeutic gene (DNA) editing.},
}
@article {pmid38368507,
year = {2024},
author = {Bai, M and Lin, W and Peng, C and Song, P and Kuang, H and Lin, J and Zhang, J and Wang, J and Chen, B and Li, H and Kong, F and Jia, G and Guan, Y},
title = {Expressing a human RNA demethylase as an assister improves gene-editing efficiency in plants.},
journal = {Molecular plant},
volume = {17},
number = {3},
pages = {363-366},
doi = {10.1016/j.molp.2024.02.010},
pmid = {38368507},
issn = {1752-9867},
mesh = {Humans ; *RNA ; *Plants/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid38424590,
year = {2024},
author = {Caforio, M and Iacovelli, S and Quintarelli, C and Locatelli, F and Folgiero, V},
title = {GMP-manufactured CRISPR/Cas9 technology as an advantageous tool to support cancer immunotherapy.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {43},
number = {1},
pages = {66},
pmid = {38424590},
issn = {1756-9966},
support = {Ministry of Health (IT)//Ministry of Health (IT)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Immunotherapy ; Immunotherapy, Adoptive ; *Neoplasms/genetics/therapy ; },
abstract = {BACKGROUND: CRISPR/Cas9 system to treat human-related diseases has achieved significant results and, even if its potential application in cancer research is improving, the application of this approach in clinical practice is still a nascent technology.<br><br>MAIN BODY: CRISPR/Cas9 technology is not yet used as a single therapy to treat tumors but it can be combined with traditional treatment strategies to provide personalized gene therapy for patients. The combination with chemotherapy, radiation and immunotherapy has been proven to be a powerful means of screening, identifying, validating and correcting tumor targets. Recently, CRISPR/Cas9 technology and CAR T-cell therapies have been integrated to open novel opportunities for the production of more efficient CAR T-cells for all patients. GMP-compatible equipment and reagents are already available for several clinical-grade systems at present, creating the basis and framework for the accelerated development of novel treatment methods.<br><br>CONCLUSION: Here we will provide a comprehensive collection of the actual GMP-grade CRISPR/Cas9-mediated approaches used to support cancer therapy highlighting how this technology is opening new opportunities for treating tumors.},
}
@article {pmid38424152,
year = {2024},
author = {Lee, SR and Lee, KL and Song, SH and Joo, MD and Lee, SH and Kang, JS and Kang, SM and Idrees, M and Kim, JW and Kong, IK},
title = {Generation of Fel d 1 chain 2 genome-edited cats by CRISPR-Cas9 system.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {4987},
pmid = {38424152},
issn = {2045-2322},
mesh = {Cats ; Animals ; Humans ; CRISPR-Cas Systems ; *Hypersensitivity/complications ; Allergens/analysis ; *Asthma/etiology ; Enzyme-Linked Immunosorbent Assay ; },
abstract = {Allergens from domestic cats (Felis catus) cause allergy-related health problems worldwide. Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma. Therefore, patients with cat allergies anticipate hypoallergenic cats. We successfully generated Fel d 1 chain 2 (CH2) genome-edited cats using the CRISPR-Cas9 system in this study. T7 endonuclease 1 assay and Sanger sequencing were used to confirm the mutation in CH2 genome-edited cats. Fel d 1 level in CH2 genome-edited cats were assessed by enzyme-linked immunosorbent assay (ELISA). Remarkably, ELISA showed that the level of Fel d 1 in the CH2 homozygous genome-edited cat (Name: Alsik) was extremely low compared with that in wild type domestic cats and could be hypoallergenic cats. Additionally, we successfully cloned the CH2 homozygous genome-edited cat using cytoplasm injection clone technology. The cloned CH2 homozygous genome-edited cat was verified using microsatellite analysis. Creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward because these cats can safely approach allergic patients.},
}
@article {pmid38418907,
year = {2024},
author = {Song, J and Zhuang, Y and Yi, C},
title = {Programmable RNA base editing via targeted modifications.},
journal = {Nature chemical biology},
volume = {20},
number = {3},
pages = {277-290},
pmid = {38418907},
issn = {1552-4469},
support = {21825701//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92153303//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA/genetics ; Mutagenesis, Site-Directed ; Genome ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editors are powerful tools in biology and hold great promise for the treatment of human diseases. Advanced DNA base editing tools, such as cytosine base editor and adenine base editor, have been developed to correct permanent mistakes in genetic material. However, undesired off-target edits would also be permanent, which poses a considerable risk for therapeutics. Alternatively, base editing at the RNA level is capable of correcting disease-causing mutations but does not lead to lasting genotoxic effects. RNA base editors offer temporary and reversible therapies and have been catching on in recent years. Here, we summarize some emerging RNA editors based on A-to-inosine, C-to-U and U-to-pseudouridine changes. We review the programmable RNA-targeting systems as well as modification enzyme-based effector proteins and highlight recent technological breakthroughs. Finally, we compare editing tools, discuss limitations and opportunities, and provide insights for the future directions of RNA base editing.},
}
@article {pmid38418617,
year = {2024},
author = {Lin, C and Zhou, J and Gao, N and Liu, R and Li, G and Wang, J and Lu, G and Shen, J},
title = {Establishing a pulmonary aspergillus fumigatus infection diagnostic platform based on RPA-CRISPR-Cas12a.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {40},
number = {4},
pages = {116},
pmid = {38418617},
issn = {1573-0972},
support = {GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; serial number 95//2021 Anhui Provincial Medical and Health Key Specialty Construction Project/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; KJ2021ZD0032//2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; 2022zhyx-C61//Research Fund of Anhui Institute of translational medicine/ ; },
mesh = {*Recombinases ; Aspergillus fumigatus/genetics ; CRISPR-Cas Systems ; *Pneumonia ; Staining and Labeling ; },
abstract = {In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 10[2] copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.},
}
@article {pmid38418450,
year = {2024},
author = {Deng, X and Sun, W and Li, X and Wang, J and Cheng, Z and Sheng, G and Wang, Y},
title = {An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1806},
pmid = {38418450},
issn = {2041-1723},
support = {31930065//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32330055//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; DNA/metabolism ; Staphylococcus aureus/genetics ; *Bacteriophages/genetics/metabolism ; },
abstract = {AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two activities remains unclear. Here, we determined the cryo-EM structure of AcrIIA15 in complex with SaCas9-sgRNA to reveal the inhibitory mechanism of the Acr's C-terminal domain (CTD) in mimicking dsDNA to block protospacer adjacent motif (PAM) recognition. For the N-terminal domain (NTD), our crystal structures of the AcrIIA15-promoter DNA show that AcrIIA15 dimerizes through its NTD to recognize double-stranded (ds) DNA. Further, AcrIIA15 can simultaneously bind to both SaCas9-sgRNA and promoter DNA, creating a supercomplex of two Cas9s bound to two CTDs converging on a dimer of the NTD bound to a dsDNA. These findings shed light on AcrIIA15's inhibitory mechanisms and its autoregulation of transcription, enhancing our understanding of phage-host interactions and CRISPR defense.},
}
@article {pmid38417646,
year = {2024},
author = {Yu, J and Tang, M and Zhou, Z and Wei, Z and Wan, F and Hou, S and Li, Q and Li, Y and Tian, L},
title = {Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy.},
journal = {Acta biomaterialia},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.actbio.2024.02.030},
pmid = {38417646},
issn = {1878-7568},
abstract = {Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.},
}
@article {pmid38386411,
year = {2024},
author = {Cao, M and Bian, X and Ji, Z and Sohail, M and Zhang, F and Linhardt, RJ and Li, B and Zhang, X},
title = {Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation.},
journal = {Analytical chemistry},
volume = {96},
number = {9},
pages = {3970-3978},
doi = {10.1021/acs.analchem.4c00403},
pmid = {38386411},
issn = {1520-6882},
mesh = {*Heparin/chemistry ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Anticoagulants/pharmacology ; Escherichia coli/metabolism ; },
abstract = {Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin's high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.},
}
@article {pmid38413594,
year = {2024},
author = {Gupta, P and Goswami, SG and Kumari, G and Saravanakumar, V and Bhargava, N and Rai, AB and Singh, P and Bhoyar, RC and Arvinden, VR and Gunda, P and Jain, S and Narayana, VK and Deolankar, SC and Prasad, TSK and Natarajan, VT and Scaria, V and Singh, S and Ramalingam, S},
title = {Development of pathophysiologically relevant models of sickle cell disease and β-thalassemia for therapeutic studies.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1794},
pmid = {38413594},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Humans ; *beta-Thalassemia/genetics/therapy ; Fetal Hemoglobin/genetics/metabolism ; *Anemia, Sickle Cell/drug therapy/genetics ; Hematopoietic Stem Cells/metabolism ; Genotype ; CRISPR-Cas Systems ; },
abstract = {Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications.},
}
@article {pmid38412943,
year = {2024},
author = {Boan, AF and Delgadin, TH and Canosa, LF and Fernandino, JI},
title = {Loss of function in somatostatin receptor 5 has no impact on the growth of medaka fish due to compensation by the other paralogs.},
journal = {General and comparative endocrinology},
volume = {351},
number = {},
pages = {114478},
doi = {10.1016/j.ygcen.2024.114478},
pmid = {38412943},
issn = {1095-6840},
abstract = {Somatic growth in vertebrates is regulated endocrinologically by the somatotropic axis, headed by the growth hormone (GH) and the insulin growth factor-I (IGF-I). Somatostatin (Sst), a peptide hormone synthesized in the hypothalamus, modulates GH actions through its receptors (Sstr). Four Sstr subtypes (Sstr 1-3 and 5) have been identified in teleosts. However, little is known about whether they have a specific function or tissue expression. The aim of this study was to determine the role of sstr2 and sstr5 in the growth of the medaka (Oryzias latipes). The assessed expression pattern across diverse tissues highlighted greater prevalence of sstr1 and sstr3 in brain, intestine and muscle than in pituitary or liver. The expression of sstr2 was high in all the tissues tested, while sstr5 was predominantly expressed in the pituitary gland. A CRISPR/Cas9 sstr5 mutant with loss of function (sstr5[-/-]) was produced. Assessment of sstr5[-/-] indicated no significant difference with the wild type regarding growth parameters such as standard length, body depth, or peduncle depth. Furthermore, the functional loss of sstr5 had no impact on the response to a nutritional challenge. The fact that several sstr subtypes were upregulated in different tissues in sstr5[-/-] medaka suggests that in the mutant fish, there may be a compensatory effect on the different tissues, predominantly by sstr1 in the liver, brain and pituitary, with sstr2 being upregulated in pituitary and liver, and sstr3 only presenting differential expression in the brain. Analysis of the sstr subtype and the sstr5[-/-] fish showed that sstr5 was not the only somatostatin receptor responsible for Sst-mediated Gh regulation.},
}
@article {pmid38411032,
year = {2024},
author = {Teng, F and Guo, F and Feng, J and Lu, Y and Qi, Y},
title = {Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.},
journal = {Insect molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/imb.12901},
pmid = {38411032},
issn = {1365-2583},
support = {2021YFC2600400//National Key Research and Development Program of China/ ; },
abstract = {Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.},
}
@article {pmid38410456,
year = {2024},
author = {Kogay, R and Wolf, YI and Koonin, EV},
title = {Defense systems and horizontal gene transfer in bacteria.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {38410456},
abstract = {Horizontal gene transfer (HGT) is a fundamental process in the evolution of prokaryotes, making major contributions to diversification and adaptation. Typically, HGT is facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages that generally impose fitness costs on their hosts. However, a substantial fraction of bacterial genes is involved in defense mechanisms that limit the propagation of MGEs, raising the possibility that they can actively restrict HGT. Here we examine whether defense systems curb HGT by exploring the connections between HGT rate and the presence of 73 defense systems in 12 bacterial species. We found that only 6 defense systems, 3 of which are different CRISPR-Cas subtypes, are associated with the reduced gene gain rate on the scale of species evolution. The hosts of such defense systems tend to have a smaller pangenome size and harbor fewer phage-related genes compared to genomes lacking these systems, suggesting that these defense mechanisms inhibit HGT by limiting the integration of prophages. We hypothesize that restriction of HGT by defense systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and fitness effect of HGT in bacterial populations.},
}
@article {pmid38409199,
year = {2024},
author = {Zheng, Y and Li, Y and Zhou, K and Li, T and VanDusen, NJ and Hua, Y},
title = {Precise genome-editing in human diseases: mechanisms, strategies and applications.},
journal = {Signal transduction and targeted therapy},
volume = {9},
number = {1},
pages = {47},
pmid = {38409199},
issn = {2059-3635},
support = {R00HL143194//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy/methods ; Genome, Human/genetics ; DNA ; },
abstract = {Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these tools has led to a revolution in the life sciences, which promises to deliver novel therapies for genetic disease. Precise genome-editing can be traced back to the 1950s with the discovery of DNA's double-helix and, after 70 years of development, has evolved from crude in vitro applications to a wide range of sophisticated capabilities, including in vivo applications. Nonetheless, precise genome-editing faces constraints such as modest efficiency, delivery challenges, and off-target effects. In this review, we explore precise genome-editing, with a focus on introduction of the landmark events in its history, various platforms, delivery systems, and applications. First, we discuss the landmark events in the history of precise genome-editing. Second, we describe the current state of precise genome-editing strategies and explain how these techniques offer unprecedented precision and versatility for modifying the human genome. Third, we introduce the current delivery systems used to deploy precise genome-editing components through DNA, RNA, and RNPs. Finally, we summarize the current applications of precise genome-editing in labeling endogenous genes, screening genetic variants, molecular recording, generating disease models, and gene therapy, including ex vivo therapy and in vivo therapy, and discuss potential future advances.},
}
@article {pmid38409124,
year = {2024},
author = {Chen, K and Stahl, EC and Kang, MH and Xu, B and Allen, R and Trinidad, M and Doudna, JA},
title = {Engineering self-deliverable ribonucleoproteins for genome editing in the brain.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1727},
pmid = {38409124},
issn = {2041-1723},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Ribonucleoproteins/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Brain/metabolism ; },
abstract = {The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.},
}
@article {pmid38409014,
year = {2024},
author = {Wang, G and Wang, F and Xu, Z and Wang, Y and Zhang, C and Zhou, Y and Hui, F and Yang, X and Nie, X and Zhang, X and Jin, S},
title = {Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {59},
pmid = {38409014},
issn = {1474-760X},
support = {2022ZD0402001-04//Science and Technology Innovation 2030/ ; 2023ZD04039-01//Ministry of Agriculture and Rural Affairs/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; 32272128//the National Natural Science Foundation of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; 2023M741299//China Postdoctoral Science Foundation/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Gossypium/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism ; Mutation ; Plants/genetics ; },
abstract = {BACKGROUND: CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly.<br><br>RESULTS: Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1[L86P] and GhTFL1[K53G+S78G] mutations, we find that the GhTFL1[L86P] mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function.<br><br>CONCLUSIONS: This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.},
}
@article {pmid38407550,
year = {2024},
author = {Kang, W and Xiao, F and Zhu, X and Ling, X and Xie, S and Li, R and Yu, P and Cao, L and Lei, C and Qiu, Y and Liu, T and Nie, Z},
title = {Engineering Anti-CRISPR Proteins to Create Cas12a Protein Switches for Activatable Genome Editing and Viral Protease Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202400599},
doi = {10.1002/anie.202400599},
pmid = {38407550},
issn = {1521-3773},
abstract = {Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR-Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti-CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry-directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease-responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3-fold). These advancements enable specific proteolysis-inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR-Cas tools for controllable gene manipulation and regulation and clinical diagnostics.},
}
@article {pmid38406926,
year = {2024},
author = {Sun, S and Yang, H and Wu, Z and Zhang, S and Xu, J and Shi, P},
title = {CRISPR/Cas systems combined with DNA nanostructures for biomedical applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4cc00290c},
pmid = {38406926},
issn = {1364-548X},
abstract = {DNA nanostructures are easy to design and construct, have good biocompatibility, and show great potential in biosensing and drug delivery. Numerous distinctive and versatile DNA nanostructures have been developed and explored for biomedical applications. In addition to DNA nanostructures that are completely assembled from DNA, composite DNA nanostructures obtained by combining DNA with other organic or inorganic materials are also widely used in related research. The CRISPR/Cas system has attracted great attention as a powerful gene editing technology and is also widely used in biomedical diagnosis. Many researchers are committed to exploring new possibilities by combining DNA nanostructures with CRISPR/Cas systems. These explorations provide support for the development of new detection methods and cargo delivery pathways, provide inspiration for improving relevant gene editing platforms, and further expand the application scope of DNA nanostructures and CRISPR/Cas systems. This paper mainly reviews the design principles and biomedical applications of CRISPR/Cas combined with DNA nanostructures based on the types of DNA nanostructures. Finally, the application status, challenges and development prospects of CRISPR/Cas combined with DNA nanostructures in detection and delivery are summarized. It is expected that this review will enable researchers to better understand the current state of the field and provide insights into the application of CRISPR/Cas systems and the development of DNA nanostructures.},
}
@article {pmid38405743,
year = {2024},
author = {Aviram, N and Shilton, AK and Lyn, NG and Reis, BS and Brivanlou, A and Marraffini, LA},
title = {The Cas10 nuclease activity relieves host dormancy to facilitate spacer acquisition and retention during type III-A CRISPR immunity.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {38405743},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
abstract = {A hallmark of CRISPR immunity is the acquisition of short viral DNA sequences, known as spacers, that are transcribed into guide RNAs to recognize complementary sequences. The staphylococcal type III-A CRISPR-Cas system uses guide RNAs to locate viral transcripts and start a response that displays two mechanisms of immunity. When immunity is triggered by an early-expressed phage RNA, degradation of viral ssDNA can cure the host from infection. In contrast, when the RNA guide targets a late-expressed transcript, defense requires the activity of Csm6, a non-specific RNase. Here we show that Csm6 triggers a growth arrest of the host that provides immunity at the population level which hinders viral propagation to allow the replication of non-infected cells. We demonstrate that this mechanism leads to defense against not only the target phage but also other viruses present in the population that fail to replicate in the arrested cells. On the other hand, dormancy limits the acquisition and retention of spacers that trigger it. We found that the ssDNase activity of type III-A systems is required for the re-growth of a subset of the arrested cells, presumably through the degradation of the phage DNA, ending target transcription and inactivating the immune response. Altogether, our work reveals a built-in mechanism within type III-A CRISPR-Cas systems that allows the exit from dormancy needed for the subsistence of spacers that provide broad-spectrum immunity.},
}
@article {pmid38331306,
year = {2024},
author = {Nakanishi, T and Yamazaki, M and Tachikawa, K and Ueta, A and Kawai, M and Ozono, K and Michigami, T},
title = {Complex intrinsic abnormalities in osteoblast lineage cells of X-linked hypophosphatemia: Analysis of human iPS cell models generated by CRISPR/Cas9-mediated gene ablation.},
journal = {Bone},
volume = {181},
number = {},
pages = {117044},
doi = {10.1016/j.bone.2024.117044},
pmid = {38331306},
issn = {1873-2763},
mesh = {Humans ; Male ; Mice ; Animals ; *Familial Hypophosphatemic Rickets/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; PHEX Phosphate Regulating Neutral Endopeptidase/genetics ; Osteoblasts/metabolism ; *Hypophosphatemia/genetics ; Fibroblast Growth Factors/metabolism ; },
abstract = {X-linked hypophosphatemia (XLH) is caused by inactivating variants of the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Although the overproduction of fibroblast growth factor 23 (FGF23) is responsible for hypophosphatemia and impaired vitamin D metabolism, the pathogenesis of XLH remains unclear. We herein generated PHEX-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene ablation to an iPS clone derived from a healthy male, and analyzed PHEX-KO iPS cells with deletions extending from exons 1 to 3 and frameshifts by inducing them to differentiate into the osteoblast lineage. We confirmed the increased production of FGF23 in osteoblast lineage cells differentiated from PHEX-KO iPS cells. In vitro mineralization was enhanced in osteoblast lineage cells from PHEX-KO iPS cells than in those from isogenic control iPS cells, which reminded us of high bone mineral density and enthesopathy in patients with XLH. The extracellular level of pyrophosphate (PPi), an inhibitor of mineralization, was elevated, and this increase appeared to be partly due to the reduced activity of tissue non-specific alkaline phosphatase (TNSALP). Osteoblast lineage cells derived from PHEX-KO iPS cells also showed the increased expression of multiple molecules such as dentine matrix protein 1, osteopontin, RUNX2, FGF receptor 1 and early growth response 1. This gene dysregulation was similar to that in the osteoblasts/osteocytes of Phex-deficient Hyp mice, suggesting that common pathogenic mechanisms are shared between human XLH and Hyp mice. Moreover, we found that the phosphorylation of CREB was markedly enhanced in osteoblast lineage cells derived from PHEX-KO iPS cells, which appeared to be associated with the up-regulation of the parathyroid hormone related protein gene. PHEX deficiency also affected the response of the ALPL gene encoding TNSALP to extracellular Pi. Collectively, these results indicate that complex intrinsic abnormalities in osteoblasts/osteocytes underlie the pathogenesis of human XLH.},
}
@article {pmid38217638,
year = {2024},
author = {Sun, M and Huang, S and Zhou, Y},
title = {Enhancing sheepgrass through genomic exploration and targeted editing.},
journal = {Science China. Life sciences},
volume = {67},
number = {3},
pages = {629-630},
pmid = {38217638},
issn = {1869-1889},
mesh = {*Poaceae/genetics ; *Genome ; Genomics ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37987939,
year = {2024},
author = {Ren, J and Hai, T and Chen, Y and Sun, K and Han, Z and Wang, J and Li, C and Wang, Q and Wang, L and Zhu, H and Yu, D and Li, W and Zhao, S},
title = {Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i[Max].},
journal = {Science China. Life sciences},
volume = {67},
number = {3},
pages = {555-564},
pmid = {37987939},
issn = {1869-1889},
mesh = {Animals ; Cattle ; Swine ; *CRISPR-Cas Systems ; *Livestock/genetics ; Gene Editing/methods ; Phenotype ; DNA ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i[Max], a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i[Max] in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12i[Max] in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i[Max] for gene editing in livestock animals and demonstrated the potential application of Cas12i[Max] in the field of animal trait improvement for agricultural production.},
}
@article {pmid38403466,
year = {2024},
author = {Mishra, A and Pandey, VP},
title = {CRISPR/Cas system: A revolutionary tool for crop improvement.},
journal = {Biotechnology journal},
volume = {19},
number = {2},
pages = {e2300298},
doi = {10.1002/biot.202300298},
pmid = {38403466},
issn = {1860-7314},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; Plant Breeding ; Gene Editing ; },
abstract = {World's population is elevating at an alarming rate thus, the rising demands of producing crops with better adaptability to biotic and abiotic stresses, superior nutritional as well as morphological qualities, and generation of high-yielding varieties have led to encourage the development of new plant breeding technologies. The availability and easy accessibility of genome sequences for a number of crop plants as well as the development of various genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has opened up possibilities to develop new varieties of crop plants with superior desirable traits. However, these approaches has limitation of being more expensive as well as having complex steps and time-consuming. The CRISPR/Cas genome editing system has been intensively studied for allowing versatile target-specific modifications of crop genome that fruitfully aid in the generation of novel varieties. It is an advanced and promising technology with the potential to meet hunger needs and contribute to food production for the ever-growing human population. This review summarizes the usage of novel CRISPR/Cas genome editing tool for targeted crop improvement in stress resistance, yield, quality and nutritional traits in the desired crop plants.},
}
@article {pmid38403433,
year = {2024},
author = {Liu, S and Liu, H and Wang, X and Shi, L},
title = {The immune system of prokaryotes: potential applications and implications for gene editing.},
journal = {Biotechnology journal},
volume = {19},
number = {2},
pages = {e2300352},
doi = {10.1002/biot.202300352},
pmid = {38403433},
issn = {1860-7314},
support = {2019CDYGYB024//Fundamental Research Funds for the Central Universities/ ; 31300726//National Natural Science Foundation of China/ ; CY200118//Chongqing Primary and Middle School Innovation Talent Training Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Bacteria/genetics ; Genetic Therapy ; Immune System ; },
abstract = {Gene therapy has revolutionized the treatment of genetic diseases. Spearheading this revolution are sophisticated genome editing methods such as TALENs, ZFNs, and CRISPR-Cas, which trace their origins back to prokaryotic immune systems. Prokaryotes have developed various antiviral defense systems to combat viral attacks and the invasion of genetic elements. The comprehension of these defense mechanisms has paved the way for the development of indispensable tools in molecular biology. Among them, restriction endonuclease originates from the innate immune system of bacteria. The CRISPR-Cas system, a widely applied genome editing technology, is derived from the prokaryotic adaptive immune system. Single-base editing is a precise editing tool based on CRISPR-Cas system that involves deamination of target base. It is worth noting that prokaryotes possess deamination enzymes as part of their defense arsenal over foreign genetic material. Furthermore, prokaryotic Argonauts (pAgo) proteins, also function in anti-phage defense, play an important role in complementing the CRISPR-Cas system by addressing certain limitations it may have. Recent studies have also shed light on the significance of Retron, a reverse transcription transposon previously showed potential in genome editing, has also come to light in the realm of prokaryotic immunity. These noteworthy findings highlight the importance of studying prokaryotic immune system for advancing genome editing techniques. Here, both the origin of prokaryotic immunity underlying aforementioned genome editing tools, and potential applications of deaminase, pAgo protein and reverse transcriptase in genome editing among prokaryotes were introduced, thus emphasizing the fundamental mechanism and significance of prokaryotic immunity.},
}
@article {pmid38403402,
year = {2024},
author = {Soykan, MN and Gunes, S},
title = {Overexpression of Klotho gene using CRISPR/Cas9 induces apoptosis and inhibits cell motility in the human colorectal cancer cells.},
journal = {Biotechnology journal},
volume = {19},
number = {2},
pages = {e2300496},
doi = {10.1002/biot.202300496},
pmid = {38403402},
issn = {1860-7314},
support = {202046A113//Eskisehir Osmangazi University Scientific Research Project Commission/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Caco-2 Cells ; Gene Editing ; *Colorectal Neoplasms/genetics/metabolism ; Apoptosis/genetics ; Cell Movement/genetics ; },
abstract = {Despite advances in early detection and treatment, colorectal cancer remains one of the leading causes of cancer-related deaths. The klotho (KL) gene plays a critical role in the development and progression of colorectal cancer. This study investigates the role of the KL gene in colorectal cancer by using the CRISPR/Cas9 system to overexpress and knock out (KO) the KL gene in human colorectal cancer cells (Caco-2). The effects of the changes were assessed by gene expression analysis, flow cytometry, scratch wound closure assays, colony formation assays, and immunofluorescence staining. Our results showed that overexpression of the KL gene increased apoptosis and decreased cell motility in cancer cells, whereas knockout of the KL gene had the opposite role. The present study elucidates the mechanisms underlying this role and highlights the potential of the CRISPR/Cas9 system as a gene editing tool in cancer research. Our data suggest that activation of the KL gene may serve as a novel therapeutic strategy and biomarker for studies in colorectal cancer.},
}
@article {pmid38403398,
year = {2024},
author = {Huang, CH and Chiu, SY and Chou, YC and Wu, KJ},
title = {A refined Uni-vector prime editing system improves genome editing outcomes in mammalian cells.},
journal = {Biotechnology journal},
volume = {19},
number = {2},
pages = {e2300353},
doi = {10.1002/biot.202300353},
pmid = {38403398},
issn = {1860-7314},
support = {MOST 108-2321-B-182A-005//National Science and Technology/Ministry of Science and Technology Summit and Frontier/ ; MOST 109-2326-B-182A-002//National Science and Technology/Ministry of Science and Technology Summit and Frontier/ ; MOST 110-2326-B-182A-004//National Science and Technology/Ministry of Science and Technology Summit and Frontier/ ; MOST 111-2326-B-182A-002//National Science and Technology/Ministry of Science and Technology Summit and Frontier/ ; OMRPG3I0011//Chang Gung Memorial Hospital/ ; OMRPG3I0012//Chang Gung Memorial Hospital/ ; OMRPG3I0013//Chang Gung Memorial Hospital/ ; NMRPG3J0671//Chang Gung Memorial Hospital/ ; NMRPG3J0672//Chang Gung Memorial Hospital/ ; NMRPG3J0673//Chang Gung Memorial Hospital/ ; CORPG3J0231//Chang Gung Memorial Hospital/ ; CORPG3J0232//Chang Gung Memorial Hospital/ ; },
mesh = {Animals ; Humans ; *Gene Editing ; HeLa Cells ; Mutation ; Transfection ; *CRISPR-Cas Systems/genetics ; Mammals ; },
abstract = {Prime editing is an advanced technology in CRISPR/Cas research with increasing numbers of improved methodologies. The original multi-vector method hampers the efficiency and precision of prime editing and also has inherent difficulty in generating homozygous mutations in mammalian cells. To overcome these technical issues, we developed a Uni-vector prime editing system, wherein the major components for prime editing were constructed in all-in-one plasmids, pPE3-pPuro and pePEmax-pPuro. The Uni-vector prime editing plasmids enhance the editing efficiency of prime editing and improved the generation of homozygous mutated mammalian cell lines. The editing efficiency is dependent of the transfection efficiency. Remarkably, the Uni-vector ePE5max system achieved an impressive editing rate approximately 79% in average, even in cell lines that are traditionally difficult to transfect, such as FaDu cell line. Furthermore, it resulted in a high frequency of homozygous knocked-in cells, with a rate of 99% in HeLa and 85% in FaDu cells. Together, our Uni-vector approach simplifies the delivery of editing components and improves the editing efficiency, especially in cells with low transfection efficiency. This approach presents an advancement in the field of prime editing.},
}
@article {pmid38402312,
year = {2024},
author = {Slaman, E and Kottenhagen, L and de Martines, W and Angenent, GC and de Maagd, RA},
title = {Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {4508},
pmid = {38402312},
issn = {2045-2322},
support = {15792//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; },
mesh = {*CRISPR-Cas Systems ; *Solanum lycopersicum/genetics ; Mutagenesis ; Gene Editing ; Mutation ; },
abstract = {Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.},
}
@article {pmid38402281,
year = {2024},
author = {Zhang, E and Neugebauer, ME and Krasnow, NA and Liu, DR},
title = {Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1697},
pmid = {38402281},
issn = {2041-1723},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; F32 GM143776/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 EB027793/EB/NIBIB NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; U19 NS132315/NS/NINDS NIH HHS/United States ; U19 NS132304/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Cytosine ; *Bacteriophages/genetics ; Escherichia coli/genetics ; },
abstract = {TadA-derived cytosine base editors (TadCBEs) enable programmable C•G-to-T•A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual A•T-to-G•C editing at certain positions and lower editing efficiencies at some sequence contexts and with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual cytosine and adenine base editor, discovering mutations at N46 and Y73 in TadA that prevent A•T-to-G•C editing and improve C•G-to-T•A editing with expanded sequence-context compatibility, respectively. In E. coli, CBE6 variants offer high C•G-to-T•A editing and no detected A•T-to-G•C editing in any sequence context. In human cells, CBE6 variants exhibit broad Cas domain compatibility and retain low off-target editing despite exceeding BE4max and previous TadCBEs in on-target editing efficiency. Finally, we show that the high selectivity of CBE6 variants is well-suited for therapeutically relevant stop codon installation without creating unwanted missense mutations from residual A•T-to-G•C editing.},
}
@article {pmid38401924,
year = {2024},
author = {Zhu, X and Zhang, J and Pan, R and Zhang, K and Dai, H},
title = {CRISPR/Cas12a-mediated entropy-driven electrochemical biosensor for detection of genetically modified maize Mon810.},
journal = {Analytica chimica acta},
volume = {1296},
number = {},
pages = {342290},
doi = {10.1016/j.aca.2024.342290},
pmid = {38401924},
issn = {1873-4324},
mesh = {*Zea mays/genetics ; CRISPR-Cas Systems ; Crops, Agricultural ; Entropy ; Plants, Genetically Modified/genetics ; DNA ; *Biosensing Techniques ; },
abstract = {Genetically modified crops (GMOs) have led to significant, if not revolutionary, agricultural advances. The development of GMOs requires necessary regulations, which depend on the detection of GMOs. A sensitive and specific biosensor for the detection of transgenic crops is crucial to improve the detection efficiency of GMOs. Here, we developed a CRISPR/Cas12a-mediated entropy-driven electrochemiluminescence (ECL) biosensor for the sensitive and specific detection of MON810, the world's most widely used transgenic insect-resistant maize. We designed two crRNAs to activate CRISPR/Cas12a, allowing it to cut non-specific single strands, and we modified the DNA tetrahedron (DT) on the surface of the gold electrode to diminish non-specific adsorption. The entropy-driven chain displacement reaction with the target DNA takes place for amplification. After optimization, the biosensor has satisfactory accuracy and selectivity, with a linear range of ECL of 1-106 fM and a limit of detection (LOD) of 3.3 fM by the 3σ method. The biosensor does not require polymerase chain reaction (PCR) amplification or complex sample processing, which dramatically improves transgenic crop detection efficiency. This new biosensor achieves rapid, sensitive, and highly specific detection of transgenic crops, and has great potential for large-scale field detection of transgenic crops.},
}
@article {pmid38400086,
year = {2024},
author = {Yu, W and Liu, J and Liu, Y and Forlenza, M and Chen, H},
title = {Application of CRISPR/Cas9 for Rapid Genome Editing of Pseudorabies Virus and Bovine Herpesvirus-1.},
journal = {Viruses},
volume = {16},
number = {2},
pages = {},
pmid = {38400086},
issn = {1999-4915},
mesh = {Animals ; *Herpesvirus 1, Suid ; Gene Editing ; CRISPR-Cas Systems ; *Herpesvirus 1, Bovine/genetics/metabolism ; *Pseudorabies/prevention &amp; control ; Glycoproteins/genetics ; },
abstract = {The CRISPR/Cas9 system is widely used to manipulate viral genomes. Although Alphaherpesvirinae genomes are large and complicated to edit, in recent years several Pseudorabies virus (PRV) mutants have been successfully generated using the CRISPR/Cas9 system. However, the application of CRISPR/Cas9 editing on another member of alpha herpesviruses, bovine herpesvirus-1 (BHV-1), is rarely reported. This paper reports a rapid and straightforward approach to manipulating herpesviruses genome using CRISPR/Cas9. The recombinant plasmids contained the left and right arm of the thymidine kinase (TK) gene of PRV or of the glycoprotein I (gI) and glycoprotein E (gE) of BHV-1. Upon the cleavage of the TK or gIgE gene by Cas9 protein, this was replaced by the enhanced green fluorescence protein (eGFP) by homologous recombination. With this approach, we generated recombinant TK-/eGFP+ PRV and gIgE-/eGFP+ BHV-1 mutants and then proceeded to characterize their biological activities in vitro and in vivo. In conclusion, we showed that alpha herpesvirus, including PRV and BHV-1, can be rapidly edited using the CRISPR/Cas9 approach paving the way to the development of animal herpesvirus vaccines.},
}
@article {pmid38400062,
year = {2024},
author = {Hyder, U and Shukla, A and Challa, A and D'Orso, I},
title = {HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies.},
journal = {Viruses},
volume = {16},
number = {2},
pages = {},
pmid = {38400062},
issn = {1999-4915},
support = {R01AI114362/NH/NIH HHS/United States ; R21AI175042/NH/NIH HHS/United States ; F99CA264296/NH/NIH HHS/United States ; },
mesh = {Humans ; Proviruses/genetics ; CRISPR-Cas Systems ; *HIV-1/genetics ; Virus Latency/genetics ; *HIV Infections ; },
abstract = {HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the "host phase" and the viral TF Tat during the "viral phase", which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.},
}
@article {pmid38358884,
year = {2024},
author = {Li, J and Wu, P and Cao, Z and Huang, G and Lu, Z and Yan, J and Zhang, H and Zhou, Y and Liu, R and Chen, H and Ma, L and Luo, M},
title = {Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.},
journal = {Cell reports},
volume = {43},
number = {2},
pages = {113765},
doi = {10.1016/j.celrep.2024.113765},
pmid = {38358884},
issn = {2211-1247},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Nucleotides ; },
abstract = {The increasing emergence of Cas9 variants has attracted broad interest, as these variants were designed to expand CRISPR applications. New Cas9 variants typically feature higher editing efficiency, improved editing specificity, or alternative PAM sequences. To select Cas9 variants and gRNAs for high-fidelity and efficient genome editing, it is crucial to systematically quantify the editing performances of gRNAs and develop prediction models based on high-quality datasets. Using synthetic gRNA-target paired libraries and next-generation sequencing, we compared the activity and specificity of gRNAs of four SpCas9 variants. The nucleotide composition in the PAM-distal region had more influence on the editing efficiency of HiFi Cas9 and LZ3 Cas9. We further developed machine learning models to predict the gRNA efficiency and specificity for the four Cas9 variants. To aid users from broad research areas, the machine learning models for the predictions of gRNA editing efficiency within human genome sites are available on our website.},
}
@article {pmid38277892,
year = {2024},
author = {Zeng, Q and Cao, J and Xie, F and Zhu, L and Wu, X and Hu, X and Chen, Z and Chen, X and Li, X and Chiang, CM and Wu, H},
title = {CRISPR-Cas9-mediated chicken prmt5 gene knockout and its critical role in interferon regulation.},
journal = {Poultry science},
volume = {103},
number = {3},
pages = {103344},
pmid = {38277892},
issn = {1525-3171},
support = {R01 CA251698/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Interferons/metabolism ; *Chickens/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques/veterinary ; Cell Line ; Mammals/metabolism ; },
abstract = {Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-β activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5[-/-] DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5[-/-] DF-1 cells had increased IFN-β production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-β production and interferon-stimulated gene expression.},
}
@article {pmid38271204,
year = {2024},
author = {Geller, E and Noble, MA and Morales, M and Gockley, J and Emera, D and Uebbing, S and Cotney, JL and Noonan, JP},
title = {Massively parallel disruption of enhancers active in human neural stem cells.},
journal = {Cell reports},
volume = {43},
number = {2},
pages = {113693},
doi = {10.1016/j.celrep.2024.113693},
pmid = {38271204},
issn = {2211-1247},
support = {R01 GM094780/GM/NIGMS NIH HHS/United States ; R01 HD102030/HD/NICHD NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; Enhancer Elements, Genetic/genetics ; *Neural Stem Cells/metabolism ; Chromatin/metabolism ; Cerebral Cortex/metabolism ; },
abstract = {Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution.},
}
@article {pmid38114809,
year = {2024},
author = {},
title = {Expanding the genome-targeting scope of a compact Cas9 using phage-assisted evolution.},
journal = {Nature chemical biology},
volume = {20},
number = {3},
pages = {275-276},
pmid = {38114809},
issn = {1552-4469},
mesh = {*CRISPR-Cas Systems/genetics ; },
}
@article {pmid38052959,
year = {2024},
author = {Qi, T and Wang, Y and Yang, Y and Gao, S and Liu, J and Huang, Q and Tian, Y and Tang, J and Zheng, WV and Wang, Y},
title = {Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM.},
journal = {Nature chemical biology},
volume = {20},
number = {3},
pages = {344-352},
pmid = {38052959},
issn = {1552-4469},
support = {82222007//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82170281//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Proprotein Convertase 9 ; Adenine ; *Bacteriophages ; Endonucleases/genetics ; },
abstract = {Compact Cas9 nucleases hold great promise for therapeutic applications. Although several compact Cas9 nucleases have been developed, many genomic loci still could not be edited due to a lack of protospacer adjacent motifs (PAMs). We previously developed a compact SlugCas9 recognizing an NNGG PAM. Here we demonstrate that SlugCas9 displays comparable activity to SpCas9. We developed a simple phage-assisted evolution to engineer SlugCas9 for unique PAM requirements. Interestingly, we generated a SlugCas9 variant (SlugCas9-NNG) that could recognize an NNG PAM, expanding the targeting scope. We further developed a SlugCas9-NNG-based adenine base editor and demonstrated that it could be delivered by a single adeno-associated virus to disrupt PCSK9 splice donor and splice acceptor. These genome editors greatly enhance our ability for in vivo genome editing.},
}
@article {pmid37735239,
year = {2024},
author = {Schmidheini, L and Mathis, N and Marquart, KF and Rothgangl, T and Kissling, L and Böck, D and Chanez, C and Wang, JP and Jinek, M and Schwank, G},
title = {Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.},
journal = {Nature chemical biology},
volume = {20},
number = {3},
pages = {333-343},
pmid = {37735239},
issn = {1552-4469},
support = {180257//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome ; },
abstract = {CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N4AH and N5HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N3VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.},
}
@article {pmid37589413,
year = {2024},
author = {Xu, T and Yang, X and Feng, X and Luo, H and Luo, C and Jia, MA and Lei, L},
title = {Sensitive and Visual Detection of Brassica Yellows Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12 Assay.},
journal = {Phytopathology},
volume = {114},
number = {2},
pages = {474-483},
doi = {10.1094/PHYTO-06-23-0195-R},
pmid = {37589413},
issn = {0031-949X},
mesh = {*Brassica ; Reverse Transcription ; CRISPR-Cas Systems ; Plant Diseases ; *Nucleic Acid Amplification Techniques ; *Molecular Diagnostic Techniques ; },
abstract = {Brassica yellows virus (BrYV) is an economically important virus on cruciferous species. In this study, a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was developed for the detection of BrYV. The limit of detection of this method reached 32.8 copies of the BrYV ORF5, which is 100-fold more sensitive than the RT-LAMP method. Moreover, there was no cross-reactivity with other rapeseed-infecting RNA viruses or poleroviruses. We dried the CRISPR/Cas12a reagent in a trehalose and pullulan mixture to retain its efficacy at the RT-LAMP temperature of 63°C in order to allow portable BrYV detection in a water bath. The entire process can be performed in about 1 h, and a positive result can be rapidly and conveniently detected using a handheld UV lamp. In the field, the RT-LAMP-CRISPR/Cas12a assay was accurate and had higher sensitivity than RT-LAMP and reverse transcription-polymerase chain reaction assays. The novel RT-LAMP-CRISPR/Cas12a assay allows convenient, portable, rapid, low-cost, highly sensitive, and specific detection of BrYV and has great potential for on-site monitoring of BrYV.},
}
@article {pmid37182685,
year = {2024},
author = {Li, X and Ren, C and Huang, A and Zhao, Y and Wang, L and Shen, H and Gao, C and Chen, B and Zhu, T and Xiong, J and Zhu, D and Huang, Y and Ding, J and Yuan, Z and Ding, W and Wang, H},
title = {PIBF1 regulates multiple gene expression via impeding long-range chromatin interaction to drive the malignant transformation of HPV16 integration epithelial cells.},
journal = {Journal of advanced research},
volume = {57},
number = {},
pages = {163-180},
doi = {10.1016/j.jare.2023.04.015},
pmid = {37182685},
issn = {2090-1224},
mesh = {Humans ; Female ; Chromatin/genetics ; Human papillomavirus 16/genetics ; *Uterine Cervical Neoplasms/genetics ; *Papillomavirus Infections/genetics ; RNA, Guide, CRISPR-Cas Systems ; Carcinogenesis ; Epithelial Cells ; Human Papillomavirus Viruses ; Gene Expression ; *Pregnancy Proteins ; Suppressor Factors, Immunologic ; },
abstract = {INTRODUCTION: Human papillomavirus (HPV) integration can induce gene expression dysregulation by destroying higher-order chromatin structure in cervical cancer.<br><br>OBJECTIVES: We established a 13q22 site-specific HPV16 gene knock-in cell model to interrogate the changes in chromatin structure at the initial stages of host cell malignant transformation.<br><br>METHODS: We designed a CRISPR-Cas9 system with sgRNA targeting 13q22 site and constructed the HPV16 gene donor. Cells were cotransfected, screened, and fluorescence sorted. The whole genome sequencing (WGS) was used to confirm the precise HPV16 gene integration site. Western blot and qRT-PCR were used to measure gene expression. In vitro and in vivo analysis were performed to estimate the tumorigenic potential of the HPV16 knock-in cell model. Combined Hi-C, chromatin immunoprecipitation and RNA sequencing analyses revealed correlations between chromatin structure and gene expression. We performed a coimmunoprecipitation assay with anti-PIBF1 antibody to identify endogenous interacting proteins. In vivo analysis was used to determine the role of PIBF1 in the tumor growth of cervical cancer cells.<br><br>RESULTS: We successfully established a 13q22 site-specific HPV16 gene knock-in cell model. We found that HPV integration promoted cell proliferation, invasion and stratified growth in vitro, and monoclonal proliferation in vivo. HPV integration divided the affected topologically associated domain (TAD) into two smaller domains, and the progesterone-induced blocking factor 1 (PIBF1) gene near the integration site was upregulated, although PIBF1 was not enriched at the domain boundary by CUT-Tag signal analysis. Moreover, PIBF1 was found to interact with the cohesin complex off chromatin to reduce contact domain formation by disrupting the cohesin ring-shaped structure, causing dysregulation of tumorigenesis-related genes. Xenograft experiments determined the role of PIBF1 in the proliferation in cervical cancer cells.<br><br>CONCLUSION: We highlight that PIBF1, a potential chromatin structure regulatory protein, is activated by HPV integration, which provides new insights into HPV integration-driven cervical carcinogenesis.},
}
@article {pmid38400039,
year = {2024},
author = {Pathak, R and Eliscovich, C and Mena, I and Cupic, A and Rutkowska, M and Chandran, K and Jangra, RK and García-Sastre, A and Singer, RH and Kalpana, GV},
title = {Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence.},
journal = {Viruses},
volume = {16},
number = {2},
pages = {},
pmid = {38400039},
issn = {1999-4915},
support = {R01 DA043169/DA/NIDA NIH HHS/United States ; S10 OD026852/OD/NIH HHS/United States ; R01 DK110063/DK/NIDDK NIH HHS/United States ; 75N93021C00014/AI/NIAID NIH HHS/United States ; P30 CA013330/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *SARS-CoV-2/genetics/metabolism ; *COVID-19/metabolism ; RNA Replication ; In Situ Hybridization, Fluorescence/methods ; Reactive Oxygen Species/metabolism ; Subgenomic RNA ; RNA, Guide, CRISPR-Cas Systems ; Fluorescent Antibody Technique ; Proteins/metabolism ; RNA, Viral/genetics/metabolism ; },
abstract = {SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19.},
}
@article {pmid38399687,
year = {2024},
author = {Calderón, IL and Barros, MJ and Fernández-Navarro, N and Acuña, LG},
title = {Detection of Nucleic Acids of the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-Based Assay.},
journal = {Microorganisms},
volume = {12},
number = {2},
pages = {},
pmid = {38399687},
issn = {2076-2607},
support = {11201070//Agencia Nacional de Investigaci&#xf3;n y Desarrollo/ ; 1221610//Agencia Nacional de Investigaci&#xf3;n y Desarrollo/ ; },
abstract = {Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.},
}
@article {pmid38395579,
year = {2024},
author = {Huang, R and Li, M and Qu, Z and Liu, Y and Lu, X and Li, R and Zou, L},
title = {Label-free fluorescence detection of mercury ions based on thymine-mercury-thymine structure and CRISPR-Cas12a.},
journal = {Food research international (Ottawa, Ont.)},
volume = {180},
number = {},
pages = {114058},
doi = {10.1016/j.foodres.2024.114058},
pmid = {38395579},
issn = {1873-7145},
mesh = {Spectrometry, Fluorescence/methods ; *Thymine/chemistry ; CRISPR-Cas Systems ; *Mercury/chemistry ; Ions/chemistry ; },
abstract = {In this work, we developed a novel label-free fluorescent sensor for the highly sensitive detection of mercury ions (Hg[2+]) based on the coordination chemistry of thymine-Hg[2+]-thymine (T-Hg[2+]-T) structures and the properties of CRISPR-Cas12a systems. Most notably, two T-rich sequences (a blocker and an activator) were designed to form stable double-stranded structures in the presence of Hg[2+] via the T-Hg[2+]-T base pairing. The formation of T-T mismatched double-stranded DNA between the blocker and the activator prevented the cleavage of G-rich sequences by Cas12a, allowing them to fold into G-quadruplex-thioflavin T complexes, resulting in significantly enhanced fluorescence. Under the optimized conditions, the developed sensor showed an excellent response for Hg[2+] detection in the linear range of 0.05 to 200 nM with a detection limit of 23 pM. Moreover, this fluorescent sensor exhibited excellent selectivity and was successfully used for the detection of Hg[2+] in real samples of Zhujiang river water and tangerine peel, demonstrating its potential in environmental monitoring and food safety applications.},
}
@article {pmid38353672,
year = {2024},
author = {Yang, R and Xie, S and Zhou, B and Guo, M and Fan, J and Su, F and Ji, Z and Chen, Y and Li, B},
title = {Postamplifying Cas12a Activation through Hybridization Chain Reaction-Triggered Fluorescent Nanocluster Formation for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS applied materials &amp; interfaces},
volume = {16},
number = {8},
pages = {9890-9899},
doi = {10.1021/acsami.3c18732},
pmid = {38353672},
issn = {1944-8252},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; Biological Assay ; Coloring Agents ; Copper ; DNA ; *Nucleic Acids ; *Biosensing Techniques ; },
abstract = {CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.},
}
@article {pmid38320372,
year = {2024},
author = {Navarro-Serna, S and Piñeiro-Silva, C and Fernández-Martín, I and Dehesa-Etxebeste, M and López de Munain, A and Gadea, J},
title = {Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production.},
journal = {Theriogenology},
volume = {218},
number = {},
pages = {111-118},
doi = {10.1016/j.theriogenology.2024.01.040},
pmid = {38320372},
issn = {1879-3231},
mesh = {Humans ; Animals ; Swine/genetics ; Animals, Genetically Modified ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/veterinary/methods ; Fertilization in Vitro/veterinary ; Oocytes ; Electroporation/veterinary/methods ; *Virus Diseases/veterinary ; *Swine Diseases/genetics ; },
abstract = {Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.},
}
@article {pmid38395113,
year = {2024},
author = {Li, X and Han, J and Yang, J and Zhang, H},
title = {The structural biology of type III CRISPR-Cas systems.},
journal = {Journal of structural biology},
volume = {216},
number = {1},
pages = {108070},
doi = {10.1016/j.jsb.2024.108070},
pmid = {38395113},
issn = {1095-8657},
abstract = {CRISPR-Cas system is an RNA-guided adaptive immune system widespread in bacteria and archaea. Among them, type III CRISPR-Cas systems are the most ancient throughout the CRISPR-Cas family, proving anti-phage defense through a crRNA-guided RNA targeting manner and possessing multiple enzymatic activities. Type III CRISPR-Cas systems comprise four typical members (type III-A to III-D) and two atypical members (type III-E and type III-F), providing immune defense through distinct mechanisms. Here, we delve into structural studies conducted on three well-characterized members: the type III-A, III-B, and III-E systems, provide an overview of the structural insights into the crRNA-guided target RNA cleavage, self/non-self discrimination, and the target RNA-dependent regulation of enzymatic subunits in the effector complex.},
}
@article {pmid38390600,
year = {2023},
author = {Lopes, R and Prasad, MK},
title = {Beyond the promise: evaluating and mitigating off-target effects in CRISPR gene editing for safer therapeutics.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1339189},
pmid = {38390600},
issn = {2296-4185},
abstract = {Over the last decade, CRISPR has revolutionized drug development due to its potential to cure genetic diseases that currently do not have any treatment. CRISPR was adapted from bacteria for gene editing in human cells in 2012 and, remarkably, only 11 years later has seen it's very first approval as a medicine for the treatment of sickle cell disease and transfusion-dependent beta-thalassemia. However, the application of CRISPR systems is associated with unintended off-target and on-target alterations (including small indels, and structural variations such as translocations, inversions and large deletions), which are a source of risk for patients and a vital concern for the development of safe therapies. In recent years, a wide range of methods has been developed to detect unwanted effects of CRISPR-Cas nuclease activity. In this review, we summarize the different methods for off-target assessment, discuss their strengths and limitations, and highlight strategies to improve the safety of CRISPR systems. Finally, we discuss their relevance and application for the pre-clinical risk assessment of CRISPR therapeutics within the current regulatory context.},
}
@article {pmid38383653,
year = {2024},
author = {Reardon, S},
title = {MEGA-CRISPR tool gives a power boost to cancer-fighting cells.},
journal = {Nature},
volume = {626},
number = {8001},
pages = {940},
pmid = {38383653},
issn = {1476-4687},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/genetics/immunology/therapy ; *RNA Editing/genetics ; *Immunotherapy, Adoptive/methods/trends ; },
}
@article {pmid38374637,
year = {2024},
author = {Ma, J and Park, SW and Kim, G and Kim, CS and Chang, HX and Chilvers, MI and Sang, H},
title = {Characterization of SsHog1 and Shk1 Using Efficient Gene Knockout Systems through Repeated Protoplasting and CRISPR/Cas9 Ribonucleoprotein Approaches in Sclerotinia sclerotiorum.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {8},
pages = {4237-4245},
doi = {10.1021/acs.jafc.3c08093},
pmid = {38374637},
issn = {1520-5118},
mesh = {Gene Knockout Techniques ; *CRISPR-Cas Systems ; Histidine Kinase/genetics ; *Ascomycota/metabolism ; },
abstract = {Sclerotinia sclerotiorum is the causal agent of sclerotinia stem rot in over 400 plant species. In a previous study, the group III histidine kinase gene of S. sclerotiorum (Shk1) revealed its involvement in iprodione and fludioxonil sensitivity and osmotic stress. To further investigate the fungicide sensitivity associated with the high-osmolarity glycerol (HOG) pathway, we functionally characterized SsHog1, which is the downstream kinase of Shk1. To generate knockout mutants, split marker transformation combined with a newly developed repeated protoplasting method and CRISPR/Cas9 ribonucleoprotein (RNP) delivery approach were used. The pure SsHog1 and Shk1 knockout mutants showed reduced sensitivity to fungicides and increased sensitivity to osmotic stress. In addition, the SsHog1 knockout mutants demonstrated reduced virulence compared to Shk1 knockout mutants and wild-type. Our results indicate that the repeated protoplasting method and RNP approach can generate genetically pure homokaryotic mutants and SsHog1 is involved in osmotic adaptation, fungicide sensitivity, and virulence in S. sclerotiorum.},
}
@article {pmid38366220,
year = {2024},
author = {Lenharo, M},
title = {Move over, CRISPR: RNA-editing therapies pick up steam.},
journal = {Nature},
volume = {626},
number = {8001},
pages = {933-934},
doi = {10.1038/d41586-024-00275-6},
pmid = {38366220},
issn = {1476-4687},
mesh = {Humans ; *Clinical Trials as Topic/legislation &amp; jurisprudence/standards ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods/trends ; *RNA/genetics ; *RNA Editing/genetics ; },
}
@article {pmid38355417,
year = {2024},
author = {Wang, W and Wang, B and Li, Q and Tian, R and Lu, X and Peng, Y and Sun, J and Bai, J and Gao, Z and Sun, X},
title = {Ultrasensitive Detection Strategy of Norovirus Based on a Dual Enhancement Strategy: CRISPR-Responsive Self-Assembled SNA and Isothermal Amplification.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {8},
pages = {4415-4425},
doi = {10.1021/acs.jafc.4c00557},
pmid = {38355417},
issn = {1520-5118},
mesh = {*Norovirus/genetics ; Gold ; *Metal Nanoparticles ; *Nucleic Acids ; Cell Nucleus ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Spherical nucleic acids (SNAs) have been used to construct various nanobiosensors with gold nanoparticles (AuNPs) as nuclei. The SNAs play a critical role in biosensing due to their various physical and chemical properties, programmability, and specificity recognition ability. In this study, CRISPR-responsive self-assembled spherical nucleic acid (CRISPR-rsSNA) detection probes were constructed by conjugating fluorescein-labeled probes to the surface of AuNPs to improve the sensing performance. Also, the mechanism of ssDNA and the role of different fluorescent groups in the self-assembly process of CRISPR-rsSNA were explored. Then, CRISPR-rsSNA and reverse transcription-recombinase polymerase amplification (RT-RPA) were combined to develop an ultrasensitive fluorescence-detection strategy for norovirus. In the presence of the virus, the target RNA sequence of the virus was transformed and amplified by RT-RPA. The resulting dsDNA activated the trans-cleavage activity of CRISPR cas12a, resulting in disintegrating the outer nucleic acid structure of the CRISPR-rsSNA at a diffusible rate, which released reporter molecules. Norovirus was quantitated by fluorescence detection. This strategy facilitated the detection of the norovirus at the attomolar level. An RT-RPA kit for norovirus detected would be developed based on this method. The proposed method would be used for the detection of different viruses just by changing the target RNA and crRNA of the CRISPR cas12a system which provided a foundation for high-throughput detection of various substances.},
}
@article {pmid38344988,
year = {2024},
author = {Liao, C and Cui, J and Gao, M and Wang, B and Ito, K and Guo, Y and Zhang, B},
title = {Dual-sgRNA CRISPRa System for Enhanced MK-7 Production and Salmonella Infection Mitigation in Bacillus subtilis natto Applied to Caco-2 Cells.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {8},
pages = {4301-4316},
doi = {10.1021/acs.jafc.3c08866},
pmid = {38344988},
issn = {1520-5118},
mesh = {Humans ; Bacillus subtilis/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Caco-2 Cells ; *Soy Foods ; Fermentation ; *Salmonella Infections/prevention &amp; control ; },
abstract = {This study optimized the menaquinone-7 (MK-7) synthetic pathways in Bacillus subtilis (B. subtilis) natto NB205, a strain that originated from natto, to enhance its MK-7 production. Utilizing mutation breeding, we developed NBMK308, a mutant strain that demonstrated a significant 117.23% increase in MK-7 production. A comprehensive transcriptome analysis identified two key genes, ispA and ispE, as being critical in MK-7 synthesis. The dual-sgRNA CRISPRa system was utilized to achieve precise regulation of ispA and ispE in the newly engineered strain, A3E3. This strategic modulation resulted in a significant enhancement of MK-7 production, achieving increases of 20.02% and 201.41% compared to traditional overexpression systems and the original strain NB205, respectively. Furthermore, the fermentation supernatant from A3E3 notably inhibited Salmonella invasion in Caco-2 cells, showcasing its potential for combating such infections. The safety of the dual-sgRNA CRISPRa system was confirmed through cell assays. The utilization of the dual-sgRNA CRISPRa system in this study was crucial for the precise regulation of key genes in MK-7 synthesis, leading to a remarkable increase in production and demonstrating additional therapeutic potential in inhibiting pathogenic infections. This approach effectively combined the advantages of microbial fermentation and biotechnology, addressing health and nutritional challenges.},
}
@article {pmid38303561,
year = {2024},
author = {Kim, JH and Yu, J and Kim, JY and Park, YJ and Bae, S and Kang, KK and Jung, YJ},
title = {Phenotypic characterization of pre-harvest sprouting resistance mutants generated by the CRISPR/Cas9-geminiviral replicon system in rice.},
journal = {BMB reports},
volume = {57},
number = {2},
pages = {79-85},
pmid = {38303561},
issn = {1976-670X},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Phenotype ; },
abstract = {Pre-harvest sprouting is a critical phenomenon involving germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. In this paper, we generated HDR mutant lines with one region SNP (C/T) and an insertion of 6 bp (GGT/GGTGGCGGC) in OsERF1 genes for pre-harvest sprouting (PHS) resistance using CRISPR/Cas9 and a geminiviral replicon system. The incidence of HDR was 2.6% in transformed calli. T1 seeds were harvested from 12 HDR-induced calli and named ERF1-hdr line. Molecular stability, key agronomic properties, physiological properties, and biochemical properties of target genes in the ERF1-hdr line were investigated for three years. The ERF1-hdr line showed significantly enhanced seed dormancy and pre-harvest sprouting resistance. qRT-PCR analysis suggested that enhanced ABA signaling resulted in a stronger phenotype of PHS resistance. These results indicate that efficient HDR can be achieved through SNP/InDel replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits. [BMB Reports 2024; 57(2): 79-85].},
}
@article {pmid38301655,
year = {2024},
author = {Baudrier, L and Benamozig, O and Langley, J and Chopra, S and Kalashnikova, T and Benaoudia, S and Singh, G and Mahoney, DJ and Wright, NAM and Billon, P},
title = {One-pot DTECT enables rapid and efficient capture of genetic signatures for precision genome editing and clinical diagnostics.},
journal = {Cell reports methods},
volume = {4},
number = {2},
pages = {100698},
doi = {10.1016/j.crmeth.2024.100698},
pmid = {38301655},
issn = {2667-2375},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mutation/genetics ; Genomics ; },
abstract = {The detection of genomic sequences and their alterations is crucial for basic research and clinical diagnostics. However, current methodologies are costly and time-consuming and require outsourcing sample preparation, processing, and analysis to genomic companies. Here, we establish One-pot DTECT, a platform that expedites the detection of genetic signatures, only requiring a short incubation of a PCR product in an optimized one-pot mixture. One-pot DTECT enables qualitative, quantitative, and visual detection of biologically relevant variants, such as cancer mutations, and nucleotide changes introduced by prime editing and base editing into cancer cells and human primary T cells. Notably, One-pot DTECT achieves quantification accuracy for targeted genetic signatures comparable with Sanger and next-generation sequencing. Furthermore, its effectiveness as a diagnostic platform is demonstrated by successfully detecting sickle cell variants in blood and saliva samples. Altogether, One-pot DTECT offers an efficient, versatile, adaptable, and cost-effective alternative to traditional methods for detecting genomic signatures.},
}
@article {pmid38265239,
year = {2024},
author = {Maroc, L and Shaker, H and Shapiro, RS},
title = {Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system.},
journal = {mSphere},
volume = {9},
number = {2},
pages = {e0076123},
pmid = {38265239},
issn = {2379-5042},
support = {RGPIN-2018-4914//Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; PJT 162195//Canadian Institutes of Health Research (CIHR)/ ; //Canada Research Chairs (Chaires de recherche du Canada)/ ; //NSERC CREATE/ ; },
mesh = {*Antifungal Agents/pharmacology/therapeutic use ; *Candida glabrata/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; Biofilms ; },
abstract = {The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.IMPORTANCENakaseomyces (formerly Candida) glabrata is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in N. glabrata. We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.},
}
@article {pmid38224450,
year = {2024},
author = {Finocchio, G and Koopal, B and Potocnik, A and Heijstek, C and Westphal, AH and Jinek, M and Swarts, DC},
title = {Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA.},
journal = {Nucleic acids research},
volume = {52},
number = {4},
pages = {2012-2029},
pmid = {38224450},
issn = {1362-4962},
support = {ERC-CoG-820152/ERC_/European Research Council/International ; 016.Veni.192.072/NWO_/Dutch Research Council/Netherlands ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Prokaryotic Cells ; Signal Transduction ; Immune System ; NAD+ Nucleosidase ; },
abstract = {In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.},
}
@article {pmid38180826,
year = {2024},
author = {Kozaeva, E and Nielsen, ZS and Nieto-Domínguez, M and Nikel, PI},
title = {The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria.},
journal = {Nucleic acids research},
volume = {52},
number = {4},
pages = {e19},
pmid = {38180826},
issn = {1362-4962},
support = {NNF17CC0026768//Novo Nordisk Foundation/ ; 713683//European Union's Horizon2020/ ; 40979//Villum Experiment program/ ; NNF20CC0035580//Novo Nordisk Foundation/ ; NNF18OC0034818//LiFe/ ; NNF21OC0067996//TARGET/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cytidine/genetics ; Adenine ; Nucleotides ; Gram-Negative Bacteria/genetics ; },
abstract = {A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.},
}
@article {pmid38078368,
year = {2024},
author = {Andrysiak, K and Machaj, G and Priesmann, D and Woźnicka, O and Martyniak, A and Ylla, G and Krüger, M and Pyza, E and Potulska-Chromik, A and Kostera-Pruszczyk, A and Łoboda, A and Stępniewski, J and Dulak, J},
title = {Dysregulated iron homeostasis in dystrophin-deficient cardiomyocytes: correction by gene editing and pharmacological treatment.},
journal = {Cardiovascular research},
volume = {120},
number = {1},
pages = {69-81},
pmid = {38078368},
issn = {1755-3245},
support = {2018/30/A/NZ3/00412//MAESTRO/ ; Z/H20/0023//National Science Centre/ ; #2019/35/B/NZ3/02817//OPUS/ ; //Biochemistry, Biophysics and Biotechnology at Jagiellonian University/ ; //Strategic Programme Excellence Initiative/ ; },
mesh = {Humans ; Dystrophin ; Myocytes, Cardiac/metabolism ; Gene Editing/methods ; Proteomics ; *Induced Pluripotent Stem Cells/metabolism ; *Muscular Dystrophy, Duchenne/drug therapy/genetics ; *Cardiomyopathies/metabolism ; Homeostasis ; Iron/metabolism/pharmacology/therapeutic use ; CRISPR-Cas Systems ; },
abstract = {AIMS: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD.<br><br>METHODS AND RESULTS: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM.<br><br>CONCLUSION: To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy.},
}
@article {pmid37991105,
year = {2024},
author = {Hu, H and Zhang, Y and Yu, F},
title = {A CRISPR/Cas9-based vector system enables the fast breeding of selection-marker-free canola with Rcr1-rendered clubroot resistance.},
journal = {Journal of experimental botany},
volume = {75},
number = {5},
pages = {1347-1363},
pmid = {37991105},
issn = {1460-2431},
support = {//Agriculture and Agri-Food Canada/ ; },
mesh = {CRISPR-Cas Systems ; Plant Breeding ; *Brassica napus/genetics ; *Brassica/genetics ; *Brassica rapa/genetics ; },
abstract = {Breeding for disease resistance in major crops is of crucial importance for global food security and sustainability. However, common biotechnologies such as traditional transgenesis or genome editing do not provide an ideal solution, whereas transgenic crops free of selection markers such as cisgenic/intragenic crops might be suitable. In this study, after cloning and functional verification of the Rcr1 gene for resistance to clubroot (Plasmodiophora brassicae), we confirmed that the genes Rcr1, Rcr2, Rcr4, and CRa from Brassica rapa crops and the resistance gene from B. napus oilseed rape cv. 'Mendel' on chromosome A03 were identical in their coding regions. We also determined that Rcr1 has a wide distribution in Brassica breeding materials and renders potent resistance against multiple representative clubroot strains in Canada. We then modified a CRISPR/Cas9-based cisgenic vector system and found that it enabled the fast breeding of selection-marker-free transgenic crops with add-on traits, with selection-marker-free canola (B. napus) germplasms with Rcr1-rendered stable resistance to clubroot disease being successfully developed within 2 years. In the B. napus background, the intragenic vector system was able to remove unwanted residue sequences from the final product with high editing efficiency, and off-target mutations were not detected. Our study demonstrates the potential of applying this breeding strategy to other crops that can be transformed by Agrobacterium. Following the streamlined working procedure, intragenic germplasms can be developed within two generations, which could significantly reduce the breeding time and labor compared to traditional introgression whilst still achieving comparable or even better breeding results.},
}
@article {pmid38390187,
year = {2024},
author = {Zhou, J and Li, Z and Seun Olajide, J and Wang, G},
title = {CRISPR/Cas-based nucleic acid detection strategies: Trends and challenges.},
journal = {Heliyon},
volume = {10},
number = {4},
pages = {e26179},
pmid = {38390187},
issn = {2405-8440},
abstract = {CRISPR/Cas systems have become integral parts of nucleic acid detection apparatus and biosensors. Various CRISPR/Cas systems such as CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, CRISPR/Cas14 and CRISPR/Cas3 utilize different mechanisms to detect or differentiate biological activities and nucleotide sequences. Usually, CRISPR/Cas-based nucleic acid detection systems are combined with polymerase chain reaction, loop-mediated isothermal amplification, recombinase polymerase amplification and transcriptional technologies for effective diagnostics. Premised on these, many CRISPR/Cas-based nucleic acid biosensors have been developed to detect nucleic acids of viral and bacterial pathogens in clinical samples, as well as other applications in life sciences including biosecurity, food safety and environmental assessment. Additionally, CRISPR/Cas-based nucleic acid detection systems have showed better specificity compared with other molecular diagnostic methods. In this review, we give an overview of various CRISPR/Cas-based nucleic acid detection methods and highlight some advances in their development and components. We also discourse some operational challenges as well as advantages and disadvantages of various systems. Finally, important considerations are offered for the improvement of CRISPR/Cas-based nucleic acid testing.},
}
@article {pmid38389535,
year = {2024},
author = {Patakova, P and Vasylkivska, M and Sedlar, K and Jureckova, K and Bezdicek, M and Lovecka, P and Branska, B and Kastanek, P and Krofta, K},
title = {Whole genome sequencing and characterization of Pantoea agglomerans DBM 3797, endophyte, isolated from fresh hop (Humulus lupulus L.).},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1305338},
pmid = {38389535},
issn = {1664-302X},
abstract = {BACKGROUND: This paper brings new information about the genome and phenotypic characteristics of Pantoea agglomerans strain DBM 3797, isolated from fresh Czech hop (Humulus lupulus) in the Saaz hop-growing region. Although P. agglomerans strains are frequently isolated from different materials, there are not usually thoroughly characterized even if they have versatile metabolism and those isolated from plants may have a considerable potential for application in agriculture as a support culture for plant growth.<br><br>METHODS: P. agglomerans DBM 3797 was cultured under aerobic and anaerobic conditions, its metabolites were analyzed by HPLC and it was tested for plant growth promotion abilities, such as phosphate solubilization, siderophore and indol-3-acetic acid productions. In addition, genomic DNA was extracted, sequenced and de novo assembly was performed. Further, genome annotation, pan-genome analysis and selected genome analyses, such as CRISPR arrays detection, antibiotic resistance and secondary metabolite genes identification were carried out.<br><br>RESULTS AND DISCUSSION: The typical appearance characteristics of the strain include the formation of symplasmata in submerged liquid culture and the formation of pale yellow colonies on agar. The genetic information of the strain (in total 4.8 Mb) is divided between a chromosome and two plasmids. The strain lacks any CRISPR-Cas system but is equipped with four restriction-modification systems. The phenotypic analysis focused on growth under both aerobic and anaerobic conditions, as well as traits associated with plant growth promotion. At both levels (genomic and phenotypic), the production of siderophores, indoleacetic acid-derived growth promoters, gluconic acid, and enzyme activities related to the degradation of complex organic compounds were found. Extracellular gluconic acid production under aerobic conditions (up to 8 g/l) is probably the result of glucose oxidation by the membrane-bound pyrroloquinoline quinone-dependent enzyme glucose dehydrogenase. The strain has a number of properties potentially beneficial to the hop plant and its closest relatives include the strains also isolated from the aerial parts of plants, yet its safety profile needs to be addressed in follow-up research.},
}
@article {pmid38388680,
year = {2024},
author = {Toufikuzzaman, M and Hassan Samee, MA and Sohel Rahman, M},
title = {CRISPR-DIPOFF: an interpretable deep learning approach for CRISPR Cas-9 off-target prediction.},
journal = {Briefings in bioinformatics},
volume = {25},
number = {2},
pages = {},
pmid = {38388680},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; *Deep Learning ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; Neural Networks, Computer ; },
abstract = {CRISPR Cas-9 is a groundbreaking genome-editing tool that harnesses bacterial defense systems to alter DNA sequences accurately. This innovative technology holds vast promise in multiple domains like biotechnology, agriculture and medicine. However, such power does not come without its own peril, and one such issue is the potential for unintended modifications (Off-Target), which highlights the need for accurate prediction and mitigation strategies. Though previous studies have demonstrated improvement in Off-Target prediction capability with the application of deep learning, they often struggle with the precision-recall trade-off, limiting their effectiveness and do not provide proper interpretation of the complex decision-making process of their models. To address these limitations, we have thoroughly explored deep learning networks, particularly the recurrent neural network based models, leveraging their established success in handling sequence data. Furthermore, we have employed genetic algorithm for hyperparameter tuning to optimize these models' performance. The results from our experiments demonstrate significant performance improvement compared with the current state-of-the-art in Off-Target prediction, highlighting the efficacy of our approach. Furthermore, leveraging the power of the integrated gradient method, we make an effort to interpret our models resulting in a detailed analysis and understanding of the underlying factors that contribute to Off-Target predictions, in particular the presence of two sub-regions in the seed region of single guide RNA which extends the established biological hypothesis of Off-Target effects. To the best of our knowledge, our model can be considered as the first model combining high efficacy, interpretability and a desirable balance between precision and recall.},
}
@article {pmid38364329,
year = {2024},
author = {Li, P and Ye, Y and Li, Y and Xie, Z and Ye, L and Huang, J},
title = {A MoS2 nanosheet-based CRISPR/Cas12a biosensor for efficient miRNA quantification for acute myocardial infarction.},
journal = {Biosensors &amp; bioelectronics},
volume = {251},
number = {},
pages = {116129},
doi = {10.1016/j.bios.2024.116129},
pmid = {38364329},
issn = {1873-4235},
mesh = {Humans ; *MicroRNAs/analysis ; Molybdenum ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Disulfides ; *Myocardial Infarction/diagnosis/genetics ; },
abstract = {Acute myocardial infarction (AMI) represents the leading cause of cardiovascular death worldwide, and it is thus pivotal to develop effective approaches for the timely detection of AMI markers, especially possessing the characteristics of antibody-free, signal amplification, and manipulation convenience. We herein construct a MoS2 nanosheet-powered CRISPR/Cas12a sensing strategy for sensitive determination of miR-499, a superior AMI biomarker to protein markers. The presence of miR-499 at a trace level is able to induce a significantly enhanced fluorescence signal in a DNA-based molecular engineering platform, which consists of CRISPR/Cas12a enzymatic reactions and MoS2 nanosheet-controllable signal reporting components. The MoS2 nanosheets were characterized by using atomic force microscopy (AFM) and transmission electron microscope (TEM). The detection feasibility was verified by using polyacrylamide gel electrophoresis (PAGE) analysis and fluorescence measurements. The detection limit is determined as 381.78 pM with the linear range from 0.1 ⅹ 10[-9] to 13.33 ⅹ 10[-9] M in a fast manner (about 30 min). Furthermore, miRNA detection in real human serum is also conducted with desirable recovery rates (89.5 %-97.6 %), which may find potential application for the clinic diagnosis. We describe herein the first example of MoS2 nanosheet-based signal amplified fluorescence sensor for effective detection of AMI-related miRNA.},
}
@article {pmid38342386,
year = {2024},
author = {Bayat, H and Farahmand, F and Tabatabaee, SH and Shams, F and Mohammadian, O and Pourmaleki, E and Rahimpour, A},
title = {Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.},
journal = {Protein expression and purification},
volume = {217},
number = {},
pages = {106445},
doi = {10.1016/j.pep.2024.106445},
pmid = {38342386},
issn = {1096-0279},
mesh = {Cricetinae ; Animals ; *Gene Editing/methods ; Cricetulus ; CHO Cells ; *CRISPR-Cas Systems ; Deoxyribonuclease I/genetics/metabolism ; DNA, Ribosomal ; Antibodies, Monoclonal/genetics/metabolism ; },
abstract = {INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus.<br><br>METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays.<br><br>RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells.<br><br>CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.},
}
@article {pmid38240497,
year = {2024},
author = {Zhang, M and Sun, S and Liang, X and Liu, Z and Yin, J and Li, Q and Yang, S},
title = {A quaternary ammonium-based nanosystem enables delivery of CRISPR/Cas9 for cancer therapy.},
journal = {Biomaterials science},
volume = {12},
number = {5},
pages = {1197-1210},
doi = {10.1039/d3bm01629c},
pmid = {38240497},
issn = {2047-4849},
mesh = {Animals ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Ammonium Compounds ; Gene Editing ; Ribonucleoproteins/genetics ; *Neoplasms/drug therapy/genetics ; },
abstract = {Genome editing mediated by CRISPR/Cas9 is an attractive weapon for cancer therapy. However, in vivo delivery of CRISPR/Cas9 components to achieve therapeutic efficiency is still challenging. Herein, a quaternary ammonium-functionalized poly(L-lysine) and a cholesterol-modified PEG (QNP) were self-assembled with a negatively charged CRISPR Cas9/sgRNA ribonucleoprotein (RNP) to form a ternary complex (QNP/RNP). Such a delivery system of QNP exhibited multiplex genome editing capabilities in vitro (e.g., the GFP gene and the PLK1 gene). In addition, QNP/RNPPLK1 containing PLK1 sgRNA led to 30.99% of genome editing efficiency in MCF-7 cells with negligible cytotoxicity of the carrier. QNP/RNPPLK1, which was capable of simultaneously inhibiting cell proliferation, mediating cell cycle arrest and downregulating expression of PLK1, held great in vitro therapeutic efficiency. Moreover, QNP/RNPPLK1 exhibited outstanding accumulation in tumors and high biocompatibility in vivo. In an MCF-7 xenograft animal model, QNP/RNPPLK1 showed excellent anti-tumor efficacy and achieved 17.75% indels ratio. This work showcases the successful delivery of CRISPR Cas9/sgRNA RNP with enhanced genome editing efficiency and provides a potential on-demand strategy for cancer therapy.},
}
@article {pmid38224812,
year = {2024},
author = {Wang, J and Torres, IM and Shang, M and Al-Armanazi, J and Dilawar, H and Hettiarachchi, DU and Paladines-Parrales, A and Chambers, B and Pottle, K and Soman, M and Su, B and Dunham, RA},
title = {One-step knock-in of two antimicrobial peptide transgenes at multiple loci of catfish by CRISPR/Cas9-mediated multiplex genome engineering.},
journal = {International journal of biological macromolecules},
volume = {260},
number = {Pt 1},
pages = {129384},
doi = {10.1016/j.ijbiomac.2024.129384},
pmid = {38224812},
issn = {1879-0003},
mesh = {Humans ; Animals ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; *Catfishes/genetics ; Gene Editing/methods ; Transgenes/genetics ; Mammals/genetics ; },
abstract = {CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As-Cath) transgene and the myostatin (two target sites: mstn1, mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % ∼ 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh, mc4r, mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms.},
}
@article {pmid38038993,
year = {2024},
author = {Li, J and Pan, W and Zhang, S and Ma, G and Li, A and Zhang, H and Liu, L},
title = {A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system.},
journal = {The Plant journal : for cell and molecular biology},
volume = {117},
number = {5},
pages = {1604-1613},
doi = {10.1111/tpj.16575},
pmid = {38038993},
issn = {1365-313X},
support = {2022YFD1201700//National Key Research and Development Program of China/ ; 32101685//National Natural Science Foundation of China/ ; },
mesh = {*Sorghum/genetics ; Plants, Genetically Modified/genetics ; Transformation, Genetic ; Gene Editing/methods ; Agrobacterium/genetics ; Edible Grain/genetics ; CRISPR-Cas Systems ; },
abstract = {Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by-effects, we investigated the impact of GRF4-GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4-GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM-WUS. As agrobacterium effectors increase the ability of T-DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4-GIF1 with helper plasmid pVS1-VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71-fold of the original method. Compared with BBM-WUS, overexpressing GRF4-GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene-editing tool based on this GRF4-GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.},
}
@article {pmid38388140,
year = {2024},
author = {Gómez-García, F and Garcia-Gonzalez, MA},
title = {Gene editing: a near future for the treatment of genetic kidney diseases.},
journal = {Kidney international},
volume = {105},
number = {3},
pages = {430-433},
doi = {10.1016/j.kint.2024.01.004},
pmid = {38388140},
issn = {1523-1755},
mesh = {Rats ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Engineering ; *Kidney Diseases/genetics/therapy ; Kidney ; },
abstract = {The study by Chen et al. is the first to apply the revolutionary genetic engineering tool, base editing, in a rat model for the treatment of primary hyperoxaluria type 1, a disease that originates in the liver but in which the kidney is the main organ affected. This commentary contextualizes and describes the gene-editing technology applied by the authors, provides an interpretation and opinion of their results, and indicates possible future applications.},
}
@article {pmid38387899,
year = {2024},
author = {Man, JC and Cheng, J and Zhao, L},
title = {[Effect of Knocking Out HOXA5 Gene by CRISPR-Cas9-Mediated Gene Editing Technique on Proliferation of Acute Myeloid Leukemia Cells].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {32},
number = {1},
pages = {52-56},
doi = {10.19746/j.cnki.issn.1009-2137.2024.01.009},
pmid = {38387899},
issn = {1009-2137},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; *Leukemia, Myeloid, Acute/metabolism ; Genes, Homeobox ; Cell Line, Tumor ; Cell Proliferation ; Homeodomain Proteins/genetics ; },
abstract = {OBJECTIVE: To construct a acute myeloid leukemia (AML) cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique, so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells, and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML.<br><br>METHODS: The expression of HOXA5 in bone marrow mononuclear cells (BMMC) of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique, and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot, and the cell proliferation was detected by CCK-8 assay.<br><br>RESULTS: Compared with non-tumor hematological patients, the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased (P <0.05). The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique, and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased (P <0.05).<br><br>CONCLUSION: HOXA5 is highly expressed in AML cells, and knocking out HOXA5 can significantly affect the proliferation ability of AML cells, which provides a new potential therapeutic target for the precise treatment of AML.},
}
@article {pmid38387850,
year = {2024},
author = {Müller, JA and Schäffler, N and Kellerer, T and Schwake, G and Ligon, TS and Rädler, JO},
title = {Kinetics of RNA-LNP delivery and protein expression.},
journal = {European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V},
volume = {},
number = {},
pages = {114222},
doi = {10.1016/j.ejpb.2024.114222},
pmid = {38387850},
issn = {1873-3441},
abstract = {Lipid nanoparticles (LNPs) employing ionizable lipids are the most advanced technology for delivery of RNA, most notably mRNA, to cells. LNPs represent well-defined core-shell particles with efficient nucleic acid encapsulation, low immunogenicity and enhanced efficacy. While much is known about the structure and activity of LNPs, less attention is given to the timing of LNP uptake, cytosolic transfer and protein expression. However, LNP kinetics is a key factor determining delivery efficiency. Hence quantitative insight into the multi-cascaded pathway of LNPs is of interest to elucidate the mechanism of delivery. Here, we review experiments as well as theoretical modeling of the timing of LNP uptake, mRNA-release and protein expression. We describe LNP delivery as a sequence of stochastic transfer processes and review a mathematical model of subsequent protein translation from mRNA. We compile probabilities and numbers obtained from time resolved microscopy. Specifically, live-cell imaging on single cell arrays (LISCA) allows for high-throughput acquisition of thousands of individual GFP reporter expression time courses. The traces yield the distribution of mRNA life-times, expression rates and expression onset. Correlation analysis reveals an inverse dependence of gene expression efficiency and transfection onset-times. Finally, we discuss why timing of mRNA release is critical in the context of codelivery of multiple nucleic acid species as in the case of mRNA co-expression or CRISPR/Cas gene editing.},
}
@article {pmid38387349,
year = {2024},
author = {Chaudhary, V and Kumar, M and Chauhan, C and Sirohi, U and Srivastav, AL and Rani, L},
title = {Strategies for mitigation of pesticides from the environment through alternative approaches: A review of recent developments and future prospects.},
journal = {Journal of environmental management},
volume = {354},
number = {},
pages = {120326},
doi = {10.1016/j.jenvman.2024.120326},
pmid = {38387349},
issn = {1095-8630},
abstract = {Chemical-based peticides are having negative impacts on both the healths of human beings and plants as well. The World Health Organisation (WHO), reported that each year, >25 million individuals in poor nations are having acute pesticide poisoning cases along with 20,000 fatal injuries at global level. Normally, only ∼0.1% of the pesticide reaches to the intended targets, and rest amount is expected to come into the food chain/environment for a longer period of time. Therefore, it is crucial to reduce the amounts of pesticides present in the soil. Physical or chemical treatments are either expensive or incapable to do so. Hence, pesticide detoxification can be achieved through bioremediation/biotechnologies, including nano-based methodologies, integrated approaches etc. These are relatively affordable, efficient and environmentally sound methods. Therefore, alternate strategies like as advanced biotechnological tools like as CRISPR Cas system, RNAi and genetic engineering for development of insects and pest resistant plants which are directly involved in the development of disease- and pest-resistant plants and indirectly reduce the use of pesticides. Omics tools and multi omics approaches like metagenomics, genomics, transcriptomics, proteomics, and metabolomics for the efficient functional gene mining and their validation for bioremediation of pesticides also discussed from the literatures. Overall, the review focuses on the most recent advancements in bioremediation methods to lessen the effects of pesticides along with the role of microorganisms in pesticides elimination. Further, pesticide detection is also a big challenge which can be done by using HPLC, GC, SERS, and LSPR ELISA etc. which have also been described in this review.},
}
@article {pmid38280363,
year = {2024},
author = {Zhang, C and Zhao, X and Huang, Z and Li, Z and Hu, J and Liu, R and Lv, Y},
title = {Highly sensitive detection of aflatoxin B1 byCRISPR/Cas12a-assisted single nanoparticle counting.},
journal = {Food chemistry},
volume = {443},
number = {},
pages = {138557},
doi = {10.1016/j.foodchem.2024.138557},
pmid = {38280363},
issn = {1873-7072},
mesh = {Aflatoxin B1 ; CRISPR-Cas Systems ; Biological Assay ; Certification ; *Nanoparticles ; *Biosensing Techniques ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.},
}
@article {pmid38253802,
year = {2024},
author = {Liu, R and Jiang, D and Yun, Y and Feng, Z and Zheng, F and Xiang, Y and Fan, H and Zhang, J},
title = {Photoactivatable Engineering of CRISPR/Cas9-Inducible DNAzyme Probe for In Situ Imaging of Nuclear Zinc Ions.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {63},
number = {10},
pages = {e202315536},
doi = {10.1002/anie.202315536},
pmid = {38253802},
issn = {1521-3773},
support = {22004063//National Natural Science Foundation of China/ ; 22274072//National Natural Science Foundation of China/ ; 21804059//National Natural Science Foundation of China/ ; 22074076//National Natural Science Foundation of China/ ; 20200303//Natural Science Foundation of Jiangsu Province/ ; 5431ZZXM2206//State Key Laboratory of Analytical Chemistry for Life Science/ ; },
mesh = {Humans ; Mice ; Animals ; *Zinc/chemistry ; *DNA, Catalytic/metabolism ; CRISPR-Cas Systems ; HeLa Cells ; RNA, Guide, CRISPR-Cas Systems ; Metals/chemistry ; Ions/metabolism ; Acids ; },
abstract = {DNAzyme-based fluorescent probes for imaging metal ions in living cells have received much attention recently. However, employing in situ metal ions imaging within subcellular organelles, such as nucleus, remains a significant challenge. We developed a three-stranded DNAzyme probe (TSDP) that contained a 20-base-pair (20-bp) recognition site of a CRISPR/Cas9, which blocks the DNAzyme activity. When Cas9, with its specialized nuclear localization function, forms an active complex with sgRNA within the cell nucleus, it cleaves the TSDP at the recognition site, resulting in the in situ formation of catalytic DNAzyme structure. With this design, the CRISPR/Cas9-inducible imaging of nuclear Zn[2+] is demonstrated in living cells. Moreover, the superiority of CRISPR-DNAzyme for spatiotemporal control imaging was demonstrated by integrating it with photoactivation strategy and Boolean logic gate for dynamic monitoring nuclear Zn[2+] in both HeLa cells and mice. Collectively, this conceptual design expands the DNAzyme toolbox for visualizing nuclear metal ions and thus provides new analytical methods for nuclear metal-associated biology.},
}
@article {pmid38385691,
year = {2024},
author = {Zhang, H and Chen, B and Wang, Z and Peng, K and Liu, Y and Wang, Z},
title = {Resensitizing tigecycline- and colistin-resistant Escherichia coli using an engineered conjugative CRISPR/Cas9 system.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0388423},
doi = {10.1128/spectrum.03884-23},
pmid = {38385691},
issn = {2165-0497},
abstract = {Tigecycline and colistin were referred to as the "last resort" antibiotics in defending against carbapenem-resistant, Gram-negative bacterial infections, and are currently widely used in clinical treatment. However, the emergence and prevalence of plasmid-mediated tet(X4) and mcr-1 genes pose a serious threat to the therapeutic application of tigecycline and colistin, respectively. In this research, a tigecycline- and colistin-resistant bacteria resensitization system was developed based on efficient and specific DNA damage caused by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Protein 9 (Cas9) nucleases. A conjugation method was used to deliver the resensitization system, which harbors two single-guide RNAs targeting tet(X4) and mcr-1 genes and constitutively expressed Cas9. The conjugation efficiency was nearly 100% after conjugation condition optimization in vitro, and the resensitivity efficiency for clinical isolates was over 90%. In addition, when performing resensitization in vivo, the resistance marker was replaced with a glutamate-based, chromosomal, plasmid-balanced lethal system to prevent the introduction of additional resistance genes in clinical settings, making this strategy a therapeutic approach to combat the in vivo spread of antibiotic resistance genes (ARGs) among bacterial pathogens. As a proof of concept, this resensitive system can significantly decrease the counts of tigecycline- and colistin-resistant bacteria to 1% in vivo. Our study demonstrates the efficacy and adaptability of CRISPR-Cas systems as powerful and programmable antimicrobials in resensitizing tet(X4)- and mcr-1-mediated, tigecycline- and colistin-resistant strains, and opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.IMPORTANCEThe emergence of plasmid-encoded tet(X4) and mcr-1 isolated from human and animal sources has affected the treatment of tigecycline and colistin, and has posed a significant threat to public health. Tigecycline and colistin are considered as the "last line of defense" for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, so there is an urgent need to find a method that can resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR/Cas9 system, which can simultaneously resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant Escherichia coli. The counts of tigecycline- and colistin-resistant bacteria decreased to 1% in vivo after the resensitization system was administered. This study opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.},
}
@article {pmid38385549,
year = {2024},
author = {Arizala, D and Arif, M},
title = {Impact of homologous recombination on core genome evolution and host adaptation of Pectobacterium parmentieri.},
journal = {Genome biology and evolution},
volume = {},
number = {},
pages = {},
doi = {10.1093/gbe/evae032},
pmid = {38385549},
issn = {1759-6653},
abstract = {Homologous recombination is a major force mechanism driving bacterial evolution, host adaptability and acquisition of novel virulence traits. Pectobacterium parmentieri is a plant bacterial pathogen distributed worldwide, primarily affecting potatoes, by causing soft rot and blackleg diseases. The goal of this investigation was to understand the impact of homologous recombination on the genomic evolution of P. parmentieri. Analysis of P. parmentieri genomes using Roary revealed a dynamic pan-genome with 3,742 core genes and over 55% accessory genome variability. Bayesian population structure analysis identified seven lineages, indicating species heterogeneity. ClonalFrameML analysis displayed 5,125 recombination events, with the lineage 4 exhibiting the highest events. fastGEAR analysis identified 486 ancestral and 941 recent recombination events ranging 43 bp - 119 kb and 36 bp - 13.96 kb, respectively, suggesting ongoing adaptation. Notably, 11% (412 genes) of the core genome underwent recent recombination, with lineage 1 as the main donor. The prevalence of recent recombination (double compared to ancient) events implies continuous adaptation, possibly driven by global potato trade. Recombination events were found in genes involved in vital cellular processes (DNA replication, DNA repair, RNA processing, homeostasis, and metabolism), pathogenicity determinants (type secretion systems, cell-wall degrading enzymes, iron scavengers, lipopolysaccharides, flagellum, etc.), antimicrobial compounds (phenazine and colicin) and even CRISPR-Cas genes. Overall, these results emphasize the potential role of homologous recombination in P. parmentieri's evolutionary dynamics, influencing host colonization, pathogenicity, adaptive immunity, and ecological fitness.},
}
@article {pmid38384479,
year = {2024},
author = {Tanaka, M and Fujikawa, R and Sekiguchi, T and Hernandez, J and Johnson, OT and Tanaka, D and Kumafuji, K and Serikawa, T and Hoang Trung, H and Hattori, K and Mashimo, T and Kuwamura, M and Gestwicki, JE and Kuramoto, T},
title = {A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.},
journal = {Frontiers in neuroscience},
volume = {18},
number = {},
pages = {1263724},
pmid = {38384479},
issn = {1662-4548},
abstract = {Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.},
}
@article {pmid38383973,
year = {2024},
author = {Bhattacharyya, P and Mehndiratta, K and Maiti, S and Chakraborty, D},
title = {Rare genetic disorders in India: Current status, challenges, and CRISPR-based therapy.},
journal = {Journal of biosciences},
volume = {49},
number = {},
pages = {},
pmid = {38383973},
issn = {0973-7138},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Rare Diseases/epidemiology/genetics/therapy ; Genetic Therapy ; India ; },
abstract = {Rare genetic diseases are a group of life-threatening disorders affecting significant populations worldwide and posing substantial challenges to healthcare systems globally. India, with its vast population, is also no exception. The country harbors millions of individuals affected by these fatal disorders, which often result from mutations in a single gene. The emergence of CRISPR-Cas9 technology, however, has ushered in a new era of hope in genetic therapies. CRISPR-based treatments hold the potential to precisely edit and correct diseasecausing mutations, offering tailored solutions for rare genetic diseases in India. This review explores the landscape of rare genetic diseases in India along with national policies and major challenges, and examines the implications of CRISPR-based therapies for potential cure. It delves into the potential of this technology in providing personalized and effective treatments. However, alongside these promising prospects, some ethical considerations, regulatory challenges, and concerns about the accessibility of CRISPR therapies are also discussed since addressing these issues is crucial for harnessing the full power of CRISPR in tackling rare genetic diseases in India. By taking a multidisciplinary approach that combines scientific advancements, ethical principles, and regulatory frameworks, these complexities can be reconciled, paving the way for innovative and impactful healthcare solutions for rare diseases in India.},
}
@article {pmid38383786,
year = {2024},
author = {Tamulaitiene, G and Sabonis, D and Sasnauskas, G and Ruksenaite, A and Silanskas, A and Avraham, C and Ofir, G and Sorek, R and Zaremba, M and Siksnys, V},
title = {Activation of Thoeris antiviral system via SIR2 effector filament assembly.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {38383786},
issn = {1476-4687},
abstract = {To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems[1-7]. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components[8-12]. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD[+] (refs. [12-14]). Although the structure of ThsA has been solved[15], the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD[+] depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.},
}
@article {pmid38383558,
year = {2024},
author = {Ding, Y and Tous, C and Choi, J and Chen, J and Wong, WW},
title = {Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1572},
pmid = {38383558},
issn = {2041-1723},
support = {R01EB031904//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R01EB029483//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R01GM129011//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 1S10RR024523-01//U.S. Department of Health &amp; Human Services | NIH | National Center for Research Resources (NCRR)/ ; 2027045//National Science Foundation (NSF)/ ; },
mesh = {Animals ; Mice ; *RNA/genetics ; *CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL (Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13 effectors that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineer Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13 effectors, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.},
}
@article {pmid38383404,
year = {2024},
author = {Phuphisut, O and Poodeepiyasawat, A and Yoonuan, T and Watthanakulpanich, D and Thawornkuno, C and Reamtong, O and Sato, M and Adisakwattana, P},
title = {Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.},
journal = {Parasites &amp; vectors},
volume = {17},
number = {1},
pages = {80},
pmid = {38383404},
issn = {1756-3305},
support = {This research project was supported by Mahidol University (Fiscal Year 2021) through Orawan Phuphisut.//Mahidol University/ ; },
mesh = {Animals ; Humans ; *Opisthorchiasis ; *Opisthorchis/genetics ; CRISPR-Cas Systems ; Recombinases/genetics ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction ; Feces ; DNA ; },
abstract = {BACKGROUND: Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces.<br><br>METHODS: To detect O.&#xa0;viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.&#xa0;viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods.<br><br>RESULTS: The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.&#xa0;viverrini DNA. On the basis of the detection limit, the assay could detect O.&#xa0;viverrini DNA at concentrations as low as 10[-1]&#xa0;ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1&#xa0;ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.&#xa0;viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections.<br><br>CONCLUSIONS: The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.&#xa0;viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.&#xa0;viverrini infection, marking an initial step in the development of point-of-care diagnosis.},
}
@article {pmid38382707,
year = {2024},
author = {Yeh, HY and Cox, NA and Hinton, A and Berrang, ME},
title = {Detection and Distribution of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in Campylobacter jejuni Isolates from Chicken Livers.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100250},
doi = {10.1016/j.jfp.2024.100250},
pmid = {38382707},
issn = {1944-9097},
abstract = {Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide linked to consumption of undercooked broiler livers. Application of bacteriophages during poultry production has been used as an alternative approach to reduce contamination of poultry meat by Campylobacter. To make this approach effective, understanding the presence of the bacteriophage sequences in the CRISPR spacers in C. jejuni is critical as they may confer bacterial resistance to bacteriophage treatment. Therefore, in this study, we explored the distribution of the CRISPR arrays from 178 C. jejuni isolated from chicken livers between January and July 2018. Genomic DNA of C. jejuni isolates was extracted, and CRISPR type 1 sequences were amplified by PCR. Amplicons were purified and sequenced by the Sanger dideoxy sequencing method. Direct repeats (DR) and spacers of CRISPR sequences were identified using the CRISPRFinder program. Further, spacer sequences were submitted to the CRISPRTarget to identify potential homology to bacteriophage types. Even though, CRISPR-Cas is reportedly not an active system in Campylobacter, a total of 155 (87%) C. jejuni isolates were found to harbor CRISPR sequences; one type of DR was identified in all 155 isolates. The CRISPR loci lengths ranged from 97 to 431 nucleotides. The numbers of spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates, that could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases showed that most spacer sequences were homologous to Campylobacter bacteriophage DA10. The results of our study provide important information relative to development of an effective bacteriophage treatment to mitigate Campylobacter during poultry production.},
}
@article {pmid38381767,
year = {2024},
author = {Sun, W and Liu, X and Song, L and Tao, L and Lai, K and Jiang, H and Xiao, H},
title = {The TTN p. Tyr4418Ter mutation causes cardiomyopathy in human and mice.},
journal = {PloS one},
volume = {19},
number = {2},
pages = {e0296802},
pmid = {38381767},
issn = {1932-6203},
mesh = {Animals ; Humans ; Infant ; Mice ; *Cardiomyopathies/genetics ; *Cardiomyopathy, Dilated ; Connectin/genetics ; Heart ; Mutation ; },
abstract = {OBJECTIVE: To generate a mouse model carrying TTNtv Y4370* simulating the newly discovered human heterozygous nonsense TTNtv c.13254T>G (p.Tyr4418Ter) to supplement and improve the functional evidence of pathogenic mutation TTNtv c.13254T>G on the pathogenic type of dilated cardiomyopathy.<br><br>METHODS: We generated 4 mice carrying TTNtv p. Y4370* through CRISPR/Cas-mediated genome engineering. Monthly serological detection, bimonthly echocardiography, and histology evaluation were carried out to observe and compare alterations of cardiac structure and function between 4 TTN+/- mice and 4 wild-type (WT) mice.<br><br>RESULTS: For the two-month-old TTN+/- mice, serum glutamic-oxalacetic transaminase (AST), lactic dehydrogenase (LDH), and creatine kinase (CK) were significantly increased, the diastolic Left Ventricular Systolic Anterior Wall (LVAW), and the LV mass markedly rose, with the left ventricular volume displaying an increasing trend and Ejection Fraction (EF) and Fractional Shortening (FS) showing a decreasing trend. Besides, the histological evaluation showed that cardiac fibrosis level and positive rate of cardiac mast cell of TTN+/- mice were obviously increased compared with WT mice.<br><br>CONCLUSIONS: TTNtv Y4370* could lead to cardiac structure and function alterations in mice, supplementing the evidence of TTNtv c.13254T>G pathogenicity in human.},
}
@article {pmid38381340,
year = {2024},
author = {Wu, H and Chen, LL},
title = {The Functional Circular RNA Screening via RfxCas13d/BSJ-gRNA System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2765},
number = {},
pages = {173-191},
pmid = {38381340},
issn = {1940-6029},
mesh = {*RNA, Circular ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Untranslated ; RNA/genetics ; Cell Cycle ; RNA, Messenger ; },
abstract = {Although discovered decades ago, functions of circular RNAs (circRNAs) produced from exon(s) back-splicing of pre-mRNAs have only been unveiled recently. As circRNAs share overlapping sequences with their cognate linear RNAs, except for the back-splicing junction sites, it is difficult to distinguish circRNAs from cognate mRNAs in functional studies. In this chapter, we describe a programmable method for the large-scale functional circRNA screening based on the RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system. This method can be applied both in vivo and in cell to explore highly expressed circRNAs that may influence cell growth, either under natural conditions or in response to environmental stimulation, without disturbing cognate linear mRNAs.},
}
@article {pmid38381083,
year = {2024},
author = {Montagud-Martínez, R and Márquez-Costa, R and Heras-Hernández, M and Dolcemascolo, R and Rodrigo, G},
title = {On the ever-growing functional versatility of the CRISPR-Cas13 system.},
journal = {Microbial biotechnology},
volume = {17},
number = {2},
pages = {e14418},
pmid = {38381083},
issn = {1751-7915},
support = {PDC2022-133941-I00//Ministerio de Ciencia e Innovaci&#xf3;n/ ; PGC2018-101410-B-I00//Ministerio de Ciencia e Innovaci&#xf3;n/ ; PRE2019-088531//Ministerio de Ciencia e Innovaci&#xf3;n/ ; 813239//European Commission/ ; JAEINT-19-00844//Consejo Superior de Investigaciones Cientificas/ ; GVA-COVID19/2021/036//Generalitat Valenciana/ ; SEJI-2020-011//Generalitat Valenciana/ ; },
mesh = {*CRISPR-Cas Systems ; *Prokaryotic Cells ; RNA ; Ribonucleoproteins ; Synthetic Biology ; },
abstract = {CRISPR-Cas systems evolved in prokaryotes to implement a powerful antiviral immune response as a result of sequence-specific targeting by ribonucleoproteins. One of such systems consists of an RNA-guided RNA endonuclease, known as CRISPR-Cas13. In very recent years, this system is being repurposed in different ways in order to decipher and engineer gene expression programmes. Here, we discuss the functional versatility of the CRISPR-Cas13 system, which includes the ability for RNA silencing, RNA editing, RNA tracking, nucleic acid detection and translation regulation. This functional palette makes the CRISPR-Cas13 system a relevant tool in the broad field of systems and synthetic biology.},
}
@article {pmid38380498,
year = {2024},
author = {He, Z and Song, C and Li, S and Dong, C and Liao, W and Xiong, Y and Yang, S and Liu, Y},
title = {Development and Application of the CRISPR-dcas13d-eIF4G Translational Regulatory System to Inhibit Ferroptosis in Calcium Oxalate Crystal-Induced Kidney Injury.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2309234},
doi = {10.1002/advs.202309234},
pmid = {38380498},
issn = {2198-3844},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; 82270797//National Natural Science Foundation of China/ ; 82070723//National Natural Science Foundation of China/ ; 2022CFC020//Natural Science Foundation of Hubei Province/ ; 2022CFB799//Natural Science Foundation of Hubei Province/ ; RCJC20221008092723011//Shenzhen Science and Technology Program/ ; JCYJ20220818102001002//Shenzhen Science and Technology Program/ ; },
abstract = {The CRISPR-Cas system, initially for DNA-level gene editing and transcription regulation, has expanded to RNA targeting with the Cas13d family, notably the RfxCas13d. This advancement allows for mRNA targeting with high specificity, particularly after catalytic inactivation, broadening the exploration of translation regulation. This study introduces a CRISPR-dCas13d-eIF4G fusion module, combining dCas13d with the eIF4G translation regulatory element, enhancing target mRNA translation levels. This module, using specially designed sgRNAs, selectively boosts protein translation in targeted tissue cells without altering transcription, leading to notable protein expression upregulation. This system is applied to a kidney stone disease model, focusing on ferroptosis-linked GPX4 gene regulation. By targeting GPX4 with sgRNAs, its protein expression is upregulated in human renal cells and mouse kidney tissue, countering ferroptosis and resisting calcium oxalate-induced cell damage, hence mitigating stone formation. This study evidences the CRISPR-dCas13d-eIF4G system's efficacy in eukaryotic cells, presenting a novel protein translation research approach and potential kidney stone disease treatment advancements.},
}
@article {pmid38380103,
year = {2024},
author = {Zeng, D and Jiao, J and Mo, T},
title = {Combination of nucleic acid amplification and CRISPR/Cas technology in pathogen detection.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1355234},
pmid = {38380103},
issn = {1664-302X},
abstract = {Major health events caused by pathogenic microorganisms are increasing, seriously jeopardizing human lives. Currently PCR and ITA are widely used for rapid testing in food, medicine, industry and agriculture. However, due to the non-specificity of the amplification process, researchers have proposed the combination of nucleic acid amplification technology with the novel technology CRISPR for detection, which improves the specificity and credibility of results. This paper summarizes the research progress of nucleic acid amplification technology in conjunction with CRISPR/Cas technology for the detection of pathogens, which provides a reference and theoretical basis for the subsequent application of nucleic acid amplification technology in the field of pathogen detection.},
}
@article {pmid38332568,
year = {2024},
author = {Nomura, T and Kim, JS and Ishikawa, M and Suzuki, K and Mochida, K},
title = {High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.},
journal = {Microbial biotechnology},
volume = {17},
number = {2},
pages = {e14393},
pmid = {38332568},
issn = {1751-7915},
support = {21K05511//Japan Science and Technology Agency Grant-in-Aid for Scientific Research (C)/ ; JPMJOP1832//Program on Open Innovation Platform with Enterprises, Research Institute and Academia/ ; JPMJSA2204//Science and Technology Research Partnership for Sustainable Development/ ; JPMJGX23B0//Green Technologies for Excellence Program/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Euglena gracilis/genetics ; Genetic Engineering ; Ribonucleoproteins/genetics ; },
abstract = {Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.},
}
@article {pmid38325660,
year = {2024},
author = {Sedeek, K and Mohammed, N and Zhou, Y and Zuccolo, A and Sanikommu, K and Kantharajappa, S and Al-Bader, N and Tashkandi, M and Wing, RA and Mahfouz, MM},
title = {Multitrait engineering of Hassawi red rice for sustainable cultivation.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {341},
number = {},
pages = {112018},
doi = {10.1016/j.plantsci.2024.112018},
pmid = {38325660},
issn = {1873-2259},
mesh = {*Oryza/genetics ; Gene Editing ; Phenotype ; Agriculture ; CRISPR-Cas Systems ; },
abstract = {Sustainable agriculture requires locally adapted varieties that produce nutritious food with limited agricultural inputs. Genome engineering represents a viable approach to develop cultivars that fulfill these criteria. For example, the red Hassawi rice, a native landrace of Saudi Arabia, tolerates local drought and high-salinity conditions and produces grain with diverse health-promoting phytochemicals. However, Hassawi has a long growth cycle, high cultivation costs, low productivity, and susceptibility to lodging. Here, to improve these undesirable traits via genome editing, we established efficient regeneration and Agrobacterium-mediated transformation protocols for Hassawi. In addition, we generated the first high-quality reference genome and targeted the key flowering repressor gene, Hd4, thus shortening the plant's lifecycle and height. Using CRISPR/Cas9 multiplexing, we simultaneously disrupted negative regulators of flowering time (Hd2, Hd4, and Hd5), grain size (GS3), grain number (GN1a), and plant height (Sd1). The resulting homozygous mutant lines flowered extremely early (∼56 days) and had shorter stems (approximately 107 cm), longer grains (by 5.1%), and more grains per plant (by 50.2%), thereby enhancing overall productivity. Furthermore, the awns of grains were 86.4% shorter compared to unedited plants. Moreover, the modified rice grain displayed improved nutritional attributes. As a result, the modified Hassawi rice combines several desirable traits that can incentivize large-scale cultivation and reduce malnutrition.},
}
@article {pmid38325289,
year = {2024},
author = {Ali, Y and Gomez-Sanchez, CE and Plonczynski, M and Naray-Fejes-Toth, A and Fejes-Toth, G and Gomez-Sanchez, EP},
title = {mTOR Regulates Mineralocorticoid Receptor Transcriptional Activity by ULK1-Dependent and -Independent Mechanisms.},
journal = {Endocrinology},
volume = {165},
number = {4},
pages = {},
pmid = {38325289},
issn = {1945-7170},
support = {R01 HL144847/HL/NHLBI NIH HHS/United States ; U54 GM115428/GM/NIGMS NIH HHS/United States ; },
mesh = {Mice ; Rats ; Animals ; Mechanistic Target of Rapamycin Complex 1/metabolism ; *Receptors, Mineralocorticoid/genetics/metabolism ; *Aldosterone ; Ligands ; Multiprotein Complexes/metabolism ; RNA, Guide, CRISPR-Cas Systems ; TOR Serine-Threonine Kinases/metabolism ; Phosphorylation ; Regulatory-Associated Protein of mTOR ; Sirolimus/pharmacology ; Transcription Factors/metabolism ; Rapamycin-Insensitive Companion of mTOR Protein/metabolism ; Autophagy-Related Protein-1 Homolog/genetics/metabolism ; },
abstract = {The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.},
}
@article {pmid38311249,
year = {2024},
author = {Haber, Z and Sharma, D and Selvaraj, KSV and Sade, N},
title = {Is CRISPR/Cas9-based multi-trait enhancement of wheat forthcoming?.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {341},
number = {},
pages = {112021},
doi = {10.1016/j.plantsci.2024.112021},
pmid = {38311249},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems ; *Triticum/genetics ; Gene Editing ; Plants, Genetically Modified/genetics ; Genes, Plant ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have been implemented in recent years in the genome editing of eukaryotes, including plants. The original system of knocking out a single gene by causing a double-strand break (DSB), followed by non-homologous end joining (NHEJ) or Homology-directed repair (HDR) has undergone many adaptations. These adaptations include employing CRISPR/Cas9 to upregulate gene expression or to cause specific small changes to the DNA sequence of the gene-of-interest. In plants, multiplexing, i.e., inducing multiple changes by CRISPR/Cas9, is extremely relevant due to the redundancy of many plant genes, and the time- and labor-consuming generation of stable transgenic plant lines via crossing. Here we discuss relevant examples of various traits, such as yield, biofortification, gluten content, abiotic stress tolerance, and biotic stress resistance, which have been successfully manipulated using CRISPR/Cas9 in plants. While existing studies have primarily focused on proving the impact of CRISPR/Cas9 on a single trait, there is a growing interest among researchers in creating a multi-stress tolerant wheat cultivar 'super wheat', to commercially and sustainably enhance wheat yields under climate change. Due to the complexity of the technical difficulties in generating multi-target CRISPR/Cas9 lines and of the interactions between stress responses, we propose enhancing already commercial local landraces with higher yield traits along with stress tolerances specific to the respective localities, instead of generating a general 'super wheat'. We hope this will serve as the sustainable solution to commercially enhancing crop yields under both stable and challenging environmental conditions.},
}
@article {pmid38306811,
year = {2024},
author = {Esmaelpourfarkhani, M and Ramezani, M and Alibolandi, M and Abnous, K and Taghdisi, SM},
title = {CRISPR-Cas12a-based colorimetric aptasensor for aflatoxin M1 detection based on oxidase-mimicking activity of flower-like MnO2 nanozymes.},
journal = {Talanta},
volume = {271},
number = {},
pages = {125729},
doi = {10.1016/j.talanta.2024.125729},
pmid = {38306811},
issn = {1873-3573},
mesh = {*Aflatoxin M1/analysis ; Oxidoreductases ; CRISPR-Cas Systems ; Colorimetry ; Manganese Compounds ; *Biosensing Techniques/methods ; Oxides ; },
abstract = {Given the highly mutagenic and carcinogenic nature of Aflatoxin M1 (AFM1), the quantity assessment of AFM1 residues in milk and dairy products is necessary to maintain consumer health and food safety. Herein, CRISPR-Cas12a-based colorimetric aptasensor was developed using the catalytic activity of flower-like nanozymes of MnO2 and trans-cleavage property of CRISPR-Cas12a system to quantitatively detect AFM1. The basis of the developed colorimetric aptasensor relies on whether or not the CRISPR-Cas12a system is activated, as well as the contrast in oxidase-mimicking capability exhibited by flower-like MnO2 nanozymes when AFM1 is absent or present. When AFM1 is not present in the sample, single-stranded DNA (ssDNA) is degraded by the activated CRISPR-Cas12a, and the solution turns into yellow due to the catalytic activity of the nanozymes. While, in the attendance of AFM1, ssDNA degradation does not occur due to the inactivation of the CRISPR-Cas12a. Therefore, with the adsorption of the ssDNA on the MnO2 nanozymes, their catalytic activity decreases, and the solution color becomes pale yellow due to less oxidation of the chromogenic substrate. In this aptasensor, the relative absorbance changes increased linearly from 6 to 160 ng L[-1], and the detection limit was 2.1 ng L[-1]. The developed aptasensor displays a selective detection performance and a practical application for quantitative analysis of AFM1 in milk samples. The results of the introduced aptasensor open up the way to design other selective and sensitive aptasensors for the detection of other mycotoxins by substitution of the used sequences.},
}
@article {pmid38277969,
year = {2024},
author = {Lin, K and Yao, K and Li, X and Li, Q and Guo, X and You, W and Ren, W and Bian, Y and Guo, J and Sun, Z and Zhang, R and Yang, X and Li, Z and Li, B},
title = {Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod).},
journal = {Talanta},
volume = {271},
number = {},
pages = {125616},
doi = {10.1016/j.talanta.2023.125616},
pmid = {38277969},
issn = {1873-3573},
mesh = {Humans ; *Nucleic Acids ; *Whooping Cough ; CRISPR-Cas Systems ; Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.},
}
@article {pmid38277968,
year = {2024},
author = {Gu, X and Tang, Q and Kang, X and Ji, H and Shi, X and Shi, L and Pan, A and Zhu, Y and Jiang, W and Zhang, J and Liu, J and Wu, M and Wu, L and Qin, Y},
title = {A portable CRISPR-Cas12a triggered photothermal biosensor for sensitive and visual detection of Staphylococcus aureus and Listeria monocytogenes.},
journal = {Talanta},
volume = {271},
number = {},
pages = {125678},
doi = {10.1016/j.talanta.2024.125678},
pmid = {38277968},
issn = {1873-3573},
mesh = {Humans ; *Listeria monocytogenes/genetics ; Staphylococcus aureus/genetics ; CRISPR-Cas Systems ; *Staphylococcal Infections ; DNA ; *Biosensing Techniques ; },
abstract = {The detection of foodborne pathogens is crucial for ensuring the maintenance of food safety. In the present study, a portable CRISPR-Cas12a triggered photothermal biosensor integrating branch hybrid chain reaction (bHCR) and DNA metallization strategy for sensitive and visual detection of foodborne pathogens was proposed. The sheared probes were utilized to block the locker probes, which enabled preventing the assembly of bHCR in the absence of target bacteria, while target bacteria can activate the cleavage of sheared probes through CRISPR-Cas12a. Therefore, the locker probes functioned as initiating chains, triggering the formation of the branching double-stranded DNA consisting of H1, H2, and H3. The silver particles, which were in situ deposited on the DNA structure, functioned as a signal factor for conducting photothermal detection. Staphylococcus aureus and Listeria monocytogenes were selected as the foodborne pathogens to verify the analytical performance of this CRISPR-Cas12a triggered photothermal sensor platform. The sensor exhibited a sensitive detection with a low detection limit of 1 CFU/mL, while the concentration ranged from 10[0] to 10[8] CFU/mL. Furthermore, this method could efficiently detect target bacteria in multiple food samples. The findings demonstrate that this strategy can serve as a valuable reference for the development of a portable platform enabling quantitative analysis, visualization, and highly sensitive detection of foodborne bacteria.},
}
@article {pmid38245957,
year = {2024},
author = {Li, H and Wang, Y and Wan, Y and Li, M and Xu, J and Wang, Q and Wu, D},
title = {Stimuli-responsive incremental DNA machine auto-catalyzed CRISPR-Cas12a feedback amplification permits ultrasensitive molecular diagnosis of esophageal cancer-related microRNA.},
journal = {Talanta},
volume = {271},
number = {},
pages = {125675},
doi = {10.1016/j.talanta.2024.125675},
pmid = {38245957},
issn = {1873-3573},
mesh = {Humans ; *MicroRNAs/genetics ; Feedback ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Esophageal Neoplasms/diagnosis/genetics ; Catalysis ; *Biosensing Techniques ; },
abstract = {Development of new diagnostic methods is essential for disease diagnosis and treatment. In this work, we present a stimuli-responsive incremental DNA machine auto-catalyzed CRISPR-Cas12a (SRI-DNA machine/CRISPR-Cas12a) feedback amplification for ultrasensitive molecular detection of miRNA-21, which is an important biomarker related closely to the initiation and development of cancers, such as esophageal cancer. Strategically, the powerful SRI-DNA machine and efficient trans-cleavage activity of the CRISPR-Cas12a system are ingeniously integrated via a rationally designed probe termed as stem-elongated functional hairpin probe (SEF-HP). The SRI-DNA machine begins with the target miRNA, the trigger of the reaction, binding complementarily to the SEF-HP, followed by autonomously performed mechanical strand replication, cleavage, and displacement circuit at multiple sites. This conversion process led to the amplified generation of numerous DNA activators that are complementary with CRISPR RNA (CrRNA). Once formed the DNA activator/CrRNA heteroduplex, the trans-cleavage activity of the CRISPR-Cas12a was activated to nonspecific cleavage of single-stranded areas of a reporter probe for fluorescence emission. Under optimal conditions, the target miRNA can be detected with a wide linear range and an excellent specificity. As a proof-of-concept, this SRI-DNA machine/CRISPR-Cas12a feedback amplification system is adaptable and scalable to higher-order artificial amplification circuits for biomarkers detection, highlighting its promising potential in early diagnosis and disease treatment.},
}
@article {pmid38142039,
year = {2024},
author = {Chen, Z and Zhang, D and Zheng, R and Yang, L and Huo, Y and Zhang, D and Fang, X and Li, Y and Xu, G and Li, D and Geng, H},
title = {In vivo base editing rescues primary hyperoxaluria type 1 in rats.},
journal = {Kidney international},
volume = {105},
number = {3},
pages = {496-507},
doi = {10.1016/j.kint.2023.11.029},
pmid = {38142039},
issn = {1523-1755},
mesh = {Humans ; Rats ; Animals ; Child ; Calcium Oxalate ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Hyperoxaluria, Primary/genetics/therapy ; Transaminases/genetics/chemistry/metabolism ; Alanine ; *Hyperoxaluria ; Mutation ; },
abstract = {Primary hyperoxaluria type 1 (PH1) is a childhood-onset autosomal recessive disease, characterized by nephrocalcinosis, multiple recurrent urinary calcium oxalate stones, and a high risk of progressive kidney damage. PH1 is caused by inherent genetic defects of the alanine glyoxylate aminotransferase (AGXT) gene. The in vivo repair of disease-causing genes was exceedingly inefficient before the invention of base editors which can efficiently introduce precisely targeted base alterations without double-strand DNA breaks. Adenine base editor (ABE) can precisely convert A·T to G·C with the assistance of specific guide RNA. Here, we demonstrated that systemic delivery of dual adeno-associated virus encoding a split-ABE8e could artificially repair 13% of the pathogenic allele in Agxt[Q84X] rats, a model of PH1, alleviating the disease phenotype. Specifically, ABE treatment partially restored the expression of alanine-glyoxylate-aminotransferase (AGT), reduced endogenous oxalate synthesis and alleviated calcium oxalate crystal deposition. Western blot and immunohistochemistry confirmed that ABE8e treatment restored AGT protein expression in hepatocytes. Moreover, the precise editing efficiency in the liver remained stable six months after treatment. Thus, our findings provided a prospect of in vivo base editing as a personalized and precise medicine for PH1 by directly correcting the mutant Agxt gene.},
}
@article {pmid38102813,
year = {2024},
author = {Zhong, D and Pan, H and Li, K and Zhou, Y and Zhao, F and Ye, L and Ruan, S and Deng, Q and Xu, J and Lu, Y},
title = {Targeted A-to-T and A-to-C base replacement in maize using an optimized adenine base editor.},
journal = {Plant biotechnology journal},
volume = {22},
number = {3},
pages = {541-543},
pmid = {38102813},
issn = {1467-7652},
support = {2021YFD1201300//National Key Research and Development Program of China/ ; 32070396//National Natural Science Foundation of China/ ; },
mesh = {*Adenine ; *Zea mays/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid37997505,
year = {2024},
author = {Wang, M and Li, S and Li, H and Song, C and Xie, W and Zuo, S and Zhou, X and Zhou, C and Ji, Z and Zhou, H},
title = {Genome editing of a dominant resistance gene for broad-spectrum resistance to bacterial diseases in rice without growth penalty.},
journal = {Plant biotechnology journal},
volume = {22},
number = {3},
pages = {529-531},
pmid = {37997505},
issn = {1467-7652},
support = {YBXM2313//Nanfan special project of the Chinese Academy of Agricultural Sciences/ ; B21HJ0215//Hainan Yazhou Bay Seed Lab/ ; },
mesh = {Gene Editing ; *Oryza/genetics/microbiology ; Genes, Dominant ; *Biological Phenomena ; *Bacterial Infections/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37942846,
year = {2024},
author = {Shang, L and Tao, J and Song, J and Wang, Y and Zhang, X and Ge, P and Li, F and Dong, H and Gai, W and Grierson, D and Ye, Z and Zhang, Y},
title = {CRISPR/Cas9-mediated mutations of FANTASTIC FOUR gene family for creating early flowering mutants in tomato.},
journal = {Plant biotechnology journal},
volume = {22},
number = {3},
pages = {774-784},
pmid = {37942846},
issn = {1467-7652},
support = {2662022YLPY001//Fundamental Research Funds for the Central Universities/ ; 2022BBA0062//Hubei Key Research &amp; Development Plan/ ; 2022BBA0066//Hubei Key Research &amp; Development Plan/ ; 2021hszd007//Key Project of Hubei Hongshan Laboratory/ ; 2021YFD1200201//National Key Research &amp; Development Plan/ ; 2022YFD1200502//National Key Research &amp; Development Plan/ ; 32372696//National Natural Science Foundation of China/ ; 31972426//National Natural Science Foundation of China/ ; 31991182//National Natural Science Foundation of China/ ; 2022021302024852//Wuhan Biological Breeding Major Project/ ; SZYJY2023022//HZAU-AGIS Cooperation Fund/ ; },
mesh = {*Solanum lycopersicum/genetics ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Mutation/genetics ; Flowers ; Gene Expression Regulation, Plant/genetics ; },
abstract = {Flowering time is of great agricultural importance and the timing and extent of flowering usually determines yield and availability of flowers, fruits and seeds. Identification of genes determining flowering has important practical applications for tomato breeding. Here we demonstrate the roles of the FANTASTIC FOUR (FAF) gene family in regulating tomato flowering time. In this plant-specific gene family, SlFAF1/2a shows a constitutive expression pattern during the transition of the shoot apical meristem (SAM) from vegetative to reproductive growth and significantly influences flowering time. Overexpressing SlFAF1/2a causes earlier flowering compared with the transformations of other genes in the FAF family. SlFAF1/2c also positively regulates tomato flowering, although to a lesser extent. The other members of the SlFAF gene family, SlFAF1/2b, SlFAF3/4a and SlFAF3/4b, are negative regulators of tomato flowering and faf1/2b, faf3/4a and faf3/4b single mutants all display early flowering. We generated a series of early flowering mutants using the CRISPR/Cas9 editing system, and the faf1/2b faf3/4a faf3/4b triple mutant flowering earliest compared with other mutants. More importantly, these mutants show no adverse effect on yield. Our results have uncovered the role of the FAF gene family in regulating tomato flowering time and generated early flowering germplasms for molecular breeding.},
}
@article {pmid37932934,
year = {2024},
author = {Zhou, S and Cai, L and Wu, H and Wang, B and Gu, B and Cui, S and Huang, X and Xu, Z and Hao, B and Hou, H and Hu, Y and Li, C and Tian, Y and Liu, X and Chen, L and Liu, S and Jiang, L and Wan, J},
title = {Fine-tuning rice heading date through multiplex editing of the regulatory regions of key genes by CRISPR-Cas9.},
journal = {Plant biotechnology journal},
volume = {22},
number = {3},
pages = {751-758},
pmid = {37932934},
issn = {1467-7652},
support = {32272115//National Natural Science Foundation of China/ ; BK20212010//Natural Science Foundation of Jiangsu Province/ ; B21HJ1004//program from Hainan Yazhou Bay Seed Lab/ ; 2022ZD04001//STI 2030 - Major Projects/ ; },
mesh = {*Oryza/metabolism ; Quantitative Trait Loci ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Promoter Regions, Genetic/genetics ; Plant Proteins/genetics/metabolism ; Flowers/genetics ; },
abstract = {Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR-Cas9 based genome editing strategy called high-efficiency multiplex promoter-targeting (HMP) to generate novel alleles at cis-regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions.},
}
@article {pmid38379787,
year = {2024},
author = {Duan, Z and Liang, Y and Sun, J and Zheng, H and Lin, T and Luo, P and Wang, M and Liu, R and Chen, Y and Guo, S and Jia, N and Xie, H and Zhou, M and Xia, M and Zhao, K and Wang, S and Liu, N and Jia, Y and Si, W and Chen, Q and Hong, Y and Tian, R and Zhu, JK},
title = {An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants.},
journal = {Innovation (Cambridge (Mass.))},
volume = {5},
number = {2},
pages = {100564},
pmid = {38379787},
issn = {2666-6758},
abstract = {The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01[HiFi] (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that Cas-SF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.},
}
@article {pmid38378658,
year = {2024},
author = {Xie, L and Feng, H and Li, Z and Li, D and Yang, X and Yuan, T and Yan, N and He, C and Zheng, J and Zuo, Z and Zheng, Y and Cao, Y and Lu, Y and Xiong, XY and Zuo, E},
title = {Undetectable off-target effects induced by FokI catalytic domain in mouse embryos.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {51},
pmid = {38378658},
issn = {1474-760X},
mesh = {Animals ; Mice ; Catalytic Domain ; *Gene Editing/methods ; *Genome ; CRISPR-Cas Systems ; },
abstract = {The FokI catalytic domain can be fused to various DNA binding architectures to improve the precision of genome editing tools. However, evaluation of off-target effects is essential for developing these tools. We use Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) to detect low-frequency off-target editing events in mouse embryos injected with FokI-based architectures. Specifically, we test FokI-heterodimers fused with TALENs, FokI homodimers fused with RYdCas9, or FokI catalytic domains alone resulting in no significant off-target effects. These FokI genome editing systems exhibit undetectable off-target effects in mouse embryos, supporting the further development of these systems for clinical applications.},
}
@article {pmid38378266,
year = {2024},
author = {},
title = {Retraction: CRISPR/Cas genome editing perspectives for barley breeding.},
journal = {Physiologia plantarum},
volume = {176},
number = {1},
pages = {e14208},
doi = {10.1111/ppl.14208},
pmid = {38378266},
issn = {1399-3054},
abstract = {Kershanskaya, O.I., Yessenbaeva, G.L., Nelidova, D.S., Karabekova, A.N. &amp; Sadullaeva, Z.N. (2022) CRISPR/Cas genome editing perspectives for barley breeding. Physiologia Plantarum, 174(3), e13686. Available from: https://doi.org/10.1111/ppl.13686. The above article published online on 1 May 2022 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the Editor, Ykä (Yrjö) Helariutta and John Wiley &amp; Sons Ltd. The retraction has been agreed following concerns raised by a third party regarding the peer review process. Further investigation by the publisher has found manipulation of the peer review process. As a result, the conclusions reported in the article are not considered reliable.},
}
@article {pmid38378112,
year = {2024},
author = {Patnaik, A and Rai, SK and Dhaked, RK},
title = {CRISPR-Cas12a assisted recombinase based strand invading isothermal amplification platform designed for targeted detection of Bacillus anthracis Sterne.},
journal = {International journal of biological macromolecules},
volume = {263},
number = {Pt 1},
pages = {130216},
doi = {10.1016/j.ijbiomac.2024.130216},
pmid = {38378112},
issn = {1879-0003},
abstract = {Detection of a pathogen is crucial prior to all prophylaxis and post exposure treatment, as it can prevent further disease manifestation. In this study, we have developed a nucleic acid pre-amplification based CRISPR diagnostic for detection and surveillance of Bacillus anthracis Sterne. Strand Invasion Based isothermal Amplification (SIBA) platform and Cas12a (CRISPR endo-nuclease) was used to develop CRISPR-SIBA, a multifaceted diagnostic platform. SIBA was employed as the isothermal pre-amplification platform. CRISPR-Cas12a based collateral trans-cleavage reaction was used to ensure and enhance the specificity of the system. Efficiency of the detection system was evaluated by detecting Bacillus anthracis Sterne in complex wastewater sample backgrounds. Previously reported, Prophage 3, Cya and Pag genes of Bacillus anthracis were used as targets for this assay. The amplification system provided reliable and specific detection readout, with a sensitivity limit of 100 colony forming units in 40 min. The endpoint fluorescence from CRISPR collateral cleavage reactions gave a detection limit of 10[5] to 10[6] CFUs. The experiments conducted in this study provide the evidence for SIBA's applicability and compatibility with CRISPR-Cas system and its efficiency to specifically detect Bacillus anthracis Sterne. CRISPR-SIBA can be translated into developing cost-effective diagnostics for pathogens in resource constrained settings.},
}
@article {pmid38374393,
year = {2024},
author = {Smidler, AL and Marrogi, E and Kauffman, J and Paton, DG and Westervelt, KA and Church, GM and Esvelt, KM and Shaw, WR and Catteruccia, F},
title = {CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {4057},
pmid = {38374393},
issn = {2045-2322},
support = {R01 AI104956/AI/NIAID NIH HHS/United States ; R00-DK102669-04/GF/NIH HHS/United States ; },
mesh = {Humans ; Animals ; Male ; Female ; *Anopheles/genetics ; Mosquito Control/methods ; Mosquito Vectors/genetics ; Semen ; RNA, Guide, CRISPR-Cas Systems ; *Infertility, Male/genetics ; *Malaria ; Mutagenesis ; Germ Cells ; },
abstract = {Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.},
}
@article {pmid38371406,
year = {2024},
author = {Saikia, B and S, R and Debbarma, J and Maharana, J and Sastry, GN and Chikkaputtaiah, C},
title = {CRISPR/Cas9-based genome editing and functional analysis of SlHyPRP1 and SlDEA1 genes of Solanum lycopersicum L. in imparting genetic tolerance to multiple stress factors.},
journal = {Frontiers in plant science},
volume = {15},
number = {},
pages = {1304381},
pmid = {38371406},
issn = {1664-462X},
abstract = {CRISPR/Cas is a breakthrough genome editing system because of its precision, target specificity, and efficiency. As a speed breeding system, it is more robust than the conventional breeding and biotechnological approaches for qualitative and quantitative trait improvement. Tomato (Solanum lycopersicum L.) is an economically important crop, but its yield and productivity have been severely impacted due to different abiotic and biotic stresses. The recently identified SlHyPRP1 and SlDEA1 are two potential negative regulatory genes in response to different abiotic (drought and salinity) and biotic stress (bacterial leaf spot and bacterial wilt) conditions in S. lycopersicum L. The present study aimed to evaluate the drought, salinity, bacterial leaf spot, and bacterial wilt tolerance response in S. lycopersicum L. crop through CRISPR/Cas9 genome editing of SlHyPRP1 and SlDEA1 and their functional analysis. The transient single- and dual-gene SlHyPRP1 and SlDEA1 CRISPR-edited plants were phenotypically better responsive to multiple stress factors taken under the study. The CRISPR-edited SlHyPRP1 and SlDEA1 plants showed a higher level of chlorophyll and proline content compared to wild-type (WT) plants under abiotic stress conditions. Reactive oxygen species accumulation and the cell death count per total area of leaves and roots under biotic stress were less in CRISPR-edited SlHyPRP1 and SlDEA1 plants compared to WT plants. The study reveals that the combined loss-of-function of SlHyPRP1 along with SlDEA1 is essential for imparting significant multi-stress tolerance (drought, salinity, bacterial leaf spot, and bacterial wilt) in S. lycopersicum L. The main feature of the study is the detailed genetic characterization of SlDEA1, a poorly studied 8CM family gene in multi-stress tolerance, through the CRISPR/Cas9 gene editing system. The study revealed the key negative regulatory role of SlDEA1 that function together as an anchor gene with SlHyPRP1 in imparting multi-stress tolerance in S. lycopersicum L. It was interesting that the present study also showed that transient CRISPR/Cas9 editing events of SlHyPRP1 and SlDEA1 genes were successfully replicated in stably generated parent-genome-edited line (GEd0) and genome-edited first-generation lines (GEd1) of S. lycopersicum L. With these upshots, the study's key findings demonstrate outstanding value in developing sustainable multi-stress tolerance in S. lycopersicum L. and other crops to cope with climate change.},
}
@article {pmid38370378,
year = {2024},
author = {Zhou, Q and Fang, L and Tang, Y and Wang, Q and Tang, X and Zhu, L and Peng, N and Wang, B and Song, W and Fu, H},
title = {Exosome-mediated delivery of artificial circular RNAs for gene therapy of bladder cancer.},
journal = {Journal of Cancer},
volume = {15},
number = {6},
pages = {1770-1778},
pmid = {38370378},
issn = {1837-9664},
abstract = {Bladder cancer (BCa) is one of the most common malignancies affecting men. Oncogenic transcription factors function as an important regulator in the progression of human cancer. In our study, we aimed to construct artificial circular non-coding RNAs (acircRNAs) consisting of three functional units that mimic the CRISPR-Cas system and elucidate its therapeutic role in bladder cancer. Additionally, the compare of the efficiency in regulating gene expression between acircRNA and CRISPR-dCas systems was performed. We connected the cDNA sequences of TFs aptamer and constructed a circRNA. To demonstrate the platform's practicality, β-catenin and NF-κB were chosen as functional targets, while T24 and 5637 cell lines served as test models. Real-time Quantitative PCR (qPCR), double luciferase assay and related phenotype assay were used to detect the expression of related genes and the therapeutic effect. To elucidate the functionality of acircRNAs, luciferase vectors capable of detecting β-catenin and NF-κB expression were employed to assess the inhibitory impact of acircRNA on β-catenin and NF-κB. Consequently, the optimal combination involving acircRNA-3 was determined. Next, qPCR assay was employed to assess the relative expression levels of target downstream genes following acircRNA treatment. The expression of c-myc and cyclin D1 were used to determine the function of β-catenin, while Bcl-XL and TRAF1 were used to determine that of NF-κB. The acircRNAs inhibited the β-catenin and NF-κB related signaling in BCa cells specifically. CD63-HuR fusion protein was used to loading acircRNA into exosomes. The results showed that acircRNA could inhibit the activity of the target transcription factors, and the inhibitory effect was better than that of CRIPSR-dCas9-KRAB. Furthermore, functional experiments demonstrated that the transfection of acircRNA in bladder cells resulted in decreased proliferation, enhanced apoptosis, and suppressed migration. In conclusion, our synthetic gene device exhibited anti-tumor regulatory capabilities and showed greater efficiency in tumor suppression compared to the CRISPR-dCas9-KRAB system. Therefore, our device provides a new strategy for cancer treatment and could be a useful strategy for cancer cells.},
}
@article {pmid38369827,
year = {2024},
author = {Wang, J and Zhao, P and Qin, M and Zhao, Y and Liu, C and Xia, X},
title = {[Advances in CRISPR sensing and detection technology].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {40},
number = {2},
pages = {367-377},
doi = {10.13345/j.cjb.230482},
pmid = {38369827},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems ; *Exosomes ; *Nucleic Acids ; RNA ; Technology ; *Biosensing Techniques ; },
abstract = {The CRISPR sensing and detection technology has the advantages of cheap, simple, portable, high sensitivity, and high specificity, therefore is regarded as the "next-generation molecular diagnostic technology". Due to the specific recognition, cis-cleavage and nonspecific trans-cleavage capabilities, CRISPR-Cas systems have been implemented for the detection of nucleic acid targets (DNA and RNA) as well as non-nucleic acid targets (e.g., proteins, exosomes, cells, and small molecules). This review summarizes the current CRISPR sensing and detection technologies in terms of the activity characteristics of different Cas proteins, with the aim to understand the advantages and development history of different CRISPR sensing and detection technologies, as well as promote its development and application. Moreover, this review summarizes the applications of various CRISPR sensing and detection technologies according to the types of detection targets, hoping to facilitate the development of novel CRISPR sensing detection technology.},
}
@article {pmid38368839,
year = {2024},
author = {Agapov, A and Baker, KS and Bedekar, P and Bhatia, RP and Blower, TR and Brockhurst, MA and Brown, C and Chong, CE and Fothergill, JL and Graham, S and Hall, JP and Maestri, A and McQuarrie, S and Olina, A and Pagliara, S and Recker, M and Richmond, A and Shaw, SJ and Szczelkun, MD and Taylor, TB and van Houte, S and Went, SC and Westra, ER and White, MF and Wright, R},
title = {Multi-layered genome defences in bacteria.},
journal = {Current opinion in microbiology},
volume = {78},
number = {},
pages = {102436},
doi = {10.1016/j.mib.2024.102436},
pmid = {38368839},
issn = {1879-0364},
abstract = {Bacteria have evolved a variety of defence mechanisms to protect against mobile genetic elements, including restriction-modification systems and CRISPR-Cas. In recent years, dozens of previously unknown defence systems (DSs) have been discovered. Notably, diverse DSs often coexist within the same genome, and some co-occur at frequencies significantly higher than would be expected by chance, implying potential synergistic interactions. Recent studies have provided evidence of defence mechanisms that enhance or complement one another. Here, we review the interactions between DSs at the mechanistic, regulatory, ecological and evolutionary levels.},
}
@article {pmid38368461,
year = {2024},
author = {Saha, A and Ahsan, M and Arantes, PR and Schmitz, M and Chanez, C and Jinek, M and Palermo, G},
title = {An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1473},
pmid = {38368461},
issn = {2041-1723},
support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; R01 GM116961/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; DNA/genetics ; Gene Editing ; Catalysis ; },
abstract = {CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.},
}
@article {pmid37266832,
year = {2024},
author = {Lotfi, M and Ashouri, A and Mojarrad, M and Mozaffari-Jovin, S and Abbaszadegan, MR},
title = {Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.},
journal = {Molecular biotechnology},
volume = {66},
number = {3},
pages = {517-530},
pmid = {37266832},
issn = {1559-0305},
support = {973542//National Institute for Medical Research Development/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; HEK293 Cells ; *beta-Thalassemia/genetics ; Gene Editing/methods ; Recombinational DNA Repair ; },
abstract = {Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.},
}
@article {pmid38368418,
year = {2024},
author = {Bamidele, N and Zhang, H and Dong, X and Cheng, H and Gaston, N and Feinzig, H and Cao, H and Kelly, K and Watts, JK and Xie, J and Gao, G and Sontheimer, EJ},
title = {Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1458},
pmid = {38368418},
issn = {2041-1723},
support = {F31 GM143879/GM/NIGMS NIH HHS/United States ; R01 GM150273/GM/NIGMS NIH HHS/United States ; R25 GM113686/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *Adenine/metabolism ; Gene Editing ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.},
}
@article {pmid38368357,
year = {2024},
author = {Zhao, JJ and Sun, XY and Tian, SN and Zhao, ZZ and Yin, MD and Zhao, M and Zhang, F and Li, SA and Yang, ZX and Wen, W and Cheng, T and Gong, A and Zhang, JP and Zhang, XB},
title = {Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.},
journal = {BMC genomics},
volume = {25},
number = {1},
pages = {189},
pmid = {38368357},
issn = {1471-2164},
support = {2019YFA0110803//National Key Research and Development Program of China/ ; 81870149//National Natural Science Foundation of China/ ; 2022-I2M-2-003//Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS)/ ; 2020-PT310-011//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; TSBICIP-KJGG-017//Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project/ ; 3332021093//CAMS Fundamental Research Funds for Central Research Institutes/ ; HH22KYZX0022//Haihe Laboratory of Cell Ecosystem Innovation Fund/ ; Z23-05//State Key Laboratory of Experimental Hematology Research Grant/ ; },
mesh = {Mice ; Animals ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Hemophilia A/genetics/therapy ; *Nanopore Sequencing ; Gene Editing/methods ; DNA ; },
abstract = {BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing.<br><br>RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements.<br><br>CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.},
}
@article {pmid38366192,
year = {2024},
author = {Dion, MB and Shah, SA and Deng, L and Thorsen, J and Stokholm, J and Krogfelt, KA and Schjørring, S and Horvath, P and Allard, A and Nielsen, DS and Petit, MA and Moineau, S},
title = {Escherichia coli CRISPR arrays from early life fecal samples preferentially target prophages.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrae005},
pmid = {38366192},
issn = {1751-7370},
abstract = {CRISPR-Cas systems are defense mechanisms against phages and other nucleic acids that invade bacteria and archaea. In Escherichia coli, it is generally accepted that CRISPR-Cas systems are inactive in laboratory conditions due to a transcriptional repressor. In natural isolates, it has been shown that CRISPR arrays remain stable over the years and that most spacer targets (protospacers) remain unknown. Here, we re-examine CRISPR arrays in natural E. coli isolates and investigate viral and bacterial genomes for spacer targets using a bioinformatics approach coupled to a unique biological dataset. We first sequenced the CRISPR1 array of 1769 E. coli isolates from the fecal samples of 639 children obtained during their first year of life. We built a network with edges between isolates that reflect the number of shared spacers. The isolates grouped into 34 modules. A search for matching spacers in bacterial genomes showed that E. coli spacers almost exclusively target prophages. While we found instances of self-targeting spacers, those involving a prophage and a spacer within the same bacterial genome were rare. The extensive search for matching spacers also expanded the library of known E. coli protospacers to 60%. Altogether, these results suggest that E. coli's CRISPR-Cas is an anti-prophage system and highlight the importance of reconsidering the criteria use to deem CRISPR-Cas systems active.},
}
@article {pmid38366022,
year = {2024},
author = {Watson, BNJ and Capria, L and Alseth, EO and Pons, BJ and Biswas, A and Lenzi, L and Buckling, A and van Houte, S and Westra, ER and Meaden, S},
title = {CRISPR-Cas in Pseudomonas aeruginosa provides transient population-level immunity against high phage exposures.},
journal = {The ISME journal},
volume = {18},
number = {1},
pages = {},
pmid = {38366022},
issn = {1751-7370},
support = {/ERC_/European Research Council/International ; BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/X010600/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
abstract = {The prokaryotic adaptive immune system, CRISPR-Cas (clustered regularly interspaced short palindromic repeats; CRISPR-associated), requires the acquisition of spacer sequences that target invading mobile genetic elements such as phages. Previous work has identified ecological variables that drive the evolution of CRISPR-based immunity of the model organism Pseudomonas aeruginosa PA14 against its phage DMS3vir, resulting in rapid phage extinction. However, it is unclear if and how stable such acquired immunity is within bacterial populations, and how this depends on the environment. Here, we examine the dynamics of CRISPR spacer acquisition and loss over a 30-day evolution experiment and identify conditions that tip the balance between long-term maintenance of immunity versus invasion of alternative resistance strategies that support phage persistence. Specifically, we find that both the initial phage dose and reinfection frequencies determine whether or not acquired CRISPR immunity is maintained in the long term, and whether or not phage can coexist with the bacteria. At the population genetics level, emergence and loss of CRISPR immunity are associated with high levels of spacer diversity that subsequently decline due to invasion of bacteria carrying pilus-associated mutations. Together, these results provide high resolution of the dynamics of CRISPR immunity acquisition and loss and demonstrate that the cumulative phage burden determines the effectiveness of CRISPR over ecologically relevant timeframes.},
}
@article {pmid38362491,
year = {2024},
author = {Macarrón Palacios, A and Korus, P and Wilkens, BGC and Heshmatpour, N and Patnaik, SR},
title = {Revolutionizing in vivo therapy with CRISPR/Cas genome editing: breakthroughs, opportunities and challenges.},
journal = {Frontiers in genome editing},
volume = {6},
number = {},
pages = {1342193},
pmid = {38362491},
issn = {2673-3439},
abstract = {Genome editing using the CRISPR/Cas system has revolutionized the field of genetic engineering, offering unprecedented opportunities for therapeutic applications in vivo. Despite the numerous ongoing clinical trials focusing on ex vivo genome editing, recent studies emphasize the therapeutic promise of in vivo gene editing using CRISPR/Cas technology. However, it is worth noting that the complete attainment of the inherent capabilities of in vivo therapy in humans is yet to be accomplished. Before the full realization of in vivo therapeutic potential, it is crucial to achieve enhanced specificity in selectively targeting defective cells while minimizing harm to healthy cells. This review examines emerging studies, focusing on CRISPR/Cas-based pre-clinical and clinical trials for innovative therapeutic approaches for a wide range of diseases. Furthermore, we emphasize targeting cancer-specific sequences target in genes associated with tumors, shedding light on the diverse strategies employed in cancer treatment. We highlight the various challenges associated with in vivo CRISPR/Cas-based cancer therapy and explore their prospective clinical translatability and the strategies employed to overcome these obstacles.},
}
@article {pmid38361756,
year = {2024},
author = {Amjad, E and Pezzani, R and Sokouti, B},
title = {A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma.},
journal = {Oncology research},
volume = {32},
number = {3},
pages = {439-461},
pmid = {38361756},
issn = {1555-3906},
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; *Liver Neoplasms/genetics/therapy/metabolism ; Genetic Therapy ; Treatment Outcome ; },
abstract = {Noncoding RNAs instruct the Cas9 nuclease to site-specifically cleave DNA in the CRISPR/Cas9 system. Despite the high incidence of hepatocellular carcinoma (HCC), the patient's outcome is poor. As a result of the emergence of therapeutic resistance in HCC patients, clinicians have faced difficulties in treating such tumor. In addition, CRISPR/Cas9 screens were used to identify genes that improve the clinical response of HCC patients. It is the objective of this article to summarize the current understanding of the use of the CRISPR/Cas9 system for the treatment of cancer, with a particular emphasis on HCC as part of the current state of knowledge. Thus, in order to locate recent developments in oncology research, we examined both the Scopus database and the PubMed database. The ability to selectively interfere with gene expression in combinatorial CRISPR/Cas9 screening can lead to the discovery of new effective HCC treatment regimens by combining clinically approved drugs. Drug resistance can be overcome with the help of the CRISPR/Cas9 system. HCC signature genes and resistance to treatment have been uncovered by genome-scale CRISPR activation screening, although this method is not without limitations. It has been extensively examined whether CRISPR can be used as a tool for disease research and gene therapy. CRISPR and its applications to tumor research, particularly in HCC, are examined in this study through a review of the literature.},
}
@article {pmid38360922,
year = {2024},
author = {Fauser, F and Kadam, BN and Arangundy-Franklin, S and Davis, JE and Vaidya, V and Schmidt, NJ and Lew, G and Xia, DF and Mureli, R and Ng, C and Zhou, Y and Scarlott, NA and Eshleman, J and Bendaña, YR and Shivak, DA and Reik, A and Li, P and Davis, GD and Miller, JC},
title = {Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1181},
pmid = {38360922},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing ; *Cytidine Deaminase/genetics/metabolism ; DNA/metabolism ; Zinc Fingers ; Cytidine/genetics ; CRISPR-Cas Systems ; },
abstract = {Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.},
}
@article {pmid38347661,
year = {2024},
author = {Zhang, Q and Gao, X and Ho, YP and Liu, M and Han, Y and Li, DL and Yuan, HM and Zhang, CY},
title = {Controllable Assembly of a Quantum Dot-Based Aptasensor Guided by CRISPR/Cas12a for Direct Measurement of Circulating Tumor Cells in Human Blood.},
journal = {Nano letters},
volume = {24},
number = {7},
pages = {2360-2368},
doi = {10.1021/acs.nanolett.3c04828},
pmid = {38347661},
issn = {1530-6992},
mesh = {Humans ; *Neoplastic Cells, Circulating/pathology ; *Quantum Dots/chemistry ; CRISPR-Cas Systems/genetics ; Liquid Biopsy ; },
abstract = {Accurate and sensitive analysis of circulating tumor cells (CTCs) in human blood provides a non-invasive approach for the evaluation of cancer metastasis and early cancer diagnosis. Herein, we demonstrate the controllable assembly of a quantum dot (QD)-based aptasensor guided by CRISPR/Cas12a for direct measurement of CTCs in human blood. We introduce a magnetic bead@activator/recognizer duplex core-shell structure to construct a multifunctional platform for the capture and direct detection of CTCs in human blood, without the need for additional CTC release and re-identification steps. Notably, the introduction of magnetic separation ensures that only a target-induced free activator can initiate the downstream catalysis, efficiently avoiding the undesired catalysis triggered by inappropriate recognition of the activator/recognizer duplex structure by crRNAs. This aptasensor achieves high CTC-capture efficiency (82.72%) and sensitive detection of CTCs with a limit of detection of 2 cells mL[-1] in human blood, holding great promise for the liquid biopsy of cancers.},
}
@article {pmid38299816,
year = {2024},
author = {Ferreira, MR and Queiroga, V and Moreira, LM},
title = {Genomic editing in Burkholderia multivorans by CRISPR/Cas9.},
journal = {Applied and environmental microbiology},
volume = {90},
number = {2},
pages = {e0225023},
pmid = {38299816},
issn = {1098-5336},
support = {UIDB/04565/2020//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia-Portugal/ ; UIDP/04565/2020//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia- Portugal/ ; LA/P/0140/2020//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia- Portugal/ ; PD/BD/150337/2019//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia- Portugal/ ; },
mesh = {Animals ; Humans ; CRISPR-Cas Systems ; *Burkholderia Infections/microbiology ; *Cystic Fibrosis/microbiology ; Gene Editing ; *Burkholderia/genetics ; *Burkholderia cepacia complex/genetics ; Genomics ; },
abstract = {Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.},
}
@article {pmid38291234,
year = {2024},
author = {Koch, L},
title = {Packaging and delivery of genome-editing tools.},
journal = {Nature reviews. Genetics},
volume = {25},
number = {3},
pages = {161},
pmid = {38291234},
issn = {1471-0064},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid38238618,
year = {2024},
author = {Ghosh, A and Maiti, S and Chakraborty, D},
title = {Emerging opportunities for gene editing therapies in India.},
journal = {Nature medicine},
volume = {30},
number = {2},
pages = {324-325},
pmid = {38238618},
issn = {1546-170X},
mesh = {*Gene Editing ; *Genetic Therapy ; CRISPR-Cas Systems/genetics ; India ; },
}
@article {pmid38199534,
year = {2024},
author = {Wang, JD and Zhang, JS and Li, XX and Wang, KJ and Li, M and Mao, YY and Wan, XH},
title = {Knockout of TGF-β receptor II by CRISPR/Cas9 delays mesenchymal transition of Lens epithelium and posterior capsule opacification.},
journal = {International journal of biological macromolecules},
volume = {259},
number = {Pt 2},
pages = {129290},
doi = {10.1016/j.ijbiomac.2024.129290},
pmid = {38199534},
issn = {1879-0003},
mesh = {Animals ; Humans ; Rabbits ; *Capsule Opacification/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Lens, Crystalline/metabolism ; Epithelial Cells ; Epithelial-Mesenchymal Transition/genetics ; Epithelium/metabolism ; Cell Movement ; Cell Proliferation ; },
abstract = {Posterior capsule opacification (PCO) is the most common postoperative complication of cataract surgery. Transforming growth factor-β (TGF-β) is related to epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) that is proven to induce PCO formation in clinical and experimental studies. In this study, CRISPR sequences targeting exon of TGF-βRII were knocked out with lentiviral transfection in LECs. Rabbits' PCO model was established and recombinant adeno-associated virus (AAV) for transferring the gRNA of TGF βRII were intravitreally injected. SgRNA inhibited TGF-βRII expression and human LECs proliferation. In TGF-βRII knockout group, LECs motility and migration were suppressed, N-cadherin and vimentin expressions were significantly decreased, whereas E-cadherin was increased. The animal model showed that TGF-βRII knockout in vivo was effective in suppressing PCO. The current study suggested that the CRISPR/Cas9 endonuclease system could suppress TGF-βRII secretion, which participates in the EMT procedure of LECs in vitro and PCO in vivo. These findings might provide a new gene-editing approach and insight into a novel therapeutic strategy for PCO.},
}
@article {pmid38185820,
year = {2024},
author = {Zhou, P and G C, B and Stolte, F and Wu, C},
title = {Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.},
journal = {Applied and environmental microbiology},
volume = {90},
number = {2},
pages = {e0166523},
pmid = {38185820},
issn = {1098-5336},
support = {DE030895//HHS | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; },
mesh = {*Fusobacterium nucleatum ; RNA, Guide, CRISPR-Cas Systems ; *Riboswitch ; Theophylline/metabolism ; Gene Silencing ; *Fusobacterium ; },
abstract = {Gene inactivation by creating in-frame deletion mutations in Fusobacterium nucleatum is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX and radD, which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes bamA and ftsZ, which are vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and Fusobacterium periodonticum. With CRISPRi's potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.},
}
@article {pmid38057426,
year = {2024},
author = {Alves, CRR and Ha, LL and Yaworski, R and Sutton, ER and Lazzarotto, CR and Christie, KA and Reilly, A and Beauvais, A and Doll, RM and de la Cruz, D and Maguire, CA and Swoboda, KJ and Tsai, SQ and Kothary, R and Kleinstiver, BP},
title = {Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy.},
journal = {Nature biomedical engineering},
volume = {8},
number = {2},
pages = {118-131},
pmid = {38057426},
issn = {2157-846X},
support = {DP2CA281401//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; P01HL142494//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; Howard M. Goodman Fellowship//Massachusetts General Hospital (MGH)/ ; R01DC017117//U.S. Department of Health &amp; Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; U01AI157189//U.S. Department of Health &amp; Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)/ ; 575466//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; Grant//Muscular Dystrophy Canada (Dystrophie musculaire Canada)/ ; PJT-156379//Gouvernement du Canada | Instituts de Recherche en Sant&#xe9; du Canada | CIHR Skin Research Training Centre (Skin Research Training Centre)/ ; },
mesh = {Mice ; Animals ; Humans ; *RNA-Binding Proteins/genetics/metabolism ; SMN Complex Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Muscular Atrophy, Spinal/genetics/therapy ; Exons/genetics ; Survival of Motor Neuron 2 Protein/genetics ; },
abstract = {Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.},
}
@article {pmid37903903,
year = {2024},
author = {},
title = {Intrinsic RNA-targeting activity limits the applicability of CRISPR-Cas13.},
journal = {Nature biomedical engineering},
volume = {8},
number = {2},
pages = {116-117},
pmid = {37903903},
issn = {2157-846X},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Viral ; },
}
@article {pmid37872368,
year = {2024},
author = {Li, Z and Li, Z and Cheng, X and Wang, S and Wang, X and Ma, S and Lu, Z and Zhang, H and Zhao, W and Chen, Z and Yao, Y and Zhang, C and Chao, L and Li, W and Fei, T},
title = {Intrinsic targeting of host RNA by Cas13 constrains its utility.},
journal = {Nature biomedical engineering},
volume = {8},
number = {2},
pages = {177-192},
pmid = {37872368},
issn = {2157-846X},
support = {32071441//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *RNA/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy ; Gene Expression Profiling ; Lentivirus/genetics ; Mammals/genetics ; },
abstract = {Cas13 can be used for the knockdown, editing, imaging or detection of RNA and for RNA-based gene therapy. Here by using RNA immunoprecipitation sequencing, transcriptome profiling, biochemical analysis, high-throughput screening and machine learning, we show that Cas13 can intrinsically target host RNA in mammalian cells through previously unappreciated mechanisms. Different from its known cis/trans RNA-cleavage activity, Cas13 can also cleave host RNA via mechanisms that are transcript-specific, independent of the sequence of CRISPR RNA and dynamically dependent on the conformational state of Cas13, as we show for several Cas13-family effectors encoded in one-vector and two-vector lentiviral systems. Moreover, host genes involved in viral processes and whose transcripts are intrinsically targeted by Cas13 contribute to constraining the lentiviral delivery and expression of Cas13. Our findings offer guidance for the appropriate use of lentiviral Cas13 systems and highlight the need for caution regarding intrinsic RNA targeting in Cas13-based applications.},
}
@article {pmid38359997,
year = {2024},
author = {Nguyen, L},
title = {RNA therapeutics for neurological disease.},
journal = {Progress in molecular biology and translational science},
volume = {203},
number = {},
pages = {165-180},
doi = {10.1016/bs.pmbts.2024.01.005},
pmid = {38359997},
issn = {1878-0814},
mesh = {Humans ; RNA/genetics ; *Nervous System Diseases/genetics/therapy ; *Alzheimer Disease ; Central Nervous System ; },
abstract = {Neurological disorders are the group of diseases that primarily affect the center nervous system, which could lead to a significant negative impact on the ability of learning new skills, speaking, breathing, walking, judging, making decision, and other essential living skills. In the last decade, neurological disorders have significantly increased their impact to our community and become the one of leading causes of disability and death. The World Health Organization has identified neurological disorders including Alzheimer's disease and other dementia as the health crisis for the modern life. Tremendous ongoing research efforts focus on understanding of disease genetics, molecular mechanisms and developing therapeutic interventions. Because of the urgent need of the effective therapeutics and the recent advances in the toolkits and understanding for developing more drug-like RNA molecules, there is a growing interest for developing RNA therapeutics for neurological disorders. This article will discuss genetics and mechanisms of neurological disorders and how RNA-based molecules have been used to develop therapeutics for this group of diseases, challenges of RNA therapeutics and future perspectives on this rising therapeutic intervention tool.},
}
@article {pmid38359984,
year = {2024},
author = {Ramakrishnan, N and Malachowski, T and Verma, P},
title = {A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions.},
journal = {Methods in cell biology},
volume = {182},
number = {},
pages = {299-312},
doi = {10.1016/bs.mcb.2023.02.005},
pmid = {38359984},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; Flow Cytometry ; *Genomics ; Genome ; Gene Editing ; },
abstract = {Probing epistasis between two genes can be a critical first step in identifying the molecular players in a cellular pathway. The advent of CRISPR-Cas mediated genetic screen has enabled studying of these genetic interactions at a genomic scale. However, when combining depletion of two genes using CRISPR Cas9, reduced targeting efficiencies due to competition for Cas loading and recombination in the cloning step have emerged as key challenges. Moreover, given conventional CRISPR screens typically involve comparison between the initial and final time point, it is difficult to parse the time kinetics with which a perturbed genetic interaction impacts viability, and it also becomes challenging to assess epistasis with essential genes. Here, we discuss a high-throughput flow-based approach to study genetic interactions. By utilizing two different Cas9 orthologs and monitoring viability at multiple time points, this approach helps to effectively mitigate the limitations of Cas9 competition and enables assessment of genetic interactions with both essential and non-essential genes at a high temporal resolution.},
}
@article {pmid38359969,
year = {2024},
author = {Lee, SW and Frankston, CM and Kim, J},
title = {Epigenome editing in cancer: Advances and challenges for potential therapeutic options.},
journal = {International review of cell and molecular biology},
volume = {383},
number = {},
pages = {191-230},
doi = {10.1016/bs.ircmb.2023.10.001},
pmid = {38359969},
issn = {1937-6448},
mesh = {Humans ; *CRISPR-Cas Systems ; Epigenome ; Epigenesis, Genetic ; Gene Editing ; Oncogenes ; *Neoplasms/genetics/therapy/pathology ; DNA Methylation ; },
abstract = {Cancers are diseases caused by genetic and non-genetic environmental factors. Epigenetic alterations, some attributed to non-genetic factors, can lead to cancer development. Epigenetic changes can occur in tumor suppressors or oncogenes, or they may contribute to global cell state changes, making cells abnormal. Recent advances in gene editing technology show potential for cancer treatment. Herein, we will discuss our current knowledge of epigenetic alterations occurring in cancer and epigenetic editing technologies that can be applied to developing therapeutic options.},
}
@article {pmid38358883,
year = {2024},
author = {Nguyen, LT and Macaluso, NC and Rakestraw, NR and Carman, DR and Pizzano, BLM and Hautamaki, RC and Rananaware, SR and Roberts, IE and Jain, PK},
title = {Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.},
journal = {Cell reports},
volume = {43},
number = {2},
pages = {113777},
doi = {10.1016/j.celrep.2024.113777},
pmid = {38358883},
issn = {2211-1247},
abstract = {There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing.},
}
@article {pmid38357966,
year = {2024},
author = {Lan, H and Shu, W and Jiang, D and Yu, L and Xu, G},
title = {Cas-based bacterial detection: recent advances and perspectives.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3an02120c},
pmid = {38357966},
issn = {1364-5528},
abstract = {Persistent bacterial infections pose a formidable threat to global health, contributing to widespread challenges in areas such as food safety, medical hygiene, and animal husbandry. Addressing this peril demands the urgent implementation of swift and highly sensitive detection methodologies suitable for point-of-care testing and large-scale screening. These methodologies play a pivotal role in the identification of pathogenic bacteria, discerning drug-resistant strains, and managing and treating diseases. Fortunately, new technology, the CRISPR/Cas system, has emerged. The clustered regularly interspaced short joint repeats (CRISPR) system, which is part of bacterial adaptive immunity, has already played a huge role in the field of gene editing. It has been employed as a diagnostic tool for virus detection, featuring high sensitivity, specificity, and single-nucleotide resolution. When applied to bacterial detection, it also surpasses expectations. In this review, we summarise recent advances in the detection of bacteria such as Mycobacterium tuberculosis (MTB), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Salmonella and Acinetobacter baumannii (A. baumannii) using the CRISPR/Cas system. We emphasize the significance and benefits of this methodology, showcasing the capability of diverse effector proteins to swiftly and precisely recognize bacterial pathogens. Furthermore, the CRISPR/Cas system exhibits promise in the identification of antibiotic-resistant strains. Nevertheless, this technology is not without challenges that need to be resolved. For example, CRISPR/Cas systems must overcome natural off-target effects and require high-quality nucleic acid samples to improve sensitivity and specificity. In addition, limited applicability due to the protospacer adjacent motif (PAM) needs to be addressed to increase its versatility. Despite the challenges, we are optimistic about the future of bacterial detection using CRISPR/Cas. We have already highlighted its potential in medical microbiology. As research progresses, this technology will revolutionize the detection of bacterial infections.},
}
@article {pmid38356346,
year = {2024},
author = {Liao, Y and Li, M and Wu, H and Liao, Y and Xin, J and Yuan, X and Li, Y and Wei, A and Zou, X and Guo, D and Xue, Z and Zhu, G and Wang, Z and Xu, P and Zhang, H and Chen, X and Du, K and Zhou, H and Xia, D and Ali, A and Wu, X},
title = {Generation of aroma in three-line hybrid rice through CRISPR/Cas9 editing of BETAINE ALDEHYDE DEHYDROGENASE2 (OsBADH2).},
journal = {Physiologia plantarum},
volume = {176},
number = {1},
pages = {e14206},
doi = {10.1111/ppl.14206},
pmid = {38356346},
issn = {1399-3054},
support = {Chang Ke Han 2021-59//Science and Technology Bureau of Changde, Hunan Province/ ; 2022JDRC0111//Science and Technology Department of Sichuan Province/ ; 2022ZDZX0016//Science and Technology Department of Sichuan Province/ ; 2023-YF08-00005-SN//Chengdu Science and Technology Bureau/ ; 2022-YF09-00036-SN//Chengdu Science and Technology Bureau/ ; },
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Odorants ; Genes, Plant ; Betaine/*analogs &amp; derivatives ; },
abstract = {Aroma or fragrance in rice is a genetically controlled trait; Its high appreciation by consumers increases the rice market price. Previous studies have revealed that the rice aroma is controlled by a specific gene called BETAINE ALDEHYDE DEHYDROGENASE (OsBADH2), and mutation of this gene leads to the accumulation of an aromatic substance 2-acetyl-1-pyrroline (2-AP). The use of genetic engineering to produce aroma in commercial and cultivated hybrids is a contemporary need for molecular breeding. The current study reports the generation of aroma in the three-line hybrid restorer line Shu-Hui-313 (SH313). We created knock-out (KO) lines of OsBADH2 through the CRISPR/Cas9. The analysis of KO lines revealed a significantly increased content of 2AP in the grains compared with the control. However, other phenotypic traits (plant height, seed setting rate, and 1000-grain weight) were significantly decreased. These KO lines were crossed with a non-aromatic three-line hybrid rice male sterile line (Rong-7-A) to produce Rong-7-You-626 (R7Y626), R7Y627 and R7Y628. The measurement of 2-AP revealed significantly increased contents in these cross combinations. We compared the content of 2-AP in tissues at the booting stage. Data revealed that young spike stalk base contained the highest content of 2-AP and can be used for identification (by simple chewing) of aromatic lines under field conditions. In conclusion, our dataset offers a genetic source and illustrates the generation of aroma in non-aromatic hybrids, and outlines a straightforward identification under field conditions.},
}
@article {pmid38356108,
year = {2024},
author = {Nweze, JE and Schweichhart, JS and Angel, R},
title = {Viral communities in millipede guts: Insights into the diversity and potential role in modulating the microbiome.},
journal = {Environmental microbiology},
volume = {26},
number = {2},
pages = {e16586},
doi = {10.1111/1462-2920.16586},
pmid = {38356108},
issn = {1462-2920},
support = {21-04987S//Czech Science Foundation (GA &#x10c;R)/ ; 19-24309Y//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; },
mesh = {*Viruses/genetics ; *Microbiota/genetics ; DNA Viruses/genetics ; *Gastrointestinal Microbiome/genetics ; DNA ; *RNA Viruses/genetics ; },
abstract = {Millipedes are important detritivores harbouring a diverse microbiome. Previous research focused on bacterial and archaeal diversity, while the virome remained neglected. We elucidated the DNA and RNA viral diversity in the hindguts of two model millipede species with distinct microbiomes: the tropical Epibolus pulchripes (methanogenic, dominated by Bacillota) and the temperate Glomeris connexa (non-methanogenic, dominated by Pseudomonadota). Based on metagenomic and metatranscriptomic assembled viral genomes, the viral communities differed markedly and preferentially infected the most abundant prokaryotic taxa. The majority of DNA viruses were Caudoviricetes (dsDNA), Cirlivirales (ssDNA) and Microviridae (ssDNA), while RNA viruses consisted of Leviviricetes (ssRNA), Potyviridae (ssRNA) and Eukaryotic viruses. A high abundance of subtypes I-C, I-B and II-C CRISPR-Cas systems was found, primarily from Pseudomonadota, Bacteroidota and Bacillota. In addition, auxiliary metabolic genes that modulate chitin degradation, vitamins and amino acid biosynthesis and sulphur metabolism were also detected. Lastly, we found low virus-to-microbe-ratios and a prevalence of lysogenic viruses, supporting a Piggyback-the-Winner dynamic in both hosts.},
}
@article {pmid38356075,
year = {2024},
author = {O'Leary, K},
title = {Gene-editing breakthrough for a rare hereditary disorder.},
journal = {Nature medicine},
volume = {},
number = {},
pages = {},
doi = {10.1038/d41591-024-00008-2},
pmid = {38356075},
issn = {1546-170X},
}
@article {pmid38354496,
year = {2024},
author = {Yang, C and Du, C and Yuan, F and Yu, P and Wang, B and Su, C and Zou, R and Wang, J and Yan, X and Sun, C and Li, H},
title = {CRISPR/Cas12a-derived ratiometric fluorescence sensor for high-sensitive Pb[2+] detection based on CDs@ZIF-8 and DNAzyme.},
journal = {Biosensors &amp; bioelectronics},
volume = {251},
number = {},
pages = {116089},
doi = {10.1016/j.bios.2024.116089},
pmid = {38354496},
issn = {1873-4235},
abstract = {Benefiting from specific target recognition and trans-cleavage capabilities, the CRISPR/Cas12a system has great application prospects in the design of highly sensitive and rapid fluorescence biosensors. The CRISPR/Cas12a-based fluorophore-quencher molecular beacons exhibit single-color emission and are easily exposed to interference from environmental factors. Herein, we design a CRISPR/Cas12a-derived ratiometric fluorescence sensor for Pb[2+] detection based on embedded carbon dots@zeolitic imidazolate framework-8 (CDs@ZIF-8) composites and DNAzyme. The functions of ZIF-8 about encapsulating red emissive CDs in the inner cavity and adsorbing DNA on the outer surface are integrated to establish dual fluorescence signals, thereby reducing the possibility of interference and improving sensing accuracy. The presence of Pb[2+] is converted into the change of activator by the GR5 DNAzyme to activate the CRISPR/Cas12a system, which provides signal amplification through multiple turnovers of side branch cutting, achieving highly sensitive detection of Pb[2+] with a low detection limit of 18 pM. This method has the advantages of simplicity, universality, and excellent quantitative ability, and has broad prospects in sensing applications.},
}
@article {pmid38354234,
year = {2024},
author = {Yang, L and Sheets, TP and Feng, Y and Yu, G and Bajgain, P and Hsu, KS and So, D and Seaman, S and Lee, J and Lin, L and Evans, CN and Guest, MR and Chari, R and St Croix, B},
title = {Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform.},
journal = {Science advances},
volume = {10},
number = {7},
pages = {eadj2445},
pmid = {38354234},
issn = {2375-2548},
mesh = {Ligands ; *Membrane Proteins/genetics ; Transcriptional Activation ; *CRISPR-Cas Systems ; },
abstract = {The majority of clinically approved drugs target proteins that are secreted or cell surface bound. However, further advances in this area have been hindered by the challenging nature of receptor deorphanization, as there are still many secreted and cell-bound proteins with unknown binding partners. Here, we developed an advanced screening platform that combines CRISPR-CAS9 guide-mediated gene activation (CRISPRa) and high-avidity bead-based selection. The CRISPRa platform incorporates serial enrichment and flow cytometry-based monitoring, resulting in substantially improved screening sensitivity for well-known yet weak interactions of the checkpoint inhibitor family. Our approach has successfully revealed that siglec-4 exerts regulatory control over T cell activation through a low affinity trans-interaction with the costimulatory receptor 4-1BB. Our highly efficient screening platform holds great promise for identifying extracellular interactions of uncharacterized receptor-ligand partners, which is essential to develop next-generation therapeutics, including additional immune checkpoint inhibitors.},
}
@article {pmid38353738,
year = {2024},
author = {Nguyen, TM and Wu, PY and Chang, CH and Huang, LF},
title = {High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {206},
pmid = {38353738},
issn = {1432-0614},
support = {111-2313-B-155-001-MY3//National Science and Technology Council/ ; 110-2313-B-155-001//National Science and Technology Council/ ; },
mesh = {Humans ; *Oryza/genetics ; Bone Morphogenetic Protein 2/genetics ; Introns ; Recombinant Proteins/genetics ; Pharmaceutical Preparations ; },
abstract = {Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3' splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 μg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter-signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. KEY POINTS: • The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 • The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins • The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity.},
}
@article {pmid36288702,
year = {2022},
author = {Wang, L and Xie, X and Lv, B and Liu, Y and Li, W and Zhang, Z and Yang, J and Yan, G and Chen, W and Zhang, C and Wang, F and Li, C and Ma, L},
title = {A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA.},
journal = {Cell reports},
volume = {41},
number = {4},
pages = {111533},
doi = {10.1016/j.celrep.2022.111533},
pmid = {36288702},
issn = {2211-1247},
mesh = {*Argonaute Proteins/metabolism ; RNA ; DNA, Single-Stranded ; RNA Cleavage ; DNA/metabolism ; Nucleotides/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.},
}
@article {pmid36208793,
year = {2022},
author = {Duckert, B and Fauvart, M and Goos, P and Stakenborg, T and Lagae, L and Braeken, D},
title = {High-definition electroporation: Precise and efficient transfection on a microelectrode array.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {352},
number = {},
pages = {61-73},
doi = {10.1016/j.jconrel.2022.10.001},
pmid = {36208793},
issn = {1873-4995},
mesh = {Microelectrodes ; *Electroporation/methods ; Transfection ; RNA, Messenger ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells.},
}
@article {pmid36183859,
year = {2022},
author = {Gan, X and Ding, D and Wang, M and Yang, Y and Sun, D and Li, W and Ding, W and Yang, F and Zhou, W and Yuan, S},
title = {DANCR deletion retards the initiation and progression of hepatocellular carcinoma based on gene knockout and patient-derived xenograft in situ hepatoma mice model.},
journal = {Cancer letters},
volume = {550},
number = {},
pages = {215930},
doi = {10.1016/j.canlet.2022.215930},
pmid = {36183859},
issn = {1872-7980},
mesh = {Animals ; *Carcinoma, Hepatocellular/genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Heterografts ; Humans ; *Liver Neoplasms/genetics/pathology ; Mice ; *MicroRNAs/genetics ; Prospective Studies ; *RNA, Long Noncoding/genetics/metabolism ; RNA, Small Interfering ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Our previous study has demonstrated that the expression level of long noncoding RNA (lncRNA)-differentiation antagonizing non-protein coding RNA (DANCR) increases in hepatocellular carcinoma (HCC), contributing to the initiation and aggravation of such kind of malignant tumor, which is recognized as a promising therapeutic target for patients with HCC. To further investigate the effect of DANCR on HCC in preclinical models, we generated a Dancr knockout (KO) mice model by Cas9/gRNA technology and a patient-derived xenograft (PDX) in situ hepatoma mice model using immunodeficient mice and utilized adeno-associated virus 8 (AAV8) delivery DANCR-shRNA system to silence the expression of DANCR in xenograft tumor. Here, we reported that Dancr expression mainly occurred in hepatocytes and its depletion significantly alleviated hepatic fibrosis in mice and showed a prospective result with smaller tumor size and fewer number of tumors in HCC preclinical mice model. Additionally, we found that the expression of Dancr in mice cirrhotic liver was positively correlated with the content of Dancr in serum. Overall, DANCR KO can inhibit the occurrence and development of HCC and is a target worthy of further study in patients with HCC.},
}
@article {pmid36146813,
year = {2022},
author = {Zawadzka, M and Andrzejewska-Romanowska, A and Gumna, J and Garfinkel, DJ and Pachulska-Wieczorek, K},
title = {Cell Compartment-Specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome.},
journal = {Viruses},
volume = {14},
number = {9},
pages = {},
pmid = {36146813},
issn = {1999-4915},
support = {R01 GM124216/GM/NIGMS NIH HHS/United States ; GM124216/NH/NIH HHS/United States ; },
mesh = {*RNA/genetics ; RNA, Messenger/metabolism ; *Retroelements ; Saccharomyces cerevisiae/genetics/metabolism ; Terminal Repeat Sequences ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The structural transitions RNAs undergo during trafficking are not well understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and those synthesized in vitro, we detected Ty1 gRNA structural alterations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, whereas the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA-RNA interactions required for retrotransposition.},
}
@article {pmid36127125,
year = {2022},
author = {Wang, J and Yang, Z and Ye, K},
title = {Methylation guide RNAs without box C/D motifs.},
journal = {RNA (New York, N.Y.)},
volume = {28},
number = {12},
pages = {1597-1605},
pmid = {36127125},
issn = {1469-9001},
mesh = {*RNA, Archaeal/genetics ; Methylation ; Base Sequence ; RNA/genetics/metabolism ; RNA, Small Nucleolar/genetics ; Nucleic Acid Conformation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The defining feature of methylation guide RNAs is two sets of box C and D motifs that form kink-turn structures specifically recognized by L7Ae family proteins. Here, we engineered a new type of methylation guide that lacks C/D motifs and requires no L7Ae for assembly and function. We determined a crystal structure of a bipartite C/D-free guide RNA in complex with Nop5, fibrillarin and substrate in the active form at 2.2 Å resolution. The stems of new guide RNAs functionally replace C/D motifs in Nop5 binding, precisely placing the substrate for site-specific modification. We also found that the bipartite architecture and association of L7Ae with C/D motifs enhance modification when association of guide RNAs or substrates is weak. Our study provides insights into the variations, robustness and possible evolutionary path of methylation guide RNAs.},
}
@article {pmid36127124,
year = {2022},
author = {Thalalla Gamage, S and Bortolin-Cavaillé, ML and Link, C and Bryson, K and Sas-Chen, A and Schwartz, S and Cavaillé, J and Meier, JL},
title = {Antisense pairing and SNORD13 structure guide RNA cytidine acetylation.},
journal = {RNA (New York, N.Y.)},
volume = {28},
number = {12},
pages = {1582-1596},
pmid = {36127124},
issn = {1469-9001},
mesh = {Humans ; Acetylation ; *Cytidine/genetics/metabolism ; RNA, Ribosomal, 18S/genetics ; RNA, Ribosomal/metabolism ; RNA, Small Nucleolar/genetics/metabolism ; Nucleotides/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {N4-acetylcytidine (ac[4]C) is an RNA nucleobase found in all domains of life. The establishment of ac[4]C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show that mutant snoRNAs can be used in combination with nucleotide resolution ac[4]C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac[4]C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.},
}
@article {pmid35941106,
year = {2022},
author = {Lisitskaya, L and Shin, Y and Agapov, A and Olina, A and Kropocheva, E and Ryazansky, S and Aravin, AA and Esyunina, D and Murakami, KS and Kulbachinskiy, A},
title = {Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4624},
pmid = {35941106},
issn = {2041-1723},
support = {R35 GM131860/GM/NIGMS NIH HHS/United States ; },
mesh = {*Argonaute Proteins/metabolism ; Bacteria/genetics ; Bacterial Proteins/genetics/metabolism ; DNA/metabolism ; Endonucleases/metabolism ; Prokaryotic Cells/metabolism ; *RNA/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.},
}
@article {pmid35914131,
year = {2022},
author = {Khatri, BS and Burt, A},
title = {A theory of resistance to multiplexed gene drive demonstrates the significant role of weakly deleterious natural genetic variation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {32},
pages = {e2200567119},
pmid = {35914131},
issn = {1091-6490},
mesh = {Alleles ; *Gene Drive Technology ; Genetic Drift ; *Genetic Variation/genetics ; Haplotypes ; *Models, Genetic ; Mutation ; Polymorphism, Single Nucleotide/genetics ; Selection, Genetic ; Stochastic Processes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Evolution of resistance is a major barrier to successful deployment of gene-drive systems to suppress natural populations, which could greatly reduce the burden of many vector-borne diseases. Multiplexed guide RNAs (gRNAs) that require resistance mutations in all target cut sites are a promising antiresistance strategy since, in principle, resistance would only arise in unrealistically large populations. Using stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) nonhomologous end-joining mutations, 2) single-nucleotide mutants arising de novo, or 3) single-nucleotide polymorphisms preexisting as standing variation. Our results explore the relative importance of these mechanisms and highlight a complexity of the mutation-selection-drift balance between haplotypes with complete resistance and those with an incomplete number of resistant alleles. We find that this leads to a phenomenon where weakly deleterious naturally occurring variants greatly amplify the probability of multisite resistance compared to de novo mutation. This key result provides design criterion for antiresistance multiplexed systems, which, in general, will need a larger number of gRNAs compared to de novo expectations. This theory may have wider application to the evolution of resistance or evolutionary rescue when multiple changes are required before selection can act.},
}
@article {pmid35779766,
year = {2022},
author = {Xiang, Y and Katrekar, D and Mali, P},
title = {Methods for recruiting endogenous and exogenous ADAR enzymes for site-specific RNA editing.},
journal = {Methods (San Diego, Calif.)},
volume = {205},
number = {},
pages = {158-166},
doi = {10.1016/j.ymeth.2022.06.011},
pmid = {35779766},
issn = {1095-9130},
support = {R01 HG009285/HG/NHGRI NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; R01 GM123313/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine/metabolism ; Adenosine Deaminase/genetics/metabolism ; *RNA Editing/genetics ; RNA, Messenger/genetics ; *RNA-Binding Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adenosine deaminases acting on RNA (ADARs) can be repurposed to achieve site-specific A-to-I RNA editing by recruiting them to a target of interest via an ADAR-recruiting guide RNA (adRNA). In this chapter, we present details towards experimental methods to enable this via two orthogonal strategies: one, via recruitment of endogenous ADARs (i.e. ADARs already natively expressed in cells); and two, via recruitment of exogenous ADARs (i.e. ADARs delivered into cells). Towards the former, we describe the use of circular adRNAs to recruit endogenous ADARs to a desired mRNA target. This results in robust, persistent and highly transcript specific editing both in vitro and in vivo. Towards the latter, we describe the use of a split-ADAR2 system, which allows for overexpression of ADAR2 variants that can be utilized to edit adenosines with high specificity, including at challenging to edit adenosines in non-preferred motifs such as those flanked by a 5' guanosine. We anticipate the described methods should facilitate RNA editing applications across research and biotechnology settings.},
}
@article {pmid35700773,
year = {2022},
author = {Chen, H and Pan, Y and Zhou, Q and Liang, C and Wong, CC and Zhou, Y and Huang, D and Liu, W and Zhai, J and Gou, H and Su, H and Zhang, X and Xu, H and Wang, Y and Kang, W and Kei Wu, WK and Yu, J},
title = {METTL3 Inhibits Antitumor Immunity by Targeting m[6]A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer.},
journal = {Gastroenterology},
volume = {163},
number = {4},
pages = {891-907},
doi = {10.1053/j.gastro.2022.06.024},
pmid = {35700773},
issn = {1528-0012},
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors ; *CD8-Positive T-Lymphocytes/metabolism ; Cell Line, Tumor ; Chemokine CXCL1 ; *Colorectal Neoplasms/pathology ; Cytokines/metabolism ; Humans ; Methyltransferases/genetics/metabolism ; Mice ; Mice, Knockout ; Phenylurea Compounds ; Receptors, Interleukin-8B/genetics/metabolism ; Triazoles ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND &amp; AIMS: N[6]-Methyladenosine (m[6]A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m[6]A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC.<br><br>METHODS: Mettl3 knockout mice, CD34[+] humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M[6]A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n&#xa0;= 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration.<br><br>RESULTS: We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4[+] and CD8[+] T cells, and eventually suppressed CRC in Apc[Min/+]Mettl3[+/-] mice, CD34[+] humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m[6]A-BHLHE41-CXCL1 axis by analysis of m[6]A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m[6]A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in&#xa0;vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in&#xa0;vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment.<br><br>CONCLUSIONS: Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m[6]A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.},
}
@article {pmid35628198,
year = {2022},
author = {Nashimoto, M},
title = {TRUE Gene Silencing.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628198},
issn = {1422-0067},
support = {the Science Research Promotion Fund//the Promotion and Mutual Aid Corporation for Private Schools of Japan/ ; },
mesh = {Endoribonucleases/metabolism ; Gene Silencing ; Humans ; *MicroRNAs/genetics ; *RNA Precursors ; RNA, Transfer/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {TRUE gene silencing is an RNA-mediated gene expression control technology and is termed after tRNase Z[L]-utilizing efficacious gene silencing. In this review, I overview the potentiality of small guide RNA (sgRNA) for TRUE gene silencing as novel therapeutics. First, I describe the physiology of tRNase Z[L] and cellular small RNA, and then sgRNA and TRUE gene silencing. An endoribonuclease, tRNase Z[L], which can efficiently remove a 3' trailer from pre-tRNA, is thought to play the role in tRNA maturation in the nucleus and mitochondria. There exist various small RNAs including miRNA and fragments from tRNA and rRNA, which can function as sgRNA, in living cells, and human cells appear to be harnessing cytosolic tRNase Z[L] for gene regulation together with these small RNAs. By utilizing the property of tRNase Z[L] to recognize and cleave micro-pre-tRNA, a pre-tRNA-like or micro-pre-tRNA-like complex, as well as pre-tRNA, tRNase Z[L] can be made to cleave any target RNA at any desired site under the direction of an artificial sgRNA that binds a target RNA and forms the pre-tRNA-like or micro-pre-tRNA-like complex. This general RNA cleavage method underlies TRUE gene silencing. Various examples of the application of TRUE gene silencing are reviewed including the application to several human cancer cells in order to induce apoptosis. Lastly, I discuss the potentiality of sgRNA as novel therapeutics for multiple myeloma.},
}
@article {pmid35507371,
year = {2022},
author = {Hunt, VM and Chen, W},
title = {A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {42},
pages = {6215-6218},
doi = {10.1039/d2cc01478e},
pmid = {35507371},
issn = {1364-548X},
mesh = {Animals ; Gene Expression ; Gene Expression Regulation ; Mammals/genetics/metabolism ; *MicroRNAs/genetics ; RNA, Messenger ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5' cap and 3' poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.},
}
@article {pmid35481478,
year = {2023},
author = {Ding, Y and Wang, M and Bu, H and Li, J and Lin, X and Xu, X},
title = {Application of an F0-based genetic assay in adult zebrafish to identify modifier genes of an inherited cardiomyopathy.},
journal = {Disease models &amp; mechanisms},
volume = {16},
number = {5},
pages = {},
pmid = {35481478},
issn = {1754-8411},
support = {R01 HL081753/HL/NHLBI NIH HHS/United States ; R01 HL107304/HL/NHLBI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/metabolism ; Animals ; Apoptosis Regulatory Proteins/genetics ; *Cardiomyopathies/genetics ; Gene Knockout Techniques ; Genes, Modifier ; *Zebrafish/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Modifier genes contribute significantly to our understanding of pathophysiology in human diseases; however, effective approaches to identify modifier genes are still lacking. Here, we aim to develop a rapid F0-based genetic assay in adult zebrafish using the bag3 gene knockout (bag3e2/e2) cardiomyopathy model as a paradigm. First, by utilizing a classic genetic breeding approach, we identified dnajb6b as a deleterious modifier gene for bag3 cardiomyopathy. Next, we established an F0-based genetic assay in adult zebrafish through injection of predicted microhomology-mediated end joining (MMEJ)-inducing single guide RNA/Cas9 protein complex. We showed that effective gene knockdown is maintained in F0 adult fish, enabling recapitulation of both salutary modifying effects of the mtor haploinsufficiency and deleterious modifying effects of the dnajb6b gene on bag3 cardiomyopathy. We finally deployed the F0-based genetic assay to screen differentially expressed genes in the bag3 cardiomyopathy model. As a result, myh9b was identified as a novel modifier gene for bag3 cardiomyopathy. Together, these data prove the feasibility of an F0 adult zebrafish-based genetic assay that can be effectively used to discover modifier genes for inherited cardiomyopathy.},
}
@article {pmid35381200,
year = {2022},
author = {Koopal, B and Potocnik, A and Mutte, SK and Aparicio-Maldonado, C and Lindhoud, S and Vervoort, JJM and Brouns, SJJ and Swarts, DC},
title = {Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA.},
journal = {Cell},
volume = {185},
number = {9},
pages = {1471-1486.e19},
pmid = {35381200},
issn = {1097-4172},
mesh = {*Argonaute Proteins/metabolism ; DNA/metabolism ; Prokaryotic Cells/cytology/metabolism/*physiology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)[+] depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.},
}
@article {pmid35294178,
year = {2022},
author = {Gao, M and Su, S and Cao, J and Xiang, S and Huang, Y and Shu, X and Ma, J and Liu, J},
title = {Targeted Manipulation of Cellular RNA m[6]A Methylation at the Single-Base Level.},
journal = {ACS chemical biology},
volume = {17},
number = {4},
pages = {854-863},
doi = {10.1021/acschembio.1c00895},
pmid = {35294178},
issn = {1554-8937},
mesh = {Methylation ; *Methyltransferases/genetics/metabolism ; *RNA/metabolism ; RNA, Messenger/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Development of tools for precise manipulation of cellular mRNA m[6]A methylation at the base level is highly required. Here, we report an RNA-guided RNA modification strategy using a fusion protein containing deactivated nuclease Cas13b and m[6]A methyltransferase METTL14, namely, dCas13b-M14, which is designedly positioned in the cytoplasm. dCas13b-M14 naturally heterodimerizes with endogenous METTL3 to form a catalytic complex to methylate specific cytoplasmic mRNA under a guide RNA (gRNA). We developed assays to screen and validate the guiding specificity of varied gRNAs at single-base resolution. With an optimum combination of dCas13b-M14 and gRNAs inside cells, we have successfully tuned methylation levels of several selected mRNA m[6]A sites. The off-target effect was evaluated by whole transcriptome m[6]A sequencing, and a very minor perturbation on the methylome was revealed. Finally, we successfully utilized the editing tool to achieve de novo methylations on five selected mRNA sites. Together, this study paves the way for studying position-dependent roles of m[6]A methylation in a particular transcript.},
}
@article {pmid35279766,
year = {2022},
author = {Hobohm, L and Koudelka, T and Bahr, FH and Truberg, J and Kapell, S and Schacht, SS and Meisinger, D and Mengel, M and Jochimsen, A and Hofmann, A and Heintz, L and Tholey, A and Voss, M},
title = {N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {3},
pages = {185},
pmid = {35279766},
issn = {1420-9071},
support = {CRC877//Deutsche Forschungsgemeinschaft/ ; project Z2//Deutsche Forschungsgemeinschaft/ ; CRC877//Deutsche Forschungsgemeinschaft/ ; junior funding//Deutsche Forschungsgemeinschaft/ ; //Department of Medicine, Kiel University/ ; //Kiel Life Science/ ; },
mesh = {Aspartic Acid Endopeptidases/deficiency/genetics/*metabolism ; Catalytic Domain/genetics ; Gene Editing ; Golgi Apparatus/*metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Mutagenesis, Site-Directed ; N-Acetylgalactosaminyltransferases/genetics/*metabolism ; *Proteolysis ; Proteome/*analysis ; Proteomics/methods ; Substrate Specificity ; Polypeptide N-acetylgalactosaminyltransferase ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Golgi membrane proteins such as glycosyltransferases and other glycan-modifying enzymes are key to glycosylation of proteins and lipids. Secretion of soluble Golgi enzymes that are released from their membrane anchor by endoprotease activity is a wide-spread yet largely unexplored phenomenon. The intramembrane protease SPPL3 can specifically cleave select Golgi enzymes, enabling their secretion and concomitantly altering global cellular glycosylation, yet the entire range of Golgi enzymes cleaved by SPPL3 under physiological conditions remains to be defined. Here, we established isogenic SPPL3-deficient HEK293 and HeLa cell lines and applied N-terminomics to identify substrates cleaved by SPPL3 and released into cell culture supernatants. With high confidence, our study identifies more than 20 substrates of SPPL3, including entirely novel substrates. Notably, our N-terminome analyses provide a comprehensive list of SPPL3 cleavage sites demonstrating that SPPL3-mediated shedding of Golgi enzymes occurs through intramembrane proteolysis. Through the use of chimeric glycosyltransferase constructs we show that transmembrane domains can determine cleavage by SPPL3. Using our cleavage site data, we surveyed public proteome data and found that SPPL3 cleavage products are present in human blood. We also generated HEK293 knock-in cells expressing the active site mutant D271A from the endogenous SPPL3 locus. Immunoblot analyses revealed that secretion of select novel substrates such as the key mucin-type O-glycosylation enzyme GALNT2 is dependent on endogenous SPPL3 protease activity. In sum, our study expands the spectrum of known physiological substrates of SPPL3 corroborating its significant role in Golgi enzyme turnover and secretion as well as in the regulation of global glycosylation pathways.},
}
@article {pmid35194183,
year = {2022},
author = {Attwaters, M},
title = {In vivo RNA base editing with circular RNAs.},
journal = {Nature reviews. Genetics},
volume = {23},
number = {4},
pages = {196-197},
pmid = {35194183},
issn = {1471-0064},
mesh = {Base Sequence ; Hydrolases ; *RNA Editing ; *RNA, Circular ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid35176627,
year = {2022},
author = {Li, Z and Kanazashi, H and Tokashiki, Y and Fujikawa, R and Okagaki, A and Katoh, S and Kojima, K and Haruna, K and Matsushita, N and Ishikawa, TO and Chen, H and Yamamura, K},
title = {TTR exon-humanized mouse optimal for verifying new therapies for FAP.},
journal = {Biochemical and biophysical research communications},
volume = {599},
number = {},
pages = {69-74},
doi = {10.1016/j.bbrc.2022.02.035},
pmid = {35176627},
issn = {1090-2104},
mesh = {Animals ; Disease Models, Animal ; Exons ; Gene Expression Regulation ; Humans ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Transgenic ; Polyneuropathies/*etiology/therapy ; Prealbumin/analysis/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.},
}
@article {pmid35145312,
year = {2022},
author = {Katrekar, D and Yen, J and Xiang, Y and Saha, A and Meluzzi, D and Savva, Y and Mali, P},
title = {Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {938-945},
pmid = {35145312},
issn = {1546-1696},
support = {R01 GM123313/GM/NIGMS NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; K01 DK119687/DK/NIDDK NIH HHS/United States ; R01 HG009285/HG/NHGRI NIH HHS/United States ; },
mesh = {Adenosine/metabolism ; Animals ; Mice ; Mice, Inbred C57BL ; RNA/genetics/metabolism ; *RNA Editing/genetics ; RNA, Circular ; RNA-Binding Proteins/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.},
}
@article {pmid35022325,
year = {2022},
author = {Meier, UT},
title = {Guide RNA acrobatics: the one-for-two shuffle.},
journal = {Genes &amp; development},
volume = {36},
number = {1-2},
pages = {1-3},
pmid = {35022325},
issn = {1549-5477},
support = {R01 HL136662/HL/NHLBI NIH HHS/United States ; },
mesh = {*Pseudouridine/genetics/metabolism ; RNA/genetics/metabolism ; RNA Processing, Post-Transcriptional ; RNA, Ribosomal/genetics ; RNA, Small Nucleolar ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {RNA modifications are crucial for the proper function of the RNAs. The sites of pseudouridines are often specified by dual hairpin guide RNAs, with one or both hairpins identifying a target uridine. In this issue of Genes &amp; Development, Jády and colleagues (pp. 70-83) identify a novel mechanism by which a single guide RNA hairpin can specify two uridines adjacent to each other or separated by 1 nt; i.e., one for two or guide RNA acrobatics.},
}
@article {pmid34980913,
year = {2022},
author = {Reautschnig, P and Wahn, N and Wettengel, J and Schulz, AE and Latifi, N and Vogel, P and Kang, TW and Pfeiffer, LS and Zarges, C and Naumann, U and Zender, L and Li, JB and Stafforst, T},
title = {CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {759-768},
pmid = {34980913},
issn = {1546-1696},
mesh = {Animals ; Base Sequence ; Mice ; RNA/metabolism ; *RNA Editing/genetics ; RNA, Messenger/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.},
}
@article {pmid34916304,
year = {2022},
author = {Jády, BE and Ketele, A and Moulis, D and Kiss, T},
title = {Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity.},
journal = {Genes &amp; development},
volume = {36},
number = {1-2},
pages = {70-83},
pmid = {34916304},
issn = {1549-5477},
mesh = {Humans ; Nucleic Acid Conformation ; *Pseudouridine/chemistry ; RNA/chemistry ; RNA, Ribosomal ; Uridine ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal "antisense" sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5'-UN-3', including the 5' substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.},
}
@article {pmid34792013,
year = {2021},
author = {Batra, K and Maan, S and Sehrawat, A},
title = {An Insight on RNA Based Therapeutics and Vaccines: Challenges and Opportunities.},
journal = {Current topics in medicinal chemistry},
volume = {21},
number = {32},
pages = {2851-2855},
doi = {10.2174/1568026621666211118095451},
pmid = {34792013},
issn = {1873-4294},
support = {SRG/2020/000229//Science and Engineering Research Board, Department of Science and Technology, Government of India for Startup research/ ; },
mesh = {Animals ; *Aptamers, Nucleotide ; Humans ; *Oligonucleotides, Antisense ; Pharmaceutical Preparations/*classification ; *RNA, Small Interfering ; *mRNA Vaccines ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In this era, RNA molecules have provided a unique opportunity to researchers all over the world for expanding their range of targets in the development of drugs. Due to the unique pharmacological as well as physicochemical characteristics of different RNA molecules such as aptamers, small interfering RNAs (siRNA), antisense oligonucleotides (ASO) and guide RNAs (gRNA), they have emerged recently as a new class of drugs. They are used for selective action on proteins and genes that were not possible to target by conventional drug molecules. These RNA molecules like guide RNAs are also components of novel gene editing mechanisms which can modify the genome nearly in all cells. Vaccines based on RNA molecules have also provided a promising alternative to conventional live attenuated vaccines. RNA based vaccines have high potency, can be rapidly developed, and have potential for manufacturing at a cheaper rate and safe administration. However, the application of these RNAs has been restricted by the high instability and inefficient in vivo delivery. Technological advancement needs to overcome these issues so that RNA based drugs targeting several diseases can be developed. This article emphasizes the potential of RNA based drugs and the major barriers associated with the development of RNA therapeutics. Additionally, the role of RNA based vaccines and their challenges in advancing this promising vaccine platform for the prevention of infectious diseases have been discussed.},
}
@article {pmid34725166,
year = {2021},
author = {Talross, GJS and Deryusheva, S and Gall, JG},
title = {Stable lariats bearing a snoRNA (slb-snoRNA) in eukaryotic cells: A level of regulation for guide RNAs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {45},
pages = {},
pmid = {34725166},
issn = {1091-6490},
support = {R01 GM033397/GM/NIGMS NIH HHS/United States ; },
mesh = {3T3 Cells ; Animals ; Female ; Gene Expression Regulation ; HeLa Cells ; Humans ; Introns ; Mice ; RNA, Circular/*metabolism ; RNA, Small Nucleolar/*metabolism ; Saccharomyces cerevisiae ; Xenopus laevis ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Small nucleolar (sno)RNAs guide posttranscriptional modifications essential for the biogenesis and function of their target. The majority of snoRNAs in higher eukaryotes are encoded within introns. They are first released from nascent transcripts in the form of a lariat and rapidly targeted by the debranching enzyme and nuclear exonucleases for linearization and further trimming. In this study, we report that some snoRNAs are encoded within unusually stable intronic RNAs. These intronic sequences can escape the debranching enzyme and accumulate as lariats. Stable lariats bearing a snoRNA, or slb-snoRNA, are associated with snoRNA binding proteins but do not guide posttranscriptional modification. While most slb-snoRNAs accumulate in the nucleus, some can be exported to the cytoplasm. We find that this export competes with snoRNA maturation. Slb-snoRNAs provide a previously unknown layer of regulation to snoRNA and snoRNA binding proteins.},
}
@article {pmid34610303,
year = {2021},
author = {Zhou, B and Zhang, Y and Li, S and Wu, L and Fejes-Toth, G and Naray-Fejes-Toth, A and Soukas, AA},
title = {Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase.},
journal = {Cell reports},
volume = {37},
number = {1},
pages = {109785},
pmid = {34610303},
issn = {2211-1247},
support = {R01 DK101522/DK/NIDDK NIH HHS/United States ; P30 DK040561/DK/NIDDK NIH HHS/United States ; K08 DK087941/DK/NIDDK NIH HHS/United States ; R56 AG068999/AG/NIA NIH HHS/United States ; P30 DK057521/DK/NIDDK NIH HHS/United States ; R01 AG058256/AG/NIA NIH HHS/United States ; },
mesh = {AMP-Activated Protein Kinases/antagonists &amp; inhibitors/*metabolism ; Animals ; Diabetes Mellitus, Type 2/pathology ; Diet, High-Fat ; Forkhead Box Protein O1/metabolism ; Glucose/metabolism ; Immediate-Early Proteins/deficiency/genetics/*metabolism ; Insulin/metabolism ; Insulin Resistance ; Liver/*metabolism ; Mechanistic Target of Rapamycin Complex 2/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Phosphorylation ; Protein Serine-Threonine Kinases/deficiency/genetics/*metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Signal Transduction ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {A hallmark of type 2 diabetes (T2D) is hepatic resistance to insulin's glucose-lowering effects. The serum- and glucocorticoid-regulated family of protein kinases (SGK) is activated downstream of mechanistic target of rapamycin complex 2 (mTORC2) in response to insulin in parallel to AKT. Surprisingly, despite an identical substrate recognition motif to AKT, which drives insulin sensitivity, pathological accumulation of SGK1 drives insulin resistance. Liver-specific Sgk1-knockout (Sgk1[Lko]) mice display improved glucose tolerance and insulin sensitivity and are protected from hepatic steatosis when fed a high-fat diet. Sgk1 promotes insulin resistance by inactivating AMP-activated protein kinase (AMPK) via phosphorylation on inhibitory site AMPKα[Ser485/491]. We demonstrate that SGK1 is dominant among SGK family kinases in regulation of insulin sensitivity, as Sgk1, Sgk2, and Sgk3 triple-knockout mice have similar increases in hepatic insulin sensitivity. In aggregate, these data suggest that targeting hepatic SGK1 may have therapeutic potential in T2D.},
}
@article {pmid34565318,
year = {2021},
author = {Baleva, MV and Piunova, UE and Chicherin, IV and Krasavina, DG and Levitskii, SA and Kamenski, PA},
title = {Yeast Translational Activator Mss51p and Human ZMYND17 - Two Proteins with a Common Origin, but Different Functions.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {9},
pages = {1151-1161},
doi = {10.1134/S0006297921090108},
pmid = {34565318},
issn = {1608-3040},
mesh = {Electron Transport Complex IV/metabolism ; Evolution, Molecular ; Gene Editing ; HeLa Cells ; Humans ; Mitochondria/enzymology/metabolism ; NADH Dehydrogenase/metabolism ; Phylogeny ; Protein Subunits/metabolism ; Proton-Translocating ATPases/metabolism ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/classification/genetics/*metabolism ; Transcription Factors/classification/deficiency/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Despite its similarity to protein biosynthesis in bacteria, translation in the mitochondria of modern eukaryotes has several unique features, such as the necessity for coordination of translation of mitochondrial mRNAs encoding proteins of the electron transport chain complexes with translation of other protein components of these complexes in the cytosol. In the mitochondria of baker's yeast Saccharomyces cerevisiae, this coordination is carried out by a system of translational activators that predominantly interact with the 5'-untranslated regions of mitochondrial mRNAs. No such system has been found in human mitochondria, except a single identified translational activator, TACO1. Here, we studied the role of the ZMYND17 gene, an ortholog of the yeast gene for the translational activator Mss51p, on the mitochondrial translation in human cells. Deletion of the ZMYND17 gene did not affect translation in the mitochondria, but led to the decrease in the cytochrome c oxidase activity and increase in the amount of free F1 subunit of ATP synthase. We also investigated the evolutionary history of Mss51p and ZMYND17 and suggested a possible mechanism for the divergence of functions of these orthologous proteins.},
}
@article {pmid34517953,
year = {2021},
author = {Ojha, N and Diaz Quiroz, JF and Rosenthal, JJC},
title = {In vitro and in cellula site-directed RNA editing using the λNDD-BoxB system.},
journal = {Methods in enzymology},
volume = {658},
number = {},
pages = {335-358},
doi = {10.1016/bs.mie.2021.06.009},
pmid = {34517953},
issn = {1557-7988},
support = {R01 NS087726/NS/NINDS NIH HHS/United States ; },
mesh = {*Adenosine Deaminase/genetics ; Humans ; Inosine ; *RNA Editing ; RNA-Binding Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-directed RNA editing (SDRE) exploits the enzymatic activity of Adenosine Deaminases Acting on RNAs (ADAR) to program changes in genetic information as it passes through RNA. ADARs convert adenosine (A) to inosine (I) through a hydrolytic deamination and since I can be read as guanosine (G) during translation, this change can regulate gene function and correct G→A genetic mutations. In SDRE, ADARs are redirected to convert user-defined A's to I's. SDRE also has certain advantages over genome editing because the changes in RNA are reversible and thus safer. In addition, ADARs are endogenously expressed in humans and therefore unlikely to provoke immunological complications when administered. Recently, a variety of systems for SDRE have been developed. Some rely on harnessing endogenously expressed ADARs and other deliver engineered versions of ADAR's catalytic domain. All systems are currently under refinement, and there are still challenges associated with raising their efficiency and specificity to levels that are adequate for therapeutics. This chapter provides a detailed protocol for in vitro and in cellula editing assays using the λNDD-BoxB system, one of the first systems developed for SDRE. The λNDD-BoxB system relies on gRNAs that are linked to the catalytic domain of human ADAR2 through a small RNA binding protein-RNA stem/loop interaction. We provide step-by-step protocols for (a) the construction of guide RNAs and editing enzyme plasmids, and (b) their use in vitro and in cellula for editing assays using a fluorescent protein-based reporter system containing a premature termination codon that can be corrected by editing.},
}
@article {pmid34475498,
year = {2021},
author = {Höfler, S and Lukat, P and Blankenfeldt, W and Carlomagno, T},
title = {Eukaryotic Box C/D methylation machinery has two non-symmetric protein assembly sites.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {17561},
pmid = {34475498},
issn = {2045-2322},
mesh = {Archaea/*genetics ; Eukaryota/*genetics ; Methylation ; Methyltransferases/*metabolism ; RNA/*genetics ; RNA, Small Nucleolar/*metabolism ; Ribonucleoproteins/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2'-OH of RNA. The central 'guide RNA' has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C'/D' motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C'/D' motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2'-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C'/D' motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2'-O-methylation enzymes.},
}
@article {pmid34385348,
year = {2021},
author = {Deryusheva, S and Talross, GJS and Gall, JG},
title = {SnoRNA guide activities: real and ambiguous.},
journal = {RNA (New York, N.Y.)},
volume = {27},
number = {11},
pages = {1363-1373},
pmid = {34385348},
issn = {1469-9001},
support = {R01 GM033397/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; Coiled Bodies/genetics/*metabolism ; Humans ; Methylation ; Pseudouridine/*metabolism ; *RNA Processing, Post-Transcriptional ; RNA, Ribosomal/genetics/metabolism ; RNA, Small Nucleolar/chemistry/genetics/*metabolism ; Spliceosomes/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2'-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.},
}
@article {pmid34326847,
year = {2021},
author = {Song, S and Manook, M and Kwun, J and Jackson, AM and Knechtle, SJ and Kelsoe, G},
title = {Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {705140},
pmid = {34326847},
issn = {1664-3224},
support = {R01 AI128832/AI/NIAID NIH HHS/United States ; U19 AI131471/AI/NIAID NIH HHS/United States ; UM1 AI109565/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antibody Specificity ; B-Lymphocyte Subsets/*immunology ; Cell Line ; Cells, Cultured ; Clonal Evolution ; Clone Cells ; Female ; Gene Rearrangement, B-Lymphocyte ; Genes, Reporter ; Graft Rejection/*immunology ; HLA Antigens/*immunology ; Histocompatibility Antigens/biosynthesis/immunology ; Humans ; Immunoglobulin G/immunology ; *Immunologic Memory ; Indicators and Reagents ; Isoantibodies/*blood/immunology ; Lymphocyte Activation ; Macaca mulatta ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Skin Transplantation ; Species Specificity ; Specific Pathogen-Free Organisms ; V(D)J Recombination ; beta 2-Microglobulin/antagonists &amp; inhibitors/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.},
}
@article {pmid34289345,
year = {2021},
author = {Evavold, CL and Hafner-Bratkovič, I and Devant, P and D'Andrea, JM and Ngwa, EM and Boršić, E and Doench, JG and LaFleur, MW and Sharpe, AH and Thiagarajah, JR and Kagan, JC},
title = {Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.},
journal = {Cell},
volume = {184},
number = {17},
pages = {4495-4511.e19},
pmid = {34289345},
issn = {1097-4172},
support = {R01 AI093589/AI/NIAID NIH HHS/United States ; R56 AI093589/AI/NIAID NIH HHS/United States ; R35 GM142683/GM/NIGMS NIH HHS/United States ; R03 DK125630/DK/NIDDK NIH HHS/United States ; R37 AI116550/AI/NIAID NIH HHS/United States ; U19 AI133524/AI/NIAID NIH HHS/United States ; F31 AI138369/AI/NIAID NIH HHS/United States ; R01 AI116550/AI/NIAID NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; K08 DK113106/DK/NIDDK NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*metabolism ; Amino Acids/metabolism ; Animals ; Cell Adhesion Molecules, Neuronal/metabolism ; Cell Line ; Genetic Testing ; Humans ; Inflammasomes/metabolism ; Intracellular Signaling Peptides and Proteins/chemistry/*metabolism ; Macrophages/metabolism ; Mechanistic Target of Rapamycin Complex 1/*metabolism ; Mechanistic Target of Rapamycin Complex 2/metabolism ; Mice, Inbred C57BL ; Mitochondria/metabolism ; Monomeric GTP-Binding Proteins/*metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Nerve Growth Factors/metabolism ; Phosphate-Binding Proteins/chemistry/*metabolism ; Protein Domains ; *Protein Multimerization ; *Pyroptosis ; Reactive Oxygen Species/metabolism ; *Signal Transduction ; TOR Serine-Threonine Kinases/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).},
}
@article {pmid34239016,
year = {2021},
author = {Banerjee, T and Takahashi, H and Subekti, DRG and Kamagata, K},
title = {Engineering of the genome editing protein Cas9 to slide along DNA.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14165},
pmid = {34239016},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; DNA/*metabolism ; *Gene Editing ; *Genetic Engineering ; HMGN Proteins/metabolism ; Models, Biological ; Mutation/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The genome editing protein Cas9 faces engineering challenges in improving off-target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy. Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA. A sliding model of Cas9 and its engineering action are discussed herein.},
}
@article {pmid34237300,
year = {2021},
author = {Wang, N and Lu, L and Cao, QF and Qian, S and Ding, J and Wang, C and Duan, H and Shen, H and Qi, J},
title = {Partial inhibition of activin receptor-like kinase 4 alleviates bladder fibrosis caused by bladder outlet obstruction.},
journal = {Experimental cell research},
volume = {406},
number = {1},
pages = {112724},
doi = {10.1016/j.yexcr.2021.112724},
pmid = {34237300},
issn = {1090-2422},
mesh = {Activin Receptors, Type I/antagonists &amp; inhibitors/*genetics/metabolism ; Animals ; Base Sequence ; Disease Models, Animal ; Extracellular Matrix/metabolism/pathology ; Gene Editing ; Gene Expression Regulation ; Humans ; JNK Mitogen-Activated Protein Kinases/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; Mitogen-Activated Protein Kinase 3/genetics/metabolism ; Phosphorylation ; Signal Transduction ; Smad2 Protein/*genetics/metabolism ; Smad3 Protein/*genetics/metabolism ; Transforming Growth Factor beta/genetics/metabolism ; Urinary Bladder/*metabolism/pathology ; Urinary Bladder Neck Obstruction/*genetics/metabolism/pathology/therapy ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The bladder undergoes profound structural alterations after bladder outlet obstruction (BOO), characterized by hypertrophy of the bladder wall and accumulation of extracellular matrix (ECM). Transforming growth factor-β (TGF-β) has been found to promote fibrosis of the bladder induced by partial bladder outlet obstruction (pBOO). Activin receptor-like kinase 4 (ALK4) is a downstream receptor of the TGF-β superfamily. However, the role of the ALK4-Smad2/3 pathway in the pathogenesis of bladder fibrosis caused by pBOO remains unknown. This study focused on learning the role of ALK4 in the process of bladder fibrosis caused by pBOO. The pBOO mice models showed that ALK4 expression was found to upregulate in the wild-type bladder 6 weeks after pBOO compared to control group. Then, mice with heterozygous knockout of the ALK4 gene (ALK4+/-) were generated. Histological analysis and Western blot (WB) results showed significant suppression of collagen expression in the bladders of ALK4+/- mice after pBOO compared with WT mice. WB also showed that ALK4+/- mice demonstrated significant suppression of phosphorylated Smad2/3 (p-Smad2/3) expression in the bladder 6 weeks after pBOO but not of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase or protein 38 (p-ERK, p-JNK, p-P38) expression. This effect might have occurred through partial inactivation of the Smad2/3 signaling pathway. In vitro, ALK4 overexpression promoted collagen production in cultured BSMCs and activated the Smad2/3 signaling pathway. Taken together, our results demonstrated that ALK4 insufficiency alleviated bladder fibrosis in a mouse model of pBOO partly by suppressing Smad2/3 activity.},
}
@article {pmid34172975,
year = {2021},
author = {Tekel, SJ and Brookhouser, N and Standage-Beier, K and Wang, X and Brafman, DA},
title = {Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment.},
journal = {Nature protocols},
volume = {16},
number = {7},
pages = {3596-3624},
pmid = {34172975},
issn = {1750-2799},
support = {R01GM121698//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R21AG056706//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01GM106081//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Adenosine/*metabolism ; Base Sequence ; Clone Cells ; Cryopreservation ; Cytosine/*metabolism ; Flow Cytometry ; Gene Editing/*methods ; *Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Plasmids/genetics ; Reproducibility of Results ; Single-Cell Analysis ; Transfection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification. Here, we outline a series of methods that employ transient reporters of editing enrichment (TREE) to facilitate the highly efficient single-base editing of human cells at precise genomic loci. Briefly, these transient reporters of editing enrichment based methods employ a transient episomal fluorescent reporter that allows for the real-time, flow-cytometry-based enrichment of cells that have had single nucleotide changes at precise genomic locations. This protocol details how these approaches can enable the rapid (~3-4 weeks) and efficient (clonal editing efficiencies >80%) generation of biallelic or multiplexed edited isogenic hPSC lines using adenosine and cytosine base editors.},
}
@article {pmid34117606,
year = {2021},
author = {Jin, S and Zhan, J and Zhou, Y},
title = {Argonaute proteins: structures and their endonuclease activity.},
journal = {Molecular biology reports},
volume = {48},
number = {5},
pages = {4837-4849},
pmid = {34117606},
issn = {1573-4978},
mesh = {*Argonaute Proteins/chemistry/genetics/metabolism ; Endonucleases/chemistry/metabolism ; Gene Editing ; Gene Silencing ; RNA, Small Interfering/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins are highly conserved and widely expressed in almost all organisms. They not only play a critical role in the biogenesis of small RNAs but also defend against invading nucleic acids via small RNA or DNA-mediated gene silencing pathways. One functional mechanism of Argonaute proteins is acting as a nucleic-acid-guided endonuclease, which can cleave targets complementary to DNA or RNA guides. The cleavage then leads to translational silencing directly or indirectly by recruiting additional silencing proteins. Here, we summarized the latest research progress in structural and biological studies of Argonaute proteins and pointed out their potential applications in the field of gene editing.},
}
@article {pmid34090973,
year = {2022},
author = {Zhang, Y and Wang, Y and Xu, L and Lou, C and Ouyang, Q and Qian, L},
title = {Paired dCas9 design as a nucleic acid detection platform for pathogenic strains.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {70-77},
doi = {10.1016/j.ymeth.2021.06.003},
pmid = {34090973},
issn = {1095-9130},
mesh = {DNA ; Luciferases ; Luminescence ; *Mycobacterium tuberculosis/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The wide application of molecular beacon probes in specific DNA detection, especially in the fast prototyping of pathogen DNA detection kits in point-of-care diagnostics, has been hindered by the nonflexible choice of target sequences and the unstable fluorophore output. We developed an in vitro DNA detection system consisting of a pair of dCas9 proteins linked to split halves of luciferase, named the Paired dCas9 (PC) reporter. Co-localization of the reporter pair to a ~46 bp target sequence defined by two single guide RNAs (sgRNAs) activated luciferase which subsequently generated highly intensified luminescent signals. Combined with an array design and statistical analyses, the PC reporter system could be programmed to access sequence information across the entire genome of the pathogenic Mycobacterium tuberculosis H37Rv strain. These findings suggest great potential for the PC reporter in effective and affordable in vitro nucleic acid detection technologies. In this article we highlighted the systems design from our previous researchworkon the PC reporter (Zhang et al, 2015)with a focuson methodology.},
}
@article {pmid34085255,
year = {2021},
author = {Czekay, DP and Schultz, SK and Kothe, U},
title = {Assaying the Molecular Determinants and Kinetics of RNA Pseudouridylation by H/ACA snoRNPs and Stand-Alone Pseudouridine Synthases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2298},
number = {},
pages = {357-378},
pmid = {34085255},
issn = {1940-6029},
mesh = {Escherichia coli/genetics ; Intramolecular Transferases/*genetics ; Kinetics ; Pseudouridine/*genetics ; RNA/*genetics ; RNA Processing, Post-Transcriptional/genetics ; Ribonucleoproteins, Small Nucleolar/*genetics ; Saccharomyces cerevisiae/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.},
}
@article {pmid34067312,
year = {2021},
author = {Perrone, D and Marchesi, E and Preti, L and Navacchia, ML},
title = {Modified Nucleosides, Nucleotides and Nucleic Acids via Click Azide-Alkyne Cycloaddition for Pharmacological Applications.},
journal = {Molecules (Basel, Switzerland)},
volume = {26},
number = {11},
pages = {},
pmid = {34067312},
issn = {1420-3049},
mesh = {Adenosine/chemistry ; Alkynes/*chemistry ; Animals ; Azides/*chemistry ; Cell Line, Tumor ; Click Chemistry ; Cycloaddition Reaction ; DNA/chemistry ; ErbB Receptors/chemistry ; Humans ; Mice ; Nucleic Acids/*chemistry ; Nucleosides/*chemistry ; Nucleotides/*chemistry ; Oligonucleotides, Antisense/chemistry ; Reproducibility of Results ; Technology, Pharmaceutical ; Triazoles/chemistry ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The click azide = alkyne 1,3-dipolar cycloaddition (click chemistry) has become the approach of choice for bioconjugations in medicinal chemistry, providing facile reaction conditions amenable to both small and biological molecules. Many nucleoside analogs are known for their marked impact in cancer therapy and for the treatment of virus diseases and new targeted oligonucleotides have been developed for different purposes. The click chemistry allowing the tolerated union between units with a wide diversity of functional groups represents a robust means of designing new hybrid compounds with an extraordinary diversity of applications. This review provides an overview of the most recent works related to the use of click chemistry methodology in the field of nucleosides, nucleotides and nucleic acids for pharmacological applications.},
}
@article {pmid33974999,
year = {2022},
author = {Dang, L and Zhou, X and Zhong, X and Yu, W and Huang, S and Liu, H and Chen, Y and Zhang, W and Yuan, L and Li, L and Huang, X and Li, G and Liu, J and Tong, G},
title = {Correction of the pathogenic mutation in TGM1 gene by adenine base editing in mutant embryos.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {175-183},
pmid = {33974999},
issn = {1525-0024},
mesh = {*Adenine ; *Gene Editing/methods ; Heterozygote ; Humans ; Mutation ; *Transglutaminases/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {A couple diagnosed as carriers for lamellar ichthyosis, an autosomal recessive rare disease, encountered two pregnancy losses. Their blood samples showed the same heterozygous c.607C>T mutation in the TGM1 gene. However, we found that about 98.4% of the sperm had mutations, suggesting possible de novo germline mutation. To explore the probability of correcting this mutation, we used two different adenine base editors (ABEs) combined with related truncated single guide RNA (sgRNA) to repair the pathogenic mutation in mutant zygotes. Our results showed that the editing efficiency was 73.8% for ABEmax-NG combined with 20-bp-length sgRNA and 78.7% for Sc-ABEmax combined with 19-bp-length sgRNA. The whole-genome sequencing (WGS) and deep sequencing analysis demonstrated precise DNA editing. This study reveals the possibility of correcting the genetic mutation in embryos with the ABE system.},
}
@article {pmid33958780,
year = {2021},
author = {Cheng, L and Li, Y and Qi, Q and Xu, P and Feng, R and Palmer, L and Chen, J and Wu, R and Yee, T and Zhang, J and Yao, Y and Sharma, A and Hardison, RC and Weiss, MJ and Cheng, Y},
title = {Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.},
journal = {Nature genetics},
volume = {53},
number = {6},
pages = {869-880},
pmid = {33958780},
issn = {1546-1718},
support = {2017093/DDCF_/Doris Duke Charitable Foundation/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; R24 DK106766/DK/NIDDK NIH HHS/United States ; R35 GM133614/GM/NIGMS NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/genetics ; Base Pairing/genetics ; Base Sequence ; Cell Line ; DNA/*genetics ; Epigenesis, Genetic ; Fetal Hemoglobin/*genetics ; Gene Editing ; *Gene Expression Regulation ; Genome, Human ; Humans ; Mutagenesis/genetics ; Nucleotides/*genetics ; Point Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA/genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; Repressor Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34[+] hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.},
}
@article {pmid33939417,
year = {2021},
author = {Doherty, EE and Wilcox, XE and van Sint Fiet, L and Kemmel, C and Turunen, JJ and Klein, B and Tantillo, DJ and Fisher, AJ and Beal, PA},
title = {Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position.},
journal = {Journal of the American Chemical Society},
volume = {143},
number = {18},
pages = {6865-6876},
pmid = {33939417},
issn = {1520-5126},
support = {P30 GM124165/GM/NIGMS NIH HHS/United States ; S10 OD021527/OD/NIH HHS/United States ; T32 GM113770/GM/NIGMS NIH HHS/United States ; },
mesh = {Cytidine/*chemistry ; Humans ; Models, Molecular ; RNA Editing ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.},
}
@article {pmid33857147,
year = {2021},
author = {Zhang, Y and Davis, L and Maizels, N},
title = {Pathways and signatures of mutagenesis at targeted DNA nicks.},
journal = {PLoS genetics},
volume = {17},
number = {4},
pages = {e1009329},
pmid = {33857147},
issn = {1553-7404},
support = {P01 CA077852/CA/NCI NIH HHS/United States ; R01 CA183967/CA/NCI NIH HHS/United States ; R21 CA190675/CA/NCI NIH HHS/United States ; },
mesh = {BRCA2 Protein/*genetics ; Cell Cycle/genetics ; DNA Breaks, Double-Stranded ; *DNA Breaks, Single-Stranded ; DNA Damage/*genetics ; DNA Helicases/genetics ; DNA Repair/genetics ; DNA-Binding Proteins/genetics ; DNA-Directed DNA Polymerase/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; INDEL Mutation/genetics ; Mad2 Proteins/genetics ; Mutagenesis/*genetics ; Nucleotidyltransferases/genetics ; Signal Transduction/genetics ; DNA Polymerase theta ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.},
}
@article {pmid33823543,
year = {2021},
author = {Trucks, S and Hanspach, G and Hengesbach, M},
title = {Eukaryote specific RNA and protein features facilitate assembly and catalysis of H/ACA snoRNPs.},
journal = {Nucleic acids research},
volume = {49},
number = {8},
pages = {4629-4642},
pmid = {33823543},
issn = {1362-4962},
mesh = {Catalysis ; Escherichia coli/metabolism ; Fluorescence Resonance Energy Transfer ; Gene Expression ; Hydro-Lyases/genetics/*metabolism ; In Vitro Techniques ; Inverted Repeat Sequences ; Microtubule-Associated Proteins/genetics/*metabolism ; Models, Molecular ; Nuclear Proteins/genetics/*metabolism ; Protein Domains ; RNA Folding ; RNA, Small Nucleolar/genetics/*metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Recombinant Proteins ; Ribonucleoproteins, Small Nuclear/genetics/*metabolism ; Ribonucleoproteins, Small Nucleolar/genetics/*metabolism ; Saccharomyces cerevisiae/enzymology/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.},
}
@article {pmid33761328,
year = {2021},
author = {Cronan, MR and Hughes, EJ and Brewer, WJ and Viswanathan, G and Hunt, EG and Singh, B and Mehra, S and Oehlers, SH and Gregory, SG and Kaushal, D and Tobin, DM},
title = {A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.},
journal = {Cell},
volume = {184},
number = {7},
pages = {1757-1774.e14},
pmid = {33761328},
issn = {1097-4172},
support = {R01 AI127715/AI/NIAID NIH HHS/United States ; R01 AI130236/AI/NIAID NIH HHS/United States ; P51 OD011133/OD/NIH HHS/United States ; R43 AI134249/AI/NIAID NIH HHS/United States ; R01 AI125517/AI/NIAID NIH HHS/United States ; R01 AI111943/AI/NIAID NIH HHS/United States ; T32 GM136627/GM/NIGMS NIH HHS/United States ; P51 OD011104/OD/NIH HHS/United States ; P30 CA014236/CA/NCI NIH HHS/United States ; P01 AG051428/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/genetics/metabolism ; Cadherins/genetics/metabolism ; Cell Differentiation ; Disease Models, Animal ; Epithelioid Cells/cytology/immunology/metabolism ; Granuloma/immunology/metabolism/*pathology ; Hematopoietic Stem Cells/cytology/metabolism ; Immunity/*physiology ; Interferon-gamma/metabolism ; Interleukin-12/metabolism ; Macrophages/cytology/immunology/metabolism ; Mycobacterium Infections, Nontuberculous/immunology/*pathology ; Mycobacterium marinum/isolation &amp; purification/physiology ; Necrosis ; Receptors, Interleukin-4/antagonists &amp; inhibitors/genetics/metabolism ; STAT6 Transcription Factor/antagonists &amp; inhibitors/genetics/metabolism ; Signal Transduction ; Zebrafish/growth &amp; development/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.},
}
@article {pmid33739974,
year = {2021},
author = {Tang, Y and Qu, Z and Lei, J and He, R and Adelson, DL and Zhu, Y and Yang, Z and Wang, D},
title = {The long noncoding RNA FRILAIR regulates strawberry fruit ripening by functioning as a noncanonical target mimic.},
journal = {PLoS genetics},
volume = {17},
number = {3},
pages = {e1009461},
pmid = {33739974},
issn = {1553-7404},
mesh = {Fragaria/*genetics/*growth &amp; development ; Fruit/*genetics ; Gene Editing ; *Gene Expression Regulation, Plant ; Genetic Association Studies ; MicroRNAs/genetics ; Models, Biological ; Phenotype ; *RNA, Long Noncoding ; RNA, Messenger/genetics ; *RNA, Plant ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Long noncoding RNAs (lncRNAs) are emerging as important regulators in plant development, but few of them have been functionally characterized in fruit ripening. Here, we have identified 25,613 lncRNAs from strawberry ripening fruits based on RNA-seq data from poly(A)-depleted libraries and rRNA-depleted libraries, most of which exhibited distinct temporal expression patterns. A novel lncRNA, FRILAIR harbours the miR397 binding site that is highly conserved in diverse strawberry species. FRILAIR overexpression promoted fruit maturation in the Falandi strawberry, which was consistent with the finding from knocking down miR397, which can guide the mRNA cleavage of both FRILAIR and LAC11a (encoding a putative laccase-11-like protein). Moreover, LAC11a mRNA levels were increased in both FRILAIR overexpressing and miR397 knockdown fruits, and accelerated fruit maturation was also found in LAC11a overexpressing fruits. Overall, our study demonstrates that FRILAIR can act as a noncanonical target mimic of miR397 to modulate the expression of LAC11a in the strawberry fruit ripening process.},
}
@article {pmid33705909,
year = {2021},
author = {Sakamoto, K and Akishiba, M and Iwata, T and Arafiles, JVV and Imanishi, M and Futaki, S},
title = {Use of homoarginine to obtain attenuated cationic membrane lytic peptides.},
journal = {Bioorganic &amp; medicinal chemistry letters},
volume = {40},
number = {},
pages = {127925},
doi = {10.1016/j.bmcl.2021.127925},
pmid = {33705909},
issn = {1464-3405},
mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/pharmacology ; Dextrans/chemistry ; Drug Carriers/*chemistry/toxicity ; Drug Liberation ; Endosomes/*metabolism ; Fluoresceins/chemistry ; Fluorescent Dyes/chemistry ; HeLa Cells ; Homoarginine/*chemistry ; Humans ; Immunoglobulin G/pharmacology ; Integrases/pharmacology ; Intracellular Membranes/*metabolism ; Liposomes/chemistry ; Peptides/*chemistry/toxicity ; Sulfonic Acids/chemistry ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Our research group has been studying the design of intracellular delivery peptides based on cationic lytic peptides. By placing negatively charged amino acids on potentially hydrophobic faces of the peptides, membrane lytic activity is attenuated on the cell surface, whereas it recovers in endosomes, enabling cytosolic delivery of proteins including antibodies. These lytic peptides generally contain multiple lysines, facilitating cell surface interaction and membrane perturbation. This study evaluated the effect of lysine-to-homoarginine substitution using HAad as a model delivery peptide. The resulting peptide had a comparable or better delivery efficacy for Cre recombinase, antibodies, and the Cas9/sgRNA complex with one-quarter of the concentration of HAad, implying that a subtle structural difference can affect delivery activity.},
}
@article {pmid33691100,
year = {2021},
author = {Chandramouly, G and Liao, S and Rusanov, T and Borisonnik, N and Calbert, ML and Kent, T and Sullivan-Reed, K and Vekariya, U and Kashkina, E and Skorski, T and Yan, H and Pomerantz, RT},
title = {Polθ promotes the repair of 5'-DNA-protein crosslinks by microhomology-mediated end-joining.},
journal = {Cell reports},
volume = {34},
number = {10},
pages = {108820},
pmid = {33691100},
issn = {2211-1247},
support = {R01 GM057962/GM/NIGMS NIH HHS/United States ; R01 GM130889/GM/NIGMS NIH HHS/United States ; R01 GM137124/GM/NIGMS NIH HHS/United States ; R01 GM115472/GM/NIGMS NIH HHS/United States ; R01 CA186238/CA/NCI NIH HHS/United States ; R01 CA244179/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Cell Line ; Cross-Linking Reagents/*pharmacology ; DNA/chemistry ; DNA End-Joining Repair/*drug effects ; DNA-Binding Proteins/genetics/metabolism ; DNA-Directed DNA Polymerase/deficiency/genetics/*metabolism ; Embryonic Stem Cells/cytology/metabolism ; Formaldehyde/pharmacology ; Humans ; Mice ; Ovum/metabolism ; Phosphoric Diester Hydrolases/genetics/metabolism ; Xenopus/growth &amp; development/metabolism ; DNA Polymerase theta ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.},
}
@article {pmid33660779,
year = {2021},
author = {Gerasimov, ES and Gasparyan, AA and Afonin, DA and Zimmer, SL and Kraeva, N and Lukeš, J and Yurchenko, V and Kolesnikov, A},
title = {Complete minicircle genome of Leptomonas pyrrhocoris reveals sources of its non-canonical mitochondrial RNA editing events.},
journal = {Nucleic acids research},
volume = {49},
number = {6},
pages = {3354-3370},
pmid = {33660779},
issn = {1362-4962},
mesh = {*Genome, Protozoan ; Phylogeny ; *RNA Editing ; RNA, Messenger/*metabolism ; RNA, Mitochondrial/*metabolism ; Transcriptome ; Trypanosomatina/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Uridine insertion/deletion (U-indel) editing of mitochondrial mRNA, unique to the protistan class Kinetoplastea, generates canonical as well as potentially non-productive editing events. While the molecular machinery and the role of the guide (g) RNAs that provide required information for U-indel editing are well understood, little is known about the forces underlying its apparently error-prone nature. Analysis of a gRNA:mRNA pair allows the dissection of editing events in a given position of a given mitochondrial transcript. A complete gRNA dataset, paired with a fully characterized mRNA population that includes non-canonically edited transcripts, would allow such an analysis to be performed globally across the mitochondrial transcriptome. To achieve this, we have assembled 67 minicircles of the insect parasite Leptomonas pyrrhocoris, with each minicircle typically encoding one gRNA located in one of two similar-sized units of different origin. From this relatively narrow set of annotated gRNAs, we have dissected all identified mitochondrial editing events in L. pyrrhocoris, the strains of which dramatically differ in the abundance of individual minicircle classes. Our results support a model in which a multitude of editing events are driven by a limited set of gRNAs, with individual gRNAs possessing an inherent ability to guide canonical and non-canonical editing.},
}
@article {pmid33592173,
year = {2021},
author = {Latorre-Muro, P and O'Malley, KE and Bennett, CF and Perry, EA and Balsa, E and Tavares, CDJ and Jedrychowski, M and Gygi, SP and Puigserver, P},
title = {A cold-stress-inducible PERK/OGT axis controls TOM70-assisted mitochondrial protein import and cristae formation.},
journal = {Cell metabolism},
volume = {33},
number = {3},
pages = {598-614.e7},
pmid = {33592173},
issn = {1932-7420},
support = {F32 GM125243/GM/NIGMS NIH HHS/United States ; F30 DE028206/DE/NIDCR NIH HHS/United States ; R01 DK081418/DK/NIDDK NIH HHS/United States ; R01 GM067945/GM/NIGMS NIH HHS/United States ; R01 DK089883/DK/NIDDK NIH HHS/United States ; },
mesh = {Adipocytes, Brown/cytology/drug effects/metabolism ; Animals ; Casein Kinase II/metabolism ; Cold Temperature ; Endoplasmic Reticulum/metabolism ; Endoplasmic Reticulum Stress ; GTP Phosphohydrolases/genetics/metabolism ; Glycosylation ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism/pathology ; Mitochondrial Precursor Protein Import Complex Proteins/genetics/*metabolism ; Mitochondrial Proteins/genetics/*metabolism ; N-Acetylglucosaminyltransferases/genetics/*metabolism ; Phosphorylation ; Protein Isoforms/genetics/metabolism ; Protein Transport ; eIF-2 Kinase/antagonists &amp; inhibitors/deficiency/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The architecture of cristae provides a spatial mitochondrial organization that contains functional respiratory complexes. Several protein components including OPA1 and MICOS complex subunits organize cristae structure, but upstream regulatory mechanisms are largely unknown. Here, in vivo and in vitro reconstitution experiments show that the endoplasmic reticulum (ER) kinase PERK promotes cristae formation by increasing TOM70-assisted mitochondrial import of MIC19, a critical subunit of the MICOS complex. Cold stress or β-adrenergic stimulation activates PERK that phosphorylates O-linked N-acetylglucosamine transferase (OGT). Phosphorylated OGT glycosylates TOM70 on Ser94, enhancing MIC19 protein import into mitochondria and promoting cristae formation and respiration. In addition, PERK-activated OGT O-GlcNAcylates and attenuates CK2α activity, which mediates TOM70 Ser94 phosphorylation and decreases MIC19 mitochondrial protein import. We have identified a cold-stress inter-organelle PERK-OGT-TOM70 axis that increases cell respiration through mitochondrial protein import and subsequent cristae formation. These studies have significant implications in cellular bioenergetics and adaptations to stress conditions.},
}
@article {pmid33483369,
year = {2021},
author = {Höfler, S and Lukat, P and Blankenfeldt, W and Carlomagno, T},
title = {High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain.},
journal = {RNA (New York, N.Y.)},
volume = {27},
number = {4},
pages = {496-512},
pmid = {33483369},
issn = {1469-9001},
mesh = {Amino Acid Sequence ; Archaeal Proteins/*chemistry/genetics/metabolism ; Binding Sites ; Chromosomal Proteins, Non-Histone/*chemistry/genetics/metabolism ; Crystallography, X-Ray ; Gene Expression ; Methylation ; Models, Molecular ; Nuclear Proteins/*chemistry/genetics/metabolism ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Pyrococcus furiosus/*genetics/metabolism ; RNA, Fungal/genetics/metabolism ; RNA, Ribosomal/genetics/metabolism ; RNA, Small Nucleolar/genetics/metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Ribonucleoproteins, Small Nuclear/chemistry/genetics/metabolism ; Ribonucleoproteins, Small Nucleolar/*chemistry/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/*chemistry/genetics/metabolism ; Sequence Alignment ; Structural Homology, Protein ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.},
}
@article {pmid33454003,
year = {2020},
author = {Watanabe, Y and Seya, D and Ihara, D and Ishii, S and Uemoto, T and Kubo, A and Arai, Y and Isomoto, Y and Nakano, A and Abe, T and Shigeta, M and Kawamura, T and Saito, Y and Ogura, T and Nakagawa, O},
title = {Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {51},
pages = {17632-17645},
pmid = {33454003},
issn = {1083-351X},
mesh = {Animals ; Arteries/growth &amp; development/metabolism ; Branchial Region/blood supply/growth &amp; development ; Cell Cycle Proteins/deficiency/genetics/*metabolism ; Cell Differentiation ; Embryo, Mammalian/metabolism ; Endothelium/cytology/*metabolism ; Female ; Humans ; Mice ; Mice, Knockout ; Morphogenesis ; Myocytes, Smooth Muscle/cytology/metabolism ; Receptors, Notch/*metabolism ; Regulatory Sequences, Nucleic Acid ; Signal Transduction ; Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.},
}
@article {pmid33440782,
year = {2021},
author = {Fish, RJ and Freire, C and Di Sanza, C and Neerman-Arbez, M},
title = {Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33440782},
issn = {1422-0067},
support = {#31003A_172864/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; Base Sequence ; *Biomarkers ; Blood Coagulation ; Blood Platelets/*metabolism ; Disease Models, Animal ; Exons ; Fibrinogen/chemistry/genetics/*metabolism ; Gene Editing ; Gene Expression ; Plasmids/genetics ; Platelet Activation ; Sequence Deletion ; Venous Thrombosis/*blood/diagnosis/*etiology ; Zebrafish ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga[-/-] larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga[+/-] larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding Aα[Δ19-56]. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα[+/Δ19-56] fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga[-/-] mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα[+/Δ19-56]. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.},
}
@article {pmid33439865,
year = {2021},
author = {Petratou, K and Spencer, SA and Kelsh, RN and Lister, JA},
title = {The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor.},
journal = {PloS one},
volume = {16},
number = {1},
pages = {e0244794},
pmid = {33439865},
issn = {1932-6203},
support = {UL1 TR000058/TR/NCATS NIH HHS/United States ; BB/ L00769X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/*genetics/metabolism ; Cell Differentiation ; Cell Lineage ; Embryo, Nonmammalian/metabolism/pathology ; Larva/growth &amp; development/metabolism ; Melanocytes/cytology/metabolism ; Multipotent Stem Cells/cytology/metabolism ; Mutagenesis ; Neural Crest/cytology/*growth &amp; development ; Pigmentation/genetics ; Zebrafish/embryology/genetics/*growth &amp; development ; Zebrafish Proteins/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of tfec mutants demonstrates that Tfec also functions in the initial specification of all three pigment cell-types, although the melanocyte and xanthophore lineages recover later. We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.},
}
@article {pmid33382574,
year = {2021},
author = {Gödecke, N and Herrmann, S and Hauser, H and Mayer-Bartschmid, A and Trautwein, M and Wirth, D},
title = {Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell Expression Heterogeneity and Ensures Robust Antibody Production.},
journal = {ACS synthetic biology},
volume = {10},
number = {1},
pages = {145-157},
doi = {10.1021/acssynbio.0c00519},
pmid = {33382574},
issn = {2161-5063},
mesh = {Animals ; Antibodies, Monoclonal/*biosynthesis/genetics ; CHO Cells ; CRISPR-Associated Protein 9/genetics ; Chromosomes/*genetics ; Cricetinae ; Cricetulus ; Gene Expression ; Recombinant Proteins/biosynthesis/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Transgenes/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The expression of endogenous genes as well as transgenes depends on regulatory elements within and surrounding genes as well as their epigenetic modifications. Members of a cloned cell population often show pronounced cell-to-cell heterogeneity with respect to the expression of a certain gene. To investigate the heterogeneity of recombinant protein expression we targeted cassettes into two preselected chromosomal hot-spots in Chinese hamster ovary (CHO) cells. Depending on the gene of interest and the design of the expression cassette, we found strong expression variability that could be reduced by epigenetic modifiers, but not by site-specific recruitment of the modulator dCas9-VPR. In particular, the implementation of ubiquitous chromatin opening elements (UCOEs) reduced cell-to-cell heterogeneity and concomitantly increased expression. The application of this method to recombinant antibody expression confirmed that rational design of cell lines for production of transgenes with predictable and high titers is a promising approach.},
}
@article {pmid33376716,
year = {2020},
author = {Cai, P and Xie, Y and Dong, M and Zhu, Q},
title = {Inhibition of MEIS3 Generates Cetuximab Resistance through c-Met and Akt.},
journal = {BioMed research international},
volume = {2020},
number = {},
pages = {2046248},
pmid = {33376716},
issn = {2314-6141},
mesh = {Antineoplastic Agents, Immunological/pharmacology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cetuximab/*pharmacology ; Colonic Neoplasms/*drug therapy ; *Drug Resistance, Neoplasm ; Homeodomain Proteins/*antagonists &amp; inhibitors/metabolism ; Humans ; Inhibitory Concentration 50 ; Proto-Oncogene Proteins c-akt/*metabolism ; Proto-Oncogene Proteins c-met/*metabolism ; Signal Transduction ; Transcription Factors/*antagonists &amp; inhibitors/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Although cetuximab has been widely used in the treatment of colon cancer, a large number of patients eventually develop drug resistance. Therefore, it is essential to clarify the mechanism of drug resistance.<br><br>METHODS: In this study, we combined in silico analysis and a single guide RNA (sgRNA) library to locate cetuximab-sensitive genes. Cell proliferation, apoptosis, and cell cycle were assessed to validate the change in cetuximab sensitivity. Finally, western blotting was performed to detect changes in epidermal growth factor (EGFR) upstream and downstream genes.<br><br>RESULTS: Using in silico analysis and the sgRNA library, MEIS3 was confirmed as the cetuximab-sensitive gene. Further experiments indicated that the expression of MEIS3 could determine the level of cetuximab. Meanwhile, MEIS3-inhibited cells were sensitive to mesenchymal epithelial transition factor (c-Met) and protein kinase B (Akt) inhibitors, which is related to the change in phosphorylation of c-Met and degradation of Akt.<br><br>CONCLUSION: MEIS3 modified the sensitivity to cetuximab through c-Met and Akt.},
}
@article {pmid33353963,
year = {2020},
author = {Álvarez, B and Mencía, M and de Lorenzo, V and Fernández, L&#xc1;},
title = {In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6436},
pmid = {33353963},
issn = {2041-1723},
mesh = {Aminohydrolases/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; DNA-Directed RNA Polymerases/metabolism ; Directed Molecular Evolution ; Escherichia coli/genetics ; *Genome, Bacterial ; Microbial Sensitivity Tests ; Mutagenesis/genetics ; Mutation/genetics ; Recombinant Fusion Proteins/*metabolism ; Viral Proteins/metabolism ; beta-Lactamases/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells. To overcome these limitations, here we report an in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. Transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP elongation. Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. While the efficiency is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.},
}
@article {pmid33333714,
year = {2020},
author = {Lisitskaya, L and Petushkov, I and Esyunina, D and Aravin, A and Kulbachinskiy, A},
title = {Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {4},
pages = {1484-1489},
doi = {10.1016/j.bbrc.2020.10.051},
pmid = {33333714},
issn = {1090-2104},
mesh = {Argonaute Proteins/chemistry/genetics/*metabolism ; Bacterial Proteins/chemistry/genetics/*metabolism ; Base Sequence ; DNA/chemistry/genetics/*metabolism ; Models, Molecular ; Nucleic Acid Conformation ; Nucleic Acid Denaturation ; Protein Binding ; Rhodobacter sphaeroides/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.},
}
@article {pmid33220920,
year = {2021},
author = {Ito, R and Morita, M and Nakano, T and Sato, I and Yokoyama, A and Sugawara, A},
title = {The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.},
journal = {Biochemical and biophysical research communications},
volume = {534},
number = {},
pages = {672-679},
doi = {10.1016/j.bbrc.2020.11.020},
pmid = {33220920},
issn = {1090-2104},
mesh = {Adrenal Cortex/drug effects/metabolism ; Aldosterone/biosynthesis ; Base Sequence ; Calcium Signaling ; Cell Line ; Cytochrome P-450 CYP11B2/*antagonists &amp; inhibitors/*genetics ; Drug Evaluation, Preclinical/methods ; Gene Editing/*methods ; Gene Expression Regulation, Enzymologic/drug effects ; Genes, Reporter ; High-Throughput Screening Assays/*methods ; Humans ; Hyperaldosteronism/drug therapy/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Steroid 11-beta-Hydroxylase/genetics ; Tacrolimus/pharmacology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca[2+]) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca[2+] signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.},
}
@article {pmid33199603,
year = {2020},
author = {Schauer, GD and Spenkelink, LM and Lewis, JS and Yurieva, O and Mueller, SH and van Oijen, AM and O'Donnell, ME},
title = {Replisome bypass of a protein-based R-loop block by Pif1.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {48},
pages = {30354-30361},
pmid = {33199603},
issn = {1091-6490},
support = {/HHMI/Howard Hughes Medical Institute/United States ; R01 GM115809/GM/NIGMS NIH HHS/United States ; R00 GM126143/GM/NIGMS NIH HHS/United States ; K99 GM126143/GM/NIGMS NIH HHS/United States ; T32 CA009673/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/metabolism ; DNA/chemistry/*genetics/*metabolism ; *DNA Replication ; Gene Editing ; Models, Biological ; Proliferating Cell Nuclear Antigen/metabolism ; Protein Binding ; *R-Loop Structures ; Telomere-Binding Proteins/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.},
}
@article {pmid33180792,
year = {2020},
author = {Marzec, M},
title = {New insights into the function of mammalian Argonaute2.},
journal = {PLoS genetics},
volume = {16},
number = {11},
pages = {e1009058},
pmid = {33180792},
issn = {1553-7404},
mesh = {Animals ; Argonaute Proteins/*genetics/*metabolism/physiology ; Humans ; Mammals/genetics/metabolism ; MicroRNAs/genetics/metabolism ; RNA, Messenger/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Uncovering the mechanisms that recognise a microRNA (miRNA) target is 1 of the biggest challenges because the Ago-miRNA complex is able to overcome different derogations of complementarity when binding targets. However, the recently solved crystallographic structure of Argonaute2 (Ago2) and a high-throughput analysis that used repurposed sequencing techniques has brought us closer to achieving this goal.},
}
@article {pmid33170095,
year = {2021},
author = {Nose, K and Hidaka, K and Yano, T and Tomita, Y and Fukuda, M},
title = {Short-Chain Guide RNA for Site-Directed A-to-I RNA Editing.},
journal = {Nucleic acid therapeutics},
volume = {31},
number = {1},
pages = {58-67},
doi = {10.1089/nat.2020.0866},
pmid = {33170095},
issn = {2159-3345},
mesh = {Adenosine Deaminase/*genetics ; Base Sequence/genetics ; Humans ; *Molecular Targeted Therapy ; Nucleic Acid Conformation ; Oligonucleotides, Antisense/*genetics ; RNA Editing/*genetics ; RNA, Messenger ; RNA-Binding Proteins/antagonists &amp; inhibitors/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-directed RNA editing is a promising genetic modification technology for therapeutic and pharmaceutical applications. We previously constructed adenosine deaminases acting on RNA (ADAR)-guiding RNAs (AD-gRNAs) that direct A-to-I RNA editing activity of native human ADAR2 into a programmable target site. In this study, we developed the short-chain AD-gRNA (shAD-gRNA) as a potential basic framework for practical RNA-editing oligonucleotides. Based on knowledge of previous AD-gRNA, shAD-gRNAs were designed to have the shortest possible sequence for the induction of editing activity. In vitro, compared to the original AD-gRNA, the shAD-gRNAs showed similar or superior editing induction activity, depending on the target RNA sequence, and had lower off-target editing activity around the target site, which is predicted to be a hotspot for off-target editing. Moreover, shAD-gRNAs achieved target RNA editing with both exogenous and endogenous human ADARs in cultured cells. Our results present shAD-gRNA as a short basic framework that would be applicable to further development for practical RNA-editing oligonucleotides.},
}
@article {pmid33144570,
year = {2020},
author = {Adolfi, A and Gantz, VM and Jasinskiene, N and Lee, HF and Hwang, K and Terradas, G and Bulger, EA and Ramaiah, A and Bennett, JB and Emerson, JJ and Marshall, JM and Bier, E and James, AA},
title = {Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5553},
pmid = {33144570},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 AI029746/AI/NIAID NIH HHS/United States ; R01 GM123303/GM/NIGMS NIH HHS/United States ; R37 AI029746/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Anopheles/*genetics ; CRISPR-Associated Protein 9/metabolism ; Female ; Genetics, Population ; Green Fluorescent Proteins/metabolism ; Heterozygote ; Inheritance Patterns/genetics ; Kynurenine 3-Monooxygenase/genetics ; Malaria/*parasitology ; Male ; Models, Genetic ; Mosaicism ; Phenotype ; Phylogeny ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.},
}
@article {pmid33122430,
year = {2020},
author = {Park, MS and Sim, G and Kehling, AC and Nakanishi, K},
title = {Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {46},
pages = {28576-28578},
pmid = {33122430},
issn = {1091-6490},
support = {R01 GM124320/GM/NIGMS NIH HHS/United States ; R01 GM138997/GM/NIGMS NIH HHS/United States ; S10 OD023582/OD/NIH HHS/United States ; },
mesh = {Argonaute Proteins/*metabolism ; Humans ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {RNA interfering is a eukaryote-specific gene silencing by 20∼23-nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3' end-shortened variants of let-7a, miR-27a, and specific miR-17-92 families that make AGO3 an extremely competent slicer, increasing target cleavage up to ∼82-fold in some instances. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.},
}
@article {pmid33111609,
year = {2021},
author = {Schmidt, A and Hanspach, G and Hengesbach, M},
title = {Structural dynamics govern substrate recruitment and catalytic turnover in H/ACA RNP pseudouridylation.},
journal = {RNA biology},
volume = {18},
number = {9},
pages = {1300-1309},
pmid = {33111609},
issn = {1555-8584},
mesh = {Archaeal Proteins/genetics/*metabolism ; Base Pairing ; Catalysis ; Pseudouridine/genetics/*metabolism ; Pyrococcus furiosus/genetics/*metabolism ; RNA, Small Nucleolar/genetics/*metabolism ; Ribonucleoproteins, Small Nucleolar/genetics/*metabolism ; Spliceosomes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.},
}
@article {pmid33092630,
year = {2020},
author = {Liu, S and Harmston, N and Glaser, TL and Wong, Y and Zhong, Z and Madan, B and Virshup, DM and Petretto, E},
title = {Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation.},
journal = {Genome medicine},
volume = {12},
number = {1},
pages = {89},
pmid = {33092630},
issn = {1756-994X},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; Gene Editing ; *Gene Expression Profiling/methods ; *Gene Expression Regulation ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Regulatory Networks ; Heterografts ; Humans ; Mice ; Neoplasms/genetics/metabolism/pathology ; Open Reading Frames ; Quantitative Trait Loci ; *RNA, Long Noncoding ; Wnt Proteins/*metabolism ; Wnt Signaling Pathway ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Wnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized.<br><br>METHODS: We comprehensively mapped Wnt-regulated lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model and examined the response of lncRNAs to Wnt inhibition between in vivo and in vitro cancer models. We further annotated and characterized these Wnt-regulated lncRNAs using existing genomic classifications (using data from FANTOM5) in the context of Wnt signaling and inferred their role in cancer pathogenesis (using GWAS and expression data from the TCGA). To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation both in vivo and in vitro.<br><br>RESULTS: We identified 3633 lncRNAs, of which 1503 were regulated by Wnt signaling in an orthotopic Wnt-addicted pancreatic cancer model. These lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. Our analysis suggested that Wnt signaling inhibition could influence the co-expression relationship of Wnt-regulated lncRNAs and their eQTL-linked protein-coding genes. Wnt-regulated lncRNAs were also implicated in specific gene networks involved in distinct biological processes that contribute to the pathogenesis of cancers. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1503) of the Wnt-regulated lncRNAs were found to modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1503) that had a functional effect on cancer cell growth.<br><br>CONCLUSIONS: Our study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling, and establishes a framework for systematic discovery of functional lncRNAs.},
}
@article {pmid33058229,
year = {2020},
author = {Huang, X and Lv, J and Li, Y and Mao, S and Li, Z and Jing, Z and Sun, Y and Zhang, X and Shen, S and Wang, X and Di, M and Ge, J and Huang, X and Zuo, E and Chi, T},
title = {Programmable C-to-U RNA editing using the human APOBEC3A deaminase.},
journal = {The EMBO journal},
volume = {39},
number = {22},
pages = {e104741},
pmid = {33058229},
issn = {1460-2075},
support = {31922048//National Natural Science Foundation of China (NSFC)/ ; //Fundamental Research Funds for Central Non-profit Scientific Institution/ ; },
mesh = {Cell Nucleus/metabolism ; Cytidine Deaminase/*genetics ; HEK293 Cells ; Humans ; Proteins/*genetics ; RNA/chemistry/metabolism ; *RNA Editing ; Transcriptome ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.},
}
@article {pmid32978258,
year = {2020},
author = {Amraei, R and Alwani, T and Ho, RX and Aryan, Z and Wang, S and Rahimi, N},
title = {Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {49},
pages = {16691-16699},
pmid = {32978258},
issn = {1083-351X},
support = {R21 CA191970/CA/NCI NIH HHS/United States ; R21 CA193958/CA/NCI NIH HHS/United States ; UL1 TR001430/TR/NCATS NIH HHS/United States ; },
mesh = {AMP-Activated Protein Kinases/*metabolism ; Amino Acid Motifs ; Animals ; *Autophagy/drug effects ; Autophagy-Related Protein-1 Homolog/metabolism ; Beclin-1/metabolism ; CD28 Antigens/chemistry/genetics/*metabolism ; *Cell Adhesion/drug effects ; HEK293 Cells ; Humans ; I-kappa B Kinase/deficiency/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/metabolism ; Lipopolysaccharides/pharmacology ; Microscopy, Fluorescence ; Microtubule-Associated Proteins/metabolism ; Phosphorylation/drug effects ; Primates ; Sirolimus/pharmacology ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser[220] The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.},
}
@article {pmid32976766,
year = {2020},
author = {Sumer, SA and Hoffmann, S and Laue, S and Campbell, B and Raedecke, K and Frajs, V and Clauss, S and Kääb, S and Janssen, JWG and Jauch, A and Laugwitz, KL and Dorn, T and Moretti, A and Rappold, GA},
title = {Precise Correction of Heterozygous SHOX2 Mutations in hiPSCs Derived from Patients with Atrial Fibrillation via Genome Editing and Sib Selection.},
journal = {Stem cell reports},
volume = {15},
number = {4},
pages = {999-1013},
pmid = {32976766},
issn = {2213-6711},
support = {788381/ERC_/European Research Council/International ; },
mesh = {Alleles ; Atrial Fibrillation/*genetics ; Base Sequence ; Clone Cells ; *Gene Editing ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Homeodomain Proteins/*genetics ; Humans ; Induced Pluripotent Stem Cells/*pathology ; Mutation/*genetics ; Recombinational DNA Repair ; Single-Cell Analysis ; Stochastic Processes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Patient-specific human induced pluripotent stem cells (hiPSCs) offer unprecedented opportunities for the investigation of multigenic disease, personalized medicine, and stem cell therapy. For heterogeneous diseases such as atrial fibrillation (AF), however, precise correction of the associated mutation is crucial. Here, we generated and corrected hiPSC lines from two AF patients carrying different heterozygous SHOX2 mutations. We developed a strategy for the scarless correction of heterozygous mutations, based on stochastic enrichment by sib selection, followed by allele quantification via digital PCR and next-generation sequencing to detect isogenic subpopulations. This allowed enriching edited cells 8- to 20-fold. The method does not require antibiotic selection or cell sorting and can be easily combined with base-and-prime editing approaches. Our strategy helps to overcome low efficiencies of homology-dependent repair in hiPSCs and facilitates the generation of isogenic control lines that represent the gold standard for modeling complex diseases in vitro.},
}
@article {pmid32958957,
year = {2021},
author = {Kim, HK and Yu, G and Park, J and Min, S and Lee, S and Yoon, S and Kim, HH},
title = {Predicting the efficiency of prime editing guide RNAs in human cells.},
journal = {Nature biotechnology},
volume = {39},
number = {2},
pages = {198-206},
pmid = {32958957},
issn = {1546-1696},
mesh = {Algorithms ; CRISPR-Associated Protein 9/metabolism ; Cell Line, Tumor ; Computer Simulation ; *Gene Editing ; HEK293 Cells ; Humans ; Machine Learning ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Prime editing enables the introduction of virtually any small-sized genetic change without requiring donor DNA or double-strand breaks. However, evaluation of prime editing efficiency requires time-consuming experiments, and the factors that affect efficiency have not been extensively investigated. In this study, we performed high-throughput evaluation of prime editor 2 (PE2) activities in human cells using 54,836 pairs of prime editing guide RNAs (pegRNAs) and their target sequences. The resulting data sets allowed us to identify factors affecting PE2 efficiency and to develop three computational models to predict pegRNA efficiency. For a given target sequence, the computational models predict efficiencies of pegRNAs with different lengths of primer binding sites and reverse transcriptase templates for edits of various types and positions. Testing the accuracy of the predictions using test data sets that were not used for training, we found Spearman's correlations between 0.47 and 0.81. Our computational models and information about factors affecting PE2 efficiency will facilitate practical application of prime editing.},
}
@article {pmid32920464,
year = {2021},
author = {Ishikawa, T and Haino, A and Ichiyanagi, T and Takahashi, M and Seki, M and Nashimoto, M},
title = {Heptamer-type small guide RNA that can shift macrophages toward the M1 state.},
journal = {Blood cells, molecules &amp; diseases},
volume = {86},
number = {},
pages = {102503},
doi = {10.1016/j.bcmd.2020.102503},
pmid = {32920464},
issn = {1096-0961},
mesh = {Animals ; Cell Line ; Cell Line, Tumor ; Cells, Cultured ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Genetic Therapy ; Humans ; Interleukin-12/genetics/immunology ; Macrophage Activation ; *Macrophages/immunology/metabolism ; Mice, Inbred NOD ; Mice, SCID ; Multiple Myeloma/genetics/immunology/*therapy ; *Tumor-Associated Macrophages/immunology/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Multiple myeloma is a refractory cancer of plasma cells. Although treatment strategies for multiple myeloma are getting improved year by year, in most cases patients relapse due to the emergence of drug-resistant mutations in the myeloma cells. The interplay between myeloma cells and tumor-associated macrophages (TAM) is important for the pathology. We thought that some heptamer-type sgRNAs for TRUE gene silencing would be able to transform TAM toward the M1 state and might become therapeutic drugs for myeloma. Here, we searched for heptamer-type sgRNAs that can shift macrophages toward the M1 state. We screened a heptamer-type sgRNA library for the ability to up-regulate IL-12b gene expression in human macrophage-like cell lines, and found three such sgRNAs. One of the sgRNAs, H12960, which also showed such ability in human fresh macrophages and mouse macrophage-like cell lines, efficiently suppressed human myeloma cell growth in SCID/NOD mice.},
}
@article {pmid32792663,
year = {2020},
author = {Kim, D and Lim, K and Kim, DE and Kim, JS},
title = {Genome-wide specificity of dCpf1 cytidine base editors.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4072},
pmid = {32792663},
issn = {2041-1723},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytidine/genetics/*metabolism ; DNA/genetics/metabolism ; Endonucleases/genetics/*metabolism ; Escherichia coli/genetics/metabolism ; Gene Editing ; Genome, Human/genetics ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Whole Genome Sequencing/*methods ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cpf1-linked base editors broaden the targeting scope of programmable cytidine deaminases by recognizing thymidine-rich protospacer-adjacent motifs (PAM) without inducing DNA double-strand breaks (DSBs). Here we present an unbiased in vitro method for identifying genome-wide off-target sites of Cpf1 base editors via whole genome sequencing. First, we treat human genomic DNA with dLbCpf1-BE ribonucleoprotein (RNP) complexes, which convert C-to-U at on-target and off-target sites and, then, with a mixture of E. coli uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, which removes uracil and produces single-strand breaks (SSBs) in vitro. Whole-genome sequencing of the resulting digested genome (Digenome-seq) reveals that, on average, dLbCpf1-BE induces 12 SSBs in vitro per crRNA in the human genome. Off-target sites with an editing frequency as low as 0.1% are successfully identified by this modified Digenome-seq method, demonstrating its high sensitivity. dLbCpf1-BEs and LbCpf1 nucleases often recognize different off-target sites, calling for independent analysis of each tool.},
}
@article {pmid32776944,
year = {2020},
author = {Zhang, L and Gao, Y and Zhang, R and Sun, F and Cheng, C and Qian, F and Duan, X and Wei, G and Sun, C and Pang, X and Chen, P and Chai, R and Yang, T and Wu, H and Liu, D},
title = {THOC1 deficiency leads to late-onset nonsyndromic hearing loss through p53-mediated hair cell apoptosis.},
journal = {PLoS genetics},
volume = {16},
number = {8},
pages = {e1008953},
pmid = {32776944},
issn = {1553-7404},
mesh = {Animals ; Apoptosis/genetics ; Benzothiazoles/pharmacology ; CRISPR-Associated Protein 9/genetics ; DNA-Binding Proteins/deficiency/*genetics ; Deafness/*genetics/pathology ; Disease Models, Animal ; Gene Expression Regulation/drug effects ; Gene Knockout Techniques ; Hair Cells, Auditory/metabolism/pathology ; Hair Cells, Auditory, Inner/*metabolism/pathology ; Humans ; Mice ; Mutation ; RNA-Binding Proteins/*genetics ; Signal Transduction/drug effects ; Toluene/analogs &amp; derivatives/pharmacology ; Tumor Suppressor Protein p53/antagonists &amp; inhibitors/*genetics ; Exome Sequencing ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.},
}
@article {pmid32721385,
year = {2020},
author = {Jin, S and Fei, H and Zhu, Z and Luo, Y and Liu, J and Gao, S and Zhang, F and Chen, YH and Wang, Y and Gao, C},
title = {Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {728-740.e6},
doi = {10.1016/j.molcel.2020.07.005},
pmid = {32721385},
issn = {1097-4164},
mesh = {Cytidine Deaminase/*genetics ; *Cytosine/chemistry ; Gene Editing/*methods ; Genes, Plant ; Humans ; Minor Histocompatibility Antigens/*genetics ; Mutation ; Oryza/*genetics ; RNA, Plant/chemistry ; Reproducibility of Results ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.},
}
@article {pmid32631830,
year = {2020},
author = {Christensen, CF and Koyama, T and Nagy, S and Danielsen, ET and Texada, MJ and Halberg, KA and Rewitz, K},
title = {Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {14},
pages = {},
pmid = {32631830},
issn = {1477-9129},
mesh = {Animals ; Body Size ; Drosophila/growth &amp; development/metabolism ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Ecdysterone/pharmacology ; Gene Expression Regulation, Developmental/drug effects ; Insect Hormones/antagonists &amp; inhibitors/genetics/*metabolism ; Larva/growth &amp; development/metabolism ; Metamorphosis, Biological ; Microscopy, Fluorescence ; Neurons/metabolism ; RNA Interference ; Receptors, Steroid/antagonists &amp; inhibitors/genetics/*metabolism ; Signal Transduction ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. Here, we show that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. We identify the Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. We find that the steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. Our findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, our findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation.},
}
@article {pmid32533916,
year = {2020},
author = {Arbab, M and Shen, MW and Mok, B and Wilson, C and Matuszek, &#x17b; and Cassa, CA and Liu, DR},
title = {Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning.},
journal = {Cell},
volume = {182},
number = {2},
pages = {463-480.e30},
pmid = {32533916},
issn = {1097-4172},
support = {R21 HG010391/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R01 HG010372/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Gene Editing/*methods ; Gene Library ; Humans ; *Machine Learning ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.},
}
@article {pmid32525891,
year = {2020},
author = {DiScala, M and Najor, MS and Yung, T and Morgan, D and Abukhdeir, AM and Cobleigh, MA},
title = {Loss of STAT6 leads to anchorage-independent growth and trastuzumab resistance in HER2+ breast cancer cells.},
journal = {PloS one},
volume = {15},
number = {6},
pages = {e0234146},
pmid = {32525891},
issn = {1932-6203},
mesh = {Antineoplastic Agents, Immunological/*pharmacology ; Breast Neoplasms/drug therapy/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Drug Resistance, Neoplasm/*drug effects/genetics ; Epithelial-Mesenchymal Transition ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Receptor, ErbB-2/genetics/*metabolism ; STAT6 Transcription Factor/deficiency/*genetics ; Trastuzumab/*pharmacology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance remains a major hurdle in the clinical management of these patients. Therefore, identifying molecular changes that cause trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Interestingly, loss of STAT6 resulted in anchorage-independent growth and changes in several genes involved in epithelial to mesenchymal transition. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer.},
}
@article {pmid32512638,
year = {2020},
author = {Le, QA and Hirata, M and Nguyen, NT and Takebayashi, K and Wittayarat, M and Sato, Y and Namula, Z and Nii, M and Tanihara, F and Otoi, T},
title = {Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes.},
journal = {Animal science journal = Nihon chikusan Gakkaiho},
volume = {91},
number = {1},
pages = {e13386},
doi = {10.1111/asj.13386},
pmid = {32512638},
issn = {1740-0929},
support = {JPMJOP1613//Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA)/International ; JP17H03938//KAKENHI/International ; JP18K12062//KAKENHI/International ; JP19K16014//KAKENHI/International ; },
mesh = {Animals ; Blastocyst ; *CRISPR-Associated Protein 9/pharmacology ; Dose-Response Relationship, Drug ; Electroporation/*methods/*veterinary ; Embryonic Development/*genetics ; *Gene Editing ; Gene Targeting/*veterinary ; Myostatin/*genetics ; Swine/*embryology/*genetics ; *Zygote ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.},
}
@article {pmid32492422,
year = {2020},
author = {Jacobs, JA and Sehgal, A},
title = {Anandamide Metabolites Protect against Seizures through the TRP Channel Water Witch in Drosophila melanogaster.},
journal = {Cell reports},
volume = {31},
number = {9},
pages = {107710},
pmid = {32492422},
issn = {2211-1247},
support = {T32 GM008076/GM/NIGMS NIH HHS/United States ; T32 HL007953/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Anticonvulsants/*therapeutic use ; Arachidonic Acids/metabolism/*therapeutic use ; Calcium/metabolism ; Disease Models, Animal ; Drosophila Proteins/deficiency/genetics/*metabolism ; Drosophila melanogaster/metabolism ; Endocannabinoids/metabolism/*therapeutic use ; Glycerides/*therapeutic use ; Polyunsaturated Alkamides/metabolism/therapeutic use ; Receptor, Cannabinoid, CB1/metabolism ; Seizures/pathology/*prevention &amp; control ; Transient Receptor Potential Channels/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Endocannabinoids protect against seizures, but their mechanism of action is still unclear, as they can have effects independent of known cannabinoid receptors. Using Drosophila melanogaster, which lacks canonical cannabinoid receptors, we report that the endocannabinoids anandamide and 2-arachidonoylglycerol protect against seizures in multiple fly seizure models. Surprisingly, inhibition of anandamide catabolism renders flies insensitive to protection by anandamide, indicating that anandamide metabolites are responsible for seizure protection. Consistent with this finding, arachidonic acid, a direct metabolite of anandamide, protects against seizures. To identify downstream effectors, we test for a role of transient receptor potential (TRP) channels and find that the TRPV1 antagonist capsazepine blocks the protective effect of anandamide. Also, a targeted genetic screen of TRP channels identifies water witch as a mediator of protection by anandamide. Using a Drosophila model, we reveal the role of arachidonic acid in seizure protection and identify a cannabinoid-receptor-1/2-independent mechanism of endocannabinoid seizure protection.},
}
@article {pmid32472108,
year = {2020},
author = {Major, F},
title = {From transient recognition to efficient silencing: a RISCky business.},
journal = {Nature structural &amp; molecular biology},
volume = {27},
number = {6},
pages = {519-520},
pmid = {32472108},
issn = {1545-9985},
mesh = {3' Untranslated Regions ; Gene Silencing ; Magnetic Resonance Spectroscopy ; RNA, Messenger/metabolism ; RNA-Induced Silencing Complex/*chemistry/genetics/*metabolism ; Sirtuin 1/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid32439758,
year = {2020},
author = {Postlmayr, A and Dumeau, CE and Wutz, A},
title = {Cdk8 is required for establishment of H3K27me3 and gene repression by Xist and mouse development.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {11},
pages = {},
pmid = {32439758},
issn = {1477-9129},
mesh = {Animals ; Cyclin-Dependent Kinase 8/genetics/*metabolism ; Cyclin-Dependent Kinases/genetics/metabolism ; Embryo, Mammalian ; Embryonic Development ; Enhancer of Zeste Homolog 2 Protein/metabolism ; Female ; Gene Editing ; Gene Expression Regulation, Developmental ; Histones/*metabolism ; Male ; Methylation ; Mice ; Mice, Transgenic ; Mouse Embryonic Stem Cells/cytology/metabolism ; Mutagenesis ; Polycomb Repressive Complex 2/metabolism ; RNA, Long Noncoding/genetics/*metabolism ; SOXB1 Transcription Factors/deficiency/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {We previously identified the cyclin dependent kinase Cdk8 as a putative silencing factor for Xist To investigate its role in X inactivation, we engineered a Cdk8 mutation in mouse embryonic stem cells (ESCs) carrying an inducible system for studying Xist function. We found that Xist repressed X-linked genes at half of the expression level in Cdk8 mutant cells, whereas they were almost completely silenced in the controls. Lack of Cdk8 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation but not PRC1 recruitment by Xist Transgenic expression of wild-type but not catalytically inactive Cdk8 restored efficient gene repression and PRC2 recruitment. Mutation of the paralogous kinase Cdk19 did not affect Xist function, and combined mutations of Cdk8 and Cdk19 resembled the Cdk8 mutation. In mice, a Cdk8 mutation caused post-implantation lethality. We observed that homozygous Cdk8 mutant female embryos showed a greater developmental delay than males on day 10.5. Together with the inefficient repression of X-linked genes in differentiating Cdk8 mutant female ESCs, these data show a requirement for Cdk8 in the initiation of X inactivation.},
}
@article {pmid32268068,
year = {2020},
author = {Li, S and Jendresen, CB and Landberg, J and Pedersen, LE and Sonnenschein, N and Jensen, SI and Nielsen, AT},
title = {Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production.},
journal = {ACS synthetic biology},
volume = {9},
number = {5},
pages = {1030-1040},
doi = {10.1021/acssynbio.9b00143},
pmid = {32268068},
issn = {2161-5063},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Gene Library ; *Genome, Bacterial ; Metabolic Engineering ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Growth decoupling can be used to optimize microbial production of biobased compounds by inhibiting excess biomass formation and redirect carbon flux from growth to product formation. However, identifying suitable genetic targets through rational design is challenging. Here, we conduct a genome-wide CRISPRi screen to discover growth switches suitable for decoupling growth and production. Using an sgRNA library covering 12 238 loci in the Escherichia coli genome, we screen for targets that inhibit growth while allowing for continued protein production. In total, we identify 1332 sgRNAs that simultaneously decrease growth and maintain or increase accumulation of GFP. The top target sibB/ibsB shows more than 5-fold increase in GFP accumulation and 45% decrease in biomass formation. Overall, our genome-wide CRISPRi screen provides key targets for growth decoupling, and the approach can be applied to improve biobased production in other microorganisms.},
}
@article {pmid32202498,
year = {2020},
author = {Graziadei, A and Gabel, F and Kirkpatrick, J and Carlomagno, T},
title = {The guide sRNA sequence determines the activity level of box C/D RNPs.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32202498},
issn = {2050-084X},
support = {FP7 ITN project RNPnet (contract number 289007//European Commission/ ; CA294/3-2//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Algorithms ; *Base Sequence ; Methylation ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation ; Ribonucleoproteins/*metabolism ; Solutions ; Structure-Activity Relationship ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {2'-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate-guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.},
}
@article {pmid32125904,
year = {2020},
author = {Xue, VW and Wong, SCC and Song, G and Cho, WCS},
title = {Promising RNA-based cancer gene therapy using extracellular vesicles for drug delivery.},
journal = {Expert opinion on biological therapy},
volume = {20},
number = {7},
pages = {767-777},
doi = {10.1080/14712598.2020.1738377},
pmid = {32125904},
issn = {1744-7682},
mesh = {Drug Carriers/*chemistry ; Extracellular Vesicles/*chemistry ; Genetic Therapy/*methods ; Humans ; MicroRNAs/chemistry/metabolism ; Neoplasms/*therapy ; RNA/chemistry/*metabolism ; RNA Interference ; RNA, Small Interfering/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: RNA-based cancer gene therapy shows potential in cancer treatment. However, the safe and efficient transfer of therapeutic RNA to target cells has always been a challenge. The ideal drug delivery system should be effective with low immunogenicity and toxicity. Besides, a high specificity of drug delivery is necessary to improve efficacy and avoid the side effects associated with tumor heterogeneity. As endogenous RNA vehicles, extracellular vesicles (EVs) have shown their advantages and potential as drug delivery systems in gene therapy.<br><br>AREAS COVERED: We summarize the performance of EVs as a drug delivery system in RNA-based cancer gene therapy and discuss the advantages, limitations, and potentials of this translational medicine. In addition, we compare the characteristics and differences of current drug delivery systems and expound the principles of selecting a drug delivery system suitable for cancer gene therapy.<br><br>EXPERT OPINION: EVs are highly biocompatible membrane structures with low cytotoxicity which provide a new choice for drug delivery in RNA-based cancer gene therapy. The specificity of engineered EVs and artificial EV-mimetics can be improved through peptide or polymer decoration. However, apart from therapeutic RNA, EVs naturally carry many molecules. This may lead to unpredictable effects and thus should be applied with caution.},
}
@article {pmid32094641,
year = {2020},
author = {Qin, R and Li, J and Liu, X and Xu, R and Yang, J and Wei, P},
title = {SpCas9-NG self-targets the sgRNA sequence in plant genome editing.},
journal = {Nature plants},
volume = {6},
number = {3},
pages = {197-201},
pmid = {32094641},
issn = {2055-0278},
mesh = {CRISPR-Associated Protein 9/*genetics ; DNA, Plant/genetics ; Gene Editing ; *Genome, Plant ; Oryza/*genetics ; Plants, Genetically Modified/genetics ; RNA, Plant/*genetics ; Streptococcus pyogenes/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Streptococcus pyogenes Cas9 (SpCas9)-NG recognizes NGN protospacer adjacent motifs and expands the scope of genome-editing tools. In this study, we found that SpCas9-NG not only targeted the genome but also efficiently self-targeted the single-guide RNA sequence in transfer DNA in transgenic plants, potentially increasing off-target risk by generating new single-guide RNAs. We further showed that the self-target effect of SpCas9-NG could be greatly alleviated by using a modified single-guide RNA scaffold starting with a GCCCC sequence.},
}
@article {pmid32051406,
year = {2020},
author = {Yamaguchi, S and Oe, A and Nishida, KM and Yamashita, K and Kajiya, A and Hirano, S and Matsumoto, N and Dohmae, N and Ishitani, R and Saito, K and Siomi, H and Nishimasu, H and Siomi, MC and Nureki, O},
title = {Crystal structure of Drosophila Piwi.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {858},
pmid = {32051406},
issn = {2041-1723},
mesh = {Animals ; Argonaute Proteins/chemistry/*classification/genetics ; Bombyx/metabolism ; Cell Line ; Crystallography, X-Ray ; DNA Transposable Elements/genetics ; Drosophila/genetics/*metabolism ; Drosophila Proteins/*chemistry/genetics ; Drosophila melanogaster/genetics/metabolism ; Gene Silencing ; Hydrogen Bonding ; Models, Molecular ; Protein Conformation ; Protein Domains ; RNA, Small Interfering/metabolism ; RNA, Untranslated ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila PIWI-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 Å resolution. A structural comparison of Piwi with other Argonautes highlights the PIWI-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons.},
}
@article {pmid32042167,
year = {2020},
author = {Gilpatrick, T and Lee, I and Graham, JE and Raimondeau, E and Bowen, R and Heron, A and Downs, B and Sukumar, S and Sedlazeck, FJ and Timp, W},
title = {Targeted nanopore sequencing with Cas9-guided adapter ligation.},
journal = {Nature biotechnology},
volume = {38},
number = {4},
pages = {433-438},
pmid = {32042167},
issn = {1546-1696},
support = {T32 CA130840/CA/NCI NIH HHS/United States ; T32 CA153952/CA/NCI NIH HHS/United States ; R01 HG009190/HG/NHGRI NIH HHS/United States ; T32 GM007057/GM/NIGMS NIH HHS/United States ; T32 GM136577/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; Cells, Cultured ; Chromosomes, Human/genetics ; Genetic Loci/genetics ; Genetic Variation ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Nanopore Sequencing/*methods ; Sequence Analysis, DNA ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods[1-4] are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.},
}
@article {pmid31988462,
year = {2020},
author = {Moretti, A and Fonteyne, L and Giesert, F and Hoppmann, P and Meier, AB and Bozoglu, T and Baehr, A and Schneider, CM and Sinnecker, D and Klett, K and Fröhlich, T and Rahman, FA and Haufe, T and Sun, S and Jurisch, V and Kessler, B and Hinkel, R and Dirschinger, R and Martens, E and Jilek, C and Graf, A and Krebs, S and Santamaria, G and Kurome, M and Zakhartchenko, V and Campbell, B and Voelse, K and Wolf, A and Ziegler, T and Reichert, S and Lee, S and Flenkenthaler, F and Dorn, T and Jeremias, I and Blum, H and Dendorfer, A and Schnieke, A and Krause, S and Walter, MC and Klymiuk, N and Laugwitz, KL and Wolf, E and Wurst, W and Kupatt, C},
title = {Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.},
journal = {Nature medicine},
volume = {26},
number = {2},
pages = {207-214},
pmid = {31988462},
issn = {1546-170X},
support = {681524/ERC_/European Research Council/International ; 788381/ERC_/European Research Council/International ; },
mesh = {Animals ; Disease Models, Animal ; Dystrophin/*genetics ; Exons ; Female ; *Frameshift Mutation ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Therapy ; Genome ; Heart Failure/genetics/therapy ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Male ; Mass Spectrometry ; Muscle, Skeletal/metabolism ; Muscles/metabolism ; Muscular Dystrophy, Duchenne/*genetics/*therapy ; Myoblasts/metabolism ; Myocytes, Cardiac/metabolism ; Proteome ; Swine ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs[1], intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. [2]) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca[2+] handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.},
}
@article {pmid31974864,
year = {2020},
author = {Su, X and Chen, W and Cai, Q and Liang, P and Chen, Y and Cong, P and Huang, J},
title = {Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.},
journal = {Science China. Life sciences},
volume = {63},
number = {7},
pages = {996-1005},
pmid = {31974864},
issn = {1869-1889},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Blastocyst ; Cytoplasm/metabolism ; Cytosine/administration &amp; dosage/*metabolism ; Embryo, Mammalian/*metabolism ; Embryonic Development/*genetics ; Female ; Fertilization ; Fertilization in Vitro ; Gene Editing ; Genome/*genetics ; Male ; Mutation ; Oocytes/*metabolism ; Swine ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.},
}
@article {pmid31964975,
year = {2020},
author = {Xie, H and Gu, Y and Wang, W and Wang, X and Ye, X and Xin, C and Lu, M and Reddy, BA and Shu, P},
title = {Silencing of SENP2 in Multiple Myeloma Induces Bortezomib Resistance by Activating NF-κB Through the Modulation of IκBα Sumoylation.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {766},
pmid = {31964975},
issn = {2045-2322},
mesh = {Bortezomib/*pharmacology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Computer Simulation ; Cysteine Endopeptidases/*genetics ; *Down-Regulation ; *Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Humans ; Multiple Myeloma/drug therapy/*genetics/metabolism ; NF-KappaB Inhibitor alpha/*metabolism ; NF-kappa B/metabolism ; Signal Transduction ; Sumoylation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The proteasome inhibitor bortezomib is the most successfully applied chemotherapeutic drug for treating multiple myeloma. However, its clinical efficacy reduced due to resistance development. The underlying molecular mechanisms of bortezomib resistance are poorly understood. In this study, by combining in silico analysis and sgRNA library based drug resistance screening assay, we identified SENP2 (Sentrin/SUMO-specific proteases-2) as a bortezomib sensitive gene and found its expression highly downregulated in bortezomib resistant multiple myeloma patient's samples. Furthermore, down regulation of SENP2 in multiple myeloma cell line RPMI8226 alleviated bortezomib induced cell proliferation inhibition and apoptosis, whereas, overexpression of SENP2 sensitized these cells to bortezomib treatment. We further demonstrate that knockdown of SENP2 in RPMI8226 cells increased SUMO2 conjugated IκBα that resulted in the activation of NF-κB. Taken together, we report that silencing of SENP2 and consequent activation of NF-κB through the modulation of IκBα sumoylation as a novel mechanism inducing bortezomib resistance in multiple myeloma.},
}
@article {pmid31959181,
year = {2020},
author = {Blechert, O and Mei, H and Zang, X and Zheng, H and Liang, G and Liu, W},
title = {Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {6},
pmid = {31959181},
issn = {1472-6750},
mesh = {CRISPR-Associated Protein 9/*metabolism ; Culture Media/chemistry ; Fungal Proteins/*genetics ; Gene Knockout Techniques ; Mutation ; Mycology/methods ; Ribonucleoproteins/metabolism ; Trichophyton/genetics/*growth &amp; development/metabolism ; Uracil/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80-90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum.<br><br>RESULTS: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing.<br><br>CONCLUSIONS: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.},
}
@article {pmid31925439,
year = {2020},
author = {Halmai, JANM and Deng, P and Gonzalez, CE and Coggins, NB and Cameron, D and Carter, JL and Buchanan, FKB and Waldo, JJ and Lock, SR and Anderson, JD and O'Geen, H and Segal, DJ and Nolta, J and Fink, KD},
title = {Artificial escape from XCI by DNA methylation editing of the CDKL5 gene.},
journal = {Nucleic acids research},
volume = {48},
number = {5},
pages = {2372-2387},
pmid = {31925439},
issn = {1362-4962},
support = {R25 GM056765/GM/NIGMS NIH HHS/United States ; T32 HL086350/HL/NHLBI NIH HHS/United States ; T32 MH112507/MH/NIMH NIH HHS/United States ; UL1 TR001860/TR/NCATS NIH HHS/United States ; KL2 TR001859/TR/NCATS NIH HHS/United States ; T32 GM099608/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Associated Protein 9/genetics/metabolism ; Catalytic Domain ; Cell Line, Tumor ; Chromosomes, Human, X/*chemistry/metabolism ; CpG Islands ; *Epigenesis, Genetic ; Gene Editing ; Gene Silencing ; Humans ; Mixed Function Oxygenases/genetics/metabolism ; Neurons/cytology/metabolism ; *Promoter Regions, Genetic ; Protein Serine-Threonine Kinases/*genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Messenger/*genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; *X Chromosome Inactivation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.},
}
@article {pmid31919243,
year = {2020},
author = {Majumder, M and Mukhopadhyay, S and Kharel, P and Gupta, R},
title = {The presence of the ACA box in archaeal H/ACA guide RNAs promotes atypical pseudouridylation.},
journal = {RNA (New York, N.Y.)},
volume = {26},
number = {4},
pages = {396-418},
pmid = {31919243},
issn = {1469-9001},
support = {R15 GM055945/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaeal Proteins/metabolism ; Haloferax volcanii/genetics ; Nucleotide Motifs ; Pseudouridine/chemistry/*metabolism ; *RNA Processing, Post-Transcriptional ; RNA, Archaeal/chemistry/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Archaea and eukaryotes, in addition to protein-only enzymes, also possess ribonucleoproteins containing an H/ACA guide RNA plus four proteins that produce pseudouridine (Ψ). Although typical conditions for these RNA-guided reactions are known, certain variant conditions allow pseudouridylation. We used mutants of the two stem-loops of the Haloferax volcanii sR-h45 RNA that guides three pseudouridylations in 23S rRNA and their target RNAs to characterize modifications under various atypical conditions. The 5' stem-loop produces Ψ2605 and the 3' stem-loop produces Ψ1940 and Ψ1942. The latter two modifications require unpaired "UVUN" (V = A, C, or G) in the target and ACA box in the guide. Ψ1942 modification requires the presence of U1940 (or Ψ1940). Ψ1940 is not produced in the Ψ1942-containing substrate, suggesting a sequential modification of the two residues. The ACA box of a single stem-loop guide is not required when typically unpaired "UN" is up to 17 bases from its position in the guide, but is needed when the distance increases to 19 bases or the N is paired. However, ANA of the H box of the double stem-loop guide is needed even for the 5' typical pseudouridylation. The most 5' unpaired U in a string of U's is converted to Ψ, and in the absence of an unpaired U, a paired U can also be modified. Certain mutants of the Cbf5 protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of nonconstitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing.},
}
@article {pmid31874824,
year = {2020},
author = {Kato, Y and Arakawa, S and Terasawa, K and Inokuchi, JI and Iwata, T and Shimizu, S and Watabe, T and Hara-Yokoyama, M},
title = {The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation.},
journal = {Bioorganic &amp; medicinal chemistry letters},
volume = {30},
number = {3},
pages = {126891},
doi = {10.1016/j.bmcl.2019.126891},
pmid = {31874824},
issn = {1464-3405},
mesh = {Animals ; Autophagy/drug effects ; Cell Line ; Ceramides/*chemistry/pharmacology ; Fibroblasts/cytology/metabolism/pathology ; Gene Editing ; Lipid Droplets/*metabolism ; Lipolysis/*drug effects ; Lysosomal-Associated Membrane Protein 2/genetics/*metabolism ; Mice ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.},
}
@article {pmid31713616,
year = {2019},
author = {Okafor, IC and Singh, D and Wang, Y and Jung, M and Wang, H and Mallon, J and Bailey, S and Lee, JK and Ha, T},
title = {Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding.},
journal = {Nucleic acids research},
volume = {47},
number = {22},
pages = {11880-11888},
pmid = {31713616},
issn = {1362-4962},
support = {F31 GM134675/GM/NIGMS NIH HHS/United States ; R01 GM097330/GM/NIGMS NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; T32 GM080189/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism/*physiology ; DNA/*chemistry/drug effects/*metabolism ; *DNA Cleavage ; DNA Helicases/physiology ; *Gain of Function Mutation ; Gene Editing/methods ; Nucleic Acid Conformation/drug effects ; Protein Engineering ; Single Molecule Imaging ; Streptococcus pyogenes/enzymology/genetics ; Substrate Specificity/drug effects/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.},
}
@article {pmid31695077,
year = {2019},
author = {Valdés, JJ and Miller, AD},
title = {New opportunities for designing effective small interfering RNAs.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16146},
pmid = {31695077},
issn = {2045-2322},
mesh = {Argonaute Proteins/chemistry/metabolism ; Base Pairing ; Crystallography, X-Ray ; Drug Design ; Humans ; Hydrolysis ; Magnesium ; Molecular Docking Simulation ; Monte Carlo Method ; Nucleic Acid Conformation ; Nucleic Acid Hybridization ; *RNA Interference ; RNA, Messenger/chemistry/metabolism ; RNA, Small Interfering/*chemistry/genetics/metabolism ; RNA, Viral/antagonists &amp; inhibitors/chemistry ; RNA-Induced Silencing Complex ; Structure-Activity Relationship ; Thermodynamics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.},
}
@article {pmid31672920,
year = {2019},
author = {Lee, HC and Zhao, YJ},
title = {Resolving the topological enigma in Ca[2+] signaling by cyclic ADP-ribose and NAADP.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {52},
pages = {19831-19843},
pmid = {31672920},
issn = {1083-351X},
mesh = {ADP-ribosyl Cyclase 1/chemistry/genetics/*metabolism ; Animals ; *Calcium Signaling ; Calcium-Binding Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Cyclic ADP-Ribose/*metabolism ; Cytoskeletal Proteins/metabolism ; Humans ; Molecular Chaperones/metabolism ; NADP/*analogs &amp; derivatives/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endolysosomal Ca[2+] stores, respectively. Both are synthesized by the CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years, it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca[2+] stores. The discovery of type III CD38, whose catalytic domain faces the cytosol, has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence; and discusses the regulators of its activity, folding, and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca[2+]-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile α and TIR motif-containing 1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38 but can catalyze the same set of multireactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca[2+]-signaling enzyme like CD38.},
}
@article {pmid31566620,
year = {2019},
author = {Winczura, K and Grzechnik, P},
title = {DNAzyme-dependent Analysis of rRNA 2'-O-Methylation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {151},
pages = {},
doi = {10.3791/59700},
pmid = {31566620},
issn = {1940-087X},
support = {200473/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Base Sequence ; Cell Nucleolus/genetics/metabolism ; DNA, Catalytic/genetics/*metabolism ; Genes, rRNA/*physiology ; Methylation ; RNA, Ribosomal/genetics/metabolism ; RNA, Small Nucleolar/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2'-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2'-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2'-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-'O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2'-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2'-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.},
}
@article {pmid31500270,
year = {2019},
author = {Deryusheva, S and Gall, JG},
title = {Small, Smaller, Smallest: Minimal Structural Requirements for a Fully Functional Box C/D Modification Guide RNA.},
journal = {Biomolecules},
volume = {9},
number = {9},
pages = {},
pmid = {31500270},
issn = {2218-273X},
support = {R01 GM033397/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Female ; Nucleic Acid Conformation ; RNA, Long Noncoding/chemistry/*metabolism ; Xenopus laevis ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-specific 2'-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.},
}
@article {pmid31311819,
year = {2019},
author = {Kelly, EK and Czekay, DP and Kothe, U},
title = {Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {10},
pages = {1393-1404},
pmid = {31311819},
issn = {1469-9001},
mesh = {*Base Pairing ; Kinetics ; Pseudouridine/*metabolism ; Ribonucleoproteins, Small Nucleolar/*metabolism ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide-substrate RNA pairs.},
}
@article {pmid31276570,
year = {2019},
author = {Wieben, ED and Aleff, RA and Basu, S and Sarangi, V and Bowman, B and McLaughlin, IJ and Mills, JR and Butz, ML and Highsmith, EW and Ida, CM and Ekholm, JM and Baratz, KH and Fautsch, MP},
title = {Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.},
journal = {PloS one},
volume = {14},
number = {7},
pages = {e0219446},
pmid = {31276570},
issn = {1932-6203},
mesh = {Alleles ; Computational Biology/methods ; Fuchs' Endothelial Dystrophy/diagnosis/*genetics ; *Gene Amplification ; Gene Editing ; Genetic Association Studies ; *Genetic Predisposition to Disease ; Genomics/methods ; Genotype ; Humans ; Phenotype ; Transcription Factor 4/*genetics ; *Trinucleotide Repeat Expansion ; Trinucleotide Repeats ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.},
}
@article {pmid31239279,
year = {2019},
author = {Serebrenik, YV and Sansbury, SE and Kumar, SS and Henao-Mejia, J and Shalem, O},
title = {Efficient and flexible tagging of endogenous genes by homology-independent intron targeting.},
journal = {Genome research},
volume = {29},
number = {8},
pages = {1322-1328},
pmid = {31239279},
issn = {1549-5469},
support = {F32 CA239499/CA/NCI NIH HHS/United States ; T32 GM008216/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; Calnexin/genetics/metabolism ; Cell Line, Tumor ; Chromobox Protein Homolog 5 ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; Exons ; Fibroblasts/cytology/metabolism ; Gene Editing/*methods ; Gene Expression ; *Genome, Human ; HEK293 Cells ; HeLa Cells ; Humans ; *Introns ; *Mutagenesis, Insertional ; Plasmids/chemistry/metabolism ; Recombinant Fusion Proteins/biosynthesis/*genetics ; Vimentin/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.},
}
@article {pmid31226499,
year = {2019},
author = {Ishikawa, T and Haino, A and Ichiyanagi, T and Seki, M and Nashimoto, M},
title = {Evaluation of double heptamer-type sgRNA as a potential therapeutic agent against multiple myeloma.},
journal = {Blood cells, molecules &amp; diseases},
volume = {79},
number = {},
pages = {102341},
doi = {10.1016/j.bcmd.2019.102341},
pmid = {31226499},
issn = {1096-0961},
mesh = {Animals ; Cell Line, Tumor ; Cell Survival/drug effects ; Cyclin D1/antagonists &amp; inhibitors/genetics ; *Drug Design ; Gene Silencing ; Heterografts/drug effects ; Humans ; Mice ; Multiple Myeloma/*drug therapy/pathology ; Proto-Oncogene Proteins c-bcl-2/antagonists &amp; inhibitors/genetics ; RNA, Messenger ; Survival Analysis ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase Z[L]-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability.},
}
@article {pmid31210215,
year = {2019},
author = {Berckman, EA and Chen, W},
title = {Exploiting dCas9 fusion proteins for dynamic assembly of synthetic metabolons.},
journal = {Chemical communications (Cambridge, England)},
volume = {55},
number = {57},
pages = {8219-8222},
pmid = {31210215},
issn = {1364-548X},
support = {T32 GM133395/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/chemistry/genetics/*metabolism ; Cellulase/chemistry/genetics/metabolism ; Cellulosomes/chemistry/metabolism ; DNA/chemistry/metabolism ; Protein Binding ; Protein Domains ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways.},
}
@article {pmid31113995,
year = {2019},
author = {Jan Vonk, P and Escobar, N and Wösten, HAB and Lugones, LG and Ohm, RA},
title = {High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7632},
pmid = {31113995},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; Fungal Proteins/genetics/metabolism ; *Gene Deletion ; Gene Targeting/*methods ; Genetic Engineering/methods ; Homologous Recombination ; Ribonucleoproteins/genetics/*metabolism ; Schizophyllum/*genetics ; Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.},
}
@article {pmid31113933,
year = {2019},
author = {Song, Z and Tu, X and Zhou, Q and Huang, J and Chen, Y and Liu, J and Lee, S and Kim, W and Nowsheen, S and Luo, K and Yuan, J and Lou, Z},
title = {A novel UCHL3 inhibitor, perifosine, enhances PARP inhibitor cytotoxicity through inhibition of homologous recombination-mediated DNA double strand break repair.},
journal = {Cell death &amp; disease},
volume = {10},
number = {6},
pages = {398},
pmid = {31113933},
issn = {2041-4889},
support = {R01 CA203971/CA/NCI NIH HHS/United States ; R01 CA217183/CA/NCI NIH HHS/United States ; R01 CA224921/CA/NCI NIH HHS/United States ; T32 GM065841/GM/NIGMS NIH HHS/United States ; P50 CA136393/CA/NCI NIH HHS/United States ; R01 CA189666/CA/NCI NIH HHS/United States ; R01 CA203561/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/drug effects ; BRCA2 Protein/metabolism ; Cell Line, Tumor ; Female ; Humans ; Mice ; Mice, Nude ; Phosphorylcholine/*analogs &amp; derivatives/pharmacology/therapeutic use ; Phthalazines/*pharmacology/therapeutic use ; Piperazines/*pharmacology/therapeutic use ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use ; Rad51 Recombinase/metabolism ; Radiation, Ionizing ; Recombinational DNA Repair/*drug effects ; Transplantation, Heterologous ; Triple Negative Breast Neoplasms/drug therapy/pathology ; Ubiquitin Thiolesterase/antagonists &amp; inhibitors/genetics/*metabolism ; Ubiquitination/drug effects ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Triple-negative breast cancer (TNBC) treatment remains a great challenge for clinical practice and novel therapeutic strategies are urgently needed. UCHL3 is a deubiquitinase that is overexpressed in TNBC and correlates with poor prognosis. UCHL3 deubiquitinates RAD51 thereby promoting the recruitment of RAD51 to DNA damage sites and augmenting DNA repair. Therefore, UCHL3 overexpression can render cancer cells resistant to DNA damage inducing chemo and radiotherapy, and targeting UCHL3 can sensitize TNBC to radiation and chemotherapy. However, small molecule inhibitors of UCHL3 are yet to be identified. Here we report that perifosine, a previously reported Akt inhibitor, can inhibit UCHL3 in vitro and in vivo. We found low dose (50 nM) perifosine inhibited UCHL3 deubiquitination activity without affecting Akt activity. Furthermore, perifosine enhanced Olaparib-induced growth inhibition in TNBC cells. Mechanistically, perifosine induced RAD51 ubiquitination and blocked the RAD51-BRCA2 interaction, which in turn decreased ionizing radiation-induced foci (IRIF) of Rad51 and, thereby, homologous recombination (HR)-mediated DNA double strand break repair. In addition, combination of perifosine and Olaparib showed synergistic antitumor activity in vivo in TNBC xenograft model. Thus, our present study provides a novel therapeutic approach to optimize PARP inhibitor treatment efficiency.},
}
@article {pmid31069393,
year = {2019},
author = {Hegge, JW and Swarts, DC and Chandradoss, SD and Cui, TJ and Kneppers, J and Jinek, M and Joo, C and van der Oost, J},
title = {DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute.},
journal = {Nucleic acids research},
volume = {47},
number = {11},
pages = {5809-5821},
pmid = {31069393},
issn = {1362-4962},
mesh = {Argonaute Proteins/genetics/*metabolism ; Bacterial Proteins/metabolism ; Clostridium butyricum/genetics/*metabolism ; DNA/*chemistry/metabolism ; *DNA Cleavage ; DNA, Single-Stranded/analysis ; Fluorescence Resonance Energy Transfer ; Gene Editing ; Gene Silencing ; Mutation ; Phylogeny ; Plasmids/metabolism ; Protein Binding ; Temperature ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.},
}
@article {pmid31065636,
year = {2019},
author = {Chen, Z and Lee, WG},
title = {A switching role of hard-uptake nanoparticles in microalgae cell electroporation.},
journal = {The Analyst},
volume = {144},
number = {11},
pages = {3581-3589},
doi = {10.1039/c9an00314b},
pmid = {31065636},
issn = {1364-5528},
mesh = {Cell Survival/drug effects ; Chlamydomonas reinhardtii ; DNA/genetics/*pharmacokinetics ; Electroporation/methods ; *Gene Transfer Techniques ; Genetic Vectors/genetics/pharmacokinetics ; Green Fluorescent Proteins/genetics ; Microalgae ; Nanoparticles/*chemistry/toxicity ; Oxazines/chemistry/toxicity ; Particle Size ; Polystyrenes/chemistry/toxicity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The microalgal cell wall is a natural barrier that limits the efficiency of gene delivery in algae genetic engineering. Here, we report the role of hard-uptake nanoparticles (huNPs) in microalgae cell electroporation to enhance the delivery of genes in Chlamydomonas reinhardtii. This role can be divided into two categories: (i) a 'transient state' for short-term behavior under confocal visualization and (ii) a 'steady state' for long-term behavior in cell culture. First, the 'transient' role of gene-huNP complexes was investigated after washing for clear confocal imaging to observe the location of huNPs after electroporation. Second, the 'steady-state' role of the gene-huNP complexes was examined after electroporation by transferring cells to a fresh, medium-rich culture environment without washing to obtain a stable cell culture. For selection of the huNPs, we used two types of nanoparticles (NPs, 250 nm and 530 nm) larger than the threshold size of electroporation uptake to avoid unwanted endocytic uptake of NPs. In the transient state, the visualization results indicate that gene-NP (250 nm) complexes were positioned on the cells and helped to deliver more genes than did the 530 nm NPs. In the steady state, the gene-NP (530 nm) complexes helped stably deliver more genes to the cells by precipitation of NPs due to gravity. We believe that these findings illustrate how gene-NP complexes function in microalgae cell electroporation and could help set up a protocol for enhanced microalgae applications associated with NPs such as environmental waste removal and biofuel production.},
}
@article {pmid30971458,
year = {2019},
author = {Shields, EJ and Petracovici, AF and Bonasio, R},
title = {lncRedibly versatile: biochemical and biological functions of long noncoding RNAs.},
journal = {The Biochemical journal},
volume = {476},
number = {7},
pages = {1083-1104},
pmid = {30971458},
issn = {1470-8728},
support = {DP2 MH107055/MH/NIMH NIH HHS/United States ; R01 GM127408/GM/NIGMS NIH HHS/United States ; T32 HD083185/HD/NICHD NIH HHS/United States ; T32 HG000046/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Biochemical Phenomena ; Chromatin/genetics/metabolism ; DNA/genetics/metabolism ; Humans ; Models, Biological ; Protein Binding ; RNA, Long Noncoding/*chemistry/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Long noncoding RNAs (lncRNAs) are transcripts that do not code for proteins, but nevertheless exert regulatory effects on various biochemical pathways, in part via interactions with proteins, DNA, and other RNAs. LncRNAs are thought to regulate transcription and other biological processes by acting, for example, as guides that target proteins to chromatin, scaffolds that facilitate protein-protein interactions and complex formation, and orchestrators of phase-separated compartments. The study of lncRNAs has reached an exciting time, as recent advances in experimental and computational methods allow for genome-wide interrogation of biochemical and biological mechanisms of these enigmatic transcripts. A better appreciation for the biochemical versatility of lncRNAs has allowed us to begin closing gaps in our knowledge of how they act in diverse cellular and organismal contexts, including development and disease.},
}
@article {pmid30943016,
year = {2019},
author = {Chen, G and Katrekar, D and Mali, P},
title = {RNA-Guided Adenosine Deaminases: Advances and Challenges for Therapeutic RNA Editing.},
journal = {Biochemistry},
volume = {58},
number = {15},
pages = {1947-1957},
doi = {10.1021/acs.biochem.9b00046},
pmid = {30943016},
issn = {1520-4995},
support = {R01 CA222826/CA/NCI NIH HHS/United States ; R01 GM123313/GM/NIGMS NIH HHS/United States ; R01 HG009285/HG/NHGRI NIH HHS/United States ; },
mesh = {Adenosine/*metabolism ; Adenosine Deaminase/genetics/*metabolism ; Animals ; Humans ; Inosine/*metabolism ; Point Mutation/genetics ; RNA/genetics/*metabolism ; *RNA Editing ; RNA-Binding Proteins/genetics/metabolism ; Transcriptome ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Targeted transcriptome engineering, in contrast to genome engineering, offers a complementary and potentially tunable and reversible strategy for cellular engineering. In this regard, adenosine to inosine (A-to-I) RNA base editing was recently engineered to make programmable base conversions on target RNAs. Similar to the DNA base editing technology, A-to-I RNA editing may offer an attractive alternative in a therapeutic setting, especially for the correction of point mutations. This Perspective introduces five currently characterized RNA editing systems and serves as a reader's guide for implementing an appropriate RNA editing strategy for applications in research or therapeutics.},
}
@article {pmid30874552,
year = {2019},
author = {Hwang, B and Lee, W and Yum, SY and Jeon, Y and Cho, N and Jang, G and Bang, D},
title = {Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1234},
pmid = {30874552},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/genetics ; Cell Differentiation/genetics ; Cell Lineage/*genetics ; Cytidine Deaminase/genetics ; DNA Barcoding, Taxonomic/*methods ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Long Interspersed Nucleotide Elements/*genetics ; Mutagenesis ; Single-Cell Analysis/methods ; Time-Lapse Imaging ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods provide new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we develop a genome editing strategy using a cytidine deaminase fused with nickase Cas9 (nCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns serve as a genetic barcode, which is induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.},
}
@article {pmid30760597,
year = {2019},
author = {Oberhofer, G and Ivy, T and Hay, BA},
title = {Cleave and Rescue, a novel selfish genetic element and general strategy for gene drive.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {13},
pages = {6250-6259},
pmid = {30760597},
issn = {1091-6490},
support = {P40 OD018537/OD/NIH HHS/United States ; T32 GM007616/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Behavior, Animal ; CRISPR-Associated Protein 9/genetics ; Drosophila melanogaster/*genetics ; Female ; Gene Drive Technology/*methods ; Gene Knockout Techniques ; Genes, Essential/*genetics ; Genes, X-Linked ; Genetics, Population ; Genotype ; Germ Cells ; Male ; Models, Genetic ; Phenotype ; Population Dynamics ; *Repetitive Sequences, Nucleic Acid ; Transgenes ; X Chromosome ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {There is great interest in being able to spread beneficial traits throughout wild populations in ways that are self-sustaining. Here, we describe a chromosomal selfish genetic element, CleaveR [Cleave and Rescue (ClvR)], able to achieve this goal. ClvR comprises two linked chromosomal components. One, germline-expressed Cas9 and guide RNAs (gRNAs)-the Cleaver-cleaves and thereby disrupts endogenous copies of a gene whose product is essential. The other, a recoded version of the essential gene resistant to cleavage and gene conversion with cleaved copies-the Rescue-provides essential gene function. ClvR enhances its transmission, and that of linked genes, by creating conditions in which progeny lacking ClvR die because they have no functional copies of the essential gene. In contrast, those who inherit ClvR survive, resulting in an increase in ClvR frequency. ClvR is predicted to spread to fixation under diverse conditions. To test these predictions, we generated a ClvR element in Drosophila melanogasterClvR[tko] is located on chromosome 3 and uses Cas9 and four gRNAs to disrupt melanogaster technical knockout (tko), an X-linked essential gene. Rescue activity is provided by tko from Drosophila virilisClvR[tko] results in germline and maternal carryover-dependent inactivation of melanogaster tko (>99% per generation); lethality caused by this loss is rescued by the virilis transgene; ClvR[tko] activities are robust to genetic diversity in strains from five continents; and uncleavable but functional melanogaster tko alleles were not observed. Finally, ClvR[tko] spreads to transgene fixation. The simplicity of ClvR suggests it may be useful for altering populations in diverse species.},
}
@article {pmid30759234,
year = {2019},
author = {Izumikawa, K and Nobe, Y and Ishikawa, H and Yamauchi, Y and Taoka, M and Sato, K and Nakayama, H and Simpson, RJ and Isobe, T and Takahashi, N},
title = {TDP-43 regulates site-specific 2'-O-methylation of U1 and U2 snRNAs via controlling the Cajal body localization of a subset of C/D scaRNAs.},
journal = {Nucleic acids research},
volume = {47},
number = {5},
pages = {2487-2505},
pmid = {30759234},
issn = {1362-4962},
mesh = {Amyotrophic Lateral Sclerosis/*genetics/pathology ; Cell Nucleolus/genetics ; Coiled Bodies/*genetics/metabolism ; Cytidine/analogs &amp; derivatives/genetics ; DNA-Binding Proteins/*genetics ; HeLa Cells ; Humans ; Molecular Chaperones ; Proteins/*genetics ; RNA, Small Nuclear/genetics ; Ribonucleoproteins, Small Nuclear/genetics ; Telomerase ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2'-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.},
}
@article {pmid30753225,
year = {2019},
author = {Mezzadra, R and de Bruijn, M and Jae, LT and Gomez-Eerland, R and Duursma, A and Scheeren, FA and Brummelkamp, TR and Schumacher, TN},
title = {SLFN11 can sensitize tumor cells towards IFN-γ-mediated T cell killing.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0212053},
pmid = {30753225},
issn = {1932-6203},
mesh = {Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; Cell Line, Tumor ; Cell Survival/drug effects ; Humans ; Interferon-gamma/*pharmacology ; Nuclear Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Quinolines/pharmacology ; RNA Interference ; RNA, Small Interfering/metabolism ; T-Lymphocytes, Cytotoxic/*immunology/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Experimental and clinical observations have highlighted the role of cytotoxic T cells in human tumor control. However, the parameters that control tumor cell sensitivity to T cell attack remain incompletely understood. To identify modulators of tumor cell sensitivity to T cell effector mechanisms, we performed a whole genome haploid screen in HAP1 cells. Selection of tumor cells by exposure to tumor-specific T cells identified components of the interferon-γ (IFN-γ) receptor (IFNGR) signaling pathway, and tumor cell killing by cytotoxic T cells was shown to be in large part mediated by the pro-apoptotic effects of IFN-γ. Notably, we identified schlafen 11 (SLFN11), a known modulator of DNA damage toxicity, as a regulator of tumor cell sensitivity to T cell-secreted IFN-γ. SLFN11 does not influence IFNGR signaling, but couples IFNGR signaling to the induction of the DNA damage response (DDR) in a context dependent fashion. In line with this role of SLFN11, loss of SLFN11 can reduce IFN-γ mediated toxicity. Collectively, our data indicate that SLFN11 can couple IFN-γ exposure of tumor cells to DDR and cellular apoptosis. Future work should reveal the mechanistic basis for the link between IFNGR signaling and DNA damage response, and identify tumor cell types in which SLFN11 contributes to the anti-tumor activity of T cells.},
}
@article {pmid30737497,
year = {2019},
author = {Katrekar, D and Chen, G and Meluzzi, D and Ganesh, A and Worlikar, A and Shih, YR and Varghese, S and Mali, P},
title = {In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.},
journal = {Nature methods},
volume = {16},
number = {3},
pages = {239-242},
pmid = {30737497},
issn = {1548-7105},
support = {R01 GM123313/GM/NIGMS NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; K01 DK119687/DK/NIDDK NIH HHS/United States ; R01 HG009285/HG/NHGRI NIH HHS/United States ; F32 DK112682/DK/NIDDK NIH HHS/United States ; },
mesh = {Adenosine Deaminase/*metabolism ; Animals ; Disease Models, Animal ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; *Point Mutation ; *RNA Editing ; RNA Splicing ; RNA, Messenger/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.},
}
@article {pmid30652305,
year = {2019},
author = {Liu, D and Li, S and Cui, Y and Tong, H and Li, S and Yan, Y},
title = {Podocan affects C2C12 myogenic differentiation by enhancing Wnt/β-catenin signaling.},
journal = {Journal of cellular physiology},
volume = {234},
number = {7},
pages = {11130-11139},
doi = {10.1002/jcp.27763},
pmid = {30652305},
issn = {1097-4652},
mesh = {Animals ; Cell Differentiation ; Gene Expression Regulation/drug effects/physiology ; Heterocyclic Compounds, 3-Ring/pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Muscle Development/*physiology ; Myoblasts/*physiology ; Proteins/genetics/*metabolism ; *Wnt Signaling Pathway ; beta Catenin/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Podocan, a small leucine-rich repeat protein, is a negative regulator of cell proliferation. In this study, we demonstrated that podocan is involved in the differentiation of C2C12 murine myoblasts. Podocan expression increased with the progression of C2C12 differentiation. As expect, siRNA-mediated podocan knockdown inhibited C2C12 differentiation, as indicated by inhibition of MYOG, MYH2, and desmin expression, as well as reductions in the differentiation and fusion indices. Overexpression of podocan using dCas9 technology promoted C2C12 cell differentiation. In addition, supplementation of culture medium with podocan influenced C2C12 differentiation. Podocan knockdown reduced Wnt/β-catenin signaling, characterized by a reduction in the nuclear translocation of β-catenin, whereas podocan overexpression had the opposite effect. Furthermore, treatment with XAV939, an inhibitor of Wnt/β-catenin, reduced the podocan-mediated promotion of C2C12 differentiation. Induction of muscle injury in mice by bupivacaine administration suggested that podocan may play a role in muscle regeneration. In summary, our results suggest that podocan is required for normal C2C12 differentiation and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.},
}
@article {pmid30622278,
year = {2019},
author = {Liang, P and Xie, X and Zhi, S and Sun, H and Zhang, X and Chen, Y and Chen, Y and Xiong, Y and Ma, W and Liu, D and Huang, J and Songyang, Z},
title = {Genome-wide profiling of adenine base editor specificity by EndoV-seq.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {67},
pmid = {30622278},
issn = {2041-1723},
mesh = {Adenine/metabolism ; Animals ; Bacterial Proteins/*metabolism ; Base Sequence/genetics ; CRISPR-Associated Proteins/*metabolism ; DNA/genetics/metabolism ; Deamination ; Deoxyribonuclease (Pyrimidine Dimer)/*metabolism ; Fibroblasts ; Gene Editing/methods ; Genome, Human/*genetics ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Inosine/metabolism ; Mice ; Multiplex Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Whole Genome Sequencing/*methods ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2-19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7-320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease.},
}
@article {pmid30610016,
year = {2019},
author = {Del Olmo, T and Lauzier, A and Normandin, C and Larcher, R and Lecours, M and Jean, D and Lessard, L and Steinberg, F and Boisvert, FM and Jean, S},
title = {APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting.},
journal = {EMBO reports},
volume = {20},
number = {2},
pages = {},
pmid = {30610016},
issn = {1469-3178},
mesh = {Clathrin/*metabolism ; DNA-(Apurinic or Apyrimidinic Site) Lyase/*metabolism ; Endonucleases/*metabolism ; Endosomes/metabolism ; Humans ; Mass Spectrometry ; Multifunctional Enzymes/*metabolism ; Protein Binding ; Protein Interaction Mapping ; Protein Interaction Maps ; Protein Transport ; rab GTP-Binding Proteins/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase-activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.},
}
@article {pmid30581135,
year = {2019},
author = {Monteleone, LR and Matthews, MM and Palumbo, CM and Thomas, JM and Zheng, Y and Chiang, Y and Fisher, AJ and Beal, PA},
title = {A Bump-Hole Approach for Directed RNA Editing.},
journal = {Cell chemical biology},
volume = {26},
number = {2},
pages = {269-277.e5},
pmid = {30581135},
issn = {2451-9448},
support = {R01 GM061115/GM/NIGMS NIH HHS/United States ; T32 GM113770/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine/chemistry ; Adenosine Deaminase/genetics/metabolism ; Cell Line ; Crystallography, X-Ray ; Gene Editing/*methods ; Humans ; Mutagenesis, Site-Directed ; Oligonucleotides/chemistry/metabolism ; Protein Structure, Tertiary ; RNA/*chemistry/metabolism ; RNA-Binding Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity.},
}
@article {pmid30527661,
year = {2019},
author = {Chang, TH and Mattei, E and Gainetdinov, I and Colpan, C and Weng, Z and Zamore, PD},
title = {Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster.},
journal = {Molecular cell},
volume = {73},
number = {2},
pages = {291-303.e6},
pmid = {30527661},
issn = {1097-4164},
support = {P01 HD078253/HD/NICHD NIH HHS/United States ; R37 GM062862/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Argonaute Proteins/genetics/metabolism ; Binding Sites ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; *DNA Transposable Elements ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/*enzymology/genetics ; Gene Expression Regulation ; Promoter Regions, Genetic ; Protein Binding ; RNA Helicases/genetics/metabolism ; RNA Polymerase II/genetics/*metabolism ; RNA, Messenger/*biosynthesis/genetics ; RNA, Small Interfering/*biosynthesis/genetics ; *Transcription, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.},
}
@article {pmid30455466,
year = {2019},
author = {Lebar, T and Verbič, A and Ljubetič, A and Jerala, R},
title = {Polarized displacement by transcription activator-like effectors for regulatory circuits.},
journal = {Nature chemical biology},
volume = {15},
number = {1},
pages = {80-87},
doi = {10.1038/s41589-018-0163-8},
pmid = {30455466},
issn = {1552-4469},
mesh = {Binding Sites ; CRISPR-Associated Protein 9/genetics ; DNA/genetics/*metabolism ; Early Growth Response Protein 1/genetics/metabolism ; *Gene Expression Regulation ; Gene Regulatory Networks ; HEK293 Cells ; Humans ; Models, Theoretical ; Transcription Activator-Like Effectors/genetics/*metabolism ; Zinc Fingers ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The interplay between DNA-binding proteins plays an important role in transcriptional regulation and could increase the precision and complexity of designed regulatory circuits. Here we show that a transcription activator-like effector (TALE) can displace another TALE protein from DNA in a highly polarized manner, displacing only the 3'- but not 5'-bound overlapping or adjacent TALE. We propose that the polarized displacement by TALEs is based on its multipartite nature of binding to DNA. The polarized TALE displacement provides strategies for the specific regulation of gene expression, for construction of all two-input Boolean genetic logic circuits based on the robust propagation of the displacement across multiple neighboring sites, for displacement of zinc finger-based transcription factors and for suppression of Cas9-gRNA-mediated genome cleavage, enriching the synthetic biology toolbox and contributing to the understanding of the underlying principles of the facilitated displacement.},
}
@article {pmid30380771,
year = {2018},
author = {Andersen, KL and Nielsen, H},
title = {Knock-Down of a Novel snoRNA in Tetrahymena Reveals a Dual Role in 5.8S rRNA Processing and Generation of a 26S rRNA Fragment.},
journal = {Biomolecules},
volume = {8},
number = {4},
pages = {},
pmid = {30380771},
issn = {2218-273X},
mesh = {Base Sequence ; Cell Survival ; Conserved Sequence ; Gene Expression Regulation ; *Gene Knockdown Techniques ; Genome ; Methylation ; Nucleic Acid Conformation ; Protozoan Proteins/chemistry/metabolism ; RNA Processing, Post-Transcriptional/*genetics ; RNA, Ribosomal/chemistry/*genetics ; RNA, Ribosomal, 5.8S/chemistry/*genetics ; RNA, Small Nucleolar/chemistry/*genetics ; Tetrahymena/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In eukaryotes, 18S, 5.8S, and 28S rRNAs are transcribed as precursor molecules that undergo extensive modification and nucleolytic processing to form the mature rRNA species. Central in the process are the small nucleolar RNAs (snoRNAs). The majority of snoRNAs guide site specific chemical modifications but a few are involved in defining pre-rRNA cleavages. Here, we describe an unusual snoRNA (TtnuCD32) belonging to the box C/D subgroup from the ciliate Tetrahymena thermophila. We show that TtnuCD32 is unlikely to function as a modification guide snoRNA and that it is critical for cell viability. Cell lines with genetic knock-down of TtnuCD32 were impaired in growth and displayed two novel and apparently unrelated phenotypes. The most prominent phenotype is the accumulation of processing intermediates of 5.8S rRNA. The second phenotype is the decrease in abundance of a ~100 nt 26S rRNA fragment of unknown function. Sequence analysis demonstrated that TtnuCD32 share features with the essential snoRNA U14 but an alternative candidate (TtnuCD25) was more closely related to other U14 sequences. This, together with the fact that the observed rRNA processing phenotypes were not similar to what has been observed in U14 depleted cells, suggests that TtnuCD32 is a U14 homolog that has gained novel functions.},
}
@article {pmid30307925,
year = {2018},
author = {Elsner, M},
title = {Barcodes galore for developmental biology.},
journal = {Nature biotechnology},
volume = {36},
number = {10},
pages = {936},
pmid = {30307925},
issn = {1546-1696},
mesh = {Animals ; Biotechnology ; Cell Lineage/genetics ; *DNA Barcoding, Taxonomic ; *Developmental Biology ; Embryonic Development/genetics ; Mice ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid30301832,
year = {2019},
author = {Deryusheva, S and Gall, JG},
title = {scaRNAs and snoRNAs: Are they limited to specific classes of substrate RNAs?.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {1},
pages = {17-22},
pmid = {30301832},
issn = {1469-9001},
support = {R01 GM033397/GM/NIGMS NIH HHS/United States ; R01 GM33397/NH/NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Coiled Bodies/*metabolism ; HeLa Cells ; Humans ; Nucleic Acid Conformation ; RNA Processing, Post-Transcriptional ; RNA, Ribosomal/chemistry/genetics/metabolism ; RNA, Small Nuclear/chemistry/genetics/metabolism ; RNA, Small Nucleolar/*chemistry/genetics/*metabolism ; Spliceosomes/genetics/metabolism ; Xenopus/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Posttranscriptional modifications of rRNA occur in the nucleolus where rRNA modification guide RNAs, or snoRNAs, concentrate. On the other hand, scaRNAs, the modification guide RNAs for spliceosomal snRNAs, concentrate in the Cajal body (CB). It is generally assumed, therefore, that snRNAs must accumulate in CBs to be modified by scaRNAs. Here we demonstrate that the evidence for the latter postulate is not consistent. In the nucleus, scaRNA localization is not limited to CBs. Furthermore, canonical scaRNAs can modify rRNAs. We suggest that the conventional view that scaRNAs function only in the CB needs revision.},
}
@article {pmid30252600,
year = {2018},
author = {Moore, AN and McWatters, DC and Hudson, AJ and Russell, AG},
title = {RNA-Seq employing a novel rRNA depletion strategy reveals a rich repertoire of snoRNAs in Euglena gracilis including box C/D and Ψ-guide RNAs targeting the modification of rRNA extremities.},
journal = {RNA biology},
volume = {15},
number = {10},
pages = {1309-1318},
pmid = {30252600},
issn = {1555-8584},
mesh = {Euglena gracilis/genetics ; Gene Library ; Nucleic Acid Conformation ; Pseudouridine/genetics ; RNA, Ribosomal/*genetics ; RNA, Small Nucleolar/classification/*genetics ; RNA, Untranslated/genetics ; *Sequence Analysis, RNA ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Previous mRNA transcriptome studies of Euglena gracilis have shown that this organism possesses a large and diverse complement of protein coding genes; however, the study of non-coding RNA classes has been limited. The natural extensive fragmentation of the E. gracilis large subunit ribosomal RNA presents additional barriers to the identification of non-coding RNAs as size-selected small RNA libraries will be dominated by rRNA sequences. In this study we have developed a strategy to significantly reduce rRNA amplification prior to RNA-Seq analysis thereby producing a ncRNA library allowing for the identification of many new E. gracilis small RNAs. Library analysis reveals 113 unique new small nucleolar (sno) RNAs and a large collection of snoRNA isoforms, as well as the first significant collection of nuclear tRNAs in this organism. A 3' end AGAUGN consensus motif and conserved structural features can now be defined for E. gracilis pseudouridine guide RNAs. snoRNAs of both classes were identified that target modification of the 3' extremities of rRNAs utilizing predicted base-pairing interactions with internally transcribed spacers (ITS), providing insight into the timing of steps in rRNA maturation. Cumulatively, this represents the most comprehensive analysis of small ncRNAs in Euglena gracilis to date.},
}
@article {pmid30218085,
year = {2018},
author = {Fujikane, R and Behm-Ansmant, I and Tillault, AS and Loegler, C and Igel-Bourguignon, V and Marguet, E and Forterre, P and Branlant, C and Motorin, Y and Charpentier, B},
title = {Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {13815},
pmid = {30218085},
issn = {2045-2322},
mesh = {Archaeal Proteins/metabolism ; Genes, Archaeal/genetics ; Intramolecular Transferases/metabolism ; Nucleic Acid Conformation ; Pseudouridine/*metabolism ; RNA/metabolism ; RNA, Archaeal/*biosynthesis/*genetics ; RNA, Ribosomal ; RNA, Transfer ; Ribonucleoproteins/metabolism ; Ribonucleoproteins, Small Nucleolar/genetics/metabolism ; Uridine/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.},
}
@article {pmid30208061,
year = {2018},
author = {Ata, H and Ekstrom, TL and Martínez-Gálvez, G and Mann, CM and Dvornikov, AV and Schaefbauer, KJ and Ma, AC and Dobbs, D and Clark, KJ and Ekker, SC},
title = {Robust activation of microhomology-mediated end joining for precision gene editing applications.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007652},
pmid = {30208061},
issn = {1553-7404},
support = {P30 DK090728/DK/NIDDK NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; R01 GM063904/GM/NIGMS NIH HHS/United States ; P30 DK084567/DK/NIDDK NIH HHS/United States ; T32 GM065841/GM/NIGMS NIH HHS/United States ; UL1 TR002377/TR/NCATS NIH HHS/United States ; },
mesh = {Algorithms ; Alleles ; Animals ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; Feasibility Studies ; Female ; Gene Editing/*methods ; Genetic Diseases, Inborn/genetics/therapy ; Genetic Therapy/methods ; HeLa Cells ; Humans ; Male ; *Models, Genetic ; Mutagenesis, Site-Directed ; Zebrafish ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.},
}
@article {pmid30183280,
year = {2018},
author = {Rauch, S and He, C and Dickinson, BC},
title = {Targeted m[6]A Reader Proteins To Study Epitranscriptomic Regulation of Single RNAs.},
journal = {Journal of the American Chemical Society},
volume = {140},
number = {38},
pages = {11974-11981},
pmid = {30183280},
issn = {1520-5126},
support = {P30 CA014599/CA/NCI NIH HHS/United States ; R35 GM119840/GM/NIGMS NIH HHS/United States ; RM1 HG008935/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenosine/*analogs &amp; derivatives/chemistry ; CRISPR-Associated Proteins/genetics/metabolism ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Nucleic Acid Hybridization ; Protein Engineering/methods ; RNA/*chemistry/genetics/*metabolism ; RNA Processing, Post-Transcriptional ; RNA-Binding Proteins/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; *Transcriptome ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Post-transcriptional gene expression regulation of RNA has emerged as a key factor that controls mammalian protein production. RNA trafficking, translation efficiency, and stability are all controlled at the transcript level. For example, in addition to the commonly known processing steps of capping, splicing, and polyadenylation, RNA can be chemically modified. In eukaryotes, N[6]-methyladenosine (m[6]A) is the most prevalent mRNA modification. While the writers, erasers, and readers for m[6]A are rapidly being uncovered and studied at the whole-cell level, their competitive interplay to regulate methylated RNA transcripts has yet to be elucidated. To address this limitation, we report the development of programmable dPspCas13b-m[6]A reader proteins to investigate the regulatory effects of specific readers on single transcripts in live cells. We fused the two most well-characterized m[6]A reader proteins, YTHDF1 and YTHDF2, to a catalytically inactive PspCas13b protein, which can target the reader to a specific RNA of interest using guide RNA (gRNA) complementarity. We then demonstrate that the fused reader proteins each retain their reported functional role on a reporter construct: YTHDF2 induces degradation and YTHDF1 enhances translation. Finally, we show that the system can target endogenous mRNA transcripts within cells, using YTHDF2 as an exemplar, where we found tethering with YTHDF2 leads to decay of the target transcript. The development of dCas13b-based tools to study the regulation of endogenous RNAs will dramatically enhance our understanding of how RNA regulation occurs at the single RNA level. Additionally, our new tools, which permit transcript-specific mediated decay or enhanced protein production, will find utility in synthetic biology applications aimed at controlling genetic information flow at the RNA level.},
}
@article {pmid30166483,
year = {2018},
author = {Farzadfard, F and Lu, TK},
title = {Emerging applications for DNA writers and molecular recorders.},
journal = {Science (New York, N.Y.)},
volume = {361},
number = {6405},
pages = {870-875},
pmid = {30166483},
issn = {1095-9203},
support = {P50 GM098792/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins ; Biosensing Techniques ; Brain Mapping ; CRISPR-Associated Protein 9 ; Cell Engineering/methods ; DNA/chemistry/genetics ; Directed Molecular Evolution/methods ; Endonucleases ; Gene Editing/*trends ; Genetic Engineering/*trends ; Genome ; Humans ; Information Storage and Retrieval/*methods ; Recombination, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Natural life is encoded by evolvable, DNA-based memory. Recent advances in dynamic genome-engineering technologies, which we collectively refer to as in vivo DNA writing, have opened new avenues for investigating and engineering biology. This Review surveys these technological advances, outlines their prospects and emerging applications, and discusses the features and current limitations of these technologies for building various genetic circuits for processing and recording information in living cells.},
}
@article {pmid30061362,
year = {2018},
author = {Lu, H and Zhang, S and Wu, J and Chen, M and Cai, MC and Fu, Y and Li, W and Wang, J and Zhao, X and Yu, Z and Ma, P and Zhuang, G},
title = {Molecular Targeted Therapies Elicit Concurrent Apoptotic and GSDME-Dependent Pyroptotic Tumor Cell Death.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {24},
number = {23},
pages = {6066-6077},
doi = {10.1158/1078-0432.CCR-18-1478},
pmid = {30061362},
issn = {1557-3265},
mesh = {Animals ; Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects/genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Disease Models, Animal ; Gene Editing ; Gene Targeting ; Humans ; Lung Neoplasms/diagnosis/drug therapy/genetics/metabolism ; Mice ; Models, Biological ; Pyroptosis/*drug effects/genetics ; Receptors, Estrogen/genetics/*metabolism ; Signal Transduction/*drug effects ; Tomography, X-Ray Computed ; Xenograft Model Antitumor Assays ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {PURPOSE: The induced death signals following oncogene inhibition underlie clinical efficacy of molecular targeted therapies against human cancer, and defects of intact cell apoptosis machinery often lead to therapeutic failure. Despite potential importance, other forms of regulated cell death triggered by pharmacologic intervention have not been systematically characterized.<br><br>EXPERIMENTAL DESIGN: Pyroptotic cell death was assessed by immunoblot analysis, phase-contrast imaging, scanning electron microscopy, and flow cytometry. Tumor tissues of patients with lung cancer were analyzed using IHC. Functional impact of pyroptosis on drug response was investigated in cell lines and xenograft models.<br><br>RESULTS: We showed that diverse small-molecule inhibitors specifically targeting KRAS-, EGFR-, or ALK-driven lung cancer uniformly elicited robust pyroptotic cell death, in addition to simultaneously invoking cellular apoptosis. Upon drug treatment, the mitochondrial intrinsic apoptotic pathway was engaged and the mobilized caspase-3 protease cleaved and activated gasdermin E (GSDME, encoded by DFNA5), which permeabilized cytoplasmic membrane and executed cell-lytic pyroptosis. GSDME displayed ubiquitous expression in various lung cancer cell lines and clinical specimens, including KRAS-mutant, EGFR-altered, and ALK-rearranged adenocarcinomas. As a result, cooccurrence and interplay of apoptosis and pyroptosis were widespread in lung cancer cells, succumbing to genotype-matched regimens. We further demonstrated that pyroptotic cell death partially contributed to the drug response in a subset of cancer models.<br><br>CONCLUSIONS: These results pinpoint GSDME-dependent pyroptosis as a previously unrecognized mechanism of action for molecular targeted agents to eradicate oncogene-addicted neoplastic cells, which may have important implications for the clinical development and optimal application of anticancer therapeutics.},
}
@article {pmid30016076,
year = {2018},
author = {Paunovska, K and Gil, CJ and Lokugamage, MP and Sago, CD and Sato, M and Lando, GN and Gamboa Castro, M and Bryksin, AV and Dahlman, JE},
title = {Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery.},
journal = {ACS nano},
volume = {12},
number = {8},
pages = {8341-8349},
pmid = {30016076},
issn = {1936-086X},
support = {T32 EB006343/EB/NIBIB NIH HHS/United States ; T32 EB021962/EB/NIBIB NIH HHS/United States ; T32 GM008433/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Cholesterol/*chemistry/metabolism ; Drug Carriers/chemistry/metabolism ; *Drug Delivery Systems ; Endothelial Cells/chemistry/metabolism ; Female ; Liver/chemistry/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nanoparticles/*chemistry/metabolism ; RNA, Small Interfering/*chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Lipid nanoparticles (LNPs) are formulated using unmodified cholesterol. However, cholesterol is naturally esterified and oxidized in vivo, and these cholesterol variants are differentially trafficked in vivo via lipoproteins including LDL and VLDL. We hypothesized that incorporating the same cholesterol variants into LNPs-which can be structurally similar to LDL and VLDL-would alter nanoparticle targeting in vivo. To test this hypothesis, we quantified how >100 LNPs made with six cholesterol variants delivered DNA barcodes to 18 cell types in wild-type, LDLR[-/-], and VLDLR[-/-] mice that were both age-matched and female. By analyzing ∼2000 in vivo drug delivery data points, we found that LNPs formulated with esterified cholesterol delivered nucleic acids more efficiently than LNPs formulated with regular or oxidized cholesterol when compared across all tested cell types in the mouse. We also identified an LNP containing cholesteryl oleate that efficiently delivered siRNA and sgRNA to liver endothelial cells in vivo. Delivery was as-or more-efficient as the same LNP made with unmodified cholesterol. Moreover, delivery to liver endothelial cells was 3 times more efficient than delivery to hepatocytes, distinguishing this oleate LNP from hepatocyte-targeting LNPs. RNA delivery can be improved by rationally selecting cholesterol variants, allowing optimization of nanoparticle targeting.},
}
@article {pmid29996105,
year = {2018},
author = {Liu, Y and Esyunina, D and Olovnikov, I and Teplova, M and Kulbachinskiy, A and Aravin, AA and Patel, DJ},
title = {Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA.},
journal = {Cell reports},
volume = {24},
number = {2},
pages = {453-462},
pmid = {29996105},
issn = {2211-1247},
support = {R00 HD057233/HD/NICHD NIH HHS/United States ; R01 GM104962/GM/NIGMS NIH HHS/United States ; DP2 OD007371/OD/NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 GM097363/GM/NIGMS NIH HHS/United States ; R01 AI068776/AI/NIAID NIH HHS/United States ; },
mesh = {Argonaute Proteins/*chemistry ; Base Pairing ; Base Sequence ; Biocatalysis ; DNA, Bacterial/*metabolism ; Models, Molecular ; Multiprotein Complexes/*chemistry ; Protein Structure, Secondary ; Rhodobacter sphaeroides/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling.},
}
@article {pmid29904106,
year = {2018},
author = {Liu, Z and Lu, Z and Yang, G and Huang, S and Li, G and Feng, S and Liu, Y and Li, J and Yu, W and Zhang, Y and Chen, J and Sun, Q and Huang, X},
title = {Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2338},
pmid = {29904106},
issn = {2041-1723},
mesh = {Adenine/chemistry ; Animals ; Animals, Newborn ; Cell Line, Tumor ; *Disease Models, Animal ; Female ; Genome, Human ; Genotype ; Green Fluorescent Proteins/chemistry ; High-Throughput Nucleotide Sequencing ; Homeodomain Proteins/*genetics ; Humans ; Male ; Mice ; Mutation ; Protein Domains ; RNA, Messenger/metabolism ; Receptors, Androgen/*genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Transcription Factors/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.},
}
@article {pmid29895326,
year = {2018},
author = {Postberg, J and Jönsson, F and Weil, PP and Bulic, A and Juranek, SA and Lipps, HJ},
title = {27nt-RNAs guide histone variant deposition via 'RNA-induced DNA replication interference' and thus transmit parental genome partitioning in Stylonychia.},
journal = {Epigenetics &amp; chromatin},
volume = {11},
number = {1},
pages = {31},
pmid = {29895326},
issn = {1756-8935},
support = {LI 231/29-1//Deutsche Forschungsgemeinschaft (DE)/International ; AOBJ: 571454//Deutsche Forschungsgemeinschaft (DE)/International ; na//Peter und Traudl Engelhorn Stiftung/International ; },
mesh = {Argonaute Proteins/metabolism ; Ciliophora/genetics/*physiology ; *DNA Replication ; Genetic Variation ; Genome, Protozoan ; Histones/*genetics ; Micronucleus, Germline/genetics ; RNA, Protozoan/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: During sexual reproduction in the unicellular ciliate Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1.<br><br>RESULTS: These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected.<br><br>CONCLUSIONS: We introduce the concept of 'RNA-induced DNA replication interference' and explain how the parental functional genome partition could become transmitted to the progeny.},
}
@article {pmid29871894,
year = {2018},
author = {De Zoysa, MD and Wu, G and Katz, R and Yu, YT},
title = {Guide-substrate base-pairing requirement for box H/ACA RNA-guided RNA pseudouridylation.},
journal = {RNA (New York, N.Y.)},
volume = {24},
number = {8},
pages = {1106-1117},
pmid = {29871894},
issn = {1469-9001},
support = {R01 GM104077/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Pairing/*genetics ; Base Sequence ; Binding Sites/genetics ; *Nucleic Acid Conformation ; Pseudouridine/*chemistry ; Saccharomyces cerevisiae/*genetics ; Spliceosomes/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Box H/ACA RNAs are a group of small RNAs found in abundance in eukaryotes (as well as in archaea). Although their sequences differ, eukaryotic box H/ACA RNAs all share the same unique hairpin-hinge-hairpin-tail structure. Almost all of them function as guides that primarily direct pseudouridylation of rRNAs and spliceosomal snRNAs at specific sites. Although box H/ACA RNA-guided pseudouridylation has been extensively studied, the detailed rules governing this reaction, especially those concerning the guide RNA-substrate RNA base-pairing interactions that determine the specificity and efficiency of pseudouridylation, are still not exactly clear. This is particularly relevant given that the lengths of the guide sequences involved in base-pairing vary from one box H/ACA RNA to another. Here, we carry out a detailed investigation into guide-substrate base-pairing interactions, and identify the minimum number of base pairs (8), required for RNA-guided pseudouridylation. In addition, we find that the pseudouridylation pocket, present in each hairpin of box H/ACA RNA, exhibits flexibility in fitting slightly different substrate sequences. Our results are consistent across three independent pseudouridylation pockets tested, suggesting that our findings are generally applicable to box H/ACA RNA-guided RNA pseudouridylation.},
}
@article {pmid29864424,
year = {2018},
author = {Wang, J and Yuan, Z and Zhang, Y},
title = {Alternative translation initiation from two in-frame start codons in DHX33 gene.},
journal = {Biochemical and biophysical research communications},
volume = {502},
number = {4},
pages = {501-507},
doi = {10.1016/j.bbrc.2018.05.201},
pmid = {29864424},
issn = {1090-2104},
mesh = {Animals ; Cells, Cultured ; *Codon, Initiator ; DEAD-box RNA Helicases/*biosynthesis/chemistry/*genetics ; Humans ; Isoenzymes/biosynthesis/chemistry/genetics ; Mice ; Mice, Knockout ; NIH 3T3 Cells ; *Peptide Chain Initiation, Translational ; Reading Frames ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {DHX33 has been shown to play key roles in promoting cell proliferation. We have previously found that DHX33 protein is a doublet. In this report, we discovered that DHX33 doublet is due to alternative translation initiation by two in-frame initiation codons. This is supported by studies from both cell lines and mouse models. DHX33 translation initiation from either AUG codon happens at equal efficiency. Short DHX33 protein has similar cellular location and functions with full-length DHX33. Our results suggest that leaky scanning normally occur in DHX33 mRNA translation, which may serve as a safeguard mechanism to ensure optimal DHX33 translation efficiency. This is the first report of DEAD/DEAH box proteins that can be regulated by alternative translation initiation.},
}
@article {pmid29804837,
year = {2018},
author = {Jain, S and Ba, Z and Zhang, Y and Dai, HQ and Alt, FW},
title = {CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning.},
journal = {Cell},
volume = {174},
number = {1},
pages = {102-116.e14},
pmid = {29804837},
issn = {1097-4172},
support = {/HHMI/Howard Hughes Medical Institute/United States ; R01 AI020047/AI/NIAID NIH HHS/United States ; R37 AI020047/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CCCTC-Binding Factor/*metabolism ; Cell Line ; Chromatin/*metabolism ; DNA, Intergenic/genetics/metabolism ; DNA-Binding Proteins/deficiency/genetics ; Homeodomain Proteins/*metabolism ; Immunoglobulin Heavy Chains/genetics/metabolism ; Immunoglobulin Variable Region/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Models, Molecular ; Mutagenesis ; Protein Sorting Signals ; Receptors, Antigen, T-Cell/genetics/metabolism ; *V(D)J Recombination ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.},
}
@article {pmid29787760,
year = {2018},
author = {Shin, JH and Jung, S and Ramakrishna, S and Kim, HH and Lee, J},
title = {In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.},
journal = {Biochemical and biophysical research communications},
volume = {502},
number = {1},
pages = {116-122},
doi = {10.1016/j.bbrc.2018.05.130},
pmid = {29787760},
issn = {1090-2104},
mesh = {Animals ; *DNA End-Joining Repair ; Disease Models, Animal ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Hydrolases/*genetics ; Mice ; Mutation ; RNA, Messenger/genetics ; Tyrosinemias/*genetics/pathology/*therapy ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence.},
}
@article {pmid29599436,
year = {2018},
author = {Gao, Y and Tan, J and Jin, J and Ma, H and Chen, X and Leger, B and Xu, J and Spagnol, ST and Dahl, KN and Levine, AS and Liu, Y and Lan, L},
title = {SIRT6 facilitates directional telomere movement upon oxidative damage.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {5407},
pmid = {29599436},
issn = {2045-2322},
support = {P30 CA047904/CA/NCI NIH HHS/United States ; R01 GM118833/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Triphosphatases/metabolism ; Cell Line ; Chromatin/chemistry/metabolism ; Chromatin Assembly and Disassembly ; Chromosomal Proteins, Non-Histone/metabolism ; DNA Damage ; DNA Repair ; Gene Editing ; Humans ; Microscopy, Fluorescence ; *Oxidative Stress ; Sirtuins/deficiency/genetics/*metabolism ; Telomere/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Oxidative damage to telomeres leads to telomere attrition and genomic instability, resulting in poor cell viability. Telomere dynamics contribute to the maintenance of telomere integrity; however, whether oxidative damage induces telomere movement and how telomere mobility is regulated remain poorly understood. Here, we show that oxidative damage at telomeres triggers directional telomere movement. The presence of the human Sir2 homolog, Sirtuin 6 (SIRT6) is required for oxidative damage-induced telomeric movement. SIRT6 knock out (KO) cells show neither damage-induced telomere movement nor chromatin decondensation at damaged telomeres; both are observed in wild type (WT) cells. A deacetylation mutant of SIRT6 increases damage-induced telomeric movement in SIRT6 KO cells as well as WT SIRT6. SIRT6 recruits the chromatin-remodeling protein SNF2H to damaged telomeres, which appears to promote chromatin decondensation independent of its deacetylase activity. Together, our results suggest that SIRT6 plays a role in the regulation of telomere movement upon oxidative damage, shedding new light onto the function of SIRT6 in telomere maintenance.},
}
@article {pmid29549325,
year = {2018},
author = {Yang, CT and Li, JM and Chu, WK and Chow, SE},
title = {Downregulation of lumican accelerates lung cancer cell invasion through p120 catenin.},
journal = {Cell death &amp; disease},
volume = {9},
number = {4},
pages = {414},
pmid = {29549325},
issn = {2041-4889},
mesh = {Actin Cytoskeleton ; Catenins/*metabolism ; Cell Line, Tumor ; Cell Movement/drug effects ; Culture Media, Conditioned/pharmacology ; Down-Regulation ; Humans ; Lumican/antagonists &amp; inhibitors/genetics/*metabolism ; Lung Neoplasms/metabolism/pathology ; Macrophages/cytology/metabolism ; Protein Binding ; RNA Interference ; RNA, Small Interfering/metabolism ; Tubulin/metabolism ; rac GTP-Binding Proteins/metabolism ; rho GTP-Binding Proteins/metabolism ; Delta Catenin ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The overexpression of lumican has been found in lung cancer cells; however, the functional role of lumican in lung cancer cells remains unclear. In this study, we found lumican functioned as a tubulin-binding protein and the depletion of lumican by transfection with its specific shRNA increased lung cancer cell invasion. Such alterations led to morphological changes and actin cytoskeleton remodeling, including the induction of membrane ruffling or protrusion and stress fiber formation, correlated with the increased activities of Rac and Rho. The downregulation of lumican was also implicated in macrophage-conditioned media (maCM)-induced cell invasion. Immunofluorescence images and immunoprecipitation assays revealed the co-localization of p120-catenin (p120ctn) and lumican. Reduction in the levels of p120ctn induced membrane ruffling and the activation of the Rho family, which accelerated cell invasion. Our data indicated that lumican is associated with microtubule-modulated p120ctn signaling, providing important insights into lung cancer progression.},
}
@article {pmid29534235,
year = {2018},
author = {Ge, P and Islam, S and Zhong, C and Zhang, S},
title = {De novo discovery of structural motifs in RNA 3D structures through clustering.},
journal = {Nucleic acids research},
volume = {46},
number = {9},
pages = {4783-4793},
pmid = {29534235},
issn = {1362-4962},
support = {R01 GM102515/GM/NIGMS NIH HHS/United States ; },
mesh = {Cluster Analysis ; Humans ; Models, Molecular ; Nucleotide Motifs ; RNA/*chemistry ; RNA, Catalytic/chemistry ; RNA, Ribosomal/chemistry ; RNA, Transfer/chemistry ; Riboswitch ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {As functional components in three-dimensional (3D) conformation of an RNA, the RNA structural motifs provide an easy way to associate the molecular architectures with their biological mechanisms. In the past years, many computational tools have been developed to search motif instances by using the existing knowledge of well-studied families. Recently, with the rapidly increasing number of resolved RNA 3D structures, there is an urgent need to discover novel motifs with the newly presented information. In this work, we classify all the loops in non-redundant RNA 3D structures to detect plausible RNA structural motif families by using a clustering pipeline. Compared with other clustering approaches, our method has two benefits: first, the underlying alignment algorithm is tolerant to the variations in 3D structures. Second, sophisticated downstream analysis has been performed to ensure the clusters are valid and easily applied to further research. The final clustering results contain many interesting new variants of known motif families, such as GNAA tetraloop, kink-turn, sarcin-ricin and T-loop. We have also discovered potential novel functional motifs conserved in ribosomal RNA, sgRNA, SRP RNA, riboswitch and ribozyme.},
}
@article {pmid29484589,
year = {2018},
author = {Jani, S and Jackson, A and Davies-Sala, C and Chiem, K and Soler-Bistué, A and Zorreguieta, A and Tolmasky, ME},
title = {Assessment of External Guide Sequences' (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1737},
number = {},
pages = {89-98},
doi = {10.1007/978-1-4939-7634-8_6},
pmid = {29484589},
issn = {1940-6029},
support = {R15 AI047115/AI/NIAID NIH HHS/United States ; T37 MD001368/MD/NIMHD NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Bacteria/drug effects/*genetics ; Cell-Penetrating Peptides/*pharmacology ; Drug Resistance, Bacterial ; Oligoribonucleotides, Antisense/genetics/*metabolism ; RNA Precursors/genetics/metabolism ; RNA, Bacterial/genetics/*metabolism ; RNA, Messenger/genetics/*metabolism ; Ribonuclease P/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5'-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.},
}
@article {pmid29482569,
year = {2018},
author = {Yu, H and Long, W and Zhang, X and Xu, K and Guo, J and Zhao, H and Li, H and Qing, Y and Pan, W and Jia, B and Zhao, HY and Huang, X and Wei, HJ},
title = {Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer.},
journal = {Journal of translational medicine},
volume = {16},
number = {1},
pages = {41},
pmid = {29482569},
issn = {1479-5876},
support = {2016ZX08009-003-006//the National Genetically Modified Organisms Breeding Major Projects/International ; 31560637//the National Natural Science Foundation of China/International ; 2014FC006//Major Program on Basic Research Projects of Yunnan Province/International ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; DNA/metabolism ; Disease Models, Animal ; Embryo, Mammalian/metabolism ; Fetus/cytology ; Fibroblasts/metabolism ; Gene Knockout Techniques ; Germ Cells/metabolism ; Growth and Development ; Laron Syndrome/*pathology ; *Nuclear Transfer Techniques ; Receptors, Somatotropin/*metabolism ; Swine ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome.<br><br>METHODS: To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing.&#xa0;GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing.<br><br>RESULTS: In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR[-47/-1], GHR[-47/-46]) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13&#xa0;months old, the average body weight of wild-type pigs varied from 0.6 to 89.5&#xa0;kg, but that of GHRKO pigs varied from only 0.9 to 37.0&#xa0;kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission.<br><br>CONCLUSION: The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome.},
}
@article {pmid29457977,
year = {2018},
author = {Hua, B and Wang, Y and Park, S and Han, KY and Singh, D and Kim, JH and Cheng, W and Ha, T},
title = {The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.},
journal = {Biochemistry},
volume = {57},
number = {10},
pages = {1572-1576},
pmid = {29457977},
issn = {1520-4995},
support = {DP2 OD008693/OD/NIH HHS/United States ; R21 AI135559/AI/NIAID NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Algorithms ; Bacterial Proteins/chemistry/*metabolism ; CRISPR-Associated Protein 9 ; DNA/metabolism ; Endonucleases/*metabolism ; Fluorescence ; Protein Binding ; Single Molecule Imaging/*methods ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.},
}
@article {pmid29379150,
year = {2018},
author = {Liu, W and Duttke, SH and Hetzel, J and Groth, M and Feng, S and Gallego-Bartolome, J and Zhong, Z and Kuo, HY and Wang, Z and Zhai, J and Chory, J and Jacobsen, SE},
title = {RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.},
journal = {Nature plants},
volume = {4},
number = {3},
pages = {181-188},
pmid = {29379150},
issn = {2055-0278},
support = {R37 GM060398/GM/NIGMS NIH HHS/United States ; R01 GM094428/GM/NIGMS NIH HHS/United States ; R01 GM052413/GM/NIGMS NIH HHS/United States ; T32 GM007240/GM/NIGMS NIH HHS/United States ; R01 GM060398/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/*metabolism ; Arabidopsis Proteins/*metabolism ; Argonaute Proteins/metabolism ; *DNA Methylation ; DNA-Directed RNA Polymerases/*metabolism ; *Gene Silencing ; Genome-Wide Association Study ; RNA, Small Nuclear/*metabolism ; Transcription, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.},
}
@article {pmid29371426,
year = {2018},
author = {Gu, B and Swigut, T and Spencley, A and Bauer, MR and Chung, M and Meyer, T and Wysocka, J},
title = {Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements.},
journal = {Science (New York, N.Y.)},
volume = {359},
number = {6379},
pages = {1050-1055},
pmid = {29371426},
issn = {1095-9203},
support = {T32 CA009302/CA/NCI NIH HHS/United States ; R01 GM112720/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P50 GM107615/GM/NIGMS NIH HHS/United States ; S10 OD018073/OD/NIH HHS/United States ; R35 GM127026/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins ; CRISPR-Associated Protein 9 ; Cell Line ; Cell Nucleus/genetics ; Endonucleases ; Mice ; Oligonucleotide Array Sequence Analysis ; RNA Polymerase II/metabolism ; *Regulatory Sequences, Nucleic Acid ; Single Molecule Imaging/*methods ; Single-Cell Analysis/*methods ; *Transcription, Genetic ; Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.},
}
@article {pmid29298376,
year = {2018},
author = {Hardcastle, T and Novosjolova, I and Kotikam, V and Cheruiyot, SK and Mutisya, D and Kennedy, SD and Egli, M and Kelley, ML and Smith, AVB and Rozners, E},
title = {A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs.},
journal = {ACS chemical biology},
volume = {13},
number = {3},
pages = {533-536},
pmid = {29298376},
issn = {1554-8937},
support = {R01 GM071461/GM/NIGMS NIH HHS/United States ; },
mesh = {Amides/*metabolism ; Argonaute Proteins ; Humans ; Models, Molecular ; Protein Conformation ; RNA Interference ; RNA, Small Interfering/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.},
}
@article {pmid29272705,
year = {2017},
author = {Chen, GR and Sive, H and Bartel, DP},
title = {A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially Rescues Severely Impaired Cleavage Found in Fish.},
journal = {Molecular cell},
volume = {68},
number = {6},
pages = {1095-1107.e5},
pmid = {29272705},
issn = {1097-4164},
support = {R01 GM061835/GM/NIGMS NIH HHS/United States ; R35 GM118135/GM/NIGMS NIH HHS/United States ; R37 GM061835/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Argonaute Proteins/genetics/*metabolism ; *Base Pair Mismatch ; Gene Knockdown Techniques ; Humans ; MicroRNAs/genetics/*metabolism ; *RNA Interference ; RNA, Messenger/*genetics/metabolism ; RNA-Induced Silencing Complex/genetics/metabolism ; Zebrafish/*genetics/physiology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The RNAi pathway provides both innate immunity and efficient gene-knockdown tools in many eukaryotic species, but curiously not in zebrafish. We discovered that RNAi is less effective in zebrafish at least partly because Argonaute2-catalyzed mRNA slicing is impaired. This defect is due to two mutations that arose in an ancestor of most teleost fish, implying that most fish lack effective RNAi. Despite lacking efficient slicing activity, these fish have retained the ability to produce miR-451, a microRNA generated by a cleavage reaction analogous to slicing. This ability is due to a G-G mismatch within the fish miR-451 precursor, which substantially enhances its cleavage. An analogous G-G mismatch (or sometimes also a G-A mismatch) enhances target slicing, despite disrupting seed pairing important for target binding. These results provide a strategy for restoring RNAi to zebrafish and reveal unanticipated opposing effects of a seed mismatch with implications for mechanism and guide-RNA design.},
}
@article {pmid29195056,
year = {2018},
author = {Kim, Y and Kang, YG and Choe, JY and Lee, D and Shin, C and Hong, SW and Lee, DK},
title = {RNA Interference-Mediated Gene Silencing by Branched Tripodal RNAs Does Not Require Dicer Processing.},
journal = {Nucleic acid therapeutics},
volume = {28},
number = {1},
pages = {44-49},
doi = {10.1089/nat.2017.0681},
pmid = {29195056},
issn = {2159-3345},
mesh = {Argonaute Proteins/*genetics/metabolism ; Base Sequence ; Cell Line ; DEAD-box RNA Helicases/deficiency/*genetics ; Genes, Reporter ; HeLa Cells ; Humans ; Luciferases/antagonists &amp; inhibitors/genetics/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; MicroRNAs/genetics/metabolism ; Nucleic Acid Conformation ; Proto-Oncogene Proteins c-met/antagonists &amp; inhibitors/genetics/metabolism ; *RNA Interference ; RNA, Small Interfering/*genetics/metabolism ; Ribonuclease III/deficiency/*genetics ; Survivin/antagonists &amp; inhibitors/genetics/metabolism ; beta Catenin/antagonists &amp; inhibitors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Specific gene silencing through RNA interference (RNAi) holds great promise as the next-generation therapeutic development platform. Previously, we have shown that branched, tripodal interfering RNA (tiRNA) structures could simultaneously trigger RNAi-mediated gene silencing of three target genes with 38 nt-long guide strands associated with Argonaute 2. Herein, we show that the branched RNA structure can trigger effective gene silencing in Dicer knockout cell line, demonstrating that the Dicer-mediated processing is not required for tiRNA activity. The finding of this study confirms the flexibility of the structure of RNAi triggers as well as the length of the guide strand in RNAi-mediated gene silencing.},
}
@article {pmid29177505,
year = {2018},
author = {Caton, EA and Kelly, EK and Kamalampeta, R and Kothe, U},
title = {Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA.},
journal = {Nucleic acids research},
volume = {46},
number = {2},
pages = {905-916},
pmid = {29177505},
issn = {1362-4962},
mesh = {Algorithms ; Base Sequence ; Binding, Competitive ; Kinetics ; Nuclear Proteins/genetics/metabolism ; Protein Binding ; Pseudouridine/*metabolism ; RNA/genetics/*metabolism ; RNA, Small Nucleolar/genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; Ribonucleoproteins/genetics/*metabolism ; Ribonucleoproteins, Small Nucleolar/genetics/metabolism ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p-Nop10p-Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.},
}
@article {pmid29099293,
year = {2018},
author = {Vallecillo-Viejo, IC and Liscovitch-Brauer, N and Montiel-Gonzalez, MF and Eisenberg, E and Rosenthal, JJC},
title = {Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.},
journal = {RNA biology},
volume = {15},
number = {1},
pages = {104-114},
pmid = {29099293},
issn = {1555-8584},
support = {R01 NS087726/NS/NINDS NIH HHS/United States ; },
mesh = {Adenosine/genetics ; Codon, Nonsense/genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Epithelial Cells/metabolism ; Humans ; Inosine/genetics ; *Mutagenesis, Site-Directed ; Mutation/genetics ; RNA Editing/*genetics ; Transcriptome/*genetics ; Transfection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.},
}
@article {pmid29042431,
year = {2017},
author = {Lawson, MJ and Camsund, D and Larsson, J and Baltekin, &#xd6; and Fange, D and Elf, J},
title = {In situ genotyping of a pooled strain library after characterizing complex phenotypes.},
journal = {Molecular systems biology},
volume = {13},
number = {10},
pages = {947},
pmid = {29042431},
issn = {1744-4292},
mesh = {Escherichia coli/*classification/*genetics ; Escherichia coli Proteins/genetics ; Gene Library ; Genotype ; Genotyping Techniques/*methods ; Microfluidic Analytical Techniques ; Phenotype ; Single Molecule Imaging/methods ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.},
}
@article {pmid28984845,
year = {2017},
author = {Azad, MTA and Bhakta, S and Tsukahara, T},
title = {Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy.},
journal = {Gene therapy},
volume = {24},
number = {12},
pages = {779-786},
pmid = {28984845},
issn = {1476-5462},
mesh = {Adenosine/genetics ; Adenosine Deaminase/*metabolism ; Alleles ; Blotting, Western ; Codon, Terminator ; *Genetic Code ; *Genetic Therapy ; Green Fluorescent Proteins/genetics ; Humans ; Inosine/genetics ; Point Mutation ; *RNA Editing ; RNA-Binding Proteins/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-directed RNA editing is an important technique for correcting gene sequences and ultimately tuning protein function. In this study, we engineered the deaminase domain of adenosine deaminase acting on RNA (ADAR1) and the MS2 system to target-specific adenosines, with the goal of correcting G-to-A mutations at the RNA level. For this purpose, the ADAR1 deaminase domain was fused downstream of the RNA-binding protein MS2, which has affinity for the MS2 RNA. To direct editing to specific targets, we designed guide RNAs complementary to target RNAs. The guide RNAs directed the ADAR1 deaminase to the desired editing site, where it converted adenosine to inosine. To provide proof of principle, we used an allele of enhanced green fluorescent protein (EGFP) bearing a mutation at the 58th amino acid (TGG), encoding Trp, into an amber (TAG) or ochre (TAA) stop codon. In HEK-293 cells, our system could convert stop codons to read-through codons, thereby turning on fluorescence. We confirmed the specificity of editing at the DNA level by restriction fragment length polymorphism analysis and sequencing, and at the protein level by western blotting. The editing efficiency of this enzyme system was ~5%. We believe that this system could be used to treat genetic diseases resulting from G-to-A point mutations.},
}
@article {pmid28939659,
year = {2018},
author = {Klum, SM and Chandradoss, SD and Schirle, NT and Joo, C and MacRae, IJ},
title = {Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition.},
journal = {The EMBO journal},
volume = {37},
number = {1},
pages = {75-88},
pmid = {28939659},
issn = {1460-2075},
support = {309509/ERC_/European Research Council/International ; R01 GM104475/GM/NIGMS NIH HHS/United States ; R01 GM115649/GM/NIGMS NIH HHS/United States ; },
mesh = {Argonaute Proteins/*chemistry/genetics/*metabolism ; Crystallography, X-Ray ; Humans ; MicroRNAs/genetics/*metabolism ; *Models, Biological ; Protein Binding ; Protein Conformation ; RNA, Messenger/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins use microRNAs (miRNAs) to identify mRNAs targeted for post-transcriptional repression. Biochemical assays have demonstrated that Argonaute functions by modulating the binding properties of its miRNA guide so that pairing to the seed region is exquisitely fast and accurate. However, the mechanisms used by Argonaute to reshape the binding properties of its small RNA guide remain poorly understood. Here, we identify a structural element, α-helix-7, in human Argonaute2 (Ago2) that is required for speed and fidelity in binding target RNAs. Biochemical, structural, and single-molecule data indicate that helix-7 acts as a molecular wedge that pivots to enforce rapid making and breaking of miRNA:target base pairs in the 3' half of the seed region. These activities allow Ago2 to rapidly dismiss off-targets and dynamically search for seed-matched sites at a rate approaching the limit of diffusion.},
}
@article {pmid28879853,
year = {2018},
author = {Brocken, DJW and Tark-Dame, M and Dame, RT},
title = {dCas9: A Versatile Tool for Epigenome Editing.},
journal = {Current issues in molecular biology},
volume = {26},
number = {},
pages = {15-32},
doi = {10.21775/cimb.026.015},
pmid = {28879853},
issn = {1467-3045},
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9 ; Chromatin/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics/metabolism ; DNA Methylation ; Endonucleases/*genetics/metabolism ; Epigenesis, Genetic ; Gene Editing/*methods ; *Genome, Human ; Histones/genetics/metabolism ; Humans ; Transcription Activator-Like Effectors/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Zinc Fingers ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The epigenome is a heritable layer of information not encoded in the DNA sequence of the genome, but in chemical modifications of DNA or histones. These chemical modifications, together with transcription factors, operate as spatiotemporal regulators of genome activity. Dissecting epigenome function requires controlled site-specific alteration of epigenetic information. Such control can be obtained using designed DNA-binding platforms associated with effector domains to function as targeted transcription factors or epigenetic modifiers. Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation, and the potential of this approach in basic research in these fields.},
}
@article {pmid28781232,
year = {2017},
author = {Elkayam, E and Faehnle, CR and Morales, M and Sun, J and Li, H and Joshua-Tor, L},
title = {Multivalent Recruitment of Human Argonaute by GW182.},
journal = {Molecular cell},
volume = {67},
number = {4},
pages = {646-658.e3},
pmid = {28781232},
issn = {1097-4164},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; R01 GM111742/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Argonaute Proteins/chemistry/genetics/*metabolism ; Autoantigens/chemistry/genetics/*metabolism ; Binding Sites ; Binding, Competitive ; Crystallography, X-Ray ; Eukaryotic Initiation Factors/chemistry/genetics/*metabolism ; *Gene Silencing ; Humans ; MicroRNAs/chemistry/genetics/*metabolism ; Molecular Docking Simulation ; Mutation ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation ; Protein Interaction Domains and Motifs ; RNA-Binding Proteins/chemistry/genetics/*metabolism ; Sf9 Cells ; Structure-Activity Relationship ; Transfection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In miRNA-mediated gene silencing, the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of the targeted mRNA. GW182 can interact with hAgo via three of the GW/WG repeats in its Argonaute-binding domain: motif-1, motif-2, and the hook motif. The structure of hAgo1 in complex with the hook motif of hGW182 reveals a "gate"-like interaction that is critical for GW182 docking into one of hAgo1's tryptophan-binding pockets. We show that hAgo1 and hAgo2 have a single GW182-binding site and that miRNA binding increases hAgo's affinity to GW182. With target binding occurring rapidly, this ensures that only mature RISC would be recruited for silencing. Finally, we show that hGW182 can recruit up to three copies of hAgo via its three GW motifs. This may explain the observed cooperativity in miRNA-mediated gene silencing.},
}
@article {pmid28780643,
year = {2017},
author = {Wang, Z and Zhu, J},
title = {MEMOIR: A Novel System for Neural Lineage Tracing.},
journal = {Neuroscience bulletin},
volume = {33},
number = {6},
pages = {763-765},
pmid = {28780643},
issn = {1995-8218},
mesh = {Animals ; CRISPR-Associated Protein 9 ; Cell Lineage ; Humans ; In Situ Hybridization, Fluorescence/*methods ; Mice ; Mutagenesis ; Neural Stem Cells/*cytology ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/methods ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid28777932,
year = {2017},
author = {Betts, JA and Moradi Marjaneh, M and Al-Ejeh, F and Lim, YC and Shi, W and Sivakumaran, H and Tropée, R and Patch, AM and Clark, MB and Bartonicek, N and Wiegmans, AP and Hillman, KM and Kaufmann, S and Bain, AL and Gloss, BS and Crawford, J and Kazakoff, S and Wani, S and Wen, SW and Day, B and Möller, A and Cloonan, N and Pearson, J and Brown, MA and Mercer, TR and Waddell, N and Khanna, KK and Dray, E and Dinger, ME and Edwards, SL and French, JD},
title = {Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.},
journal = {American journal of human genetics},
volume = {101},
number = {2},
pages = {255-266},
pmid = {28777932},
issn = {1537-6605},
mesh = {Breast Neoplasms/*genetics ; Cell Line, Tumor ; Chromatin/metabolism ; Chromosomes, Human, Pair 11/*genetics ; Cyclin D1/*genetics ; DNA Breaks, Double-Stranded ; DNA Damage/genetics ; DNA Repair/*genetics ; Enhancer Elements, Genetic/genetics ; Estrogens/metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease/genetics ; Humans ; MCF-7 Cells ; Polymorphism, Single Nucleotide/genetics ; Promoter Regions, Genetic/genetics ; RNA Interference ; RNA, Long Noncoding/*genetics ; RNA, Small Interfering/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.},
}
@article {pmid28751638,
year = {2017},
author = {Xiong, T and Meister, GE and Workman, RE and Kato, NC and Spellberg, MJ and Turker, F and Timp, W and Ostermeier, M and Novina, CD},
title = {Targeted DNA methylation in human cells using engineered dCas9-methyltransferases.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {6732},
pmid = {28751638},
issn = {2045-2322},
support = {DP1 DK105602/DK/NIDDK NIH HHS/United States ; },
mesh = {Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9/*genetics/metabolism ; CpG Islands ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; *DNA Methylation ; Escherichia coli/genetics/metabolism ; Gene Editing/*methods ; Genetic Vectors/chemistry/metabolism ; HEK293 Cells ; Humans ; Kinetics ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Engineering/*methods ; Protein Interaction Domains and Motifs ; Recombinant Fusion Proteins/genetics/metabolism ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.},
}
@article {pmid28712504,
year = {2017},
author = {Weeks, DP and Yang, B},
title = {Preface.},
journal = {Progress in molecular biology and translational science},
volume = {149},
number = {},
pages = {xiii-xiv},
doi = {10.1016/S1877-1173(17)30107-2},
pmid = {28712504},
issn = {1878-0814},
mesh = {Animals ; *Gene Editing ; Mice ; Plants/genetics ; Plants, Genetically Modified ; RNA, Catalytic/metabolism ; Stress, Physiological/genetics ; Zinc Finger Nucleases ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid28592872,
year = {2017},
author = {Hu, YW and Wu, XM and Ren, SS and Cao, L and Nie, P and Chang, MX},
title = {NOD1 deficiency impairs CD44a/Lck as well as PI3K/Akt pathway.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {2979},
pmid = {28592872},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; Computational Biology/methods ; Gene Expression ; Gene Expression Profiling ; Gene Knockout Techniques ; Genes, MHC Class I ; Histocompatibility Antigens Class II/genetics ; Hyaluronan Receptors/genetics/*metabolism ; Immune System/immunology/metabolism ; Larva ; Models, Biological ; Nod1 Signaling Adaptor Protein/*deficiency ; Phosphatidylinositol 3-Kinases/*metabolism ; Proto-Oncogene Proteins c-akt/*metabolism ; *Signal Transduction ; Zebrafish ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Pattern recognition receptors (PRRs) are crucial for host defense and tissue homeostasis against infecting pathogens. PRRs are highly conserved cross species, suggesting their key roles in fundamental biological processes. Though much have been learned for NOD1 receptor in the innate and adaptive immune responses, the roles of NOD1 during embryonic and larval stages remain poorly understood. Here, we report that NOD1 is necessary for the modulation of PI3K-Akt pathway and larval survival in zebrafish. Transcriptome analysis revealed that the significantly enriched pathways in NOD1 [-/-] zebrafish larvae were mainly involved in metabolism and immune system processes. Biochemical analysis demonstrated that NOD1 was required for the expression of CD44a that, in turn, activated the PI3K-Akt pathway during larval development. Conversely, over-expression of CD44a in NOD1-deficient zebrafish restored the modulation of the PI3K-Akt pathway and improved larval survival. Collectively, our work indicates that NOD1 plays a previously undetected protective role in larval survival through CD44a-mediated activation of the PI3K-Akt signaling.},
}
@article {pmid28591791,
year = {2017},
author = {Haraszti, RA and Roux, L and Coles, AH and Turanov, AA and Alterman, JF and Echeverria, D and Godinho, BMDC and Aronin, N and Khvorova, A},
title = {5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.},
journal = {Nucleic acids research},
volume = {45},
number = {13},
pages = {7581-7592},
pmid = {28591791},
issn = {1362-4962},
support = {R01 GM108803/GM/NIGMS NIH HHS/United States ; S10 OD020012/OD/NIH HHS/United States ; },
mesh = {Animals ; Exoribonucleases/metabolism ; Female ; Gene Silencing ; HeLa Cells ; Humans ; Hydrophobic and Hydrophilic Interactions ; Kidney/metabolism ; Liver/metabolism ; Mice ; Models, Molecular ; Nucleic Acid Conformation ; Organophosphonates/*pharmacology ; Phosphorylation ; RNA Stability/drug effects ; RNA, Small Interfering/chemistry/genetics/*metabolism ; RNA-Induced Silencing Complex/chemistry/genetics/metabolism ; Tissue Distribution ; Vinyl Compounds/*pharmacology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {5΄-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5΄-vinylphosphonate confers novel properties to siRNAs. Specifically, 5΄-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5΄-to-3΄ exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5΄-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5΄-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5΄-phosphate is removed within 2 hours. The 5΄-phosphate mimic 5΄-(E)-vinylphosphonate stabilizes the 5΄ end of the guide strand by protecting it from phosphatases and 5΄-to-3΄ exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5΄-(E)-vinylphosphonate stabilizes 5΄ phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.},
}
@article {pmid28432181,
year = {2017},
author = {Deryusheva, S and Gall, JG},
title = {Dual nature of pseudouridylation in U2 snRNA: Pus1p-dependent and Pus1p-independent activities in yeasts and higher eukaryotes.},
journal = {RNA (New York, N.Y.)},
volume = {23},
number = {7},
pages = {1060-1067},
pmid = {28432181},
issn = {1469-9001},
support = {R01 GM033397/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Caenorhabditis elegans/genetics/metabolism ; Drosophila melanogaster/genetics/metabolism ; Gene Knockout Techniques ; Intramolecular Transferases/genetics/*metabolism ; Mice ; Pseudouridine/metabolism ; RNA, Fungal/metabolism ; RNA, Small Nuclear/chemistry/*metabolism ; Schizosaccharomyces/genetics/metabolism ; Xenopus/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved post-transcriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast Saccharomyces cerevisiae and mouse. Furthermore, in S. cerevisiae, Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system, we tested enzymatic activity of Pus1p from the fission yeast Schizosaccharomyces pombe, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the frog Xenopus tropicalis We demonstrated that Pus1p from C. elegans has no enzymatic activity on U2 snRNA when expressed in yeast cells, whereas in similar experiments, position 44 in yeast U2 snRNA (equivalent to position 43 in vertebrates) is a genuine substrate for Pus1p from S. cerevisiae, S. pombe, Drosophila, Xenopus, and mouse. However, when we analyzed U2 snRNAs from Pus1 knockout mice and the pus1Δ S. pombe strain, we could not detect any changes in their modification patterns when compared to wild-type U2 snRNAs. In S. pombe, we found a novel box H/ACA RNA encoded downstream from the RPC10 gene and experimentally verified its guide RNA activity for positioning Ψ43 and Ψ44 in U2 snRNA. In vertebrates, we showed that SCARNA8 (also known as U92 scaRNA) is a guide for U2-Ψ43 in addition to its previously established targets U2-Ψ34/Ψ44.},
}
@article {pmid28379609,
year = {2017},
author = {Li, C and Liu, C and Qi, X and Wu, Y and Fei, X and Mao, L and Cheng, B and Li, X and Xie, C},
title = {RNA-guided Cas9 as an in vivo desired-target mutator in maize.},
journal = {Plant biotechnology journal},
volume = {15},
number = {12},
pages = {1566-1576},
pmid = {28379609},
issn = {1467-7652},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Protein 9 ; Endonucleases/genetics ; Gene Editing ; Inheritance Patterns ; *Mutation ; Mutation Rate ; Plant Breeding/*methods ; Plants, Genetically Modified/genetics ; Sequence Analysis, DNA ; Zea mays/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.},
}
@article {pmid28298224,
year = {2017},
author = {Jensen, ED and Ferreira, R and Jakočiūnas, T and Arsovska, D and Zhang, J and Ding, L and Smith, JD and David, F and Nielsen, J and Jensen, MK and Keasling, JD},
title = {Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies.},
journal = {Microbial cell factories},
volume = {16},
number = {1},
pages = {46},
pmid = {28298224},
issn = {1475-2859},
mesh = {Biosynthetic Pathways/genetics ; CRISPR-Associated Proteins/genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/genetics/*metabolism ; Promoter Regions, Genetic ; Saccharomyces cerevisiae/*genetics ; Synthetic Biology/methods ; Terpenes/metabolism ; *Transcription, Genetic ; Triglycerides/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.<br><br>RESULTS: In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.<br><br>CONCLUSION: Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.},
}
@article {pmid28231446,
year = {2017},
author = {},
title = {How Will Kinetics and Thermodynamics Inform Our Future Efforts to Understand and Build Biological Systems?.},
journal = {Cell systems},
volume = {4},
number = {2},
pages = {144-146},
doi = {10.1016/j.cels.2017.02.005},
pmid = {28231446},
issn = {2405-4712},
mesh = {Cell-Free System ; Kinetics ; Machine Learning ; *Models, Biological ; Thermodynamics ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid28148949,
year = {2017},
author = {Fukuda, M and Umeno, H and Nose, K and Nishitarumizu, A and Noguchi, R and Nakagawa, H},
title = {Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {41478},
pmid = {28148949},
issn = {2045-2322},
mesh = {Adenosine/*metabolism ; Adenosine Deaminase/chemistry/metabolism ; Base Sequence ; Codon/genetics ; HEK293 Cells ; Humans ; Inosine/*metabolism ; Intracellular Space/*metabolism ; *Mutagenesis, Site-Directed ; Protein Domains ; RNA Editing/*genetics ; RNA-Binding Proteins/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.},
}
@article {pmid28144031,
year = {2017},
author = {Lewis, CJ and Pan, T and Kalsotra, A},
title = {RNA modifications and structures cooperate to guide RNA-protein interactions.},
journal = {Nature reviews. Molecular cell biology},
volume = {18},
number = {3},
pages = {202-210},
pmid = {28144031},
issn = {1471-0080},
support = {R01 GM113194/GM/NIGMS NIH HHS/United States ; R01 HL126845/HL/NHLBI NIH HHS/United States ; },
mesh = {Nucleic Acid Conformation ; Protein Biosynthesis ; Protein Splicing ; Proteins/*genetics/metabolism ; RNA Stability ; RNA, Messenger/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {An emerging body of evidence indicates that post-transcriptional gene regulation relies not only on the sequence of mRNAs but also on their folding into intricate secondary structures and on the chemical modifications of the RNA bases. These features, which are highly dynamic and interdependent, exert direct control over the transcriptome and thereby influence many aspects of cell function. Here, we consider how the coupling of RNA modifications and structures shapes RNA-protein interactions at different steps of the gene expression process.},
}
@article {pmid28139645,
year = {2017},
author = {Ren, R and Deng, L and Xue, Y and Suzuki, K and Zhang, W and Yu, Y and Wu, J and Sun, L and Gong, X and Luan, H and Yang, F and Ju, Z and Ren, X and Wang, S and Tang, H and Geng, L and Zhang, W and Li, J and Qiao, J and Xu, T and Qu, J and Liu, GH},
title = {Visualization of aging-associated chromatin alterations with an engineered TALE system.},
journal = {Cell research},
volume = {27},
number = {4},
pages = {483-504},
pmid = {28139645},
issn = {1748-7838},
support = {K99 CA158055/CA/NCI NIH HHS/United States ; R00 CA158055/CA/NCI NIH HHS/United States ; P30 CA086862/CA/NCI NIH HHS/United States ; R01 CA200673/CA/NCI NIH HHS/United States ; R01 CA203834/CA/NCI NIH HHS/United States ; },
mesh = {Aging/*genetics ; Animals ; CRISPR-Associated Proteins/metabolism ; Cell Differentiation ; Cell Line ; Cellular Senescence ; Centromere/metabolism ; Chromatin/*genetics ; DNA, Ribosomal/metabolism ; *Genetic Engineering ; Genetic Loci ; Humans ; Imaging, Three-Dimensional ; Mesenchymal Stem Cells/cytology ; Mice, Inbred C57BL ; Mitosis ; Nucleolus Organizer Region/genetics ; Oocytes/cytology ; Repetitive Sequences, Nucleic Acid/genetics ; Telomerase/metabolism ; Telomere/metabolism ; Thioredoxins/metabolism ; Transcription Activator-Like Effector Nucleases/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Visualization of specific genomic loci in live cells is a prerequisite for the investigation of dynamic changes in chromatin architecture during diverse biological processes, such as cellular aging. However, current precision genomic imaging methods are hampered by the lack of fluorescent probes with high specificity and signal-to-noise contrast. We find that conventional transcription activator-like effectors (TALEs) tend to form protein aggregates, thereby compromising their performance in imaging applications. Through screening, we found that fusing thioredoxin with TALEs prevented aggregate formation, unlocking the full power of TALE-based genomic imaging. Using thioredoxin-fused TALEs (TTALEs), we achieved high-quality imaging at various genomic loci and observed aging-associated (epi) genomic alterations at telomeres and centromeres in human and mouse premature aging models. Importantly, we identified attrition of ribosomal DNA repeats as a molecular marker for human aging. Our study establishes a simple and robust imaging method for precisely monitoring chromatin dynamics in vitro and in vivo.},
}
@article {pmid28072976,
year = {2017},
author = {Bian, XC and Yang, ZL and Feng, HL and Zhao, XM and Gu, B and Li, J and Sun, H and Liu, YQ},
title = {[Establishment and validation of human cancer cell lines with stable Cas9 expression].},
journal = {Zhonghua bing li xue za zhi = Chinese journal of pathology},
volume = {46},
number = {1},
pages = {43-48},
doi = {10.3760/cma.j.issn.0529-5807.2017.01.010},
pmid = {28072976},
issn = {0529-5807},
mesh = {CRISPR-Associated Proteins/*metabolism ; Cell Line, Tumor/*metabolism ; *Gene Editing ; Humans ; Neoplasm Proteins/*metabolism ; Repressor Proteins/genetics ; Transfection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions: Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.},
}
@article {pmid28031372,
year = {2017},
author = {Gumienny, R and Jedlinski, DJ and Schmidt, A and Gypas, F and Martin, G and Vina-Vilaseca, A and Zavolan, M},
title = {High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq.},
journal = {Nucleic acids research},
volume = {45},
number = {5},
pages = {2341-2353},
pmid = {28031372},
issn = {1362-4962},
mesh = {*Algorithms ; Base Sequence ; Cross-Linking Reagents/chemistry ; Databases, Genetic ; High-Throughput Nucleotide Sequencing/*methods ; Immunoprecipitation ; Methylation ; Protein Binding ; RNA, Ribosomal, 18S/genetics/metabolism ; RNA, Ribosomal, 28S/genetics/metabolism ; RNA, Small Nucleolar/*genetics/metabolism ; Ribonucleoproteins, Small Nucleolar/*genetics/metabolism ; Ribose/metabolism ; Software ; *Transcriptome ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {High-throughput sequencing has greatly facilitated the discovery of long and short non-coding RNAs (ncRNAs), which frequently guide ribonucleoprotein complexes to RNA targets, to modulate their metabolism and expression. However, for many ncRNAs, the targets remain to be discovered. In this study, we developed computational methods to map C/D box snoRNA target sites using data from core small nucleolar ribonucleoprotein crosslinking and immunoprecipitation and from transcriptome-wide mapping of 2΄-O-ribose methylation sites. We thereby assigned the snoRNA guide to a known methylation site in the 18S rRNA, we uncovered a novel partially methylated site in the 28S ribosomal RNA, and we captured a site in the 28S rRNA in interaction with multiple snoRNAs. Although we also captured mRNAs in interaction with snoRNAs, we did not detect 2΄-O-methylation of these targets. Our study provides an integrated approach to the comprehensive characterization of 2΄-O-methylation targets of snoRNAs in species beyond those in which these interactions have been traditionally studied and contributes to the rapidly developing field of 'epitranscriptomics'.},
}
@article {pmid27907896,
year = {2017},
author = {Wettengel, J and Reautschnig, P and Geisler, S and Kahle, PJ and Stafforst, T},
title = {Harnessing human ADAR2 for RNA repair - Recoding a PINK1 mutation rescues mitophagy.},
journal = {Nucleic acids research},
volume = {45},
number = {5},
pages = {2797-2808},
pmid = {27907896},
issn = {1362-4962},
mesh = {Adenosine Deaminase/*metabolism ; Codon, Nonsense ; Genome ; HEK293 Cells ; HeLa Cells ; Humans ; *Mitophagy ; *Point Mutation ; Protein Kinases/*genetics ; *RNA Editing ; RNA, Messenger/metabolism ; RNA-Binding Proteins/*metabolism ; Transfection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-directed A-to-I RNA editing is a technology for re-programming genetic information at the RNA-level. We describe here the first design of genetically encodable guideRNAs that enable the re-addressing of human ADAR2 toward specific sites in user-defined mRNA targets. Up to 65% editing yield has been achieved in cell culture for the recoding of a premature Stop codon (UAG) into tryptophan (UIG). In the targeted gene, editing was very specific. We applied the technology to recode a recessive loss-of-function mutation in PINK1 (W437X) in HeLa cells and showed functional rescue of PINK1/Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. In contrast to other editing strategies, this approach requires no artificial protein. Our novel guideRNAs may allow for the development of a platform technology that requires only the administration or expression of a guideRNA to recode genetic information, with high potential for application in biology and medicine.},
}
@article {pmid27903888,
year = {2017},
author = {Elkayam, E and Parmar, R and Brown, CR and Willoughby, JL and Theile, CS and Manoharan, M and Joshua-Tor, L},
title = {siRNA carrying an (E)-vinylphosphonate moiety at the 5΄ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2.},
journal = {Nucleic acids research},
volume = {45},
number = {6},
pages = {3528-3536},
pmid = {27903888},
issn = {1362-4962},
support = {P30 CA045508/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Argonaute Proteins/*chemistry/*metabolism ; Binding Sites ; Humans ; Mice ; Models, Molecular ; Organophosphonates/*chemistry ; *RNA Interference ; RNA, Small Interfering/*chemistry/*metabolism ; Receptors, Albumin/genetics/metabolism ; Vinyl Compounds/*chemistry ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5΄- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5΄- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5΄-(E)-vinylphosphonate (5΄-E-VP) guide RNA at 2.5-Å resolution. The structure demonstrates how the 5΄ binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5΄-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5΄-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.},
}
@article {pmid27851737,
year = {2016},
author = {Hayashi, R and Schnabl, J and Handler, D and Mohn, F and Ameres, SL and Brennecke, J},
title = {Genetic and mechanistic diversity of piRNA 3'-end formation.},
journal = {Nature},
volume = {539},
number = {7630},
pages = {588-592},
pmid = {27851737},
issn = {1476-4687},
support = {338252/ERC_/European Research Council/International ; W 1207/FWF_/Austrian Science Fund FWF/Austria ; Y 733/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Animals ; Argonaute Proteins/metabolism ; Cytoplasm/metabolism ; Drosophila Proteins/deficiency/*metabolism ; Drosophila melanogaster/enzymology/*genetics/metabolism ; Endoribonucleases/deficiency/metabolism ; Exoribonucleases/deficiency/metabolism ; Female ; Nuclear Proteins/metabolism ; Peptide Initiation Factors/metabolism ; RNA Processing, Post-Transcriptional ; RNA, Small Interfering/*biosynthesis/chemistry/*genetics/metabolism ; Transcription, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD-a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. Formation of piRNA 3' ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3'-end formation.},
}
@article {pmid27741323,
year = {2016},
author = {Willkomm, S and Zander, A and Grohmann, D and Restle, T},
title = {Mechanistic Insights into Archaeal and Human Argonaute Substrate Binding and Cleavage Properties.},
journal = {PloS one},
volume = {11},
number = {10},
pages = {e0164695},
pmid = {27741323},
issn = {1932-6203},
mesh = {Argonaute Proteins/chemistry/*metabolism ; DNA/metabolism ; DNA Cleavage ; Humans ; Kinetics ; Methanocaldococcus/genetics/*metabolism ; Oligonucleotides/chemistry/metabolism ; Protein Binding ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation.},
}
@article {pmid27625427,
year = {2016},
author = {Yang, Z and Lin, J and Ye, K},
title = {Box C/D guide RNAs recognize a maximum of 10 nt of substrates.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {113},
number = {39},
pages = {10878-10883},
pmid = {27625427},
issn = {1091-6490},
mesh = {Animals ; Arabidopsis/metabolism ; Archaea/metabolism ; Base Sequence ; Humans ; Mice ; Nucleotides/*metabolism ; Oryza/genetics ; Ribonucleoproteins/chemistry/metabolism ; Saccharomyces cerevisiae/metabolism ; Schizosaccharomyces/metabolism ; Static Electricity ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D' and boxes C' to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide-substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C' side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.},
}
@article {pmid27557710,
year = {2016},
author = {Montiel-González, MF and Vallecillo-Viejo, IC and Rosenthal, JJ},
title = {An efficient system for selectively altering genetic information within mRNAs.},
journal = {Nucleic acids research},
volume = {44},
number = {21},
pages = {e157},
pmid = {27557710},
issn = {1362-4962},
support = {P20 GM103642/GM/NIGMS NIH HHS/United States ; R01 NS064259/NS/NINDS NIH HHS/United States ; R01 NS087726/NS/NINDS NIH HHS/United States ; G12 MD007600/MD/NIMHD NIH HHS/United States ; },
mesh = {Adenosine Deaminase/chemistry/genetics ; Calibration ; Codon, Nonsense ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Genes, Reporter ; *Genetic Techniques ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; Humans ; Mutation ; Peptides/genetics ; Pichia/genetics ; Protein Domains ; *RNA Editing ; RNA, Messenger/*genetics ; RNA-Binding Proteins/chemistry/genetics ; Tryptophan/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A: G premature termination codon (PTC) to tryptophan (U G: G) was improved from ∼11 % to ∼70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.},
}
@article {pmid27546791,
year = {2016},
author = {Cuella-Martin, R and Oliveira, C and Lockstone, HE and Snellenberg, S and Grolmusova, N and Chapman, JR},
title = {53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms.},
journal = {Molecular cell},
volume = {64},
number = {1},
pages = {51-64},
pmid = {27546791},
issn = {1097-4164},
support = {19270/CRUK_/Cancer Research UK/United Kingdom ; C52690/A19270/CRUK_/Cancer Research UK/United Kingdom ; C5255/A18085/CRUK_/Cancer Research UK/United Kingdom ; 090532/Z/09/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Bacterial Proteins/genetics/metabolism ; Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9 ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Breaks, Double-Stranded ; *DNA Repair ; Endonucleases/genetics/metabolism ; Gamma Rays ; Gene Editing ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Promoter Regions, Genetic ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Signal Transduction ; Tumor Suppressor Protein p53/chemistry/*genetics/metabolism ; Tumor Suppressor p53-Binding Protein 1/chemistry/*genetics/metabolism ; Ubiquitin Thiolesterase/chemistry/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice.},
}
@article {pmid27325485,
year = {2016},
author = {Miyoshi, T and Ito, K and Murakami, R and Uchiumi, T},
title = {Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {11846},
pmid = {27325485},
issn = {2041-1723},
mesh = {Argonaute Proteins/*chemistry/genetics/*metabolism ; Bacterial Proteins/chemistry/genetics/metabolism ; Base Pairing ; Base Sequence ; Binding Sites ; Crystallography, X-Ray ; DNA/chemistry/genetics/metabolism ; Escherichia coli/genetics ; Gene Expression Regulation, Bacterial ; Gene Silencing/*physiology ; Models, Molecular ; Nucleic Acid Conformation ; Nucleic Acid Heteroduplexes/*chemistry/*metabolism ; Protein Conformation ; Protein Domains ; Protein Interaction Maps ; Recombinant Proteins ; Rhodobacter sphaeroides/genetics/*metabolism ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.},
}
@article {pmid27285342,
year = {2016},
author = {Haino, A and Ishikawa, T and Seki, M and Nashimoto, M},
title = {TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {112},
pages = {},
pmid = {27285342},
issn = {1940-087X},
mesh = {Apoptosis ; Gene Library ; *Gene Silencing ; Humans ; Neoplasms ; Transfection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {TRUE gene silencing (termed after tRNase Z(L)-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3' processing endoribonuclease, tRNase Z(L), to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.},
}
@article {pmid27140916,
year = {2016},
author = {Braff, JL and Yaung, SJ and Esvelt, KM and Church, GM},
title = {Characterizing Cas9 Protospacer-Adjacent Motifs with High-Throughput Sequencing of Library Depletion Experiments.},
journal = {Cold Spring Harbor protocols},
volume = {2016},
number = {5},
pages = {},
doi = {10.1101/pdb.prot090183},
pmid = {27140916},
issn = {1559-6095},
mesh = {Endonucleases/*genetics/*metabolism ; *Genetic Testing ; Plasmids ; Selection, Genetic ; Sequence Analysis, DNA ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {This protocol outlines a general approach for characterizing the protospacer-adjacent motifs (PAMs) of Cas9 orthologs. It uses a three-plasmid system: One plasmid carries Cas9 and its tracrRNA, a second targeting vector contains the spacer and repeat, and the third plasmid encodes the targeted sequence (as the protospacer) with varying PAM sequences. It leverages the Cas9 nuclease activity to cleave and destroy plasmids that bear a compatible PAM. The level of depletion of a library of targeted plasmids after Cas9-mediated selection can then be assessed by deep sequencing to reveal candidate PAMs for downstream validation.},
}
@article {pmid27138038,
year = {2016},
author = {EauClaire, SF and Zhang, J and Rivera, CG and Huang, LL},
title = {Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {43},
number = {7},
pages = {1001-1015},
pmid = {27138038},
issn = {1476-5535},
mesh = {Amino Acid Transport Systems, Basic/genetics/metabolism ; Base Sequence ; Biosynthetic Pathways/*genetics ; Culture Media/chemistry ; DNA Fragmentation ; Fungal Proteins/*genetics/metabolism ; Gene Deletion ; Gene Expression Regulation, Fungal ; Genetic Loci ; *Genome, Fungal ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; beta Carotene/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The yeast Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butanediol, isoprenoids, and other chemicals. The construction of a successful production strain usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect the metabolic fluxes for the conversion of sugars and other feed stocks into the desired product. RNA-guided Cas9 based genome editing has been demonstrated in many prokaryotic and eukaryotic hosts including S. cerevisiae, in which it has been additionally exploited as a tool for metabolic engineering. To extend the utilization of RNA-guided Cas9 as a metabolic pathway building tool, we demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the beta-carotene biosynthetic pathway. Furthermore, we generated a combinatorial strain library for the beta-carotene biosynthetic pathway, directly integrated into the yeast genome to create a diverse library of strains. This enabled the screening of combinatorial libraries in stable chromosomally integrated strains for rapid improvements of product titers. This combinatorial approach for pathway assembly will significantly accelerate the current speed of metabolic engineering for S. cerevisiae as an industrial platform, and increase the number of strains that can be simultaneously evaluated for enzyme screening, expression optimization and protein engineering to achieve the titer, rate and yield necessary for the commercialization of new industrial fermentation products.},
}
@article {pmid27101517,
year = {2016},
author = {Jiang, W and Zhu, TF},
title = {Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments.},
journal = {Nature protocols},
volume = {11},
number = {5},
pages = {960-975},
pmid = {27101517},
issn = {1750-2799},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Protein 9 ; *Chromosomes, Bacterial ; Cloning, Molecular/*methods ; Endonucleases/genetics ; Escherichia coli/genetics ; Genetic Vectors ; Genome, Bacterial/*genetics ; Genomics/methods ; Synthetic Biology/methods ; Workflow ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Cloning of long microbial genomic sequences is an essential tool in synthetic biology and genome engineering. Such long sequences are often difficult to obtain directly by traditional PCR or restriction enzyme digestion, and therefore the cloning of these sequences has remained a technical obstacle in molecular biology. Based on the in vitro application of RNA-guided Cas9 nuclease, the method of Cas9-assisted targeting of chromosome segments (CATCH) cleaves target DNA in vitro from intact bacterial chromosomes embedded in agarose plugs, which can be subsequently ligated with cloning vector through Gibson assembly. Here we describe an optimized protocol of CATCH cloning for the targeted cloning of long genomic sequences of up to 100 kb from microorganisms. The protocol uses standard laboratory equipment and takes ∼8 h of bench time over several days, and it may potentially simplify and accelerate efforts to isolate and clone large gene clusters from microorganisms.},
}
@article {pmid27040163,
year = {2016},
author = {Conrad, T and Albrecht, AS and de Melo Costa, VR and Sauer, S and Meierhofer, D and Ørom, UA},
title = {Serial interactome capture of the human cell nucleus.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {11212},
pmid = {27040163},
issn = {2041-1723},
mesh = {Cell Nucleus/genetics/*metabolism/radiation effects ; DNA/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation ; Gene Regulatory Networks ; HEK293 Cells ; HeLa Cells ; Humans ; K562 Cells ; Protein Interaction Mapping ; RNA, Messenger/genetics/*metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Signal Transduction ; Transcription Factors/genetics/*metabolism ; Ultraviolet Rays ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.},
}
@article {pmid26965260,
year = {2016},
author = {Huang, C and Wu, G and Yu, YT},
title = {Purification and Functional Reconstitution of Box H/ACA Ribonucleoprotein Particles.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1421},
number = {},
pages = {97-109},
pmid = {26965260},
issn = {1940-6029},
support = {R01 GM104077/GM/NIGMS NIH HHS/United States ; R21 AG039559/AG/NIA NIH HHS/United States ; AG039559/AG/NIA NIH HHS/United States ; GM104077/GM/NIGMS NIH HHS/United States ; },
mesh = {Cell Culture Techniques/methods ; Fungal Proteins/genetics/isolation &amp; purification/*metabolism ; Immunoprecipitation/methods ; Pseudouridine/genetics/*metabolism ; RNA Processing, Post-Transcriptional ; Ribonucleoproteins/genetics/isolation &amp; purification/*metabolism ; Transcription, Genetic ; Yeasts/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Pseudouridylation is the most abundant and widespread RNA modification, and it plays an important role in modulating the structure and function of RNA. In eukaryotes and archaea, RNA pseudouridylation is catalyzed largely by box H/ACA ribonucleoproteins (RNPs), a distinct group of RNA-protein complexes each consisting of a unique RNA and four common proteins. The RNA component of the complex serves as a guide that base-pairs with its substrate RNA and specifies the target uridine to be modified. In order to systematically study the function and mechanism of pseudouridylation, it is desirable to have a reconstitution system in which biochemically purified/reconstituted box H/ACA RNPs are capable of introducing pseudouridines into an RNA at any target site. Here, we describe a method for the reconstitution of functional box H/ACA RNPs using designer box H/ACA guide RNAs, which in principle can be adopted to reconstitute other RNA-protein complexes as well.},
}
@article {pmid26712743,
year = {2015},
author = {Kalia, M and Willkomm, S and Claussen, JC and Restle, T and Bonvin, AM},
title = {Novel Insights into Guide RNA 5'-Nucleoside/Tide Binding by Human Argonaute 2.},
journal = {International journal of molecular sciences},
volume = {17},
number = {1},
pages = {},
pmid = {26712743},
issn = {1422-0067},
mesh = {Amino Acid Sequence ; Argonaute Proteins/*chemistry/metabolism ; Base Sequence ; Binding Sites ; Humans ; *Molecular Dynamics Simulation ; Molecular Sequence Data ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The human Argonaute 2 (hAgo2) protein is a key player of RNA interference (RNAi). Upon complex formation with small non-coding RNAs, the protein initially interacts with the 5'-end of a given guide RNA through multiple interactions within the MID domain. This interaction has been reported to show a strong bias for U and A over C and G at the 5'-position. Performing molecular dynamics simulations of binary hAgo2/OH-guide-RNA complexes, we show that hAgo2 is a highly flexible protein capable of binding to guide strands with all four possible 5'-bases. Especially, in the case of C and G this is associated with rather large individual conformational rearrangements affecting the MID, PAZ and even the N-terminal domains to different degrees. Moreover, a 5'-G induces domain motions in the protein, which trigger a previously unreported interaction between the 5'-base and the L2 linker domain. Combining our in silico analyses with biochemical studies of recombinant hAgo2, we find that, contrary to previous observations, hAgo2 is capable of functionally accommodating guide strands regardless of the 5'-base.},
}
@article {pmid26707469,
year = {2016},
author = {Raitskin, O and Patron, NJ},
title = {Multi-gene engineering in plants with RNA-guided Cas9 nuclease.},
journal = {Current opinion in biotechnology},
volume = {37},
number = {},
pages = {69-75},
doi = {10.1016/j.copbio.2015.11.008},
pmid = {26707469},
issn = {1879-0429},
support = {BB/L014130/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Endonucleases/genetics/*metabolism ; Gene Expression ; *Genetic Engineering ; Humans ; Plants/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The use of RNA-guided Cas9 endonuclease for the concurrent engineering of multiple genes has been demonstrated in a number of plant species. Although Cas9 is a large monomeric protein, the single guide RNA (sgRNA) that directs it to a specific DNA target sequence is small and easy to reprogram. It is therefore relatively simple to produce numerous sgRNAs to target multiple endogenous sequences. Several approaches to express multiple sgRNAs and Cas9 in plants for the purpose of simultaneous editing or transcriptional regulation of many genes have recently been reported.},
}
@article {pmid26628643,
year = {2016},
author = {Slaymaker, IM and Gao, L and Zetsche, B and Scott, DA and Yan, WX and Zhang, F},
title = {Rationally engineered Cas9 nucleases with improved specificity.},
journal = {Science (New York, N.Y.)},
volume = {351},
number = {6268},
pages = {84-88},
pmid = {26628643},
issn = {1095-9203},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; 5DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; 1R01MH110049/MH/NIMH NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; T32 GM008313/GM/NIGMS NIH HHS/United States ; T32GM007753/GM/NIGMS NIH HHS/United States ; 5R01DK097768-03/DK/NIDDK NIH HHS/United States ; },
mesh = {Bacterial Proteins/*chemistry/genetics ; *DNA Cleavage ; Endonucleases/*chemistry/genetics ; Humans ; Mutagenesis ; Point Mutation ; Protein Conformation ; *Protein Engineering ; Streptococcus pyogenes/*enzymology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.},
}
@article {pmid26592935,
year = {2015},
author = {Jo, MH and Song, JJ and Hohng, S},
title = {Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.},
journal = {BMB reports},
volume = {48},
number = {12},
pages = {643-644},
doi = {10.5483/bmbrep.2015.48.12.235},
pmid = {26592935},
issn = {1976-670X},
mesh = {Argonaute Proteins/chemistry/genetics/*metabolism ; Fluorescence Resonance Energy Transfer/methods ; Humans ; MicroRNAs/metabolism ; Optical Imaging/methods ; RNA Interference ; RNA, Small Interfering/chemistry/genetics/metabolism ; RNA-Induced Silencing Complex/chemistry/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.},
}
@article {pmid26240384,
year = {2015},
author = {Toffano-Nioche, C and Gautheret, D and Leclerc, F},
title = {Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea.},
journal = {Nucleic acids research},
volume = {43},
number = {16},
pages = {7744-7761},
pmid = {26240384},
issn = {1362-4962},
mesh = {Base Pairing ; Euryarchaeota/genetics ; Gene Expression Regulation, Archaeal ; Models, Molecular ; Nucleotide Motifs ; Pyrobaculum/*genetics ; Pyrococcus abyssi/*genetics ; RNA Folding ; RNA, Archaeal/*chemistry/metabolism ; RNA, Small Nucleolar/*chemistry/metabolism ; RNA, Transfer/metabolism ; Structure-Activity Relationship ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.},
}
@article {pmid26238202,
year = {2015},
author = {Tamura, M and Kawano, M and Sato, M and Nashimoto, M},
title = {Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing.},
journal = {Molecular medicine reports},
volume = {12},
number = {4},
pages = {6365-6369},
doi = {10.3892/mmr.2015.4160},
pmid = {26238202},
issn = {1791-3004},
mesh = {Apoptosis/drug effects ; Cell Line, Tumor ; Chlorpromazine/pharmacology ; *Endocytosis/drug effects ; Endoplasmic Reticulum/drug effects/metabolism ; *Gene Silencing ; Golgi Apparatus/drug effects/metabolism ; HEK293 Cells ; HL-60 Cells ; Humans ; Leukemia/genetics ; Nystatin/pharmacology ; RNA, Messenger/genetics/metabolism ; Transfection ; bcl-2-Associated X Protein/genetics/metabolism ; beta-Cyclodextrins/pharmacology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl‑2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl‑β‑cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis‑inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin‑, caveolae‑ and raft‑independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis‑inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA‑mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically‑targeted and potent sgRNA drugs.},
}
@article {pmid26140592,
year = {2015},
author = {Salomon, WE and Jolly, SM and Moore, MJ and Zamore, PD and Serebrov, V},
title = {Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides.},
journal = {Cell},
volume = {162},
number = {1},
pages = {84-95},
pmid = {26140592},
issn = {1097-4172},
support = {GM65236/GM/NIGMS NIH HHS/United States ; R01 GM053007/GM/NIGMS NIH HHS/United States ; R01 GM062862/GM/NIGMS NIH HHS/United States ; T32 GM107000/GM/NIGMS NIH HHS/United States ; R01 GM065236/GM/NIGMS NIH HHS/United States ; GM62862/GM/NIGMS NIH HHS/United States ; GM053007/GM/NIGMS NIH HHS/United States ; R37 GM062862/GM/NIGMS NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Argonaute Proteins/chemistry/*metabolism ; Bacterial Proteins/metabolism ; Mice ; MicroRNAs/*metabolism ; Molecular Imaging ; *Nucleic Acid Hybridization ; RNA-Induced Silencing Complex/metabolism ; Thermodynamics ; Thermus thermophilus/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA.},
}
@article {pmid26123697,
year = {2015},
author = {Hentze, JL and Carlsson, MA and Kondo, S and Nässel, DR and Rewitz, KF},
title = {The Neuropeptide Allatostatin A Regulates Metabolism and Feeding Decisions in Drosophila.},
journal = {Scientific reports},
volume = {5},
number = {},
pages = {11680},
pmid = {26123697},
issn = {2045-2322},
mesh = {Animals ; Drosophila/*metabolism ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Eating ; Energy Metabolism ; Female ; Insect Hormones/antagonists &amp; inhibitors/genetics/metabolism ; Lipid Metabolism ; Male ; Neurons/metabolism ; Neuropeptides/antagonists &amp; inhibitors/genetics/*metabolism ; Oligopeptides/antagonists &amp; inhibitors/genetics/metabolism ; Pyrrolidonecarboxylic Acid/analogs &amp; derivatives/antagonists &amp; inhibitors/metabolism ; RNA Interference ; RNA, Messenger/metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Neuropeptide/antagonists &amp; inhibitors/genetics/metabolism ; Signal Transduction ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Coordinating metabolism and feeding is important to avoid obesity and metabolic diseases, yet the underlying mechanisms, balancing nutrient intake and metabolic expenditure, are poorly understood. Several mechanisms controlling these processes are conserved in Drosophila, where homeostasis and energy mobilization are regulated by the glucagon-related adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). Here, we provide evidence that the Drosophila neuropeptide Allatostatin A (AstA) regulates AKH and DILP signaling. The AstA receptor gene, Dar-2, is expressed in both the insulin and AKH producing cells. Silencing of Dar-2 in these cells results in changes in gene expression and physiology associated with reduced DILP and AKH signaling and animals lacking AstA accumulate high lipid levels. This suggests that AstA is regulating the balance between DILP and AKH, believed to be important for the maintenance of nutrient homeostasis in response to changing ratios of dietary sugar and protein. Furthermore, AstA and Dar-2 are regulated differentially by dietary carbohydrates and protein and AstA-neuronal activity modulates feeding choices between these types of nutrients. Our results suggest that AstA is involved in assigning value to these nutrients to coordinate metabolic and feeding decisions, responses that are important to balance food intake according to metabolic needs.},
}
@article {pmid26076094,
year = {2015},
author = {Kim, SJ and Lee, CH and Lee, SW},
title = {Targeting the MicroRNA Passenger Strand for Regulating Therapeutic Transgenes.},
journal = {Nucleic acid therapeutics},
volume = {25},
number = {4},
pages = {209-218},
doi = {10.1089/nat.2015.0543},
pmid = {26076094},
issn = {2159-3345},
mesh = {Base Pair Mismatch ; Base Pairing ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Genes, Reporter ; HeLa Cells ; Hep G2 Cells ; Humans ; Jurkat Cells ; Luciferases/genetics/metabolism ; MCF-7 Cells ; MicroRNAs/*genetics/metabolism ; RNA, Antisense/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; *Transgenes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Gene therapy strategies have been developed, which can tissue or disease specifically regulate expression of exogenous transgenes by means of endogenous microRNA (miRNA) activity. However, the use of an endogenous guide strand to regulate an exogenous transgene could affect expression of endogenous miRNA target genes. In this study, we developed a new regulatory system of exogenous transgene expression by targeting the passenger strand. We constructed reporter constructs harboring miRNA-122 guide or passenger target sites with perfect or imperfect complementarity. We observed downregulation of an exogenous transgene harboring the miRNA-122 target sites against either the guide or passenger strand in cells expressing the cognate miRNA or cells stably expressing the miRNA target site. Moreover, the transgene activity as well as the gene expression level increased specifically by intracellular introduction of the antisense RNA against the corresponding strand. Endogenous target gene expression was induced by the transgene construct harboring the miRNA guide strand target sites, but not the passenger strand target sites. Importantly, the therapeutic transgene activity was efficiently regulated by targeting the passenger strand. These results suggested that an approach to passenger strand-regulated expression of therapeutic transgenes could be applied more safely as a therapeutic tool.},
}
@article {pmid25978870,
year = {2015},
author = {Kanchiswamy, CN and Malnoy, M and Velasco, R and Kim, JS and Viola, R},
title = {Non-GMO genetically edited crop plants.},
journal = {Trends in biotechnology},
volume = {33},
number = {9},
pages = {489-491},
doi = {10.1016/j.tibtech.2015.04.002},
pmid = {25978870},
issn = {1879-3096},
mesh = {Agriculture ; *Crops, Agricultural ; *Plants, Genetically Modified ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is also likely to bypass genetically modified organism (GMO) legislation as the genome editing complex is degraded in the recipient cells.},
}
@article {pmid25977554,
year = {2015},
author = {Han, BW and Wang, W and Li, C and Weng, Z and Zamore, PD},
title = {Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production.},
journal = {Science (New York, N.Y.)},
volume = {348},
number = {6236},
pages = {817-821},
pmid = {25977554},
issn = {1095-9203},
support = {GM65236/GM/NIGMS NIH HHS/United States ; HG007000/HG/NHGRI NIH HHS/United States ; R01 GM062862/GM/NIGMS NIH HHS/United States ; R01 GM065236/GM/NIGMS NIH HHS/United States ; GM62862/GM/NIGMS NIH HHS/United States ; R37 GM062862/GM/NIGMS NIH HHS/United States ; U41 HG007000/HG/NHGRI NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Argonaute Proteins/genetics/*metabolism ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/genetics/*metabolism ; Endoribonucleases/genetics/*metabolism ; Female ; Germ Cells/metabolism ; Male ; Metabolic Networks and Pathways ; Mice ; Ovary/metabolism ; Peptide Initiation Factors/genetics/*metabolism ; *RNA Cleavage ; RNA, Small Interfering/biosynthesis/*metabolism ; *Retroelements ; Testis/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {PIWI-interacting RNAs (piRNAs) protect the animal germ line by silencing transposons. Primary piRNAs, generated from transcripts of genomic transposon "junkyards" (piRNA clusters), are amplified by the "ping-pong" pathway, yielding secondary piRNAs. We report that secondary piRNAs, bound to the PIWI protein Ago3, can initiate primary piRNA production from cleaved transposon RNAs. The first ~26 nucleotides (nt) of each cleaved RNA becomes a secondary piRNA, but the subsequent ~26 nt become the first in a series of phased primary piRNAs that bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. The ping-pong pathway increases only the abundance of piRNAs, whereas production of phased primary piRNAs from cleaved transposon RNAs adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence.},
}
@article {pmid25977553,
year = {2015},
author = {Mohn, F and Handler, D and Brennecke, J},
title = {Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis.},
journal = {Science (New York, N.Y.)},
volume = {348},
number = {6236},
pages = {812-817},
pmid = {25977553},
issn = {1095-9203},
support = {260711/ERC_/European Research Council/International ; Y 510/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Animals ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/*enzymology/genetics ; Endoribonucleases/genetics/*metabolism ; Evolution, Molecular ; Female ; Germ Cells/enzymology ; Male ; Mice ; Ovary/enzymology ; *RNA Cleavage ; RNA, Small Interfering/biosynthesis/*metabolism ; RNA-Binding Proteins/genetics ; Testis/enzymology ; *Transcription, Genetic ; Uridine/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends.},
}
@article {pmid25977536,
year = {2015},
author = {Siomi, H and Siomi, MC},
title = {RNA. Phased piRNAs tackle transposons.},
journal = {Science (New York, N.Y.)},
volume = {348},
number = {6236},
pages = {756-757},
doi = {10.1126/science.aab3004},
pmid = {25977536},
issn = {1095-9203},
mesh = {Animals ; Argonaute Proteins/*metabolism ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/*enzymology/*metabolism ; Endoribonucleases/*metabolism ; Female ; Male ; Peptide Initiation Factors/*metabolism ; *RNA Cleavage ; RNA, Small Interfering/*metabolism ; *Retroelements ; *Transcription, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid25957684,
year = {2015},
author = {Zheng, S and Vuong, BQ and Vaidyanathan, B and Lin, JY and Huang, FT and Chaudhuri, J},
title = {Non-coding RNA Generated following Lariat Debranching Mediates Targeting of AID to DNA.},
journal = {Cell},
volume = {161},
number = {4},
pages = {762-773},
pmid = {25957684},
issn = {1097-4172},
support = {G12 MD007603/MD/NIMHD NIH HHS/United States ; G12 RR003060/RR/NCRR NIH HHS/United States ; R01 AI072194/AI/NIAID NIH HHS/United States ; 1R01AI072194/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Cytidine Deaminase/*metabolism ; G-Quadruplexes ; *Immunoglobulin Class Switching ; Introns ; Maltose-Binding Proteins/metabolism ; Mice ; RNA Processing, Post-Transcriptional ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Transcription through immunoglobulin switch (S) regions is essential for class switch recombination (CSR), but no molecular function of the transcripts has been described. Likewise, recruitment of activation-induced cytidine deaminase (AID) to S regions is critical for CSR; however, the underlying mechanism has not been fully elucidated. Here, we demonstrate that intronic switch RNA acts in trans to target AID to S region DNA. AID binds directly to switch RNA through G-quadruplexes formed by the RNA molecules. Disruption of this interaction by mutation of a key residue in the putative RNA-binding domain of AID impairs recruitment of AID to S region DNA, thereby abolishing CSR. Additionally, inhibition of RNA lariat processing leads to loss of AID localization to S regions and compromises CSR; both defects can be rescued by exogenous expression of switch transcripts in a sequence-specific manner. These studies uncover an RNA-mediated mechanism of targeting AID to DNA.},
}
@article {pmid25822791,
year = {2015},
author = {Iwasaki, S and Sasaki, HM and Sakaguchi, Y and Suzuki, T and Tadakuma, H and Tomari, Y},
title = {Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex.},
journal = {Nature},
volume = {521},
number = {7553},
pages = {533-536},
pmid = {25822791},
issn = {1476-4687},
mesh = {Animals ; Argonaute Proteins/metabolism ; Drosophila Proteins/metabolism ; Drosophila melanogaster/*enzymology/*genetics ; Heat-Shock Proteins/metabolism ; In Vitro Techniques ; Janus Kinases/metabolism ; Protein Binding ; RNA Helicases/metabolism ; *RNA Interference ; RNA, Small Interfering/genetics/metabolism ; RNA-Binding Proteins/metabolism ; RNA-Induced Silencing Complex/*chemistry/*metabolism ; Ribonuclease III/metabolism ; Transcription Factors/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2-R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2-R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2-R2D2-siRNA complex on Ago2, in a manner dependent on recognition of the 5'-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2-R2D2 and thereby assemble RISC.},
}
@article {pmid25730490,
year = {2015},
author = {Chavez, A and Scheiman, J and Vora, S and Pruitt, BW and Tuttle, M and P R Iyer, E and Lin, S and Kiani, S and Guzman, CD and Wiegand, DJ and Ter-Ovanesyan, D and Braff, JL and Davidsohn, N and Housden, BE and Perrimon, N and Weiss, R and Aach, J and Collins, JJ and Church, GM},
title = {Highly efficient Cas9-mediated transcriptional programming.},
journal = {Nature methods},
volume = {12},
number = {4},
pages = {326-328},
pmid = {25730490},
issn = {1548-7105},
support = {P50 HG005550/HG/NHGRI NIH HHS/United States ; T32 GM007598/GM/NIGMS NIH HHS/United States ; R01 CA173712/CA/NCI NIH HHS/United States ; T32 CA009216/CA/NCI NIH HHS/United States ; 5T32CA009216-34/CA/NCI NIH HHS/United States ; },
mesh = {Cell Differentiation/genetics ; *Endonucleases/genetics ; *Genetic Techniques ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Neurons/cytology ; Staphylococcus aureus ; *Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).},
}
@article {pmid25730316,
year = {2015},
author = {Ninomiya, S and Kawano, M and Abe, T and Ishikawa, T and Takahashi, M and Tamura, M and Takahashi, Y and Nashimoto, M},
title = {Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.},
journal = {PloS one},
volume = {10},
number = {3},
pages = {e0118631},
pmid = {25730316},
issn = {1932-6203},
mesh = {Base Sequence ; Cells, Cultured ; Endoribonucleases/blood ; High-Throughput Nucleotide Sequencing ; Humans ; Jurkat Cells ; Leukocytes, Mononuclear/cytology/*metabolism ; Multiple Myeloma/blood/pathology ; Nucleic Acid Conformation ; RNA/analysis/blood/isolation &amp; purification ; Sequence Analysis, RNA ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Several pieces of evidence suggest that small RNA degradation products together with tRNase ZL appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5'-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase ZL, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5-40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5-40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 5'-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase ZL and sgRNA may be extended intercellularly.},
}
@article {pmid25616362,
year = {2014},
author = {Spenkuch, F and Motorin, Y and Helm, M},
title = {Pseudouridine: still mysterious, but never a fake (uridine)!.},
journal = {RNA biology},
volume = {11},
number = {12},
pages = {1540-1554},
pmid = {25616362},
issn = {1555-8584},
mesh = {Escherichia coli/genetics/metabolism ; Humans ; Intramolecular Transferases/genetics/*metabolism ; Isoenzymes/genetics/metabolism ; Nucleic Acid Conformation ; Pseudouridine/*metabolism ; RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA, Mitochondrial ; RNA, Ribosomal/genetics/metabolism ; RNA, Transfer, Amino Acid-Specific/chemistry/genetics/*metabolism ; Ribonucleoproteins, Small Nuclear/genetics/metabolism ; Ribosomes/chemistry/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Uridine/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA. Although Ψ was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of Ψ synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.},
}
@article {pmid25590339,
year = {2014},
author = {Yu, YT and Meier, UT},
title = {RNA-guided isomerization of uridine to pseudouridine--pseudouridylation.},
journal = {RNA biology},
volume = {11},
number = {12},
pages = {1483-1494},
pmid = {25590339},
issn = {1555-8584},
support = {AG039559/AG/NIA NIH HHS/United States ; R01 GM104077/GM/NIGMS NIH HHS/United States ; GM097752/GM/NIGMS NIH HHS/United States ; R01 GM097752/GM/NIGMS NIH HHS/United States ; GM104077/GM/NIGMS NIH HHS/United States ; },
mesh = {Dyskeratosis Congenita/genetics/*metabolism/pathology ; Humans ; Isomerism ; Male ; Mutation ; Nucleic Acid Conformation ; Prostatic Neoplasms/genetics/*metabolism/pathology ; Pseudouridine/*metabolism ; *RNA Processing, Post-Transcriptional ; RNA, Ribosomal/genetics/metabolism ; RNA, Small Nuclear/genetics/metabolism ; RNA, Transfer, Amino Acid-Specific/genetics/metabolism ; Ribonucleoproteins, Small Nuclear/genetics/metabolism ; Uridine/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Box H/ACA ribonucleoproteins (RNPs), each consisting of one unique guide RNA and 4 common core proteins, constitute a family of complex enzymes that catalyze, in an RNA-guided manner, the isomerization of uridines to pseudouridines (Ψs) in RNAs, a reaction known as pseudouridylation. Over the years, box H/ACA RNPs have been extensively studied revealing many important aspects of these RNA modifying machines. In this review, we focus on the composition, structure, and biogenesis of H/ACA RNPs. We explain the mechanism of how this enzyme family recognizes and specifies its target uridine in a substrate RNA. We discuss the substrates of box H/ACA RNPs, focusing on rRNA (rRNA) and spliceosomal small nuclear RNA (snRNA). We describe the modification product Ψ and its contribution to RNA function. Finally, we consider possible mechanisms of the bone marrow failure syndrome dyskeratosis congenita and of prostate and other cancers linked to mutations in H/ACA RNPs.},
}
@article {pmid25437003,
year = {2014},
author = {Iizuka, S and Oridate, N and Nashimoto, M and Fukuda, S and Tamura, M},
title = {Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.},
journal = {PloS one},
volume = {9},
number = {12},
pages = {e114121},
pmid = {25437003},
issn = {1932-6203},
mesh = {Apoptosis ; Carcinoma, Squamous Cell/*genetics/pathology/*therapy ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1/analysis/*genetics ; *Gene Silencing ; Genetic Therapy ; Head and Neck Neoplasms/*genetics/pathology/*therapy ; Humans ; RNA, Messenger/*genetics ; Squamous Cell Carcinoma of Head and Neck ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs.},
}
@article {pmid25398344,
year = {2014},
author = {Renouf, B and Piganeau, M and Ghezraoui, H and Jasin, M and Brunet, E},
title = {Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.},
journal = {Methods in enzymology},
volume = {546},
number = {},
pages = {251-271},
pmid = {25398344},
issn = {1557-7988},
support = {R01 GM054668/GM/NIGMS NIH HHS/United States ; GM054668/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; Cell Line ; *DNA Breaks, Double-Stranded ; Deoxyribonuclease I/genetics/*metabolism ; Humans ; Molecular Sequence Data ; Neoplasms/*genetics ; Plasmids/genetics ; Polymerase Chain Reaction/methods ; Transfection/methods ; *Translocation, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Recurrent chromosomal translocations are found in numerous tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci. Using different PCR-based methods, we also explain how to quantify translocation frequency and to analyze breakpoint junctions in the cells of interest. In addition, PCR detection of translocations is used as a very sensitive method to detect off-target effects, which has general utility.},
}
@article {pmid25339675,
year = {2014},
author = {Reyes, LM and Estrada, JL and Wang, ZY and Blosser, RJ and Smith, RF and Sidner, RA and Paris, LL and Blankenship, RL and Ray, CN and Miner, AC and Tector, M and Tector, AJ},
title = {Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {193},
number = {11},
pages = {5751-5757},
pmid = {25339675},
issn = {1550-6606},
support = {C06 RR010601/RR/NCRR NIH HHS/United States ; C06RR10601-01/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; Cells, Cultured ; Endonucleases/metabolism ; Fibroblasts/*physiology ; Gene Knockdown Techniques/*methods ; Genetic Engineering ; Graft Rejection ; Histocompatibility Antigens Class I/*genetics ; Models, Animal ; Nuclear Transfer Techniques ; Swine/genetics/*immunology ; T-Lymphocytes/*immunology ; *Transplantation, Heterologous ; Virus Diseases/*immunology ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.},
}
@article {pmid25281382,
year = {2014},
author = {Zhang, GC and Kong, II and Kim, H and Liu, JJ and Cate, JH and Jin, YS},
title = {Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease.},
journal = {Applied and environmental microbiology},
volume = {80},
number = {24},
pages = {7694-7701},
pmid = {25281382},
issn = {1098-5336},
mesh = {3-Isopropylmalate Dehydrogenase/genetics/metabolism ; Aldose-Ketose Isomerases/genetics/metabolism ; Autotrophic Processes ; Endonucleases/metabolism ; Hydro-Lyases/genetics/metabolism ; Industrial Microbiology ; *Metabolic Engineering ; Plasmids/genetics ; Polyploidy ; Saccharomyces cerevisiae/*genetics/growth &amp; development/*metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.},
}
@article {pmid25124321,
year = {2014},
author = {Fazio, S and Tavori, H},
title = {Peeking into a cool future: genome editing to delete PCSK9 and control hypercholesterolemia in a single shot.},
journal = {Circulation research},
volume = {115},
number = {5},
pages = {472-474},
pmid = {25124321},
issn = {1524-4571},
support = {R01 HL057986/HL/NHLBI NIH HHS/United States ; R01 HL106845/HL/NHLBI NIH HHS/United States ; R01-HL057986/HL/NHLBI NIH HHS/United States ; R01-HL106845/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Liver/*enzymology ; Male ; *Mutation ; Proprotein Convertase 9 ; Proprotein Convertases/*genetics ; *RNA Editing ; Serine Endopeptidases/*genetics ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid25064679,
year = {2014},
author = {Wang, X and Yuan, Y and Zhou, Q and Wan, H and Wang, M and Zhou, Q and Zhao, XY and Sha, J},
title = {RNA guided genome editing in mouse germ-line stem cells.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {41},
number = {7},
pages = {409-411},
doi = {10.1016/j.jgg.2014.06.001},
pmid = {25064679},
issn = {1673-8527},
mesh = {Animals ; Base Sequence ; Genetic Engineering/*methods ; Male ; Mice ; Spermatozoa/*cytology ; Stem Cells/*metabolism ; Testis/cytology ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid24875628,
year = {2014},
author = {Lee, JS and Kwak, SJ and Kim, J and Kim, AK and Noh, HM and Kim, JS and Yu, K},
title = {RNA-guided genome editing in Drosophila with the purified Cas9 protein.},
journal = {G3 (Bethesda, Md.)},
volume = {4},
number = {7},
pages = {1291-1295},
pmid = {24875628},
issn = {2160-1836},
mesh = {Animals ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA-Binding Proteins/genetics ; Drosophila/genetics ; Drosophila Proteins/*genetics/metabolism ; *Genome ; Germ-Line Mutation ; RNA Editing ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay.},
}
@article {pmid24657904,
year = {2014},
author = {Xue, Z and Ren, M and Wu, M and Dai, J and Rong, YS and Gao, G},
title = {Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {4},
number = {5},
pages = {925-929},
pmid = {24657904},
issn = {2160-1836},
support = {//Intramural NIH HHS/United States ; },
mesh = {Animals ; *Animals, Genetically Modified ; Bacterial Proteins/*genetics ; Deoxyribonucleases/*genetics ; Drosophila/*genetics ; Gene Expression ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Gene Order ; Gene Targeting ; Germ-Line Mutation ; Homologous Recombination ; Microinjections ; Transgenes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.},
}
@article {pmid38353732,
year = {2024},
author = {Kovalev, MA and Davletshin, AI and Karpov, DS},
title = {Engineering Cas9: next generation of genomic editors.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {209},
pmid = {38353732},
issn = {1432-0614},
support = {22-14-00377//Russian Science Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Genomics ; Mutagenesis ; Mutation ; CRISPR-Associated Protein 9/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9. KEY POINTS: • The next-generation Cas9-based editors are more active than in the first one. • PAM-relaxed variants of Cas9 are improved by increased specificity and activity. • Less mutagenic and immunogenic variants of Cas9 are created.},
}
@article {pmid38353624,
year = {2024},
author = {da, JAT},
title = {"CRISPR" Mutations: Inaccurate Linguistic Variations and Misrepresentation of the CRISPR Acronym.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {3-4},
doi = {10.1089/crispr.2024.0005},
pmid = {38353624},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Mutation ; },
}
@article {pmid38353623,
year = {2024},
author = {Bonnycastle, LL and Swift, AJ and Mansell, EC and Lee, A and Winnicki, E and Li, ES and Robertson, CC and Parsons, VA and Huynh, T and Krilow, C and Mohlke, KL and Erdos, MR and Narisu, N and Collins, FS},
title = {Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {53-67},
doi = {10.1089/crispr.2023.0066},
pmid = {38353623},
issn = {2573-1602},
mesh = {Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Diabetes Mellitus, Type 2 ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {We developed an efficient CRISPR prime editing protocol and generated isogenic-induced pluripotent stem cell (iPSC) lines carrying heterozygous or homozygous alleles for putatively causal single nucleotide variants at six type 2 diabetes loci (ABCC8, MTNR1B, TCF7L2, HNF4A, CAMK1D, and GCK). Our two-step sequence-based approach to first identify transfected cell pools with the highest fraction of edited cells significantly reduced the downstream efforts to isolate single clones of edited cells. We found that prime editing can make targeted genetic changes in iPSC and optimization of system components and guide RNA designs that were critical to achieve acceptable efficiency. Systems utilizing PEmax, epegRNA modifications, and MLH1dn provided significant benefit, producing editing efficiencies of 36-73%. Editing success and pegRNA design optimization required for each variant differed depending on the sequence at the target site. With attention to design, prime editing is a promising approach to generate isogenic iPSC lines, enabling the study of specific genetic changes in a common genetic background.},
}
@article {pmid38353622,
year = {2024},
author = {Barrangou, R and Davies, K},
title = {CRISPR Momentum in the Clinic and the Field.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {1-2},
doi = {10.1089/crispr.2024.29172.editorial},
pmid = {38353622},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid38353621,
year = {2024},
author = {Li, Z and Lan, J and Shi, X and Lu, T and Hu, X and Liu, X and Chen, Y and He, Z},
title = {Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {29-40},
doi = {10.1089/crispr.2023.0034},
pmid = {38353621},
issn = {2573-1602},
mesh = {Animals ; Swine/genetics ; *Receptor, Melanocortin, Type 1/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Mutation ; INDEL Mutation/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.},
}
@article {pmid38353620,
year = {2024},
author = {},
title = {Acknowledgment of Reviewers 2023.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {69},
doi = {10.1089/crispr.2024.29169.ack},
pmid = {38353620},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid38353619,
year = {2024},
author = {Gray, V and Thomas, U and Davies, K},
title = {Warrior Spirit: An Interview with Victoria Gray, Sickle Cell Pioneer.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {5-11},
doi = {10.1089/crispr.2024.29171.vgr},
pmid = {38353619},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; *Anemia, Sickle Cell/genetics/therapy ; },
}
@article {pmid38353618,
year = {2024},
author = {Li, R and Xu, Y and Wu, F and Peng, Z and Chan, J and Zhang, L},
title = {Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (Haliotis discus hannai).},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {41-52},
doi = {10.1089/crispr.2023.0070},
pmid = {38353618},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Tubulin ; Opsins ; },
abstract = {The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene β-tubulin and light-sensitive transmembrane protein r-opsin as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of β-tubulin and r-opsin genes from injected larvae identified various genomic long-fragment deletions. In situ hybridization showed gene expression patterns of β-tubulin and r-opsin were significantly altered in the mosaic mutants. Knocking out β-tubulin in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of r-opsin in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.},
}
@article {pmid38353617,
year = {2024},
author = {Burger, BT and Beaton, BP and Campbell, MA and Brett, BT and Rohrer, MS and Plummer, S and Barnes, D and Jiang, K and Naswa, S and Lange, J and Ott, A and Alger, E and Rincon, G and Rounsley, S and Betthauser, J and Mtango, NR and Benne, JA and Hammerand, J and Durfee, CJ and Rotolo, ML and Cameron, P and Lied, AM and Irby, MJ and Nyer, DB and Fuller, CK and Gradia, S and Kanner, SB and Park, KE and Waters, J and Simpson, S and Telugu, BP and Salgado, BC and Brandariz-Nuñez, A and Rowland, RRR and Culbertson, M and Rice, E and Cigan, AM},
title = {Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.},
journal = {The CRISPR journal},
volume = {7},
number = {1},
pages = {12-28},
doi = {10.1089/crispr.2023.0061},
pmid = {38353617},
issn = {2573-1602},
mesh = {Animals ; Swine ; *Porcine respiratory and reproductive syndrome virus/genetics ; *Porcine Reproductive and Respiratory Syndrome/genetics ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Gene Editing ; Livestock ; },
abstract = {Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.},
}
@article {pmid38326929,
year = {2024},
author = {Liang, Y and Gao, S and Qi, X and Valentovich, LN and An, Y},
title = {Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.},
journal = {ACS synthetic biology},
volume = {13},
number = {2},
pages = {428-448},
doi = {10.1021/acssynbio.3c00685},
pmid = {38326929},
issn = {2161-5063},
mesh = {*Gene Editing ; *Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Metabolic Engineering ; DNA/metabolism ; },
abstract = {The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.},
}
@article {pmid38325664,
year = {2024},
author = {Walther, J and Porenta, D and Wilbie, D and Seinen, C and Benne, N and Yang, Q and de Jong, OG and Lei, Z and Mastrobattista, E},
title = {Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo.},
journal = {European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V},
volume = {196},
number = {},
pages = {114207},
doi = {10.1016/j.ejpb.2024.114207},
pmid = {38325664},
issn = {1873-3441},
mesh = {Humans ; Female ; Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics ; HEK293 Cells ; Tissue Distribution ; *Nanoparticles ; DNA ; *Liposomes ; },
abstract = {The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.},
}
@article {pmid38300409,
year = {2024},
author = {Choi, SY and Romero-Calle, DX and Cho, HG and Bae, HW and Cho, YH},
title = {Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {62},
number = {1},
pages = {1-10},
pmid = {38300409},
issn = {1976-3794},
mesh = {*Bacteriophages/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Plasmids/genetics ; Recombination, Genetic ; },
abstract = {Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.},
}
@article {pmid38281457,
year = {2024},
author = {Yan, Q and Li, D and Jia, S and Yang, J and Ma, J},
title = {Novel gene-based therapeutic approaches for the management of hepatic complications in diabetes: Reviewing recent advances.},
journal = {Journal of diabetes and its complications},
volume = {38},
number = {2},
pages = {108688},
doi = {10.1016/j.jdiacomp.2024.108688},
pmid = {38281457},
issn = {1873-460X},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Diabetes Mellitus/genetics ; Insulin/genetics ; },
abstract = {Diabetes mellitus is a chronic metabolic disorder marked by hyperglycemia and systemic complications, including hepatic dysfunction, significantly contributing to disease progression and morbidity. This article reviews recent advances in gene-based therapeutic strategies targeting hepatic complications in diabetes, offering a promising approach for precision medicine by addressing underlying molecular mechanisms. Traditional treatments for hepatic complications in diabetes often manage symptoms rather than molecular causes, showing limited efficacy. Gene-based therapies are poised to correct dysfunctional pathways and restore hepatic function. Fundamental gene therapy approaches include gene silencing via small interfering RNAs (siRNAs) to target hepatic glucose production, lipid metabolism, and inflammation. Viral vectors can restore insulin sensitivity and reduce oxidative stress in diabetic livers. Genome editing, especially CRISPR-Cas9, allows the precise modification of disease-associated genes, offering immense potential for hepatic complication treatment. Strategies using CRISPR-Cas9 to enhance insulin receptor expression and modulate aberrant lipid regulatory genes are explored. Safety challenges in gene-based therapies, such as off-target effects and immune responses, are discussed. Advances in nanoparticle-based delivery systems and targeted gene editing techniques offer solutions to enhance specificity and minimize adverse effects. In conclusion, gene-based therapeutic approaches are a transformative direction in managing hepatic complications in diabetes. Further research is needed to optimize efficacy, safety, and long-term outcomes. Nevertheless, these innovative strategies promise to improve the lives of individuals with diabetes by addressing hepatic dysfunction's genetic root causes.},
}
@article {pmid38243901,
year = {2024},
author = {Fang, H and Zhao, J and Zhao, X and Dong, N and Zhao, Y and Zhang, D},
title = {Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9.},
journal = {ACS synthetic biology},
volume = {13},
number = {2},
pages = {613-623},
doi = {10.1021/acssynbio.3c00585},
pmid = {38243901},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Plasmids/genetics ; },
abstract = {The introduction of complex biosynthetic pathways into the hosts' chromosomes is gaining attention with the development of synthetic biology. While CRISPR-Cas9 has been widely employed for gene knock-in, the process of multigene insertion remains cumbersome due to laborious and empirical gene cloning procedures. To address this, we devised a standardized iterative genome editing system for Escherichia coli, harnessing the power of CRISPR-Cas9 and MetClo assembly. This comprehensive toolkit comprises two fundamental elements based on the Golden Gate standard for modular assembly of sgRNA or CRISPR arrays and donor DNAs. We achieved a gene insertion efficiency of up to 100%, targeting a single locus. Expression of tracrRNA using a strong promoter enhances multiplex genomic insertion efficiency to 7.3%, compared with 0.76% when a native promoter is used. To demonstrate the robust capabilities of this genome editing toolbox, we successfully integrated 5-10 genes from the coenzyme B12 biosynthetic pathway ranging from 5.3 to 8 Kb in length into the chromosome of E. coli chassis cells, resulting in 14 antibiotic-free, plasmid-free producers. Following an extensive screening process involving genes from diverse sources, cistronic design modifications, and chromosome repositioning, we obtained a recombinant strain yielding 1.49 mg L[-1] coenzyme B12, the highest known titer achieved by using E. coli as the producer. Illuminating its user-friendliness, this genome editing system is an exceedingly versatile tool for expediently integrating complex biosynthetic pathway genes into hosts' genomes, thus facilitating pathway optimization for chemical production.},
}
@article {pmid38240694,
year = {2024},
author = {Kim, SH and Park, JH and Shin, S and Shin, S and Chun, D and Kim, YG and Yoo, J and You, WK and Lee, JS and Lee, GM},
title = {Genome-Wide CRISPR/Cas9 Screening Unveils a Novel Target ATF7IP-SETDB1 Complex for Enhancing Difficult-to-Express Protein Production.},
journal = {ACS synthetic biology},
volume = {13},
number = {2},
pages = {634-647},
doi = {10.1021/acssynbio.3c00646},
pmid = {38240694},
issn = {2161-5063},
mesh = {Cricetinae ; Animals ; Cricetulus ; *CRISPR-Cas Systems/genetics ; CHO Cells ; *Genome ; Protein Processing, Post-Translational ; Antibodies, Monoclonal/metabolism ; },
abstract = {With the emerging novel biotherapeutics that are typically difficult-to-express (DTE), improvement is required for high-yield production. To identify novel targets that can enhance DTE protein production, we performed genome-wide fluorescence-activated cell sorting (FACS)-based clustered regularly interspaced short palindromic repeats (CRISPR) knockout screening in bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cells. The screen identified the two highest-scoring genes, Atf7ip and Setdb1, which are the binding partners for H3K9me3-mediated transcriptional repression. The ATF7IP-SETDB1 complex knockout in bsAb-producing CHO cells suppressed cell growth but enhanced productivity by up to 2.7-fold. Decreased H3K9me3 levels and an increased transcriptional expression level of the transgene were also observed. Furthermore, perturbation of the ATF7IP-SETDB1 complex in monoclonal antibody (mAb)-producing CHO cells led to substantial improvements in mAb production, increasing the productivity by up to 3.9-fold without affecting the product quality. Taken together, the genome-wide FACS-based CRISPR screen identified promising targets associated with histone methylation, whose perturbation enhanced the productivity by unlocking the transgene expression.},
}
@article {pmid38181781,
year = {2024},
author = {Neidemire-Colley, L and Khanal, S and Braunreiter, KM and Gao, Y and Kumar, R and Snyder, KJ and Weber, MA and Surana, S and Toirov, O and Karunasiri, M and Duszynski, ME and Chi, M and Malik, P and Kalyan, S and Chan, WK and Naeimi Kararoudi, M and Choe, HK and Garzon, R and Ranganathan, P},
title = {CRISPR/Cas9 deletion of MIR155HG in human T cells reduces incidence and severity of acute GVHD in a xenogeneic model.},
journal = {Blood advances},
volume = {8},
number = {4},
pages = {947-958},
doi = {10.1182/bloodadvances.2023010570},
pmid = {38181781},
issn = {2473-9537},
support = {R01 CA252469/CA/NCI NIH HHS/United States ; R01 HL163849/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; Mice ; Animals ; Incidence ; CRISPR-Cas Systems ; Mice, Inbred NOD ; Mice, SCID ; *Graft vs Host Disease/prevention &amp; control ; Mice, Knockout ; *MicroRNAs/genetics ; },
abstract = {Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). Using preclinical mouse models of disease, previous work in our laboratory has linked microRNA-155 (miR-155) to the development of acute GVHD. Transplantation of donor T cells from miR-155 host gene (MIR155HG) knockout mice prevented acute GVHD in multiple murine models of disease while maintaining critical graft-versus-leukemia (GVL) response, necessary for relapse prevention. In this study, we used clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 genome editing to delete miR-155 in primary T cells (MIR155HGΔexon3) from human donors, resulting in stable and sustained reduction in expression of miR-155. Using the xenogeneic model of acute GVHD, we show that NOD/SCID/IL2rγnull (NSG) mice receiving MIR155HGΔexon3 human T cells provide protection from lethal acute GVHD compared with mice that received human T cells with intact miR-155. MIR155HGΔexon3 human T cells persist in the recipients displaying decreased proliferation potential, reduced pathogenic T helper-1 cell population, and infiltration into GVHD target organs, such as the liver and skin. Importantly, MIR155HGΔexon3 human T cells retain GVL response significantly improving survival in an in vivo model of xeno-GVL. Altogether, we show that CRISPR/Cas9-mediated deletion of MIR155HG in primary human donor T cells is an innovative approach to generate allogeneic donor T cells that provide protection from lethal GVHD while maintaining robust antileukemic response.},
}
@article {pmid38145493,
year = {2024},
author = {Sato, R and Nanasato, Y and Takata, N and Nagano, S and Fukatsu, E and Fujino, T and Yamaguchi, K and Moriguchi, Y and Shigenobu, S and Suzuki, Y and Kasahara, M and Ueno, S},
title = {Efficient selection of a biallelic and nonchimeric gene-edited tree using Oxford Nanopore Technologies sequencing.},
journal = {Tree physiology},
volume = {44},
number = {2},
pages = {},
doi = {10.1093/treephys/tpad158},
pmid = {38145493},
issn = {1758-4469},
support = {JPJ009841//Ministry of Agriculture, Forestry and Fisheries commissioned project/ ; 19H03022//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Trees/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Nanopores ; Plants ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease system is a versatile and essential biotechnological tool in the life sciences that allows efficient genome editing. When generating gene-edited trees, T0-generation plants are often used for subsequent analysis because of the time that is required to obtain the desired mutants via crossing. However, T0-generation plants exhibit various unexpected mutations, which emphasizes the need to identify mutants with expected mutation patterns. The two critical checkpoints in this process are to confirm the expected mutation patterns in both alleles and to exclude somatic chimeric plants. In this study, we generated gene-edited Cryptomeria japonica plants and established a method to determine chimerism and mutation patterns using fragment analysis and Oxford Nanopore Technologies (ONT)-based amplicon sequencing. In the first screening, fragment analysis, i.e., indel detection via amplicon analysis, was used to predict indel mutation patterns in both alleles and to discriminate somatic chimeric plants in 188 candidate mutants. In the second screening, we precisely determined the mutation patterns and chimerism in the mutants using ONT-based amplicon sequencing, where confirmation of both alleles can be achieved using allele-specific markers flanking the single guide RNA target site. In the present study, a bioinformatic analysis procedure was developed and provided for the rapid and accurate determination of DNA mutation patterns using ONT-based amplicon sequencing. As ONT amplicon sequencing has a low running cost compared with other long-read analysis methods, such as PacBio, it is a powerful tool in plant genetics and biotechnology to select gene-edited plants with expected indel patterns in the T0-generation.},
}
@article {pmid38013500,
year = {2024},
author = {Kim, D and Kim, SH and Yoon, C and Lee, GM},
title = {Genome-wide CRISPR/Cas9 knockout screening to mitigate cell growth inhibition induced by histone deacetylase inhibitors in recombinant CHO cells.},
journal = {Biotechnology and bioengineering},
volume = {121},
number = {3},
pages = {931-941},
doi = {10.1002/bit.28611},
pmid = {38013500},
issn = {1097-0290},
support = {SRFC-MA1901-09//Samsung Research Funding Center of Samsung Electronics/ ; },
mesh = {Cricetinae ; Animals ; Cricetulus ; CHO Cells ; *CRISPR-Cas Systems ; *Histone Deacetylase Inhibitors/pharmacology ; Cell Proliferation ; },
abstract = {Histone deacetylase inhibitors (iHDACs) have been extensively studied as enhancers of therapeutic protein production in recombinant Chinese hamster ovary (CHO) (rCHO) cell cultures. However, the addition of iHDACs reduces the viable cell concentration (VCC) in rCHO cell cultures, thereby reducing their potential to enhance therapeutic protein production. To mitigate the negative effects of iHDACs on VCC, screening using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based single-gene knockout (KO) library in rCHO cells was performed in the presence of CI994, a member of iHDACs, and 10 potential KO genes that enhanced the VCC of CI994-treated rCHO cells were identified. Among these, Bcor was validated as a promising KO target that improved VCC without negatively affecting the specific productivity in the presence of CI994. Bcor KO increased the VCC and therapeutic protein concentrations in both batch and fed-batch cultures in the presence of CI994. Taken together, these findings highlight the potential of the whole-genome CRISPR/Cas9-based single-gene KO cell library to identify KO target genes for the development of iHDAC-resistant rCHO cells for enhanced therapeutic protein production.},
}
@article {pmid37516348,
year = {2024},
author = {Chen, Y and Luo, X and Kang, R and Cui, K and Ou, J and Zhang, X and Liang, P},
title = {Current therapies for osteoarthritis and prospects of CRISPR-based genome, epigenome, and RNA editing in osteoarthritis treatment.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {51},
number = {2},
pages = {159-183},
doi = {10.1016/j.jgg.2023.07.007},
pmid = {37516348},
issn = {1673-8527},
mesh = {Humans ; *Gene Editing/methods ; Epigenome ; Quality of Life ; RNA Editing ; *Osteoarthritis/genetics/therapy ; CRISPR-Cas Systems/genetics ; },
abstract = {Osteoarthritis (OA) is one of the most common degenerative joint diseases worldwide, causing pain, disability, and decreased quality of life. The balance between regeneration and inflammation-induced degradation results in multiple etiologies and complex pathogenesis of OA. Currently, there is a lack of effective therapeutic strategies for OA treatment. With the development of CRISPR-based genome, epigenome, and RNA editing tools, OA treatment has been improved by targeting genetic risk factors, activating chondrogenic elements, and modulating inflammatory regulators. Supported by cell therapy and in vivo delivery vectors, genome, epigenome, and RNA editing tools may provide a promising approach for personalized OA therapy. This review summarizes CRISPR-based genome, epigenome, and RNA editing tools that can be applied to the treatment of OA and provides insights into the development of CRISPR-based therapeutics for OA treatment. Moreover, in-depth evaluations of the efficacy and safety of these tools in human OA treatment are needed.},
}
@article {pmid37142705,
year = {2024},
author = {Davis, JR and Banskota, S and Levy, JM and Newby, GA and Wang, X and Anzalone, AV and Nelson, AT and Chen, PJ and Hennes, AD and An, M and Roh, H and Randolph, PB and Musunuru, K and Liu, DR},
title = {Efficient prime editing in mouse brain, liver and heart with dual AAVs.},
journal = {Nature biotechnology},
volume = {42},
number = {2},
pages = {253-264},
pmid = {37142705},
issn = {1546-1696},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R35 HL145203/HL/NHLBI NIH HHS/United States ; UG3 AI150551/AI/NIAID NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; R01 HL148769/HL/NHLBI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems ; Liver/metabolism ; Hepatocytes/metabolism ; Brain ; CRISPR-Cas Systems ; },
abstract = {Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.},
}
@article {pmid37095350,
year = {2024},
author = {Sun, C and Lei, Y and Li, B and Gao, Q and Li, Y and Cao, W and Yang, C and Li, H and Wang, Z and Li, Y and Wang, Y and Liu, J and Zhao, KT and Gao, C},
title = {Precise integration of large DNA sequences in plant genomes using PrimeRoot editors.},
journal = {Nature biotechnology},
volume = {42},
number = {2},
pages = {316-327},
pmid = {37095350},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems/genetics ; Base Sequence ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Genome, Plant/genetics ; Gene Editing/methods ; Plants/genetics ; DNA/metabolism ; *Oryza/genetics/metabolism ; },
abstract = {A technique for chromosomal insertion of large DNA segments is much needed in plant breeding and synthetic biology to facilitate the introduction of desired agronomic traits and signaling and metabolic pathways. Here we describe PrimeRoot, a genome editing approach to generate targeted precise large DNA insertions in plants. Third-generation PrimeRoot editors employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genomes. We demonstrate the use of PrimeRoot to accurately introduce gene regulatory elements in rice. In this study, we also integrated a gene cassette comprising PigmR, which confers rice blast resistance driven by an Act1 promoter, into a predicted genomic safe harbor site of Kitaake rice and obtain edited plants harboring the expected insertion with an efficiency of 6.3%. We found that these rice plants have increased blast resistance. These results establish PrimeRoot as a promising approach to precisely insert large segments of DNA in plants.},
}
@article {pmid38352835,
year = {2024},
author = {Kargbo, RB},
title = {Innovations in Precision Oncology: Biomarker-Driven Approaches for Enhanced Cancer Therapy.},
journal = {ACS medicinal chemistry letters},
volume = {15},
number = {2},
pages = {169-170},
pmid = {38352835},
issn = {1948-5875},
abstract = {Precision oncology is revolutionized by targeted therapies like talazoparib, a PARP inhibitor, leveraging advanced understanding of DNA repair mechanisms such as ribonucleotide excision repair and homologous recombination repair. CRISPR-Cas technology has been pivotal in unraveling these pathways, facilitating personalized treatment strategies. The identification of genomic loss of heterozygosity as a biomarker targets HRR-deficient cancers, enhancing talazoparib's efficacy. These breakthroughs represent a significant advancement in precision medicine, offering more effective, individualized cancer therapies.},
}
@article {pmid38352559,
year = {2024},
author = {Bukhari, H and Nithianandam, V and Battaglia, RA and Cicalo, A and Sarkar, S and Comjean, A and Hu, Y and Leventhal, MJ and Dong, X and Feany, MB},
title = {Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.02.02.578624},
pmid = {38352559},
abstract = {Missense mutations in the gene encoding the microtubule-associated protein tau cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human tau in experimental model organisms, including Drosophila , have been described. These models replicate key features of the human disease, but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas mediated gene editing to model frontotemporal dementia caused by the tau P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for tau P251L display age-dependent neurodegeneration, metabolic defects and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies we performed single-cell RNA sequencing on approximately 130,000 cells from brains of tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified and non-cell autonomously in glial cells. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell-type specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy, which faithfully recapitulates the genetic context and phenotypic features of the human disease and use the results of comprehensive single cell sequencing analysis to outline pathways of neurotoxicity and highlight the role of non-cell autonomous changes in glia.},
}
@article {pmid38352317,
year = {2024},
author = {Xu, S and Neupane, S and Wang, H and Pham, TP and Snyman, M and Huynh, TV and Wang, L},
title = {Efficient CRISPR genome editing and integrative genomic analyses reveal the mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.01.29.577787},
pmid = {38352317},
abstract = {Despite the revolutionary impacts of CRISPR-Cas gene editing systems, the effective and widespread use of CRISPR technologies in emerging model organisms still faces significant challenges. These include the inefficiency in generating heritable mutations at the organismal level, limited knowledge about the genomic consequences of gene editing, and an inadequate understanding of the inheritance patterns of CRISPR-Cas-induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for CRISPR editing in the microcrustacean Daphnia pulex ; 2) assessing the editing efficiency of Cas9 and Cas12a nucleases, examining mutation inheritance patterns, and analyzing the local and global mutation spectrum in the scarlet mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet underlying their swimming behavior changes. Our reengineered CRISPR microinjection method results in efficient biallelic editing with both nucleases. While indels are dominant in Cas-induced mutations, a few on-site large deletions (>1kb) are observed, most likely caused by microhomology-mediated end joining repair. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progenies. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes (e.g., NMDA1, ABAT, CNTNAP2) involved in human neurodegenerative diseases. This study expands our understanding of the dynamics of gene editing in the tractable model organism Daphnia and highlights its promising potential as a neurological disease model.},
}
@article {pmid38352164,
year = {2024},
author = {Mandal, S and Ghorai, M and Anand, U and Roy, D and Kant, N and Mishra, T and Mane, AB and Jha, NK and Lal, MK and Tiwari, RK and Kumar, M and Radha, and Ghosh, A and Bhattacharjee, R and Proćków, J and Dey, A},
title = {Corrigendum: Cytokinins: a genetic target for increasing yield potential in the CRISPR era.},
journal = {Frontiers in genetics},
volume = {15},
number = {},
pages = {1360528},
doi = {10.3389/fgene.2024.1360528},
pmid = {38352164},
issn = {1664-8021},
abstract = {[This corrects the article DOI: 10.3389/fgene.2022.883930.].},
}
@article {pmid38351451,
year = {2024},
author = {Obermeier, M and Rogiers, V and Vanhaecke, T and Baert, Y},
title = {Lipofection-Based Delivery of CRISPR/Cas9 Ribonucleoprotein for Gene Editing in Male Germline Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2770},
number = {},
pages = {123-134},
pmid = {38351451},
issn = {1940-6029},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Ribonucleoproteins/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Germ Cells/metabolism ; },
abstract = {Gene editing in the murine germline is a valuable approach to investigate germ cell maturation and generate mouse models. Several studies demonstrated that CRISPR/Cas9 alters the genome of cultured male mouse germline stem cells delivered by electroporation of plasmids. Recently, we showed proof-of-principle that gene knockout can be effectively targeted in mouse germline stem cells by lipofecting Cas9:gRNA ribonucleoproteins. In this protocol, we describe a simple, fast, and cheap workflow for gene editing via the lipofection of non-integrative ribonucleoproteins in murine male germline stem cells.},
}
@article {pmid38350240,
year = {2024},
author = {Kim, H and Jang, H and Song, J and Lee, SM and Lee, S and Kwon, HJ and Kim, S and Kang, T and Park, HG},
title = {A CRISPR/Cas12 trans-cleavage reporter enabling label-free colorimetric detection of SARS-CoV-2 and its variants.},
journal = {Biosensors &amp; bioelectronics},
volume = {251},
number = {},
pages = {116102},
doi = {10.1016/j.bios.2024.116102},
pmid = {38350240},
issn = {1873-4235},
abstract = {We present a label-free colorimetric CRISPR/Cas-based method enabling affordable molecular diagnostics for SARS-CoV-2. This technique utilizes 3,3'-diethylthiadicarbocyanine iodide (DISC2(5)) which exhibits a distinct color transition from purple to blue when it forms dimers by inserting into the duplex of the thymidine adenine (TA) repeat sequence. Loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) was used to amplify target samples, which were subsequently subjected to the CRISPR/Cas12a system. The target amplicons would activate Cas12a to degrade nearby TA repeat sequences, preserving DISC2(5) in its free form to display purple as opposed to blue in the absence of the target. Based on this design approach, SARS-CoV-2 RNA was colorimetrically detected very sensitively down to 2 copies/μL, and delta and omicron variants of SARS-CoV-2 were also successfully identified. The practical diagnostic utility of this method was further validated by reliably identifying 179 clinical samples including 20 variant samples with 100% clinical sensitivity and specificity. This technique has the potential to become a promising CRISPR-based colorimetric platform for molecular diagnostics of a wide range of target pathogens.},
}
@article {pmid38284796,
year = {2024},
author = {Xia, R and Ouyang, N and Wang, T and Zhuang, Y and Miao, P},
title = {Electrochemical detection of glutathione based on accelerated CRISPR/Cas12a trans-cleavage with MnO2 nanosheets.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {15},
pages = {2034-2037},
doi = {10.1039/d3cc06186h},
pmid = {38284796},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; Oxides ; Manganese Compounds ; Glutathione/analysis ; *Biosensing Techniques ; },
abstract = {The CRISPR/Cas12a system is accelerated by glutathione-mediated reduction of MnO2 nanosheets. By monitoring the trans-cleavage of the DNA probe, an electrochemical method for glutathione assay is fabricated, with the detection limit of 3.5 pM. It provides a promising tool for plasma analysis with satisfactory performance, indicating the broad application prospects of this glutathione assay.},
}
@article {pmid38263276,
year = {2024},
author = {Huang, ME and Qin, Y and Shang, Y and Hao, Q and Zhan, C and Lian, C and Luo, S and Liu, LD and Zhang, S and Zhang, Y and Wo, Y and Li, N and Wu, S and Gui, T and Wang, B and Luo, Y and Cai, Y and Liu, X and Xu, Z and Dai, P and Li, S and Zhang, L and Dong, J and Wang, J and Zheng, X and Xu, Y and Sun, Y and Wu, W and Yeap, LS and Meng, FL},
title = {C-to-G editing generates double-strand breaks causing deletion, transversion and translocation.},
journal = {Nature cell biology},
volume = {26},
number = {2},
pages = {294-304},
pmid = {38263276},
issn = {1476-4679},
support = {32325019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31970880//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32090040//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32170884//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31722020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 81861138014//National Natural Science Foundation of China (National Science Foundation of China)/ ; HS2021SHZX001//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 23XD1424000//Natural Science Foundation of Shanghai (Natural Science Foundation of Shanghai Municipality)/ ; JCTD-2020-17//Chinese Academy of Sciences (CAS)/ ; },
mesh = {Humans ; *Translocation, Genetic ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair/genetics ; CRISPR-Cas Systems ; *Alkanesulfonic Acids ; },
abstract = {Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.},
}
@article {pmid38349963,
year = {2024},
author = {Ghouneimy, A and Ali, Z and Aman, R and Jiang, W and Aouida, M and Mahfouz, M},
title = {CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00655},
pmid = {38349963},
issn = {2161-5063},
abstract = {The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a CRISPR-Cas multiplexed diagnostic assay (CRISPRD) to detect cervical cancer-causing hrHPVs in one reaction (one-pot assay). We harnessed the compatibility of thermostable AapCas12b, TccCas13a, and HheCas13a nucleases with isothermal amplification and successfully detected HPV16 and HPV18, along with an internal control in a single-pot assay with a limit of detection of 10 copies and 100% specificity. This platform offers a rapid and practical solution for the multiplex detection of hrHPVs, which may facilitate large-scale hrHPV point-of-care screening. Furthermore, the CRISPRD platform programmability enables it to be adapted for the multiplex detection of any two nucleic acid biomarkers as well as internal control.},
}
@article {pmid38349260,
year = {2024},
author = {Tang, S and Zhu, H and Sheng, L and Mu, Q and Wang, Y and Xu, K and Zhou, M and Xu, Z and Wu, A and Ouyang, G},
title = {CALCRL knockdown suppresses cancer stemness and chemoresistance in acute myeloid leukemia with FLT3-ITD and DNM3TA-R882 double mutations.},
journal = {Drug development research},
volume = {85},
number = {1},
pages = {e22137},
doi = {10.1002/ddr.22137},
pmid = {38349260},
issn = {1098-2299},
support = {//Medical Health Science and Technology Project of Zhejiang Provincial Health Commission/ ; //Ningbo Yinzhou District Agricultural and Social Development Science and Technology project/ ; //Natural Science Foundation of Ningbo Municipality/ ; },
mesh = {Animals ; Humans ; *Receptors, Calcitonin ; Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Leukemia, Myeloid, Acute/drug therapy/genetics ; Mutation ; Cytarabine ; fms-Like Tyrosine Kinase 3/genetics ; Calcitonin Receptor-Like Protein ; },
abstract = {Acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (FLT3-ITD) and DNA methyltransferase 3A (DNMT3A) R882 double mutations had a worse prognosis compared with AML with FLT3-ITD or DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL) in AML with FLT3-ITD and DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITD and DNMT3A-R882 double-mutated AML cell model. Quantitative real-time PCR and Western blot assay were carried out to examine corresponding gene and protein expression. Methylation of CALCRL promoter was measured by methylation-specific PCR (MSP). Cell viability, colony formation, flow cytometry, and sphere formation assays were conducted to determine cell proliferation, apoptosis, and stemness. MOLM13 cells were exposed to stepwise increasing concentrations of cytarabine (Ara-C) to generate MOLM13/Ara-C cells. An in vivo AML animal model was established, and the tumor volume and weight were recorded. TUNEL assay was adopted to examine cell apoptosis in tumor tissues. DNMT3A-R882 mutation upregulated the expression of CALCRL while downregulated the DNA methylation level of CALCRL in MOLM13 cells. CALCRL knockdown greatly inhibited cell proliferation, promoted apoptosis and repressed cell stemness, accompanied with the downregulated Oct4, SOX2, and Nanog in DNMT3A-R882-mutated MOLM13 cells and MOLM13/Ara-C cells. Furthermore, CALCRL knockdown restricted tumor growth and the chemoresistance of AML in vivo, as well as inducing cell apoptosis in tumor tissues. Together, these data reveal that CALCRL is a vital regulator of leukemia cell survival and resistance to chemotherapy, suggesting CALCRL as a promising therapeutic target for the treatment of FTL3-ITD and DNMT3A-R882 double-mutated AML.},
}
@article {pmid38346980,
year = {2024},
author = {Pavličev, M and McDonough-Goldstein, CE and Zupan, AM and Muglia, L and Hu, YC and Kong, F and Monangi, N and Dagdas, G and Zupančič, N and Maziarz, J and Sinner, D and Zhang, G and Wagner, G and Muglia, L},
title = {A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1152},
pmid = {38346980},
issn = {2041-1723},
mesh = {Pregnancy ; Female ; Humans ; Animals ; Mice ; *Endometriosis/genetics/metabolism ; Alleles ; Endometrium/metabolism ; Estrogens/metabolism ; *Neoplasms/genetics ; Wnt4 Protein/genetics ; },
abstract = {The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.},
}
@article {pmid38346683,
year = {2024},
author = {Boob, AG and Zhu, Z and Intasian, P and Jain, M and Petrov, VA and Lane, ST and Tan, SI and Xun, G and Zhao, H},
title = {CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae062},
pmid = {38346683},
issn = {1362-4962},
support = {DE-SC0018420//U.S. Department of Energy/ ; },
abstract = {The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.},
}
@article {pmid38346597,
year = {2024},
author = {Cui, Y and Qu, X},
title = {CRISPR-Cas systems of lactic acid bacteria and applications in food science.},
journal = {Biotechnology advances},
volume = {71},
number = {},
pages = {108323},
doi = {10.1016/j.biotechadv.2024.108323},
pmid = {38346597},
issn = {1873-1899},
abstract = {CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) systems are widely distributed in lactic acid bacteria (LAB), contributing to their RNA-mediated adaptive defense immunity. The CRISPR-Cas-based genetic tools have exhibited powerful capability. It has been highly utilized in different organisms, accelerating the development of life science. The review summarized the components, adaptive immunity mechanisms, and classification of CRISPR-Cas systems; analyzed the distribution and characteristics of CRISPR-Cas system in LAB. The review focuses on the development of CRISPR-Cas-based genetic tools in LAB for providing latest development and future trend. The diverse and broad applications of CRISPR-Cas systems in food/probiotic industry are introduced. LAB harbor a plenty of CRISPR-Cas systems, which contribute to generate safer and more robust strains with increased resistance against bacteriophage and prevent the dissemination of plasmids carrying antibiotic-resistance markers. Furthermore, the CRISPR-Cas system from LAB could be used to exploit novel, flexible, programmable genome editing tools of native host and other organisms, resolving the limitation of genetic operation of some LAB species, increasing the important biological functions of probiotics, improving the adaptation of probiotics in complex environments, and inhibiting the growth of foodborne pathogens. The development of the genetic tools based on CRISPR-Cas system in LAB, especially the endogenous CRISPR-Cas system, will open new avenues for precise regulation, rational design, and flexible application of LAB.},
}
@article {pmid38346278,
year = {2024},
author = {Zhou, L and Simonian, AL},
title = {CRISPR/Cas Technology: The Unique Synthetic Biology Genome-Editing Tool Shifting the Paradigm in Viral Diagnostics, Defense, and Therapeutics.},
journal = {Annual review of biomedical engineering},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-bioeng-081723-013033},
pmid = {38346278},
issn = {1545-4274},
abstract = {The emergence of the COVID-19 pandemic has starkly exposed our significantly limited ability to promptly identify and respond to emergent biological threats. Consequently, there is an urgent need to advance biotechnological methods for addressing both known and unforeseen biological hazards. Recently, the CRISPR/Cas system has revolutionized genetic engineering, enabling precise and efficient synthetic biology applications. Therefore, this review aims to provide a comprehensive introduction to the fundamental principles underlying the CRISPR/Cas system and assess the advantages and limitations of various CRISPR/Cas-based techniques applicable to the detection of, defense against, and treatment of viral infections. These techniques include viral diagnostics, the development of antiviral vaccines, B cell engineering for antibody production, viral activation/interference, and epigenetic modifications. Furthermore, this review delves into the challenges and bioethical considerations associated with use of the CRISPR/Cas system. With the continuous evolution of technology, the CRISPR/Cas system holds considerable promise for addressing both existing and unforeseen biological threats. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 26 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}
@article {pmid38345258,
year = {2024},
author = {Xiao, Y and Yuan, Y and Yadlapalli, S},
title = {One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {203},
pages = {},
doi = {10.3791/64729},
pmid = {38345258},
issn = {1940-087X},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Drosophila/genetics ; Plasmids ; },
abstract = {The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.},
}
@article {pmid38345242,
year = {2024},
author = {Choi, N and Schlücker, S},
title = {Convergence of Surface-Enhanced Raman Scattering with Molecular Diagnostics: A Perspective on Future Directions.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.3c11370},
pmid = {38345242},
issn = {1936-086X},
abstract = {Molecular diagnostics (MD) is widely employed in multiple scientific disciplines, such as oncology, pathogen detection, forensic investigations, and the pharmaceutical industry. Techniques such as polymerase chain reaction (PCR) revolutionized the rapid and accurate identification of nucleic acids (DNA, RNA). More recently, CRISPR and its CRISPR-associated protein (Cas) have been a ground-breaking discovery that is the latest revolution in molecular biology, including MD. Surface-enhanced Raman scattering (SERS) is a very attractive alternative to fluorescence as the currently most widely used optical readout in MD. In this Perspective, milestones in the development of MD, SERS-PCR, and next-generation approaches to MD, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), are briefly summarized. Our perspective on the future convergence of SERS with MD is focused on SERS-based CRISPR/Cas (SERS-CRISPR) since we anticipate many promising applications in this rapidly emerging field. We predict that major future developments will exploit the advantages of real-time monitoring with the superior brightness, photostability, and spectral multiplexing potential of SERS nanotags in an automated workflow for rapid assays under isothermal, amplification-free conditions.},
}
@article {pmid38345028,
year = {2024},
author = {Poore, CP and Wei, S and Chen, B and Low, SW and Tan, JSQ and Lee, AT and Nilius, B and Liao, P},
title = {In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel.},
journal = {Journal of drug targeting},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/1061186X.2024.2313522},
pmid = {38345028},
issn = {1029-2330},
abstract = {BACKGROUND: Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4.<br><br>METHODS: We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence.<br><br>RESULTS: In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment.<br><br>CONCLUSION: M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.},
}
@article {pmid38344439,
year = {2024},
author = {Allemailem, KS},
title = {Recent Advances in Understanding the Molecular Mechanisms of Multidrug Resistance and Novel Approaches of CRISPR/Cas9-Based Genome-Editing to Combat This Health Emergency.},
journal = {International journal of nanomedicine},
volume = {19},
number = {},
pages = {1125-1143},
pmid = {38344439},
issn = {1178-2013},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; CRISPR-Associated Protein 9 ; Bacteria/genetics ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics ; },
abstract = {The rapid spread of multidrug resistance (MDR), due to abusive use of antibiotics has led to global health emergency, causing substantial morbidity and mortality. Bacteria attain MDR by different means such as antibiotic modification/degradation, target protection/modification/bypass, and enhanced efflux mechanisms. The classical approaches of counteracting MDR bacteria are expensive and time-consuming, thus, it is highly significant to understand the molecular mechanisms of this resistance to curb the problem from core level. The revolutionary approach of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated sequence 9 (CRISPR/Cas9), considered as a next-generation genome-editing tool presents an innovative opportunity to precisely target and edit bacterial genome to alter their MDR strategy. Different bacteria possessing antibiotic resistance genes such as mecA, ermB, ramR, tetA, mqrB and blaKPC that have been targeted by CRISPR/Cas9 to re-sensitize these pathogens against antibiotics, such as methicillin, erythromycin, tigecycline, colistin and carbapenem, respectively. The CRISPR/Cas9 from S. pyogenes is the most widely studied genome-editing tool, consisting of a Cas9 DNA endonuclease associated with tracrRNA and crRNA, which can be systematically coupled as sgRNA. The targeting strategies of CRISPR/Cas9 to bacterial cells is mediated through phage, plasmids, vesicles and nanoparticles. However, the targeting approaches of this genome-editing tool to specific bacteria is a challenging task and still remains at a very preliminary stage due to numerous obstacles awaiting to be solved. This review elaborates some recent updates about the molecular mechanisms of antibiotic resistance and the innovative role of CRISPR/Cas9 system in modulating these resistance mechanisms. Furthermore, the delivery approaches of this genome-editing system in bacterial cells are discussed. In addition, some challenges and future prospects are also described.},
}
@article {pmid38343627,
year = {2024},
author = {Bleriot, I and Pacios, O and Blasco, L and Fernández-García, L and López, M and Ortiz-Cartagena, C and Barrio-Pujante, A and García-Contreras, R and Pirnay, JP and Wood, TK and Tomás, M},
title = {Improving phage therapy by evasion of phage resistance mechanisms.},
journal = {JAC-antimicrobial resistance},
volume = {6},
number = {1},
pages = {dlae017},
pmid = {38343627},
issn = {2632-1823},
abstract = {Antibiotic failure is one of the most worrisome threats to global health. Among the new therapeutic efforts that are being explored, the use of bacteriophages (viruses that kill bacteria), also known as 'phages', is being extensively studied as a strategy to target bacterial pathogens. However, one of the main drawbacks of phage therapy is the plethora of defence mechanisms that bacteria use to defend themselves against phages. This review aims to summarize the therapeutic approaches that are being evaluated to overcome the bacterial defence systems, including the most innovative therapeutic approaches applied: circumvention of phage receptor mutations; modification of prophages; targeting of CRISPR-Cas systems and the biofilm matrix; engineering of safer and more efficacious phages; and inhibition of the anti-persister strategies used by bacteria.},
}
@article {pmid38340341,
year = {2024},
author = {Irmisch, P and Mogila, I and Samatanga, B and Tamulaitis, G and Seidel, R},
title = {Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae080},
pmid = {38340341},
issn = {1362-4962},
support = {GA 724863/ERC_/European Research Council/International ; },
abstract = {The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.},
}
@article {pmid38340191,
year = {2024},
author = {Leng, F and Zhou, G and Shi, R and Liu, C and Lin, Y and Yu, X and Zhang, Y and He, X and Liu, Z and Sun, M and Bao, F and Hu, Y and He, Y},
title = {Development of PEG-mediated genetic transformation and gene editing system of Bryum argenteum as an abiotic stress tolerance model plant.},
journal = {Plant cell reports},
volume = {43},
number = {3},
pages = {63},
pmid = {38340191},
issn = {1432-203X},
support = {91631109 to Y.He//Major Research Plan of National Nature Science Foundation of China/ ; No.31970658 Y.He//General Program of National Natural Science Foundation of China/ ; },
mesh = {Gene Editing ; *Bryopsida/genetics ; *Bryophyta/genetics ; Stress, Physiological/genetics ; Transformation, Genetic ; CRISPR-Cas Systems/genetics ; Protoplasts ; },
abstract = {To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.},
}
@article {pmid38297130,
year = {2024},
author = {Li, Y and Ran, Q and Duan, Q and Jin, J and Wang, Y and Yu, L and Wang, C and Zhu, Z and Chen, X and Weng, L and Li, Z and Wang, J and Wu, Q and Wang, H and Tian, H and Song, S and Shan, Z and Zhai, Q and Qin, H and Chen, S and Fang, L and Yin, H and Zhou, H and Jiang, X and Wang, P},
title = {7-Dehydrocholesterol dictates ferroptosis sensitivity.},
journal = {Nature},
volume = {626},
number = {7998},
pages = {411-418},
pmid = {38297130},
issn = {1476-4687},
mesh = {Humans ; Cell Membrane/metabolism ; Cholesterol/biosynthesis/metabolism ; CRISPR-Cas Systems/genetics ; *Dehydrocholesterols/metabolism ; *Ferroptosis ; Genome, Human ; Kidney Diseases/metabolism ; Mitochondrial Membranes/metabolism ; Neoplasm Metastasis ; Neoplasms/metabolism/pathology ; Phospholipids/metabolism ; Reperfusion Injury/metabolism ; },
abstract = {Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer[1-3], degenerative disorders[4] and organ ischaemia-reperfusion injury (IRI)[5,6]. Here, using genome-wide CRISPR-Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)-an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1, CYP51A1, EBP and SC5D, function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI.},
}
@article {pmid38215750,
year = {2024},
author = {Pacini, C and Duncan, E and Gonçalves, E and Gilbert, J and Bhosle, S and Horswell, S and Karakoc, E and Lightfoot, H and Curry, E and Muyas, F and Bouaboula, M and Pedamallu, CS and Cortes-Ciriano, I and Behan, FM and Zalmas, LP and Barthorpe, A and Francies, H and Rowley, S and Pollard, J and Beltrao, P and Parts, L and Iorio, F and Garnett, MJ},
title = {A comprehensive clinically informed map of dependencies in cancer cells and framework for target prioritization.},
journal = {Cancer cell},
volume = {42},
number = {2},
pages = {301-316.e9},
doi = {10.1016/j.ccell.2023.12.016},
pmid = {38215750},
issn = {1878-3686},
mesh = {Humans ; *Genetic Testing ; Phenotype ; Drug Discovery ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; CRISPR-Cas Systems ; },
abstract = {Genetic screens in cancer cell lines inform gene function and drug discovery. More comprehensive screen datasets with multi-omics data are needed to enhance opportunities to functionally map genetic vulnerabilities. Here, we construct a second-generation map of cancer dependencies by annotating 930 cancer cell lines with multi-omic data and analyze relationships between molecular markers and cancer dependencies derived from CRISPR-Cas9 screens. We identify dependency-associated gene expression markers beyond driver genes, and observe many gene addiction relationships driven by gain of function rather than synthetic lethal effects. By combining clinically informed dependency-marker associations with protein-protein interaction networks, we identify 370 anti-cancer priority targets for 27 cancer types, many of which have network-based evidence of a functional link with a marker in a cancer type. Mapping these targets to sequenced tumor cohorts identifies tractable targets in different cancer types. This target prioritization map enhances understanding of gene dependencies and identifies candidate anti-cancer targets for drug development.},
}
@article {pmid38344320,
year = {2022},
author = {Javadi, M and Sazegar, H and Doosti, A},
title = {Impact of NOX4 Knockout by CRISPR/Cas9 on the MCF-7, HCA-7 and UM-RC-6 Cancer Cells.},
journal = {Iranian journal of biotechnology},
volume = {20},
number = {4},
pages = {e3115},
pmid = {38344320},
issn = {1728-3043},
abstract = {BACKGROUND: The second most common cause of mortality is cancer. Increased NOX4 expression is linked to cancer development and metastasis. However, the significance of NOX4 in cell growth and assault, remains unclear.<br><br>OBJECTIVE: This study aimed to evaluate the effect of NOX4 knockouts in MCF7, UM-RC-6, HCA-7 cell lines.<br><br>MATERIALS AND METHODS: The NOX4 gene was knocked out in MCF7, UM-RC-6, and HCA-7 cell lines through using CRISPR Cas-9 genetic engineering techniques. After transfection, the CRISPR Cas-9 cassette, the T7 endonuclease I, qPCR, and western blotting assay detected the NOX4 knockouts. MTT and Annexin assessed the percentage of cell proliferation and apoptosis. Real-time PCR was used to measure the expression of pro- and anti-apoptotic genes.<br><br>RESULTS: Occurrence of NOX4 gene knockout in the examined cell lines, was confirmed by q-PCR and Western blot (P<0.001). The NOX4-deleted cell lines with increased sub-G1 caused lowered cell proliferation and population at S / G2/ M phases. In Vitro, NOX4 silencing caused lowered expressions of anti-apoptosis genes BCL-2 and SURVIVIN&#xa0;(P<0.0001), leading to increased tendency of apoptosis in the cell lines (P<0.0001) of the apoptotic genes BAX, P53, FAS. Additionally, the MTT and Annexin results of the target gene NOX4 knockout inhibited proliferation, increased mortality rates (P<0.01), and increased apoptosis.<br><br>CONCLUSION: The findings of this study indicate that using NOX4 as a target can have therapeutic value for creating potential treatments against breast, colorectal, and kidney cancers which shows a need for a deeper understanding of the biology of these cancers with direct clinical outcomes for developing novel treatment strategies.},
}
@article {pmid38339188,
year = {2024},
author = {Li, S and Lao, J and Sun, Y and Hua, X and Lin, P and Wang, F and Shen, G and Zhao, P and Xia, Q},
title = {CRISPR/Cas9-Mediated Editing of BmEcKL1 Gene Sequence Affected Silk Gland Development of Silkworms (Bombyx mori).},
journal = {International journal of molecular sciences},
volume = {25},
number = {3},
pages = {},
pmid = {38339188},
issn = {1422-0067},
support = {2022YFD1201600//National Key Research and Development Program of China/ ; 32030103//National Natural Science Foundation of China/ ; 32172798//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Bombyx/metabolism ; Silk/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Insect Proteins/genetics/metabolism ; },
abstract = {The silkworm (Bombyx mori) has served humankind through silk protein production. However, traditional sericulture and the silk industry have encountered considerable bottlenecks and must rely on major technological breakthroughs to keep up with the current rapid developments. The adoption of gene editing technology has nevertheless brought new hope to traditional sericulture and the silk industry. The long period and low efficiency of traditional genetic breeding methods to obtain high silk-yielding silkworm strains have hindered the development of the sericulture industry; the use of gene editing technology to specifically control the expression of genes related to silk gland development or silk protein synthesis is beneficial for obtaining silkworm strains with excellent traits. In this study, BmEcKL1 was specifically knocked out in the middle (MSGs) and posterior (PSGs) silk glands using CRISPR/Cas9 technology, and ΔBmEcKL1-MSG and ΔBmEcKL1-PSG strains with improved MSGs and PSGs and increased silk production were obtained. This work identifies and proves that BmEcKL1 directly or indirectly participates in silk gland development and silk protein synthesis, providing new perspectives for investigating silk gland development and silk protein synthesis mechanisms in silkworms, which is of great significance for selecting and breeding high silk-yielding silkworm varieties.},
}
@article {pmid38338978,
year = {2024},
author = {Salman, A and McClements, ME and MacLaren, RE},
title = {CRISPR Manipulation of Age-Related Macular Degeneration Haplotypes in the Complement System: Potential Future Therapeutic Applications/Avenues.},
journal = {International journal of molecular sciences},
volume = {25},
number = {3},
pages = {},
pmid = {38338978},
issn = {1422-0067},
mesh = {Humans ; Aged ; Haplotypes ; *Complement Factor B ; *Macular Degeneration/genetics/therapy/pathology ; Complement Activation/genetics ; Risk Factors ; Polymorphism, Single Nucleotide ; },
abstract = {Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among the elderly in the developed world. Whilst AMD is a multifactorial disease, the involvement of the complement system in its pathology is well documented, with single-nucleotide polymorphisms (SNPs) in different complement genes representing an increased risk factor. With several complement inhibitors explored in clinical trials showing limited success, patients with AMD are still without a reliable treatment option. This indicates that there is still a gap of knowledge in the functional implications and manipulation of the complement system in AMD, hindering the progress towards translational treatments. Since the discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool, the field of molecular biology has been revolutionised. Genetic variants in the complement system have long been associated with an increased risk of AMD, and a variety of haplotypes have been identified to be predisposing/protective, with variation in complement genes believed to be the trigger for dysregulation of the cascade leading to inflammation. AMD-haplotypes (SNPs) alter specific aspects of the activation and regulation of the complement cascade, providing valuable insights into the pathogenic mechanisms of AMD with important diagnostic and therapeutic implications. The effect of targeting these AMD-related SNPs on the regulation of the complement cascade has been poorly explored, and the CRISPR/Cas system provides an ideal tool with which to explore this avenue. Current research concentrates on the association events of specific AMD-related SNPs in complement genes without looking into the effect of targeting these SNPs and therefore influencing the complement system in AMD pathogenesis. This review will explore the current understanding of manipulating the complement system in AMD pathogenesis utilising the genomic manipulation powers of the CRISPR/Cas systems. A number of AMD-related SNPs in different complement factor genes will be explored, with a particular emphasis on factor H (CFH), factor B (CFB), and complement C3 (C3).},
}
@article {pmid38338898,
year = {2024},
author = {Evmenov, K and Pustogarov, N and Panteleev, D and Safin, A and Alkalaeva, E},
title = {An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein.},
journal = {International journal of molecular sciences},
volume = {25},
number = {3},
pages = {},
pmid = {38338898},
issn = {1422-0067},
support = {075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Ribonucleoproteins/genetics/metabolism ; Gene Editing/methods ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).},
}
@article {pmid38336977,
year = {2024},
author = {Kim, CH and Lee, WJ and Oh, Y and Lee, Y and Lee, HK and Seong, JB and Lim, KS and Park, SJ and Huh, JW and Kim, YH and Kim, KM and Hur, JK and Lee, SH},
title = {Utilization of nicking properties of CRISPR-Cas12a effector for genome editing.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {3352},
pmid = {38336977},
issn = {2045-2322},
support = {KGM5282322, KGM4562323, KGM5382221//the Korea Research Institute of Bioscience and Biotechnology (KRIBB; Research Initiative Program)/ ; KGM5282322, KGM4562323, KGM5382221//the Korea Research Institute of Bioscience and Biotechnology (KRIBB; Research Initiative Program)/ ; KGM5282322, KGM4562323, KGM5382221//the Korea Research Institute of Bioscience and Biotechnology (KRIBB; Research Initiative Program)/ ; 2022R1A2C4001609, 2020R1C1C1010869, 2021M3A9I4024452//Korean Ministry of Education, Science and Technology/ ; 2022R1A2C4001609, 2020R1C1C1010869, 2021M3A9I4024452//Korean Ministry of Education, Science and Technology/ ; 2022R1A2C4001609, 2020R1C1C1010869, 2021M3A9I4024452//Korean Ministry of Education, Science and Technology/ ; (22A0203L1)//the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health &amp; Welfare)./ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; Mutation ; DNA Breaks, Double-Stranded ; },
abstract = {The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.},
}
@article {pmid38336900,
year = {2024},
author = {Lin, JY and Liu, YC and Tseng, YH and Chan, MT and Chang, CC},
title = {TALE-based organellar genome editing and gene expression in plants.},
journal = {Plant cell reports},
volume = {43},
number = {3},
pages = {61},
pmid = {38336900},
issn = {1432-203X},
support = {NSTC 112-2313-B-006-004//National Science and Technology Council/ ; },
mesh = {*Gene Editing/methods ; *Transcription Activator-Like Effectors/genetics ; CRISPR-Cas Systems/genetics ; Plants/genetics ; Organelles/genetics ; Gene Expression ; Genome, Plant/genetics ; },
abstract = {TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.},
}
@article {pmid38335532,
year = {2024},
author = {Chen, F and Du, H and Tao, M and Xu, L and Wang, C and White, JC and Wang, Z and Xing, B},
title = {Nitrogen-Doped Carbon Dots Facilitate CRISPR/Cas for Reducing Antibiotic Resistance Genes in the Environment.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c08558},
pmid = {38335532},
issn = {1520-5118},
abstract = {The continued acquisition and propagation of antibiotic resistance genes (ARGs) in the environment confound efforts to manage the global rise in antibiotic resistance. Here, CRISPR-Cas9/sgRNAs carried by nitrogen-doped carbon dots (NCDs) were developed to precisely target multi-"high-risk" ARGs (tet, cat, and aph(3')-Ia) commonly detected in the environment. NCDs facilitated the delivery of Cas9/sgRNAs to Escherichia coli (E. coli) without cytotoxicity, achieving sustained elimination of target ARGs. The elimination was optimized using different weight ratios of NCDs and Cas9 protein (1:1, 1:20, and 1:40), and Cas9/multi sgRNAs were designed to achieve multi-cleavage of ARGs in either a single strain or mixed populations. Importantly, NCDs successfully facilitated Cas9/multi sgRNAs for resensitization of antibiotic-resistant bacteria in soil (approaching 50%), whereas Cas9/multi sgRNAs alone were inactivated in the complex environment. This work highlights the potential of a fast and precise strategy to minimize the reservoir of antibiotic resistance in agricultural system.},
}
@article {pmid38334670,
year = {2024},
author = {Gao, S and He, L and Lam, CK and Taylor, MRG and Mestroni, L and Lombardi, R and Chen, SN},
title = {Filamin C Deficiency Impairs Sarcomere Stability and Activates Focal Adhesion Kinase through PDGFRA Signaling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.},
journal = {Cells},
volume = {13},
number = {3},
pages = {},
pmid = {38334670},
issn = {2073-4409},
support = {R01HL147064/NH/NIH HHS/United States ; R01 HL147064/HL/NHLBI NIH HHS/United States ; T32 HL007822/HL/NHLBI NIH HHS/United States ; R01 HL164634/HL/NHLBI NIH HHS/United States ; T32 5T32HL007822-23/NH/NIH HHS/United States ; },
mesh = {Humans ; Myocytes, Cardiac/metabolism ; Sarcomeres/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Focal Adhesion Protein-Tyrosine Kinases/metabolism ; Filamins/genetics/metabolism ; *Cardiomyopathies/metabolism ; Receptor Protein-Tyrosine Kinases/metabolism ; Signal Transduction ; },
abstract = {Truncating mutations in filamin C (FLNC) are associated with dilated cardiomyopathy and arrhythmogenic cardiomyopathy. FLNC is an actin-binding protein and is known to interact with transmembrane and structural proteins; hence, the ablation of FLNC in cardiomyocytes is expected to dysregulate cell adhesion, cytoskeletal organization, sarcomere structural integrity, and likely nuclear function. Our previous study showed that the transcriptional profiles of FLNC homozygous deletions in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly comparable to the transcriptome profiles of hiPSC-CMs from patients with FLNC truncating mutations. Therefore, in this study, we used CRISPR-Cas-engineered hiPSC-derived FLNC knockout cardiac myocytes as a model of FLNC cardiomyopathy to determine pathogenic mechanisms and to examine structural changes caused by FLNC deficiency. RNA sequencing data indicated the significant upregulation of focal adhesion signaling and the dysregulation of thin filament genes in FLNC-knockout (FLNC[KO]) hiPSC-CMs compared to isogenic hiPSC-CMs. Furthermore, our findings suggest that the complete loss of FLNC in cardiomyocytes led to cytoskeletal defects and the activation of focal adhesion kinase. Pharmacological inhibition of PDGFRA signaling using crenolanib (an FDA-approved drug) reduced focal adhesion kinase activation and partially normalized the focal adhesion signaling pathway. The findings from this study suggest the opportunity in repurposing FDA-approved drug as a therapeutic strategy to treat FLNC cardiomyopathy.},
}
@article {pmid38334653,
year = {2024},
author = {Aoki, K and Yamasaki, M and Umezono, R and Hamamoto, T and Kamachi, Y},
title = {Systematic Comparison of Computational Tools for Sanger Sequencing-Based Genome Editing Analysis.},
journal = {Cells},
volume = {13},
number = {3},
pages = {},
pmid = {38334653},
issn = {2073-4409},
support = {JSPS KAKENHI 21K06202//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; INDEL Mutation/genetics ; Genome/genetics ; Genomics ; },
abstract = {Successful genome editing depends on the cleavage efficiency of programmable nucleases (PNs) such as the CRISPR-Cas system. Various methods have been developed to assess the efficiency of PNs, most of which estimate the occurrence of indels caused by PN-induced double-strand breaks. In these methods, PN genomic target sites are amplified through PCR, and the resulting PCR products are subsequently analyzed using Sanger sequencing, high-throughput sequencing, or mismatch detection assays. Among these methods, Sanger sequencing of PCR products followed by indel analysis using online web tools has gained popularity due to its user-friendly nature. This approach estimates indel frequencies by computationally analyzing sequencing trace data. However, the accuracy of these computational tools remains uncertain. In this study, we compared the performance of four web tools, TIDE, ICE, DECODR, and SeqScreener, using artificial sequencing templates with predetermined indels. Our results demonstrated that these tools were able to estimate indel frequency with acceptable accuracy when the indels were simple and contained only a few base changes. However, the estimated values became more variable among the tools when the sequencing templates contained more complex indels or knock-in sequences. Moreover, although these tools effectively estimated the net indel sizes, their capability to deconvolute indel sequences exhibited variability with certain limitations. These findings underscore the importance of judiciously selecting and using an appropriate tool with caution, depending on the type of genome editing being performed.},
}
@article {pmid38334640,
year = {2024},
author = {Fu, L and Wang, S and Liu, L and Shibata, Y and Okada, M and Luu, N and Shi, YB},
title = {Simplifying Genotyping of Mutants from Genome Editing with a Parallel qPCR-Based iGenotype Index.},
journal = {Cells},
volume = {13},
number = {3},
pages = {},
pmid = {38334640},
issn = {2073-4409},
support = {Intramural Research Program of NICHD/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Genotype ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; DNA Repair ; },
abstract = {Targeted genome editing is a powerful tool in reverse genetic studies of gene function in many aspects of biological and pathological processes. The CRISPR/Cas system or engineered endonucleases such as ZFNs and TALENs are the most widely used genome editing tools that are introduced into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, triggering cellular DNA repair through either homologous recombination or non-homologous end joining (NHEJ). DNA repair through the NHEJ mechanism is usually error-prone, leading to point mutations or indels (insertions and deletions) within the targeted region. Some of the mutations in embryos are germline transmissible, thus providing an effective way to generate model organisms with targeted gene mutations. However, point mutations and short indels are difficult to be effectively genotyped, often requiring time-consuming and costly DNA sequencing to obtain reliable results. Here, we developed a parallel qPCR assay in combination with an iGenotype index to allow simple and reliable genotyping. The genotype-associated iGenotype indexes converged to three simple genotype-specific constant values (1, 0, -1) regardless of allele-specific primers used in the parallel qPCR assays or gene mutations at wide ranges of PCR template concentrations, thus resulting in clear genotype-specific cutoffs, established through statistical analysis, for genotype identification. While we established such a genotyping assay in the Xenopus tropicalis model, the approach should be applicable to genotyping of any organism or cells and can be potentially used for large-scale, automated genotyping.},
}
@article {pmid38334140,
year = {2024},
author = {van Beljouw, SPB and Brouns, SJJ},
title = {CRISPR-controlled proteases.},
journal = {Biochemical Society transactions},
volume = {},
number = {},
pages = {},
doi = {10.1042/BST20230962},
pmid = {38334140},
issn = {1470-8752},
abstract = {With the discovery of CRISPR-controlled proteases, CRISPR-Cas has moved beyond mere nucleic acid targeting into the territory of targeted protein cleavage. Here, we review the understanding of Craspase, the best-studied member of the growing CRISPR RNA-guided protease family. We recollect the original bioinformatic prediction and early experimental characterizations; evaluate some of the mechanistic structural intricacies and emerging biotechnology; discuss open questions and unexplained mysteries; and indicate future directions for the rapidly moving field of the CRISPR proteases.},
}
@article {pmid38333010,
year = {2024},
author = {Rao, Y and Su, R and Wu, C and Yang, G and Fu, R and Wu, J and Liang, J and Liu, J and Jiang, Z and Xu, C and Huang, L},
title = {Marine fungus Aspergillus c1. sp metabolite activates the HSF1/PGC-1α axis, inducing a thermogenic program for treating obesity.},
journal = {Frontiers in pharmacology},
volume = {15},
number = {},
pages = {1320040},
pmid = {38333010},
issn = {1663-9812},
abstract = {Background and aims: Obesity is one of the most prevalent diseases worldwide with less ideal approved agents in clinic. Activating the HSF1/PGC-1α axis in adipose tissues has been reported to induce thermogenesis in mice, which presents a promising therapeutic avenue for obesity treatment. The present study aimed to identified novel natural HSF1 activator and evaluated the therapeutic effects of the newly discovered compound on obesity-associated metabolic disorders and the molecular mechanisms of these effects. Methods: Our previous reported HSF1/PGC-1α activator screening system was used to identify novel natural HSF1 activator. The PGC-1α luciferase activity, immunoblot, protein nuclear-translocation, immunofluorescence, chromatin immunoprecipitation assays were used to evaluate the activity of compound HN-001 in activating HSF1. The experiments of mitochondrial number measurement, TG assay and imaging, cellular metabolic assay, gene assays, and CRISPR/Cas 9 were applied for investigating the metabolic effect of HN-001 in C3H10-T1/2 adipocytes. The in vivo anti-obesity efficacies and beneficial metabolic effects of HN-001 were evaluated by performing body and fat mass quantification, plasma chemical analysis, GTT, ITT, cold tolerance test, thermogenesis analysis. Results: HN-001 dose- and time-dependently activated HSF1 and induced HSF1 nuclear translocation, resulting in an enhancement in binding with the gene Pgc-1α. This improvement induced activation of adipose thermogenesis and enhancement of mitochondrial oxidation capacity, thus inhibiting adipocyte maturation. Deletion of HSF1 in adipocytes impaired mitochondrial oxidation and abolished the above beneficial metabolic effects of HN-001, including adipocyte browning induction, improvements in mitogenesis and oxidation capacity, and lipid-lowering ability. In mice, HN-001 treatment efficiently alleviated diet-induced obesity and metabolic disorders. These changes were associated with increased body temperature in mice and activation of the HSF1/PGC-1α axis in adipose tissues. UCP1 expression and mitochondrial biogenesis were increased in both white and brown adipose tissues of HN-001-treated mice. Conclusion: These data indicate that HN-001 may have therapeutic potential for obesity-related metabolic diseases by increasing the capacity of energy expenditure in adipose tissues through a mechanism involving the HSF1/PGC-1α axis, which shed new light on the development of novel anti-obesity agents derived from marine sources.},
}
@article {pmid38332767,
year = {2024},
author = {Ahmad, A and Hoffman, NE and Jones, MGK and Zhang, B},
title = {Editorial: Frontiers in global regulatory landscape of CRISPR-edited plants.},
journal = {Frontiers in plant science},
volume = {15},
number = {},
pages = {1367698},
doi = {10.3389/fpls.2024.1367698},
pmid = {38332767},
issn = {1664-462X},
}
@article {pmid38324621,
year = {2024},
author = {Meng, Q and Wei, L and Ma, K and Shi, M and Lin, X and Ho, JWK and Li, Y and Zhang, X},
title = {scDecouple: decoupling cellular response from infected proportion bias in scCRISPR-seq.},
journal = {Briefings in bioinformatics},
volume = {25},
number = {2},
pages = {},
pmid = {38324621},
issn = {1477-4054},
support = {2019YFA0906700//National Key R&amp;D Program of China/ ; 62373210//National Natural Science Foundation of China/ ; 2021Z11JCQ020//Tsinghua University Initiative Scientific Research Program/ ; Z210010//Beijing Natural Science Foundation/ ; 2020Z99CFG006//Tsinghua University Spring Breeze Fund/ ; //Innovation and Technology Commission of Hong Kong/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *High-Throughput Screening Assays ; },
abstract = {Single-cell clustered regularly interspaced short palindromic repeats-sequencing (scCRISPR-seq) is an emerging high-throughput CRISPR screening technology where the true cellular response to perturbation is coupled with infected proportion bias of guide RNAs (gRNAs) across different cell clusters. The mixing of these effects introduces noise into scCRISPR-seq data analysis and thus obstacles to relevant studies. We developed scDecouple to decouple true cellular response of perturbation from the influence of infected proportion bias. scDecouple first models the distribution of gene expression profiles in perturbed cells and then iteratively finds the maximum likelihood of cell cluster proportions as well as the cellular response for each gRNA. We demonstrated its performance in a series of simulation experiments. By applying scDecouple to real scCRISPR-seq data, we found that scDecouple enhances the identification of biologically perturbation-related genes. scDecouple can benefit scCRISPR-seq data analysis, especially in the case of heterogeneous samples or complex gRNA libraries.},
}
@article {pmid38305901,
year = {2024},
author = {Ke, Y and Zhang, S and Gao, Y and Chen, X and He, J and Chen, Y and Zhang, Q and Ding, X},
title = {Engineered CRISPRa System for Precise and Multiplex Gene Regulation Using a Hybrid dCas12a Variant and Hairpin-Spacer crRNAs.},
journal = {Analytical chemistry},
volume = {96},
number = {6},
pages = {2637-2642},
doi = {10.1021/acs.analchem.3c05318},
pmid = {38305901},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Endonucleases/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a nucleases have emerged as a promising alternative to CRISPR-Cas9 in gene editing and expression regulation. However, the adoption of Cas12a has been hindered due to general off-target activities and limited efficiency. Here, we utilized a hybrid engineered Cas12a variant and hairpin-spacer crRNAs (h-CAP) to enhance the specificity and efficiency of the CRISPR-Cas12a system. Leveraging the h-CAP strategy, we demonstrate both single-base-specific and multiplex gene expression regulation in human cells.},
}
@article {pmid38305871,
year = {2024},
author = {Li, QN and Wang, DX and Chen, DY and Lyu, JA and Wang, YX and Wu, SL and Jiang, HX and Kong, DM},
title = {Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics.},
journal = {Analytical chemistry},
volume = {96},
number = {6},
pages = {2692-2701},
doi = {10.1021/acs.analchem.3c05497},
pmid = {38305871},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; Survivin/genetics ; Biomarkers ; Point-of-Care Testing ; *Nucleic Acids ; *Biosensing Techniques ; },
abstract = {In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.},
}
@article {pmid38290431,
year = {2024},
author = {Kasputis, T and He, Y and Ci, Q and Chen, J},
title = {On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System.},
journal = {Analytical chemistry},
volume = {96},
number = {6},
pages = {2676-2683},
doi = {10.1021/acs.analchem.3c05459},
pmid = {38290431},
issn = {1520-6882},
mesh = {Humans ; *Graphite ; CRISPR-Cas Systems/genetics ; *Sepsis/diagnosis ; Bacteria ; Coloring Agents ; *Bacterial Infections ; Oxides ; *Biosensing Techniques ; },
abstract = {Sepsis is an extremely dangerous medical condition that emanates from the body's response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsis-inducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsis-inducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 10[3] CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resource-limited settings, expediting the response for patients at risk of sepsis.},
}
@article {pmid38217497,
year = {2024},
author = {Xia, X and Chen, Q and Zuo, T and Liang, Z and Xu, G and Wei, F and Yang, J and Hu, Q and Zhao, Z and Tang, BZ and Cen, Y},
title = {DNA Robots for CRISPR/Cas12a Activity Management and Universal Platforms for Biosensing.},
journal = {Analytical chemistry},
volume = {96},
number = {6},
pages = {2620-2627},
doi = {10.1021/acs.analchem.3c05210},
pmid = {38217497},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; *Robotics ; Colorimetry ; DNA/genetics ; Excision Repair ; *Biosensing Techniques ; },
abstract = {The CRISPR/Cas12a system is a revolutionary genome editing technique that is widely employed in biosensing and molecular diagnostics. However, there are few reports on precisely managing the trans-cleavage activity of Cas12a by simple modification since the traditional methods to manage Cas12a often require difficult and rigorous regulation of core components. Hence, we developed a novel CRISPR/Cas12a regulatory mechanism, named DNA Robots for Enzyme Activity Management (DREAM), by introducing two simple DNA robots, apurinic/apyrimidinic site (AP site) or nick on target activator. First, we revealed the mechanism of how the DREAM strategy precisely regulated Cas12a through different binding affinities. Second, the DREAM strategy was found to improve the selectivity of Cas12a for identifying base mismatch. Third, a modular biosensor for base excision repair enzymes based on the DREAM strategy was developed by utilizing diversified generation ways of DNA robots, and a multi-signal output platform such as fluorescence, colorimetry, and visual lateral flow strip was constructed. Furthermore, we extended logic sensing circuits to overcome the barrier that Cas12a could not detect simultaneously in a single tube. Overall, the DREAM strategy not only provided new prospects for programmable Cas12a biosensing systems but also enabled portable, specific, and humanized detection with great potential for molecular diagnostics.},
}
@article {pmid38191205,
year = {2024},
author = {Ma, S and Zhang, T and Wang, R and Wang, P and Liu, Y and Chang, J and Wang, A and Lan, X and Sun, L and Sun, H and Shi, R and Lu, W and Liu, D and Zhang, N and Hu, W and Wang, X and Xing, W and Jia, L and Xia, Q},
title = {High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori.},
journal = {Genome research},
volume = {34},
number = {1},
pages = {134-144},
doi = {10.1101/gr.278297.123},
pmid = {38191205},
issn = {1549-5469},
mesh = {Animals ; *Bombyx/genetics ; RNA, Guide, CRISPR-Cas Systems ; Mutagenesis ; Gene Editing/methods ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems ; },
abstract = {Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR-Cas9-induced library in a nonmodel multicellular organism, Bombyx mori We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollutant cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.},
}
@article {pmid38103518,
year = {2024},
author = {Leclerc, D and Siroky, MD and Miller, SM},
title = {Next-generation biological vector platforms for in vivo delivery of genome editing agents.},
journal = {Current opinion in biotechnology},
volume = {85},
number = {},
pages = {103040},
doi = {10.1016/j.copbio.2023.103040},
pmid = {38103518},
issn = {1879-0429},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; Mammals ; },
abstract = {CRISPR-based genome editing holds promise for addressing genetic disease, infectious disease, and cancer and has rapidly advanced from primary research to clinical trials in recent years. However, the lack of safe and potent in vivo delivery methods for CRISPR components has limited most ongoing clinical trials to ex vivo gene therapy. Effective CRISPR in vivo genome editing necessitates an effective vehicle ensuring target cell transduction while minimizing off-target effects, toxicity, and immune reactions. In this review, we examine promising biological-derived platforms to deliver DNA editing agents in vivo and the engineering thereof, encompassing potent viral-based vehicles, flexible protein nanocages, and mammalian-derived particles.},
}
@article {pmid38100153,
year = {2024},
author = {Liu, J and Wei, X and Zhang, Y and Ran, Y and Qu, B and Wang, C and Zhao, F and Zhang, L},
title = {dCas9-guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by using Staphylococcus aureus.},
journal = {Cell biology international},
volume = {48},
number = {3},
pages = {300-310},
doi = {10.1002/cbin.12106},
pmid = {38100153},
issn = {1095-8355},
support = {31501033//National Natural Science Foundation of China/ ; },
mesh = {Female ; Animals ; Cattle ; Humans ; RNA, Guide, CRISPR-Cas Systems ; Milk Proteins/metabolism ; Staphylococcus aureus/genetics/metabolism ; *Mastitis, Bovine/genetics/metabolism ; *Staphylococcal Infections/veterinary/metabolism ; Demethylation ; Mammary Glands, Animal/metabolism ; Epithelial Cells/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; },
abstract = {Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.},
}
@article {pmid38058125,
year = {2024},
author = {Rankin, AE and Fox, E and Chisholm, T and Lantz, N and Rajan, A and Phillips, W and Griffin, E and Harper, J and Suhr, C and Tan, M and Wang, J and Yang, A and Kim, ES and Ankrah, NKA and Chakraborty, P and Lam, ACK and Laws, ME and Lee, J and Park, KK and Wesel, E and Covert, PH and Kockel, L and Park, S and Kim, SK},
title = {Simplified homology-assisted CRISPR for gene editing in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {14},
number = {2},
pages = {},
pmid = {38058125},
issn = {2160-1836},
support = {P30 DK116074/DK/NIDDK NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; U41 HG000739/HG/NHGRI NIH HHS/United States ; R01 DK107507/GF/NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; *Drosophila/genetics ; Transgenes ; Genome ; CRISPR-Cas Systems ; },
abstract = {In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.},
}
@article {pmid38053334,
year = {2024},
author = {Cattaneo, C and Enzo, E and De Rosa, L and Sercia, L and Consiglio, F and Forcato, M and Bicciato, S and Paiardini, A and Basso, G and Tagliafico, E and Paganelli, A and Fiorentini, C and Magnoni, C and Latella, MC and De Luca, M},
title = {Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {2},
pages = {372-383},
pmid = {38053334},
issn = {1525-0024},
mesh = {Humans ; *Epidermolysis Bullosa Simplex/genetics/therapy/metabolism ; Alleles ; CRISPR-Cas Systems ; Keratinocytes/metabolism ; Mutation ; Stem Cells/metabolism ; },
abstract = {Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.},
}
@article {pmid38006899,
year = {2024},
author = {Schambach, A and Buchholz, CJ and Torres-Ruiz, R and Cichutek, K and Morgan, M and Trapani, I and Büning, H},
title = {A new age of precision gene therapy.},
journal = {Lancet (London, England)},
volume = {403},
number = {10426},
pages = {568-582},
doi = {10.1016/S0140-6736(23)01952-9},
pmid = {38006899},
issn = {1474-547X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; },
abstract = {Gene therapy has become a clinical reality as market-approved advanced therapy medicinal products for the treatment of distinct monogenetic diseases and B-cell malignancies. This Therapeutic Review aims to explain how progress in genome editing technologies offers the possibility to expand both therapeutic options and the types of diseases that will become treatable. To frame these impressive advances in the context of modern medicine, we incorporate examples from human clinical trials into our discussion on how genome editing will complement currently available strategies in gene therapy, which still mainly rely on gene addition strategies. Furthermore, safety considerations and ethical implications, including the issue of accessibility, are addressed as these crucial parameters will define the impact that gene therapy in general and genome editing in particular will have on how we treat patients in the near future.},
}
@article {pmid37963801,
year = {2024},
author = {Cao, Y and Tian, Y and Huang, J and Xu, L and Fan, Z and Pan, Z and Chen, S and Gao, Y and Wei, L and Zheng, S and Zhang, X and Yu, Y and Ren, F},
title = {CRISPR/Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection.},
journal = {Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi},
volume = {57},
number = {1},
pages = {118-127},
doi = {10.1016/j.jmii.2023.10.010},
pmid = {37963801},
issn = {1995-9133},
mesh = {Humans ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Klebsiella pneumoniae/genetics/metabolism ; CRISPR-Cas Systems ; Microbial Sensitivity Tests ; beta-Lactamases/genetics/metabolism ; Carbapenems/pharmacology/therapeutic use ; *Carbapenem-Resistant Enterobacteriaceae/genetics ; *Klebsiella Infections/drug therapy ; },
abstract = {BACKGROUND/PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner.<br><br>METHODS: K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-&#x3b2;-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation.<br><br>RESULTS: Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/&#x3bc;L with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 10[1] copies/&#x3bc;L with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1&#xa0;CFU/cm[2] KP could be detected.<br><br>CONCLUSION: We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.},
}
@article {pmid37870061,
year = {2023},
author = {Bajwa, KK and Punetha, M and Kumar, D and Yadav, PS and Long, CR and Selokar, NL},
title = {Electroporation-based CRISPR gene editing in adult buffalo fibroblast cells.},
journal = {Animal biotechnology},
volume = {34},
number = {9},
pages = {5055-5066},
doi = {10.1080/10495398.2023.2271030},
pmid = {37870061},
issn = {1532-2378},
mesh = {Animals ; *Gene Editing/methods ; *Buffaloes/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation ; Transfection ; Fibroblasts ; DNA ; CRISPR-Cas Systems/genetics ; },
abstract = {Electroporation is a widely used method for delivering CRISPR components into cells; however, it presents challenges when applied to difficult-to-transfect cells like adult buffalo fibroblasts. In this study, the ITGB2 gene (encoding the CD18 protein), plays vital for cellular adhesion and immune responses, was selected for editing experiments. To optimize electroporation conditions, we investigated parameters such as electric field strength, pulse duration, plasmid DNA amount, cuvette type, and cell type. The best transfection rates were obtained in a 4 mm gap cuvette with a single 20-millisecond pulse of 300 V using a 10 μg of all-in-one CRISPR plasmid for 10[6] cells in 100 μL of electroporation buffer. Increasing DNA quantity enhanced transfection rates but compromised cell viability. The 4 mm cuvette gap had high transfection rates than the 2 mm gap, and newborn cells exhibited higher transfection rates than adult cells. We achieved transfection rates of 10-12% with a cell viability of 25-30% for adult fibroblast cells. Subsequently, successfully edited the ITGB2 gene with a 30% editing efficiency, confirmed through various analysis methods, including T7E1 assay, TIDE and ICE analysis, and TA cloning. In conclusion, electroporation conditions reported here can edit buffalo gene(s) for various biotechnological research applications.},
}
@article {pmid37845337,
year = {2023},
author = {Xiong, X and Liu, K and Li, Z and Xia, FN and Ruan, XM and He, X and Li, JF},
title = {Split complementation of base editors to minimize off-target edits.},
journal = {Nature plants},
volume = {9},
number = {11},
pages = {1832-1847},
pmid = {37845337},
issn = {2055-0278},
mesh = {Humans ; *Gene Editing ; *RNA, Guide, CRISPR-Cas Systems ; RNA ; Deoxyribonuclease I/genetics ; CRISPR-Cas Systems ; *Alkanesulfonic Acids ; },
abstract = {Base editors (BEs) empower the efficient installation of beneficial or corrective point mutations in crop and human genomes. However, conventional BEs can induce unpredictable guide RNA (gRNA)-independent off-target edits in the genome and transcriptome due to spurious activities of BE-enclosing deaminases, and current improvements mostly rely on deaminase-specific mutagenesis or exogenous regulators. Here we developed a split deaminase for safe editing (SAFE) system applicable to BEs containing distinct cytidine or adenosine deaminases, with no need of external regulators. In SAFE, a BE was properly split at a deaminase domain embedded inside a Cas9 nickase, simultaneously fragmenting and deactivating both the deaminase and the Cas9 nickase. The gRNA-conditioned BE reassembly conferred robust on-target editing in plant, human and yeast cells, while minimizing both gRNA-independent and gRNA-dependent off-target DNA/RNA edits. SAFE also substantially increased product purity by eliminating indels. Altogether, SAFE provides a generalizable solution for BEs to suppress off-target editing and improve on-target performance.},
}
@article {pmid36946758,
year = {2023},
author = {Guo, X and Geng, L and Jiang, C and Yao, W and Jin, J and Liu, Z and Mu, Y},
title = {Multiplexed genome engineering for porcine fetal fibroblasts with gRNA-tRNA arrays based on CRISPR/Cas9.},
journal = {Animal biotechnology},
volume = {34},
number = {9},
pages = {4703-4712},
doi = {10.1080/10495398.2023.2187402},
pmid = {36946758},
issn = {1532-2378},
mesh = {Swine/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; RNA, Transfer/genetics ; Fibroblasts ; },
abstract = {Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR-Cas9 gRNA-tRNA array (GTR-CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the IGFBP3, MSTN, MC4R, and SOCS2 genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.},
}
@article {pmid36905152,
year = {2023},
author = {Yum, SY and Choi, W and Kim, S and Jang, G and Koo, O},
title = {Identification AAVS1-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9.},
journal = {Animal biotechnology},
volume = {34},
number = {9},
pages = {4730-4735},
doi = {10.1080/10495398.2023.2187408},
pmid = {36905152},
issn = {1532-2378},
mesh = {Animals ; Swine/genetics ; Humans ; Mice ; *Recombinases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Animals, Genetically Modified/metabolism ; Gene Targeting ; },
abstract = {Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.},
}
@article {pmid36877952,
year = {2023},
author = {Sundaresan, Y and Yacoub, S and Kodati, B and Amankwa, CE and Raola, A and Zode, G},
title = {Therapeutic applications of CRISPR/Cas9 gene editing technology for the treatment of ocular diseases.},
journal = {The FEBS journal},
volume = {290},
number = {22},
pages = {5248-5269},
pmid = {36877952},
issn = {1742-4658},
support = {R01 EY026177/EY/NEI NIH HHS/United States ; R01 EY030366/EY/NEI NIH HHS/United States ; EY030366/EY/NEI NIH HHS/United States ; EY026177/EY/NEI NIH HHS/United States ; EY026177/EY/NEI NIH HHS/United States ; EY030366/EY/NEI NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; Genomics ; Genome ; },
abstract = {Ocular diseases are a highly heterogeneous group of phenotypes, caused by a spectrum of genetic variants and environmental factors that exhibit diverse clinical symptoms. As a result of its anatomical location, structure and immune privilege, the eye is an ideal system to assess and validate novel genetic therapies. Advances in genome editing have revolutionized the field of biomedical science, enabling researchers to understand the biology behind disease mechanisms and allow the treatment of several health conditions, including ocular pathologies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing facilitates efficient and specific genetic modifications in the nucleic acid sequence, resulting in permanent changes at the genomic level. This approach has advantages over other treatment strategies and is promising for the treatment of various genetic and non-genetic ocular conditions. This review provides an overview of the CRISPR/CRISPR-associated protein 9 (Cas9) system and summarizes recent advances in the therapeutic application of CRISPR/Cas9 for the treatment of various ocular pathologies, as well as future challenges.},
}
@article {pmid36797492,
year = {2023},
author = {Koeppel, J and Weller, J and Peets, EM and Pallaseni, A and Kuzmin, I and Raudvere, U and Peterson, H and Liberante, FG and Parts, L},
title = {Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants.},
journal = {Nature biotechnology},
volume = {41},
number = {10},
pages = {1446-1456},
pmid = {36797492},
issn = {1546-1696},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Humans ; *DNA Repair/genetics ; *DNA Transposable Elements ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Most short sequences can be precisely written into a selected genomic target using prime editing; however, it remains unclear what factors govern insertion. We design a library of 3,604 sequences of various lengths and measure the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems in varying DNA repair contexts. We find that length, nucleotide composition and secondary structure of the insertion sequence all affect insertion rates. We also discover that the 3' flap nucleases TREX1 and TREX2 suppress the insertion of longer sequences. Combining the sequence and repair features into a machine learning model, we can predict relative frequency of insertions into a site with R = 0.70. Finally, we demonstrate how our accurate prediction and user-friendly software help choose codon variants of common fusion tags that insert at high efficiency, and provide a catalog of empirically determined insertion rates for over a hundred useful sequences.},
}
@article {pmid35507885,
year = {2023},
author = {Hung, SW and Chuang, CK and Wong, CH and Yen, CH and Peng, SH and Yang, C and Chen, MC and Yang, TS and Tu, CF},
title = {Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault.},
journal = {Animal biotechnology},
volume = {34},
number = {9},
pages = {4196-4209},
doi = {10.1080/10495398.2022.2062602},
pmid = {35507885},
issn = {1532-2378},
mesh = {Humans ; Swine ; Animals ; Female ; Pregnancy ; *Porcine Reproductive and Respiratory Syndrome/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Leukocytes, Mononuclear ; Microinjections ; Placenta ; RNA, Guide, CRISPR-Cas Systems ; *Porcine respiratory and reproductive syndrome virus/genetics ; Macrophages/metabolism ; Cytoplasm ; *Swine Diseases ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.},
}
@article {pmid38332517,
year = {2024},
author = {Yang, L and Liu, Z and Sun, J and Chen, Z and Gao, F and Guo, Y},
title = {Adenine base editor-based correction of the cardiac pathogenic Lmna c.1621C &gt; T mutation in murine hearts.},
journal = {Journal of cellular and molecular medicine},
volume = {28},
number = {4},
pages = {e18145},
pmid = {38332517},
issn = {1582-4934},
support = {2022YFA1104800//National Key Research and Development Program of China/ ; 32100660//National Natural Science Foundation of China/ ; 82100349//National Natural Science Foundation of China/ ; 82170367//National Natural Science Foundation of China/ ; 82222006//National Natural Science Foundation of China/ ; 7232094//Beijing Natural Science Foundation/ ; 20220484024//Beijing Nova Program/ ; Z211100002121003//Beijing Nova Program/ ; },
mesh = {Mice ; Animals ; Gene Editing ; Adenine ; RNA, Guide, CRISPR-Cas Systems ; Mutation/genetics ; Genetic Therapy ; },
abstract = {Base editors are emerging as powerful tools to correct single-nucleotide variants and treat genetic diseases. In particular, the adenine base editors (ABEs) exhibit robust and accurate adenine-to-guanidine editing capacity and have entered the clinical stage for cardiovascular therapy. Despite the tremendous progress using ABEs to treat heart diseases, a standard technical route toward successful ABE-based therapy remains to be fully established. In this study, we harnessed adeno-associated virus (AAV) and a mouse model carrying the cardiomyopathy-causing Lmna c.1621C > T mutation to demonstrate key steps and concerns in designing a cardiac ABE experiment in vivo. We found DeepABE as a reliable deep-learning-based model to predict ABE editing outcomes in the heart. Screening of sgRNAs for a Cas9 mutant with relieved protospacer adjacent motif (PAM) allowed the reduction of bystander editing. The ABE editing efficiency can be significantly enhanced by modifying the TadA and Cas9 variants, which are core components of ABEs. The ABE systems can be delivered into the heart via either dual AAV or all-in-one AAV vectors. Together, this study showcased crucial technical considerations in designing an ABE system for the heart and pointed out major challenges in further improvement of this new technology for gene therapy.},
}
@article {pmid38332115,
year = {2024},
author = {Sankar, A and Y S, RK and Singh, A and Roy, R and Shukla, R and Verma, B},
title = {Next-Generation Therapeutics for Rare Genetic Disorders.},
journal = {Mutagenesis},
volume = {},
number = {},
pages = {},
doi = {10.1093/mutage/geae002},
pmid = {38332115},
issn = {1464-3804},
abstract = {The therapeutic potential of the human genome has been explored through the development of next-generation therapeutics, which have had a high impact on treating genetic disorders. Classical treatments have traditionally focused on common diseases that require repeated treatments. However, with the recent advancements in the development of nucleic acids, utilizing DNA and RNA to modify or correct gene expression in genetic disorders, there has been a paradigm shift in the treatment of rare diseases, offering more potential one-time cure options. Advanced technologies that use CRISPR-Cas 9, antisense oligonucleotides, siRNA, miRNA, and aptamers are promising tools that have achieved successful breakthroughs in the treatment of various genetic disorders. The advancement in the chemistry of these molecules has improved their efficacy, reduced toxicity, and expanded their clinical use across a wide range of tissues in various categories of human disorders. However, challenges persist regarding the safety and efficacy of these advanced technologies in translating into clinical practice. This review mainly focuses on the potential therapies for rare genetic diseases and considers how next-generation techniques enable drug development to achieve long-lasting curative effects through gene inhibition, replacement, and editing.},
}
@article {pmid38331711,
year = {2024},
author = {Dong, H},
title = {Application of genome editing techniques to regulate gene expression in crops.},
journal = {BMC plant biology},
volume = {24},
number = {1},
pages = {100},
pmid = {38331711},
issn = {1471-2229},
support = {B22C10213//Project of Hainan Yazhou Bay Seed Laboratory/ ; },
abstract = {BACKGROUND: Enhanced agricultural production is urgently required to meet the food demands of the increasing global population. Abundant genetic diversity is expected to accelerate crop development. In particular, the development of the CRISPR/Cas genome editing technology has greatly enhanced our ability to improve crop's genetic diversity through direct artificial gene modification. However, recent studies have shown that most crop improvement efforts using CRISPR/Cas techniques have mainly focused on the coding regions, and there is a relatively lack of studies on the regulatory regions of gene expression.<br><br>RESULTS: This review briefly summarizes the development of CRISPR/Cas system in the beginning. Subsequently, the importance of gene regulatory regions in plants is discussed. The review focuses on recent developments and applications of mutations in regulatory regions via CRISPR/Cas techniques in crop breeding.<br><br>CONCLUSION: Finally, an outline of perspectives for future crop breeding using genome editing technologies is provided. This review provides new research insights for crop improvement using genome editing techniques.},
}
@article {pmid38331551,
year = {2024},
author = {Zhang, F and Hao, D and Liu, R and Wang, J and Sang, Y and Wang, S and Wang, X},
title = {Preparation and recognition mechanism study of an scFv targeting chloramphenicol for a hybridization chain reaction-CRISPR/Cas12a amplified fluoroimmunoassay.},
journal = {Analytica chimica acta},
volume = {1293},
number = {},
pages = {342283},
doi = {10.1016/j.aca.2024.342283},
pmid = {38331551},
issn = {1873-4324},
mesh = {Humans ; Chloramphenicol ; CRISPR-Cas Systems ; HEK293 Cells ; Nucleic Acid Hybridization ; Fluoroimmunoassay ; Antibodies ; Biosensing Techniques ; },
abstract = {Recombinant antibody-based immunoassays have emerged as crucial techniques for detecting antibiotic residues in food samples. Developing a stable recombinant antibody production system and enhancing detection sensitivity are crucial for their biosensing applications. Here, we bioengineered a single-chain fragment variable (scFv) antibody to target chloramphenicol (CAP) using both Bacillus subtilis and HEK 293 systems, with the HEK 293-derived scFv demonstrating superior sensitivity. Computational chemistry analyses indicated that ASP-99 and ASN-102 residues in the scFv play key roles in antibody recognition, and the hydroxyl group near the benzene ring of the target molecule is critical for in antibody binding. Furthermore, we enhanced the scFv's biosensing sensitivity using an HCR-CRISPR/Cas12a amplification strategy in a streptavidin-based immunoassay. In the dual-step amplification process, detection limits for CAP in the HCR and HCR-CRISPR/Cas12a stages were significantly reduced to 55.23 pg/mL and 3.31 pg/mL, respectively. These findings introduce an effective method for developing CAP-specific scFv antibodies and also propose a multi-amplification strategy to increase immunoassay sensitivity. Additionally, theoretical studies also offer valuable guidance in CAP hapten design and genetic engineering for antibody modification.},
}
@article {pmid38330622,
year = {2024},
author = {Papaioannou, V},
title = {The road to gene manipulation in the mouse: Jean Brachet Memorial Lecture of the International Society of Differentiation (delivered June 21, 2023 at Cold Spring Harbor Laboratory).},
journal = {Differentiation; research in biological diversity},
volume = {136},
number = {},
pages = {100753},
doi = {10.1016/j.diff.2024.100753},
pmid = {38330622},
issn = {1432-0436},
abstract = {Genetic manipulation in mammals has progressed rapidly in the past decade with the advent of CRISPR-Cas gene editing tools, promising profound impacts on the understanding of human development, health and disease. However, many years of research in divergent fields of experimental embryology, genetics, reproduction, molecular biology and transgenic technology laid the groundwork and have played critical roles for this progress. This article details various threads of research and the central role of the laboratory mouse that came together in reaching this point, all from the perspective of a scientist whose research was deeply immersed in the field.},
}
@article {pmid38329578,
year = {2024},
author = {Miroshnichenko, D and Timerbaev, V and Divashuk, M and Pushin, A and Alekseeva, V and Kroupin, P and Bazhenov, M and Samarina, M and Ermolaev, A and Karlov, G and Dolgov, S},
title = {CRISPR/Cas9-mediated мultiplexed multi-allelic mutagenesis of genes located on A, B and R subgenomes of hexaploid triticale.},
journal = {Plant cell reports},
volume = {43},
number = {3},
pages = {59},
pmid = {38329578},
issn = {1432-203X},
support = {agreement #075-15-2019-1667//KGC-ARRIAB/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Triticale ; Mutagenesis/genetics ; },
abstract = {The first-time generation of hexaploid triticale plants harbouring variable panels of novel mutations in gene families involved in starch biosynthesis has been achieved by the subgenome-independent multiplexed CRISPR/Cas9-mediated editing.},
}
@article {pmid38328448,
year = {2024},
author = {Duan, C and Liu, Y and Liu, Y and Liu, L and Cai, M and Zhang, R and Zeng, Q and Koonin, EV and Krupovic, M and Li, M},
title = {Diversity of Bathyarchaeia viruses in metagenomes and virus-encoded CRISPR system components.},
journal = {ISME communications},
volume = {4},
number = {1},
pages = {ycad011},
pmid = {38328448},
issn = {2730-6151},
abstract = {Bathyarchaeia represent a class of archaea common and abundant in sedimentary ecosystems. Here we report 56 metagenome-assembled genomes of Bathyarchaeia viruses identified in metagenomes from different environments. Gene sharing network and phylogenomic analyses led to the proposal of four virus families, including viruses of the realms Duplodnaviria and Adnaviria, and archaea-specific spindle-shaped viruses. Genomic analyses uncovered diverse CRISPR elements in these viruses. Viruses of the proposed family "Fuxiviridae" harbor an atypical Type IV-B CRISPR-Cas system and a Cas4 protein that might interfere with host immunity. Viruses of the family "Chiyouviridae" encode a Cas2-like endonuclease and two mini-CRISPR arrays, one with a repeat identical to that in the host CRISPR array, potentially allowing the virus to recruit the host CRISPR adaptation machinery to acquire spacers that could contribute to competition with other mobile genetic elements or to inhibit host defenses. These findings present an outline of the Bathyarchaeia virome and offer a glimpse into their counter-defense mechanisms.},
}
@article {pmid38277710,
year = {2024},
author = {Houweling, PJ and Crossman, V and Tiong, CF and Coles, CA and Taylor, RL and Clayton, JS and Graham, A and Vlahos, K and Howden, SE and North, KN},
title = {Generation of a human ACTA1-tdTomato reporter iPSC line using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {75},
number = {},
pages = {103313},
doi = {10.1016/j.scr.2024.103313},
pmid = {38277710},
issn = {1876-7753},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Induced Pluripotent Stem Cells/metabolism ; Actins/genetics/metabolism ; Muscle, Skeletal/metabolism ; *Red Fluorescent Protein ; },
abstract = {We used gene editing to introduce DNA sequences encoding the tdTomato fluorescent protein into the α -skeletal actin 1 (ACTA1) locus to develop an ACTA1-tdTomato induced pluripotent stem cell reporter line for monitoring differentiation of skeletal muscle. This cell line will be used to better understand skeletal muscle maturation and development in vitro as well as provide a useful tool for drug screening and the evaluation of novel therapeutics for the treatment of skeletal muscle disease.},
}
@article {pmid38217996,
year = {2024},
author = {Binder, S and Ramachandran, H and Hildebrandt, B and Dobner, J and Rossi, A},
title = {Prime-Editing of human ACTB in induced pluripotent stem cells to model human ACTB Loss-of-Function diseases and compensatory mechanisms.},
journal = {Stem cell research},
volume = {75},
number = {},
pages = {103304},
doi = {10.1016/j.scr.2024.103304},
pmid = {38217996},
issn = {1876-7753},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism ; Actins/genetics/metabolism ; Mutation ; Cell Differentiation ; Intellectual Disability/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Beta-actin (ACTB) heterozygous loss-of-function mutations are associated with pleiotropic developmental disorders entailing intellectual disability and frequent organ malformations in affected individuals. We generated two CRISPR/Cas9 prime-edited human induced pluripotent stem cell (iPSC) lines, IUFi004-A-1 and IUFi004-A-2, carrying a heterozygous missense mutation in exon 4 of ACTB. Mutant iPSCs exhibited normal cell morphology and genomic integrity, maintained expression of pluripotency markers, and differentiated into the three primary germ layers. The mutants offer a valuable platform for examining the molecular and functional consequences of ACTB haploinsufficiency, developing effective treatments, and exploring mechanisms underlying phenotypic variability and genetic compensation observed in monogenic diseases.},
}
@article {pmid38211411,
year = {2024},
author = {Han, HW and Im, YS and Kim, YO and Kwak, S and Cho, SJ},
title = {Generation of a CAG-EGFP tagged cell line (KSCBi017-A-2) from human induced pluripotent stem cells using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {75},
number = {},
pages = {103303},
doi = {10.1016/j.scr.2024.103303},
pmid = {38211411},
issn = {1876-7753},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Green Fluorescent Proteins/metabolism ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.},
}
@article {pmid38180813,
year = {2024},
author = {Miyamoto, H and Kobayashi, H and Kishima, N and Yamazaki, K and Hamamichi, S and Uno, N and Abe, S and Hiramuki, Y and Kazuki, K and Tomizuka, K and Kazuki, Y},
title = {Rapid human genomic DNA cloning into mouse artificial chromosome via direct chromosome transfer from human iPSC and CRISPR/Cas9-mediated translocation.},
journal = {Nucleic acids research},
volume = {52},
number = {3},
pages = {1498-1511},
pmid = {38180813},
issn = {1362-4962},
support = {JPMJCR18S4//JST/ ; //CREST/ ; JP23ama121046//AMED/ ; JP23am0401002//Science and Technology Platform Program for Advanced Biological Medicine from AMED/ ; JP23bm1123038//AMED/ ; 21-101//Joint Research of the Exploratory Research Center on Life and Living Systems/ ; },
mesh = {Cricetinae ; Mice ; Humans ; Animals ; Cricetulus ; Induced Pluripotent Stem Cells ; Translocation, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Chromosomes, Artificial ; Gene Editing ; DNA ; Cloning, Molecular ; },
abstract = {A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.},
}
@article {pmid37981496,
year = {2024},
author = {Ahmar, S and Usman, B and Hensel, G and Jung, KH and Gruszka, D},
title = {CRISPR enables sustainable cereal production for a greener future.},
journal = {Trends in plant science},
volume = {29},
number = {2},
pages = {179-195},
doi = {10.1016/j.tplants.2023.10.016},
pmid = {37981496},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems/genetics ; Edible Grain/genetics ; Artificial Intelligence ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.},
}
@article {pmid37838519,
year = {2024},
author = {Wang, C and Wang, K and Kou, Y},
title = {Genome editing creates disease-resistant crops without yield penalties.},
journal = {Trends in plant science},
volume = {29},
number = {2},
pages = {114-116},
doi = {10.1016/j.tplants.2023.10.004},
pmid = {37838519},
issn = {1878-4372},
mesh = {Gene Editing ; CRISPR-Cas Systems ; Genome, Plant/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Application of disease-resistant varieties is the most effective and environmentally friendly way to control crop diseases. However, there is often a trade-off between disease resistance and yield. Several recent studies have demonstrated that genome-editing technology brings a new strategy for generating disease-resistant crops without yield penalties.},
}
@article {pmid38328240,
year = {2024},
author = {Madugula, SS and Pujar, P and Bharani, N and Wang, S and Jayasinghe-Arachchige, VM and Pham, T and Mashburn, D and Artilis, M and Liu, J},
title = {Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.01.22.576286},
pmid = {38328240},
abstract = {The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations like large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In the current study, we aim to elucidate the unique protein attributes associated with Cas9 and Cas12 families and identify the features that distinguish each family from the other. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,495 features) encoding various physiochemical, topological, constitutional, and coevolutionary information of Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and Non-Cas proteins. All the models were evaluated rigorously on the test and independent datasets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 95% and 97% on their respective independent datasets, while the multiclass classifier achieved a high F1 score of 0.97. We observed that Quasi-sequence-order descriptors like Schneider-lag descriptors and Composition descriptors like charge, volume, and polarizability are essential for the Cas12 family. More interestingly, we discovered that Amino Acid Composition descriptors, especially the Tripeptide Composition (TPC) descriptors, are important for the Cas9 family. Four of the identified important descriptors of Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all the Cas9 proteins and were located within different catalytically important domains of the Cas9 protein structure. Among these four tripeptides, tripeptides DHI and HHA are well-known to be involved in the DNA cleavage activity of the Cas9 protein. We therefore propose the the other two tripeptides, PWN and PYY, may also be essential for the Cas9 family. Our identified important descriptors enhanced the understanding of the catalytic mechanisms of Cas9 and Cas12 proteins and provide valuable insights into design of novel Cas systems to achieve enhanced gene-editing properties.},
}
@article {pmid38326330,
year = {2024},
author = {Cautereels, C and Smets, J and De Saeger, J and Cool, L and Zhu, Y and Zimmermann, A and Steensels, J and Gorkovskiy, A and Jacobs, TB and Verstrepen, KJ},
title = {Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {1113},
pmid = {38326330},
issn = {2041-1723},
support = {1S25923N, 1SC2422N//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; 12W3918N, 12W3921N//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; HBC.2020.2623//Agentschap Innoveren en Ondernemen (Flanders Innovation &amp; Entrepreneurship)/ ; 202008440364//CSC | CHINA/UNESCO - the Great Wall Fellowship (UNESCO/People&apos;s Republic of China (The Great Wall))/ ; },
mesh = {*Integrases/genetics/metabolism ; *Recombination, Genetic ; Escherichia coli/genetics/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Recombinases/metabolism ; DNA/metabolism ; },
abstract = {Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.},
}
@article {pmid38325828,
year = {2024},
author = {Matsumoto, N and Miyano, M and Abe, T and Kashima, T and Kato-Ishikura, E and Inoue, KI and Liu, J and Kiyonari, H and Takeuchi, H and Ikegaya, Y},
title = {Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.},
journal = {Biological &amp; pharmaceutical bulletin},
volume = {47},
number = {2},
pages = {394-398},
doi = {10.1248/bpb.b23-00598},
pmid = {38325828},
issn = {1347-5215},
mesh = {Animals ; Rats ; *Gene Editing ; *CRISPR-Cas Systems ; Dopamine Plasma Membrane Transport Proteins/genetics/metabolism ; Red Fluorescent Protein ; Dopaminergic Neurons/metabolism ; },
abstract = {Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.},
}
@article {pmid38324086,
year = {2024},
author = {Zhou, W and Li, Y and Liu, G and Qin, W and Wei, D and Wang, F and Gao, B},
title = {CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {197},
pmid = {38324086},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; DNA ; Green Fluorescent Proteins ; Lycopene ; RNA, Guide, CRISPR-Cas Systems ; *Saccharomycetales ; },
abstract = {Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: • Rapid marker recycling based on the visualization of a green fluorescent protein • One-step multifragment integration and large fragment knockout in the genome • A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.},
}
@article {pmid38323815,
year = {2024},
author = {Irfan, M and Solbiati, J and Duran-Pinedo, A and Rocha, FG and Gibson, FC and Frias-Lopez, J},
title = {A Porphyromonas gingivalis hypothetical protein controlled by the type I-C CRISPR-Cas system is a novel adhesin important in virulence.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0123123},
doi = {10.1128/msystems.01231-23},
pmid = {38323815},
issn = {2379-5077},
abstract = {The ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen, uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis. Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans' most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P. gingivalis and impacts the ability of P. gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms.},
}
@article {pmid38320438,
year = {2024},
author = {Saadh, MJ and Allela, OQB and Sattay, ZJ and Al Zuhairi, RAH and Ahmad, H and Eldesoky, GE and Adil, M and Ali, MS},
title = {Deciphering the functional landscape and therapeutic implications of noncoding RNAs in the TGF-β signaling pathway in colorectal cancer: A comprehensive review.},
journal = {Pathology, research and practice},
volume = {255},
number = {},
pages = {155158},
doi = {10.1016/j.prp.2024.155158},
pmid = {38320438},
issn = {1618-0631},
abstract = {Colorectal cancer (CRC) remains a major global health concern, necessitating an in-depth exploration of the intricate molecular mechanisms underlying its progression and potential therapeutic interventions. Transforming Growth Factor-β (TGF-β) signaling, a pivotal pathway implicated in CRC plays a dual role as a tumor suppressor in the early stages and a promoter of tumor progression in later stages. Recent research has shed light on the critical involvement of noncoding RNAs (ncRNAs) in modulating the TGF-β signaling pathway, introducing a new layer of complexity to our understanding of CRC pathogenesis. This comprehensive review synthesizes the current state of knowledge regarding the function and therapeutic potential of various classes of ncRNAs, including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), in the context of TGF-β signaling in CRC. The intricate interplay between these ncRNAs and key components of the TGF-β pathway is dissected, revealing regulatory networks that contribute to the dynamic balance between tumor suppression and promotion. Emphasis is placed on how dysregulation of specific ncRNAs can disrupt this delicate equilibrium, fostering CRC initiation, progression, and metastasis. Moreover, the review provides a critical appraisal of the emerging therapeutic strategies targeting ncRNAs associated with TGF-β signaling in CRC. The potential of these ncRNAs as diagnostic and prognostic biomarkers is discussed, highlighting their clinical relevance. Additionally, the challenges and prospects of developing RNA-based therapeutics, such as RNA interference and CRISPR/Cas-based approaches, are explored in the context of modulating TGF-β signaling for CRC treatment. In conclusion, this review offers a comprehensive overview of the intricate interplay between ncRNAs and the TGF-β signaling pathway in CRC. By unraveling the functional significance of these regulatory elements, we gain valuable insights into the molecular landscape of CRC, paving the way for the development of novel and targeted therapeutic interventions aimed at modulating the TGF-β signaling cascade through the manipulation of ncRNAs.},
}
@article {pmid38319094,
year = {2024},
author = {Han, X and Chang, L and Chen, H and Zhao, J and Tian, F and Ross, RP and Stanton, C and van Sinderen, D and Chen, W and Yang, B},
title = {Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0207423},
doi = {10.1128/aem.02074-23},
pmid = {38319094},
issn = {1098-5336},
abstract = {Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.},
}
@article {pmid38319081,
year = {2024},
author = {Sun, Q and Lin, H and Li, Y and Yuan, L and Li, B and Ma, Y and Wang, H and Deng, X and Chen, H and Tang, S},
title = {A photocontrolled one-pot isothermal amplification and CRISPR-Cas12a assay for rapid detection of SARS-CoV-2 Omicron variants.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0364523},
doi = {10.1128/spectrum.03645-23},
pmid = {38319081},
issn = {2165-0497},
abstract = {CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensitivity of 97.3% and a specificity of 100.0% and showed a concordance of 98.3% with Sanger sequencing results.IMPORTANCEWe successfully developed one-pot recombinase polymerase amplification/CRISPR-Cas12a genotyping assay by adapting photocontrolled CRISPR-Cas technology to optimize the conditions of nucleic acid amplification and CRISPR-Cas12a-mediated detection. This innovative approach was able to quickly distinguish severe acute respiratory syndrome coronavirus 2 Omicron variants and can be readily modified for detecting any nucleic acid mutations. The assay system demonstrates excellent clinical performance, including rapid detection, user-friendly operations, and minimized risk of contamination, which highlights its promising potential as a point-of-care testing for wide applications in resource-limiting settings.},
}
@article {pmid38317186,
year = {2024},
author = {Wu, J and Tan, HY and Chan, YT and Lu, Y and Feng, Z and Yuan, H and Zhang, C and Feng, Y and Wang, N},
title = {PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {43},
number = {1},
pages = {42},
pmid = {38317186},
issn = {1756-9966},
support = {17119621//Research Grants Committee (RGC) of Hong Kong, HKSAR/ ; 17121419//Research Grants Committee (RGC) of Hong Kong, HKSAR/ ; 18192141//the Health and Medical Research Fund/ ; 19200701//the Health and Medical Research Fund/ ; 19201951//the Health and Medical Research Fund/ ; PRP/028/22FX//Innovation and Technology Fund/ ; ITC RC/IHK/4/7//the Health@InnoHK Initiative Fund of the Hong Kong Special Administrative Region Government/ ; },
mesh = {Humans ; *Liver Neoplasms/pathology ; *Carcinoma, Hepatocellular/pathology ; Hedgehog Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Transformation, Neoplastic/metabolism ; Carcinogenesis/metabolism ; Choline/metabolism ; Neoplastic Stem Cells/metabolism ; Cell Cycle Proteins/metabolism ; Adaptor Proteins, Signal Transducing/metabolism ; },
abstract = {BACKGROUND: Par-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of PARD3 in hepatic tumorigenesis.<br><br>METHODS: The essential role of PARD3 in mediating hepatic tumorigenesis was assessed in diet-induced spontaneous liver tumour and syngeneic tumour models. The mechanism of PARD3 was delineated by bulk and single-cell RNA sequencing. The clinical significance of PARD3 was identified by tissue array analysis.<br><br>RESULTS: PARD3 was overexpressed in tumour tissues and PARD3 overexpression was positively correlated with high tumour stage as well as the poor prognosis in patients. In models of spontaneous liver cancer induced by choline-deficient, amino acid-defined (CDAA) and methionine-choline-deficient (MCD) diets, upregulation of PARD3 was induced specifically at the tumorigenesis stage rather than other early stages of liver disease progression. Site-directed knockout of PARD3 using an adeno-associated virus 8 (AAV8)-delivered CRISPR/Cas9 single-guide RNA (sgRNA) plasmid blocked hepatic tumorigenesis, while PARD3 overexpression accelerated liver tumour progression. In particular, single-cell sequencing analysis suggested that PARD3 was enriched in primitive tumour cells and its overexpression enhanced tumour-initiating cell (TICs). Overexpression of PARD3 maintained the self-renewal ability of the CD133[+] TIC population within hepatocellular carcinoma (HCC) cells and promoted the in vitro and in vivo tumorigenicity of CD133[+] TICs. Transcriptome analysis revealed that Sonic Hedgehog (SHH) signalling was activated in PARD3-overexpressing CD133[+] TICs. Mechanistically, PARD3 interacted with aPKC to further activate SHH signalling and downstream stemness-related genes. Suppression of SHH signalling and aPKC expression attenuated the in vitro and in vivo tumorigenicity of PARD3-overexpressing CD133[+] TICs. Tissue array analysis revealed that PARD3 expression was positively associated with the phosphorylation of aPKC, SOX2 and Gli1 and that the combination of these markers could be used to stratify HCC patients into two clusters with different clinicopathological characteristics and overall survival prognoses. The natural compound berberine was selected as a potent suppressor of PARD3 expression and could be used as a preventive agent for liver cancer that completely blocks diet-induced hepatic tumorigenesis in a PARD3-dependent manner.<br><br>CONCLUSION: This study revealed PARD3 as a potential preventive target of liver tumorigenesis via TIC regulation.},
}
@article {pmid38315256,
year = {2024},
author = {Sui, Z and Wu, Q and Geng, J and Xiao, J and Huang, D},
title = {CRISPR/Cas9-mediated efficient white genome editing in the black soldier fly Hermetia illucens.},
journal = {Molecular genetics and genomics : MGG},
volume = {299},
number = {1},
pages = {5},
pmid = {38315256},
issn = {1617-4623},
support = {31970440//National Natural Science Foundation of China/ ; 31830084//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Diptera/genetics ; RNA, Guide, CRISPR-Cas Systems ; Mutation ; },
abstract = {The CRISPR/Cas9 system is the most straightforward genome-editing technology to date, enabling genetic engineering in many insects, including the black soldier fly, Hermetia illucens. The white gene plays a significant role in the multifarious life activities of insects, especially the pigmentation of the eyes. In this study, the white gene of H. illucens (Hiwhite) was cloned, identified, and bioinformatically analysed for the first time. Using quantitative real-time polymerase chain reaction (qPCR), we found that the white gene was expressed in the whole body of the adult flies, particularly in Malpighian tubules and compound eyes. Furthermore, we utilised CRISPR/Cas9-mediated genome-editing technology to successfully generate heritable Hiwhite mutants using two single guide RNAs. During Hiwhite genome editing, we determined the timing, method, and needle-pulling parameters for embryo microinjection by observing early embryonic developmental features. We used the CasOT program to obtain highly specific guide RNAs (gRNAs) at the genome-wide level. According to the phenotypes of Hiwhite knockout strains, the pigmentation of larval stemmata, imaginal compound eyes, and ocelli differed from those of the wild type. These phenotypes were similar to those observed in other insects harbouring white gene mutations. In conclusion, our results described a detailed white genome editing process in black soldier flies, which lays a solid foundation for intensive research on the pigmentation pathway of the eyes and provides a methodological basis for further genome engineering applications in black soldier flies.},
}
@article {pmid38314924,
year = {2024},
author = {Miyamoto, K and Abe, G and Kawakami, K and Tamura, K and Ansai, S},
title = {The dwarf neon rainbowfish Melanotaenia praecox, a small spiny-rayed fish with potential as a new Acanthomorpha model fish: II. Establishment of a microinjection procedure for genetic engineering.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {},
number = {},
pages = {},
doi = {10.1002/dvdy.698},
pmid = {38314924},
issn = {1097-0177},
support = {2022036015//Takeda Science Fundation/ ; 22K06232//Japan Society for the Promotion of Science/ ; 20H04854//Japan Society for the Promotion of Science/ ; 22H02627//Japan Society for the Promotion of Science/ ; 21K19202//Japan Society for the Promotion of Science/ ; 21H05//Japan Society for the Promotion of Science/ ; },
abstract = {BACKGROUND: Rainbowfish is a clade of colorful freshwater fish. Melanotaenia praecox is a small rainbowfish species with biological characteristics that make it potentially useful as an experimental model species. We anticipate that M. praecox could become a new model used in various fields, such as ecology, evolution, and developmental biology. However, few previous studies have described experimental set-ups needed to understand the molecular and genetic mechanisms within this species.<br><br>RESULTS: We describe detailed procedures for genetic engineering in the rainbowfish M. praecox. By using these procedures, we successfully demonstrated CRISPR/Cas-mediated knockout and Tol2 transposon-mediated transgenesis in this species. Regarding the CRISPR/Cas system, we disrupted the tyrosinase gene and then showed that injected embryos lacked pigmentation over much of their body. We also demonstrated that a Tol2 construct, including a GFP gene driven by a ubiquitous promoter, was efficiently integrated into the genome of M. praecox embryos.<br><br>CONCLUSIONS: The establishment of procedures for genetic engineering in M. praecox enables investigation of the genetic mechanisms behind a broad range of biological phenomena in this species. Thus, we suggest that M. praecox can be used as a new model species in various experimental biology fields.},
}
@article {pmid38312197,
year = {2023},
author = {Divya, K and Thangaraj, M and Krishna Radhika, N},
title = {CRISPR/Cas9: an advanced platform for root and tuber crops improvement.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1242510},
pmid = {38312197},
issn = {2673-3439},
abstract = {Root and tuber crops (RTCs), which include cassava, potato, sweet potato, and yams, principally function as staple crops for a considerable fraction of the world population, in addition to their diverse applications in nutrition, industry, and bioenergy sectors. Even then, RTCs are an underutilized group considering their potential as industrial raw material. Complexities in conventional RTC improvement programs curb the extensive exploitation of the potentials of this group of crop species for food, energy production, value addition, and sustainable development. Now, with the advent of whole-genome sequencing, sufficient sequence data are available for cassava, sweet potato, and potato. These genomic resources provide enormous scope for the improvement of tuber crops, to make them better suited for agronomic and industrial applications. There has been remarkable progress in RTC improvement through the deployment of new strategies like gene editing over the last decade. This review brings out the major areas where CRISPR/Cas technology has improved tuber crops. Strategies for genetic transformation of RTCs with CRISPR/Cas9 constructs and regeneration of edited lines and the bottlenecks encountered in their establishment are also discussed. Certain attributes of tuber crops requiring focus in future research along with putative editing targets are also indicated. Altogether, this review provides a comprehensive account of developments achieved, future lines of research, bottlenecks, and major experimental concerns regarding the establishment of CRISPR/Cas9-based gene editing in RTCs.},
}
@article {pmid38311724,
year = {2024},
author = {Ninyio, N and Schmitt, K and Sergon, G and Nilsson, C and Andersson, S and Scherbak, N},
title = {Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9.},
journal = {Microbial cell factories},
volume = {23},
number = {1},
pages = {39},
pmid = {38311724},
issn = {1475-2859},
mesh = {Animals ; Humans ; Epitopes ; Escherichia coli/genetics ; *HIV-1/genetics ; CRISPR-Cas Systems ; HIV Antibodies ; *Vaccines ; },
abstract = {BACKGROUND: Mucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.<br><br>RESULTS: The HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) - rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3&#xa0;&#xb5;g/10[8] cfu.<br><br>CONCLUSIONS: The CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.},
}
@article {pmid38310944,
year = {2024},
author = {Zhang, D and Gan, Y and Le, L and Pu, L},
title = {Epigenetic variation in maize agronomical traits for breeding and trait improvement.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2024.01.006},
pmid = {38310944},
issn = {1673-8527},
abstract = {Epigenetics-mediated breeding (Epibreeding) involves engineering crop traits and stress responses through targeted manipulation of key epigenetic features to enhance agricultural productivity. While conventional breeding methods raise concerns about reduced genetic diversity, epibreeding propels crop improvement through epigenetic variations that regulate gene expression, ultimately impacting crop yield. Epigenetic regulation in crops encompasses various modes, including histone modification, DNA modification, RNA modification, non-coding RNA, and chromatin remodeling. This review summarizes the epigenetic mechanisms underlying major agronomic traits in maize and identifies candidate epigenetic landmarks in the maize breeding process. We propose a novel strategy for improving maize yield through epibreeding, combining CRISPR/Cas-based epigenome editing technology and Synthetic Epigenetics (SynEpi). Finally, we discuss the challenges and opportunities associated with enhancing maize traits through epibreeding.},
}
@article {pmid38310457,
year = {2024},
author = {Upreeti, A and Mukherjee, S},
title = {Therapeutic Potential of CRISPR/Cas in Hashimoto's Thyroiditis: A Comprehensive Review.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115665232266508231210154930},
pmid = {38310457},
issn = {1875-5631},
abstract = {Hashimoto's thyroiditis (HT) is a commonly occurring illness of autoimmune endocrine origin. It is usually present in the pediatric age group along with other well-known diseases, such as type 1 insulin-dependent diabetes. The defining feature of this disease is the immune-- mediated attack on the thyroid gland resulting in the destruction of thyroid tissues and cells. Given that HT frequently affects family members, it is well-recognized that individuals are genetically predisposed to this disease. Patients with HT also display a significantly increased risk for several different cancers, justifying the eminent need for the development of therapies for managing and treating HT. Gene editing has made several advancements in the field of molecular biology and has turned out to become a promising approach to correct several autoimmune diseases. Currently, CRISPR/Cas, a nuclease-based editing technique, is publicized as a promising tool for curing several genetic diseases and cancers. However, very limited research has been conducted as of now on autoimmune disease management and cure via CRISPR/Cas technique. This review provides an account of the potential candidate genes associated with Hashimoto's thyroiditis, and only a few animal and human models have been generated via the CRISPR/Cas gene editing technique. Mouse models of autoimmune thyroiditis generated through the CRISPR/Cas gene editing technique by targeting the candidate genes will provide us with a deeper insight into the pathophysiology of HT and further pave the way for the immunomodulation of HT via gene editing.},
}
@article {pmid38308076,
year = {2024},
author = {Gopikrishnan, M and Haryini, S and C, GPD},
title = {Emerging strategies and therapeutic innovations for combating drug resistance in Staphylococcus aureus strains: A comprehensive review.},
journal = {Journal of basic microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1002/jobm.202300579},
pmid = {38308076},
issn = {1521-4028},
support = {OMI/11/2020-ECD-1//Indian Council of Medical Research (ICMR)/ ; },
abstract = {In recent years, antibiotic therapy has encountered significant challenges due to the rapid emergence of multidrug resistance among bacteria responsible for life-threatening illnesses, creating uncertainty about the future management of infectious diseases. The escalation of antimicrobial resistance in the post-COVID era compared to the pre-COVID era has raised global concern. The prevalence of nosocomial-related infections, especially outbreaks of drug-resistant strains of Staphylococcus aureus, have been reported worldwide, with India being a notable hotspot for such occurrences. Various virulence factors and mutations characterize nosocomial infections involving S. aureus. The lack of proper alternative treatments leading to increased drug resistance emphasizes the need to investigate and examine recent research to combat future pandemics. In the current genomics era, the application of advanced technologies such as next-generation sequencing (NGS), machine learning (ML), and quantum computing (QC) for genomic analysis and resistance prediction has significantly increased the pace of diagnosing drug-resistant pathogens and insights into genetic intricacies. Despite prompt diagnosis, the elimination of drug-resistant infections remains unattainable in the absence of effective alternative therapies. Researchers are exploring various alternative therapeutic approaches, including phage therapy, antimicrobial peptides, photodynamic therapy, vaccines, host-directed therapies, and more. The proposed review mainly focuses on the resistance journey of S. aureus over the past decade, detailing its resistance mechanisms, prevalence in the subcontinent, innovations in rapid diagnosis of the drug-resistant strains, including the applicants of NGS and ML application along with QC, it helps to design alternative novel therapeutics approaches against S. aureus infection.},
}
@article {pmid38306418,
year = {2024},
author = {Li, X and Wirtz, T and Weber, T and Lebedin, M and Lowenstein, ED and Sommermann, T and Zach, A and Yasuda, T and de la Rosa, K and Chu, VT and Schulte, JH and Müller, I and Kocks, C and Rajewsky, K},
title = {Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis.},
journal = {Science immunology},
volume = {9},
number = {92},
pages = {eadi0042},
doi = {10.1126/sciimmunol.adi0042},
pmid = {38306418},
issn = {2470-9468},
mesh = {Animals ; Mice ; Humans ; *Lymphohistiocytosis, Hemophagocytic/genetics/therapy ; CRISPR-Cas Systems ; *Epstein-Barr Virus Infections/genetics/therapy ; Memory T Cells ; Herpesvirus 4, Human ; Membrane Proteins/genetics ; },
abstract = {Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus-based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus-triggered FHL2, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D)-whose deficiency causes the FHL subtype FHL3-in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells.},
}
@article {pmid38305919,
year = {2024},
author = {Ng, HM and Gondo, T and Tanaka, H and Akashi, R},
title = {CRISPR/Cas9-mediated knockout of NYC1 gene enhances chlorophyll retention and reduces tillering in Zoysia matrella (L.) Merrill.},
journal = {Plant cell reports},
volume = {43},
number = {2},
pages = {50},
pmid = {38305919},
issn = {1432-203X},
support = {17K07608 (2017-2019)//Japan Society for the Promotion of Science/ ; 23K05524 (2023-2026)//Japan Society for the Promotion of Science/ ; 118 (2022)//Ito Foundation/ ; 32 (2023)//Ito Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Genome, Plant ; Chlorophyll A ; Plant Breeding ; Gene Editing ; Poaceae/genetics ; Chlorophyll ; },
abstract = {Genome editing by CRISPR/Cas9 can be applied to Z. matrella 'Wakaba', and knockout mutants of ZmNYC1 gene exhibited stay-green phenotype and reduced tillering. Zoysia matrella is a widely used C4 warm-season turfgrass for landscaping, golf courses, and sports fields. Here, we used the CRISPR/Cas9 system to target the Non-Yellow Coloring1 (ZmNYC1) gene in the highly heterozygous allotetraploid Z. matrella 'Wakaba', aiming to generate a novel stay-green variety. Of 441 Agrobacterium-infected calli, 22 (5.0%) were transformed, and 14 of these (63.6%) showed targeted mutations through cleaved amplified polymorphic sequences analysis. Sequencing analysis revealed mutations mostly consisting of 1 or 2 bp indels, occurring 2 to 4 bp upstream of the PAM sequence. Regenerated plants exhibited five ZmNYC1 target locus genotypes, including homozygous mutants with a complete knockout of all four alleles in the T0 generation. Under dark treatment, ZmNYC1-mutated plants displayed suppressed chlorophyll b (Chl b) degradation, leading to higher chlorophyll content and Chl b, with a lower chlorophyll a/chlorophyll b ratio compared to the wild type (WT). However, the ZmNYC1 mutation also inhibited plant growth in homozygous mutant genotypes, exhibiting reduced tillering compared to WT. Additionally, during winter simulation, mutant with a complete knockout retained greenness longer than the WT. This is the first successful use of CRISPR/Cas9 genome editing in zoysiagrass. The mutants of the ZmNYC1 gene would serve as valuable breeding material for developing improved zoysiagrass varieties that can maintain their green color for longer periods, even during winter dormancy.},
}
@article {pmid38304712,
year = {2024},
author = {Chen, P and Wang, S and Li, H and Qi, X and Hou, Y and Ma, T},
title = {Comparative genomic analyses of Cutibacterium granulosum provide insights into genomic diversity.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1343227},
pmid = {38304712},
issn = {1664-302X},
abstract = {Cutibacterium granulosum, a commensal bacterium found on human skin, formerly known as Propionibacterium granulosum, rarely causes infections and is generally considered non-pathogenic. Recent research has revealed the transferability of the multidrug-resistant plasmid pTZC1 between C. granulosum and Cutibacterium acnes, the latter being an opportunistic pathogen in surgical site infections. However, there is a noticeable lack of research on the genome of C. granulosum, and the genetic landscape of this species remains largely uncharted. We investigated the genomic features and evolutionary structure of C. granulosum by analyzing a total of 30 Metagenome-Assembled Genomes (MAGs) and isolate genomes retrieved from public databases, as well as those generated in this study. A pan-genome of 6,077 genes was identified for C. granulosum. Remarkably, the 'cloud genes' constituted 62.38% of the pan-genome. Genes associated with mobilome: prophages, transposons [X], defense mechanisms [V] and replication, recombination and repair [L] were enriched in the cloud genome. Phylogenomic analysis revealed two distinct mono-clades, highlighting the genomic diversity of C. granulosum. The genomic diversity was further confirmed by the distribution of Average Nucleotide Identity (ANI) values. The functional profiles analysis of C. granulosum unveiled a wide range of potential Antibiotic Resistance Genes (ARGs) and virulence factors, suggesting its potential tolerance to various environmental challenges. Subtype I-E of the CRISPR-Cas system was the most abundant in these genomes, a feature also detected in C. acnes genomes. Given the widespread distribution of C. granulosum strains within skin microbiome, our findings make a substantial contribution to our broader understanding of the genetic diversity, which may open new avenues for investigating the mechanisms and treatment of conditions such as acne vulgaris.},
}
@article {pmid38304452,
year = {2023},
author = {Nourozi, M and Nazarain-Firouzabadi, F and Ismaili, A and Ahmadvand, R and Poormazaheri, H},
title = {CRISPR/Cas StNRL1 gene knockout increases resistance to late blight and susceptibility to early blight in potato.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1278127},
pmid = {38304452},
issn = {1664-462X},
abstract = {With the development of genome editing technologies, editing susceptible genes is a promising method to modify plants for resistance to stress. NPH3/RPT2-LIKE1 protein (NRL1) interacts with effector Pi02860 of Phytophthora infestans and creates a protein complex, promoting the proteasome-mediated degradation of the guanine nucleotide exchange factor SWAP70. SWAP70, as a positive regulator, enhances cell death triggered by the perception of the P. infestans pathogen-associated molecular pattern (PAMP) INF1. Using a clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a construct was made to introduce four guide RNAs into the potato cultivar Agria. A total of 60 putative transgenic lines were regenerated, in which 10 transgenic lines with deletions were selected and analyzed. A mutant line with a four-allelic knockdown of StNRL1 gene was obtained, showing an ~90% reduction in StNRL1 expression level, resulting in enhanced resistance to P. infestans. Surprisingly, mutant lines were susceptible to Alternaria alternata, suggesting that StNRL1 may play a role as a resistance gene; hence, silencing StNRL1 enhances resistance to P. infestans.},
}
@article {pmid38052131,
year = {2024},
author = {Takahashi, K and Galloway, KE},
title = {RNA-based controllers for engineering gene and cell therapies.},
journal = {Current opinion in biotechnology},
volume = {85},
number = {},
pages = {103026},
doi = {10.1016/j.copbio.2023.103026},
pmid = {38052131},
issn = {1879-0429},
mesh = {*RNA/genetics ; *Genetic Engineering ; Gene Expression Regulation ; Cell- and Tissue-Based Therapy ; Genetic Vectors ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Engineered RNA-based genetic controllers provide compact, tunable, post-transcriptional gene regulation. As RNA devices are generally small, these devices are portable to DNA and RNA viral vectors. RNA tools have recently expanded to allow reading and editing of endogenous RNAs for profiling and programming of transcriptional states. With their expanded capabilities and highly compact, modular, and programmable nature, RNA-based controllers will support greater safety, efficacy, and performance in gene and cell-based therapies. In this review, we highlight RNA-based controllers and their potential as user-guided and autonomous systems for control of gene and cell-based therapies.},
}
@article {pmid38303616,
year = {2023},
author = {Foo, N and Adesanya, O and Nielsen, J and Nicol, D},
title = {The Patent Landscape for CRISPR Genome Editing in Australia.},
journal = {Journal of law and medicine},
volume = {30},
number = {2},
pages = {286-309},
pmid = {38303616},
issn = {1320-159X},
mesh = {*Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Australia ; },
abstract = {Although Australia has a proud record of health and medical research, it finds less traction when it comes to innovative product development. Patent filings are recognised as one of the measures of national innovation, and this is one measure where Australian innovators are falling short. We examined whether there may be discrete pockets of innovation in particular areas of technology where Australian researchers are making significant contributions. This study used patent filings as a measure of innovation and used clustered regularly interspaced short palindromic repeat (CRISPR) genome editing as a case study. We found a rich patent landscape, with filings for general methods and compositions and for specific diseases. However, the contribution by Australian applicants was small, with only four out of 519 filings. This indicates that navigating the CRISPR patent landscape to secure freedom to operate is likely to be complex for Australian innovators in this field.},
}
@article {pmid38303094,
year = {2024},
author = {Guo, Y and Xue, Z and Gong, M and Jin, S and Wu, X and Liu, W},
title = {CRISPR-TE: a web-based tool to generate single guide RNAs targeting transposable elements.},
journal = {Mobile DNA},
volume = {15},
number = {1},
pages = {3},
pmid = {38303094},
issn = {1759-8753},
abstract = {BACKGROUND: The CRISPR/Cas systems have emerged as powerful tools in genome engineering. Recent studies highlighting the crucial role of transposable elements (TEs) have stimulated research interest in manipulating these elements to understand their functions. However, designing single guide RNAs (sgRNAs) that are specific and efficient for TE manipulation is a significant challenge, given their sequence repetitiveness and high copy numbers. While various sgRNA design tools have been developed for gene editing, an optimized sgRNA designer for TE manipulation has yet to be established.<br><br>RESULTS: We present CRISPR-TE, a web-based application featuring an accessible graphical user interface, available at https://www.crisprte.cn/ ,&#xa0;and currently tailored to the human and mouse genomes.&#xa0;CRISPR-TE identifies all potential sgRNAs for TEs and provides a comprehensive solution for efficient TE targeting at both the single copy and subfamily levels. Our analysis shows that sgRNAs targeting TEs can more effectively target evolutionarily young TEs with conserved sequences at the subfamily level.<br><br>CONCLUSIONS: CRISPR-TE offers a versatile framework for designing sgRNAs for TE targeting. CRISPR-TE is publicly accessible at https://www.crisprte.cn/ as an online web service and the source code of CRISPR-TE is available at https://github.com/WanluLiuLab/CRISPRTE/ .},
}
@article {pmid38302906,
year = {2024},
author = {Duddy, G and Courtis, K and Horwood, J and Olsen, J and Horsler, H and Hodgson, T and Varsani-Brown, S and Abdullah, A and Denti, L and Lane, H and Delaqua, F and Janzen, J and Strom, M and Rosewell, I and Crawley, K and Davies, B},
title = {Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.},
journal = {BMC biology},
volume = {22},
number = {1},
pages = {26},
pmid = {38302906},
issn = {1741-7007},
support = {CC0199/CRUK_/Cancer Research UK/United Kingdom ; CC0199/WT_/Wellcome Trust/United Kingdom ; CC0199/ARC_/Arthritis Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; CC0199/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Dependovirus/genetics ; Reproducibility of Results ; Zygote ; Gene Targeting ; Gene Knock-In Techniques/methods ; },
abstract = {BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route.<br><br>RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods.<br><br>CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7&#xa0;kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.},
}
@article {pmid38302235,
year = {2024},
author = {Saretzki, G},
title = {Measuring telomerase activity using TRAP assays.},
journal = {Methods in cell biology},
volume = {181},
number = {},
pages = {127-149},
doi = {10.1016/bs.mcb.2022.12.009},
pmid = {38302235},
issn = {0091-679X},
mesh = {*Telomerase/genetics/analysis/metabolism ; Telomere/chemistry/metabolism ; Real-Time Polymerase Chain Reaction ; },
abstract = {Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.},
}
@article {pmid38301007,
year = {2024},
author = {Steens, JA and Bravo, JPK and Salazar, CRP and Yildiz, C and Amieiro, AM and Köstlbacher, S and Prinsen, SHP and Andres, AS and Patinios, C and Bardis, A and Barendregt, A and Scheltema, RA and Ettema, TJG and van der Oost, J and Taylor, DW and Staals, RHJ},
title = {Type III-B CRISPR-Cas cascade of proteolytic cleavages.},
journal = {Science (New York, N.Y.)},
volume = {383},
number = {6682},
pages = {512-519},
doi = {10.1126/science.adk0378},
pmid = {38301007},
issn = {1095-9203},
mesh = {*Bacterial Proteins/chemistry/genetics ; *Caspases/chemistry/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; RNA/metabolism ; *Proteolysis ; *Myxococcales/enzymology/genetics ; Protein Domains ; },
abstract = {The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas-based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities.},
}
@article {pmid38300351,
year = {2024},
author = {Yamamoto, K and Tochikawa, S and Miura, Y and Matsunobu, S and Hirose, Y and Eki, T},
title = {Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {188},
pmid = {38300351},
issn = {1432-0614},
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; DNA Damage ; Mitomycin ; Luciferases ; DNA ; },
abstract = {Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.},
}
@article {pmid38288550,
year = {2024},
author = {Hii, ARK and Qi, X and Wu, Z},
title = {Advanced strategies for CRISPR/Cas9 delivery and applications in gene editing, therapy, and cancer detection using nanoparticles and nanocarriers.},
journal = {Journal of materials chemistry. B},
volume = {12},
number = {6},
pages = {1467-1489},
doi = {10.1039/d3tb01850d},
pmid = {38288550},
issn = {2050-7518},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Therapy/methods ; *Neoplasms/genetics ; *Nanoparticles ; },
abstract = {Cancer remains one of the deadliest diseases, and is characterised by the uncontrolled growth of modified human cells. Unlike infectious diseases, cancer does not originate from foreign agents. Though a variety of diagnostic procedures are available; their cost-effectiveness and accessibility create significant hurdles. Non-specific cancer symptoms further complicate early detection, leading to belated recognition of certain cancer. The lack of reliable biomarkers hampers effective treatment, as chemotherapy, radiation therapy, and surgery often result in poor outcomes and high recurrence rates. Genetic and epigenetic mutations play a crucial role in cancer pathogenesis, necessitating the development of alternate treatment methods. The advent of CRISPR/Cas9 technology has transformed molecular biology and exhibits potential for gene modification and therapy in various cancer types. Nonetheless, obstacles such as safe transport, off-target consequences, and potency must be overcome before widespread clinical use. Notably, this review delves into the multifaceted landscape of cancer research, highlighting the pivotal role of nanoparticles in advancing CRISPR/Cas9-based cancer interventions. By addressing the challenges associated with cancer diagnosis and treatment, this integrated approach paves the way for innovative solutions and improved patient outcomes.},
}
@article {pmid38278939,
year = {2024},
author = {Ledford, H},
title = {CRISPR-edited crops break new ground in Africa.},
journal = {Nature},
volume = {626},
number = {7998},
pages = {245-246},
doi = {10.1038/d41586-024-00176-8},
pmid = {38278939},
issn = {1476-4687},
mesh = {Africa ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Gene Editing ; *Plants, Genetically Modified/genetics ; *Pest Control, Biological/methods/trends ; },
}
@article {pmid38228008,
year = {2024},
author = {Ma, Y and Wei, H and Wang, Y and Cheng, X and Chen, H and Yang, X and Zhang, H and Rong, Z and Wang, S},
title = {Efficient magnetic enrichment cascade single-step RPA-CRISPR/Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples.},
journal = {Journal of hazardous materials},
volume = {465},
number = {},
pages = {133494},
doi = {10.1016/j.jhazmat.2024.133494},
pmid = {38228008},
issn = {1873-3336},
mesh = {*Recombinases ; *Staphylococcus aureus ; CRISPR-Cas Systems ; Biological Assay ; Magnetic Phenomena ; Nucleic Acid Amplification Techniques ; },
abstract = {Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that could cause severe food poisoning. Thus, rapid, efficient, and ultrasensitive detection of S. aureus in food samples is urgently needed. Here, we report an efficient magnetic enrichment cascade single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the ultrasensitive detection of S. aureus. Magnetic beads (MBs) functionalized with S. aureus-specific antibodies were initially used for S. aureus enrichment from the complex matrix, with 98% capture efficiency in 5 min and 100-fold sensitivity improvement compared with unenriched S. aureus. Next, a single-step RPA-CRISPR/Cas12a-based diagnostic system with optimized extraction-free bacteria lysis was constructed. This assay could detect as low as 1 copy/μL (five copies/reaction) of extracted DNA template and 10 CFU/mL of S. aureus within 40 min. Furthermore, the assay could effectively detect S. aureus in real food samples such as lake water, orange juice, pork, and lettuce, with concordant results to qPCR assays. The proposed cascade signal-amplification assay eliminates the need for lengthy bacterial culture and complex sample preparation steps. Hence, the proposed assay shows great application potential for rapid, efficient, and ultrasensitive detection of pathogens in real food samples.},
}
@article {pmid38199209,
year = {2024},
author = {Luo, Y and Chen, Y and Xie, H and Zhu, W and Zhang, G},
title = {Interpretable CRISPR/Cas9 off-target activities with mismatches and indels prediction using BERT.},
journal = {Computers in biology and medicine},
volume = {169},
number = {},
pages = {107932},
doi = {10.1016/j.compbiomed.2024.107932},
pmid = {38199209},
issn = {1879-0534},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *CRISPR-Cas Systems ; Gene Editing ; Genome ; Genomics ; },
abstract = {Off-target effects of CRISPR/Cas9 can lead to suboptimal genome editing outcomes. Numerous deep learning-based approaches have achieved excellent performance for off-target prediction; however, few can predict the off-target activities with both mismatches and indels between single guide RNA (sgRNA) and target DNA sequence pair. In addition, data imbalance is a common pitfall for off-target prediction. Moreover, due to the complexity of genomic contexts, generating an interpretable model also remains challenged. To address these issues, firstly we developed a BERT-based model called CRISPR-BERT for enhancing the prediction of off-target activities with both mismatches and indels. Secondly, we proposed an adaptive batch-wise class balancing strategy to combat the noise exists in imbalanced off-target data. Finally, we applied a visualization approach for investigating the generalizable nucleotide position-dependent patterns of sgRNA-DNA pair for off-target activity. In our comprehensive comparison to existing methods on five mismatches-only datasets and two mismatches-and-indels datasets, CRISPR-BERT achieved the best performance in terms of AUROC and PRAUC. Besides, the visualization analysis demonstrated how implicit knowledge learned by CRISPR-BERT facilitates off-target prediction, which shows potential in model interpretability. Collectively, CRISPR-BERT provides an accurate and interpretable framework for off-target prediction, further contributes to sgRNA optimization in practical use for improved target specificity in CRISPR/Cas9 genome editing. The source code is available at https://github.com/BrokenStringx/CRISPR-BERT.},
}
@article {pmid38197659,
year = {2024},
author = {Qu, H and Zhang, W and Li, J and Fu, Q and Li, X and Wang, M and Fu, G and Cui, J},
title = {A rapid and sensitive CRISPR-Cas12a for the detection of Fusobacterium nucleatum.},
journal = {Microbiology spectrum},
volume = {12},
number = {2},
pages = {e0362923},
pmid = {38197659},
issn = {2165-0497},
mesh = {Humans ; *Fusobacterium nucleatum ; CRISPR-Cas Systems ; Gastrointestinal Tract ; Health Status ; *Communicable Diseases ; Recombinases ; },
abstract = {Fusobacterium nucleatum (Fn), as a conditional pathogen, can cause a range of oral and gastrointestinal diseases. However, existing clinical detection methods require expensive equipment and complex procedures, which are inconvenient for large-scale screening in epidemiological research. The purpose of this study was to establish a reliable, rapid, and inexpensive detection method based on CRISPR/Cas12a technology for the detection of Fn. Specific recombinase polymerase amplification (RPA) primer sequences and crRNA sequences were designed based on the nusG gene of Fn. Subsequently, a fluorescence assay and a lateral flow immunoassay were established using the RPA and CRISPR-Cas12a system (RPA-CRISPR-Cas12a). Sensitivity validation revealed a limit of detection of 5 copies/µL. This method could distinguish Fn from other pathogens with excellent specificity. Furthermore, the RPA-CRISPR-Cas12a assay was highly consistent with the classical quantitative real-time PCR method when testing periodontal pocket samples. This makes it a promising method for the detection of Fn and has the potential to play an increasingly important role in infectious disease testing.IMPORTANCEFusobacterium nucleatum (Fn) naturally exists in the microbial communities of the oral and gastrointestinal tracts of healthy individuals and can cause inflammatory diseases in the oral and gastrointestinal tracts. Recent studies have shown that Fn is closely associated with the occurrence and development of gastrointestinal cancer. Therefore, the detection of Fn is very important. Unlike the existing clinical detection methods, this study established a fluorescence-based assay and lateral flow immunoassay based on the RPA and CRISPR-Cas12a system (RPA-CRISPR-Cas12a), which is fast, reliable, and inexpensive and can complete the detection within 30-40 minutes. This makes it a promising method for the detection of Fn and has the potential to play an increasingly important role in infectious disease testing.},
}
@article {pmid37989654,
year = {2024},
author = {Morris, JA and Sun, JS and Sanjana, NE},
title = {Next-generation forward genetic screens: uniting high-throughput perturbations with single-cell analysis.},
journal = {Trends in genetics : TIG},
volume = {40},
number = {2},
pages = {118-133},
doi = {10.1016/j.tig.2023.10.012},
pmid = {37989654},
issn = {0168-9525},
support = {R01 AI176601/AI/NIAID NIH HHS/United States ; R01 CA279135/CA/NCI NIH HHS/United States ; R01 HG012790/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; Genetic Testing ; Single-Cell Analysis/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbations or combinations of perturbations impact the cellular transcriptome, proteome, and epigenome. We review technologies that pair CRISPR screens with single-cell multiomics and the unique opportunities afforded by extending pooled screens using deep multimodal phenotyping.},
}
@article {pmid37899674,
year = {2024},
author = {Bai, X and Yu, K and Xiong, S and Chen, J and Yang, Y and Ye, X and Yao, H and Wang, F and Fang, Q and Song, Q and Ye, G},
title = {CRISPR/Cas9-mediated mutagenesis of the white gene in an ectoparasitic wasp, Habrobracon hebetor.},
journal = {Pest management science},
volume = {80},
number = {3},
pages = {1219-1227},
doi = {10.1002/ps.7851},
pmid = {37899674},
issn = {1526-4998},
support = {31830074//Key Program of National Natural Science Foundation of China/ ; U21A20225//Regional Joint Fund for Innovation and Development of NSFC/ ; 2016RA4008//Program for Chinese Innovation Team in Key Areas of Science and Technology of Ministry of Science and Technology of the People's Republic of China/ ; },
mesh = {Animals ; Female ; *Wasps/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Mutagenesis ; DNA ; },
abstract = {BACKGROUND: The ectoparasitic wasp Habrobracon hebetor (Hymenoptera, Braconidae) can parasitize various species of lepidopteran pests. To maximize its potential for biological control, it is necessary to investigate its gene function through genome engineering.<br><br>RESULTS: To test the effectiveness of genome engineering system in H. hebetor, we injected the mixture of clustered regularly interspaced short palindromic repeats (CRISPR) -associated (Cas) 9 protein and single guide RNA(s) targeting gene white into embryos. The resulting mutants display a phenotype of eye pigment loss. The phenotype was caused by small indel and is heritable. Then, we compared some biological parameters between wildtype and mutant, and found there were no significant differences in other parameters except for the offspring female rate and adult longevity. In addition, cocoons could be used to extract genomic DNA for genotype during the gene editing process without causing unnecessary harm to H. hebetor.<br><br>CONCLUSION: Our results demonstrate that the CRISPR/Cas9 system can be used for H. hebetor genome editing and it does not adversely affect biological parameters of the parasitoid wasps. We also provide a feasible non-invasive genotype detection method using genomic DNA extracted from cocoons. Our study introduces a novel tool and method for studying gene function in H. hebetor, and may contribute to better application of H. hebetor in biocontrol. &#xa9; 2023 Society of Chemical Industry.},
}
@article {pmid37874890,
year = {2024},
author = {Sun, X and Lei, R and Zhang, H and Chen, W and Jia, Q and Guo, X and Zhang, Y and Wu, P and Wang, X},
title = {Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/CRISPR-Cas12a method for potential on-site disease diagnosis.},
journal = {Pest management science},
volume = {80},
number = {3},
pages = {1168-1181},
doi = {10.1002/ps.7847},
pmid = {37874890},
issn = {1526-4998},
support = {//Basic Scientific Research Foundation of Chinese Academy of Inspection and Quarantine/ ; //National Key Research and Development Program of China/ ; //the Subject Construction Project of Dalian University/ ; },
mesh = {Humans ; *Recombinases ; *Glycine max ; CRISPR-Cas Systems ; Biological Assay ; DNA ; Nucleic Acid Amplification Techniques ; },
abstract = {BACKGROUND: Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora.<br><br>RESULTS: The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12&#x2009;days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60&#x2009;min.<br><br>CONCLUSION: The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic beads or rapid DNA extraction, isothermal nucleic acid amplification at 39&#x2009;&#xb0;C, CRISPR-Cas12a cleavage reaction at 37&#x2009;&#xb0;C, and lateral flow assay or endpoint fluorescence visualization at room temperature. The RPA and CRISPR-Cas12a reagents can be preloaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR-Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, and has promising applications for rapid on-site disease screening, port inspection, or controlling fungal pathogen transmission in crop. &#xa9; 2023 Society of Chemical Industry.},
}
@article {pmid37605534,
year = {2023},
author = {House, IG and Derrick, EB and Sek, K and Chen, AXY and Li, J and Lai, J and Todd, KL and Munoz, I and Michie, J and Chan, CW and Huang, YK and Chan, JD and Petley, EV and Tong, J and Nguyen, D and Engel, S and Savas, P and Hogg, SJ and Vervoort, SJ and Kearney, CJ and Burr, ML and Lam, EYN and Gilan, O and Bedoui, S and Johnstone, RW and Dawson, MA and Loi, S and Darcy, PK and Beavis, PA},
title = {CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity.},
journal = {Cell reports},
volume = {42},
number = {8},
pages = {113014},
doi = {10.1016/j.celrep.2023.113014},
pmid = {37605534},
issn = {2211-1247},
mesh = {Feedback ; *CRISPR-Cas Systems ; *Immune Checkpoint Inhibitors ; Suppressor of Cytokine Signaling Proteins/genetics ; Signal Transduction ; },
abstract = {CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.},
}
@article {pmid38300346,
year = {2024},
author = {Yu, Y and Zhang, Y and Chen, X and Li, W and Wang, Z and Mi, Q and Zhang, J},
title = {Bi-functionality of glyoxal caged nucleic acid coupled with CRISPR/Cas12a system for Hg[2+] determination.},
journal = {Mikrochimica acta},
volume = {191},
number = {2},
pages = {120},
pmid = {38300346},
issn = {1436-5073},
support = {2018YFC160440//Special National Key Research and Development Plan/ ; 20392001800//Science and Technology Commission of Shanghai Municipality/ ; 31671923//National Natural Science Foundation of China/ ; },
mesh = {*Nucleic Acids ; CRISPR-Cas Systems ; RNA ; *Mercury ; Glyoxal ; },
abstract = {A highly sensitive and selective fluorescence method has been conducted for the detection of Hg[2+] based on aminophenylboronic acid-modified carboxyl magnetic beads (CMB@APBA) and CRISPR/Cas12a system mediated by glyoxal caged nucleic acid (gcDNA). As a bi-functional DNA linker, gcDNA offers advantages of simultaneous recognition by boronic acid and complementary DNA/RNA. Under acidic condition, gcDNA can be immobilized on CMB@APBA through the formation of borate ester bond. The formed boric acid-esterified gcDNA can further bind with complementary CRISPR RNA through A-T base pairing to activate Cas12a with kcat/Km ratio of 3.4 × 10[7] s[-1] M[-1], allowing for amplified signal. Hg[2+] can specifically combine with CMB@APBA, resulting in the release of gcDNA from CMB@APBA and the following inhibition on the activation of CRISPR/Cas12a system around magnetic bead. Under optimal conditions, the method exhibits a linear range from 20 to 250 nM, with a detection limit of 2.72 nM. The proposed method can detect Hg[2+] in milk and tea beverages, providing a great significance for on-site monitoring of Hg[2+] contamination in food.},
}
@article {pmid38298016,
year = {2024},
author = {Yahsi, B and Palaz, F and Dincer, P},
title = {Applications of CRISPR Epigenome Editors in Tumor Immunology and Autoimmunity.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00524},
pmid = {38298016},
issn = {2161-5063},
abstract = {Over the past decade, CRISPR-Cas systems have become indispensable tools for genetic engineering and have been used in clinical trials for various diseases. Beyond genome editing, CRISPR-Cas systems can also be used for performing programmable epigenetic modifications. Recent efforts in enhancing CRISPR-based epigenome modifiers have yielded potent tools enabling targeted DNA methylation/demethylation capable of sustaining epigenetic memory through numerous cell divisions. Moreover, it has been understood that during chronic inflammatory states, including cancer, T cells encounter a state called T cell exhaustion that involves elevated inhibitory receptors (e.g., LAG-3, TIM3, PD-1, CD39) and reduced effector T cell-related protein levels (IFN-γ, granzyme B, and perforin). Importantly, epigenetic dysregulation has been identified as one of the key drivers of T cell exhaustion, and it remains one of the biggest obstacles in the field of immunotherapy and decreases the efficiency of chimeric antigen receptor T (CAR-T) cell therapy. Similarly, autoimmune diseases exhibit epigenetically dysfunctional regulatory T (Treg) cells. For instance, FOXP3 intronic regions, known as conserved noncoding sequences, display hypomethylation in healthy states but hypermethylation in pathological contexts. Therefore, the reversal of epigenetic dysregulation in cancer and autoimmune diseases using CRISPR-based epigenome modifiers has important therapeutic implications. In this review, we outline the progressive refinement of CRISPR-based epigenome modifiers and explore their potential therapeutic applications in tumor immunology and autoimmunity.},
}
@article {pmid38297069,
year = {2024},
author = {Sharp, BM and Jiang, Q and Kim, P and Chen, H},
title = {Inactivation of phosphodiesterase-4B gene in rat nucleus accumbens shell by CRISPR/Cas9 or positive allosteric modulation of the protein affects the motivation to chronically self-administer nicotine.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {2562},
pmid = {38297069},
issn = {2045-2322},
support = {U01 DA053672/DA/NIDA NIH HHS/United States ; },
mesh = {Humans ; Rats ; Female ; Animals ; *Nicotine ; *Nucleus Accumbens/metabolism ; CRISPR-Cas Systems ; Motivation ; Cyclic Nucleotide Phosphodiesterases, Type 4/genetics/metabolism ; Genome-Wide Association Study ; Rats, Long-Evans ; RNA, Guide, CRISPR-Cas Systems ; Self Administration/methods ; },
abstract = {Large scale human genome wide association studies (GWAS) have identified a growing pool of genes associated with cigarette smoking. One of the most prominent, phosphodiesterase-4B (PDE4B), has been associated with multiple smoking phenotypes. Although PDE4B modulates the half-life of neuronal cAMP, its precise role in smoking behaviors is unknown. To address this knowledge gap, we used a reverse translational approach. We inactivated PDE4B in bilateral medial nucleus accumbens shell (NAcs) neurons by injecting AAV containing a specific gRNA in female transgenic Cas9+ Long Evans rats. These rats then were given 23-h chronic access to nicotine intravenous self-administration (IVSA) under a schedule of increasing fixed ratios (FR). With the increased effort required at FR7, nicotine SA (i.e. active presses and drug infusions) declined significantly in controls, whereas it was maintained in the mutagenized group. A progressive ratio (PR) study also showed significantly greater cumulative nicotine infusions in the PDE4B-edited group. Hence, we hypothesized that enhanced PDE4B protein activity would reduce nicotine IVSA. A positive allosteric modulator, 2-(3-(4-chloro-3-fluorophenyl)-5-ethyl-1H-1,2,4-triazol-1-yl)-N-(3,5-dichlorobenzyl)acetamide (MR-L2), was microinfused into NAcs bilaterally at FR3 or FR5; in both cohorts, MR-L2 acutely reduced nicotine IVSA. In summary, these studies show that the activity of PDE4B regulates the capacity of NAcs to maintain nicotine IVSA in face of the cost of increasing work. This finding and the results of the PR study indicate that PDE4B affects the motivation to obtain nicotine. These reverse translational studies in rats provide insight into the motivational effects of NAcs PDE4B that advance our understanding of the smoking behaviors mapped in human GWAS.},
}
@article {pmid38296979,
year = {2024},
author = {Zhang, Z and Rong, X and Xie, T and Li, Z and Song, H and Zhen, S and Wang, H and Wu, J and Jaffrey, SR and Li, X},
title = {Fluorogenic CRISPR for genomic DNA imaging.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {934},
pmid = {38296979},
issn = {2041-1723},
support = {R35 NS111631/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; DNA/genetics ; Genome ; Genomics ; },
abstract = {Genomic DNA exhibits high heterogeneity in terms of its dynamic within the nucleus, its structure and functional roles. CRISPR-based imaging approaches can image genomic loci in living cells. However, conventional CRISPR-based tools involve expressing constitutively fluorescent proteins, resulting in high background and nonspecific nucleolar signal. Here, we construct fluorogenic CRISPR (fCRISPR) to overcome these issues. fCRISPR is designed with dCas9, an engineered sgRNA, and a fluorogenic protein. Fluorogenic proteins are degraded unless they are bound to specific RNA hairpins. These hairpins are inserted into sgRNA, resulting in dCas9: sgRNA: fluorogenic protein ternary complexes that enable fluorogenic DNA imaging. With fCRISPR, we image various genomic DNA in different human cells with high signal-to-noise ratio and sensitivity. Furthermore, fCRISPR tracks chromosomes dynamics and length. fCRISPR also allows DNA double-strand breaks (DSBs) and repair to be tracked in real time. Taken together, fCRISPR offers a high-contrast and sensitive platform for imaging genomic loci.},
}
@article {pmid38277772,
year = {2024},
author = {Liu, X and Yang, H and Liu, J and Liu, K and Jin, L and Zhang, Y and Khan, MR and Zhong, K and Cao, J and He, Q and Xia, X and Deng, R},
title = {In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria.},
journal = {Analytical chemistry},
volume = {96},
number = {5},
pages = {2032-2040},
doi = {10.1021/acs.analchem.3c04532},
pmid = {38277772},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; Alleles ; In Situ Hybridization, Fluorescence ; *Quinolones/pharmacology ; *Salmonella enterica/genetics ; Polymerase Chain Reaction ; },
abstract = {In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.},
}
@article {pmid38205958,
year = {2024},
author = {Örmälä-Tiznado, A-M and Allander, L and Maatallah, M and Kabir, MH and Brisse, S and Sandegren, L and Patpatia, S and Coorens, M and Giske, CG},
title = {Molecular characteristics, fitness, and virulence of high-risk and non-high-risk clones of carbapenemase-producing Klebsiella pneumoniae.},
journal = {Microbiology spectrum},
volume = {12},
number = {2},
pages = {e0403622},
pmid = {38205958},
issn = {2165-0497},
support = {2016-02889//Vetenskapsr&#xe5;det (VR)/ ; },
abstract = {Extensively drug-resistant (XDR) Klebsiella pneumoniae inflict a notable burden on healthcare worldwide. Of specific concern are strains producing carbapenem-hydrolyzing enzymes, as the therapeutic options for these strains are still very limited. Specific sequence types of K. pneumoniae have been noted for their epidemic occurrence globally, but the mechanisms behind the success of specific clones remain unclear. Herein, we have characterized 20 high-risk clones (HiRCs) and 10 non-HiRCs of XDR K. pneumoniae, exploring factors connected to the epidemiological success of some clones. Isolates were subjected to core genome multilocus sequence typing analysis to determine the clonal relationships of the isolates and subsequently characterized with regard to features known to be linked to overall bacterial fitness and virulence. The genomes were analyzed in silico for capsule types, O antigens, virulence factors, antimicrobial resistance genes, prophages, and CRISPR-Cas loci. In vitro growth experiments were conducted to retrieve proxies for absolute and relative fitness for 11 HiRC and 9 non-HiRC isolates selected based on the clonal groups they belonged to, and infections in a Galleria mellonella insect model were used to evaluate the virulence of the isolates in vivo. This study did not find evidence that virulence factors, prophages, CRISPR-Cas loci, or fitness measured in vitro alone would contribute to the global epidemiological success of specific clones of carbapenemase-producing XDR K. pneumoniae. However, this study did find the HiRC group to be more virulent than the non-HiRC group when measured in vivo in a model with G. mellonella. This suggests that the virulence and epidemiological success of certain clones of K. pneumoniae cannot be explained by individual traits investigated in this study and thus warrant further experiments in the future.IMPORTANCEHerein, we explored potential explanations for the successfulness of some epidemic or high-risk clones of carbapenemase-producing Klebsiella pneumoniae. We found differences in mortality in a larva model but found no clear genomic differences in known virulence markers. Most of the research on virulence in K. pneumoniae has been focused on hypervirulent strains, but here, we try to understand differences within the group of highly resistant strains. The results from the larva virulence model could be used to design experiments in higher animals. Moreover, the data could provide further support to a differentiated infection control approach against extensively drug-resistant strains, based on their classification as high-risk clones.},
}
@article {pmid37930476,
year = {2024},
author = {Liang, SQ and Xue, W},
title = {All types of base conversions allowed by base editors.},
journal = {Science China. Life sciences},
volume = {67},
number = {2},
pages = {431-433},
pmid = {37930476},
issn = {1869-1889},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid37606848,
year = {2024},
author = {Chen, Z and Li, X and Zhang, Q and Sun, W and Song, X and Zhang, X and Huang, X and Sun, B},
title = {Enlarged DNA unwinding by Nme2Cas9 permits a broadened base editing window beyond the protospacer.},
journal = {Science China. Life sciences},
volume = {67},
number = {2},
pages = {424-427},
pmid = {37606848},
issn = {1869-1889},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; DNA/genetics ; },
}
@article {pmid36515893,
year = {2024},
author = {Wu, H and Wang, Y and Wang, H},
title = {Workflow for Performing Genetic Manipulation in Human Trophoblast Stem Cells Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2767},
number = {},
pages = {53-62},
pmid = {36515893},
issn = {1940-6029},
mesh = {Female ; Pregnancy ; Humans ; *CRISPR-Cas Systems/genetics ; Workflow ; *Placenta ; Stem Cells ; Trophoblasts ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based tool kits greatly facilitate the investigation of gene function, especially gene editing and transcriptional regulation technology. Human trophoblast stem cell (hTSC) is a promising cell model for simulating the development and function of human placental trophoblast cells. Here, we provide an optimized workflow for efficient genome editing and transcriptional activation in hTSCs using a lentivirus-dependent method. We also present methods to evaluate the gene targeted mutations and transcriptional activation.},
}
@article {pmid38296939,
year = {2024},
author = {Zhao, M and Cheng, X and Shao, P and Dong, Y and Wu, Y and Xiao, L and Cui, Z and Sun, X and Gao, C and Chen, J and Huang, Z and Zhang, J},
title = {Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {950},
pmid = {38296939},
issn = {2041-1723},
support = {31771550//National Natural Science Foundation of China (National Science Foundation of China)/ ; 81972267//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31870821//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230058//National Natural Science Foundation of China (National Science Foundation of China)/ ; 81973273//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Female ; Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Protoplasts ; RNA, Guide, CRISPR-Cas Systems ; Tumor-Associated Macrophages ; Escherichia coli/genetics ; *Neoplasms/genetics/therapy ; Immunotherapy ; Phospholipids ; Tumor Microenvironment ; },
abstract = {The CRISPR-Cas9 system offers substantial potential for cancer therapy by enabling precise manipulation of key genes involved in tumorigenesis and immune response. Despite its promise, the system faces critical challenges, including the preservation of cell viability post-editing and ensuring safe in vivo delivery. To address these issues, this study develops an in vivo CRISPR-Cas9 system targeting tumor-associated macrophages (TAMs). We employ bacterial protoplast-derived nanovesicles (NVs) modified with pH-responsive PEG-conjugated phospholipid derivatives and galactosamine-conjugated phospholipid derivatives tailored for TAM targeting. Utilizing plasmid-transformed E. coli protoplasts as production platforms, we successfully load NVs with two key components: a Cas9-sgRNA ribonucleoprotein targeting Pik3cg, a pivotal molecular switch of macrophage polarization, and bacterial CpG-rich DNA fragments, acting as potent TLR9 ligands. This NV-based, self-assembly approach shows promise for scalable clinical production. Our strategy remodels the tumor microenvironment by stabilizing an M1-like phenotype in TAMs, thus inhibiting tumor growth in female mice. This in vivo CRISPR-Cas9 technology opens avenues for cancer immunotherapy, overcoming challenges related to cell viability and safe, precise in vivo delivery.},
}
@article {pmid38296696,
year = {2024},
author = {Lin, LC and Kao, CY and Chang, SC and Hidrosollo, JH and Lu, JJ},
title = {Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types.},
journal = {Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmii.2024.01.005},
pmid = {38296696},
issn = {1995-9133},
abstract = {BACKGROUND AND PURPOSE: Our previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates.<br><br>METHODS: A comparative analysis of 36&#xa0;S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152&#xa0;S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations.<br><br>RESULTS: Forty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36&#xa0;S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates.<br><br>CONCLUSION: Our findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.},
}
@article {pmid38294975,
year = {2024},
author = {Longhurst, HJ and Lindsay, K and Petersen, RS and Fijen, LM and Gurugama, P and Maag, D and Butler, JS and Shah, MY and Golden, A and Xu, Y and Boiselle, C and Vogel, JD and Abdelhady, AM and Maitland, ML and McKee, MD and Seitzer, J and Han, BW and Soukamneuth, S and Leonard, J and Sepp-Lorenzino, L and Clark, ED and Lebwohl, D and Cohn, DM},
title = {CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema.},
journal = {The New England journal of medicine},
volume = {390},
number = {5},
pages = {432-441},
doi = {10.1056/NEJMoa2309149},
pmid = {38294975},
issn = {1533-4406},
mesh = {Adult ; Humans ; Angioedema ; *Angioedemas, Hereditary/blood/drug therapy/genetics ; Complement C1 Inhibitor Protein/therapeutic use ; *CRISPR-Cas Systems ; Dose-Response Relationship, Drug ; *Gene Editing/methods ; Plasma Kallikrein/genetics ; Treatment Outcome ; },
abstract = {BACKGROUND: Hereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose.<br><br>METHODS: In this phase 1 dose-escalation portion of a combined phase 1-2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks.<br><br>RESULTS: Three patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of -67% in the 25-mg group, -84% in the 50-mg group, and -95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was -91% in the 25-mg group, -97% in the 50-mg group, and -80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was -95%.<br><br>CONCLUSIONS: In this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.).},
}
@article {pmid38294941,
year = {2024},
author = {Prezza, G and Liao, C and Reichardt, S and Beisel, CL and Westermann, AJ},
title = {CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {6},
pages = {e2311323121},
doi = {10.1073/pnas.2311323121},
pmid = {38294941},
issn = {1091-6490},
support = {Starting Grant 101040214//EC | European Research Council (ERC)/ ; Consolidator Grant 865973//EC | European Research Council (ERC)/ ; },
mesh = {Humans ; *Bacteroides thetaiotaomicron/genetics ; RNA, Guide, CRISPR-Cas Systems ; Bile ; RNA, Bacterial/genetics ; *RNA, Small Untranslated/genetics ; },
abstract = {Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.},
}
@article {pmid38293790,
year = {2024},
author = {Bartosik, M and Moranova, L and Izadi, N and Strmiskova, J and Sebuyoya, R and Holcakova, J and Hrstka, R},
title = {Advanced technologies towards improved HPV diagnostics.},
journal = {Journal of medical virology},
volume = {96},
number = {2},
pages = {e29409},
doi = {10.1002/jmv.29409},
pmid = {38293790},
issn = {1096-9071},
support = {//Ministerstvo Zdravotnictv&#xed; Cesk&#xe9; Republiky/ ; //Ministerstvo &#x160;kolstv&#xed;, Ml&#xe1;de&#x17e;e a Telov&#xfd;chovy/ ; //Agentura Pro Zdravotnick&#xfd; V&#xfd;zkum Cesk&#xe9; Republiky/ ; },
mesh = {Female ; Humans ; Human Papillomavirus Viruses ; *Papillomavirus Infections/complications ; Papillomaviridae/genetics ; *Uterine Cervical Neoplasms ; Technology ; },
abstract = {Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.},
}
@article {pmid38293027,
year = {2024},
author = {Kwon, T and Artiaga, BL and McDowell, CD and Whitworth, KM and Wells, KD and Prather, RS and Delhon, G and Cigan, M and White, SN and Retallick, J and Gaudreault, NN and Morozov, I and Richt, JA},
title = {Gene editing of pigs to control influenza A virus infections.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.01.15.575771},
pmid = {38293027},
abstract = {Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro . IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to WT pigs. Our findings could support the commercial use of GE pigs to minimize (i) the economic losses caused by IAV infection in pigs, and (ii) the emergence of novel IAVs with pandemic potential through genetic reassortment in the "mixing vessel", the pig.},
}
@article {pmid38291236,
year = {2024},
author = {Mayo-Muñoz, D and Pinilla-Redondo, R and Camara-Wilpert, S and Birkholz, N and Fineran, PC},
title = {Inhibitors of bacterial immune systems: discovery, mechanisms and applications.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {38291236},
issn = {1471-0064},
abstract = {To contend with the diversity and ubiquity of bacteriophages and other mobile genetic elements, bacteria have developed an arsenal of immune defence mechanisms. Bacterial defences include CRISPR-Cas, restriction-modification and a growing list of mechanistically diverse systems, which constitute the bacterial 'immune system'. As a response, bacteriophages and mobile genetic elements have evolved direct and indirect mechanisms to circumvent or block bacterial defence pathways and ensure successful infection. Recent advances in methodological and computational approaches, as well as the increasing availability of genome sequences, have boosted the discovery of direct inhibitors of bacterial defence systems. In this Review, we discuss methods for the discovery of direct inhibitors, their diverse mechanisms of action and perspectives on their emerging applications in biotechnology and beyond.},
}
@article {pmid38291210,
year = {2024},
author = {Bahl, E and Jyoti, A and Singh, A and Siddqui, A and Upadhyay, SK and Jain, D and Shah, MP and Saxena, J},
title = {Nanomaterials for intelligent CRISPR-Cas tools: improving environment sustainability.},
journal = {Environmental science and pollution research international},
volume = {},
number = {},
pages = {},
pmid = {38291210},
issn = {1614-7499},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a desirable gene modification tool covering a wide area in various sectors of medicine, agriculture, and microbial biotechnology. The role of this incredible genetic engineering technology has been extensively investigated; however, it remains formidable with cargo choices, nonspecific delivery, and insertional mutagenesis. Various nanomaterials including lipid, polymeric, and inorganic are being used to deliver the CRISPR-Cas system. Progress in nanomaterials could potentially address these challenges by accelerating precision targeting, cost-effectiveness, and one-step delivery. In this review, we highlighted the advances in nanotechnology and nanomaterials as smart delivery systems for CRISPR-Cas so as to ameliorate applications for environmental remediation including biomedical research and healthcare, strategies for mitigating antimicrobial resistance, and to be used as nanofertilizers for enhancing crop growth, and reducing the environmental impact of traditional fertilizers. The timely co-evolution of nanotechnology and CRISPR technologies has contributed to smart novel nanostructure hybrids for improving the onerous tasks of environmental remediation and biological sustainability.},
}
@article {pmid38291084,
year = {2024},
author = {Lin, K and Chang, YC and Billmann, M and Ward, HN and Le, K and Hassan, AZ and Bhojoo, U and Chan, K and Costanzo, M and Moffat, J and Boone, C and Bielinsky, AK and Myers, CL},
title = {A scalable platform for efficient CRISPR-Cas9 chemical-genetic screens of DNA damage-inducing compounds.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {2508},
pmid = {38291084},
issn = {2045-2322},
support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; UL1 TR002494/TR/NCATS NIH HHS/United States ; F30 CA257227/CA/NCI NIH HHS/United States ; 5F30CA257227-02/CA/NCI NIH HHS/United States ; R35 GM141805/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Genome ; Genetic Testing ; DNA Damage ; },
abstract = {Current approaches to define chemical-genetic interactions (CGIs) in human cell lines are resource-intensive. We designed a scalable chemical-genetic screening platform by generating a DNA damage response (DDR)-focused custom sgRNA library targeting 1011 genes with 3033 sgRNAs. We performed five proof-of-principle compound screens and found that the compounds' known modes-of-action (MoA) were enriched among the compounds' CGIs. These scalable screens recapitulated expected CGIs at a comparable signal-to-noise ratio (SNR) relative to genome-wide screens. Furthermore, time-resolved CGIs, captured by sequencing screens at various time points, suggested an unexpected, late interstrand-crosslinking (ICL) repair pathway response to camptothecin-induced DNA damage. Our approach can facilitate screening compounds at scale with 20-fold fewer resources than commonly used genome-wide libraries and produce biologically informative CGI profiles.},
}
@article {pmid38176521,
year = {2024},
author = {Yang, H and Liu, A and Ma, F and Gao, X and Wang, K and Wang, Y},
title = {Establishment of portable Pseudomonas aeruginosa detection platform based on one-tube CRISPR/Cas12a combined with recombinase polymerase amplification technology.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {554},
number = {},
pages = {117760},
doi = {10.1016/j.cca.2024.117760},
pmid = {38176521},
issn = {1873-3492},
mesh = {Humans ; *Recombinases ; *Pseudomonas aeruginosa/genetics ; CRISPR-Cas Systems/genetics ; Nucleotidyltransferases ; Technology ; Aerosols ; Nucleic Acid Amplification Techniques ; },
abstract = {Pseudomonas aeruginosa, a common Gram-negative bacterium, is associated with diverse diseases. Its increasing resistance to antibiotics presents challenges in clinical treatment. The predominant diagnostic approach involves conventional biochemical cultures, known for their time and labor intensiveness. Despite progress in isothermal amplification studies, limitations persist, including reliance on specialized equipment, intricate primer design, and aerosol contamination. Therefore, there is a demand for enhanced clinical assays. This study successfully combined RPA and CRISPR/Cas12a techniques. Through a series of experiments involving the design and screening of lasB crRNA, the creation of lasB RPA primers, and the establishment of a streamlined RPA-CRISPR/Cas12a assay, the study developed a one-tube detection method targeting P. aeruginosa's lasB gene. The assay demonstrated inclusive behavior across standard and 21 isolates, while specifically discerning P. aeruginosa from diverse strains. Sensitivity reached 15.9 CFU/reaction. Clinical validation revealed a 97.62% concordance with traditional methods. The one-tube assay's protocol mitigated aerosol contamination. Offering precision, specificity, and sensitivity, this method shows promise for field applications in resource-scarce regions, enabling early detection and improved management of P. aeruginosa infections.},
}
@article {pmid38154756,
year = {2024},
author = {Chang, PK},
title = {Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {170},
number = {},
pages = {103863},
doi = {10.1016/j.fgb.2023.103863},
pmid = {38154756},
issn = {1096-0937},
mesh = {Humans ; *Aflatoxins/genetics ; Aspergillus flavus/genetics/metabolism ; CRISPR-Cas Systems ; Commerce ; Internationality ; Multigene Family ; DNA/metabolism ; *Indoles ; *Peptides, Cyclic ; },
abstract = {Aspergillus flavus produces hepatocarcinogenic aflatoxin that adversely impacts human and animal health and international trade. A promising means to manage preharvest aflatoxin contamination of crops is biological control, which employs non-aflatoxigenic A. flavus isolates possessing defective aflatoxin gene clusters to outcompete field toxigenic populations. However, these isolates often produce other toxic metabolites. The CRISPR/Cas9 technology has greatly advanced genome editing and gene functional studies. Its use in deleting large chromosomal segments of filamentous fungi is rarely reported. A system of dual CRISPR/Cas9 combined with a 60-nucleotide donor DNA that allowed removal of A. flavus gene clusters involved in production of harmful specialized metabolites was established. It efficiently deleted a 102-kb segment containing both aflatoxin and cyclopiazonic acid gene clusters from toxigenic A. flavus morphotypes, L-type and S-type. It further deleted the 27-kb ustiloxin B gene cluster of a resulting L-type mutant. Overall efficiencies of deletion ranged from 66.6 % to 85.6 % and efficiencies of deletions repaired by a single copy of donor DNA ranged from 50.5 % to 72.7 %. To determine the capacity of this technique, a pigment-screening setup based on absence of aspergillic acid gene cluster was devised. Chromosomal segments of 201 kb and 301 kb were deleted with efficiencies of 57.7 % to 69.2 %, respectively. This system used natural A. flavus isolates as recipients, eliminated a forced-recycling step to produce recipients for next round deletion, and generated maker-free deletants with sequences predefined by donor DNA. The research provides a method for creating genuine atoxigenic biocontrol strains friendly for field trial release.},
}
@article {pmid38062876,
year = {2024},
author = {Liang, N and He, J and Yan, J and Han, X and Zhang, X and Niu, Y and Sha, W and Li, J},
title = {DBC1 maintains skeletal muscle integrity by enhancing myogenesis and preventing myofibre wasting.},
journal = {Journal of cachexia, sarcopenia and muscle},
volume = {15},
number = {1},
pages = {255-269},
pmid = {38062876},
issn = {2190-6009},
support = {2021-I2M-1-050//CAMS Innovation Fund for Medical Sciences/ ; 2022-I2M-1-012//CAMS Innovation Fund for Medical Sciences/ ; 2022YFA1103803//National Key R&amp;D Program of China/ ; 2022-JKCS-14//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; 2060204//State Key Laboratory Special Fund/ ; },
mesh = {Male ; Mice ; Animals ; Mice, Inbred C57BL ; *RNA, Guide, CRISPR-Cas Systems ; *Muscle, Skeletal/pathology ; Muscular Atrophy/pathology ; Cachexia/pathology ; Muscle Development ; },
abstract = {BACKGROUND: Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying mechanisms remain poorly understood, and specific approved medications are currently unavailable. Deleted in breast cancer 1 (DBC1) is a well-known regulator of senescence, metabolism or apoptosis. Recent reports suggest that DBC1 may also potentially regulate muscle function, as mice lacking DBC1 exhibit weakness and limpness. However, the function of DBC1 in skeletal muscle and its associated molecular mechanisms remain unknown, thus prompting the focus of this study.<br><br>METHODS: Tibialis anterior (TA) muscle-specific DBC1 knockdown C57BL/6J male mice were generated through a single injection of 2.00 E&#xa0;+&#xa0;11&#xa0;vg of adeno-associated virus 9 delivering single-guide RNA for DBC1. Grip strength and endurance were assessed 2&#xa0;months later, followed by skeletal muscle harvest. Muscle atrophy model was generated by cast immobilization of the mouse hindlimb for 2&#xa0;weeks. Molecular markers of atrophy were probed in muscles upon termination. Cardiotoxin (CTX) was injected in TA muscles of DBC1 knockdown mice, and muscle regeneration was assessed by immunohistochemistry, quantitative PCR and western blotting. DBC1 knockdown C2C12 cells and myotubes were investigated using immunofluorescence staining, Seahorse, immunohistology, fluorescence-activated cell sorting and RNA-sequencing analyses.<br><br>RESULTS: DBC1 knockdown in skeletal muscle of young mice led to signatures of muscle atrophy, including a 28% reduction in muscle grip force (P&#xa0;=&#xa0;0.023), a 54.4% reduction in running distance (P&#xa0;=&#xa0;0.002), a 14.3% reduction in muscle mass (P&#xa0;=&#xa0;0.007) and significantly smaller myofibre cross-sectional areas (P&#xa0;<&#xa0;0.0001). DBC1 levels decrease in age-related or limb immobilization-induced atrophic mouse muscles and overexpress DBC1-attenuated atrophic phenotypes in these mice. Muscle regeneration was hampered in mice with CTX-induced muscle injury by DBC1 knockdown, as evidenced by reductions in myofibre cross-sectional areas of regenerating myofibres with centralized nuclei (P&#xa0;<&#xa0;0.0001), percentages of MyoG[+] nuclei (P&#xa0;<&#xa0;0.0001) and fusion index (P&#xa0;<&#xa0;0.0001). DBC1 transcriptionally regulated mouse double minute 2 (MDM2), which mediated ubiquitination and degradation of forkhead box O3 (FOXO3). Increased FOXO3 proteins hampered myogenesis in DBC1 knockdown satellite cells by compromising around 50% of mitochondrial functions (P&#xa0;<&#xa0;0.001) and exacerbated atrophy in DBC1 knockdown myofibres by activating the ubiquitin-proteasome and autophagy-lysosome pathways.<br><br>CONCLUSIONS: DBC1 is essential in maintaining skeletal muscle integrity by protecting against myofibres wasting and enhancing muscle regeneration via FOXO3. This research highlights the significance of DBC1 for healthy skeletal muscle function and its connection to muscular atrophy.},
}
@article {pmid38029964,
year = {2024},
author = {Nakanishi, K},
title = {When Argonaute takes out the ribonuclease sword.},
journal = {The Journal of biological chemistry},
volume = {300},
number = {1},
pages = {105499},
pmid = {38029964},
issn = {1083-351X},
support = {R01 GM124320/GM/NIGMS NIH HHS/United States ; R01 GM138997/GM/NIGMS NIH HHS/United States ; R21 AI178624/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; RNA, Small Interfering/metabolism ; *Ribonucleases/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Argonaute Proteins/genetics/metabolism ; RNA-Induced Silencing Complex/metabolism ; *MicroRNAs/genetics ; },
abstract = {Argonaute (AGO) proteins in all three domains of life form ribonucleoprotein or deoxyribonucleoprotein complexes by loading a guide RNA or DNA, respectively. Since all AGOs retain a PIWI domain that takes an RNase H fold, the ancestor was likely an endoribonuclease (i.e., a slicer). In animals, most miRNA-mediated gene silencing occurs slicer independently. However, the slicer activity of AGO is indispensable in specific events, such as development and differentiation, which are critical for vertebrates and thus cannot be replaced by the slicer-independent regulation. This review highlights the distinctions in catalytic activation mechanisms among slicing-competent AGOs, shedding light on the roles of two metal ions in target recognition and cleavage. The precision of the target specificity by the RNA-induced silencing complexes is reevaluated and redefined. The possible coevolutionary relationship between slicer-independent gene regulation and AGO-binding protein, GW182, is also explored. These discussions reveal that numerous captivating questions remain unanswered regarding the timing and manner in which AGOs employ their slicing activity.},
}
@article {pmid37932446,
year = {2024},
author = {Yang, H and Patel, DJ},
title = {Fanzors: Striking expansion of RNA-guided endonucleases to eukaryotes.},
journal = {Cell research},
volume = {34},
number = {2},
pages = {99-100},
pmid = {37932446},
issn = {1748-7838},
mesh = {*RNA ; *Eukaryota/genetics/metabolism ; Endonucleases/genetics ; CRISPR-Cas Systems ; },
}
@article {pmid38287906,
year = {2024},
author = {Han, Y and Zhou, J and Zhang, R and Liang, Y and Lai, L and Li, Z},
title = {Genome-edited rabbits: Unleashing the potential of a promising experimental animal model across diverse diseases.},
journal = {Zoological research},
volume = {45},
number = {2},
pages = {253-262},
doi = {10.24272/j.issn.2095-8137.2023.201},
pmid = {38287906},
issn = {2095-8137},
mesh = {Humans ; Rabbits ; Animals ; Mice ; CRISPR-Cas Systems ; *Veterinary Drugs ; Gene Editing/veterinary ; Models, Animal ; *Biomedical Research ; },
abstract = {Animal models are extensively used in all aspects of biomedical research, with substantial contributions to our understanding of diseases, the development of pharmaceuticals, and the exploration of gene functions. The field of genome modification in rabbits has progressed slowly. However, recent advancements, particularly in CRISPR/Cas9-related technologies, have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases, including cardiovascular disorders, immunodeficiencies, aging-related ailments, neurological diseases, and ophthalmic pathologies. These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice. This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine, underscoring their impact and future potential in translational medicine.},
}
@article {pmid38287808,
year = {2024},
author = {Chanda, S and Lepikhov, K and Dahlem, C and Schymik, HS and Hoppstädter, J and Geber, AK and Wagner, K and Kessler, SM and Empting, M and Kiemer, AK},
title = {Gene Editing and Small Molecule Inhibitors of the RNA Binding Protein IGF2BP2/IMP2 Show its Potential as an Anti-Cancer Drug Target.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {29},
number = {1},
pages = {41},
doi = {10.31083/j.fbl2901041},
pmid = {38287808},
issn = {2768-6698},
support = {DFG, KI702//German Research Foundation/ ; },
mesh = {Humans ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Liver Neoplasms ; *Antineoplastic Agents ; RNA-Binding Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: The RNA-binding protein IGF2BP2/IMP2/VICKZ2/p62 is an oncofetal protein that is overexpressed in several cancer entities. Employing IMP2 knockout colorectal cancer cells, we could show the important role of IMP2 in several hallmarks of cancer. This study aimed to functionally characterize IMP2 in lung (A549, LLC1) and hepatocellular carcinoma (HepG2, Huh7) cell lines to assess its role as a potential target for these cancer entities.<br><br>METHODS: IMP2 knockouts were generated by CRISPR/Cas9 and its variant approach prime editing; the editing efficiency of two single guide RNAs (sgRNAs) was verified via next-generation sequencing. We studied the effect of IMP2 knockout on cell proliferation, colony formation, and migration and employed small-molecule inhibitors of IMP2.<br><br>RESULTS: Despite multiple attempts, it was not possible to generate IMP2 biallelic knockouts in A549 and Huh7 cells. Both sgRNAs showed good editing efficiency. However, edited cells lost their ability to proliferate. The attempt to generate an IMP2 biallelic knockout in LLC1 cells using CRISPR/Cas9 was successful. Monoallelic knockout cell lines of IMP2 showed a reduction in 2D cell proliferation and reduced migration. In 3D cultures, a change in morphology from compact spheroids to loose aggregates and a distinct reduction in the colony formation ability of the IMP2 knockouts was observed, an effect that was mimicked by previously identified IMP2 inhibitor compounds that also showed an inhibitory effect on colony formation.<br><br>CONCLUSIONS: Our in vitro target validation supports that IMP2 is essential for tumor cell proliferation, migration, and colony formation in several cancer entities.},
}
@article {pmid38287498,
year = {2024},
author = {Fukuoka, M and Kodama, T and Murai, K and Hikita, H and Sometani, E and Sung, J and Shimoda, A and Shigeno, S and Motooka, D and Nishio, A and Furuta, K and Tatsumi, T and Yusa, K and Takehara, T},
title = {Genome-wide loss-of-function genetic screen identifies INSIG2 as the vulnerability of hepatitis B virus-integrated hepatoma cells.},
journal = {Cancer science},
volume = {},
number = {},
pages = {},
doi = {10.1111/cas.16070},
pmid = {38287498},
issn = {1349-7006},
support = {JP23ama221410//Japan Agency for Medical Research and Development/ ; JP23fk0210110//Japan Agency for Medical Research and Development/ ; JP23fk0210131//Japan Agency for Medical Research and Development/ ; JP23fk0310501//Japan Agency for Medical Research and Development/ ; JP23fk0310512//Japan Agency for Medical Research and Development/ ; JP23fk0310524//Japan Agency for Medical Research and Development/ ; },
abstract = {There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G1 -to-S transition in HepG2.2.15 cells. CDK2 inhibitor suppressed cell cycle progression in HepG2.2.15 cells and INSIG2 inhibition did not suppress cell proliferation in the presence of CDK2 inhibitor. In conclusion, INSIG2 inhibition induced cell cycle arrest in HBV-integrated hepatoma cells in a CDK2-dependent manner, and thus INSIG2 might be a vulnerability factor for HBV-associated liver cancer.},
}
@article {pmid38284970,
year = {2024},
author = {Sultana, S and Azlan, A and Mohd Desa, MN and Mahyudin, NA and Anburaj, A},
title = {A review of CRISPR-Cas and PCR-based methods for the detection of animal species in the food chain-current challenges and future prospects.},
journal = {Food additives &amp; contaminants. Part A, Chemistry, analysis, control, exposure &amp; risk assessment},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/19440049.2024.2304577},
pmid = {38284970},
issn = {1944-0057},
abstract = {Regular testing and systematic investigation play a vital role to ensure product safety. Until now, the existing food authentication techniques have been based on proteins, lipids, and nucleic acid-based assays. Among various deoxyribonucleic acid (DNA)-based methods, the recently developed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based bio-sensing is an innovative and fast-expanding technology. The CRISPR/Cas-9 is known as Clustered Regularly Interspaced Short Palindromic Repeats due to the flexibility and simplicity of the CRISPR/Cas9 site-specific editing tool has been applied in many biological research areas such as Gene therapy, cell line development, discovering mechanisms of disease, and drug discovery. Nowadays, the CRISPR-Cas system has also been introduced into food authentication via detecting DNA barcodes of poultry and livestock both in processed and unprocessed food samples. This review documents various DNA based approaches, in an accessible format. Future CRISPR technologies are forecast while challenges are outlined.},
}
@article {pmid38284691,
year = {2024},
author = {Bakhtiyari, N and Farajnia, S and Ghasemali, S and Farajnia, S and Pourmohammad, A and Saeidvafa, S},
title = {Strategies to Overcome Antimicrobial Resistance in Nosocomial Infections, A Review and Update.},
journal = {Infectious disorders drug targets},
volume = {},
number = {},
pages = {},
doi = {10.2174/0118715265276529231214105423},
pmid = {38284691},
issn = {2212-3989},
abstract = {Nosocomial infections, also known as healthcare-associated infections, are a signif-icant global concern due to their strong association with high mortality and morbidity in both developed and developing countries. These infections are caused by a variety of pathogens, particularly the ESKAPE group of bacteria, which includes the six pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudo-monas aeruginosa, and Enterobacter spp. These bacteria have demonstrated noteworthy re-sistance to different antibiotics. Antimicrobial resistance mechanisms can manifest in various forms, including restricting drug uptake, modifying drug targets, inactivating drugs, active drug efflux, and biofilm formation. Accordingly, various strategies have been developed to combat antibiotic-resistant bacteria. These strategies encompass the development of new antibiotics, the utilization of bacterio-phages that specifically target these bacteria, antimicrobial combination therapy and the use of peptides or enzymes that target the genomes or essential proteins of resistant bacteria. Among promising approaches to overcome antibiotic resistance, the CRISPR/Cas system stands out and offers many advantages. This system enables precise and efficient editing of genetic material at specific locations in the genome. Functioning as a bacterial "adaptive im-mune system," the CRISPR/Cas system recognizes, degrades, and remembers foreign DNA sequences through the use of spacer DNA segments that are transcribed into CRISPR RNAs (crRNA). This paper has focused on nosocomial infections, specifically the pathogens involved in hospi-tal infections, the mechanisms underlying bacterial resistance, and the strategies currently em-ployed to address this issue. Special emphasis has been placed on the application of CRISPR/Cas technology for overcoming antimicrobial resistance.},
}
@article {pmid38283851,
year = {2024},
author = {Shum, C and Han, SY and Thiruvahindrapuram, B and Wang, Z and de Rijke, J and Zhang, B and Sundberg, M and Chen, C and Buttermore, ED and Makhortova, N and Howe, J and Sahin, M and Scherer, SW},
title = {Combining Off-flow, a Nextflow-coded program, and whole genome sequencing reveals unintended genetic variation in CRISPR/Cas-edited iPSCs.},
journal = {Computational and structural biotechnology journal},
volume = {23},
number = {},
pages = {638-647},
pmid = {38283851},
issn = {2001-0370},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.},
}
@article {pmid38281656,
year = {2024},
author = {Goetzl, EJ and Alpert, J},
title = {CRISPR-Cas Gene Editing to the Genetic Rescue: CRISPR-Cas Gene Editing.},
journal = {The American journal of medicine},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.amjmed.2024.01.006},
pmid = {38281656},
issn = {1555-7162},
}
@article {pmid38281430,
year = {2024},
author = {Zhao, C and Yang, Z and Hu, T and Liu, J and Zhao, Y and Leng, D and Yang, K and An, G},
title = {CRISPR-Cas12a based target recognition initiated duplex-specific nuclease enhanced fluorescence and colorimetric analysis of cell-free DNA (cfDNA).},
journal = {Talanta},
volume = {271},
number = {},
pages = {125717},
doi = {10.1016/j.talanta.2024.125717},
pmid = {38281430},
issn = {1873-3573},
abstract = {The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers.},
}
@article {pmid38281301,
year = {2024},
author = {Ikram, M and Rauf, A and Rao, MJ and Maqsood, MFK and Bakhsh, MZM and Ullah, M and Batool, M and Mehran, M and Tahira, M},
title = {CRISPR-Cas9 based molecular breeding in crop plants: a review.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {227},
pmid = {38281301},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *DNA Shuffling ; Genome-Wide Association Study ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; Plant Breeding/methods ; },
abstract = {Traditional crop breeding techniques are not quickly boosting yields to fulfill the expanding population needs. Long crop lifespans hinder the ability of plant breeding to develop superior crop varieties. Due to the arduous crossing, selecting, and challenging processes, it can take decades to establish new varieties with desired agronomic traits. Develop new plant varieties instantly to reduce hunger and improve food security. As a result of the adoption of conventional agricultural techniques, crop genetic diversity has decreased over time. Several traditional and molecular techniques, such as genetic selection, mutant breeding, somaclonal variation, genome-wide association studies, and others, have improved agronomic traits associated with agricultural plant productivity, quality, and resistance to biotic and abiotic stresses. In addition, modern genome editing approaches based on programmable nucleases, CRISPR, and Cas9 proteins have escorted an exciting new era of plant breeding. Plant breeders and scientists worldwide rely on cutting-edge techniques like quick breeding, genome editing tools, and high-throughput phenotyping to boost crop breeding output. This review compiles discoveries in numerous areas of crop breeding, such as using genome editing tools to accelerate the breeding process and create yearly crop generations with the desired features, to describe the shift from conventional to modern plant breeding techniques.},
}
@article {pmid38281263,
year = {2024},
author = {Szekely, O and Rangadurai, AK and Gu, S and Manghrani, A and Guseva, S and Al-Hashimi, HM},
title = {NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like G·T/U in RNA:DNA hybrids: implications for the fidelity of transcription and CRISPR/Cas9 gene editing.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae027},
pmid = {38281263},
issn = {1362-4962},
support = {R01GM089846/NH/NIH HHS/United States ; },
abstract = {Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G·T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R1ρ experiments, we show that dG·rU and dT·rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol·T/U $ \mathbin{\lower.3ex\hbox{$\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}
\vphantom{_{\vbox to.5ex{\vss}
}
}
}
$}
}
$ G·Tenol/Uenol) and anionic (G·T-/U-) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG·rU- formed with a ten-fold higher propensity relative to dT-·rG and dG·dT- and this could be attributed to the lower pKa (ΔpKa ∼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G·T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G·U versus G·T, and indicate that anionic Watson-Crick-like G·U- could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.},
}
@article {pmid38281060,
year = {2024},
author = {Denner, J},
title = {Porcine endogenous retroviruses in xenotransplantation.},
journal = {Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association},
volume = {},
number = {},
pages = {},
doi = {10.1093/ndt/gfae023},
pmid = {38281060},
issn = {1460-2385},
abstract = {Xenotransplantation using pig cells, tissues or organs is under development to alleviate the shortage of human donor organs. Meanwhile remarkably long survival times of pig organs in non-human primates were reported as well as the functionality of pig kidneys and hearts in brain-dead humans. Most importantly, two transplantations of pig hearts in patients were performed with survival times of the patients of 8 and 6 weeks. Xenotransplantation may be associated with the transmission of porcine microorganisms including viruses to the recipient. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated like other viruses can. PERVs are able to infect certain human cells and pose therefore a risk for xenotransplantation. It is well known that retroviruses are able to induce tumors and immunodeficiencies. However, until now, PERV was not transmitted in all infection experiments using small animals and non-human primates, in all preclinical xenotransplantation trials in non-human primates and in all clinical trials in humans. In addition, several strategies including antiretrovirals, PERV-specific siRNA, vaccines and genome editing using CRISPR/Cas have been developed to prevent PERV transmission.},
}
@article {pmid38280977,
year = {2024},
author = {Fan, P and Wang, H and Zhao, F and Zhang, T and Li, J and Sun, X and Yu, Y and Xiong, H and Lai, L and Sui, T},
title = {Targeted mutagenesis in mice via an engineered AsCas12f1 system.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {81},
number = {1},
pages = {63},
pmid = {38280977},
issn = {1420-9071},
support = {32101226//National Natural Science Foundation of China/ ; },
mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Streptococcus pyogenes ; Gene Editing ; Mutagenesis ; },
abstract = {SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.},
}
@article {pmid38260645,
year = {2024},
author = {Burman, N and Belukhina, S and Depardieu, F and Wilkinson, RA and Skutel, M and Santiago-Frangos, A and Graham, AB and Livenskyi, A and Chechenina, A and Morozova, N and Zahl, T and Henriques, WS and Buyukyoruk, M and Rouillon, C and Shyrokova, L and Kurata, T and Hauryliuk, V and Severinov, K and Groseille, J and Thierry, A and Koszul, R and Tesson, F and Bernheim, A and Bikard, D and Wiedenheft, B and Isaev, A},
title = {Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {38260645},
support = {R01 GM129325/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; F31 GM153146/GM/NIGMS NIH HHS/United States ; K99 GM147842/GM/NIGMS NIH HHS/United States ; P20 GM103474/GM/NIGMS NIH HHS/United States ; },
abstract = {Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNA[Lys]. Phage T5 subverts PARIS immunity through expression of a tRNA[Lys] variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.},
}
@article {pmid37697004,
year = {2024},
author = {Su, M and Li, F and Wang, Y and Gao, Y and Lan, W and Shao, Z and Zhu, C and Tang, N and Gan, J and Wu, Z and Ji, Q},
title = {Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity.},
journal = {Nature chemical biology},
volume = {20},
number = {2},
pages = {180-189},
pmid = {37697004},
issn = {1552-4469},
support = {22207074//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/chemistry ; Genome ; Endonucleases/genetics/metabolism ; Gene Editing ; },
abstract = {CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.},
}
@article {pmid37003532,
year = {2024},
author = {Ying, X and Huang, Y and Liu, B and Hu, W and Ji, D and Chen, C and Zhang, H and Liang, Y and Lv, Y and Ji, W},
title = {Targeted m[6]A demethylation of ITGA6 mRNA by a multisite dCasRx-m[6]A editor inhibits bladder cancer development.},
journal = {Journal of advanced research},
volume = {56},
number = {},
pages = {57-68},
doi = {10.1016/j.jare.2023.03.010},
pmid = {37003532},
issn = {2090-1224},
mesh = {Humans ; Mice ; Animals ; Integrin alpha6/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Mice, Nude ; Cell Line, Tumor ; *RNA, Guide, CRISPR-Cas Systems ; *Urinary Bladder Neoplasms/genetics/pathology ; Demethylation ; Methyltransferases/genetics/metabolism ; },
abstract = {INTRODUCTION: N6-methyladenosine (m[6]A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m[6]A modification. However, the therapeutic effect of targeting ITGA6 multisite m[6]A modifications in BCa remains unknown.<br><br>OBJECTIVES: We aim to develop a multisite dCasRx- m[6]A editor for assessing the effects of the multisite dCasRx-m[6]A editor targeted m[6]A demethylation of ITGA6 mRNA in BC growth and progression.<br><br>METHODS: The multisite dCasRx- m[6]A editor was generated by cloning. m[6]A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m[6]A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m[6]A editor in BC growth and progression.<br><br>RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m[6]A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or &#x3b1;-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m[6]A sites. The results confirmed the dCasRx-m[6]A editor modified m[6]A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m[6]A demethylation of ITGA6 mRNA by the multisite dCasRx-m[6]A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth.<br><br>CONCLUSION: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m[6]A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m[6]A modifications.},
}
@article {pmid38280857,
year = {2024},
author = {Li, Z and Guo, R and Sun, X and Li, G and Shao, Z and Huo, X and Yang, R and Liu, X and Cao, X and Zhang, H and Zhang, W and Zhang, X and Ma, S and Zhang, M and Liu, Y and Yao, Y and Shi, J and Yang, H and Hu, C and Zhou, Y and Xu, C},
title = {Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {831},
pmid = {38280857},
issn = {2041-1723},
mesh = {Mice ; Animals ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Tyrosinemias/genetics/therapy ; Mammals ; },
abstract = {Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.},
}
@article {pmid38280845,
year = {2024},
author = {Zhang, H and Ma, J and Wu, Z and Chen, X and Qian, Y and Chen, W and Wang, Z and Zhang, Y and Zhu, H and Huang, X and Ji, Q},
title = {BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {825},
pmid = {38280845},
issn = {2041-1723},
support = {22277078, 22077083//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Exodeoxyribonucleases/genetics ; Escherichia coli/genetics/metabolism ; DNA/genetics ; *Escherichia coli Proteins/genetics/metabolism ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB, a 3'→5' DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3'→5' DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering.},
}
@article {pmid38280789,
year = {2024},
author = {Wang, S and Liu, Y and Liu, R and Xie, L and Yang, H and Ge, S and Yu, J},
title = {Strand displacement amplification triggered 3D DNA roller assisted CRISPR/Cas12a electrochemiluminescence cascaded signal amplification for sensitive detection of Ec-16S rDNA.},
journal = {Analytica chimica acta},
volume = {1291},
number = {},
pages = {342213},
doi = {10.1016/j.aca.2024.342213},
pmid = {38280789},
issn = {1873-4324},
mesh = {DNA, Ribosomal ; *Manganese Compounds ; CRISPR-Cas Systems/genetics ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; Oxides ; *Biosensing Techniques/methods ; Escherichia coli ; },
abstract = {BACKGROUND: Escherichia coli can cause gastrointestinal infection, urinary tract infection and other infectious diseases. Accurate detection of Escherichia coli 16S rDNA (Ec-16S rDNA) in clinical practice is of great significance for the identification and treatment of related diseases. At present, there are various types of sensors that can achieve accurate detection of Ec-16S rDNA. Electrochemiluminescence (ECL) has attracted considerable attention from researchers, which causes excellent performance in bioanalysis. Based on the previous research, it is significance to develop a novel, sensitive and efficient ECL biosensor.<br><br>RESULTS: In this work, an ECL biosensor for the detection of Ec-16S rDNA was constructed by integrating CRISPR/Cas12a technology with the cascade signal amplification strategy consisting of strand displacement amplification (SDA) and dual-particle three-dimensional (3D) DNA rollers. The amplification products of SDA triggered the operation of the DNA rollers, and the products generated by the DNA rollers activated CRISPR/Cas12a to cleave the signal probe, thereby realizing the change of the ECL signal. The cascade amplification strategy realized the exponential amplification of the target signal and greatly improved the sensitivity. Manganese dioxide nanoflowers (MnO2 NFs) as a co-reaction promoter effectively enhanced the ECL intensity of tin disulfide quantum dots (SnS2 QDs). A new ternary ECL system (SnS2 QDs/S2O8[2-]/MnO2 NFs) was prepared, which made the change of ECL intensity of biosensor more significant. The proposed biosensor had a response range of 100 aM-10&#xa0;nM and a detection limit of 27.29 aM (S/N&#xa0;=&#xa0;3).<br><br>SIGNIFICANCE AND NOVELTY: Herein, the cascade signal amplification strategy formed by SDA and dual-particle 3D DNA rollers enabled the ECL biosensor to have high sensitivity and low detection limit. At the same time, the cascade signal amplification strategy was integrated with CRISPR/Cas12a to enable the biosensor to efficiently detect the target. It can provide a new idea for the detection of Ec-16S rDNA in disease diagnosis and clinical analysis.},
}
@article {pmid38280781,
year = {2024},
author = {Ding, S and Yuan, Y and Dong, J and Du, F and Cui, X and Shi, Z and Tang, Z},
title = {Leveraging CRISPR/Cas12 signal amplifier to sensitive detection of apurinic/apyrimidinic endonuclease 1 and high-throughput inhibitor screening.},
journal = {Analytica chimica acta},
volume = {1291},
number = {},
pages = {342212},
doi = {10.1016/j.aca.2024.342212},
pmid = {38280781},
issn = {1873-4324},
mesh = {*High-Throughput Screening Assays ; *CRISPR-Cas Systems ; DNA Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism ; Endonucleases/metabolism ; },
abstract = {As an essential protein in DNA repair, apurinic/apyrimidinic endonuclease 1 (APE1) plays multiple critical functions in maintaining homeostasis, making it a significant biomarker and therapeutic target for many disorders. Here, we describe a simple method to detect APE1 based on the Releasing-Extension-Signal amplification Test (REST) strategy that leverages the dsDNA as the activator to fully unlock the trans-cleavage activity of CRISPR/Cas12a. This assay provides a rapid and specific APE1 detection with a detection limit down to 1.05 × 10[-5] U/mL. We also combined this method with an automated pipetting platform and a microplate reader for high-throughput screening of potential inhibitors of APE1. Besides, by changing the modification on the probe, the REST strategy was easily repurposed to detect various DNA glycosylases. Taken together, the simplicity and robustness of the method offer a new choice for APE1 detection and inhibitor screening, showing great potential in practical use. Furthermore, the REST strategy devised in this study provides a new example of applying CRISPR/Cas12a signal amplifier to non-nucleic acid biosensing and inhibitor screening, which broadens the CRISPR-Dx toolbox.},
}
@article {pmid38253889,
year = {2024},
author = {DeJulius, CR and Walton, BL and Colazo, JM and d'Arcy, R and Francini, N and Brunger, JM and Duvall, CL},
title = {Engineering approaches for RNA-based and cell-based osteoarthritis therapies.},
journal = {Nature reviews. Rheumatology},
volume = {20},
number = {2},
pages = {81-100},
pmid = {38253889},
issn = {1759-4804},
mesh = {Humans ; *CRISPR-Cas Systems ; RNA ; Quality of Life ; Gene Editing ; *Osteoarthritis/genetics/therapy ; },
abstract = {Osteoarthritis (OA) is a chronic, debilitating disease that substantially impairs the quality of life of affected individuals. The underlying mechanisms of OA are diverse and are becoming increasingly understood at the systemic, tissue, cellular and gene levels. However, the pharmacological therapies available remain limited, owing to drug delivery barriers, and consist mainly of broadly immunosuppressive regimens, such as corticosteroids, that provide only short-term palliative benefits and do not alter disease progression. Engineered RNA-based and cell-based therapies developed with synthetic chemistry and biology tools provide promise for future OA treatments with durable, efficacious mechanisms of action that can specifically target the underlying drivers of pathology. This Review highlights emerging classes of RNA-based technologies that hold potential for OA therapies, including small interfering RNA for gene silencing, microRNA and anti-microRNA for multi-gene regulation, mRNA for gene supplementation, and RNA-guided gene-editing platforms such as CRISPR-Cas9. Various cell-engineering strategies are also examined that potentiate disease-dependent, spatiotemporally regulated production of therapeutic molecules, and a conceptual framework is presented for their application as OA treatments. In summary, this Review highlights modern genetic medicines that have been clinically approved for other diseases, in addition to emerging genome and cellular engineering approaches, with the goal of emphasizing their potential as transformative OA treatments.},
}
@article {pmid36905150,
year = {2023},
author = {Shandilya, UK and Sharma, A and Sodhi, M and Mukesh, M},
title = {Editing of HSF-1 and Na/K-ATPase α1 subunit by CRISPR/Cas9 reduces thermal tolerance of bovine skin fibroblasts to heat shock in vitro.},
journal = {Animal biotechnology},
volume = {34},
number = {8},
pages = {3626-3636},
doi = {10.1080/10495398.2023.2187403},
pmid = {36905150},
issn = {1532-2378},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; Heat Shock Transcription Factors/genetics ; *Heat-Shock Response/genetics ; Cell Line ; Fibroblasts/metabolism ; Adenosine Triphosphatases/genetics/metabolism ; },
abstract = {A follow-up to our previous findings, the present study was planned to evaluate the role of Na/K-ATPase alpha1-subunit (ATP1A1) gene in heat shock tolerance. The primary fibroblast culture was established using ear pinna tissue samples of Sahiwal cattle (Bos indicus). The knockout cell lines of Na/K-ATP1A1 and HSF-1 (heat shock factor-1, as a positive control) genes were developed by CRISPR/Cas9 method and the gene-editing was confirmed by the genomic cleavage detection assay. The two knockout cell lines (ATP1A1 and HSF-1) and wild-type fibroblasts were exposed to heat shock at 42 °C in vitro and different cellular parameters viz., apoptosis, proliferation, mitochondrial membrane potential (ΔΨm), oxidative stress, along with expression pattern of heat-responsive genes were studied. The results showed that in vitro heat shock given to knockout fibroblast cells of both ATP1A1 and HSF-1 genes resulted in decreased cell viability, while increasing the apoptosis rate, membrane depolarization, and ROS levels. However, the overall impact was more in HSF-1 knockout cells as compared to ATP1A1 knockout cells. Taken together, these results indicated that the ATP1A1 gene plays a critical role as HSF-1 under heat stress and helps cells to cope with heat shock.},
}
@article {pmid38279306,
year = {2024},
author = {Kovalev, MA and Gladysh, NS and Bogdanova, AS and Bolsheva, NL and Popchenko, MI and Kudryavtseva, AV},
title = {Editing Metabolism, Sex, and Microbiome: How Can We Help Poplar Resist Pathogens?.},
journal = {International journal of molecular sciences},
volume = {25},
number = {2},
pages = {},
pmid = {38279306},
issn = {1422-0067},
support = {22-14-00404//Russian Science Foundation/ ; 075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *Populus/genetics/metabolism ; Genotype ; CRISPR-Cas Systems ; Phenotype ; Gene Editing ; Plants, Genetically Modified/genetics ; },
abstract = {Poplar (Populus) is a genus of woody plants of great economic value. Due to the growing economic importance of poplar, there is a need to ensure its stable growth by increasing its resistance to pathogens. Genetic engineering can create organisms with improved traits faster than traditional methods, and with the development of CRISPR/Cas-based genome editing systems, scientists have a new highly effective tool for creating valuable genotypes. In this review, we summarize the latest research data on poplar diseases, the biology of their pathogens and how these plants resist pathogens. In the final section, we propose to plant male or mixed poplar populations; consider the genes of the MLO group, transcription factors of the WRKY and MYB families and defensive proteins BbChit1, LJAMP2, MsrA2 and PtDef as the most promising targets for genetic engineering; and also pay attention to the possibility of microbiome engineering.},
}
@article {pmid38278531,
year = {2024},
author = {Im, SH and Robby, AI and Choi, H and Chung, JY and Kim, YS and Park, SY and Chung, HJ},
title = {A Wireless, CRISPR-Polymer Dot Electrochemical Sensor for the Diagnosis of Bacterial Pneumonia and Multi-Drug Resistance.},
journal = {ACS applied materials &amp; interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.3c17151},
pmid = {38278531},
issn = {1944-8252},
abstract = {Rapid and accurate diagnosis is crucial for managing the global health threat posed by multidrug-resistant bacterial infections; however, current methods have limitations in either being time-consuming, labor-intensive, or requiring instruments with high costs. Addressing these challenges, we introduce a wireless electrochemical sensor integrating the CRISPR/Cas system with electroconductive polymer dot (PD) nanoparticles to rapidly detect bacterial pathogens from human sputum. To enhance the electroconductive properties, we synthesized copper-ion-immobilized PD (PD-Cu), followed by conjugation of the deactivated Cas9 protein (dCas9) onto PD-Cu-coated Si electrodes to generate the dCas9-PD-Cu sensor. The dCas9-PD-Cu sensor integrated with isothermal amplification can specifically detect target nucleic acids of multidrug-resistant bacteria, such as the antibiotic resistance genes kpc-2 and mecA. The dCas9-PD-Cu sensor exhibits high sensitivity, allowing for the detection of ∼54 femtograms of target nucleic acids, based on measuring the changes in resistivity of the Si electrodes through target capture by dCas9. Furthermore, a wireless sensing platform of the dCas9-PD-Cu sensor was established using a Bluetooth module and a microcontroller unit for detection using a smartphone. We demonstrate the feasibility of the platform in diagnosing multidrug-resistant bacterial pneumonia in patients' sputum samples, achieving 92% accuracy. The current study presents a versatile biosensor platform that can overcome the limitations of conventional diagnostics in the clinic.},
}
@article {pmid38273342,
year = {2024},
author = {Liu, XL and Xie, J and Xie, ZN and Zhong, C and Liu, H and Zhang, SH and Jin, J},
title = {Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.},
journal = {Microbial cell factories},
volume = {23},
number = {1},
pages = {34},
pmid = {38273342},
issn = {1475-2859},
mesh = {Tandem Mass Spectrometry/methods ; Squalene Monooxygenase/genetics/metabolism ; *Wolfiporia/genetics/metabolism ; Molecular Docking Simulation ; Squalene ; CRISPR-Cas Systems ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Triterpenes/metabolism ; *Phytosterols ; },
abstract = {BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids.<br><br>RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach.<br><br>CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.},
}
@article {pmid38273037,
year = {2024},
author = {Bayat, H and Mirahmadi, M and Azarshin, Z and Ohadi, H and Delbari, A and Ohadi, M},
title = {CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {2136},
pmid = {38273037},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Microsatellite Repeats ; Neurons/metabolism ; Membrane Glycoproteins/metabolism ; Nerve Tissue Proteins/metabolism ; },
abstract = {The human neuron-specific gene, GPM6B (Glycoprotein membrane 6B), is considered a key gene in neural cell functionality. This gene contains an exceptionally long and strictly monomorphic short tandem repeat (STR) of 9-repeats, (GA)9. STRs in regulatory regions, may impact on the expression of nearby genes. We used CRISPR-based tool to delete this GA-repeat in NT2 cells, and analyzed the consequence of this deletion on GPM6B expression. Subsequently, the edited cells were induced to differentiate into neural cells, using retinoic acid (RA) treatment. Deletion of the GA-repeat significantly decreased the expression of GPM6B at the RNA (p < 0.05) and protein (40%) levels. Compared to the control cells, the edited cells showed dramatic decrease of the astrocyte and neural cell markers, including GFAP (0.77-fold), TUBB3 (0.57-fold), and MAP2 (0.2-fold). Subsequent sorting of the edited cells showed an increased number of NES (p < 0.01), but a decreased number of GFAP (p < 0.001), TUBB3 (p < 0.05), and MAP2 (p < 0.01), compared to the control cells. In conclusion, CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B, led to decreased expression of this gene, which in turn, disrupted differentiation of NT2 cells into neural cells.},
}
@article {pmid38272895,
year = {2024},
author = {Anderson, MAE and Gonzalez, E and Edgington, MP and Ang, JXD and Purusothaman, DK and Shackleford, L and Nevard, K and Verkuijl, SAN and Harvey-Samuel, T and Leftwich, PT and Esvelt, K and Alphey, L},
title = {A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {729},
pmid = {38272895},
issn = {2041-1723},
support = {N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; N66001-17-2-4054//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 110117/Z/15/Z//Wellcome Trust (Wellcome)/ ; 110117/Z/15/Z//Wellcome Trust (Wellcome)/ ; 110117/Z/15/Z//Wellcome Trust (Wellcome)/ ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Aedes/genetics ; *Gene Drive Technology ; RNA, Guide, CRISPR-Cas Systems ; *Insecticides ; *Zika Virus Infection/genetics ; *Zika Virus/genetics ; },
abstract = {Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.},
}
@article {pmid38241798,
year = {2024},
author = {Wang, J and Jiang, H and Chen, Y and Zhu, X and Wu, Q and Chen, W and Zhao, Q and Wang, J and Qin, P},
title = {CRISPR/Cas9-mediated SERS/colorimetric dual-mode lateral flow platform combined with smartphone for rapid and sensitive detection of Staphylococcus aureus.},
journal = {Biosensors &amp; bioelectronics},
volume = {249},
number = {},
pages = {116046},
doi = {10.1016/j.bios.2024.116046},
pmid = {38241798},
issn = {1873-4235},
mesh = {Humans ; Staphylococcus aureus/genetics ; Colorimetry ; Smartphone ; CRISPR-Cas Systems/genetics ; *Metal Nanoparticles ; *Biosensing Techniques ; Spectrum Analysis, Raman/methods ; *Staphylococcal Infections/diagnosis ; Gold ; },
abstract = {Pathogenic bacteria infections pose a significant threat to global public health, making the development of rapid and reliable detection methods urgent. Here, we developed a surface-enhanced Raman scattering (SERS) and colorimetric dual-mode platform, termed smartphone-integrated CRISPR/Cas9-mediated lateral flow strip (SCC-LFS), and applied it to the ultrasensitive detection of Staphylococcus aureus (S. aureus). Strategically, functionalized silver-coated gold nanostar (AuNS@Ag) was prepared and used as the labeling material for LFS assay. In the presence of S. aureus, target gene-induced amplicons can be accurately recognized and unwound by the user-defined CRISPR/Cas9 system, forming intermediate bridges that bind many AuNS@Ag to the test line (T-line) of the strip. As a result, the T-line was colored and a recognizable SERS signal was obtained using a smartphone-integrated portable Raman spectrometer. This design not only maintains the simplicity of visual readout, but also integrates the quantitative capability of SERS, enabling the user to flexibly select the assay mode as needed. With this method, S. aureus down to 1 CFU/mL can be detected by both colorimetric and SERS modes, which is better than most existing methods. By incorporating a rapid extraction procedure, the entire assay can be completed in 45 min. The robustness and practicality of the method were further demonstrated by various real samples, indicating its considerable potential toward reliable screening of S. aureus.},
}
@article {pmid38178651,
year = {2024},
author = {Park, CS and Habib, O and Lee, Y and Hur, JK},
title = {Applications of CRISPR technologies to the development of gene and cell therapy.},
journal = {BMB reports},
volume = {57},
number = {1},
pages = {2-11},
pmid = {38178651},
issn = {1976-670X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Engineering ; Technology ; Cell- and Tissue-Based Therapy ; },
abstract = {Advancements in gene and cell therapy have resulted in novel therapeutics for diseases previously considered incurable or challenging to treat. Among the various contributing technologies, genome editing stands out as one of the most crucial for the progress in gene and cell therapy. The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the subsequent evolution of genetic engineering technology have markedly expanded the field of target-specific gene editing. Originally studied in the immune systems of bacteria and archaea, the CRISPR system has demonstrated wide applicability to effective genome editing of various biological systems including human cells. The development of CRISPR-based base editing has enabled directional cytosine-tothymine and adenine-to-guanine substitutions of select DNA bases at the target locus. Subsequent advances in prime editing further elevated the flexibility of the edit multiple consecutive bases to desired sequences. The recent CRISPR technologies also have been actively utilized for the development of in vivo and ex vivo gene and cell therapies. We anticipate that the medical applications of CRISPR will rapidly progress to provide unprecedented possibilities to develop novel therapeutics towards various diseases. [BMB Reports 2024; 57(1): 2-11].},
}
@article {pmid38178650,
year = {2024},
author = {Park, JC and Bae, S},
title = {Current status of genome editing technologies: special issue of BMB Reports in 2024.},
journal = {BMB reports},
volume = {57},
number = {1},
pages = {1},
pmid = {38178650},
issn = {1976-670X},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Plants/genetics ; DNA ; Technology ; },
abstract = {Since the identification of DNA as a genetic material, manipulating DNA in various organisms has been a long standing dream of humanity. In pursuit of this objective, technologies to edit genome have been extensively developed over the recent decades. The emergence of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems enabled site-specific DNA cleavage in a programmable manner. Furthermore, the advent of base editors (BEs) and prime editors (PEs) has enabled base conversion and insertion/deletion with a high accuracy. In addition to the editing of genomic DNA in the nucleus, attempts to manipulate circular DNAs in organelle are currently ongoing. These technologies are bringing major progress in diverse fields including the engineering of cells, livestock, and plants as well as therapeutic gene correction in humans. In this special issue, we aim to cover the recent advances in genome editing technology and its applications in therapeutics, breed improvement in plants and livestock, RNA recording, and protein evolution. [BMB Reports 2024; 57(1): 1].},
}
@article {pmid38105495,
year = {2024},
author = {Rawal, P and Tripathi, DM and Hemati, H and Kumar, J and Tyagi, P and Sarin, SK and Nain, V and Kaur, S},
title = {Targeted HBx gene editing by CRISPR/Cas9 system effectively reduces epithelial to mesenchymal transition and HBV replication in hepatoma cells.},
journal = {Liver international : official journal of the International Association for the Study of the Liver},
volume = {44},
number = {2},
pages = {614-624},
doi = {10.1111/liv.15805},
pmid = {38105495},
issn = {1478-3231},
support = {//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics ; Hepatitis B virus/genetics ; *Liver Neoplasms/genetics ; Hepatitis B Surface Antigens/genetics ; Gene Editing ; CRISPR-Cas Systems ; Epithelial-Mesenchymal Transition/genetics ; RNA, Guide, CRISPR-Cas Systems ; DNA, Circular ; Virus Replication ; Hep G2 Cells ; *Hepatitis B ; },
abstract = {BACKGROUND AND AIMS: Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV-induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC.<br><br>METHODOLOGY AND RESULTS: We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in&#xa0;vitro effects on tumour properties of HepG2-2.15. Full length HBx gene was excised using HBx-CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx-CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx-CRISPR treated cells as compared to controls in both two- and three- dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx-CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated.<br><br>CONCLUSION: Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV-HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV-induced HCC.},
}
@article {pmid38053294,
year = {2024},
author = {Lee, Y and Oh, Y and Lee, SH},
title = {Recent advances in genome engineering by CRISPR technology.},
journal = {BMB reports},
volume = {57},
number = {1},
pages = {12-18},
pmid = {38053294},
issn = {1976-670X},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome/genetics ; Technology ; },
abstract = {Due to the development of CRISPR technology, the era of effective editing of target genes has arrived. However, the offtarget problem that occurs when recognizing target DNA due to the inherent nature of CRISPR components remains the biggest task to be overcome in the future. In this review, the principle of inducing such unintended off-target editing is analyzed from the structural aspect of CRISPR, and the methodology that has been developed to reduce off-target editing until now is summarized. [BMB Reports 2024; 57(1): 12-18].},
}
@article {pmid38053293,
year = {2024},
author = {Han, AR and Shin, HR and Kwon, J and Lee, SB and Lee, SE and Kim, EY and Kweon, J and Chang, EJ and Kim, Y and Kim, SW},
title = {Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells.},
journal = {BMB reports},
volume = {57},
number = {1},
pages = {60-65},
pmid = {38053293},
issn = {1976-670X},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Ribonucleoproteins/genetics/metabolism ; DNA ; *Mesenchymal Stem Cells/metabolism ; },
abstract = {The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. [BMB Reports 2024; 57(1): 60-65].},
}
@article {pmid38053291,
year = {2024},
author = {Hong, C and Han, JH and Hwang, GH and Bae, S and Seo, PJ},
title = {Genome-wide in-locus epitope tagging of Arabidopsis proteins using prime editors.},
journal = {BMB reports},
volume = {57},
number = {1},
pages = {66-70},
pmid = {38053291},
issn = {1976-670X},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Arabidopsis Proteins/genetics ; DNA/genetics ; },
abstract = {Prime editors (PEs), which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusion proteins programmed with prime editing guide RNAs (pegRNAs), can not only edit bases but also install transversions, insertions, or deletions without both donor DNA and double-strand breaks at the target DNA. As the demand for in-locus tagging is increasing, to reflect gene expression dynamics influenced by endogenous genomic contexts, we demonstrated that PEs can be used to introduce the hemagglutinin (HA) epitope tag to a target gene locus, enabling molecular and biochemical studies using in-locus tagged plants. To promote genome-wide in-locus tagging, we also implemented a publicly available database that designs pegRNAs for in-locus tagging of all the Arabidopsis genes. [BMB Reports 2024; 57(1): 66-70].},
}
@article {pmid37991148,
year = {2024},
author = {Liu, X and Wang, Y and Wang, H and He, Y and Song, Y and Li, Z and Li, M and Wei, C and Dong, Y and Xue, L and Zhang, J and Zhu, JK and Wang, M},
title = {Generating herbicide resistant and dwarf rice germplasms through precise sequence insertion or replacement.},
journal = {Plant biotechnology journal},
volume = {22},
number = {2},
pages = {293-295},
pmid = {37991148},
issn = {1467-7652},
support = {2021BEB04075//Key Research and Development Program of Ningxia/ ; ZDXM04//Nanfan special project, CAAS/ ; ZDXM23014//Nanfan special project, CAAS/ ; 2021YFA1300404//National Key Research and Development Program of China/ ; 31901046//National Natural Science Foundation of China/ ; 32188102//National Natural Science Foundation of China/ ; 32271524//National Natural Science Foundation of China/ ; 2022AAC03007//Natural Science Foundation of Ningxia Province/ ; 2020272//the Youth Innovation Promotion Association, CAS/ ; },
mesh = {*Herbicides ; *Oryza/genetics ; Mutagenesis, Insertional ; Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid37823527,
year = {2024},
author = {Holme, IB and Ingvardsen, CR and Dionisio, G and Podzimska-Sroka, D and Kristiansen, K and Feilberg, A and Brinch-Pedersen, H},
title = {CRISPR/Cas9-mediated mutation of Eil1 transcription factor genes affects exogenous ethylene tolerance and early flower senescence in Campanula portenschlagiana.},
journal = {Plant biotechnology journal},
volume = {22},
number = {2},
pages = {484-496},
pmid = {37823527},
issn = {1467-7652},
support = {GUDP grant no. 34009-15-1010//The Danish Agricultural Agency (Ministry of Food, Agriculture and Fisheries)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Plant Senescence ; Ethylenes/metabolism ; Mutation/genetics ; Transcription Factors/genetics ; *Campanulaceae/metabolism ; Flowers/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; },
abstract = {Improving tolerance to ethylene-induced early senescence of flowers and fruits is of major economic importance for the ornamental and food industry. Genetic modifications of genes in the ethylene-signalling pathway have frequently resulted in increased tolerance but often with unwanted side effects. Here, we used CRISPR/Cas9 to knockout the function of two CpEil1 genes expressed in flowers of the diploid ornamental plant Campanula portenschlagiana. The ethylene tolerance in flowers of the primary mutants with knockout of only one or all four alleles clearly showed increased tolerance to exogenous ethylene, although lower tolerance was obtained with one compared to four mutated alleles. The allele dosage effect was confirmed in progenies where flowers of plants with zero, one, two, three and four mutated alleles showed increasing ethylene tolerance. Mutation of the Cpeil1 alleles had no significant effect on flower longevity and endogenous flower ethylene level, indicating that CpEil1 is not involved in age-dependent senescence of flowers. The study suggests focus on EIN3/Eils expressed in the organs subjected to early senescence for obtaining tolerance towards exogenous ethylene. Furthermore, the observed allelic dosage effect constitutes a key handle for a gradual regulation of sensitivity towards exogenous ethylene, simultaneously monitoring possibly unwanted side effects.},
}
@article {pmid37816147,
year = {2024},
author = {Wang, Y and Li, X and Liu, M and Zhou, Y and Li, F},
title = {Guide RNA scaffold variants enabled easy cloning of large gRNA cluster for multiplexed gene editing.},
journal = {Plant biotechnology journal},
volume = {22},
number = {2},
pages = {460-471},
pmid = {37816147},
issn = {1467-7652},
support = {2018YFD1000800//National Key Research and Development Program of China/ ; 32272491//National Natural Science Foundation of China/ ; 91940301//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Cloning, Molecular ; },
abstract = {Cas9 protein-mediated gene editing has revolutionized genetic manipulation in most organisms. There are many cases where multiplexed gene editing is needed. Cas9 is capable of multiplex gene editing when expressed with multiple guide RNAs. Conventional cloning methods for multiplexed gene editing vector is not efficient due to repeated use of a single-guide RNA scaffold and inefficient ligation. In this study, we conducted structure-guided mutagenesis and random mutagenesis on the original sgRNA scaffold and identified a large number of functional sgRNA scaffold variants. With these scaffold variants and different tRNAs, fusion polymerase chain reaction protocol was developed to rapidly synthesize spacer-scaffold-tRNA-spacer units with up to 9 targets. In conjunction with golden gate cloning, gene editing vectors with up to 24 target sites were efficiently cloned in one-step cloning. One such gene editing vector targeting 12 genes in tomato were tested in stable transformation and 10 out of the 12 genes were found mutated in a single transgenic line. To facilitate the application of multiplexed gene editing using these scaffold variants and tRNAs from different species, a webserver was created to generate primer sets and provide template sequences for the synthesis of large sgRNA expression units based on the user-supplied target sequences and species.},
}
@article {pmid38272874,
year = {2024},
author = {Ajore, R and Mattsson, J and Pertesi, M and Ekdahl, L and Ali, Z and Hansson, M and Nilsson, B},
title = {Genome-wide CRISPR/Cas9 screen identifies regulators of BCMA expression on multiple myeloma cells.},
journal = {Blood cancer journal},
volume = {14},
number = {1},
pages = {21},
pmid = {38272874},
issn = {2044-5385},
mesh = {Humans ; *Multiple Myeloma/genetics/metabolism ; B-Cell Maturation Antigen ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
}
@article {pmid38272227,
year = {2024},
author = {Sharrar, A and Arake de Tacca, L and Meacham, Z and Staples-Ager, J and Collingwood, T and Rabuka, D and Schelle, M},
title = {Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {105685},
doi = {10.1016/j.jbc.2024.105685},
pmid = {38272227},
issn = {1083-351X},
abstract = {The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We engineer the nuclease (termed AiEvo2) for increased specificity, PAM recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntington's patient derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 CAR-T therapy from primary human T cells using multiplexed simultaneous editing and CAR insertion. To further ensure precise editing, we engineered an anti-CRISPR protein (ErAcr) to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel ErAcr protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.},
}
@article {pmid38270707,
year = {2024},
author = {Bharati, J and Kumar, S and Mohan, NH and Pegu, SR and Borah, S and Gupta, VK and Sarkar, M},
title = {CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {195},
pmid = {38270707},
issn = {1573-4978},
support = {IXX15063//Indian Council of Agricultural Research/ ; BT/PR24689/NER/95/812/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Female ; Swine ; Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Luteal Cells ; Vascular Endothelial Growth Factor A/genetics ; Annexin A5 ; },
abstract = {BACKGROUND: The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production.<br><br>METHOD AND RESULTS: The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p&#x2009;<&#x2009;0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p&#x2009;<&#x2009;0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p&#x2009;<&#x2009;0.05) upon KO, which was further validated by the significant (p&#x2009;<&#x2009;0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study.<br><br>CONCLUSION: The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.},
}
@article {pmid38269913,
year = {2024},
author = {Jain, Y and Izzath, FAM and Wilson, LOW and Bauer, DC},
title = {Data Visualization of CRISPR-Cas9 Guide RNA Design Tools.},
journal = {Studies in health technology and informatics},
volume = {310},
number = {},
pages = {770-774},
doi = {10.3233/SHTI231069},
pmid = {38269913},
issn = {1879-8365},
mesh = {*Data Visualization ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Engineering ; Genomics ; },
abstract = {With the advancement of genomic engineering and genetic modification techniques, the uptake of computational tools to design guide RNA increased drastically. Searching for genomic targets to design guides with maximum on-target activity (efficiency) and minimum off-target activity (specificity) is now an essential part of genome editing experiments. Today, a variety of tools exist that allow the search of genomic targets and let users customize their search parameters to better suit their experiments. Here we present an overview of different ways to visualize these searched CRISPR target sites along with specific downstream information like primer design, restriction enzyme activity and mutational outcome prediction after a double-stranded break. We discuss the importance of a good visualization summary to interpret information along with different ways to represent similar information effectively.},
}
@article {pmid38167890,
year = {2024},
author = {Wang, H and Su, A and Chang, J and Liu, X and Liang, C and Xu, S},
title = {Sensitive detection of genetically modified maize based on a CRISPR/Cas12a system.},
journal = {The Analyst},
volume = {149},
number = {3},
pages = {836-845},
doi = {10.1039/d3an01788e},
pmid = {38167890},
issn = {1364-5528},
mesh = {Plants, Genetically Modified/genetics ; *Zea mays/genetics ; *CRISPR-Cas Systems/genetics ; Transgenes ; },
abstract = {With the vigorous development of biotechnology, genetically modified organisms (GMOs) have become more and more common. In order to effectively supervise and administrate them, the rapid and accurate detection of GMOs is urgently demanded. Here, GMO gene-specific sensing methods based on colorimetry and surface-enhanced Raman scattering (SERS) were proposed based on the lateral branch cleavage function of the CRISPR/Cas12a system. Two transgenes, pCaMV35S and M810 Cry1Ab, were chosen as targets for transgenic crops. By using these methods, we performed transgenic detection on five types of maize leaves and successfully distinguished transgenic from non-transgenic samples. The colorimetric method is rapid, economical and available for field detection. The SERS approach, giving a higher sensitivity to 100 fM, is more suitable for laboratory application scenarios. This study explores practical transgenic detection approaches and will be valuable for the supervision of GMOs.},
}
@article {pmid38131397,
year = {2024},
author = {Yu, HM and Liang, GX and Wang, HY and Hang, XM and Wang, HH and Peng, JX and Wang, L},
title = {A MnO2 nanosheet-mediated CRISPR/Cas12a system for the detection of organophosphorus pesticides in environmental water.},
journal = {The Analyst},
volume = {149},
number = {3},
pages = {729-734},
doi = {10.1039/d3an02020g},
pmid = {38131397},
issn = {1364-5528},
mesh = {*Pesticides/analysis ; Organophosphorus Compounds ; Acetylcholinesterase/genetics/metabolism ; CRISPR-Cas Systems ; Dichlorvos ; Water ; Manganese Compounds ; Oxides ; Acetylcholine ; *Biosensing Techniques/methods ; },
abstract = {Nowadays, easy, convenient, and sensitive sensing strategies are still critical for organophosphorus pesticides in environmental water samples. Herein, a novel organophosphorus pesticide (OP) assay based on acetylcholinesterase (AChE) and a MnO2 nanosheet-mediated CRISPR/Cas12a reaction is reported. The single-strand DNA (ssDNA) activator of CRISPR/Cas12a was simply adsorbed on the MnO2 nanosheets as the nanoswitches of the assay. In the absence of target OPs, AChE hydrolyzed acetylcholine (ATCh) to thiocholine (TCh), which reduced the MnO2 nanosheets to Mn[2+], resulting in the release of the activator followed by activation of the CRISPR/Cas12a system. The activated Cas12a thereafter nonspecifically cleaved the FAM/BHQ1-labeled ssDNA (FQ-reporter), producing a fluorescence signal. Upon the addition of target OPs, the hydrolysis of ATCh by AChE was inhibited owing to OPs combining with AChE, and thus effective quantification of OPs could be achieved by measuring the fluorescence changes of the system. As a proof of concept, dichlorvos (DDVP) was chosen as a model OP analyte to address the feasibility of the proposed method. Attributed to the excellent trans-cleavage activity of Cas12a, the fluorescent biosensor exhibits a satisfactory limit of detection (LOD) for DDVP at 0.135 ng mL[-1]. In addition, the excellent recoveries for the detection of DDVP in environmental water samples demonstrate the applicability of the proposed assay in real sample research.},
}
@article {pmid38047496,
year = {2024},
author = {Sun, X and Wang, J and Mou, C and Shi, K and Bao, W and Chen, Z},
title = {Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line.},
journal = {Biotechnology journal},
volume = {19},
number = {1},
pages = {e2300389},
doi = {10.1002/biot.202300389},
pmid = {38047496},
issn = {1860-7314},
support = {2021YFD1800104//National Key Research and Development Program of China/ ; AB21238003//Guangxi Key Research and Development Plan/ ; CX (21) 2014//Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province/ ; YZ2022187//Yangzhou City and Yangzhou University Corporation/ ; D18007//111 Project/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Virus Replication/genetics ; Gene Editing ; Antiviral Agents/pharmacology ; },
abstract = {Antiviral vaccines for pig diseases are essential to prevent epidemic outbreaks. However, their production is often hindered by inefficient manufacturing processes that yield lower quantities of the vaccine. To accelerate the progress of various areas of bioproduction, we have considered the necessity of enhancing viral replication efficiency by optimizing ST (swine testicular) cell lines that are commonly utilized in virus manufacturing. CRISPR/Cas9 gene-editing technology were utilized to create IRF3 or IRF7 knockout cell lines that facilitate high-titer viral stock production. Compared to the parental cell lines, the ST IRF3/7 KO cell line displayed a compromised antiviral response to a panel of viruses (Porcine epidemic diarrhea virus, Senecavirus A, Parainfluenza virus 5, and Getah virus), as evidenced by decreased expression of interferon and certain antiviral factors. The inhibition of these responses led to heightened viral replication and increased cytopathic effects, ultimately promoting apoptosis. As a result, the development of these cell lines offers a more efficient approach for biopharmaceutical companies to boost their virus production and reduce associated costs.},
}
@article {pmid38010373,
year = {2024},
author = {Li, J and Fan, G and Sakari, M and Tsukahara, T},
title = {Improvement of C-to-U RNA editing using an artificial MS2-APOBEC system.},
journal = {Biotechnology journal},
volume = {19},
number = {1},
pages = {e2300321},
doi = {10.1002/biot.202300321},
pmid = {38010373},
issn = {1860-7314},
support = {17H02204//Japan Society for the Promotion of Science/ ; 18K19288//Japan Society for the Promotion of Science/ ; 21H02067//Japan Society for the Promotion of Science/ ; },
mesh = {*RNA Editing/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Proteins/metabolism ; RNA ; *Cytidine Deaminase ; },
abstract = {RNA cytidine deamination (C-to-U editing) has been achieved using the MS2-apolipoprotein B-editing catalytic polypeptide-like (APOBEC)1 editing system. Here, we fused the cytidine deaminase (CDA) enzymes APOBEC3A and APOBEC3G with the MS2 system and examined their RNA editing efficiencies in transfected HEK 293T cells. Given the single-stranded RNA preferences of APOBEC3A and APOBEC3G, we designed unconventional guide RNAs that induced a loop at the target sequence, allowing the target to form a single-stranded structure. Because APOBEC3A and APOBEC3G have different base preferences (5'-TC and 5'-CC, respectively), we introduced the D317W mutation into APOBEC3G to convert its base preference to that of APOBEC3A. Upon co-transfection with a guide RNA that induced the formation of a 14 nt loop on the target sequence, MS2-fused APOBEC3A and APOBEC3G showed high editing efficiency. While the D317W mutation of APOBEC3G led to a slight improvement in editing efficiency, the difference was not statistically significant. These findings indicate that APOBEC3A and APOBEC3G can induce C-to-U RNA editing when transfected with a loop guide RNA. Moreover, the editing efficiency of APOBEC3G can be enhanced by site-specific mutation to alter the base preference. Overall, our results demonstrate that the MS2 system can fuse and catalyze reactions with different enzymes, suggesting that it holds an even greater potential for RNA editing than is utilized currently.},
}
@article {pmid37882473,
year = {2024},
author = {Haraguchi, S and Dang-Nguyen, TQ and Kikuchi, K and Somfai, T},
title = {Electroporation-mediated genome editing in vitrified/warmed porcine zygotes obtained in vitro.},
journal = {Molecular reproduction and development},
volume = {91},
number = {1},
pages = {e23712},
doi = {10.1002/mrd.23712},
pmid = {37882473},
issn = {1098-2795},
support = {//Japan Society for the Promotion of Science/ ; },
mesh = {Swine/genetics ; Animals ; *Gene Editing ; *Zygote/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Embryonic Development ; Electroporation ; Blastocyst/metabolism ; Cryopreservation ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) system is the most efficient and widely used technology for genome editing in all sorts of organisms, including livestock animals. Here, we examined the feasibility of CRISPR/Cas9-derived genome editing (GE) in vitrified porcine zygotes, where the flexible planning of experiments in time and space is expected. OCT4 and CD46 genes were targeted, and the Cas9/sgRNA ribonucleoprotein complexes (RNP) were electroporated into zygotes at 2 h after warming. Vitrification or GE alone did not significantly reduce the developmental rates to the blastocyst stage. However, vitrification followed by GE significantly reduced blastocyst development. Sequencing analysis of the resultant blastocysts revealed efficient GE for both OCT4 (nonvitrified: 91.0%, vitrified: 95.1%) and CD46 (nonvitrified: 94.5%, vitrified: 93.2%), with no significant difference among them. Immunocytochemical analysis showed that GE-blastocysts lacked detectable proteins. They were smaller in size, and the cell numbers were significantly reduced compared with the control (p < 0.01). Finally, we demonstrated that double GE efficiently occurs (100%) when the OCT4-RNP and CD46-RNP are simultaneously introduced into zygotes after vitrification/warming. This is the first demonstration that vitrified porcine zygotes can be used in GE as efficiently as nonvitrified ones.},
}
@article {pmid38269199,
year = {2023},
author = {Nasseri, S and Parsa, S and Vahabzadeh, Z and Baban, B and Khademerfan, MB and Nikkhoo, B and Rastegar Khosravi, M and Bahrami, S and Fathi, F},
title = {CRISPR/Cas9-Induced Fam83h Knock-out Leads to Impaired Wnt/β-Catenin Pathway and Altered Expression of Tooth Mineralization Genes in Mice.},
journal = {Iranian journal of biotechnology},
volume = {21},
number = {4},
pages = {e3673},
pmid = {38269199},
issn = {1728-3043},
abstract = {BACKGROUND: Dental enamel formation is a complex process that is regulated by various genes. One such gene, Family With Sequence Similarity 83 Member H (Fam83h), has been identified as an essential factor for dental enamel formation. Additionally, Fam83h has been found to be potentially linked to the Wnt/β-catenin pathway.<br><br>OBJECTIVES: This study aimed to investigate the effects of the Fam83h knockout gene on mineralization and formation of teeth, along with mediators of the Wnt/&#x3b2;-catenin pathway as a development aspect in mice.<br><br>MATERIALS AND METHODS: To confirm the Fam83h-KnockOut mice, both Sanger sequencing and Western blot methods were used. then used qPCR to measure the expression levels of genes related to tooth mineralization and formation of dental root, including Fam20a, Dspp, Dmp1, Enam, Ambn, Sppl2a, Mmp20, and Wnt/&#x3b2;-catenin pathway mediators, in both the Fam83h-Knockout and wild-type mice at 5, 11 and 18 days of age. also the expression level of Fgf10 and mediators of the Wnt/&#x3b2;-catenin pathway was measured in the skin of both Knockout and wild-type mice using qPCR. A histological assessment was then performed to further investigate the results.<br><br>RESULTS: A significant reduction in the expression levels of Ambn, Mmp20, Dspp, and Fgf10 in the dental root of Fam83h-Knockout mice compared to their wild-type counterparts was demonstrated by our results, indicating potential disruptions in tooth development. Significant down-regulation of CK1a, CK1e, and &#x3b2;-catenin in the dental root of Fam83h-Knockout mice was associated with a reduction in mineralization and formation-related gene. Additionally, the skin analysis of Fam83h-Knockout mice revealed reduced levels of Fgf10, CK1a, CK1e, and &#x3b2;-catenin. Further histological assessment confirmed that the concurrent reduction of Fgf10 expression level and Wnt/&#x3b2;-catenin genes were associated with alterations in hair follicle maturation.<br><br>CONCLUSIONS: The concurrent reduction in the expression level of both Wnt/&#x3b2;-catenin mediators and mineralization-related genes, resulting in the disruption of dental mineralization and formation, was caused by the deficiency of Fam83h. Our findings suggest a cumulative effect and multi-factorial interplay between Fam83h, Wnt/&#x392;-Catenin signaling, and dental mineralization-related genes subsequently, during the dental formation process.},
}
@article {pmid38269016,
year = {2023},
author = {Onaran Acar, B and Cengız, G and Goncuoglu, M},
title = {Vancomycin-variable enterococci in sheep and cattle isolates and whole-genome sequencing analysis of isolates harboring vanM and vanB genes.},
journal = {Iranian journal of veterinary research},
volume = {24},
number = {3},
pages = {182-192},
pmid = {38269016},
issn = {1728-1997},
abstract = {BACKGROUND: Vancomycin resistance encoded by the vanA/B/M genes in enterococci is clinically important because of the transmission of these genes between bacteria. While vancomycin resistance is determined by detecting only vanA and vanB genes by routine analyses, failure to detect vanM resistance causes vancomycin resistance to be overlooked, and clinically appropriate treatment cannot be provided.<br><br>AIMS: The study aimed to examine the presence of vanM-positive enterococcal isolates in Ankara, Turkey, and to have detailed information about them with sequence analyses.<br><br>METHODS: Caecal samples were collected from sheep and cattle during slaughter at different slaughterhouses in Ankara, Turkey. Enterococci isolates were identified, confirmed, and analyzed for the presence of vanA/B/M genes. Antibiotic resistance profiles of isolates were determined by the broth microdilution method. A whole genome sequence analysis of the isolates harboring the vanM and vanB genes was performed.<br><br>RESULTS: 13.7% of enterococcal isolates were determined as Enterococcus faecium and Enterococcus faecalis. 15% of these isolates contained vanB, and 40% were vanM-positive. S98b and C32 isolates were determined to contain 16 CRISPR-Cas elements. 80% of the enterococci isolates were resistant to nitrofurantoin and 15% to ciprofloxacin. The first vanM-positive vancomycin-variable enterococci (VVE) isolates from food-producing animals were identified, and the S98b strain has been assigned to Genbank with the accession number CP104083.1.<br><br>CONCLUSION: Therefore, new studies are needed to facilitate the identification of vanM-resistant enterococci and VVE strains.},
}
@article {pmid38266982,
year = {2024},
author = {Karneyeva, K and Kolesnik, M and Livenskyi, A and Zgoda, V and Zubarev, V and Trofimova, A and Artamonova, D and Ispolatov, Y and Severinov, K},
title = {Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168448},
doi = {10.1016/j.jmb.2024.168448},
pmid = {38266982},
issn = {1089-8638},
abstract = {Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity.},
}
@article {pmid38266942,
year = {2024},
author = {Sun, Q and Zhang, H and Ding, F and Gao, X and Zhu, Z and Yang, C},
title = {Development of ionizable lipid nanoparticles and a lyophilized formulation for potent CRISPR-Cas9 delivery and genome editing.},
journal = {International journal of pharmaceutics},
volume = {652},
number = {},
pages = {123845},
doi = {10.1016/j.ijpharm.2024.123845},
pmid = {38266942},
issn = {1873-3476},
abstract = {CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.},
}
@article {pmid38266238,
year = {2024},
author = {Qu, Z and Zhang, B and Kong, L and Zhang, Y and Zhao, Y and Gong, Y and Gao, X and Feng, M and Zhang, J and Yan, L},
title = {Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL-induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway.},
journal = {Journal of leukocyte biology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jleuko/qiae011},
pmid = {38266238},
issn = {1938-3673},
abstract = {The overactivation of the osteoclasts is a crucial pathological factor in the development of osteoporosis. Myeloid zinc finger 1 (MZF1), belonging to the scan-zinc finger family, plays a significant role in various processes associated with tumor malignant progression and acts as an essential transcription factor regulating osteoblast expression. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis. First, we established MZF1-deficient female mice and evaluated the femur bone phenotype by Micro-CT and histological staining. Our findings indicate that MZF1-/- mice exhibited a low bone mass osteoporosis phenotype. RANKL could independently induce the differentiation of RAW264.7 cells into osteoclasts, and we found that the expression level of MZF1 protein decreased gradually. Then, the CRISPR/Cas 9 gene editing technique was used to build a RAW264.7 cell model with MZF1 knockout, and RANKL was used to independently induce MZF1-/- and WT cells to differentiate into mature osteoclasts. Tartrate-resistant acid phosphatase (TRAP) staining and F-actin fluorescence results showed that the MZF1-/- group produced more TRAP positive mature osteoclasts and larger actin rings. The expression of osteoclast-associated genes (including TRAP, CTSK, c-Fos and NFATc1) was evaluated by RT-qPCR and Western blot. The expression of key genes of osteoclast differentiation in MZF1-/- group was significantly increased. Furthermore, we found that cell viability was increased in the early stages of RANKL-induced cell differentiation in the MZF1-/- group cells. We examined some prevalent ferroptosis markers, including malondialdehyde, glutathione, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis. The results suggest that MZF1 may be involved in osteoclast differentiation by regulating RANKL-induced ferroptosis of osteoclasts. Collectively, our findings shed light on the essential involvement of MZF1 in the regulation of osteoclastogenesis in osteoporosis and provide insights into its potential underlying mechanism.},
}
@article {pmid38262608,
year = {2024},
author = {Siedentop, B and Rüegg, D and Bonhoeffer, S and Chabas, H},
title = {My host's enemy is my enemy: plasmids carrying CRISPR-Cas as a defence against phages.},
journal = {Proceedings. Biological sciences},
volume = {291},
number = {2015},
pages = {20232449},
pmid = {38262608},
issn = {1471-2954},
mesh = {*CRISPR-Cas Systems ; Plasmids ; *Bacteriophages ; Disease Outbreaks ; Ecology ; },
abstract = {Bacteria are infected by mobile genetic elements like plasmids and virulent phages, and those infections significantly impact bacterial ecology and evolution. Recent discoveries reveal that some plasmids carry anti-phage immune systems like CRISPR-Cas, suggesting that plasmids may participate in the coevolutionary arms race between virulent phages and bacteria. Intuitively, this seems reasonable as virulent phages kill the plasmid's obligate host. However, the efficiency of CRISPR-Cas systems carried by plasmids can be expected to be lower than those carried by the chromosome due to continuous segregation loss, creating susceptible cells for phage amplification. To evaluate the anti-phage protection efficiency of CRISPR-Cas on plasmids, we develop a stochastic model describing the dynamics of a virulent phage infection against which a conjugative plasmid defends using CRISPR-Cas. We show that CRISPR-Cas on plasmids provides robust protection, except in limited parameter sets. In these cases, high segregation loss favours phage outbreaks by generating a population of defenceless cells on which the phage can evolve and escape CRISPR-Cas immunity. We show that the phage's ability to exploit segregation loss depends strongly on the evolvability of both CRISPR-Cas and the phage itself.},
}
@article {pmid38261989,
year = {2024},
author = {Berríos, KN and Barka, A and Gill, J and Serrano, JC and Bailer, PF and Parker, JB and Evitt, NH and Gajula, KS and Shi, J and Kohli, RM},
title = {Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae024},
pmid = {38261989},
issn = {1362-4962},
support = {R01-GM138908/GF/NIH HHS/United States ; },
abstract = {The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.},
}
@article {pmid38261981,
year = {2024},
author = {Bigelyte, G and Duchovska, B and Zedaveinyte, R and Sasnauskas, G and Sinkunas, T and Dalgediene, I and Tamulaitiene, G and Silanskas, A and Kazlauskas, D and Valančauskas, L and Madariaga-Marcos, J and Seidel, R and Siksnys, V and Karvelis, T},
title = {Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae016},
pmid = {38261981},
issn = {1362-4962},
support = {S-MIP-21-8//Research Council of Lithuania/ ; 02-002-P-0001//Central Project Management Agency, Lithuania/ ; //Lithuanian Research Library Consortium (LMBA)/ ; },
abstract = {Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.},
}
@article {pmid38261110,
year = {2024},
author = {Shao, J and Peng, B and Zhang, Y and Yan, X and Yao, X and Hu, X and Li, L and Fu, X and Zheng, H and Tang, K},
title = {A high-efficient protoplast transient system for screening gene editing elements in Salvia miltiorrhiza.},
journal = {Plant cell reports},
volume = {43},
number = {2},
pages = {45},
pmid = {38261110},
issn = {1432-203X},
support = {2018YFA0900600//Key Technologies Research and Development Program/ ; },
mesh = {Humans ; *Salvia miltiorrhiza/genetics ; Gene Editing ; Protoplasts ; RNA, Guide, CRISPR-Cas Systems ; Cell Wall ; },
abstract = {A high-efficiency protoplast transient system was devised to screen genome editing elements in Salvia miltiorrhiza. Medicinal plants with high-value pharmaceutical ingredients have attracted research attention due to their beneficial effects on human health. Cell wall-free protoplasts of plants can be used to evaluate the efficiency of genome editing mutagenesis. The capabilities of gene editing in medicinal plants remain to be fully explored owing to their complex genetic background and shortfall of suitable transformation. Here, we took the Salvia miltiorrhiza as a representative example for developing a method to screen favorable gene editing elements with high editing efficiency in medical plants by a PEG-mediated protoplast transformation. Results indicated that using the endogenous SmU6.1 of S. miltiorrhiza to drive sgRNA and the plant codon-optimized Cas9 driven by the promoter SlEF1α can enhance the efficiency of editing. In summary, we uncover an efficacious transient method for screening editing elements and shed new light on increasing gene editing efficiency in medicinal plants.},
}
@article {pmid38260726,
year = {2023},
author = {Dixit, S and Kumar, A and Srinivasan, K and Vincent, PMDR and Ramu Krishnan, N},
title = {Advancing genome editing with artificial intelligence: opportunities, challenges, and future directions.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1335901},
pmid = {38260726},
issn = {2296-4185},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing (GED) technologies have unlocked exciting possibilities for understanding genes and improving medical treatments. On the other hand, Artificial intelligence (AI) helps genome editing achieve more precision, efficiency, and affordability in tackling various diseases, like Sickle cell anemia or Thalassemia. AI models have been in use for designing guide RNAs (gRNAs) for CRISPR-Cas systems. Tools like DeepCRISPR, CRISTA, and DeepHF have the capability to predict optimal guide RNAs (gRNAs) for a specified target sequence. These predictions take into account multiple factors, including genomic context, Cas protein type, desired mutation type, on-target/off-target scores, potential off-target sites, and the potential impacts of genome editing on gene function and cell phenotype. These models aid in optimizing different genome editing technologies, such as base, prime, and epigenome editing, which are advanced techniques to introduce precise and programmable changes to DNA sequences without relying on the homology-directed repair pathway or donor DNA templates. Furthermore, AI, in collaboration with genome editing and precision medicine, enables personalized treatments based on genetic profiles. AI analyzes patients' genomic data to identify mutations, variations, and biomarkers associated with different diseases like Cancer, Diabetes, Alzheimer's, etc. However, several challenges persist, including high costs, off-target editing, suitable delivery methods for CRISPR cargoes, improving editing efficiency, and ensuring safety in clinical applications. This review explores AI's contribution to improving CRISPR-based genome editing technologies and addresses existing challenges. It also discusses potential areas for future research in AI-driven CRISPR-based genome editing technologies. The integration of AI and genome editing opens up new possibilities for genetics, biomedicine, and healthcare, with significant implications for human health.},
}
@article {pmid38260716,
year = {2024},
author = {Asmamaw Mengstie, M and Teshome Azezew, M and Asmamaw Dejenie, T and Teshome, AA and Tadele Admasu, F and Behaile Teklemariam, A and Tilahun Mulu, A and Mekonnen Agidew, M and Adugna, DG and Geremew, H and Abebe, EC},
title = {Recent Advancements in Reducing the Off-Target Effect of CRISPR-Cas9 Genome Editing.},
journal = {Biologics : targets &amp; therapy},
volume = {18},
number = {},
pages = {21-28},
pmid = {38260716},
issn = {1177-5475},
abstract = {The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)) and the associated protein (Cas9) system, a young but well-studied genome-editing tool, holds plausible solutions to a wide range of genetic disorders. The single-guide RNA (sgRNA) with a 20-base user-defined spacer sequence and the Cas9 endonuclease form the core of the CRISPR-Cas9 system. This sgRNA can direct the Cas9 nuclease to any genomic region that includes a protospacer adjacent motif (PAM) just downstream and matches the spacer sequence. The current challenge in the clinical applications of CRISPR-Cas9 genome-editing technology is the potential off-target effects that can cause DNA cleavage at the incorrect sites. Off-target genome editing confuses and diminishes the therapeutic potential of CRISPR-Cas9 in addition to potentially casting doubt on scientific findings regarding the activities of genes. In this review, we summarize the recent technological advancements in reducing the off-target effect of CRISPR-Cas9 genome editing.},
}
@article {pmid38259970,
year = {2023},
author = {Ma, L and Zhu, M and Meng, Q and Wang, Y and Wang, X},
title = {Real-time detection of Seneca Valley virus by one-tube RPA-CRISPR/Cas12a assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1305222},
pmid = {38259970},
issn = {2235-2988},
mesh = {Animals ; Swine ; *Recombinases ; CRISPR-Cas Systems ; Nucleotidyltransferases ; *Picornaviridae ; },
abstract = {INTRODUCTION: Senecavirus A (SVA) is a highly contagious virus that causes vesicular disease in pigs. At present, laboratory detection methods, such as virus isolation and polymerase chain reaction (PCR), required precision instruments and qualified personnel, making them unsuitable for point-of-care tests (POCT). Fortunately, the emergence of CRISPR/Cas system has provided new opportunities for fast and efficient pathogen detection.<br><br>METHODS: This study successfully developed a precise and sensitive detection platform for diagnosing SVA by combining the CRISPR system with recombinase polymerase amplification (RPA).<br><br>RESULTS: The minimum detection limit of the assay was 10 copies of the SVA genome. Meanwhile, the assay demonstrated high specificity. To validate the effectiveness of this system, we tested 85 swine clinical samples and found that the fluorescence method had a 100% coincidence rate compared to RT-qPCR.<br><br>DISCUSSION: Overall, the RPA-CRISPR/Cas12a assay established in our study is a highly effective method for detecting SVA and holds great potential for practical applications in the resource-limited settings.},
}
@article {pmid38198738,
year = {2024},
author = {Yang, WW and Zhao, ML and Liu, ML and Liang, WB and Zhong, X and Zhuo, Y},
title = {Circular DNAzyme-Switched CRISPR/Cas12a Assay for Electrochemiluminescent Response of Demethylase Activity.},
journal = {ACS sensors},
volume = {9},
number = {1},
pages = {344-350},
doi = {10.1021/acssensors.3c02025},
pmid = {38198738},
issn = {2379-3694},
mesh = {*DNA, Catalytic/chemistry ; CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Silver ; DNA/chemistry ; RNA ; },
abstract = {DNA nanostructure provides powerful tools for DNA demethylase activity detection, but its stability has been significantly challenged. By virtue of circular DNA with resistance to exonuclease degradation, herein, the circular DNAzyme duplex with artificial methylated modification was constructed to identify the target and output the DNA activators to drive the CRISPR/Cas12a, constructing an "on-off-on" electrochemiluminescence (ECL) biosensor for monitoring the activity of the O[6]-methylguanine-DNA methyltransferase (MGMT). Specifically, the circular DNAzyme duplex consisted of the chimeric RNA-DNA substrate ring with double activator sequences and two single-stranded DNAzymes, whose catalytic domains were premodified with the methyl groups. When the MGMT was present, the methylated DNAzymes were repaired and restored the catalytic activity to cleave the chimeric RNA-DNA substrates, followed by the output of DNA activators to initiate the CRISPR/Cas12a. Subsequently, the ECL signals of silver nanoparticle-modified SnO2 nanospheres (Ag@SnO2) were recovered by releasing the ferrocene-labeled quenching probes (Fc-DNA) from the electrode surface because of the trans-cleavage activity of CRISPR/Cas12a, thus achieving the specific and sensitive ECL detection of MGMT from 2.5 × 10[-4] to 2.5 × 10[2] ng/mL with a low limit (9.69 × 10[-5] ng/mL). This strategy affords novel ideas and insights into research on how to project stable nucleic acid probes to detect DNA demethylases beyond traditional methods.},
}
@article {pmid38171463,
year = {2024},
author = {De Rouck, S and Mocchetti, A and Dermauw, W and Van Leeuwen, T},
title = {SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods.},
journal = {Insect biochemistry and molecular biology},
volume = {165},
number = {},
pages = {104068},
doi = {10.1016/j.ibmb.2023.104068},
pmid = {38171463},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; *Arthropods/genetics ; Gene Editing/methods ; *Tetranychidae/genetics ; },
abstract = {The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes - phytoene desaturase, CYP384A1 and Antennapedia - but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods.},
}
@article {pmid38104453,
year = {2024},
author = {Zhen, D and Zhang, S and Yang, A and Ma, Q and Deng, Z and Fang, J and Cai, Q and He, J},
title = {A supersensitive electrochemical sensor based on RCA amplification-assisted "silver chain"-linked gold interdigital electrodes and CRISPR/Cas9 for the detection of Staphylococcus aureus in food.},
journal = {Food chemistry},
volume = {440},
number = {},
pages = {138197},
doi = {10.1016/j.foodchem.2023.138197},
pmid = {38104453},
issn = {1873-7072},
mesh = {*Gold ; Staphylococcus aureus/genetics ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; *Biosensing Techniques ; Electrodes ; Limit of Detection ; Electrochemical Techniques ; },
abstract = {With the rising emphasis on food safety, technology to rapidly identify Staphylococcus aureus (S. aureus) is of great significance. Herein, we developed a novel electrochemical biosensor based on the CRISPR/Cas9 system and rolling circle amplification (RCA)-assisted "silver chain"-linked gold interdigital electrodes (Au-IDE). This sensor utilizes RCA to create DNA long chains that span the Au-IDE, and CRISPR/Cas9 as a recognition component to recognize capture/target dsDNA. Additionally, we used silver staining technology to improve detection sensitivity. Then, we detected S. aureus through impedance changes that occurred when the silver chain between the Au-IDE was connected or broke, with a limit of detection (LOD) of 7 CFU/mL and a detection time of 1.5 h. Lastly, we successfully employed this sensor to detect S. aureus in real food samples, making it a promising tool for food monitoring.},
}
@article {pmid38085648,
year = {2024},
author = {Yu, Y and Li, Q and Shi, W and Yang, Y and He, H and Dai, J and Mao, G and Ma, Y},
title = {Programmable Aptasensor for Regulating CRISPR/Cas12a Activity.},
journal = {ACS sensors},
volume = {9},
number = {1},
pages = {244-250},
doi = {10.1021/acssensors.3c01881},
pmid = {38085648},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Oligonucleotides ; DNA, Single-Stranded ; Nucleotides ; Adenosine Triphosphate ; },
abstract = {CRISPR-mediated aptasensors have gained prevalence for detecting non-nucleic acid targets. However, there is an urgent need to develop an easily customizable design to improve the signal-to-noise ratio, enhance universality, and expand the detection range. In this article, we report a CRISPR-mediated programmable aptasensor (CPAS) platform. The platform includes single-stranded DNA comprising the aptamer sequence, locker DNA, and a crRNA recognition region, forming a hairpin structure through complementary hybridization. With T4 DNA polymerase, the crRNA recognition region was transformed into a complete double-stranded DNA through stem-loop extension, thereby activating the trans-cleavage activity of Cas 12a and generating fluorescence signals. The specific binding between the target molecule and aptamer disrupted the formation of the hairpin structure, altering the fluorescence signals. Notably, the CPAS platform allows for easy customization by simply changing the aptamer sequence and locker DNA, without entailing adjustments to the crRNA. The optimal number of bases in the locker DNA was determined to be seven nucleotides for the SARS-CoV-2 spike (S) protein and four nucleotides for ATP. The CPAS platform exhibited high sensitivity for S protein and ATP detection. Integration with a lateral flow assay enabled sensitive detection within 1 h, revealing its excellent potential for portable analysis.},
}
@article {pmid38042369,
year = {2024},
author = {Katz, LS and Brill, G and Wang, P and Lambertini, L and Zhang, P and Haldeman, JM and Liu, H and Newgard, CB and Stewart, AF and Garcia-Ocaña, A and Scott, DK},
title = {Transcriptional activation of the Myc gene by glucose in β-cells requires a ChREBP-dependent 3-D chromatin interaction between the Myc and Pvt1 genes.},
journal = {Molecular metabolism},
volume = {79},
number = {},
pages = {101848},
pmid = {38042369},
issn = {2212-8778},
mesh = {Rats ; Animals ; Transcriptional Activation/genetics ; *Glucose/metabolism ; *Genes, myc ; RNA, Guide, CRISPR-Cas Systems ; Chromatin/genetics ; Transcription Factors/metabolism ; DNA ; },
abstract = {OBJECTIVE: All forms of diabetes result from insufficient functional β-cell mass. Thus, achieving the therapeutic goal of expanding β-cell mass requires a better mechanistic understanding of how β-cells proliferate. Glucose is a natural β-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood.<br><br>METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE.<br><br>RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated &#x3b2;-cell proliferation.<br><br>CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.},
}
@article {pmid38041940,
year = {2024},
author = {Shi, R and Chen, C and Zhao, S and Yuan, H and Zhao, J and Zhao, H},
title = {Stem cell therapy with CRISPR/Cas9-mediated MALAT1 delivery modulates miR-142 and rescues wound healing in rats with age-associated diabetic foot ulcers.},
journal = {Archives of gerontology and geriatrics},
volume = {118},
number = {},
pages = {105283},
doi = {10.1016/j.archger.2023.105283},
pmid = {38041940},
issn = {1872-6976},
mesh = {Rats ; Humans ; Animals ; Aged ; *Diabetic Foot/genetics/therapy ; *RNA, Long Noncoding/genetics ; *Diabetes Mellitus, Experimental/complications/genetics/pathology ; CRISPR-Cas Systems ; Quality of Life ; Wound Healing/genetics ; Stem Cell Transplantation/adverse effects ; *MicroRNAs/genetics ; },
abstract = {BACKGROUND: Diabetic foot ulcer (DFU) is a serious diabetes complication, significantly impacting the quality of life, particularly in the elderly. Age-associated DFUs pose additional challenges due to impaired healing mechanisms. Our study aims to explore the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a miR-142 sponge in repairing diabetic rat foot ulcer tissue under age-associated diabetes, offering a new theoretical basis and therapeutic target for preventing and treating diabetic vascular disease in the elderly.<br><br>METHODS: Using qPCR, we analyzed MALAT1 and miR-142 expression in EPCs and hUC-MSCs. Targetscan predicted potential interaction targets for MALAT1 and miR-142, confirmed by dual luciferase reporter gene assay. An age-associated diabetic rat model was established using Streptozotocin (STZ) injection. Hypoxia, apoptosis, and angiogenesis-related proteins were assessed through Western Blot. In vitro, miR-142 inhibition and MALAT1 overexpression promoted foot ulcer healing in diabetic rats.<br><br>RESULTS: MALAT1 acted as a miR-142 sponge, downregulated in hUC-MSCs under high glucose, relevant to age-associated diabetic foot ulcers. MiR-142 negatively regulated SIRT1 and Nrf2. In vitro experiments demonstrated potential significance for age-related DFU treatment.<br><br>CONCLUSIONS: MALAT1 in human umbilical cord mesenchymal stem cells expedited foot ulcer healing in diabetic rats, particularly in age-associated diabetes, through miR-142 sponge activity. These findings offer insights for novel therapeutic strategies targeting elderly diabetic foot ulcers, emphasizing exogenous stem cell transplantation's potential in effective DFU treatment for the elderly.},
}
@article {pmid38032126,
year = {2024},
author = {Sun, L and Alariqi, M and Wang, Y and Wang, Q and Xu, Z and Zafar, MN and Yang, G and Jia, R and Hussain, A and Chen, Y and Ding, X and Zhou, J and Wang, G and Wang, F and Li, J and Zou, J and Zhu, X and Yu, L and Sun, Y and Liang, S and Hui, F and Chen, L and Guo, W and Wang, Y and Zhu, H and Lindsey, K and Nie, X and Zhang, X and Jin, S},
title = {Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {4},
pages = {e2306157},
pmid = {38032126},
issn = {2198-3844},
support = {32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; 2022ZD0402001-04//Science and Technology Innovation 2030/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; 32272128//National Natural Science Fund of China/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Insecta ; },
abstract = {Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca[2+] ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca[2+] and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.},
}
@article {pmid37849226,
year = {2024},
author = {Li, Z and Han, Z},
title = {Advancements of the CRISPR/Cas9 System in the Treatment of Liver Cancer.},
journal = {Current protein &amp; peptide science},
volume = {25},
number = {2},
pages = {154-162},
doi = {10.2174/0113892037238265231006051215},
pmid = {37849226},
issn = {1875-5550},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; *Liver Neoplasms/genetics/therapy ; },
abstract = {In recent years, the CRISPR/Cas9 system has become a rapidly advancing gene editing technology with significant advantages in various fields, particularly biomedicine. Liver cancer is a severe malignancy that threatens human health and is primarily treated with surgery, radiotherapy, and chemotherapy. However, surgery may not be suitable for advanced cases of liver cancer with distant metastases. Moreover, radiotherapy and chemotherapy have low specificity and numerous side effects that limit their effectiveness; therefore, more effective and safer treatments are required. With the advancement of the biomolecular mechanism of cancer, CRISPR/Cas9 gene editing technology has been widely used in the study of liver cancer to gain insights into gene functions, establish tumor models, screen tumor phenotype-related genes, and perform gene therapy. This review outlines the research progress of CRISPR/Cas9 gene editing technology in the treatment of liver cancer and provides a relevant theoretical basis for its research and application in the treatment of liver cancer.},
}
@article {pmid37733908,
year = {2024},
author = {Hattori, T and Fundora, KA and Hamamoto, K and Opozda, DM and Liang, X and Liu, X and Zhang, J and Uzun, Y and Takahashi, Y and Wang, HG},
title = {ER stress elicits non-canonical CASP8 (caspase 8) activation on autophagosomal membranes to induce apoptosis.},
journal = {Autophagy},
volume = {20},
number = {2},
pages = {349-364},
pmid = {37733908},
issn = {1554-8635},
support = {R01 CA222349/CA/NCI NIH HHS/United States ; R01 GM127954/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Caspase 8/genetics ; *NF-kappa B/metabolism ; Autophagy ; RNA, Guide, CRISPR-Cas Systems ; Apoptosis/genetics ; Endoplasmic Reticulum Stress ; Endosomal Sorting Complexes Required for Transport/metabolism ; *Neoplasms ; },
abstract = {The VPS37A gene encodes a subunit of the endosomal sorting complex required for transport (ESCRT)-I complex that is frequently lost in a wide variety of human solid cancers. We have previously demonstrated the role of VPS37A in directing the ESCRT membrane scission machinery to seal the phagophore for autophagosome completion. Here, we report that VPS37A-deficient cells exhibit an accumulation of the apoptotic initiator CASP8 (caspase 8) on the phagophore and are primed to undergo rapid apoptosis through the intracellular death-inducing signaling complex (iDISC)-mediated CASP8 activation upon exposure to endoplasmic reticulum (ER) stress. Using CRISPR-Cas9 gene editing and comparative transcriptome analysis, we identified the ATF4-mediated stress response pathway as a crucial mediator to elicit iDISC-mediated apoptosis following the inhibition of autophagosome closure. Notably, ATF4-mediated iDISC activation occurred independently of the death receptor TNFRSF10B/DR5 upregulation but required the pro-apoptotic transcriptional factor DDIT3/CHOP to enhance the mitochondrial amplification pathway for full-activation of CASP8 in VPS37A-deficient cells stimulated with ER stress inducers. Our analysis also revealed the upregulation of NFKB/NF-kB signaling as a potential mechanism responsible for restraining iDISC activation and promoting cell survival upon VPS37A depletion. These findings have important implications for the future development of new strategies to treat human cancers, especially those with VPS37A loss.Abbreviations: ATG: autophagy related; BMS: BMS-345541; CASP: caspase; CHMP: charged multivesicular body protein; DKO: double knockout; Dox: doxycycline; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; gRNA: guide RNA; GSEA: gene set enrichment analysis; GSK157: GSK2656157; iDISC: intracellular death-inducing signaling complex; IKK: inhibitor of NFKB kinase; IPA: ingenuity pathway analysis; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NFKB/NF-kB: nuclear factor kappa B; OZ: 5Z-7-oxozeaenol; RNA-seq: RNA sequencing; UPR: unfolded protein response; TFT: transcription factor target; THG: thapsigargin; TUN: tunicamycin; VPS: vacuolar protein sorting.},
}
@article {pmid37690471,
year = {2024},
author = {Nikitchina, N and Ulashchik, E and Shmanai, V and Heckel, AM and Tarassov, I and Mazunin, I and Entelis, N},
title = {Targeting of CRISPR-Cas12a crRNAs into human mitochondria.},
journal = {Biochimie},
volume = {217},
number = {},
pages = {74-85},
doi = {10.1016/j.biochi.2023.09.006},
pmid = {37690471},
issn = {1638-6183},
mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Mitochondria/genetics ; DNA, Mitochondrial/genetics ; *Mitochondrial Diseases/genetics ; },
abstract = {Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3'-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.},
}
@article {pmid37561581,
year = {2023},
author = {Kabra, M and Shahi, PK and Wang, Y and Sinha, D and Spillane, A and Newby, GA and Saxena, S and Tong, Y and Chang, Y and Abdeen, AA and Edwards, KL and Theisen, CO and Liu, DR and Gamm, DM and Gong, S and Saha, K and Pattnaik, BR},
title = {Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy.},
journal = {The Journal of clinical investigation},
volume = {133},
number = {19},
pages = {},
pmid = {37561581},
issn = {1558-8238},
support = {R24 EY032434/EY/NEI NIH HHS/United States ; R01 EY024995/EY/NEI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; S10 OD026957/OD/NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; P30 EY016665/EY/NEI NIH HHS/United States ; },
mesh = {Mice ; Animals ; Humans ; Child ; Gene Editing ; *Channelopathies/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells ; Retina ; Retinal Pigment Epithelium ; Mutation ; RNA, Messenger ; },
abstract = {Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors' delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+ΔR), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders.},
}
@article {pmid38259920,
year = {2023},
author = {Prado, GS and Rocha, DC and Dos Santos, LN and Contiliani, DF and Nobile, PM and Martinati-Schenk, JC and Padilha, L and Maluf, MP and Lubini, G and Pereira, TC and Monteiro-Vitorello, CB and Creste, S and Boscariol-Camargo, RL and Takita, MA and Cristofani-Yaly, M and de Souza, AA},
title = {CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1331258},
pmid = {38259920},
issn = {1664-462X},
abstract = {Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.},
}
@article {pmid38259224,
year = {2024},
author = {Li, B and Zhai, G and Dong, Y and Wang, L and Ma, P},
title = {Recent progress on the CRISPR/Cas system in optical biosensors.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3ay02147e},
pmid = {38259224},
issn = {1759-9679},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are adaptive immune systems unique to archaea and bacteria, with the characteristics of targeted recognition and gene editing to resist the invasion of foreign nucleic acids. Biosensors combined with the CRISPR/Cas system and optical detection technology have attracted much attention in medical diagnoses, food safety, agricultural progress, and environmental monitoring owing to their good sensitivity, high selectivity, and fast detection efficiency. In this review, we introduce the mechanism of CRISPR/Cas systems and developments in this area, followed by summarizing recent progress on CRISPR/Cas system-based optical biosensors combined with colorimetric, fluorescence, electrochemiluminescence and surface-enhanced Raman scattering optical techniques in various fields. Finally, we discuss the challenges and future perspectives of CRISPR/Cas systems in optical biosensors.},
}
@article {pmid38257946,
year = {2024},
author = {Li, J and Wu, S and Zhang, K and Sun, X and Lin, W and Wang, C and Lin, S},
title = {Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects.},
journal = {Microorganisms},
volume = {12},
number = {1},
pages = {},
pmid = {38257946},
issn = {2076-2607},
support = {4980.01//Gordon and Betty Moore Foundation/ ; 42276096//National Natural Science Foundation of China/ ; 2022YFC3105301//National Key Research and Development Program of China/ ; 2022YFC3102003//National Key Research and Development Program of China/ ; 422QN265//Natural Science Foundation of Hainan Province/ ; },
abstract = {Initially discovered over 35 years ago in the bacterium Escherichia coli as a defense system against invasion of viral (or other exogenous) DNA into the genome, CRISPR/Cas has ushered in a new era of functional genetics and served as a versatile genetic tool in all branches of life science. CRISPR/Cas has revolutionized the methodology of gene knockout with simplicity and rapidity, but it is also powerful for gene knock-in and gene modification. In the field of marine biology and ecology, this tool has been instrumental in the functional characterization of 'dark' genes and the documentation of the functional differentiation of gene paralogs. Powerful as it is, challenges exist that have hindered the advances in functional genetics in some important lineages. This review examines the status of applications of CRISPR/Cas in marine research and assesses the prospect of quickly expanding the deployment of this powerful tool to address the myriad fundamental marine biology and biological oceanography questions.},
}
@article {pmid38257926,
year = {2024},
author = {Jia, J and Lu, SE},
title = {Comparative Genome Analyses Provide Insight into the Antimicrobial Activity of Endophytic Burkholderia.},
journal = {Microorganisms},
volume = {12},
number = {1},
pages = {},
pmid = {38257926},
issn = {2076-2607},
support = {MIS-401260//United States Department of Agriculture/ ; },
abstract = {Endophytic bacteria are endosymbionts that colonize a portion of plants without harming the plant for at least a part of its life cycle. Bacterial endophytes play an essential role in promoting plant growth using multiple mechanisms. The genus Burkholderia is an important member among endophytes and encompasses bacterial species with high genetic versatility and adaptability. In this study, the endophytic characteristics of Burkholderia species are investigated via comparative genomic analyses of several endophytic Burkholderia strains with pathogenic Burkholderia strains. A group of bacterial genes was identified and predicted as the putative endophytic behavior genes of Burkholderia. Multiple antimicrobial biosynthesis genes were observed in these endophytic bacteria; however, certain important pathogenic and virulence genes were absent. The majority of resistome genes were distributed relatively evenly among the endophytic and pathogenic bacteria. All known types of secretion systems were found in the studied bacteria. This includes T3SS and T4SS, which were previously thought to be disproportionately represented in endophytes. Additionally, questionable CRISPR-Cas systems with an orphan CRISPR array were prevalent, suggesting that intact CRISPR-Cas systems may not exist in symbiotes of Burkholderia. This research not only sheds light on the antimicrobial activities that contribute to biocontrol but also expands our understanding of genomic variations in Burkholderia's endophytic and pathogenic bacteria.},
}
@article {pmid38256875,
year = {2023},
author = {Hasselbeck, S and Cheng, X},
title = {Molecular Marvels: Small Molecules Paving the Way for Enhanced Gene Therapy.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {17},
number = {1},
pages = {},
doi = {10.3390/ph17010041},
pmid = {38256875},
issn = {1424-8247},
abstract = {In the rapidly evolving landscape of genetic engineering, the advent of CRISPR-Cas technologies has catalyzed a paradigm shift, empowering scientists to manipulate the genetic code with unprecedented accuracy and efficiency. Despite the remarkable capabilities inherent to CRISPR-Cas systems, recent advancements have witnessed the integration of small molecules to augment their functionality, introducing new dimensions to the precision and versatility of gene editing applications. This review delves into the synergy between CRISPR-Cas technologies based specifically on Cas9 and small-molecule drugs, elucidating the pivotal role of chemicals in optimizing target specificity and editing efficiency. By examining a diverse array of applications, ranging from therapeutic interventions to agricultural advancements, we explore how the judicious use of chemicals enhances the precision of CRISPR-Cas9-mediated genetic modifications. In this review, we emphasize the significance of small-molecule drugs in fine-tuning the CRISPR-Cas9 machinery, which allows researchers to exert meticulous control over the editing process. We delve into the mechanisms through which these chemicals bolster target specificity, mitigate off-target effects, and contribute to the overall refinement of gene editing outcomes. Additionally, we discuss the potential of chemical integration in expanding the scope of CRISPR-Cas9 technologies, enabling tailored solutions for diverse genetic manipulation challenges. As CRISPR-Cas9 technologies continue to evolve, the integration of small-molecule drugs emerges as a crucial avenue for advancing the precision and applicability of gene editing techniques. This review not only synthesizes current knowledge but also highlights future prospects, paving the way for a deeper understanding of the synergistic interplay between CRISPR-Cas9 systems and chemical modulators in the pursuit of more controlled and efficient genetic modifications.},
}
@article {pmid38256391,
year = {2024},
author = {Shabbir, MAB and Ul-Rahman, A and Iftikhar, MR and Rasheed, M and Maan, MK and Sattar, A and Ahmad, M and Khan, FA and Ahmad, W and Riaz, MI and Aslam, HB},
title = {Exploring the Interplay of the CRISPR-CAS System with Antibiotic Resistance in Staphylococcus aureus: A Poultry Meat Study from Lahore, Pakistan.},
journal = {Medicina (Kaunas, Lithuania)},
volume = {60},
number = {1},
pages = {},
doi = {10.3390/medicina60010130},
pmid = {38256391},
issn = {1648-9144},
mesh = {Humans ; Animals ; Pakistan ; *Staphylococcus aureus/genetics ; CRISPR-Cas Systems/genetics ; Phylogeny ; Poultry ; *Staphylococcal Infections ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Microbial/genetics ; },
abstract = {Staphylococcus aureus is one of the major pathogens responsible for causing food poisoning worldwide. The emergence of antibiotic resistance in this bacterium is influenced by various factors. Among them, bacterial acquired defense systems described as clustered regularly interspaced short palindromic repeats (CRISPR)-cas system might be involved in antibiotic resistance development in bacteria. The current study was designed to assess the prevalence of S. aureus and its antibiotic resistance profile and identify the relationship of the CRISPR-cas system with antimicrobial resistance, followed by phylogenetic analysis. Total samples (n = 188) of poultry meat were collected from the poultry bird market of Lahore, Punjab, Pakistan. We used both phenotypic (antibiotic disc diffusion) and genotypic methods (PCR) to identify multi-drug resistant (MDR) strains of S. aureus. Additionally, the role of the CRISPR-Cas system in the isolated MDR S. aureus was also assessed. In addition, real-time quantitative PCR (qRT-PCR) was used to evaluate the association of the CRISPR-cas system with antimicrobial resistance. All of the S. aureus isolates showed 100% resistance against erythromycin, 97.5% were resistant to tetracycline, and 75% were resistant to methicillin. Eleven isolates were MDR in the current study. The CRISPR system was found in all MDR isolates, and fifteen spacers were identified within the CRISPR locus. Furthermore, MDR S. aureus isolates and the standard strain showed higher expression levels of CRISPR-associated genes. The correlation of said system with MDR isolates points to foreign gene acquisition by horizontal transfer. Current knowledge could be utilized to tackle antibiotic-resistant bacteria, mainly S. aureus.},
}
@article {pmid38256061,
year = {2024},
author = {Hammad, R and Alzubi, J and Rhiel, M and Chmielewski, KO and Mosti, L and Rositzka, J and Heugel, M and Lawrenz, J and Pennucci, V and Gläser, B and Fischer, J and Schambach, A and Moritz, T and Lachmann, N and Cornu, TI and Mussolino, C and Schäfer, R and Cathomen, T},
title = {CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.},
journal = {International journal of molecular sciences},
volume = {25},
number = {2},
pages = {},
doi = {10.3390/ijms25020985},
pmid = {38256061},
issn = {1422-0067},
support = {01EK1602 (iMACnet)//Federal Ministry of Education and Research/ ; Open Access Publication Fund//freiburg/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Pluripotent Stem Cells ; *Induced Pluripotent Stem Cells ; Hematopoietic Stem Cells ; Alleles ; },
abstract = {The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.},
}
@article {pmid38093011,
year = {2024},
author = {Schmidt, R and Ward, CC and Dajani, R and Armour-Garb, Z and Ota, M and Allain, V and Hernandez, R and Layeghi, M and Xing, G and Goudy, L and Dorovskyi, D and Wang, C and Chen, YY and Ye, CJ and Shy, BR and Gilbert, LA and Eyquem, J and Pritchard, JK and Dodgson, SE and Marson, A},
title = {Base-editing mutagenesis maps alleles to tune human T cell functions.},
journal = {Nature},
volume = {625},
number = {7996},
pages = {805-812},
pmid = {38093011},
issn = {1476-4687},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Alleles ; RNA, Guide, CRISPR-Cas Systems ; T-Lymphocytes ; Mutagenesis/genetics ; Amino Acids/genetics ; },
abstract = {CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies[1-6]. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency[7,8]. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements[9] (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.},
}
@article {pmid38048327,
year = {2024},
author = {Lu, X and Xiao, J and Wang, L and Zhu, B and Huang, F},
title = {The nuclease-associated short prokaryotic Argonaute system nonspecifically degrades DNA upon activation by target recognition.},
journal = {Nucleic acids research},
volume = {52},
number = {2},
pages = {844-855},
pmid = {38048327},
issn = {1362-4962},
support = {32150009//National Natural Science Foundation of China/ ; JCYJ20210324115811032//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; DNA/genetics/metabolism ; Prokaryotic Cells/metabolism ; *Nucleic Acids/metabolism ; RNA/metabolism ; Endonucleases/metabolism ; Argonaute Proteins/metabolism ; },
abstract = {Prokaryotic Argonautes (pAgos) play a vital role in host defense by utilizing short nucleic acid guides to recognize and target complementary nucleic acids. Despite being the majority of pAgos, short pAgos have only recently received attention. Short pAgos are often associated with proteins containing an APAZ domain and a nuclease domain including DUF4365, SMEK, or HNH domain. In contrast to long pAgos that specifically cleave the target DNA, our study demonstrates that the short pAgo from Thermocrispum municipal, along with its associated DUF4365-APAZ protein, forms a heterodimeric complex. Upon RNA-guided target DNA recognition, this complex is activated to nonspecifically cleave DNA. Additionally, we found that the TmuRE-Ago complex shows a preference for 5'-OH guide RNA, specifically requires a uridine nucleotide at the 5' end of the guide RNA, and is sensitive to single-nucleotide mismatches between the guide RNA and target DNA. Based on its catalytic properties, our study has established a novel nucleic acid detection method and demonstrated its feasibility. This study not only expands our understanding of the defense mechanism employed by short pAgo systems but also suggests their potential applications in nucleic acid detection.},
}
@article {pmid38033325,
year = {2024},
author = {Zhang, H and Kelly, K and Lee, J and Echeverria, D and Cooper, D and Panwala, R and Amrani, N and Chen, Z and Gaston, N and Wagh, A and Newby, GA and Xie, J and Liu, DR and Gao, G and Wolfe, SA and Khvorova, A and Watts, JK and Sontheimer, EJ},
title = {Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.},
journal = {Nucleic acids research},
volume = {52},
number = {2},
pages = {977-997},
pmid = {38033325},
issn = {1362-4962},
support = {UG3AI150551/NH/NIH HHS/United States ; UH3 TR002668/TR/NCATS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; S10 OD020012/CD/ODCDC CDC HHS/United States ; UG3AI150551/NH/NIH HHS/United States ; UH3 TR002668/TR/NCATS NIH HHS/United States ; S10 OD020012/CD/ODCDC CDC HHS/United States ; },
mesh = {Animals ; Mice ; *Gene Editing ; *RNA, Guide, CRISPR-Cas Systems ; Tissue Distribution ; RNA/genetics ; Oligonucleotides ; },
abstract = {Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.},
}
@article {pmid38033324,
year = {2024},
author = {Molina Vargas, AM and Sinha, S and Osborn, R and Arantes, PR and Patel, A and Dewhurst, S and Hardy, DJ and Cameron, A and Palermo, G and O'Connell, MR},
title = {New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity.},
journal = {Nucleic acids research},
volume = {52},
number = {2},
pages = {921-939},
pmid = {38033324},
issn = {1362-4962},
support = {R35 GM133462/GM/NIGMS NIH HHS/United States ; R35GM133462/NH/NIH HHS/United States ; R35GM133462/NH/NIH HHS/United States ; },
mesh = {*RNA/genetics ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; },
abstract = {An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.},
}
@article {pmid38008286,
year = {2024},
author = {Tachikawa, K and Shimizu, T and Imai, T and Ko, R and Kawai, Y and Omae, Y and Tokunaga, K and Frith, MC and Yamano, Y and Mitsuhashi, S},
title = {Cost-Effective Cas9-Mediated Targeted Sequencing of Spinocerebellar Ataxia Repeat Expansions.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {26},
number = {2},
pages = {85-95},
doi = {10.1016/j.jmoldx.2023.10.004},
pmid = {38008286},
issn = {1943-7811},
mesh = {Humans ; *CRISPR-Cas Systems ; Cost-Benefit Analysis ; *Spinocerebellar Ataxias/diagnosis/genetics ; Microsatellite Repeats/genetics ; Whole Genome Sequencing ; High-Throughput Nucleotide Sequencing ; },
abstract = {Hereditary repeat diseases are caused by an abnormal expansion of short tandem repeats in the genome. Among them, spinocerebellar ataxia (SCA) is a heterogeneous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. This study developed a cost-effective SCA repeat panel [Flongle SCA repeat panel sequencing (FLO-SCAp)] using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, Flongle. This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) values of coverage and on-target rate were 39.5 (12 to 72) and 11.6% (7.5% to 16.5%), respectively. This approach was validated by comparing repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing. A high correlation was observed between FLO-SCAp and short-read whole-genome sequencing when the repeat length was ≤250 bp (r = 0.98; P < 0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.},
}
@article {pmid37994786,
year = {2024},
author = {Weischedel, J and Higgins, L and Rogers, S and Gramalla-Schmitz, A and Wyrzykowska, P and Borgoni, S and MacCarthy, T and Chahwan, R},
title = {Modular cytosine base editing promotes epigenomic and genomic modifications.},
journal = {Nucleic acids research},
volume = {52},
number = {2},
pages = {e8},
pmid = {37994786},
issn = {1362-4962},
support = {310030_212553//SNF/ ; 22B140//Novartis Foundation/ ; 41309//Vontobel Stiftung/ ; F-41309-01-01//UZH/ ; //UZH-URPP/ ; //Translational Cancer Research/ ; //Swiss Excellence Scholarship/ ; //Functional Genomics Center Zurich/ ; //ETH Zurich/ ; },
mesh = {*Gene Editing ; *Epigenomics ; Cytosine/chemistry ; RNA/genetics ; Cytosine Deaminase/genetics ; CRISPR-Cas Systems ; },
abstract = {Prokaryotic and eukaryotic adaptive immunity differ considerably. Yet, their fundamental mechanisms of gene editing via Cas9 and activation-induced deaminase (AID), respectively, can be conveniently complimentary. Cas9 is an RNA targeted dual nuclease expressed in several bacterial species. AID is a cytosine deaminase expressed in germinal centre B cells to mediate genomic antibody diversification. AID can also mediate epigenomic reprogramming via active DNA demethylation. It is known that sequence motifs, nucleic acid structures, and associated co-factors affect AID activity. But despite repeated attempts, deciphering AID's intrinsic catalytic activities and harnessing its targeted recruitment to DNA is still intractable. Even recent cytosine base editors are unable to fully recapitulate AID's genomic and epigenomic editing properties. Here, we describe the first instance of a modular AID-based editor that recapitulates the full spectrum of genomic and epigenomic editing activity. Our 'Swiss army knife' toolbox will help better understand AID biology per se as well as improve targeted genomic and epigenomic editing.},
}
@article {pmid37783228,
year = {2024},
author = {Gao, X and Shang, K and Zhu, K and Wang, L and Mu, Z and Fu, X and Yu, X and Qin, B and Zhu, H and Ding, W and Cui, S},
title = {Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute.},
journal = {Nature},
volume = {625},
number = {7996},
pages = {822-831},
pmid = {37783228},
issn = {1476-4687},
mesh = {*Nucleic Acids ; NAD+ Nucleosidase/metabolism ; Cryoelectron Microscopy ; RNA, Guide, CRISPR-Cas Systems ; DNA/metabolism ; RNA ; Argonaute Proteins/metabolism ; },
abstract = {Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids[1,2]. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA-target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA-DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a 'roadblock' that prevents the guide RNA 5' ends and the target DNA 3' ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA-DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.},
}
@article {pmid37673833,
year = {2024},
author = {Amanzholova, M and Shaizadinova, A and Bulashev, A and Abeldenov, S},
title = {Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay.},
journal = {Veterinary research communications},
volume = {48},
number = {1},
pages = {291-300},
pmid = {37673833},
issn = {1573-7446},
support = {AP09259771//Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan/ ; BR10764944//Ministry of Agriculture of the Republic of Kazakhstan/ ; },
mesh = {Cattle ; Female ; Animals ; Staphylococcus aureus/genetics ; *Mastitis, Bovine/diagnosis ; Milk ; CRISPR-Cas Systems ; *Cattle Diseases/genetics ; },
abstract = {Bovine mastitis, a common and costly disease in dairy cattle, is primarily caused by Staphylococcus aureus. Timely and accurate detection of this pathogen is crucial for effective disease management. In this study, we developed and validated a novel molecular diagnostic assay based on the CRISPR/Cas12a system coupled with Recombinase Polymerase Amplification (RPA) and Loop-Mediated Isothermal Amplification (LAMP). We utilized specific primers targeting the nucleotide sequences of the S.aureus genes of interest, such as nuc and sea. RPA/LAMP reactions were performed under optimized conditions, and the resulting products were subsequently subjected to CRISPR/Cas12a detection. The CRISPR/Cas12a assay successfully detected the target nuc and sea genes, with a limit of detection of 10[4] and 10[2] gene copies per reaction, respectively. All 13 S.aureus clinical isolates were identified by RPA-CRISPR/Cas12a assay. The total reaction time is approximately 1 h. The assay demonstrated high sensitivity for the detection of S.aureus in both laboratory and clinical samples.},
}
@article {pmid38255897,
year = {2024},
author = {Shumega, AR and Pavlov, YI and Chirinskaite, AV and Rubel, AA and Inge-Vechtomov, SG and Stepchenkova, EI},
title = {CRISPR/Cas9 as a Mutagenic Factor.},
journal = {International journal of molecular sciences},
volume = {25},
number = {2},
pages = {},
doi = {10.3390/ijms25020823},
pmid = {38255897},
issn = {1422-0067},
support = {20-14-00148-&#x41f;//Russian Science Foundation/ ; },
mesh = {*Mutagens ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Mutagenesis/genetics ; Mutation ; },
abstract = {The discovery of the CRISPR/Cas9 microbial adaptive immune system has revolutionized the field of genetics, by greatly enhancing the capacity for genome editing. CRISPR/Cas9-based editing starts with DNA breaks (or other lesions) predominantly at target sites and, unfortunately, at off-target genome sites. DNA repair systems differing in accuracy participate in establishing desired genetic changes but also introduce unwanted mutations, that may lead to hereditary, oncological, and other diseases. New approaches to alleviate the risks associated with genome editing include attenuating the off-target activity of editing complex through the use of modified forms of Cas9 nuclease and single guide RNA (sgRNA), improving delivery methods for sgRNA/Cas9 complex, and directing DNA lesions caused by the sgRNA/Cas9 to non-mutagenic repair pathways. Here, we have described CRISPR/Cas9 as a new powerful mutagenic factor, discussed its mutagenic properties, and reviewed factors influencing the mutagenic activity of CRISPR/Cas9.},
}
@article {pmid38255224,
year = {2024},
author = {Kruglova, N and Shepelev, M},
title = {Increasing Gene Editing Efficiency via CRISPR/Cas9- or Cas12a-Mediated Knock-In in Primary Human T Cells.},
journal = {Biomedicines},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/biomedicines12010119},
pmid = {38255224},
issn = {2227-9059},
support = {22-15-00381//Russian Science Foundation/ ; 075-15-2019-1661//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {T lymphocytes represent a promising target for genome editing. They are primarily modified to recognize and kill tumor cells or to withstand HIV infection. In most studies, T cell genome editing is performed using the CRISPR/Cas technology. Although this technology is easily programmable and widely accessible, its efficiency of T cell genome editing was initially low. Several crucial improvements were made in the components of the CRISPR/Cas technology and their delivery methods, as well as in the culturing conditions of T cells, before a reasonable editing level suitable for clinical applications was achieved. In this review, we summarize and describe the aforementioned parameters that affect human T cell editing efficiency using the CRISPR/Cas technology, with a special focus on gene knock-in.},
}
@article {pmid38254824,
year = {2024},
author = {Harfmann, M and Schröder, T and Głów, D and Jung, M and Uhde, A and Kröger, N and Horn, S and Riecken, K and Fehse, B and Ayuk, FA},
title = {CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture.},
journal = {Cancers},
volume = {16},
number = {2},
pages = {},
doi = {10.3390/cancers16020334},
pmid = {38254824},
issn = {2072-6694},
abstract = {BACKGROUND: CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment.<br><br>METHODS: CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45[ko] effector cells, CD45[ko]/CD45CAR-T and CD45[ko]/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays.<br><br>RESULTS: As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45[ko]/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45[ko]/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45[ko] cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45[ko]/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro.<br><br>CONCLUSIONS: We report the efficient production of highly and durably active CD45[ko]/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45[ko]/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.},
}
@article {pmid38254649,
year = {2023},
author = {Oscorbin, IP and Filipenko, ML},
title = {A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of Anoxybacillus flavithermus.},
journal = {Biomolecules},
volume = {14},
number = {1},
pages = {},
doi = {10.3390/biom14010049},
pmid = {38254649},
issn = {2218-273X},
support = {22-24-01136//Russian Science Foundation/ ; },
mesh = {*RNA-Directed DNA Polymerase/genetics ; Introns/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Anoxybacillus ; },
abstract = {Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, Anoxybacillus flavithermus, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn[2+] ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs.},
}
@article {pmid38091988,
year = {2024},
author = {Nuttle, X and Burt, ND and Currall, B and Moysés-Oliveira, M and Mohajeri, K and Bhavsar, R and Lucente, D and Yadav, R and Tai, DJC and Gusella, JF and Talkowski, ME},
title = {Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries.},
journal = {Cell reports methods},
volume = {4},
number = {1},
pages = {100672},
doi = {10.1016/j.crmeth.2023.100672},
pmid = {38091988},
issn = {2667-2375},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Mutation ; *Induced Pluripotent Stem Cells ; Genomics ; },
abstract = {New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.},
}
@article {pmid38253589,
year = {2024},
author = {Yu, J and Shin, J and Yu, J and Kim, J and Yu, D and Heo, WD},
title = {Programmable RNA base editing with photoactivatable CRISPR-Cas13.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {673},
pmid = {38253589},
issn = {2041-1723},
mesh = {Animals ; Mice ; *RNA/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; RNA Editing/genetics ; RNA Interference ; Mammals ; },
abstract = {CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.},
}
@article {pmid38252823,
year = {2024},
author = {Yang, DD and Rusch, LM and Widney, KA and Morgenthaler, AB and Copley, SD},
title = {Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {5},
pages = {e2316834121},
doi = {10.1073/pnas.2316834121},
pmid = {38252823},
issn = {1091-6490},
support = {R01GM124365//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Library ; Carbon ; Acetates ; },
abstract = {CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.},
}
@article {pmid38251733,
year = {2024},
author = {Kong, H and Yi, K and Mintz, RL and Wang, B and Xu, Y and Lao, YH and Tao, Y and Li, M},
title = {CRISPR/Cas detection with nanodevices: moving deeper into liquid biopsy.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3cc05375j},
pmid = {38251733},
issn = {1364-548X},
abstract = {The emerging field of liquid biopsy has garnered significant interest in precision diagnostics, offering a non-invasive and repetitive method for analyzing bodily fluids to procure real-time diagnostic data. The precision and accuracy offered by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) technology have advanced and broadened the applications of liquid biopsy. Significantly, when combined with swiftly advancing nanotechnology, CRISPR/Cas-mediated nanodevices show vast potential in precise liquid biopsy applications. However, persistent challenges are still associated with off-target effects, and the current platforms also constrain the performance of the assays. In this review, we highlight the merits of CRISPR/Cas systems in liquid biopsy, tracing the development of CRISPR/Cas systems and their current applications in disease diagnosis particularly in liquid biopsies. We also outline ongoing efforts to design nanoscale devices with improved sensing and readout capabilities, aiming to enhance the performance of CRISPR/Cas detectors in liquid biopsy. Finally, we identify the critical obstacles hindering the widespread adoption of CRISPR/Cas liquid biopsy and explore potential solutions. This feature article presents a comprehensive overview of CRISPR/Cas-mediated liquid biopsies, emphasizing the progress in integrating nanodevices to improve specificity and sensitivity. It also sheds light on future research directions in employing nanodevices for CRISPR/Cas-based liquid biopsies in the realm of precision medicine.},
}
@article {pmid38249803,
year = {2023},
author = {Zhang, Y and Yu, W and Wang, M and Zhang, L and Li, P},
title = {Nanozyme-assisted amplification-free CRISPR/Cas system realizes visual detection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1327498},
pmid = {38249803},
issn = {2296-4185},
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated) system has proven to be a powerful tool for nucleic acid detection due to its inherent advantages of effective nucleic acid identification and editing capabilities, and is therefore known as the next-generation of molecular diagnostic technology. However, the detection technologies based on CRISPR/Cas systems require preamplification of target analytes; that is, target gene amplification steps through isothermal amplification or PCR before detection to increase target analyte concentrations. This creates a number of testing limitations, such as extended testing time and the need for more sophisticated testing instruments. To overcome the above limitations, various amplification-free assay strategies based on CRISPR/Cas systems have been explored as alternatives, which omit the preamplification step to increase the concentrations of the target analytes. Nanozymes play a pivotal role in enhancing the sensitivity of CRISPR-based detection, enabling visual and rapid CRISPR assays. The utilization of nanozyme exceptional enzyme-like catalytic activity holds great promise for signal amplification in both electrochemical and optical domains, encompassing strategies for electrochemical signal sensors and colorimetric signal sensors. Rather than relying on converting a single detection target analyte into multiple analytes, these methods focus on signal amplification, the main mechanism of which involves the ability to form a large number of reporter molecules or to improve the performance of the sensor. This exploitation of nanozymes for signal amplification results in the heightened sensitivity and accuracy of detection outcomes. In addition to the strategies that improve sensor performance through the application of nanozymes, additional methods are needed to achieve visual signal amplification strategies without preamplification processes. Herein, we review the strategies for improving CRISPR/Cas systems that do not require preamplification, providing a simple, intuitive and preamplification-free CRISPR/Cas system detection platform by improving in-system one-step amplification programs, or enhancing nanozyme-mediated signal amplification strategies.},
}
@article {pmid38248424,
year = {2024},
author = {Kim, Y and Nam, D and Lee, ES and Kim, S and Cha, BS and Park, KS},
title = {Aptamer-Based Switching System for Communication of Non-Interacting Proteins.},
journal = {Biosensors},
volume = {14},
number = {1},
pages = {},
doi = {10.3390/bios14010047},
pmid = {38248424},
issn = {2079-6374},
support = {NRF-2020R1C1C1012275//National Research Foundation of Korea/ ; LCA Experts Training Graduate Program for Knowledge-based Environmental Service//Ministry of Environment, Korea/ ; },
mesh = {*Oligonucleotides ; *Antibodies ; Communication ; RNA ; Reaction Time ; },
abstract = {Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. In this study, we devised an aptamer-based switching system that enables communication between non-interacting proteins. As a proof of concept, two proteins, Cas13a and T7 RNA polymerase (T7 RNAP), were rationally connected using an aptamer that specifically binds to T7 RNAP. The proposed switching system can be modulated in both signal-on and signal-off manners and its responsiveness to the target activator can be controlled by adjusting the reaction time. This study paves the way for the expansion of biological networks by mediating interactions between proteins using aptamers.},
}
@article {pmid38248403,
year = {2024},
author = {Park, DH and Haizan, I and Ahn, MJ and Choi, MY and Kim, MJ and Choi, JH},
title = {One-Pot CRISPR-Cas12a-Based Viral DNA Detection via HRP-Enriched Extended ssDNA-Modified Au@Fe3O4 Nanoparticles.},
journal = {Biosensors},
volume = {14},
number = {1},
pages = {},
doi = {10.3390/bios14010026},
pmid = {38248403},
issn = {2079-6374},
support = {NRF-2022R1C1C1008797//National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded ; DNA, Viral ; *Nanoparticles ; Pandemics ; },
abstract = {In the context of virus outbreaks, the need for early and accurate diagnosis has become increasingly urgent. In addition to being crucial for effective disease control, timely and precise detection of viral infections is also necessary for the implementation of essential public health measures, especially during pandemics. Among these measures, point-of-care testing (POCT) stands out as a powerful approach with the potential to revolutionize the landscape of viral diagnosis. In this study, we developed a one-pot clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based viral DNA detection system tailored for POCT; this method utilizes multi-enzyme-modified Au@Fe3O4 nanoparticles. As an alternative to nucleic acid amplification, our method uses single-stranded DNA elongation to facilitate multi-enzyme modification; this guarantees heightened sensitivity and expedites the diagnostic process. We achieved a satisfactory limit of detection of 0.25 nM, demonstrating the remarkable sensitivity of the method without the need for sophisticated equipment. The incorporation of Au@Fe3O4 magnetic nanoparticles facilitates sample separation, further streamlining the workflow and reinforcing the simplicity of our method. This integrated approach offers a practical solution for sensitive viral DNA detection in POCT scenarios, advancing the field of rapid and accurate diagnostics.},
}
@article {pmid38248386,
year = {2023},
author = {Chen, Y and Jiang, H and Liu, X and Wang, X},
title = {Engineered Electrochemiluminescence Biosensors for Monitoring Heavy Metal Ions: Current Status and Prospects.},
journal = {Biosensors},
volume = {14},
number = {1},
pages = {},
doi = {10.3390/bios14010009},
pmid = {38248386},
issn = {2079-6374},
support = {82061148012//National Natural Science Foundation of China/ ; 82027806//National Natural Science Foundation of China/ ; 21974019//National Natural Science Foundation of China/ ; 82372220//National Natural Science Foundation of China/ ; BE2019716//Primary Research &amp; Development Plan of Jiangsu Province/ ; },
mesh = {*Metals, Heavy ; Biometry ; Carbon ; Ions ; *Nanostructures ; },
abstract = {Metal ion contamination has serious impacts on environmental and biological health, so it is crucial to effectively monitor the levels of these metal ions. With the continuous progression of optoelectronic nanotechnology and biometrics, the emerging electrochemiluminescence (ECL) biosensing technology has not only proven its simplicity, but also showcased its utility and remarkable sensitivity in engineered monitoring of residual heavy metal contaminants. This comprehensive review begins by introducing the composition, advantages, and detection principles of ECL biosensors, and delving into the engineered aspects. Furthermore, it explores two signal amplification methods: biometric element-based strategies (e.g., HCR, RCA, EDC, and CRISPR/Cas) and nanomaterial (NM)-based amplification, including quantum dots, metal nanoclusters, carbon-based nanomaterials, and porous nanomaterials. Ultimately, this review envisions future research trends and engineered technological enhancements of ECL biosensors to meet the surging demand for metal ion monitoring.},
}
@article {pmid38247853,
year = {2024},
author = {Cenni, V and Evangelisti, C and Santi, S and Sabatelli, P and Neri, S and Cavallo, M and Lattanzi, G and Mattioli, E},
title = {Desmin and Plectin Recruitment to the Nucleus and Nuclei Orientation Are Lost in Emery-Dreifuss Muscular Dystrophy Myoblasts Subjected to Mechanical Stimulation.},
journal = {Cells},
volume = {13},
number = {2},
pages = {},
doi = {10.3390/cells13020162},
pmid = {38247853},
issn = {2073-4409},
support = {DSB.AD007.201//AIDMED, CRISPR-CAS/ ; GAE-P00000147//Associazione Alessandra Proietti/ ; cod. ECS_00000033 - CUP B89I22000650001//ECOSISTER Project/ ; prot. 201742SBXA_005//PRIN MUR/ ; Prot. 20223WFJJ3//PRIN MUR ADIPOAGE/ ; 2019-004426-24//AIFA TREAT-LMNA/ ; },
mesh = {Humans ; Desmin ; *Lamin Type A ; Plectin ; Myoblasts ; *Muscular Dystrophy, Emery-Dreifuss/genetics ; },
abstract = {In muscle cells subjected to mechanical stimulation, LINC complex and cytoskeletal proteins are basic to preserve cellular architecture and maintain nuclei orientation and positioning. In this context, the role of lamin A/C remains mostly elusive. This study demonstrates that in human myoblasts subjected to mechanical stretching, lamin A/C recruits desmin and plectin to the nuclear periphery, allowing a proper spatial orientation of the nuclei. Interestingly, in Emery-Dreifuss Muscular Dystrophy (EDMD2) myoblasts exposed to mechanical stretching, the recruitment of desmin and plectin to the nucleus and nuclear orientation were impaired, suggesting that a functional lamin A/C is crucial for the response to mechanical strain. While describing a new mechanism of action headed by lamin A/C, these findings show a structural alteration that could be involved in the onset of the muscle defects observed in muscular laminopathies.},
}
@article {pmid38177854,
year = {2024},
author = {Birney, E},
title = {A society-wide conversation is needed about germline genome editing using CRISPR.},
journal = {Nature medicine},
volume = {30},
number = {1},
pages = {30-32},
pmid = {38177854},
issn = {1546-170X},
mesh = {*Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Germ Cells ; },
}
@article {pmid38170544,
year = {2024},
author = {Harris, E},
title = {Sickle Cell Disease Approvals Include First CRISPR Gene Editing Therapy.},
journal = {JAMA},
volume = {331},
number = {4},
pages = {280},
doi = {10.1001/jama.2023.26113},
pmid = {38170544},
issn = {1538-3598},
mesh = {Humans ; *Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Genetic Therapy ; },
}
@article {pmid38165777,
year = {2024},
author = {Hu, Y and Liu, L and Wang, C and Zhou, J and Liu, R and Lv, Y},
title = {CRISPR-Cas12a-enhanced mass spectrometric DNA nanomachine for HPV-16 detection in human serum.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {8},
pages = {976-979},
doi = {10.1039/d3cc05949a},
pmid = {38165777},
issn = {1364-548X},
mesh = {Humans ; Female ; *Uterine Cervical Neoplasms/diagnosis ; Human papillomavirus 16/genetics ; Sensitivity and Specificity ; CRISPR-Cas Systems ; DNA, Viral/analysis ; Mass Spectrometry ; *Biosensing Techniques ; },
abstract = {Cervical cancer is the second most harmful cancer to women, while human papillomavirus (HPV)-16 is the main cause of cervical cancer. Early virus screening can reduce the risk of death, but is sometimes challenged by the low concentration and complicated matrix in physiological samples such as human serum. Herein, we report a CRISPR-Cas12a-enhanced mass spectrometric 3D DNA nanomachine for HPV-16 detection in human serum. Thanks to the high sensitivity of the CRISPR-Cas12a-enhanced 3D DNA nanomachine, and the high matrix-tolerance of elemental mass spectrometry, the proposed method was successfully applied for direct human serum analysis with a low detection limit of 1.2 pM.},
}
@article {pmid38129236,
year = {2024},
author = {Zou, J and Huang, Y and Gao, C and Wang, K},
title = {Unlocking crop diversity: Enhancing variations through genome editing.},
journal = {Science bulletin},
volume = {69},
number = {3},
pages = {281-284},
doi = {10.1016/j.scib.2023.12.013},
pmid = {38129236},
issn = {2095-9281},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; },
}
@article {pmid37050882,
year = {2023},
author = {Gerber, A and van Otterdijk, S and Bruggeman, FJ and Tutucci, E},
title = {Understanding spatiotemporal coupling of gene expression using single molecule RNA imaging technologies.},
journal = {Transcription},
volume = {14},
number = {3-5},
pages = {105-126},
doi = {10.1080/21541264.2023.2199669},
pmid = {37050882},
issn = {2154-1272},
mesh = {*RNA/genetics ; In Situ Hybridization, Fluorescence/methods ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/metabolism ; Protein Biosynthesis ; },
abstract = {Across all kingdoms of life, gene regulatory mechanisms underlie cellular adaptation to ever-changing environments. Regulation of gene expression adjusts protein synthesis and, in turn, cellular growth. Messenger RNAs are key molecules in the process of gene expression. Our ability to quantitatively measure mRNA expression in single cells has improved tremendously over the past decades. This revealed an unexpected coordination between the steps that control the life of an mRNA, from transcription to degradation. Here, we provide an overview of the state-of-the-art imaging approaches for measurement and quantitative understanding of gene expression, starting from the early visualizations of single genes by electron microscopy to current fluorescence-based approaches in single cells, including live-cell RNA-imaging approaches to FISH-based spatial transcriptomics across model organisms. We also highlight how these methods have shaped our current understanding of the spatiotemporal coupling between transcriptional and post-transcriptional events in prokaryotes. We conclude by discussing future challenges of this multidisciplinary field.Abbreviations: mRNA: messenger RNA; rRNA: ribosomal rDNA; tRNA: transfer RNA; sRNA: small RNA; FISH: fluorescence in situ hybridization; RNP: ribonucleoprotein; smFISH: single RNA molecule FISH; smiFISH: single molecule inexpensive FISH; HCR-FISH: Hybridization Chain-Reaction-FISH; RCA: Rolling Circle Amplification; seqFISH: Sequential FISH; MERFISH: Multiplexed error robust FISH; UTR: Untranslated region; RBP: RNA binding protein; FP: fluorescent protein; eGFP: enhanced GFP, MCP: MS2 coat protein; PCP: PP7 coat protein; MB: Molecular beacons; sgRNA: single guide RNA.},
}
@article {pmid38247619,
year = {2024},
author = {Jangid, H and Kumar, D and Kumar, G and Kumar, R and Mamidi, N},
title = {An Emerging Foodborne Pathogen Spotlight: A Bibliometric Analysis and Scholarly Review of Escherichia coli O157 Research.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {13},
number = {1},
pages = {},
doi = {10.3390/antibiotics13010060},
pmid = {38247619},
issn = {2079-6382},
abstract = {Foodborne infections pose a substantial global threat, causing an estimated 600 million illnesses and resulting in approximately 420,000 deaths annually. Among the diverse array of pathogens implicated in these infections, Escherichia coli (E. coli), specifically the O157 strain (E. coli O157), emerges as a prominent pathogen associated with severe outbreaks. This study employs a comprehensive bibliometric analysis and scholarly review focused on E. coli O157 research. The bibliometric analysis highlights the significant role played by the United States in the E. coli O157 research domain. Further exploration underscores the noteworthy contributions of the researcher Doyle MP, whose body of work, consisting of 84 documents and an impressive H-Index of 49, reflects their substantial impact in the field. Recent research trends indicate a discernible shift towards innovative detection methods, exemplified by the adoption of CRISPR-CAS and Loop-Mediated Isothermal Amplification. Moreover, high-throughput whole-genome sequencing techniques are gaining prominence for the expeditious analysis of pathogenic E. coli strains. Scientists are increasingly exploring antimicrobial agents, including phage therapy, to address the challenges posed by antibiotic-resistant E. coli strains, thereby addressing critical concerns related to multi-drug resistance. This comprehensive analysis provides vital insights into the dynamic landscape of E. coli O157 research. It serves as a valuable resource for researchers, policymakers, and healthcare professionals dedicated to mitigating E. coli O157 outbreaks and advancing global public health strategies.},
}
@article {pmid38247505,
year = {2024},
author = {Guo, H and Rogg, M and Keller, J and Scherzinger, AK and Jäckel, J and Meyer, C and Sammarco, A and Helmstädter, M and Gorka, O and Groß, O and Schell, C and Bechtel-Walz, W},
title = {ADP-Ribosylation Factor-Interacting Protein 2 Acts as a Novel Regulator of Mitophagy and Autophagy in Podocytes in Diabetic Nephropathy.},
journal = {Antioxidants (Basel, Switzerland)},
volume = {13},
number = {1},
pages = {},
doi = {10.3390/antiox13010081},
pmid = {38247505},
issn = {2076-3921},
support = {BE 6135/3//Deutsche Forschungsgemeinschaft/ ; N/A//Berta-Ottenstein-Program for Advanced Clinician Scientists, Faculty of Medicine, University of Freiburg/ ; BEC2198/21//Forschungskommission of the Medizinische Fakult&#xe4;t Universit&#xe4;t Freiburg/ ; FP-MC-CIG 293568//Marie Curie Career Integration Grant/ ; N/A//Margarete von Wrangell Program Ministerium f&#xfc;r Wissenschaft, Forschung und Kunst des Landes Baden-W&#xfc;rttemberg/ ; N/A//Chinese Scholarship Counsil/ ; 431984000//Deutsche Forschungsgemeinschaft/ ; 256073931//Deutsche Forschungsgemeinschaft/ ; },
abstract = {(1) Background: Differentiated podocytes are particularly vulnerable to oxidative stress and cellular waste products. The disease-related loss of postmitotic podocytes is a direct indicator of renal disease progression and aging. Podocytes use highly specific regulated networks of autophagy and endocytosis that counteract the increasing number of damaged protein aggregates and help maintain cellular homeostasis. Here, we demonstrate that ARFIP2 is a regulator of autophagy and mitophagy in podocytes both in vitro and in vivo. (2) Methods: In a recent molecular regulatory network analysis of mouse glomeruli, we identified ADP-ribosylation factor-interacting protein 2 (Arfip2), a cytoskeletal regulator and cofactor of ATG9-mediated autophagosome formation, to be differentially expressed with age. We generated an Arfip2-deficient immortalized podocyte cell line using the CRISPR/Cas technique to investigate the significance of Arfip2 for renal homeostasis in vitro. For the in vivo analyses of Arfip2 deficiency, we used a mouse model of Streptozotozin-induced type I diabetes and investigated physiological data and (patho)histological (ultra)structural modifications. (3) Results: ARFIP2 deficiency in immortalized human podocytes impedes autophagy. Beyond this, ARFIP2 deficiency in human podocytes interferes with ATG9A trafficking and the PINK1-Parkin pathway, leading to the compromised fission of mitochondria and short-term increase in mitochondrial respiration and induction of mitophagy. In diabetic mice, Arfip2 deficiency deteriorates autophagy and leads to foot process effacement, histopathological changes, and early albuminuria. (4) Conclusions: In summary, we show that ARFIP2 is a novel regulator of autophagy and mitochondrial homeostasis in podocytes by facilitating ATG9A trafficking during PINK1/Parkin-regulated mitophagy.},
}
@article {pmid38246942,
year = {2024},
author = {Jeon, C and Chung, MY and Lee, JM},
title = {Reassessing the contribution of TOMATO AGAMOUS-LIKE1 to fruit ripening by CRISPR/Cas9 mutagenesis.},
journal = {Plant cell reports},
volume = {43},
number = {2},
pages = {41},
pmid = {38246942},
issn = {1432-203X},
support = {2021R1A2C2093789//National Research Foundation of Korea/ ; },
mesh = {*Solanum lycopersicum/genetics ; Fruit/genetics ; CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; Mutation/genetics ; },
abstract = {CRISPR/Cas9-edited TOMATO AGAMOUS-LIKE1 (TAGL1) provided new insights into fruit ripening. TOMATO AGAMOUS LIKE 1 (TAGL1) has been identified as playing a key role in the process of tomato fruit development and ripening. We have re-evaluated the functions of TAGL1 using CRISPR/Cas9 mutagenesis. Three KO mutants contained frameshift mutations resulting in premature termination codons due to a 1 bp insertion. TAGL1-KO mutants exhibited dark immature fruits and orange ripening fruits. The fruit shape was characterized by a prominent pointed tip at the end and the pericarp thickness was significantly thinner. TAGL1-KO mutants showed reduced ethylene biosynthesis, increased firmness, and delayed onset of ripening. The chlorophyll content of TAGL1-KO mutants was higher in the mature green stage and the lycopene content of TAGL1-KO mutants in the ripening stage was lower compared to the WT. ACS2, ACS4, ACO1, ACO3, PG2a, PL, PME, EXP1, and PSY1 in the mutants were significantly down-regulated during ripening. Ripening fruits in the double mutant of rin and tagl1 showed a more extreme phenotype than the rin mutant suggesting that the double mutation acts synergistically during ripening. TAGL1-targeted mutagenesis by CRISPR/Cas9 strengthens its regulatory functions controlling ripening parameters and provides new insights into fruit ripening.},
}
@article {pmid38246741,
year = {2024},
author = {Fu, X and Sun, J and Yu, B and Ye, Y and Sheng, L and Ji, J and Zheng, J and Fan, M and Shao, J and Sun, X},
title = {Investigating enzyme kinetics and fluorescence sensing strategy of CRISPR/Cas12a for foodborne pathogenic bacteria.},
journal = {Analytica chimica acta},
volume = {1290},
number = {},
pages = {342203},
doi = {10.1016/j.aca.2024.342203},
pmid = {38246741},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems ; Fluorescence ; Bacteria ; Coloring Agents ; Fluorescein ; *Staphylococcal Infections ; Staphylococcus aureus/genetics ; },
abstract = {Foodborne pathogenic bacteria are widespread in various foods, whose cross-contamination and re-contamination are critical influences on food safety. Rapid, accurate, and sensitive detection of foodborne pathogenic bacteria remains a topic of concern. CRISPR/Cas12a can recognize double-stranded DNA directly, showing great potential in nucleic acid detection. However, few studies have investigated the cleavage properties of CRISPR/Cas12a. In this study, the trans-cleavage properties of LbCas12a and AsCas12a were investigated to construct the detection methods for foodborne pathogenic bacteria. The highly sensitive fluorescent strategies for foodborne pathogens were constructed by analyzing the cleavage rates and properties of substrates at different substrate concentrations. Cas12a was activated in the presence of foodborne pathogenic target sequence was present, resulting in the cleavage of a single-stranded reporter ssDNA co-labelled by fluorescein quencher and fluorescein. The sensitivity and specificity of the Cas12a fluorescent strategy was investigated with Salmonella and Staphylococcus aureus as examples. The results showed that AsCas12a was slightly more capable of trans-cleavage than LbCas12a. The detection limits of AsCas12a for Salmonella and Staphylococcus aureus were 24.9 CFU mL[-1] and 1.50 CFU mL[-1], respectively. In all the seven bacteria, Staphylococcus aureus and Salmonella were accurately discriminated. The study provided a basis for constructing and improving the CRISPR/Cas12a fluorescence strategies. The AsCas12a-based detection strategy is expected to be a promising method for field detection.},
}
@article {pmid38246560,
year = {2024},
author = {Mudde, AC and Kuo, CY and Kohn, DB and Booth, C},
title = {What a Clinician Needs to Know about Genome Editing: Status and Opportunities for Inborn Errors of Immunity.},
journal = {The journal of allergy and clinical immunology. In practice},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jaip.2024.01.019},
pmid = {38246560},
issn = {2213-2201},
abstract = {During the past twenty years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector mediated gene addition, inborn errors of immunity (IEIs) are excellent candidate diseases for a corrective autologous haematopoietic stem cell gene editing strategy. Research on gene editing for IEIs is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of IEIs. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.},
}
@article {pmid38245560,
year = {2024},
author = {Duan, N and Hand, E and Pheko, M and Sharma, S and Emiola, A},
title = {Structure-guided discovery of anti-CRISPR and anti-phage defense proteins.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {649},
pmid = {38245560},
issn = {2041-1723},
mesh = {*Bacteriophages/metabolism ; CRISPR-Cas Systems ; Viral Proteins/genetics/metabolism ; Bacteria/genetics ; Bacteroidetes/metabolism ; },
abstract = {Bacteria use a variety of defense systems to protect themselves from phage infection. In turn, phages have evolved diverse counter-defense measures to overcome host defenses. Here, we use protein structural similarity and gene co-occurrence analyses to screen >66 million viral protein sequences and >330,000 metagenome-assembled genomes for the identification of anti-phage and counter-defense systems. We predict structures for ~300,000 proteins and perform large-scale, pairwise comparison to known anti-CRISPR (Acr) and anti-phage proteins to identify structural homologs that otherwise may not be uncovered using primary sequence search. This way, we identify a Bacteroidota phage Acr protein that inhibits Cas12a, and an Akkermansia muciniphila anti-phage defense protein, termed BxaP. Gene bxaP is found in loci encoding Bacteriophage Exclusion (BREX) and restriction-modification defense systems, but confers immunity independently. Our work highlights the advantage of combining protein structural features and gene co-localization information in studying host-phage interactions.},
}
@article {pmid38244227,
year = {2024},
author = {Martins, PMM and Granato, LM and Morgan, T and Nalin, JL and Takita, MA and Alfenas-Zerbini, P and de Souza, AA},
title = {Analysis of CRISPR-Cas loci distribution in Xanthomonas citri and its possible control by the quorum sensing system.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnae005},
pmid = {38244227},
issn = {1574-6968},
abstract = {Xanthomonas is an important genus of plant-associated bacteria that causes significant yield losses of economically important crops worldwide. Different approaches have assessed genetic diversity and evolutionary interrelationships among the Xanthomonas species. However, information from clustered regularly interspaced short palindromic repeats (CRISPR) has yet to be explored. In this work, we analyzed the architecture of CRISPR-Cas loci and presented a sequence similarity-based clustering of conserved Cas proteins in different species of Xanthomonas. Although absent in many investigated genomes, Xanthomonas harbors subtype I-C and I-F CRISPR-Cas systems. The most represented species, Xanthomonas citri, presents a great diversity of genome sequences with an uneven distribution of CRISPR-Cas systems among the subspecies/pathovars. Only Xanthomonas citri subsp. citri and Xanthomonas citri pv. punicae have these systems, exclusively of subtype I-C system. Moreover, the most likely targets of the X. citri CRISPR spacers are viruses (phages). At the same time, few are plasmids, indicating that CRISPR/Cas system is possibly a mechanism to control the invasion of foreign DNA. We also showed in X. citri susbp. citri that the cas genes are regulated by the diffusible signal factor (DSF), the quorum sensing (QS) signal molecule, according to cell density increases, and under environmental stress like starvation. These results suggest that the regulation of CRISPR-Cas by QS occurs to activate the gene expression only during phage infection or due to environmental stresses, avoiding a possible reduction in fitness. Although more studies are needed, CRISPR-Cas systems may have been selected in the Xanthomonas genus throughout evolution, according to the cost-benefit of protecting against biological threats and fitness maintenance in challenging conditions.},
}
@article {pmid38243871,
year = {2024},
author = {Ma, L and Ma, Y and Gao, Q and Liu, S and Zhu, Z and Shi, X and Dai, F and Reis, RL and Kundu, SC and Cai, K and Xiao, B},
title = {Mulberry Leaf Lipid Nanoparticles: a Naturally Targeted CRISPR/Cas9 Oral Delivery Platform for Alleviation of Colon Diseases.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2307247},
doi = {10.1002/smll.202307247},
pmid = {38243871},
issn = {1613-6829},
support = {82072060//National Natural Science Foundation of China/ ; 82360110//National Natural Science Foundation of China/ ; 22008201//National Natural Science Foundation of China/ ; 20212BDH81019//Science and Technology Department of Jiangxi Province/ ; 20224BAB206073//Science and Technology Department of Jiangxi Province/ ; SWU-XDPY22006//Fundamental Research Funds for the Central Universities/ ; SWU-KQ22075//Fundamental Research Funds for the Central Universities/ ; 2205012980212766//Venture &amp; Innovation Support Program for Chongqing Overseas Returnees/ ; 2022NSCQ-JQX5279//Science Fund for Distinguished Young Scholars of Chongqing Municipality/ ; },
abstract = {Oral treatment of colon diseases with the CRISPR/Cas9 system has been hampered by the lack of a safe and efficient delivery platform. Overexpressed CD98 plays a crucial role in the progression of ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC). In this study, lipid nanoparticles (LNPs) derived from mulberry leaves are functionalized with Pluronic copolymers and optimized to deliver the CRISPR/Cas gene editing machinery for CD98 knockdown. The obtained LNPs possessed a hydrodynamic diameter of 267.2 nm, a narrow size distribution, and a negative surface charge (-25.6 mV). Incorporating Pluronic F127 into LNPs improved their stability in the gastrointestinal tract and facilitated their penetration through the colonic mucus barrier. The galactose end groups promoted endocytosis of the LNPs by macrophages via asialoglycoprotein receptor-mediated endocytosis, with a transfection efficiency of 2.2-fold higher than Lipofectamine 6000. The LNPs significantly decreased CD98 expression, down-regulated pro-inflammatory cytokines (TNF-α and IL-6), up-regulated anti-inflammatory factors (IL-10), and polarized macrophages to M2 phenotype. Oral administration of LNPs mitigated UC and CAC by alleviating inflammation, restoring the colonic barrier, and modulating intestinal microbiota. As the first oral CRISPR/Cas9 delivery LNP, this system offers a precise and efficient platform for the oral treatment of colon diseases.},
}
@article {pmid38243310,
year = {2024},
author = {Heo, SJ and Enriquez, LD and Federman, S and Chang, AY and Mace, R and Shevade, K and Nguyen, P and Litterman, AJ and Shafer, S and Przybyla, L and Chow, ED},
title = {Compact CRISPR genetic screens enabled by improved guide RNA library cloning.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {25},
pmid = {38243310},
issn = {1474-760X},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; Gene Editing ; Cloning, Molecular ; },
abstract = {CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.},
}
@article {pmid38242128,
year = {2024},
author = {Hu, C and Myers, MT and Zhou, X and Hou, Z and Lozen, ML and Nam, KH and Zhang, Y and Ke, A},
title = {Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2023.12.034},
pmid = {38242128},
issn = {1097-4164},
abstract = {Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.},
}
@article {pmid38240803,
year = {2024},
author = {Gong, Y and Li, S and Liu, Q and Chen, F and Shao, Y},
title = {CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {154},
pmid = {38240803},
issn = {1432-0614},
support = {32272288//National Natural Science Foundation of China/ ; 31671835//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing ; *Monascus/genetics/metabolism ; CRISPR-Cas Systems ; Gene Targeting/methods ; Lovastatin/metabolism ; Agrobacterium tumefaciens/genetics ; DNA/metabolism ; },
abstract = {Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (10[6] protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.},
}
@article {pmid38239723,
year = {2023},
author = {Andriyanov, PA and Kashina, DD and Menshikova, AN},
title = {Genomic analysis of multidrug-resistant Delftia tsuruhatensis isolated from raw bovine milk.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1321122},
pmid = {38239723},
issn = {1664-302X},
abstract = {Delftia tsuruhatensis is a gram-negative, aerobic bacterium mostly known as an organic pollutant degrading and growth-promoting microorganism. However, it recently emerged as an opportunistic human pathogen. To date, the source of D. tsuruhatensis infection is not clear. The majority of studies of D. tsuruhatensis have focused on environmental or clinical strains, while investigations of D. tsuruhatensis strains isolated from food sources are limited. In the present study, we report the case of D. tsuruhatensis isolation from raw bovine milk. Classical bacteriology approaches, as well as next-generation sequencing and comparative genomics, were used to characterize the features of the D. tsuruhatensis MR-6/3H strain. The MR-6/3H strain was resistant to 19 antimicrobials among 23 tested, including all aminoglycosides, phenicol, trimethoprim-sulfamethoxazole, and almost all β-lactams. Phylogenetically, the MR-6/3H was close to clinical origin strains, including those previously isolated in Russia. Comparative genomics revealed the presence of putative antimicrobial resistance genes in the MR-6/3H isolate, mostly associated with efflux systems. Notably, genus-specific OXA-926-like β-lactamase was also detected. In all, 27 putative virulence factors were predicted, the majority of which were associated with motility, adherence, stress survival, siderophore synthesis, and immunomodulation. In the MR-6/3H genome, the five prophage regions were identified, including two with intact levels. Integrons and CRISPR-Cas systems were not detected in the MR-6/3H isolate. Thus, our findings suggest that raw milk can be the potential source of and transmission route for the dissemination of multidrug-resistant D. tsuruhatensis.},
}
@article {pmid38239625,
year = {2023},
author = {Dampier, W and Berman, R and Nonnemacher, MR and Wigdahl, B},
title = {Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1248982},
pmid = {38239625},
issn = {2673-3439},
abstract = {Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability. Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential. Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV. Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.},
}
@article {pmid38239344,
year = {2023},
author = {Li, X and Chen, Z and Ye, W and Yu, J and Zhang, X and Li, Y and Niu, Y and Ran, S and Wang, S and Luo, Z and Zhao, J and Hao, Y and Zong, J and Xia, C and Xia, J and Wu, J},
title = {High-throughput CRISPR technology: a novel horizon for solid organ transplantation.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1295523},
pmid = {38239344},
issn = {1664-3224},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {Organ transplantation is the gold standard therapy for end-stage organ failure. However, the shortage of available grafts and long-term graft dysfunction remain the primary barriers to organ transplantation. Exploring approaches to solve these issues is urgent, and CRISPR/Cas9-based transcriptome editing provides one potential solution. Furthermore, combining CRISPR/Cas9-based gene editing with an ex vivo organ perfusion system would enable pre-implantation transcriptome editing of grafts. How to determine effective intervention targets becomes a new problem. Fortunately, the advent of high-throughput CRISPR screening has dramatically accelerated the effective targets. This review summarizes the current advancements, utilization, and workflow of CRISPR screening in various immune and non-immune cells. It also discusses the ongoing applications of CRISPR/Cas-based gene editing in transplantation and the prospective applications of CRISPR screening in solid organ transplantation.},
}
@article {pmid38233866,
year = {2024},
author = {Li, J and Kong, D and Ke, Y and Zeng, W and Miki, D},
title = {Application of multiple sgRNAs boosts efficiency of CRISPR/Cas9-mediated gene targeting in Arabidopsis.},
journal = {BMC biology},
volume = {22},
number = {1},
pages = {6},
pmid = {38233866},
issn = {1741-7007},
support = {20ZR1467000//Shanghai Science and Technology Development Foundation/ ; 23WZ2500800//Shanghai Science and Technology Development Foundation/ ; G202201355L//Foreign Expert Project/ ; },
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Protein-Tyrosine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; Gene Targeting/methods ; },
abstract = {BACKGROUND: Precise gene targeting (GT) is a powerful tool for heritable precision genome engineering, enabling knock-in or replacement of the endogenous sequence via homologous recombination. We recently established a CRISPR/Cas9-mediated approach for heritable GT in Arabidopsis thaliana (Arabidopsis) and rice and reported that the double-strand breaks (DSBs) frequency of Cas9 influences the GT efficiency. However, the relationship between DSBs and GT at the same locus was not examined. Furthermore, it has never been investigated whether an increase in the number of copies of sgRNAs or the use of multiple sgRNAs would improve the efficiency of GT.<br><br>RESULTS: Here, we achieved precise GT at endogenous&#xa0;loci Embryo Defective 2410 (EMB2410) and Repressor of Silencing 1 (ROS1) using the sequential transformation strategy and the combination of sgRNAs. We show that increasing of sgRNAs copy number elevates both DSBs and GT efficiency. On the other hand, application of multiple sgRNAs does not always enhance GT efficiency. Our results also suggested that some inefficient sgRNAs would play a role as a helper to facilitate other sgRNAs DSBs activity.<br><br>CONCLUSIONS: The results of this study clearly show that DSB efficiency, rather than mutation pattern, is one of the most important key factors determining GT efficiency. This study provides new insights into the relationship between sgRNAs, DSBs, and GTs and the molecular mechanisms of CRISPR/Cas9-mediated GTs in plants.},
}
@article {pmid38233864,
year = {2024},
author = {Ding, X and Shi, MX and Liu, D and Cao, JX and Zhang, KX and Zhang, RD and Zhang, LP and Ai, KX and Su, B and Zhang, J},
title = {Transformation to small cell lung cancer is irrespective of EGFR and accelerated by SMAD4-mediated ASCL1 transcription independently of RB1 in non-small cell lung cancer.},
journal = {Cell communication and signaling : CCS},
volume = {22},
number = {1},
pages = {45},
pmid = {38233864},
issn = {1478-811X},
support = {19140901402, 17ZR1423500 and 201409003600//Science and Technology Commission of Shanghai Municipality/ ; 19140901402, 17ZR1423500 and 201409003600//Science and Technology Commission of Shanghai Municipality/ ; 82272897//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; *Small Cell Lung Carcinoma/genetics/drug therapy/pathology ; *Lung Neoplasms/pathology ; ErbB Receptors/genetics ; Mutation/genetics ; Protein Kinase Inhibitors/pharmacology ; Basic Helix-Loop-Helix Transcription Factors/genetics/therapeutic use ; Smad4 Protein/genetics ; Ubiquitin-Protein Ligases/genetics ; Retinoblastoma Binding Proteins/genetics ; },
abstract = {OBJECTIVES: Histological transformation to small cell lung cancer (SCLC) has been identified as a mechanism of TKIs resistance in EGFR-mutant non-small cell lung cancer (NSCLC). We aim to explore the prevalence of transformation in EGFR-wildtype NSCLC and the mechanism of SCLC transformation, which are rarely understood.<br><br>METHODS: We reviewed 1474 NSCLC patients to investigate the NSCLC-to-SCLC transformed cases and the basic clinical characteristics, driver gene status and disease course of them. To explore the potential functional genes in SCLC transformation, we obtained pre- and post-transformation specimens and subjected them to a multigene NGS panel involving 416 cancer-related genes. To validate the putative gene function, we established knocked-out models by CRISPR-Cas 9 in HCC827 and A549-TP53[-/-] cells and investigated the effects on tumor growth, drug sensitivity and neuroendocrine phenotype in vitro and in vivo. We also detected the expression level of protein and mRNA to explore the molecular mechanism involved.<br><br>RESULTS: We firstly reported an incidence rate of 9.73% (11/113) of SCLC transformation in EGFR-wildtype NSCLC and demonstrated that SCLC transformation is irrespective of EGFR mutation status (P&#x2009;=&#x2009;0.16). We sequenced 8 paired tumors and identified a series of mutant genes specially in transformed SCLC such as SMAD4, RICTOR and RET. We firstly demonstrated that SMAD4 deficiency can accelerate SCLC transition by inducing neuroendocrine phenotype regardless of RB1 status in TP53-deficient NSCLC cells. Further mechanical experiments identified the SMAD4 can regulate ASCL1 transcription competitively with Myc in NSCLC cells and Myc inhibitor acts as a potential subsequent treatment agent.<br><br>CONCLUSIONS: Transformation to SCLC is irrespective of EFGR status and can be accelerated by SMAD4 in non-small cell lung cancer. Myc inhibitor acts as a potential therapeutic drug for SMAD4-mediated resistant lung cancer. Video Abstract.},
}
@article {pmid38232570,
year = {2024},
author = {Lei, X and Cao, S and Liu, T and Wu, Y and Yu, S},
title = {Non-canonical CRISPR/Cas12a-based technology: A novel horizon for biosensing in nucleic acid detection.},
journal = {Talanta},
volume = {271},
number = {},
pages = {125663},
doi = {10.1016/j.talanta.2024.125663},
pmid = {38232570},
issn = {1873-3573},
abstract = {Nucleic acids are essential biomarkers in molecular diagnostics. The CRISPR/Cas system has been widely used for nucleic acid detection. Moreover, canonical CRISPR/Cas12a based biosensors can specifically recognize and cleave target DNA, as well as single-strand DNA serving as reporter probe, which have become a super star in recent years in the field of nucleic acid detection due to its high specificity, universal programmability and simple operation. However, canonical CRISPR/Cas12a based biosensors are hard to meet the requirements of higher sensitivity, higher specificity, higher efficiency, larger target scope, easier operation, multiplexing, low cost and diversified signal reading. Then, advanced non-canonical CRISPR/Cas12a based biosensors emerge. In this review, applications of non-canonical CRISPR/Cas12a-based biosensors in nucleic acid detection are summarized. And the principles, peculiarities, performances and perspectives of these non-canonical CRISPR/Cas12a based biosensors are also discussed.},
}
@article {pmid38231831,
year = {2023},
author = {Banks, J},
title = {The Gene Editing Juggernaut is Picking Up Speed.},
journal = {IEEE pulse},
volume = {14},
number = {5},
pages = {23-26},
doi = {10.1109/MPULS.2023.3344054},
pmid = {38231831},
issn = {2154-2317},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; },
abstract = {CRISPR-Cas9, the tool for editing genes by precisely cutting DNA and letting the body's natural DNA repair processes take over, deservedly led to Nobel prizes in 2020 for its pioneers, Emmanuelle Charpentier and Jennifer Doudna. Since their breakthroughs in 2012, the technology has moved forward in leaps and bounds, and techniques to manipulate genes that were once the realm of science fiction are becoming very much science "fact."},
}
@article {pmid38231658,
year = {2024},
author = {Philippidis, A},
title = {CASGEVY Makes History as FDA Approves First CRISPR/Cas9 Genome Edited Therapy.},
journal = {Human gene therapy},
volume = {35},
number = {1-2},
pages = {1-4},
doi = {10.1089/hum.2023.29263.bfs},
pmid = {38231658},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid38231433,
year = {2024},
author = {Yang, Y and Gong, F and Shan, X and Tan, Z and Zhou, F and Ji, X and Xiang, M and Wang, F and He, Z},
title = {Amplification-free detection of Mpox virus DNA using Cas12a and multiple crRNAs.},
journal = {Mikrochimica acta},
volume = {191},
number = {2},
pages = {102},
pmid = {38231433},
issn = {1436-5073},
mesh = {Humans ; *Monkeypox virus ; CRISPR-Cas Systems ; *Mpox (monkeypox) ; RNA, Guide, CRISPR-Cas Systems ; DNA, Viral/genetics ; DNA Viruses ; RNA ; },
abstract = {Mpox virus (MPXV) is a zoonotic DNA virus that caused human Mpox, leading to the 2022 global outbreak. MPXV infections can cause a number of clinical syndromes, which increases public health threats. Therefore, it is necessary to develop an effective and reliable method for infection prevention and control of epidemic. Here, a Cas12a-based direct detection assay for MPXV DNA is established without the need for amplification. By targeting the envelope protein gene (B6R) of MPXV, four CRISPR RNAs (crRNAs) are designed. When MPXV DNA is introduced, every Cas12a/crRNA complex can target a different site of the same MPXV gene. Concomitantly, the trans-cleavage activity of Cas12a is triggered to cleave the DNA reporter probes, releasing a fluorescence signal. Due to the application of multiple crRNAs, the amount of active Cas12a increases. Thus, more DNA reporter probes are cleaved. As a consequence, the detection signals are accumulated, which improves the limit of detection (LOD) and the detection speed. The LOD of the multiple crRNA system can be improved to ~ 0.16 pM, which is a decrease of the LOD by approximately ~ 27 times compared with the individual crRNA reactions. Furthermore, using multiple crRNAs increases the specificity of the assay. Given the outstanding performance, this assay has great potential for Mpox diagnosis.},
}
@article {pmid38230675,
year = {2024},
author = {Hongdan, G and Yao, D and Qiang, C and Meng, H and Xiaorong, L and Zhihao, X and Dongli, M},
title = {A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus.},
journal = {The Journal of international medical research},
volume = {52},
number = {1},
pages = {3000605231223083},
pmid = {38230675},
issn = {1473-2300},
mesh = {Child ; Humans ; *CRISPR-Cas Systems/genetics ; Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Respiratory Syncytial Virus, Human/genetics/metabolism ; Adenoviridae/genetics ; },
abstract = {OBJECTIVE: Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously.<br><br>METHODS: The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control.<br><br>RESULTS: RSV and ADV could be detected at levels as low as 10[4] copies/mL and 10[3] copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency.<br><br>CONCLUSION: This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.},
}
@article {pmid38225854,
year = {2024},
author = {Raza, S and Poria, R and Kala, D and Sharma, N and Sharma, AK and Florien, N and Tuli, HS and Kaushal, A and Gupta, S},
title = {Innovations in dengue virus detection: An overview of conventional and electrochemical biosensor approaches.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.2553},
pmid = {38225854},
issn = {1470-8744},
abstract = {Globally, people are in great threat due to the highly spreading of viral infectious diseases. Every year like 100-300 million cases of infections are found, and among them, above 80% are not recognized and irrelevant. Dengue virus (DENV) is an arbovirus infection that currently infects people most frequently. DENV encompasses four viral serotypes, and they each express comparable sign. From a mild febrile sickness to a potentially fatal dengue hemorrhagic fever, dengue can induce a variety of symptoms. Presently, the globe is being challenged by the untimely identification of dengue infection. Therefore, this review summarizes advances in the detection of dengue from conventional methods (nucleic acid-based, polymerase chain reaction-based, and serological approaches) to novel biosensors. This work illustrates an extensive study of the current designs and fabrication approaches involved in the formation of electrochemical biosensors for untimely identifications of dengue. Additionally, in electrochemical sensing of DENV, we skimmed through significances of biorecognition molecules like lectins, nucleic acid, and antibodies. The introduction of emerging techniques such as the CRISPR/Cas' system and their integration with biosensing platforms has also been summarized. Furthermore, the review revealed the importance of electrochemical approach compared with traditional diagnostic methods.},
}
@article {pmid38225637,
year = {2024},
author = {Cooper, SE and Coelho, MA and Strauss, ME and Gontarczyk, AM and Wu, Q and Garnett, MJ and Marioni, JC and Bassett, AR},
title = {scSNV-seq: high-throughput phenotyping of single nucleotide variants by coupled single-cell genotyping and transcriptomics.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {20},
pmid = {38225637},
issn = {1474-760X},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Genotype ; *RNA, Guide, CRISPR-Cas Systems ; *Gene Expression Profiling ; Transcriptome ; Nucleotides ; Single-Cell Analysis ; High-Throughput Nucleotide Sequencing ; },
abstract = {CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations including single nucleotide variants (SNVs) associated with diseases. Interpretation of these data is currently limited as genotypes cannot be accurately inferred from guide RNA identity alone. scSNV-seq overcomes this limitation by coupling single-cell genotyping and transcriptomics of the same cells enabling accurate and high-throughput screening of SNVs. Analysis of variants across the JAK1 gene with scSNV-seq demonstrates the importance of determining the precise genetic perturbation and accurately classifies clinically observed missense variants into three functional categories: benign, loss of function, and separation of function.},
}
@article {pmid38225194,
year = {2024},
author = {Hesping, E and Boddey, JA},
title = {Whole-genome CRISPR screens to understand Apicomplexan-host interactions.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.15221},
pmid = {38225194},
issn = {1365-2958},
support = {//Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS/ ; 1176955//National Health and Medical Research Council/ ; },
abstract = {Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole-genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite-host interactions.},
}
@article {pmid38222822,
year = {2024},
author = {Lee, SY and Kang, B and Venkatesh, J and Lee, JH and Lee, S and Kim, JM and Back, S and Kwon, JK and Kang, BC},
title = {Development of virus-induced genome editing methods in Solanaceous crops.},
journal = {Horticulture research},
volume = {11},
number = {1},
pages = {uhad233},
pmid = {38222822},
issn = {2662-6810},
abstract = {Genome editing (GE) using CRISPR/Cas systems has revolutionized plant mutagenesis. However, conventional transgene-mediated GE methods have limitations due to the time-consuming generation of stable transgenic lines expressing the Cas9/single guide RNA (sgRNA) module through tissue cultures. Virus-induced genome editing (VIGE) systems have been successfully employed in model plants, such as Arabidopsis thaliana and Nicotiana spp. In this study, we developed two VIGE methods for Solanaceous plants. First, we used the tobacco rattle virus (TRV) vector to deliver sgRNAs into a transgenic tomato (Solanum lycopersicum) line of cultivar Micro-Tom expressing Cas9. Second, we devised a transgene-free GE method based on a potato virus X (PVX) vector to deliver Cas9 and sgRNAs. We designed and cloned sgRNAs targeting Phytoene desaturase in the VIGE vectors and determined optimal conditions for VIGE. We evaluated VIGE efficiency through deep sequencing of the target gene after viral vector inoculation, detecting 40.3% and 36.5% mutation rates for TRV- and PVX-mediated GE, respectively. To improve editing efficiency, we applied a 37°C heat treatment, which increased the editing efficiency by 33% to 46% and 56% to 76% for TRV- and PVX-mediated VIGE, respectively. To obtain edited plants, we subjected inoculated cotyledons to tissue culture, yielding successful editing events. We also demonstrated that PVX-mediated GE can be applied to other Solanaceous crops, such as potato (Solanum tuberosum) and eggplant (Solanum melongena). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.},
}
@article {pmid38222286,
year = {2023},
author = {Kharbikar, L and Konwarh, R and Chakraborty, M and Nandanwar, S and Marathe, A and Yele, Y and Ghosh, PK and Sanan-Mishra, N and Singh, AP},
title = {3Bs of CRISPR-Cas mediated genome editing in plants: exploring the basics, bioinformatics and biosafety landscape.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {29},
number = {12},
pages = {1825-1850},
pmid = {38222286},
issn = {0971-5894},
abstract = {UNLABELLED: The recent thrust in research has projected the type II clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) system as an avant-garde plant genome editing tool. It facilitates the induction of site-specific double-stranded DNA cleavage by the RNA-guided DNA endonuclease (RGEN), Cas9. Elimination, addition, or alteration of sections in DNA sequence besides the creation of a knockout genotype (CRISPRko) is aided by the CRISPR-Cas9 system in its wild form (wtCas9). The inactivation of the nuclease domain generates a dead Cas9 (dCas9), which is capable of targeting genomic DNA without scissoring it. The dCas9 system can be engineered by fusing it with different effectors to facilitate transcriptional activation (CRISPRa) and transcriptional interference (CRISPRi). CRISPR-Cas thus holds tremendous prospects as a genome-manipulating stratagem for a wide gamut of crops. In this article, we present a brief on the fundamentals and the general workflow of the CRISPR-Cas system followed by an overview of the prospects of bioinformatics in propelling CRISPR-Cas research with a special thrust on the available databases and algorithms/web-accessible applications that have aided in increasing the usage and efficiency of editing. The article also provides an update on the current regulatory landscape in different countries on the CRISPR-Cas edited plants to emphasize the far-reaching impact of the genomic editing technology.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-023-01397-3.},
}
@article {pmid38220093,
year = {2024},
author = {Laksana, C and Sophiphun, O and Chanprame, S},
title = {Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {111987},
doi = {10.1016/j.plantsci.2024.111987},
pmid = {38220093},
issn = {1873-2259},
abstract = {Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1[st] target site; two insertions, one deletion, and one base substitution for the 2[nd] target site; and one base substitution and insertion for the 3[rd] target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production.},
}
@article {pmid38141396,
year = {2024},
author = {You, H and Jones, MK},
title = {Harnessing CRISPR-based molecular diagnosis in the fight against malaria.},
journal = {EBioMedicine},
volume = {99},
number = {},
pages = {104927},
pmid = {38141396},
issn = {2352-3964},
mesh = {Humans ; *Malaria/diagnosis/genetics ; CRISPR-Cas Systems ; },
}
@article {pmid38141039,
year = {2024},
author = {Babaei, M and Thomsen, PT and Pastor, MC and Jensen, MK and Borodina, I},
title = {Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets.},
journal = {ACS synthetic biology},
volume = {13},
number = {1},
pages = {168-182},
doi = {10.1021/acssynbio.3c00396},
pmid = {38141039},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Levodopa/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Tyrosine/genetics/metabolism ; },
abstract = {Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.g., amino acids) that can be screened either directly or by artificial biosensors, with low-throughput targeted validation of the molecule of interest to uncover nonintuitive beneficial metabolic engineering targets and combinations hereof. Using this workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and l-DOPA production from two large 4k gRNA libraries each deregulating 1000 metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library plasmids for individual regulatory targets improving the production of l-tyrosine-derived betaxanthins, identifying 30 targets that increased intracellular betaxanthin content 3.5-5.7 fold. Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased the secreted titer by up to 15%. To investigate whether any of the six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and subjected it to our proposed coupled workflow. The combination of regulating PYC1 and NTH2 simultaneously resulted in the highest (threefold) improvement of the betaxanthin content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a l-DOPA producing strain, identifying 10 targets that increased the secreted titer by up to 89%, further validating our screening by proxy workflow. This coupled approach is useful for strain development in the absence of direct HTP screening assays for products of interest.},
}
@article {pmid38131200,
year = {2024},
author = {Laurette, P and Cao, C and Ramanujam, D and Schwaderer, M and Lueneburg, T and Kuss, S and Weiss, L and Dilshat, R and Furlong, EEM and Rezende, F and Engelhardt, S and Gilsbach, R},
title = {In Vivo Silencing of Regulatory Elements Using a Single AAV-CRISPRi Vector.},
journal = {Circulation research},
volume = {134},
number = {2},
pages = {223-225},
doi = {10.1161/CIRCRESAHA.123.323854},
pmid = {38131200},
issn = {1524-4571},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Regulatory Sequences, Nucleic Acid ; Genetic Vectors ; Dependovirus/genetics ; },
}
@article {pmid38085703,
year = {2024},
author = {Garza Elizondo, AM and Chappell, J},
title = {Targeted Transcriptional Activation Using a CRISPR-Associated Transposon System.},
journal = {ACS synthetic biology},
volume = {13},
number = {1},
pages = {328-336},
doi = {10.1021/acssynbio.3c00563},
pmid = {38085703},
issn = {2161-5063},
mesh = {Transcriptional Activation/genetics ; *Escherichia coli/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Vibrio cholerae/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; },
abstract = {Synthetic perturbation of gene expression is central to our ability to reliably uncover genotype-phenotype relationships in microbes. Here, we present a novel transcription activation strategy that uses the Vibrio cholerae CRISPR-Associated Transposon (CAST) system to selectively insert promoter elements upstream of genes of interest. Through this strategy, we show robust activation of both recombinant and endogenous genes across the Escherichia coli chromosome. We then demonstrate the precise tuning of expression levels by exchanging the promoter elements being inserted. Finally, we demonstrate that CAST activation can be used to synthetically induce ampicillin-resistant phenotypes in E. coli.},
}
@article {pmid38061052,
year = {2024},
author = {Tang, N and Wu, Z and Gao, Y and Chen, W and Wang, Z and Su, M and Ji, W and Ji, Q},
title = {Molecular Basis and Genome Editing Applications of a Compact Eubacterium ventriosum CRISPR-Cas9 System.},
journal = {ACS synthetic biology},
volume = {13},
number = {1},
pages = {269-281},
doi = {10.1021/acssynbio.3c00501},
pmid = {38061052},
issn = {2161-5063},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; DNA/genetics ; *Eubacterium ; },
abstract = {CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A Eubacterium ventriosum CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox.},
}
@article {pmid38218299,
year = {2024},
author = {Zhang, H and Shi, M and Ma, X and Liu, M and Wang, N and Lu, Q and Li, Z and Zhao, Y and Zhao, H and Chen, H and Zhang, H and Jiang, T and Ouyang, S and Huo, Y and Bi, L},
title = {Type-III-A structure of mycobacteria CRISPR-Csm complexes involving atypical crRNAs.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {129331},
doi = {10.1016/j.ijbiomac.2024.129331},
pmid = {38218299},
issn = {1879-0003},
abstract = {Tuberculosis (TB), a leading cause of mortality globally, is a chronic infectious disease caused by Mycobacterium tuberculosis that primarily infiltrates the lung. The mature crRNAs in M. tuberculosis transcribed from the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus exhibit an atypical structure featured with 5' and 3' repeat tags at both ends of the intact crRNA, in contrast to typical Type-III-A crRNAs that possess 5' repeat tags and partial crRNA sequences. However, this structural peculiarity particularly concerning the specific binding characteristics of the 3' repeat end within the mature crRNA within the Csm complex, has not been comprehensively elucidated. Here, our Mycobacteria CRISPR-Csm complexes structure represents the largest Csm complex reported to date. It incorporates an atypical Type-III-A CRISPR RNA (crRNA) (46 nt) with 5' 8-nt and 3' 4-nt repeat sequences in the stoichiometry of Mycobacteria Csm1125364151. The PAM-independent single-stranded RNAs (ssRNAs) are the most suitable substrate for the Csm complex. The 3'-repeat end trimming of mature crRNA was not necessary for its cleavage activity in Type-III-A Csm complex. Our work broadens our understanding of the Type-III-A Csm complex and identifies another mature crRNA processing mechanism in the Type-III-A CRISPR-Cas system based on structural biology.},
}
@article {pmid38217650,
year = {2024},
author = {Prezza, G and Westermann, AJ},
title = {CRISPR Interference-Based Functional Small RNA Genomics.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2741},
number = {},
pages = {101-116},
pmid = {38217650},
issn = {1940-6029},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Genomics/methods ; RNA, Untranslated/genetics ; RNA, Bacterial/genetics ; Bacteria/genetics ; *RNA, Small Untranslated/genetics ; },
abstract = {Small RNAs (sRNAs) are versatile regulators universally present in species across the prokaryotic kingdom, yet their functional characterization remains a major bottleneck. Gene inactivation through random transposon insertion has proven extremely valuable in discovering hidden gene functions. However, this approach is biased toward long genes and usually results in the underrepresentation of sRNA mutants. In contrast, CRISPR interference (CRISPRi) harnesses guide RNAs to recruit cleavage-deficient Cas nucleases to specific DNA loci. The ensuing steric hindrance inhibits RNA polymerase assembly at-or migration along-predefined genes, allowing for targeted knockdown screens without major length bias. In this chapter, we provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs. Using the abundant microbiota species Bacteroides thetaiotaomicron as a model, we describe the design and generation of a guide library targeting the full intergenic sRNA repertoire of this organism and its application to identify sRNA knockdown-associated fitness effects. Our protocol is generic and thus suitable for the systematic assessment of sRNA-associated phenotypes in a wide range of bacterial species and experimental conditions. We expect CRISPRi-based functional genomics to boost sRNA research in understudied bacterial taxa, for instance, members of the gut microbiota.},
}
@article {pmid38216797,
year = {2024},
author = {Iqbal, S and Begum, F},
title = {Identification and characterization of integrated prophages and CRISPR-Cas system in Bacillus subtilis RS10 genome.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {},
number = {},
pages = {},
pmid = {38216797},
issn = {1678-4405},
abstract = {Bacteriophages have been extensively investigated due to their prominent role in the virulence and resistance of pathogenic bacteria. However, little attention has been given to the non-pathogenic Bacillus phages, and their role in the ecological bacteria genome is overlooked. In the present study, we characterized two Bacillus phages with a linear DNA genome of 33.6 kb with 44.83% GC contents and 129.3 kb with 34.70% GC contents. A total of 46 and 175 putative coding DNA sequences (CDS) were identified in prophage 1 (P1) and prophage 2 (P2), respectively, with no tRNA genes. Comparative genome sequence analysis revealed that P1 shares eight CDS with phage Jimmer 2 (NC-041976), and phage Osiris (NC-028969), and six with phage phi CT9441A (NC-029022). On the other hand, P2 showed high similarity with Bacill_SPbeta_NC_001884 and Bacillus phage phi 105. Further, genome analysis indicates several horizontal gene transfer events in both phages during the evolution process. In addition, we detected two CRISPR-Cas systems for the first time in B. subtilis. The identified CRISPR system consists of 24 and 25 direct repeats and integrase coding genes, while the cas gene which encodes Cas protein involved in the cleavage of a target sequence is missing. These findings will expand the current knowledge of soil phages as well as help to develop a new perspective for investigating more ecological phages to understand their role in bacterial communities and diversity.},
}
@article {pmid38216559,
year = {2024},
author = {Kuo, HC and Prupes, J and Chou, CW and Finkelstein, IJ},
title = {Massively parallel profiling of RNA-targeting CRISPR-Cas13d.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {498},
pmid = {38216559},
issn = {2041-1723},
support = {R01 GM124141/GM/NIGMS NIH HHS/United States ; },
mesh = {*RNA/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; },
abstract = {CRISPR-Cas13d cleaves RNA and is used in vivo and for diagnostics. However, a systematic understanding of its RNA binding and cleavage specificity is lacking. Here, we describe an RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) for measuring the binding affinity for > 10,000 RNAs containing structural perturbations and other alterations relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d reveals that it does not require a protospacer flanking sequence but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is penalized by mismatches in the distal crRNA-target RNA region, while alterations in the proximal region inhibit nuclease activity. A biophysical model built from these data reveals that target recognition initiates in the distal end of the target RNA. Using this model, we design crRNAs that can differentiate between SARS-CoV-2 variants by modulating nuclease activation. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme.},
}
@article {pmid38215900,
year = {2024},
author = {Manske, K and Dreßler, L and Fräßle, SP and Effenberger, M and Tschulik, C and Cletiu, V and Benke, E and Wagner, M and Schober, K and Müller, TR and Stemberger, C and Germeroth, L and Busch, DH and Poltorak, MP},
title = {Miniaturized CAR knocked onto CD3ε extends TCR function with CAR specificity under control of endogenous TCR signaling cascade.},
journal = {Journal of immunological methods},
volume = {526},
number = {},
pages = {113617},
doi = {10.1016/j.jim.2024.113617},
pmid = {38215900},
issn = {1872-7905},
abstract = {Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.},
}
@article {pmid38213321,
year = {2024},
author = {Gil, M and Hamann, CA and Brunger, JM and Gama, V},
title = {Engineering a CRISPRoff Platform to Modulate Expression of Myeloid Cell Leukemia (MCL-1) in Committed Oligodendrocyte Neural Precursor Cells.},
journal = {Bio-protocol},
volume = {14},
number = {1},
pages = {e4913},
pmid = {38213321},
issn = {2331-8325},
abstract = {In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems. Key features • Generation of an inducible CRISPRoff Sleeping Beauty transposon system. • Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation. • Strategy to downregulate an essential gene at different stages of oligodendrocyte development.},
}
@article {pmid38212640,
year = {2024},
author = {Herring-Nicholas, A and Dimig, H and Roesing, MR and Josephs, EA},
title = {Selection of extended CRISPR RNAs with enhanced targeting and specificity.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {86},
pmid = {38212640},
issn = {2399-3642},
support = {R21 EB033595/EB/NIBIB NIH HHS/United States ; R35 GM133483/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; RNA ; Genetic Therapy ; },
abstract = {As CRISPR effectors like Cas9 increasingly enter clinical trials for therapeutic gene editing, a future for personalized medicine will require efficient methods to protect individuals from the potential of off-target mutations that may also occur at specific sequences in their genomes that are similar to the therapeutic target. A Cas9 enzyme's ability to recognize their targets (and off-targets) are determined by the sequence of their RNA-cofactors (their guide RNAs or gRNAs). Here, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5' nucleotide extensions near its DNA-targeting segment-a modification that can increase gene editing specificity by orders of magnitude-to identify extended gRNAs (x-gRNAs) that effectively block any activity at those off-target sites while still maintaining strong activity at their intended targets. X-gRNAs that have been selected for specific target / off-target pairs can significantly out-perform other methods that reduce Cas9 off-target activity overall, like using Cas9 variants engineered for higher specificity in general, and we demonstrate their effectiveness in clinically-relevant gRNAs. Our streamlined approach to efficiently identify highly specific and active x-gRNAs provides a way to move beyond a one-size-fits-all model of high-fidelity CRISPR for safer and more effective personalized gene therapies.},
}
@article {pmid38211976,
year = {2024},
author = {Martínez-Trejo, A and Ruiz-Ruiz, JM and Gonzalez-Avila, LU and Saldaña-Padilla, A and Hernández-Cortez, C and de Jesús Colmenero-Solís, R and Bello-López, JM and Castro-Escarpulli, G},
title = {The CRISPR-Cas system in clinical strains of Acinetobacter baumannii: an in-silico analysis.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/lambio/ovae003},
pmid = {38211976},
issn = {1472-765X},
abstract = {Acinetobacter baumannii is a relevant bacterium due to its high resistance profiles. It is well known that antimicrobial resistance is primarily linked to mutations and the acquisition of external genomic material, such as plasmids or phages which the CRISPR-Cas system was related. It is known that the system can influence the acquisition of foreign genetic material and play a role in various physiological pathways. In this study, we conducted an in-silico analysis using 91 fully assembled genomes of clinical strains obtained from the NCBI database. Among the analyzed genomes, the I-F1 subtype of the CRISPR-Cas system was detected showcasing variations in architecture and phylogeny. Using bioinformatic tools we determined the presence, distribution, and specific characteristics of the CRISPR-Cas system we found possible association of the system with resistance genes but no with virulence determinants. Analysis of the system's components, including spacer sequences, suggests its potential role in protecting against phage infections, highlighting its protective function.},
}
@article {pmid38211734,
year = {2024},
author = {Zahedipour, F and Zahedipour, F and Zamani, P and Jaafari, MR and Sahebkar, A},
title = {Harnessing CRISPR technology for viral therapeutics and vaccines: from preclinical studies to clinical applications.},
journal = {Virus research},
volume = {341},
number = {},
pages = {199314},
doi = {10.1016/j.virusres.2024.199314},
pmid = {38211734},
issn = {1872-7492},
abstract = {The CRISPR/Cas system, identified as a type of bacterial adaptive immune system, have attracted significant attention due to its remarkable ability to precisely detect and eliminate foreign genetic material and nucleic acids. Expanding upon these inherent capabilities, recent investigations have unveiled the potential of reprogrammed CRISPR/Cas 9, 12, and 13 systems for treating viral infections associated with human diseases, specifically targeting DNA and RNA viruses, respectively. Of particular interest is the RNA virus responsible for the recent global outbreak of coronavirus disease 2019 (COVID-19), which presents a substantial public health risk, coupled with limited efficacy of current prophylactic and therapeutic techniques. In this regard, the utilization of CRISPR/Cas technology offers a promising gene editing approach to overcome the limitations of conventional methods in managing viral infections. This comprehensive review provides an overview of the latest CRISPR/Cas-based therapeutic and vaccine strategies employed to combat human viral infections. Additionally, we discuss significant challenges and offer insights into the future prospects of this cutting-edge gene editing technology.},
}
@article {pmid38203615,
year = {2023},
author = {Davletshin, AI and Matveeva, AA and Bachurin, SS and Karpov, DS and Garbuz, DG},
title = {Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain.},
journal = {International journal of molecular sciences},
volume = {25},
number = {1},
pages = {},
pmid = {38203615},
issn = {1422-0067},
support = {22-14-00377//Russian Science Foundation/ ; 075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; Mutagenesis ; Mutation ; *Chromatin ; Amino Acids ; },
abstract = {CRISPR/Cas systems are used for genome editing, both in basic science and in biotechnology. However, CRISPR/Cas editors have several limitations, including insufficient specificity leading to "off-targets" and the dependence of activity on chromatin state. A number of highly specific Cas9 variants have now been obtained, but most of them are characterized by reduced activity on eukaryotic chromatin. We identified a spatial cluster of amino acid residues in the PAM-recognizing domain of Streptococcus pyogenes Cas9, whose mutations restore the activity of one of the highly specific forms of SpyCas9 without reducing its activity in Saccharomyces cerevisiae. In addition, one of these new mutations also increases the efficiency of SpyCas9-mediated editing of a site localized on the stable nucleosome. The improved Cas9 variants we obtained, which are capable of editing hard-to-reach regions of the yeast genome, may help in both basic research and yeast biotechnological applications.},
}
@article {pmid38182795,
year = {2024},
author = {Torella, L and Klermund, J and Bilbao-Arribas, M and Tamayo, I and Andrieux, G and Chmielewski, KO and Vales, A and Olagüe, C and Moreno-Luqui, D and Raimondi, I and Abad, A and Torrens-Baile, J and Salido, E and Huarte, M and Hernaez, M and Boerries, M and Cathomen, T and Zabaleta, N and Gonzalez-Aseguinolaza, G},
title = {Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.},
journal = {EMBO molecular medicine},
volume = {16},
number = {1},
pages = {112-131},
pmid = {38182795},
issn = {1757-4684},
support = {RTI2018-101936-B-I00//MEC | Spanish National Plan for Scientific and Technical Research and Innovation (Plan Estatal de Investigaci&#xf3;n Cient&#xed;fica y T&#xe9;cnica y de Innovaci&#xf3;n)/ ; TERAV Network RD21/0017/0001//MEC | Instituto de Salud Carlos III (ISCIII)/ ; CA311/4-1 and CRC1160,Project ID 256073931- A07//German Research Foundation/ ; CRC/TRR167 (ID 259373024-Z01),CRC1453 (ID 431984000-S1),and CRC1479 (ID: 441891347- S1)//German Research Foundation/ ; MIRACUM-FKZ 01ZZ1801B//Bundesministerium f&#xfc;r Bildung und Forschung (BMBF)/ ; EkoEstMed-FKZ 01ZZ2015//Bundesministerium f&#xfc;r Bildung und Forschung (BMBF)/ ; AAVolution EC project number 101071041//European Commission (EC)/ ; GenE-HumDi CA21113//European Commission (EC)/ ; },
mesh = {Humans ; Animals ; Mice ; *CRISPR-Cas Systems ; Deoxyribonuclease I/genetics/metabolism ; Gene Editing ; *Hyperoxaluria, Primary/genetics/therapy ; },
abstract = {The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.},
}
@article {pmid38112450,
year = {2024},
author = {Peng, L and Fang, T and Cai, Q and Li, H and Li, H and Sun, H and Zhu, M and Dai, L and Shao, Y and Cai, L},
title = {Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction.},
journal = {Journal of clinical microbiology},
volume = {62},
number = {1},
pages = {e0092323},
pmid = {38112450},
issn = {1098-660X},
support = {LGC22H200013//Zhejiang Provincial Natural Science Foundation of China/ ; Z20220032//Hangzhou Medical Health Science and Technology Project/ ; ZD20210018//Hangzhou Medical Health Science and Technology Project/ ; HHQN 2022001//Hangzhou Red Cross Hospital Youth Fund/ ; },
mesh = {Humans ; *Mycobacterium tuberculosis/genetics ; Sputum/microbiology ; Cross-Priming ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; *Tuberculosis/diagnosis/microbiology ; },
abstract = {In this study, we successfully established a new One-Pot method, named TB One-Pot, for detecting Mtb in sputum by combining CRISPR-cas12b-mediated trans-cleavage with cross-priming amplification (CPA). Our study evaluated the diagnostic performance of TB One-Pot in clinical sputum samples for tuberculosis. The findings provide evidence for the potential of TB One-Pot as a diagnostic tool for tuberculosis.},
}
@article {pmid37061991,
year = {2024},
author = {Sahu, A and Verma, R and Gupta, U and Kashyap, S and Sanyal, I},
title = {An Overview of Targeted Genome Editing Strategies for Reducing the Biosynthesis of Phytic Acid: an Anti-nutrient in Crop Plants.},
journal = {Molecular biotechnology},
volume = {66},
number = {1},
pages = {11-25},
pmid = {37061991},
issn = {1559-0305},
mesh = {*Gene Editing ; *Phytic Acid/metabolism ; Crops, Agricultural/genetics ; Nutrients ; Lipids ; CRISPR-Cas Systems ; },
abstract = {Anti-nutrients are substances either found naturally or are of synthetic origin, which leads to the inactivation of nutrients and limits their utilization in metabolic processes. Phytic acid is classified as an anti-nutrient, as it has a strong binding affinity with most minerals like Fe, Zn, Mg, Ca, Mn, and Cd and impairs their proper metabolism. Removing anti-nutrients from cereal grains may enable the bioavailability of both macro- and micronutrients which is the desired goal of genetic engineering tools for the betterment of agronomic traits. Several strategies have been adopted to minimize phytic acid content in plants. Pursuing the molecular strategies, there are several studies, which result in the decrement of the total phytic acid content in grains of major as well as minor crops. Biosynthesis of phytic acid mainly takes place in the seed comprising lipid-dependent and lipid-independent pathways, involving various enzymes. Furthermore, some studies show that interruption of these enzymes may involve the pleiotropic effect. However, using modern biotechnological approaches, undesirable agronomic traits can be removed. This review presents an overview of different genes encoding the various enzymes involved in the biosynthetic pathway of phytic acid which is being targeted for its reduction. It also, highlights and enumerates the variety of potential applications of genome editing tools such as TALEN, ZFN, and CRISPR/Cas9 to knock out the desired genes, and RNAi for their silencing.},
}
@article {pmid36991112,
year = {2024},
author = {Lampe, GD and King, RT and Halpin-Healy, TS and Klompe, SE and Hogan, MI and Vo, PLH and Tang, S and Chavez, A and Sternberg, SH},
title = {Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases.},
journal = {Nature biotechnology},
volume = {42},
number = {1},
pages = {87-98},
pmid = {36991112},
issn = {1546-1696},
support = {DP2 HG011650/HG/NHGRI NIH HHS/United States ; F31 HL167530/HL/NHLBI NIH HHS/United States ; T32 GM145440/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Transposases/genetics ; Plasmids ; DNA ; Genome ; Gene Editing ; },
abstract = {Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.},
}
@article {pmid38203503,
year = {2023},
author = {Bartosh, UI and Dome, AS and Zhukova, NV and Karitskaya, PE and Stepanov, GA},
title = {CRISPR/Cas9 as a New Antiviral Strategy for Treating Hepatitis Viral Infections.},
journal = {International journal of molecular sciences},
volume = {25},
number = {1},
pages = {},
pmid = {38203503},
issn = {1422-0067},
support = {122022100238-7//Russian state-funded project for ICBFM SB RAS/ ; },
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Carcinoma, Hepatocellular ; *Liver Neoplasms ; RNA, Guide, CRISPR-Cas Systems ; Hepatitis Viruses ; *Hepatitis A ; *Hepatitis C ; Antiviral Agents/pharmacology/therapeutic use ; },
abstract = {Hepatitis is an inflammatory liver disease primarily caused by hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. The chronic forms of hepatitis resulting from HBV and HCV infections can progress to cirrhosis or hepatocellular carcinoma (HCC), while acute hepatitis can lead to acute liver failure, sometimes resulting in fatality. Viral hepatitis was responsible for over 1 million reported deaths annually. The treatment of hepatitis caused by viral infections currently involves the use of interferon-α (IFN-α), nucleoside inhibitors, and reverse transcriptase inhibitors (for HBV). However, these methods do not always lead to a complete cure for viral infections, and chronic forms of the disease pose significant treatment challenges. These facts underscore the urgent need to explore novel drug developments for the treatment of viral hepatitis. The discovery of the CRISPR/Cas9 system and the subsequent development of various modifications of this system have represented a groundbreaking advance in the quest for innovative strategies in the treatment of viral infections. This technology enables the targeted disruption of specific regions of the genome of infectious agents or the direct manipulation of cellular factors involved in viral replication by introducing a double-strand DNA break, which is targeted by guide RNA (spacer). This review provides a comprehensive summary of our current knowledge regarding the application of the CRISPR/Cas system in the regulation of viral infections caused by HAV, HBV, and HCV. It also highlights new strategies for drug development aimed at addressing both acute and chronic forms of viral hepatitis.},
}
@article {pmid38203173,
year = {2023},
author = {Blau, K and Gallert, C},
title = {Prophage Carriage and Genetic Diversity within Environmental Isolates of Clostridioides difficile.},
journal = {International journal of molecular sciences},
volume = {25},
number = {1},
pages = {},
pmid = {38203173},
issn = {1422-0067},
support = {GA 546/13-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Humans ; Prophages/genetics ; Clostridioides ; *Clostridioides difficile/genetics ; Phylogeny ; *Bacteriophages/genetics ; Genetic Variation ; },
abstract = {Clostridioides difficile is an important human pathogen causing antibiotic-associated diarrhoea worldwide. Besides using antibiotics for treatment, the interest in bacteriophages as an alternative therapeutic option has increased. Prophage abundance and genetic diversity are well-documented in clinical strains, but the carriage of prophages in environmental strains of C. difficile has not yet been explored. Thus, the prevalence and genetic diversity of integrated prophages in the genomes of 166 environmental C. difficile isolates were identified. In addition, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were determined in the genomes of prophage regions. Predicted prophages and CRISPR-Cas systems were identified by using the PHASTER web server and CRISPRCasFinder, respectively. Phylogenetic relationships among predicated prophages were also constructed based on phage-related genes, terminase large (TerL) subunits and LysM. Among 372 intact prophages, the predominant prophages were phiCDHM1, phiCDHM19, phiMMP01, phiCD506, phiCD27, phiCD211, phiMMP03, and phiC2, followed by phiMMP02, phiCDKM9, phiCD6356, phiCDKM15, and phiCD505. Two newly discovered siphoviruses, phiSM101- and phivB_CpeS-CP51-like Clostridium phages, were identified in two C. difficile genomes. Most prophages were found in sequence types (STs) ST11, ST3, ST8, ST109, and ST2, followed by ST6, ST17, ST4, ST5, ST44, and ST58. An obvious correlation was found between prophage types and STs/ribotypes. Most predicated prophages carry CRISPR arrays. Some prophages carry several gene products, such as accessory gene regulator (Agr), putative spore protease, and abortive infection (Abi) systems. This study shows that prophage carriage, along with genetic diversity and their CRISPR arrays, may play a role in the biology, lifestyle, and fitness of their host strains.},
}
@article {pmid38201231,
year = {2023},
author = {Kim, IS},
title = {DNA Barcoding Technology for Lineage Recording and Tracing to Resolve Cell Fate Determination.},
journal = {Cells},
volume = {13},
number = {1},
pages = {},
pmid = {38201231},
issn = {2073-4409},
support = {NRF-2021R1A5A2030333//National Research Foundation of Korea/ ; SSTF-BA2102-08//Samsung Science and Technology Foundation/ ; },
mesh = {*DNA Barcoding, Taxonomic ; Phylogeny ; Cell Differentiation ; *DNA ; Technology ; },
abstract = {In various biological contexts, cells receive signals and stimuli that prompt them to change their current state, leading to transitions into a future state. This change underlies the processes of development, tissue maintenance, immune response, and the pathogenesis of various diseases. Following the path of cells from their initial identity to their current state reveals how cells adapt to their surroundings and undergo transformations to attain adjusted cellular states. DNA-based molecular barcoding technology enables the documentation of a phylogenetic tree and the deterministic events of cell lineages, providing the mechanisms and timing of cell lineage commitment that can either promote homeostasis or lead to cellular dysregulation. This review comprehensively presents recently emerging molecular recording technologies that utilize CRISPR/Cas systems, base editing, recombination, and innate variable sequences in the genome. Detailing their underlying principles, applications, and constraints paves the way for the lineage tracing of every cell within complex biological systems, encompassing the hidden steps and intermediate states of organism development and disease progression.},
}
@article {pmid38164718,
year = {2024},
author = {Qi, L and Liu, J and Liu, S and Liu, Y and Xiao, Y and Zhang, Z and Zhou, W and Jiang, Y and Fang, X},
title = {Ultrasensitive Point-of-Care Detection of Protein Markers Using an Aptamer-CRISPR/Cas12a-Regulated Liquid Crystal Sensor (ALICS).},
journal = {Analytical chemistry},
volume = {96},
number = {2},
pages = {866-875},
doi = {10.1021/acs.analchem.3c04492},
pmid = {38164718},
issn = {1520-6882},
mesh = {Point-of-Care Systems ; CRISPR-Cas Systems ; *Liquid Crystals ; RNA, Guide, CRISPR-Cas Systems ; Oligonucleotides ; DNA Probes ; *Biosensing Techniques ; },
abstract = {Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.},
}
@article {pmid38200865,
year = {2023},
author = {Punetha, M and Kumar, D and Saini, S and Chaudhary, S and Bajwa, KK and Sharma, S and Mangal, M and Yadav, PS and Green, JA and Whitworth, K and Datta, TK},
title = {Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.},
journal = {Animals : an open access journal from MDPI},
volume = {14},
number = {1},
pages = {},
pmid = {38200865},
issn = {2076-2615},
support = {NASF/GTR-8025/2020-21//NASF/ ; SERB/SRG/2021/00131//SERB/ ; },
abstract = {Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.},
}
@article {pmid38171948,
year = {2024},
author = {Keshavan, N and Minczuk, M and Viscomi, C and Rahman, S},
title = {Gene therapy for mitochondrial disorders.},
journal = {Journal of inherited metabolic disease},
volume = {47},
number = {1},
pages = {145-175},
doi = {10.1002/jimd.12699},
pmid = {38171948},
issn = {1573-2665},
support = {/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; Animals ; Mice ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; DNA, Mitochondrial/genetics ; Endonucleases/genetics/metabolism ; *Mitochondrial Diseases/genetics/therapy ; },
abstract = {In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.},
}
@article {pmid38070592,
year = {2024},
author = {Alejandra Llanes-Cuesta, M and Hoi, V and Ha, R and Tan, H and Imamul Islam, M and Eftekharpour, E and Wang, JF},
title = {Redox Protein Thioredoxin Mediates Neurite Outgrowth in Primary Cultured Mouse Cerebral Cortical Neurons.},
journal = {Neuroscience},
volume = {537},
number = {},
pages = {165-173},
doi = {10.1016/j.neuroscience.2023.12.002},
pmid = {38070592},
issn = {1873-7544},
mesh = {Mice ; Humans ; Animals ; *RNA, Guide, CRISPR-Cas Systems ; Cyclic AMP Response Element-Binding Protein/metabolism ; Hydrogen Peroxide/metabolism ; *Neuroblastoma/metabolism ; Thioredoxins/metabolism ; Neurons/metabolism ; Oxidation-Reduction ; Neuronal Outgrowth ; },
abstract = {Thioredoxin system plays an important role in maintaining the cellular redox balance. Recent evidence suggests that thioredoxin (Trx) system may promote cell survival and neuroprotection. In this study, we explored the role of thioredoxin system in neuronal differentiation using a primary mouse cortical neuronal cell culture. First, Trx and Trx reductase (TrxR) protein levels were analyzed in cultured neurons from 1 to 32 days in vitro (DIV). The result showed that Trx and TrxR protein levels time-dependently increased in the neuron cell culture from 1 to 18 DIV. To establish the role of Trx in neuronal differentiation, Trx gene expression was knockdown in cultured neurons using Trx sgRNA CRISPR/Cas9 technology. Treatment with CRISPR/Cas9/Trx sgRNA decreased Trx protein levels and caused a reduction in dendritic outgrowth and branching of cultured neurons. Then, primary cortical neurons were treated with the Trx inhibitor PX12 to block Trx reducing activity. Treatment with PX12 also reduced dendritic outgrowth and branching. Furthermore, PX12 treatment reduced the ratio of phosphorylated cyclic AMP response element-binding protein (CREB)/total CREB protein levels. To investigate whether CREB phosphorylation is redox regulated, SH-SY5Y cells were treated with H2O2, which reduced phosphorylated CREB protein levels and increased CREB thiol oxidation. However, treatment with CB3, a Trx-mimetic tripeptide, rescued H2O2-decreased CREB phosphorylation. Our results suggest that Trx regulates neuronal differentiation and maturation of primary mouse cortical neurons by targeting CREB neurotrophic pathway. Trx may regulate CREB activation by maintaining the cellular redox balance.},
}
@article {pmid37994543,
year = {2024},
author = {Ma, S and Wang, F and Xuejing, Z and Liping, Q and Xueping, G and Lu, X and Qi, Q},
title = {Repurposing endogenous type II CRISPR-Cas9 system for genome editing in Streptococcus thermophilus.},
journal = {Biotechnology and bioengineering},
volume = {121},
number = {2},
pages = {749-756},
doi = {10.1002/bit.28608},
pmid = {37994543},
issn = {1097-0290},
support = {2021YFC2100500//National Key Research and Development Program of China/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Streptococcus thermophilus/genetics ; DNA ; },
abstract = {Streptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II-A CRISPR-Cas9, by delivering a self-targeting CRISPR array with DNA repair template into S. thermophilus LMD-9. We achieved 785-bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1-kb DNA repair template was provided. While providing with 1.5-kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234-bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%-100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single-gene knockout mutants with efficiencies of 29%-80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD-9. Altogether, repurposing the native type II-A CRISPR-Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications.},
}
@article {pmid37542151,
year = {2024},
author = {Narváez-Pérez, LF and Paz-Bermúdez, F and Avalos-Fuentes, JA and Campos-Romo, A and Florán-Garduño, B and Segovia, J},
title = {CRISPR/sgRNA-directed synergistic activation mediator (SAM) as a therapeutic tool for Parkinson´s disease.},
journal = {Gene therapy},
volume = {31},
number = {1-2},
pages = {31-44},
pmid = {37542151},
issn = {1476-5462},
mesh = {Rats ; Animals ; *Parkinson Disease/genetics/therapy/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Oxidopamine ; Rats, Sprague-Dawley ; *Neurodegenerative Diseases ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dopamine/metabolism ; Corpus Striatum/metabolism ; Tyrosine 3-Monooxygenase/genetics/metabolism/pharmacology ; Substantia Nigra/metabolism ; },
abstract = {Parkinson`s disease (PD) is the second most prevalent neurodegenerative disease, and different gene therapy strategies have been used as experimental treatments. As a proof-of-concept for the treatment of PD, we used SAM, a CRISPR gene activation system, to activate the endogenous tyrosine hydroxylase gene (th) of astrocytes to produce dopamine (DA) in the striatum of 6-OHDA-lesioned rats. Potential sgRNAs within the rat th promoter region were tested, and the expression of the Th protein was determined in the C6 glial cell line. Employing pseudo-lentivirus, the SAM complex and the selected sgRNA were transferred into cultures of rat astrocytes, and gene expression and Th protein synthesis were ascertained; furthermore, DA release into the culture medium was determined by HPLC. The DA-producing astrocytes were implanted into the striatum of 6-OHDA hemiparkinsonian rats. We observed motor behavior improvement in the lesioned rats that received DA-astrocytes compared to lesioned rats receiving astrocytes that did not produce DA. Our data indicate that the SAM-induced expression of the astrocyte´s endogenous th gene can generate DA-producing astrocytes that effectively reduce the motor asymmetry induced by the lesion.},
}
@article {pmid38200316,
year = {2024},
author = {Baca, CF and Yu, Y and Rostøl, JT and Majumder, P and Patel, DJ and Marraffini, LA},
title = {The CRISPR effector Cam1 mediates membrane depolarization for phage defence.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {38200316},
issn = {1476-4687},
abstract = {Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors[1-5]. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain[6,7]. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.},
}
@article {pmid38200015,
year = {2024},
author = {Zhen, X and Xu, X and Ye, L and Xie, S and Huang, Z and Yang, S and Wang, Y and Li, J and Long, F and Ouyang, S},
title = {Structural basis of antiphage immunity generated by a prokaryotic Argonaute-associated SPARSA system.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {450},
pmid = {38200015},
issn = {2041-1723},
support = {82225028,82172287//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22173020//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {NAD ; RNA, Guide, CRISPR-Cas Systems ; Argonaute Proteins/genetics ; *Bacteriophages/genetics ; *Nucleic Acids ; },
abstract = {Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of prokaryotic counterparts (pAgo) are less well known. Recently, short pAgos in conjunction with the associated TIR or Sir2 (SPARTA or SPARSA) were found to serve as antiviral systems to combat phage infections. Herein, we present the cryo-EM structures of nicotinamide adenine dinucleotide (NAD[+])-bound SPARSA with and without nucleic acids at resolutions of 3.1 Å and 3.6 Å, respectively. Our results reveal that the APAZ (Analogue of PAZ) domain and the short pAgo form a featured architecture similar to the long pAgo to accommodate nucleic acids. We further identified the key residues for NAD[+] binding and elucidated the structural basis for guide RNA and target DNA recognition. Using structural comparisons, molecular dynamics simulations, and biochemical experiments, we proposed a putative mechanism for NAD[+] hydrolysis in which an H186 loop mediates nucleophilic attack by catalytic water molecules. Overall, our study provides mechanistic insight into the antiphage role of the SPARSA system.},
}
@article {pmid38197893,
year = {2024},
author = {Mahfouz, M},
title = {Revolutionizing Point-of-Care Diagnostics via CRISPR Systems.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00763},
pmid = {38197893},
issn = {2161-5063},
}
@article {pmid38195902,
year = {2024},
author = {Jalota, K and Sharma, V and Agarwal, C and Jindal, S},
title = {Eco-friendly approaches to phytochemical production: elicitation and beyond.},
journal = {Natural products and bioprospecting},
volume = {14},
number = {1},
pages = {5},
pmid = {38195902},
issn = {2192-2195},
abstract = {Highly ameliorated phytochemicals from plants are recognized to have numerous beneficial effects on human health. However, obtaining secondary metabolites directly from wild plants is posing a great threat to endangered plant species due to their over exploitation. Moreover, due to complicated structure and stereospecificity chemical synthesis of these compounds is a troublesome procedure. As a result, sustainable and ecofriendly in vitro strategy has been adopted for phytochemicals production. But, lack of fully differentiated cells lowers down cultured cells productivity. Consequently, for enhancing yield of metabolites produced by cultured plant cells a variety of methodologies has been followed one such approach includes elicitation of culture medium that provoke stress responses in plants enhancing synthesis and storage of bioactive compounds. Nevertheless, for conclusive breakthrough in synthesizing bioactive compounds at commercial level in-depth knowledge regarding metabolic responses to elicitation in plant cell cultures is needed. However, technological advancement has led to development of molecular based approaches like metabolic engineering and synthetic biology which can serve as promising path for phytochemicals synthesis. This review article deals with classification, stimulating effect of elicitors on cultured cells, parameters of elicitors and action mechanism in plants, modern approaches like metabolic engineering for future advances.},
}
@article {pmid38195874,
year = {2024},
author = {Marshall, M},
title = {How CRISPR could yield the next blockbuster crop.},
journal = {Nature},
volume = {625},
number = {7994},
pages = {230-232},
pmid = {38195874},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing/methods/trends ; Genes, Plant ; Indigenous Peoples ; *Plants/genetics ; },
}
@article {pmid38192601,
year = {2024},
author = {Guo, K and Zhao, J and Fang, S and Zhang, Q and Nie, L and Zhao, W},
title = {The effects of different rootstocks on aroma components, activities and genes expression of aroma-related enzymes in oriental melon fruit.},
journal = {PeerJ},
volume = {12},
number = {},
pages = {e16704},
pmid = {38192601},
issn = {2167-8359},
mesh = {Odorants ; Fruit/genetics ; *Cucumis melo ; Alcohol Dehydrogenase ; Smell/genetics ; *Cucurbita ; },
abstract = {Grafting is widely applied in the cultivation of melon. In this study, 'Qinmi No.1' (Cucumis melo L.(QG)) and 'Ribenxuesong' (Cucurbita maxima Duch. (RG)) were used as rootstocks for 'Qingxin Yangjiaocui' (Cucumis melo L.). The results showed that grafting with muskmelon rootstocks had no significant effect on fruit aroma, but grafting with pumpkin rootstocks significantly reduced the odor intensity and odor preference scores of melon fruits. Compared with the fruits from self-grafted plants (SG), four new aromatic volatiles with a sweet smell were detected, the alcohol dehydrogenase (ADH) activity was significantly decreased at 30 DAP, but unaffected at 42 DAP in QG fruits. There was no difference for alcohol acetyltransferase (AAT) activity between QG and SG fruits. The expression level of CmADH2 was significantly higher at 30 DAP and 42 DAP, but CmAAT2 was significantly lower at 42 DAP in QG fruits compared with SG fruits. In RG fruits, the main aroma compounds including butanoic acid ethyl ester, 2-methyl-2-butene-1-al, and 2-methylheptan-1-al were absent, while the volatile compounds with unpleasant odor characteristics including trans, cis-2,6-nonadien-1-ol, (E,E)-2,4-heptadienal, octanoic acid, and styrene were detected. Compared with SG fruits, 1-nonanol and 1-heptanol with green odor characteristics were significantly increased, but eucalyptol and farnesene with fruity aroma characteristics were significantly decreased in RG fruits. The ADH activity of RG fruits was significantly lower than that of SG fruits at 30 DAP and the AAT activity was significantly lower than that of SG fruits at 42 DAP. In addition, the expression levels of CmADH and CmAAT homologs in RG fruits were significantly lower than those in SG or QG fruits. These results show that grafting with pumpkin rootstocks affected the main aroma components, reduced ADH and AAT activities, and down-regulated the expression levels of CmADHs and CmAATs in the melon fruits. This study reveals the mechanism of different rootstocks on melon fruit aroma quality, and lays a theoretical foundation for the selection of rootstocks in melon production. Future studies using overexpression or CRISPR/CAS system to obtain stable transgenic lines of genes encoding key aromatic volatiles, would be promising to effectively improve the flavor quality of melon.},
}
@article {pmid38183888,
year = {2024},
author = {Duboscq-Bidot, L and Hoareau, B and Ader, F and Fontaine, V and Villard, E},
title = {Generation of CRISPR-Cas9 edited human induced pluripotent stem cell line carrying BAG3 V468M mutation in its BAG domain.},
journal = {Stem cell research},
volume = {74},
number = {},
pages = {103294},
doi = {10.1016/j.scr.2023.103294},
pmid = {38183888},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Apoptosis Regulatory Proteins/genetics ; Adaptor Proteins, Signal Transducing/genetics ; Mutation/genetics ; *Cardiomyopathy, Dilated/genetics ; },
abstract = {BCL2-Associated Athanogene 3 (BAG3) gene was identified mutated in patients with dilated cardiomyopathy (DCM), an important cause of heart failure and premature death. BAG3 is a cytoprotective co-chaperonne protein involved in many cellular process with a central role in the maintenance of protostasis. We generated two human induced pluripotent stem cell lines (hiPSc), one carrying the heterozygous, the other the homozygous p.V468M mutation identified in DCM familial cases. All lines expressed pluripotent markers, had normal karyotype, and differentiated into derivatives of the three germ layers. Sudies of hiPSc derived cardiomyocytes will help to understand the role of BAG3 in DCM.},
}
@article {pmid38118206,
year = {2024},
author = {Sun, W and Li, X and Dong, J and Zhou, Y},
title = {Generation of a TAB2 knockout hESC line (WAe009-A-Z) derived from H9 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {74},
number = {},
pages = {103284},
doi = {10.1016/j.scr.2023.103284},
pmid = {38118206},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Signal Transduction ; Adaptor Proteins, Signal Transducing/genetics ; },
abstract = {TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediate protein that connects TNFR1 and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 has been proved clinically relevant to congenital heart defects (CHD) and cardiomyopathy. In this study, we created a TAB2 knockout human embryonic stem cell line by CRISPR/Cas9 technology. The WAe009-A-Z cell line displayed stem cell morphology, pluripotency and normal karyotype, which could develop into three germ layers in vitro.},
}
@article {pmid38104430,
year = {2024},
author = {Schreiber, MK and Zafeiriou, MP},
title = {Generation of Pelizaeus-Merzbacher disease (PMD) mutant (PLP1-C33Y) in induced pluripotent stem cell (iPSC) by CRISPR/Cas9 genome editing.},
journal = {Stem cell research},
volume = {74},
number = {},
pages = {103276},
doi = {10.1016/j.scr.2023.103276},
pmid = {38104430},
issn = {1876-7753},
mesh = {Humans ; *Pelizaeus-Merzbacher Disease/genetics ; Myelin Proteolipid Protein/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; },
abstract = {Genetic alterations in the PLP1 gene, i.e. point mutations and duplications, are associated with demyelinating disease Pelizaeus-Merzbacher. Here, we describe the generation of a human iPSC line harboring a PLP1 variant in codon 33 which leads to an amino acid change from cysteine to tyrosine. The established PLP1[C33Y] iPSC line enables the study of PMD pathophysiology by investigating various cell types and -characteristics in our developed protocol for bioengineered neuronal organoids (BENOs)[1].},
}
@article {pmid38103335,
year = {2024},
author = {Qiu, B and Zheng, W and Zhong, Y and Liu, H and Yu, J and Luo, Y and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {74},
number = {},
pages = {103283},
doi = {10.1016/j.scr.2023.103283},
pmid = {38103335},
issn = {1876-7753},
mesh = {Mice ; Humans ; Animals ; *Diphtheria Toxin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Integrases/metabolism ; Mice, Transgenic ; Homologous Recombination ; Cell Line ; },
abstract = {Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor-mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA-mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs.},
}
@article {pmid38100916,
year = {2024},
author = {Lahm, H and Singh, NK and Gottmann, I and Doppler, SA and Dzilic, E and Preisler, H and Schneider, S and Lange, R and Krane, M and Dreßen, M},
title = {Insertion of a FLAG-tag sequence at the end of exon 9 of the TBX5 gene in three induced pluripotent stem cell lines (DHMi004-A-4, DHMi004-A-5, DHMi004-A-6) by CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {74},
number = {},
pages = {103261},
doi = {10.1016/j.scr.2023.103261},
pmid = {38100916},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Homologous Recombination ; Exons/genetics ; },
abstract = {The identification of TBX5-related regulatory sequences in genes essential for heart development is hampered by the absence of antibodies which allow precipitation of TBX5:DNA complexes. Employing CRISPR/Cas9 technology, we have inserted a FLAG-tag sequence at the end of exon 9 of the TBX5 gene prior to the stop codon by homologous recombination. The translated TBX5-FLAG fusion protein of the three iPSC lines can effectively be precipitated by anti-FLAG antibodies and, thus, allow the detection of specific TBX5-binding sites and their associated genes.},
}
@article {pmid38078721,
year = {2024},
author = {Wang, Y and Tang, Y and Chen, Y and Yu, G and Zhang, X and Yang, L and Zhao, C and Wang, P and Gao, S},
title = {Ultrasensitive one-pot detection of monkeypox virus with RPA and CRISPR in a sucrose-aided multiphase aqueous system.},
journal = {Microbiology spectrum},
volume = {12},
number = {1},
pages = {e0226723},
pmid = {38078721},
issn = {2165-0497},
support = {2022M721665//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 2022ZB358//Jiangsu Funding Program for Excellent Postdoctoral Talent of China/ ; },
mesh = {Humans ; *Monkeypox virus/genetics ; *Mpox (monkeypox) ; Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Outbreaks ; Hydrolases ; Sucrose ; Recombinases ; CRISPR-Cas Systems ; },
abstract = {The monkeypox virus was declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) and continues to cause infection cases worldwide. Given the risk of virus evolution, it is essential to identify monkeypox virus infection in a timely manner to prevent outbreaks. This study establishes a novel one-pot recombinase polymerase amplification-Clustered Regularly Interspaced Short Palindromic Repeats (RPA-CRISPR) assay for monkeypox virus with an ultra-high sensitivity. The assay shows good specificity, accuracy, and the rapidness and convenience important for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox, which is useful for the prevention of an epidemic.},
}
@article {pmid38078715,
year = {2024},
author = {Wang, H and Li, H and Tang, B and Ye, C and Han, M and Teng, L and Yue, M and Li, Y},
title = {Fast and sensitive differential diagnosis of pseudorabies virus-infected versus pseudorabies virus-vaccinated swine using CRISPR-Cas12a.},
journal = {Microbiology spectrum},
volume = {12},
number = {1},
pages = {e0261723},
pmid = {38078715},
issn = {2165-0497},
support = {2022YFD1800100, 2019YFE0103900//MOST | National Key Research and Development Program of China (NKPs)/ ; 32102620//MOST | National Natural Science Foundation of China (NSFC)/ ; 31872837//MOST | National Natural Science Foundation of China (NSFC)/ ; LZ22C180004//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; 2021JJLH0083//Joint Project of Hainan Provincial Science and Technology Program and Sanya Yazhou Bay Science and Technology/ ; },
mesh = {Animals ; Swine ; *Herpesvirus 1, Suid/genetics ; *Pseudorabies/diagnosis/prevention &amp; control ; CRISPR-Cas Systems ; Diagnosis, Differential ; *Swine Diseases/diagnosis/prevention &amp; control ; Vaccines, Attenuated ; Antibodies, Viral ; },
abstract = {Pseudorabies virus (PRV) causes high mortality and miscarriage rates in the infected swine, and the eradication policy coupled with large-scale vaccination of live attenuated vaccines has been adopted globally against PRV. Differential diagnosis of the vaccinated and infected swine is highly demanded. Our multienzyme isothermal rapid amplification (MIRA)-Cas12a detection method described in this study can diagnose PRV with a superior sensitivity comparable to the quantitative PCR (qPCR) and a competitive detection speed (only half the time as qPCR needs). The portable feature and the simple procedure of MIRA-Cas12a make it easier to deploy for clinical diagnosis, even in resource-limited settings. The MIRA-Cas12a method would provide immediate and accurate diagnostic information for policymakers to respond promptly.},
}
@article {pmid38064043,
year = {2024},
author = {Shen, Q and Yan, F and Li, YW and Wang, J and Ji, J and Yan, WX and He, DC and Song, P and Shi, TQ},
title = {Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology.},
journal = {Biotechnology letters},
volume = {46},
number = {1},
pages = {37-46},
pmid = {38064043},
issn = {1573-6776},
support = {2021YFC2104300//National key research and development program of China/ ; BK20210573//Natural Science Foundation of Jiangsu Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Yarrowia/genetics/metabolism ; Arachidonic Acid/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Metabolic Engineering ; },
abstract = {Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.},
}
@article {pmid38054713,
year = {2024},
author = {St Pierre, J and Roberts, J and Alam, MA and Shields, RC},
title = {Construction of an arrayed CRISPRi library as a resource for essential gene function studies in Streptococcus mutans.},
journal = {Microbiology spectrum},
volume = {12},
number = {1},
pages = {e0314923},
pmid = {38054713},
issn = {2165-0497},
support = {P20 GM103429/GM/NIGMS NIH HHS/United States ; R03 DE029882/DE/NIDCR NIH HHS/United States ; },
mesh = {*Streptococcus mutans/genetics ; *Genes, Essential ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; CRISPR-Cas Systems ; },
abstract = {The construction of arrayed mutant libraries has advanced the field of bacterial genetics by allowing researchers to more efficiently study the exact function and importance of encoded genes. In this study, we constructed an arrayed clustered regularly interspaced short palindromic repeats interference (CRISPRi) library, known as S treptococcus mutans arrayed CRISPRi (SNAP), as a resource to study >250 essential and growth-supporting genes in Streptococcus mutans. SNAP will be made available to the research community, and we anticipate that its distribution will lead to high-quality, high-throughput, and reproducible studies of essential genes.},
}
@article {pmid38051195,
year = {2023},
author = {Green, EI and Jaouen, E and Klug, D and Proveti Olmo, R and Gautier, A and Blandin, S and Marois, E},
title = {A population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {38051195},
issn = {2050-084X},
support = {ANR-19-CE35-0007-01//ANR/ ; ANR-11-LABX-0024//ANR/ ; #ANR-11-EQPX-0022//ANR/ ; #KL 3251/1-1//DFG/ ; ANR-19-CE35-0007-01//Agence Nationale de la Recherche/ ; ANR-11-LABX-0024//Agence Nationale de la Recherche/ ; ANR-11-EQPX-0022//Agence Nationale de la Recherche/ ; KL 3251/1-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Animals ; Mice ; *Anopheles/genetics/parasitology ; *Gene Drive Technology ; *Antimalarials/pharmacology ; Mosquito Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems ; Plasmodium falciparum/genetics ; Plasmodium berghei/genetics ; *Lipoproteins ; },
abstract = {Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.},
}
@article {pmid38018989,
year = {2024},
author = {Chen, L and Peirano, G and Yen, K and Wang, B and Terlecky, A and DeVinney, R and Kreiswirth, BN and Pitout, JDD},
title = {CRISPR-Cas9-mediated IncF plasmid curing in extraintestinal pathogenic Escherichia coli.},
journal = {Microbiology spectrum},
volume = {12},
number = {1},
pages = {e0369223},
pmid = {38018989},
issn = {2165-0497},
support = {R01 AI090155/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Extraintestinal Pathogenic Escherichia coli ; *Escherichia coli Infections/microbiology ; CRISPR-Cas Systems ; Plasmids/genetics ; Anti-Bacterial Agents ; },
abstract = {Understanding the role of IncF plasmids in the success of drug-resistant bacteria has far-reaching implications for tackling antibiotic resistance. The study's use of a novel CRISPR-Cas9-mediated plasmid-curing system provides a precision tool for dissecting the specific impact of IncF plasmids on ExPEC clones, especially high-risk, multidrug-resistant strains like ST131, ST1193, and ST410. The study offers a crucial stepping stone for future research into understanding how these plasmids influence more complex aspects of bacterial behavior, such as cell invasion and in vivo fitness.},
}
@article {pmid38014967,
year = {2024},
author = {Zhang, H and Zhao, H and Cao, L and Yu, B and Wei, J and Pan, G and Bao, J and Zhou, Z},
title = {Harnessing multiplex crRNA enables an amplification-free/CRISPR-Cas12a-based diagnostic methodology for Nosema bombycis.},
journal = {Microbiology spectrum},
volume = {12},
number = {1},
pages = {e0301423},
pmid = {38014967},
issn = {2165-0497},
support = {XDJK2020B005//Central Universities in China/ ; cstc2018jcyj-yszxX0012//CSTC | Natural Science Foundation of Chongqing Municipality ()/ ; cx2018087//Venture and Innovation Support Program for Chongqing Overseas Returnees (Venture &amp; Innovation Support Program for Chongqing Overseas Returnees)/ ; 31802141//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Microsporidiosis ; *Nosema/genetics ; *Bombyx ; },
abstract = {The multiplex-crRNA CRISPR/Cas12a detection method saves hands-on time, reduces the risk of aerosol pollution, and can be directly applied to detecting silkworms infected with Nosema bombycis. This study provides a new approach for the inspection and quarantine of silkworm pébrine disease in sericulture and provides a new method for the detection of other pathogens.},
}
@article {pmid38007613,
year = {2024},
author = {Farley, KO and Forbes, CA and Shaw, NC and Kuzminski, E and Ward, M and Baynam, G and Lassmann, T and Fear, VS},
title = {CRISPR-Cas9-generated PTCHD1 2489T&gt;G stem cells recapitulate patient phenotype when undergoing neural induction.},
journal = {HGG advances},
volume = {5},
number = {1},
pages = {100257},
pmid = {38007613},
issn = {2666-2477},
mesh = {Child ; Humans ; *Autism Spectrum Disorder/diagnosis ; CRISPR-Cas Systems/genetics ; Delayed Diagnosis ; Phenotype ; Stem Cells/metabolism ; Membrane Proteins/genetics ; },
abstract = {An estimated 3.5%-5.9% of the global population live with rare diseases, and approximately 80% of these diseases have a genetic cause. Rare genetic diseases are difficult to diagnose, with some affected individuals experiencing diagnostic delays of 5-30 years. Next-generation sequencing has improved clinical diagnostic rates to 33%-48%. In a majority of cases, novel variants potentially causing the disease are discovered. These variants require functional validation in specialist laboratories, resulting in a diagnostic delay. In the interim, the finding is classified as a genetic variant of uncertain significance (VUS) and the affected individual remains undiagnosed. A VUS (PTCHD1 c. 2489T>G) was identified in a child with autistic behavior, global developmental delay, and hypotonia. Loss of function mutations in PTCHD1 are associated with autism spectrum disorder and intellectual disability; however, the molecular function of PTCHD1 and its role in neurodevelopmental disease is unknown. Here, we apply CRISPR gene editing and induced pluripotent stem cell (iPSC) neural disease modeling to assess the variant. During differentiation from iPSCs to neural progenitors, we detect subtle but significant gene signatures in synaptic transmission and muscle contraction pathways. Our work supports the causal link between the genetic variant and the child's phenotype, providing evidence for the variant to be considered a pathogenic variant according to the American College of Medical Genetics and Genomics guidelines. In addition, our study provides molecular data on the role of PTCHD1 in the context of other neurodevelopmental disorders.},
}
@article {pmid37922902,
year = {2024},
author = {Brooks, DL and Whittaker, MN and Said, H and Dwivedi, G and Qu, P and Musunuru, K and Ahrens-Nicklas, RC and Alameh, MG and Wang, X},
title = {A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant.},
journal = {HGG advances},
volume = {5},
number = {1},
pages = {100253},
doi = {10.1016/j.xhgg.2023.100253},
pmid = {37922902},
issn = {2666-2477},
support = {R35 HL145203/HL/NHLBI NIH HHS/United States ; },
mesh = {Mice ; Animals ; Gene Editing ; RNA, Messenger/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Phenylketonurias/genetics ; *Phenylalanine Hydroxylase/genetics ; Adenine ; *Liposomes ; *Nanoparticles ; },
abstract = {The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs in vitro, followed by assessment in c.1222C>T humanized mice in vivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.},
}
@article {pmid37907826,
year = {2024},
author = {Arroyo-Olarte, R and Mejía-Muñoz, A and León-Cabrera, S},
title = {Expanded Alternatives of CRISPR-Cas9 Applications in Immunotherapy of Colorectal Cancer.},
journal = {Molecular diagnosis &amp; therapy},
volume = {28},
number = {1},
pages = {69-86},
pmid = {37907826},
issn = {1179-2000},
support = {A1S23944//Consejo Nacional de Ciencia y Tecnolog&#xed;a/ ; IN215421//Direcci&#xf3;n General de Asuntos del Personal Acad&#xe9;mico, Universidad Nacional Aut&#xf3;noma de M&#xe9;xico/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Immunotherapy ; Genetic Therapy ; *Colorectal Neoplasms/genetics/therapy ; },
abstract = {Immunotherapy for colorectal cancer (CRC) is limited to patients with advanced disease who have already undergone first-line chemotherapy and whose tumors exhibit microsatellite instability. Novel technical strategies are required to enhance therapeutic options and achieve a more robust immunological response. Therefore, exploring gene analysis and manipulation at the molecular level can further accelerate the development of advanced technologies to address these challenges. The emergence of advanced genome editing technology, particularly of clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9, holds promise in expanding the boundaries of cancer immunotherapy. In this manuscript, we provide a comprehensive review of the applications and perspectives of CRISPR technology in improving the design, generation, and efficiency of current immunotherapies, focusing on solid tumors such as colorectal cancer, where these approaches have not been as successful as in hematological conditions.},
}
@article {pmid37004637,
year = {2024},
author = {Lu, J and Tong, Y and Dong, R and Yang, Y and Hu, W and Zhang, M and Liu, Q and Zhao, S and Adams, JH and Qin, L and Chen, X},
title = {Large DNA fragment knock-in and sequential gene editing in Plasmodium falciparum: a preliminary study using suicide-rescue-based CRISPR/Cas9 system.},
journal = {Molecular and cellular biochemistry},
volume = {479},
number = {1},
pages = {99-107},
pmid = {37004637},
issn = {1573-4919},
support = {R01 AI117017/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Plasmodium falciparum/genetics ; DNA ; *Malaria/genetics ; },
abstract = {CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.},
}
@article {pmid38191764,
year = {2024},
author = {Ilahibaks, NF and Kluiver, TA and de Jong, OG and de Jager, SCA and Schiffelers, RM and Vader, P and Peng, WC and Lei, Z and Sluijter, JPG},
title = {Extracellular vesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin-kexin type 9 (Pcsk9) in primary mouse hepatocytes.},
journal = {Journal of extracellular vesicles},
volume = {13},
number = {1},
pages = {e12389},
pmid = {38191764},
issn = {2001-3078},
support = {116002016/ZONMW_/ZonMw/Netherlands ; 725229/ERC_/European Research Council/International ; },
mesh = {Animals ; Mice ; *Extracellular Vesicles ; CRISPR-Cas Systems ; Cholesterol, LDL ; Proprotein Convertase 9/genetics ; Tacrolimus Binding Protein 1A ; Sirolimus ; Hepatocytes ; Subtilisins ; },
abstract = {The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.},
}
@article {pmid38190500,
year = {2024},
author = {Tan, W and Zhang, C and Cheng, S and Hu, X and Wang, M and Xian, Y},
title = {DNA Gate-Based CRISPR-Cas Exponential Amplification System for Ultrasensitive Small Extracellular Vesicle Detection to Enhance Breast Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c04873},
pmid = {38190500},
issn = {1520-6882},
abstract = {Tumor-derived small extracellular vesicles (tEVs) as potential biomarkers possess abundant surface proteins closely related to parent cells, which are crucial for noninvasive cancer diagnosis. However, tEVs exhibit phenotype heterogeneity and low abundance, posing a significant challenge for multiplex detection with a high sensitivity. Herein, we developed a DNA gate-based exponential amplification CRISPR-Cas (DGEAC) system for accurate and ultrasensitive detection of tEVs, which can greatly improve the accuracy of breast cancer (BC) diagnosis. Based on the coexpression of CD63 and vascular endothelial growth factor (VEGF) on BC-derived tEVs, we developed a dual-aptamer-based AND gate fluorescent probe by proximity hybridization. By integrating the target recognition and trans-cleavage activity of Cas12a, an autocatalysis-driven exponential amplification circuit was developed for ultrasensitive detection of CD63 and VEGF proteins on tEVs, which could avoid false negative signals from single protein or other interfering proteins. We achieved highly sensitive detection of tEVs over a linear range from 1.75 × 10[3] to 3.5 × 10[8] particles/mL with a detection limit as low as 1.02 × 10[3] particles/mL. Furthermore, the DGEAC system can distinguish tEVs from tEVs derived from different BC cell lines, including MDA-MB-231, MCF-7, SKBR3, and MCF-10A. Compared to linear amplification (AUC 90.0%), the DGEAC system effectively differentiates BC in different stages (AUC 98.3%).},
}
@article {pmid38188023,
year = {2023},
author = {Lei, P and Ju, Y and Peng, F and Luo, J},
title = {Applications and advancements of CRISPR-Cas in the treatment of lung cancer.},
journal = {Frontiers in cell and developmental biology},
volume = {11},
number = {},
pages = {1295084},
pmid = {38188023},
issn = {2296-634X},
abstract = {Lung cancer is one of the most malignant diseases and a major contributor to cancer-related deaths worldwide due to the deficiency of early diagnosis and effective therapy that are of great importance for patient prognosis and quality of life. Over the past decade, the advent of clustered regularly interspaced short palindromic repeats/CRISPR associated protein (CRISPR/Cas) system has significantly propelled the progress of both fundamental research and clinical trials of lung cancer. In this review, we review the current applications of the CRISPR/Cas system in diagnosis, target identification, and treatment resistance of lung cancer. Furthermore, we summarize the development of lung cancer animal models and delivery methods based on CRISPR system, providing novel insights into clinical diagnosis and treatment strategies of lung cancer.},
}
@article {pmid38186450,
year = {2023},
author = {Mondal, R and Brahmbhatt, N and Sandhu, SK and Shah, H and Vashi, M and Gandhi, SK and Patel, P},
title = {Applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a Genetic Scalpel for the Treatment of Cancer: A Translational Narrative Review.},
journal = {Cureus},
volume = {15},
number = {12},
pages = {e50031},
pmid = {38186450},
issn = {2168-8184},
abstract = {Cancer remains a global health challenge with high prevalence and mortality rates, imposing significant financial and emotional burdens on affected families. However, hope lies in genetic manipulation, with a focus on innovative approaches to combat genetically linked cancers. Clustered regularly interspaced short palindromic repeats (CRISPR), an adaptive immune system found in various bacteria and archaea, hold immense potential. We searched articles on PubMed Central, Medline, and PubMed databases indexed journals. The keywords from the research topic, i.e., "CRISPR AND genetic therapy," were searched, and we found 3397 articles. Following this, we explored the medical subject headings (MeSH) glossary and created a search strategy "Clustered Regularly Interspaced Short Palindromic Repeats"[Mesh] AND "Genetic Therapy"[Majr] and after applying a variety of filters we included 30 studies in our review. CRISPR consists of unique spacers and CRISPR-associated (Cas) genes, operating through acquisition, CRISPR ribonucleic acid (crRNA) biogenesis, and target interference phases. The type II CRISPR-Cas9 system is a well-researched avenue for gene editing, with Cas9 cleaving specific genomic regions and initiating deoxyribonucleic acid (DNA) repair mechanisms. Cancer results from genetic alterations, leading to tumor development with properties like metastasis. CRISPR/Cas9 offers precise genome editing to inhibit tumor formation by removing specific genomic sequences and promoting DNA repair. Challenges in CRISPR's use for cancer therapy, including delivery methods, cell adaptation, and ethical concerns, are recognized. Beyond cancer, CRISPR finds diverse applications in infectious diseases and non-cancerous conditions, signifying its transformative potential in modern medicine. CRISPR technology represents a groundbreaking frontier in cancer therapy and beyond, offering insights into genetic editing, disease mechanisms, and potential cures. Despite challenges and ethical considerations, precise genome editing promises improved cancer treatments and innovative medical interventions in the future.},
}
@article {pmid38182395,
year = {2024},
author = {Liu, R and Wang, X and Wang, S and Xie, L and Zhao, P and Li, L and Ge, S and Yu, J},
title = {Rolling circle amplification assisted CRISPR/Cas12a dual-cleavage photoelectrochemical biosensor for highly sensitive detection of miRNA-21.},
journal = {Analytica chimica acta},
volume = {1287},
number = {},
pages = {342125},
doi = {10.1016/j.aca.2023.342125},
pmid = {38182395},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems/genetics ; *Biomedical Research ; Cobalt ; *MicroRNAs ; },
abstract = {BACKGROUND: MicroRNA-21 has been determined to be the only microRNA overexpressed in 11 types of solid tumors, making it an excellent candidate as a biomarker for disease diagnosis and therapy. Photoelectrochemical (PEC) biosensors have been widely used for quantification of microRNA-21. However, most PEC biosensing processes still suffer from some problems, such as the difficulty of avoiding the influence of interferents in complex matrices and the false-positive signals. There is a pressing need for establishing a sensitive and stable PEC method to detect microRNA-21.<br><br>RESULTS: Herein, a nicking endonuclease-mediated rolling circle amplification (RCA)-assisted CRISPR/Cas12a PEC biosensor was fabricated for ultrasensitive detection of microRNA-21. The p-p type heterojunction PbS QDs/Co3O4 polyhedra were prepared as the quencher, thus the initial PEC signal attained the "off" state. Furthermore, the target was specifically identified and amplified by the RCA process. Then, its product single-stranded DNA S1 activated the cis- and trans-cleavage abilities of CRISPR/Cas12a, leading to almost all of the PbS QDs/Co3O4 polyhedra to leave the electrode surface, the p-n semiconductor quenching effect to be disrupted, and the signal achieving the "super-on" state. This pattern of PEC signal changed from "off" to "on" eliminated the interference of false-positive signals. The proposed PEC biosensor presented a satisfactory linear relationship ranging from 1&#xa0;fM to 10&#xa0;nM with a detection limit of 0.76&#xa0;fM (3 Sb/N).<br><br>SIGNIFICANCE AND NOVELTY: With innovatively synthesized PbS QDs/Co3O4 polyhedra as the effective quencher for PEC signal, the CRISPR/Cas12a dual-cleavage PEC biosensor possessed excellent selectivity, stability and repeatability. Furthermore, the detection of various miRNAs can be realized by changing the relevant base sequences in the constructed PEC biosensor. It also provides a powerful strategy for early clinical diagnosis and biomedical research.},
}
@article {pmid38182385,
year = {2024},
author = {Wang, X and Zheng, D and Wang, C and Xue, D and Wang, Q and Xia, J},
title = {Harnessing intermolecular G-quadruplex-based spatial confinement effect for accelerated activation of CRISPR/Cas12a empowers ultra-sensitive detection of PML/RARA fusion genes.},
journal = {Analytica chimica acta},
volume = {1287},
number = {},
pages = {342108},
doi = {10.1016/j.aca.2023.342108},
pmid = {38182385},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Disease Progression ; *Endonucleases ; Protein Isoforms ; },
abstract = {Accurate detection and classification of the three isoforms of PML/RARA genomic fragments are crucial for predicting disease progression, stratifying risk, and administering precise drug therapies in acute promyelocytic leukemia (APL). In this study, we have developed a highly specific nucleic acid detection platform capable of quantifying the long isoform of the three main PML-RARA isoforms at a constant temperature. This platform integrates the strengths of the CRISPR/Cas12a nuclease-based method and the rolling circle amplification (RCA) technique. Notably, the RCA-assisted CRISPR/Cas12a trans-cleavage system incorporates a spatial confinement effect by utilizing intermolecular G-quadruplex structures. This innovative design effectively enhances the local concentration of CRISPR/Cas12a, thereby accelerating its cleaving efficiency towards reporter nucleic acids and enabling the detection of PML/RARA fusion gene expression through spectroscopy. The robust detection of PML/RARA fusion gene from human serum samples validates the reliability and potential of this platform in the screening, diagnosis, and prognosis of APL cases. Our findings present an approach that holds significant potential for the further development of the robust CRISPR/Cas sensor system, offering a rapid and adaptable paradigm for APL diagnosis.},
}
@article {pmid38182384,
year = {2024},
author = {Zhu, F and Zhao, Q},
title = {CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin.},
journal = {Analytica chimica acta},
volume = {1287},
number = {},
pages = {342106},
doi = {10.1016/j.aca.2023.342106},
pmid = {38182384},
issn = {1873-4324},
mesh = {*Thrombin ; *CRISPR-Cas Systems ; Antibodies ; Biological Assay ; DNA ; },
abstract = {BACKGROUND: Thrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited.<br><br>RESULTS: Here we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media.<br><br>SIGNIFICANCE: This assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.},
}
@article {pmid38036077,
year = {2024},
author = {Zhao, L and Fang, S and Ma, Y and Ren, J and Hao, L and Wang, L and Yang, J and Lu, X and Yang, L and Wang, G},
title = {Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro.},
journal = {Gene},
volume = {896},
number = {},
pages = {148038},
doi = {10.1016/j.gene.2023.148038},
pmid = {38036077},
issn = {1879-0038},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; *Hemophilia A/genetics/therapy ; CRISPR-Associated Protein 9/genetics ; },
abstract = {BACKGROUND: Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease.<br><br>METHODS: Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression.<br><br>RESULTS: We identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro.<br><br>CONCLUSIONS: Knock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.},
}
@article {pmid38015471,
year = {2024},
author = {Ko, SC and Woo, HM},
title = {CRISPR-dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria.},
journal = {Nucleic acids research},
volume = {52},
number = {1},
pages = {492-506},
pmid = {38015471},
issn = {1362-4962},
support = {2022R1A2C2008242//Ministry of Science and ICT/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Messenger/genetics/metabolism ; Escherichia coli/genetics/metabolism ; RNA, Bacterial/genetics/metabolism ; *RNA, Small Untranslated/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacterial small RNAs (sRNAs) function in post-transcriptional regulatory responses to environmental changes. However, the lack of eukaryotic RNA interference-like machinery in bacteria has limited the systematic engineering of RNA repression. Here, we report the development of clustered regularly interspaced short palindromic repeats (CRISPR)-guided dead CRIPSR-associated protein 13a (dCas13a) ribonucleoprotein that utilizes programmable CRISPR RNAs (crRNAs) to repress trans-acting and cis-acting sRNA as the target, altering regulatory mechanisms and stress-related phenotypes. In addition, we implemented a modular loop engineering of the crRNA to promote modular repression of the target gene with 92% knockdown efficiency and a single base-pair mismatch specificity. With the engineered crRNAs, we achieved targetable single-gene repression in the polycistronic operon. For metabolic application, 102 crRNAs were constructed in the biofoundry and used for screening novel knockdown sRNA targets to improve lycopene (colored antioxidant) production in Escherichia coli. The CRISPR-dCas13a system will assist as a valuable systematic tool for the discovery of novel sRNAs and the fine-tuning of bacterial RNA repression in both scientific and industrial applications.},
}
@article {pmid38000387,
year = {2024},
author = {Swain, T and Pflueger, C and Freytag, S and Poppe, D and Pflueger, J and Nguyen, TV and Li, JK and Lister, R},
title = {A modular dCas9-based recruitment platform for combinatorial epigenome editing.},
journal = {Nucleic acids research},
volume = {52},
number = {1},
pages = {474-491},
pmid = {38000387},
issn = {1362-4962},
support = {GNT1069830//National Health and Medical Research Council/ ; GNT1178460//NHMRC/ ; DP701101609//Australian Research Council/ ; //Silvia and Charles Viertel&#xa0;Senior Medical Research Fellowship/ ; //Howard Hughes Medical Institute International Research Scholarship/ ; RPG66-21//Raine Medical Research Foundation/ ; //Margaret and Terry Orr Memorial/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Epigenome ; Gene Expression Regulation ; Chromatin/genetics ; Epigenesis, Genetic ; },
abstract = {Targeted epigenome editing tools allow precise manipulation and investigation of genome modifications, however they often display high context dependency and variable efficacy between target genes and cell types. While systems that simultaneously recruit multiple distinct 'effector' chromatin regulators can improve efficacy, they generally lack control over effector composition and spatial organisation. To overcome this we have created a modular combinatorial epigenome editing platform, called SSSavi. This system is an interchangeable and reconfigurable docking platform fused to dCas9 that enables simultaneous recruitment of up to four different effectors, allowing precise control of effector composition and spatial ordering. We demonstrate the activity and specificity of the SSSavi system and, by testing it against existing multi-effector targeting systems, demonstrate its comparable efficacy. Furthermore, we demonstrate the importance of the spatial ordering of the recruited effectors for effective transcriptional regulation. Together, the SSSavi system enables exploration of combinatorial effector co-recruitment to enhance manipulation of chromatin contexts previously resistant to targeted editing.},
}
@article {pmid37985205,
year = {2024},
author = {Ito, Y and Inoue, S and Nakashima, T and Zhang, H and Li, Y and Kasuya, H and Matsukawa, T and Wu, Z and Yoshikawa, T and Kataoka, M and Ishikawa, T and Kagoya, Y},
title = {Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells.},
journal = {Nucleic acids research},
volume = {52},
number = {1},
pages = {141-153},
pmid = {37985205},
issn = {1362-4962},
support = {JP22bm0704066//Japan Agency for Medical Research and Development/ ; JPMJFR2060//Japan Science and Technology Agency/ ; JP20H03543//Japan Society for the Promotion of Science/ ; //Aichi Cancer Center Joint Research Project on Priority Areas/ ; //Princess Takamatsunomiya Cancer Research Foundation/ ; //Takeda Science Foundation/ ; //Uehara Memorial Foundation/ ; //Astellas Foundation for Research on Metabolic Disorders/ ; //SGH Foundation/ ; //Kobayashi Foundation for Cancer Research/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; T-Lymphocytes ; },
abstract = {Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.},
}
@article {pmid38182383,
year = {2024},
author = {Hao, J and Jia, M and Liu, Y and Lv, Z and Chen, J and Xiong, W and Zeng, Z},
title = {Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.},
journal = {Analytica chimica acta},
volume = {1287},
number = {},
pages = {342101},
doi = {10.1016/j.aca.2023.342101},
pmid = {38182383},
issn = {1873-4324},
mesh = {Mice ; Animals ; Swine ; *Recombinases ; *Haemophilus parasuis/genetics ; CRISPR-Cas Systems ; Biological Assay ; Cross Reactions ; },
abstract = {BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions.<br><br>RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8&#xa0;h earlier than traditional PCR. Compared to qPCR, our detection results were 100&#xa0;% accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35&#xa0;min.<br><br>SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.},
}
@article {pmid38181979,
year = {2024},
author = {Storz, U},
title = {The CRISPR Cas patent files, part 2: Is Cpf1/Cas12a a less conflict- prone alternative to Cas9 ?.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2023.12.016},
pmid = {38181979},
issn = {1873-4863},
abstract = {CRISPR Cpf1/Cas12a has been discussed as a less conflict prone alternative, patent-wise, to Cas9. This article investigates whether or not this assumption is correct, and comes to the conclusion that the promise that CRISPR Cpf1/Cas12 would make things easier, and be less conflict-prone, is fragile.},
}
@article {pmid38180237,
year = {2024},
author = {Li, J and Zhang, K and Lin, G and Li, J},
title = {CRISPR-Cas system: A promising tool for rapid detection of SARS-CoV-2 variants.},
journal = {Journal of medical virology},
volume = {96},
number = {1},
pages = {e29356},
doi = {10.1002/jmv.29356},
pmid = {38180237},
issn = {1096-9071},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; Mutation ; },
abstract = {COVID-19, caused by SARS-CoV-2, remains a global health crisis. The emergence of multiple variants with enhanced characteristics necessitates their detection and monitoring. Genome sequencing, the gold standard, faces implementation challenges due to complexity, cost, and limited throughput. The CRISPR-Cas system offers promising potential for rapid variant detection, with advantages such as speed, sensitivity, specificity, and programmability. This review provides an in-depth examination of the applications of CRISPR-Cas in mutation detection specifically for SARS-CoV-2. It begins by introducing SARS-CoV-2 and existing variant detection platforms. The principles of the CRISPR-Cas system are then clarified, followed by an exploration of three CRISPR-Cas-based mutation detection platforms, which are evaluated from different perspectives. The review discusses strategies for mutation site selection and the utilization of CRISPR-Cas, offering valuable insights for the development of mutation detection methods. Furthermore, a critical analysis of the clinical applications, advantages, disadvantages, challenges, and prospects of the CRISPR-Cas system is provided.},
}
@article {pmid38177728,
year = {2023},
author = {Wen, DJ and Theodoris, CV},
title = {Interpretable model of CRISPR-Cas9 enzymatic reactions.},
journal = {Nature computational science},
volume = {3},
number = {12},
pages = {1011-1012},
pmid = {38177728},
issn = {2662-8457},
mesh = {*CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid38177723,
year = {2023},
author = {Zhang, Z and Lamson, AR and Shelley, M and Troyanskaya, O},
title = {Interpretable neural architecture search and transfer learning for understanding CRISPR-Cas9 off-target enzymatic reactions.},
journal = {Nature computational science},
volume = {3},
number = {12},
pages = {1056-1066},
pmid = {38177723},
issn = {2662-8457},
support = {R01GM071966//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM071966//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 395506//Simons Foundation/ ; 395506//Simons Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Neural Networks, Computer ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; Machine Learning ; },
abstract = {Finely tuned enzymatic pathways control cellular processes, and their dysregulation can lead to disease. Developing predictive and interpretable models for these pathways is challenging because of the complexity of the pathways and of the cellular and genomic contexts. Here we introduce Elektrum, a deep learning framework that addresses these challenges with data-driven and biophysically interpretable models for determining the kinetics of biochemical systems. First, it uses in vitro kinetic assays to rapidly hypothesize an ensemble of high-quality kinetically interpretable neural networks (KINNs) that predict reaction rates. It then employs a transfer learning step, where the KINNs are inserted as intermediary layers into deeper convolutional neural networks, fine-tuning the predictions for reaction-dependent in vivo outcomes. We apply Elektrum to predict CRISPR-Cas9 off-target editing probabilities and demonstrate that Elektrum achieves improved performance, regularizes neural network architectures and maintains physical interpretability.},
}
@article {pmid38177114,
year = {2024},
author = {Yu, Y and Farooq, MS and Eberhart Meessen, S and Jiang, Y and Kato, D and Zhan, T and Weiss, C and Seger, R and Kang, W and Zhang, X and Yu, J and Ebert, MPA and Burgermeister, E},
title = {Nuclear pore protein POM121 regulates subcellular localization and transcriptional activity of PPARγ.},
journal = {Cell death &amp; disease},
volume = {15},
number = {1},
pages = {7},
pmid = {38177114},
issn = {2041-4889},
support = {Bu2285//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2727//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2727//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ICON//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2727//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TMR//Universit&#xe4;t Heidelberg (University of Heidelberg)/ ; IMIM//Universit&#xe4;t Heidelberg (University of Heidelberg)/ ; Merck HD InnovationCall//Merck KGaA/ ; },
mesh = {Humans ; *Nuclear Pore/metabolism ; PPAR gamma/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Nuclear Pore Complex Proteins/metabolism ; Transcription Factors/metabolism ; *Neoplasms/metabolism ; Membrane Glycoproteins/metabolism ; },
abstract = {Manipulation of the subcellular localization of transcription factors by preventing their shuttling via the nuclear pore complex (NPC) emerges as a novel therapeutic strategy against cancer. One transmembrane component of the NPC is POM121, encoded by a tandem gene locus POM121A/C on chromosome 7. Overexpression of POM121 is associated with metabolic diseases (e.g., diabetes) and unfavorable clinical outcome in patients with colorectal cancer (CRC). Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor with anti-diabetic and anti-tumoral efficacy. It is inhibited by export from the nucleus to the cytosol via the RAS-RAF-MEK1/2-ERK1/2 signaling pathway, a major oncogenic driver of CRC. We therefore hypothesized that POM121 participates in the transport of PPARγ across the NPC to regulate its transcriptional activity on genes involved in metabolic and tumor control. We found that POM121A/C mRNA was enriched and POM121 protein co-expressed with PPARγ in tissues from CRC patients conferring poor prognosis. Its interactome was predicted to include proteins responsible for tumor metabolism and immunity, and in-silico modeling provided insights into potential 3D structures of POM121. A peptide region downstream of the nuclear localization sequence (NLS) of POM121 was identified as a cytoplasmic interactor of PPARγ. POM121 positivity correlated with the cytoplasmic localization of PPARγ in patients with KRAS mutant CRC. In contrast, POM121A/C silencing by CRISPR/Cas9 sgRNA or siRNA enforced nuclear accumulation of PPARγ and activated PPARγ target genes promoting lipid metabolism and cell cycle arrest resulting in reduced proliferation of human CRC cells. Our data suggest the POM121-PPARγ axis as a potential drugable target in CRC.},
}
@article {pmid38156660,
year = {2024},
author = {Zhang, X and Li, Z and Yang, L and Hu, B and Zheng, Q and Man, J and Cao, J},
title = {CRISPR/Cas12a-Derived Photoelectrochemical Aptasensor Based on Au Nanoparticle-Attached CdS/UiO-66-NH2 Heterostructures for the Rapid and Sensitive Detection of Ochratoxin A.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {1},
pages = {874-882},
doi = {10.1021/acs.jafc.3c09106},
pmid = {38156660},
issn = {1520-5118},
mesh = {*Biosensing Techniques/methods ; Gold/chemistry ; CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; *Aptamers, Nucleotide/chemistry ; DNA, Single-Stranded ; Limit of Detection ; Electrochemical Techniques/methods ; },
abstract = {The sensitive and accurate detection of ochratoxin A (OTA) is crucial for public health due to its high toxicity. Herein, using Au nanoparticle (NP)-attached CdS/UiO-66-NH2 heterostructures as photoactive materials, a photoelectrochemical (PEC) aptasensor was presented for the ultrasensitive assay of OTA based on a competitive displacement reaction triggering the trans-cleavage ability of CRISPR/Cas12a. In this sensing strategy, methylene blue-labeled single-stranded DNA (MB-ssDNA) was immobilized on the Au NPs/CdS/UiO-66-NH2 electrode to accelerate the separation of the photogenerated carrier, thus producing a significantly increased PEC response. In the presence of OTA, it specifically bound with the aptamer (Apt) and resulted in the release of the activation chain, triggering the trans-cleavage characteristics of CRISPR/Cas12a. MB-ssDNA was cut randomly on the electrode surface to convert the PEC signal from the "on" to the "off" state, thereby achieving a quantitative and accurate detection of OTA. The CRISPR/Cas12a-derived PEC aptasensor exhibited excellent sensitivity and specificity, with a linear range from 100 to 50 ng/mL and a detection limit of 38 fg/mL. Overall, the proposed aptasensor could provide a rapid, accurate, and sensitive method for the determination of OTA in actual samples.},
}
@article {pmid38134022,
year = {2024},
author = {Qiao, Z and Xue, L and Sun, M and Ma, N and Shi, H and Yang, W and Cheong, LZ and Huang, X and Xiong, Y},
title = {Dual-Functional Tetrahedron Multivalent Aptamer Assisted Amplification-Free CRISPR/Cas12a Assay for Sensitive Detection of Salmonella.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {1},
pages = {857-864},
doi = {10.1021/acs.jafc.3c07582},
pmid = {38134022},
issn = {1520-5118},
mesh = {Humans ; *CRISPR-Cas Systems ; Salmonella/genetics ; Oligonucleotides ; Biological Assay ; Disease Outbreaks ; *Biosensing Techniques ; Nucleic Acid Amplification Techniques ; },
abstract = {Salmonellosis continues to impose a significant economic burden globally. Rapid and sensitive detection of Salmonella is crucial to preventing the outbreaks of foodborne illnesses, yet it remains a formidable challenge. Herein, a dual-functional tetrahedron multivalent aptamer assisted amplification-free CRISPR/Cas12a assay was developed for Salmonella detection. In the system, the aptamer was programmatically assembled on the tetrahedral DNA nanostructure to fabricate a multivalent aptamer (TDN-multiApt), which displayed a 3.5-fold enhanced avidity over the monovalent aptamer and possessed four CRISPR/Cas12a targeting fragments to amplify signal. Therefore, TDN-multiApt could directly activate Cas12a to achieve the second signal amplification without any nucleic acid amplification. By virtue of the synergism of high avidity and cascaded signal amplifications, the proposed method allowed the ultrasensitive detection of Salmonella as low as 7 cfu mL[-1]. Meanwhile, this novel platform also exhibited excellent specificity against target bacteria and performed well in the detection of various samples, indicating its potential application in real samples.},
}
@article {pmid38177471,
year = {2022},
author = {Shang, S and Cai, XS and Qi, LS},
title = {Computation empowers CRISPR discovery and technology.},
journal = {Nature computational science},
volume = {2},
number = {9},
pages = {533-535},
pmid = {38177471},
issn = {2662-8457},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Technology ; },
}
@article {pmid38175477,
year = {2024},
author = {Janezic, S and Garneau, JR and Monot, M},
title = {Comparative Genomics of Clostridioides difficile.},
journal = {Advances in experimental medicine and biology},
volume = {1435},
number = {},
pages = {199-218},
pmid = {38175477},
issn = {0065-2598},
mesh = {*Clostridioides ; *Clostridioides difficile/genetics ; Base Composition ; Phylogeny ; RNA, Ribosomal, 16S ; Sequence Analysis, DNA ; Genomics ; },
abstract = {Clostridioides difficile, a Gram-positive spore-forming anaerobic bacterium, has rapidly emerged as the leading cause of nosocomial diarrhoea in hospitals. The availability of large numbers of genome sequences, mainly due to the use of next-generation sequencing methods, has undoubtedly shown their immense advantages in the determination of C. difficile population structure. The implementation of fine-scale comparative genomic approaches has paved the way for global transmission and recurrence studies, as well as more targeted studies, such as the PaLoc or CRISPR/Cas systems. In this chapter, we provide an overview of recent and significant findings on C. difficile using comparative genomic studies with implications for epidemiology, infection control and understanding of the evolution of C. difficile.},
}
@article {pmid38174858,
year = {2024},
author = {Chen, JL and Leeder, WM and Morais, P and Adachi, H and Yu, YT},
title = {Pseudouridylation-mediated gene expression modulation.},
journal = {The Biochemical journal},
volume = {481},
number = {1},
pages = {1-16},
doi = {10.1042/BCJ20230096},
pmid = {38174858},
issn = {1470-8728},
mesh = {*Pseudouridine/genetics/metabolism ; *RNA Precursors/metabolism ; RNA, Guide, CRISPR-Cas Systems ; RNA/metabolism ; RNA Processing, Post-Transcriptional ; Protein Biosynthesis ; },
abstract = {RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA splicing and the modulation of premature termination codon (PTC) readthrough. This review provides insights into its mechanisms, functions, and potential therapeutic applications. It examines the mechanisms governing RNA-guided pseudouridylation, emphasizing the roles of guide RNAs and pseudouridine synthases in catalyzing uridine-to-pseudouridine conversion. A key focus is the impact of RNA-guided pseudouridylation of U2 small nuclear RNA on pre-mRNA splicing, encompassing its influence on branch site recognition and spliceosome assembly. Additionally, the review discusses the emerging role of RNA-guided pseudouridylation in regulating PTC readthrough, impacting translation termination and genetic disorders. Finally, it explores the therapeutic potential of pseudouridine modifications, offering insights into potential treatments for genetic diseases and cancer and the development of mRNA vaccine.},
}
@article {pmid38173969,
year = {2023},
author = {Xu, Z and Chen, S and Wu, W and Wen, Y and Cao, H},
title = {Type I CRISPR-Cas-mediated microbial gene editing and regulation.},
journal = {AIMS microbiology},
volume = {9},
number = {4},
pages = {780-800},
pmid = {38173969},
issn = {2471-1888},
abstract = {There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.},
}
@article {pmid38172811,
year = {2024},
author = {Kwon, HJ and Lee, J and Kwon, SJ and Lee, HS},
title = {Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP.},
journal = {Microbial cell factories},
volume = {23},
number = {1},
pages = {6},
pmid = {38172811},
issn = {1475-2859},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Editing ; Clostridium/genetics/metabolism ; Metabolic Engineering ; },
abstract = {BACKGROUND: Clostridium sp. AWRP (AWRP) is a novel acetogenic bacterium isolated under high partial pressure of carbon monoxide (CO) and can be one of promising candidates for alcohol production from carbon oxides. Compared to model strains such as C. ljungdahlii and C. autoethanogenum, however, genetic manipulation of AWRP has not been established, preventing studies on its physiological characteristics and metabolic engineering.<br><br>RESULTS: We were able to demonstrate the genetic domestication of AWRP, including transformation of shuttle plasmids, promoter characterization, and genome editing. From the conjugation experiment with E. coli S17-1, among the four replicons tested (pCB102, pAM&#x3b2;1, pIP404, and pIM13), three replicated in AWRP but pCB102 was the only one that could be transferred by electroporation. DNA methylation in E. coli significantly influenced transformation efficiencies in AWRP: the highest transformation efficiencies (10[2]-10[3] CFU/&#xb5;g) were achieved with unmethylated plasmid DNA. Determination of strengths of several clostridial promoters enabled the establishment of a CRISPR/Cas12a genome editing system based on Acidaminococcus sp. BV3L6 cas12a gene; interestingly, the commonly used CRISPR/Cas9 system did not work in AWRP, although it expressed the weakest promoter (C. acetobutylicum Pptb) tested. This system was successfully employed for the single gene deletion (xylB and pyrE) and double deletion of two prophage gene clusters.<br><br>CONCLUSIONS: The presented genome editing system allowed us to achieve several genome manipulations, including double deletion of two large prophage groups. The genetic toolbox developed in this study will offer a chance for deeper studies on Clostridium sp. AWRP for syngas fermentation and carbon dioxide (CO2) sequestration.},
}
@article {pmid38109169,
year = {2024},
author = {Yuan, J and Wang, L and Huang, L and He, K and Wang, H and Xu, X and Su, B and Wang, J},
title = {CRISPR-Cas12a-Mediated Hue-Recognition Lateral Flow Assay for Point-of-Need Detection of Salmonella.},
journal = {Analytical chemistry},
volume = {96},
number = {1},
pages = {220-228},
doi = {10.1021/acs.analchem.3c03753},
pmid = {38109169},
issn = {1520-6882},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Salmonella typhimurium/genetics ; Biological Assay ; Chickens ; Food ; Nucleic Acid Amplification Techniques ; },
abstract = {Nucleic acid detection of pathogens in a point-of-need (PON) manner is of great significance yet remains challenging for sensitive and accurate visual discrimination. Here, we report a CRISPR-Cas12a-mediated lateral flow assay for PON detection of Salmonella typhimurium (S.ty) that is a prevailing pathogen disseminated through tainted food. The variation of the fluorescence color of the test line is exploited to interpret the results, enabling the discrimination between positive and negative samples on the basis of a hue-recognition mechanism. By leveraging the cleavage activity of Cas12a and hue-recognition readout, the assay facilitated by recombinase polymerase amplification can yield a visual detection limit of 1 copy μL[-1] for S.ty genomic DNA within 1 h. The assay also displays a high specificity toward S.ty in fresh chicken samples, as well as a sensitivity 10-fold better than that of the commercial test strip. Moreover, a semiquantitative detection of S.ty ranging from 0 to 4 × 10[3] CFU/mL by the naked eye is made possible, thanks to the easily discernible color change of the test line. This approach provides an easy, rapid, accurate, and user-friendly solution for the PON detection of Salmonella and other pathogens.},
}
@article {pmid38172625,
year = {2024},
author = {Vill, AC and Rice, EJ and De Vlaminck, I and Danko, CG and Brito, IL},
title = {Precision run-on sequencing (PRO-seq) for microbiome transcriptomics.},
journal = {Nature microbiology},
volume = {9},
number = {1},
pages = {241-250},
pmid = {38172625},
issn = {2058-5276},
support = {R01 HG010346/HG/NHGRI NIH HHS/United States ; R01 HG009309/HG/NHGRI NIH HHS/United States ; R01 HG010346/HG/NHGRI NIH HHS/United States ; R01 HG009309/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems ; DNA-Directed RNA Polymerases/genetics ; RNA/genetics ; Gene Expression Profiling ; },
abstract = {Bacteria respond to environmental stimuli through precise regulation of transcription initiation and elongation. Bulk RNA sequencing primarily characterizes mature transcripts, so to identify actively transcribed loci we need to capture RNA polymerase (RNAP) complexed with nascent RNA. However, such capture methods have only previously been applied to culturable, genetically tractable organisms such as E. coli and B. subtilis. Here we apply precision run-on sequencing (PRO-seq) to profile nascent transcription in cultured E. coli and diverse uncultured bacteria. We demonstrate that PRO-seq can characterize the transcription of small, structured, or post-transcriptionally modified RNAs, which are often absent from bulk RNA-seq libraries. Applying PRO-seq to the human microbiome highlights taxon-specific RNAP pause motifs and pause-site distributions across non-coding RNA loci that reflect structure-coincident pausing. We also uncover concurrent transcription and cleavage of CRISPR guide RNAs and transfer RNAs. We demonstrate the utility of PRO-seq for exploring transcriptional dynamics in diverse microbial communities.},
}
@article {pmid38170285,
year = {2024},
author = {Luo, Q and Zhang, C and Deng, X and Liu, D and Pan, X and Gong, Y and Tang, Q and Zhang, K and Liao, X},
title = {A CRISPR-Cas12a-based electrochemical biosensor for the detection of microphthalmia-associated transcription factor.},
journal = {Mikrochimica acta},
volume = {191},
number = {1},
pages = {73},
pmid = {38170285},
issn = {1436-5073},
support = {2023r129//the Startup Foundation for Introducing Talent of NUIST/ ; 21964018//the National Natural Science Foundation of China/ ; 2020GXNSFAA259068//the Natural Science Foundation of Guangxi Province/ ; 21-220-06-202205//the Guangxi key laboratory of basic and translational research of Bone and joint Degenerative Disease/ ; },
mesh = {Humans ; *Microphthalmia-Associated Transcription Factor/genetics ; *CRISPR-Cas Systems ; Metallocenes ; Gold ; DNA/genetics ; },
abstract = {A novel electrochemical biosensor that combines the CRISPR-Cas12a system with a gold electrode is reported for the rapid and sensitive detection of microphthalmia-associated transcription factor (MITF). The biosensor consists of a gold electrode modified with DNA1, which contains the target sequence of MITF and is labeled with ferrocene, an electroactive molecule. The biosensor also includes hairpin DNA, which has a binding site for MITF and can hybridize with helper DNA to form a double-stranded complex that activates CRISPR-Cas12a. When MITF is present, it binds to hairpin DNA and prevents its hybridization with helper DNA, thus inhibiting CRISPR-Cas12a activity and preserving the DPV signal of ferrocene. When MITF is absent, hairpin DNA hybridizes with helper DNA and activates CRISPR-Cas12a, which cleaves DNA1 and releases ferrocene, thus reducing the DPV signal. The biosensor can detect MITF with high sensitivity (with an LOD of 8.14 fM), specificity, and accuracy in various samples, such as cell nuclear extracts and human serum. The biosensor can also diagnose and monitor melanocyte-related diseases and melanin production. This work provides a simple, fast, sensitive, and cost-effective biosensor for MITF detection and a valuable tool for applications in genetic testing, disease diagnosis, and drug screening.},
}
@article {pmid38169468,
year = {2024},
author = {Baik, R and Cromer, MK and Glenn, SE and Vakulskas, CA and Chmielewski, KO and Dudek, AM and Feist, WN and Klermund, J and Shipp, S and Cathomen, T and Dever, DP and Porteus, MH},
title = {Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {111},
pmid = {38169468},
issn = {2041-1723},
mesh = {Humans ; Animals ; Mice ; *Gene Editing/methods ; Hematopoietic Stem Cells/metabolism ; *Hematopoietic Stem Cell Transplantation ; Recombinant Proteins/metabolism ; Peptides/metabolism ; CRISPR-Cas Systems ; },
abstract = {Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.},
}
@article {pmid38168957,
year = {2024},
author = {},
title = {A CRISPR-based system for temporal, combinatorial gene knockout in mice.},
journal = {Nature immunology},
volume = {25},
number = {1},
pages = {15-16},
pmid = {38168957},
issn = {1529-2916},
mesh = {Animals ; Mice ; Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice, Knockout ; },
}
@article {pmid38167825,
year = {2024},
author = {Kijner, S and Ennis, D and Shmorak, S and Florentin, A and Yassour, M},
title = {CRISPR-Cas-based identification of a sialylated human milk oligosaccharides utilization cluster in the infant gut commensal Bacteroides dorei.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {105},
pmid = {38167825},
issn = {2041-1723},
support = {2660/18//Israel Science Foundation (ISF)/ ; },
mesh = {Infant ; Humans ; *Milk, Human ; *CRISPR-Cas Systems/genetics ; Oligosaccharides ; Bacteroides/genetics ; },
abstract = {The infant gut microbiome is impacted by early-life feeding, as human milk oligosaccharides (HMOs) found in breastmilk cannot be digested by infants and serve as nutrients for their gut bacteria. While the vast majority of HMO-utilization research has focused on Bifidobacterium species, recent studies have suggested additional HMO-utilizers, mostly Bacteroides, yet their utilization mechanism is poorly characterized. Here, we investigate Bacteroides dorei isolates from breastfed-infants and identify that polysaccharide utilization locus (PUL) 33 enables B. dorei to utilize sialylated HMOs. We perform transcriptional profiling and identity upregulated genes when growing on sialylated HMOs. Using CRISPR-Cas12 to knock-out four PUL33 genes, combined with complementation assays, we identify GH33 as the critical gene in PUL33 for sialylated HMO-utilization. This demonstration of an HMO-utilization system by Bacteroides species isolated from infants opens the way to further characterization of additional such systems, to better understand HMO-utilization in the infant gut.},
}
@article {pmid38167305,
year = {2024},
author = {Klose, SM and Legione, AR and Monotti, I and Bushell, RN and Sugiyama, T and Browning, GF and Vaz, PK},
title = {Genomic characterization of Mycoplasma edwardii isolated from a dog bite induced cat wound reveals multiple horizontal gene transfer events and loss of the CRISPR/Cas system.},
journal = {Journal of medical microbiology},
volume = {73},
number = {1},
pages = {},
doi = {10.1099/jmm.0.001788},
pmid = {38167305},
issn = {1473-5644},
mesh = {Dogs ; Cats ; Animals ; *Mycoplasma Infections/veterinary/microbiology ; CRISPR-Cas Systems ; Gene Transfer, Horizontal ; *Mycoplasma/genetics ; Genomics ; },
abstract = {A domestic short hair cat (Felis catus) suffering from a purulent wound infection resulting from a dog bite was sampled for bacterial culture and isolation as the wound had been unresponsive to prolonged antimicrobial treatment. A mycoplasma was isolated from the wound. Whole genome sequencing of the isolate was performed using short-read Illumina and long-read Oxford Nanopore chemistry, and the organism was identified as Mycoplasma edwardii. Comparison of the genome sequence of the isolate to a reference M. edwardii genome sequence (canid isolate) identified the loss of several key bacterial factors involved in genome editing, as well the insertion of several novel ORFs most closely related to those found in other canine mycoplasmas, specifically Mycoplasma canis, M. cynos, M. molare and M. maculosa. This is only the second known report of disease caused by M. edwardii in a non-canid species, and the first report of it infecting and causing clinical disease in a cat.},
}
@article {pmid38165033,
year = {2024},
author = {Smith, JE and Granzier, H},
title = {In vivo gene editing of CAMKIID: out with the bad and in with the good.},
journal = {The Journal of clinical investigation},
volume = {134},
number = {1},
pages = {},
pmid = {38165033},
issn = {1558-8238},
support = {R35 HL144998/HL/NHLBI NIH HHS/United States ; },
mesh = {Mice ; Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Genetic Therapy/methods ; DNA ; },
abstract = {The ability to change an organism's DNA through gene editing is of great importance for the prevention and treatment of genetic and acquired diseases. Rapid progress has been made during the last decade due to the discovery and refinement of CRISPR/Cas9 as an accurate, fast, and reliable genome editing technique. In this issue of the JCI, Lebek et al. present the culmination from a line of work in the Olson laboratory focused on in vivo gene editing of CAMK2D. The paper presents a combined state-of-the-art gene therapy approach that demonstrates how gene therapy can yield cardioprotection in a mouse model and takes notable steps toward potential applicability in patients.},
}
@article {pmid38164146,
year = {2024},
author = {Tang, D and Yan, Y and Li, Y and Li, Y and Tian, J and Yang, L and Ding, H and Bashir, G and Zhou, H and Ding, Q and Tao, R and Zhang, S and Wang, Z and Wu, S},
title = {Targeting DAD1 gene with CRISPR-Cas9 system transmucosally delivered by fluorinated polylysine nanoparticles for bladder cancer intravesical gene therapy.},
journal = {Theranostics},
volume = {14},
number = {1},
pages = {203-219},
pmid = {38164146},
issn = {1838-7640},
mesh = {Mice ; Animals ; Humans ; Urinary Bladder/pathology ; Polylysine/metabolism ; CRISPR-Cas Systems/genetics ; *Urinary Bladder Neoplasms/therapy/drug therapy ; *Nanoparticles ; Genetic Therapy ; },
abstract = {Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.},
}
@article {pmid38164106,
year = {2024},
author = {Komninakis, SV and Domingues, W and Sanabani, S and Folgosi, VA and Barbosa, IN and Casseb, J},
title = {CRISPR/CAS as a powerful tool for HIV cure - a review.},
journal = {AIDS research and human retroviruses},
volume = {},
number = {},
pages = {},
doi = {10.1089/AID.2022.0148},
pmid = {38164106},
issn = {1931-8405},
abstract = {Despite care and the availability of effective antiretroviral treatment, some people living with HIV (PLHIV) suffer from neurocognitive disorders associated with the human immunodeficiency virus (HAND) that is a major problem in their quality of life. The different types of HAND can be characterized into asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and the most severe form known as HIV-associated dementia (HAD). Little is known about the mechanisms of HAND, but it is accepted that it is correlated with the infection of astrocytes, microglial cells and macrophages in the human brain. The formation of a viral reservoir that remains dormant as a provirus in resting CD4+ T lymphocytes, and of refuge tissues, such as the brain is a major contributor to HIV eradication. In recent years, a new set of tools has emerged: gene editing based on the CRISPR/Cas9 system, which can alter genomic segments by insertion, deletion, and replacement and has great therapeutic potential. This technology has been used in research to treat HIV and appears to offer hope for a possible cure for HIV infection and perhaps the prevention of HAND. This approach has the potential to directly impact the quality of life of HIV-infected individuals, which is a very important topic to be known and discussed.},
}
@article {pmid38160083,
year = {2023},
author = {Yang, F and Zheng, K and Yao, Y},
title = {China's regulatory change toward genome-edited crops.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.12.008},
pmid = {38160083},
issn = {1879-3096},
abstract = {China's newly issued genome-edited crop safety assessment guidelines do not entirely deviate from the existing genetically modified regulatory regimes. However, there is an urgent need to reform the genome-edited regulatory framework based on scientific values and a novel regulatory philosophy that balances diverse values to enhance public trust.},
}
@article {pmid38157889,
year = {2023},
author = {Kis, A and Polgári, D and Dalmadi, &#xc1; and Ahmad, I and Rakszegi, M and Sági, L and Csorba, T and Havelda, Z},
title = {Targeted mutations in the GW2.1 gene modulate grain traits and induce yield loss in barley.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {340},
number = {},
pages = {111968},
doi = {10.1016/j.plantsci.2023.111968},
pmid = {38157889},
issn = {1873-2259},
abstract = {Grain Width and Weight 2 (GW2) is an E3-ubiquitin ligase-encoding gene that negatively regulates the size and weight of the grain in cereal species. Therefore, disabling GW2 gene activity was suggested for enhancing crop productivity. We show here that CRISPR/Cas-mediated mutagenesis of the barley GW2.1 homologue results in the development of elongated grains and increased protein content. At the same time, GW2.1 loss of function induces a significant grain yield deficit caused by reduced spike numbers and low grain setting. We also show that the converse effect caused by GW2.1 absence on crop yield and protein content is largely independent of cultivation conditions. These findings indicate that the barley GW2.1 gene is necessary for the optimization between yield and grain traits. Altogether, our data show that the loss of GW2.1 gene activity in barley is associated with pleiotropic effects negatively affecting the development of generative organs and consequently the grain production. Our findings contribute to the better understanding of grain development and the utilisation of GW2.1 control in quantitative and qualitative genetic improvement of barley.},
}
@article {pmid38104562,
year = {2024},
author = {Han, R},
title = {First in vivo base-editing trial shows promise.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {1},
pages = {1-2},
doi = {10.1016/j.ymthe.2023.12.001},
pmid = {38104562},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid38036737,
year = {2023},
author = {Yoon, DE and Kim, NR and Park, SJ and Jeong, TY and Eun, B and Cho, Y and Lim, SY and Lee, H and Seong, JK and Kim, K},
title = {Precise base editing without unintended indels in human cells and mouse primary myoblasts.},
journal = {Experimental &amp; molecular medicine},
volume = {55},
number = {12},
pages = {2586-2595},
pmid = {38036737},
issn = {2092-6413},
support = {NRF-2020M3A9D5A01082439//National Research Foundation of Korea (NRF)/ ; NRF-2023R1A2C2004222//National Research Foundation of Korea (NRF)/ ; RS-2023-00261905//National Research Foundation of Korea (NRF)/ ; RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; },
mesh = {Humans ; Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; INDEL Mutation ; Chromatin ; Nucleotides ; Peptides ; },
abstract = {Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.},
}
@article {pmid38012387,
year = {2024},
author = {Mullard, A},
title = {CRISPR gets the glory in landmark approval, but haemoglobin research made it possible.},
journal = {Nature reviews. Drug discovery},
volume = {23},
number = {1},
pages = {14-15},
doi = {10.1038/d41573-023-00188-5},
pmid = {38012387},
issn = {1474-1784},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; },
}
@article {pmid37952084,
year = {2024},
author = {Badon, IW and Oh, Y and Kim, HJ and Lee, SH},
title = {Recent application of CRISPR-Cas12 and OMEGA system for genome editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {1},
pages = {32-43},
doi = {10.1016/j.ymthe.2023.11.013},
pmid = {37952084},
issn = {1525-0024},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Phylogeny ; Bacteria/genetics ; Endonucleases/metabolism ; },
abstract = {In 2012, it was discovered that precise gene editing could be induced in target DNA using the reprogrammable characteristics of the CRISPR system. Since then, several studies have investigated the potential of the CRISPR system to edit various biological organisms. For the typical CRISPR system obtained from bacteria and archaea, many application studies have been conducted and have spread to various fields. To date, orthologs with various characteristics other than CRISPR-Cas9 have been discovered and are being intensively studied in the field of gene editing. CRISPR-Cas12 and its varied orthologs are representative examples of genome editing tools and have superior properties in terms of in vivo target gene editing compared with Cas9. Recently, TnpB and Fanzor of the OMEGA (obligate mobile element guided activity) system were identified to be the ancestor of CRISPR-Cas12 on the basis of phylogenetic analysis. Notably, the compact sizes of Cas12 and OMEGA endonucleases allow adeno-associated virus (AAV) delivery; hence, they are set to challenge Cas9 for in vivo gene therapy. This review is focused on these RNA-guided reprogrammable endonucleases: their structure, biochemistry, off-target effects, and applications in therapeutic gene editing.},
}
@article {pmid37889037,
year = {2024},
author = {Gökbağ, B and Tang, S and Fan, K and Cheng, L and Yu, L and Zhao, Y and Li, L},
title = {SLKB: synthetic lethality knowledge base.},
journal = {Nucleic acids research},
volume = {52},
number = {D1},
pages = {D1418-D1428},
pmid = {37889037},
issn = {1362-4962},
support = {//Ohio State University/ ; },
mesh = {Humans ; *Synthetic Lethal Mutations ; RNA, Guide, CRISPR-Cas Systems ; Knowledge Bases ; *Neoplasms/genetics ; },
abstract = {Emerging CRISPR-Cas9 technology permits synthetic lethality (SL) screening of large number of gene pairs from gene combination double knockout (CDKO) experiments. However, the poor integration and annotation of CDKO SL data in current SL databases limit their utility, and diverse methods of calculating SL scores prohibit their comparison. To overcome these shortcomings, we have developed SL knowledge base (SLKB) that incorporates data of 11 CDKO experiments in 22 cell lines, 16,059 SL gene pairs and 264,424 non-SL gene pairs. Additionally, within SLKB, we have implemented five SL calculation methods: median score with and without background control normalization (Median-B/NB), sgRNA-derived score (sgRNA-B/NB), Horlbeck score, GEMINI score and MAGeCK score. The five scores have demonstrated a mere 1.21% overlap among their top 10% SL gene pairs, reflecting high diversity. Users can browse SL networks and assess the impact of scoring methods using Venn diagrams. The SL network generated from all data in SLKB shows a greater likelihood of SL gene pair connectivity with other SL gene pairs than non-SL pairs. Comparison of SL networks between two cell lines demonstrated greater likelihood to share SL hub genes than SL gene pairs. SLKB website and pipeline can be freely accessed at https://slkb.osubmi.org and https://slkb.docs.osubmi.org/, respectively.},
}
@article {pmid37500408,
year = {2024},
author = {Teng, Y and Jiang, T and Yan, Y},
title = {The expanded CRISPR toolbox for constructing microbial cell factories.},
journal = {Trends in biotechnology},
volume = {42},
number = {1},
pages = {104-118},
doi = {10.1016/j.tibtech.2023.06.012},
pmid = {37500408},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {Microbial cell factories (MCFs) convert low-cost carbon sources into valuable compounds. The CRISPR/Cas9 system has revolutionized MCF construction as a remarkable genome editing tool with unprecedented programmability. Recently, the CRISPR toolbox has been significantly expanded through the exploration of new CRISPR systems, the engineering of Cas effectors, and the incorporation of other effectors, enabling multi-level regulation and gene editing free of double-strand breaks. This expanded CRISPR toolbox powerfully promotes MCF construction by facilitating pathway construction, enzyme engineering, flux redistribution, and metabolic burden control. In this article, we summarize different CRISPR tool designs and their applications in MCF construction for gene editing, transcriptional regulation, and enzyme modulation. Finally, we also discuss future perspectives for the development and application of the CRISPR toolbox.},
}
@article {pmid37482468,
year = {2024},
author = {Cao, M and Li, B and Zhang, X},
title = {Anti-CRISPR with non-protein substances.},
journal = {Trends in biotechnology},
volume = {42},
number = {1},
pages = {14-16},
doi = {10.1016/j.tibtech.2023.07.002},
pmid = {37482468},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; },
abstract = {Therapeutics based on clustered regularly interspaced short palindromic repeats (CRISPR) have gained significant attention as a promising synthetic biology technique, but there are concerns about the potential for persistent activation of CRISPR-associated protein (Cas) and subsequent off-target effects. This forum focuses on advances in anti-CRISPR studies based on non-protein substances in the hope of developing effective anti-CRISPR strategies to mitigate these concerns.},
}
@article {pmid38156005,
year = {2023},
author = {Muhammad, N and Avila, F and Nedashkovskaya, OI and Kim, SG},
title = {Three novel marine species of the genus Reichenbachiella exhibiting degradation of complex polysaccharides.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1265676},
pmid = {38156005},
issn = {1664-302X},
abstract = {Three novel strains designated ABR2-5[T], BKB1-1[T], and WSW4-B4[T] belonging to the genus Reichenbachiella of the phylum Bacteroidota were isolated from algae and mud samples collected in the West Sea, Korea. All three strains were enriched for genes encoding up to 216 carbohydrate-active enzymes (CAZymes), which participate in the degradation of agar, alginate, carrageenan, laminarin, and starch. The 16S rRNA sequence similarities among the three novel isolates were 94.0%-94.7%, and against all three existing species in the genus Reichenbachiella they were 93.6%-97.2%. The genome sizes of the strains ABR2-5[T], BKB1-1[T], and WSW4-B4[T] were 5.5, 4.4, and 5.0 Mb, respectively, and the GC content ranged from 41.1%-42.0%. The average nucleotide identity and the digital DNA-DNA hybridization values of each novel strain within the isolates and all existing species in the genus Reichenbachiella were in a range of 69.2%-75.5% and 17.7-18.9%, respectively, supporting the creation of three new species. The three novel strains exhibited a distinctive fatty acid profile characterized by elevated levels of iso-C15:0 (37.7%-47.4%) and C16:1 ω5c (14.4%-22.9%). Specifically, strain ABR2-5[T] displayed an additional higher proportion of C16:0 (13.0%). The polar lipids were phosphatidylethanolamine, unidentified lipids, aminolipids, and glycolipids. Menaquinone-7 was identified as the respiratory quinone of the isolates. A comparative genome analysis was performed using the KEGG, RAST, antiSMASH, CRISPRCasFinder, dbCAN, and dbCAN-PUL servers and CRISPRcasIdentifier software. The results revealed that the isolates harbored many key genes involved in central metabolism for the synthesis of essential amino acids and vitamins, hydrolytic enzymes, carotenoid pigments, and antimicrobial compounds. The KEGG analysis showed that the three isolates possessed a complete pathway of dissimilatory nitrate reduction to ammonium (DNRA), which is involved in the conservation of bioavailable nitrogen within the ecosystem. Moreover, all the strains possessed genes that participated in the metabolism of heavy metals, including arsenic, copper, cobalt, ferrous, and manganese. All three isolated strains contain the class 2 type II subtype C1 CRISPR-Cas system in their genomes. The distinguished phenotypic, chemotaxonomic, and genomic characteristics led us to propose that the three strains represent three novel species in the genus Reichenbachiella: R. ulvae sp. nov. (ABR2-5[T] = KCTC 82990[T] = JCM 35839[T]), R. agarivorans sp. nov. (BKB1-1[T] = KCTC 82964[T] = JCM 35840[T]), and R. carrageenanivorans sp. nov. (WSW4-B4[T] = KCTC 82706[T] = JCM 35841[T]).},
}
@article {pmid38154710,
year = {2023},
author = {Shrikrishna, NS and Mahari, S and Gandhi, S},
title = {Sensing of trans-cleavage activity of CRISPR/Cas12a for detection of Salmonella.},
journal = {International journal of biological macromolecules},
volume = {258},
number = {Pt 2},
pages = {128979},
doi = {10.1016/j.ijbiomac.2023.128979},
pmid = {38154710},
issn = {1879-0003},
abstract = {Salmonella typhimurium (S. typhi) a predominant foodborne pathogen, significantly impacting global public health. Therefore, timely diagnosis is imperative to safeguard overall human health. To address this, we developed a novel CRISPR/Cas12a-mediated electrochemical detection system (biosensor) for targeting the SifA gene of S. typhi. To construct the biosensor, we utilized a screen-printed gold electrode (SPGE) as an electrochemical transducer and CRISPR/Cas12a for detection of SifA gene of S. typhi. The developed electrochemical biosensor exhibited an exceptional detection limit of 0.634 ± 0.029 pM, which was determined through differential pulse voltammetry (DPV) by utilizing a potentiostat. We compared the fabricated biosensor with gold standard RT-PCR and the visual detection limit of SifA was found to be 10 μM (in spiked buffer samples). The lower detection limit of fabricated biosensor provides an upper edge over the RT-PCR. Further, the fabricated biosensor also has the potential to serve as a rapid, stable, efficient, and early detection tool for S. typhi, offering promising advancements in diagnostic realms.},
}
@article {pmid38153530,
year = {2023},
author = {Bravo-Vázquez, LA and Méndez-García, A and Chamu-García, V and Rodríguez, AL and Bandyopadhyay, A and Paul, S},
title = {The applications of CRISPR/Cas-mediated microRNA and lncRNA editing in plant biology: shaping the future of plant non-coding RNA research.},
journal = {Planta},
volume = {259},
number = {2},
pages = {32},
pmid = {38153530},
issn = {1432-2048},
mesh = {*RNA, Long Noncoding/genetics ; *MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; RNA, Untranslated/genetics ; Genomics ; },
abstract = {CRISPR/Cas technology has greatly facilitated plant non-coding RNA (ncRNA) biology research, establishing itself as a promising tool for ncRNA functional characterization and ncRNA-mediated plant improvement. Throughout the last decade, the promising genome editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas; CRISPR/Cas) has allowed unprecedented advances in the field of plant functional genomics and crop improvement. Even though CRISPR/Cas-mediated genome editing system has been widely used to elucidate the biological significance of a number of plant protein-coding genes, this technology has been barely applied in the functional analysis of those non-coding RNAs (ncRNAs) that modulate gene expression, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Nevertheless, compelling findings indicate that CRISPR/Cas-based ncRNA editing has remarkable potential for deciphering the biological roles of ncRNAs in plants, as well as for plant breeding. For instance, it has been demonstrated that CRISPR/Cas tool could overcome the challenges associated with other approaches employed in functional genomic studies (e.g., incomplete knockdown and off-target activity). Thus, in this review article, we discuss the current status and progress of CRISPR/Cas-mediated ncRNA editing in plant science in order to provide novel prospects for further assessment and validation of the biological activities of plant ncRNAs and to enhance the development of ncRNA-centered protocols for crop improvement.},
}
@article {pmid38153181,
year = {2023},
author = {Wagenbach, M and Vicente, JJ and Wagenbach, W and Wordeman, L},
title = {Production of CRISPR-Cas9 Transgenic Cell Lines for Knocksideways Studies.},
journal = {Current protocols},
volume = {3},
number = {12},
pages = {e965},
pmid = {38153181},
issn = {2691-1299},
support = {R01 GM069429/GM/NIGMS NIH HHS/United States ; R01 GM145567/GM/NIGMS NIH HHS/United States ; 2319918/NH/NIH HHS/United States ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; Animals, Genetically Modified ; Cell Line ; Sirolimus ; },
abstract = {Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin-relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off-target effects are minimized by testing for the most efficient target site using a split-GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split-GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners.},
}
@article {pmid38153158,
year = {2023},
author = {Littleford-Colquhoun, B and Kartzinel, TR},
title = {A CRISPR-based strategy for targeted sequencing in biodiversity science.},
journal = {Molecular ecology resources},
volume = {},
number = {},
pages = {e13920},
doi = {10.1111/1755-0998.13920},
pmid = {38153158},
issn = {1755-0998},
support = {//Institute at Brown for Environment and Society seed award/ ; NSF DEB-2046797//National Science Foundation/ ; },
abstract = {Many applications in molecular ecology require the ability to match specific DNA sequences from single- or mixed-species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target-specific enrichment capabilities of CRISPR-Cas systems may offer advantages in some applications. We identified 54,837 CRISPR-Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single- and mixed-species samples, which yielded mean chloroplast sequence lengths of 2,530-11,367 bp, depending on the experiment. In comparison to mixed-species experiments, single-species experiments yielded more on-target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed-species experiments yielded sufficient data to provide ≥48-fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL-P6 marker. Prior work developed CRISPR-based enrichment protocols for long-read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short-read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR-based analyses of mixed-species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.},
}
@article {pmid38150477,
year = {2024},
author = {Gao, S and Guan, H and Bloomer, H and Wich, D and Song, D and Khirallah, J and Ye, Z and Zhao, Y and Chen, M and Xu, C and Liu, L and Xu, Q},
title = {Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {2},
pages = {e2308415120},
doi = {10.1073/pnas.2308415120},
pmid = {38150477},
issn = {1091-6490},
mesh = {Humans ; *Base Pairing ; *CRISPR-Cas Systems ; DNA/genetics/chemistry ; *DNA, Z-Form/genetics ; *Gene Editing/methods ; RNA/genetics ; RNA, Guide, CRISPR-Cas Systems ; Thymine/chemistry ; },
abstract = {Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.},
}
@article {pmid38147207,
year = {2024},
author = {Lossius, C and Kresinsky, A and Quiet, L and Müller, JP},
title = {CRISPR/Cas9-Mediated Modification of PTP Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2743},
number = {},
pages = {43-56},
pmid = {38147207},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Transcriptional Activation ; Mammals ; },
abstract = {Alteration of protein tyrosine phosphatase (PTP) gene expression is a commonly used approach to experimentally analyze their function in the cell physiology of mammalian cells. Here, exemplified for receptor-type PTPRJ (Dep-1, CD148) and PPTRC (CD45), we provide the CRISPR/Cas9-mediated approaches for their inactivation and transcriptional activation using genome editing. These methods are generally applicable to any other protein of interest.},
}
@article {pmid38147133,
year = {2023},
author = {Jiang, T and Li, Y and Hong, W and Lin, M},
title = {A robust CRISPR interference gene repression system in Vibrio parahaemolyticus.},
journal = {Archives of microbiology},
volume = {206},
number = {1},
pages = {41},
pmid = {38147133},
issn = {1432-072X},
support = {XB2021139//Fujian University of Traditional Chinese Medicine University Clinical Special Project Program/ ; },
mesh = {*Vibrio parahaemolyticus/genetics ; RNA, Guide, CRISPR-Cas Systems ; Aquaculture ; Arabinose ; Drug Discovery ; },
abstract = {Vibrio parahaemolyticus is a significant cause of seafood-associated gastroenteritis and pestilence in aquaculture worldwide. Despite extensive research, strategies for protein depletion in this pathogen remain limited. Herein, we constructed a new CRISPR interference (CRISPRi) system for gene repression based on the combination of a shuttle vector pVv3 and the nuclease-null Cas9 variant (dead Cas9, or dCas9) from Streptococcus pyrogens. This CRISPRi is induced by adding both IPTG and arabinose. We showed that gene repression is scalable via the use of multiple sgRNAs. We also demonstrated that this gene repression can be precisely tuned by adjusting the amount of two different inducers and can be reversed by removing the inducers. This system provides a simple approach for selective gene repression on a genome-wide scale in V. parahaemolyticus. Application of this system will dramatically accelerate investigations of this bacterium, including studies of physiology, pathogenesis, and drug target discovery.},
}
@article {pmid38145378,
year = {2023},
author = {Van Heest, AE and Deng, F and Zhao, RT and Harzallah, NS and Fleming, HE and Bhatia, SN and Hao, L},
title = {CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {202},
pages = {},
doi = {10.3791/66189},
pmid = {38145378},
issn = {1940-087X},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Urinalysis ; *Body Fluids ; Biomarkers ; DNA/genetics ; },
abstract = {Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.},
}
@article {pmid38058090,
year = {2023},
author = {Panichella, G and Aimo, A},
title = {CRISPR-Cas9 for the Treatment of Transthyretin Cardiac Amyloidosis.},
journal = {Current pharmaceutical design},
volume = {29},
number = {39},
pages = {3166-3169},
doi = {10.2174/0113816128267417231127070901},
pmid = {38058090},
issn = {1873-4286},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Prealbumin/genetics ; *Amyloidosis ; Gene Editing ; },
}
@article {pmid38052295,
year = {2024},
author = {Mochii, M and Akizuki, K and Ossaka, H and Kagawa, N and Umesono, Y and Suzuki, KT},
title = {A CRISPR-Cas9-mediated versatile method for targeted integration of a fluorescent protein gene to visualize endogenous gene expression in Xenopus laevis.},
journal = {Developmental biology},
volume = {506},
number = {},
pages = {42-51},
doi = {10.1016/j.ydbio.2023.11.010},
pmid = {38052295},
issn = {1095-564X},
mesh = {Animals ; Xenopus laevis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Expression ; DNA ; },
abstract = {Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several reports regarding targeted integration techniques in Xenopus, there is still room for improvement of them, especially in creating reporter lines that rely on endogenous regulatory enhancers/promoters. We developed a CRISPR-Cas9-based simple method to efficiently introduce a fluorescent protein gene into 5' untranslated regions (5'UTRs) of target genes in Xenopus laevis. A donor plasmid DNA encoding an enhanced green fluorescent protein (eGFP) flanked by a genomic fragment ranging from 66 bp to 878 bp including target 5'UTR was co-injected into fertilized eggs with a single guide RNA and Cas9 protein. Injections for krt12.2.L, myod1.S, sox2.L or brevican.S resulted in embryos expressing eGFP fluorescence in a tissue-specific manner, recapitulating endogenous expression of target genes. Integrations of the donor DNA into the target regions were examined by genotyping PCR for the eGFP-expressing embryos. The rate of embryos expressing the specific eGFP varied from 2.1% to 13.2% depending on the target locus and length of the genomic fragment in the donor plasmids. Germline transmission of an integrated DNA was observed. This simple method provides a powerful tool for exploring gene expression and function in developmental and regeneration research in X. laevis.},
}
@article {pmid37978800,
year = {2024},
author = {Banerjee, P and Voth, GA},
title = {Conformational transitions of the HIV-1 Gag polyprotein upon multimerization and gRNA binding.},
journal = {Biophysical journal},
volume = {123},
number = {1},
pages = {42-56},
doi = {10.1016/j.bpj.2023.11.017},
pmid = {37978800},
issn = {1542-0086},
support = {U54 AI170855/AI/NIAID NIH HHS/United States ; },
mesh = {*Gene Products, gag/chemistry/genetics/metabolism ; *HIV-1/metabolism ; RNA, Viral/chemistry ; Virus Assembly ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; gag Gene Products, Human Immunodeficiency Virus/chemistry/metabolism ; },
abstract = {During the HIV-1 assembly process, the Gag polyprotein multimerizes at the producer cell plasma membrane, resulting in the formation of spherical immature virus particles. Gag-genomic RNA (gRNA) interactions play a crucial role in the multimerization process, which is yet to be fully understood. We performed large-scale all-atom molecular dynamics simulations of membrane-bound full-length Gag dimer, hexamer, and 18-mer. The inter-domain dynamic correlation of Gag, quantified by the heterogeneous elastic network model applied to the simulated trajectories, is observed to be altered by implicit gRNA binding, as well as the multimerization state of the Gag. The lateral dynamics of our simulated membrane-bound Gag proteins, with and without gRNA binding, agree with prior experimental data and help to validate our simulation models and methods. The gRNA binding is observed to affect mainly the SP1 domain of the 18-mer and the matrix-capsid linker domain of the hexamer. In the absence of gRNA binding, the independent dynamical motion of the nucleocapsid domain results in a collapsed state of the dimeric Gag. Unlike stable SP1 helices in the six-helix bundle, without IP6 binding, the SP1 domain undergoes a spontaneous helix-to-coil transition in the dimeric Gag. Together, our findings reveal conformational switches of Gag at different stages of the multimerization process and predict that the gRNA binding reinforces an efficient binding surface of Gag for multimerization, and also regulates the dynamic organization of the local membrane region itself.},
}
@article {pmid37951433,
year = {2024},
author = {Bhadauria, V and Han, T and Li, G and Ma, W and Zhang, M and Yang, J and Zhao, W and Peng, YL},
title = {A gln-tRNA-based CRISPR/Cas9 knockout system enables the functional characterization of genes in the genetically recalcitrant brassica anthracnose fungus Colletotrichum higginsianum.},
journal = {International journal of biological macromolecules},
volume = {254},
number = {Pt 3},
pages = {127953},
doi = {10.1016/j.ijbiomac.2023.127953},
pmid = {37951433},
issn = {1879-0003},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; *Colletotrichum ; DNA/metabolism ; },
abstract = {Colletotrichum higginsianum causes anthracnose disease in brassicas. The availability of the C. higginsianum genome has paved the way for the genome-wide exploration of genes associated with virulence/pathogenicity. However, delimiting the biological functions of these genes remains an arduous task due to the recalcitrance of C. higginsianum to genetic manipulations. Here, we report a CRISPR/Cas9-based system that can knock out the genes in C. higginsianum with a staggering 100% homologous recombination frequency (HRF). The system comprises two vectors: pCas9-Ch_tRp-sgRNA, in which a C. higginsianum glutaminyl-tRNA drives the expression of sgRNA, and pCE-Zero-HPT carrying a donor DNA cassette containing the marker gene HPT flanked by homology arms. Upon co-transformation of the C. higginsianum protoplasts, pCas9-Ch_tRp-sgRNA causes a DNA double-strand break in the targeted gene, followed by homology-directed replacement of the gene with HPT by pCE-Zero-HPT, thereby generating loss-of-function mutants. Using the system, we generated the knockout mutants of two effector candidates (ChBas3 and OBR06881) with a 100% HRF. Interestingly, the ΔChBas3 and ΔOBR06881 mutants did not seem to affect the C. higginsianum infection of Arabidopsis thaliana. Altogether, the CRISPR/Cas9 system developed in the study enables the targeted deletion of genes, including effectors, in C. higginsianum, thus determining their biological functions.},
}
@article {pmid38144710,
year = {2023},
author = {Bhuyan, SJ and Kumar, M and Ramrao Devde, P and Rai, AC and Mishra, AK and Singh, PK and Siddique, KHM},
title = {Progress in gene editing tools, implications and success in plants: a review.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1272678},
pmid = {38144710},
issn = {2673-3439},
abstract = {Genetic modifications are made through diverse mutagenesis techniques for crop improvement programs. Among these mutagenesis tools, the traditional methods involve chemical and radiation-induced mutagenesis, resulting in off-target and unintended mutations in the genome. However, recent advances have introduced site-directed nucleases (SDNs) for gene editing, significantly reducing off-target changes in the genome compared to induced mutagenesis and naturally occurring mutations in breeding populations. SDNs have revolutionized genetic engineering, enabling precise gene editing in recent decades. One widely used method, homology-directed repair (HDR), has been effective for accurate base substitution and gene alterations in some plant species. However, its application has been limited due to the inefficiency of HDR in plant cells and the prevalence of the error-prone repair pathway known as non-homologous end joining (NHEJ). The discovery of CRISPR-Cas has been a game-changer in this field. This system induces mutations by creating double-strand breaks (DSBs) in the genome and repairing them through associated repair pathways like NHEJ. As a result, the CRISPR-Cas system has been extensively used to transform plants for gene function analysis and to enhance desirable traits. Researchers have made significant progress in genetic engineering in recent years, particularly in understanding the CRISPR-Cas mechanism. This has led to various CRISPR-Cas variants, including CRISPR-Cas13, CRISPR interference, CRISPR activation, base editors, primes editors, and CRASPASE, a new CRISPR-Cas system for genetic engineering that cleaves proteins. Moreover, gene editing technologies like the prime editor and base editor approaches offer excellent opportunities for plant genome engineering. These cutting-edge tools have opened up new avenues for rapidly manipulating plant genomes. This review article provides a comprehensive overview of the current state of plant genetic engineering, focusing on recently developed tools for gene alteration and their potential applications in plant research.},
}
@article {pmid38142864,
year = {2023},
author = {Riedl, M and Bordone, L and Revenko, A and Newman, KB and Cohn, DM},
title = {Clinical Progress in Hepatic Targeting for Novel Prophylactic Therapies in Hereditary Angioedema.},
journal = {The journal of allergy and clinical immunology. In practice},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jaip.2023.12.025},
pmid = {38142864},
issn = {2213-2201},
abstract = {Hereditary angioedema (HAE) is typically caused by a deficiency of the protease inhibitor C1 inhibitor (C1INH). The absence of C1INH activity on plasma kallikrein and factor XIIa leads to overproduction of the vasoactive peptide bradykinin, with resulting angioedema. As the primary site of C1INH and prekallikrein production, the liver is being recognized as an important therapeutic target in HAE, leading to development of hepatic-focused treatment strategies such as GalNAc-conjugated antisense technology and gene modification. This paper reviews currently available data on hepatic-focused interventions for HAE that have advanced into human trials. Donidalorsen is an investigational GalNAc3-conjugated antisense oligonucleotide that binds to prekallikrein mRNA in the liver and reduces the expression of prekallikrein. Phase 2 data with subcutaneous donidalorsen demonstrated a significant reduction in HAE attack rate compared with placebo. Phase 3 trials are currently underway. ADX-324 is a GalNAc3-conjugated siRNA being investigated in HAE. BMN 331 is an investigational AAV5-based gene therapy vector that expresses wild-type human C1INH and is targeted to hepatocytes. A single intravenous dose of BMN 331 is intended to replace the defective SERPING1 gene and enable patients to produce functional C1INH. A first-in-human phase 1/2 study is ongoing with BMN 331. NTLA-2002 is an investigational in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based therapy designed to knock out the prekallikrein-coding KLKB1 gene in hepatocytes; a phase 1/2 study is ongoing. Findings from these and other ongoing studies are highly anticipated with the expectation of expanding the array of treatment options in HAE.},
}
@article {pmid38087988,
year = {2024},
author = {Sundar Rajan, V and Wypijewska Del Nogal, A and Levin, S and Wilhelmsson, LM and Westerlund, F},
title = {Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues.},
journal = {Nanoscale},
volume = {16},
number = {2},
pages = {752-764},
pmid = {38087988},
issn = {2040-3372},
mesh = {Humans ; *SARS-CoV-2 ; RNA, Viral/chemistry ; Base Sequence ; Nucleic Acid Conformation ; Optical Tweezers ; *COVID-19 ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The parasitic nature of the SARS-CoV-2 virus demands selective packaging of its RNA genome (gRNA) from the abundance of other nucleic acids present in infected cells. Despite increasing evidence that stem-loop 4 (SL4) of the gRNA 5' UTR is involved in the initiation of this process by binding the nucleocapsid (N) protein, little is known about its conformational dynamics. Here, we unravel the stability, dynamics and (un)folding pathways of SL4 using optical tweezers and a base analogue, tC[O], that provides a local and subtle increase in base stacking without perturbing hydrogen bonding. We find that SL4 (un)folds mainly in a single step or through an intermediate, encompassing nucleotides from the central U bulge to the hairpin loop. Due to an upper-stem CU mismatch, SL4 is prone to misfold, the extent of which can be tuned by incorporating tC[O] at different positions. Our study contributes to a better understanding of SARS-CoV-2 packaging and the design of drugs targeting SL4. We also highlight the generalizability of using base analogues in optical tweezers experiments for probing intramolecular states and conformational transitions of various nucleic acids at the level of single molecules and with base-pair resolution.},
}
@article {pmid38082131,
year = {2024},
author = {Thompson, T},
title = {How CRISPR gene editing could help treat Alzheimer's.},
journal = {Nature},
volume = {625},
number = {7993},
pages = {13-14},
pmid = {38082131},
issn = {1476-4687},
mesh = {Humans ; *Alzheimer Disease/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; Mutation ; },
}
@article {pmid38012416,
year = {2024},
author = {LaFleur, MW and Lemmen, AM and Streeter, ISL and Nguyen, TH and Milling, LE and Derosia, NM and Hoffman, ZM and Gillis, JE and Tjokrosurjo, Q and Markson, SC and Huang, AY and Anekal, PV and Montero Llopis, P and Haining, WN and Doench, JG and Sharpe, AH},
title = {X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system.},
journal = {Nature immunology},
volume = {25},
number = {1},
pages = {178-188},
pmid = {38012416},
issn = {1529-2916},
support = {P01 AI108545/AI/NIAID NIH HHS/United States ; U54 CA225088/CA/NCI NIH HHS/United States ; P01 AI108545/AI/NIAID NIH HHS/United States ; U54 CA225088/CA/NCI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics ; Mice, Knockout ; Immune System ; Gene Editing ; },
abstract = {Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.},
}
@article {pmid37935845,
year = {2024},
author = {Yu, Z and Liu, H and Chen, Z and Shao, Y and Wang, Z and Cheng, F and Zhang, Y and Wang, Z and Tu, J and Song, X and Qi, K},
title = {LAMP assay coupled with a CRISPR/Cas12a system for the rapid and ultrasensitive detection of porcine circovirus-like virus in the field.},
journal = {Analytical and bioanalytical chemistry},
volume = {416},
number = {2},
pages = {363-372},
pmid = {37935845},
issn = {1618-2650},
support = {31972642//the Natural Science Foundation of China/ ; },
mesh = {Swine ; Animals ; *Circovirus/genetics ; CRISPR-Cas Systems ; *Swine Diseases/diagnosis ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; *Viruses ; },
abstract = {A recent outbreak of porcine circovirus-like virus (PCLV), a virus that may be associated with porcine diarrhea, has been reported in swine herds in China. The virus is spreading rapidly, causing huge economic losses to the swine farming industry. To achieve the rapid, inexpensive, and sensitive detection of PCLV, we combined loop-mediated isothermal amplification (LAMP) and the CRISPR/Cas12a system, whose fluorescence intensity readout can detect PCLV ORF4 gene levels as low as 10 copies. To overcome the need for sophisticated equipment, lateral flow strip reading technology was introduced for the first time in a LAMP-Cas12a-based system to detect PCLV. The lateral flow strip (LFS) results were readout by the naked eye, and the method was highly sensitive with a detection limit of 10 copies, with a detection time of about 60 min. In addition, the method is highly specific and has no cross-reactivity with other related viruses. In conclusion, LAMP-CRISPR/Cas12a-based assays have the advantages of rapidity, accuracy, portability, low cost, and visualization of the results. They therefore have great potential, especially for areas where specialized equipment is lacking, and can expect to be an ideal method for early diagnosis and on-site detection of PCLV.},
}
@article {pmid37819132,
year = {2023},
author = {Seeger, C},
title = {A CRISPR-based system to investigate HBV cccDNA biology.},
journal = {Journal of virology},
volume = {97},
number = {10},
pages = {e0118523},
pmid = {37819132},
issn = {1098-5514},
support = {P30 CA006927/CA/NCI NIH HHS/United States ; R01 AI130022/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *DNA, Circular/genetics ; DNA, Viral/genetics ; *Hepatitis B virus/genetics ; Hepatitis B, Chronic ; Virus Replication/genetics ; *CRISPR-Cas Systems ; },
abstract = {Hepatitis B virus cccDNA is the key target for the necessary development of antiviral therapies aimed at curing chronic hepatitis B. The CRISPR-based system to produce covalently closed circular (cccDNA)-like extrachromosomal DNAs described in this report enables large-scale screens of chemical libraries to identify drug candidates with the potential to permanently inactivate cccDNA. Moreover, this approach permits investigations on unresolved problems as described in this report concerning cccDNA biology including mechanisms of SMC5/6-dependent transcriptional silencing and the contributions of the SMC5/6 complex to cccDNA stability in resting and dividing hepatocytes.},
}
@article {pmid36450762,
year = {2022},
author = {Tiyaboonchai, A and Vonada, A and Posey, J and Pelz, C and Wakefield, L and Grompe, M},
title = {Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7391},
pmid = {36450762},
issn = {2041-1723},
support = {T32 GM142619/GM/NIGMS NIH HHS/United States ; F32 DK117516/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Homologous Recombination ; *RNA, Catalytic/genetics ; Transgenes ; },
abstract = {Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.},
}
@article {pmid36410036,
year = {2022},
author = {Goyon, A and Nguyen, D and Boulanouar, S and Yehl, P and Zhang, K},
title = {Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level.},
journal = {Analytical chemistry},
volume = {94},
number = {48},
pages = {16960-16966},
doi = {10.1021/acs.analchem.2c04681},
pmid = {36410036},
issn = {1520-6882},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Tandem Mass Spectrometry ; Nucleotides ; Gene Editing/methods ; Chromatography, Liquid ; },
abstract = {The chemistry of guide RNA (gRNA) affects the performance of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technique. However, the literature is very scarce about the study of gRNA degradation and in particular at the single nucleotide level. In this work, we developed a workflow to characterize the impurities of large RNAs at the single nucleotide level and identified the residues prone to degradation. Our strategy involves (i) the reduction of RNA length, (ii) a chromatographic mode able to capture subtle changes in impurity polarity, and (iii) a streamlined data treatment. To illustrate the approach, stressed gRNA samples were analyzed by coupling an immobilized ribonuclease T1 cartridge to a hydrophilic interaction liquid chromatography (HILIC) column hyphenated with tandem mass spectrometry (MS/MS). Critical findings were made possible by the presented technology. In particular, the desulfurization of phosphorothioate (PS) linkages was the major degradation observed at the single nucleotide level while no change in purity profile could be observed when using conventional ion-pairing reversed-phase (IPRP) liquid chromatography. To our knowledge, this is the first time that several impurity types are screened for a large RNA molecule using an automated online digestion analysis approach.},
}
@article {pmid36375249,
year = {2022},
author = {Tabata, T and Masumura, Y and Higo, S and Kunimatsu, S and Kameda, S and Inoue, H and Okuno, S and Ogawa, S and Takashima, S and Watanabe, M and Miyagawa, S and Hikoso, S and Sakata, Y},
title = {Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption.},
journal = {Biochemical and biophysical research communications},
volume = {637},
number = {},
pages = {40-49},
doi = {10.1016/j.bbrc.2022.10.088},
pmid = {36375249},
issn = {1090-2104},
mesh = {Mice ; Animals ; *Calcium/metabolism ; Kinetics ; Myocytes, Cardiac/metabolism ; Gene Editing/methods ; Calcium Channels, L-Type/genetics/metabolism ; *Cardiomyopathy, Dilated/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Kinetic analysis of intracellular calcium (Ca[2+]) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca[2+] kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca[2+] kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca[2+] amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.},
}
@article {pmid36320101,
year = {2022},
author = {Ali, Y and Gomez-Sanchez, EP and Gomez-Sanchez, CE},
title = {Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human Adrenocortical Carcinoma Cells.},
journal = {Endocrinology},
volume = {164},
number = {1},
pages = {},
pmid = {36320101},
issn = {1945-7170},
support = {R01 HL144847/HL/NHLBI NIH HHS/United States ; U54 GM115428/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Angiotensin II/pharmacology/metabolism ; Aldosterone/metabolism ; *Adrenocortical Carcinoma ; Hydrocortisone/metabolism ; Sirolimus/pharmacology ; RNA, Guide, CRISPR-Cas Systems ; *Adrenal Cortex Neoplasms/drug therapy ; TOR Serine-Threonine Kinases ; },
abstract = {BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined.<br><br>METHODS: HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA.<br><br>RESULTS: The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3&#x3b2;-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production.<br><br>CONCLUSION: mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.},
}
@article {pmid36318971,
year = {2022},
author = {Metzger, D and Miller, K and Lyon, W and Migliozzi, R and Pangburn, HA and Saldanha, R},
title = {Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure.},
journal = {ACS synthetic biology},
volume = {11},
number = {11},
pages = {3657-3668},
doi = {10.1021/acssynbio.2c00214},
pmid = {36318971},
issn = {2161-5063},
mesh = {Humans ; *Anthrax/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Promoter Regions, Genetic/genetics ; Receptors, Peptide/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transcription Factors/genetics ; },
abstract = {Anthrax infection is caused byBacillus anthracis, a bacterium that once established within the host releases lethal toxin (LeTx). Anthrax LeTx is internalized by the capillary morphogenesis protein 2/anthrax toxin receptor 2 (CMG2/ANTXR2) cell surface receptor on mammalian cells. Once inside the cell, LeTx cleaves mitogen-activated protein kinases (MAPKs), ultimately leading to cell death. Previous reports have shown that decreased expression of ANTXR2 reduces cell susceptibility to LeTx. By ablating the ANTXR2 gene in cells in vitro, we observed complete resistance to LeTx-induced cell death. Here, we directed CRISPR/dCas9-based tools to the ANTXR2 promoter to modulate ANTXR2 expression without altering the underlying gene sequence in human cell lines that express the receptor at high levels. We hypothesized that downregulating the expression of the ANTXR2 gene at the genomic level would mitigate the impact of toxin exposure. In one epigenetic editing approach, we employed the fusion of DNMT3A DNA methyltransferase and dCas9 (dCas9-DNMT3A) to methylate CpGs within the CpG island of the ANTXR2 promoter and found this repressed ANTXR2 gene expression resulting in significant resistance to LeTx-induced cell death. Furthermore, by multiplexing gRNAs to direct dCas9-DNMT3A to multiple sites in the ANTXR2 promoter, we applied a broader distribution of CpG methylation along the gene promoter resulting in enhanced repression and resistance to LeTx. In parallel, we directed the dCas9-KRAB-MeCP2 transcriptional repressor to the ANTXR2 promoter to quickly and robustly repress ANTXR2 expression. With this approach, in as little as two weeks, we created resistance to LeTx at a similar level to ANTXR2 gene-ablated cells. Overall, we present a transcriptional tuning approach to inhibit the effects of LeTx and provide a framework to repress toxin-binding cell surface receptors.},
}
@article {pmid36288281,
year = {2022},
author = {Ottaviano, G and Georgiadis, C and Gkazi, SA and Syed, F and Zhan, H and Etuk, A and Preece, R and Chu, J and Kubat, A and Adams, S and Veys, P and Vora, A and Rao, K and Qasim, W and , },
title = {Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia.},
journal = {Science translational medicine},
volume = {14},
number = {668},
pages = {eabq3010},
doi = {10.1126/scitranslmed.abq3010},
pmid = {36288281},
issn = {1946-6242},
support = {MR/S019022/1/MRC_/Medical Research Council/United Kingdom ; MRF_MRF-045-0003-RG-GEOR-C0907/MRF/MRF/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {Child ; Humans ; Alemtuzumab ; Antigens, CD19/metabolism ; Cyclophosphamide ; *Graft vs Host Disease/metabolism ; Immunotherapy, Adoptive ; *Leukemia, B-Cell ; *Leukemia, Lymphocytic, Chronic, B-Cell/metabolism ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/metabolism ; *Receptors, Chimeric Antigen/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; T-Lymphocytes ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Genome editing of allogeneic T cells can provide "off-the-shelf" alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator-like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3' long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 10[6] to 2.0 × 10[6] CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.},
}
@article {pmid36202979,
year = {2022},
author = {Fabrick, JA and Heu, CC and LeRoy, DM and DeGain, BA and Yelich, AJ and Unnithan, GC and Wu, Y and Li, X and Carrière, Y and Tabashnik, BE},
title = {Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {16706},
pmid = {36202979},
issn = {2045-2322},
mesh = {ATP-Binding Cassette Transporters/genetics/metabolism ; Adenosine Triphosphate/metabolism ; Animals ; *Bacillus thuringiensis/genetics/metabolism ; Bacillus thuringiensis Toxins ; Bacterial Proteins/genetics/metabolism ; Crops, Agricultural/genetics ; Endotoxins/genetics/metabolism ; Hemolysin Proteins/genetics/metabolism/pharmacology ; Humans ; Insecticide Resistance/genetics ; Larva/genetics ; *Moths/genetics/metabolism ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Zea mays/genetics ; },
abstract = {Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.},
}
@article {pmid36194264,
year = {2022},
author = {Jain, G and Ertesvåg, H},
title = {Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {21},
pages = {7129-7138},
pmid = {36194264},
issn = {1432-0614},
support = {239001//Norges Forskningsr&#xe5;d/ ; 274691//Norges Forskningsr&#xe5;d/ ; },
mesh = {Mutagenesis, Site-Directed ; *Mycolic Acids/metabolism ; Plasmids/genetics ; Recombinases/genetics ; *Rhodococcus/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Triglycerides/metabolism ; },
abstract = {Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria.},
}
@article {pmid36137145,
year = {2022},
author = {Chaiyadet, S and Tangkawattana, S and Smout, MJ and Ittiprasert, W and Mann, VH and Deenonpoe, R and Arunsan, P and Loukas, A and Brindley, PJ and Laha, T},
title = {Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.},
journal = {PLoS pathogens},
volume = {18},
number = {9},
pages = {e1010839},
pmid = {36137145},
issn = {1553-7374},
support = {R01 CA164719/CA/NCI NIH HHS/United States ; 107475/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Bile Duct Neoplasms/genetics/parasitology ; Bile Ducts, Intrahepatic/metabolism/parasitology/pathology ; *Cholangiocarcinoma/genetics/parasitology ; Cricetinae ; *Fasciola hepatica/genetics/metabolism ; Fibrosis ; Granulins/metabolism ; Intercellular Signaling Peptides and Proteins ; *Nitrosamines ; *Opisthorchiasis/complications/parasitology/pathology ; *Opisthorchis/genetics/metabolism ; Persistent Infection ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.},
}
@article {pmid36085316,
year = {2022},
author = {David, SR and Maheshwaram, SK and Shet, D and Lakshminarayana, MB and Soni, GV},
title = {Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {15243},
pmid = {36085316},
issn = {2045-2322},
mesh = {*CRISPR-Associated Protein 9 ; *DNA ; Electrophoretic Mobility Shift Assay ; RNA, Guide, CRISPR-Cas Systems/genetics ; Temperature ; },
abstract = {The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR-Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min[-1] at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9-DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR-Cas9 technology.},
}
@article {pmid36085107,
year = {2022},
author = {Ortega, MS and Bickhart, DM and Lockhart, KN and Null, DJ and Hutchison, JL and McClure, JC and Cole, JB},
title = {Truncation of IFT80 causes early embryonic loss in Holstein cattle associated with Holstein haplotype 2.},
journal = {Journal of dairy science},
volume = {105},
number = {11},
pages = {9001-9011},
doi = {10.3168/jds.2022-21853},
pmid = {36085107},
issn = {1525-3198},
mesh = {Cattle ; Male ; Animals ; Mice ; Haplotypes ; *Hedgehog Proteins/genetics/metabolism ; *Codon, Nonsense ; RNA, Guide, CRISPR-Cas Systems ; Homozygote ; Carrier Proteins ; },
abstract = {Recessive alleles represent genetic risk in populations that have undergone bottleneck events. We present a comprehensive framework for identification and validation of these genetic defects, including haplotype-based detection, variant selection from sequence data, and validation using knockout embryos. Holstein haplotype 2 (HH2), which causes embryonic death, was used to demonstrate the approach. Holstein haplotype 2 was identified using a deficiency-of-homozygotes approach and confirmed to negatively affect conception rate and stillbirths. Five carriers were present in a group of 183 sequenced Holstein bulls selected to maximize the coverage of unique haplotypes. Three variants concordant with haplotype calls were found in HH2: a high-priority frameshift mutation resulting, and 2 low-priority variants (1 synonymous variant, 1 premature stop codon). The frameshift in intraflagellar 80 (IFT80) was confirmed in a separate group of Holsteins from the 1000 Bull Genomes Project that shared no animals with the discovery set. IFT80-null embryos were generated by truncating the IFT80 transcript at exon 2 or 11 using a CRISPR-Cas9 system. Abattoir-derived oocytes were fertilized in vitro, and zygotes were injected at the one-cell stage either with a guide RNA and CAS9 mRNA complex (n = 100) or Cas9 mRNA (control, n = 100) before return to culture, and replicated 3 times. IFT80 is activated at the 8-cell stage, and IFT80-null embryos arrested at this stage of development, which is consistent with data from mouse hypomorphs and HH2 carrier-to-carrier matings. This frameshift in IFT80 on chromosome 1 at 107,172,615 bp (p.Leu381fs) disrupts WNT and hedgehog signaling, and is responsible for the death of homozygous embryos.},
}
@article {pmid36044643,
year = {2022},
author = {Moreno-Giménez, E and Selma, S and Calvache, C and Orzáez, D},
title = {GB_SynP: A Modular dCas9-Regulated Synthetic Promoter Collection for Fine-Tuned Recombinant Gene Expression in Plants.},
journal = {ACS synthetic biology},
volume = {11},
number = {9},
pages = {3037-3048},
pmid = {36044643},
issn = {2161-5063},
mesh = {5' Untranslated Regions ; Gene Expression ; *Plants/genetics ; Promoter Regions, Genetic/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Trans-Activators/genetics ; },
abstract = {Programmable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5'UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an autoluminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.},
}
@article {pmid36037371,
year = {2022},
author = {Qin, G and Yang, J and Zhao, C and Ren, J and Qu, X},
title = {Manipulating complex chromatin folding via CRISPR-guided bioorthogonal chemistry.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {36},
pages = {e2204725119},
pmid = {36037371},
issn = {1091-6490},
mesh = {*Chromatin/genetics ; *Chromatin Assembly and Disassembly ; Chromosomes ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Enhancer Elements, Genetic ; Genome ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Precise manipulation of chromatin folding is important for understanding the relationship between the three-dimensional genome and nuclear function. Existing tools can reversibly establish individual chromatin loops but fail to manipulate two or more chromatin loops. Here, we engineer a powerful CRISPR system which can manipulate multiple chromatin contacts using bioorthogonal reactions, termed the bioorthogonal reaction-mediated programmable chromatin loop (BPCL) system. The multiinput BPCL system employs engineered single-guide RNAs recognized by discrete bioorthogonal adaptors to independently and dynamically control different chromatin loops formation without cross-talk in the same cell or to establish hubs of multiway chromatin contacts. We use the BPCL system to successfully juxtapose the pluripotency gene promoters to enhancers and activate their endogenous expression. BPCL enables us to independently engineer multiway chromatin contacts without cross-talk, which provides a way to precisely dissect the high complexity and dynamic nature of chromatin folding.},
}
@article {pmid36012441,
year = {2022},
author = {Walflor, HSM and Lucena, ARC and Tuon, FF and Medeiros, LCS and Faoro, H},
title = {Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.},
journal = {International journal of molecular sciences},
volume = {23},
number = {16},
pages = {},
pmid = {36012441},
issn = {1422-0067},
support = {424410/2018-4//National Council for Scientific and Technological Development/ ; VPPCB- 07-FIO-18-2-38//Oswaldo Cruz Foundation/ ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial/genetics ; Escherichia coli ; *Escherichia coli Infections/microbiology ; *Fosfomycin/pharmacology ; Humans ; Microbial Sensitivity Tests ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems ; beta-Lactamases/genetics ; },
abstract = {Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.},
}
@article {pmid36009033,
year = {2022},
author = {Yamamoto, R and Sato, G and Amai, T and Ueda, M and Kuroda, K},
title = {Development of Artificial System to Induce Chromatin Loosening in Saccharomyces cerevisiae.},
journal = {Biomolecules},
volume = {12},
number = {8},
pages = {},
pmid = {36009033},
issn = {2218-273X},
mesh = {Acetylation ; Chromatin/genetics/metabolism ; Euchromatin/metabolism ; Heterochromatin ; Histones/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus.},
}
@article {pmid35993750,
year = {2022},
author = {Xia, Y and Hu, J and Li, X and Zheng, S and Wang, G and Tan, S and Zou, Z and Ling, Q and Yang, F and Fan, X},
title = {Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {186},
pages = {},
doi = {10.3791/63949},
pmid = {35993750},
issn = {1940-087X},
mesh = {Animals ; Cardiac Myosins/genetics ; *Cardiomyopathy, Hypertrophic/genetics ; *Cardiomyopathy, Hypertrophic, Familial/diagnosis/genetics ; Mice ; Mice, Inbred C57BL ; Mutation ; Myosin Heavy Chains/genetics ; Pedigree ; Phenotype ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM remains elusive. We previously identified a myosin heavy chain 7 (MYH7) gene heterozygous variant, NM_000257.4: c.G2468A (p.G823E), in a large Chinese Han family with HCM. In this family, variant G823E cosegregates with an autosomal dominant disorder. This variant is located in the lever arm domain of the neck region of the MYH7 protein and is highly conserved among homologous myosins and species. To verify the pathogenicity of the G823E variant, we produced a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus with CRISPR/Cas9-mediated genome engineering. We designed gRNA targeting vectors and donor oligonucleotides (with targeting sequences flanked by 134 bp of homology). The p.G823E (GGG to GAG) site in the donor oligonucleotide was introduced into exon 23 of MYH7 by homology-directed repair. A silenced p.R819 (AGG to CGA) was also inserted to prevent gRNA binding and re-cleavage of the sequence after homology-directed repair. Echocardiography revealed left ventricular posterior wall (LVPW) hypertrophy with systole in MYH7 G823E/- mice at 2 months of age. These results were likewise validated by histological analysis (Figure 3). These results demonstrate that the G823E variant plays an important role in the pathogenesis of HCM. Our findings enrich the spectrum of MYH7 variants linked to familial HCM and may provide guidance for genetic counseling and prenatal diagnosis in this Chinese family.},
}
@article {pmid35952970,
year = {2022},
author = {García-Huerta, E and Espinoza-Corona, S and Lagunas-Rangel, FA and Bazan-Tejeda, ML and Vazquez-Cobix, Y and Ortega-Pierres, MG and Bermúdez-Cruz, RM},
title = {Implementation of a tunable t-CRISPRi system for gene regulation in Giardia duodenalis.},
journal = {Plasmid},
volume = {122},
number = {},
pages = {102641},
doi = {10.1016/j.plasmid.2022.102641},
pmid = {35952970},
issn = {1095-9890},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Doxycycline ; *Giardia lamblia/genetics/metabolism ; Plasmids ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Tubulin ; },
abstract = {Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.},
}
@article {pmid35931202,
year = {2022},
author = {Anaganti, N and Chattopadhyay, A and Poirier, JT and Hussain, MM},
title = {Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein.},
journal = {Journal of lipid research},
volume = {63},
number = {9},
pages = {100257},
pmid = {35931202},
issn = {1539-7262},
support = {P30 DK020541/DK/NIDDK NIH HHS/United States ; },
mesh = {Apolipoprotein B-48/metabolism ; Apolipoproteins B/chemistry/genetics ; *Carcinoma, Hepatocellular/genetics ; Carrier Proteins ; Cell Line ; DNA, Complementary ; Humans ; Lipoproteins/metabolism ; *Liver Neoplasms/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger ; Ribonucleoproteins ; Triglycerides/metabolism ; },
abstract = {The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.},
}
@article {pmid35869699,
year = {2022},
author = {Sène, MA and Xia, Y and Kamen, AA},
title = {From functional genomics of vero cells to CRISPR-based genomic deletion for improved viral production rates.},
journal = {Biotechnology and bioengineering},
volume = {119},
number = {10},
pages = {2794-2805},
pmid = {35869699},
issn = {1097-0290},
mesh = {Animals ; *Antiviral Agents ; Chlorocebus aethiops ; Genomics ; RNA, Guide, CRISPR-Cas Systems ; Vero Cells ; *Virus Replication/genetics ; },
abstract = {Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high-throughput vaccine manufacturing platforms is the lack of understanding of virus-host interactions during infection and cell-based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology-based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70-fold and an 87-fold increase of total viral particles productions in ISG15[-/-] Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV-GFP.},
}
@article {pmid35740988,
year = {2022},
author = {Mayes, CM and Santarpia, J},
title = {Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43.},
journal = {Cells},
volume = {11},
number = {12},
pages = {},
pmid = {35740988},
issn = {2073-4409},
mesh = {*Coronavirus ; *Coronavirus Infections ; *Coronavirus OC43, Human/genetics ; Humans ; Polymorphism, Single Nucleotide ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Viral/genetics ; },
abstract = {Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus's replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments.},
}
@article {pmid35700449,
year = {2022},
author = {Stamatiadis, P and Cosemans, G and Boel, A and Menten, B and De Sutter, P and Stoop, D and Chuva de Sousa Lopes, SM and Lluis, F and Coucke, P and Heindryckx, B},
title = {TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.},
journal = {Human reproduction (Oxford, England)},
volume = {37},
number = {8},
pages = {1760-1773},
doi = {10.1093/humrep/deac138},
pmid = {35700449},
issn = {1460-2350},
mesh = {Blastocyst/metabolism ; CDX2 Transcription Factor/genetics/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Embryonic Development/physiology ; Female ; GATA3 Transcription Factor/genetics/metabolism ; Humans ; *In Vitro Oocyte Maturation Techniques ; Male ; Muscle Proteins/genetics/metabolism ; Pregnancy ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; Semen/metabolism ; TEA Domain Transcription Factors ; Transcription Factors/genetics/metabolism ; },
abstract = {STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse?<br><br>SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner.<br><br>WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet.<br><br>STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n&#x2009;=&#x2009;164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n&#x2009;=&#x2009;26) and non-injected media-control embryos (n&#x2009;=&#x2009;51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n&#x2009;=&#x2009;55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n&#x2009;=&#x2009;45) and media control (n&#x2009;=&#x2009;36) embryos were cultured for an extended period of 8.5&#x2009;days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n&#x2009;=&#x2009;74) to target TEAD4 or used as media-control (n&#x2009;=&#x2009;33). The editing efficiency was successfully assessed in n&#x2009;=&#x2009;25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n&#x2009;=&#x2009;10) and non-injected media-control embryos (n&#x2009;=&#x2009;29).<br><br>A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4&#x2009;days in mouse or 6.5&#x2009;days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5&#x2009;days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis.<br><br>NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5&#x2009;days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n&#x2009;=&#x2009;74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n&#x2009;=&#x2009;10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human.<br><br>LARGE SCALE DATA: N/A.<br><br>CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes.<br><br>Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for &#x223c;15% of human conceptions.<br><br>The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722&#x2009;N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests.<br><br>TRIAL REGISTRATION NUMBER: N/A.},
}
@article {pmid35690588,
year = {2022},
author = {Aliu, E and Lee, K and Wang, K},
title = {CRISPR RNA-guided integrase enables high-efficiency targeted genome engineering in Agrobacterium tumefaciens.},
journal = {Plant biotechnology journal},
volume = {20},
number = {10},
pages = {1916-1927},
pmid = {35690588},
issn = {1467-7652},
mesh = {*Agrobacterium tumefaciens/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements ; DNA, Bacterial ; Integrases/genetics ; RNA ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter-kingdom gene transfer capability made Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such as Agrobacterium-host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA-guided integrase system for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA-Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre-loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.},
}
@article {pmid35485960,
year = {2022},
author = {Yelina, NE and Holland, D and Gonzalez-Jorge, S and Hirsz, D and Yang, Z and Henderson, IR},
title = {Coexpression of MEIOTIC-TOPOISOMERASE VIB-dCas9 with guide RNAs specific to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {7},
pages = {},
pmid = {35485960},
issn = {2160-1836},
support = {BB/N007557/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; Crossing Over, Genetic ; Homologous Recombination ; Meiosis/genetics ; Plant Breeding ; Plants/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {During meiosis, homologous chromosomes pair and recombine, which can result in reciprocal crossovers that increase genetic diversity. Crossovers are unevenly distributed along eukaryote chromosomes and show repression in heterochromatin and the centromeres. Within the chromosome arms, crossovers are often concentrated in hotspots, which are typically in the kilobase range. The uneven distribution of crossovers along chromosomes, together with their low number per meiosis, creates a limitation during crop breeding, where recombination can be beneficial. Therefore, targeting crossovers to specific genome locations has the potential to accelerate crop improvement. In plants, meiotic crossovers are initiated by DNA double-strand breaks that are catalyzed by SPO11 complexes, which consist of 2 catalytic (SPO11-1 and SPO11-2) and 2 noncatalytic subunits (MTOPVIB). We used the model plant Arabidopsis thaliana to coexpress an MTOPVIB-dCas9 fusion protein with guide RNAs specific to the 3a crossover hotspot. We observed that this was insufficient to significantly change meiotic crossover frequency or pattern within 3a. We discuss the implications of our findings for targeting meiotic recombination within plant genomes.},
}
@article {pmid35428787,
year = {2022},
author = {Drobna-Śledzińska, M and Maćkowska-Maślak, N and Jaksik, R and Dąbek, P and Witt, M and Dawidowska, M},
title = {CRISPRi for specific inhibition of miRNA clusters and miRNAs with high sequence homology.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {6297},
pmid = {35428787},
issn = {2045-2322},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *MicroRNAs/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Sequence Homology ; Transcription Initiation Site ; },
abstract = {miRNAs form a class of noncoding RNAs, involved in post-transcriptional regulation of gene expression, broadly studied for their involvement in physiological and pathological context. Inhibition of mature miRNA transcripts, commonly used in miRNA loss-of-function experiments, may not be specific in case of miRNAs with high sequence homology, e.g. miRNAs from the same seed family. Phenotypic effects of miRNA repression might be biased by the repression of highly similar miRNAs. Another challenge is simultaneous inhibition of multiple miRNAs encoded within policistronic clusters, potentially co-regulating common biological processes. To elucidate roles of miRNA clusters and miRNAs with high sequence homology, it is of key importance to selectively repress only the miRNAs of interest. Targeting miRNAs on genomic level with CRISPR/dCas9-based methods is an attractive alternative to blocking mature miRNAs. Yet, so far no clear guidelines on the design of CRISPR inhibition (CRISPRi) experiments, specifically for miRNA repression, have been proposed. To address this need, here we propose a strategy for effective inhibition of miRNAs and miRNA clusters using CRISPRi. We provide clues on how to approach the challenges in using CRISPR/dCas in miRNA studies, which include prediction of miRNA transcription start sites (TSSs) and the design of single guide RNAs (sgRNAs). The strategy implements three TSS prediction online tools, dedicated specifically for miRNAs: miRStart, FANTOM 5 miRNA atlas, DIANA-miRGen, and CRISPOR tool for sgRNAs design; it includes testing and selection of optimal sgRNAs. We demonstrate that compared to siRNA/shRNA-based miRNA silencing, CRISPRi improves the repression specificity for miRNAs with highly similar sequence and contribute to higher uniformity of the effects of silencing the whole miRNA clusters. This strategy may be adapted for CRISPR-mediated activation (CRISPRa) of miRNA expression.},
}
@article {pmid35409332,
year = {2022},
author = {Ou-Yang, H and Yang, SH and Chen, W and Yang, SH and Cidem, A and Sung, LY and Chen, CM},
title = {Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals.},
journal = {International journal of molecular sciences},
volume = {23},
number = {7},
pages = {},
pmid = {35409332},
issn = {1422-0067},
support = {MOST-108-2313-B-005-039-MY3//The Ministry of Science and Technology of Taiwan/ ; MOE-111-S-0023-A//The Ministry of Education of Taiwan/ ; },
mesh = {Animals ; *DNA, Cruciform ; Homologous Recombination ; Mice ; Mice, Transgenic ; NIH 3T3 Cells ; *RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery.},
}
@article {pmid35398175,
year = {2022},
author = {Rad, FT and Gargari, BN and Ghorbian, S and Farsani, ZS and Sharifi, R},
title = {Inhibiting the growth of melanoma cells via hTERT gene editing using CRISPR-dCas9-dnmt3a system.},
journal = {Gene},
volume = {828},
number = {},
pages = {146477},
doi = {10.1016/j.gene.2022.146477},
pmid = {35398175},
issn = {1879-0038},
mesh = {Gene Editing/methods ; Hexadimethrine Bromide/metabolism ; Humans ; *Melanoma/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Telomerase/genetics/metabolism ; Transfection ; },
abstract = {CRISPR-Cas9 gene-editing technology has pushed the boundaries of genetic modification. The principle of this method is based on the purposeful defense system of DNA degradation and will be one of the most powerful instruments for gene editing shortly. The purpose of this study was to evaluate the capability of this approach to manage melanoma cells. The present study used EF1a-hsaCas9-U6-gRNA as a hybrid vector of sgRNA and Cas9 for the transfection of A-375 melanoma cells. Transfection efficiency was enhanced by examining the two concentrations of 4 and 8 µg/mL of hexadimethrine bromide (trade name Polybrene). The existence of Cas9 in transfected cells was detected by flow cytometry. The expression level of the metabisulfite-modified hTERT gene was measured by real-time PCR technique. The presence of telomerase in cells was determined by flow cytometry and western blotting analysis. The hTERT gene promoter methylation was also evaluated by HRM assay. Finally, the induction of apoptosis in transfected A375 cells was assessed using flow cytometry. The results showed that the presence of gRNA significantly increased the transfection efficiency (up to about 7.75 times higher). The hTERT expression levels in A-375 cells were significantly decreased at different concentrations of Polybrene (in a dose-dependent manner) and various amounts of transfection (P < 0.05). The expression of hTERT in basal cells was not significantly different from the group transfected without gRNA (P˃0.05) but was significantly higher than the group transfected with gRNA (P < 0.05). The results of flow cytometry and western blotting analysis showed a decrease in hTERT level compared to cells transfected without gRNA as well as basal cells. The methylation of hTERT gene promoter in the cells transfected with gRNA at a concentration of 80 μg/mL in the presence of both 4 μg/mL and 8 μg/mL of Polybrene was significantly increased compared to those transfected without sRNA (P < 0.05). The flow cytometry results indicated no significant difference in the induction of apoptosis in the transfected cells compared to the basal cells (P < 0.05). Evidence suggests that the designed CRISPR/Cas9 system reduces the expression of the hTERT gene and telomerase presence, thereby inhibiting the growth of melanoma cells.},
}
@article {pmid35383205,
year = {2022},
author = {Piao, X and Meng, D and Zhang, X and Song, Q and Lv, H and Jia, Y},
title = {Dual-gRNA approach with limited off-target effect corrects C9ORF72 repeat expansion in vivo.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {5672},
pmid = {35383205},
issn = {2045-2322},
mesh = {*Amyotrophic Lateral Sclerosis/genetics ; C9orf72 Protein/genetics ; DNA Repeat Expansion ; *Frontotemporal Dementia/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {C9ORF72 GGGGCC repeat expansion is the most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia, which generates abnormal DNA and RNA structures and produces toxic proteins. Recently, efficacy of CRISPR/Cas9-mediated editing has been proven in treatment of disease. However, DNA low complexity surrounding C9ORF72 expansion increases the off-target risks. Here we provide a dual-gRNA design outside of the low complexity region which enables us to remove the repeat DNA in a 'cutting-deletion-fusion' manner with a high fusion efficiency (50%). Our dual-gRNA design limits off-target effect and does not significantly affect C9ORF72 expression. In neurons carrying patient C9ORF72 expansion, our approach removes the repeat DNA and corrects the RNA foci in vitro and in vivo. Therefore, we conclude that our proof-of-concept design correct C9ORF72 repeat expansion, which may have potential therapeutic value for the patients.},
}
@article {pmid35188200,
year = {2022},
author = {LaManna, LM and Parulekar, MS and Maliga, P},
title = {Multiple sgRNAs for one-step inactivation of the duplicated acetyl-coenzyme A carboxylase 2 (ACC2) genes in Brassica napus.},
journal = {Plant physiology},
volume = {189},
number = {1},
pages = {178-187},
pmid = {35188200},
issn = {1532-2548},
mesh = {Acetyl-CoA Carboxylase/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *Brassica napus ; Coenzyme A ; RNA, Guide, CRISPR-Cas Systems ; Spectinomycin ; },
abstract = {Efficient plastid transformation in Arabidopsis (Arabidopsis thaliana) requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast acetyl-coenzyme A carboxylase (ACCase) encoded in the acetyl-coenzyme A carboxylase 2 (ACC2) nuclear gene. To obtain plastid transformation-competent oilseed rape (Brassica napus), we inactivated all nuclear encoded, chloroplast targeted ACCase copies using CRISPR-Cas9. Brassica napus (2n = 38, AACC) is a recent interspecific hybrid of Brassica rapa (2n = 20, AA) and B. oleracea (2n = 18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single-guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric egg cell promoter 1.2 (EC1.2p) known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologs in a single step, each of the two vectors carried four sgRNAs. Four T0 transgenic lines were obtained by Agrobacterium tumefaciens-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T1 progeny, in seven of which no wild-type (WT) copy remained. The B. napus T2 seedlings lacking WT ACC2 gene copies exhibited a spectinomycin hypersensitive phenotype, suggesting that they will be a useful resource for chloroplast genome transformation.},
}
@article {pmid35166837,
year = {2022},
author = {Lin, P and Shen, G and Guo, K and Qin, S and Pu, Q and Wang, Z and Gao, P and Xia, Z and Khan, N and Jiang, J and Xia, Q and Wu, M},
title = {Type III CRISPR-based RNA editing for programmable control of SARS-CoV-2 and human coronaviruses.},
journal = {Nucleic acids research},
volume = {50},
number = {8},
pages = {e47},
pmid = {35166837},
issn = {1362-4962},
support = {R01 AI109317/AI/NIAID NIH HHS/United States ; R01 AI138203/AI/NIAID NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Antiviral Agents ; *COVID-19/therapy ; Humans ; Mice ; Pandemics/prevention &amp; control ; RNA Editing/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *SARS-CoV-2/genetics ; },
abstract = {Gene-editing technologies, including the widespread usage of CRISPR endonucleases, have the potential for clinical treatments of various human diseases. Due to the rapid mutations of SARS-CoV-2, specific and effective prevention and treatment by CRISPR toolkits for coronavirus disease 2019 (COVID-19) are urgently needed to control the current pandemic spread. Here, we designed Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) as a therapeutic to combat SARS-CoV-2 infection. We provided a proof of principle demonstration that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses. We report that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice. Moreover, bioinformatics screening of gRNAs along RNA sequences reveals that a group of five gRNAs (hCoV-gRNAs) could potentially target 99.98% of human coronaviruses. TEAR-CoV also exerted specific targeting and cleavage of common human coronaviruses. The fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.},
}
@article {pmid35086559,
year = {2022},
author = {Grodzki, M and Bluhm, AP and Schaefer, M and Tagmount, A and Russo, M and Sobh, A and Rafiee, R and Vulpe, CD and Karst, SM and Norris, MH},
title = {Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.},
journal = {Genome medicine},
volume = {14},
number = {1},
pages = {10},
pmid = {35086559},
issn = {1756-994X},
support = {R01 AI123144/AI/NIAID NIH HHS/United States ; Clinical and Translational Science Institute//University of Florida/ ; R01AI123144//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {Animals ; Antiviral Agents/pharmacology/therapeutic use ; COVID-19/pathology/virology ; Chlorocebus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Exoribonucleases/antagonists &amp; inhibitors/genetics/metabolism ; Gene Editing/methods ; *Genome, Viral ; HEK293 Cells ; Host-Pathogen Interactions/drug effects/*genetics ; Humans ; Microtubule-Associated Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Proteins/antagonists &amp; inhibitors/genetics/metabolism ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Small Interfering/metabolism ; SARS-CoV-2/drug effects/*genetics/physiology ; Vero Cells ; Virus Replication/genetics ; COVID-19 Drug Treatment ; },
abstract = {BACKGROUND: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design.<br><br>METHODS: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings.<br><br>RESULTS: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens.<br><br>CONCLUSIONS: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.},
}
@article {pmid35044397,
year = {2022},
author = {Bengtson, M and Bharadwaj, M and Franch, O and van der Torre, J and Meerdink, V and Schallig, H and Dekker, C},
title = {CRISPR-dCas9 based DNA detection scheme for diagnostics in resource-limited settings.},
journal = {Nanoscale},
volume = {14},
number = {5},
pages = {1885-1895},
pmid = {35044397},
issn = {2040-3372},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Nucleic Acid Amplification Techniques ; Point-of-Care Testing ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.},
}
@article {pmid35022620,
year = {2022},
author = {Feldman, D and Funk, L and Le, A and Carlson, RJ and Leiken, MD and Tsai, F and Soong, B and Singh, A and Blainey, PC},
title = {Pooled genetic perturbation screens with image-based phenotypes.},
journal = {Nature protocols},
volume = {17},
number = {2},
pages = {476-512},
pmid = {35022620},
issn = {1750-2799},
support = {P50 HG006193/HG/NHGRI NIH HHS/United States ; R01 HG009283/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >10[6] perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.},
}
@article {pmid34932902,
year = {2022},
author = {Goyon, A and Scott, B and Kurita, K and Maschinot, C and Meyer, K and Yehl, P and Zhang, K},
title = {On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System.},
journal = {Analytical chemistry},
volume = {94},
number = {2},
pages = {1169-1177},
doi = {10.1021/acs.analchem.1c04350},
pmid = {34932902},
issn = {1520-6882},
mesh = {Chromatography, Liquid/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA, Guide, CRISPR-Cas Systems ; *Ribonucleases ; Tandem Mass Spectrometry ; },
abstract = {In this study, for the first time, the automated digestion and sequencing of an RNA molecule via the use of immobilized RNase cartridges attached to a multidimensional liquid chromatography (LC)-mass spectrometry (MS) system are presented. We first developed an on-line digestion-HILIC two-dimensional (2D)-LC-MS method in order to sequence CRISPR guide RNAs for gene editing. Three RNases (T1, A, and U2) were immobilized on polyetheretherketone cartridges, and their performance was evaluated. Ultrafast digestions were performed within 2.3 min with the on-line approach versus 30 min via the conventional off-line approach. The higher sequence coverage was achieved by the RNase T1 (71%), which is the same as the off-line mode. A 20-fold reduction in the gRNA sample amount was achieved with the on-line digestion approach (6.5 μg) in comparison to that with the off-line approach (130 μg). In the second step, a three-dimensional (3D)-LC-MS method was developed for the sequencing of fractions collected on-line across the main peak and the partially separated tail by the reference ion-pairing RPLC method. Additional insights were gained in order to better understand the cause of the main peak tailing.},
}
@article {pmid34872615,
year = {2021},
author = {Kim, HS and Grimes, SM and Hooker, AC and Lau, BT and Ji, HP},
title = {Single-cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {331},
pmid = {34872615},
issn = {1474-760X},
support = {R01 HG006137/HG/NHGRI NIH HHS/United States ; R33 CA247700/CA/NCI NIH HHS/United States ; R35 HG011292/HG/NHGRI NIH HHS/United States ; P01 HG000205/HG/NHGRI NIH HHS/United States ; },
mesh = {Alternative Splicing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Exons ; Genomics ; HEK293 Cells ; Humans ; Nanopore Sequencing/*methods ; Neoplasm Proteins ; Protein Isoforms/genetics/*metabolism ; RNA Isoforms/genetics/metabolism ; RNA Splicing ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Receptors for Activated C Kinase ; Transcriptome ; },
abstract = {We developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.},
}
@article {pmid34860007,
year = {2021},
author = {Otto, M and Skrekas, C and Gossing, M and Gustafsson, J and Siewers, V and David, F},
title = {Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries.},
journal = {ACS synthetic biology},
volume = {10},
number = {12},
pages = {3461-3474},
pmid = {34860007},
issn = {2161-5063},
mesh = {Cloning, Molecular ; Gene Library ; Genetic Engineering/methods ; Genomics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.},
}
@article {pmid34853281,
year = {2021},
author = {Sato, Y},
title = {Development of Lipid Nanoparticles for the Delivery of Macromolecules Based on the Molecular Design of pH-Sensitive Cationic Lipids.},
journal = {Chemical &amp; pharmaceutical bulletin},
volume = {69},
number = {12},
pages = {1141-1159},
doi = {10.1248/cpb.c21-00705},
pmid = {34853281},
issn = {1347-5223},
mesh = {COVID-19/immunology/prevention &amp; control/virology ; Cations/chemistry ; Drug Carriers/*chemistry ; Hydrogen-Ion Concentration ; Lipids/*chemistry ; Liposomes/*chemistry ; Nanoparticles/*chemistry ; RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism ; RNA, Small Interfering/chemistry/metabolism ; SARS-CoV-2/isolation &amp; purification ; mRNA Vaccines/chemistry/metabolism ; },
abstract = {Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.},
}
@article {pmid34792162,
year = {2021},
author = {Aldag, P and Welzel, F and Jakob, L and Schmidbauer, A and Rutkauskas, M and Fettes, F and Grohmann, D and Seidel, R},
title = {Probing the stability of the SpCas9-DNA complex after cleavage.},
journal = {Nucleic acids research},
volume = {49},
number = {21},
pages = {12411-12421},
pmid = {34792162},
issn = {1362-4962},
mesh = {Algorithms ; CRISPR-Associated Protein 9/genetics/*metabolism ; DNA/genetics/*metabolism ; *DNA Cleavage ; Enzyme Stability/genetics ; Magnetics ; Mutation ; Optical Tweezers ; Protein Binding ; R-Loop Structures/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/*enzymology/genetics ; },
abstract = {CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.},
}
@article {pmid34743703,
year = {2021},
author = {Yang, Z and Li, L and Turkoz, A and Chen, P and Harari-Steinfeld, R and Bobbin, M and Stefanson, O and Choi, H and Pietrobon, V and Alphson, B and Goswami, A and Balan, V and Kearney, A and Patel, D and Yang, J and Inel, D and Vinod, V and Cesano, A and Wang, B and Roh, KH and Qi, LS and Marincola, FM},
title = {Contextual reprogramming of CAR-T cells for treatment of HER2[+] cancers.},
journal = {Journal of translational medicine},
volume = {19},
number = {1},
pages = {459},
pmid = {34743703},
issn = {1479-5876},
mesh = {CD28 Antigens/genetics ; Cell Line, Tumor ; Humans ; Immunotherapy, Adoptive ; *Neoplasms ; RNA, Guide, CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; *Single-Chain Antibodies ; T-Lymphocytes ; },
abstract = {BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities.<br><br>METHODS: To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3&#x3b6; co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS.<br><br>RESULTS: Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2[+] FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product.<br><br>CONCLUSIONS: As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.},
}
@article {pmid34672952,
year = {2021},
author = {Hussmann, JA and Ling, J and Ravisankar, P and Yan, J and Cirincione, A and Xu, A and Simpson, D and Yang, D and Bothmer, A and Cotta-Ramusino, C and Weissman, JS and Adamson, B},
title = {Mapping the genetic landscape of DNA double-strand break repair.},
journal = {Cell},
volume = {184},
number = {22},
pages = {5653-5669.e25},
pmid = {34672952},
issn = {1097-4172},
support = {R35 GM138167/GM/NIGMS NIH HHS/United States ; P30 CA072720/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 HG003284/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/metabolism ; Cell Line ; Cluster Analysis ; *DNA Breaks, Double-Stranded ; DNA Repair/genetics ; Gene Editing ; Gene Expression Regulation ; Genome, Human ; *Genomics ; Humans ; Phenotype ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Reproducibility of Results ; },
abstract = {Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.},
}
@article {pmid34623476,
year = {2022},
author = {Kumar, M and Ayzenshtat, D and Marko, A and Bocobza, S},
title = {Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA-sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco.},
journal = {Plant cell reports},
volume = {41},
number = {1},
pages = {175-194},
pmid = {34623476},
issn = {1432-203X},
support = {20-01-0209//ministry of agriculture and rural development/ ; },
mesh = {DNA, Bacterial/genetics/metabolism ; DNA, Plant/*genetics/metabolism ; Gene Editing/*methods ; *Genome, Plant ; Plant Proteins/*genetics/metabolism ; Promoter Regions, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA, Plant/*genetics/metabolism ; RNA, Transfer/genetics/metabolism ; Tetraploidy ; Nicotiana/*genetics ; Ubiquitins/genetics/metabolism ; },
abstract = {Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA-gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos. While gene-editing methodologies, such as CRISPR-Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA-sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.},
}
@article {pmid34580281,
year = {2021},
author = {Huang, YT and Cheng, AC and Tang, HC and Huang, GC and Cai, L and Lin, TH and Wu, KJ and Tseng, PH and Wang, GG and Chen, WY},
title = {USP7 facilitates SMAD3 autoregulation to repress cancer progression in p53-deficient lung cancer.},
journal = {Cell death &amp; disease},
volume = {12},
number = {10},
pages = {880},
pmid = {34580281},
issn = {2041-4889},
support = {107-2320-B-010-024-MY3//Ministry of Science and Technology, Taiwan (Ministry of Science and Technology of Taiwan)/ ; },
mesh = {Animals ; Base Sequence ; Cell Line, Tumor ; *Disease Progression ; Down-Regulation ; Enhancer Elements, Genetic/genetics ; Gene Deletion ; Gene Expression Regulation, Neoplastic ; Genetic Loci ; HEK293 Cells ; *Homeostasis ; Humans ; Luciferases/metabolism ; Lung Neoplasms/genetics/*metabolism/*pathology ; Male ; Mice, Inbred C57BL ; Models, Biological ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Smad3 Protein/*metabolism ; Tumor Suppressor Protein p53/*deficiency/metabolism ; Ubiquitin-Specific Peptidase 7/*metabolism ; },
abstract = {USP7, one of the most abundant ubiquitin-specific proteases (USP), plays multifaceted roles in many cellular events, including oncogenic pathways. Accumulated studies have suggested that USP7, through modulating the MDM2/MDMX-p53 pathway, is a promising target for cancer treatment; however, little is known about the function of USP7 in p53-deficient tumors. Here we report that USP7 regulates the autoregulation of SMAD3, a key regulator of transforming growth factor β (TGFβ) signaling, that represses the cell progression of p53-deficient lung cancer. CRISPR/Cas9-mediated inactivation of USP7 in p53-deficient lung cancer H1299 line resulted in advanced cell proliferation in vitro and in xenograft tumor in vivo. Genome-wide analyses (ChIP-seq and RNA-seq) of USP7 KO H1299 cells reveal a dramatic reduction of SMAD3 autoregulation, including decreased gene expression and blunted function of associated super-enhancer (SE). Furthermore, biochemical assays show that SMAD3 is conjugated by mono-ubiquitin, which negatively regulates the DNA-binding function of SMAD3, in USP7 KO cells. In addition, cell-free and cell-based analyses further demonstrate that the deubiquitinase activity of USP7 mediates the removal of mono-ubiquitin from SMAD3 and facilitates the DNA-binding of SMAD3-SMAD4 dimer at SMAD3 locus, and thus enhance the autoregulation of SMAD3. Collectively, our study identified a novel mechanism by which USP7, through catalyzing the SMAD3 de-monoubiquitination, facilitates the positive autoregulation of SMAD3, and represses the cancer progression of p53-deficient lung cancer.},
}
@article {pmid34555894,
year = {2021},
author = {Li, C and Zhou, J and Rao, S and Du, G and Liu, S},
title = {Visualized Multigene Editing System for Aspergillus niger.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2607-2616},
doi = {10.1021/acssynbio.1c00231},
pmid = {34555894},
issn = {2161-5063},
mesh = {Aspergillus niger/*genetics ; DNA, Fungal/genetics ; Gene Editing/*methods ; *Genes, Fungal ; Pigments, Biological/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Transfer/genetics ; },
abstract = {The resistance markers could ensure the entry of the CRISPR/Cas9 system into Aspergillus niger cells instead of gene editing. To increase the efficiency of positive colony screening on the primary transformation plates, we designed a visualized multigene editing system (VMS) via a unique tRNA-guide RNA (gRNA) array containing the gRNAs of a pigment gene albA and target genes. Disruption of albA produces white colonies, and the sequences of the endogenous tRNA[Ala], tRNA[Phe], tRNA[Arg], tRNA[Ile], and tRNA[Leu] enhance gRNA release. The disruption efficiencies of multigene were analyzed in the A. niger strain AG11 using ammA, amyA, prtT, kusA, and glaA as reporters. In white colonies on the primary transformation plates, the disruption rates of one-, two-, three-, four-, and five-target genes reached 89.2, 70.91, 50, 22.41, and 4.17%, respectively. The VMS developed here provides an effective method for screening homokaryotic multigene editing strains of A. niger.},
}
@article {pmid34551006,
year = {2021},
author = {Matchett-Oates, L and Braich, S and Spangenberg, GC and Rochfort, S and Cogan, NOI},
title = {In silico analysis enabling informed design for genome editing in medicinal cannabis; gene families and variant characterisation.},
journal = {PloS one},
volume = {16},
number = {9},
pages = {e0257413},
pmid = {34551006},
issn = {1932-6203},
mesh = {Cannabinoids/biosynthesis ; Cannabis/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Copy Number Variations ; Gene Editing/*methods ; *Genome, Plant ; Polymorphism, Single Nucleotide ; RNA, Guide, CRISPR-Cas Systems/metabolism ; User-Computer Interface ; },
abstract = {BACKGROUND: Cannabis has been used worldwide for centuries for industrial, recreational and medicinal use, however, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. This study proposes and develops an in silico best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis.<br><br>RESULTS: A large dataset of reference genomes was accessed and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variance and highly polymorphic gene sequences exist in the genome making genome editing using CRISPR, Zinc Fingers and TALENs technically difficult. Evaluation of allele or additional gene copies was determined through nucleotide and amino acid alignments with comparative sequence analysis performed. From determined gene copy number and presence of SNPs, multiple online CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways to create knockouts for further investigation. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites.<br><br>CONCLUSIONS: Using this framework, the approach has wider applications to all plant species regardless of ploidy number or highly homologous gene sequences.<br><br>SIGNIFICANCE STATEMENT: Using this framework, a best-practice approach to genome editing is possible in all plant species, including cannabis, delivering a comprehensive in silico evaluation of the cannabinoid pathway diversity from a large set of whole genome sequences. Identification of SNP variants across all genes could improve genome editing potentially leading to novel applications across multiple disciplines, including agriculture and medicine.},
}
@article {pmid34533878,
year = {2021},
author = {Oesinghaus, L and Simmel, FC},
title = {Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a*.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {44},
pages = {23894-23902},
pmid = {34533878},
issn = {1521-3773},
mesh = {Animals ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; Cell Line ; Endodeoxyribonucleases/*genetics/metabolism ; Fibroblasts/*metabolism ; Gene Expression Regulation/genetics ; HEK293 Cells ; Humans ; Mice ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Spatiotemporal control of the activity of CRISPR-associated (Cas) proteins is of considerable interest for basic research and therapeutics. Here, we show that conditional guide RNAs (gRNAs) for Cas12a can be transcribed in mammalian cells by RNA polymerase II, followed by activation via input-dependent processing of the 3' tail of the gRNA transcript. We demonstrate processing using an RNA strand displacement mechanism, as well as microRNA-dependent processing, and cleavage by a guanine-responsive ribozyme. We further demonstrate that Cas12a along with several independently switchable gRNAs can be compactly integrated on a single transcript using stabilizing RNA triplexes, providing a route towards Cas12a-based gene regulation constructs with multi-input switching capabilities. The principle is shown to work in HEK and mouse fibroblast cells using luminescence, fluorescence, and is also demonstrated for the conditional upregulation of an endogenous gene.},
}
@article {pmid34499031,
year = {2021},
author = {Magnusson, JP and Rios, AR and Wu, L and Qi, LS},
title = {Enhanced Cas12a multi-gene regulation using a CRISPR array separator.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34499031},
issn = {2050-084X},
support = {U01 DK127405/DK/NIDDK NIH HHS/United States ; U01 EB021240/EB/NIBIB NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/*genetics ; *Gene Expression Regulation ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; Humans ; Operon ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.},
}
@article {pmid34453037,
year = {2021},
author = {Lai, X and Liu, J and Zou, Z and Wang, Y and Wang, Y and Liu, X and Huang, W and Ma, Y and Chen, Q and Li, F and Wu, G and Li, W and Wang, W and Yuan, Y and Jiang, B},
title = {SOX10 ablation severely impairs the generation of postmigratory neural crest from human pluripotent stem cells.},
journal = {Cell death &amp; disease},
volume = {12},
number = {9},
pages = {814},
pmid = {34453037},
issn = {2041-4889},
mesh = {Apoptosis/genetics ; Base Sequence ; Biomarkers/metabolism ; Cell Differentiation/genetics ; *Cell Movement/genetics ; Cell Shape/genetics ; Epithelial Cells/metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Neural Crest/*cytology ; Neurons/cytology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; SOXE Transcription Factors/deficiency/*metabolism ; Schwann Cells/cytology ; },
abstract = {Animal studies have indicated that SOX10 is one of the key transcription factors regulating the proliferation, migration and differentiation of multipotent neural crest (NC), and mutation of SOX10 in humans may lead to type 4 Waardenburg syndrome (WS). However, the exact role of SOX10 in human NC development and the underlying molecular mechanisms of SOX10-related human diseases remain poorly understood due to the lack of appropriate human model systems. In this study, we successfully generated SOX10-knockout human induced pluripotent stem cells (SOX10[-/-] hiPSCs) by the CRISPR-Cas9 gene editing tool. We found that loss of SOX10 significantly inhibited the generation of p75[high]HNK1[+]/CD49D[+] postmigratory neural crest stem cells (NCSCs) and upregulated the cell apoptosis rate during NC commitment from hiPSCs. Moreover, we discovered that both the neuronal and glial differentiation capacities of SOX10[-/-] NCSCs were severely compromised. Intriguingly, we showed that SOX10[-/-] hiPSCs generated markedly more TFAP2C[+]nonneural ectoderm cells (NNE) than control hiPSCs during neural crest differentiation. Our results indicate that SOX10 is crucial for the transition of premigratory cells to migrating NC and is vital for NC survival. Taken together, these results provide new insights into the function of SOX10 in human NC development, and the SOX10-knockout hiPSC lines may serve as a valuable cell model to study the pathogenesis of SOX10-related human neurocristopathies.},
}
@article {pmid34425866,
year = {2021},
author = {Sun, M and Wang, Y and Zheng, C and Wei, Y and Hou, J and Zhang, P and He, W and Lv, X and Ding, Y and Liang, H and Hon, CC and Chen, X and Xu, H and Chen, Y},
title = {Systematic functional interrogation of human pseudogenes using CRISPRi.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {240},
pmid = {34425866},
issn = {1474-760X},
support = {R37 CA228304/CA/NCI NIH HHS/United States ; P50 CA186784/CA/NCI NIH HHS/United States ; R01 HL146642/HL/NHLBI NIH HHS/United States ; R35 GM137927/GM/NIGMS NIH HHS/United States ; R01 GM130838/GM/NIGMS NIH HHS/United States ; R01 NS117668/NS/NINDS NIH HHS/United States ; },
mesh = {Breast Neoplasms/genetics ; Cell Nucleus/genetics ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computational Biology ; Forkhead Box Protein M1/metabolism ; Gene Expression Regulation, Neoplastic ; Hepatocyte Nuclear Factor 3-alpha/metabolism ; Humans ; MCF-7 Cells ; Promoter Regions, Genetic/genetics ; Protein Binding ; Pseudogenes/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Up-Regulation/genetics ; },
abstract = {BACKGROUND: The human genome encodes over 14,000 pseudogenes that are evolutionary relics of protein-coding genes and commonly considered as nonfunctional. Emerging evidence suggests that some pseudogenes may exert important functions. However, to what extent human pseudogenes are functionally relevant remains unclear. There has been no large-scale characterization of pseudogene function because of technical challenges, including high sequence similarity between pseudogene and parent genes, and poor annotation of transcription start sites.<br><br>RESULTS: To overcome these technical obstacles, we develop an integrated computational pipeline to design the first genome-wide library of CRISPR interference (CRISPRi) single-guide RNAs (sgRNAs) that target human pseudogene promoter-proximal regions. We perform the first pseudogene-focused CRISPRi screen in luminal A breast cancer cells and reveal approximately 70 pseudogenes that affect breast cancer cell fitness. Among the top hits, we identify a cancer-testis unitary pseudogene, MGAT4EP, that is predominantly localized in the nucleus and interacts with FOXA1, a key regulator in luminal A breast cancer. By enhancing the promoter binding of FOXA1, MGAT4EP upregulates the expression of oncogenic transcription factor FOXM1. Integrative analyses of multi-omic data from the Cancer Genome Atlas (TCGA) reveal many unitary pseudogenes whose expressions are significantly dysregulated and/or associated with overall/relapse-free survival of patients in diverse cancer types.<br><br>CONCLUSIONS: Our study represents the first large-scale study characterizing pseudogene function. Our findings suggest the importance of nuclear function of unitary pseudogenes and underscore their underappreciated roles in human diseases. The functional genomic resources developed here will greatly facilitate the study of human pseudogene function.},
}
@article {pmid34405212,
year = {2021},
author = {Tao, XX and Wang, B and Zuo, SK and Yu, Y},
title = {[Identification of mouse lines with HA-tagged prostaglandin receptors].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {73},
number = {4},
pages = {559-570},
pmid = {34405212},
issn = {0371-0874},
mesh = {Animals ; Mice ; Oocytes ; Plasmids ; *RNA, Guide, CRISPR-Cas Systems ; *Receptors, Prostaglandin ; },
abstract = {Prostaglandins are a class of poly-unsaturated fatty acids-derived bioactive lipids with important physiological function by binding to specific receptors. Prostaglandin receptors lack specific antibodies, which greatly impedes the research on our understanding of the signaling of prostaglandins. The aim of this study was to identify nine mouse lines with amino terminal (-NH2, -N) HA-tagged prostaglandin receptors by using the combination of artificial sperm and CRISPR-Cas9 technology. The guide RNA expression plasmid and labeled targeting vector plasmids were transferred into "artificial sperm cells". The "artificial sperm cells" containing labeled proteins were selected and injected into mouse oocytes, and implanted into pseudopregnant mice to obtain labeled mice. The genomic DNA of the prostaglandin receptor tagged mice was extracted, and the genotypes of mice were detected by PCR method. We also isolated mouse peritoneal macrophages to verify the protein expression of HA-labeled prostaglandin receptor by Western blot. Specific DNA bands were amplified in prostaglandin receptor labeled mice, and specific HA protein bands were detected in macrophage proteins, which was not detected in wild type mice. In summary, we successfully constructed 9 mouse lines with HA-tagged prostaglandin receptors, providing a powerful tool for further study of the pathophysiological functions of prostaglandin signaling both in vivo and in vitro.},
}
@article {pmid34383464,
year = {2021},
author = {Lehr, FX and Kuzembayeva, A and Bailey, ME and Kleindienst, W and Kabisch, J and Koeppl, H},
title = {Functionalizing Cell-Free Systems with CRISPR-Associated Proteins: Application to RNA-Based Circuit Engineering.},
journal = {ACS synthetic biology},
volume = {10},
number = {9},
pages = {2138-2150},
doi = {10.1021/acssynbio.0c00386},
pmid = {34383464},
issn = {2161-5063},
mesh = {5' Untranslated Regions ; CRISPR-Associated Proteins/*genetics ; Cell-Free System ; Escherichia coli/genetics ; Genetic Engineering/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Logic ; Protein Biosynthesis/genetics ; RNA/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Messenger/metabolism ; },
abstract = {Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far, the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged a recent protocol for producing an E. coli-based cell-free expression system with two CRISPR-associated proteins, Csy4 and dCas9, expressed prior to harvest. We found that pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrated the benefits and versatility of dCas9 and Csy4 through the use of RNA circuitry based on a combination of single guide RNAs, small transcriptional activator RNAs, and toehold switches. For instance, we show that Csy4 processing increased 4-fold the dynamic range of a previously published AND-logic gate. Additionally, blending the CRISPR-enhanced extracts enabled us to reduce leakage in a multiple inputs gate, and to extend the type of Boolean functions available for RNA-based circuits, such as NAND-logic. Finally, we reported the use of simultaneous transcriptional and translational reporters in our RNA-based circuits. In particular, the AND-gate mRNA and protein levels were able to be independently monitored in response to transcriptional and translational activators. We hope this work will facilitate the adoption of advanced processing tools for RNA-based circuit prototyping in a cell-free environment.},
}
@article {pmid34353917,
year = {2021},
author = {Feng, W and Cao, Z and Lim, PX and Zhao, H and Luo, H and Mao, N and Lee, YS and Rivera, AA and Choi, D and Wu, C and Han, T and Romero, R and de Stanchina, E and Carver, BS and Wang, Q and Jasin, M and Sawyers, CL},
title = {Rapid interrogation of cancer cell of origin through CRISPR editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {32},
pages = {},
pmid = {34353917},
issn = {1091-6490},
support = {R01 CA193837/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P50 CA092629/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA155169/CA/NCI NIH HHS/United States ; U54 CA224079/CA/NCI NIH HHS/United States ; R35 CA253174/CA/NCI NIH HHS/United States ; P50 CA069568/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/metabolism ; Animals ; CRISPR-Associated Protein 9/genetics ; *Chromosome Deletion ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Epithelial Cells ; Gene Editing/*methods ; Genes, Tumor Suppressor ; Humans ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Organoids ; Prostate/*cytology ; Prostatic Neoplasms/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems ; Ribonucleoproteins/genetics ; Transcriptional Regulator ERG/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.},
}
@article {pmid34326544,
year = {2021},
author = {Reilly, SK and Gosai, SJ and Gutierrez, A and Mackay-Smith, A and Ulirsch, JC and Kanai, M and Mouri, K and Berenzy, D and Kales, S and Butler, GM and Gladden-Young, A and Bhuiyan, RM and Stitzel, ML and Finucane, HK and Sabeti, PC and Tewhey, R},
title = {Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR-FlowFISH.},
journal = {Nature genetics},
volume = {53},
number = {8},
pages = {1166-1176},
pmid = {34326544},
issn = {1546-1718},
support = {R00 HG008179/HG/NHGRI NIH HHS/United States ; T32 GM007226/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R00 HG010669/HG/NHGRI NIH HHS/United States ; K99 HG008179/HG/NHGRI NIH HHS/United States ; UM1 HG009435/HG/NHGRI NIH HHS/United States ; F32 HG009226/HG/NHGRI NIH HHS/United States ; K99 HG010669/HG/NHGRI NIH HHS/United States ; R01 AI151051/AI/NIAID NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics ; Bayes Theorem ; Clustered Regularly Interspaced Short Palindromic Repeats ; Delta-5 Fatty Acid Desaturase ; Deoxyribonuclease I/genetics/metabolism ; Fatty Acid Desaturases/genetics ; Flow Cytometry ; GATA1 Transcription Factor/genetics ; Humans ; In Situ Hybridization, Fluorescence/*methods ; K562 Cells ; LIM Domain Proteins/genetics ; Models, Genetic ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins/genetics ; Quantitative Trait Loci ; RNA, Guide, CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR-FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene and display activating and/or silencing effects. At the cholesterol-level-associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominate causal variants and, importantly, identify their target genes.},
}
@article {pmid34281556,
year = {2021},
author = {Wu, T and Wang, Y and Xiao, T and Ai, Y and Li, J and Zeng, YA and Yu, QC},
title = {Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {42},
pmid = {34281556},
issn = {1472-6750},
mesh = {ADAM17 Protein/genetics/*metabolism ; Base Sequence ; Cell Line ; Down-Regulation ; Endothelial Protein C Receptor/*genetics/metabolism ; Gene Library ; Genetic Vectors/genetics/metabolism ; Humans ; Lentivirus/*genetics/metabolism ; Mammary Glands, Human/*enzymology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/β-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins.<br><br>RESULTS: We established a screening system which combines CRISPR-Cas9 guided gene disruption&#xa0;with fluorescence activated cell sorting&#xa0;technique (FACS). CommaD&#x3b2; (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression.<br><br>CONCLUSION: We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.},
}
@article {pmid34213093,
year = {2021},
author = {Chen, ZZ and Wang, JY and Kang, Y and Yang, QY and Gu, XY and Zhi, DL and Yan, L and Long, CZ and Shen, B and Niu, YY},
title = {PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys.},
journal = {Zoological research},
volume = {42},
number = {4},
pages = {469-477},
pmid = {34213093},
issn = {2095-8137},
mesh = {Animals ; Animals, Newborn ; CRISPR-Associated Protein 9/genetics/metabolism ; *Disease Models, Animal ; Embryo Culture Techniques ; Embryo Transfer ; Fibroblasts/physiology ; Frameshift Mutation ; Gene Expression Regulation ; Macaca fascicularis/embryology/*genetics ; Monkey Diseases/genetics ; Mutation ; Parkinson Disease/genetics/*veterinary ; Protein Kinases/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Mutations of PTEN-induced kinase I (PINK1) cause early-onset Parkinson's disease (PD) with selective neurodegeneration in humans. However, current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients. This suggests that generating PINK1 disease models in non-human primates (NHPs) that are close to humans is essential to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both of which can reduce off-target effects without compromising on-target editing, are two optimized strategies in the CRISPR/Cas9 system for establishing disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target the PINK1 gene. We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA. However, western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts. We further reprogramed mutant fibroblasts into induced pluripotent stem cells (iPSCs), which showed similar ability to differentiate into dopamine (DA) neurons. Taken together, our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.},
}
@article {pmid34203807,
year = {2021},
author = {Song, B and Yang, S and Hwang, GH and Yu, J and Bae, S},
title = {Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage.},
journal = {International journal of molecular sciences},
volume = {22},
number = {12},
pages = {},
pmid = {34203807},
issn = {1422-0067},
support = {2021R1A2C3012908//National Research Foundation of Korea/ ; },
mesh = {Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/metabolism ; *DNA Cleavage ; DNA End-Joining Repair/*genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Mutation/genetics ; Oligonucleotides/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Deletion/genetics ; },
abstract = {Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.},
}
@article {pmid34174443,
year = {2022},
author = {Xue, Y and Hu, X and Wang, D and Li, D and Li, Y and Wang, F and Huang, M and Gu, X and Xu, Z and Zhou, J and Wang, J and Chai, R and Shen, J and Chen, ZY and Li, GL and Yang, H and Li, H and Zuo, E and Shu, Y},
title = {Gene editing in a Myo6 semi-dominant mouse model rescues auditory function.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {105-118},
pmid = {34174443},
issn = {1525-0024},
support = {R01 DC016875/DC/NIDCD NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Disease Models, Animal ; Evoked Potentials, Auditory, Brain Stem/genetics ; *Gene Editing ; Hearing ; *Hearing Loss/genetics/therapy ; Humans ; Mice ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Myosin VI（MYO6） is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6 gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6 mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated in vivo delivery of Staphylococcus aureus Cas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6[WT/C442Y] mouse model that recapitulated the phenotypes of human patients. The in vivo editing efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6[C442Y] is 4.05% on average in Myo6[WT/C442Y] mice, which was ∼17-fold greater than editing efficiency of Myo6[WT] alleles. Rescue of auditory function was observed up to 5 months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6[WT/C442Y] mice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for in vivo genome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.},
}
@article {pmid34167459,
year = {2021},
author = {Cui, Y and Wang, Z and Köster, J and Liao, X and Peng, S and Tang, T and Huang, C and Yang, C},
title = {VISPR-online: a web-based interactive tool to visualize CRISPR screening experiments.},
journal = {BMC bioinformatics},
volume = {22},
number = {1},
pages = {344},
pmid = {34167459},
issn = {1471-2105},
support = {2020YFA0709800//Key Technologies Research and Development Program/ ; 2018YFB0204301//Key Technologies Research and Development Program (CN)/ ; 61972408//National Outstanding Youth Science Fund Project of National Natural Science Foundation of China/ ; 61772543//National Natural Science Foundation of China-Yunnan Joint Fund/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Internet ; RNA, Guide, CRISPR-Cas Systems ; Research ; *Software ; },
abstract = {BACKGROUND: VISPR is an interactive visualization and analysis framework for CRISPR screening experiments. However, it only supports the output of MAGeCK, and requires installation and manual configuration. Furthermore, VISPR is designed to run on a single computer, and data sharing between collaborators is challenging.<br><br>RESULTS: To make the tool easily accessible to the community, we present VISPR-online, a web-based general application allowing users to visualize, explore, and share CRISPR screening data online with a few simple steps. VISPR-online provides an exploration of screening results and visualization of read count changes. Apart from MAGeCK, VISPR-online supports two more popular CRISPR screening analysis tools: BAGEL and JACKS. It provides an interactive environment for exploring gene essentiality, viewing guide RNA (gRNA) locations, and allowing users to resume and share screening results.<br><br>CONCLUSIONS: VISPR-online allows users to visualize, explore and share CRISPR screening data online. It is freely available at http://vispr-online.weililab.org , while the source code is available at https://github.com/lemoncyb/VISPR-online .},
}
@article {pmid34082781,
year = {2021},
author = {Park, SJ and Jeong, TY and Shin, SK and Yoon, DE and Lim, SY and Kim, SP and Choi, J and Lee, H and Hong, JI and Ahn, J and Seong, JK and Kim, K},
title = {Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {170},
pmid = {34082781},
issn = {1474-760X},
mesh = {Animals ; Base Sequence ; Cell Line ; Cells/*metabolism ; Chromatin/metabolism ; Embryo, Mammalian/*metabolism ; *Gene Editing ; Humans ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mutagenesis/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editors, novel genome-editing tools consisting of a CRISPR-Cas9 nickase and an engineered reverse transcriptase, can induce targeted mutagenesis. Nevertheless, much effort is required to optimize and improve the efficiency of prime-editing. Herein, we introduce two strategies to improve the editing efficiency using proximal dead sgRNA and chromatin-modulating peptides. We used enhanced prime-editing to generate Igf2 mutant mice with editing frequencies of up to 47% and observed germline transmission, no off-target effects, and a dwarf phenotype. This improved prime-editing method can be efficiently applied to cell research and to generate mouse models.},
}
@article {pmid34078888,
year = {2021},
author = {Buchman, A and Shriner, I and Yang, T and Liu, J and Antoshechkin, I and Marshall, JM and Perry, MW and Akbari, OS},
title = {Engineered reproductively isolated species drive reversible population replacement.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3281},
pmid = {34078888},
issn = {2041-1723},
support = {R00 EY027016/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/deficiency/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila melanogaster/*genetics/metabolism ; Female ; Gene Drive Technology/*methods ; Gene Flow ; *Genes, Lethal ; *Genetic Speciation ; INDEL Mutation ; Male ; *Population Dynamics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Reproductive Isolation ; },
abstract = {Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.},
}
@article {pmid34041503,
year = {2021},
author = {Layden, HM and Eleuteri, NA and Hiebert, SW and Stengel, KR},
title = {A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100530},
pmid = {34041503},
issn = {2666-1667},
support = {F31 HL156565/HL/NHLBI NIH HHS/United States ; R01 CA178030/CA/NCI NIH HHS/United States ; R01 CA164605/CA/NCI NIH HHS/United States ; P30 DK058404/DK/NIDDK NIH HHS/United States ; F31 HL156565/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Cells, Cultured ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Transcription Factors/analysis/chemistry/genetics/metabolism ; *Transcription, Genetic/genetics/physiology ; },
abstract = {Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors. For complete details on the use and execution of this protocol, please refer to Stengel et al., 2021).},
}
@article {pmid33948577,
year = {2021},
author = {Uchida, N and Li, L and Nassehi, T and Drysdale, CM and Yapundich, M and Gamer, J and Haro-Mora, JJ and Demirci, S and Leonard, A and Bonifacino, AC and Krouse, AE and Linde, NS and Allen, C and Peshwa, MV and De Ravin, SS and Donahue, RE and Malech, HL and Tisdale, JF},
title = {Preclinical evaluation for engraftment of CD34[+] cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.},
journal = {Cell reports. Medicine},
volume = {2},
number = {4},
pages = {100247},
pmid = {33948577},
issn = {2666-3791},
mesh = {Anemia, Sickle Cell/*genetics ; Animals ; Antigens, CD34/*metabolism ; Gene Editing/methods ; Gene Targeting/methods ; Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells/*metabolism ; Hemoglobin, Sickle/genetics ; Heterografts/*immunology ; Humans ; Macaca mulatta/*genetics ; Mice ; RNA, Guide, CRISPR-Cas Systems/metabolism ; beta-Globins/genetics ; },
abstract = {Sickle cell disease (SCD) is caused by a 20A > T mutation in the β-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34[+] cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34[+] cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus β-to-βs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34[+] cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34[+] HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.},
}
@article {pmid33934834,
year = {2021},
author = {Ghavami, S and Pandi, A},
title = {CRISPR interference and its applications.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {123-140},
doi = {10.1016/bs.pmbts.2021.01.007},
pmid = {33934834},
issn = {1878-0814},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Transcription Factors ; },
abstract = {Sequence-specific control of gene expression is a powerful tool for identifying and studying gene functions and cellular processes. CRISPR interference (CRISPRi) is an RNA-based method for highly specific silencing of the transcription in prokaryotic or eukaryotic cells. The typical CRISPRi system is a type II CRISPR (clustered regularly interspaced palindromic repeats) machinery of Streptococcus pyogenes. CRISPRi requires two main components: A catalytically inactivated Cas9, namely dCas9 and a guide RNA (sgRNA). These two components associate and form a DNA recognition complex. The dCas9/sgRNA complex then specifically binds to the target DNA complementary with the sgRNA and sterically prevents the association of the promoter or transcription factors with their trans-acting sequences or blocks the transcription elongation. This chapter discusses CRISPRi structure, mechanism and its applications.},
}
@article {pmid33924938,
year = {2021},
author = {Brackett, K and Mungale, A and Lopez-Isidro, M and Proctor, DA and Najarro, G and Arias, C},
title = {CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi's Sarcoma-Associated Herpesvirus-Infected Cells.},
journal = {Viruses},
volume = {13},
number = {5},
pages = {},
pmid = {33924938},
issn = {1999-4915},
mesh = {Cell Line ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Expression Regulation, Viral ; *Gene Silencing ; Genes, Reporter ; Genes, Viral ; Herpesviridae Infections/virology ; Herpesvirus 8, Human/*genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems ; Virus Activation/*genetics ; Virus Latency/*genetics ; },
abstract = {Uncovering viral gene functions requires the modulation of gene expression through overexpression or loss-of-function. CRISPR interference (CRISPRi), a modification of the CRISPR-Cas9 gene editing technology, allows specific and efficient transcriptional silencing without genetic ablation. CRISPRi has been used to silence eukaryotic and prokaryotic genes at the single-gene and genome-wide levels. Here, we report the use of CRISPRi to silence latent and lytic viral genes, with an efficiency of ~80-90%, in epithelial and B-cells carrying multiple copies of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome. Our results validate CRISPRi for the analysis of KSHV viral elements, providing a functional genomics tool for studying virus-host interactions.},
}
@article {pmid33914736,
year = {2021},
author = {Keatinge, M and Tsarouchas, TM and Munir, T and Porter, NJ and Larraz, J and Gianni, D and Tsai, HH and Becker, CG and Lyons, DA and Becker, T},
title = {CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.},
journal = {PLoS genetics},
volume = {17},
number = {4},
pages = {e1009515},
pmid = {33914736},
issn = {1553-7404},
support = {/WT_/Wellcome Trust/United Kingdom ; 102836/Z/13/Z/WT_/Wellcome Trust/United Kingdom ; MR/R001049/1/MRC_/Medical Research Council/United Kingdom ; BB/R003742/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Axons/metabolism/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Macrophages/metabolism ; Osteonectin/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Recovery of Function/genetics ; Regeneration/*genetics ; Spinal Cord/growth &amp; development/pathology ; Spinal Cord Injuries/genetics/pathology/therapy ; Spinal Cord Regeneration/*genetics/physiology ; Transforming Growth Factor beta1/*genetics ; Transforming Growth Factor beta3/genetics ; Zebrafish/genetics/growth &amp; development ; Zebrafish Proteins/*genetics ; },
abstract = {Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.},
}
@article {pmid33820980,
year = {2021},
author = {Balderston, S and Taulbee, JJ and Celaya, E and Fung, K and Jiao, A and Smith, K and Hajian, R and Gasiunas, G and Kutanovas, S and Kim, D and Parkinson, J and Dickerson, K and Ripoll, JJ and Peytavi, R and Lu, HW and Barron, F and Goldsmith, BR and Collins, PG and Conboy, IM and Siksnys, V and Aran, K},
title = {Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.},
journal = {Nature biomedical engineering},
volume = {5},
number = {7},
pages = {713-725},
pmid = {33820980},
issn = {2157-846X},
mesh = {Anemia, Sickle Cell/genetics/pathology ; Biosensing Techniques/instrumentation/*methods ; CRISPR-Associated Protein 9/chemistry/*metabolism ; DNA/*genetics/metabolism ; Genome, Human ; Graphite/chemistry ; Heterozygote ; Homozygote ; Humans ; Immobilized Proteins/chemistry/metabolism ; *Polymorphism, Single Nucleotide ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Superoxide Dismutase-1/genetics ; Transistors, Electronic ; },
abstract = {Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.},
}
@article {pmid33720123,
year = {2021},
author = {See, JE and Shin, HR and Jang, G and Kweon, J and Kim, Y},
title = {Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {168},
pages = {},
doi = {10.3791/61557},
pmid = {33720123},
issn = {1940-087X},
mesh = {BRCA1 Protein/*genetics ; Base Sequence ; Breast Neoplasms/genetics ; CRISPR-Associated Protein 9/metabolism ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; *Gene Editing ; *Genetic Variation ; High-Throughput Nucleotide Sequencing ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.},
}
@article {pmid33649229,
year = {2021},
author = {Qiu, M and Glass, Z and Chen, J and Haas, M and Jin, X and Zhao, X and Rui, X and Ye, Z and Li, Y and Zhang, F and Xu, Q},
title = {Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {10},
pages = {},
pmid = {33649229},
issn = {1091-6490},
support = {S10 OD021624/OD/NIH HHS/United States ; U24 HG010423/HG/NHGRI NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {Angiopoietin-Like Protein 3 ; *Angiopoietin-like Proteins/genetics/metabolism ; Animals ; CRISPR-Associated Protein 9/*genetics ; *Drug Carriers/chemistry/pharmacokinetics/pharmacology ; Female ; *Gene Editing ; *Lipids/chemistry/pharmacokinetics/pharmacology ; Liver/*metabolism ; Mice ; Mice, Inbred BALB C ; Nanoparticles/*chemistry ; Organ Specificity ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/pharmacokinetics/pharmacology ; *RNA, Messenger/chemistry/genetics/pharmacokinetics/pharmacology ; },
abstract = {Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9-based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.},
}
@article {pmid33625849,
year = {2021},
author = {Choo, XY and Lim, YM and Katwadi, K and Yap, L and Tryggvason, K and Sun, AX and Li, S and Handoko, L and Ouyang, JF and Rackham, OJL},
title = {Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments.},
journal = {ACS synthetic biology},
volume = {10},
number = {3},
pages = {640-645},
doi = {10.1021/acssynbio.0c00499},
pmid = {33625849},
issn = {2161-5063},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Human Embryonic Stem Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Messenger/metabolism ; Single-Cell Analysis/*methods ; Transcription Factors/genetics/metabolism ; },
abstract = {The combination of single-cell RNA sequencing with CRISPR inhibition/activation provides a high-throughput approach to simultaneously study the effects of hundreds if not thousands of gene perturbations in a single experiment. One recent development in CRISPR-based single-cell techniques introduces a feature barcoding technology that allows for the simultaneous capture of mRNA and guide RNA (gRNA) from the same cell. This is achieved by introducing a capture sequence, whose complement can be incorporated into each gRNA and that can be used to amplify these features prior to sequencing. However, because the technology is in its infancy, there is little information available on how such experimental parameters can be optimized. To overcome this, we varied the capture sequence, capture sequence position, and gRNA backbone to identify an optimal gRNA scaffold for CRISPR activation gene perturbation studies. We provide a report on our screening approach along with our observations and recommendations for future use.},
}
@article {pmid33570447,
year = {2021},
author = {Nuñez-Muñoz, L and Vargas-Hernández, B and Hinojosa-Moya, J and Ruiz-Medrano, R and Xoconostle-Cázares, B},
title = {Plant drought tolerance provided through genome editing of the trehalase gene.},
journal = {Plant signaling &amp; behavior},
volume = {16},
number = {4},
pages = {1877005},
pmid = {33570447},
issn = {1559-2324},
mesh = {Adaptation, Physiological/*genetics ; Amino Acid Sequence ; Arabidopsis/*genetics/*physiology ; Base Sequence ; Computer Simulation ; DNA, Bacterial/genetics ; *Droughts ; *Gene Editing ; Gene Expression Regulation, Plant ; Gene Silencing ; *Genes, Plant ; Genetic Vectors/metabolism ; Molecular Docking Simulation ; Mutation/genetics ; Phenotype ; Phylogeny ; Plant Leaves/genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic/genetics ; Protein Domains ; RNA, Guide, CRISPR-Cas Systems/genetics ; Substrate Specificity ; Nicotiana/genetics ; Transformation, Genetic ; Trehalase/chemistry/*genetics/metabolism ; Zea mays/genetics ; },
abstract = {Drought is one of the main abiotic factors that affect agricultural productivity, jeopardizing food security. Modern biotechnology is a useful tool for the generation of stress-tolerant crops, but its release and field-testing involves complex regulatory frameworks. However, gene editing technology mediated by the CRISPR/Cas9 system is a suitable strategy for plant breeding, which can lead to precise and specific modifications in the plant genome. The aim of the present work is to produce drought-tolerant plant varieties by modifying the trehalase gene. Furthermore, a new vector platform was developed to edit monocot and dicot genomes, by modifying vectors adding a streptomycin resistance marker for use with the hypervirulent Agrobacterium tumefaciens AGL1 strain. The gRNA design was based on the trehalase sequence in several species of the genus Selaginella that show drought tolerance. Arabidopsis thaliana carrying editions in the trehalase substrate-binding domain showed a higher tolerance to drought stress. In addition, a transient transformation system for gene editing in maize leaves was characterized.},
}
@article {pmid33539780,
year = {2021},
author = {Park, J and Lim, JM and Jung, I and Heo, SJ and Park, J and Chang, Y and Kim, HK and Jung, D and Yu, JH and Min, S and Yoon, S and Cho, SR and Park, T and Kim, HH},
title = {Recording of elapsed time and temporal information about biological events using Cas9.},
journal = {Cell},
volume = {184},
number = {4},
pages = {1047-1063.e23},
doi = {10.1016/j.cell.2021.01.014},
pmid = {33539780},
issn = {1097-4172},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; Cellular Microenvironment ; Computer Simulation ; HEK293 Cells ; Half-Life ; Humans ; INDEL Mutation/genetics ; Inflammation/pathology ; Integrases/metabolism ; Male ; Mice, Nude ; Promoter Regions, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Time Factors ; },
abstract = {DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."},
}
@article {pmid33433623,
year = {2021},
author = {Huang, RS and Lai, MC and Shih, HA and Lin, S},
title = {A robust platform for expansion and genome editing of primary human natural killer cells.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {3},
pages = {},
pmid = {33433623},
issn = {1540-9538},
mesh = {Amino Acid Sequence ; Biomarkers/metabolism ; CRISPR-Associated Protein 9/metabolism ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Chromosomes, Human/genetics ; Cryopreservation ; Cytotoxicity, Immunologic ; DNA/metabolism ; Feeder Cells/cytology ; *Gene Editing ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Killer Cells, Natural/*metabolism ; Lymphocyte Activation/immunology ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Ribonucleoproteins/metabolism ; Translocation, Genetic ; },
abstract = {Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.},
}
@article {pmid33372040,
year = {2021},
author = {Dai, M and Boudreault, J and Wang, N and Poulet, S and Daliah, G and Yan, G and Moamer, A and Burgos, SA and Sabri, S and Ali, S and Lebrun, JJ},
title = {Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways.},
journal = {Cancer research},
volume = {81},
number = {5},
pages = {1332-1346},
doi = {10.1158/0008-5472.CAN-20-2121},
pmid = {33372040},
issn = {1538-7445},
support = {8117//CIHR/Canada ; },
mesh = {Animals ; Breast Neoplasms/genetics/pathology ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cyclin-Dependent Kinase 4/*genetics/metabolism ; Cyclin-Dependent Kinase 6/*genetics/metabolism ; DNA Repair/*physiology ; DNA Replication/*physiology ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Lung Neoplasms/genetics/secondary ; Male ; Mice, SCID ; Neoplasms/genetics/*pathology ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/pharmacology ; Xenograft Model Antitumor Assays ; },
abstract = {Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.},
}
@article {pmid33274853,
year = {2021},
author = {Manzano, I and Taylor, N and Csordas, M and Vezeau, GE and Salis, HM and Zydney, AL},
title = {Purification of Cas9-RNA complexes by ultrafiltration.},
journal = {Biotechnology progress},
volume = {37},
number = {2},
pages = {e3104},
doi = {10.1002/btpr.3104},
pmid = {33274853},
issn = {1520-6033},
support = {1505592//Division of Chemical, Bioengineering, Environmental, and Transport Systems/ ; Seed-2019//Pennsylvania State University/ ; },
mesh = {CRISPR-Associated Protein 9/chemistry/*isolation &amp; purification/metabolism ; Chromatography, Affinity/*methods ; Escherichia coli/genetics/*metabolism ; Histidine/*chemistry ; RNA, Guide, CRISPR-Cas Systems/*isolation &amp; purification ; Streptococcus pyogenes/*enzymology/genetics ; Ultrafiltration/*methods ; },
abstract = {The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.},
}
@article {pmid33247547,
year = {2021},
author = {Liu, J and Luo, T and Xue, Y and Mao, L and Stang, PJ and Wang, M},
title = {Hierarchical Self-assembly of Discrete Metal-Organic Cages into Supramolecular Nanoparticles for Intracellular Protein Delivery.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {10},
pages = {5429-5435},
doi = {10.1002/anie.202013904},
pmid = {33247547},
issn = {1521-3773},
mesh = {Adamantane/analogs &amp; derivatives/metabolism ; Antineoplastic Agents/metabolism/pharmacology ; CRISPR-Associated Protein 9/genetics/metabolism ; Drug Carriers/chemical synthesis/*chemistry/metabolism ; Endocytosis/physiology ; Gene Editing/methods ; Genome, Human ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Integrases/genetics/metabolism ; Metal-Organic Frameworks/chemical synthesis/*chemistry/metabolism ; Nanoparticles/*chemistry/metabolism ; Polyethyleneimine/analogs &amp; derivatives/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Ribonuclease, Pancreatic/metabolism/pharmacology ; Ribonucleoproteins/genetics/metabolism ; beta-Cyclodextrins/chemical synthesis/chemistry/metabolism ; },
abstract = {Hierarchical self-assembly (HAS) is a powerful approach to create supramolecular nanostructures for biomedical applications. This potency, however, is generally challenged by the difficulty of controlling the HAS of biomacromolecules and the functionality of resulted HAS nanostructures. Herein, we report a modular approach for controlling the HAS of discrete metal-organic cages (MOC) into supramolecular nanoparticles, and its potential for intracellular protein delivery and cell-fate specification. The hierarchical coordination-driven self-assembly of adamantane-functionalized M12 L24 MOC (Ada-MOC) and the host-guest interaction of Ada-MOC with β-cyclodextrin-conjugated polyethylenimine (PEI-βCD) afford supramolecular nanoparticles in a controllable manner. HAS maintains high efficiency and orthogonality in the presence of protein, enabling the encapsulation of protein into the nanoparticles for intracellular protein delivery for therapeutic application and CRISPR/Cas9 genome editing.},
}
@article {pmid33231079,
year = {2020},
author = {Henningsen, J and Schwarz-Schilling, M and Leibl, A and Gutiérrez, JN and Sagredo, S and Simmel, FC},
title = {Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback Loop Containing dCas9-sgRNA.},
journal = {ACS synthetic biology},
volume = {9},
number = {12},
pages = {3377-3387},
doi = {10.1021/acssynbio.0c00438},
pmid = {33231079},
issn = {2161-5063},
mesh = {Circadian Rhythm/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics/growth &amp; development/metabolism ; *Feedback, Physiological ; Gene Editing/*methods ; Microfluidics ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; Single-Cell Analysis ; Stochastic Processes ; },
abstract = {Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the "repressilator" can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over several days. With a period of ≈14 bacterial generations, our oscillator is similar in speed as previously reported oscillators. Using an information-theoretic approach for the analysis of the single cell data, the potential of the circuit to act as a synthetic pacemaker for cellular processes is evaluated. We also observe that the oscillator appears to affect cellular growth, leading to variations in growth rate with the oscillator's frequency.},
}
@article {pmid33122788,
year = {2020},
author = {Borrás, T and Cowley, DO and Asokan, P and Pandya, K},
title = {Generation of a Matrix Gla (Mgp) floxed mouse, followed by conditional knockout, uncovers a new Mgp function in the eye.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18583},
pmid = {33122788},
issn = {2045-2322},
support = {EY11906 (TB)/NH/NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 EY026220/EY/NEI NIH HHS/United States ; EY030608 (TB)/NH/NIH HHS/United States ; EY026220 (TB)/NH/NIH HHS/United States ; P30 CA016086 (DOC)/NH/NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Calcium-Binding Proteins/*physiology ; Extracellular Matrix Proteins/*physiology ; Eye/*physiopathology ; Female ; Glaucoma/physiopathology ; Integrases/metabolism ; *Intraocular Pressure ; Mice ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage ; Trabecular Meshwork/*physiopathology ; Matrix Gla Protein ; },
abstract = {The ability to ablate a gene in a given tissue by generating a conditional knockout (cKO) is crucial for determining its function in the targeted tissue. Such tissue-specific ablation is even more critical when the gene's conventional knockout (KO) is lethal, which precludes studying the consequences of its deletion in other tissues. Therefore, here we describe a successful strategy that generated a Matrix Gla floxed mouse (Mgp.floxed) by the CRISPR/Cas9 system, that subsequently allowed the generation of cKOs by local viral delivery of the Cre-recombinase enzyme. MGP is a well-established inhibitor of calcification gene, highly expressed in arteries' smooth muscle cells and chondrocytes. MGP is also one of the most abundant genes in the trabecular meshwork, the eye tissue responsible for maintenance of intraocular pressure (IOP) and development of Glaucoma. Our strategy entailed one-step injection of two gRNAs, Cas9 protein and a long-single-stranded-circular DNA donor vector (lsscDNA, 6.7 kb) containing two loxP sites in cis and 900-700 bp 5'/3' homology arms. Ocular intracameral injection of Mgp.floxed mice with a Cre-adenovirus, led to an Mgp.TMcKO mouse which developed elevated IOP. Our study discovered a new role for the Mgp gene as a keeper of physiological IOP in the eye.},
}
@article {pmid33122087,
year = {2021},
author = {Chen, Q and Zhang, Y and Yin, H},
title = {Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {246-258},
doi = {10.1016/j.addr.2020.10.014},
pmid = {33122087},
issn = {1872-8294},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Therapy ; RNA, Guide, CRISPR-Cas Systems/*genetics ; RNA, Messenger/*genetics ; },
abstract = {The discovery and applications of clustered regularly interspaced short palindromic repeat (CRISPR) systems have revolutionized our ability to track and manipulate specific nucleic acid sequences in many cell types of various organisms. The robustness and simplicity of these platforms have rapidly extended their applications from basic research to the development of therapeutics. However, many hurdles remain on the path to translation of the CRISPR systems to therapeutic applications: efficient delivery, detectable off-target effects, potential immunogenicity, and others. Chemical modifications provide a variety of protection options for guide RNA, Cas9 mRNA and donor templates. For example, chemically modified gRNA demonstrated enhanced on-target editing efficiency, minimized immune response and decreased off-target genome editing. In this review, we summarize the use of chemically modified nucleotides for CRISPR-mediated genome editing and emphasize open questions that remain to be addressed in clinical applications.},
}
@article {pmid33095043,
year = {2020},
author = {Pfitzner, C and White, MA and Piltz, SG and Scherer, M and Adikusuma, F and Hughes, JN and Thomas, PQ},
title = {Progress Toward Zygotic and Germline Gene Drives in Mice.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {388-397},
doi = {10.1089/crispr.2020.0050},
pmid = {33095043},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; *Gene Drive Technology ; Gene Expression Regulation ; *Genes, Synthetic ; Male ; *Meiosis ; Mice ; Mice, Transgenic ; Models, Animal ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Recombinational DNA Repair ; *Zygote ; },
abstract = {CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in Mus musculus by constructing "split drive" systems where gRNA expression occurs on a separate chromosome to Cas9, which is under the control of either a zygotic (CAG) or germline (Vasa) promoter. While both systems generated double-strand breaks at their intended target site in vivo, no homology-directed repair between chromosomes ("homing") was detectable. Our data indicate that robust and specific Cas9 expression during meiosis is a critical requirement for the generation of efficient CRISPR-based synthetic gene drives in rodents.},
}
@article {pmid33086621,
year = {2020},
author = {Kapitansky, O and Karmon, G and Sragovich, S and Hadar, A and Shahoha, M and Jaljuli, I and Bikovski, L and Giladi, E and Palovics, R and Iram, T and Gozes, I},
title = {Single Cell ADNP Predictive of Human Muscle Disorders: Mouse Knockdown Results in Muscle Wasting.},
journal = {Cells},
volume = {9},
number = {10},
pages = {},
pmid = {33086621},
issn = {2073-4409},
mesh = {Adult ; Animals ; Base Sequence ; Behavior, Animal ; Child ; Female ; Gait ; Gene Expression Regulation ; *Gene Knockdown Techniques ; Homeodomain Proteins/genetics/*metabolism ; Humans ; Male ; Mice ; Mice, Knockout ; Motor Activity ; Muscles/metabolism/*pathology ; Muscular Diseases/*metabolism/*pathology ; NIH 3T3 Cells ; Naphthoquinones ; Nerve Tissue Proteins/genetics/*metabolism ; Neuromuscular Junction/metabolism/pathology ; Physical Conditioning, Animal ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; *Single-Cell Analysis ; Stem Cells/metabolism ; Wasting Syndrome/metabolism/*pathology ; },
abstract = {Activity-dependent neuroprotective protein (ADNP) mutations are linked with cognitive dysfunctions characterizing the autistic-like ADNP syndrome patients, who also suffer from delayed motor maturation. We thus hypothesized that ADNP is deregulated in versatile myopathies and that local ADNP muscle deficiency results in myopathy, treatable by the ADNP fragment NAP. Here, single-cell transcriptomics identified ADNP as a major constituent of the developing human muscle. ADNP transcript concentrations further predicted multiple human muscle diseases, with concentrations negatively correlated with the ADNP target interacting protein, microtubule end protein 1 (EB1). Reverting back to modeling at the single-cell level of the male mouse transcriptome, Adnp mRNA concentrations age-dependently correlated with motor disease as well as with sexual maturation gene transcripts, while Adnp expressing limb muscle cells significantly decreased with aging. Mouse Adnp heterozygous deficiency exhibited muscle microtubule reduction and myosin light chain (Myl2) deregulation coupled with motor dysfunction. CRISPR knockdown of adult gastrocnemius muscle Adnp in a Cas9 mouse resulted in treadmill (male) and gait (female) dysfunctions that were specifically ameliorated by treatment with the ADNP snippet, microtubule interacting, Myl2-regulating, NAP (CP201). Taken together, our studies provide new hope for personalized diagnosis/therapeutics in versatile myopathies.},
}
@article {pmid33051688,
year = {2021},
author = {Gooden, AA and Evans, CN and Sheets, TP and Clapp, ME and Chari, R},
title = {dbGuide: a database of functionally validated guide RNAs for genome editing in human and mouse cells.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D871-D876},
pmid = {33051688},
issn = {1362-4962},
mesh = {Animals ; Cells/*metabolism ; *Databases, Genetic ; *Gene Editing ; *Genome, Human ; Humans ; Mice ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Reproducibility of Results ; User-Computer Interface ; },
abstract = {With the technology's accessibility and ease of use, CRISPR has been employed widely in many different organisms and experimental settings. As a result, thousands of publications have used CRISPR to make specific genetic perturbations, establishing in itself a resource of validated guide RNA sequences. While numerous computational tools to assist in the design and identification of candidate guide RNAs exist, these are still just at best predictions and generally, researchers inevitably will test multiple sequences for functional activity. Here, we present dbGuide (https://sgrnascorer.cancer.gov/dbguide), a database of functionally validated guide RNA sequences for CRISPR/Cas9-based knockout in human and mouse. Our database not only contains computationally determined candidate guide RNA sequences, but of even greater value, over 4000 sequences which have been functionally validated either through direct amplicon sequencing or manual curation of literature from over 1000 publications. Finally, our established framework will allow for continual addition of newly published and experimentally validated guide RNA sequences for CRISPR/Cas9-based knockout as well as incorporation of sequences from different gene editing systems, additional species and other types of site-specific functionalities such as base editing, gene activation, repression and epigenetic modification.},
}
@article {pmid33050877,
year = {2020},
author = {Gerasimova, SV and Hertig, C and Korotkova, AM and Kolosovskaya, EV and Otto, I and Hiekel, S and Kochetov, AV and Khlestkina, EK and Kumlehn, J},
title = {Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene.},
journal = {BMC plant biology},
volume = {20},
number = {Suppl 1},
pages = {255},
pmid = {33050877},
issn = {1471-2229},
mesh = {CRISPR-Associated Protein 9/*metabolism ; Edible Grain/genetics ; Gene Knockout Techniques ; Gene Targeting ; Hordeum/anatomy &amp; histology/*genetics ; Phenotype ; Plant Proteins/*metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Transcription Factors/*metabolism ; },
abstract = {BACKGROUND: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition.<br><br>RESULTS: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype.<br><br>CONCLUSIONS: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high &#x3b2;-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.},
}
@article {pmid33043306,
year = {2020},
author = {Hunker, AC and Zweifel, LS},
title = {Protocol to Design, Clone, and Validate sgRNAs for In Vivo Reverse Genetic Studies.},
journal = {STAR protocols},
volume = {1},
number = {2},
pages = {},
pmid = {33043306},
issn = {2666-1667},
support = {R01 MH104450/MH/NIMH NIH HHS/United States ; P30 DA048736/DA/NIDA NIH HHS/United States ; F31 MH116549/MH/NIMH NIH HHS/United States ; T32 GM007270/GM/NIGMS NIH HHS/United States ; R01 DA044315/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; Cells, Cultured ; Cloning, Molecular/*methods ; Mice ; Mutagenesis ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Rats ; Reproducibility of Results ; *Reverse Genetics/methods/standards ; },
abstract = {AAV-CRISPR/Cas9 permits gene mutagenesis in the adult CNS. Current methods determining In Vivo on-target mutagenesis have been limited by the ability to isolate virally transduced cells. This protocol optimizes a workflow for the design, cloning, and validation of sgRNAs delivered by AAVs In Vivo that can be applied to any target gene in the CNS of rat or mouse model systems and can be adapted to Cre or Flp driver lines using AAV-FLEX-SaCas9-sgRNA or AAV-FLEXfrt-SaCas9-sgRNA, respectively. For complete details on the use and execution of this protocol, please refer to Hunker et al. (2020).},
}
@article {pmid33016195,
year = {2020},
author = {Nyberg, KG and Nguyen, JQ and Kwon, YJ and Blythe, S and Beitel, GJ and Carthew, R},
title = {A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila.},
journal = {Fly},
volume = {14},
number = {1-4},
pages = {34-48},
pmid = {33016195},
issn = {1933-6942},
support = {R35 GM118144/GM/NIGMS NIH HHS/United States ; R01 GM108964/GM/NIGMS NIH HHS/United States ; P40 OD010949/OD/NIH HHS/United States ; F32 GM122349/GM/NIGMS NIH HHS/United States ; P41 RR006009/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; Chromosomes ; Drosophila melanogaster/embryology/*genetics ; Gene Editing/*methods ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.},
}
@article {pmid32978126,
year = {2020},
author = {Banta, AB and Enright, AL and Siletti, C and Peters, JM},
title = {A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {23},
pages = {},
pmid = {32978126},
issn = {1098-5336},
support = {T32 GM008349/GM/NIGMS NIH HHS/United States ; T32 GM135066/GM/NIGMS NIH HHS/United States ; },
mesh = {Biofuels/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genes, Bacterial ; Genetic Association Studies/*methods ; RNA, Bacterial ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Zymomonas/*genetics/metabolism ; },
abstract = {Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes-including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.IMPORTANCE Biofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next-generation biofuels like isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to explore gene function in Z. mobilis We characterize genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to isobutanol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.},
}
@article {pmid32955044,
year = {2020},
author = {Qiao, J and Lin, S and Sun, W and Ma, L and Liu, Y},
title = {A method for the quantitative detection of Cas12a ribonucleoproteins.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {83},
pages = {12616-12619},
doi = {10.1039/d0cc04019c},
pmid = {32955044},
issn = {1364-548X},
mesh = {Bacterial Proteins/*analysis/metabolism ; CRISPR-Associated Proteins/*analysis/metabolism ; Endodeoxyribonucleases/*analysis/metabolism ; Endonucleases/metabolism ; HEK293 Cells ; Humans ; Limit of Detection ; RNA/metabolism ; RNA, Double-Stranded/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Real-Time Polymerase Chain Reaction/*methods ; },
abstract = {Cas12a ribonucleoprotein (RNP) is an RNA-guided CRISPR-associated nuclease used widely for genome editing and molecular diagnostics. Conventional detection methods rely on adopting antibody-based reagents that are expensive and lack scalability, and, moreover, only detect Cas12 enzyme rather than RNP, which is the true effector. Here, we describe a method for the rapid and quantitative detection of the effective Cas12a RNPs by the combined use of anti-CRISPR protein AcrVA1 and stem-loop RT-qPCR, achieving a limit of detection (LOD) of 1 fM in reaction buffer and 0.1 pM under biologically representative conditions.},
}
@article {pmid32949493,
year = {2020},
author = {Xu, XS and Bulger, EA and Gantz, VM and Klanseck, C and Heimler, SR and Auradkar, A and Bennett, JB and Miller, LA and Leahy, S and Juste, SS and Buchman, A and Akbari, OS and Marshall, JM and Bier, E},
title = {Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.},
journal = {Molecular cell},
volume = {80},
number = {2},
pages = {246-262.e4},
doi = {10.1016/j.molcel.2020.09.003},
pmid = {32949493},
issn = {1097-4164},
support = {DP5 OD023098/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; Chromosomes/genetics ; Drosophila melanogaster/genetics ; Female ; *Gene Deletion ; *Gene Drive Technology ; Green Fluorescent Proteins/metabolism ; Inheritance Patterns/genetics ; Mutagenesis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transgenes ; },
abstract = {CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.},
}
@article {pmid32948788,
year = {2020},
author = {Kohama, Y and Higo, S and Masumura, Y and Shiba, M and Kondo, T and Ishizu, T and Higo, T and Nakamura, S and Kameda, S and Tabata, T and Inoue, H and Motooka, D and Okuzaki, D and Takashima, S and Miyagawa, S and Sawa, Y and Hikoso, S and Sakata, Y},
title = {Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {15348},
pmid = {32948788},
issn = {2045-2322},
mesh = {Animals ; BRCA2 Protein/genetics ; Cardiac Myosins/genetics ; Cell Differentiation/genetics ; Cells, Cultured ; Dependovirus/*genetics ; Fanconi Anemia/genetics/metabolism ; Fanconi Anemia Complementation Group A Protein/genetics ; *Gene Transfer Techniques ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/cytology/physiology ; Male ; Mice, Transgenic ; Myocytes, Cardiac/cytology/*physiology ; Myosin Light Chains/genetics ; RNA, Guide, CRISPR-Cas Systems ; S Phase/*physiology ; Transgenes ; },
abstract = {Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.},
}
@article {pmid32945949,
year = {2021},
author = {Kim, H and Choi, J},
title = {A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {1059-1070},
pmid = {32945949},
issn = {1432-203X},
support = {grant No. 2018R1A2B6006233//The Basic Science Research Program of National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology/ ; grant No. WISET-2019-674ho//The National Research Foundation of Korea and the Center for Women in Science, Engineering, and Technology/ ; grant No. PJ01477602//The New Breeding Technologies Development Program, Rural Development Administration (RDA), Republic of Korea/ ; },
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cotyledon/genetics ; Endodeoxyribonucleases/genetics ; Gene Editing/*methods ; Plants, Genetically Modified ; Protoplasts/cytology ; RNA, Guide, CRISPR-Cas Systems ; Reproducibility of Results ; Ribonucleoproteins/*genetics ; Glycine max/*cytology/*genetics ; },
abstract = {Calli protoplasts isolated from three soybean cultivars are useful tools to evaluate guide RNAs for clustered regularly interspaced short palindromic repeats (CRISPR)-based precise gene editing. A type V CRISPR effector, LbCpf1(Cas12a) from Lachnospiraceae bacterium ND 2006, has been used for precision editing of the plant genome. We report that callus-derived protoplasts from three soybeans, including Glycine Max var. Williams 82 and two Korean cultivars (Kwangan and Daewon) represent efficient systems for the screening of active crRNA for CRISPR/LbCpf1. CRISPR/LbCpf1 ribonucleoproteins (RNPs) were delivered as complexes of purified endonucleases mixed with designed crRNA to simultaneously edit target genes of GlymaFAD2-1A and GlymaFAD2-1B transfected into three soybean protoplasts including genome-sequenced Williams 82 with cultivars, Kwangan and Daewon. Previously, we reported that nine crRNAs designed for LbCpf1 exhibited varying degrees of editing efficacy for two FAD2 genes. Among the nine crRNAs, the LbCpf1-crRNA3 complexes showed the highest efficiency in soybean cotyledon protoplasts. The new screening systems of callus protoplasts from three soybeans have been successfully used to transfect GFP-tagged markers and CRISPR/LbCpf1 RNPs. The callus protoplasts confirm that the LbCpf1-crRNA3 complex is an active crRNA for LbCpf1 to edit two FAD2 genes similar to cotyledon protoplasts. These results demonstrate that soybean callus protoplast-based CRISPR/crRNA selection is a new and practical tool to screen the efficacy of crRNAs and a prerequisite for progressive regeneration of the edited soybean.},
}
@article {pmid32807909,
year = {2020},
author = {Gayet, RV and de Puig, H and English, MA and Soenksen, LR and Nguyen, PQ and Mao, AS and Angenent-Mari, NM and Collins, JJ},
title = {Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release.},
journal = {Nature protocols},
volume = {15},
number = {9},
pages = {3030-3063},
pmid = {32807909},
issn = {1750-2799},
mesh = {Acrylic Resins/chemistry ; Bacterial Proteins/metabolism ; Base Sequence ; CRISPR-Associated Proteins/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/chemistry/genetics ; *Diagnostic Techniques and Procedures ; Drug Delivery Systems/*methods ; *Drug Liberation ; Endodeoxyribonucleases/metabolism ; Humans ; K562 Cells ; Polyethylene Glycols/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; Smart Materials/*chemistry ; },
abstract = {Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid-responsive materials using the CRISPR-associated nuclease Cas12a as a user-programmable sensor and material actuator. This approach improves on the sensitivity of current DNA-responsive materials while enabling their rapid repurposing toward new sequence targets. Here, we provide a comprehensive resource for the design, synthesis and actuation of CRISPR-responsive hydrogels. First, we provide guidelines for the synthesis of Cas12a guide RNAs (gRNAs) for in vitro applications. We then outline methods for the synthesis of both polyethylene glycol-DNA (PEG-DNA) and polyacrylamide-DNA (PA-DNA) hydrogels, as well as their controlled degradation using Cas12a for the release of cargos, including small molecules, enzymes, nanoparticles and living cells within hours. Finally, we detail the design and assembly of microfluidic paper-based devices that use Cas12a-sensitive hydrogels to convert DNA inputs into a variety of visual and electronic readouts for use in diagnostics. Following the initial validation of the gRNA and Cas12a components (1 d), the synthesis and testing of either PEG-DNA or PA-DNA hydrogels require 3-4 d of laboratory time. Optional extensions, including the release of primary human cells or the design of the paper-based diagnostic, require an additional 2-3 d each.},
}
@article {pmid32761301,
year = {2020},
author = {Ji, J and Xu, MX and Qian, TY and Zhu, SZ and Jiang, F and Liu, ZX and Xu, WS and Zhou, J and Xiao, MB},
title = {The AKR1B1 inhibitor epalrestat suppresses the progression of cervical cancer.},
journal = {Molecular biology reports},
volume = {47},
number = {8},
pages = {6091-6103},
pmid = {32761301},
issn = {1573-4978},
support = {BE2019692//Key Research and Development Plan of Jiangsu Province/ ; 2019M661909//Postdoctoral Science Foundation of China/ ; MS22018006//the Social Development Foundation of Nantong City/ ; MS12019018//the Social Development Foundation of Nantong City/ ; MS12019020//the Social Development Foundation of Nantong City/ ; Tfj 18006//Teaching Research Project of Affiliated Hospital of Nantong University/ ; },
mesh = {Aldehyde Reductase/antagonists &amp; inhibitors/genetics/*physiology ; Cell Division/drug effects ; Cell Movement/drug effects ; Enzyme Inhibitors/*pharmacology ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Gene Ontology ; HeLa Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Proteins/antagonists &amp; inhibitors/genetics/*physiology ; RNA, Guide, CRISPR-Cas Systems/genetics/pharmacology ; Rhodanine/*analogs &amp; derivatives/pharmacology ; Thiazolidines/*pharmacology ; Tumor Stem Cell Assay ; Uterine Cervical Neoplasms/*drug therapy/pathology ; },
abstract = {Cervical cancer is the leading cause of cancer-related death among women worldwide. Identifying an effective treatment with fewer side effects is imperative, because all of the current treatments have unique disadvantages. Aldo-keto reductase family 1 member B1 (AKR1B1) is highly expressed in various cancers and is associated with tumor development, but has not been studied in cervical cancer. In the current study, we used CRISPR/Cas9 technology to establish a stable HeLa cell line with AKR1B1 knockout. In vitro, AKR1B1 knockout inhibited the proliferation, migration and invasion of HeLa cells, providing evidence that AKR1B1 is an innovative therapeutic target. Notably, the clinically used epalrestat, an inhibitor of aldose reductases, including AKR1B1, had the same effect as AKR1B1 knockout on HeLa cells. This result suggests that epalrestat could be used in the clinical treatment of cervical cancer, a prospect that undoubtedly requires further research. Moreover, aiming to determine the underlying regulatory mechanism of AKR1B1, we screened a series of differentially regulated genes (DEGs) by RNA sequencing and verified selected DEGs by quantitative RT-PCR. In addition, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs revealed a correlation between AKR1B1 and cancer. In summary, epalrestat inhibits the progression of cervical cancer by inhibiting AKR1B1, and thus may be a new drug for the clinical treatment of cervical cancer.},
}
@article {pmid32665336,
year = {2020},
author = {Cody, WB and Scholthof, HB},
title = {Native Processing of Single Guide RNA Transcripts to Create Catalytic Cas9/Single Guide RNA Complexes in Planta.},
journal = {Plant physiology},
volume = {184},
number = {2},
pages = {1194-1206},
pmid = {32665336},
issn = {1532-2548},
mesh = {*Catalysis ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; RNA Precursors/*genetics ; *RNA, Guide, CRISPR-Cas Systems ; Nicotiana/*genetics ; Transcriptional Activation/*genetics ; },
abstract = {The present CRISPR/Cas9 gene editing dogma for single guide RNA (sgRNA) delivery is based on the premise that 5'-and 3'-nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides at the 5' and 3' termini of sgRNAs. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5', but not 3', proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5' overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript, it also produced indels when delivered with Cas9. These results reveal that 5' auto-processing of progenitor sgRNAs occurs natively in plants. Toward a possible mechanism for the perceived auto-processing, we found, using in vitro-generated RNAs and those isolated from plants, that the 5' to 3' exoribonuclease XRN1 can degrade elongated progenitor sgRNAs, whereas the mature sgRNA end products are resistant. Comparisons with other studies suggest that sgRNA auto-processing may be a phenomenon not unique to plants, but present in other eukaryotes as well.},
}
@article {pmid32641830,
year = {2020},
author = {Mok, BY and de Moraes, MH and Zeng, J and Bosch, DE and Kotrys, AV and Raguram, A and Hsu, F and Radey, MC and Peterson, SB and Mootha, VK and Mougous, JD and Liu, DR},
title = {A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.},
journal = {Nature},
volume = {583},
number = {7817},
pages = {631-637},
pmid = {32641830},
issn = {1476-4687},
support = {P30 DK089507/DK/NIDDK NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R35 GM122455/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 AI080609/AI/NIAID NIH HHS/United States ; T32 GM095450/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Toxins/chemistry/genetics/*metabolism ; Base Sequence ; Burkholderia cenocepacia/enzymology/genetics ; Cell Respiration/genetics ; Cytidine/metabolism ; Cytidine Deaminase/chemistry/genetics/*metabolism ; DNA, Mitochondrial/*genetics ; Gene Editing/*methods ; Genes, Mitochondrial/*genetics ; Genome, Mitochondrial/genetics ; HEK293 Cells ; Humans ; Mitochondria/*genetics ; Mitochondrial Diseases/genetics/therapy ; Mutation ; Oxidative Phosphorylation ; Protein Engineering ; RNA, Guide, CRISPR-Cas Systems/genetics ; Substrate Specificity ; Type VI Secretion Systems/metabolism ; },
abstract = {Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques[1,2]. Because previously described cytidine deaminases operate on single-stranded nucleic acids[3], their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria[4]. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases[9,10].Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.},
}
@article {pmid32584896,
year = {2020},
author = {Raisner, R and Bainer, R and Haverty, PM and Benedetti, KL and Gascoigne, KE},
title = {Super-enhancer acquisition drives oncogene expression in triple negative breast cancer.},
journal = {PloS one},
volume = {15},
number = {6},
pages = {e0235343},
pmid = {32584896},
issn = {1932-6203},
support = {//CIHR/Canada ; },
mesh = {Cell Line, Tumor ; Chromatin Immunoprecipitation ; Enhancer Elements, Genetic/*genetics ; Female ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Histones/chemistry/genetics/metabolism ; Humans ; Membrane Proteins/genetics/metabolism ; Oncogenes/*genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Triple Negative Breast Neoplasms/genetics/*pathology ; },
abstract = {Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFβ pseudo-receptor BAMBI.},
}
@article {pmid32513233,
year = {2020},
author = {Song, Q and Ni, K and Liu, M and Li, Y and Wang, L and Wang, Y and Liu, Y and Yu, Z and Qi, Y and Lu, Z and Ma, L},
title = {Direct-seq: programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {136},
pmid = {32513233},
issn = {1474-760X},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Profiling ; Genotyping Techniques ; HEK293 Cells ; Humans ; K562 Cells ; *RNA, Guide, CRISPR-Cas Systems ; *Sequence Analysis, RNA ; *Single-Cell Analysis ; },
abstract = {CRISPR-based genome perturbation provides a new avenue to conveniently change DNA sequences, transcription, and epigenetic modifications in genetic screens. However, it remains challenging to assay the complex molecular readouts after perturbation at high resolution and at scale. By introducing an A/G mixed capture sequence into the gRNA scaffold, we demonstrate that gRNA transcripts could be directly reverse transcribed by poly (dT) primer together with the endogenous mRNA, followed by high-content molecular phenotyping in scRNA-seq (Direct-seq). With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow.},
}
@article {pmid32510061,
year = {2020},
author = {Ling, X and Gao, X and Chang, L and Chen, H and Shi, X and Liu, T},
title = {Rational design of minimum CRISPR guide RNA by site-specific Cas9-RNA conjugation.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {54},
pages = {7515-7518},
doi = {10.1039/d0cc01432j},
pmid = {32510061},
issn = {1364-548X},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/chemistry/*metabolism ; Crystallography, X-Ray ; Gene Editing/methods ; HEK293 Cells ; Humans ; Nucleic Acid Conformation ; RNA, Guide, CRISPR-Cas Systems/chemistry/*metabolism ; Research Design ; Streptococcus pyogenes/metabolism ; },
abstract = {The CRISPR-Cas9 system enables facile and efficient genome engineering in living cells and organisms. We report a Cas9-RNA conjugation strategy to afford minimal crRNA containing only the guide sequence for the target gene, which may simplify and reduce the cost for large-scale and high-throughput crRNA synthesis and lead to new insights into the design of CRISPR family complexes for diverse purposes.},
}
@article {pmid32485092,
year = {2020},
author = {Anderson, DP and Benns, HJ and Tate, EW and Child, MA},
title = {CRISPR-TAPE: protein-centric CRISPR guide design for targeted proteome engineering.},
journal = {Molecular systems biology},
volume = {16},
number = {6},
pages = {e9475},
pmid = {32485092},
issn = {1744-4292},
support = {BB/M011178/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 202553/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Algorithms ; Automation ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; *Protein Engineering ; Proteins/*metabolism ; Proteome/*metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; User-Computer Interface ; },
abstract = {Rational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. To address this, we have developed CRISPR-TAPE, a protein-centric gRNA design algorithm that allows users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time-consuming post hoc curation, simplifying gRNA outputs and reducing CPU times.},
}
@article {pmid32467238,
year = {2020},
author = {Chen, HM and Yao, X and Ren, Q and Chang, CC and Liu, LY and Miyares, RL and Lee, T},
title = {Enhanced Golic+: highly effective CRISPR gene targeting and transgene HACKing in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {11},
pages = {},
doi = {10.1242/dev.181974},
pmid = {32467238},
issn = {1477-9129},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Animals, Genetically Modified/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drosophila/genetics/*metabolism ; Drosophila Proteins/genetics/metabolism ; Female ; Gene Targeting/*methods ; Germ Cells/cytology/metabolism ; Male ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Transcription Factors/genetics/metabolism ; Transgenes/*genetics ; },
abstract = {Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here, we deliver a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+-based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila.},
}
@article {pmid32394775,
year = {2020},
author = {Guerriero, ML and Corrigan, A and Bornot, A and Firth, M and O'Shea, P and Ross-Thriepland, D and Peel, S},
title = {Delivering Robust Candidates to the Drug Pipeline through Computational Analysis of Arrayed CRISPR Screens.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {6},
pages = {646-654},
doi = {10.1177/2472555220921132},
pmid = {32394775},
issn = {2472-5560},
mesh = {Biomarkers/analysis ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Computational Biology ; Drug Discovery ; Gene Library ; Genome, Human/genetics ; High-Throughput Screening Assays/*trends ; Humans ; Molecular Imaging/trends ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Genome-wide arrayed CRISPR screening is a powerful method for drug target identification as it enables exploration of the effect of individual gene perturbations using diverse highly multiplexed functional and phenotypic assays. Using high-content imaging, we can measure changes in biomarker expression, intracellular localization, and cell morphology. Here we present the computational pipeline we have developed to support the analysis and interpretation of arrayed CRISPR screens. This includes evaluating the quality of guide RNA libraries, performing image analysis, evaluating assay results quality, data processing, hit identification, ranking, visualization, and biological interpretation.},
}
@article {pmid32379958,
year = {2020},
author = {Liu, D and Johnson, VM and Pakrasi, HB},
title = {A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803.},
journal = {ACS synthetic biology},
volume = {9},
number = {6},
pages = {1441-1449},
doi = {10.1021/acssynbio.0c00106},
pmid = {32379958},
issn = {2161-5063},
support = {T32 EB014855/EB/NIBIB NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Gene Expression/drug effects ; Photosystem II Protein Complex/*genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Rhamnose/pharmacology ; Riboswitch ; Synechocystis/*metabolism ; },
abstract = {The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, P rhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.},
}
@article {pmid32367370,
year = {2020},
author = {McDougall, WM and Kandpal, M and Perreira, JM and Brass, AL},
title = {Discovery of Zika Virus Dependency and Restriction Factors Using Flow-Based Arrayed CRISPR Screening for Identification of Targets (FACS-IT).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2142},
number = {},
pages = {215-234},
doi = {10.1007/978-1-0716-0581-3_17},
pmid = {32367370},
issn = {1940-6029},
mesh = {A549 Cells ; CRISPR-Associated Protein 9/metabolism ; Cell Culture Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Flow Cytometry ; Gene Knockout Techniques ; Genetic Testing/*methods ; HEK293 Cells ; High-Throughput Screening Assays/*methods ; Humans ; Phenotype ; RNA, Guide, CRISPR-Cas Systems/genetics ; Viral Load/methods ; Virulence Factors/genetics/isolation &amp; purification ; Virus Cultivation/methods ; Zika Virus/*genetics/*pathogenicity/physiology ; Zika Virus Infection/*diagnosis/genetics/pathology ; },
abstract = {The discovery and implementation of CRISPR/Cas9 tools in pooled genetic screens have allowed for the rapid, high-fidelity identification of host-virus interactions. However, pooled CRISPR screening has significant limitations in its ability both to perform cell biology and plate reader-based screens and to find alleles that result in intermediate-strength phenotypes. Here we introduce an arrayed CRISPR screening method, FACS-IT, which allows researchers to use high content imaging analysis, plate reader assays, cell supernatant characterization, and percent infectivity to characterize CRISPR-mediated gene disruptions causing both moderate and extreme phenotypic changes. By using flow sorting capabilities and CRISPR libraries that are widely available, FACS-IT overcomes both the significant limitation of pooled screening approaches and the prohibitive costs of large-scale arrayed CRISPR reagents. In doing so, FACS-IT will enable researchers to creatively use CRISPR screening to obtain a deeper understanding of biology across a wide range of fields and applications.},
}
@article {pmid32353322,
year = {2020},
author = {Wang, P and Xu, L and Gao, Y and Han, R},
title = {BEON: A Functional Fluorescence Reporter for Quantification and Enrichment of Adenine Base-Editing Activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {7},
pages = {1696-1705},
pmid = {32353322},
issn = {1525-0024},
support = {R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {Adenine/*metabolism ; Adenosine Deaminase/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; Cell Line ; Codon, Terminator ; Escherichia coli Proteins/*genetics/metabolism ; Gene Editing ; Genes, Reporter ; Green Fluorescent Proteins/genetics/*metabolism ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Recombinant Fusion Proteins/genetics/metabolism ; Transfection ; },
abstract = {Adenine base editor (ABE) is a new generation of genome-editing technology through fusion of Cas9 nickase with an evolved E. coli TadA (TadA∗) and holds great promise as novel genome-editing therapeutics for treating genetic disorders. ABEs can directly convert A-T to G-C in specific genomic DNA targets without introducing double-strand breaks (DSBs). We recently showed that computer program-assisted analysis of Sanger sequencing traces can be used as a low-cost and rapid alternative of deep sequencing to assess base-editing outcomes. Here we developed a rapid fluorescence-based reporter assay (Base Editing ON [BEON]) to quantify ABE efficiency. The assay relies on the restoration of the downstream green fluorescent protein (GFP) in ABE-mediated editing of a stop codon located within the guide RNA (gRNA). We showed that this assay can be used to screen for effective ABE variants, characterize the protospacer adjacent motif (PAM) requirement of a novel NNG-targeting ABE based on ScCas9, and enrich for edited cells. Finally, we demonstrated that the reporter assay allowed us to assess the feasibility of ABE editing to correct point mutations associated with dysferlinopathy. Taken together, the BEON assay would facilitate and simplify the studies with ABEs.},
}
@article {pmid32348718,
year = {2020},
author = {Li, A and Tanner, MR and Lee, CM and Hurley, AE and De Giorgi, M and Jarrett, KE and Davis, TH and Doerfler, AM and Bao, G and Beeton, C and Lagor, WR},
title = {AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {6},
pages = {1432-1441},
pmid = {32348718},
issn = {1525-0024},
support = {UG3 HL151545/HL/NHLBI NIH HHS/United States ; T32 HL007676/HL/NHLBI NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; U42 OD026645/OD/NIH HHS/United States ; T32 GM008231/GM/NIGMS NIH HHS/United States ; S10 RR024574/RR/NCRR NIH HHS/United States ; 19POST34430092/AHA_/American Heart Association-American Stroke Association/United States ; },
mesh = {Animals ; Biomarkers ; CRISPR-Associated Protein 9/adverse effects/*immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics ; Gene Editing/*methods ; Gene Expression ; Gene Order ; Genetic Vectors/*genetics ; Hepatocytes/metabolism ; Humans ; Immunization ; Immunologic Memory ; Immunophenotyping ; Mice ; RNA, Guide, CRISPR-Cas Systems ; T-Lymphocyte Subsets/immunology/metabolism ; Transgenes ; },
abstract = {Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8[+] T cells in the liver. This cytotoxic T cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, and complete elimination of genome-edited cells, and was followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR-Cas9-based in vivo genome editing in the liver.},
}
@article {pmid32348672,
year = {2020},
author = {Lim, H and Jun, S and Park, M and Lim, J and Jeong, J and Lee, JH and Bang, D},
title = {Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System.},
journal = {ACS synthetic biology},
volume = {9},
number = {5},
pages = {1003-1009},
doi = {10.1021/acssynbio.0c00002},
pmid = {32348672},
issn = {2161-5063},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Gene Library ; Genome, Bacterial ; Mutation ; Plasmids/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by coutilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.},
}
@article {pmid32342086,
year = {2020},
author = {Ye, Y and Zhang, X and Xie, F and Xu, B and Xie, P and Yang, T and Shi, Q and Zhang, CY and Zhang, Y and Chen, J and Jiang, X and Li, J},
title = {An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells.},
journal = {Biomaterials science},
volume = {8},
number = {10},
pages = {2966-2976},
doi = {10.1039/d0bm00427h},
pmid = {32342086},
issn = {2047-4849},
mesh = {*Bioengineering ; CRISPR-Associated Protein 9/*metabolism ; Exosomes/*metabolism ; *Gene Editing ; Gene Transfer Techniques ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {CRISPR-Cas9 is a versatile genome-editing technology that is a promising gene therapy tactic. However, the delivery of CRISPR-Cas9 is still a major obstacle to its broader clinical application. Here, we confirm that the components of CRISPR-Cas9-sgRNA and Cas9 protein-can be packaged into exosomes, where sgRNA and Cas9 protein exist as a sgRNA:Cas9 ribonucleoprotein complex. Although exosomal CRISPR-Cas9 components can be delivered into recipient cells, they are not adequate to abrogate the target gene in recipient cells. To solve this, we engineered a functionalized exosome (M-CRISPR-Cas9 exosome) that could encapsulate CRISPR-Cas9 components more efficiently. To improve the loading efficiency of Cas9 proteins into exosomes, we artificially engineered exosomes by fusing GFP and GFP nanobody with exosomal membrane protein CD63 and Cas9 protein, respectively. Therefore, Cas9 proteins could be captured selectively and efficiently loaded into exosomes due to the affinity of GFP-GFP nanobody rather than random loading. sgRNA and Cas9 protein exist as a complex in functionalized exosomes and can be delivered into recipient cells. To show the function of modified exosomes-delivered CRISPR-Cas9 components in recipient cells visually, we generated a reporter cell line (A549stop-DsRed) that produced a red fluorescent signal when the stop element was deleted by the sgRNA-guided endonuclease. Using A549stop-DsRed reporter cells, we showed that modified exosomes loaded with CRISPR-Cas9 components abrogated the target gene more efficiently in recipient cells. Our study reports an alternative tactic for CRISPR-Cas9 delivery.},
}
@article {pmid32210731,
year = {2020},
author = {Zhan, H and Xiao, L and Li, A and Yao, L and Cai, Z and Liu, Y},
title = {Engineering Cellular Signal Sensors based on CRISPR-sgRNA Reconstruction Approaches.},
journal = {International journal of biological sciences},
volume = {16},
number = {8},
pages = {1441-1449},
pmid = {32210731},
issn = {1449-2288},
mesh = {Animals ; Cell Line ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Regulatory Networks ; Genetic Therapy ; Genetic Vectors ; Genome ; Humans ; Light ; Mice ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Riboswitch ; Signal Transduction ; Synthetic Biology ; },
abstract = {The discovery of the CRISPR systems has enriched the application of gene therapy and biotechnology. As a type of robust and simple toolbox, the CRISPR system has greatly promoted the development of cellular signal sensors at the genomic level. Although CRISPR systems have demonstrated that they can be used in eukaryotic and even mammalian cells after extraction from prokaryotic cells, controlling their gene-editing activity remains a challenge. Here we summarize the advantages and disadvantages of building a CRIRPR-based signal sensor through sgRNA reconstruction, as well as possible ways to reprogram the signal network of cells. We also propose how to further improve the design of the current signal sensors based on sgRNA-riboswitch. We believe that the development of these technologies and the construction of platforms can further promote the development of environment detection, disease diagnosis, and gene therapy by means of synthetic biology.},
}
@article {pmid32156728,
year = {2020},
author = {Raffeiner, P and Hart, JR and García-Caballero, D and Bar-Peled, L and Weinberg, MS and Vogt, PK},
title = {An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {12},
pages = {6571-6579},
pmid = {32156728},
issn = {1091-6490},
support = {K99 CA215249/CA/NCI NIH HHS/United States ; R35 CA197582/CA/NCI NIH HHS/United States ; R50 CA243899/CA/NCI NIH HHS/United States ; },
mesh = {Aptamers, Nucleotide ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry/genetics/*metabolism ; CRISPR-Associated Protein 9/genetics ; Capsid Proteins/metabolism ; Cell Line, Tumor ; Cell Proliferation/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Promoter Regions, Genetic ; Protein Domains ; Proto-Oncogene Proteins c-myc/*metabolism ; RNA, Guide, CRISPR-Cas Systems ; RNA, Long Noncoding/*genetics/metabolism ; Repressor Proteins/chemistry/genetics/*metabolism ; Transcription, Genetic ; },
abstract = {MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.},
}
@article {pmid32156270,
year = {2020},
author = {Ding, T and Huang, C and Liang, Z and Ma, X and Wang, N and Huo, YX},
title = {Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {63},
pmid = {32156270},
issn = {1475-2859},
support = {grant No. 21676026//National Natural Science Foundation of China/ ; grant No. 91951113//National Natural Science Foundation of China/ ; grant No. 2019GXRC033//Innovation Team Project of Colleges and Universities in Jinan/ ; },
mesh = {Cloning, Molecular/*methods ; Escherichia coli/*genetics ; Gene Editing/*methods ; Plasmids/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Recombination, Genetic ; Sequence Deletion ; },
abstract = {BACKGROUND: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system.<br><br>RESULTS: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100&#xa0;kb with an efficiency of 83.33% was achieved in Escherichia coli.<br><br>CONCLUSIONS: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.},
}
@article {pmid32134913,
year = {2020},
author = {Weuring, WJ and Singh, S and Volkers, L and Rook, MB and van 't Slot, RH and Bosma, M and Inserra, M and Vetter, I and Verhoeven-Duif, NM and Braun, KPJ and Rivara, M and Koeleman, BPC},
title = {NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome.},
journal = {PloS one},
volume = {15},
number = {3},
pages = {e0219106},
pmid = {32134913},
issn = {1932-6203},
mesh = {Animals ; Anticonvulsants/pharmacology ; Disease Models, Animal ; Epilepsies, Myoclonic/metabolism/*pathology ; Humans ; Locomotion/drug effects ; Morpholinos/metabolism ; Mutagenesis ; NAV1.1 Voltage-Gated Sodium Channel/chemistry/genetics/*metabolism ; NAV1.6 Voltage-Gated Sodium Channel/chemistry/genetics/*metabolism ; Neurons/drug effects/metabolism ; Pentylenetetrazole/pharmacology ; Phenotype ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Messenger/metabolism ; Voltage-Gated Sodium Channel Agonists/pharmacology ; Voltage-Gated Sodium Channel Blockers/pharmacology ; Zebrafish ; Zebrafish Proteins/chemistry/genetics/*metabolism ; gamma-Aminobutyric Acid/metabolism ; },
abstract = {Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.},
}
@article {pmid32122365,
year = {2020},
author = {Imkeller, K and Ambrosi, G and Boutros, M and Huber, W},
title = {gscreend: modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {53},
pmid = {32122365},
issn = {1474-760X},
mesh = {Animals ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene-Environment Interaction ; Genome-Wide Association Study/*methods ; Genomics/*methods ; Genotype ; HCT116 Cells ; HEK293 Cells ; Humans ; Mice ; Models, Genetic ; RNA, Guide, CRISPR-Cas Systems/chemistry/genetics ; *Software ; },
abstract = {Pooled CRISPR screens are a powerful tool to probe genotype-phenotype relationships at genome-wide scale. However, criteria for optimal design are missing, and it remains unclear how experimental parameters affect results. Here, we report that random decreases in gRNA abundance are more likely than increases due to bottle-neck effects during the cell proliferation phase. Failure to consider this asymmetry leads to loss of detection power. We provide a new statistical test that addresses this problem and improves hit detection at reduced experiment size. The method is implemented in the R package gscreend, which is available at http://bioconductor.org/packages/gscreend.},
}
@article {pmid32020125,
year = {2020},
author = {Reardon, S},
title = {Step aside CRISPR, RNA editing is taking off.},
journal = {Nature},
volume = {578},
number = {7793},
pages = {24-27},
pmid = {32020125},
issn = {1476-4687},
mesh = {Adenosine Deaminase ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Point Mutation ; RNA ; *RNA Editing ; RNA, Guide, CRISPR-Cas Systems ; },
}
@article {pmid32012858,
year = {2020},
author = {Biagioni, A and Laurenzana, A and Chillà, A and Del Rosso, M and Andreucci, E and Poteti, M and Bani, D and Guasti, D and Fibbi, G and Margheri, F},
title = {uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines.},
journal = {Cells},
volume = {9},
number = {2},
pages = {},
pmid = {32012858},
issn = {2073-4409},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/metabolism ; Cell Line, Tumor ; Cell Respiration/genetics ; Colonic Neoplasms/genetics/*metabolism/ultrastructure ; Deoxyribonuclease I/metabolism ; Fluorescence ; Gene Expression Regulation, Neoplastic ; *Gene Knockout Techniques ; *Glycolysis/genetics ; Humans ; Lactic Acid/metabolism ; Melanoma/genetics/*metabolism/ultrastructure ; Mitochondria/metabolism/ultrastructure ; Organelle Biogenesis ; *Oxidative Phosphorylation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Receptors, Urokinase Plasminogen Activator/genetics/*metabolism ; Stress, Physiological ; },
abstract = {Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB.},
}
@article {pmid32001098,
year = {2020},
author = {Marzec, M and Brąszewska-Zalewska, A and Hensel, G},
title = {Prime Editing: A New Way for Genome Editing.},
journal = {Trends in cell biology},
volume = {30},
number = {4},
pages = {257-259},
doi = {10.1016/j.tcb.2020.01.004},
pmid = {32001098},
issn = {1879-3088},
mesh = {DNA/genetics ; *Gene Editing ; Humans ; Hybridization, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise and efficient use of genome editing tools are hampered by the introduction of DNA double-strand breaks, donor DNA templates, or homology-directed repair. A recent study expands the genome editing toolbox with the introduction of prime editing, which overcomes previous challenges and introduces insertions, deletions, and all putative 12 types of base-to-base conversions in human cells.},
}
@article {pmid31978124,
year = {2020},
author = {Naim, F and Shand, K and Hayashi, S and O'Brien, M and McGree, J and Johnson, AAT and Dugdale, B and Waterhouse, PM},
title = {Are the current gRNA ranking prediction algorithms useful for genome editing in plants?.},
journal = {PloS one},
volume = {15},
number = {1},
pages = {e0227994},
pmid = {31978124},
issn = {1932-6203},
mesh = {*Algorithms ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; *Gene Editing ; *Genome, Plant ; Plant Leaves/genetics ; Plants/*genetics ; Plants, Genetically Modified ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Nicotiana/genetics ; Transgenes ; },
abstract = {Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.},
}
@article {pmid31967454,
year = {2020},
author = {Kuhn, J and Lin, Y and Krhac Levacic, A and Al Danaf, N and Peng, L and Höhn, M and Lamb, DC and Wagner, E and Lächelt, U},
title = {Delivery of Cas9/sgRNA Ribonucleoprotein Complexes via Hydroxystearyl Oligoamino Amides.},
journal = {Bioconjugate chemistry},
volume = {31},
number = {3},
pages = {729-742},
doi = {10.1021/acs.bioconjchem.9b00853},
pmid = {31967454},
issn = {1520-4812},
mesh = {Amides/*chemistry ; CRISPR-Associated Protein 9/*metabolism ; Cell Line ; Drug Carriers/*chemistry ; Endosomes/metabolism ; Humans ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; Ribonucleoproteins/*chemistry/*metabolism ; },
abstract = {The programmable endonuclease activity and simple usage of CRISPR/Cas9 have revolutionized the field of genome editing. The binding of single guide RNA (sgRNA) by the Cas9 protein results in the formation of negatively charged ribonucleoprotein (RNP) complexes. The presence of this functional complex inside cells is imperative for the intended specific genome modifications. The direct intracellular delivery of Cas9/sgRNA RNP complexes is of great advantage. In this work, a compound library of sequence-defined oligo(ethylenamino) amides containing structural motifs for stable nanoparticle formation, cellular uptake, and endosomal release was used for the screening and development of suitable Cas9 RNP delivery vehicles. Lipid-containing oligoaminoamides (lipo-OAAs) were identified as the most efficient carriers for intracellular Cas9/sgRNA delivery and gene disruption. Fluorescence correlation spectroscopy measurements indicated that the lipo-OAAs only interact with sgRNA-loaded Cas9 protein, which suggests exclusive ionic interaction with the negatively charged RNPs. The type of contained fatty acid turned out to have a critical impact on the knock out efficiency: the presence of one hydroxy group in the fatty acid dramatically changes the properties and performance of the resulting Cas9/sgRNA lipo-OAA complexes. The lipo-OAA-containing hydroxy-stearic acid (OHSteA) was superior to the analogues with saturated or unsaturated fatty acids without hydroxylation; it formed smaller and more defined nanoparticles with Cas9/sgRNA and improved the cellular uptake and endosomal release, which altogether resulted in an increased nuclear association and the highest gene knock out levels. The efficient and adaptable delivery platform has high potential for the future development of therapeutics based on precise genome modifications.},
}
@article {pmid31900425,
year = {2020},
author = {Li, Z and Zhang, H and Xiao, R and Chang, L},
title = {Cryo-EM structure of a type I-F CRISPR RNA guided surveillance complex bound to transposition protein TniQ.},
journal = {Cell research},
volume = {30},
number = {2},
pages = {179-181},
pmid = {31900425},
issn = {1748-7838},
support = {R01 GM138675/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/*metabolism/*ultrastructure ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Cryoelectron Microscopy ; DNA Transposable Elements/genetics ; Models, Molecular ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; Vibrio cholerae/genetics ; },
}
@article {pmid31848794,
year = {2020},
author = {Zhang, X and Kang, L and Zhang, Q and Meng, Q and Pan, Y and Yu, Z and Shi, N and Jackson, S and Zhang, X and Wang, H and Tor, M and Hong, Y},
title = {An RNAi suppressor activates in planta virus-mediated gene editing.},
journal = {Functional &amp; integrative genomics},
volume = {20},
number = {4},
pages = {471-477},
doi = {10.1007/s10142-019-00730-y},
pmid = {31848794},
issn = {1438-7948},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing/*methods ; Potexvirus/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA, Small Interfering/*genetics/metabolism ; Nicotiana/*genetics ; },
abstract = {RNA-guided CRISPR/Cas9 technology has been developed for gene/genome editing (GE) in organisms across kingdoms. However, in planta delivery of the two core GE components, Cas9 and small guide RNA (sgRNA), often involves time-consuming and labor-intensive production of transgenic plants. Here we show that Foxtail mosaic virus, a monocot- and dicot-infecting potexvirus, can simultaneously express Cas9, sgRNA, and an RNAi suppressor to efficiently induce GE in Nicotiana benthamiana through a transgenic plant-free manner.},
}
@article {pmid31833031,
year = {2020},
author = {García-Tuñón, I and Vuelta, E and Lozano, L and Herrero, M and Méndez, L and Palomero-Hernandez, J and Pérez-Caro, M and Pérez-García, J and González-Sarmiento, R and Sánchez-Martín, M},
title = {Establishment of a conditional Nomo1 mouse model by CRISPR/Cas9 technology.},
journal = {Molecular biology reports},
volume = {47},
number = {2},
pages = {1381-1391},
pmid = {31833031},
issn = {1573-4978},
support = {PI16/01920//Instituto de Salud Carlos III/ ; },
mesh = {Alleles ; Animals ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Disease Models, Animal ; Exons/genetics ; Integrases/metabolism ; Membrane Proteins/*genetics/metabolism ; Mice, Inbred C57BL ; Mice, Transgenic ; Mosaicism ; Mutation/genetics ; Nodal Protein/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1[f/f]). Nomo1[f/f] mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1[f/f] mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1[f/f] mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.},
}
@article {pmid31823436,
year = {2020},
author = {Beier, S and Ulpinnis, C and Schwalbe, M and Münch, T and Hoffie, R and Koeppel, I and Hertig, C and Budhagatapalli, N and Hiekel, S and Pathi, KM and Hensel, G and Grosse, M and Chamas, S and Gerasimova, S and Kumlehn, J and Scholz, U and Schmutzer, T},
title = {Kmasker plants - a tool for assessing complex sequence space in plant species.},
journal = {The Plant journal : for cell and molecular biology},
volume = {102},
number = {3},
pages = {631-642},
doi = {10.1111/tpj.14645},
pmid = {31823436},
issn = {1365-313X},
mesh = {Algorithms ; Gene Editing ; Genome, Plant/*genetics ; Genomics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Analysis, DNA ; Software ; },
abstract = {Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.},
}
@article {pmid31822517,
year = {2020},
author = {Chen, CL and Rodiger, J and Chung, V and Viswanatha, R and Mohr, SE and Hu, Y and Perrimon, N},
title = {SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {2},
pages = {489-494},
pmid = {31822517},
issn = {2160-1836},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; R21 ES025615/ES/NIEHS NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Diptera ; Gene Editing/*methods ; Humans ; Internet ; Mice ; *Polymorphism, Single Nucleotide ; *RNA, Guide, CRISPR-Cas Systems ; Rats ; Software ; Zebrafish ; },
abstract = {CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.},
}
@article {pmid31819258,
year = {2020},
author = {Nguyen, DN and Roth, TL and Li, PJ and Chen, PA and Apathy, R and Mamedov, MR and Vo, LT and Tobin, VR and Goodman, D and Shifrut, E and Bluestone, JA and Puck, JM and Szoka, FC and Marson, A},
title = {Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency.},
journal = {Nature biotechnology},
volume = {38},
number = {1},
pages = {44-49},
pmid = {31819258},
issn = {1546-1696},
support = {DP3 DK111914/DK/NIDDK NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; T32 AI007641/AI/NIAID NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; R01 DK119979/DK/NIDDK NIH HHS/United States ; },
mesh = {Adult ; CRISPR-Associated Protein 9/*metabolism ; Gene Editing ; Humans ; Nanoparticles/chemistry ; Polymers/*chemistry ; Protein Stability ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies[1-5]. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3[+]) T cells, CD8[+] T cells, CD4[+] T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived[6] hematopoietic stem progenitor cells (HSPCs).},
}
@article {pmid31802632,
year = {2020},
author = {Ganguly, J and Martin-Pascual, M and van Kranenburg, R},
title = {CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313.},
journal = {Microbial biotechnology},
volume = {13},
number = {2},
pages = {339-349},
pmid = {31802632},
issn = {1751-7915},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; Metabolic Networks and Pathways ; *RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Hungateiclostridium thermocellum DSM 1313 has biotechnological potential as a whole-cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been hampered due to the lack of simple and high-throughput genome engineering tools. Recently in our research group, a thermophilic bacterial CRISPR-Cas9-based system has been developed as a transcriptional suppression tool for regulation of gene expression. We applied ThermoCas9-based CRISPR interference (CRISPRi) to repress the H. thermocellum central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes. The effects of repression on target genes were studied based on transcriptional expression and product formation. Single-guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62% respectively. This resulted in 24% and 17% decrease in lactate and acetate production, correspondingly. Hence, the CRISPRi approach for H. thermocellum to downregulate metabolic genes can be used for remodelling of metabolic pathways without the requisite for genome engineering. These data established for the first time the feasibility of employing CRISPRi-mediated gene repression of metabolic genes in H. thermocellum DSM 1313.},
}
@article {pmid31797132,
year = {2020},
author = {Ouyang, L and Ma, M and Li, L},
title = {An efficient transgene-free DNA-editing system for Arabidopsis using a fluorescent marker.},
journal = {Biotechnology letters},
volume = {42},
number = {2},
pages = {313-318},
doi = {10.1007/s10529-019-02778-z},
pmid = {31797132},
issn = {1573-6776},
support = {31470677//National Natural Science Foundation of China/ ; 2017A030307017//Natural Science Foundation of Guangdong Province/ ; 2017A030303087//the Science and Technology Tackle Key Project of Guangdong Province/ ; Key project of basic research and applied research of Guangdong Province//NO/ ; },
mesh = {Arabidopsis/genetics/*growth &amp; development ; Gene Editing ; Genetic Markers ; Luminescent Proteins/genetics/*metabolism ; MicroRNAs/*genetics/metabolism ; Mutation ; RNA, Guide, CRISPR-Cas Systems ; Transgenes ; Red Fluorescent Protein ; },
abstract = {OBJECTIVE: To obtain transgene-free progeny by constructing a DNA editing system with a fluorescent screening marker gene and two pairs of single-guide RNAs to simultaneously recognize two different sites in the target gene encoding Arabidopsis microRNA(miR)160A RESULTS: The T1 seeds with red fluorescence were easily identified and were selected to verify that the proportion of miR160A knockout mutants reached approximately 50%. Seeds with no fluorescence in the T2 generation were selected and screened for homozygous mutants. In the T2 generation plants, the Cas9 fragment was not detected by polymerase chain reaction. The traits of the homozygous mutants were stably inherited by the T2 population.<br><br>CONCLUSIONS: A highly efficient DNA-editing construct was successfully developed and can be used as a plant genome site-specific editing tool that may be useful for improving plant genetic resources.},
}
@article {pmid31784727,
year = {2019},
author = {Fulco, CP and Nasser, J and Jones, TR and Munson, G and Bergman, DT and Subramanian, V and Grossman, SR and Anyoha, R and Doughty, BR and Patwardhan, TA and Nguyen, TH and Kane, M and Perez, EM and Durand, NC and Lareau, CA and Stamenova, EK and Aiden, EL and Lander, ES and Engreitz, JM},
title = {Activity-by-contact model of enhancer-promoter regulation from thousands of CRISPR perturbations.},
journal = {Nature genetics},
volume = {51},
number = {12},
pages = {1664-1669},
pmid = {31784727},
issn = {1546-1718},
support = {U01 HL130010/HL/NHLBI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; K99 HG009917/HG/NHGRI NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; UM1 HG009375/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Enhancer Elements, Genetic ; GATA1 Transcription Factor/genetics ; Gene Expression Regulation ; Histone Deacetylase 6/genetics ; Humans ; In Situ Hybridization, Fluorescence ; K562 Cells ; Mice ; Models, Genetic ; *Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases[1-4]. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types[5,6]. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.},
}
@article {pmid31658298,
year = {2019},
author = {Soubeyrand, S and Lau, P and Peters, V and McPherson, R},
title = {Off-target effects of CRISPRa on interleukin-6 expression.},
journal = {PloS one},
volume = {14},
number = {10},
pages = {e0224113},
pmid = {31658298},
issn = {1932-6203},
support = {//CIHR/Canada ; },
mesh = {Binding Sites ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Profiling/*methods ; Gene Expression Regulation/drug effects ; HEK293 Cells ; HeLa Cells ; Humans ; Interleukin-6/*genetics ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/*pharmacology ; RNA, Long Noncoding/*genetics ; Transcriptional Activation ; Up-Regulation ; },
abstract = {Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology.},
}
@article {pmid31612609,
year = {2019},
author = {Marzec, M and Hensel, G},
title = {More precise, more universal and more specific - the next generation of RNA-guided endonucleases for genome editing.},
journal = {The FEBS journal},
volume = {286},
number = {23},
pages = {4657-4660},
doi = {10.1111/febs.15079},
pmid = {31612609},
issn = {1742-4658},
support = {FKZ 031B0547//German Federal Ministry of Education and Research/International ; CZ.02.1.01./0.0/0.0/16_019/0000827//Czech Science Foundation/International ; SPP 813103381//Czech Science Foundation/International ; },
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {CRISPR is a prokaryotic defence system that was adapted as a tool for genome editing and has become one of the most important discoveries of this century. CRISPR-associated endonucleases cleave DNA at precise sites, which are marked by complementary short-guided RNA. The recently developed versions of endonucleases are compatible with a broad range of PAM motifs, have a higher specificity and enable a specific nucleotide to be replaced.},
}
@article {pmid31591552,
year = {2019},
author = {Reis, AC and Halper, SM and Vezeau, GE and Cetnar, DP and Hossain, A and Clauer, PR and Salis, HM},
title = {Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays.},
journal = {Nature biotechnology},
volume = {37},
number = {11},
pages = {1294-1301},
pmid = {31591552},
issn = {1546-1696},
mesh = {Amino Acids/biosynthesis ; CRISPR-Associated Protein 9/metabolism ; Escherichia coli/*genetics ; Escherichia coli Proteins/*genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Machine Learning ; Metabolic Engineering ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Succinic Acid/metabolism ; Synthetic Biology ; },
abstract = {Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.},
}
@article {pmid31586051,
year = {2019},
author = {Hamilton, TA and Pellegrino, GM and Therrien, JA and Ham, DT and Bartlett, PC and Karas, BJ and Gloor, GB and Edgell, DR},
title = {Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4544},
pmid = {31586051},
issn = {2041-1723},
support = {PJT-159708//CIHR/Canada ; },
mesh = {Anti-Infective Agents/*administration &amp; dosage ; Biofilms/drug effects ; CRISPR-Associated Protein 9/*administration &amp; dosage/genetics ; Coculture Techniques ; *Conjugation, Genetic ; Drug Delivery Systems/*methods ; Escherichia coli/drug effects/genetics ; *Gene Transfer Techniques ; Microbial Sensitivity Tests ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae ; Salmonella enterica/drug effects/genetics ; },
abstract = {The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing.},
}
@article {pmid31578372,
year = {2019},
author = {Simonneau, C and Yang, J and Kong, X and Kilker, R and Edelstein, L and Fortina, P and Londin, E and Horowitz, A},
title = {Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14238},
pmid = {31578372},
issn = {2045-2322},
support = {R01 HL128234/HL/NHLBI NIH HHS/United States ; R01 HL119984/HL/NHLBI NIH HHS/United States ; P30 CA56036//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/International ; },
mesh = {Adherens Junctions/physiology ; Cell Line, Transformed ; Clone Cells/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Collagen/metabolism ; Endothelial Cells/*cytology/metabolism ; Fibronectins/metabolism ; Flow Cytometry/methods ; Fluorescent Dyes/analysis ; Gelatin ; Gene Library ; Genome-Wide Association Study/instrumentation/*methods ; High-Throughput Screening Assays/instrumentation/*methods ; Humans ; Male ; Miniaturization ; Permeability/drug effects ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Repressor Proteins/genetics ; Signal Transduction/genetics ; Thrombin/metabolism/pharmacology ; Tight Junctions/physiology ; Transcription, Genetic ; Vascular Endothelial Growth Factor A/pharmacology ; },
abstract = {The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer's intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 μm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.},
}
@article {pmid31577419,
year = {2019},
author = {Ferreira, R and Skrekas, C and Hedin, A and Sánchez, BJ and Siewers, V and Nielsen, J and David, F},
title = {Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation.},
journal = {ACS synthetic biology},
volume = {8},
number = {11},
pages = {2457-2463},
doi = {10.1021/acssynbio.9b00258},
pmid = {31577419},
issn = {2161-5063},
mesh = {Acetyl Coenzyme A/metabolism ; Biosensing Techniques ; CRISPR-Associated Protein 9/*genetics ; Carbon/*metabolism ; Computer Simulation ; Cytosol/metabolism ; Ethanol/metabolism ; Gene Expression Regulation, Fungal ; Genes, Fungal ; Glucose/metabolism ; Malonyl Coenzyme A/metabolism ; Metabolic Engineering/*methods ; Metabolic Flux Analysis/*methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae/*genetics/*metabolism ; Synthetic Biology/methods ; Transcription, Genetic ; },
abstract = {Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes toward increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-scale metabolic model, and a gRNA library targeting a total of 168 selected genes was designed. After multiple rounds of fluorescence-activated cell sorting and library sequencing, the gRNAs that were functional and increased flux toward malonyl-CoA were assessed for their efficiency to enhance 3-hydroxypropionic acid (3-HP) production. 3-HP production was significantly improved upon fine-tuning genes involved in providing malonyl-CoA precursors, cofactor supply, as well as chromatin remodeling.},
}
@article {pmid31534539,
year = {2019},
author = {Hsu, MN and Liao, HT and Truong, VA and Huang, KL and Yu, FJ and Chen, HH and Nguyen, TKN and Makarevich, P and Parfyonova, Y and Hu, YC},
title = {CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.},
journal = {Theranostics},
volume = {9},
number = {21},
pages = {6099-6111},
pmid = {31534539},
issn = {1838-7640},
mesh = {Adipocytes/physiology ; Adipose Tissue ; Animals ; Axons/physiology ; Baculoviridae/genetics ; Cell Movement ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Mesenchymal Stem Cells ; Nerve Growth Factors/genetics/*metabolism ; *Nerve Regeneration ; Neurons/physiology ; Peripheral Nerves/physiology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Rats ; Rats, Sprague-Dawley ; Recovery of Function ; Schwann Cells/physiology ; Streptococcus pyogenes/enzymology/genetics ; },
abstract = {Background: Peripheral nerve regeneration requires coordinated functions of neurotrophic factors and neuronal cells. CRISPR activation (CRISPRa) is a powerful tool that exploits inactive Cas9 (dCas9), single guide RNA (sgRNA) and transcription activator for gene activation, but has yet to be harnessed for tissue regeneration. Methods: We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes (BDNF, GDNF and NGF). The hybrid BV was used to transduce rat adipose-derived stem cells (ASC) and functionalize the ASC sheets. We further implanted the ASC sheets into sciatic nerve injury sites in rats. Results: Transduction of rat ASC with the hybrid BV vector enabled robust, simultaneous and prolonged activation of the 3 neurotrophic factors for at least 21 days. The CRISPRa-engineered ASC sheets were able to promote Schwann cell (SC) migration, neuron proliferation and neurite outgrowth in vitro. The CRISPRa-engineered ASC sheets further enhanced in vivo functional recovery, nerve reinnervation, axon regeneration and remyelination. Conclusion: These data collectively implicated the potentials of the hybrid BV-delivered CRISPRa system for multiplexed activation of endogenous neurotrophic factor genes in ASC sheets to promote peripheral nerve regeneration.},
}
@article {pmid31442407,
year = {2019},
author = {Dong, MB and Wang, G and Chow, RD and Ye, L and Zhu, L and Dai, X and Park, JJ and Kim, HR and Errami, Y and Guzman, CD and Zhou, X and Chen, KY and Renauer, PA and Du, Y and Shen, J and Lam, SZ and Zhou, JJ and Lannin, DR and Herbst, RS and Chen, S},
title = {Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells.},
journal = {Cell},
volume = {178},
number = {5},
pages = {1189-1204.e23},
pmid = {31442407},
issn = {1097-4172},
support = {P50 CA121974/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; RF1 DA048811/DA/NIDA NIH HHS/United States ; S10 OD018521/OD/NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; T32 GM007499/GM/NIGMS NIH HHS/United States ; UL1 TR001863/TR/NCATS NIH HHS/United States ; T32 HD007149/HD/NICHD NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; P50 CA196530/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/pathology/therapy ; CD8-Positive T-Lymphocytes/cytology/immunology/*metabolism ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytokines/genetics/metabolism ; Female ; Humans ; Immunologic Memory ; Immunotherapy ; Male ; Mice ; Mice, Knockout ; NF-kappa B/metabolism ; Programmed Cell Death 1 Receptor/metabolism ; RNA Helicases/deficiency/*genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Transcriptome ; },
abstract = {CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.},
}
@article {pmid31383454,
year = {2019},
author = {Villamizar, O and Waters, SA and Scott, T and Saayman, S and Grepo, N and Urak, R and Davis, A and Jaffe, A and Morris, KV},
title = {Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {10},
pages = {1737-1748},
pmid = {31383454},
issn = {1525-0024},
support = {P30 CA033572/CA/NCI NIH HHS/United States ; R01 DK104681/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/metabolism ; Cell Line ; Cell Membrane/metabolism ; Cell-Penetrating Peptides/genetics ; Cystic Fibrosis/*genetics/metabolism/therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/*metabolism ; Epithelial Cells/cytology/metabolism ; Humans ; Molecular Targeted Therapy/*methods ; Nanoparticles/chemistry ; Nasal Mucosa/cytology/*metabolism ; RNA, Guide, CRISPR-Cas Systems/pharmacology ; RNA, Long Noncoding/genetics ; Transcriptional Activation ; tat Gene Products, Human Immunodeficiency Virus/genetics ; },
abstract = {Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.},
}
@article {pmid31361218,
year = {2019},
author = {Zuo, Z and Zolekar, A and Babu, K and Lin, VJ and Hayatshahi, HS and Rajan, R and Wang, YC and Liu, J},
title = {Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31361218},
issn = {2050-084X},
support = {Start-up Fund and Faculty Pilot Grant//University of North Texas (UNT) Health Science Center/International ; Start-up Fund and Basic Research Seed Grant//University of North Texas Health Science Center/International ; P20GM103640/GM/NIGMS NIH HHS/United States ; P20 GM103640/GM/NIGMS NIH HHS/United States ; Program for Professor of Special Appointment at Shanghai Institutions of Higher Learning//Shanghai Municipal Education Commission/International ; Start-up Fund and Faculty Pilot Grant//University of North Texas Health Science Center/International ; Start-up Fund and Basic Research Seed Grant//University of North Texas (UNT) Health Science Center/International ; MCB-1716423//National Science Foundation/International ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/*metabolism ; Catalytic Domain ; DNA/chemistry/*metabolism ; Molecular Dynamics Simulation ; Protein Binding ; Protein Conformation ; RNA, Guide, CRISPR-Cas Systems/chemistry/*metabolism ; Streptococcus pyogenes/*enzymology ; },
abstract = {The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.},
}
@article {pmid31327755,
year = {2019},
author = {Nance, ME and Shi, R and Hakim, CH and Wasala, NB and Yue, Y and Pan, X and Zhang, T and Robinson, CA and Duan, SX and Yao, G and Yang, NN and Chen, SJ and Wagner, KR and Gersbach, CA and Duan, D},
title = {AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {9},
pages = {1568-1585},
pmid = {31327755},
issn = {1525-0024},
support = {R01 AR069085/AR/NIAMS NIH HHS/United States ; R01 GM063732/GM/NIGMS NIH HHS/United States ; R01 GM117059/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics ; Disease Models, Animal ; Dystrophin/chemistry/*genetics ; *Gene Editing ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Vectors/*genetics ; Mice ; Mice, Knockout ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Duchenne/genetics/therapy ; Myoblasts/*metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Regeneration ; Satellite Cells, Skeletal Muscle/metabolism ; Transduction, Genetic ; },
abstract = {CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7[+] MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7[+] MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin[+] cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.},
}
@article {pmid31320398,
year = {2019},
author = {Widom, JR and Rai, V and Rohlman, CE and Walter, NG},
title = {Versatile transcription control based on reversible dCas9 binding.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {11},
pages = {1457-1469},
pmid = {31320398},
issn = {1469-9001},
support = {K99 GM120457/GM/NIGMS NIH HHS/United States ; R00 GM120457/GM/NIGMS NIH HHS/United States ; R01 GM122803/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/enzymology ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/*metabolism ; Catalysis ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/enzymology ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Templates, Genetic ; *Transcription, Genetic ; },
abstract = {The ability to control transcription in a time-dependent manner in vitro promises numerous applications in molecular biology and nanotechnology. Here we demonstrate an approach that enables precise, independent control over the production of multiple RNA transcripts in vitro using single guide RNA (sgRNA)-directed transcription blockades by catalytically dead Streptococcus pyogenes CRISPR-Cas9 enzyme (dCas9). We show that when bound to a DNA template, the dCas9:sgRNA complex forms a robust blockade to transcription by RNA polymerases (RNAPs) from bacteriophages SP6, T3, and T7 (>99.5% efficiency), and a partial blockade to transcription by Escherichia coli RNAP (∼70% efficiency). We find that all three bacteriophage RNAPs dissociate from the DNA template upon encountering the dCas9 blockade, while E. coli RNAP stays bound for at least the 90-min duration of our experiments. The blockade maintains >95% efficiency when four mismatches are introduced into the 5' end of the sgRNA target sequence. Notably, when using such a mismatched blockade, production of specific RNA species can be activated on demand by addition of a double-stranded competitor DNA perfectly matching the sgRNA. This strategy enables the independent production of multiple RNA species in a temporally controlled fashion from the same DNA template, demonstrating a new approach for transcription control.},
}
@article {pmid31303383,
year = {2019},
author = {Vredevoogd, DW and Kuilman, T and Ligtenberg, MA and Boshuizen, J and Stecker, KE and de Bruijn, B and Krijgsman, O and Huang, X and Kenski, JCN and Lacroix, R and Mezzadra, R and Gomez-Eerland, R and Yildiz, M and Dagidir, I and Apriamashvili, G and Zandhuis, N and van der Noort, V and Visser, NL and Blank, CU and Altelaar, M and Schumacher, TN and Peeper, DS},
title = {Augmenting Immunotherapy Impact by Lowering Tumor TNF Cytotoxicity Threshold.},
journal = {Cell},
volume = {178},
number = {3},
pages = {585-599.e15},
doi = {10.1016/j.cell.2019.06.014},
pmid = {31303383},
issn = {1097-4172},
mesh = {Animals ; Apoptosis/drug effects ; CD8-Positive T-Lymphocytes/cytology/*immunology/metabolism ; Cell Line, Tumor ; Humans ; *Immunotherapy ; Inhibitor of Apoptosis Proteins/metabolism ; Interferon-gamma/metabolism ; Kaplan-Meier Estimate ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasms/mortality/therapy ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; Receptors, Interferon/deficiency/genetics ; Signal Transduction/drug effects ; TNF Receptor-Associated Factor 2/deficiency/genetics ; Tumor Necrosis Factor-alpha/*metabolism/pharmacology ; Interferon gamma Receptor ; },
abstract = {New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.},
}
@article {pmid31273916,
year = {2019},
author = {Hu, J and Li, S and Li, Z and Li, H and Song, W and Zhao, H and Lai, J and Xia, L and Li, D and Zhang, Y},
title = {A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize.},
journal = {Molecular plant pathology},
volume = {20},
number = {10},
pages = {1463-1474},
pmid = {31273916},
issn = {1364-3703},
mesh = {Mutagenesis ; Plant Viruses/*genetics/physiology ; Plants, Genetically Modified/*metabolism/*virology ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Triticum/metabolism/virology ; Zea mays/metabolism/virology ; },
abstract = {Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.},
}
@article {pmid31271518,
year = {2019},
author = {Chen, R and Huang, H and Liu, H and Xi, J and Ning, J and Zeng, W and Shen, C and Zhang, T and Yu, G and Xu, Q and Chen, X and Wang, J and Lu, F},
title = {Friend or Foe? Evidence Indicates Endogenous Exosomes Can Deliver Functional gRNA and Cas9 Protein.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {15},
number = {38},
pages = {e1902686},
doi = {10.1002/smll.201902686},
pmid = {31271518},
issn = {1613-6829},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; Cell Line, Tumor ; DNA, Viral/genetics ; Exosomes/*metabolism ; Gene Editing ; HeLa Cells ; Hepatitis B virus/genetics ; Humans ; Papillomaviridae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/associated nuclease (Cas) system is an efficient gene editing tool. In this study, it is found that both single guide RNA (gRNA) and Cas9 protein could be exported from the CRISPR/Cas9-expressing cells by endogenous exosomes independently. Further experiments demonstrate that these naturally produced endogenous exosomes could be used as a vehicle to deliver the functional Cas9 and hepatitis B virus (HBV)-specific gRNA to cut HBV DNA transfected in HuH7 cells or human papilloma virus (HPV)-specific gRNA to cut the integrated HPV DNA in HeLa cells, respectively. In conclusion, this study indicates the potential of endogenous exosomes as a safe and effective delivery vehicle of the functional gRNA and Cas9 protein. Meanwhile, the endogenous exosomes-mediated delivery of gene editing activity to adjacent and distant cells or tissues may further complicate the off-target and safety concerns about the CRISPR/Cas9 system.},
}
@article {pmid31235883,
year = {2019},
author = {Kim, JH and Rege, M and Valeri, J and Dunagin, MC and Metzger, A and Titus, KR and Gilgenast, TG and Gong, W and Beagan, JA and Raj, A and Phillips-Cremins, JE},
title = {LADL: light-activated dynamic looping for endogenous gene expression control.},
journal = {Nature methods},
volume = {16},
number = {7},
pages = {633-639},
pmid = {31235883},
issn = {1548-7105},
support = {DP2 MH110247/MH/NIMH NIH HHS/United States ; U01 HL129998/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Carrier Proteins/genetics ; Cells, Cultured ; DNA-Binding Proteins ; *Gene Expression Regulation ; Kruppel-Like Factor 4 ; Kruppel-Like Transcription Factors/genetics ; Light ; Male ; Mice ; Nerve Tissue Proteins/genetics ; Promoter Regions, Genetic ; *Protein Engineering ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.},
}
@article {pmid31215123,
year = {2019},
author = {Liu, J and Chang, J and Jiang, Y and Meng, X and Sun, T and Mao, L and Xu, Q and Wang, M},
title = {Fast and Efficient CRISPR/Cas9 Genome Editing In Vivo Enabled by Bioreducible Lipid and Messenger RNA Nanoparticles.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {31},
number = {33},
pages = {e1902575},
pmid = {31215123},
issn = {1521-4095},
support = {21621062//National Natural Science Foundation of China/ ; 21435007//National Natural Science Foundation of China/ ; R21 EB024041/EB/NIBIB NIH HHS/United States ; 21790391//National Natural Science Foundation of China/ ; 21778056//National Natural Science Foundation of China/ ; 2017YFA0208100//National Key Research and Development Program of China/ ; 2016YFA0200104//National Key Research and Development Program of China/ ; NIH R21 EB024041/GF/NIH HHS/United States ; 21790390//National Natural Science Foundation of China/ ; UG3 TR002636/TR/NCATS NIH HHS/United States ; UG3 TR002636-01/GF/NIH HHS/United States ; },
mesh = {Animals ; Biological Transport ; CRISPR-Associated Protein 9/*genetics ; Cell Line, Tumor ; Gene Editing/*methods ; Gene Knockdown Techniques/methods ; Gene Transfer Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Lipids/*chemistry ; Mice ; Nanoparticles/*chemistry ; Oxidation-Reduction ; Proprotein Convertase 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/*chemistry ; RNA, Messenger/administration &amp; dosage/*chemistry ; },
abstract = {A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA-O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA-O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.},
}
@article {pmid31110232,
year = {2019},
author = {Duan, B and Zhou, C and Zhu, C and Yu, Y and Li, G and Zhang, S and Zhang, C and Ye, X and Ma, H and Qu, S and Zhang, Z and Wang, P and Sun, S and Liu, Q},
title = {Model-based understanding of single-cell CRISPR screening.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2233},
pmid = {31110232},
issn = {2041-1723},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computational Biology/methods ; Datasets as Topic ; Feasibility Studies ; Gene Expression Profiling/methods ; Gene Knockdown Techniques ; Humans ; Jurkat Cells ; K562 Cells ; *Models, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/*methods ; },
abstract = {The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data.},
}
@article {pmid31103783,
year = {2019},
author = {Wang, X and Ma, S and Liu, Y and Lu, W and Sun, L and Zhao, P and Xia, Q},
title = {Transcriptional repression of endogenous genes in BmE cells using CRISPRi system.},
journal = {Insect biochemistry and molecular biology},
volume = {111},
number = {},
pages = {103172},
doi = {10.1016/j.ibmb.2019.05.007},
pmid = {31103783},
issn = {1879-0240},
mesh = {Animals ; Bombyx/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression ; Gene Silencing ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems ; *Transcription, Genetic ; },
abstract = {Recent advancements in genetic engineering technology have led to the development of CRISPR interference (CRISPRi) as a precise tool for regulating gene expression. When CRISPR/dCas9 is fused with transcriptional repressors, the system can robustly silence endogenous gene expression. The CRISPR/Cas9 tool is a promising alternative in organisms (e.g., Bombyx mori) that do not respond to traditional gene suppression techniques, such as RNA interference (RNAi). However, transcriptional repressors remain poorly categorized in multiple cell types and species, leading to difficulties in optimizing performance and efficiency. Here, we tested CRISPRi usability and efficiency in Bombyx mori cells (BmE). We fused dCas9 to five transcriptional repressors including KRAB, Hairy, SID, SRDX, and ERD. All five constructs were efficient in BmE cells. In a proof-of-concept experiment, we showed that CRISPRi acting on BmSoxE (a gene involved in cell proliferation) could generate similar phenotypes as RNAi gene suppression. Moreover, CRISPRi has fewer off-target effects. Through co-transfection of BmE cells with sgRNAs, we also demonstrated that dCas9 could simultaneously repress the expression of multiple genes. Furthermore, we identified sgRNA distance from transcriptional start site (TSS) and the dCas9: sgRNA ratio as the two limiting factors of CRISPRi efficiency. Our results demonstrated that CRISPR/dCas9 is a viable and rapid alternative for functional investigations of the B. mori genome and perhaps other Lepidoptera insects.},
}
@article {pmid31064995,
year = {2019},
author = {Oesinghaus, L and Simmel, FC},
title = {Switching the activity of Cas12a using guide RNA strand displacement circuits.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2092},
pmid = {31064995},
issn = {2041-1723},
support = {694410//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/International ; 031L0011//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/International ; },
mesh = {Acidaminococcus/genetics ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/*genetics/metabolism ; Escherichia coli/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Transformation, Bacterial ; },
abstract = {The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.},
}
@article {pmid31059138,
year = {2019},
author = {Gao, Q and Xu, WY and Yan, T and Fang, XD and Cao, Q and Zhang, ZJ and Ding, ZH and Wang, Y and Wang, XB},
title = {Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.},
journal = {The New phytologist},
volume = {223},
number = {4},
pages = {2120-2133},
doi = {10.1111/nph.15889},
pmid = {31059138},
issn = {1469-8137},
mesh = {Animals ; Base Sequence ; DNA, Complementary/genetics ; Edible Grain/*genetics/*virology ; Gene Editing ; Genetic Vectors/metabolism ; *Genome, Plant ; *Genomics ; Glucuronidase/metabolism ; Hemiptera/*virology ; Hordeum/ultrastructure/virology ; Plant Leaves/virology ; Plant Viruses/*physiology/ultrastructure ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Rhabdoviridae/*physiology/ultrastructure ; Nicotiana/ultrastructure/virology ; },
abstract = {Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid β-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.},
}
@article {pmid30995470,
year = {2019},
author = {Genga, RMJ and Kernfeld, EM and Parsi, KM and Parsons, TJ and Ziller, MJ and Maehr, R},
title = {Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development.},
journal = {Cell reports},
volume = {27},
number = {3},
pages = {708-718.e10},
pmid = {30995470},
issn = {2211-1247},
support = {DP3 DK111898/DK/NIDDK NIH HHS/United States ; R01 AI132963/AI/NIAID NIH HHS/United States ; U01 DK104218/DK/NIDDK NIH HHS/United States ; },
mesh = {Cell Differentiation ; Chromatin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endoderm/cytology/metabolism ; Hepatocyte Nuclear Factor 3-beta/antagonists &amp; inhibitors/genetics/metabolism ; Human Embryonic Stem Cells/cytology/metabolism ; Humans ; Pluripotent Stem Cells/cytology/metabolism ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; SOXF Transcription Factors/genetics/metabolism ; Signal Transduction ; Single-Cell Analysis ; Smad4 Protein/genetics/metabolism ; Transcription Factors/antagonists &amp; inhibitors/genetics/*metabolism ; Transforming Growth Factor beta/metabolism ; },
abstract = {Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFβ signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.},
}
@article {pmid30985763,
year = {2019},
author = {Luo, H and Sobh, A and Vulpe, CD and Brewer, E and Dovat, S and Qiu, Y and Huang, S},
title = {HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {145},
pages = {},
pmid = {30985763},
issn = {1940-087X},
support = {R01 CA204044/CA/NCI NIH HHS/United States ; R01 DK110108/DK/NIDDK NIH HHS/United States ; R01 HL141950/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CCCTC-Binding Factor/*metabolism ; Chromatin ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Gene Expression Regulation ; Gene Library ; *Genes, Homeobox ; Humans ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.},
}
@article {pmid30940799,
year = {2019},
author = {Knapp, DJHF and Michaels, YS and Jamilly, M and Ferry, QRV and Barbosa, H and Milne, TA and Fulga, TA},
title = {Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1490},
pmid = {30940799},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; MC_UU_00016/6/MRC_/Medical Research Council/United Kingdom ; MC_UU_12009/6/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; Gene Editing ; Gene Expression Regulation ; Humans ; Nucleic Acid Conformation ; Promoter Regions, Genetic ; RNA Polymerase II/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/chemistry/*genetics/metabolism ; RNA, Transfer/chemistry/*genetics/metabolism ; },
abstract = {Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5', and 3' processing efficiencies across a library of tRNA variants. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.},
}
@article {pmid30912053,
year = {2019},
author = {Skipper, KA and Mikkelsen, JG},
title = {Toward In Vivo Gene Therapy Using CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {293-306},
doi = {10.1007/978-1-4939-9170-9_18},
pmid = {30912053},
issn = {1940-6029},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Dependovirus/genetics ; *Gene Editing ; Genetic Therapy/*methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {CRISPR, a revolutionizing technology allowing researchers to navigate in and edit the genome, is moving on the fast track toward clinical use for ex vivo correction of disease-causing mutations in stem cells. As we await the first trials utilizing ex vivo CRISPR editing, implementation of CRISPR-based gene editing as an in vivo treatment directly in patients still remains an ultimate challenge. However, quickly accumulating evidence has provided proof-of-concept for efficacious editing in vivo. Attempts to edit genes directly in animals have largely relied on classical vector systems based on virus-based delivery of gene cassettes encoding the Cas9 endonuclease and single guide RNA, the key components of the CRISPR system. However, whereas persistent gene expression has been the primary goal of gene therapy for decades, things may be different in the case of CRISPR delivery. Is short-term presence of the CRISPR components perhaps sufficient for efficacy and ideal for safety?-and are strategies needed for restricting immune recognition of the bacteria-derived editing tool? Here, while answers to these questions still blow in the wind, we review prominent examples of genome editing with focus on targeting of genes with CRISPR in liver, muscles, and eyes of the mouse.},
}
@article {pmid30912043,
year = {2019},
author = {Fakhiri, J and Nickl, M and Grimm, D},
title = {Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (AAV) Vectors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {111-126},
doi = {10.1007/978-1-4939-9170-9_8},
pmid = {30912043},
issn = {1940-6029},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Dependovirus/*genetics ; Gene Editing ; Genetic Vectors/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Genome editing reagents including the recently introduced CRISPR/Cas9 system have become established and widely used molecular tools to answer fundamental biological questions and to target and treat genetic diseases. The CRISPR system, originally derived from bacteria and archaea, can be delivered into cells using different techniques, comprising (1) transfection of mRNA or plasmid DNA, (2) electroporation of plasmid DNA or the Cas9 protein in a complex with a g(uide)RNA, or (3) use of nonviral or viral vectors. Among the latter, Adeno-associated viruses (AAVs) are particularly attractive owing to many favorable traits: (1) their apathogenicity and episomal persistence, (2) the ease of virus production and purification, (3) the safe handling under lowest biosafety level 1 conditions, and (4) the availability of numerous natural serotypes and synthetic capsid variants with distinct cell specificities. Here, we describe a fast and simple protocol for small-scale packaging of CRISPR/Cas9 components into AAV vectors. To showcase its potential, we employ this method for screening of gRNAs targeting the murine miR-122 locus in Hepa1-6 cells (using AAV serotype 6, AAV6) or the 5'LTR of the human immunodeficiency virus (HIV) in HeLaP4-NLtr cells (using a synthetic AAV9 variant). We furthermore provide a detailed protocol for large-scale production of purified AAV/CRISPR vector stocks that permit higher cleavage efficiencies in vitro and are suitable for direct in vivo applications.},
}
@article {pmid30912040,
year = {2019},
author = {Lin, L and Luo, Y},
title = {Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {67-77},
doi = {10.1007/978-1-4939-9170-9_5},
pmid = {30912040},
issn = {1940-6029},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Breaks, Double-Stranded ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. Thus, experimentally validating gRNA activity is still the gold standard in defining the best gRNAs for gene editing experiments. One such approach for experimentally selecting gRNAs with high activity is fluorescent surrogate reporter vectors. We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.},
}
@article {pmid30912036,
year = {2019},
author = {Pallarès Masmitjà, M and Knödlseder, N and Güell, M},
title = {CRISPR-gRNA Design.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {3-11},
doi = {10.1007/978-1-4939-9170-9_1},
pmid = {30912036},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Gene editing has great therapeutic impact, being of interest for many scientists worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has been adapted for gene editing to serve as an efficient, rapid, and cost-effective tool. To fulfill CRISPR experiment's goals, two components are important: an endonuclease and a gRNA. The most commonly used endonucleases are Cpf1 and Cas9 and are described in depth in this chapter. The gRNA targets the genome site to be edited, giving great importance to its design to obtain increased efficiency and decreased off-target events. In this chapter, we describe different tools to design suitable gRNAs for a variety of experimental purposes.},
}
@article {pmid30871638,
year = {2019},
author = {Fukushima, HS and Takeda, H and Nakamura, R},
title = {Targeted in vivo epigenome editing of H3K27me3.},
journal = {Epigenetics &amp; chromatin},
volume = {12},
number = {1},
pages = {17},
pmid = {30871638},
issn = {1756-8935},
mesh = {Animals ; Embryo, Nonmammalian ; Enhancer of Zeste Homolog 2 Protein/*metabolism ; *Epigenesis, Genetic ; Fish Proteins/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Developmental ; Histones/*metabolism ; Methylation ; Oryzias/genetics/*metabolism ; *Protein Processing, Post-Translational ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Epigenetic modifications have a central role in transcriptional regulation. While several studies using next-generation sequencing have revealed genome-wide associations between epigenetic modifications and transcriptional states, a direct causal relationship at specific genomic loci has not been fully demonstrated, due to a lack of technology for targeted manipulation of epigenetic modifications. Recently, epigenome editing techniques based on the CRISPR-Cas9 system have been reported to directly manipulate specific modifications at precise genomic regions. However, the number of editable modifications as well as studies applying these techniques in vivo is still limited.<br><br>RESULTS: Here, we report direct modification of the epigenome in medaka (Japanese killifish, Oryzias latipes) embryos. Specifically, we developed a method to ectopically induce the repressive histone modification, H3K27me3 in a locus-specific manner, using a fusion construct of Oryzias latipes H3K27 methyltransferase Ezh2 (olEzh2) and dCas9 (dCas9-olEzh2). Co-injection of dCas9-olEzh2 mRNA with single guide RNAs (sgRNAs) into one-cell-stage embryos induced specific H3K27me3 accumulation at the targeted loci and induced downregulation of gene expression.<br><br>CONCLUSION: In this study, we established the in vivo epigenome editing of H3K27me3 using medaka embryos. The locus-specific manipulation of the epigenome in living organisms will lead to a previously inaccessible understanding of the role of epigenetic modifications in development and disease.},
}
@article {pmid30866794,
year = {2019},
author = {Merritt, BB and Cheung, LS},
title = {GRIBCG: a software for selection of sgRNAs in the design of balancer chromosomes.},
journal = {BMC bioinformatics},
volume = {20},
number = {1},
pages = {122},
pmid = {30866794},
issn = {1471-2105},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; Chromosome Inversion ; Chromosomes, Insect/*genetics ; Drosophila/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics/*isolation &amp; purification ; *Software ; Streptococcus pyogenes ; },
abstract = {BACKGROUND: Balancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research.<br><br>RESULTS: Here, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes.<br><br>CONCLUSION: GRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.},
}
@article {pmid30846198,
year = {2019},
author = {Moon, SB and Kim, DY and Ko, JH and Kim, JS and Kim, YS},
title = {Improving CRISPR Genome Editing by Engineering Guide RNAs.},
journal = {Trends in biotechnology},
volume = {37},
number = {8},
pages = {870-881},
doi = {10.1016/j.tibtech.2019.01.009},
pmid = {30846198},
issn = {1879-3096},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Endoribonucleases/*metabolism ; Gene Editing/*methods/trends ; Genetic Engineering/*methods/trends ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Substrate Specificity ; },
abstract = {CRISPR technology is a two-component gene editing system in which the effector protein induces genetic alterations with the aid of a gene targeting guide RNA. Guide RNA can be produced through chemical synthesis, in vitro transcription, or intracellular transcription. Guide RNAs can be engineered to have chemical modifications, alterations in the spacer length, sequence modifications, fusion of RNA or DNA components, and incorporation of deoxynucleotides. Engineered guide RNA can improve genome editing efficiency and target specificity, regulation of biological toxicity, sensitive and specific molecular imaging, multiplexing, and editing flexibility. Therefore, engineered guide RNA will enable more specific, efficient, and safe gene editing, ultimately improving the clinical benefits of gene therapy.},
}
@article {pmid30833658,
year = {2019},
author = {Kim, D and Kim, DE and Lee, G and Cho, SI and Kim, JS},
title = {Genome-wide target specificity of CRISPR RNA-guided adenine base editors.},
journal = {Nature biotechnology},
volume = {37},
number = {4},
pages = {430-435},
pmid = {30833658},
issn = {1546-1696},
mesh = {Adenine/*metabolism ; Aminohydrolases/metabolism ; Animals ; Base Sequence ; Binding Sites/genetics ; Biotechnology ; CRISPR-Associated Protein 9/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine Deaminase/metabolism ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Whole Genome Sequencing ; },
abstract = {Adenine base editors[1] enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants[1-4]. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing[5-8], to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites containing inosines, the products of adenine deamination, we treat human genomic DNA with an adenine base editor 7.10 protein-guide RNA complex and either endonuclease V or a combination of human alkyladenine DNA glycosylase and endonuclease VIII in vitro. Digenome-seq detects adenine base editor off-target sites with a substitution frequency of 0.1% or more. We show that adenine base editor 7.10, the cytosine base editor BE3, and unmodified CRISPR-associated protein 9 (Cas9) often recognize different off-target sites, highlighting the need for independent assessments of their genome-wide specificities[6]. Using targeted sequencing, we also show that use of preassembled adenine base editor ribonucleoproteins, modified guide RNAs[5,8-11], and Sniper/Cas9 (ref. [12]) reduces adenine base editor off-target activity in human cells.},
}
@article {pmid30820813,
year = {2019},
author = {Hirata, M and Tanihara, F and Wittayarat, M and Hirano, T and Nguyen, NT and Le, QA and Namula, Z and Nii, M and Otoi, T},
title = {Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes.},
journal = {In vitro cellular &amp; developmental biology. Animal},
volume = {55},
number = {4},
pages = {237-242},
pmid = {30820813},
issn = {1543-706X},
mesh = {Animals ; Blastocyst/metabolism ; CRISPR-Associated Protein 9/*metabolism ; *Cell Differentiation ; Electroporation/*methods ; Embryonic Development ; *Gene Editing ; *Genome ; Mutation/*genetics ; Mutation Rate ; Oocytes/*metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Swine ; Zygote/*metabolism ; },
abstract = {The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.},
}
@article {pmid30816279,
year = {2019},
author = {Zhang, Y and Ozono, S and Yao, W and Tobiume, M and Yamaoka, S and Kishigami, S and Fujita, H and Tokunaga, K},
title = {CRISPR-mediated activation of endogenous BST-2/tetherin expression inhibits wild-type HIV-1 production.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3134},
pmid = {30816279},
issn = {2045-2322},
mesh = {Antigens, CD/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; GPI-Linked Proteins/genetics ; Gene Expression ; Gene Targeting ; HEK293 Cells ; HIV Infections/*genetics/pathology/virology ; HIV-1/*physiology ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Transcriptional Activation ; *Virus Replication ; },
abstract = {The CRISPR technology not only can knock out target genes by using the RNA-guided Cas9 nuclease but also can activate their expression when a nuclease-deficient Cas9 (dCas9) is employed. Using the latter function, we here show the effect of the CRISPR-mediated pinpoint activation of endogenous expression of BST-2 (also known as tetherin), a virus restriction factor with a broad antiviral spectrum. Single-guide RNA (sgRNA) sequences targeting the BST-2 promoter were selected by promoter assays. Potential sgRNAs and dCas9 fused to the VP64 transactivation domain, along with an accessory transcriptional activator complex, were introduced into cells by lentiviral transduction. Increased expression of BST-2 mRNA in transduced cells was confirmed by real-time RT-PCR. Cells in which BST-2 expression was highly enhanced showed the effective inhibition of HIV-1 production and replication even in the presence of the viral antagonist Vpu against BST-2. These findings confirm that the physiological stoichiometry between host restriction factors and viral antagonists may determine the outcome of the battle with viruses.},
}
@article {pmid30705318,
year = {2019},
author = {Kanavarioti, A},
title = {HPLC methods for purity evaluation of man-made single-stranded RNAs.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1019},
pmid = {30705318},
issn = {2045-2322},
support = {R43 HG010051/HG/NHGRI NIH HHS/United States ; },
mesh = {Chromatography, High Pressure Liquid/*methods ; Nucleic Acid Conformation ; Nucleic Acid Denaturation ; RNA/*analysis/chemistry ; RNA Stability ; RNA, Guide, CRISPR-Cas Systems/analysis/chemistry ; RNA, Messenger/analysis/chemistry ; RNA, Transfer/analysis/chemistry ; },
abstract = {Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences.},
}
@article {pmid30683138,
year = {2019},
author = {Fortin, JP and Tan, J and Gascoigne, KE and Haverty, PM and Forrest, WF and Costa, MR and Martin, SE},
title = {Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {21},
pmid = {30683138},
issn = {1474-760X},
mesh = {Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Targeting ; Genomics/*methods ; Humans ; Myosin Light Chains/genetics ; Neoplasms/genetics ; *RNA, Guide, CRISPR-Cas Systems ; SOX9 Transcription Factor/genetics ; SOXE Transcription Factors/genetics ; },
abstract = {BACKGROUND: Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed.<br><br>RESULTS: In this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We find that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks. Lastly, we also find that single nucleotide polymorphisms located in protospacer regions can impair on-target activity as a result of mismatch tolerance.<br><br>CONCLUSION: We show the impact of multi-target effects on estimating cancer cell dependencies and the impact of off-target effects caused by mismatch tolerance in sgRNA-DNA binding.},
}
@article {pmid30635953,
year = {2019},
author = {Nakamura, S and Ishihara, M and Ando, N and Watanabe, S and Sakurai, T and Sato, M},
title = {Transplacental delivery of genome editing components causes mutations in embryonic cardiomyocytes of mid-gestational murine fetuses.},
journal = {IUBMB life},
volume = {71},
number = {7},
pages = {835-844},
doi = {10.1002/iub.2004},
pmid = {30635953},
issn = {1521-6551},
mesh = {Animals ; CRISPR-Associated Protein 9/administration &amp; dosage/genetics ; Cells, Cultured ; Female ; Fetus/*physiology ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors ; Genome ; Green Fluorescent Proteins/*genetics ; Heart/*embryology/physiology ; Male ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; *Mutation ; Myocytes, Cardiac/cytology/*physiology ; Plasmids ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/*genetics ; Transgenes ; },
abstract = {Genome editing, as exemplified by CRISPR/Cas9, is now recognized as a powerful tool for the engineering of endogenous target genes. It employs only two components, namely, Cas9 in the form of DNA, mRNA, or protein; and guide RNA (gRNA), which is specific to a target gene. When these components are transferred to cells, they create insertion/deletion mutations (indels) within a target gene. Therefore, when fetuses within the uteri of pregnant murine females are exposed to these reagents, fetal cells incorporating them should show mutations in the target gene. To examine a possible genome editing of fetal cells in vivo, we intravenously administered a solution containing plasmid DNA-FuGENE complex to pregnant wild-type female mice [which had been successfully mated with enhanced green fluorescent protein (EGFP)-expressing male transgenic mice] on day 12.5 of gestation. The plasmid DNA induces the expression of gRNA, which was targeted at the EGFP cDNA, and that of the Cas9 gene. All fetuses in the pregnant females should express EGFP systemically, since they are heterozygous (Tg/+) for the transgene. Thus, the delivery of CRISPR system targeted at EGFP in the fetuses will cause a reduced expression of EGFP as a result of the genome editing of EGFP genomic sequence. Of the 24 fetuses isolated from three pregnant females 2 days after gene delivery, 3 were found to have reduced fluorescence in their hearts. Genotyping of the dissected hearts revealed the presence of the transgene construct (Cas9 gene) in all the samples. Furthermore, all the three samples exhibited mutations at the target loci, although normal cells were also present. Thus, transplacental delivery of gene editing components may be a useful tool for developing animal models with heart disorder for heart-related disease research, and gene therapy in congenital heart defects such as hypertrophic cardiomyopathy (HCM). © 2019 IUBMB Life, 9999(9999):1-10, 2019.},
}
@article {pmid30565136,
year = {2019},
author = {Quinton, RJ and Ganem, NJ},
title = {CRISPR-Mediated Approaches to Regulate YAP/TAZ Levels.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1893},
number = {},
pages = {203-214},
pmid = {30565136},
issn = {1940-6029},
support = {R01 GM117150/GM/NIGMS NIH HHS/United States ; //CIHR/Canada ; },
mesh = {Acyltransferases ; Cell Cycle Proteins ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Genetic Vectors ; Hippo Signaling Pathway ; Humans ; Nuclear Proteins/*genetics/metabolism ; Protein Serine-Threonine Kinases/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Signal Transduction ; Transcription Factors/*genetics/metabolism ; },
abstract = {The advent of CRISPR has revolutionized genomic engineering, and harnessing its power to regulate levels of the transcriptional co-activators YAP and TAZ represents an exciting new opportunity in the field of Hippo signaling. Initially repurposed from the microbial immune system to perform highly specific gene knockouts, CRISPR technology has now been expanded to modulate the transcriptional activity of any gene of interest in mammalian systems. Here, we describe strategies to employ CRISPR to genetically knock out the genes encoding for YAP (YAP1) or TAZ (WWTR1) in mammalian cell lines, as well as briefly outline an approach for utilizing CRISPR to transcriptionally modulate YAP/TAZ levels.},
}
@article {pmid30521567,
year = {2018},
author = {Danilo, B and Perrot, L and Botton, E and Nogué, F and Mazier, M},
title = {The DFR locus: A smart landing pad for targeted transgene insertion in tomato.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0208395},
pmid = {30521567},
issn = {1932-6203},
mesh = {Agrobacterium/genetics ; Anthocyanins/biosynthesis ; *Gene Editing ; Solanum lycopersicum/*anatomy &amp; histology/*genetics/growth &amp; development/microbiology ; Plant Proteins/*genetics ; Plants, Genetically Modified/anatomy &amp; histology/growth &amp; development/microbiology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Deletion ; Transformation, Bacterial ; Transgenes ; },
abstract = {Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.},
}
@article {pmid30518686,
year = {2018},
author = {Hakim, CH and Wasala, NB and Nelson, CE and Wasala, LP and Yue, Y and Louderman, JA and Lessa, TB and Dai, A and Zhang, K and Jenkins, GJ and Nance, ME and Pan, X and Kodippili, K and Yang, NN and Chen, SJ and Gersbach, CA and Duan, D},
title = {AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.},
journal = {JCI insight},
volume = {3},
number = {23},
pages = {},
pmid = {30518686},
issn = {2379-3708},
support = {R01 AR069085/AR/NIAMS NIH HHS/United States ; R01 GM063732/GM/NIGMS NIH HHS/United States ; R01 GM117059/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus ; Disease Models, Animal ; Dystrophin/*genetics ; Female ; Fibrosis ; *Gene Editing ; Genetic Therapy ; Genetic Vectors ; Male ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Duchenne/*genetics/pathology ; Mutation ; Myocardium/metabolism/pathology ; Neuromuscular Diseases ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.},
}
@article {pmid30458109,
year = {2018},
author = {Hand, TH and Das, A and Roth, MO and Smith, CL and Jean-Baptiste, UL and Li, H},
title = {Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity.},
journal = {ACS synthetic biology},
volume = {7},
number = {12},
pages = {2908-2917},
pmid = {30458109},
issn = {2161-5063},
support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; },
mesh = {Actinomycetales/*metabolism ; Base Pair Mismatch ; CRISPR-Associated Protein 9/chemistry/genetics/*metabolism ; Directed Molecular Evolution ; Gene Editing/*methods ; Mutagenesis ; Phosphates/*chemistry ; Plasmids/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/isolation &amp; purification/metabolism ; Recombinant Proteins/biosynthesis/chemistry/isolation &amp; purification ; Substrate Specificity ; },
abstract = {Despite being utilized widely in genome sciences, CRISPR-Cas9 remains limited in achieving high fidelity in cleaving DNA. A better understanding of the molecular basis of Cas9 holds the key to improve Cas9-based tools. We employed direct evolution and in vitro characterizations to explore structural parameters that impact the specificity of the thermophilic Cas9 from Acidothermus cellulolyticus (AceCas9). By identifying variants that are able to cleave mismatched protospacers within the seed region, we found a critical role of the phosphate lock residues in substrate specificity in a manner that depends on their sizes and charges. Removal of the negative charge from the phosphate lock residues significantly decreases sensitivity to the guide-DNA mismatches. An increase in size of the substituted residues further reduces the sensitivity to mismatches at the first position of the protospacer. Our findings identify the phosphate lock residues as an important site for tuning the specificity and catalytic efficiency of Cas9.},
}
@article {pmid30394729,
year = {2018},
author = {Sago, CD and Lokugamage, MP and Islam, FZ and Krupczak, BR and Sato, M and Dahlman, JE},
title = {Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution.},
journal = {Journal of the American Chemical Society},
volume = {140},
number = {49},
pages = {17095-17105},
pmid = {30394729},
issn = {1520-5126},
support = {R01 DE026941/DE/NIDCR NIH HHS/United States ; T32 EB021962/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD/genetics ; Bone Marrow/*metabolism ; Cell Adhesion Molecules/genetics ; Computational Biology ; Directed Molecular Evolution ; Drug Carriers/*chemistry/metabolism ; Endothelial Cells/metabolism ; Gene Silencing ; Mice ; Nanoparticles/*chemistry/metabolism ; Phosphatidylcholines/chemistry/metabolism ; Polyethylene Glycols/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/*pharmacology ; RNA, Small Interfering/genetics/*pharmacology ; },
abstract = {Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g., CRISPR) therapies. However, siRNA and sgRNA have not been delivered to BMECs using systemically administered nanoparticles. Given that in vitro nanoparticle screens have not identified nanoparticles with BMEC tropism, we developed a system to quantify how >100 different nanoparticles deliver siRNA in a single mouse. This is the first barcoding system capable of quantifying functional cytosolic siRNA delivery (where the siRNA drug is active), distinguishing it from in vivo screens that quantify biodistribution (where the siRNA drug went). Combining this approach with bioinformatics, we performed in vivo directed evolution, and identified BM1, a lipid nanoparticle (LNP) that delivers siRNA and sgRNA to BMECs. Interestingly, chemical analysis revealed BMEC tropism was not related to LNP size; tropism changed with the structure of poly(ethylene glycol), as well as the presence of cholesterol. These results suggest that significant changes to vascular targeting can be imparted to a LNP by making simple changes to its chemical composition, rather than using active targeting ligands. BM1 is the first nanoparticle to efficiently deliver siRNA and sgRNA to BMECs in vivo, demonstrating that this functional in vivo screen can identify nanoparticles with novel tropism in vivo. More generally, in vivo screening may help reveal the complex relationship between nanoparticle structure and tropism, thereby helping scientists understand how simple chemical changes control nanoparticle targeting.},
}
@article {pmid30365048,
year = {2019},
author = {Tang, B and Zhang, Y and Zhang, W and Zhu, Y and Yuan, S},
title = {Deletion of FOXL2 by CRISPR promotes cell cycle G0/G1 restriction in KGN cells.},
journal = {International journal of molecular medicine},
volume = {43},
number = {1},
pages = {567-574},
doi = {10.3892/ijmm.2018.3956},
pmid = {30365048},
issn = {1791-244X},
mesh = {Apoptosis ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; *Cell Cycle ; Cell Line ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Forkhead Box Protein L2/*metabolism ; *Gene Deletion ; Humans ; RNA Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Reproducibility of Results ; },
abstract = {Forkhead box L2 (FOXL2), a member of the forkhead family of transcription factors, is important in eyelid and ovary differentiation. Although the function of FOXL2 in organogenesis has been investigated, the detailed mechanisms by which FOXL2 mediates cellular process remain to be fully elucidated. Few FOXL2‑knockout cell lines have been reported, which has limited molecular mechanism investigations. CRISPR is a novel gene editing technique that has been widely used in human genetic diseases. In the present study, FOXL2 was disrupted using clustered regularly interspaced short palindromic repeats (CRISPR), and screening of a stable knockout cell line was performed in human ovarian granulosa KGN cells. Three sites (F404, F425 and F446) around the ATG start codon on the FOXL2 DNA sequence were constructed in a guide RNA lentivirus. Targeting F425 was most efficient, and western blot analysis and DNA sequencing of the resulting cells suggested that both FOXL2 alleles were fully disrupted. In addition, flow cytometry results indicated that the knockout of FOXL2 restricted cell cycle progression at the G0/G1 phase. In addition, the expression levels of cell cycle mediators cyclin D1 and cyclin‑dependent kinase 4 were reduced. These results confirmed that FOXL2 disruption in KGN cells is associated with the cell cycle attenuation.},
}
@article {pmid30346403,
year = {2018},
author = {Nageshwaran, S and Chavez, A and Cher Yeo, N and Guo, X and Lance-Byrne, A and Tung, A and Collins, JJ and Church, GM},
title = {CRISPR Guide RNA Cloning for Mammalian Systems.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {140},
pages = {},
pmid = {30346403},
issn = {1940-087X},
support = {RM1 HG008525/HG/NHGRI NIH HHS/United States ; T32 CA009216/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.},
}
@article {pmid30296940,
year = {2018},
author = {Daley, TP and Lin, Z and Lin, X and Liu, Y and Wong, WH and Qi, LS},
title = {CRISPhieRmix: a hierarchical mixture model for CRISPR pooled screens.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {159},
pmid = {30296940},
issn = {1474-760X},
support = {R01DA036858//National Institutes of Health (US)/International ; R01HG007834//National Institutes of Health (US)/International ; R01 GM109836/GM/NIGMS NIH HHS/United States ; OD017887//National Institutes of Health (US)/International ; DP5 OD017887/OD/NIH HHS/United States ; U01 EB021240/EB/NIBIB NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; U01EB021240/NH/NIH HHS/United States ; R01 HG007834/HG/NHGRI NIH HHS/United States ; R01GM109836//National Institutes of Health (US)/International ; },
mesh = {Algorithms ; Area Under Curve ; Bayes Theorem ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computer Simulation ; Databases, Genetic ; Genes, Essential ; *Genetic Testing ; *Models, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; ROC Curve ; },
abstract = {Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens. We discuss key issues in the analysis and design of screens, particularly the number of guides needed for faithful full discovery.},
}
@article {pmid30265865,
year = {2019},
author = {Galizi, R and Jaramillo, A},
title = {Engineering CRISPR guide RNA riboswitches for in vivo applications.},
journal = {Current opinion in biotechnology},
volume = {55},
number = {},
pages = {103-113},
doi = {10.1016/j.copbio.2018.08.007},
pmid = {30265865},
issn = {1879-0429},
support = {BB/M017982/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P020615/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Genetic Engineering/*methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Reproducibility of Results ; Riboswitch ; },
abstract = {CRISPR-based genome editing provides a simple and scalable toolbox for a variety of therapeutic and biotechnology applications. Whilst the fundamental properties of CRISPR proved easily transferable from the native prokaryotic hosts to eukaryotic and multicellular organisms, the tight control of the CRISPR-editing activity remains a major challenge. Here we summarise recent developments of CRISPR and riboswitch technologies and recommend novel functionalised synthetic-gRNA (sgRNA) designs to achieve inducible and spatiotemporal regulation of CRISPR-based genetic editors in response to cellular or extracellular stimuli. We believe that future advances of these tools will have major implications for both basic and applied research, spanning from fundamental genetic studies and synthetic biology to genetic editing and gene therapy.},
}
@article {pmid30252595,
year = {2019},
author = {Liao, C and Slotkowski, RA and Achmedov, T and Beisel, CL},
title = {The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays.},
journal = {RNA biology},
volume = {16},
number = {4},
pages = {404-412},
pmid = {30252595},
issn = {1555-8584},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Cleavage ; DNA, Intergenic/genetics ; Francisella/*genetics ; Nucleic Acid Conformation ; RNA Processing, Post-Transcriptional/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Rho Factor/metabolism ; Terminal Repeat Sequences/*genetics ; Transcription Termination, Genetic ; },
abstract = {The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.},
}
@article {pmid30152993,
year = {2018},
author = {Zeng, J and Teo, J and Banerjee, A and Chapman, TW and Kim, J and Sarpeshkar, R},
title = {A Synthetic Microbial Operational Amplifier.},
journal = {ACS synthetic biology},
volume = {7},
number = {9},
pages = {2007-2013},
doi = {10.1021/acssynbio.8b00138},
pmid = {30152993},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; Carboxylic Ester Hydrolases/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Synthetic Biology/*methods ; Transcription, Genetic/drug effects ; },
abstract = {Synthetic biology has created oscillators, latches, logic gates, logarithmically linear circuits, and load drivers that have electronic analogs in living cells. The ubiquitous operational amplifier, which allows circuits to operate robustly and precisely has not been built with biomolecular parts. As in electronics, a biological operational-amplifier could greatly improve the predictability of circuits despite noise and variability, a problem that all cellular circuits face. Here, we show how to create a synthetic three-stage inducer-input operational amplifier with a fast CRISPR-based differential-input push-pull stage, a slow transcription-and-translation amplification stage, and a fast-enzymatic output stage. Our "Bio-OpAmp" uses only 5 proteins including dCas9. It expands the toolkit of fundamental analog circuits in synthetic biology and provides a simple circuit motif for robust and precise molecular homeostasis.},
}
@article {pmid30138555,
year = {2018},
author = {Dominguez-Monedero, A and Davies, JA},
title = {Tamoxifen- and Mifepristone-Inducible Versions of CRISPR Effectors, Cas9 and Cpf1.},
journal = {ACS synthetic biology},
volume = {7},
number = {9},
pages = {2160-2169},
doi = {10.1021/acssynbio.8b00145},
pmid = {30138555},
issn = {2161-5063},
support = {BB/M018040/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/*genetics ; Gene Editing/*methods ; Gene Expression/*drug effects ; Genes, Reporter ; HEK293 Cells ; Humans ; Mifepristone/*pharmacology ; Mutagenesis ; Plasmids/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Receptors, Estrogen/genetics/metabolism ; Recombinant Fusion Proteins/biosynthesis/genetics ; Tamoxifen/*pharmacology ; },
abstract = {Methods for making specific modifications to the genomes of living cells are powerful research tools. Two methods currently dominate, CRISPR and Cre recombinase. CRISPR has the advantage that it can act on unmodified target genes; Cre has the advantage of being available in drug-inducible versions, allowing temporal control, but it requires engineering ("floxing") of the target gene. Here, we have combined these advantages by constructing drug (tamoxifen/mifepristone)-inducible Cas9 and Cpf1 CRISPR effectors. We demonstrate their low background activity and robust activation with drugs, by using gRNAs to target them to TetR, in a cell carrying a Tet-repressed reporter gene. As well as being useful in their own right, the research tools generated here will pave the way to making further drug-inducible effector proteins.},
}
@article {pmid30086710,
year = {2018},
author = {Blighe, K and DeDionisio, L and Christie, KA and Chawes, B and Shareef, S and Kakouli-Duarte, T and Chao-Shern, C and Harding, V and Kelly, RS and Castellano, L and Stebbing, J and Lasky-Su, JA and Nesbit, MA and Moore, CBT},
title = {Gene editing in the context of an increasingly complex genome.},
journal = {BMC genomics},
volume = {19},
number = {1},
pages = {595},
pmid = {30086710},
issn = {1471-2164},
support = {NIHR-RP-011-053/DH_/Department of Health/United Kingdom ; },
mesh = {Gene Editing/*methods ; Genetic Engineering ; Genetic Variation ; *Genome, Human ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule -and how these relate to regulation- have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin.In this review, we chart the course of the major advances in the biomedical sciences in the era pre- and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA- RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms.Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.},
}
@article {pmid30068653,
year = {2018},
author = {Wang, Z and Wang, W and Cui, YC and Pan, Q and Zhu, W and Gendron, P and Guo, F and Cen, S and Witcher, M and Liang, C},
title = {HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site.},
journal = {Journal of virology},
volume = {92},
number = {20},
pages = {},
pmid = {30068653},
issn = {1098-5514},
support = {IBC150405//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; Cell Line ; DNA, Viral/genetics/*metabolism ; *Gene Editing ; HIV-1/*genetics ; Humans ; Mutagenesis, Insertional ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; Sequence Deletion ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3 [Lys] in each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCE The results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3 [Lys] during reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.},
}
@article {pmid30021068,
year = {2018},
author = {Menn, DJ and Pradhan, S and Kiani, S and Wang, X},
title = {Fluorescent Guide RNAs Facilitate Development of Layered Pol II-Driven CRISPR Circuits.},
journal = {ACS synthetic biology},
volume = {7},
number = {8},
pages = {1929-1936},
doi = {10.1021/acssynbio.8b00153},
pmid = {30021068},
issn = {2161-5063},
support = {R01 GM106081/GM/NIGMS NIH HHS/United States ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; RNA Polymerase II/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/*chemistry/*metabolism ; Synthetic Biology/methods ; },
abstract = {Efficient clustered regularly interspaced short palindromic repeat (CRISPR) guide RNA (gRNA) expression from RNA Polymerase II (Pol II) promoters will aid in construction of complex CRISPR-based synthetic gene networks. Yet, we require tools to properly visualize gRNA directly to quantitatively study the corresponding network behavior. To address this need, we employed a fluorescent gRNA (fgRNA) to visualize synthetic CRISPR network dynamics without affecting gRNA functionality. We show that studying gRNA dynamics directly enables circuit modification and improvement of network function in Pol II-driven CRISPR circuits. This approach generates information necessary for optimizing the overall function of these networks and provides insight into the hurdles remaining in Pol II-regulated gRNA expression.},
}
@article {pmid30011268,
year = {2018},
author = {Wienert, B and Shin, J and Zelin, E and Pestal, K and Corn, JE},
title = {In vitro-transcribed guide RNAs trigger an innate immune response via the RIG-I pathway.},
journal = {PLoS biology},
volume = {16},
number = {7},
pages = {e2005840},
pmid = {30011268},
issn = {1545-7885},
mesh = {Cell Line ; Cytosol/metabolism ; DEAD Box Protein 58/*metabolism ; Humans ; Immunity, Innate/*genetics ; Interferon Type I/metabolism ; Models, Biological ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Receptors, Immunologic ; *Transcription, Genetic ; },
abstract = {Clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While editing of immortalized cell lines has become relatively easy, editing of therapeutically relevant primary cells and tissues can remain challenging. One recent advancement is the delivery of a Cas9 protein and an in vitro-transcribed (IVT) guide RNA (gRNA) as a precomplexed ribonucleoprotein (RNP). This approach allows editing of primary cells such as T cells and hematopoietic stem cells, but the consequences beyond genome editing of introducing foreign Cas9 RNPs into mammalian cells are not fully understood. Here, we show that the IVT gRNAs commonly used by many laboratories for RNP editing trigger a potent innate immune response that is similar to canonical immune-stimulating ligands. IVT gRNAs are recognized in the cytosol through the retinoic acid-inducible gene I (RIG-I) pathway but not the melanoma differentiation-associated gene 5 (MDA5) pathway, thereby triggering a type I interferon response. Removal of the 5'-triphosphate from gRNAs ameliorates inflammatory signaling and prevents the loss of viability associated with genome editing in hematopoietic stem cells. The potential for Cas9 RNP editing to induce a potent antiviral response indicates that care must be taken when designing therapeutic strategies to edit primary cells.},
}
@article {pmid29980686,
year = {2018},
author = {Mir, A and Alterman, JF and Hassler, MR and Debacker, AJ and Hudgens, E and Echeverria, D and Brodsky, MH and Khvorova, A and Watts, JK and Sontheimer, EJ},
title = {Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2641},
pmid = {29980686},
issn = {2041-1723},
support = {S10 OD020012/OD/NIH HHS/United States ; A-10199//CHDI Foundation (CHDI)/International ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genetic Loci ; Genome, Human/*genetics ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/*metabolism ; },
abstract = {RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2'-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.},
}
@article {pmid29980666,
year = {2018},
author = {Weltner, J and Balboa, D and Katayama, S and Bespalov, M and Krjutškov, K and Jouhilahti, EM and Trokovic, R and Kere, J and Otonkoski, T},
title = {Human pluripotent reprogramming with CRISPR activators.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2643},
pmid = {29980666},
issn = {2041-1723},
mesh = {Alu Elements/genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; Cellular Reprogramming/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Embryo, Mammalian/metabolism ; Fibroblasts/metabolism ; Gene Expression Regulation, Developmental ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Kruppel-Like Factor 4 ; Male ; Nanog Homeobox Protein/metabolism ; Neural Stem Cells/metabolism ; Nucleotide Motifs/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Transcription, Genetic ; },
abstract = {CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.},
}
@article {pmid29856956,
year = {2018},
author = {Kim, MY and Yu, KR and Kenderian, SS and Ruella, M and Chen, S and Shin, TH and Aljanahi, AA and Schreeder, D and Klichinsky, M and Shestova, O and Kozlowski, MS and Cummins, KD and Shan, X and Shestov, M and Bagg, A and Morrissette, JJD and Sekhri, P and Lazzarotto, CR and Calvo, KR and Kuhns, DB and Donahue, RE and Behbehani, GK and Tsai, SQ and Dunbar, CE and Gill, S},
title = {Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.},
journal = {Cell},
volume = {173},
number = {6},
pages = {1439-1453.e19},
pmid = {29856956},
issn = {1097-4172},
support = {P01 CA214278/CA/NCI NIH HHS/United States ; Z01 HL002339-16/ImNIH/Intramural NIH HHS/United States ; K99 CA212302/CA/NCI NIH HHS/United States ; R00 CA212302/CA/NCI NIH HHS/United States ; Z01 HL002339-17/ImNIH/Intramural NIH HHS/United States ; P30 CA016058/CA/NCI NIH HHS/United States ; K08 CA194256/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Cell Differentiation ; Cell Line, Tumor ; Cell Lineage ; Electroporation ; Female ; Hematopoiesis ; Hematopoietic Stem Cells/*cytology ; Humans ; Immunotherapy/*methods ; Leukemia, Myeloid, Acute/immunology/*therapy ; Macaca mulatta ; Male ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Neoplasm Transplantation ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Reactive Oxygen Species ; Sialic Acid Binding Ig-like Lectin 3/*genetics ; T-Lymphocytes/cytology/*immunology ; },
abstract = {The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.},
}
@article {pmid29754817,
year = {2018},
author = {Wei, Z and Yoshihara, E and He, N and Hah, N and Fan, W and Pinto, AFM and Huddy, T and Wang, Y and Ross, B and Estepa, G and Dai, Y and Ding, N and Sherman, MH and Fang, S and Zhao, X and Liddle, C and Atkins, AR and Yu, RT and Downes, M and Evans, RM},
title = {Vitamin D Switches BAF Complexes to Protect β Cells.},
journal = {Cell},
volume = {173},
number = {5},
pages = {1135-1149.e15},
pmid = {29754817},
issn = {1097-4172},
support = {P01 HL088093/HL/NHLBI NIH HHS/United States ; S10 OD021815/OD/NIH HHS/United States ; R01 HL105278/HL/NHLBI NIH HHS/United States ; P42 ES010337/ES/NIEHS NIH HHS/United States ; R37 DK057978/DK/NIDDK NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 DK057978/DK/NIDDK NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Calcitriol/analogs &amp; derivatives/pharmacology ; Chromatin Assembly and Disassembly ; Chromosomal Proteins, Non-Histone/*metabolism ; Diabetes Mellitus, Experimental/chemically induced/metabolism/pathology ; Humans ; Insulin/blood/metabolism ; Insulin-Secreting Cells/cytology/*drug effects/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; Mutagenesis, Site-Directed ; Oxidative Phosphorylation/drug effects ; Protein Binding ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Small Interfering/metabolism ; Receptors, Calcitriol/antagonists &amp; inhibitors/genetics/*metabolism ; Transcription Factors/antagonists &amp; inhibitors/genetics/*metabolism ; Transcription, Genetic/drug effects ; Vitamin D/*pharmacology ; },
abstract = {A primary cause of disease progression in type 2 diabetes (T2D) is β cell dysfunction due to inflammatory stress and insulin resistance. However, preventing β cell exhaustion under diabetic conditions is a major therapeutic challenge. Here, we identify the vitamin D receptor (VDR) as a key modulator of inflammation and β cell survival. Alternative recognition of an acetylated lysine in VDR by bromodomain proteins BRD7 and BRD9 directs association to PBAF and BAF chromatin remodeling complexes, respectively. Mechanistically, ligand promotes VDR association with PBAF to effect genome-wide changes in chromatin accessibility and enhancer landscape, resulting in an anti-inflammatory response. Importantly, pharmacological inhibition of BRD9 promotes PBAF-VDR association to restore β cell function and ameliorate hyperglycemia in murine T2D models. These studies reveal an unrecognized VDR-dependent transcriptional program underpinning β cell survival and identifies the VDR:PBAF/BAF association as a potential therapeutic target for T2D.},
}
@article {pmid29754232,
year = {2018},
author = {Gapinske, M and Tague, N and Winter, J and Underhill, GH and Perez-Pinera, P},
title = {Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1772},
number = {},
pages = {233-248},
doi = {10.1007/978-1-4939-7795-6_13},
pmid = {29754232},
issn = {1940-6029},
mesh = {Cell Line, Tumor ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Endonucleases/*genetics ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Genetic Engineering/methods ; Genetic Vectors/*genetics ; Genome/genetics ; HCT116 Cells ; Homozygote ; Humans ; Jagged-1 Protein/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Deletion/*genetics ; },
abstract = {Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.},
}
@article {pmid29748595,
year = {2018},
author = {Koo, T and Park, SW and Jo, DH and Kim, D and Kim, JH and Cho, HY and Kim, J and Kim, JH and Kim, JS},
title = {CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1855},
pmid = {29748595},
issn = {2041-1723},
mesh = {Animals ; Bacterial Proteins/administration &amp; dosage/genetics ; Cell Line ; Choroidal Neovascularization/etiology/genetics/pathology/*therapy ; Clostridiales/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Endonucleases/administration &amp; dosage/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/*administration &amp; dosage/genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; Intravitreal Injections ; Lasers/adverse effects ; Macular Degeneration/etiology/genetics/pathology/*therapy ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; Retinal Pigment Epithelium/blood supply/pathology/radiation effects ; Specific Pathogen-Free Organisms ; Treatment Outcome ; Vascular Endothelial Growth Factor A/genetics ; },
abstract = {LbCpf1, derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications. Here we show that LbCpf1 can be used for therapeutic gene editing in a mouse model of age-related macular degeneration (AMD). The intravitreal delivery of LbCpf1, targeted to two angiogenesis-associated genes encoding vascular endothelial growth factor A (Vegfa) and hypoxia inducing factor 1a (Hif1a), using adeno-associated virus, led to efficient gene disruption with no apparent off-target effects in the retina and retinal pigment epithelium (RPE) cells. Importantly, LbCpf1 targeted to Vegfa or Hif1a in RPE cells reduced the area of laser-induced choroidal neovascularization as efficiently as aflibercept, an anti-VEGF drug currently used in the clinic, without inducing cone dysfunction. Unlike aflibercept, LbCpf1 targeted to Vegfa or Hif1a achieved a long-term therapeutic effect on CNV, potentially avoiding repetitive injections. Taken together, these results indicate that LbCpf1-mediated in vivo genome editing to ablate pathologic angiogenesis provides an effective strategy for the treatment of AMD and other neovascularization-associated diseases.},
}
@article {pmid29735714,
year = {2018},
author = {Singh, D and Mallon, J and Poddar, A and Wang, Y and Tippana, R and Yang, O and Bailey, S and Ha, T},
title = {Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {21},
pages = {5444-5449},
pmid = {29735714},
issn = {1091-6490},
support = {R01 GM112659/GM/NIGMS NIH HHS/United States ; T32 GM080189/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM097330/GM/NIGMS NIH HHS/United States ; R01 GM065367/GM/NIGMS NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; },
mesh = {Acidaminococcus/*enzymology ; Bacterial Proteins/chemistry/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Cleavage ; DNA, Bacterial/chemistry/*genetics/metabolism ; DNA, Single-Stranded/chemistry/*genetics/metabolism ; Endonucleases/chemistry/*genetics/metabolism ; Genome, Bacterial ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; RNA, Bacterial/chemistry/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/chemistry/*genetics/metabolism ; },
abstract = {CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.},
}
@article {pmid29704201,
year = {2019},
author = {Raikwar, SP and Thangavel, R and Dubova, I and Selvakumar, GP and Ahmed, ME and Kempuraj, D and Zaheer, SA and Iyer, SS and Zaheer, A},
title = {Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer's Disease Therapeutic Target.},
journal = {Molecular neurobiology},
volume = {56},
number = {1},
pages = {378-393},
pmid = {29704201},
issn = {1559-1182},
support = {I01 BX002477/BX/BLRD VA/United States ; R01 AG048205/AG/NIA NIH HHS/United States ; I01BX002477//U.S. Department of Veterans Affairs/ ; AG048205//National Institute on Aging/ ; },
mesh = {Alzheimer Disease/*drug therapy ; Animals ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; Cell Line ; DNA Mutational Analysis ; Dependovirus/metabolism ; *Gene Editing ; Glia Maturation Factor/*genetics/metabolism ; Lentivirus/metabolism ; MAP Kinase Signaling System ; Mice ; Microglia/*metabolism ; *Molecular Targeted Therapy ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Transduction, Genetic ; },
abstract = {Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.},
}
@article {pmid29685355,
year = {2018},
author = {Lee, SS and Shin, H and Jo, S and Lee, SM and Um, Y and Woo, HM},
title = {Rapid identification of unknown carboxyl esterase activity in Corynebacterium glutamicum using RNA-guided CRISPR interference.},
journal = {Enzyme and microbial technology},
volume = {114},
number = {},
pages = {63-68},
doi = {10.1016/j.enzmictec.2018.04.004},
pmid = {29685355},
issn = {1879-0909},
mesh = {Acetates/metabolism ; Bacterial Proteins/*genetics/metabolism ; Carboxylesterase/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/*enzymology/*genetics ; *RNA Interference ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance production of value-added chemicals in Corynebacterium glutamicum as an industrial host. In this study, the RNA-guided CRISPR interference (CRISPRi) was applied to rapidly identify of unknown genes for native esterase activity in C. glutamicum. Combining with the carboxyl esterase (MekB) protein sequence alignment, two target genes (the cg0961 and cg0754) were selected for the CRISPRi application to investigate the possible native esterase in C. glutamicum. The recombinant strain with repressed expression of the cg0961 gene exhibited almost no capability on degradation of methyl acetate as a substrate of carboxyl esterase. This result was also confirmed in the cg0961 gene deletion mutant. Thus, we concluded that Cg0961 plays a major role of the native carboxyl esterase activity in C. glutamicum. In addition, CRISPRi demonstrated an application for gene identification and its function as another genetic tool for metabolic engineering in C. glutamicum.},
}
@article {pmid29651054,
year = {2018},
author = {Canver, MC and Haeussler, M and Bauer, DE and Orkin, SH and Sanjana, NE and Shalem, O and Yuan, GC and Zhang, F and Concordet, JP and Pinello, L},
title = {Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments.},
journal = {Nature protocols},
volume = {13},
number = {5},
pages = {946-986},
pmid = {29651054},
issn = {1750-2799},
support = {F30 DK103359/DK/NIDDK NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; U41 HG002371/HG/NHGRI NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; Endonucleases/*metabolism ; Gene Editing/*methods ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.},
}
@article {pmid29650467,
year = {2018},
author = {McMahon, MA and Prakash, TP and Cleveland, DW and Bennett, CF and Rahdar, M},
title = {Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {5},
pages = {1228-1240},
pmid = {29650467},
issn = {1525-0024},
support = {R01 GM074150/GM/NIGMS NIH HHS/United States ; R01 NS027036/NS/NINDS NIH HHS/United States ; R35 GM122476/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Binding Sites ; Cell Line ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*metabolism ; *Gene Editing ; Gene Targeting ; Genetic Variation ; Humans ; Mammals ; Molecular Conformation ; Protein Binding ; *RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed in vivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity. Modified crRNAs were designed with the same modifications that provide protection against nucleases and enable wide distribution in vivo. We show crRNAs with chemically modified terminal nucleotides are exonuclease resistant while retaining gene-editing activity. Chemically modified or DNA-substituted nucleotides at select positions and up to 70% of the crRNA DNA specificity region are also well tolerated. In addition, gene-editing activity is maintained with phosphorothioate backbone substitutions in the crRNA DNA specificity region. Finally, we demonstrate that 42-mer synthetic crRNAs from the similar CRISPR-Cas9 system are taken up by cells, an attractive property for in vivo delivery. Our study is the first to show that chemically modified crRNAs of the CRISPR-Cpf1 system can functionally replace and mediate comparable gene editing to the natural crRNA, which holds the potential for enhancing both viral- and non-viral-mediated in vivo gene editing.},
}
@article {pmid29622042,
year = {2018},
author = {Wang, J and Zhao, P and Li, Y and Xu, L and Tian, P},
title = {Engineering CRISPR interference system in Klebsiella pneumoniae for attenuating lactic acid synthesis.},
journal = {Microbial cell factories},
volume = {17},
number = {1},
pages = {56},
pmid = {29622042},
issn = {1475-2859},
support = {2015AA021003//National High Technology Research and Development Program/ ; 21276014//National Natural Science Foundation of China/ ; 21476011//National Natural Science Foundation of China/ ; },
mesh = {Bioreactors ; Butylene Glycols/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Silencing ; Green Fluorescent Proteins/genetics ; Klebsiella pneumoniae/*genetics/*metabolism ; L-Lactate Dehydrogenase/genetics ; Lactic Acid/analogs &amp; derivatives/*biosynthesis ; Metabolic Engineering/*methods ; Propylene Glycols/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid.<br><br>RESULTS: Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in&#xa0;K.&#xa0;pneumoniae. EGFP was recruited as a reporter of this CRISPRi system. Fluorescence assay of this CRISPRi system showed that enhanced green fluorescent protein (EGFP) expression level was repressed by 85-90%. To further test this CRISPRi system, guide RNAs were designed to individually or simultaneously target four lactate-producing enzyme genes. Results showed that all lactate-producing enzyme genes were significantly repressed. Notably, D-lactate dehydrogenase (ldhA)&#xa0;was shown to be the most influential enzyme for lactic acid formation in micro-aerobic conditions, as inhibiting ldhA&#xa0;alone led to lactic acid level similar to simultaneously repressing four genes. In shake flask cultivation, the strain coexpressing puuC (an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde to 3-HP) and dCas9-sgRNA inhibiting ldhA produced 1.37-fold 3-HP relative to the reference strain. Furthermore, in bioreactor cultivation, this CRISPRi strain inhibiting ldhA produced 36.7&#xa0;g/L 3-HP, but only generated 1&#xa0;g/L lactic acid. Clearly, this engineered CRISPRi system largely simplified downstream separation of 3-HP from its isomer lactic acid, an extreme challenge for 3-HP bioprocess.<br><br>CONCLUSIONS: This study offers a deep understanding of lactic acid metabolism in diverse species, and we believe that this CRISPRi system will facilitate biomanufacturing and functional genome studies of K.&#xa0;pneumoniae or beyond.},
}
@article {pmid29615501,
year = {2018},
author = {Kujoth, GC and Sullivan, TD and Merkhofer, R and Lee, TJ and Wang, H and Brandhorst, T and Wüthrich, M and Klein, BS},
title = {CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis.},
journal = {mBio},
volume = {9},
number = {2},
pages = {},
pmid = {29615501},
issn = {2150-7511},
support = {R37 AI035681/AI/NIAID NIH HHS/United States ; T32 GM008692/GM/NIGMS NIH HHS/United States ; R21 AI123758/AI/NIAID NIH HHS/United States ; T32 AI055397/AI/NIAID NIH HHS/United States ; F30 MD011547/MD/NIMHD NIH HHS/United States ; R01 AI035681/AI/NIAID NIH HHS/United States ; R01 AI093553/AI/NIAID NIH HHS/United States ; R01 AI040996/AI/NIAID NIH HHS/United States ; R01 AI130411/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Blastomyces/genetics/*growth &amp; development/*metabolism ; Blastomycosis/microbiology ; CRISPR-Associated Protein 9/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colony Count, Microbial ; Disease Models, Animal ; Gene Editing/*methods ; *Genetic Fitness ; Metabolic Networks and Pathways/genetics ; Mice ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Zinc/*metabolism ; },
abstract = {Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo IMPORTANCE Blastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.},
}
@article {pmid29605708,
year = {2018},
author = {Georgiadis, C and Preece, R and Nickolay, L and Etuk, A and Petrova, A and Ladon, D and Danyi, A and Humphryes-Kirilov, N and Ajetunmobi, A and Kim, D and Kim, JS and Qasim, W},
title = {Long Terminal Repeat CRISPR-CAR-Coupled "Universal" T Cells Mediate Potent Anti-leukemic Effects.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {5},
pages = {1215-1227},
pmid = {29605708},
issn = {1525-0024},
support = {RP-2014-05-007/DH_/Department of Health/United Kingdom ; BTRU-2014-10074//Department of Health/United Kingdom ; IS-BRC-1215-20012//Department of Health/United Kingdom ; },
mesh = {Animals ; Antigens, CD19/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; *Gene Editing ; Gene Order ; Genetic Vectors/genetics ; Humans ; Immunophenotyping ; Immunotherapy, Adoptive/methods ; In Situ Hybridization, Fluorescence ; Lentivirus/genetics ; Leukemia/genetics/immunology/therapy ; RNA, Guide, CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/*genetics ; Receptors, Chimeric Antigen ; T-Lymphocytes/*immunology/*metabolism ; *Terminal Repeat Sequences ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR[+] (>99%) TCR[-] populations by automated magnetic separation. Molecular analyses, including NGS, WGS, and Digenome-seq, verified on-target specificity with no evidence of predicted off-target events. Robust anti-leukemic effects were demonstrated in humanized immunodeficient mice and were sustained longer than by conventional CAR[+]TCR[+] T cells. Terminal-TRAC (TT) CAR T cells offer the possibility of a pre-manufactured, non-HLA-matched CAR cell therapy and will be evaluated in phase 1 trials against B cell malignancies shortly.},
}
@article {pmid29598153,
year = {2018},
author = {Zhou, M and Hu, Z and Qiu, L and Zhou, T and Feng, M and Hu, Q and Zeng, B and Li, Z and Sun, Q and Wu, Y and Liu, X and Wu, L and Liang, D},
title = {Seamless Genetic Conversion of SMN2 to SMN1 via CRISPR/Cpf1 and Single-Stranded Oligodeoxynucleotides in Spinal Muscular Atrophy Patient-Specific Induced Pluripotent Stem Cells.},
journal = {Human gene therapy},
volume = {29},
number = {11},
pages = {1252-1263},
doi = {10.1089/hum.2017.255},
pmid = {29598153},
issn = {1557-7422},
mesh = {Base Sequence ; Cell Differentiation ; Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Conversion ; Genotype ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Motor Neurons/metabolism ; Muscular Atrophy, Spinal/*genetics ; Oligodeoxyribonucleotides/*metabolism ; Proto-Oncogene Proteins c-myc/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Survival of Motor Neuron 1 Protein/*genetics ; Survival of Motor Neuron 2 Protein/*genetics ; Young Adult ; },
abstract = {Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.},
}
@article {pmid29577908,
year = {2018},
author = {Hamidian, A and Vaapil, M and von Stedingk, K and Fujita, T and Persson, CU and Eriksson, P and Veerla, S and De Preter, K and Speleman, F and Fujii, H and Påhlman, S and Mohlin, S},
title = {Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator.},
journal = {Biochemical and biophysical research communications},
volume = {499},
number = {2},
pages = {291-298},
doi = {10.1016/j.bbrc.2018.03.150},
pmid = {29577908},
issn = {1090-2104},
mesh = {Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Cell Hypoxia/genetics ; Cell Line, Tumor ; Chromatin/metabolism ; Chromatin Immunoprecipitation/*methods ; DNA/*metabolism ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; *Genetic Engineering ; Homeodomain Proteins/genetics/*metabolism ; Humans ; Mass Spectrometry/*methods ; Mice ; Neuroblastoma/genetics/pathology ; *Promoter Regions, Genetic ; Proto-Oncogene Proteins c-myc/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Messenger/genetics/metabolism ; Rapamycin-Insensitive Companion of mTOR Protein/genetics/metabolism ; Reproducibility of Results ; Transcription Factors/genetics/*metabolism ; },
abstract = {Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.},
}
@article {pmid29549971,
year = {2018},
author = {Li, Y and Yang, T and Yu, Y and Shi, N and Yang, L and Glass, Z and Bolinger, J and Finkel, IJ and Li, W and Xu, Q},
title = {Combinatorial library of chalcogen-containing lipidoids for intracellular delivery of genome-editing proteins.},
journal = {Biomaterials},
volume = {178},
number = {},
pages = {652-662},
doi = {10.1016/j.biomaterials.2018.03.011},
pmid = {29549971},
issn = {1878-5905},
mesh = {Animals ; Cell Death ; Cell Survival ; Chalcogens/*chemistry ; *Combinatorial Chemistry Techniques ; *Gene Editing ; *Gene Transfer Techniques ; Green Fluorescent Proteins/*genetics ; HeLa Cells ; Humans ; Integrases/metabolism ; Intracellular Space/*metabolism ; Lipids/*chemistry ; Mice ; Nanoparticles/chemistry/ultrastructure ; Proton Magnetic Resonance Spectroscopy ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Recombination, Genetic/genetics ; Structure-Activity Relationship ; },
abstract = {Protein based therapeutics with high specificities and low off-target effects are used for transient and accurate manipulation of cell functions. However, developing safe and efficient carriers for intracellular delivery of active therapeutic proteins is a long-standing challenge. Here we report a combinatorial library of chalcogen (O, S, Se) containing lipidoid nanoparticles (LNPs) as efficient nanocarriers for intracellular delivery of negatively supercharged Cre recombinase ((-30)GFP-Cre) and anionic Cas9:single-guide RNA (Cas9:sgRNA) ribonucleoprotein (RNP) for genome editing. The structure-activity relationship between the lipidoids and intracellular protein delivery efficiencies was explored and it was demonstrated that the newly developed LNPs are effective for gene recombination in vivo.},
}
@article {pmid29535977,
year = {2018},
author = {Ouellette, SP},
title = {Feasibility of a Conditional Knockout System for Chlamydia Based on CRISPR Interference.},
journal = {Frontiers in cellular and infection microbiology},
volume = {8},
number = {},
pages = {59},
pmid = {29535977},
issn = {2235-2988},
support = {R35 GM124798/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/*genetics/*metabolism ; CRISPR-Associated Protein 9/genetics ; Cell Line ; Chlamydia/*genetics/*metabolism ; Chlamydia trachomatis/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics ; Fibroblasts ; Gene Expression Regulation, Bacterial ; Gene Knockout Techniques ; Mice ; Mycobacterium tuberculosis ; Phosphoproteins/*genetics/*metabolism ; Plasmids/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Serogroup ; Staphylococcus aureus/genetics ; *Transformation, Bacterial ; },
abstract = {Chlamydia is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of C. trachomatis with an exogenous plasmid, genetic manipulation of Chlamydia remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of Chlamydia may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi) based on the catalytically inactive Cas9 variant (dCas9) of Staphylococcus aureus to inducibly, and reversibly, repress gene expression in C. trachomatis. CRISPRi has been developed and used successfully in a variety of bacterial organisms including E. coli and Mycobacterium tuberculosis. I first describe the creation of a single plasmid system for CRISPRi in Chlamydia, targeted to a non-essential gene, incA, that expresses a dispensable inclusion membrane protein. Control transformations of C. trachomatis serovar L2 with plasmids encoding only the dCas9, under the control of an inducible promoter, or only the guide RNA (gRNA) targeted to the 5' UTR of incA, expressed constitutively, failed to prevent expression of IncA. Importantly, expression of dCas9 alone did not have a deleterious effect on chlamydiae. Transformation of C. trachomatis with a plasmid combining the dCas9 and a gRNA targeting incA and induction of expression of the dCas9 resulted in the reversible inhibition of IncA expression. Consequently, conditional knockout mediated by CRISPRi is feasible in Chlamydia. Potential improvements and experimental concerns in using the system are also discussed.},
}
@article {pmid29507191,
year = {2018},
author = {Tsui, C and Inouye, C and Levy, M and Lu, A and Florens, L and Washburn, MP and Tjian, R},
title = {dCas9-targeted locus-specific protein isolation method identifies histone gene regulators.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {12},
pages = {E2734-E2741},
pmid = {29507191},
issn = {1091-6490},
support = {U01 DA047729/DA/NIDA NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {3' Untranslated Regions ; Animals ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9 ; Chromatin/genetics/*isolation &amp; purification ; Chromatin Immunoprecipitation ; Chromosomal Proteins, Non-Histone/*genetics/metabolism ; Drosophila Proteins/*genetics/metabolism ; Drosophila melanogaster/genetics ; Endonucleases/*genetics/metabolism ; Gene Expression ; Genes, Regulator/*genetics ; Histones/*genetics/metabolism ; Humans ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA-Binding Proteins/*genetics/metabolism ; Recombinant Proteins/genetics/metabolism ; },
abstract = {Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called "CLASP" (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors. Our CLASP method, coupled to mass spectrometry and functional screens, can be efficiently adapted for isolating associated regulatory factors in an unbiased manner targeting multiple genomic loci across different cell types. Here, we applied our method to isolate the Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3' UTRs.},
}
@article {pmid29480524,
year = {2018},
author = {Fujita, T and Kitaura, F and Oji, A and Tanigawa, N and Yuno, M and Ikawa, M and Taniuchi, I and Fujii, H},
title = {Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {23},
number = {4},
pages = {318-325},
doi = {10.1111/gtc.12573},
pmid = {29480524},
issn = {1365-2443},
mesh = {Animals ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CRISPR-Associated Protein 9/genetics/*metabolism ; Cells, Cultured ; Chromatin Immunoprecipitation/*methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation ; Genetic Engineering/*methods ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4[+] T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.},
}
@article {pmid29476918,
year = {2018},
author = {Yan, F and Kuang, Y and Ren, B and Wang, J and Zhang, D and Lin, H and Yang, B and Zhou, X and Zhou, H},
title = {Highly Efficient A·T to G·C Base Editing by Cas9n-Guided tRNA Adenosine Deaminase in Rice.},
journal = {Molecular plant},
volume = {11},
number = {4},
pages = {631-634},
doi = {10.1016/j.molp.2018.02.008},
pmid = {29476918},
issn = {1752-9867},
mesh = {Adenosine Deaminase/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; Gene Editing/*methods ; *Mutation ; Oryza/enzymology/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; RNA, Transfer/*genetics ; },
}
@article {pmid29462720,
year = {2018},
author = {Ding, D and Chen, K and Chen, Y and Li, H and Xie, K},
title = {Engineering Introns to Express RNA Guides for Cas9- and Cpf1-Mediated Multiplex Genome Editing.},
journal = {Molecular plant},
volume = {11},
number = {4},
pages = {542-552},
doi = {10.1016/j.molp.2018.02.005},
pmid = {29462720},
issn = {1752-9867},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/*metabolism ; Gene Editing/*methods ; Gene Expression ; *Genomics ; Introns/*genetics ; Oryza/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) system has emerged as the revolutionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector (e.g., Cas9 and Cpf1) to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs (gRNA) and CRISPR RNAs (crRNA) from introns of Cas9 and Cpf1. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpf1 to cleave the spliced intron that contains tRNA-gRNA polycistron or crRNA-crRNA array. We demonstrated that the tRNA-gRNA intron is able to fuse with Cas9 as one gene. Such a hybrid gene could be expressed using one polymerase II promoter, and exhibited high efficiency and robustness in simultaneously targeting multiple sites. We also implemented this strategy in Cpf1-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpf1 and intronic crRNA array exhibited remarkably increased efficiency compared with the conventional Cpf1 vectors. Taken together, this study presents a method to express CRISPR reagents from one hybrid gene to increase genome-editing efficiency and capacity. Owing to its simplicity and versatility, this method could be broadly used to develop sophisticated CRISPR tools in eukaryotes.},
}
@article {pmid29456084,
year = {2018},
author = {Liu, XS and Wu, H and Krzisch, M and Wu, X and Graef, J and Muffat, J and Hnisz, D and Li, CH and Yuan, B and Xu, C and Li, Y and Vershkov, D and Cacace, A and Young, RA and Jaenisch, R},
title = {Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene.},
journal = {Cell},
volume = {172},
number = {5},
pages = {979-992.e6},
pmid = {29456084},
issn = {1097-4172},
support = {R01 GM123511/GM/NIGMS NIH HHS/United States ; R01 MH104610/MH/NIMH NIH HHS/United States ; R01 GM114864/GM/NIGMS NIH HHS/United States ; R01 NS088538/NS/NINDS NIH HHS/United States ; R37 CA084198/CA/NCI NIH HHS/United States ; R37 HD045022/HD/NICHD NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R01 HD045022/HD/NICHD NIH HHS/United States ; R01 CA084198/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; DNA Methylation/*genetics ; Epigenesis, Genetic ; Fragile X Mental Retardation Protein/*genetics ; Fragile X Syndrome/*genetics ; *Gene Editing ; HEK293 Cells ; Heterochromatin/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Kinetics ; Male ; Mice ; Neurons/metabolism/*pathology ; Phenotype ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.},
}
@article {pmid29438517,
year = {2018},
author = {Juergens, H and Varela, JA and Gorter de Vries, AR and Perli, T and Gast, VJM and Gyurchev, NY and Rajkumar, AS and Mans, R and Pronk, JT and Morrissey, JP and Daran, JG},
title = {Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.},
journal = {FEMS yeast research},
volume = {18},
number = {3},
pages = {},
pmid = {29438517},
issn = {1567-1364},
mesh = {CRISPR-Associated Protein 9/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fungal Proteins/genetics ; Gene Deletion ; *Gene Editing ; Gene Expression ; Kluyveromyces/*genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Saccharomycetales/*genetics ; },
abstract = {While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.},
}
@article {pmid29404407,
year = {2018},
author = {Long, C and Li, H and Tiburcy, M and Rodriguez-Caycedo, C and Kyrychenko, V and Zhou, H and Zhang, Y and Min, YL and Shelton, JM and Mammen, PPA and Liaw, NY and Zimmermann, WH and Bassel-Duby, R and Schneider, JW and Olson, EN},
title = {Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing.},
journal = {Science advances},
volume = {4},
number = {1},
pages = {eaap9004},
pmid = {29404407},
issn = {2375-2548},
support = {R01 HL077439/HL/NHLBI NIH HHS/United States ; R01 DK099653/DK/NIDDK NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Base Sequence ; Dystrophin/genetics ; Exons/genetics ; *Gene Editing ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Muscular Dystrophy, Duchenne/*genetics ; Mutation/*genetics ; Myocardium/*pathology ; Myocytes, Cardiac/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Messenger/genetics/metabolism ; Tissue Engineering/*methods ; },
abstract = {Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). Most of these mutations are clustered in "hotspots." There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.},
}
@article {pmid29286403,
year = {2017},
author = {Köferle, A and Stricker, SH},
title = {A Universal Protocol for Large-scale gRNA Library Production from any DNA Source.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {130},
pages = {},
pmid = {29286403},
issn = {1940-087X},
mesh = {Animals ; DNA/*genetics ; *Gene Library ; Humans ; Mice ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.},
}
@article {pmid29282854,
year = {2018},
author = {Wang, P and Zhang, L and Zheng, W and Cong, L and Guo, Z and Xie, Y and Wang, L and Tang, R and Feng, Q and Hamada, Y and Gonda, K and Hu, Z and Wu, X and Jiang, X},
title = {Thermo-triggered Release of CRISPR-Cas9 System by Lipid-Encapsulated Gold Nanoparticles for Tumor Therapy.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {57},
number = {6},
pages = {1491-1496},
doi = {10.1002/anie.201708689},
pmid = {29282854},
issn = {1521-3773},
mesh = {Animals ; Apoptosis/radiation effects ; CRISPR-Associated Protein 9/*genetics ; Cell Cycle Proteins/genetics ; Cell Line, Tumor ; Gene Transfer Techniques ; Glutathione/chemistry ; Gold/*chemistry ; Humans ; Hyperthermia, Induced ; Lasers ; Lipids/*chemistry ; Melanoma, Experimental/pathology/*therapy ; Metal Nanoparticles/*chemistry ; Mice ; Microscopy, Confocal ; Peptide Fragments/chemistry ; Plasmids/genetics/*therapeutic use ; Protein Serine-Threonine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Surface Plasmon Resonance ; Polo-Like Kinase 1 ; },
abstract = {CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.},
}
@article {pmid29211736,
year = {2017},
author = {Adikusuma, F and Pfitzner, C and Thomas, PQ},
title = {Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression.},
journal = {PloS one},
volume = {12},
number = {12},
pages = {e0187236},
pmid = {29211736},
issn = {1932-6203},
mesh = {Animals ; Cells, Cultured ; Cloning, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells/metabolism ; Genetic Vectors ; Mice ; Plasmids ; Polymerase Chain Reaction ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.},
}
@article {pmid29179261,
year = {2017},
author = {Park, DS and Yoon, M and Kweon, J and Jang, AH and Kim, Y and Choi, SC},
title = {Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos.},
journal = {Molecules and cells},
volume = {40},
number = {11},
pages = {823-827},
pmid = {29179261},
issn = {0219-1032},
mesh = {Albinism, Oculocutaneous/*genetics ; Amino Acid Substitution ; Animals ; Cytidine Deaminase/*metabolism ; Gene Editing/methods ; Monophenol Monooxygenase/*genetics ; Mutation Rate ; Phenotype ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Xenopus Proteins/genetics ; Xenopus laevis/*embryology/genetics ; },
abstract = {Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.},
}
@article {pmid29151576,
year = {2017},
author = {Rueda, FO and Bista, M and Newton, MD and Goeppert, AU and Cuomo, ME and Gordon, E and Kröner, F and Read, JA and Wrigley, JD and Rueda, D and Taylor, BJM},
title = {Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1610},
pmid = {29151576},
issn = {2041-1723},
support = {MC_UP_1102/5/MRC_/Medical Research Council/United Kingdom ; //Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/*chemistry/metabolism ; Biocatalysis ; CRISPR-Associated Protein 9 ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*chemistry/genetics ; Endonucleases/*chemistry/metabolism ; Nucleic Acid Conformation ; RNA/*chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/*chemistry/genetics ; },
abstract = {The CRISPR-Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA-RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.},
}
@article {pmid29091290,
year = {2018},
author = {Wangensteen, KJ and Wang, YJ and Dou, Z and Wang, AW and Mosleh-Shirazi, E and Horlbeck, MA and Gilbert, LA and Weissman, JS and Berger, SL and Kaestner, KH},
title = {Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform.},
journal = {Hepatology (Baltimore, Md.)},
volume = {68},
number = {2},
pages = {663-676},
pmid = {29091290},
issn = {1527-3350},
support = {U01 CA217882/CA/NCI NIH HHS/United States ; R00 CA204602/CA/NCI NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; P30 DK050306/DK/NIDDK NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; R01 DK102667/DK/NIDDK NIH HHS/United States ; R01 CA249929/CA/NCI NIH HHS/United States ; T32 DK007066/DK/NIDDK NIH HHS/United States ; P30 DK019525/DK/NIDDK NIH HHS/United States ; K08 DK106478/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Blotting, Western ; Carcinogenesis/*genetics ; Carcinoma, Hepatocellular/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression Regulation, Neoplastic/genetics ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing ; Immunohistochemistry ; Liver/metabolism/pathology ; Liver Neoplasms/*genetics ; Mice ; Oncogenes ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Real-Time Polymerase Chain Reaction ; Transcriptional Activation ; },
abstract = {UNLABELLED: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors.<br><br>CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).},
}
@article {pmid29058669,
year = {2017},
author = {Howe, FS and Russell, A and Lamstaes, AR and El-Sagheer, A and Nair, A and Brown, T and Mellor, J},
title = {CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape.},
journal = {eLife},
volume = {6},
number = {},
pages = {},
pmid = {29058669},
issn = {2050-084X},
support = {BB/J001694/2//Biotechnology and Biological Sciences Research Council/United Kingdom ; 209897/Z/17/Z//Wellcome Trust/United Kingdom ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9 ; DNA, Fungal/*metabolism ; Endonucleases/genetics/*metabolism ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; *Transcription, Genetic ; },
abstract = {CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9-binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.},
}
@article {pmid28956384,
year = {2017},
author = {Chang, L and Sun, C and Chen, X and Yang, W and Zhang, J and Zhang, Y and Yuan, W and Zhu, X},
title = {[Knocking out of human DNAH2 gene in U2OS cells by CRISPR/Cas9n double nick system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {33},
number = {2},
pages = {284-293},
doi = {10.13345/j.cjb.160273},
pmid = {28956384},
issn = {1000-3061},
mesh = {Axonemal Dyneins/*genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Knockout Techniques ; Genetic Vectors ; Humans ; Plasmids ; RNA, Guide, CRISPR-Cas Systems ; Transfection ; },
abstract = {To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.},
}
@article {pmid28938163,
year = {2017},
author = {Liang, C and Li, F and Wang, L and Zhang, ZK and Wang, C and He, B and Li, J and Chen, Z and Shaikh, AB and Liu, J and Wu, X and Peng, S and Dang, L and Guo, B and He, X and Au, DWT and Lu, C and Zhu, H and Zhang, BT and Lu, A and Zhang, G},
title = {Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma.},
journal = {Biomaterials},
volume = {147},
number = {},
pages = {68-85},
doi = {10.1016/j.biomaterials.2017.09.015},
pmid = {28938163},
issn = {1878-5905},
mesh = {Animals ; Aptamers, Nucleotide/*chemistry ; Bone Neoplasms/pathology/*therapy ; CRISPR-Associated Protein 9/*genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Fluorescent Dyes/chemistry ; Gene Editing ; Gene Transfer Techniques ; Genetic Therapy ; Humans ; Liposomes ; Male ; Mice ; Mice, Inbred BALB C ; Osteosarcoma/pathology/*therapy ; Particle Size ; Polyethylene Glycols/chemistry ; Polyethyleneimine/analogs &amp; derivatives/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; Surface Properties ; Vascular Endothelial Growth Factor A/*genetics/metabolism ; Wound Healing/drug effects ; },
abstract = {Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment.},
}
@article {pmid28785016,
year = {2017},
author = {Scott, T and Moyo, B and Nicholson, S and Maepa, MB and Watashi, K and Ely, A and Weinberg, MS and Arbuthnot, P},
title = {ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {7401},
pmid = {28785016},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; Dependovirus/*genetics ; Gene Editing ; Genetic Vectors/pharmacology ; Hep G2 Cells ; Hepatitis B/virology ; Hepatitis B virus/*drug effects/genetics/physiology ; Humans ; Mutagenesis, Site-Directed ; Open Reading Frames ; RNA, Guide, CRISPR-Cas Systems/chemical synthesis/*genetics ; Staphylococcus aureus/metabolism ; Virus Replication/drug effects ; },
abstract = {Management of infection with hepatitis B virus (HBV) remains a global health problem. Persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is responsible for modest curative efficacy of currently licensed drugs. Novel gene editing technologies, such as those based on CRISPR/Cas9, provide the means for permanently disabling cccDNA. However, efficient delivery of antiviral sequences to infected hepatocytes is challenging. A limiting factor is the large size of sequences encoding Cas9 from Streptococcus pyogenes, and resultant incompatibility with the popular single stranded adeno-associated viral vectors (ssAAVs). We thus explored the utility of ssAAVs for delivery of engineered CRISPR/Cas9 of Staphylococcus aureus (Sa), which is encoded by shorter DNA sequences. Short guide RNAs (sgRNAs) were designed with cognates in the S open reading frame of HBV and incorporated into AAVs that also encoded SaCas9. Intended targeted mutation of HBV DNA was observed after transduction of cells with the all-in-one vectors. Efficacy against HBV-infected hNTCP-HepG2 cells indicated that inactivation of cccDNA was successful. Analysis of likely off-target mutagenesis revealed no unintended sequence changes. Use of ssAAVs to deliver all components required to disable cccDNA by SaCas9 is novel and the technology has curative potential for HBV infection.},
}
@article {pmid28724960,
year = {2017},
author = {Xie, C and Chen, YL and Wang, DF and Wang, YL and Zhang, TP and Li, H and Liang, F and Zhao, Y and Zhang, GY},
title = {SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {5795},
pmid = {28724960},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression ; MicroRNAs/genetics/*metabolism ; Promoter Regions, Genetic ; RNA Polymerase II/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; },
abstract = {CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5'- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.},
}
@article {pmid28724352,
year = {2017},
author = {Jia, G and Wang, X and Xiao, G},
title = {A permutation-based non-parametric analysis of CRISPR screen data.},
journal = {BMC genomics},
volume = {18},
number = {1},
pages = {545},
pmid = {28724352},
issn = {1471-2164},
support = {R15 GM113157/GM/NIGMS NIH HHS/United States ; },
mesh = {*Algorithms ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Statistics, Nonparametric ; },
abstract = {BACKGROUND: Clustered regularly-interspaced short palindromic repeats (CRISPR) screens are usually implemented in cultured cells to identify genes with critical functions. Although several methods have been developed or adapted to analyze CRISPR screening data, no single specific algorithm has gained popularity. Thus, rigorous procedures are needed to overcome the shortcomings of existing algorithms.<br><br>METHODS: We developed a Permutation-Based Non-Parametric Analysis (PBNPA) algorithm, which computes p-values at the gene level by permuting sgRNA labels, and thus it avoids restrictive distributional assumptions. Although PBNPA is designed to analyze CRISPR data, it can also be applied to analyze genetic screens implemented with siRNAs or shRNAs and drug screens.<br><br>RESULTS: We compared the performance of PBNPA with competing methods on simulated data as well as on real data. PBNPA outperformed recent methods designed for CRISPR screen analysis, as well as methods used for analyzing other functional genomics screens, in terms of Receiver Operating Characteristics (ROC) curves and False Discovery Rate (FDR) control for simulated data under various settings. Remarkably, the PBNPA algorithm showed better consistency and FDR control on published real data as well.<br><br>CONCLUSIONS: PBNPA yields more consistent and reliable results than its competitors, especially when the data quality is low. R package of PBNPA is available at: https://cran.r-project.org/web/packages/PBNPA/ .},
}
@article {pmid28705897,
year = {2017},
author = {Servetnick, MD and Steinworth, B and Babonis, LS and Simmons, D and Salinas-Saavedra, M and Martindale, MQ},
title = {Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral-aboral patterning.},
journal = {Development (Cambridge, England)},
volume = {144},
number = {16},
pages = {2951-2960},
pmid = {28705897},
issn = {1477-9129},
support = {R01 GM093116/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Endoderm/*embryology ; Fetal Proteins/genetics/metabolism ; Immunohistochemistry ; In Situ Hybridization ; Pharynx/*embryology ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Sea Anemones/*embryology/*metabolism ; T-Box Domain Proteins/genetics/metabolism ; Wnt Proteins/genetics/metabolism ; },
abstract = {The mesoderm is a key novelty in animal evolution, although we understand little of how the mesoderm arose. brachyury, the founding member of the T-box gene family, is a key gene in chordate mesoderm development. However, the brachyury gene was present in the common ancestor of fungi and animals long before mesoderm appeared. To explore ancestral roles of brachyury prior to the evolution of definitive mesoderm, we excised the gene using CRISPR/Cas9 in the diploblastic cnidarian Nematostella vectensis Nvbrachyury is normally expressed in precursors of the pharynx, which separates endoderm from ectoderm. In knockout embryos, the pharynx does not form, embryos fail to elongate, and endoderm organization, ectodermal cell polarity and patterning along the oral-aboral axis are disrupted. Expression of many genes both inside and outside the Nvbrachyury expression domain is affected, including downregulation of Wnt genes at the oral pole. Our results point to an ancient role for brachyury in morphogenesis, cell polarity and the patterning of both ectodermal and endodermal derivatives along the primary body axis.},
}
@article {pmid28692085,
year = {2017},
author = {Du, Z and Uversky, VN},
title = {Functional roles of intrinsic disorder in CRISPR-associated protein Cas9.},
journal = {Molecular bioSystems},
volume = {13},
number = {9},
pages = {1770-1780},
doi = {10.1039/c7mb00279c},
pmid = {28692085},
issn = {1742-2051},
mesh = {Actinomyces/enzymology ; Binding Sites ; CRISPR-Associated Proteins/*chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Intrinsically Disordered Proteins/*chemistry/metabolism ; Models, Molecular ; Protein Binding ; Protein Conformation ; RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism ; Structure-Activity Relationship ; },
abstract = {Protein intrinsic disorder is an important characteristic commonly detected in multifunctional or RNA- and DNA-binding proteins. Due to their high conformational flexibility and solvent accessibility, intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) execute diverse functions including interaction with multiple partners, and are frequently subjected to various post-translational modifications. Recent studies on the components comprising the CRISPR (clustered regularly interspaced short palindromic repeats) system have elucidated the crystal structure of Cas9 proteins and the mechanism by which the Cas9-sgRNA complex recognizes and cleaves its target DNA. Yet the extent and functional implications of intrinsic disorder in the Cas9 protein have never been fully assessed. Here, we present a comprehensive computational analysis based on both sequence and structural data in an attempt to investigate the roles of IDPRs in the functioning of Cas9 proteins of different origin. We conclude that among the functional roles of IDPRs in Cas9 proteins are recognition of the target DNA and mediation of nucleic acid and protein binding.},
}
@article {pmid28643251,
year = {2017},
author = {Kaneko, T},
title = {Genome Editing in Mouse and Rat by Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1630},
number = {},
pages = {81-89},
doi = {10.1007/978-1-4939-7128-2_7},
pmid = {28643251},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Proteins/metabolism ; Electroporation/*methods ; Endonucleases/*metabolism ; Female ; Gene Editing/*methods ; Male ; Mice ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems/genetics ; Rats ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases/metabolism ; },
abstract = {Many knock-out/knock-in mouse and rat strains have been produced by genome editing techniques using engineered endonucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Microinjection of engineered endonucleases into pronuclear-stage embryos is required to produce genome-edited rodents and the development of easy, rapid, and high-efficiency methods that do not require special skills such as microinjection is needed. This chapter presents a new technique called Technique for Animal Knockout system by Electroporation (TAKE), which produces genome-edited rodents by direct introduction of engineered endonucleases into intact embryos using electroporation.},
}
@article {pmid28643250,
year = {2017},
author = {Noda, T and Oji, A and Ikawa, M},
title = {Genome Editing in Mouse Zygotes and Embryonic Stem Cells by Introducing SgRNA/Cas9 Expressing Plasmids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1630},
number = {},
pages = {67-80},
doi = {10.1007/978-1-4939-7128-2_6},
pmid = {28643250},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Proteins/genetics/metabolism ; Chimera/genetics ; Embryonic Stem Cells/*cytology ; Gene Editing/*methods ; Mice ; Microinjections ; Mosaicism ; Phenotype ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Zygote/*cytology ; },
abstract = {In mammalian cells, genome editing with the single guide RNA (sgRNA)/Cas9 complex allows for high targeting efficiency within a relatively short time frame with the added benefits of being low cost and easy to design. sgRNA/Cas9-mediated editing in mouse zygotes has accelerated the analysis of gene functions and the generation of mouse models of human diseases. Despite the benefits, this method still suffers from several problems, such as mosaicism in the founder generation which complicates genotyping and phenotypical analyses, and the low efficiency of more complicated genome editing. Thus, we recently established the system for genome editing in embryonic stem (ES) cells and its application for chimeric analysis in mice. In this section, we introduce the procedure for genome editing in mouse zygotes and ES cells.},
}
@article {pmid28562584,
year = {2017},
author = {Stella, S and Alcón, P and Montoya, G},
title = {Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.},
journal = {Nature},
volume = {546},
number = {7659},
pages = {559-563},
pmid = {28562584},
issn = {1476-4687},
mesh = {Acidaminococcus/enzymology ; Adenosine Triphosphate/metabolism ; Base Pairing ; Crystallography, X-Ray ; DNA/genetics/*metabolism ; *DNA Cleavage ; Endonucleases/*chemistry/*metabolism ; Francisella/*enzymology ; Gene Editing ; Gram-Positive Bacteria/enzymology ; Lysine/metabolism ; Models, Molecular ; Protein Domains ; Protein Engineering ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; Substrate Specificity ; },
abstract = {Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.},
}
@article {pmid28511701,
year = {2017},
author = {Quadros, RM and Miura, H and Harms, DW and Akatsuka, H and Sato, T and Aida, T and Redder, R and Richardson, GP and Inagaki, Y and Sakai, D and Buckley, SM and Seshacharyulu, P and Batra, SK and Behlke, MA and Zeiner, SA and Jacobi, AM and Izu, Y and Thoreson, WB and Urness, LD and Mansour, SL and Ohtsuka, M and Gurumurthy, CB},
title = {Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.},
journal = {Genome biology},
volume = {18},
number = {1},
pages = {92},
pmid = {28511701},
issn = {1474-760X},
support = {P30 CA036727/CA/NCI NIH HHS/United States ; P20 GM103471/GM/NIGMS NIH HHS/United States ; R01 EY010542/EY/NEI NIH HHS/United States ; 087377//Wellcome Trust/United Kingdom ; P30 GM110768/GM/NIGMS NIH HHS/United States ; R21 DC014779/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/genetics/metabolism ; Endonucleases/genetics/metabolism ; Founder Effect ; Gene Editing/*methods ; Genes, Reporter ; Genetic Loci ; Integrases/genetics/metabolism ; Mice ; Mice, Transgenic/*genetics/growth &amp; development ; Microinjections ; Mutagenesis, Insertional/*methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Recombinational DNA Repair ; Ribonucleoproteins/*genetics/metabolism ; Zygote/growth &amp; development/metabolism ; },
abstract = {BACKGROUND: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.<br><br>RESULTS: Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA&#x2009;+&#x2009;tracrRNA&#x2009;+&#x2009;Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring.<br><br>CONCLUSIONS: Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.},
}
@article {pmid28411216,
year = {2017},
author = {Zhao, C and Shu, X and Sun, B},
title = {Construction of a Gene Knockdown System Based on Catalytically Inactive ("Dead") Cas9 (dCas9) in Staphylococcus aureus.},
journal = {Applied and environmental microbiology},
volume = {83},
number = {12},
pages = {},
pmid = {28411216},
issn = {1098-5336},
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9 ; Endonucleases/genetics/metabolism ; Gene Knockdown Techniques/instrumentation/*methods ; Gene Silencing ; Operon ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/*genetics/metabolism ; },
abstract = {There has been an absence of an efficient method of gene knockdown in the important human pathogen Staphylococcus aureus like RNA interference in eukaryotes. The previously developed antisense RNA technology is mainly applied for forward genetic screening but is rather limited in specific gene knockdown because of the lack of rational antisense RNA design strategies. Here we report an efficient and specific system for gene knockdown in S. aureus based on the type II clustered regularly interspaced short palindromic repeat (CRISPR) system from Streptococcus pyogenes We can achieve gene silencing with the coexpression of dCas9, an RNA-guided DNA binding protein, and a small guide RNA complementary to the target gene. With this system, we have successfully silenced diverse sets of genes varying in size and expression level in different S. aureus strains. This system exhibited high-efficiency knockdown of both essential and nonessential genes, and its effect is inducible and reversible. In addition, the system can repress the expression of multiple genes simultaneously and silence an entire operon or part of it. This RNA-guided DNA targeting system thus provides a simple, rapid, and affordable method for selective gene knockdown in S. aureus IMPORTANCE Staphylococcus aureus is an important human and animal pathogen that can cause a diversity of infectious diseases. Molecular genetic study of S. aureus has provided an avenue for the understanding of its virulence, pathogenesis, and drug resistance, leading to the discovery of new therapies for the treatment of staphylococcal infections. However, methodologies developed for genetic manipulation of S. aureus usually involve either low efficiency or laborious procedures. Here we report an RNA-guided system for gene knockdown in S. aureus and show its high efficiency and simplicity for selective gene silencing in different strains of S. aureus This simple, rapid, and affordable system may serve as a promising tool for functional gene study in S. aureus, especially for the study of essential genes, thus facilitating the understanding of this pathogen and its interaction with its hosts.},
}
@article {pmid28374750,
year = {2017},
author = {Wu, D and Guan, X and Zhu, Y and Ren, K and Huang, Z},
title = {Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA.},
journal = {Cell research},
volume = {27},
number = {5},
pages = {705-708},
pmid = {28374750},
issn = {1748-7838},
mesh = {Base Sequence ; CRISPR-Associated Proteins/chemistry/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/metabolism ; Models, Molecular ; *Nucleic Acid Conformation ; Nucleotide Motifs/*genetics ; Protein Domains ; RNA, Guide, CRISPR-Cas Systems/chemistry/*genetics/metabolism ; },
}
@article {pmid28288556,
year = {2017},
author = {Hough, SH and Kancleris, K and Brody, L and Humphryes-Kirilov, N and Wolanski, J and Dunaway, K and Ajetunmobi, A and Dillard, V},
title = {Guide Picker is a comprehensive design tool for visualizing and selecting guides for CRISPR experiments.},
journal = {BMC bioinformatics},
volume = {18},
number = {1},
pages = {167},
pmid = {28288556},
issn = {1471-2105},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Humans ; Internet ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; *User-Computer Interface ; },
abstract = {BACKGROUND: Guide Picker (https://www.deskgen.com/guide-picker/) serves as a meta tool for designing CRISPR experiments by presenting ten different guide RNA scoring functions in one simple graphical interface. It allows investigators to simultaneously visualize and sort through every guide targeting the protein-coding regions of any mouse or human gene.<br><br>RESULTS: Utilizing a multidimensional graphical display featuring two plots and four axes, Guide Picker can analyze all guides while filtering based on four different criteria at a time. Guide Picker further facilitates the CRISPR design process by using pre-computed scores for all guides, thereby offering rapid guide RNA generation and selection.<br><br>CONCLUSIONS: The ease-of-use of Guide Picker complements CRISPR itself, matching a powerful and modular biological system with a flexible online web tool that can be used in a variety of genome editing experimental contexts.},
}
@article {pmid28287608,
year = {2017},
author = {Shi, ZD and Soh, CL and Zhu, Z and Huangfu, D},
title = {Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {121},
pages = {},
pmid = {28287608},
issn = {1940-087X},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 DK096239/DK/NIDDK NIH HHS/United States ; },
mesh = {Cell Differentiation ; DNA End-Joining Repair ; *Gene Editing ; Gene Knockout Techniques ; Humans ; Pancreas/*growth &amp; development/metabolism ; Phenotype ; Pluripotent Stem Cells/*cytology ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic β-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.},
}
@article {pmid28256578,
year = {2017},
author = {Ferry, QR and Lyutova, R and Fulga, TA},
title = {Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14633},
pmid = {28256578},
issn = {2041-1723},
support = {G0902418/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Algorithms ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Flow Cytometry ; Gene Regulatory Networks/genetics ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Ligands ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Transcriptional Activation/*genetics ; Transfection ; },
abstract = {CRISPR-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene. Here we report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells. This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 5' end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators. By replacing the SBH loop with ligand-controlled RNA-cleaving units, we demonstrate conditional activation of quiescent sgRNAs programmed to respond to genetically encoded or externally delivered triggers. We use this system to couple multiple synthetic and endogenous target genes with specific inducers, and assemble gene regulatory modules demonstrating parallel and orthogonal transcriptional programs. We anticipate that this 'plug and play' approach will be a valuable addition to the synthetic biology toolkit, facilitating the understanding of natural gene circuits and the design of cell-based therapeutic strategies.},
}
@article {pmid28253259,
year = {2017},
author = {Pulido-Quetglas, C and Aparicio-Prat, E and Arnan, C and Polidori, T and Hermoso, T and Palumbo, E and Ponomarenko, J and Guigo, R and Johnson, R},
title = {Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.},
journal = {PLoS computational biology},
volume = {13},
number = {3},
pages = {e1005341},
pmid = {28253259},
issn = {1553-7358},
support = {R01 MH101814/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*genetics ; *Gene Deletion ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; RNA Editing/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.},
}
@article {pmid28250269,
year = {2017},
author = {Machitani, M and Sakurai, F and Wakabayashi, K and Nakatani, K and Takayama, K and Tachibana, M and Mizuguchi, H},
title = {Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression.},
journal = {Biological &amp; pharmaceutical bulletin},
volume = {40},
number = {3},
pages = {272-277},
doi = {10.1248/bpb.b16-00700},
pmid = {28250269},
issn = {1347-5215},
mesh = {A549 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Efficiency ; Endonucleases/metabolism ; Genetic Engineering/*methods ; Genetic Vectors/*immunology ; Genome ; Genotype ; Humans ; Interferon Type I/*biosynthesis/metabolism ; *Mutagenesis ; Mutation ; Plasmids ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome engineering technology is a powerful tool for generation of cells and animals with engineered mutations in their genomes. In order to introduce the CRISPR/Cas9 system into target cells, nonviral and viral vectors are often used; however, such vectors trigger innate immune responses associated with production of type I interferons (IFNs). We have recently demonstrated that type I IFNs inhibit short-hairpin RNA-mediated gene silencing, which led us to hypothesize that type I IFNs may also inhibit CRISPR/Cas9-mediated genome mutagenesis. Here we investigated this hypothesis. A single-strand annealing assay using a reporter plasmid demonstrated that CRISPR/Cas9-mediated cleavage efficiencies of the target double-stranded DNA were significantly reduced by IFNα. A mismatch recognition nuclease-dependent genotyping assay also demonstrated that IFNα reduced insertion or deletion (indel) mutation levels by approximately half. Treatment with IFNα did not alter Cas9 protein expression levels, whereas the copy numbers of guide RNA (gRNA) were significantly reduced by IFNα stimulation. These results indicate that type I IFNs significantly reduce gRNA expression levels following introduction of the CRISPR/Cas9 system in the cells, leading to a reduction in the efficiencies of CRISPR/Cas9-mediated genome mutagenesis. Our findings provide important clues for the achievement of efficient genome engineering using the CRISPR/Cas9 system.},
}
@article {pmid29750482,
year = {2017},
author = {Wang, Y and Xiao, T and Zhu, X and Zhao, X and Wei, D and Zhu, X},
title = {[Identification, cloning and functional verification of U6 promoter from Cryptococcus neoformans].},
journal = {Wei sheng wu xue bao = Acta microbiologica Sinica},
volume = {57},
number = {2},
pages = {197-208},
pmid = {29750482},
issn = {0001-6209},
mesh = {Base Sequence ; *Cloning, Molecular ; Cryptococcus neoformans/*genetics/metabolism ; Gene Library ; Humans ; Molecular Sequence Data ; *Promoter Regions, Genetic ; RNA Interference ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA, Small Nuclear/*genetics/metabolism ; },
abstract = {OBJECTIVE: To identify and clone the polymerase Ⅲ U6 promoter from Cryptococcus neoformans (CnU6 promoter), and verify if CnU6 promoter can effectively transcribe shRNA and gRNA of CRISPR/Cas9 system.<br><br>METHODS: Combining the C. neoformans genome information published in GenBank database and RNA-seq library data from our laboratory, we obtained the U6 RNA sequence with high transcriptional level by bioinformatics analysis. The putative CnU6 promoter was ligated upstream of shRNA and gRNA by EasyGeno and overlapping PCR respectively. Based on shRNA-mediated target gene silence phenotype by RNAi and gene mutation by gRNA-guided Cas9 nuclease mediated target sites editing by CRISPR/Cas9 system, we could identify if CnU6 promoter could drive the transcription of short RNA.<br><br>RESULTS: CnU6 promoter could drive the transcribtion of shRNA, which could silence the target gene, and gRNA, which could guide Cas9 nuclease to cut the target site.<br><br>CONCLUSION: The CnU6 promoter from C. neoformans was successfully identified and cloned, which could drive the transcription of shRNA and gRNA efficiently.},
}
@article {pmid28099430,
year = {2017},
author = {Datlinger, P and Rendeiro, AF and Schmidl, C and Krausgruber, T and Traxler, P and Klughammer, J and Schuster, LC and Kuchler, A and Alpar, D and Bock, C},
title = {Pooled CRISPR screening with single-cell transcriptome readout.},
journal = {Nature methods},
volume = {14},
number = {3},
pages = {297-301},
pmid = {28099430},
issn = {1548-7105},
support = {679146/ERC_/European Research Council/International ; I 1626/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Cell Line ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression Profiling/*methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/methods ; Transcriptome/*genetics ; },
abstract = {CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.},
}
@article {pmid27995572,
year = {2018},
author = {Lin, H and Hu, H and Duan, W and Liu, Y and Tan, G and Li, Z and Liu, Y and Deng, B and Song, X and Wang, W and Wen, D and Wang, Y and Li, C},
title = {Intramuscular Delivery of scAAV9-hIGF1 Prolongs Survival in the hSOD1[G93A] ALS Mouse Model via Upregulation of D-Amino Acid Oxidase.},
journal = {Molecular neurobiology},
volume = {55},
number = {1},
pages = {682-695},
pmid = {27995572},
issn = {1559-1182},
support = {H0912-81171210//the Natural Science Foundation of China/International ; C090301-30870882//National Natural Science Foundation of China/International ; 0647007D//Science and Technological department of Hebei Province/International ; },
mesh = {Amyotrophic Lateral Sclerosis/*pathology ; Animals ; Apoptosis ; D-Amino-Acid Oxidase/*metabolism ; Dependovirus/*metabolism ; Disease Models, Animal ; Disease Progression ; Female ; Gene Transfer Techniques ; Humans ; Injections, Intramuscular ; Insulin-Like Growth Factor I/*administration &amp; dosage ; Male ; Mice, Transgenic ; Motor Neurons/metabolism ; Muscle, Skeletal/metabolism ; Muscular Atrophy/pathology ; Phenotype ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Serine/metabolism ; Superoxide Dismutase/*metabolism ; Survival Analysis ; Transduction, Genetic ; *Up-Regulation ; },
abstract = {Self-complementary adeno-associated viral vector 9 (scAAV9) has been confirmed to be an efficient AAV serotype for gene transfer to the central nervous system (CNS). Neurotrophic factors have been considered to be therapeutic targets for amyotrophic lateral sclerosis (ALS). In the present study, we intramuscularly injected scAAV9 encoding human insulin-like growth factor 1 (hIGF1) into an hSOD1[G93A] ALS mouse model. We observed that scAAV9-hIGF1 significantly reduced the loss of motor neurons of the anterior horn in the lumbar spinal cord and delayed muscle atrophy in ALS mice. Importantly, IGF1 significantly delayed disease onset and prolonged the life span of ALS mice. In addition, scAAV9-hIGF1 protected motor neurons from apoptosis through upregulation of D-amino acid oxidase (DAO), which controls the level of D-serine. Moreover, to further verify these results, we used CRISPR-Cas9 system to target the central nervous system knockdown of IGF1. This experiment supported the continued investigation of neurotrophic factor gene therapies targeting the central nervous system as a potential treatment for ALS.},
}
@article {pmid27984733,
year = {2016},
author = {Adamson, B and Norman, TM and Jost, M and Cho, MY and Nuñez, JK and Chen, Y and Villalta, JE and Gilbert, LA and Horlbeck, MA and Hein, MY and Pak, RA and Gray, AN and Gross, CA and Dixit, A and Parnas, O and Regev, A and Weissman, JS},
title = {A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.},
journal = {Cell},
volume = {167},
number = {7},
pages = {1867-1882.e21},
pmid = {27984733},
issn = {1097-4172},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; R00 CA204602/CA/NCI NIH HHS/United States ; R35 GM118061/GM/NIGMS NIH HHS/United States ; K99 CA204602/CA/NCI NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; R01 GM102790/GM/NIGMS NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; F32 GM116331/GM/NIGMS NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; R01 AG041826/AG/NIA NIH HHS/United States ; P50 HG006193/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endoribonucleases ; Feedback ; Humans ; Models, Molecular ; Protein Serine-Threonine Kinases ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/*methods ; Transcription, Genetic ; Unfolded Protein Response ; },
abstract = {Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.},
}
@article {pmid27896535,
year = {2017},
author = {Cooper, CA and Challagulla, A and Jenkins, KA and Wise, TG and O'Neil, TE and Morris, KR and Tizard, ML and Doran, TJ},
title = {Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).},
journal = {Transgenic research},
volume = {26},
number = {3},
pages = {331-347},
pmid = {27896535},
issn = {1573-9368},
mesh = {Animals ; *Animals, Genetically Modified ; Chick Embryo ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics ; Insemination, Artificial ; Male ; RNA, Guide, CRISPR-Cas Systems ; Spermatozoa/*physiology ; Transcription Factors/genetics ; Transfection/*methods ; },
abstract = {Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.},
}
@article {pmid27874072,
year = {2016},
author = {Zuo, Z and Liu, J},
title = {Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations.},
journal = {Scientific reports},
volume = {5},
number = {},
pages = {37584},
pmid = {27874072},
issn = {2045-2322},
mesh = {Bacterial Proteins/*chemistry/genetics ; CRISPR-Associated Protein 9 ; Catalytic Domain ; Cations, Monovalent ; DNA/*chemistry/genetics ; *DNA Cleavage ; Endonucleases/*chemistry/genetics ; Magnesium/chemistry ; *Molecular Dynamics Simulation ; RNA, Guide, CRISPR-Cas Systems/chemistry ; },
abstract = {The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg[2+] ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg[2+] bound. The simulation results showed that binding of two Mg[2+] ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.},
}
@article {pmid27858321,
year = {2017},
author = {Nie, Y and Cong, P and Liu, X and Wang, M and Chen, Y and He, Z},
title = {Development of a bacterial-based negative selection system for rapid screening of active single guide RNAs.},
journal = {Biotechnology letters},
volume = {39},
number = {3},
pages = {351-358},
doi = {10.1007/s10529-016-2259-0},
pmid = {27858321},
issn = {1573-6776},
mesh = {Animals ; Base Sequence ; Escherichia coli/*genetics ; Genetic Testing/*methods ; HEK293 Cells ; Humans ; INDEL Mutation/genetics ; Mice ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Sus scrofa ; },
abstract = {OBJECTIVES: To develop an in vitro method for rapid evaluation of the capability of a designed single guide RNAs (sgRNAs) to guide Cas9 nucleases to cleave target loci in mammalian cells.<br><br>RESULTS: We constructed a Cas9/sgRNA plasmid with two SP6 promoters to simultaneously express Cas9 nuclease and the sgRNA and a negative selection plasmid harbouring a target site of the sgRNA. After co-transforming chemically competent E. coli DH5&#x3b1; cells with the two plasmids, the transformants were plated at a low density on two LB plates: one containing only ampicillin and the other containing both ampicillin and chloramphenicol. The colony-count on the ampicillin&#xa0;+&#xa0;chloramphenicol plate was compared with that on the ampicillin-only plate to calculate the survival percentage. The survival % was negatively correlated with the genome editing efficiency of the sgRNA in mammalian cells evaluated by a T7 endonuclease 1 (T7E1) assay (r ranged from -0.8 to -0.92). This system eliminates the need for cell culture, transfection, FACS sorting, PCR and T7E1 nuclease treatment, and significantly reduces the cost of screening for active sgRNAs, especially in the case of large-scale screening.<br><br>CONCLUSIONS: We have developed a bacterial-based negative selection system for rapid screening of active sgRNAs in mammalian cells at a very low cost.},
}
@article {pmid27808171,
year = {2016},
author = {Kurata, M and Rathe, SK and Bailey, NJ and Aumann, NK and Jones, JM and Veldhuijzen, GW and Moriarity, BS and Largaespada, DA},
title = {Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.},
journal = {Scientific reports},
volume = {6},
number = {},
pages = {36199},
pmid = {27808171},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; Cell Line ; Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytarabine/pharmacology/*therapeutic use ; DNA, Complementary/genetics ; Deoxycytidine Kinase/genetics ; Dexamethasone/pharmacology ; Drug Resistance, Neoplasm/*genetics ; Equilibrative Nucleoside Transporter 1/genetics ; *Gene Library ; Genetic Loci ; *Genetic Testing ; *Genome, Human ; Glucocorticoids/pharmacology ; Humans ; Inhibitory Concentration 50 ; Leukemia, Myeloid, Acute/*drug therapy/enzymology/genetics ; Mice ; Mutation/genetics ; Prednisolone/pharmacology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Receptors, Glucocorticoid/metabolism ; Reproducibility of Results ; U937 Cells ; },
abstract = {Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.},
}
@article {pmid27661255,
year = {2016},
author = {Horlbeck, MA and Gilbert, LA and Villalta, JE and Adamson, B and Pak, RA and Chen, Y and Fields, AP and Park, CY and Corn, JE and Kampmann, M and Weissman, JS},
title = {Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.},
journal = {eLife},
volume = {5},
number = {},
pages = {},
pmid = {27661255},
issn = {2050-084X},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; T32 GM008284/GM/NIGMS NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; K99 CA204602/CA/NCI NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/*metabolism ; CRISPR-Associated Protein 9 ; Chromosome Mapping ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*metabolism ; Gene Targeting/*methods ; Humans ; Mice ; Nucleosomes/*metabolism ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; },
abstract = {We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.},
}
@article {pmid27804953,
year = {2016},
author = {Lim, Y and Bak, SY and Sung, K and Jeong, E and Lee, SH and Kim, JS and Bae, S and Kim, SK},
title = {Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {13350},
pmid = {27804953},
issn = {2041-1723},
mesh = {CRISPR-Associated Proteins/*metabolism ; DNA/chemistry/metabolism ; Endonucleases/chemistry/*metabolism ; Fluorescence Resonance Energy Transfer ; Models, Biological ; Nucleic Acid Conformation ; Nucleic Acid Heteroduplexes/chemistry/metabolism ; Protein Conformation ; RNA, Guide, CRISPR-Cas Systems/*chemistry/*metabolism ; },
abstract = {The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.},
}
@article {pmid27798611,
year = {2016},
author = {Hess, GT and Frésard, L and Han, K and Lee, CH and Li, A and Cimprich, KA and Montgomery, SB and Bassik, MC},
title = {Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.},
journal = {Nature methods},
volume = {13},
number = {12},
pages = {1036-1042},
pmid = {27798611},
issn = {1548-7105},
support = {DP2 HD084069/HD/NICHD NIH HHS/United States ; R01 HG008150/HG/NHGRI NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; T32 GM007790/GM/NIGMS NIH HHS/United States ; S10 RR025518/RR/NCRR NIH HHS/United States ; R01 ES016486/ES/NIEHS NIH HHS/United States ; },
mesh = {Bortezomib/pharmacology ; CRISPR-Associated Proteins/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytidine Deaminase/genetics ; Directed Molecular Evolution/*methods ; Drug Resistance/genetics ; Green Fluorescent Proteins/genetics ; Humans ; K562 Cells ; Levivirus/genetics ; *Point Mutation ; Proteasome Endopeptidase Complex/genetics ; Protein Engineering/*methods ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.},
}
@article {pmid27723754,
year = {2016},
author = {Ma, Y and Zhang, J and Yin, W and Zhang, Z and Song, Y and Chang, X},
title = {Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells.},
journal = {Nature methods},
volume = {13},
number = {12},
pages = {1029-1035},
pmid = {27723754},
issn = {1548-7105},
mesh = {CRISPR-Associated Proteins/chemistry/*genetics ; Cell Culture Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytidine Deaminase/chemistry/*genetics ; Drug Resistance, Neoplasm/genetics ; Fusion Proteins, bcr-abl/genetics ; Gene Targeting ; Green Fluorescent Proteins/chemistry/genetics ; HEK293 Cells ; Humans ; Imatinib Mesylate/pharmacology ; K562 Cells ; *Mutagenesis, Site-Directed ; Point Mutation ; *RNA, Guide, CRISPR-Cas Systems ; Recombinant Fusion Proteins/chemistry/*genetics ; },
abstract = {A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.},
}
@article {pmid27402906,
year = {2016},
author = {Ma, J and Köster, J and Qin, Q and Hu, S and Li, W and Chen, C and Cao, Q and Wang, J and Mei, S and Liu, Q and Xu, H and Liu, XS},
title = {CRISPR-DO for genome-wide CRISPR design and optimization.},
journal = {Bioinformatics (Oxford, England)},
volume = {32},
number = {21},
pages = {3336-3338},
pmid = {27402906},
issn = {1367-4811},
support = {R01 HG008728/HG/NHGRI NIH HHS/United States ; R01 HG008927/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Computational Biology ; DNA ; Exons ; *Gene Editing ; *Genome ; Humans ; Mice ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {MOTIVATION: Despite the growing popularity in using CRISPR/Cas9 technology for genome editing and gene knockout, its performance still relies on well-designed single guide RNAs (sgRNA). In this study, we propose a web application for the Design and Optimization (CRISPR-DO) of guide sequences that target both coding and non-coding regions in spCas9 CRISPR system across human, mouse, zebrafish, fly and worm genomes. CRISPR-DO uses a computational sequence model to predict sgRNA efficiency, and employs a specificity scoring function to evaluate the potential of off-target effect. It also provides information on functional conservation of target sequences, as well as the overlaps with exons, putative regulatory sequences and single-nucleotide polymorphisms (SNPs). The web application has a user-friendly genome-browser interface to facilitate the selection of the best target DNA sequences for experimental design.<br><br>CRISPR-DO is available at http://cistrome.org/crispr/ CONTACT: qiliu@tongji.edu.cn or hanxu@jimmy.harvard.edu or xsliu@jimmy.harvard.eduSupplementary information: Supplementary data are available at Bioinformatics online.},
}
@article {pmid27768202,
year = {2016},
author = {Yiu, G and Tieu, E and Nguyen, AT and Wong, B and Smit-McBride, Z},
title = {Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {57},
number = {13},
pages = {5490-5497},
pmid = {27768202},
issn = {1552-5783},
support = {K08 EY026101/EY/NEI NIH HHS/United States ; P30 EY012576/EY/NEI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9 ; Cells, Cultured ; Choroidal Neovascularization/genetics/metabolism/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*genetics ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Humans ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Real-Time Polymerase Chain Reaction ; Retinal Pigment Epithelium/cytology/*metabolism ; Vascular Endothelial Growth Factor A/biosynthesis/*genetics ; },
abstract = {PURPOSE: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.<br><br>METHODS: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay.<br><br>RESULTS: Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% &#xb1; 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% &#xb1; 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% &#xb1; 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected.<br><br>CONCLUSIONS: The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.},
}
@article {pmid27708104,
year = {2016},
author = {Sanjana, NE and Wright, J and Zheng, K and Shalem, O and Fontanillas, P and Joung, J and Cheng, C and Regev, A and Zhang, F},
title = {High-resolution interrogation of functional elements in the noncoding genome.},
journal = {Science (New York, N.Y.)},
volume = {353},
number = {6307},
pages = {1545-1549},
pmid = {27708104},
issn = {1095-9203},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; K99 HG008171/HG/NHGRI NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; F32 DK096822/DK/NIDDK NIH HHS/United States ; R00 HG008171/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/pharmacology/therapeutic use ; Bacterial Proteins ; CRISPR-Associated Protein 9 ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cullin Proteins/*genetics ; Drug Resistance, Neoplasm/genetics ; Endonucleases ; Enhancer Elements, Genetic/genetics/*physiology ; *Gene Expression Regulation ; Gene Expression Regulation, Neoplastic ; Genes, Neurofibromatosis 1 ; Genes, Neurofibromatosis 2 ; Genetic Loci ; *Genome, Human ; Genomics/*methods ; Humans ; Indoles/pharmacology/therapeutic use ; Melanoma/drug therapy/genetics ; Mutagenesis ; Proto-Oncogene Proteins B-raf/antagonists &amp; inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sulfonamides/pharmacology/therapeutic use ; Vemurafenib ; },
abstract = {The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ~18,000 single guide RNAs targeting >700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.},
}
@article {pmid27576906,
year = {2017},
author = {Gebler, C and Lohoff, T and Paszkowski-Rogacz, M and Mircetic, J and Chakraborty, D and Camgoz, A and Hamann, MV and Theis, M and Thiede, C and Buchholz, F},
title = {Inactivation of Cancer Mutations Utilizing CRISPR/Cas9.},
journal = {Journal of the National Cancer Institute},
volume = {109},
number = {1},
pages = {},
pmid = {27576906},
issn = {1460-2105},
mesh = {*Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9 ; Carcinoma/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Colonic Neoplasms/*genetics ; Computational Biology ; DNA Cleavage ; *Endonucleases/genetics/metabolism ; Genes, Reporter ; Green Fluorescent Proteins/genetics ; HeLa Cells ; Humans ; Leukemia, Myeloid, Acute/*genetics ; Mutation/*genetics ; Nuclear Proteins/genetics ; Nucleophosmin ; Proto-Oncogene Proteins B-raf/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Transfection ; },
abstract = {Although whole-genome sequencing has uncovered a large number of mutations that drive tumorigenesis, functional ratification for most mutations remains sparse. Here, we present an approach to test functional relevance of tumor mutations employing CRISPR/Cas9. Combining comprehensive sgRNA design and an efficient reporter assay to nominate efficient and selective sgRNAs, we establish a pipeline to dissect roles of cancer mutations with potential applicability to personalized medicine and future therapeutic use.},
}
@article {pmid27418421,
year = {2017},
author = {Chuai, GH and Wang, QL and Liu, Q},
title = {In Silico Meets In Vivo: Towards Computational CRISPR-Based sgRNA Design.},
journal = {Trends in biotechnology},
volume = {35},
number = {1},
pages = {12-21},
doi = {10.1016/j.tibtech.2016.06.008},
pmid = {27418421},
issn = {1879-3096},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computer Simulation ; Gene Editing/*methods ; Gene Targeting/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Models, Genetic ; RNA Editing/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Sequence Analysis, RNA/*methods ; },
abstract = {CRISPR-based genome editing has been widely implemented in various cell types. In silico single guide RNA (sgRNA) design is a key step for successful gene editing using the CRISPR system, and continuing efforts are aimed at refining in silico sgRNA design with high on-target efficacy and reduced off-target effects. Many sgRNA design tools are available, but careful assessments of their application scenarios and performance benchmarks across different types of genome-editing data are needed. Efficient in silico models can be built that integrate current heterogeneous genome-editing data to derive unbiased sgRNA design rules and identify key features for improving sgRNA design. Comprehensive evaluation of on-target and off-target effects of sgRNA will allow more precise genome editing and gene therapies using the CRISPR system.},
}
@article {pmid27209600,
year = {2016},
author = {Yang, Y and Liu, S and Cheng, Y and Nie, L and Lv, C and Wang, G and Zhang, Y and Hao, L},
title = {Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter.},
journal = {Applied biochemistry and biotechnology},
volume = {180},
number = {4},
pages = {655-667},
doi = {10.1007/s12010-016-2122-8},
pmid = {27209600},
issn = {1559-0291},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Proteins/*metabolism ; DNA Repair ; Fetus/cytology ; Fibroblasts/metabolism ; Fluorescent Dyes/*metabolism ; Gene Targeting ; *Genes, Reporter ; Genetic Vectors/metabolism ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; Sus scrofa ; },
abstract = {The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.},
}
@article {pmid27198218,
year = {2016},
author = {Fu, BX and St Onge, RP and Fire, AZ and Smith, JD},
title = {Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo.},
journal = {Nucleic acids research},
volume = {44},
number = {11},
pages = {5365-5377},
pmid = {27198218},
issn = {1362-4962},
support = {P01 HG000205/HG/NHGRI NIH HHS/United States ; R01 GM037706/GM/NIGMS NIH HHS/United States ; T32 GM007790/GM/NIGMS NIH HHS/United States ; U01 GM110706/GM/NIGMS NIH HHS/United States ; },
mesh = {*Base Pair Mismatch ; Base Sequence ; Endonucleases/*metabolism ; Genetic Variation ; RNA, Guide, CRISPR-Cas Systems/*genetics/*metabolism ; Recombination, Genetic ; Substrate Specificity ; },
abstract = {Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.},
}
@article {pmid27178736,
year = {2016},
author = {Vad-Nielsen, J and Lin, L and Bolund, L and Nielsen, AL and Luo, Y},
title = {Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {73},
number = {22},
pages = {4315-4325},
pmid = {27178736},
issn = {1420-9071},
mesh = {Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Fluorescent Dyes/metabolism ; Gene Deletion ; *Gene Expression ; Gene Targeting/*methods ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Loci ; Humans ; Plasmids/metabolism ; Polymerase Chain Reaction ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Reproducibility of Results ; },
abstract = {The engineered CRISPR/Cas9 technology has developed as the most efficient and broadly used genome editing tool. However, simultaneously targeting multiple genes (or genomic loci) in the same individual cells using CRISPR/Cas9 remain one technical challenge. In this article, we have developed a Golden Gate Assembly method for the generation of CRISPR gRNA expression arrays, thus enabling simultaneous gene targeting. Using this method, the generation of CRISPR gRNA expression array can be accomplished in 2 weeks, and contains up to 30 gRNA expression cassettes. We demonstrated in the study that simultaneously targeting 10 genomic loci or simultaneously inhibition of multiple endogenous genes could be achieved using the multiplexed gRNA expression array vector in human cells. The complete set of plasmids is available through the non-profit plasmid repository Addgene.},
}
@article {pmid27159246,
year = {2016},
author = {Brown, A and Woods, WS and Perez-Pinera, P},
title = {Multiplexed Targeted Genome Engineering Using a Universal Nuclease-Assisted Vector Integration System.},
journal = {ACS synthetic biology},
volume = {5},
number = {7},
pages = {582-588},
doi = {10.1021/acssynbio.6b00056},
pmid = {27159246},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Protein 9 ; Cortactin/genetics ; Endonucleases/genetics ; Gene Editing ; Gene Knockout Techniques/*methods ; Genetic Engineering/*methods ; Genetic Vectors ; Genome ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics ; Green Fluorescent Proteins/genetics ; HCT116 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems ; Transcription Activator-Like Effector Nucleases ; Transfection ; Transgenes ; },
abstract = {Engineered nucleases are capable of efficiently modifying complex genomes through introduction of targeted double-strand breaks. However, mammalian genome engineering remains limited by low efficiency of heterologous DNA integration at target sites, which is typically performed through homologous recombination, a complex, ineffective and costly process. In this study, we developed a multiplexable and universal nuclease-assisted vector integration system for rapid generation of gene knock outs using selection that does not require customized targeting vectors, thereby minimizing the cost and time frame needed for gene editing. Importantly, this system is capable of remodeling native mammalian genomes through integration of DNA, up to 50 kb, enabling rapid generation and screening of multigene knockouts from a single transfection. These results support that nuclease assisted vector integration is a robust tool for genome-scale gene editing that will facilitate diverse applications in synthetic biology and gene therapy.},
}
@article {pmid27075976,
year = {2016},
author = {Singh, AM and Perry, DW and Steffey, VVA and Miller, K and Allison, DW},
title = {Decoding the Epigenetic Heterogeneity of Human Pluripotent Stem Cells with Seamless Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1516},
number = {},
pages = {153-169},
doi = {10.1007/7651_2016_324},
pmid = {27075976},
issn = {1940-6029},
mesh = {Cell Differentiation/genetics ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cyclin-Dependent Kinase 2/genetics ; Epigenesis, Genetic/genetics ; Gene Editing/*methods ; Genetic Heterogeneity ; Histone-Lysine N-Methyltransferase/*genetics ; Human Embryonic Stem Cells/*cytology ; Humans ; Pluripotent Stem Cells/*cytology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase, MLL2 (KMT2B), during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here, we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically, by utilizing dimeric CRISPR RNA-guided nucleases, RFNs (commercially known as the NextGEN™ CRISPR), in combination with an excision-only piggyBac™ transposase, we demonstrate how to generate a point mutation of threonine-542, a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection, and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless "donor-excision" approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations, as the use of ssODN necessitate additional mutations in the donor PAM sequence, along with extensive cloning efforts. The method described here therefore provides the highest targeting efficiency with the lowest "off-target" mutation rates possible, while removing the labor-intensive efforts associated with screening thousands of clones. In sum, this chapter describes how seamless gene editing may be utilized to examine stem cell heterogeneity of epigenetic marks, but is also widely applicable for performing precise genetic manipulations in numerous other cell types.},
}
@article {pmid27072506,
year = {2016},
author = {Bassalo, MC and Garst, AD and Halweg-Edwards, AL and Grau, WC and Domaille, DW and Mutalik, VK and Arkin, AP and Gill, RT},
title = {Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli.},
journal = {ACS synthetic biology},
volume = {5},
number = {7},
pages = {561-568},
doi = {10.1021/acssynbio.5b00187},
pmid = {27072506},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; Butanols/metabolism ; CRISPR-Associated Protein 9 ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Escherichia coli/*genetics/*metabolism ; Escherichia coli Proteins/genetics ; Genetic Engineering/methods ; Genome, Bacterial ; Green Fluorescent Proteins/genetics/metabolism ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways ; MutS DNA Mismatch-Binding Protein/genetics ; Mutation ; RNA, Guide, CRISPR-Cas Systems ; Reproducibility of Results ; },
abstract = {Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.},
}
@article {pmid27052803,
year = {2016},
author = {Choi, JG and Dang, Y and Abraham, S and Ma, H and Zhang, J and Guo, H and Cai, Y and Mikkelsen, JG and Wu, H and Shankar, P and Manjunath, N},
title = {Lentivirus pre-packed with Cas9 protein for safer gene editing.},
journal = {Gene therapy},
volume = {23},
number = {7},
pages = {627-633},
pmid = {27052803},
issn = {1476-5462},
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9 ; Chromosomes, Human, Pair 4/genetics ; Endonucleases/*genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors/genetics ; HEK293 Cells ; HIV/genetics ; Humans ; Jurkat Cells ; Lentivirus/*genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Receptors, CCR5/genetics/metabolism ; Terminal Repeat Sequences ; Virus Assembly ; },
abstract = {The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.},
}
@article {pmid27032928,
year = {2016},
author = {Zeng, B and Zhan, S and Wang, Y and Huang, Y and Xu, J and Liu, Q and Li, Z and Huang, Y and Tan, A},
title = {Expansion of CRISPR targeting sites in Bombyx mori.},
journal = {Insect biochemistry and molecular biology},
volume = {72},
number = {},
pages = {31-40},
doi = {10.1016/j.ibmb.2016.03.006},
pmid = {27032928},
issn = {1879-0240},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Nonmammalian ; Endonucleases/genetics/metabolism ; Gene Targeting ; Green Fluorescent Proteins/genetics ; Mutagenesis ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.},
}
@article {pmid27025950,
year = {2016},
author = {Cheng, J and Roden, CA and Pan, W and Zhu, S and Baccei, A and Pan, X and Jiang, T and Kluger, Y and Weissman, SM and Guo, S and Flavell, RA and Ding, Y and Lu, J},
title = {A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {11178},
pmid = {27025950},
issn = {2041-1723},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; R01 CA149109/CA/NCI NIH HHS/United States ; R01 GM099811/GM/NIGMS NIH HHS/United States ; R01GM099811/GM/NIGMS NIH HHS/United States ; P30 CA016359/CA/NCI NIH HHS/United States ; R01CA149109/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9 ; Cell Line ; Chromosome Mapping/*methods ; Cloning, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/metabolism ; DNA Restriction Enzymes/genetics/metabolism ; Endonucleases/genetics/metabolism ; Gene Library ; *Genome ; Humans ; Mice ; MicroRNAs/*genetics/metabolism ; Oligonucleotide Array Sequence Analysis ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; *Untranslated Regions ; },
abstract = {Clustered regularly-interspaced palindromic repeats (CRISPR)-based genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify genetic regulators. Applying CRISPR screens to interrogate functional elements in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. Here we present a Molecular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a proof-of-principle screen that identifies novel cis-regulatory domains for miR-142 biogenesis. The Molecular Chipper approach utilizes a combination of random fragmentation and a type III restriction enzyme to derive a densely covering sgRNA library from input DNA. Applying this approach to 17 microRNAs and their flanking regions and with a reporter for miR-142 activity, we identify both the pre-miR-142 region and two previously unrecognized cis-domains important for miR-142 biogenesis, with the latter regulating miR-142 processing. This strategy will be useful for identifying functional noncoding elements in mammalian genomes.},
}
@article {pmid26990633,
year = {2016},
author = {Yin, C and Zhang, T and Li, F and Yang, F and Putatunda, R and Young, WB and Khalili, K and Hu, W and Zhang, Y},
title = {Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS.},
journal = {AIDS (London, England)},
volume = {30},
number = {8},
pages = {1163-1174},
pmid = {26990633},
issn = {1473-5571},
support = {T32 MH079785/MH/NIMH NIH HHS/United States ; R01 DK075964/DK/NIDDK NIH HHS/United States ; R01 NS087971/NS/NINDS NIH HHS/United States ; P30 MH092177/MH/NIMH NIH HHS/United States ; R21 MH100949/MH/NIMH NIH HHS/United States ; },
mesh = {Acquired Immunodeficiency Syndrome/therapy/*virology ; Anti-HIV Agents/*metabolism ; Biological Products/*metabolism ; Computational Biology ; *Gene Expression Regulation, Viral ; Genes, Reporter ; *Genetic Testing ; Genome, Viral ; Genotyping Techniques ; HEK293 Cells ; HIV-1/*drug effects/genetics ; Humans ; Luciferases/analysis/genetics ; Polymerase Chain Reaction ; Proviruses/drug effects/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/*metabolism ; Recombination, Genetic ; Virus Latency/drug effects ; },
abstract = {OBJECTIVE: There is an urgent need for the development of HIV-1 genome eradication strategies that lead to a permanent cure for HIV-1/AIDS. We previously reported that four guide RNAs (gRNAs) targeting HIV-1 long terminal repeats (LTR) effectively eradicated the entire HIV-1 genome. In this study, we sought to identify the best gRNAs targeting HIV-1 LTR and viral structural region and optimize gRNA pairing that can efficiently eradicate the HIV-1 genome.<br><br>DESIGN: Highly specific gRNAs were designed using bioinformatics tools, and their capacity of guiding CRISPR-associated system 9 to cleave HIV-1 proviral DNA was evaluated using high-throughput HIV-1 luciferase reporter assay and rapid Direct-PCR genotyping.<br><br>METHODS: The target seed sequences for each gRNA were cloned into lentiviral vectors. HEK293T cells were cotransfected with a pEcoHIV-NL4-3-firefly-luciferase reporter vector (1&#x200a;:&#x200a;20) over lentiviral vectors carrying CRISPR-associated system 9 and single gRNA or various combinations of gRNAs. The EcoHIV DNA cleaving efficiency was evaluated by Direct-PCR genotyping, and the EcoHIV transcription/replication activity was examined by a luciferase reporter assay.<br><br>RESULTS: Most of the designed gRNAs are effective to eliminate the predicted HIV-1 genome sequence between the selected two target sites. This is evidenced by the presence of PCR genotypic deletion/insertion and the decrease of luciferase reporter activity. In particular, a combination of viral structural gRNAs with LTR gRNAs provided a higher efficiency of genome eradication and an easier approach for PCR genotyping.<br><br>CONCLUSION: Our screening strategy can specifically and effectively identify gRNAs targeting HIV-1 LTR and structural region to excise proviral HIV-1 from the host genome.},
}
@article {pmid26960129,
year = {2016},
author = {Olieric, V and Weinert, T and Finke, AD and Anders, C and Li, D and Olieric, N and Borca, CN and Steinmetz, MO and Caffrey, M and Jinek, M and Wang, M},
title = {Data-collection strategy for challenging native SAD phasing.},
journal = {Acta crystallographica. Section D, Structural biology},
volume = {72},
number = {Pt 3},
pages = {421-429},
pmid = {26960129},
issn = {2059-7983},
mesh = {Animals ; CRISPR-Associated Proteins/chemistry ; Cattle ; Crystallography, X-Ray/*methods ; DNA/chemistry ; Diacylglycerol Kinase/chemistry ; Escherichia coli/chemistry/enzymology ; Models, Molecular ; Protein Conformation ; Proteins/*chemistry ; RNA, Guide, CRISPR-Cas Systems/chemistry ; Rats ; Staphylococcus/chemistry/enzymology ; Stathmin/chemistry ; Tubulin/chemistry ; },
abstract = {Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.},
}
@article {pmid26879140,
year = {2016},
author = {Zhang, H and Zhang, X and Fan, C and Xie, Q and Xu, C and Zhao, Q and Liu, Y and Wu, X and Zhang, H},
title = {A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.},
journal = {Biochemical and biophysical research communications},
volume = {471},
number = {4},
pages = {528-532},
doi = {10.1016/j.bbrc.2016.02.041},
pmid = {26879140},
issn = {1090-2104},
mesh = {Bacterial Proteins ; CRISPR-Associated Protein 9 ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; Gene Knockout Techniques ; *Genetic Engineering ; HEK293 Cells ; HT29 Cells ; Humans ; Lentivirus ; Luciferases/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics/*isolation &amp; purification ; *Selection, Genetic ; Transfection ; },
abstract = {CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs.},
}
@article {pmid26837577,
year = {2016},
author = {Lee, YJ and Hoynes-O'Connor, A and Leong, MC and Moon, TS},
title = {Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.},
journal = {Nucleic acids research},
volume = {44},
number = {5},
pages = {2462-2473},
pmid = {26837577},
issn = {1362-4962},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Design ; Escherichia coli/*genetics/metabolism ; *Gene Expression Regulation, Bacterial ; Gene Targeting/methods ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Nucleic Acid Hybridization ; Plasmids/chemistry/metabolism ; RNA, Antisense/chemical synthesis/*metabolism ; RNA, Guide, CRISPR-Cas Systems/*antagonists &amp; inhibitors/genetics ; Streptococcus pyogenes/*genetics/metabolism ; },
abstract = {A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.},
}
@article {pmid26824432,
year = {2016},
author = {Farasat, I and Salis, HM},
title = {A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation.},
journal = {PLoS computational biology},
volume = {12},
number = {1},
pages = {e1004724},
pmid = {26824432},
issn = {1553-7358},
mesh = {Bacteriophage lambda/genetics ; CRISPR-Associated Proteins/*chemistry/*genetics/metabolism ; Computer Simulation ; DNA/chemistry/genetics/metabolism ; Gene Expression Regulation/*genetics/physiology ; Genome/genetics ; *Models, Genetic ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/chemistry/*genetics/metabolism ; },
abstract = {The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9's off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits.},
}
@article {pmid26729910,
year = {2016},
author = {Du, D and Qi, LS},
title = {CRISPR Technology for Genome Activation and Repression in Mammalian Cells.},
journal = {Cold Spring Harbor protocols},
volume = {2016},
number = {1},
pages = {pdb.prot090175},
doi = {10.1101/pdb.prot090175},
pmid = {26729910},
issn = {1559-6095},
mesh = {Animals ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; *Databases, Genetic ; Genome/*physiology ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/physiology ; Transcription, Genetic ; },
abstract = {Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.},
}
@article {pmid26589814,
year = {2015},
author = {Rahdar, M and McMahon, MA and Prakash, TP and Swayze, EE and Bennett, CF and Cleveland, DW},
title = {Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {112},
number = {51},
pages = {E7110-7},
pmid = {26589814},
issn = {1091-6490},
support = {R01 NS027036/NS/NINDS NIH HHS/United States ; R01-GM 074150/GM/NIGMS NIH HHS/United States ; F32 GM109657/GM/NIGMS NIH HHS/United States ; R01 GM074150/GM/NIGMS NIH HHS/United States ; R01-NS27036/NS/NINDS NIH HHS/United States ; F32-GM109657/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Synthetic ; Genetic Engineering ; *Genome, Human ; HEK293 Cells ; Humans ; Models, Genetic ; Molecular Sequence Data ; Nucleic Acid Conformation ; RNA/chemistry/genetics/metabolism ; *RNA Editing ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.},
}
@article {pmid26538064,
year = {2015},
author = {Zhang, Y and Yin, C and Zhang, T and Li, F and Yang, W and Kaminski, R and Fagan, PR and Putatunda, R and Young, WB and Khalili, K and Hu, W},
title = {CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs.},
journal = {Scientific reports},
volume = {5},
number = {},
pages = {16277},
pmid = {26538064},
issn = {2045-2322},
support = {T32 MH079785/MH/NIMH NIH HHS/United States ; R01 MH101041/MH/NIMH NIH HHS/United States ; R01 DK075964/DK/NIDDK NIH HHS/United States ; R01NS087971/NS/NINDS NIH HHS/United States ; R01 NS087971/NS/NINDS NIH HHS/United States ; P30 MH092177/MH/NIMH NIH HHS/United States ; R01DK075964/DK/NIDDK NIH HHS/United States ; R01MH101041/MH/NIMH NIH HHS/United States ; P30MH092177/MH/NIMH NIH HHS/United States ; R21 MH100949/MH/NIMH NIH HHS/United States ; },
mesh = {Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; HIV Infections/*virology ; HIV Long Terminal Repeat/genetics ; HIV-1/*genetics ; Humans ; Jurkat Cells ; Proviruses/genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; T-Lymphocytes/virology ; Virus Activation/*genetics ; Virus Latency/*genetics ; },
abstract = {Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a "shock and kill" strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs.},
}
@article {pmid26436575,
year = {2015},
author = {Dahlman, JE and Abudayyeh, OO and Joung, J and Gootenberg, JS and Zhang, F and Konermann, S},
title = {Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease.},
journal = {Nature biotechnology},
volume = {33},
number = {11},
pages = {1159-1161},
pmid = {26436575},
issn = {1546-1696},
support = {DP1 MH100706/MH/NIMH NIH HHS/United States ; 1DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9 ; Endonucleases/*genetics ; Gene Knockout Techniques/*methods ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, can activate gene expression using an active Streptococcus pyogenes Cas9 nuclease, without inducing double-stranded breaks. We use these 'dead RNAs' to perform orthogonal gene knockout and transcriptional activation in human cells.},
}
@article {pmid26324940,
year = {2015},
author = {Deng, W and Shi, X and Tjian, R and Lionnet, T and Singer, RH},
title = {CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {112},
number = {38},
pages = {11870-11875},
pmid = {26324940},
issn = {1091-6490},
support = {//Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Binding Sites ; CRISPR-Associated Proteins/*metabolism ; Carbocyanines/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Color ; DNA/metabolism ; Electrophoretic Mobility Shift Assay ; Embryo, Mammalian/cytology ; Fibroblasts/metabolism ; Fluorescent Dyes/metabolism ; *Genetic Loci ; Humans ; In Situ Hybridization, Fluorescence ; Mice ; RNA, Guide, CRISPR-Cas Systems ; *Staining and Labeling ; },
abstract = {Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.},
}
@article {pmid26297141,
year = {2015},
author = {Lowder, LG and Zhang, D and Baltes, NJ and Paul, JW and Tang, X and Zheng, X and Voytas, DF and Hsieh, TF and Zhang, Y and Qi, Y},
title = {A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.},
journal = {Plant physiology},
volume = {169},
number = {2},
pages = {971-985},
pmid = {26297141},
issn = {1532-2548},
mesh = {Arabidopsis/genetics ; Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9 ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Methylation ; DNA, Bacterial ; Endonucleases/*genetics ; *Gene Expression Regulation, Plant ; Genetic Engineering/*methods ; Genome, Plant ; Genomic Imprinting ; Mutagenesis ; Oryza/genetics ; Plant Proteins/genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems ; Nicotiana/genetics ; },
abstract = {The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.},
}
@article {pmid26294043,
year = {2015},
author = {Li, Z and Liu, ZB and Xing, A and Moon, BP and Koellhoffer, JP and Huang, L and Ward, RT and Clifton, E and Falco, SC and Cigan, AM},
title = {Cas9-Guide RNA Directed Genome Editing in Soybean.},
journal = {Plant physiology},
volume = {169},
number = {2},
pages = {960-970},
pmid = {26294043},
issn = {1532-2548},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Protein 9 ; DNA End-Joining Repair ; Endonucleases/genetics ; Genetic Engineering/*methods ; Genome, Plant ; Homologous Recombination ; Mutagenesis, Insertional ; Plant Proteins/genetics ; Plants, Genetically Modified ; RNA Editing ; *RNA, Guide, CRISPR-Cas Systems ; Glycine max/drug effects/*genetics ; Sulfonamides/pharmacology ; Triazines/pharmacology ; },
abstract = {Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.},
}
@article {pmid26269544,
year = {2015},
author = {Svitashev, S and Young, JK and Schwartz, C and Gao, H and Falco, SC and Cigan, AM},
title = {Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA.},
journal = {Plant physiology},
volume = {169},
number = {2},
pages = {931-945},
pmid = {26269544},
issn = {1532-2548},
mesh = {Acetolactate Synthase/genetics ; Agrobacterium/genetics ; Amino Acid Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Molecular Sequence Data ; Mutagenesis, Insertional/methods ; Mutation ; Plant Breeding/methods ; Plant Proteins/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Zea mays/*genetics ; },
abstract = {Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.},
}
@article {pmid26126518,
year = {2015},
author = {Fine, EJ and Appleton, CM and White, DE and Brown, MT and Deshmukh, H and Kemp, ML and Bao, G},
title = {Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes.},
journal = {Scientific reports},
volume = {5},
number = {},
pages = {10777},
pmid = {26126518},
issn = {2045-2322},
mesh = {Amino Acid Sequence ; Base Sequence ; Blotting, Western ; CRISPR-Associated Proteins/chemistry/*metabolism ; Cell Line ; Endonucleases/metabolism ; Genetic Vectors/*metabolism ; Humans ; Molecular Sequence Data ; RNA, Guide, CRISPR-Cas Systems/genetics ; Receptors, CCR5/*genetics ; Sequence Analysis, DNA ; Streptococcus pyogenes/metabolism ; Trans-Splicing/*genetics ; beta-Globins/*genetics ; },
abstract = {CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9) displayed ~35% of the nuclease activity compared with the wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had substantially decreased activity at lower dosing levels. The greatly reduced open reading frame length of the tsSpCas9 relative to wtSpCas9 potentially allows for more complex and longer genetic elements to be packaged into an AAV vector including tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions to SpCas9. For unknown reasons, the tsSpCas9 system did not work in all cell types tested. The use of protein trans-splicing may help facilitate exciting new avenues of research and therapeutic applications through AAV-based delivery of CRISPR/Cas9 systems.},
}
@article {pmid26063738,
year = {2015},
author = {Xu, H and Xiao, T and Chen, CH and Li, W and Meyer, CA and Wu, Q and Wu, D and Cong, L and Zhang, F and Liu, JS and Brown, M and Liu, XS},
title = {Sequence determinants of improved CRISPR sgRNA design.},
journal = {Genome research},
volume = {25},
number = {8},
pages = {1147-1157},
pmid = {26063738},
issn = {1549-5469},
support = {U01 CA180980/CA/NCI NIH HHS/United States ; P50 CA090381/CA/NCI NIH HHS/United States ; R01 GM113242/GM/NIGMS NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; R01 HG008728/HG/NHGRI NIH HHS/United States ; R01 GM113242-01/GM/NIGMS NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; DNA/analysis ; Gene Knockout Techniques ; HL-60 Cells ; Humans ; Models, Genetic ; Mutation Rate ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; },
abstract = {The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.},
}
@article {pmid26050089,
year = {2015},
author = {Wang, J and Li, X and Zhao, Y and Li, J and Zhou, Q and Liu, Z},
title = {Generation of cell-type-specific gene mutations by expressing the sgRNA of the CRISPR system from the RNA polymerase II promoters.},
journal = {Protein &amp; cell},
volume = {6},
number = {9},
pages = {689-692},
pmid = {26050089},
issn = {1674-8018},
mesh = {Animals ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression ; Genetic Engineering/*methods ; Mice ; Molecular Sequence Data ; *Mutation ; Promoter Regions, Genetic/*genetics ; RNA Polymerase II/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
}
@article {pmid25827874,
year = {2015},
author = {Fujita, T and Fujii, H},
title = {Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1288},
number = {},
pages = {43-52},
doi = {10.1007/978-1-4939-2474-5_4},
pmid = {25827874},
issn = {1940-6029},
mesh = {Chromatin/*genetics/*metabolism ; *Chromatin Immunoprecipitation/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression ; Genetic Vectors/genetics ; *Genome ; *High-Throughput Nucleotide Sequencing/methods ; Mass Spectrometry ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we describe engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using the CRISPR system, for purification of specific genomic regions retaining molecular interactions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP combined with mass spectrometry (enChIP-MS), next-generation sequencing (enChIP-Seq), and RNA-Seq (enChIP-RNA-Seq) can identify proteins, other genomic regions, and RNA, respectively, that interact with the target genomic region.},
}
@article {pmid25740494,
year = {2015},
author = {Flowers, GP and Crews, CM},
title = {Generating and identifying axolotls with targeted mutations using Cas9 RNA-guided nuclease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1290},
number = {},
pages = {279-295},
doi = {10.1007/978-1-4939-2495-0_22},
pmid = {25740494},
issn = {1940-6029},
mesh = {Ambystoma mexicanum/embryology/*genetics ; Animals ; DNA/genetics/isolation &amp; purification ; Deoxyribonucleases/*metabolism ; Embryo, Nonmammalian/metabolism ; Endonucleases/*genetics ; Female ; Male ; Microinjections ; Mutagenesis, Site-Directed/*methods ; *Mutation ; Polymerase Chain Reaction ; RNA, Guide, CRISPR-Cas Systems/*genetics ; RNA, Messenger/biosynthesis/genetics ; },
abstract = {The CRISPR/Cas9 RNA-guided nuclease now enables a reverse genetics approach to investigate the function of genes of interest during regeneration in the axolotl. The process of generating the constructs necessary for targeting a gene of interest is considerably less labor intensive than for other methods of targeted mutagenesis such as Zinc finger nucleases or Transcription activator-like effector nucleases. Here, we describe the identification of targetable sequences in the gene of interest, the construction of unique guide RNAs, the microinjection of these RNAs with Cas9-encoding mRNA, the selection of well-injected animals, and an inexpensive, PCR-based method for identifying highly mutagenized animals.},
}
@article {pmid25732831,
year = {2015},
author = {Aubrey, BJ and Kelly, GL and Kueh, AJ and Brennan, MS and O'Connor, L and Milla, L and Wilcox, S and Tai, L and Strasser, A and Herold, MJ},
title = {An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo.},
journal = {Cell reports},
volume = {10},
number = {8},
pages = {1422-1432},
doi = {10.1016/j.celrep.2015.02.002},
pmid = {25732831},
issn = {2211-1247},
mesh = {Animals ; Apoptosis Regulatory Proteins/genetics/metabolism ; Base Sequence ; Bcl-2-Like Protein 11 ; Cell Line ; Cell Survival/drug effects ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA End-Joining Repair ; Doxycycline/pharmacology ; Genes, Essential/*genetics ; Genes, Neoplasm/*genetics ; Humans ; Lentivirus/*genetics ; Membrane Proteins/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Transgenic ; Mutation ; Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism ; Neoplasms/*genetics/metabolism/pathology ; Proto-Oncogene Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; Transplantation, Heterologous ; Tumor Suppressor Protein p53/deficiency/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.},
}
@article {pmid25712100,
year = {2015},
author = {O'Geen, H and Henry, IM and Bhakta, MS and Meckler, JF and Segal, DJ},
title = {A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.},
journal = {Nucleic acids research},
volume = {43},
number = {6},
pages = {3389-3404},
pmid = {25712100},
issn = {1362-4962},
support = {GM097073/GM/NIGMS NIH HHS/United States ; RR 026825/RR/NCRR NIH HHS/United States ; HL086350/HL/NHLBI NIH HHS/United States ; T32 HL086350/HL/NHLBI NIH HHS/United States ; RR12088/RR/NCRR NIH HHS/United States ; R21 HG006761/HG/NHGRI NIH HHS/United States ; CA0933730/CA/NCI NIH HHS/United States ; HG006761/HG/NHGRI NIH HHS/United States ; RR12964/RR/NCRR NIH HHS/United States ; R01 GM097073/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; Base Sequence ; Binding Sites/genetics ; CRISPR-Associated Proteins/*genetics/*metabolism ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome-Wide Association Study ; INDEL Mutation ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.},
}
@article {pmid25664545,
year = {2015},
author = {Kim, D and Bae, S and Park, J and Kim, E and Kim, S and Yu, HR and Hwang, J and Kim, JI and Kim, JS},
title = {Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.},
journal = {Nature methods},
volume = {12},
number = {3},
pages = {237-43, 1 p following 243},
pmid = {25664545},
issn = {1548-7105},
mesh = {Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics/metabolism ; Genome, Human ; Haploidy ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Limit of Detection ; Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Sensitivity and Specificity ; },
abstract = {Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.},
}
@article {pmid25585508,
year = {2015},
author = {Randau, L},
title = {Evolution of small guide RNA genes in hyperthermophilic archaea.},
journal = {Annals of the New York Academy of Sciences},
volume = {1341},
number = {},
pages = {188-193},
doi = {10.1111/nyas.12643},
pmid = {25585508},
issn = {1749-6632},
mesh = {Archaea/*genetics ; Base Sequence ; *Evolution, Molecular ; Gene Expression Regulation, Archaeal ; Genome, Archaeal/genetics ; Hot Temperature ; Models, Genetic ; RNA, Archaeal/*genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; },
abstract = {Profiling the RNA production in hyperthermophilic archaea revealed an abundance of small RNA-guided processes near the upper temperature limit of life. Archaea utilize the base-pairing ability of RNA guide sequences to target ribosomal RNAs, transfer RNAs, messenger RNAs, and viral genomes. Cellular processes that are guided by small RNAs include the modification of RNA molecules, trans-splicing, gene regulation, and RNA and DNA degradation. Here, a brief overview of our knowledge on small guide RNA genes in archaeal genomes is provided and examples of their putative roles in genome evolution are described.},
}
@article {pmid25545896,
year = {2015},
author = {Osborn, MJ and Gabriel, R and Webber, BR and DeFeo, AP and McElroy, AN and Jarjour, J and Starker, CG and Wagner, JE and Joung, JK and Voytas, DF and von Kalle, C and Schmidt, M and Blazar, BR and Tolar, J},
title = {Fanconi anemia gene editing by the CRISPR/Cas9 system.},
journal = {Human gene therapy},
volume = {26},
number = {2},
pages = {114-126},
pmid = {25545896},
issn = {1557-7422},
support = {T32 HD060536/HD/NICHD NIH HHS/United States ; UL1TR000114/TR/NCATS NIH HHS/United States ; T32 HL007062/HL/NHLBI NIH HHS/United States ; P01 CA065493/CA/NCI NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; 8UL1TR000114-02/TR/NCATS NIH HHS/United States ; UL1 TR000114/TR/NCATS NIH HHS/United States ; KL2 TR000113/TR/NCATS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Deoxyribonuclease I/genetics/*metabolism ; Electroporation ; Fanconi Anemia/genetics/metabolism/pathology/therapy ; Fanconi Anemia Complementation Group C Protein/genetics/*metabolism ; Fibroblasts/*metabolism/pathology ; Gene Expression ; Genetic Engineering ; Genetic Loci ; *Genome, Human ; Humans ; Lipids ; Molecular Sequence Data ; Molecular Targeted Therapy ; Plasmids/chemistry/metabolism ; Polymerase Chain Reaction/methods ; Primary Cell Culture ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/chemistry/enzymology ; Transfection ; },
abstract = {Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.},
}
@article {pmid25541967,
year = {2014},
author = {Wang, W and Ye, C and Liu, J and Zhang, D and Kimata, JT and Zhou, P},
title = {CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.},
journal = {PloS one},
volume = {9},
number = {12},
pages = {e115987},
pmid = {25541967},
issn = {1932-6203},
support = {P30 AI036211/AI/NIAID NIH HHS/United States ; R01 AI106574/AI/NIAID NIH HHS/United States ; AI106574/AI/NIAID NIH HHS/United States ; P30AI036211/AI/NIAID NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Proteins/*genetics/therapeutic use ; Cell Line ; Genetic Engineering ; Genetic Therapy ; Genetic Vectors/*genetics/therapeutic use ; HIV Infections/genetics/*therapy ; HIV-1/*physiology ; Humans ; Lentivirus/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics/therapeutic use ; Receptors, CCR5/*genetics ; Transduction, Genetic ; Virus Internalization ; },
abstract = {CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.},
}
@article {pmid25517468,
year = {2014},
author = {Mandal, PK and Ferreira, LM and Collins, R and Meissner, TB and Boutwell, CL and Friesen, M and Vrbanac, V and Garrison, BS and Stortchevoi, A and Bryder, D and Musunuru, K and Brand, H and Tager, AM and Allen, TM and Talkowski, ME and Rossi, DJ and Cowan, CA},
title = {Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.},
journal = {Cell stem cell},
volume = {15},
number = {5},
pages = {643-652},
pmid = {25517468},
issn = {1875-9777},
support = {R01HL107630/HL/NHLBI NIH HHS/United States ; U01 HL100408/HL/NHLBI NIH HHS/United States ; P01-AI104715/AI/NIAID NIH HHS/United States ; R00 MH095867/MH/NIMH NIH HHS/United States ; K99 MH095867/MH/NIMH NIH HHS/United States ; U01DK072473/DK/NIDDK NIH HHS/United States ; U01 DK072473/DK/NIDDK NIH HHS/United States ; R01 HL107630/HL/NHLBI NIH HHS/United States ; U01HL100408/HL/NHLBI NIH HHS/United States ; P01 AI104715/AI/NIAID NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; U01 HL107440/HL/NHLBI NIH HHS/United States ; R01DK097768/DK/NIDDK NIH HHS/United States ; MH095867/MH/NIMH NIH HHS/United States ; P30 AI060354/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD34/metabolism ; CRISPR-Associated Proteins/*metabolism ; Cell Lineage ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Deletion ; Gene Targeting ; Genetic Loci ; Genome, Human/genetics ; Hematopoietic Stem Cells/cytology/*metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; RNA Editing/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Receptors, CCR5/metabolism ; },
abstract = {Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.},
}
@article {pmid25497837,
year = {2014},
author = {Lin, S and Staahl, BT and Alla, RK and Doudna, JA},
title = {Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.},
journal = {eLife},
volume = {3},
number = {},
pages = {e04766},
pmid = {25497837},
issn = {2050-084X},
mesh = {Base Sequence ; Cell Cycle/genetics ; Cell Survival ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/*genetics/metabolism ; DNA Breaks, Double-Stranded ; Embryo, Mammalian ; Embryonic Stem Cells/cytology/metabolism ; Endonucleases/*genetics/metabolism ; Fibroblasts/cytology/metabolism ; Gene Expression ; Gene Transfer Techniques ; Genetic Engineering/methods ; *Genome, Human ; HEK293 Cells ; Humans ; Infant, Newborn ; Molecular Sequence Data ; RNA, Guide, CRISPR-Cas Systems/chemistry/*genetics/metabolism ; *Recombinational DNA Repair ; Sequence Analysis, DNA ; Signal Transduction ; Staphylococcus/chemistry/enzymology ; Time Factors ; },
abstract = {The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.},
}
@article {pmid25437567,
year = {2014},
author = {Ren, X and Yang, Z and Xu, J and Sun, J and Mao, D and Hu, Y and Yang, SJ and Qiao, HH and Wang, X and Hu, Q and Deng, P and Liu, LP and Ji, JY and Li, JB and Ni, JQ},
title = {Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.},
journal = {Cell reports},
volume = {9},
number = {3},
pages = {1151-1162},
pmid = {25437567},
issn = {2211-1247},
support = {R01 DK095013/DK/NIDDK NIH HHS/United States ; R01 GM102484/GM/NIGMS NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; R01GM102484/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Base Composition/genetics ; Base Sequence ; CRISPR-Associated Proteins/*metabolism ; Chromobox Protein Homolog 5 ; Chromosomal Proteins, Non-Histone/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Repair/genetics ; Drosophila melanogaster/*genetics ; Germ Cells/metabolism ; Inheritance Patterns/genetics ; Injections ; Molecular Sequence Data ; Mutagenesis/genetics ; Mutation/genetics ; Mutation Rate ; Nucleotides/genetics ; Organ Specificity ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; },
abstract = {The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.},
}
@article {pmid25418680,
year = {2015},
author = {Smith, C and Abalde-Atristain, L and He, C and Brodsky, BR and Braunstein, EM and Chaudhari, P and Jang, YY and Cheng, L and Ye, Z},
title = {Efficient and allele-specific genome editing of disease loci in human iPSCs.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {23},
number = {3},
pages = {570-577},
pmid = {25418680},
issn = {1525-0024},
support = {T32 GM007814/GM/NIGMS NIH HHS/United States ; U01 HL107446/HL/NHLBI NIH HHS/United States ; T32 HL007525/HL/NHLBI NIH HHS/United States ; 2R01-HL073781/HL/NHLBI NIH HHS/United States ; R01 HL073781/HL/NHLBI NIH HHS/United States ; K12 HL087169/HL/NHLBI NIH HHS/United States ; U01-HL107446/HL/NHLBI NIH HHS/United States ; },
mesh = {*Alleles ; Bacterial Proteins/genetics/metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; Endonucleases/genetics/metabolism ; Gene Expression ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/*metabolism/pathology ; Janus Kinase 2/genetics/metabolism ; Molecular Sequence Data ; Mutation ; Myeloproliferative Disorders/genetics/metabolism/pathology ; *Quantitative Trait Loci ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Recombinational DNA Repair ; alpha 1-Antitrypsin/genetics/metabolism ; },
abstract = {Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.},
}
@article {pmid25398353,
year = {2014},
author = {Li, JF and Zhang, D and Sheen, J},
title = {Cas9-based genome editing in Arabidopsis and tobacco.},
journal = {Methods in enzymology},
volume = {546},
number = {},
pages = {459-472},
doi = {10.1016/B978-0-12-801185-0.00022-2},
pmid = {25398353},
issn = {1557-7988},
support = {R01 GM60493/GM/NIGMS NIH HHS/United States ; R01 GM70567/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/genetics ; Base Sequence ; CRISPR-Associated Proteins/genetics/*metabolism ; Endonucleases/genetics/*metabolism ; Genetic Engineering/methods ; Genome, Plant ; Homologous Recombination ; Molecular Sequence Data ; Mutagenesis ; Protoplasts/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/*enzymology/genetics ; Nicotiana/genetics ; },
abstract = {Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.},
}
@article {pmid25398333,
year = {2014},
author = {Anders, C and Jinek, M},
title = {In vitro enzymology of Cas9.},
journal = {Methods in enzymology},
volume = {546},
number = {},
pages = {1-20},
pmid = {25398333},
issn = {1557-7988},
support = {337284/ERC_/European Research Council/International ; },
mesh = {Base Sequence ; CRISPR-Associated Proteins/*genetics/isolation &amp; purification/metabolism ; Cell Line ; Cloning, Molecular/*methods ; DNA Cleavage ; Endonucleases/*genetics/isolation &amp; purification/metabolism ; Molecular Sequence Data ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Recombinant Proteins/genetics/isolation &amp; purification/metabolism ; Streptococcus pyogenes/*enzymology/*genetics/metabolism ; Transcription, Genetic ; Transcriptome ; Transformation, Genetic ; },
abstract = {Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.},
}
@article {pmid25398078,
year = {2014},
author = {Lawhorn, IE and Ferreira, JP and Wang, CL},
title = {Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53.},
journal = {PloS one},
volume = {9},
number = {11},
pages = {e113232},
pmid = {25398078},
issn = {1932-6203},
support = {R21 AG040360/AG/NIA NIH HHS/United States ; S10RR025518-01/RR/NCRR NIH HHS/United States ; 5R21AG040360-02/AG/NIA NIH HHS/United States ; S10 RR027431/RR/NCRR NIH HHS/United States ; S10RR027431-01/RR/NCRR NIH HHS/United States ; S10 RR025518/RR/NCRR NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genetic Vectors/metabolism ; HEK293 Cells ; Humans ; RNA, Guide, CRISPR-Cas Systems/*metabolism ; RNA, Messenger/metabolism ; Real-Time Polymerase Chain Reaction ; Transcription Initiation Site ; Tumor Suppressor Protein p53/genetics/*metabolism ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.},
}
@article {pmid25378133,
year = {2015},
author = {Tan, EP and Li, Y and Velasco-Herrera, Mdel C and Yusa, K and Bradley, A},
title = {Off-target assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {53},
number = {2},
pages = {225-236},
doi = {10.1002/dvg.22835},
pmid = {25378133},
issn = {1526-968X},
support = {098051/WT_/Wellcome Trust/United Kingdom ; 14356/CRUK_/Cancer Research UK/United Kingdom ; WT077187/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Bacterial Proteins/genetics ; Base Sequence ; CRISPR-Associated Protein 9 ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; DNA Repair ; Embryonic Stem Cells/*physiology ; Endonucleases/genetics ; Gene Targeting ; *Genetic Engineering ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*physiology ; Male ; Mice ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The CRISPR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA. In a Cas9-based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human-induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near complementarity.},
}
@article {pmid25362885,
year = {2015},
author = {He, Z and Proudfoot, C and Mileham, AJ and McLaren, DG and Whitelaw, CB and Lillico, SG},
title = {Highly efficient targeted chromosome deletions using CRISPR/Cas9.},
journal = {Biotechnology and bioengineering},
volume = {112},
number = {5},
pages = {1060-1064},
doi = {10.1002/bit.25490},
pmid = {25362885},
issn = {1097-0290},
support = {BBS/E/D/05251442/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/05251443/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/05251444/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/05251445/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Base Sequence ; Cell Line ; *Chromosome Deletion ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; *DNA Breaks, Double-Stranded ; Endonucleases/genetics ; Gene Targeting/*methods ; Genetic Engineering/methods ; Humans ; Molecular Sequence Data ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user-specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment. Predetermined genomic DNA segments ranging from several-hundred base pairs to 1 Mbp can be precisely deleted at frequencies of 1-10%, with no apparent correlation between the size of the deleted fragment and the deletion frequency. The high efficiency of this technique holds promise for large genomic deletions that could be useful in generation of cell and animal models with engineered chromosomes.},
}
@article {pmid25352017,
year = {2014},
author = {Jacobs, JZ and Ciccaglione, KM and Tournier, V and Zaratiegui, M},
title = {Implementation of the CRISPR-Cas9 system in fission yeast.},
journal = {Nature communications},
volume = {5},
number = {},
pages = {5344},
pmid = {25352017},
issn = {2041-1723},
support = {R01 GM105831/GM/NIGMS NIH HHS/United States ; T32 GM008339/GM/NIGMS NIH HHS/United States ; 1R01GM105831/GM/NIGMS NIH HHS/United States ; 5T32GM008339/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Epitope Mapping ; Gene Expression Regulation, Fungal ; Genetic Vectors/metabolism ; Mutagenesis/genetics ; Mutation/genetics ; Promoter Regions, Genetic ; RNA, Fungal/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Schizosaccharomyces/*genetics ; },
abstract = {Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence. Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast.},
}
@article {pmid25303670,
year = {2014},
author = {Munoz, IM and Szyniarowski, P and Toth, R and Rouse, J and Lachaud, C},
title = {Improved genome editing in human cell lines using the CRISPR method.},
journal = {PloS one},
volume = {9},
number = {10},
pages = {e109752},
pmid = {25303670},
issn = {1932-6203},
support = {MC_U127070192/MRC_/Medical Research Council/United Kingdom ; MC_UU_12016/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Proteins/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Targeting/*methods ; *Genome ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.},
}
@article {pmid25299451,
year = {2014},
author = {Fan, Z and Li, W and Lee, SR and Meng, Q and Shi, B and Bunch, TD and White, KL and Kong, IK and Wang, Z},
title = {Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.},
journal = {PloS one},
volume = {9},
number = {10},
pages = {e109755},
pmid = {25299451},
issn = {1932-6203},
mesh = {Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Associated Proteins/*genetics/metabolism ; Cell Nucleus/genetics/metabolism ; Cytosol/metabolism ; Endonucleases/*genetics/metabolism ; Epithelial Cells/cytology/metabolism ; Gene Targeting/*methods ; Genetic Engineering/methods ; KCNQ1 Potassium Channel/genetics/metabolism ; Kidney/cytology/metabolism ; Mesocricetus/*genetics ; Microinjections ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Plasmids/administration &amp; dosage/genetics ; Protein Phosphatase 1/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA, Messenger/administration &amp; dosage/genetics ; STAT2 Transcription Factor/genetics/metabolism ; },
abstract = {The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.},
}
@article {pmid25239977,
year = {2014},
author = {Jiang, W and Brueggeman, AJ and Horken, KM and Plucinak, TM and Weeks, DP},
title = {Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.},
journal = {Eukaryotic cell},
volume = {13},
number = {11},
pages = {1465-1469},
pmid = {25239977},
issn = {1535-9786},
mesh = {Base Sequence ; Chlamydomonas reinhardtii/*genetics ; Cinnamates/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Plant/analysis/genetics ; Drug Resistance/genetics ; Gene Targeting/methods ; Hygromycin B/analogs &amp; derivatives/pharmacology ; RNA, Guide, CRISPR-Cas Systems/*genetics ; RNA, Plant/analysis/*genetics ; Sequence Analysis, DNA ; Tacrolimus Binding Protein 1A/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.},
}
@article {pmid25193712,
year = {2014},
author = {Zhang, JH and Pandey, M and Kahler, JF and Loshakov, A and Harris, B and Dagur, PK and Mo, YY and Simonds, WF},
title = {Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter.},
journal = {Journal of biotechnology},
volume = {189},
number = {},
pages = {1-8},
pmid = {25193712},
issn = {1873-4863},
support = {R01 CA154989/CA/NCI NIH HHS/United States ; Z99 DK999999//Intramural NIH HHS/United States ; },
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics ; High-Throughput Nucleotide Sequencing ; Humans ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Genomic engineering by the guide RNA (gRNA)-directed CRISPR/CAS9 is rapidly becoming a method of choice for various biological systems. However, pressing concerns remain regarding its off-target activities and wide variations in efficacies. While next generation sequencing (NGS) has been primarily used to evaluate the efficacies and off-target activities of gRNAs, it only detects the imperfectly repaired double strand DNA breaks (DSB) by the error-prone non-homologous end joining (NHEJ) mechanism and may not faithfully represent the DSB activities because the efficiency of NHEJ-mediated repair varies depending on the local chromatin environment. Here we describe a reporter system for unbiased detection and comparison of DSB activities that promises to improve the chance of success in genomic engineering and to facilitate large-scale screening of CAS9 activities and gRNA libraries. Additionally, we demonstrated that the tolerances to mismatches between a gRNA and the corresponding target DNA can occur at any position of the gRNA, and depend on both specific gRNA sequences and CAS9 constructs used.},
}
@article {pmid25166277,
year = {2014},
author = {Li, K and Wang, G and Andersen, T and Zhou, P and Pu, WT},
title = {Optimization of genome engineering approaches with the CRISPR/Cas9 system.},
journal = {PloS one},
volume = {9},
number = {8},
pages = {e105779},
pmid = {25166277},
issn = {1932-6203},
support = {U01 HL098188/HL/NHLBI NIH HHS/United States ; U01HL098188/HL/NHLBI NIH HHS/United States ; T32 HL007572/HL/NHLBI NIH HHS/United States ; U01HL098166/HL/NHLBI NIH HHS/United States ; U01 HL098166/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/*methods ; Genome ; Mice ; Mutagenesis ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.},
}
@article {pmid25147920,
year = {2014},
author = {Waldrip, ZJ and Byrum, SD and Storey, AJ and Gao, J and Byrd, AK and Mackintosh, SG and Wahls, WP and Taverna, SD and Raney, KD and Tackett, AJ},
title = {A CRISPR-based approach for proteomic analysis of a single genomic locus.},
journal = {Epigenetics},
volume = {9},
number = {9},
pages = {1207-1211},
pmid = {25147920},
issn = {1559-2308},
support = {UL1 RR029884/RR/NCRR NIH HHS/United States ; P20 GM103429/GM/NIGMS NIH HHS/United States ; R33CA173264/CA/NCI NIH HHS/United States ; R01 GM098922/GM/NIGMS NIH HHS/United States ; P20GM103429/GM/NIGMS NIH HHS/United States ; P30 GM103450/GM/NIGMS NIH HHS/United States ; R01GM098922/GM/NIGMS NIH HHS/United States ; R33 CA173264/CA/NCI NIH HHS/United States ; R01 GM081766/GM/NIGMS NIH HHS/United States ; P30GM103450/GM/NIGMS NIH HHS/United States ; R01GM106024/GM/NIGMS NIH HHS/United States ; R01 GM106024/GM/NIGMS NIH HHS/United States ; R01GM081766/GM/NIGMS NIH HHS/United States ; UL1RR029884/RR/NCRR NIH HHS/United States ; },
mesh = {Chromatin/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genetic Loci ; Histones/*metabolism ; Mass Spectrometry ; Protein Processing, Post-Translational ; Proteome/genetics/*metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Saccharomyces cerevisiae/*metabolism ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; },
abstract = {Any given chromosomal activity (e.g., transcription) is governed predominantly by the local epiproteome. However, defining local epiproteomes has been limited by a lack of effective technologies to isolate discrete sections of chromatin and to identify with precision specific proteins and histone posttranslational modifications (PTMs). We report the use of the Cas9 and guide RNA (gRNA) components of the CRISPR system for gRNA-directed purification of a discrete section of chromatin. Quantitative mass spectrometry provides for unambiguous identification of proteins and histone PTMs specifically associated with the enriched chromatin. This CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach revealed changes in the local epiproteome of a promoter during activation of transcription. CRISPR-ChAP-MS thus has broad applications for discovering molecular components and dynamic regulation of any in vivo activity at a given chromosomal location.},
}
@article {pmid25128437,
year = {2014},
author = {Ren, X and Yang, Z and Mao, D and Chang, Z and Qiao, HH and Wang, X and Sun, J and Hu, Q and Cui, Y and Liu, LP and Ji, JY and Xu, J and Ni, JQ},
title = {Performance of the Cas9 nickase system in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {4},
number = {10},
pages = {1955-1962},
pmid = {25128437},
issn = {2160-1836},
support = {R01 DK095013/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/metabolism ; Argonaute Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Repair ; Deoxyribonuclease I/genetics/*metabolism ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/*enzymology/growth &amp; development ; Embryo, Nonmammalian/metabolism ; Genetic Vectors/genetics/metabolism ; Mutagenesis ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9(D10A) nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.},
}
@article {pmid25105359,
year = {2014},
author = {Ranganathan, V and Wahlin, K and Maruotti, J and Zack, DJ},
title = {Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.},
journal = {Nature communications},
volume = {5},
number = {},
pages = {4516},
pmid = {25105359},
issn = {2041-1723},
support = {5T32EY007143/EY/NEI NIH HHS/United States ; 5P30EY001765/EY/NEI NIH HHS/United States ; R01 EY009769/EY/NEI NIH HHS/United States ; P30 EY001765/EY/NEI NIH HHS/United States ; T32 EY007143/EY/NEI NIH HHS/United States ; R01EY009769/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/*genetics ; Base Sequence ; Binding Sites ; Cattle ; Chickens ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Embryonic Stem Cells/cytology ; Genetic Engineering/methods ; Genome, Human ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; Mice ; Molecular Sequence Data ; Mutation ; Nucleotides/chemistry ; Plasmids/metabolism ; *Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Rats ; Streptococcus pyogenes ; Zebrafish ; },
abstract = {The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.},
}
@article {pmid24956386,
year = {2014},
author = {Xie, S and Shen, B and Zhang, C and Huang, X and Zhang, Y},
title = {sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.},
journal = {PloS one},
volume = {9},
number = {6},
pages = {e100448},
pmid = {24956386},
issn = {1932-6203},
mesh = {*Algorithms ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computational Biology/*methods ; DNA Cleavage ; Humans ; Molecular Sequence Data ; RNA Editing/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; Sequence Homology, Nucleic Acid ; *Software ; },
abstract = {Although the CRISPR/Cas9/sgRNA system efficiently cleaves intracellular DNA at desired target sites, major concerns remain on potential "off-target" cleavage that may occur throughout the whole genome. In order to improve CRISPR-Cas9 specificity for targeted genome editing and transcriptional control, we describe a bioinformatics tool "sgRNAcas9", which is a software package developed for fast design of CRISPR sgRNA with minimized off-target effects. This package consists of programs to perform a search for CRISPR target sites (protospacers) with user-defined parameters, predict genome-wide Cas9 potential off-target cleavage sites (POT), classify the POT into three categories, batch-design oligonucleotides for constructing 20-nt (nucleotides) or truncated sgRNA expression vectors, extract desired length nucleotide sequences flanking the on- or off-target cleavage sites for designing PCR primer pairs to validate the mutations by T7E1 cleavage assay. Importantly, by identifying potential off-target sites in silico, the sgRNAcas9 allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of sgRNA for genome editing applications. sgRNAcas9 software package is publicly available at BiooTools website (www.biootools.com) under the terms of the GNU General Public License.},
}
@article {pmid24916110,
year = {2014},
author = {Ding, Q and Strong, A and Patel, KM and Ng, SL and Gosis, BS and Regan, SN and Cowan, CA and Rader, DJ and Musunuru, K},
title = {Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing.},
journal = {Circulation research},
volume = {115},
number = {5},
pages = {488-492},
pmid = {24916110},
issn = {1524-4571},
support = {U01-HL100408/HL/NHLBI NIH HHS/United States ; R01-DK097768/DK/NIDDK NIH HHS/United States ; R01 HL109489/HL/NHLBI NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; P30 DK019525/DK/NIDDK NIH HHS/United States ; R01-HL109489/HL/NHLBI NIH HHS/United States ; },
mesh = {3T3-L1 Cells ; Adenoviridae/genetics ; Animals ; Biomarkers/blood ; Cardiovascular Diseases/enzymology/genetics/prevention &amp; control ; Cholesterol/blood ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; Genotype ; Liver/*enzymology ; Male ; Mice ; Mice, Inbred C57BL ; *Mutation ; Phenotype ; Proprotein Convertase 9 ; Proprotein Convertases/blood/*genetics ; Protective Factors ; *RNA Editing ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Receptors, LDL/metabolism ; Serine Endopeptidases/blood/*genetics ; Time Factors ; Transfection ; },
abstract = {RATIONALE: Individuals with naturally occurring loss-of-function proprotein convertase subtilisin/kexin type 9 (PCSK9) mutations experience reduced low-density lipoprotein cholesterol levels and protection against cardiovascular disease.<br><br>OBJECTIVE: The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo.<br><br>METHODS AND RESULTS: We used adenovirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that <3 to 4 days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic low-density lipoprotein receptor levels, and decreased plasma cholesterol levels (by 35-40%). No off-target mutagenesis was detected in 10 selected sites.<br><br>CONCLUSIONS: Genome editing with the CRISPR-CRISPR-associated 9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.},
}
@article {pmid24873830,
year = {2014},
author = {Gagnon, JA and Valen, E and Thyme, SB and Huang, P and Akhmetova, L and Pauli, A and Montague, TG and Zimmerman, S and Richter, C and Schier, AF},
title = {Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.},
journal = {PloS one},
volume = {9},
number = {5},
pages = {e98186},
pmid = {24873830},
issn = {1932-6203},
support = {R01 HG005111/HG/NHGRI NIH HHS/United States ; K99 HD076935/HD/NICHD NIH HHS/United States ; R21 HD072733/HD/NICHD NIH HHS/United States ; R01 GM056211/GM/NIGMS NIH HHS/United States ; R01 HL109525/HL/NHLBI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Gene Frequency ; Humans ; INDEL Mutation ; *Mutagenesis ; Mutation Rate ; Oligonucleotides/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism ; Zebrafish/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.},
}
@article {pmid24827782,
year = {2014},
author = {Ronda, C and Pedersen, LE and Hansen, HG and Kallehauge, TB and Betenbaugh, MJ and Nielsen, AT and Kildegaard, HF},
title = {Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool.},
journal = {Biotechnology and bioengineering},
volume = {111},
number = {8},
pages = {1604-1616},
pmid = {24827782},
issn = {1097-0290},
mesh = {Animals ; Base Sequence ; CHO Cells/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cricetinae ; Cricetulus ; Endonucleases/genetics/metabolism ; Fucosyltransferases/*genetics ; Gene Knockout Techniques/*methods ; Genome ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; INDEL Mutation ; Internet ; Molecular Chaperones/*genetics ; Molecular Sequence Data ; *RNA Editing ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.},
}
@article {pmid24719468,
year = {2014},
author = {Lei, Y and Lu, L and Liu, HY and Li, S and Xing, F and Chen, LL},
title = {CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants.},
journal = {Molecular plant},
volume = {7},
number = {9},
pages = {1494-1496},
doi = {10.1093/mp/ssu044},
pmid = {24719468},
issn = {1752-9867},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/*methods ; Genome, Plant/genetics ; *Internet ; Plants/*genetics ; RNA, Guide, CRISPR-Cas Systems/*genetics ; User-Computer Interface ; },
}
@article {pmid24710347,
year = {2014},
author = {Jia, H and Wang, N},
title = {Targeted genome editing of sweet orange using Cas9/sgRNA.},
journal = {PloS one},
volume = {9},
number = {4},
pages = {e93806},
pmid = {24710347},
issn = {1932-6203},
mesh = {Citrus sinensis/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Targeting/*methods ; Genetic Engineering/*methods ; RNA, Guide, CRISPR-Cas Systems/*genetics ; },
abstract = {Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.},
}
@article {pmid24487629,
year = {2014},
author = {Zhao, Y and Dai, Z and Liang, Y and Yin, M and Ma, K and He, M and Ouyang, H and Teng, CB},
title = {Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system.},
journal = {Scientific reports},
volume = {4},
number = {},
pages = {3943},
pmid = {24487629},
issn = {2045-2322},
mesh = {3T3 Cells ; Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9 ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/metabolism ; Gene Expression ; Gene Targeting/*methods ; Green Fluorescent Proteins/genetics ; Mice ; MicroRNAs/*antagonists &amp; inhibitors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Small Interfering/*antagonists &amp; inhibitors/genetics ; Recombinant Fusion Proteins/biosynthesis/genetics ; Swine ; },
abstract = {Here, we report a convenient and efficient miRNA inhibition strategy employing the CRISPR system. Using specifically designed gRNAs, miRNA gene has been cut at a single site by Cas9, resulting in knockdown of the miRNA in murine cells. Using a modified CRISPR interference system (CRISPRi), inactive Cas9 can reversibly prevent the expression of both monocistronic miRNAs and polycistronic miRNA clusters. Furthermore, CRISPR/CRISPRi is also capable of suppressing genes in porcine cells.},
}
@article {pmid38142668,
year = {2024},
author = {Liu, Z and Xu, J and Huang, S and Dai, W and Zhang, W and Li, L and Xiao, X and Wu, T},
title = {Gene point mutation information translation and detection: Leveraging single base extension and CRISPR/Cas12a.},
journal = {Biosensors &amp; bioelectronics},
volume = {247},
number = {},
pages = {115936},
doi = {10.1016/j.bios.2023.115936},
pmid = {38142668},
issn = {1873-4235},
mesh = {Humans ; Point Mutation ; Mutation ; CRISPR-Cas Systems/genetics ; ErbB Receptors ; *Lung Neoplasms ; Protein Kinase Inhibitors ; *Biosensing Techniques ; },
abstract = {Gene point mutations play a significant role in the development of cancer. Therefore, developing a sensitive, specific, and universally applicable method for detecting gene point mutation is crucial for clinical diagnosis, prognosis, and cancer treatment. Recently, gene point mutation detection methods based on CRISPR/Cas12a detection have emerged. However, existing methods generally lack universality and specificity. In this study, we have developed a CRISPR/Cas12a-based method that combines improved allele-specific polymerase chain reaction and single base extension to translate the point mutation information in the target dsDNA into length information in ssDNA activators to overcome the limitations associated with PAM sequences in the CRISPR/Cas12a system. Our method achieved a detection limit of 0.002% for clinically significant EGFR T790M mutation. The CRISPR/Cas12a system we constructed demonstrates high sensitivity, specificity, and universality in detecting gene point mutations, making it a promising tool for clinical cancer screening.},
}
@article {pmid38142459,
year = {2023},
author = {Yu, L and Marchisio, MA},
title = {Scaffold RNA engineering in type V CRISPR-Cas systems: a potent way to enhance gene expression in the yeast Saccharomyces cerevisiae.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad1216},
pmid = {38142459},
issn = {1362-4962},
abstract = {New, orthogonal transcription factors in eukaryotic cells have been realized by engineering nuclease-deficient CRISPR-associated proteins and/or their guide RNAs. In this work, we present a new kind of orthogonal transcriptional activators, in Saccharomyces cerevisiae, made by turning type V CRISPR RNA into a scaffold RNA (ScRNA) able to recruit a variable number of VP64 activation domains. The activator arises from the complex between the synthetic ScRNA and DNase-deficient type V Cas proteins: dCas12e and denAsCas12a. The transcription activation achieved via the newly engineered dCas:ScRNA system is up to 4.7-fold higher than that obtained with the direct fusion of VP64 to Cas proteins. The new transcription factors have been proven to be functional in circuits such as Boolean gates, converters, multiplex-gene and metabolic-pathway activation. Our results extend the CRISPR-Cas-based technology with a new effective tool that only demands RNA engineering and improves the current design of transcription factors based on type V Cas proteins.},
}
@article {pmid38141444,
year = {2024},
author = {Arshad, F and Abdillah, AN and Shivanand, P and Ahmed, MU},
title = {CeO2 nanozyme mediated RPA/CRISPR-Cas12a dual-mode biosensor for detection of invA gene in Salmonella.},
journal = {Biosensors &amp; bioelectronics},
volume = {247},
number = {},
pages = {115940},
doi = {10.1016/j.bios.2023.115940},
pmid = {38141444},
issn = {1873-4235},
mesh = {Cattle ; Animals ; *Recombinases ; CRISPR-Cas Systems/genetics ; Hydrogen Peroxide ; *Biosensing Techniques ; DNA, Bacterial ; },
abstract = {This study reports a novel biosensing system that leverages recombinase polymerase amplification (RPA) in conjunction with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology, integrated with a nanozyme (NZ) based on cerium dioxide (CeO2). With the integration of CeO2 NZ, a dual-mode detection platform could be developed for Salmonella detection using fluorometric and colourimetric assays. The CRISPR/Cas12a system, when activated in the presence of target DNA, could cleave the FAM-labelled probe to lead to a fluorometric response. Also, when the CeO2 NZ was introduced in the presence of H2O2, a colourimetric response was generated, directly proportional to the concentration of target DNA present. We hypothesise that adding highly reactive H2O2 within the post-CRISPR/Cas12a reaction system allows for increased release of hydroxyl free radicals within the mixture. Thus, the double recognition through NZ and the CRISPR/Cas12a system provided enhanced selectivity and sensitivity to the method. The proposed biosensor could successfully detect Salmonella at concentrations as low as 0.88 pg/μL and 1.28 pg/μL for fluorometric and colourimetric responses, respectively. Furthermore, the developed biosensor could be applied in real sample analysis of raw food samples (chicken, egg, and beef) to give a good recovery in the spiked food samples with varying concentrations of cultured bacterial DNA.},
}
@article {pmid37856214,
year = {2024},
author = {Lebek, S and Caravia, XM and Straub, LG and Alzhanov, D and Tan, W and Li, H and McAnally, JR and Chen, K and Xu, L and Scherer, PE and Liu, N and Bassel-Duby, R and Olson, EN},
title = {CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model.},
journal = {The Journal of clinical investigation},
volume = {134},
number = {1},
pages = {},
pmid = {37856214},
issn = {1558-8238},
mesh = {Mice ; Animals ; Humans ; CRISPR-Cas Systems ; Gene Editing ; Heart ; *Cardiomyopathies/genetics ; *Cardiovascular Diseases/genetics ; },
abstract = {Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.},
}
@article {pmid37642157,
year = {2023},
author = {Yang, B and Li, J and Yan, J and Zhang, K and Ouyang, Z and Lu, Y and Wei, H and Li, Q and Yao, X and Lu, S and Hong, Y and Wang, X and Guo, L},
title = {Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield.},
journal = {Journal of integrative plant biology},
volume = {65},
number = {11},
pages = {2421-2436},
doi = {10.1111/jipb.13560},
pmid = {37642157},
issn = {1744-7909},
mesh = {*Brassica napus/growth &amp; development ; *Type C Phospholipases/metabolism ; *Sphingolipids/metabolism ; *Phospholipids/metabolism ; CRISPR-Cas Systems ; Plant Proteins/metabolism ; Seeds/growth &amp; development ; Gene Knockout Techniques ; },
abstract = {Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.},
}
@article {pmid38140636,
year = {2023},
author = {Kasianchuk, N and Dobrowolska, K and Harkava, S and Bretcan, A and Zarębska-Michaluk, D and Jaroszewicz, J and Flisiak, R and Rzymski, P},
title = {Gene-Editing and RNA Interference in Treating Hepatitis B: A Review.},
journal = {Viruses},
volume = {15},
number = {12},
pages = {},
pmid = {38140636},
issn = {1999-4915},
mesh = {Humans ; RNA Interference ; CRISPR-Cas Systems ; Genetic Therapy ; DNA, Viral/genetics ; *Hepatitis B/prevention &amp; control/genetics ; *Hepatitis B, Chronic/prevention &amp; control/genetics ; Hepatitis B virus/genetics/metabolism ; Antiviral Agents/therapeutic use/metabolism ; },
abstract = {The hepatitis B virus (HBV) continues to cause substantial health and economic burdens, and its target of elimination may not be reached in 2030 without further efforts in diagnostics, non-pharmaceutical prevention measures, vaccination, and treatment. Current therapeutic options in chronic HBV, based on interferons and/or nucleos(t)ide analogs, suppress the virus replication but do not eliminate the pathogen and suffer from several constraints. This paper reviews the progress on biotechnological approaches in functional and definitive HBV treatments, including gene-editing tools, i.e., zinc-finger proteins, transcription activator-like effector nucleases, and CRISPR/Cas9, as well as therapeutics based on RNA interference. The advantages and challenges of these approaches are also discussed. Although the safety and efficacy of gene-editing tools in HBV therapies are yet to be demonstrated, they show promise for the revitalization of a much-needed advance in the field and offer viral eradication. Particular hopes are related to CRISPR/Cas9; however, therapeutics employing this system are yet to enter the clinical testing phases. In contrast, a number of candidates based on RNA interference, intending to confer a functional cure, have already been introduced to human studies. However, larger and longer trials are required to assess their efficacy and safety. Considering that prevention is always superior to treatment, it is essential to pursue global efforts in HBV vaccination.},
}
@article {pmid38139836,
year = {2023},
author = {Adamiec-Organisciok, M and Wegrzyn, M and Cienciala, L and Sojka, D and Nackiewicz, J and Skonieczna, M},
title = {Compensative Resistance to Erastin-Induced Ferroptosis in GPX4 Knock-Out Mutants in HCT116 Cell Lines.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {16},
number = {12},
pages = {},
pmid = {38139836},
issn = {1424-8247},
support = {02/040/BKM23/1041//Silesian University of Technology/ ; 02/040/BK_23/1035//Silesian University of Technology/ ; Project-Based Learning - PBL (The Excellence Initiative - Research University program) No. 54/2020//Silesian University of Technology/ ; POWR.03.05.00-00-Z098/17//European Union/ ; },
abstract = {Ferroptosis results from the accumulation of oxidized and damaged lipids which then leads to programmed cell death. This programmed process is iron-dependent, and as a fundamental biological process, plays a crucial role in tissue homeostasis. The ferroptosis molecular pathway depends on self-regulatory genes: GPX4; TFRC; ACSL4; FSP1; SLC7A11, and PROM2. Some of them were considered here as ferro-sensitive or ferro-resistance markers. We examined the impact of GPX4 gene knock-out, using the CRISPR/Cas-9 technique, on ferroptosis induction in the HCT116 colorectal cancer cell line. The results confirmed that cells lacking the GPX4 gene (GPX4 KO) should be more susceptible to ferroptosis after erastin treatment. However, the decrease in cell viability was not as significant as we initially assumed. Based on the lipid peroxidation markers profile and RT-qPCR gene expression analysis, we revealed the activation of an alternative antioxidant system supporting GPX4 KO cells, mostly for cellular ferroptotic death avoidance. Increased expression of FSP1 and PRDX1 genes in knock-out mutants was associated with their function-recognized here as ferroptosis suppressors. For such reasons, studies on the role of GPX4 and other crucial genes from the ferroptotic pathway should be explored. Despite promising prospects, the utilization of ferroptosis mechanisms in cancer therapy remains at the stage of experimental and in vitro preclinical studies.},
}
@article {pmid38139398,
year = {2023},
author = {Liu, B and Zhong, X and Liu, Z and Guan, X and Wang, Q and Qi, R and Zhou, X and Huang, J},
title = {Probiotic Potential and Safety Assessment of Lactiplantibacillus plantarum cqf-43 and Whole-Genome Sequence Analysis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {24},
pages = {},
pmid = {38139398},
issn = {1422-0067},
support = {cstc2021jxjl00018//Chongqing Research Institute of Performance Incentive and Guidance Project/ ; CARS-35//modern agricultural industrial technology construction, Chongqing/ ; },
mesh = {Animals ; Female ; Swine ; Mice ; *Genome, Bacterial ; RNA, Ribosomal, 16S ; Phylogeny ; Anti-Bacterial Agents ; *Probiotics ; Gelatinases/genetics ; Sequence Analysis ; },
abstract = {This study reports the whole-genome sequence of Lactiplantibacillus plantarum cqf-43 isolated from healthy sow feces. Based on genomic analysis, we performed a comprehensive safety assessment of strain cqf-43, using both in vitro and in vivo experiments, and explored its probiotic potential. The total genome length measures 3,169,201 bp, boasting a GC content of 44.59%. Through phylogenetic analyses, leveraging both 16S rRNA gene and whole-genome sequences, we confidently categorize strain cqf-43 as a member of Lactiplantibacillus. Genome annotation using Prokka unveiled a total of 3141 genes, encompassing 2990 protein-coding sequences, 71 tRNAs, 16 rRNAs, and 1 tmRNA. Functional annotations derived from COG and KEGG databases highlighted a significant abundance of genes related to metabolism, with a notable emphasis on carbohydrate utilization. The genome also revealed the presence of prophage regions and CRISPR-Cas regions while lacking virulence and toxin genes. Screening for antibiotic resistance genes via the CARD database yielded no detectable transferable resistance genes, effectively eliminating the potential for harmful gene transfer. It is worth highlighting that the virulence factors identified via the VFDB database primarily contribute to bolstering pathogen resilience in hostile environments. This characteristic is particularly advantageous for probiotics. Furthermore, the genome is devoid of menacing genes such as hemolysin, gelatinase, and biogenic amine-producing genes. Our investigation also unveiled the presence of three unannotated secondary metabolite biosynthetic gene clusters, as detected by the online tool antiSMASH, suggesting a great deal of unknown potential for this strain. Rigorous in vitro experiments confirmed tolerance of strain cqf-43 in the intestinal environment, its antimicrobial efficacy, sensitivity to antibiotics, absence of hemolysis and gelatinase activity, and its inability to produce biogenic amines. In addition, a 28-day oral toxicity test showed that the strain cqf-43 did not pose a health hazard in mice, further establishing it as a safe strain.},
}
@article {pmid38139149,
year = {2023},
author = {Karpov, DS and Sosnovtseva, AO and Pylina, SV and Bastrich, AN and Petrova, DA and Kovalev, MA and Shuvalova, AI and Eremkina, AK and Mokrysheva, NG},
title = {Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".},
journal = {International journal of molecular sciences},
volume = {24},
number = {24},
pages = {},
pmid = {38139149},
issn = {1422-0067},
support = {agreement no. 075-15-2022-310//the Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *Diabetes Mellitus, Type 1/genetics/therapy ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Insulin/genetics ; Cell- and Tissue-Based Therapy ; },
abstract = {Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing β-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to β-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a "Trojan horse".},
}
@article {pmid38137031,
year = {2023},
author = {Oppert, B and Dossey, AT and Chu, FC and Šatović-Vukšić, E and Plohl, M and Smith, TPL and Koren, S and Olmstead, ML and Leierer, D and Ragan, G and Johnston, JS},
title = {The Genome of the Yellow Mealworm, Tenebrio molitor: It's Bigger Than You Think.},
journal = {Genes},
volume = {14},
number = {12},
pages = {},
pmid = {38137031},
issn = {2073-4425},
support = {Contract No. 140D6318C0055//United States Department of Defense/ ; },
mesh = {Animals ; Male ; Humans ; *Tenebrio/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Eye Color ; Animal Feed/analysis ; Larva/metabolism ; },
abstract = {BACKGROUND: Insects are a sustainable source of protein for human food and animal feed. We present a genome assembly, CRISPR gene editing, and life stage-specific transcriptomes for the yellow mealworm, Tenebrio molitor, one of the most intensively farmed insects worldwide.<br><br>METHODS: Long and short reads and long-range data were obtained from a T. molitor male pupa. Sequencing transcripts from 12 T. molitor life stages resulted in 279 million reads for gene prediction and genetic engineering. A unique plasmid delivery system containing guide RNAs targeting the eye color gene vermilion flanking the muscle actin gene promoter and EGFP marker was used in CRISPR/Cas9 transformation.<br><br>RESULTS: The assembly is approximately 53% of the genome size of 756.8 &#xb1; 9.6 Mb, measured using flow cytometry. Assembly was complicated by a satellitome of at least 11 highly conserved satDNAs occupying 28% of the genome. The injection of the plasmid into embryos resulted in knock-out of Tm vermilion and knock-in of EGFP.<br><br>CONCLUSIONS: The genome of T. molitor is longer than current assemblies (including ours) due to a substantial amount (26.5%) of only one highly abundant satellite DNA sequence. Genetic sequences and transformation tools for an insect important to the food and feed industries will promote the sustainable utilization of mealworms and other farmed insects.},
}
@article {pmid38070238,
year = {2024},
author = {Hu, X and Li, J and Li, YT and Zhang, Y and Xiao, MM and Zhang, Z and Liu, Y and Zhang, ZY and Zhang, GJ},
title = {Plug-and-play smart transistor bio-chips implementing point-of-care diagnosis of AMI with modified CRISPR/Cas12a system.},
journal = {Biosensors &amp; bioelectronics},
volume = {246},
number = {},
pages = {115909},
doi = {10.1016/j.bios.2023.115909},
pmid = {38070238},
issn = {1873-4235},
mesh = {Humans ; Point-of-Care Systems ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; Troponin I ; *Myocardial Infarction/diagnosis/genetics ; },
abstract = {The point-of-care diagnosis of acute myocardial infarction (AMI), an extremely lethal disease with only a few hours of golden rescue time, is significant and urgently required. Here, we describe a plug-and-play carbon nanotube field effect transistor (CNT-FET) bio-chip supported with a smart portable readout for ultrasensitive and on-site testing of cardiac troponin I (cTnI), which is one of the most specific and valuable biomarkers of AMI. A modified clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system, featuring the G-triplex structured reporter, was first combined with the CNT-FET to realize non-nucleic acid detection. Such a unique CNT-FET biosensor achieved the high sensitivity (LOD: 0.33 fg/mL), which is expected to give timely warning in the early stage of myocardial injury. In addition, a bilayer gate dielectric consisting of Y2O3/HfO2, employed into the passivation process, enabled the high environmental stability and repeatability of CNT-FET. More importantly, the homemade compact chip readout forged a field-deployable cTnI analytical tool, realizing "plasma-to-answer" performance for AMI patients in point-of-care testing scenarios. The developed technology holds promise to help doctors make clinical decisions faster, especially in remote areas.},
}
@article {pmid38069633,
year = {2023},
author = {Tian, Z and Octaviani, S and Huang, J},
title = {Unraveling therapeutic targets in acute myeloid leukemia through multiplexed genome editing CRISPR screening.},
journal = {Expert opinion on therapeutic targets},
volume = {27},
number = {12},
pages = {1173-1176},
doi = {10.1080/14728222.2023.2293751},
pmid = {38069633},
issn = {1744-7631},
mesh = {Humans ; *Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Leukemia, Myeloid, Acute/genetics/therapy ; CRISPR-Cas Systems ; },
}
@article {pmid38065367,
year = {2024},
author = {Mahato, RK and Bhattacharya, S and Khullar, N and Sidhu, IS and Reddy, PH and Bhatti, GK and Bhatti, JS},
title = {Targeting long non-coding RNAs in cancer therapy using CRISPR-Cas9 technology: A novel paradigm for precision oncology.},
journal = {Journal of biotechnology},
volume = {379},
number = {},
pages = {98-119},
doi = {10.1016/j.jbiotec.2023.12.003},
pmid = {38065367},
issn = {1873-4863},
mesh = {Humans ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems/genetics ; *RNA, Long Noncoding/genetics ; Precision Medicine ; Genetic Therapy ; Gene Editing ; },
abstract = {Cancer is the second leading cause of death worldwide, despite recent advances in its identification and management. To improve cancer patient diagnosis and care, it is necessary to identify new biomarkers and molecular targets. In recent years, long non-coding RNAs (lncRNAs) have surfaced as important contributors to various cellular activities, with growing proof indicating their substantial role in the genesis, development, and spread of cancer. Their unique expression profiles within specific tissues and their wide-ranging functionalities make lncRNAs excellent candidates for potential therapeutic intervention in cancer management. They are implicated in multiple hallmarks of cancer, such as uncontrolled proliferation, angiogenesis, and immune evasion. This review article explores the innovative application of CRISPR-Cas9 technology in targeting lncRNAs as a cancer therapeutic strategy. The CRISPR-Cas9 system has been widely applied in functional genomics, gene therapy, and cancer research, offering a versatile platform for lncRNA targeting. CRISPR-Cas9-mediated targeting of lncRNAs can be achieved through CRISPR interference, activation or the complete knockout of lncRNA loci. Combining CRISPR-Cas9 technology with high-throughput functional genomics makes it possible to identify lncRNAs critical for the survival of specific cancer subtypes, opening the door for tailored treatments and personalised cancer therapies. CRISPR-Cas9-mediated lncRNA targeting with other cutting-edge cancer therapies, such as immunotherapy and targeted molecular therapeutics can be used to overcome the drug resistance in cancer. The synergy of lncRNA research and CRISPR-Cas9 technology presents immense potential for individualized cancer treatment, offering renewed hope in the battle against this disease.},
}
@article {pmid38064994,
year = {2024},
author = {Sun, T and Wang, W and Wang, F and Shen, W and Geng, L and Zhang, Y and Bi, M and Gong, T and Liu, C and Guo, C and Yao, Z and Wang, T and Bai, J},
title = {A novel universal small-molecule detection platform based on antibody-controlled Cas12a switching.},
journal = {Biosensors &amp; bioelectronics},
volume = {246},
number = {},
pages = {115897},
doi = {10.1016/j.bios.2023.115897},
pmid = {38064994},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques ; Antibodies ; Biological Assay ; Biotin ; },
abstract = {Molecular diagnostics play an important role in illness detection, prevention, and treatment, and are vital in point-of-care test. In this investigation, a novel CRISPR/Cas12a based small-molecule detection platform was developed using Antibody-Controlled Cas12a Biosensor (ACCBOR), in which antibody would control the trans-cleavage activity of CRISPR/Cas12a. In this system, small-molecule was labeled around the PAM sites of no target sequence(NTS), and antibody would bind on the labeled molecule to prevent the combination of CRISPR/Cas12a, resulting the decrease of trans-cleavage activity. Biotin-, digoxin-, 25-hydroxyvitamin D3 (25-OH-VD3)-labeled NTS and corresponding binding protein were separately used to verify its preformance, showing great universality. Finally, one-pot detection of 25-OH-VD3 was developed, exhibiting high sensitivity and excellent specificity. The limit of detection could be 259.86 pg/mL in serum within 30 min. This assay platform also has the advantages of low cost, easy operation (one-pot method), and fast detection (∼30 min), would be a new possibilities for the highly sensitive detection of other small-molecule targets.},
}
@article {pmid38042054,
year = {2024},
author = {Yin, Y and Wen, J and Wen, M and Fu, X and Ke, G and Zhang, XB},
title = {The design strategies for CRISPR-based biosensing: Target recognition, signal conversion, and signal amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {246},
number = {},
pages = {115839},
doi = {10.1016/j.bios.2023.115839},
pmid = {38042054},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; Colorimetry ; Coloring Agents ; CRISPR-Cas Systems/genetics ; },
abstract = {Rapid, sensitive and selective biosensing is highly important for analyzing biological targets and dynamic physiological processes in cells and living organisms. As an emerging tool, clustered regularly interspaced short palindromic repeats (CRISPR) system is featured with excellent complementary-dependent cleavage and efficient trans-cleavage ability. These merits enable CRISPR system to improve the specificity, sensitivity, and speed for molecular detection. Herein, the structures and functions of several CRISPR proteins for biosensing are summarized in depth. Moreover, the strategies of target recognition, signal conversion, and signal amplification for CRISPR-based biosensing were highlighted from the perspective of biosensor design principles. The state-of-art applications and recent advances of CRISPR system are then outlined, with emphasis on their fluorescent, electrochemical, colorimetric, and applications in POCT technology. Finally, the current challenges and future prospects of this frontier research area are discussed.},
}
@article {pmid38040136,
year = {2024},
author = {Nadarajapillai, K and Jung, S and Sellaththurai, S and Ganeshalingam, S and Kim, MJ and Lee, J},
title = {CRISPR/Cas9-mediated knockout of tnf-α1 in zebrafish reduces disease resistance after Edwardsiella piscicida bacterial infection.},
journal = {Fish &amp; shellfish immunology},
volume = {144},
number = {},
pages = {109249},
doi = {10.1016/j.fsi.2023.109249},
pmid = {38040136},
issn = {1095-9947},
mesh = {Animals ; Disease Resistance/genetics ; Zebrafish ; Tumor Necrosis Factor-alpha/genetics/metabolism ; *Enterobacteriaceae Infections ; CRISPR-Cas Systems ; *Edwardsiella/physiology ; Cytokines/genetics ; *Fish Diseases ; Bacterial Proteins/genetics ; },
abstract = {Tumor necrosis factor (TNF) is an important cytokine involved in immune responses to bacterial infections in vertebrates, including fish. Although Tnf-α is a well-studied cytokine, there are contradictory findings about Tnf-α function following bacterial infection. In this study, we analyzed the expression and function of the Tnf-α-type I isoform (Tnf-α1) in zebrafish by knockout experiments using the CRISPR/Cas9 gene-editing tool. The open reading frame of tnf-α1 encodes a 25.82 kDa protein with 234 amino acids (aa). The expression of tnf-α1 in the early stages of zebrafish was observed from the 2-cell stage. Adult zebrafish spleens showed the highest expression of tnf-α1. To evaluate the function of Tnf-α1, an 8 bp deletion in the target region, resulting in a short truncated protein of 55 aa, was used to create the tnf-α1 knockout mutant. The pattern of downstream gene expression in 7-day larvae in wild-type (WT) and tnf-α1 knockout fish was examined. We also verified the fish mortality rate after Edwardsiella piscicida challenge and found that it was much higher in tnf-α1 knockout fish than in WT fish. Additionally, downstream gene expression analyses after E. piscicida exposure revealed a distinct expression pattern in tnf-α1 knockout fish compared to that in WT fish. Overall, our study using tnf-α1 deletion in zebrafish confirmed that Tnf-α1 is critical for immune regulation during bacterial infection.},
}
@article {pmid38029710,
year = {2024},
author = {Ahamed, MA and Khalid, MAU and Dong, M and Politza, AJ and Zhang, Z and Kshirsagar, A and Liu, T and Guan, W},
title = {Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {246},
number = {},
pages = {115866},
doi = {10.1016/j.bios.2023.115866},
pmid = {38029710},
issn = {1873-4235},
mesh = {Humans ; Recombinases ; *Mpox (monkeypox) ; *Nanopores ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; },
abstract = {Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 μL reaction system. By integrating 2 μL of RPA amplifications into a 20 μL CRISPR reaction, we attained an overall LoD of 16 copies/μL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care.},
}
@article {pmid37982370,
year = {2024},
author = {Zeng, W and Zheng, L and Li, Y and Yang, J and Mao, T and Zhang, J and Liu, Y and Ning, J and Zhang, T and Huang, H and Chen, X and Lu, F},
title = {Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration.},
journal = {Emerging microbes &amp; infections},
volume = {13},
number = {1},
pages = {2284286},
doi = {10.1080/22221751.2023.2284286},
pmid = {37982370},
issn = {2222-1751},
mesh = {Animals ; Mice ; *Hepatitis B virus/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism/pharmacology ; DNA, Circular/genetics/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; DNA, Viral/genetics/metabolism ; *Hepatitis B/genetics ; Virus Replication ; },
abstract = {The persistence of HBV covalently closed circular DNA (cccDNA) and HBV integration into the host genome in infected hepatocytes pose significant challenges to the cure of chronic HBV infection. Although CRISPR/Cas9-mediated genome editing shows promise for targeted clearance of viral genomes, a safe and efficient delivery method is currently lacking. Here, we developed a novel approach by combining light-induced heterodimerization and protein acylation to enhance the loading efficiency of Cas9 protein into extracellular vesicles (EVs). Moreover, vesicular stomatitis virus-glycoprotein (VSV-G) was incorporated onto the EVs membrane, significantly facilitating the endosomal escape of Cas9 protein and increasing its gene editing activity in recipient cells. Our results demonstrated that engineered EVs containing Cas9/gRNA and VSV-G can effectively reduce viral antigens and cccDNA levels in the HBV-replicating and infected cell models. Notably, we also confirmed the antiviral activity and high safety of the engineered EVs in the HBV-replicating mouse model generated by hydrodynamic injection and the HBV transgenic mouse model. In conclusion, engineered EVs could successfully mediate functional CRISPR/Cas9 delivery both in vitro and in vivo, leading to the clearance of episomal cccDNA and integrated viral DNA fragments, and providing a novel therapeutic approach for curing chronic HBV infection.},
}
@article {pmid38137006,
year = {2023},
author = {Gonzalez-Salinas, F and Herrera-Gamboa, J and Rojo, R and Trevino, V},
title = {Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.},
journal = {Genes},
volume = {14},
number = {12},
pages = {},
pmid = {38137006},
issn = {2073-4425},
support = {TecBASE//Tecnol&#xf3;gico de Monterrey/ ; 814825//Consejo Nacional de Humanidades, Ciencias y Tecnolog&#xed;as/ ; },
mesh = {Humans ; Female ; MCF-7 Cells ; *CRISPR-Cas Systems ; *Breast Neoplasms/genetics ; Phosphatidylinositol 3-Kinases/metabolism ; Transcription Factors/genetics/metabolism ; Antigens, Neoplasm/genetics ; Neoplasm Proteins/genetics ; },
abstract = {Breast cancer is one of the leading causes of death in women around the world. Over time, many genes and mutations that are associated with the development of this disease have been identified. However, the specific role of many genes has not yet been fully elucidated. Higher ARID4B expression has been identified as a risk factor for diverse cancer types. Silencing experiments also showed that ARID4B is associated with developing cancer-associated characteristics. However, no transcriptomic studies have shown the overall cellular effect of loss of function in breast cancer in humans. This study addresses the impact of loss-of-function mutations in breast cancer MCF-7 cells. Using the CRISPR/Cas9 system, we generated mutations that caused heterozygous truncated proteins, isolating three monoclonal lines carrying insertions and deletions in ARID4B. We observed reduced proliferation and migration in in vitro experiments. In addition, from RNA-seq assays, a differential expression analysis shows known and novel deregulated cancer-associate pathways in mutated cells supporting the impact of ARID4B. For example, we found the AKT-PI3K pathway to be altered at the transcript level but through different genes than those reported for ARID4B. Our transcriptomic results also suggest new insights into the role of ARID4B in aggressiveness by the epithelial-to-mesenchymal transition and TGF-β pathways and in metabolism through cholesterol and mevalonate pathways. We also performed exome sequencing to show that no off-target effects were apparent. In conclusion, the ARID4B gene is associated with some aggressive phenotypes in breast cancer cells.},
}
@article {pmid38136999,
year = {2023},
author = {Kumam, Y and Trick, HN and Vara Prasad, PV and Jugulam, M},
title = {Transformative Approaches for Sustainable Weed Management: The Power of Gene Drive and CRISPR-Cas9.},
journal = {Genes},
volume = {14},
number = {12},
pages = {},
pmid = {38136999},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; *Gene Drive Technology/methods ; Ecosystem ; Weed Control/methods ; Plant Weeds/genetics ; },
abstract = {Weeds can negatively impact crop yields and the ecosystem's health. While many weed management strategies have been developed and deployed, there is a greater need for the development of sustainable methods for employing integrated weed management. Gene drive systems can be used as one of the approaches to suppress the aggressive growth and reproductive behavior of weeds, although their efficacy is yet to be tested. Their popularity in insect pest management has increased, however, with the advent of CRISPR-Cas9 technology, which provides specificity and precision in editing the target gene. This review focuses on the different types of gene drive systems, including the use of CRISPR-Cas9-based systems and their success stories in pest management, while also exploring their possible applications in weed species. Factors that govern the success of a gene drive system in weeds, including the mode of reproduction, the availability of weed genome databases, and well-established transformation protocols are also discussed. Importantly, the risks associated with the release of weed populations with gene drive-bearing alleles into wild populations are also examined, along with the importance of addressing ecological consequences and ethical concerns.},
}
@article {pmid38136570,
year = {2023},
author = {Motoche-Monar, C and Ordoñez, JE and Chang, O and Gonzales-Zubiate, FA},
title = {gRNA Design: How Its Evolution Impacted on CRISPR/Cas9 Systems Refinement.},
journal = {Biomolecules},
volume = {13},
number = {12},
pages = {},
pmid = {38136570},
issn = {2218-273X},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; Algorithms ; Machine Learning ; },
abstract = {Over the past decade, genetic engineering has witnessed a revolution with the emergence of a relatively new genetic editing tool based on RNA-guided nucleases: the CRISPR/Cas9 system. Since the first report in 1987 and characterization in 2007 as a bacterial defense mechanism, this system has garnered immense interest and research attention. CRISPR systems provide immunity to bacteria against invading genetic material; however, with specific modifications in sequence and structure, it becomes a precise editing system capable of modifying the genomes of a wide range of organisms. The refinement of these modifications encompasses diverse approaches, including the development of more accurate nucleases, understanding of the cellular context and epigenetic conditions, and the re-designing guide RNAs (gRNAs). Considering the critical importance of the correct performance of CRISPR/Cas9 systems, our scope will emphasize the latter approach. Hence, we present an overview of the past and the most recent guide RNA web-based design tools, highlighting the evolution of their computational architecture and gRNA characteristics over the years. Our study explains computational approaches that use machine learning techniques, neural networks, and gRNA/target interactions data to enable predictions and classifications. This review could open the door to a dynamic community that uses up-to-date algorithms to optimize and create promising gRNAs, suitable for modern CRISPR/Cas9 engineering.},
}
@article {pmid37792967,
year = {2024},
author = {An, X and Zhang, S and Jiang, Y and Liu, X and Fang, C and Wang, J and Zhao, L and Hou, Q and Zhang, J and Wan, X},
title = {CRISPR/Cas9-based genome editing of 14 lipid metabolic genes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.},
journal = {Plant biotechnology journal},
volume = {22},
number = {1},
pages = {216-232},
pmid = {37792967},
issn = {1467-7652},
support = {BAIC02-2023//Beijing Innovation Consortium of Agriculture Research System/ ; Z201100006820114//Beijing Nova Program/ ; Z191100004019005//Beijing Science and Technology Plan Program/ ; 06500136//Fundamental Research Funds for the Central Universities of China/ ; 2021YFF1000302//National Key Research and Development Program of China/ ; 2022YFF1003501//National Key Research and Development Program of China/ ; 31971958//National Natural Science Foundation of China/ ; },
mesh = {Zea mays/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Molecular Docking Simulation ; Pyrones/metabolism ; Plant Breeding ; *Arabidopsis/genetics ; Lipids ; Pollen/genetics/metabolism ; *Infertility/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.},
}
@article {pmid37769010,
year = {2024},
author = {Bi, W and Liu, J and Li, Y and He, Z and Chen, Y and Zhao, T and Liang, X and Wang, X and Meng, X and Dou, D and Xu, G},
title = {CRISPR/Cas9-guided editing of a novel susceptibility gene in potato improves Phytophthora resistance without growth penalty.},
journal = {Plant biotechnology journal},
volume = {22},
number = {1},
pages = {4-6},
pmid = {37769010},
issn = {1467-7652},
support = {32000200//National Natural Science Foundation of China/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; *Phytophthora/genetics ; Gene Editing ; },
}
@article {pmid38135742,
year = {2023},
author = {Fang, Z and Xu, M and Shen, S and Sun, W and Yu, Q and Wu, Q and Xiang, L and Weng, Q},
title = {Prediction and characterization of prophages of Stenotrophomonas maltophilia reveals a remarkable phylogenetic diversity of prophages.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {22941},
pmid = {38135742},
issn = {2045-2322},
support = {U1812401//Joint Fund of the National Natural Science Foundation of China and the Karst Science Research Center of Guizhou Province/ ; },
mesh = {*Prophages/genetics ; Phylogeny ; *Stenotrophomonas maltophilia/genetics ; Genomics ; Gene Transfer, Horizontal ; },
abstract = {Prophages, which enables bacterial hosts to acquire novel traits, and increase genetic variation and evolutionary innovation, are considered to be one of the greatest drivers of bacterial diversity and evolution. Stenotrophomonas maltophilia is widely distributed and one of the most important multidrug resistant bacteria in hospitals. However, the distribution and genetic diversity of S. maltophilia prophages have not been elucidated. In this study, putative prophages were predicted in S. maltophilia genomes by using virus prediction tools, and the genetic diversity and phylogeny of S. maltophilia and the prophages they harbor were further analyzed. A total of 356 prophage regions were predicted from 88 S. maltophilia genomes. Among them, 144 were intact prophages, but 77.09% of the intact prophages did not match any known phage sequences in the public database. The number of prophage carried by S. maltophilia is related to its host habitat and is an important factor affecting the size of the host genome, but it is not related to the genetic diversity of the prophage. The prediction of auxiliary genes encoded by prophage showed that antibiotic resistance genes was not predicted for any of the prophages except for one questionable prophage, while 53 virulence genes and 169 carbohydrate active enzymes were predicted from 11.24 and 44.1% prophages, respectively. Most of the prophages (72.29%) mediated horizontal gene transfer of S. maltophilia genome, but only involved in 6.25% of the horizontal gene transfer events. In addition, CRISPR prediction indicated 97.75% S. maltophilia strains contained the CRISPR-Cas system containing 818 spacer sequences. However, these spacer sequences did not match any known S. maltophilia phages, and only a few S. maltophilia prophages. Comparative genomic analysis revealed a highly conserved and syntenic organization with genomic rearrangement between the prophages and the known related S. maltophilia phages. Our results indicate a high prevalence and genetic diversity of prophages in the genome of S. maltophilia, as well as the presence of a large number of uncharacterized phages. It provides an important complement to understanding the diversity and biological characteristics of phages, as well as the interactions and evolution between bacteria and phages.},
}
@article {pmid38135367,
year = {2024},
author = {The Lancet Haematology, },
title = {The dawn of the CRISPR/Cas9 gene therapy era.},
journal = {The Lancet. Haematology},
volume = {11},
number = {1},
pages = {e1},
doi = {10.1016/S2352-3026(23)00372-1},
pmid = {38135367},
issn = {2352-3026},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; },
}
@article {pmid38134886,
year = {2023},
author = {Ren, X and Yang, H and Nierenberg, JL and Sun, Y and Chen, J and Beaman, C and Pham, T and Nobuhara, M and Takagi, MA and Narayan, V and Li, Y and Ziv, E and Shen, Y},
title = {High-throughput PRIME-editing screens identify functional DNA variants in the human genome.},
journal = {Molecular cell},
volume = {83},
number = {24},
pages = {4633-4645.e9},
doi = {10.1016/j.molcel.2023.11.021},
pmid = {38134886},
issn = {1097-4164},
mesh = {Humans ; *Genome, Human/genetics ; *Genome-Wide Association Study ; Reproducibility of Results ; Regulatory Sequences, Nucleic Acid ; DNA ; Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and single-base resolution manner remains challenging. Here, we develop a pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated single-base resolution analysis. Next, we applied PRIME to functionally characterize 1,304 genome-wide association study (GWAS)-identified non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate that PRIME is capable of characterizing genetic variants at single-base resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.},
}
@article {pmid38007952,
year = {2024},
author = {Yin, X and Yang, L and Sun, X and Zheng, Q and Piao, Y and Hu, B and Zhang, X and Cao, J},
title = {Development and validation of sensitive and rapid CRISPR/Cas12-based PCR method to detect hazelnut in unlabeled products.},
journal = {Food chemistry},
volume = {438},
number = {},
pages = {137952},
doi = {10.1016/j.foodchem.2023.137952},
pmid = {38007952},
issn = {1873-7072},
mesh = {Humans ; *Plant Proteins/analysis ; *Corylus/genetics ; CRISPR-Cas Systems ; Food Analysis/methods ; Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction/methods ; },
abstract = {Hazelnut, one of the most popular tree nuts, is widely found in processed food and even very small amounts can trigger severe allergic reactions in susceptible people. Herein, we developed a sensitive and rapid method based on CRISPR and qPCR capable of detecting low-abundance hazelnut in processed food. The assay, known as CRISPR-based nucleic acid test method (Crinac) can detect 1 % of hazelnut in a mixture and allows the species to be identified in a complex processed sample. The detection process can be completed within 60 min. Contributed to amplification via PCR and CRISPR/Cas12a, enables end-fluorescence measurement for the quantification of hazelnut, thus reducing assay time and eliminating the need for costly real-time fluorescence PCR instruments. The assay based on CRISPR/Cas12 and PCR has potential as a sensitive and reliable analytical tool for the detection of food authenticity.},
}
@article {pmid37366599,
year = {2023},
author = {Gomez, K and Duran, P and Tonello, R and Allen, HN and Boinon, L and Calderon-Rivera, A and Loya-López, S and Nelson, TS and Ran, D and Moutal, A and Bunnett, NW and Khanna, R},
title = {Neuropilin-1 is essential for vascular endothelial growth factor A-mediated increase of sensory neuron activity and development of pain-like behaviors.},
journal = {Pain},
volume = {164},
number = {12},
pages = {2696-2710},
pmid = {37366599},
issn = {1872-6623},
support = {R01 DA042852/DA/NIDA NIH HHS/United States ; R01 NS102722/NS/NINDS NIH HHS/United States ; R41 NS122545/NS/NINDS NIH HHS/United States ; R61 NS126026/NS/NINDS NIH HHS/United States ; },
mesh = {Rats ; Male ; Female ; Animals ; *Vascular Endothelial Growth Factor A/metabolism ; Neuropilin-1/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Sensory Receptor Cells/metabolism ; *Neuralgia/metabolism ; Hyperalgesia/metabolism ; Ganglia, Spinal/metabolism ; Sodium/metabolism ; },
abstract = {Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain through the enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. Nrp-1 is expressed in both peptidergic and nonpeptidergic sensory neurons. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was used to knockdown NRP-1. Neuropilin-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. Neuropilin-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented spinal nerve injury-induced mechanical allodynia and thermal hyperalgesia in both male and female rats. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system.},
}
@article {pmid38134884,
year = {2023},
author = {Gopala Krishna, V and Gautsch, VG and D'Angiolella, V},
title = {The case of the missing Ks: Base editor screen to assess cellular fitness at single lysines.},
journal = {Molecular cell},
volume = {83},
number = {24},
pages = {4442-4444},
doi = {10.1016/j.molcel.2023.11.037},
pmid = {38134884},
issn = {1097-4164},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Adenine ; },
abstract = {In this issue of Molecular Cell, Bao et al.[1] set out to elucidate "functional lysines" in the genome using adenine base editors. The study reveals several cases of alteration of functions that previous canonical CRISPR-Cas9 screens were unable to detect.},
}
@article {pmid38134625,
year = {2023},
author = {Xu, S and Wang, X and Wu, C and Zhu, X and Deng, X and Wu, Y and Liu, M and Huang, X and Wu, L and Huang, H},
title = {MscI restriction enzyme cooperating recombinase-aided isothermal amplification for the ultrasensitive and rapid detection of low-abundance EGFR mutations on microfluidic chip.},
journal = {Biosensors &amp; bioelectronics},
volume = {247},
number = {},
pages = {115925},
doi = {10.1016/j.bios.2023.115925},
pmid = {38134625},
issn = {1873-4235},
abstract = {The detection of low-abundance mutation genes of the epidermal growth factor receptor (EGFR) exon 21 (EGFR L858R) plays a crucial role in the diagnosis of non-small cell lung cancer (NSCLC), as it enables early cancer detection and facilitates the development of treatment strategies. A detection platform was developed by combining the MscI restriction enzyme with the recombinase-aided isothermal amplification (RAA) technique (MRE-RAA). During the RAA process, "TGG^CCA" site of the wild-type genes was cleaved by the MscI restriction enzyme, while only the low-abundance mutation genes underwent amplification. Notably, when the RAA product was combined with CRISPR-Cas system, the sensitivity of detecting the EGFR L858R mutation increased by up to 1000-fold for addition of the MscI restriction enzyme. This achievement marked the first instance of attaining an analytical sensitivity of 0.001%. Furthermore, a disk-shaped microfluidic chip was developed to automate pretreatment while concurrently analyzing four blood samples. The microfluidic features of the chip include DNA extraction, MRE-RAA, and CRISPR-based detection. The fluorescence signal is employed for detection in the microfluidic chip, which is visible to the naked eye upon exposure to blue light irradiation. Furthermore, this platform has the capability to facilitate early diagnosis for various types of cancer by enabling high-sensitivity detection of low-abundance mutation genes.},
}
@article {pmid37993335,
year = {2023},
author = {Zheng, B and Niu, L and Xu, H and Yang, Y and Chen, Y and Wang, C and Chen, W and Huang, W},
title = {Engineering redirected NF-κB/OIP5 expression programs to enhance tumor responses to chemotherapy in bladder cancer.},
journal = {Science bulletin},
volume = {68},
number = {24},
pages = {3207-3224},
doi = {10.1016/j.scib.2023.11.027},
pmid = {37993335},
issn = {2095-9281},
mesh = {Humans ; *NF-kappa B/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Urinary Bladder Neoplasms/drug therapy ; Vincristine/pharmacology ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Nuclear factor kappa-B (NF-κB), a pivotal transcriptional regulator, plays a crucial role in modulating downstream genes implicated in tumor drug resistance. We establish a programmable system within bladder cancer cells to tailor drug responses by employing a synthetic clustered regularly interspaced short palindromic repeats (CRISPR)-based expression strategy that emulates natural transcriptional regulators. Our investigation uncovers the functional significance of Opa-interacting protein 5 (OIP5), upregulated upon NF-κB activation, as a key regulator governing drug-resistance to vincristine (VCR) treatment in bladder cancer. Through engineered guide RNAs (sgRNAs) targeting OIP5 to integrate NF-κB aptamers, we construct a modular scaffold RNA that encodes both the target locus and regulatory functionality. This engineered CRISPR scaffold RNA effectively responds to VCR stimulus by binding with activated NF-κB. Intriguingly, it redirects NF-κB to attenuate OIP5 expression-a reversal of its original role-while concurrently obstructing multiple NF-κB-mediated drug resistance pathways. This dual action thwarts drug resistance development. Further enhancing therapeutic potential, we develop a versatile nanoparticle system capable of co-delivering CRISPR scaffold RNAs and VCR. This synergistic approach demonstrates potent anti-tumor effects in both in vitro and in vivo settings. Our nanoparticle-mediated combination presents a compelling proof-of-concept, showcasing the utility of engineered CRISPR-based synthetic expression programs to reconfigure cellular drug responses and heighten tumor cell susceptibility to chemotherapy.},
}
@article {pmid37989676,
year = {2023},
author = {Wang, X and Rao, Q and Lu, Z and Deng, X and Shen, R and Wang, R and Dong, W and Qi, X and Jin, Z and Tang, Y and Du, D},
title = {Rapid and sensitive Cas13a/Cas12a-based one-pot dual-target strategy to detect monkeypox virus and its co-infected viruses.},
journal = {Science bulletin},
volume = {68},
number = {24},
pages = {3142-3148},
doi = {10.1016/j.scib.2023.11.023},
pmid = {37989676},
issn = {2095-9281},
mesh = {*Monkeypox virus/genetics ; *CRISPR-Cas Systems ; },
}
@article {pmid37973465,
year = {2023},
author = {Fu, Y and He, X and Gao, XD and Li, F and Ge, S and Yang, Z and Fan, X},
title = {Prime editing: current advances and therapeutic opportunities in human diseases.},
journal = {Science bulletin},
volume = {68},
number = {24},
pages = {3278-3291},
doi = {10.1016/j.scib.2023.11.015},
pmid = {37973465},
issn = {2095-9281},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Mutation ; Point Mutation ; },
abstract = {Gene editing ushers in a new era of disease treatment since many genetic diseases are caused by base-pair mutations in genomic DNA. With the rapid development of genome editing technology, novel editing tools such as base editing and prime editing (PE) have attracted public attention, heralding a great leap forward in this field. PE, in particular, is characterized by no need for double-strand breaks (DSBs) or homology sequence templates with variable application scenarios, including point mutations as well as insertions or deletions. With higher editing efficiency and fewer byproducts than traditional editing tools, PE holds great promise as a therapeutic strategy for human diseases. Subsequently, a growing demand for the standard construction of PE system has spawned numerous easy-to-access internet resources and tools for personalized prime editing guide RNA (pegRNA) design and off-target site prediction. In this review, we mainly introduce the innovation and evolutionary strategy of PE systems and the auxiliary tools for PE design and analysis. Additionally, its application and future potential in the clinical field have been summarized and envisaged.},
}
@article {pmid37874946,
year = {2023},
author = {Li, X and Gui, S and Gui, R and Li, J and Huang, R and Hu, M and Luo, XJ and Nie, X},
title = {Multifunctional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-Based Nanobomb against Carbapenem-Resistant Acinetobacter baumannii Infection through Cascade Reaction and Amplification Synergistic Effect.},
journal = {ACS nano},
volume = {17},
number = {24},
pages = {24632-24653},
doi = {10.1021/acsnano.3c03267},
pmid = {37874946},
issn = {1936-086X},
mesh = {Animals ; Mice ; *Acinetobacter baumannii/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Acinetobacter Infections/drug therapy/genetics/microbiology ; Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Carbapenems/pharmacology/therapeutic use ; Imipenem/pharmacology/therapeutic use ; Microbial Sensitivity Tests ; },
abstract = {Carbapenems have been considered to be the preferred antibiotics against Acinetobacter baumannii thus far. However, carbapenem-resistant Acinetobacter baumannii (CRAB) has gradually escalated worldwide, and it frequently causes respiratory and bloodstream infections. Its resistance may lead to high mortality. Thus, there is an urgent need to develop antibacterial drugs. In our research, the pH-sensitive sgRNA-I/L@ZS nanosystem delivered imipenem and better released it in infected tissues to synergistically damage bacteria with nanoparticles. Gene editing of the CRISPR-Cas9 nanosystem amplified the synergistic effect by reversing the drug-resistance of imipenem. Nitric oxide, which l-arginine reacted with ROS to produce in cascade reaction and bacterial infection sites, was beneficial to heal the infected tissues and induce bacteria death for further enhancing antibacterial effects. In addition, this nanocomposite influenced host-bacteria interactions and restrained and destroyed biofilms. The sgRNA-I/L@ZS nanosystem, similar to a nanobomb, was a high-efficiency bactericide against CRAB. Eventually, in acute pneumonia and peritonitis mouse models, the sgRNA-I/L@ZS nanosystem could combat bacteria and protect tissues from infection. It had marked suppressive effects on inflammation and promoted healing and proliferation of infected tissues. This multifunctional nanosystem is expected to be an effective antibacterial agent in the clinic based on good biocompatibility and no toxic side effects. Therefore, developing the nanocomposites will take a favorable step toward solving intractable public health issues.},
}
@article {pmid37017106,
year = {2023},
author = {Rangari, SK and Kaur Sudha, M and Kaur, H and Uppal, N and Singh, G and Vikal, Y and Sharma, P},
title = {DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize.},
journal = {GM crops &amp; food},
volume = {14},
number = {1},
pages = {1-7},
doi = {10.1080/21645698.2023.2197303},
pmid = {37017106},
issn = {2164-5701},
mesh = {*Gene Editing/methods ; *Zea mays/genetics ; RNA, Guide, CRISPR-Cas Systems ; Frameshift Mutation ; DNA ; CRISPR-Cas Systems/genetics ; },
abstract = {Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger's sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.},
}
@article {pmid38134336,
year = {2023},
author = {Devarajan, A},
title = {Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00596},
pmid = {38134336},
issn = {2161-5063},
abstract = {CRISPR-Cas9 and Cre recombinase, two tools extensively used for genome interrogation, have catalyzed key breakthroughs in our understanding of complex biological processes and diseases. However, the immense complexity of biological systems and off-target effects hinder clinical applications, necessitating the development of platforms to control gene editing over spatial and temporal dimensions. Among the strategies developed for inducible control, light is particularly attractive as it is noninvasive and affords high spatiotemporal resolution. The principles for optical control of Cas9 and Cre recombinase are broadly similar and involve photocaged enzymes and small molecules, engineered split- and single-chain constructs, light-induced expression, and delivery by light-responsive nanocarriers. Few systems enable spatiotemporal control with a high dynamic range without loss of wild-type editing efficiencies. Such systems posit the promise of light-activatable systems in the clinic. While the prospect of clinical applications is palpably exciting, optimization and extensive preclinical validation are warranted. Judicious integration of optically activated CRISPR and Cre, tailored for the desired application, may help to bridge the "bench-to-bedside" gap in therapeutic gene editing.},
}
@article {pmid38134284,
year = {2023},
author = {Huth, T and Dreher, EC and Lemke, S and Fritzsche, S and Sugiyanto, RN and Castven, D and Ibberson, D and Sticht, C and Eiteneuer, E and Jauch, A and Pusch, S and Albrecht, T and Goeppert, B and Candia, J and Wang, XW and Ji, J and Marquardt, JU and Nahnsen, S and Schirmacher, P and Roessler, S},
title = {Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.},
journal = {Science advances},
volume = {9},
number = {51},
pages = {eadh1442},
pmid = {38134284},
issn = {2375-2548},
mesh = {Humans ; *Synthetic Lethal Mutations ; *Liver Neoplasms/genetics/pathology ; Chromosome Deletion ; Chromosome Aberrations ; Chromosomes ; CRISPR-Cas Systems ; },
abstract = {Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.},
}
@article {pmid38131794,
year = {2023},
author = {Safenkova, IV and Samokhvalov, AV and Serebrennikova, KV and Eremin, SA and Zherdev, AV and Dzantiev, BB},
title = {DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts.},
journal = {Biosensors},
volume = {13},
number = {12},
pages = {},
pmid = {38131794},
issn = {2079-6374},
support = {075-15-2022-318//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*Salts ; CRISPR-Cas Systems ; Reproducibility of Results ; DNA ; DNA, Single-Stranded ; Fluorescein ; *Biosensing Techniques ; },
abstract = {CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg[2+], Ca[2+], Ba[2+]), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg[2+]. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes.},
}
@article {pmid38131781,
year = {2023},
author = {Bhardwaj, P and Nanaware, NS and Behera, SP and Kulkarni, S and Deval, H and Kumar, R and Dwivedi, GR and Kant, R and Singh, R},
title = {CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus.},
journal = {Biosensors},
volume = {13},
number = {12},
pages = {},
pmid = {38131781},
issn = {2079-6374},
support = {Intramural//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Scrub Typhus/diagnosis/genetics ; CRISPR-Cas Systems ; Sensitivity and Specificity ; *Orientia tsutsugamushi/genetics ; DNA ; },
abstract = {Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.},
}
@article {pmid38129658,
year = {2023},
author = {Liu, J and Li, W and Jin, X and Lin, F and Han, J and Zhang, Y},
title = {Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {1300},
pmid = {38129658},
issn = {2399-3642},
support = {82388201//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31801158//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Genome ; Fluorescence ; },
abstract = {CRISPR-mediated knock-in (KI) technology opens a new era of fluorescent-protein labeling in zebrafish, a preferred model organism for in vivo imaging. We described here an optimized zebrafish gene-tagging strategy, which enables easy and high-efficiency KI, ensures high odds of obtaining seamless KI germlines and is suitable for wide applications. Plasmid donors for 3'-labeling were optimized by shortening the microhomologous arms and by reducing the number and reversing the sequence of the consensus Cas9/sgRNA binding sites. To allow for scar-less KI across the genome, linearized dsDNA donors with 5'-chemical modifications were generated and successfully incorporated into our method. To refine the germline screen workflow and expedite the screen process, we combined fluorescence enrichment and caudal-fin junction-PCR. Furthermore, to trace proteins expressed at a low abundance, we developed a fluorescent signal amplifier using the transcriptional activation strategy. Together, our strategies enable efficient gene-tagging and sensitive expression detection for almost every gene in zebrafish.},
}
@article {pmid38128483,
year = {2023},
author = {Sashittal, P and Schmidt, H and Chan, M and Raphael, BJ},
title = {Startle: A star homoplasy approach for CRISPR-Cas9 lineage tracing.},
journal = {Cell systems},
volume = {14},
number = {12},
pages = {1113-1121.e9},
doi = {10.1016/j.cels.2023.11.005},
pmid = {38128483},
issn = {2405-4720},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Phylogeny ; *Gene Editing/methods ; Cell Lineage/genetics ; Mutation ; },
abstract = {CRISPR-Cas9-based genome editing combined with single-cell sequencing enables the tracing of the history of cell divisions, or cellular lineage, in tissues and whole organisms. Although standard phylogenetic approaches may be applied to reconstruct cellular lineage trees from this data, the unique features of the CRISPR-Cas9 editing process motivate the development of specialized models that describe the evolution of CRISPR-Cas9-induced mutations. Here, we introduce the "star homoplasy" evolutionary model that constrains a phylogenetic character to mutate at most once along a lineage, capturing the "non-modifiability" property of CRISPR-Cas9 mutations. We derive a combinatorial characterization of star homoplasy phylogenies and use this characterization to develop an algorithm, "Startle", that computes a maximum parsimony star homoplasy phylogeny. We demonstrate that Startle infers more accurate phylogenies on simulated lineage tracing data compared with existing methods and finds parsimonious phylogenies with fewer metastatic migrations on lineage tracing data from mouse metastatic lung adenocarcinoma.},
}
@article {pmid38128320,
year = {2023},
author = {Chen, H and Feng, Y and Liu, F and Tan, C and Xu, N and Jiang, Y and Tan, Y},
title = {Universal smartphone-assisted label-free CRISPR/Cas12a-DNAzyme chemiluminescence biosensing platform for on-site detection of nucleic acid and non-nucleic acid targets.},
journal = {Biosensors &amp; bioelectronics},
volume = {247},
number = {},
pages = {115929},
doi = {10.1016/j.bios.2023.115929},
pmid = {38128320},
issn = {1873-4235},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) system enables sensitive and specific detection of biomolecules, thanks to its programmability, high fidelity, and powerful signal amplification capabilities. Herein, a universal smartphone-assisted label-free G-quadruplex (G4) DNAzyme-based chemiluminescence CRISPR/Cas12a biosensing platform (G4CLCas) is firstly described that achieves on-site, ultrasensitive visual detection of nucleic acid and non-nucleic acid targets. The G4CLCas-based sensing platform relies on Cas12a trans-cleavage activation that triggers the cleavage of the G4 DNAzyme, resulting in chemiluminescence signals off/on compared to that of the control. Chemiluminescence signals are captured as images that are quantitatively analyzed and visualized using a smartphone-assisted imaging cartridge. Under optimal conditions, G4CLCas achieves a low limit of detection (LOD) of 8.6 aM (∼5.2 copies/μL) for monkeypox virus (MPXV) DNA within the linear concentration range of 10-300 aM and can accurately quantify viral DNA in spiked samples. G4CLCas can also detect non-nucleic acid targets, whereby it achieves a low LOD value of 84.3 nM for adenosine triphosphate (ATP) within the linear concentration range of 2-2000 μM. Here, a label-free, portable, on-site CRISPR/Cas12a chemiluminescence biosensing platform based on the G4 DNAzyme substrates is proposed with potential applications in clinical detection and bioanalytical chemistry research.},
}
@article {pmid38127746,
year = {2023},
author = {Cohen, J},
title = {'Gene drive' may help fight viruses with viruses.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6677},
pages = {1337-1338},
doi = {10.1126/science.adn6357},
pmid = {38127746},
issn = {1095-9203},
mesh = {Mice ; Animals ; *Herpesviridae/genetics ; *Gene Drive Technology ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {CRISPR-engineered herpesviruses can speed spread of genes to viral relatives in mice.},
}
@article {pmid38126782,
year = {2023},
author = {Geslewitz, WE and Cardenas, A and Zhou, X and Zhang, Y and Criss, AK and Seifert, HS},
title = {Development and implementation of a Type I-C CRISPR-based programmable repression system for Neisseria gonorrhoeae.},
journal = {mBio},
volume = {},
number = {},
pages = {e0302523},
doi = {10.1128/mbio.03025-23},
pmid = {38126782},
issn = {2150-7511},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have proven instrumental in genetically manipulating many eukaryotic and prokaryotic organisms. Despite its usefulness, a CRISPR system had yet to be developed for use in Neisseria gonorrhoeae (Gc), a bacterium that is the main etiological agent of gonorrhea infection. Here, we developed a programmable and IPTG-inducible Type I-C CRISPR interference (CRISPRi) system derived from the commensal species Neisseria lactamica as a gene repression system in Gc. As opposed to generating genetic knockouts, the Type I-C CRISPRi system allows us to block transcription of specific genes without generating deletions in the DNA. We explored the properties of this system and found that a minimal spacer array is sufficient for gene repression while also facilitating efficient spacer reprogramming. Importantly, we also show that we can use CRISPRi to knockdown genes that are essential to Gc that cannot normally be knocked out under laboratory settings. Gc encodes ~800 essential genes, many of which have no predicted function. We predict that this Type I-C CRISPRi system can be used to help categorize gene functions and perhaps contribute to the development of novel therapeutics for gonorrhea.},
}
@article {pmid38126308,
year = {2023},
author = {Wang, B and Xu, YT and Zhang, TY and Wang, HY and Zhang, X and Wu, ZQ and Zhao, WW and Chen, HY and Xu, JJ},
title = {An Ultrasensitive and Efficient microRNA Nanosensor Empowered by the CRISPR/Cas Confined in a Nanopore.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.3c03723},
pmid = {38126308},
issn = {1530-6992},
abstract = {This work presents a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-nanopipette nano-electrochemistry (Cas = CRISPR-associated proteins) capable of ultrasensitive microRNA detection. Nanoconfinement of the CRISPR/Cas13a within a nanopipette leads to a high catalytic efficacy of ca. 169 times higher than that in bulk electrolyte, contributing to the amplified electrochemical responses. CRISPR/Cas13a-enabled detection of representative microRNA-25 achieves a low limit of detection down to 10 aM. Practical application of this method is further demonstrated for single-cell and real human serum detection. Its general applicability is validated by addressing microRNA-141 and the SARS-CoV-2 RNA gene fragment. This work introduces a new CRISPR/Cas-empowered nanotechnology for ultrasensitive nano-electrochemistry and bioanalysis.},
}
@article {pmid38126277,
year = {2023},
author = {Clarkson, J and Yip, SH and Porteous, R and Kauff, A and Heather, AK and Herbison, AE},
title = {CRISPR-Cas9 knockdown of ESR1 in preoptic GABA-kisspeptin neurons suppresses the preovulatory surge and estrous cycles in female mice.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {38126277},
issn = {2050-084X},
support = {/WT_/Wellcome Trust/United Kingdom ; 10.35802/212242/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Mice ; Female ; Animals ; *Kisspeptins/metabolism ; *CRISPR-Cas Systems ; Gonadotropin-Releasing Hormone/metabolism ; GABAergic Neurons/metabolism ; Estrous Cycle/physiology ; gamma-Aminobutyric Acid/metabolism ; },
abstract = {Evidence suggests that estradiol-sensing preoptic area GABA neurons are involved in the preovulatory surge mechanism necessary for ovulation. In vivo CRISPR-Cas9 editing was used to achieve a 60-70% knockdown in estrogen receptor alpha (ESR1) expression by GABA neurons located within the regions of the rostral periventricular area of the third ventricle (RP3V) and medial preoptic nuclei (MPN) in adult female mice. Mice exhibited variable reproductive phenotypes with the only significant finding being mice with bilateral ESR1 deletion in RP3V GABA neurons having reduced cFos expression in gonadotropin-releasing hormone (GnRH) neurons at the time of the surge. One sub-population of RP3V GABA neurons expresses kisspeptin. Re-grouping ESR1-edited mice on the basis of their RP3V kisspeptin expression revealed a highly consistent phenotype; mice with a near-complete loss of kisspeptin immunoreactivity displayed constant estrus and failed to exhibit surge activation but retained pulsatile luteinizing hormone (LH) secretion. These observations demonstrate that ESR1-expressing GABA-kisspeptin neurons in the RP3V are essential for the murine preovulatory LH surge mechanism.},
}
@article {pmid38125580,
year = {2023},
author = {Panahi, B and Dehganzad, B and Nami, Y},
title = {CRISPR-Cas systems feature and targeting phages diversity in Lacticaseibacillus rhamnosus strains.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1281307},
pmid = {38125580},
issn = {1664-302X},
abstract = {One of the most important adaptive immune systems in bacteria against phages is clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes. In this investigation, an approach based on genome mining was employed to characterize the CRISPR-Cas systems of Lacticaseibacillus rhamnosus strains. The analysis involved retrieving complete genome sequences of L. rhamnosus strains, and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Following this, an analysis of homology in spacer sequences from identified CRISPR arrays was carried out to investigate and characterize the range of target phages. The findings revealed that 106 strains possessed valid CRISPR-Cas structures (comprising CRISPR loci and Cas genes), constituting 45% of the examined L. rhamnosus strains. The diversity observed in the CRISPR-Cas systems indicated that all identified systems belonged to subtype II-A. Analyzing the homology of spacer sequences with phage and prophage genomes discovered that strains possessing only CRISPR-Cas subtype II targeted a broader spectrum of foreign phages. In summary, this study suggests that while there is not significant diversity among the CRISPR-Cas systems identified in L. rhamnosus strains, there exists notable variation in subtype II-A systems between L. rhamnosus and other lactobacilli. The diverse nature of these CRISPR-Cas systems underscores their natural activity and importance in adaptive immunity.},
}
@article {pmid38122983,
year = {2023},
author = {Mondol, SM and Islam, MR and Rakhi, NN and Shakil, SK and Islam, I and Mustary, JF and Amiruzzaman, and Shahjalal, HM and Gomes, DJ and Rahaman, MM},
title = {Unveiling a High-Risk Epidemic Clone (ST 357) of 'Difficult to Treat Extensively Drug-Resistant' (DT-XDR) Pseudomonas aeruginosa from a burn patient in Bangladesh: A Resilient Beast Revealing Co-existence of Four Classes of Beta Lactamases.},
journal = {Journal of global antimicrobial resistance},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgar.2023.11.014},
pmid = {38122983},
issn = {2213-7173},
abstract = {OBJECTIVES: Pseudomonas aeruginosa (P. aeruginosa) stands out as a key culprit in the colonization of burn wounds, instigating grave infections of heightened severity. In this study, we have performed comparative whole genome analysis of a difficult to treat extensively drug resistant (DT-XDR) P. aeruginosa isolated from a burn patient in order to elucidate genomic diversity, molecular patterns, mechanisms and genes responsible for conferring antimicrobial resistance and virulence.<br><br>METHOD: P. aeruginosa SHNIBPS206 was isolated from an infected burn wound of a critically injured burn patient. Whole genome sequencing was carried out and annotated with Prokka. Sequence type, serotype, antimicrobial resistance genes and mechanisms, virulence genes, metal resistance genes and CRISPR/Cas systems were investigated. Later, pangenome analysis was carried out to find out genomic diversity.<br><br>RESULT: P. aeruginosa SHNIBPS206 (MLST 357, Serotype O11)) was resistant to 14 antibiotics including carbapenems and harboured all four classes of beta lactamase producing genes: Class A (blaPME-1, blaVEB-9), Class B (blaNDM-1), Class C (blaPDC-11) and Class D (blaOXA-846). Mutational analysis of Porin D gave valuable insights. Several efflux pump, virulence and metal resistance genes were also detected. Pangenome analysis revealed high genomic diversity among different strains of P. aeruginosa.<br><br>CONCLUSION: To our knowledge, this is the first report of an extensively drug resistant ST 357 P. aeruginosa from Bangladesh, which is an epidemic high-risk P. aeruginosa clone. Further research and in-depth comprehensive studies are required to investigate the prevalence of such high-risk clone of P. aeruginosa in Bangladesh.},
}
@article {pmid38117659,
year = {2023},
author = {Sun, Y and Hiew, SH and Miserez, A},
title = {Bioinspired Squid Peptides─A Tale of Curiosity-Driven Research Leading to Unforeseen Biomedical Applications.},
journal = {Accounts of chemical research},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.accounts.3c00685},
pmid = {38117659},
issn = {1520-4898},
abstract = {ConspectusThe molecular design of many peptide-based materials originates from structural proteins identified in living organisms. Prominent examples that have garnered broad interdisciplinary research interest (chemistry, materials science, bioengineering, etc.) include elastin, silk, or mussel adhesive proteins. The critical first steps in this type of research are to identify a convenient model system of interest followed by sequencing the prevailing proteins from which these biological structures are assembled. In our laboratory, the main model systems for many years have been the hard biotools of cephalopods, particularly their parrot-like tough beak and their sucker ring teeth (SRT) embedded within the sucker cuptions that line the interior surfaces of their arms and tentacles. Unlike the majority of biological hard tissues, these structures are devoid of biominerals and consist of protein/polysaccharide biomolecular composites (the beak) or, in the case of SRT, are entirely made of proteins that are assembled by supramolecular interactions.In this Account, we chronicle our journey into the discovery of these intriguing biological materials. We initially focus on their excellent mechanical robustness followed by the identification and sequencing of the structural proteins from which they are built, using the latest "omics" techniques including next-generation sequencing and high-throughput proteomics. A common feature of these proteins is their modular architecture at the molecular level consisting of short peptide repeats. We describe the molecular design of these peptide building blocks, highlighting the consensus motifs identified to play a key role in biofabrication and in regulating the mechanical properties of the macroscopic biological material. Structure/property relationships unveiled through advanced spectroscopic and scattering techniques, including Raman, infrared, circular dichroism, and NMR spectroscopies as well as wide-angle and small-angle X-ray scattering, are also discussed.We then present recent developments in exploiting the discovered molecular designs to engineer peptides and their conjugates for promising biomedical applications. One example includes short peptide hydrogels that self-assemble entirely under aqueous conditions and simultaneously encapsulate large macromolecules during the gelation process. A second example involves peptide coacervate microdroplets produced by liquid-liquid phase separation. These microdroplets are capable of recruiting and delivering large macromolecular therapeutics (genes, mRNA, proteins, peptides, CRISPR/Cas 9 modalities, etc.) into mammalian cells, which introduces exciting prospects in cancer, gene, and immune therapies.This Account also serves as a testament to how curiosity-driven explorations, which may lack an obvious practical goal initially, can lead to discoveries with unexpected and promising translational potential.},
}
@article {pmid38117128,
year = {2023},
author = {Chen, Q and Wu, J and Tang, C and Wang, Y},
title = {CRISPR-based platforms for the specific and dual detection of defoliating/nondefoliating strains of Verticillium dahliae.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.7940},
pmid = {38117128},
issn = {1526-4998},
abstract = {BACKGROUND: Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt disease on more than 400 plant species worldwide. Due to its broad host range and its ability to survive long term in the soil, there are few effective control measures for V. dahliae once it has become established. Accurate, sensitive, and rapid detection of V. dahliae is crucial for limiting pathogen entry into new regional environments and early management of Verticillium wilt.<br><br>RESULTS: In this study, we developed a method to detect V. dahliae based on recombinase polymerase amplification (RPA) and CRISPR/Cas technology and used fluorescence and lateral flow test strips to monitor the outcomes. Through the establishment and optimization of RPA-CRISPR/Cas13a detection, the sensitivity of the fluorescence method was 1 aM for genomic DNA (gDNA) within 20 min, while the sensitivity of the lateral flow strip method was 100 aM for gDNA in 30 min. The field applicability of RPA-CRISPR/Cas13a was also validated by the detection of V. dahliae on smoke trees (Cotinus coggygria) in Xiangshan Park, Beijing, China. Finally, diplex detection for defoliating and nondefoliating pathotypes of V. dahliae was established by combining CRISPR-Cas12a/Cas13a with specific target genes.<br><br>CONCLUSION: Taken together, this study achieved rapid, sensitive, and accurate detection of V. dahliae and the differentiation of defoliating and nondefoliating pathotypes and provides potential for field-deployable diagnostic tools for rapid and ultrasensitive detection. This article is protected by copyright. All rights reserved.},
}
@article {pmid38091991,
year = {2023},
author = {Wei, J and Lotfy, P and Faizi, K and Baungaard, S and Gibson, E and Wang, E and Slabodkin, H and Kinnaman, E and Chandrasekaran, S and Kitano, H and Durrant, MG and Duffy, CV and Pawluk, A and Hsu, PD and Konermann, S},
title = {Deep learning and CRISPR-Cas13d ortholog discovery for optimized RNA targeting.},
journal = {Cell systems},
volume = {14},
number = {12},
pages = {1087-1102.e13},
doi = {10.1016/j.cels.2023.11.006},
pmid = {38091991},
issn = {2405-4720},
mesh = {Animals ; Humans ; *RNA/genetics ; CRISPR-Cas Systems/genetics ; *Deep Learning ; RNA, Guide, CRISPR-Cas Systems ; Transcriptome ; Mammals/genetics ; },
abstract = {Effective and precise mammalian transcriptome engineering technologies are needed to accelerate biological discovery and RNA therapeutics. Despite the promise of programmable CRISPR-Cas13 ribonucleases, their utility has been hampered by an incomplete understanding of guide RNA design rules and cellular toxicity resulting from off-target or collateral RNA cleavage. Here, we quantified the performance of over 127,000 RfxCas13d (CasRx) guide RNAs and systematically evaluated seven machine learning models to build a guide efficiency prediction algorithm orthogonally validated across multiple human cell types. Deep learning model interpretation revealed preferred sequence motifs and secondary features for highly efficient guides. We next identified and screened 46 novel Cas13d orthologs, finding that DjCas13d achieves low cellular toxicity and high specificity-even when targeting abundant transcripts in sensitive cell types, including stem cells and neurons. Our Cas13d guide efficiency model was successfully generalized to DjCas13d, illustrating the power of combining machine learning with ortholog discovery to advance RNA targeting in human cells.},
}
@article {pmid38010352,
year = {2023},
author = {Cao, G and Yang, N and Xiong, Y and Shi, M and Wang, L and Nie, F and Huo, D and Hou, C},
title = {Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays.},
journal = {ACS sensors},
volume = {8},
number = {12},
pages = {4655-4663},
doi = {10.1021/acssensors.3c01686},
pmid = {38010352},
issn = {2379-3694},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems/genetics ; Mutation ; DNA, Single-Stranded/genetics ; *Mycobacterium tuberculosis ; Rifampin ; },
abstract = {Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the "TTTN". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA not only achieved efficient amplification but also converted dsDNA to single-stranded DNA (ssDNA) without complicated steps. The ssDNA products activated the trans-cleavage activity of Cas12a, outputting signals. The application of Asy-RPA completely freed Cas12a from the PAM, which can be more widely used in nucleic acid detection, such as lumpy skin disease virus, with an actual detection limit as low as 1.21 × 10[1] copies·μL[-1]. More importantly, Cas12a was intolerant to mutations on ssDNA. This provided technical support for the detection and identification of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples. The detection and availability of different treatment options for treatment-resistant and WT-TB were significant for the elimination of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a was suitable for the detection of various viruses and bacteria and was a boon for the detection of dsDNA without recognizable PAM.},
}
@article {pmid37995688,
year = {2023},
author = {Bao, Y and Pan, Q and Xu, P and Liu, Z and Zhang, Z and Liu, Y and Xu, Y and Yu, Y and Zhou, Z and Wei, W},
title = {Unbiased interrogation of functional lysine residues in human proteome.},
journal = {Molecular cell},
volume = {83},
number = {24},
pages = {4614-4632.e6},
doi = {10.1016/j.molcel.2023.10.033},
pmid = {37995688},
issn = {1097-4164},
mesh = {Humans ; *Proteome/genetics ; *Lysine/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Codon ; },
abstract = {CRISPR screens have empowered the high-throughput dissection of gene functions; however, more explicit genetic elements, such as codons of amino acids, require thorough interrogation. Here, we establish a CRISPR strategy for unbiasedly probing functional amino acid residues at the genome scale. By coupling adenine base editors and barcoded sgRNAs, we target 215,689 out of 611,267 (35%) lysine codons, involving 85% of the total protein-coding genes. We identify 1,572 lysine codons whose mutations perturb human cell fitness, with many of them implicated in cancer. These codons are then mirrored to gene knockout screen data to provide functional insights into the role of lysine residues in cellular fitness. Mining these data, we uncover a CUL3-centric regulatory network in which lysine residues of CUL3 CRL complex proteins control cell fitness by specifying protein-protein interactions. Our study offers a general strategy for interrogating genetic elements and provides functional insights into the human proteome.},
}
@article {pmid37973463,
year = {2023},
author = {Yang, L and Chen, J},
title = {Expanding genome editing scopes with artificial intelligence.},
journal = {Science bulletin},
volume = {68},
number = {23},
pages = {2881-2883},
doi = {10.1016/j.scib.2023.10.025},
pmid = {37973463},
issn = {2095-9281},
mesh = {*Gene Editing ; *Artificial Intelligence ; CRISPR-Cas Systems ; },
}
@article {pmid37865316,
year = {2023},
author = {Park, E and Yang, CR and Raghuram, V and Chen, L and Chou, CL and Knepper, MA},
title = {Using CRISPR-Cas9/phosphoproteomics to identify substrates of calcium/calmodulin-dependent kinase 2δ.},
journal = {The Journal of biological chemistry},
volume = {299},
number = {12},
pages = {105371},
doi = {10.1016/j.jbc.2023.105371},
pmid = {37865316},
issn = {1083-351X},
mesh = {*Aquaporin 2/genetics/chemistry/metabolism ; Calcium/metabolism ; CRISPR-Cas Systems ; Calmodulin/metabolism ; Chromatography, Liquid ; *Kidney Tubules, Collecting/metabolism ; Tandem Mass Spectrometry ; RNA, Guide, CRISPR-Cas Systems ; Vasopressins/metabolism ; Phosphorylation ; Calcium-Calmodulin-Dependent Protein Kinases/metabolism ; },
abstract = {Ca[2+]/Calmodulin-dependent protein kinase 2 (CAMK2) family proteins are involved in the regulation of cellular processes in a variety of tissues including brain, heart, liver, and kidney. One member, CAMK2δ (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water excretion through regulation of the water channel aquaporin-2 (AQP2). To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d and carried out LC-MS/MS-based quantitative phosphoproteomics. Specifically, we used CRISPR/Cas9 with two different guide RNAs targeting the CAMK2D catalytic domain to create multiple CAMK2D KO cell lines. AQP2 protein abundance was lower in the CAMK2D KO cells than in CAMK2D-intact controls. AQP2 phosphorylation at Ser256 and Ser269 (normalized for total AQP2) was decreased. However, trafficking of AQP2 to and from the apical plasma membrane was sustained. Large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1 nM, 30 min) allowed quantification of 11,570 phosphosites of which 169 were significantly decreased, while 206 were increased in abundance in CAMK2D KO clones. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of -(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous in vitro phosphorylation studies using recombinant CAMK2D. Thirty five of the significantly downregulated phosphorylation sites in CAMK2D KO cells had exactly this motif and are judged to be likely direct CAMK2D targets. This adds to the list of known CAMK2D target proteins found in prior reductionist studies.},
}
@article {pmid37772866,
year = {2023},
author = {Li, YG and Kishida, K and Ogawa-Kishida, N and Christie, PJ},
title = {Ligand-displaying Escherichia coli cells and minicells for programmable delivery of toxic payloads via type IV secretion systems.},
journal = {mBio},
volume = {14},
number = {5},
pages = {e0214323},
pmid = {37772866},
issn = {2150-7511},
support = {R35 GM131892/GM/NIGMS NIH HHS/United States ; },
mesh = {*Escherichia coli ; *Type IV Secretion Systems ; Ligands ; Plasmids/genetics ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems ; },
abstract = {The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize the urgent need for alternative antibacterial strategies. We engineered Escherichia coli to conjugatively transfer plasmids to specific E. coli and Pseudomonas aeruginosa recipient cells through the surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for the selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled programmed delivery system (PDS), Nb-displaying E. coli donors with different conjugation systems and mobilizable pCrispr plasmids suppressed the growth of Ag-displaying recipient cells to significantly greater extents than unpaired recipients. We also showed that anucleate minicells armed with conjugation machines and pCrispr plasmids were highly effective in killing E. coli recipients. Together, our findings suggest that bacteria or minicells armed with PDSs may prove highly effective as an adjunct or alternative to antibiotics for antimicrobial intervention.},
}
@article {pmid37770768,
year = {2024},
author = {Mathey-Andrews, N},
title = {Prime editing GEMMs to model cancer mutations.},
journal = {Nature reviews. Cancer},
volume = {24},
number = {1},
pages = {2},
pmid = {37770768},
issn = {1474-1768},
mesh = {Humans ; Mutation ; *Gene Editing ; CRISPR-Cas Systems ; *Neoplasms/genetics ; },
}
@article {pmid38116030,
year = {2023},
author = {Kath, J and Franke, C and Drosdek, V and Du, W and Glaser, V and Fuster-Garcia, C and Stein, M and Zittel, T and Schulenberg, S and Porter, CE and Andersch, L and Künkele, A and Alcaniz, J and Hoffmann, J and Abken, H and Abou-El-Enein, M and Pruß, A and Suzuki, M and Cathomen, T and Stripecke, R and Volk, HD and Reinke, P and Schmueck-Henneresse, M and Wagner, DL},
title = {Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.11.10.565518},
pmid = {38116030},
abstract = {UNLABELLED: Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.<br><br>KEY POINTS: Integration of &#x3b6;-deficient CARs into CD3 &#x3b6; gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 &#x3b6;-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.},
}
@article {pmid38115051,
year = {2023},
author = {Zheng, L and Jiang, Y and Huang, F and Wu, Q and Lou, Y},
title = {A colorimetric, photothermal, and fluorescent triple-mode CRISPR/cas biosensor for drug-resistance bacteria detection.},
journal = {Journal of nanobiotechnology},
volume = {21},
number = {1},
pages = {493},
pmid = {38115051},
issn = {1477-3155},
support = {LQ21H200008//Zhejiang Provincial Natural Science Foundation of China/ ; Y2020108//Science and Technology Bureau of Wenzhou/ ; ZR2020QB107//Shandong Provincial Natural Science Foundation/ ; 2018ZX10201001-009//National Major Infectious Disease Prevention Projects/ ; },
mesh = {*CRISPR-Cas Systems ; Colorimetry ; Hydrogen Peroxide ; Bacteria/genetics ; Coloring Agents ; DNA, Single-Stranded ; *Biosensing Techniques ; },
abstract = {A multimodal analytical strategy utilizing different modalities to cross-validate each other, can effectively minimize false positives or negatives and ensure the accuracy of detection results. Herein, we establish a colorimetric, photothermal, and fluorescent triple modal CRISPR/Cas12a detection platform (CPF-CRISPR). An MNPs-ssDNA-HRP signal probe is designed to act as a substrate to trigger three signal outputs. In the presence of the DNA target, MNPs-ssDNA-HRP is cleaved by the activated CRISPR/Cas12a, resulting in the release of HRP and generating short DNA strands with 3-terminal hydroxyl on magnetic beads. The released HRP subsequently catalyzed TMB-H2O2 reaction and oxidized TMB is used for colorimetric and photothermal signal detection. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), the remaining short DNA strands are used as primers to form poly-T and function as scaffolds to form copper nanoclusters for fluorescent signal output. To verify the practical application of CPF-CRISPR, we employed MRSA as a model. The results demonstrate the platform's high accuracy and sensitivity, with a limit of detection of 10[1] CFU/mL when combined with recombinase polymerase amplification. Therefore, by harnessing the programmability of CRISPR/Cas12a, the biosensor has the potential to detect various drug-resistant bacteria, demonstrating significant practical applicability.},
}
@article {pmid38114895,
year = {2023},
author = {Butt, L and Unnersjö-Jess, D and Reilly, D and Hahnfeldt, R and Rinschen, MM and Bozek, K and Schermer, B and Benzing, T and Höhne, M},
title = {In vivo characterization of a podocyte-expressed short podocin isoform.},
journal = {BMC nephrology},
volume = {24},
number = {1},
pages = {378},
pmid = {38114895},
issn = {1471-2369},
support = {KFO 329, BR4917/3, INST 1856/71-1 FUGG//Deutsche Forschungsgemeinschaft/ ; KFO 329, BR4917/3, INST 1856/71-1 FUGG//Deutsche Forschungsgemeinschaft/ ; KFO 329, BR4917/3, INST 1856/71-1 FUGG//Deutsche Forschungsgemeinschaft/ ; KFO 329, BR4917/3, INST 1856/71-1 FUGG//Deutsche Forschungsgemeinschaft/ ; KFO 329, BR4917/3, INST 1856/71-1 FUGG//Deutsche Forschungsgemeinschaft/ ; Project No: 2019_KollegSE.04//Else Kr&#xf6;ner-Fresenius-Stiftung,Germany/ ; Project No: 2019_KollegSE.04//Eva Luise und Horst K&#xf6;hler Stiftung/ ; NNF19OC0056043//Novo Nordisk Fonden/ ; Young Researcher Fellowship//Carlsbergfondet/ ; 311-8.03.03.02-147635//North Rhine-Westphalia return program/ ; 01ZX1917B//Bundesministerium f&#xfc;r Bildung und Forschung/ ; BMBF 01-GM1901E//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
mesh = {Humans ; Animals ; Mice ; *Podocytes/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; *Nephrotic Syndrome/genetics/metabolism ; RNA, Messenger/metabolism ; },
abstract = {The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocin[Δexon5]) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocin[Δexon5] and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocin[wildtype] and podocin[Δexon5]. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocin[Δexon5] were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocin[Δexon5], whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocin[Δexon5] were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.},
}
@article {pmid38114787,
year = {2023},
author = {Ertl, H},
title = {Efficient computation reveals rare CRISPR-Cas systems.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {38114787},
issn = {1471-0064},
}
@article {pmid38114763,
year = {2023},
author = {Jiang, Y and Qian, X and Zheng, M and Deng, K and Li, C},
title = {Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors.},
journal = {Mikrochimica acta},
volume = {191},
number = {1},
pages = {43},
pmid = {38114763},
issn = {1436-5073},
support = {2021RC5028//Science and Technology Innovation Program of Hunan Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Transcription Factors ; DNA, Single-Stranded ; RNA ; },
abstract = {An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin. According to the finding, a CRISPR/Cas12a-based strategy coupled with a signal reported system was designed for transcription factors detection. By using mono-labeled ssDNA (single-stranded DNA) as reporter and two newly synthesized N-C (nitrogen-doped carbon) nanosheets as scavenger to eliminate the fluorescent background, the strategy realized the detection of NF-ĸB p50 (p50 subunit of nuclear factor kappa-B) with a linear detection range of 0.8 - 2000.0 pM and a LOD of 0.5 pM. The discovery of "enhancement and inactivation effect" not only deepened insight into CRISPR/Cas12a but also broadened the practical application of CRISPR/Cas systems for the molecular detection and disease diagnostics.},
}
@article {pmid38114601,
year = {2023},
author = {Hofeichner, J and Gahr, BM and Huber, M and Boos, A and Rottbauer, W and Just, S},
title = {CRISPR/Cas9-mediated nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {22679},
pmid = {38114601},
issn = {2045-2322},
support = {JU2859/7-1//Deutsche Forschungsgemeinschaft/ ; JU2859/9-1//Deutsche Forschungsgemeinschaft/ ; e:Med-SYMBOL-HF grant #01ZX1407A//German Federal Ministry of Education and Research/ ; e:Med-coNfirm grant #01ZX1708C//German Federal Ministry of Education and Research/ ; },
mesh = {Humans ; Animals ; Mice ; *Zebrafish/genetics/metabolism ; *Microfilament Proteins/metabolism ; CRISPR-Cas Systems ; Muscle Contraction/genetics ; Muscle, Skeletal/metabolism ; Zebrafish Proteins/genetics/metabolism ; },
abstract = {Nexilin (NEXN) plays a crucial role in stabilizing the sarcomeric Z-disk of striated muscle fibers and, when mutated, leads to dilated cardiomyopathy in humans. Due to its early neonatal lethality in mice, the detailed impact of the constitutive homozygous NEXN knockout on heart and skeletal muscle morphology and function is insufficiently investigated. Here, we characterized a constitutive homozygous CRISPR/Cas9-mediated nexn knockout zebrafish model. We found that Nexn deficient embryos developed significantly reduced cardiac contractility and under stressed conditions also impaired skeletal muscle organization whereas skeletal muscle function seemed not to be affected. Remarkably, in contrast to nexn morphants, CRISPR/Cas9 nexn[-/-] knockout embryos showed a milder phenotype without the development of a pronounced pericardial edema or blood congestion. nexn-specific expression analysis as well as whole transcriptome profiling suggest some degree of compensatory mechanisms. Transcripts of numerous essential sarcomeric proteins were massively induced and may mediate a sarcomere stabilizing function in nexn[-/-] knockout embryos. Our findings demonstrate the successful generation and characterization of a constitutive homozygous nexn knockout line enabling the detailed investigation of the role of nexn on heart and skeletal muscle development and function as well as to assess putative compensatory mechanisms induced by the loss of Nexn.},
}
@article {pmid38111883,
year = {2023},
author = {Spencer, KP and Burger, JT and Campa, M},
title = {CRISPR-based resistance to grapevine virus A.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1296251},
pmid = {38111883},
issn = {1664-462X},
abstract = {INTRODUCTION: Grapevine (Vitis vinifera) is an important fruit crop which contributes significantly to the agricultural sector worldwide. Grapevine viruses are widespread and cause serious diseases which impact the quality and quantity of crop yields. More than 80 viruses plague grapevine, with RNA viruses constituting the largest of these. A recent extension to the clustered regularly interspaced, short palindromic repeat (CRISPR) armory is the Cas13 effector, which exclusively targets single-strand RNA. CRISPR/Cas has been implemented as a defense mechanism in plants, against both DNA and RNA viruses, by being programmed to directly target and cleave the viral genomes. The efficacy of the CRISPR/Cas tool in plants is dependent on efficient delivery of its components into plant cells.<br><br>METHODS: To this end, the aim of this study was to use the recent Cas13d variant from Ruminococcus flavefaciens (CasRx) to target the RNA virus, grapevine virus A (GVA). GVA naturally infects grapevine, but can infect the model plant Nicotiana benthamiana, making it a helpful model to study virus infection in grapevine. gRNAs were designed against the coat protein (CP) gene of GVA. N. benthamiana plants expressing CasRx were co-infiltrated with GVA, and with a tobacco rattle virus (TRV)-gRNA expression vector, harbouring a CP gRNA.<br><br>RESULTS AND DISCUSSION: Results indicated more consistent GVA reductions, specifically gRNA CP-T2, which demonstrated a significant negative correlation with GVA accumulation, as well as multiple gRNA co-infiltrations which similarly showed reduced GVA titre. By establishing a virus-targeting defense system in plants, efficient virus interference mechanisms can be established and applied to major crops, such as grapevine.},
}
@article {pmid38111351,
year = {2023},
author = {Choudhary, A and Goodman, C},
title = {Genome Editing: The Future Begins!.},
journal = {Biochemistry},
volume = {62},
number = {24},
pages = {3453-3454},
doi = {10.1021/acs.biochem.3c00671},
pmid = {38111351},
issn = {1520-4995},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Forecasting ; },
}
@article {pmid38109552,
year = {2023},
author = {Oechslin, N and Da Silva, N and Ankavay, M and Moradpour, D and Gouttenoire, J},
title = {A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {52},
pages = {e2307423120},
doi = {10.1073/pnas.2307423120},
pmid = {38109552},
issn = {1091-6490},
support = {31003A_179424//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (SNF)/ ; 31003A_207477//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (SNF)/ ; 18C140//Novartis Foundation/ ; },
mesh = {Humans ; *Hepatitis E virus/genetics ; *Hepatitis E ; CRISPR-Cas Systems ; Endosomes/genetics/metabolism ; Virus Replication/genetics ; RNA, Viral/genetics ; },
abstract = {Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.},
}
@article {pmid38109298,
year = {2023},
author = {},
title = {Correction to 'CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes'.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad1228},
pmid = {38109298},
issn = {1362-4962},
}
@article {pmid37991367,
year = {2023},
author = {Chang, J and Parent, LJ},
title = {HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.},
journal = {Journal of virology},
volume = {97},
number = {12},
pages = {e0117923},
pmid = {37991367},
issn = {1098-5514},
support = {T32 CA060395/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; HeLa Cells ; *Euchromatin/metabolism ; *HIV-1/genetics/metabolism ; Histone Code ; RNA, Guide, CRISPR-Cas Systems ; Virus Assembly ; },
abstract = {The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.},
}
@article {pmid37936350,
year = {2023},
author = {Wang, J and Maschietto, F and Qiu, T and Arantes, PR and Skeens, E and Palermo, G and Lisi, GP and Batista, VS},
title = {Substrate-independent activation pathways of the CRISPR-Cas9 HNH nuclease.},
journal = {Biophysical journal},
volume = {122},
number = {24},
pages = {4635-4644},
doi = {10.1016/j.bpj.2023.11.005},
pmid = {37936350},
issn = {1542-0086},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Molecular Dynamics Simulation ; Hydrogen/metabolism ; Amides ; },
abstract = {A hallmark of tightly regulated high-fidelity enzymes is that they become activated only after encountering cognate substrates, often by an induced-fit mechanism rather than conformational selection. Upon analysis of molecular dynamics trajectories, we recently discovered that the Cas9 HNH domain exists in three conformations: 1) Y836 (which is two residues away from the catalytic D839 and H840 residues) is hydrogen bonded to the D829 backbone amide, 2) Y836 is hydrogen bonded to the backbone amide of D861 (which is one residue away from the third catalytic residue N863), and 3) Y836 is not hydrogen bonded to either residue. Each of the three conformers differs from the active state of HNH. The conversion between the inactive and active states involves a local unfolding-refolding process that displaces the Cα and side chain of the catalytic N863 residue by ∼5 Å and ∼10 Å, respectively. In this study, we report the two largest principal components of coordinate variance of the HNH domain throughout molecular dynamics trajectories to establish the interconversion pathways of these conformations. We show that conformation 2 is an obligate step between conformations 1 and 3, which are not directly interconvertible without conformation 2. The loss of hydrogen bonding of the Y836 side chain in conformation 3 likely plays an essential role in activation during local unfolding-refolding of an α-helix containing the catalytic N863. Three single Lys-to-Ala mutants appear to eliminate this substrate-independent activation pathway of the wild-type HNH nuclease, thereby enhancing the fidelity of HNH cleavage.},
}
@article {pmid37794167,
year = {2024},
author = {Kim, JS and Chen, J},
title = {Base editing of organellar DNA with programmable deaminases.},
journal = {Nature reviews. Molecular cell biology},
volume = {25},
number = {1},
pages = {34-45},
pmid = {37794167},
issn = {1471-0080},
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; DNA, Mitochondrial/genetics ; Mutation ; Mitochondria/genetics ; },
abstract = {Mitochondria and chloroplasts are organelles that include their own genomes, which encode key genes for ATP production and carbon dioxide fixation, respectively. Mutations in mitochondrial DNA can cause diverse genetic disorders and are also linked to ageing and age-related diseases, including cancer. Targeted editing of organellar DNA should be useful for studying organellar genes and developing novel therapeutics, but it has been hindered by lack of efficient tools in living cells. Recently, CRISPR-free, protein-only base editors, such as double-stranded DNA deaminase toxin A-derived cytosine base editors (DdCBEs) and adenine base editors (ABEs), have been developed, which enable targeted organellar DNA editing in human cell lines, animals and plants. In this Review, we present programmable deaminases developed for base editing of organellar DNA in vitro and discuss mitochondrial DNA editing in animals, and plastid genome (plastome) editing in plants. We also discuss precision and efficiency limitations of these tools and propose improvements for therapeutic, agricultural and environmental applications.},
}
@article {pmid38108964,
year = {2024},
author = {Fung, C and Miles, LB and Bryson-Richardson, RJ and Bird, PI},
title = {Manipulation of Proteostasis Networks in Transgenic ZAAT Zebrafish via CRISPR-Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2750},
number = {},
pages = {19-32},
pmid = {38108964},
issn = {1940-6029},
mesh = {Humans ; Animals ; Mice ; *Proteostasis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Zebrafish/genetics ; Animals, Genetically Modified ; *Perciformes ; },
abstract = {The CRISPR-Cas9 genome editing system is used to induce mutations in genes of interest resulting in the loss of functional protein. A transgenic zebrafish α1-antitrypsin deficiency (AATD) model displays an unusual phenotype, in that it lacks the hepatic accumulation of the misfolding Z α1-antitrypsin (ZAAT) evident in human and mouse models. Here we describe the application of the CRISPR-Cas9 system to generate mutant zebrafish with defects in key proteostasis networks likely to be involved in the hepatic processing of ZAAT in this model. We describe the targeting of the atf6a and man1b1 genes as examples.},
}
@article {pmid38108903,
year = {2023},
author = {Freitas-Alves, NS and Moreira-Pinto, CE and Arraes, FBM and Costa, LSL and de Abreu, RA and Moreira, VJV and Lourenço-Tessutti, IT and Pinheiro, DH and Lisei-de-Sa, ME and Paes-de-Melo, B and Pereira, BM and Guimaraes, PM and Brasileiro, ACM and de Almeida-Engler, J and Soccol, CR and Morgante, CV and Basso, MF and Grossi-de-Sa, MF},
title = {An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.},
journal = {Planta},
volume = {259},
number = {1},
pages = {23},
pmid = {38108903},
issn = {1432-2048},
support = {Project Sv.922/18//Cofecub/ ; },
mesh = {Animals ; *Glycine max/genetics ; RNA, Guide, CRISPR-Cas Systems ; Biological Assay ; Cotyledon ; *Nematoda/genetics ; },
abstract = {The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.},
}
@article {pmid38108547,
year = {2023},
author = {Macchia, E and Torricelli, F and Caputo, M and Sarcina, L and Scandurra, C and Bollella, P and Catacchio, M and Piscitelli, M and Franco, CD and Scamarcio, G and Torsi, L},
title = {Point-of-care Ultra-Portable Single-Molecule Bioassays for One-Health.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {},
number = {},
pages = {e2309705},
doi = {10.1002/adma.202309705},
pmid = {38108547},
issn = {1521-4095},
abstract = {Screening asymptomatic organisms (humans, animals, plants) with a high-diagnostic accuracy using point-of-care-testing (POCT) technologies, though still visionary holds great potential. Convenient surveillance requires easy-to-use, cost-effective, ultra-portable but highly reliable, in-vitro-diagnostic devices that are ready for use wherever they are needed. Currently, there are not yet such devices available on the market, but there are a couple more promising technologies developed at readiness-level 5: the Clustered-Regularly-Interspaced-Short-Palindromic-Repeats/CRISPR lateral-flow-strip tests and the Single-Molecule-with-a-large-Transistor/SiMoT bioelectronic palmar devices. They both hold key features delineated by the World-Health-Organization for POCT systems and show an occurrence of false-positive and false-negative errors <1-5% resulting in diagnostic-selectivity and sensitivity >95-99%, while limit-of-detections are of few markers. CRISPR-strip is a molecular assay that, combined with enzymatic amplification, can detect down to few copies of DNA/RNA markers in blood while SiMoT immunometric and molecular test can detect down to a single oligonucleotide, protein marker, or pathogens in 0.1 mL of blood, saliva, and olive-sap. These technologies can prospectively enable the systematic and reliable surveillance of asymptomatic ones prior to worsening/proliferation of illnesses allowing for timely diagnosis and swift prognosis. This could establish a proactive healthcare ecosystem that results in effective treatments for all living organisms generating diffuse and well-being at efficient costs. This article is protected by copyright. All rights reserved.},
}
@article {pmid38108521,
year = {2023},
author = {De Paolo, R and Munagala, U and Cucco, F and Sarti, S and Pitto, L and Martignano, F and Conticello, SG and Poliseno, L},
title = {Modified Cas9-Guided Oxford Nanopore Technology Sequencing Uncovers Single and Multiple Transgene Insertion Sites in a Zebrafish Melanoma Model.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {489-492},
doi = {10.1089/crispr.2023.0062},
pmid = {38108521},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Melanoma/genetics ; *Nanopores ; Zebrafish/genetics ; Gene Editing ; },
}
@article {pmid38108520,
year = {2023},
author = {Urnov, F},
title = {Special Issue: CRISPR Trials.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {488},
doi = {10.1089/crispr.2023.29166.cfp2},
pmid = {38108520},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid38056451,
year = {2023},
author = {Mao, Q and Ye, Q and Xu, Y and Jiang, J and Fan, Y and Zhuang, L and Liu, G and Wang, T and Zhang, Z and Feng, T and Kong, S and Lu, J and Zhang, H and Wang, H and Lin, CP},
title = {Murine trophoblast organoids as a model for trophoblast development and CRISPR-Cas9 screening.},
journal = {Developmental cell},
volume = {58},
number = {24},
pages = {2992-3008.e7},
doi = {10.1016/j.devcel.2023.11.007},
pmid = {38056451},
issn = {1878-1551},
mesh = {Pregnancy ; Female ; Mice ; Animals ; *Placenta ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Trophoblasts ; Cell Differentiation ; Organoids ; Mammals ; },
abstract = {The placenta becomes one of the most diversified organs during placental mammal radiation. The main in vitro model for studying mouse trophoblast development is the 2D differentiation model of trophoblast stem cells, which is highly skewed to certain lineages and thus hampers systematic screens. Here, we established culture conditions for the establishment, maintenance, and differentiation of murine trophoblast organoids. Murine trophoblast organoids under the maintenance condition contain stem cell-like populations, whereas differentiated organoids possess various trophoblasts resembling placental ones in vivo. Ablation of Nubpl or Gcm1 in trophoblast organoids recapitulated their deficiency phenotypes in vivo, suggesting that those organoids are valid in vitro models for trophoblast development. Importantly, we performed an efficient CRISPR-Cas9 screening in mouse trophoblast organoids using a focused sgRNA (single guide RNA) library targeting G protein-coupled receptors. Together, our results establish an organoid model to investigate mouse trophoblast development and a practicable approach to performing forward screening in trophoblast lineages.},
}
@article {pmid38019145,
year = {2023},
author = {Bao, M and Dollery, SJ and Yuqing, F and Tobin, GJ and Du, K},
title = {Micropillar enhanced FRET-CRISPR biosensor for nucleic acid detection.},
journal = {Lab on a chip},
volume = {24},
number = {1},
pages = {47-55},
doi = {10.1039/d3lc00780d},
pmid = {38019145},
issn = {1473-0189},
mesh = {Fluorescence Resonance Energy Transfer ; Biological Assay ; Lab-On-A-Chip Devices ; Technology ; *Nucleic Acids ; *Biosensing Techniques ; CRISPR-Cas Systems ; },
abstract = {CRISPR technology has gained widespread adoption for pathogen detection due to its exceptional sensitivity and specificity. Although recent studies have investigated the potential of high-aspect-ratio microstructures in enhancing biochemical applications, their application in CRISPR-based detection has been relatively rare. In this study, we developed a FRET-based biosensor in combination with high-aspect-ratio microstructures and Cas12a-mediated trans-cleavage for detecting HPV 16 DNA fragments. Remarkably, our results show that micropillars with higher density exhibit superior molecular binding capabilities, leading to a tenfold increase in detection sensitivity. Furthermore, we investigated the effectiveness of two surface chemical treatment methods for enhancing the developed FRET assay. A simple and effective approach was also developed to mitigate bubble generation in microfluidic devices, a crucial issue in biochemical reactions within such devices. Overall, this work introduces a novel approach using micropillars for CRISPR-based viral detection and provides valuable insights into optimizing biochemical reactions within microfluidic devices.},
}
@article {pmid37976326,
year = {2023},
author = {Malbranke, C and Rostain, W and Depardieu, F and Cocco, S and Monasson, R and Bikard, D},
title = {Computational design of novel Cas9 PAM-interacting domains using evolution-based modelling and structural quality assessment.},
journal = {PLoS computational biology},
volume = {19},
number = {11},
pages = {e1011621},
pmid = {37976326},
issn = {1553-7358},
mesh = {*CRISPR-Cas Systems ; *Proteins/genetics/chemistry ; Amino Acid Sequence ; Machine Learning ; Learning ; },
abstract = {We present here an approach to protein design that combines (i) scarce functional information such as experimental data (ii) evolutionary information learned from a natural sequence variants and (iii) physics-grounded modeling. Using a Restricted Boltzmann Machine (RBM), we learn a sequence model of a protein family. We use semi-supervision to leverage available functional information during the RBM training. We then propose a strategy to explore the protein representation space that can be informed by external models such as an empirical force-field method (FoldX). Our approach is applied to a domain of the Cas9 protein responsible for recognition of a short DNA motif. We experimentally assess the functionality of 71 variants generated to explore a range of RBM and FoldX energies. Sequences with as many as 50 differences (20% of the protein domain) to the wild-type retained functionality. Overall, 21/71 sequences designed with our method were functional. Interestingly, 6/71 sequences showed an improved activity in comparison with the original wild-type protein sequence. These results demonstrate the interest in further exploring the synergies between machine-learning of protein sequence representations and physics grounded modeling strategies informed by structural information.},
}
@article {pmid37955062,
year = {2023},
author = {Liu, Y and Liu, W and Wang, B},
title = {Engineering CRISPR guide RNAs for programmable RNA sensors.},
journal = {Biochemical Society transactions},
volume = {51},
number = {6},
pages = {2061-2070},
doi = {10.1042/BST20221486},
pmid = {37955062},
issn = {1470-8752},
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Base Pairing ; },
abstract = {As the most valuable feature of the CRISPR system, the programmability based on Watson-Crick base pairing has been widely exploited in engineering RNA sensors. The base pairing in these systems offers a connection between the RNA of interest and the CRISPR effector, providing a highly specific mechanism for RNA detection both in vivo and in vitro. In the last decade, despite the many successful RNA sensing approaches developed during the era of CRISPR explosion, a deeper understanding of the characteristics of CRISPR systems and the continuous expansion of the CRISPR family members indicates that the CRISPR-based RNA sensor remains a promising area from which a variety of new functions and applications can be engineered. Here, we present a systematic overview of the various strategies of engineering CRISPR gRNA for programmable RNA detection with an aim to clarify the role of gRNA's programmability among the present limitations and future development of CRISPR-enabled RNA sensors.},
}
@article {pmid37848710,
year = {2023},
author = {Tian, R and Li, Y and Zhao, H and Lyu, W and Zhao, J and Wang, X and Lu, H and Xu, H and Ren, W and Tan, QQ and Shi, Q and Wang, GD and Zhang, YP and Lai, L and Mi, J and Jiang, YH and Zhang, YQ},
title = {Modeling SHANK3-associated autism spectrum disorder in Beagle dogs via CRISPR/Cas9 gene editing.},
journal = {Molecular psychiatry},
volume = {28},
number = {9},
pages = {3739-3750},
pmid = {37848710},
issn = {1476-5578},
mesh = {Dogs ; Humans ; Animals ; Mice ; *Autism Spectrum Disorder/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; Nerve Tissue Proteins/genetics/metabolism ; Primates/genetics/metabolism ; Disease Models, Animal ; Microfilament Proteins/genetics ; },
abstract = {Despite intensive studies in modeling neuropsychiatric disorders especially autism spectrum disorder (ASD) in animals, many challenges remain. Genetic mutant mice have contributed substantially to the current understanding of the molecular and neural circuit mechanisms underlying ASD. However, the translational value of ASD mouse models in preclinical studies is limited to certain aspects of the disease due to the apparent differences in brain and behavior between rodents and humans. Non-human primates have been used to model ASD in recent years. However, a low reproduction rate due to a long reproductive cycle and a single birth per pregnancy, and an extremely high cost prohibit a wide use of them in preclinical studies. Canine model is an appealing alternative because of its complex and effective dog-human social interactions. In contrast to non-human primates, dog has comparable drug metabolism as humans and a high reproduction rate. In this study, we aimed to model ASD in experimental dogs by manipulating the Shank3 gene as SHANK3 mutations are one of most replicated genetic defects identified from ASD patients. Using CRISPR/Cas9 gene editing, we successfully generated and characterized multiple lines of Beagle Shank3 (bShank3) mutants that have been propagated for a few generations. We developed and validated a battery of behavioral assays that can be used in controlled experimental setting for mutant dogs. bShank3 mutants exhibited distinct and robust social behavior deficits including social withdrawal and reduced social interactions with humans, and heightened anxiety in different experimental settings (n = 27 for wild-type controls and n = 44 for mutants). We demonstrate the feasibility of producing a large number of mutant animals in a reasonable time frame. The robust and unique behavioral findings support the validity and value of a canine model to investigate the pathophysiology and develop treatments for ASD and potentially other psychiatric disorders.},
}
@article {pmid37819140,
year = {2023},
author = {Joehnk, B and Ali, N and Voorhies, M and Walcott, K and Sil, A},
title = {Recyclable CRISPR/Cas9-mediated gene disruption and deletions in Histoplasma.},
journal = {mSphere},
volume = {8},
number = {6},
pages = {e0037023},
pmid = {37819140},
issn = {2379-5042},
support = {2R37AI066224//HHS | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Histoplasma/genetics ; Homologous Recombination ; Mutagenesis, Site-Directed ; Mutagenesis, Insertional ; Mammals ; },
abstract = {Histoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.},
}
@article {pmid37500934,
year = {2023},
author = {Au, TY and Arudkumar, J and Assavarittirong, C and Benjamin, S},
title = {Killing two birds with one stone: CRISPR/Cas9 CCR5 knockout hematopoietic stem cells transplantation to treat patients with HIV infection and hematological malignancies concurrently.},
journal = {Clinical and experimental medicine},
volume = {23},
number = {8},
pages = {4163-4175},
pmid = {37500934},
issn = {1591-9528},
mesh = {Animals ; Humans ; *HIV Infections/complications/therapy/genetics ; CRISPR-Cas Systems ; *HIV-1/metabolism ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; *Hematologic Neoplasms ; Receptors, CCR5/genetics/metabolism ; },
abstract = {Human immunodeficiency virus (HIV) is known to cause hematological malignancy. Hematopoietic stem cell transplantation (HPSCT) is an advanced treatment for that. Currently, there are three successful HIV-eliminated cases, and two received HPSCT from CCR5-absent donors. It is well established that the CCR5 protein on the cell surface assists human immunodeficiency virus entry. Preliminary studies have revealed that knocking out CCR5 and/or CXCR4 may inhibit the viral entry of HIV, which may prove promising in the further development of HIV treatment options. Herein, we suggest performing autologous or allogeneic HSCT with CCR5 KO hematopoietic stem cells in patients who suffer from complicated HIV conditions, particularly drug-resistant HIV or a concurrent diagnosis of HIV with lymphoma/leukemia, to achieve complete HIV remission. Nevertheless, at the clinical forefront of CRISPR-HIV technology, more efforts should be directed to advance nonhuman primate (NHP) models for studies of HIV pathogenesis and off-target assessments within this system. CRISPR-Cas9 knock out of host HSCT-expressing CCR5 or CXCR4 may confer HIV-resistance, which when applied to bedside therapeutics in an allogeneic or autologous manner can warrant a permanent and effective treatment outcome.},
}
@article {pmid38108519,
year = {2023},
author = {Newsom, SN and Wang, DS and Rostami, S and Schuster, I and Parameshwaran, HP and Joseph, YG and Qin, PZ and Liu, J and Rajan, R},
title = {Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {527-542},
doi = {10.1089/crispr.2023.0022},
pmid = {38108519},
issn = {2573-1602},
mesh = {Catalytic Domain ; *DNA Cleavage ; *Gene Editing ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Endonucleases ; Streptococcus pyogenes/genetics ; },
abstract = {To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9[H982A]) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn[2+]-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn[2+], but not Mg[2+], produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.},
}
@article {pmid38108518,
year = {2023},
author = {Dalla Benetta, E and López-Denman, AJ and Li, HH and Masri, RA and Brogan, DJ and Bui, M and Yang, T and Li, M and Dunn, M and Klein, MJ and Jackson, S and Catalan, K and Blasdell, KR and Tng, P and Antoshechkin, I and Alphey, LS and Paradkar, PN and Akbari, OS},
title = {Engineered Antiviral Sensor Targets Infected Mosquitoes.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {543-556},
doi = {10.1089/crispr.2023.0056},
pmid = {38108518},
issn = {2573-1602},
mesh = {Animals ; *Culicidae ; CRISPR-Cas Systems/genetics ; Gene Editing ; Mosquito Vectors/genetics ; RNA, Viral/genetics ; Antiviral Agents/pharmacology ; },
abstract = {Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.},
}
@article {pmid38108517,
year = {2023},
author = {Chen, Z and Kwan, SY and Mir, A and Hazeltine, M and Shin, M and Liang, SQ and Chan, IL and Kelly, K and Ghanta, KS and Gaston, N and Cao, Y and Xie, J and Gao, G and Xue, W and Sontheimer, EJ and Watts, JK},
title = {A Fluorescent Reporter Mouse for In Vivo Assessment of Genome Editing with Diverse Cas Nucleases and Prime Editors.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {570-582},
doi = {10.1089/crispr.2023.0048},
pmid = {38108517},
issn = {2573-1602},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Endonucleases/genetics ; Technology ; },
abstract = {CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.},
}
@article {pmid38108516,
year = {2023},
author = {Burnight, ER and Wiley, LA and Mullin, NK and Adur, MK and Lang, MJ and Cranston, CM and Jiao, C and Russell, SR and Sohn, EH and Han, IC and Ross, JW and Stone, EM and Mullins, RF and Tucker, BA},
title = {CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {502-513},
doi = {10.1089/crispr.2023.0039},
pmid = {38108516},
issn = {2573-1602},
mesh = {Humans ; Animals ; Swine ; *Rhodopsin/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Retinitis Pigmentosa/genetics/therapy ; Alleles ; },
abstract = {Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.},
}
@article {pmid38104668,
year = {2023},
author = {Singh, VK and Ahmed, S and Saini, DK and Gahlaut, V and Chauhan, S and Khandare, K and Kumar, A and Sharma, PK and Kumar, J},
title = {Manipulating epigenetic diversity in crop plants: Techniques, challenges and opportunities.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {},
number = {},
pages = {130544},
doi = {10.1016/j.bbagen.2023.130544},
pmid = {38104668},
issn = {1872-8006},
abstract = {Epigenetic modifications act as conductors of inheritable alterations in gene expression, all while keeping the DNA sequence intact, thereby playing a pivotal role in shaping plant growth and development. This review article presents an overview of techniques employed to investigate and manipulate epigenetic diversity in crop plants, focusing on both naturally occurring and artificially induced epialleles. The significance of epigenetic modifications in facilitating adaptive responses is explored through the examination of how various biotic and abiotic stresses impact them. Further, environmental chemicals are explored for their role in inducing epigenetic changes, particularly focusing on inhibitors of DNA methylation like 5-AzaC and zebularine, as well as inhibitors of histone deacetylation including trichostatin A and sodium butyrate. The review delves into various approaches for generating epialleles, including tissue culture techniques, mutagenesis, and grafting, elucidating their potential to induce heritable epigenetic modifications in plants. In addition, the ground breaking CRISPR/Cas is emphasized for its accuracy in targeting specific epigenetic changes. This presents a potent tools for deciphering the intricacies of epigenetic mechanisms. Furthermore, the intricate relationship between epigenetic modifications and non-coding RNA expression, including siRNAs and miRNAs, is investigated. The emerging role of exo-RNAi in epigenetic regulation is also introduced, unveiling its promising potential for future applications. The article concludes by addressing the opportunities and challenges presented by these techniques, emphasizing their implications for crop improvement. Conclusively, this extensive review provides valuable insights into the intricate realm of epigenetic changes, illuminating their significance in phenotypic plasticity and their potential in advancing crop improvement.},
}
@article {pmid38104450,
year = {2023},
author = {Shi, X and Li, H and Yao, S and Ding, Y and Lin, X and Xu, H and Liu, Y and Zhao, C and Zhang, T and Wang, J},
title = {A CRISPR/Cas12a-assisted bacteria quantification platform combined with magnetic covalent organic frameworks and hybridization chain reaction.},
journal = {Food chemistry},
volume = {440},
number = {},
pages = {138196},
doi = {10.1016/j.foodchem.2023.138196},
pmid = {38104450},
issn = {1873-7072},
abstract = {The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans-cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.},
}
@article {pmid38104156,
year = {2023},
author = {Pan, X and Li, H and Putta, P and Zhang, X},
title = {LinRace: cell division history reconstruction of single cells using paired lineage barcode and gene expression data.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {8388},
pmid = {38104156},
issn = {2041-1723},
support = {R35GM143070//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DBI-2145736//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; DBI-2019771//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Lineage/genetics ; Cell Division/genetics ; Gene Expression ; },
abstract = {Lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes in single cells, which allows for inference of cell lineage and cell types at the whole organism level. While most state-of-the-art methods for lineage reconstruction utilize only the lineage barcode data, methods that incorporate gene expressions are emerging. Effectively incorporating the gene expression data requires a reasonable model of how gene expression data changes along generations of divisions. Here, we present LinRace (Lineage Reconstruction with asymmetric cell division model), which integrates lineage barcode and gene expression data using asymmetric cell division model and infers cell lineages and ancestral cell states using Neighbor-Joining and maximum-likelihood heuristics. On both simulated and real data, LinRace outputs more accurate cell division trees than existing methods. With inferred ancestral states, LinRace can also show how a progenitor cell generates a large population of cells with various functionalities.},
}
@article {pmid38103430,
year = {2023},
author = {Booncherd, K and Sreebun, S and Pasomboon, P and Boonanuntanasarn, S},
title = {Effects of CRISPR/Cas9-mediated dnd1 knockout impairs gonadal development in striped catfish.},
journal = {Animal : an international journal of animal bioscience},
volume = {18},
number = {1},
pages = {101039},
doi = {10.1016/j.animal.2023.101039},
pmid = {38103430},
issn = {1751-732X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.},
}
@article {pmid38101163,
year = {2023},
author = {Castillo, M and Guevara, G and Baldanta, S and Rodríguez, PS and Agudo, L and Nogales, J and Carrasco, AD and Arribas-Aguilar, F and Pérez-Pérez, J and García, JL and Galán, B and Navarro Llorens, JM},
title = {Characterization of Limnospira platensis PCC 9108 R-M and CRISPR-Cas systems.},
journal = {Microbiological research},
volume = {279},
number = {},
pages = {127572},
doi = {10.1016/j.micres.2023.127572},
pmid = {38101163},
issn = {1618-0623},
abstract = {The filamentous cyanobacterium Limnospira platensis, formerly known as Arthrospira platensis or spirulina, is one of the most commercially important species of microalgae. Due to its high nutritional value, pharmacological and industrial applications it is extensively cultivated on a large commercial scale. Despite its widespread use, its precise manipulation is still under development due to the lack of effective genetic protocols. Genetic transformation of Limnospira has been attempted but the methods reported have not been generally reproducible in other laboratories. Knowledge of the transformation defense mechanisms is essential for understanding its physiology and for broadening their applications. With the aim to understand more about the genetic defenses of L. platensis, in this work we have identified the restriction-modification and CRISPR-Cas systems and we have cloned and characterized thirteen methylases. In parallel, we have also characterized the methylome and orphan methyltransferases using genome-wide analysis of DNA methylation patterns and RNA-seq. The identification and characterization of these enzymes will be a valuable resource to know how this strain avoids being genetically manipulated and for further genomics studies.},
}
@article {pmid38099671,
year = {2023},
author = {, },
title = {Retraction for Mustafa and Makhawi, "SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases".},
journal = {Journal of clinical microbiology},
volume = {},
number = {},
pages = {e0152123},
doi = {10.1128/jcm.01521-23},
pmid = {38099671},
issn = {1098-660X},
}
@article {pmid38055961,
year = {2023},
author = {Zhao, R and Tang, Y and Song, D and Liu, M and Li, B},
title = {CRISPR/Cas12a-Responsive Hydrogels for Conjugation-Free and Universal Indicator Release in Colorimetric Detection.},
journal = {Analytical chemistry},
volume = {95},
number = {50},
pages = {18522-18529},
doi = {10.1021/acs.analchem.3c03900},
pmid = {38055961},
issn = {1520-6882},
mesh = {Humans ; Female ; Pregnancy ; *CRISPR-Cas Systems/genetics ; Colorimetry ; Amylases ; Hydrogels ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; },
abstract = {Recent advances have demonstrated the significant potential and advantages to repurpose existing point-of-care reactions/devices to realize portable detection of nonoriginal targets, e.g., pathogen genes. However, pursuing this aim usually requires protein indicator-nucleic acid conjugation via a covalent bond, which may bring drawbacks such as high cost, complicated procedure, and annoying component rebuilding. Herein, we developed a conjugation-free, effective, and universal detection platform called CRIs-gel (CRISPR/Cas12a-Responsive Indicators@RCA hydrogels). Various protein indicators are pre-encapsulated into the hydrogels made of effective and high-yield rolling circle amplification (RCA). Upon a targeting sequence binding with its antisense crRNA, CRISPR/Cas12a starts its trans-cleavage activity to crush the hydrogel, which may directly release the indicator for downstream readout. Two proteins, amylase (GA) and human chorionic gonadotropin (hCG), are successfully used as model indicators to trigger the downstream amylum-I2 color change and pregnancy test strip response. After coupling with upstream isothermal nucleic acid amplification, both portable readouts may detect as few as 2 copies/μL genetic sequences of influenza A virus (FluA), human papilloma virus (HPV), SARS-CoV-2, and influenza B virus (FluB). This conjugation-free CRIs-gel platform is thus simple, sensitive, and universal and can provide innovative insights for portable point-of-care testing (POCT) development.},
}
@article {pmid37914398,
year = {2023},
author = {Krishnan, A and Ali, LM and Prabhu, SG and Pillai, VN and Chameettachal, A and Vivet-Boudou, V and Bernacchi, S and Mustafa, F and Marquet, R and Rizvi, TA},
title = {Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging.},
journal = {RNA (New York, N.Y.)},
volume = {30},
number = {1},
pages = {68-88},
doi = {10.1261/rna.079840.123},
pmid = {37914398},
issn = {1469-9001},
mesh = {Animals ; Cats ; Humans ; *Immunodeficiency Virus, Feline/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; RNA, Viral/chemistry ; Binding Sites ; Genomics ; Virus Assembly/genetics ; },
abstract = {The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5' end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U[85]CUG[88] stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U[85]CUG[88] stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80-92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U[85]CUG[88] sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50[Gag] on WT and mutant U[85]CUG[88] ψ RNAs led to reduced levels of Pr50[Gag] binding to mutant U[85]CUG[88] ψ RNAs, indicating that the U[85]CUG[88] stretch is crucial for ψ RNA-Pr50[Gag] interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.},
}
@article {pmid37729550,
year = {2023},
author = {Tuma, J and Chen, YJ and Collins, MG and Paul, A and Li, J and Han, H and Sharma, R and Murthy, N and Lee, HY},
title = {Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection.},
journal = {Biochemistry},
volume = {62},
number = {24},
pages = {3533-3547},
doi = {10.1021/acs.biochem.3c00371},
pmid = {37729550},
issn = {1520-4995},
support = {R01 MH125979/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems ; Brain ; RNA, Messenger/genetics ; *Nervous System Diseases ; *Nanoparticles ; RNA, Small Interfering ; },
abstract = {Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.},
}
@article {pmid37421290,
year = {2023},
author = {Cheng, L and Xiong, W and Li, S and Wang, G and Zhou, J and Li, H},
title = {CRISPR-Cas9 screening identified lethal genes enriched in necroptosis pathway and of prognosis significance in osteosarcoma.},
journal = {The journal of gene medicine},
volume = {25},
number = {12},
pages = {e3563},
doi = {10.1002/jgm.3563},
pmid = {37421290},
issn = {1521-2254},
support = {HKZ [2022] No. 146//Nanchang Science and Technology Bureau's/ ; GZYYKJZ [2022] No. 6//Young and middle-aged backbone talents of traditional Chinese medicine in Jiangxi Province (the fourth batch)/ ; },
mesh = {Humans ; Genes, Lethal ; CRISPR-Cas Systems ; Necroptosis/genetics ; *Osteosarcoma/diagnosis/genetics ; *Bone Neoplasms/diagnosis/genetics ; },
abstract = {BACKGROUND: The present study aimed to identify indispensable genes associated with tumor cell viability according to the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) datasets, which may support new therapeutic targets for patients with osteosarcoma.<br><br>METHODS: The transcriptome patterns between tumor and normal tissues, which were obtained from the Therapeutically Applicable Research to Generate Effective Treatments dataset, were overlapped with the genomics associated with cell viability screened by CRISPR-Cas9 technology. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses were employed to determine enrichment pathways related to lethal genes. Least absolute shrinkage and selection operator (LASSO) regression was employed to construct a risk model related to lethal genes for predicting clinical outcomes of osteosarcoma. Univariate and multivariate Cox regressions were conducted to assess the prognostic value of this feature. Weighted gene co-expression network analysis was performed to identify modules associated with patients with high-risk score.<br><br>RESULTS: In total, 34 lethal genes were identified in this investigation. These genes were enriched in the necroptosis pathway. The risk model based on LASSO regression algorithm distinguishes patients with high-risk score from patients with low-risk score. Compared with low-risk patients, high-risk patients showed a shorter overall survival rate in both the training and validation sets. The time-dependent receiver operating characteristic curves of 1, 3 and 5&#xa0;years displayed that the risk score has great prediction performance. The necroptosis pathway represents the main difference in biological behavior between the high-risk group and the low-risk group. Meanwhile, CDK6 and SMARCB1 may serve as important targets for detecting osteosarcoma progression.<br><br>CONCLUSIONS: The present study developed a predictive model that outperformed classical clinicopathological parameters for predicting the clinical outcomes of osteosarcoma patients and identified specific lethal genes, including CDK6 and SMARCB1, as well as the necroptosis pathway. These findings may serve as potential targets for future osteosarcoma treatments.},
}
@article {pmid36306429,
year = {2023},
author = {Patange, S and Maragh, S},
title = {Fire Burn and Cauldron Bubble: What Is in Your Genome Editing Brew?.},
journal = {Biochemistry},
volume = {62},
number = {24},
pages = {3500-3511},
doi = {10.1021/acs.biochem.2c00431},
pmid = {36306429},
issn = {1520-4995},
mesh = {Humans ; *Gene Editing ; RNA ; *Burns ; CRISPR-Cas Systems ; },
abstract = {Genome editing is a rapidly evolving biotechnology with the potential to transform many sectors of industry such as agriculture, biomanufacturing, and medicine. This technology is enabled by an ever-growing portfolio of biomolecular reagents that span the central dogma, from DNA to RNA to protein. In this paper, we draw from our unique perspective as the National Metrology Institute of the United States to bring attention to the importance of understanding and reporting genome editing formulations accurately and promoting concepts to verify successful delivery into cells. Achieving the correct understanding may be hindered by the way units, quantities, and stoichiometries are reported in the field. We highlight the variability in how editing formulations are reported in the literature and examine how a reference molecule could be used to verify the delivery of a reagent into cells. We provide recommendations on how more accurate reporting of editing formulations and more careful verification of the steps in an editing experiment can help set baseline expectations of reagent performance, toward the aim of enabling genome editing studies to be more reproducible. We conclude with a future outlook on technologies that can further our control and enable our understanding of genome editing outcomes at the single-cell level.},
}
@article {pmid36049184,
year = {2023},
author = {Tou, CJ and Kleinstiver, BP},
title = {Recent Advances in Double-Strand Break-Free Kilobase-Scale Genome Editing Technologies.},
journal = {Biochemistry},
volume = {62},
number = {24},
pages = {3493-3499},
pmid = {36049184},
issn = {1520-4995},
support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; },
mesh = {*Gene Editing ; *DNA Breaks, Double-Stranded ; Genome ; Mutation ; CRISPR-Cas Systems ; DNA Repair ; },
abstract = {Genome editing approaches have transformed the ability to make user-defined changes to genomes in both ex vivo and in vivo contexts. Despite the abundant development of technologies that permit the installation of nucleotide-level changes, until recently, larger-scale sequence edits via technologies independent of DNA double-strand breaks (DSBs) had remained less explored. Here, we review recent advances toward DSB-free technologies that enable kilobase-scale modifications including insertions, deletions, inversions, replacements, and others. These technologies provide new capabilities for users, while offering hope for the simplification of putative therapeutic strategies by moving away from small mutation-specific edits and toward more generalizable kilobase-scale approaches.},
}
@article {pmid35436085,
year = {2023},
author = {Ryan, DE and Diamant-Levi, T and Steinfeld, I and Taussig, D and Visal-Shah, S and Thakker, S and Lunstad, BD and Kaiser, RJ and McCaffrey, R and Ortiz, M and Townsend, J and Welch, WRW and Singh, M and Curry, B and Dellinger, DJ and Bruhn, L},
title = {Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.},
journal = {Biochemistry},
volume = {62},
number = {24},
pages = {3512-3520},
doi = {10.1021/acs.biochem.1c00768},
pmid = {35436085},
issn = {1520-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Phosphonoacetic Acid ; Gene Editing/methods ; RNA, Messenger ; },
abstract = {CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.},
}
@article {pmid35325535,
year = {2023},
author = {Brogan, DJ and Akbari, OS},
title = {CRISPR Diagnostics: Advances toward the Point of Care.},
journal = {Biochemistry},
volume = {62},
number = {24},
pages = {3488-3492},
pmid = {35325535},
issn = {1520-4995},
support = {DP2 AI152071/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Point-of-Care Systems ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Pandemics ; *COVID-19/diagnosis ; },
abstract = {CRISPR diagnostics have recently emerged as powerful diagnostic tools for the rapid detection of infections. The ultimate goal is to develop these diagnostics for the point of care, where patients quickly receive and easily interpret results. Although they are in their infancy, the COVID-19 pandemic has accelerated innovation of CRISPR diagnostics and led to an explosion of improvements to these systems. Challenges that have impeded the implementation at the point of care have been addressed, and CRISPR diagnostics have been dramatically simplified. Here we outline recent developments and advancements in CRISPR diagnostics that have pushed these technologies to the point of care.},
}
@article {pmid38099659,
year = {2023},
author = {Xu, P-X and Ren, H-Y and Zhao, N and Jin, X-J and Wen, B-H and Qin, T},
title = {Distribution characteristics of the Legionella CRISPR-Cas system and its regulatory mechanism underpinning phenotypic function.},
journal = {Infection and immunity},
volume = {},
number = {},
pages = {e0022923},
doi = {10.1128/iai.00229-23},
pmid = {38099659},
issn = {1098-5522},
abstract = {Legionella is a common intracellular parasitic bacterium that infects humans via the respiratory tract, causing Legionnaires' disease, with fever and pneumonia as the main symptoms. The emergence of highly virulent and azithromycin-resistant Legionella pneumophila is a major challenge in clinical anti-infective therapy. The CRISPR-Cas acquired immune system provides immune defense against foreign nucleic acids and regulates strain biological functions. However, the distribution of the CRISPR-Cas system in Legionella and how it regulates gene expression in L. pneumophila remain unclear. Herein, we assessed 915 Legionella whole-genome sequences to determine the distribution characteristics of the CRISPR-Cas system and constructed gene deletion mutants to explore the regulation of the system based on growth ability in vitro, antibiotic sensitivity, and intracellular proliferation of L. pneumophila. The CRISPR-Cas system in Legionella was predominantly Type II-B and was mainly concentrated in the genome of L. pneumophila ST1 strains. The Type II-B CRISPR-Cas system showed no effect on the strain's growth ability in vitro but significantly reduced resistance to azithromycin and decreased proliferation ability due to regulation of the lpeAB efflux pump and the Dot/Icm type IV secretion system. Thus, the Type II-B CRISPR-Cas system plays a crucial role in regulating the virulence of L. pneumophila. This expands our understanding of drug resistance and pathogenicity in Legionella, provides a scientific basis for the prevention of Legionnaires' disease outbreaks and the rational use of clinical drugs, and facilitates effective treatment of Legionnaires' disease.},
}
@article {pmid38099496,
year = {2023},
author = {Schlabach, MR and Lin, S and Collester, ZR and Wrocklage, C and Shenker, S and Calnan, C and Xu, T and Gannon, HS and Williams, LJ and Thompson, F and Dunbar, PR and LaMothe, RA and Garrett, TE and Colletti, N and Hohmann, AF and Tubo, NJ and Bullock, CP and Le Mercier, I and Sofjan, K and Merkin, JJ and Keegan, S and Kryukov, GV and Dugopolski, C and Stegmeier, F and Wong, K and Sharp, FA and Cadzow, L and Benson, MJ},
title = {Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens.},
journal = {The Journal of clinical investigation},
volume = {133},
number = {24},
pages = {},
pmid = {38099496},
issn = {1558-8238},
mesh = {Humans ; Animals ; Mice ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Lymphocytes, Tumor-Infiltrating ; Immunotherapy, Adoptive ; *Neoplasms/genetics ; Gene Editing ; Suppressor of Cytokine Signaling 1 Protein/genetics ; },
abstract = {Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.},
}
@article {pmid38096814,
year = {2023},
author = {Lou, YC and Chen, L and Borges, AL and West-Roberts, J and Firek, BA and Morowitz, MJ and Banfield, JF},
title = {Infant gut DNA bacteriophage strain persistence during the first 3 years of life.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2023.11.015},
pmid = {38096814},
issn = {1934-6069},
abstract = {Bacteriophages are key components of gut microbiomes, yet the phage colonization process in the infant gut remains uncertain. Here, we establish a large phage sequence database and use strain-resolved analyses to investigate DNA phage succession in infants throughout the first 3 years of life. Analysis of 819 fecal metagenomes collected from 28 full-term and 24 preterm infants and their mothers revealed that early-life phageome richness increases over time and reaches adult-like complexity by age 3. Approximately 9% of early phage colonizers, which are mostly maternally transmitted and infect Bacteroides, persist for 3 years and are more prevalent in full-term than in preterm infants. Although rare, phages with stop codon reassignment are more likely to persist than non-recoded phages and generally display an increase in in-frame reassigned stop codons over 3 years. Overall, maternal seeding, stop codon reassignment, host CRISPR-Cas locus prevalence, and diverse phage populations contribute to stable viral colonization.},
}
@article {pmid38096061,
year = {2023},
author = {Nievergelt, AP and Diener, DR and Bogdanova, A and Brown, T and Pigino, G},
title = {Protocol for precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.},
journal = {STAR protocols},
volume = {5},
number = {1},
pages = {102774},
doi = {10.1016/j.xpro.2023.102774},
pmid = {38096061},
issn = {2666-1667},
abstract = {CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols. For complete details on the use and execution of this protocol, please refer to Nievergelt et al. (2023).[1].},
}
@article {pmid38095804,
year = {2023},
author = {Larouche, M and Khoury, E and Brisson, D and Gaudet, D},
title = {Inhibition of Angiopoietin-Like Protein 3 or 3/8 Complex and ApoC-III in Severe Hypertriglyceridemia.},
journal = {Current atherosclerosis reports},
volume = {},
number = {},
pages = {},
pmid = {38095804},
issn = {1534-6242},
abstract = {PURPOSE OF REVIEW: The role of the inhibition of ANGPTL3 in severe or refractory hypercholesterolemia is well documented, less in severe hyperTG. This review focuses on the preclinical and clinical development of ApoC-III inhibitors and ANGPTL3, 4, and 3/8 complex inhibitors for the treatment of severe or refractory forms of hypertriglyceridemia to prevent cardiovascular disease or other morbidities.<br><br>RECENT FINDINGS: APOC3 and ANGPTL3 became targets for drug development following the identification of naturally occurring loss of function variants in families with a favorable lipid profile and low cardiovascular risk. The inhibition of ANGPTL3 covers a broad spectrum of lipid disorders from severe hypercholesterolemia to severe hypertriglyceridemia, while the inhibition of ApoC-III can treat hypertriglyceridemia regardless of the severity. Preclinical and clinical data suggest that ApoC-III inhibitors, ANGPTL3 inhibitors, and inhibitors of the ANGPTL3/8 complex that is formed postprandially are highly effective for the treatment of severe or refractory hypertriglyceridemia. Inhibition of ANGPTL3 or the ANGPTL3/8 complex upregulates LPL and facilitates the hydrolysis and clearance of triglyceride-rich lipoproteins (TRL) (LPL-dependent mechanisms), whereas ApoC-III inhibitors contribute to the management and clearance of TRL through both LPL-dependent and LPL-independent mechanisms making it possible to successfully lower TG in subjects completely lacking LPL (familial chylomicronemia syndrome). Most of these agents are biologicals including monoclonal antibodies (mAb), antisense nucleotides (ASO), small interfering RNA (siRNA), or CRISPR-cas gene editing strategies.},
}
@article {pmid38095724,
year = {2023},
author = {Zhang, L and Xu, X and Cao, L and Zhu, Z and Ding, Y and Jiang, H and Li, B and Liu, J},
title = {Multi-aptamer-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of S. pneumoniae and S. aureus.},
journal = {Mikrochimica acta},
volume = {191},
number = {1},
pages = {29},
pmid = {38095724},
issn = {1436-5073},
support = {22ZDYF1033//Sichuan Province Science and Technology Support Program/ ; 22ZYZF0007//central government guides local science and technology development funds to be directed transfer payment projects/ ; 2022NSFSC1426//National Natural Science Foundation of China/ ; },
mesh = {Staphylococcus aureus/genetics ; *Methicillin-Resistant Staphylococcus aureus ; *Aptamers, Nucleotide/genetics ; Streptococcus pneumoniae/genetics ; Bacteria ; },
abstract = {A novel nucleic acid aptamer nanoprobes-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of Streptococcus pneumoniae and Staphylococcus aureus is presented. In this fluorescence assay system, utilizing the hairpin allosteric effect caused by the aptamer binding to the target bacteria, the detection of S. pneumoniae is first achieved through changes in fluorescence due to FRET. Subsequently, a Cas12a protein mixture is added to detect S. aureus. The amplified output signal is triggered by two methods to ensure the sensitivity of the method: the synergistic FRET effect is achieved by the assembly of multi-aptamer through the conjugation of streptavidin-biotin, and the trans-cleavage function of CRISPR/Cas 12a. Under the optimized conditions, the proposed hairpin allosteric aptasensor could achieve high sensitivity (a detection limit of 135 cfu/mL) and broad-concentration quantification (dynamic range of 10[3]-10[7] cfu/mL) of S. pneumoniae. The aptamer-assisted CRISPR system for S. aureus detection showed good linearity (R[2] = 0.996) in the concentration range 10[2]-10[8] cfu/mL, with a detection limit of 39 cfu/mL. No cross-reactivity with other foodborne pathogenic bacteria was observed in both systems. Taking only 55 min, this method of multiple pathogen detection proved to be promising.},
}
@article {pmid38094634,
year = {2023},
author = {Dziuba, A and Dzierżak, S and Sodo, A and Wawszczak-Kasza, M and Zegadło, K and Białek, J and Zych, N and Kiebzak, W and Matykiewicz, J and Głuszek, S and Adamus-Białek, W},
title = {Comparative study of virulence potential, phylogenetic origin, CRISPR-Cas regions and drug resistance of Escherichia coli isolates from urine and other clinical materials.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1289683},
pmid = {38094634},
issn = {1664-302X},
abstract = {INTRODUCTION: Urinary tract infections (UTI), among which the main etiological factor is uropathogenic Escherichia coli (UPEC, E. coli), remain an important issue for clinicians. The aim of the study was to demonstrate clear differences in the pathogenic properties of urine-derived E. coli compared to other extraintestinal E. coli clinical isolates (derived from: blood, lower respiratory tracts, sputum, reproductive tract, body fluids, perianal pus, other pus, wound, postoperative wound and other sources).<br><br>METHODS: The collection of 784 E. coli isolates was collected from various materials of hospitalized patients. They were analyzed in terms of virulence-associated genes (papC, sfaD/sfaE, cnf1, usp., fimG/H, hlyA), belonging to phylogenetic groups and the presence of CRISPR-Cas regions using PCR. In addition, the epidemiological data and the antibiotic resistance profiles provided by the hospital's microbiology department were included for statistical analyses.<br><br>RESULTS: Urine-derived E. coli showed significantly greater virulence potential compared to other isolates, but they were generally unremarkable in terms of drug resistance. The isolates most often belonged to phylogenetic group B2. Drug resistance was negatively correlated with CRISPR 2 presence and high average virulence score, but positively correlated with CRISPR 4 presence. To the best of our knowledge, we are the first to report significant differences in sputum-derived isolates-they revealed the lowest virulence potential and, at the same time, the highest drug resistance.<br><br>DISCUSSION: In conclusion, we demonstrated significant differences of urinary-derived E. coli compared to other clinical E. coli isolates. We would like to suggest excluding penicillins from use in E. coli infection at this time and monitoring strains with a high pathogenicity potential.},
}
@article {pmid38093832,
year = {2023},
author = {Hasanzadeh, A and Ebadati, A and Dastanpour, L and Aref, AR and Sahandi Zangabad, P and Kalbasi, A and Dai, X and Mehta, G and Ghasemi, A and Fatahi, Y and Joshi, S and Hamblin, MR and Karimi, M},
title = {Applications of Innovation Technologies for Personalized Cancer Medicine: Stem Cells and Gene-Editing Tools.},
journal = {ACS pharmacology &amp; translational science},
volume = {6},
number = {12},
pages = {1758-1779},
pmid = {38093832},
issn = {2575-9108},
abstract = {Personalized medicine is a new approach toward safer and even cheaper treatments with minimal side effects and toxicity. Planning a therapy based on individual properties causes an effective result in a patient's treatment, especially in a complex disease such as cancer. The benefits of personalized medicine include not only early diagnosis with high accuracy but also a more appropriate and effective therapeutic approach based on the unique clinical, genetic, and epigenetic features and biomarker profiles of a specific patient's disease. In order to achieve personalized cancer therapy, understanding cancer biology plays an important role. One of the crucial applications of personalized medicine that has gained consideration more recently due to its capability in developing disease therapy is related to the field of stem cells. We review various applications of pluripotent, somatic, and cancer stem cells in personalized medicine, including targeted cancer therapy, cancer modeling, diagnostics, and drug screening. CRISPR-Cas gene-editing technology is then discussed as a state-of-the-art biotechnological advance with substantial impacts on medical and therapeutic applications. As part of this section, the role of CRISPR-Cas genome editing in recent cancer studies is reviewed as a further example of personalized medicine application.},
}
@article {pmid38052051,
year = {2023},
author = {Angelopoulou, L and Stylianopoulou, E and Tegopoulos, K and Farmakioti, I and Grigoriou, M and Skavdis, G},
title = {A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {514-526},
doi = {10.1089/crispr.2023.0047},
pmid = {38052051},
issn = {2573-1602},
mesh = {Polymorphism, Restriction Fragment Length ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Polymerase Chain Reaction/methods ; Mutagenesis/genetics ; },
abstract = {As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.},
}
@article {pmid38052047,
year = {2023},
author = {Barrangou, R},
title = {First Drug Approval Rises the CRISPR Tide.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {487},
doi = {10.1089/crispr.2023.29168.editorial},
pmid = {38052047},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drug Approval ; },
}
@article {pmid37917075,
year = {2023},
author = {Thakkar, N and Hejzlarova, A and Brabec, V and Dolezel, D},
title = {Germline Editing of Drosophila Using CRISPR-Cas9-Based Cytosine and Adenine Base Editors.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {557-569},
doi = {10.1089/crispr.2023.0026},
pmid = {37917075},
issn = {2573-1602},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Drosophila/genetics ; Drosophila melanogaster/genetics ; Cytosine ; Adenine ; RNA, Guide, CRISPR-Cas Systems ; Germ Cells ; },
abstract = {Target-AID, BE3, and ABE7.10 base editors fused to the catalytically modified Cas9 and xCas9(3.7) were tested for germline editing of the fruit fly Drosophila melanogaster. We developed a guide RNA-expressing construct, white-4gRNA, targeting splice sites in the white gene, an X-chromosome located gene. Using white-4gRNA flies and transgenic lines expressing Target-AID, BE3, and ABE7.10 base editors, we tested the efficiency of stable germline gene editing at three different temperatures. Classical Cas9 generating insertions/deletions by non-homologous end joining served as a reference. Our data indicate that gene editing is most efficient at 28°C, the highest temperature suitable for fruit flies. Finally, we created a new allele of the core circadian clock gene timeless using Target-AID. This base edited mutant allele tim[SS308-9FL] had a disrupted circadian clock with a period of ∼29 h. The white-4gRNA expressing fly can be used to test new generations of base editors for future applications in Drosophila.},
}
@article {pmid38093107,
year = {2023},
author = {O'Leary, K},
title = {Base editors in the clinic.},
journal = {Nature medicine},
volume = {29},
number = {12},
pages = {2972},
doi = {10.1038/s41591-023-02708-7},
pmid = {38093107},
issn = {1546-170X},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid38090672,
year = {2023},
author = {Shi, M and Zhang, H and Fleming, J and Wei, W and Chen, H and Dai, X and Liu, Y and Li, C and Ran, F and Wu, Z and Wang, Y and Zhang, X and Zhang, H and Bi, L},
title = {EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis.},
journal = {Frontiers in molecular biosciences},
volume = {10},
number = {},
pages = {1261613},
pmid = {38090672},
issn = {2296-889X},
abstract = {Introduction: Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored. Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system. Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions. Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.},
}
@article {pmid38089461,
year = {2023},
author = {Adefisoye, MA and Olaniran, AO},
title = {Antimicrobial resistance expansion in pathogens: a review of current mitigation strategies and advances towards innovative therapy.},
journal = {JAC-antimicrobial resistance},
volume = {5},
number = {6},
pages = {dlad127},
pmid = {38089461},
issn = {2632-1823},
abstract = {The escalating problem of antimicrobial resistance (AMR) proliferation in clinically important pathogens has become one of the biggest threats to human health and the global economy. Previous studies have estimated AMR-associated deaths and disability-adjusted life-years (DALYs) in many countries with a view to presenting a clearer picture of the global burden of AMR-related diseases. Recently, several novel strategies have been advanced to combat resistance spread. These include efflux activity inhibition, closing of mutant selection window (MSW), biofilm disruption, lytic bacteriophage particles, nanoantibiotics, engineered antimicrobial peptides, and the CRISPR-Cas9 gene-editing technique. The single or integrated deployment of these strategies has shown potentialities towards mitigating resistance and contributing to valuable therapeutic outcomes. Correspondingly, the new paradigm of personalized medicine demands innovative interventions such as improved and accurate point-of-care diagnosis and treatment to curtail AMR. The CRISPR-Cas system is a novel and highly promising nucleic acid detection and manipulating technology with the potential for application in the control of AMR. This review thus considers the specifics of some of the AMR-mitigating strategies, while noting their drawbacks, and discusses the advances in the CRISPR-based technology as an important point-of-care tool for tracking and curbing AMR in our fight against a looming 'post-antibiotic' era.},
}
@article {pmid38085335,
year = {2023},
author = {Gouda, MNR and Jeevan, H and Shashank, HG},
title = {CRISPR/Cas9: a cutting-edge solution for combatting the fall armyworm, Spodoptera frugiperda.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {13},
pmid = {38085335},
issn = {1573-4978},
mesh = {Animals ; Spodoptera/genetics ; *CRISPR-Cas Systems/genetics ; *Insecticides/pharmacology ; Insecticide Resistance/genetics ; Phenotype ; Larva/genetics ; },
abstract = {The utilization of CRISPR/Cas9 in Spodoptera frugiperda, commonly known as fall armyworm, presents a groundbreaking avenue for pest management. With its ability to precisely modify the insect's genome, CRISPR/Cas9 offers innovative strategies to combat this destructive pest. The application of CRISPR/Cas9 in S. frugiperda holds immense potential. It enables the identification and functional analysis of key genes associated with its behavior, development, and insecticide resistance. This knowledge can unveil novel target sites for more effective and specific insecticides. Additionally, CRISPR/Cas9 can facilitate the development of population control methods by disrupting vital genes essential for survival. However, challenges such as off-target effects and the efficient delivery of CRISPR/Cas9 components remain. Addressing these obstacles is vital to ensure accurate and reliable results. Furthermore, ethical considerations, biosafety protocols, and regulatory frameworks must be integral to the adoption of this technology. Looking forward, CRISPR/Cas9-based gene drive systems hold the potential to promulgate desirable genetic traits within S. frugiperda populations, offering a sustainable and eco-friendly approach. This could curtail their reproductive capabilities or make them more susceptible to certain interventions. In conclusion, CRISPR/Cas9 presents a transformative platform for precise and targeted pest management in S. frugiperda. By deciphering the insect's genetic makeup and developing innovative strategies, we can mitigate the devastating impact of fall armyworm on agriculture while ensuring environmental sustainability.},
}
@article {pmid38076855,
year = {2023},
author = {Wiegand, T and Hoffmann, FT and Walker, MWG and Tang, S and Richard, E and Le, HC and Meers, C and Sternberg, SH},
title = {Emergence of RNA-guided transcription factors via domestication of transposon-encoded TnpB nucleases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {38076855},
support = {F32 GM143924/GM/NIGMS NIH HHS/United States ; T32 GM145440/GM/NIGMS NIH HHS/United States ; },
abstract = {Transposon-encoded tnpB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination[1-4]. This widespread gene family was repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12[5,6]. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans[7]. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility[8], phage susceptibility[9], and host immunity[10]. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of genes encoded by transposable elements, and reveals that RNA-guided transcription factors emerged long before the development of dCas9-based editors.},
}
@article {pmid37971665,
year = {2023},
author = {Ben-Zvi, I and Karasik, D and Ackert-Bicknell, CL},
title = {Zebrafish as a Model for Osteoporosis: Functional Validations of Genome-Wide Association Studies.},
journal = {Current osteoporosis reports},
volume = {21},
number = {6},
pages = {650-659},
pmid = {37971665},
issn = {1544-2241},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; Genome-Wide Association Study ; CRISPR-Cas Systems ; *Osteoporosis/genetics ; Mammals/genetics ; },
abstract = {PURPOSE OF REVIEW: GWAS, as a largely correlational analysis, requires in vitro or in vivo validation. Zebrafish (Danio rerio) have many advantages for studying the genetics of human diseases. Since gene editing in zebrafish has been highly valuable for studying embryonic skeletal developmental processes that are prenatally or perinatally lethal in mammalian models, we are reviewing pros and cons of this model.<br><br>RECENT FINDINGS: The true power for the use of zebrafish is the ease by which the genome can be edited, especially using the CRISPR/Cas9 system. Gene editing, followed by phenotyping, for complex traits such as BMD, is beneficial, but the major physiological differences between the fish and mammals must be considered. Like mammals, zebrafish do have main bone cells; thus, both in vivo stem cell analyses and in vivo imaging are doable. Yet, the "long" bones of fish are peculiar, and their bone cavities do not contain bone marrow. Partial duplication of the zebrafish genome should be taken into account. Overall, small fish toolkit can provide unmatched opportunities for genetic modifications and morphological investigation as a follow-up to human-first discovery.},
}
@article {pmid37931323,
year = {2023},
author = {Li, B and Qu, L and Yang, J},
title = {RNA-Guided RNA Modifications: Biogenesis, Functions, and Applications.},
journal = {Accounts of chemical research},
volume = {56},
number = {22},
pages = {3198-3210},
doi = {10.1021/acs.accounts.3c00474},
pmid = {37931323},
issn = {1520-4898},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *RNA/chemistry ; RNA Processing, Post-Transcriptional ; },
abstract = {ConspectusPost-transcriptional modifications are ubiquitous in both protein-coding and noncoding RNAs (ncRNAs), playing crucial functional roles in diverse biological processes across all kingdoms of life. These RNA modifications can be achieved through two distinct mechanisms: RNA-independent and RNA-guided (also known as RNA-dependent). In the RNA-independent mechanism, modifications are directly introduced onto RNA molecules by enzymes without the involvement of other RNA molecules, while the cellular RNA-guided RNA modification system exists in the form of RNA-protein complexes, wherein one guide RNA collaborates with a set of proteins, including the modifying enzyme. The primary function of guide RNAs lies in their ability to bind to complementary regions within the target RNAs, orchestrating the installation of specific modifications. Both mechanisms offer unique advantages and are critical to the diverse and dynamic landscape of RNA modifications. RNA-independent modifications provide rapid and direct modification of RNA molecules, while RNA-guided mechanisms offer precise and programmable means to introduce modifications at specific RNA sites. Recently, emerging evidence has shed light on RNA-guided RNA modifications as a captivating area of research, providing precise and programmable control over RNA sequences and functions.In this Account, we focus on RNA modifications synthesized in an RNA-guided manner, including 2'-O-methylated nucleotides (Nm), pseudouridine (Ψ), N[4]-acetylcytidine (ac[4]C), and inosine (I). This Account sheds light on the intricate processes of biogenesis and elucidates the regulatory roles of these modifications in RNA metabolism. These roles include pivotal functions such as RNA stability, translation, and splicing, where each modification contributes to the diverse and finely tuned regulatory landscape of RNA biology. In addition to elucidating the biogenesis and functions of these modifications, we also provide an overview of high-throughput methods and their underlying biochemical principles used for the transcriptome-wide investigation of these modifications and their fundamental interactions in RNA-guided systems. This includes exploring RNA-protein interactions and RNA-RNA interactions, which play crucial roles in the dynamic regulatory networks of RNA-guided modifications. The ever-advancing methodologies have greatly enhanced our understanding of the dynamic and widespread nature of RNA-guided RNA modifications and their regulatory functions. Furthermore, the applications of RNA-guided RNA modifications are discussed, illuminating their potential in diverse fields. From basic research to gene therapy, the programmable nature of RNA-guided modifications presents exciting opportunities for manipulating gene expression and developing innovative therapeutic strategies.},
}
@article {pmid37690529,
year = {2023},
author = {Wang, L and Kulthinee, S and Slate-Romano, J and Zhao, T and Shanmugam, H and Dubielecka, PM and Zhang, LX and Qin, G and Zhuang, S and Chin, YE and Zhao, TC},
title = {Inhibition of integrin alpha v/beta 5 mitigates the protective effect induced by irisin in hemorrhage.},
journal = {Experimental and molecular pathology},
volume = {134},
number = {},
pages = {104869},
doi = {10.1016/j.yexmp.2023.104869},
pmid = {37690529},
issn = {1096-0945},
mesh = {Humans ; Mice ; Animals ; *Fibronectins/genetics/pharmacology ; *Integrin alphaV ; RNA, Guide, CRISPR-Cas Systems ; Hemorrhage ; },
abstract = {INTRODUCTION: Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvβ5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvβ5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvβ5 contributes to the effects of irisin during the hemorrhagic response.<br><br>METHODS: Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35-45&#xa0;mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5&#xa0; &#x3bc;g/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin &#x3b1;v&#x3b2;5, or control RGDS (1&#xa0;mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin &#x3b2;5 with nanoparticle delivery of integrin &#x3b2;5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues.<br><br>RESULTS: Hemorrhage induced reduction of integrin &#x3b1;v&#x3b2;5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin &#x3b2;5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-&#x3b1; (TNF-&#x3b1;) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin &#x3b2;5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin &#x3b1;v&#x3b2;5, or knockdown of integrin &#x3b2;5.<br><br>CONCLUSION: Integrin &#x3b1;v&#x3b2;5 plays an important role for irisin in modulating the protective effect during hemorrhage.},
}
@article {pmid38083710,
year = {2023},
author = {Li, Y and Deng, F and Zhang, C and Lin, X and Goldys, E},
title = {Integrated RPA-CRISPR/Cas12a system towards Point-of-Care H. pylori detection.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2023},
number = {},
pages = {1-4},
doi = {10.1109/EMBC40787.2023.10340749},
pmid = {38083710},
issn = {2694-0604},
mesh = {Humans ; *Point-of-Care Systems ; *Helicobacter pylori/genetics ; CRISPR-Cas Systems ; Heating ; DNA ; },
abstract = {The rapidly advanced CRISPR/Cas biosensing technology provides unprecedent potential for the development of novel biosensing systems. It provides a new approach for realizing rapid, sensitivity and highly specific pathogen nucleic acid detection, with the capability to combine other technologies, including Polymerase Chain Reaction or isothermal amplifications. The detection of Helicobacter pylori (H. pylori), one of the most common human pathogens to cause various gastroduodenal diseases, has also been explored with the assistance of CRISPR/Cas systems. However, gaps still remain for the development of end-user friendly sensing systems.In this study, a combined RPA-CRISPR/Cas12a biosensing system has been established. It shown the capability to quantitively detect the presence of H. pylori genome DNA with 4 orders of magnitude linear range, and sensitivity of 1.4 copies/µL. The overall reaction can be done within 45 mins at room temperature, which eliminates the needs for heating instrumentation. In addition, with the addition of pullulan as a protective reagent, the potential of storing CRISPR/Cas12a system reagents by using a freeze-dry approach has also been demonstrated.Clinical Relevance - This study represents a novel exploration to applying CRISPR/Cas12a-based biosensing technology to the detection of pathogen DNA with improved potential for the development of Point-of-Care diagnostics. This critical aspect of our technology will contribute to address the newly emerged pathogenic threats and support public health systems.},
}
@article {pmid38083573,
year = {2023},
author = {Deng, F and Li, Y and Sang, R and Zhang, C and Hall, T and Yang, D and Goldys, E},
title = {RNA reporter based CRISPR/Cas12a biosensing platform for sensitive detection of circulating tumor DNA.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2023},
number = {},
pages = {1-4},
doi = {10.1109/EMBC40787.2023.10340759},
pmid = {38083573},
issn = {2694-0604},
mesh = {RNA ; *Circulating Tumor DNA ; CRISPR-Cas Systems ; *Nucleic Acids ; Fluorescence ; },
abstract = {CRISPR/Cas biotechnology provides an exceptional platform for biosensor development. To date, the reported CRISPR/Cas biosensing systems have shown extraordinary performance for nucleic acids, small molecules, small proteins and microorganism detection. The CRISPR/Cas12a biosensing system, as a typical example, has been well established and applied for both nucleic acids and non-nucleic acids target detection. However, all established CRISPR/Cas12a biosensing systems are based on DNA reporters, which potentially limits further application.In this study, we established an RNA reporter based CRISPR/Cas12a biosensing system. A basic biosensing system was evaluated, and the limit of detection was found to be 1 nM. Afterwards, we optimized this biosensing system using both temperature and chemical enhancers. The final optimal biosensing system (with DTT &amp; 37°C) shows fluorescence signal increased by a factor of ~10 compared with the basic system. The optimal biosensing system was further applied for the detection of circulating tumor DNA (ctDNA), which shows over 4 orders of magnitude detection range from 1pM to 25 nM, with the limit of detection of 1pM. This RNA reporter based CRISPR/Cas12a biosensing system provides an effective platform for nucleic acids quantification.Clinical Relevance- This research provides a novel approach for ctDNA diagnostics, which is an attractive biomarker for noninvasive monitoring of tumor growth, response, and spread.},
}
@article {pmid38082155,
year = {2023},
author = {Burkhardt, UE and Fritsch, EF},
title = {Serving up whatever you wish: CRISPR-base editing generates novel cancer-restricted antigens for immunotherapy.},
journal = {Genes and immunity},
volume = {24},
number = {6},
pages = {292-294},
pmid = {38082155},
issn = {1476-5470},
mesh = {Humans ; *Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Immunotherapy ; *Neoplasms/genetics/therapy ; },
}
@article {pmid38082079,
year = {2023},
author = {},
title = {First in vivo base editing lowers cholesterol.},
journal = {Nature biotechnology},
volume = {41},
number = {12},
pages = {1665},
doi = {10.1038/s41587-023-02070-6},
pmid = {38082079},
issn = {1546-1696},
mesh = {*Gene Editing ; *Cholesterol ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid38081875,
year = {2023},
author = {Nandy, K and Babu, D and Rani, S and Joshi, G and Ijee, S and George, A and Palani, D and Premkumar, C and Rajesh, P and Vijayanand, S and David, E and Murugesan, M and Velayudhan, SR},
title = {Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {21953},
pmid = {38081875},
issn = {2045-2322},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Adenine/metabolism ; Mutation ; },
abstract = {The preferred method for disease modeling using induced pluripotent stem cells (iPSCs) is to generate isogenic cell lines by correcting or introducing pathogenic mutations. Base editing enables the precise installation of point mutations at specific genomic locations without the need for deleterious double-strand breaks used in the CRISPR-Cas9 gene editing methods. We created a bulk population of iPSCs that homogeneously express ABE8e adenine base editor enzyme under a doxycycline-inducible expression system at the AAVS1 safe harbor locus. These cells enabled fast, efficient and inducible gene editing at targeted genomic regions, eliminating the need for single-cell cloning and screening to identify those with homozygous mutations. We could achieve multiplex genomic editing by creating homozygous mutations in very high efficiencies at four independent genomic loci simultaneously in AAVS1-iABE8e iPSCs, which is highly challenging with previously described methods. The inducible ABE8e expression system allows editing of the genes of interest within a specific time window, enabling temporal control of gene editing to study the cell or lineage-specific functions of genes and their molecular pathways. In summary, the inducible ABE8e system provides a fast, efficient and versatile gene-editing tool for disease modeling and functional genomic studies.},
}
@article {pmid38081800,
year = {2023},
author = {Lu, Y and Wen, J and Wang, C and Wang, M and Jiang, F and Miao, L and Xu, M and Li, Y and Chen, X and Chen, Y},
title = {Mesophilic Argonaute-Based Single Polystyrene Sphere Aptamer Fluorescence Platform for the Multiplexed and Ultrasensitive Detection of Non-Nucleic Acid Targets.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2308424},
doi = {10.1002/smll.202308424},
pmid = {38081800},
issn = {1613-6829},
support = {2022YFF0607900//National Key Research and Development Program of China/ ; 2022BBA0057//Key R&amp;D Program of Hubei Province/ ; KF-202201//Open Project of Key Laboratory of Detection Technology of Focus Chemical Hazards in Animal-derived Food for State Market Regulation/ ; 32172293//National Natural Science Foundation of China/ ; },
abstract = {The rapid, simultaneous, and accurate identification of multiple non-nucleic acid targets in clinical or food samples at room temperature is essential for public health. Argonautes (Agos) are guided, programmable, target-activated, next-generation nucleic acid endonucleases that could realize one-pot and multiplexed detection using a single enzyme, which cannot be achieved with CRISPR/Cas. However, currently reported thermophilic Ago-based multi-detection sensors are mainly employed in the detection of nucleic acids. Herein, this work proposes a Mesophilic Argonaute Report-based single millimeter Polystyrene Sphere (MARPS) multiplex detection platform for the simultaneous analysis of non-nucleic acid targets. The aptamer is utilized as the recognition element, and a single millimeter-sized polystyrene sphere (PSmm) with a large concentration of guide DNA on the surface served as the microreactor. These are combined with precise Clostridium butyricum Ago (CbAgo) cleavage and exonuclease I (Exo I) signal amplification to achieve the efficient and sensitive recognition of non-nucleic acid targets, such as mycotoxins (<60 pg mL[-1]) and pathogenic bacteria (<10[2] cfu mL[-1]). The novel MARPS platform is the first to use mesophilic Agos for the multiplex detection of non-nucleic acid targets, overcoming the limitations of CRISPR/Cas in this regard and representing a major advancement in non-nucleic acid target detection using a gene-editing-based system.},
}
@article {pmid38076861,
year = {2023},
author = {Nguyen, GT and Schelling, MA and Buscher, KA and Sritharan, A and Sashital, DG},
title = {CRISPR-Cas12a exhibits metal-dependent specificity switching.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.11.29.569287},
pmid = {38076861},
abstract = {Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg [2+] concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg [2+] concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg [2+] concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can unwind and bind seed mutant targets more rapidly at low Mg [2+] concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg [2+] concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.},
}
@article {pmid38076770,
year = {2023},
author = {Das, S and Sathee, L},
title = {miRNA mediated regulation of nitrogen response and nitrogen use efficiency of plants: the case of wheat.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {29},
number = {10},
pages = {1371-1394},
pmid = {38076770},
issn = {0971-5894},
abstract = {Nitrogen (N) is needed for plant growth and development and is the major limiting nutrient due to its higher demand in agricultural production globally. The use of N fertilizers has increased considerably in recent years to achieve higher cereal yields. High N inputs coupled with declining N use efficiency (NUE) result in the degradation of the environment. Plants have developed multidimensional strategies in response to changes in N availability in soil. These strategies include N stress-induced responses such as changes in gene expression patterns. Several N stress-induced genes and other regulatory factors, such as microRNAs (miRNAs), have been identified in different plant species, opening a new avenue of research in plant biology. This review presents a general overview of miRNA-mediated regulation of N response and NUE. Further, the in-silico target predictions and the predicted miRNA-gene network for nutrient metabolism/homeostasis in wheat provide novel insights. The information on N-regulated miRNAs and the differentially expressed target transcripts are necessary resources for genetic improvement of NUE by genome editing.},
}
@article {pmid38076202,
year = {2023},
author = {Zhou, J and Ren, XM and Wang, X and Li, Z and J Xian, C},
title = {Recent advances and challenges of the use of the CRISPR/Cas system as a non-nucleic acid molecular diagnostic.},
journal = {Heliyon},
volume = {9},
number = {12},
pages = {e22767},
pmid = {38076202},
issn = {2405-8440},
abstract = {The clustered regularly interspaced short palindromic DNA sequence repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) systems are currently applied not only as a gene editing tool but also as a novel molecular diagnostic technique. The CRISPR/Cas systems have emerged as an efficient molecular diagnostic system that can detect nucleic acids, proteins and small molecule compounds, by converting a non-nucleic acid into a nucleic acid signal of Cas-identifiable and keeping inherent properties of high sensitivity and specificity. While its multiple advantages for nucleic acid detection have been widely published in excellent reviews, there have been no systematic analyses and reviews on the principles and characteristics of CRISPR/Cas-based diagnostic systems for non-nucleic acids. The present work reviewed the basic process, principles, characteristics, strategies, recent advances, and challenges of CRISPR/Cas-based molecular diagnostic methods for detecting non-nucleic acids, which may provide a basis or some references for future development and application as molecular diagnostic tools.},
}
@article {pmid38075036,
year = {2023},
author = {Miao, M and Li, S and Yuan, J and Liu, P and Fang, X and Zhang, C and Zhang, X and Li, F},
title = {CRISPR/Cas9-mediated gene mutation of EcIAG leads to sex reversal in the male ridgetail white prawn Exopalaemon carinicauda.},
journal = {Frontiers in endocrinology},
volume = {14},
number = {},
pages = {1266641},
pmid = {38075036},
issn = {1664-2392},
mesh = {Humans ; Animals ; Male ; Female ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Androgens/metabolism ; Sex Differentiation/genetics ; *Palaemonidae/genetics/metabolism ; Mutation ; },
abstract = {In the culture of crustaceans, most species show sexual dimorphism. Monosex culture is an effective approach to achieve high yield and economic value, especially for decapods of high value. Previous studies have developed some sex control strategies such as manual segregation, manipulation of male androgenic gland and knockdown of the male sexual differentiation switch gene encoding insulin-like androgenic gland hormone (IAG) in decapods. However, these methods could not generate hereditable changes. Genetic manipulation to achieve sex reversal individuals is absent up to now. In the present study, the gene encoding IAG (EcIAG) was identified in the ridgetail white prawn Exopalaemon carinicauda. Sequence analysis showed that EcIAG encoded conserved amino acid structure like IAGs in other decapod species. CRISPR/Cas9-mediated genome editing technology was used to knock out EcIAG. Two sgRNAs targeting the second exon of EcIAG were designed and microinjected into the prawn zygotes or the embryos at the first cleavage with commercial Cas9 protein. EcIAG in three genetic males was knocked out in both chromosome sets, which successfully generated sex reversal and phenotypic female characters. The results suggest that CRISPR/Cas9-mediated genome editing technology is an effective way to develop sex manipulation technology and contribute to monosex aquaculture in crustaceans.},
}
@article {pmid38071078,
year = {2023},
author = {Fang, S and Wang, G and Yang, LH},
title = {[Advances in AAV-CRISPR/Cas9-Mediated Hemophilia A Gene Therapy --Review].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {31},
number = {6},
pages = {1890-1893},
doi = {10.19746/j.cnki.issn.1009-2137.2023.06.045},
pmid = {38071078},
issn = {1009-2137},
mesh = {Humans ; *Hemophilia A/genetics/therapy ; CRISPR-Cas Systems ; *Hemophilia B/genetics/therapy ; Gene Editing ; Genetic Therapy ; Genetic Vectors ; },
abstract = {Hemophilia A(HA) is an X-linked recessive bleeding disorder caused by mutations in coagulation factor VIII. Nowadays, exogenous coagulation factor replacement therapy is the main treatment. With the continuous development of gene therapy, new research directions have been provided for the treatment of hemophilia A. CRISPR-Cas9 technology was applied to select suitable target sites, and mediate the targeted knock-in and efficient expression of exogenous B-domain-deleted FⅧ variant gene through corresponding vectors for the treatment of hemophilia A.CRISPR-Cas9 technology is an emerging gene editing tool with great efficiency, safety and effectiveness, and has been widely used in hemophilia gene therapy research. This paper reviews the vector selection, construction of therapeutic genes, gene editing technology and selection of expression target sites for hemophilia A gene therapy at this stage.},
}
@article {pmid38070114,
year = {2024},
author = {Andreu-Saumell, I and Rodriguez-Garcia, A and Guedan, S},
title = {Genome Editing in CAR-T Cells Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2748},
number = {},
pages = {151-165},
pmid = {38070114},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; T-Lymphocytes ; *Neoplasms/genetics ; },
abstract = {CAR-T cell therapy is revolutionizing the treatment of hematologic malignancies. However, there are still many challenges ahead before CAR-T cells can be used effectively to treat solid tumors and certain hematologic cancers, such as T-cell malignancies. Next-generation CAR-T cells containing further genetic modifications are being developed to overcome some of the current limitations of this therapy. In this regard, genome editing is being explored to knock out or knock in genes with the goal of enhancing CAR-T cell efficacy or increasing access. In this chapter, we describe in detail a protocol to knock out genes on CAR-T cells using CRISPR-Cas9 technology. Among various gene editing protocols, due to its simplicity, versatility, and reduced toxicity, we focused on the electroporation of ribonucleoprotein complexes containing the Cas9 protein together with sgRNA. All together, these protocols allow for the design of the knockout strategy, CAR-T cell expansion and genome editing, and analysis of knockout efficiency.},
}
@article {pmid38069437,
year = {2023},
author = {Prokhorova, D and Matveeva, A and Zakabunin, A and Ryabchenko, A and Stepanov, G},
title = {Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069437},
issn = {1422-0067},
support = {122022100238-7//Russian state-funded project for ICBFM SB RAS/ ; 21-64-00017//Russian Science Foundation/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Transfection ; Mammals/genetics ; },
abstract = {At present, there are many strategies to improve the activity of CRISPR/Cas9. A well-known and effective approach is guide RNA modification. Many chemical guide RNA modifications have been studied, whereas naturally occurring RNA modifications are largely unexplored. N1-methylpseudouridine (m1Ψ) is an RNA base modification widely used in mRNA therapy, and it holds great promise for application in genome editing systems. The present study focuses on investigating the effect of N1-methylpseudouridine on the functioning of CRISPR/Cas9. In vitro cleavage assays helped determine the level of m1Ψ guide RNA modification that is sufficient to cleave the target substrate. By analyzing FAM-labeled dsDNA substrate cleavage, we calculated the kinetic parameters and the specificity scores of modified guide RNAs. Neon transfection and digital PCR enabled us to assess the activity of modified guide RNAs in mammalian cells. Our study shows that the presence of m1Ψ in guide RNAs can help preserve on-target genome editing while significantly reducing the off-target effects of CRISPR/Cas9 in vitro. We also demonstrate that Cas9 complexes with guide RNAs containing m1Ψ allow for genome editing in human cells. Thus, the incorporation of m1Ψ into guide RNAs supports CRISPR/Cas9 activity both in vitro and in cells.},
}
@article {pmid38069264,
year = {2023},
author = {Chandra, D and Cho, K and Pham, HA and Lee, JY and Han, O},
title = {Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069264},
issn = {1422-0067},
support = {RS-2023-00248217//Rural Development Administration/ ; , 2023R1A2C1002936//National Research Foundation of Korea/ ; },
mesh = {*Glutens/metabolism ; Seed Storage Proteins/genetics ; *Oryza/metabolism ; Down-Regulation/genetics ; CRISPR-Cas Systems ; Gene Editing ; Plant Proteins/genetics/metabolism ; Edible Grain/genetics/metabolism ; Seeds/metabolism ; Starch/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.},
}
@article {pmid38069142,
year = {2023},
author = {Lebedeva, MA and Dobychkina, DA and Lutova, LA},
title = {CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069142},
issn = {1422-0067},
support = {&#x2116; 075-15-2022-322//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Plant Root Nodulation/genetics ; Root Nodules, Plant/metabolism ; Nitrates/metabolism ; Symbiosis/genetics ; CRISPR-Cas Systems/genetics ; *Medicago truncatula/metabolism ; Nitrogen/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; *Rhizobium/metabolism ; Plant Roots/metabolism ; },
abstract = {Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume-rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.},
}
@article {pmid38069102,
year = {2023},
author = {Park, JH and Kim, H},
title = {Harnessing CRISPR/Cas9 for Enhanced Disease Resistance in Hot Peppers: A Comparative Study on CaMLO2-Gene-Editing Efficiency across Six Cultivars.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069102},
issn = {1422-0067},
support = {Project No. PJ01654303//The New Breeding Technologies Development Program, Rural Development Administration (RDA), Republic of Korea/ ; Grant No. 2022R1A2C1010869//The Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Capsicum/microbiology ; Disease Resistance/genetics ; RNA, Guide, CRISPR-Cas Systems ; Fungi/genetics ; },
abstract = {The Capsicum annuum Mildew Locus O (CaMLO2) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated CaMLO2 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/CaMLO2single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/CaMLO2sgRNA2-RNP systems by extending their application from the bell pepper 'Dempsey' and the hot pepper 'CM334' to six commercial hot pepper cultivars. Across the six cultivars, CaMLO2sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas CaMLO2sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/CaMLO2sgRNA1-RNP system to achieve efficient gene editing at the CaMLO2 locus in different Capsicum annuum cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with CaMLO2-mediated enhanced disease resistance.},
}
@article {pmid38069090,
year = {2023},
author = {Huang, Z and Habib, A and Zhao, G and Ding, X},
title = {CRISPR-Cas9 Mediated Stable Expression of Exogenous Proteins in the CHO Cell Line through Site-Specific Integration.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069090},
issn = {1422-0067},
mesh = {Cricetinae ; Animals ; Humans ; CHO Cells ; Cricetulus ; *CRISPR-Cas Systems ; *Serum Albumin, Human ; Recombinant Proteins ; },
abstract = {Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression.},
}
@article {pmid38066536,
year = {2023},
author = {Mehryar, MM and Shi, X and Li, J and Wu, Q},
title = {DNA polymerases in precise and predictable CRISPR/Cas9-mediated chromosomal rearrangements.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {288},
pmid = {38066536},
issn = {1741-7007},
support = {2022YFC3400200//National Key R&amp;D Program of China/ ; (31630039//the National Natural Science Foundation of China/ ; 19JC1412500//the Science and Technology Commission of Shanghai Municipality/ ; 21DZ2210200//the Science and Technology Commission of Shanghai Municipality/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; DNA/genetics ; DNA-Directed DNA Polymerase/genetics ; },
abstract = {BACKGROUND: Recent studies have shown that, owning to its cohesive cleavage, Cas9-mediated CRISPR gene editing outcomes at junctions of chromosomal rearrangements or DNA-fragment editing are precise and predictable; however, the underlying mechanisms are poorly understood due to lack of suitable assay system and analysis tool.<br><br>RESULTS: Here we developed a customized computer program to take account of staggered or cohesive Cas9 cleavage and to rapidly process large volumes of junctional sequencing reads from chromosomal rearrangements or DNA-fragment editing, including DNA-fragment inversions, duplications, and deletions. We also established a sensitive assay system using HPRT1 and DCK as reporters for cell growth during DNA-fragment editing by Cas9 with dual sgRNAs and found prominent large resections or long deletions at junctions of chromosomal rearrangements. In addition, we found that knockdown of PolQ (encoding Pol&#x3b8; polymerase), which has a prominent role in theta-mediated end joining (TMEJ) or microhomology-mediated end joining (MMEJ), results in increased large resections but decreased small deletions. We also found that the mechanisms for generating small deletions of 1bp and >1bp during DNA-fragment editing are different with regard to their opposite dependencies on Pol&#x3b8; and Pol&#x3bb; (encoded by the PolL gene). Specifically, Pol&#x3b8; suppresses 1bp deletions but promotes >1bp deletions, whereas Pol&#x3bb; promotes 1bp deletions but suppresses >1bp deletions. Finally, we found that Pol&#x3bb; is the main DNA polymerase responsible for fill-in of the 5' overhangs of staggered Cas9 cleavage ends.<br><br>CONCLUSIONS: These findings contribute to our understanding of the molecular mechanisms of CRISPR/Cas9-mediated DNA-fragment editing and have important implications for controllable, precise, and predictable gene editing.},
}
@article {pmid38062143,
year = {2023},
author = {Ledford, H},
title = {CRISPR 2.0: a new wave of gene editors heads for clinical trials.},
journal = {Nature},
volume = {624},
number = {7991},
pages = {234-235},
doi = {10.1038/d41586-023-03797-7},
pmid = {38062143},
issn = {1476-4687},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid38049144,
year = {2023},
author = {Hamilton, TA and Joris, BR and Shrestha, A and Browne, TS and Rodrigue, S and Karas, BJ and Gloor, GB and Edgell, DR},
title = {De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials.},
journal = {ACS synthetic biology},
volume = {12},
number = {12},
pages = {3578-3590},
doi = {10.1021/acssynbio.3c00319},
pmid = {38049144},
issn = {2161-5063},
mesh = {Humans ; *Escherichia coli/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Conjugation, Genetic/genetics ; Plasmids/genetics ; *Anti-Infective Agents ; },
abstract = {Metagenomic sequences represent an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequences requires the identification of suitable candidate systems as starting scaffolds for de novo DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic "trash bin" for genes associated with conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut data set representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugation genes with a differential distribution in human cohorts. We synthesized de novo an entire Citrobacter spp. conjugative system of 54 kb containing at least 47 genes and assembled it into a plasmid, pCitro. We found that pCitro conjugates from Escherichia coli to Citrobacter rodentium with a 30-fold higher frequency than to E. coli, and is compatible with Citrobacter resident plasmids. Mutations in the traV and traY conjugation components of pCitro inhibited conjugation. We showed that pCitro can be repurposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and Citrobacter-specific sgRNAs to kill C. rodentium. Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.},
}
@article {pmid38048294,
year = {2023},
author = {Fletcher, J and O'Connor-Moneley, J and Frawley, D and Flanagan, PR and Alaalm, L and Menendez-Manjon, P and Estevez, SV and Hendricks, S and Woodruff, AL and Buscaino, A and Anderson, MZ and Sullivan, DJ and Moran, GP},
title = {Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function.},
journal = {PLoS genetics},
volume = {19},
number = {12},
pages = {e1011082},
pmid = {38048294},
issn = {1553-7404},
support = {F31 AI167576/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Candida albicans/metabolism ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Mutagenesis ; Phenotype ; Gene Expression Regulation, Fungal ; Gene Deletion ; },
abstract = {The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOβ2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.},
}
@article {pmid38039555,
year = {2023},
author = {Xu, Z and Dong, Y and Ma, N and Zhu, X and Zhang, X and Yin, H and Chen, S and Zhu, JJ and Tian, Y and Min, Q},
title = {Confinement in Dual-Chain-Locked DNA Origami Nanocages Programs Marker-Responsive Delivery of CRISPR/Cas9 Ribonucleoproteins.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {49},
pages = {26557-26568},
doi = {10.1021/jacs.3c04074},
pmid = {38039555},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems ; Ribonucleoproteins ; DNA ; *MicroRNAs ; Adenosine Triphosphate ; },
abstract = {Delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) offers a powerful tool for therapeutic genome editing. However, precise manipulation of CRISPR/Cas9 RNPs to switch the machinery on and off according to diverse disease microenvironments remains challenging. Here, we present dual-chain-locked DNA origami nanocages (DL-DONCs) that can confine Cas9 RNPs in the inner cavity for efficient cargo delivery and dual-marker-responsive genome editing in the specified pathological states. By engineering of ATP or miRNA-21-responsive dsDNAs as chain locks on the DONCs, the permeability of nanocages and accessibility of encapsulated Cas9 RNPs can be finely regulated. The resulting DL-DONCs enabled steric protection of bioactive Cas9 RNPs from premature release and deactivation during transportation while dismounting the dual chain locks in response to molecular triggers after internalization into tumor cells, facilitating the escape of Cas9 RNPs from the confinement for gene editing. Due to the dual-marker-dominated uncaging mechanism, the gene editing efficiency could be exclusively determined by the combined level of ATP and miRNA-21 in the target cellular environment. By targeting the tumor-associated PLK-1 gene, the DL-DONCs-enveloped Cas9 RNPs have demonstrated superior inhibitory effects on the proliferation of tumor cells in vitro and in vivo. The developed DL-DONCs provide a custom-made platform for the precise manipulation of Cas9 RNPs, which can be potentially applied to on-demand gene editing for classified therapy in response to arbitrary disease-associated biomolecules.},
}
@article {pmid38035530,
year = {2023},
author = {Gargano, C and Vartanian-Grimaldi, JS and Hovhannisyan, Y and Joanne, P and Agbulut, O and Li, Z and Parlakian, A and Decaux, JF},
title = {Generation of induced pluripotent stem cell line (TMOi001-A-11) carrying a homozygous deletion in the synemin gene using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103254},
doi = {10.1016/j.scr.2023.103254},
pmid = {38035530},
issn = {1876-7753},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Homozygote ; Sequence Deletion ; *Cardiomyopathies/genetics/metabolism ; },
abstract = {A number of genetic variants in the SYNM gene encoding for the intermediate filament synemin have been reported in patients with cardiomyopathies, skeletal myopathies, cancer and certain neurodegenerative disorders. To better understand its role, we generated a human induced pluripotent stem cell line with a homozygous deletion in the SYNM gene by CRISPR/Cas9 genome editing. The synemin-knockout human induced pluripotent stem cells exhibit typical morphology of pluripotent cells, expression of pluripotency markers, normal karyotype and differentiation capacity in the three germ layers. This line will allow us to investigate the role of synemin in cardiomyopathy upon differentiation into beating cardiomyocytes.},
}
@article {pmid38033235,
year = {2023},
author = {Jiang, T and Teng, Y and Li, C and Gan, Q and Zhang, J and Zou, Y and Desai, BK and Yan, Y},
title = {Establishing Tunable Genetic Logic Gates with Versatile Dynamic Performance by Varying Regulatory Parameters.},
journal = {ACS synthetic biology},
volume = {12},
number = {12},
pages = {3730-3742},
doi = {10.1021/acssynbio.3c00554},
pmid = {38033235},
issn = {2161-5063},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Promoter Regions, Genetic/genetics ; *Logic ; },
abstract = {Genetic logic gates can be employed in metabolic engineering and synthetic biology to regulate gene expression based on diverse inputs. Design of tunable genetic logic gates with versatile dynamic performance is essential for expanding the usability of these toolsets. Here, using the p-coumaric acid biosensor system as a proof-of-concept, we initially investigated the parameters influencing the buffer (BUF) genetic logic gates. Subsequently, integrating binding sequences from the p-coumaric acid biosensor system and tetR or lacI regulation systems into a constitutive promoter yielded AND genetic logic gates. Additionally, characterized antisense RNAs (asRNAs) or single guide RNAs (sgRNAs) with various repression efficiencies were combined with BUF gates to construct a suite of p-coumaric acid-triggered NOT genetic logic gates. Finally, the designed BUF and NOT gates were combined to construct bifunctional genetic circuits that were subjected to orthogonality evaluation. The genetic logic gates established in this study can serve as valuable tools in future applications of metabolic engineering and synthetic biology.},
}
@article {pmid38029556,
year = {2023},
author = {Denman, CR and Tran, HD and Shin, MK and Ryu, J and Kuhn, B and Jo, J},
title = {Generation of a human embryonic stem cell reporter line, TMEM119-EGFP, for the visualisation of in vitro differentiated human microglia.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103264},
doi = {10.1016/j.scr.2023.103264},
pmid = {38029556},
issn = {1876-7753},
mesh = {Humans ; *Microglia/metabolism ; *Human Embryonic Stem Cells ; Cell Line ; Cell Differentiation ; Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Transmembrane protein 119 (TMEM119) is a recently identified microglia marker that is not expressed by other immune cells. Using CRISPR/Cas9 technology, we introduced enhanced green fluorescence protein (EGFP), into the H9 WA-09 human embryonic stem cell line, directly before the TMEM119 stop codon. Sanger sequencing confirmed successful insertion of the EGFP sequence. The newly created cell line expressed a normal morphology and karyotype, several pluripotency markers, and the ability to differentiate into all three germ layers. H9-TMEM119-EGFP can be used to provide a deeper understanding of the role of TMEM119 in microglia by monitoring its expression under different experimental conditions.},
}
@article {pmid38011612,
year = {2023},
author = {Pyhtila, B and Kasowitz, S and Leeson, R and Barrangou, R},
title = {The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene.},
journal = {The CRISPR journal},
volume = {6},
number = {6},
pages = {493-501},
doi = {10.1089/crispr.2023.0059},
pmid = {38011612},
issn = {2573-1602},
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Plants/genetics ; Genome ; Plasmids/genetics ; Mammals/genetics ; },
abstract = {CRISPR-based technologies have rapidly enabled the democratization of genome editing in academic institutions through distribution by Addgene over the past decade. Recently, several distribution milestones have been reached, with a collection of >15,000 plasmids deposited by >1,000 laboratories spanning ∼40 countries now shipped 300,000 times to ∼5,000 organizations traversing ∼100 countries. Yet, both deposits of and requests for CRISPR plasmids continue to rise for this disruptive technology. Distribution patterns revealed robust demand for three distinct classes of CRISPR effectors, namely nucleases (e.g., Cas9 and Cas12), modulators (deactivated CRISPR nucleases fused to transcriptional regulators and epigenome modifiers), and chimeric effectors (Cas proteins fused to enzymes carrying out other activities such as deamination, reverse transcription, transposition, and integration). Yearly deposits over the past decade are requested in near-even proportions, reflecting continuous technological development and requests for novel constructs. Though it is unclear whether the slowing rate of requests is inherent to a pandemic operational lag or a transition from emerging to mature technology, it is noteworthy that the relative proportion of requests from plasmids deposited in the previous year remains stable, suggesting robust development of novel tools concurrent with continued adoption of editing, base editing, prime editing, and more. Predictably, most requested plasmids are designed for mammalian genome manipulation, presumably for medical research and human health pursuits, reflecting investments in therapeutic applications. Concurrently, requests for plant and microbial constructs are on the rise, especially in regions of the world more reliant on local agricultural inputs and focused on food and feed applications, illustrating continued diversification of genome editing applications.},
}
@article {pmid38006676,
year = {2023},
author = {Rhode, J and Hagenau, L and Beimdiek, J and Ullmann, R and Hossain, F and Tzvetkova, A and Jensen, LR and Kuss, AW},
title = {Generation of two iPSC lines (MHHi001-A-12 and MHHi001-A-13) carrying biallelic truncating mutations at the 3'-end of SRCAP using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103249},
doi = {10.1016/j.scr.2023.103249},
pmid = {38006676},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Cell Line ; Adenosine Triphosphatases/genetics/metabolism ; },
abstract = {Non-Floating Harbour Syndrome (FLHS) neurodevelopmental disorder (NDD) is a recently described disorder caused by mutations in certain regions of the SRCAP gene. We generated two iPSC lines that contain truncating mutation on both alleles at the 3'-end of SRCAP using CRISPR/Cas9 technology. Both cell lines are pluripotent, differentiate into the 3 germ layers and contain no genomic aberrations or off-target modifications. The cell lines form part of a human disease model to investigate the effects of truncating mutations in different regions of SRCAP.},
}
@article {pmid37995437,
year = {2023},
author = {Walz, K and Janz, A and Klopocki, E and Gerull, B},
title = {Generation of a CRISPR/Cas9-edited Plakoglobin (JUP) knock-out (JMUi001-A-4) iPSC line to model the cardiac phenotype of arrhythmogenic cardiomyopathy.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103240},
doi = {10.1016/j.scr.2023.103240},
pmid = {37995437},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; gamma Catenin/genetics/metabolism ; *Arrhythmogenic Right Ventricular Dysplasia/genetics ; Myocytes, Cardiac/metabolism ; Phenotype ; },
abstract = {Arrhythmogenic cardiomyopathy (ACM) represents the cardiac phenotype of Naxos disease, an autosomal recessive disease with an additional cutaneous phenotype. ACM is mainly caused by mutated desmosomal proteins, which are part of cardiac adherens junctions and provide mechanical and electrical stability. Here, we generated a knock-out (KO) of the junctional protein Plakoglobin (JUP-KO; JMUi001-A-4) using the CRISPR/Cas9 system in healthy control induced pluripotent stem cells (iPSCs, (JMUi001-A). JUP-KO iPSCs maintained pluripotency, differentiation potential and genomic integrity and provide an in vitro system modelling ACM when differentiated into cardiomyocytes.},
}
@article {pmid37992565,
year = {2023},
author = {Zhao, K and Zhang, H and Cui, J and Zhang, Y and Zhou, T and Long, B},
title = {Generation of an NSD2-deficient human embryonic stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103255},
doi = {10.1016/j.scr.2023.103255},
pmid = {37992565},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; Embryonic Stem Cells/metabolism ; Cell Line ; },
abstract = {NSD2 is a histone methyltransferase (HMT) and is involved in the epigenetic regulation of hematopoiesis and hematological cancers. To understand and illustrate the precise roles of NSD2 in hematopoietic development, here we constructed a human embryonic stem cell (hESC) line with knockout of NSD2 using CRISPR/Cas9-mediated gene targeting. The cell line maintained typical stem cell morphology and normal karyotype. Furthermore, the pluripotency of the cell line was evidenced by high expression level of pluripotency genes and differentiation potential into three germ layers. The cell line provides a good model for studying roles of NSD2 in embryonic development, especially hematopoiesis.},
}
@article {pmid37989316,
year = {2023},
author = {Han, W and Qiu, HY and Sun, S and Fu, ZC and Wang, GQ and Qian, X and Wang, L and Zhai, X and Wei, J and Wang, Y and Guo, YL and Cao, GH and Ji, RJ and Zhang, YZ and Ma, H and Wang, H and Zhao, M and Wu, J and Bi, L and Chen, QB and Li, Z and Yu, L and Mou, X and Yin, H and Yang, L and Chen, J and Yang, B and Zhang, Y},
title = {Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.},
journal = {Cell stem cell},
volume = {30},
number = {12},
pages = {1624-1639.e8},
doi = {10.1016/j.stem.2023.10.007},
pmid = {37989316},
issn = {1875-9777},
mesh = {Humans ; *Gene Editing ; Fetal Hemoglobin/genetics/metabolism ; gamma-Globins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation/genetics ; *Hemoglobinopathies/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; Transcription Factors/metabolism ; },
abstract = {Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating β-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and β-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating β-hemoglobinopathies.},
}
@article {pmid37988619,
year = {2023},
author = {Venkataraman, M and Yñigez-Gutierrez, A and Infante, V and MacIntyre, A and Fernandes-Júnior, PI and Ané, JM and Pfleger, B},
title = {Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.},
journal = {ACS synthetic biology},
volume = {12},
number = {12},
pages = {3623-3634},
doi = {10.1021/acssynbio.3c00414},
pmid = {37988619},
issn = {2161-5063},
support = {T32 GM135066/GM/NIGMS NIH HHS/United States ; },
mesh = {*Nitrogen ; *Soil ; Synthetic Biology ; Plasmids/genetics ; Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.},
}
@article {pmid37984032,
year = {2023},
author = {Maierhof, SK and Schinke, C and Cernoch, J and Hew, L and Bruske, LP and Fernandez Vallone, V and Fischer, K and Stachelscheid, H and Huehnchen, P and Endres, M and Diecke, S and Telugu, NS and Boehmerle, W},
title = {Generation of an NCS1 gene knockout human induced pluripotent stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103253},
doi = {10.1016/j.scr.2023.103253},
pmid = {37984032},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Gene Knockout Techniques ; Calcium/metabolism ; Gene Editing ; },
abstract = {NCS1 (Neuronal calcium sensor protein 1) encodes a highly conserved calcium binding protein abundantly expressed in neurons. It modulates intracellular calcium homeostasis, calcium-dependent signaling pathways as well as neuronal transmission and plasticity. Here, we generated a NCS1 knockout human induced pluripotent stem cell (hiPSC) line using CRISPR-Cas9 genome editing. It shows regular expression of pluripotent markers, normal iPSC morphology and karyotype as well as no detectable off-target effects on top 6 potentially affected genes. This newly generated cell line constitutes a valuable tool for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological disease.},
}
@article {pmid37983481,
year = {2023},
author = {Zhong, Z and Li, Z and Yang, J and Wang, Q},
title = {Unified Model to Predict gRNA Efficiency across Diverse Cell Lines and CRISPR-Cas9 Systems.},
journal = {Journal of chemical information and modeling},
volume = {63},
number = {23},
pages = {7320-7329},
doi = {10.1021/acs.jcim.3c01339},
pmid = {37983481},
issn = {1549-960X},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Cell Line ; Machine Learning ; },
abstract = {Computationally predicting the efficiency of a guide RNA (gRNA) from its sequence is crucial to designing the CRISPR-Cas9 system. Currently, machine learning (ML)-based models are widely used for such predictions. However, these ML models often show performance imbalance when applied to multiple data sets from diverse sources, hindering the practical utilization of these tools. To address this issue, we propose a Michaelis-Menten theoretical framework that integrates information from multiple data sets. We demonstrate that the binding free energy can serve as a useful invariant that bridges the data from different experimental setups. Building upon this framework, we develop a new ML model called Uni-deepSG. This model exhibits broad applicability on 27 data sets with different cell types, Cas9 variants, and gRNA designs. Our work confirms the existence of a generalized model for predicting gRNA efficiency and lays the theoretical groundwork necessary to finalize such a model.},
}
@article {pmid37977144,
year = {2023},
author = {Namipashaki, A and Pugsley, K and Liu, X and Abrehart, K and Lim, SM and Sun, G and Herold, MJ and Polo, JM and Bellgrove, MA and Hawi, Z},
title = {Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling.},
journal = {Stem cell reports},
volume = {18},
number = {12},
pages = {2515-2527},
pmid = {37977144},
issn = {2213-6711},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; Cell Line ; Cloning, Molecular ; Gene Editing/methods ; },
abstract = {The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method's utility in stem cell disease modeling.},
}
@article {pmid37941139,
year = {2023},
author = {Cheng, X and Li, X and Kang, Y and Zhang, D and Yu, Q and Chen, J and Li, X and Du, L and Yang, T and Gong, Y and Yi, M and Zhang, S and Zhu, S and Ding, S and Cheng, W},
title = {Rapid in situ RNA imaging based on Cas12a thrusting strand displacement reaction.},
journal = {Nucleic acids research},
volume = {51},
number = {22},
pages = {e111},
pmid = {37941139},
issn = {1362-4962},
support = {82372334//National Natural Science Foundation of China/ ; cstc2019jcyjjqX0028//Chongqing Science Fund for Distinguished Young Scholars/ ; CXQT20013//Foundation for Innovative Research Groups of Chongqing Higher Education Institutions/ ; CQYC20200303107//Chongqing Talents-Innovation Leading Talents Project/ ; cstc2021jcyj-bsh X0153//Natural Science Foundation of Chongqing/ ; 472020320220007//First Clinical College of Chongqing Medical University/ ; },
mesh = {*RNA/genetics ; CRISPR-Cas Systems ; DNA/genetics/chemistry ; Sensitivity and Specificity ; In Situ Hybridization ; Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; },
abstract = {RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.},
}
@article {pmid37932516,
year = {2023},
author = {Chen, Y and Lu, T and Liu, Y and Liu, Y and Bai, S and Chen, Q and Zhao, B and Wu, X},
title = {Establishment of SLC7A11-knockout mouse and its preliminary investigation in melanoma.},
journal = {In vitro cellular &amp; developmental biology. Animal},
volume = {59},
number = {9},
pages = {729-737},
pmid = {37932516},
issn = {1543-706X},
support = {Grant no. 32172722//National Natural Science Foundation of China/ ; Grant no. X20220652//Innovation and Entrepreneurship Talent Project of Lanzhou/ ; },
mesh = {Mice ; Animals ; *Melanoma/genetics ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems ; Cell Line ; Cystine/metabolism ; *Rodent Diseases ; },
abstract = {Solute carrier family 7 member 11 (SLC7A11)/xCT is an amino acid transporter that mediates the cystine uptake and glutamate export, participates in several malignant tumors' progression. However, the role of SLC7A11 on the occurrence and development of melanoma still remains unclear. Here, the transcribed mRNA encoding for Cas9 and sgRNA targeting SLC7A11 in vitro were microinjected into zygotes, to establish the SLC7A11 knockout (KO) mice (SLC7A11[-/-]). Further, we conducted melanoma-bearing mice using the metastatic melanoma cell line (B16-F10) to observe the melanoma development. There was no off-target in KO mice detected by T7E1 cleavage assay. The results showed that the tumor volume of KO mice was significantly lower than that of SLC7A11[+/+] (WT) mice at 8d, 10d, 12d, 14d, and 16d (P < 0.05). The tumors of WT appeared to more disorganized morphology, more unbalanced nuclear-cytoplasmic ratio, less defined boundary, and increased tumor necrosis. And after SLC7A11 deletion, the expression of CXCL9 and TLR6 were significantly up-regulated, and that of NOS2 and CCL8 were significantly down-regulated (P < 0.01). Additionally, Ki67 immunostaining revealed lower proliferating cells in the tumors of SLC7A11 KO mice compared to WT mice. In summary, the deletion of SLC7A11 significantly inhibited the development of melanoma. Our results provide direct evidence to identify SLC7A11 as a novel target for molecular therapy and prognosis judgment of melanoma.},
}
@article {pmid37932451,
year = {2023},
author = {Zheng, C and Wei, Y and Zhang, P and Lin, K and He, D and Teng, H and Manyam, G and Zhang, Z and Liu, W and Lee, HRL and Tang, X and He, W and Islam, N and Jain, A and Chiu, Y and Cao, S and Diao, Y and Meyer-Gauen, S and Höök, M and Malovannaya, A and Li, W and Hu, M and Wang, W and Xu, H and Kopetz, S and Chen, Y},
title = {CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.},
journal = {Nature structural &amp; molecular biology},
volume = {30},
number = {12},
pages = {1878-1892},
pmid = {37932451},
issn = {1545-9985},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; P50 CA221707/CA/NCI NIH HHS/United States ; R35 GM137927/GM/NIGMS NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; R01 GM130838/GM/NIGMS NIH HHS/United States ; P50 CA127001/CA/NCI NIH HHS/United States ; R01 NS117668/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Humans ; Open Reading Frames/genetics ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; Proteome/genetics ; },
abstract = {Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.},
}
@article {pmid37931537,
year = {2023},
author = {Ruan, Y and Tian, X and Li, X and Zhang, Y},
title = {Establishment of a human TLR4 compound heterozygous knockout hESC line (WAe009-A-N) to model toll-like receptor 4 deficiency by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {73},
number = {},
pages = {103225},
doi = {10.1016/j.scr.2023.103225},
pmid = {37931537},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Toll-Like Receptor 4/genetics/metabolism ; Cell Line ; Embryonic Stem Cells/metabolism ; },
abstract = {Toll-like receptor 4 (TLR4) is a pattern recognition receptor (PRRS) and an important protective immune receptor. TLR4 deficiency can lead to Inflammatory bowel disease. To explore the role of TLR4, we used CRISPR/Cas9 system to produce TLR4 compound heterozygous knockout embryonic stem cells in H9 cell line. The WAe009-A-N has a compound heterozygous 7 bp deletion/8 bp deletion in TLR4 exon 3, which resulted in a frameshift in the translation of TLR4, and TLR4 protein wasn't detectable in this cell line. In addition, the WAe009-A-N with normal karyotype can express pluripotent markers and differentiate into three germ layers in vitro.},
}
@article {pmid37930830,
year = {2023},
author = {Song, G and Tian, C and Li, J and Zhang, F and Peng, Y and Gao, X and Tian, Y},
title = {Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system.},
journal = {Nucleic acids research},
volume = {51},
number = {22},
pages = {12381-12396},
pmid = {37930830},
issn = {1362-4962},
support = {2022YFF0710700//National Key R&amp;D Program of China/ ; 32270567//National Natural Science Foundation of China/ ; KFJ-BRP-017-55//Biological Resources Program of Chinese Academy of Sciences/ ; 2018QNRC001//Young Elite Scientist Sponsorship Program/ ; },
mesh = {Humans ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Plasmids/genetics ; Bacterial Proteins/metabolism ; Escherichia coli/genetics/metabolism ; },
abstract = {Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.},
}
@article {pmid37924981,
year = {2023},
author = {Mansour, HM and El-Khatib, AS},
title = {Exploring Parkinson-associated kinases for CRISPR/Cas9-based gene editing: beyond alpha-synuclein.},
journal = {Ageing research reviews},
volume = {92},
number = {},
pages = {102114},
doi = {10.1016/j.arr.2023.102114},
pmid = {37924981},
issn = {1872-9649},
mesh = {Humans ; alpha-Synuclein/genetics ; Gene Editing ; *Parkinson Disease/genetics/therapy ; CRISPR-Cas Systems/genetics ; *Neurodegenerative Diseases/genetics ; Protein Kinase C/genetics ; },
abstract = {Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substania nigra (SN) and is associated with progressive motor impairment. PD is classified into familial and sporadic forms. The first genetic association studies in PD reported the involvement of Synuclein alpha (SNCA) mutations in the pathobiology of familial PD. Subsequent studies suggested mutations in PTEN-induced putative kinase 1 (PINK1), PARKIN, leucine repeat kinase-2 (LRRK2), and DJ-1 causing familial PD. In addition, kinase dysregulation has been embroiled in the pathogenesis of PD. The genome-editing mechanism CRISPR (clustered regularly interspaced short palindromic repeats) has recently influenced industry and scientific discoveries and is expected to expedite neurodegenerative disease research. This review will discuss the structure, function, and history of the CRISPR/Cas9 genome editing system. Moreover, it summarizes genes-encoding kinases involved in PD pathogenesis and targeted by CRISPR/Cas9 technology, including LRRK2, PINK1, Protein kinase C-delta (PKC-γ), and adenosine monophosphate-activated protein kinase (AMPK). We provide an overview of novel kinases to be targeted by the CRISPR/Cas9 system such as G-protein coupled receptor kinases (GRKs), cyclin-G-associated kinases (GAKs), cyclin-dependent kinase 5 (CDK5), Ataxia telangiectasia mutated (ATM), c-ABL, and rearranged during transfection (RET) receptors. Additionally, we will explain the off-target effects of CRISPR/Cas9 system and how to address them. Also, we will shed light on the associated challenges and future directions that are enabling the efficient use of CRISPR/Cas9 technology in kinases research in PD. In conclusion, gene editing, in addition to gene therapy, might be a possible promising strategy for PD therapy.},
}
@article {pmid37906116,
year = {2023},
author = {Tang, W and Tong, T and Wang, H and Lu, X and Yang, C and Wu, Y and Wang, Y and Liu, J and Ding, B},
title = {A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {62},
number = {51},
pages = {e202315093},
doi = {10.1002/anie.202315093},
pmid = {37906116},
issn = {1521-3773},
support = {2021YFA1200302//National Key R&amp;D Program of China/ ; 22025201//National Natural Science Foundation of China/ ; 22077023//National Natural Science Foundation of China/ ; 22377020//National Natural Science Foundation of China/ ; 21721002//National Natural Science Foundation of China/ ; XDB36000000//Strategic Priority Research Program of Chinese Academy of Sciences/ ; YSBR-036//CAS Project for Young Scientists in Basic Research/ ; //Sponsored by Beijing Nova Program/ ; //Youth Innovation Promotion Association CAS/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genetic Therapy ; DNA/genetics ; },
abstract = {DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.},
}
@article {pmid37839785,
year = {2024},
author = {Keating, M and Hagle, R and Osorio-Méndez, D and Rodriguez-Parks, A and Almutawa, SI and Kang, J},
title = {A robust knock-in approach using a minimal promoter and a minicircle.},
journal = {Developmental biology},
volume = {505},
number = {},
pages = {24-33},
doi = {10.1016/j.ydbio.2023.10.002},
pmid = {37839785},
issn = {1095-564X},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Zebrafish/genetics ; Gene Knock-In Techniques ; Animals, Genetically Modified ; Gene Editing ; },
abstract = {Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.},
}
@article {pmid37833064,
year = {2024},
author = {Lotfi, M and Butler, AE and Sukhorukov, VN and Sahebkar, A},
title = {Application of CRISPR-Cas9 technology in diabetes research.},
journal = {Diabetic medicine : a journal of the British Diabetic Association},
volume = {41},
number = {1},
pages = {e15240},
doi = {10.1111/dme.15240},
pmid = {37833064},
issn = {1464-5491},
support = {23-65-10014//Russian Science Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Diabetes Mellitus, Type 2/genetics/therapy ; Gene Editing ; Recombinational DNA Repair ; DNA End-Joining Repair ; },
abstract = {Diabetes is a chronic disorder with rapidly increasing prevalence that is a major global issue of our current era. There are two major types of diabetes. Polygenic forms of diabetes include type 1 diabetes (T1D) and type 2 diabetes (T2D) and its monogenic forms are maturity-onset diabetes of the young (MODY) and neonatal diabetes mellitus (NDM). There are no permanent therapeutic approaches for diabetes and current therapies rely on regular administration of various drugs or insulin injection. Recently, gene editing strategies have offered new promise for treating genetic disorders. Targeted genome editing is a fast-growing technology, recruiting programmable nucleases to specifically modify target genomic sequences. These targeted nucleases generate double-strand breaks at target regions in the genome, which induce cellular repair pathways including non-homologous end joining (NHEJ) and homology-directed repair (HDR). Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a novel gene-editing system, permitting precise genome modification. CRISPR/Cas9 has great potential for various applications in diabetic research such as gene screening, generation of diabetic animal models and treatment. In this article, gene-editing strategies are summarized with a focus on the CRISPR/Cas9 approach in diabetes research.},
}
@article {pmid37830810,
year = {2023},
author = {},
title = {CRISPR-Cas9 Gene Editing in T Cells Induces Site-Specific Chromosome Loss.},
journal = {Cancer discovery},
volume = {13},
number = {12},
pages = {OF16},
doi = {10.1158/2159-8290.CD-RW2023-163},
pmid = {37830810},
issn = {2159-8290},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; T-Lymphocytes ; Chromosome Deletion ; Chromosomes ; },
abstract = {CRISPR-Cas9 gene editing in T cells can lead to partial or complete loss of the targeted chromosome.},
}
@article {pmid37805711,
year = {2023},
author = {Thomas, SP and Domm, JM and van Vloten, JP and Xu, L and Vadivel, A and Yates, JGE and Pei, Y and Ingrao, J and van Lieshout, LP and Jackson, SR and Minott, JA and Achuthan, A and Mehrani, Y and McAusland, TM and Zhang, W and Karimi, K and Vaughan, AE and de Jong, J and Kang, MH and Thebaud, B and Wootton, SK},
title = {A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {12},
pages = {3457-3477},
doi = {10.1016/j.ymthe.2023.10.002},
pmid = {37805711},
issn = {1525-0024},
mesh = {Mice ; Animals ; *Gene Editing ; *Doxycycline ; Dependovirus/genetics ; Genetic Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems ; Lung/metabolism ; Surface-Active Agents/metabolism ; CRISPR-Cas Systems ; },
abstract = {Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 10[11] vg donor template and 1 × 10[11] vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 10[12] vg SP-B-donor template and 5 × 10[11] vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.},
}
@article {pmid37716439,
year = {2023},
author = {Shahwar, D and Ahn, N and Kim, D and Ahn, W and Park, Y},
title = {Mutagenesis-based plant breeding approaches and genome engineering: A review focused on tomato.},
journal = {Mutation research. Reviews in mutation research},
volume = {792},
number = {},
pages = {108473},
doi = {10.1016/j.mrrev.2023.108473},
pmid = {37716439},
issn = {1388-2139},
mesh = {Humans ; *Solanum lycopersicum/genetics ; Genetic Engineering/methods ; Plant Breeding/methods ; Genome, Plant/genetics ; Crops, Agricultural/genetics ; Mutagenesis/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Breeding is the most important and efficient method for crop improvement involving repeated modification of the genetic makeup of a plant population over many generations. In this review, various accessible breeding approaches, such as conventional breeding and mutation breeding (physical and chemical mutagenesis and insertional mutagenesis), are discussed with respect to the actual impact of research on the economic improvement of tomato agriculture. Tomatoes are among the most economically important fruit crops consumed worldwide because of their high nutritional content and health-related benefits. Additionally, we summarize mutation-based mapping approaches, including Mutmap and MutChromeSeq, for the efficient mapping of several genes identified by random indel mutations that are beneficial for crop improvement. Difficulties and challenges in the adaptation of new genome editing techniques that provide opportunities to demonstrate precise mutations are also addressed. Lastly, this review focuses on various effective and convenient genome editing tools, such as RNA interference (RNAi), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), and their potential for the improvement of numerous desirable traits to allow the development of better varieties of tomato and other horticultural crops.},
}
@article {pmid37684549,
year = {2023},
author = {Lee, TW and Hunter, FW and Tsai, P and Print, CG and Wilson, WR and Jamieson, SMF},
title = {Clonal dynamics limits detection of selection in tumour xenograft CRISPR/Cas9 screens.},
journal = {Cancer gene therapy},
volume = {30},
number = {12},
pages = {1610-1623},
pmid = {37684549},
issn = {1476-5500},
support = {1115022//Auckland Medical Research Foundation (AMRF)/ ; 14/538//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 19/433//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; },
mesh = {Humans ; Mice ; Animals ; Aged ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Heterografts ; RNA, Guide, CRISPR-Cas Systems ; Clone Cells ; },
abstract = {Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are edited in vitro and inoculated into mice to form tumours, allow evaluation of gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. To improve our understanding of the key parameters for success with this method, we investigated the choice of cell line, mouse host, tumour harvesting timepoint and guide RNA (gRNA) library size. We found that high gRNA (80-95%) representation was maintained in a HCT116 subline transduced with the GeCKOv2 whole-genome gRNA library and transplanted into NSG mice when tumours were harvested at early (14 d) but not late time points (38-43 d). The decreased representation in older tumours was accompanied by large increases in variance in gRNA read counts, with notable expansion of a small number of random clones in each sample. The variable clonal dynamics resulted in a high level of 'noise' that limited the detection of gRNA-based selection. Using simulated datasets derived from our experimental data, we show that considerable reductions in count variance would be achieved with smaller library sizes. Based on our findings, we suggest a pathway to rationally design adequately powered in vivo CRISPR screens for successful evaluation of gene function.},
}
@article {pmid37647419,
year = {2024},
author = {Wang, H and Ai, L and Xia, Y and Wang, G and Xiong, Z and Song, X},
title = {Software-based screening for efficient sgRNAs in Lactococcus lactis.},
journal = {Journal of the science of food and agriculture},
volume = {104},
number = {2},
pages = {1200-1206},
doi = {10.1002/jsfa.12946},
pmid = {37647419},
issn = {1097-0010},
support = {32025029//National Science Foundation for Distinguished Young Scholars/ ; 2021-Y06//CIFST-Yili Foundation of Health Science/ ; 2101070007800120//Shanghai Education committee scientific research innovation projects/ ; 32101928//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Lactococcus lactis/genetics ; Software ; },
abstract = {BACKGROUND: The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria according to the demands of the user.<br><br>RESULTS: We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR - and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results.<br><br>CONCLUSION: Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results - a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. Software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. &#xa9; 2023 Society of Chemical Industry.},
}
@article {pmid37559489,
year = {2023},
author = {Watanabe, T and Ochi, Y and Kajihara, R and Ichikawa, K and Ezaki, R and Matsuzaki, M and Horiuchi, H},
title = {Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.},
journal = {Biotechnology journal},
volume = {18},
number = {12},
pages = {e2300328},
doi = {10.1002/biot.202300328},
pmid = {37559489},
issn = {1860-7314},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems/genetics ; Heparin ; Transfection ; Gene Editing/methods ; Germ Cells ; },
abstract = {Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p < 0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine 2000 and our PGC medium were the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them.},
}
@article {pmid37542640,
year = {2023},
author = {Zheng, H and Zhao, J and Wang, D and Fu, ZQ},
title = {Rising from the dead: the power of genome editing.},
journal = {Science China. Life sciences},
volume = {66},
number = {12},
pages = {2949-2951},
pmid = {37542640},
issn = {1869-1889},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid37505431,
year = {2023},
author = {Qi, Y and Zhang, Y and Tian, S and Zong, R and Yan, X and Wang, Y and Wang, Y and Zhao, J},
title = {An optimized prime editing system for efficient modification of the pig genome.},
journal = {Science China. Life sciences},
volume = {66},
number = {12},
pages = {2851-2861},
pmid = {37505431},
issn = {1869-1889},
mesh = {Mice ; Animals ; Swine ; *Thymine ; *Animals, Domestic ; Transgenes ; Agriculture ; Cytosine ; Gene Editing ; Histone Deacetylase Inhibitors ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.},
}
@article {pmid38075777,
year = {2023},
author = {Kesarwani, P and Vora, DS and Sundar, D},
title = {Improved Machine Learning-Based Model for the Classification of Off-Targets in the CRISPR/Cpf1 System.},
journal = {ACS omega},
volume = {8},
number = {48},
pages = {45578-45588},
pmid = {38075777},
issn = {2470-1343},
abstract = {Targeted nucleases are widely used for altering the specific location of the genome with precision. The endonucleases facilitate efficient genome editing via designing a guide RNA (gRNA) consisting of a 20-nucleotide target sequence. gRNA preferably binds to the target location, but the on- and off-target activities of gRNAs vary widely. The off-target activity due to mismatch tolerance in the CRISPR-Cas system is a major factor inhibiting its clinical applications. Ensuring on-target efficiency and minimizing off-targets for a target sequence are the major objectives of this study. A pipeline has been designed to predict potential off-target sites in the human genome for a target sequence, and a multilayer perceptron (MLP) has been used to predict the cleavage efficiency of the potential off-target sites. An MLP-based classifier was trained with sequence- and base-dependent binding energy-associated features for AsCpf1 and LbCpf1 to predict the target efficiencies. Positional preferences of nucleotides, distribution of mismatches, and classification-dependent feature importance between high-activity and low-activity off-targets were also studied. Positional preference of nucleotides revealed that thymine is highly disfavored at positions adjacent to Protospacer Adjacent Motif (PAM), whereas guanine is favored in high-activity off-targets. Mismatch distribution analysis revealed that mismatches were more prominent in the trunk region (16, 17, 18 nucleotides from PAM sequence), and the promiscuous region and transition type mismatch were more preferred at 16, 17, and 18 nucleotides positions. The distribution of mismatches was a distinctive feature between high-activity and low-activity off-targets. Thermodynamics-associated features such as low to moderate melting temperature of the nonseed region and base-dependent PAM binding energy were predicted as best predictors by the multilayer perceptron for high-activity off-targets. GC content, some types of dinucleotide frequencies, number of bulges, and mismatches in the seed and trunk regions were other characteristic features between high-activity and low-activity off-targets for both LbCpf1 and AsCpf1.},
}
@article {pmid38073045,
year = {2023},
author = {Li, HD and Fang, GH and Ye, BC and Yin, BC},
title = {RNase H-Driven crRNA Switch Circuits for Rapid and Sensitive Detection of Various Analytical Targets.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c04267},
pmid = {38073045},
issn = {1520-6882},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target preamplification to improve sensitivity and detection limits. Here, we propose a generic crRNA switch circuit-regulated CRISPR/Cas sensor for the sensitive detection of various targets. The crRNA switch is engineered and designed in a blocked state but can be activated in the presence of triggers, which are target-induced association DNA to initiate the trans-cleavage activity of Cas12a for signal reporting. Additionally, RNase H is introduced to specifically hydrolyze RNA duplexed with the DNA trigger, resulting in the regeneration of the trigger to activate more crRNA switches. Such a combination provides a generic and sensitive strategy for the effective sensing of the p53 sequence, thrombin, and adenosine triphosphate. The design is incorporated with nucleic acid nanotechnology and extensively broadens the application scope of the CRISPR technology in biosensing.},
}
@article {pmid38072634,
year = {2023},
author = {Su, X and Jin, X and Wang, Z and Duan, S},
title = {Unraveling Exogenous DNA Processing in Cas4-Lacking Crispr Systems: A Novel Bypass Pathway Explored.},
journal = {Advanced biology},
volume = {},
number = {},
pages = {e2300454},
doi = {10.1002/adbi.202300454},
pmid = {38072634},
issn = {2701-0198},
support = {210000-581835//Qiantang Scholars Fund in Hangzhou City University/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are widely distributed adaptive immune systems found in prokaryotes. The process involves three main stages: adaptation, expression, and interference. While the adaptation stage has been extensively studied, there is still an incomplete understanding of the mechanisms underlying the capture, trimming, and integration of exogenous DNA. For instance, Cas4, a CRISPR-Cas protein with endonuclease activity, is responsible for selecting and processing protospacer adjacent motif (PAM) sequences. However, some CRISPR isoforms lack Cas4 activity, relying on other enzymes for adaptive immunity. Recently, Wang et al. presented a novel model of exogenous DNA processing in a type I-E CRISPR system lacking Cas4 in a Nature article. This model integrates protospacer processing into CRISPR arrays through fine-tuned synthases formed by DnaQ-like exonuclease (DEDDh) and Cas1-Cas2 complexes. Their study introduces a novel model, shedding new light on the evolution of CRISPR adaptive immunity. This perspective comprehensively examines the fundamental process of CRISPR adaptive immunity, detailing both the classical pathway mediated by Cas4 and the alternative pathway mediated by DEDDh. Furthermore, a thorough evaluation of Wang et al.'s work is conducted, highlighting its strengths, weaknesses, and existing research challenges.},
}
@article {pmid38069533,
year = {2023},
author = {Pérez, AA and Tobin, A and Stechly, JV and Ferrante, JA and Hunter, ME},
title = {A minimally invasive, field-applicable CRISPR/Cas biosensor to aid in the detection of Pseudogymnoascus destructans, the causative fungal agent of white-nose syndrome in bats.},
journal = {Molecular ecology resources},
volume = {},
number = {},
pages = {},
doi = {10.1111/1755-0998.13902},
pmid = {38069533},
issn = {1755-0998},
support = {//U.S. Geological Survey/ ; },
abstract = {The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd-positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd-positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.},
}
@article {pmid38069443,
year = {2023},
author = {Matveeva, A and Ryabchenko, A and Petrova, V and Prokhorova, D and Zhuravlev, E and Zakabunin, A and Tikunov, A and Stepanov, G},
title = {Expression and Functional Analysis of the Compact Thermophilic Anoxybacillus flavithermus Cas9 Nuclease.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069443},
issn = {1422-0067},
support = {075-15-2021-1085//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools, as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The subclass of thermophilic Cas9 nucleases is actively expanding due to the advances in genome sequencing allowing for the meticulous examination of various microorganisms' genomes in search of the novel CRISPR systems. The most prominent thermophilic Cas9 effectors known to date are GeoCas9, ThermoCas9, IgnaviCas9, AceCas9, and others. These nucleases are characterized by a varying temperature range of the activity and stringent PAM preferences; thus, further diversification of the naturally occurring thermophilic Cas9 subclass presents an intriguing task. This study focuses on generating a construct to express a compact Cas9 nuclease (AnoCas9) from the thermophilic microorganism Anoxybacillus flavithermus displaying the nuclease activity in the 37-60 °C range and the PAM preference of 5'-NNNNCDAA-3' in vitro. Here, we highlight the close relation of AnoCas9 to the GeoCas9 family of compact thermophilic Cas9 effectors. AnoCas9, beyond broadening the repertoire of Cas9 nucleases, suggests application in areas requiring the presence of thermostable CRISPR/Cas systems in vitro, such as sequencing libraries' enrichment, allele-specific isothermal PCR, and others.},
}
@article {pmid38069429,
year = {2023},
author = {Gunitseva, N and Evteeva, M and Korzhenkov, A and Patrushev, M},
title = {A New RNA-Dependent Cas12g Nuclease.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069429},
issn = {1422-0067},
support = {agreement number 075-15-2021-1062//The study was supported by grants from the Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of runaway mutant variants. One of the most promising applications is in the treatment of genetic disorders caused by mutations in RNA molecules. In this study, we investigate a previously undescribed Cas12g nuclease which was found in metagenomes from promising thermophilic microbial communities during the expedition to the Republic of North Ossetia-Alania in 2020. The method outlined in this study can be applied to other Cas orthologs and variants, leading to a better understanding of the CRISPR-Cas system and its enzymatic activities. The cis-cleavage activity of the new type V-G Cas effector was indicated by in vitro RNA cleavage experiments. While CRISPR-Cas systems are known for their high specificity, there is still a risk of unintended cleavage of nontargeted RNA molecules. Ultimately, the search for new genome editing tools and the study of their properties will remove barriers to research in this area. With continued research and development, we may be able to unlock their full potential.},
}
@article {pmid38069367,
year = {2023},
author = {Petrova, IO and Smirnikhina, SA},
title = {The Development, Optimization and Future of Prime Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069367},
issn = {1422-0067},
support = {state assignment from the Ministry of Education and Science of Russia for the Research Centre for Medical Genetics//Ministry of Education and Science of Russia for the Research Centre for Medical Genetics/ ; },
abstract = {Prime editing is a rapidly developing method of CRISPR/Cas-based genome editing. The increasing number of novel PE applications and improved versions demands constant analysis and evaluation. The present review covers the mechanism of prime editing, the optimization of the method and the possible next step in the evolution of CRISPR/Cas9-associated genome editing. The basic components of a prime editing system are a prime editor fusion protein, consisting of nickase and reverse transcriptase, and prime editing guide RNA, consisting of a protospacer, scaffold, primer binding site and reverse transcription template. Some prime editing systems include other parts, such as additional RNA molecules. All of these components were optimized to achieve better efficiency for different target organisms and/or compactization for viral delivery. Insights into prime editing mechanisms allowed us to increase the efficiency by recruiting mismatch repair inhibitors. However, the next step in prime editing evolution requires the incorporation of new mechanisms. Prime editors combined with integrases allow us to combine the precision of prime editing with the target insertion of large, several-kilobase-long DNA fragments.},
}
@article {pmid38069353,
year = {2023},
author = {Nguyen, HM and Watanabe, S and Sharmin, S and Kawaguchi, T and Tan, XE and Wannigama, DL and Cui, L},
title = {RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069353},
issn = {1422-0067},
abstract = {RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.},
}
@article {pmid38069255,
year = {2023},
author = {Kim, J},
title = {Nucleic Acid-Based Approaches to Tackle KRAS Mutant Cancers.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38069255},
issn = {1422-0067},
support = {GCU- 202110510001//Gachon University research fund/ ; 2022R1F1A10750851112982076870101//National Research Foundation of Korea (NRF) grant/ ; },
abstract = {Activating mutations in KRAS are highly relevant to various cancers, driving persistent efforts toward the development of drugs that can effectively inhibit KRAS activity. Previously, KRAS was considered 'undruggable'; however, the recent advances in our understanding of RNA and nucleic acid chemistry and delivery formulations have sparked a paradigm shift in the approach to KRAS inhibition. We are currently witnessing a large wave of next-generation drugs for KRAS mutant cancers-nucleic acid-based therapeutics. In this review, we discuss the current progress in targeting KRAS mutant tumors and outline significant developments in nucleic acid-based strategies. We delve into their mechanisms of action, address existing challenges, and offer insights into the current clinical trial status of these approaches. We aim to provide a thorough understanding of the potential of nucleic acid-based strategies in the field of KRAS mutant cancer therapeutics.},
}
@article {pmid38068981,
year = {2023},
author = {Martín-Valmaseda, M and Devin, SR and Ortuño-Hernández, G and Pérez-Caselles, C and Mahdavi, SME and Bujdoso, G and Salazar, JA and Martínez-Gómez, P and Alburquerque, N},
title = {CRISPR/Cas as a Genome-Editing Technique in Fruit Tree Breeding.},
journal = {International journal of molecular sciences},
volume = {24},
number = {23},
pages = {},
pmid = {38068981},
issn = {1422-0067},
abstract = {CRISPR (short for "Clustered Regularly Interspaced Short Palindromic Repeats") is a technology that research scientists use to selectively modify the DNA of living organisms. CRISPR was adapted for use in the laboratory from the naturally occurring genome-editing systems found in bacteria. In this work, we reviewed the methods used to introduce CRISPR/Cas-mediated genome editing into fruit species, as well as the impacts of the application of this technology to activate and knock out target genes in different fruit tree species, including on tree development, yield, fruit quality, and tolerance to biotic and abiotic stresses. The application of this gene-editing technology could allow the development of new generations of fruit crops with improved traits by targeting different genetic segments or even could facilitate the introduction of traits into elite cultivars without changing other traits. However, currently, the scarcity of efficient regeneration and transformation protocols in some species, the fact that many of those procedures are genotype-dependent, and the convenience of segregating the transgenic parts of the CRISPR system represent the main handicaps limiting the potential of genetic editing techniques for fruit trees. Finally, the latest news on the legislation and regulations about the use of plants modified using CRISPR/Cas systems has been also discussed.},
}
@article {pmid38068598,
year = {2023},
author = {Komal, J and Desai, HR and Samal, I and Mastinu, A and Patel, RD and Kumar, PVD and Majhi, PK and Mahanta, DK and Bhoi, TK},
title = {Unveiling the Genetic Symphony: Harnessing CRISPR-Cas Genome Editing for Effective Insect Pest Management.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {23},
pages = {},
pmid = {38068598},
issn = {2223-7747},
abstract = {Phytophagous insects pose a significant threat to global crop yield and food security. The need for increased agricultural output while reducing dependence on harmful synthetic insecticides necessitates the implementation of innovative methods. The utilization of CRISPR-Cas (Clustered regularly interspaced short palindromic repeats) technology to develop insect pest-resistant plants is believed to be a highly effective approach in reducing production expenses and enhancing the profitability of farms. Insect genome research provides vital insights into gene functions, allowing for a better knowledge of insect biology, adaptability, and the development of targeted pest management and disease prevention measures. The CRISPR-Cas gene editing technique has the capability to modify the DNA of insects, either to trigger a gene drive or to overcome their resistance to specific insecticides. The advancements in CRISPR technology and its various applications have shown potential in developing insect-resistant varieties of plants and other strategies for effective pest management through a sustainable approach. This could have significant consequences for ensuring food security. This approach involves using genome editing to create modified insects or crop plants. The article critically analyzed and discussed the potential and challenges associated with exploring and utilizing CRISPR-Cas technology for reducing insect pest pressure in crop plants.},
}
@article {pmid38067110,
year = {2023},
author = {Ali, R and Alhaj Sulaiman, A and Memon, B and Pradhan, S and Algethami, M and Aouida, M and McKay, G and Madhusudan, S and Abdelalim, EM and Ramotar, D},
title = {Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.},
journal = {Cells},
volume = {12},
number = {23},
pages = {},
pmid = {38067110},
issn = {2073-4409},
abstract = {Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.},
}
@article {pmid38066376,
year = {2024},
author = {Hill, CM and Hatoum-Aslan, A},
title = {Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2734},
number = {},
pages = {279-299},
pmid = {38066376},
issn = {1940-6029},
abstract = {The functional characterization of "hypothetical" phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications-the strictly lytic variety-are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.},
}
@article {pmid38064533,
year = {2023},
author = {Meacham, Z and de Tacca, LA and Bondy-Denomy, J and Rabuka, D and Schelle, M},
title = {Cas9 degradation in human cells using phage anti-CRISPR proteins.},
journal = {PLoS biology},
volume = {21},
number = {12},
pages = {e3002431},
doi = {10.1371/journal.pbio.3002431},
pmid = {38064533},
issn = {1545-7885},
abstract = {Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here, we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. AcrIIA1-induced degradation of SpyCas9 is abolished by mutations in AcrIIA1 that break a direct physical interaction between the 2 proteins. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.},
}
@article {pmid38059344,
year = {2023},
author = {Smith, LM and Hampton, HG and Yevstigneyeva, MS and Mahler, M and Paquet, ZSM and Fineran, PC},
title = {CRISPR-Cas immunity is repressed by the LysR-type transcriptional regulator PigU.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad1165},
pmid = {38059344},
issn = {1362-4962},
support = {//Royal Society of New Zealand/ ; //University of Otago Doctoral Scholarships/ ; },
abstract = {Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.},
}
@article {pmid38058394,
year = {2023},
author = {Kenarkoohi, A and Abdoli, A and Rostamzad, A and Rashnavadi, M and Naserifar, R and Abdi, J and Shams, M and Bozorgomid, A and Saeb, S and Al-Fahad, D and Khezri, K and Falahi, S},
title = {Presence of CRISPR CAS-Like Sequences as a Proposed Mechanism for Horizontal Genetic Exchanges between Trichomonas vaginalis and Its Associated Virus: A Comparative Genomic Analysis with the First Report of a Putative CRISPR CAS Structures in Eukaryotic Cells.},
journal = {BioMed research international},
volume = {2023},
number = {},
pages = {8069559},
pmid = {38058394},
issn = {2314-6141},
mesh = {*Trichomonas vaginalis/genetics ; Satellite Viruses/genetics ; CRISPR-Cas Systems/genetics ; Eukaryotic Cells ; Genomics ; Archaea/genetics ; DNA Transposable Elements ; },
abstract = {INTRODUCTION: Trichomonas vaginalis genome is among the largest genome size and coding capacities. Combinations of gene duplications, transposon, repeated sequences, and lateral gene transfers (LGTs) have contributed to the unexpected large genomic size and diversity. This study is aimed at investigating genomic exchange and seeking for presence of the CRISPR CAS system as one of the possible mechanisms for some level of genetic exchange. Material and Methods. In this comparative analysis, 398 publicly available Trichomonas vaginalis complete genomes were investigated for the presence of CRISPR CAS. Spacer sequences were also analyzed for their origin using BLAST.<br><br>RESULTS: We identified a CRISPR CAS (Cas3). CRISPR spacers are highly similar to transposable genetic elements such as viruses of protozoan parasites, especially megavirals, some transposons, and, interestingly, papillomavirus and HIV-1 in a few cases. Discussion. There is a striking similarity between the prokaryotes/Archaean CRISPR and what we find as eukaryotic CRISPR. About 5-10% of the 398 T. vaginalis possess a CRISPR structure.<br><br>CONCLUSION: According to sequences and their organization, we assume that these repeated sequences and spacer, along with their mentioned features, could be the eukaryotic homolog of prokaryotes and Archaean CRISPR systems and may involve in a process similar to the CRISPR function.},
}
@article {pmid38057266,
year = {2023},
author = {Karunarathne, S and Walker, E and Sharma, D and Li, C and Han, Y},
title = {Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance.},
journal = {Journal of Zhejiang University. Science. B},
volume = {24},
number = {12},
pages = {1069-1092},
doi = {10.1631/jzus.B2200552},
pmid = {38057266},
issn = {1862-1783},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Hordeum/genetics ; Crops, Agricultural/genetics ; Stress, Physiological/genetics ; },
abstract = {Abiotic stresses, predominately drought, heat, salinity, cold, and waterlogging, adversely affect cereal crops. They limit barley production worldwide and cause huge economic losses. In barley, functional genes under various stresses have been identified over the years and genetic improvement to stress tolerance has taken a new turn with the introduction of modern gene-editing platforms. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a robust and versatile tool for precise mutation creation and trait improvement. In this review, we highlight the stress-affected regions and the corresponding economic losses among the main barley producers. We collate about 150 key genes associated with stress tolerance and combine them into a single physical map for potential breeding practices. We also overview the applications of precise base editing, prime editing, and multiplexing technologies for targeted trait modification, and discuss current challenges including high-throughput mutant genotyping and genotype dependency in genetic transformation to promote commercial breeding. The listed genes counteract key stresses such as drought, salinity, and nutrient deficiency, and the potential application of the respective gene-editing technologies will provide insight into barley improvement for climate resilience.},
}
@article {pmid38057050,
year = {2024},
author = {Cui, J and Luo, Q and Wei, C and Deng, X and Liang, H and Wei, J and Gong, Y and Tang, Q and Zhang, K and Liao, X},
title = {Electrochemical biosensing for E.coli detection based on triple helix DNA inhibition of CRISPR/Cas12a cleavage activity.},
journal = {Analytica chimica acta},
volume = {1285},
number = {},
pages = {342028},
doi = {10.1016/j.aca.2023.342028},
pmid = {38057050},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; DNA/genetics ; *Nucleic Acids ; Oligonucleotides ; Antibodies ; Escherichia coli/genetics ; *Biosensing Techniques ; },
abstract = {BACKGROUND: Escherichia coli (E.coli) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract, posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety.<br><br>RESULTS: We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli. Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity.<br><br>SIGNIFICANCE: With a minimal detection limit of 5.02&#xa0;CFU/mL and a linear range of 1&#xa0;&#xd7;&#xa0;10[2] - 1&#xa0;&#xd7;&#xa0;10[7]&#xa0;CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.},
}
@article {pmid38054656,
year = {2023},
author = {Wang, R and Mao, X and Xu, J and Yao, P and Jiang, J and Li, Q and Wang, F},
title = {Engineering of the LAMP-CRISPR/Cas12b platform for Chlamydia psittaci detection.},
journal = {Journal of medical microbiology},
volume = {72},
number = {12},
pages = {},
doi = {10.1099/jmm.0.001781},
pmid = {38054656},
issn = {1473-5644},
mesh = {Animals ; Adult ; Humans ; *Chlamydophila psittaci/genetics ; *Psittacosis/diagnosis ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Zoonoses ; },
abstract = {Introduction. Chlamydia psittaci (C. psittaci) is a zoonotic infection, that causes psittacosis (parrot fever) in humans, leading to severe clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, in rare cases, death.Gap Statement. Rapid, sensitive and specific detection of C. psittaci facilitates timely diagnosis and treatment of patients.Aim. This study aimed to engineer the LAMP-CRISPR/Cas12b platform for C. psittaci detection.Methodology. The loop-mediated isothermal amplification (LAMP) technique and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 12b (CRISPR-Cas12b) assay were combined to establish two-step and one-tube LAMP-CRISPR/Cas12b reaction systems, respectively, for rapidly detecting C. psittaci.Results. The two-step and one-tube LAMP-CRISPR/Cas12b assay could complete detection within 1 h. No cross-reactivity was observed from non-C. psittaci templates with specific LAMP amplification primers and single-guide RNA (sgRNA) targeting the highly conserved short fragment CPSIT_0429 gene of C. psittaci. The detection limits of the two-step and one-tube LAMP-CRISPR/Cas12b reaction were 10[2] aM and 10[3] aM, respectively. The results were consistent with qPCR for nucleic acid detection in 160 clinical samples, including 80 suspected C. psittaci samples, kept in the laboratory.Conclusions. The LAMP-CRISPR/Cas12b assay developed in this study provides a sensitive and specific method for rapidly detecting C. psittaci and offers technical support for its rapid diagnosis.},
}
@article {pmid38053292,
year = {2023},
author = {Shelake, RM and Pramanik, D and Kim, JY},
title = {CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution.},
journal = {BMB reports},
volume = {},
number = {},
pages = {},
pmid = {38053292},
issn = {1976-670X},
abstract = {Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements.},
}
@article {pmid38053290,
year = {2023},
author = {Oh, GS and An, S and Kim, S},
title = {Harnessing CRISPR-Cas adaptation for RNA recording and beyond.},
journal = {BMB reports},
volume = {},
number = {},
pages = {},
pmid = {38053290},
issn = {1976-670X},
abstract = {Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNAguided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies.},
}
@article {pmid38052872,
year = {2023},
author = {Kulhankova, K and Traore, S and Cheng, X and Benk-Fortin, H and Hallée, S and Harvey, M and Roberge, J and Couture, F and Kohli, S and Gross, TJ and Meyerholz, DK and Rettig, GR and Thommandru, B and Kurgan, G and Wohlford-Lenane, C and Hartigan-O'Connor, DJ and Yates, BP and Newby, GA and Liu, DR and Tarantal, AF and Guay, D and McCray, PB},
title = {Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {8051},
pmid = {38052872},
issn = {2041-1723},
support = {U24 HG010423/HG/NHGRI NIH HHS/United States ; P01 HL152960/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Humans ; Mice ; Macaca mulatta/metabolism ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Epithelial Cells/metabolism ; Respiratory Mucosa/metabolism ; Ribonucleoproteins/metabolism ; Peptides/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.},
}
@article {pmid38052854,
year = {2023},
author = {Ramos, A and Koch, CE and Liu-Lupo, Y and Hellinger, RD and Kyung, T and Abbott, KL and Fröse, J and Goulet, D and Gordon, KS and Eidell, KP and Leclerc, P and Whittaker, CA and Larson, RC and Muscato, AJ and Yates, KB and Dubrot, J and Doench, JG and Regev, A and Vander Heiden, MG and Maus, MV and Manguso, RT and Birnbaum, ME and Hemann, MT},
title = {Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {8048},
pmid = {38052854},
issn = {2041-1723},
support = {R21-AI151827//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; Animals ; Mice ; *Receptors, Chimeric Antigen ; RNA, Guide, CRISPR-Cas Systems ; Immunotherapy, Adoptive ; T-Lymphocytes ; *Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics/therapy ; *Leukemia ; *Burkitt Lymphoma ; Tumor Microenvironment ; },
abstract = {CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.},
}
@article {pmid38051715,
year = {2023},
author = {Balke, I and Silamikelis, I and Radovica-Spalvina, I and Zeltina, V and Resevica, G and Fridmanis, D and Zeltins, A},
title = {Ryegrass mottle virus complete genome determination and development of infectious cDNA by combining two methods- 3' RACE and RNA-Seq.},
journal = {PloS one},
volume = {18},
number = {12},
pages = {e0287278},
pmid = {38051715},
issn = {1932-6203},
mesh = {DNA, Complementary/genetics ; *Lolium/genetics ; RNA-Seq ; Reproducibility of Results ; RNA, Guide, CRISPR-Cas Systems ; *RNA Viruses/genetics ; Genome, Viral ; *Viruses/genetics ; RNA, Viral/genetics ; Open Reading Frames/genetics ; },
abstract = {Ryegrass mottle virus (RGMoV; genus: Sobemovirus) is a single-stranded positive RNA virus with a 30 nm viral particle size. It exhibits T = 3 symmetry with 180 coat protein (CP) subunits forming a viral structure. The RGMoV genome comprises five open reading frames that encode P1, Px, a membrane-anchored 3C-like serine protease, a viral genome-linked protein, P16, an RNA-dependent RNA polymerase, and CP. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in the deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was used to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, viral infection was not induced when the transcribed gRNA was introduced into oat plants, indicating the potential absence of certain sequences in either the 5' or 3' untranslated regions (UTR) or both. The complete sequence of the 3' UTR was determined through 3' end RACE, while the 5' UTR was identified using high-throughput sequencing (HTS)-RNA-Seq to resolve the potential absences. Only the icDNA vector containing the newly identified UTR sequences proved infectious, resulting in typical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infections in oat plants after at least one passage. The successful generation of icDNA highlighted the synergistic potential of utilizing both methods when a single approach failed. Furthermore, this study demonstrated the reliability of HTS as a method for determining the complete genome sequence of viral genomes.},
}
@article {pmid38050977,
year = {2023},
author = {Malla, RR and Middela, K},
title = {CRISPR-Based Approaches for Cancer Immunotherapy.},
journal = {Critical reviews in oncogenesis},
volume = {28},
number = {4},
pages = {1-14},
doi = {10.1615/CritRevOncog.2023048723},
pmid = {38050977},
issn = {0893-9675},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Immunotherapy ; *Neoplasms/genetics/therapy ; DNA ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful gene editing tool that has the potential to revolutionize cancer treatment. It allows for precise and efficient editing of specific genes that drive cancer growth and progression. CRISPR-based approaches gene knock-out, which deletes specific genes or sequences of DNA within a cancer cell, and gene knock-in, which inserts new sequences of DNA into a cancer cell to identify potential targets for cancer therapy. Further, genome-wide CRISPR-Cas9-based screens identify specific markers for diagnosis of cancers. Recently, immunotherapy has become a highly efficient strategy for the treatment of cancer. The use of CRISPR in cancer immunotherapy is focused on enhancing the function of T cells, making them more effective at attacking cancer cells and inactivating the immune evasion mechanisms of cancer cells. It has the potential to generate CAR-T cells, which are T cells that have been genetically engineered to target and attack cancer cells specifically. This review uncovers the latest developments in CRISPR-based gene editing strategies and delivery of their components in cancer cells. In addition, the applications of CRISPR in cancer immune therapy are discussed. Overall, this review helps to explore the potential of CRISPR-based strategies in cancer immune therapy in clinical settings.},
}
@article {pmid38049764,
year = {2023},
author = {Tian, X and Teo, WFA and Wee, WY and Yang, Y and Ahmed, H and Jakubovics, NS and Choo, SW and Tan, GYA},
title = {Genome characterization and taxonomy of Actinomyces acetigenes sp. nov., and Actinomyces stomatis sp. nov., previously isolated from the human oral cavity.},
journal = {BMC genomics},
volume = {24},
number = {1},
pages = {734},
pmid = {38049764},
issn = {1471-2164},
support = {WB20211227000125//Wenzhou Municipal Key Laboratory for Applied Biomedical and the Biopharmaceutical Informatics/ ; WB20210429000008//Zhejiang Bioinformatics International Science and Technology Cooperation Center at Wenzhou-Kean University/ ; 5000105//The high-level talent recruitment program for academic and research platform construction from Wenzhou-Kean University/ ; },
mesh = {Humans ; Female ; *Actinomyces/genetics ; Phylogeny ; Sequence Analysis, DNA ; RNA, Ribosomal, 16S/genetics ; *Mouth ; Nucleic Acid Hybridization ; Nucleotides ; DNA ; DNA, Bacterial/genetics ; Bacterial Typing Techniques ; Fatty Acids/chemistry ; },
abstract = {BACKGROUND: Actinomyces strains are commonly found as part of the normal microflora on human tissue surfaces, including the oropharynx, gastrointestinal tract, and female genital tract. Understanding the diversity and characterization of Actinomyces species is crucial for human health, as they play an important role in dental plaque formation and biofilm-related infections. Two Actinomyces strains ATCC 49340[ T] and ATCC 51655[ T] have been utilized in various studies, but their accurate species classification and description remain unresolved.<br><br>RESULTS: To investigate the genomic properties and taxonomic status of these strains, we employed both 16S rRNA Sanger sequencing and whole-genome sequencing using the Illumina HiSeq X Ten platform with PE151 (paired-end) sequencing. Our analyses revealed that the draft genome of Actinomyces acetigenes ATCC 49340[&#xa0;T] was 3.27 Mbp with a 68.0% GC content, and Actinomyces stomatis ATCC 51655[&#xa0;T] has a genome size of 3.08 Mbp with a 68.1% GC content. Multi-locus (atpA, rpoB, pgi, metG, gltA, gyrA, and core genome SNPs) sequence analysis supported the phylogenetic placement of strains ATCC 51655[&#xa0;T] and ATCC 49340[&#xa0;T] as independent lineages. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) analyses indicated that both strains represented novel Actinomyces species, with values below the threshold for species demarcation (70% dDDH, 95% ANI and AAI). Pangenome analysis identified 5,731 gene clusters with strains ATCC 49340[&#xa0;T] and ATCC 51655[&#xa0;T] possessing 1,515 and 1,518 unique gene clusters, respectively. Additionally, genomic islands (GIs) prediction uncovered 24 putative GIs in strain ATCC 49340[&#xa0;T] and 16 in strain ATCC 51655[&#xa0;T], contributing to their genetic diversity and potential adaptive capabilities. Pathogenicity analysis highlighted the potential human pathogenicity risk associated with both strains, with several virulence-associated factors identified. CRISPR-Cas analysis exposed the presence of CRISPR and Cas genes in both strains, indicating these strains might evolve a robust defense mechanism against them.<br><br>CONCLUSION: This study supports the classification of strains ATCC 49340[&#xa0;T] and ATCC 51655[&#xa0;T] as novel species within the Actinomyces, in which the name Actinomyces acetigenes sp. nov. (type strain ATCC 49340[&#xa0;T]&#x2009;=&#x2009;VPI D163E-3[&#xa0;T]&#x2009;=&#x2009;CCUG 34286[&#xa0;T]&#xa0;=&#x2009;CCUG 35339&#xa0;[T]) and Actinomyces stomatis sp. nov. (type strain ATCC 51655[&#xa0;T]&#x2009;=&#x2009;PK606[T]&#x2009;=&#x2009;CCUG 33930[&#xa0;T]) are proposed.},
}
@article {pmid38049763,
year = {2023},
author = {Tao, S and Hu, C and Fang, Y and Zhang, H and Xu, Y and Zheng, L and Chen, L and Liang, W},
title = {Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system.},
journal = {BMC microbiology},
volume = {23},
number = {1},
pages = {380},
pmid = {38049763},
issn = {1471-2180},
support = {2022z2202022//the Project of the key R &amp; D program of 2022 year of Ningbo Science and Technology Bureau/ ; 2023j020//Key Project of Ningbo Municipal Science and Technology Bureau/ ; },
mesh = {*Vancomycin/pharmacology ; *RNA, Guide, CRISPR-Cas Systems ; Vancomycin Resistance/genetics ; CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; Plasmids/genetics ; Bacterial Proteins/genetics ; },
abstract = {OBJECTIVE: The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.<br><br>METHODS: Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioM&#xe9;rieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.<br><br>RESULTS: In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.<br><br>CONCLUSION: In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.},
}
@article {pmid38049415,
year = {2023},
author = {Yin, DP and Zhang, H and Teng, H and Zhang, D and Chen, P and Xie, L and Liu, JS},
title = {Overexpressed Gαi1 exerts pro-tumorigenic activity in nasopharyngeal carcinoma.},
journal = {Cell death &amp; disease},
volume = {14},
number = {12},
pages = {792},
pmid = {38049415},
issn = {2041-4889},
support = {82002850//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Mice ; Humans ; Nasopharyngeal Carcinoma/genetics/pathology ; *Proto-Oncogene Proteins c-akt/genetics/metabolism ; Mice, Nude ; RNA, Guide, CRISPR-Cas Systems ; Signal Transduction ; *Nasopharyngeal Neoplasms/pathology ; TOR Serine-Threonine Kinases/genetics/metabolism ; Cell Proliferation/genetics ; Transcription Factors/pharmacology ; RNA, Small Interfering/pharmacology ; Cell Line, Tumor ; },
abstract = {The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate that Gαi1 transcripts' number in NPC tissues is significantly higher than that in the normal nasal epithelial tissues. Its overexpression correlates with poor survival in certain NPC patients. Moreover, Gαi1 is significantly upregulated in NPC tissues of local primary patients and in different primary human NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in primary nasal epithelial cells. Genetic silencing (via shRNA strategy) or knockout (via CRISPR-sgRNA method) of Gαi1 substantially suppressed viability, proliferation, cell cycle progression, and migration in primary NPC cells, causing significant caspase-apoptosis activation. Contrarily, ectopic Gαi1 expression exerted pro-tumorigenic activity and strengthened cell proliferation and migration in primary NPC cells. Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated after Gαi1 shRNA or KO in primary NPC cells, but strengthened following Gαi1 overexpression. In Gαi1-silenced primary NPC cells, a S473D constitutively-active mutant Akt1 (caAkt1) restored Akt-S6K phosphorylation and ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Bioinformatics analyses proposed zinc finger protein 384 (ZNF384) as a potential transcription factor of Gαi1. In primary NPC cells, ZNF384 shRNA or knockout (via CRISPR-sgRNA method) decreased Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated it. Importantly, there was an increased binding between ZNF384 protein and the Gαi1 promoter in human NPC tissues and different NPC cells. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing adeno-associated virus (AAV) impeded subcutaneous NPC xenograft growth in nude mice. Gαi1 downregulation, Akt-mTOR inactivation, and apoptosis induction were detected in Gαi1-silenced NPC xenograft tissues. Gαi1 KO also effectively inhibited the growth of NPC xenografts in nude mice. Together, overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation.},
}
@article {pmid38048395,
year = {2023},
author = {Feng, W and Bogomolovas, J and Wang, L and Li, M and Chen, J},
title = {ALPK3 Functions as a Pseudokinase.},
journal = {Circulation},
volume = {148},
number = {23},
pages = {1911-1913},
pmid = {38048395},
issn = {1524-4539},
support = {R01 HL144872/HL/NHLBI NIH HHS/United States ; R01 HL146759/HL/NHLBI NIH HHS/United States ; R01 HL155826/HL/NHLBI NIH HHS/United States ; R01 HL158981/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Protein Kinases/genetics ; Mutation ; *CRISPR-Cas Systems ; },
}
@article {pmid38047242,
year = {2023},
author = {Störtz, F and Mak, JK and Minary, P},
title = {piCRISPR: Physically informed deep learning models for CRISPR/Cas9 off-target cleavage prediction.},
journal = {Artificial intelligence in the life sciences},
volume = {3},
number = {},
pages = {None},
pmid = {38047242},
issn = {2667-3185},
abstract = {CRISPR/Cas programmable nuclease systems have become ubiquitous in the field of gene editing. With progressing development, applications in in vivo therapeutic gene editing are increasingly within reach, yet limited by possible adverse side effects from unwanted edits. Recent years have thus seen continuous development of off-target prediction algorithms trained on in vitro cleavage assay data gained from immortalised cell lines. It has been shown that in contrast to experimental epigenetic features, computed physically informed features are so far underutilised despite bearing considerably larger correlation with cleavage activity. Here, we implement state-of-the-art deep learning algorithms and feature encodings for off-target prediction with emphasis on physically informed features that capture the biological environment of the cleavage site, hence terming our approach piCRISPR. Features were gained from the large, diverse crisprSQL off-target cleavage dataset. We find that our best-performing models highlight the importance of sequence context and chromatin accessibility for cleavage prediction and compare favourably with literature standard prediction performance. We further show that our novel, environmentally sensitive features are crucial to accurate prediction on sequence-identical locus pairs, making them highly relevant for clinical guide design. The source code and trained models can be found ready to use at github.com/florianst/picrispr.},
}
@article {pmid38042139,
year = {2023},
author = {Wang, L and Cao, S and Li, L and Cao, L and Zhao, Z and Huang, T and Li, J and Zhang, X and Li, X and Zhang, N and Wang, X and Gong, P},
title = {Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system.},
journal = {Talanta},
volume = {269},
number = {},
pages = {125413},
doi = {10.1016/j.talanta.2023.125413},
pmid = {38042139},
issn = {1873-3573},
abstract = {Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.},
}
@article {pmid38041553,
year = {2023},
author = {Schmitz, M and Querques, I},
title = {DNA on the move: mechanisms, functions and applications of transposable elements.},
journal = {FEBS open bio},
volume = {},
number = {},
pages = {},
doi = {10.1002/2211-5463.13743},
pmid = {38041553},
issn = {2211-5463},
abstract = {Transposons are mobile genetic elements that have invaded all domains of life by moving between and within their host genomes. Due to their mobility (or transposition), transposons facilitate horizontal gene transfer in bacteria and foster the evolution of new molecular functions in prokaryotes and eukaryotes. As transposition can lead to detrimental genomic rearrangements, organisms have evolved a multitude of molecular strategies to control transposons, including genome defence mechanisms provided by CRISPR-Cas systems. Apart from their biological impacts on genomes, DNA transposons have been leveraged as efficient gene insertion vectors in basic research, transgenesis and gene therapy. However, the close to random insertion profile of transposon-based tools limits their programmability and safety. Despite recent advances brought by the development of CRISPR-associated genome editing nucleases, a strategy for efficient insertion of large, multi-kilobase transgenes at user-defined genomic sites is currently challenging. The discovery and experimental characterization of bacterial CRISPR-associated transposons (CASTs) led to the attractive hypothesis that these systems could be repurposed as programmable, site-specific gene integration technologies. Here, we provide a broad overview of the molecular mechanisms underpinning DNA transposition and of its biological and technological impact. The second focus of the article is to describe recent mechanistic and functional analyses of CAST transposition. Finally, current challenges and desired future advances of CAST-based genome engineering applications are briefly discussed.},
}
@article {pmid38041146,
year = {2023},
author = {Zhang, J and Xu, Y and Wang, M and Li, X and Liu, Z and Kuang, D and Deng, Z and Ou, HY and Qu, J},
title = {Mobilizable plasmids drive the spread of antimicrobial resistance genes and virulence genes in Klebsiella pneumoniae.},
journal = {Genome medicine},
volume = {15},
number = {1},
pages = {106},
pmid = {38041146},
issn = {1756-994X},
support = {2018YFE0102400//National Key Research and Development Program of China/ ; 32070572//National Natural Science Foundation of China/ ; 82000011//National Natural Science Foundation of China/ ; 23YF1436100//Science and Technology Commission of Shanghai Municipality/ ; 19JC1413000//Science and Technology Commission of Shanghai Municipality/ ; },
mesh = {Humans ; *Klebsiella pneumoniae/genetics ; *Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; Escherichia coli/genetics ; Carbapenems ; beta-Lactamases/genetics ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis.<br><br>METHODS: Carbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements.<br><br>RESULTS: Our results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR-Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae.<br><br>CONCLUSIONS: Our work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR-Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.},
}
@article {pmid38038847,
year = {2024},
author = {Cavanaugh, C and Hesson, J and Mathieu, J},
title = {Genomic Engineering of Induced Pluripotent Stem Cell-Derived Cardiomyocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2735},
number = {},
pages = {129-143},
pmid = {38038847},
issn = {1940-6029},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac ; Mutation ; *Cardiomyopathies/genetics ; Genomics ; CRISPR-Cas Systems ; },
abstract = {Recent advances in patient-derived induced Pluripotent Stem Cell (iPSC) generation, improvement of cardiomyocyte-directed differentiation protocols, and the availability of new genome editing techniques have opened up new avenues for disease modeling of cardiomyopathies. Patients with cardiomyopathies often harbor a single-base substitution believed to be linked to the disease phenotype. Somatic cells derived from patients can be efficiently reprogrammed into iPSCs and subsequently engineered. The targeting of a precise mutation can be achieved by the introduction of double stranded breaks with CRISPR-Cas9 and by homology-directed repair when using a DNA donor template. This allows for the correction of a mutation in a patient iPSC line to generate an isogenic control. In addition, key mutations associated with cardiomyopathies can be introduced in an iPSC line derived from a healthy individual using the same techniques. In this chapter, we describe in detail how to engineer pluripotent stem cells to model cardiomyopathy in a dish using CRISPR-Cas9 technology.},
}
@article {pmid38036926,
year = {2023},
author = {Liu, R and Yao, J and Zhou, S and Yang, J and Zhang, Y and Yang, X and Li, L and Zhang, Y and Zhuang, Y and Yang, Y and Chen, X},
title = {Spatiotemporal control of RNA metabolism and CRISPR-Cas functions using engineered photoswitchable RNA-binding proteins.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {38036926},
issn = {1750-2799},
abstract = {RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.},
}
@article {pmid38036097,
year = {2023},
author = {Becker, HJ and Yamazaki, S},
title = {Understanding Genetic Heterogeneity in Gene-Edited HSC Products.},
journal = {Experimental hematology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.exphem.2023.11.007},
pmid = {38036097},
issn = {1873-2399},
abstract = {CRISPR/Cas gene editing has transformed genetic research and is poised to drive the next generation of gene therapies targeting hematopoietic stem cells (HSCs). However, the installation of the 'desired' edit is most often only achieved in a minor subset of alleles. The array of cellular pathways triggered by gene editing tools produces a broad spectrum of 'undesired' editing outcomes, including short insertions and deletions (indels) and chromosome rearrangements, leading to considerable genetic heterogeneity in gene edited HSC populations. This heterogeneity may undermine the effect of the genetic intervention, since only a subset of cells will carry the intended modification. Also, undesired mutations represent a potential safety concern as gene editing advances toward broader clinical use. Here, we will review the different sources of 'undesired' edits and will discuss strategies for their mitigation and control.},
}
@article {pmid38034561,
year = {2023},
author = {Nadi, R and Juan-Vicente, L and Mateo-Bonmatí, E and Micol, JL},
title = {The unequal functional redundancy of Arabidopsis INCURVATA11 and CUPULIFORMIS2 is not dependent on genetic background.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1239093},
pmid = {38034561},
issn = {1664-462X},
abstract = {The paralogous genes INCURVATA11 (ICU11) and CUPULIFORMIS2 (CP2) encode components of the epigenetic machinery in Arabidopsis and belong to the 2-oxoglutarate and Fe (II)-dependent dioxygenase superfamily. We previously inferred unequal functional redundancy between ICU11 and CP2 from a study of the synergistic phenotypes of the double mutant and sesquimutant combinations of icu11 and cp2 mutations, although they represented mixed genetic backgrounds. To avoid potential confounding effects arising from different genetic backgrounds, we generated the icu11-5 and icu11-6 mutants via CRISPR/Cas genome editing in the Col-0 background and crossed them to cp2 mutants in Col-0. The resulting mutants exhibited a postembryonic-lethal phenotype reminiscent of strong embryonic flower (emf) mutants. Double mutants involving icu11-5 and mutations affecting epigenetic machinery components displayed synergistic phenotypes, whereas cp2-3 did not besides icu11-5. Our results confirmed the unequal functional redundancy between ICU11 and CP2 and demonstrated that it is not allele or genetic background specific. An increase in sucrose content in the culture medium partially rescued the post-germinative lethality of icu11 cp2 double mutants and sesquimutants, facilitating the study of their morphological phenotypes throughout their life cycle, which include floral organ homeotic transformations. We thus established that the ICU11-CP2 module is required for proper flower organ identity.},
}
@article {pmid38034032,
year = {2023},
author = {Cavazza, A and Hendel, A and Bak, RO and Rio, P and Güell, M and Lainšček, D and Arechavala-Gomeza, V and Peng, L and Hapil, FZ and Harvey, J and Ortega, FG and Gonzalez-Martinez, C and Lederer, CW and Mikkelsen, K and Gasiunas, G and Kalter, N and Gonçalves, MAFV and Petersen, J and Garanto, A and Montoliu, L and Maresca, M and Seemann, SE and Gorodkin, J and Mazini, L and Sanchez, R and Rodriguez-Madoz, JR and Maldonado-Pérez, N and Laura, T and Schmueck-Henneresse, M and Maccalli, C and Grünewald, J and Carmona, G and Kachamakova-Trojanowska, N and Miccio, A and Martin, F and Turchiano, G and Cathomen, T and Luo, Y and Tsai, SQ and Benabdellah, K and , },
title = {Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.},
journal = {Molecular therapy. Nucleic acids},
volume = {34},
number = {},
pages = {102066},
pmid = {38034032},
issn = {2162-2531},
abstract = {The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.},
}
@article {pmid38033326,
year = {2023},
author = {Jungfer, K and Sigg, A and Jinek, M},
title = {Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad1102},
pmid = {38033326},
issn = {1362-4962},
support = {ERC-CoG-820152/ERC_/European Research Council/International ; },
abstract = {Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications.},
}
@article {pmid37996748,
year = {2023},
author = {Reardon, S},
title = {'Treasure trove' of new CRISPR systems holds promise for genome editing.},
journal = {Nature},
volume = {624},
number = {7990},
pages = {17-18},
pmid = {37996748},
issn = {1476-4687},
mesh = {*Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37905414,
year = {2023},
author = {Kim, EP and Kim, DY and Park, C and Yoo, SM and Lee, MS and Kim, GA},
title = {Effects of klotho protein or klotho knockdown in porcine oocytes at different stages.},
journal = {Zygote (Cambridge, England)},
volume = {31},
number = {6},
pages = {577-581},
doi = {10.1017/S096719942300045X},
pmid = {37905414},
issn = {1469-8730},
mesh = {Pregnancy ; Animals ; Female ; Swine ; *RNA, Guide, CRISPR-Cas Systems ; *Oocytes/physiology ; Blastocyst ; Embryonic Development/genetics ; Parthenogenesis ; },
abstract = {Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.},
}
@article {pmid36973619,
year = {2023},
author = {Khanna, K and Ohri, P and Bhardwaj, R},
title = {Nanotechnology and CRISPR/Cas9 system for sustainable agriculture.},
journal = {Environmental science and pollution research international},
volume = {30},
number = {56},
pages = {118049-118064},
pmid = {36973619},
issn = {1614-7499},
mesh = {*CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Agriculture ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), a genome editing tool, has gained a tremendous position due to its therapeutic efficacy, ability to counteract abiotic/biotic stresses in plants, environmental remediation and sustainable agriculture with the aim of food security. This is mainly due to their potential of precised genome modification and numerous genetic engineering protocols with versatility as well as simplicity. This technique is quite useful for crop refinement and overcoming the agricultural losses and regaining the soil fertility hampered by hazardous chemicals. Since CRISPR/Cas9 has been widely accepted in genome editing in plants, however, their revolutionised nature and progress enable genetic engineers to face numerous challenges in plant biotechnology. Therefore, nanoparticles have addressed these challenges and improved cargo delivery and genomic editing processes. Henceforth, this barrier prevents CRISPR-based genetic engineering in plants in order to show efficacy in full potential and eliminate all the barriers. This advancement accelerates the genome editing process and its applications in plant biotechnology enable us to sustain and feed the massive population under varying environments. Genome editing tools using CRISPR/Cas9 and nanotechnology are advantageous that produce transgenic-free plants that overcome global food demands. Here, in this review, we have aimed towards the mechanisms/delivery systems linked with CRISPR/Cas9 system. We have elaborated on the applications of CRISPR/Cas9 and nanotechnology-based systems for sustainable agriculture. Moreover, the challenges and limitations associated with genome editing and delivery systems have also been discussed with a special emphasis on crop improvement.},
}
@article {pmid38033317,
year = {2023},
author = {Sinha, S and Molina Vargas, AM and Arantes, PR and Patel, A and O'Connell, MR and Palermo, G},
title = {Unveiling the RNA-mediated allosteric activation discloses functional hotspots in CRISPR-Cas13a.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad1127},
pmid = {38033317},
issn = {1362-4962},
support = {R35 GM133462/GM/NIGMS NIH HHS/United States ; /NH/NIH HHS/United States ; R01GM141329/NH/NIH HHS/United States ; },
abstract = {Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.},
}
@article {pmid38033086,
year = {2023},
author = {Mogila, I and Tamulaitiene, G and Keda, K and Timinskas, A and Ruksenaite, A and Sasnauskas, G and Venclovas, &#x10c; and Siksnys, V and Tamulaitis, G},
title = {Ribosomal stalk-captured CARF-RelE ribonuclease inhibits translation following CRISPR signaling.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6674},
pages = {1036-1041},
doi = {10.1126/science.adj2107},
pmid = {38033086},
issn = {1095-9203},
mesh = {*Ribonucleases/chemistry ; Signal Transduction ; Bacteria/genetics ; Bacterial Proteins/chemistry ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry ; },
abstract = {Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cAn) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cAn-dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA4), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.},
}
@article {pmid38032088,
year = {2023},
author = {Nagorska, A and Zaucker, A and Lambert, F and Inman, A and Toral-Perez, S and Gorodkin, J and Wan, Y and Smutny, M and Sampath, K},
title = {Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.},
journal = {Development (Cambridge, England)},
volume = {150},
number = {23},
pages = {},
doi = {10.1242/dev.201657},
pmid = {38032088},
issn = {1477-9129},
support = {/WT_/Wellcome Trust/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Humans ; *Zebrafish ; 3' Untranslated Regions ; *Regulon/genetics ; Morphogenesis/genetics ; Heart Valves ; Zebrafish Proteins/genetics/metabolism ; Proprotein Convertases/genetics/metabolism ; },
abstract = {Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.},
}
@article {pmid38028586,
year = {2023},
author = {Misra, V and Mall, AK and Pandey, H and Srivastava, S and Sharma, A},
title = {Advancements and prospects of CRISPR/Cas9 technologies for abiotic and biotic stresses in sugar beet.},
journal = {Frontiers in genetics},
volume = {14},
number = {},
pages = {1235855},
pmid = {38028586},
issn = {1664-8021},
abstract = {Sugar beet is a crop with high sucrose content, known for sugar production and recently being considered as an emerging raw material for bioethanol production. This crop is also utilized as cattle feed, mainly when animal green fodder is scarce. Bioethanol and hydrogen gas production from this crop is an essential source of clean energy. Environmental stresses (abiotic/biotic) severely affect the productivity of this crop. Over the past few decades, the molecular mechanisms of biotic and abiotic stress responses in sugar beet have been investigated using next-generation sequencing, gene editing/silencing, and over-expression approaches. This information can be efficiently utilized through CRISPR/Cas 9 technology to mitigate the effects of abiotic and biotic stresses in sugar beet cultivation. This review highlights the potential use of CRISPR/Cas 9 technology for abiotic and biotic stress management in sugar beet. Beet genes known to be involved in response to alkaline, cold, and heavy metal stresses can be precisely modified via CRISPR/Cas 9 technology for enhancing sugar beet's resilience to abiotic stresses with minimal off-target effects. Similarly, CRISPR/Cas 9 technology can help generate insect-resistant sugar beet varieties by targeting susceptibility-related genes, whereas incorporating Cry1Ab and Cry1C genes may provide defense against lepidopteron insects. Overall, CRISPR/Cas 9 technology may help enhance sugar beet's adaptability to challenging environments, ensuring sustainable, high-yield production.},
}
@article {pmid38028200,
year = {2023},
author = {Budzko, L and Hoffa-Sobiech, K and Jackowiak, P and Figlerowicz, M},
title = {Engineered deaminases as a key component of DNA and RNA editing tools.},
journal = {Molecular therapy. Nucleic acids},
volume = {34},
number = {},
pages = {102062},
pmid = {38028200},
issn = {2162-2531},
abstract = {Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at specific sites in either DNA or RNA by combining a targeting module (such as a catalytically impaired CRISPR-Cas component) and an effector module (most often a deaminase). Deaminase-based molecular tools are already being utilized in a wide spectrum of therapeutic and research applications; however, their medical and biotechnological potential seems to be much greater. Recent reports indicate that the further development of nucleic acid editing systems depends largely on our ability to engineer the substrate specificity and catalytic activity of the editors themselves. In this review, we summarize the current trends and achievements in deaminase engineering. The presented data indicate that the potential of these enzymes has not yet been fully revealed or understood. Several examples show that even relatively minor changes in the structure of deaminases can give them completely new and unique properties.},
}
@article {pmid37995212,
year = {2023},
author = {Papkou, A and Garcia-Pastor, L and Escudero, JA and Wagner, A},
title = {A rugged yet easily navigable fitness landscape.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6673},
pages = {eadh3860},
doi = {10.1126/science.adh3860},
pmid = {37995212},
issn = {1095-9203},
mesh = {*Genetic Fitness ; Models, Genetic ; Mutation ; *Tetrahydrofolate Dehydrogenase/chemistry/genetics ; *Escherichia coli Proteins/chemistry/genetics ; Gene Editing ; CRISPR-Cas Systems ; Selection, Genetic ; Computer Simulation ; },
abstract = {Fitness landscape theory predicts that rugged landscapes with multiple peaks impair Darwinian evolution, but experimental evidence is limited. In this study, we used genome editing to map the fitness of >260,000 genotypes of the key metabolic enzyme dihydrofolate reductase in the presence of the antibiotic trimethoprim, which targets this enzyme. The resulting landscape is highly rugged and harbors 514 fitness peaks. However, its highest peaks are accessible to evolving populations via abundant fitness-increasing paths. Different peaks share large basins of attraction that render the outcome of adaptive evolution highly contingent on chance events. Our work shows that ruggedness need not be an obstacle to Darwinian evolution but can reduce its predictability. If true in general, the complexity of optimization problems on realistic landscapes may require reappraisal.},
}
@article {pmid37202920,
year = {2023},
author = {Ma, YF and Zhang, MQ and Gong, LL and Liu, XZ and Long, GJ and Guo, H and Hull, JJ and Dewer, Y and He, M and He, P},
title = {Efficient nanoparticle-based CRISPR-Cas13d induced mRNA disruption of an eye pigmentation gene in the white-backed planthopper, Sogatella furcifera.},
journal = {Insect science},
volume = {30},
number = {6},
pages = {1552-1564},
doi = {10.1111/1744-7917.13203},
pmid = {37202920},
issn = {1744-7917},
support = {31860617//National&#xa0; Natural Science Foundation of China/ ; 32260671//National&#xa0; Natural Science Foundation of China/ ; 2017-33//Scientific Research&#xa0; Foundation of Guizhou University of China/ ; [2019]05//Program of&#xa0; Talent Cultivation of Guizhou University/ ; QKH[2017]2956//National Natural&#xa0; Science Foundation of China/ ; 111Program,D20023//Program of Introducing Talents&#xa0; of Discipline to Universities of China/ ; QianjiaoheKYnumber(2020)004//Frontiers Science Center&#xa0; for Asymmetric Synthesis and Medicinal Molecules, Department of Education, Guizhou Province/ ; QKH[2017]2956//Science and Technology Support of Guizhou province/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; RNA, Messenger/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Hemiptera/genetics ; RNA/genetics ; Insecta/genetics ; Pigmentation/genetics ; *Nanoparticles ; },
abstract = {The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) system has driven gene manipulation technology to a new era with applications reported in organisms that span the tree of life. The utility of CRISPR-mediated editing was further expanded to mRNA following identification of the RNA-targeting Cas13 family of smaller endonuclease proteins. Application of this family to insect research, however, has been more limited. In this study, the smallest Cas13 family member, Cas13d, and guide RNAs (gRNAs) were complexed with a versatile nanomaterial (star polycation, SPc) to generate a proof-of-concept RNA-editing platform capable of disrupting mRNA expression of the eye pigmentation gene tryptophan 2,3-dioxygenase (SfTO) in white-backed planthoppers (WBPHs). The resulting red-eye phenotype was present in 19.76% (with SPc) and 22.99% (without SPc) of the treatment groups and was comparable to the red-eye phenotype generated following conventional RNA interference knockdown (22.22%). Furthermore, the Cas13/gRNA phenotype manifested more quickly than RNA interference. Consistent with the expected Cas13d mechanism, SfTO transcript levels were significantly reduced. Taken together, the results indicate that the SPc-CRISPR-Cas13d/gRNA complex negatively impacted expression of the target gene. These findings confirm the utility of this novel mRNA disruption system in insects and lay the foundation for further development of these tools in the implementation of green agricultural pest management tactics.},
}
@article {pmid36413117,
year = {2023},
author = {Martin, PB and Holbrook, SE and Hicks, AN and Hines, TJ and Bogdanik, LP and Burgess, RW and Cox, GA},
title = {Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.},
journal = {Human molecular genetics},
volume = {32},
number = {8},
pages = {1276-1288},
pmid = {36413117},
issn = {1460-2083},
support = {R01 NS102414/NS/NINDS NIH HHS/United States ; R24 NS098523/NH/NIH HHS/United States ; },
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Alleles ; Amino Acid Substitution ; Axons/metabolism/pathology ; *Charcot-Marie-Tooth Disease/classification/complications/genetics ; CRISPR-Cas Systems ; *Disease Models, Animal ; Disease Progression ; Gene Knock-In Techniques ; Mice, Inbred C57BL ; Motor Neurons/metabolism/pathology ; Motor Skills Disorders/complications ; Muscle Weakness/complications ; Mutation ; Myelin Sheath/metabolism ; Neuromuscular Junction/metabolism/pathology ; Reproducibility of Results ; Sensory Receptor Cells/metabolism/pathology ; Weight Loss ; },
abstract = {Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.},
}
@article {pmid37995578,
year = {2023},
author = {Bi, M and Wang, Z and Cheng, K and Cui, Y and He, Y and Ma, J and Qi, M},
title = {Construction of transcription factor mutagenesis population in tomato using a pooled CRISPR/Cas9 plasmid library.},
journal = {Plant physiology and biochemistry : PPB},
volume = {205},
number = {},
pages = {108094},
doi = {10.1016/j.plaphy.2023.108094},
pmid = {37995578},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Solanum lycopersicum/genetics ; Transcription Factors/genetics ; Mutagenesis ; Plasmids ; },
abstract = {Adequate mutant materials are the prerequisite for conducting gene function research or screening novel functional genes in plants. The strategy of constructing a large-scale mutant population using the pooled CRISPR/Cas9-sgRNA library has been implemented in several crops. However, the effective application of this CRISPR/Cas9 large-scale screening strategy to tomato remains to be attempted. Here, we identified 990 transcription factors in the tomato genome, designed and synthesized a CRISPR/Cas9 plasmid library containing 4379 sgRNAs. Using this pooled library, 487 T0 positive plants were obtained, among which 92 plants harbored a single sgRNA sequence, targeting 65 different transcription factors, with a mutation rate of 23%. In the T0 mutant population, the occurrence of homozygous and biallelic mutations was observed at higher frequencies. Additionally, the utilization of a small-scale CRISPR/Cas9 library targeting 30 transcription factors could enhance the efficacy of single sgRNA recognition in positive plants, increasing it from 19% to 42%. Phenotypic characterization of several mutants identified from the mutant population demonstrated the utility of our CRISPR/Cas9 mutant library. Taken together, our study offers insights into the implementation and optimization of CRISPR/Cas9-mediated large-scale knockout library in tomato.},
}
@article {pmid37790407,
year = {2023},
author = {Bell, RT and Sahakyan, H and Makarova, KS and Wolf, YI and Koonin, EV},
title = {CoCoNuTs: A diverse subclass of Type IV restriction systems predicted to target RNA.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.07.31.551357},
pmid = {37790407},
abstract = {A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote CoCoNuTs (coiled-coil nuclease tandems) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with 3 distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial tmRNA, YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. The CoCoNuTs, together with the ancillary restriction factors, might employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.},
}
@article {pmid37767888,
year = {2023},
author = {Liao, T and Xu, X and Wu, J and Xie, Y and Yan, J},
title = {Increased expression levels of DLX5 inhibit the development of the nervous system.},
journal = {International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience},
volume = {83},
number = {8},
pages = {728-739},
doi = {10.1002/jdn.10300},
pmid = {37767888},
issn = {1873-474X},
support = {2020Y9134//Joint Funds for the Innovation of Science and Technology, Fujian Province/ ; 2020Y9161//Joint Funds for the Innovation of Science and Technology, Fujian Province/ ; 82001575//National Natural Science Foundation of China/ ; 2020J01334//Natural Science Foundation of Fujian Province of China/ ; },
mesh = {Animals ; Humans ; Female ; *Homeodomain Proteins/genetics/metabolism ; Zebrafish/metabolism ; *Pre-Eclampsia ; RNA, Guide, CRISPR-Cas Systems ; Brain/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {INTRODUCTION: Preeclampsia is a hypertensive disorder of pregnancy. DLX5 plays an important role in the migration and differentiation of subglobus pallidus precursor cells.<br><br>METHODS: We established a zebrafish line expressing high levels of DLX5 and investigated changes in behavior and development of the nervous system.<br><br>RESULTS: The ratios of brain volume area to whole body area at 96&#x2009;hpf zebrafish in the experimental group (gRNA&#x2009;+&#x2009;CasRx) were significantly lower than the WT group and the negative control group (casRx) (P&#x2009;<&#x2009;0.01). Behavioral trajectory distances and movement speeds exhibited by the 6th day of development in zebrafish in the experimental group (gRNA&#x2009;+&#x2009;CasRx) were significantly shorter (P&#x2009;<&#x2009;0.01) and lower (P&#x2009;<&#x2009;0.05) than the negative control group (gRNA&#x2009;+&#x2009;CasRx), respectively.<br><br>CONCLUSIONS: Data suggested that the increased expression levels of DLX5 can inhibit brain volume development and behavioral activities in zebrafish. Maybe the high expression levels of DLX5 in the pathological state of preeclampsia can inhibit the development of the nervous system in offspring.},
}
@article {pmid38023829,
year = {2023},
author = {Suprasanna, P and Klimek-Chodacka, M and Jain, SM},
title = {Editorial: CRISPR tools, technology development, and application.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1329780},
pmid = {38023829},
issn = {1664-462X},
}
@article {pmid38020120,
year = {2023},
author = {Zhang, S and Wang, Y and Mao, D and Wang, Y and Zhang, H and Pan, Y and Wang, Y and Teng, S and Huang, P},
title = {Current trends of clinical trials involving CRISPR/Cas systems.},
journal = {Frontiers in medicine},
volume = {10},
number = {},
pages = {1292452},
pmid = {38020120},
issn = {2296-858X},
abstract = {The CRISPR/Cas9 system is a powerful genome editing tool that has made enormous impacts on next-generation molecular diagnostics and therapeutics, especially for genetic disorders that traditional therapies cannot cure. Currently, CRISPR-based gene editing is widely applied in basic, preclinical, and clinical studies. In this review, we attempt to identify trends in clinical studies involving CRISPR techniques to gain insights into the improvement and contribution of CRISPR/Cas technologies compared to traditional modified modalities. The review of clinical trials is focused on the applications of the CRISPR/Cas systems in the treatment of cancer, hematological, endocrine, and immune system diseases, as well as in diagnostics. The scientific basis underlined is analyzed. In addition, the challenges of CRISPR application in disease therapies and recent advances that expand and improve CRISPR applications in precision medicine are discussed.},
}
@article {pmid38019812,
year = {2023},
author = {Maltseva, EA and Vasil'eva, IA and Moor, NA and Kim, DV and Dyrkheeva, NS and Kutuzov, MM and Vokhtantsev, IP and Kulishova, LM and Zharkov, DO and Lavrik, OI},
title = {Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.},
journal = {PloS one},
volume = {18},
number = {11},
pages = {e0294683},
pmid = {38019812},
issn = {1932-6203},
mesh = {Humans ; *CRISPR-Cas Systems ; *Poly(ADP-ribose) Polymerases/genetics/metabolism ; DNA Repair ; DNA Damage ; DNA/genetics/metabolism ; DNA Breaks ; RNA ; },
abstract = {CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.},
}
@article {pmid38019742,
year = {2023},
author = {Blicharska, D and Szućko-Kociuba, I and Filip, E and Orłowska, A and Skuza, L},
title = {[PRIME EDITING - a new method of editing genes].},
journal = {Postepy biochemii},
volume = {69},
number = {3},
pages = {146-158},
doi = {10.18388/pb.2021_494},
pmid = {38019742},
issn = {0032-5422},
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Genome ; DNA ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {The Prime Editing method introduces the expected manipulations within a given genome with a Cas9-nicase and pegRNA structure and a reverse transcriptase, which is responsible for the synthesis of the segment, which is then incorporated into the edited strand. This technique is based on the previously discovered CRISPR/Cas9 method. It differs from CRISPR/Cas9 in the absence of double cracks within the DNA helix, which is due to its complex structure, including the presence of additional elements, i. e. the reverse transcriptase and the matrix within the pegRNA. PE is used to modify the DNA double helix. The work deals mainly with the creation and improvement as well as testing of the modern Prime Editing method. Information on the structure and functioning of the system is provided, as well as the research carried out so far with the use of PE, carried out within the genomes of cells derived from plant, animal, and human organisms, is described. The paper also contains information on the potential benefits and hopes related to the use of this innovative method.},
}
@article {pmid37995623,
year = {2024},
author = {Zhang, T and Xie, Z and Zheng, X and Liang, Y and Lu, Y and Zhong, H and Qian, F and Zhu, Y and Sun, R and Sheng, Y and Hu, J},
title = {CRISPR-Cas12a powered hybrid nanoparticle for extracellular vesicle aggregation and in-situ microRNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {245},
number = {},
pages = {115856},
doi = {10.1016/j.bios.2023.115856},
pmid = {37995623},
issn = {1873-4235},
mesh = {Humans ; *MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; *Nanoparticles ; *Extracellular Vesicles/genetics ; },
abstract = {Efficient extracellular vesicle (EV) enrichment and timely internal RNA detection for cancer diagnostics are highly desirable and remain a challenge. Here, we report a rapid EV aggregation induced in-situ microRNA detection technology based on cationic lipid-polymer hybrid nanoparticles encapsulating cascade system of catalytic hairpin assembly and CRISPR-Cas12a (CLHN-CCC), allowing for EV enrichment in three-dimensional space and in-situ detection of internal microRNAs in one step within 30 min. The enrichment efficiency (>90%) of CLHN-CCC is demonstrated in artificial EVs, cell-secreted EVs and serum EVs, which is 5-fold higher than that of traditional ultracentrifugation. The sensitive detection of artificial EVs and internal miR-1290 was achieved with the limit of detection of 10 particles/μL and 0.07 amol, respectively. After lyophilization, CLHN-CCC shows no obvious loss of performance within 6 months, making it much more robust and user friendly. This technique could sensitively (sensitivity = 92.9%) and selectively (selectivity = 85.7%) identify low amount miR-1290 in serum EVs, distinguishing early-stage pancreatic cancer patients from healthy subjects, showing high potential for clinical applications.},
}
@article {pmid37916874,
year = {2023},
author = {Canarutto, D and Asperti, C and Vavassori, V and Porcellini, S and Rovelli, E and Paulis, M and Ferrari, S and Varesi, A and Fiumara, M and Jacob, A and Sergi Sergi, L and Visigalli, I and Ferrua, F and González-Granado, LI and Lougaris, V and Finocchi, A and Villa, A and Radrizzani, M and Naldini, L},
title = {Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4[+] T-cells for the treatment of Hyper IgM1.},
journal = {The EMBO journal},
volume = {42},
number = {23},
pages = {e114188},
pmid = {37916874},
issn = {1460-2075},
support = {JTC-2017/EDSCID//E-Rare/ ; TTPGE0322TT//Fondazione Telethon ETS/ ; GENESPIRE.E//Genespire Srl/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; T-Lymphocytes ; CD4-Positive T-Lymphocytes ; },
abstract = {Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4[+] T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.},
}
@article {pmid37910509,
year = {2023},
author = {Wang, J and Lin, J and Chen, Y and Liu, J and Zheng, Q and Deng, M and Wang, R and Zhang, Y and Feng, S and Xu, Z and Ye, W and Hu, Y and Duan, J and Lin, Y and Dai, J and Chen, Y and Li, Y and Luo, T and Chen, Q and Lu, Z},
title = {An ultra-compact promoter drives widespread neuronal expression in mouse and monkey brains.},
journal = {Cell reports},
volume = {42},
number = {11},
pages = {113348},
doi = {10.1016/j.celrep.2023.113348},
pmid = {37910509},
issn = {2211-1247},
mesh = {Mice ; Animals ; Humans ; *RNA, Guide, CRISPR-Cas Systems ; *Neurons/metabolism ; Brain/metabolism ; Neuroglia/metabolism ; Genetic Therapy ; Genetic Vectors/genetics ; Dependovirus/genetics/metabolism ; },
abstract = {Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type selectivity. However, whether ultra-compact promoters can offer neuron-specific expression is unclear. Here, we report the development of an extremely short promoter that enables selective gene expression in neurons, but not glial cells, in the brain. The promoter sequence originates from the human CALM1 gene and is only 120 bp in size. The CALM1 promoter (pCALM1) embedded in an adeno-associated virus (AAV) genome directed broad reporter expression in excitatory and inhibitory neurons in mouse and monkey brains. Moreover, pCALM1, when inserted into an all-in-one AAV vector expressing SpCas9 and sgRNA, drives constitutive and conditional in vivo gene editing in neurons and elicits functional alterations. These data demonstrate the ability of pCALM1 to conduct restricted neuronal gene expression, illustrating the feasibility of ultra-miniature promoters for targeting brain-cell subtypes.},
}
@article {pmid37906593,
year = {2023},
author = {Qiu, H and Li, G and Yuan, J and Yang, D and Ma, Y and Wang, F and Dai, Y and Chang, X},
title = {Efficient exon skipping by base-editor-mediated abrogation of exonic splicing enhancers.},
journal = {Cell reports},
volume = {42},
number = {11},
pages = {113340},
doi = {10.1016/j.celrep.2023.113340},
pmid = {37906593},
issn = {2211-1247},
mesh = {Humans ; *Dystrophin/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics/metabolism ; RNA Splicing/genetics ; Exons/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.},
}
@article {pmid36905356,
year = {2023},
author = {Liu, J and Chen, Y and Nong, B and Luo, X and Cui, K and Li, Z and Zhang, P and Tan, W and Yang, Y and Ma, W and Liang, P and Songyang, Z},
title = {CRISPR-assisted transcription activation by phase-separation proteins.},
journal = {Protein &amp; cell},
volume = {14},
number = {12},
pages = {874-887},
pmid = {36905356},
issn = {1674-8018},
mesh = {Humans ; Transcriptional Activation ; *RNA, Guide, CRISPR-Cas Systems ; *Gene Expression Regulation ; CRISPR-Cas Systems/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.},
}
@article {pmid38017546,
year = {2023},
author = {Hao, W and Chen, Z and Tang, J and Yang, R and Gao, WQ and Xu, H},
title = {hnRNPA2B1 promotes the occurrence and progression of hepatocellular carcinoma by downregulating PCK1 mRNA via a m6A RNA methylation manner.},
journal = {Journal of translational medicine},
volume = {21},
number = {1},
pages = {861},
pmid = {38017546},
issn = {1479-5876},
support = {81630073//National Natural Science Foundation of China/ ; 81972343//National Natural Science Foundation of China/ ; 31571399//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Humans ; Mice ; Carcinogenesis/genetics ; *Carcinoma, Hepatocellular/pathology ; Cell Line, Tumor ; Intracellular Signaling Peptides and Proteins/metabolism ; *Liver Neoplasms/pathology ; Methylation ; Phosphoenolpyruvate Carboxykinase (GTP)/genetics/metabolism ; RNA/metabolism ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/genetics ; },
abstract = {BACKGROUND: N6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.<br><br>METHODS: Multiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.<br><br>RESULTS: hnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.<br><br>CONCLUSION: Our study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.},
}
@article {pmid38017385,
year = {2023},
author = {Yang, Y and Mei, H and Han, X and Zhang, X and Cheng, J and Zhang, Z and Wang, H and Xu, H},
title = {Synthetic CRISPR/dCas9-KRAB system driven by specific PSA promoter suppresses malignant biological behavior of prostate cancer cells through negative feedback inhibition of PSA expression.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {96},
pmid = {38017385},
issn = {1689-1392},
support = {81902557//National Natural Science Foundation of China/ ; 2019YFA0906000//National Key R&amp;D Program of China/ ; SZSM202111007//Sanming Project of Medicine in Shenzhen/ ; SZXK020//Shenzhen Key Medical Discipline Construction Fund/ ; JCYJ20190806164616292//Shenzhen Municipal Science and Technology Innovation Council/ ; },
mesh = {Humans ; Male ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Prostate-Specific Antigen/genetics ; Prostate ; RNA, Guide, CRISPR-Cas Systems ; Feedback ; *Prostatic Neoplasms/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {PSA is a type of proto-oncogene that is specifically and highly expressed in embryonic and prostate cancer cells, but not expressed in normal prostate tissue cells. The specific expression of prostate-specific antigen (PSA) is found to be related with the conditional transcriptional regulation of its promoter. Clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-KRAB is a newly developed transcriptional regulatory system that inhibits gene expression by interupting the DNA transcription process. Induction of CRISPR-dCas9-KRAB expression through the PSA promoter may help feedback inhibition of cellular PSA gene expression via single guide RNA (sgRNA), thereby monitoring and suppressing the malignant state of tumor cells. In this study, we examined the transcriptional activity of the PSA promoter in different prostate cancer cells and normal prostate epithelial cells and determined that it is indeed a prostate cancer cell-specific promoter.Then we constructed the CRISPR-dCas9-KRAB system driven by the PSA promoter, which can inhibit PSA gene expression in the prostate cancer cells at the transcriptional level, and therefore supress the malignant growth and migration of prostate cancer cells and promote their apoptosis in vitro. This study provides a potentially effective anti-cancer strategy for gene therapy of prostate cancer.},
}
@article {pmid38016350,
year = {2023},
author = {Tan, J and Shen, M and Chai, N and Liu, Q and Liu, YG and Zhu, Q},
title = {Genome editing for plant synthetic metabolic engineering and developmental regulation.},
journal = {Journal of plant physiology},
volume = {291},
number = {},
pages = {154141},
doi = {10.1016/j.jplph.2023.154141},
pmid = {38016350},
issn = {1618-1328},
abstract = {Plant metabolism and development are a reflection of the orderly expression of genetic information intertwined with the environment interactions. Genome editing is the cornerstone for scientists to modify endogenous genes or introduce exogenous functional genes and metabolic pathways, holding immense potential applications in molecular breeding and biosynthesis. Over the course of nearly a decade of development, genome editing has advanced significantly beyond the simple cutting of double-stranded DNA, now enabling precise base and fragment replacements, regulation of gene expression and translation, as well as epigenetic modifications. However, the utilization of genome editing in plant synthetic metabolic engineering and developmental regulation remains exploratory. Here, we provide an introduction and a comprehensive overview of the editing attributes associated with various CRISPR/Cas tools, along with diverse strategies for the meticulous control of plant metabolic pathways and developments. Furthermore, we discuss the limitations of current approaches and future prospects for genome editing-driven plant breeding.},
}
@article {pmid38015466,
year = {2023},
author = {Wimmer, F and Englert, F and Wandera, KG and Alkhnbashi, OS and Collins, SP and Backofen, R and Beisel, CL},
title = {Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad1097},
pmid = {38015466},
issn = {1362-4962},
support = {BA 2168/11-2//Deutsche Forschungsgemeinschaft/ ; CIBSS - EXC-2189//Germany's Excellence Strategy/ ; },
abstract = {CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.},
}
@article {pmid37990877,
year = {2023},
author = {Fadul, SM and Arshad, A and Mehmood, R},
title = {CRISPR-based epigenome editing: mechanisms and applications.},
journal = {Epigenomics},
volume = {15},
number = {21},
pages = {1137-1155},
doi = {10.2217/epi-2023-0281},
pmid = {37990877},
issn = {1750-192X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Epigenome ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Epigenomic anomalies contribute significantly to the development of numerous human disorders. The development of epigenetic research tools is essential for understanding how epigenetic marks contribute to gene expression. A gene-editing technique known as CRISPR (clustered regularly interspaced short palindromic repeats) typically targets a particular DNA sequence using a guide RNA (gRNA). CRISPR/Cas9 technology has been remodeled for epigenome editing by generating a 'dead' Cas9 protein (dCas9) that lacks nuclease activity and juxtaposing it with an epigenetic effector domain. Based on fusion partners of dCas9, a specific epigenetic state can be achieved. CRISPR-based epigenome editing has widespread application in drug screening, cancer treatment and regenerative medicine. This paper discusses the tools developed for CRISPR-based epigenome editing and their applications.},
}
@article {pmid38013928,
year = {2022},
author = {Deol, P and Madhwal, A and Sharma, G and Kaushik, R and Malik, YS},
title = {CRISPR use in diagnosis and therapy for COVID-19.},
journal = {Methods in microbiology},
volume = {50},
number = {},
pages = {123-150},
pmid = {38013928},
issn = {0580-9517},
abstract = {Since the beginning of the COVID-19 pandemic, many diagnostic approaches (RT-qPCR, RAPID, LFA) have been adopted, with RT-qPCR being the most popular/gold standard. But, one of the major problems of COVID-19 diagnostics is the presentation of a wide range of symptoms which varies among different patients and needs early diagnosis for better management. Even though RT-qPCR is a precise molecular technique false negative results may be obtained. On the other hand, CRISPR-based SARS-CoV-2 detection approaches are cost and time efficient, highly sensitive and specific, and do not require sophisticated instruments. Moreover, they also show promise for increased scalability and diagnostic tests can be carried out at the point-of-care (POC). The CRISPR can be customized to the target of any genomic region of interest within the desired genome possessing a broad range of other applications and has been efficiently implemented for diagnosis of SARS-CoV-2. The CRISPR/Cas systems provide the specific gene targeting with immense potential to develop new generation diagnostics and therapeutics. Moreover, with the CRISPR/Cas based therapeutics, multiplexing is possible, where different sgRNAs or crRNAs can be guided to more than one target within the same gene thus decreasing the possibility of viral escape mutants. As an exceptionally efficient tool CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) systems can be implemented to target a broad range of ssRNA viruses that can be used for both, diagnosis and treatment for a variety of viral diseases including SARS-CoV-2. However, the efficacy and safety of the CRISPR-based therapeutics needs to be assessed in pre-clinical and clinical settings. Although the CRISPR biotechnologies are not very helpful to control the present pandemic of COVID-19 it is hopeful that the limitations of the CRISPR/Cas system can be overcome in the near future. The CRISPR based strategies may lead to a new era in the field of disease diagnosis and therapeutic development that would make us better prepared for future viral threats.},
}
@article {pmid38012557,
year = {2023},
author = {Javadi, M and Sazegar, H and Doosti, A},
title = {Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells.},
journal = {BMC cancer},
volume = {23},
number = {1},
pages = {1155},
pmid = {38012557},
issn = {1471-2407},
mesh = {Humans ; Female ; *Gene Editing/methods ; *Breast Neoplasms/genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Transfection ; NADPH Oxidase 4/genetics ; },
abstract = {BACKGROUND: The increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study.<br><br>METHODS: The predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified.<br><br>RESULTS: There were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR&#x2009;=&#x2009;1.93, 95% CI 1.49-2.49, P&#x2009;<&#x2009;0.001) and a higher percentage of lymph node metastases (OR&#x2009;=&#x2009;3.22, 95% CI 2.18-4.29, P&#x2009;<&#x2009;0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P&#x2009;<&#x2009;0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P&#x2009;<&#x2009;0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with "CRISPR HDR" or "CRISPR excision." Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment.<br><br>CONCLUSIONS: The NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.},
}
@article {pmid38012215,
year = {2023},
author = {Nan, X and Hardinge, P and Hoehn, S and Dighe, SN and Ukeri, J and Pease, DF and Griffin, J and Warrington, JI and Saud, Z and Hottinger, E and Webster, G and Jones, D and Kille, P and Weightman, A and Stanton, R and Castell, OK and Murray, JAH and Jurkowski, TP},
title = {VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {20832},
pmid = {38012215},
issn = {2045-2322},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/V028448/1/MRC_/Medical Research Council/United Kingdom ; BB/W003562/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis/epidemiology ; Wastewater ; Pandemics ; Wastewater-Based Epidemiological Monitoring ; Point-of-Care Testing ; },
abstract = {The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.},
}
@article {pmid38012108,
year = {2023},
author = {Tripathi, JN and Ntui, VO and Tripathi, L},
title = {Precision genetics tools for genetic improvement of banana.},
journal = {The plant genome},
volume = {},
number = {},
pages = {e20416},
doi = {10.1002/tpg2.20416},
pmid = {38012108},
issn = {1940-3372},
support = {//United States Agency for International Development (USAID)/ ; },
abstract = {Banana is an important food security crop for millions of people in the tropics but it faces challenges from diseases and pests. Traditional breeding methods have limitations, prompting the exploration of precision genetic tools like genetic modification and genome editing. Extensive efforts using transgenic approaches have been made to develop improved banana varieties with resistance to banana Xanthomonas wilt, Fusarium wilt, and nematodes. However, these efforts should be extended for other pests, diseases, and abiotic stresses. The commercialization of transgenic crops still faces continuous challenges with regulatory and public acceptance. Genome editing, particularly CRISPR/Cas, offers precise modifications to the banana genome and has been successfully applied in the improvement of banana. Targeting specific genes can contribute to the development of improved banana varieties with enhanced resistance to various biotic and abiotic constraints. This review discusses recent advances in banana improvement achieved through genetic modification and genome editing.},
}
@article {pmid38010835,
year = {2023},
author = {He, Y and Shao, S and Chen, J},
title = {High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.3c02158},
pmid = {38010835},
issn = {2379-3694},
abstract = {Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 10[3] copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.},
}
@article {pmid38010820,
year = {2023},
author = {Thomsen, MK},
title = {The double-edge sword of CRISPR application for in vivo studies.},
journal = {Oncotarget},
volume = {14},
number = {},
pages = {919-920},
pmid = {38010820},
issn = {1949-2553},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; CRISPR-Cas Systems ; Dependovirus/genetics ; },
}
@article {pmid38009957,
year = {2023},
author = {Liu, J and Jaffe, AL and Chen, L and Bor, B and Banfield, JF},
title = {Host translation machinery is not a barrier to phages that interact with both CPR and non-CPR bacteria.},
journal = {mBio},
volume = {},
number = {},
pages = {e0176623},
doi = {10.1128/mbio.01766-23},
pmid = {38009957},
issn = {2150-7511},
abstract = {Here, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.},
}
@article {pmid38009936,
year = {2023},
author = {Muzyukina, P and Shkaruta, A and Guzman, NM and Andreani, J and Borges, AL and Bondy-Denomy, J and Maikova, A and Semenova, E and Severinov, K and Soutourina, O},
title = {Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0040123},
doi = {10.1128/msphere.00401-23},
pmid = {38009936},
issn = {2379-5042},
abstract = {Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.},
}
@article {pmid38008027,
year = {2023},
author = {Wang, Q and Ren, Y and Meng, T and Yang, X and Lu, L and Yang, H and Hou, H and Negahdary, M and Wan, Y and Song, F and Li, J},
title = {Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic.},
journal = {Talanta},
volume = {269},
number = {},
pages = {125458},
doi = {10.1016/j.talanta.2023.125458},
pmid = {38008027},
issn = {1873-3573},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.},
}
@article {pmid38007799,
year = {2023},
author = {Feng, Q and Li, J},
title = {Construction and Validation of a CRISPR/dCpf1-Based Transcriptional Regulatory System in Saccharomyces Cerevisiae.},
journal = {Studies in health technology and informatics},
volume = {308},
number = {},
pages = {680-688},
doi = {10.3233/SHTI230900},
pmid = {38007799},
issn = {1879-8365},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene Expression Regulation ; },
abstract = {Saccharomyces cerevisiae has been extensively studied and applied as a model microbial platform for efficient and sustainable production of biofuels, chemicals and natural products. In recent years, the application of CRISPR system in genome editing and transcriptional regulation has greatly improved the efficiency of microbial genome editing. To achieve transcriptional regulation the CRISPR/dCpf1-mediated CRISPRa/i transcriptional regulation system was constructed in this study. By constructing crRNA arrays and adding different activation proteins, repression efficiencies from 62% to 177% for the CRISPRi system and activation efficiencies from 154% to 320% for the CRISPRa system were achieved in the eGFP gene reporter system.},
}
@article {pmid38006503,
year = {2024},
author = {Chung, T and Merrill, JR and Lyons, SK},
title = {CRISPR/Cas for PET Reporter Gene Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2729},
number = {},
pages = {285-301},
pmid = {38006503},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Genes, Reporter ; Transgenes ; *Genome ; Gene Knock-In Techniques ; Genetic Engineering ; },
abstract = {The relatively recent discovery of CRISPR/Cas has led to a revolution in our ability to efficiently manipulate the genome of eukaryotic cells. We describe here a protocol that employs CRISPR technology to precisely knock-in a PET imaging reporter transgene into a specific genetic locus of interest. Resulting transcription of the targeted reporter will more accurately mimic physiologic expression of the endogenous allele than conventional approaches, and so this method has the potential to become an efficient way to generate a new generation of "gold-standard" reporter transgenes. We break down the protocol into three experimental stages: how to identify the genomic location that the reporter transgene will be inserted, how to practically insert the reporter transgene into the genome, and how to screen resultant clones for the correct targeted event.},
}
@article {pmid37977400,
year = {2024},
author = {Cao, H and Mao, K and Zhang, H and Wu, Q and Ju, H and Feng, X},
title = {Thermal stability and micrdose-based coupling CRISPR/Cas12a biosensor for amplification-free detection of hgcA gene in paddy soil.},
journal = {The Science of the total environment},
volume = {909},
number = {},
pages = {168536},
doi = {10.1016/j.scitotenv.2023.168536},
pmid = {37977400},
issn = {1879-1026},
mesh = {CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Biological Assay ; *Methylmercury Compounds ; Soil ; *Biosensing Techniques ; },
abstract = {The lack of point-of-use (POU) methods hinders the utilization of the hgcA gene to rapidly evaluate methylmercury risks. CRISPR/Cas12a is a promising technology, but shortcomings such as low sensitivity, a strict reaction temperature and high background signal limit its further utilization. Here, a thermally stable microsystem-based CRISPR/Cas12a biosensor was constructed to achieve POU analysis for hgcA. First, three target gRNAs were designed to recognize hgcA. Then, a microsystem was developed to eliminate the background signal. Next, the effect of temperature on the activity of the Cas12a-gRNA complex was explored and its thermal stability was discovered. After that, coupling gRNA assay was introduced to improve sensitivity, exhibiting a limit of detection as low as 0.49 pM with a linear range of 0.98-125 pM, and a recovery rate between 90 and 110 % for hgcA. The biosensor was finally utilized to assess hgcA abundance in paddy soil, and high abundance of hgcA was found in these paddy soil samples. This study not only systematically explored the influence of temperature and microsystem on CRISPR/Cas12a, providing vital references for other novel CRISPR-based detection methods, but also applied the CRISPR-based analytical method to the field of environmental geochemistry for the first time, demonstrating enormous potential for POU detection in this field.},
}
@article {pmid37922912,
year = {2023},
author = {Sasaki-Honda, M and Akatsuka, K and Sawai, T},
title = {Is epigenome editing non-inheritable? Implications for ethics and the regulation of human applications.},
journal = {Stem cell reports},
volume = {18},
number = {11},
pages = {2005-2009},
pmid = {37922912},
issn = {2213-6711},
mesh = {Animals ; Humans ; *Epigenome ; *Epigenesis, Genetic ; Epigenomics ; Gene Editing ; CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {Epigenome editing offers ethical advantages with non-inheritable gene expression control. However, concerns arise regarding potential transgenerational effects in humans. Ethical and regulatory evaluation is crucial, considering recent advancements and enhanced understanding of transgenerational epigenetics in both mammals and humans.},
}
@article {pmid37884601,
year = {2023},
author = {Crunkhorn, S},
title = {Engineering a compact genome-editing tool.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {12},
pages = {953},
doi = {10.1038/d41573-023-00170-1},
pmid = {37884601},
issn = {1474-1784},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Genetic Engineering ; },
}
@article {pmid36840708,
year = {2023},
author = {Diaz Quiroz, JF and Ojha, N and Shayhidin, EE and De Silva, D and Dabney, J and Lancaster, A and Coull, J and Milstein, S and Fraley, AW and Brown, CR and Rosenthal, JJC},
title = {Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.},
journal = {Nucleic acids research},
volume = {51},
number = {7},
pages = {e41},
pmid = {36840708},
issn = {1362-4962},
mesh = {*RNA Editing/genetics ; Base Sequence ; Mutation ; Codon, Nonsense ; RNA, Protozoan/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.},
}
@article {pmid36522352,
year = {2022},
author = {Wang, Y and Cottle, WT and Wang, H and Gavrilov, M and Zou, RS and Pham, MT and Yegnasubramanian, S and Bailey, S and Ha, T},
title = {Achieving single nucleotide sensitivity in direct hybridization genome imaging.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7776},
pmid = {36522352},
issn = {2041-1723},
support = {R01 GM097330/GM/NIGMS NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; T32 GM007445/GM/NIGMS NIH HHS/United States ; },
mesh = {*Nucleotides ; In Situ Hybridization, Fluorescence ; Nucleic Acid Hybridization ; *DNA/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.},
}
@article {pmid38003586,
year = {2023},
author = {Kang, KK and Cho, YG},
title = {Genetic Analysis Based on CRISPR/Cas9 Technology in Plants.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003586},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Plants/genetics ; Genetic Engineering ; Genome, Plant ; },
abstract = {Genome-editing technology is a type of genetic engineering in which DNA is inserted into, replaced in, or deleted from the genome using artificially engineered nucleases or genetic scissors [...].},
}
@article {pmid38003514,
year = {2023},
author = {Ding, S and Liu, J and Han, X and Tang, M},
title = {CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003514},
issn = {1422-0067},
support = {82200677//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; RNA ; *Neoplasms/genetics/therapy ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.},
}
@article {pmid38003431,
year = {2023},
author = {Movahedi, A and Aghaei-Dargiri, S and Li, H and Zhuge, Q and Sun, W},
title = {CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003431},
issn = {1422-0067},
support = {CX2019030//Weibo Sun/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Containment of Biohazards ; Plant Breeding ; Crops, Agricultural/genetics ; Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants.},
}
@article {pmid38003351,
year = {2023},
author = {Davis-Anderson, K and Micheva-Viteva, S and Solomon, E and Hovde, B and Cirigliano, E and Harris, J and Twary, S and Iyer, R},
title = {CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003351},
issn = {1422-0067},
support = {20190167DR//Los Alamos National Security (United States)/ ; HDTRA1-1-38382//Defence Threat Reduction Agency DTRA-JSTO/ ; },
mesh = {*Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Motor Neurons/metabolism ; Neurogenesis ; },
abstract = {Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.},
}
@article {pmid38003326,
year = {2023},
author = {Nagy, B and Öktem, A and Ferenc, G and Ungor, D and Kalac, A and Kelemen-Valkony, I and Fodor, E and Nagy, I and Dudits, D and Ayaydin, F},
title = {CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003326},
issn = {1422-0067},
support = {RRF-2.3.1-21-2022-00007//National Research, Development and Innovation Office of the Hungarian Government/ ; 739593//European Union's Horizon 2020 research and innovation program/ ; },
mesh = {CRISPR-Cas Systems/genetics ; Protoplasts ; Zea mays/genetics ; *Nucleic Acids ; Polymers ; RNA, Guide, CRISPR-Cas Systems ; Mutagenesis ; Gene Editing/methods ; Green Fluorescent Proteins/genetics ; Oligonucleotides ; *Nanoparticles ; },
abstract = {Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.},
}
@article {pmid37856955,
year = {2024},
author = {Mao, K and Zhang, H and Ran, F and Cao, H and Feng, R and Du, W and Li, X and Yang, Z},
title = {Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater.},
journal = {Journal of hazardous materials},
volume = {462},
number = {},
pages = {132793},
doi = {10.1016/j.jhazmat.2023.132793},
pmid = {37856955},
issn = {1873-3336},
mesh = {*Wastewater ; CRISPR-Cas Systems ; Anti-Bacterial Agents ; Drug Resistance, Microbial/genetics ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; },
abstract = {Wastewater is among the main sources of antibiotic resistance genes (ARGs) in the environment, but effective methods to quickly assess ARGs on-site in wastewater are lacking. Here, using the typical ARG ermB as the target, we report a portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification (LAMP) for the detection of ARGs. Six primers of LAMP and the crRNA of CRISPR/Cas12a were first designed to be preamplification with LAMP and lead Cas12a to recognize the ermB via base pairing. Due to the trans-cleavage activity of CRISPR/Cas12a after amplicon recognition, ssDNA probes modified with reporter molecules were used to implement a visual assay with lateral flow test strips and fluorescence. After a simple nucleic acid extraction with magnetic beads, the constructed biosensor possesses excellent sensitivity and selectivity as low as 2.75 × 10[3] copies/μL using fluorescence and later flow strips in wastewater. We further evaluated the community-wide prevalence of ermB in wastewater influent and found high mass loads of ermB during different months. This user-friendly and low-cost biosensor is applicable for rapid on-site ARG detection, providing a potential point-of-use method for rapid assessments of ARG abundance in wastewater from large city areas with many wastewater treatment plants and in resource-limited rural areas.},
}
@article {pmid37794102,
year = {2023},
author = {Tulkens, D and Boelens, M and Naert, T and Carron, M and Demuynck, S and Dewaele, S and Van Isterdael, G and Creytens, D and Pieters, T and Goossens, S and Van Vlierberghe, P and Vleminckx, K},
title = {Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis.},
journal = {Leukemia},
volume = {37},
number = {12},
pages = {2404-2413},
pmid = {37794102},
issn = {1476-5551},
support = {01G01115//Universiteit Gent (UGent)/ ; 3G0A6922//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; 3G0D8716//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; 3F021818//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; },
mesh = {Animals ; Humans ; Histone Methyltransferases/genetics ; Xenopus/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Enhancer of Zeste Homolog 2 Protein/genetics ; Mutation ; *Leukemia, Myeloid, Acute ; },
abstract = {CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.},
}
@article {pmid37722684,
year = {2023},
author = {Raban, R and Marshall, JM and Hay, BA and Akbari, OS},
title = {Manipulating the Destiny of Wild Populations Using CRISPR.},
journal = {Annual review of genetics},
volume = {57},
number = {},
pages = {361-390},
doi = {10.1146/annurev-genet-031623-105059},
pmid = {37722684},
issn = {1545-2948},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genome ; },
abstract = {Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.},
}
@article {pmid38003266,
year = {2023},
author = {Tyumentseva, M and Tyumentsev, A and Akimkin, V},
title = {CRISPR/Cas9 Landscape: Current State and Future Perspectives.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003266},
issn = {1422-0067},
support = {Agreement No. 075-15-2019-1666//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Breeding ; Gene Editing ; CRISPR-Associated Protein 9/genetics ; Biotechnology ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is focused on the current CRISPR/Cas9 landscape, e.g., on Cas9 variants with improved properties, on Cas9-derived and fusion proteins, on Cas9 delivery methods, on pre-existing immunity against CRISPR/Cas9 proteins, anti-CRISPR proteins, and their possible roles in CRISPR/Cas9 function improvement. Moreover, this review presents a detailed outline of CRISPR/Cas9-based diagnostics and therapeutic approaches. Finally, the review addresses the future expansion of genome editors' toolbox with Cas9 orthologs and other CRISPR/Cas proteins.},
}
@article {pmid38003255,
year = {2023},
author = {Mohammadian Gol, T and Kim, M and Sinn, R and Ureña-Bailén, G and Stegmeyer, S and Gratz, PG and Zahedipour, F and Roig-Merino, A and Antony, JS and Mezger, M},
title = {CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.},
journal = {International journal of molecular sciences},
volume = {24},
number = {22},
pages = {},
pmid = {38003255},
issn = {1422-0067},
support = {//Stefan Morsch Stiftung/ ; N&#xb0;. 440-0-0)//Clinician Scientist Program (/ ; //F&#xf6;rderverein f&#xfc;r krebskranke Kinder T&#xfc;bingen e.V./ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems ; Killer Cells, Natural ; Antigens, CD/metabolism ; },
abstract = {Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.},
}
@article {pmid38001333,
year = {2023},
author = {Güngör, B and Biró, JB and Domonkos, &#xc1; and Horváth, B and Kaló, P},
title = {Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {20676},
pmid = {38001333},
issn = {2045-2322},
support = {OTKA 119652//Hungarian National Research Fund/National Research, Development and Innovation Office/ ; 129547//Hungarian National Research Fund/National Research, Development and Innovation Office/ ; 132646//Hungarian National Research Fund/National Research, Development and Innovation Office/ ; HUN17-03//Collaborative Research Programme ICGEB/ ; },
mesh = {*Medicago truncatula/metabolism ; Cysteine/metabolism ; CRISPR-Cas Systems/genetics ; Mutagenesis ; Peptides/metabolism ; *Sinorhizobium meliloti/genetics ; Symbiosis/genetics ; Nitrogen Fixation/genetics ; Root Nodules, Plant/microbiology ; },
abstract = {The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.},
}
@article {pmid37935852,
year = {2023},
author = {Witkowsky, L and Norstad, M and Glynn, AR and Kliegman, M},
title = {Towards affordable CRISPR genomic therapies: a task force convened by the Innovative Genomics Institute.},
journal = {Gene therapy},
volume = {30},
number = {10-11},
pages = {747-752},
pmid = {37935852},
issn = {1476-5462},
support = {2021364//Doris Duke Charitable Foundation (DDCF)/ ; 22-06759//Laura and John Arnold Foundation (Arnold Foundation)/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genomics ; CRISPR-Cas Systems ; },
}
@article {pmid38001092,
year = {2023},
author = {Li, Y and Wu, Y and Xu, R and Guo, J and Quan, F and Zhang, Y and Huang, D and Pei, Y and Gao, H and Liu, W and Liu, J and Zhang, Z and Deng, R and Shi, J and Zhang, K},
title = {In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {7722},
pmid = {38001092},
issn = {2041-1723},
support = {No. 22122409//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Mutation ; DNA, Mitochondrial/genetics ; Mitochondria/genetics ; *Neoplasms/genetics ; },
abstract = {Mutations in mitochondrial DNA (mtDNA) play critical roles in many human diseases. In vivo visualization of cells bearing mtDNA mutations is important for resolving the complexity of these diseases, which remains challenging. Here we develop an integrated nano Cas12a sensor (InCasor) and show its utility for efficient imaging of mtDNA mutations in live cells and tumor-bearing mouse models. We co-deliver Cas12a/crRNA, fluorophore-quencher reporters and Mg[2+] into mitochondria. This process enables the activation of Cas12a's trans-cleavage by targeting mtDNA, which efficiently cleave reporters to generate fluorescent signals for robustly sensing and reporting single-nucleotide variations (SNVs) in cells. Since engineered crRNA significantly increase Cas12a's sensitivity to mismatches in mtDNA, we can identify tumor tissue and metastases by visualizing cells with mutant mtDNAs in vivo using InCasor. This CRISPR imaging nanoprobe holds potential for applications in mtDNA mutation-related basic research, diagnostics and gene therapies.},
}
@article {pmid37998328,
year = {2023},
author = {Kim, Y and Lee, HM},
title = {CRISPR-Cas System Is an Effective Tool for Identifying Drug Combinations That Provide Synergistic Therapeutic Potential in Cancers.},
journal = {Cells},
volume = {12},
number = {22},
pages = {},
pmid = {37998328},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/drug therapy/genetics ; Drug Combinations ; },
abstract = {Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising applications. However, identifying and implementing effective drug combinations are critical for achieving favorable treatment outcomes. Given the enormous possibilities of combinations, a rational approach is required to predict the impact of drug combinations. Thus, CRISPR-Cas-based and other approaches, such as high-throughput pharmacological and genetic screening approaches, have been used to identify possible drug combinations. In particular, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool that enables us to efficiently identify possible drug combinations that can improve treatment outcomes by reducing the total search space. In this review, we discuss the rational approaches to identifying, examining, and predicting drug combinations and their impact.},
}
@article {pmid37998150,
year = {2023},
author = {Yun, D and Jung, C},
title = {MiRNA-Responsive CRISPR-Cas System via a DNA Regulator.},
journal = {Biosensors},
volume = {13},
number = {11},
pages = {},
pmid = {37998150},
issn = {2079-6374},
support = {20009356//Ministry of Trade, Industry and Energy/ ; 2022M3A9B6082670//Ministry of Science and ICT/ ; 2021R1C1C1004147//Ministry of Science and ICT/ ; },
mesh = {*CRISPR-Cas Systems ; *MicroRNAs ; Gene Editing/methods ; DNA/chemistry ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) genome editing technology is widely used for gene editing because it provides versatility in genetic manipulation. Several methods for regulating CRISPR activity already exist for accurate editing, but these require complex engineering. Thus, a simple and convenient regulatory system is required. In this study, we devised a CRISPR activation system using a DNA regulator that can be activated by miRNAs. The designed regulator was divided into two parts. The inhibition component consisted of the protospacer-adjacent motif (PAM) and seed sequence, which are important for Cas9 target recognition and bind to the ribonucleoprotein (RNP) complex for inhibition. The miRNA recognition component has a single-stranded toehold DNA for target miRNA binding and a partial double-stranded DNA complementary to the remaining miRNA sequence. In the presence of target miRNAs, the structure of the regulator is disrupted by the miRNAs, leading to its dissociation from the RNP complex and subsequent restoration of CRISPR activity. This method is easy to design and can be applied to various miRNAs via simple sequence manipulation. Therefore, this strategy provides a general platform for controlled genome editing.},
}
@article {pmid37998138,
year = {2023},
author = {Jeung, JH and Han, H and Lee, CY and Ahn, JK},
title = {CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.},
journal = {Biosensors},
volume = {13},
number = {11},
pages = {},
pmid = {37998138},
issn = {2079-6374},
support = {kitech EO-23-0005//Korea Institute of Industrial Technology/ ; 321104-3//Ministry of Agriculture, Food and Rural Affairs/ ; },
mesh = {*CRISPR-Cas Systems ; Phosphodiesterase I/genetics ; Exodeoxyribonucleases ; Limit of Detection ; DNA ; DNA Probes ; *Biosensing Techniques/methods ; },
abstract = {This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5'-end of a hairpin probe (HP), forming a blunt end. When the 3'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.},
}
@article {pmid37997999,
year = {2023},
author = {Joshi, A and Yang, SY and Song, HG and Min, J and Lee, JH},
title = {Genetic Databases and Gene Editing Tools for Enhancing Crop Resistance against Abiotic Stress.},
journal = {Biology},
volume = {12},
number = {11},
pages = {},
pmid = {37997999},
issn = {2079-7737},
support = {RS-2021-RD009903//Rural Development Administration/ ; 2023 research fund//Jeonbuk National University/ ; },
abstract = {Abiotic stresses extensively reduce agricultural crop production globally. Traditional breeding technology has been the fundamental approach used to cope with abiotic stresses. The development of gene editing technology for modifying genes responsible for the stresses and the related genetic networks has established the foundation for sustainable agriculture against environmental stress. Integrated approaches based on functional genomics and transcriptomics are now expanding the opportunities to elucidate the molecular mechanisms underlying abiotic stress responses. This review summarizes some of the features and weblinks of plant genome databases related to abiotic stress genes utilized for improving crops. The gene-editing tool based on clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized stress tolerance research due to its simplicity, versatility, adaptability, flexibility, and broader applications. However, off-target and low cleavage efficiency hinder the successful application of CRISPR/Cas systems. Computational tools have been developed for designing highly competent gRNA with better cleavage efficiency. This powerful genome editing tool offers tremendous crop improvement opportunities, overcoming conventional breeding techniques' shortcomings. Furthermore, we also discuss the mechanistic insights of the CRISPR/Cas9-based genome editing technology. This review focused on the current advances in understanding plant species' abiotic stress response mechanism and applying the CRISPR/Cas system genome editing technology to develop crop resilience against drought, salinity, temperature, heavy metals, and herbicides.},
}
@article {pmid37997875,
year = {2023},
author = {Cercenado, E},
title = {What are the most relevant publications in Clinical Microbiology in the last two years?.},
journal = {Revista espanola de quimioterapia : publicacion oficial de la Sociedad Espanola de Quimioterapia},
volume = {36 Suppl 1},
number = {},
pages = {64-67},
doi = {10.37201/req/s01.15.2023},
pmid = {37997875},
issn = {1988-9518},
mesh = {Humans ; *Artificial Intelligence ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Flow Cytometry ; Drug Combinations ; Ceftazidime ; Microbial Sensitivity Tests ; beta-Lactamases ; },
abstract = {This minireview describes some of the articles published in the last two years related to innovative technologies including CRISPR-Cas, surface-enhanced Raman spectroscopy, microfluidics, flow cytometry, Fourier transform infrared spectroscopy, and artificial intelligence and their application to microbiological diagnosis, molecular typing and antimicrobial susceptibility testing. In addition, some articles related to resistance to new antimicrobials (ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and cefiderocol) are also described.},
}
@article {pmid37996158,
year = {2023},
author = {Qiao, Z and Xue, L and Sun, M and Zhang, M and Chen, M and Xu, X and Yang, W and Wang, R},
title = {Highly sensitive detection of Salmonella based on dual-functional HCR-mediated multivalent aptamer and amplification-free CRISPR/Cas12a system.},
journal = {Analytica chimica acta},
volume = {1284},
number = {},
pages = {341998},
doi = {10.1016/j.aca.2023.341998},
pmid = {37996158},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/genetics/chemistry ; DNA/chemistry ; Nucleic Acid Hybridization ; Salmonella/genetics ; *Biosensing Techniques/methods ; },
abstract = {BACKGROUND: Salmonella infection severely threatens human health and causes substantial medical and financial concerns. Sensitive and specific detection of Salmonella in food samples is crucial but remains challenging. While some traditional assays for S. typhimurium are reliable, they suffer from various limitations, such as being time-consuming (culture-based methods), involving intricate nucleic molecular extraction (polymerization chain reaction, PCR), and exhibiting inadequate sensitivity (enzyme-linked immunosorbent assay, ELISA). In this case, it is essential to establish a rapid, simple-operation, and sensitive method for monitoring S. typhimurium to preserve food quality and prevent contamination.<br><br>RESULT: Herein, an amplification-free detection method for Salmonella was developed by coupling the aptamer magnetic separation with dual-functional HCR (hybridization chain reaction)-scaffold multivalent aptamer and the activity of CRISPR/Cas12a. In the detection system, the dual-functional HCR-scaffold multivalent aptamer with high binding affinity and specificity was fabricated in advance by assembling numerous Salmonella specific aptamers on the long HCR products. In addition to the enhanced affinity, the HCR-multiApt also contains a massive amount of repeated CRISPR-targetable DNA units in its HCR scaffold, which could trigger the trans-cleavage activity of Cas12a. In the presence of target bacteria, the HCR-scaffold multivalent aptamer could attach on the surface of bacteria effectively and amplified the signal of bacteria into CRISPR/Cas12a based fluorescent readout. The proposed detection system allowed for ultrasensitive detection of Salmonella in a linear range from 10[0] to 10[7]&#xa0;cfu&#xa0;mL[-1] with a LOD (limit of detection) of 2&#xa0;cfu&#xa0;mL[-1].<br><br>SIGNIFICANCE: The novel dual-functional HCR-multiApt presents a simple and powerful strategy for improving the aptamer binding affinity toward Salmonella. Simultaneously, integrating this dual-functional HCR-multiApt with the CRISPR/Cas12a system significantly enhances the sensitivity by cascade signal amplification in a nucleic acids amplification-free way. Finally, leveraging the versatility of the aptamer, this highly sensitive method can be further extended for application in the detection of other bacteria, food safety monitoring, or clinical diagnostics.},
}
@article {pmid37996055,
year = {2023},
author = {Tu, W and Hu, X and Wan, R and Xiao, X and Shen, Y and Srikaram, P and Avvaru, SN and Yang, F and Pi, F and Zhou, Y and Wan, M and Gao, P},
title = {Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jconrel.2023.11.034},
pmid = {37996055},
issn = {1873-4995},
abstract = {Our previous studies have shown that miR-511-3p treatment has a beneficial effect in alleviating allergic airway inflammation. Here, we sought to explore its therapeutic potential in animal models and gain a deeper understanding of its therapeutic value for asthma. miR-511-3p knockout mice (miR-511-3p[-/-]) were generated by CRISPR/Cas and showed exacerbated airway hyper-responsiveness and Th2-associated allergic airway inflammation compared with wild-type (WT) mice after exposed to cockroach allergen. RNA nanoparticles with mannose decorated EV-miR-511-3p were also created by loading miR-511-3p mimics into the mannose decorated EVs with engineered RNA nanoparticle PRNA-3WJ (Man-EV-miR-511-3p). Intra-tracheal inhalation of Man-EV-miR-511-3p, which could effectively penetrate the airway mucus barrier and deliver functional miR-511-3p to lung macrophages, successfully reversed the increased airway inflammation observed in miR-511-3p[-/-] mice. Through microarray analysis, complement C3 (C3) was identified as one of the major targets of miR-511-3p. C3 was increased in LPS-treated macrophages but decreased after miR-511-3p treatment. Consistent with these findings, C3 expression was elevated in the lung macrophages of an asthma mouse model but decreased in mice treated with miR-511-3p. Further experiments, including miRNA-mRNA pulldown and luciferase reporter assays, confirmed that miR-511-3p directly binds to C3 and activates the C3 gene. Thus, miR-511-3p represents a promising therapeutic target for asthma, and RNA nanotechnology reprogrammed EVs are efficient carriers for miRNA delivery for disease treatment.},
}
@article {pmid37995242,
year = {2023},
author = {Altae-Tran, H and Kannan, S and Suberski, AJ and Mears, KS and Demircioglu, FE and Moeller, L and Kocalar, S and Oshiro, R and Makarova, KS and Macrae, RK and Koonin, EV and Zhang, F},
title = {Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6673},
pages = {eadi1910},
doi = {10.1126/science.adi1910},
pmid = {37995242},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Biotechnology ; RNA ; },
abstract = {Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity. We experimentally characterized three HNH nuclease-containing CRISPR systems, including the first type IV system with a specified interference mechanism, and engineered them for genome editing. We also identified and characterized a candidate type VII system, which we show acts on RNA. This work opens new avenues for harnessing CRISPR and for the broader exploration of the vast functional diversity of microbial proteins.},
}
@article {pmid37995060,
year = {2023},
author = {Jibrilla, M and Raji, H and Okeke, MI},
title = {Survey of attitude to human genome modification in Nigeria.},
journal = {Journal of community genetics},
volume = {},
number = {},
pages = {},
pmid = {37995060},
issn = {1868-310X},
abstract = {Gene editing and mitochondrial replacement therapy (MRT) are biotechnologies used to modify the host nuclear and mitochondrial DNA, respectively. Gene editing is the modification of a region of the host genome using site-specific nucleases, in particular the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Heritable and somatic genome editing (HGE and SGE) are used in gene therapy. MRT is a technique used to substitute the defective mitochondria in the recipient embryo with a female donor healthy mitochondrion in order to prevent the inheritance of mothers' defective mitochondria resulting in the change of mitochondria of the entire generation to come. To evaluate the perception of the Nigerian citizens on human genome modification, two survey forms were created and distributed in-person and majorly online. There was a total of 268 responses, 188 from the public and 80 from health workers and bio-scientists. The results showed poor knowledge about gene editing and MRT by the Nigerian public, but its use to prevent and cure inherited diseases was supported. Morality and religion have great influence on the attitude of Nigerians towards genome modification, but the influence of religion and morality is not unequivocal. Multiple regression analysis of Nigerian public responses shows that gender (females), age (19-30 years), monthly income (NGN 0 to 30,000), and level of education (tertiary) are significantly associated with approval of human genome editing, but the survey of health workers and bio-scientists shows no significant association except for females who approve and Muslims who disapprove of human genome editing.},
}
@article {pmid37993945,
year = {2023},
author = {Hu, Y and Liu, L and Jiang, Q and Fang, W and Chen, Y and Hong, Y and Zhai, X},
title = {CRISPR/Cas9: a powerful tool in colorectal cancer research.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {42},
number = {1},
pages = {308},
pmid = {37993945},
issn = {1756-9966},
mesh = {Animals ; Mice ; Humans ; *CRISPR-Cas Systems ; Genetic Therapy ; Oncogenes ; *Colorectal Neoplasms/genetics/therapy ; },
abstract = {Colorectal cancer (CRC) is one of the most common malignant cancers worldwide and seriously threatens human health. The clustered regulatory interspaced short palindromic repeat/CRISPR-associate nuclease 9 (CRISPR/Cas9) system is an adaptive immune system of bacteria or archaea. Since its introduction, research into various aspects of treatment approaches for CRC has been accelerated, including investigation of the oncogenes, tumor suppressor genes (TSGs), drug resistance genes, target genes, mouse model construction, and especially in genome-wide library screening. Furthermore, the CRISPR/Cas9 system can be utilized for gene therapy for CRC, specifically involving in the molecular targeted drug delivery or targeted knockout in vivo. In this review, we elucidate the mechanism of the CRISPR/Cas9 system and its comprehensive applications in CRC. Additionally, we discussed the issue of off-target effects associated with CRISPR/Cas9, which serves to restrict its practical application. Future research on CRC should in-depth and systematically utilize the CRISPR/Cas9 system thereby achieving clinical practice.},
}
@article {pmid37993930,
year = {2023},
author = {Kouroukli, AG and Rajaram, N and Bashtrykov, P and Kretzmer, H and Siebert, R and Jeltsch, A and Bens, S},
title = {Targeting oncogenic TERT promoter variants by allele-specific epigenome editing.},
journal = {Clinical epigenetics},
volume = {15},
number = {1},
pages = {183},
pmid = {37993930},
issn = {1868-7083},
support = {ID09//Baden-W&#xfc;rttemberg Stiftung gGmbH/ ; ID09//Baden-W&#xfc;rttemberg Stiftung gGmbH/ ; },
mesh = {Humans ; Alleles ; DNA Methylation ; Epigenome ; RNA, Guide, CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Lung Neoplasms ; Nucleotides ; Mutation ; *Telomerase/genetics ; },
abstract = {BACKGROUND: Activation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.<br><br>RESULTS: Sequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.<br><br>CONCLUSIONS: We applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.},
}
@article {pmid37993613,
year = {2023},
author = {Song, Y},
title = {RNA-based anti-CRISPRs.},
journal = {Nature chemical biology},
volume = {19},
number = {12},
pages = {1433},
doi = {10.1038/s41589-023-01500-5},
pmid = {37993613},
issn = {1552-4469},
mesh = {*RNA/genetics ; CRISPR-Cas Systems ; *Bacteriophages/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
}
@article {pmid37993299,
year = {2023},
author = {Zaman, QU and Raza, A and Lozano-Juste, J and Chao, L and Jones, MGK and Wang, HF and Varshney, RK},
title = {Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.10.007},
pmid = {37993299},
issn = {1879-3096},
abstract = {The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.},
}
@article {pmid37992834,
year = {2023},
author = {Wen, J and Deng, H and He, D and Yuan, Y},
title = {Dual-functional DNAzyme powered CRISPR-Cas12a sensor for ultrasensitive and high-throughput detection of Pb[2+] in freshwater.},
journal = {The Science of the total environment},
volume = {911},
number = {},
pages = {168708},
doi = {10.1016/j.scitotenv.2023.168708},
pmid = {37992834},
issn = {1879-1026},
abstract = {Freshwater lead pollution has posed severe threat to the environment and human health, underscoring the urgent necessity for accurate and user-friendly detection methods. Herein, we introduce a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) sensor for highly sensitive Pb[2+] detection. To accomplish this, we designed a dual-functional deoxyribozyme (df-DNAzyme) probe that functions as an activator for the CRISPR-Cas12a system while also recognizing Pb[2+]. The df-DNAzyme probe was subsequently combined with gold nanoparticles (AuNPs) to fabricate a DNAzyme/AuNP nanoprobe, facilitating the activation of CRISPR-Cas12a in a one-to-multiple manner. Upon exposure to Pb[2+], the df-DNAzyme is cleaved, causing disintegration of the DNAzyme/AuNP nanoprobe from magnetic beads. The degraded DNAzyme/AuNP containing multiple double-stranded DNA activators efficiently triggers CRISPR-Cas12a activity, initiating cleavage of fluorescence-quenched reporter DNA and generating amplified signals accordingly. The amplified fluorescence signal is accurately quantified using a quantitative polymerase chain reaction (qPCR) instrument capable of measuring 96 or 384 samples simultaneously at the microliter scale. This technique demonstrates ultra-sensitive detection capability for Pb[2+] at concentrations as low as 1 pg/L within a range from 1 pg/L to 10 μg/L, surpassing limits set by World Health Organization (WHO) and United States Environmental Protection Agency (US EPA) guidelines. This study offers an ultrasensitive and high-throughput method for the detection of Pb[2+] in freshwater, thereby advancing a novel approach towards the development of precise and convenient techniques for detecting harmful contaminants.},
}
@article {pmid37992756,
year = {2023},
author = {Yirmiya, E and Leavitt, A and Lu, A and Ragucci, AE and Avraham, C and Osterman, I and Garb, J and Antine, SP and Mooney, SE and Hobbs, SJ and Kranzusch, PJ and Amitai, G and Sorek, R},
title = {Phages overcome bacterial immunity via diverse anti-defence proteins.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-023-06869-w},
pmid = {37992756},
issn = {1476-4687},
abstract = {It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems[1-4], but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and discovered four distinct families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2, and Had1 efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.},
}
@article {pmid37984376,
year = {2023},
author = {Limaye, A and Cho, K and Hall, B and Khillan, JS and Kulkarni, AB},
title = {Genotyping Protocols for Genetically Engineered Mice.},
journal = {Current protocols},
volume = {3},
number = {11},
pages = {e929},
doi = {10.1002/cpz1.929},
pmid = {37984376},
issn = {2691-1299},
mesh = {Humans ; Mice ; Animals ; Genotype ; Endopeptidase K/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Mice, Knockout ; *DNA/genetics ; Disease Models, Animal ; Mammals/genetics ; },
abstract = {Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice.},
}
@article {pmid37978173,
year = {2023},
author = {Rueff, AS and van Raaphorst, R and Aggarwal, SD and Santos-Moreno, J and Laloux, G and Schaerli, Y and Weiser, JN and Veening, JW},
title = {Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {7454},
pmid = {37978173},
issn = {2041-1723},
support = {R01 AI150893/AI/NIAID NIH HHS/United States ; R37 AI038446/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Streptococcus pneumoniae/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Phenotype ; Biological Variation, Population ; },
abstract = {Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.},
}
@article {pmid37977780,
year = {2023},
author = {Liang, P and Lv, B and Chen, K and Qiao, W and Li, D},
title = {An ultrasensitive Cd[2+] detection biosensor based on DNAzyme and CRISPR/Cas12a coupled with hybridization chain reaction.},
journal = {Analytica chimica acta},
volume = {1283},
number = {},
pages = {341950},
doi = {10.1016/j.aca.2023.341950},
pmid = {37977780},
issn = {1873-4324},
mesh = {Humans ; *DNA, Catalytic ; CRISPR-Cas Systems ; Cadmium ; Nucleic Acid Hybridization ; Biological Assay ; *Biosensing Techniques ; },
abstract = {The detection of cadmium is essential because it poses a significant threat to human health and the environment. Recent advancements in biosensors that detect nonnucleic-acid targets using CRISPR/Cas12a in combination with aptamers or DNAzymes show promising performance. Herein, we integrated DNAzyme, hybridization chain reaction (HCR) and CRISPR/Cas12a into a single biosensor for the first time and realized the ultrasensitive detection of Cd[2+]. A single phosphorothioate ribonucleobase (rA)-containing oligonucleotide (PS substrate) and a Cd[2+]-specific DNAzyme (Cdzyme) are used for Cd[2][+] recognition, releasing short single-stranded DNA. Then, the HCR is triggered by the cleavage products for signal transduction and amplification. Next, the trans-cleavage activity of Cas12a is activated due to the presence of crRNA complementary strands and PAM sites in the HCR products. As a result, FQ-reporters are cleaved, and the fluorescence values can be easily read using a fluorometer, allowing Cd[2][[+]] quantification by measuring the fluorescent signal. The Cd[2][[+]] detection biosensor is ultrasensitive with a detection limit of 1.25 pM. Moreover, the biosensor shows great stability under different pH and various anion conditions. The proposed sensor was utilized for environmental water sample detection, demonstrating the dependability of the detection system. Considering the high sensitivity and reliable performance of the assay, it could be further used in environmental monitoring. In addition, the design strategy reported in this study could extend the application of CRISPR/Cas12a in heavy metal detection.},
}
@article {pmid37975774,
year = {2023},
author = {Benarroch, L},
title = {[CRIPSR-Cas9: A therapeutic strategy for laminopathies?].},
journal = {Medecine sciences : M/S},
volume = {39 Hors s&#xe9;rie n&#xb0; 1},
number = {},
pages = {65},
doi = {10.1051/medsci/2023139},
pmid = {37975774},
issn = {1958-5381},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; *Laminopathies ; Lamin Type A/genetics ; },
}
@article {pmid37957349,
year = {2023},
author = {Naddaf, M},
title = {First trial of 'base editing' in humans lowers cholesterol - but raises safety concerns.},
journal = {Nature},
volume = {623},
number = {7988},
pages = {671-672},
pmid = {37957349},
issn = {1476-4687},
mesh = {Humans ; *Cholesterol ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37956622,
year = {2024},
author = {Yudin Kharismasari, C and Irkham, and Zein, MIHL and Hardianto, A and Nur Zakiyyah, S and Umar Ibrahim, A and Ozsoz, M and Wahyuni Hartati, Y},
title = {CRISPR/Cas12-based electrochemical biosensors for clinical diagnostic and food monitoring.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {155},
number = {},
pages = {108600},
doi = {10.1016/j.bioelechem.2023.108600},
pmid = {37956622},
issn = {1878-562X},
mesh = {CRISPR-Cas Systems/genetics ; DNA/genetics/chemistry ; *Nucleic Acids ; *Biosensing Techniques/methods ; Electrochemical Techniques ; },
abstract = {Each organism has a unique sequence of nitrogenous bases in in the form of DNA or RNA which distinguish them from other organisms. This characteristic makes nucleic acid-based detection extremely selective and compare to other molecular techniques. In recent years, several nucleic acid-based detection technology methods have been developed, one of which is the electrochemical biosensor. Electrochemical biosensors are known to have high sensitivity and accuracy. In addition, the ease of miniaturization of this electrochemical technique has garnered interest from many researchers. On the other hand, the CRISPR/Cas12 method has been widely used in detecting nucleic acids due to its highly selective nature. The CRISPR/Cas12 method is also reported to increase the sensitivity of electrochemical biosensors through the utilization of modified electrodes. The electrodes can be modified according to detection needs so that the biosensor's performance can be improved. This review discusses the application of CRISPR/Cas12-based electrochemical biosensors, as well as various electrode modifications that have been successfully used to improve the performance of these biosensors in the clinical and food monitoring fields.},
}
@article {pmid37930278,
year = {2023},
author = {Shaw, WM and Khalil, AS and Ellis, T},
title = {A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3393-3405},
pmid = {37930278},
issn = {2161-5063},
support = {R01 EB029483/EB/NIBIB NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Ecosystem ; Biotechnology ; Plasmids/genetics ; },
abstract = {Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast Saccharomyces cerevisiae, an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.},
}
@article {pmid37922816,
year = {2024},
author = {Chen, X and Huang, C and Zhang, J and Hu, Q and Wang, D and You, Q and Guo, Y and Chen, H and Xu, J and Hu, M},
title = {Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection.},
journal = {Talanta},
volume = {268},
number = {Pt 1},
pages = {125350},
doi = {10.1016/j.talanta.2023.125350},
pmid = {37922816},
issn = {1873-3573},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *RNA, Long Noncoding ; *MicroRNAs/genetics ; *Biosensing Techniques ; },
abstract = {Some non-coding RNAs are abnormally expressed during the occurrence and development of diseases, so it is necessary to develop analytical methods that can specifically and sensitively detect them. In typical CRISPR/Cas12a system, a complete crRNA that can recognize single-stranded or double-stranded DNA is necessary to activate its trans-cleavage activity, which limits its direct application in RNA detection. Here, we prospectively find that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, and a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) was proposed based on this. This system can detect non-coding RNA to pM-level (10 pM for miRNA-21). To expand the application of this system, we combined it with HCR and CHA to establish a detection platform for non-coding RNA. The results show that the proposed method can specifically detect RNA to fM-level (2.5 fM for miRNA-21, 8.98 fM for miR-128-3p, and 81.6 fM for lncRNA PACER). The spiked recovery rates of miRNA-21, miR-128-3p, and lncRNA PACER in normal human serum were in range from 104.7 to 109.4 %, indicating the proposed method owns good applicability. In general, this MCM-CRISPR/Cas12a system further breaks the limitations of the typical CRISPR/Cas12a system that cannot be directly used for non-coding RNA detection. Besides, its combination with HCR and CHA achieves highly sensitive detection of non-coding RNA.},
}
@article {pmid37924448,
year = {2023},
author = {Feng, Q and Hao, S and Fang, P and Zhang, P and Sheng, X},
title = {Role of GPX4 inhibition-mediated ferroptosis in the chemoresistance of ovarian cancer to Taxol in vitro.},
journal = {Molecular biology reports},
volume = {50},
number = {12},
pages = {10189-10198},
pmid = {37924448},
issn = {1573-4978},
support = {SZLY2017030//Shenzhen Healthcare Research Project/ ; SZSM201812075//Sanming Project of Medicine in Shenzhen/ ; JCYJ20210324134413038//Shenzhen Science and Technology Innovation Program/ ; 82103124//National Natural Science Foundation of China/ ; },
mesh = {Humans ; Female ; Paclitaxel/pharmacology ; Apoptosis ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/pharmacology ; *Ferroptosis ; Cell Line, Tumor ; *Ovarian Neoplasms/drug therapy/genetics ; Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Ovarian cancer remains a common gynecological tumor and the fifth leading cause of death worldwide. Taxol-based chemotherapy is a standard approach to the treatment of ovarian cancer. Glutathione peroxidase 4 (GPX4) is the key regulator of ferroptosis, which is an important form of cell death. Here, we investigate the effect of GPX4 inhibition-mediated ferroptosis on the sensitivity of ovarian cancer cells to Taxol.<br><br>METHODS AND RESULTS: A2780/PTX and OVCAR-3/PTX Taxol-resistant ovarian cancer cells were established, and stable GPX4 knockout cell lines were generated via lentivirus GPX4-sgRNA. The GPX4 expression level, the apoptosis rate and cell viability were analyzed. The levels of ferroptosis-related factor indicators such as malondialdehyde (MDA) and reactive oxygen species (ROS) were measured. The results showed that the GPX4 protein and mRNA levels were increased in the Taxol-resistant cells. Moreover, GPX4 knockout reduced cell viability and inhibited the colony formation rate. In addition, we found that GPX4 inhibition increased Taxol sensitivity by inducing ferroptosis.<br><br>CONCLUSIONS: In summary, our studies reveal that GPX4 inhibition promotes ferroptosis and increases the sensitivity of ovarian cancer cells to Taxol in vitro.},
}
@article {pmid37870387,
year = {2023},
author = {Li, T and Cheng, N},
title = {Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors.},
journal = {ACS sensors},
volume = {8},
number = {11},
pages = {3988-4007},
doi = {10.1021/acssensors.3c01338},
pmid = {37870387},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems ; *Point-of-Care Systems ; Biological Assay ; Colorimetry ; Microfluidics ; },
abstract = {Point-of-care (POC) detection is getting more and more attention in many fields due to its accuracy and on-site test property. The CRISPR/Cas12a system is endowed with excellent sensitivity, target identification specificity, and signal amplification ability in biosensing because of its unique trans-cleavage ability. As a result, a lot of research has been made to develop CRISPR/Cas12a-based biosensors. In this review, we focused on signal readout strategies and summarized recent sensitivity-improving strategies in fluorescence, colorimetric, and electrochemical signaling. Then we introduced novel portability-improving strategies based on lateral flow assays (LFAs), microfluidic chips, simplified instruments, and one-pot design. In the end, we also provide our outlook for the future development of CRISPR/Cas12a biosensors.},
}
@article {pmid37848112,
year = {2023},
author = {Wang, Z and Yuan, H and Yang, L and Ma, L and Zhang, Y and Deng, J and Li, X and Xiao, W and Li, Z and Qiu, J and Ouyang, H and Pang, D},
title = {Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.},
journal = {International journal of biological macromolecules},
volume = {253},
number = {Pt 7},
pages = {127418},
doi = {10.1016/j.ijbiomac.2023.127418},
pmid = {37848112},
issn = {1879-0003},
mesh = {Animals ; Mice ; Humans ; *Proprotein Convertase 9/metabolism ; *CRISPR-Cas Systems/genetics ; Adenine/metabolism ; Gene Editing ; DNA/genetics ; Ubiquitin-Protein Ligases/metabolism ; },
abstract = {Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.},
}
@article {pmid37820936,
year = {2023},
author = {Wang, Z and Zhang, Y and Kang, Z and Mao, H},
title = {Improvement of wheat drought tolerance through editing of TaATX4 by CRISPR/Cas9.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {50},
number = {11},
pages = {913-916},
doi = {10.1016/j.jgg.2023.10.001},
pmid = {37820936},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems/genetics ; *Triticum/genetics ; Drought Resistance ; Gene Editing ; Genome, Plant ; },
}
@article {pmid37783810,
year = {2023},
author = {Carvalho, T},
title = {Discontinued CRISPR gene therapy for sickle-cell disease improves symptoms.},
journal = {Nature medicine},
volume = {29},
number = {11},
pages = {2669-2670},
doi = {10.1038/d41591-023-00088-6},
pmid = {37783810},
issn = {1546-170X},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems/genetics ; Genetic Therapy ; Gene Editing ; },
}
@article {pmid37758106,
year = {2023},
author = {Harisa, GI and Faris, TM and Sherif, AY and Alzhrani, RF and Alanazi, SA and Kohaf, NA and Alanazi, FK},
title = {Gene-editing technology, from macromolecule therapeutics to organ transplantation: Applications, limitations, and prospective uses.},
journal = {International journal of biological macromolecules},
volume = {253},
number = {Pt 5},
pages = {127055},
doi = {10.1016/j.ijbiomac.2023.127055},
pmid = {37758106},
issn = {1879-0003},
mesh = {Animals ; Humans ; Swine ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; Genetic Therapy ; *Organ Transplantation ; },
abstract = {Gene editing technologies (GETs) could induce gene knockdown or gene knockout for biomedical applications. The clinical success of gene silence by RNAi therapies pays attention to other GETs as therapeutic approaches. This review aims to highlight GETs, categories, mechanisms, challenges, current use, and prospective applications. The different academic search engines, electronic databases, and bibliographies of selected articles were used in the preparation of this review with a focus on the fundamental considerations. The present results revealed that, among GETs, CRISPR/Cas9 has higher editing efficiency and targeting specificity compared to other GETs to insert, delete, modify, or replace the gene at a specific location in the host genome. Therefore, CRISPR/Cas9 is talented in the production of molecular, tissue, cell, and organ therapies. Consequently, GETs could be used in the discovery of innovative therapeutics for genetic diseases, pandemics, cancer, hopeless diseases, and organ failure. Specifically, GETs have been used to produce gene-modified animals to spare human organ failure. Genetically modified pigs are used in clinical trials as a source of heart, liver, kidneys, and lungs for xenotransplantation (XT) in humans. Viral, non-viral, and hybrid vectors have been utilized for the delivery of GETs with some limitations. Therefore, extracellular vesicles (EVs) are proposed as intelligent and future cargoes for GETs delivery in clinical applications. This study concluded that GETs are promising for the production of molecular, cellular, and organ therapies. The use of GETs as XT is still in the early stage as well and they have ethical and biosafety issues.},
}
@article {pmid37709005,
year = {2023},
author = {Kim, SH and Shin, S and Baek, M and Xiong, K and Karottki, KJC and Hefzi, H and Grav, LM and Pedersen, LE and Kildegaard, HF and Lewis, NE and Lee, JS and Lee, GM},
title = {Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.},
journal = {Metabolic engineering},
volume = {80},
number = {},
pages = {66-77},
doi = {10.1016/j.ymben.2023.09.006},
pmid = {37709005},
issn = {1096-7184},
mesh = {Cricetinae ; Animals ; Cricetulus ; CHO Cells ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Genome ; Antibodies, Monoclonal ; },
abstract = {Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.},
}
@article {pmid37992599,
year = {2023},
author = {Theuerkauf, SA and Herrera-Carrillo, E and John, F and Zinser, LJ and Molina, MA and Riechert, V and Thalheimer, FB and Börner, K and Grimm, D and Chlanda, P and Berkhout, B and Buchholz, CJ},
title = {AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.},
journal = {Biomaterials},
volume = {303},
number = {},
pages = {122399},
doi = {10.1016/j.biomaterials.2023.122399},
pmid = {37992599},
issn = {1878-5905},
abstract = {Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.},
}
@article {pmid37992484,
year = {2023},
author = {Wu, C and Yue, Y and Huang, B and Ji, H and Wu, L and Huang, H},
title = {CRISPR-powered microfluidic biosensor for preamplification-free detection of ochratoxin A.},
journal = {Talanta},
volume = {269},
number = {},
pages = {125414},
doi = {10.1016/j.talanta.2023.125414},
pmid = {37992484},
issn = {1873-3573},
abstract = {The CRISPR technology, which does not require complex instruments, expensive reagents or professional operators, has attracted a lot of attention. When utilizing the CRISPR-Cas system for detection, the pre-amplification step is often necessary to enhance sensitivity. However, this approach tends to introduce complexity and prolong the time required. To address this issue, we employed Pd@PCN-222 nanozyme to label single-stranded DNA, referred to as Pd@PCN-222 CRISPR nanozyme, which serves as the reporter of the CRISPR system. Pd@PCN-222 nanozyme possess exceptional catalytic activity for the reduction of H2O2. Compared with traditional electrochemical probe ferrocene and methylene blue without catalytic activity, there is a significant amplification of the electrochemical signal. So the need for pre-amplification was eliminated. In this study, we constructed a CRISPR-Cas system for ochratoxin A, utilizing the Pd@PCN-222 CRISPR nanozyme to amplified signal avoiding pre-amplification with outstanding detection of 1.21 pg/mL. Furthermore, we developed a microfluidic electrochemical chip for the on-site detection of ochratoxin A. This achievement holds significant promise in establishing a practical on-site detection platform for identifying food safety hazards.},
}
@article {pmid37987913,
year = {2024},
author = {Nagalakshmi, U and Meier, N and Dinesh-Kumar, SP},
title = {Virus-Induced Heritable Gene Editing in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2724},
number = {},
pages = {273-288},
pmid = {37987913},
issn = {1940-6029},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Tobacco/genetics ; *Arabidopsis/genetics ; Endonucleases ; },
abstract = {Gene editing using clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease is an excellent tool for assessing gene function in plants. However, delivery of CRISPR/Cas-editing components into plant cells is still a major bottleneck and requires tissue culture-based approaches and regeneration of plants. To overcome this limitation, several plant viral vectors have recently been engineered to deliver single-guide RNA (sgRNA) targets into SpCas9-expressing plants. Here, we describe an optimized, step-by-step protocol based on the tobacco rattle virus (TRV)-based vector system to deliver sgRNAs fused to mobile tRNA sequences for efficient heritable editing in Nicotiana benthamiana and Arabidopsis thaliana model systems. The protocol described here could be adopted to study the function of any gene of interest.},
}
@article {pmid37974039,
year = {2023},
author = {Wong, C},
title = {UK first to approve CRISPR treatment for diseases: what you need to know.},
journal = {Nature},
volume = {623},
number = {7988},
pages = {676-677},
doi = {10.1038/d41586-023-03590-6},
pmid = {37974039},
issn = {1476-4687},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; *beta-Thalassemia/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Gene Editing/economics/legislation &amp; jurisprudence/trends ; United Kingdom ; Humans ; },
}
@article {pmid37986642,
year = {2023},
author = {Behrouzian Fard, G and Ahmadi, MH and Gholamin, M and Amirfakhrian, R and Saberi Teimourian, E and Karimi, MA and Hosseini Bafghi, M},
title = {CRISPR-Cas9 technology: As an efficient genome modification tool in the cancer diagnosis and treatment.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28603},
pmid = {37986642},
issn = {1097-0290},
abstract = {Cancer is the second most common cause of death globally and is a major public health concern. Managing this disease is difficult due to its multiple stages and numerous genetic and epigenetic changes. Traditional cancer diagnosis and treatment methods have limitations, making it crucial to develop new modalities to combat the increasing burden of cancer. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has transformed genetic engineering due to its simplicity, specificity, low cytotoxicity, and cost-effectiveness. It has been proposed as an effective technology to enhance cancer diagnosis and treatment strategies. This article presents the most recent discoveries regarding the structure, mechanism, and delivery methods of the highly powerful genome editing tool, CRISPR-Cas9. In terms of diagnosis, the article examines the role of CRISPR-Cas9 in detecting microRNAs and DNA methylation, and discusses two popular gene detection techniques that utilize the CRISPR-Cas system: DNA endonuclease-targeted CRISPR trans reporter and specific high sensitivity enzymatic reporter unlocking. Regarding treatment, the article explores several genes that have been identified and modified by CRISPR-Cas9 for effective tumorigenesis of common cancers such as breast, lung, and colorectal cancer. The present review also addresses the challenges and ethical issues associated with using CRISPR-Cas9 as a diagnostic and therapeutic tool. Despite some limitations, CRISPR-Cas9-based cancer diagnosis has the potential to become the next generation of cancer diagnostic tools, and the continuous progress of CRISPR-Cas9 can greatly aid in cancer treatment.},
}
@article {pmid37985550,
year = {2023},
author = {Ramos, JN and Araújo, MRB and Sant'Anna, LO and Bokermann, S and Camargo, CH and Prates, FD and Sacchi, CT and Vieira, VV and Campos, KR and Santos, MBN and Viana, MVC and Azevedo, V and Aburjaile, FF and de Mattos-Guaraldi, AL and Dos Santos, LS},
title = {Molecular characterization and whole-genome sequencing of Corynebacterium diphtheriae causing skin lesion.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {37985550},
issn = {1435-4373},
abstract = {We present a case of skin lesion caused by nontoxigenic Corynebacterium diphtheriae. Genomic taxonomy analyses corroborated the preliminary identification provided by mass spectrometry. The strain showed a susceptible phenotype with increased exposure to penicillin, the first drug of choice for the treatment. An empty type 1 class integron carrying only the sul1 gene, which encodes sulfonamide resistance, was found flanked by transposases. Virulence factors involved in adherence and iron uptake, as well as the CRISPR-Cas system, were predicted. MLST analysis revealed the ST-681, previously reported in French Guiana, a European territory.},
}
@article {pmid37984988,
year = {2024},
author = {Geng, K and Merino, LG and Veiga, RG and Sommerauer, C and Epperlein, J and Brinkman, EK and Kutter, C},
title = {Intrinsic deletion at 10q23.31, including the PTEN gene locus, is aggravated upon CRISPR-Cas9-mediated genome engineering in HAP1 cells mimicking cancer profiles.},
journal = {Life science alliance},
volume = {7},
number = {2},
pages = {},
pmid = {37984988},
issn = {2575-1077},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; Chromosome Structures ; Phenotype ; *Neoplasms/genetics ; PTEN Phosphohydrolase/genetics ; },
abstract = {The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9-modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9-modified cells, which could impact cancer research.},
}
@article {pmid37984590,
year = {2023},
author = {Storz, U},
title = {The CRISPR Cas patent files, part 1: Cas9 - where to we stand at the 10 year halftime ?.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2023.11.003},
pmid = {37984590},
issn = {1873-4863},
abstract = {CRISPR Cas9 has turned out to be one of the most influential technologies in the life sciences. However, ferocious patent debates and an unclear licensing situation makes access to this technology difficult for Small and medium enterprises. This article gives an overview of the status quo 10 years after the seminal patents were filed.},
}
@article {pmid37984056,
year = {2023},
author = {Puchta, H and Houben, A},
title = {Plant chromosome engineering - past, present and future.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.19414},
pmid = {37984056},
issn = {1469-8137},
support = {//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
abstract = {Spontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X-ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double-strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing.},
}
@article {pmid37983496,
year = {2023},
author = {Altae-Tran, H and Shmakov, SA and Makarova, KS and Wolf, YI and Kannan, S and Zhang, F and Koonin, EV},
title = {Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {48},
pages = {e2308224120},
doi = {10.1073/pnas.2308224120},
pmid = {37983496},
issn = {1091-6490},
support = {NA//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {The TnpB proteins are transposon-associated RNA-guided nucleases that are among the most abundant proteins encoded in bacterial and archaeal genomes, but whose functions in the transposon life cycle remain unknown. TnpB appears to be the evolutionary ancestor of Cas12, the effector nuclease of type V CRISPR-Cas systems. We performed a comprehensive census of TnpBs in archaeal and bacterial genomes and constructed a phylogenetic tree on which we mapped various features of these proteins. In multiple branches of the tree, the catalytic site of the TnpB nuclease is rearranged, demonstrating structural and probably biochemical malleability of this enzyme. We identified numerous cases of apparent recruitment of TnpB for other functions of which the most common is the evolution of type V CRISPR-Cas effectors on about 50 independent occasions. In many other cases of more radical exaptation, the catalytic site of the TnpB nuclease is apparently inactivated, suggesting a regulatory function, whereas in others, the activity appears to be retained, indicating that the recruited TnpB functions as a nuclease, for example, as a toxin. These findings demonstrate remarkable evolutionary malleability of the TnpB scaffold and provide extensive opportunities for further exploration of RNA-guided biological systems as well as multiple applications.},
}
@article {pmid37948952,
year = {2024},
author = {Gong, T and Liao, L and Jiang, B and Yuan, R and Xiang, Y},
title = {Ag[+]-stabilized DNA triplex coupled with catalytic hairpin assembly and CRISPR/Cas12a amplifications for sensitive metallothionein assay.},
journal = {Talanta},
volume = {268},
number = {Pt 2},
pages = {125392},
doi = {10.1016/j.talanta.2023.125392},
pmid = {37948952},
issn = {1873-3573},
mesh = {Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; DNA/genetics/chemistry ; DNA, Single-Stranded/genetics ; DNA Probes/genetics/chemistry ; Biomarkers ; },
abstract = {Metallothionein (MT) is a protein biomarker secreted by liver in response to the treatment for heavy metal toxicity and oncological diseases. On the basis of a new Ag[+]-stabilized DNA triplex probe (Ag[+]-SDTP), we establish a fluorescent biosensing system for high sensitivity detection of MT by combining catalytic hairpin assembly (CHA) and the CRISPR/Cas12a signal enhancements. The MT analyte complexes with Ag[+] in Ag[+]-SDTP to disrupt the triplex structure and to release the ssDNA strands, which trigger subsequent CHA formation of many protospacer adjacent motif (PAM)-containing dsDNAs from two hairpins. Cas12a/crRNA further recognizes these PAM sequences to activate its trans-catalytic activity to cyclically cleave the fluorescently quenched ssDNA reporters to recovery drastically amplified fluorescence for detecting MT down to 0.34 nM within the dynamic range of 1∼800 nM. Moreover, the sensing method is able to selectively discriminate MT from other non-specific molecules and can realize low level detection of MT in diluted human serums, manifesting its potentiality for monitoring the disease-specific MT biomarker at trace levels.},
}
@article {pmid37837800,
year = {2023},
author = {Jin, S and Yong, H and Liu, Y and Bao, W},
title = {CRISPR/Cas9-mediated high-mobility group A2 knockout inhibits cell proliferation and invasion in papillary thyroid carcinoma cells.},
journal = {Advances in medical sciences},
volume = {68},
number = {2},
pages = {409-416},
doi = {10.1016/j.advms.2023.10.001},
pmid = {37837800},
issn = {1898-4002},
mesh = {Humans ; Thyroid Cancer, Papillary/genetics/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Thyroid Neoplasms/metabolism ; Cell Proliferation/genetics ; Necrosis/genetics ; Gene Expression Regulation, Neoplastic ; *MicroRNAs/genetics ; Neoplasm Invasiveness/genetics ; },
abstract = {PURPOSE: Metastasis and recurrence are the prognostic risk factor in patients with thyroid carcinoma. High-mobility group A2 (HMGA2) protein plays a crucial role in papillary thyroid carcinoma (PTC) metastasis. The aim of this study was to investigate the mechanisms underlying the HMGA2 effect on PTC cell proliferation and invasion.<br><br>MATERIALS AND METHODS: We used the CRISPR/Cas9 system to perform knockout of the HMGA2 gene in the human PTC cell line TPC-1. The knockout monoclonal cells were screened and verified by PCR analysis and genomic sequencing. Cell proliferation was examined after the knockout of the HMGA2 gene using cell counting kit-8 (CCK-8) assays. Furthermore, cell migration and invasion after the knockout were examined by cell scratch tests. Additionally, the changes in cell cycle and apoptosis after the knockout were detected by flow cytometry.<br><br>RESULTS: The results of the PCR analysis and the genomic sequencing confirmed that the human PTC TPC-1&#xa0;&#x200b;cell line with knockout of HMGA2 gene was successfully established. The knockout of the HMGA2 gene significantly reduced the cell proliferation, growth, and invasion. Meanwhile, the knockout of the HMGA2 gene delayed the conversion of the G2/M phase and promoted cell necrosis.<br><br>CONCLUSION: The CRISPR/Cas9-mediated HMGA2 knockout in the TPC-1&#xa0;&#x200b;cell line inhibited cell proliferation and invasion, which might be due to the blockage of the cell cycle in the G2/M phase and the promotion of cell necrosis.},
}
@article {pmid37802457,
year = {2023},
author = {Xin, Q and Jia, H and Wang, B and Pan, L},
title = {CRISPR-dCpf1 mediated whole genome crRNA inhibition library for high-throughput screening of growth characteristic genes in Bacillus amyloliquefaciens LB1ba02.},
journal = {International journal of biological macromolecules},
volume = {253},
number = {Pt 5},
pages = {127179},
doi = {10.1016/j.ijbiomac.2023.127179},
pmid = {37802457},
issn = {1879-0003},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Screening Assays ; Operon ; },
abstract = {Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.},
}
@article {pmid37683750,
year = {2023},
author = {Zhang, S and Wang, B and Li, Q and Hui, W and Yang, L and Wang, Z and Zhang, W and Yue, F and Liu, N and Li, H and Lu, F and Zhang, K and Zeng, Q and Wu, AM},
title = {CRISPR/Cas9 mutated p-coumaroyl shikimate 3'-hydroxylase 3 gene in Populus tomentosa reveals lignin functioning on supporting tree upright.},
journal = {International journal of biological macromolecules},
volume = {253},
number = {Pt 3},
pages = {126762},
doi = {10.1016/j.ijbiomac.2023.126762},
pmid = {37683750},
issn = {1879-0003},
mesh = {*Lignin/metabolism ; Mixed Function Oxygenases/metabolism ; Trees ; *Populus/metabolism ; CRISPR-Cas Systems/genetics ; Flavonoids/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/metabolism ; },
abstract = {The lignin plays one of the most important roles in plant secondary metabolism. However, it is still unclear how lignin can contribute to the impressive height of wood growth. In this study, C3'H, a rate-limiting enzyme of the lignin pathway, was used as the target gene. C3'H3 was knocked out by CRISPR/Cas9 in Populus tomentosa. Compared with wild-type popular trees, c3'h3 mutants exhibited dwarf phenotypes, collapsed xylem vessels, weakened phloem thickening, decreased hydraulic conductivity and photosynthetic efficiency, and reduced auxin content, except for reduced total lignin content and significantly increased H-subunit lignin. In the c3'h3 mutant, the flavonoid biosynthesis genes CHS, CHI, F3H, DFR, ANR, and LAR were upregulated, and flavonoid metabolite accumulations were detected, indicating that decreasing the lignin biosynthesis pathway enhanced flavonoid metabolic flux. Furthermore, flavonoid metabolites, such as naringenin and hesperetin, were largely increased, while higher hesperetin content suppressed plant cell division. Thus, studying the c3'h3 mutant allows us to deduce that lignin deficiency suppresses tree growth and leads to the dwarf phenotype due to collapsed xylem and thickened phloem, limiting material exchanges and transport.},
}
@article {pmid37982648,
year = {2023},
author = {Liu, Y and Binda, CS and Berkhout, B and Das, AT},
title = {CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0133423},
doi = {10.1128/jvi.01334-23},
pmid = {37982648},
issn = {1098-5514},
abstract = {Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches.},
}
@article {pmid37980404,
year = {2023},
author = {Moon, J and Liu, C},
title = {Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {7504},
pmid = {37980404},
issn = {2041-1723},
support = {U01 AI148306/AI/NIAID NIH HHS/United States ; U01 CA269147/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; RNA, Guide, CRISPR-Cas Systems ; *MicroRNAs/genetics ; Biological Assay ; *Nucleic Acids ; Technology ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification and the lack of quantitative ability. Here, we develop an asymmetric CRISPR assay for cascade signal amplification detection of nucleic acids by leveraging the asymmetric trans-cleavage behavior of competitive crRNA. We discover that the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a can induce cascade signal amplification, significantly improving the target detection signal. In addition, we find that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, enabling direct RNA detection by Cas12a. Based on these findings, we apply our asymmetric CRISPR assay to quantitatively detect microRNA without the need for pre-amplification, achieving a detection sensitivity of 856 aM. Moreover, using this method, we analyze and quantify miR-19a biomarker in plasma samples from bladder cancer patients. This asymmetric CRISPR assay has the potential to be widely applied for simple and sensitive nucleic acid detection in various diagnostic settings.},
}
@article {pmid37980345,
year = {2023},
author = {Chen, Q and Chuai, G and Zhang, H and Tang, J and Duan, L and Guan, H and Li, W and Li, W and Wen, J and Zuo, E and Zhang, Q and Liu, Q},
title = {Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {7521},
pmid = {37980345},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; DNA/genetics ; },
abstract = {The powerful CRISPR genome editing system is hindered by its off-target effects, and existing computational tools achieved limited performance in genome-wide off-target prediction due to the lack of deep understanding of the CRISPR molecular mechanism. In this study, we propose to incorporate molecular dynamics (MD) simulations in the computational analysis of CRISPR system, and present CRISOT, an integrated tool suite containing four related modules, i.e., CRISOT-FP, CRISOT-Score, CRISOT-Spec, CRISORT-Opti for RNA-DNA molecular interaction fingerprint generation, genome-wide CRISPR off-target prediction, sgRNA specificity evaluation and sgRNA optimization of Cas9 system respectively. Our comprehensive computational and experimental tests reveal that CRISOT outperforms existing tools with extensive in silico validations and proof-of-concept experimental validations. In addition, CRISOT shows potential in accurately predicting off-target effects of the base editors and prime editors, indicating that the derived RNA-DNA molecular interaction fingerprint captures the underlying mechanisms of RNA-DNA interaction among distinct CRISPR systems. Collectively, CRISOT provides an efficient and generalizable framework for genome-wide CRISPR off-target prediction, evaluation and sgRNA optimization for improved targeting specificity in CRISPR genome editing.},
}
@article {pmid37947312,
year = {2023},
author = {Chen, R and Zhao, J and Han, M and Dong, Y and Jiang, F and Chen, Y},
title = {DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {46},
pages = {18037-18045},
doi = {10.1021/acs.jafc.3c06530},
pmid = {37947312},
issn = {1520-5118},
mesh = {Argonaute Proteins ; CRISPR-Cas Systems ; *Bacteriophages ; *Nucleic Acids ; DNA ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; },
abstract = {A novel method for detecting low levels of viable foodborne pathogens, specifically Salmonella typhimurium (S. typhimurium), has been developed. Traditional nucleic acid assay, such as polymerase chain reaction (PCR), often requires complex DNA extraction and amplification, making it challenging to differentiate between viable and nonviable pathogens. This assay employed a phage as the recognition element to precisely identify and lyse viable S. typhimurium that can undergo DNA extraction. It combined the efficient trans-cleavage activities of CRISPR/Cas12a with the specific cleavage advantages of Argonaute proteins, enabling ultrasensitive detection. This double-enzyme-mediated nucleic acid test can accurately distinguish viable and nonviable S. typhimurium with a detection limit of 23 CFU/mL without DNA amplification. The method was successfully applied to common food samples, producing results consistent with quantitative PCR tests. This work provides a promising platform for easily detecting viable foodborne pathogens with high sensitivity without the need for DNA extraction and amplification.},
}
@article {pmid37922572,
year = {2023},
author = {Viswan, A and Yoshikawa, C and Yamagishi, A and Furuhata, Y and Kato, Y and Yamazaki, T and Nakamura, C},
title = {Efficient genome editing by controlled release of Cas9 ribonucleoprotein in plant cytosol using polymer-modified microneedle array.},
journal = {Biochemical and biophysical research communications},
volume = {686},
number = {},
pages = {149179},
doi = {10.1016/j.bbrc.2023.149179},
pmid = {37922572},
issn = {1090-2104},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Ribonucleoproteins/genetics/metabolism ; Cytosol/metabolism ; Delayed-Action Preparations ; DNA ; Genome, Plant/genetics ; },
abstract = {Direct delivery of genome-editing proteins into plant tissues could be useful in obtaining DNA-free genome-edited crops obviating the need for backcrossing to remove vector-derived DNA from the host genome as in the case of genetically modified organisms generated using DNA vector. Previously, we successfully delivered Cas9 ribonucleoprotein (RNP) into plant tissue by inserting microneedle array (MNA) physisorbed with Cas9 RNPs. Here, to enhance protein delivery and improve genome-editing efficiency, we introduced a bioactive polymer DMA/HPA/NHS modification to the MNA, which allowed strong bonding between the proteins and MNA. Compared with other modifying agents, this MNA modification resulted in better release of immobilized protein in a plant cytosol-mimicking environment. The delivery of Cas9 RNPs in Arabidopsis thaliana reporter plants was improved from 4 out of 17 leaf tissues when using unmodified MNAs to 9 out of 17 when using the polymer-modified MNAs. Further improvements in delivery efficiency can be envisaged by optimizing the polymer modification conditions, which could have significant implications for the development of more effective plant genome editing techniques.},
}
@article {pmid37844014,
year = {2023},
author = {Pradhan, SK and Karuppannasamy, A and Sujatha, PM and Nagaraja, BC and Narayanappa, AC and Chalapathi, P and Dhawane, Y and Bynakal, S and Riegler, M and Maligeppagol, M and Ramasamy, A},
title = {Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of white gene in melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) produced white eye phenotype.},
journal = {Archives of insect biochemistry and physiology},
volume = {114},
number = {4},
pages = {e22059},
doi = {10.1002/arch.22059},
pmid = {37844014},
issn = {1520-6327},
support = {//Centre for Agricultural Bioinformatics/ ; },
mesh = {Animals ; *Tephritidae/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; *Cucurbitaceae/genetics ; Microinjections ; Phenotype ; Ribonucleoproteins/genetics ; },
abstract = {Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNA to target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.},
}
@article {pmid37657567,
year = {2023},
author = {Ashok, K and Bhargava, CN and Asokan, R and Pradeep, C and Kennedy, JS and Manamohan, M and Rai, A},
title = {CRISPR/Cas9 mediated mutagenesis of the major sex pheromone gene, acyl-CoA delta-9 desaturase (DES9) in Fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).},
journal = {International journal of biological macromolecules},
volume = {253},
number = {Pt 2},
pages = {126557},
doi = {10.1016/j.ijbiomac.2023.126557},
pmid = {37657567},
issn = {1879-0003},
mesh = {Female ; Male ; Animals ; Spodoptera/genetics ; *Sex Attractants/genetics ; RNA, Guide, CRISPR-Cas Systems ; Stearoyl-CoA Desaturase/genetics ; CRISPR-Cas Systems/genetics ; *Moths/genetics ; Mutagenesis ; },
abstract = {The Fall armyworm, Spodoptera frugiperda is a significant global pest causing serious yield loss on several staple crops. In this regard, this pest defies several management approaches based on chemicals, Bt transgenics etc., requiring effective alternatives. Recently CRISPR/Cas9 mediated genome editing has opened up newer avenues to establish functions of various target genes before employing them for further application. The virgin female moths of S. frugiperda emit sex pheromones to draw conspecific males. Therefore, we have edited the key pheromone synthesis gene, fatty acyl-CoA Delta-9 desaturase (DES9) of the Indian population of S. frugiperda. In order to achieve a larger deletion of the DES9, we have designed two single guide RNA (sgRNA) in sense and antisense direction targeting the first exon instead of a single guide RNA. The sgRNA caused site-specific knockout with a larger deletion which impacted the mating. Crossing studies between wild male and mutant female resulted in no fecundity, while fecundity was normal when mutant male crossed with the wild female. This indicates that mating disruption is stronger in females where DES9 is mutated. The current work is the first of its kind to show that DES9 gene editing impacted the likelihood of mating in S. frugiperda.},
}
@article {pmid37978935,
year = {2023},
author = {Li, Y and Liu, Y and Tang, X and Qiao, J and Kou, J and Man, S and Zhu, L and Ma, L},
title = {CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.3c01463},
pmid = {37978935},
issn = {2379-3694},
abstract = {CRISPR/Cas system is becoming an increasingly influential technology that has been repositioned in nucleic acid detection. A preamplification step is usually required to improve the sensitivity of CRISPR/Cas-based detection. The striking biological features of CRISPR/Cas, including programmability, high sensitivity and sequence specificity, and single-base resolution. More strikingly, the target-activated trans-cleavage could act as a biocatalytic signal transductor and amplifier, thereby empowering it to potentially perform nucleic acid detection without a preamplification step. The reports of such work are on the rise, which is not only scientifically significant but also promising for futuristic end-user applications. This review started with the introduction of the detection methods of nucleic acids and the CRISPR/Cas-based diagnostics (CRISPR-Dx). Next, we objectively discussed the pros and cons of preamplification steps for CRISPR-Dx. We then illustrated and highlighted the recently developed strategies for CRISPR/Cas-powered amplification-free detection that can be realized through the uses of ultralocalized reactors, cascade reactions, ultrasensitive detection systems, or others. Lastly, the challenges and futuristic perspectives were proposed. It can be expected that this work not only makes the researchers better understand the current strategies for this emerging field, but also provides insight for designing novel CRISPR-Dx without a preamplification step to win practicable use in the near future.},
}
@article {pmid37977804,
year = {2023},
author = {Wang, W and Geng, L and Zhang, Y and Shen, W and Bi, M and Gong, T and Hu, Z and Guo, C and Wang, T and Sun, T},
title = {Development of antibody-aptamer sandwich-like immunosensor based on RCA and Nicked-PAM CRISPR/Cas12a system for the ultra-sensitive detection of a biomarker.},
journal = {Analytica chimica acta},
volume = {1283},
number = {},
pages = {341849},
doi = {10.1016/j.aca.2023.341849},
pmid = {37977804},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; Immunoassay ; Antibodies ; Biomarkers ; C-Reactive Protein ; Oligonucleotides ; },
abstract = {Biomarkers are the most sensitive reactants and early indicators of many kinds of diseases. The development of highly sensitive and simple techniques to quantify them is challenging. In this study, based on rolling cycle amplification (RCA) and the Nicked PAM/CRISPR-Cas12a system (RNPC) as a signal reporter, a sandwich-type method was developed using antibody@magnetic beads and aptamer for the high-sensitive detection of the C-reactive protein (CRP). The antibody-antigen (target)-aptamer sandwich-like reaction was coupled to RCA, which can produce hundreds of similar binding sites and are discriminated by CRISPR/Cas12a for signal amplification. The ultrasensitivity is achieved based on the dual-signal enhancing strategy, which involves the special recognition of aptamers, RCA, and trans-cleavage of CRISPR/Cas12a. By incorporating the CRISPR/Cas12a system with cleaved PAM, the nonspecific amplification of the RCA reaction alone was greatly reduced, and the dual signal output of RCA and Cas12a improved the detection sensitivity. Our assay can be performed only in two steps. The first step takes only 20 min of target capture, followed by a one-pot reaction, where the target concentration can be obtained by fluorescence values as long as there are 37 °C reaction conditions. Under optimal conditions, this system detected CRP with high sensitivity. The fabricated biosensor showed detection limits of 0.40 pg/mL in phosphate-buffered saline and 0.73 pg/mL in diluted human serum and a broad linear dynamic range of 1.28 pg/mL to 100 ng/mL within a total readout time of 90 min. The method could be used to perform multi-step signal amplification, which can help in the ultrasensitive detection of other proteins. Overall, the proposed biosensor might be used as an immunosensor biosensor platform.},
}
@article {pmid37975669,
year = {2023},
author = {Lammens, E-M and Volke, DC and Schroven, K and Voet, M and Kerremans, A and Lavigne, R and Hendrix, H},
title = {A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0270723},
doi = {10.1128/spectrum.02707-23},
pmid = {37975669},
issn = {2165-0497},
abstract = {The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.},
}
@article {pmid37974198,
year = {2023},
author = {Ozyerli-Goknar, E and Kala, EY and Aksu, AC and Bulut, I and Cingöz, A and Nizamuddin, S and Biniossek, M and Seker-Polat, F and Morova, T and Aztekin, C and Kung, SHY and Syed, H and Tuncbag, N and Gönen, M and Philpott, M and Cribbs, AP and Acilan, C and Lack, NA and Onder, TT and Timmers, HTM and Bagci-Onder, T},
title = {Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.},
journal = {Cell communication and signaling : CCS},
volume = {21},
number = {1},
pages = {328},
pmid = {37974198},
issn = {1478-811X},
support = {MR/V010182/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; Cell Survival ; *Nuclear Proteins/metabolism ; *Glioblastoma/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; DNA-Binding Proteins/metabolism ; Transcription Factors/genetics/metabolism ; Kinesins/genetics/metabolism ; },
abstract = {BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers.<br><br>METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers.<br><br>RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&amp;RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L.<br><br>CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.},
}
@article {pmid37972178,
year = {2023},
author = {Dhingra, Y and Sashital, DG},
title = {A tool for more specific DNA integration.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6672},
pages = {768-769},
doi = {10.1126/science.adl0863},
pmid = {37972178},
issn = {1095-9203},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Gene Editing ; DNA Transposable Elements ; },
abstract = {The efficiency of targeted DNA insertion by CRISPR transposons is improved.},
}
@article {pmid37972161,
year = {2023},
author = {George, JT and Acree, C and Park, JU and Kong, M and Wiegand, T and Pignot, YL and Kellogg, EH and Greene, EC and Sternberg, SH},
title = {Mechanism of target site selection by type V-K CRISPR-associated transposases.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6672},
pages = {eadj8543},
doi = {10.1126/science.adj8543},
pmid = {37972161},
issn = {1095-9203},
mesh = {*DNA Transposable Elements ; Transposases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy ; RNA ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.},
}
@article {pmid37924654,
year = {2024},
author = {Li, Y and Cai, M and Zhang, W and Liu, Y and Yuan, X and Han, N and Li, J and Jin, S and Ding, C},
title = {Cas12a-based direct visualization of nanoparticle-stabilized fluorescence signal for multiplex detection of DNA methylation biomarkers.},
journal = {Biosensors &amp; bioelectronics},
volume = {244},
number = {},
pages = {115810},
doi = {10.1016/j.bios.2023.115810},
pmid = {37924654},
issn = {1873-4235},
mesh = {DNA Methylation ; CRISPR-Cas Systems/genetics ; Reproducibility of Results ; *Biosensing Techniques ; Biomarkers ; *Nucleic Acids ; *Nanoparticles ; Nucleic Acid Amplification Techniques ; },
abstract = {The CRISPR-Cas12a RNA-guided complexes hold immense promise for nucleic acid detection. However, limitations arise from their specificity in detecting off-targets and the stability of the signal molecules. Here, we have developed a platform that integrates multiplex amplification and nanomolecular-reporting signals, allowing us to detect various clinically relevant nucleic acid targets with enhanced stability, sensitivity, and visual interpretation. Through the electrostatic co-assembly of the Oligo reporter with oppositely charged nanoparticles, we observed a significant enhancement in its stability in low-pollution environments, reaching up to a threefold increase compared to the original version. Additionally, the fluorescence efficiency was expanded by three orders of magnitude, broadening the detection range considerably. Utilizing a multiplex strategy, this assay can accomplish simultaneous detection of multiple targets and single-point indication detection of nine specific targets. This significant advancement heightened the sensitivity of disease screening and improved the accuracy of diagnosing disease-related changes. We tested this assay in a colorectal cancer model, demonstrating that it can identify DNA methylation features at the aM-level within 40-60 min. Validation using clinical samples yielded consistent results with qPCR and bisulfite sequencing, affirming the assay's reliability and potential for clinical applications.},
}
@article {pmid37698072,
year = {2023},
author = {Lin, Z and Chen, L and Tang, S and Zhao, M and Li, T and You, J and You, C and Li, B and Zhao, Q and Zhang, D and Wang, J and Shen, Z and Song, X and Zhang, S and Cao, X},
title = {Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).},
journal = {Journal of integrative plant biology},
volume = {65},
number = {11},
pages = {2416-2420},
doi = {10.1111/jipb.13567},
pmid = {37698072},
issn = {1744-7909},
support = {2021ZD0031//Key Projects in Science and Technology of Inner Mongolia/ ; XDA26030202//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; CAS (2022096)//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Poaceae/genetics ; Agrobacterium/genetics ; },
abstract = {The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.},
}
@article {pmid37972155,
year = {2023},
author = {Kaiser, J},
title = {Rewriting DNA in the body lowers cholesterol.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6672},
pages = {751},
doi = {10.1126/science.adm9506},
pmid = {37972155},
issn = {1095-9203},
mesh = {Humans ; *DNA/genetics ; *Cholesterol ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Verve Therapeutics says its base-editing approach may help prevent heart disease in many people.},
}
@article {pmid37971255,
year = {2023},
author = {Martínez-Alvarez, L and Ramond, J-B and Vikram, S and León-Sobrino, C and Maggs-Kölling, G and Cowan, DA},
title = {With a pinch of salt: metagenomic insights into Namib Desert salt pan microbial mats and halites reveal functionally adapted and competitive communities.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0062923},
doi = {10.1128/aem.00629-23},
pmid = {37971255},
issn = {1098-5336},
abstract = {The hyperarid Namib Desert is one of the oldest deserts on Earth. It contains multiple clusters of playas which are saline-rich springs surrounded by halite evaporites. Playas are of great ecological importance, and their indigenous (poly)extremophilic microorganisms are potentially involved in the precipitation of minerals such as carbonates and sulfates and have been of great biotechnological importance. While there has been a considerable amount of microbial ecology research performed on various Namib Desert edaphic microbiomes, little is known about the microbial communities inhabiting its multiple playas. In this work, we provide a comprehensive taxonomic and functional potential characterization of the microbial, including viral, communities of sediment mats and halites from two distant salt pans of the Namib Desert, contributing toward a better understanding of the ecology of this biome.},
}
@article {pmid37968435,
year = {2023},
author = {Cui, C and Chen, TH},
title = {CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity.},
journal = {Mikrochimica acta},
volume = {190},
number = {12},
pages = {468},
pmid = {37968435},
issn = {1436-5073},
support = {11217820//Hong Kong Research Grants Council/ ; N_CityU119/19//Hong Kong Research Grants Council/ ; JCYJ20210324134006017//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 9678242//City University of Hong Kong/ ; 7020072//City University of Hong Kong/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Glycosylases ; DNA/genetics/metabolism ; Guanine ; },
abstract = {Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 μU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.},
}
@article {pmid37966867,
year = {2023},
author = {Guo, J and Zhu, Y and Miao, P},
title = {Nano-Impact Electrochemical Biosensing Based on a CRISPR-Responsive DNA Hydrogel.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.3c03448},
pmid = {37966867},
issn = {1530-6992},
abstract = {Nano-impact electrochemistry (NIE) enables simple, rapid, and high-throughput biocoupling and biomolecular recognition. However, the low effective collision frequency limits the sensitivity. In this study, we propose a novel NIE sensing strategy amplified by the CRISPR-responsive DNA hydrogel and cascade DNA assembly. By controlling the phase transition of DNA hydrogel and the self-electrolysis of silver nanoparticles, we can obtain significant electrochemical responses. The whole process includes target miRNA-induced strand displacement amplification, catalytic hairpin assembly, and CRISPR/Cas trans-cutting. Thus, ultrahigh sensitivity is promised. This NIE biosensing strategy achieves a limit of detection as low as 4.21 aM for miR-141 and demonstrates a high specificity for practical applications. It may have wide applicability in nucleic acid sensing and shows great potential in disease diagnosis.},
}
@article {pmid37965049,
year = {2023},
author = {Koller, F and Cieslak, M},
title = {A perspective from the EU: unintended genetic changes in plants caused by NGT-their relevance for a comprehensive molecular characterisation and risk assessment.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1276226},
pmid = {37965049},
issn = {2296-4185},
abstract = {Several regions in the world are currently holding discussions in regard to the regulation of new genomic techniques (NGTs) and their application in agriculture. The European Commission, for instance, is proposing the introduction of specific regulation for NGT plants. Various questions need to be answered including e.g., the extent to which NGT-induced intended and unintended genetic modifications must be subjected to a mandatory risk assessment as part of an approval procedure. This review mostly focuses on findings in regard to unintended genetic changes that can be caused by the application of NGTs. More specifically, the review deals with the application of the nuclease CRISPR/Cas, which is currently the most important tool for developing NGT plants, and its potential to introduce double strand breaks (DSBs) at a targeted DNA sequence. For this purpose, we identified the differences in comparison to non-targeted mutagenesis methods used in conventional breeding. The review concludes that unintended genetic changes caused by NGT processes are relevant to risk assessment. Due to the technical characteristics of NGTs, the sites of the unintended changes, their genomic context and their frequency (in regard to specific sites) mean that the resulting gene combinations (intended or unintended) may be unlikely to occur with conventional methods. This, in turn, implies that the biological effects (phenotypes) can also be different and may cause risks to health and the environment. Therefore, we conclude that the assessment of intended as well as unintended genetic changes should be part of a mandatory comprehensive molecular characterisation and risk assessment of NGT plants that are meant for environmental releases or for market authorisation.},
}
@article {pmid37965048,
year = {2023},
author = {Dhokane, D and Shaikh, A and Yadav, A and Giri, N and Bandyopadhyay, A and Dasgupta, S and Bhadra, B},
title = {CRISPR-based bioengineering in microalgae for production of industrially important biomolecules.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1267826},
pmid = {37965048},
issn = {2296-4185},
abstract = {Microalgae, as photosynthetic organisms, have the potential to produce biomolecules for use in food, feed, cosmetics, nutraceuticals, fuel, and other applications. Faster growth rates and higher protein and lipid content make microalgae a popular chassis for many industrial applications. However, challenges such as low productivity and high production costs have limited their commercialization. To overcome these challenges, bioengineering approaches such as genetic engineering, metabolic engineering, and synthetic biology have been employed to improve the productivity and quality of microalgae-based products. Genetic engineering employing genome editing tools like CRISPR/Cas allows precise and targeted genetic modifications. CRISPR/Cas systems are presently used to modify the genetic makeup of microalgae for enhanced production of specific biomolecules. However, these tools are yet to be explored explicitly in microalgae owing to some limitations. Despite the progress made in CRISPR-based bioengineering approaches, there is still a need for further research to optimize the production of microalgae-based products. This includes improving the efficiency of genome editing tools, understanding the regulatory mechanisms of microalgal metabolism, and optimizing growth conditions and cultivation strategies. Additionally, addressing the ethical, social, and environmental concerns associated with genetic modification of microalgae is crucial for the responsible development and commercialization of microalgae-based products. This review summarizes the advancements of CRISPR-based bioengineering for production of industrially important biomolecules and provides key considerations to use CRISPR/Cas systems in microalgae. The review will help researchers to understand the progress and to initiate genome editing experiments in microalgae.},
}
@article {pmid37962270,
year = {2023},
author = {Labuschagne, M},
title = {Biofortification to improve food security.},
journal = {Emerging topics in life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1042/ETLS20230066},
pmid = {37962270},
issn = {2397-8554},
abstract = {Crop biofortification has significantly progressed in the last few decades. The first biofortification success was quality protein maize, leading to double the amount of the essential amino acids lysine and tryptophan. This was followed by biofortification of staple crops such as maize, wheat, rice, legumes and cassava for nutrients such as Fe and Zn and provitamin A. These crops have reached millions of households, especially in the developing regions of the world. The development and release of these biofortified crops through conventional breeding generally took 8-10 years. To speed up the process, molecular markers, genome-wide association studies and genomic selection have been incorporated into breeding efforts. Genetic engineering has the potential to increase the efficiency of crop biofortification through multi-nutrient biofortification in a short timespan and to combine biofortification with climate resilience. Regulatory issues still prevent the dissemination of genetically modified crops in many countries. This could be overcome by CRISPR-Cas-mediated genome editing, as it seems that many countries will regulate products of genome editing less strictly than transgenic crops. Effective policies on national or regional level are needed for the sustainable production of biofortified crops. The availability of affordable quality biofortified seed and other inputs should be ensured through local seed systems, which will increase the production and adoption of biofortified crops. There is scope to expand the crops and the range of nutrients for biofortification. Genetic engineering should be combined with conventional breeding as a approach for future improvement of multi-nutrient crops.},
}
@article {pmid37958714,
year = {2023},
author = {Wang, L and Sun, J and Liu, Z and Zheng, Q and Wang, G},
title = {Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining.},
journal = {International journal of molecular sciences},
volume = {24},
number = {21},
pages = {},
pmid = {37958714},
issn = {1422-0067},
support = {No. 30500-5195126//Inner Mongolia University Start-Up Found/ ; No.10000-21311201/004//Inner Mongolia University High-level Talents Award] under Grant/ ; },
mesh = {Animals ; *Chickens/genetics ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; 3' Untranslated Regions ; Gene Knock-In Techniques ; Transgenes ; Gene Editing ; },
abstract = {Precision exogenous gene knock-in is an attractive field for transgenic Gallus gallus (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3'UTR of GAPDH near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in GAPDH could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of GAPDH, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3'UTR of GAPDH on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (DAZL) and actin beta (ACTB) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.},
}
@article {pmid37958206,
year = {2023},
author = {Li, X and Zhong, Y and Qiao, Y and Li, H and Hu, X and Imani, S and Zheng, S and Li, J},
title = {Advances and Challenges in Cytomegalovirus Detection Methods for Liver Transplant Donors.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {13},
number = {21},
pages = {},
pmid = {37958206},
issn = {2075-4418},
support = {202311842021X//Xiaoping Li/ ; ZDKJ2019009//Shusen Zheng/ ; LGF21H030006//Shusen Zheng/ ; JNL-2022002A, JNL-2022023C//Jianhui Li/ ; KFJJ2023005//Shusen Zheng/ ; },
abstract = {Cytomegalovirus (CMV) infection is a highly prevalent opportunistic infection among liver transplant recipients. When the liver donor is infected with CMV, there is a risk of transmission to the recipient, leading to CMV infection. To improve the postoperative outcome of liver transplantation, it is crucial to shift the focus of CMV detection to the donor and achieve early diagnosis, as well as implement effective preventative and therapeutic measures. However, the commonly used CMV detection methods in the past had limitations that prevented their early and accurate diagnosis in liver transplant donors. This review focuses on the latest advancements in CMV detection methods that can potentially be applied to liver transplant donors. The objective is to compare and evaluate their clinical utility, thereby providing guidance and support for rapid and accurate diagnosis of CMV infection in the clinic. The clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR-Cas) system-based assay emerges as a promising method for detecting the virus, offering great prospects for early and expedient CMV infection diagnosis in clinical settings.},
}
@article {pmid37957729,
year = {2023},
author = {Zhou, X and Wang, S and Ma, Y and Li, Y and Deng, G and Shi, J and Wang, X},
title = {Rapid detection of avian influenza virus based on CRISPR-Cas12a.},
journal = {Virology journal},
volume = {20},
number = {1},
pages = {261},
pmid = {37957729},
issn = {1743-422X},
support = {2016YFD0500800//National Key Research and Development Program of China/ ; LH2023C050//Heilongjiang Provincial Natural Science Foundation of China/ ; },
mesh = {Animals ; *Influenza in Birds/diagnosis ; CRISPR-Cas Systems ; *Influenza A virus ; Birds ; Poultry ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction/methods ; Newcastle disease virus/genetics ; RNA ; },
abstract = {BACKGROUND: Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations.<br><br>METHODS: The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1-H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV).<br><br>RESULTS: The results showed that the method was able to detect AIV and that the detection limit can reach 6.7&#xa0;copies/&#x3bc;L and 12&#xa0;copies/&#x3bc;L for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field.<br><br>CONCLUSION: An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.},
}
@article {pmid37957716,
year = {2023},
author = {Bustos, FJ and Pandian, S and Haensgen, H and Zhao, JP and Strouf, H and Heidenreich, M and Swiech, L and Deverman, BE and Gradinaru, V and Zhang, F and Constantine-Paton, M},
title = {Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {232},
pmid = {37957716},
issn = {1741-7007},
support = {R03 EY014420/EY/NEI NIH HHS/United States ; R01-EY014074/EY/NEI NIH HHS/United States ; R01-EY014420/EY/NEI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Autism Spectrum Disorder/genetics ; DNA Copy Number Variations ; RNA, Guide, CRISPR-Cas Systems ; Synaptic Transmission/genetics ; Genomics ; },
abstract = {BACKGROUND: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies.<br><br>RESULTS: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700&#xa0;bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300&#xa0;bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD.<br><br>CONCLUSIONS: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.},
}
@article {pmid37957573,
year = {2023},
author = {Dutta, B and Halder, U and Chitikineni, A and Varshney, RK and Bandopadhyay, R},
title = {Delving into the lifestyle of Sundarban Wetland resident, biofilm producing, halotolerant Salinicoccus roseus: a comparative genomics-based intervention.},
journal = {BMC genomics},
volume = {24},
number = {1},
pages = {681},
pmid = {37957573},
issn = {1471-2164},
mesh = {DNA, Bacterial/genetics ; *Wetlands ; *Ecosystem ; Genomics ; Biofilms ; Phylogeny ; Genome, Bacterial ; },
abstract = {BACKGROUND: Microbial community played an essential role in ecosystem processes, be it mangrove wetland or other intertidal ecologies. Several enzymatic activities like hydrolases are effective ecological indicators of soil microbial function. So far, little is known on halophilic bacterial contribution and function on a genomic viewpoint of Indian Sundarban Wetland. Considering the above mentioned issues, the aims of this study was to understand the life style, metabolic functionalities and genomic features of the isolated bacterium, Salinicoccus roseus strain RF1H. A comparative genome-based study of S. roseus has not been reported yet. Henceforth, we have considered the inclusion of the intra-species genome comparison of S. roseus to gain insight into the high degree of variation in the genome of strain RF1H among others.<br><br>RESULTS: Salinicoccus roseus strain RF1H is a pink-red pigmented, Gram-positive and non-motile cocci. The bacterium exhibited high salt tolerance (up to 15% NaCl), antibiotic resistance, biofilm formation and secretion of extracellular hydrolytic enzymes. The circular genome was approximately 2.62978&#xa0;Mb in size, encoding 574 predicted genes with GC content 49.5%. Presence of genomic elements (prophages, transposable elements, CRISPR-Cas system) represented bacterial virulence and multidrug-resistance. Furthermore, genes associated with salt tolerance, temperature adaptation and DNA repair system were distributed in 17 genomic islands. Genes related to hydrocarbon degradation manifested metabolic capability of the bacterium for potential biotechnological applications. A comparative pangenome analysis revealed two-component response regulator, modified C4-dicarboxylate transport system and osmotic stress regulated ATP-binding proteins. Presence of genes encoding arginine decarboxylase (ADC) enzyme being involved in biofilm formation was reported from the genome. In silico study revealed the protein is thermostable and made up with&#x2009;~&#x2009;415 amino acids, and hydrophilic in nature. Three motifs appeared to be evolutionary conserved in all Salinicoccus sequences.<br><br>CONCLUSION: The first report of whole genome analysis of Salinicoccus roseus strain RF1H provided information of metabolic functionalities, biofilm formation, resistance mechanism and adaptation strategies to thrive in climate-change induced vulnerable spot like Sundarban. Comparative genome analysis highlighted the unique genome content that contributed the strain's adaptability. The biomolecules produced during metabolism are important sources of compounds with potential beneficial applications in pharmaceuticals.},
}
@article {pmid37934952,
year = {2023},
author = {Guo, Q and Yan, Y and Zhang, Z and Xu, B and Bangash, HL and Sui, X and Yang, Y and Zhou, Z and Zhao, S and Peng, N},
title = {Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome Editing Tool.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3487-3496},
doi = {10.1021/acssynbio.3c00450},
pmid = {37934952},
issn = {2161-5063},
mesh = {*Gene Editing ; *Limosilactobacillus reuteri/genetics ; Endonucleases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Using genetically tractable probiotics to engineer live biotherapeutic products (LBPs) for disease treatment is urgently needed. Limosilactobacillus reuteri is an important vertebrate gut symbiont, which has great potential for developing LBPs. However, in L. reuteri, synthetic biology work is largely limited by the long editing cycle. In this study, we identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03 and found the endogenous Cas9 (LrCas9) recognizing a broad protospacer-adjacent motif (PAM) sequence (3'-NDR; N = A, G, T, C; D = A, G, T; R = A, G). We reprogrammed the LrCas9 for efficient gene deletion (95.46%), point mutation (86.36%), large fragment deletion (40 kb), and gene integration (1743 bp, 73.9%), which uncovered the function of the repeated conserved domains in mucus-binding protein. Moreover, we analyzed the distribution of endogenous endonucleases in 304 strains of L. reuteri and found the existence of programmable endonucleases in 98.36% of L. reuteri strains suggesting the potential to reprogram endogenous endonucleases for genetic manipulation in the majority of L. reuteri strains. In conclusion, this study highlights the development of a new probiotic chassis based on endogenous endonucleases in L. reuteri 03, which paves the way for the development of genome editing tools for functional genetic studies in other L. reuteri. We believe that the development of an endogenous endonuclease-based genetic tool will greatly facilitate the construction of LBPs.},
}
@article {pmid37924725,
year = {2023},
author = {Lu, Y and Chan, YT and Wu, J and Feng, Z and Yuan, H and Li, Q and Xing, T and Xu, L and Zhang, C and Tan, HY and Lee, TK and Feng, Y and Wang, N},
title = {CRISPR/Cas9 screens unravel miR-3689a-3p regulating sorafenib resistance in hepatocellular carcinoma via suppressing CCS/SOD1-dependent mitochondrial oxidative stress.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {71},
number = {},
pages = {101015},
doi = {10.1016/j.drup.2023.101015},
pmid = {37924725},
issn = {1532-2084},
mesh = {Humans ; *Carcinoma, Hepatocellular/drug therapy/genetics ; Sorafenib/pharmacology/therapeutic use ; Superoxide Dismutase-1 ; CRISPR-Cas Systems ; Copper ; Proteomics ; *Liver Neoplasms/drug therapy/genetics ; *MicroRNAs/genetics ; Superoxide Dismutase/genetics ; Oxidative Stress/genetics ; },
abstract = {AIMS: Therapeutic outcome of sorafenib in hepatocellular carcinoma (HCC) is undermined by the development of drug resistance. This study aimed to identify the critical microRNA (miRNA) which is responsible for sorafenib resistance at the genomic level.<br><br>METHODS: CRISPR/Cas9 screen followed by gain- and loss-of-function assays both in vitro and in vivo were applied to identify the role of miR-3689a-3p in mediating sorafenib response in HCC. The upstream and downstream molecules of miR-3689a-3p and their mechanism of action were investigated.<br><br>RESULTS: CRISPR/Cas9 screening identified miR-3689a-3p was the most up-regulated miRNA in sorafenib sensitive HCC. Knockdown of miR-3689a-3p significantly increased sorafenib resistance, while its overexpression sensitized HCC response to sorafenib treatment. Proteomic analysis revealed that the effect of miR-3689a-3p was related to the copper-dependent mitochondrial superoxide dismutase type 1 (SOD1) activity. Mechanistically, miR-3689a-3p targeted the 3'UTR of the intracellular copper chaperone for superoxide dismutase (CCS) and suppressed its expression. As a result, miR-3689a-3p disrupted the intracellular copper trafficking and reduced SOD1-mediated scavenge of mitochondrial oxidative stress that eventually caused HCC cell death in response to sorafenib treatment. CCS overexpression blunted sorafenib response in HCC. Clinically, miR-3689a-3p was down-regulated in HCC and predicted favorable prognosis for HCC patients.<br><br>CONCLUSION: Our findings provide comprehensive evidence for miR-3689a-3p as a positive regulator and potential druggable target for improving sorafenib treatment in HCC.},
}
@article {pmid37906167,
year = {2023},
author = {Liu, F and Qiao, K and Meng, W and Liu, J and Gao, Y and Zhu, J},
title = {Construction of a CRISPR Interference System for Gene Knockdown in Stenotrophomonas maltophilia AGS-1 from Aerobic Granular Sludge.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3497-3504},
doi = {10.1021/acssynbio.3c00480},
pmid = {37906167},
issn = {2161-5063},
mesh = {Gene Knockdown Techniques ; *Stenotrophomonas maltophilia/genetics ; Sewage ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {To identify the function of attachment genes involved in biofilm formation in Stenotrophomonas maltophilia AGS-1 isolated from aerobic granular sludge, an effective gene molecular tool is needed. We developed a two-plasmid CRISPRi system in Stenotrophomonas maltophilia AGS-1. One plasmid expressed dCas9 protein with the l-arabinose inducible promoter, and the other plasmid contained the sgRNA cassette complementary to the target gene. Under control of the araC-inducible promoter, this system exhibited little leaky basal expression and highly induced expression that silenced endogenous and exogenous genes with reversible knockdown. This system achieved up to 211-fold suppression for mCherry expression on the nontemplate strand compared to the template strand (91-fold). The utility of the developed CRISPRi platform was also characterized by suppressing the xanA and rpfF genes. The expression of these two genes was rapidly depleted and the adhesion ability decreased, which demonstrated that the modulation of either gene was an important factor for biofilm formation of the AGS-1 strain. The system also tested the ability to simultaneously silence transcriptional suppression of multiple targeted genes, an entire operon, or part of it. Lastly, the use of CRISPRi allowed us to dissect the gene intricacies involved in flagellar biosynthesis. Collectively, these results demonstrated that the CRISPRi system was a simple, feasible, and controllable manipulation system of gene expression in the AGS-1 strain.},
}
@article {pmid37881961,
year = {2023},
author = {Deng, M and Wu, Y and Lv, X and Liu, L and Li, J and Du, G and Chen, J and Liu, Y},
title = {Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3443-3453},
doi = {10.1021/acssynbio.3c00494},
pmid = {37881961},
issn = {2161-5063},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Carrier Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The industrial yeast Komagataella phaffii is a highly effective platform for heterologous protein production, owing to its high protein expression and secretion capacity. Heterologous genes and proteins are involved in multiple processes, including transcription, translation, protein folding, modification, transportation, and degradation; however, engineering these proteins and genes is challenging due to inefficient genome editing techniques. We employed Pseudomonas aeruginosa phage single-stranded DNA-annealing protein (SSAP) PapRecT and P. aeruginosa single-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitated K. phaffii CRISPR-based genome engineering. Specifically, a host-independent method was developed by expressing sgRNA with PapRecT-PaSSB in a single plasmid, with which only a 50 bp short homologous arm (HA) reached a 100% positive rate for CRISPR-based gene insertion, reaching 18 colony-forming units (CFU) per μg of donor DNA. Single deletion using 1000 bp HA attained 100%, reaching 68 CFUs per μg of donor DNA. Using this efficient CRISPR-based genome editing tool, we integrated three genes (INO4, GAL4-like, and PAB1) at three different loci for overexpression to realize the collaborative regulation of human-lactalbumin (α-LA) production. Specifically, we strengthened phospholipid biosynthesis to facilitate endoplasmic reticulum membrane formation and enhanced recombinant protein transcription and translation by overexpressing transcription and translation factors. The final production of α-LA in the 3 L fermentation reached 113.4 mg L[-1], two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in K. phaffii. The CRISPR-based genome editing method developed in this study is suitable for the synergistic multiple-site engineering of protein and biochemical biosynthesis pathways to improve the biomanufacturing efficiency.},
}
@article {pmid37873982,
year = {2023},
author = {Cazier, AP and Irvin, OM and Chávez, LS and Dalvi, S and Abraham, H and Wickramanayake, N and Yellayi, S and Blazeck, J},
title = {A Rapid Antibody Enhancement Platform in Saccharomyces cerevisiae Using an Improved, Diversifying CRISPR Base Editor.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3287-3300},
doi = {10.1021/acssynbio.3c00299},
pmid = {37873982},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Antibodies/genetics ; DNA ; },
abstract = {The yeast Saccharomyces cerevisiae is commonly used to interrogate and screen protein variants and to perform directed evolution studies to develop proteins with enhanced features. While several techniques have been described that help enable the use of yeast for directed evolution, there remains a need to increase their speed and ease of use. Here we present yDBE, a yeast diversifying base editor that functions in vivo and employs a CRISPR-dCas9-directed cytidine deaminase base editor to diversify DNA in a targeted, rapid, and high-breadth manner. To develop yDBE, we enhanced the mutation rate of an initial base editor by employing improved deaminase variants and characterizing several scaffolded guide constructs. We then demonstrate the ability of the yDBE platform to improve the affinity of a displayed antibody scFv, rapidly generating diversified libraries and isolating improved binders via cell sorting. By performing high-throughput sequencing analysis of the high-activity yDBE, we show that it enables a mutation rate of 2.13 × 10[-4] substitutions/bp/generation over a window of 100 bp. As yDBE functions entirely in vivo and can be easily programmed to diversify nearly any such window of DNA, we posit that it can be a powerful tool for facilitating a variety of directed evolution experiments.},
}
@article {pmid37864048,
year = {2023},
author = {},
title = {Split complementation of base editors reduces off-target edits.},
journal = {Nature plants},
volume = {9},
number = {11},
pages = {1787-1788},
pmid = {37864048},
issn = {2055-0278},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid37843014,
year = {2023},
author = {Guesdon, G and Gourgues, G and Rideau, F and Ipoutcha, T and Manso-Silván, L and Jules, M and Sirand-Pugnet, P and Blanchard, A and Lartigue, C},
title = {Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3252-3266},
doi = {10.1021/acssynbio.3c00248},
pmid = {37843014},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems/genetics ; Phylogeny ; Genome, Bacterial/genetics ; DNA ; Cloning, Molecular ; Gene Editing/methods ; },
abstract = {The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.},
}
@article {pmid37830328,
year = {2023},
author = {Yang, P and Yang, J and Lin, T and Liu, Q and Yin, Y and Chen, D and Yang, S},
title = {Efficient Genome Editing in Most Staphylococcus aureus by Using the Restriction-Modification System Silent CRISPR-Cas9 Toolkit.},
journal = {ACS synthetic biology},
volume = {12},
number = {11},
pages = {3340-3351},
doi = {10.1021/acssynbio.3c00339},
pmid = {37830328},
issn = {2161-5063},
mesh = {Humans ; *Gene Editing ; *Staphylococcus aureus/genetics ; CRISPR-Cas Systems/genetics ; DNA Restriction-Modification Enzymes/genetics ; Plasmids/genetics ; },
abstract = {Staphylococcus aureus is a clinically important pathogen that threatens human health due to its strong pathogenicity and drug resistance, leading to meningitis, endocarditis, and skin and soft tissue infections. Genetic manipulation in S. aureus is a powerful approach for characterizing the molecular mechanisms of bacterial drug resistance, pathogenicity, and virulence. However, a strong restriction barrier presents a major obstacle to the extensive utilization of genetic manipulation tools in clinical isolates of S. aureus. Here, we constructed a restriction-modification (RM) system silent CRISPR-Cas9 toolkit that synonymously eliminated the type I RM targets of S. aureus from plasmids, downsized plasmids using minicircle technology, and combined with a plasmid artificial modification (PAM) method to circumvent the type II RM system. The RM-silent CRISPR-Cas9 toolkit enables a significant improvement in transformation (10[5]-10[6] transformants per microgram plasmid in strains we tested) and high-success efficiency editing for gene deletion (knockout strain obtained in one-round electroporation) in a wide range of S. aureus species including clinical isolates of unknown genetic background. The RM-silent CRISPR-Cas9 toolkits could expedite the process of mutant construction in most S. aureus strains, and this approach could be applied to the design of other genetic toolkit plasmids for utilization in a wider range of S. aureus strains.},
}
@article {pmid37723364,
year = {2023},
author = {Lotfi, M and Morshedi Rad, D and Mashhadi, SS and Ashouri, A and Mojarrad, M and Mozaffari-Jovin, S and Farrokhi, S and Hashemi, M and Lotfi, M and Ebrahimi Warkiani, M and Abbaszadegan, MR},
title = {Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.},
journal = {Stem cell reviews and reports},
volume = {19},
number = {8},
pages = {2576-2596},
pmid = {37723364},
issn = {2629-3277},
support = {DP200101860//Australian Research Council/ ; 2021CDF1148//Cancer Institute NSW/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Pluripotent Stem Cells ; Cell Differentiation ; Stem Cell Transplantation ; },
abstract = {Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.},
}
@article {pmid37653182,
year = {2023},
author = {Ge, N and Liu, M and Li, R and Allen, NM and Galvin, J and Shen, S and O'Brien, T and Prendiville, TW},
title = {Using Ribonucleoprotein-based CRISPR/Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5A[±]).},
journal = {Stem cell reviews and reports},
volume = {19},
number = {8},
pages = {2774-2789},
pmid = {37653182},
issn = {2629-3277},
mesh = {Humans ; Nucleotides ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; *Long QT Syndrome/genetics ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A[±]) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.},
}
@article {pmid37256239,
year = {2023},
author = {Huang, C and Zhao, Y and Ye, Q and Gleason, J and Rousseva, V and Stout, B and Lin, S and Hariri, R and Zhang, X and He, S},
title = {Characterization of CRISPR/Cas9-edited human placental allogenic stromal cells with low tissue factor expression and reduced thrombotic effects.},
journal = {Cytotherapy},
volume = {25},
number = {12},
pages = {1265-1270.e2},
doi = {10.1016/j.jcyt.2023.04.013},
pmid = {37256239},
issn = {1477-2566},
mesh = {Female ; Pregnancy ; Humans ; *Thromboplastin/genetics ; CRISPR-Cas Systems/genetics ; Placenta ; Stromal Cells ; *Thrombosis ; },
abstract = {The tissue factor (TF/CD142) expressed by mesenchymal stromal cells (MSCs) has been regarded as a safety concern in clinical applications as it may trigger thrombosis when MSCs administered intravenously. Human placental allogenic stromal cells (ASCs) are culture-expanded, undifferentiated MSC-like cells derived from full-term postpartum placenta and possess immunomodulatory and pro-angiogenic activities, however, express TF. Here we performed CRISPR/Cas9-mediated TF gene knock out (TFKO) in ASCs, leading to significantly lower TF expression, activity and thrombotic effects. ASCs' characteristics including expansion, expression of phenotypic markers and secretory profile remained unchanged in edited cells, and their immunomodulatory activities, which are functionally relevant to therapeutic applications, were not affected upon TFKO. Taken together, this study provides a feasible strategy which could improve the clinical safety features of MSC-based cell therapy by CRISRP/Cas9-mediated TF gene knock out.},
}
@article {pmid37970179,
year = {2021},
author = {Son, YE and Park, HS},
title = {Genetic Manipulation and Transformation Methods for Aspergillus spp.},
journal = {Mycobiology},
volume = {49},
number = {2},
pages = {95-104},
pmid = {37970179},
issn = {1229-8093},
abstract = {Species of the genus Aspergillus have a variety of effects on humans and have been considered industrial cell factories due to their prominent ability for manufacturing several products such as heterologous proteins, secondary metabolites, and organic acids. Scientists are trying to improve fungal strains and re-design metabolic processes through advanced genetic manipulation techniques and gene delivery systems to enhance their industrial efficiency and utility. In this review, we describe the current status of the genetic manipulation techniques and transformation methods for species of the genus Aspergillus. The host strains, selective markers, and experimental materials required for the genetic manipulation and fungal transformation are described in detail. Furthermore, the advantages and disadvantages of these techniques are described.},
}
@article {pmid37957275,
year = {2023},
author = {Duan, Y and Szlam, F and Hu, Y and Chen, W and Li, R and Ke, Y and Sniecinski, R and Salaita, K},
title = {Detection of cellular traction forces via the force-triggered Cas12a-mediated catalytic cleavage of a fluorogenic reporter strand.},
journal = {Nature biomedical engineering},
volume = {7},
number = {11},
pages = {1404-1418},
pmid = {37957275},
issn = {2157-846X},
support = {5R01GM131099-04//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DMR 1905947//National Science Foundation (NSF)/ ; RM1GM145394//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 23POST1028975//American Heart Association (American Heart Association, Inc.)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Traction ; Cell Adhesion/physiology ; Proteins ; Carrier Proteins ; },
abstract = {Molecular forces generated by cell receptors are infrequent and transient, and hence difficult to detect. Here we report an assay that leverages the CRISPR-associated protein 12a (Cas12a) to amplify the detection of cellular traction forces generated by as few as 50 adherent cells. The assay involves the immobilization of a DNA duplex modified with a ligand specific for a cell receptor. Traction forces of tens of piconewtons trigger the dehybridization of the duplex, exposing a cryptic Cas12-activating strand that sets off the indiscriminate Cas12-mediated cleavage of a fluorogenic reporter strand. We used the assay to perform hundreds of force measurements using human platelets from a single blood draw to extract individualized dose-response curves and half-maximal inhibitory concentrations for a panel of antiplatelet drugs. For seven patients who had undergone cardiopulmonary bypass, platelet dysfunction strongly correlated with the need for platelet transfusion to limit bleeding. The Cas12a-mediated detection of cellular traction forces may be used to assess cell state, and to screen for genes, cell-adhesion ligands, drugs or metabolites that modulate cell mechanics.},
}
@article {pmid37955405,
year = {2023},
author = {Mendoza, BJ and Zheng, X and Clements, JC and Cotter, C and Trinh, CT},
title = {Potency of CRISPR-Cas Antifungals Is Enhanced by Cotargeting DNA Repair and Growth Regulatory Machinery at the Genetic Level.},
journal = {ACS infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsinfecdis.3c00342},
pmid = {37955405},
issn = {2373-8227},
abstract = {The emergence of virulent, resistant, and rapidly evolving fungal pathogens poses a significant threat to public health, agriculture, and the environment. Targeting cellular processes with standard small-molecule intervention may be effective but requires long development times and is prone to antibiotic resistance. To overcome the current limitations of antibiotic development and treatment, this study harnesses CRISPR-Cas systems as antifungals by capitalizing on their adaptability, specificity, and efficiency in target design. The conventional design of CRISPR-Cas antimicrobials, based on induction of DNA double-strand breaks (DSBs), is potentially less effective in fungi due to robust eukaryotic DNA repair machinery. Here, we report a novel design principle to formulate more effective CRISPR-Cas antifungals by cotargeting essential genes with DNA repair defensive genes that remove the fungi's ability to repair the DSB sites of essential genes. By evaluating this design on the model fungus Saccharomyces cerevisiae, we demonstrated that essential and defensive gene cotargeting is more effective than either essential or defensive gene targeting alone. The top-performing CRISPR-Cas antifungals performed as effectively as the antibiotic Geneticin. A gene cotargeting interaction analysis revealed that cotargeting essential genes with RAD52 involved in homologous recombination (HR) was the most synergistic combination. Fast growth kinetics of S. cerevisiae induced resistance to CRISPR-Cas antifungals, where genetic mutations mostly occurred in defensive genes and guide RNA sequences.},
}
@article {pmid37954164,
year = {2023},
author = {Nix, JL and Schettini, GP and Speckhart, SL and Ealy, AD and Biase, FH},
title = {Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART).},
journal = {PNAS nexus},
volume = {2},
number = {11},
pages = {pgad343},
pmid = {37954164},
issn = {2752-6542},
abstract = {CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A RNPs targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1,000 nucleotides long. Electroporation of Cas13a RNPs prevents the production of the corresponding proteins. We electroporated Cas9D10A RNPs targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a RNPs targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P < 0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (False Discovery Rate, FDR < 0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is a key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.},
}
@article {pmid37953351,
year = {2023},
author = {Veluchamy, A and Teles, K and Fischle, W},
title = {CRISPR-broad: combined design of multi-targeting gRNAs and broad, multiplex target finding.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {19717},
pmid = {37953351},
issn = {2045-2322},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Caenorhabditis elegans/genetics ; Gene Editing ; DNA ; Genome, Plant ; },
abstract = {In CRISPR-Cas and related nuclease-mediated genome editing, target recognition is based on guide RNAs (gRNAs) that are complementary to selected DNA regions. While single site targeting is fundamental for localized genome editing, targeting to expanded and multiple chromosome elements is desirable for various biological applications such as genome mapping and epigenome editing that make use of different fusion proteins with enzymatically dead Cas9. The current gRNA design tools are not suitable for this task, as these are optimized for defining single gRNAs for unique loci. Here, we introduce CRISPR-broad, a standalone, open-source application that defines gRNAs with multiple but specific targets in large continuous or spread regions of the genome, as defined by the user. This ability to identify multi-targeting gRNAs and corresponding multiple targetable regions in genomes is based on a novel aggregate gRNA scoring derived from on-target windows and off-target sites. Applying the new tool to the genomes of two model species, C. elegans and H. sapiens, we verified its efficiency in determining multi-targeting gRNAs and ranking potential target regions optimized for broad targeting. Further, we demonstrated the general usability of CRISPR-broad by cellular mapping of a large human genome element using dCas9 fused to green fluorescent protein.},
}
@article {pmid37951952,
year = {2023},
author = {Ma, B and Lu, C and Wang, Y and Yu, J and Zhao, K and Xue, R and Ren, H and Lv, X and Pan, R and Zhang, J and Zhu, Y and Xu, J},
title = {A genomic catalogue of soil microbiomes boosts mining of biodiversity and genetic resources.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {7318},
pmid = {37951952},
issn = {2041-1723},
mesh = {*Soil ; *Microbiota/genetics ; Metagenome/genetics ; Biodiversity ; Genomics ; Soil Microbiology ; },
abstract = {Soil harbors a vast expanse of unidentified microbes, termed as microbial dark matter, presenting an untapped reservo)ir of microbial biodiversity and genetic resources, but has yet to be fully explored. In this study, we conduct a large-scale excavation of soil microbial dark matter by reconstructing 40,039 metagenome-assembled genome bins (the SMAG catalogue) from 3304 soil metagenomes. We identify 16,530 of 21,077 species-level genome bins (SGBs) as unknown SGBs (uSGBs), which expand archaeal and bacterial diversity across the tree of life. We also illustrate the pivotal role of uSGBs in augmenting soil microbiome's functional landscape and intra-species genome diversity, providing large proportions of the 43,169 biosynthetic gene clusters and 8545 CRISPR-Cas genes. Additionally, we determine that uSGBs contributed 84.6% of previously unexplored viral-host associations from the SMAG catalogue. The SMAG catalogue provides an useful genomic resource for further studies investigating soil microbial biodiversity and genetic resources.},
}
@article {pmid37951948,
year = {2023},
author = {Wei, T and Sun, Y and Cheng, Q and Chatterjee, S and Traylor, Z and Johnson, LT and Coquelin, ML and Wang, J and Torres, MJ and Lian, X and Wang, X and Xiao, Y and Hodges, CA and Siegwart, DJ},
title = {Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {7322},
pmid = {37951948},
issn = {2041-1723},
support = {P30CA142543//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; SIEGWA18XX0//Cystic Fibrosis Foundation (CF Foundation)/ ; HODGES19R1//Cystic Fibrosis Foundation (CF Foundation)/ ; RP160157//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; },
mesh = {Humans ; Mice ; Animals ; *Cystic Fibrosis/therapy/drug therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Lung/metabolism ; RNA, Messenger/genetics/therapeutic use ; },
abstract = {Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.},
}
@article {pmid37724007,
year = {2023},
author = {Gustafson, DL and Viola, LO and Towers, CG and Das, S and Duval, DL and Van Eaton, KM},
title = {Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.},
journal = {Veterinary and comparative oncology},
volume = {21},
number = {4},
pages = {726-738},
doi = {10.1111/vco.12937},
pmid = {37724007},
issn = {1476-5829},
support = {R01CA190170/CA/NCI NIH HHS/United States ; R00CA245187/CA/NCI NIH HHS/United States ; R01CA190170/CA/NCI NIH HHS/United States ; R00CA245187/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Dogs ; Humans ; *Dog Diseases/drug therapy ; RNA, Guide, CRISPR-Cas Systems ; Hydroxychloroquine/pharmacology ; *Antineoplastic Agents/pharmacology ; Cell Line, Tumor ; *Osteosarcoma/drug therapy/veterinary ; Autophagy ; },
abstract = {Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm) values ranging from 4.6-15.8 μM and 2.1-5.1 μM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.},
}
@article {pmid37571976,
year = {2023},
author = {Zhang, A and Shan, T and Sun, Y and Chen, Z and Hu, J and Hu, Z and Ming, Z and Zhu, Z and Li, X and He, J and Liu, S and Jiang, L and Dong, X and Wu, Y and Wang, Y and Liu, Y and Li, C and Wan, J},
title = {Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors.},
journal = {Plant biotechnology journal},
volume = {21},
number = {12},
pages = {2597-2610},
pmid = {37571976},
issn = {1467-7652},
support = {JSSCBS20210272//Doctor of Entrepreneurship and Innovation in Jiangsu Province/ ; B21HJ1004//Hainan Yazhou Bay Seed Laboratory/ ; cx(21)1002//Jiangsu Agricultural Science and Technology Innovation Fund/ ; //Ministry of Agriculture and Rural Affairs of China/ ; BK20212010//Natural Science Foundation of Jiangsu Province/ ; KYCX22_0734//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; 2020QNRC001//Young Elite Scientists Sponsorship Program by CAST/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Oryza/genetics ; Plant Breeding ; },
abstract = {CRISPR-based directed evolution is an effective breeding biotechnology to improve agronomic traits in plants. However, its gene diversification is still limited using individual single guide RNA. We described here a multiplexed orthogonal base editor (MoBE), and a randomly multiplexed sgRNAs assembly strategy to maximize gene diversification. MoBE could induce efficiently orthogonal ABE (<36.6%), CBE (<36.0%), and A&amp;CBE (<37.6%) on different targets, while the sgRNA assembling strategy randomized base editing events on various targets. With respective 130 and 84 targets from each strand of the 34th exon of rice acetyl-coenzyme A carboxylase (OsACC), we observed the target-scaffold combination types up to 27 294 in randomly dual and randomly triple sgRNA libraries. We further performed directed evolution of OsACC using MoBE and randomly dual sgRNA libraries in rice, and obtained single or linked mutations of stronger herbicide resistance. These strategies are useful for in situ directed evolution of functional genes and may accelerate trait improvement in rice.},
}
@article {pmid36876933,
year = {2023},
author = {Zheng, S and Liang, F and Zhang, Y and Fei, JF and Qin, W and Liu, Y},
title = {Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {192},
pages = {},
doi = {10.3791/64977},
pmid = {36876933},
issn = {1940-087X},
mesh = {Animals ; Humans ; *Gene Editing ; *Zebrafish ; Adenine ; Drug Evaluation, Preclinical ; Guanine ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.},
}
@article {pmid36727818,
year = {2023},
author = {Li, T and Zhang, L and Lu, T and Zhu, T and Feng, C and Gao, N and Liu, F and Yu, J and Chen, K and Zhong, J and Tang, Q and Zhang, Q and Deng, X and Ren, J and Zeng, J and Zhou, H and Zhu, J},
title = {Engineered Extracellular Vesicle-Delivered CRISPR/CasRx as a Novel RNA Editing Tool.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {10},
pages = {e2206517},
pmid = {36727818},
issn = {2198-3844},
support = {2018YFA0107900//Chinese Academy of Science/ ; 92168103//Chinese Academy of Science/ ; 32171417//Chinese Academy of Science/ ; 82001140//Chinese Academy of Science/ ; 2019CXJQ01//Chinese Academy of Science/ ; 2018YFA0107900//Ministry of Science and Technology of China/ ; 92168103//National Natural Science Foundation of China/ ; 32171417//National Natural Science Foundation of China/ ; 82001140//National Natural Science Foundation of China/ ; 2019CXJQ01//Shanghai Municipal Government/ ; //Peak Disciplines (Type IV) of Institutions of Higher Leaning in Shanghai/ ; },
mesh = {*RNA Editing/genetics ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Engineered extracellular vesicles (EVs) are considered excellent delivery vehicles for a variety of therapeutic agents, including nucleic acids, proteins, drugs, and nanomaterials. Recently, several studies have indicated that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) delivered by EVs enable efficient DNA editing. However, an RNA editing tool delivered by EVs is still unavailable. Here, a signal peptide-optimized and EVs-delivered guide RNA (gRNA) and CRISPR/CasRx (Cas13d) system capable of rapidly inhibiting the expression of targeted genes with quick catabolism after performing their functions is developed. EVs with CRISPR/CasRx and tandem gRNAs targeting pivotal cytokines are further packed whose levels increase substantially over the course of acute inflammatory diseases and find that these engineered EVs inhibit macrophage activation in vitro. More importantly, this system attenuates lipopolysaccharide (LPS)-triggered acute lung injury and sepsis in the acute phase, mitigating organ damage and improving the prognosis in vivo. In summary, a potent tool is provided for short-acting RNA editing, which could be a powerful therapeutic platform for the treatment of acute diseases.},
}
@article {pmid37951882,
year = {2023},
author = {Li, Y and Wang, X and Xu, R and Wang, T and Zhang, D and Qian, W},
title = {Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.},
journal = {BMC microbiology},
volume = {23},
number = {1},
pages = {333},
pmid = {37951882},
issn = {1471-2180},
mesh = {Humans ; *CRISPR-Cas Systems ; *Biological Assay ; Coloring Agents ; Nucleotidyltransferases ; Recombinases ; },
abstract = {Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.},
}
@article {pmid37749866,
year = {2023},
author = {Navaridas, R and Vidal-Sabanés, M and Ruiz-Mitjana, A and Altés, G and Perramon-Güell, A and Yeramian, A and Egea, J and Encinas, M and Gatius, S and Matias-Guiu, X and Dolcet, X},
title = {In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {32},
pages = {e2303134},
pmid = {37749866},
issn = {2198-3844},
support = {PID2019-104734RB-I00//Spanish Ministry of Science and Innovation/ ; FPU18/04480//Spanish Ministry of Universities/ ; },
mesh = {Humans ; Female ; Mice ; Animals ; Tumor Suppressor Protein p53/genetics/metabolism ; Epithelial-Mesenchymal Transition ; CRISPR-Cas Systems/genetics ; *Endometrial Neoplasms/genetics/metabolism/pathology ; *Uterine Neoplasms/genetics/pathology ; *Carcinosarcoma/genetics/pathology ; },
abstract = {Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.},
}
@article {pmid37605962,
year = {2023},
author = {Zeng, L and Zheng, S and Stejskal, V and Opit, G and Aulicky, R and Li, Z},
title = {New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a.},
journal = {Pest management science},
volume = {79},
number = {12},
pages = {5304-5311},
doi = {10.1002/ps.7739},
pmid = {37605962},
issn = {1526-4998},
support = {2022YFC2601500//National Key Research and Development Program of China/ ; CARS-02-32//the earmarked fund for China Agriculture Research System/ ; SYND-2022-15//Sanya Yazhou Bay Science and Technology City Administration funded project/ ; MZE-RO0423//the Ministry of Agriculture of the Czech Republic institutional support/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Recombinases ; *Coleoptera/genetics ; DNA ; },
abstract = {BACKGROUND: Khapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system.<br><br>RESULTS: We designed and selected the first khapra beetle-specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA&#x2009;=&#x2009;100&#x2009;nM/500&#x2009;nM). With only a 37&#x2009;&#xb0;C-heat-source and a blue light torch, RPA and CRISPR/CAS12a-based visualization assays can be completed within 40&#x2009;min to differentiate between khapra beetle and nine similar Dermestidae species. After DNA extraction using a kit (4-5&#x2009;h) or a simple method (5&#x2009;min), the specific amplicons were obtained after a 15&#x2009;min RPA reaction at 37&#x2009;&#xb0;C, followed by a 15&#x2009;min color reaction under 37&#x2009;&#xb0;C in dark conditions using a CRISPR/CAS12a system and a fluorescent probe (5'-FAM/3'-BHQ1 labeled). This method is ingenious to low levels of DNA (10[-1] &#x2009;ng&#x2009;&#x3bc;L[-1]) and meets the sensitivity requirements for detecting a single khapra beetle's egg (&#x2248;0.7&#x2009;mm).<br><br>CONCLUSION: Our specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37&#x2009;&#xb0;C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management, and provides value as a reference for monitoring and identification of other pests. &#xa9; 2023 Society of Chemical Industry.},
}
@article {pmid37528583,
year = {2023},
author = {Wu, X and Ren, B and Liu, L and Qiu, S and Li, X and Li, P and Yan, F and Lin, H and Zhou, X and Zhang, D and Zhou, H},
title = {Adenine base editor incorporating the N-methylpurine DNA glycosylase MPGv3 enables efficient A-to-K base editing in rice.},
journal = {Plant communications},
volume = {4},
number = {6},
pages = {100668},
doi = {10.1016/j.xplc.2023.100668},
pmid = {37528583},
issn = {2590-3462},
mesh = {Gene Editing ; *Oryza/genetics ; Adenine ; CRISPR-Cas Systems ; *DNA Glycosylases/genetics ; },
}
@article {pmid37950006,
year = {2023},
author = {Hodgson, J},
title = {Drug pipeline 3Q23 - ERT, bispecifics and CRISPR in sickle cell disease.},
journal = {Nature biotechnology},
volume = {41},
number = {11},
pages = {1498-1500},
doi = {10.1038/s41587-023-02015-z},
pmid = {37950006},
issn = {1546-1696},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Anemia, Sickle Cell/drug therapy/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37949766,
year = {2023},
author = {Stella, G and Marraffini, L},
title = {Type III CRISPR-Cas: beyond the Cas10 effector complex.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2023.10.006},
pmid = {37949766},
issn = {0968-0004},
abstract = {Type III CRISPR-Cas loci encode some of the most abundant, yet complex, immune systems of prokaryotes. They are composed of a Cas10 complex that uses an RNA guide to recognize transcripts from bacteriophage and plasmid invaders. Target recognition triggers three activities within this complex: ssDNA degradation, synthesis of cyclic oligoadenylates (cOA) that act as second messengers to activate CARF-domain effectors, and cleavage of target RNA. This review covers recent research in type III CRISPR-Cas systems that looked beyond the activity of the canonical Cas10 complexes towards: (i) ancillary nucleases and understanding how they provide defense by sensing cOA molecules; (ii) ring nucleases and their role in regulating cOA production; and (iii) CRISPR-associated proteases, including the function of the Craspase complex in a transcriptional response to phage infection.},
}
@article {pmid37947658,
year = {2023},
author = {Dibas, A and Rhiel, M and Patel, VB and Andrieux, G and Boerries, M and Cornu, TI and Alzubi, J and Cathomen, T},
title = {Cell-Based Models of 'Cytokine Release Syndrome' Endorse CD40L and Granulocyte-Macrophage Colony-Stimulating Factor Knockout in Chimeric Antigen Receptor T Cells as Mitigation Strategy.},
journal = {Cells},
volume = {12},
number = {21},
pages = {},
doi = {10.3390/cells12212581},
pmid = {37947658},
issn = {2073-4409},
support = {CARAT-667980, geneTIGA-101057438//European Union/ ; EkoEstMed-01ZZ2015, MIRACUM-01ZZ1801B//German Federal Ministry of Education and Research/ ; scholarship 91686634//German Academic Exchange Service/ ; },
mesh = {Humans ; Animals ; Mice ; *Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; *Receptors, Chimeric Antigen/genetics ; CD40 Ligand ; Cytokine Release Syndrome ; Interleukin-6 ; Mice, Knockout ; T-Lymphocytes ; },
abstract = {While chimeric antigen receptor (CAR) T cell therapy has shown promising outcomes among patients with hematologic malignancies, it has also been associated with undesirable side-effects such as cytokine release syndrome (CRS). CRS is triggered by CAR T-cell-based activation of monocytes, which are stimulated via the CD40L-CD40R axis or via uptake of GM-CSF to secrete proinflammatory cytokines. Mouse models have been used to model CRS, but working with them is labor-intensive and they are not amenable to screening approaches. To overcome this challenge, we established two simple cell-based CRS in vitro models that entail the co-culturing of leukemic B cells with CD19-targeting CAR T cells and primary monocytes from the same donor. Upon antigen encounter, CAR T cells upregulated CD40L and released GM-CSF which in turn stimulated the monocytes to secrete IL-6. To endorse these models, we demonstrated that neutralizing antibodies or genetic disruption of the CD40L and/or CSF2 loci in CAR T cells using CRISPR-Cas technology significantly reduced IL-6 secretion by bystander monocytes without affecting the cytolytic activity of the engineered lymphocytes in vitro. Overall, our cell-based models were able to recapitulate CRS in vitro, allowing us to validate mitigation strategies based on antibodies or genome editing.},
}
@article {pmid37947521,
year = {2023},
author = {Zhang, Y and Kubiak, AM and Bailey, TS and Claessen, L and Hittmeyer, P and Dubois, L and Theys, J and Lambin, P},
title = {Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0245923},
doi = {10.1128/spectrum.02459-23},
pmid = {37947521},
issn = {2165-0497},
abstract = {Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes, the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes (Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia (Clostridium butyricum and Clostridium sporogenes) to produce near-infrared fluorescence from biliverdin and hemin.IMPORTANCEContinued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.},
}
@article {pmid37946200,
year = {2023},
author = {Zhao, D and Gao, X and Zhou, J and Li, J and Qian, Y and Wang, D and Niu, W and Zhang, T and Hu, M and Xiong, H and Lai, L and Li, Z},
title = {Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {250},
pmid = {37946200},
issn = {1741-7007},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics ; Adenine/chemistry ; Gene Editing/methods ; DNA/genetics ; Mammals/genetics ; },
abstract = {BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency.<br><br>RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797[Smu] and Nme2ABE8e-797[-C], respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice.<br><br>CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.},
}
@article {pmid37921444,
year = {2023},
author = {Escalona-Noguero, C and Alarcón-Iniesta, H and López-Valls, M and Del Carpio, LP and Piulats, JM and Somoza, &#xc1; and Sot, B},
title = {Detection of the Uveal Melanoma-Associated Mutation GNAQ Q209P from Liquid Biopsy Using CRISPR/Cas12a Technology.},
journal = {Analytical chemistry},
volume = {95},
number = {45},
pages = {16692-16700},
doi = {10.1021/acs.analchem.3c03460},
pmid = {37921444},
issn = {1520-6882},
mesh = {Humans ; *GTP-Binding Protein alpha Subunits, Gq-G11/genetics/metabolism ; *GTP-Binding Protein alpha Subunits/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; },
abstract = {Uveal melanoma (UM) is a rare ocular tumor characterized by high metastasis risk and poor prognosis. The in-depth characterization of UM's molecular profile is critical for better disease classification and prognosis. Furthermore, the development of detection tools to monitor UM evolution upon treatment is of great interest for designing optimal therapeutic strategies. However, commonly used techniques, such as ddPCR or NGS, are costly, and they involve sophisticated equipment and complex experimental design. The development of alternative sensing methods that are fast, simple, and inexpensive would be of great benefit to improve UM's diagnosis and management, especially when combined with liquid biopsy. Samples from liquid biopsy can be obtained with minimal invasiveness, and the detection of circulating tumor DNA (ctDNA) in UM patients' plasma has proven useful for the diagnosis of metastasis, prognosis prediction, and disease monitoring. In this context, CRISPR/Cas12a-derived molecular sensors, thanks to their high specificity and sensitivity and their potential for point of care diagnosis, are particularly interesting. Here, we developed a CRISPR/Cas12a-based approach for the specific detection of the UM-related mutation GNAQ Q209P that relies on the design of highly specific crRNAs. Coupled with allele-specific PCR, it constitutes a sensitive platform for liquid biopsy detection, capable of sensing GNAQ Q209P in plasma samples with a low ctDNA concentration and fractional abundance. Finally, our method was validated using plasma samples from metastatic UM patients.},
}
@article {pmid37828409,
year = {2023},
author = {Wang, J and Le Gall, J and Frock, RL and Strick, TR},
title = {Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.},
journal = {Nature structural &amp; molecular biology},
volume = {30},
number = {11},
pages = {1707-1718},
pmid = {37828409},
issn = {1545-9985},
mesh = {*CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; DNA/genetics ; Genome ; Streptococcus pyogenes/metabolism ; Gene Editing ; },
abstract = {Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3' overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.},
}
@article {pmid37710013,
year = {2023},
author = {Santiago-Frangos, A and Henriques, WS and Wiegand, T and Gauvin, CC and Buyukyoruk, M and Graham, AB and Wilkinson, RA and Triem, L and Neselu, K and Eng, ET and Lander, GC and Wiedenheft, B},
title = {Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays.},
journal = {Nature structural &amp; molecular biology},
volume = {30},
number = {11},
pages = {1675-1685},
pmid = {37710013},
issn = {1545-9985},
support = {R01 GM129325/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; U24 GM129539/GM/NIGMS NIH HHS/United States ; K99 GM147842/GM/NIGMS NIH HHS/United States ; P20 GM103474/GM/NIGMS NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Associated Proteins/metabolism ; DNA/chemistry ; Binding Sites ; Plasmids ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560-kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1-Cas2/3) and non-Cas proteins (for example, integration host factor) fold 150 base pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5'-GT. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA.},
}
@article {pmid37945231,
year = {2023},
author = {Wang, Z and Zhang, R and Pei, Y and Wu, W and Hu, Z and Zuo, Y},
title = {The knockout of the nicotinic acetylcholine receptor subunit gene α1 (nAChR α1) through CRISPR/Cas9 technology exposes its involvement in the resistance of Spodoptera exigua to insecticides.},
journal = {Pesticide biochemistry and physiology},
volume = {196},
number = {},
pages = {105616},
doi = {10.1016/j.pestbp.2023.105616},
pmid = {37945231},
issn = {1095-9939},
mesh = {Animals ; *Insecticides/pharmacology ; Spodoptera/metabolism ; *Receptors, Nicotinic/genetics/metabolism ; CRISPR-Cas Systems ; Technology ; },
abstract = {Insect nicotinic acetylcholine receptors (nAChRs) are the directed targets of many insecticides. However, there have been no reports on the molecular characterization of the nAChR gene family or the causal association between nAChR α1 and resistance to insecticides in S. exigua, which is a significant agricultural pest. In this study, we identified a total of 9 candidate nAChR subunits in S. exigua, namely nAChR α1-α7 and nAChR β1-β2. For functional validation roles of Seα1 in insecticide resistance of S. exigua, we introduced a ∼ 1041-bp deletion of the Seα1 gene in a homozygous mutant strain (Seα1-KO) by CRISPR/Cas9 genome editing system, resulting in a premature truncation of the Seα1 protein and the subsequent loss of functional transmembrane (TM) 3 and TM4 elements. Compared with WH-S strain (wild-type strain), the Seα1-KO strain exhibited 2.62-folds resistant to trifluoropyrimidine, 8.3-folds resistant to dimehypo, and 5.28-folds resistant to dinotefuran, but no significant change in susceptibility to emamectin benzoate, spinetoram, lambda-cyhalothrin, permethrin and chlorpyrifos. Thus, this study has laid a solid foundation for investigating the role of nAChRs in S. exigua, and provides evidence for the crucial involvement of the α1 subunit in the mechanism of trifluoropyrimidine, dimehypo, and dinotefuran in S. exigua. Moreover, it provides a reference for the value of Seα1 subunit and its homologues in other species as insecticide targets.},
}
@article {pmid37944631,
year = {2023},
author = {Levchenko, O and Panchuk, I and Kochergin-Nikitsky, K and Petrova, I and Nagieva, S and Pilkin, M and Yakovlev, I and Smirnikhina, S and Deev, R and Lavrov, A},
title = {Unexpected extra exon skipping in the DYSF gene during restoring the reading frame by CRISPR/Cas9.},
journal = {Bio Systems},
volume = {235},
number = {},
pages = {105072},
doi = {10.1016/j.biosystems.2023.105072},
pmid = {37944631},
issn = {1872-8324},
abstract = {The DYSF gene encoding dysferlin protein is one of the largest and has many transcripts. Pathogenic variants in the gene can lead to various types of myopathies, which makes it a good object for studying the events occurring in it during genome editing by the CRISPR/Cas method. In this study, we evaluated the possibility of permanent skipping of exons 3-4, and 26-27 which deletion does not violate the reading frame and allows to eliminate truncated variants within exons. Editing was performed with simultaneous transfection of two sgRNA- and sa/spCas9-containing plasmids on HEK293T cell cultures and healthy donor myoblasts. Skipping of exons 3-4 was performed by destroying the splicing acceptor sites, and exons 26-27 by cuts in the flanking exons with the corresponding deletion in the DNA. Some unexpected results were obtained, when exons 26-27 were skipped, exon 30 was also absent in the transcript, although it is not alternatively spliced and is normally present in all transcripts. This event indicates that DNA changes near splicing sites can affect adjacent exons and the whole gene. However, this fact requires further study.},
}
@article {pmid37943774,
year = {2023},
author = {Hasan, MN and Hyodo, T and Biswas, M and Rahman, ML and Mihara, Y and Karnan, S and Ota, A and Tsuzuki, S and Hosokawa, Y and Konishi, H},
title = {Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.},
journal = {PloS one},
volume = {18},
number = {11},
pages = {e0294146},
pmid = {37943774},
issn = {1932-6203},
mesh = {Humans ; *Gene Editing/methods ; Flow Cytometry ; CRISPR-Cas Systems/genetics ; *Neural Cell Adhesion Molecule L1/genetics ; Cell Line ; },
abstract = {CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.},
}
@article {pmid37941664,
year = {2023},
author = {Roychowdhury, R and Ballén-Taborda, C and Chaturvedi, P},
title = {Editorial: Characterizing and improving traits for resilient crop development.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1307327},
pmid = {37941664},
issn = {1664-462X},
}
@article {pmid37877218,
year = {2023},
author = {Maghsoud, Y and Jayasinghe-Arachchige, VM and Kumari, P and Cisneros, GA and Liu, J},
title = {Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate Off-Target Effects.},
journal = {Journal of chemical information and modeling},
volume = {63},
number = {21},
pages = {6834-6850},
doi = {10.1021/acs.jcim.3c01284},
pmid = {37877218},
issn = {1549-960X},
mesh = {*CRISPR-Cas Systems ; *Molecular Dynamics Simulation ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; DNA/chemistry ; RNA/chemistry ; Endonucleases/chemistry/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool using Cas family proteins. Two magnesium-dependent nuclease domains of the Cas9 enzyme, termed HNH and RuvC, are responsible for cleaving the target DNA (t-DNA) and nontarget DNA strands, respectively. The HNH domain is believed to determine the DNA cleavage activity of both endonuclease domains and is sensitive to complementary RNA-DNA base pairing. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights into improving its specificity by reducing off-target cleavages. Here, we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics and coupled quantum mechanics/molecular mechanics simulations to study two possible mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G at the fifth position from the protospacer adjacent motif region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Based on these simulations, our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic roles for the two studied systems, including K253, K263, R820, K896, and K913.},
}
@article {pmid37718653,
year = {2023},
author = {Muñoz-Pujol, G and Ugarteburu, O and Segur-Bailach, E and Moliner, S and Jurado, S and Garrabou, G and Guitart-Mampel, M and García-Villoria, J and Artuch, R and Fons, C and Ribes, A and Tort, F},
title = {CRISPR/Cas9-based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders.},
journal = {Journal of inherited metabolic disease},
volume = {46},
number = {6},
pages = {1029-1042},
doi = {10.1002/jimd.12681},
pmid = {37718653},
issn = {1573-2665},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Virulence ; Genomics ; *Metabolic Diseases/genetics ; },
abstract = {The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9-based strategy for knock-in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD-associated genes, including seven reported disease-causing variants and four benign/likely benign variants. Overall, 11 knock-in cell models were generated via homology-directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock-in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non-pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders.},
}
@article {pmid37467014,
year = {2023},
author = {Samanta, A and George, N and Arnaoutova, I and Chen, HD and Mansfield, BC and Hart, C and Carlo, T and Chou, JY},
title = {CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia.},
journal = {Journal of inherited metabolic disease},
volume = {46},
number = {6},
pages = {1147-1158},
doi = {10.1002/jimd.12660},
pmid = {37467014},
issn = {1573-2665},
mesh = {Mice ; Animals ; *Oligonucleotides/metabolism ; CRISPR-Cas Systems ; *Glycogen Storage Disease Type I/genetics/therapy/metabolism ; Liver/metabolism ; Glucose-6-Phosphatase/genetics/metabolism ; },
abstract = {Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.},
}
@article {pmid37940844,
year = {2023},
author = {Ramos, JN and Araújo, MRB and Baio, PVP and Sant'Anna, LO and Veras, JFC and Vieira, &#xc9;MD and Sousa, M&#xc2;B and Camargo, CH and Sacchi, CT and Campos, KR and Santos, MBN and Bokermann, S and Alvim, LB and Sanches Dos Santos, L and de Mattos-Guaraldi, AL and Vieira, VV},
title = {Molecular characterization and phylogenetic analysis of the first Corynebacterium rouxii strains isolated in Brazil: a recent member of Corynebacterium diphtheriae complex.},
journal = {BMC genomic data},
volume = {24},
number = {1},
pages = {65},
pmid = {37940844},
issn = {2730-6844},
support = {E-26/202.088/2020; E-26/205.901/2022; E26/211.554/2019; E-26/210.285/2021//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; E-26/202.088/2020; E-26/205.901/2022; E26/211.554/2019; E-26/210.285/2021//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; E-26/202.088/2020; E-26/205.901/2022; E26/211.554/2019; E-26/210.285/2021//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; E-26/202.088/2020; E-26/205.901/2022; E26/211.554/2019; E-26/210.285/2021//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; 309948/2018-5//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico - CNPq/ ; SR-2/UERJ//Sub-Reitoria de P&#xf3;s-Gradua&#xe7;&#xe3;o e Pesquisa da Universidade do Estado do Rio de Janeiro/ ; VPPCB-007-FIO-18-2-103//Programa Inova Fiocruz/ ; },
mesh = {*Corynebacterium diphtheriae/genetics ; Phylogeny ; Brazil ; Multilocus Sequence Typing ; Corynebacterium/genetics ; },
abstract = {BACKGROUND: Corynebacterium diphtheriae complex was formed by the species C. diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in the recent past. In addition to C. diphtheriae, C. ulcerans and C. pseudotuberculosis species can carry the tox gene, which encodes diphtheria toxin. Currently, three new species have been included in the complex: Corynebacterium rouxii, Corynebacterium silvaticum, and Corynebacterium belfantii. C. rouxii is derived from the ancient Belfanti biovar of C. diptheriae. We provide the complete genome sequences of two non-toxigenic strains C. rouxii isolated from a cat with a purulent infection in Brazil. The taxonomic status and sequence type, as well as the presence of resistance and virulence genes, and CRISPR-Cas system were additionally defined.<br><br>RESULTS: The genomes showed an average size of 2.4&#xa0;Mb and 53.2% GC content, similar to the type strain of the species deposited in Genbank/NCBI. Strains were identified as C. rouxii by the rMLST database, with 95% identity. ANI and DDH in silico were consistent with values above the proposed cut-off points for species limit, corroborating the identification of the strains as C. rouxii. MLST analyses revealed a new ST, which differs from ST-537 only by the fusA allele. No horizontal transfer resistance gene was predicted in both genomes and no mutation was detected in the constitutive genes gyrA and rpoB. Some mutations were found in the seven penicillin-binding proteins (PBPs) detected. The tox gene was not found, but its regulatory gene dtxR was present. Among the predicted virulence genes are those involved in iron uptake and adherence, in addition to the DIP0733 protein involved in epithelial cell adhesion and invasion. The CRISPR-Cas type I-E system was detected in both genomes, with 16 spacer sequences each. Of them, half are unknown according to the databases used, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids.<br><br>CONCLUSIONS: This is the first genomic study of C. rouxii reported in Brazil. Here we performed taxonomic analysis and the prediction of virulence factors. The genomic analyses performed in this study may help to understand the potential pathogenesis of non-toxigenic C. rouxii strains.},
}
@article {pmid37938759,
year = {2023},
author = {Du Toit, A},
title = {Sabotage of CRISPR-Cas.},
journal = {Nature reviews. Microbiology},
volume = {},
number = {},
pages = {},
pmid = {37938759},
issn = {1740-1534},
}
@article {pmid37933907,
year = {2023},
author = {Wang, Y and Wu, L and Yu, X and Wang, G and Pan, T and Huang, Z and Cui, T and Huang, T and Huang, Z and Nie, L and Qian, C},
title = {Development of a rapid, sensitive detection method for SARS-CoV-2 and influenza virus based on recombinase polymerase amplification combined with CRISPR-Cas12a assay.},
journal = {Journal of medical virology},
volume = {95},
number = {11},
pages = {e29215},
doi = {10.1002/jmv.29215},
pmid = {37933907},
issn = {1096-9071},
mesh = {Humans ; Recombinases ; SARS-CoV-2 ; *Influenza, Human ; CRISPR-Cas Systems ; *COVID-19 ; Nucleotidyltransferases ; *Orthomyxoviridae ; Nucleic Acid Amplification Techniques ; },
abstract = {Respiratory tract infections are associated with the most common diseases transmitted among people and remain a huge threat to global public health. Rapid and sensitive diagnosis of causative agents is critical for timely treatment and disease control. Here, we developed a novel method based on recombinase polymerase amplification (RPA) combined with CRISPR-Cas12a to detect three viral pathogens, including SARS-CoV-2, influenza A, and influenza B, which cause similar symptom complexes of flu cold in the respiratory tract. The detection method can be completed within 1 h, which is faster than other standard detection methods, and the limit of detection is approximately 10[2] copies/μL. Additionally, this detection system is highly specific and there is no cross-reactivity with other common respiratory tract pathogens. Based on this assay, we further developed a more simplified RPA/CRISPR-Cas12a system combined with lateral flow assay on a manual microfluidic chip, which can simultaneously detect these three viruses. This low-cost detection system is rapid and sensitive, which could be applied in the field and resource-limited areas without bulky and expensive instruments, providing powerful tools for the point-of-care diagnostic.},
}
@article {pmid37932818,
year = {2023},
author = {Schmitt, LT and Schneider, A and Posorski, J and Lansing, F and Jelicic, M and Jain, M and Sayed, S and Buchholz, F and Sürün, D},
title = {Quantification of evolved DNA-editing enzymes at scale with DEQSeq.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {254},
pmid = {37932818},
issn = {1474-760X},
support = {742133/ERC_/European Research Council/International ; },
mesh = {*Recombinases/genetics/metabolism ; *Directed Molecular Evolution/methods ; Gene Editing/methods ; DNA ; CRISPR-Cas Systems ; },
abstract = {We introduce DEQSeq, a nanopore sequencing approach that rationalizes the selection of favorable genome editing enzymes from directed molecular evolution experiments. With the ability to capture full-length sequences, editing efficiencies, and specificities from thousands of evolved enzymes simultaneously, DEQSeq streamlines the process of identifying the most valuable variants for further study and application. We apply DEQSeq to evolved libraries of Cas12f-ABEs and designer-recombinases, identifying variants with improved properties for future applications. Our results demonstrate that DEQSeq is a powerful tool for accelerating enzyme discovery and advancing genome editing research.},
}
@article {pmid37932698,
year = {2023},
author = {Ahmed, NA and Khattab, RA and Ragab, YM and Hassan, M},
title = {Safety assessment of Enterococcus lactis strains complemented with comparative genomics analysis reveals probiotic and safety characteristics of the entire species.},
journal = {BMC genomics},
volume = {24},
number = {1},
pages = {667},
pmid = {37932698},
issn = {1471-2164},
mesh = {Humans ; Caco-2 Cells ; Microbial Sensitivity Tests ; Enterococcus/genetics ; Anti-Bacterial Agents ; *Probiotics ; Genomics ; *Enterococcus faecium/genetics ; },
abstract = {BACKGROUND: The gut microbiota is considered a rich source for potential novel probiotics. Enterococcus genus is a normal component of a healthy gut microbiota, suggesting its vital role. Nosocomial infections caused mainly by E. facalis and E. faecium have been attributed to the plasticity of the Enterococcus genomes. In this study, we assessed the probiotic and safety characteristics of two E. lactis strains isolated from the human gut microbiota using in-vitro and in silico approaches. Additionally, the safety of the E. lactis species was evaluated using comparative genomics analysis.<br><br>RESULTS: The two E. lactis strains 10NA and 50NA showed resistance to bile salts and acid tolerance with antibacterial activity against Escherichia coli, Salmonella typhi, and Clostridioides difficile. For safety assays, the two strains did not display any type of hemolysis on blood agar, and the survival of Caco-2 cells was not significantly different (P-value&#x2009;>&#x2009;0.05) compared to the control using cell free supernatants at 100% (v/v), 50% (v/v), 10% (v/v), and 5% (v/v) concentrations. Regarding antibiotic susceptibility, both strains were sensitive to vancomycin, tetracycline, and chloramphenicol. Comprehensive whole-genome analysis revealed no concerning associations between virulence or antibiotic resistance genes and any of the identified mobile genetic elements. Comparative genome analysis with closely related E. faecium species genomes revealed the distinctive genomic safety of the E. lactis species.<br><br>CONCLUSIONS: Our two E. lactis strains showed promising probiotic properties in-vitro. Their genomes were devoid of any transferable antibiotic resistance genes. In silico comparative analysis confirmed the safety of the E. lactis species. These results suggest that E. lactis species could be a potential source for safer Enterococcus probiotic supplements.},
}
@article {pmid37932335,
year = {2023},
author = {Huang, S and Wang, X and Chen, X and Liu, X and Xu, Q and Zhang, L and Huang, G and Wu, J},
title = {Rapid and sensitive detection of Pseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {19199},
pmid = {37932335},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Pseudomonas aeruginosa ; *Acinetobacter baumannii ; *Household Articles ; *Manipulation, Osteopathic ; },
abstract = {CRISPR based technologies have been used for fast and sensitive detection of pathogens. To test the possibility of CRISPR based detection strategy in Pseudomonas aeruginosa infections, a combined method of recombinase polymerase amplification followed by Cas12a-mediated detection via fluorescence reader or lateral flow biosensor (named Cas12a-RCFL) has been established in this study. The Cas12a-RCFL can detect as low as 50 CFU/mL Pseudomonas aeruginosa. The whole detection process can be finished within one hour with satisfied detection specificity. Cas12a-RCFL also shows good sensitivity of detecting Pseudomonas aeruginosa inStaphylococcus aureus and Acinetobacter baumannii contaminated samples. For the detection of 22 clinical samples, Cas12a-RCFL matches with PCR sequencing result exactly without DNA purification. This Cas12a-RCFL is rapid and sensitive with low cost, which shows good quality to be adopted as a point-of-care testing method.},
}
@article {pmid37918326,
year = {2023},
author = {Niinuma, S and Wake, Y and Nakagawa, Y and Kaneko, T},
title = {Importance of nuclear localization signal-fused Cas9 in the production of genome-edited mice via embryo electroporation.},
journal = {Biochemical and biophysical research communications},
volume = {685},
number = {},
pages = {149140},
doi = {10.1016/j.bbrc.2023.149140},
pmid = {37918326},
issn = {1090-2104},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Nuclear Localization Signals/genetics ; Mice, Knockout ; Gene Editing/methods ; Electroporation/methods ; },
abstract = {Previously, to generate genome-edited animals by introducing CRISPR-associated protein 9 (Cas9) into embryos, we developed the Technique for Animal Knockout system by Electroporation (TAKE). Additionally, by fluorescently labeling Cas9, we successfully visualized the Cas9 introduced into the pronuclei of embryos; however, whether Cas9 was introduced directly into the pronuclei by electric pulse or transferred from the cytoplasm by nuclear localization signal (NLS) remained unknown. Herein, we evaluated the localization of Cas9 with (Cas9-NLS) or without NLS (Cas9-noNLS) in mice embryos following electroporation by fusing them with GFP. Furthermore, we visually studied their effects on genome-editing rates in offspring by targeting tyrosinase gene. Fluorescence intensity in pronuclei of Cas9-NLS-electroporated embryos and genome-editing rates of offspring were significantly higher than those of Cas9-noNLS-electroporated embryos. Furthermore, fluorescence in Cas9-NLS-electroporated embryos in which pronuclei had not yet appeared 2.5 h after insemination was observed in the pronuclei of embryos appearing 3.5 h after electroporation. We demonstrated the effective transportation of Cas9 from the cytoplasm to pronuclei by the NLS following TAKE, which resulted in increased genome-editing rates in offspring. The TAKE along with fluorescently labeled nucleases can be used to verify nuclease delivery into individual embryos prior to embryo transfer for efficiently producing genome-edited animals.},
}
@article {pmid37853195,
year = {2023},
author = {Kraus, C and Sontheimer, EJ},
title = {Viruses use RNA decoys to thwart CRISPR defences.},
journal = {Nature},
volume = {623},
number = {7987},
pages = {490-491},
pmid = {37853195},
issn = {1476-4687},
mesh = {*RNA ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Viruses/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37843111,
year = {2023},
author = {Yuan, B and Zhang, S and Song, L and Chen, J and Cao, J and Qiu, J and Qiu, Z and Chen, J and Zhao, XM and Cheng, TL},
title = {Engineering of cytosine base editors with DNA damage minimization and editing scope diversification.},
journal = {Nucleic acids research},
volume = {51},
number = {20},
pages = {e105},
pmid = {37843111},
issn = {1362-4962},
support = {2019YFA0111000//National Key R&amp;D Program of China/ ; 20ZR1403100//Natural Science Foundation of Shanghai/ ; 20JC1419500//Shanghai Municipal Science and Technology/ ; LG-QS-202203-08//Lingang Laboratory/ ; 31600826//National Natural Science Foundation of China/ ; B18015//111 Project of China/ ; 2018SHZDZX01 to X.M.Z.//Shanghai Municipal Science and Technology Major Project/ ; XDB32060202//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; //Fundamental Research Funds for the Central Universities/ ; 31625013//Natural Science Foundation of China/ ; 19XD1404300//Program of Shanghai Academic Research Leader/ ; SHSMU-ZDCX20211100//Innovative research team of high-level local universities in Shanghai/ ; //Ruijin Hospital Shanghai Jiao Tong University School of Medicine/ ; },
mesh = {Humans ; *Gene Editing ; *Cytosine ; RNA/genetics ; DNA Damage ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Cytosine base editors (CBEs), which enable precise C-to-T substitutions, have been restricted by potential safety risks, including DNA off-target edits, RNA off-target edits and additional genotoxicity such as DNA damages induced by double-strand breaks (DSBs). Though DNA and RNA off-target edits have been ameliorated via various strategies, evaluation and minimization of DSB-associated DNA damage risks for most CBEs remain to be resolved. Here we demonstrate that YE1, an engineered CBE variant with minimized DNA and RNA off-target edits, could induce prominent DSB-associated DNA damage risks, manifested as γH2AX accumulation in human cells. We then perform deaminase engineering for two deaminases lamprey LjCDA1 and human APOBEC3A, and generate divergent CBE variants with eliminated DSB-associated DNA damage risks, in addition to minimized DNA/RNA off-target edits. Furthermore, the editing scopes and sequence preferences of APOBEC3A-derived CBEs could be further diversified by internal fusion strategy. Taken together, this study provides updated evaluation platform for DSB-associated DNA damage risks of CBEs and further generates a series of safer toolkits with diversified editing signatures to expand their applications.},
}
@article {pmid37811564,
year = {2023},
author = {Lin, Z and Sun, B and Yang, X and Jiang, Y and Wu, S and Lv, B and Pan, Y and Zhang, Q and Wang, X and Xiang, G and Lou, Y and Xiao, X},
title = {Infectious Disease Diagnosis and Pathogen Identification Platform Based on Multiplex Recombinase Polymerase Amplification-Assisted CRISPR-Cas12a System.},
journal = {ACS infectious diseases},
volume = {9},
number = {11},
pages = {2306-2315},
doi = {10.1021/acsinfecdis.3c00381},
pmid = {37811564},
issn = {2373-8227},
mesh = {Humans ; *Hand, Foot and Mouth Disease ; CRISPR-Cas Systems ; Recombinases ; *Influenza A Virus, H1N1 Subtype ; Influenza A Virus, H3N2 Subtype ; Sensitivity and Specificity ; Nucleotidyltransferases ; Multiplex Polymerase Chain Reaction ; *Nucleic Acids ; },
abstract = {Controlling and mitigating infectious diseases caused by multiple pathogens or pathogens with several subtypes require multiplex nucleic acid detection platforms that can detect several target genes rapidly, specifically, sensitively, and simultaneously. Here, we develop a detection platform, termed Multiplex Assay of RPA and Collateral Effect of Cas12a-based System (MARPLES), based on multiplex nucleic acid amplification and Cas12a ssDNase activation to diagnose these diseases and identify their pathogens. We use the clinical specimens of hand, foot, and mouth disease (HFMD) and influenza A to evaluate the feasibility of MARPLES in diagnosing the disease and identifying the pathogen, respectively, and find that MARPLES can accurately diagnose the HFMD associated with enterovirus 71, coxsackievirus A16 (CVA16), CVA6, or CVA10 and identify the exact types of H1N1 and H3N2 in an hour, showing high sensitivity and specificity and 100% predictive agreement with qRT-PCR. Collectively, our findings demonstrate that MARPLES is a promising multiplex nucleic acid detection platform for disease diagnosis and pathogen identification.},
}
@article {pmid37734427,
year = {2023},
author = {Tang, Y and Du, E and Wang, G and Qin, F and Meng, Z and Dai, L and Wang, Y and Ren, S},
title = {A negative feedback loop centered on SMAD3 expression in transforming growth factor β1-induced corneal myofibroblast differentiation.},
journal = {Experimental eye research},
volume = {236},
number = {},
pages = {109654},
doi = {10.1016/j.exer.2023.109654},
pmid = {37734427},
issn = {1096-0007},
mesh = {Humans ; *Transforming Growth Factor beta1/metabolism ; Myofibroblasts/metabolism ; Smad3 Protein/metabolism ; Feedback ; RNA, Guide, CRISPR-Cas Systems ; Cells, Cultured ; Fibroblasts/metabolism ; *Lentivirus Infections/metabolism ; Transforming Growth Factor beta/metabolism ; },
abstract = {SMAD3 downregulation is documented in transforming growth factor β1 (TGF-β1)-induced corneal fibroblasts differentiation to myofibroblasts ("fibroTOmyoDiff") or corneal wound healing. However, the exact regulatory mechanism of TGF-β1/SMAD3 pathway in this context remains unclear. Here, we investigated the role and related mechanism of SMAD3 down-regulation in TGF-β1-induced human corneal fibroTOmyoDiff. By detecting expression changes of SMAD family during this process, we demonstrated that SMAD3 protein expression was dramatically decreased in the process and the decrease occurred mainly in SMAD3 gene transcription. Furthermore, SMAD3 overexpression using lentivirus infection and knockdown using sgRNA lentivirus infection or siRNAs revealed that SMAD3 overexpression enhanced TGF-β1-induced corneal fibroTOmyoDiff and vice versa. In addition, specific siRNAs and inhibitors targeting particular signaling pathway were used to figure out the intracellular signaling pathway regulating SMAD3, and the result showed that the decease of SMAD3 induced by TGF-β1 stimulation in human corneal fibroblasts (HCFs) was strikingly prevented by SMAD4 knockdown or p38 signaling inhibitor SB203580 treatment. Collectively, these results demonstrate that, in TGF-β1 induced corneal fibroTOmyoDiff, down-regulation of SMAD3 expression regulated by SMAD4 and p38 signaling pathways forms a negative feedback loop of TGFβ signaling to avoid excessive activation of the signaling, which suggest that SMAD3 may be a key target for corneal fibrosis treatment.},
}
@article {pmid37700173,
year = {2023},
author = {Chen, Y and Xiang, H and Jia, L and Yang, Q and Zhang, J and Jiang, J and Zeng, W and Deng, L and Jin, J and Gao, Q and Li, X},
title = {High-throughput creation of Nicotiana tabacum gene-targeted mutants based on CRISPR/Cas9.},
journal = {Plant cell reports},
volume = {42},
number = {12},
pages = {2039-2042},
pmid = {37700173},
issn = {1432-203X},
support = {110202101034//Creation and Identification of Editing Material Library of Tobacco Transporter and Enzyme Related Genes in Red flower Mammoth Gold/ ; 2022JY03//Development of Tagless Gene Editing Technology and Breeding Application of Low Nicotine Editing Materials/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Tobacco/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; },
abstract = {4382 available sgRNAs targeting 1060 tobacco genes were obtained, and 10,682 targeted mutants were created using high-throughput methods. Four optimization experiments were established to solve problems encountered during genetic transformation.},
}
@article {pmid37624911,
year = {2023},
author = {Adams, D and Algalarrondo, V and Echaniz-Laguna, A},
title = {Hereditary transthyretin amyloidosis in the era of RNA interference, antisense oligonucleotide, and CRISPR-Cas9 treatments.},
journal = {Blood},
volume = {142},
number = {19},
pages = {1600-1612},
doi = {10.1182/blood.2023019884},
pmid = {37624911},
issn = {1528-0020},
mesh = {Adult ; Humans ; *Oligonucleotides, Antisense/genetics/therapeutic use ; RNA Interference ; Quality of Life ; CRISPR-Cas Systems ; *Amyloid Neuropathies, Familial/therapy/drug therapy ; Oligonucleotides ; RNA ; },
abstract = {Hereditary transthyretin amyloidosis (ATTRv) is a rare autosomal dominant adult-onset disorder caused by point mutations in the transthyretin (TTR) gene encoding TTR, also known as prealbumin. ATTRv survival ranges from 3 to 10 years, and peripheral nervous system and heart are usually the 2 main tissues affected, although central nervous system and eye may also be involved. Because the liver is the main TTR protein secretor organ, it has been the main target of treatments developed these last years, including liver transplantation, which has been shown to significantly increase survival in a subset of patients carrying the so-called "early-onset Val30Met" TTR gene mutation. More recently, treatments targeting hepatic TTR RNA have been developed. Hepatic TTR RNA targeting is performed using RNA interference (RNAi) and antisense oligonucleotide (ASO) technologies involving lipid nanoparticle carriers or N-acetylgalactosamine fragments. RNAi and ASO treatments induce an 80% decrease in TTR liver production for a period of 1 to 12 weeks. ASO and RNAi phase 3 trials in patients with TTR-related polyneuropathy have shown a positive impact on neuropathy clinical scores and quality of life end points, and delayed RNAi treatment negatively affects survival. Clinical trials specifically investigating RNAi therapy in TTR cardiomyopathy are underway. Hepatic RNA targeting has revolutionized ATTRv treatment and may allow for the transforming a fatal disease into a treatable disorder. Because retina and choroid plexus secrete limited quantities of TTR protein, both tissues are now seen as the next targets for fully controlling the disease.},
}
@article {pmid37938683,
year = {2022},
author = {Marasco, R and Fusi, M and Ramond, JB and Van Goethem, MW and Seferji, K and Maggs-Kölling, G and Cowan, DA and Daffonchio, D},
title = {The plant rhizosheath-root niche is an edaphic "mini-oasis" in hyperarid deserts with enhanced microbial competition.},
journal = {ISME communications},
volume = {2},
number = {1},
pages = {47},
pmid = {37938683},
issn = {2730-6151},
abstract = {Plants have evolved unique morphological and developmental adaptations to cope with the abiotic stresses imposed by (hyper)arid environments. Such adaptations include the formation of rhizosheath-root system in which mutualistic plant-soil microbiome associations are established: the plant provides a nutrient-rich and shielded environment to microorganisms, which in return improve plant-fitness through plant growth promoting services. We hypothesized that the rhizosheath-root systems represent refuge niches and resource islands for the desert edaphic microbial communities. As a corollary, we posited that microorganisms compete intensively to colonize such "oasis" and only those beneficial microorganisms improving host fitness are preferentially selected by plant. Our results show that the belowground rhizosheath-root micro-environment is largely more hospitable than the surrounding gravel plain soil with higher nutrient and humidity contents, and cooler temperatures. By combining metabarcoding and shotgun metagenomics, we demonstrated that edaphic microbial biomass and community stability increased from the non-vegetated soils to the rhizosheath-root system. Concomitantly, non-vegetated soil communities favored autotrophy lifestyle while those associated with the plant niches were mainly heterotrophs and enriched in microbial plant growth promoting capacities. An intense inter-taxon microbial competition is involved in the colonization and homeostasis of the rhizosheath zone, as documented by significant enrichment of antibiotic resistance genes and CRISPR-Cas motifs. Altogether, our results demonstrate that rhizosheath-root systems are "edaphic mini-oases" and microbial diversity hotspots in hyperarid deserts. However, to colonize such refuge niches, the desert soil microorganisms compete intensively and are therefore prepared to outcompete potential rivals.},
}
@article {pmid37932102,
year = {2023},
author = {Papaioannou, VE and Behringer, RR},
title = {Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.over107957},
pmid = {37932102},
issn = {1559-6095},
abstract = {Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells. For the planning stages of making different types of alleles, we first consider general strategies and then provide details specific to either homologous recombination in ES cells or making alleles by gene editing with CRISPR-Cas in preimplantation embryos. The types of alleles considered are null or knockout alleles, reporter gene knock-in alleles, point mutations, and conditional null alleles.},
}
@article {pmid37932094,
year = {2023},
author = {Papaioannou, VE and Behringer, RR},
title = {Recovering a Targeted Mutation in Mice from Embryonic Stem Cell Chimeras or CRISPR-Cas Founders.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.over107959},
pmid = {37932094},
issn = {1559-6095},
abstract = {Following the production of chimeras from targeted embryonic stem (ES) cells or obtaining founders from CRISPR-Cas gene editing in preimplantation embryos, the desired targeted mutation must be recovered and established in the heterozygous state in a strain or stock of mice for further study. The breeding schemes for ES chimeras and CRISPR-Cas founders differ. For ES cell chimeras, we discuss the relative benefits of breeding from male or female chimeras. We discuss the importance of genetic background and provide practical advice for putting the mutation on inbred or outbred backgrounds or producing a coisogenic strain. For CRISPR-Cas founders, which will most likely be mosaic for different mutations, initial breeding strategies are discussed to maintain a desired genetic background at the same time as producing progeny to segregate different alleles. Strategies for testing the progeny to recognize indels, missense mutations, and knock-in mutations are discussed. In the event that ES cell chimeras or CRISPR-Cas founders produce no offspring or fail to transmit the mutant allele(s), there is a troubleshooting guide to pinpoint the problem. If heterozygous offspring from the chimeras or founders are normal, fertile, and of both sexes, the analysis of homozygous effects of the mutation can now begin; if not, possible dominant effects are considered.},
}
@article {pmid37932093,
year = {2023},
author = {Papaioannou, VE and Behringer, RR},
title = {Strategies for the Production and Phenotypic Analysis of Mutations in the Mouse.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.over107955},
pmid = {37932093},
issn = {1559-6095},
abstract = {The laboratory mouse is one of the primary model organisms for human biology and genetics. The production and phenotypic analysis of mutations in the mouse has made great strides with targeted mutagenesis in embryonic stem (ES) cells and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing in preimplantation embryos. Mutations in many genes are now available or can be easily produced. For anyone planning to use mutational analysis for a research program, this article introduces an overall strategy for obtaining or generating a mutation and systematically analyzing the phenotype.},
}
@article {pmid37932089,
year = {2023},
author = {Papaioannou, VE and Behringer, RR},
title = {Phenotypic Analysis of Dominant Mutant Effects in Mice.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.over107978},
pmid = {37932089},
issn = {1559-6095},
abstract = {Dominant effects of a mutation may show up at any time during a mutational analysis, including during the early stages of an embryonic stem (ES) cell gene targeting experiment. Here, we discuss the mechanisms of dominant and semidominant effects and how they might appear if they show up in heterozygous ES cells, in ES cell chimeras, or in heterozygous progeny of chimeras. Similarly, dominant effects may be seen in mice heterozygous for CRISPR-Cas-targeted, -induced, or spontaneous mutations. If the dominant effects prevent the germline transmission of ES cells or cause fertility problems in heterozygotes, they can severely limit further analysis of the mutation. Ways to circumvent such reproductive problems are presented. The special case of imprinted genes, which may be functionally hemizygous and present a different phenotype when inherited from the mother than when inherited from the father, is discussed.},
}
@article {pmid37932087,
year = {2023},
author = {Papaioannou, VE and Behringer, RR},
title = {Embryonic Stem Cell Gene Targeting and Chimera Production in Mice.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.over107958},
pmid = {37932087},
issn = {1559-6095},
abstract = {Producing a custom gene mutation in embryonic stem (ES) cells, whether through homologous recombination or CRISPR-Cas gene editing, is the first step along the way to getting the mutation into live mice. However, there are a number of additional steps along the way, each presenting technical challenges. Here, we provide a guide for troubleshooting when the results are not as expected and to distinguish technical problems from possible biological effects of the mutation. From the isolation of clonal lines of targeted ES cells through the production of ES cell chimeras with the targeted ES cell clone, we discuss common technical problems and their most likely causes and solutions. We also provide guidance for situations where the mutation has a phenotype in the form of a dominant effect on ES cells or in chimeras.},
}
@article {pmid37819494,
year = {2023},
author = {Tamizi, AA and Md-Yusof, AA and Mohd-Zim, NA and Nazaruddin, NH and Sekeli, R and Zainuddin, Z and Samsulrizal, NH},
title = {Agrobacterium-mediated in planta transformation of cut coleoptile: a new, simplified, and tissue culture-independent method to deliver the CRISPR/Cas9 system in rice.},
journal = {Molecular biology reports},
volume = {50},
number = {11},
pages = {9353-9366},
pmid = {37819494},
issn = {1573-4978},
support = {FRGS/1/2019/STG05/UIAM/03/8//Ministry of Higher Education, Malaysia/ ; RMK-12 Mega Project: P-502, Sub-project D//Institut Penyelidikan dan Kemajuan Pertanian Malaysia/ ; MDI/SM/PL/312/001 Jld.21//Institut Penyelidikan dan Kemajuan Pertanian Malaysia/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Oryza/genetics/metabolism ; Cotyledon/genetics ; Tissue Culture Techniques/methods ; Plants, Genetically Modified/genetics ; Agrobacterium tumefaciens/genetics ; },
abstract = {BACKGROUND: Agrobacterium-mediated transformation and particle bombardment are the two common approaches for genome editing in plant species using CRISPR/Cas9 system. Both methods require careful manipulations of undifferentiated cells and tissue culture to regenerate the potentially edited plants. However, tissue culture techniques are laborious and time-consuming.<br><br>METHODS AND RESULTS: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR&#xa0;219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25&#x2009;&#xb1;&#x2009;2&#xa0;&#xb0;C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants.<br><br>CONCLUSION: This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.},
}
@article {pmid37805953,
year = {2023},
author = {Liu, J and Zhu, J and Zhang, Q and Lv, R and Liu, H},
title = {Establishing a one-step marker-free CRISPR/Cas9 system for industrial Aspergillus niger using counter-selectable marker Ang-ace2.},
journal = {Biotechnology letters},
volume = {45},
number = {11-12},
pages = {1477-1485},
pmid = {37805953},
issn = {1573-6776},
support = {2021YFC2100700//the National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Aspergillus niger/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {OBJECTIVES: To develop a one-step, marker-free CRISPR/Cas9 system for highly efficient genome editing in industrial Aspergillus niger, with a short genetic operation cycle.<br><br>RESULTS: Firstly, evaluation of different promoters for sgRNA expression revealed tRNA[Gly15] as the most efficient, achieving a remarkable 100% gene editing efficiency. Furthermore, a counter-selectable marker, Ang-ace2, was identified for A. niger. Subsequently, a CRISPR/Cas9 plasmid was developed, utilizing a truncated AMA1 element and the Ang-ace2 conditional expression cassette driven by a Tet-on promoter. In the presence of doxycycline, the plasmid demonstrated a 33% loss efficiency in the progeny of A. niger spores after a single generation, resulting in a shortened genetic operation cycle of 16&#xa0;days for CRISPR/Cas9.<br><br>CONCLUSIONS: The one-step marker-free CRISPR/Cas9 system was successfully developed in industrial A. niger, allowing for efficient gene editing while simultaneously reducing the editing time.},
}
@article {pmid37750448,
year = {2023},
author = {Hassan, AZ and Ward, HN and Rahman, M and Billmann, M and Lee, Y and Myers, CL},
title = {Dimensionality reduction methods for extracting functional networks from large-scale CRISPR screens.},
journal = {Molecular systems biology},
volume = {19},
number = {11},
pages = {e11657},
pmid = {37750448},
issn = {1744-4292},
support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; R01HG005853/NH/NIH HHS/United States ; R01HG005084/NH/NIH HHS/United States ; T32GM008347/NH/NIH HHS/United States ; R01HG005084/NH/NIH HHS/United States ; R01HG005853/NH/NIH HHS/United States ; T32GM008347/NH/NIH HHS/United States ; },
mesh = {Humans ; Cell Line, Tumor ; *Oncogenes ; *Gene Regulatory Networks ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-Cas9 screens facilitate the discovery of gene functional relationships and phenotype-specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole-genome CRISPR screens aimed at identifying cancer-specific genetic dependencies across human cell lines. A mitochondria-associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co-essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods-autoencoders, robust, and classical principal component analyses (PCA)-for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel "onion" normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low-dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction-based normalization tools.},
}
@article {pmid37722240,
year = {2023},
author = {Bigini, F and Lee, SH and Sun, YJ and Sun, Y and Mahajan, VB},
title = {Unleashing the potential of CRISPR multiplexing: Harnessing Cas12 and Cas13 for precise gene modulation in eye diseases.},
journal = {Vision research},
volume = {213},
number = {},
pages = {108317},
doi = {10.1016/j.visres.2023.108317},
pmid = {37722240},
issn = {1878-5646},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; RNA, Messenger ; Eye ; *Eye Diseases/genetics/therapy ; },
abstract = {Gene therapy is a flourishing field with the potential to revolutionize the treatment of genetic diseases. The emergence of CRISPR-Cas9 has significantly advanced targeted and efficient genome editing. Although CRISPR-Cas9 has demonstrated promising potential applications in various genetic disorders, it faces limitations in simultaneously targeting multiple genes. Novel CRISPR systems, such as Cas12 and Cas13, have been developed to overcome these challenges, enabling multiplexing and providing unique advantages. Cas13, in particular, targets mRNA instead of genomic DNA, permitting precise gene expression control and mitigating off-target effects. This review investigates the potential of Cas12 and Cas13 in ocular gene therapy applications, such as suppression of inflammation and cell death. In addition, the capabilities of Cas12 and Cas13 are explored in addressing potential targets related with disease mechanisms such as aberrant isoforms, mitochondrial genes, cis-regulatory sequences, modifier genes, and long non-coding RNAs. Anatomical accessibility and relative immune privilege of the eye provide an ideal organ system for evaluating these novel techniques' efficacy and safety. By targeting multiple genes concurrently, CRISPR-Cas12 and Cas13 systems hold promise for treating a range of ocular disorders, including glaucoma, retinal dystrophies, and age-related macular degeneration. Nonetheless, additional refinement is required to ascertain the safety and efficacy of these approaches in ocular disease treatments. Thus, the development of Cas12 and Cas13 systems marks a significant advancement in gene therapy, offering the potential to devise effective treatments for ocular disorders.},
}
@article {pmid37632456,
year = {2023},
author = {Bohrer, LR and Stone, NE and Wright, AT and Han, S and Sicher, I and Sulchek, TA and Mullins, RF and Tucker, BA},
title = {CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing.},
journal = {Stem cells (Dayton, Ohio)},
volume = {41},
number = {11},
pages = {1037-1046},
pmid = {37632456},
issn = {1549-4918},
support = {R01 EY026008/EY/NEI NIH HHS/United States ; R01 EY026008/NH/NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Microfluidics ; Transfection ; },
abstract = {Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.},
}
@article {pmid37407493,
year = {2023},
author = {Sakai, Y and Okabe, Y and Itai, G and Shiozawa, S},
title = {An efficient evaluation system for factors affecting the genome editing efficiency in mouse.},
journal = {Experimental animals},
volume = {72},
number = {4},
pages = {526-534},
doi = {10.1538/expanim.23-0045},
pmid = {37407493},
issn = {1881-7122},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems ; Mutation ; Genotype ; },
abstract = {Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.},
}
@article {pmid37932077,
year = {2023},
author = {Papaioannou, VE and Behringer, RR},
title = {Obtaining or Generating Gene Mutations in Mice.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.over107956},
pmid = {37932077},
issn = {1559-6095},
abstract = {The starting point in a mutational analysis of gene function is obtaining or producing a mutant. Here different methods of obtaining mouse mutants are discussed, including screening for spontaneous mutants, screening for mutants following chemical or X-ray mutagenesis, and producing mutations through targeted manipulation of the genome. Manipulation of the genome can be random, as in different types of insertional mutagenesis. Alternatively, targeted manipulation such as gene targeting using homologous recombination in embryonic stem (ES) cells or gene editing by CRISPR-Cas can be used to produce custom mutations in a specific gene. The basic methods are outlined, and the advantages and disadvantages of homologous recombination and CRISPR-Cas gene editing are discussed. Resources for obtaining mutations that already exist are provided. If, for your planned study, no suitable mutations are available, there is advice about what you should know about your gene of interest before embarking on a gene targeting experiment.},
}
@article {pmid37930004,
year = {2023},
author = {Yang, Z and Zhang, Y and Du, S and Zhao, Q and Huang, X and Wu, R and Yan, Q and Han, X and Cao, S and Chang, Y-F and Wen, Y},
title = {Upregulation of occludin by cytolethal distending toxin facilitates Glaesserella parasuis adhesion to respiratory tract cells.},
journal = {Infection and immunity},
volume = {},
number = {},
pages = {e0035123},
doi = {10.1128/iai.00351-23},
pmid = {37930004},
issn = {1098-5522},
abstract = {Virulent Glaesserella parasuis may engender systemic infection characterized by fibrinous polyserositis and pneumonia. G. parasuis causes systemic disease through upper respiratory tract infection, but the mechanism has not been fully characterized. Tight junction (TJ) proteins maintain the integrity and impermeability of the epithelial barriers. In this work, we applied the recombinant cytolethal distending toxin (CDT) holotoxin and cdt-deficient mutants to assess whether CDT interacted with TJ proteins of airway tract cells. Our results indicated that CDT induced the TJ occludin (OCLN) expression in newborn pig tracheal epithelial cells within the first 3 hours of bacterial infection, followed by a significant decrease. Overexpression of OCLN in target cells made them more susceptible to G. parasuis adhesion, whereas ablation of OCLN expression by CRISPR/Cas 9 gene editing technology in target cells decreased their susceptibility to bacterial adhesion. In addition, CDT treatment could upregulate the OCLN levels in the lung tissue of C57/BL6 mice. In summary, highly virulent G. parasuis strain SC1401 stimulated the tight junction expression, resulting in higher bacterial adhesion to respiratory tract cells, and this process is closely related to CDT. Our results may provide novel insights into G. parasuis infection and CDT-mediated pathogenesis.},
}
@article {pmid37903930,
year = {2023},
author = {Ledford, H},
title = {Is CRISPR safe? Genome editing gets its first FDA scrutiny.},
journal = {Nature},
volume = {623},
number = {7986},
pages = {234-235},
pmid = {37903930},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/legislation &amp; jurisprudence/methods/standards ; *United States Food and Drug Administration ; United States ; },
}
@article {pmid37813991,
year = {2023},
author = {},
title = {Combining compact human protein domains with CRISPR systems for robust gene activation.},
journal = {Nature methods},
volume = {20},
number = {11},
pages = {1635-1636},
pmid = {37813991},
issn = {1548-7105},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Protein Domains ; *Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid37770710,
year = {2023},
author = {Zhou, Y and Luo, K and Liang, L and Chen, M and He, X},
title = {A new Bayesian factor analysis method improves detection of genes and biological processes affected by perturbations in single-cell CRISPR screening.},
journal = {Nature methods},
volume = {20},
number = {11},
pages = {1693-1703},
pmid = {37770710},
issn = {1548-7105},
support = {R01MH110531//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01MH116281//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01HG010773//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01GM126553//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01HG011883//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 2016307//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CD8-Positive T-Lymphocytes ; Bayes Theorem ; Transcriptome ; *Biological Phenomena ; CRISPR-Cas Systems ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) screening coupled with single-cell RNA sequencing has emerged as a powerful tool to characterize the effects of genetic perturbations on the whole transcriptome at a single-cell level. However, due to its sparsity and complex structure, analysis of single-cell CRISPR screening data is challenging. In particular, standard differential expression analysis methods are often underpowered to detect genes affected by CRISPR perturbations. We developed a statistical method for such data, called guided sparse factor analysis (GSFA). GSFA infers latent factors that represent coregulated genes or gene modules; by borrowing information from these factors, it infers the effects of genetic perturbations on individual genes. We demonstrated through extensive simulation studies that GSFA detects perturbation effects with much higher power than state-of-the-art methods. Using single-cell CRISPR data from human CD8[+] T cells and neural progenitor cells, we showed that GSFA identified biologically relevant gene modules and specific genes affected by CRISPR perturbations, many of which were missed by existing methods, providing new insights into the functions of genes involved in T cell activation and neurodevelopment.},
}
@article {pmid37741267,
year = {2024},
author = {Wang, H and Su, A and Bao, C and Liang, C and Xu, W and Chang, J and Xu, S},
title = {A CRISPR/Cas12a-SERS platform for amplification-free detection of African swine fever virus genes.},
journal = {Talanta},
volume = {267},
number = {},
pages = {125225},
doi = {10.1016/j.talanta.2023.125225},
pmid = {37741267},
issn = {1873-3573},
mesh = {Animals ; Swine ; *African Swine Fever Virus/genetics ; CRISPR-Cas Systems ; DNA, Single-Stranded ; *Nucleic Acids ; *Biosensing Techniques ; },
abstract = {A surface-enhanced Raman scattering (SERS) strategy combined with a CRISPR/Cas12a system is designed for the amplification-free gene detection of African swine fever virus (ASFV). A SERS sensing probe was fabricated by conjugating plasmonic SERS tags on the magnetic bead (MB) surface with an single-stranded DNA (ssDNA) as a linker. The target ASFV gene-activated Cas12a protein starts the trans-cleavage function on the linker ssDNA, which causes the release of SERS tags, leading to a decrease of the SERS signal detected above the collective MBs. Two signal enhancement strategies were adopted to improve the liquid-phase detection sensitivity arriving at the fM level. One is the unlimited trans-cleavage function of the Cas12a protein, and the other is the magnetic-induced collection of probes that can significantly gather the analytes from the solution to the laser spot and provide SERS hotspots during SERS measurement. Detection range is from 100 nM to 10 fM without the gene amplification steps. This sensing method achieved the SERS detection of ASFV gene in the serum system and the extracted nucleic acids in viral samples with high sensitivity and selectivity at a relative standard deviation of <8%. This sensing platform is mainly in use for site inspection and quick testing of gene samples.},
}
@article {pmid37734291,
year = {2024},
author = {Wang, L and Sun, J and Zhao, J and Bai, J and Zhang, Y and Zhu, Y and Zhang, W and Wang, C and Langford, PR and Liu, S and Li, G},
title = {A CRISPR-Cas12a-based platform facilitates the detection and serotyping of Streptococcus suis serotype 2.},
journal = {Talanta},
volume = {267},
number = {},
pages = {125202},
doi = {10.1016/j.talanta.2023.125202},
pmid = {37734291},
issn = {1873-3573},
mesh = {Humans ; Swine ; Animals ; Serotyping/methods ; Serogroup ; *Streptococcus suis/genetics ; CRISPR-Cas Systems ; *Streptococcal Infections/diagnosis/veterinary/epidemiology ; },
abstract = {Streptococcus suis serotype 2 is an economically important zoonotic pathogen that causes septicemia, arthritis, and meningitis in pigs and humans. S. suis serotype 2 is responsible for substantial economic losses to the swine industry and poses a serious threat to public health, and accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a high-fidelity detection and serotyping platform for S. suis serotype 2 based on recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system called Cards-SSJ/K. Cards-SSJ had a detection limit of 10 CFU, takes <60 min, and no cross-reaction was found with other S. suis serotypes, closely related Streptococcus spp., or common pig pathogens, and Cards-SSK could differentiate serotype 2 from serotype 1/2. Results from Cards-SSJ and qPCR were equivalent in detecting S. suis serotype 2 in tissue samples. Analysis indicated that despite a relatively high reagent cost compared to PCR and qPCR, Cards-SSJ was less time-consuming and had low requirements for equipment and personnel. Thus, it is an excellent method for point-of-care detection for S. suis serotype 2.},
}
@article {pmid37924727,
year = {2023},
author = {Wu, X and Zhang, Y and Jiang, X and Ma, T and Guo, Y and Wu, X and Guo, Y and Cheng, X},
title = {Considerations in engineering viral vectors for genome editing in plants.},
journal = {Virology},
volume = {589},
number = {},
pages = {109922},
doi = {10.1016/j.virol.2023.109922},
pmid = {37924727},
issn = {1096-0341},
abstract = {Plant viruses have been engineered to express proteins and induce gene silencing for decades. Recently, plant viruses have also been used to deliver components into plant cells for genome editing, a technique called virus-induced genome editing (VIGE). Although more than a dozen plant viruses have been engineered into VIGE vectors and VIGE has been successfully accomplished in some plant species, application of VIGE to crops that are difficult to tissue culture and/or have low regeneration efficiency is still tough. This paper discusses factors to consider for an ideal VIGE vector, including insertion capacity for foreign DNA, vertical transmission ability, expression level of the target gene, stability of foreign DNA insertion, and biosafety. We also proposed a step-by-step schedule for excavating the suitable viral vector for VIGE.},
}
@article {pmid37924393,
year = {2023},
author = {Qian, Y and Wang, D and Niu, W and Shi, Z and Wu, M and Zhao, D and Li, J and Gao, X and Zhang, Z and Lai, L and Li, Z},
title = {Development of a highly efficient prime editor system in mice and rabbits.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {80},
number = {11},
pages = {346},
pmid = {37924393},
issn = {1420-9071},
support = {31970574//The National Natural Science Foundation of China/ ; },
mesh = {Rabbits ; Animals ; Mice ; Binding Sites ; *Gene Editing ; Models, Animal ; Mutation ; Temperature ; *CRISPR-Cas Systems/genetics ; },
abstract = {The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.},
}
@article {pmid37921129,
year = {2023},
author = {Kah Man, L and An Gie, O and Chian Huey, M and Yong, P and Siik Kee, L and Cheng Ze, L and Ming Yu, T and Foo, JB and Hamzah, S},
title = {The Cutting-edge of CRISPR for Cancer Treatment and its Future Prospects.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010258617231020062637},
pmid = {37921129},
issn = {1873-4316},
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a versatile technology that allows precise modification of genes. One of its most promising applications is in cancer treatment. By targeting and editing specific genes involved in cancer development and progression, CRISPR has the potential to become a powerful tool in the fight against cancer. This review aims to assess the recent progress in CRISPR technology for cancer research and to examine the obstacles and potential strategies to address them. The two most commonly used CRISPR systems for gene editing are CRISPR/Cas9 and CRISPR/Cas12a. CRISPR/Cas9 employs different repairing systems, including homologous recombination (HR) and nonhomologous end joining (NHEJ), to introduce precise modifications to the target genes. However, off-target effects and low editing efficiency are some of the main challenges associated with this technology. To overcome these issues, researchers are exploring new delivery methods and developing CRISPR/Cas systems with improved specificity. Moreover, there are ethical concerns surrounding using CRISPR in gene editing, including the potential for unintended consequences and the creation of genetically modified organisms. It is important to address these issues through rigorous testing and strict regulations. Despite these challenges, the potential benefits of CRISPR in cancer therapy cannot be overlooked. By introducing precise modifications to cancer cells, CRISPR could offer a targeted and effective treatment option for patients with different types of cancer. Further investigation and development of CRISPR technology are necessary to overcome the existing challenges and harness its full potential in cancer therapy.},
}
@article {pmid37920811,
year = {2023},
author = {Gumustop, I and Ortakci, F},
title = {Comparative genomics of Loigolactobacillus coryniformis with an emphasis on L. coryniformis strain FOL-19 isolated from cheese.},
journal = {Computational and structural biotechnology journal},
volume = {21},
number = {},
pages = {5111-5124},
pmid = {37920811},
issn = {2001-0370},
abstract = {Loigolactobacillus coryniformis is a member of lactic acid bacteria isolated from various ecological niches. We isolated a novel L. coryniformis strain FOL-19 from artisanal Tulum cheese and performed the whole-genome sequencing for FOL-19. Then, genomic characterization of FOL-19 against ten available whole genome sequences of the same species isolated from kimchi, silage, fermented meat, air of cowshed, dairy, and pheasant chyme was performed to uncover the genetic diversity and biotechnological potential of overall species. The average genome size of 2.93 ± 0.1 Mb, GC content of 42.96% ± 0.002, number of CDS of 2905 ± 165, number of tRNA of 56 ± 10, and number of CRISPR elements of 6.55 ± 1.83 was found. Both Type I and II Cas clusters were observed in L. coryniformis. No bacteriocin biosynthesis gene clusters were found. All strains harbored at least one plasmid except KCTC 3167. All strains were predicted to carry multiple IS elements. The most common origin of the IS elements was belong to Lactiplantibacillus plantarum. Comparative genomic analysis of L. coryniformis revealed hypervariability at the strain level and the presence of CRISPR/Cas suggests that L. coryniformis holds a promising potential for being a reservoir for new CRISPR-based tools. All L. coryniformis strains except PH-1 were predicted to harbor pdu and cbi-cob-hem gene clusters encoding industrially relevant traits of reuterin and cobalamin biosynthesis, respectively. These findings put a step forward for the genomic characterization of L. coryniformis strains for biotechnological applications via genome-guided strain selection to identify industrially relevant traits.},
}
@article {pmid37920190,
year = {2023},
author = {Matinvafa, MA and Makani, S and Parsasharif, N and Zahed, MA and Movahed, E and Ghiasvand, S},
title = {CRISPR-Cas technology secures sustainability through its applications: a review in green biotechnology.},
journal = {3 Biotech},
volume = {13},
number = {11},
pages = {383},
pmid = {37920190},
issn = {2190-572X},
abstract = {The CRISPR-Cas system's applications in biotechnology offer a promising avenue for addressing pressing global challenges, such as climate change, environmental pollution, the energy crisis, and the food crisis, thereby advancing sustainability. The ever-growing demand for food due to the projected population of around 9.6 billion by 2050 requires innovation in agriculture. CRISPR-Cas technology emerges as a powerful solution, enhancing crop varieties, optimizing yields, and improving resilience to stressors. It offers multiple gene editing, base editing, and prime editing, surpassing conventional methods. CRISPR-Cas introduces disease and herbicide resistance, high-yielding, drought-tolerant, and water-efficient crops to address rising water utilization and to improve the efficiency of agricultural practices which promise food sustainability and revolutionize agriculture for the benefit of future generations. The application of CRISPR-Cas technology extends beyond agriculture to address environmental challenges. With the adverse impacts of climate change and pollution endangering ecosystems, there is a growing need for sustainable solutions. The technology's potential in carbon capture and reduction through bio-sequestration is a pivotal strategy for combating climate change. Genomic advancements allow for the development of genetically modified organisms, optimizing biofuel and biomaterial production, and contributing to a renewable and sustainable energy future. This study reviews the multifaceted applications of CRISPR-Cas technology in the agricultural and environmental fields and emphasizes its potential to secure a sustainable future.},
}
@article {pmid37919649,
year = {2023},
author = {Bui, TP and Le, H and Ta, DT and Nguyen, CX and Le, NT and Tran, TT and Van Nguyen, P and Stacey, G and Stacey, MG and Pham, NB and Chu, HH and Do, PT},
title = {Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system.},
journal = {BMC plant biology},
volume = {23},
number = {1},
pages = {533},
pmid = {37919649},
issn = {1471-2229},
mesh = {*Soybeans/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Mutation ; Fungi ; Plant Diseases/genetics/microbiology ; Disease Resistance/genetics ; },
abstract = {BACKGROUND: Powdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance.<br><br>RESULTS: A dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants.<br><br>CONCLUSIONS: Our results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing&#xa0;the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.},
}
@article {pmid37883656,
year = {2023},
author = {Fu, J and Zhang, L and Long, Y and Liu, Z and Meng, G and Zhao, H and Su, X and Shi, S},
title = {Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein.},
journal = {Analytical chemistry},
volume = {95},
number = {44},
pages = {16089-16097},
doi = {10.1021/acs.analchem.3c01861},
pmid = {37883656},
issn = {1520-6882},
mesh = {*RNA, Viral ; *Nucleotidyltransferases ; Computer Simulation ; Nucleotides ; Recombinases ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Thanks to its ease, speed, and sensitivity, CRISPR-based nucleic acid detection has been increasingly explored for molecular diagnostics. However, one of its major limitations is lack of multiplexing capability because the detection relies on the trans-cleavage activity of the Cas protein, which necessitates the use of multiple orthogonal Cas proteins for multiplex detection. Here we report the development of a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein (Cas12a). This method, termed as CRISPR-TMSD, integrates the toehold-mediated strand displacement (TMSD) reaction, and the cis-cleavage activity of the Cas protein and multiplexed detection are achieved using a single Cas protein owing to the use of target-specific reporters. A set of computational simulation toolkits was used to design the TMSD reporter, allowing for highly sensitive and specific identification of target sequences. In combination with the recombinase polymerase amplification (RPA), the detection limit can reach as low as 1 copy/μL. As proof of concept, CRISPR-TMSD was subsequently used to detect an oncogenic gene and SARS-CoV-2 RNA with a single-nucleotide resolution. This work represents a conceptually new strategy for designing a CRISPR-based diagnostic system and has great potential to expand the application of CRISPR-based diagnostics.},
}
@article {pmid37883554,
year = {2023},
author = {Lin, L and DeMartino, J and Wang, D and van Son, GJF and van der Linden, R and Begthel, H and Korving, J and Andersson-Rolf, A and van den Brink, S and Lopez-Iglesias, C and van de Wetering, WJ and Balwierz, A and Margaritis, T and van de Wetering, M and Peters, PJ and Drost, J and van Es, JH and Clevers, H},
title = {Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6669},
pages = {451-458},
doi = {10.1126/science.adi2246},
pmid = {37883554},
issn = {1095-9203},
mesh = {Humans ; Cell Differentiation/genetics ; *CRISPR-Cas Systems ; *Enteroendocrine Cells/cytology/metabolism ; *Gene Expression Regulation ; Organoids ; *Zinc Fingers ; Adult ; *Cell Lineage/genetics ; *Repressor Proteins/genetics/metabolism ; },
abstract = {Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.},
}
@article {pmid37798358,
year = {2023},
author = {Pankratz, D and Gomez, NO and Nielsen, A and Mustafayeva, A and Gür, M and Arce-Rodriguez, F and Nikel, PI and Häussler, S and Arce-Rodriguez, A},
title = {An expanded CRISPR-Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates.},
journal = {Nature protocols},
volume = {18},
number = {11},
pages = {3253-3288},
pmid = {37798358},
issn = {1750-2799},
support = {SPP 1879//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ZN3428//Nieders&#xe4;chsische Ministerium f&#xfc;r Wissenschaft und Kultur (Lower Saxony Ministry of Science and Culture)/ ; NNF18OC0033946//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF20CC0035580//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF21OC0067996//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF18OC0034818//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; 8021-00039B//Det Frie Forskningsr&#xe5;d (Danish Council for Independent Research)/ ; 814418//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*CRISPR-Cas Systems ; *Pseudomonas aeruginosa/genetics ; DNA, Single-Stranded ; Gene Editing/methods ; Genetic Engineering/methods ; },
abstract = {Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.},
}
@article {pmid37794072,
year = {2023},
author = {Han, W and Gao, BQ and Zhu, J and He, Z and Li, J and Yang, L and Chen, J},
title = {Design and application of the transformer base editor in mammalian cells and mice.},
journal = {Nature protocols},
volume = {18},
number = {11},
pages = {3194-3228},
pmid = {37794072},
issn = {1750-2799},
support = {2018YFA0801401//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2019YFA0802804//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 21JC1404600//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 20PJ1410200//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 81872305//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31925011//National Natural Science Foundation of China (National Science Foundation of China)/ ; NK2022010207//Ministry of Agriculture and Rural Affairs of the People's Republic of China (MOA)/ ; 21JC1404600//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 20PJ1410200//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism ; Mutation ; Mammals/genetics ; },
abstract = {Fusing apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like cytidine deaminase with catalytically impaired Cas proteins (e.g., nCas9 or dCas9) provides a novel gene-editing technology, base editing, that grants targeted base substitutions with high efficiency. However, genome-wide and transcriptome-wide off-target mutations are observed in base editing, which raises safety concerns regarding therapeutic applications. Previously, we developed a new base editing system, the transformer base editor (tBE), to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations both in mammalian cells and in mice. Here we describe a detailed protocol for the design and application of the tBE. Steps for designing single-guide RNA (sgRNA) and helper sgRNA pairs, making constructs, determining the genome-wide and transcriptome-wide off-target mutations, producing the tBE-containing adeno-associated viruses, delivering adeno-associated viruses into mice and examining the in vivo editing effects are included in this protocol. High-precision base editing by the tBE can be completed within 2-3 weeks (in mammalian cells) or within 6-8 weeks (in mice), with sgRNA-helper sgRNA pairs. The whole process can be collaboratively accomplished by researchers using standard techniques from molecular biology, bioinformatics and mouse husbandry.},
}
@article {pmid37712250,
year = {2023},
author = {Lebek, S and Caravia, XM and Chemello, F and Tan, W and McAnally, JR and Chen, K and Xu, L and Liu, N and Bassel-Duby, R and Olson, EN},
title = {Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice.},
journal = {Circulation},
volume = {148},
number = {19},
pages = {1490-1504},
doi = {10.1161/CIRCULATIONAHA.123.065117},
pmid = {37712250},
issn = {1524-4539},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Mice ; Humans ; Animals ; Gene Editing ; CRISPR-Cas Systems ; Mice, Knockout ; *Induced Pluripotent Stem Cells/metabolism ; *Heart Failure ; Myocytes, Cardiac/metabolism ; Phosphorylation ; Fibrosis ; Adenine ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism ; },
abstract = {BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca[2+]/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders.<br><br>METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKII&#x3b4;. We generated mice harboring a phospho-resistant CaMKII&#x3b4; mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKII&#x3b4; gene editing was assessed using deep amplicon sequencing. Cellular Ca[2+] homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes.<br><br>RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKII&#x3b4; phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKII&#x3b4; phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKII&#x3b4;, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKII&#x3b4; compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca[2+] transients and an increased frequency of arrhythmias after chronic &#x3b2;-adrenergic stress, CaMKII&#x3b4;-edited cardiomyocytes were protected from these adverse responses.<br><br>CONCLUSIONS: Ablation of CaMKII&#x3b4; autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.},
}
@article {pmid37706265,
year = {2023},
author = {Ning, L and Xi, J and Zi, Y and Chen, M and Zou, Q and Zhou, X and Tang, C},
title = {Prospects and challenges of CRISPR/Cas9 gene-editing technology in cancer research.},
journal = {Clinical genetics},
volume = {104},
number = {6},
pages = {613-624},
doi = {10.1111/cge.14424},
pmid = {37706265},
issn = {1399-0004},
support = {2022KQNCX095//Youth Innovation Project of Guangdong Province University/ ; 2021B1212040016//The Science and Technology Planning Project of Guangdong Province/ ; 2022YFA1105403//National Key Research and Development Program of China Stem Cell and Translational Research/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; Immunotherapy ; *Neoplasms/genetics ; },
abstract = {Cancer, one of the leading causes of death, usually commences and progresses as a result of a series of gene mutations and dysregulation of expression. With the development of clustered regularly interspaced palindromic repeat (CRISPR)/Cas9 gene-editing technology, it is possible to edit and then decode the functions of cancer-related gene mutations, markedly advance the research of biological mechanisms and treatment of cancer. This review summarizes the mechanism and development of CRISPR/Cas9 gene-editing technology in recent years and describes its potential application in cancer-related research, such as the establishment of human tumor disease models, gene therapy and immunotherapy. The challenges and future development directions are highlighted to provide a reference for exploring pathological mechanisms and potential treatment protocols of cancer.},
}
@article {pmid37906052,
year = {2023},
author = {Wang, X and Hu, Z and Zhang, W and Wu, S and Hao, Y and Xiao, X and Li, J and Yu, X and Yang, C and Wang, J and Zhang, H and Ma, F and Shi, W and Wang, J and Lei, X and Zhang, X and He, S},
title = {Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16.},
journal = {The Journal of cell biology},
volume = {222},
number = {12},
pages = {},
pmid = {37906052},
issn = {1540-8140},
support = {31830051//National Natural Science Foundation of China/ ; 2022YFC2502700//National Key Research and Development Program of China/ ; 2022-I2M-2-004//Chinese Academy of Medical Sciences/ ; 3332022077//Peking Union Medical College/ ; //Priority Academic Program Development of the Jiangsu Higher Education Institutes/ ; BM2013003//Jiangsu Key Laboratory of Neuropsychiatric Diseases/ ; 2019M650563//China Postdoctoral Science Foundation/ ; BK20160314//Natural Science Foundation of Jiangsu Province/ ; //National Center of Technology Innovation for Biopharmaceuticals/ ; //Suzhou Institute of Systems Medicine/ ; SZS2022005//Suzhou Municipal Key Laboratory/ ; },
mesh = {Animals ; Child, Preschool ; Mice ; Apoptosis ; *Enterovirus A, Human/genetics ; Lysosomes ; CRISPR-Cas Systems ; *Enterovirus/genetics ; Pyroptosis ; Virus Replication ; *Viral Nonstructural Proteins/genetics ; },
abstract = {Enterovirus 71 (EV71) and Coxsackie A16 (CVA16) are two major causative agents of hand, foot, and mouth disease (HFMD) in young children. However, the mechanisms regulating the replication and pathogenesis of EV71/CVA16 remain incompletely understood. We performed a genome-wide CRISPR-Cas9 knockout screen and identified Ragulator as a mediator of EV71-induced apoptosis and pyroptosis. The Ragulator-Rag complex is required for EV71 and CVA16 replication. Upon infection, the Ragulator-Rag complex recruits viral 3D protein to the lysosomal surface through the interaction between 3D and RagB. Disruption of the lysosome-tethered Ragulator-Rag-3D complex significantly impairs the replication of EV71/CVA16. We discovered a novel EV71 inhibitor, ZHSI-1, which interacts with 3D and significantly reduces the lysosomal tethering of 3D. ZHSI-1 treatment significantly represses replication of EV71/CVA16 as well as virus-induced pyroptosis associated with viral pathogenesis. Importantly, ZHSI-1 treatment effectively protects against EV71 infection in neonatal and young mice. Thus, our study indicates that targeting lysosome-tethered Ragulator-Rag-3D may be an effective therapeutic strategy for HFMD.},
}
@article {pmid37724511,
year = {2023},
author = {Chiurillo, MA and Ahmed, M and González, C and Raja, A and Lander, N},
title = {Gene editing of putative cAMP and Ca[2+] -regulated proteins using an efficient cloning-free CRISPR/Cas9 system in Trypanosoma cruzi.},
journal = {The Journal of eukaryotic microbiology},
volume = {70},
number = {6},
pages = {e12999},
doi = {10.1111/jeu.12999},
pmid = {37724511},
issn = {1550-7408},
support = {23IPA1054779//American Heart Association/ ; R00AI137322//U.S. National Institutes of Health/ ; //Latino Faculty Association of University of Cincinnati/ ; },
mesh = {Humans ; *Trypanosoma cruzi/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Chagas Disease/parasitology ; Protozoan Proteins/genetics/metabolism ; },
abstract = {Trypanosoma cruzi, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca[2+]) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics. Although the use of CRISPR/Cas9 technology prompted reverse genetics approaches for functional analysis in T. cruzi, it is still necessary to expand the toolbox for genome editing in this parasite, as for example to perform multigene analysis. Here we used an efficient T7RNAP/Cas9 strategy to tag and delete three genes predicted to be involved in cAMP and Ca[2+] signaling pathways: a putative Ca[2+] /calmodulin-dependent protein kinase (CAMK), Flagellar Member 6 (FLAM6) and Cyclic nucleotide-binding domain/C2 domain-containing protein (CC2CP). We endogenously tagged these three genes and determined the subcellular localization of the tagged proteins. Furthermore, the strategy used to knockout these genes allows us to presume that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results will open new venues for future research on the role of these proteins in T. cruzi.},
}
@article {pmid37522631,
year = {2023},
author = {Liu, Y and Ma, D and Constabel, CP},
title = {CRISPR/Cas9 Disruption of MYB134 and MYB115 in Transgenic Poplar Leads to Differential Reduction of Proanthocyanidin Synthesis in Roots and Leaves.},
journal = {Plant &amp; cell physiology},
volume = {64},
number = {10},
pages = {1189-1203},
doi = {10.1093/pcp/pcad086},
pmid = {37522631},
issn = {1471-9053},
support = {RGPIN-2020-06646//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Proanthocyanidins/metabolism ; *Populus/genetics/metabolism ; CRISPR-Cas Systems ; Transcription Factors/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Proanthocyanidins (PAs) are common specialized metabolites and particularly abundant in trees and woody plants. In poplar (Populus spp.), PA biosynthesis is stress-induced and regulated by two previously studied transcription factors MYB115 and MYB134. To determine the relative contribution of these regulators to PA biosynthesis, we created single- and double-knockout (KO) mutants for both genes in transgenic poplars using CRISPR/Cas9. Knocking out either MYB134 or MYB115 showed reduced PA accumulation and downregulated flavonoid genes in leaves, but MYB134 disruption had the greatest impact and reduced PAs to 30% of controls. In roots, by contrast, only the MYB134/MYB115 double-KOs showed a significant change in PA concentration. The loss of PAs paralleled the lower expression of PA biosynthesis genes and concentrations of flavan-3-ol PA precursors catechin and epicatechin. Interestingly, salicinoids were also affected in double-KOs, with distinct patterns in roots and shoots. We conclude that the regulatory pathways for PA biosynthesis differ in poplar leaves and roots. The residual PA content in the double-KO plants indicates that other transcription factors must also be involved in control of the PA pathway.},
}
@article {pmid37919000,
year = {2024},
author = {Mun, SY and Lee, W and Lee, SY and Chang, JY and Chang, HC},
title = {Pediococcus inopinatus with a well-developed CRISPR-Cas system dominates in long-term fermented kimchi, Mukeunji.},
journal = {Food microbiology},
volume = {117},
number = {},
pages = {104385},
doi = {10.1016/j.fm.2023.104385},
pmid = {37919000},
issn = {1095-9998},
mesh = {CRISPR-Cas Systems ; *Lactobacillales ; Pediococcus/genetics ; *Fermented Foods ; },
abstract = {Kimchi is produced through a low-temperature fermentation without pre-sterilization, resulting in a heterogeneous microbial community. As fermentation progresses, dominant lactic acid bacteria (LAB) species emerge and undergo a transition process. In this study, LAB were isolated from Mukeunji, a long-term fermented kimchi that is in the final stage of kimchi fermentation process. It was confirmed, through culture-dependent and independent analysis, as well as metagenome analysis, that Pediococcus inopinatus are generally dominant in long-term fermented kimchi. Comparative analysis of the de novo assembled whole genome of P. inopinatus with other kimchi LAB revealed that this species has a well-developed clustered regularly interspaced short palindromic repeats (CRISPR) system. The CRISPR system of P. inopinatus has an additional copy of the csa3 gene, a transcription factor for cas genes. Indeed, this species not only highly expresses cas1 and cas2, which induce spacer acquisition, but also has many diverse spacers that are actively expressed. These findings indicate that the well-developed CRISPR-Cas system is enabling P. inopinatus to dominate in long-fermented kimchi. Overall, this study revealed that LAB with a robust defense system dominate in the final stage of kimchi fermentation and presented a model for the succession mechanism of kimchi LAB.},
}
@article {pmid37918963,
year = {2024},
author = {Ondra, M and Lenart, L and Centorame, A and Dumut, DC and He, A and Zaidi, SSZ and Hanrahan, JW and De Sanctis, JB and Radzioch, D and Hajduch, M},
title = {CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane.},
journal = {Life science alliance},
volume = {7},
number = {1},
pages = {},
pmid = {37918963},
issn = {2575-1077},
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Cystic Fibrosis/genetics/metabolism ; Cell Membrane/metabolism ; Cell Line ; },
abstract = {CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.},
}
@article {pmid37917353,
year = {2023},
author = {Zhang, X and Li, H and Zhao, W and Xu, J and Wang, S and Yu, R},
title = {Development of a separation platform comprising magnetic beads combined with the CRISPR/Cas12a system enabling ultrasensitive and rapid detection of miRNA-21.},
journal = {Mikrochimica acta},
volume = {190},
number = {11},
pages = {458},
pmid = {37917353},
issn = {1436-5073},
support = {22164011//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Fluorescence ; *MicroRNAs ; Oligonucleotides ; },
abstract = {A separation platform has been developed mediated by a combination of magnetic beads and the CRISPR/Cas12a system to achieve ultrasensitive and rapid detection of miRNA-21 at a low level. In this system, with the assistance of an auxiliary probe, the target miRNA-21 can be specifically combined with three-stranded probes to initiate the SDR reaction. Abundant aptamer A3 was added to the solution that can activate the CRISPR/Cas12a system and initiate the trans-cleavage reaction to recover the fluorescence signal. Using magnetic beads to mediate the separation considerably greatly improves the signal conversion efficiency and detection sensitivity. At the 492 nm excitation wavelength, and 502-650 nm scan range, through analyzing the fluorescence peak intensity at 520 nm, the biosensor's determination range and limit of detection is 8 fM-250 nM and 2.42 fM, respectively, and the RSD is 19.03-37.80. Compared with other biosensors, the biosensor developed exhibited superior performance and the signal recovered excellently in 1% human serum and the LOD is 12.12 fM. This method provides a novel highly sensitive scheme for detecting miRNA .},
}
@article {pmid37916531,
year = {2023},
author = {Mousavi, SM and Hashemi, SA and Kalashgrani, MY and Rahmanian, V and Riazi, M and Omidifar, N and Althomali, RH and Rahman, MM and Chiang, WH and Gholami, A},
title = {Recent Progress in Prompt Molecular Detection of Exosomes Using CRISPR/Cas and Microfluidic-Assisted Approaches Toward Smart Cancer Diagnosis and Analysis.},
journal = {ChemMedChem},
volume = {},
number = {},
pages = {e202300359},
doi = {10.1002/cmdc.202300359},
pmid = {37916531},
issn = {1860-7187},
abstract = {Nano exosomes are essential indicators of molecular mechanisms involved in interacting with cancer cells and the tumor environment. The nano exosomes, as nanostructures based on lipids and nucleic acids, provide a communication pathway for information transfer by transporting biomolecules from the target cell to other cells. Importantly, these extracellular vesicles are released into the blood flow by the most invading cells, i.e., cancer cells; thereby, they could be considered a promising specific biomarker for cancer diagnosis. The CRISPR-Cas systems, as a genome editing approach, provide the inactivation possibility of genes or even the complete removal of the gene from the cell without affecting intracellular mechanisms. These practical systems provide vital information about the involved factors in cancer development that could lead to more effective cancer treatment. Meanwhile, microfluidic approaches can also significantly benefit cancer research due to their proper sensitivity, high throughput, low material consumption, low cost, and advanced spatial and temporal control. Thereby, employing CRISPR-Cas- and microfluidic-based approaches toward nano exosome monitoring could be considered a valuable source of information for cancer therapy and diagnosis. This review assessed the recent progress in these promising diagnosis approaches toward accurate cancer therapy and in-depth study of cancer cell behavior.},
}
@article {pmid37914776,
year = {2023},
author = {Zorz, J and Paquette, AJ and Gillis, T and Kouris, A and Khot, V and Demirkaya, C and De La Hoz Siegler, H and Strous, M and Vadlamani, A},
title = {Coordinated proteome change precedes cell lysis and death in a mat-forming cyanobacterium.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
pmid = {37914776},
issn = {1751-7370},
abstract = {Cyanobacteria form dense multicellular communities that experience transient conditions in terms of access to light and oxygen. These systems are productive but also undergo substantial biomass turnover through cell death, supplementing heightened heterotrophic respiration. Here we use metagenomics and metaproteomics to survey the molecular response of a mat-forming cyanobacterium undergoing mass cell lysis after exposure to dark and anoxic conditions. A lack of evidence for viral, bacterial, or eukaryotic antagonism contradicts commonly held beliefs on the causative agent for cyanobacterial death during dense growth. Instead, proteogenomics data indicated that lysis likely resulted from a genetically programmed response triggered by a failure to maintain osmotic pressure in the wake of severe energy limitation. Cyanobacterial DNA was rapidly degraded, yet cyanobacterial proteins remained abundant. A subset of proteins, including enzymes involved in amino acid metabolism, peptidases, toxin-antitoxin systems, and a potentially self-targeting CRISPR-Cas system, were upregulated upon lysis, indicating possible involvement in the programmed cell death response. We propose this natural form of cell death could provide new pathways for controlling harmful algal blooms and for sustainable bioproduct production.},
}
@article {pmid37913834,
year = {2023},
author = {Yeung, CC and Garva, R and Pickard, A and Lu, Y and Mallikarjun, V and Swift, J and Taylor, SH and Rai, J and Eyre, DR and Chaturvedi, M and Itoh, Y and Meng, QJ and Mauch, C and Zigrino, P and Kadler, KE},
title = {Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.matbio.2023.10.002},
pmid = {37913834},
issn = {1569-1802},
abstract = {The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.},
}
@article {pmid37910550,
year = {2023},
author = {Angelo, L and Vaillant, A and Blanchet, M and Labonté, P},
title = {Pangenomic antiviral effect of REP 2139 in CRISPR/Cas9 engineered cell lines expressing hepatitis B virus surface antigen.},
journal = {PloS one},
volume = {18},
number = {11},
pages = {e0293167},
pmid = {37910550},
issn = {1932-6203},
mesh = {Humans ; Hepatitis B Surface Antigens ; Hepatitis B virus ; CRISPR-Cas Systems/genetics ; Antiviral Agents/therapeutic use ; Polymers/metabolism ; *Nucleic Acids/metabolism ; *Hepatitis B, Chronic/drug therapy ; Cell Line ; *Liver Neoplasms/genetics ; *Vaccines/therapeutic use ; Antigens, Surface/metabolism ; *Hepatitis B/drug therapy ; },
abstract = {Chronic hepatitis B remains a global health problem with 296 million people living with chronic HBV infection and being at risk of developing cirrhosis and hepatocellular carcinoma. Non-infectious subviral particles (SVP) are produced in large excess over infectious Dane particles in patients and are the major source of Hepatitis B surface antigen (HBsAg). They are thought to exhaust the immune system, and it is generally considered that functional cure requires the clearance of HBsAg from blood of patient. Nucleic acid polymers (NAPs) antiviral activity lead to the inhibition of HBsAg release, resulting in rapid clearance of HBsAg from circulation in vivo. However, their efficacy has only been demonstrated in limited genotypes in small scale clinical trials. HBV exists as nine main genotypes (A to I). In this study, the HBsAg ORFs from the most prevalent genotypes (A, B, C, D, E, G), which account for over 96% of human cases, were inserted into the AAVS1 safe-harbor of HepG2 cells using CRISPR/Cas9 knock-in. A cell line producing the D144A vaccine escape mutant was also engineered. The secretion of HBsAg was confirmed into these new genotype cell lines (GCLs) and the antiviral activity of the NAP REP 2139 was then assessed. The results demonstrate that REP 2139 exerts an antiviral effect in all genotypes and serotypes tested in this study, including the vaccine escape mutant, suggesting a pangenomic effect of the NAPs.},
}
@article {pmid37910191,
year = {2023},
author = {Wang, D and Wang, D and Liao, K and Zhang, B and Li, S and Liu, M and Lv, L and Xue, F},
title = {Optical detection using CRISPR-Cas12a of Helicobacter pylori for veterinary applications.},
journal = {Mikrochimica acta},
volume = {190},
number = {11},
pages = {455},
pmid = {37910191},
issn = {1436-5073},
support = {(2021YFD1800500)//National Key Research and Development Program of China/ ; (2020YFA0910200)//National Key Research and Development Program of China/ ; ZDYF2022XDNY248//Hainan Provincial Department of Science and Technology/ ; CX (21)2038//Jiangsu Agricultural Science and Technology Independent Innovation Fund/ ; NAUSY-ZD08//Research Institute of Sanya Nanjing Agricultural University/ ; },
mesh = {Animals ; Cats ; Dogs ; *Helicobacter pylori/genetics ; *Cat Diseases/genetics ; CRISPR-Cas Systems ; *Helicobacter Infections/diagnosis/veterinary/genetics ; *Dog Diseases/genetics ; },
abstract = {Helicobacter pylori (H. pylori) is a zoonotic gastric microorganism capable of efficient interspecies transmission. Domesticated companion animals, particularly dogs and cats, serve as natural reservoirs for H. pylori. This phenomenon facilitates the extensive dissemination of H. pylori among households with pets. Hence, the prompt and precise identification of H. pylori in companion animals holds paramount importance for the well-being of both animals and their owners. With the assistance of Multienzyme Isothermal Rapid Amplification (MIRA) and CRISPR-Cas12a system, we successfully crafted a highly adaptable optical detection platform for H. pylori. Three sensor systems with corresponding visual interpretations were proposed. This study demonstrated a rapid turnaround time of approximately 45 min from DNA extraction to the result display. Moreover, this platform topped germiculture and real-time PCR in terms of sensitivity or efficiency in clinical diagnoses of 66 samples. This platform possesses significant potential as a versatile approach and represents the premiere application of CRISPR for the non-invasive detection of H. pylori in companion animals, thereby mitigating the dissemination of H. pylori among household members.},
}
@article {pmid37909049,
year = {2023},
author = {Lin, J and Alfastsen, L and Bhoobalan-Chitty, Y and Peng, X},
title = {Molecular basis for inhibition of type III-B CRISPR-Cas by an archaeal viral anti-CRISPR protein.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2023.10.003},
pmid = {37909049},
issn = {1934-6069},
abstract = {Despite a wide presence of type III clustered regularly interspaced short palindromic repeats, CRISPR-associated (CRISPR-Cas) in archaea and bacteria, very few anti-CRISPR (Acr) proteins inhibiting type III immunity have been identified, and even less is known about their inhibition mechanism. Here, we present the discovery of a type III CRISPR-Cas inhibitor, AcrIIIB2, encoded by Sulfolobus virus S. islandicus rod-shaped virus 3 (SIRV3). AcrIIIB2 inhibits type III-B CRISPR-Cas immune response to protospacers encoded in middle/late-expressed viral genes. Investigation of the interactions between S. islandicus type III-B CRISPR-Cas Cmr-α-related proteins and AcrIIIB2 reveals that the Acr does not bind to Csx1 but rather interacts with the Cmr-α effector complex. Furthermore, in vitro assays demonstrate that AcrIIIB2 can block the dissociation of cleaved target RNA from the Cmr-α complex, thereby inhibiting the Cmr-α turnover, thus preventing host cellular dormancy and further viral genome degradation by the type III-B CRISPR-Cas immunity.},
}
@article {pmid37908969,
year = {2023},
author = {Angulo, J and Astin, CP and Bauer, O and Blash, KJ and Bowen, NM and Chukwudinma, NJ and DiNofrio, AS and Faletti, DO and Ghulam, AM and Gusinde-Duffy, CM and Horace, KJ and Ingram, AM and Isaack, KE and Jeong, G and Kiser, RJ and Kobylanski, JS and Long, MR and Manning, GA and Morales, JM and Nguyen, KH and Pham, RT and Phillips, MH and Reel, TW and Seo, JE and Vo, HD and Wukoson, AM and Yeary, KA and Zheng, GY and Lukowitz, W},
title = {CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1251557},
pmid = {37908969},
issn = {2673-3439},
abstract = {Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.},
}
@article {pmid37908833,
year = {2023},
author = {Vu, BN and Vu, TV and Yoo, JY and Nguyen, NT and Ko, KS and Kim, JY and Lee, KO},
title = {CRISPR-Cas-mediated unfolded protein response control for enhancing plant stress resistance.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1271368},
pmid = {37908833},
issn = {1664-462X},
abstract = {Plants consistently encounter environmental stresses that negatively affect their growth and development. To mitigate these challenges, plants have developed a range of adaptive strategies, including the unfolded protein response (UPR), which enables them to manage endoplasmic reticulum (ER) stress resulting from various adverse conditions. The CRISPR-Cas system has emerged as a powerful tool for plant biotechnology, with the potential to improve plant tolerance and resistance to biotic and abiotic stresses, as well as enhance crop productivity and quality by targeting specific genes, including those related to the UPR. This review highlights recent advancements in UPR signaling pathways and CRISPR-Cas technology, with a particular focus on the use of CRISPR-Cas in studying plant UPR. We also explore prospective applications of CRISPR-Cas in engineering UPR-related genes for crop improvement. The integration of CRISPR-Cas technology into plant biotechnology holds the promise to revolutionize agriculture by producing crops with enhanced resistance to environmental stresses, increased productivity, and improved quality traits.},
}
@article {pmid37908718,
year = {2023},
author = {Wu, Y and Wang, Y and Zhou, J and Wang, J and Zhan, Q and Wang, Q and Yang, E and Jin, W and Tong, F and Zhao, J and Hong, B and Liu, J and Kang, C},
title = {Universal theranostic CRISPR/Cas13a RNA-editing system for glioma.},
journal = {Theranostics},
volume = {13},
number = {15},
pages = {5305-5321},
pmid = {37908718},
issn = {1838-7640},
mesh = {Humans ; Animals ; Mice ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Nude ; Precision Medicine ; CRISPR-Cas Systems/genetics ; RNA ; *Glioma/diagnosis/genetics/therapy ; },
abstract = {Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for in vivo molecular diagnoses. Methods: A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and in vitro biological validation methods. For in vivo imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice. Results: The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us in vivo imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice. Conclusions: Our work demonstrated the clinical potential of a Cas13a-based in vivo imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma.},
}
@article {pmid37813236,
year = {2023},
author = {Kevadiya, BD and Islam, F and Deol, P and Zaman, LA and Mosselhy, DA and Ashaduzzaman, M and Bajwa, N and Routhu, NK and Singh, PA and Dawre, S and Vora, LK and Nahid, S and Mathur, D and Nayan, MU and Baldi, A and Kothari, R and Patel, TA and Madan, J and Gounani, Z and Bariwal, J and Hettie, KS and Gendelman, HE},
title = {Delivery of gene editing therapeutics.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {54},
number = {},
pages = {102711},
doi = {10.1016/j.nano.2023.102711},
pmid = {37813236},
issn = {1549-9642},
support = {R01 NS034239/NS/NINDS NIH HHS/United States ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Gene Transfer Techniques ; Genetic Therapy ; },
abstract = {For the past decades, gene editing demonstrated the potential to attenuate each of the root causes of genetic, infectious, immune, cancerous, and degenerative disorders. More recently, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR-Cas9) editing proved effective for editing genomic, cancerous, or microbial DNA to limit disease onset or spread. However, the strategies to deliver CRISPR-Cas9 cargos and elicit protective immune responses requires safe delivery to disease targeted cells and tissues. While viral vector-based systems and viral particles demonstrate high efficiency and stable transgene expression, each are limited in their packaging capacities and secondary untoward immune responses. In contrast, the nonviral vector lipid nanoparticles were successfully used for as vaccine and therapeutic deliverables. Herein, we highlight each available gene delivery systems for treating and preventing a broad range of infectious, inflammatory, genetic, and degenerative diseases. STATEMENT OF SIGNIFICANCE: CRISPR-Cas9 gene editing for disease treatment and prevention is an emerging field that can change the outcome of many chronic debilitating disorders.},
}
@article {pmid37813160,
year = {2023},
author = {Tiyaboonchai, A and Wakefield, L and Vonada, A and May, CL and Dorrell, C and Enicks, D and Sairavi, A and Kaestner, KH and Grompe, M},
title = {In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9.},
journal = {Developmental biology},
volume = {504},
number = {},
pages = {120-127},
doi = {10.1016/j.ydbio.2023.09.011},
pmid = {37813160},
issn = {1095-564X},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Keratin-14/genetics/metabolism ; *RNA Polymerase II/genetics/metabolism ; Integrases/genetics ; Mice, Transgenic ; Promoter Regions, Genetic/genetics ; },
abstract = {The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on RNA polymerase II promoter driven self-cleaving guide RNAs (scgRNA) to achieve tissue-specificity. To demonstrate proof-of-principle for this approach a transgenic mouse was generated harbouring a knock-in of a scgRNA into the Cytokeratin 14 (Krt14) locus. Krt14 expression marks the stem cells of squamous epithelium in the skin and oral mucosa. The scgRNA targets a Stop cassette preceding a fluorescent reporter in the Ai9-tdtomato mouse. Ai9-tdtomato reporter mice harbouring this allele along with a spCas9 transgene demonstrated precise marking of the Krt14 lineage. We conclude that RNA polymerase II promoter driven scgRNAs enable the use of CRISPR/spCas9 for genetic lineage tracing.},
}
@article {pmid37769384,
year = {2023},
author = {Chatrousse, L and Poullion, T and El-Kassar, L and Giraud-Triboult, K and Boissart, C and Sanatine, P and Sommer, P and Benchoua, A},
title = {Establishment of heterozygous and homozygous SHANK3 knockout clonal pluripotent stem cells from the parental hESC line SA001 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {72},
number = {},
pages = {103209},
doi = {10.1016/j.scr.2023.103209},
pmid = {37769384},
issn = {1876-7753},
mesh = {Humans ; Male ; *Human Embryonic Stem Cells/metabolism ; *Autism Spectrum Disorder/genetics ; CRISPR-Cas Systems/genetics ; Nerve Tissue Proteins/genetics/metabolism ; Clone Cells/metabolism ; },
abstract = {Phelan-McDermid syndrome (PMS) is a rare genetic disease characterized by a global developmental delay with autism spectrum disorder. PMS is caused by loss of function mutations in the SHANK3 gene leading to SHANK3 protein haploinsufficiency. This study describes the generation of isogenic clones produced from one male human embryonic stem cell line with deletions in SHANK3, in a heterozygous or homozygous manner, using CRISPR/Cas9 indel methodology. Differentiation of these clones into different neuronal lineages will help understanding PMS etiology and find treatments for PMD patients. (85/100 words).},
}
@article {pmid37741309,
year = {2023},
author = {Zhou, Z and Wang, P and Wang, Q and Dong, Z and Chen, X and Zhuo, R and Wu, R and Liu, Y and Yang, L and Liu, M},
title = {SASH1 contributes to glial cell migration in the early development of the central nervous system.},
journal = {Developmental biology},
volume = {504},
number = {},
pages = {49-57},
doi = {10.1016/j.ydbio.2023.09.006},
pmid = {37741309},
issn = {1095-564X},
mesh = {Animals ; Humans ; *Zebrafish/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Central Nervous System/metabolism ; Cell Movement/genetics ; RNA, Messenger ; Mammals/metabolism ; },
abstract = {SAM and SH3 domain-containing 1 (SASH1), a member of the SLy protein family, is a tumor suppressor gene that has been studied for its association with various cancers. SASH1 is highly expressed in the mammalian central nervous system, particularly in glial cells, and is expressed in the central nervous system during zebrafish embryo development. However, SASH1's role in brain development has rarely been investigated. In this study, Morpholino oligonucleotides (MO) were used to down-regulate sash1a expression in zebrafish to observe morphological changes in the brain. Three transgenic zebrafish lines, Tg(gfap:eGFP), Tg(hb9:eGFP), and Tg(coro1a:eGFP) were selected to observe changes in glial cells, neurons, and immune cells after sash1a knockdown. Our results showed that the number of microglia residing in the developmental brain was reduced, whereas the axonal growth of caudal primary motor neurons was unaffected by sash1a downregulation. And more significantly, the gfap [+] glia presented abnormal arrangements and disordered orientations in sash1a morphants. The similar phenotype was verified in the mutation induced by the injection of cas9 mRNA and sash1a sgRNA. We further performed behavioral experiments in zebrafish larvae that had been injected with sash1a MO at one-cell stage, and found them exhibiting abnormal behavior trajectories. Moreover, injecting the human SASH1 mRNA rescued these phenomena in sash1a MO zebrafish. In summary, our study revealed that the downregulation of SASH1 leads to malformations in the embryonic brain and disorganization of glial cell marshalling, suggesting that SASH1 plays an important role in the migration of glial cells during embryonic brain development.},
}
@article {pmid37722184,
year = {2023},
author = {Zou, Q and Lu, Y and Qing, B and Li, N and Zhou, T and Pan, J and Zhang, X and Zhang, X and Chen, Y and Sun, SK},
title = {Photoactivatable base editors for spatiotemporally controlled genome editing in vivo.},
journal = {Biomaterials},
volume = {302},
number = {},
pages = {122328},
doi = {10.1016/j.biomaterials.2023.122328},
pmid = {37722184},
issn = {1878-5905},
mesh = {Mice ; Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Genome ; Adenine ; Light ; },
abstract = {CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.},
}
@article {pmid37708613,
year = {2023},
author = {Cao, Y and Tang, L and Su, J and Wang, H and Liang, P and Rong, K and Gong, T},
title = {Generation of a TRPV1 knockout human pluripotent stem cell line (WAe009-A-U) using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {72},
number = {},
pages = {103202},
doi = {10.1016/j.scr.2023.103202},
pmid = {37708613},
issn = {1876-7753},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Nociceptors/metabolism ; Cell Line ; TRPV Cation Channels/genetics/metabolism ; *Pluripotent Stem Cells/metabolism ; *Human Embryonic Stem Cells/metabolism ; },
abstract = {The transient receptor potential vanilloid subfamily 1 (TRPV1) is a polymodal nociceptor that is highly expressed in sensory nerves. Activation of TRPV1 receptors excites primary afferent nociceptors by opening cation channels, allowing the influx of Na[+] and Ca[2+] ions into the cytoplasm. Here, a TRPV1 knockout human embryonic stem cell line was generated using the CRISPR/Cas9 genome-editing technology to further study the function of TRPV1. The cell line confirmed with normal pluripotency and karyotype.},
}
@article {pmid37690432,
year = {2023},
author = {Han, HJ and Kim, JH},
title = {KSCBi005-A-8(hiPSC-PD-L1KO), a PD-L1 knockout human induced pluripotent stem cell line for demonstrating the role of the PD-1/PD-L1 axis.},
journal = {Stem cell research},
volume = {72},
number = {},
pages = {103196},
doi = {10.1016/j.scr.2023.103196},
pmid = {37690432},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; Programmed Cell Death 1 Receptor/genetics/metabolism ; B7-H1 Antigen/genetics/metabolism ; Ligands ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; },
abstract = {Programmed Cell Death Ligand 1 (PD-L1) is a trans-membrane protein that attenuates the host immune response to tumor cells. PD-L1 ligand on the surface of the cancer binds to PD-1 transmembrane receptors on T cells and exhausts T cells function. Using CRISPR-Cas9 genome editing, we generated biallelic PD-L1 mutants in human induced pluripotent stem cells (hiPSCs). The PD-L1 homozygous-knockout hiPSCs retained their normal morphology, gene expression, and in vivo differentiation potential.},
}
@article {pmid37096780,
year = {2023},
author = {Xu, L and Zeng, Q and Liang, L and Yang, Z and Qu, M and Li, H and Zhang, B and Zhang, J and Yuan, X and Chen, L and Fan, Z and He, L and Nan, X and Yue, W and Xie, X and Pei, X},
title = {Generation of Rh D-negative blood using CRISPR/Cas9.},
journal = {Cell proliferation},
volume = {56},
number = {11},
pages = {e13486},
pmid = {37096780},
issn = {1365-2184},
support = {202002030025//Science and Technology Program of Guangzhou, China/ ; 32200589//National Nature Science Foundation of China/ ; 201904010378//The Guangzhou Scientific Research Program/ ; 2017YFA0103100//The National Key Research and Development Program of China/ ; 2017YFA0103103//The National Key Research and Development Program of China/ ; 2017YFA0103104//The National Key Research and Development Program of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Endothelial Cells ; Erythrocytes/metabolism ; Gene Editing/methods ; Cell Line ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD[-/-] cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD[-/-] derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.},
}
@article {pmid37907567,
year = {2023},
author = {Witz, A and Dardare, J and Francois, A and Husson, M and Rouyer, M and Demange, J and Merlin, JL and Gilson, P and Harlé, A},
title = {CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {18741},
pmid = {37907567},
issn = {2045-2322},
mesh = {Humans ; BRCA1 Protein/genetics ; BRCA2 Protein/genetics ; *Adenocarcinoma/genetics ; CRISPR-Cas Systems ; *Pancreatic Neoplasms/drug therapy/genetics/pathology ; Phthalazines/pharmacology ; Germ-Line Mutation ; Mutation ; MCF-7 Cells ; },
abstract = {Pancreatic cancer is one of the most aggressive diseases with a very poor outcome. Olaparib, a PARP inhibitor, as maintenance therapy showed benefits in patients with metastatic pancreatic adenocarcinoma bearing germline BRCA1/2 mutations. However, germline BRCA mutation has been described in only 4-7% of patients with pancreatic adenocarcinoma. A CRISPR/Cas9-mediated system was used to knock-in the c.763G > T p.(Glu255*) and c.2133C > A p.(Cys711*) mutations in cell lines to obtain truncated BRCA1 and BRCA2 proteins, respectively. A CRISPR/Cas9 ribonucleoprotein complex was assembled for each mutation and transfected into two pancreatic cell lines (T3M4 and Capan-2) and into a breast cancer cell lines (MCF7) as control. BRCA protein levels were significantly decreased in all BRCA-depleted cells (P < 0.05), proving the transfection efficiency of our CRISPR/Cas9 systems. As expected, the calculated olaparib IC50 were significantly reduced for all cell lines harbored BRCA1 or BRCA2 mutations compared to wild-type BRCA1/2 cells (P < 0.01). Furthermore, we observed a higher induction of apoptosis after 72 h olaparib treatment in BRCA-depleted cells than in wild-type cells. This strategy might offer new insights into the management of patients with pancreatic cancer and open up new perspectives based on the in vivo use of CRISPR/Cas9 strategy.},
}
@article {pmid37905201,
year = {2022},
author = {Vu, TV and Nguyen, NT and Kim, J and Das, S and Lee, J and Kim, JY},
title = {The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato.},
journal = {Biodesign research},
volume = {2022},
number = {},
pages = {0001},
pmid = {37905201},
issn = {2693-1257},
abstract = {Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA (gRNA) has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs (pegRNAs) negatively affected the cleaving activity of the Cas9 nuclease. The self-complementarity between the primer binding sequences (PBSs) and spacer sequence might pose risks to the activity of the Cas9 complex. However, modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein (SlMBP21), alcobaca (SlALC), and acetolactate synthase 1 (SlALS1) loci. Our data show challenges of the PE approach in tomato, indicating that a further improvement of the PE system for successful applications is demanded, such as the use of improved expression systems for enriching active PE complexes.},
}
@article {pmid37904281,
year = {2023},
author = {Yoon, DE and Lee, H and Kim, K},
title = {Recent Research Trends in Stem Cells Using CRISPR/Cas-Based Genome Editing Methods.},
journal = {International journal of stem cells},
volume = {},
number = {},
pages = {},
doi = {10.15283/ijsc23030},
pmid = {37904281},
issn = {2005-3606},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system, a rapidly advancing genome editing technology, allows DNA alterations into the genome of organisms. Gene editing using the CRISPR system enables more precise and diverse editing, such as single nucleotide conversion, precise knock-in of target sequences or genes, chromosomal rearrangement, or gene disruption by simple cutting. Moreover, CRISPR systems comprising transcriptional activators/repressors can be used for epigenetic regulation without DNA damage. Stem cell DNA engineering based on gene editing tools has enormous potential to provide clues regarding the pathogenesis of diseases and to study the mechanisms and treatments of incurable diseases. Here, we review the latest trends in stem cell research using various CRISPR/Cas technologies and discuss their future prospects in treating various diseases.},
}
@article {pmid37904134,
year = {2023},
author = {Guna, A and Page, KR and Replogle, JM and Esantsi, TK and Wang, ML and Weissman, JS and Voorhees, RM},
title = {A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens.},
journal = {BMC genomics},
volume = {24},
number = {1},
pages = {651},
pmid = {37904134},
issn = {1471-2164},
support = {NS115380//NIH F31 Ruth L. Kirchstein National Research Service Award/ ; 2RM1 HG009490-06//Center for Genome Editing and Recording/ ; 2019L/LT000858//Human Frontier Science Program/ ; DP2GM137412//NIH's National Institute of General Medical Sciences/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; Genome ; CRISPR-Cas Systems ; },
abstract = {Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.},
}
@article {pmid37901282,
year = {2023},
author = {Li, G and Zhang, Y and Dailey, M and Qi, Y},
title = {Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1251903},
pmid = {37901282},
issn = {2673-3439},
abstract = {Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T0 plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0 plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.},
}
@article {pmid37900415,
year = {2023},
author = {Chavada, J and Muneshwar, KN and Ghulaxe, Y and Wani, M and Sarda, PP and Huse, S},
title = {Antibiotic Resistance: Challenges and Strategies in Combating Infections.},
journal = {Cureus},
volume = {15},
number = {9},
pages = {e46013},
pmid = {37900415},
issn = {2168-8184},
abstract = {From a broader perspective, antibiotic or antimicrobial resistance is still evolving and spreading internationally. Infectious diseases have become more complex and often impossible to cure, increasing morbidity and mortality. Despite the failure of conventional, standard antimicrobial therapy, no new class of antibiotics has been developed in the last 20 years, which results in various cutting-edge and other tactics that can be used to encounter these disease-causing microorganisms with antibiotic resistance. In the continued fight against bacterial infections, there is an urgent requirement for new antibiotics and other antimicrobials. Antibiotic resistance is inevitable, and pharmaceutical companies consistently show little interest in funding novel antibiotic research. Some methods are being used as a possible replacement for conventional antibiotics. Combination therapy, methods that target the proteins or enzymes that cause antimicrobial resistance and bacterial resistance, systems for delivery of the drug, physicochemical approaches, and informal ways, such as the CRISPR-Cas system, are some of these approaches. These various approaches influence how multi-drug-resistant organisms are handled in human clinical settings.},
}
@article {pmid37846511,
year = {2023},
author = {Fan, H and Luo, SH and Zhu, Y and Shi, J and Yin, F and Li, J},
title = {trans-Cleavage of the CRISPR-Cas12a-aptamer system for one-step antigen detection.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {88},
pages = {13151-13154},
doi = {10.1039/d3cc04532c},
pmid = {37846511},
issn = {1364-548X},
mesh = {Male ; Humans ; CRISPR-Cas Systems/genetics ; Prostate-Specific Antigen ; Coloring Agents ; Oligonucleotides ; *Prostatic Neoplasms/diagnosis/genetics ; *Biosensing Techniques ; },
abstract = {Rapid detection of prostate-specific antigen (PSA) is pivotal for the early screening of prostate cancer (PCa). Here, we devise a one-step, amplification-free fluorescent detection strategy for PSA, employing the trans-cleavage principle of a CRISPR-Cas12a-aptamer system. This method offers a linear range of 0.31-5 ng mL[-1] and a detection limit of 0.16 ng mL[-1]. The high-confidence quantification of PSA is demonstrated through the analysis of real samples, effectively distinguishing between PCa patients and healthy individuals.},
}
@article {pmid37827223,
year = {2023},
author = {Fu, X and Li, J and Wu, Y and Mao, C and Jiang, Y},
title = {PAR2 deficiency tunes inflammatory microenvironment to magnify STING signalling for mitigating cancer metastasis via anionic CRISPR/Cas9 nanoparticles.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {363},
number = {},
pages = {733-746},
doi = {10.1016/j.jconrel.2023.10.017},
pmid = {37827223},
issn = {1873-4995},
mesh = {Humans ; Female ; CRISPR-Cas Systems ; Receptor, PAR-2/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; *Nanoparticles ; *Breast Neoplasms/genetics ; Tumor Microenvironment ; },
abstract = {Metastasis is one of the most significant causes for deterioration of breast cancer, contributing to the clinical failure of anti-tumour drugs. Excessive inflammatory responses intensively promote the occurrence and development of tumour, while protease-activated receptor 2 (PAR2) as a cell membrane receptor actively participates in both tumour cell functions and inflammatory responses. However, rare investigations linked PAR2-mediated inflammatory environment to tumour progression. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is an emerging and powerful gene editing technique and can be applied for probing the new role of PAR2 in breast cancer metastasis, but it still needs the development of an efficient and safe delivery system. This work constructed anionic bovine serum albumin (BSA) nanoparticles to encapsulate CRISPR/Cas9 plasmid encoding PAR2 sgRNA and Cas9 (tBSA/Cas9-PAR2) for triggering PAR2 deficiency. tBSA/Cas9-PAR2 remarkably promoted CRISPR/Cas9 to enter and transfect both inflammatory and cancer cells, initiating precise PAR2 gene editing in vitro and in vivo. PAR2 deficiency by tBSA/Cas9-PAR2 effectively suppressed NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome signalling in inflammatory microenvironment to magnify stimulator of interferon genes (STING) signalling, reactive oxygen species (ROS) accumulation and epithelial-mesenchymal transition (EMT) reversal, consequently preventing breast cancer metastasis. Therefore, this study not only demonstrated the involvement and underlying mechanism of PAR2 in tumour progression via modulating inflammatory microenvironment, but also suggested PAR2 deficiency by tBSA/Cas9-PAR2 as an attractive therapeutic strategy candidate for breast cancer metastasis.},
}
@article {pmid37519063,
year = {2023},
author = {Kazimierska, M and Podralska, M and Żurawek, M and Woźniak, T and Kasprzyk, ME and Sura, W and Łosiewski, W and Ziółkowska-Suchanek, I and Kluiver, J and van den Berg, A and Rozwadowska, N and Dzikiewicz-Krawczyk, A},
title = {CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.},
journal = {Molecular oncology},
volume = {17},
number = {11},
pages = {2295-2313},
pmid = {37519063},
issn = {1878-0261},
support = {952304//H2020 Spreading Excellence and Widening Participation/ ; 2016/23/D/NZ1/01611//Narodowe Centrum Nauki/ ; 2017/26/D/NZ1/01234//Narodowe Centrum Nauki/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Cell Line ; Transcription Factors/metabolism ; Gene Expression Regulation ; *Neoplasms/genetics ; },
abstract = {The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.},
}
@article {pmid37897710,
year = {2023},
author = {Hamed, SM and Mohamed, HO and Ashour, HM and Fahmy, LI},
title = {Comparative genomic analysis of strong biofilm-forming Klebsiella pneumoniae isolates uncovers novel ISEcp1-mediated chromosomal integration of a full plasmid-like sequence.},
journal = {Infectious diseases (London, England)},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/23744235.2023.2272624},
pmid = {37897710},
issn = {2374-4243},
abstract = {BACKGROUND: The goal of the current study was to elucidate the genomic background of biofilm formation in Klebsiella pneumoniae.<br><br>METHODS: Clinical isolates were screened for biofilm formation using the crystal violet assay. Antimicrobial resistance (AMR) profiles were assessed by disk diffusion and broth microdilution tests. Biofilm formation was correlated to virulence and resistance genes screened by PCR. Draft genomes of three isolates that form strong biofilm were generated by Illumina sequencing.<br><br>RESULTS: Only the siderophore-coding gene iutA was significantly associated with more pronounced biofilm formation. ST1399-KL43-O1/O2v1 and ST11-KL15-O4 were assigned to the multidrug-resistant strain K21 and the extensively drug-resistant strain K237, respectively. ST1999-KL38-O12 was assigned to K57. Correlated with CRISPR/Cas distribution, more plasmid replicons and prophage sequences were identified in K21 and K237 compared to K57. The acquired AMR genes (blaOXA-48, rmtF, aac(6')-Ib and qnrB) and (blaNDM-1, blaCTX-M, aph(3')-VI, qnrS, and aac(6')-Ib-cr) were found in K237 and K21, respectively. The latter showed a novel ISEcp1-mediated chromosomal integration of replicon type IncM1 plasmid-like structure harboring blaCTX-M-14 and aph(3')-VI that uniquely interrupted rcsC. The plasmid-mediated heavy metal resistance genes merACDEPRT and arsABCDR were spotted in K21, which also exclusively carried the acquired virulence genes mrkABCDF and the hypervirulence-associated genes iucABCD-iutA, and rmpA/A2. Pangenome analysis revealed NTUH-K2044 accessory genes most frequently shared with K21.<br><br>CONCLUSIONS: While less virulent to Galleria mellonella than ST1999 (K57), the strong biofilm former, multidrug-resistant, NDM-producer K. pneumoniae K21 (ST1399-KL43-O1/O2v1) carries a novel chromosomally integrated plasmid-like structure and hypervirulence-associated genes and represents a serious threat to countries in the area.},
}
@article {pmid37896818,
year = {2023},
author = {Xiao, Y and Fei, D and Li, M and Ma, Y and Ma, M},
title = {Establishment and Application of CRISPR-Cas12a-Based Recombinase Polymerase Amplification and a Lateral Flow Dipstick and Fluorescence for the Detection and Distinction of Deformed Wing Virus Types A and B.},
journal = {Viruses},
volume = {15},
number = {10},
pages = {},
pmid = {37896818},
issn = {1999-4915},
support = {The role and mechanism of M6A modification in the replication of Chinese sacbrood virus and RNAi immune regulation//Mingxiao Ma/ ; Screening of structural proteins of deformed wing virus (DWV) with host interaction proteins and their role in infection//Dongliang Fei/ ; H2022043//Ming Li: Horizontal Project of Jinzhou Medical University/ ; },
mesh = {Bees ; Animals ; *Recombinases/genetics ; CRISPR-Cas Systems ; Fluorescence ; *RNA Viruses/genetics ; },
abstract = {Deformed wing virus (DWV) is one of the important pathogens of the honey bee (Apis mellifera), which consists of three master variants: types A, B, and C. Among them, DWV types A (DWV-A) and B (DWV-B) are the most prevalent variants in honey bee colonies and have been linked to colony decline. DWV-A and DWV-B have different virulence, but it is difficult to distinguish them via traditional methods. In this study, we established a visual detection assay for DWV-A and DWV-B using recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 12a fluorescence system (RPA-CRISPR-Cas12a-LFD). The limit of detection of this system was ~6.5 × 10[0] and 6.2 × 10[1] copies/μL for DWV-A and DWV-B, respectively. The assays were specific and non-cross-reactive against other bee viruses, and the results could be visualized within 1 h. The assays were validated by extracting cDNA from 36 clinical samples of bees that were suspected to be infected with DWV. The findings were consistent with those of traditional reverse transcription-quantitative polymerase chain reaction, and the RPA-CRISPR-Cas12a assay showed the specific, sensitive, simple, and appropriate detection of DWV-A and DWV-B. This method can facilitate the visual and qualitative detection of DWV-A and DWV-B as well as the monitoring of different subtypes, thereby providing potentially better control and preventing current and future DWV outbreaks.},
}
@article {pmid37896260,
year = {2023},
author = {Öktem, M and Mastrobattista, E and de Jong, OG},
title = {Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction.},
journal = {Pharmaceutics},
volume = {15},
number = {10},
pages = {},
pmid = {37896260},
issn = {1999-4923},
support = {n/a//Turkish Ministry of Education/ ; },
abstract = {The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.},
}
@article {pmid37896028,
year = {2023},
author = {Tsakirpaloglou, N and Septiningsih, EM and Thomson, MJ},
title = {Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {20},
pages = {},
pmid = {37896028},
issn = {2223-7747},
support = {2020-67013-31811//United States Department of Agriculture/ ; n/a//Texas A&amp;M AgriLife Research/ ; n/a//Texas A&amp;M H.M. Beachell Endowed Chair for Rice Improvement/ ; },
abstract = {With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of gene expression. Likewise, new trait improvement approaches can take advantage of targeted editing to improve stress tolerance, disease resistance, and nutritional traits. However, several key steps in the process can prove tricky for researchers who might be new to plant genome editing. Here, we present step-by-step guidelines and best practices for a crop genome editing pipeline that should help to improve the rate of success. Important factors in the process include proper target sequence analysis and single guide RNA (sgRNA) design, sequencing of the target site in the genotypes of interest, performing an in vitro CRISPR/Cas9 ribonucleoprotein (RNP) assay to validate the designed sgRNAs, preparing the transformation constructs, considering a protoplast editing step as further validation, and, finally, stable plant transformation and mutation detection by Sanger and/or next-generation sequencing. With these detailed guidelines, a new user should be able to quickly set up a genome editing pipeline in their crop of interest and start making progress with the different CRISPR/Cas-based editing variants for gene validation and trait improvement purposes.},
}
@article {pmid37895260,
year = {2023},
author = {Ullah, N and Yang, N and Guan, Z and Xiang, K and Wang, Y and Diaby, M and Chen, C and Gao, B and Song, C},
title = {Comparative Analysis and Phylogenetic Insights of Cas14-Homology Proteins in Bacteria and Archaea.},
journal = {Genes},
volume = {14},
number = {10},
pages = {},
pmid = {37895260},
issn = {2073-4425},
support = {31671313//The National Natural Science Foundation of China/ ; 32271508//The National Natural Science Foundation of China/ ; xxxxxx//High-end Talent Support Program of Yangzhou University to Chengyi Song/ ; },
mesh = {*Archaea/genetics ; Phylogeny ; Bacteria/genetics ; *CRISPR-Associated Proteins ; },
abstract = {Type-V-F Cas12f proteins, also known as Cas14, have drawn significant interest within the diverse CRISPR-Cas nucleases due to their compact size. This study involves analyzing and comparing Cas14-homology proteins in prokaryotic genomes through mining, sequence comparisons, a phylogenetic analysis, and an array/repeat analysis. In our analysis, we identified and mined a total of 93 Cas14-homology proteins that ranged in size from 344 aa to 843 aa. The majority of the Cas14-homology proteins discovered in this analysis were found within the Firmicutes group, which contained 37 species, representing 42% of all the Cas14-homology proteins identified. In archaea, the DPANN group had the highest number of species containing Cas14-homology proteins, a total of three species. The phylogenetic analysis results demonstrate the division of Cas14-homology proteins into three clades: Cas14-A, Cas14-B, and Cas14-U. Extensive similarity was observed at the C-terminal end (CTD) through a domain comparison of the three clades, suggesting a potentially shared mechanism of action due to the presence of cutting domains in that region. Additionally, a sequence similarity analysis of all the identified Cas14 sequences indicated a low level of similarity (18%) between the protein variants. The analysis of repeats/arrays in the extended nucleotide sequences of the identified Cas14-homology proteins highlighted that 44 out of the total mined proteins possessed CRISPR-associated repeats, with 20 of them being specific to Cas14. Our study contributes to the increased understanding of Cas14 proteins across prokaryotic genomes. These homologous proteins have the potential for future applications in the mining and engineering of Cas14 proteins.},
}
@article {pmid37895206,
year = {2023},
author = {Nejat, S and Menikdiwela, KR and Efotte, A and Scoggin, S and Vandanmagsar, B and Thornalley, PJ and Dehbi, M and Moustaid-Moussa, N},
title = {Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.},
journal = {Genes},
volume = {14},
number = {10},
pages = {},
pmid = {37895206},
issn = {2073-4425},
mesh = {Animals ; Female ; Male ; Mice ; Body Weight/genetics ; CRISPR-Cas Systems/genetics ; *Diabetes Mellitus, Type 2/genetics/metabolism ; Diet, High-Fat/adverse effects ; Glucose/metabolism ; HSP40 Heat-Shock Proteins/genetics/metabolism ; Insulin/genetics/metabolism ; *Insulin Resistance/genetics ; Mice, Knockout ; Obesity/genetics/metabolism ; Phenotype ; RNA, Messenger ; },
abstract = {Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D.},
}
@article {pmid37869009,
year = {2023},
author = {Kubiak, AM and Claessen, L and Zhang, Y and Khazaie, K and Bailey, TS},
title = {Refined control of CRISPR-Cas9 gene editing in Clostridium sporogenes: the creation of recombinant strains for therapeutic applications.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1241632},
pmid = {37869009},
issn = {1664-3224},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; *Neoplasms/genetics ; Cytokines/genetics ; },
abstract = {Despite considerable clinical success, the potential of cancer immunotherapy is restricted by a lack of tumour-targeting strategies. Treatment requires systemic delivery of cytokines or antibodies at high levels to achieve clinically effective doses at malignant sites. This is exacerbated by poor penetration of tumour tissue by therapeutic antibodies. High-grade immune-related adverse events (irAEs) occur in a significant number of patients (5-15%, cancer- and therapeutic-dependent) that can lead to lifelong issues and can exclude from treatment patients with pre-existing autoimmune diseases. Tumour-homing bacteria, genetically engineered to produce therapeutics, is one of the approaches that seeks to mitigate these drawbacks. The ability of Clostridium sporogenes to form spores that are unable to germinate in the presence of oxygen (typical of healthy tissue) offers a unique advantage over other vectors. However, the limited utility of existing gene editing tools hinders the development of therapeutic strains. To overcome the limitations of previous systems, expression of the Cas9 protein and the gRNA was controlled using tetracycline inducible promoters. Furthermore, the components of the system were divided across two plasmids, improving the efficiency of cloning and conjugation. Genome integrated therapeutic genes were assayed biochemically and in cell-based functional assays. The potency of these strains was further improved through rationally-conceived gene knock-outs. The new system was validated by demonstrating the efficient addition and deletion of large sequences from the genome. This included the creation of recombinant strains expressing two pro-inflammatory cytokines, interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF), and a pro-drug converting enzyme (PCE). A comparative, temporal in vitro analysis of the integrant strains and their plasmid-based equivalents revealed a substantial reduction of cytokine activity in chromosome-based constructs. To compensate for this loss, a 7.6 kb operon of proteolytic genes was deleted from the genome. The resultant knock-out strains showed an 8- to 10-fold increase in cytokine activity compared to parental strains.},
}
@article {pmid37864244,
year = {2023},
author = {Rajaram, N and Kouroukli, AG and Bens, S and Bashtrykov, P and Jeltsch, A},
title = {Development of super-specific epigenome editing by targeted allele-specific DNA methylation.},
journal = {Epigenetics &amp; chromatin},
volume = {16},
number = {1},
pages = {41},
pmid = {37864244},
issn = {1756-8935},
support = {Epigenetics program, ID09//Baden-W&#xfc;rttemberg Stiftung/ ; Epigenetics program, ID09//Baden-W&#xfc;rttemberg Stiftung/ ; Epigenetics program, ID09//Baden-W&#xfc;rttemberg Stiftung/ ; Epigenetics program, ID09//Baden-W&#xfc;rttemberg Stiftung/ ; },
mesh = {Humans ; *DNA Methylation ; *RNA, Guide, CRISPR-Cas Systems ; Epigenesis, Genetic ; Alleles ; HEK293 Cells ; Epigenome ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {BACKGROUND: Epigenome editing refers to the targeted reprogramming of genomic loci using an EpiEditor which may consist of an sgRNA/dCas9 complex that recruits DNMT3A/3L to the target locus. Methylation of the locus can lead to a modulation of gene expression. Allele-specific DNA methylation (ASM) refers to the targeted methylation delivery only to one allele of a locus. In the context of diseases caused by a dominant mutation, the selective DNA methylation of the mutant allele could be used to repress its expression but retain the functionality of the normal gene.<br><br>RESULTS: To set up allele-specific targeted DNA methylation, target regions were selected from hypomethylated CGIs bearing a heterozygous SNP in their promoters in the HEK293 cell line. We aimed at delivering maximum DNA methylation with highest allelic specificity in the targeted regions. Placing SNPs in the PAM or seed regions of the sgRNA, we designed 24 different sgRNAs targeting single alleles in 14 different gene loci. We achieved efficient ASM in multiple cases, such as ISG15, MSH6, GPD1L, MRPL52, PDE8A, NARF, DAP3, and GSPT1, which in best cases led to five to tenfold stronger average DNA methylation at the on-target allele and absolute differences in the DNA methylation gain at on- and off-target alleles of&#x2009;>&#x2009;50%. In general, loci with the allele discriminatory SNP positioned in the PAM region showed higher success rate of ASM and better specificity. Highest DNA methylation was observed on day 3 after transfection followed by a gradual decline. In selected cases, ASM was stable up to 11 days in HEK293 cells and it led up to a 3.6-fold change in allelic expression ratios.<br><br>CONCLUSIONS: We successfully delivered ASM at multiple genomic loci with high specificity, efficiency and stability. This form of super-specific epigenome editing could find applications in the treatment of diseases caused by dominant mutations, because it allows silencing of the mutant allele without repression of the expression of the normal allele thereby minimizing potential side-effects of the treatment.},
}
@article {pmid37845542,
year = {2023},
author = {Wang, J and Wang, X and Li, B and Zhang, K and Mao, J},
title = {Entropy-driven reactions for controlling CRISPR/Cas12a and constructing an electrochemical biosensor for cardiac biomarkers detection.},
journal = {Mikrochimica acta},
volume = {190},
number = {11},
pages = {440},
pmid = {37845542},
issn = {1436-5073},
support = {81904055//Young Scientists Fund/ ; },
mesh = {Humans ; Entropy ; *CRISPR-Cas Systems ; Calibration ; *DNA, Single-Stranded ; Oligonucleotides ; Biomarkers ; RNA ; },
abstract = {An electrochemical biosensor is reported for controlling CRISPR/Cas12a activity through the utilization of entropy-driven reactions, alongside the construction of a highly sensitive biosensor for B-type natriuretic peptide (BNP) detection. In the biosensor, entropy-driven reactions are employed to regulate the activity of CRISPR/Cas12a - a gene editing tool - capable of nonspecific cleavage of single-stranded DNA (ssDNA). The biosensor architecture encompasses an electrode that is modified with ssDNA probes designed to hybridize with target BNP aptamers. These aptamers, furnished with labeled ssDNA triggers, facilitate the activation of CRISPR/Cas12a through interaction with its guide RNA. Upon the presence of BNP, it associates with the aptamers, subsequently liberating the triggers that instigate the entropy-driven reactions. As a consequence of these reactions, more stable duplexes emerge between the triggers and guide RNA, thereby activating CRISPR/Cas12a. The activated CRISPR/Cas12a subsequently executes cleavage of ssDNA probes residing on the electrode surface, culminating in the generation of an electrochemical signal directly (the calibration plots of differential pulse voltammetric detection were acquired at a working potential of 0.2 V (vs. ref. electrode)) proportional to the BNP concentration. Validation of the biosensor's performance is undertaken, wherein BNP detection is demonstrated in both buffer and human serum samples. Evident in the findings is the biosensor's discernible sensitivity and specificity for BNP detection, exemplified by a detection limit of 13.53 fM and a lack of interference originating from other cardiac biomarkers, respectively. Furthermore, the biosensor's potential to discriminate between healthy individuals and those afflicted by heart failure, predicated on distinctive BNP levels, is illustrated.},
}
@article {pmid37834313,
year = {2023},
author = {Cai, R and Lv, R and Shi, X and Yang, G and Jin, J},
title = {CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation.},
journal = {International journal of molecular sciences},
volume = {24},
number = {19},
pages = {},
pmid = {37834313},
issn = {1422-0067},
support = {2023-JC-QN-0243//Natural Science Foundation of Shaanxi Province/ ; 2021YFF1000602 and 2021YFD1301200//the National Key Research and Development Program of China/ ; CARS-35//the China Agriculture Research System/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; Epigenesis, Genetic ; DNA ; },
abstract = {CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity of Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, the changes to the DNA sequence that are mediated by CRISPR/Cas9 affect the structures and stability of the genome, which may affect the accuracy of results. Mutations in the RuvC and HNH regions of the Cas9 protein lead to the inactivation of Cas9 into dCas9 with no endonuclease activity. Despite the loss of endonuclease activity, dCas9 can still bind the DNA strand using guide RNA. Recently, proteins with active/inhibitory effects have been linked to the end of the dCas9 protein to form fusion proteins with transcriptional active/inhibitory effects, named CRISPRa and CRISPRi, respectively. These CRISPR tools mediate the transcription activity of protein-coding and non-coding genes by regulating the chromosomal modification states of target gene promoters, enhancers, and other functional elements. Here, we highlight the epigenetic mechanisms and applications of the common CRISPR/dCas9 tools, by which we hope to provide a reference for future related gene regulation, gene function, high-throughput target gene screening, and disease treatment.},
}
@article {pmid37834295,
year = {2023},
author = {Camargo, JA and Viana, NI and Pimenta, R and Guimarães, VR and Dos Santos, GA and Candido, P and Ghazarian, V and Romão, P and Silva, IA and Birbrair, A and Srougi, M and Nahas, WC and Leite, KR and Trarbach, EB and Reis, ST},
title = {The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer.},
journal = {International journal of molecular sciences},
volume = {24},
number = {19},
pages = {},
pmid = {37834295},
issn = {1422-0067},
support = {2018/19906-3//S&#xe3;o Paulo Research Foundation/ ; 2019/00156-7//S&#xe3;o Paulo Research Foundation/ ; 2019/19138-9//S&#xe3;o Paulo Research Foundation/ ; 2017/17362-3//S&#xe3;o Paulo Research Foundation/ ; },
mesh = {Male ; Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; Matrix Metalloproteinase 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; bcl-2-Associated X Protein/metabolism ; *Prostatic Neoplasms/genetics/pathology ; *MicroRNAs/genetics/metabolism ; TOR Serine-Threonine Kinases/metabolism ; GPI-Linked Proteins/genetics/metabolism ; *Immediate-Early Proteins/genetics ; Tumor Suppressor Proteins/genetics ; RNA-Binding Proteins/metabolism ; },
abstract = {Prostate cancer (PCa) has a high prevalence and represents an important health problem, with an increased risk of metastasis. With the advance of CRISPR-Cas9 genome editing, new possibilities have been created for investigating PCa. The technique is effective in knockout oncogenes, reducing tumor resistance. MMP9 and miR-21 target genes are associated with PCa progression; therefore, we evaluated the MMP-9 and miR-21 targets in PCa using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) of MMP9 and miR-21 sequences were inserted into a PX-330 plasmid, and transfected in DU145 and PC-3 PCa cell lines. MMP9 and RECK expression was assessed by qPCR, WB, and IF. The miR-21 targets, integrins, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed with Annexin5, 7-AAD and Ki67 markers. Invasion assays were performed with Matrigel. The miR-21 CRISPR-Cas9-edited cells upregulated RECK, MARCKS, BTG2, and PDCD4. CDH1, ITGB3 and ITGB1 were increased in MMP9 and miR-21 CRISPR-Cas9-edited cells. Increased BAX and decreased mTOR were observed in MMP9 and miR-21 CRISPR-Cas9-edited cells. Reduced cell proliferation, increased apoptosis and low invasion in MMP9 and miR-21 edited cells was observed, compared to Scramble. CRISPR-Cas9-edited cells of miR-21 and MMP9 attenuate cell proliferation, invasion and stimulate apoptosis, impeding PCa evolution.},
}
@article {pmid37833763,
year = {2023},
author = {Dang, S and Sui, H and Zhang, S and Wu, D and Chen, Z and Zhai, J and Bai, M},
title = {CRISPR-Cas12a test strip (CRISPR/CAST) package: In-situ detection of Brucella from infected livestock.},
journal = {BMC veterinary research},
volume = {19},
number = {1},
pages = {202},
pmid = {37833763},
issn = {1746-6148},
mesh = {Animals ; Cattle ; Sheep/genetics ; Livestock ; CRISPR-Cas Systems ; *Brucellosis/diagnosis/veterinary ; Brucella abortus ; DNA ; *Nucleic Acids ; *Cattle Diseases/genetics ; *Sheep Diseases/diagnosis/genetics ; },
abstract = {BACKGROUND: Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out.<br><br>RESULTS: We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16&#xa0;M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30&#xa0;min under isothermal temperature conditions, with a sensitivity of 10 copies/&#x3bc;l. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive).<br><br>CONCLUSIONS: The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30&#xa0;min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics.},
}
@article {pmid37827629,
year = {2023},
author = {Deng, L and Zhou, S and Dong, J and Liu, Y and Huang, Z and Sun, H and Jin, L and Huo, D and Hou, C},
title = {CRISPR/Cas12a and primer-assisted rolling circle amplification integrated ultra-sensitive dual-signal sensing platform for EGFR 19 detection.},
journal = {Analytica chimica acta},
volume = {1279},
number = {},
pages = {341755},
doi = {10.1016/j.aca.2023.341755},
pmid = {37827629},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Coloring Agents ; ErbB Receptors/genetics ; *Biosensing Techniques/methods ; },
abstract = {Herein, we integrated CRISPR/Cas12a with primer-assisted rolling circle amplification (PARCA) to specifically detect EGFR 19 from the genome. We fused the method into fluorescent and electrochemical detection systems forming a stable and sensitive dual-signal sensing platform. The fluorescent detection system stably detected EGFR 19 in a linear range from 500 fM to 10 nM with an ultra-low background signal. The electrochemical detection system possessed a detection limit as low as 42 aM due to the introduction of nanomaterial UIO-66-NH2. The dual-signal sensing platform showed superior performance in complex serum samples and real cell genomes and provided a flexible and dynamic approach for the ultra-sensitive detection of EGFR 19.},
}
@article {pmid37824705,
year = {2023},
author = {Chen, Y and Zhang, Y and Luo, S and Yang, X and Liu, C and Zhang, Q and Liu, Y and Zhang, X},
title = {Foldback-crRNA-Enhanced CRISPR/Cas13a System (FCECas13a) Enables Direct Detection of Ultrashort sncRNA.},
journal = {Analytical chemistry},
volume = {95},
number = {42},
pages = {15606-15613},
doi = {10.1021/acs.analchem.3c02687},
pmid = {37824705},
issn = {1520-6882},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; *RNA, Small Untranslated ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Nucleotides ; },
abstract = {The CRISPR/Cas13a system has promising applications in clinical small noncoding RNA (sncRNA) detection because it is free from the interference of genomic DNA. However, detecting ultrashort sncRNAs (less than 20 nucleotides) has been challenging because the Cas13a nuclease requires longer crRNA-target RNA hybrids to be activated. Here, we report the development of a foldback-crRNA-enhanced CRISPR/Cas13a (FCECas13a) system that overcomes the limitations of the current CRISPR/Cas13a system in detecting ultrashort sncRNAs. The FCECas13a system employs a 3'-terminal foldback crRNA that hybridizes with the target ultrashort sncRNA, forming a double strand that "tricks" the Cas13a nuclease into activating the HEPN structural domain and generating trans-cleavage activity. The FCECas13a system can accurately detect miRNA720 (a sncRNA currently known as tRNA-derived small RNA), which is only 17 nucleotides long and has a concentration as low as 15 fM within 20 min. This FCECas13a system opens new avenues for ultrashort sncRNA detection with significant implications for basic biological research, disease prognosis, and molecular diagnosis.},
}
@article {pmid37824660,
year = {2023},
author = {Cohen, J},
title = {In 'proof of concept,' CRISPR-edited chickens shrug off flu.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6667},
pages = {140-141},
doi = {10.1126/science.adl2973},
pmid = {37824660},
issn = {1095-9203},
mesh = {Animals ; *Chickens/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Edits to one gene can't completely stop infections, however.},
}
@article {pmid37796686,
year = {2023},
author = {Domont, J and Thiblet, M and Etienne, A and Santos, HAD and Cadalen, T and Hance, P and Gagneul, D and Hilbert, JL and Rambaud, C},
title = {CRISPR/Cas9-Targeted Mutagenesis of CiGAS and CiGAO to Reduce Bitterness in Chicory (Cichorium intybus L.).},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {28},
number = {9},
pages = {201},
doi = {10.31083/j.fbl2809201},
pmid = {37796686},
issn = {2768-6698},
support = {//R&#xe9;gion des Hauts-de-France/ ; },
mesh = {*Chicory/genetics ; CRISPR-Cas Systems/genetics ; Taste/genetics ; Mutagenesis ; },
abstract = {BACKGROUND: Chicory (Cichorium intybus L.), a member of the Asteraceae family, is known for its numerous health benefits, including its prebiotic, digestive, antioxidant or anti-inflammatory effects. Used as a coffee substitute, chicory roots is also appreciated for its bitterness, which can prove to be a disadvantage for other uses in food. The bitterness of chicory is largely linked to the presence of sesquiterpene lactones (STLs) in the roots.<br><br>METHODS: In order to create less bitter industrial chicory varieties, CRISPR/Cas9 technology was used to inhibit the first two genes of the STL biosynthetic pathway: germacrene A synthase (CiGAS), short form, and germacrene A oxidase (CiGAO). To determine the impact of these reductions on the perception of bitterness, a sensory analysis of 13 field-grown chicories genotypes, contrasting for their STL composition, allowed the construction of obtain a bitterness scale by correlating STL content with perceived bitterness. The edited chicories were positioned on this scale according to their STL content.<br><br>RESULTS: Biallelic mutations in two of the copies of CiGAS-short form or in the CiGAO gene led to a reduction in STL content of edited chicories and a reduction in bitterness, or even an absence of perception, was obtained for some mutants.<br><br>CONCLUSIONS: The use of the CRISPR/Cas9 tool as well as the choice of targets therefore makes it possible to modulate the bitterness of chicory.},
}
@article {pmid37795981,
year = {2023},
author = {Wang, H and Wang, S and Wang, H and Tang, F and Chen, D and Liang, Y and Li, Z},
title = {Amplification-free detection of telomerase activity at the single-cell level via Cas12a-lighting-up single microbeads (Cas12a-LSMBs).},
journal = {Lab on a chip},
volume = {23},
number = {21},
pages = {4674-4679},
doi = {10.1039/d3lc00598d},
pmid = {37795981},
issn = {1473-0189},
mesh = {*Telomerase ; CRISPR-Cas Systems ; Microspheres ; DNA, Single-Stranded ; Fluorescence ; *Biosensing Techniques ; },
abstract = {Telomerase overexpresses in almost all cancer cells and has been deemed a universal biomarker for cancer diagnosis and therapy. However, simple and ultrasensitive detection of telomerase activity in single-cells is still a huge challenge. Herein, we wish to report Cas12a-lighting up single microbeads (Cas12a-LSMBs) for ultrasensitive detection of telomerase activity without nucleic acid amplification. In this platform, single-strand DNA reporter (ssDNA reporter)-functionalized single-microbeads (functionalized-SMBs) are employed as a reactor for the trans-cleavage of telomerase-activated CRISPR/Cas12a as well as a reporting unit for fluorescence signal enrichment and visualization. Due to the space-confined effect and signal enrichment mechanism on the surface of the functionalized SMBs, the Cas12a-LSMBs can accurately detect telomerase activity in crude cell lysates with high specificity. Importantly, we have demonstrated that the Cas12a-LSMBs are a reliable and practical tool to detect telomerase activity in single cells and investigate cellular heterogeneity of telomerase activity from cell-to-cell variations.},
}
@article {pmid37794568,
year = {2023},
author = {Dai, Z},
title = {Novel genetic tools improve Penicillium expansum patulin synthase production in Aspergillus niger.},
journal = {The FEBS journal},
volume = {290},
number = {21},
pages = {5094-5097},
doi = {10.1111/febs.16956},
pmid = {37794568},
issn = {1742-4658},
support = {DE-NL0030038//Bioenergy Technologies Office (BETO) of Department of Energy/ ; },
mesh = {Gene Editing/methods ; Aspergillus niger/genetics ; *Patulin/genetics/metabolism ; *Penicillium/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Since the first CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system was developed for creating double-stranded DNA breaks, it has been adapted and improved for different biotechnological applications. In this issue of The FEBS Journal, Arentshorst et al. developed a novel approach to enhance transgene expression of a specific protein, patulin synthase (PatE) from Penicillium expansum, in the important industrial filamentous fungus Aspergillus niger. Their technique involved the disruption of selected genes with counter-effects on targeted protein production and simultaneous integration of glucoamylase landing sites into the disrupted gene locus such as protease regulator (prtT) in an ATP-dependent DNA helicase II subunit 1 (kusA or ku70)-deletion strain. Multiple copies of the PatE transgene expression cassette were introduced by CRISPR-Cas9-mediated insertion. The purified PatE was further used for structural and functional studies, and the technique laid the foundation for elevating the overall production of various proteins or chemicals in those industrially important fungi.},
}
@article {pmid37778776,
year = {2023},
author = {Zhang, H and Tu, T},
title = {Targeting Hepatitis B Virus DNA Using Designer Gene Editors.},
journal = {Clinics in liver disease},
volume = {27},
number = {4},
pages = {895-916},
doi = {10.1016/j.cld.2023.05.006},
pmid = {37778776},
issn = {1557-8224},
mesh = {Humans ; *Hepatitis B, Chronic/therapy ; Hepatitis B virus/genetics ; CRISPR-Cas Systems ; DNA, Viral/genetics ; DNA, Circular/genetics/metabolism/pharmacology ; *Hepatitis B ; Virus Replication ; },
abstract = {Chronic hepatitis B virus (HBV) infection is a serious disease that currently has no cure. Key forms of HBV include covalently closed circular DNA, which mediates chronic persistence, and integrated DNA, which contributes to immune evasion and carcinogenesis. These forms are not targeted by current therapies; however, gene editing technologies have emerged as promising tools for disrupting HBV DNA. Gene editor-induced double-stranded breaks at precise locations within the HBV genome can induce effects ranging from inactivation of target genes to complete degradation of the target genome. Although promising, several challenges remain in efficacy and safety that require solutions.},
}
@article {pmid37723222,
year = {2023},
author = {Madigan, V and Zhang, F and Dahlman, JE},
title = {Drug delivery systems for CRISPR-based genome editors.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {11},
pages = {875-894},
pmid = {37723222},
issn = {1474-1784},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; Drug Delivery Systems ; Proteins/genetics ; },
abstract = {CRISPR-based drugs can theoretically manipulate any genetic target. In practice, however, these drugs must enter the desired cell without eliciting an unwanted immune response, so a delivery system is often required. Here, we review drug delivery systems for CRISPR-based genome editors, focusing on adeno-associated viruses and lipid nanoparticles. After describing how these systems are engineered and their subsequent characterization in preclinical animal models, we highlight data from recent clinical trials. Preclinical targeting mediated by polymers, proteins, including virus-like particles, and other vehicles that may deliver CRISPR systems in the future is also discussed.},
}
@article {pmid37695141,
year = {2023},
author = {Chneiweiss, H},
title = {[Genome editing for the clinics: First successes and new issues].},
journal = {Medecine sciences : M/S},
volume = {39},
number = {8-9},
pages = {589-590},
doi = {10.1051/medsci/2023110},
pmid = {37695141},
issn = {1958-5381},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Genetic Therapy ; },
}
@article {pmid37616014,
year = {2023},
author = {Zhu, X and Xu, W and Liu, B and Zhan, Y and Xia, T},
title = {Adaptation of high-efficiency CRISPR/Cas9-based multiplex genome editing system in white lupin by using endogenous promoters.},
journal = {Physiologia plantarum},
volume = {175},
number = {4},
pages = {e13976},
doi = {10.1111/ppl.13976},
pmid = {37616014},
issn = {1399-3054},
support = {2020J01548//Natural Science Foundation of Fujian Province/ ; 31761130073//National Natural Science Foundation of China/ ; 31872169//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Lupinus/genetics ; Mutation/genetics ; },
abstract = {White lupin (Lupinus albus L.) is an important crop with high phosphorus (P) use efficiency; however, technologies for its functional genomic and molecular analyses are limited. Cluster regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been applied to gene editing and function genomics in many crops, but its application in white lupin has not been well documented. Here, we adapted the CRISPR/Cas9-based multiplex genome editing system by using the native U3/U6 and ubiquitin (UBQ) promoters to drive sgRNAs and Cas9. Two target sites (T1 and T2) within the Lalb_Chr05g0223881 gene, encoding a putative trehalase, were selected to validate its efficacy in white lupin based on the Agrobacterium rhizogenes-mediated transformation. We found that the T0 hairy roots were efficiently mutated at T1 and T2 with a frequency of 6.25%-35% and 50%-92.31%, respectively. The mutation types include nucleotide insertion, deletion, substitution, and complicated variant. Simultaneous mutations of the two targets were also observed with a range of 6.25%-35%. The combination of LaU6.6 promoter for sgRNA and LaUBQ12 promoter for Cas9 generated the highest frequency of homozygous/biallelic mutations at 38.46%. In addition, the target-sgRNA sequence also contributes to the editing efficiency of the CRISPR/Cas9 system in white lupin. In conclusion, our results expand the toolbox of the CRISPR/Cas9 system and benefit the basic research in white lupin.},
}
@article {pmid37335926,
year = {2023},
author = {Arentshorst, M and Kooloth Valappil, P and Mózsik, L and Regensburg-Tuïnk, TJG and Seekles, SJ and Tjallinks, G and Fraaije, MW and Visser, J and Ram, AFJ},
title = {A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.},
journal = {The FEBS journal},
volume = {290},
number = {21},
pages = {5127-5140},
doi = {10.1111/febs.16891},
pmid = {37335926},
issn = {1742-4658},
support = {ENPPS.LIFT.019.037//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; },
mesh = {*Aspergillus niger/genetics ; *CRISPR-Cas Systems ; Glucan 1,4-alpha-Glucosidase/genetics/metabolism ; Gene Editing ; },
abstract = {The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 μg·mL[-1] PatE protein in the culture medium with a purity just under 90%.},
}
@article {pmid37895127,
year = {2023},
author = {Shahriari, Z and Su, X and Zheng, K and Zhang, Z},
title = {Advances and Prospects of Virus-Resistant Breeding in Tomatoes.},
journal = {International journal of molecular sciences},
volume = {24},
number = {20},
pages = {},
pmid = {37895127},
issn = {1422-0067},
support = {202205AR070001//Yunnan Seed Laboratory/ ; 202202AE090022//Major Science and Technology Program of Yunnan Province/ ; 32160620//National Natural Science Foundation of China/ ; 31660508//National Natural Science Foundation of China/ ; },
mesh = {*Solanum lycopersicum/genetics ; Disease Resistance/genetics ; Plant Breeding ; *Plant Viruses ; Antiviral Agents ; Plant Diseases/genetics ; },
abstract = {Plant viruses are the main pathogens which cause significant quality and yield losses in tomato crops. The important viruses that infect tomatoes worldwide belong to five genera: Begomovirus, Orthotospovirus, Tobamovirus, Potyvirus, and Crinivirus. Tomato resistance genes against viruses, including Ty gene resistance against begomoviruses, Sw gene resistance against orthotospoviruses, Tm gene resistance against tobamoviruses, and Pot 1 gene resistance against potyviruses, have been identified from wild germplasm and introduced into cultivated cultivars via hybrid breeding. However, these resistance genes mainly exhibit qualitative resistance mediated by single genes, which cannot protect against virus mutations, recombination, mixed-infection, or emerging viruses, thus posing a great challenge to tomato antiviral breeding. Based on the epidemic characteristics of tomato viruses, we propose that future studies on tomato virus resistance breeding should focus on rapidly, safely, and efficiently creating broad-spectrum germplasm materials resistant to multiple viruses. Accordingly, we summarized and analyzed the advantages and characteristics of the three tomato antiviral breeding strategies, including marker-assisted selection (MAS)-based hybrid breeding, RNA interference (RNAi)-based transgenic breeding, and CRISPR/Cas-based gene editing. Finally, we highlighted the challenges and provided suggestions for improving tomato antiviral breeding in the future using the three breeding strategies.},
}
@article {pmid37894996,
year = {2023},
author = {Li, H and Song, K and Li, B and Zhang, X and Wang, D and Dong, S and Yang, L},
title = {CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {20},
pages = {},
pmid = {37894996},
issn = {1422-0067},
support = {SDAIT-25-01//the Foundation of Innovation Team Project for Modern Agricultural Industrious Technology System of Shandong Province/ ; YDZX2022123//Special Funds for Local Scientific and Technological Development Guided by the Central Gov-ernment/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Camellia sinensis/genetics ; Genome, Plant ; Gene Editing/methods ; },
abstract = {CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.},
}
@article {pmid37894990,
year = {2023},
author = {Xia, Y and Li, Y and Shen, W and Yang, H and Chen, X},
title = {CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges.},
journal = {International journal of molecular sciences},
volume = {24},
number = {20},
pages = {},
pmid = {37894990},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics/metabolism ; Gene Editing/methods ; Biotechnology ; Bioengineering ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable resilience against diverse stressors, and distinctive regulatory mechanisms, have emerged as a highly promising alternative for diverse industrial applications. This comprehensive review serves to encapsulate the prevailing gene editing methodologies and their associated applications within the traditional industrial microorganism, Saccharomyces cerevisiae. Additionally, it delineates the current panorama of non-conventional yeast strains, accentuating their latent potential in the realm of industrial and biotechnological utilization. Within this discourse, we also contemplate the potential value these tools offer alongside the attendant challenges they pose.},
}
@article {pmid37894981,
year = {2023},
author = {Nakamura, S and Morohoshi, K and Inada, E and Sato, Y and Watanabe, S and Saitoh, I and Sato, M},
title = {Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups.},
journal = {International journal of molecular sciences},
volume = {24},
number = {20},
pages = {},
pmid = {37894981},
issn = {1422-0067},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals, Newborn ; Zygote ; },
abstract = {Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. However, this approach requires a long and laborious task. Recently, many researchers have attempted to overcome these limitations by generating somatic mutations in the adult stage through tail vein injection or local administration of CRISPR reagents, as a new strategy called "in vivo somatic cell genome editing". This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. The newborn is an ideal stage to perform in vivo somatic cell genome editing because it is immune-privileged, easily accessible, and only a small amount of CRISPR reagents is required to achieve somatic cell genome editing throughout the entire body, owing to its small size. In this review, we summarize in vivo genome engineering strategies that have been successfully demonstrated in newborns. We also report successful in vivo genome editing through the neonatal introduction of genome editing reagents into various sites in newborns (as exemplified by intravenous injection via the facial vein), which will be helpful for creating models for genetic diseases or treating many genetic diseases.},
}
@article {pmid37874380,
year = {2023},
author = {Dutta, TK and Ray, S and Phani, V},
title = {The status of the CRISPR/Cas9 research in plant-nematode interactions.},
journal = {Planta},
volume = {258},
number = {6},
pages = {103},
pmid = {37874380},
issn = {1432-2048},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Nematoda ; Gene Editing ; Gene Silencing ; Crops, Agricultural/genetics ; },
abstract = {As an important biotic stressor, plant-parasitic nematodes afflict global crop productivity. Deployment of CRISPR/Cas9 system that selectively knock out host susceptibility genes conferred improved nematode tolerance in crop plants. As an important biotic stressor, plant-parasitic nematodes cause a considerable yield decline in crop plants that eventually contributes to a negative impact on global food security. Being obligate plant parasites, the root-knot and cyst nematodes maintain an intricate and sophisticated relationship with their host plants by hijacking the host's physiological and metabolic pathways for their own benefit. Significant progress has been made toward developing RNAi-based transgenic crops that confer nematode resistance. However, the strategy of host-induced gene silencing that targets nematode effectors is likely to fail because the induced silencing of effectors (which interact with plant R genes) may lead to the development of nematode phenotypes that break resistance. Lately, the CRISPR/Cas9-based genome editing system has been deployed to achieve host resistance against bacteria, fungi, and viruses. In these studies, host susceptibility (S) genes were knocked out to achieve resistance via loss of susceptibility. As the S genes are recessively inherited in plants, induced mutations of the S genes are likely to be long-lasting and confer broad-spectrum resistance. A number of S genes contributing to plant susceptibility to nematodes have been identified in Arabidopsis thaliana, rice, tomato, cucumber, and soybean. A few of these S genes were targeted for CRISPR/Cas9-based knockout experiments to improve nematode tolerance in crop plants. Nevertheless, the CRISPR/Cas9 system was mostly utilized to interrogate the molecular basis of plant-nematode interactions rather than direct research toward achieving tolerance in crop plants. The current standalone article summarizes the progress made so far on CRISPR/Cas9 research in plant-nematode interactions.},
}
@article {pmid37868976,
year = {2023},
author = {Audouze-Chaud, J and Mathews, JA and Crome, SQ},
title = {Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1275413},
pmid = {37868976},
issn = {1664-3224},
mesh = {Humans ; *Immunity, Innate ; *CRISPR-Cas Systems ; Killer Cells, Natural ; Gene Editing ; },
abstract = {Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) 'helper' ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s.},
}
@article {pmid37830594,
year = {2023},
author = {Velatooru, LR and Hu, CH and Bijani, P and Wang, X and Bojaxhi, P and Chen, H and Duvic, M and Ni, X},
title = {New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.},
journal = {Cells},
volume = {12},
number = {19},
pages = {},
pmid = {37830594},
issn = {2073-4409},
mesh = {Humans ; Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems ; *Lymphoma, T-Cell, Cutaneous/metabolism ; *Sezary Syndrome ; RNA, Small Interfering ; *Skin Neoplasms/pathology ; Janus Kinase 3/genetics/metabolism ; },
abstract = {Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.},
}
@article {pmid37824835,
year = {2023},
author = {Barrangou, R},
title = {The CRISPR Toolbox: The End of the Beginning.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {403},
doi = {10.1089/crispr.2023.29167.editorial},
pmid = {37824835},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
}
@article {pmid37824833,
year = {2023},
author = {Urnov, F},
title = {Special Issue: CRISPR Trials.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {404},
doi = {10.1089/crispr.2023.29166.cfp},
pmid = {37824833},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37824068,
year = {2024},
author = {Wang, X and Chen, Q and Lackford, B and Hu, G},
title = {Dissecting the Role of the Ccr4-Not Deadenylase Complex in Pluripotency and Differentiation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2723},
number = {},
pages = {125-141},
pmid = {37824068},
issn = {1940-6029},
mesh = {Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems ; *Transcription Factors/genetics ; Cell Differentiation/genetics ; },
abstract = {In this chapter, we describe methods and assays to examine the role of the Ccr4-Not mRNA deadenylase complex in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. We present the following procedures: sgRNA design and cloning, the culture of mouse ESCs, transfection and colony picking, genotyping, generation of Cnot3 conditional deletion ESCs, assessment of Cnot3 deletion efficiency, and examination of the impact on ESC maintenance and differentiation. The above approach and protocols can also be readily applied to study other Ccr4-Not subunits in ESC fate regulation, thereby facilitating the systematic dissection of Ccr4-Not function in stem cells and development.},
}
@article {pmid37821497,
year = {2023},
author = {Sanz Juste, S and Okamoto, EM and Nguyen, C and Feng, X and López Del Amo, V},
title = {Next-generation CRISPR gene-drive systems using Cas12a nuclease.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6388},
pmid = {37821497},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; Agriculture ; Endonucleases/genetics ; Alleles ; },
abstract = {One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. However, all current gene drives employ a Cas9 nuclease that is constitutively active, impeding our control over their propagation abilities and limiting the generation of alternative gene drive arrangements. Yet, other nucleases such as the temperature sensitive Cas12a have not been explored for gene drive designs in insects. To address this, we herein present a proof-of-concept gene-drive system driven by Cas12a that can be regulated via temperature modulation. Furthermore, we combined Cas9 and Cas12a to build double gene drives capable of simultaneously spreading two independent engineered alleles. The development of Cas12a-mediated gene drives provides an innovative option for designing next-generation vector control strategies to combat disease vectors and agricultural pests.},
}
@article {pmid37819747,
year = {2023},
author = {Li, QN and Wang, DX and Han, GM and Liu, B and Tang, AN and Kong, DM},
title = {Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing.},
journal = {Analytical chemistry},
volume = {95},
number = {42},
pages = {15725-15735},
doi = {10.1021/acs.analchem.3c03131},
pmid = {37819747},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; Biological Assay ; Image Processing, Computer-Assisted ; *Nucleic Acids ; Oligonucleotides ; *Biosensing Techniques ; },
abstract = {The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 μM, and 8.3 × 10[-5] U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.},
}
@article {pmid37819512,
year = {2024},
author = {Yu, MA and Banta, AB and Ward, RD and Prasad, NK and Kwon, MS and Rosenberg, OS and Peters, JM},
title = {Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2721},
number = {},
pages = {13-32},
pmid = {37819512},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Pseudomonas aeruginosa/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Silencing ; Phenotype ; },
abstract = {CRISPR interference (CRISPRi) is a robust gene silencing technique that is ideal for targeting essential and conditionally essential (CE) genes. CRISPRi is especially valuable for investigating gene function in pathogens such as P. aeruginosa where essential and CE genes underlie clinically important phenotypes such as antibiotic susceptibility and virulence. To facilitate the use of CRISPRi in diverse bacteria-including P. aeruginosa-we developed a suite of modular, mobilizable, and integrating vectors we call, "Mobile-CRISPRi." We further optimized Mobile-CRISPRi for use in P. aeruginosa mouse models of acute lung infection by expressing the CRISPRi machinery at low levels constitutively, enabling partial knockdown of essential and CE genes without the need for an exogenous inducer. Here, we describe protocols for creating Mobile-CRISPRi knockdown strains and testing their phenotypes in a mouse pneumonia model of P. aeruginosa infection. In addition, we provide comprehensive guide RNA designs to target genes in common laboratory strains of P. aeruginosa and other Pseudomonas species.},
}
@article {pmid37806043,
year = {2023},
author = {Wei, B and Wang, J and Dai, L and Chen, B and Zhang, K},
title = {Characterization of synthetic guide ribonucleic acids through hydrophilic interaction chromatography coupled with mass spectrometry.},
journal = {Journal of chromatography. A},
volume = {1710},
number = {},
pages = {464414},
doi = {10.1016/j.chroma.2023.464414},
pmid = {37806043},
issn = {1873-3778},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Mass Spectrometry ; Chromatography, Liquid/methods ; *Oligonucleotides/analysis ; Hydrophobic and Hydrophilic Interactions ; },
abstract = {In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.},
}
@article {pmid37803452,
year = {2023},
author = {Guo, T and Chen, GQ and Li, XF and Wang, M and Liu, KM and Yang, XY and Liu, SC and Feng, YL and Liu, PY and Lin, H and Xie, AY},
title = {Small extrachromosomal circular DNA harboring targeted tumor suppressor gene mutations supports intratumor heterogeneity in mouse liver cancer induced by multiplexed CRISPR/Cas9.},
journal = {Genome medicine},
volume = {15},
number = {1},
pages = {80},
pmid = {37803452},
issn = {1756-994X},
support = {2015C03047//Department of Science and Technology of Zhejiang Province/ ; 31870806//National Natural Science Foundation of China/ ; 32071439//National Natural Science Foundation of China/ ; 202204A05//Department of Science and Technology of Hangzhou/ ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Liver Neoplasms/genetics ; Gene Editing ; Mutation ; Genes, Tumor Suppressor ; DNA ; Genomic Instability ; DNA, Circular ; },
abstract = {BACKGROUND: Primary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer.<br><br>METHODS: CRISPR/Cas9-based targeted somatic multiplex-mutagenesis was used to target 34 tumor suppressor genes (TSGs) for induction of primary liver tumors in mice. Target site mutations in tumor cells were analyzed and compared between single-cell clones and their subclones, between different time points of cell proliferation, and between parental clones and single-cell clones derived from mouse subcutaneous allografts. Genomic instability and generation of extrachromosomal circular DNA (eccDNA) was explored as a potential mechanism underlying the oscillation of target site mutations in these liver tumor cells.<br><br>RESULTS: After efficiently inducing autochthonous liver tumors in mice within 30-60&#xa0;days, analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of IGH in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of eccDNA harboring targeted TSG mutations in these tumor cells.<br><br>CONCLUSIONS: Small eccDNAs carrying TSG mutations may serve as an important source supporting intratumor heterogeneity and tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.},
}
@article {pmid37802026,
year = {2023},
author = {Newton, MD and Losito, M and Smith, QM and Parnandi, N and Taylor, BJ and Akcakaya, P and Maresca, M and van Eijk, P and Reed, SH and Boulton, SJ and King, GA and Cuomo, ME and Rueda, DS},
title = {Negative DNA supercoiling induces genome-wide Cas9 off-target activity.},
journal = {Molecular cell},
volume = {83},
number = {19},
pages = {3533-3545.e5},
doi = {10.1016/j.molcel.2023.09.008},
pmid = {37802026},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome ; DNA/genetics ; Optical Tweezers ; },
abstract = {CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.},
}
@article {pmid37798384,
year = {2023},
author = {An, Y and Talwar, CS and Park, KH and Ahn, WC and Lee, SJ and Go, SR and Cho, JH and Kim, DY and Kim, YS and Cho, S and Kim, JH and Kim, TJ and Woo, EJ},
title = {Design of hypoxia responsive CRISPR-Cas9 for target gene regulation.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {16763},
pmid = {37798384},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; CRISPR-Associated Protein 9/genetics ; Gene Editing ; Hypoxia/genetics ; *Neoplasms/genetics ; },
abstract = {The CRISPR-Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR-Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.},
}
@article {pmid37794590,
year = {2023},
author = {Tsuchida, CA and Brandes, N and Bueno, R and Trinidad, M and Mazumder, T and Yu, B and Hwang, B and Chang, C and Liu, J and Sun, Y and Hopkins, CR and Parker, KR and Qi, Y and Hofman, L and Satpathy, AT and Stadtmauer, EA and Cate, JHD and Eyquem, J and Fraietta, JA and June, CH and Chang, HY and Ye, CJ and Doudna, JA},
title = {Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.},
journal = {Cell},
volume = {186},
number = {21},
pages = {4567-4582.e20},
doi = {10.1016/j.cell.2023.08.041},
pmid = {37794590},
issn = {1097-4172},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Gene Editing/methods ; DNA Damage ; Chromosomes ; },
abstract = {CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.},
}
@article {pmid37494719,
year = {2023},
author = {Miao, R and Jahn, M and Shabestary, K and Peltier, G and Hudson, EP},
title = {CRISPR interference screens reveal growth-robustness tradeoffs in Synechocystis sp. PCC 6803 across growth conditions.},
journal = {The Plant cell},
volume = {35},
number = {11},
pages = {3937-3956},
pmid = {37494719},
issn = {1532-298X},
support = {NNF20OC0061469//Novo Nordisk Foundation/ ; 2020-04329//Swedish Research Council/ ; SSF ARC19-0051//Swedish Foundation for Strategic Research/ ; 2015-939//Swedish Research Council Formas/ ; 1789//Federation of European Microbiological Societies/ ; },
mesh = {*Synechocystis/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Photosynthesis/genetics ; Gene Expression ; Light ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and noncoding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in 1 condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive data set assigns condition-specific importance to many previously unannotated genes and suggests additional functions for central metabolic enzymes. Phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, and the small protein CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion proximal to the PAM site. Fitness data for all genes in all conditions are compiled in an interactive web application.},
}
@article {pmid37894253,
year = {2023},
author = {Bano, S and Hassan, N and Rafiq, M and Hassan, F and Rehman, M and Iqbal, N and Ali, H and Hasan, F and Kang, YQ},
title = {Biofilms as Battlefield Armor for Bacteria against Antibiotics: Challenges and Combating Strategies.},
journal = {Microorganisms},
volume = {11},
number = {10},
pages = {},
pmid = {37894253},
issn = {2076-2607},
abstract = {Bacterial biofilms are formed by communities, which are encased in a matrix of extracellular polymeric substances (EPS). Notably, bacteria in biofilms display a set of 'emergent properties' that vary considerably from free-living bacterial cells. Biofilms help bacteria to survive under multiple stressful conditions such as providing immunity against antibiotics. Apart from the provision of multi-layered defense for enabling poor antibiotic absorption and adaptive persistor cells, biofilms utilize their extracellular components, e.g., extracellular DNA (eDNA), chemical-like catalase, various genes and their regulators to combat antibiotics. The response of biofilms depends on the type of antibiotic that comes into contact with biofilms. For example, excessive production of eDNA exerts resistance against cell wall and DNA targeting antibiotics and the release of antagonist chemicals neutralizes cell membrane inhibitors, whereas the induction of protein and folic acid antibiotics inside cells is lowered by mutating genes and their regulators. Here, we review the current state of knowledge of biofilm-based resistance to various antibiotic classes in bacteria and genes responsible for biofilm development, and the key role of quorum sensing in developing biofilms and antibiotic resistance is also discussed. In this review, we also highlight new and modified techniques such as CRISPR/Cas, nanotechnology and bacteriophage therapy. These technologies might be useful to eliminate pathogens residing in biofilms by combating biofilm-induced antibiotic resistance and making this world free of antibiotic resistance.},
}
@article {pmid37894094,
year = {2023},
author = {Chen, JHK and Leung, HY and Wong, CMC and Yuen, KY and Cheng, VCC},
title = {Prevalence and Characteristics of Invasive Staphylococcus argenteus among Patients with Bacteremia in Hong Kong.},
journal = {Microorganisms},
volume = {11},
number = {10},
pages = {},
pmid = {37894094},
issn = {2076-2607},
support = {CID-HKU1-16//Health and Medical Research Fund, , Health Bureau, Hong Kong SAR Government/ ; },
abstract = {Staphylococcus argenteus is a novel Staphylococcus species derived from Staphylococcus aureus. Information on the prevalence and genetic characteristics of invasive S. argenteus in Asia is limited. In this study, 275 invasive S. aureus complex strains were retrieved from blood culture specimens in Hong Kong and re-analyzed using MALDI-TOF mass spectrometry and an in-house multiplex real-time PCR for S. argenteus. The prevalence of invasive S. argenteus in Hong Kong was found to be 4.0% (11/275). These strains were primarily susceptible to commonly used antibiotics, except penicillin. Whole-genome sequencing revealed the circulation of three S. argenteus genotypes (ST-2250, ST-1223, and ST-2854) in Hong Kong, with ST-2250 and ST-1223 being the predominant genotypes. The local ST-2250 and ST-1223 strains showed close phylogenetic relationships with isolates from mainland China. Antimicrobial-resistant genes (fosB, tet-38, mepA, blaI, blaZ) could be found in nearly all local S. argenteus strains. The ST-1223 and ST-2250 genotypes carried multiple staphylococcal enterotoxin genes that could cause food poisoning and toxic shock syndrome. The CRISPR/Cas locus was observed only in the ST-2250 strains. This study provides the first report on the molecular epidemiology of invasive S. argenteus in Hong Kong, and further analysis is needed to understand its transmission reservoir.},
}
@article {pmid37864194,
year = {2023},
author = {Wang, M and Ding, F and Wang, H and Li, L and Dai, Y and Sun, Z and Li, N},
title = {Versatile generation of precise gene edits in bovines using SEGCPN.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {226},
pmid = {37864194},
issn = {1741-7007},
mesh = {Female ; Animals ; Cattle/genetics ; Male ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Gene Targeting/methods ; Gene Knockout Techniques ; Point Mutation ; },
abstract = {BACKGROUND: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals.<br><br>RESULTS: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb &#x3b1;-casein gene was replaced with a 2.6-kb human &#x3b1;-lactalbumin gene.<br><br>CONCLUSIONS: In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.},
}
@article {pmid37862369,
year = {2023},
author = {Kelishadi, M and Shahsavarani, H and Tabarraei, A and Shokrgozar, MA and Teimoori-Toolabi, L and Azadmanesh, K},
title = {The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53.},
journal = {PloS one},
volume = {18},
number = {10},
pages = {e0286231},
pmid = {37862369},
issn = {1932-6203},
mesh = {Animals ; *Herpesvirus 1, Human/genetics/metabolism ; CRISPR-Cas Systems ; Chickens/metabolism ; Tumor Suppressor Protein p53/genetics/metabolism ; Chorioallantoic Membrane/metabolism ; *Neoplasms/genetics/therapy ; *Oncolytic Viruses/genetics ; },
abstract = {Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.},
}
@article {pmid37856015,
year = {2024},
author = {Zaragosi, LE and Gouleau, A and Delin, M and Lebrigand, K and Arguel, MJ and Girard-Riboulleau, C and Rios, G and Redman, E and Plaisant, M and Waldmann, R and Magnone, V and Marcet, B and Barbry, P and Ponzio, G},
title = {Combination of CRISPR-Cas9-RNP and Single-Cell RNAseq to Identify Cell State-Specific FOXJ1 Functions in the Human Airway Epithelium.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2725},
number = {},
pages = {1-25},
pmid = {37856015},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Ecosystem ; RNA, Guide, CRISPR-Cas Systems ; Single-Cell Gene Expression Analysis ; Epithelial Cells ; Epithelium/metabolism ; Bronchi ; Forkhead Transcription Factors/genetics/metabolism ; },
abstract = {The study of the airway epithelium in vitro is routinely performed using air-liquid culture (ALI) models from nasal or bronchial basal cells. These 3D experimental models allow to follow the regeneration steps of fully differentiated mucociliary epithelium and to study gene function by performing gene invalidation. Recent progress made with CRISPR-based techniques has overcome the experimental difficulty of this approach, by a direct transfection of ribonucleoprotein complexes combining a mix of synthetic small guide RNAs (sgRNAs) and recombinant Cas9. The approach shows more than 95% efficiency and does not require any selection step. A limitation of this approach is that it generates cell populations that contain heterogeneous deletions, which makes the evaluation of invalidation efficiency difficult. We have successfully used Flongle sequencing (Nanopore) to quantify the number of distinct deletions. We describe the use of CRISPR-Cas9 RNP in combination with single-cell RNA sequencing to functionally characterize the impact of gene invalidation in ALI cultures. The complex ecosystem of the airway epithelium, composed of many cell types, makes single-cell approaches particularly relevant to study cell type, or cell state-specific events. This protocol describes the invalidation of FOXJ1 in ALI cultures through the following steps: (1) Establishment of basal cell cultures from nasal turbinates, (2) CRISPR-Cas9 RNP invalidation of FOXJ1, (3) Quantification of FOXJ1 invalidation efficiency by Nanopore sequencing, (4) Dissociation of ALI cultures and single-cell RNAseq, (5) Analysis of single-cell RNAseq data from FOXJ1-invalidated cells.We confirm here that FOXJ1 invalidation impairs the final differentiation step of multiciliated cells and provides a framework to explore other gene functions.},
}
@article {pmid37497643,
year = {2023},
author = {Samach, A and Mafessoni, F and Gross, O and Melamed-Bessudo, C and Filler-Hayut, S and Dahan-Meir, T and Amsellem, Z and Pawlowski, WP and Levy, AA},
title = {CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.},
journal = {The Plant cell},
volume = {35},
number = {11},
pages = {3957-3972},
pmid = {37497643},
issn = {1532-298X},
support = {1027/14//Israel Science Foundation/ ; 2016//Minerva Foundation/ ; FR-39-2020//BARD/ ; },
mesh = {*Chromothripsis ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Homologous Recombination ; DNA End-Joining Repair ; },
abstract = {DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.},
}
@article {pmid36097701,
year = {2023},
author = {Miao, J and Lan, T and Guo, H and Wang, J and Zhang, G and Wang, Z and Yang, P and Li, H and Zhang, C and Wang, Y and Li, XM and Miao, M},
title = {Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system.},
journal = {Animal models and experimental medicine},
volume = {6},
number = {5},
pages = {489-498},
pmid = {36097701},
issn = {2576-2095},
support = {202300410259//Natural Science Foundation of Henan Province/ ; 202001043//Henan Postdoctoral Science Foundation/ ; 2021T140184//China Postdoctoral Science Foundation/ ; },
mesh = {Animals ; Cricetinae ; *NF-kappa B/metabolism ; Mesocricetus ; *CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; },
abstract = {BACKGROUND: SHARPIN (SHANK-associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF-κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system.<br><br>METHODS: A single-guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping. SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase-quantitative polymerase chain reaction.<br><br>RESULTS: All the offspring harbored germline-transmitted SHARPIN mutations. Compared with wild-type hamsters, SHARPIN protein was undetectable in SHARPIN[-/-] hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN[-/-] hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF-&#x3ba;B and phosphorylation of NF-&#x3ba;B and I&#x3ba;B protein significantly diminished in SHARPIN[-/-] animals.<br><br>CONCLUSIONS: A novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.},
}
@article {pmid37894063,
year = {2023},
author = {Rabaan, AA and Al Fares, MA and Almaghaslah, M and Alpakistany, T and Al Kaabi, NA and Alshamrani, SA and Alshehri, AA and Almazni, IA and Saif, A and Hakami, AR and Khamis, F and Alfaresi, M and Alsalem, Z and Alsoliabi, ZA and Al Amri, KAS and Hassoueh, AK and Mohapatra, RK and Arteaga-Livias, K and Alissa, M},
title = {Application of CRISPR-Cas System to Mitigate Superbug Infections.},
journal = {Microorganisms},
volume = {11},
number = {10},
pages = {},
pmid = {37894063},
issn = {2076-2607},
support = {PSAU/2023/R/1444//Prince Sattam Bin Abdulaziz University/ ; },
abstract = {Multidrug resistance in bacterial strains known as superbugs is estimated to cause fatal infections worldwide. Migration and urbanization have resulted in overcrowding and inadequate sanitation, contributing to a high risk of superbug infections within and between different communities. The CRISPR-Cas system, mainly type II, has been projected as a robust tool to precisely edit drug-resistant bacterial genomes to combat antibiotic-resistant bacterial strains effectively. To entirely opt for its potential, advanced development in the CRISPR-Cas system is needed to reduce toxicity and promote efficacy in gene-editing applications. This might involve base-editing techniques used to produce point mutations. These methods employ designed Cas9 variations, such as the adenine base editor (ABE) and the cytidine base editor (CBE), to directly edit single base pairs without causing DSBs. The CBE and ABE could change a target base pair into a different one (for example, G-C to A-T or C-G to A-T). In this review, we addressed the limitations of the CRISPR/Cas system and explored strategies for circumventing these limitations by applying diverse base-editing techniques. Furthermore, we also discussed recent research showcasing the ability of base editors to eliminate drug-resistant microbes.},
}
@article {pmid37892243,
year = {2023},
author = {Chen, BC and Chen, YZ and Lin, HY},
title = {An Introduced RNA-Only Approach for Plasmid Curing via the CRISPR-Cpf1 System in Saccharomyces cerevisiae.},
journal = {Biomolecules},
volume = {13},
number = {10},
pages = {},
pmid = {37892243},
issn = {2218-273X},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *RNA/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Plasmids/genetics ; },
abstract = {The CRISPR-Cas system has been widely used for genome editing due to its convenience, simplicity and flexibility. Using a plasmid-carrying Cas protein and crRNA or sgRNA expression cassettes is an efficient strategy in the CRISPR-Cas genome editing system. However, the plasmid remains in the cells after genome editing. Development of general plasmid-curing strategies is necessary. Based on our previous CRISPR-Cpf1 genome-editing system in Saccharomyces cerevisiae, the crRNA, designed for the replication origin of the CRISPR-Cpf1 plasmid, and the ssDNA, as a template for homologous recombination, were introduced for plasmid curing. The efficiency of the plasmid curing was 96 ± 4%. In addition, we further simplified the plasmid curing system by transforming only one crRNA into S. cerevisiae, and the curing efficiency was about 70%. In summary, we have developed a CRISPR-mediated plasmid-curing system. The RNA-only plasmid curing system is fast and easy. This plasmid curing strategy can be applied in broad hosts by designing crRNA specific for the replication origin of the plasmid. The plasmid curing system via CRISPR-Cas editing technology can be applied to produce traceless products without foreign genes and to perform iterative processes in multiple rounds of genome editing.},
}
@article {pmid37891676,
year = {2023},
author = {Ramezannia, Z and Shamekh, A and Bannazadeh Baghi, H},
title = {CRISPR-Cas system to discover host-virus interactions in Flaviviridae.},
journal = {Virology journal},
volume = {20},
number = {1},
pages = {247},
pmid = {37891676},
issn = {1743-422X},
mesh = {Humans ; CRISPR-Cas Systems ; Host Microbial Interactions ; *Flaviviridae/genetics ; Gene Editing ; *Viruses/genetics ; },
abstract = {The Flaviviridae virus family members cause severe human diseases and are responsible for considerable mortality and morbidity worldwide. Therefore, researchers have conducted genetic screens to enhance insight into viral dependency and develop potential anti-viral strategies to treat and prevent these infections. The host factors identified by the clustered regularly interspaced short palindromic repeats (CRISPR) system can be potential targets for drug development. Meanwhile, CRISPR technology can be efficiently used to treat viral diseases as it targets both DNA and RNA. This paper discusses the host factors related to the life cycle of viruses of this family that were recently discovered using the CRISPR system. It also explores the role of immune factors and recent advances in gene editing in treating flavivirus-related diseases. The ever-increasing advancements of this technology may promise new therapeutic approaches with unique capabilities, surpassing the traditional methods of drug production and treatment.},
}
@article {pmid37891182,
year = {2023},
author = {Allen, D and Knop, O and Itkowitz, B and Kalter, N and Rosenberg, M and Iancu, O and Beider, K and Lee, YN and Nagler, A and Somech, R and Hendel, A},
title = {CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6771},
pmid = {37891182},
issn = {2041-1723},
support = {755758//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 3115/19//Israel Science Foundation (ISF)/ ; 2031/19//Israel Science Foundation (ISF)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Hematopoietic Stem Cells/metabolism ; Gene Editing/methods ; Gene Expression Regulation ; *Hematopoietic Stem Cell Transplantation ; DNA-Binding Proteins/metabolism ; Nuclear Proteins/metabolism ; },
abstract = {RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient's own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3[+]TCRαβ[+] and CD3[+]TCRγδ[+] T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.},
}
@article {pmid37889074,
year = {2023},
author = {Yan, Y and Zheng, J and Zhang, X and Yin, Y},
title = {dbAPIS: a database of anti-prokaryotic immune system genes.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad932},
pmid = {37889074},
issn = {1362-4962},
support = {R21AI171952/NH/NIH HHS/United States ; },
abstract = {Anti-prokaryotic immune system (APIS) proteins, typically encoded by phages, prophages, and plasmids, inhibit prokaryotic immune systems (e.g. restriction modification, toxin-antitoxin, CRISPR-Cas). A growing number of APIS genes have been characterized and dispersed in the literature. Here we developed dbAPIS (https://bcb.unl.edu/dbAPIS), as the first literature curated data repository for experimentally verified APIS genes and their associated protein families. The key features of dbAPIS include: (i) experimentally verified APIS genes with their protein sequences, functional annotation, PDB or AlphaFold predicted structures, genomic context, sequence and structural homologs from different microbiome/virome databases; (ii) classification of APIS proteins into sequence-based families and construction of hidden Markov models (HMMs); (iii) user-friendly web interface for data browsing by the inhibited immune system types or by the hosts, and functions for searching and batch downloading of pre-computed data; (iv) Inclusion of all types of APIS proteins (except for anti-CRISPRs) that inhibit a variety of prokaryotic defense systems (e.g. RM, TA, CBASS, Thoeris, Gabija). The current release of dbAPIS contains 41 verified APIS proteins and ∼4400 sequence homologs of 92 families and 38 clans. dbAPIS will facilitate the discovery of novel anti-defense genes and genomic islands in phages, by providing a user-friendly data repository and a web resource for an easy homology search against known APIS proteins.},
}
@article {pmid37889041,
year = {2023},
author = {Adler, BA and Trinidad, MI and Bellieny-Rabelo, D and Zhang, E and Karp, HM and Skopintsev, P and Thornton, BW and Weissman, RF and Yoon, PH and Chen, L and Hessler, T and Eggers, AR and Colognori, D and Boger, R and Doherty, EE and Tsuchida, CA and Tran, RV and Hofman, L and Shi, H and Wasko, KM and Zhou, Z and Xia, C and Al-Shimary, MJ and Patel, JR and Thomas, VCJX and Pattali, R and Kan, MJ and Vardapetyan, A and Yang, A and Lahiri, A and Maxwell, MF and Murdock, AG and Ramit, GC and Henderson, HR and Calvert, RW and Bamert, RS and Knott, GJ and Lapinaite, A and Pausch, P and Cofsky, JC and Sontheimer, EJ and Wiedenheft, B and Fineran, PC and Brouns, SJJ and Sashital, DG and Thomas, BC and Brown, CT and Goltsman, DSA and Barrangou, R and Siksnys, V and Banfield, JF and Savage, DF and Doudna, JA},
title = {CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad890},
pmid = {37889041},
issn = {1362-4962},
support = {U01AI142817/NH/NIH HHS/United States ; DP2GM149550/NH/NIH HHS/United States ; R35GM134867/NH/NIH HHS/United States ; 101003229/ERC_/European Research Council/International ; GM140876/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
abstract = {CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.},
}
@article {pmid37888561,
year = {2023},
author = {Flores-Contreras, EA and Carrasco-González, JA and Linhares, DCL and Corzo, CA and Campos-Villalobos, JI and Henao-Díaz, A and Melchor-Martínez, EM and Iqbal, HMN and González-González, RB and Parra-Saldívar, R and González-González, E},
title = {Emergent Molecular Techniques Applied to the Detection of Porcine Viruses.},
journal = {Veterinary sciences},
volume = {10},
number = {10},
pages = {},
pmid = {37888561},
issn = {2306-7381},
abstract = {Molecular diagnostic tests have evolved very rapidly in the field of human health, especially with the arrival of the recent pandemic caused by the SARS-CoV-2 virus. However, the animal sector is constantly neglected, even though accurate detection by molecular tools could represent economic advantages by preventing the spread of viruses. In this regard, the swine industry is of great interest. The main viruses that affect the swine industry are described in this review, including African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and porcine circovirus (PCV), which have been effectively detected by different molecular tools in recent times. Here, we describe the rationale of molecular techniques such as multiplex PCR, isothermal methods (LAMP, NASBA, RPA, and PSR) and novel methods such as CRISPR-Cas and microfluidics platforms. Successful molecular diagnostic developments are presented by highlighting their most important findings. Finally, we describe the barriers that hinder the large-scale development of affordable, accessible, rapid, and easy-to-use molecular diagnostic tests. The evolution of diagnostic techniques is critical to prevent the spread of viruses and the development of viral reservoirs in the swine industry that impact the possible development of future pandemics and the world economy.},
}
@article {pmid37887310,
year = {2023},
author = {Preta, G},
title = {Development of New Genome Editing Tools for the Treatment of Hyperlipidemia.},
journal = {Cells},
volume = {12},
number = {20},
pages = {},
pmid = {37887310},
issn = {2073-4409},
support = {01.2.2-CPVA-V-716-01-0001//European Regional Development Fund/ ; },
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; *Hyperlipidemias/genetics/therapy ; *Dyslipidemias/genetics ; Genetic Therapy/adverse effects ; },
abstract = {Hyperlipidemia is a medical condition characterized by high levels of lipids in the blood. It is often associated with an increased risk of cardiovascular diseases such as heart attacks and strokes. Traditional treatment approaches for hyperlipidemia involve lifestyle modifications, dietary changes, and the use of medications like statins. Recent advancements in genome editing technologies, including CRISPR-Cas9, have opened up new possibilities for the treatment of this condition. This review provides a general overview of the main target genes involved in lipid metabolism and highlights the progress made during recent years towards the development of new treatments for dyslipidemia.},
}
@article {pmid37887288,
year = {2023},
author = {Geoffroy, M and Pili, L and Buffa, V and Caroff, M and Bigot, A and Gicquel, E and Rouby, G and Richard, I and Fragnoud, R},
title = {CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy.},
journal = {Cells},
volume = {12},
number = {20},
pages = {},
pmid = {37887288},
issn = {2073-4409},
mesh = {Animals ; Humans ; *Pentosyltransferases/genetics ; Muscle, Skeletal/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Dystroglycans/metabolism ; *Muscular Dystrophies, Limb-Girdle/metabolism ; Cell Line ; Genetic Therapy/methods ; },
abstract = {Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products.},
}
@article {pmid37884905,
year = {2023},
author = {Cheng, Y and Li, Y and Yang, J and He, H and Zhang, X and Liu, J and Yang, X},
title = {Multiplex CRISPR-Cas9 knockout of EIL3, EIL4, and EIN2L advances soybean flowering time and pod set.},
journal = {BMC plant biology},
volume = {23},
number = {1},
pages = {519},
pmid = {37884905},
issn = {1471-2229},
support = {No. 32172079//National Natural Science Foundation of China/ ; No. 20200201113JC//Department of Science and Technology of Jilin Province/ ; },
mesh = {*Soybeans/metabolism ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Ethylenes/metabolism ; },
abstract = {BACKGROUND: Ethylene inhibitor treatment of soybean promotes flower bud differentiation and early flowering, suggested that there is a close relationship between ethylene signaling and soybean growth and development. The short-lived ETHYLENE INSENSITIVE2 (EIN2) and ETHYLENE INSENSITIVE3 (EIN3) proteins play central roles in plant development. The objective of this study was carried out gene editing of EIL family members in soybeans and to examine the effects on soybean yield and other markers of growth.<br><br>METHODS AND RESULTS: By editing key-node genes in the ethylene signaling pathway using a multi-sgRNA-in-one strategy, we obtained a series of gene edited lines with variable edit combinations among 15 target genes. EIL3, EIL4, and EIN2L were editable genes favored by the T0 soybean lines. Pot experiments also show that the early flowering stage R1 of the EIL3, EIL4, and EIN2L triple mutant was 7.05 d earlier than that of the wild-type control. The yield of the triple mutant was also increased, being 1.65-fold higher than that of the control. Comparative RNA-seq revealed that sucrose synthase, AUX28, MADS3, type-III polyketide synthase A/B, ABC transporter G family member 26, tetraketide alpha-pyrone reductase, and fatty acyl-CoA reductase 2 may be involved in regulating early flowering and high-yield phenotypes in triple mutant soybean plants.<br><br>CONCLUSION: Our results provide a scientific basis for genetic modification to promote the development of earlier-flowering and higher-yielding soybean cultivars.},
}
@article {pmid37883564,
year = {2023},
author = {Tang, S and Sternberg, SH},
title = {Genome editing with retroelements.},
journal = {Science (New York, N.Y.)},
volume = {382},
number = {6669},
pages = {370-371},
doi = {10.1126/science.adi3183},
pmid = {37883564},
issn = {1095-9203},
mesh = {*Gene Editing ; *Retroelements ; RNA ; CRISPR-Cas Systems ; },
abstract = {RNA-guided DNA writing enzymes offer promise for programmable gene insertion.},
}
@article {pmid37875911,
year = {2023},
author = {Meyers, S and Demeyer, S and Cools, J},
title = {CRISPR screening in hematology research: from bulk to single-cell level.},
journal = {Journal of hematology &amp; oncology},
volume = {16},
number = {1},
pages = {107},
pmid = {37875911},
issn = {1756-8722},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Testing ; },
abstract = {The CRISPR genome editing technology has revolutionized the way gene function is studied. Genome editing can be achieved in single genes or for thousands of genes simultaneously in sensitive genetic screens. While conventional genetic screens are limited to bulk measurements of cell behavior, recent developments in single-cell technologies make it possible to combine CRISPR screening with single-cell profiling. In this way, cell behavior and gene expression can be monitored simultaneously, with the additional possibility of including data on chromatin accessibility and protein levels. Moreover, the availability of various Cas proteins leading to inactivation, activation, or other effects on gene function further broadens the scope of such screens. The integration of single-cell multi-omics approaches with CRISPR screening open the path to high-content information on the impact of genetic perturbations at single-cell resolution. Current limitations in cell throughput and data density need to be taken into consideration, but new technologies are rapidly evolving and are likely to easily overcome these limitations. In this review, we discuss the use of bulk CRISPR screening in hematology research, as well as the emergence of single-cell CRISPR screening and its added value to the field.},
}
@article {pmid37864134,
year = {2023},
author = {Wang, KT and Adler, CE},
title = {CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians.},
journal = {BMC genomics},
volume = {24},
number = {1},
pages = {625},
pmid = {37864134},
issn = {1471-2164},
support = {R01 GM139933/GM/NIGMS NIH HHS/United States ; GM139933/NH/NIH HHS/United States ; },
mesh = {Animals ; RNA, Ribosomal, 16S/genetics ; *Planarians/genetics ; CRISPR-Cas Systems ; Sequence Analysis, RNA/methods ; RNA/genetics ; RNA, Messenger/genetics ; Single-Cell Analysis ; Gene Expression Profiling/methods ; RNA, Ribosomal/genetics ; },
abstract = {BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) relies on PCR amplification to retrieve information from vanishingly small amounts of starting material. To selectively enrich mRNA from abundant non-polyadenylated transcripts, poly(A) selection is a key step during library preparation. However, some transcripts, such as mitochondrial genes, can escape this elimination and overwhelm libraries. Often, these transcripts are removed in silico, but whether physical depletion improves detection of rare transcripts in single cells is unclear.<br><br>RESULTS: We find that a single 16S ribosomal RNA is widely enriched in planarian scRNA-seq datasets, independent of the library preparation method. To deplete this transcript from scRNA-seq libraries, we design 30 single-guide RNAs spanning its length. To evaluate the effects of depletion, we perform a side-by-side comparison of the effects of eliminating the 16S transcript and find a substantial increase in the number of genes detected per cell, coupled with virtually complete loss of the 16S RNA. Moreover, we systematically determine that library complexity increases with a limited number of PCR cycles following CRISPR treatment. When compared to in silico depletion of 16S, physically removing it reduces dropout rates, retrieves more clusters, and reveals more differentially expressed genes.<br><br>CONCLUSIONS: Our results show that abundant transcripts reduce the retrieval of informative transcripts in scRNA-seq and distort the analysis. Physical removal of these contaminants enables the detection of rare transcripts at lower sequencing depth, and also outperforms in silico depletion. Importantly, this method can be easily customized to deplete any abundant transcript from scRNA-seq libraries.},
}
@article {pmid37848032,
year = {2023},
author = {Zhao, F and Hu, Y and Fan, Z and Huang, B and Wei, L and Xie, Y and Huang, Y and Mei, S and Wang, L and Wang, L and Ai, B and Fang, J and Liang, C and Xu, F and Tan, W and Guo, F},
title = {Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay.},
journal = {Cell reports methods},
volume = {3},
number = {10},
pages = {100620},
doi = {10.1016/j.crmeth.2023.100620},
pmid = {37848032},
issn = {2667-2375},
mesh = {Humans ; Recombinases/genetics ; CRISPR-Cas Systems/genetics ; Monkeypox virus ; DNA, Viral/genetics ; *Monkeypox ; Nucleotidyltransferases ; *Orthopoxvirus ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Mpox is caused by a zoonotic virus belonging to the Orthopoxvirus genus and the Poxviridae family. In this study, we develop a recombinase polymerase amplification (RPA)-coupled CRISPR-Cas12a detection assay for the mpox virus. We design and test a series of CRISPR-derived RNAs(crRNAs) targeting the conserved D6R and E9L genes for orthopoxvirus and the unique N3R and N4R genes for mpox viruses. D6R crRNA-1 exhibits the most robust activity in detecting orthopoxviruses, and N4R crRNA-2 is able to distinguish the mpox virus from other orthopoxviruses. The Cas12a/crRNA assay alone presents a detection limit of 10[8] copies of viral DNA, whereas coupling RPA increases the detection limit to 1-10 copies. The one-tube RPA-Cas12a assay can, therefore, detect viral DNA as low as 1 copy within 30 min and holds the promise of providing point-of-care detection for mpox viral infection.},
}
@article {pmid37832631,
year = {2023},
author = {Sahu, S and Poplawska, M and Lim, SH and Dutta, D},
title = {CRISPR-based precision medicine for hematologic disorders: Advancements, challenges, and prospects.},
journal = {Life sciences},
volume = {333},
number = {},
pages = {122165},
doi = {10.1016/j.lfs.2023.122165},
pmid = {37832631},
issn = {1879-0631},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Precision Medicine ; Gene Editing ; Genome ; *Hematologic Diseases/genetics/therapy ; Endonucleases/genetics ; },
abstract = {The development of programmable nucleases to introduce defined alterations in genomic sequences has been a powerful tool for precision medicine. While several nucleases such as zinc-finger nucleases (ZFN), transcriptor activator-like effector nucleases (TALEN), and meganucleases have been explored, the advent of CRISPR/Cas9 technology has revolutionized the field of genome engineering. In addition to disease modeling, the CRISPR/Cas9 technology has contributed to safer and more effective treatment strategies for hematologic diseases and personalized T-cell-based therapies. Here we discuss the applications of the CRISPR technology in the treatment of hematologic diseases, their efficacy, and ongoing clinical trials. We examine the obstacles to their successful use and the approaches investigated to overcome these challenges. Finally, we provide our perspectives to improve this genome editing tool for targeted therapies.},
}
@article {pmid37824834,
year = {2023},
author = {Zhang, P and Abel, L and Casanova, JL and Yang, R},
title = {Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An Experimental and Computational Approach for Analyzing CRISPR-Edited Cells.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {462-472},
pmid = {37824834},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genotype ; CRISPR-Associated Protein 9/genetics ; Genome ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) creates double-stranded breaks, the repair of which generates indels around the target sites. These repairs can be mono-/multi-allelic, and the editing is often random and sometimes prolonged, resulting in considerable intercellular heterogeneity. The genotyping of CRISPR-Cas9-edited cells is challenging and the traditional genotyping methods are laborious. We introduce here a streamlined experimental and computational protocol for genotyping CRISPR-Cas9 genome-edited cells including cost-effective multiplexed sequencing and the software Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE). In this approach, CRISPR-Cas9-edited products are sequenced in great depth, then GMUSCLE quantitatively and qualitatively identifies the genotypes, which enable the selection and investigation of cell clones with genotypes of interest. We validate the protocol and software by performing CRISPR-Cas9-mediated disruption on interferon-α/β receptor alpha, multiplexed sequencing, and identifying the genotypes simultaneously for 20 cell clones. Besides the multiplexed sequencing ability of this protocol, GMUSCLE is also applicable for the sequencing data from bulk cell populations. GMUSCLE is publicly available at our HGIDSOFT server and GitHub.},
}
@article {pmid37757856,
year = {2023},
author = {Zhao, R and Luo, W and Wu, Y and Zhang, L and Liu, X and Li, J and Yang, Y and Wang, L and Wang, L and Han, X and Wang, Z and Zhang, J and Lv, K and Chen, T and Xie, G},
title = {Unmodificated stepless regulation of CRISPR/Cas12a multi-performance.},
journal = {Nucleic acids research},
volume = {51},
number = {19},
pages = {10795-10807},
pmid = {37757856},
issn = {1362-4962},
support = {2022YFC2603800//National Key R&amp;D Program of China/ ; 82172369//National Natural Science Foundation of China/ ; cstc2021jcyj-msxmX0326//Natural Science Foundation of Chongqing/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Endonucleases/genetics ; RNA/genetics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.},
}
@article {pmid37747760,
year = {2023},
author = {McQuarrie, S and Athukoralage, JS and McMahon, SA and Graham, S and Ackermann, K and Bode, BE and White, MF and Gloster, TM},
title = {Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open.},
journal = {Nucleic acids research},
volume = {51},
number = {19},
pages = {10590-10605},
doi = {10.1093/nar/gkad739},
pmid = {37747760},
issn = {1362-4962},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 101018608/ERC_/European Research Council/International ; },
mesh = {*Ribonucleases/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Second Messenger Systems ; Catalytic Domain ; Nucleotides, Cyclic ; CRISPR-Cas Systems ; },
abstract = {Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.},
}
@article {pmid37738140,
year = {2023},
author = {Noshay, JM and Walker, T and Alexander, WG and Klingeman, DM and Romero, J and Walker, AM and Prates, E and Eckert, C and Irle, S and Kainer, D and Jacobson, DA},
title = {Quantum biological insights into CRISPR-Cas9 sgRNA efficiency from explainable-AI driven feature engineering.},
journal = {Nucleic acids research},
volume = {51},
number = {19},
pages = {10147-10161},
pmid = {37738140},
issn = {1362-4962},
support = {FWP ERKPA17//Biological and Environmental Research/ ; //Office of Biological and Environmental Research in the DOE Office of Science/ ; DE-AC05-00OR45678//U.S. Department of Energy/ ; FWP ERKP123//U.S. Department of Energy, Office of Science, through the Genomic Science Program, Office of Biological and Environmental Research/ ; DE-AC05-00OR22725//Office of Science of the U.S. Department of Energy/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Artificial Intelligence ; Escherichia coli/genetics ; DNA ; Gene Editing ; },
abstract = {CRISPR-Cas9 tools have transformed genetic manipulation capabilities in the laboratory. Empirical rules-of-thumb have been developed for only a narrow range of model organisms, and mechanistic underpinnings for sgRNA efficiency remain poorly understood. This work establishes a novel feature set and new public resource, produced with quantum chemical tensors, for interpreting and predicting sgRNA efficiency. Feature engineering for sgRNA efficiency is performed using an explainable-artificial intelligence model: iterative Random Forest (iRF). By encoding quantitative attributes of position-specific sequences for Escherichia coli sgRNAs, we identify important traits for sgRNA design in bacterial species. Additionally, we show that expanding positional encoding to quantum descriptors of base-pair, dimer, trimer, and tetramer sequences captures intricate interactions in local and neighboring nucleotides of the target DNA. These features highlight variation in CRISPR-Cas9 sgRNA dynamics between E. coli and H. sapiens genomes. These novel encodings of sgRNAs enhance our understanding of the elaborate quantum biological processes involved in CRISPR-Cas9 machinery.},
}
@article {pmid37718311,
year = {2023},
author = {Liu, X and Zhao, Z and Li, W and Li, Y and Yang, Q and Liu, N and Chen, Y and Yin, L},
title = {Engineering Nucleotidoproteins for Base-Pairing-Assisted Cytosolic Delivery and Genome Editing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {62},
number = {45},
pages = {e202307664},
doi = {10.1002/anie.202307664},
pmid = {37718311},
issn = {1521-3773},
support = {BK20220245//Natural Science Foundation of Jiangsu Province/ ; 82241008//National Natural Science Foundation of China/ ; 52273144//National Natural Science Foundation of China/ ; 52325305//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Base Pairing ; Proteins/genetics ; Endosomes ; Mammals ; },
abstract = {Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.},
}
@article {pmid37676985,
year = {2023},
author = {Molugu, K and Khajanchi, N and Lazzarotto, CR and Tsai, SQ and Saha, K},
title = {Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {473-485},
pmid = {37676985},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; },
abstract = {Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.},
}
@article {pmid37672599,
year = {2023},
author = {Rieffer, AE and Chen, Y and Salamango, DJ and Moraes, SN and Harris, RS},
title = {APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {430-446},
pmid = {37672599},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; Cytidine Deaminase/genetics ; Minor Histocompatibility Antigens/genetics ; },
abstract = {Precision genome editing has become a reality with the discovery of base editors. Cytosine base editor (CBE) technologies are improving rapidly but are mostly optimized for TC dinucleotide targets. Here, we report the development and implementation of APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels (ARSENEL) in living cells. The ARSENEL panel is comprised of four constructs that quantitatively report editing of each of the four dinucleotide motifs (AC/CC/GC/TC) through real-time accumulation of eGFP fluorescence. Editing rates of APOBEC3Bctd and AIDΔC CBEs reflect established mechanistic preferences with intrinsic biases to TC and GC, respectively. Twelve different (new and established) base editors are tested here using this system with a full-length APOBEC3B CBE showing the greatest on-target TC specificity and an APOBEC3A construct showing the highest editing efficiency. In addition, ARSENEL enables real-time assessment of natural and synthetic APOBEC inhibitors with the most potent to-date being the large subunit of the Epstein-Barr virus ribonucleotide reductase. These reporters have the potential to play important roles in research and development as precision genome engineering technologies progress toward achieving maximal specificity and efficiency.},
}
@article {pmid37887134,
year = {2023},
author = {Liu, Y and Xia, W and Zhao, W and Hao, P and Wang, Z and Yu, X and Shentu, X and Sun, K},
title = {RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses.},
journal = {Biosensors},
volume = {13},
number = {10},
pages = {},
pmid = {37887134},
issn = {2079-6374},
support = {2022C02047//Key Research Program of Zhejiang Province of China/ ; 32102165//National Natural Science Foundation of China/ ; LQ22C140003//Zhejiang Provincial Natural Science Foundation/ ; },
abstract = {The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.},
}
@article {pmid37886655,
year = {2023},
author = {Fischer, K and Schnieke, A},
title = {How genome editing changed the world of large animal research.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1272687},
pmid = {37886655},
issn = {2673-3439},
abstract = {The first genetically modified large animals were developed in 1985 by microinjection to increase the growth of agricultural livestock such as pigs. Since then, it has been a difficult trail due to the lack of genetic tools. Although methods and technologies were developed quickly for the main experimental mammal, the mouse, e.g., efficient pronuclear microinjection, gene targeting in embryonic stem cells, and omics data, most of it was-and in part still is-lacking when it comes to livestock. Over the next few decades, progress in genetic engineering of large animals was driven less by research for agriculture but more for biomedical applications, such as the production of pharmaceutical proteins in the milk of sheep, goats, or cows, xeno-organ transplantation, and modeling human diseases. Available technologies determined if a desired animal model could be realized, and efficiencies were generally low. Presented here is a short review of how genome editing tools, specifically CRISPR/Cas, have impacted the large animal field in recent years. Although there will be a focus on genome engineering of pigs for biomedical applications, the general principles and experimental approaches also apply to other livestock species or applications.},
}
@article {pmid37885449,
year = {2023},
author = {Mao, X and Xu, M and Luo, S and Yang, Y and Zhong, J and Zhou, J and Fan, H and Li, X and Chen, Z},
title = {Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1273988},
pmid = {37885449},
issn = {2296-4185},
abstract = {In the realm of pathogen detection, isothermal amplification technology has emerged as a swift, precise, and sensitive alternative to conventional PCR. This paper explores the fundamental principles of recombinase polymerase amplification (RPA) and recombinase-aid amplification (RAA) and reviews the current status of integrating the CRISPR-Cas system with RPA/RAA techniques. Furthermore, this paper explores the confluence of isothermal amplification and CRISPR-Cas technology, providing a comprehensive review and enhancements of existing combined methodologies such as SHERLOCK and DETECTR. We investigate the practical applications of RPA/RAA in conjunction with CRISPR-Cas for pathogen detection, highlighting how this integrated approach significantly advances both research and clinical implementation in the field. This paper aims to provide readers with a concise understanding of the fusion of RPA/RAA and CRISPR-Cas technology, offering insights into their clinical utility, ongoing enhancements, and the promising prospects of this integrated approach in pathogen detection.},
}
@article {pmid37884864,
year = {2023},
author = {Tao, S and Chen, H and Li, N and Fang, Y and Zhang, H and Xu, Y and Chen, L and Liang, W},
title = {Elimination of blaKPC-2-mediated carbapenem resistance in Escherichia coli by CRISPR-Cas9 system.},
journal = {BMC microbiology},
volume = {23},
number = {1},
pages = {310},
pmid = {37884864},
issn = {1471-2180},
support = {2022z2202022//the Project of the key R &amp; D program of 2022 year of Ningbo Science and Technology Bureau/ ; 2023j020//Key Project of Ningbo Municipal Science and Technology Bureau/ ; },
abstract = {OBJECTIVE: The purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the blaKPC-2 gene by curing the blaKPC-2-harboring plasmid of carbapenem-resistant using the CRISPR-Cas9 system.<br><br>METHODS: The single guide RNA (sgRNA) specifically targeted to the blaKPC-2 gene was designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-sgRNA(blaKPC-2) was transformed into Escherichia coli (E.coli) carrying pET24-blaKPC-2. The elimination efficiency in strains was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by E-test strips (bioM&#xe9;rieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.<br><br>RESULTS: In the present study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the blaKPC-2 gene. PCR and qPCR results indicated that prokaryotic CRISPR-Cas9 plasmid transforming drug-resistant bacteria can efficiently clear blaKPC-2-harboring plasmids. In addition, the drug susceptibility test results showed that the bacterial resistance to imipenem was significantly reduced and allowed the resistant model bacteria to restore susceptibility to antibiotics after the blaKPC-2-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system.<br><br>CONCLUSION: In conclusion, our study demonstrated that the one plasmid-mediated CRISPR-Cas9 system can be used as a novel tool to remove resistance plasmids and re-sensitize the recipient bacteria to antibiotics. This strategy provided a great potential to counteract the ever-worsening spread of the blaKPC-2 gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.},
}
@article {pmid37884479,
year = {2023},
author = {Feng, S and Zhang, Y and Gao, Y and Liu, Y and Wang, Y and Han, X and Zhang, T and Song, Y},
title = {A Gene-Editable Palladium-Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202313968},
doi = {10.1002/anie.202313968},
pmid = {37884479},
issn = {1521-3773},
abstract = {Macrophage phagocytosis of tumor cells has emerged as an attractive strategy for tumor therapy. Nevertheless, immunosuppressive M2 macrophages in the tumor microenvironment and the high expression of anti-phagocytic signals from tumor cells impede therapeutic efficacy. To address these issues and improve the management of malignant tumors, in this study we developed a gene-editable palladium-based bioorthogonal nanoplatform, consisting of CRISPR/Cas9 gene editing system-linked Pd nanoclusters, and a hyaluronic acid surface layer (HBPdC). This HBPdC nanoplatform exhibited satisfactory tumor-targeting efficiency and triggered Fenton-like reactions in the tumor microenvironment to generate reactive oxygen species for chemodynamic therapy and macrophage M1 polarization, which directly eliminated tumor cells, and stimulated the antitumor response of macrophages. HBPdC could reprogram tumor cells through gene editing to reduce the expression of CD47 and adipocyte plasma membrane-associated protein, thereby promoting their recognition and phagocytosis by macrophages. Moreover, HBPdC induced the activation of sequestered prodrugs via bioorthogonal catalysis, enabling chemotherapy and thereby enhancing tumor cell death. Importantly, the Pd nanoclusters of HBPdC were sufficiently cleared through basic metabolic pathways, confirming their biocompatibility and biosafety. Therefore, by promoting macrophage phagocytosis, the HBPdC system developed herein, represents a highly promising antitumor toolset for cancer therapy applications.},
}
@article {pmid37880225,
year = {2023},
author = {Liu, J and Wang, FZ and Li, C and Li, Y and Li, JF},
title = {Hidden prevalence of deletion-inversion bi-alleles in CRISPR-mediated deletions of tandemly arrayed genes in plants.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6787},
pmid = {37880225},
issn = {2041-1723},
support = {31900305//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Genes, Plant/genetics ; Alleles ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Homozygote ; Prevalence ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Sequence Deletion ; Mutation ; Chromosome Inversion ; },
abstract = {Tandemly arrayed genes (TAGs) with functional redundancy and chromosomal linkage constitute 14 ~ 35% in sequenced plant genomes. The multiplex CRISPR system is the tool of choice for creating targeted TAG deletions. Here, we show that up to ~80% of CRISPR-mediated TAG knockout alleles in Arabidopsis and rice are deletion-inversion (delinver) bi-alleles, which are easily misidentified as homozygous deletion alleles by routine PCR-based genotyping. This can lead to misinterpretation of experimental data and production of progenies with genetic heterogeneity in an unnoticed manner. In ~2,650 transgenic events, delinver mutation frequencies are predominantly correlated with deletion frequencies but unrelated to chromosomal locations or deletion sizes. Delinver mutations also occur frequently at genomic non-TAG loci during multiplexed CRISPR editing. Our work raises the alarm about delinver mutations as common unwanted products of targeted TAG deletions in plants and helps prevent false interpretation of plant TAG functions due to this hidden genotype issue.},
}
@article {pmid37878144,
year = {2023},
author = {Zeng, S and Lei, S and Qu, C and Wang, Y and Teng, S and Huang, P},
title = {CRISPR/Cas-based gene editing in therapeutic strategies for beta-thalassemia.},
journal = {Human genetics},
volume = {},
number = {},
pages = {},
pmid = {37878144},
issn = {1432-1203},
support = {31771624//National Natural Science Foundation of China/ ; },
abstract = {Beta-thalassemia (β-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of β-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for β-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy. For allogeneic HSCT, human lymphocyte antigen (HLA)-matched donors are rarely available, and acute graft-versus-host disease (GVHD) may occur after the transplantation. Thus, these conventional treatments are facing significant challenges. In recent years, with the advent and advancement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) gene editing technology, precise genome editing has achieved encouraging successes in basic and clinical studies for treating various genetic disorders, including β-thalassemia. Target gene-edited autogeneic HSCT helps patients avoid graft rejection and GVHD, making it a promising curative therapy for transfusion-dependent β-thalassemia (TDT). In this review, we introduce the development and mechanisms of CRISPR/Cas9. Recent advances on feasible strategies of CRISPR/Cas9 targeting three globin genes (HBB, HBG, and HBA) and targeting cell selections for β-thalassemia therapy are highlighted. Current CRISPR-based clinical trials in the treatment of β-thalassemia are summarized, which are focused on γ-globin reactivation and fetal hemoglobin reproduction in hematopoietic stem cells. Lastly, the applications of other promising CRISPR-based technologies, such as base editing and prime editing, in treating β-thalassemia and the limitations of the CRISPR/Cas system in therapeutic applications are discussed.},
}
@article {pmid37877474,
year = {2023},
author = {Peng, R and Chen, X and Xu, F and Hailstone, R and Men, Y and Du, K},
title = {Pneumatic nano-sieve for CRISPR-based detection of drug-resistant bacteria.},
journal = {Nanoscale horizons},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3nh00365e},
pmid = {37877474},
issn = {2055-6764},
abstract = {The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieved an on-chip concentration factor of ∼15-fold for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU mL[-1]. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.},
}
@article {pmid37877401,
year = {2023},
author = {Yang, H and Liu, Z and Lü, W and Wang, F and Zhang, Y},
title = {[Construction and validation of sheep VASA gene knock-in vector based on CRISPR/Cas9 system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {10},
pages = {4219-4233},
doi = {10.13345/j.cjb.220963},
pmid = {37877401},
issn = {1872-2075},
mesh = {Male ; Animals ; Sheep/genetics ; *CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques ; *RNA, Guide, CRISPR-Cas Systems ; Plasmids ; Germ Cells ; },
abstract = {This study aimed to explore the expression changes of VASA gene in sheep testis development and to construct VASA gene knock-in vector to prepare for the study on the differentiation of sheep germ cells in vitro. The testicular tissues of 3-month-old (3M) and 9-month-old (9M) sheep which represent immature and mature stages, respectively, were collected. The differential expression of VASA gene was analyzed by quantitative real-time PCR (qPCR) and Western blotting, and the location of VASA gene was detected by immunohistochemistry. The sgRNA targeting the VASA gene was designed and homologous recombination vectors were constructed by PCR. Subsequently, plasmids were transferred into sheep ear fibroblasts. The VASA gene was activated in combination with CRISPR/dCas9 technology to further verify the efficiency of the vector. The results showed that the expression level of VASA gene increased significantly with the development of sheep testis (P < 0.01), and was mainly located in spermatocytes and round spermatids. The knock-in vector of VASA gene was constructed by CRISPR/Cas9 system, and the Cas9-gRNA vector and pEGFP-PGK puro-VASA vector were transfected into ear fibroblasts. After CRISPR/dCas9 system was activated, ear fibroblasts successfully expressed VASA gene. The results suggest that VASA gene plays a potential function in sheep testicular development and spermatogenesis, and the VASA gene knock-in vector can be constructed in vitro through the CRISPR/Cas9 system. Our results provided effective research tools for further research of germ cell development and differentiation.},
}
@article {pmid37877400,
year = {2023},
author = {Ma, B and Cui, J and Qian, H and Zhang, X and Yang, S and Zhang, Q and Han, Y and Zhang, Z and Wang, J and Xu, K},
title = {[A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {10},
pages = {4204-4218},
doi = {10.13345/j.cjb.220954},
pmid = {37877400},
issn = {1872-2075},
mesh = {Humans ; Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Homologous Recombination ; DNA ; },
abstract = {During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.},
}
@article {pmid37877393,
year = {2023},
author = {Yang, Y and Liu, Y and Chen, M and Li, S and Lu, X and He, Y and Zhang, K and Zou, Q},
title = {[Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {10},
pages = {4098-4107},
doi = {10.13345/j.cjb.230092},
pmid = {37877393},
issn = {1872-2075},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; Sirolimus/pharmacology/metabolism ; Caspase 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Cell Differentiation ; Puromycin/metabolism ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.},
}
@article {pmid37877383,
year = {2023},
author = {Zhao, W and Huang, G and Zhu, X and Bi, Y and Tang, D},
title = {[Application of single base editing technique in pig genetic improvement: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {10},
pages = {3936-3947},
doi = {10.13345/j.cjb.230291},
pmid = {37877383},
issn = {1872-2075},
mesh = {Animals ; Swine/genetics ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Traditional pig breeding has a long cycle and high cost, and there is an urgent need to use new technologies to revitalize the pig breeding industry. The recently emerged CRISPR/Cas9 genome editing technique shows great potential in pig genetic improvement, and has since become a research hotspot. Base editor is a new base editing technology developed based on the CRISPR/Cas9 system, which can achieve targeted mutation of a single base. CRISPR/Cas9 technology is easy to operate and simple to design, but it can lead to DNA double strand breaks, unstable gene structures, and random insertion and deletion of genes, which greatly restricts the application of this technique. Different from CRISPR/Cas9 technique, the single base editing technique does not produce double strand breaks. Therefore, it has higher accuracy and safety for genome editing, and is expected to advance the pig genetic breeding applications. This review summarized the working principle and shortcomings of CRISPR/Cas9 technique, the development and advantages of single base editing, the principles and application characteristics of different base editors and their applications in pig genetic improvement, with the aim to facilitate genome editing-assisted genetic breeding of pig.},
}
@article {pmid37877083,
year = {2023},
author = {Ahmad, A and Jamil, A and Munawar, N},
title = {GMOs or non-GMOs? The CRISPR Conundrum.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1232938},
pmid = {37877083},
issn = {1664-462X},
abstract = {CRISPR-Cas9, the "genetic scissors", is being presaged as a revolutionary technology, having tremendous potential to create designer crops by introducing precise and targeted modifications in the genome to achieve global food security in the face of climate change and increasing population. Traditional genetic engineering relies on random and unpredictable insertion of isolated genes or foreign DNA elements into the plant genome. However, CRISPR-Cas based gene editing does not necessarily involve inserting a foreign DNA element into the plant genome from different species but introducing new traits by precisely altering the existing genes. CRISPR edited crops are touching markets, however, the world community is divided over whether these crops should be considered genetically modified (GM) or non-GM. Classification of CRISPR edited crops, especially transgene free crops as traditional GM crops, will significantly affect their future and public acceptance in some regions. Therefore, the future of the CRISPR edited crops is depending upon their regulation as GM or non-GMs, and their public perception. Here we briefly discuss how CRISPR edited crops are different from traditional genetically modified crops. In addition, we discuss different CRISPR reagents and their delivery tools to produce transgene-free CRISPR edited crops. Moreover, we also summarize the regulatory classification of CRISPR modifications and how different countries are regulating CRISPR edited crops. We summarize that the controversy of CRISPR-edited plants as GM or non-GM will continue until a universal, transparent, and scalable regulatory framework for CRISPR-edited plants will be introduced worldwide, with increased public awareness by involving all stakeholders.},
}
@article {pmid37875936,
year = {2023},
author = {Zhang, YR and Yin, TL and Zhou, LQ},
title = {CRISPR/Cas9 technology: applications in oocytes and early embryos.},
journal = {Journal of translational medicine},
volume = {21},
number = {1},
pages = {746},
pmid = {37875936},
issn = {1479-5876},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Oocytes ; Embryo, Mammalian ; Embryonic Development/genetics ; Mammals ; },
abstract = {CRISPR/Cas9, a highly versatile genome-editing tool, has garnered significant attention in recent years. Despite the unique characteristics of oocytes and early embryos compared to other cell types, this technology has been increasing used in mammalian reproduction. In this comprehensive review, we elucidate the fundamental principles of CRISPR/Cas9-related methodologies and explore their wide-ranging applications in deciphering molecular intricacies during oocyte and early embryo development as well as in addressing associated diseases. However, it is imperative to acknowledge the limitations inherent to these technologies, including the potential for off-target effects, as well as the ethical concerns surrounding the manipulation of human embryos. Thus, a judicious and thoughtful approach is warranted. Regardless of these challenges, CRISPR/Cas9 technology undeniably represents a formidable tool for genome and epigenome manipulation within oocytes and early embryos. Continuous refinements in this field are poised to fortify its future prospects and applications.},
}
@article {pmid37874448,
year = {2023},
author = {Sagarbarria, MGS and Caraan, JAM and Layos, AJG},
title = {Usefulness of current sgRNA design guidelines and in vitro cleavage assays for plant CRISPR/Cas genome editing: a case targeting the polyphenol oxidase gene family in eggplant (Solanum melongena L.).},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {37874448},
issn = {1573-9368},
support = {N91832A//Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development/ ; },
abstract = {The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed in the development of new crop varieties that can withstand the agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to the multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA before in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs.},
}
@article {pmid37873636,
year = {2023},
author = {Ebrahimi, S and Khosravi, MA and Raz, A and Karimipoor, M and Parvizi, P},
title = {CRISPR-Cas Technology as a Revolutionary Genome Editing tool: Mechanisms and Biomedical Applications.},
journal = {Iranian biomedical journal},
volume = {},
number = {},
pages = {},
doi = {10.52547/ibj.3898},
pmid = {37873636},
issn = {2008-823X},
abstract = {Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.},
}
@article {pmid37871213,
year = {2023},
author = {Fang, C and Yang, M and Tang, Y and Zhang, L and Zhao, H and Ni, H and Chen, Q and Meng, F and Jiang, J},
title = {Dynamics of cis-regulatory sequences and transcriptional divergence of duplicated genes in soybean.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {44},
pages = {e2303836120},
doi = {10.1073/pnas.2303836120},
pmid = {37871213},
issn = {1091-6490},
support = {2021YFF1001202//National Key R &amp; D Program of China/ ; 32172032//MOST | National Natural Science Foundation of China (NSFC)/ ; CARS-04//National Project of China/ ; MCB-1412948//National Science Foundation (NSF)/ ; ISO-2029959//National Science Foundation (NSF)/ ; },
abstract = {Transcriptional divergence of duplicated genes after whole genome duplication (WGD) has been described in many plant lineages and is often associated with subgenome dominance, a genome-wide mechanism. However, it is unknown what underlies the transcriptional divergence of duplicated genes in polyploid species that lack subgenome dominance. Soybean is a paleotetraploid with a WGD that occurred 5 to 13 Mya. Approximately 50% of the duplicated genes retained from this WGD exhibit transcriptional divergence. We developed accessible chromatin region (ACR) datasets from leaf, flower, and seed tissues using MNase-hypersensitivity sequencing. We validated enhancer function of several ACRs associated with known genes using CRISPR/Cas9-mediated genome editing. The ACR datasets were used to examine and correlate the transcriptional patterns of 17,111 pairs of duplicated genes in different tissues. We demonstrate that ACR dynamics are correlated with divergence of both expression level and tissue specificity of individual gene pairs. Gain or loss of flanking ACRs and mutation of cis-regulatory elements (CREs) within the ACRs can change the balance of the expression level and/or tissue specificity of the duplicated genes. Analysis of DNA sequences associated with ACRs revealed that the extensive sequence rearrangement after the WGD reshaped the CRE landscape, which appears to play a key role in the transcriptional divergence of duplicated genes in soybean. This may represent a general mechanism for transcriptional divergence of duplicated genes in polyploids that lack subgenome dominance.},
}
@article {pmid37869712,
year = {2023},
author = {Wu, Y and Feng, S and Sun, Z and Hu, Y and Jia, X and Zeng, B},
title = {An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1249841},
pmid = {37869712},
issn = {2296-4185},
abstract = {Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.},
}
@article {pmid37853119,
year = {2023},
author = {Chi, H and Hoikkala, V and Grüschow, S and Graham, S and Shirran, S and White, MF},
title = {Antiviral type III CRISPR signalling via conjugation of ATP and SAM.},
journal = {Nature},
volume = {622},
number = {7984},
pages = {826-833},
pmid = {37853119},
issn = {1476-4687},
mesh = {*Adenosine Triphosphate/metabolism ; *Bacteroides fragilis/enzymology/genetics/immunology ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics/immunology/physiology ; Endonucleases/chemistry/metabolism ; *Escherichia coli/genetics/growth &amp; development/immunology/metabolism ; Membrane Proteins/genetics/metabolism ; Phosphoric Diester Hydrolases/genetics/metabolism ; RNA/immunology/metabolism ; *S-Adenosylmethionine/metabolism ; *Second Messenger Systems ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements[1,2]. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain[3-5] or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors[6-9]. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases[10]. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.},
}
@article {pmid37555565,
year = {2023},
author = {Merkulov, P and Gvaramiya, S and Dudnikov, M and Komakhin, R and Omarov, M and Kocheshkova, A and Konstantinov, Z and Soloviev, A and Karlov, G and Divashuk, M and Kirov, I},
title = {Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants.},
journal = {Journal of integrative plant biology},
volume = {65},
number = {10},
pages = {2242-2261},
doi = {10.1111/jipb.13555},
pmid = {37555565},
issn = {1744-7909},
support = {075-15-2019-1667//Ministry of Science and Higher Education of the Russian Federation/ ; 20-74-10055//Russian Science Foundation/ ; },
mesh = {DNA Methylation/genetics ; *Nanopore Sequencing ; CRISPR-Cas Systems ; *Hemorrhagic Fever, Ebola/genetics ; *Arabidopsis/genetics ; Plants, Genetically Modified/genetics ; DNA Transposable Elements/genetics ; },
abstract = {Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ (EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40× sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 (decrease in DNA methylation 1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.},
}
@article {pmid37351728,
year = {2023},
author = {Taway, K and Dachphun, I and Vuttipongchaikij, S and Suttangkakul, A},
title = {Evaluation of cucumber UBL5 promoter as a tool for transgene expression and genome editing in plants.},
journal = {Transgenic research},
volume = {32},
number = {5},
pages = {437-449},
pmid = {37351728},
issn = {1573-9368},
support = {Graduate School Fellowship Program//Kasetsart University/ ; NRCT5-RSA63002-02//National Research Council of Thailand/ ; Kasetsart University Reinventing University Program 2021//Thailand Science Research and Innovation/ ; CRP6405030740//Agricultural Research Development Agency/ ; },
mesh = {Gene Editing/methods ; *Cucumis sativus/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Plants/genetics ; Transgenes ; *Arabidopsis/genetics ; },
abstract = {Transgene expression and genome editing can help improve cucumber varieties to better respond to climate change. This study aimed to evaluate the applicability of the CsUBL5 promoter in transgene expression and genome editing in cucumber. The CsUBL5 promoter was cloned and analyzed to identify cis-elements that respond to abiotic signals, hormones, signal molecules, and nutrient treatments. 5' deletion constructs of the promoter were tested for their ability to drive GUS reporter expression in cucumber cotyledons, Arabidopsis seedlings, and tobacco leaves, and their response to various treatments including SA, light, drought, IAA, and GA was determined. The results showed that the CsUBL5 promoter effectively drove transgene expression in these plants, and their expressions under treatments were consistent with the predicted cis-elements, with some exceptions. Furthermore, the pCsUBL5-749 deletion construct can improve genome editing efficiency in cucumber when driving Cas9 expression. The editing efficiency of two sgRNAs targeting the ATG6 gene in cucumber was up to 4.6-fold higher using pCsUBL5-749 compared to a rice UBI promoter, although the effects of changing promoter on the editing efficiency is sgRNA specific. These findings highlight the potential utility of the CsUBL5 promoter for improving cucumber varieties through genetic engineering and genome editing. It also demonstrates the importance of modulating Cas9 expression to increase genome editing efficiency in cucumbers.},
}
@article {pmid37330986,
year = {2023},
author = {Abeuova, L and Kali, B and Tussipkan, D and Akhmetollayeva, A and Ramankulov, Y and Manabayeva, S},
title = {CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato.},
journal = {Transgenic research},
volume = {32},
number = {5},
pages = {383-397},
pmid = {37330986},
issn = {1573-9368},
mesh = {CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Starch Synthase/genetics ; Mutagenesis/genetics ; },
abstract = {CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.},
}
@article {pmid37864303,
year = {2023},
author = {Pietralla, J and Capdeville, N and Schindele, P and Puchta, H},
title = {Optimizing ErCas12a for efficient gene editing in Arabidopsis thaliana.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14194},
pmid = {37864303},
issn = {1467-7652},
abstract = {The ErCas12a nuclease, also known as MAD7, is part of a CRISPR/Cas system from Eubacterium rectale and distantly related to Cas12a nucleases. As it shares only 31% sequence homology with the commonly used AsCas12a, its intellectual property may not be covered by the granted patent rights for Cas12a nucleases. Thus, ErCas12a became an attractive alternative for practical applications. However, the editing efficiency of ErCas12a is strongly target sequence- and temperature-dependent. Therefore, optimization of the enzyme activity through protein engineering is especially attractive for its application in plants, as they are cultivated at lower temperatures. Based on the knowledge obtained from the optimization of Cas12a nucleases, we opted to improve the gene editing efficiency of ErCas12a by introducing analogous amino acid exchanges. Interestingly, neither of these mutations analogous to those in the enhanced or Ultra versions of AsCas12a resulted in significant editing enhancement of ErCas12a in Arabidopsis thaliana. However, two different mutations, V156R and K172R, in putative alpha helical structures of the enzyme showed a detectable improvement in editing. By combining these two mutations, we obtained an improved ErCas12a (imErCas12a) variant, showing several-fold increase in activity in comparison to the wild-type enzyme in Arabidopsis. This variant yields strong editing efficiencies at 22 °C which could be further increased by raising the cultivation temperature to 28 °C and even enabled editing of formerly inaccessible targets. Additionally, no enhanced off-site activity was detected. Thus, imErCas12a is an economically attractive and efficient alternative to other CRISPR/Cas systems for plant genome engineering.},
}
@article {pmid37861059,
year = {2023},
author = {Claeys, H and Neyrinck, E and Dumoulin, L and Pharazyn, A and Verstichele, A and Pauwels, L and Nuccio, ML and Van Ex, F},
title = {Coordinated gene upregulation in maize through CRISPR/Cas-mediated enhancer insertion.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14191},
pmid = {37861059},
issn = {1467-7652},
support = {HBC.2018.2242//Agentschap Innoveren en Ondernemen/ ; },
}
@article {pmid37858564,
year = {2023},
author = {Song, YL and He, XL and Li, Y and Wang, M and Jiang, M and Xu, L and Yu, X},
title = {Homogeneous detection of viral nucleic acid via selective recognition proximity ligation and signal amplification with T7 transcription and CRISPR/Cas12a system.},
journal = {Analytica chimica acta},
volume = {1280},
number = {},
pages = {341881},
doi = {10.1016/j.aca.2023.341881},
pmid = {37858564},
issn = {1873-4324},
mesh = {*Nucleic Acids ; CRISPR-Cas Systems ; DNA Ligases ; Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; },
abstract = {The synthetic biology has employed the synthetic gene networks through engineering to construct various functions in biological systems. However, the use of gene circuits to create sensors for detecting low-abundance targets has been limited due to the lack of signal amplification strategies beyond direct output of detection signals. To address this issue, we introduce a novel method utilizing Selective Recognition Proximity Ligation and signal amplification with T7 Transcription and CRISPR/Cas12a system (SRPL-TraCs), which permits the incorporation of cell-free gene circuits with signal amplification and enables the construction of high-order cascade signal amplification strategy to detect biomarkers in homogeneous systems. Specifically, the SRPL-TraCs utilizes selective recognition proximity ligation with high-fidelity T4 DNA ligase and generates a unique crRNA via T7 transcription, along with target-activated Cas12a/crRNA system to achieve excellent specificity for HIV-1 DNA. With this straightforward synthetic biology-based method, the proposed SRPL-TraCs has the potential to detect numerous other interesting targets beyond the nucleic acids.},
}
@article {pmid37858552,
year = {2023},
author = {Zhu, Y and Liu, J and Liu, S and Zhu, X and Wu, J and Zhou, Q and He, J and Wang, H and Gao, W},
title = {CRISPR/Cas12a-assisted visible fluorescence for pseudo dual nucleic acid detection based on an integrated chip.},
journal = {Analytica chimica acta},
volume = {1280},
number = {},
pages = {341860},
doi = {10.1016/j.aca.2023.341860},
pmid = {37858552},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; Fluorescence ; Drug Contamination ; Light ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; },
abstract = {BACKGROUND: A false negative result is one of the major problems in nucleic acid detection. Failure to screen positive samples for pathogens or viruses poses a risk to public health. This situation will lead to more serious consequences for infectious pathogens or viruses. At present, the common solution is to introduce exogenous or endogenous internal control. Because it amplifies and is detected separately from the target gene, it cannot avoid false negative results caused by DNA extraction failure or reagent inactivation. There is an urgent need for a simple and reliable method to solve the false negative problem of nucleic acid detection.<br><br>RESULTS: We established a chip and an on-chip detection method for the integrated detection of target genes and internal control using the CRISPR system in LAMP amplification products. The chip is processed from a low-cost PMMA board and has three chambers and some channels. After adding the sample, the chip only needs to be rotated twice, and the sample enters three chambers successively depending on its gravity for dual LAMP reaction and CRISPR detections. With a portable LED blue light exciter, visual fluorescence detection is realized. Whether the detection result is positive, negative, or invalid can be determined according to the fluorescence in the CRISPR chamber for target gene and CRISPR chamber for internal control. In this study, the detection of Salmonella enterica in Fenneropenaeus chinensis was taken as an example. The results showed good specificity and sensitivity. It could detect as low as 15 copies/&#x3bc;L of Salmonella enterica.<br><br>SIGNIFICANCE: The on-chip detection solves the problem of aerosol contamination and false negative results. It has the advantages of high sensitivity, high specificity, high accuracy, and low cost. This research will advance the development of nucleic acid detection technology, providing a new and reliable strategy for POCT detection of pathogenic bacteria and viruses.},
}
@article {pmid37857619,
year = {2023},
author = {Sun, H and Wang, Z and Shen, L and Feng, Y and Han, L and Qian, X and Meng, R and Ji, K and Liang, D and Zhou, F and Lou, X and Zhang, J and Shen, B},
title = {Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6625},
pmid = {37857619},
issn = {2041-1723},
mesh = {*Gene Editing ; *Mitochondria/metabolism ; Mutation ; DNA, Mitochondrial/genetics ; Cytidine Deaminase/genetics ; CRISPR-Cas Systems ; Cytosine ; },
abstract = {DddA-derived cytosine base editors (DdCBEs) greatly facilitated the basic and therapeutic research of mitochondrial DNA mutation diseases. Here we devise a saturated spacer library and successfully identify seven DddA homologs by performing high-throughput sequencing based screen. DddAs of Streptomyces sp. BK438 and Lachnospiraceae bacterium sunii NSJ-8 display high deaminase activity with a strong GC context preference, and DddA of Ruminococcus sp. AF17-6 is highly compatible to AC context. We also find that different split sites result in wide divergence on off-target activity and context preference of DdCBEs derived from these DddA homologs. Additionally, we demonstrate the orthogonality between DddA and DddIA, and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddIA. The current study presents a comprehensive and unbiased strategy for screening and characterizing dsDNA cytidine deaminases, and expands the toolbox for mtDNA editing, providing additional insights for optimizing dsDNA base editors.},
}
@article {pmid37853129,
year = {2023},
author = {Camara-Wilpert, S and Mayo-Muñoz, D and Russel, J and Fagerlund, RD and Madsen, JS and Fineran, PC and Sørensen, SJ and Pinilla-Redondo, R},
title = {Bacteriophages suppress CRISPR-Cas immunity using RNA-based anti-CRISPRs.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {37853129},
issn = {1476-4687},
abstract = {Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids[1]. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity[2,3]. Here we unveil a distinct type of CRISPR-Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units[4]. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR-Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR-Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes[5]. Functional testing of nine candidates spanning the two CRISPR-Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications[6].},
}
@article {pmid37853101,
year = {2023},
author = {Sastre, D and Zafar, F and Torres, CAM and Piper, D and Kirik, D and Sanders, LH and Qi, LS and Schüle, B},
title = {Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson's disease.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {17796},
pmid = {37853101},
issn = {2045-2322},
support = {R01 NS119528/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; alpha-Synuclein/genetics/metabolism ; *Parkinson Disease ; *Methicillin-Resistant Staphylococcus aureus/genetics ; Staphylococcus aureus/genetics ; DNA, Mitochondrial/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Stem Cells/metabolism ; Oxidative Stress/genetics ; },
abstract = {Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.},
}
@article {pmid37730998,
year = {2023},
author = {Santinha, AJ and Klingler, E and Kuhn, M and Farouni, R and Lagler, S and Kalamakis, G and Lischetti, U and Jabaudon, D and Platt, RJ},
title = {Transcriptional linkage analysis with in vivo AAV-Perturb-seq.},
journal = {Nature},
volume = {622},
number = {7982},
pages = {367-375},
pmid = {37730998},
issn = {1476-4687},
mesh = {Animals ; Humans ; Mice ; *Dependovirus/genetics ; *Gene Editing ; *Genetic Association Studies/methods ; Neurons/metabolism ; Phenotype ; Prefrontal Cortex/metabolism ; *Transcription, Genetic/genetics ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; DiGeorge Syndrome/drug therapy/genetics ; Disease Models, Animal ; RNA Processing, Post-Transcriptional ; Synapses/pathology ; Genetic Predisposition to Disease ; },
abstract = {The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner[1,2]. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome[3,4] genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.},
}
@article {pmid37852951,
year = {2023},
author = {Liu, P and Foiret, J and Situ, Y and Zhang, N and Kare, AJ and Wu, B and Raie, MN and Ferrara, KW and Qi, LS},
title = {Sonogenetic control of multiplexed genome regulation and base editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6575},
pmid = {37852951},
issn = {2041-1723},
support = {R01 CA253316/CA/NCI NIH HHS/United States ; R01 CA266470/CA/NCI NIH HHS/United States ; R21 AG077193/AG/NIA NIH HHS/United States ; R01 CA112356/CA/NCI NIH HHS/United States ; R21 CA270609/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Gene Editing ; *Genome/genetics ; Genetic Therapy ; Epigenome ; Genes, Reporter ; CRISPR-Cas Systems/genetics ; },
abstract = {Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.},
}
@article {pmid37852949,
year = {2023},
author = {Riedmayr, LM and Hinrichsmeyer, KS and Thalhammer, SB and Mittas, DM and Karguth, N and Otify, DY and Böhm, S and Weber, VJ and Bartoschek, MD and Splith, V and Brümmer, M and Ferreira, R and Boon, N and Wögenstein, GM and Grimm, C and Wijnholds, J and Mehlfeld, V and Michalakis, S and Fenske, S and Biel, M and Becirovic, E},
title = {mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6578},
pmid = {37852949},
issn = {2041-1723},
support = {BE 4830/2-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BE4830/5-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; MI 1238/4-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; FE 1929/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BI 484/7-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 310030E_213089//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 310030_212190//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Humans ; Animals ; Mice ; *Trans-Splicing/genetics ; *Gene Editing ; Genetic Therapy ; Stargardt Disease ; Genetic Vectors/genetics ; Dependovirus/genetics/metabolism ; ATP-Binding Cassette Transporters/metabolism ; },
abstract = {Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.},
}
@article {pmid37850639,
year = {2023},
author = {Liu, J and Li, Q and Wang, X and Liu, Z and Ye, Q and Liu, T and Pan, S and Peng, N},
title = {An archaeal virus-encoded anti-CRISPR protein inhibits type III-B immunity by inhibiting Cas RNP complex turnover.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad804},
pmid = {37850639},
issn = {1362-4962},
support = {2022YFA0912200//National Key Research and Development Program of China/ ; 32270090//National Natural Science Foundation of China/ ; 2021hszd013//Foundation of Hubei Hongshan Laboratory/ ; AMLKF202203//State Key Laboratory of Agricultural Microbiology/ ; },
abstract = {CRISPR-Cas systems are widespread in prokaryotes and provide adaptive immune against viral infection. Viruses encode a type of proteins called anti-CRISPR to evade the immunity. Here, we identify an archaeal virus-encoded anti-CRISPR protein, AcrIIIB2, that inhibits Type III-B immunity. We find that AcrIIIB2 inhibits Type III-B CRISPR-Cas immunity in vivo regardless of viral early or middle-/late-expressed genes to be targeted. We also demonstrate that AcrIIIB2 interacts with Cmr4α subunit, forming a complex with target RNA and Cmr-α ribonucleoprotein complex (RNP). Furtherly, we discover that AcrIIIB2 inhibits the RNase activity, ssDNase activity and cOA synthesis activity of Cmr-α RNP in vitro under a higher target RNA-to-Cmr-α RNP ratio and has no effect on Cmr-α activities at the target RNA-to-Cmr-α RNP ratio of 1. Our results suggest that once the target RNA is cleaved by Cmr-α RNP, AcrIIIB2 probably inhibits the disassociation of cleaved target RNA, therefore blocking the access of other target RNA substrates. Together, our findings highlight the multiple functions of a novel anti-CRISPR protein on inhibition of the most complicated CRISPR-Cas system targeting the genes involved in the whole life cycle of viruses.},
}
@article {pmid37758944,
year = {2023},
author = {Meng, X and Yao, D and Imaizumi, K and Chen, X and Kelley, KW and Reis, N and Thete, MV and Arjun McKinney, A and Kulkarni, S and Panagiotakos, G and Bassik, MC and Pașca, SP},
title = {Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment.},
journal = {Nature},
volume = {622},
number = {7982},
pages = {359-366},
pmid = {37758944},
issn = {1476-4687},
mesh = {Female ; Humans ; Infant, Newborn ; Pregnancy ; Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Interneurons/cytology/metabolism/pathology ; *Neurodevelopmental Disorders/genetics/pathology ; Organoids/cytology/embryology/growth &amp; development/metabolism/pathology ; Endoplasmic Reticulum/metabolism ; Prosencephalon/cytology/embryology/growth &amp; development/metabolism/pathology ; Active Transport, Cell Nucleus ; },
abstract = {The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain[1-3], which has been challenging to study at inaccessible stages of late gestation and early postnatal human development[4]. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development[5], but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids[6]. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.},
}
@article {pmid37849926,
year = {2023},
author = {Bost, J and Recalde, A and Waßmer, B and Wagner, A and Siebers, B and Albers, SV},
title = {Application of the endogenous CRISPR-Cas type I-D system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1254891},
pmid = {37849926},
issn = {1664-302X},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems are widely distributed among bacteria and archaea. In this study, we demonstrate the successful utilization of the type I-D CRISPR-Cas system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Given its extreme growth conditions characterized by a temperature of 75°C and pH 3, an uracil auxotrophic selection system was previously established, providing a basis for our investigations. We developed a novel plasmid specifically designed for genome editing, which incorporates a mini-CRISPR array that can be induced using xylose, resulting in targeted DNA cleavage. Additionally, we integrated a gene encoding the β-galactosidase of Saccharolobus solfataricus into the plasmid, enabling blue-white screening and facilitating the mutant screening process. Through the introduction of donor DNA containing genomic modifications into the plasmid, we successfully generated deletion mutants and point mutations in the genome of S. acidocaldarius. Exploiting the PAM (protospacer adjacent motif) dependence of type I systems, we experimentally confirmed the functionality of three different PAMs (CCA, GTA, and TCA) through a self-targeting assessment assay and the gene deletion of upsE. Our findings elucidate the application of the endogenous Type I-D CRISPR-Cas system for genetic engineering in S. acidocaldarius, thus expanding its genetic toolbox.},
}
@article {pmid37850135,
year = {2022},
author = {Melesse Vergara, M and Labbé, J and Tannous, J},
title = {Reflection on the Challenges, Accomplishments, and New Frontiers of Gene Drives.},
journal = {Biodesign research},
volume = {2022},
number = {},
pages = {9853416},
pmid = {37850135},
issn = {2693-1257},
abstract = {Ongoing pest and disease outbreaks pose a serious threat to human, crop, and animal lives, emphasizing the need for constant genetic discoveries that could serve as mitigation strategies. Gene drives are genetic engineering approaches discovered decades ago that may allow quick, super-Mendelian dissemination of genetic modifications in wild populations, offering hopes for medicine, agriculture, and ecology in combating diseases. Following its first discovery, several naturally occurring selfish genetic elements were identified and several gene drive mechanisms that could attain relatively high threshold population replacement have been proposed. This review provides a comprehensive overview of the recent advances in gene drive research with a particular emphasis on CRISPR-Cas gene drives, the technology that has revolutionized the process of genome engineering. Herein, we discuss the benefits and caveats of this technology and place it within the context of natural gene drives discovered to date and various synthetic drives engineered. Later, we elaborate on the strategies for designing synthetic drive systems to address resistance issues and prevent them from altering the entire wild populations. Lastly, we highlight the major applications of synthetic CRISPR-based gene drives in different living organisms, including plants, animals, and microorganisms.},
}
@article {pmid37556411,
year = {2023},
author = {Tijaro-Bulla, S and Osman, EA and St Laurent, CD and McCord, KA and Macauley, MS and Gibbs, JM},
title = {Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.},
journal = {ACS chemical biology},
volume = {18},
number = {10},
pages = {2156-2162},
doi = {10.1021/acschembio.3c00140},
pmid = {37556411},
issn = {1554-8937},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Nucleotides ; },
abstract = {CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide-alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2'-O-Me group and a phosphorothioate backbone in varying number at both the 5'- and 3'-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.},
}
@article {pmid36861834,
year = {2023},
author = {Lane-Reticker, SK and Kessler, EA and Muscato, AJ and Kim, SY and Doench, JG and Yates, KB and Manguso, RT and Dubrot, J},
title = {Protocol for in vivo CRISPR screening using selective CRISPR antigen removal lentiviral vectors.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102082},
pmid = {36861834},
issn = {2666-1667},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Library ; Genome ; Cell Line ; },
abstract = {Recognition of Cas9 and other proteins encoded in delivery vectors has limited CRISPR technology in vivo. Here, we present a protocol for genome engineering using selective CRISPR antigen removal (SCAR) lentiviral vectors in Renca mouse model. This protocol describes how to conduct an in vivo genetic screen with a sgRNA library and SCAR vectors that can be applied to different cell lines and contexts. For complete details on the use and execution of this protocol, please refer to Dubrot et al. (2021).[1].},
}
@article {pmid36853714,
year = {2023},
author = {Huber, A and Dijkstra, C and Ernst, M and Eissmann, MF},
title = {Generation of gene-of-interest knockouts in murine organoids using CRISPR-Cas9.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102076},
pmid = {36853714},
issn = {2666-1667},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Organoids ; Blotting, Western ; Clone Cells ; },
abstract = {Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here, we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes, organoid cell sorting, and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis.},
}
@article {pmid36853689,
year = {2023},
author = {Ren, X and Takagi, MA and Shen, Y},
title = {Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102084},
pmid = {36853689},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Clone Cells ; Recombinational DNA Repair ; },
abstract = {Allelic tagging of endogenous genes enables studying gene function and transcriptional control in the native genomic context. Here, we present an efficient protocol for bi-allelic tagging of protein-coding genes with fluorescent reporters in human iPSCs using the CRISPR-Cas9-mediated homology-directed repair. We detail steps for design, cloning, electroporation, and single-cell clone isolation and validation. The tagging strategy described in this protocol is readily applicable for knockin of other reporters in diverse cell types for biomedical research.},
}
@article {pmid36853660,
year = {2023},
author = {Cassidy, A and Pelletier, S},
title = {CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102116},
pmid = {36853660},
issn = {2666-1667},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Alleles ; Introns/genetics ; Exons/genetics ; },
abstract = {In this protocol, we describe the generation of conditional alleles in mice using the DECAI (DEgradation based on Cre-regulated Artificial Intron) approach. We detail steps for the CRISPR-mediated insertion of the short DECAI cassette within exon 3 of Scyl1 and the functional validation of alleles at genomic, transcriptomic, and protein levels. This strategy simplifies the process of generating mice with conditional alleles. For complete details on the use and execution of this protocol, please refer to Cassidy et al. (2022).[1].},
}
@article {pmid36640369,
year = {2023},
author = {Kurashina, M and Mizumoto, K},
title = {Targeting endogenous proteins for spatial and temporal knockdown using auxin-inducible degron in Caenorhabditis elegans.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102028},
pmid = {36640369},
issn = {2666-1667},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Indoleacetic Acids/pharmacology ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {The auxin-inducible degron (AID) provides reversible, spatiotemporal control for the knockdown of target proteins. Here, we present a protocol for AID-mediated protein knockdown in Caenorhabditis elegans. We describe steps for generating the knock-in mutants using two CRISPR-Cas9 genome editing techniques and preparing the auxin-containing nematode growth media (NGM) plates. We also detail AID-mediated spatiotemporal protein knockdown. For complete details on the use and execution of this protocol, please refer to Kurashina et al. (2021).[1].},
}
@article {pmid36598849,
year = {2023},
author = {Chan, DKH and Collins, SD and Buczacki, SJA},
title = {Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {101978},
pmid = {36598849},
issn = {2666-1667},
mesh = {Humans ; Adult ; Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Organoids ; Coloring Agents ; RNA/genetics ; },
abstract = {While readily achieved in cell lines, the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here, we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein.},
}
@article {pmid35644856,
year = {2023},
author = {Zhao, J and Hu, H and Zhou, H and Zhang, J and Wang, L and Wang, R},
title = {Reactive oxygen signaling molecule inducible regulation of CRISPR-Cas9 gene editing.},
journal = {Cell biology and toxicology},
volume = {39},
number = {5},
pages = {2421-2429},
pmid = {35644856},
issn = {1573-6822},
support = {startup//Tongji Medical College, Huazhong University of Science and Technology/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Hydrogen Peroxide/pharmacology ; CRISPR-Associated Protein 9/genetics ; },
abstract = {We report development of a controllable gene editing tool that boronated gRNA, simply generated in situ, could regulate binding of gRNA molecules with either Cas9 endonuclease or target genes, thus serving as a modulator that can control CRISPR-Cas9 gene editing. Subsequent treatment with H2O2 facilitates the restoration of gene editing ability of the boronated gRNA to the level of using untreated gRNA. This is one of the few cases using small molecule to regulate CRISPR-Cas9 gene editing, which is a complement to the light approach, displaying great application potential. We develop a controllable gene editing tools based on the CRISPR-Cas9 gene editing system. This tool can be regulated by oxidative small molecule, i.e., H2O2. Compared with the light method, the application scope of our CRISPR-Cas9 systems have been widened with the small-molecule-triggered approaches, preventing the potential damage of cells or organism caused by UV light. In addition, the gain-of-function tools are expanding the gene code expansion for mechanistic studies of target enzymes since it provides a positive route to evaluate the activity of a given enzyme in dynamic and inversible regulation of targeting cellular processes.},
}
@article {pmid37849904,
year = {2020},
author = {Hassan, MM and Yuan, G and Chen, JG and Tuskan, GA and Yang, X},
title = {Prime Editing Technology and Its Prospects for Future Applications in Plant Biology Research.},
journal = {Biodesign research},
volume = {2020},
number = {},
pages = {9350905},
pmid = {37849904},
issn = {2693-1257},
abstract = {Many applications in plant biology requires editing genomes accurately including correcting point mutations, incorporation of single-nucleotide polymorphisms (SNPs), and introduction of multinucleotide insertion/deletions (indels) into a predetermined position in the genome. These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems, which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence. However, low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient. Another kind of technology known as base editing can perform precise editing; however, only certain types of modifications can be obtained, e.g., C/G-to-T/A and A/T-to-G/C. Recently, a new type of genome-editing technology, referred to as "prime editing," has been developed, which can achieve various types of editing such as any base-to-base conversion, including both transitions (C→T, G→A, A→G, and T→C) and transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A, and T→G), as well as small indels without the requirement for inducing double-stranded break in the DNA. Because prime editing has wide flexibility to achieve different types of edits in the genome, it holds a great potential for developing superior crops for various purposes, such as increasing yield, providing resistance to various abiotic and biotic stresses, and improving quality of plant product. In this review, we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.},
}
@article {pmid37849898,
year = {2020},
author = {Abraham, PE and Labbé, JL and McBride, AA},
title = {Advancing How We Learn from Biodesign to Mitigate Risks with Next-Generation Genome Engineering.},
journal = {Biodesign research},
volume = {2020},
number = {},
pages = {9429650},
pmid = {37849898},
issn = {2693-1257},
abstract = {In the last decade, the unprecedented simplicity and flexibility of the CRISPR-Cas system has made it the dominant transformative tool in gene and genome editing. However, this democratized technology is both a boon and a bane, for which we have yet to understand the full potential to investigate and rewrite genomes (also named "genome biodesign"). Rapid CRISPR advances in a range of applications in basic research, agriculture, and clinical applications pose new risks and raise several biosecurity concerns. In such a fast-moving field of research, we emphasize the importance of properly communicating the quality and accuracy of results and recommend new reporting requirements for results derived from next-generation genome engineering.},
}
@article {pmid37849464,
year = {2023},
author = {Niu, C and Liu, J and Xing, X and Zhang, C},
title = {Exploring the Trans-Cleavage Activity with Rolling Circle Amplification for Fast Detection of miRNA.},
journal = {Biodesign research},
volume = {5},
number = {},
pages = {0010},
pmid = {37849464},
issn = {2693-1257},
abstract = {MicroRNAs (miRNAs) are a class of endogenous short noncoding RNA. They regulate gene expression and function, essential to biological processes. It is necessary to develop an efficient detection method to determine these valuable biomarkers for the diagnosis of cancers. In this paper, we proposed a general and rapid method for sensitive and quantitative detection of miRNA by combining CRISPR-Cas12a and rolling circle amplification (RCA) with the precircularized probe. Eventually, the detection of miRNA-21 could be completed in 70 min with a limit of detection of 8.1 pM with high specificity. The reaction time was reduced by almost 4 h from more than 5 h to 70 min, which makes detection more efficient. This design improves the efficiency of CRISPR-Cas and RCA-based sensing strategy and shows great potential in lab-based detection and point-of-care test.},
}
@article {pmid37849462,
year = {2023},
author = {Liu, L and Helal, SE and Peng, N},
title = {CRISPR-Cas-Based Engineering of Probiotics.},
journal = {Biodesign research},
volume = {5},
number = {},
pages = {0017},
pmid = {37849462},
issn = {2693-1257},
abstract = {Probiotics are the treasure of the microbiology fields. They have been widely used in the food industry, clinical treatment, and other fields. The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic's developed applications. In recent years, various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics. As important genome editing tools, CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics. The high efficiency, flexibility, and specificity are the advantages of using CRISPR-Cas systems. Here, we summarize the classification and distribution of CRISPR-Cas systems in probiotics, as well as the editing tools developed on the basis of them. Then, we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems. Finally, we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.},
}
@article {pmid37847697,
year = {2023},
author = {O'Brien, A and Bauer, DC and Burgio, G},
title = {Predicting CRISPR-Cas12a guide efficiency for targeting using machine learning.},
journal = {PloS one},
volume = {18},
number = {10},
pages = {e0292924},
pmid = {37847697},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Endonucleases/genetics ; RNA ; Nucleotides ; },
abstract = {Genome editing through the development of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas technology has revolutionized many fields in biology. Beyond Cas9 nucleases, Cas12a (formerly Cpf1) has emerged as a promising alternative to Cas9 for editing AT-rich genomes. Despite the promises, guide RNA efficiency prediction through computational tools search still lacks accuracy. Through a computational meta-analysis, here we report that Cas12a target and off-target cleavage behavior are a factor of nucleotide bias combined with nucleotide mismatches relative to the protospacer adjacent motif (PAM) site. These features helped to train a Random Forest machine learning model to improve the accuracy by at least 15% over existing algorithms to predict guide RNA efficiency for the Cas12a enzyme. Despite the progresses, our report underscores the need for more representative datasets and further benchmarking to reliably and accurately predict guide RNA efficiency and off-target effects for Cas12a enzymes.},
}
@article {pmid37847464,
year = {2023},
author = {Hejlesen, R and Kjær-Sørensen, K and Fago, A and Oxvig, C},
title = {Generation and validation of a myoglobin knockout zebrafish model.},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {37847464},
issn = {1573-9368},
support = {NNF19OC0057938//Novo Nordisk Fonden, Denmark/ ; },
abstract = {Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.},
}
@article {pmid37846029,
year = {2023},
author = {Goren, MG and Mahata, T and Qimron, U},
title = {An efficient, scarless, selection-free technology for phage engineering.},
journal = {RNA biology},
volume = {20},
number = {1},
pages = {830-835},
pmid = {37846029},
issn = {1555-8584},
mesh = {*Bacteriophages/genetics ; Escherichia coli/genetics ; CRISPR-Cas Systems ; Mutation ; Technology ; },
abstract = {Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50-201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text].},
}
@article {pmid37841202,
year = {2023},
author = {Dubey, AK and Mostafavi, E},
title = {Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy.},
journal = {Frontiers in chemistry},
volume = {11},
number = {},
pages = {1259435},
pmid = {37841202},
issn = {2296-2646},
abstract = {The use of biomaterials in delivering CRISPR/Cas9 for gene therapy in infectious diseases holds tremendous potential. This innovative approach combines the advantages of CRISPR/Cas9 with the protective properties of biomaterials, enabling accurate and efficient gene editing while enhancing safety. Biomaterials play a vital role in shielding CRISPR/Cas9 components, such as lipid nanoparticles or viral vectors, from immunological processes and degradation, extending their effectiveness. By utilizing the flexibility of biomaterials, tailored systems can be designed to address specific genetic diseases, paving the way for personalized therapeutics. Furthermore, this delivery method offers promising avenues in combating viral illnesses by precisely modifying pathogen genomes, and reducing their pathogenicity. Biomaterials facilitate site-specific gene modifications, ensuring effective delivery to infected cells while minimizing off-target effects. However, challenges remain, including optimizing delivery efficiency, reducing off-target effects, ensuring long-term safety, and establishing scalable production techniques. Thorough research, pre-clinical investigations, and rigorous safety evaluations are imperative for successful translation from the laboratory to clinical applications. In this review, we discussed how CRISPR/Cas9 delivery using biomaterials revolutionizes gene therapy and infectious disease treatment, offering precise and safe editing capabilities with the potential to significantly improve human health and quality of life.},
}
@article {pmid37837547,
year = {2023},
author = {Yadav, B and Majhi, A and Phagna, K and Meena, MK and Ram, H},
title = {Negative regulators of grain yield and mineral contents in rice: potential targets for CRISPR-Cas9-mediated genome editing.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {4},
pages = {317},
pmid = {37837547},
issn = {1438-7948},
mesh = {Gene Editing ; CRISPR-Cas Systems ; *Oryza/genetics/metabolism ; Phenotype ; Edible Grain/genetics ; *MicroRNAs/metabolism ; Genome, Plant ; },
abstract = {Rice is a major global staple food crop, and improving its grain yield and nutritional quality has been a major thrust research area since last decades. Yield and nutritional quality are complex traits which are controlled by multiple signaling pathways. Sincere efforts during past decades of research have identified several key genetic and molecular regulators that governed these complex traits. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene knockout approaches has accelerated the development of improved varieties; however, finding out target gene with negative regulatory function in particular trait without giving any pleiotropic effect remains a challenge. Here, we have reviewed past and recent literature and identified important negative regulators of grain yield and mineral contents which could be potential targets for CRISPR-Cas9-mediated gene knockout. Additionally, we have also compiled a list of microRNAs (miRNAs), which target positive regulators of grain yield, plant stress tolerance, and grain mineral contents. Knocking out these miRNAs could help to increase expression of such positive regulators and thus improve the plant trait. The knowledge presented in this review would help to further accelerate the CRISPR-Cas9-mediated trait improvement in rice.},
}
@article {pmid37834394,
year = {2023},
author = {Jia, Y and Yang, J and Chen, Y and Liu, Y and Jin, Y and Wang, C and Gong, B and Zhao, Q},
title = {Identification of NCAPG as an Essential Gene for Neuroblastoma Employing CRISPR-Cas9 Screening Database and Experimental Verification.},
journal = {International journal of molecular sciences},
volume = {24},
number = {19},
pages = {},
pmid = {37834394},
issn = {1422-0067},
support = {2018YFC1313000//National Key Research and Development Program of China/ ; 2018YFC1313001//National Key Research and Development Program of China/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; },
mesh = {Child ; Humans ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Genes, Essential ; Cell Cycle Proteins/metabolism ; *Neuroblastoma/metabolism ; },
abstract = {Neuroblastoma is the most common extracranial solid tumor in children. Patients with neuroblastoma have a poor prognosis. The development of therapy targets and the ability to predict prognosis will be enhanced through further exploration of the genetically related genes of neuroblastoma. The present investigation utilized CRISPR-Cas9 genome-wide screening based on the DepMap database to determine essential genes for neuroblastoma cells' continued survival. WGCNA analysis was used to determine the progression-related genes, and a prognostic signature was constructed. The signature gene, NCAPG, was downregulated in neuroblastoma cells to explore its impact on various cellular processes. This research used DepMap and WGCNA to pinpoint 45 progression-related essential genes for neuroblastoma. A risk signature comprising NCAPG and MAD2L1 was established. The suppression of NCAPG prevented neuroblastoma cells from proliferating, migrating, and invading. The results of flow cytometric analysis demonstrated that NCAPG inhibition caused cell cycle arrest during the G2 and S phases and the activation of apoptosis. Additionally, NCAPG downregulation activated the p53-mediated apoptotic pathway, inducing cell apoptosis. The present work showed that NCAPG knockdown reduced neuroblastoma cell progression and may serve as a basis for further investigation into diagnostic indicators and therapy targets for neuroblastoma.},
}
@article {pmid37834283,
year = {2023},
author = {Wu, K and Xu, C and Li, T and Ma, H and Gong, J and Li, X and Sun, X and Hu, X},
title = {Application of Nanotechnology in Plant Genetic Engineering.},
journal = {International journal of molecular sciences},
volume = {24},
number = {19},
pages = {},
pmid = {37834283},
issn = {1422-0067},
support = {No. LR23C150001//Zhejiang Provincial Natural Science Foundation of China/ ; LQ23C150003//Zhejiang Provincial Natural Science Foundation of China/ ; No. 32102318//National Natural Science Foundation of China/ ; },
mesh = {Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Gene Editing ; Genome, Plant ; Edible Grain/genetics ; Nanotechnology ; Plant Breeding ; },
abstract = {The ever-increasing food requirement with globally growing population demands advanced agricultural practices to improve grain yield, to gain crop resilience under unpredictable extreme weather, and to reduce production loss caused by insects and pathogens. To fulfill such requests, genome engineering technology has been applied to various plant species. To date, several generations of genome engineering methods have been developed. Among these methods, the new mainstream technology is clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases. One of the most important processes in genome engineering is to deliver gene cassettes into plant cells. Conventionally used systems have several shortcomings, such as being labor- and time-consuming procedures, potential tissue damage, and low transformation efficiency. Taking advantage of nanotechnology, the nanoparticle-mediated gene delivery method presents technical superiority over conventional approaches due to its high efficiency and adaptability in different plant species. In this review, we summarize the evolution of plant biomolecular delivery methods and discussed their characteristics as well as limitations. We focused on the cutting-edge nanotechnology-based delivery system, and reviewed different types of nanoparticles, preparation of nanomaterials, mechanism of nanoparticle transport, and advanced application in plant genome engineering. On the basis of established methods, we concluded that the combination of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies can accelerate crop improvement efficiently in the future.},
}
@article {pmid37834150,
year = {2023},
author = {Tabassum, T and Pietrogrande, G and Healy, M and Wolvetang, EJ},
title = {CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.},
journal = {International journal of molecular sciences},
volume = {24},
number = {19},
pages = {},
pmid = {37834150},
issn = {1422-0067},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Homologous Recombination ; DNA Repair/genetics ; Genome ; Mammals ; },
abstract = {DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.},
}
@article {pmid37833288,
year = {2023},
author = {Sadremomtaz, A and Glass, RF and Guerrero, JE and LaJeunesse, DR and Josephs, EA and Zadegan, R},
title = {Digital data storage on DNA tape using CRISPR base editors.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6472},
pmid = {37833288},
issn = {2041-1723},
support = {R35 GM133483/GM/NIGMS NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA/genetics ; Gene Editing/methods ; DNA Replication ; Information Storage and Retrieval ; CRISPR-Cas Systems/genetics ; },
abstract = {While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school's logo and the title of this study on the DNA tapes.},
}
@article {pmid37832938,
year = {2023},
author = {Aroca, A and García, I},
title = {Advances in plant molecular biology: towards new challenges.},
journal = {Journal of experimental botany},
volume = {74},
number = {19},
pages = {5949-5954},
pmid = {37832938},
issn = {1460-2431},
support = {PID2019-109785GB-I00//AEI/ ; PROYEXCEL_00177 and P20_00030//Junta de Andaluc&#xed;a/ ; },
mesh = {*Plants/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Genome, Plant ; Plant Breeding ; },
}
@article {pmid37831441,
year = {2023},
author = {Huang, S and Zhang, Q and Zhao, M and Wang, X and Zhang, Y and Gan, B and Zhang, P},
title = {The deubiquitinase ZRANB1 is an E3 ubiquitin ligase for SLC7A11 and regulates ferroptotic resistance.},
journal = {The Journal of cell biology},
volume = {222},
number = {11},
pages = {},
pmid = {37831441},
issn = {1540-8140},
support = {81874116//National Natural Science Foundation of China/ ; 2021YFA1201200//National Key Research and Development Program of China/ ; 2017YFE0129100//Key Program for International S&amp;T Cooperation Projects of China/ ; //Huazhong University of Science and Technology/ ; //National Young Talents Program of China/ ; },
mesh = {Humans ; *Ubiquitin-Protein Ligases/genetics ; RNA, Guide, CRISPR-Cas Systems ; Lipid Peroxidation ; Glutathione ; *Neoplasms ; Deubiquitinating Enzymes ; Amino Acid Transport System y+/genetics ; },
abstract = {The dependency of cancer cells on iron increases their susceptibility to ferroptosis, thus providing new opportunities for patients with treatment-resistant tumors. However, we show that lipid peroxidation, a hallmark of ferroptosis, was found in various areas of patient samples, indicating the potential resistance of ferroptosis. Using whole deubiquitinases (DUBs) sgRNA screening, we found that loss of ZRANB1 confers cancer cell resistance to ferroptosis. Intriguingly, functional studies revealed that ZRANB1 ubiquitinates and represses SLC7A11 expression as an E3 ubiquitin ligase and that ZRANB1 inhibits glutathione (GSH) synthesis through SLC7A11 degradation, leading to elevated lipid peroxidation and ferroptosis. Deletion of the region (residues 463-584) abolishes the E3 activity of ZRANB1. Moreover, we show that ZRANB1 has lower expression in tumors, which is positively correlated with lipid peroxidation. Collectively, our results demonstrate the role of ZRANB1 in ferroptosis resistance and unveil mechanisms involving modulation of E3 ligase activity through an unconventional catalytic domain.},
}
@article {pmid37823804,
year = {2023},
author = {Horgan, RW},
title = {Cure Rare Disease: An Initiative to Enable N of 1 Gene Editing.},
journal = {Human gene therapy},
volume = {34},
number = {19-20},
pages = {980-981},
doi = {10.1089/hum.2023.29248.rwh},
pmid = {37823804},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing ; *Rare Diseases/genetics/therapy ; CRISPR-Cas Systems ; Genetic Therapy ; },
}
@article {pmid37821613,
year = {2023},
author = {Kozlov, M},
title = {Monkey survives for two years after gene-edited pig-kidney transplant.},
journal = {Nature},
volume = {622},
number = {7983},
pages = {437-438},
pmid = {37821613},
issn = {1476-4687},
mesh = {Animals ; Swine ; *Gene Editing ; Haplorhini ; *Kidney Transplantation ; CRISPR-Cas Systems/genetics ; Genome, Plant ; },
}
@article {pmid37813387,
year = {2023},
author = {Barbier, I and Kusumawardhani, H and Chauhan, L and Harlapur, PV and Jolly, MK and Schaerli, Y},
title = {Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in E. coli: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects.},
journal = {ACS synthetic biology},
volume = {12},
number = {10},
pages = {3064-3071},
doi = {10.1021/acssynbio.3c00375},
pmid = {37813387},
issn = {2161-5063},
mesh = {*Escherichia coli/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Synthetic ; RNA/metabolism ; },
abstract = {Gene expression control based on clustered regularly interspaced short palindromic repeats (CRISPR) has emerged as a powerful approach for constructing synthetic gene circuits. While the use of CRISPR interference (CRISPRi) is already well-established in prokaryotic circuits, CRISPR activation (CRISPRa) is less mature, and a combination of the two in the same circuits is only just emerging. Here, we report that combining CRISPRi with SoxS-based CRISPRa in Escherichia coli can lead to context-dependent effects due to different affinities in the formation of CRISPRa and CRISPRi complexes, resulting in loss of predictable behavior. We show that this effect can be avoided by using the same scaffold guide RNA structure for both complexes.},
}
@article {pmid37811735,
year = {2023},
author = {Chen, H and Zhou, T and Li, S and Feng, J and Li, W and Li, L and Zhou, X and Wang, M and Li, F and Zhao, X and Ren, L},
title = {Living Magnetotactic Microrobots Based on Bacteria with a Surface-Displayed CRISPR/Cas12a System for Penaeus Viruses Detection.},
journal = {ACS applied materials &amp; interfaces},
volume = {15},
number = {41},
pages = {47930-47938},
doi = {10.1021/acsami.3c09690},
pmid = {37811735},
issn = {1944-8252},
mesh = {Animals ; *Penaeidae ; CRISPR-Cas Systems/genetics ; Bacteria/genetics ; *Nucleic Acids ; *Viruses ; },
abstract = {Bacterial microrobots are an emerging living material in the field of diagnostics. However, it is an important challenge to make bacterial microrobots with both controlled motility and specific functions. Herein, magnetically driven diagnostic bacterial microrobots are prepared by standardized and modular synthetic biology methods. To ensure mobility, the Mms6 protein is displayed on the surface of bacteria and is exploited for magnetic biomineralization. This gives the bacterial microrobot the ability to cruise flexibly and rapidly with a magnetization intensity up to about 18.65 emu g[-1]. To achieve the diagnostic function, the Cas12a protein is displayed on the bacterial surface and is used for aquatic pathogen nucleic acid detection. This allows the bacterial microrobot to achieve sensitive, rapid, and accurate on-site nucleic acid detection, with detection limits of 8 copies μL[-1] for decapod iridescent virus 1 (DIV1) and 7 copies μL[-1] for white spot syndrome virus (WSSV). In particular, the diagnostic results based on the bacterial microrobots remained consistent with the gold standard test results when tested on shrimp tissue. This approach is a flexible and customizable strategy for building bacterial microrobots, providing a reliable and versatile solution for the design of bacterial microrobots.},
}
@article {pmid37807760,
year = {2023},
author = {Liu, Q and Meng, G and Wang, M and Li, X and Liu, M and Wang, F and Yang, Y and Dong, C},
title = {Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {41},
pages = {15249-15260},
doi = {10.1021/acs.jafc.3c05131},
pmid = {37807760},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Cordyceps/genetics ; Disease Resistance/genetics ; Plant Breeding ; Gene Editing/methods ; },
abstract = {Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.},
}
@article {pmid37807677,
year = {2023},
author = {Wang, S and Xue, Y and Zhang, P and Yan, Q and Li, Y and Jiang, Z},
title = {CRISPR/Cas9 System-Mediated Multi-copy Expression of an Alkaline Serine Protease in Aspergillus niger for the Production of XOD-Inhibitory Peptides.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {41},
pages = {15194-15203},
doi = {10.1021/acs.jafc.3c04138},
pmid = {37807677},
issn = {1520-5118},
mesh = {*Aspergillus niger/genetics/metabolism ; *Xanthine Oxidase/metabolism ; Serine Proteases/genetics/metabolism ; CRISPR-Cas Systems ; Serine/metabolism ; Peptides/genetics/metabolism ; Serine Endopeptidases/genetics/metabolism ; },
abstract = {CRISPR/Cas9 system-mediated multi-copy expression of an alkaline serine protease (AoproS8) from Aspergillus oryzae was successfully built in Aspergillus niger. Furthermore, AoproS8 was continuously knocked in the glaA, amyA, and aamy gene loci in A. niger to construct multi-copy expression strains. The yield of the AoproS8 3.0 strain was 2.1 times higher than that of the AoproS8 1.0 strain. Then, a high protease activity of 11,023.2 U/mL with a protein concentration of 10.8 mg/mL was obtained through fed-batch fermentation in a 5 L fermenter. This is the first report on the high-level expression of alkaline serine proteases in A. niger. AoproS8 showed optimal activity at pH 9.0 and 40 °C. It was used for the production of xanthine oxidase (XOD)-inhibitory peptides from eight food processing protein by-products. Among them, the duck hemoglobin hydrolysates showed the highest XOD-inhibitory activity with an IC50 value of 2.39 mg/mL. Thus, our work provides a useful way for efficient expression of proteases in A. niger and high-value utilization of protein by-products.},
}
@article {pmid37791744,
year = {2023},
author = {D'Agnese, E and Chase, D and Andruszkiewicz-Allan, E},
title = {ISOTHERMAL RECOMBINANT POLYMERASE AMPLIFICATION AND CRIPSR(CAS12A) ASSAY DETECTION OF RENIBACTERIUM SALMONINARUM AS AN EXAMPLE FOR WILDLIFE PATHOGEN DETECTION IN ENVIRONMENTAL DNA SAMPLES.},
journal = {Journal of wildlife diseases},
volume = {59},
number = {4},
pages = {545-556},
doi = {10.7589/JWD-D-22-00128},
pmid = {37791744},
issn = {1943-3700},
mesh = {Animals ; Animals, Wild ; *DNA, Environmental ; CRISPR-Cas Systems ; Ecosystem ; *Micrococcaceae/genetics ; *Kidney Diseases/microbiology/veterinary ; Salmon/genetics/microbiology ; Water ; *Fish Diseases/diagnosis/microbiology ; },
abstract = {Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon (Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum, is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20-40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.},
}
@article {pmid37782626,
year = {2023},
author = {Shigemori, H and Fujita, S and Tamiya, E and Wakida, SI and Nagai, H},
title = {Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex Double-Stranded DNA Detection.},
journal = {Bioconjugate chemistry},
volume = {34},
number = {10},
pages = {1754-1765},
pmid = {37782626},
issn = {1520-4812},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; RNA ; DNA, Single-Stranded/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12 (Cas12) system is attracting interest for its potential as a next-generation nucleic acid detection tool. The system can recognize double-stranded DNA (dsDNA) based on Cas12-CRISPR RNA (crRNA) and induce signal transduction by collateral cleavage. This property is expected to simplify comprehensive genotyping. Here, we report a solid-phase collateral cleavage (SPCC) reaction by CRISPR/Cas12 and its application toward one-pot multiplex dsDNA detection with minimal operational steps. In the sensor, Cas12-crRNA and single-stranded DNA (ssDNA) are immobilized on the sensing surface and act as enzyme and reporter substrates, respectively. We also report a dual-target dsDNA sensor prepared by immobilizing Cas12-crRNA and a fluorophore-labeled ssDNA reporter on separate spots. When a spot captures a target dsDNA sequence, it cleaves the ssDNA reporter on the same spot and reduces its fluorescence by 42.1-57.3%. Crucially, spots targeting different sequences do not show a reduction in fluorescence, thus confirming the one-pot multiplex dsDNA detection by SPCC. Furthermore, the sequence specificity has a two-base resolution, and the detectable concentration for the target dsDNA is at least 10[-9] M. In the future, the SPCC-based sensor array could achieve one-pot comprehensive genotyping by using an array spotter as a reagent-immobilizing method.},
}
@article {pmid37775147,
year = {2023},
author = {Zhang, G and Luo, Y and Dai, X and Dai, Z},
title = {Benchmarking deep learning methods for predicting CRISPR/Cas9 sgRNA on- and off-target activities.},
journal = {Briefings in bioinformatics},
volume = {24},
number = {6},
pages = {},
doi = {10.1093/bib/bbad333},
pmid = {37775147},
issn = {1477-4054},
support = {62103249//National Natural Science Foundation of China/ ; 2022A1515011720//Guangdong Basic, Applied Basic Research Foundation/ ; NTF20032//STU Scientific Research Foundation for Talents/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; Benchmarking ; *Deep Learning ; },
abstract = {In silico design of single guide RNA (sgRNA) plays a critical role in clustered regularly interspaced, short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Continuous efforts are aimed at improving sgRNA design with efficient on-target activity and reduced off-target mutations. In the last 5 years, an increasing number of deep learning-based methods have achieved breakthrough performance in predicting sgRNA on- and off-target activities. Nevertheless, it is worthwhile to systematically evaluate these methods for their predictive abilities. In this review, we conducted a systematic survey on the progress in prediction of on- and off-target editing. We investigated the performances of 10 mainstream deep learning-based on-target predictors using nine public datasets with different sample sizes. We found that in most scenarios, these methods showed superior predictive power on large- and medium-scale datasets than on small-scale datasets. In addition, we performed unbiased experiments to provide in-depth comparison of eight representative approaches for off-target prediction on 12 publicly available datasets with various imbalanced ratios of positive/negative samples. Most methods showed excellent performance on balanced datasets but have much room for improvement on moderate- and severe-imbalanced datasets. This study provides comprehensive perspectives on CRISPR/Cas9 sgRNA on- and off-target activity prediction and improvement for method development.},
}
@article {pmid37768786,
year = {2023},
author = {Ganesan, V and Monteiro, L and Pedada, D and Stohr, A and Blenner, M},
title = {High-Efficiency Multiplexed Cytosine Base Editors for Natural Product Synthesis in Yarrowia lipolytica.},
journal = {ACS synthetic biology},
volume = {12},
number = {10},
pages = {3082-3091},
doi = {10.1021/acssynbio.3c00435},
pmid = {37768786},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Yarrowia/genetics/metabolism ; Cytosine/metabolism ; Canavanine/genetics/metabolism ; Gene Editing ; },
abstract = {Yarrowia lipolytica is an industrial host with a high fatty acid flux. Even though CRISPR-based tools have accelerated its metabolic engineering, there remains a need to develop tools for rapid multiplexed strain engineering to accelerate the design-build-test-learn cycle. Base editors have the potential to perform high-efficiency multiplexed gene editing because they do not depend upon double-stranded DNA breaks. Here, we identified that base editors are less toxic than CRISPR-Cas9 for multiplexed gene editing. We increased the editing efficiency by removing the extra nucleotides between tRNA and gRNA and increasing the base editor and gRNA copy number in a Ku70 deficient strain. We achieved five multiplexed gene editing in the ΔKu70 strain at 42% efficiency. Initially, we were unsuccessful at performing multiplexed base editing in NHEJ competent strain; however, we increased the editing efficiency by using a co-selection approach to enrich base editing events. Base editor-mediated canavanine gene (CAN1) knockout provided resistance to the import of canavanine, which enriched the base editing in other unrelated genetic loci. We performed multiplexed editing of up to three genes at 40% efficiency in the Po1f strain through the CAN1 co-selection approach. Finally, we demonstrated the application of multiplexed cytosine base editor for rapid multigene knockout to increase naringenin production by 2-fold from glucose or glycerol as a carbon source.},
}
@article {pmid37759224,
year = {2023},
author = {Tan, K and Lu, W and Chen, F and Shi, H and Ma, Y and Chen, Z and Wu, W and Lv, Z and Mo, J},
title = {CRISPR-Cas9 knockout screening identifies KIAA1429 as an essential gene in Ewing sarcoma.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {42},
number = {1},
pages = {250},
pmid = {37759224},
issn = {1756-9966},
mesh = {Animals ; Humans ; Child ; *Sarcoma, Ewing/genetics/pathology ; Genes, Essential ; CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Models, Animal ; Cell Proliferation ; },
abstract = {BACKGROUND: Ewing sarcoma (ES) is an aggressive childhood bone and soft tissue cancer. KIAA1429 is one type of N6-methyladenosine (m6A) writer that plays a tumor-progressive role in various cancers, but the role of KIAA1429 in ES remains to be elucidated. The aim of the study was to investigate the role of KIAA1429 in ES.<br><br>METHODS: We performed a multi-omic screen including CRISPR-Cas9 functional genomic and transcriptomic approaches, and identified that KIAA1429 played a significant role in ES progression. Gene knockdown, quantitative real-time PCR (Q-RT-PCR), immunoblotting, CellTiter-Glo assays, clonogenic assays, a subcutaneous xenograft model and immunohistochemistry were used to assess the functional role of KIAA1429 in ES. We mainly conducted RNA sequencing (RNA-seq) in ES cells to analyze the downstream regulatory mechanism of KIAA1429. An integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq indicated the upstream regulatory mechanism of KIAA1429.<br><br>RESULTS: In vitro and in vivo CRISPR-Cas9 knockout screening identified KIAA1429 as an ES-dependent gene. Genetic suppression of KIAA1429 inhibited ES cell proliferation and tumorigenicity both in vitro and in vivo.&#xa0;Further studies revealed that KIAA1429 promotes ES tumorigenesis by regulating the ribosome-associated cell cycle and cancer-related inflammation. Interestingly, we found that STAT3 was a target of KIAA1429 and that a STAT3 inhibitor reduced KIAA1429 transcript levels, indicating positive feedback between KIAA1429 and STAT3. Finally, we found that NKX2-2 bound to the KIAA1429 promoter and transactivated KIAA1429.<br><br>CONCLUSION: Our study systematically analyzed ES-dependent epigenetic/transcriptional regulatory genes and identified KIAA1429 as a biomarker of tumor progression in ES, providing a potential therapeutic target for treating ES.},
}
@article {pmid37725011,
year = {2023},
author = {Mahmood, MA},
title = {Efficient A·T-to-C·G Base Editing via Adenine Transversion Editors.},
journal = {Cellular reprogramming},
volume = {25},
number = {5},
pages = {187-189},
doi = {10.1089/cell.2023.0094},
pmid = {37725011},
issn = {2152-4998},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics/metabolism ; Adenine ; Cytosine ; },
abstract = {Generating A-to-C transversions to correct defective alleles or introduce novel alleles has posed significant challenges. However, two recent studies focusing on adenine transversions have achieved successful A-to-C transversions in mouse embryos and plant cell. These remarkable accomplishments notably broaden the range of base editing and their applications both in fundamental research and in therapeutics.},
}
@article {pmid37723203,
year = {2023},
author = {Huang, X and Jia, H and Xu, J and Wang, Y and Wen, J and Wang, N},
title = {Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy.},
journal = {Nature plants},
volume = {9},
number = {10},
pages = {1591-1597},
pmid = {37723203},
issn = {2055-0278},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; Transgenes ; Plants, Genetically Modified/genetics ; *Herbicides ; Genome, Plant ; },
abstract = {Transgene-free plant genome editing in the T0 generation is highly desirable but challenging[1,2]. Here we achieved such a goal using a co-editing strategy via Agrobacterium-mediated transient expression of cytosine base editor to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, Cas12a/CRISPR RNA for editing gene(s) of interest, and green fluorescent protein for selecting transgene-free transformants. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 1.9% to 42.1% in tomato, tobacco, potato and citrus. This co-editing strategy is particularly useful for transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation.},
}
@article {pmid37722085,
year = {2023},
author = {Ma, JX and He, WY and Hua, HM and Zhu, Q and Zheng, GS and Zimin, AA and Wang, WF and Lu, YH},
title = {Development of a CRISPR/Cas9[D10A] Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces.},
journal = {ACS synthetic biology},
volume = {12},
number = {10},
pages = {3114-3123},
doi = {10.1021/acssynbio.3c00466},
pmid = {37722085},
issn = {2161-5063},
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Streptomyces/genetics/metabolism ; DNA ; *Actinomycetales/metabolism ; },
abstract = {Streptomycetes have a strong ability to produce a vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools have greatly facilitated strain improvement for target NP overproduction as well as novel NP discovery in Streptomyces. However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many Streptomyces strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9[D10A] nickase (nCas9)-based genome editing tool in the model strain Streptomyces coelicolor M145. We showed that in the presence of both targeting sgRNA and Cas proteins, utilization of nCas9 instead of Cas9 significantly reduced the toxicity to the host and greatly enhanced cell survival. Using this tool, we achieved deletion of single genes and gene clusters with efficiencies of 87-100 and 63-87%, and simultaneous deletion of two genes or gene clusters with efficiencies of 47 and 43%, respectively. The editing efficiency of nCas9 is comparable to that of the Cas9-mediated editing tool. Finally, the nCas9-based editing tool was successfully applied for genome editing in the industrial rapamycin-producing strain Streptomyces rapamycinicus, in which CRISPR/Cas9 cannot work well. We achieved the deletion of three tested genes with an efficiency of 27.2-30%. Collectively, the CRISPR/nCas9-based editing tool offers a convenient and efficient genetic modification system for the engineering of streptomycetes, particularly those with low DNA transformation efficiency.},
}
@article {pmid37714059,
year = {2023},
author = {Wu, X and Zhao, Y and Guo, C and Liu, C and Zhang, Q and Chen, Y and Liu, Y and Zhang, X},
title = {RatioCRISPR: A ratiometric biochip based on CRISPR/Cas12a for automated and multiplexed detection of heteroplasmic SNPs in mitochondrial DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {241},
number = {},
pages = {115676},
doi = {10.1016/j.bios.2023.115676},
pmid = {37714059},
issn = {1873-4235},
mesh = {*DNA, Mitochondrial/genetics ; Polymorphism, Single Nucleotide/genetics ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; Mitochondria ; Mutation ; },
abstract = {Mitochondrial genetic diseases are often characterized by heteroplasmic single nucleotide polymorphisms (SNPs) where both wild-type (WT) and mutant-type (MT) coexist, making detection of accurate SNP abundance critical for diagnosis. Here, we present RatioCRISPR, an automated ratiometric biochip sensor based on the CRISPR/Cas12a system for detecting multiple heteroplasmic SNPs in mitochondrial DNA (mtDNA). The ratiometric sensor output is only influenced by the relative abundance of WT and MT, with minimal impact from sample concentration. Biochips allow the simultaneous detection of multiple SNP sites for more accurate disease diagnosis. RatioCRISPR can accurately detect 8 samples simultaneously within 25 min with a limit of detection (LOD) of 15.7 aM. We successfully detected 13 simulated samples of three mtDNA point mutations (m.3460G>A, m.11778G>A, and m.14484T>C), which lead to Leber's hereditary optic neuropathy (LHON) and set a threshold (60%) of heteroplasmy to evaluate disease risk. This automated and accurate biosensor has broad applications in diagnosing multiple SNPs, especially those with heteroplasmic variations, making it an advanced and convenient tool for mtDNA disease diagnosis.},
}
@article {pmid37713292,
year = {2023},
author = {Richardson, RR and Steyert, M and Khim, SN and Crutcher, GW and Brandenburg, C and Robertson, CD and Romanowski, AJ and Inen, J and Altas, B and Poulopoulos, A},
title = {Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {447-461},
doi = {10.1089/crispr.2023.0036},
pmid = {37713292},
issn = {2573-1602},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; DNA Repair/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[[HE]], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.},
}
@article {pmid37710395,
year = {2023},
author = {Yin, Y and Xie, W and Xiong, M and Gao, Y and Liu, Q and Han, D and Ke, G and Zhang, XB},
title = {FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {62},
number = {44},
pages = {e202309837},
doi = {10.1002/anie.202309837},
pmid = {37710395},
issn = {1521-3773},
support = {22122403//National Natural Science Foundation of China/ ; 21890744//National Natural Science Foundation of China/ ; 21974087//National Natural Science Foundation of China/ ; 2021JJ10012//Natural Science Foundation of Hunan Province/ ; CX20220391//Postgraduate Scientific Research Innovation Project of Hunan Province/ ; },
mesh = {Cell Membrane ; DNA ; Membrane Fluidity ; Oligonucleotides ; Bioengineering ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; *Neoplasms/genetics ; },
abstract = {The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.},
}
@article {pmid37622407,
year = {2023},
author = {Zhao, J and Jiang, Y and Hoffmann, A and Tan, Y and Xiao, L},
title = {SeBLOS2 knockout via CRISPR/Cas9 leads to the loss of larval integument coloration in Spodoptera exigua (Lepidoptera: Noctuidae).},
journal = {Archives of insect biochemistry and physiology},
volume = {114},
number = {3},
pages = {e22040},
doi = {10.1002/arch.22040},
pmid = {37622407},
issn = {1520-6327},
support = {CX(21)3088//Jiangsu Agricultural Science and Technology Independent Innovation Fund/ ; CX(22)5003//Jiangsu Agricultural Science and Technology Independent Innovation Fund/ ; 31901889//National Natural Science Foundation of China/ ; 202108320141//China Scholarship Council/ ; CX(21)3088, CX(22)5003//Jiangsu Agricultural Science and Technology Independent Innovation Fund/ ; },
mesh = {Animals ; Spodoptera/genetics ; Larva/genetics ; *CRISPR-Cas Systems ; *Integumentary System ; Genotype ; },
abstract = {CRISPR/Cas9 technology is a precise and powerful tool for functionally exploring insect genes. The present study tested CRISPR/Cas9 as a way of undertaking effective gene mutagenesis in an important agricultural pest, the beet armyworm Spodoptera exigua. Based on a S. exigua transcriptome database, the entire complementary DNA sequence of SeBLOS2 encoding 140 amino acid residues was cloned. The gene was highly expressed in late larval stages (L3-L5). Using the CRISPR/Cas9 method, SeBLOS2 was knocked out by altering two sites in the coding region. This resulted in 70%-74% of the G0 generation (L4-L5) larvae displaying mosaic translucent integument. Four different mutations occurred at SeBLOS2-specific target sites, as demonstrated by further polymerase chain reaction-based genotypic analysis. Homozygote mutant L3 larvae were obtained in the G1 generation, with complete loss of white stripes and spots on their larval integument. These results demonstrate a crucial role of SeBLOS2 in integument pigmentation and suggest that the gene can act as a suitable nonlethal marker for functional research on genes in S. exigua and other Lepidopteran pests.},
}
@article {pmid37615574,
year = {2023},
author = {Zhang, L and He, W and Fu, R and Wang, S and Chen, Y and Xu, H},
title = {Guide-specific loss of efficiency and off-target reduction with Cas9 variants.},
journal = {Nucleic acids research},
volume = {51},
number = {18},
pages = {9880-9893},
pmid = {37615574},
issn = {1362-4962},
support = {R01 NS117668/NS/NINDS NIH HHS/United States ; R35GM137927/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Mutation ; Gene Editing ; },
abstract = {High-fidelity clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA-target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against wild-type SpCas9, we found that ∼20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15-18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in the REC3 domain account for the loss of efficiency. We also observed various degrees of sequence-dependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer learning-based computational framework for the prediction of on-target efficiency and off-target effects with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.},
}
@article {pmid37615478,
year = {2023},
author = {Liu, K and Sakuraba, Y and Ohtsuki, N and Yang, M and Ueda, Y and Yanagisawa, S},
title = {CRISPR/Cas9-mediated elimination of OsHHO3, a transcriptional repressor of three AMMONIUM TRANSPORTER1 genes, improves nitrogen use efficiency in rice.},
journal = {Plant biotechnology journal},
volume = {21},
number = {11},
pages = {2169-2172},
pmid = {37615478},
issn = {1467-7652},
support = {JPMJCR15O5//Core Research for Evolutional Science and Technology/ ; 22H04977//he Japan Society for the Promotion of Science KAKENHI/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Oryza/genetics/metabolism ; *Ammonium Compounds ; Nitrogen/metabolism ; Transcription Factors/genetics ; },
}
@article {pmid37535444,
year = {2023},
author = {Zhang, T and Zhang, W and Ding, C and Hu, Z and Fan, C and Zhang, J and Li, Z and Diao, S and Shen, L and Zhang, B and Liu, G and Su, X},
title = {A breeding strategy for improving drought and salt tolerance of poplar based on CRISPR/Cas9.},
journal = {Plant biotechnology journal},
volume = {21},
number = {11},
pages = {2160-2162},
pmid = {37535444},
issn = {1467-7652},
mesh = {*Salt Tolerance/genetics ; CRISPR-Cas Systems/genetics ; Droughts ; Plant Breeding ; Stress, Physiological ; Plant Proteins/genetics ; *Populus/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; },
}
@article {pmid37522511,
year = {2023},
author = {Yonehara, K and Kumakura, N and Motoyama, T and Ishihama, N and Dallery, JF and O'Connell, R and Shirasu, K},
title = {Efficient multiple gene knockout in Colletotrichum higginsianum via CRISPR/Cas9 ribonucleoprotein and URA3-based marker recycling.},
journal = {Molecular plant pathology},
volume = {24},
number = {11},
pages = {1451-1464},
pmid = {37522511},
issn = {1364-3703},
support = {JPMJAX20B4//Japan Science and Technology Agency/ ; 19KK0397//Japan Society for the Promotion of Science/ ; 23K05158//Japan Society for the Promotion of Science/ ; JP18K14440//Japan Society for the Promotion of Science/ ; JP20H05909//Japan Society for the Promotion of Science/ ; JP20K15500//Japan Society for the Promotion of Science/ ; JP22H00364//Japan Society for the Promotion of Science/ ; },
mesh = {Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics/microbiology ; Virulence Factors/genetics ; *Colletotrichum/genetics ; },
abstract = {Colletotrichum higginsianum is a hemibiotrophic pathogen that causes anthracnose disease on crucifer hosts, including Arabidopsis thaliana. Despite the availability of genomic and transcriptomic information and the ability to transform both organisms, identifying C. higginsianum genes involved in virulence has been challenging due to recalcitrance to gene targeting and redundancy of virulence factors. To overcome these obstacles, we developed an efficient method for multiple gene disruption in C. higginsianum by combining CRISPR/Cas9 and a URA3-based marker recycling system. Our method significantly increased the efficiency of gene knockout via homologous recombination by introducing genomic DNA double-strand breaks. We demonstrated the applicability of the URA3-based marker recycling system for multiple gene targeting in the same strain. Using our technology, we successfully targeted two melanin biosynthesis genes, SCD1 and PKS1, which resulted in deficiency in melanization and loss of pathogenicity in the mutants. Our findings demonstrate the effectiveness of our methods in analysing virulence factors in C. higginsianum, thus accelerating research on plant-fungus interactions.},
}
@article {pmid37486520,
year = {2023},
author = {Gonzalez, EA and Nader, H and Siebert, M and Suarez, DA and Alméciga-Díaz, CJ and Baldo, G},
title = {Genome Editing Tools for Lysosomal Storage Disorders.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {127-155},
pmid = {37486520},
issn = {0065-2598},
mesh = {Humans ; *Gene Editing ; Genetic Therapy ; Genome ; *Lysosomal Storage Diseases/genetics/therapy ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing has multiple applications in the biomedical field. They can be used to modify genomes at specific locations, being able to either delete, reduce, or even enhance gene transcription and protein expression. Here, we summarize applications of genome editing used in the field of lysosomal disorders. We focus on the development of cell lines for study of disease pathogenesis, drug discovery, and pathogenicity of specific variants. Furthermore, we highlight the main studies that use gene editing as a gene therapy platform for these disorders, both in preclinical and clinical studies. We conclude that gene editing has been able to change quickly the scenario of these disorders, allowing the development of new therapies and improving the knowledge on disease pathogenesis. Should they confirm their hype, the first gene editing-based products for lysosomal disorders could be available in the next years.},
}
@article {pmid37486519,
year = {2023},
author = {Burle-Caldas, GA and Grazielle-Silva, V and Faustino, LP and Teixeira, SMR},
title = {CRISPR Genome Editing and the Study of Chagas Disease.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {111-125},
pmid = {37486519},
issn = {0065-2598},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Chagas Disease/genetics/parasitology ; *Trypanosoma cruzi/genetics ; },
abstract = {Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an illness that affects 6-8 million people worldwide and is responsible for approximately 50,000 deaths per year. Despite intense research efforts on Chagas disease and its causative agent, there is still a lack of effective treatments or strategies for disease control. Although significant progress has been made toward the elucidation of molecular mechanisms involved in host-parasite interactions, particularly immune evasion mechanisms, a deeper understanding of these processes has been hindered by a lack of efficient genetic manipulation protocols. One major challenge is the fact that several parasite virulence factors are encoded by multigene families, which constitute a distinctive feature of the T. cruzi genome. The recent advent of the CRISPR/Cas9 technology represented an enormous breakthrough in the studies involving T. cruzi genetic manipulation compared to previous protocols that are poorly efficient and required a long generation time to develop parasite mutants. Since the first publication of CRISPR gene editing in T. cruzi, in 2014, different groups have used distinct protocols to generated knockout mutants, parasites overexpressing a protein or expressing proteins with sequence tags inserted in the endogenous gene. Importantly, CRISPR gene editing allowed generation of parasite mutants with gene disruption in multi-copy gene families. We described four main strategies used to edit the T. cruzi genome and summarized a large list of studies performed by different groups in the past 7 years that are addressing several mechanisms involved with parasite proliferation, differentiation, and survival strategies within its different hosts.},
}
@article {pmid37486513,
year = {2023},
author = {Contiliani, DF and Moraes, VN and Passos, GA and Pereira, TC},
title = {What Is the CRISPR System and How It Is Used?.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {1-11},
pmid = {37486513},
issn = {0065-2598},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/metabolism ; RNA ; DNA Breaks, Double-Stranded ; DNA/genetics ; },
abstract = {CRISPR is a revolutionary gene editing technology that has enabled scientists worldwide to explore the cell's genetic blueprint in an unprecedented easy way. In this chapter, we will briefly present the history behind the development of this innovative tool, how it emerged from a natural bacterial mechanism for antiviral defense, its key components (Cas9 endonuclease and single guide RNA), mode of action (DNA cleavage and repair via NHEJ or HDR), and versatility (acting on single- or double-stranded DNA or RNA) for diverse purposes beyond gene editing such as stochastic marking, digital encoding, high-fidelity SNP genotyping, programmed chromosome fission/fusion, gene mapping, nucleic acid detection, regulation of gene expression, DNA/RNA labeling or tracking, and more.},
}
@article {pmid37479866,
year = {2023},
author = {Nishiguchi, M and Futamura, N and Endo, M and Mikami, M and Toki, S and Katahata, SI and Ohmiya, Y and Konagaya, KI and Nanasato, Y and Taniguchi, T and Maruyama, TE},
title = {CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11779},
pmid = {37479866},
issn = {2045-2322},
support = {JP20H03037//JSPS KAKENHI/ ; },
mesh = {Humans ; *Rhinitis, Allergic, Seasonal/genetics ; Trees ; *Cryptomeria/genetics ; CRISPR-Cas Systems ; Pollen/genetics ; *Arabidopsis ; },
abstract = {Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.},
}
@article {pmid37432138,
year = {2023},
author = {Wang, Y and Fu, L and Tao, D and Han, X and Xu, B and Deng, M and Li, S and Zhao, C and Li, X and Zhao, S and Gong, P and Yang, Y and Khazalwa, EM and Ma, Y and Ruan, J and Li, C and Xie, S},
title = {Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2051-2060},
doi = {10.1021/acssynbio.3c00089},
pmid = {37432138},
issn = {2161-5063},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Point-of-Care Systems ; Biological Assay ; DNA, Single-Stranded/genetics ; RNA ; Nucleic Acid Amplification Techniques ; },
abstract = {The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.},
}
@article {pmid37422184,
year = {2023},
author = {Van Hove, B and De Wannemaeker, L and Missiaen, I and Maertens, J and De Mey, M},
title = {Taming CRISPRi: Dynamic range tuning through guide RNA diversion.},
journal = {New biotechnology},
volume = {77},
number = {},
pages = {50-57},
doi = {10.1016/j.nbt.2023.07.001},
pmid = {37422184},
issn = {1876-4347},
mesh = {*CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; RNA ; },
abstract = {CRISPRi is a powerful technique to repress gene expression in a targeted and highly efficient manner. However, this potency is a double-edged sword in inducible systems, as even leaky expression of guide RNA results in a repression phenotype, complicating applications such as dynamic metabolic engineering. We evaluated three methods to enhance the controllability of CRISPRi by modulating the level of free and DNA-bound guide RNA complexes. Overall repression can be attenuated through rationally designed mismatches in the reversibility determining region of the guide RNA sequence; decoy target sites can selectively modulate repression at low levels of induction; and the implementation of feedback control not only enhances the linearity of induction, but broadens the dynamic range of the output as well. Furthermore, feedback control significantly enhances the recovery rate after induction is removed. Used in combination, these techniques enable the fine-tuning of CRISPRi to meet restrictions imposed by the target and match the input signal required for induction.},
}
@article {pmid37402100,
year = {2023},
author = {Aquino-Jarquin, G},
title = {Compelling Reasons to Perform Basic and Pre-clinical Research on Germline Genome Editing.},
journal = {Stem cell reviews and reports},
volume = {19},
number = {7},
pages = {2554-2555},
pmid = {37402100},
issn = {2629-3277},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Germ Cells ; },
}
@article {pmid37311684,
year = {2023},
author = {Nishimura, M and Takeyama, H and Hosokawa, M},
title = {Enhancing the sensitivity of bacterial single-cell RNA sequencing using RamDA-seq and Cas9-based rRNA depletion.},
journal = {Journal of bioscience and bioengineering},
volume = {136},
number = {2},
pages = {152-158},
doi = {10.1016/j.jbiosc.2023.05.010},
pmid = {37311684},
issn = {1347-4421},
mesh = {*Escherichia coli/genetics ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, RNA/methods ; RNA, Ribosomal ; Gene Expression Profiling/methods ; Bacteria/genetics ; Single-Cell Analysis/methods ; },
abstract = {Bacterial populations exhibit heterogeneity in gene expression, which facilitates their survival and adaptation to unstable and unpredictable environments through the bet-hedging strategy. However, unraveling the rare subpopulations and heterogeneity in gene expression using population-level gene expression analysis remains a challenging task. Single-cell RNA sequencing (scRNA-seq) has the potential to identify rare subpopulations and capture heterogeneity in bacterial populations, but standard methods for scRNA-seq in bacteria are still under development, mainly due to differences in mRNA abundance and structure between eukaryotic and prokaryotic organisms. In this study, we present a hybrid approach that combines random displacement amplification sequencing (RamDA-seq) with Cas9-based rRNA depletion for scRNA-seq in bacteria. This approach allows cDNA amplification and subsequent sequencing library preparation from low-abundance bacterial RNAs. We evaluated its sequenced read proportion, gene detection sensitivity, and gene expression patterns from the dilution series of total RNA or the sorted single Escherichia coli cells. Our results demonstrated the detection of more than 1000 genes, about 24% of the genes in the E. coli genome, from single cells with less sequencing effort compared to conventional methods. We observed gene expression clusters between different cellular proliferation states or heat shock treatment. The approach demonstrated high detection sensitivity in gene expression analysis compared to current bacterial scRNA-seq methods and proved to be an invaluable tool for understanding the ecology of bacterial populations and capturing the heterogeneity of bacterial gene expression.},
}
@article {pmid37309240,
year = {2023},
author = {Chen, Q and An, B and Peng, X and Wu, Y and Peng, M and Zhang, C and He, Y and Sang, H and Kong, Q},
title = {Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans.},
journal = {Journal of basic microbiology},
volume = {63},
number = {10},
pages = {1095-1105},
doi = {10.1002/jobm.202300102},
pmid = {37309240},
issn = {1521-4028},
support = {CXTDA2017038//Jiangsu Dermatology Innovation Team Foundation/ ; },
mesh = {*Cryptococcus neoformans/genetics/metabolism ; RNA Interference ; CRISPR-Cas Systems ; Melanins ; Dihydroxyphenylalanine ; RNA, Small Interfering ; },
abstract = {The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR-Cas9. The RNAi system was constructed by pSilencer 4.1-CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR-Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real-time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR-Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR-Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait-regulating genes of other serotypes of C. neoformans.},
}
@article {pmid37290639,
year = {2023},
author = {Chacko, L and Chaudhary, A and Singh, B and Dewanjee, S and Kandimalla, R},
title = {CRISPR-Cas9 in Alzheimer's disease: Therapeutic trends, modalities, and challenges.},
journal = {Drug discovery today},
volume = {28},
number = {8},
pages = {103652},
doi = {10.1016/j.drudis.2023.103652},
pmid = {37290639},
issn = {1878-5832},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Alzheimer Disease/drug therapy/genetics ; Genetic Therapy ; },
abstract = {Alzheimer's disease (AD) is a progressive neurodegenerative disorder with no known cure, which has prompted the exploration of novel therapeutic approaches. The clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) tool has generated significant interest for its potential in AD therapeutics by correcting faulty genes. Our report comprehensively reviews emerging applications for CRISPR-Cas9 in developing in vitro and in vivo models for AD research and therapeutics. We further assess its ability to identify and validate genetic markers and potential therapeutic targets for AD. Moreover, we review the current challenges and delivery strategies for the in vivo application of CRISPR-Cas9 in AD therapeutics.},
}
@article {pmid37269452,
year = {2023},
author = {Tang, G and Man, H and Wang, J and Zou, J and Zhao, J and Han, J},
title = {An oxidoreductase gene CtnD involved in citrinin biosynthesis in Monascus purpureus verified by CRISPR/Cas9 gene editing and overexpression.},
journal = {Mycotoxin research},
volume = {39},
number = {3},
pages = {247-259},
pmid = {37269452},
issn = {1867-1632},
support = {qiankehe support 2019-2776//Science and Technology Program of Guizhou Province/ ; qiankehe basic 2019-1035//Science and Technology Program of Guizhou Province/ ; },
mesh = {*Citrinin/metabolism ; *Monascus/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Oxidoreductases/genetics/metabolism ; Pigments, Biological/metabolism ; },
abstract = {Monascus produces a kind of mycotoxin, citrinin, whose synthetic pathway is still not entirely clear. The function of CtnD, a putative oxidoreductase located upstream of pksCT in the citrinin gene cluster, has not been reported. In this study, the CtnD overexpressed strain and the Cas9 constitutively expressed chassis strain were obtained by genetic transformation mediated by Agrobacterium tumefaciens. The pyrG and CtnD double gene-edited strains were then obtained by transforming the protoplasts of the Cas9 chassis strain with in vitro sgRNAs. The results showed that overexpression of CtnD resulted in significant increases in citrinin content of more than 31.7% and 67.7% in the mycelium and fermented broth, respectively. The edited CtnD caused citrinin levels to be reduced by more than 91% in the mycelium and 98% in the fermented broth, respectively. It was shown that CtnD is a key enzyme involved in citrinin biosynthesis. RNA-Seq and RT-qPCR showed that the overexpression of CtnD had no significant effect on the expression of CtnA, CtnB, CtnE, and CtnF but led to distinct changes in the expression of acyl-CoA thioesterase and two MFS transporters, which may play an unknown role in citrinin metabolism. This study is the first to report the important function of CtnD in M. purpureus through a combination of CRISPR/Cas9 editing and overexpression.},
}
@article {pmid37243510,
year = {2023},
author = {Perroud, PF and Guyon-Debast, A and Casacuberta, JM and Paul, W and Pichon, JP and Comeau, D and Nogué, F},
title = {Improved prime editing allows for routine predictable gene editing in Physcomitrium patens.},
journal = {Journal of experimental botany},
volume = {74},
number = {19},
pages = {6176-6187},
pmid = {37243510},
issn = {1460-2431},
support = {//Limagrain Europe/ ; //CRISPLUS/ ; //The Institut Jean-Pierre Bourgin/ ; //LabEx Saclay Plant Sciences/ ; (ANR)-10-LABX-0040-SPS//Agence Nationale de la Recherche/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA-Directed DNA Polymerase ; },
abstract = {Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3' extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.},
}
@article {pmid37014117,
year = {2023},
author = {Karmacharya, A and Li, D and Leng, Y and Shi, G and Liu, Z and Yang, S and Du, Y and Dai, W and Zhong, S},
title = {Targeting Disease Susceptibility Genes in Wheat Through wide Hybridization with Maize Expressing Cas9 and Guide RNA.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {36},
number = {9},
pages = {554-557},
doi = {10.1094/MPMI-01-23-0004-SC},
pmid = {37014117},
issn = {0894-0282},
mesh = {*CRISPR-Cas Systems/genetics ; *Triticum/genetics ; Zea mays/genetics ; Disease Susceptibility ; RNA ; },
abstract = {Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight and tan spot, Septoria nodorum blotch, and spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15 to 33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool, not only for targeting susceptibility genes to improve disease resistance without regulatory issues, but also for understanding gene function in wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.},
}
@article {pmid36853667,
year = {2023},
author = {Flumens, D and Campillo-Davo, D and Janssens, I and Roex, G and De Waele, J and Anguille, S and Lion, E},
title = {One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102112},
pmid = {36853667},
issn = {2666-1667},
mesh = {Humans ; *Receptors, Antigen, T-Cell, alpha-beta/genetics ; *CD8-Positive T-Lymphocytes/metabolism ; RNA ; CRISPR-Cas Systems/genetics ; Electroporation ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRβ chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8[+] T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRα and TCRβ constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.},
}
@article {pmid36574341,
year = {2023},
author = {Teng, ACT and Tavassoli, M and Shrestha, S and Marks, RM and McFadden, MJ and Evagelou, SL and Lindsay, K and Vandenbelt, A and Li, W and Ivakine, E and Cohn, R and Santerre, JP and Gramolini, AO},
title = {An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {101933},
pmid = {36574341},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Staphylococcus aureus/genetics ; Cost-Benefit Analysis ; Reproducibility of Results ; },
abstract = {Here, we describe a protocol for purifying functional clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Staphylococcus aureus within 24 h and over 90% purity. SaCas9 purification begins with immobilized metal affinity chromatography, followed by cation exchange chromatography, and ended with centrifugal concentrators. The simplicity, cost-effectiveness, and reproducibility of such protocols will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating CRISPR research.},
}
@article {pmid36516053,
year = {2023},
author = {Laprie-Sentenac, M and Cretet-Rodeschini, C and Menger, L},
title = {Optimized protocol to generate genome-wide inactivated Cas9-expressing murine T cells.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {101922},
pmid = {36516053},
issn = {2666-1667},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; T-Lymphocytes ; Gene Library ; Retroviridae/genetics ; },
abstract = {In vivo genome-wide CRISPR screens in primary T cells allow the systematic and unbiased identification of non-redundant regulatory mechanisms shaping immune responses. Here, we present an optimized protocol for efficient generation of a pool of genome-wide inactivated Cas9-expressing T cells using a retroviral library of sgRNA. We detail the process of large-scale viral production and library integration in activated murine T cells as well as the two-step PCR approach for sgRNA recovery and abundance evaluation. For complete details on the use and execution of this protocol, please refer to Sutra Del Galy et al. (2020).},
}
@article {pmid34971344,
year = {2023},
author = {Qi-Xin, G and Lu, X and Hao, B and Guo-Hong, C and Guo-Bin, C},
title = {PAZ domain is critical for spermatogenesis in chicken.},
journal = {Animal biotechnology},
volume = {34},
number = {4},
pages = {1102-1111},
doi = {10.1080/10495398.2021.2011742},
pmid = {34971344},
issn = {1532-2378},
mesh = {Female ; Male ; Animals ; *Chickens/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Semen ; Spermatogenesis/genetics ; Spermatozoa ; CRISPR-Cas Systems ; },
abstract = {Piwi-like protein 1 (PIWIL1) plays a crucial role in stem cell proliferation, embryogenesis, growth, and development. We aimed to unravel the function of PIWIL1 and its Piwi/Argonaute/Zwille (PAZ) domain in chicken embryogenesis. The expression of PIWI1 at different stages of spermatogenesis was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the PAZ domain was mutated based on its 3D structure model using the clustered regularly interspaced short palindromic repeats Cas9 (CRISPR/Cas9) technology. The results indicated that PIWIL1 mRNA was specifically expressed in spermatogonium cells undergoing meiosis. After targeting the PAZ domain (300-370 amino acid residues), we obtained two mutant DF-1 cell clones with 23-bp and 8-bp deletions. Injection of the pCMV-Cas9-puro-sgRNA-2 construct into 2.5-day embryos resulted in generation of 19 different PAZ mutants (13 males and 6 females), which showed delayed hatching, reduced quality of semen, and decreased expression of PIWIL1 and SOX2 at embryonic days 5 and 18. However, we could not obtain PAZ double knockout (KO) chickens by crossing of the F0 generation, suggesting that PAZ double KO may halt embryonic development. Our results indicate that PIWIL1 plays an important role in meiosis and that PAZ mutations can lead to decreased sperm quality, whereas its double KO may arrest embryogenesis in chicken.},
}
@article {pmid32707002,
year = {2023},
author = {Challagulla, A and Jenkins, KA and O'Neil, TE and Morris, KR and Wise, TG and Tizard, ML and Bean, AGD and Schat, KA and Doran, TJ},
title = {Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.},
journal = {Animal biotechnology},
volume = {34},
number = {4},
pages = {775-784},
doi = {10.1080/10495398.2020.1789869},
pmid = {32707002},
issn = {1532-2378},
mesh = {Animals ; Chick Embryo ; Female ; Male ; *Chickens/genetics ; *CRISPR-Cas Systems/genetics ; Transfection ; Animals, Genetically Modified/genetics ; Gene Editing/methods ; Germ Cells/metabolism ; },
abstract = {Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.},
}
@article {pmid37840739,
year = {2023},
author = {Han, R and Hong, Y and Xu, R and Guo, W and Zhang, M and Lu, Z and Han, Q and Mo, Z and Dan, X and Li, Y},
title = {Genomic evidence of genetic diversity and functional evolution in Flavobacterium columnare.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1240471},
pmid = {37840739},
issn = {1664-302X},
abstract = {Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish. Columnaris disease can cause heavy economic losses in aquaculture. In this study, whole-genome sequencing was used to characterize this pathogen. F. columnare isolate AH-01 had a circular chromosome and plasmid that encoded a total of 3,022 genes. Isolate GX-01 only had a circular chromosome and encoded 2,965 genes. Genomic islands, prophage regions, and CRISPR/Cas systems were identified in both genomes. Both genomes presented evidence of gene variation and horizontal transfer, both of which are the essential components of genetic diversity, genome plasticity, and functional evolution. Single-gene phylogeny and comparative genome analyses were performed to investigate the variation and evolution of this pathogen. Genetic analysis of 16S rRNA and housekeeping gene sequences significantly clustered 55 F. columnare isolates into four clades. The intragroup identity of the 16S rRNA gene exceeded 99%, while the intergroup identity was below the species delineation threshold. We discovered significant translocation, inversion, and rearrangement events that influenced local synteny within each group. Notably, the observed alignments varied considerably among all the studied groups. The core genomes of all strains with available sequences comprised 747 genes, corresponding to approximately 25% of the genome. Core genome multilocus sequence typing, genome-wide orthology and phylogenetic analyses, and average nucleotide identity suggested that the currently existing F. columnare was an assemblage of several distinct species, with levels of divergence at least equivalent to those between recognized bacterial species. The present investigation provided genomic evidence of gene variation and horizontal transfer, which were the basis of genetic diversity, genome plasticity, and functional evolution. The findings supported a proposed new taxonomic perspective on F. columnare.},
}
@article {pmid37839151,
year = {2023},
author = {De Pablo-Moreno, JA and Miguel-Batuecas, A and Rodríguez-Merchán, EC and Liras, A},
title = {Treatment of congenital coagulopathies, from biologic to biotechnological drugs: The relevance of gene editing (CRISPR/Cas).},
journal = {Thrombosis research},
volume = {231},
number = {},
pages = {99-111},
doi = {10.1016/j.thromres.2023.10.001},
pmid = {37839151},
issn = {1879-2472},
abstract = {Congenital coagulopathies have, throughout the history of medicine, been a focus of scientific study and of great interest as they constitute an alteration of one of the most important and conserved pathways of evolution. The first therapeutic strategies developed to address them were aimed at restoring the blood components lost during hemorrhage by administering whole blood or plasma. Later on, the use of cryoprecipitates was a significant breakthrough as it made it possible to decrease the volumes of blood infused. In the 1970' and 80', clotting factor concentrates became the treatment and, from the 1990's to the present day, recombinant factors -with increasingly longer half-lives- have taken over as the treatment of choice for certain coagulopathies in a seamless yet momentous transition from biological to biotechnological drugs. The beginning of this century, however, saw the emergence of new advanced (gene and cell) treatments, which are currently transforming the therapeutic landscape. The possibility to use cells and viruses as well as specific or bispecific antibodies as medicines is likely to spark a revolution in the world of pharmacology where therapies will be individualized and have long-term effects. Specifically, attention is nowadays focused on the development of gene editing strategies, chiefly those based on CRISPR/Cas technology. Rare coagulopathies such as hemophilia A and B, or even ultra-rare ones such as factor V deficiency, could be among those deriving the greatest benefit from these new developments.},
}
@article {pmid36814841,
year = {2023},
author = {Zhang, W and Wang, R and Kong, D and Peng, F and Chen, M and Zeng, W and Giaume, F and He, S and Zhang, H and Wang, Z and Kyozuka, J and Zhu, JK and Fornara, F and Miki, D},
title = {Precise and heritable gene targeting in rice using a sequential transformation strategy.},
journal = {Cell reports methods},
volume = {3},
number = {1},
pages = {100389},
pmid = {36814841},
issn = {2667-2375},
mesh = {CRISPR-Cas Systems/genetics ; *Oryza/genetics ; RNA, Guide, CRISPR-Cas Systems ; Plant Breeding ; Gene Targeting ; *Arabidopsis/genetics ; },
abstract = {Gene targeting (GT) is a powerful tool for modifying endogenous genomic sequences of interest, such as sequence replacement and gene knockin. Although the efficiency of GT is extremely low in higher plants, engineered sequence-specific nucleases (SSNs)-mediated double-strand breaks (DSBs) can improve GT frequency. We recently reported a CRISPR-Cas9-mediated approach for heritable GT in Arabidopsis, called the "sequential transformation" strategy. For efficient establishment of GT via the sequential transformation method, strong Cas9 activity and robust DSBs are required in the plant cells being infected with Agrobacterium carrying sgRNA and donor DNA. Accordingly, we generated two independent parental lines with maize Ubiquitin 1 promoter-driven Cas9 and established sequential transformation-mediated GT in the Japonica rice cultivar Oryza sativa Nipponbare. We achieved precise GFP knockin into the endogenous OsFTL1 and OsROS1a loci. We believe that our GT technology could be widely utilized in rice research and breeding applications.},
}
@article {pmid36814834,
year = {2023},
author = {Vinceti, A and De Lucia, RR and Cremaschi, P and Perron, U and Karakoc, E and Mauri, L and Fernandez, C and Kluczynski, KH and Anderson, DS and Iorio, F},
title = {An interactive web application for processing, correcting, and visualizing genome-wide pooled CRISPR-Cas9 screens.},
journal = {Cell reports methods},
volume = {3},
number = {1},
pages = {100373},
pmid = {36814834},
issn = {2667-2375},
mesh = {*CRISPR-Cas Systems/genetics ; *Genome ; Genomics/methods ; Software ; },
abstract = {A limitation of pooled CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes arising from copy-number-amplified genomics regions. To solve this issue, we previously developed CRISPRcleanR: a computational method implemented as R/python package and in a dockerized version. CRISPRcleanR detects and corrects biased responses to CRISPR-Cas9 targeting in an unsupervised fashion, accurately reducing false-positive signals while maintaining sensitivity in identifying relevant genetic dependencies. Here, we present CRISPRcleanR [WebApp] , a web application enabling access to CRISPRcleanR through an intuitive interface. CRISPRcleanR [WebApp] removes the complexity of R/python language user interactions; provides user-friendly access to a complete analytical pipeline, not requiring any data pre-processing and generating gene-level summaries of essentiality with associated statistical scores; and offers a range of interactively explorable plots while supporting a more comprehensive range of CRISPR guide RNAs' libraries than the original package. CRISPRcleanR [WebApp] is available at https://crisprcleanr-webapp.fht.org/.},
}
@article {pmid37662258,
year = {2023},
author = {Hall, AN and Hall, BW and Kinney, KJ and Olsen, GG and Banta, AB and Noguera, DR and Donohue, TJ and Peters, JM},
title = {Tools for Genetic Engineering and Gene Expression Control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37662258},
support = {T32 GM007133/GM/NIGMS NIH HHS/United States ; },
abstract = {Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7 integration with promoters from our library, we establish CRISPR interference systems for N. aromaticivorans and R. sphaeroides that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.},
}
@article {pmid37546762,
year = {2023},
author = {Sinan, S and Appleby, NM and Russell, R},
title = {Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37546762},
abstract = {CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the affinities of both the precursor and mature forms of the crRNA, while deletion of an upstream sequence had no significant effect on affinity of the pre-crRNA. After processing, the mature crRNA remains very tightly bound to Cas12a, with a half-life of ~1 day and a Kd value of 60 pM. Addition of a 5'-phosphoryl group, which is normally lost during the processing reaction as the scissile phosphate, tightens binding of the mature crRNA by ~10-fold by accelerating binding and slowing dissociation. Using a direct competition assay, we found that pre-crRNA binding specificity is robust to other changes in RNA sequence, including tested changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in some genome editing applications.},
}
@article {pmid36747634,
year = {2023},
author = {Benetta, ED and López-Denman, AJ and Li, HH and Masri, RA and Brogan, DJ and Bui, M and Yang, T and Li, M and Dunn, M and Klein, MJ and Jackson, S and Catalan, K and Blasdell, KR and Tng, P and Antoshechkin, I and Alphey, LS and Paradkar, PN and Akbari, OS},
title = {Engineered Antiviral Sensor Targets Infected Mosquitoes.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {36747634},
abstract = {Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.},
}
@article {pmid37838676,
year = {2023},
author = {Liu, C and Yue, Y and Xue, Y and Zhou, C and Ma, Y},
title = {CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {211},
pmid = {37838676},
issn = {1475-2859},
support = {2018YFA0900200//National Key R&amp;D Program of China/ ; 31870789//National Natural Science Foundation of China/ ; },
abstract = {BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis.<br><br>RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50&#xa0;kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system.<br><br>CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.},
}
@article {pmid37836188,
year = {2023},
author = {Bulle, M and Sheri, V and Aileni, M and Zhang, B},
title = {Chloroplast Genome Engineering: A Plausible Approach to Combat Chili Thrips and Other Agronomic Insect Pests of Crops.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {19},
pages = {},
pmid = {37836188},
issn = {2223-7747},
abstract = {The world population's growing demand for food is expected to increase dramatically by 2050. The agronomic productivity for food is severely affected due to biotic and abiotic constraints. At a global level, insect pests alone account for ~20% loss in crop yield every year. Deployment of noxious chemical pesticides to control insect pests always has a threatening effect on human health and environmental sustainability. Consequently, this necessitates for the establishment of innovative, environmentally friendly, cost-effective, and alternative means to mitigate insect pest management strategies. According to a recent study, using chloroplasts engineered with double-strand RNA (dsRNA) is novel successful combinatorial strategy deployed to effectively control the most vexing pest, the western flower thrips (WFT: Frankliniella occidentalis). Such biotechnological avenues allowed us to recapitulate the recent progress of research methods, such as RNAi, CRISPR/Cas, mini chromosomes, and RNA-binding proteins with plastid engineering for a plausible approach to effectively mitigate agronomic insect pests. We further discussed the significance of the maternal inheritance of the chloroplast, which is the major advantage of chloroplast genome engineering.},
}
@article {pmid37834437,
year = {2023},
author = {Sun, K and Liu, Y and Zhao, W and Ma, B and Zhang, M and Yu, X and Ye, Z},
title = {Prokaryotic Argonaute Proteins: A New Frontier in Point-of-Care Viral Diagnostics.},
journal = {International journal of molecular sciences},
volume = {24},
number = {19},
pages = {},
pmid = {37834437},
issn = {1422-0067},
support = {2021YFF0600800//GRANTS FROM THE NATIONAL KEY RESEARCH AND DEVELOPMENT PROGRAMS OF CHINA/ ; },
abstract = {The recent pandemic of SARS-CoV-2 has underscored the critical need for rapid and precise viral detection technologies. Point-of-care (POC) technologies, which offer immediate and accurate testing at or near the site of patient care, have become a cornerstone of modern medicine. Prokaryotic Argonaute proteins (pAgo), proficient in recognizing target RNA or DNA with complementary sequences, have emerged as potential game-changers. pAgo present several advantages over the currently popular CRISPR/Cas systems-based POC diagnostics, including the absence of a PAM sequence requirement, the use of shorter nucleic acid molecules as guides, and a smaller protein size. This review provides a comprehensive overview of pAgo protein detection platforms and critically assesses their potential in the field of viral POC diagnostics. The objective is to catalyze further research and innovation in pAgo nucleic acid detection and diagnostics, ultimately facilitating the creation of enhanced diagnostic tools for clinic viral infections in POC settings.},
}
@article {pmid37833755,
year = {2023},
author = {Zheng, Y and Fu, H and Chen, J and Li, J and Bian, Y and Hu, P and Lei, L and Liu, Y and Yang, J and Peng, W},
title = {Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {208},
pmid = {37833755},
issn = {1475-2859},
support = {2019BJH021//The Innovation Base for Introducing Talents of Discipline of Hubei Province/ ; 2019BJH021//The Innovation Base for Introducing Talents of Discipline of Hubei Province/ ; SKLBEE2020021//Open Project Funding of the State Key Laboratory of Biocatalysis and Enzyme Engineering/ ; SKLBEE2020021//Open Project Funding of the State Key Laboratory of Biocatalysis and Enzyme Engineering/ ; SKLBEE2020021//Open Project Funding of the State Key Laboratory of Biocatalysis and Enzyme Engineering/ ; 2021YFC2100300//The National Key Research and Development Program of China/ ; 2021YFC2100300//The National Key Research and Development Program of China/ ; 2021BCA113//The Key R&amp;D projects in Hubei Province/ ; },
abstract = {BACKGROUND: Zymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis.<br><br>RESULTS: We constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong Pgap promoter from a plasmid could render the Z. mobilis ZM4 cells sufficient sensitivity to 4-CP. The clmPheS-expressing cells were assayed to be extremely sensitive to 0.2 mM 4-CP. Subsequently, the clmPheS-assisted counterselection endowed fast curing of genome engineering plasmids immediately after obtaining the desired mutants, shortening the time of every two rounds of multiplex chromosome editing by at least 9 days, and enabled the development of a strategy for scarless modification of the native Z. mobilis ZM4 plasmids.<br><br>CONCLUSIONS: This study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.},
}
@article {pmid37833505,
year = {2023},
author = {Trasanidou, D and Potocnik, A and Barendse, P and Mohanraju, P and Bouzetos, E and Karpouzis, E and Desmet, A and van Kranenburg, R and van der Oost, J and Staals, RHJ and Mougiakos, I},
title = {Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {1042},
pmid = {37833505},
issn = {2399-3642},
support = {FZM 083-2/2018-2019//Alexander S. Onassis Public Benefit Foundation (Onassis Foundation)/ ; },
abstract = {Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.},
}
@article {pmid37833198,
year = {2023},
author = {Schmitt, DS and Siegel, SD and Selle, K},
title = {Applications of designer phage encoding recombinant gene payloads.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.09.008},
pmid = {37833198},
issn = {1879-3096},
abstract = {Advances in genetic engineering, synthetic biology, and DNA sequencing have transformed the re-emergent therapeutic bacteriophage field. The increasing rate of multidrug resistant (MDR) infections and the speed at which new bacteriophages can be isolated, sequenced, characterized, and engineered has reinvigorated phage therapy and unlocked new applications of phages for modulating bacteria. The methods used to genetically engineer bacteriophages are undergoing significant development, but identification of heterologous gene payloads with desirable activity and determination of their impact on bacteria or human cells in translationally relevant applications remain underexplored areas. Here, we discuss and categorize recombinant gene payloads for their potential outcome on phage-bacteria interactions when genetically engineered into phage genomes for expression in their bacterial hosts.},
}
@article {pmid37827669,
year = {2023},
author = {Zhu, Y and Zheng, X and Zhou, S and Xiao, W and Sun, X and Zhou, J and Qian, F and Zhang, T and Sheng, Y and Hu, J},
title = {A dual amplification-based CRISPR/Cas12a biosensor for sensitive detection of miRNA in prostate cancer.},
journal = {Analytica chimica acta},
volume = {1279},
number = {},
pages = {341769},
doi = {10.1016/j.aca.2023.341769},
pmid = {37827669},
issn = {1873-4324},
abstract = {MicroRNA (miRNA) has gained significant attention as a potential biomarker for cancer clinics, and there is an urgent need for developing sensing strategies with high selectivity, sensitivity, and low background. In vitro diagnosis based on Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated protein (CRISPR/Cas) technology could simplify the detection procedure, improve sensitivity and selectivity, and has broad application prospects as the next-generation molecular diagnosis technology. We propose a novel dual signal amplification strategy, called CENTER, which integrates the CRISPR/Cas12a system, an entropy-driven DNA signaling network, and strand displacement amplification to achieve ultrasensitive detection of miR-141, a potential marker for prostate cancer. The experimental results demonstrate that CENTER can distinguish single nucleotide mutations, and the strategy exhibits a good linear calibration curve ranging from 100 aM to 1 pM. Due to dual signal amplification, the detection limit is as low as 34 aM. We proposed a method for identifying miR-141 expressed in human serum and successfully distinguished between prostate cancer patients (n = 20) and healthy individuals (n = 15) with an impressive accuracy of 94%. Overall, CENTER shows great promise for the detection of miRNA.},
}
@article {pmid37824016,
year = {2024},
author = {Madariaga-Marcos, J and Aldag, P and Kauert, DJ and Seidel, R},
title = {Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2694},
number = {},
pages = {421-449},
pmid = {37824016},
issn = {1940-6029},
abstract = {Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conformations, interactions, and activity of biomolecular complexes. Here, we describe a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule fluorescence spectroscopy and force/twisting experiments. We apply the setup to reveal the DNA interactions of the CRISPR-Cas surveillance complex Cascade. Single-molecule fluorescence of a labeled Cascade allows to follow the DNA association and dissociation of the protein. Simultaneously, the magnetic tweezers probe the DNA unwinding during R-loop formation by the bound Cascade complexes. Furthermore, the setup supports observation of 1D diffusion of protein complexes on stretched DNA molecules. This technique can be applied to study a vast range of protein-DNA interactions.},
}
@article {pmid37823193,
year = {2023},
author = {Saxena, V and Arregui, S and Zhang, S and Canas, J and Qin, X and Hains, DS and Schwaderer, AL},
title = {Generation of Atp6v1g3-Cre mice for investigation of intercalated cells and the collecting duct.},
journal = {American journal of physiology. Renal physiology},
volume = {},
number = {},
pages = {},
doi = {10.1152/ajprenal.00137.2023},
pmid = {37823193},
issn = {1522-1466},
support = {R01DK106286//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; },
abstract = {Kidney intercalated cells (IC) cells maintain acid-base homeostasis and recent studies demonstrate they function in the kidney's innate defense. To study kidney innate immune function, ICs have been enriched using vacuolar ATPase (V-ATPase) B1 subunit (Atp6v1b1)-Cre (B1-Cre) mice. Although Atp6v1b1 is considered kidney specific, it is expressed in multiple organ systems, both in mice and humans, raising the possibility of off target effects when using the Cre-lox system. We have recently shown using single cell RNA sequencing (scRNAseq) that the gene that codes for the V-ATPase G3 subunit (mouse gene: Atp6v1g3; human gene: ATP6V1G3; protein abbreviation: G3) mRNA is selectively enriched in murine or human kidney intercalated cells. In this study, we generated Atp6v1g3-Cre (G3-Cre) reporter mice using CRISPR/CAS technology and crossed it with Tdtomato[flox/flox] mice. The resultant G3-Cre[+]Tdt[+] progeny was evaluated for kidney specificity in multiple tissues and found to be highly specific to kidney cells with minimal or no expression in other organs evaluated as compared to B1-Cre mice. Tdt[+] cells were flow sorted and were enriched for IC marker genes on RT-PCR analysis. Next, we crossed these mice to ihCD59 mice to generate IC depletion mouse model (G3-Cre[+]ihCD59[+/+]). ICs were depleted in these mice using intermedilysin (ILY) which resulted in lower blood pH, suggestive of a distal renal tubular acidosis (dRTA) phenotype. The G3-Cre mice were healthy, bred normally and produce regular sized litter. Thus, this new "IC reporter" mice can be a useful tool to study ICs.},
}
@article {pmid37822083,
year = {2023},
author = {Lv, P and Su, F and Chen, F and Yan, C and Xia, D and Sun, H and Li, S and Duan, Z and Ma, C and Zhang, H and Wang, M and Niu, X and Zhu, JK and Zhang, J},
title = {Genome editing in rice using CRISPR/Cas12i3.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14192},
pmid = {37822083},
issn = {1467-7652},
support = {2022LZGC001//Key Research and Development Program of Shandong Province/ ; 2021LZGC012//Key Research and Development Program of Shandong Province/ ; },
abstract = {The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.},
}
@article {pmid37821806,
year = {2023},
author = {Zhang, X and He, X and Zhang, Y and Chen, L and Pan, Z and Huang, Y and Li, H},
title = {A new method for the detection of Mycobacterium tuberculosis based on the CRISPR/Cas system.},
journal = {BMC infectious diseases},
volume = {23},
number = {1},
pages = {680},
pmid = {37821806},
issn = {1471-2334},
abstract = {OBJECT: Mycobacterium tuberculosis (MTB) is a bacterium that can cause zoonoses by aerosol transmission. Tuberculosis (TB) caused by MTB heavily burdens world public health security. Developing efficient, specific, convenient, and inexpensive MTB assays are essential for preventing and controlling TB.<br><br>METHODS: In this study, we established a specific detection method for MTB using the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) system, combined with recombinase mediated isothermal nucleic acid amplification (RAA) to improve the sensitivity of the detection system and achieve "two-level" amplification of the detection signal. The sensitivity and specificity of RAA combined with the CRISPR/Cas system were analyzed. Using BACTEC 960 culture as the gold standard for detecting MTB, we established the TB-CRISPR technique by testing 504 samples from patients with suspected TB.<br><br>RESULTS: MTB H37Ra could be seen as low as 3.13 CFU/mL by the CRISPR-Cas12a system targeting IS6110. With BACTEC960 culture (120 positives and 384 negatives) as the gold standard, the sensitivity of the TB-CRISPR technique was 0.883 (0.809-0.932), and the specificity was 0.940 (0.910-0.961). According to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) reached 0.944 (0.914-0.975) within 95% CI. The positive likelihood ratio (PLR) was 14.747 (9.870-22.035), and the negative likelihood ratio (NLR) was 0.124 (0.076-0.203). The positive predictive value (PPV) was 0.822 (0.742-0.881), and the negative predictive value (NPV) was 0.963 (0.937-0.979).<br><br>CONCLUSION: TB-CRISPR plays an essential role in the molecular diagnosis of TB. The whole detection time is less than 1.5&#xa0;h. It is easy to operate and does not need complex instruments. It is of great significance for the rapid detection of MTB and the clinical diagnosis of TB.},
}
@article {pmid37820858,
year = {2023},
author = {Godbole, N and Quinn, A and Carrion, F and Pelosi, E and Salomon, C},
title = {Extracellular vesicles as a potential delivery platform for CRISPR-Cas based therapy in epithelial ovarian cancer.},
journal = {Seminars in cancer biology},
volume = {96},
number = {},
pages = {64-81},
doi = {10.1016/j.semcancer.2023.10.002},
pmid = {37820858},
issn = {1096-3650},
abstract = {Ovarian Cancer (OC) is the most common gynecological malignancy and the eighth most diagnosed cancer in females worldwide. Presently, it ranks as the fifth leading cause of cancer-related mortality among patients globally. Major factors contributing to the lethality of OC worldwide include delayed diagnosis, chemotherapy resistance, high metastatic rates, and the heterogeneity of subtypes. Despite continuous efforts to develop novel targeted therapies and chemotherapeutic agents, challenges persist in the form of OC resistance and recurrence. In the last decade, CRISPR-Cas-based genome editing has emerged as a powerful tool for modifying genetic and epigenetic mechanisms, holding potential for treating numerous diseases. However, a significant challenge for therapeutic applications of CRISPR-Cas technology is the absence of an optimal vehicle for delivering CRISPR molecular machinery into targeted cells or tissues. Recently, extracellular vesicles (EVs) have gained traction as potential delivery vehicles for various therapeutic agents. These heterogeneous, membrane-derived vesicles are released by nearly all cells into extracellular spaces. They carry a molecular cargo of proteins and nucleic acids within their intraluminal space, encased by a cholesterol-rich phospholipid bilayer membrane. EVs actively engage in cell-to-cell communication by delivering cargo to both neighboring and distant cells. Their inherent ability to shield molecular cargo from degradation and cross biological barriers positions them ideally for delivering CRISPR-Cas ribonucleoproteins (RNP) to target cells. Furthermore, they exhibit higher biocompatibility, lower immunogenicity, and reduced toxicity compared to classical delivery platforms such as adeno-associated virus, lentiviruses, and synthetic nanoparticles. This review explores the potential of employing different CRISPR-Cas systems to target specific genes in OC, while also discussing various methods for engineering EVs to load CRISPR components and enhance their targeting capabilities.},
}
@article {pmid37819690,
year = {2023},
author = {Yang, Z and Mao, S and Wang, L and Fu, S and Dong, Y and Jaffrezic-Renault, N and Guo, Z},
title = {CRISPR/Cas and Argonaute-Based Biosensors for Pathogen Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.3c01232},
pmid = {37819690},
issn = {2379-3694},
abstract = {Over the past few decades, pathogens have posed a threat to human security, and rapid identification of pathogens should be one of the ideal methods to prevent major public health security outbreaks. Therefore, there is an urgent need for highly sensitive and specific approaches to identify and quantify pathogens. Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas systems and Argonaute (Ago) belong to the Microbial Defense Systems (MDS). The guided, programmable, and targeted activation of nucleases by both of them is leading the way to a new generation of pathogens detection. We compare these two nucleases in terms of similarities and differences. In addition, we discuss future challenges and prospects for the development of the CRISPR/Cas systems and Argonaute (Ago) biosensors, especially electrochemical biosensors. This review is expected to afford researchers entering this multidisciplinary field useful guidance and to provide inspiration for the development of more innovative electrochemical biosensors for pathogens detection.},
}
@article {pmid37819511,
year = {2024},
author = {Chen, W and Ji, Q},
title = {CRISPR/Cas9-based Genome Editing of Pseudomonas aeruginosa.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2721},
number = {},
pages = {3-12},
pmid = {37819511},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Pseudomonas aeruginosa/genetics ; Point Mutation ; Recombinational DNA Repair ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been developed as a robust genome engineering tool in a variety of organisms attributed to its high efficiency and versatility. In this chapter, we described the detailed procedures of CRISPR-Cas9-based genetic manipulation in Pseudomonas aeruginosa, including precise gene deletion and insertion via Cas9-mediated DNA double-strand break and homologous recombination repair. In addition, we provided a detailed protocol for cytidine base editor, a highly efficient gene inactivation and point mutation tool in Pseudomonas aeruginosa.},
}
@article {pmid37819098,
year = {2023},
author = {Shimomura, Y and Sugi, Y and Kume, A and Tanaka, W and Yoshihara, T and Matsuura, T and Komiya, Y and Ogata, Y and Suda, W and Hattori, M and Higurashi, T and Nakajima, A and Matsumoto, M},
title = {Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR-Cas region.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0512322},
doi = {10.1128/spectrum.05123-22},
pmid = {37819098},
issn = {2165-0497},
abstract = {Fusobacterium nucleatum is associated with colorectal cancer (CRC), and identical F. nucleatum strains seemed to be detected in 75% of patients who exhibited the presence of F. nucleatum in both CRC and saliva samples in our previous study; however, the validation of strain identity and the development of a method for strain-level discrimination are required for etiological studies. We confirmed that F. nucleatum isolates obtained from CRC and saliva samples derived from patients with CRC originated from their identical strains using whole-genome sequencing. We evaluated the hypervariable region of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) system in F. nucleatum strains isolated from the CRC and saliva specimens of CRC patients to develop a method for genotyping this bacterium at the strain level. The developed method consisted of two simple PCR steps to amplify the different lengths and sequences of the CRISPR-Cas regions from F. nucleatum strains using specific but common primers. This method could successfully detect identical strains present in both cryopreserved CRC samples and saliva obtained from the same CRC patient. Dynamic monitoring of F. nucleatum strains in saliva obtained from patients with colorectal adenoma before and after oral care showed interindividual variability in F. nucleatum strains during oral care. This study provided a simple and rapid method for comprehensively identifying F. nucleatum at the strain level in clinical samples, leading to a paradigm shift in CRC research, such as the investigation of pathogenic F. nucleatum strains and monitoring of pathogenic strains in clinical trials for preventing CRC recurrence. (This study was registered in the UMIN Clinical Trials Registry under ID UMIN000016229.) IMPORTANCE Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.},
}
@article {pmid37819096,
year = {2023},
author = {Khot, V and Strous, M and Dong, X and Kiesser, AK},
title = {Viral diversity and dynamics and CRISPR-Cas-mediated immunity in a robust alkaliphilic cyanobacterial consortium.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0221723},
doi = {10.1128/spectrum.02217-23},
pmid = {37819096},
issn = {2165-0497},
abstract = {In many industries, from food to biofuels, contamination of production systems with predators is a costly problem and requires the maintenance of sterile operating conditions. In this study, we look at the robustness of one such alkaliphilic consortium, comprised largely of a cyanobacterium Candidatus Phormidium alkaliphilum, to viral predation. This consortium has existed without a community crash for several years in laboratory and pilot-scale environments. We look at clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems and viral dynamics in this consortium at four conditions using metagenomic analyses. Results show that while there are active viral members in this community, viral predation of the cyanobacteria is low and does not affect the community dynamics. The multiple CRISPR arrays within the Phormidium were found to be static following the initial lab establishment of consortium. Multiple cryptic CRISPR-Cas systems were detected with uncertain viral protection capacity. Our results suggest that the dynamics of potential viruses and CRISPR-Cas-mediated immunity likely play an important role in the initial establishment of consortia and may continue to support the functional robustness of engineered microbial communities throughout biotechnology applications. IMPORTANCE Biotechnology applications utilizing the function of microbial communities have become increasingly important solutions as we strive for sustainable applications. Although viral infections are known to have a significant impact on microbial turnover and nutrient cycling, viral dynamics have remained largely overlooked in these engineered communities. Predatory perturbations to the functional stability of these microbial biotechnology applications must be investigated in order to design more robust applications. In this study, we closely examine virus-microbe dynamics in a model microbial community used in a biotechnology application. Our findings suggest that viral dynamics change significantly with environmental conditions and that microbial immunity may play an important role in maintaining functional stability. We present this study as a comprehensive template for other researchers interested in exploring predatory dynamics in engineered microbial communities.},
}
@article {pmid37818780,
year = {2023},
author = {Lin, Q and Takebayashi, K and Torigoe, N and Liu, B and Namula, Z and Hirata, M and Tanihara, F and Nagahara, M and Otoi, T},
title = {Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos.},
journal = {Animal science journal = Nihon chikusan Gakkaiho},
volume = {94},
number = {1},
pages = {e13878},
doi = {10.1111/asj.13878},
pmid = {37818780},
issn = {1740-0929},
support = {JP22K19896//Japan Society for the Promotion of Science/ ; JP22H02499//Japan Society for the Promotion of Science/ ; },
abstract = {The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.},
}
@article {pmid37817254,
year = {2023},
author = {Feng, S and Xie, X and Liu, J and Li, A and Wang, Q and Guo, D and Li, S and Li, Y and Wang, Z and Guo, T and Zhou, J and Tang, DYY and Show, PL},
title = {A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles.},
journal = {Journal of nanobiotechnology},
volume = {21},
number = {1},
pages = {370},
pmid = {37817254},
issn = {1477-3155},
support = {No. U1804112//National Natural Science Foundation of China/ ; 23ZX005//Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 232300421164//Natural Science Foundation of Henan Province/ ; },
abstract = {Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.},
}
@article {pmid37816270,
year = {2023},
author = {Shelake, RM and Jadhav, AM and Bhosale, PB and Kim, JY},
title = {Unlocking secrets of nature's chemists: Potential of CRISPR/Cas-based tools in plant metabolic engineering for customized nutraceutical and medicinal profiles.},
journal = {Plant physiology and biochemistry : PPB},
volume = {203},
number = {},
pages = {108070},
doi = {10.1016/j.plaphy.2023.108070},
pmid = {37816270},
issn = {1873-2690},
abstract = {Plant species have evolved diverse metabolic pathways to effectively respond to internal and external signals throughout their life cycle, allowing adaptation to their sessile and phototropic nature. These pathways selectively activate specific metabolic processes, producing plant secondary metabolites (PSMs) governed by genetic and environmental factors. Humans have utilized PSM-enriched plant sources for millennia in medicine and nutraceuticals. Recent technological advances have significantly contributed to discovering metabolic pathways and related genes involved in the biosynthesis of specific PSM in different food crops and medicinal plants. Consequently, there is a growing demand for plant materials rich in nutrients and bioactive compounds, marketed as "superfoods". To meet the industrial demand for superfoods and therapeutic PSMs, modern methods such as system biology, omics, synthetic biology, and genome editing (GE) play a crucial role in identifying the molecular players, limiting steps, and regulatory circuitry involved in PSM production. Among these methods, clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR/Cas) is the most widely used system for plant GE due to its simple design, flexibility, precision, and multiplexing capabilities. Utilizing the CRISPR-based toolbox for metabolic engineering (ME) offers an ideal solution for developing plants with tailored preventive (nutraceuticals) and curative (therapeutic) metabolic profiles in an ecofriendly way. This review discusses recent advances in understanding the multifactorial regulation of metabolic pathways, the application of CRISPR-based tools for plant ME, and the potential research areas for enhancing plant metabolic profiles.},
}
@article {pmid37816156,
year = {2023},
author = {Gu, L and Zhang, R and Fan, X and Wang, Y and Ma, K and Jiang, J and Li, G and Wang, H and Fan, F and Zhang, X},
title = {Development of CRISPR/Cas9-Based Genome Editing Tools for Polyploid Yeast Cyberlindnera jadinii and Its Application in Engineering Heterologous Steroid-Producing Strains.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00278},
pmid = {37816156},
issn = {2161-5063},
abstract = {In this study, a suite of efficient CRISPR/Cas9 tools was developed to overcome the genetic manipulation challenges posed by the polyploid genome of industrial yeast Cyberlindnera jadinii. The developed CRISPR/Cas9 system can achieve a 100% single-gene knockdown efficiency in strain NBRC0988. Moreover, the integration of a single exogenous gene into the target locus using a 50 bp homology arm achieved near-100% efficiency. The efficiency of simultaneous integration of three genes into the chromosome is strongly influenced by the length of the homology arm, with the highest integration efficiency of 62.5% obtained when selecting a homology arm of about 500 bp. By utilizing the CRISPR/Cas system, this study demonstrated the potential of C. jadinii in producing heterologous sterols. Through shake-flask fermentation, the engineered strains produced 92.1 and 81.8 mg/L of campesterol and cholesterol, respectively. Furthermore, the production levels of these two sterols were further enhanced through high-cell-density fed-batch fermentation in a 5 L bioreactor. The highest titer of campesterol reached 807 mg/L [biomass OD600 = 294, productivity of 6.73 mg/(L·h)]. The titer of cholesterol reached 1.52 g/L [biomass OD600 = 380, productivity of 9.06 mg/(L·h)], marking the first gram-scale production of steroidal compounds in C. jadinii.},
}
@article {pmid37813990,
year = {2023},
author = {Mahata, B and Cabrera, A and Brenner, DA and Guerra-Resendez, RS and Li, J and Goell, J and Wang, K and Guo, Y and Escobar, M and Parthasarathy, AK and Szadowski, H and Bedford, G and Reed, DR and Kim, S and Hilton, IB},
title = {Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control.},
journal = {Nature methods},
volume = {},
number = {},
pages = {},
pmid = {37813990},
issn = {1548-7105},
support = {R35GM143532//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21EB030772//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R56HG012206//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
abstract = {Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR-Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR-dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.},
}
@article {pmid37808752,
year = {2023},
author = {Upreti, C and Kumar, P and Durso, LM and Palmer, KL},
title = {CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.09.26.559507},
pmid = {37808752},
abstract = {UNLABELLED: The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system CRISPR-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its anti-plasmid efficacy in an agricultural niche - manure. We show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. CRISPR-Cas was found to be an effective barrier against resistance plasmid transfer in manure, with improved effect as time progressed. CRISPR-based antimicrobials to cure resistant E. faecalis of erythromycin resistance was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting, and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a OneHealth approach.<br><br>IMPORTANCE: Antibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas - a bacterial defense system - to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium - Enterococcus faecalis . We show that CRISPR-Cas is prevalent, functional in manure, and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas based strategies for control of antibiotic resistance in agricultural settings.},
}
@article {pmid37808693,
year = {2023},
author = {Alseth, EO and Custodio, R and Sundius, SA and Kuske, RA and Brown, SP and Westra, ER},
title = {The impact of phage and phage resistance on microbial community dynamics.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.09.26.559468},
pmid = {37808693},
abstract = {Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. Yet little is known about how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes either Pseudomonas aeruginosa wild type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Our results show that the microbial community structure is drastically altered by the addition of phage, with Acinetobacter baumannii becoming the dominant species and P. aeruginosa being driven nearly extinct, whereas P. aeruginosa outcompetes the other species in the absence of phage. Moreover, we find that a P. aeruginosa strain with the ability to evolve CRISPR-based resistance generally does better when in the presence of A. baumannii , but that this benefit is largely lost over time as phage is driven extinct. Combined, our data highlight how phage-targeting a dominant species allows for the competitive release of the strongest competitor whilst also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.},
}
@article {pmid37808520,
year = {2023},
author = {Li, X and Zhong, J and Li, H and Qiao, Y and Mao, X and Fan, H and Zhong, Y and Imani, S and Zheng, S and Li, J},
title = {Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid.},
journal = {Frontiers in molecular biosciences},
volume = {10},
number = {},
pages = {1260883},
pmid = {37808520},
issn = {2296-889X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are widely used as gene editing tools in biology, microbiology, and other fields. CRISPR is composed of highly conserved repetitive sequences and spacer sequences in tandem. The spacer sequence has homology with foreign nucleic acids such as viruses and plasmids; Cas effector proteins have endonucleases, and become a hotspot in the field of molecular diagnosis because they recognize and cut specific DNA or RNA sequences. Researchers have developed many diagnostic platforms with high sensitivity, high specificity, and low cost by using Cas proteins (Cas9, Cas12, Cas13, Cas14, etc.) in combination with signal amplification and transformation technologies (fluorescence method, lateral flow technology, etc.), providing a new way for rapid detection of pathogen nucleic acid. This paper introduces the biological mechanism and classification of CRISPR-Cas technology, summarizes the existing rapid detection technology for pathogen nucleic acid based on the trans cleavage activity of Cas, describes its characteristics, functions, and application scenarios, and prospects the future application of this technology.},
}
@article {pmid37806898,
year = {2023},
author = {Li, Y and Jia, J and Man, S and Ye, S and Ma, L},
title = {Emerging programmable nuclease-based detection for food safety.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.09.006},
pmid = {37806898},
issn = {1879-3096},
abstract = {Food safety issues are an important challenge across the world. Programmable nucleases are emerging as new tools because of their significant biological advantages. This forum article provides an overview of recent advances and challenges in the novel paradigm of programmable nuclease-based detection for food safety.},
}
@article {pmid36160051,
year = {2022},
author = {Haideri, T and Howells, A and Jiang, Y and Yang, J and Bao, X and Lian, XL},
title = {Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells.},
journal = {Cell reports methods},
volume = {2},
number = {9},
pages = {100290},
pmid = {36160051},
issn = {2667-2375},
support = {R01 AR072731/AR/NIAMS NIH HHS/United States ; R21 EB026035/EB/NIBIB NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics ; *Pluripotent Stem Cells ; Plasmids ; },
abstract = {CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs.},
}
@article {pmid36160047,
year = {2022},
author = {Cai, H and Winslow, MM},
title = {A new system for multiplexed mosaic analysis of gene function in the mouse.},
journal = {Cell reports methods},
volume = {2},
number = {9},
pages = {100295},
pmid = {36160047},
issn = {2667-2375},
support = {R01 CA230025/CA/NCI NIH HHS/United States ; R01 CA231253/CA/NCI NIH HHS/United States ; R01 CA123249/CA/NCI NIH HHS/United States ; },
mesh = {Mice ; Animals ; Mice, Transgenic ; *Mosaicism ; *Integrases/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In a recent issue of Cell, Liu et al. present an innovative mouse model system in which Cre/lox stochastically turns on transgenic expression of one out of up to 100 sgRNAs in somatic cells, creating genetic mosaicism that enables the multiplexed assessment of gene function in vivo.},
}
@article {pmid37805851,
year = {2023},
author = {Zhang, J and Yang, J and Meng, Y and Xing, S and Liu, Q and Li, M},
title = {[Construction of a 10rolGLP-1-expressing glucose-lowing Saccharomyces cerevisiae by CRISPR/Cas9 technique].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {9},
pages = {3747-3756},
doi = {10.13345/j.cjb.220870},
pmid = {37805851},
issn = {1872-2075},
mesh = {Mice ; Animals ; *Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems ; Glucose/metabolism ; Blood Glucose/metabolism ; *Diabetes Mellitus, Type 2/genetics/therapy ; Hypoglycemic Agents/metabolism ; },
abstract = {To develop a novel glucose-lowering biomedicine with potential benefits in the treatment of type 2 diabetes, we used the 10rolGLP-1 gene previously constructed in our laboratory and the CRISPR/Cas9 genome editing technique to create an engineered Saccharomyces cerevisiae strain. The gRNA expression vector pYES2-gRNA, the donor vector pNK1-L-PGK-10rolGLP-1-R and the Cas9 expression vector pGADT7-Cas9 were constructed and co-transformed into S. cerevisiae INVSc1 strain, with the PGK-10rolGLP-1 expressing unit specifically knocked in through homologous recombination. Finally, an S. cerevisiae strain highly expressing the 10rolGLP-1 with glucose-lowering activity was obtained. SDS-PAGE and Western blotting results confirmed that two recombinant strains of S. cerevisiae stably expressed the 10rolGLP-1 and exhibited the desired glucose-lowering property when orally administered to mice. Hypoglycemic experiment results showed that the recombinant hypoglycemic S. cerevisiae strain offered a highly hypoglycemic effect on the diabetic mouse model, and the blood glucose decline was adagio, which can avoid the dangerous consequences caused by rapid decline in blood glucose. Moreover, the body weight and other symptoms such as polyuria also improved significantly, indicating that the orally hypoglycemic S. cerevisiae strain that we constructed may develop into an effective, safe, economic, practical and ideal functional food for type 2 diabetes mellitus treatment.},
}
@article {pmid37805850,
year = {2023},
author = {Qi, S and Li, X and Zhou, D and Huang, J},
title = {[Construction of a stable centromere protein F overexpression cell model of hepatocellular carcinoma using CRISPR activation system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {9},
pages = {3738-3746},
doi = {10.13345/j.cjb.220656},
pmid = {37805850},
issn = {1872-2075},
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics ; *Liver Neoplasms/genetics ; RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Hygromycin B ; },
abstract = {Current studies have shown that centromere protein F (CENPF) was overexpressed in hepatocellular carcinoma (HCC) and might be involved in the pathogenesis of HCC. Specifically, due to the very large molecular weight (358 kDa) of CENPF full length protein, only CENPF knock-down, but not overexpression models, were applied currently to explore the carcinogenicity of CENPF in HCC. Whether CENPF overexpression is a cause or an effect in HCC remains to be illustrated. We aimed to establish a CENPF overexpression cell model using CRISPR/dCas9 synergistic activation mediator (SAM) system with lentiMPHv2 and lentiSAMv2 vectors to explore the role of CENPF overexpression in HCC. Single guide RNAs (sgRNAs) that specifically identify the transcription initiation site of CENPF gene were synthesized and inserted into the lentiSAMv2 plasmid. Huh-7 and HCCLM3 cells were first transduced with lentiMPHv2 and then selected with hygromycin B. The cells were then transduced with lentiSAMv2 carrying specific sgRNA for CENPF gene, followed by blasticidin S selection. The mRNA and protein detection results of Huh-7 and HCCLM3 cells screened by hygromycin B and blasticidin S showed that the endogenous overexpression of CENPF can be induced by sgRNA1 and sgRNA4, especially by sgRNA4. By using the CRISPR/dCas9 technique, stable cell models with overexpressed CENPF were successfully constructed to explore the role of CENPF in tumorigenesis, which provides a reference for the construction of cell models overexpressing large molecular weight protein.},
}
@article {pmid37803599,
year = {2023},
author = {Zhou, J and Wang, TY and Lan, Z and Yang, HJ and Ye, XJ and Min, R and Wang, ZH and Huang, Q and Cao, J and Gao, YE and Wang, WL and Sun, XL and Zhang, Y},
title = {Strategy of functional nucleic acids-mediated isothermal amplification for detection of foodborne microbial contaminants: A review.},
journal = {Food research international (Ottawa, Ont.)},
volume = {173},
number = {Pt 1},
pages = {113286},
doi = {10.1016/j.foodres.2023.113286},
pmid = {37803599},
issn = {1873-7145},
abstract = {Foodborne microbial contamination (FMC) is the leading cause of food poisoning and foodborne illness. The foodborne microbial detection methods based on isothermal amplification have high sensitivity and short detection time, and functional nucleic acids (FNAs) could extend the detectable object of isothermal amplification to mycotoxins. Therefore, the strategy of FNAs-mediated isothermal amplification has been emergingly applied in biosensors for foodborne microbial contaminants detection, making biosensors more sensitive with lower cost and less dependent on nanomaterials for signal output. Here, the mechanism of six isothermal amplification technologies and their application in detecting FMC is firstly introduced. Then the strategy of FNAs-mediated isothermal amplification is systematically discussed from perspectives of FNAs' versatility including recognition elements (Aptamer, DNAzyme), programming tools (DNA tweezer, DNA walker and CRISPR-Cas) and signal units (G-quadruplex, FNAs-based nanomaterials). Finally, challenges and prospects are presented in terms of addressing the issue of nonspecific amplification reaction, developing better FNAs-based sensing elements and eliminating food matrix effects.},
}
@article {pmid37797871,
year = {2023},
author = {Anand, V and Prabhakaran, HS and Prakash, A and Hussain, MS and Kumar, M},
title = {Differential processing of CRISPR RNA by LinCas5c and LinCas6 of Leptospira.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1867},
number = {12},
pages = {130469},
doi = {10.1016/j.bbagen.2023.130469},
pmid = {37797871},
issn = {1872-8006},
abstract = {Leptospira interrogans serovar Copenhageni's genome harbors two CRISPR-Cas systems belonging to subtypes I-B and I-C. However, in L. interrogans, the subtype I-C locus lacks an array component essential for assembling an interference complex. Thus, the reason for sustaining the expense of a cluster of cas genes (I-C) is obscure. Type I-C (previously Dvulg) is the only CRISPR subtype that engages Cas5c, a Cas5 variant, to process precursor CRISPR-RNA (pre-crRNA). In this study, thus, the recombinant Cas5c (rLinCas5c) of L.interrogans and its mutant variants were cloned, expressed, and purified. The purified rLinCas5c is illustrated as metal-independent, sequence, and size-specific cleavage on repeat RNA and pre-crRNA of subtype I-B or orphan CRISPR array. However, the Cas6-bound mature crRNA of subtype I-B fends off from the rLinCas5c activity. In addition, rLinCas5c holds metal and size-dependent DNase activity. The bioinformatics analysis of LinCas5c inferred that it belongs to the subgroup Cas5c-B. Substitution of Phe141 with a more conserved His residue and deletion of unique (β1'-β2') insertions usher a gain of rLinCas5c activity over nucleic acid. Overall, our results uncover the functional diversity of Cas5c ribonucleases and infer an incognito auxiliary role in the absence of a cognate CRISPR array.},
}
@article {pmid37797813,
year = {2023},
author = {Aquino-Jarquin, G},
title = {Genome and transcriptome engineering by compact and versatile CRISPR-Cas systems.},
journal = {Drug discovery today},
volume = {},
number = {},
pages = {103793},
doi = {10.1016/j.drudis.2023.103793},
pmid = {37797813},
issn = {1878-5832},
abstract = {Comparative genomics has enabled the discovery of tiny clustered regularly interspaced short palindromic repeat (CRISPR) bacterial immune system effectors with enormous potential for manipulating eukaryotic genomes. Recently, smaller Cas proteins, including miniature Cas9, Cas12, and Cas13 proteins, have been identified and validated as efficient genome editing and base editing tools in human cells. The compact size of these novel CRISPR effectors is highly desirable for generating CRISPR-based therapeutic approaches, mainly to overcome in vivo delivery constraints, providing a promising opportunity for editing pathogenic mutations of clinical relevance and knocking down RNAs in human cells without inducing chromosomal insertions or genome alterations. Thus, these tiny CRISPR-Cas systems represent new and highly programmable, specific, and efficient platforms, which expand the CRISPR toolkit for potential therapeutic opportunities. Teaser: The compact size of the novel CRISPR effectors is highly appropriate for generating CRISPR-based therapeutic modalities due to the viral packaging capacity of these nucleases for in vivo delivery.},
}
@article {pmid37797781,
year = {2023},
author = {Harsij, Z and Ghafoorzadeh, Z and Goharian, E},
title = {The CRISPR Revolution: Unraveling the mysteries of Life's genetic code.},
journal = {Gene},
volume = {892},
number = {},
pages = {147870},
doi = {10.1016/j.gene.2023.147870},
pmid = {37797781},
issn = {1879-0038},
abstract = {A biotechnological revolution is triggered by CRISPR-Cas systems' variety, measured quality, and proficiency. Identifying nucleic acid biomarkers, one of the methods that use CRISPR for diagnosis, is an extremely sensitive diagnostic method.A broad range of infectious and noninfecting diseases, mutations, and CRISPR deletions associated with genetic disorders have been detected using diagnostics. Furthermore, this technology is used to test proteins and micromolecules. We focus on how Cas proteins can be used to detect diseases in genes, agriculture, and cancer therapy. Furthermore, CRISPR technology has many negative impacts on the health of living organisms, environmental and population structures in spite of its numerous contributions to biomedical science. Therefore, an investigation into the impact of genome editing on nontargeted species is important for these reasons. CRISPR in the future is briefly discussed towards the end of this review.},
}
@article {pmid37796250,
year = {2023},
author = {Jung, H and Lee, D and Lee, S and Kong, HJ and Park, J and Seo, YS},
title = {Comparative genomic analysis of Chryseobacterium species: deep insights into plant-growth-promoting and halotolerant capacities.},
journal = {Microbial genomics},
volume = {9},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001108},
pmid = {37796250},
issn = {2057-5858},
mesh = {*Chryseobacterium/genetics ; Phylogeny ; Comparative Genomic Hybridization ; Genomics ; Soil ; },
abstract = {Members of the genus Chryseobacterium have attracted great interest as beneficial bacteria that can promote plant growth and biocontrol. Given the recent risks of climate change, it is important to develop tolerance strategies for efficient applications of plant-beneficial bacteria in saline environments. However, the genetic determinants of plant-growth-promoting and halotolerance effects in Chryseobacterium have not yet been investigated at the genomic level. Here, a comparative genomic analysis was conducted with seven Chryseobacterium species. Phylogenetic and phylogenomic analyses revealed niche-specific evolutionary distances between soil and freshwater Chryseobacterium species, consistent with differences in genomic statistics, indicating that the freshwater bacteria have smaller genome sizes and fewer genes than the soil bacteria. Phosphorus- and zinc-cycling genes (required for nutrient acquisition in plants) were universally present in all species, whereas nitrification and sulphite reduction genes (required for nitrogen- and sulphur-cycling, respectively) were distributed only in soil bacteria. A pan-genome containing 6842 gene clusters was constructed, which reflected the general features of the core, accessory and unique genomes. Halotolerant species with an accessory genome shared a Kdp potassium transporter and biosynthetic pathways for branched-chain amino acids and the carotenoid lycopene, which are associated with countermeasures against salt stress. Protein-protein interaction network analysis was used to define the genetic determinants of Chryseobacterium salivictor NBC122 that reduce salt damage in bacteria and plants. Sixteen hub genes comprised the aromatic compound degradation and Por secretion systems, which are required to cope with complex stresses associated with saline environments. Horizontal gene transfer and CRISPR-Cas analyses indicated that C. salivictor NBC122 underwent more evolutionary events when interacting with different environments. These findings provide deep insights into genomic adaptation to dynamic interactions between plant-growth-promoting Chryseobacterium and salt stress.},
}
@article {pmid37694511,
year = {2023},
author = {Pan, M and Champer, J},
title = {Making waves: Comparative analysis of gene drive spread characteristics in a continuous space model.},
journal = {Molecular ecology},
volume = {32},
number = {20},
pages = {5673-5694},
doi = {10.1111/mec.17131},
pmid = {37694511},
issn = {1365-294X},
mesh = {Humans ; *Gene Drive Technology/methods ; CRISPR-Cas Systems ; },
abstract = {With their ability to rapidly increase in frequency, gene drives can be used to modify or suppress target populations after an initial release of drive individuals. Recent advances have revealed many possibilities for different types of drives, and several of these have been realized in experiments. These drives have advantages and disadvantages related to their ease of construction, confinement and capacity to be used for modification or suppression. Though characteristics of these drives have been explored in modelling studies, assessment in continuous space environments has been limited, often focusing on outcomes rather than fundamental properties. Here, we conduct a comparative analysis of many different gene drive types that have the capacity to form a wave of advance in continuous space using individual-based simulations in continuous space. We evaluate the drive wave speed as a function of drive performance and ecological parameters, which reveals substantial differences between drive performance in panmictic versus spatial environments. In particular, we find that suppression drive waves are uniquely vulnerable to fitness costs and undesired CRISPR cleavage activity in embryos by maternal deposition. Some drives, however, retain robust performance even with widely varying efficiency parameters. To gain a better understanding of drive waves, we compare their panmictic performance and find that the rate of wild-type allele removal is correlated with drive wave speed, though this is also affected by other factors. Overall, our results provide a useful resource for understanding the performance of drives in spatially continuous environments, which may be most representative of potential drive deployment in many relevant scenarios.},
}
@article {pmid37795915,
year = {2023},
author = {Koutsogiannis, Z and Mina, JG and Albus, CA and Kol, MA and Holthuis, JCM and Pohl, E and Denny, PW},
title = {Toxoplasma ceramide synthases: Gene duplication, functional divergence, and roles in parasite fitness.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {37},
number = {11},
pages = {e23229},
doi = {10.1096/fj.202201603RRR},
pmid = {37795915},
issn = {1530-6860},
support = {//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo (FAPESP)/ ; BB/M024156/1//UKRI|Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/T003987/1//UKRI|Biotechnology and Biological Sciences Research Council (BBSRC)/ ; MR/P027989/1//UKRI|Medical Research Council (MRC)/ ; },
abstract = {Toxoplasma gondii is an obligate, intracellular apicomplexan protozoan parasite of both humans and animals that can cause fetal damage and abortion and severe disease in the immunosuppressed. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localization and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2. Interestingly, we observed that while TgCerS1 was a fully functional orthologue of the yeast ceramide synthase (Lag1p) capable of catalyzing the conversion of sphinganine to ceramide, in contrast TgCerS2 was catalytically inactive. Furthermore, genomic deletion of TgCerS1 using CRISPR/Cas-9 led to viable but slow-growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of TgCerS2 was only accessible utilizing the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this "pseudo" ceramide synthase, TgCerS2, has a considerably greater role in parasite fitness than its catalytically active orthologue (TgCerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa may have arisen through gene duplication. However, in the Apicomplexa the duplicated copy is hypothesized to have subsequently evolved into a non-functional "pseudo" ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites.},
}
@article {pmid37795780,
year = {2023},
author = {Basu, S and Bhattacharya, D and Pramanik, A and Saha, M and Mukherjee, J},
title = {In-silico whole-genome sequence analysis of a halotolerant filamentous mangrove cyanobacterium revealed CRISPR-Cas systems with unique properties.},
journal = {Journal of phycology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jpy.13393},
pmid = {37795780},
issn = {1529-8817},
support = {PURSE Phase II//Department of Science and Technology, Ministry of Science and Technology, India/ ; 200510616803//University Grants Commission/ ; },
abstract = {Novel CRISPR systems capable of cleaving both DNA and RNA are progressively emerging as attractive tools for genome manipulation of prokaryotic and eukaryotic organisms. We report specific characteristics of CRISPR systems present in Oxynema aestuarii AP17, a halotolerant, filamentous cyanobacterium and the second known member of the Oxynema genus. In-silico analyses of its whole-genome sequence revealed the presence of multiple Type I and Type III CRISPR loci with one Type I-G system previously unreported in cyanobacteria. We further identified the leader sequences at the 5' end of multiple CRISPR loci, many of which were distinct from previously reported cyanobacterial CRISPR leaders. Phylogenetic analyses of the O. aestuarii AP17 Cas1 proteins revealed two protein sequences that were unique and distantly related to other cyanobacterial Cas1 protein sequences. Our findings are significant because novel Class 1 CRISPR systems possess multi-subunit effectors and are highly flexible for repurposing by protein domain fusions made to the effector complex. Additionally, Type III CRISPRs are particularly useful for genome editing in certain extremophiles for which mesophilic Type II CRISPRs are ineffective.},
}
@article {pmid37795042,
year = {2023},
author = {Allemailem, KS and Alsahli, MA and Almatroudi, A and Alrumaihi, F and Al Abdulmonem, W and Moawad, AA and Alwanian, WM and Almansour, NM and Rahmani, AH and Khan, AA},
title = {Innovative Strategies of Reprogramming Immune System Cells by Targeting CRISPR/Cas9-Based Genome-Editing Tools: A New Era of Cancer Management.},
journal = {International journal of nanomedicine},
volume = {18},
number = {},
pages = {5531-5559},
pmid = {37795042},
issn = {1178-2013},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Immunotherapy, Adoptive ; T-Lymphocytes ; *Neoplasms/genetics/therapy ; },
abstract = {The recent developments in the study of clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system have revolutionized the art of genome-editing and its applications for cellular differentiation and immune response behavior. This technology has further helped in understanding the mysteries of cancer progression and possible designing of novel antitumor immunotherapies. CRISPR/Cas9-based genome-editing is now often used to engineer universal T-cells, equipped with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR). In addition, this technology is used in cytokine stimulation, antibody designing, natural killer (NK) cell transfer, and to overcome immune checkpoints. The innovative potential of CRISPR/Cas9 in preparing the building blocks of adoptive cell transfer (ACT) immunotherapy has opened a new window of antitumor immunotherapy and some of them have gained FDA approval. The manipulation of immunogenetic regulators has opened a new interface for designing, implementation and interpretation of CRISPR/Cas9-based screening in immuno-oncology. Several cancers like lymphoma, melanoma, lung, and liver malignancies have been treated with this strategy, once thought to be impossible. The safe and efficient delivery of CRISPR/Cas9 system within the immune cells for the genome-editing strategy is a challenging task which needs to be sorted out for efficient immunotherapy. Several targeting approaches like virus-mediated, electroporation, microinjection and nanoformulation-based methods have been used, but each procedure offers some limitations. Here, we elaborate the recent updates of cancer management through immunotherapy in partnership with CRISPR/Cas9 technology. Further, some innovative methods of targeting this genome-editing system within the immune system cells for reprogramming them, as a novel strategy of anticancer immunotherapy is elaborated. In addition, future prospects and clinical trials are also discussed.},
}
@article {pmid37794706,
year = {2023},
author = {Vu, TV and Nguyen, NT and Kim, J and Hong, JC and Kim, JY},
title = {Prime editing: Mechanism insight and recent applications in plants.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14188},
pmid = {37794706},
issn = {1467-7652},
support = {2020M3A9I4038352//National Research Foundation of Korea/ ; 2020R1A6A1A03044344//National Research Foundation of Korea/ ; 2021R1A5A8029490//National Research Foundation of Korea/ ; 2022R1A2C3010331//National Research Foundation of Korea/ ; PJ01686702//Rural Development Administration (RDA), Korea/ ; },
abstract = {Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.},
}
@article {pmid37794046,
year = {2023},
author = {Zhao, L and Koseki, SRT and Silverstein, RA and Amrani, N and Peng, C and Kramme, C and Savic, N and Pacesa, M and Rodríguez, TC and Stan, T and Tysinger, E and Hong, L and Yudistyra, V and Ponnapati, MR and Jacobson, JM and Church, GM and Jakimo, N and Truant, R and Jinek, M and Kleinstiver, BP and Sontheimer, EJ and Chatterjee, P},
title = {PAM-flexible genome editing with an engineered chimeric Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6175},
pmid = {37794046},
issn = {2041-1723},
support = {/HHMI/Howard Hughes Medical Institute/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome ; },
abstract = {CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.},
}
@article {pmid37794017,
year = {2023},
author = {Yang, Z and Li, Z and Li, B and Bu, R and Tan, GY and Wang, Z and Yan, H and Xin, Z and Zhang, G and Li, M and Xiang, H and Zhang, L and Wang, W},
title = {A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6193},
pmid = {37794017},
issn = {2041-1723},
abstract = {Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency (~ 10[8] CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.},
}
@article {pmid37792418,
year = {2023},
author = {Srivastava, P and Prasad, D},
title = {Human Norovirus Detection: How Much Are We Prepared?.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {},
doi = {10.1089/fpd.2023.0024},
pmid = {37792418},
issn = {1556-7125},
abstract = {Norovirus (NoV) is known to be the second nonbacterial enteric pathogen after rotavirus that causes acute gastroenteritis. They can be spread from person to person through fecal-oral routes. Infection can lead to severe diarrhea, causing stomach pain, vomiting, and nausea. Rapid detection of NoV can control huge economic and productive losses. Genotyping various emerging NoV strains is important to compare the severity among different strains. Conventional immunological and molecular methods have evolved and contributed to developing detection techniques. Immunological (enzyme-linked immunosorbent assay) and molecular detection (reverse transcriptase polymerase chain reaction [RT-PCR], RT-quantitative PCR, loop-mediated isothermal amplification, nucleic acid sequence-based alignment, recombinase polymerase amplification) methods have been mainly used. The development of biosensors using aptasensor, affinity peptides, nanoparticles, microfluidics, and so on, are currently the most researched topics. The availability of next-generation sequencing technologies has greatly influenced the diagnosis of NoV. The complementation of advanced technologies is helpful in identification of new variants. In this study, techniques that are useful in detecting NoV are discussed. This review has investigated the availability of recent methods used in the detection, present status, and futuristic plan of action in case of outbreak and pandemic.},
}
@article {pmid37791541,
year = {2023},
author = {Felgate, H and Sethi, D and Faust, K and Kiy, C and Härtel, C and Rupp, J and Clifford, R and Dean, R and Tremlett, C and Wain, J and Langridge, G and Clarke, P and Page, AJ and Webber, MA},
title = {Characterisation of neonatal Staphylococcus capitis NRCS-A isolates compared with non NRCS-A Staphylococcus capitis from neonates and adults.},
journal = {Microbial genomics},
volume = {9},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001106},
pmid = {37791541},
issn = {2057-5858},
mesh = {Infant ; Infant, Newborn ; Adult ; Humans ; *Staphylococcus capitis/genetics ; *Staphylococcal Infections/epidemiology/drug therapy ; Anti-Bacterial Agents/therapeutic use ; *Sepsis ; Intensive Care Units, Neonatal ; },
abstract = {Staphylococcus capitis is a frequent cause of late-onset sepsis in neonates admitted to Neonatal Intensive Care Units (NICU). One clone of S. capitis, NRCS-A has been isolated from NICUs globally although the reasons for the global success of this clone are not well understood.We analysed a collection of S. capitis colonising babies admitted to two NICUs, one in the UK and one in Germany as well as corresponding pathological clinical isolates. Genome analysis identified a population structure of three groups; non-NRCS-A isolates, NRCS-A isolates, and a group of 'proto NRCS-A' - isolates closely related to NRCS-A but not associated with neonatal infection. All bloodstream isolates belonged to the NRCS-A group and were indistinguishable from strains carried on the skin or in the gut. NRCS-A isolates showed increased tolerance to chlorhexidine and antibiotics relative to the other S. capitis as well as enhanced ability to grow at higher pH values. Analysis of the pangenome of 138 isolates identified characteristic nsr and tarJ genes in both the NRCS-A and proto groups. A CRISPR-cas system was only seen in NRCS-A isolates which also showed enrichment of genes for metal acquisition and transport.We found evidence for transmission of S. capitis NRCS-A within NICU, with related isolates shared between babies and multiple acquisitions by some babies. Our data show NRCS-A strains commonly colonise uninfected babies in NICU representing a potential reservoir for potential infection. This work provides more evidence that adaptation to survive in the gut and on skin facilitates spread of NRCS-A, and that metal acquisition and tolerance may be important to the biology of NRCS-A. Understanding how NRCS-A survives in NICUs can help develop infection control procedures against this clone.},
}
@article {pmid37642716,
year = {2023},
author = {Chen, QH and Qian, YD and Niu, YJ and Hu, CY and Meng, YH},
title = {Characterization of an efficient CRISPR-iCas9 system in Yarrowia lipolytica for the biosynthesis of carotenoids.},
journal = {Applied microbiology and biotechnology},
volume = {107},
number = {20},
pages = {6299-6313},
pmid = {37642716},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; Carotenoids ; *Yarrowia/genetics ; Gene Editing/methods ; beta Carotene ; },
abstract = {The application of clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas9) technology in the genetic modification of Yarrowia lipolytica is challenged by low efficiency and low throughput. Here, a highly efficient CRISPR-iCas9 (with D147Y and P411T mutants) genetic manipulation tool was established for Y. lipolytica, which was further utilized to integrate carotene synthetic key genes and significantly improve the target product yield. First, CRISPR-iCas9 could shorten the time of genetic modification and enable the rapid knockout of nonsense suppressors. iCas9 can lead to more than 98% knockout efficiency for NHEJ-mediated repair after optimal target disruption of a single gene, 100% knockout efficiency for a single gene-guided version, and more than 80% knockout efficiency for multiple genes simultaneously in Y. lipolytica. Subsequently, this technology allowed for rapid one-step integration of large fragments (up to 9902-bp) of genes into chromosomes. Finally, YL-ABTG and YL-ABTG2Z were further constructed through CRISPR-iCas9 integration of key genes in a one-step process, resulting in a maximum β-carotene and zeaxanthin content of 3.12 mg/g and 2.33 mg/g dry cell weight, respectively. Therefore, CRISPR-iCas9 technology provides a feasible approach to genetic modification for efficient biosynthesis of biological compounds in Y. lipolytica. KEY POINTS: • iCas9 with D147Y and P411T mutants improved the CRISPR efficiency in Y. lipolytica. • CRISPR-iCas9 achieved efficient gene knockout and integration in Y. lipolytica. • CRISPR-iCas9 rapidly modified Y. lipolytica for carotenoid bioproduction.},
}
@article {pmid37562287,
year = {2023},
author = {Gao, F and Li, P and Yin, Y and Du, X and Cao, G and Wu, S and Zhao, Y},
title = {Molecular breeding of livestock for disease resistance.},
journal = {Virology},
volume = {587},
number = {},
pages = {109862},
doi = {10.1016/j.virol.2023.109862},
pmid = {37562287},
issn = {1096-0341},
mesh = {Animals ; Swine ; Disease Resistance/genetics ; Livestock ; DNA Shuffling ; *African Swine Fever Virus ; *Porcine respiratory and reproductive syndrome virus ; CRISPR-Cas Systems ; },
abstract = {Animal infectious diseases pose a significant threat to the global agriculture and biomedicine industries, leading to significant economic losses and public health risks. The emergence and spread of viral infections such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and avian influenza virus (AIV) have highlighted the need for innovative approaches to develop resilient and disease-resistant animal populations. Gene editing technologies, such as CRISPR/Cas9, offer a promising avenue for generating animals with enhanced disease resistance. This review summarizes recent advances in molecular breeding strategies for generating disease-resistant animals, focusing on the development of disease-resistant livestock. We also highlight the potential applications of genome-wide CRISPR/Cas9 library screening and base editors in producing precise gene modified livestock for disease resistance in the future. Overall, gene editing technologies have the potential to revolutionize animal breeding and improve animal health and welfare.},
}
@article {pmid37791490,
year = {2023},
author = {Bennis, NX and Anderson, JP and Kok, SMC and Daran, JG},
title = {Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foad043},
pmid = {37791490},
issn = {1567-1364},
abstract = {ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae were defined as a 21 nt spacer flanked by 19 nt direct repeats expressed from either RNApolII or III promoters, achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multi-spacer crRNA array using the RNA self-processing capability, which allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is established as a valuable addition to the genetic toolbox for S. cerevisiae.},
}
@article {pmid37790334,
year = {2023},
author = {Enustun, E and Armbruster, EG and Lee, J and Zhang, S and Yee, BA and Gu, Y and Deep, A and Naritomi, JT and Liang, Q and Aigner, S and Adler, BA and Cress, BF and Doudna, JA and Chaikeeratisak, V and Cleveland, DW and Ghassemian, M and Yeo, GW and Pogliano, J and Corbett, KD},
title = {A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37790334},
abstract = {Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro , and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro . Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.},
}
@article {pmid37789479,
year = {2023},
author = {Ma, Z and Ma, L and Zhou, J},
title = {Applications of CRISPR/Cas genome editing in economically important fruit crops: recent advances and future directions.},
journal = {Molecular horticulture},
volume = {3},
number = {1},
pages = {1},
pmid = {37789479},
issn = {2730-9401},
abstract = {Fruit crops, consist of climacteric and non-climacteric fruits, are the major sources of nutrients and fiber for human diet. Since 2013, CRISPR/Cas (Clustered Regularly Interspersed Short Palindromic Repeats and CRISPR-Associated Protein) genome editing system has been widely employed in different plants, leading to unprecedented progress in the genetic improvement of many agronomically important fruit crops. Here, we summarize latest advancements in CRISPR/Cas genome editing of fruit crops, including efforts to decipher the mechanisms behind plant development and plant immunity, We also highlight the potential challenges and improvements in the application of genome editing tools to fruit crops, including optimizing the expression of CRISPR/Cas cassette, improving the delivery efficiency of CRISPR/Cas reagents, increasing the specificity of genome editing, and optimizing the transformation and regeneration system. In addition, we propose the perspectives on the application of genome editing in crop breeding especially in fruit crops and highlight the potential challenges. It is worth noting that efforts to manipulate fruit crops with genome editing systems are urgently needed for fruit crops breeding and demonstration.},
}
@article {pmid37788375,
year = {2023},
author = {Kulkarni, A and Tanga, S and Karmakar, A and Hota, A and Maji, B},
title = {CRISPR-Based Precision Molecular Diagnostics for Disease Detection and Surveillance.},
journal = {ACS applied bio materials},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsabm.3c00439},
pmid = {37788375},
issn = {2576-6422},
abstract = {Sensitive, rapid, and portable molecular diagnostics is the future of disease surveillance, containment, and therapy. The recent SARS-CoV-2 pandemic has reminded us of the vulnerability of lives from ever-evolving pathogens. At the same time, it has provided opportunities to bridge the gap by translating basic molecular biology into therapeutic tools. One such molecular biology technique is CRISPR (clustered regularly interspaced short palindromic repeat) which has revolutionized the field of molecular diagnostics at the need of the hour. The use of CRISPR-Cas systems has been widespread in biology research due to the ease of performing genetic manipulations. In 2012, CRISPR-Cas systems were, for the first time, shown to be reprogrammable, i.e., capable of performing sequence-specific gene editing. This discovery catapulted the field of CRISPR-Cas research and opened many unexplored avenues in the field of gene editing, from basic research to therapeutics. One such field that benefitted greatly from this discovery was molecular diagnostics, as using CRISPR-Cas technologies enabled existing diagnostic methods to become more sensitive, accurate, and portable, a necessity in disease control. This Review aims to capture some of the trajectories and advances made in this arena and provides a comprehensive understanding of the methods and their potential use as point-of-care diagnostics.},
}
@article {pmid37772705,
year = {2023},
author = {Skoufou-Papoutsaki, N and Adler, S and D'Santos, P and Mannion, L and Mehmed, S and Kemp, R and Smith, A and Perrone, F and Nayak, K and Russell, A and Zilbauer, M and Winton, DJ},
title = {Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR.},
journal = {Disease models &amp; mechanisms},
volume = {16},
number = {10},
pages = {},
doi = {10.1242/dmm.050279},
pmid = {37772705},
issn = {1754-8411},
support = {103805/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Ribonucleoproteins/metabolism ; Gene Editing/methods ; Organoids/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.},
}
@article {pmid37714158,
year = {2023},
author = {Yoshimatsu, S and Okahara, J and Yoshie, J and Igarashi, Y and Nakajima, R and Sanosaka, T and Qian, E and Sato, T and Kobayashi, H and Morimoto, S and Kishi, N and Pillis, DM and Malik, P and Noce, T and Okano, H},
title = {Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing.},
journal = {Cell reports methods},
volume = {3},
number = {9},
pages = {100590},
pmid = {37714158},
issn = {2667-2375},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Tyrosine 3-Monooxygenase/genetics ; Primates/genetics ; Mammals/genetics ; },
abstract = {Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications.},
}
@article {pmid37781628,
year = {2023},
author = {Duan, C and Liu, Y and Liu, Y and Liu, L and Cai, M and Zhang, R and Zeng, Q and Koonin, EV and Krupovic, M and Li, M},
title = {Diversity of Bathyarchaeia viruses in metagenomes and virus-encoded CRISPR system components.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.08.24.554615},
pmid = {37781628},
abstract = {Bathyarchaeia represent a class of archaea common and abundant in sedimentary ecosystems. The virome of Bathyarchaeia so far has not been characterized. Here we report 56 metagenome-assembled genomes of Bathyarchaeia viruses identified in metagenomes from different environments. Gene sharing network and phylogenomic analyses led to the proposal of four virus families, including viruses of the realms Duplodnaviria and Adnaviria , and archaea-specific spindle-shaped viruses. Genomic analyses uncovered diverse CRISPR elements in these viruses. Viruses of the proposed family ' Fuxiviridae ' harbor an atypical type IV-B CRISPR-Cas system and a Cas4 protein that might interfere with host immunity. Viruses of the family ' Chiyouviridae ' encode a Cas2-like endonuclease and two mini-CRISPR arrays, one with a repeat identical to that in the host CRISPR array, potentially allowing the virus to recruit the host CRISPR adaptation machinery to acquire spacers that could contribute to competition with other mobile genetic elements or to inhibition of host defenses. These findings present an outline of the Bathyarchaeia virome and offer a glimpse into their counter-defense mechanisms.},
}
@article {pmid37781609,
year = {2023},
author = {Goldberg, GW and Kogenaru, M and Keegan, S and Haase, MAB and Kagermazova, L and Arias, MA and Onyebeke, K and Adams, S and Fenyö, D and Noyes, MB and Boeke, JD},
title = {Engineered transcription-associated Cas9 targeting in eukaryotic cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.09.18.558319},
pmid = {37781609},
abstract = {DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Tr anscription- a ssociated C as9 T argeting (TraCT). Engineered TraCT is enabled when suboptimal PAM interactions limit basal activity in vivo and when one or more nascent RNA substrates are still tethered to the actively transcribing target DNA in cis . We further show that this phenomenon can be exploited for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes RNA binding in cis as a mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotes.},
}
@article {pmid37780490,
year = {2023},
author = {Movahedi, A and Pucker, B and Kadkhodaei, S},
title = {Editorial: Genomics and gene editing of orphan plants.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1277625},
pmid = {37780490},
issn = {1664-462X},
}
@article {pmid37778114,
year = {2023},
author = {Yadav, S and Kalwan, G and Meena, S and Gill, SS and Yadava, YK and Gaikwad, K and Jain, PK},
title = {Unravelling the due importance of pseudogenes and their resurrection in plants.},
journal = {Plant physiology and biochemistry : PPB},
volume = {203},
number = {},
pages = {108062},
doi = {10.1016/j.plaphy.2023.108062},
pmid = {37778114},
issn = {1873-2690},
abstract = {The complexities of a genome are underpinned to the vast expanses of the intergenic region, which constitutes ∼97-98% of the genome. This region is essentially composed of what is colloquially referred to as the "junk DNA" and is composed of various elements like transposons, repeats, pseudogenes, etc. The latter have long been considered as dead elements merely contributing to transcriptional noise in the genome. Many studies now describe the previously unknown regulatory functions of these genes. Recent advances in the Next-generation sequencing (NGS) technologies have allowed unprecedented access to these regions. With the availability of whole genome sequences of more than 788 different plant species in past 20 years, genome annotation has become feasible like never before. Different bioinformatic pipelines are available for the identification of pseudogenes. However, still little is known about their biological functions. The functional validation of these genes remains challenging and research in this area is still in infancy, particularly in plants. CRISPR/Cas-based genome editing could provide solutions to understand the biological roles of these genes by allowing creation of precise edits within these genes. The possibility of pseudogene reactivation or resurrection as has been demonstrated in a few studies might open new avenues of genetic manipulation to yield a desirable phenotype. This review aims at comprehensively summarizing the progress made with regards to the identification of pseudogenes and understanding their biological functions in plants.},
}
@article {pmid37778080,
year = {2023},
author = {Pastuszka, A and Rousseau, GM and Somerville, V and Levesque, S and Fiset, JP and Goulet, A and Doyon, Y and Moineau, S},
title = {Dairy phages escape CRISPR defence of Streptococcus thermophilus via the anti-CRISPR AcrIIA3.},
journal = {International journal of food microbiology},
volume = {407},
number = {},
pages = {110414},
doi = {10.1016/j.ijfoodmicro.2023.110414},
pmid = {37778080},
issn = {1879-3460},
abstract = {Bacterial community collapse due to phage infection is a major risk in cheese making processes. As virulent phages are ubiquitous and diverse in milk fermentation factories, the use of phage-resistant lactic acid bacteria (LAB) is essential to obtain high-quality fermented dairy products. The LAB species Streptococcus thermophilus contains two type II-A CRISPR-Cas systems (CRISPR1 and CRISPR3) that can effectively protect against phage infection. However, virulent streptococcal phages carrying anti-CRISPR proteins (ACR) that block the activity of CRISPR-Cas systems have emerged in yogurt and cheese environments. For example, phages carrying AcrIIA5 can impede both CRISPR1 and CRISPR3 systems, while AcrIIA6 stops only CRISPR1. Here, we explore the activity and diversity of a third streptococcal phage anti-CRISPR protein, namely AcrIIA3. We were able to demonstrate that AcrIIA3 is efficiently active against the CRISPR3-Cas system of S. thermophilus. We used AlphaFold2 to infer the structure of AcrIIA3 and we predicted that this new family of functional ACR in virulent streptococcal phages has a new α-helical fold, with no previously identified structural homologs. Because ACR proteins are being explored as modulators in genome editing applications, we also tested AcrIIA3 against SpCas9. We found that AcrIIA3 could block SpCas9 in bacteria but not in human cells. Understanding the diversity and functioning of anti-defence mechanisms will be of importance in the design of long-term stable starter cultures.},
}
@article {pmid37777530,
year = {2023},
author = {Thürkauf, M and Lin, S and Oliveri, F and Grimm, D and Platt, RJ and Rüegg, MA},
title = {Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6116},
pmid = {37777530},
issn = {2041-1723},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Deletion ; RNA, Guide, CRISPR-Cas Systems ; Mice, Knockout ; Muscle Fibers, Skeletal ; },
abstract = {Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.},
}
@article {pmid37776859,
year = {2023},
author = {Hino, T and Omura, SN and Nakagawa, R and Togashi, T and Takeda, SN and Hiramoto, T and Tasaka, S and Hirano, H and Tokuyama, T and Uosaki, H and Ishiguro, S and Kagieva, M and Yamano, H and Ozaki, Y and Motooka, D and Mori, H and Kirita, Y and Kise, Y and Itoh, Y and Matoba, S and Aburatani, H and Yachie, N and Karvelis, T and Siksnys, V and Ohmori, T and Hoshino, A and Nureki, O},
title = {An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2023.08.031},
pmid = {37776859},
issn = {1097-4172},
abstract = {SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.},
}
@article {pmid37775783,
year = {2023},
author = {Xie, D and Gu, B and Liu, Y and Ye, P and Ma, Y and Wen, T and Song, X and Zhao, Z},
title = {Efficient targeted recombination with CRISPR/Cas9 in hybrids of Caenorhabditis nematodes with suppressed recombination.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {203},
pmid = {37775783},
issn = {1741-7007},
support = {N_HKBU201/18//Hong Kong Baptist University/ ; HKBU12101520//Hong Kong Baptist University/ ; HKBU12101522//Hong Kong Baptist University/ ; GHP/176/21SZ//Innovation and Technology Commission - Hong Kong/ ; },
mesh = {Animals ; *Caenorhabditis/genetics ; CRISPR-Cas Systems ; Chromosome Mapping ; Genome ; Recombination, Genetic ; },
abstract = {BACKGROUND: Homology-based recombination (HR) is the cornerstone of genetic mapping. However, a lack of sufficient sequence homology or the presence of a genomic rearrangement prevents HR through crossing, which inhibits genetic mapping in relevant genomic regions. This is particularly true in species hybrids whose genomic sequences are highly divergent along with various genome arrangements, making the mapping of genetic loci, such as hybrid incompatibility (HI) loci, through crossing impractical. We previously mapped tens of HI loci between two nematodes, Caenorhabditis briggsae and C. nigoni, through the repeated backcrossing of GFP-linked C. briggsae fragments into C. nigoni. However, the median introgression size was over 7 Mb, indicating apparent HR suppression and preventing the subsequent cloning of the causative gene underlying a given HI phenotype. Therefore, a robust method that permits recombination independent of sequence homology is desperately desired.<br><br>RESULTS: Here, we report a method of highly efficient targeted recombination (TR) induced by CRISPR/Cas9 with dual guide RNAs (gRNAs), which circumvents the HR suppression in hybrids between the two species. We demonstrated that a single gRNA was able to induce efficient TR between highly homologous sequences only in the F1 hybrids but not in the hybrids that carry a GFP-linked C. briggsae fragment in an otherwise C. nigoni background. We achieved highly efficient TR, regardless of sequence homology or genetic background, when dual gRNAs were used that each specifically targeted one parental chromosome. We further showed that dual gRNAs were able to induce efficient TR within genomic regions that had undergone inversion, in which HR-based recombination was expected to be suppressed, supporting the idea that dual-gRNA-induced TR can be achieved through nonhomology-based end joining between two parental chromosomes.<br><br>CONCLUSIONS: Recombination suppression can be circumvented through CRISPR/Cas9 with dual gRNAs, regardless of sequence homology or the genetic background of the species hybrid. This method is expected to be applicable to other situations in which recombination is suppressed in interspecies or intrapopulation hybrids.},
}
@article {pmid37749340,
year = {2023},
author = {Ledford, H},
title = {How Dolly the sheep's legacy lives on: CRISPR cattle and cloned camels.},
journal = {Nature},
volume = {622},
number = {7981},
pages = {18-19},
doi = {10.1038/d41586-023-02921-x},
pmid = {37749340},
issn = {1476-4687},
mesh = {Animals ; *Cattle/genetics ; *Camelus/genetics ; *CRISPR-Cas Systems/genetics ; *Sheep/genetics ; *Gene Editing/trends/veterinary ; *Cloning, Organism/trends/veterinary ; },
}
@article {pmid37774634,
year = {2023},
author = {Kharnaior, P and Tamang, JP},
title = {Microbiome and metabolome in home-made fermented soybean foods of India revealed by metagenome-assembled genomes and metabolomics.},
journal = {International journal of food microbiology},
volume = {407},
number = {},
pages = {110417},
doi = {10.1016/j.ijfoodmicro.2023.110417},
pmid = {37774634},
issn = {1879-3460},
abstract = {Grep-chhurpi, peha, peron namsing and peruñyaan are lesser-known home-made fermented soybean foods prepared by the native people of Arunachal Pradesh in India. Present work aims to study the microbiome, their functional annotations, metabolites and recovery of metagenome-assembled genomes (MAGs) in these four fermented soybean foods. Metagenomes revealed the dominance of bacteria (97.80 %) with minor traces of viruses, eukaryotes and archaea. Bacillota is the most abundant phylum with Bacillus subtilis as the abundant species. Metagenome also revealed the abundance of lactic acid bacteria such as Enterococcus casseliflavus, Enterococcus faecium, Mammaliicoccus sciuri and Staphylococcus saprophyticus in all samples. B. subtilis was the major species found in all products. Predictive metabolic pathways showed the abundance of genes associated with metabolisms. Metabolomics analysis revealed both targeted and untargeted metabolites, which suggested their role in flavour development and therapeutic properties. High-quality MAGs, identified as B. subtilis, Enterococcus faecalis, Pediococcus acidilactici and B. velezensis, showed the presence of several biomarkers corresponding to various bio-functional properties. Gene clusters of secondary metabolites (antimicrobial peptides) and CRISPR-Cas systems were detected in all MAGs. This present work also provides key elements related to the cultivability of identified species of MAGs for future use as starter cultures in fermented soybean food product development. Additionally, comparison of microbiome and metabolites of grep-chhurpi, peron namsing and peruñyaan with that of other fermented soybean foods of Asia revealed a distinct difference.},
}
@article {pmid37773156,
year = {2023},
author = {Zhong, Z and Liu, G and Tang, Z and Xiang, S and Yang, L and Huang, L and He, Y and Fan, T and Liu, S and Zheng, X and Zhang, T and Qi, Y and Huang, J and Zhang, Y},
title = {Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {6102},
pmid = {37773156},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant/genetics ; *Oryza/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Among CRISPR-Cas genome editing systems, Streptococcus pyogenes Cas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probiotic Lactobacillus rhamnosus. We have confirmed the predicted 5'-NGAAA-3' PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9's A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond.},
}
@article {pmid37771787,
year = {2023},
author = {Waldeck, K and Van Zuylekom, J and Cullinane, C and Gulati, T and Simpson, KJ and Tothill, RW and Blyth, B and Hicks, RJ},
title = {A genome-wide CRISPR/Cas9 screen identifies DNA-PK as a sensitiser to [177]Lutetium-DOTA-octreotate radionuclide therapy.},
journal = {Theranostics},
volume = {13},
number = {14},
pages = {4745-4761},
pmid = {37771787},
issn = {1838-7640},
mesh = {Animals ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; DNA ; *Lutetium/metabolism ; *Neuroendocrine Tumors/genetics/radiotherapy ; Octreotide/therapeutic use ; Radioisotopes/therapeutic use ; },
abstract = {Peptide receptor radionuclide therapy (PRRT) using [177]Lutetium-DOTA-octreotate (LuTate) for neuroendocrine tumours (NET) is now an approved treatment available in many countries, though primary or secondary resistance continue to limit its effectiveness or durability. We hypothesised that a genome-wide CRISPR/Cas9 screen would identify key mediators of response to LuTate and gene targets that might offer opportunities for novel combination therapies for NET patients. Methods: We utilised a genome-wide CRISPR-Cas9 screen in LuTate-treated cells to identify genes that impact on the sensitivity or resistance of cells to LuTate. Hits were validated through single-gene knockout. LuTate-resistant cells were assessed to confirm LuTate uptake and retention, and persistence of somatostatin receptor 2 (SSTR2) expression. Gene knockouts conferring LuTate sensitivity were further characterised by pharmacological sensitisation using specific inhibitors and in vivo analysis of the efficacy of these inhibitors in combination with LuTate. Results: The CRISPR-Cas9 screen identified several potential targets for both resistance and sensitivity to PRRT. Two gene knockouts which conferred LuTate resistance in vitro, ARRB2 and MVP, have potential mechanisms related to LuTate binding and retention, and modulation of DNA-damage repair (DDR) pathways, respectively. The screen showed that sensitivity to LuTate treatment in vitro can be conferred by the loss of a variety of genes involved in DDR pathways, with loss of genes involved in Non-Homologous End-Joining (NHEJ) being the most lethal. Loss of the key NHEJ gene, PRKDC (DNA-PK), either by gene loss or inhibition by two different inhibitors, resulted in significantly reduced cell survival upon exposure of cells to LuTate. In SSTR2-positive xenograft-bearing mice, the combination of nedisertib (a DNA-PK specific inhibitor) and LuTate produced a more robust control of tumour growth and increased survival compared to LuTate alone. Conclusions: DDR pathways are critical for sensing and repairing radiation-induced DNA damage, and our study shows that regulation of DDR pathways may be involved in both resistance and sensitivity to PRRT. Additionally, the use of a DNA-PK inhibitor in combination with LuTate PRRT significantly improves the efficacy of the treatment in pre-clinical models, providing further evidence for the clinical efficacy of this combination.},
}
@article {pmid37771576,
year = {2023},
author = {Liu, Y and Ge, H and Marchisio, MA},
title = {Hybrid Boolean gates show that Cas12c controls transcription activation effectively in the yeast S. cerevisiae.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1267174},
pmid = {37771576},
issn = {2296-4185},
abstract = {Among CRISPR-Cas systems, type V CRISPR-Cas12c is of significant interest because Cas12c recognizes a very simple PAM (TN) and has the ability to silence gene expression without cleaving the DNA. We studied how new transcription factors for the yeast Saccharomyces cerevisiae can be built on Cas12c. We found that, upon fusion to a strong activation domain, Cas12c is an efficient activator. Its functionality was proved as a component of hybrid Boolean gates, i.e., logic circuits that mix transcriptional and translational control (the latter reached via tetracycline-responsive riboswitches). Moreover, Cas12c activity can be strongly inhibited by the anti-CRISPR AcrVA1 protein. Thus, Cas12c has the potential to be a new tool to control the activation of gene expression within yeast synthetic gene circuits.},
}
@article {pmid37770400,
year = {2023},
author = {Tkachenko, AA and Borshchevskaya, LN and Sineoky, SP and Gordeeva, TL},
title = {CRISPR/Cas9-Mediated Genome Editing of the Komagataella phaffii to Obtain a Phytase-Producer Markerless Strain.},
journal = {Biochemistry. Biokhimiia},
volume = {88},
number = {9},
pages = {1338-1346},
doi = {10.1134/S0006297923090134},
pmid = {37770400},
issn = {1608-3040},
mesh = {Gene Editing/methods ; *6-Phytase/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Saccharomycetales/genetics ; },
abstract = {Using CRISPR/Cas9 system, the recipient strains K. phaffii VKPM Y-5013 (His[-] phenotype) and K. phaffii VKPM Y-5014 (Leu[-] phenotype) were derived from the K. phaffii VKPM Y-4287 strain, which has a high expression potential. Based on the developed recipient strains, markerless producers of heterologous proteins could be obtained. Efficiency of the gene inactivation with different variants of sgRNA ranged from 65 to 98% and from 15 to 72% for the HIS4 and LEU2 genes, respectively. The recipient strains retained growth characteristics of the parent strain and exhibited high expression potential, as estimated by the production of heterologous phytase from Citrobacter gillenii. Average productivity of the transformants based on the K. phaffii VKPM Y-5013 and K. phaffii VKPM Y-5014 strains was 2.1 and 2.0 times higher than productivity of the transformants of the commercial K. phaffii GS115 strain. Method for sequential integration of genetic material into genome of the K. phaffii VKPM Y-5013 strain was proposed. A highly effective multicopy markerless strain producing C. gillenii phytase was obtained.},
}
@article {pmid37769783,
year = {2023},
author = {Yu, X and Huo, G and Yu, J and Li, H and Li, J},
title = {Prime editing: Its systematic optimization and current applications in disease treatment and agricultural breeding.},
journal = {International journal of biological macromolecules},
volume = {253},
number = {Pt 4},
pages = {127025},
doi = {10.1016/j.ijbiomac.2023.127025},
pmid = {37769783},
issn = {1879-0003},
abstract = {CRISPR/Cas-mediated genome-editing technology has accelerated the development of the life sciences. Prime editing has raised genome editing to a new level because it allows for all 12 types of base substitutions, targeted insertions and deletions, large DNA fragment integration, and even combinations of these edits without generating DNA double-strand breaks. This versatile and game-changing technology has successfully been applied to human cells and plants, and it currently plays important roles in basic research, gene therapy, and crop breeding. Although prime editing has substantially expanded the range of possibilities for genome editing, its efficiency requires improvement. In this review, we briefly introduce prime editing and highlight recent optimizations that have improved the efficiency of prime editors. We also describe how the dual-pegRNA strategy has expanded current editing capabilities, and we summarize the potential of prime editing in treating mammalian diseases and improving crop breeding. Finally, we discuss the limitations of current prime editors and future prospects for optimizing these editors.},
}
@article {pmid37703002,
year = {2023},
author = {Harris, E},
title = {Stem Cell Gene Editing Improved Sickle Cell Disease Outcomes.},
journal = {JAMA},
volume = {330},
number = {13},
pages = {1218},
doi = {10.1001/jama.2023.17177},
pmid = {37703002},
issn = {1538-3598},
mesh = {Humans ; Gene Editing ; *Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Stem Cells ; *Hematopoietic Stem Cell Transplantation ; },
}
@article {pmid37544501,
year = {2023},
author = {Singh, P and Ali, SA and Kumar, S and Mohanty, AK},
title = {CRISPR-Cas9 based knockout of S100A8 in mammary epithelial cells enhances cell proliferation and triggers oncogenic transformation via the PI3K-Akt pathway: Insights from a deep proteomic analysis.},
journal = {Journal of proteomics},
volume = {288},
number = {},
pages = {104981},
doi = {10.1016/j.jprot.2023.104981},
pmid = {37544501},
issn = {1876-7737},
mesh = {Humans ; *Proto-Oncogene Proteins c-akt/metabolism ; *Phosphatidylinositol 3-Kinases/metabolism/pharmacology ; CRISPR-Cas Systems ; Proteomics ; Cell Line, Tumor ; Calgranulin A/metabolism ; Cell Proliferation ; Epithelial Cells/metabolism ; Epithelial-Mesenchymal Transition ; Cell Movement ; },
abstract = {S100A8 is a calcium-binding protein with multiple functions, including being a chemoattractant for phagocytes and playing a key role in the inflammatory response. Its expression has been shown to influence epithelial-mesenchymal transition (EMT) and metastasis in colorectal cancer. However, the role of S100A8 in cell proliferation and differentiation remains unknown. In this study, we used the CRISPR-Cas9 system to knock out S100A8 in healthy mammary epithelial cells and investigated the resulting changes in proteome profiling and signaling pathways. Our results showed that S100A8 knockout led to an increase in cell proliferation and migration, reduced cell-cell adhesion, and increased apoptosis compared to wildtype cells. Proteomics data indicated that S100A8 significantly affects cell cycle progression, cell proliferation, and cell survival through the PI3K-Akt pathway. Furthermore, our findings suggest that S100A8 function is associated with Pten expression, a negative regulator of the PI3K-Akt pathway. These results indicate that S100A8 dysregulation in healthy cells can lead to altered cellular physiology and higher proliferation, similar to cancerous growth. Therefore, maintaining S100A8 expression is critical for preserving healthy cell physiology. This study provides novel insights into the role of S100A8 in cell proliferation and differentiation and its potential relevance to cancer biology. SIGNIFICANCE: The study suggests that maintaining S100A8 expression is critical for preserving healthy cell physiology, and dysregulation of S100A8 in healthy cells can lead to altered cellular physiology and higher proliferation, similar to cancerous growth. Therefore, targeting the PI3K-Akt pathway or regulating Pten expression, a negative regulator of the PI3K-Akt pathway, may be potential strategies for cancer treatment by controlling S100A8 dysregulation. Additionally, S100A8 and S100A9 have been shown to promote metastasis of breast carcinoma by forming a metastatic milieu. However, the differential expression of S100A8 in tumors and its dual effects of antitumor and protumor make the relationship between S100A8 and tumors complicated. Currently, most research focuses on the function of S100A8 as a secretory protein in the microenvironment of tumors, and its function inside healthy cells without forming dimers remains unclear. Furthermore, the study provides insight into the role of S100A8 in cell proliferation and differentiation, which may have implications for other diseases beyond cancer. The functional role of S100A8 in normal mammary epithelial cells remains completely uncertain. Therefore, the objective of this study is to investigate the function of S100A8 on proliferation in mammary epithelial cells after its deletion and to elucidate the underlying proteins involved in downstream signaling. Our findings indicate that the deletion of S100A8 leads to excessive proliferation in normal mammary epithelial cells, reduces apoptosis, and affects cell-cell adhesion molecules required for cellular communication, resulting in a cancer-like phenotype.},
}
@article {pmid37218079,
year = {2023},
author = {Nakazawa, T and Yamaguchi, I and Zhang, Y and Saka, C and Wu, H and Kayama, K and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9.},
journal = {Environmental microbiology},
volume = {25},
number = {10},
pages = {1909-1924},
doi = {10.1111/1462-2920.16427},
pmid = {37218079},
issn = {1462-2920},
support = {19H03017//Grants-in-Aid for Scientific Research (KAKENHI)/ ; 19F19095//Grants-in-Aid for Scientific Research (KAKENHI)/ ; 21K18224//Grants-in-Aid for Scientific Research (KAKENHI)/ ; 19K22332//Grants-in-Aid for Scientific Research (KAKENHI)/ ; 18KK0178//Grants-in-Aid for Scientific Research (KAKENHI)/ ; 18H02254//Grants-in-Aid for Scientific Research (KAKENHI)/ ; 120209920//JSPS Bilateral Program (JPJSBP)/ ; 120208402//JSPS Bilateral Program (JPJSBP)/ ; },
mesh = {*Pleurotus/genetics/metabolism ; Lignin/metabolism ; CRISPR-Cas Systems ; Peroxidases/genetics/metabolism ; Cell Wall/metabolism ; },
abstract = {Lignin-modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white-rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long-standing issue, we examined the lignin-degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple-gene mutant was generated from a monokaryotic wild-type strain PC9 using plasmid-based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple-gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple-gene mutants were generated. The lignin-degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple-gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple-gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.},
}
@article {pmid36724073,
year = {2023},
author = {Yang, J and Guo, F and Chin, HS and Chen, GB and Ang, CH and Lin, Q and Hong, W and Fu, NY},
title = {Sequential genome-wide CRISPR-Cas9 screens identify genes regulating cell-surface expression of tetraspanins.},
journal = {Cell reports},
volume = {42},
number = {2},
pages = {112065},
doi = {10.1016/j.celrep.2023.112065},
pmid = {36724073},
issn = {2211-1247},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Tetraspanins/genetics/metabolism ; Cell Membrane/metabolism ; Membrane Proteins/genetics/metabolism ; *Neoplasms/genetics ; },
abstract = {Tetraspanins, a superfamily of membrane proteins, mediate diverse biological processes through tetraspanin-enriched microdomains in the plasma membrane. However, how their cell-surface presentation is controlled remains unclear. To identify the regulators of tetraspanin trafficking, we conduct sequential genome-wide loss-of-function CRISPR-Cas9 screens based on cell-surface expression of a tetraspanin member, TSPAN8. Several genes potentially involved in endoplasmic reticulum (ER) targeting, different biological processes in the Golgi apparatus, and protein trafficking are identified and functionally validated. Importantly, we find that biantennary N-glycans generated by MGAT1/2, but not more complex glycan structures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intramembrane-cleaving protease reported previously to act as a sheddase of multiple glycan-modifying enzymes, controls cell-surface tetraspanin expression through a mechanism associated with lacto-series glycolipid biosynthesis. Our study provides critical insights into the molecular regulation of cell-surface presentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion.},
}
@article {pmid36701230,
year = {2023},
author = {Schimmel, J and Muñoz-Subirana, N and Kool, H and van Schendel, R and van der Vlies, S and Kamp, JA and de Vrij, FMS and Kushner, SA and Smith, GCM and Boulton, SJ and Tijsterman, M},
title = {Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways.},
journal = {Cell reports},
volume = {42},
number = {2},
pages = {112019},
doi = {10.1016/j.celrep.2023.112019},
pmid = {36701230},
issn = {2211-1247},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Mutation/genetics ; DNA End-Joining Repair ; },
abstract = {Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.},
}
@article {pmid37769296,
year = {2023},
author = {Kim, H and Gu, C and Mustfa, SA and Martella, DA and Wang, C and Wang, Y and Chiappini, C},
title = {CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells.},
journal = {ACS applied materials &amp; interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.3c07918},
pmid = {37769296},
issn = {1944-8252},
support = {MC_PC_16048/MRC_/Medical Research Council/United Kingdom ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.},
}
@article {pmid37766991,
year = {2023},
author = {Li, D and Chen, Y and Huang, F and Wang, J and Li, X and Yang, Y},
title = {CRISPRe: An innate transcriptional enhancer for endogenous genes in CRISPR-Cas immunity.},
journal = {iScience},
volume = {26},
number = {10},
pages = {107814},
pmid = {37766991},
issn = {2589-0042},
abstract = {CRISPR-Cas system has been repurposed to the promising strategy of CRISPR-based transcriptional interference/activation (CRISPRi/CRISPRa) without eliciting DNA breaks that enables Cas complex a block for transcription initiation or elongation, which greatly expands its application fields and values. However, loss of Cas nuclease ability, especially the endogenous nuclease, may affect genome stability seriously. Here, we found a transcriptional enhancer for genes (CRISPRe) in type I-C system of industrial strain Ketogulonicigenium vulgare by maintaining the natural activity of Cas3 nuclease and introducing the specific motifs that do not trigger immunity. CRISPRe greatly improved the expression of heterologous and endogenous genes and the biosynthesis of products by facilitating transcriptional elongation. Besides, the mechanism for pyrroloquinoline quinone (PQQ) biosynthesis regulated by coupling transcriptional-translational elongation in operon was elucidated. Hence, we enrich the toolbox for CRISPR-Cas system and provide a new framework for gene regulation at transcription.},
}
@article {pmid37766348,
year = {2023},
author = {Monod, A and Koch, C and Jindra, C and Haspeslagh, M and Howald, D and Wenker, C and Gerber, V and Rottenberg, S and Hahn, K},
title = {CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts.},
journal = {Viruses},
volume = {15},
number = {9},
pages = {},
pmid = {37766348},
issn = {1999-4915},
support = {33-890//Swiss Armed Forces, Veterinary Department and ISME Equine Research/ ; },
mesh = {Animals ; Horses ; *CRISPR-Cas Systems ; *Skin Neoplasms ; Oncogenes ; Fibroblasts ; Gene Targeting ; },
abstract = {Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis.},
}
@article {pmid37764155,
year = {2023},
author = {Jo, SJ and Kwon, J and Kim, SG and Lee, SJ},
title = {The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era.},
journal = {Microorganisms},
volume = {11},
number = {9},
pages = {},
pmid = {37764155},
issn = {2076-2607},
support = {2022R1I1A1A01069698//National Research Foundation of Korea/ ; },
abstract = {Amid the escalating challenges of antibiotic resistance, bacterial infections have emerged as a global threat. Bacteriophages (phages), viral entities capable of selectively infecting bacteria, are gaining momentum as promising alternatives to traditional antibiotics. Their distinctive attributes, including host specificity, inherent self-amplification, and potential synergy with antibiotics, render them compelling candidates. Phage engineering, a burgeoning discipline, involves the strategic modification of bacteriophages to enhance their therapeutic potential and broaden their applications. The integration of CRISPR-Cas systems facilitates precise genetic modifications, enabling phages to serve as carriers of functional genes/proteins, thereby enhancing diagnostics, drug delivery, and therapy. Phage engineering holds promise in transforming precision medicine, addressing antibiotic resistance, and advancing diverse applications. Emphasizing the profound therapeutic potential of phages, this review underscores their pivotal role in combatting bacterial diseases and highlights their significance in the post-antibiotic era.},
}
@article {pmid37764119,
year = {2023},
author = {Wang, C and Yang, Y and Tang, S and Liu, Y and Wei, Y and Wan, X and Liu, Y and Zhang, Z and Sunkang, Y},
title = {Comparison of Structural Features of CRISPR-Cas Systems in Thermophilic Bacteria.},
journal = {Microorganisms},
volume = {11},
number = {9},
pages = {},
pmid = {37764119},
issn = {2076-2607},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) is an adaptive immune system that defends most archaea and many bacteria from foreign DNA, such as phages, viruses, and plasmids. The link between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria remains unclear. To investigate the relationship between the structural characteristics, diversity, and distribution properties of the CRISPR-Cas system and the optimum growth temperature in thermophilic bacteria, genomes of 61 species of thermophilic bacteria with complete genome sequences were downloaded from GenBank in this study. We used CRISPRFinder to extensively study CRISPR structures and CRISPR-associated genes (cas) from thermophilic bacteria. We statistically analyzed the association between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria. The results revealed that 59 strains of 61 thermophilic bacteria had at least one CRISPR locus, accounting for 96.72% of the total. Additionally, a total of 362 CRISPR loci, 209 entirely distinct repetitive sequences, 131 cas genes, and 7744 spacer sequences were discovered. The average number of CRISPR loci and the average minimum free energy (MFE) of the RNA secondary structure of repeat sequences were positively correlated with temperature whereas the average length of CRISPR loci and the average number of spacers were negatively correlated. The temperature did not affect the average number of CRISPR loci, the average length of repeats, or the guanine-cytosine (GC) content of repeats. The average number of CRISPR loci, the average length of the repeats, and the GC content of the repeats did not reflect temperature dependence. This study may provide a new basis for the study of the thermophilic bacterial adaptation mechanisms of thermophilic bacteria.},
}
@article {pmid37762662,
year = {2023},
author = {Yang, Y and Zhang, Y and Sun, Z and Shen, Z and Li, Y and Guo, Y and Feng, Y and Sun, S and Guo, M and Hu, Z and Yan, C},
title = {Knocking Out OsAAP11 to Improve Rice Grain Quality Using CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762662},
issn = {1422-0067},
support = {BK20210798, BE2022365-2, BE2022335, JBGS [2021]001, BE2021334-1//Jiangsu Province Government/ ; 2023M733005//China Postdoctoral Science Foundation/ ; 32301827//National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Agriculture ; Cooking ; Edible Grain/genetics ; },
abstract = {The demand for rice grain quality, particularly in terms of eating and cooking quality, is increasingly concerning at present. However, the limited availability of rice-quality-related gene resources and time-consuming and inefficient traditional breeding methods have severely hindered the pace of rice grain quality improvement. Exploring novel methods for improving rice grain quality and creating new germplasms is an urgent problem that needs to be addressed. In this study, an amino-acid-transporter-encoding gene OsAAP11 (Os11g0195600) mainly expressed in endosperm was selected as the target for gene editing using the CRISPR/Cas9 system in three japonica genetic backgrounds (Wuyungeng30, Nangeng9108, and Yanggeng158, hereafter referred to as WYG30, NG9108, and YG158). We successfully obtained homozygous osaap11 mutants without transgenic insertion. Subsequently, we conducted comprehensive investigations on the agronomic traits, rice grain quality traits, and transcriptomic analysis of these mutants. The results demonstrate that loss of OsAAP11 function led to a reduced amino acid content and total protein content in grains without affecting the agronomic traits of the plants; meanwhile, it significantly increased the peak viscosity, holding viscosity, and final viscosity values during the cooking process, thereby enhancing the eating and cooking quality. This study not only provides valuable genetic resources and fundamental materials for improving rice grain quality but also provides novel technical support for the rapid enhancement of rice grain quality.},
}
@article {pmid37762535,
year = {2023},
author = {Hryhorowicz, M and Lipiński, D and Zeyland, J},
title = {Evolution of CRISPR/Cas Systems for Precise Genome Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762535},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Technology ; },
abstract = {The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.},
}
@article {pmid37611838,
year = {2023},
author = {Khan, A and Paneerselvam, N and Lawson, BR},
title = {Antiretrovirals to CCR5 CRISPR/Cas9 gene editing - A paradigm shift chasing an HIV cure.},
journal = {Clinical immunology (Orlando, Fla.)},
volume = {255},
number = {},
pages = {109741},
doi = {10.1016/j.clim.2023.109741},
pmid = {37611838},
issn = {1521-7035},
support = {R01 AI150381/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; Receptors, CCR5/genetics ; *HIV Infections/therapy/drug therapy ; *HIV-1/genetics ; Anti-Retroviral Agents/therapeutic use ; },
abstract = {The evolution of drug-resistant viral strains and anatomical and cellular reservoirs of HIV pose significant clinical challenges to antiretroviral therapy. CCR5 is a coreceptor critical for HIV host cell fusion, and a homozygous 32-bp gene deletion (∆32) leads to its loss of function. Interestingly, an allogeneic HSCT from an HIV-negative ∆32 donor to an HIV-1-infected recipient demonstrated a curative approach by rendering the recipient's blood cells resistant to viral entry. Ex vivo gene editing tools, such as CRISPR/Cas9, hold tremendous promise in generating allogeneic HSC grafts that can potentially replace allogeneic ∆32 HSCTs. Here, we review antiretroviral therapeutic challenges, clinical successes, and failures of allogeneic and allogeneic ∆32 HSCTs, and newer exciting developments within CCR5 editing using CRISPR/Cas9 in the search to cure HIV.},
}
@article {pmid37402253,
year = {2023},
author = {Cui, Y and Cao, Q and Li, Y and He, M and Liu, X},
title = {Advances in cis-element- and natural variation-mediated transcriptional regulation and applications in gene editing of major crops.},
journal = {Journal of experimental botany},
volume = {74},
number = {18},
pages = {5441-5457},
doi = {10.1093/jxb/erad248},
pmid = {37402253},
issn = {1460-2431},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Prospective Studies ; Genome, Plant/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Gene Expression Regulation ; *Oryza/genetics ; Trans-Activators/genetics ; },
abstract = {Transcriptional regulation is crucial to control of gene expression. Both spatio-temporal expression patterns and expression levels of genes are determined by the interaction between cis-acting elements and trans-acting factors. Numerous studies have focused on the trans-acting factors that mediate transcriptional regulatory networks. However, cis-acting elements, such as enhancers, silencers, transposons, and natural variations in the genome, are also vital for gene expression regulation and could be utilized by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing to improve crop quality and yield. In this review, we discuss current understanding of cis-element-mediated transcriptional regulation in major crops, including rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays), as well as the latest advancements in gene editing techniques and their applications in crops to highlight prospective strategies for crop breeding.},
}
@article {pmid37762436,
year = {2023},
author = {Zhang, J and Liang, X and Zhang, H and Ishfaq, S and Xi, K and Zhou, X and Yang, X and Guo, W},
title = {Rapid and Sensitive Detection of Toxigenic Fusarium asiaticum Integrating Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Techniques.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762436},
issn = {1422-0067},
support = {2022YFD1400100//National Key Research and Development Program of China/ ; 2022YFE0139500//National Key Research and Development Program of China/ ; },
mesh = {Recombinases ; *Fusarium/genetics ; CRISPR-Cas Systems ; Nucleotidyltransferases ; *Keratoconjunctivitis ; },
abstract = {Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. Both Fusarium graminearum and F. asiaticum are the causal agents of FHB in China. F. asiaticum is the predominant species in the Middle-Lower Reaches of the Yangtze River (MLRYR) and southwest China. Therefore, detecting F. asiaticum in a timely manner is crucial for controlling the disease and preventing mycotoxins from entering the food chain. Here, we combined rapid genomic DNA extraction, recombinase polymerase amplification, Cas12a cleavage, and lateral flow detection techniques to develop a method for the rapid detection of F. asiaticum. The reaction conditions were optimized to provide a rapid, sensitive, and cost-effective method for F. asiaticum detection. The optimized method demonstrated exceptional specificity in detecting F. asiaticum while not detecting any of the 14 other Fusarium strains and 3 non-Fusarium species. Additionally, it could detect F. asiaticum DNA at concentrations as low as 20 ag/μL, allowing for the diagnosis of F. asiaticum infection in maize and wheat kernels even after 3 days of inoculation. The developed assay will provide an efficient and robust detection platform to accelerate plant pathogen detection.},
}
@article {pmid37759711,
year = {2023},
author = {Michalski, MN and Williams, BO},
title = {The Past, Present, and Future of Genetically Engineered Mouse Models for Skeletal Biology.},
journal = {Biomolecules},
volume = {13},
number = {9},
pages = {},
pmid = {37759711},
issn = {2218-273X},
support = {K08 DE031039/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Musculoskeletal Diseases ; Biology ; },
abstract = {The ability to create genetically engineered mouse models (GEMMs) has exponentially increased our understanding of many areas of biology. Musculoskeletal biology is no exception. In this review, we will first discuss the historical development of GEMMs and how these developments have influenced musculoskeletal disease research. This review will also update our 2008 review that appeared in BONEKey, a journal that is no longer readily available online. We will first review the historical development of GEMMs in general, followed by a particular emphasis on the ability to perform tissue-specific (conditional) knockouts focusing on musculoskeletal tissues. We will then discuss how the development of CRISPR/Cas-based technologies during the last decade has revolutionized the generation of GEMMs.},
}
@article {pmid37758954,
year = {2023},
author = {Meers, C and Le, HC and Pesari, SR and Hoffmann, FT and Walker, MWG and Gezelle, J and Tang, S and Sternberg, SH},
title = {Transposon-encoded nucleases use guide RNAs to promote their selfish spread.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {37758954},
issn = {1476-4687},
abstract = {Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance[1]. IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase[2], but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively[3,4]. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases[5,6], but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence.},
}
@article {pmid37674079,
year = {2023},
author = {Wang, C and Zheng, X and Zhang, J and Jiang, X and Wang, J and Li, Y and Li, X and Shen, G and Peng, J and Zheng, P and Gu, Y and Chen, J and Lin, M and Deng, C and Gao, H and Lu, Z and Zhao, Y and Luo, M},
title = {CD300ld on neutrophils is required for tumour-driven immune suppression.},
journal = {Nature},
volume = {621},
number = {7980},
pages = {830-839},
pmid = {37674079},
issn = {1476-4687},
mesh = {Animals ; Humans ; Mice ; CRISPR-Cas Systems ; Disease Progression ; Gene Editing ; Immunotherapy ; *Myeloid-Derived Suppressor Cells/immunology/pathology ; *Neoplasms/immunology/pathology ; *Neutrophils/immunology/pathology ; *Receptors, Immunologic/immunology ; Survival Analysis ; T-Lymphocytes/cytology/immunology/pathology ; Tumor Microenvironment ; Lymphocyte Activation ; },
abstract = {The immune-suppressive tumour microenvironment represents a major obstacle to effective immunotherapy[1,2]. Pathologically activated neutrophils, also known as polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), are a critical component of the tumour microenvironment and have crucial roles in tumour progression and therapy resistance[2-4]. Identification of the key molecules on PMN-MDSCs is required to selectively target these cells for tumour treatment. Here, we performed an in vivo CRISPR-Cas9 screen in a tumour mouse model and identified CD300ld as a top candidate of tumour-favouring receptors. CD300ld is specifically expressed in normal neutrophils and is upregulated in PMN-MDSCs upon tumour-bearing. CD300ld knockout inhibits the development of multiple tumour types in a PMN-MDSC-dependent manner. CD300ld is required for the recruitment of PMN-MDSCs into tumours and their function to suppress T cell activation. CD300ld acts via the STAT3-S100A8/A9 axis, and knockout of Cd300ld reverses the tumour immune-suppressive microenvironment. CD300ld is upregulated in human cancers and shows an unfavourable correlation with patient survival. Blocking CD300ld activity inhibits tumour development and has synergistic effects with anti-PD1. Our study identifies CD300ld as a critical immune suppressor present on PMN-MDSCs, being required for tumour immune resistance and providing a potential target for cancer immunotherapy.},
}
@article {pmid37749638,
year = {2023},
author = {Sun, G and Zhao, S and Fan, Z and Wang, Y and Liu, H and Cao, H and Sun, G and Huang, T and Cai, H and Pan, H and Rong, D and Gao, Y and Tang, W},
title = {CHSY1 promotes CD8[+] T cell exhaustion through activation of succinate metabolism pathway leading to colorectal cancer liver metastasis based on CRISPR/Cas9 screening.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {42},
number = {1},
pages = {248},
pmid = {37749638},
issn = {1756-9966},
support = {Grant No.YNRCQN0325//Hospital talent special fund/ ; },
mesh = {Humans ; Animals ; Mice ; Early Detection of Cancer ; CRISPR-Cas Systems ; Phosphatidylinositol 3-Kinases ; T-Cell Exhaustion ; *Liver Neoplasms/genetics ; *Artemisinins ; CD8-Positive T-Lymphocytes ; *Colorectal Neoplasms/genetics ; Tumor Microenvironment ; Glucuronosyltransferase ; *N-Acetylgalactosaminyltransferases ; Multifunctional Enzymes ; },
abstract = {BACKGROUND: The most common site of metastasis in colorectal cancer (CRC) is the liver and liver metastases occur in more than 50% of patients during diagnosis or treatment. The occurrence of metastasis depends on a series of events known as the invasive-metastasis cascade. Currently, the underlying genes and pathways regulating metastasis initiation in the liver microenvironment are unknown.<br><br>METHODS: We performed systematic CRISPR/Cas9 screening using an in vivo mouse model of CRC liver metastasis to identify key regulators of CRC metastasis. We present the full results of this screen,which included a list of genes that promote or repress CRC liver colonization. By silencing these genes individually, we found that chondroitin sulfate synthase 1 (CHSY1) may be involved in CRC metastasis. We verified the function of CHSY1 and its involvement in liver metastasis of CRC through in vivo and in vitro experiments.<br><br>RESULT: The results of TCGA and CRISPR/Cas9 showed that CHSY1 was overexpressed in CRC primary and liver metastasis tissues and indicated a worse clinical prognosis. In vitro and in vivo experiments confirmed that CHSY1 facilitated the liver metastasis of CRC and CHSY1 induced CD8[+] T cell exhaustion and upregulated PD-L1 expression. The metabolomic analysis indicated that CHSY1 promoted CD8[+] T cell exhaustion by activating the succinate metabolism pathway leading to liver metastasis of CRC. Artemisinin as a CHSY1 inhibitor reduced liver metastasis and enhanced the effect of anti-PD1 in CRC. PLGA-loaded Artemisinin and ICG probe reduced liver metastasis and increased the efficiency of anti-PD1 treatment in CRC.<br><br>CONCLUSION: CHSY1 could promote CD8[+] T cell exhaustion through activation of the succinate metabolic and PI3K/AKT/HIF1A pathway, leading to CRC liver metastasis. The combination of CHSY1 knockdown and anti-PD1 contributes to synergistic resistance to CRC liver metastasis. Artemisinin significantly inhibits CHSY1 activity and in combination with anti-PD1 could synergistically treat CRC liver metastases. This study provides new targets and specific strategies for the treatment of CRC liver metastases, bringing new hope and benefits to patients.},
}
@article {pmid37745481,
year = {2023},
author = {da Silva, JF and Tou, CJ and King, EM and Eller, ML and Ma, L and Rufino-Ramos, D and Kleinstiver, BP},
title = {Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.09.12.557440},
pmid = {37745481},
abstract = {Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the "click"-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether "click DNA" (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.},
}
@article {pmid37744931,
year = {2023},
author = {Wu, Z and Yu, L and Shi, W and Ma, J},
title = {Argonaute protein-based nucleic acid detection technology.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1255716},
pmid = {37744931},
issn = {1664-302X},
abstract = {It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with nucleic acid shearing activity that exhibit specific recognition properties beyond CRISPR-Cas nucleases, which are highly researched but restricted PAM sequence recognition. Therefore, research on Ago protein-mediated nucleic acid detection technology has attracted significant attention from researchers in recent years. Using Ago proteins in developing nucleic acid detection platforms can enable efficient, convenient, and rapid nucleic acid detection and pathogen diagnosis, which is of great importance for human life and health and technological development. In this article, we introduce the structure and function of Argonaute proteins and discuss the latest advances in their use in nucleic acid detection.},
}
@article {pmid37744827,
year = {2023},
author = {León, E and Ortiz, V and Pérez, A and Téllez, J and Díaz, GJ and Ramírez H, MH and Contreras R, LE},
title = {Anti-SpCas9 IgY Polyclonal Antibodies Production for CRISPR Research Use.},
journal = {ACS omega},
volume = {8},
number = {37},
pages = {33809-33818},
pmid = {37744827},
issn = {2470-1343},
abstract = {The CRISPR/Cas adaptative immune system has been harnessed as an RNA-guided, programmable genome editing tool, allowing for diverse biotechnological applications. The implementation of the system relies on the ability to detect the Cas9 protein in biological samples. This task is facilitated by employing antibodies, which exhibit several advantageous features and applications in the context of tropical neglected diseases. This study reports a one-month immunization scheme with the Cas9 protein fromStreptococcus pyogenes to produce IgY polyclonal antibodies (anti-SpCas9), which can be rapidly isolated by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively. Immunodetection assays indicate that the antibodies are highly sensitive, specific, and useful for detecting the SpCas9 protein in promastigotes ofLeishmania braziliensisexpressing exogenous SpCas9. Thus, the simple method for producing anti-SpCas9 IgY antibodies will accelerate CRISPR/Cas-based studies in Leishmania spp. This approach serves as a valuable research tool in this parasite model and holds the potential for wide application in various other biological samples, promoting the implementation of the system. In fact, a bioinformatics approach based on the identification of antigenic determinants in the SpCas9 protein suggests the possibility of using the anti-SpCas9 IgY antibodies in applications such as Prime and Base editing.},
}
@article {pmid37744550,
year = {2023},
author = {Karimi, Z and Ranjbar, R and Najafi, A},
title = {CRISPR Typing of Clinical Strains of Salmonella spp. Isolated in Tehran, Iran.},
journal = {Iranian journal of public health},
volume = {52},
number = {8},
pages = {1758-1763},
pmid = {37744550},
issn = {2251-6093},
abstract = {BACKGROUND: Salmonella is one of the most leading causes of food-born infection and death among infants and people with the poor immunity system. Because Salmonella spp. have diversity in the genome composition and pathogenicity, access to rapid identification and genotyping is necessary to control of salmonellosis. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) typing is a genotyping method that checks these variable sequences in the bacterial genome in a specific species. This study aimed to differentiate Salmonella strains using CRISPR region.<br><br>METHODS: Salmonella isolates, previously identified via standard microbiological and molecular tests, were subjected to the study. Bacterial DNA was extracted and PCR was done using specific primers. The different PCR products were sequenced and the repeats patterns were used to identify additional or degenerate repeat clusters in the CRISPR region. All different sequences were analyzed using CRISPRtionary tool for dendrogram generation using the binary file.<br><br>RESULTS: Overall, 119 strains of various Salmonella serovars were used. The result showed unique CRISPR and diversity in spacer both in sequence and the number. Analysis of the extracted sequence and band patterns illustrated that, except for S. infantis, both S. enteritidis and S. typhimurium isolates were classified as a separate cluster.<br><br>CONCLUSION: CRISPR genotyping could provide serotype/spacers dictionary and it is performed at low cost and high speed in comparison to the other typing methods. Therefore, the assessment of CRISPR and spacer content can be considered as a powerful and practical discriminatory method for subtyping of Salmonella isolates.},
}
@article {pmid37743659,
year = {2023},
author = {Vasileva, A and Selkova, P and Arseniev, A and Abramova, M and Shcheglova, N and Musharova, O and Mizgirev, I and Artamonova, T and Khodorkovskii, M and Severinov, K and Fedorova, I},
title = {Characterization of CoCas9 nuclease from Capnocytophaga ochracea.},
journal = {RNA biology},
volume = {20},
number = {1},
pages = {750-759},
pmid = {37743659},
issn = {1555-8584},
abstract = {Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.},
}
@article {pmid37650323,
year = {2023},
author = {Singh Angom, R and Wang, Y and Wang, E and Dutta, SK and Mukhopadhyay, D},
title = {Conditional, Tissue-Specific CRISPR/Cas9 Vector System in Zebrafish Reveals the Role of Nrp1b in Heart Regeneration.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {43},
number = {10},
pages = {1921-1934},
doi = {10.1161/ATVBAHA.123.319189},
pmid = {37650323},
issn = {1524-4636},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; *Myocytes, Cardiac/physiology ; Neuropilin-1/genetics ; *Regeneration ; Ventricular Remodeling ; *Zebrafish/genetics ; *Zebrafish Proteins/physiology ; },
abstract = {BACKGROUND: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. Neuropilins play crucial roles in zebrafish heart regeneration, heart failure in mice, and electrical remodeling after myocardial infarction in rats. But the cell-specific functions of nrp1 have not been described before. In this study, we have investigated the role of nrp1 isoforms, including nrp1a and nrp1b, in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts.<br><br>METHODS: In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cmlc2 promoter drives Cas9 expression to silence the nrp1 gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts.<br><br>RESULTS: We observed that both the isoforms (nrp1a and nrp1b) are upregulated after the cryoinjury. Interestingly, the nrp1b knockout significantly delayed heart regeneration and impaired cardiac function in the adult zebrafish after cryoinjury, demonstrated by reduced heart rate, ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury.<br><br>CONCLUSIONS: To our knowledge, this study is novel where we have reported a heat-shock-mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.},
}
@article {pmid37589141,
year = {2023},
author = {Whittaker, MN and Testa, LC and Quigley, A and Jindal, I and Cortez-Alvarado, SV and Qu, P and Yang, Y and Alameh, MG and Musunuru, K and Wang, X},
title = {Epigenome Editing Durability Varies Widely Across Cardiovascular Disease Target Genes.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {43},
number = {10},
pages = {2075-2077},
pmid = {37589141},
issn = {1524-4636},
support = {R01 HL148769/HL/NHLBI NIH HHS/United States ; R35 HL145203/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Epigenome ; *Cardiovascular Diseases/genetics/therapy ; Epigenesis, Genetic ; CRISPR-Cas Systems ; Gene Editing ; DNA Methylation ; },
}
@article {pmid37742320,
year = {2023},
author = {Terradas, G and Macias, VM and Peterson, H and McKeand, S and Krawczyk, G and Rasgon, JL},
title = {The development and expansion of in vivo germline editing technologies in arthropods: Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and beyond.},
journal = {Integrative and comparative biology},
volume = {},
number = {},
pages = {},
doi = {10.1093/icb/icad123},
pmid = {37742320},
issn = {1557-7023},
abstract = {In the past 20 years, sequencing technologies have led to easy access to genomic data from non-model organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene editing vectors into early-stage embryos have been used for arthropod transgenesis and the discovery of CRISPR-Cas technologies allowed for targeted mutagenesis and the creation of knock-outs or knock-ins in arthropods. Receptor-Mediated Ovary transduction of Cargo (ReMOT Control) acts as an alternative to embryonic microinjections, which requires expensive equipment and extensive hands-on training. ReMOT Control's main advantage is its ease of use coupled with the ability to hypothetically target any vitellogenic species, as injections are administered to the egg-laying adult rather than embryos. After its initial application in the mosquito Aedes aegypti, ReMOT Control has successfully produced mutants not only for mosquitoes but for multiple arthropod species from diverse orders, such as ticks, mites, wasps, beetles, and true bugs, and is being extended to crustaceans, demonstrating the versatility of the technique. In this review, we discuss the current state of ReMOT Control from its proof-of-concept to the advances and challenges on the application across species after 5 years since its development, including novel extensions of the technique such as DIPA-CRISPR.},
}
@article {pmid37741886,
year = {2023},
author = {Yu, Q and Van Minsel, P and Galle, E and Thienpont, B},
title = {GiRAFR improves gRNA detection and annotation in single-cell CRISPR screens.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {975},
pmid = {37741886},
issn = {2399-3642},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Down-Regulation ; *RNA, Guide, CRISPR-Cas Systems ; Research Personnel ; Software ; },
abstract = {Novel methods that combine single cell RNA-seq with CRISPR screens enable high-throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software is however lacking to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes. Here, we describe a CRISPR droplet sequencing (CROP-seq) dataset. During analysis, we observed that the most commonly used method fails to detect mutant gRNAs. We therefore developed a python tool to identify and characterize intact and mutant gRNAs, called GiRAFR. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells, attenuated downregulation of target genes, as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 35% of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens.},
}
@article {pmid37741566,
year = {2023},
author = {Zhou, X and Zhao, Y and Ni, P and Ni, Z and Sun, Q and Zong, Y},
title = {CRISPR-mediated acceleration of wheat improvement: advances and perspectives.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2023.09.007},
pmid = {37741566},
issn = {1673-8527},
abstract = {Common wheat (Triticum aestivum) is one of the most widely cultivated and consumed crops globally. In the face of limited arable land and climate changes, it is a great challenge to maintain current and increased future wheat production. Enhancing agronomic traits in wheat by introducing mutations across all three homoeologous copies of each gene has proven to be a difficult task due to its large genome with high repetition. However, CRISPR/Cas genome editing technologies offer a powerful means of precisely manipulating the genomes of crop species, thereby opening up new possibilities for biotechnology and breeding. In this review, we first focus on the development and optimization of current CRISPR-based genome editing tools in wheat, emphasizing recent breakthroughs in precise and multiplex genome editing. We then describe the general procedure of wheat genome editing and highlight different methods to deliver the necessary genome editing reagents into wheat cells. Furthermore, we summarize the recent applications and advancements of CRISPR/Cas technologies for wheat improvement. Lastly, we discuss the remaining challenges specific to wheat genome editing and its future prospects.},
}
@article {pmid37741424,
year = {2023},
author = {Lv, X and Li, Y and Xiu, X and Liao, C and Xu, Y and Liu, Y and Li, J and Du, G and Liu, L},
title = {CRISPR genetic toolkits of classical food microorganisms: Current state and future prospects.},
journal = {Biotechnology advances},
volume = {69},
number = {},
pages = {108261},
doi = {10.1016/j.biotechadv.2023.108261},
pmid = {37741424},
issn = {1873-1899},
abstract = {Production of food-related products using microorganisms in an environmentally friendly manner is a crucial solution to global food safety and environmental pollution issues. Traditional microbial modification methods rely on artificial selection or natural mutations, which require time for repeated screening and reproduction, leading to unstable results. Therefore, it is imperative to develop rapid, efficient, and precise microbial modification technologies. This review summarizes recent advances in the construction of gene editing and metabolic regulation toolkits based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) systems and their applications in reconstructing food microorganism metabolic networks. The development and application of gene editing toolkits from single-site gene editing to multi-site and genome-scale gene editing was also introduced. Moreover, it presented a detailed introduction to CRISPR interference, CRISPR activation, and logic circuit toolkits for metabolic network regulation. Moreover, the current challenges and future prospects for developing CRISPR genetic toolkits were also discussed.},
}
@article {pmid37739561,
year = {2023},
author = {Paul, SS},
title = {Phage engineering for development of diagnostic tools.},
journal = {Progress in molecular biology and translational science},
volume = {200},
number = {},
pages = {91-101},
doi = {10.1016/bs.pmbts.2023.04.004},
pmid = {37739561},
issn = {1878-0814},
mesh = {Humans ; *Technology ; *Bacteriophages/genetics ; Biological Assay ; Gene Editing ; },
abstract = {The bacteriophages rely on the host cell to provide energy and resources for their own replication. Antibody-based diagnostic tests rely on the antibody and the biomarker interactions. Since, most of these diagnostic tools employ the use of antibodies; hence, they require intensive storage protocols at cold conditions and incur high time and capital cost due to their production and purification process. Phage-based diagnostics can overcome this limitation. Bacteriophages, have been used as emerging tools for the detection of various pathogens. Rapid phage-mediated detection assays have become commercial diagnostic tools. Conventional method and new cloning approaches have been followed to specifically detect a disease- causing microbial strains. This review discusses use of Phage typing as diagnostic tools, phage-based detection methods, and their usage for signal amplification. Design rules for reporter phage engineering are also discussed followed by different engineering platforms for phage genome editing. We also discuss recent examples of how phage research is influencing the recent advances in the development of phage-based diagnostics for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays.},
}
@article {pmid37736272,
year = {2023},
author = {Yin, G and Wang, XH and Sun, Y},
title = {Recent advances in CRISPR-Cas system for the treatment of genetic hearing loss.},
journal = {American journal of stem cells},
volume = {12},
number = {3},
pages = {37-50},
pmid = {37736272},
issn = {2160-4150},
abstract = {Genetic hearing loss has emerged as a significant public health concern that demands attention. Among the various treatment strategies, gene therapy based on gene editing technology is considered the most promising approach for addressing genetic hearing loss by repairing or eliminating mutated genes. The advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has revolutionized gene therapy through its remarkable gene editing capabilities. This system has been extensively employed in mammalian gene editing and is currently being evaluated through clinical trials. Against this backdrop, this review aims to provide an overview of recent advances in utilizing the CRISPR-Cas system to treat genetic hearing loss. Additionally, we delve into the primary challenges and prospects associated with the current application of this system in addressing genetic hearing loss.},
}
@article {pmid37733197,
year = {2023},
author = {Raudstein, M and Kjærner-Semb, E and Barvik, M and Broll, S and Straume, AH and Edvardsen, RB},
title = {In vivo CRISPR/LbCas12a-mediated knock-in and knock-out in Atlantic salmon (Salmo salar L.).},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {37733197},
issn = {1573-9368},
support = {301602//Norges Forskningsr&#xe5;d/ ; 301602//Norges Forskningsr&#xe5;d/ ; },
abstract = {Genome editing using the CRISPR/Cas system offers the potential to enhance current breeding programs and introduce desirable genetic traits, including disease resistance, in salmon aquaculture. Several nucleases are available using this system, displaying differences regarding structure, cleavage, and PAM requirement. Cas9 is well established in Atlantic salmon, but Cas12a has yet to be tested in vivo in this species. In the present work, we microinjected salmon embryos with LbCas12a ribonucleoprotein complexes targeting the pigmentation gene solute carrier family 45 member 2 (slc45a2). Using CRISPR/LbCas12a, we were able to knock-out slc45a2 and knock-in a FLAG sequence element by providing single-stranded DNA templates. High-throughput sequencing revealed perfect HDR rates up to 34.3% and 54.9% in individual larvae using either target or non-target strand template design, respectively. In this work, we demonstrate the in vivo application of CRISPR/LbCas12a in Atlantic salmon, expanding the toolbox for editing the genome of this important aquaculture species.},
}
@article {pmid37723277,
year = {2023},
author = {Ledford, H},
title = {Super-precise CRISPR tool enters US clinical trials for the first time.},
journal = {Nature},
volume = {621},
number = {7980},
pages = {667-668},
pmid = {37723277},
issn = {1476-4687},
mesh = {United States ; *Clinical Trials as Topic ; *Gene Editing/methods/standards ; *CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods/standards ; Leukemia/therapy ; Humans ; },
}
@article {pmid37494933,
year = {2023},
author = {Roberts, MA and Deol, KK and Mathiowetz, AJ and Lange, M and Leto, DE and Stevenson, J and Hashemi, SH and Morgens, DW and Easter, E and Heydari, K and Nalls, MA and Bassik, MC and Kampmann, M and Kopito, RR and Faghri, F and Olzmann, JA},
title = {Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.},
journal = {Developmental cell},
volume = {58},
number = {18},
pages = {1782-1800.e10},
pmid = {37494933},
issn = {1878-1551},
support = {F32 GM113370/GM/NIGMS NIH HHS/United States ; R01 DK128099/DK/NIDDK NIH HHS/United States ; F31 DK121477/DK/NIDDK NIH HHS/United States ; R01 GM074874/GM/NIGMS NIH HHS/United States ; R01 GM112948/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Perilipin-2/genetics/metabolism ; *Lipid Droplets/metabolism ; *CRISPR-Cas Systems/genetics ; Lipid Metabolism/genetics ; Cell Line ; },
abstract = {Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.},
}
@article {pmid37731160,
year = {2023},
author = {Hussain, A and Akram, S and Ahmad, D and Rehman, M and Ahmed, A and Ali, SA},
title = {Molecular Assessment and Validation of the Selected Enterococcal Strains as Probiotics.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {37731160},
issn = {1867-1314},
support = {NRPU-20-1339/R&amp;D/09//Higher Education Commision, Pakistan/ ; },
abstract = {Probiotics are live microorganisms which confer health benefits to the host. Lactic acid bacteria (LAB) are used as probiotics since decades. Enterococci being the member of LAB have proven probiotic strains; therefore, this study was aimed at finding out the potential probiotic candidates from the pool of locally isolated strains. For initial screening, one hundred and twenty-two strains were selected and subjected to different confirmatory and phenotypic tests to choose the best strains that have potential probiotic criteria, i.e., no potential virulence traits, antibiotic resistance, and having tolerance properties. Keeping this criterion, only eleven strains (n = 11) were selected for further assessment. All virulence traits such as production of hemolysin, gelatinase, biofilm, and DNase were performed and not found in the tested strains. The molecular assessment indicates the presence of few virulence-associated genes in Enterococcus faecalis strains with variable frequency. The phenotypic and genotypic assessments of antibiotic resistance profile indicate that the selected strain was susceptible to ten commonly used antibiotics, and there were no transferrable antibiotic resistance genes. The presence of CRISPR-Cas genes also confirmed the absence of antibiotic resistance genes. Various enterocin-producing genes like EntP, EntB, EntA, and EntQ were also identified in the selected strains which make them promising probiotic lead strains. Different tolerance assays like acid, NaCl, and gastric juice tolerance that mimic host conditions was also evaluated by providing artificial conditions. Cellular adhesion and aggregation properties like auto- and co-aggregation were also checked and their results reflect all in the favor of lead probiotic strains.},
}
@article {pmid37696181,
year = {2023},
author = {Liu, Y and Fan, R and Yi, J and Cui, Q and Cui, C},
title = {A fusion framework of deep learning and machine learning for predicting sgRNA cleavage efficiency.},
journal = {Computers in biology and medicine},
volume = {165},
number = {},
pages = {107476},
doi = {10.1016/j.compbiomed.2023.107476},
pmid = {37696181},
issn = {1879-0534},
mesh = {*Deep Learning ; RNA, Guide, CRISPR-Cas Systems ; Machine Learning ; Neural Networks, Computer ; },
abstract = {CRISPR/Cas9 system is a powerful tool for genome editing. Numerous studies have shown that sgRNAs can strongly affect the efficiency of editing. However, it is still not clear what rules should be followed for designing sgRNA with high cleavage efficiency. At present, several machine learning or deep learning methods have been developed to predict the cleavage efficiency of sgRNAs, however, the prediction accuracy of these tools is still not satisfactory. Here we propose a fusion framework of deep learning and machine learning, which first deals with the primary sequence and secondary structure features of the sgRNAs using both convolutional neural network (CNN) and recurrent neural network (RNN), and then uses the features extracted by the deep neural network to train a conventional machine learning model with LGBM. As a result, the new approach overwhelmed previous methods. The Spearman's correlation coefficient between predicted and measured sgRNA cleavage efficiency of our model (0.917) is improved by over 5% compared with the most advanced method (0.865), and the mean square error reduces from 7.89 × 10[-3] to 4.75 × 10[-3]. Finally, we developed an online tool, CRISep (http://www.cuilab.cn/CRISep), to evaluate the availability of sgRNAs based on our models.},
}
@article {pmid37669145,
year = {2023},
author = {Wang, X and Li, Y and Wang, X and Sandoval, DM and He, Z and A, S and Sáez, IL and Wang, W},
title = {Guanidyl-Rich Poly(β Amino Ester)s for Universal Functional Cytosolic Protein Delivery and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 Ribonucleoprotein Based Gene Editing.},
journal = {ACS nano},
volume = {17},
number = {18},
pages = {17799-17810},
doi = {10.1021/acsnano.3c03269},
pmid = {37669145},
issn = {1936-086X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; HeLa Cells ; Green Fluorescent Proteins ; Esters ; Guanidine ; Guanidines ; },
abstract = {Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(β amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.},
}
@article {pmid37593879,
year = {2023},
author = {Zhai, Y and Yang, L and Zheng, W and Wang, Q and Zhu, Z and Han, F and Hao, Y and Ma, S and Cheng, G},
title = {A precise design strategy for a cell-derived extracellular matrix based on CRISPR/Cas9 for regulating neural stem cell function.},
journal = {Biomaterials science},
volume = {11},
number = {19},
pages = {6537-6544},
doi = {10.1039/d2bm01466a},
pmid = {37593879},
issn = {2047-4849},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Extracellular Matrix/metabolism ; Heparan Sulfate Proteoglycans ; Heparitin Sulfate/metabolism ; *Neural Stem Cells/metabolism ; },
abstract = {The extracellular matrix (ECM) is a natural microenvironment pivotal for stem cell survival, as well as proliferation, differentiation and metastasis, composed of a variety of biological molecular complexes secreted by resident cells in tissues and organs. Heparan sulfate proteoglycan (HSPG) is a type of ECM protein that contains one or more covalently attached heparan sulfate chains. Heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains of great importance in development and normal physiology. Herein, we constructed endogenous HSPG2 overexpression in mouse embryonic fibroblasts based on the CRISPR/Cas9 synergistic activation mediator system and then fabricated a cell-derived HSPG2 functional ECM (ECM[HSPG2]). The ECM[HSPG2] is capable of enriching basic fibroblast growth factor (bFGF), which binds more strongly than the negative control ECM. With a growing bFGF concentration, ECM[HSPG2] could better maintain neural stem cell (NSCs) stemness and promote NSC proliferation and differentiation in culture. These findings provide a precise design strategy for producing a specific cell-derived ECM for biomaterials in research and regenerative medicine.},
}
@article {pmid37562129,
year = {2023},
author = {Zou, K and Wang, F and Zhang, Z and Zhou, Y and Li, P and Wang, D and Zhu, M and Jia, C and Wei, Z},
title = {Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells.},
journal = {Poultry science},
volume = {102},
number = {10},
pages = {102970},
pmid = {37562129},
issn = {1525-3171},
mesh = {Animals ; Gene Knockout Techniques/veterinary ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Chickens/genetics ; Endonucleases/genetics ; },
abstract = {The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.},
}
@article {pmid37494991,
year = {2023},
author = {Huang, T and Li, L and Li, J and Li, X and Li, S and Wang, X and Zhang, N and Yu, Y and Zhang, X and Zhao, Z and Guo, Y and Cao, L and Gong, P},
title = {Rapid, sensitive, and visual detection of Clonorchis sinensis with an RPA-CRISPR/Cas12a-based dual readout portable platform.},
journal = {International journal of biological macromolecules},
volume = {249},
number = {},
pages = {125967},
doi = {10.1016/j.ijbiomac.2023.125967},
pmid = {37494991},
issn = {1879-0003},
mesh = {Animals ; Humans ; *Clonorchis sinensis/genetics ; CRISPR-Cas Systems/genetics ; Cross Reactions ; Food ; Liver ; },
abstract = {Clonorchis sinensis is a food-borne parasite that parasitizes the liver and bile ducts of humans and many animals. This parasite exerts a high burden due to diverse hepatobiliary morbidities (e.g., cholangitis, cholecystitis, cholelithiasis, and cholangiocarcinoma), and an effective detection strategy is urgently needed. CRISPR/Cas12a exhibits nonspecific trans-cleavage activity upon binding to its specific target and has been widely used for nucleic acid detection. In this study, an RPA-CRISPR/Cas12a-based dual readout portable detection platform was established, which shows high sensitivity (one copy/μl) and specificity (no cross-reactivity with common pathogens) by rapid preamplification and combines lateral flow strips and visual fluorescence for visualization of results by the naked eye within 1 h. Moreover, 50 human fecal swabs and 50 fish flesh samples were detected by this platform and nested PCR. The CRISPR/Cas12a-based dual readout portable platform showed 10.0 % (5/50) C. sinensis-positive samples in human fecal swabs and 28.0 % (14/50) in fish flesh, which was consistent with the results of nested PCR. The results demonstrate that our portable platform has the advantages of stability, sensitivity, accuracy, and low equipment requirements. Furthermore, we provide novel point-of-care testing (POCT) for clinical use in remote rural and resource-constrained areas.},
}
@article {pmid37485817,
year = {2023},
author = {Beha, MJ and Kim, JC and Im, SH and Kim, Y and Yang, S and Lee, J and Nam, YR and Lee, H and Park, HS and Chung, HJ},
title = {Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {27},
pages = {e2302253},
pmid = {37485817},
issn = {2198-3844},
support = {2021R1A2C2011763//National Research Foundation of Korea/ ; 2021M3E5E3080383//National Research Foundation of Korea/ ; 2022R1A4A5028131//National Research Foundation of Korea/ ; 2023R1A2C1005945//National Research Foundation of Korea/ ; HI22C2010//Ministry of Health and Welfare/ ; SSTF-BA1702-09//Samsung Science and Technology Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Pharmaceutical Preparations ; Nanomedicine ; Gene Editing ; },
abstract = {Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.},
}
@article {pmid37452759,
year = {2023},
author = {Chikmagalur Nagaraja, B and Karuppannasamy, A and Ramasamy, A and Cholenahalli Narayanappa, A and Chalapathi, P and Maligeppagol, M},
title = {CRISPR/Cas9-mediated mutagenesis of Sex lethal (Sxl) gene impacts fertility of the Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).},
journal = {Archives of insect biochemistry and physiology},
volume = {114},
number = {2},
pages = {1-15},
doi = {10.1002/arch.22035},
pmid = {37452759},
issn = {1520-6327},
support = {//ICAR CABin project/ ; },
mesh = {Male ; Animals ; Spodoptera/genetics ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Fertility/genetics ; Mutagenesis ; Larva ; Zea mays ; },
abstract = {Identification of novel approaches for managing the global pest, the Fall armyworm, Spodoptera frugiperda, is the need of the hour, as it defies many management strategies including synthetic chemicals, Bt transgenics, and so on. Recently CRISPR/Cas9-based genome editing opened up newer avenues to design novel pest management strategies such as precision-guided sterile insect technique (pgSIT). In this regard, genes governing sex determination, egg reproduction, and spermatogenesis could be the prime targets for genome editing. This requires validation of the target genes, preferably by a nontransgenic DNA-free editing, before the final application. One such important gene regulating sex determination in Drosophila is the Sex lethal (Sxl). However, the function of Sxl is not highly conserved in other insects and, in particular, we are beginning to comprehend its role in Lepidoptera with only one reference available in Spodoptera litura till date. In the present study, we have edited the sxl gene of S. frugiperda through the delivery of ribonucleoprotein complex (sgRNA + Cas9) at G0 stage embryo, targeting the conserved region of all the documented five splice variants. Results clearly showed that editing of sxl gene impacted the overall fecundity and hatching rate. Therefore, Sxl could be one of the target genes for developing pgSIT approach for the management of S. frugiperda.},
}
@article {pmid37519013,
year = {2023},
author = {Balmas, E and Sozza, F and Bottini, S and Ratto, ML and Savorè, G and Becca, S and Snijders, KE and Bertero, A},
title = {Manipulating and studying gene function in human pluripotent stem cell models.},
journal = {FEBS letters},
volume = {597},
number = {18},
pages = {2250-2287},
doi = {10.1002/1873-3468.14709},
pmid = {37519013},
issn = {1873-3468},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Pluripotent Stem Cells ; Transfection ; Biomarkers/metabolism ; },
abstract = {Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss-, gain-, and change-of-function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single-cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.},
}
@article {pmid37730702,
year = {2023},
author = {Deng, X and Osikpa, E and Yang, J and Oladeji, SJ and Smith, J and Gao, X and Gao, Y},
title = {Structural basis for the activation of a compact CRISPR-Cas13 nuclease.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5845},
pmid = {37730702},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Dependovirus/genetics ; Endonucleases/genetics ; RNA ; },
abstract = {The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.},
}
@article {pmid37729124,
year = {2023},
author = {Liu, M and Li, Z and Chen, J and Lin, J and Lu, Q and Ye, Y and Zhang, H and Zhang, B and Ouyang, S},
title = {Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage.},
journal = {PLoS genetics},
volume = {19},
number = {9},
pages = {e1010930},
pmid = {37729124},
issn = {1553-7404},
mesh = {RNA Cleavage ; *RNA, Guide, CRISPR-Cas Systems ; *Endonucleases/genetics ; Endoribonucleases ; RNA/genetics ; },
abstract = {Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.},
}
@article {pmid37728624,
year = {2023},
author = {Huang, Y and Chen, M and Hu, G and Wu, B and He, M},
title = {Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37728624},
issn = {1432-0614},
abstract = {Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.},
}
@article {pmid37728251,
year = {2023},
author = {Sparmann, A and Vogel, J},
title = {RNA-based medicine: from molecular mechanisms to therapy.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {e114760},
doi = {10.15252/embj.2023114760},
pmid = {37728251},
issn = {1460-2075},
support = {//Elite Network Bavaria/Elitenetzwerk Bayern/ ; },
abstract = {RNA-based therapeutics have the potential to revolutionize the treatment and prevention of human diseases. While early research faced setbacks, it established the basis for breakthroughs in RNA-based drug design that culminated in the extraordinarily fast development of mRNA vaccines to combat the COVID-19 pandemic. We have now reached a pivotal moment where RNA medicines are poised to make a broad impact in the clinic. In this review, we present an overview of different RNA-based strategies to generate novel therapeutics, including antisense and RNAi-based mechanisms, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Using three rare genetic diseases as examples, we highlight the opportunities, but also the challenges to wide-ranging applications of this class of drugs.},
}
@article {pmid37727048,
year = {2023},
author = {Zhou, S and Zhu, S and Huang, Z and Chen, J and Li, J and Yang, M and Jin, L and Huo, D and Hou, C},
title = {Target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a for simultaneous detection of HPV16 and HPV18.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3cc04223e},
pmid = {37727048},
issn = {1364-548X},
abstract = {Simultaneous detection of multiple targets can provide important data support for clinical diagnosis and treatment. Here, we report a facile isothermal assay based on target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a (TM-RCT/Cas12a-Cas13a). Through facile one-step amplification (TM-RCT), two target DNAs are converted to RNA amplified products. The simultaneous detection of HPV16 and HPV18 is then achieved by combining two CRISPR/Cas systems. This system shows excellent sensing performance and provides a universal method for simultaneous detection.},
}
@article {pmid37726790,
year = {2023},
author = {Danti, L and Lundin, K and Sepponen, K and Yohannes, DA and Kere, J and Tuuri, T and Tapanainen, JS},
title = {CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate.},
journal = {Journal of ovarian research},
volume = {16},
number = {1},
pages = {194},
pmid = {37726790},
issn = {1757-2215},
support = {813707//European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie/ ; },
mesh = {Animals ; Humans ; Female ; Male ; *Human Embryonic Stem Cells ; CRISPR-Cas Systems ; Steroidogenic Factor 1/genetics ; Cell Differentiation/genetics ; Cytochrome P450 Family 17 ; },
abstract = {The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms' tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.},
}
@article {pmid37572348,
year = {2023},
author = {Cachera, P and Olsson, H and Coumou, H and Jensen, ML and Sánchez, BJ and Strucko, T and van den Broek, M and Daran, JM and Jensen, MK and Sonnenschein, N and Lisby, M and Mortensen, UH},
title = {CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries.},
journal = {Nucleic acids research},
volume = {51},
number = {17},
pages = {e91},
pmid = {37572348},
issn = {1362-4962},
support = {//European Union's Horizon 2020/ ; 722287//Marie Sk&#x142;odowska-Curie/ ; 087-2012-3//Danish National Advanced Technology Foundation/ ; DNRF115//Danish National Research Foundation/ ; NNF19SA0035438//Novo Nordisk Foundation Bioscience Ph.D. Programme/ ; NNF17SA0031362//Fermentation-based Biomanufacturing Initiative/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; Betaxanthins ; Reproducibility of Results ; Gene Editing/methods ; Genetic Engineering/methods ; },
abstract = {Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.},
}
@article {pmid37568030,
year = {2023},
author = {Gruber, K and Melton, L},
title = {CRISPR upgrades insect proteins for feed.},
journal = {Nature biotechnology},
volume = {41},
number = {8},
pages = {1038-1040},
doi = {10.1038/s41587-023-01902-9},
pmid = {37568030},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Insect Proteins/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37560931,
year = {2023},
author = {Marsic, T and Gundra, SR and Wang, Q and Aman, R and Mahas, A and Mahfouz, MM},
title = {Programmable site-specific DNA double-strand breaks via PNA-assisted prokaryotic Argonautes.},
journal = {Nucleic acids research},
volume = {51},
number = {17},
pages = {9491-9506},
pmid = {37560931},
issn = {1362-4962},
support = {//KAUST Smart Health Initiative (KSHI)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Peptide Nucleic Acids/genetics ; Gene Editing ; Genome ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.},
}
@article {pmid37500264,
year = {2023},
author = {Igarashi, T and Katayama, T and Maruyama, JI},
title = {CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae industrial strains.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {87},
number = {10},
pages = {1236-1248},
doi = {10.1093/bbb/zbad101},
pmid = {37500264},
issn = {1347-6947},
support = {23H04547//Grant-in-Aid for Transformative Research Areas/ ; //Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Soy Foods ; Aspergillus/genetics ; },
abstract = {Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.},
}
@article {pmid36931733,
year = {2023},
author = {Sun, R and Raban, R and Akbari, OS},
title = {Generating Aedes aegypti Mutant Strains with Transgenic Cas9.},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {9},
pages = {671-678},
doi = {10.1101/pdb.prot108085},
pmid = {36931733},
issn = {1559-6095},
support = {R01 AI151004/AI/NIAID NIH HHS/United States ; R01 GM132825/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Aedes/genetics ; Animals, Genetically Modified ; Recombinational DNA Repair ; },
abstract = {Here, we provide a protocol for generating Aedes aegypti mutant strains via end-joining (EJ) or homology-directed repair (HDR) mechanisms using genetically encoded Cas9.},
}
@article {pmid35637909,
year = {2022},
author = {Shirai, Y and Piulachs, MD and Belles, X and Daimon, T},
title = {DIPA-CRISPR is a simple and accessible method for insect gene editing.},
journal = {Cell reports methods},
volume = {2},
number = {5},
pages = {100215},
pmid = {35637909},
issn = {2667-2375},
mesh = {Animals ; Female ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Insecta/genetics ; },
abstract = {Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed "direct parental" CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.},
}
@article {pmid37725519,
year = {2023},
author = {Saini, H and Thakur, R and Gill, R and Tyagi, K and Goswami, M},
title = {CRISPR/Cas9-gene editing approaches in plant breeding.},
journal = {GM crops &amp; food},
volume = {14},
number = {1},
pages = {1-17},
pmid = {37725519},
issn = {2164-5701},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Plant Breeding ; Agriculture ; Blotting, Southern ; },
abstract = {CRISPR/Cas9 gene editing system is recently developed robust genome editing technology for accelerating plant breeding. Various modifications of this editing system have been established for adaptability in plant varieties as well as for its improved efficiency and portability. This review provides an in-depth look at the various strategies for synthesizing gRNAs for efficient delivery in plant cells, including chemical synthesis and in vitro transcription. It also covers traditional analytical tools and emerging developments in detection methods to analyze CRISPR/Cas9 mediated mutation in plant breeding. Additionally, the review outlines the various analytical tools which are used to detect and analyze CRISPR/Cas9 mediated mutations, such as next-generation sequencing, restriction enzyme analysis, and southern blotting. Finally, the review discusses emerging detection methods, including digital PCR and qPCR. Hence, CRISPR/Cas9 has great potential for transforming agriculture and opening avenues for new advancements in the system for gene editing in plants.},
}
@article {pmid37723839,
year = {2023},
author = {Whitley, JA and Cai, H},
title = {Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.},
journal = {Journal of extracellular vesicles},
volume = {12},
number = {9},
pages = {e12343},
pmid = {37723839},
issn = {2001-3078},
support = {U01 CA225784-01/CA/NCI NIH HHS/United States ; R21 AI157831/AI/NIAID NIH HHS/United States ; R21 AI171944/AI/NIAID NIH HHS/United States ; },
mesh = {*Extracellular Vesicles ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Cell Membrane ; Genetic Therapy ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex-based CRISPR systems have gained momentum due to their reduction of off-target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell-derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs-based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus-like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA-coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single-chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs-based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs-based delivery of CRISPR RNP in gene therapy and disease treatment.},
}
@article {pmid37723612,
year = {2023},
author = {Rashid, SJ and Nale, JY and Millard, AD and Clokie, MRJ},
title = {Novel ribotype/sequence type associations and diverse CRISPR-Cas systems in environmental Clostridioides difficile strains from Northern Iraq.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnad091},
pmid = {37723612},
issn = {1574-6968},
abstract = {The environment is a natural reservoir of Clostridioides difficile and here, we aimed to isolate the pathogen from seven locations in Northern Iraq. Four of the sites yielded thirty-one isolates (ten from soils, twenty-one from sediments) which together represent ribotypes 001 (five), 010 (five), 011 (two), 035 (two), 091 (eight) and 604 (nine). Twenty-five of the isolates (∼81%) are non-toxigenic while 6 (∼19%) encode the toxin A and B genes. The genomes of eleven selected isolates represent six sequence types: ST-3 (two), ST-15 (one), ST-107 (five), ST-137 (one), ST-177 (one) and ST-181 (one). Five novel ribotype/sequence type (RT/ST) associations: RT011/ST-137, RT035/ST-107, RT091/ST-107, RT604/ST-177 and RT604/ST-181 were identified and the first three are linked to ribotypes previously uncharacterised by multilocus sequence typing (MLST). Nine of the genomes belong to Clade 1 and two are closely related to the cryptic C-I clade. Diverse multiple prophages and CRISPR-Cas systems (class 1 subtype I-B1 and class 2 type V CRISPR-Cas systems) with spacers identical to other C. difficile phages and plasmids were detected in the genomes. Our data show the broader diversity that exists within environmental C. difficile strains from a much less studied location and their potential role in evolution and emergence of new strains.},
}
@article {pmid37722021,
year = {2023},
author = {Zhu, F and Zhao, Q},
title = {CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c02834},
pmid = {37722021},
issn = {1520-6882},
abstract = {CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high trans-cleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive small-molecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of small-molecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.},
}
@article {pmid37720722,
year = {2023},
author = {Menchaca, A},
title = {Assisted Reproductive Technologies (ART) and genome editing to support a sustainable livestock.},
journal = {Animal reproduction},
volume = {20},
number = {2},
pages = {e20230074},
pmid = {37720722},
issn = {1984-3143},
abstract = {This article provides an overview of assisted reproductive technologies (ART) and genome engineering to improve livestock production systems for the contribution of global sustainability. Most ruminant production systems are conducted on grassland conditions, as is the case of South American countries that are leaders in meat and milk production worldwide with a well-established grass-feed livestock. These systems have many strengths from an environmental perspective and consumer preferences but requires certain improvements to enhance resource efficiency. Reproductive performance is one of the main challenges particularly in cow-calf operations that usually are conducted under adverse conditions and thus ART can make a great contribution. Fixed-time artificial insemination is applied in South America in large scale programs as 20 to 30% of cows receive this technology every year in each country, with greater calving rate and significant herd genetic gain occurred in this region. Sexed semen has also been increasingly implemented, enhancing resource efficiency by a) obtaining desired female replacement and improving animal welfare by avoiding newborn male sacrifice in dairy industry, or b) alternatively producing male calves for beef industry. In vitro embryo production has been massively applied, with this region showing the greatest number of embryos produced worldwide leading to significant improvement in herd genetics and productivity. Although the contribution of these technologies is considerable, further improvements will be required for a significant livestock transformation and novel biotechnologies such as genome editing are already available. Through the CRISPR/Cas-based system it is possible to enhance food yield and quality, avoid animal welfare concerns, overcome animal health threats, and control pests and invasive species harming food production. In summary, a significant enhancement in livestock productivity and resource efficiency can be made through reproductive technologies and genome editing, improving at the same time profitability for farmers, and global food security and sustainability.},
}
@article {pmid37720320,
year = {2023},
author = {Li, X and Zhu, S and Zhang, X and Ren, Y and He, J and Zhou, J and Yin, L and Wang, G and Zhong, T and Wang, L and Xiao, Y and Zhu, C and Yin, C and Yu, X},
title = {Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1215466},
pmid = {37720320},
issn = {2296-4185},
abstract = {The rapid diagnosis of pathogenic infections plays a vital role in disease prevention, control, and public health safety. Recombinase-aided amplification (RAA) is an innovative isothermal nucleic acid amplification technology capable of fast DNA or RNA amplification at low temperatures. RAA offers advantages such as simplicity, speed, precision, energy efficiency, and convenient operation. This technology relies on four essential components: recombinase, single-stranded DNA-binding protein (SSB), DNA polymerase, and deoxyribonucleoside triphosphates, which collectively replace the laborious thermal cycling process of traditional polymerase chain reaction (PCR). In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system, a groundbreaking genome engineering tool, has garnered widespread attention across biotechnology, agriculture, and medicine. Increasingly, researchers have integrated the recombinase polymerase amplification system (or RAA system) with CRISPR technology, enabling more convenient and intuitive determination of detection results. This integration has significantly expanded the application of RAA in pathogen detection. The step-by-step operation of these two systems has been successfully employed for molecular diagnosis of pathogenic microbes, while the single-tube one-step method holds promise for efficient pathogen detection. This paper provides a comprehensive review of RAA combined with CRISPR-Cas and its applications in pathogen detection, aiming to serve as a valuable reference for further research in related fields.},
}
@article {pmid37670689,
year = {2023},
author = {Frion, J and Meller, A and Marbach, G and Lévesque, D and Roucou, X and Boisvert, FM},
title = {CRISPR/Cas9-mediated knockout of the ubiquitin variant UbKEKS reveals a role in regulating nucleolar structures and composition.},
journal = {Biology open},
volume = {12},
number = {9},
pages = {},
doi = {10.1242/bio.059984},
pmid = {37670689},
issn = {2046-6390},
support = {398925/CAPMC/CIHR/Canada ; },
mesh = {*Ubiquitin/genetics ; *CRISPR-Cas Systems ; Ubiquitins ; Ubiquitination ; Apoptosis ; },
abstract = {Ubiquitination is a post-translational modification responsible for one of the most complex multilayered communication and regulation systems in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Recently discovered ubiquitin variant UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. Depletion of UbKEKS induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. Using data-independent acquisition mass spectrometry and microscopy, we show that despite not impacting protein stability, UbKEKS is required to maintain a normal nucleolar organization. The absence of UbKEKS increases nucleoli's size and accentuate their circularity while disrupting dense fibrillar component and fibrillar centre structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Lack of UbKEKS favours nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis observed by flow cytometry and real-time monitoring. Overall, these results identify the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the ubiquitination system.},
}
@article {pmid37336378,
year = {2023},
author = {Ma, W and Yang, J and Ding, J and Duan, C and Zhao, W and Peng, YL and Bhadauria, V},
title = {CRISPR/Cas9-mediated deletion of large chromosomal segments identifies a minichromosome modulating the Colletotrichum graminicola virulence on maize.},
journal = {International journal of biological macromolecules},
volume = {245},
number = {},
pages = {125462},
doi = {10.1016/j.ijbiomac.2023.125462},
pmid = {37336378},
issn = {1879-0003},
mesh = {Virulence/genetics ; *Zea mays/genetics/microbiology ; *CRISPR-Cas Systems/genetics ; DNA ; },
abstract = {Colletotrichum graminicola causes anthracnose on maize, an economically significant disease worldwide. To decipher how the pathogen controls its virulence/pathogenicity on maize at the minichromosomal level, we sequenced the genome and transcriptome of the C. graminicola strain T1-3-3. The 61.91 Mb genome contains three transcriptionally repressed, full-length strain-specific minichromosomes (<1 Mb; Chr11 through Chr13). A CRISPR/Cas9-based system was developed to knock out large chromosomal segments; it involved the generation of multiple simultaneous DNA double-strand breaks across a targeted genomic region, followed by homology-directed replacement thereof with a donor DNA template carrying the selectable marker hygromycin phosphotransferase gene flanked by homologous sequence arms of the targeted region. Using this system, we obtained distinct mutants functionally nullisomic for individual minichromosomes. Only the ΔChr12 mutant lacking the 498.44 Kb genomic region carrying all of the 31 genes of Chr12 exhibited attenuated virulence on maize and was indistinguishable from T1-3-3 in fungal growth and conidiation, indicating that Chr12 is a conditionally dispensable minichromosome and imparts full virulence to C. graminicola on maize. The CRISPR/Cas9-mediated genome editing system developed in this study will enable the determination of the biological functions of minichromosomes or large chromosomal segments in fungal plant pathogens.},
}
@article {pmid37719218,
year = {2023},
author = {Muthusamy, M and Son, S and Park, SR and Lee, SI},
title = {Heat shock factor binding protein BrHSBP1 regulates seed and pod development in Brassica rapa.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1232736},
pmid = {37719218},
issn = {1664-462X},
abstract = {Plant heat shock factor binding proteins (HSBPs) are well known for their implication in the negative regulation of heat stress response (HSR) pathways. Herein, we report on the hitherto unknown functions of HSBP1 in Brassica rapa (BrHSBP1). BrHBSP1 was found to be predominant in flower buds and young leaves, while its segmental duplicate, BrHSBP1-like, was abundant in green siliques. Exposure to abiotic stress conditions, such as heat, drought, cold, and H2O2, and to phytohormones was found to differentially regulate BrHSBP1. The activity of BrHSBP1-GFP fusion proteins revealed their cellular localization in nuclei and cytosols. Transgenic overexpression of BrHSBP1 (BrHSBP1[OX]) improved pod and seed sizes, while CRISPR-Cas BrHSBP1 knock-out mutants (Brhsbp1_KO) were associated with aborted seed and pod development. The transcriptomic signatures of BrHSBP1[OX] and Brhsbp1_KO lines revealed that 360 and 2381 genes, respectively, were differentially expressed (Log2FC≥2, padj<0.05) expressed relative to control lines. In particular, developmental processes, including plant reproductive structure development (RSD)-related genes, were relatively downregulated in Brhsbp1_KO. Furthermore, yeast two-hybrid assays confirmed that BrHSBP1 can physically bind to RSD and other genes. Taking the findings together, it is clear that BrHSBP1 is involved in seed development via the modulation of RSD genes. Our findings represent the addition of a new regulatory player in seed and pod development in B. rapa.},
}
@article {pmid37718766,
year = {2023},
author = {Yousefi-Najafabadi, Z and Mehmandoostli, Z and Asgari, Y and Kaboli, S and Falak, R and Kardar, GA},
title = {Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy.},
journal = {Cell journal},
volume = {25},
number = {9},
pages = {633-644},
doi = {10.22074/cellj.2023.1999548.1269},
pmid = {37718766},
issn = {2228-5806},
abstract = {OBJECTIVE: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME).<br><br>MATERIALS AND METHODS: In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes.<br><br>RESULTS: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control.<br><br>CONCLUSION: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.},
}
@article {pmid37717422,
year = {2023},
author = {Dong, J and Wu, X and Hu, Q and Sun, C and Li, J and Song, P and Su, Y and Zhou, L},
title = {An immobilization-free electrochemical biosensor based on CRISPR/Cas13a and FAM-RNA-MB for simultaneous detection of multiple pathogens.},
journal = {Biosensors &amp; bioelectronics},
volume = {241},
number = {},
pages = {115673},
doi = {10.1016/j.bios.2023.115673},
pmid = {37717422},
issn = {1873-4235},
abstract = {To better respond to biosecurity issues, we need to build good technology and material reserves for pathogenic microorganism screening. Here, we designed an electrochemical/optical signal probe with a common fluorophore and an electrochemically active group, breaking the previous perception that the signal probe is composed of a fluorophore and a quenching group and realizing the response of three signals: electrochemistry, fluorescence, and direct observation. Then, we proposed a homogeneous electrochemical nucleic acid detection system based on CRISPR/Cas named "HELEN-CR" by integrating free electrochemical/optical signal probes and Cas13a cleavage, achieving a limit of detection of 1 pM within 25 min. To improve the detection sensitivity, we applied recombinase polymerase amplification to amplify the target nucleic acid, achieving a limit of detection of 30 zM within 45 min. Complemented by our self-developed multi-chamber microfluidic chip and portable electrochemical instrument, simultaneous detection of multiple pathogens can be achieved within 50 min, facilitating minimally trained personnel to obtain detection results quickly in a difficult environment. This study proposes a simple, scalable, and general idea and solution for the rapid detection of pathogenic microorganisms and biosecurity monitoring.},
}
@article {pmid37716509,
year = {2023},
author = {Saha, K and Subramenium Ganapathy, A and Wang, A and Arumugam, P and Michael Morris, N and Harris, L and Yochum, G and Koltun, W and Perdew, GH and Nighot, M and Ma, T and Nighot, P},
title = {Alpha-tocopherylquinone-mediated activation of the Aryl Hydrocarbon Receptor regulates the production of inflammation-inducing cytokines and ameliorates intestinal inflammation.},
journal = {Mucosal immunology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.mucimm.2023.09.003},
pmid = {37716509},
issn = {1935-3456},
abstract = {This study investigated the role of Alpha-tocopherylquinone (TQ) in regulating the intestinal immune system and the underlying mechanisms. In the experimental dextran sodium sulfate and T cell-mediated colitis models, TQ significantly reduced the mRNA levels of IL-6, IL-1β, IL17A, IL-23, and TNF-α, and the abundance of pro-inflammatory macrophages, Th17 cells and ILC3s in the colons of WT mice. TQ also prevented LPS-induced activation of NFκB and Stat-3 pathway in the human macrophage U937 cells. Pharmacological inhibition or CRISPR-Cas-9-mediated knockout of Aryl hydrocarbon Receptor (AHR) prevented the anti-inflammatory effects of TQ in the LPS-treated U937 cells. Furthermore, TQ reduced the mRNA levels of the LPS-induced proinflammatory cytokines in the WT but not Ahr[-/-] mice splenocytes. TQ also reduced IL-6R protein levels and IL-6-induced Stat-3 activation in Jurkat cells and in-vitro differentiation of Th17 cells from WT, but not Ahr[-/-] mice naïve T cells. Additionally, TQ prevented the pro-inflammatory effects of LPS on macrophages and stimulation of T cells in human PBMCs and significantly reduced the abundance of TNF-α, IL-1β, and IL-6[hi] inflammatory macrophages and Th17 cells in surgically resected Crohn's disease (CD) tissue. Our study shows that TQ is a naturally occurring, non-toxic, and effective immune modulator that activates AhR and suppresses the Stat-3-NFkB signaling.},
}
@article {pmid37693568,
year = {2023},
author = {Nemudraia, A and Nemudryi, A and Wiedenheft, B},
title = {Repair of CRISPR-guided RNA breaks enables site-specific RNA editing in human cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37693568},
abstract = {UNLABELLED: Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. Here, we show that site-specific RNA breaks generated with RNA-targeting CRISPR complexes are repaired in human cells, and this repair can be used for programmable deletions in human transcripts that restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.<br><br>ONE-SENTENCE SUMMARY: CRISPR-guided RNA breaks are repaired in human cells, and this RNA repair can be used for programmable editing of human transcriptomes.},
}
@article {pmid37672770,
year = {2023},
author = {Naveed, M and Aqib Shabbir, M and Aziz, T and Hurraira, HM and Fatima Zaidi, S and Athar, R and Chattha, HA and Alharbi, M and Alshammari, A and Alasmari, AF},
title = {CRISPR-Cas9 guided rna based model for the treatment of Amyotrophic Lateral Sclerosis: A progressive neurodegenerative disorder.},
journal = {Acta biochimica Polonica},
volume = {70},
number = {3},
pages = {643-653},
doi = {10.18388/abp.2020_6789},
pmid = {37672770},
issn = {1734-154X},
mesh = {Humans ; *Amyotrophic Lateral Sclerosis/genetics/therapy ; CRISPR-Cas Systems/genetics ; Gene Editing ; Muscles ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that leads to the degeneration of motor neurons and the weakening of muscles. Despite extensive research efforts, there is currently no cure for ALS and existing treatments only address its symptoms. To address this unmet medical need, genome editing technologies, such as CRISPR-Cas9, have emerged as a promising solution for the development of new treatments for ALS. Studies have shown that CRISPR-Cas9-guided RNAs have the potential to provide accurate and effective silencing in the genetic disease of ALS. Results have demonstrated a 67% on-target score and a 98% off-target score with GC content within the range of 40-60%. This is further validated by the correlation between the gRNA's structural accuracy and the minimum free energy. The use of CRISPR-Cas9 provides a unique opportunity to target this disease at the molecular level, offering hope for the development of a more effective treatment. In silico and computational therapeutic approaches for ALS suggest that the CRISPR-Cas9 protein holds promise as a future treatment candidate. The CRISPR mechanism and the specificity of gRNA provide a novel therapeutic approach for this genetic disease, offering new hope to those affected by ALS. This study highlights the potential of CRISPR-Cas9 as a promising solution for the development of new treatments for ALS. Further research is required to validate these findings in preclinical and clinical trials and to establish the safety and efficacy of this approach in the treatment of ALS.},
}
@article {pmid37658843,
year = {2023},
author = {Duarte, F and Vachey, G and Caron, NS and Sipion, M and Rey, M and Perrier, AL and Hayden, MR and Déglon, N},
title = {Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders.},
journal = {Human gene therapy},
volume = {34},
number = {17-18},
pages = {958-974},
doi = {10.1089/hum.2023.109},
pmid = {37658843},
issn = {1557-7422},
mesh = {Humans ; Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems ; HEK293 Cells ; Exons/genetics ; Alleles ; *Central Nervous System Diseases ; *Huntington Disease/genetics/therapy ; Central Nervous System ; },
abstract = {Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.},
}
@article {pmid37254814,
year = {2023},
author = {Eroglu, M and Yu, B and Derry, WB},
title = {Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans.},
journal = {The FEBS journal},
volume = {290},
number = {18},
pages = {4429-4439},
doi = {10.1111/febs.16876},
pmid = {37254814},
issn = {1742-4658},
support = {PJT 165837//CIHR/Canada ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Oligonucleotides/genetics ; DNA ; DNA, Single-Stranded/genetics ; },
abstract = {Highly efficient generation of deletions, substitutions, and small insertions (up to ~ 150 bp) into the Caenorhabditis elegans genome by CRISPR/Cas9 has been facilitated by the use of single-stranded oligonucleotide donors as repair templates. However, insertion of larger sequences such as fluorescent markers and other functional domains remains challenging due to uncertainty of optimal performance between single-stranded or double-stranded repair templates and labor-intensive as well as inefficient protocols for their preparations. Here, we simplify the generation of long ssDNA as donors in CRISPR/Cas9. High yields of ssDNA can be rapidly generated using a standard PCR followed by a single enzymatic digest with lambda exonuclease. Comparison of long ssDNA donors obtained using this method to dsDNA demonstrates orders of magnitude increased insertion frequency for ssDNA donors. This can be leveraged to simultaneously generate multiple large insertions as well as successful edits without the use of selection or co-conversion (co-CRISPR) markers when necessary. Our approach complements the CRISPR/Cas9 toolkit for C. elegans to enable highly efficient insertion of longer sequences with a simple, standardized, and labor-minimal protocol.},
}
@article {pmid37226388,
year = {2023},
author = {Chu, J and Romero, A and Taulbee, J and Aran, K},
title = {Development of Single Molecule Techniques for Sensing and Manipulation of CRISPR and Polymerase Enzymes.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {19},
number = {38},
pages = {e2300328},
doi = {10.1002/smll.202300328},
pmid = {37226388},
issn = {1613-6829},
support = {1R01HL161361-01/NH/NIH HHS/United States ; 1R01HL161361-01/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Biotechnology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and polymerases are powerful enzymes and their diverse applications in genomics, proteomics, and transcriptomics have revolutionized the biotechnology industry today. CRISPR has been widely adopted for genomic editing applications and Polymerases can efficiently amplify genomic transcripts via polymerase chain reaction (PCR). Further investigations into these enzymes can reveal specific details about their mechanisms that greatly expand their use. Single-molecule techniques are an effective way to probe enzymatic mechanisms because they may resolve intermediary conformations and states with greater detail than ensemble or bulk biosensing techniques. This review discusses various techniques for sensing and manipulation of single biomolecules that can help facilitate and expedite these discoveries. Each platform is categorized as optical, mechanical, or electronic. The methods, operating principles, outputs, and utility of each technique are briefly introduced, followed by a discussion of their applications to monitor and control CRISPR and Polymerases at the single molecule level, and closing with a brief overview of their limitations and future prospects.},
}
@article {pmid37715442,
year = {2023},
author = {Vasques Raposo, J and Rodrigues Carvalho Barros, L and Ribas Torres, L and Barbosa da Silva Pinto, R and De Oliveira Lopes, A and Mello de Souza, E and Hernan Bonamino, M and Salete de Paula, V},
title = {CRISPR/Cas-9 vector system: targets UL-39 and inhibits Simplexvirus humanalpha1 (HSV-1) replication in vitro.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {69},
number = {7},
pages = {19-23},
doi = {10.14715/cmb/2023.69.7.3},
pmid = {37715442},
issn = {1165-158X},
mesh = {Chlorocebus aethiops ; Animals ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Vero Cells ; *Herpesvirus 1, Human/genetics ; Biological Assay ; },
abstract = {Simplexvirus humanalpha1 (HSV-1) affects approximately 67% of the world's population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into the plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyses revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas 9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.},
}
@article {pmid37715317,
year = {2023},
author = {Chan, WT and Garcillán-Barcia, MP and Yeo, CC and Espinosa, M},
title = {Type II bacterial toxin-antitoxins: hypotheses, facts, and the newfound plethora of the PezAT system.},
journal = {FEMS microbiology reviews},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsre/fuad052},
pmid = {37715317},
issn = {1574-6976},
abstract = {Toxin-antitoxin (TA) systems are entities found in the prokaryotic genomes, with eight reported types. Type II, the best characterised, is comprised of two genes organised as an operon. Whereas toxins impair growth, the cognate antitoxin neutralises its activity. TAs appeared to be involved in plasmid maintenance, persistence, virulence, and defence against bacteriophages. Most Type II toxins target the bacterial translational machinery. They seem to be antecessors of Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) RNases, minimal nucleotidyltransferase domains, or CRISPR-Cas systems. Four TAs encoded by Streptococcus pneumoniae, RelBE, YefMYoeB, Phd-Doc, and HicAB, belong to HEPN-RNases. The fifth is represented by PezAT/Epsilon-Zeta. PezT/Zeta toxins phosphorylate the peptidoglycan precursors, thereby blocking cell wall synthesis. We explore the body of knowledge (facts) and hypotheses procured for Type II TAs and analyse the data accumulated on the PezAT family. Bioinformatics analyses showed that homologues of PezT/Zeta toxin are abundantly distributed among 14 bacterial phyla mostly in Proteobacteria (48%), Firmicutes (27%), and Actinobacteria (18%), showing the widespread distribution of this TA. The pezAT locus was found to be mainly chromosomally encoded whereas its homologue, the tripartite omega-epsilon-zeta locus, was found mostly on plasmids. We found several orphan pezT/zeta toxins, unaccompanied by a cognate antitoxin.},
}
@article {pmid37715312,
year = {2023},
author = {Xi, Y and Zhao, J and Zhang, J and Jin, Y and Yang, H and Duan, G and Chen, S and Long, J},
title = {Analysis of the features of 105 confirmed CRISPR loci in 487 Klebsiella variicola.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/lambio/ovad108},
pmid = {37715312},
issn = {1472-765X},
abstract = {Klebsiella variicola (K. variicola), an emerging human pathogen, poses a threat to public health. The horizontal gene transfer (HGT) of plasmids is an important driver of the emergence of multiple antibiotic-resistant K. variicola. Clustered regularly interspersed short palindromic repeats (CRISPR) coupled with CRISPR-associated genes (CRISPR/Cas) constitute an adaptive immune system in bacteria, and can provide acquired immunity against HGT. However, the information about the CRISPR/Cas system in K. variicola is still limited. In this study, 487 genomes of K. variicola obtained from the National Center for Biotechnology Information database were used to analyze the characteristics of CRISPR/Cas systems. Approximately 21.56% of genomes (105/487) harbor at least one confirmed CRISPR array. Three types of CRISPR/Cas systems, namely, the types I-E, I-E*, and IV-A systems, were identified among 105 strains. Spacer origin analysis further revealed that approximately one-third of spacers significantly match plasmids or phages, which demonstrates the implication of CRISPR/Cas systems in controlling HGT. Moreover, spacers in K. variicola tend to target mobile genetic elements from K. pneumoniae. This finding provides new evidence of the interaction of K. variicola and K. pneumoniae during their evolution. Collectively, our results provide valuable insights into the role of CRISPR/Cas systems in K. variicola.},
}
@article {pmid37714365,
year = {2023},
author = {Fadaie, M and Dianat-Moghadam, H and Ghafouri, E and Naderi, S and Darvishali, MH and Ghovvati, M and Khanahmad, H and Boshtam, M and Makvandi, P},
title = {Unraveling the potential of M13 phages in biomedicine: Advancing drug nanodelivery and gene therapy.},
journal = {Environmental research},
volume = {},
number = {},
pages = {117132},
doi = {10.1016/j.envres.2023.117132},
pmid = {37714365},
issn = {1096-0953},
abstract = {M13 phages possessing filamentous phage genomes offer the benefits of selective display of molecular moieties and delivery of therapeutic agent payloads with a tolerable safety profile. M13 phage-displayed technology for resembling antigen portions led to the discovery of mimetic epitopes that applied to antibody-based therapy and could be useful in the design of anticancer vaccines. To date, the excremental experiences have engaged the M13 phage in the development of innovative biosensors for detecting biospecies, biomolecules, and human cells with an acceptable limit of detection. Addressing the emergence of antibiotic-resistant bacteria, M13 phages are potent for packaging the programmed gene editing tools, such as CRISPR/Cas, to target multiple antimicrobial genes. Moreover, their display potential in combination with nanoparticles inspires new approaches for engineering targeted theragnostic platforms targeting multiple cellular biomarkers in vivo. In this review, we present the available data on optimizing the use of bacteriophages with a focus on the to date experiences with M13 phages, either as monoagent or as part of combination regimens in the practices of biosensors, vaccines, bactericidal, modeling of specific antigen epitopes, and phage-guided nanoparticles for drug delivery systems. Despite increasing research interest, a deep understanding of the underlying biological and genetic behaviors of M13 phages is needed to enable the full potential of these bioagents in biomedicine, as discussed here. We also discuss some of the challenges that have thus far limited the development and practical marketing of M13 phages.},
}
@article {pmid37713428,
year = {2023},
author = {Watson, BNJ and Pursey, E and Gandon, S and Westra, ER},
title = {Transient eco-evolutionary dynamics early in a phage epidemic have strong and lasting impact on the long-term evolution of bacterial defences.},
journal = {PLoS biology},
volume = {21},
number = {9},
pages = {e3002122},
doi = {10.1371/journal.pbio.3002122},
pmid = {37713428},
issn = {1545-7885},
abstract = {Organisms have evolved a range of constitutive (always active) and inducible (elicited by parasites) defence mechanisms, but we have limited understanding of what drives the evolution of these orthogonal defence strategies. Bacteria and their phages offer a tractable system to study this: Bacteria can acquire constitutive resistance by mutation of the phage receptor (surface mutation, sm) or induced resistance through their CRISPR-Cas-adaptive immune system. Using a combination of theory and experiments, we demonstrate that the mechanism that establishes first has a strong advantage because it weakens selection for the alternative resistance mechanism. As a consequence, ecological factors that alter the relative frequencies at which the different resistances are acquired have a strong and lasting impact: High growth conditions promote the evolution of sm resistance by increasing the influx of receptor mutation events during the early stages of the epidemic, whereas a high infection risk during this stage of the epidemic promotes the evolution of CRISPR immunity, since it fuels the (infection-dependent) acquisition of CRISPR immunity. This work highlights the strong and lasting impact of the transient evolutionary dynamics during the early stages of an epidemic on the long-term evolution of constitutive and induced defences, which may be leveraged to manipulate phage resistance evolution in clinical and applied settings.},
}
@article {pmid37676272,
year = {2023},
author = {Luo, P and Huang, X and Luo, F and Chen, Z and Chen, Y and Lin, C and Wang, J and Qiu, B and Lin, Z},
title = {Low-Background Signal-On Homogeneous Electrochemiluminescence Biosensor for Hepatitis B Virus Detection Based on the Regulation of the Length of DNA Modified on the Nanoparticles by CRISPR/Cas12a and Hybridization Chain Reaction.},
journal = {Analytical chemistry},
volume = {95},
number = {37},
pages = {14127-14134},
doi = {10.1021/acs.analchem.3c03141},
pmid = {37676272},
issn = {1520-6882},
mesh = {Hepatitis B virus/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; *Ruthenium ; Silicon Dioxide ; DNA ; *Nanoparticles ; },
abstract = {In this work, combined with the high amplification efficiency of hybridization chain reaction (HCR), high specificity of the CRISPR/Cas12a system, and convenience of the homogeneous electrochemiluminescence (ECL) assay based on the regulation of negative charge on the reporting probes, a sensitive ECL biosensor for hepatitis B virus DNA (chosen as a model target) had been developed. The initiator chain trigger DNA that can induce HCR amplification is modified on the surface of ruthenium bipyridine-doped silica nanoparticles (Ru@SiO2 NPs) first, and large amounts of negative charges modified on the particles were achieved through the HCR amplification reaction. The efficiency of the nanoparticles reaching the negatively charged working electrode can be regulated and realize the change of the ECL signal. In addition, long DNA on the surface of the luminescent body may prevent the coreactant from entering the pore to react with ruthenium bipyridine. These factors combine to produce a low-background system. The presence of the target can activate the CRISPR/Cas12a system and make trigger DNA disappear from the nanoparticle surface, and strong ECL can be detected. The sensor does not require a complex electrode modification; therefore, it has better reproducibility. Additionally, due to dual signal amplification, the sensor has a high sensitivity. In the range of 10 fM to 10 nM, the ECL intensity exhibits a strong linear relationship with the logarithm of the target concentration, and the detection limit is 7.41 fM. This sensor has shown high accuracy in detecting clinical samples, which holds significant potential for application in clinical testing.},
}
@article {pmid37668549,
year = {2023},
author = {Jia, Z and Zhang, Y and Zhang, C and Wei, X and Zhang, M},
title = {Biosensing Intestinal Alkaline Phosphatase by Pregnancy Test Strips Based on Target-Triggered CRISPR-Cas12a Activity to Monitor Intestinal Inflammation.},
journal = {Analytical chemistry},
volume = {95},
number = {37},
pages = {14111-14118},
doi = {10.1021/acs.analchem.3c03099},
pmid = {37668549},
issn = {1520-6882},
mesh = {Female ; Pregnancy ; Animals ; Mice ; Alkaline Phosphatase ; CRISPR-Cas Systems ; *Inflammatory Bowel Diseases/diagnosis ; *Colitis ; DNA, Single-Stranded ; *Pregnancy Tests ; Inflammation/diagnosis ; },
abstract = {With an increasing incidence worldwide, inflammatory bowel disease (IBD) is a chronic inflammatory disease affecting the gastrointestinal tract, which impairs the life quality of patients. Therefore, it is of great significance to construct a sensitive, simple, and convenient biosensor to analyze IBD-associated biomarkers for an auxiliary diagnosis of IBD. Intestinal alkaline phosphatase (IAP), expressed by the intestinal epithelium, is an endogenous protein that is thought to play a vital role in maintaining intestinal homeostasis and is considered a potential biomarker for IBD. Here, an IAP detection method was developed using pregnancy test strips by dephosphorylation. Initially, a double-stranded DNA (dsDNA) was designed to respond to IAP and acted as an activator of Cas12a. In the presence of IAP, the designed dsDNA was not digested by lambda exonuclease (λ exo), which hybridized to the Cas12a-crRNA duplex and resulted in the activation of the trans-cleavage of Cas12a. Further, the activated Cas12a cleaved the single-strand DNA (ssDNA) linker in the MBs-ssDNA-hCG probe, triggering the release of hCG. With magnetic separation, the released hCG could be quantitatively detected by pregnancy test strips. IAP levels were analyzed in feces from colitis and healthy mice by pregnancy test strips. The results showed that the IAP level of colitis mice (3.89 ± 1.92 U/L) was much lower than that of healthy mice (39.64 ± 24.93 U/L), indicating the correlation between IAP and intestinal inflammation. Taken together, a sensitive, user-friendly detection assay based on pregnancy test strips was constructed to monitor IAP and used as an auxiliary diagnostic approach for IBD in a clinical scene.},
}
@article {pmid37712284,
year = {2023},
author = {Huang, D and Zhao, Y and Fang, M and Shen, P and Xu, H and He, Y and Chen, S and Si, Z and Xu, Z},
title = {Magnetofluid-integrated biosensors based on DNase-dead Cas12a for visual point-of-care testing of HIV-1 by an up and down chip.},
journal = {Lab on a chip},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3lc00558e},
pmid = {37712284},
issn = {1473-0189},
abstract = {The CRISPR Cas system, as a novel nucleic acid detection tool, is often hindered by cumbersome experimental procedures, complicated reagent transfer processes, and associated aerosol pollution risks. In this study, an integrated nucleic acid detection platform named "up and down chip" was developed, which combined RT-RAA technology for nucleic acid amplification, DNase-dead Cas12a-modified magnetic beads for specific recognition of target nucleic acid, and HRP-TMB chromogenic reaction for signal output in different chambers of a single microfluidic chip. The magnetic beads were migrated in an up-and-down manner between different chambers through magnetic driving, achieving a "sample-in, result-out" detection mode. By introducing a homemade heating box for temperature control during the reaction and using the naked eye or a smartphone APP for color-based signal reading, no professional or precise instruments were required in this platform. Using this platform, highly sensitive detection of the HIV-1 genome as low as 250 copies (CPs) per mL was achieved within 100 min while maintaining good detection performance against common variants as well as excellent specificity and anti-interference ability. In addition, compared with qRT-PCR, it also exhibited good accuracy for 56 spiked plasma samples, indicating its promising potential for clinical application.},
}
@article {pmid37711452,
year = {2023},
author = {Xiao, J and Li, J and Quan, S and Wang, Y and Jiang, G and Wang, Y and Huang, H and Jiao, W and Shen, A},
title = {Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1233353},
pmid = {37711452},
issn = {2296-4185},
abstract = {Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies μL[-1], and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.},
}
@article {pmid37711183,
year = {2023},
author = {Deng, B and Xue, J},
title = {HIV infection detection using CRISPR/Cas systems: Present and future prospects.},
journal = {Computational and structural biotechnology journal},
volume = {21},
number = {},
pages = {4409-4423},
pmid = {37711183},
issn = {2001-0370},
abstract = {Human immunodeficiency virus (HIV) infection poses substantial medical risks to global public health. An essential strategy to combat the HIV epidemic is timely and effective virus testing. CRISPR-based assays combine the highly compatible CRISPR system with different elements, yielding portability, digitization capabilities, low economic burden and low operational thresholds. The application of CRISPR-based assays has demonstrated rapid, accurate, and accessible means of pathogen testing, suggesting great potential as point-of-care (POC) assays. This review outlines the different types of CRISPR/Cas systems based on Cas proteins and their applications for the detection of HIV. Additionally, we also offer an overview of future perspectives on CRISPR-based methods for HIV detection, including advances in nucleic acid amplification-free testing, improved personal testing, and refined testing for HIV genotypes and drug-resistant strains.},
}
@article {pmid37710076,
year = {2023},
author = {He, Q and Lei, X and Liu, Y and Wang, X and Ji, N and Yin, H and Wang, H and Zhang, H and Yu, G},
title = {Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202300401},
doi = {10.1002/cbic.202300401},
pmid = {37710076},
issn = {1439-7633},
abstract = {RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in the nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs with concentrations down to 25 pM, which could be further improved by approximately 15,000-fold (~ 2 fM) through incorporating the recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems of various species, potentially providing an easily adaptable platform for clinical diagnosis.},
}
@article {pmid37709482,
year = {2023},
author = {Liu, Y and Liu, H and Yu, G and Sun, W and Aizaz, M and Yang, G and Chen, L},
title = {One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus.},
journal = {Analytica chimica acta},
volume = {1278},
number = {},
pages = {341757},
doi = {10.1016/j.aca.2023.341757},
pmid = {37709482},
issn = {1873-4324},
mesh = {Humans ; *Methicillin-Resistant Staphylococcus aureus/genetics ; CRISPR-Cas Systems ; Environmental Pollution ; Escherichia coli ; Point-of-Care Testing ; },
abstract = {At present, methicillin-resistant Staphylococcus aureus (MRSA) has caused a serious impact on a global scale. The infection and carrier rate of MRSA in the community is increasing year by year, but there is still no convenient detection system for on-site rapid detection. It is very important to select a rapid detection system to accurately and quickly detect patients infected with MRSA. We have developed a high-efficient single-tube detection platform based on RPA and CRISPR reaction system to detect the genes of mecA and clfA of MRSA. Using this detection platform, visual MRSA detection could be achieved in 30 min. It was observed that this detection platform was capable to successfully detect the target genomic as low as 5 copies μL[-1], and the reaction was completed in one step without opening the lid. This detection platform could only detect MRSA, but not other common clinical pathogenic bacteria, such as Salmonella, Pseudomonas aeruginosa, Staphylococcus xylosus, Aeromonas hydrophila, Escherichia coli and Staphylococcus warneri, indicated its satisfactory selectivity for MRSA without interference from other bacteria. The results of clinical samples show that the platform has outstanding advantages in sensitivity, specificity and identification of methicillin resistance. The entire reaction can be completed in one step in the handheld instrument without opening the cover, avoiding aerosol pollution during the reaction. The detection platform combined with handheld instruments will have great application potential in point-of-care testing.},
}
@article {pmid37709481,
year = {2023},
author = {Shi, K and Chen, J and Li, Y and Li, Q and Song, J and Yi, Z and Li, D and Zhang, J},
title = {Hg[2+]-triggered cascade strand displacement assisted CRISPR-Cas12a for Hg[2+] quantitative detection using a portable glucose meter.},
journal = {Analytica chimica acta},
volume = {1278},
number = {},
pages = {341756},
doi = {10.1016/j.aca.2023.341756},
pmid = {37709481},
issn = {1873-4324},
mesh = {*Glucose ; CRISPR-Cas Systems ; Catalysis ; *Mercury ; Sucrase ; },
abstract = {CRISPR-Cas12a is a powerful and programmable tool that has revolutionized the field of biosensing. However, the construction of a CRISPR-Cas12a-mediated portable system for on-site and quantitative detection of mercury ion (Hg[2+]) has yet to be explored. By integrating a target-triggered cascade toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a, we herein construct a portable on-site biosensor for the quantitative, sensitive, and selective detection of Hg[2+] with a glucose meter. The Hg[2+] initiates two cascade TSDRs through the T-Hg[2+]-T interaction to produce multiple double-stranded DNAs that can activate Cas12a's trans-cleavage activity. The Cas12a cleaves the sucrase-modified DNA on the electrode, resulting in the liberation of sucrase into the solution. The freed sucrase can catalyze sucrose to generate glucose, which can be quantitatively monitored by a glucometer. The developed portable biosensor provides a dynamic range of 5 orders of magnitude with a detection limit of 40 fM. This biosensor also displays excellent selectivity and stability for detecting Hg[2+]. Moreover, environmental water samples are utilized to further verify the robustness and effectiveness of the developed biosensor, highlighting its potential application in environmental monitoring and food safety analysis.},
}
@article {pmid37709435,
year = {2023},
author = {Ren, D and Chen, Q and Xia, X and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {CRISPR/Cas12a-based fluorescence aptasensor integrated with two-dimensional cobalt oxyhydroxide nanosheets for IFN-γ detection.},
journal = {Analytica chimica acta},
volume = {1278},
number = {},
pages = {341750},
doi = {10.1016/j.aca.2023.341750},
pmid = {37709435},
issn = {1873-4324},
mesh = {*Interferon-gamma ; *CRISPR-Cas Systems ; DNA, Complementary ; Cytokines ; Oligonucleotides ; },
abstract = {Cytokine storm (CS) is a risky immune overreaction accompanied by significant elevations of pro-inflammatory cytokines including interferon-γ (IFN-γ), interleukin and tumor necrosis factor. Sensitive detection of cytokine is conducive to studying CS progress and diagnosing infectious diseases. In this study, we developed a tandem system combining aptamer, strand displacement amplification (SDA), CRISPR/Cas12a, and cobalt oxyhydroxide nanosheets (termed Apt-SCN tandem system) as a signal-amplified platform for IFN-γ detection. Owing to the stronger affinity, target IFN-γ bound specifically to the aptamer from aptamer-complementary DNA (Apt-cDNA) duplex. The cDNA released from the Apt-cDNA duplex initiated SDA, resulting in the generation of double-stranded DNA products that could activate the trans-cleavage activity of CRISPR/Cas12a. The activated CRISPR/Cas12a further cleaved FAM-labeled single-stranded DNA probe, preventing it from adhering to the cobalt oxyhydroxide nanosheets and recovering the fluorescence signal. Sensitive fluorometric analysis of IFN-γ was successfully performed with detection limit as low as 0.37 nM. Unlike traditional protein analysis methods, Apt-SCN tandem system incorporates multiple signal amplification techniques and may also be applicable for other cytokines assay. This study was the initial study to utilize SDA and CRISPR/Cas12a to detect IFN-γ, showing great potential for cytokines clinical assay and CS prevention.},
}
@article {pmid37708031,
year = {2023},
author = {Li, M and Li, D and Lin, L and Wang, P and Zhao, W},
title = {Precise Interference of RNA-Protein Interaction by CRISPR-Cas13-Mediated Peptide Competition.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00287},
pmid = {37708031},
issn = {2161-5063},
abstract = {RNA-protein interactions are essential nodes of cellular regulatory circuits and play critical roles in normal physiology and disease. However, the precise roles of individual RNA-protein interactions remain elusive. Here we report a method for precise interference of endogenous RNA interacting with the RNA binding protein (RBP). TTP is an RBP that recognizes the AU-rich element (ARE) of mRNA via the binding domain TZF and represses gene expression. We engineer Cas13b, a class 2 type VI CRISPR-Cas endonuclease that exclusively targets RNA, to direct the peptide of TZF to the binding site and compete with endogenous TTP. We show that this tool specifically interferes with TTP interacting with the PIM1 and IL-2 3' UTR under the guidance of the gRNA specific for the AREs. Further, precise interference with the TTP-PIM1 interaction exerts a distinct effect on cell proliferation compared to transcriptome-wide interference. Thus, our work establishes a tool for deep understanding of RNA-RBP interactions.},
}
@article {pmid37707449,
year = {2023},
author = {Farooq, A and Lee, M and Han, S and Jung, GY and Kim, SJ and Jung, MY},
title = {Kinetic, genomic, and physiological analysis reveals diversity in the ecological adaptation and metabolic potential of Brachybacterium equifaecis sp. nov. isolated from horse feces.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0504822},
doi = {10.1128/spectrum.05048-22},
pmid = {37707449},
issn = {2165-0497},
abstract = {Brachybacterium species have been identified in various ecological niches and belong to the family Dermabacteriaceae within the phylum Actinobacteria. In this study, we isolated a novel Brachybacterium equifaecis JHP9 strain from horse feces and compared its kinetic, biochemical, and genomic features with those of other Brachybacterium strains. Moreover, comparative genomic analysis using publicly available Brachybacterium genomes was performed to determine the properties involved in their ecological adaptation and metabolic potential. Novel species delineation was determined phylogenetically through 16S rRNA gene similarity (up to 97.9%), average nucleotide identity (79.5-82.5%), average amino acid identity (66.7-75.8%), and in silico DNA-DNA hybridization (23.7-27.9) using closely related strains. This study also presents the first report of the kinetic properties of Brachybacterium species. Most of the Brachybacterium strains displayed high oxygen (K m(app) =1.6-24.2 µM) and glucose (K m(app) =0.73-1.22 µM) affinities, which may manifest niche adaptations. Various carbohydrate metabolisms under aerobic and anaerobic conditions, antibiotic resistance, mobile genetic elements, carbohydrate-active enzymes, lactic acid production, and the clustered regularly interspaced short palindromic repeats-Cas and bacteriophage exclusion systems were observed in the genotypic and/or phenotypic properties of Brachybacterium species, suggesting their genome flexibility, defense mechanisms, and adaptability. Our study contributes to the knowledge of the kinetic, physiological, and genomic properties of Brachybacterium species, including the novel JHP9 strain, which advocates for their tolerant and thriving nature in various environments, leading to their ecological adaptation. IMPORTANCE Basic physiological and genomic properties of most of the Brachybacterium isolates have been studied; however, the ability of this bacterium to adapt to diverse environments, which may demonstrate its role in niche differentiation, is to be identified yet. Therefore, here, we explored cellular kinetics, metabolic diversity, and ecological adaptation/defensive properties of the novel Brachybacterium strain through physiological and comparative genomic analysis. In addition, we presented the first report examining Brachybacterium kinetics, indicating that all strains of Brachybacterium, including the novel one, have high oxygen and glucose affinity. Furthermore, the comparative genomic analysis also revealed that the novel bacterium contains versatile genomic properties, which provide the novel bacterium with significant competitive advantages. Thus, in-depth genotypic and phenotypic analysis with kinetic properties at the species level of this genus is beneficial in clarifying its differential characteristics, conferring the ability to inhabit diverse ecological niches.},
}
@article {pmid37643152,
year = {2023},
author = {Teng, Y and Wang, J and Jiang, T and Zou, Y and Yan, Y},
title = {Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria.},
journal = {ACS synthetic biology},
volume = {12},
number = {9},
pages = {2764-2772},
doi = {10.1021/acssynbio.3c00366},
pmid = {37643152},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics ; CRISPR-Associated Protein 9 ; Gene Expression Regulation, Bacterial ; Gene Editing ; },
abstract = {The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.},
}
@article {pmid37602730,
year = {2023},
author = {Gussak, A and Ferrando, ML and Schrama, M and van Baarlen, P and Wells, JM},
title = {Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology.},
journal = {ACS synthetic biology},
volume = {12},
number = {9},
pages = {2546-2560},
doi = {10.1021/acssynbio.3c00110},
pmid = {37602730},
issn = {2161-5063},
mesh = {Humans ; Animals ; Swine ; *Streptococcus suis/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Technology ; Anti-Bacterial Agents ; },
abstract = {Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.},
}
@article {pmid37584634,
year = {2023},
author = {Dykstra, CB and Pyne, ME and Martin, VJJ},
title = {CRAPS: Chromosomal-Repair-Assisted Pathway Shuffling in Yeast.},
journal = {ACS synthetic biology},
volume = {12},
number = {9},
pages = {2578-2587},
doi = {10.1021/acssynbio.3c00170},
pmid = {37584634},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Carotenoids/metabolism ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multistep biochemical pathway. Current pathway assembly workflows involve combining genetic parts ex vivo and assembling one pathway configuration per tube or well. Here, we present CRAPS, Chromosomal-Repair-Assisted Pathway Shuffling, an in vivo pathway engineering technique that enables the self-assembly of one pathway configuration per cell. CRAPS leverages the yeast chromosomal repair pathway and utilizes a pool of inactive, chromosomally integrated orthologous gene variants corresponding to a target multistep pathway. Supplying gRNAs to the CRAPS host activates the expression of one gene variant per pathway step, resulting in a unique pathway configuration in each cell. We deployed CRAPS to build more than 1000 theoretical combinations of a four-step carotenoid biosynthesis network. Sampling the CRAPS pathway space yielded strains with distinct color phenotypes and carotenoid product profiles. We anticipate that CRAPS will expedite strain engineering campaigns by enabling the generation and sampling of vast biochemical spaces.},
}
@article {pmid37399127,
year = {2023},
author = {Shi, L and Li, X and Xue, L and Zhang, J and Huang, B and Sun, Z and Zhang, Z and Dai, X and Han, S and Dong, W and Zhang, X},
title = {Creation of herbicide-resistance in allotetraploid peanut using CRISPR/Cas9-meditated cytosine base-editing.},
journal = {Plant biotechnology journal},
volume = {21},
number = {10},
pages = {1923-1925},
pmid = {37399127},
issn = {1467-7652},
support = {201300111000//the Key Project of Science and Technology of Henan Province/ ; 221100110300//the Key Project of Science and Technology of Henan Province/ ; CARS-13//China Agriculture Research System of MOF and MARA/ ; 2022YFD1200400//National Key Research and Development Program of China/ ; S2012-5//the Henan Province Agriculture Research System/ ; 222301420026//the Joint Funds of Natural Science Foundation of Henan Province/ ; 212102110256//the Programs for Science and Technology Development of Henan Province/ ; },
mesh = {Arachis/genetics ; CRISPR-Cas Systems/genetics ; Cytosine ; *Fabaceae ; *Herbicides ; Gene Editing ; },
}
@article {pmid37390886,
year = {2023},
author = {Cui, J and Wen, D and Wang, L and Meng, C and Wang, Y and Zhao, Z and Wu, C},
title = {CRISPR/Cas9-induced asap1a and asap1b co-knockout mutant zebrafish displayed abnormal embryonic development and impaired neutrophil migration.},
journal = {Gene expression patterns : GEP},
volume = {49},
number = {},
pages = {119331},
doi = {10.1016/j.gep.2023.119331},
pmid = {37390886},
issn = {1872-7298},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Neutrophils ; Protein Isoforms/genetics ; Embryonic Development ; },
abstract = {ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.},
}
@article {pmid35357269,
year = {2023},
author = {Deb, R and Chaudhary, P and De, S},
title = {CRISPR/cas9 cassette targeting Escherichia coli [bla]CTX-M specific gene of mastitis cow milk origin can alter the antibiotic resistant phenotype for cefotaxime.},
journal = {Animal biotechnology},
volume = {34},
number = {5},
pages = {1849-1854},
doi = {10.1080/10495398.2022.2053695},
pmid = {35357269},
issn = {1532-2378},
mesh = {Cattle ; Female ; Animals ; Anti-Bacterial Agents/pharmacology ; Cefotaxime/pharmacology ; Escherichia coli/genetics/metabolism ; *Escherichia coli Infections/veterinary/epidemiology/genetics ; Milk/metabolism ; CRISPR-Cas Systems/genetics ; Phenotype ; beta-Lactamases/genetics/metabolism ; beta-Lactams ; *Mastitis/genetics ; *Cattle Diseases/genetics ; },
abstract = {CTX-M beta-lactamases are one of the most important extended spectrum beta-lactamase (ESBL) resistance enzymes found in E. coli. In the present study, 59% of E. coli isolates from mastitis cow milk were reported to be positive for ESBL types. The prevalence of beta-lactam (β-lactam) antibiotic resistance was reported to be 84%, 72.7%, 52.27%, 50%, and 45.4% for cefotaxime, cefepime, cefuroxime, oxacillin, and cephalexine, respectively. The [bla]CTX-M gene was found in 65% (n = 17) of the E. coli isolates when they were genotyped. Further, the use of a CRISPR/cas9 cassette to target the E. coli [bla]CTX-M gene revealed changes in antibiotic phenotypes for cefotaxime.},
}
@article {pmid37704762,
year = {2023},
author = {Li, C and Fleck, JS and Martins-Costa, C and Burkard, TR and Themann, J and Stuempflen, M and Peer, AM and Vertesy, &#xc1; and Littleboy, JB and Esk, C and Elling, U and Kasprian, G and Corsini, NS and Treutlein, B and Knoblich, JA},
title = {Single-cell brain organoid screening identifies developmental defects in autism.},
journal = {Nature},
volume = {621},
number = {7978},
pages = {373-380},
pmid = {37704762},
issn = {1476-4687},
mesh = {Humans ; *Autism Spectrum Disorder/complications/genetics/pathology ; Autistic Disorder/complications/genetics/pathology ; *Brain/cytology/metabolism ; Cell Lineage/genetics ; Chromatin/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; *Developmental Disabilities/complications/genetics/pathology ; Gene Editing ; Loss of Function Mutation ; Mosaicism ; Neurons/metabolism/pathology ; *Organoids/cytology/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Single-Cell Gene Expression Analysis ; Transcription, Genetic ; },
abstract = {The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders[1-4]. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.},
}
@article {pmid37703922,
year = {2023},
author = {Nami, Y and Rostampour, M and Panahi, B},
title = {CRISPR-Cas systems and diversity of targeting phages in Lactobacillus johnsonii strains; insights from genome mining approach.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {114},
number = {},
pages = {105500},
doi = {10.1016/j.meegid.2023.105500},
pmid = {37703922},
issn = {1567-7257},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system. These genes protect bacteria from being eaten by bacteriophages. In this study, CRISPR-Cas systems were characterized using a genomic approach. For this purpose, genome sequences of Lactobacillus johnsonii strains were retrieved, and the diversity, occurrence, and evolution of the CRISPR-Cas systems were analyzed. Then, homology analyses of spacer sequences in identified CRISPR arrays were performed to analyze and characterize the diversity of target phages and plasmids. Finally, the evolutionary paths of spaceromes in each subtype of CRISPR arrays were performed using acquisition and deletion events surveyed under the selective pressure of foreign plasmids and phages. Results showed that 138 strains contain valid CRISPR-Cas structures (CRISPR loci together with the Cas genes) in their genomes, which accounted for about 17% of the L. johnsonii studied strains belonging to subtypes II-A, I-E, and I-C. Moreover, results indicated that some specific groups of plasmids were targeted with specific CRISPR array systems. Homology analysis of spacer sequences with phage genomes also revealed that spacers of strains that harbored only CRISPR-Cas subtype-II targeted a greater diversity of foreign phages. In conclusion, the current study indicates that there is great diversity between the CRISPR-Cas systems identified in L. johnsonii strains. Such diverse CRISPR-Cas systems indicate that these systems are naturally active and important in terms of adaptive immunity and evolutionary relationships.},
}
@article {pmid37699682,
year = {2022},
author = {Yang, C and Dong, X and Zhang, X and Bi, C},
title = {[Application of genome editing technology in industrial microorganisms: current status and perspectives].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {11},
pages = {4132-4145},
doi = {10.13345/j.cjb.220566},
pmid = {37699682},
issn = {1872-2075},
mesh = {*Gene Editing ; *Biotechnology ; Endonucleases ; Escherichia coli/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {Precise and efficient manipulation of gene expression or rewriting genome sequence is the research hotspots of genome editing, and it is also the core enabling technology contributing to the rapid development of industrial biotechnology. Genome editing technology has experienced three stages of development, from zinc finger nuclease (ZFNs), to transcription activator like effector nuclease (TALEN) and Cas nuclease. Currently, vigorous development of CRISPR/Cas has enabled researchers establish a series of first-generation and second-generation Cas-based genome editing technologies. This contributed to the establishment and optimization for prokaryotic chassis such as Escherichia coli or eukaryotic chassis such as Saccharomyces cerevisiae. This paper summarizes the current development and application of industrial biotechnology using conventional chassis cells, and prospects future development trend with the aim to facilitate researchers to optimize industrial biotechnology and its potential applications.},
}
@article {pmid37699034,
year = {2023},
author = {Sheng, H and Wu, S and Xue, Y and Zhao, W and Caplan, AB and Hovde, CJ and Minnich, SA},
title = {Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.},
journal = {PloS one},
volume = {18},
number = {9},
pages = {e0291520},
pmid = {37699034},
issn = {1932-6203},
support = {P20 GM103408/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; CRISPR-Cas Systems/genetics ; Engineering ; *Gastroenteritis ; *Enterohemorrhagic Escherichia coli/genetics ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Microbial ; *Escherichia coli O157 ; },
abstract = {Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.},
}
@article {pmid37698688,
year = {2023},
author = {Shakoor, S and Rao, AQ and Ajmal, S and Yasmeen, A and Khan, MAU and Sadaqat, S and Ashraf, NM and Wolter, F and Pacher, M and Husnain, T},
title = {Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.},
journal = {Planta},
volume = {258},
number = {4},
pages = {79},
pmid = {37698688},
issn = {1432-2048},
mesh = {Viral Load ; *Gossypium/genetics ; *CRISPR-Cas Systems/genetics ; Proteomics ; DNA ; },
abstract = {Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (βC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (βC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited βC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.},
}
@article {pmid37697159,
year = {2023},
author = {Singh, A and Pandey, H and Pandey, S and Lal, D and Chauhan, D and Aparna, and Antre, SH and B, S and Kumar, A},
title = {Drought stress in maize: stress perception to molecular response and strategies for its improvement.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {4},
pages = {296},
pmid = {37697159},
issn = {1438-7948},
mesh = {*Droughts ; *Zea mays/genetics ; Plant Breeding ; Drought Resistance ; Crops, Agricultural/genetics ; Perception ; },
abstract = {Given the future demand for food crops, increasing crop productivity in drought-prone rainfed areas has become essential. Drought-tolerant varieties are warranted to solve this problem in major crops, with drought tolerance as a high-priority trait for future research. Maize is one such crop affected by drought stress, which limits production, resulting in substantial economic losses. It became a more serious issue due to global climate change. The most drought sensitive among all stages of maize is the reproductive stages and the most important for overall maize production. The exact molecular basis of reproductive drought sensitivity remains unclear due to genes' complex regulation of drought stress. Understanding the molecular biology and signaling of the unexplored area of reproductive drought tolerance will provide an opportunity to develop climate-smart drought-tolerant next-generation maize cultivars. In recent decades, significant progress has been made in maize to understand the drought tolerance mechanism. However, improving maize drought tolerance through breeding is ineffective due to the complex nature and multigenic control of drought traits. With the help of advanced breeding techniques, molecular genetics, and a precision genome editing approach like CRISPR-Cas, candidate genes for drought-tolerant maize can be identified and targeted. This review summarizes the effects of drought stress on each growth stage of maize, potential genes, and transcription factors that determine drought tolerance. In addition, we discussed drought stress sensing, its molecular mechanisms, different approaches to developing drought-resistant maize varieties, and how molecular breeding and genome editing will help with the current unpredictable climate change.},
}
@article {pmid37696879,
year = {2023},
author = {Przybyszewska-Podstawka, A and Czapiński, J and Kałafut, J and Rivero-Müller, A},
title = {Synthetic circuits based on split Cas9 to detect cellular events.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {14988},
pmid = {37696879},
issn = {2045-2322},
support = {DEC-2015/17/B/NZ1/01777//Narodowe Centrum Nauki/ ; DEC-2017/25/B/NZ4/02364//Narodowym Centrum Nauki/ ; },
mesh = {*Epithelial-Mesenchymal Transition/genetics ; *CRISPR-Cas Systems ; Epithelial Cells ; Giant Cells ; Acclimatization ; },
abstract = {Synthetic biology involves the engineering of logic circuit gates that process different inputs to produce specific outputs, enabling the creation or control of biological functions. While CRISPR has become the tool of choice in molecular biology due to its RNA-guided targetability to other nucleic acids, it has not been frequently applied to logic gates beyond those controlling the guide RNA (gRNA). In this study, we present an adaptation of split Cas9 to generate logic gates capable of sensing biological events, leveraging a Cas9 reporter (EGxxFP) to detect occurrences such as cancer cell origin, epithelial to mesenchymal transition (EMT), and cell-cell fusion. First, we positioned the complementing halves of split Cas9 under different promoters-one specific to cancer cells of epithelial origin (phCEA) and the other a universal promoter. The use of self-assembling inteins facilitated the reconstitution of the Cas9 halves. Consequently, only cancer cells with an epithelial origin activated the reporter, exhibiting green fluorescence. Subsequently, we explored whether this system could detect biological processes such as epithelial to mesenchymal transition (EMT). To achieve this, we designed a logic gate where one half of Cas9 is expressed under the phCEA, while the other is activated by TWIST1. The results showed that cells undergoing EMT effectively activated the reporter. Next, we combined the two inputs (epithelial origin and EMT) to create a new logic gate, where only cancer epithelial cells undergoing EMT activated the reporter. Lastly, we applied the split-Cas9 logic gate as a sensor of cell-cell fusion, both in induced and naturally occurring scenarios. Each cell type expressed one half of split Cas9, and the induction of fusion resulted in the appearance of multinucleated syncytia and the fluorescent reporter. The simplicity of the split Cas9 system presented here allows for its integration into various cellular processes, not only as a sensor but also as an actuator.},
}
@article {pmid37696787,
year = {2023},
author = {Auradkar, A and Guichard, A and Kaduwal, S and Sneider, M and Bier, E},
title = {tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5587},
pmid = {37696787},
issn = {2041-1723},
support = {R01 GM117321/GM/NIGMS NIH HHS/United States ; R01 AI162911/AI/NIAID NIH HHS/United States ; R01 GM144608/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; Gene Knockdown Techniques ; CRISPR-Associated Protein 9/genetics ; DNA Cleavage ; Mammals ; },
abstract = {CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14-15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.},
}
@article {pmid37695079,
year = {2023},
author = {Jiang, T and Liu, R and Shen, J},
title = {CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.},
journal = {Journal of medical virology},
volume = {95},
number = {9},
pages = {e29090},
doi = {10.1002/jmv.29090},
pmid = {37695079},
issn = {1096-9071},
support = {2022zhyx-C61//Research Fund of Anhui Institute of Translational Medicine/ ; },
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; DNA ; Nucleotidyltransferases ; RNA ; },
abstract = {The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.},
}
@article {pmid37694285,
year = {2023},
author = {Wang, J and Zhao, YT and Zhang, L and Dubielecka, PM and Qin, G and Chin, YE and Gower, AC and Zhuang, S and Liu, PY and Zhao, TC},
title = {Irisin deficiency exacerbates diet-induced insulin resistance and cardiac dysfunction in type II diabetes in mice.},
journal = {American journal of physiology. Cell physiology},
volume = {},
number = {},
pages = {},
doi = {10.1152/ajpcell.00232.2023},
pmid = {37694285},
issn = {1522-1563},
support = {R01 HL115265/HL/NHLBI NIH HHS/United States ; },
abstract = {Myokine Irisin is involved in the regulation of a variety of physiological conditions, metabolism, and survival. We and others have demonstrated that recombinant irisin contributes critically to modulation of insulin resistance and the improvement of cardiac function. However, whether deletion of irisin will regulates cardiac function and insulin sensitivity in type II diabetes remains unclear. We utilized the CRISPR/Cas-9 genome-editing system to delete irisin globally in mice and high fat diet (HFD)-induced type II diabetes model. We found that irisin deficiency did not result in developmental abnormality during adult stage, which illustrates normal cardiac function and insulin sensitivity in the absence of stress. The ultrastructural analysis of the transmission electronic microscope (TEM) indicated that deletion of irisin did not change the morphology of mitochondria in myocardium. Gene expression profiling showed that several key signaling pathways related to integrin signaling, extracellular matrix and insulin-like growth factors signaling were coordinately downregulated by deletion of irisin. When mice were fed with a HFD for sixteen weeks, ablation of irisin in mice exposed to HFD resulted in much severer insulin resistance, metabolic derangements, profound cardiac dysfunction and hypertrophic response and remodeling as compared with wild type control. Taken together, our results indicate that the loss of irisin exacerbates insulin resistance, metabolic disorders, and cardiac dysfunction in response to a high fat diet and promoted myocardial remodeling and hypertrophic response. This evidence reveals the molecular evidence and the critical role of irisin in modulating insulin resistance and cardiac function in type II diabetes.},
}
@article {pmid37694158,
year = {2023},
author = {Davis, DJ and McNew, JF and Maresca-Fichter, H and Chen, K and Telugu, BP and Bryda, EC},
title = {Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1256451},
pmid = {37694158},
issn = {2673-3439},
abstract = {Recent advances in CRISPR-Cas genome editing technology have been instrumental in improving the efficiency to produce genetically modified animal models. In this study we have combined four very promising approaches to come up with a highly effective pipeline to produce knock-in mouse and rat models. The four combined methods include: AAV-mediated DNA delivery, single-stranded DNA donor templates, 2-cell embryo modification, and CRISPR-Cas ribonucleoprotein (RNP) electroporation. Using this new combined approach, we were able to produce successfully targeted knock-in rat models containing either Cre or Flp recombinase sequences with knock-in efficiencies over 90%. Furthermore, we were able to produce a knock-in mouse model containing a Cre recombinase targeted insertion with over 50% knock-in efficiency directly comparing efficiencies to other commonly used approaches. Our modified AAV-mediated DNA delivery with 2-cell embryo CRISPR-Cas9 RNP electroporation technique has proven to be highly effective for generating both knock-in mouse and knock-in rat models.},
}
@article {pmid37669587,
year = {2023},
author = {Wang, H and Sun, Y and Zhou, Y and Liu, Y and Chen, S and Sun, W and Zhang, Z and Guo, J and Yang, C and Li, Z and Chen, L},
title = {Unamplified system for sensitive and typing detection of ASFV by the cascade platform that CRISPR-Cas12a combined with graphene field-effect transistor.},
journal = {Biosensors &amp; bioelectronics},
volume = {240},
number = {},
pages = {115637},
doi = {10.1016/j.bios.2023.115637},
pmid = {37669587},
issn = {1873-4235},
mesh = {CRISPR-Cas Systems/genetics ; *Graphite ; *Biosensing Techniques ; Electricity ; Real-Time Polymerase Chain Reaction ; },
abstract = {At present, the 100% case fatality and the cross-infection of virus strains make the ASFV 's harm to society continue to expand. The absence of an effective commercial vaccine poses early detection remains the most effective means of curbing ASFV infection. Here, we report a cascaded detection platform based on the CRISPR-Cas12a system combined with graphene field-effect transistor sensors. The cascade platform could detect ASFV as low as 0.5 aM within 30 min and achieve typing of wild and vaccine strains of ASFV in a single detection system. The evaluation of 16 clinical samples proved that, compared with the gold standard Real-time PCR method, this platform has outstanding advantages in sensitivity, specificity and typing. Combining CRISPR-Cas12a's high specificity with the bipolar electric field effect of graphene field-effect transistor, the cascade platform is expected to achieve clinical application in the field of DNA disease detection, and provides a new direction for multi-strain disease typing.},
}
@article {pmid37648854,
year = {2023},
author = {Mamedov, MR and Vedova, S and Freimer, JW and Sahu, AD and Ramesh, A and Arce, MM and Meringa, AD and Ota, M and Chen, PA and Hanspers, K and Nguyen, VQ and Takeshima, KA and Rios, AC and Pritchard, JK and Kuball, J and Sebestyen, Z and Adams, EJ and Marson, A},
title = {CRISPR screens decode cancer cell pathways that trigger γδ T cell detection.},
journal = {Nature},
volume = {621},
number = {7977},
pages = {188-195},
pmid = {37648854},
issn = {1476-4687},
mesh = {Humans ; AMP-Activated Protein Kinases/genetics/metabolism ; Cell Line ; Cell Membrane/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; *Neoplasms/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, gamma-delta/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; },
abstract = {γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background[1-5]. γδ T cells can sense conserved cell stress signals prevalent in transformed cells[2,3], although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells[4]-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. [6-8]), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.},
}
@article {pmid37625413,
year = {2023},
author = {Kita, Y and Okuzaki, Y and Naoe, Y and Lee, J and Bang, U and Okawa, N and Ichiki, A and Jonouchi, T and Sakurai, H and Kojima, Y and Hotta, A},
title = {Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs.},
journal = {Stem cell reports},
volume = {18},
number = {9},
pages = {1753-1765},
doi = {10.1016/j.stemcr.2023.07.007},
pmid = {37625413},
issn = {2213-6711},
mesh = {Humans ; *Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Binding Sites ; Exons/genetics ; },
abstract = {To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.},
}
@article {pmid37622284,
year = {2023},
author = {Xu, H and Pan, R and Huang, W and Zhu, X},
title = {Label-free dual-mode sensing platform based on target-regulated CRISPR-Cas12a activity for ochratoxin A in Morinda officinalis.},
journal = {Analytical methods : advancing methods and applications},
volume = {15},
number = {35},
pages = {4518-4523},
doi = {10.1039/d3ay01025b},
pmid = {37622284},
issn = {1759-9679},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Morinda ; *Ochratoxins ; *Mycotoxins ; *Rubiaceae ; Oligonucleotides ; },
abstract = {Many traditional Chinese herbs are susceptible to ochratoxin A (OTA), a potent mycotoxin, which causes serious effects on the quality of the herb and on people's health. The development of methods to detect OTA is extremely important. Most methods for detecting OTA are based on a single-signal output mode, which might be easily influenced by complex environmental conditions. In this research, by taking advantage of the cleavage of DNA by target-induced CRISPR-Cas12a activity and the difference in electrostatic force of DNA to different charge electrochemiluminescent (ECL) and electrochemical (EC) probes, a biosensor is developed for the detection of OTA. First, the CRISPR-Cas12a system consists of a well-designed crRNA, its complementary strand (also as an aptamer for OTA), and Cas12a. Without the target, this CRISPR-Cas12a system is in the "activated stage", which digests hairpin DNA on the electrode, resulting in a weak ECL signal and strong current response. With the introduction of OTA bound with the aptamer, CRISPR-Cas12a activity is inhibited ("locked stage"). Thus, hairpin DNA remained intact on the electrode, resulting in recovery of the ECL signal and attenuation of the current intensity. As a result, this label-free dual-mode sensing platform realizes an assay for OTA in Morinda officinalis. This target-regulated CRISPR-Cas12a activity-sensing platform with dual-mode output not only provides high sensitivity (due to the CRISPR-Cas12a system), but also has good anti-interference ability against complex substrates (due to dual-mode output), and exhibits a broad range of prospects for application.},
}
@article {pmid37598442,
year = {2024},
author = {Gong, S and Song, K and Zhang, S and Zhou, P and Pan, W and Li, N and Tang, B},
title = {CRISPR-Cas12a-mediated dual-enzyme cascade amplification for sensitive colorimetric detection of HPV-16 target and ATP.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125050},
doi = {10.1016/j.talanta.2023.125050},
pmid = {37598442},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; Colorimetry ; Human papillomavirus 16/genetics ; Catalysis ; Glucose Oxidase ; *Nucleic Acids ; Adenosine Triphosphate ; },
abstract = {The establishment of sensitive and facile colorimetric platform based on the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is of great significance for in vitro diagnosis. Herein, we develop a dual-enzyme cascade amplification strategy based on CRISPR-Cas12a and glucose oxidase (GOx) for instrument-free and sensitive detection of target analytes. HPV-16 DNA as the model nucleic acid target directly initiated CRISPR-Cas12a-based signal transduction, resulting in the enzymatic cleavage of ssDNA linker and the release of GOx from magnetic nanoparticles 1 (MNPs1). Following simple magnetic separation, the supernatant containing GOx was taken out and used to catalyze the substrate, resulting in a visually detectable color change. The detection limit (LOD) of HPV-16 DNA was as low as 1 pM, and the entire process could be completed within 70 min without the need for expensive equipment. Notably, the dual-enzyme cascade amplification strategy was successfully applied to the detection of non-nucleic acid targets, such as ATP, via a simple signal transduction process. The visual LOD for ATP detection reaches 2.5 μM. The approach provides a robust, sensitive and reliable point-of-care biosensing platform for the detection of target analytes.},
}
@article {pmid37579676,
year = {2024},
author = {Wu, Y and Pei, J and Li, Y and Wang, G and Li, L and Liu, J and Tian, G},
title = {High-sensitive and rapid electrochemical detection of miRNA-31 in saliva using Cas12a-based 3D nano-harvester with improved trans-cleavage efficiency.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125066},
doi = {10.1016/j.talanta.2023.125066},
pmid = {37579676},
issn = {1873-3573},
mesh = {Humans ; Saliva ; CRISPR-Cas Systems ; *Carcinoma, Squamous Cell ; *Mouth Neoplasms/diagnosis/genetics ; Squamous Cell Carcinoma of Head and Neck ; *Head and Neck Neoplasms ; *MicroRNAs ; *Biosensing Techniques ; },
abstract = {Salivary miRNA-31 is a reliable diagnostic marker for early-stage oral squamous cell carcinoma (OSCC), but accurate detection of miRNA-31 in saliva samples is a challenge because of its low level and high sequence homology. The CRISPR/Cas12a system has the exceptional potential to enable simple nucleic acid analysis but suffers from low speed and sensitivity. To achieve rapid and high-sensitive detection of miRNA-31 using the CRISPR/Cas12a system, a Cas12a-based nano-harvester activated by a polymerase-driven DNA walker, named as dual 3D nanorobots, was developed. The target walked rapidly on the surface of DNA hairpin-modified magnetic nanoparticles driven by DNA polymerase, generating numerous double-strand DNA (dsDNA). Then, the Cas12a bound to the generated dsDNA for activating its trans-cleavage activity, forming 3D nano-harvester. Subsequently, the harvester cut and released methylene blue-labeled DNA hairpins immobilized on the sensing interface, leading to the change in electrochemical signal. We found that the trans-cleavage activity of the harvester was higher than the conventional CRISPR/Cas12a system. The developed dual 3D nanorobots could enable rapid (detection time within 60 min), high-sensitive (detection limit of femtomolar), and specific analysis of miRNA-31 in saliva samples. Thus, our established electrochemical biosensing strategy has great potential for early diagnosis of OSCC.},
}
@article {pmid37567119,
year = {2024},
author = {Peng, Y and Xue, P and Wang, R and Shang, H and Yao, B and Zheng, Z and Yan, C and Chen, W and Xu, J},
title = {Engineering of an adaptive tandem CRISPR/Cas12a molecular amplifier permits robust analysis of Vibrio parahaemolyticus.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125061},
doi = {10.1016/j.talanta.2023.125061},
pmid = {37567119},
issn = {1873-3573},
mesh = {Humans ; *Vibrio parahaemolyticus/genetics ; CRISPR-Cas Systems/genetics ; Coloring Agents ; DNA Nucleotidylexotransferase ; Food Safety ; Recombinases ; Nucleic Acid Amplification Techniques ; },
abstract = {Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans-cleavage of a reporter molecule. By combining these processes, the target genomic DNA extracted from V. parahaemolyticus is able to activate many CRISPR/Cas12a units (CRISPR/Cas12a[n]) simultaneously, resulting in a greatly amplified target signal to indicate the presence and concentration of V. parahaemolyticus. This unique model offers more advantages compared to traditional amplification models that use one RPA amplicon to activate one CRISPR/Cas12a unit. Under optimized conditions, our method enables the detection of target V. parahaemolyticus within a linear range of 1 × 10[2]-1 × 10[7] CFU/mL, with an impressive limit of detection as low as 12.4 CFU/mL. It is conceivable that the adaptive tandem CRISPR/Cas12a molecular amplifier could be adapted as routine diagnostic kits in future for in-field detection of pathogens.},
}
@article {pmid37428871,
year = {2023},
author = {Li, BE and Li, GY and Cai, W and Zhu, Q and Seruggia, D and Fujiwara, Y and Vakoc, CR and Orkin, SH},
title = {In vivo CRISPR/Cas9 screening identifies Pbrm1 as a regulator of myeloid leukemia development in mice.},
journal = {Blood advances},
volume = {7},
number = {18},
pages = {5281-5293},
doi = {10.1182/bloodadvances.2022009455},
pmid = {37428871},
issn = {2473-9537},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Disease Progression ; Gene Editing ; *Leukemia, Myeloid/genetics ; Mutation ; *DNA-Binding Proteins/genetics ; *Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin-remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.},
}
@article {pmid37376803,
year = {2023},
author = {Qing, D and Chen, W and Huang, S and Li, J and Pan, Y and Zhou, W and Liang, Q and Yuan, J and Gan, D and Chen, L and Chen, L and Huang, J and Zhou, Y and Dai, G and Deng, G},
title = {Editing of rice (Oryza sativa L.) OsMKK3 gene using CRISPR/Cas9 decreases grain length by modulating the expression of photosystem components.},
journal = {Proteomics},
volume = {23},
number = {18},
pages = {e2200538},
doi = {10.1002/pmic.202200538},
pmid = {37376803},
issn = {1615-9861},
support = {U20A2032//National Natural Science Foundation of China/ ; 2023GXNSFDA026060//Guangxi Natural Science Foundation/ ; 2021GXNSFAA220083//Guangxi Natural Science Foundation/ ; },
mesh = {*Oryza/metabolism ; Proteomics/methods ; CRISPR-Cas Systems/genetics ; Edible Grain/metabolism ; Photosynthesis/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.},
}
@article {pmid37693076,
year = {2022},
author = {Jinka, C and Sainath, C and Babu, S and Chennupati, AC and Muppidi, LP and Krishnan, M and Sekar, G and Chinnaiyan, M and Andugula, SK},
title = {CRISPR-Cas9 gene editing and human diseases.},
journal = {Bioinformation},
volume = {18},
number = {11},
pages = {1081-1086},
pmid = {37693076},
issn = {0973-2063},
abstract = {CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effective ex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importance of the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic (non-viral) and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specific mRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this review illustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting future researchers to adopt more clinical trials on this breakthrough delivery system.},
}
@article {pmid37691844,
year = {2023},
author = {Hegde, S and Rauch, HE and Hughes, GL and Shariat, N},
title = {Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome.},
journal = {Access microbiology},
volume = {5},
number = {8},
pages = {},
pmid = {37691844},
issn = {2516-8290},
abstract = {The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.},
}
@article {pmid37691410,
year = {2023},
author = {Qiao, J and Zhao, Z and Li, Y and Lu, M and Man, S and Ye, S and Zhang, Q and Ma, L},
title = {Recent advances of food safety detection by nucleic acid isothermal amplification integrated with CRISPR/Cas.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-22},
doi = {10.1080/10408398.2023.2246558},
pmid = {37691410},
issn = {1549-7852},
abstract = {Food safety problems have become one of the most important public health issues worldwide. Therefore, the development of rapid, effective and robust detection is of great importance. Amongst a range of methods, nucleic acid isothermal amplification (NAIA) plays a great role in food safety detection. However, the widespread application remains limited due to a few shortcomings. CRISPR/Cas system has emerged as a powerful tool in nucleic acid detection, which could be readily integrated with NAIA to improve the detection sensitivity, specificity, adaptability versatility and dependability. However, currently there was a lack of a comprehensive summary regarding the integration of NAIA and CRISPR/Cas in the field of food safety detection. In this review, the recent advances in food safety detection based on CRISPR/Cas-integrated NAIA were comprehensively reviewed. To begin with, the development of NAIA was summarized. Then, the types and working principles of CRISPR/Cas were introduced. The applications of the integration of NAIA and CRISPR/Cas for food safety were mainly introduced and objectively discussed. Lastly, current challenges and future opportunities were proposed. In summary, this technology is expected to become an important approach for food safety detection, leading to a safer and more reliable food industry.},
}
@article {pmid37689895,
year = {2023},
author = {Shangpliang, HNJ and Tamang, JP},
title = {Metagenomics and metagenome-assembled genomes mining of health benefits in jalebi batter, a naturally fermented cereal-based food of India.},
journal = {Food research international (Ottawa, Ont.)},
volume = {172},
number = {},
pages = {113130},
doi = {10.1016/j.foodres.2023.113130},
pmid = {37689895},
issn = {1873-7145},
mesh = {*Metagenome ; *Edible Grain ; Metagenomics ; India ; },
abstract = {Jalebi is one of the oldest Indian traditional fermented wheat-based confectioneries. Since jalebi is prepared by natural fermentation, diverse microbial community is expected to play bio-functional activities. Due to limited studies, information on microbial community structure in jalebi is unknown. Hence, the present study is aimed to profile the microbial community in jalebi by shotgun metagenomics and also to predict putative probiotic and functional genes by metagenome-assembled genome (MAG). Bacteria were the most abundant domain (91.91%) under which Bacillota was the most abundant phylum (82%). The most abundant species was Lapidilactobacillus dextrinicus followed by several species of lactic acid bacteria, acetic acid bacteria including few yeasts. Lap. dextrinicus was also significantly abundant in jalebi when compared to similar fermented wheat-based sourdough. Additionally, Lap. bayanensis, Pediococcus stilesii, and yeast- Candida glabrata, Gluconobacter japonicus, Pichia kudriavzevii, Wickerhamomyces anomalus were only detected in jalebi, which are not detected in sourdough. Few viruses and archaea were detected with < 1 % abundance. In silico screening of genes from the abundant species was mined using both KEGG and EggNOG database for putative health beneficial attributes. Circular genomes of five high-quality MAGs, identified as Lapidilactobacillus dextrinicus, Enterococcus hirae, Pediococcus stilesii, Acetobacter indonesiensis and Acetobacter cibinongensis, were constructed separately and putative genes were mapped and annotated. The CRISPR/Cas gene clusters in the genomes of four MAGs except Acetobacter cibinongensis were detected. MAGs also showed several secondary metabolites. Since, the identified MAGs have different putative genes for bio-functional properties, this may pave the way to selectively culture the uncultivated putative microbes for jalebi production. We believe this is the first report on metagenomic and MAGs of jalebi.},
}
@article {pmid37658470,
year = {2023},
author = {Xiang, X and Xing, G and Liu, Y and Wen, Q and Wei, Y and Lu, J and Chen, Y and Ji, Y and Chen, S and Liu, T and Shang, Y},
title = {Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {36},
pages = {13518-13526},
doi = {10.1021/acs.jafc.3c03799},
pmid = {37658470},
issn = {1520-5118},
mesh = {*Immunomagnetic Separation ; *Salmonella typhimurium/genetics ; CRISPR-Cas Systems ; Microfluidics ; Antibodies ; },
abstract = {A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 10[2] CFU/mL and a mean recovery of about 106% in the spiked milk sample.},
}
@article {pmid37646615,
year = {2023},
author = {Liu, X and Cao, Z and Wang, W and Zou, C and Wang, Y and Pan, L and Jia, B and Zhang, K and Zhang, W and Li, W and Hao, Q and Zhang, Y and Zhang, W and Xue, X and Lin, W and Li, M and Gu, J},
title = {Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma.},
journal = {ACS nano},
volume = {17},
number = {17},
pages = {16432-16447},
doi = {10.1021/acsnano.2c12857},
pmid = {37646615},
issn = {1936-086X},
mesh = {Animals ; Mice ; *Glioblastoma/genetics/radiotherapy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Glioma ; *Extracellular Vesicles/genetics ; Glutathione ; },
abstract = {Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an in vivo loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.},
}
@article {pmid37595030,
year = {2023},
author = {Wang, T and Chen, G and Zhang, S and Li, D and Wei, G and Zhao, X and Liu, Y and Ding, D and Zhang, X},
title = {Steerable Microneedles Enabling Deep Delivery of Photosensitizers and CRISPR/Cas9 Systems for Effective Combination Cancer Therapy.},
journal = {Nano letters},
volume = {23},
number = {17},
pages = {7990-7999},
doi = {10.1021/acs.nanolett.3c01914},
pmid = {37595030},
issn = {1530-6992},
mesh = {*Photosensitizing Agents ; CRISPR-Cas Systems/genetics ; Combined Modality Therapy ; Phototherapy ; Lysosomes ; *Neoplasms ; },
abstract = {Although gene therapy has shown prospects in treating triple-negative breast cancer, it is insufficient to treat such a malignant tumor. Herein, nanoparticles (NPs)-embedded dissolving microneedles (IR780-PL/pFBXO44@MNs) with steerable and flectional property were developed to achieve the codelivery of FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and hydrophobic photosensitizers. For improved NP penetration in tumor tissue, collagenase@MNs were preapplied to degrade the tumor matrix. Under light irradiation, IR780 exhibited remarkable phototherapy, while the escape efficiency of NPs from lysosomes was improved. pFBXO44 was subsequently released in tumor cell cytoplasm via reducing the disulfide bonds of NPs, which could specifically knock out the FBXO44 gene to inhibit the migration and invasion of tumor cells. As a result, tumor cells were eradicated, and lung metastasis was effectively suppressed. This micelle-incorporated microneedle platform broadens the potential of combining gene editing and photo synergistic cancer therapy.},
}
@article {pmid37584415,
year = {2023},
author = {Sun, Y and Xu, X and Chen, L and Chew, WL and Ping, Y and Miserez, A},
title = {Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery.},
journal = {ACS nano},
volume = {17},
number = {17},
pages = {16597-16606},
doi = {10.1021/acsnano.3c02669},
pmid = {37584415},
issn = {1936-086X},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Reproducibility of Results ; Peptides/genetics ; Oxidation-Reduction ; },
abstract = {CRISPR/Cas9-based genome editing tools have enormous potential for the development of various therapeutic treatments due to their reliability and broad applicability. A central requirement of CRISPR/Cas9 is the efficient intracellular delivery of the editing machinery, which remains a well-recognized challenge, notably to deliver Cas9 in its native protein form. Herein, a phase-separating peptide with intracellular redox-triggered release properties is employed to encapsulate and deliver all three forms of CRISRP-Cas9 editing machinery, namely, pDNA, mRNA/sgRNA, and the ribonucleoprotein complex. These modalities are readily recruited within peptide coacervates during liquid-liquid phase separation by simple mixing and exhibit higher transfection and editing efficiency compared to highly optimized commercially available transfection reagents currently used for genome editing.},
}
@article {pmid37582231,
year = {2023},
author = {Fletcher, RB and Stokes, LD and Kelly, IB and Henderson, KM and Vallecillo-Viejo, IC and Colazo, JM and Wong, BV and Yu, F and d'Arcy, R and Struthers, MN and Evans, BC and Ayers, J and Castanon, M and Weirich, MJ and Reilly, SK and Patel, SS and Ivanova, YI and Silvera Batista, CA and Weiss, SM and Gersbach, CA and Brunger, JM and Duvall, CL},
title = {Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.},
journal = {ACS nano},
volume = {17},
number = {17},
pages = {16412-16431},
doi = {10.1021/acsnano.2c12261},
pmid = {37582231},
issn = {1936-086X},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Silicon ; Porosity ; Polymers ; *Nanoparticles ; },
abstract = {The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.},
}
@article {pmid37087571,
year = {2023},
author = {Wu, Y and Xiao, N and Cai, Y and Yang, Q and Yu, L and Chen, Z and Shi, W and Liu, J and Pan, C and Li, Y and Zhang, X and Zhou, C and Huang, N and Ji, H and Zhu, S and Li, A},
title = {CRISPR-Cas9-mediated editing of the OsHPPD 3' UTR confers enhanced resistance to HPPD-inhibiting herbicides in rice.},
journal = {Plant communications},
volume = {4},
number = {5},
pages = {100605},
doi = {10.1016/j.xplc.2023.100605},
pmid = {37087571},
issn = {2590-3462},
mesh = {CRISPR-Cas Systems/genetics ; *Oryza/genetics ; 3' Untranslated Regions ; *Herbicides/pharmacology ; },
abstract = {This study reports the creation of herbicide-resistant rice lines via CRISPR-Cas9-mediated editing of the 3' UTR of OsHPPD. Resistance index calculations revealed that two resistant lines, TS8-2[#]-10 and TS8-8[#]-6, exhibited 4.8-fold and 3.7-fold greater resistance to HPPD-inhibiting herbicides compared with the wild type, YG3012.},
}
@article {pmid36702901,
year = {2023},
author = {},
title = {CLASH enables large-scale parallel knock-in for cell engineering.},
journal = {Nature biotechnology},
volume = {41},
number = {9},
pages = {1202-1203},
pmid = {36702901},
issn = {1546-1696},
mesh = {*Cell Engineering ; Gene Knock-In Techniques ; *Genetic Engineering ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid36702900,
year = {2023},
author = {Dai, X and Park, JJ and Du, Y and Na, Z and Lam, SZ and Chow, RD and Renauer, PA and Gu, J and Xin, S and Chu, Z and Liao, C and Clark, P and Zhao, H and Slavoff, S and Chen, S},
title = {Massively parallel knock-in engineering of human T cells.},
journal = {Nature biotechnology},
volume = {41},
number = {9},
pages = {1239-1255},
pmid = {36702900},
issn = {1546-1696},
support = {R01GM122984//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2CA238295//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01CA231112//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R33CA225498//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1RF1DA048811//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; U54CA209992//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; W81XWH-21-1-0514//U.S. Department of Defense (United States Department of Defense)/ ; },
mesh = {Humans ; *Bacterial Proteins/genetics ; *Gene Editing/methods ; CD4-Positive T-Lymphocytes/metabolism ; RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.},
}
@article {pmid36693989,
year = {2023},
author = {},
title = {CRISPR-Csm for eukaryotic RNA knockdown and imaging without toxicity.},
journal = {Nature biotechnology},
volume = {41},
number = {9},
pages = {1204-1205},
pmid = {36693989},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; RNA ; Eukaryota/genetics ; *CRISPR-Associated Proteins/metabolism ; RNA, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37689118,
year = {2023},
author = {Zhou, J and Liu, S and Xie, B and Wang, W and Xu, N and Xu, A and Dong, W and Jiang, M},
title = {Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding.},
journal = {Bioresource technology},
volume = {388},
number = {},
pages = {129716},
doi = {10.1016/j.biortech.2023.129716},
pmid = {37689118},
issn = {1873-2976},
abstract = {Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.},
}
@article {pmid37688677,
year = {2023},
author = {Shaheen, N and Ahmad, S and Alghamdi, SS and Rehman, HM and Javed, MA and Tabassum, J and Shao, G},
title = {CRISPR-Cas System, a Possible "Savior" of Rice Threatened by Climate Change: An Updated Review.},
journal = {Rice (New York, N.Y.)},
volume = {16},
number = {1},
pages = {39},
pmid = {37688677},
issn = {1939-8425},
support = {2021PE0AC05//Intelligent technology and platform development for rice breeding/ ; },
abstract = {Climate change has significantly affected agriculture production, particularly the rice crop that is consumed by almost half of the world's population and contributes significantly to global food security. Rice is vulnerable to several abiotic and biotic stresses such as drought, heat, salinity, heavy metals, rice blast, and bacterial blight that cause huge yield losses in rice, thus threatening food security worldwide. In this regard, several plant breeding and biotechnological techniques have been used to raise such rice varieties that could tackle climate changes. Nowadays, gene editing (GE) technology has revolutionized crop improvement. Among GE technology, CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) system has emerged as one of the most convenient, robust, cost-effective, and less labor-intensive system due to which it has got more popularity among plant researchers, especially rice breeders and geneticists. Since 2013 (the year of first application of CRISPR/Cas-based GE system in rice), several trait-specific climate-resilient rice lines have been developed using CRISPR/Cas-based GE tools. Earlier, several reports have been published confirming the successful application of GE tools for rice improvement. However, this review particularly aims to provide an updated and well-synthesized brief discussion based on the recent studies (from 2020 to present) on the applications of GE tools, particularly CRISPR-based systems for developing CRISPR rice to tackle the current alarming situation of climate change, worldwide. Moreover, potential limitations and technical bottlenecks in the development of CRISPR rice, and prospects are also discussed.},
}
@article {pmid37687368,
year = {2023},
author = {Tang, Q and Wang, X and Jin, X and Peng, J and Zhang, H and Wang, Y},
title = {CRISPR/Cas Technology Revolutionizes Crop Breeding.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {17},
pages = {},
pmid = {37687368},
issn = {2223-7747},
abstract = {Crop breeding is an important global strategy to meet sustainable food demand. CRISPR/Cas is a most promising gene-editing technology for rapid and precise generation of novel germplasm and promoting the development of a series of new breeding techniques, which will certainly lead to the transformation of agricultural innovation. In this review, we summarize recent advances of CRISPR/Cas technology in gene function analyses and the generation of new germplasms with increased yield, improved product quality, and enhanced resistance to biotic and abiotic stress. We highlight their applications and breakthroughs in agriculture, including crop de novo domestication, decoupling the gene pleiotropy tradeoff, crop hybrid seed conventional production, hybrid rice asexual reproduction, and double haploid breeding; the continuous development and application of these technologies will undoubtedly usher in a new era for crop breeding. Moreover, the challenges and development of CRISPR/Cas technology in crops are also discussed.},
}
@article {pmid37624847,
year = {2023},
author = {Jirawannaporn, S and Limothai, U and Tachaboon, S and Dinhuzen, J and Kiatamornrak, P and Chaisuriyong, W and Srisawat, N},
title = {The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis.},
journal = {PLoS neglected tropical diseases},
volume = {17},
number = {8},
pages = {e0011596},
pmid = {37624847},
issn = {1935-2735},
mesh = {Humans ; Recombinases ; *Leptospira/genetics ; CRISPR-Cas Systems ; *Leptospirosis/diagnosis ; Antibodies, Bacterial ; Fever ; Immunoglobulin M ; },
abstract = {BACKGROUND: Lack of available sensitive point-of-care testing is one of the primary obstacles to the rapid diagnosis of leptospirosis. The purpose of this study was to test the performance of two point-of-care tests, a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) fluorescence-based diagnostic assay (FBDA), a Leptospira immunoglobulin M (IgM) rapid diagnostic test (RDT), and the two tests combined.<br><br>For the diagnosis of 171 clinical samples, a recombinase polymerase amplification (RPA)-CRISPR/Cas12a FBDA for whole blood and Leptospira IgM RDT (Medical Science Public Health, Thailand) for serum were used. The confirmed cases were determined by using any positive qPCR, microscopic agglutination test (MAT), and culture results. Diagnostic accuracy was assessed on the first day of enrollment and stratified by the day after symptom onset. The overall sensitivity of the Leptospira IgM RDT and RPA-CRISPR/Cas12a FBDA was 55.66% and 60.38%, respectively. When the two tests were combined, the sensitivity rose to 84.91%. The specificity of each test was 63.08% and 100%, respectively, and 63.08% when combined. The sensitivity of the Leptospira IgM RDT rose on days 4-6 after the onset of fever, while the RPA-CRISPR/Cas12a FBDA continued to decrease. When the two tests were combined, the sensitivity was over 80% at different days post-onset of fever.<br><br>CONCLUSIONS/SIGNIFICANCE: The combination of Leptospira IgM RDT and RPA-CRISPR/Cas12 FBDA exhibited significant sensitivity for the detection of leptospires at various days after the onset of fever, thereby reducing the likelihood of misdiagnosis. The combination of these assays may be suitable for early leptospirosis screening in situations with limited resources.},
}
@article {pmid37623910,
year = {2023},
author = {Wang, H and Li, YL and Fan, YJ and Dong, JX and Ren, X and Ma, H and Wu, D and Gao, ZF and Wei, Q and Xia, F},
title = {DNA Tile and Invading Stacking Primer-Assisted CRISPR-Cas12a Multiple Amplification System for Entropy-Driven Electrochemical Detection of MicroRNA with Tunable Sensitivity.},
journal = {Analytical chemistry},
volume = {95},
number = {36},
pages = {13659-13667},
doi = {10.1021/acs.analchem.3c02603},
pmid = {37623910},
issn = {1520-6882},
mesh = {Entropy ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Electrodes ; *MicroRNAs/genetics ; },
abstract = {Conventional electrochemical detection of microRNA (miRNA) encounters issues of poor sensitivity and fixed dynamic range. Here, we report a DNA tile and invading stacking primer-assisted CRISPR-Cas12a multiple amplification strategy to construct an entropy-controlled electrochemical biosensor for the detection of miRNA with tunable sensitivity and dynamic range. To amplify the signal, a cascade amplification of the CRISPR-Cas12a system along with invading stacking primer signal amplification (ISPSA) was designed to detect trace amounts of miRNA-31 (miR-31). The target miR-31 could activate ISPSA and produce numerous DNAs, triggering the cleavage of the single-stranded linker probe (LP) that connects a methylene blue-labeled DNA tile with a DNA tetrahedron to form a Y-shaped DNA scaffold on the electrode. Based on the decrease of current, miR-31 can be accurately and efficiently detected. Impressively, by changing the loop length of the LP, it is possible to finely tune the entropic contribution while keeping the enthalpic contribution constant. This strategy has shown a tunable limit of detection for miRNA from 0.31 fM to 0.56 pM, as well as a dynamic range from ∼2200-fold to ∼270,000-fold. Moreover, it demonstrated satisfactory results in identifying cancer cells with a high expression of miR-31. Our strategy broadens the application of conventional electrochemical biosensing and provides a tunable strategy for detecting miRNAs at varying concentrations.},
}
@article {pmid37529831,
year = {2023},
author = {Liang, Z and Wei, S and Wu, Y and Guo, Y and Zhang, B and Yang, H},
title = {Temporally gene knockout using heat shock-inducible genome-editing system in plants.},
journal = {The plant genome},
volume = {16},
number = {3},
pages = {e20376},
doi = {10.1002/tpg2.20376},
pmid = {37529831},
issn = {1940-3372},
support = {32170410//National Natural Science Foundation of China/ ; 202204051001019//Science and Technology Innovation Young Talent Team of Shanxi Province/ ; 2022YFF1002802//National Key Research and Development Program of China/ ; },
mesh = {Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Genome, Plant ; Heat-Shock Response ; Immunoglobulin E/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) has emerged as a powerful tool to generate targeted loss-of-function mutations for functional genomic studies. As a next step, tools to generate genome modifications in a spatially and temporally precise manner will enable researchers to further dissect gene function. Here, we present two heat shock-inducible genome-editing (IGE) systems that efficiently edit target genes when the system is induced, thus allowing us to target specific developmental stages. For this conditional editing system, we chose the natural heat-inducible promoter from heat-shock protein 18.2 (HSP18.2) from Arabidopsis thaliana and the synthetic heat-inducible promoter heat shock-response element HSE-COR15A to drive the expression of Cas9. We tested these two IGE systems in Arabidopsis using cyclic or continuous heat-shock treatments at the seedling and bolting stages. A real-time quantitative polymerase chain reaction analysis revealed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE-COR15A IGE system upon both cyclic and continuous treatments. By targeting brassinosteroid-insensitive 1 (BRI1) and phytoene desaturase (PDS), we demonstrate that both cyclic and continuous heat inductions successfully activated the HSP18.2 IGE system at the two developmental stages, resulting in highly efficient targeted mutagenesis and clear phenotypic outcomes. By contrast, the HSE-COR15A IGE system was only induced at the seedling stage and was less effective than the HSP18.2 IGE system in terms of mutagenesis frequencies. The presented heat shock-IGE systems can be conditionally induced to efficiently inactivate genes at any developmental stage and are uniquely suited for the dissection and systematic characterization of essential genes.},
}
@article {pmid37686249,
year = {2023},
author = {Huang, S and Dai, R and Zhang, Z and Zhang, H and Zhang, M and Li, Z and Zhao, K and Xiong, W and Cheng, S and Wang, B and Wan, Y},
title = {CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686249},
issn = {1422-0067},
support = {322MS032//Hainan Provincial Natural Science Foundation of China/ ; ZY2022HN01//The Central Government Guides Local Science and Technology Development Projects/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Diagnostic Imaging ; Gene Editing ; },
abstract = {CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.},
}
@article {pmid37686137,
year = {2023},
author = {Xiong, Y and Xi, X and Xiang, Y and Li, S and Liu, H and Su, Y and He, R and Xiong, C and Xu, B and Wang, X and Fu, L and Zhao, C and Han, X and Li, X and Xie, S and Ruan, J},
title = {CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686137},
issn = {1422-0067},
support = {No. 32072685//National Natural Science Foundation of China/ ; },
mesh = {Humans ; Animals ; Swine ; *Encephalitis Virus, Japanese/genetics ; Introns/genetics ; RNA, Guide, CRISPR-Cas Systems ; Calreticulin ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Encephalitis Viruses, Japanese ; Mutation ; Antiviral Agents ; Cytosine ; Mammals ; },
abstract = {The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.},
}
@article {pmid37686051,
year = {2023},
author = {Huang, W and Zheng, A and Huang, H and Chen, Z and Ma, J and Li, X and Liang, Q and Li, L and Liu, R and Huang, Z and Qin, Y and Tang, Y and Li, H and Zhang, F and Wang, Q and Sun, B},
title = {Effects of sgRNAs, Promoters, and Explants on the Gene Editing Efficiency of the CRISPR/Cas9 System in Chinese Kale.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686051},
issn = {1422-0067},
support = {32072586, 31500247, 32172593//National Natural Science Foundation of China/ ; 2022NSFSC1689//Natural Science Foundation of Sichuan Province/ ; sccxtd-2023-05//Project of New Varieties Breeding of Sichuan Vegetable Innovation Team/ ; },
mesh = {*Brassica/genetics ; Gene Editing ; *RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The CRISPR/Cas9 system is extensively used for plant gene editing. This study developed an efficient CRISPR/Cas9 system for Chinese kale using multiple sgRNAs and two promoters to create various CRISPR/Cas9 vectors. These vectors targeted BoaZDS and BoaCRTISO in Chinese kale protoplasts and cotyledons. Transient transformation of Chinese kale protoplasts was assessed for editing efficiency at three BoaZDS sites. Notably, sgRNA: Z2 achieved the highest efficiency (90%). Efficiency reached 100% when two sgRNAs targeted BoaZDS with a deletion of a large fragment (576 bp) between them. However, simultaneous targeting of BoaZDS and BoaCRTISO yielded lower efficiency. Transformation of cotyledons led to Chinese kale mutants with albino phenotypes for boazds mutants and orange-mottled phenotypes for boacrtiso mutants. The mutation efficiency of 35S-CRISPR/Cas9 (92.59%) exceeded YAO-CRISPR/Cas9 (70.97%) in protoplasts, and YAO-CRISPR/Cas9 (96.49%) surpassed 35S-CRISPR/Cas9 (58%) in cotyledons. These findings introduce a strategy for enhancing CRISPR/Cas9 editing efficiency in Chinese kale.},
}
@article {pmid37686014,
year = {2023},
author = {Schneider, P and Wander, P and Arentsen-Peters, STCJM and Vrenken, KS and Rockx-Brouwer, D and Adriaanse, FRS and Hoeve, V and Paassen, I and Drost, J and Pieters, R and Stam, RW},
title = {CRISPR-Cas9 Library Screening Identifies Novel Molecular Vulnerabilities in KMT2A-Rearranged Acute Lymphoblastic Leukemia.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686014},
issn = {1422-0067},
support = {Not applicable//Foundation KiKa/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Library ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics ; Transcription Factors ; Cell Line ; Antigens, Neoplasm ; Neoplasm Proteins ; },
abstract = {In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.},
}
@article {pmid37686009,
year = {2023},
author = {Lu, X and Zhang, M and Li, G and Zhang, S and Zhang, J and Fu, X and Sun, F},
title = {Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686009},
issn = {1422-0067},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Medicine ; *Cystic Fibrosis ; Endonucleases ; *Nucleic Acids ; },
abstract = {The rapid advancements in gene therapy have opened up new possibilities for treating genetic disorders, including Duchenne muscular dystrophy, thalassemia, cystic fibrosis, hemophilia, and familial hypercholesterolemia. The utilization of the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has revolutionized the field of gene therapy by enabling precise targeting of genes. In recent years, CRISPR/Cas9 has demonstrated remarkable efficacy in treating cancer and genetic diseases. However, the susceptibility of nucleic acid drugs to degradation by nucleic acid endonucleases necessitates the development of functional vectors capable of protecting the nucleic acids from enzymatic degradation while ensuring safety and effectiveness. This review explores the biomedical potential of non-viral vector-based CRISPR/Cas9 systems for treating genetic diseases. Furthermore, it provides a comprehensive overview of recent advances in viral and non-viral vector-based gene therapy for genetic disorders, including preclinical and clinical study insights. Additionally, the review analyzes the current limitations of these delivery systems and proposes avenues for developing novel nano-delivery platforms.},
}
@article {pmid37685921,
year = {2023},
author = {Lee, YR and Ko, KS and Lee, HE and Lee, ES and Han, K and Yoo, JY and Vu, BN and Choi, HN and Lee, YN and Hong, JC and Lee, KO and Kim, DS},
title = {CRISPR/Cas9-Mediated HY5 Gene Editing Reduces Growth Inhibition in Chinese Cabbage (Brassica rapa) under ER Stress.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37685921},
issn = {1422-0067},
support = {PJ01626602//Cooperative Research Program for Agriculture Science and Technology Development/ ; },
mesh = {*Brassica rapa/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Hypocotyl ; RNA, Guide, CRISPR-Cas Systems ; Reactive Oxygen Species ; Crops, Agricultural ; Tunicamycin ; },
abstract = {Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops.},
}
@article {pmid37684555,
year = {2023},
author = {Zhao, J and Xi, Y and Zhang, J and Jin, Y and Yang, H and Duan, G and Chen, S and Long, J},
title = {Characterization and diversity of CRISPR/Cas systems in Klebsiella oxytoca.},
journal = {Molecular genetics and genomics : MGG},
volume = {},
number = {},
pages = {},
pmid = {37684555},
issn = {1617-4623},
support = {2022M712859//China Postdoctoral Science Foundation/ ; 2018ZX10301407//National Science and Technology Specific Projects/ ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system is a crucial adaptive immune system for bacteria to resist foreign DNA infection. In this study, we investigated the prevalence and diversity of CRISPR/Cas systems in 175 Klebsiella oxytoca (K. oxytoca) strains. Specifically, 58.86% (103/175) of these strains possessed at least one confirmed CRISPR locus. Two CRISPR/Cas system types, I-F and IV-A3, were identified in 69 strains. Type I-F system was the most prevalent in this species, which correlated well with MLST. Differently, type IV-A3 system was randomly distributed. Moreover, the type IV-A3 system was separated into two subgroups, with subgroup-specific cas genes and repeat sequences. In addition, spacer origin analysis revealed that approximately one-fifth of type I-F spacers and one-third of type IV-A3 spacers had a significant match to MGEs. The phage tail tape measure protein and conjunctive transfer system protein were important targets of type I-F and IV-A3 systems in K. oxytoca, respectively. PAM sequences were inferred to be 5'-NCC-3' for type I-F, 5'-AAG-3' for subgroup IV-A3-a, and 5'-AAN-3' for subgroup IV-A3-b. Collectively, our findings will shed light on the prevalence, diversity, and functional effects of the CRISPR/Cas system in K. oxytoca.},
}
@article {pmid37684258,
year = {2023},
author = {Brockman, QR and Scherer, A and McGivney, GR and Gutierrez, WR and Rytlewski, J and Sheehan, A and Warrier, A and Laverty, EA and Roughton, G and Carnevale, NC and Knepper-Adrian, V and Dodd, RD},
title = {Discrepancies in indel software resolution with somatic CRISPR/Cas9 tumorigenesis models.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {14798},
pmid = {37684258},
issn = {2045-2322},
support = {T32 GM0677954/NH/NIH HHS/United States ; T32 HL07734/NH/NIH HHS/United States ; R01 NS119322/NH/NIH HHS/United States ; P30 CA086862/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Carcinogenesis/genetics ; Cell Transformation, Neoplastic ; Cell Line ; Software ; },
abstract = {CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.},
}
@article {pmid37683609,
year = {2023},
author = {Patinios, C and Beisel, CL},
title = {For the CRISPR Fan(zor)atics: RNA-guided DNA endonucleases discovered in eukaryotes.},
journal = {Molecular cell},
volume = {83},
number = {17},
pages = {3046-3048},
doi = {10.1016/j.molcel.2023.08.019},
pmid = {37683609},
issn = {1097-4164},
mesh = {*Eukaryota/genetics ; *Deoxyribonuclease I ; Endonucleases/genetics ; RNA ; DNA/genetics ; },
abstract = {RNA-guided DNA endonucleases such as those from CRISPR-Cas systems were considered limited to prokaryotes. Saito et al.[1] reveal that distant eukaryotic relatives of Cas nucleases, called Fanzors, also function as RNA-guided DNA endonucleases and can be harnessed for genome editing.},
}
@article {pmid37682467,
year = {2023},
author = {Pan, C and Qi, Y},
title = {Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2698},
number = {},
pages = {27-40},
pmid = {37682467},
issn = {1940-6029},
mesh = {*Arabidopsis/genetics ; RNA, Guide, CRISPR-Cas Systems ; Agrobacterium ; CRISPR-Cas Systems/genetics ; RNA ; },
abstract = {The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures on the sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.},
}
@article {pmid37679413,
year = {2023},
author = {Nakamura, A and Yano, T and Mitsuda, N and Furubayashi, M and Ito, S and Sugano, SS and Terakawa, T},
title = {The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {14205},
pmid = {37679413},
issn = {2045-2322},
mesh = {Animals ; *Oryza/genetics ; CRISPR-Cas Systems ; Gene Editing ; Sonication ; Vibrissae ; Plant Breeding ; *Callosities ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYβ genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYβ mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.},
}
@article {pmid37679324,
year = {2023},
author = {Ham, DT and Browne, TS and Banglorewala, PN and Wilson, TL and Michael, RK and Gloor, GB and Edgell, DR},
title = {A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5514},
pmid = {37679324},
issn = {2041-1723},
support = {PJT-159708//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Data Accuracy ; Endonucleases ; Machine Learning ; },
abstract = {The CRISPR/Cas9 nuclease from Streptococcus pyogenes (SpCas9) can be used with single guide RNAs (sgRNAs) as a sequence-specific antimicrobial agent and as a genome-engineering tool. However, current bacterial sgRNA activity models struggle with accurate predictions and do not generalize well, possibly because the underlying datasets used to train the models do not accurately measure SpCas9/sgRNA activity and cannot distinguish on-target cleavage from toxicity. Here, we solve this problem by using a two-plasmid positive selection system to generate high-quality data that more accurately reports on SpCas9/sgRNA cleavage and that separates activity from toxicity. We develop a machine learning architecture (crisprHAL) that can be trained on existing datasets, that shows marked improvements in sgRNA activity prediction accuracy when transfer learning is used with small amounts of high-quality data, and that can generalize predictions to different bacteria. The crisprHAL model recapitulates known SpCas9/sgRNA-target DNA interactions and provides a pathway to a generalizable sgRNA bacterial activity prediction tool that will enable accurate antimicrobial and genome engineering applications.},
}
@article {pmid37678882,
year = {2023},
author = {Haldrup, J and Andersen, S and Labial, ARL and Wolff, JH and Frandsen, FP and Skov, TW and Rovsing, AB and Nielsen, I and Jakobsen, TS and Askou, AL and Thomsen, MK and Corydon, TJ and Thomsen, EA and Mikkelsen, JG},
title = {Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad676},
pmid = {37678882},
issn = {1362-4962},
support = {R324-2019-1832//Lundbeck Foundation/ ; 8056-00010A//Innovation Fund/ ; //Synoptik Foundation/ ; 38189//Velux Foundation/ ; },
abstract = {Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.},
}
@article {pmid37677025,
year = {2023},
author = {Montiel-Manriquez, R and Castro-Hernández, C and Arriaga-Canon, C and Herrera, LA},
title = {Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {199},
pages = {},
doi = {10.3791/65124},
pmid = {37677025},
issn = {1940-087X},
mesh = {Male ; Humans ; *Oligonucleotides, Antisense/genetics ; *RNA, Long Noncoding/genetics ; Cell Nucleus ; Oligonucleotides ; Cell Line ; },
abstract = {Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.},
}
@article {pmid37595863,
year = {2023},
author = {Wang, Y and Yang, T and Liu, G and Xie, L and Guo, J and Xiong, W},
title = {Application of CRISPR/Cas12a in the rapid detection of pathogens.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {548},
number = {},
pages = {117520},
doi = {10.1016/j.cca.2023.117520},
pmid = {37595863},
issn = {1873-3492},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The combination of clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an effective gene-editing instrument. Among them, the CRISPR-Cas12a system forms a DNA-cleavage-capable complex with crRNA and exerts its trans-cleavage activity by recognising the PAM site on the target pathogen's gene. After amplifying the pathogenic gene, display materials such as fluorescent probes are added to the detection system, along with the advantages of rapid detection and high sensitivity of the CRISPR system, so that pathogenic bacteria can be diagnosed with greater speed and precision. This article reviews the mechanism of CRISPR-Cas12a in rapid detection, as well as its progress in the rapid detection of pathogenic bacteria in conjunction with various molecular biology techniques, in order to provide a foundation for the future development of a more effective detection platform.},
}
@article {pmid37591461,
year = {2023},
author = {Lin, YQ and Feng, KK and Lu, JY and Le, JQ and Li, WL and Zhang, BC and Li, CL and Song, XH and Tong, LW and Shao, JW},
title = {CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {361},
number = {},
pages = {727-749},
doi = {10.1016/j.jconrel.2023.08.028},
pmid = {37591461},
issn = {1873-4995},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Technology ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.},
}
@article {pmid37522352,
year = {2023},
author = {Singh, J and Liu, KG and Allen, A and Jiang, W and Qin, PZ},
title = {A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.},
journal = {Nucleic acids research},
volume = {51},
number = {16},
pages = {8730-8743},
pmid = {37522352},
issn = {1362-4962},
support = {T32 GM118289/GM/NIGMS NIH HHS/United States ; R01GM124413/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; RNA/genetics ; },
abstract = {CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.},
}
@article {pmid37503841,
year = {2023},
author = {Olsen, NS and Nielsen, TK and Cui, L and Dedon, P and Neve, H and Hansen, LH and Kot, W},
title = {A novel Queuovirinae lineage of Pseudomonas aeruginosa phages encode dPreQ0 DNA modifications with a single GA motif that provide restriction and CRISPR Cas9 protection in vitro.},
journal = {Nucleic acids research},
volume = {51},
number = {16},
pages = {8663-8676},
pmid = {37503841},
issn = {1362-4962},
support = {RGP0024/2018//Human Frontier Science Program/ ; 17595//Villum Experiment Grant/ ; //National Research Foundation of Singapore/ ; //Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group/ ; },
mesh = {Humans ; *Pseudomonas Phages/genetics ; Pseudomonas aeruginosa ; CRISPR-Cas Systems ; DNA/genetics ; *Bacteriophages/genetics ; },
abstract = {Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2'-deoxyguanosine (G) with dPreQ0, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ0 modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ0 biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.},
}
@article {pmid36822625,
year = {2023},
author = {Li, G and Zhu, X and Wang, Y and Ma, H and Wang, Y and Wu, H and Li, X and Wang, Y and Gao, J and Chen, X and Huang, X and Yao, Y and Hu, X},
title = {Transcription-wide impact by RESCUE-induced off-target single-nucleotide variants in mammalian cells.},
journal = {Journal of molecular cell biology},
volume = {15},
number = {2},
pages = {},
pmid = {36822625},
issn = {1759-4685},
support = {2016ZX08009003-006//Ministry of Agri-culture of China/ ; 2020SKLAB6-24//National Key Laboratory Open Fund Project/ ; 02020200//Zhejiang University/ ; 32071347//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mutation ; *MicroRNAs/genetics ; RNA, Messenger ; Nucleotides ; CRISPR-Cas Systems ; Mammals ; },
abstract = {RNA base editing is a promising tool in precise molecular therapy. Currently, there are two widely used RNA base editors, REPAIR and RESCUE. REPAIR only facilitates A-to-I conversions, while RESCUE performs both A-to-I and C-to-U conversions. Thus, RESCUE can generate twice the number of mutations compared to REPAIR. However, transcription-wide impact due to RESCUE-induced off-target single-nucleotide variants (SNVs) is not fully appreciated. Therefore, to determine the off-target effects of RESCUE-mediated editing, we employed transcription-wide sequencing on cells edited by RESCUE. The SNVs showed different off-target effects on mRNA, circRNA, lncRNA, and miRNA expression patterns and their interacting networks. Our results illustrate the transcription-wide impact of RESCUE-induced off-target SNVs and highlight the need for careful characterization of the off-target impact by this editing platform.},
}
@article {pmid37676518,
year = {2022},
author = {Gentzel, IN and Ohlson, EW and Redinbaugh, MG and Wang, GL},
title = {VIGE: virus-induced genome editing for improving abiotic and biotic stress traits in plants.},
journal = {Stress biology},
volume = {2},
number = {1},
pages = {2},
pmid = {37676518},
issn = {2731-0450},
abstract = {Agricultural production is hampered by disease, pests, and environmental stresses. To minimize yield loss, it is important to develop crop cultivars with resistance or tolerance to their respective biotic and abiotic constraints. Transformation techniques are not optimized for many species and desirable cultivars may not be amenable to genetic transformation, necessitating inferior cultivar usage and time-consuming introgression through backcrossing to the preferred variety. Overcoming these limitations will greatly facilitate the development of disease, insect, and abiotic stress tolerant crops. One such avenue for rapid crop improvement is the development of viral systems to deliver CRISPR/Cas-based genome editing technology to plants to generate targeted beneficial mutations. Viral delivery of genomic editing constructs can theoretically be applied to span the entire host range of the virus utilized, circumventing the challenges associated with traditional transformation and breeding techniques. Here we explore the types of viruses that have been optimized for CRISPR/Cas9 delivery, the phenotypic outcomes achieved in recent studies, and discuss the future potential of this rapidly advancing technology.},
}
@article {pmid37674114,
year = {2023},
author = {Wang, M and Chen, M and Wu, X and Huang, X and Yu, B},
title = {CRISPR applications in cancer diagnosis and treatment.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {73},
pmid = {37674114},
issn = {1689-1392},
support = {SZSM201812059//Shenzhen Sanming Project/ ; SZXK040//Shenzhen Key Medical Discipline Construction Fund/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Neoplasms/diagnosis/genetics/therapy ; },
abstract = {Cancer remains a significant global health challenge, necessitating the exploration of novel and more precise therapeutic options beyond conventional treatments. In this regard, clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as highly promising tools for clinical gene editing applications. The CRISPR family encompasses diverse CRISPR-associated (Cas) proteins that possess the ability to recognize specific target sequences. The initial CRISPR system consisted of the Cas9 protein and a single-guide RNA, which guide Cas9 to the desired target sequence, facilitating precise double-stranded cleavage. In addition to the traditional cis-cleavage activity, the more recently discovered Cas12 and Cas13 proteins exhibit trans-cleavage activity, which expands their potential applications in cancer diagnosis. In this review, we provide an overview of the functional characteristics of Cas9, Cas12, and Cas13. Furthermore, we highlight the latest advancements and applications of these CRISPR systems in cancer gene therapy and molecular diagnosis. We also emphasize the importance of understanding the strengths and limitations of each CRISPR system to maximize their clinical utility. By providing a comprehensive overview of the current state of CRISPR technology in cancer research, we aim to inspire further exploration and innovation in this rapidly evolving field.},
}
@article {pmid37673883,
year = {2023},
author = {Bestas, B and Wimberger, S and Degtev, D and Madsen, A and Rottner, AK and Karlsson, F and Naumenko, S and Callahan, M and Touza, JL and Francescatto, M and Möller, CI and Badertscher, L and Li, S and Cerboni, S and Selfjord, N and Ericson, E and Gordon, E and Firth, M and Chylinski, K and Taheri-Ghahfarokhi, A and Bohlooly-Y, M and Snowden, M and Pangalos, M and Nuttall, B and Akcakaya, P and Sienski, G and Maresca, M},
title = {A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5474},
pmid = {37673883},
issn = {2041-1723},
mesh = {Animals ; Mice ; *Translocation, Genetic ; *Proprotein Convertase 9/genetics ; CRISPR-Cas Systems/genetics ; Mutation ; Endonucleases/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.},
}
@article {pmid37673355,
year = {2023},
author = {Yao, S and Wu, X and Li, Y and Song, Y and Wang, C and Zhang, G and Feng, J},
title = {Harnessing the Native Type I-F CRISPR-Cas System of Acinetobacter baumannii for Genome Editing and Gene Repression.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {106962},
doi = {10.1016/j.ijantimicag.2023.106962},
pmid = {37673355},
issn = {1872-7913},
abstract = {The rapid emergence of infections caused by MDR Acinetobacter baumannii poses a serious threat to global public health. Therefore, it has become important to obtain a deeper understanding of the mechanisms of multidrug resistance and pathogenesis of A. baumannii. However, there are still relatively few genetic engineering tools for A. baumannii. Although A. baumannii possesses Type I-F CRISPR-Cas systems, they have not yet been used for genetic modifications. Here, we developed a single plasmid-mediated native Type I-F CRISPR-Cas system for gene editing and gene regulation in A. baumannii. The protospacer adjacent motif (PAM) sequence was identified as 5'-NCC-3' by analysis of the CRISPR array. Through introduction of the RecAb homologous recombination system, we significantly increased the knockout efficiency of the oxyR gene from 12.5% to 75.0% in A. baumannii. To investigate transcriptional inhibition by the Type I-F CRISPR system, we deleted the gene encoding its Cas2-3 nuclease and repurposed the native Type I-F Cascade effector to regulate transcription of alcohol dehydrogenase gene adh4. The level of adh4 transcription was inhibited by up to 900-fold compared to the control. The Cascade transcriptional module was also successfully applied in a clinical Klebsiella pneumoniae isolate. This study proposed a tool for future exploration of the genetic characteristics of A. baumannii or other clinical strains.},
}
@article {pmid37671884,
year = {2023},
author = {Misra, CS and Pandey, N and Appukuttan, D and Rath, D},
title = {Effective gene silencing using type I-E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0520422},
doi = {10.1128/spectrum.05204-22},
pmid = {37671884},
issn = {2165-0497},
abstract = {The extremely radiation-resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting, with its multiploid genome presenting an additional challenge. In view of this, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for the expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A dCas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post-irradiation. Targeting the Radiation and Desiccation Response Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPR interference tool developed in this study will facilitate the study of essential genes, hypothetical genes, and cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further, the tool can be extended for implementing high-throughput approaches in such studies. IMPORTANCE Deinococcus radiodurans is a microbe that exhibits a very high degree of radiation resistance. In addition, it is also identified as an organism of industrial importance. We report the development of a gene-knockdown system in this organism by engineering a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-Cascade system. We used this system to silence an assayable acid phosphatase gene, phoN to 10% of its activity. The study further shows the application of the Cascade system to target an essential gene ssb, that caused poor recovery from radiation. We demonstrate the utility of CRISPR-Cascade to study the role of a regulatory cis-element in radiation response as well as for multi-gene silencing. This easy-to-implement CRISPR interference system would provide an effective tool for better understanding of complex phenomena such as radiation response in D. radiodurans and may also enhance the potential of this microbe for industrial application.},
}
@article {pmid37671018,
year = {2023},
author = {Nievergelt, AP and Diener, DR and Bogdanova, A and Brown, T and Pigino, G},
title = {Efficient precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.},
journal = {Cell reports methods},
volume = {3},
number = {8},
pages = {100562},
pmid = {37671018},
issn = {2667-2375},
mesh = {*Chlamydomonas reinhardtii ; CRISPR-Cas Systems ; *Chlamydomonas ; Culture ; Electroporation ; },
abstract = {CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed.},
}
@article {pmid37669948,
year = {2023},
author = {Rananaware, SR and Vesco, EK and Shoemaker, GM and Anekar, SS and Sandoval, LSW and Meister, KS and Macaluso, NC and Nguyen, LT and Jain, PK},
title = {Programmable RNA detection with CRISPR-Cas12a.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5409},
pmid = {37669948},
issn = {2041-1723},
support = {R21 AI168795/AI/NIAID NIH HHS/United States ; R35 GM147788/GM/NIGMS NIH HHS/United States ; R21 AI156321/AI/NIAID NIH HHS/United States ; },
mesh = {*RNA ; *CRISPR-Cas Systems ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems ; Recombination, Genetic ; },
abstract = {Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.},
}
@article {pmid37668921,
year = {2024},
author = {Shiraki, T and Kawakami, K},
title = {Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2707},
number = {},
pages = {305-318},
pmid = {37668921},
issn = {1940-6029},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems ; *Zebrafish/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics ; Frameshift Mutation ; },
abstract = {The controlled expression of Cas9 and/or sgRNA in transgenic zebrafish made it possible to knock out a gene in a spatially and/or temporally controlled manner. This transgenic approach can be more useful if multiple sgRNAs are efficiently expressed since we can improve the biallelic frame-shift mutation rate and circumvent the functional redundancy of genes and genetic compensation. We developed the tRNA-based system to express multiple functional sgRNAs from a single transcript in zebrafish and found that it is applicable to the transgenic expression of multiple sgRNAs. In this chapter, we describe a procedure for the generation of plasmids containing multiple sgRNAs flanked by tRNAs and a method to induce multiple CRISPR/Cas9-mediated genome modifications in transgenic zebrafish.},
}
@article {pmid37668219,
year = {2023},
author = {Kang, YJ and Kim, JH and Lee, GH and Ha, HJ and Park, YH and Hong, E and Park, HH},
title = {The structure of AcrIC9 revealing the putative inhibitory mechanism of AcrIC9 against the type IC CRISPR-Cas system.},
journal = {IUCrJ},
volume = {10},
number = {Pt 5},
pages = {624-634},
pmid = {37668219},
issn = {2052-2525},
support = {2018R1A4A1023822//National Research Foundation of Korea/ ; 2021R1A2C3003331//National Research Foundation of Korea/ ; 20020237//Ministry of Trade, Industry and Energy, Korea Evaluation Institute of Industrial Technology/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Bacteriophages ; Polymers ; Protein Domains ; *Rhodobacter capsulatus/genetics ; },
abstract = {CRISPR-Cas systems are known to be part of the bacterial adaptive immune system that provides resistance against intruders such as viruses, phages and other mobile genetic elements. To combat this bacterial defense mechanism, phages encode inhibitors called Acrs (anti-CRISPR proteins) that can suppress them. AcrIC9 is the most recently identified member of the AcrIC family that inhibits the type IC CRISPR-Cas system. Here, the crystal structure of AcrIC9 from Rhodobacter capsulatus is reported, which comprises a novel fold made of three central antiparallel β-strands surrounded by three α-helixes, a structure that has not been detected before. It is also shown that AcrIC9 can form a dimer via disulfide bonds generated by the Cys69 residue. Finally, it is revealed that AcrIC9 directly binds to the type IC cascade. Analysis and comparison of its structure with structural homologs indicate that AcrIC9 belongs to DNA-mimic Acrs that directly bind to the cascade complex and hinder the target DNA from binding to the cascade.},
}
@article {pmid37666438,
year = {2023},
author = {Misra, G and Qaisar, S and Singh, P},
title = {CRISPR based therapeutic targeting of signaling pathways in breast cancer.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {},
number = {},
pages = {166872},
doi = {10.1016/j.bbadis.2023.166872},
pmid = {37666438},
issn = {1879-260X},
abstract = {Breast cancer represents the prevailing malignancy in females and contributing towards mortality on a global scale. There are innumerable anomalous genes that are responsible in the multi factorial carcinogenesis pathway. Although several disease-causing mutations have been detected, therapy frequently focuses on attenuating the manifestation of the disease rather than harmonizing the mutation in the target area. However, the emergence of the CRISPR-Cas system as a DNA modifying tool has proved to be a promising gene editing tool for breast cancer therapeutics. Henceforth, in this review, we have discussed different signaling pathways that have been targeted by the CRISPR-Cas system and explored their role in breast cancer therapies. We have also tried to comprehensively cover CRISPR-Cas9 mediated gene knockout in breast cancer. In this review many alterations in breast cancer cells that affect cell signaling pathways have also been described. This review will be a valuable addition to providing comprehensive knowledge of CRISPR-Cas mediated therapeutic targeting in breast cancer.},
}
@article {pmid37666372,
year = {2023},
author = {Ahmar, S and Hensel, G and Gruszka, D},
title = {CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals - current status, improvements, and perspectives.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108248},
doi = {10.1016/j.biotechadv.2023.108248},
pmid = {37666372},
issn = {1873-1899},
abstract = {Cereal crops, including triticeae species (barley, wheat, rye), as well as edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop varieties were developed mainly for optimal environmental conditions. Thus, it has become evident that taking into account the ongoing climate changes, currently a priority should be given to developing new stress-tolerant cereal cultivars. It is necessary to enhance the accuracy of methods and time required to generate new cereal cultivars with the desired features to adapt to climate change and keep up with the world population expansion. The CRISPR/Cas9 system has been developed as a powerful and versatile genome editing tool to achieve desirable traits, such as developing high-yielding, stress-tolerant, and disease-resistant transgene-free lines in major cereals. Despite recent advances, the CRISPR/Cas9 application in cereals faces several challenges, including a significant amount of time required to develop transgene-free lines, laboriousness, and a limited number of genotypes that may be used for the transformation and in vitro regeneration. Additionally, developing elite lines through genome editing has been restricted in many countries, especially Europe and New Zealand, due to a lack of flexibility in GMO regulations. This review provides a comprehensive update to researchers interested in improving cereals using gene-editing technologies, such as CRISPR/Cas9. We will review some critical and recent studies on crop improvements and their contributing factors to superior cereals through gene-editing technologies.},
}
@article {pmid37651830,
year = {2023},
author = {Rivera-Sánchez, P and Søndergaard, L and Wathikthinnakon, M and B D Magnusson, H and Frederiksen, HR and Aabæk Hammer, F and Taleb, R and Christian Cassidy, C and Tranholm Bruun, M and Tümer, Z and Holst, B and Brasch-Andersen, C and Møller, RS and Freude, K and Chandrasekaran, A},
title = {Generation of eight hiPSCs lines from two pathogenic variants in CACNA1A using the CRISPR-Cas9 gene editing technology.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103193},
doi = {10.1016/j.scr.2023.103193},
pmid = {37651830},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Induced Pluripotent Stem Cells ; Calcium ; Cell Differentiation ; Calcium Channels ; },
abstract = {Developmental and epileptic encephalopathies (DEEs) are rare severe neurodevelopmental disorders with a cumulative incidence of 1:6.000 live births. Many epileptic conditions arise from single nucleotide variants in CACNA1A (calcium voltage-gated channel subunit alpha1 A), encoding the CaV2.1 calcium channel subunit. Human induced pluripotent stem cells (hiPSCs) are an optimal choice for modeling DEEs, as they can be differentiated in vitro into diverse neuronal subpopulations. Here, we report the generation of hiPSC lines with two pathogenic CACNA1A variants c.1767C > T, p. (Arg589Cys), referred to as R589C and c. 2139G > A, p.(Ala713Thr), referred to as A713T, previously associated with epilepsy. The variants were introduced into a hiPSC line from a healthy individual via CRISPR-Cas9 gene editing technology.},
}
@article {pmid37540965,
year = {2023},
author = {Schuurmans, IME and Wu, KM and van Karnebeek, CDM and Nadif Kasri, N and Garanto, A},
title = {Generation of an induced pluripotent stem cell line carrying biallelic deletions (SCTCi019-B) in ALDH7A1 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103173},
doi = {10.1016/j.scr.2023.103173},
pmid = {37540965},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Aldehyde Dehydrogenase/genetics/metabolism ; *Epilepsy/genetics ; Mutation ; },
abstract = {Biallelic pathogenic variants in ALDH7A1 are associated with pyridoxine-dependent epilepsy (PDE). ALDH7A1 encodes for the third enzyme of the lysine catabolism pathway. In this study a human isogenic ALDH7A1 knock-out iPSC line was created using CRISPR/Cas9 technology. One clone (SCTCi019-B) with biallelic deletions in ALDH7A1 was obtained and fully characterized, showing expression of pluripotency markers, a normal karyotype and no off-targets. Human-based models derived from this iPSC line will contribute to gain insights in the molecular mechanism of disease underlying PDE.},
}
@article {pmid37495752,
year = {2023},
author = {Rosello, M and Serafini, M and Concordet, JP and Del Bene, F},
title = {Precise mutagenesis in zebrafish using cytosine base editors.},
journal = {Nature protocols},
volume = {18},
number = {9},
pages = {2794-2813},
pmid = {37495752},
issn = {1750-2799},
support = {ANR-18-IAHU-0001//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-20-CE17-0020-02//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-11-INBS-0014-TEFOR//Agence Nationale de la Recherche (French National Research Agency)/ ; MND202003011485//Fondation pour la Recherche M&#xe9;dicale (Foundation for Medical Research in France)/ ; ECO20170637481//Fondation pour la Recherche M&#xe9;dicale (Foundation for Medical Research in France)/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Cytosine ; Mutagenesis ; },
abstract = {Base editing is a powerful CRISPR-based technology for introducing precise substitutions into the genome. This technology greatly advances mutagenesis possibilities in vivo, particularly in zebrafish, for which the generation of precise point mutations is still challenging. Zebrafish have emerged as an important model for genetic studies and in vivo disease modeling. With the development of different base editor variants that recognize protospacer-adjacent motifs (PAMs) other than the classical 5'-NGG-3' PAM, it is now possible to design and test several guide RNAs to find the most efficient way to precisely introduce the desired substitution. Here, we describe the experimental design strategies and protocols for cytosine base editing in zebrafish, from guide RNA design and selection of base editor variants to generation of the zebrafish mutant line carrying the substitution of interest. By using co-selection by introducing a loss-of-function mutation in genes necessary for the formation of pigments, injected embryos with highly efficient base editing can be directly analyzed to determine the phenotypic impact of the targeted substitution. The generation of mutant embryos after base editor injections in zebrafish can be completed within 2 weeks.},
}
@article {pmid37481965,
year = {2023},
author = {Jeong, J and Lee, D and Park, BC and Lee, TH and Park, SW},
title = {Establishment of a TNFRSF11B knock-out human induced pluripotent stem cell line (KSCBi002-B-2) via CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103167},
doi = {10.1016/j.scr.2023.103167},
pmid = {37481965},
issn = {1876-7753},
mesh = {Humans ; Osteoprotegerin/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Osteitis Deformans/genetics/metabolism ; },
abstract = {A TNFRSF11B (TNF Receptor Superfamily Member 11b) gene encodes a soluble decoy receptor, osteoprotegerin (OPG), which has a key role in repressing osteoclast differentiation. In this report, we generated a biallelic knock-out hiPSC line for the TNFRSF11B gene via CRISPR/Cas9. When TNFRSF11B Knock-out hiPSCs were differentiated into mesenchymal progenitor cells (MPCs), the expression level of OPG was significantly decreased compared to normal hiPSC-derived MPCs. This knock-out hiPSCs will provide a chance to study Paget disease of bone 5 (juvenile Paget disease).},
}
@article {pmid37474806,
year = {2023},
author = {Riesenberg, S and Kanis, P and Macak, D and Wollny, D and Düsterhöft, D and Kowalewski, J and Helmbrecht, N and Maricic, T and Pääbo, S},
title = {Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.},
journal = {Nature methods},
volume = {20},
number = {9},
pages = {1388-1399},
pmid = {37474806},
issn = {1548-7105},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA ; },
abstract = {Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.},
}
@article {pmid37473460,
year = {2023},
author = {Ben Yacoub, T and Letellier, C and Wohlschlegel, J and Condroyer, C and Slembrouck-Brec, A and Goureau, O and Zeitz, C and Audo, I},
title = {Generation of gene corrected human isogenic iPSC lines (IDVi003-A_CR13, IDVi003-A_CR21, IDVi003-A_CR24) from an inherited retinal dystrophy patient-derived IPSC line ITM2B-5286-3 (IDVi003-A) carrying the ITM2B c.782A &gt; C variant using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103166},
doi = {10.1016/j.scr.2023.103166},
pmid = {37473460},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Retinal Dystrophies/genetics/metabolism ; Cell Differentiation ; Mutation ; Adaptor Proteins, Signal Transducing/genetics ; },
abstract = {The ITM2B-related retinal dystrophy (ITM2B-RD) was identified within patients carrying the autosomal dominant variant [c.782A > C, p.(Glu261Ala)] in ITM2B from whom induced pluripotent stem cell (IPSC) lines were previously generated. Here, we report the generation of three isogenic control iPSC lines from the derived affected subject cell line (ITM2B-5286-3) using CRISPR/Cas9 engineering. The three generated lines express pluripotency markers, can be differentiated into the three germ layers and present a normal karyotype. The generated iPSC lines can be used to study the implications of ITM2B-RD variant in vitro.},
}
@article {pmid37429070,
year = {2023},
author = {Lu, F and Gao, Y and Li, E},
title = {Generation of a FLNA knockout hESC line (WAe009-A-P) to model cardiac valvular dysplasia using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103162},
doi = {10.1016/j.scr.2023.103162},
pmid = {37429070},
issn = {1876-7753},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Cell Line ; Mutation ; Heart Valves ; },
abstract = {The FLNA gene encodes the cytoskeletal protein filamin A which plays a key role in the structure and function of the cardiac valves. Truncating FLNA mutations are associated with cardiac valvular dysplasia. To further understand the exact role of FLNA in this disease, we have generated a human FLNA knockout cell line from H9 using CRISPR/Cas9 technology in this study. This cell line WAe009-A-P has a 2 bp deletion in the exon 2 of FLNA gene which resulted in a frameshift in the translation of FLNA and no FLNA protein was detected in this cell line. Moreover, WAe009-A-P also expressed pluripotency markers, had a normal female karyotype (46XX) and maintained the ability to differentiate into the three germ layers in vitro.},
}
@article {pmid37406498,
year = {2023},
author = {Alowaysi, M and Al-Shehri, M and Baadhaim, M and AlZahrani, H and Aboalola, D and Daghestani, M and Hashem, H and Aljahdali, R and Salem, R and Alharbi, A and Muharraq, M and Alghamdi, K and Alsobiy, F and Zia, A and Lehmann, R and Tegner, J and Alsayegh, K},
title = {Generation of myoglobin (MB)-knockout human embryonic stem cell (hESC) line (KAIMRCe002-A-1S) using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103158},
doi = {10.1016/j.scr.2023.103158},
pmid = {37406498},
issn = {1876-7753},
mesh = {Humans ; *Myoglobin/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Technology ; },
abstract = {Myoglobin (MB) is a cytoplasmic hemoprotein that is predominantly expressed in the heart and oxidative myofibers of skeletal muscle. It has been demonstrated that MB binds to oxygen and promotes its diffusion for energy production in the mitochondria. Recently, MB was found to be expressed in different forms of malignant tumors and cancer cell lines. Further studies using gene disruption technology will enhance the understanding of MB's role in human cardiovascular biology and cancers. Here, we describe the generation of a homozygous MB knockout in human embryonic stem cells (hESC-MB[-/-]) via CRISPR/Cas9 to study MB function in human biology and diseases.},
}
@article {pmid37393721,
year = {2023},
author = {Amin, G and Castañeda, SL and Zabalegui, F and Belli, C and Atorrasagasti, C and Miriuka, SG and Moro, LN},
title = {Generation of two edited iPSCs lines by CRISPR/Cas9 with point mutations in PKP2 gene for arrhythmogenic cardiomyopathy in vitro modeling.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103157},
doi = {10.1016/j.scr.2023.103157},
pmid = {37393721},
issn = {1876-7753},
mesh = {Humans ; Point Mutation ; *Induced Pluripotent Stem Cells/metabolism ; *Arrhythmogenic Right Ventricular Dysplasia/genetics ; CRISPR-Cas Systems/genetics ; *Cardiomyopathies/genetics ; Mutation/genetics ; Plakophilins/genetics/metabolism ; },
abstract = {The arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease characterized by the progressive replacement of contractile myocardium by fibro-fatty adipose tissue, that generates ventricular arrhythmias and sudden death in patients. The ACM has a genetic origin with alterations in desmosomal genes with the most commonly mutated being the PKP2 gene. We generated two CRISPR/Cas9 edited iPSCs lines, one iPSC line with a point mutation in PKP2 reported in patients with ACM and another iPSC line with a premature stop codon to knock-out the same gene.},
}
@article {pmid37343428,
year = {2023},
author = {Thongsin, N and Suwanpitak, S and Wattanapanitch, M},
title = {CRISPR-Cas9-mediated disruption of B2M and CIITA genes eliminates HLA class I and II expression in human induced pluripotent stem cells (MUSIi001-A-2).},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103138},
doi = {10.1016/j.scr.2023.103138},
pmid = {37343428},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; Graft Rejection ; Cell Line ; },
abstract = {Cell-based therapy offers great promise for treating degenerative diseases. Although autologous cell-based therapy is ideal, it may be impractical due to the high manufacturing cost and long production time. Allogeneic cell-based therapy offers a more cost-effective alternative; however, the risk of graft rejection is a major concern. Here, we generated HLA class-I and -II null iPSC line by knocking out CIITA gene in the B2M-knockout MUSIi001-A-1 cell line using CRISPR/Cas9 system. The MUSIi001-A-2 line provides a valuable model for studying immunological responses against allogeneic T cells and serves as a prototype for developing specific cell types for future cell-based therapy.},
}
@article {pmid37331109,
year = {2023},
author = {Boissart, C and Chatrousse, L and Poullion, T and El-Kassar, L and Giraud-Triboult, K and Benchoua, A},
title = {CRISPR/Cas9-mediated generation of human embryonic stem cell sub-lines with HPRT1 gene knockout to model Lesch Nyhan disease.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103144},
doi = {10.1016/j.scr.2023.103144},
pmid = {37331109},
issn = {1876-7753},
mesh = {Humans ; Male ; *Lesch-Nyhan Syndrome/genetics/metabolism ; Hypoxanthine Phosphoribosyltransferase/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Human Embryonic Stem Cells/metabolism ; },
abstract = {Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder.},
}
@article {pmid37327621,
year = {2023},
author = {Jiang, M and Tang, C and Luo, X and Zhou, X and Chen, M and Chi, Y and Lai, L and Zou, Q},
title = {Generation of a homozygous RANGRF knockout hiPSC line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103136},
doi = {10.1016/j.scr.2023.103136},
pmid = {37327621},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Homozygote ; Mutation ; Gene Editing ; },
abstract = {The RAN Guanine Nucleotide Release Factor (RANGRF) gene encodes the protein MOG1, which binds to Nav1.5 and facilitates its transport to the cell membrane. Nav1.5 mutations have been linked to various cardiac arrhythmias and cardiomyopathy. To investigate the role of RANGRF in this process, we utilized the CRISPR/Cas9 gene editing system to generate a homozygous RANGRF knockout hiPSC line. The availability of the cell line will prove to be an invaluable asset in the study of disease mechanisms and the testing of gene therapies for cardiomyopathy.},
}
@article {pmid36997812,
year = {2023},
author = {Paenkaew, S and Jaito, N and Pradit, W and Chomdej, S and Nganvongpanit, K and Siengdee, P and Buddhachat, K},
title = {RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.},
journal = {Veterinary research communications},
volume = {47},
number = {3},
pages = {1601-1613},
pmid = {36997812},
issn = {1573-7446},
support = {N42A650332//National Research Council of Thailand/ ; R2565A060//Faculty of Science, Naresuan University, Thailand/ ; R2566C051//Global and Frontier Research University Fund, Naresuan University/ ; },
mesh = {Dogs ; Animals ; Ehrlichia canis/genetics ; *Anaplasmosis/diagnosis/epidemiology/genetics ; CRISPR-Cas Systems ; Recombinases/genetics ; Thailand ; RNA, Ribosomal, 16S/genetics ; *Ehrlichiosis/diagnosis/veterinary/genetics ; *Dog Diseases/diagnosis/epidemiology ; },
abstract = {Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance.},
}
@article {pmid37663930,
year = {2023},
author = {Tang, D and Jia, T and Luo, Y and Mou, B and Cheng, J and Qi, S and Yao, S and Su, Z and Yu, Y and Chen, Q},
title = {DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.},
journal = {Innovation (Cambridge (Mass.))},
volume = {4},
number = {5},
pages = {100495},
pmid = {37663930},
issn = {2666-6758},
abstract = {In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.},
}
@article {pmid37662968,
year = {2023},
author = {Han, H and Yang, Y and Jiao, Y and Qi, H and Han, Z and Wang, L and Dong, L and Tian, J and Vanhaesebroeck, B and Li, X and Liu, J and Ma, G and Lei, H},
title = {Leverage of nuclease-deficient CasX for preventing pathological angiogenesis.},
journal = {Molecular therapy. Nucleic acids},
volume = {33},
number = {},
pages = {738-748},
pmid = {37662968},
issn = {2162-2531},
abstract = {Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.},
}
@article {pmid37662220,
year = {2023},
author = {Szekely, O and Rangadurai, AK and Gu, S and Manghrani, A and Guseva, S and Al-Hashimi, HM},
title = {NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like G·T/U in RNA:DNA hybrids: Implications for the fidelity of transcription and CRISPR/Cas9 gene editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.08.24.554670},
pmid = {37662220},
abstract = {Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G•T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R 1ρ experiments, we show that dG•rU and dT•rG mismatches in two RNA:DNA hybrids transiently form tautomeric (G [enol] •T/U ⇄G•T [enol] /U [enol]) and anionic (G•T [-] /U [-]) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG•rU [-] formed with a ten-fold higher propensity relative to dT [-] •rG and dG•dT [-] and this could be attributed to the lower pK a (Δ pK a ∼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G•T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G•U versus G•T, and indicate that anionic Watson-Crick-like G•U [-] could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.},
}
@article {pmid37624706,
year = {2023},
author = {Wang, Y and Xia, X and Wu, M and Sun, Q and Zhang, W and Qiu, Y and Deng, R and Luo, A},
title = {Species-Level Monitoring of Key Bacteria in Fermentation Processes Using Single-Nucleotide Resolved Nucleic Acid Assays Based on CRISPR/Cas12.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {35},
pages = {13147-13155},
doi = {10.1021/acs.jafc.3c04775},
pmid = {37624706},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; Fermentation ; Bacteria ; *Bacillus ; *Bacillus amyloliquefaciens ; Nucleotides ; *Nucleic Acids ; },
abstract = {Microorganisms can determine the flavor and quality of fermented food, such as Baijiu, which is produced via Daqu fermentation. Therefore, monitoring key microorganisms during fermentation is important for ensuring high-quality fermented food. Here, we report a single-nucleotide resolved nucleic acid assay based on the CRISPR/Cas12 system, enabling the quantification of Bacillus amyloliquefaciens, a key microorganism in Daqu fermentation at the species level. The assay employs an amplification-refractory mutation system derived from PCR to analyze minor genetic differences between different Bacillus species. The utilization of CRISPR/Cas12 further guaranties the specificity of identifying the PCR amplicon and enables the quantification of Bacillus amyloliquefaciens via end-measurement fluorescence. Compared to conventional qPCR, the assay allows for species-level detection of bacteria, thus enabling the precise detection of the Bacillus strain that yields high-level 2,3,5,6-tetramethylpyrazine. The assay promises the precise monitoring of bacterial growth and contribution to flavor during Daqu fermentation, thus facilitating fermented food quality control.},
}
@article {pmid37579713,
year = {2023},
author = {Wu, Z and Sun, DW and Pu, H},
title = {CRISPR/Cas12a and G-quadruplex DNAzyme-driven multimodal biosensor for visual detection of Aflatoxin B1.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {302},
number = {},
pages = {123121},
doi = {10.1016/j.saa.2023.123121},
pmid = {37579713},
issn = {1873-3557},
mesh = {Humans ; Aflatoxin B1/analysis ; DNA, Complementary/chemistry/genetics ; *DNA, Catalytic ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; Food Contamination/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; Coloring Agents ; },
abstract = {Aflatoxin B1 (AFB1) contamination severely threatens human and animal health, it is thus critical to construct a strategy for its rapid, accurate, and visual detection. Herein, a multimodal biosensor was proposed based on CRISPR/Cas12a cleaved G-quadruplex (G4) for AFB1 detection. Briefly, specific binding of AFB1 to the aptamer occupied the binding site of the complementary DNA (cDNA), and cDNA then activated Cas12a to cleave G4 into fragments. Meanwhile, the intact G4-DNAzyme could catalyze 3, 3', 5, 5'-tetramethylbenzidine (TMB) to form colourimetric/SERS/fluorescent signal-enhanced TMBox, and the yellow solution produced by TMBox under acidic conditions could be integrated with a smartphone application for visual detection. The colourimetric/SERS/fluorescent biosensor yielded detection limits of 0.85, 0.79, and 1.65 pg·mL[-1], respectively, and was applied for detecting AFB1 in peanut, maize, and badam samples. The method is suitable for visual detection in naturally contaminated peanut samples and has prospective applications in the food industry.},
}
@article {pmid37662134,
year = {2023},
author = {Kannadasan, AB and Sumantran, VN and Vaidyanathan, R},
title = {A Global Comprehensive Study of the Distribution of Type I-E and Type I-E* CRISPR-Cas Systems in Klebsiella pneumoniae.},
journal = {Indian journal of community medicine : official publication of Indian Association of Preventive &amp; Social Medicine},
volume = {48},
number = {4},
pages = {567-572},
pmid = {37662134},
issn = {0970-0218},
abstract = {BACKGROUND: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems are the short DNA sequences and RNA-dependent nuclease involved in the adaptive immunity in bacteria and archaea. The type of CRISPR-Cas system influences antibiotic susceptibility in Klebsiella pneumoniae. Here, our objective was to study the diversity of CRISPR-Cas system in the genome of K. pneumoniae from the available whole genome sequencing (WGS) data.<br><br>MATERIAL AND METHODS: We identified the CRISPR-Cas systems of K. pneumoniae using the CRISPR-CasFinder database. The complete genome sequence and its submission details were obtained from the National Center for Biotechnology Information (NCBI) database.<br><br>RESULTS: A total of 1607 K. pneumoniae whole genome sequences were analyzed. The major contributors of WGS data of K. pneumoniae were China (26.6%), United States (21.5%), Australia (10%), South Korea (8%), India (5.5%), and United Kingdom (4.9%). Out of 1607 genomes analyzed, almost one-fourth were CRISPR-Cas positive (403/1607) and three-fourth were CRISPR-Cas negative (1204/1607). Among CRISPR-Cas positive strains, 220 belonged to type I-E* and 183 were type I-E. Furthermore, type I-E* CRISPR-Cas systems were significantly higher in Asia (P < 0.001), whereas type I-E were significantly higher in Europe (P < 0.01). Among countries, typically, type I-E* strains were found to be higher in China (P < 0.01) and India (P < 0.01), whereas type I-E strains were higher in Germany (P < 0.01).<br><br>CONCLUSION: Hence, it is important to know the type of CRISPR-Cas systems in K. pneumoniae strains across the countries and it can help to understand the diversity of CRISPR-Cas systems worldwide.},
}
@article {pmid37662004,
year = {2023},
author = {Qian, Y and Zhou, D and Li, M and Zhao, Y and Liu, H and Yang, L and Ying, Z and Huang, G},
title = {Application of CRISPR-Cas system in the diagnosis and therapy of ESKAPE infections.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1223696},
pmid = {37662004},
issn = {2235-2988},
mesh = {Humans ; CRISPR-Cas Systems ; *Acinetobacter baumannii/genetics ; Anti-Bacterial Agents/therapeutic use ; *Cross Infection/diagnosis ; *Enterococcus faecium ; },
abstract = {Antimicrobial-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens represent a global threat to human health. ESKAPE pathogens are the most common opportunistic pathogens in nosocomial infections, and a considerable number of their clinical isolates are not susceptible to conventional antimicrobial therapy. Therefore, innovative therapeutic strategies that can effectively deal with ESKAPE pathogens will bring huge social and economic benefits and ease the suffering of tens of thousands of patients. Among these strategies, CRISPR (clustered regularly interspaced short palindromic repeats) system has received extra attention due to its high specificity. Regrettably, there is currently no direct CRISPR-system-based anti-infective treatment. This paper reviews the applications of CRISPR-Cas system in the study of ESKAPE pathogens, aiming to provide directions for the research of ideal new drugs and provide a reference for solving a series of problems caused by multidrug-resistant bacteria (MDR) in the post-antibiotic era. However, most research is still far from clinical application.},
}
@article {pmid37659410,
year = {2023},
author = {Liu, C and Wang, R and Li, J and Cheng, F and Shu, X and Zhao, H and Xue, Q and Yu, H and Wu, A and Wang, L and Hu, S and Zhang, Y and Yang, J and Xiang, H and Li, M},
title = {Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2023.08.005},
pmid = {37659410},
issn = {1934-6069},
abstract = {CRISPR RNAs (crRNAs) and Cas proteins work together to provide prokaryotes with adaptive immunity against genetic invaders like bacteriophages and plasmids. However, the coordination of crRNA production and cas expression remains poorly understood. Here, we demonstrate that widespread modulatory mini-CRISPRs encode cas-regulating RNAs (CreRs) that mediate autorepression of type I-B, I-E, and V-A Cas proteins, based on their limited complementarity to cas promoters. This autorepression not only reduces autoimmune risks but also responds to changes in the abundance of canonical crRNAs that compete with CreR for Cas proteins. Furthermore, the CreR-guided autorepression of Cas proteins can be alleviated or even subverted by diverse bacteriophage anti-CRISPR (Acr) proteins that inhibit Cas effectors, which, in turn, promotes the generation of new Cas proteins. Our findings reveal a general RNA-guided autorepression paradigm for diverse Cas effectors, shedding light on the intricate self-coordination of CRISPR-Cas and its transcriptional counterstrategy against Acr proteins.},
}
@article {pmid37657665,
year = {2023},
author = {Redhead, C and Taye, N and Hubmacher, D},
title = {En route towards a personalized medicine approach: Innovative therapeutic modalities for connective tissue disorders.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.matbio.2023.08.005},
pmid = {37657665},
issn = {1569-1802},
abstract = {Connective tissue disorders can be caused by pathogenic variants (mutations) in genes encoding extracellular matrix (ECM) proteins. Such disorders typically manifest during development or postnatal growth and result in significant morbidity and mortality. The development of curative treatments for connective tissue disorders is hampered in part by the inability of many mature connective tissues to efficiently regenerate. To be most effective, therapeutic strategies designed to preserve or restore tissue function will likely need to be initiated during phases of significant endogenous connective tissue remodeling and organ sculpting postnatally and directly target the underlying ECM protein mutations. With recent advances in whole exome sequencing, in-vitro and in-vivo disease modeling, and the development of mutation-specific molecular therapeutic modalities, it is now feasible to directly correct disease-causing mutations underlying connective tissue disorders and ameliorate their pathogenic consequences. These technological advances may lead to potentially curative personalized medicine approaches for connective tissue disorders that have previously been considered incurable. In this review, we highlight innovative therapeutic modalities including gene replacement, exon skipping, DNA/mRNA editing, and pharmacological approaches that were used to preserve or restore tissue function in the context of connective tissue disorders. Inherent to a successful application of these approaches is the need to further the understanding of mechanisms that regulate ECM formation and homeostasis, and to decipher how individual mutations in ECM proteins compromise ECM and connective tissue development and function.},
}
@article {pmid37611446,
year = {2023},
author = {Lin, C and Chen, F and Huang, D and Li, W and He, C and Tang, Y and Li, X and Liu, C and Han, L and Yang, Y and Zhu, Y and Chen, R and Shi, Y and Xia, C and Yan, Z and Du, H and Huang, L},
title = {A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.},
journal = {Biosensors &amp; bioelectronics},
volume = {239},
number = {},
pages = {115609},
doi = {10.1016/j.bios.2023.115609},
pmid = {37611446},
issn = {1873-4235},
mesh = {Humans ; CRISPR-Cas Systems ; *COVID-19/diagnosis ; SARS-CoV-2 ; *Biosensing Techniques ; DNA ; *RNA Viruses ; },
abstract = {Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/μL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/μL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.},
}
@article {pmid37597500,
year = {2023},
author = {Hu, H and Dong, K and Yan, B and Mu, Y and Liao, Y and Zhang, L and Guo, S and Xiao, X and Wang, X},
title = {Highly-sensitive and homogenous detection of 8-oxoguanine based DNA oxidative damage by a CRISPR-enhanced structure-switching aptamer assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {239},
number = {},
pages = {115588},
doi = {10.1016/j.bios.2023.115588},
pmid = {37597500},
issn = {1873-4235},
mesh = {Male ; Humans ; *Biosensing Techniques ; Semen ; Biological Assay ; Oligonucleotides ; Oxidative Stress ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {8-oxoguanine (8-oxoG) based DNA damage is the most common type of DNA damage which greatly affect gene expression. Therefore, accurate quantification of 8-oxoG based DNA damage is of high clinical significance. However, current methods for 8-oxoG detection struggle to balance convenience, low cost, and sensitivity. Herein, we have proposed and investigated the shortened crRNA mode of CRISPR-Cas12a system and greatly enhanced its signal-to-noise ratio. Taking advantages of the shortened crRNA mode, we further developed a CRISPR-enhanced structure-switching aptamer assay (CESA) for 8-oxoG. The analytical performance of CESA was thoroughly investigated via detecting free 8-oxoG and 8-oxoG on gDNA. The CESA displayed impressive sensitivity for free 8-oxoG, with detection and quantification limits of 32.3 pM and 0.107 nM. These limits modestly rose to 64.5 pM and 0.215 nM when examining 8-oxoG on gDNA. To demonstrate the clinical practicability and significance of the CESA system, we further applied it to measuring 8-oxoG levels in 7 plasma samples (Cervical carcinoma, 11.87 ± 0.69 nM VS. Healthy control, 2.66 ± 0.42 nM), 24 seminal plasma samples (Asthenospermia, 22.29 ± 7.48 nM VS. Normal sperm, 9.75 ± 3.59 nM), 10 breast-tissue gDNA samples (Breast cancer, 2.77 ± 0.63 nM/μg VS. Healthy control, 0.41 ± 0.09 nM/μg), and 24 sperm gDNA samples (Asthenospermia, 28.62 ± 4.84 VS. Normal sperm, 16.67 ± 3.31). This work not only proposes a novel design paradigm of shortened crRNA for developing CRISPR-Cas12a based biosensors but also offers a powerful tool for detecting 8-oxoG based DNA damage.},
}
@article {pmid37589291,
year = {2023},
author = {Johnson, CJ and Kulkarni, A and Buxton, WJ and Hui, TY and Kayastha, A and Khoja, AA and Leandre, J and Mehta, VV and Ostrowski, L and Pareizs, EG and Scotto, RL and Vargas, V and Vellingiri, RM and Verzino, G and Vohra, R and Wakade, SC and Winkeljohn, VM and Winkeljohn, VM and Rotterman, TM and Stolfi, A},
title = {Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.},
journal = {Biology open},
volume = {12},
number = {9},
pages = {},
doi = {10.1242/bio.060002},
pmid = {37589291},
issn = {2046-6390},
support = {K99 NS126576/NS/NINDS NIH HHS/United States ; R01 GM143326/GM/NIGMS NIH HHS/United States ; R01GM143326/NH/NIH HHS/United States ; K99NS126576/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Ciona ; Larva/genetics ; Metamorphosis, Biological/genetics ; Sensory Receptor Cells ; },
abstract = {Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate 'capstone' research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.},
}
@article {pmid37552860,
year = {2023},
author = {Li, Y and Cooper, BH and Liu, Y and Wu, D and Zhang, X and Rohs, R and Qin, PZ},
title = {CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid.},
journal = {Biochemistry},
volume = {62},
number = {17},
pages = {2541-2548},
doi = {10.1021/acs.biochem.3c00250},
pmid = {37552860},
issn = {1520-4995},
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; Nucleotides ; DNA/genetics ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (kcat) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between kcat and the PAM+(17-20) DNA duplex dissociation free energy (ΔGNN(17-20)[0]), with sequences having smaller ΔGNN(17-20)[0] showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.},
}
@article {pmid37356052,
year = {2023},
author = {Zheng, R and Zhang, L and Parvin, R and Su, L and Chi, J and Shi, K and Ye, F and Huang, X},
title = {Progress and Perspective of CRISPR-Cas9 Technology in Translational Medicine.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {25},
pages = {e2300195},
pmid = {37356052},
issn = {2198-3844},
support = {2022C03007//Key Research and Development Plan of Zhejiang Province "Spearhead"/ ; 2020ZY0036//Wenzhou Major Science and Technology Innovation Tackling Project/ ; LGF22C100003//Zhejiang Provincial Natural Science Foundation of China/ ; WIUCASQD2021012//Wenzhou high-level innovation team : Development and application team of functional liver cancer-on-a-chip program of Wenzhou Institute, University of Chinese Academy of Sciences/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Translational Science, Biomedical ; Gene Editing/methods ; Genetic Therapy/methods ; Genomics ; },
abstract = {Translational medicine aims to improve human health by exploring potential treatment methods developed during basic scientific research and applying them to the treatment of patients in clinical settings. The advanced perceptions of gene functions have remarkably revolutionized clinical treatment strategies for target agents. However, the progress in gene editing therapy has been hindered due to the severe off-target effects and limited editing sites. Fortunately, the development in the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system has renewed hope for gene therapy field. The CRISPR-Cas9 system can fulfill various simple or complex purposes, including gene knockout, knock-in, activation, interference, base editing, and sequence detection. Accordingly, the CRISPR-Cas9 system is adaptable to translational medicine, which calls for the alteration of genomic sequences. This review aims to present the latest CRISPR-Cas9 technology achievements and prospect to translational medicine advances. The principle and characterization of the CRISPR-Cas9 system are firstly introduced. The authors then focus on recent pre-clinical and clinical research directions, including the construction of disease models, disease-related gene screening and regulation, and disease treatment and diagnosis for multiple refractory diseases. Finally, some clinical challenges including off-target effects, in vivo vectors, and ethical problems, and future perspective are also discussed.},
}
@article {pmid37288647,
year = {2023},
author = {Meng, W and Qiao, K and Liu, F and Gao, X and Hu, X and Liu, J and Gao, Y and Zhu, J},
title = {Construction and application of a new CRISPR/Cas12a system in Stenotrophomonas AGS-1 from aerobic granular sludge.},
journal = {Biotechnology journal},
volume = {18},
number = {9},
pages = {e2200596},
doi = {10.1002/biot.202200596},
pmid = {37288647},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; *Sewage ; Gene Editing/methods ; Plasmids ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Aerobic granular sludge (AGS) is a microbial aggregate with a biofilm structure. Thus, investigating AGS in the aspect of biofilm and microbial attachment at the genetic level would help to reveal the mechanism of granule biofilm formation. In this work, a two-plasmid clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)12a genome editing system was constructed to identify attachment genes for the first time in Stenotrophomonas AGS-1 from AGS. One plasmid contained a Cas12a cassette driven by an arabinose-inducible promoter, and another contained the specific crRNA and homologous arms (HAs). Acidaminococcus sp. Cas12a (AsCas12a) was adopted and proven to have mild toxicity (compared to Cas9) and strong cleavage activity for AGS-1. CRISPR/Cas12a-mediated rmlA knockout decreased attachment ability by 38.26%. Overexpression of rmlA in AGS-1 resulted in an increase of 30.33% in attachment ability. These results showed that the modulation of rmlA was an important factor for the biofilm formation of AGS-1. Moreover, two other genes (xanB and rpfF) were knocked out by CRISPR/Cas12a and identified as attachment-related genes in AGS-1. Also, this system could achieve point mutations. These data indicated that the CRISPR/Cas12a system could be an effective molecular platform for attachment gene function identification, which would be useful for the development of AGS in wastewater treatment.},
}
@article {pmid37657377,
year = {2023},
author = {Long, K and Cao, G and Qiu, Y and Yang, N and Chen, J and Yang, M and Hou, C and Huo, D},
title = {Hybridization chain reaction circuit controller: CRISPR/Cas12a conversion amplifier for miRNA-21 sensitive detection.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125130},
doi = {10.1016/j.talanta.2023.125130},
pmid = {37657377},
issn = {1873-3573},
abstract = {MicroRNA (miRNA) is crucial to the diagnose of various diseases. However, the accurate detection of miRNA has been challenging due to its short length and low abundance. Here, we designed a hybridization chain reaction (HCR) circuit controller to initiate the CRISPR/Cas12a conversion amplifier (HCR-Cas12a controller) for sensitive detection of miRNA-21 (miR-21). In the HCR, pre-crRNA was encapsulated in a hairpin structure until the miR-21 was present. Afterward, Cas12a fully exerted its RNase activity to self-mature pre-crRNA. Then, the trans-cleavage activity of Cas12a was initiated by activator. This results in the conversion of biological signals to fluorescent signal. During HCR-Cas12a controller, the circuit formed quickly, while the Cas12a system worked in a short time. The miR-21 was ultra-sensitively detected with the wide detection range of 1 fM - 100 nM, and the calculated limit of detection was 75.4 aM. The sensitivity was an order of magnitude lower than the standard method. The formation of HCR at room temperature does not require a thermal cycler. Additionally, Cas12a can work without the need for precise or expensive instruments. Therefore, our proposed method was suitable for low-resource settings, and provided a technical basis for sensitive detection of miRNA in low concentration range.},
}
@article {pmid37462508,
year = {2023},
author = {Son, W and Chung, KW},
title = {Targeted recombination of homologous chromosomes using CRISPR-Cas9.},
journal = {FEBS open bio},
volume = {13},
number = {9},
pages = {1658-1666},
pmid = {37462508},
issn = {2211-5463},
support = {NRF-2019R1A2C1087547//National Research Foundation of Korea/ ; NRF-2021R1A4A2001389//National Research Foundation of Korea/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Homologous Recombination/genetics ; Mutagenesis ; Drosophila/genetics ; Chromosomes ; },
abstract = {CRISPR mutagenesis is an efficient way to disrupt specific target genes in many model organisms. We previously devised a targeted CRISPR recombination method to generate intragenic recombinants of alleles in Drosophila. Here, we assessed the applicability of CRISPR targeting-induced recombination to different genetic loci. We compared the ectopic recombination rates in the male germline by CRISPR targeting at two neighboring genetic loci within the genomic region that consists of the repressed chromatin domain of the Lobe gene, and the transcriptionally active domain of PRAS40. Targeting around the transcription initiation of PRAS40 resulted in higher recombination rates of homologous chromosomes than targeting at the Lobe intron. Based on the efficient homologous recombination by CRISPR targeting observed around transcriptionally active loci, we further investigated targeted recombination between P-elements that are inserted at different genomic locations. Male recombination by CRISPR targeting of P-elements located proximally and distally to the ebony gene produced recombinants deficient for the intervening region of ebony transcription. Taken together, we suggest that targeted homologous recombination by CRISPR targeting may have specific genetic applications, such as generation of allelic combinations or chromosomal variations.},
}
@article {pmid37381714,
year = {2023},
author = {Fu, D and Yan, J and Zhang, Z and Liu, Y and Ma, X and Ding, J and Yang, S and Zhao, R and Chang, A and Gao, C and Liu, J and Zhao, T and Wang, X and Huang, C and Gao, S and Ma, Y and Tang, B and Feng, Y and Wang, H and Hao, J},
title = {Nuclear PLD1 combined with NPM1 induces gemcitabine resistance through tumorigenic IL7R in pancreatic adenocarcinoma.},
journal = {Cancer biology &amp; medicine},
volume = {20},
number = {8},
pages = {599-626},
pmid = {37381714},
issn = {2095-3941},
mesh = {Humans ; Gemcitabine ; *Pancreatic Neoplasms/drug therapy/genetics ; STAT5 Transcription Factor/metabolism/pharmacology ; Deoxycytidine/pharmacology/therapeutic use ; Receptors, Interleukin-7/metabolism ; *Adenocarcinoma/drug therapy/genetics ; RNA, Guide, CRISPR-Cas Systems ; Antimetabolites, Antineoplastic/pharmacology/therapeutic use ; Drug Resistance, Neoplasm/genetics ; *Carcinoma, Pancreatic Ductal/drug therapy/genetics/pathology ; Nuclear Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; },
abstract = {OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%. Of PDAC patients, 15%-20% are eligible for radical surgery. Gemcitabine is an important chemotherapeutic agent for patients with PDAC; however, the efficacy of gemcitabine is limited due to resistance. Therefore, reducing gemcitabine resistance is essential for improving survival of patients with PDAC. Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC.<br><br>METHODS: We constructed a human genome-wide CRISPRa/dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment. Then, co-IP, ChIP, ChIP-seq, transcriptome sequencing, and qPCR were used to determine the specific mechanism by which phospholipase D1 (PLD1) confers resistance to gemcitabine.<br><br>RESULTS: PLD1 combines with nucleophosmin 1 (NPM1) and triggers NPM1 nuclear translocation, where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor (IL7R) expression. Upon interleukin 7 (IL-7) binding, IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein, BCL-2, and induce gemcitabine resistance. The PLD1 inhibitor, Vu0155069, targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells.<br><br>CONCLUSIONS: PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1, further promoting the downstream JAK1/STAT5/Bcl-2 pathway. Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity.},
}
@article {pmid37656446,
year = {2023},
author = {Li, Y and Zhao, Z and Liu, Y and Wang, N and Man, S and Ma, L and Wang, S},
title = {CRISPR/Cas System: The Accelerator for the Development of Non-nucleic Acid Target Detection in Food Safety.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c03619},
pmid = {37656446},
issn = {1520-5118},
abstract = {Non-nucleic acid targets have posed a serious challenge to food safety. The detection of non-nucleic acid targets can enable us to monitor food contamination in a timely manner. In recent years, the CRISPR/Cas system has been extensively explored in biosensing. However, there is a lack of a summary of CRISPR/Cas-powered detection tailored to non-nucleic acid targets involved in food safety. This review comprehensively summarizes the recent advances on the construction of CRISPR/Cas-powered detection and the promising applications in the field of food safety related non-nucleic acid targets. The current challenges and futuristic perspectives are also proposed accordingly. The rapidly evolving CRISPR/Cas system has provided a powerful propellant for non-nucleic acid target detection via integration with aptamer and/or DNAzyme. Compared with traditional analytical methods, CRISPR/Cas-powered detection is conceptually novel, essentially eliminates the dependence on large instruments, and also demonstrates the capability for rapid, accurate, sensitive, and on-site testing.},
}
@article {pmid37654098,
year = {2023},
author = {Gawlitt, S and Liao, C and Achmedov, T and Beisel, CL},
title = {Shortened CRISPR-Cas9 arrays enable multiplexed gene targeting in bacteria from a smaller DNA footprint.},
journal = {RNA biology},
volume = {20},
number = {1},
pages = {666-680},
doi = {10.1080/15476286.2023.2247247},
pmid = {37654098},
issn = {1555-8584},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Footprinting ; Escherichia coli/genetics ; Gene Targeting ; Bacteria/genetics ; Endonucleases ; },
abstract = {CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies.},
}
@article {pmid37647403,
year = {2023},
author = {Song, N and Chu, Y and Li, S and Dong, Y and Fan, X and Tang, J and Guo, Y and Teng, G and Yao, C and Yang, D},
title = {Cascade dynamic assembly/disassembly of DNA nanoframework enabling the controlled delivery of CRISPR-Cas9 system.},
journal = {Science advances},
volume = {9},
number = {35},
pages = {eadi3602},
pmid = {37647403},
issn = {2375-2548},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *DNA/genetics ; Acrylamide ; CRISPR-Associated Protein 9/genetics ; Cystamine ; Ribonucleoproteins ; },
abstract = {CRISPR-Cas9 has been explored as a therapeutic agent for down-regulating target genes; the controlled delivery of Cas9 ribonucleoprotein (RNP) is essential for therapeutic efficacy and remains a challenge. Here, we report cascade dynamic assembly/disassembly of DNA nanoframework (NF) that enables the controlled delivery of Cas9 RNP. NF was prepared with acrylamide-modified DNA that initiated cascade hybridization chain reaction (HCR). Through an HCR, single-guide RNA was incorporated to NF; simultaneously, the internal space of NF was expanded, facilitating the loading of Cas9 protein. NF was designed with hydrophilic acylamino and hydrophobic isopropyl, allowing dynamic swelling and aggregation. The responsive release of Cas9 RNP was realized by introducing disulfide bond-containing N,N-bis(acryloyl)cystamine that was specifically in response to glutathione of cancer cells, triggering the complete disassembly of NF. In vitro and in vivo investigations demonstrated the high gene editing efficiency in cancer cells, the hypotoxicity in normal cells, and notable antitumor efficacy in a breast cancer mouse model.},
}
@article {pmid37646679,
year = {2023},
author = {Sharma, A and Boelens, JJ and Cancio, M and Hankins, JS and Bhad, P and Azizy, M and Lewandowski, A and Zhao, X and Chitnis, S and Peddinti, R and Zheng, Y and Kapoor, N and Ciceri, F and Maclachlan, T and Yang, Y and Liu, Y and Yuan, J and Naumann, U and Yu, VWC and Stevenson, SC and De Vita, S and LaBelle, JL},
title = {CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.},
journal = {The New England journal of medicine},
volume = {389},
number = {9},
pages = {820-832},
doi = {10.1056/NEJMoa2215643},
pmid = {37646679},
issn = {1533-4406},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Fetal Hemoglobin/genetics ; *Anemia, Sickle Cell/genetics/therapy ; Erythrocytes ; Hemoglobin, Sickle ; Antigens, CD34 ; },
abstract = {BACKGROUND: Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.<br><br>METHODS: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923.<br><br>RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period.<br><br>CONCLUSIONS: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).},
}
@article {pmid37646520,
year = {2023},
author = {Chen, S and Cao, H and Xu, Z and Huang, J and Liu, Z and Li, T and Duan, C and Wu, W and Wen, Y and Zhang, LH and Xu, Z},
title = {A type I-F CRISPRi system unveils the novel role of CzcR in modulating multidrug resistance of Pseudomonas aeruginosa.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0112323},
doi = {10.1128/spectrum.01123-23},
pmid = {37646520},
issn = {2165-0497},
abstract = {Pseudomonas aeruginosa has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn[2+])-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of P. aeruginosa to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in P. aeruginosa. IMPORTANCE P. aeruginosa is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn[2+] and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in P. aeruginosa and important clues for precise anti-pseudomonal therapies.},
}
@article {pmid37645763,
year = {2023},
author = {Borrajo, J and Javanmardi, K and Griffin, J and Martin, SJS and Yao, D and Hill, K and Blainey, PC and Al-Shayeb, B},
title = {Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37645763},
support = {DP2 HL141005/HL/NHLBI NIH HHS/United States ; },
abstract = {UNLABELLED: Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts in situ by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.<br><br>ONE-SENTENCE SUMMARY: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.},
}
@article {pmid37645720,
year = {2023},
author = {Peng, R and Chen, X and Xu, F and Hailstone, R and Men, Y and Du, K},
title = {Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37645720},
abstract = {The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieve an on-chip concentration factor of 15 for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU/mL. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.},
}
@article {pmid37642743,
year = {2023},
author = {Curtin, K and Wang, J and Fike, BJ and Binkley, B and Li, P},
title = {A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing.},
journal = {Biomedical microdevices},
volume = {25},
number = {3},
pages = {34},
pmid = {37642743},
issn = {1572-8781},
support = {R01GM135432/GM/NIGMS NIH HHS/United States ; R01GM135432/GM/NIGMS NIH HHS/United States ; R01GM135432/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; Microfluidics ; Printing, Three-Dimensional ; },
abstract = {Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL[- 1] level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.},
}
@article {pmid37573509,
year = {2023},
author = {Li, S and Mereby, SA and Rothstein, M and Johnson, MR and Brack, BJ and Mallarino, R},
title = {TIGER: Single-step in vivo genome editing in a non-traditional rodent.},
journal = {Cell reports},
volume = {42},
number = {8},
pages = {112980},
doi = {10.1016/j.celrep.2023.112980},
pmid = {37573509},
issn = {2211-1247},
mesh = {Female ; Pregnancy ; Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Rodentia/genetics ; },
abstract = {Rodents are taxonomically diverse and have evolved a variety of traits. A mechanistic understanding of such traits has remained elusive, however, largely because genome editing in non-traditional model species remains challenging. Here, using the African striped mouse (Rhabdomys pumilio), we describe TIGER (targeted in vivo genome editing in rodents), a method that relies on a simple intraoviductal injecting technique and uses recombinant adeno-associated viruses (rAAVs) as the sole vehicle to deliver reagents into pregnant females. We demonstrate that TIGER generates knockout and knockin (up to 3 kb) lines with high efficiency. Moreover, we engineer a double-cleaving repair rAAV template and find that it significantly increases knockin frequency and germline transmission rates. Lastly, we show that an oversized double-cleaving rAAV template leads to an insertion of 3.8 kb. Thus, TIGER constitutes an attractive alternative to traditional ex vivo genome-editing methods and has the potential to be extended to a broad range of species.},
}
@article {pmid37561631,
year = {2023},
author = {Liu, Z and Zheng, X and Chen, J and Zheng, L and Ma, Z and Chen, L and Deng, M and Tang, H and Zhou, L and Kang, T and Wu, Y and Liu, Z},
title = {NFYC-37 promotes tumor growth by activating the mevalonate pathway in bladder cancer.},
journal = {Cell reports},
volume = {42},
number = {8},
pages = {112963},
doi = {10.1016/j.celrep.2023.112963},
pmid = {37561631},
issn = {2211-1247},
mesh = {Humans ; Mevalonic Acid/metabolism ; *Hydroxymethylglutaryl-CoA Reductase Inhibitors ; RNA, Guide, CRISPR-Cas Systems ; *Urinary Bladder Neoplasms/genetics/pathology ; Transcription Factors/metabolism ; },
abstract = {Dysregulation of transcription is a hallmark of cancer, including bladder cancer (BLCA). CRISPR-Cas9 screening using a lentivirus library with single guide RNAs (sgRNAs) targeting human transcription factors and chromatin modifiers is used to reveal genes critical for the proliferation and survival of BLCA cells. As a result, the nuclear transcription factor Y subunit gamma (NFYC)-37, but not NFYC-50, is observed to promote cell proliferation and tumor growth in BLCA. Mechanistically, NFYC-37 interacts with CBP and SREBP2 to activate mevalonate pathway transcription, promoting cholesterol biosynthesis. However, NFYC-50 recruits more of the arginine methyltransferase CARM1 than NFYC-37 to methylate CBP, which prevents the CBP-SREBP2 interaction and subsequently inhibits the mevalonate pathway. Importantly, statins targeting the mevalonate pathway can suppress NFYC-37-induced cell proliferation and tumor growth, indicating the need for conducting a clinical trial with statins for treating patients with BLCA and high NFYC-37 levels, as most patients with BLCA have high NFYC-37 levels.},
}
@article {pmid37544379,
year = {2023},
author = {Agrawal, P and Harish, V and Mohd, S and Singh, SK and Tewari, D and Tatiparthi, R and Harshita, and Vishwas, S and Sutrapu, S and Dua, K and Gulati, M},
title = {Role of CRISPR/Cas9 in the treatment of Duchenne muscular dystrophy and its delivery strategies.},
journal = {Life sciences},
volume = {330},
number = {},
pages = {122003},
doi = {10.1016/j.lfs.2023.122003},
pmid = {37544379},
issn = {1879-0631},
mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; Mutation ; Exons ; },
abstract = {Duchenne muscular dystrophy (DMD) is a neuromuscular disorder brought on by mutations in the DMD gene, which prevent muscle cells from expressing the dystrophin protein. CRISPR/Cas9 technology has evolved as potential option to treat DMD due to its ability to permanently skip exons, restoring the disrupted DMD reading frame and leading to dystrophin restoration. Even though, having potential to treat DMD, the delivery, safety and efficacy of this technology is still challenging. Several delivery methods, including viral vectors, nanoparticles, and electroporation, have been explored to deliver CRISPR/Cas9 to the targeted cells. Despite the potential of CRISPR/Cas9 technology in the treatment of DMD, several limitations need to be addressed. The off-target effects of CRISPR/Cas9 are a major concern that needs to be addressed to avoid unintended mutations. The delivery of CRISPR/Cas9 to the target cells and the immune response due to the viral vectors used for delivery are a few other limitations. The clinical trials of CRISPR/Cas9 for DMD provide valuable insights into the safety and efficacy of this technology in humans and the limitations that need to be known. Therefore, in this review we insightfully discussed the challenges and limitations of CRISPR/Cas9 in the treatment of DMD and delivery strategies used, and the ongoing efforts to overcome these challenges and restore dystrophin expression in DMD patients in the ongoing trials.},
}
@article {pmid37486124,
year = {2023},
author = {Garrigues, S and Peng, M and Kun, RS and de Vries, RP},
title = {Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9.},
journal = {mBio},
volume = {14},
number = {4},
pages = {e0066823},
pmid = {37486124},
issn = {2150-7511},
support = {15807//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Genetic Engineering ; Aspergillus niger/genetics ; },
abstract = {CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain ΔkusA. Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci-known as off-target mutations-could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.},
}
@article {pmid36931732,
year = {2023},
author = {Sun, R and Raban, R and Akbari, OS},
title = {CRISPR-Cas9 Methods and Key Considerations in the Production of Aedes aegypti Mutant Strains.},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {9},
pages = {pdb.top107693},
doi = {10.1101/pdb.top107693},
pmid = {36931732},
issn = {1559-6095},
mesh = {Animals ; Humans ; CRISPR-Cas Systems ; *Aedes/genetics ; *Yellow Fever/genetics/prevention &amp; control ; Gene Editing/methods ; Animals, Genetically Modified ; },
abstract = {Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti, which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.},
}
@article {pmid35736243,
year = {2023},
author = {Geisert, RD and Johns, DN and Pfeiffer, CA and Sullivan, RM and Lucas, CG and Simintiras, CA and Redel, BK and Wells, KD and Spencer, TE and Prather, RS},
title = {Gene editing provides a tool to investigate genes involved in reproduction of pigs.},
journal = {Molecular reproduction and development},
volume = {90},
number = {7},
pages = {459-468},
doi = {10.1002/mrd.23620},
pmid = {35736243},
issn = {1098-2795},
support = {2017-67015-26451//National Institute of Food and Agriculture/ ; },
mesh = {Pregnancy ; Female ; Animals ; Swine/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Reproduction/genetics ; Endometrium/metabolism ; },
abstract = {CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.},
}
@article {pmid37642232,
year = {2023},
author = {Li, J and Tang, C and Liang, G and Tian, H and Lai, G and Wu, Y and Liu, S and Zhang, W and Liu, S and Shao, H},
title = {Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 System: Factors Affecting Precision Gene Editing Efficiency and Optimization Strategies.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2023.115},
pmid = {37642232},
issn = {1557-7422},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)- CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provided novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools.},
}
@article {pmid37641539,
year = {2023},
author = {Li, Y and Huang, B and Chen, J and Huang, L and Xu, J and Wang, Y and Cui, G and Zhao, H and Xin, B and Song, W and Zhu, JK and Lai, J},
title = {Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14122},
pmid = {37641539},
issn = {1467-7652},
support = {B21HJ0509//the Hainan Yazhou Bay Seed Lab/ ; SYND-2022-03//the Yazhou Bay Science and Technology City Administration/ ; SYND-2022-06//the Yazhou Bay Science and Technology City Administration/ ; },
abstract = {The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V systems typically introducing small indels, and type I system mediating long-range deletions. However, the precision of type I systems for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C system with Cas11c for plant genome editing. The Dvu I-C system was efficient to introduce controllable large fragment deletion up to at least 20 kb using paired crRNAs. The paired-crRNAs design also improved the controllability of deletions for the type I-E system. Dvu I-C system was sensitive to spacer length and mismatch, which was benefit for target specificity. In addition, we showed that the Dvu I-C system was efficient for generating stable transgenic lines in maize and rice with the editing efficiency up to 86.67%. Overall, Dvu I-C system we developed here is powerful for achieving controllable large fragment deletions.},
}
@article {pmid37641170,
year = {2023},
author = {Chen, K and Wang, Y},
title = {CRISPR/Cas systems for in situ imaging of intracellular nucleic acids: Concepts and applications.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28543},
pmid = {37641170},
issn = {1097-0290},
support = {//National Natural Science Foundation of China/ ; //Beijing Hospitals Authority Youth Programme/ ; 82002198//National Science Foundation of China/ ; },
abstract = {Accurate and precise localization of intracellular nucleic acids is crucial for regulating genetic information transcription and diagnosing diseases. Although intracellular nucleic acid imaging methods are available for various cell types, their widespread utilization is impeded by the intricate nature of the process and its exorbitant cost. Recently, numerous intracellular nucleic acid labeling techniques based on clustered regularly interspaced short palindromic repeats (CRISPR) have been established due to their modularity, flexibility, and specificity. In this work, we present various CRISPR methods that are currently employed for visualizing intracellular genomic sequences and RNA, based on their detection principles and application scenarios. Furthermore, we discuss the advantages and drawbacks of the existing CRISPR imaging methods, as well as future research directions. We anticipate that with continued refinement, more advanced CRISPR-based imaging techniques can be developed to better elucidate the localization and dynamics of intracellular nucleic acids, thereby providing a powerful tool for molecular biology research and clinical molecular pathology diagnosis.},
}
@article {pmid37637458,
year = {2023},
author = {Liu, S and Huang, S and Li, F and Sun, Y and Fu, J and Xiao, F and Jia, N and Huang, X and Sun, C and Zhou, J and Wang, Y and Qu, D},
title = {Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1239269},
pmid = {37637458},
issn = {2235-2988},
mesh = {*Recombinases ; *Pseudomonas aeruginosa/genetics ; CRISPR-Cas Systems ; Nucleotidyltransferases ; Hospitals ; },
abstract = {Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 12a (Cas12a) biosensing system and was termed P. aeruginosa-CRISPR-RPA assay. The P. aeruginosa-CRISPR-RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non-P. aeruginosa strains, and the clinical samples. As a result, the P. aeruginosa-CRISPR-RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42°C for 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by P. aeruginosa-CRISPR-RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa-CRISPR-RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa-CRISPR-RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings.},
}
@article {pmid37637117,
year = {2023},
author = {Dong, Y and Huang, Y and Fan, H and Song, L and An, X and Xu, S and Li, M and Tong, Y},
title = {Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1230775},
pmid = {37637117},
issn = {1664-302X},
abstract = {Phage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel phage from fermentation substrates with BL21(DE3) as the host, named TR1. It showed a typical podovirus morphology with a head diameter of 51.46 ± 2.04 nm and a tail length of 9.31 ± 2.77 nm. The burst size of phage TR1 was 151 PFU/cell, suggesting its strong fecundity in the fermentation system. Additionally, whole-genome sequencing revealed that phage TR1 has a DNA genome of 44,099 bp in length with a 43.8% GC content, encoding a total of 68 open reading frames. Comparative genomics and phylogenetic analysis designated this phage to be a new species of the genus Christensenvirus. To counteract phage TR1, we employed the CRISPR/Cas9 system-based strategy and constructed two phage-resistant E. coli strains, BL21-C and BL21-T, based on conserved genes. Both EOP assays and growth curves indicated strong phage resistance of the recombinant strains, without affecting cell growth. Therefore, this study aimed to provide a resilient strategy to respond to ever-changing phages and ongoing phage-host arm race in industrial fermentation environments by the personalized design of spacers in the recombinant CRISPR/Cas system-containing plasmid. More importantly, our research sparks the use of phage defense mechanism to prevent phage contamination in extensive biotechnological applications.},
}
@article {pmid37315207,
year = {2023},
author = {Sretenovic, S and Green, Y and Wu, Y and Cheng, Y and Zhang, T and Van Eck, J and Qi, Y},
title = {Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.},
journal = {Plant physiology},
volume = {193},
number = {1},
pages = {291-303},
doi = {10.1093/plphys/kiad347},
pmid = {37315207},
issn = {1532-2548},
support = {2018-33522-28789//U.S. Department of Agriculture Biotechnology Risk Assessment Grant Program/ ; IOS-2029889//National Science Foundation Plant Genome Research Program/ ; //Foundation for Food and Agriculture Research Fellow/ ; },
mesh = {*Solanum lycopersicum/genetics ; Transcriptome/genetics ; Adenine/metabolism ; Mutation/genetics ; Gene Editing ; RNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.},
}
@article {pmid37294917,
year = {2023},
author = {Vavassori, V and Ferrari, S and Beretta, S and Asperti, C and Albano, L and Annoni, A and Gaddoni, C and Varesi, A and Soldi, M and Cuomo, A and Bonaldi, T and Radrizzani, M and Merelli, I and Naldini, L},
title = {Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.},
journal = {Blood},
volume = {142},
number = {9},
pages = {812-826},
doi = {10.1182/blood.2022019333},
pmid = {37294917},
issn = {1528-0020},
mesh = {Humans ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; RNA/metabolism ; CRISPR-Cas Systems ; },
abstract = {Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.},
}
@article {pmid37636103,
year = {2023},
author = {Ashraf, S and Ahmad, A and Khan, SH and Jamil, A and Sadia, B and Brown, JK},
title = {LbCas12a mediated suppression of Cotton leaf curl Multan virus.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1233295},
pmid = {37636103},
issn = {1664-462X},
abstract = {Begomoviruses are contagious and severely affect commercially important fiber and food crops. Cotton leaf curl Multan virus (CLCuMuV) is one of the most dominant specie of Begomovirus and a major constraint on cotton yield in Pakistan. Currently, the field of plant genome editing is being revolutionized by the CRISPR/Cas system applications such as base editing, prime editing and CRISPR based gene drives. CRISPR/Cas9 system has successfully been used against biotic and abiotic plant stresses with proof-of-concept studies in both model and crop plants. CRISPR/Cas12 and CRISPR/Cas13 have recently been applied in plant sciences for basic and applied research. In this study, we used a novel approach, multiplexed crRNA-based Cas12a toolbox to target the different ORFs of the CLCuMuV genome at multiple sites simultaneously. This method successfully eliminated the symptoms of CLCuMuV in Nicotiana benthamiana and Nicotiana tabacum. Three individual crRNAs were designed from the CLCuMuV genome, targeting the specific sites of four different ORFs (C1, V1 and overlapping region of C2 and C3). The Cas12a-based construct Cas12a-MV was designed through Golden Gate three-way cloning for precise editing of CLCuMuV genome. Cas12a-MV construct was confirmed through whole genome sequencing using the primers Ubi-intron-F1 and M13-R1. Transient assays were performed in 4 weeks old Nicotiana benthamiana plants, through the agroinfiltration method. Sanger sequencing indicated that the Cas12a-MV constructs made a considerable mutations at the target sites of the viral genome. In addition, TIDE analysis of Sanger sequencing results showed the editing efficiency of crRNA1 (21.7%), crRNA2 (24.9%) and crRNA3 (55.6%). Furthermore, the Cas12a-MV construct was stably transformed into Nicotiana tabacum through the leaf disc method to evaluate the potential of transgenic plants against CLCuMuV. For transgene analysis, the DNA of transgenic plants of Nicotiana tabacum was subjected to PCR to amplify Cas12a genes with specific primers. Infectious clones were agro-inoculated in transgenic and non-transgenic plants (control) for the infectivity assay. The transgenic plants containing Cas12a-MV showed rare symptoms and remained healthy compared to control plants with severe symptoms. The transgenic plants containing Cas12a-MV showed a significant reduction in virus accumulation (0.05) as compared to control plants (1.0). The results demonstrated the potential use of the multiplex LbCas12a system to develop virus resistance in model and crop plants against begomoviruses.},
}
@article {pmid37636063,
year = {2023},
author = {Wei, XY and Liu, L and Hu, H and Jia, HJ and Bu, LK and Pei, DS},
title = {Ultra-sensitive detection of ecologically rare fish from eDNA samples based on the RPA-CRISPR/Cas12a technology.},
journal = {iScience},
volume = {26},
number = {9},
pages = {107519},
pmid = {37636063},
issn = {2589-0042},
abstract = {Environmental DNA (eDNA) research holds great promise for improving biodiversity science and conservation efforts by enabling worldwide species censuses in near real-time. Current eDNA methods face challenges in detecting low-abundance ecologically important species. In this study, we used isothermal recombinase polymerase amplification (RPA)-CRISPR/Cas detection to test Ctenopharyngodon idella. RPA-CRISPR-Cas12a detected 6.0 eDNA copies/μL within 35 min. Ecologically rare species were identified in the Three Gorges Reservoir Area (TGRA) using functional distinctiveness and geographical restrictiveness, with seven fish species (9%) classified as potentially ecologically rare including three species in this investigation. RPA-CRISPR/Cas12a-FQ outperformed high-throughput sequencing (HTS) and qPCR in detecting low-abundance eDNA (AUC = 0.883∗∗). A significant linear correlation (R[2] = 0.682∗∗) between RPA-CRISPR/Cas12a-FQ and HTS quantification suggests its potential for predicting species abundance and enhancing eDNA-based fish biodiversity monitoring. This study highlights the value of RPA-CRISPR/Cas12a-FQ as a tool for advancing eDNA research and conservation efforts.},
}
@article {pmid37580593,
year = {2023},
author = {Reysenbach, AL and Terns, MP},
title = {CRISPR-influenced symbiosis.},
journal = {Nature microbiology},
volume = {8},
number = {9},
pages = {1611-1612},
pmid = {37580593},
issn = {2058-5276},
mesh = {*Symbiosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
}
@article {pmid37491642,
year = {2023},
author = {Kiani, L},
title = {New CRISPR-based strategies for Alzheimer disease.},
journal = {Nature reviews. Neurology},
volume = {19},
number = {9},
pages = {507},
doi = {10.1038/s41582-023-00856-5},
pmid = {37491642},
issn = {1759-4766},
mesh = {Humans ; *Alzheimer Disease/genetics/therapy ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37343700,
year = {2023},
author = {Song, Z and Zhang, G and Huang, S and Liu, Y and Li, G and Zhou, X and Sun, J and Gao, P and Chen, Y and Huang, X and Liu, J and Wang, X},
title = {PE-STOP: A versatile tool for installing nonsense substitutions amenable for precise reversion.},
journal = {The Journal of biological chemistry},
volume = {299},
number = {8},
pages = {104942},
pmid = {37343700},
issn = {1083-351X},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *Codon, Nonsense ; Gene Silencing ; Codon, Terminator/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {The rapid advances in genome editing technologies have revolutionized the study of gene functions in cell or animal models. The recent generation of double-stranded DNA cleavage-independent base editors has been suitably adapted for interrogation of protein-coding genes on the basis of introducing premature stop codons or disabling the start codons. However, such versions of stop/start codon-oriented genetic tools still present limitations on their versatility, base-level precision, and target specificity. Here, we exploit a newly developed prime editor (PE) that differs from base editors by its adoption of a reverse transcriptase activity, which enables incorporation of various types of precise edits templated by a specialized prime editing guide RNA. Based on such a versatile platform, we established a prime editing-empowered method (PE-STOP) for installation of nonsense substitutions, providing a complementary approach to the present gene-targeting tools. PE-STOP is bioinformatically predicted to feature substantially expanded coverage in the genome space. In practice, PE-STOP introduces stop codons with good efficiencies in human embryonic kidney 293T and N2a cells (with medians of 29% [ten sites] and 25% [four sites] editing efficiencies, respectively), while exhibiting minimal off-target effects and high on-target precision. Furthermore, given the fact that PE installs prime editing guide RNA-templated mutations, we introduce a unique strategy for precise genetic rescue of PE-STOP-dependent nonsense mutation via the same PE platform. Altogether, the present work demonstrates a versatile and specific tool for gene inactivation and for functional interrogation of nonsense mutations.},
}
@article {pmid37186122,
year = {2023},
author = {Guo, Z and Hu, YH and Feng, GS and Valenzuela Ripoll, C and Li, ZZ and Cai, SD and Wang, QQ and Luo, WW and Li, Q and Liang, LY and Wu, ZK and Zhang, JG and Javaheri, A and Wang, L and Lu, J and Liu, PQ},
title = {JMJD6 protects against isoproterenol-induced cardiac hypertrophy via inhibition of NF-κB activation by demethylating R149 of the p65 subunit.},
journal = {Acta pharmacologica Sinica},
volume = {44},
number = {9},
pages = {1777-1789},
pmid = {37186122},
issn = {1745-7254},
mesh = {Rats ; Animals ; *NF-kappa B/metabolism ; Isoproterenol/toxicity ; *Heart Failure/metabolism ; Histones/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Rats, Sprague-Dawley ; Stroke Volume ; Cardiomegaly/chemically induced/prevention &amp; control/drug therapy ; Myocytes, Cardiac/metabolism ; },
abstract = {Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. In this study we investigated the role of a histone arginine demethylase, Jumonji C domain-containing protein 6 (JMJD6) in pathological cardiac hypertrophy. Cardiac hypertrophy was induced in rats by subcutaneous injection of isoproterenol (ISO, 1.2 mg·kg[-1]·d[-1]) for a week. At the end of the experiment, the rats underwent echocardiography, followed by euthanasia and heart collection. We found that JMJD6 levels were compensatorily increased in ISO-induced hypertrophic cardiac tissues, but reduced in patients with heart failure with reduced ejection fraction (HFrEF). Furthermore, we demonstrated that JMJD6 overexpression significantly attenuated ISO-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) evidenced by the decreased cardiomyocyte surface area and hypertrophic genes expression. Cardiac-specific JMJD6 overexpression in rats protected the hearts against ISO-induced cardiac hypertrophy and fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) exacerbated ISO-induced hypertrophic responses in NRCMs. We revealed that JMJD6 interacted with NF-κB p65 in cytoplasm and reduced nuclear levels of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 bound to p65 and demethylated p65 at the R149 residue to inhibit the nuclear translocation of p65, thus inactivating NF-κB signaling and protecting against pathological cardiac hypertrophy. In addition, we found that JMJD6 demethylated histone H3R8, which might be a new histone substrate of JMJD6. These results suggest that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.},
}
@article {pmid37636024,
year = {2023},
author = {Nouri Nojadeh, J and Bildiren Eryilmaz, NS and Ergüder, BI},
title = {CRISPR/Cas9 genome editing for neurodegenerative diseases.},
journal = {EXCLI journal},
volume = {22},
number = {},
pages = {567-582},
pmid = {37636024},
issn = {1611-2156},
abstract = {Gene therapy has emerged as a promising therapeutic strategy for various conditions, including blood disorders, ocular disease, cancer, and nervous system disorders. The advent of gene editing techniques has facilitated the ability of researchers to specifically target and modify the eukaryotic cell genome, making it a valuable tool for gene therapy. This can be performed through either in vivo or ex vivo approaches. Gene editing tools, such as zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR-Cas-associated nucleases, can be employed for gene therapy purposes. Among these tools, CRISPR-Cas-based gene editing stands out because of its ability to introduce heritable genome changes by designing short guide RNAs. This review aims to provide an overview of CRISPR-Cas technology and summarizes the latest research on the application of CRISPR/Cas9 genome editing technology for the treatment of the most prevalent neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, and Spinocerebellar ataxia.},
}
@article {pmid37633620,
year = {2023},
author = {Liu, Z and Liu, J and Yang, Z and Zhu, L and Zhu, Z and Huang, H and Jiang, L},
title = {Endogenous CRISPR-Cas mediated in situ genome editing: State-of-the-art and the road ahead for engineering prokaryotes.},
journal = {Biotechnology advances},
volume = {68},
number = {},
pages = {108241},
doi = {10.1016/j.biotechadv.2023.108241},
pmid = {37633620},
issn = {1873-1899},
abstract = {The CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing in various prokaryotes. However, the perturbation of DNA homeostasis and the inherent toxicity of Cas9/12a proteins could easily lead to cell death, which led to the development of endogenous CRISPR-Cas systems. Programming the widespread endogenous CRISPR-Cas systems for in situ genome editing represents a promising tool in prokaryotes, especially in genetically intractable species. Here, this review briefly summarizes the advances of endogenous CRISPR-Cas-mediated genome editing, covering aspects of establishing and optimizing the genetic tools. In particular, this review presents the application of different types of endogenous CRISPR-Cas tools for strain engineering, including genome editing and genetic regulation. Notably, this review also provides a detailed discussion of the transposon-associated CRISPR-Cas systems, and the programmable RNA-guided transposition using endogenous CRISPR-Cas systems to enable editing of microbial communities for understanding and control. Therefore, they will be a powerful tool for targeted genetic manipulation. Overall, this review will not only facilitate the development of standard genetic manipulation tools for non-model prokaryotes but will also enable more non-model prokaryotes to be genetically tractable.},
}
@article {pmid37633433,
year = {2023},
author = {Zhao, F and Ding, X and Liu, Z and Yan, X and Chen, Y and Jiang, Y and Chen, S and Wang, Y and Kang, T and Xie, C and He, M and Zheng, J},
title = {Application of CRISPR/Cas9-based genome editing in ecotoxicology.},
journal = {Environmental pollution (Barking, Essex : 1987)},
volume = {336},
number = {},
pages = {122458},
doi = {10.1016/j.envpol.2023.122458},
pmid = {37633433},
issn = {1873-6424},
abstract = {Chemicals are widely used and released into the environment, and their degradation, accumulation, migration, and transformation processes in the environment can pose a threat to the ecosystem. The advancement in analytical methods with high-throughput screening of biomolecules has revolutionized the way toxicologists used to explore the effects of chemicals on organisms. CRISPR/Cas is a newly developed tool, widely used in the exploration of basic science and biologically engineered products given its high efficiency and low cost. For example, it can edit target genes efficiently, and save loss of the crop yield caused by environmental pollution as well as gain a better understanding of the toxicity mechanisms from various chemicals. This review briefly introduces the development history of CRISPR/Cas and summarizes the current application of CRISPR/Cas in ecotoxicology, including its application on improving crop yield and drug resistance towards agricultural pollution, antibiotic pollution and other threats. The benefits by applying the CRISPR/Cas9 system in conventional toxicity mechanism studies are fully demonstrated here together with its foreseeable expansions in other area of ecotoxicology. Finally, the prospects and disadvantages of CRISPR/Cas system in the field of ecotoxicology are also discussed.},
}
@article {pmid37631249,
year = {2023},
author = {Titze-de-Almeida, SS and Titze-de-Almeida, R},
title = {Progress in circRNA-Targeted Therapy in Experimental Parkinson's Disease.},
journal = {Pharmaceutics},
volume = {15},
number = {8},
pages = {},
pmid = {37631249},
issn = {1999-4923},
abstract = {Circular RNAs (circRNAs) are single-stranded RNA molecules often circularized by backsplicing. Growing evidence implicates circRNAs in the underlying mechanisms of various diseases, such as Alzheimer's and Parkinson's disease (PD)-the first and second most prevalent neurodegenerative disorders. In this sense, circSNCA, circHIPK2, circHIPK3, and circSLC8A1 are circRNAs that have been related to the neurodegenerative process of PD. Gain-of-function and loss-of-function studies on circRNAs have shed light on their roles in the pathobiology of various diseases. Gain-of-function approaches typically employ viral or non-viral vectors that hyperexpress RNA sequences capable of circularizing to form the specific circRNA under investigation. In contrast, loss-of-function studies utilize CRISPR/Cas systems, antisense oligonucleotides (ASOs), or RNAi techniques to knock down the target circRNA. The role of aberrantly expressed circRNAs in brain pathology has raised a critical question: could circRNAs serve as viable targets for neuroprotective treatments? Translating any oligonucleotide-based therapy, including those targeting circRNAs, involves developing adequate brain delivery systems, minimizing off-target effects, and addressing the high costs of treatment. Nonetheless, RNAi-based FDA-approved drugs have entered the market, and circRNAs have attracted significant attention and investment from major pharmaceutical companies. Spanning from bench to bedside, circRNAs present a vast opportunity in biotechnology for oligonucleotide-based therapies designed to slow or even halt the progression of neurodegenerative diseases.},
}
@article {pmid37630620,
year = {2023},
author = {Yadalam, PK and Arumuganainar, D and Anegundi, RV and Shrivastava, D and Alftaikhah, SAA and Almutairi, HA and Alobaida, MA and Alkaberi, AA and Srivastava, KC},
title = {CRISPR-Cas-Based Adaptive Immunity Mediates Phage Resistance in Periodontal Red Complex Pathogens.},
journal = {Microorganisms},
volume = {11},
number = {8},
pages = {},
pmid = {37630620},
issn = {2076-2607},
abstract = {Periodontal diseases are polymicrobial immune-inflammatory diseases that can severely destroy tooth-supporting structures. The critical bacteria responsible for this destruction include red complex bacteria such as Porphoromonas gingivalis, Tanerella forsythia and Treponema denticola. These organisms have developed adaptive immune mechanisms against bacteriophages/viruses, plasmids and transposons through clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). The CRISPR-Cas system contributes to adaptive immunity, and this acquired genetic immune system of bacteria may contribute to moderating the microbiome of chronic periodontitis. The current research examined the role of the CRISPR-Cas system of red complex bacteria in the dysbiosis of oral bacteriophages in periodontitis. Whole-genome sequences of red complex bacteria were obtained and investigated for CRISPR using the CRISPR identification tool. Repeated spacer sequences were analyzed for homologous sequences in the bacteriophage genome and viromes using BLAST algorithms. The results of the BLAST spacer analysis for T. denticola spacers had a 100% score (e value with a bacillus phage), and the results for T. forsthyia and P. gingivalis had a 56% score with a pectophage and cellulophage (e value: 0.21), respectively. The machine learning model of the identified red complex CRISPR sequences predicts with area an under the curve (AUC) accuracy of 100 percent, indicating phage inhibition. These results infer that red complex bacteria could significantly inhibit viruses and phages with CRISPR immune sequences. Therefore, the role of viruses and bacteriophages in modulating sub-gingival bacterial growth in periodontitis is limited or questionable.},
}
@article {pmid37630508,
year = {2023},
author = {Alkompoz, AK and Hamed, SM and Zaid, ASA and Almangour, TA and Al-Agamy, MH and Aboshanab, KM},
title = {Correlation of CRISPR/Cas and Antimicrobial Resistance in Klebsiella pneumoniae Clinical Isolates Recovered from Patients in Egypt Compared to Global Strains.},
journal = {Microorganisms},
volume = {11},
number = {8},
pages = {},
pmid = {37630508},
issn = {2076-2607},
support = {RGP-038//Deanship of Scientific Research at King Saud University/ ; },
abstract = {The CRISPR/Cas system has been long known to interfere with the acquisition of foreign genetic elements and was recommended as a tool for fighting antimicrobial resistance. The current study aimed to explore the prevalence of the CRISPR/Cas system in Klebsiella pneumoniae isolates recovered from patients in Egypt in comparison to global strains and correlate the CRISPR/Cas to susceptibility to antimicrobial agents. A total of 181 clinical isolates were PCR-screened for cas and selected antimicrobial resistance genes (ARGs). In parallel, 888 complete genome sequences were retrieved from the NCBI database for in silico analysis. CRISPR/Cas was found in 46 (25.4%) isolates, comprising 18.8% type I-E and 6.6% type I-E*. Multidrug resistance (MDR) and extensive drug resistance (XDR) were found in 73.5% and 25.4% of the isolates, respectively. More than 95% of the CRISPR/Cas-bearing isolates were MDR (65.2%) or XDR (32.6%). No significant difference was found in the susceptibility to the tested antimicrobial agents among the CRISPR/Cas-positive and -negative isolates. The same finding was obtained for the majority of the screened ARGs. Among the published genomes, 23.2% carried CRISPR/Cas, with a higher share of I-E* (12.8%). They were confined to specific sequence types (STs), most commonly ST147, ST23, ST15, and ST14. More plasmids and ARGs were carried by the CRISPR/Cas-negative group than others, but their distribution in the two groups was not significantly different. The prevalence of some ARGs, such as blaKPC, blaTEM, and rmtB, was significantly higher among the genomes of the CRISPR/Cas-negative strains. A weak, nonsignificant positive correlation was found between the number of spacers and the number of resistance plasmids and ARGs. In conclusion, the correlation between CRISPR/Cas and susceptibility to antimicrobial agents or bearing resistance plasmids and ARGs was found to be nonsignificant. Plasmid-targeting spacers might not be naturally captured by CRISPR/Cas. Spacer match analysis is recommended to provide a clearer image of the exact behavior of CRISPR/Cas towards resistance plasmids.},
}
@article {pmid37629024,
year = {2023},
author = {Yin, X and Harmancey, R and McPherson, DD and Kim, H and Huang, SL},
title = {Liposome-Based Carriers for CRISPR Genome Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {16},
pages = {},
pmid = {37629024},
issn = {1422-0067},
support = {HL135092, McPherson/NH/NIH HHS/United States ; NS098918, Booker and Huang/NH/NIH HHS/United States ; },
mesh = {*Gene Editing ; *Liposomes ; RNA, Guide, CRISPR-Cas Systems ; Biotechnology ; Cues ; },
abstract = {The CRISPR-based genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR), has sparked renewed interest in gene therapy. This interest is accompanied by the development of single-guide RNAs (sgRNAs), which enable the introduction of desired genetic modifications at the targeted site when used alongside the CRISPR components. However, the efficient delivery of CRISPR/Cas remains a challenge. Successful gene editing relies on the development of a delivery strategy that can effectively deliver the CRISPR cargo to the target site. To overcome this obstacle, researchers have extensively explored non-viral, viral, and physical methods for targeted delivery of CRISPR/Cas9 and a guide RNA (gRNA) into cells and tissues. Among those methods, liposomes offer a promising approach to enhance the delivery of CRISPR/Cas and gRNA. Liposomes facilitate endosomal escape and leverage various stimuli such as light, pH, ultrasound, and environmental cues to provide both spatial and temporal control of cargo release. Thus, the combination of the CRISPR-based system with liposome delivery technology enables precise and efficient genetic modifications in cells and tissues. This approach has numerous applications in basic research, biotechnology, and therapeutic interventions. For instance, it can be employed to correct genetic mutations associated with inherited diseases and other disorders or to modify immune cells to enhance their disease-fighting capabilities. In summary, liposome-based CRISPR genome editing provides a valuable tool for achieving precise and efficient genetic modifications. This review discusses future directions and opportunities to further advance this rapidly evolving field.},
}
@article {pmid37432544,
year = {2023},
author = {Banerjee, S and Mukherjee, A and Kundu, A},
title = {The current scenario and future perspectives of transgenic oilseed mustard by CRISPR-Cas9.},
journal = {Molecular biology reports},
volume = {50},
number = {9},
pages = {7705-7728},
pmid = {37432544},
issn = {1573-4978},
support = {TAR/2021/000233//Department of Science and Technology, Government of West Bengal/ ; },
mesh = {*Mustard Plant/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genome, Plant ; *Brassica napus/genetics ; },
abstract = {PURPOSE: Production of a designer crop having added attributes is the primary goal of all plant biotechnologists. Specifically, development of a crop with a simple biotechnological approach and at a rapid pace is most desirable. Genetic engineering enables us to displace genes among species. The newly incorporated foreign gene(s) in the host genome can create a new trait(s) by regulating the genotypes and/or phenotypes. The advent of the CRISPR-Cas9 tools has enabled the modification of a plant genome easily by introducing mutation or replacing genomic fragment. Oilseed mustard varieties (e.g., Brassica juncea, Brassica nigra, Brassica napus, and Brassica carinata) are one such plants, which have been transformed with different genes isolated from the wide range of species. Current reports proved that the yield and value of oilseed mustard has been tremendously improved by the introduction of stably inherited new traits such as insect and herbicide resistance. However, the genetic transformation of oilseed mustard remains incompetent due to lack of potential plant transformation systems. To solve numerous complications involved in genetically modified oilseed mustard crop varieties regeneration procedures, scientific research is being conducted to rectify the unwanted complications. Thus, this study provides a broader overview of the present status of new traits introduced in each mentioned varieties of oilseed mustard plant by different genetical engineering tools, especially CRISPR-Cas9, which will be useful to improve the transformation system of oilseed mustard crop plants.<br><br>METHODS: This review presents recent improvements made in oilseed mustard genetic engineering methodologies by using CRISPR-Cas9 tools, present status of new traits introduced in oilseed mustard plant varieties.<br><br>RESULTS: The review highlighted that the transgenic oilseed mustard production is a challenging process and the transgenic varieties of oilseed mustard provide a powerful tool for enhanced mustard yield. Over expression studies and silencing of desired genes provide functional importance of genes involved in mustard growth and development under different biotic and abiotic stress conditions. Thus, it can be expected that in near future CRISPR can contribute enormously in improving the mustard plant's architecture and develop stress resilient oilseed mustard plant species.},
}
@article {pmid37403571,
year = {2023},
author = {Donà, M and Bradamante, G and Bogojevic, Z and Gutzat, R and Streubel, S and Mosiolek, M and Dolan, L and Mittelsten Scheid, O},
title = {A versatile CRISPR-based system for lineage tracing in living plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {115},
number = {5},
pages = {1169-1184},
doi = {10.1111/tpj.16378},
pmid = {37403571},
issn = {1365-313X},
support = {WWTF WWTF LS13-057//Vienna Science and Technology Fund/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Mutation ; *Frameshift Mutation ; Phenotype ; Cell Lineage/genetics ; },
abstract = {Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.},
}
@article {pmid36694315,
year = {2023},
author = {Liu, J and Shang, X and Yao, B and Zhang, Y and Huang, S and Guo, Y and Wang, X},
title = {Design and Construction of Carboxylesterase 2c Gene Knockout Rats by CRISPR/Cas9.},
journal = {Current drug metabolism},
volume = {24},
number = {3},
pages = {190-199},
doi = {10.2174/1389200224666230123140919},
pmid = {36694315},
issn = {1875-5453},
support = {011//East China Normal University/ ; 18430760400//Science and Technology Commission of Shanghai Municipality/ ; 2019CNECNUPI02//Shanghai Science and Technology Development Foundation/ ; 81773808//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Rats ; Humans ; Animals ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Diltiazem/metabolism ; Liver/metabolism ; Aspirin/metabolism ; },
abstract = {BACKGROUND: Carboxylesterase 2 (CES2) is mainly distributed in the human liver and gut, and plays an active role in the metabolic activation of many prodrugs and lipid metabolism. Although CES2 is of great significance, there are still few animal models related to CES2.<br><br>OBJECTIVES: This research aims to construct Ces2c gene knockout (KO) rats and further study the function of CES2.<br><br>METHODS: CRISPR/Cas9 gene editing technology was used to target and cleave the rat Ces2c gene. Compensatory effects of major CES subtypes both in the liver and small intestine of KO rats were detected at mRNA levels. Meanwhile, diltiazem and aspirin were used as substrates to test the metabolic capacity of Ces2c in KO rats.<br><br>RESULTS: This Ces2c KO rat model showed normal growth and breeding without off-target effects. The metabolic function of Ces2c KO rats was verified by the metabolic study of CES2 substrates in vitro. The results showed that the metabolic capacity of diltiazem in KO rats was weakened, while the metabolic ability of aspirin did not change significantly. In addition, the serum physiological indexes showed that the Ces2c deletion did not affect the liver function of rats..<br><br>CONCLUSION: The Ces2c KO rat model was successfully constructed by CRISPR/Cas9 system. This rat model can not only be used as an important tool to study the drug metabolism mediated by CES2, but also as an important animal model to study the physiological function of CES2.},
}
@article {pmid37628816,
year = {2023},
author = {Liesenhoff, C and Paulus, SM and Havertz, C and Geerlof, A and Priglinger, S and Priglinger, CS and Ohlmann, A},
title = {Endogenous Galectin-1 Modulates Cell Biological Properties of Immortalized Retinal Pigment Epithelial Cells In Vitro.},
journal = {International journal of molecular sciences},
volume = {24},
number = {16},
pages = {},
pmid = {37628816},
issn = {1422-0067},
support = {OH 214/4-3; PR 1248/2-3//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Humans ; *Galectin 1/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Actins ; Epithelial Cells ; Retinal Pigments ; },
abstract = {In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1[-/-]) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1[-/-] cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1[-/-] cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1[-/-] cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype.},
}
@article {pmid37628594,
year = {2023},
author = {Sauvagère, S and Siatka, C},
title = {CRISPR-Cas: 'The Multipurpose Molecular Tool' for Gene Therapy and Diagnosis.},
journal = {Genes},
volume = {14},
number = {8},
pages = {},
pmid = {37628594},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems/genetics ; *Genetic Therapy ; DNA Damage ; Technology ; },
abstract = {Since the discovery of the CRISPR-Cas engineering system in 2012, several approaches for using this innovative molecular tool in therapeutic strategies and even diagnosis have been investigated. The use of this tool requires a global approach to DNA damage processes and repair systems in cells. The diversity in the functions of various Cas proteins allows for the use of this technology in clinical applications and trials. Wide variants of Cas12 and Cas13 are exploited using the collateral effect in many diagnostic applications. Even though this tool is well known, its use still raises real-world ethical and regulatory questions.},
}
@article {pmid37626297,
year = {2023},
author = {Jia, T and Yuan, F and Tao, J and Wang, G and Zhang, X and Zhang, B and Li, H},
title = {CRISPR/Cas13d targeting GZMA in PARs pathway regulates the function of osteoclasts in chronic apical periodontitis.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {70},
pmid = {37626297},
issn = {1689-1392},
support = {2022M723863//Project funded by China Postdoctoral Science Foundation/ ; 2017YFA0106200//National Key Research and Development Program of China/ ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Osteoclasts ; Apoptosis/genetics ; RNA, Guide, CRISPR-Cas Systems ; Transforming Growth Factor beta ; *Periapical Periodontitis/genetics ; Granzymes ; },
abstract = {Chronic apical periodontitis is a prevalent oral disease characterized by bone loss, and its underlying mechanisms remain unclear. This study aimed to investigate the role and mechanism of the serine protease GZMA in osteoclasts during chronic apical periodontitis. To address this, we employed crRNA/Cas13d to inhibit GZMA expression and examined its impact on osteoclast behavior. Our findings revealed that GZMA plays a significant role in promoting osteoclast cell proliferation while inhibiting cell apoptosis. Additionally, the inhibition of GZMA led to a notable increase in miR-25-3p expression, which, in turn, downregulated the expression of TGF-β. Consequently, the reduction in TGF-β expression led to a decrease in PAR1 expression within the PARs pathway. These results suggest that GZMA might serve as a promising therapeutic target for the treatment of chronic apical periodontitis. Furthermore, our study highlights the potential of targeting GZMA using crRNA/Cas13d as a valuable approach for future therapeutic interventions.},
}
@article {pmid37626063,
year = {2023},
author = {Tsai, HH and Kao, HJ and Kuo, MW and Lin, CH and Chang, CM and Chen, YY and Chen, HH and Kwok, PY and Yu, AL and Yu, J},
title = {Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5183},
pmid = {37626063},
issn = {2041-1723},
support = {OMRPG3C0048//Chang Gung Medical Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; Cell Line ; },
abstract = {CRISPR-Cas9 genome editing has promising therapeutic potential for genetic diseases and cancers, but safety could be a concern. Here we use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing and in comparison to four parental cell lines. In addition to the previously reported large structural variants at on-target sites, we identify heretofore unexpected large chromosomal deletions (91.2 and 136 Kb) at atypical non-homologous off-target sites without sequence similarity to the sgRNA in two edited lines. The observed large structural variants induced by CRISPR-Cas9 editing in dividing cells may result in pathogenic consequences and thus limit the usefulness of the CRISPR-Cas9 editing system for disease modeling and gene therapy. In this work, our whole genomic analysis may provide a valuable strategy to ensure genome integrity after genomic editing to minimize the risk of unintended effects in research and clinical applications.},
}
@article {pmid37624748,
year = {2023},
author = {Lira, C and Cunha, DP and Vasconcelos, ZFM},
title = {Biotechnological advances in gene therapy of hematopoietic stem cells: Systematic review and meta-analysis.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2022.237},
pmid = {37624748},
issn = {1557-7422},
abstract = {Gene therapy (GT) has emerged as a promising treatment option for disorders in the hematopoietic system, particularly primary immunodeficiencies (PID). Hematopoietic stem cells (HSC) have gained attention due to their ability to support long-term hematopoiesis. In this study, we present a summary of research evaluating the most effective method of gene editing in HSC for translational medicine. We conducted a systematic literature search in various databases, including Cochrane, LILACs, Scielo, and Pubmed (MEDLINE), covering the period from January 1989 to June 10th, 2023. Our aim was to identify articles that assessed the efficiency of gene editing in HSC and clinical trials focusing on PID. Our research protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; registration number CRD42022349850). Out of the 470 studies identified in our search, 77 met the inclusion criteria. Among these, 61 studies were included in strategy 1 (gene therapy using HSC - GT-HSC) of the systematic review (SR). We performed a meta-analysis on 17 of these studies. Additionally, 16 studies were categorized under strategy 2 (clinical trials for PID). While clinical trials have demonstrated the potential benefits of GT-HSC, the safety and efficacy of gene editing still pose significant challenges. Various viral and non-viral approaches for gene delivery have been explored in basic and clinical research, with viral vectors being the most commonly used method in HSC therapeutics. Although promising, recent technologies like CRISPR/Cas are not yet ready for efficient long-term restoration of the immune system as a whole.},
}
@article {pmid37623635,
year = {2023},
author = {Wu, Y and Li, X and Dong, L and Liu, T and Tang, Z and Lin, R and Norvienyeku, J and Xing, M},
title = {A New Insight into 6-Pentyl-2H-pyran-2-one against Peronophythora litchii via TOR Pathway.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {9},
number = {8},
pages = {},
doi = {10.3390/jof9080863},
pmid = {37623635},
issn = {2309-608X},
support = {No. 32260649//the National Natural Science Foundation of China/ ; 320RC482//the Natural Science Foundation of Hainan Province/ ; },
abstract = {The litchi downy blight disease of litchi caused by Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.},
}
@article {pmid37622910,
year = {2023},
author = {Burkin, KM and Ivanov, AV and Zherdev, AV and Dzantiev, BB and Safenkova, IV},
title = {A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity.},
journal = {Biosensors},
volume = {13},
number = {8},
pages = {},
pmid = {37622910},
issn = {2079-6374},
support = {//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *COVID-19 ; CRISPR-Cas Systems ; SARS-CoV-2/genetics ; DNA Probes ; Antibodies ; DNA, Single-Stranded ; },
abstract = {CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.},
}
@article {pmid37622855,
year = {2023},
author = {Kwak, N and Park, BJ and Song, YJ},
title = {A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V.},
journal = {Biosensors},
volume = {13},
number = {8},
pages = {},
pmid = {37622855},
issn = {2079-6374},
support = {HI22C0286//Korea Health Industry Development Institute/Republic of Korea ; 202110510001//Gachon University/ ; },
mesh = {Humans ; Animals ; *Encephalitis Virus, Japanese/genetics ; CRISPR-Cas Systems ; Antiviral Agents ; China ; Genotype ; },
abstract = {The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 10[2] copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR.},
}
@article {pmid37622352,
year = {2023},
author = {Zhou, Y and Ruan, Z and Fang, C and Chen, X and Xu, H and Wang, Z and Yuan, Z},
title = {[Bioconversion of C1 gases and genetic engineering modification of gas-utilizing microorganisms].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {8},
pages = {3125-3142},
doi = {10.13345/j.cjb.220738},
pmid = {37622352},
issn = {1872-2075},
mesh = {*Gene Editing ; *Gases ; Carbon Dioxide ; Genetic Engineering ; Cloning, Molecular ; },
abstract = {C1 gases including CO, CO2 and CH4, are mainly derived from terrestrial biological activities, industrial waste gas and gasification syngas. Particularly, CO2 and CH4 are two of the most important greenhouse gases contributing to climate change. Bioconversion of C1 gases is not only a promising solution to addressing the problem of waste gases emission, but also a novel route to produce fuels or chemicals. In the past few years, C1-gas-utilizing microorganisms have drawn much attention and a variety of gene-editing technologies have been applied to improve their product yields or to expand product portfolios. This article reviewed the biological characteristics, aerobic or anaerobic metabolic pathways as well as the metabolic products of methanotrophs, autotrophic acetogens, and carboxydotrophic bacteria. In addition, gene-editing technologies (e.g. gene interruption technology using homologous recombination, group Ⅱ intron ClosTron technology, CRISPR/Cas gene editing and phage recombinase-mediated efficient integration of large DNA fragments) and their application in these C1-gas-utilizing microorganisms were also summarized.},
}
@article {pmid37620620,
year = {2023},
author = {Zhan, X and Tu, Z and Song, W and Nie, B and Li, S and Zhang, J and Zhang, F},
title = {Cas13a-based multiplex RNA targeting for potato virus Y.},
journal = {Planta},
volume = {258},
number = {4},
pages = {70},
pmid = {37620620},
issn = {1432-2048},
support = {32271912//National Natural Science Foundation of China/ ; 32271546//National Natural Science Foundation of China/ ; },
mesh = {*Potyvirus/genetics ; RNA ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; RNA Processing, Post-Transcriptional ; },
abstract = {The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR-Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA-gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants.},
}
@article {pmid37620295,
year = {2023},
author = {Deng, C and Li, S and Liu, Y and Bao, W and Xu, C and Zheng, W and Wang, M and Ma, X},
title = {Split-Cas9-based targeted gene editing and nanobody-mediated proteolysis-targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells.},
journal = {Clinical and translational medicine},
volume = {13},
number = {8},
pages = {e1382},
pmid = {37620295},
issn = {2001-1326},
support = {2018YFA0902804//National Key Research and Development Project of China/ ; 31670944//National Natural Science Foundation/ ; 81673345//National Natural Science Foundation/ ; 31870861//National Natural Science Foundation/ ; 17431904600//Science and Technology Innovation Action Plan of Shanghai/ ; },
mesh = {Humans ; Animals ; Survivin/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Proteolysis ; Apoptosis/genetics ; Disease Models, Animal ; *Neoplasms/genetics/therapy ; },
abstract = {BACKGROUND: Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary.<br><br>METHODS: Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression.<br><br>RESULTS: Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined.<br><br>CONCLUSIONS: In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.},
}
@article {pmid37488781,
year = {2023},
author = {Miranda, JA and Fenner, K and McKinzie, PB and Dobrovolsky, VN and Revollo, JR},
title = {Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.},
journal = {Environmental and molecular mutagenesis},
volume = {64},
number = {7},
pages = {374-381},
doi = {10.1002/em.22566},
pmid = {37488781},
issn = {1098-2280},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Escherichia coli/genetics ; Mutation ; DNA/genetics ; },
abstract = {DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10[-8] mutations per base pair. In this proof-of-principle study, we evaluated whether this technique could also detect the on- and off-target mutations generated by a cytosine-to-thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on-target mutant allele fractions ranging from ~7% to ~63%, depending on the single-guide RNA (sgRNA) used, while no on-target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3-fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on- and off-target mutations in cell populations that have undergone genome editing.},
}
@article {pmid37475654,
year = {2023},
author = {Choi, H and Park, SW and Oh, J and Kim, CS and Sung, GH and Sang, H},
title = {Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.},
journal = {FEMS microbiology letters},
volume = {370},
number = {},
pages = {},
doi = {10.1093/femsle/fnad072},
pmid = {37475654},
issn = {1574-6968},
support = {PJ01653303//Rural Development Administration/ ; 2020R1C1C1010108//National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems ; *Cordyceps/genetics ; Ribonucleoproteins/genetics ; Mutation ; },
abstract = {Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.},
}
@article {pmid37471041,
year = {2023},
author = {Liu, W and Zuo, S and Shao, Y and Bi, K and Zhao, J and Huang, L and Xu, Z and Lian, J},
title = {Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.},
journal = {Nucleic acids research},
volume = {51},
number = {15},
pages = {8293-8307},
pmid = {37471041},
issn = {1362-4962},
support = {2021YFC2103200//National Key Research and Development Program of China/ ; 22278361//National Natural Science Foundation of China/ ; LR20B060003//Natural Science Foundation of Zhejiang Province/ ; 226-2023-00015//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics/metabolism ; Metabolic Engineering/methods ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.},
}
@article {pmid37468054,
year = {2023},
author = {Wang, L and Fu, D and Weng, S and Xu, H and Liu, L and Guo, C and Ren, Y and Liu, Z and Han, X},
title = {Genome-scale CRISPR-Cas9 screening stratifies pancreatic cancer with distinct outcomes and immunotherapeutic efficacy.},
journal = {Cellular signalling},
volume = {110},
number = {},
pages = {110811},
doi = {10.1016/j.cellsig.2023.110811},
pmid = {37468054},
issn = {1873-3913},
mesh = {Humans ; *Early Detection of Cancer ; CRISPR-Cas Systems/genetics ; *Pancreatic Neoplasms/diagnosis/genetics/therapy ; Immunotherapy ; },
abstract = {Pancreatic cancer (PC) was featured by dramatic heterogeneity and dismal outcomes. An ideal classification strategy capable of achieving risk stratification and individualized treatment is urgently needed to significantly improve prognosis. In this study, using the 105 prognostic cancer essential genes identified by genome-scale CRISPR-Cas9 screening and univariate Cox analysis, we established and verified three heterogeneous subtypes via non-negative matrix factorization (NMF) and nearest template prediction (NTP) algorithms in the TCGA-PAAD cohort (176 samples) and four multi-center cohorts (233 samples), respectively. Among them, C1 with the worst prognosis was enriched in immune-related pathways, possessed superior infiltration abundance of immune cells and immune checkpoint molecules expression, and might be most sensitive to immunotherapy. C3, owing a moderate prognosis, might be featured by proliferative biological function, and despite its highest immunogenicity, the defects in antigen processing and presentation ability coupled with barren immune environment render it ineffective for immunotherapy, while it had potential sensitivity to paclitaxel and methotrexate. Besides, C2 harbored the best prognosis and was characterized by metabolism-related functions. These results could deepen our understanding of PC molecular heterogeneity and provide a trustworthy reference for prognostic stratification management and precision medicine in clinical practice.},
}
@article {pmid37464068,
year = {2023},
author = {Neldeborg, S and Soerensen, JF and Møller, CT and Bill, M and Gao, Z and Bak, RO and Holm, K and Sorensen, B and Nyegaard, M and Luo, Y and Hokland, P and Stougaard, M and Ludvigsen, M and Holm, CK},
title = {Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.},
journal = {Leukemia},
volume = {37},
number = {9},
pages = {1792-1801},
pmid = {37464068},
issn = {1476-5551},
support = {NA//Karen Elise Jensens Fond (Karen Elise Jensen Foundation)/ ; },
mesh = {Animals ; Mice ; RUNX1 Translocation Partner 1 Protein/genetics ; *Translocation, Genetic ; Introns/genetics ; Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Tumor Burden ; CRISPR-Cas Systems ; *Leukemia, Myeloid, Acute/genetics/therapy ; Cell Proliferation ; Oncogene Proteins, Fusion/genetics/metabolism ; },
abstract = {Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.},
}
@article {pmid37369005,
year = {2023},
author = {Dickson, KA and Field, N and Blackman, T and Ma, Y and Xie, T and Kurangil, E and Idrees, S and Rathnayake, SNH and Mahbub, RM and Faiz, A and Marsh, DJ},
title = {CRISPR single base-editing: in silico predictions to variant clonal cell lines.},
journal = {Human molecular genetics},
volume = {32},
number = {17},
pages = {2704-2716},
doi = {10.1093/hmg/ddad105},
pmid = {37369005},
issn = {1460-2083},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line ; Adenine/metabolism ; Cytosine/metabolism ; },
abstract = {Engineering single base edits using CRISPR technology including specific deaminases and single-guide RNA (sgRNA) is a rapidly evolving field. Different types of base edits can be constructed, with cytidine base editors (CBEs) facilitating transition of C-to-T variants, adenine base editors (ABEs) enabling transition of A-to-G variants, C-to-G transversion base editors (CGBEs) and recently adenine transversion editors (AYBE) that create A-to-C and A-to-T variants. The base-editing machine learning algorithm BE-Hive predicts which sgRNA and base editor combinations have the strongest likelihood of achieving desired base edits. We have used BE-Hive and TP53 mutation data from The Cancer Genome Atlas (TCGA) ovarian cancer cohort to predict which mutations can be engineered, or reverted to wild-type (WT) sequence, using CBEs, ABEs or CGBEs. We have developed and automated a ranking system to assist in selecting optimally designed sgRNA that considers the presence of a suitable protospacer adjacent motif (PAM), the frequency of predicted bystander edits, editing efficiency and target base change. We have generated single constructs containing ABE or CBE editing machinery, an sgRNA cloning backbone and an enhanced green fluorescent protein tag (EGFP), removing the need for co-transfection of multiple plasmids. We have tested our ranking system and new plasmid constructs to engineer the p53 mutants Y220C, R282W and R248Q into WT p53 cells and shown that these mutants cannot activate four p53 target genes, mimicking the behaviour of endogenous p53 mutations. This field will continue to rapidly progress, requiring new strategies such as we propose to ensure desired base-editing outcomes.},
}
@article {pmid37345937,
year = {2023},
author = {Reichard, WD and Smith, SE and Robertson, JB},
title = {BLINCAR: a reusable bioluminescent and Cas9-based genetic toolset for repeatedly modifying wild-type Scheffersomyces stipitis.},
journal = {mSphere},
volume = {8},
number = {4},
pages = {e0022423},
pmid = {37345937},
issn = {2379-5042},
mesh = {*Xylose ; *Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; Cellulose ; Ethanol ; Anti-Bacterial Agents ; },
abstract = {Scheffersomyces stipitis is a yeast that robustly ferments the 5-carbon sugar xylose, making the yeast a valuable candidate for lignocellulosic ethanol fermentation. However, the non-canonical codon usage of S. stipitis is an obstacle for implementing molecular tools that were developed for other yeast species, thereby limiting the molecular toolset available for S. stipitis. Here, we developed a series of molecular tools for S. stipitis including BLINCAR, a Bio-Luminescent Indicator that is Nullified by Cas9-Actuated Recombination, which can be used repeatedly to add different exogenous DNA payloads to the wild-type S. stipitis genome or used repeatedly to remove multiple native S. stipitis genes from the wild-type genome. Through the use of BLINCAR tools, one first produces antibiotic-resistant, bioluminescent colonies of S. stipitis whose bioluminescence highlights those clones that have been genetically modified; then second, once candidate clones have been confirmed, one uses a transient Cas9-producing plasmid to nullify the antibiotic resistance and bioluminescent markers from the prior introduction, thereby producing non-bioluminescent colonies that highlight those clones which have been re-sensitized to the antibiotic and are therefore susceptible to another round of BLINCAR implementation. IMPORTANCE Cellulose and hemicellulose that comprise a large portion of sawdust, leaves, and grass can be valuable sources of fermentable sugars for ethanol production. However, some of the sugars liberated from hemicellulose (like xylose) are not easily fermented using conventional glucose-fermenting yeast like Saccharomyces cerevisiae, so engineering robust xylose-fermenting yeast that is not inhibited by other components liberated from cellulose/hemicellulose will be important for maximizing yield and making lignocellulosic ethanol fermentation cost efficient. The yeast Scheffersomyces stipitis is one such yeast that can ferment xylose; however, it possesses several barriers to genetic manipulation. It is difficult to transform, has only a few antibiotic resistance markers, and uses an alternative genetic code from most other organisms. We developed a genetic toolset for S. stipitis that lowers these barriers and allows a user to deliver and/or delete multiple genetic elements to/from the wild-type genome, thereby expanding S. stipitis's potential.},
}
@article {pmid36958601,
year = {2023},
author = {Liang, Z and Wu, Y and Guo, Y and Wei, S},
title = {Addition of the T5 exonuclease increases the prime editing efficiency in plants.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {50},
number = {8},
pages = {582-588},
doi = {10.1016/j.jgg.2023.03.008},
pmid = {36958601},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Mutation ; Plants, Genetically Modified/genetics ; },
abstract = {Prime editing (PE) is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates, but is limited by low editing efficiency. We previously fused the M-MLV reverse transcriptase to the Cas9 nickase, generating the PE2 (v1) system, but the editing efficiency of this system is still low. Here we develop different versions of PE2 by adding the 5'-to-3' exonuclease at different positions of the nCas9-M-MLV RT fusion protein. PE2 (v2), in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions, deletions, and insertions at several genomic sites by 1.7- to 2.9-fold in plant cells compared to PE2 (v1). The improved editing efficiency of PE2 (v2) is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1, PE-P2, and PPE systems. Using PE2 (v2), we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites. The PE efficiency is further improved by combining PE2 (v2) and dual-pegRNAs. Taken together, the increased genome editing efficiency of PE2 (v2) developed in this study may enhance the applications of PE in plants.},
}
@article {pmid36878350,
year = {2023},
author = {Ryu, J and Adashi, EY and Hennebold, JD},
title = {The history, use, and challenges of therapeutic somatic cell and germline gene editing.},
journal = {Fertility and sterility},
volume = {120},
number = {3 Pt 1},
pages = {528-538},
doi = {10.1016/j.fertnstert.2023.02.040},
pmid = {36878350},
issn = {1556-5653},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Mutation ; Germ Cells ; Precision Medicine ; },
abstract = {The advent of directed gene-editing technologies now over 10 years ago ushered in a new era of precision medicine wherein specific disease-causing mutations can be corrected. In parallel with developing new gene-editing platforms, optimizing their efficiency and delivery has been remarkable. With their development, there has been interest in using gene-editing systems for correcting disease mutations in differentiated somatic cells ex vivo or in vivo or for germline gene editing in gametes or 1-cell embryos to potentially limit genetic diseases in the offspring and in future generations. This review details the development and history of the current gene-editing systems and the advantages and challenges in their use for somatic cell and germline gene editing.},
}
@article {pmid36781946,
year = {2023},
author = {Yaméogo, P and Gérard, C and Majeau, N and Tremblay, JP},
title = {Removal of the GAA repeat in the heart of a Friedreich's ataxia mouse model using CjCas9.},
journal = {Gene therapy},
volume = {30},
number = {7-8},
pages = {612-619},
pmid = {36781946},
issn = {1476-5462},
support = {//CIHR/Canada ; },
mesh = {Mice ; Animals ; *Friedreich Ataxia/genetics/therapy/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Most Friedreich ataxia (FRDA) cases are caused by the elongation of the GAA repeat (GAAr) sequence in the first intron of the FXN gene, leading to a decrease of the frataxin protein expression. Deletion of this GAAr with CRISPR/Cas9 technology leads to an increase in frataxin expression in vitro. We are therefore aiming to develop FRDA treatment based on the deletion of GAAr with CRISPR/Cas9 technology using a single AAV expressing a small Cas9 (CjCas9) and two single guide RNAs (sgRNAs) targeting the FXN gene. This AAV was intraperitoneally administrated to YG8sR (250-300 GAAr) and to YG8-800 (800 GAAr) mice. DNA and RNA were extracted from different organs a month later. PCR amplification of part of intron 1 of the FXN gene detected some GAAr deletion in some cells in heart and liver of both mouse models, but the editing rate was not sufficient to cause an increase in frataxin mRNA in the heart. However, the correlation observed between the editing rate and the distribution of AAV suggests a possible therapy based on the removal of the GAAr with a better delivery tool of the CRISPR/Cas9 system.},
}
@article {pmid34744169,
year = {2023},
author = {Simhadri, VL and McGill, JR and Sauna, ZE},
title = {Endotoxin contamination in commercially available Cas9 proteins potentially induces T-cell mediated responses.},
journal = {Gene therapy},
volume = {30},
number = {7-8},
pages = {575-580},
pmid = {34744169},
issn = {1476-5462},
support = {/FD/FDA HHS/United States ; },
mesh = {*T-Lymphocytes ; *CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Interferon-gamma/genetics ; Endotoxins/genetics ; },
abstract = {Immune responses to Cas proteins have been demonstrated recently and these may prove to be an impediment to their clinical use in gene editing. To make meaningful assessments of Cas9 immunogenicity during drug development and licensure it is imperative the reagents are free of impurities that could affect in vitro assessments of immunogenicity. Here we address the issue of endotoxin levels in laboratory grade Cas9 proteins used to measure T-cell memory responses. Many of these reagents have not been developed for immunogenicity assays, are or microbial origin and carry varying levels of endotoxin. The use of these reagents, off the shelf, without measuring endotoxin levels is likely to introduce incorrect estimates of the prevalence of memory T-cell responses in research studies. We demonstrate wide variation in endotoxin levels in Cas9 proteins from seven suppliers. Different lots from the same supplier also contained varying levels of endotoxin. ELISPOT assays showed similar large variations in the interferon-γ signals. Finally, when we carried out endotoxin depletion in four Cas9 proteins with strong signals in the ELISPOT assay, we found dampening of the interferon-γ signals.},
}
@article {pmid37619525,
year = {2023},
author = {Jose, B and Punetha, M and Tripathi, MK and Khanna, S and Yadav, V and Singh, AK and Kumar, B and Singh, K and Chouhan, VS and Sarkar, M},
title = {CRISPR/Cas mediated disruption of BMPR-1B gene and introduction of FecB mutation into the Caprine embryos using Easi-CRISPR strategy.},
journal = {Theriogenology},
volume = {211},
number = {},
pages = {125-133},
doi = {10.1016/j.theriogenology.2023.08.008},
pmid = {37619525},
issn = {1879-3231},
abstract = {Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1-2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments.},
}
@article {pmid37615340,
year = {2023},
author = {Luo, W and Zhang, Z and Zhou, D and Jiang, Y and Yang, J and He, B and Yu, H and Song, Y},
title = {Deep Tumor Penetration of CRISPR-Cas System for Photothermal-Sensitized Immunotherapy via Probiotics.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.3c02061},
pmid = {37615340},
issn = {1530-6992},
abstract = {Since central cells are more malignant and aggressive in solid tumors, improving penetration of therapeutic agents and activating immunity in tumor centers exhibit great potential in cancer therapies. Here, polydopamine-coated Escherichia coli Nissle 1917 (EcN) bearing CRISPR-Cas9 plasmid-loaded liposomes (Lipo-P) are applied for enhanced immunotherapy in deep tumors through activation of innate and adaptive immunity simultaneously. After accumulation in the tumor center through hypoxia targeting, Lipo-P could be detached under the reduction of reactive oxygen species (ROS)-responsive linkers, lowering the thermal resistance of cancer cells via Hsp90α depletion. Owing to that, heating induced by polydopamine upon near-infrared irradiation could achieve effective tumor ablation. Furthermore, mild photothermal therapy induces immunogenic cell death, as bacterial infections in tumor tissues trigger innate immunity. This bacteria-assisted approach provides a promising photothermal-sensitized immunotherapy in deep tumors.},
}
@article {pmid37612816,
year = {2023},
author = {Yin, W and Zhuang, J and Li, J and Xia, L and Hu, K and Yin, J and Mu, Y},
title = {Digital Recombinase Polymerase Amplification, Digital Loop-Mediated Isothermal Amplification, and Digital CRISPR-Cas Assisted Assay: Current Status, Challenges, and Perspectives.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2303398},
doi = {10.1002/smll.202303398},
pmid = {37612816},
issn = {1613-6829},
support = {62201497//National Natural Science Foundation of China/ ; LQ22F010005//Zhejiang Provincial Natural Science Foundation of China/ ; 2023RC053//Zhejiang Provincial Medical and Health Technology/ ; },
abstract = {Digital nucleic acid detection based on microfluidics technology can quantify the initial amount of nucleic acid in the sample with low equipment requirements and simple operations, which can be widely used in clinical and in vitro diagnosis. Recently, isothermal amplification technologies such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR-Cas) assisted technologies have become a hot spot of attention and state-of-the-art digital nucleic acid chips have provided a powerful tool for these technologies. Herein, isothermal amplification technologies including RPA, LAMP, and CRISPR-Cas assisted methods, based on digital nucleic acid microfluidics chips recently, have been reviewed. Moreover, the challenges of digital isothermal amplification and possible strategies to address them are discussed. Finally, future directions of digital isothermal amplification technology, such as microfluidic chip and device manufacturing, multiplex detection, and one-pot detection, are outlined.},
}
@article {pmid37612728,
year = {2023},
author = {Krill-Burger, JM and Dempster, JM and Borah, AA and Paolella, BR and Root, DE and Golub, TR and Boehm, JS and Hahn, WC and McFarland, JM and Vazquez, F and Tsherniak, A},
title = {Partial gene suppression improves identification of cancer vulnerabilities when CRISPR-Cas9 knockout is pan-lethal.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {192},
pmid = {37612728},
issn = {1474-760X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Genetic Techniques ; RNA Interference ; Cell Line ; },
abstract = {BACKGROUND: Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies-genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines.<br><br>RESULTS: We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line's dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies.<br><br>CONCLUSIONS: Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map.},
}
@article {pmid37610064,
year = {2023},
author = {Li, Y and Huang, C and Liu, Y and Zeng, J and Yu, H and Tong, Z and Yuan, X and Sui, X and Fang, D and Xiao, B and Zhao, S and Yuan, C},
title = {CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14159},
pmid = {37610064},
issn = {1467-7652},
support = {31860499//National Natural Science Foundation of China/ ; 2022530000241014//YNTC/ ; 2019530000241002//YNTC/ ; },
abstract = {CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.},
}
@article {pmid37609704,
year = {2023},
author = {Ma, X and Zhou, F and Yang, D and Chen, Y and Li, M and Wang, P},
title = {miRNA Detection for Prostate Cancer Diagnosis by miRoll-Cas: miRNA Rolling Circle Transcription for CRISPR-Cas Assay.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c02231},
pmid = {37609704},
issn = {1520-6882},
abstract = {Micro-RNA (miRNA) emerges as a promising type of biomarker for cancer diagnosis. There is an urgent need for developing rapid, convenient, and precise miRNA detection methods that may be conducted with limited laboratory facilities, especially in underdeveloped areas. Herein, we developed a miRNA detection method termed miRoll-Cas, where miRNA is first amplified by rolling circle transcription and then subject to CRISPR-Cas13a assay. Using miRoll-Cas, we realized the sensitive detection of multiple cancer-relevant miRNA markers (miR21, miR141, and Let7b) and specifically identified other variants of the Let7 family, which can accurately discriminate prostate cancer patients from healthy people. We envision that miRoll-Cas may be readily translated to clinical applications in the diagnosis of a variety of diseases beyond cancer.},
}
@article {pmid37608945,
year = {2023},
author = {Wang, L and Chen, X and Pan, F and Yao, G and Chen, J},
title = {Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1205765},
pmid = {37608945},
issn = {1664-302X},
abstract = {Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10[-5] ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.},
}
@article {pmid37608792,
year = {2023},
author = {Liu, P and Huang, L and Song, CQ},
title = {Editorial: Genome editing applications of CRISPR/Cas9 in metabolic diseases, hormonal system and cancer research.},
journal = {Frontiers in endocrinology},
volume = {14},
number = {},
pages = {1256966},
pmid = {37608792},
issn = {1664-2392},
mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; *Neoplasms/genetics/therapy ; *Metabolic Diseases/genetics/therapy ; Research ; },
}
@article {pmid37607619,
year = {2023},
author = {Dhingra, Y and Sashital, DG},
title = {Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {105178},
doi = {10.1016/j.jbc.2023.105178},
pmid = {37607619},
issn = {1083-351X},
abstract = {CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn[2+] dependent, Cas1 preferentially uses Mg[2+] over Mn[2+]. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.},
}
@article {pmid37606708,
year = {2023},
author = {Gautam, R and Shukla, P and Kirti, PB},
title = {Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {136},
number = {9},
pages = {195},
pmid = {37606708},
issn = {1432-2242},
mesh = {Male ; Humans ; Cytoplasm ; *Seeds/genetics ; Fertility ; Pollen ; *Infertility, Male ; },
abstract = {The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.},
}
@article {pmid37486064,
year = {2023},
author = {Bielczyk-Maczynska, E and Sharma, D and Blencowe, M and Saliba Gustafsson, P and Gloudemans, MJ and Yang, X and Carcamo-Orive, I and Wabitsch, M and Svensson, KJ and Park, CY and Quertermous, T and Knowles, JW and Li, J},
title = {A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.},
journal = {American journal of physiology. Cell physiology},
volume = {325},
number = {3},
pages = {C648-C660},
doi = {10.1152/ajpcell.00148.2023},
pmid = {37486064},
issn = {1522-1563},
support = {R01 DK125260/DK/NIDDK NIH HHS/United States ; UM1 HG011972/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; Adipogenesis/genetics ; Adipocytes/metabolism ; Cell Line ; *Metabolic Diseases/metabolism ; Cell Differentiation/genetics ; },
abstract = {CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW &amp; NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.},
}
@article {pmid36877372,
year = {2023},
author = {Dawes, P and Murray, LF and Olson, MN and Barton, NJ and Smullen, M and Suresh, M and Yan, G and Zhang, Y and Fernandez-Fontaine, A and English, J and Uddin, M and Pak, C and Church, GM and Chan, Y and Lim, ET},
title = {oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids.},
journal = {Human genetics},
volume = {142},
number = {8},
pages = {1281-1291},
pmid = {36877372},
issn = {1432-1203},
support = {R01 MH113279/MH/NIMH NIH HHS/United States ; RM1 HG008525/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Organoids ; Genotype ; },
abstract = {Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin[+] cells and decreased proportions of TRA-1-60[+] cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.},
}
@article {pmid37605748,
year = {2023},
author = {Tong, C and Liang, Y and Zhang, Z and Wang, S and Zheng, X and Liu, Q and Song, B},
title = {Review of knockout technology approaches in bacterial drug resistance research.},
journal = {PeerJ},
volume = {11},
number = {},
pages = {e15790},
pmid = {37605748},
issn = {2167-8359},
mesh = {*Craniocerebral Trauma ; Drug Resistance, Bacterial/genetics ; Gene Knockout Techniques ; Genes, Bacterial ; Technology ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; Animals ; },
abstract = {Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.},
}
@article {pmid37605597,
year = {2023},
author = {S Teixeira, LF and Bellini, MH},
title = {NF-ĸΒ1 knockout reduces IL6 expression under hypoxia in renal cell carcinoma.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {69},
number = {6},
pages = {8-14},
doi = {10.14715/cmb/2023.69.6.2},
pmid = {37605597},
issn = {1165-158X},
mesh = {Adult ; Humans ; *Carcinoma, Renal Cell/genetics ; Interleukin-6/genetics ; NF-kappa B/genetics ; *Kidney Neoplasms/genetics ; *Craniocerebral Trauma ; Hypoxia ; },
abstract = {Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer, accounting for more than 90% of all renal neoplasms. Clear cell RCC (ccRCC) is the most common subtype of RCC. Most patients with ccRCC have a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a protein that downregulates various intracellular proteins, including hypoxia-inducible factor (HIF). Many molecules have been identified to be responsible for the aggressive phenotype of ccRCC, including the transcription factor nuclear factor kappa B (NF-кB). The increase in NF-кB activity observed in RCC is correlated with an increase in angiogenesis markers, such as interleukin 6 (IL-6). In recent years, several groups have demonstrated the functional role of NF-кB1 in RCC tumorigenicity. Herein, we used the CRISPR/Cas-9 technique to obtain an NF-кB1 knockout-human renal adenocarcinoma cell line. Expression of IL-6 at the mRNA and protein levels was analyzed under normoxia and hypoxia by real time-polymerase chain reaction and multiplex assay, respectively. The CRISPR/Cas9 technique was effective in producing 786-0 knockout cells for NF-κB1 (p105/p50), as confirmed by western blot analysis. Suppression of p50 expression in 786-0 single guide RNA (sg)1, 786-0 sg2 and 786-0 sg3 cells downregulated IL-6 mRNA and protein expression under normoxia and hypoxia. The observed decrease in the differential expression of IL-6 in hypoxia/normoxia is suggestive of a change in cellular responsiveness to hypoxia with respect to IL-6.},
}
@article {pmid37603175,
year = {2023},
author = {Mencarelli, G and Pieroni, B and Murphy, KM and Gargaro, M},
title = {Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2700},
number = {},
pages = {77-92},
pmid = {37603175},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *CD40 Antigens ; Cytokines ; Genes, MHC Class II ; Pathogen-Associated Molecular Pattern Molecules ; Dendritic Cells ; },
abstract = {Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.},
}
@article {pmid37601766,
year = {2023},
author = {Mazloum, A and Karagyaur, M and Chernyshev, R and van Schalkwyk, A and Jun, M and Qiang, F and Sprygin, A},
title = {Post-genomic era in agriculture and veterinary science: successful and proposed application of genetic targeting technologies.},
journal = {Frontiers in veterinary science},
volume = {10},
number = {},
pages = {1180621},
pmid = {37601766},
issn = {2297-1769},
abstract = {Gene editing tools have become an indispensable part of research into the fundamental aspects of cell biology. With a vast body of literature having been generated based on next generation sequencing technologies, keeping track of this ever-growing body of information remains challenging. This necessitates the translation of genomic data into tangible applications. In order to address this objective, the generated Next Generation Sequencing (NGS) data forms the basis for targeted genome editing strategies, employing known enzymes of various cellular machinery, in generating organisms with specifically selected phenotypes. This review focuses primarily on CRISPR/Cas9 technology in the context of its advantages over Zinc finger proteins (ZNF) and Transcription activator-like effector nucleases (TALEN) and meganucleases mutagenesis strategies, for use in agricultural and veterinary applications. This review will describe the application of CRISPR/Cas9 in creating modified organisms with custom-made properties, without the undesired non-targeted effects associated with virus vector vaccines and bioactive molecules produced in bacterial systems. Examples of the successful and unsuccessful applications of this technology to plants, animals and microorganisms are provided, as well as an in-depth look into possible future trends and applications in vaccine development, disease resistance and enhanced phenotypic traits will be discussed.},
}
@article {pmid37598400,
year = {2023},
author = {Hu, S and Chen, Y and Xie, D and Xu, K and Fu, Y and Chi, W and Liu, H and Huang, J},
title = {Nme2 Cas9-mediated therapeutic editing in inhibiting angiogenesis after wet age-related macular degeneration onset.},
journal = {Clinical and translational medicine},
volume = {13},
number = {8},
pages = {e1383},
pmid = {37598400},
issn = {2001-1326},
support = {31971365//National Natural Science Foundation of China/ ; 32001063//National Natural Science Foundation of China/ ; 82271688//National Natural Science Foundation of China/ ; 2019BT02Y276//Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program/ ; 2020B1515120090//Guangdong Basic and Applied Basic Research Foundation/ ; 2023A1515010176//Guangdong Basic and Applied Basic Research Foundation/ ; 2023A04J1952//Guangzhou Science and Technology Planning Project/ ; 23ptpy59//Fundamental Research Funds for the Central Universities, Sun Yat-sen University/ ; },
mesh = {Aged ; Humans ; *Vascular Endothelial Growth Factor A/genetics ; CRISPR-Cas Systems/genetics ; *Macular Degeneration/genetics/therapy ; Immunotherapy ; Gene Editing ; },
abstract = {BACKGROUND: Age-related macular degeneration (AMD), particularly wet AMD characterised by choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) pathway contributes to CNV pathogenesis. Previous gene editing research indicated that disrupting these genes in retinal pigment epithelial cells could have a preventive effect on CNV progression. However, no studies have yet been conducted using gene editing to disrupt VEGF signalling after CNV induction for therapeutic validation, which is critical to the clinical application of wet AMD gene editing therapies.<br><br>METHOD: Here, we employed the single-adeno-associated virus-mediated Nme2 Cas9 to disrupt key molecules in VEGF signalling, Hif1&#x3b1;, Vegfa and Vegfr2 after inducing CNV and estimated their therapeutic effects.<br><br>RESULTS: We found that Nme2 Cas9 made efficient editing in target genes up to 71.8% post 11 days in vivo. And only Nme2 Cas9-Vegfa treatment during the early stage of CNV development reduced the CNV lesion area by 49.5%, compared to the negative control, while Nme2 Cas9-Hif1&#x3b1; or Nme2 Cas9-Vegfr2 treatment did not show therapeutic effect. Besides, no off-target effects were observed in Nme2 Cas9-mediated gene editing in vivo.<br><br>CONCLUSIONS: This study provides proof-of-concept possibility of employing Nme2 Cas9 for potential anti-angiogenesis therapy in wet AMD.},
}
@article {pmid37596335,
year = {2023},
author = {Yuzbashev, TV and Yuzbasheva, EY and Melkina, OE and Patel, D and Bubnov, D and Dietz, H and Ledesma-Amaro, R},
title = {A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {858},
pmid = {37596335},
issn = {2399-3642},
support = {BB/R01602X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering ; Engineering ; Escherichia coli/genetics ; Gene Library ; },
abstract = {CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid.},
}
@article {pmid37595758,
year = {2023},
author = {Dettmer-Monaco, V and Weißert, K and Ammann, S and Monaco, G and Lei, L and Gräßel, L and Rhiel, M and Rositzka, J and Kaufmann, MM and Geiger, K and Andrieux, G and Lao, J and Thoulass, G and Schell, C and Boerries, M and Illert, AL and Cornu, TI and Ehl, S and Aichele, P and Cathomen, T},
title = {Gene editing of hematopoietic stem cells restores T cell response in familial hemophagocytic lymphohistiocytosis.},
journal = {The Journal of allergy and clinical immunology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jaci.2023.08.003},
pmid = {37595758},
issn = {1097-6825},
abstract = {BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for ∼30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene, which result in impaired degranulation of cytotoxic vesicles and hence compromised T and NK cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality.<br><br>OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site (cSD) in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV).<br><br>METHODS: We employed CRISPR-Cas technology to delete the disease-underlying mutation in HSCs, and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and CAST-Seq based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection.<br><br>RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wildtype cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH.<br><br>CONCLUSION: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T cell response in the absence of genotoxicity-associated clonal outgrowth.},
}
@article {pmid37595547,
year = {2023},
author = {Wilbanks, B and Pearson, K and Maher, LJ},
title = {A non-rational approach to optimized targeting of CRISPR-Cas9 complexes.},
journal = {Cell chemical biology},
volume = {30},
number = {8},
pages = {855-857},
doi = {10.1016/j.chembiol.2023.07.012},
pmid = {37595547},
issn = {2451-9448},
mesh = {*CRISPR-Cas Systems/genetics ; *Genomics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In this issue of Cell Chemical Biology, Bush et al.[1] report an in vitro selection method for optimizing CRISPR-Cas9 single-guide RNAs. This approach may be useful in targeting previously intractable genomic sequences. The results also provide insights into which positions in single-guide RNAs are most amenable to modification.},
}
@article {pmid37594269,
year = {2023},
author = {Barrangou, R},
title = {CRISPR Milestones for Sustainable Agriculture and Forestry.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {303-304},
doi = {10.1089/crispr.2023.29163.editorial},
pmid = {37594269},
issn = {2573-1602},
mesh = {*Forestry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid35474898,
year = {2021},
author = {Kramme, C and Plesa, AM and Wang, HH and Wolf, B and Smela, MP and Guo, X and Kohman, RE and Chatterjee, P and Church, GM},
title = {An integrated pipeline for mammalian genetic screening.},
journal = {Cell reports methods},
volume = {1},
number = {6},
pages = {100082},
pmid = {35474898},
issn = {2667-2375},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mammals/genetics ; Gene Library ; Genetic Testing ; },
abstract = {With the recent advancements in genome editing, next-generation sequencing (NGS), and scalable cloning techniques, scientists can now conduct genetic screens at unprecedented levels of scale and precision. With such a multitude of technologies, there is a need for a simple yet comprehensive pipeline to enable systematic mammalian genetic screening. In this study, we develop unique algorithms for target identification and a toxin-less Gateway cloning tool, termed MegaGate, for library cloning which, when combined with existing genetic perturbation methods and NGS-coupled readouts, enable versatile engineering of relevant mammalian cell lines. Our integrated pipeline for sequencing-based target ascertainment and modular perturbation screening (STAMPScreen) can thus be utilized for a host of cell state engineering applications.},
}
@article {pmid34935002,
year = {2021},
author = {Xiong, K and Karottki, KJC and Hefzi, H and Li, S and Grav, LM and Li, S and Spahn, P and Lee, JS and Ventina, I and Lee, GM and Lewis, NE and Kildegaard, HF and Pedersen, LE},
title = {An optimized genome-wide, virus-free CRISPR screen for mammalian cells.},
journal = {Cell reports methods},
volume = {1},
number = {4},
pages = {},
pmid = {34935002},
issn = {2667-2375},
support = {R35 GM119850/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Cricetinae ; *CRISPR-Cas Systems/genetics ; CHO Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetulus ; Genome ; Lentivirus/genetics ; },
abstract = {Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability.},
}
@article {pmid37594268,
year = {2023},
author = {Karvelis, T and Siksnys, V},
title = {Fanzors: Mysterious TnpB-Like Bacterial Transposon-Related RNA-Guided DNA Nucleases of Eukaryotes.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {310-312},
doi = {10.1089/crispr.2023.29164.tka},
pmid = {37594268},
issn = {2573-1602},
mesh = {*Eukaryota ; *CRISPR-Cas Systems ; Gene Editing ; RNA ; },
}
@article {pmid37594267,
year = {2023},
author = {Willmann, MR},
title = {CRISPR and the Plant Pathologists' Holy Grail.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {308-309},
doi = {10.1089/crispr.2023.29165.mwi},
pmid = {37594267},
issn = {2573-1602},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Pathologists ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37593347,
year = {2023},
author = {Zhang, M and Zhu, Z and Xun, G and Zhao, H},
title = {To Cut or not to Cut: Next-generation Genome Editors for Precision Genome Engineering.},
journal = {Current opinion in biomedical engineering},
volume = {28},
number = {},
pages = {},
pmid = {37593347},
issn = {2468-4511},
support = {U54 DK107965/DK/NIDDK NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; },
abstract = {Since the original report of repurposing the CRISPR/Cas9 system for genome engineering, the past decade has witnessed profound improvement in our ability to efficiently manipulate the mammalian genome. However, significant challenges lie ahead that hinder the translation of CRISPR-based gene editing technologies into safe and effective therapeutics. The CRISPR systems often have a limited target scope due to PAM restrictions, and the off-target activity also poses serious risks for therapeutic applications. Moreover, the first-generation genome editors typically achieve desired genomic modifications by inducing double-strand breaks (DSBs) at target site(s). Despite being highly efficient, this "cut and fix" strategy is less favorable in clinical settings due to drawbacks associated with the nuclease-induced DSBs. In this review, we focus on recent advances that help address these challenges, including the engineering and discovery of novel CRISPR/Cas systems with improved functionalities and the development of DSB-free genome editors.},
}
@article {pmid37591976,
year = {2023},
author = {Zhan, X and Liu, W and Nie, B and Zhang, F and Zhang, J},
title = {Cas13d-mediated multiplex RNA targeting confers a broad-spectrum resistance against RNA viruses in potato.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {855},
pmid = {37591976},
issn = {2399-3642},
support = {32271912//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31600408//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {RNA ; *Solanum tuberosum/genetics ; *RNA Viruses ; RNA Processing, Post-Transcriptional ; Archaea ; Crops, Agricultural ; },
abstract = {CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses.},
}
@article {pmid37591832,
year = {2023},
author = {Geurts, MH and Gandhi, S and Boretto, MG and Akkerman, N and Derks, LLM and van Son, G and Celotti, M and Harshuk-Shabso, S and Peci, F and Begthel, H and Hendriks, D and Schürmann, P and Andersson-Rolf, A and Chuva de Sousa Lopes, SM and van Es, JH and van Boxtel, R and Clevers, H},
title = {One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4998},
pmid = {37591832},
issn = {2041-1723},
support = {C6307/A29058//Cancer Research UK (CRUK)/ ; C6307/A29058//Cancer Research UK (CRUK)/ ; },
mesh = {Adult ; Humans ; *RNA, Guide, CRISPR-Cas Systems ; Oncogenes ; Carcinogenesis/genetics ; Cell Transformation, Neoplastic ; *Adult Stem Cells ; Organoids ; },
abstract = {Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.},
}
@article {pmid37591406,
year = {2023},
author = {Peng, K and Chen, X and Pei, K and Wang, X and Ma, X and Liang, C and Dong, Q and Liu, Z and Han, M and Liu, G and Yang, H and Zheng, M and Liu, G and Gao, M},
title = {Lipodystrophic gene Agpat2 deficiency aggravates hyperlipidemia and atherosclerosis in Ldlr[-/-] mice.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {},
number = {},
pages = {166850},
doi = {10.1016/j.bbadis.2023.166850},
pmid = {37591406},
issn = {1879-260X},
abstract = {AIMS: Dysfunction of adipose tissue increases the risk of cardiovascular disease. It was well established that obesity aggravates atherosclerosis, but the effect of adipose tissue loss on atherosclerosis has been less studied. Agpat2 is the first causative gene of congenital generalized lipodystrophy (CGL), but the role of Agpat2 on atherosclerosis has not been reported. Hypertriglyceridemia is one of the clinical manifestations of CGL patients, but it is usually absent in CGL mouse model on a normal diet. This study will investigate the effect of Agpat2 on hyperlipidemia and atherosclerosis.<br><br>METHODS AND RESULTS: In this study, Agpat2 knockout (Agpat2[-/-]) mice were generated using CRISPR/Cas system, which showed severe loss of adipose tissue and fatty liver, consistent with previous reports. Agpat2[-/-] mice were then crossed with hypercholesterolemic and atherosclerotic prone LDL receptor knockout (Ldlr[-/-]) mice to obtain double knockout mouse model (Agpat2[-/-]Ldlr[-/-]). Plasma lipid profile, insulin resistance, fatty liver, and atherosclerotic lesions were observed after 12&#x202f;weeks of the atherogenic high-fat diet (HFD) feeding. We found that compared with Ldlr[-/-] mice, Agpat2[-/-]Ldlr[-/-] mice showed significantly higher plasma total cholesterol and triglycerides after HFD feeding. Agpat2[-/-]Ldlr[-/-] mice also developed hyperglycemia and hyperinsulinemia, with increased pancreatic islet area. The liver weight of Agpat2[-/-]Ldlr[-/-] mice was about 4 times higher than that of Ldlr[-/-] mice. The liver lipid deposition was severe and Sirius red staining showed liver fibrosis. In addition, in Agpat2[-/-]Ldlr[-/-] mice, the area of atherosclerotic lesions in aortic arch and aortic root was significantly increased.<br><br>CONCLUSIONS: Our results show that Agpat2 deficiency led to more severe hyperlipidemia, liver fibrosis and aggravation of atherosclerosis in Ldlr[-/-] mice. This study provided additional insights into the role of adipose tissue in hyperlipidemia and atherosclerosis.},
}
@article {pmid37402217,
year = {2023},
author = {Tripathi, L and Ntui, VO and Tripathi, JN and Norman, D and Crawford, J},
title = {A new and novel high-fidelity genome editing tool for banana using Cas-CLOVER.},
journal = {Plant biotechnology journal},
volume = {21},
number = {9},
pages = {1731-1733},
pmid = {37402217},
issn = {1467-7652},
support = {//CGIAR Research Program for root, tubers ad banana/ ; },
mesh = {*Gene Editing ; *Musa/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
}
@article {pmid37378559,
year = {2023},
author = {Jiang, T and Hu, X and Shen, J},
title = {Establishment of a Novel Detection Platform for Clostridioides difficile Toxin Genes Based on Orthogonal CRISPR.},
journal = {Microbiology spectrum},
volume = {11},
number = {4},
pages = {e0188623},
pmid = {37378559},
issn = {2165-0497},
support = {Grant No. GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Departtment of Education/ ; Grant No. AHWJ2021a011//2021 Anhui Provincial Health and Health Commission Key Scientific Research Project/ ; },
mesh = {*Clostridioides difficile/isolation &amp; purification ; *Clostridium Infections/diagnosis ; *CRISPR-Cas Systems ; *Genetic Techniques ; Chromatography, Affinity/methods ; Sensitivity and Specificity ; Humans ; },
abstract = {Clostridioides difficile is one of the leading pathogens causing nosocomial infection. The infection can range from mild to severe, and rapid identification is pivotal for early clinical diagnosis and appropriate treatment. Here, a genetic testing platform for toxins, referred to as OC-MAB (orthogonal CRISPR system combined with multiple recombinase polymerase amplification [RPA]), was developed to detect the C. difficile toxin genes tcdA and tcdB. While recognizing the amplified products of the tcdA gene and the tcdB gene, Cas13a and Cas12a could activate their cleavage activities to cut labeled RNA and DNA probes, respectively. The cleaved products were subsequently identified by dual-channel fluorescence using a quantitative PCR (qPCR) instrument. Finally, they could also be combined with labeled antibodies on immunochromatographic test strips to achieve visual detection. The OC-MAB platform exhibited ultrahigh sensitivity in detecting the tcdA and tcdB genes at levels of as low as 10[2] to 10[1] copies/mL. When testing 72 clinical stool samples, the sensitivity (95% confidence interval [CI], 0.90, 1) and specificity (95% CI, 0.84, 1) of the single-tube method based on the fluorescence readout was 100%, with a positive predictive value (PPA) value of 100% (95% CI, 0.90, 1) and a negative predictive value (NPA) value of 100% (95% CI, 0.84, 1), compared to the results of qPCR. Likewise, the sensitivity of the 2-step method based on the test strip readout was 100% (95% CI, 0.90, 1), while the specificity was 96.3% (95% CI, 0.79, 0.99), with a PPA of 98% (95% CI, 0.87, 0.99) and an NPA of 100% (95% CI, 0.90, 1). In short, orthogonal CRISPR technology is a promising tool for the detection of C. difficile toxin genes. IMPORTANCE C. difficile is currently the primary causative agent of hospital-acquired antibiotic-induced diarrhea, and timely and accurate diagnosis is crucial for hospital-acquired infection control and epidemiological investigation. Here, a new method for the identification of C. difficile was developed based on the recently popular CRISPR technology, and an orthogonal CRISPR dual system was utilized for the simultaneous detection of toxin genes A and B. It also uses a currently rare CRISPR dual-target lateral flow strip with powerful color-changing capabilities, which is appropriate for point-of-care testing (POCT).},
}
@article {pmid37323119,
year = {2023},
author = {Liu, X and Yu, Y and Yao, W and Yin, Z and Wang, Y and Huang, Z and Zhou, JQ and Liu, J and Lu, X and Wang, F and Zhang, G and Chen, G and Xiao, Y and Deng, H and Tang, W},
title = {CRISPR/Cas9-mediated simultaneous mutation of three salicylic acid 5-hydroxylase (OsS5H) genes confers broad-spectrum disease resistance in rice.},
journal = {Plant biotechnology journal},
volume = {21},
number = {9},
pages = {1873-1886},
pmid = {37323119},
issn = {1467-7652},
support = {32172078//National Natural Science Foundation of China/ ; U22A20502//National Natural Science Foundation of China/ ; 2021JJ40238//Natural Science Foundation of Hunan Province/ ; 2022JJ30285//Natural Science Foundation of Hunan Province/ ; 2021JC0007//the Science and Technology Innovation Program of Hunan Province/ ; 2021NK1001//the Science and Technology Innovation Program of Hunan Province/ ; 2023NK1010//the Science and Technology Innovation Program of Hunan Province/ ; },
mesh = {Disease Resistance/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Salicylic Acid/pharmacology/metabolism ; Mutation/genetics ; *Oryza/metabolism ; Plant Diseases/microbiology ; *Xanthomonas ; Gene Expression Regulation, Plant ; },
abstract = {Salicylic acid (SA) is an essential plant hormone that plays critical roles in basal defence and amplification of local immune responses and establishes resistance against various pathogens. However, the comprehensive knowledge of the salicylic acid 5-hydroxylase (S5H) in rice-pathogen interaction is still elusive. Here, we reported that three OsS5H homologues displayed salicylic acid 5-hydroxylase activity, converting SA into 2,5-dihydroxybenzoic acid (2,5-DHBA). OsS5H1, OsS5H2, and OsS5H3 were preferentially expressed in rice leaves at heading stage and responded quickly to exogenous SA treatment. We found that bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) strongly induced the expression of OsS5H1, OsS5H2, and OsS5H3. Rice plants overexpressing OsS5H1, OsS5H2, and OsS5H3 showed significantly decreased SA contents and increased 2,5-DHBA levels, and were more susceptible to bacterial blight and rice blast. A simple single guide RNA (sgRNA) was designed to create oss5h1oss5h2oss5h3 triple mutants through CRISPR/Cas9-mediated gene mutagenesis. The oss5h1oss5h2oss5h3 exhibited stronger resistance to Xoo than single oss5h mutants. And oss5h1oss5h2oss5h3 plants displayed enhanced rice blast resistance. The conferred pathogen resistance in oss5h1oss5h2oss5h3 was attributed to the significantly upregulation of OsWRKY45 and pathogenesis-related (PR) genes. Besides, flg22-induced reactive oxygen species (ROS) burst was enhanced in oss5h1oss5h2oss5h3. Collectively, our study provides a fast and effective approach to generate rice varieties with broad-spectrum disease resistance through OsS5H gene editing.},
}
@article {pmid37590503,
year = {2023},
author = {Craft, J and Truong, T and Penn, BH},
title = {High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {198},
pages = {},
doi = {10.3791/65264},
pmid = {37590503},
issn = {1940-087X},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Macrophages ; Cell Line ; Electroporation ; },
abstract = {Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.},
}
@article {pmid37590316,
year = {2023},
author = {Dardiry, M and Eberhard, G and Witte, H and Rödelsperger, C and Lightfoot, JW and Sommer, RJ},
title = {Divergent combinations of cis-regulatory elements control the evolution of phenotypic plasticity.},
journal = {PLoS biology},
volume = {21},
number = {8},
pages = {e3002270},
doi = {10.1371/journal.pbio.3002270},
pmid = {37590316},
issn = {1545-7885},
abstract = {The widespread occurrence of phenotypic plasticity across all domains of life demonstrates its evolutionary significance. However, how plasticity itself evolves and how it contributes to evolution is poorly understood. Here, we investigate the predatory nematode Pristionchus pacificus with its feeding structure plasticity using recombinant-inbred-line and quantitative-trait-locus (QTL) analyses between natural isolates. We show that a single QTL at a core developmental gene controls the expression of the cannibalistic morph. This QTL is composed of several cis-regulatory elements. Through CRISPR/Cas-9 engineering, we identify copy number variation of potential transcription factor binding sites that interacts with a single intronic nucleotide polymorphism. Another intronic element eliminates gene expression altogether, mimicking knockouts of the locus. Comparisons of additional isolates further support the rapid evolution of these cis-regulatory elements. Finally, an independent QTL study reveals evidence for parallel evolution at the same locus. Thus, combinations of cis-regulatory elements shape plastic trait expression and control nematode cannibalism.},
}
@article {pmid37588594,
year = {2023},
author = {van der Wal, YA and Nordli, H and Akandwanaho, A and Greiner-Tollersrud, L and Kool, J and Jørgensen, JB},
title = {CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1214912},
pmid = {37588594},
issn = {1664-3224},
mesh = {Animals ; *Salmonidae/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Cell Line ; Salmon/genetics ; *Infectious pancreatic necrosis virus ; Mammals ; },
abstract = {BACKGROUND: Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.<br><br>METHODS: By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.<br><br>RESULTS: We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.<br><br>CONCLUSION: Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.},
}
@article {pmid37588264,
year = {2023},
author = {Huang, S and Baskin, JM},
title = {Adding a Chemical Biology Twist to CRISPR Screening.},
journal = {Israel journal of chemistry},
volume = {63},
number = {1-2},
pages = {},
pmid = {37588264},
issn = {0021-2148},
support = {R01 GM131101/GM/NIGMS NIH HHS/United States ; R01 GM143367/GM/NIGMS NIH HHS/United States ; T32 GM138826/GM/NIGMS NIH HHS/United States ; },
abstract = {In less than a decade, CRISPR screening has revolutionized forward genetics and cell and molecular biology. Advances in screening technologies, including sgRNA libraries, Cas9-expressing cell lines, and streamlined sequencing pipelines, have democratized pooled CRISPR screens at genome-wide scale. Initially, many such screens were survival-based, identifying essential genes in physiological or perturbed processes. With the application of new chemical biology tools to CRISPR screening, the phenotypic space is no longer limited to live/dead selection or screening for levels of conventional fluorescent protein reporters. Further, the resolution has been increased from cell populations to single cells or even the subcellular level. We highlight advances in pooled CRISPR screening, powered by chemical biology, that have expanded phenotypic space, resolution, scope, and scalability as well as strengthened the CRISPR/Cas enzyme toolkit to enable biological hypothesis generation and discovery.},
}
@article {pmid37587688,
year = {2023},
author = {Li, X and Liao, F and Gao, J and Song, G and Zhang, C and Ji, N and Wang, X and Wen, J and He, J and Wei, Y and Zhang, H and Li, Z and Yu, G and Yin, H},
title = {Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad669},
pmid = {37587688},
issn = {1362-4962},
support = {2022YFC3400400//National Key R&amp;D Program of China/ ; 32200496//National Natural Science Foundation of China/ ; 2022KJ192//Scientific Research Program of Tianjin Municipal Education Commission/ ; ZRMS2022000096//Hubei Provincial Natural Science Foundation/ ; //Scientific Research Program of Hubei Provincial Department of Education/ ; },
abstract = {CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.},
}
@article {pmid37587435,
year = {2023},
author = {Liu, W and Wang, X and Liu, R and Liao, Y and Peng, Z and Jiang, H and Jing, Q and Xing, Y},
title = {Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.},
journal = {BMC biotechnology},
volume = {23},
number = {1},
pages = {29},
pmid = {37587435},
issn = {1472-6750},
support = {2016ZX08006-003//National Science and Technology Major Project/ ; 31771372//National Natural Science Foundation of China/ ; },
mesh = {Swine ; Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Fetus ; Electroporation ; Fibroblasts ; },
abstract = {BACKGROUND: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips.<br><br>METHODS: We firstly delivered a 13&#xa0;kb Cas9-EGFP and a 3.5&#xa0;kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13&#xa0;kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector&#x2122; P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs.<br><br>RESULTS: Notably lower transfection efficiencies were observed when delivering the 13&#xa0;kb vector than delivering the 3.5&#xa0;kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113&#x2009;+&#x2009;CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13&#xa0;kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113&#x2009;+&#x2009;CA-137 and CA-137&#x2009;+&#x2009;CA-137 programs showed significantly superior (P&#x2009;<&#x2009;0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer.<br><br>CONCLUSIONS: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector&#x2122; system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector&#x2122; P3 buffer.},
}
@article {pmid37584388,
year = {2023},
author = {Doering, L and Cornean, A and Thumberger, T and Benjaminsen, J and Wittbrodt, B and Kellner, T and Hammouda, OT and Gorenflo, M and Wittbrodt, J and Gierten, J},
title = {CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease.},
journal = {Disease models &amp; mechanisms},
volume = {16},
number = {8},
pages = {},
doi = {10.1242/dmm.049811},
pmid = {37584388},
issn = {1754-8411},
support = {810172/ERC_/European Research Council/International ; },
mesh = {Humans ; Child ; *Gene Editing ; Mutation/genetics ; Point Mutation ; Transcription Factors/metabolism ; *Heart Defects, Congenital/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.},
}
@article {pmid37584144,
year = {2023},
author = {Chen, L and Deng, L and Sun, W and Liu, J and Zhang, T and Li, S},
title = {[Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {7},
pages = {3003-3014},
doi = {10.13345/j.cjb.221052},
pmid = {37584144},
issn = {1872-2075},
mesh = {Animals ; Male ; Female ; Mice ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Rad51 Recombinase/genetics ; Mice, Inbred C57BL ; Disease Models, Animal ; Recombination, Genetic ; },
abstract = {The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.},
}
@article {pmid37552581,
year = {2023},
author = {Torello Pianale, L and Olsson, L},
title = {ScEnSor Kit for Saccharomyces cerevisiae Engineering and Biosensor-Driven Investigation of the Intracellular Environment.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2493-2497},
doi = {10.1021/acssynbio.3c00124},
pmid = {37552581},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems ; Plasmids/genetics ; Genome, Fungal ; },
abstract = {In this study, the three-step build-transform-assess toolbox for real-time monitoring of the yeast intracellular environment has been expanded and upgraded to the two-module ScEnSor (S. cerevisiae Engineering + Biosensor) Kit. The Biosensor Module includes eight fluorescent reporters for the intracellular environment; three of them (unfolded protein response, pyruvate metabolism, and ethanol consumption) were newly implemented to complement the original five. The Genome-Integration Module comprises a set of backbone plasmids for the assembly of 1-6 transcriptional units (each consisting of promoter, coding sequence, and terminator) for efficient marker-free single-locus genome integration (in HO and/or X2 loci). Altogether, the ScEnSor Kit enables rapid and easy construction of strains with new transcriptional units as well as high-throughput investigation of the yeast intracellular environment.},
}
@article {pmid37531174,
year = {2023},
author = {Yan, H and Han, S and Hughes, S and Zeng, Y},
title = {Extraction-free, one-pot CRISPR/Cas12a detection of microRNAs directly from extracellular vesicles.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {67},
pages = {10165-10168},
doi = {10.1039/d3cc02982d},
pmid = {37531174},
issn = {1364-548X},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; *Extracellular Vesicles ; },
abstract = {Current methods for extracellular vesicle (EV) miRNA analysis mostly require RNA extraction, which results in a multi-step, time-consuming workflow. This study reports an extraction-free method that combines thermolysis treatment of EVs with a one-pot EXTRA-CRISPR assay, enabling the vastly simplified analysis of EV miRNAs with a comparable performance to that of the extraction-based assays.},
}
@article {pmid37523223,
year = {2023},
author = {Li, G and Qi, Y},
title = {CRISPR Empowers Tree Bioengineering for a Sustainable Future.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {305-307},
doi = {10.1089/crispr.2023.29161.gli},
pmid = {37523223},
issn = {2573-1602},
mesh = {*Trees ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Bioengineering ; },
}
@article {pmid37505910,
year = {2023},
author = {Ji, H and Xiong, W and Guo, S and Wang, S and Xing, X and Tian, T and Zhou, X},
title = {Isonitrile-Tetrazine Click-and-Release Chemistry for Controlling RNA-Guided Nucleic Acid Cleavage.},
journal = {ACS chemical biology},
volume = {18},
number = {8},
pages = {1829-1837},
doi = {10.1021/acschembio.3c00255},
pmid = {37505910},
issn = {1554-8937},
mesh = {Humans ; *RNA ; RNA, Guide, CRISPR-Cas Systems ; *Heterocyclic Compounds ; Gene Editing ; Click Chemistry ; },
abstract = {With the increasing demand for the regulation of CRISPR systems, a considerable number of studies have been conducted to control their excessive activity levels. In this context, we propose a method that involves a bioorthogonal cleavage reaction between isonitrile and tetrazine to modulate the cleavage activity of the CRISPR system. Importantly, isonitrile demonstrates significant potential for modifying sgRNAs, making it a promising candidate for bioorthogonal reactions, a phenomenon that has not been previously reported. Our approach utilizes the 3-isocyanopropyl-carbonate group as a caging group to deactivate the CRISPR systems, while tetrazine acts as an activator to restore their activities. Through the implementation of post-synthetic modifications and click-and-release chemistry, we have successfully achieved the regulation of RNA-guided nucleic acid cleavage, which holds great promise for controlling gene editing in human cells.},
}
@article {pmid37486342,
year = {2023},
author = {Gossing, M and Limeta, A and Skrekas, C and Wigglesworth, M and Davis, A and Siewers, V and David, F},
title = {Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2271-2277},
doi = {10.1021/acssynbio.2c00689},
pmid = {37486342},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics ; *Mutation Rate ; CRISPR-Cas Systems/genetics ; DNA ; DNA-Directed DNA Polymerase/genetics ; RNA ; Mutation ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymerase─called yEvolvR─was recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.},
}
@article {pmid37486333,
year = {2023},
author = {Skrekas, C and Limeta, A and Siewers, V and David, F},
title = {Targeted In Vivo Mutagenesis in Yeast Using CRISPR/Cas9 and Hyperactive Cytidine and Adenine Deaminases.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2278-2289},
doi = {10.1021/acssynbio.2c00690},
pmid = {37486333},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; Mutagenesis, Site-Directed ; Gene Editing ; },
abstract = {Directed evolution is a preferred strategy to improve the function of proteins such as enzymes that act as bottlenecks in metabolic pathways. Common directed evolution approaches rely on error-prone PCR-based libraries where the number of possible variants is usually limited by cellular transformation efficiencies. Targeted in vivo mutagenesis can advance directed evolution approaches and help to overcome limitations in library generation. In the current study, we aimed to develop a high-efficiency time-controllable targeted mutagenesis toolkit in the yeast Saccharomyces cerevisiae by employing the CRISPR/Cas9 technology. To that end, we fused the dCas9 protein with hyperactive variants of adenine and cytidine deaminases aiming to create an inducible CRISPR-based mutagenesis tool targeting a specific DNA sequence in vivo with extended editing windows and high mutagenesis efficiency. We also investigated the effect of guide RNA multiplexing on the mutagenesis efficiency both phenotypically and on the DNA level.},
}
@article {pmid37429825,
year = {2023},
author = {Abdelhady, AM and Phillips, JA and Xu, Y and Stroh, M},
title = {Clinical Pharmacology and Translational Considerations in the Development of CRISPR-Based Therapies.},
journal = {Clinical pharmacology and therapeutics},
volume = {114},
number = {3},
pages = {591-603},
doi = {10.1002/cpt.3000},
pmid = {37429825},
issn = {1532-6535},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pharmacology, Clinical ; Gene Editing/methods ; },
abstract = {Genome editing holds the potential for curative treatments of human disease, however, clinical realization has proven to be a challenging journey with incremental progress made up until recently. Over the last decade, advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have provided the necessary breakthrough for genome editing in the clinic. The progress of investigational CRISPR therapies from bench to bedside reflects the culmination of multiple advances occurring in parallel, several of which intersect with clinical pharmacology and translation. Directing the CRISPR therapy to the intended site of action has necessitated novel delivery platforms, and this has resulted in special considerations for the complete characterization of distribution, metabolism, and excretion, as well as immunogenicity. Once at the site of action, CRISPR therapies aim to make permanent alterations to the genome and achieve therapeutically relevant effects with a single dose. This fundamental aspect of the mechanism of action for CRISPR therapies results in new considerations for clinical translation and dose selection. Early advances in model-informed development of CRISPR therapies have incorporated key facets of the mechanism of action and have captured hallmark features of clinical pharmacokinetics and pharmacodynamics from phase I investigations. Given the recent emergence of CRISPR therapies in clinical development, the landscape continues to evolve rapidly with ample opportunity for continued innovation. Here, we provide a snapshot of selected topics in clinical pharmacology and translation that has supported the advance of systemically administered in vivo and ex vivo CRISPR-based investigational therapies in the clinic.},
}
@article {pmid37406248,
year = {2023},
author = {Monte, DFM},
title = {My CRISPR Story: Back to Brazil.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {313-315},
doi = {10.1089/crispr.2023.0032},
pmid = {37406248},
issn = {2573-1602},
mesh = {Brazil ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37402223,
year = {2023},
author = {Whitford, CM and Gren, T and Palazzotto, E and Lee, SY and Tong, Y and Weber, T},
title = {Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2353-2366},
doi = {10.1021/acssynbio.3c00188},
pmid = {37402223},
issn = {2161-5063},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Actinomycetales/genetics ; Systems Analysis ; },
abstract = {CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor. We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of Streptomyces chassis and beyond.},
}
@article {pmid37347931,
year = {2023},
author = {Lamothe, G and Carbonneau, J and Joly Beauparlant, C and Vincent, T and Quessy, P and Guedon, A and Kobinger, G and Lemay, JF and Boivin, G and Droit, A and Turgeon, N and Tremblay, JP},
title = {Rapid and Technically Simple Detection of SARS-CoV-2 Variants Using CRISPR Cas12 and Cas13.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {369-385},
doi = {10.1089/crispr.2023.0007},
pmid = {37347931},
issn = {2573-1602},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic tests. We also optimized a strategy to modify loop-mediated isothermal amplification amplicons for downstream use with Cas12 and Cas13 for future multiplexing. The close ancestry of the BA.1 and BA.2 variants of SARS-CoV-2 (Omicron) made these excellent candidates for the development of a first test using this workflow. With a quick turnaround time and low requirements for laboratory equipment, the test we have created is ideally suited for settings such as mobile clinics lacking equipment such as Next-Generation Sequencers or Sanger Sequencers and the personnel to run these devices.},
}
@article {pmid37335046,
year = {2023},
author = {Luo, M and Ma, J and Cheng, X and Wu, S and Bartels, DJ and Guay, D and Engelhardt, JF and Liu, X},
title = {Genome Editing in Ferret Airway Epithelia Mediated by CRISPR/Nucleases Delivered with Amphiphilic Shuttle Peptides.},
journal = {Human gene therapy},
volume = {34},
number = {15-16},
pages = {705-718},
doi = {10.1089/hum.2023.016},
pmid = {37335046},
issn = {1557-7422},
support = {R01 HL165404/HL/NHLBI NIH HHS/United States ; P01 HL152960/HL/NHLBI NIH HHS/United States ; 75N92019C00010/HL/NHLBI NIH HHS/United States ; P30 DK054759/DK/NIDDK NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; Ferrets/genetics ; Epithelium ; Peptides/genetics ; *Lung Diseases/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene editing strategies are attractive for treating genetic pulmonary diseases such as cystic fibrosis (CF). However, challenges have included the development of safe and effective vector systems for gene editing of airway epithelia and model systems to report their efficiency and durability. The domestic ferret (Mustela putorius furo) has a high degree of conservation in lung cellular anatomy with humans, and has served as an excellent model for many types of lung diseases, including CF. In this study, we evaluated the efficiency of amphiphilic shuttle peptide S10 for protein delivery and gene editing using SpCas9, and AsCas12a (Cpf1) ribonucleoproteins (RNPs). These approaches were evaluated in proliferating ferret airway basal cells, polarized airway epithelia in vitro, and lungs in vivo, by accessing the editing efficiency using reporter ferrets and measuring indels at the ferret CFTR locus. Our results demonstrate that shuttle peptides efficiently enable delivery of reporter proteins/peptides and gene editing SpCas9 or Cpf1 RNP complexes to ferret airway epithelial cells in vitro and in vivo. We measured S10 delivery efficiency of green fluorescent protein (GFP)-nuclear localization signal (NLS) protein or SpCas9 RNP into ferret airway basal cells and fully differentiated ciliated and nonciliated epithelial cells in vitro. In vitro and in vivo gene editing efficiencies were determined by Cas/LoxP-gRNA RNP-mediated conversion of a ROSA-TG Cre recombinase reporter using transgenic primary cells and ferrets. S10/Cas9 RNP was more effective, relative to S10/Cpf1 RNP at gene editing of the ROSA-TG locus. Intratracheal lung delivery of the S10 shuttle combined with GFP-NLS protein or D-Retro-Inverso (DRI)-NLS peptide demonstrated efficiencies of protein delivery that were ∼3-fold or 14-fold greater, respectively, than the efficiency of gene editing at the ROSA-TG locus using S10/Cas9/LoxP-gRNA. Cpf1 RNPs was less effective than SpCas9 at gene editing of LoxP locus. These data demonstrate the feasibility of shuttle peptide delivery of Cas RNPs to the ferret airways and the potential utility for developing ex vivo stem cell-based and in vivo gene editing therapies for genetic pulmonary diseases such as CF.},
}
@article {pmid37307061,
year = {2023},
author = {Chen, YH and Sharma, S and Bewg, WP and Xue, LJ and Gizelbach, CR and Tsai, CJ},
title = {Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {339-349},
doi = {10.1089/crispr.2022.0096},
pmid = {37307061},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Populus/genetics ; DNA Copy Number Variations ; Mutagenesis ; Translocation, Genetic/genetics ; },
abstract = {The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.},
}
@article {pmid37276175,
year = {2023},
author = {Huang, L and Yang, C and Chen, Y and Deng, H and Liao, Z and Xiao, H},
title = {CRISPR-Mediated Base Editing: Promises and Challenges for a Viable Oncotherapy Strategy.},
journal = {Human gene therapy},
volume = {34},
number = {15-16},
pages = {669-681},
doi = {10.1089/hum.2023.045},
pmid = {37276175},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {Base editing technology, developed from the CRISPR/Cas9 system, is able to efficiently implement single-base substitutions at specific DNA or RNA sites without generating double-strand breaks with precision and efficiency. Point mutations account for 58% of disease-causing genetic mutations in humans, and single nucleotide variants are an important cause of tumorigenesis, and the advent of base editors offers new hope for the study or treatment of such diseases. Although it has some limitations, base editors have been continuously improved in terms of editing efficiency, specificity, and product purity since their development. In this review, we examine the main base editing technologies and discuss their applications and prospects in tumor research and therapy, as well as elaborate on their mode of delivery.},
}
@article {pmid36912815,
year = {2023},
author = {Mayes, CM and Santarpia, JL},
title = {Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {359-368},
doi = {10.1089/crispr.2022.0095},
pmid = {36912815},
issn = {2573-1602},
mesh = {Humans ; SARS-CoV-2/genetics ; *Coronavirus 229E, Human/genetics ; *Coronavirus OC43, Human/genetics ; *COVID-19/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.},
}
@article {pmid37584115,
year = {2023},
author = {Xue, S and Wang, S and Liu, L and Zhong, Q and Cheng, Z and Xiao, S},
title = {[Precision gene editing technologies based on CRISPR/Cas9: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {7},
pages = {2566-2578},
doi = {10.13345/j.cjb.220879},
pmid = {37584115},
issn = {1872-2075},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Mutation ; Genome ; },
abstract = {Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.},
}
@article {pmid37583711,
year = {2023},
author = {Wang, Z and Wang, Z and Zhang, F and Wu, L},
title = {Thermus thermophilus Argonaute-based signal amplifier for highly sensitive and specific microRNA detection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1221943},
pmid = {37583711},
issn = {2296-4185},
abstract = {The prokaryote-derived gene defense system as a new generation of nucleic acid detection tool exhibits impressive performance in the field of molecular diagnosis. Prokaryotic Argonaute (Ago) is a CRISPR-associated protein that is guided by a short DNA (gDNA) and then efficiently cleaves gDNA-complementary nucleic acids and presents unique characteristics that are different from the CRISPR/Cas system. However, the application of Ago in biosensing is still relatively scarce, and many properties of Ago need to be further clarified. In this study, we aim to systematically explore the properties of Thermus thermophilus Argonaute (TtAgo), including the dependence of TtAgo activity on guide DNA (gDNA) length, substrates' length, and the position of gDNA complementary region on the substrate. Based on these properties, we constructed an exonuclease III-assisted target-recycled amplification system (exoAgo) for sensitive miRNA detection. The result showed that exoAgo can be used for miRNA profiling with a detection limit of 12.2 pM and single-base-resolution and keep good performance for the detection of complex samples, which indicates that Ago has great application potential in the detection of nucleic acids. In conclusion, this study will provide guidance for further development and utilization of Ago in the field of biosensing.},
}
@article {pmid37582836,
year = {2023},
author = {Tao, Y and Lamas, V and Du, W and Zhu, W and Li, Y and Whittaker, MN and Zuris, JA and Thompson, DB and Rameshbabu, AP and Shu, Y and Gao, X and Hu, JH and Pei, C and Kong, WJ and Liu, X and Wu, H and Kleinstiver, BP and Liu, DR and Chen, ZY},
title = {Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4928},
pmid = {37582836},
issn = {2041-1723},
support = {UG3 AI150551/AI/NIAID NIH HHS/United States ; R01 DC012115/DC/NIDCD NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 DC019404/DC/NIDCD NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; U24 HG010423/HG/NHGRI NIH HHS/United States ; UH3 TR002636/TR/NCATS NIH HHS/United States ; R01 DC016875/DC/NIDCD NIH HHS/United States ; R01 DC005575/DC/NIDCD NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Ribonucleoproteins/genetics ; Liposomes ; RNA, Guide, CRISPR-Cas Systems ; *Hearing Loss/genetics/therapy ; *Deafness/genetics ; },
abstract = {Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2[Obl/+], with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.},
}
@article {pmid37137337,
year = {2023},
author = {Nizan, S and Amitzur, A and Dahan-Meir, T and Benichou, JIC and Bar-Ziv, A and Perl-Treves, R},
title = {Mutagenesis of the melon Prv gene by CRISPR/Cas9 breaks papaya ringspot virus resistance and generates an autoimmune allele with constitutive defense responses.},
journal = {Journal of experimental botany},
volume = {74},
number = {15},
pages = {4579-4596},
pmid = {37137337},
issn = {1460-2431},
mesh = {*Disease Resistance/genetics ; Alleles ; *Cucurbitaceae/genetics ; CRISPR-Cas Systems ; Mutagenesis ; Plant Diseases/genetics ; },
abstract = {The majority of plant disease resistance (R) genes encode nucleotide binding-leucine-rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154, and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels, and defense gene expression. The autoimmune phenotype observed at 25 °C proved to be temperature dependent, being suppressed at 32 °C. This is a first report on the successful application of CRISPR/Cas9 to confirm R gene function in melon. Such validation opens up new opportunities for molecular breeding of disease resistance in this important vegetable crop.},
}
@article {pmid37580710,
year = {2023},
author = {Zhao, N and Weng, S and Liu, Z and Xu, H and Ren, Y and Guo, C and Liu, L and Zhang, Z and Ji, Y and Han, X},
title = {CRISPR-Cas9 identifies growth-related subtypes of glioblastoma with therapeutical significance through cell line knockdown.},
journal = {BMC cancer},
volume = {23},
number = {1},
pages = {749},
pmid = {37580710},
issn = {1471-2407},
support = {81702465//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Glioblastoma/genetics/therapy/metabolism ; DNA Copy Number Variations ; CRISPR-Cas Systems ; *Brain Neoplasms/genetics/therapy/metabolism ; Cell Line ; ErbB Receptors/metabolism ; Prognosis ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is a type of highly malignant brain tumor that is known for its significant intratumoral heterogeneity, meaning that there can be a high degree of variability within the tumor tissue. Despite the identification of several subtypes of GBM in recent years, there remains to explore a classification based on genes related to proliferation and growth.<br><br>METHODS: The growth-related genes of GBM were identified by CRISPR-Cas9 and univariate Cox regression analysis. The expression of these genes in the Cancer Genome Atlas cohort (TCGA) was used to construct growth-related genes subtypes (GGSs) via consensus clustering. Validation of this subtyping was performed using the nearest template prediction (NTP) algorithm in two independent Gene Expression Omnibus (GEO) cohorts and the ZZ cohort. Additionally, copy number variations, biological functions, and potential drugs were analyzed for each of the different subtypes separately.<br><br>RESULTS: Our research established multicenter-validated GGSs. GGS1 exhibits the poorest prognosis, with the highest frequency of chr 7 gain &amp; chr 10 loss, and the lowest frequency of chr 19 &amp; 20 co-gain. Additionally, GGS1 displays the highest expression of EGFR. Furthermore, it is significantly enriched in metabolic, stemness, proliferation, and signaling pathways. Besides we showed that Foretinib may be a potential therapeutic agent for GGS1, the worst prognostic subtype, through data screening and in vitro experiments. GGS2 has a moderate prognosis, with a slightly higher proportion of chr 7 gain &amp; chr 10 loss, and the highest proportion of chr 19 &amp; 20 co-gain. The prognosis of GGS3 is the best, with the least chr 7 gain &amp; 10 loss and EGFR expression.<br><br>CONCLUSIONS: These results enhance our understanding of the heterogeneity of GBM and offer insights for stratified management and precise treatment of GBM patients.},
}
@article {pmid37580703,
year = {2023},
author = {Qureshi, A and Connolly, JB},
title = {Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control Anopheles gambiae the mosquito vector of human malaria.},
journal = {Malaria journal},
volume = {22},
number = {1},
pages = {234},
pmid = {37580703},
issn = {1475-2875},
mesh = {Animals ; Humans ; Mosquito Vectors/genetics ; *Anopheles/genetics ; CRISPR-Cas Systems ; *Gene Drive Technology/methods ; Allergens/genetics ; *Malaria ; *MicroRNAs ; },
abstract = {BACKGROUND: Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxF[CRISPRh] transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxF[CRISPRh] previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity.<br><br>METHODS: Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived.<br><br>RESULTS: While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxF[CRISPRh] transgenic proteins from environmental releases was assessed as negligible.<br><br>CONCLUSIONS: Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxF[CRISPRh] were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.},
}
@article {pmid37580318,
year = {2023},
author = {Wimberger, S and Akrap, N and Firth, M and Brengdahl, J and Engberg, S and Schwinn, MK and Slater, MR and Lundin, A and Hsieh, PP and Li, S and Cerboni, S and Sumner, J and Bestas, B and Schiffthaler, B and Magnusson, B and Di Castro, S and Iyer, P and Bohlooly-Y, M and Machleidt, T and Rees, S and Engkvist, O and Norris, T and Cadogan, EB and Forment, JV and Šviković, S and Akcakaya, P and Taheri-Ghahfarokhi, A and Maresca, M},
title = {Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4761},
pmid = {37580318},
issn = {2041-1723},
support = {765269//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Protein Kinases/genetics ; DNA Repair/genetics ; DNA/genetics ; },
abstract = {Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.},
}
@article {pmid37577534,
year = {2023},
author = {Hu, C and Myers, MT and Zhou, X and Hou, Z and Lozen, ML and Zhang, Y and Ke, A},
title = {Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.08.05.552134},
pmid = {37577534},
abstract = {Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets, and the nuclease-helicase Cas3 to degrade them. Among seven subtypes, Type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here we use four cryo-electron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling efficient NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9, that strongly inhibit N. lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through direct competition, whereas AcrIC9 achieves so through allosteric inhibition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for controllable Type I CRISPR genome engineering.},
}
@article {pmid37573099,
year = {2023},
author = {Zeng, Q and Zhou, M and Hu, Z and Deng, W and Li, Z and Wu, L and Liang, D},
title = {Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA.},
journal = {Analytica chimica acta},
volume = {1276},
number = {},
pages = {341622},
doi = {10.1016/j.aca.2023.341622},
pmid = {37573099},
issn = {1873-4324},
mesh = {Humans ; Female ; *RNA ; DNA, Single-Stranded/genetics ; CRISPR-Cas Systems ; *Nucleic Acids ; Biological Assay ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR-Cas12a RNA-guided complexes have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, they are limited by the complexity of the operation, risk of carry-over contamination, and degradation of CRISPR RNA (crRNA). In this study, a Cas12a-based single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA)-mediated one-step diagnostic method (CasDOS) was established to overcome these drawbacks. mD-crRNA consisted of wild-type crRNA (Wt-crRNA) with ssDNA extensions at the 3' and 5' ends. Compared to Wt-crRNA, mD-crRNA exhibited a 100-1000-fold increase in sensitivity in the one-step assay, reducing the cis-cleavage activity of Cas12a to avoid excessive cleavage of the target DNA in the early stages of the reaction, leading to increased amplification and accumulation of the target amplicons, and improved the speed and intensity of the generated fluorescence signal. The detectability of CasDOS was 16.6 aM for the constructed plasmids of Streptococcus agalactiae (GBS), human papillomavirus type 16 (HPV16), and type 18 (HPV18). In clinical trials, CasDOS achieved 100% accuracy in identifying the known genotypes of the five HPV DNA samples. Moreover, CasDOS showed complete concordance with the qPCR results for GBS detection in ten vaginal or cervical swab samples, with a turnaround time from sampling to results within 30 min. In addition, mD-crRNA remained stable after Ribonuclease R treatment, suggesting that it might be more suitable as a raw material for the CRISPR detection kit. In conclusion, we have developed a universal, rapid, and highly sensitive one-step CRISPR detection assay.},
}
@article {pmid37569693,
year = {2023},
author = {Wei, W and Chen, ZN and Wang, K},
title = {CRISPR/Cas9: A Powerful Strategy to Improve CAR-T Cell Persistence.},
journal = {International journal of molecular sciences},
volume = {24},
number = {15},
pages = {},
pmid = {37569693},
issn = {1422-0067},
support = {20220309//the Young Talent fund of the University Association for Science and Technology in Shaanxi, China/ ; },
mesh = {Humans ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Gene Editing ; Neoplasm Recurrence, Local/genetics ; Immunotherapy, Adoptive ; *Neoplasms/genetics/therapy ; },
abstract = {As an emerging treatment strategy for malignant tumors, chimeric antigen receptor T (CAR-T) cell therapy has been widely used in clinical practice, and its efficacy has been markedly improved in the past decade. However, the clinical effect of CAR-T therapy is not so satisfying, especially in solid tumors. Even in hematologic malignancies, a proportion of patients eventually relapse after receiving CAR-T cell infusions, owing to the poor expansion and persistence of CAR-T cells. Recently, CRISPR/Cas9 technology has provided an effective approach to promoting the proliferation and persistence of CAR-T cells in the body. This technology has been utilized in CAR-T cells to generate a memory phenotype, reduce exhaustion, and screen new targets to improve the anti-tumor potential. In this review, we aim to describe the major causes limiting the persistence of CAR-T cells in patients and discuss the application of CRISPR/Cas9 in promoting CAR-T cell persistence and its anti-tumor function. Finally, we investigate clinical trials for CRISPR/Cas9-engineered CAR-T cells for the treatment of cancer.},
}
@article {pmid37569333,
year = {2023},
author = {Stepchenkova, EI and Zadorsky, SP and Shumega, AR and Aksenova, AY},
title = {Practical Approaches for the Yeast Saccharomyces cerevisiae Genome Modification.},
journal = {International journal of molecular sciences},
volume = {24},
number = {15},
pages = {},
pmid = {37569333},
issn = {1422-0067},
support = {20-15-00081//Russian Science Foundation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {The yeast S. cerevisiae is a unique genetic object for which a wide range of relatively simple, inexpensive, and non-time-consuming methods have been developed that allow the performing of a wide variety of genome modifications. Among the latter, one can mention point mutations, disruptions and deletions of particular genes and regions of chromosomes, insertion of cassettes for the expression of heterologous genes, targeted chromosomal rearrangements such as translocations and inversions, directed changes in the karyotype (loss or duplication of particular chromosomes, changes in the level of ploidy), mating-type changes, etc. Classical yeast genome manipulations have been advanced with CRISPR/Cas9 technology in recent years that allow for the generation of multiple simultaneous changes in the yeast genome. In this review we discuss practical applications of both the classical yeast genome modification methods as well as CRISPR/Cas9 technology. In addition, we review methods for ploidy changes, including aneuploid generation, methods for mating type switching and directed DSB. Combined with a description of useful selective markers and transformation techniques, this work represents a nearly complete guide to yeast genome modification.},
}
@article {pmid37569295,
year = {2023},
author = {Bekalu, ZE and Panting, M and Bæksted Holme, I and Brinch-Pedersen, H},
title = {Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {15},
pages = {},
pmid = {37569295},
issn = {1422-0067},
support = {8055-00038B, ReTraQue project//Innovation Fund Denmark/ ; },
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; Edible Grain/genetics ; Genotype ; Phenotype ; Genome, Plant ; CRISPR-Cas Systems ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.},
}
@article {pmid37563692,
year = {2023},
author = {Nakazawa, T and Morimoto, T and Maeoka, R and Matsuda, R and Nakamura, M and Nishimura, F and Ouji, N and Yamada, S and Nakagawa, I and Park, YS and Ito, T and Nakase, H and Tsujimura, T},
title = {CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {42},
number = {1},
pages = {205},
pmid = {37563692},
issn = {1756-9966},
support = {22K09213//Japan Society for the Promotion of Science/ ; 22K16667//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; Humans ; Mice ; *Brain Neoplasms/genetics/therapy ; *Carcinoma, Intraductal, Noninfiltrating ; CRISPR-Cas Systems ; *Glioblastoma/genetics/therapy ; *Hematopoietic Stem Cell Transplantation ; Killer Cells, Natural ; *Suppressor of Cytokine Signaling Proteins/genetics ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor and has "immunologically cold" features. Changing GBM to an "immunologically hot" tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs.<br><br>METHODS: The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further&#xa0;detected in vivo antitumor effects using xenograft brain tumor mice.<br><br>RESULTS: We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)&#x264; and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice.<br><br>CONCLUSION: The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.},
}
@article {pmid37440203,
year = {2023},
author = {Giacca, M},
title = {Fulfilling the Promise of RNA Therapies for Cardiac Repair and Regeneration.},
journal = {Stem cells translational medicine},
volume = {12},
number = {8},
pages = {527-535},
pmid = {37440203},
issn = {2157-6580},
support = {787971/ERC_/European Research Council/International ; },
mesh = {*Heart/physiology ; Regeneration ; *RNA, Antisense/therapeutic use ; *Aptamers, Nucleotide/therapeutic use ; RNA Interference/drug effects ; *RNA, Guide, CRISPR-Cas Systems/therapeutic use ; Gene Editing ; Humans ; Animals ; },
abstract = {The progressive appreciation that multiple types of RNAs regulate virtually all aspects of tissue function and the availability of effective tools to deliver RNAs in vivo now offers unprecedented possibilities for obtaining RNA-based therapeutics. For the heart, RNA therapies can be developed that stimulate endogenous repair after cardiac damage. Applications in this area include acute cardioprotection after ischemia or cancer chemotherapy, therapeutic angiogenesis to promote new blood vessel formation, regeneration to form new cardiac mass, and editing of mutations to cure inherited cardiac disease. While the potential of RNA therapeutics for all these conditions is exciting, the field is still in its infancy. A number of roadblocks need to be overcome for RNA therapies to become effective, in particular, related to the problem of delivering RNA medicines into the cells and targeting them specifically to the heart.},
}
@article {pmid37429772,
year = {2023},
author = {Islam, T and Kasfy, SH},
title = {CRISPR enables heritable genome editing in planta.},
journal = {Trends in genetics : TIG},
volume = {39},
number = {9},
pages = {646-648},
doi = {10.1016/j.tig.2023.06.009},
pmid = {37429772},
issn = {0168-9525},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Plant/genetics ; Plant Breeding ; },
abstract = {Recent exciting developments in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing showcase its potential to rapidly and efficiently edit genomes in planta, eliminating long processes of tissue culture and extensive breeding for crop improvement. These new methods offer heritable transgene-free edits in one generation, making them an attractive option for improving commercially important crops.},
}
@article {pmid37379644,
year = {2023},
author = {Fan, M and Bao, Y and Berkhout, B and Herrera-Carrillo, E},
title = {CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {165},
number = {},
pages = {115046},
doi = {10.1016/j.biopha.2023.115046},
pmid = {37379644},
issn = {1950-6007},
mesh = {Animals ; Humans ; *Proviruses/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *HIV Infections/genetics ; T-Lymphocytes/metabolism ; DNA, Viral/genetics ; Endonucleases/genetics/metabolism ; Cell Culture Techniques ; Mammals ; },
abstract = {BACKGROUND: The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a.<br><br>METHODS: We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells.<br><br>FINDINGS: We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such "hypermutated" HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation.<br><br>INTERPRETATION: These results provide in vitro "proof of concept' for CRISPR-Cas12b mediated HIV-1 inactivation.},
}
@article {pmid37183575,
year = {2023},
author = {Song, N and Chu, Y and Tang, J and Yang, D},
title = {Lipid-, Inorganic-, Polymer-, and DNA-Based Nanocarriers for Delivery of the CRISPR/Cas9 system.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {24},
number = {16},
pages = {e202300180},
doi = {10.1002/cbic.202300180},
pmid = {37183575},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems/genetics ; *Polymers/metabolism ; Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; Lipids ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system has been widely explored for the precise manipulation of target DNA and has enabled efficient genomic editing in cells. Recently, CRISPR/Cas9 has shown promising potential in biomedical applications, including disease treatment, transcriptional regulation and genome-wide screening. Despite these exciting achievements, efficient and controlled delivery of the CRISPR/Cas9 system has remained a critical obstacle to its further application. Herein, we elaborate on the three delivery forms of the CRISPR/Cas9 system, and discuss the composition, advantages and limitations of these forms. Then we provide a comprehensive overview of the carriers of the system, and focus on the nonviral nanocarriers in chemical methods that facilitate efficient and controlled delivery of the CRISPR/Cas9 system. Finally, we discuss the challenges and prospects of the delivery methods of the CRISPR/Cas9 system in depth, and propose strategies to address the intracellular and extracellular barriers to delivery in clinical applications.},
}
@article {pmid37039773,
year = {2023},
author = {Hamaker, NK and Lee, KH},
title = {High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.},
journal = {Biotechnology and bioengineering},
volume = {120},
number = {9},
pages = {2419-2440},
doi = {10.1002/bit.28393},
pmid = {37039773},
issn = {1097-0290},
support = {T32 GM008550/GM/NIGMS NIH HHS/United States ; },
mesh = {Cricetinae ; Animals ; CHO Cells ; Cricetulus ; *DNA Repair/genetics ; *CRISPR-Associated Protein 9/genetics ; Recombinant Proteins/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.},
}
@article {pmid36947856,
year = {2023},
author = {Ranzau, BL and Rallapalli, KL and Evanoff, M and Paesani, F and Komor, AC},
title = {The Wild-Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence-Specific DNA Base Editing.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {24},
number = {16},
pages = {e202200788},
doi = {10.1002/cbic.202200788},
pmid = {36947856},
issn = {1439-7633},
support = {R35 GM138317/GM/NIGMS NIH HHS/United States ; T32 GM007240/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; },
mesh = {*Adenosine Deaminase/metabolism ; *Gene Editing ; RNA, Transfer/chemistry ; DNA/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Base editors are genome editing tools that enable site-specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA-deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild-type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.},
}
@article {pmid36938677,
year = {2023},
author = {Leitner, K and Motheramgari, K and Borth, N and Marx, N},
title = {Nanopore Cas9-targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells.},
journal = {Biotechnology and bioengineering},
volume = {120},
number = {9},
pages = {2403-2418},
doi = {10.1002/bit.28382},
pmid = {36938677},
issn = {1097-0290},
mesh = {Cricetinae ; Animals ; Cricetulus ; CHO Cells ; CRISPR-Cas Systems ; *Nanopore Sequencing ; *Nanopores ; Transgenes/genetics ; },
abstract = {The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution. In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on-target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification-seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.},
}
@article {pmid37582546,
year = {2022},
author = {Feeney, O},
title = {Genetics and Justice, Non-Ideal Theory and the Role of Patents: The Case of CRISPR-Cas9.},
journal = {European journal of health law},
volume = {29},
number = {3-5},
pages = {543-561},
doi = {10.1163/15718093-bja10075},
pmid = {37582546},
issn = {1571-8093},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Social Justice ; Biotechnology ; },
abstract = {There are ongoing concerns of social justice regarding inequalities in the distribution of access to potential genome editing technologies. Working within non-ideal theory, Colin Farrelly advances a justification for the use of patents to speed up the arrival of safe and effective interventions for all, including the socially disadvantaged. This paper argues that such success is less assured when one considers the actual functioning of patents and the practical implications of the patent system in the context of biotechnological innovations. I suggest that non-ideal theoretical approaches risk reverting back to a form of ideal theory if they simply refer to such real-world constraints - e.g. patents - but do not critically assess and fully examine how such constraints manifest themselves in practice. I highlight some considerations that would be important in order to develop and foster a more robust non-ideal approach to justice in biotechnological developments.},
}
@article {pmid37582540,
year = {2022},
author = {Slokenberga, S},
title = {What Would It Take to Enable Germline Editing in Europe for Medical Purposes?.},
journal = {European journal of health law},
volume = {29},
number = {3-5},
pages = {521-542},
doi = {10.1163/15718093-bja10074},
pmid = {37582540},
issn = {1571-8093},
mesh = {Humans ; Europe ; *Gene Editing ; Human Rights ; *Medicine ; Germ Cells ; },
abstract = {Commonly, the regulation on germline editing in Europe is described through the two prohibitions: the prohibition set out in Article 13 of the Convention for the protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine and the prohibition that is set out in the EU Clinical Trials Regulation. These prohibitions reflect the European regional position regarding the ethical and legal questions raised by the technology, and an unwillingness to enable such interventions in Europe. Simultaneously, these prohibitions have been shaped prior to the recent breakthroughs in the field, such as the discovery of the CRISPR-Cas technology, which has initiated a new era in the field. This contribution examines what it would take to enable human germline gene editing in Europe for medical purposes. It scrutinises in detail the content and context of the existing bans, as well as mechanisms to lift them. It argues that the bans that are prescribed by each of the European regional legal orders are embedded in strong structures, composed of values and principles. For the human germline gene editing to be enabled in Europe for health-related purposes, the approach to these values and principles needs to change. Only then can the machinery to lift the bans lead to a change.},
}
@article {pmid37582538,
year = {2022},
author = {Slokenberga, S and Minssen, T and Nordberg, A},
title = {Governing, Protecting, and Regulating the Future of Genome Editing: The Significance of ELSPI Perspectives.},
journal = {European journal of health law},
volume = {29},
number = {3-5},
pages = {327-340},
doi = {10.1163/15718093-bja10076},
pmid = {37582538},
issn = {1571-8093},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Genome, Plant ; },
}
@article {pmid37563544,
year = {2023},
author = {Dutta, TK and Vashisth, N and Ray, S and Phani, V and Chinnusamy, V and Sirohi, A},
title = {Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy.},
journal = {BMC plant biology},
volume = {23},
number = {1},
pages = {390},
pmid = {37563544},
issn = {1471-2229},
mesh = {Animals ; Gene Editing/methods ; *Arabidopsis/genetics/parasitology ; CRISPR-Cas Systems ; *Tylenchoidea ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: Plant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease.<br><br>RESULTS: Initially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1-3&#xa0;bp deletions and 1&#xa0;bp insertions adjacent to the PAM site, a long deletion of approximately 161&#xa0;bp was documented in the T0 generation. Phenotypic analysis of homozygous, 'transgene-free' T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants.<br><br>CONCLUSION: Our results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.},
}
@article {pmid37562410,
year = {2023},
author = {Schraivogel, D and Steinmetz, LM and Parts, L},
title = {Pooled Genome-Scale CRISPR Screens in Single Cells.},
journal = {Annual review of genetics},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-genet-072920-013842},
pmid = {37562410},
issn = {1545-2948},
abstract = {Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades. Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}
@article {pmid37561739,
year = {2023},
author = {Kay, GA and Patterson, EI and Hughes, GL and Lord, JS and Reimer, LJ},
title = {Knockdown resistance allele L1014F introduced by CRISPR/Cas9 is not associated with altered vector competence of Anopheles gambiae for o'nyong nyong virus.},
journal = {PloS one},
volume = {18},
number = {8},
pages = {e0288994},
pmid = {37561739},
issn = {1932-6203},
support = {N013514//MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; O'nyong-nyong Virus ; *Anopheles/genetics ; Alleles ; CRISPR-Cas Systems/genetics ; Mosquito Vectors/genetics ; Insecticide Resistance/genetics ; *Insecticides ; },
abstract = {Knockdown resistance (kdr) alleles conferring resistance to pyrethroid insecticides are widespread amongst vector populations. Previous research has suggested that these alleles are associated with changes in the vector competence of mosquitoes for arboviruses and Plasmodium, however non-target genetic differences between mosquito strains may have had a confounding effect. Here, to minimise genetic differences, the laboratory Anopheles gambiae Kisumu strain was compared to a CRISPR/Cas9 homozygous kdr L1014F mutant Kisumu-kdr line in order to examine associations with vector competence for o'nyong nyong virus (ONNV). Mosquitoes were infected using either blood feeds or intrathoracic microinjections. There were no significant differences in the prevalence of virus in mosquito body parts between kdr mutant and wildtype lines from either oral or intrathoracic injection routes. The ONNV titre was significantly higher in the legs of the wildtype strain at 7dpi following intrathoracic microinjection, but no other significant differences in viral titre were detected. ONNV was not detected in the saliva of mosquitoes from either strain. Our findings from per os infections suggest that the kdr L1014F allele is not associated with altered infection prevalence for ONNV, a key component of vector competence.},
}
@article {pmid37558681,
year = {2023},
author = {Yasumoto, S and Muranaka, T},
title = {Foreign DNA detection in genome-edited potatoes by high-throughput sequencing.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {12246},
pmid = {37558681},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; Computer Simulation ; Plant Breeding ; Gene Editing/methods ; DNA ; High-Throughput Nucleotide Sequencing/methods ; Nucleotides ; },
abstract = {Genome editing is a powerful breeding technique that introduces mutations into specific gene sequences in genomes. For genome editing in higher plants, nucleotides for artificial nuclease (e.g. TALEN or CRISPR-Cas9) are transiently or stably introduced into the plant cells. After the introduction of mutations by artificial nucleases, it is necessary to select lines that do not contain the foreign nucleotides to overcome GMO regulation; however, there is still no widely legally authorized and approved method for detecting foreign genes in genome-edited crops. Recently, k-mer analysis based on next-generation sequencing (NGS) was proposed as a new method for detecting foreign DNA in genome-edited agricultural products. Compared to conventional methods, such as PCR and Southern hybridization, in principle, this method can detect short DNA fragments with high accuracy. However, this method has not yet been applied to genome-edited potatoes. In this study, we evaluated the feasibility of k-mer analysis in tetraploid potatoes by computer simulation, and also evaluated whether the k-mer method can detect foreign genes with high accuracy by analyzing samples of genome-edited potatoes. We show that when NGS data (at a depth of × 30 the genome size) are used, the k-mer method can correctly detect foreign genes in the potato genome even with the insertion of DNA fragments of 20 nt in length. Based on these findings, we expect that k-mer analysis will be one of the main methods for detecting foreign genes in genome-edited potatoes.},
}
@article {pmid37531567,
year = {2023},
author = {Fei, X and Lei, C and Ren, W and Liu, X and Liu, C},
title = {Regulating the trans-Cleavage Activity of CRISPR/Cas12a by Using an Elongation-Caged Single-Stranded DNA Activator and the Biosensing Applications.},
journal = {Analytical chemistry},
volume = {95},
number = {32},
pages = {12169-12176},
doi = {10.1021/acs.analchem.3c02471},
pmid = {37531567},
issn = {1520-6882},
mesh = {*DNA, Single-Stranded/genetics ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Endodeoxyribonucleases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Biosensing Techniques ; },
abstract = {The CRISPR/Cas12a system exhibits extraordinary capability in the field of biosensing and molecular diagnosis due to its trans-cleavage ability. However, it is still desirable for precise control and programmable regulation of Cas12a trans-cleavage activity to promote the in-depth studies and application expansion of Cas12a-based sensing platforms. In this work, we have developed a new and robust CRISPR/Cas12a regulation mechanism by endowing the activator with the function of caging crRNA ingeniously. Specifically, we constructed an integrated elongation-caged activator (EL-activator) by extending the ssDNA activator on the 3'-end. We found that appending only about 8 nt that is complementary to the crRNA repeat region is enough to cage the crRNA spacer/repeat region, thus effectively inhibiting Cas12a trans-cleavage activity. The inner inhibition mechanism was further uncovered after a thorough investigation, demonstrating that the EL-activator works by impeding the conformation of crRNA required for Cas12a recognition and destroying its affinity with Cas12a. By further switching on the elongated moiety on the EL-activator using target biomarkers, the blocked trans-cleavage activity of Cas12a can be rapidly recovered. Finally, a versatile sensing platform was established based on the EL-activator regulation mechanism, expanding the conventional Cas12a system that only directly recognizes DNA to the direct detection of enzymes and RNA biomarkers. This work has enriched the CRISPR/Cas12a regulation toolbox and expanded its sensing applications.},
}
@article {pmid37527175,
year = {2023},
author = {Dong, J and Li, X and Zhou, S and Liu, Y and Deng, L and Chen, J and Hou, J and Hou, C and Huo, D},
title = {CRISPR/Cas12a-Powered EC/FL Dual-Mode Controlled-Release Homogeneous Biosensor for Ultrasensitive and Cross-Validated Detection of Messenger Ribonucleic Acid.},
journal = {Analytical chemistry},
volume = {95},
number = {32},
pages = {12122-12130},
doi = {10.1021/acs.analchem.3c02335},
pmid = {37527175},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Delayed-Action Preparations ; RNA, Messenger/genetics ; RNA ; Coloring Agents ; DNA, Single-Stranded/genetics ; Endonucleases ; Serine Proteinase Inhibitors ; *Biosensing Techniques ; },
abstract = {Accurate detection of cancer-associated mRNAs is beneficial to early diagnosis and potential treatment of cancer. Herein, for the first time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent (EC/FL) dual-mode controlled-release homogeneous biosensor for mRNA detection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as the signal probe, while survivin mRNA was chosen as the target RNA. In the presence of the target mRNA, the nicking endonuclease-mediated rolling circle amplification (NEM-RCA) was triggered to produce significant amounts of ssDNA, activating the collateral activity of Cas12a toward the surrounding single-stranded DNA. Thus, the ssDNA P1 completely complementary to ssDNA P2 was cleaved, resulting in that the ssDNA P2 bio-gate on Fe3O4-NH2 could not be opened due to electrostatic interactions. As a result, there was no or only a little MB in the supernatant after magnetic separation, and the measured EC/FL signal was exceedingly weak. On the contrary, the ssDNA P2 bio-gate was opened, enabling MB to be released into the supernatant, and generating an obvious EC/FL signal. Benefiting from the accuracy of EC/FL dual-mode cross-verification, high amplification efficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and high loading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity and single-base mismatch specificity. More importantly, the practical applicability of this dual-mode strategy was confirmed by mRNA quantification in complex serum environments and tumor cell lysates, providing a new way for developing a powerful disease diagnosis tool.},
}
@article {pmid37516291,
year = {2023},
author = {Minet, C and Chantal, I and Berthier, D},
title = {Recent advances in genome editing of bloodstream forms of Trypanosoma congolense using CRISPR-Cas9 ribonucleoproteins: Proof of concept.},
journal = {Experimental parasitology},
volume = {252},
number = {},
pages = {108589},
doi = {10.1016/j.exppara.2023.108589},
pmid = {37516291},
issn = {1090-2449},
mesh = {Animals ; Cattle ; *Trypanosoma congolense/genetics ; CRISPR-Cas Systems ; Gene Editing ; Ribonucleoproteins/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Trypanosomiasis, African/prevention &amp; control/veterinary ; *Trypanosoma/genetics ; *Trypanosoma brucei brucei/genetics ; },
abstract = {African Animal Trypanosomosis (AAT or Nagana) is a vector-borne disease caused by Trypanosomatidae, genus Trypanosoma. The disease is transmitted by the bite of infected hematophagous insects, mainly tsetse flies but also other blood-sucking insects including stomoxes and tabanids. Although many trypanosome species infect animals, the main agents responsible for this disease with a strong socio-economic and veterinary health impact are Trypanosoma congolense (T. congolense or Tc), Trypanosoma vivax (T.vivax), and to a lesser extent, Trypanosoma brucei brucei (T.brucei brucei or Tbb). These parasites mainly infect livestock, including cattle, in sub-Saharan Africa, with major repercussions in terms of animal productivity and poverty for populations which are often already very poor. As there is currently no vaccine, the fight against the disease is primarily based on diagnosis, treatment and vector control. To develop new tools (particularly therapeutic tools) to fight against the disease, we need to know both the biology and the genes involved in the pathogenicity and virulence of the parasites. To date, unlike for Trypanosoma brucei (T.brucei) or Trypanosoma cruzi (T.cruzi), genome editing tools has been relatively little used to study T. congolense. We present an efficient, reproducible and stable CRISPR-Cas9 genome editing system for use in Tc bloodstream forms (Tc-BSF). This plasmid-free system is based on transient expression of Cas9 protein and the use of a ribonucleoprotein formed by the Cas9 and sgRNA complex. This is the first proof of concept of genome editing using CRISPR-Cas9 ribonucleoproteins on Tc-BSF. This adapted protocol enriches the "toolbox" for the functional study of genes of interest in blood forms of the Trypanosoma congolense. This proof of concept is an important step for the scientific community working on the study of trypanosomes and opens up new perspectives for the control of and fight against animal trypanosomosis.},
}
@article {pmid37470475,
year = {2023},
author = {Schubert, L and Le, AT and Hinz, TK and Navarro, AC and Nelson-Taylor, SK and Nemenoff, RA and Heasley, LE and Doebele, RC},
title = {A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.},
journal = {Biology open},
volume = {12},
number = {8},
pages = {},
doi = {10.1242/bio.059994},
pmid = {37470475},
issn = {2046-6390},
support = {P50CA058187/NH/NIH HHS/United States ; /CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems ; *Oncogenes ; Protein Kinase Inhibitors/pharmacology ; },
abstract = {CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.},
}
@article {pmid37029320,
year = {2023},
author = {Alyateem, G and Wade, HM and Bickert, AA and Lipsey, CC and Mondal, P and Smith, MD and Labib, RM and Mock, BA and Robey, RW and Gottesman, MM},
title = {Use of CRISPR-based screens to identify mechanisms of chemotherapy resistance.},
journal = {Cancer gene therapy},
volume = {30},
number = {8},
pages = {1043-1050},
pmid = {37029320},
issn = {1476-5500},
support = {n/a//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; RNA, Guide, CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology/therapeutic use ; *Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Despite the development of new classes of targeted anti-cancer drugs, the curative treatment of metastatic solid tumors remains out of reach owing to the development of resistance to current chemotherapeutics. Although many mechanisms of drug resistance have been described, there is still a general lack of understanding of the many means by which cancer cells elude otherwise effective chemotherapy. The traditional strategy of isolating resistant clones in vitro, defining their mechanism of resistance, and testing to see whether these mechanisms play a role in clinical drug resistance is time-consuming and in many cases falls short of providing clinically relevant information. In this review, we summarize the use of CRISPR technology, including the promise and pitfalls, to generate libraries of cancer cells carrying sgRNAs that define novel mechanisms of resistance. The existing strategies using CRISPR knockout, activation, and inhibition screens, and combinations of these approaches are described. In addition, specialized approaches to identify more than one gene that may be contributing to resistance, as occurs in synthetic lethality, are described. Although these CRISPR-based approaches to cataloguing drug resistance genes in cancer cells are just beginning to be utilized, appropriately used they promise to accelerate understanding of drug resistance in cancer.},
}
@article {pmid37557170,
year = {2023},
author = {Wang, S and Gabel, C and Siddique, R and Klose, T and Chang, L},
title = {Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2023.07.010},
pmid = {37557170},
issn = {1097-4172},
abstract = {Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.},
}
@article {pmid37554703,
year = {2023},
author = {Anuradha, and Kumari, M and Zinta, G and Chauhan, R and Kumar, A and Singh, S and Singh, S},
title = {Genetic resources and breeding approaches for improvement of amaranth (Amaranthus spp.) and quinoa (Chenopodium quinoa).},
journal = {Frontiers in nutrition},
volume = {10},
number = {},
pages = {1129723},
pmid = {37554703},
issn = {2296-861X},
abstract = {Nowadays, the human population is more concerned about their diet and very specific in choosing their food sources to ensure a healthy lifestyle and avoid diseases. So people are shifting to more smart nutritious food choices other than regular cereals and staple foods they have been eating for a long time. Pseudocereals, especially, amaranth and quinoa, are important alternatives to traditional cereals due to comparatively higher nutrition, essential minerals, amino acids, and zero gluten. Both Amaranchaceae crops are low-input demanding and hardy plants tolerant to stress, drought, and salinity conditions. Thus, these crops may benefit developing countries that follow subsistence agriculture and have limited farming resources. However, these are underutilized orphan crops, and the efforts to improve them by reducing their saponin content remain ignored for a long time. Furthermore, these crops have very rich variability, but the progress of their genetic gain for getting high-yielding genotypes is slow. Realizing problems in traditional cereals and opting for crop diversification to tackle climate change, research should be focused on the genetic improvement for low saponin, nutritionally rich, tolerant to biotic and abiotic stresses, location-specific photoperiod, and high yielding varietal development of amaranth and quinoa to expand their commercial cultivation. The latest technologies that can accelerate the breeding to improve yield and quality in these crops are much behind and slower than the already established major crops of the world. We could learn from past mistakes and utilize the latest trends such as CRISPR/Cas, TILLING, and RNA interference (RNAi) technology to improve these pseudocereals genetically. Hence, the study reviewed important nutrition quality traits, morphological descriptors, their breeding behavior, available genetic resources, and breeding approaches for these crops to shed light on future breeding strategies to develop superior genotypes.},
}
@article {pmid37551560,
year = {2023},
author = {Wu, F and Lu, C and Hu, W and Guo, X and Chen, J and Luo, Z},
title = {Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination.},
journal = {Journal of Zhejiang University. Science. B},
volume = {24},
number = {8},
pages = {749-754},
doi = {10.1631/jzus.B2200705},
pmid = {37551560},
issn = {1862-1783},
support = {KYCX2021-038//the Priority Academic Program Development of Jiangsu Higher Education Institutions of China, the Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; CG2232//the Lianyungang Science and Technology Program/ ; },
mesh = {*Vibrio parahaemolyticus/genetics ; Uracil-DNA Glycosidase/genetics ; Hot Temperature ; CRISPR-Cas Systems ; Food Safety ; },
abstract = {Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.},
}
@article {pmid37550505,
year = {2023},
author = {Watts, EA and Garrett, SC and Catchpole, RJ and Clark, LM and Sanders, TJ and Marshall, CJ and Wenck, BR and Vickerman, RL and Santangelo, TJ and Fuchs, R and Robb, B and Olson, S and Graveley, BR and Terns, MP},
title = {Histones direct site-specific CRISPR spacer acquisition in model archaeon.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {37550505},
issn = {2058-5276},
support = {R35GM118160//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35GM143963//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; RO1GM100329//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35GM118140//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.},
}
@article {pmid37550295,
year = {2023},
author = {Henn, D and Zhao, D and Sivaraj, D and Trotsyuk, A and Bonham, CA and Fischer, KS and Kehl, T and Fehlmann, T and Greco, AH and Kussie, HC and Moortgat Illouz, SE and Padmanabhan, J and Barrera, JA and Kneser, U and Lenhof, HP and Januszyk, M and Levi, B and Keller, A and Longaker, MT and Chen, K and Qi, LS and Gurtner, GC},
title = {Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4729},
pmid = {37550295},
issn = {2041-1723},
support = {S10 OD025212/OD/NIH HHS/United States ; S10 OD021763/OD/NIH HHS/United States ; R21 AG077193/AG/NIA NIH HHS/United States ; P30 DK116074/DK/NIDDK NIH HHS/United States ; S10 OD018220/OD/NIH HHS/United States ; },
mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; Wound Healing/genetics ; Genes, myc ; Gene Editing ; *Craniocerebral Trauma ; Dendritic Cells ; },
abstract = {Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.},
}
@article {pmid37549968,
year = {2023},
author = {Chen, YH and Lu, J and Yang, X and Huang, LC and Zhang, CQ and Liu, QQ and Li, QF},
title = {Gene editing of non-coding regulatory DNA and its application in crop improvement.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erad313},
pmid = {37549968},
issn = {1460-2431},
abstract = {The development of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has provided precise and efficient strategies to edit target genes and generate transgene-free crops. Significant progress has been made in the editing of protein coding genes; however, studies on the editing of non-coding DNA with regulatory roles lags far behind. Non-coding regulatory DNAs, including long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and cis-regulatory elements (CREs), play crucial roles in regulating plant growth and development. Therefore, the combination of CRISPR/Cas technology and non-coding regulatory DNA has great potential to generate novel alleles that affect various agronomic traits of crops, thus providing valuable genetic resources for crop breeding. Herein, we review recent advances in the roles of non-coding regulatory DNA, attempts to edit non-coding regulatory DNA for crop improvement, and potential application of novel editing tools in modulating non-coding regulatory DNA. Finally, the existing problems, possible solutions, and future applications of gene editing of non-coding regulatory DNA in modern crop breeding practice are also discussed.},
}
@article {pmid37547711,
year = {2023},
author = {Ye, M and Yao, M and Li, C and Gong, M},
title = {Salt and osmotic stress can improve the editing efficiency of CRISPR/Cas9-mediated genome editing system in potato.},
journal = {PeerJ},
volume = {11},
number = {},
pages = {e15771},
pmid = {37547711},
issn = {2167-8359},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; Sodium Chloride/pharmacology ; Osmotic Pressure ; },
abstract = {CRISPR/Cas9-mediated genome editing technology has been widely used for the study of gene function in crops, but the differences between species have led to widely varying genome editing efficiencies. The present study utilized a potato hairy root genetic transformation system and incorporated a rapid assay with GFP as a screening marker. The results clearly demonstrated that salt and osmotic stress induced by NaCl (10 to 50 mM) and mannitol (50 to 200 mM) treatments significantly increased the positive rates of genetic transformation mediated by A. rhizogenes and the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato. However, it was observed that the regeneration of potato roots was partially inhibited as a result. The analysis of CRISPR/Cas9-mediated mutation types revealed that chimeras accounted for the largest proportion, ranging from 62.50% to 100%. Moreover, the application of salt and osmotic stress resulted in an increased probability of null mutations in potato. Notably, the highest rate of null mutations, reaching 37.5%, was observed at a NaCl concentration of 10 mM. Three potential off-target sites were sequenced and no off-targeting was found. In conclusion, the application of appropriate salt and osmotic stress significantly improved the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato, with no observed off-target effects. However, there was a trade-off as the regeneration of potato roots was partially inhibited. Overall, these findings present a new and convenient approach to enhance the genome editing efficiency of the CRISPR/Cas9-mediated gene editing system in potato.},
}
@article {pmid37547075,
year = {2023},
author = {Wang, J and Tomar, D and Martin, TG and Dubey, S and Dubey, PK and Song, J and Landesberg, G and McCormick, MG and Myers, VD and Merali, S and Merali, C and Lemster, B and McTiernan, CF and Khalili, K and Madesh, M and Cheung, JY and Kirk, JA and Feldman, AM},
title = {Bag3 Regulates Mitochondrial Function and the Inflammasome Through Canonical and Noncanonical Pathways in the Heart.},
journal = {JACC. Basic to translational science},
volume = {8},
number = {7},
pages = {820-839},
pmid = {37547075},
issn = {2452-302X},
support = {K99 DK120876/DK/NIDDK NIH HHS/United States ; R00 DK120876/DK/NIDDK NIH HHS/United States ; },
abstract = {B-cell lymphoma 2-associated athanogene-3 (Bag3) is expressed in all animal species, with Bag3 levels being most prominent in the heart, the skeletal muscle, the central nervous system, and in many cancers. Preclinical studies of Bag3 biology have focused on animals that have developed compromised cardiac function; however, the present studies were performed to identify the pathways perturbed in the heart even before the occurrence of clinical signs of dilatation and failure of the heart. These studies show that hearts carrying variants that knockout one allele of BAG3 have significant alterations in multiple cellular pathways including apoptosis, autophagy, mitochondrial homeostasis, and the inflammasome.},
}
@article {pmid37547003,
year = {2023},
author = {McLean, ZL and Gao, D and Correia, K and Roy, JCL and Shibata, S and Farnum, IN and Valdepenas-Mellor, Z and Rapuru, M and Morini, E and Ruliera, J and Gillis, T and Lucente, D and Kleinstiver, BP and Lee, JM and MacDonald, ME and Wheeler, VC and Pinto, RM and Gusella, JF},
title = {PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37547003},
support = {R01 NS091161/NS/NINDS NIH HHS/United States ; R01 NS126420/NS/NINDS NIH HHS/United States ; R01 NS119471/NS/NINDS NIH HHS/United States ; R01 NS105709/NS/NINDS NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; R01 NS049206/NS/NINDS NIH HHS/United States ; },
abstract = {Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1 , a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1 , making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.},
}
@article {pmid37523811,
year = {2023},
author = {Jiang, C and Zheng, X and Lin, L and Li, X and Li, X and Liao, Y and Jia, W and Shu, B},
title = {CRISPR Cas12a-mediated amplification-free digital DNA assay improves the diagnosis and surveillance of Nasopharyngeal carcinoma.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115546},
doi = {10.1016/j.bios.2023.115546},
pmid = {37523811},
issn = {1873-4235},
mesh = {Humans ; Nasopharyngeal Carcinoma/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; *Epstein-Barr Virus Infections ; *Nasopharyngeal Neoplasms/diagnosis/genetics/pathology ; *Carcinoma ; Case-Control Studies ; Herpesvirus 4, Human/genetics ; DNA, Viral/genetics ; *Biosensing Techniques ; Real-Time Polymerase Chain Reaction ; },
abstract = {Sensitive and accurate cell-free plasma Epstein-Barr virus (EBV) DNA measurement is essential in the routine diagnosis, monitoring and treatment of Nasopharyngeal Carcinoma (NPC). This measurement in commercial and in-house assay are commonly based on real-time quantitative PCR (qPCR) method, which requires reference materials for standardization and lack quantitative precision due to amplification bias or cross-contamination. To address these issues, we developed a CRISPR/Cas12a-mediated amplification-free digital DNA assay, which targets the repetitive sequences of EBV DNA and utilizes the cis-cleavage activity of CRISPR-Cas12a prior to droplet generation. By this mean, more activated Cas12a-crRNA duplexes could be produced for subsequent target detection and counting, thus improving the performance in detecting low EBV DNA load. We demonstrated that it was more robust than conventional qPCR for detecting plasma EBV DNA in a case-control study of 208 participants, especially when the target concentrations were around the diagnostic cut-off value for NPC. More importantly, this assay allowed a more accurate diagnosis of early-stage NPC, with an area under the curve (AUC) of 0.9883 (versus 0.7682 for qPCR). Furthermore, its absolute quantification capability enabled dynamic monitoring of EBV load in NPC patients during initial diagnosis, treatment, and recurrence, thereby potentially improving disease management and prognosis. Taken together, our results demonstrate that this amplification-free digital assay has the potential to be a robust tool to improve the diagnosis and surveillance of NPC.},
}
@article {pmid37459685,
year = {2023},
author = {Alon, DM and Mittelman, K and Stibbe, E and Countryman, S and Stodieck, L and Doraisingam, S and Leal Martin, DM and Hamo, ER and Pines, G and Burstein, D},
title = {CRISPR-based genetic diagnostics in microgravity.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115479},
doi = {10.1016/j.bios.2023.115479},
pmid = {37459685},
issn = {1873-4235},
mesh = {Humans ; *Weightlessness ; *Biosensing Techniques ; Astronauts ; Genomics ; Recombinases ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {Monitoring astronauts' health during space missions poses many challenges, including rapid assessment of crew health conditions. Sensitive genetic diagnostics are crucial for examining crew members and the spacecraft environment. CRISPR-Cas12a, coupled with isothermal amplification, has proven to be a promising biosensing system for rapid, on-site detection of genomic targets. However, the efficiency and sensitivity of CRISPR-based diagnostics have never been tested in microgravity. We tested the use of recombinase polymerase amplification (RPA) coupled with the collateral cleavage activity of Cas12a for genetic diagnostics onboard the International Space Station. We explored the detection sensitivity of amplified and unamplified target DNA. By coupling RPA with Cas12a, we identified targets in attomolar concentrations. We further assessed the reactions' stability following long-term storage. Our results demonstrate that CRISPR-based detection is a powerful tool for on-site genetic diagnostics in microgravity, and can be further utilized for long-term space endeavors to improve astronauts' health and well-being.},
}
@article {pmid37451024,
year = {2023},
author = {Shen, J and Chen, Z and Xie, R and Li, J and Liu, C and He, Y and Ma, X and Yang, H and Xie, Z},
title = {CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115523},
doi = {10.1016/j.bios.2023.115523},
pmid = {37451024},
issn = {1873-4235},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Microfluidics ; SARS-CoV-2/genetics ; *Biosensing Techniques ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; COVID-19 Testing ; },
abstract = {Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H1N1, H3N2, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.},
}
@article {pmid37423067,
year = {2023},
author = {Yuan, Q and Mao, D and Tang, X and Liu, C and Zhang, R and Deng, J and Zhu, X and Li, W and Man, Q and Sun, F},
title = {Biological effect abundance analysis of hemolytic pathogens based on engineered biomimetic sensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115502},
doi = {10.1016/j.bios.2023.115502},
pmid = {37423067},
issn = {1873-4235},
mesh = {Humans ; *Biomimetics ; Hemolysis ; *Biosensing Techniques ; Biological Assay ; Enzyme-Linked Immunosorbent Assay ; CRISPR-Cas Systems ; },
abstract = {Conventional pathogen detection strategies based on the molecular structure or chemical characteristics of biomarkers can only provide the "physical abundance" of microorganisms, but cannot reflect the "biological effect abundance" in the true sense. To address this issue, we report an erythrocyte membrane-encapsulated biomimetic sensor cascaded with CRISPR-Cas12a (EMSCC). Taking hemolytic pathogens as the target model, we first constructed an erythrocyte membrane-encapsulated biomimetic sensor (EMS). Only hemolytic pathogens with biological effects can disrupt the erythrocyte membrane (EM), resulting in signal generation. Then the signal was amplified by cascading CRISPR-Cas12a, and more than 6.67 × 10[4]-fold improvement in detection sensitivity compared to traditional erythrocyte hemolysis assay was achieved. Notably, compared with polymerase chain reaction (PCR) or enzyme linked immunosorbent assay (ELISA)-based quantification methods, EMSCC can sensitively respond to the pathogenicity change of pathogens. For the detection of simulated clinical samples based on EMSCC, we obtained an accuracy of 95% in 40 samples, demonstrating its potential clinical value.},
}
@article {pmid37395412,
year = {2023},
author = {Liu, Z and Jillette, N and Robson, P and Cheng, AW},
title = {Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.},
journal = {Nucleic acids research},
volume = {51},
number = {14},
pages = {e77},
pmid = {37395412},
issn = {1362-4962},
support = {R01-HG009900/HG/NHGRI NIH HHS/United States ; R01-HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems/genetics ; Transcriptome ; RNA Processing, Post-Transcriptional ; RNA Splicing ; Gene Editing/methods ; },
abstract = {RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.},
}
@article {pmid37395400,
year = {2023},
author = {Wang, WJ and Lin, J and Wu, CQ and Luo, AL and Xing, X and Xu, L},
title = {Establishing artificial gene connections through RNA displacement-assembly-controlled CRISPR/Cas9 function.},
journal = {Nucleic acids research},
volume = {51},
number = {14},
pages = {7691-7703},
pmid = {37395400},
issn = {1362-4962},
support = {21977122//National Natural Science Foundation of China/ ; 2020YFA0211200//National Key R&amp;D Program of China/ ; 2022B1515020047//Outstanding Youth Project of Guangdong Natural Science Foundation/ ; 2021A1515010253//General Project of Natural Science Foundation of Guangdong Province/ ; 202102020523//General Project of Natural Science Foundation of Guangzhou/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Genes, Synthetic ; Gene Regulatory Networks/genetics ; *MicroRNAs ; RNA, Messenger ; Gene Editing ; Mammals/genetics ; },
abstract = {Construction of synthetic circuits that can reprogram genetic networks and signal pathways is a long-term goal for manipulation of biosystems. However, it is still highly challenging to build artificial genetic communications among endogenous RNA species due to their sequence independence and structural diversities. Here we report an RNA-based synthetic circuit that can establish regulatory linkages between expression of endogenous genes in both Escherichiacoli and mammalian cells. This design employs a displacement-assembly approach to modulate the activity of guide RNA for function control of CRISPR/Cas9. Our experiments demonstrate the great effectiveness of this RNA circuit for building artificial connections between expression of originally unrelated genes. Both exogenous and naturally occurring RNAs, including small/microRNAs and long mRNAs, are capable of controlling expression of another endogenous gene through this approach. Moreover, an artificial signal pathway inside mammalian cells is also successfully established to control cell apoptosis through our designed synthetic circuit. This study provides a general strategy for constructing synthetic RNA circuits, which can introduce artificial connections into the genetic networks of mammalian cells and alter the cellular phenotypes.},
}
@article {pmid37354728,
year = {2023},
author = {Awasthi, P and Mishra, AK and Kocábek, T and Nath, VS and Mishra, S and Hazzouri, KM and Sudalaimuthuasari, N and Stajner, N and Jakše, J and Krofta, K and Hájek, T and Amiri, KM},
title = {CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus).},
journal = {Plant physiology and biochemistry : PPB},
volume = {201},
number = {},
pages = {107851},
doi = {10.1016/j.plaphy.2023.107851},
pmid = {37354728},
issn = {1873-2690},
mesh = {*Humulus/genetics ; CRISPR-Cas Systems ; Gene Expression Profiling ; Transcriptome ; Genomics ; },
abstract = {Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.},
}
@article {pmid37328396,
year = {2023},
author = {Barka, A and Kohli, RM and Shi, J},
title = {CRISPR tiling screen reveals cancer epigenetic 'Goldilocks' state.},
journal = {Trends in pharmacological sciences},
volume = {44},
number = {9},
pages = {555-557},
doi = {10.1016/j.tips.2023.05.007},
pmid = {37328396},
issn = {1873-3735},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neoplasms/genetics ; Epigenomics ; Epigenesis, Genetic ; CRISPR-Cas Systems ; },
abstract = {CRISPR tiling screens offer an efficient way to identify gain-of-function mutations in targets of cancer therapy. Recently, by utilizing these screens, Kwok et al. unexpectedly discovered mutations conferring drug addiction in lymphoma, revealing a requirement for a 'just right' window of histone methylation crucial for cancer survival.},
}
@article {pmid36646933,
year = {2023},
author = {Mathis, N and Allam, A and Kissling, L and Marquart, KF and Schmidheini, L and Solari, C and Balázs, Z and Krauthammer, M and Schwank, G},
title = {Predicting prime editing efficiency and product purity by deep learning.},
journal = {Nature biotechnology},
volume = {41},
number = {8},
pages = {1151-1159},
pmid = {36646933},
issn = {1546-1696},
support = {185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_185293//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 201184//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Humans ; *Deep Learning ; Gene Editing ; Hepatocytes ; Mutation ; Neural Networks, Computer ; CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing is a versatile genome editing tool but requires experimental optimization of the prime editing guide RNA (pegRNA) to achieve high editing efficiency. Here we conducted a high-throughput screen to analyze prime editing outcomes of 92,423 pegRNAs on a highly diverse set of 13,349 human pathogenic mutations that include base substitutions, insertions and deletions. Based on this dataset, we identified sequence context features that influence prime editing and trained PRIDICT (prime editing guide prediction), an attention-based bidirectional recurrent neural network. PRIDICT reliably predicts editing rates for all small-sized genetic changes with a Spearman's R of 0.85 and 0.78 for intended and unintended edits, respectively. We validated PRIDICT on endogenous editing sites as well as an external dataset and showed that pegRNAs with high (>70) versus low (<70) PRIDICT scores showed substantially increased prime editing efficiencies in different cell types in vitro (12-fold) and in hepatocytes in vivo (tenfold), highlighting the value of PRIDICT for basic and for translational research applications.},
}
@article {pmid36604543,
year = {2023},
author = {Jiao, C and Reckstadt, C and König, F and Homberger, C and Yu, J and Vogel, J and Westermann, AJ and Sharma, CM and Beisel, CL},
title = {RNA recording in single bacterial cells using reprogrammed tracrRNAs.},
journal = {Nature biotechnology},
volume = {41},
number = {8},
pages = {1107-1116},
pmid = {36604543},
issn = {1546-1696},
support = {INST93/1105-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BE 6703/1-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 865973//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 865973/ERC_/European Research Council/International ; SH 580/9-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Bacteria/genetics/metabolism ; DNA/genetics ; Gene Editing ; RNA, Bacterial/genetics ; },
abstract = {Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.},
}
@article {pmid37546822,
year = {2023},
author = {Sinha, S and Molina Vargas, AM and Arantes, PR and Patel, A and O'Connell, MR and Palermo, G},
title = {RNA-mediated Allosteric Activation in CRISPR-Cas13a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.07.27.550797},
pmid = {37546822},
support = {R35 GM133462/GM/NIGMS NIH HHS/United States ; },
abstract = {Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.},
}
@article {pmid37545761,
year = {2023},
author = {Kocsisova, Z and Coneva, V},
title = {Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1209586},
pmid = {37545761},
issn = {2673-3439},
abstract = {Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind's ability to deliver precise genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools.},
}
@article {pmid37433390,
year = {2023},
author = {Gibney, TV and Favichia, M and Latifi, L and Medwig-Kinney, TN and Matus, DQ and McIntyre, DC and Arrigo, AB and Branham, KR and Bubrig, LT and Ghaddar, A and Jiranek, JA and Liu, KE and Marcucci, CG and Porter, RJ and Pani, AM},
title = {A simple method to dramatically increase C. elegans germline microinjection efficiency.},
journal = {Developmental biology},
volume = {502},
number = {},
pages = {63-67},
doi = {10.1016/j.ydbio.2023.07.003},
pmid = {37433390},
issn = {1095-564X},
support = {R35 GM142880/GM/NIGMS NIH HHS/United States ; R35 GM137975/GM/NIGMS NIH HHS/United States ; R35 GM141886/GM/NIGMS NIH HHS/United States ; F31 HD100091/HD/NICHD NIH HHS/United States ; T32 GM007267/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; Microinjections/methods ; Animals, Genetically Modified ; *Germ Cells ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.},
}
@article {pmid37545694,
year = {2023},
author = {Khlidj, Y},
title = {What did CRISPR-Cas9 accomplish in its first 10 years?.},
journal = {Biochemia medica},
volume = {33},
number = {3},
pages = {030601},
pmid = {37545694},
issn = {1846-7482},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; },
abstract = {It's been 10 years now from the debut of clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) era in which gene engineering has never been so accessible, precise and efficient. This technology, like a refined surgical procedure, has offered the ability of removing different types of disease causing mutations and restoring key proteins activity with ease of outperforming the previous resembling methods: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Additionally, CRISPR-Cas9 systems can systematically introduce genetic sequences to the specific sites in the human genome allowing to stimulate desired functions such as anti-tumoral and anti-infectious faculties. The present brief review provides an updated resume of CRISPR-Cas9's top achievements from its first appearance to the current date focusing on the breakthrough research including in vitro, in vivo and human studies. This enables the evaluation of the previous phase 'the proof-of-concept phase' and marks the beginning of the next phase which will probably bring a spate of clinical trials.},
}
@article {pmid37490481,
year = {2023},
author = {Li, Y and Wang, X and He, Z and Johnson, M and A, S and Lara-Sáez, I and Lyu, J and Wang, W},
title = {3D Macrocyclic Structure Boosted Gene Delivery: Multi-Cyclic Poly(β-Amino Ester)s from Step Growth Polymerization.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {31},
pages = {17187-17200},
doi = {10.1021/jacs.3c04191},
pmid = {37490481},
issn = {1520-5126},
mesh = {Polymerization ; *RNA, Guide, CRISPR-Cas Systems ; *Gene Transfer Techniques ; Polymers/chemistry ; CRISPR-Cas Systems ; },
abstract = {The topological structures of polymers play a critical role in determining their gene delivery efficiency. Exploring novel polymeric structures as gene delivery vectors is thus of great interest. In this work, a new generation of multi-cyclic poly(β-amino ester)s (CPAEs) with unique topology structure was synthesized for the first time via step growth polymerization. Through controlling the occurrence stage of cyclization, three types of CPAEs with rings of different sizes and topologies were obtained. In vitro experiments demonstrated that the CPAEs with macro rings (MCPAEs) significantly boosted the transgene expression comparing to their branched counterparts. Moreover, the MCPAE vector with optimized terminal group efficiently delivered the CRISPR plasmid coding both Staphylococcus aureus Cas9 nuclease and dual guide sgRNAs for gene editing therapy.},
}
@article {pmid37490029,
year = {2023},
author = {Jia, ZQ and Zhang, SG and Wang, Y and Pan, JH and Liu, FF and Zhan, EL and Fouad, EA and Fu, YL and Pan, QR and Zhao, CQ},
title = {Physiological Function of RDL1 and RDL2 Subunits of the Ionotropic GABA Receptor in the Spodoptera litura with the CRISPR/Cas9 System In Vivo.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {31},
pages = {11875-11883},
pmid = {37490029},
issn = {1520-5118},
mesh = {Animals ; *Receptors, GABA/genetics/metabolism ; Spodoptera/physiology ; *CRISPR-Cas Systems ; Larva/genetics/metabolism ; Dieldrin ; gamma-Aminobutyric Acid ; },
abstract = {In insect ionotropic γ-aminobutyric acid receptor (iGABAR) subunits, only resistance to dieldrin (RDL) can be individually and functionally expressed in vitro. In lepidopteran, two to three RDL subtypes are identified; however, their physiological roles have not been distinguished in vivo. In this study, SlRdl1 and SlRdl2 of S. litura were individually knocked out using CRISPR/Cas9, respectively. The mortality and larval and pupal duration of KOSlRdl1 and KOSlRdl2 were increased. The flight time and distance were increased by 43.30%-80.66% and 58.96%-198.22%, respectively, in KOSlRdl1. The GABA-induced current was significantly decreased by 53.57%-74.28% and 46.91%-63.34% in the ventral nerve cord, and the GABA titer was significantly reduced by 17.65%-28.05% and 19.85%-42.46% in KOSlRdl1 and KOSlRdl2, respectively. In conclusion, SlRdl1 and SlRdl2 are necessary for the transmission of GABA-induced neural signals; however, only SlRdl1 could regulate the flight capability of S. litura. Our results provided a new avenue to study lepidopteran iGABARs.},
}
@article {pmid37545273,
year = {2023},
author = {Nie, X and Wang, D and Pan, Y and Lü, P and Yang, Y},
title = {Discovery, classification and application of the CPISPR-Cas13 system.},
journal = {Technology and health care : official journal of the European Society for Engineering and Medicine},
volume = {},
number = {},
pages = {},
doi = {10.3233/THC-230258},
pmid = {37545273},
issn = {1878-7401},
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is an acquired immune system of bacteria and archaea. Continued research has resulted in the identification of other Cas13 proteins.<br><br>OBJECTIVE: This review briefly describes the discovery, classification, and application of the CRISPR-Cas13 system, including recent technological advances in addition to factors affecting system performance.<br><br>METHODS: Cas13-based molecular therapy of human, animal, and plant transcriptomes was discussed, including regulation of gene expression to combat pathogenic RNA viruses. In addition, the latest progress, potential shortcomings, and challenges of the CRISPR-Cas system for treatment of animal and plant diseases are reviewed.<br><br>RESULTS: The CRISPR-Cas system VI is characterized by two RNA-guided higher eukaryotes and prokaryotes nucleotide-binding domains. CRISPR RNA can cleave specific RNA through the interaction between the stem-loop rich chain of uracil residues and the Cas13a protein. The CRISPR-Cas13 system has been applied for gene editing in animal and plant cells, in addition to biological detection via accurate targeting of single-stranded RNA.<br><br>CONCLUSION: The CRISPR-Cas13 system offers a high-throughput and convenient technology for detection of viruses and potentially the development of anti-cancer drugs in the near future.},
}
@article {pmid37541970,
year = {2023},
author = {Elsharawy, H and Refat, M},
title = {CRISPR/Cas9 genome editing in wheat: enhancing quality and productivity for global food security-a review.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {3},
pages = {265},
pmid = {37541970},
issn = {1438-7948},
mesh = {Humans ; *Gene Editing/methods ; *Triticum/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genome, Plant ; Crops, Agricultural/genetics ; Edible Grain/genetics ; },
abstract = {Wheat (Triticum aestivum L.) is an important cereal crop that is grown all over the world for food and industrial purposes. Wheat is essential to the human diet due to its rich content of necessary amino acids, minerals, vitamins, and calories. Various wheat breeding techniques have been utilized to improve its quality, productivity, and resistance to biotic and abiotic stress impairing production. However, these techniques are expensive, demanding, and time-consuming. Additionally, these techniques need multiple generations to provide the desired results, and the improved traits could be lost over time. To overcome these challenges, researchers have developed various genome editing tools to improve the quality and quantity of cereal crops, including wheat. Genome editing technologies evolve quickly. Nowadays, single or multiple mutations can be enabled and targeted at specific loci in the plant genome, allowing controlled removal of undesirable features or insertion of advantageous ones. Clustered, regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) is a powerful genome editing tool that can be effectively used for precise genome editing of wheat and other crops. This review aims to provide a comprehensive understanding of this technology's potential applications to enhance wheat's quality and productivity. It will first explore the function of CRISPR/Cas9 in preserving the adaptive immunity of prokaryotic organisms, followed by a discussion of its current applications in wheat breeding.},
}
@article {pmid37540372,
year = {2023},
author = {Xu, Y and Gan, ES and Ito, T},
title = {Misexpression Approaches for the Manipulation of Flower Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2686},
number = {},
pages = {429-451},
pmid = {37540372},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Transcriptional Activation ; Phenotype ; *Plants/genetics ; Flowers/genetics ; },
abstract = {The generation of dominant gain-of-function mutants through activation tagging is a forward genetic approach that can be applied to study the mechanisms of flower development, complementing the screening of loss-of-function mutants. In addition, the functions of genes of interest can be further analyzed through reverse genetics. A commonly used method is gene overexpression, where ectopic expression can result in an opposite phenotype to that caused by a loss-of-function mutation. When overexpression is detrimental, the misexpression of a gene using tissue-specific promoters can be useful to study spatial-specific function. As flower development is a multistep process, it can be advantageous to control gene expression, or its protein product activity, in a temporal and/or spatial manner. This has been made possible through several inducible promoter systems as well as inducible proteins by constructing chimeric fusions between the ligand-binding domain of the glucocorticoid receptor (GR) and the protein of interest. The recently introduced CRISPR-Cas9-based platform provides a new way of bioengineering transcriptional regulators in plants. By fusing a catalytically inactive dCas9 with functional activation or repression domains, the CRISPR-Cas9 module can achieve transcriptional activation or repression of endogenous genes. All these methods allow us to genetically manipulate gene expression during flower development. In this chapter, we describe methods to produce the expression constructs, method of screening, and more general applications of the techniques.},
}
@article {pmid37538843,
year = {2023},
author = {Zhu, J and Ju, Y and Zhou, X and Chen, T and Zhuge, X and Dai, J},
title = {Epidemiological characteristics of SHV, cmlv, and FosA6-producing carbapenem-resistant Klebsiella pneumoniae based on whole genome sequences in Jiangsu, China.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1219733},
pmid = {37538843},
issn = {1664-302X},
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly those with high virulence, cause invasive disease in clinical settings. An epidemiological investigation was conducted on the evolution, virulence, and antimicrobial resistance of CRKP isolates in two tertiary teaching hospitals in Jiangsu, China from November 2020 to December 2021. There were 31 different CRKP strains discovered. We performed whole genome sequencing (WGS) on 13 SHV, cmlv, and FosA6-producing CRKP to reveal molecular characteristics. Five ST15/ST11 isolates had CRISPR-Cas systems. By conjugation tests, KPC-2 can be transmitted horizontally to E. coil. A conjugative pHN7A8-related multi-resistance plasmid (KPC-2, blaCTX-M-65, blaTEM-1, fosA3, catII, and rmtB) was first discovered in CRKP clinical isolates. Using bacteriological testing, a serum killing assay, and an infection model with Galleria mellonella, three ST11-K64 KPC-2 generating carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) were identified. These strains harbored a virulent plasmid and an IncFII-family pKPC/pHN7A8 conjugative plasmid, which led to hypervirulence and resistance. One of these CR-hvKPs, which co-harbored KPC-2, NDM-6, SHV-182, SHV-64, and blaCTX-M-122 genes, was first discovered. Importantly, this CR-hvKP strain also produced biofilm and had non-inferior fitness. The widespread use of ceftazidime/avibactam might provide this CR-hvKP with a selective advantage; hence, immediate action is required to stop its dissemination. Another important finding is the novel ST6136 in K. pneumoniae. Finally, the sterilization efficiency rates of Fe2C nanoparticles in CRKP were more than 98%. Furthermore, our novel antibacterial Fe2C nanoparticles may also provide a therapeutic strategy for infections.},
}
@article {pmid37493946,
year = {2023},
author = {Ma, X and Zhang, Y and Qiao, X and Yuan, Y and Sheng, Q and Yue, T},
title = {Target-Induced AIE Effect Coupled with CRISPR/Cas12a System Dual-Signal Biosensing for the Ultrasensitive Detection of Gliotoxin.},
journal = {Analytical chemistry},
volume = {95},
number = {31},
pages = {11723-11731},
doi = {10.1021/acs.analchem.3c01760},
pmid = {37493946},
issn = {1520-6882},
mesh = {*Gliotoxin ; CRISPR-Cas Systems ; *Mycotoxins ; Oligonucleotides ; DNA, Single-Stranded ; *Biosensing Techniques ; },
abstract = {Here, a novel rapid and ultrasensitive aptamer biosensor was designed for target-induced activation of AIE effect and followed by the activation of Crispr Cas12a (LbCpf1)-mediated cleavage to achieve dual-signal detection. The prepared DNA building blocks contain the target aptamer, ssDNA-Fc, and Activator1. In this system, the activation mode was divided into two steps. First, when the target interacts with the aptamers, the DNA building blocks would be disintegrated rapidly, releasing a mass of Ac1, generating ETTC-dsDNA aggregated to produce a fluorescence signal by the AIE effect. Second, with the release of Ac2, LbCpf1-crRNA was activated, which greatly improves the ssDNA-Fc cleavage efficiency to render signal amplification and ultrasensitive detection of the target. Satisfactorily, using this approach to detect gliotoxin, optimal conditions for detection was achieved for reducing the detection time to 55 min, achieving a low detection limit of 2.4 fM and a satisfactory linear in the range of 50 fM to 1 nM, which addressed the shortcoming of a weak electrochemical signal in previous sensors. The water-insoluble AIE material was coupled with DNA to obtain water-soluble ETTC-dsDNA and successfully introduced into the sensor system, with a low detection limit of 5.6 fM. Subsequently, the biosensor combined with handheld electrochemical workstation was successfully applied in the detection of gliotoxin in five actual samples, with a detection range of 32.0 to 2.09 × 10[8] pM. This strategy not only provides a novel and effective detection platform for mycotoxins in complex food matrices but also opens a promising avenue for various molecules detection in imaging and disease diagnosis.},
}
@article {pmid37488217,
year = {2023},
author = {Huang, Z and Kaller, M and Hermeking, H},
title = {CRISPR/Cas9-mediated inactivation of miR-34a and miR-34b/c in HCT116 colorectal cancer cells: comprehensive characterization after exposure to 5-FU reveals EMT and autophagy as key processes regulated by miR-34.},
journal = {Cell death and differentiation},
volume = {30},
number = {8},
pages = {2017-2034},
pmid = {37488217},
issn = {1476-5403},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; HCT116 Cells ; Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Colorectal Neoplasms/drug therapy/genetics/metabolism ; Fluorouracil/pharmacology ; Autophagy/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {The miR-34a and miR-34b/c encoding genes represent direct targets of the p53 transcription factor, and presumably mediate part of the tumor suppressive effects of p53. Here, we sought to determine their functional relevance by inactivating miR-34a and/or miR-34b/c using a CRISPR/Cas9 approach in the colorectal cancer (CRC) cell line HCT116. Concomitant deletion of miR-34a and miR-34b/c resulted in significantly reduced suppression of proliferation after p53 activation, enhanced migration, invasion and EMT, as well as reduced sensitivity to chemotherapeutics, increased stress-induced autophagic flux, decreased apoptosis and upregulation of autophagy-related genes after 5-FU treatment. However, inactivation of singular miR-34a or miR-34b/c had little effects on the aforementioned processes. RNA-Seq analysis revealed that concomitant deletion of miR-34a/b/c caused EMT signature enrichment, impaired gene repression by the p53-DREAM pathway and elevated autophagy after 5-FU treatment. A gene signature comprised of mRNAs significantly upregulated after combined inactivation of miR-34a and miR-34b/c showed a significant association with the invasive colon cancer subtype CMS4 and poor overall survival in two CRC patient cohorts, and with 5-FU resistance in CRC cell lines. In miR-34a/b/c-deficient cells the upregulated miR-34 target FOXM1 directly induced p62 and ATG9A, which increased autophagy and consequently attenuated apoptosis and rendered the miR-34a/b/c-KO cells more resistant to 5-FU. Inhibition of autophagy by depletion of ATG9A or chloroquine re-sensitized miR-34a/b/c-deficient HCT116 cells to 5-FU. In summary, our findings show a complementary role of miR-34a and miR-34b/c in the regulation of EMT and autophagy which may be relevant for CRC therapy in the future.},
}
@article {pmid37468714,
year = {2023},
author = {Coogan, M and Xing, D and Su, B and Alston, V and Johnson, A and Khan, M and Khalil, K and Elaswad, A and Li, S and Wang, J and Lu, C and Wang, W and Hettiarachchi, D and Shang, M and Hasin, T and Qin, Z and Cone, R and Butts, IAE and Dunham, RA},
title = {CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.},
journal = {Transgenic research},
volume = {32},
number = {4},
pages = {251-264},
pmid = {37468714},
issn = {1573-9368},
support = {2015-67015-23488//National Institute of Food and Agriculture/ ; },
mesh = {Animals ; *Ictaluridae/genetics ; Fatty Acid Elongases/genetics ; CRISPR-Cas Systems/genetics ; *Carps ; RNA, Guide, CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; *Oncorhynchus/genetics ; },
abstract = {Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.},
}
@article {pmid37460719,
year = {2023},
author = {},
title = {A method to map single-cell lineages in the mouse brain by CRISPR-based barcoding.},
journal = {Nature methods},
volume = {20},
number = {8},
pages = {1139-1140},
pmid = {37460719},
issn = {1548-7105},
mesh = {Animals ; Mice ; Cell Lineage ; *CRISPR-Cas Systems/genetics ; *Gene Expression Profiling ; Brain ; DNA Barcoding, Taxonomic ; },
}
@article {pmid37431292,
year = {2023},
author = {Sun, Z and Ren, M and Shan, B and Yang, Q and Zhao, Z and Liu, X and Yin, L},
title = {One-pot synthesis of dynamically cross-linked polymers for serum-resistant nucleic acid delivery.},
journal = {Biomaterials science},
volume = {11},
number = {16},
pages = {5653-5662},
doi = {10.1039/d3bm00685a},
pmid = {37431292},
issn = {2047-4849},
mesh = {*Polymers/chemistry ; *Nucleic Acids ; Gene Transfer Techniques ; RNA, Guide, CRISPR-Cas Systems ; Transfection ; Polyethyleneimine/chemistry ; DNA/genetics ; Plasmids ; Polyphenols ; },
abstract = {Cationic polymers used for nucleic acid delivery often suffer from complicated syntheses, undesired intracellular cargo release and low serum stability. Herein, a series of ternary polymers were synthesized via facile green chemistry to achieve efficient plasmid DNA and mRNA delivery in serum. During the one-pot synthesis of the ternary polymer, acetylphenylboric acid (APBA), polyphenol and low-molecular weight polyethyleneimine (PEI 1.8k) were dynamically cross-linked with each other due to formation of an imine between PEI 1.8k and APBA and formation of a boronate ester between APBA and polyphenol. Series of polyphenols, including ellagic acid (EA), epigallocatechin gallate (EGCG), nordihydroguaiaretic acid (NDGA), rutin (RT) and rosmarinic acid (RA), and APBA molecules, including 2-acetylphenylboric acid (2-APBA), 3-acetylphenylboric acid (3-APBA) and 4-acetylphenylboric acid (4-APBA), were screened and the best-performing ternary polymer, 2-PEI-RT, constructed from RT and 2-APBA, was identified. The ternary polymer featured efficient DNA condensation to favor cellular internalization, and the acidic environment in endolysosomes triggered effective degradation of the polymer to promote cargo release. Thus, 2-PEI-RT showed robust plasmid DNA transfection efficiencies in various tumor cells in serum, outperforming the commercial reagent PEI 25k by 1-3 orders of magnitude. Moreover, 2-PEI-RT mediated efficient cytosolic delivery of Cas9-mRNA/sgRNA to enable pronounced CRISPR-Cas9 genome editing in vitro. Such a facile and robust platform holds great potential for non-viral nucleic acid delivery and gene therapy.},
}
@article {pmid37381725,
year = {2023},
author = {Zheng, Q and Wang, W and Zhou, Y and Mo, J and Chang, X and Zha, Z and Zha, L},
title = {Synthetic nanoparticles for the delivery of CRISPR/Cas9 gene editing system: classification and biomedical applications.},
journal = {Biomaterials science},
volume = {11},
number = {16},
pages = {5361-5389},
doi = {10.1039/d3bm00788j},
pmid = {37381725},
issn = {2047-4849},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Nanoparticles ; *Neoplasms/genetics ; },
abstract = {Gene editing has great potential in biomedical research including disease diagnosis and treatment. Clustered regularly interspaced short palindromic repeats (CRISPR) is the most straightforward and cost-effective method. The efficient and precise delivery of CRISPR can impact the specificity and efficacy of gene editing. In recent years, synthetic nanoparticles have been discovered as effective CRISPR/Cas9 delivery vehicles. We categorized synthetic nanoparticles for CRISPR/Cas9 delivery and discribed their advantages and disadvantages. Further, the building blocks of different kinds of nanoparticles and their applications in cells/tissues, cancer and other diseases were described in detail. Finally, the challenges encountered in the clinical application of CRISPR/Cas9 delivery materials were discussed, and potential solutions were provided regarding efficiency and biosafety issues.},
}
@article {pmid37166587,
year = {2023},
author = {Drapal, M and Enfissi, EMA and Almeida, J and Rapacz, E and Nogueira, M and Fraser, PD},
title = {The potential of metabolomics in assessing global compositional changes resulting from the application of CRISPR/Cas9 technologies.},
journal = {Transgenic research},
volume = {32},
number = {4},
pages = {265-278},
pmid = {37166587},
issn = {1573-9368},
support = {760331//Horizon 2020 Framework Programme/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding ; Metabolomics ; },
abstract = {Exhaustive analysis of genetically modified crops over multiple decades has increased societal confidence in the technology. New Plant Breeding Techniques are now emerging with improved precision and the ability to generate products containing no foreign DNA and mimic/replicate conventionally bred varieties. In the present study, metabolomic analysis was used to compare (i) tobacco genotypes with and without the CRISPR associated protein 9 (Cas9), (ii) tobacco lines with the edited and non-edited DE-ETIOLATED-1 gene without phenotype and (iii) leaf and fruit tissue from stable non-edited tomato progeny with and without the Cas9. In all cases, multivariate analysis based on the difference test using LC-HRMS/MS and GC-MS data indicated no significant difference in their metabolomes. The variations in metabolome composition that were evident could be associated with the processes of tissue culture regeneration and/or transformation (e.g. interaction with Agrobacterium). Metabolites responsible for the variance included quantitative changes of abundant, well characterised metabolites such as phenolics (e.g. chlorogenic acid) and several common sugars such as fructose. This study provides fundamental data on the characterisation of gene edited crops, that are important for the evaluation of the technology and its assessment. The approach also suggests that metabolomics could contribute to routine product-based analysis of crops/foods generated from New Plant Breeding approaches.},
}
@article {pmid37533651,
year = {2023},
author = {Ittiprasert, W and Moescheid, MF and Chaparro, C and Mann, VH and Quack, T and Rodpai, R and Miller, A and Wisitpongpun, P and Buakaew, W and Mentink-Kane, M and Schmid, S and Popratiloff, A and Grevelding, CG and Grunau, C and Brindley, PJ},
title = {Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.},
journal = {Cell reports methods},
volume = {3},
number = {7},
pages = {100535},
pmid = {37533651},
issn = {2667-2375},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Humans ; *Schistosoma mansoni/genetics ; Transgenes/genetics ; Animals, Genetically Modified/genetics ; *Gene Editing ; },
abstract = {The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.},
}
@article {pmid37533644,
year = {2023},
author = {Vaitsiankova, A and Thakar, T and Ciccia, A},
title = {Base-editing screens illuminate variant effects in human hematopoiesis.},
journal = {Cell reports methods},
volume = {3},
number = {7},
pages = {100541},
pmid = {37533644},
issn = {2667-2375},
support = {R01 CA197774/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Hematopoiesis/genetics ; },
abstract = {In a recent issue of Cell, Martin-Rufino et al. develop a strategy for performing high-throughput base-editing CRISPR screens coupled with single-cell readouts in the context of human hematopoiesis. Through a series of proof-of-principle experiments, the authors demonstrate the potential of base-editing screens for the study and treatment of hematological disorders.},
}
@article {pmid37532731,
year = {2023},
author = {Huang, Y and Qin, G and Cui, T and Zhao, C and Ren, J and Qu, X},
title = {A bimetallic nanoplatform for STING activation and CRISPR/Cas mediated depletion of the methionine transporter in cancer cells restores anti-tumor immune responses.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4647},
pmid = {37532731},
issn = {2041-1723},
mesh = {Female ; Mice ; Animals ; *CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; *Neoplasms/therapy/drug therapy ; Immunotherapy ; Methionine/metabolism ; Immunity ; },
abstract = {Lack of sufficient cytotoxic T lymphocytes (CD8[+] T cells) infiltration and dysfunctional state of CD8[+] T cells are considered enormous obstacles to antitumor immunity. Herein, we construct a synergistic nanoplatform to promote CD8[+] T cell infiltration in tumors while restoring T cell function by regulating methionine metabolism and activating the STING innate immune pathway. The CRISPR/Cas9 system down-regulates the methionine transporter SLC43A2 and restricts the methionine uptake by tumor cells, thereby relieving the methionine competition pressure of T cells; simultaneously, the released nutrition metal ions activate the cGAS/STING pathway. In this work, the described nanoplatform can enhance the effect of immunotherapy in preclinical cancer models in female mice, enhancing STING pathway mediated immunity and facilitating the development of amino acid metabolic intervention-based cancer therapy.},
}
@article {pmid37531786,
year = {2023},
author = {Mehmandar-Oskuie, A and Jahankhani, K and Rostamlou, A and Arabi, S and Sadat Razavi, Z and Mardi, A},
title = {Molecular landscape of LncRNAs in bladder cancer: From drug resistance to novel LncRNA-based therapeutic strategies.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {165},
number = {},
pages = {115242},
doi = {10.1016/j.biopha.2023.115242},
pmid = {37531786},
issn = {1950-6007},
abstract = {Bladder cancer (BC) is a common and serious type of cancer that ranks among the top ten most prevalent malignancies worldwide. Due to the high occurrence rate of BC, the aggressive nature of cancer cells, and their resistance to medication, managing this disease has become a growing challenge in clinical care. Long noncoding RNAs (lncRNAs) are a group of RNA transcripts that do not code for proteins and are more than 200 nucleotides in length. They play a significant role in controlling cellular pathways and molecular interactions during the onset, development and progression of different types of cancers. Recent advancements in high-throughput gene sequencing technology have led to the identification of various differentially expressed lncRNAs in BC, which indicate abnormal expression. In this review, we summarize that these lncRNAs have been found to impact several functions related to the development of BC, including proliferation, cell growth, migration, metastasis, apoptosis, epithelial-mesenchymal transition, and chemo- and radio-resistance. Additionally, lncRNAs may improve prognosis prediction for BC patients, indicating a future use for them as prognostic and diagnostic biomarkers for BC patients. This review highlights that genetic tools and anti-tumor agents, such as CRISPR/Cas systems, siRNA, shRNA, antisense oligonucleotides, and vectors, have been created for use in preclinical cancer models. This has led to a growing interest in using lncRNAs based on positive research findings.},
}
@article {pmid37528660,
year = {2023},
author = {Zhu, Y and Zhang, M and Guo, S and Xu, H and Jie, Z and Tao, SC},
title = {CRISPR-based diagnostics of different biomolecules from nucleic acids, proteins, and small molecules to exosomes.},
journal = {Acta biochimica et biophysica Sinica},
volume = {},
number = {},
pages = {},
doi = {10.3724/abbs.2023134},
pmid = {37528660},
issn = {1745-7270},
abstract = {CRISPR-based detection technologies have been widely explored for molecular diagnostics. However, the challenge lies in converting the signal of different biomolecules, such as nucleic acids, proteins, small molecules, exosomes, and ions, into a CRISPR-based nucleic acid detection signal. Understanding the detection of different biomolecules using CRISPR technology can aid in the development of practical and promising detection approaches. Unfortunately, existing reviews rarely provide an overview of CRISPR-based molecular diagnostics from the perspective of different biomolecules. Herein, we first introduce the principles and characteristics of various CRISPR nucleases for molecular diagnostics. Then, we focus on summarizing and evaluating the latest advancements in CRISPR-based detection of different biomolecules. Through a comparison of different methods of amplification and signal readout, we discuss how general detection methods can be integrated with CRISPR. Finally, we conclude by identifying opportunities for the improvement of CRISPR in quantitative, amplification-free, multiplex, all-in-one, and point-of-care testing (POCT) purposes.},
}
@article {pmid37526970,
year = {2023},
author = {Shangguan, Q and White, MF},
title = {Repurposing the atypical type I-G CRISPR system for bacterial genome engineering.},
journal = {Microbiology (Reading, England)},
volume = {169},
number = {8},
pages = {},
doi = {10.1099/mic.0.001373},
pmid = {37526970},
issn = {1465-2080},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Escherichia coli/genetics/metabolism ; DNA Replication ; DNA ; CRISPR-Cas Systems ; DNA Helicases/genetics/metabolism ; Genome, Bacterial/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long-range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.},
}
@article {pmid37526217,
year = {2023},
author = {Daskalakis, V and Papapetros, S},
title = {Engineering salt-tolerant Cas12f1 variants for gene-editing applications.},
journal = {Journal of biomolecular structure &amp; dynamics},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/07391102.2023.2240418},
pmid = {37526217},
issn = {1538-0254},
abstract = {CRISPR has revolutionized the field of genome editing in life sciences by serving as a versatile and state-of-the-art tool. Cas12f1 is a small nuclease of the bacterial immunity CRISPR system with an ideal size for cellular delivery, in contrast to CRISPR-associated (Cas) proteins like Cas9 or Cas12. However, Cas12f1 works best at low salt concentrations. In this study, we find that the plasticity of certain Cas12f1 regions (K196-Y202 and I452-L515) is negatively affected by increased salt concentrations. On this line, key protein domains (REC1, WED, Nuc, lid) that are involved in the DNA-target recognition and the activation of the catalytic RuvC domain are in turn also affected. We suggest that salt concentration should be taken in to consideration for activity assessments of Cas engineered variants, especially if the mutations are on the protospacer adjacent motif interacting domain. The results can be exploited for the engineering of Cas variants and the assessment of their activity at varying salt concentrations. We propose that the K198Q mutation can restore at great degree the compromised plasticity and could potentially lead to salt-tolerant Cas12f1 variants. The methodology can be also employed for the study of biomolecules in terms of their salinity tolerance.Communicated by Ramaswamy H. Sarma.},
}
@article {pmid37526140,
year = {2023},
author = {Chen, X and McAndrew, MJ and Lapinaite, A},
title = {Unlocking the secrets of ABEs: the molecular mechanism behind their specificity.},
journal = {Biochemical Society transactions},
volume = {},
number = {},
pages = {},
doi = {10.1042/BST20221508},
pmid = {37526140},
issn = {1470-8752},
abstract = {CRISPR-Cas, the bacterial immune systems, have transformed the field of genome editing by providing efficient, easily programmable, and accessible tools for targeted genome editing. DNA base editors (BE) are state-of-the-art CRISPR-based technology, allowing for targeted modifications of individual nucleobases within the genome. Among the BEs, adenine base editors (ABEs) have shown great potential due to their ability to convert A-to-G with high efficiency. However, current ABEs have limitations in terms of their specificity and targeting range. In this review, we provide an overview of the molecular mechanism of ABEs, with a focus on the mechanism of deoxyadenosine deamination by evolved tRNA-specific adenosine deaminase (TadA). We discuss how mutations and adjustments introduced via both directed evolution as well as rational design have improved ABE efficiency and specificity. This review offers insights into the molecular mechanism of ABEs, providing a roadmap for future developments in the precision genome editing field.},
}
@article {pmid37525893,
year = {2023},
author = {Chen, Y and Ping, Y},
title = {Development of CRISPR/Cas Delivery Systems for In Vivo Precision Genome Editing.},
journal = {Accounts of chemical research},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.accounts.3c00279},
pmid = {37525893},
issn = {1520-4898},
abstract = {ConspectusClustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) is emerging as a powerful genome-editing tool, enabling precise and targeted modifications of virtually any genomic sequence in living cells. These technologies have potential therapeutic applications for cancers, metabolic diseases, and genetic disorders. However, several major challenges hinder the full realization of their potential. Specifically, CRISPR-Cas9 gene editors, whether delivered as plasmid DNA, mRNA/sgRNA, or ribonucleoprotein (RNP), exhibit poor membrane permeability, restricting their access to the intracellular genome, where the editing occurs. Additionally, these editors lack tissue or organ specificity, raising concerns about off-target editing at the tissue level that causes unwanted genotoxicity. Though a range of delivery carriers has been developed to deliver Cas9 editors, their effectiveness is often limited by a number of barriers at both the extracellular and intracellular levels. Moreover, the prolonged activity of Cas9 increases the risk of off-target editing at the genomic level. Therefore, it is crucial to develop efficient delivery vectors, along with molecular switches to safely regulate Cas9 activity.In this Account, we summarize our recent achievements in developing different types of materials that can efficiently deliver the plasmid DNA encoding Cas9 protein and single-guide RNA (sgRNA), or Cas9 RNP into cells to highlight the design considerations of carriers for safe and efficient delivery in vitro and in vivo. After elucidating the chemical and physical factors that are responsible for encapsulating and delivering these biomacromolecules, we further elucidate how we design the biodegradable polymeric carriers using dynamic disulfide chemistry, emphasize their safe and efficient delivery features for genome-editing biomacromolecules, and also introduce the integration of the intracellular delivery of genome-editing biomacromolecules with microneedle-based transdermal delivery to promote therapeutic genome editing for inflammatory skin disorders. Finally, we review how we exploit optical, chemical, and genetic switches to control the Cas9 activity in conjunction with targeted delivery to address the spatiotemporal specificity of gene editing in vivo and demonstrate their precision therapy against cancer and colitis treatment as proof-of-concept examples. In the final part, we will summarize the progress we have made and propose the future directions that may impact the field based on our own research outcomes.},
}
@article {pmid37478847,
year = {2023},
author = {Zhao, Y and Tabet, D and Rubio Contreras, D and Lao, L and Kousholt, AN and Weile, J and Melo, H and Hoeg, L and Feng, S and Coté, AG and Lin, ZY and Setiaputra, D and Jonkers, J and Gingras, AC and Gómez Herreros, F and Roth, FP and Durocher, D},
title = {Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens.},
journal = {Molecular cell},
volume = {83},
number = {15},
pages = {2792-2809.e9},
doi = {10.1016/j.molcel.2023.06.025},
pmid = {37478847},
issn = {1097-4164},
support = {FDN159926/HG/NHGRI NIH HHS/United States ; UM1 HG011989/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Damage ; Mutation ; DNA Repair ; Phenotype ; },
abstract = {To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.},
}
@article {pmid37414921,
year = {2023},
author = {Weidenauer, K and Schmidt, C and Rohde, C and Pauli, C and Blank, MF and Heid, D and Waclawiczek, A and Corbacioglu, A and Göllner, S and Lotze, M and Vierbaum, L and Renders, S and Krijgsveld, J and Raffel, S and Sauer, T and Trumpp, A and Pabst, C and Müller-Tidow, C and Janssen, M},
title = {The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.},
journal = {Leukemia},
volume = {37},
number = {8},
pages = {1611-1625},
pmid = {37414921},
issn = {1476-5551},
support = {2021.145.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; DJCLS 04 PSD/2020//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung (Deutsche Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung)/ ; },
mesh = {Humans ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; *Azacitidine/therapeutic use ; Bridged Bicyclo Compounds, Heterocyclic/therapeutic use ; *Leukemia, Myeloid, Acute/drug therapy/genetics ; Ribosomal Protein S6 Kinases ; Ribosomal Protein S6 Kinases, 90-kDa ; RNA, Guide, CRISPR-Cas Systems ; *Drug Resistance, Neoplasm ; },
abstract = {Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.},
}
@article {pmid37403358,
year = {2023},
author = {Stahl, EC and Sabo, JK and Kang, MH and Allen, R and Applegate, E and Kim, SE and Kwon, Y and Seth, A and Lemus, N and Salinas-Rios, V and Soczek, KM and Trinidad, M and Vo, LT and Jeans, C and Wozniak, A and Morris, T and Kimberlin, A and Foti, T and Savage, DF and Doudna, JA},
title = {Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {8},
pages = {2422-2438},
doi = {10.1016/j.ymthe.2023.06.019},
pmid = {37403358},
issn = {1525-0024},
support = {F32 GM140637/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems ; Ribonucleoproteins/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Brain/metabolism ; },
abstract = {Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.},
}
@article {pmid37402371,
year = {2023},
author = {Chen, W and Ma, J and Wu, Z and Wang, Z and Zhang, H and Fu, W and Pan, D and Shi, J and Ji, Q},
title = {Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors.},
journal = {Molecular cell},
volume = {83},
number = {15},
pages = {2768-2780.e6},
doi = {10.1016/j.molcel.2023.06.014},
pmid = {37402371},
issn = {1097-4164},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Endonucleases/genetics ; DNA/genetics ; RNA ; },
abstract = {Type V CRISPR-associated systems (Cas)12 family nucleases are considered to have evolved from transposon-associated TnpB, and several of these nucleases have been engineered as versatile genome editors. Despite the conserved RNA-guided DNA-cleaving functionality, these Cas12 nucleases differ markedly from the currently identified ancestor TnpB in aspects such as guide RNA origination, effector complex composition, and protospacer adjacent motif (PAM) specificity, suggesting the presence of earlier evolutionary intermediates that could be mined to develop advanced genome manipulation biotechnologies. Using evolutionary and biochemical analyses, we identify that the miniature type V-U4 nuclease (referred to as Cas12n, 400-700 amino acids) is likely the earliest evolutionary intermediate between TnpB and large type V CRISPR systems. We demonstrate that with the exception of CRISPR array emergence, CRISPR-Cas12n shares several similar characteristics with TnpB-ωRNA, including a miniature and likely monomeric nuclease for DNA targeting, origination of guide RNA from nuclease coding sequence, and generation of a small sticky end following DNA cleavage. Cas12n nucleases recognize a unique 5'-AAN PAM sequence, of which the A nucleotide at the -2 position is also required for TnpB. Moreover, we demonstrate the robust genome-editing capacity of Cas12n in bacteria and engineer a highly efficient CRISPR-Cas12n (termed Cas12Pro) with up to 80% indel efficiency in human cells. The engineered Cas12Pro enables base editing in human cells. Our results further expand the understanding regarding type V CRISPR evolutionary mechanisms and enrich the miniature CRISPR toolbox for therapeutic applications.},
}
@article {pmid37376733,
year = {2023},
author = {Bekaert, B and Boel, A and De Witte, L and Vandenberghe, W and Popovic, M and Stamatiadis, P and Cosemans, G and Tordeurs, L and De Loore, AM and Chuva de Sousa Lopes, SM and De Sutter, P and Stoop, D and Coucke, P and Menten, B and Heindryckx, B},
title = {Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {8},
pages = {2326-2341},
doi = {10.1016/j.ymthe.2023.06.013},
pmid = {37376733},
issn = {1525-0024},
mesh = {Humans ; Male ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Semen ; Mutation ; Alleles ; Chromosomes ; },
abstract = {Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.},
}
@article {pmid37131068,
year = {2023},
author = {Gupta, SK and Vishwakarma, NK and Malakar, P and Vanspati, P and Sharma, NK and Chattopadhyay, D},
title = {Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing.},
journal = {Protoplasma},
volume = {260},
number = {5},
pages = {1437-1451},
pmid = {37131068},
issn = {1615-6102},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Cicer/genetics ; Codon ; Agrobacterium tumefaciens ; Genome, Plant/genetics ; },
abstract = {Chickpea is considered recalcitrant to in vitro tissue culture amongst all edible legumes. The clustered, regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop, which is rich in nutrients and protein. However, generating stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation protocols. As an attempt to solve this problem, we developed a modified and optimized protocol for chickpea transformation. This study transformed the single cotyledon half-embryo explants using CaMV35S promoter to drive two marker genes (β-glucuronidase gene; GUS and green fluorescent protein; GFP) through binary vectors pBI101.2 and modified pGWB2, respectively. These vectors were delivered in the explants through three different strains of Agrobacterium tumefaciens, viz., GV3101, EHA105, and LBA4404. We found better efficiency with the strain GV3101 (17.56%) compared with two other strains, i.e., 8.54 and 5.43%, respectively. We recorded better regeneration frequencies in plant tissue culture for the constructs GUS and GFP, i.e., 20.54% and 18.09%, respectively. The GV3101 was further used for the transformation of the genome editing construct. For the development of genome-edited plants, we used this modified protocol. We also used a modified binary vector pPZP200 by introducing a CaMV35S-driven chickpea codon-optimized SpCas9 gene. The promoter of the Medicago truncatula U6.1 snRNA gene was used to drive the guide RNA cassettes. This cassette targeted and edited the chickpea phytoene desaturase (CaPDS) gene. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. This study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea PDS gene using an improved chickpea transformation protocol for the first time.},
}
@article {pmid37529278,
year = {2022},
author = {Santiago, JG},
title = {Inconsistent treatments of the kinetics of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) impair assessment of its diagnostic potential.},
journal = {QRB discovery},
volume = {3},
number = {},
pages = {e9},
pmid = {37529278},
issn = {2633-2892},
abstract = {The scientific and technological advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is one of the most exciting developments of the past decade, particularly in the field of gene editing. The technology has two essential components, (1) a guide RNA to match a targeted gene and (2) a CRISPR-associated protein (e.g. Cas 9, Cas12 or Cas13) that acts as an endonuclease to specifically cut DNA. This specificity and reconfigurable nature of CRISPR has also spurred intense academic and commercial interest in the development of CRISPR-based molecular diagnostics. CRISPR Cas12 and Cas13 orthologs are most commonly applied to diagnostics, and these cleave and become activated by DNA and RNA targets, respectively. Despite the intense research interest, the limits of detection (LoDs) and applications of CRISP-based diagnostics remain an open question. A major reason for this is that reports of kinetic rates have been widely inconsistent, and the vast majority of these reports contain gross errors including violations of basic conservation and kinetic rate laws. It is the intent of this Perspective to bring attention to these issues and to identify potential improvements in the manner in which CRISPR kinetic rates and assay LoDs are reported and compared. The CRISPR field would benefit from verifications of self-consistency of data, providing sufficient data for reproduction of experiments, and, in the case of reports of novel assay LoDs, concurrent reporting of the associated kinetic rate constants. The early development of CRISPR-based diagnostics calls for self-reflection and urges us to proceed with caution.},
}
@article {pmid37524043,
year = {2023},
author = {Tian, B and Wang, Y and Tang, W and Chen, J and Zhang, J and Xue, S and Zheng, S and Cheng, G and Gu, B and Chen, M},
title = {Tandem CRISPR nucleases-based lateral flow assay for amplification-free miRNA detection via the designed "locked RNA/DNA" as fuels.},
journal = {Talanta},
volume = {266},
number = {Pt 1},
pages = {124995},
doi = {10.1016/j.talanta.2023.124995},
pmid = {37524043},
issn = {1873-3573},
abstract = {Currently, available biosensors based on CRISPR/Cas typically depend on coupling with nucleic acid amplification technologies to enhance their sensitivity. However, this approach often involves intricate amplification processes, which could be time-consuming and susceptible to contamination. In addition, signal readouts often require sophisticated and cumbersome equipment, obstructing the applicability of CRISPR/Cas assays in resource-limited regions. Herein, a tandem CRISPR/Cas13a/Cas12a mechanism (tanCRISPR) has been developed via the designed "Locked RNA/DNA" probe as fuels for the trans-cleavage nucleic acid of Cas13a and activated nucleic acid of Cas12a. Meanwhile, a lateral flow assay (LFA) is designed to combine with this tandem CRISPR/Cas13a/Cas12a mechanism (termed tanCRISPR-LFA), realizing the portable monitoring of miRNA-21. The tanCRISPR could realize the limit of detection at pM levels (266 folds lower than Cas13a-based miRNA testing alone) without the resort to target amplification procedures. Furthermore, the miRNA-21 levels of MDA-MB-231 cell extracts are sensed by tanCRISPR-LFA, which is comparable to qRT-PCR. With the virtues of portability, rapidity, sensitivity, and low cost, tanCRISPR-LFA is amenable for CRISPR/Cas-based biosensing and potential applications in the clinical diagnosis of miRNA-associated diseases.},
}
@article {pmid37523020,
year = {2023},
author = {Saakre, M and Jaiswal, S and Rathinam, M and Raman, KV and Tilgam, J and Paul, K and Sreevathsa, R and Pattanayak, D},
title = {Host-Delivered RNA Interference for Durable Pest Resistance in Plants: Advanced Methods, Challenges, and Applications.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37523020},
issn = {1559-0305},
abstract = {Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effective plant protection strategy to attain durable insect-pest resistance in crops. Development of transgenic plant using novel strategies to achieve durable resistance against the target insect.},
}
@article {pmid37478130,
year = {2023},
author = {Feng, H and Bavister, G and Gribble, KE and Mark Welch, DB},
title = {Highly efficient CRISPR-mediated gene editing in a rotifer.},
journal = {PLoS biology},
volume = {21},
number = {7},
pages = {e3001888},
pmid = {37478130},
issn = {1545-7885},
support = {R21 AG067034/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Female ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9 ; *Rotifera/genetics ; DNA Repair ; },
abstract = {Rotifers have been studied in the laboratory and field for over 100 years in investigations of microevolution, ecological dynamics, and ecotoxicology. In recent years, rotifers have emerged as a model system for modern studies of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines in rotifers, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least 1 CRISPR-induced mutation, with many apparently mutated at both alleles. In addition, we achieved precise CRISPR-mediated knock-in of a stop codon cassette in the mlh3 locus, with half of injected mothers producing F2 offspring with an insertion of the cassette. Thus, this protocol produces knockout and knock-in CRISPR/Cas9 editing with high efficiency, to further advance rotifers as a model system for biological discovery.},
}
@article {pmid37295292,
year = {2023},
author = {Cheng, Y and Wang, H and Li, M},
title = {The promise of CRISPR/Cas9 technology in diabetes mellitus therapy: How gene editing is revolutionizing diabetes research and treatment.},
journal = {Journal of diabetes and its complications},
volume = {37},
number = {8},
pages = {108524},
doi = {10.1016/j.jdiacomp.2023.108524},
pmid = {37295292},
issn = {1873-460X},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Genetic Therapy ; Stem Cells ; *Diabetes Mellitus/genetics/therapy ; },
abstract = {Diabetes mellitus is a metabolic disease, characterized by chronic hyperglycemia caused by an abnormality in insulin secretion or action. Millions of people across the world are affected by diabetes mellitus which has serious implications for their health. Over the past few decades, diabetes has become a major cause of mortality and morbidity across the world due to its rapid prevalence. Treatment for diabetes that focuses on insulin secretion and sensitization can lead to unwanted side effects and/or poor compliance, as well as treatment failure. A promising way to treat diabetes is through gene-editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). However, issues such as efficiency and off-target effects have hindered the use of these technologies. In this review, we summarize what we know today about CRISPR/Cas9 technology's therapeutic potential for treating diabetes. We discuss how different strategies are employed, including cell-based therapies (such as stem cells and brown adipocytes), targeting critical genes involved in diabetes pathogenesis, and discussing the challenges and limitations associated with this technology. A novel and powerful treatment approach to diabetes and other diseases can be found with CRISPR/Cas9 technology, and further research should be carried out in this field.},
}
@article {pmid37201284,
year = {2023},
author = {Sanchez-Muñoz, R},
title = {Efficient cut and paste: directional oligodeoxynucleotide-based targeted insertion (DOTI) as a precise genome-editing method.},
journal = {The Plant cell},
volume = {35},
number = {8},
pages = {2697-2698},
pmid = {37201284},
issn = {1532-298X},
mesh = {*Oligodeoxyribonucleotides/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37522927,
year = {2023},
author = {Yigider, E and Taspinar, MS and Agar, G},
title = {Advances in bread wheat production through CRISPR/Cas9 technology: a comprehensive review of quality and other aspects.},
journal = {Planta},
volume = {258},
number = {3},
pages = {55},
pmid = {37522927},
issn = {1432-2048},
mesh = {Humans ; *Triticum/genetics ; *Bread ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Gene Editing ; },
abstract = {This review provides a comprehensive overview of the CRISPR/Cas9 technique and the research areas of this gene editing tool in improving wheat quality. Wheat (Triticum aestivum L.), the basic nutrition for most of the human population, contributes 20% of the daily energy needed because of its, carbohydrate, essential amino acids, minerals, protein, and vitamin content. Wheat varieties that produce high yields and have enhanced nutritional quality will be required to fulfill future demands. Hexaploid wheat has A, B, and D genomes and includes three like but not identical copies of genes that influence important yield and quality. CRISPR/Cas9, which allows multiplex genome editing provides major opportunities in genome editing studies of plants, especially complicated genomes such as wheat. In this overview, we discuss the CRISPR/Cas9 technique, which is credited with bringing about a paradigm shift in genome editing studies. We also provide a summary of recent research utilizing CRISPR/Cas9 to investigate yield, quality, resistance to biotic/abiotic stress, and hybrid seed production. In addition, we provide a synopsis of the laboratory experience-based solution alternatives as well as the potential obstacles for wheat CRISPR studies. Although wheat's extensive genome and complicated polyploid structure previously slowed wheat genetic engineering and breeding progress, effective CRISPR/Cas9 systems are now successfully used to boost wheat development.},
}
@article {pmid36959722,
year = {2023},
author = {Nakazawa, T and Inoue, C and Morimoto, R and Nguyen, DX and Bao, D and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.},
journal = {Environmental microbiology},
volume = {25},
number = {8},
pages = {1393-1408},
doi = {10.1111/1462-2920.16372},
pmid = {36959722},
issn = {1462-2920},
support = {//Institute for Fermentation, Osaka/ ; 20F20092//Japan Society for the Promotion of Science/ ; 120208402//Japan Society for the Promotion of Science/ ; 120209920//Japan Society for the Promotion of Science/ ; 18H02254//Japan Society for the Promotion of Science/ ; 18KK0178//Japan Society for the Promotion of Science/ ; 19H03017//Japan Society for the Promotion of Science/ ; 19K22332//Japan Society for the Promotion of Science/ ; 21K18224//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; //Kansai Research Foundation for Technology Promotion/ ; },
mesh = {Lignin/metabolism ; CRISPR-Cas Systems ; *Polyporales/metabolism ; *Pleurotus/genetics/metabolism ; },
abstract = {White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.},
}
@article {pmid36828577,
year = {2023},
author = {Krug, J and Richter, A and Englert, C},
title = {Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA).},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {8},
pages = {107744},
doi = {10.1101/pdb.prot107744},
pmid = {36828577},
issn = {1559-6095},
mesh = {Animals ; *CRISPR-Cas Systems ; Larva/genetics ; Genotype ; Mutation ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of Nothobranchius furzeri embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).},
}
@article {pmid37522916,
year = {2023},
author = {Bora, J and Dey, A and Lyngdoh, AR and Dhasmana, A and Ranjan, A and Kishore, S and Rustagi, S and Tuli, HS and Chauhan, A and Rath, P and Malik, S},
title = {A critical review on therapeutic approaches of CRISPR-Cas9 in diabetes mellitus.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {},
number = {},
pages = {},
pmid = {37522916},
issn = {1432-1912},
abstract = {Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology.},
}
@article {pmid37521929,
year = {2023},
author = {May, D and Sanchez, S and Gilby, J and Altpeter, F},
title = {Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass (Paspalum notatum Flüggé) using CRISPR/Cas9.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1225775},
pmid = {37521929},
issn = {1664-462X},
abstract = {Polyploidy is common among grasses (Poaceae) and poses challenges for conventional breeding. Genome editing technology circumvents crossing and selfing, enabling targeted modifications to multiple gene copies in a single generation while maintaining the heterozygous context of many polyploid genomes. Bahiagrass (Paspalum notatum Flüggé; 2n=4x=40) is an apomictic, tetraploid C4 species that is widely grown in the southeastern United States as forage in beef cattle production and utility turf. The chlorophyll biosynthesis gene magnesium chelatase (MgCh) was selected as a rapid readout target for establishing genome editing in tetraploid bahiagrass. Vectors containing sgRNAs, Cas9 and nptII were delivered to callus cultures by biolistics. Edited plants were characterized through PCR-based assays and DNA sequencing, and mutagenesis frequencies as high as 99% of Illumina reads were observed. Sequencing of wild type (WT) bahiagrass revealed a high level of sequence variation in MgCh likely due to the presence of at least two copies with possibly eight different alleles, including pseudogenes. MgCh mutants exhibited visible chlorophyll depletion with up to 82% reductions in leaf greenness. Two lines displayed progression of editing over time which was linked to somatic editing. Apomictic progeny of a chimeric MgCh editing event were obtained and allowed identification of uniformly edited progeny plants among a range of chlorophyll depletion phenotypes. Sanger sequencing of a highly edited mutant revealed elevated frequency of a WT allele, probably due to frequent homology-directed repair (HDR). To our knowledge these experiments comprise the first report of genome editing applied in perennial, warm-season turf or forage grasses. This technology will accelerate bahiagrass cultivar development.},
}
@article {pmid37521915,
year = {2023},
author = {Farinati, S and Draga, S and Betto, A and Palumbo, F and Vannozzi, A and Lucchin, M and Barcaccia, G},
title = {Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1223861},
pmid = {37521915},
issn = {1664-462X},
abstract = {Plant male sterility (MS) represents the inability of the plant to generate functional anthers, pollen, or male gametes. Developing MS lines represents one of the most important challenges in plant breeding programs, since the establishment of MS lines is a major goal in F1 hybrid production. For these reasons, MS lines have been developed in several species of economic interest, particularly in horticultural crops and ornamental plants. Over the years, MS has been accomplished through many different techniques ranging from approaches based on cross-mediated conventional breeding methods, to advanced devices based on knowledge of genetics and genomics to the most advanced molecular technologies based on genome editing (GE). GE methods, in particular gene knockout mediated by CRISPR/Cas-related tools, have resulted in flexible and successful strategic ideas used to alter the function of key genes, regulating numerous biological processes including MS. These precision breeding technologies are less time-consuming and can accelerate the creation of new genetic variability with the accumulation of favorable alleles, able to dramatically change the biological process and resulting in a potential efficiency of cultivar development bypassing sexual crosses. The main goal of this manuscript is to provide a general overview of insights and advances into plant male sterility, focusing the attention on the recent new breeding GE-based applications capable of inducing MS by targeting specific nuclear genic loci. A summary of the mechanisms underlying the recent CRISPR technology and relative success applications are described for the main crop and ornamental species. The future challenges and new potential applications of CRISPR/Cas systems in MS mutant production and other potential opportunities will be discussed, as generating CRISPR-edited DNA-free by transient transformation system and transgenerational gene editing for introducing desirable alleles and for precision breeding strategies.},
}
@article {pmid37521328,
year = {2023},
author = {Li, T and Chen, Y and Chen, Z and Hao, Y and Liang, M and Liu, Y and Ou, G and Zhang, H and Tang, Y and Hao, Y and Wageh, S and Al-Hartomy, OA and Kalam, A and Zhang, B and Shi, X and Li, X and Zhang, H},
title = {Early and Sensitive Detection of Pathogens for Public Health and Biosafety: An Example of Surveillance and Genotyping of SARS-CoV-2 in Sewage Water by Cas12a-Facilitated Portable Plasmonic Biosensor.},
journal = {Research (Washington, D.C.)},
volume = {6},
number = {},
pages = {0205},
pmid = {37521328},
issn = {2639-5274},
abstract = {Infectious diseases severely threaten public health and global biosafety. In addition to transmission through the air, pathogenic microorganisms have also been detected in environmental liquid samples, such as sewage water. Conventional biochemical detection methodologies are time-consuming and cost-ineffective, and their detection limits hinder early diagnosis. In the present study, ultrafine plasmonic fiber probes with a diameter of 125 μm are fabricated for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-12a-mediated sensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Single-stranded DNA exposed on the fiber surface is trans-cleaved by the Cas12a enzyme to release gold nanoparticles that are immobilized onto the fiber surface, causing a sharp reduction in the surface plasmon resonance (SPR) wavelength. The proposed fiber probe is virus-specific with the limit of detection of ~2,300 copies/ml, and genomic copy numbers can be reflected as shifts in wavelengths. A total of 21 sewage water samples have been examined, and the data obtained are consistent with those of quantitative polymerase chain reaction (qPCR). In addition, the Omicron variant and its mutation sites have been fast detected using S gene-specific Cas12a. This study provides an accurate and convenient approach for the real-time surveillance of microbial contamination in sewage water.},
}
@article {pmid37482301,
year = {2023},
author = {Ayub, N and Soto, G},
title = {Multiple challenges in the development of commercial crops using CRISPR/Cas technology.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {335},
number = {},
pages = {111809},
doi = {10.1016/j.plantsci.2023.111809},
pmid = {37482301},
issn = {1873-2259},
abstract = {The CRISPR/Cas system is a highly efficient and versatile tool for editing plant genomes, with the potential to accelerate breeding programs and improve the sustainability of food production. Nevertheless, technical limitations delay the rapid spread of the CRISPR/Cas system benefits in agriculture. The natural features of plant species, including reproductive behavior, ploidy levels, genetic diversity, and generation times, can significantly impact the introgression of edited traits into elite germplasms. The production and selection of edited events require the same level of effort as those of their transgenic equivalents. Additionally, edited alleles tend to be recessive or not fully dominant, which differs from dominant transgenic events. To accelerate the introgression of edited events into conventional and transgenic varieties, we suggest utilizing edits on single-copy genes that induce dominant mutations. In the absence of new, simple traits that provide exceptional economic benefits for large companies, like herbicide tolerance in transgenic crops, we propose the emergence of particular public grants for edited variety productions, especially when the introgression shows a high level of technical feasibility. In the context of climate change, these public actions must be taken quickly to alleviate significant reductions in crop production.},
}
@article {pmid36897256,
year = {2023},
author = {Li, LZ and Yang, K and Jing, Y and Fan, Y and Jiang, X and Wang, S and Liu, GH and Qu, J and Ma, S and Zhang, W},
title = {CRISPR-based screening identifies XPO7 as a positive regulator of senescence.},
journal = {Protein &amp; cell},
volume = {14},
number = {8},
pages = {623-628},
pmid = {36897256},
issn = {1674-8018},
mesh = {*Cell Nucleus ; *Cellular Senescence/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37520667,
year = {2023},
author = {Wang, H and Wang, R and Yang, J and Feng, Y and Xu, S and Pei, QG},
title = {Interactions of Fibroblast Subtypes Influence Osteoclastogenesis and Alveolar Bone Destruction in Periodontitis.},
journal = {Journal of inflammation research},
volume = {16},
number = {},
pages = {3143-3156},
pmid = {37520667},
issn = {1178-7031},
abstract = {BACKGROUND: To analyze the fibroblasts subtypes in the gingival tissues of healthy controls, gingivitis and periodontitis patients, as well as the effects of interaction between subtypes on alveolar bone destruction.<br><br>METHODS: Gingival tissues were divided into three groups according to clinical and radiographic examination, and the immunostaining of EDA+FN was assessed. Fibroblasts from gingiva developed colony formation units (CFUs) and induced Trap+MNCs. The expression of osteoclastogenesis-related genes was assessed by real-time PCR. Variances in the gene profiles of CFUs were identified by principal component analysis, and cluster analysis divided CFUs into subtypes. The induction of Trap+MNCs and gene expression were compared among individual or cocultured subtypes. The fibroblast subtypes exerted critical effect on Trap+MNCs formation were selected and edited by CRISPR/Cas to investigate the influence on osteoclastogenesis in the periodontitis in mice.<br><br>RESULTS: Most periodontitis samples exhibited intensive EDA+FN staining (P < 0.05), and these fibroblasts also induced most Trap+MNCs among three groups; consistently, fibroblasts from periodontitis highly expressed genes facilitating osteoclastogenesis. According to gene profiles and osteoclastogenic induction, four clusters of CFUs were identified. The proportion of clusters was significantly different (P < 0.05) among three groups, and their interaction influenced osteoclastogenic induction. Although Cluster 4 induced less osteoclasts, it enhanced the effects of Clusters 1 and 3 on Trap+MNCs formation (P < 0.05). EDA knockout in Cluster 4 abrogated this promotion (P < 0.05), and decreased osteoclasts and alveolar bone destruction in experimental periodontitis (P < 0.05).<br><br>CONCLUSION: Heterogeneous fibroblast subtypes affect the switch or development of periodontitis. A subtype (Cluster 4) played important role during alveolar bone destruction, by regulating other subtypes via EDA+FN paracrine.},
}
@article {pmid37518013,
year = {2023},
author = {Wang, X and Cheng, M and Yang, S and Xing, C and Li, Q and Zhu, Y and Ji, Y and Du, Y},
title = {CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood.},
journal = {Parasites &amp; vectors},
volume = {16},
number = {1},
pages = {256},
pmid = {37518013},
issn = {1756-3305},
support = {2021YFC2301100//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Humans ; Mice ; Swine ; CRISPR-Cas Systems ; *Toxoplasmosis/parasitology ; *Toxoplasma/genetics ; Polymerase Chain Reaction ; Sensitivity and Specificity ; DNA, Protozoan/analysis ; },
abstract = {BACKGROUND: Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.<br><br>METHODS: This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529&#xa0;bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.<br><br>RESULTS: The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/&#x3bc;l for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5&#xa0;days post infection (dpi).<br><br>CONCLUSIONS: This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.},
}
@article {pmid37517824,
year = {2023},
author = {Wan, X and Chen, J and Wu, Y and Chen, Z and Liu, Y and Li, T and Sun, J and Zhang, T and Zhou, F and Huang, X and Li, Y and Wang, X and Sun, X},
title = {Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using CRISPR/Cas12a Detection.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {25},
number = {8},
pages = {540-554},
doi = {10.1016/j.jmoldx.2023.04.006},
pmid = {37517824},
issn = {1943-7811},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Optic Atrophy, Hereditary, Leber/diagnosis/genetics ; DNA, Mitochondrial/genetics ; Mitochondria/genetics ; Mutation ; },
abstract = {Leber hereditary optic neuropathy (LHON) is the most common maternally inherited mitochondrial disease, with >90% of cases harboring one of three point variants (m.3460G>A, m.11778G>A, and m.14484T>C). Rapid and sensitive diagnosis of LHON variants is urgently needed for early diagnosis and timely treatment after onset, which is currently limited. Herein, we adapted the Cas12a-based DNA detection platform for LHON mitochondrial variant diagnosis. Single-strand guide CRISPR RNAs and enzymatic recombinase amplification primers were first screened, the CRISPR/Cas12a system was then optimized with restriction enzymes, and finally compared with Sanger sequencing and next-generation sequencing (NGS) in multicenter clinical samples. This approach can be completed within 30 minutes using only one drop of blood and could reach a sensitivity of 1% of heteroplasmy. Among the 182 multicenter clinical samples, the CRISPR/Cas12a detection system showed high consistency with Sanger sequencing and NGS in both specificity and sensitivity. Notably, a sample harboring a de novo 3.78% m.11778G>A variant detected by NGS, but not by Sanger sequencing, was successfully confirmed using the CRISPR/Cas12a assay, which proved the effectiveness of our method. Overall, our CRISPR/Cas12a detection system provides an alternative for rapid, convenient, and sensitive detection of LHON variants, exhibiting great potential for clinical practice.},
}
@article {pmid37517615,
year = {2023},
author = {Nabi, N and Singh, S and Saffeullah, P},
title = {An updated review on distribution, biosynthesis and pharmacological effects of artemisinin: A wonder drug.},
journal = {Phytochemistry},
volume = {},
number = {},
pages = {113798},
doi = {10.1016/j.phytochem.2023.113798},
pmid = {37517615},
issn = {1873-3700},
abstract = {Plant-based drugs have been used for centuries for treating different ailments. Malaria, one of the prevalent threats in many parts of the world, is treated mainly by artemisinin-based drugs derived from plants of genus Artemisia. However, the distribution of artemisinin is restricted to a few species of the genus; besides, its yield depends on ontogeny and the plant's geographical location. Here, we review the studies focusing on biosynthesis and distributional pattern of artemisinin production in species of the genus Artemisia. We also discussed various agronomic and in vitro methods and molecular approaches to increase the yield of artemisinin. We have summarized different mechanisms of artemisinin involved in its anti-malarial, anti-cancer, anti-inflammatory and anti-viral activities (like against Covid-19). Overall the current review provides a synopsis of a global view of the distribution of artemisinin, its biosynthesis, and pharmacological potential in treating various diseases like malaria, cancer, and coronavirus, which may provoke future research efforts in drug development. Nevertheless, long-term trials and molecular approaches, like CRISPR-Cas, are required for in-depth research.},
}
@article {pmid37516612,
year = {2023},
author = {Talebian, S and Dehghani, F and Weiss, PS and Conde, J},
title = {Evolution of CRISPR-enabled biosensors for amplification-free nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.07.004},
pmid = {37516612},
issn = {1879-3096},
abstract = {CRISPR biosensors enable rapid and accurate detection of nucleic acids without resorting to target amplification. Specifically, these systems facilitate the simultaneous detection of multiple nucleic acid targets with single-base specificity. This is an invaluable asset that can ultimately facilitate accurate diagnoses of biologically complex diseases.},
}
@article {pmid37516409,
year = {2023},
author = {Popovitz, J and Sharma, R and Hoshyar, R and Soo Kim, B and Murthy, N and Lee, K},
title = {Gene editing therapeutics based on mRNA delivery.},
journal = {Advanced drug delivery reviews},
volume = {},
number = {},
pages = {115026},
doi = {10.1016/j.addr.2023.115026},
pmid = {37516409},
issn = {1872-8294},
abstract = {The field of gene editing has received much attention in recent years due to its immense therapeutic potential. In particular, gene editing therapeutics, such as the CRISPR-Cas systems, base editors, and other emerging gene editors, offer the opportunity to address previously untreatable disorders. This review aims to summarize the therapeutic applications of gene editing based on mRNA delivery. We introduce gene editing therapeutics using mRNA and focus on engineering and improvement of gene editing technology. We subsequently examine ex vivo and in vivo gene editing techniques and conclude with an exploration of the next generation of CRISPR and base editing systems.},
}
@article {pmid37515526,
year = {2023},
author = {Singh, M and Misra, CS and Bindal, G and Rangu, SS and Rath, D},
title = {CRISPR-Cas12a assisted specific detection of mpox virus.},
journal = {Journal of medical virology},
volume = {95},
number = {8},
pages = {e28974},
doi = {10.1002/jmv.28974},
pmid = {37515526},
issn = {1096-9071},
mesh = {Humans ; CRISPR-Cas Systems ; Monkeypox virus ; *Monkeypox ; *Orthopoxvirus/genetics ; Biological Assay ; },
abstract = {Mpox virus, a member of genus Orthopoxvirus, causes rash and flu-like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease-based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/μl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.},
}
@article {pmid37390747,
year = {2023},
author = {Hua, Z and Xu, K and Xiao, W and Shu, C and Li, N and Li, K and Gu, H and Zhu, Z and Zhang, L and Ren, H and Zeng, Q and Yin, Y and Bi, Y},
title = {Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs.},
journal = {Biochemical and biophysical research communications},
volume = {673},
number = {},
pages = {145-152},
doi = {10.1016/j.bbrc.2023.06.053},
pmid = {37390747},
issn = {1090-2104},
mesh = {Animals ; Swine ; *RNA, Guide, CRISPR-Cas Systems ; Gene Knockout Techniques ; *Myostatin/genetics ; Hyperplasia/genetics/pathology ; Muscle Fibers, Skeletal ; Muscle, Skeletal/pathology ; Adipocytes ; },
abstract = {Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN[-/-]). Growth testing revealed that both the growth rate and average daily weight gain of MST[-/-] pigs were greater than those of wild-type (MSTN[+/+]) pigs. Slaughter data demonstrated that the lean ratio of MSTN[-/-] pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN[+/+] pigs. Haematoxylin-eosin staining indicated that the increased leanness of MSTN[-/-] pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN[-/-] pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN[-/-] pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.},
}
@article {pmid37513832,
year = {2023},
author = {Junaid, M and Thirapanmethee, K and Khuntayaporn, P and Chomnawang, MT},
title = {CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {16},
number = {7},
pages = {},
pmid = {37513832},
issn = {1424-8247},
support = {Mullika Chomnawang//Mahidol University/ ; },
abstract = {Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.},
}
@article {pmid37513399,
year = {2023},
author = {Li, Q and Li, S and Fang, J and Yang, C and Zhao, X and Wang, Q and Zhou, W and Zheng, W},
title = {Artemisinin Confers Neuroprotection against 6-OHDA-Induced Neuronal Injury In Vitro and In Vivo through Activation of the ERK1/2 Pathway.},
journal = {Molecules (Basel, Switzerland)},
volume = {28},
number = {14},
pages = {},
pmid = {37513399},
issn = {1420-3049},
support = {32070969//National Natural Science Foundation of China/ ; 0127/2019/A3, 0113/2018/A3 and 0038/2020/AMJ//Science and Technology Development Fund, Macau SAR/ ; 2022-Natural Science Foundation//Guangdong Provincial Funding Committee for Basic and Applied Fundamental Research/ ; LY21H280010//Natural Science Foundation of Zhejiang Province/ ; 2021KY136//Medical and health Science and Technology Project of Zhejiang Province/ ; MYRG2018-00134-FHS and MYRG2020-00158-FSH//University of Macau/ ; },
mesh = {Rats ; Humans ; Mice ; Animals ; *Parkinson Disease/drug therapy/metabolism ; MAP Kinase Signaling System ; Oxidopamine/adverse effects ; *Neuroprotective Agents/therapeutic use ; Neuroprotection ; *Neurodegenerative Diseases/drug therapy ; RNA, Guide, CRISPR-Cas Systems ; *Neuroblastoma/drug therapy ; Apoptosis ; *Artemisinins/metabolism ; Dopaminergic Neurons ; },
abstract = {Parkinson's disease (PD) is an age-related, progressive neurodegenerative disease characterized by the gradual and massive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). We have recently reported that artemisinin, an FDA-approved first-line antimalarial drug, possesses a neuroprotective effect. However, the effects and underlying mechanisms of artemisinin on Parkinson's disease remain to be elucidated. In this study, we investigated the neuroprotective effects of artemisinin on 6-OHDA and MPP[+] in neuronal cells and animal models, as well as the underlying mechanisms. Our results showed that artemisinin significantly attenuated the loss of cell viability, LDH release, elevated levels of reactive oxygen species (ROS), the collapse of the mitochondria trans-membrane potential and cell apoptosis in PC12 cells. Western blot results showed that artemisinin stimulated the phosphorylation of ERK1/2, its upstream signaling proteins c-Raf and MEK and its downstream target CREB in PC12 cells in a time- and concentration-dependent manner. In addition, the protective effect of artemisinin was significantly reduced when the ERK pathway was blocked using the ERK pathway inhibitor PD98059 or when the expression of ERK was knocked down using sgRNA. These results indicate the essential role of ERK in the protective effect of artemisinin. Similar results were obtained in SH-SY5Y cells and primary cultured neurons treated with 6-OHDA, as well as in cellular models of MPP[+] injury. More interestingly, artemisinin attenuated PD-like behavior deficit in mice injected with 6-OHDA evaluated by behavioral tests including swimming test, pole-test, open field exploration and rotarod tests. Moreover, artemisinin also stimulated the phosphorylation of ERK1/2, inhibited apoptosis, and rescued dopaminergic neurons in SNc of these animals. Application of ERK pathway inhibitor PD98059 blocked the protective effect of artemisinin in mice during testing. Taking these results together, it was indicated that artemisinin preserves neuroprotective effects against 6-OHDA and MPP[+] induced injury both in vitro and in vivo by the stimulation of the ERK1/2 signaling pathway. Our findings support the potential therapeutic effect of artemisinin in the prevention and treatment of Parkinson's disease.},
}
@article {pmid37512821,
year = {2023},
author = {Reyes-Castillo, PA and González-Vázquez, R and Torres-Maravilla, E and Bautista-Hernández, JI and Zúñiga-León, E and Leyte-Lugo, M and Mateos-Sánchez, L and Mendoza-Pérez, F and Gutiérrez-Nava, MA and Reyes-Pavón, D and Azaola-Espinosa, A and Mayorga-Reyes, L},
title = {Bifidobacterium longum LBUX23 Isolated from Feces of a Newborn; Potential Probiotic Properties and Genomic Characterization.},
journal = {Microorganisms},
volume = {11},
number = {7},
pages = {},
pmid = {37512821},
issn = {2076-2607},
abstract = {Bifidobacterium longum is considered a microorganism with probiotic potential, which has been extensively studied, but these probiotic effects are strain dependent. This work aims to characterize the probiotic potential, based on the biochemical and genomic functionality, of B. longum LBUX23, isolated from neonates' feces. B. longum LBUX23 contains one circular genome of 2,287,838 bp with a G+C content of 60.05%, no plasmids, no CRISPR-Cas operon, possesses 56 tRNAs, 9 rRNAs, 1 tmRNA and 1776 coding sequences (CDSs). It has chromosomally encoded resistance genes to ampicillin and dicloxacillin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and to some emergent pathogen's clinical strains. B. longum LBUX23 was able to utilize lactose, sucrose, fructooligosaccharides (FOS), and lactulose. The maximum peak of bacterial growth was observed in sucrose and FOS at 6 h; in lactose and lactulose, it was shown at 8 h. B. longum LBUX23 can survive in gastrointestinal conditions (pH 4 to 7). A decrease in survival (96.5 and 93.8%) was observed at pH 3 and 3.5 during 120 min. argC, argH, and dapA genes could be involved in this tolerance. B. longum LBUX23 can also survive under primary and secondary glyco- or tauro-conjugated bile salts, and a mixture of bile salts due to the high extracellular bile salt hydrolase (BSH) activity (67.3 %), in taurocholic acid followed by taurodeoxycholic acid (48.5%), glycocholic acid (47.1%), oxgall (44.3%), and glycodeoxycholic acid (29.7%) probably due to the presence of the cbh and gnlE genes which form an operon (start: 119573 and end: 123812). Low BSH activity was determined intracellularly (<7%), particularly in glycocholic acid; no intracellular activity was shown. B. longum LBUX23 showed antioxidant effects in DPPH radical, mainly in intact cells (27.4%). In the case of hydroxyl radical scavenging capacity, cell debris showed the highest reduction (72.5%). In the cell-free extract, superoxide anion radical scavenging capacity was higher (90.5%). The genome of B. longum LBUX23 contains PNPOx, AhpC, Bcp, trxA, and trxB genes, which could be involved in this activity. Regarding adherence, it showed adherence up to 5% to Caco-2 cells. B. longum LBUX23 showed in vitro potential probiotic properties, mainly in BSH activity and antioxidant capacity, which indicates that it could be a good candidate for antioxidant or anti-cholesterol tests using in vivo models.},
}
@article {pmid37512736,
year = {2023},
author = {Romero Deza, AA and Schaumburg, F and Berli, CLA},
title = {Valveless On-Chip Aliquoting for Molecular Diagnosis.},
journal = {Micromachines},
volume = {14},
number = {7},
pages = {},
pmid = {37512736},
issn = {2072-666X},
support = {PIP 11220200101392CO//National Scientific and Technical Research Council/ ; },
abstract = {The detection of nucleic acids as specific markers of infectious diseases is commonly implemented in molecular biology laboratories. The translation of these benchtop assays to a lab-on-a-chip format demands huge efforts of integration and automation. The present work is motivated by a strong requirement often posed by molecular assays that combine isothermal amplification and CRISPR/Cas-based detection: after amplification, a 2-8 microliter aliquot of the reaction products must be taken for the subsequent reaction. In order to fulfill this technical problem, we have designed and prototyped a microfluidic device that is able to meter and aliquot in the required range during the stepped assay. The operation is achieved by integrating a porous material that retains the desired amount of liquid after removing the excess reaction products, an innovative solution that avoids valving and external actuation. The prototypes were calibrated and experimentally tested to demonstrate the overall performance (general fluidics, metering, aliquoting, mixing and reaction). The proposed aliquoting method is fully compatible with additional functions, such as sample concentration or reagent storage, and could be further employed in alternative applications beyond molecular diagnosis.},
}
@article {pmid37511831,
year = {2023},
author = {Yadav, RK and Tripathi, MK and Tiwari, S and Tripathi, N and Asati, R and Chauhan, S and Tiwari, PN and Payasi, DK},
title = {Genome Editing and Improvement of Abiotic Stress Tolerance in Crop Plants.},
journal = {Life (Basel, Switzerland)},
volume = {13},
number = {7},
pages = {},
pmid = {37511831},
issn = {2075-1729},
abstract = {Genome editing aims to revolutionise plant breeding and could assist in safeguarding the global food supply. The inclusion of a 12-40 bp recognition site makes mega nucleases the first tools utilized for genome editing and first generation gene-editing tools. Zinc finger nucleases (ZFNs) are the second gene-editing technique, and because they create double-stranded breaks, they are more dependable and effective. ZFNs were the original designed nuclease-based approach of genome editing. The Cys2-His2 zinc finger domain's discovery made this technique possible. Clustered regularly interspaced short palindromic repeats (CRISPR) are utilized to improve genetics, boost biomass production, increase nutrient usage efficiency, and develop disease resistance. Plant genomes can be effectively modified using genome-editing technologies to enhance characteristics without introducing foreign DNA into the genome. Next-generation plant breeding will soon be defined by these exact breeding methods. There is abroad promise that genome-edited crops will be essential in the years to come for improving the sustainability and climate-change resilience of food systems. This method also has great potential for enhancing crops' resistance to various abiotic stressors. In this review paper, we summarize the most recent findings about the mechanism of abiotic stress response in crop plants and the use of the CRISPR/Cas mediated gene-editing systems to improve tolerance to stresses including drought, salinity, cold, heat, and heavy metals.},
}
@article {pmid37511604,
year = {2023},
author = {Zhang, Y and Mo, Y and Han, L and Sun, Z and Xu, W},
title = {Exploring Transcriptional Regulation of Hyperaccumulation in Sedum plumbizincicola through Integrated Transcriptome Analysis and CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {24},
number = {14},
pages = {},
pmid = {37511604},
issn = {1422-0067},
support = {32172666//National Natural Science Foundation of China/ ; },
mesh = {Cadmium/toxicity/metabolism ; *Sedum/metabolism ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Transcription Factors/genetics ; *Soil Pollutants ; Biodegradation, Environmental ; Plant Roots/genetics/metabolism ; },
abstract = {The cadmium hyperaccumulator Sedum plumbizincicola has remarkable abilities for cadmium (Cd) transport, accumulation and detoxification, but the transcriptional regulation mechanisms responsible for its Cd hyperaccumulation remain unknown. To address this knowledge gap, we conducted a comparative transcriptome study between S. plumbizincicola and the non-hyperaccumulating ecotype (NHE) of Sedum alfredii with or without Cd treatment. Our results revealed many differentially expressed genes involved in heavy metal transport and detoxification that were abundantly expressed in S. plumbizincicola. Additionally, we identified a large number of differentially expressed transcription factor genes, highlighting the complexity of transcriptional regulatory networks. We further screened four transcription factor genes that were highly expressed in the roots of S. plumbizincicola as candidate genes for creating CRISPR/Cas9 knockout mutations. Among these, the SpARR11 and SpMYB84 mutant lines exhibited decreased Cd accumulation in their aboveground parts, suggesting that these two transcription factors may play a role in the regulation of the Cd hyperaccumulation in S. plumbizincicola. Although further research will be required to determine the precise targeted genes of these transcription factors, combined transcriptome analysis and CRISPR/Cas9 technology provides unprecedented opportunities for identifying transcription factors related to Cd hyperaccumulation and contributes to the understanding of the transcriptional regulation mechanism of hyperaccumulation in S. plumbizincicola.},
}
@article {pmid37511168,
year = {2023},
author = {Wichmann, M and Maire, CL and Nuppenau, N and Habiballa, M and Uhde, A and Kolbe, K and Schröder, T and Lamszus, K and Fehse, B and Głów, D},
title = {Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs.},
journal = {International journal of molecular sciences},
volume = {24},
number = {14},
pages = {},
pmid = {37511168},
issn = {1422-0067},
support = {SFB841/SP2 (BF)//Deutsche Forschungsgemeinschaft/ ; DZG Innovation Fund "Gene and Cell Therapy": DZG 01.202 (BF)//German Center for Infection Research/ ; MD grant to MW//C3i-Hamburg Graduate School/ ; },
mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; Retroviridae/genetics ; Gene Editing/methods ; Lentivirus/genetics ; *Nanoparticles ; },
abstract = {The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.},
}
@article {pmid37511044,
year = {2023},
author = {Peixoto, J and Príncipe, C and Pestana, A and Osório, H and Pinto, MT and Prazeres, H and Soares, P and Lima, RT},
title = {Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {24},
number = {14},
pages = {},
pmid = {37511044},
issn = {1422-0067},
support = {PTDC/MEC-ONC/31520/2017//FEEI, FEDER through COMPETE 2020 -POCI, Portugal 2020, and by Portuguese funds through FCT/Minist&#xe9;rio da Ci&#xea;ncia, Tecnologia e Ensino Superior/ ; POCI-01-0145-FEDER-028779 (PTDC/BIA-MIC/28779/2017)//FEEI, FEDER through COMPETE 2020 -POCI, Portugal 2020, and by Portuguese funds through FCT/Minist&#xe9;rio da Ci&#xea;ncia, Tecnologia e Ensino Superior/ ; project "Institute for Research and Innovation in Health Sciences" (UID/BIM/04293/2019)//FEEI, FEDER through COMPETE 2020 -POCI, Portugal 2020, and by Portuguese funds through FCT/Minist&#xe9;rio da Ci&#xea;ncia, Tecnologia e Ensino Superior/ ; "Cancer Research on Therapy Resistance: From Basic Mechanisms to Novel Targets"-NORTE-01-0145-FEDER-000051//Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF/ ; The Porto Comprehensive Cancer Center" with the reference NORTE-01-0145-FEDER-072678 - Cons&#xf3;rcio PORTO.CCC - Porto.Comprehensive Cancer Center Raquel Seruca//European Regional Development Fund/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infra-structures of Strategic Relevance/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Glioblastoma/genetics ; Proteomics ; Gene Editing/methods ; Receptors, LDL/genetics ; },
abstract = {LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.},
}
@article {pmid37510242,
year = {2023},
author = {Parra-Sánchez, &#xc1; and Antequera-Zambrano, L and Martínez-Navarrete, G and Zorrilla-Muñoz, V and Paz, JL and Alvarado, YJ and González-Paz, L and Fernández, E},
title = {Comparative Analysis of CRISPR-Cas Systems in Pseudomonas Genomes.},
journal = {Genes},
volume = {14},
number = {7},
pages = {},
pmid = {37510242},
issn = {2073-4425},
mesh = {Humans ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Pseudomonas/genetics ; Plasmids ; RNA ; },
abstract = {Pseudomonas is a bacterial genus with some saprophytic species from land and others associated with opportunistic infections in humans and animals. Factors such as pathogenicity or metabolic aspects have been related to CRISPR-Cas, and in silico studies into it have focused more on the clinical and non-environmental setting. This work aimed to perform an in silico analysis of the CRISPR-Cas systems present in Pseudomonas genomes. It analyzed 275 complete genomic sequences of Pseudomonas taken from the NCBI database. CRISPR loci were obtained from CRISPRdb. The genes associated with CRISPR (cas) and CAS proteins, and the origin and diversity of spacer sequences, were identified and compared by BLAST. The presence of self-targeting sequences, PAMs, and the conservation of DRs were visualized using WebLogo 3.6. The CRISPR-like RNA secondary structure prediction was analyzed using RNAFold and MFold. CRISPR structures were identified in 19.6% of Pseudomonas species. In all, 113 typical CRISPR arrays with 18 putative cas were found, as were 2050 spacers, of which 52% showed homology to bacteriophages, 26% to chromosomes, and 22% to plasmids. No potential self-targeting was detected within the CRISPR array. All the found DRs can form thermodynamically stable secondary RNA structures. The comparison of the CRISPR/Cas system can help understand the environmental adaptability of each evolutionary lineage of clinically and environmentally relevant species, providing data support for bacterial typing, traceability, analysis, and exploration of unconventional CRISPR.},
}
@article {pmid37510232,
year = {2023},
author = {Kanai, M and Hikino, K and Mano, S},
title = {Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).},
journal = {Genes},
volume = {14},
number = {7},
pages = {},
pmid = {37510232},
issn = {2073-4425},
mesh = {*Ricinus/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Seeds/genetics ; *Ricin/genetics/metabolism ; Cloning, Molecular ; },
abstract = {Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.},
}
@article {pmid37508483,
year = {2023},
author = {Wang, JH and Wu, SJ and Li, Y and Zhao, Y and Liu, ZM and Deng, SL and Lian, ZX},
title = {Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase.},
journal = {Cells},
volume = {12},
number = {14},
pages = {},
pmid = {37508483},
issn = {2073-4409},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Butyrylcholinesterase/genetics ; Goats/genetics ; Transfection ; },
abstract = {The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.},
}
@article {pmid37506757,
year = {2023},
author = {Muzyukina, P and Soutourina, O},
title = {CRISPR genotyping methods: Tracing the evolution from spoligotyping to machine learning.},
journal = {Biochimie},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.biochi.2023.07.017},
pmid = {37506757},
issn = {1638-6183},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with adaptive immunity defenses against foreign genetic invaders. The identification of CRISPR-Cas function is among the most impactful discoveries of recent decades that have shaped the development of genome editing in various organisms paving the way for a plethora of promising applications in biotechnology and health. Even before the discovery of CRISPR-Cas biological role, the particular structure of CRISPR loci has been explored for epidemiological genotyping of bacterial pathogens. CRISPR-Cas loci are arranged in CRISPR arrays of mostly identical direct repeats intercalated with invader-derived spacers and an operon of cas genes encoding the Cas protein components. Each small CRISPR RNA (crRNA) encoded within the CRISPR array constitutes a key functional unit of this RNA-based CRISPR-Cas defense system guiding the Cas effector proteins toward the foreign nucleic acids for their destruction. The information acquired from prior invader encounters and stored within CRISPR arrays turns out to be extremely valuable in tracing the microevolution and epidemiology of major bacterial pathogens. We review here the history of CRISPR-based typing strategies highlighting the first PCR-based methods that have set the stage for recent developments of high-throughput sequencing and machine learning-based approaches. A great amount of whole genome sequencing and metagenomic data accumulated in recent years opens up new avenues for combining experimental and computational approaches of high-resolution CRISPR-based typing.},
}
@article {pmid37506507,
year = {2023},
author = {Tanny, T and Sallam, M and Soda, N and Nguyen, NT and Alam, M and Shiddiky, MJA},
title = {CRISPR/Cas-Based Diagnostics in Agricultural Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c00913},
pmid = {37506507},
issn = {1520-5118},
abstract = {Pests and disease-causing pathogens frequently impede agricultural production. An early and efficient diagnostic tool is crucial for effective disease management. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas) have recently been harnessed to develop diagnostic tools. The CRISPR/Cas system, composed of the Cas endonuclease and guide RNA, enables precise identification and cleavage of the target nucleic acids. The inherent sensitivity, high specificity, and rapid assay time of the CRISPR/Cas system make it an effective alternative for diagnosing plant pathogens and identifying genetically modified crops. Furthermore, its potential for multiplexing and suitability for point-of-care testing at the field level provide advantages over traditional diagnostic systems such as RT-PCR, LAMP, and NGS. In this review, we discuss the recent developments in CRISPR/Cas based diagnostics and their implications in various agricultural applications. We have also emphasized the major challenges with possible solutions and provided insights into future perspectives and potential applications of the CRISPR/Cas system in agriculture.},
}
@article {pmid37504099,
year = {2023},
author = {Ivanov, AV and Safenkova, IV and Zherdev, AV and Wan, Y and Dzantiev, BB},
title = {Comparison of Single-Stranded DNA Probes Conjugated with Magnetic Particles for Trans-Cleavage in Cas12a-Based Biosensors.},
journal = {Biosensors},
volume = {13},
number = {7},
pages = {},
pmid = {37504099},
issn = {2079-6374},
support = {075-15-2022-318//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*DNA, Single-Stranded ; CRISPR-Cas Systems ; DNA ; *Biosensing Techniques ; Magnetic Phenomena ; },
abstract = {Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5') and biotin (3'). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP-ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets' detection of D. solani and E. amylovora. The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes.},
}
@article {pmid37503583,
year = {2023},
author = {Wang, BZ and Zhang, C and Zhang, JL and Sun, J},
title = {Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA.},
journal = {Yi chuan = Hereditas},
volume = {45},
number = {7},
pages = {593-601},
doi = {10.16288/j.yczz.23-099},
pmid = {37503583},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR- associated protein 9) system, a highly efficient, simple, and easy genome editing technology, offers significant potential for genetic engineering and has been commonly applied in gene function studies in Drosophila melanogaster. However, when using CRISPR/Cas9 system to edit Drosophila melanogaster gene, Cas9 and sgRNA expression elements exist in different Drosophila melanogaster individuals, and Cas9 and sgRNA must be integrated into an individual through a complex genetic hybridization process, which has a long and complex operation cycle In this study, on the basis of the CRISPR/Cas9 system, we introduced the tRNA-sgRNA system and triplex elements, used triplex elements to link Cas9 and tRNA-sgRNA genes, stabilized the end of Cas9 mRNA after single transcript cutting, and made the expression of both Cas9 protein and sgRNA with a single transcript a reality. And as we obtained the corresponding phenotypic progeny in one hybridization, genetic manipulation was simplified. We found that conditional knockout of the white(w) gene in the Drosophila melanogaster eye and the broad(br) gene in the adult wing disc resulted in corresponding phenotypes that matched expectations using our new conditional gene editing system. So the significant advances in this new conditional gene editing system over the existing CRISPR/Cas9 system are that it is more efficient, extendable, and easy to use.},
}
@article {pmid37498808,
year = {2023},
author = {Hausjell, CS and Klausberger, M and Ernst, W and Grabherr, R},
title = {Evaluation of an inducible knockout system in insect cells based on co-infection and CRISPR/Cas9.},
journal = {PloS one},
volume = {18},
number = {7},
pages = {e0289178},
pmid = {37498808},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; *Coinfection/genetics ; RNA, Guide, CRISPR-Cas Systems ; Baculoviridae/genetics ; Insecta/genetics ; },
abstract = {Due to comparably high product titers and low production costs, the baculovirus/insect cell expression system is considered a versatile production platform in the biopharmaceutical industry. Its excellence in producing complex multimeric protein assemblies, including virus-like particles (VLPs), which are considered promising vaccine candidates to counter emerging viral threats, made the system even more attractive. However, the co-formation of budded baculovirus during VLP production poses a severe challenge to downstream processing. In order to reduce the amount of budded baculovirus in the expression supernatant we developed an inducible knockout system based on CRISPR/Cas9 and co-infection with two baculoviral vectors: one bringing along the Cas9 nuclease and the other one having incorporated the sequence for sgRNA expression. With our set-up high titer viruses can be generated separately, as only when both viruses infect cells simultaneously a knockout can occur. When budding essential genes gp64 and vp80 were targeted for knockout, we measured a reduction in baculovirus titer by over 90%. However, as a consequence, we also determined lower overall eYFP fluorescence intensity showing reduced recombinant protein production, indicating that further improvements in engineering as well as purification are required in order to ultimately minimize costs and timeframes for vaccine production utilizing the baculovirus/insect cell expression system.},
}
@article {pmid37458780,
year = {2023},
author = {Jayaprakash, P and Barroso, L and Vajente, M and Maestroni, L and Louis, EJ and Morrissey, JP and Branduardi, P},
title = {CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.},
journal = {FEMS yeast research},
volume = {23},
number = {},
pages = {},
pmid = {37458780},
issn = {1567-1364},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Chromosomes ; Loss of Heterozygosity ; },
abstract = {The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.},
}
@article {pmid37402133,
year = {2023},
author = {Yu, L and Peng, Y and Sheng, M and Wang, Q and Huang, J and Yang, X},
title = {Sensitive and Amplification-Free Electrochemiluminescence Biosensor for HPV-16 Detection Based on CRISPR/Cas12a and DNA Tetrahedron Nanostructures.},
journal = {ACS sensors},
volume = {8},
number = {7},
pages = {2852-2858},
doi = {10.1021/acssensors.3c00806},
pmid = {37402133},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems ; Human papillomavirus 16/genetics ; Gold/chemistry ; *Metal Nanoparticles/chemistry ; Luminescent Measurements/methods ; DNA/genetics/chemistry ; *Biosensing Techniques/methods ; *Nanostructures/chemistry ; },
abstract = {Rapid and accurate detection of biomarkers was very important for early screening and treatment of diseases. Herein, a sensitive and amplification-free electrochemiluminescence (ECL) biosensor based on CRISPR/Cas12a and DNA tetrahedron nanostructures (TDNs) was constructed. Briefly, 3D TDN was self-assembled on the Au nanoparticle-deposited glassy carbon electrode surface to construct the biosensing interface. The presence of the target would activate the trans-cleavage activity of Cas12a-crRNA duplex to cleave the single-stranded DNA signal probe on the vertex of TDN, causing the Ru(bpy)3[2+] to fall from the electrode surface and weakened the ECL signal. Thus, the CRISPR/Cas12a system transduced the change of target concentration into an ECL signal enabling the detection of HPV-16. The specific recognition of CRISPR/Cas12a to HPV-16 made the biosensor have good selectivity, while the TDN-modified sensing interface could reduce the cleaving steric resistance and improve the cleaving performance of CRISPR/Cas12a. In addition, the pretreated biosensor could complete sample detection within 100 min with a detection limit of 8.86 fM, indicating that the developed biosensor possesses the potential application prospect for fast and sensitive nucleic acid detection.},
}
@article {pmid37220911,
year = {2023},
author = {Dasgupta, S and LaDu, JK and Garcia, GR and Li, S and Tomono-Duval, K and Rericha, Y and Huang, L and Tanguay, RL},
title = {A CRISPR-Cas9 mutation in sox9b long intergenic noncoding RNA (slincR) affects zebrafish development, behavior, and regeneration.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {194},
number = {2},
pages = {153-166},
pmid = {37220911},
issn = {1096-0929},
support = {P30 ES030287/ES/NIEHS NIH HHS/United States ; R01 ES030017/ES/NIEHS NIH HHS/United States ; P42 ES016465/ES/NIEHS NIH HHS/United States ; R35 ES031709/ES/NIEHS NIH HHS/United States ; R01-ES030017/ES/NIEHS NIH HHS/United States ; /NH/NIH HHS/United States ; },
mesh = {Animals ; Zebrafish ; Zebrafish Proteins/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; CRISPR-Cas Systems ; Receptors, Aryl Hydrocarbon/metabolism ; *Polychlorinated Dibenzodioxins ; Mutation ; RNA, Messenger/metabolism ; Regeneration ; },
abstract = {The role of long noncoding RNAs (lncRNAs) regulators of toxicological responses to environmental chemicals is gaining prominence. Previously, our laboratory discovered an lncRNA, sox9b long intergenic noncoding RNA (slincR), that is activated by multiple ligands of aryl hydrocarbon receptor (AHR). Within this study, we designed a CRISPR-Cas9-mediated slincR zebrafish mutant line to better understand its biological function in presence or absence of a model AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slincRosu3 line contains an 18 bp insertion within the slincR sequence that changes its predicted mRNA secondary structure. Toxicological profiling showed that slincRosu3 is equally or more sensitive to TCDD for morphological and behavioral phenotypes. Embryonic mRNA-sequencing showed differential responses of 499 or 908 genes in slincRosu3 in absence or presence of TCDD Specifically, unexposed slincRosu3 embryos showed disruptions in metabolic pathways, suggesting an endogenous role for slincR. slincRosu3 embryos also had repressed mRNA levels of sox9b-a transcription factor that slincR is known to negatively regulate. Hence, we studied cartilage development and regenerative capacity-both processes partially regulated by sox9b. Cartilage development was disrupted in slincRosu3 embryos both in presence and absence of TCDD. slincRosu3 embryos also displayed a lack of regenerative capacity of amputated tail fins, accompanied by a lack of cell proliferation. In summary, using a novel slincR mutant line, we show that a mutation in slincR can have widespread impacts on gene expression and structural development endogenously and limited, but significant impacts in presence of AHR induction that further highlights its importance in the developmental process.},
}
@article {pmid36894722,
year = {2023},
author = {Kim, YH and Kim, N and Okafor, I and Choi, S and Min, S and Lee, J and Bae, SM and Choi, K and Choi, J and Harihar, V and Kim, Y and Kim, JS and Kleinstiver, BP and Lee, JK and Ha, T and Kim, HH},
title = {Sniper2L is a high-fidelity Cas9 variant with high activity.},
journal = {Nature chemical biology},
volume = {19},
number = {8},
pages = {972-980},
pmid = {36894722},
issn = {1552-4469},
support = {2022R1C1C2004229//National Research Foundation of Korea (NRF)/ ; 20012443//Ministry of Trade, Industry and Energy (Ministry of Trade, Industry and Energy, Korea)/ ; HI21C1314//Ministry of Health and Welfare (Ministry of Health, Welfare and Family Affairs)/ ; R35GM122569//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 2022R1A3B1078084//National Research Foundation of Korea (NRF)/ ; 2018R1A5A2025079//National Research Foundation of Korea (NRF)/ ; R35 GM122569/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; DNA/genetics ; },
abstract = {Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.},
}
@article {pmid36879061,
year = {2023},
author = {Wei, CT and Popp, NA and Peleg, O and Powell, RL and Borenstein, E and Maly, DJ and Fowler, DM},
title = {A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.},
journal = {Nature chemical biology},
volume = {19},
number = {8},
pages = {981-991},
pmid = {36879061},
issn = {1552-4469},
support = {F30 HL151075/HL/NHLBI NIH HHS/United States ; R01 GM145011/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Kinetics ; Nucleotides ; Adenine ; },
abstract = {CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.},
}
@article {pmid37505340,
year = {2023},
author = {Ma, J and Zhang, P and Zheng, M and Wang, B and Gao, P and Qu, L and Zheng, F},
title = {A strain of Vibrio alginolyticus isolated from Azumapecten farreri and its pathogenic mechanism using CRISPR-Cas9 technology.},
journal = {Biotechnology letters},
volume = {},
number = {},
pages = {},
pmid = {37505340},
issn = {1573-6776},
support = {2019GHY112003//the Key Research and Development Program of Shandong Province/ ; No. ZR2016DM18//the National Natural Science Foundation of Shandong Province/ ; No. 2017YFC1404504//the National Key R&amp;D Program of China/ ; },
abstract = {Scallops have become an important aquaculture species in China because they contain high-quality protein, and scallops are important health food that combines multiple effects and high economic benefits. However, scallop aquaculture is perennially threatened by various pathogenic Vibrio species, leading to great economic losses. We obtained a strain of pathogenic bacteria, identified as Vibrio alginolyticus, from the diseased Azumapecten farreri in the scallop farming area of Huangdao District in 2018, and V. alginolyticus is one of the major shellfish pathogens. We showed that V. alginolyticus was isolated and identified as a pathogen in A. farreri for the first time. In this study, we evaluated its morphology and performed a phylogenetic analysis based on 16S rRNA gene sequencing. In addition, we performed a preliminary analysis of its pathogenic mechanisms. The Hfq protein in V. alginolyticus is an important RNA-binding protein in the quorum-sensing system that not only affects the sensitivity of Vibrio to environmental stress but also regulates a variety of functions, such as cell membrane formation, motility, and virulence towards the host. However, its effect on the pathogenesis of V. alginolyticus to A. farreri is unclear. To further investigate the pathogenic mechanism of the Hfq protein in V. alginolyticus to A. farreri, we used the CRISPR-Cas9 system to target and deplete the hfq gene fragment in V. alginolyticus and obtained the mutant strain V. ΔHfq[-]. We found that the peripheral flagellum of the mutant strain was lost, which reduced the motility of V. alginolyticus. Therefore, the deletion of target genes by the CRISPR/Cas9 genome editing system confirmed that the Hfq protein played a key role in reducing the ability of V. alginolyticus to infect A. farreri. In conclusion, our current findings provided valuable insights into the healthy culture of scallops.},
}
@article {pmid37504649,
year = {2023},
author = {Yu, Y and Li, T and Guo, M and Xiong, R and Yan, D and Chen, P},
title = {Possible Regulation of Larval Juvenile Hormone Titers in Bombyx mori by BmFAMeT6.},
journal = {Insects},
volume = {14},
number = {7},
pages = {},
pmid = {37504649},
issn = {2075-4450},
abstract = {Juvenile hormone (JH) plays a vital role in the growth, development, and reproduction of insects and other arthropods. Previous experiments have suggested that BmFAMeT6 could affect the duration of the silk moth's larval stage. In this study, we established the BmFAMeT6 overexpression strain and BmFAMeT6 knockout strain using the GAL4/UAS binary hybrid system and CRISPR/Cas 9 system, respectively, and found that the larval stage of the overexpression strain was shorter, while the knockout strain was longer. Our results exhibited that both the JH titers and BmKr-h1 levels in the larvae of the third instar were reduced significantly by BmFAMeT6 overexpression, but were increased obviously by BmFAMeT6 knockout. In addition, injection of farnesoic acid induced changes in the JH I and JH II levels in the hemolymphs of larvae. This study is the first to directly reveal the role of BmFAMeT6 in the regulation of insect JH titers and the relationship between farnesoic acid and JH (JH I and JH II). This provides a new perspective on regulating the growth and development of insects such as Bombyx mori.},
}
@article {pmid37504509,
year = {2023},
author = {de Dieu Habimana, J and Mukama, O and Amissah, OB and Sun, Y and Karangwa, E and Liu, Y and Mugisha, S and Cheng, N and Wang, L and Chen, J and Deng, S and Huang, R and Li, Z},
title = {A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c01876},
pmid = {37504509},
issn = {1520-6882},
abstract = {The CRISPR/Cas systems offer a programmable platform for nucleic acid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) have demonstrated the ability to target nucleic acids with greater accuracy and flexibility. However, due to the configuration of the reporter and the underlying labeling mechanism, almost all reported CRISPR-Dx rely on a single-option readout, resulting in limitations in end-point result readouts. This is also associated with high reagent consumption and delays in diagnostic reports due to protocol differences. Herein, we report for the first time a rationally designed Cas12a-based multimodal universal reporter (CAMURE) with improved sensitivity that harnesses a dual-mode reporting system, facilitating options in end-point readouts. Through systematic configurations and optimizations, our novel universal reporter achieved a 10-fold sensitivity enhancement compared to the DETECTR reporter. Our unique and versatile reporter could be paired with various readouts, conveying the same diagnostic results. We applied our novel reporter for the detection of staphylococcal enterotoxin A due to its high implication in staphylococcal food poisoning. Integrated with loop-mediated isothermal amplification, our multimodal reporter achieved 10 CFU/mL sensitivity and excellent specificity using a real-time fluorimeter, in-tube fluorescence, and lateral flow strip readouts. We also propose, using artificially contaminated milk samples, a fast (2-5 min) Triton X-100 DNA extraction approach with a comparable yield to the commercial extraction kit. Our CAMURE could be leveraged to detect all gene-encoding SEs by simply reprogramming the guide RNA and could also be applied to the detection of other infections and disease biomarkers.},
}
@article {pmid37504071,
year = {2023},
author = {Liu, CC and Dai, Y},
title = {Application of CRISPR Cas Systems for Biosensing.},
journal = {Biosensors},
volume = {13},
number = {7},
pages = {},
pmid = {37504071},
issn = {2079-6374},
abstract = {The essential properties of a biosensor are its sensitivity and selectivity to detect, monitor and quantify the biomarker(s) for the interests of medicine [...].},
}
@article {pmid37503092,
year = {2023},
author = {George, JT and Acree, C and Park, JU and Kong, M and Wiegand, T and Pignot, YL and Kellogg, EH and Greene, EC and Sternberg, SH},
title = {Mechanism of target site selection by type V-K CRISPR-associated transposases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.07.14.548620},
pmid = {37503092},
abstract = {Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsB's preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.},
}
@article {pmid37502928,
year = {2023},
author = {Hu, K and Chou, CW and Wilke, CO and Finkelstein, IJ},
title = {Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37502928},
abstract = {CRISPR-associated transposons (CASTs) co-opt CRISPR-Cas proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. Exploiting new spacers in defense-associated CRISPR arrays explains how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.},
}
@article {pmid37502876,
year = {2023},
author = {Ahmed, NM and Joglekar, P and Deming, C and , and Lemon, KP and Kong, HH and Segre, JA and Conlan, S},
title = {Genomic characterization of the C. tuberculostearicum species complex, a ubiquitous member of the human skin microbiome.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37502876},
abstract = {UNLABELLED: Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A &amp; B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates are largely syntenic and carry a diversity of methylation patterns, plasmids and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species.<br><br>IMPORTANCE: Amplicon sequencing data combined with isolate whole genome sequencing has expanded our understanding of Corynebacterium on the skin. Human skin is characterized by a diverse collection of Corynebacterium species but C. tuberculostearicum predominates many sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex including a potentially new species which we are calling C. hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomics and growth in skin-like media. This isolate collection and high-quality genome resource sets the stage for developing engineered strains for both basic and translational clinical studies. Microbiomes are shaped by taxa that are both characteristic to those sites and functionally important to that community. The genus Corynebacterium is one such taxa for the human skin and nares. Foundational studies using 16S rRNA gene sequencing and shotgun metagenomics by our lab (1, 2) and others (3) have established Corynebacterium as common members of the skin microbiome. While Corynebacterium have been positively correlated with the resolution of dysbiosis associated with eczema flares (4), the importance of the Corynebacterium spp. is less defined for skin disease severity in primary immune deficient patients (5, 6). Corynebacterium spp. are predominant members of the human aerodigestive tract microbiome (nares, oral cavity and respiratory tract) (3) and participate in microbe-microbe interactions with members of nasal microbiome (7, 8). Corynebacterium have been shown to engage with the host immune system, specifically C. accolens -promoted IL23-dependent inflammation in mice on a high-fat diet (9). C. bovis and C. mastiditis have been shown to predominate the microbiome of a ADAM10-deficient mouse model (10) as well as an ADAM17-deficient mouse model of eczema (11). Finally, C. tuberculostearicum has been shown to induce inflammation in human epidermal keratinocyte cell cultures (12). These studies establish Corynebacterium spp. as key members of the skin microbiome capable of both microbe-microbe and microbe-host interactions. A critical resource for understanding the biology of Corynebacterium on the skin is a robust collection of complete reference genomes, including isolates collected from a variety of individuals and body sites. Previously published genome collections from skin- or nares-resident species include Staphylococcus epidermidis (13), Cutibacterium acnes (14) and the recent comparative analysis of Dolosigranulum pigrum (15). Of note, while emerging bioinformatic methods and pipelines are now being employed to extract nearly-complete genomes (MAGs) from metagenomic assemblies of skin samples (16), MAGs are not yet a substitute for genomes from cultured isolates to understand strain level or pangenomic diversity. In addition to functional prediction, comparative genomics is increasingly being used to augment conventional microbiological methods to define or redefine taxonomic boundaries (17, 18), as well as describe the full extent of diversity within these boundaries (19). A pangenome, which encompasses the complete set of genes present within a set of genome sequences, enables the characterization of gene-level heterogeneity within a taxonomic group. The pangenome is commonly subdivided into the 'core' genome, referring to genes present in all strains, and the 'accessory' or 'dispensable' genome, referring to those present in only one or some isolates. (The accessory pangenome can be further subdivided to reflect a wider range of gene uniqueness, e.g. singletons.) Thorough characterization of taxa is limited by the availability of representative and high-quality genome assemblies. Unfortunately, with the exceptions of clinically relevant Corynebacterium spp. (e.g. , C. diphtheriae , C. striatum and C. pseudotuberculosis), the genus is inadequately sequenced, with 75% of species having fewer than six genomes. This includes common skin-associated species like C. tuberculostearicum with just five unique isolate genomes, only two of which are from skin. This work seeks first to characterize the distribution of Corynebacterium across 14 skin sites from 23 healthy volunteers. The second goal of this work focuses on what we identify as the predominant skin Corynebacterium species, C. tuberculostearicum . We have sequenced 23 distinct C. tuberculostearicum strains (n=40 genomes before dereplication), a five-fold increase in the number of publicly available, unique genomes (n=5). In addition to short-read assemblies, we generated five complete genomes which, along with the type strain (DSM44922), demonstrate that C. tuberculostearicum genomes are largely syntenic and carry a number of methylation systems as well as a CRISPR/Cas system. Genes from the C. tuberculostearicum genomes in our collection fall into 5451 gene clusters comprising the species pangenome. This expanded pangenome, as compared to existing public references, improved the mapping of C. tuberculostearicum metagenomic reads from unrelated healthy volunteers. In addition, we have identified a distinct C. tuberculostearicum clade that is highly enriched on the feet that may represent a new species, tentatively designated Corynebacterium hallux .},
}
@article {pmid37501535,
year = {2023},
author = {Pavia, MJ and Finn, D and Macedo-Tafur, F and Tello-Espinoza, R and Penaccio, C and Bouskill, N and Cadillo-Quiroz, H},
title = {Genes and genome-resolved metagenomics reveal the microbial functional make up of Amazon peatlands under geochemical gradients.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.16469},
pmid = {37501535},
issn = {1462-2920},
support = {CAREER-1749252//National Science Foundation/ ; DE-AC02-05CH11231//the US Department of Energy/ ; },
abstract = {The Pastaza-Marañón Foreland Basin (PMFB) holds the most extensive tropical peatland area in South America. PMFB peatlands store ~7.07 Gt of organic carbon interacting with multiple microbial heterotrophic, methanogenic, and other aerobic/anaerobic respirations. Little is understood about the contribution of distinct microbial community members inhabiting tropical peatlands. Here, we studied the metagenomes of three geochemically distinct peatlands spanning minerotrophic, mixed, and ombrotrophic conditions. Using gene- and genome-centric approaches, we evaluate the functional potential of the underlying microbial communities. Abundance analyses show significant differences in C, N, P, and S acquisition genes. Furthermore, community interactions mediated by toxin-antitoxin and CRISPR-Cas systems were enriched in oligotrophic soils, suggesting that non-metabolic interactions may exert additional controls in low-nutrient environments. Additionally, we reconstructed 519 metagenome-assembled genomes spanning 28 phyla. Our analyses detail key differences across the geochemical gradient in the predicted microbial populations involved in degradation of organic matter, and the cycling of N and S. Notably, we observed differences in the nitric oxide (NO) reduction strategies between sites with high and low N2 O fluxes and found phyla putatively capable of both NO and sulfate reduction. Our findings detail how gene abundances and microbial populations are influenced by geochemical differences in tropical peatlands.},
}
@article {pmid37500801,
year = {2023},
author = {Esser, SP and Rahlff, J and Zhao, W and Predl, M and Plewka, J and Sures, K and Wimmer, F and Lee, J and Adam, PS and McGonigle, J and Turzynski, V and Banas, I and Schwank, K and Krupovic, M and Bornemann, TLV and Figueroa-Gonzalez, PA and Jarett, J and Rattei, T and Amano, Y and Blaby, IK and Cheng, JF and Brazelton, WJ and Beisel, CL and Woyke, T and Zhang, Y and Probst, AJ},
title = {A predicted CRISPR-mediated symbiosis between uncultivated archaea.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {37500801},
issn = {2058-5276},
support = {PR1603/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; RA3432/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BE6703/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 1553211//National Science Foundation (NSF)/ ; },
abstract = {CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.},
}
@article {pmid37498289,
year = {2023},
author = {Sánchez-Costa, M and Gola, S and Rodríguez-Sáiz, M and Barredo, JL and Hidalgo, A and Berenguer, J},
title = {From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.},
journal = {Microbial biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1751-7915.14290},
pmid = {37498289},
issn = {1751-7915},
support = {GA 685474-2//H2020 European Research Council/ ; PID2019-109073RB-100//Ministerio de Ciencia e Innovaci&#xf3;n/ ; },
abstract = {Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc[2] 155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long- and short-massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long-reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR-Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high-quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.},
}
@article {pmid37496635,
year = {2023},
author = {Jiang, C and She, Q and Wang, H},
title = {Editorial: Insights in genome editing tools and mechanisms: 2022.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1240576},
pmid = {37496635},
issn = {2673-3439},
}
@article {pmid37495710,
year = {2023},
author = {Kim, C and Cnaani, A and Kültz, D},
title = {Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {12086},
pmid = {37495710},
issn = {2045-2322},
mesh = {Animals ; *Tilapia/metabolism ; CRISPR-Cas Systems/genetics ; Osmoregulation ; Gene Expression Regulation ; Cell Line ; },
abstract = {MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.},
}
@article {pmid37494395,
year = {2023},
author = {Sun, W and Cheng, Z and Wang, J and Yang, J and Li, X and Wang, J and Chen, M and Yang, X and Sheng, G and Lou, J and Wang, Y},
title = {AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {31},
pages = {e2303675120},
doi = {10.1073/pnas.2303675120},
pmid = {37494395},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; R-Loop Structures ; RNA, Guide, CRISPR-Cas Systems ; DNA/metabolism ; *Bacteriophages/genetics ; Gene Editing ; },
abstract = {Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Haemophilus parainfluenzae Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.},
}
@article {pmid37493508,
year = {2023},
author = {Ricci, L and Selma-Royo, M and Golzato, D and Nabinejad, A and Servais, C and Armanini, F and Asnicar, F and Pinto, F and Tamburini, S and Segata, N},
title = {Draft genome sequence of a representative strain of the Catenibacterium genus isolated from human feces.},
journal = {Microbiology resource announcements},
volume = {},
number = {},
pages = {e0032923},
doi = {10.1128/MRA.00329-23},
pmid = {37493508},
issn = {2576-098X},
abstract = {A strain from a previously undescribed species belonging to the Catenibacterium genus was isolated from the stool of a healthy volunteer. The strain is strictly anaerobic, and the genome encodes a CRISPR-Cas system and genes related to trimethylamine production.},
}
@article {pmid37491415,
year = {2023},
author = {Matrishin, CB and Haase, EM and Dewhirst, FE and Mark Welch, JL and Miranda-Sanchez, F and Chen, T and MacFarland, DC and Kauffman, KM},
title = {Phages are unrecognized players in the ecology of the oral pathogen Porphyromonas gingivalis.},
journal = {Microbiome},
volume = {11},
number = {1},
pages = {161},
pmid = {37491415},
issn = {2049-2618},
support = {T32DE023526/DE/NIDCR NIH HHS/United States ; R01DE016937/DE/NIDCR NIH HHS/United States ; R01DE016937/DE/NIDCR NIH HHS/United States ; R01DE016937/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Bacteriophages/genetics ; Porphyromonas gingivalis/genetics ; Prophages/genetics ; *Periodontal Diseases ; Base Sequence ; },
abstract = {BACKGROUND: Porphyromonas gingivalis (hereafter "Pg") is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg.<br><br>RESULTS: Here we systematically addressed the question of whether Pg are infected by phages-and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures.<br><br>CONCLUSION: This work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly. Video Abstract.},
}
@article {pmid37490214,
year = {2023},
author = {Chen, T and Chen, Z and Zhang, H and Li, Y and Yao, L and Zeng, B and Zhang, Z},
title = {Development of a CRISPR/Cpf1 system for multiplex gene editing in Aspergillus oryzae.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {37490214},
issn = {1874-9356},
abstract = {CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.},
}
@article {pmid37489761,
year = {2023},
author = {Li, H and Song, W and Li, H and Cui, J and Xie, Y and Wu, B and Chen, R},
title = {Advances in isothermal nucleic acid amplification methods for hepatitis B virus detection.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3an00700f},
pmid = {37489761},
issn = {1364-5528},
abstract = {Hepatitis B virus (HBV) infection is a major global health problem of widespread concern. Clinically, serological assays are the most widely used diagnostic tests for HBV infection, with the presence of HBsAg in the serum being indicative of acute and chronic hepatitis B infection. However, increased identification of HBV DNA positive but HBsAg negative cases has greatly promoted the use of molecular assays for more accurate HBV diagnosis. Over the past few decades, especially since the outbreak of COVID-19, significant advancements have been made in the techniques and devices for nucleic acid testing (NAT). Nowadays, the mainstream NAT techniques can broadly be split into two categories: PCR-based methods and non-PCR-based isothermal amplification methods. As achieving point-of-care testing (POCT) or on-site testing is an important development tendency for the next-generation NAT, non-PCR-based isothermal amplification methods like nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) have garnered significant attention in recent years. In this review, we provide a comprehensive overview of the nucleic acid isothermal amplification technologies currently used for HBV detection. The analytical performances of different methods are compared and their integration with microfluidics, lateral flow assays, and CRISPR/Cas systems is also discussed.},
}
@article {pmid37489056,
year = {2023},
author = {Ma, J and Li, X and Lou, C and Lin, X and Zhang, Z and Chen, D and Yang, S},
title = {Utility of CRISPR/Cas mediated electrochemical biosensors.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3ay00903c},
pmid = {37489056},
issn = {1759-9679},
abstract = {Electrochemical biosensors represent a class of sensors that employ biological materials as sensitive elements, electrodes as conversion elements, and potential or current as detection signals. The integration of CRISPR/Cas systems into electrochemical biosensors holds immense potential, offering enhanced versatility, heightened sensitivity and specificity, reduced recovery time, and the ability to capture and identify analytes at low concentrations. In this review, we provided a succinct summary of the fundamental principles underlying electrochemical biosensors and CRISPR/Cas systems, and new progress of electrochemical biosensors based on CRISPR/Cas systems in virus, bacteria, and cancer detections. Besides, we discussed its pros and cons, present gaps, potential problem-solvers, and future prospects. To sum up, CRISPR/Cas mediated electrochemical biosensors will surely benefit us a lot in the detection of cells and microorganisms, and of course in other promising fields.},
}
@article {pmid37487103,
year = {2023},
author = {Choi, W and Wu, H and Yserentant, K and Huang, B and Cheng, Y},
title = {Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {31},
pages = {e2302471120},
doi = {10.1073/pnas.2302471120},
pmid = {37487103},
issn = {1091-6490},
support = {S10 OD021741/OD/NIH HHS/United States ; R35 GM140847/GM/NIGMS NIH HHS/United States ; S10 OD020054/OD/NIH HHS/United States ; R01 GM131641/GM/NIGMS NIH HHS/United States ; R01 GM124334/GM/NIGMS NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; S10 OD026881/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; HEK293 Cells ; Transfection ; Green Fluorescent Proteins/metabolism ; *Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/metabolism ; Gene Editing ; },
abstract = {CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.},
}
@article {pmid37486522,
year = {2023},
author = {Oliveira, VC and Roballo, KCS and Mariano Junior, CG and Ambrósio, CE},
title = {Gene Editing Technologies Targeting TFAM and Its Relation to Mitochondrial Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {173-189},
pmid = {37486522},
issn = {0065-2598},
abstract = {Mitochondria are organelles present in the cytoplasm of eukaryotic cells; they play a key role in adenosine triphosphate (ATP) synthesis and oxidative phosphorylation. Mitochondria have their own DNA, mitochondrial DNA (mtDNA), keeping the function of the mitochondria. Mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters is and considered essential in mtDNA replication and transcription. More recently, TFAM has been shown to play a central role in the maintenance and regulation of mitochondrial copy number, inflammatory response, expression regulation, and mitochondrial genome activity. Gene editing tools such as the CRISPR-Cas 9 technique, TALENs, and other gene editing tools have been used to investigate the role of TFAM in mitochondrial mechanics and biogenesis as well as its correlation to mitochondrial disorders. Thus this chapter brings a summary of mitochondria function, dysfunction, the importance of TFAM in the maintenance of mitochondria, and state of the art of gene editing tools involving TFAM and mtDNA.},
}
@article {pmid37310200,
year = {2023},
author = {Chen, H and Yang, QL and Xu, JX and Deng, X and Zhang, YJ and Liu, T and Rots, MG and Xu, GL and Huang, KY},
title = {Efficient methods for multiple types of precise gene-editing in Chlamydomonas.},
journal = {The Plant journal : for cell and molecular biology},
volume = {115},
number = {3},
pages = {846-865},
doi = {10.1111/tpj.16265},
pmid = {37310200},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; *Chlamydomonas/genetics ; Gene Editing/methods ; *Chlamydomonas reinhardtii/genetics ; },
abstract = {Precise gene-editing using CRISPR/Cas9 technology remains a long-standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi-type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low-expression genes (CrTET1 and CrKU80), the introduction of a FLAG-HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live-cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3'-UTR of MAA7 and VIPP1 resulted in a stable knock-down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.},
}
@article {pmid37486521,
year = {2023},
author = {Paiva, IM and Damasceno, S and Cunha, TM},
title = {CRISPR Libraries and Whole-Genome Screening to Identify Essential Factors for Viral Infections.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {157-172},
pmid = {37486521},
issn = {0065-2598},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Pandemics ; *COVID-19/genetics ; *Virus Diseases/diagnosis/genetics ; Gene Editing ; },
abstract = {The CRISPR-Cas9 system has revolutionized genetics and offers a simple and inexpensive way of generating perturbation that results in gene repression, activation, or editing. The advances in this technique make possible the development of CRISPR libraries which consist of a set of sgRNAs to cause perturbations in several genes in the same cell population. The use of libraries raised the CRISPR-Cas9 technique to a genomic scale and provides a powerful approach for identifying previously unknown molecular mechanisms and pathways involved in a specific phenotype or biological process. More specifically, the CRISPRko libraries (set of sgRNAs for gene knockout) and their high-throughput screenings are widely used in research with viral agents, and it was enlarged even more with the COVID-19 pandemic. With this chapter, we aim to point out how this tool helps in understanding virus-host relationships, such as the mechanisms of virus entry into the cell, the essential factors for its replication, and the cellular pathways involved in the response against the pathogen. The chapter also provided some practical considerations for each step of an experimentation using these tools that include choosing the library and screening type, the target cell, the viral strain, the library amplification and guaranteeing its coverage, the strategies for the gene screening pipeline by bioinformatics, and finally, target validation. To conclude, it was presented a table reviewing the last updates in the research for antiviral therapies using CRISPR libraries.},
}
@article {pmid37486517,
year = {2023},
author = {Fuziwara, CS and Kimura, ET},
title = {Using CRISPR/Cas9 to Edit a Thyroid Cancer Cell Line.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {73-84},
pmid = {37486517},
issn = {0065-2598},
mesh = {Humans ; CRISPR-Cas Systems ; *Thyroid Neoplasms/genetics ; *MicroRNAs/genetics ; Cell Line, Tumor ; *RNA, Long Noncoding/genetics ; Gene Editing ; },
abstract = {Thyroid cancer is the most prevalent endocrine malignancy, comprising multiple types of cancer, with distinct clinical-pathological characteristics. The oncogenesis of thyroid cancer is related to genetic alterations in MAPK signaling that induce proliferation and modulate noncoding genes, such as microRNAs and long noncoding RNAs. In this context, CRISPR/Cas9 emerges as a potential tool to modify gene sequence and modulate gene expression in thyroid cancer cell lines. In this chapter, we explore some of the current studies in which researchers have applied CRISPR/Cas9 in vitro to investigate thyroid cancer biology (Fig. 5.1).},
}
@article {pmid37486516,
year = {2023},
author = {Bonamino, MH and Correia, EM},
title = {The CRISPR/Cas System in Human Cancer.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {59-71},
pmid = {37486516},
issn = {0065-2598},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; Models, Animal ; *Neoplasms/genetics/therapy ; },
abstract = {The use of CRISPR as a genetic editing tool modified the oncology field from its basic to applied research for opening a simple, fast, and cheaper way to manipulate the genome. This chapter reviews some of the major uses of this technique for in vitro- and in vivo-based biological screenings, for cellular and animal model generation, and new derivative tools applied to cancer research. CRISPR has opened new frontiers increasing the knowledge about cancer, pointing to new solutions to overcome several challenges to better understand the disease and design better treatments.},
}
@article {pmid37486515,
year = {2023},
author = {Furtado, CLM and da Silva Santos, R and Sales, SLA and Teixeira, LPR and Pessoa, CD&#xd3;},
title = {Long Non-coding RNAs and CRISPR-Cas Edition in Tumorigenesis.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {41-58},
pmid = {37486515},
issn = {0065-2598},
abstract = {Long non-coding RNAs (lncRNAs) are one of the most abundant and heterogeneous transcripts with key roles in chromatin remodeling and gene regulation at the transcriptional and post-transcriptional levels. Due to their role in cell growth and differentiation, lncRNAs have emerged as an important biomarker in cancer diagnosis, prognosis, and targeted treatment. Recent studies have focused on elucidating lncRNA function during malignant transformation, tumor progression and drug resistance. The advent of the CRISPR system has made it possible to precisely edit complex genomic loci such as lncRNAs. Thus, we summarized the advances in CRISPR-Cas approaches for functional studies of lncRNAs including gene knockout, knockdown, overexpression and RNA targeting in tumorigenesis and drug resistance. Additionally, we highlighted the perspectives and potential applications of CRISPR approaches to treat cancer, as an emerging and promising target therapy.},
}
@article {pmid37409577,
year = {2023},
author = {Wang, Z and Xu, C and Yu, C and Si, Z and Huang, D and Shen, P and Fang, M and Xu, Z},
title = {Integration of a CRISPR Cas12a-assisted multicolor biosensor and a micropipette tip enables visible point-of-care testing of foodborne Vibrio vulnificus.},
journal = {The Analyst},
volume = {148},
number = {15},
pages = {3509-3517},
doi = {10.1039/d3an00714f},
pmid = {37409577},
issn = {1364-5528},
mesh = {*Vibrio vulnificus/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Foodborne pathogens cause numerous food safety problems, and as a virulent bacterium falling under this category, Vibrio vulnificus (V. vulnificus) poses a huge threat to public health. The conventional methods used for the detection of V. vulnificus, including culture-based and molecular detection methods, have a variety of drawbacks, including being time-consuming and labor-intensive, the requirement of large-scale equipment, and the lack of professional operators. This paper establishes a visible detection platform for V. vulnificus based on CRISPR/Cas12a, which is integrated with nucleic acid isothermal amplification and β-galactosidase-catalyzed visible color reaction. The specific vvhA gene and a conservative segment in the 16S rDNA gene of the Vibrio genus were selected as the detection targets. By using spectrum analysis, this CRISPR detection platform achieved sensitive detection of V. vulnificus (1 CFU per reaction) with high specificity. Through the color transformation system, as low as 1 CFU per reaction of V. vulnificus in both bacterial solution and artificially contaminated seafood could be visibly observed with the naked eye. Furthermore, the consistency between our assay and the qPCR assay in the detection of V. vulnificus spiked seafood was confirmed. In general, this visible detection platform is user-friendly, accurate, portable, and equipment-free, and is expected to provide a powerful supplement in point-of-care testing of V. vulnificus and also holds good promise for future application in foodborne pathogen detection.},
}
@article {pmid37368499,
year = {2023},
author = {Trujillo Rodríguez, L and Ellington, AJ and Reisch, CR and Chevrette, MG},
title = {CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {1989-2003},
pmid = {37368499},
issn = {2161-5063},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Transposases/genetics ; DNA Transposable Elements/genetics ; Proteobacteria/genetics ; Mutagenesis/genetics ; Gene Editing ; Bacteria/genetics ; RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.},
}
@article {pmid37344596,
year = {2023},
author = {Abdel-Hafiz, HA and Schafer, JM and Chen, X and Xiao, T and Gauntner, TD and Li, Z and Theodorescu, D},
title = {Y chromosome loss in cancer drives growth by evasion of adaptive immunity.},
journal = {Nature},
volume = {619},
number = {7970},
pages = {624-631},
pmid = {37344596},
issn = {1476-4687},
mesh = {Animals ; Humans ; Mice ; *CD8-Positive T-Lymphocytes/immunology/pathology ; *Chromosome Deletion ; *Chromosomes, Human, Y/genetics ; Proteomics ; Tumor Microenvironment/immunology ; *Urinary Bladder Neoplasms/genetics/immunology/pathology/therapy ; *Tumor Escape/genetics/immunology ; Gene Expression Profiling ; Genomics ; Prognosis ; CRISPR-Cas Systems ; Gene Editing ; In Vitro Techniques ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Flow Cytometry ; Immunotherapy ; },
abstract = {Loss of the Y chromosome (LOY) is observed in multiple cancer types, including 10-40% of bladder cancers[1-6], but its clinical and biological significance is unknown. Here, using genomic and transcriptomic studies, we report that LOY correlates with poor prognoses in patients with bladder cancer. We performed in-depth studies of naturally occurring LOY mutant bladder cancer cells as well as those with targeted deletion of Y chromosome by CRISPR-Cas9. Y-positive (Y[+]) and Y-negative (Y[-]) tumours grew similarly in vitro, whereas Y[-] tumours were more aggressive than Y[+] tumours in immune-competent hosts in a T cell-dependent manner. High-dimensional flow cytometric analyses demonstrated that Y[-] tumours promote striking dysfunction or exhaustion of CD8[+] T cells in the tumour microenvironment. These findings were validated using single-nuclei RNA sequencing and spatial proteomic evaluation of human bladder cancers. Of note, compared with Y[+] tumours, Y[-] tumours exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Together, these results demonstrate that cancer cells with LOY mutations alter T cell function, promoting T cell exhaustion and sensitizing them to PD-1-targeted immunotherapy. This work provides insights into the basic biology of LOY mutation and potential biomarkers for improving cancer immunotherapy.},
}
@article {pmid37486514,
year = {2023},
author = {Monteiro, CJ and Heery, DM and Whitchurch, JB},
title = {Modern Approaches to Mouse Genome Editing Using the CRISPR-Cas Toolbox and Their Applications in Functional Genomics and Translational Research.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {13-40},
pmid = {37486514},
issn = {0065-2598},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Genomics ; Genome/genetics ; },
abstract = {Mice have been used in biological research for over a century, and their immense contribution to scientific breakthroughs can be seen across all research disciplines, with some of the main beneficiaries being the fields of medicine and life sciences. Genetically engineered mouse models (GEMMs), along with other model organisms, are fundamentally important research tools frequently utilised to enhance our understanding of pathophysiology and biological mechanisms behind disease. In the 1980s, it became possible to precisely edit the mouse genome to create gene knockout and knock-in mice, although with low efficacy. Recent advances utilising CRISPR-Cas technologies have considerably improved our ability to do this with ease and precision, while also allowing the generation of desired genetic variants from single nucleotide substitutions to large insertions/deletions. It is now quick and relatively easy to genetically edit somatic cells which were previously more recalcitrant to traditional approaches. Further refinements have created a 'CRISPR toolkit' that has expanded the use of CRISPR-Cas beyond gene knock-ins and knockouts. In this chapter, we review some of the latest applications of CRISPR-Cas technologies in GEMMs, including nuclease-dead Cas9 systems for activation or repression of gene expression, base editing and prime editing. We also discuss improvements in Cas9 specificity, targeting efficacy and delivery methods in mice. Throughout, we provide examples wherein CRISPR-Cas technologies have been applied to target clinically relevant genes in preclinical GEMMs, both to generate humanised models and for experimental gene therapy research.},
}
@article {pmid37486077,
year = {2023},
author = {Kotapati, KV and Hamid, A and Ateka, EM and Pappu, H},
title = {CRISPR/Cas, transcriptomics, and RNA interference in virus disease management.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-01-23-0002-V},
pmid = {37486077},
issn = {0031-949X},
abstract = {Plant viruses infect a wide range of commercially important crop plants and cause significant crop production losses worldwide. Numerous alterations in plant physiology related to the reprogramming of gene expression may result from viral infections. While conventional IPM-based strategies have been effective in reducing the impact of several viral diseases, continued emergence of new viruses and strains, expanding host ranges, and emergence of resistance breaking strains necessitate a sustained effort toward the development and application of new approaches for virus management that would complement existing tactics. RNA interference-based techniques, and more recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies have paved the way for precise targeting of viral transcripts and manipulation of viral genomes and host factors. In-depth knowledge of the molecular mechanisms underlying the development of disease would further expand the applicability of these recent methods. Advances in next generation/high throughput sequencing have made possible more intensive studies into host-virus interactions. Utilizing the omics data and its application has the potential to expedite in fast tracking traditional plant breeding methods, as well as applying modern molecular tools for trait enhancement, including virus resistance. Here, we summarize the recent developments in CRISPR/Cas system, transcriptomics, endogenous RNA interference and exogenous application of dsRNA in virus disease management.},
}
@article {pmid37427455,
year = {2023},
author = {Jiang, L and Zhou, B and Qian, H and Wang, H and Wang, Y and Fan, W and Zheng, G and Ge, J},
title = {Cell-type-specific CRISPRization of mitochondrial DNA using bifunctional biodegradable silica nanoparticles.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {60},
pages = {9251-9254},
doi = {10.1039/d3cc01778h},
pmid = {37427455},
issn = {1364-548X},
mesh = {*DNA, Mitochondrial/genetics ; CRISPR-Cas Systems ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Nanoparticles ; },
abstract = {We report cell-type-specific and CRISPR/Cas9-mediated mtDNA editing platform by using bifunctional biodegradable silica nanoparticles, which were capable of selective intracellular delivery to CD44-overexpressed cells and subsequent mitochondrial localization, followed by glutathione-responsive biodegradation and release of Cas9/sgRNA to realize precise mtDNA editing.},
}
@article {pmid37380765,
year = {2023},
author = {Soden, ME and Yee, JX and Zweifel, LS},
title = {Circuit coordination of opposing neuropeptide and neurotransmitter signals.},
journal = {Nature},
volume = {619},
number = {7969},
pages = {332-337},
pmid = {37380765},
issn = {1476-4687},
mesh = {*Brain/cytology/metabolism ; Calcium/metabolism ; CRISPR-Cas Systems ; Dopamine/metabolism ; Dopaminergic Neurons/metabolism ; GABAergic Neurons ; gamma-Aminobutyric Acid/metabolism ; Gene Editing ; Hypothalamic Area, Lateral/cytology/metabolism ; *Neural Pathways ; *Neurotensin/metabolism ; *Neurotransmitter Agents/metabolism ; Receptors, Neurotensin/metabolism ; *Signal Transduction ; Ventral Tegmental Area/cytology/metabolism ; },
abstract = {Fast-acting neurotransmitters and slow, modulatory neuropeptides are co-released from neurons in the central nervous system, albeit from distinct synaptic vesicles[1]. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions-for example, stimulatory versus inhibitory-work together to exert control of neural circuit output remain unclear. This has been difficult to resolve owing to the inability to selectively isolate these signalling pathways in a cell- and circuit-specific manner. Here we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate CRISPR-Cas9 mutagenesis[2] of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. We demonstrate that neurons within the lateral hypothalamus that produce the stimulatory neuropeptide neurotensin and the inhibitory neurotransmitter GABA (γ-aminobutyric acid) utilize these signals to coordinately activate dopamine-producing neurons of the ventral tegmental area. We show that GABA release from lateral hypothalamus neurotensin neurons inhibits GABA neurons within the ventral tegmental area, disinhibiting dopamine neurons and causing a rapid rise in calcium, whereas neurotensin directly generates a slow inactivating calcium signal in dopamine neurons that is dependent on the expression of neurotensin receptor 1 (Ntsr1). We further show that these two signals work together to regulate dopamine neuron responses to maximize behavioural responding. Thus, a neurotransmitter and a neuropeptide with opposing signals can act on distinct timescales through different cell types to enhance circuit output and optimize behaviour.},
}
@article {pmid37282836,
year = {2023},
author = {Ma, P and Wang, Q and Luo, X and Mao, L and Wang, Z and Ye, E and Loh, XJ and Li, Z and Wu, YL},
title = {Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery.},
journal = {Biomaterials science},
volume = {11},
number = {15},
pages = {5078-5094},
doi = {10.1039/d3bm00529a},
pmid = {37282836},
issn = {2047-4849},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Therapy ; Gene Transfer Techniques ; Endonucleases/genetics/metabolism ; Polymers ; },
abstract = {Non-viral polymeric vectors with good biocompatibility have been recently explored as delivery systems for clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases. In this review, based on current limitations and critical barriers, we summarize the advantages of stimulus-responsive polymeric delivery vectors (i.e., pH, redox, or enzymes) towards controllable CRISPR/Cas9 genome editing system delivery as well as the advances in using stimulus-responsive CRISPR/Cas9 polymeric carriers towards cancer treatment. Last but not least, the key challenges and promising development strategies of stimulus-responsive polymeric vector designs for CRISPR/Cas9 systems will also be discussed.},
}
@article {pmid37128708,
year = {2023},
author = {Liu, L and Xue, Y and Luo, J and Han, M and Liu, X and Jiang, T and Zhao, Y and Xu, Y and Ma, C},
title = {Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.},
journal = {Plant biotechnology journal},
volume = {21},
number = {8},
pages = {1519-1521},
pmid = {37128708},
issn = {1467-7652},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Chrysanthemum/genetics ; Genes, Reporter ; Flowers/genetics ; },
}
@article {pmid37485554,
year = {2023},
author = {Yang, J and Yang, K and Du, S and Luo, W and Wang, C and Liu, H and Liu, K and Zhang, Z and Gao, Y and Han, X and Song, Y},
title = {Bioorthogonal Reaction-Mediated Tumor-Selective Delivery of CRISPR/Cas9 System for Dual-Targeted Cancer Immunotherapy.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202306863},
doi = {10.1002/anie.202306863},
pmid = {37485554},
issn = {1521-3773},
abstract = {CRISPR system-assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor-targeting delivery strategy based on bioorthogonal reactions for dual-targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS-STING pathway was achieved simultaneously through tumor microenvironment (TME)-biodegradable hollow manganese dioxide (H-MnO2) nano-platform. Subsequently, CRISPR/Cas9 system-loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.},
}
@article {pmid37483607,
year = {2023},
author = {Chang, JC and Wang, CY and Lin, S},
title = {Interrogation of human microglial phagocytosis by CRISPR genome editing.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1169725},
pmid = {37483607},
issn = {1664-3224},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Microglia ; *Glioblastoma/genetics ; Amyloid beta-Peptides ; Phagocytosis/genetics ; },
abstract = {BACKGROUND: Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3.<br><br>METHODS: We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent A&#x3b2; and glioblastoma phagocytosis assays.<br><br>RESULTS: Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled A&#x3b2;1-42 peptides. Confocal microscopy further confirmed the localization of A&#x3b2;1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells.<br><br>CONCLUSION: CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of A&#x3b2; and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.},
}
@article {pmid37483272,
year = {2023},
author = {Xiao, Y and Zhang, Z and Yin, S and Ma, X},
title = {Nanoplasmonic biosensors for precision medicine.},
journal = {Frontiers in chemistry},
volume = {11},
number = {},
pages = {1209744},
pmid = {37483272},
issn = {2296-2646},
abstract = {Nanoplasmonic biosensors have a huge boost for precision medicine, which allows doctors to better understand diseases at the molecular level and to improve the earlier diagnosis and develop treatment programs. Unlike traditional biosensors, nanoplasmonic biosensors meet the global health industry's need for low-cost, rapid and portable aspects, while offering multiplexing, high sensitivity and real-time detection. In this review, we describe the common detection schemes used based on localized plasmon resonance (LSPR) and highlight three sensing classes based on LSPR. Then, we present the recent applications of nanoplasmonic in other sensing methods such as isothermal amplification, CRISPR/Cas systems, lab on a chip and enzyme-linked immunosorbent assay. The advantages of nanoplasmonic-based integrated sensing for multiple methods are discussed. Finally, we review the current applications of nanoplasmonic biosensors in precision medicine, such as DNA mutation, vaccine evaluation and drug delivery. The obstacles faced by nanoplasmonic biosensors and the current countermeasures are discussed.},
}
@article {pmid37481734,
year = {2023},
author = {Liang, Y and Zhang, J and Xu, C and Wang, J and Han, W and Yang, J and Wu, S and An, J and Liu, J and Zhang, Z and Shi, J and Zhang, K},
title = {Biomimetic Mineralized CRISPR/Cas RNA Nanoparticles for Efficient Tumor-Specific Multiplex Gene Editing.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.3c04116},
pmid = {37481734},
issn = {1936-086X},
abstract = {CRISPR/Cas9 systems have great potential to achieve sophisticated gene therapy and cell engineering by editing multiple genomic loci. However, to achieve efficient multiplex gene editing, the delivery system needs adequate capacity to transfect all CRISPR/Cas9 RNA species at the required stoichiometry into the cytosol of each individual cell. Herein, inspired by biomineralization in nature, we develop an all-in-one biomimetic mineralized CRISPR/Cas9 RNA delivery system. This system allows for precise control over the coencapsulation ratio between Cas9 mRNA and multiple sgRNAs, while also exhibiting a high RNA loading capacity. In addition, it enhances the storage stability of RNA at 4 °C for up to one month, and the surface of the nanoparticles can be easily functionalized for precise targeting of RNA nanoparticles in vivo at nonliver sites. Based on the above characteristics, as a proof-of-concept, our system was able to achieve significant gene-editing at each target gene (Survivin: 31.9%, PLK1: 24.41%, HPV: 23.2%) and promote apoptosis of HeLa cells in the mouse model, inhibiting tumor growth without obvious off-target effects in liver tissue. This system addresses various challenges associated with multicomponent RNA delivery in vivo, providing an innovative strategy for the RNA-based CRISPR/Cas9 gene editing.},
}
@article {pmid37481551,
year = {2023},
author = {Wu, J and Huang, Y and Ding, X and Kang, L and Wang, X and Li, D and Cheng, W and Liu, G and Xue, J and Ding, S},
title = {CPA-Cas12a-based lateral flow strip for portable assay of Methicillin-resistant Staphylococcus aureus in clinical sample.},
journal = {Journal of nanobiotechnology},
volume = {21},
number = {1},
pages = {234},
pmid = {37481551},
issn = {1477-3155},
support = {KJQN202200431//the Project of Science and Technology Research Program of Chongqing Municipal Education Commission/ ; KJQN202000444//the Project of Science and Technology Research Program of Chongqing Municipal Education Commission/ ; //the Talents Project of University-Town Hospital of Chongqing Medical University/ ; cstc2019jcyj-msxmX0294//the Natural Science Foundation of Chongqing/ ; cstc2020jcyj-msxmX0190//the Natural Science Foundation of Chongqing/ ; cstc2019jcyj-msxmX0179//the Natural Science Foundation of Chongqing/ ; 20200154//the Project of the Basic and Frontier Funds in the Yuzhong District of Chongqing/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus ; CRISPR-Cas Systems ; Cross-Priming ; Staphylococcus aureus ; Anti-Bacterial Agents/pharmacology ; Biological Assay ; },
abstract = {The rapid and accurate identification of methicillin-resistant Staphylococcus aureus at an early antibiotic therapy stage would be benefit to disease diagnosis and antibiotic selection. Herein, we integrated cross-priming amplification (CPA) and CRISPR/Cas 12a (designated as CPA-Cas 12a) systems to establish a sensitive and efficient lateral flow assay to detect methicillin-resistant Staphylococcus aureus. This assay relies on the CPA isothermal nucleic acid amplification strategy which can amplify the DNA extracted from Staphylococcus aureus and accompanying the indiscriminately trans-cleavage process of Cas 12a/CrRNA duplex after recognizing specific sequence. Taking the advantage of reporter and high turnover Cas 12a activity, a dramatic change in response was achieved to produce a significant increase in the analytical sensitivity. The signal conversion and output were realized using a lateral flow strip to achieve field-deployable detection. Furthermore, this bioassay was accommodated with a microfluidic device to realize automatically portable detection. This proposed assay completed within 30 min with the detection limit of 5 CFU mL[-1], was verified by testing bacterial suspension and 202 clinical samples. Given the high sensitivity, specificity and efficiency, this colorimetric readout assay through strip could be further promoted to the clinical diagnosis, clinical medication of multidrug-resistant bacteria.},
}
@article {pmid37474872,
year = {2023},
author = {Wu, P and Zhang, M and Xue, X and Ding, P and Ye, L},
title = {Dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase-tethered magnetic microspheres for colorimetric detection of microcystin-LR.},
journal = {Mikrochimica acta},
volume = {190},
number = {8},
pages = {314},
pmid = {37474872},
issn = {1436-5073},
mesh = {Microspheres ; *CRISPR-Cas Systems ; Horseradish Peroxidase/metabolism ; *Colorimetry/methods ; Hydrogen Peroxide/metabolism ; DNA, Complementary ; Magnetic Phenomena ; },
abstract = {A novel dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase (HRP) was developed for colorimetric determination of MC-LR. This dual-amplification was accomplished by combining the nuclease activity of CRISPR-Cas12a with the redox activity of HRP. HRP linked to magnetic beads through an ssDNA (MB-ssDNA-HRP) was used to induce a color change of the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 chromogenic substrate solution. Specific binding of MC-LR with its aptamer initiated the release of a complementary DNA (cDNA), which was designed to activate the trans-cleavage activity of CRISPR-Cas12a. Upon activation, Cas12a cut the ssDNA linker in MB-ssDNA-HRP, causing a reduction of HRP on the magnetic beads. Consequently, the UV-Vis absorbance of the HRP-catalyzed reaction was decreased. The dual-signal amplification facilitated by CRISPR-Cas12a and HRP enabled the colorimetric detection of MC-LR in the range 0.01 to 50 ng·mL[-1] with a limit of detection (LOD) of 4.53 pg·mL[-1]. The practicability of the developed colorimetric method was demonstrated by detecting different levels of MC-LR in spiked real water samples. The recoveries ranged from 86.2 to 118.5% and the relative standard deviation (RSD) was 8.4 to 17.6%. This work provides new inspiration for the construction of effective signal amplification platforms and demonstrates a simple and user-friendly colorimetric method for determination of trace MC-LR.},
}
@article {pmid37474569,
year = {2023},
author = {Sheng, Y and Wang, H and Ou, Y and Wu, Y and Ding, W and Tao, M and Lin, S and Deng, Z and Bai, L and Kang, Q},
title = {Insertion sequence transposition inactivates CRISPR-Cas immunity.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4366},
pmid = {37474569},
issn = {2041-1723},
abstract = {CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.},
}
@article {pmid37254611,
year = {2023},
author = {Li, YJ and Gu, JM and Ma, S and Xu, Y and Liu, M and Zhang, C and Liu, X and Wang, GF},
title = {Genome editing of the susceptibility gene ZmNANMT confers multiple disease resistance without agronomic penalty in maize.},
journal = {Plant biotechnology journal},
volume = {21},
number = {8},
pages = {1525-1527},
pmid = {37254611},
issn = {1467-7652},
mesh = {*Gene Editing ; *Zea mays/genetics ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Genome, Plant/genetics ; },
}
@article {pmid36949241,
year = {2023},
author = {Maurya, VK and Ying, Y and Lanza, DG and Heaney, JD and Lydon, JP},
title = {A CRISPR/Cas9-engineered mouse carrying a conditional knockout allele for the early growth response-1 transcription factor.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {61},
number = {3-4},
pages = {e23515},
doi = {10.1002/dvg.23515},
pmid = {36949241},
issn = {1526-968X},
support = {P30 CA125123/CA/NCI NIH HHS/United States ; R01 HD042311/HD/NICHD NIH HHS/United States ; },
mesh = {Mice ; Female ; Animals ; *Transcription Factors/genetics ; Alleles ; *CRISPR-Cas Systems ; Exons ; },
abstract = {Early growth response 1 (EGR1) mediates transcriptional programs that are indispensable for cell division, differentiation, and apoptosis in numerous physiologies and pathophysiologies. Whole-body EGR1 knockouts in mice (Egr1[KO]) have advanced our understanding of EGR1 function in an in vivo context. To extend the utility of the mouse to investigate EGR1 responses in a tissue- and/or cell-type-specific manner, we generated a mouse model in which exon 2 of the mouse Egr1 gene is floxed by CRISPR/Cas9 engineering. The floxed Egr1 alleles (Egr1[f/f]) are designed to enable spatiotemporal control of Cre-mediated EGR1 ablation in the mouse. To confirm that the Egr1[f/f] alleles can be abrogated using a Cre driver, we crossed the Egr1[f/f] mouse with a global Cre driver to generate the Egr1 conditional knockout (Egr1[d/d]) mouse in which EGR1 expression is ablated in all tissues. Genetic and protein analysis confirmed the absence of exon 2 and loss of EGR1 expression in the Egr1[d/d] mouse, respectively. Moreover, the Egr1[d/d] female exhibits overt reproductive phenotypes previously reported for the Egr1[KO] mouse. Therefore, studies described in this short technical report underscore the potential utility of the murine Egr1 floxed allele to further resolve EGR1 function at a tissue- and/or cell-type-specific level.},
}
@article {pmid37470431,
year = {2023},
author = {Mendoza, CS and Findlay, AC and Judelson, H},
title = {An LbCas12a variant and elevated incubation temperatures enhance the rate of gene editing in the oomycete Phytophthora infestans.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {},
number = {},
pages = {},
doi = {10.1094/MPMI-05-23-0072-SC},
pmid = {37470431},
issn = {0894-0282},
abstract = {CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about ten-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R), which had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures.},
}
@article {pmid37469443,
year = {2023},
author = {Lee, M},
title = {Deep learning in CRISPR-Cas systems: a review of recent studies.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1226182},
pmid = {37469443},
issn = {2296-4185},
abstract = {In genetic engineering, the revolutionary CRISPR-Cas system has proven to be a vital tool for precise genome editing. Simultaneously, the emergence and rapid evolution of deep learning methodologies has provided an impetus to the scientific exploration of genomic data. These concurrent advancements mandate regular investigation of the state-of-the-art, particularly given the pace of recent developments. This review focuses on the significant progress achieved during 2019-2023 in the utilization of deep learning for predicting guide RNA (gRNA) activity in the CRISPR-Cas system, a key element determining the effectiveness and specificity of genome editing procedures. In this paper, an analytical overview of contemporary research is provided, with emphasis placed on the amalgamation of artificial intelligence and genetic engineering. The importance of our review is underscored by the necessity to comprehend the rapidly evolving deep learning methodologies and their potential impact on the effectiveness of the CRISPR-Cas system. By analyzing recent literature, this review highlights the achievements and emerging trends in the integration of deep learning with the CRISPR-Cas systems, thus contributing to the future direction of this essential interdisciplinary research area.},
}
@article {pmid37468545,
year = {2023},
author = {Higashitani, Y and Horie, K},
title = {Long-read sequence analysis of MMEJ-mediated CRISPR genome editing reveals complex on-target vector insertions that may escape standard PCR-based quality control.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11652},
pmid = {37468545},
issn = {2045-2322},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Alleles ; Polymerase Chain Reaction ; Sequence Analysis ; },
abstract = {CRISPR genome editing is a powerful tool for elucidating biological functions. To modify the genome as intended, it is essential to understand the various modes of recombination that can occur. In this study, we report complex vector insertions that were identified during the generation of conditional alleles by CRISPR editing using microhomology-mediated end joining (MMEJ). The targeting vector contained two loxP sequences and flanking 40-bp microhomologies. The genomic regions corresponding to the loxP sequences were cleaved with Cas9 in mouse embryonic stem cells. PCR screening for targeted recombination revealed a high frequency of bands of a larger size than expected. Nanopore sequencing of these bands revealed complex vector insertions mediated not only by MMEJ but also by non-homologous end joining and homologous recombination in at least 17% of the clones. A new band appeared upon improving the PCR conditions, suggesting the presence of unintentionally modified alleles that escape standard PCR screening. This prompted us to characterize the recombination of each allele of the genome-edited clones using heterozygous single nucleotide polymorphisms, leading to confirmation of the presence of homozygous alleles. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unintended, on-target vector insertion.},
}
@article {pmid37467473,
year = {2023},
author = {Durut, N and Kornienko, AE and Schmidt, HA and Lettner, N and Donà, M and Nordborg, M and Scheid, OM},
title = {Long non-coding RNAs contribute to DNA damage resistance in Arabidopsis thaliana.},
journal = {Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/genetics/iyad135},
pmid = {37467473},
issn = {1943-2631},
abstract = {Efficient repair of DNA lesions is essential for faithful transmission of genetic information between somatic cells and for genome integrity across generations. Plants have multiple, partially redundant and overlapping DNA repair pathways, probably due to the less constricted germline and the inevitable exposure to light including higher energy wavelengths. Many proteins involved in DNA repair and their mode of actions are well described. In contrast, a role for DNA damage-associated RNA components, evident from many other organisms, is less well understood. Here, we have challenged young Arabidopsis thaliana plants with two different types of genotoxic stress and performed de novo assembly and transcriptome analysis. We identified three long non-coding RNAs (lncRNAs) that are lowly or not expressed under regular conditions but up-regulated or induced by DNA damage. We generated CRISPR/Cas deletion mutants and found that the absence of the lncRNAs impairs the recovery capacity of the plants from genotoxic stress. The genetic loci are highly conserved among world-wide distributed Arabidopsis accessions and within related species in the Brassicaceae group. Together, these results suggest that the lncRNAs have a conserved function in connection with DNA damage and provide a basis for a mechanistic analysis of their role.},
}
@article {pmid37466850,
year = {2023},
author = {Pandya, K and Jagani, D and Singh, N},
title = {CRISPR-Cas Systems: Programmable Nuclease Revolutionizing the Molecular Diagnosis.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37466850},
issn = {1559-0305},
abstract = {CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.},
}
@article {pmid37466441,
year = {2023},
author = {Ortiz-Cartagena, C and Pablo-Marcos, D and Fernández-García, L and Blasco, L and Pacios, O and Bleriot, I and Siller, M and López, M and Fernández, J and Aracil, B and Fraile-Ribot, PA and García-Fernández, S and Fernández-Cuenca, F and Hernández-García, M and Cantón, R and Calvo-Montes, J and Tomás, M},
title = {CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0132923},
doi = {10.1128/spectrum.01329-23},
pmid = {37466441},
issn = {2165-0497},
abstract = {Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction.},
}
@article {pmid37464237,
year = {2023},
author = {Mercer, M and Bhargava, V and Goldstein, CD and Buszczak, M},
title = {Targeting Endogenous Loci That Function in Drosophila Germline Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2677},
number = {},
pages = {99-112},
pmid = {37464237},
issn = {1940-6029},
support = {R35 GM144043/GM/NIGMS NIH HHS/United States ; R01 GM127569/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; Germ Cells ; Stem Cells ; },
abstract = {CRISPR-Cas9 genome editing technology can be used to manipulate the genome of Drosophila melanogaster. The ability to delete genes, make specific mutations, add tags, or make other genetic manipulations is useful for studying germline stem cell biology. In this chapter, we will describe a method to use CRISPR-Cas9 genome editing technology to make knock-out and knock-in flies. We will cover everything from guideRNA (gRNA) and donor plasmid design and cloning to screening for positive edits.},
}
@article {pmid37464161,
year = {2023},
author = {Totorikaguena, L and Olabarrieta, E},
title = {CRISPR/Cas9 as a Simple Technique for the Generation of Murine Knockout Models for Neuropsychiatric Diseases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2687},
number = {},
pages = {45-55},
pmid = {37464161},
issn = {1940-6029},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; *Gene Editing/methods ; Mutation ; },
abstract = {The development of schizophrenia-like rodent models is still a major challenge for the study of this mental disorder. Schizophrenia and other neuropsychiatric disorders are thought to be triggered by multiple factors, and furthermore, the genetic component of schizophrenia is highly complex. The edition of one single gene for mimicking some of the symptoms of the disorder could cause unintended mutations that could influence animal's behavior making it difficult to study. Since 2013, CRISPR-Cas gene-editing technology has been a great improvement in the specificity of transgenic model generation because of its speed, efficiency, cost, and apparent ease. This protocol describes a simple method to generate a knockout mouse model using CRISPR technology, which can be applied to any gene presumably involved in the development of schizophrenia and other neuropsychiatric disorders.},
}
@article {pmid37428865,
year = {2023},
author = {Duan, Y and Tan, Y and Chen, X and Pei, X and Li, M},
title = {Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2147-2156},
doi = {10.1021/acssynbio.3c00229},
pmid = {37428865},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Cytosine/metabolism ; Adenine/metabolism ; Genomics ; Fungi/genetics ; },
abstract = {Random base editing is regarded as a fundamental method for accelerating the genomic evolution in both scientific research and industrial applications. In this study, we designed a modular interaction-based dual base editor (MIDBE) that assembled a DNA helicase and various base editors through dockerin/cohesin-mediated protein-protein interactions, resulting in a self-assembled MIDBE complex capable of editing bases at any locus in the genome. The base editing type of MIDBE can be readily controlled by the induction of cytidine or/and adenine deaminase gene expression. MIDBE exhibited the highest editing efficiency 2.3 × 10[3] times greater than the native genomic mutation rate. To evaluate the potential of MIDBE in genomic evolution, we developed a removable plasmid-based MIDBE tool, which led to a remarkable 977.1% increase of lovastatin production in Monascus purpureus HJ11. MIDBE represents the first biological tool for generating and accumulating base mutations in Monascus chromosome and also offers a bottom-up strategy for designing the base editor.},
}
@article {pmid37404005,
year = {2023},
author = {Shen, JY and Zhao, Q and He, QL},
title = {Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {1908-1923},
doi = {10.1021/acssynbio.3c00099},
pmid = {37404005},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; Fungi/genetics ; Genes, Fungal ; },
abstract = {Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.},
}
@article {pmid37379837,
year = {2023},
author = {Huang, J and Lin, Q and Fei, H and He, Z and Xu, H and Li, Y and Qu, K and Han, P and Gao, Q and Li, B and Liu, G and Zhang, L and Hu, J and Zhang, R and Zuo, E and Luo, Y and Ran, Y and Qiu, JL and Zhao, KT and Gao, C},
title = {Discovery of deaminase functions by structure-based protein clustering.},
journal = {Cell},
volume = {186},
number = {15},
pages = {3182-3195.e14},
doi = {10.1016/j.cell.2023.05.041},
pmid = {37379837},
issn = {1097-4172},
mesh = {*Gene Editing ; *Proteins/metabolism ; Cytidine Deaminase/genetics/metabolism ; DNA ; CRISPR-Cas Systems ; Cytosine/metabolism ; },
abstract = {The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.},
}
@article {pmid37358911,
year = {2023},
author = {Liu, Y and Zhang, Z and Zuo, N and Jiang, W and Gu, Y},
title = {Programmable Acetylation Modification of Bacterial Proteins by a Cas12a-Guided Acetyltransferase.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2135-2146},
doi = {10.1021/acssynbio.3c00211},
pmid = {37358911},
issn = {2161-5063},
mesh = {*Bacterial Proteins/genetics/metabolism ; Acetylation ; *Acetyltransferases/metabolism ; CRISPR-Cas Systems ; Lysine/metabolism ; Protein Processing, Post-Translational ; Polyesters/metabolism ; },
abstract = {Protein lysine acetylation (PLA) is a crucial post-translational modification in organisms that regulates a variety of metabolic and physiological activities. Many advances have been made in PLA-related research; however, the quick and accurate identification of causal relationships between specific protein acetylation events and phenotypic outcomes at the proteome level remains challenging due to the lack of efficient targeted modification techniques. In this study, based on the characteristics of transcription-translation coupling in bacteria, we designed and constructed an in situ targeted protein acetylation (TPA) system integrating the dCas12a protein, guiding element crRNA, and bacterial acetylase At2. Rapid identification of multiple independent protein acetylation and cell phenotypic analyses in Gram-negative Escherichia coli and Gram-positive Clostridium ljungdahlii demonstrated that TPA is a specific and efficient targeting tool for protein modification studies and engineering.},
}
@article {pmid37348112,
year = {2023},
author = {Yang, S and Zhu, J and Zhou, X and Zhang, J and Li, Q and Bian, F and Zhu, J and Yan, T and Wang, X and Zhang, Y and Yang, J and Jiang, Y and Yang, S},
title = {RNA-Guided DNA Transposition in Corynebacterium glutamicum and Bacillus subtilis.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2198-2202},
doi = {10.1021/acssynbio.3c00193},
pmid = {37348112},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; *Corynebacterium glutamicum/genetics ; RNA ; Gene Editing ; },
abstract = {Recently discovered CRISPR-associated transposases (CASTs) have been implemented as useful multiplexed genome editing tools, albeit only in a small group of bacteria. We demonstrated that the type I-F CAST from Vibrio cholerae could induce RNA-guided transposition in Bacillus subtilis and Corynebacterium glutamicum with efficiencies of 0.00018% and 0.027%, respectively. Lowering the culturing temperature to 16 °C in rich media increased the insertion efficiency to 3.64% in B. subtilis. By adding a positive selection against spectinomycin in the cargo DNA, up to 9 kb of the DNA fragment could be integrated at the target site with a 13.4% efficiency in C. glutamicum, which was difficult using the conventional approach. The successful implementation of CAST in these two academically important and industrial-relevant Firmicutes shows its great potential in a wide variety of bacteria and could be further optimized for multiplexed genome editing.},
}
@article {pmid37326023,
year = {2023},
author = {Zhao, Y and Coelho, C and Lauer, S and Majewski, M and Laurent, JM and Brosh, R and Boeke, JD},
title = {CREEPY: CRISPR-mediated editing of synthetic episomes in yeast.},
journal = {Nucleic acids research},
volume = {51},
number = {13},
pages = {e72},
pmid = {37326023},
issn = {1362-4962},
support = {RM1 HG009491/HG/NHGRI NIH HHS/United States ; 1RM1HG009491/GF/NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plasmids/genetics ; DNA ; },
abstract = {Use of synthetic genomics to design and build 'big' DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. Saccharomyces cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics.},
}
@article {pmid37264902,
year = {2023},
author = {Killelea, T and Dimude, JU and He, L and Stewart, AL and Kemm, FE and Radovčić, M and Ivančić-Baće, I and Rudolph, CJ and Bolt, EL},
title = {Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation.},
journal = {Nucleic acids research},
volume = {51},
number = {13},
pages = {6914-6926},
pmid = {37264902},
issn = {1362-4962},
support = {BB/T006625-1//BBSRC/ ; IP-2016-06-8861//Croatian Science Foundation/ ; //University of Nottingham/ ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/metabolism ; *CRISPR-Associated Proteins/metabolism ; *Escherichia coli Proteins/metabolism ; DNA/chemistry ; CRISPR-Cas Systems ; },
abstract = {Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid of pre-existing immunity 'interference' complexes-by mechanisms that are not clear. Using E. coli we show that the chaperone DnaK inhibits DNA binding and integration by Cas1-Cas2, and inhibits naive adaptation in cells that results from chromosomal self-targeting. Inhibition of naive adaptation was reversed by deleting DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage λ) protein. We also imaged fluorescently labelled Cas1 in living cells, observing that Cas1 foci depend on active DNA replication, and are much increased in frequency in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naive adaptation from DNA self-targeting, until DnaK is triggered to release Cas1-Cas2 to target MGE DNA.},
}
@article {pmid37246708,
year = {2023},
author = {Ponnienselvan, K and Liu, P and Nyalile, T and Oikemus, S and Maitland, SA and Lawson, ND and Luban, J and Wolfe, SA},
title = {Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency.},
journal = {Nucleic acids research},
volume = {51},
number = {13},
pages = {6966-6980},
pmid = {37246708},
issn = {1362-4962},
support = {UG3 TR002668/TR/NCATS NIH HHS/United States ; R35HL140017/NH/NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Zebrafish/genetics ; Binding Sites ; *Cold Temperature ; Cold-Shock Response ; Ribonucleoproteins ; Gene Editing ; CRISPR-Cas Systems/genetics ; Mammals ; },
abstract = {Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.},
}
@article {pmid37464084,
year = {2023},
author = {Mehta, D},
title = {EU proposal on CRISPR-edited crops is welcome - but not enough.},
journal = {Nature},
volume = {619},
number = {7970},
pages = {437},
doi = {10.1038/d41586-023-02328-8},
pmid = {37464084},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; *Crop Production/legislation &amp; jurisprudence/methods ; *Crops, Agricultural/genetics ; *Gene Editing/legislation &amp; jurisprudence ; *Plants, Genetically Modified/genetics ; *European Union ; },
}
@article {pmid37464007,
year = {2023},
author = {Zhou, Y and Wang, L and Lu, Z and Yu, Z and Ma, L},
title = {Optimized minimal genome-wide human sgRNA library.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11569},
pmid = {37464007},
issn = {2045-2322},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; *Genome, Human ; Gene Library ; Genomic Library ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screening is revolting the genetic analysis of a cellular or molecular phenotype in question but is challenged by the large size of single-guide RNA (sgRNA) library. Here we designed a minimal genome-wide human sgRNA library, H-mLib, which is composed of 21,159 sgRNA pairs assembled based on a dedicated selection strategy from all potential SpCas9/sgRNAs in the human genome. These sgRNA pairs were cloned into a dual-gRNA vector each targeting one gene, resulting in a compact library size nearly identical to the number of human protein-coding genes. The performance of the H-mLib was benchmarked to other CRISPR libraries in a proliferation screening conducted in K562 cells. We also identified groups of core essential genes and cell-type specific essential genes by comparing the screening results from the K562 and Jurkat cells. Together, the H-mLib exemplified high specificity and sensitivity in identifying essential genes while containing minimal library complexity, emphasizing its advantages and applications in CRISPR screening with limited cell numbers.},
}
@article {pmid37463886,
year = {2023},
author = {Kang, Y and Yu, Y and Liu, Y and Pan, Y and Zhang, R and Ren, D and Cai, Z and Ma, J and Xiong, X and Zhang, Q and Zhang, C and Tu, R},
title = {Identification of USP29 as a key regulator of nucleotide biosynthesis in neuroblastoma through integrative analysis of multi-omics data.},
journal = {Cancer biology &amp; therapy},
volume = {24},
number = {1},
pages = {2237200},
pmid = {37463886},
issn = {1555-8576},
mesh = {Humans ; *Multiomics ; RNA, Guide, CRISPR-Cas Systems ; *Neuroblastoma/pathology ; Glycolysis ; Glucose ; Cell Line, Tumor ; N-Myc Proto-Oncogene Protein/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Ubiquitin-Specific Proteases/metabolism ; },
abstract = {Cancer cells show enhanced nucleotide biosynthesis, which is essential for their unlimited proliferation, but the underlying mechanisms are not entirely clear. Ubiquitin specific peptidase 29 (USP29) was reported to sustain neuroblastoma progression by promoting glycolysis and glutamine catabolism; however, its potential role in regulating nucleotide biosynthesis in tumor cells remains unknown. In this study, we depleted endogenous USP29 in MYCN-amplified neuroblastoma SK-N-BE2 cells by sgRNAs and conducted metabolomic analysis in cells with or without USP29 depletion, we found that USP29 deficiency caused a disorder of intermediates involved in glycolysis and nucleotide biosynthesis. De novo nucleotide biosynthesis was analyzed using [13]C6 glucose as a tracer under normoxia and hypoxia. The results indicated that USP29-depleted cells showed inhibition of nucleotide anabolic intermediates derived from glucose, and this inhibition was more significant under hypoxic conditions. Analysis of RNA sequencing data in SK-N-BE2 cells demonstrated that USP29 promoted the gene expression of metabolic enzymes involved in nucleotide anabolism, probably by regulating MYC and E2F downstream pathways. These findings indicated that USP29 is a key regulator of nucleotide biosynthesis in tumor cells.},
}
@article {pmid37463216,
year = {2023},
author = {Alba Burbano, D and Cardiff, RAL and Tickman, BI and Kiattisewee, C and Maranas, CJ and Zalatan, JG and Carothers, JM},
title = {Engineering activatable promoters for scalable and multi-input CRISPRa/i circuits.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {30},
pages = {e2220358120},
doi = {10.1073/pnas.2220358120},
pmid = {37463216},
issn = {1091-6490},
mesh = {Promoter Regions, Genetic/genetics ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics ; Gene Regulatory Networks ; CRISPR-Cas Systems/genetics ; },
abstract = {Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.},
}
@article {pmid37460924,
year = {2023},
author = {Hussen, BM and Rasul, MF and Abdullah, SR and Hidayat, HJ and Faraj, GSH and Ali, FA and Salihi, A and Baniahmad, A and Ghafouri-Fard, S and Rahman, M and Glassy, MC and Branicki, W and Taheri, M},
title = {Targeting miRNA by CRISPR/Cas in cancer: advantages and challenges.},
journal = {Military Medical Research},
volume = {10},
number = {1},
pages = {32},
pmid = {37460924},
issn = {2054-9369},
mesh = {Humans ; *MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Neoplasms/genetics/therapy ; },
abstract = {Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade. In the study of cancer, the CRISPR/CRISPR-associated protein (Cas) system opens new avenues into issues that were once unknown in our knowledge of the noncoding genome, tumor heterogeneity, and precision medicines. CRISPR/Cas-based gene-editing technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs (miRNAs). However, the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities. This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy. Furthermore, we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.},
}
@article {pmid37460897,
year = {2023},
author = {Omura, SN and Nakagawa, R and Südfeld, C and Villegas Warren, R and Wu, WY and Hirano, H and Laffeber, C and Kusakizako, T and Kise, Y and Lebbink, JHG and Itoh, Y and van der Oost, J and Nureki, O},
title = {Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {37460897},
issn = {1545-9985},
abstract = {RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.},
}
@article {pmid37460713,
year = {2023},
author = {Zhang, J and Zhang, L and Zhang, C and Yang, Y and Liu, H and Li, L and Zhang, S and Li, X and Liu, X and Liu, Y and Wang, J and Yang, G and Xia, Q and Wang, W and Yang, J},
title = {Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {37460713},
issn = {1869-1889},
abstract = {Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T1 plants. This system was used to confer Z-abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z-abienol were detected in the leaves of homozygous T1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.},
}
@article {pmid37460548,
year = {2023},
author = {Yue, Q and Meng, J and Qiu, Y and Yin, M and Zhang, L and Zhou, W and An, Z and Liu, Z and Yuan, Q and Sun, W and Li, C and Zhao, H and Molnár, I and Xu, Y and Shi, S},
title = {A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4267},
pmid = {37460548},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Saccharomyces cerevisiae/genetics ; Fungi/genetics ; },
abstract = {Synthetic biology requires efficient systems that support the well-coordinated co-expression of multiple genes. Here, we discover a 9-bp nucleotide sequence that enables efficient polycistronic gene expression in yeasts and filamentous fungi. Coupling polycistronic expression to multiplexed, markerless, CRISPR/Cas9-based genome editing, we develop a strategy termed HACKing (Highly efficient and Accessible system by CracKing genes into the genome) for the assembly of multigene pathways. HACKing allows the expression level of each enzyme to be precalibrated by linking their translation to those of host proteins with predetermined abundances under the desired fermentation conditions. We validate HACKing by rapidly constructing highly efficient Saccharomyces cerevisiae cell factories that express 13 biosynthetic genes, and produce model endogenous (1,090.41 ± 80.92 mg L[-1] squalene) or heterologous (1.04 ± 0.02 mg L[-1] mogrol) terpenoid products. Thus, HACKing addresses the need of synthetic biology for predictability, simplicity, scalability, and speed upon fungal pathway engineering for valuable metabolites.},
}
@article {pmid37459160,
year = {2023},
author = {Collins, AJ and Whitaker, RJ},
title = {CRISPR Comparison Toolkit: Rapid Identification, Visualization, and Analysis of CRISPR Array Diversity.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0080},
pmid = {37459160},
issn = {2573-1602},
abstract = {CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, no tools are currently available to automate the process of reconstructing strain histories using CRISPR spacers. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. The CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. The CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.},
}
@article {pmid37395536,
year = {2023},
author = {Lin, Y and Luo, X and Burghardt, T and Dorrer, S and Höhn, M and Wagner, E and Lächelt, U},
title = {Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {28},
pages = {15171-15179},
doi = {10.1021/jacs.3c01902},
pmid = {37395536},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems/genetics ; *Ribonucleoproteins ; Evolution, Chemical ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.},
}
@article {pmid37279283,
year = {2023},
author = {Liu, Q and Chen, Y and Hu, S and Liu, W and Xie, D and Yang, X and Huang, W and Liu, S and Chen, X and Liu, H and Huang, J},
title = {Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.},
journal = {Human gene therapy},
volume = {34},
number = {13-14},
pages = {629-638},
doi = {10.1089/hum.2023.055},
pmid = {37279283},
issn = {1557-7422},
mesh = {Humans ; Animals ; Mice ; *Dependovirus/genetics/metabolism ; *Cytosine/metabolism ; HeLa Cells ; Gene Editing ; Transfection ; CRISPR-Cas Systems ; },
abstract = {The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.},
}
@article {pmid37265143,
year = {2023},
author = {Liu, Z and Huang, L and Deng, H and Chen, Y and Xiao, H},
title = {Characteristics, Recombination Methods, and Applications Progresses of Split-Cas9 System.},
journal = {Human gene therapy},
volume = {34},
number = {13-14},
pages = {594-604},
doi = {10.1089/hum.2022.223},
pmid = {37265143},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; Genetic Vectors/genetics ; Recombination, Genetic ; },
abstract = {CRISPR technology has been used to revolutionize various facets of life sciences because of its potent gene editing capabilities. In particular, CRISPR technology is anticipated to be used to cure congenital disorders, and malignant cancers brought on by gene mutation. In this article, we introduce a Split-Cas9 system, in which Cas9 protein is split into two or more parts and recombined in cells to function specific induction circumstances. Split-Cas9 system can improve the therapeutic index of CRISPR technology by splitting Cas9 proteins into small fragments, thus enhancing their compatibility with virus vectors and precise temporal and spatial control. This article examined the combination mode of Split-Cas9 system, contrasted the differences in its split sites and activity efficiency, and discussed the use and clinical transformation in vivo and in vitro.},
}
@article {pmid37211656,
year = {2023},
author = {Ashok, K and Bhargava, CN and Asokan, R and Pradeep, C and Kennedy, JS and Rai, A and Manamohan, M},
title = {First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).},
journal = {Archives of insect biochemistry and physiology},
volume = {113},
number = {4},
pages = {e22024},
doi = {10.1002/arch.22024},
pmid = {37211656},
issn = {1520-6327},
support = {//Centre for Agricultural Bioinformatics/ ; },
mesh = {Female ; Male ; Animals ; *Tephritidae/genetics ; CRISPR-Cas Systems ; Pupa/genetics ; Drosophila ; Genomics ; },
abstract = {The Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive pest of quarantine importance affecting the global fruit trade. In managing B. dorsalis, methods like cultural, biological, chemical, sterile insect technique (SIT), and semiochemical-mediated attract-and-kill are in use with varying success. The SIT approach is the method of choice for a chemical-free, long-term suppression of B. dorsalis, followed in many countries across the globe. The nonspecific mutations caused by irradiation affect the overall fitness of flies, thus requiring a more precise method for a heritable, fitness-not-compromising approach. In this regard, CRISPR/Cas9-mediated genome editing enables the creation of mutations at the precise genomic location/s through RNA-guided dsDNA cleavage. Of late, DNA-free editing employing ribonucleoprotein complex (RNP) is preferred to validate the target genes at G0 stage embryos in insects. It requires characterizing genomic edits from adults after completing their life cycle, which may entail a few days to months, depending on longevity. Additionally, edit characterization is required from each individual, as edits are unique. Therefore, all RNP-microinjected individuals must be maintained until the end of their life cycle, irrespective of editing. To overcome this impediment, we predetermine the genomic edits from the shed tissues, such as pupal cases, to maintain only edited individuals. In this study, we have shown the utility of pupal cases from five males and females of B. dorsalis to predetermine the genomic edits, which corroborated the edits from the respective adults.},
}
@article {pmid37457727,
year = {2023},
author = {Martín-Adrados, B and Wculek, SK and Fernández-Bravo, S and Torres-Ruiz, R and Valle-Noguera, A and Gomez-Sánchez, MJ and Hernández-Walias, JC and Ferreira, FM and Corraliza, AM and Sancho, D and Esteban, V and Rodriguez-Perales, S and Cruz-Adalia, A and Nakaya, HI and Salas, A and Bernardo, D and Campos-Martín, Y and Martínez-Zamorano, E and Muñoz-López, D and Gómez Del Moral, M and Cubero, FJ and Blumberg, RS and Martínez-Naves, E},
title = {Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1185517},
pmid = {37457727},
issn = {1664-3224},
mesh = {Humans ; *Colitis, Ulcerative/pathology ; Caco-2 Cells ; Thapsigargin ; Endoplasmic Reticulum Stress/genetics ; *Inflammatory Bowel Diseases/metabolism ; Epithelial Cells/metabolism ; Hydroxymethylglutaryl-CoA Synthase ; },
abstract = {INTRODUCTION: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease.<br><br>METHODS: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells.<br><br>RESULTS: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines.<br><br>CONCLUSION: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.},
}
@article {pmid37455748,
year = {2023},
author = {Qi, L and Shi, M and Zhu, FC and Lian, CA and He, LS},
title = {Genomic evidence for the first symbiotic Deferribacterota, a novel gut symbiont from the deep-sea hydrothermal vent shrimp Rimicaris kairei.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1179935},
pmid = {37455748},
issn = {1664-302X},
abstract = {The genus Rimicaris is the dominant organism living in hydrothermal vents. However, little research has been done on the functions of their intestinal flora. Here, we investigated the potential functions of Deferribacterota, which is dominant in the intestine of Rimicaris kairei from the Central Indian Ridge. In total, six metagenome-assembled genomes (MAGs) of Deferribacterota were obtained using the metagenomic approach. The six Deferribacterota MAGs (Def-MAGs) were clustered into a new branch in the phylogenetic tree. The six Def-MAGs were further classified into three species, including one new order and two new genera, based on the results of phylogenetic analysis, relative evolutionary divergence (RED), average nucleotide identity (ANI), average amino acid identity (AAI) and DNA-DNA hybridization (DDH) values. The results of the energy metabolism study showed that these bacteria can use a variety of carbon sources, such as glycogen, sucrose, salicin, arbutin, glucose, cellobiose, and maltose. These bacteria have a type II secretion system and effector proteins that can transport some intracellular toxins to the extracellular compartment and a type V CRISPR-Cas system that can defend against various invasions. In addition, cofactors such as biotin, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) synthesized by R. kairei gut Deferribacterota may also assist their host in surviving under extreme conditions. Taken together, the potential function of Deferribacterota in the hydrothermal R. kairei gut suggests its long-term coevolution with the host.},
}
@article {pmid37455550,
year = {2023},
author = {Zhang, J and Zhang, Y and Khanal, S and Cao, D and Zhao, J and Dang, X and Nguyen, LNT and Schank, M and Wu, XY and Jiang, Y and Ning, S and Wang, L and El Gazzar, M and Moorman, JP and Guo, H and Yao, ZQ},
title = {Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication.},
journal = {Journal of medical virology},
volume = {95},
number = {7},
pages = {e28952},
doi = {10.1002/jmv.28952},
pmid = {37455550},
issn = {1096-9071},
support = {R21 AI157909/AI/NIAID NIH HHS/United States ; R15 AG069544/AG/NIA NIH HHS/United States ; S10 OD021572/OD/NIH HHS/United States ; },
mesh = {Humans ; DNA, Viral/genetics/metabolism ; CRISPR-Cas Systems ; Hepatitis B virus/genetics ; *Hepatitis B ; *Hepatitis B, Chronic ; Virus Replication ; RNA/metabolism/pharmacology ; DNA, Circular/genetics ; },
abstract = {The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.},
}
@article {pmid37451948,
year = {2023},
author = {Graver, BA and Chakravarty, N and Solomon, KV},
title = {Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.06.010},
pmid = {37451948},
issn = {1879-3096},
abstract = {Prokaryotic Argonautes (pAgos) are an emerging class of programmable endonucleases that are believed to be more flexible than existing CRISPR-Cas systems and have significant potential for biotechnology. Current applications of pAgos include a myriad of molecular diagnostics and in vitro DNA assembly tools. However, efforts have historically been centered on thermophilic pAgo variants. To enable in vivo biotechnological applications such as gene editing, focus has shifted to pAgos from mesophilic organisms. We discuss what is known of pAgos, how they are being developed for various applications, and strategies to overcome current challenges to in vivo applications in prokaryotes and eukaryotes.},
}
@article {pmid37451945,
year = {2023},
author = {Chowdhry, R and Lu, SZ and Lee, S and Godhulayyagari, S and Ebrahimi, SB and Samanta, D},
title = {Enhancing CRISPR/Cas systems with nanotechnology.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.06.005},
pmid = {37451945},
issn = {1879-3096},
abstract = {CRISPR/Cas systems have revolutionized biology and medicine, and have led to new paradigms in disease diagnostics and therapeutics. However, these complexes suffer from key limitations regarding barriers to cellular entry, stability in biological environments, and off-target effects. Integrating nanotechnology with CRISPR/Cas systems has emerged as a promising strategy to overcome these challenges and has further unlocked structures that accumulate preferentially in tissues of interest, have tunable pharmacological properties, and are activated in response to desired stimuli. Nanomaterials can also enhance CRISPR/Cas-mediated detection platforms by enabling faster, more sensitive, and convenient readouts. We highlight recent advances in this rapidly growing field. We also outline areas that need further development to fully realize the potential of CRISPR technologies.},
}
@article {pmid37446383,
year = {2023},
author = {Ormazabal, ME and Pavan, E and Vaena, E and Ferino, D and Biasizzo, J and Mucci, JM and Serra, F and Cifù, A and Scarpa, M and Rozenfeld, PA and Dardis, AE},
title = {Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446383},
issn = {1422-0067},
mesh = {Humans ; *Gaucher Disease/pathology ; Osteogenesis ; Monocytes/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1β and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.},
}
@article {pmid37446368,
year = {2023},
author = {Shirazi Parsa, H and Sabet, MS and Moieni, A and Shojaeiyan, A and Dogimont, C and Boualem, A and Bendahmane, A},
title = {CRISPR/Cas9-Mediated Cytosine Base Editing Using an Improved Transformation Procedure in Melon (Cucumis melo L.).},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446368},
issn = {1422-0067},
support = {101095736/ERC_/European Research Council/International ; },
mesh = {Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Cucumis melo/genetics ; *Cucurbitaceae/genetics ; Plants/genetics ; },
abstract = {Melon is a recalcitrant plant for stable genetic transformation. Various protocols have been tried to improve melon transformation efficiency; however, it remains significantly low compared to other plants such as tomato. In this study, the primary focus was on the optimization of key parameters during the inoculation and co-culture steps of the genetic transformation protocol. Our results showed that immersing the explants in the inoculation medium for 20 min significantly enhanced transformation efficiency. During the co-culture step, the use of filer paper, 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and a temperature of 24 °C significantly enhanced the melon transformation efficiency. Furthermore, the impact of different ethylene inhibitors and absorbers on the transformation efficiency of various melon varieties was explored. Our findings revealed that the use of these compounds led to a significant improvement in the transformation efficiency of the tested melon varieties. Subsequently, using our improved protocol and reporter-gene construct, diploid transgenic melons successfully generated. The efficiency of plant genetic transformation ranged from 3.73 to 4.83%. Expanding the scope of our investigation, the optimized protocol was applied to generate stable gene-edited melon lines using the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated cytosine base editor and obtained melon lines with editions (C-to-T and C-to-G) in the eukaryotic translation initiation factor 4E, CmeIF4E gene. In conclusion, the optimized melon transformation protocol, along with the utilization of the CRISPR/Cas9-mediated cytosine base editor, provides a reliable framework for functional gene engineering in melon. These advancements hold significant promise for furthering genetic research and facilitating crop improvement in this economically important plant species.},
}
@article {pmid37440632,
year = {2023},
author = {Sulis, DB and Jiang, X and Yang, C and Marques, BM and Matthews, ML and Miller, Z and Lan, K and Cofre-Vega, C and Liu, B and Sun, R and Sederoff, H and Bing, RG and Sun, X and Williams, CM and Jameel, H and Phillips, R and Chang, HM and Peszlen, I and Huang, YY and Li, W and Kelly, RM and Sederoff, RR and Chiang, VL and Barrangou, R and Wang, JP},
title = {Multiplex CRISPR editing of wood for sustainable fiber production.},
journal = {Science (New York, N.Y.)},
volume = {381},
number = {6654},
pages = {216-221},
doi = {10.1126/science.add4514},
pmid = {37440632},
issn = {1095-9203},
mesh = {Carbohydrates/analysis ; *Gene Editing ; *Lignin/genetics ; *Wood/genetics ; CRISPR-Cas Systems ; *Populus/genetics ; Paper ; Sustainable Growth ; },
abstract = {The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.},
}
@article {pmid37413791,
year = {2023},
author = {Ji, Z and Zhou, B and Shang, Z and Liu, S and Li, X and Zhang, X and Li, B},
title = {Active CRISPR-Cas12a on Hydrophilic Metal-Organic Frameworks: A Nanobiocomposite with High Stability and Activity for Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {95},
number = {28},
pages = {10580-10587},
doi = {10.1021/acs.analchem.3c00400},
pmid = {37413791},
issn = {1520-6882},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; *Metal-Organic Frameworks ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; *Biosensing Techniques ; },
abstract = {CRISPR-Cas12a is an accurate and responsive biosensing technique, but its limited stability has restricted its widespread applications. To address this, we propose a strategy using metal-organic frameworks (MOFs) to protect Cas12a from harsh environments. After screening multiple candidate MOFs, it was found that hydrophilic MAF-7 is highly compatible with Cas12a, and the as-formed Cas12a-on-MAF-7 (COM) not only retains high enzymatic activity but also possesses excellent tolerance to heat, salt, and organic solvents. Further investigation showed that COM can serve as an analytical component for nucleic acid detection, resulting in an ultrasensitive assay for SARS-CoV-2 RNA detection with a detection limit of 1 copy. This is the first successful attempt to create an active Cas12a nanobiocomposite that functions as a biosensor without the need for shell deconstruction or enzyme release.},
}
@article {pmid37392174,
year = {2023},
author = {Xu, J and Liu, Z and Zhang, Z and Wu, T},
title = {Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity.},
journal = {Analytical chemistry},
volume = {95},
number = {28},
pages = {10664-10669},
doi = {10.1021/acs.analchem.3c01307},
pmid = {37392174},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Fluorescent Dyes ; Nucleotides ; *Biosensing Techniques ; },
abstract = {The trans-cleavage activity of Cas12a has been widely used with various applications. Here, we report that the trans-cleavage activity of Cas12a can be significantly affected by the fluorescent probe length and reaction buffer. The optimal probe length for Cas12a is found to be 15 nucleotides, and the optimal buffer is NEBuffer 4. Compared to the popularly used reaction conditions, the activity of Cas12a is improved by about 50-fold. In addition, the detection limit of Cas12a for DNA targets has been reduced by nearly three orders of magnitude. Our method provides a powerful tool for Cas12a trans-cleavage activity applications.},
}
@article {pmid36896979,
year = {2023},
author = {Liu, T and Ji, J and Cheng, Y and Zhang, S and Wang, Z and Duan, K and Wang, Y},
title = {CRISPR/Cas9-mediated editing of GmTAP1 confers enhanced resistance to Phytophthora sojae in soybean.},
journal = {Journal of integrative plant biology},
volume = {65},
number = {7},
pages = {1609-1612},
doi = {10.1111/jipb.13476},
pmid = {36896979},
issn = {1744-7909},
support = {31721004//National Natural Science Foundation of China/ ; 31901957//National Natural Science Foundation of China/ ; 32172499//National Natural Science Foundation of China/ ; },
mesh = {*Soybeans/genetics ; *Phytophthora ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Plant Diseases/genetics ; },
abstract = {Soybean root rot disease caused by Phytophthora sojae seriously constrains soybean yield. Knocking out the susceptibility gene GmTAP1 in soybean created new soybean lines resistant to several P. sojae strains and these lines showed no agronomic penalties in the field.},
}
@article {pmid36037289,
year = {2023},
author = {Katsuda, T and Cure, H and Sussman, J and Simeonov, KP and Krapp, C and Arany, Z and Grompe, M and Stanger, BZ},
title = {Rapid in vivo multiplexed editing (RIME) of the adult mouse liver.},
journal = {Hepatology (Baltimore, Md.)},
volume = {78},
number = {2},
pages = {486-502},
doi = {10.1002/hep.32759},
pmid = {36037289},
issn = {1527-3350},
support = {R01 DK107667/DK/NIDDK NIH HHS/United States ; DK083355/DK/NIDDK NIH HHS/United States ; //Biesecker Pediatric Liver Foundation/Children's Hospital of Philadelphia/ ; },
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Liver ; Mammals ; },
abstract = {BACKGROUND AND AIMS: Assessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or perizygotic embryos. These approaches are time-consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a rapid in vivo multiplexed editing (RIME) method and provide proof of concept of this system.<br><br>APPROACH AND RESULTS: RIME, a system wherein CRISPR/caspase 9 technology, paired with adeno-associated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver. The method is quick, requiring as little as 1&#x2009;month from conceptualization to knockout, and highly efficient, enabling editing in >95% of target cells. To highlight its use, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation.<br><br>CONCLUSIONS: RIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo .},
}
@article {pmid37446306,
year = {2023},
author = {Shah, R and van den Berk, PCM and Pritchard, CEJ and Song, JY and Kreft, M and Pilzecker, B and Jacobs, H},
title = {A C57BL/6J Fancg-KO Mouse Model Generated by CRISPR/Cas9 Partially Captures the Human Phenotype.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446306},
issn = {1422-0067},
support = {KWF NKI-2017- 10032 and KWF NKI-2017-10796 to Heinz Jacobs//Dutch Cancer Society/ ; },
mesh = {Humans ; Animals ; Mice ; *Cisplatin/metabolism ; *Fanconi Anemia/genetics/metabolism ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; DNA-Binding Proteins/metabolism ; Fibroblasts/metabolism ; Mitomycin ; Phenotype ; Fanconi Anemia Complementation Group G Protein/genetics ; },
abstract = {Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.},
}
@article {pmid36893939,
year = {2023},
author = {Smits, JPH and van den Brink, NJM and Meesters, LD and Hamdaoui, H and Niehues, H and Jansen, PAM and van Vlijmen-Willems, IMJJ and Rodijk-Olthuis, D and Evrard, C and Poumay, Y and van Geel, M and Hendriks, WJAJ and Schalkwijk, J and Zeeuwen, PLJM and van den Bogaard, EH},
title = {Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes.},
journal = {The Journal of investigative dermatology},
volume = {143},
number = {8},
pages = {1520-1528.e5},
doi = {10.1016/j.jid.2023.02.021},
pmid = {36893939},
issn = {1523-1747},
mesh = {Humans ; *CRISPR-Cas Systems ; *Intermediate Filament Proteins/metabolism ; Filaggrin Proteins ; Keratinocytes/metabolism ; Phenotype ; },
abstract = {Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease.},
}
@article {pmid35882048,
year = {2023},
author = {Vora, DS and Jaiswal, AK and Sundar, D},
title = {Implementing accelerated dynamics to unravel the effects of high-fidelity Cas9 mutants on target DNA and guide RNA hybrid stability.},
journal = {Journal of biomolecular structure &amp; dynamics},
volume = {41},
number = {13},
pages = {6178-6190},
doi = {10.1080/07391102.2022.2103032},
pmid = {35882048},
issn = {1538-0254},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; Mutation ; Genome ; RNA/genetics ; },
abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR) and its associated nuclease (Cas9) offers a unique and easily reprogrammable system for editing eukaryotic genomes. Cas9 is guided to the target by an RNA strand, and precise edits are created by introducing double-stranded breaks. However, nuclease activity of Cas9 is also triggered at other sites other than the target sit, which is a major limitation for various applications. Cas9 variants have been designed to improve the efficacy of the tool by introducing certain mutations. However, the on-target activity of such Cas9 variants is often seen as compromised. Hence, understanding the sub-molecular differences in the variants is essential to elucidate the factors that contribute to efficiency. The study reveals distortions in the PAM-distal regions of the nucleic hybrids as well as changes in the interactions between the Cas9 variants and RNA-DNA hybrid, contributing to the explanation for differences in on-target activity.Communicated by Ramaswamy H. Sarma.},
}
@article {pmid37446265,
year = {2023},
author = {Han, Y and Yang, J and Wu, H and Liu, F and Qin, B and Li, R},
title = {Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446265},
issn = {1422-0067},
mesh = {*Gene Editing ; *Oryza/metabolism ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Plant Breeding ; Gene Expression Profiling ; Plant Leaves/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.},
}
@article {pmid37445761,
year = {2023},
author = {Xiao, Y and Zhang, Y and Xie, F and Olsen, RH and Shi, L and Li, L},
title = {Inhibition of Plasmid Conjugation in Escherichia coli by Targeting rbsB Gene Using CRISPRi System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37445761},
issn = {1422-0067},
mesh = {Humans ; *Escherichia coli/metabolism ; *RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plasmids/genetics ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; },
abstract = {Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.},
}
@article {pmid37445725,
year = {2023},
author = {Chohra, I and Giri, S and Malgrange, B},
title = {Generation of a Well-Characterized Homozygous Chromodomain-Helicase-DNA-Binding Protein 4[G1003D] Mutant hESC Line Using CRISPR/eCas9 (ULIEGEe001-A-1).},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37445725},
issn = {1422-0067},
support = {R.FNRS.4649//Fund for Scientific Research/ ; 22/003//Fonds leon Fredericq/ ; FSR-R.RCFRA.3775//University of Li&#xe8;ge/ ; },
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line ; Homozygote ; DNA-Binding Proteins/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {The chromatin remodeler Chromodomain-helicase-DNA-binding protein 4 (CHD4) is crucial for the development of multiple organ systems. Functional mutations of CHD4 have recently been described in a developmental disorder, namely Siffrim-Hitz-Weiss syndrome (SIHIWES). Herein, we have generated a homozygous CHD4G1003D hESC line (WAe025-A-1) using CRISPR/eCas9-based gene editing in the WA-25 hESC line. The edited hESC line maintains normal karyotype, pluripotency, and ability to differentiate into three germ layers. This cell line will be a valuable resource for studying the functional role of CHD4 during the development and disease modeling of SIHIWES in vitro.},
}
@article {pmid37443815,
year = {2023},
author = {Zhang, T and Yao, H and Wang, H and Sui, T},
title = {Development of Woolly Hair and Hairlessness in a CRISPR-Engineered Mutant Mouse Model with KRT71 Mutations.},
journal = {Cells},
volume = {12},
number = {13},
pages = {},
pmid = {37443815},
issn = {2073-4409},
mesh = {Child, Preschool ; Humans ; Animals ; Mice ; Mice, Nude ; *RNA, Guide, CRISPR-Cas Systems ; *Hair Diseases/genetics ; Hair ; Mutation/genetics ; },
abstract = {Hypotrichosis simplex (HS) and woolly hair (WH) are rare and monogenic disorders of hair loss. HS, characterized by a diffuse loss of hair, usually begins in early childhood and progresses into adulthood. WH displays strong coiled hair involving a localized area of the scalp or covering the entire side. Mutations in the keratin K71(KRT71) gene have been reported to underlie HS and WH. Here, we report the generation of a mouse model of HS and WH by the co-injection of Cas9 mRNA and sgRNA, targeting exon6 into mouse zygotes. The Krt71-knockout (KO) mice displayed the typical phenotypes, including Krt71 protein expression deletion and curly hair in their full body. Moreover, we found that mice in 3-5 weeks showed a new phenomenon of the complete shedding of hair, which was similar to nude mice. However, we discovered that the mice exhibited no immune deficiency, which was a typical feature of nude mice. To our knowledge, this novel mouse model generated by the CRISPR/Cas9 system mimicked woolly hair and could be valuable for hair disorder studies.},
}
@article {pmid37443646,
year = {2023},
author = {Chen, SJ and Rai, CI and Wang, SC and Chen, YC},
title = {Point-of-Care Testing for Infectious Diseases Based on Class 2 CRISPR/Cas Technology.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {13},
number = {13},
pages = {},
pmid = {37443646},
issn = {2075-4418},
abstract = {The early detection of infectious diseases and microorganisms is critical for effective disease treatment, control, and prevention. Currently, nucleic acid testing and antigen-antibody serum reaction are the two methods most commonly used for the detection of infectious diseases. The former is highly accurate, specific, and sensitive, but it is time-consuming, expensive, and has special technician and instrument requirements. The latter is rapid and economical, but it may not be accurate and sensitive enough. Therefore, it is necessary to develop a quick and on-site diagnostic test for point-of-care testing (POCT) to enable the clinical detection of infectious diseases that is accurate, sensitive, convenient, cheap, and portable. Here, CRISPR/Cas-based detection methods are detailed and discussed in depth. The powerful capacity of these methods will facilitate the development of diagnostic tools for POCT, though they still have some limitations. This review explores and highlights POCT based on the class 2 CRISPR/Cas assay, such as Cas12 and Cas13 proteins, for the detection of infectious diseases. We also provide an outlook on perspectives, multi-application scenarios, clinical applications, and limitations for POCT based on class 2 CRISPR/Cas technology.},
}
@article {pmid37443005,
year = {2023},
author = {Dirkx, N and Weuring, WJ and De Vriendt, E and Smal, N and van de Vondervoort, J and van 't Slot, R and Koetsier, M and Zonnekein, N and De Pooter, T and Weckhuysen, S and Koeleman, BPC},
title = {Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {156},
pmid = {37443005},
issn = {1741-7007},
support = {1616091 (MING)//Vrienden WKZ/ ; 1861419N//FWO-FKM/ ; G041821N//FWO-FKM/ ; 1S59221N//FWO-SB/ ; TreatKCNQ//European Joint Programme on Rare Disease JTC 2020/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Peptide Elongation Factor 1/genetics ; Plasmids/genetics ; *RNA, Small Untranslated ; KCNQ2 Potassium Channel/genetics ; NAV1.1 Voltage-Gated Sodium Channel/genetics ; },
abstract = {BACKGROUND: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.<br><br>RESULTS: pAIO-EF1&#x3b1;-PE2 and pAIO-EF1&#x3b1;-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1&#x3b1;-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1&#x3b1;-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.<br><br>CONCLUSION: Taken together, our study shows an improved efficacy of EF-1&#x3b1; driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.},
}
@article {pmid37442412,
year = {2023},
author = {Wang, F and Zhang, C and Xu, H and Zeng, W and Ma, L and Li, Z},
title = {Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168197},
doi = {10.1016/j.jmb.2023.168197},
pmid = {37442412},
issn = {1089-8638},
abstract = {The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a failed to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.},
}
@article {pmid37441240,
year = {2023},
author = {Guo, Y and Xia, H and Dai, T and Liu, T and Shamoun, SF and CuiPing, W},
title = {CRISPR/Cas12a-based approaches for efficient and accurate detection of Phytophthora ramorum.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1218105},
pmid = {37441240},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Phytophthora/genetics ; DNA ; Europe ; North America ; },
abstract = {INTRODUCTION: Phytophthora ramorum is a quarantine pathogen that causes leaf blight and shoot dieback of the crown, bark cankers and death on a number of both ornamental and forest trees, especially in North America and northern Europe, where it has produced severe outbreaks. Symptoms caused by P. ramorum can be confused with those by other Phytophthora and fungal species. Early and accurate detection of the causal pathogen P. ramorum is crucial for effective prevention and control of Sudden Oak Death.<br><br>METHODS: In this study, we developed a P. ramorum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPACRISPR/ Cas12a).<br><br>RESULTS: This novel method can be utilized for the molecular identification of P. ramorum under UV light and readout coming from fluorophores, and can specifically detect P. ramorum at DNA concentrations as low as 100 pg within 25 min at 37&#xb0;C.<br><br>DISCUSSION: We have developed a simple, rapid, sensitive, unaided-eye visualization, RPA CRISPR/Cas12a-based detection system for the molecular identification of P. ramorum that does not require technical expertise or expensive ancillary equipment. And this system is sensitive for both standard laboratory samples and samples from the field.},
}
@article {pmid37440893,
year = {2023},
author = {Brenner, E and Sreevatsan, S},
title = {Cold Cas: reevaluating the occurrence of CRISPR/Cas systems in Mycobacteriaceae.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1204838},
pmid = {37440893},
issn = {1664-302X},
abstract = {Bacterial CRISPR/Cas systems target foreign genetic elements such as phages and regulate gene expression by some pathogens, even in the host. The system is a marker for evolutionary history and has been used for inferences in Mycobacterium tuberculosis for 30 years. However, knowledge about mycobacterial CRISPR/Cas systems remains limited. It is believed that Type III-A Cas systems are exclusive to Mycobacterium canettii and the M. tuberculosis complex (MTBC) of organisms and that very few of the >200 diverse species of non-tuberculous mycobacteria (NTM) possess any CRISPR/Cas system. This study sought unreported CRISPR/Cas loci across NTM to better understand mycobacterial evolution, particularly in species phylogenetically near the MTBC. An analysis of available mycobacterial genomes revealed that Cas systems are widespread across Mycobacteriaceae and that some species contain multiple types. The phylogeny of Cas loci shows scattered presence in many NTM, with variation even within species, suggesting gains/losses of these loci occur frequently. Cas Type III-A systems were identified in pathogenic Mycobacterium heckeshornense and the geological environmental isolate Mycobacterium SM1. In summary, mycobacterial CRISPR/Cas systems are numerous, Type III-A systems are unreliable as markers for MTBC evolution, and mycobacterial horizontal gene transfer appears to be a frequent source of genetic variation.},
}
@article {pmid37440088,
year = {2023},
author = {da Costa, BL and Li, Y and Levi, SR and Tsang, SH and Quinn, PMJ},
title = {Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G&gt;T Mutation.},
journal = {Advances in experimental medicine and biology},
volume = {1415},
number = {},
pages = {571-576},
pmid = {37440088},
issn = {0065-2598},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; DNA Copy Number Variations ; *Retinitis Pigmentosa/genetics/therapy ; Mutation ; Eye Proteins/genetics ; Membrane Proteins/genetics ; Nerve Tissue Proteins/genetics ; },
abstract = {Mutations in the Crumbs-homologue-1 (CRB1) gene lead to a spectrum of severe inherited retinal diseases, including retinitis pigmentosa (RP). The establishment of a genotype-phenotype correlation in CRB1 patients has been difficult due to the substantial variability and phenotypic overlap between CRB1-associated diseases. This phenotypic modulation may be due to several factors, including genetic modifiers, deep intronic mutations, isoform diversity, and copy number variations. Induced pluripotent stem cell (iPSC)-derived patient retinal organoids are novel tools that can provide sensitive, quantitative, and scalable phenotypic assays. CRB1 RP patient iPSC-derived retinal organoids have shown reproducible phenotypes compared to healthy retinal organoids. However, having genetically defined iPSC isogenic controls that take into account potential phenotypic modulation is crucial. In this study, we generated iPSC from an early-onset CRB1 patient and developed a correction strategy for the c.2480G>T, p.(Gly827Val) CRB1 mutation using CRISPR/Cas9-mediated homology-directed repair.},
}
@article {pmid37440022,
year = {2023},
author = {Tsai, YT and da Costa, BL and Caruso, SM and Nolan, ND and Levi, SR and Tsang, SH and Quinn, PMJ},
title = {Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.},
journal = {Advances in experimental medicine and biology},
volume = {1415},
number = {},
pages = {109-114},
pmid = {37440022},
issn = {0065-2598},
mesh = {Humans ; Animals ; Mice ; *RNA-Directed DNA Polymerase/genetics/metabolism ; *Avian Myeloblastosis Virus/genetics/metabolism ; HEK293 Cells ; Gene Editing ; Moloney murine leukemia virus/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.},
}
@article {pmid37439822,
year = {2023},
author = {Durán-Vinet, B and Araya-Castro, K and Zaiko, A and Pochon, X and Wood, SA and Stanton, JL and Jeunen, GJ and Scriver, M and Kardailsky, A and Chao, TC and Ban, DK and Moarefian, M and Aran, K and Gemmell, NJ},
title = {CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2023.0019},
pmid = {37439822},
issn = {2573-1602},
abstract = {Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.},
}
@article {pmid37438488,
year = {2023},
author = {Sowbhagya, R and Muktha, H and Ramakrishnaiah, TN and Surendra, AS and Tanvi, Y and Nivitha, K and Rajashekara, S},
title = {CRISPR/Cas-mediated genome editing in mice for the development of drug delivery mechanism.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {37438488},
issn = {1573-4978},
abstract = {BACKGROUND: To manipulate particular locations in the bacterial genome, researchers have recently resorted to a group of unique sequences in bacterial genomes that are responsible for safeguarding bacteria against bacteriophages. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are two such systems, each of which consists of an RNA component and an enzyme component.<br><br>METHODS AND RESULTS: This review focuses primarily on how CRISPR/Cas9 technology can be used to make models to study human diseases in mice. Creating RNA molecules that direct endonucleases to a specific position in the genome are crucial for achieving a specific genetic modification. CRISPR/Cas9 technology has allowed scientists to edit the genome with greater precision than ever before. Researchers can use knock-in and knock-out methods to model human diseases such as Neurological, cardiovascular disease, and cancer.<br><br>CONCLUSIONS: In terms of developing innovative methods to discover ailments for diseases/disorders, improved CRISPR/Cas9 technology will provide easier access to valuable novel animal models.},
}
@article {pmid37437865,
year = {2023},
author = {Nayak, V and Patra, S and Singh, KR and Ganguly, B and Kumar, DN and Panda, D and Maurya, GK and Singh, J and Majhi, S and Sharma, R and Pandey, SS and Singh, RP and Kerry, RG},
title = {Advancement in precision diagnosis and therapeutic for triple-negative breast cancer: Harnessing diagnostic potential of CRISPR-cas &amp; engineered CAR T-cells mediated therapeutics.},
journal = {Environmental research},
volume = {235},
number = {},
pages = {116573},
doi = {10.1016/j.envres.2023.116573},
pmid = {37437865},
issn = {1096-0953},
abstract = {Cancer is characterized by uncontrolled cell growth, disrupted regulatory pathways, and the accumulation of genetic mutations. These mutations across different types of cancer lead to disruptions in signaling pathways and alterations in protein expression related to cellular growth and proliferation. This review highlights the AKT signaling cascade and the retinoblastoma protein (pRb) regulating cascade as promising for novel nanotheranostic interventions. Through synergizing state-of-the-art gene editing tools like the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system with nanomaterials and targeting AKT, there is potential to enhance cancer diagnostics significantly. Furthermore, the integration of modified CAR-T cells into multifunctional nanodelivery systems offers a promising approach for targeted cancer inhibition, including the eradication of cancer stem cells (CSCs). Within the context of highly aggressive and metastatic Triple-negative Breast Cancer (TNBC), this review specifically focuses on devising innovative nanotheranostics. For both pre-clinical and post-clinical TNBC detection, the utilization of the CRISPR-Cas system, guided by RNA (gRNA) and coupled with a fluorescent reporter specifically designed to detect TNBC's mutated sequence, could be promising. Additionally, a cutting-edge approach involving the engineering of TNBC-specific iCAR and syn-Notch CAR T-cells, combined with the co-delivery of a hybrid polymeric nano-liposome encapsulating a conditionally replicative adenoviral vector (CRAdV) against CSCs, could present an intriguing intervention strategy. This review thus paves the way for exciting advancements in the field of nanotheranostics for the treatment of TNBC and beyond.},
}
@article {pmid37436979,
year = {2023},
author = {Hansen, SL and Larsen, HL and Pikkupeura, LM and Maciag, G and Guiu, J and Müller, I and Clement, DL and Mueller, C and Johansen, JV and Helin, K and Lerdrup, M and Jensen, KB},
title = {An organoid-based CRISPR-Cas9 screen for regulators of intestinal epithelial maturation and cell fate.},
journal = {Science advances},
volume = {9},
number = {28},
pages = {eadg4055},
pmid = {37436979},
issn = {2375-2548},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Fetus ; *Adult Stem Cells ; Organoids ; },
abstract = {Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.},
}
@article {pmid37436915,
year = {2023},
author = {Wei, Y and Feng, LJ and Yuan, XZ and Wang, SG and Xia, PF},
title = {Developing a Base Editing System for Marine Roseobacter Clade Bacteria.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00259},
pmid = {37436915},
issn = {2161-5063},
abstract = {The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the β-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.},
}
@article {pmid37435997,
year = {2023},
author = {Calorenni, P and Leonardi, AA and Sciuto, EL and Rizzo, MG and Faro, MJL and Fazio, B and Irrera, A and Conoci, S},
title = {PCR-Free Innovative Strategies for SARS-CoV-2 Detection.},
journal = {Advanced healthcare materials},
volume = {},
number = {},
pages = {e2300512},
doi = {10.1002/adhm.202300512},
pmid = {37435997},
issn = {2192-2659},
abstract = {The pandemic outbreak caused by SARS-CoV-2 coronavirus brought a crucial issue in Public Health causing up to now more than 600 million of infected people and 6.5 million of deaths. Conventional diagnostic methods are based on quantitative reverse transcription polymerase chain reaction (RT-qPCR assay) and immuno-detection (ELISA assay). However, despite these techniques have the advantages of being standardized and consolidated, they keep some main limitations in terms of accuracy (immunoassays), time/cost consumption of analysis, the need for qualified personnel and lab constrain (molecular assays). There is crucial the need to develop new diagnostic approaches for accurate, fast and portable viral detection and quantification. Among these, PCR-free biosensors represent the most appealing solution since they can allow molecular detection without the complexity of the PCR. This will enable the possibility to be integrated in portable and low-cost systems for massive and decentralized screening of SARS-CoV-2 in a point-of-care (PoC) format, pointing to achieve a performant identification and control of infection. In this review, the most recent approaches for the SARS-CoV-2 PCR-free detection are reported, describing both the instrumental and methodological features, and highlighting their suitability for a PoC application. This article is protected by copyright. All rights reserved.},
}
@article {pmid37390772,
year = {2023},
author = {Keng, CT and Yogarajah, T and Lee, RCH and Muhammad, IBH and Chia, BS and Vasandani, SR and Lim, DS and Guo, K and Wong, YH and Mok, CK and Chu, JJH and Chew, WL},
title = {AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.},
journal = {EBioMedicine},
volume = {93},
number = {},
pages = {104682},
doi = {10.1016/j.ebiom.2023.104682},
pmid = {37390772},
issn = {2352-3964},
mesh = {Humans ; Mice ; Animals ; CRISPR-Cas Systems ; Dependovirus/genetics ; *COVID-19/genetics ; *Enterovirus/genetics ; *Enterovirus A, Human/genetics ; },
abstract = {BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice.<br><br>METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in&#xa0;vitro viral plaque assay and in&#xa0;vivo EV-A71 lethally-infected mouse model.<br><br>FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model.<br><br>INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections.<br><br>FUNDING: Agency for Science, Technology and Research (A&#x2217;STAR) Assured Research Budget, A&#x2217;STAR Central Research Fund UIBR SC18/21-1089UI, A&#x2217;STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.},
}
@article {pmid37353926,
year = {2023},
author = {Philippe, C and Cornuault, JK and de Melo, AG and Morin-Pelchat, R and Jolicoeur, AP and Moineau, S},
title = {The never-ending battle between lactic acid bacteria and their phages.},
journal = {FEMS microbiology reviews},
volume = {47},
number = {4},
pages = {},
doi = {10.1093/femsre/fuad035},
pmid = {37353926},
issn = {1574-6976},
support = {//Natural Sciences and Engineering Research Council of Canada/ ; },
abstract = {Over the past few decades, the interest in lactic acid bacteria (LAB) has been steadily growing. This is mainly due to their industrial use, their health benefits as probiotic bacteria and their ecological importance in host-related microbiota. Phage infection represents a significant risk for the production and industrial use of LAB. This created the need to study the various means of defense put in place by LAB to resist their viral enemies, as well as the countermeasures evolved by phages to overcome these defenses. In this review, we discuss defense systems that LAB employ to resist phage infections. We also describe how phages counter these mechanisms through diverse and sophisticated strategies. Furthermore, we discuss the way phage-host interactions shape each other's evolution. The recent discovery of numerous novel defense systems in other bacteria promises a new dawn for phage research in LAB.},
}
@article {pmid37343658,
year = {2023},
author = {Fatma, Z and Tan, SI and Boob, AG and Zhao, H},
title = {A landing pad system for multicopy gene integration in Issatchenkia orientalis.},
journal = {Metabolic engineering},
volume = {78},
number = {},
pages = {200-208},
doi = {10.1016/j.ymben.2023.06.010},
pmid = {37343658},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Saccharomyces cerevisiae/genetics ; Gene Editing/methods ; Succinates ; },
abstract = {The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user's choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.},
}
@article {pmid37279759,
year = {2023},
author = {Tian, R and Cao, C and He, D and Dong, D and Sun, L and Liu, J and Chen, Y and Wang, Y and Huang, Z and Li, L and Jin, Z and Huang, Z and Xie, H and Zhao, T and Zhong, C and Hong, Y and Hu, Z},
title = {Massively parallel CRISPR off-target detection enables rapid off-target prediction model building.},
journal = {Med (New York, N.Y.)},
volume = {4},
number = {7},
pages = {478-492.e6},
doi = {10.1016/j.medj.2023.05.005},
pmid = {37279759},
issn = {2666-6340},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Papillomavirus Infections/genetics ; Gene Editing/methods ; Genome ; },
abstract = {BACKGROUND: CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds tremendous potential in clinical translation. However, the off-target effect has always been a major concern.<br><br>METHODS: Here, we have developed a novel sensitive and specific off-target detection method, AID-seq (adaptor-mediated off-target identification by sequencing), that can comprehensively and faithfully detect the low-frequency off targets generated by different CRISPR nucleases (including Cas9 and Cas12a).<br><br>FINDINGS: Based on AID-seq, we developed a pooled strategy to simultaneously identify the on/off targets of multiple gRNAs, as well as using mixed human and human papillomavirus (HPV) genomes to screen the most efficient and safe targets from 416 HPV gRNA candidates for antiviral therapy. Moreover, we used the pooled strategy with 2,069 single-guide RNAs (sgRNAs) at a pool size of about 500 to profile the properties of our newly discovered CRISPR, FrCas9. Importantly, we successfully built an off-target detection model using these off-target data via the CRISPR-Net deep learning method (area under the receiver operating characteristic curve [AUROC]&#xa0;= 0.97, area under the precision recall curve [AUPRC]&#xa0;= 0.29).<br><br>CONCLUSIONS: To our knowledge, AID-seq is the most sensitive and specific in&#xa0;vitro off-target detection method to date. And the pooled AID-seq strategy can be used as a rapid and high-throughput platform to select the best sgRNAs and characterize the properties of new CRISPRs.<br><br>FUNDING: This work was supported by The National Natural Science Foundation of China (grant nos. 32171465 and 82102392), the General Program of Natural Science Foundation of Guangdong Province of China (grant no. 2021A1515012438), Guangdong Basic and Applied Basic Research Foundation (grant no. 2020A1515110170), and the National Ten Thousand Plan-Young Top Talents of China (grant no. 80000-41180002).},
}
@article {pmid37263857,
year = {2023},
author = {Qin, H and Xu, W and Yao, K},
title = {CRISPR-based genome editing in disease treatment.},
journal = {Trends in molecular medicine},
volume = {29},
number = {8},
pages = {673-674},
doi = {10.1016/j.molmed.2023.05.003},
pmid = {37263857},
issn = {1471-499X},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid37244368,
year = {2023},
author = {Chen, JP and Gong, JS and Su, C and Li, H and Xu, ZH and Shi, JS},
title = {Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.},
journal = {Metabolic engineering},
volume = {78},
number = {},
pages = {99-114},
doi = {10.1016/j.ymben.2023.05.007},
pmid = {37244368},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Synthetic Biology ; Gene Editing ; Protein Engineering ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins. Accounting for the accumulated knowledge of the pivotal factors in the solubility and folding stability of proteins, this review focuses on advanced technologies and tools of protein engineering, protein quality control systems, and the redesign of expression platforms in prokaryotic expression systems, as well as advances of the cell-free expression technologies for membrane proteins production.},
}
@article {pmid37236858,
year = {2023},
author = {Pan, C and Qi, Y},
title = {PrimeRoot for targeted large DNA insertion in plants.},
journal = {Trends in plant science},
volume = {28},
number = {8},
pages = {870-872},
doi = {10.1016/j.tplants.2023.05.002},
pmid = {37236858},
issn = {1878-4372},
mesh = {*Plant Breeding ; *Plants/genetics ; Gene Editing ; Genome, Plant/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized plant breeding through targeted genome and transcriptome modifications. However, accurate insertion of large DNA cargoes remains challenging. Recently, Sun and colleagues introduced PrimeRoot, a groundbreaking technology that enables precise and targeted integration of large DNA cargoes into plant genomes with remarkable efficiency and accuracy.},
}
@article {pmid36967299,
year = {2023},
author = {Tanaka, M and Nakamura, T},
title = {Targeting epigenetic aberrations of sarcoma in CRISPR era.},
journal = {Genes, chromosomes &amp; cancer},
volume = {62},
number = {9},
pages = {510-525},
doi = {10.1002/gcc.23142},
pmid = {36967299},
issn = {1098-2264},
support = {17cmA106609//Japan Agency for Medical Research and Development/ ; 20cm0106277//Japan Agency for Medical Research and Development/ ; 22ama221206//Japan Agency for Medical Research and Development/ ; 21ak0101170//Japan Agency for Medical Research and Development/ ; 26250029//Japan Society for the Promotion of Science/ ; 16KM07131//Japan Society for the Promotion of Science/ ; 19K07702//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Methylation ; *Sarcoma/genetics ; Epigenesis, Genetic ; Tumor Microenvironment ; },
abstract = {Sarcomas are rare malignancies that exhibit diverse biological, genetic, morphological, and clinical characteristics. Genetic alterations, such as gene fusions, mutations in transcriptional machinery components, histones, and DNA methylation regulatory molecules, play an essential role in sarcomagenesis. These mutations induce and/or cooperate with specific epigenetic aberrations required for the growth and maintenance of sarcomas. Appropriate mouse models have been developed to clarify the significance of genetic and epigenetic interactions in sarcomas. Studies using the mouse models for human sarcomas have demonstrated major advances in our understanding the developmental processes as well as tumor microenvironment of sarcomas. Recent technological progresses in epigenome editing will not only improve the studies using animal models but also provide a direct clue for epigenetic therapies. In this manuscript, we review important epigenetic aberrations in sarcomas and their representative mouse models, current methods of epigenetic editing using CRISPR/dCas9 systems, and potential applications in sarcoma studies and therapeutics.},
}
@article {pmid36965130,
year = {2023},
author = {Vanoli, F and Antonescu, CR},
title = {Modeling sarcoma relevant translocations using CRISPR-Cas9 in human embryonic stem derived mesenchymal precursors.},
journal = {Genes, chromosomes &amp; cancer},
volume = {62},
number = {9},
pages = {501-509},
doi = {10.1002/gcc.23141},
pmid = {36965130},
issn = {1098-2264},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA217694/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA217694/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Translocation, Genetic ; CRISPR-Cas Systems ; *Sarcoma/genetics ; Gene Editing ; Gene Rearrangement ; },
abstract = {The role of cancer relevant translocations in tumorigenesis has been historically hampered by the lack of faithful in vitro and in vivo models. The development of the latest genome editing tools (e.g., CRISPR-Cas9) allowed modeling of various chromosomal translocations with different effects on proliferation and transformation capacity depending on the cell line used and secondary genetic alterations. The cellular context is particularly relevant in the case of oncogenic fusions expressed in sarcomas whose histogenesis remain uncertain. Moreover, recent studies have emphasized the increased frequency of gene fusion promiscuity across different mesenchymal tumor entities, which are clinicopathologically unrelated. This review provides a summary of different strategies utilized to generate cancer models with a focus on fusion-driven mesenchymal neoplasia.},
}
@article {pmid36959711,
year = {2023},
author = {Draper, GM and Panken, DJ and Largaespada, DA},
title = {Modeling human cancer predisposition syndromes using CRISPR/Cas9 in human cell line models.},
journal = {Genes, chromosomes &amp; cancer},
volume = {62},
number = {9},
pages = {493-500},
doi = {10.1002/gcc.23140},
pmid = {36959711},
issn = {1098-2264},
support = {R01 NS115438/NS/NINDS NIH HHS/United States ; UH3 CA244687/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Syndrome ; Gene Editing ; Disease Susceptibility ; Cell Line ; *Neoplasms/genetics ; },
abstract = {The advancement of CRISPR mediated gene engineering provides an opportunity to improve upon preclinical human cell line models of cancer predisposing syndromes. This review focuses on using CRISPR/Cas9 genome editing tools to model various human cancer predisposition syndromes. We examine the genetic mutations associated with neurofibromatosis type 1, Li-Fraumeni syndrome, Gorlin syndrome, BRCA mutant breast and ovarian cancers, and APC mutant cancers. Furthermore, we discuss the possibilities of using next-generation CRISPR-derived precision gene editing tools to introduce a variety of genetic lesions into human cell lines. The goal is to improve the quality of preclinical models surrounding these cancer predisposition syndromes through dissecting the effects of these mutations on the development of cancer and to provide new insights into the underlying mechanisms of these cancer predisposition syndromes. These studies demonstrate the continued utility and improvement of CRISPR/Cas9-induced human cell line models in studying the genetic basis of cancer.},
}
@article {pmid36593416,
year = {2023},
author = {Hu, J and Gao, C},
title = {CRISPR-edited plants by grafting.},
journal = {Nature biotechnology},
volume = {41},
number = {7},
pages = {909-910},
pmid = {36593416},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Plants ; CRISPR-Cas Systems/genetics ; Gene Editing ; Plants, Genetically Modified/genetics ; },
}
@article {pmid36593415,
year = {2023},
author = {Yang, L and Machin, F and Wang, S and Saplaoura, E and Kragler, F},
title = {Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks.},
journal = {Nature biotechnology},
volume = {41},
number = {7},
pages = {958-967},
pmid = {36593415},
issn = {1546-1696},
support = {810131//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 810131//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; Transgenes/genetics ; },
abstract = {Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a lengthy process of outcrossing to eliminate CRISPR-Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.},
}
@article {pmid36593413,
year = {2023},
author = {Tou, CJ and Orr, B and Kleinstiver, BP},
title = {Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases.},
journal = {Nature biotechnology},
volume = {41},
number = {7},
pages = {968-979},
pmid = {36593413},
issn = {1546-1696},
support = {2020295403//National Science Foundation (NSF)/ ; Howard M. Goodman Fellowship//Massachusetts General Hospital (MGH)/ ; },
mesh = {Humans ; *DNA Transposable Elements ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transposases/genetics/metabolism ; Plasmids ; Endonucleases/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5' nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.},
}
@article {pmid35932337,
year = {2023},
author = {de Mélo, AHF and Nunes, AL and Carvalho, PH and da Silva, MF and Teixeira, GS and Goldbeck, R},
title = {Evaluation of Saccharomyces cerevisiae modified via CRISPR/Cas9 as a cellulosic platform microorganism in simultaneously saccharification and fermentation processes.},
journal = {Bioprocess and biosystems engineering},
volume = {46},
number = {8},
pages = {1111-1119},
pmid = {35932337},
issn = {1615-7605},
support = {001//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 307014/2020-7//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 2015/20630-4//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2016/04602-3//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/08542-7//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; },
mesh = {*Cellulose/metabolism ; Fermentation ; Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems ; *Saccharum/metabolism ; Ethanol/metabolism ; },
abstract = {The nonrenewable character and deleterious effects of fossil fuels foster the need for cleaner and more inexhaustible energy sources, such as bioethanol. Especially from lignocellulosic biomasses. However, the economic viability of this product in the market depends on process optimization and cost reduction. This research applied a sequential experimental project to investigate the process of enzymatic saccharification and simultaneous fermentation to produce ethanol with sugarcane bagasse. The differential of the work was the application of the strain of Saccharomyces cerevisiae AGY001 which was improved by evolutionary engineering to become thermotolerant and by a heterologous expression based on genomic integration by CRISPR/Cas9 to produce endoglucanase and β-glucosidase (AsENDO-AsBGL). The maximum ethanol yield found was 89% of the maximum theoretical yield (released sugars), obtained at temperature concentrations, sugarcane bagasse and inoculum at 40 °C, 16.5%, and 4.0 g/L, respectively (12.5 FPU/g bagasse). The mathematical model obtained can predict approximately 83% of the data set with 95% confidence. Therefore, these findings demonstrated the potential of sugarcane bagasse and S. cerevisiae AGY001 strain (CRISPR/Cas9 modified) in bioethanol production without the need for impractical selection media on an industrial scale, in addition to providing useful insights for the development of SSF processes.},
}
@article {pmid37435043,
year = {2023},
author = {Dorset, SR and Bak, RO},
title = {The p53 challenge of hematopoietic stem cell gene editing.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {30},
number = {},
pages = {83-89},
pmid = {37435043},
issn = {2329-0501},
abstract = {Ex vivo gene editing in hematopoietic stem and progenitor cells (HSPCs) represents a promising curative treatment strategy for monogenic blood disorders. Gene editing using the homology-directed repair (HDR) pathway enables precise genetic modifications ranging from single base pair correction to replacement or insertion of large DNA segments. Hence, HDR-based gene editing could facilitate broad application of gene editing across monogenic disorders, but the technology still faces challenges for clinical translation. Among these, recent studies demonstrate induction of a DNA damage response (DDR) and p53 activation caused by DNA double-strand breaks and exposure to recombinant adeno-associated virus vector repair templates, resulting in reduced proliferation, engraftment, and clonogenic capacity of edited HSPCs. While different mitigation strategies can reduce this DDR, more research is needed on this phenomenon to ensure safe and efficient implementation of HDR-based gene editing in the clinic.},
}
@article {pmid37434184,
year = {2023},
author = {Qian, Y and Wang, D and Niu, W and Zhao, D and Li, J and Liu, Z and Gao, X and Han, Y and Lai, L and Li, Z},
title = {A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {155},
pmid = {37434184},
issn = {1741-7007},
support = {31970574//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Humans ; Mice ; Adenine ; HEK293 Cells ; *Proprotein Convertase 9 ; *RNA, Guide, CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size.<br><br>RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (&#x394;174-296) and HNH (&#x394;786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes.<br><br>CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.},
}
@article {pmid37434066,
year = {2023},
author = {Liang, Y and Chen, F and Wang, K and Lai, L},
title = {Base editors: development and applications in biomedicine.},
journal = {Frontiers of medicine},
volume = {},
number = {},
pages = {},
pmid = {37434066},
issn = {2095-0225},
abstract = {Base editor (BE) is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase, enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break (DSB) or requiring donor DNA templates in living cells. Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems, such as CRISPR/Cas9, as the DSB induced by Cas9 will cause severe damage to the genome. Thus, base editors have important applications in the field of biomedicine, including gene function investigation, directed protein evolution, genetic lineage tracing, disease modeling, and gene therapy. Since the development of the two main base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), scientists have developed more than 100 optimized base editors with improved editing efficiency, precision, specificity, targeting scope, and capacity to be delivered in vivo, greatly enhancing their application potential in biomedicine. Here, we review the recent development of base editors, summarize their applications in the biomedical field, and discuss future perspectives and challenges for therapeutic applications.},
}
@article {pmid37429890,
year = {2023},
author = {Eom, H and Choi, YJ and Nandre, R and Han, HG and Kim, S and Kim, M and Oh, YL and Nakazawa, T and Honda, Y and Ro, HS},
title = {The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11133},
pmid = {37429890},
issn = {2045-2322},
mesh = {*Reishi/genetics ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Plant Breeding ; DNA, Mitochondrial ; *Agaricales ; Ribonucleoproteins/genetics ; },
abstract = {Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.},
}
@article {pmid37429857,
year = {2023},
author = {Cullot, G and Boutin, J and Fayet, S and Prat, F and Rosier, J and Cappellen, D and Lamrissi, I and Pennamen, P and Bouron, J and Amintas, S and Thibault, C and Moranvillier, I and Laharanne, E and Merlio, JP and Guyonnet-Duperat, V and Blouin, JM and Richard, E and Dabernat, S and Moreau-Gaudry, F and Bedel, A},
title = {Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4072},
pmid = {37429857},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Tumor Suppressor Protein p53/genetics ; Cell Cycle Checkpoints/genetics ; Cell Division ; Cell Separation ; RNA ; },
abstract = {The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.},
}
@article {pmid37306056,
year = {2023},
author = {Impens, L and Lorenzo, CD and Vandeputte, W and Wytynck, P and Debray, K and Haeghebaert, J and Herwegh, D and Jacobs, TB and Ruttink, T and Nelissen, H and Inzé, D and Pauwels, L},
title = {Combining multiplex gene editing and doubled haploid technology in maize.},
journal = {The New phytologist},
volume = {239},
number = {4},
pages = {1521-1532},
doi = {10.1111/nph.19021},
pmid = {37306056},
issn = {1469-8137},
mesh = {*Gene Editing ; *Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Haploidy ; Plants, Genetically Modified ; },
abstract = {A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.},
}
@article {pmid36596724,
year = {2023},
author = {Kim, JS and Lee, JH and Jeon, SR and Kim, Y and Jeon, SH and Wu, HG},
title = {Identification of Genes Involved in EGF-induced Apoptosis Using CRISPR/Cas9 Knockout Screening: Implications for Novel Therapeutic Targets in EGFR-Overexpressing Cancers.},
journal = {Cancer research and treatment},
volume = {55},
number = {3},
pages = {737-745},
doi = {10.4143/crt.2022.1414},
pmid = {36596724},
issn = {2005-9256},
support = {2019R1F1A1040583//National Research Foundation of Korea/ ; 2020M2D9A2092373//National Research Foundation of Korea/ ; 2021R1A2C1095168//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; },
mesh = {Humans ; *Epidermal Growth Factor/genetics/pharmacology ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Early Detection of Cancer ; ErbB Receptors/genetics/metabolism ; *Neoplasms/drug therapy/genetics ; Apoptosis/genetics ; Calcium-Binding Proteins/genetics/metabolism ; },
abstract = {PURPOSE: Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)-overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen.<br><br>MATERIALS AND METHODS: Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription-quantitative polymerase chain reaction.<br><br>RESULTS: We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment.<br><br>CONCLUSION: The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.},
}
@article {pmid37427945,
year = {2023},
author = {Xu, MF and Chen, J and Xu, Y and Zhang, JL and Zhou, Y and He, JJ and Wu, S and Wei, YL and She, ZY},
title = {Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {196},
pages = {},
doi = {10.3791/65476},
pmid = {37427945},
issn = {1940-087X},
mesh = {Humans ; HeLa Cells ; *Protein Serine-Threonine Kinases/genetics ; *CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Kinetochores/metabolism ; Mitosis/genetics ; Spindle Apparatus/metabolism ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E[-/-] HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors. In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.},
}
@article {pmid37427920,
year = {2023},
author = {Grausam, KB and Breunig, JJ},
title = {Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {196},
pages = {},
doi = {10.3791/65286},
pmid = {37427920},
issn = {1940-087X},
mesh = {Mice ; Animals ; *Neural Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Electroporation/methods ; Plasmids/genetics ; *Brain Neoplasms/genetics/therapy/metabolism ; DNA/genetics ; Mutation ; },
abstract = {Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.},
}
@article {pmid37426982,
year = {2023},
author = {Choudhary, N and Tandi, D and Verma, RK and Yadav, VK and Dhingra, N and Ghosh, T and Choudhary, M and Gaur, RK and Abdellatif, MH and Gacem, A and Eltayeb, LB and Alqahtani, MS and Yadav, KK and Jeon, BH},
title = {A comprehensive appraisal of mechanism of anti-CRISPR proteins: an advanced genome editor to amend the CRISPR gene editing.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1164461},
pmid = {37426982},
issn = {1664-462X},
abstract = {The development of precise and controlled CRISPR-Cas tools has been made possible by the discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs). The Acr protein has the ability to control off-targeted mutations and impede Cas protein-editing operations. Acr can help with selective breeding, which could help plants and animals improve their valuable features. In this review, the Acr protein-based inhibitory mechanisms that have been adopted by several Acrs, such as (a) the interruption of CRISPR-Cas complex assembly, (b) interference with target DNA binding, (c) blocking of target DNA/RNA cleavage, and (d) enzymatic modification or degradation of signalling molecules, were discussed. In addition, this review emphasizes the applications of Acr proteins in the plant research.},
}
@article {pmid37343558,
year = {2023},
author = {Ahuja, N and Hwaun, E and Pungor, JR and Rafiq, R and Nemes, S and Sakmar, T and Vogt, MA and Grasse, B and Diaz Quiroz, J and Montague, TG and Null, RW and Dallis, DN and Gavriouchkina, D and Marletaz, F and Abbo, L and Rokhsar, DS and Niell, CM and Soltesz, I and Albertin, CB and Rosenthal, JJC},
title = {Creation of an albino squid line by CRISPR-Cas9 and its application for in vivo functional imaging of neural activity.},
journal = {Current biology : CB},
volume = {33},
number = {13},
pages = {2774-2783.e5},
doi = {10.1016/j.cub.2023.05.066},
pmid = {37343558},
issn = {1879-0445},
support = {R35 GM147273/GM/NIGMS NIH HHS/United States ; RF1 NS118466/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Decapodiformes/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Genome ; },
abstract = {Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca[2+] signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.},
}
@article {pmid37060177,
year = {2023},
author = {Cheng, Y and Zhang, Y and Li, G and Fang, H and Sretenovic, S and Fan, A and Li, J and Xu, J and Que, Q and Qi, Y},
title = {CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice.},
journal = {Plant communications},
volume = {4},
number = {4},
pages = {100601},
doi = {10.1016/j.xplc.2023.100601},
pmid = {37060177},
issn = {2590-3462},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; },
}
@article {pmid37056050,
year = {2023},
author = {Yu, L and Li, Z and Ding, X and Alariqi, M and Zhang, C and Zhu, X and Fan, S and Zhu, L and Zhang, X and Jin, S},
title = {Developing an efficient CRISPR-dCas9-TV-derived transcriptional activation system to create three novel cotton germplasm materials.},
journal = {Plant communications},
volume = {4},
number = {4},
pages = {100600},
doi = {10.1016/j.xplc.2023.100600},
pmid = {37056050},
issn = {2590-3462},
mesh = {Transcriptional Activation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid37426019,
year = {2023},
author = {Fernandes, LGV and Teixeira, AF and Nascimento, ALTO},
title = {Evaluation of Leptospira interrogans knockdown mutants for LipL32, LipL41, LipL21, and OmpL1 proteins.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1199660},
pmid = {37426019},
issn = {1664-302X},
abstract = {INTRODUCTION: Leptospirosis is a worldwide zoonosis caused by pathogenic and virulent species of the genus Leptospira, whose pathophysiology and virulence factors remain widely unexplored. Recently, the application of CRISPR interference (CRISPRi) has allowed the specific and rapid gene silencing of major leptospiral proteins, favoring the elucidation of their role in bacterial basic biology, host-pathogen interaction and virulence. Episomally expressed dead Cas9 from the Streptococcus pyogenes CRISPR/Cas system (dCas9) and single-guide RNA recognize and block transcription of the target gene by base pairing, dictated by the sequence contained in the 5' 20-nt sequence of the sgRNA.<br><br>METHODS: In this work, we tailored plasmids for silencing the major proteins of L. interrogans serovar Copenhageni strain Fiocruz L1-130, namely LipL32, LipL41, LipL21 and OmpL1. Double- and triple-gene silencing by in tandem sgRNA cassettes were also achieved, despite plasmid instability.<br><br>RESULTS: OmpL1 silencing resulted in a lethal phenotype, in both L. interrogans and saprophyte L. biflexa, suggesting its essential role in leptospiral biology. Mutants were confirmed and evaluated regarding interaction with host molecules, including extracellular matrix (ECM) and plasma components, and despite the dominant abundance of the studied proteins in the leptospiral membrane, protein silencing mostly resulted in unaltered interactions, either because they intrinsically display low affinity to the molecules assayed or by a compensation mechanism, where other proteins could be upregulated to fill the niche left by protein silencing, a feature previously described for the LipL32 mutant. Evaluation of the mutants in the hamster model confirms the augmented virulence of the LipL32 mutant, as hinted previously. The essential role of LipL21 in acute disease was demonstrated, since the LipL21 knockdown mutants were avirulent in the animal model, and even though mutants could still colonize the kidneys, they were found in markedly lower numbers in the animals' liver. Taking advantage of higher bacterial burden in LipL32 mutant-infected organs, protein silencing was demonstrated in vivo directly in leptospires present in organ homogenates.<br><br>DISCUSSION: CRISPRi is now a well-established, attractive genetic tool that can be applied for exploring leptospiral virulence factors, leading to the rational for designing more effective subunit or even chimeric recombinant vaccines.},
}
@article {pmid37425722,
year = {2023},
author = {Pallaseni, A and Peets, EM and Girling, G and Crepaldi, L and Kuzmin, I and Raudvere, U and Peterson, H and Serçin, &#xd6; and Mardin, BR and Kosicki, M and Parts, L},
title = {The interplay of DNA repair context with target sequence predictably biasses Cas9-generated mutations.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.06.28.546891},
pmid = {37425722},
abstract = {The genome engineering capability of the CRISPR/Cas system depends on the DNA repair machinery to generate the final outcome. Several genes can have an impact on mutations created, but their exact function and contribution to the result of the repair are not completely characterised. This lack of knowledge has limited the ability to comprehend and regulate the editing outcomes. Here, we measure how the absence of 21 repair genes changes the mutation outcomes of Cas9-generated cuts at 2,812 synthetic target sequences in mouse embryonic stem cells. Absence of key non-homologous end joining genes Lig4, Xrcc4, and Xlf abolished small insertions and deletions, while disabling key microhomology-mediated repair genes Nbn and Polq reduced frequency of longer deletions. Complex alleles of combined insertion and deletions were preferentially generated in the absence of Xrcc6. We further discover finer structure in the outcome frequency changes for single nucleotide insertions and deletions between large microhomologies that are differentially modulated by the knockouts. We use the knowledge of the reproducible variation across repair milieus to build predictive models of Cas9 editing results that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of CRISPR/Cas9-generated mutations.},
}
@article {pmid37425362,
year = {2023},
author = {Poulalier-Delavelle, M and Baker, JP and Millard, J and Winzer, K and Minton, NP},
title = {Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1213236},
pmid = {37425362},
issn = {2296-4185},
abstract = {Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction-modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems.},
}
@article {pmid37424467,
year = {2023},
author = {Miguel, L and Gervais, J and Nicolas, G and Lecourtois, M},
title = {SorLA Protective Function Is Restored by Improving SorLA Protein Maturation in a Subset of Alzheimer's Disease-Associated SORL1 Missense Variants.},
journal = {Journal of Alzheimer's disease : JAD},
volume = {},
number = {},
pages = {},
doi = {10.3233/JAD-230211},
pmid = {37424467},
issn = {1875-8908},
abstract = {SORL1 loss of function is associated with Alzheimer's disease (AD) risk through increased Aβ peptide secretion. We expressed 10 maturation-defective rare missense SORL1 variants in HEK cells and showed that decreasing growing temperature led to a significant increase in the maturation of the encoded protein SorLA for 6/10. In edited hiPSC carrying two of these variants, maturation of the protein was restored partially by decreasing the culture temperature and was associated with concomitant decrease in Aβ secretion. Correcting SorLA maturation in the context of maturation-defective missense variants could thus be a relevant strategy to improve SorLA protective function against AD.},
}
@article {pmid37424252,
year = {2023},
author = {Si, XX and Zhang, Q and Luo, ZD and Zhang, ZY and Wang, Y and Shao, LP},
title = {A Rapid and Accurate CRISPR/Cas12b-Mediated Genotyping Assay for the Methylenetetrahydrofolate Reductase Gene Polymorphism C677T.},
journal = {Biomedical and environmental sciences : BES},
volume = {36},
number = {6},
pages = {561-565},
doi = {10.3967/bes2023.070},
pmid = {37424252},
issn = {2214-0190},
mesh = {Humans ; *Methylenetetrahydrofolate Reductase (NADPH2)/genetics/metabolism ; Genotype ; *CRISPR-Cas Systems ; Polymorphism, Genetic ; Genetic Predisposition to Disease ; Polymorphism, Single Nucleotide ; Gene Frequency ; },
}
@article {pmid37422628,
year = {2023},
author = {Bahrami, E and Schmid, JP and Jurinovic, V and Becker, M and Wirth, AK and Ludwig, R and Kreissig, S and Duque Angel, TV and Amend, D and Hunt, K and Öllinger, R and Rad, R and Frenz, JM and Solovey, M and Ziemann, F and Mann, M and Vick, B and Wichmann, C and Herold, T and Jayavelu, AK and Jeremias, I},
title = {Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo.},
journal = {Molecular cancer},
volume = {22},
number = {1},
pages = {107},
pmid = {37422628},
issn = {1476-4598},
support = {DJCLS 10 R/2021//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung/ ; DJCLS 15 R/2021//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung/ ; 681524//European Research Council Consolidator Grant/ ; },
mesh = {Humans ; Mice ; Animals ; ADAM10 Protein/genetics/metabolism ; *Proteomics ; CRISPR-Cas Systems ; Membrane Proteins/genetics/metabolism ; *Leukemia/genetics ; Disease Models, Animal ; Tumor Microenvironment ; Amyloid Precursor Protein Secretases/genetics/metabolism ; },
abstract = {BACKGROUND: Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells.<br><br>METHODS: To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR&#x2012;Cas9 pipeline in PDX models in vivo.<br><br>RESULTS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo.<br><br>CONCLUSIONS: These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.},
}
@article {pmid37418936,
year = {2023},
author = {Guo, H and Zhang, Y and Kong, F and Wang, C and Chen, S and Wang, J and Wang, D},
title = {A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani.},
journal = {Ecotoxicology and environmental safety},
volume = {263},
number = {},
pages = {115220},
doi = {10.1016/j.ecoenv.2023.115220},
pmid = {37418936},
issn = {1090-2414},
abstract = {Alternaria solani (A. solani), the causal agent of early blight in potatoes, poses a serious and persistent threat to potato production worldwide. Therefore, developing a method that can accurately detect A. solani in the early stage to avoid further spread is urgent. However, the conventional PCR-based method is not appropriate for application in the fields. Recently, the CRISPR-Cas system has been developed for nucleic acids analysis at point-of-care. Here, we propose a gold nanoparticles-based visual assay combining loop-mediated isothermal amplification with CRISPR-Cas12a to detect A. solani. After optimization, the method could detect 10[-3] ng/μL genomic gene of A. solani. The specificity of the method was confirmed by discriminating A. solani from other three highly homologous pathogens. We also developed a portable device that could be used in the fields. By integrating with the smartphone readout, this platform holds significant potential in high-throughput detection of multiple pathogens in the fields.},
}
@article {pmid37416774,
year = {2023},
author = {Hu, M and Bodnar, B and Zhang, Y and Xie, F and Li, F and Li, S and Zhao, J and Zhao, R and Gedupoori, N and Mo, Y and Lin, L and Li, X and Meng, W and Yang, X and Wang, H and Barbe, MF and Srinivasan, S and Bethea, JR and Mo, X and Xu, H and Hu, W},
title = {Defective neurite elongation and branching in Nibp/Trappc9 deficient zebrafish and mice.},
journal = {International journal of biological sciences},
volume = {19},
number = {10},
pages = {3226-3248},
pmid = {37416774},
issn = {1449-2288},
mesh = {Animals ; Mice ; *Intellectual Disability/genetics/metabolism ; *Microcephaly/genetics/metabolism ; Neurites/physiology ; Neurons/metabolism ; Zebrafish ; },
abstract = {Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome, characterized by microcephaly and intellectual disability, and caused by mutations of NIBP/TRAPPC9, a crucial and unique member of TRAPPII. To investigate the neural cellular/molecular mechanisms underlying microcephaly, we developed Nibp/Trappc9-deficient animal models using different techniques, including morpholino knockdown and CRISPR/Cas mutation in zebrafish and Cre/LoxP-mediated gene targeting in mice. Nibp/Trappc9 deficiency impaired the stability of the TRAPPII complex at actin filaments and microtubules of neurites and growth cones. This deficiency also impaired elongation and branching of neuronal dendrites and axons, without significant effects on neurite initiation or neural cell number/types in embryonic and adult brains. The positive correlation of TRAPPII stability and neurite elongation/branching suggests a potential role for TRAPPII in regulating neurite morphology. These results provide novel genetic/molecular evidence to define patients with a type of non-syndromic autosomal recessive intellectual disability and highlight the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies.},
}
@article {pmid37415116,
year = {2023},
author = {Hu, Y and Zhang, H and Guo, Z and Zhou, J and Zhang, W and Gong, M and Wu, J},
title = {CKM and TERT dual promoters drive CRISPR-dCas9 to specifically inhibit the malignant behavior of osteosarcoma cells.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {52},
pmid = {37415116},
issn = {1689-1392},
mesh = {Mice ; Animals ; Mice, Nude ; *Osteosarcoma/genetics/therapy/metabolism ; Promoter Regions, Genetic/genetics ; Muscles/metabolism/pathology ; *Bone Neoplasms/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Improvements in treatment and chemotherapy have increased the survival rate of osteosarcoma, but overall efficacy remains low, highlighting the need for new gene therapy methods. Clustered regularly interspaced short palindromic repeats-deactivated Cas9 (CRISPR-dCas9) technology offers a promising strategy, but targeting osteosarcoma cells precisely is a challenge. We designed a system to achieve specific expression of CRISPR-dCas9-KRAB in osteosarcoma cells by using the creatine kinase muscle (CKM) promoter to drive dCas9-KRAB and the telomerase reverse transcriptase (TERT) promoter to drive single guide (sg)RNA expression. We inhibited the MDM2 proto-oncogene using this system in vitro, which efficiently inhibited the malignant behavior of osteosarcoma cells and induced apoptosis without affecting normal cells. In vivo experiments demonstrated that this system effectively inhibited the growth of subcutaneously transplanted tumors in nude mice. These findings provide a new method for precise identification and intervention of osteosarcoma with significant implications for the development of gene therapy methods for other cancers. Future research should focus on optimizing this system for clinical translation.},
}
@article {pmid37286821,
year = {2023},
author = {Abid, T and Goodale, AB and Kalani, Z and Wyatt, M and Gonzalez, EM and Zhou, KN and Qian, K and Novikov, D and Condurat, AL and Bandopadhayay, P and Piccioni, F and Persky, NS and Root, DE},
title = {Genome-wide pooled CRISPR screening in neurospheres.},
journal = {Nature protocols},
volume = {18},
number = {7},
pages = {2014-2031},
pmid = {37286821},
issn = {1750-2799},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Genome ; Cell Line ; *Neoplasms ; },
abstract = {Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8-12 weeks from the initial assay development tests to deconvolution of the sequencing data.},
}
@article {pmid37277562,
year = {2023},
author = {Lei, Z and Meng, H and Rao, X and Zhao, H and Yi, C},
title = {Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors.},
journal = {Nature protocols},
volume = {18},
number = {7},
pages = {2221-2255},
pmid = {37277562},
issn = {1750-2799},
mesh = {*Gene Editing/methods ; *Biotin ; Cytosine ; Genome ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.},
}
@article {pmid37231266,
year = {2023},
author = {Han, D and Xiao, Q and Wang, Y and Zhang, H and Dong, X and Li, G and Kong, X and Wang, S and Song, J and Zhang, W and Zhou, J and Bi, L and Yuan, Y and Shi, L and Zhong, N and Yang, H and Zhou, Y},
title = {Development of miniature base editors using engineered IscB nickase.},
journal = {Nature methods},
volume = {20},
number = {7},
pages = {1029-1036},
pmid = {37231266},
issn = {1548-7105},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; *Deoxyribonuclease I/genetics/metabolism ; Gene Editing ; Cytosine ; RNA/genetics ; Mammals/genetics/metabolism ; },
abstract = {As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.},
}
@article {pmid37188955,
year = {2023},
author = {Seo, SY and Min, S and Lee, S and Seo, JH and Park, J and Kim, HK and Song, M and Baek, D and Cho, SR and Kim, HH},
title = {Massively parallel evaluation and computational prediction of the activities and specificities of 17 small Cas9s.},
journal = {Nature methods},
volume = {20},
number = {7},
pages = {999-1009},
pmid = {37188955},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {Recently, various small Cas9 orthologs and variants have been reported for use in in vivo delivery applications. Although small Cas9s are particularly suited for this purpose, selecting the most optimal small Cas9 for use at a specific target sequence continues to be challenging. Here, to this end, we have systematically compared the activities of 17 small Cas9s for thousands of target sequences. For each small Cas9, we have characterized the protospacer adjacent motif and determined optimal single guide RNA expression formats and scaffold sequence. High-throughput comparative analyses revealed distinct high- and low-activity groups of small Cas9s. We also developed DeepSmallCas9, a set of computational models predicting the activities of the small Cas9s at matched and mismatched target sequences. Together, this analysis and these computational models provide a useful guide for researchers to select the most suitable small Cas9 for specific applications.},
}
@article {pmid37156748,
year = {2023},
author = {Radoua, A and Pernon, B and Pernet, N and Jean, C and Elmallah, M and Guerrache, A and Constantinescu, AA and Hadj Hamou, S and Devy, J and Micheau, O},
title = {ptARgenOM-A Flexible Vector For CRISPR/CAS9 Nonviral Delivery.},
journal = {Small methods},
volume = {7},
number = {7},
pages = {e2300069},
doi = {10.1002/smtd.202300069},
pmid = {37156748},
issn = {2366-9608},
support = {ANR-22-LCV1-0005-01//Agence Nationale de la Recherche/ ; ANR-11LABX-0021-01//Agence Nationale de la Recherche/ ; ANR-15-IDEX-0003//Agence Nationale de la Recherche/ ; 777995//Horizon 2020/ ; 01U.2020//Conf&#xe9;rence de Coordination Interr&#xe9;gionale Est de la Ligue contre le Cancer, comit&#xe9; de C&#xf4;te d'Or/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cell Line ; Mammals/genetics ; },
abstract = {Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.},
}
@article {pmid37415091,
year = {2023},
author = {Nguyen, MT and Kim, SA and Cheng, YY and Hong, SH and Jin, YS and Han, NS},
title = {A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes.},
journal = {Journal of microbiology and biotechnology},
volume = {33},
number = {9},
pages = {1-10},
doi = {10.4014/jmb.2305.05010},
pmid = {37415091},
issn = {1738-8872},
abstract = {The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/μg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.},
}
@article {pmid37415009,
year = {2023},
author = {Kauert, DJ and Madariaga-Marcos, J and Rutkauskas, M and Wulfken, A and Songailiene, I and Sinkunas, T and Siksnys, V and Seidel, R},
title = {The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {37415009},
issn = {1545-9985},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA-DNA hybrid.},
}
@article {pmid37413959,
year = {2023},
author = {Volke, DC and Orsi, E and Nikel, PI},
title = {Emergent CRISPR-Cas-based technologies for engineering non-model bacteria.},
journal = {Current opinion in microbiology},
volume = {75},
number = {},
pages = {102353},
doi = {10.1016/j.mib.2023.102353},
pmid = {37413959},
issn = {1879-0364},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) technologies brought a transformative change in the way bacterial genomes are edited, and a plethora of studies contributed to developing multiple tools based on these approaches. Prokaryotic biotechnology benefited from the implementation of such genome engineering strategies, with an increasing number of non-model bacterial species becoming genetically tractable. In this review, we summarize the recent trends in engineering non-model microbes using CRISPR-Cas technologies, discussing their potential in supporting cell factory design towards biotechnological applications. These efforts include, among other examples, genome modifications as well as tunable transcriptional regulation (both positive and negative). Moreover, we examine how CRISPR-Cas toolkits for engineering non-model organisms enabled the exploitation of emergent biotechnological processes (e.g. native and synthetic assimilation of on-carbon substrates). Finally, we discuss our slant on the future of bacterial genome engineering for domesticating non-model organisms in light of the most recent advances in the ever-expanding CRISPR-Cas field.},
}
@article {pmid37410611,
year = {2023},
author = {Shetty, VP and Akshay, SD and Rai, P and Deekshit, VK},
title = {Integrons as the potential targets for combating multidrug resistance in Enterobacteriaceae using CRISPR- Cas9 technique.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxad137},
pmid = {37410611},
issn = {1365-2672},
abstract = {The emergence of multi-drug resistance (MDR) to pan-drug resistance (PDR) in Enterobacteriaceae has made treatment extremely challenging. Genetic mutations and horizontal gene transfer (HGT) through mobile genetic elements (MGEs) were frequently associated mechanisms of drug resistance in pathogens. However, transposons, plasmids, and integrons transfer MDR genes in bacterium via HGT much faster. Integrons are dsDNA segment that plays a crucial role in the adaptation and evolution of bacteria. They contain multiple gene cassettes that code for antibiotic resistance determinants that are expressed by a single promoter (Pc). Integrons are the cause of drug resistance in Enterobacteriaceae. Although alternatives to antibiotics such as bacteriophages, phage proteins, antimicrobial peptides, and natural compounds have been widely used to treat MDR infections, there have been limited efforts to reverse the antibiotic resistance ability of bacteria. Thus, silencing the genes harboured on MGEs achieved by Gene Editing Techniques (GETs) might prevent the spread of MDR. One such GETs, which has a simple design, good repeatability, low cost, and high efficiency, is CRISPR- Cas9 system. Thus, this review is a first of the kind that focuses on utilizing the structure of an integron to make it an ideal target for GETs like CRISPR- Cas9 systems.},
}
@article {pmid37410507,
year = {2023},
author = {Gennery, AR},
title = {Gene silencing on a WHIM.},
journal = {Blood},
volume = {142},
number = {1},
pages = {1-2},
doi = {10.1182/blood.2023020478},
pmid = {37410507},
issn = {1528-0020},
mesh = {Mice ; Animals ; Alleles ; CRISPR-Cas Systems ; *Primary Immunodeficiency Diseases/genetics ; *Immunologic Deficiency Syndromes/genetics ; *Warts/genetics ; Genetic Therapy ; },
}
@article {pmid37410233,
year = {2023},
author = {Mantwill, K and Nawroth, R},
title = {Genome-Wide CRISPR Screening for the Identification of Therapy Resistance-Associated Genes in Urothelial Carcinoma.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2684},
number = {},
pages = {155-165},
pmid = {37410233},
issn = {1940-6029},
mesh = {Humans ; *Carcinoma, Transitional Cell/genetics ; CRISPR-Cas Systems ; *Urinary Bladder Neoplasms/genetics ; Genome ; RNA Interference ; },
abstract = {The application of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology with pooled guide RNA libraries enables genome-wide screening, which has some advantages over other screening methods using chemical DNA mutagens for inducing genetic changes, RNA interference, or arrayed screens. Here we describe the use of genome-wide knockout and transcriptional activation screening enabling the CRISPR-Cas9 system to discover resistance mechanisms to CDK4/6 inhibition in bladder cancer along with next-generation sequencing (NGS) analysis. We will describe the approach for transcriptional activation in the bladder cancer cell line T24 and provide guidance on critical points during the experimental workflow.},
}
@article {pmid37406505,
year = {2023},
author = {Babalola, BA and Akinsuyi, OS and Folajimi, EO and Olujimi, F and Otunba, AA and Chikere, B and Adewumagun, IA and Adetobi, TE},
title = {Exploring the future of SARS-CoV-2 treatment after the first two years of the pandemic: A comparative study of alternative therapeutics.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {165},
number = {},
pages = {115099},
doi = {10.1016/j.biopha.2023.115099},
pmid = {37406505},
issn = {1950-6007},
abstract = {One of the most pressing challenges associated with SARS-CoV-2 treatment is the emergence of new variants that may be more transmissible, cause more severe disease, or be resistant to current treatments and vaccines. The emergence of SARS-CoV-2 has led to a global pandemic, resulting in millions of deaths worldwide. Various strategies have been employed to combat the virus, including neutralizing monoclonal antibodies (mAbs), CRISPR/Cas13, and antisense oligonucleotides (ASOs). While vaccines and small molecules have proven to be an effective means of preventing severe COVID-19 and reducing transmission rates, the emergence of new virus variants poses a challenge to their effectiveness. Monoclonal antibodies have shown promise in treating early-stage COVID-19, but their effectiveness is limited in severe cases and the emergence of new variants may reduce their binding affinity. CRISPR/Cas13 has shown potential in targeting essential viral genes, but its efficiency, specificity, and delivery to the site of infection are major limitations. ASOs have also been shown to be effective in targeting viral RNA, but they face similar challenges to CRISPR/Cas13 in terms of delivery and potential off-target effects. In conclusion, a combination of these strategies may provide a more effective means of combating SARS-CoV-2, and future research should focus on improving their efficiency, specificity, and delivery to the site of infection. It is evident that the continued research and development of these alternative therapies will be essential in the ongoing fight against SARS-CoV-2 and its potential future variants.},
}
@article {pmid37405658,
year = {2023},
author = {Schatz, S and van Dijk, FH and Dubiel, AE and Cantz, T and Eggenschwiler, R and Stitz, J},
title = {Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2681},
number = {},
pages = {361-371},
pmid = {37405658},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Receptor, Nerve Growth Factor/genetics ; HEK293 Cells ; Genetic Vectors/genetics ; Receptors, Nerve Growth Factor/genetics ; Nerve Tissue Proteins/genetics ; },
abstract = {Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.},
}
@article {pmid37404143,
year = {2023},
author = {Mikkelsen, K and Bowring, JZ and Ng, YK and Svanberg Frisinger, F and Maglegaard, JK and Li, Q and Sieber, RN and Petersen, A and Andersen, PS and Rostøl, JT and Høyland-Kroghsbo, NM and Ingmer, H},
title = {An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCCmec Cassette.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0127723},
doi = {10.1128/spectrum.01277-23},
pmid = {37404143},
issn = {2165-0497},
abstract = {CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCCmec) type V(5C2&amp;5), conferring β-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCCmec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus, CRISPR-Cas is rare, but when present, it is located within the SCCmec element, which encodes resistance to methicillin and other β-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCCmec elements in different species of non-S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus. Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.},
}
@article {pmid37403986,
year = {2023},
author = {Khairkhah, N and Bolhassani, A and Rajaei, F and Najafipour, R},
title = {Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice.},
journal = {Journal of medical virology},
volume = {95},
number = {7},
pages = {e28934},
doi = {10.1002/jmv.28934},
pmid = {37403986},
issn = {1096-9071},
mesh = {Mice ; Animals ; Humans ; *Human papillomavirus 16/genetics ; *CRISPR-Cas Systems ; Antimicrobial Peptides ; Mice, Inbred C57BL ; Oncogenes ; },
abstract = {Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.},
}
@article {pmid37402755,
year = {2023},
author = {Su, H and Wang, Y and Xu, J and Omar, AA and Grosser, JW and Calovic, M and Zhang, L and Feng, Y and Vakulskas, CA and Wang, N},
title = {Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {3957},
pmid = {37402755},
issn = {2041-1723},
mesh = {*Citrus sinensis/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Plant Breeding ; *Citrus/genetics ; *Xanthomonas/genetics ; Plant Diseases/genetics ; },
abstract = {Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.},
}
@article {pmid37390819,
year = {2023},
author = {Lue, NZ and Liau, BB},
title = {Base editor screens for in situ mutational scanning at scale.},
journal = {Molecular cell},
volume = {83},
number = {13},
pages = {2167-2187},
pmid = {37390819},
issn = {1097-4164},
support = {DP2 GM137494/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mutation ; Proteome/genetics ; Amino Acid Sequence ; },
abstract = {A fundamental challenge in biology is understanding the molecular details of protein function. How mutations alter protein activity, regulation, and response to drugs is of critical importance to human health. Recent years have seen the emergence of pooled base editor screens for in situ mutational scanning: the interrogation of protein sequence-function relationships by directly perturbing endogenous proteins in live cells. These studies have revealed the effects of disease-associated mutations, discovered novel drug resistance mechanisms, and generated biochemical insights into protein function. Here, we discuss how this "base editor scanning" approach has been applied to diverse biological questions, compare it with alternative techniques, and describe the emerging challenges that must be addressed to maximize its utility. Given its broad applicability toward profiling mutations across the proteome, base editor scanning promises to revolutionize the investigation of proteins in their native contexts.},
}
@article {pmid37271098,
year = {2023},
author = {Alshammari, M and Ahmad, A and AlKhulaifi, M and Al Farraj, D and Alsudir, S and Alarawi, M and Takashi, G and Alyamani, E},
title = {Reduction of biofilm formation of Escherichia coli by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter.},
journal = {Journal of infection and public health},
volume = {16},
number = {8},
pages = {1174-1183},
doi = {10.1016/j.jiph.2023.05.026},
pmid = {37271098},
issn = {1876-035X},
mesh = {Humans ; *Quorum Sensing/genetics ; *Escherichia coli/genetics ; Urinary Catheters ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gentian Violet/metabolism ; Biofilms ; Bacterial Proteins/genetics ; },
abstract = {BACKGROUND: Escherichia coli is a common cause of biofilm-associated urinary tract infections (UTIs). Biofilm formation in E. coli is responsible for various indwelling medical device-associated infections, including catheter-associated urinary tract infections (CAUTIs). This study aimed to reduce biofilm formation of E. coli ATCC 25922 by knocking out genes involved in quorum sensing (QS) (luxS) and adhesion (fimH and bolA) using the CRISPR/Cas9-HDR approach.<br><br>METHOD: Single-guide RNAs (sgRNAs) were designed to target luxS, fimH and bolA genes. Donor DNA for homologous recombination was constructed to provide accurate repairs of double-strand breaks (DSBs). A biofilm quantification assay (crystal violet assay) was performed to quantify the biofilm formation of mutant and wild-type strains. Morphological changes in biofilm architecture were confirmed by scanning electron microscopy (SEM). Further application of the biofilm formation of mutant and wild-type strains on urinary catheter was tested.<br><br>RESULTS: Crystal violet assay showed that the biofilm formation of &#x394;fimH, &#x394;luxS, and &#x394;bolA strains was significantly reduced compared to the wild-type strain (P value<0.001). The percentage of biofilm reduction of mutant strains was as follows: &#x394;luxS1 77.51 %, &#x394;fimH1 78.37 %, &#x394;fimH2 84.17 %, &#x394;bolA1 78.24 %, and &#x394;bolA2 75.39 %. Microscopic analysis showed that all mutant strains lack extracellular polymeric substances (EPS) production compared to the wild-type strain, which was embedded in its EPS matrix. The adherence, cell aggregation, and biofilm formation of wild-type strain on urinary catheters were significantly higher compared to &#x394;fimH, &#x394;luxS and &#x394;bolA strains.<br><br>CONCLUSION: Altogether, our results demonstrated that the knockout of luxS, fimH, and bolA genes reduced EPS matrix production, which is considered the main factor in the development, maturation, and maintenance of the integrity of biofilm. This pathway could be a potential strategy to disrupt E. coli biofilm-associated UTIs. This study suggests that CRISPR/Cas9-HDR system may provide an efficient and site-specific gene editing approach that exhibits a possible antibiofilm strategy through intervention with the QS mechanism and adhesion property to suppress biofilm formation associated with UTI catheter infections.},
}
@article {pmid37144468,
year = {2023},
author = {She, R and Luo, J and Weissman, JS},
title = {Translational fidelity screens in mammalian cells reveal eIF3 and eIF4G2 as regulators of start codon selectivity.},
journal = {Nucleic acids research},
volume = {51},
number = {12},
pages = {6355-6369},
pmid = {37144468},
issn = {1362-4962},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Humans ; Codon, Initiator/metabolism ; *RNA, Guide, CRISPR-Cas Systems ; *Eukaryotic Initiation Factor-3/genetics/metabolism ; NF-kappa B/genetics/metabolism ; Ribosomes/metabolism ; Peptide Chain Initiation, Translational ; Protein Biosynthesis ; },
abstract = {The translation initiation machinery and the ribosome orchestrate a highly dynamic scanning process to distinguish proper start codons from surrounding nucleotide sequences. Here, we performed genome-wide CRISPRi screens in human K562 cells to systematically identify modulators of the frequency of translation initiation at near-cognate start codons. We observed that depletion of any eIF3 core subunit promoted near-cognate start codon usage, though sensitivity thresholds of each subunit to sgRNA-mediated depletion varied considerably. Double sgRNA depletion experiments suggested that enhanced near-cognate usage in eIF3D depleted cells required canonical eIF4E cap-binding and was not driven by eIF2A or eIF2D-dependent leucine tRNA initiation. We further characterized the effects of eIF3D depletion and found that the N-terminus of eIF3D was strictly required for accurate start codon selection, whereas disruption of the cap-binding properties of eIF3D had no effect. Lastly, depletion of eIF3D activated TNFα signaling via NF-κB and the interferon gamma response. Similar transcriptional profiles were observed upon knockdown of eIF1A and eIF4G2, which also promoted near-cognate start codon usage, suggesting that enhanced near-cognate usage could potentially contribute to NF-κB activation. Our study thus provides new avenues to study the mechanisms and consequences of alternative start codon usage.},
}
@article {pmid36928087,
year = {2023},
author = {Gao, JL and Owusu-Ansah, A and Yang, A and Yim, E and McDermott, DH and Jacobs, P and Majumdar, S and Choi, U and Sweeney, CL and Malech, HL and Murphy, PM},
title = {CRISPR/Cas9-mediated Cxcr4 disease allele inactivation for gene therapy in a mouse model of WHIM syndrome.},
journal = {Blood},
volume = {142},
number = {1},
pages = {23-32},
doi = {10.1182/blood.2022019142},
pmid = {36928087},
issn = {1528-0020},
mesh = {Mice ; Animals ; Alleles ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Immunologic Deficiency Syndromes/genetics/therapy ; *Warts/genetics/therapy ; Genetic Therapy ; Receptors, CXCR4/genetics ; },
abstract = {WHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4+/+) and WHIM (Cxcr4+/w) HSCs and that a patient with WHIM was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4+/w and Cxcr4+/+ alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wild-type allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge, this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.},
}
@article {pmid37402738,
year = {2023},
author = {Ryan, EC and Huggins, LM and Podlevsky, JD},
title = {The engineered single guide RNA structure as a biomarker for gene-editing reagent exposure.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {10804},
pmid = {37402738},
issn = {2045-2322},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; *Bacteria/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; RNA/genetics ; Biomarkers ; },
abstract = {CRISPR arrays and CRISPR-associated (Cas) proteins comprise a prevalent adaptive immune system in bacteria and archaea. These systems defend against exogenous parasitic mobile genetic elements. The adaption of single effector CRISPR-Cas systems has massively facilitated gene-editing due to the reprogrammable guide RNA. The guide RNA affords little priming space for conventional PCR-based nucleic acid tests without foreknowledge of the spacer sequence. Further impeding detection of gene-editor exposure, these systems are derived from human microflora and pathogens (Staphylococcus pyogenes, Streptococcus aureus, etc.) that contaminate human patient samples. The single guide RNA-formed from the CRISPR RNA (crRNA) and transactivating RNA (tracrRNA)-harbors a variable tetraloop sequence between the two RNA segments, complicating PCR assays. Identical single effector Cas proteins are used for gene-editing and naturally by bacteria. Antibodies raised against these Cas proteins are unable to distinguish CRISPR-Cas gene-editors from bacterial contaminant. To overcome the high potential for false positives, we have developed a DNA displacement assay to specifically detect gene-editors. We leveraged the single guide RNA structure as an engineered moiety for gene-editor exposure that does not cross-react with bacterial CRISPRs. Our assay has been validated for five common CRISPR systems and functions in complex sample matrices.},
}
@article {pmid37402375,
year = {2023},
author = {Amundson, KK and Roux, S and Shelton, JL and Wilkins, MJ},
title = {Long-term CRISPR locus dynamics and stable host-virus co-existence in subsurface fractured shales.},
journal = {Current biology : CB},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cub.2023.06.033},
pmid = {37402375},
issn = {1879-0445},
abstract = {Viruses are the most ubiquitous biological entities on Earth. Even so, elucidating the impact of viruses on microbial communities and associated ecosystem processes often requires identification of unambiguous host-virus linkages-an undeniable challenge in many ecosystems. Subsurface fractured shales present a unique opportunity to first make these strong linkages via spacers in CRISPR-Cas arrays and subsequently reveal complex long-term host-virus dynamics. Here, we sampled two replicated sets of fractured shale wells for nearly 800 days, resulting in 78 metagenomes from temporal sampling of six wells in the Denver-Julesburg Basin (Colorado, USA). At the community level, there was strong evidence for CRISPR-Cas defense systems being used through time and likely in response to viral interactions. Within our host genomes, represented by 202 unique MAGs, we also saw that CRISPR-Cas systems were widely encoded. Together, spacers from host CRISPR loci facilitated 2,110 CRISPR-based viral linkages across 90 host MAGs spanning 25 phyla. We observed less redundancy in host-viral linkages and fewer spacers associated with hosts from the older, more established wells, possibly reflecting enrichment of more beneficial spacers through time. Leveraging temporal patterns of host-virus linkages across differing well ages, we report how host-virus co-existence dynamics develop and converge through time, possibly reflecting selection for viruses that can evade host CRISPR-Cas systems. Together, our findings shed light on the complexities of host-virus interactions as well as long-term dynamics of CRISPR-Cas defense among diverse microbial populations.},
}
@article {pmid37401603,
year = {2023},
author = {Fan, X and Wang, J and Liang, L and Liu, R},
title = {[Multiplex gene editing and regulation techniques based on CRISPR/Cas system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {6},
pages = {2449-2464},
doi = {10.13345/j.cjb.220989},
pmid = {37401603},
issn = {1872-2075},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Bacteria/genetics ; Archaea ; Bioengineering ; },
abstract = {The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.},
}
@article {pmid37166046,
year = {2023},
author = {Sahel, DK and Vora, LK and Saraswat, A and Sharma, S and Monpara, J and D'Souza, AA and Mishra, D and Tryphena, KP and Kawakita, S and Khan, S and Azhar, M and Khatri, DK and Patel, K and Singh Thakur, RR},
title = {CRISPR/Cas9 Genome Editing for Tissue-Specific In Vivo Targeting: Nanomaterials and Translational Perspective.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {19},
pages = {e2207512},
pmid = {37166046},
issn = {2198-3844},
mesh = {United States ; Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; RNA, Messenger ; *Nanostructures ; },
abstract = {Clustered randomly interspaced short palindromic repeats (CRISPRs) and its associated endonuclease protein, i.e., Cas9, have been discovered as an immune system in bacteria and archaea; nevertheless, they are now being adopted as mainstream biotechnological/molecular scissors that can modulate ample genetic and nongenetic diseases via insertion/deletion, epigenome editing, messenger RNA editing, CRISPR interference, etc. Many Food and Drug Administration-approved and ongoing clinical trials on CRISPR adopt ex vivo strategies, wherein the gene editing is performed ex vivo, followed by reimplantation to the patients. However, the in vivo delivery of the CRISPR components is still under preclinical surveillance. This review has summarized the nonviral nanodelivery strategies for gene editing using CRISPR/Cas9 and its recent advancements, strategic points of view, challenges, and future aspects for tissue-specific in vivo delivery of CRISPR/Cas9 components using nanomaterials.},
}
@article {pmid37083231,
year = {2023},
author = {Zhao, Y and Li, Y and Wang, F and Gan, X and Zheng, T and Chen, M and Wei, L and Chen, J and Yu, C},
title = {CES1-Triggered Liver-Specific Cargo Release of CRISPR/Cas9 Elements by Cationic Triadic Copolymeric Nanoparticles Targeting Gene Editing of PCSK9 for Hyperlipidemia Amelioration.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {19},
pages = {e2300502},
pmid = {37083231},
issn = {2198-3844},
support = {cstc2020jscx-msxmX0070//Technological Innovation and Application Development/ ; 82000539//National Natural Science Foundation of China/ ; R13015//Postdoctoral Launch Project of Chongqing Medical University/ ; YXY2021BSH01//Discipline Talent Training Program in the Chongqing Medical University College of Pharmacy/ ; CSTB2022NSCQ-BHX0681//Postdoctoral Program of the Chongqing Natural Science Foundation/ ; cstc2020jcyj-bshX0080//Postdoctoral Program of the Chongqing Natural Science Foundation/ ; BJRC2023//Outstanding Graduate Student Cultivation Program of Chongqing Medical University/ ; },
mesh = {Animals ; Mice ; Gene Editing/methods ; Proprotein Convertase 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Hyperlipidemias/therapy/genetics ; Liver/metabolism ; *Nanoparticles ; RNA, Messenger ; Cholesterol ; Carboxylic Ester Hydrolases/genetics ; },
abstract = {The broad application of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing tools is hindered by challenges in the efficient delivery of its two components into specific cells and intracytoplasmic release. Herein, a novel copolymer for delivery of Cas9-mRNA/ single-guide RNA (Cas9-mRNA/sgRNA) in vitro and vivo, using carboxylesterase-responsive cationic triadic copolymeric nanoparticles targeted proprotein convertase subtilisin/kexin type 9 (PCSK9) for hyperlipidemia amelioration is reported. A dimethyl biguanide derivative is designed and synthesized to form cationic block, and copolymerization onto prepolymer with propyl methacrylate, to fabricate a triadic copolymer mPEG-b-P(Met/n-PMA). The copolymer can self-assemble with Cas9-mRNA/sgRNA, indicating the excellent potential of nanoparticles to form a delivery carrier. This vehicle can efficiently release RNA in response to the hepatocytes carboxylesterase for genome editing. It was demonstrated that the mPEG-b-P(Met/n-PMA)/Cas9 mRNA/sgRNA nanoparticles effectively accumulated in hepatocytes, lead to the inhibition of PCSK9, and lowered the levels of Low-density lipoprotein cholesterol and total cholesterol in mouse serum down 20% of nontreatment. Interestingly, the nanoparticles even enable multiple functions in the regulation of blood glucose and weight. This study establishes a novel method to achieve complex CRISPR components stable loading, safe delivery, and fixed-point release, which expand the application of CRISPR delivery systems.},
}
@article {pmid37400712,
year = {2023},
author = {Vijayakumar, VE and Venkataraman, K},
title = {A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37400712},
issn = {1559-0305},
support = {5/3/8/9/ITR-F/2018//Indian Council of Medical Research/ ; IFC/7126/Hemophilia//Indo-French Centre for the Promotion of Advanced Research/ ; },
abstract = {The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins.},
}
@article {pmid37400536,
year = {2023},
author = {Wu, T and Liu, C and Zou, S and Lyu, R and Yang, B and Yan, H and Zhao, M and Tang, W},
title = {An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {37400536},
issn = {1552-4469},
support = {RSG-22-043-01-ET//American Cancer Society (American Cancer Society, Inc.)/ ; },
abstract = {Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.},
}
@article {pmid37399654,
year = {2023},
author = {Garg, D and Samota, MK and Kontis, N and Patel, N and Bala, S and Rosado, AS},
title = {Revolutionizing biofuel generation: Unleashing the power of CRISPR-Cas mediated gene editing of extremophiles.},
journal = {Microbiological research},
volume = {274},
number = {},
pages = {127443},
doi = {10.1016/j.micres.2023.127443},
pmid = {37399654},
issn = {1618-0623},
abstract = {Molecular biology techniques like gene editing have altered the specific genes in micro-organisms to increase their efficiency to produce biofuels. This review paper investigates the outcomes of Clustered regularly interspaced short palindromic repeats (CRISPR) for gene editing in extremophilic micro-organisms to produce biofuel. Commercial production of biofuel from lignocellulosic waste is limited due to various constraints. A potential strategy to enhance the capability of extremophiles to produce biofuel is gene-editing via CRISPR-Cas technology. The efficiency of intracellular enzymes like cellulase, hemicellulose in extremophilic bacteria, fungi and microalgae has been increased by alteration of genes associated with enzymatic activity and thermotolerance. extremophilic microbes like Thermococcus kodakarensis, Thermotoga maritima, Thermus thermophilus, Pyrococcus furiosus and Sulfolobus sp. are explored for biofuel production. The conversion of lignocellulosic biomass into biofuels involves pretreatment, hydrolysis and fermentation. The challenges like off-target effect associated with use of extremophiles for biofuel production is also addressed. The appropriate regulations are required to maximize effectiveness while minimizing off-target cleavage, as well as the total biosafety of this technique. The latest discovery of the CRISPR-Cas system should provide a new channel in the creation of microbial biorefineries through site- specific gene editing that might boost the generation of biofuels from extremophiles. Overall, this review study highlights the potential for genome editing methods to improve the potential of extremophiles to produce biofuel, opening the door to more effective and environmentally friendly biofuel production methods.},
}
@article {pmid37398651,
year = {2023},
author = {Vredevoogd, DW and Peeper, DS},
title = {Heterogeneity in functional genetic screens: friend or foe?.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1162706},
pmid = {37398651},
issn = {1664-3224},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; },
abstract = {Functional genetic screens to uncover tumor-intrinsic nodes of immune resistance have uncovered numerous mechanisms by which tumors evade our immune system. However, due to technical limitations, tumor heterogeneity is imperfectly captured with many of these analyses. Here, we provide an overview of the nature and sources of heterogeneity that are relevant for tumor-immune interactions. We argue that this heterogeneity may actually contribute to the discovery of novel mechanisms of immune evasion, given a sufficiently large and heterogeneous set of input data. Taking advantage of tumor cell heterogeneity, we provide proof-of-concept analyses of mechanisms of TNF resistance. Thus, consideration of tumor heterogeneity is imperative to increase our understanding of immune resistance mechanisms.},
}
@article {pmid37396453,
year = {2023},
author = {Hao, N and Donnelly, AJ and Dodd, IB and Shearwin, KE},
title = {When push comes to shove - RNA polymerase and DNA-bound protein roadblocks.},
journal = {Biophysical reviews},
volume = {15},
number = {3},
pages = {355-366},
pmid = {37396453},
issn = {1867-2450},
abstract = {In recent years, transcriptional roadblocking has emerged as a crucial regulatory mechanism in gene expression, whereby other DNA-bound obstacles can block the progression of transcribing RNA polymerase (RNAP), leading to RNAP pausing and ultimately dissociation from the DNA template. In this review, we discuss the mechanisms by which transcriptional roadblocks can impede RNAP progression, as well as how RNAP can overcome these obstacles to continue transcription. We examine different DNA-binding proteins involved in transcriptional roadblocking and their biophysical properties that determine their effectiveness in blocking RNAP progression. The catalytically dead CRISPR-Cas (dCas) protein is used as an example of an engineered programmable roadblock, and the current literature in understanding the polarity of dCas roadblocking is also discussed. Finally, we delve into a stochastic model of transcriptional roadblocking and highlight the importance of transcription factor binding kinetics and its resistance to dislodgement by an elongating RNAP in determining the strength of a roadblock.},
}
@article {pmid37158253,
year = {2023},
author = {Woźniak, T and Sura, W and Kazimierska, M and Kasprzyk, ME and Podralska, M and Dzikiewicz-Krawczyk, A},
title = {TransCRISPR-sgRNA design tool for CRISPR/Cas9 experiments targeting specific sequence motifs.},
journal = {Nucleic acids research},
volume = {51},
number = {W1},
pages = {W577-W586},
pmid = {37158253},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Genome ; Genomics ; Binding Sites/genetics ; Gene Editing ; },
abstract = {Eukaryotic genomes contain several types of recurrent sequence motifs, e.g. transcription factor motifs, miRNA binding sites, repetitive elements. CRISPR/Cas9 can facilitate identification and study of crucial motifs. We present transCRISPR, the first online tool dedicated to search for sequence motifs in the user-provided genomic regions and design optimal sgRNAs targeting them. Users can obtain sgRNAs for chosen motifs, for up to tens of thousands of target regions in 30 genomes, either for the Cas9 or dCas9 system. TransCRISPR provides user-friendly tables and visualizations, summarizing features of identified motifs and designed sgRNAs such as genomic localization, quality scores, closest transcription start sites and others. Experimental validation of sgRNAs for MYC binding sites designed with transCRISPR confirmed efficient disruption of the targeted motifs and effect on expression of MYC-regulated genes. TransCRISPR is available from https://transcrispr.igcz.poznan.pl/transcrispr/.},
}
@article {pmid37002710,
year = {2023},
author = {Sandoval-Quintana, E and Stangl, C and Huang, L and Renkens, I and Duran, R and van Haaften, G and Monroe, G and Lauga, B and Cagnon, C},
title = {CRISPR-Cas9 enrichment, a new strategy in microbial metagenomics to investigate complex genomic regions: The case of an environmental integron.},
journal = {Molecular ecology resources},
volume = {23},
number = {6},
pages = {1288-1298},
doi = {10.1111/1755-0998.13798},
pmid = {37002710},
issn = {1755-0998},
support = {06SEST09//Agence Nationale de la Recherche/ ; CESA-2011-006 01//Agence Nationale de la Recherche/ ; //E2S-UPPA/ ; STF-8420//European Molecular Biology Organization/ ; //Minist&#xe8;re de l'Enseignement Sup&#xe9;rieur et de la Recherche Scientifique/ ; 184.034.019//The Netherlands X-omics Initiative/ ; },
mesh = {*Integrons/genetics ; *Metagenomics ; CRISPR-Cas Systems ; Bacteria/genetics ; Integrases/genetics ; },
abstract = {Environmental integrons are ubiquitous in natural microbial communities, but they are mostly uncharacterized and their role remains elusive. Thus far, research has been hindered by methodological limitations. Here, we successfully used an innovative approach combining CRISPR-Cas9 enrichment with long-read nanopore sequencing to target, in a complex microbial community, a putative adaptive environmental integron, InOPS, and to unravel its complete structure and genetic context. A contig of 20 kb was recovered containing the complete integron from the microbial metagenome of oil-contaminated coastal sediments. InOPS exhibited typical integron features. The integrase, closely related to integrases of marine Desulfobacterota, possessed all the elements of a functional integron integrase. The gene cassettes harboured mostly unknown functions hampering inferences about their ecological importance. Moreover, the putative InOPS host, likely a hydrocarbonoclastic marine bacteria, raises questions as to the adaptive potential of InOPS in response to oil contamination. Finally, several mobile genetic elements were intertwined with InOPS highlighting likely genomic plasticity, and providing a source of genetic novelty. This case study showed the power of CRISPR-Cas9 enrichment to elucidate the structure and context of specific DNA regions for which only a short sequence is known. This method is a new tool for environmental microbiologists working with complex microbial communities to target low abundant, large or repetitive genetic structures that are difficult to obtain by classical metagenomics. More precisely, here, it offers new perspectives to comprehensively assess the eco-evolutionary significance of environmental integrons.},
}
@article {pmid37396379,
year = {2023},
author = {Bauer, R and Haider, D and Grempels, A and Roscher, R and Mauerer, S and Spellerberg, B},
title = {Diversity of CRISPR-Cas type II-A systems in Streptococcus anginosus.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1188671},
pmid = {37396379},
issn = {1664-302X},
abstract = {Streptococcus anginosus is a commensal Streptococcal species that is often associated with invasive bacterial infections. However, little is known about its molecular genetic background. Many Streptococcal species, including S. anginosus, harbor clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. A CRISPR-Cas type II-A system as well as a type II-C system have been reported for this species. To characterize the CRISPR-Cas type II systems of S. anginosus in more detail, we conducted a phylogenetic analysis of Cas9 sequences from CRISPR-Cas type II systems with a special focus on streptococci and S. anginosus. In addition, a phylogenetic analysis of S. anginosus strains based on housekeeping genes included in MLST analysis, was performed. All analyzed Cas9 sequences of S. anginosus clustered with the Cas9 sequences of CRISPR type II-A systems, including the Cas9 sequences of S. anginosus strains reported to harbor a type II-C system. The Cas9 genes of the CRISPR-Cas type II-C systems of other bacterial species separated into a different cluster. Moreover, analyzing the CRISPR loci found in S. anginosus, two distinct csn2 genes could be detected, a short form showing high similarity to the canonical form of the csn2 gene present in S. pyogenes. The second CRISPR type II locus of S. anginosus contained a longer variant of csn2 with close similarities to a csn2 gene that has previously been described in Streptococcus thermophilus. Since CRISPR-Cas type II-C systems do not contain a csn2 gene, the S. anginosus strains reported to have a CRISPR-Cas type II-C system appear to carry a variation of CRISPR-Cas type II-A harboring a long variant of csn2.},
}
@article {pmid37396349,
year = {2023},
author = {Chen, F and Wang, D and Lu, T and Li, S},
title = {Identification of a novel type II-C Cas9 from the fish pathogen Flavobacterium psychrophilum.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1181303},
pmid = {37396349},
issn = {1664-302X},
abstract = {Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish worldwide. As an important fish pathogen, F. psychrophilum is frequently exposed to multiple invading genetic elements in natural environments. Endonuclease Cas9 provides bacteria with adaptive interference against invading genetic elements. Previous studies revealed that several F. psychrophilum strains harbored a type II-C Cas9 called Fp1Cas9, but little is known about the potential role of this endonuclease against invading genetic elements. In this work, we identified a gene encoding a novel type II-C Cas9 called Fp2Cas9 from F. psychrophilum strain CN46. Through bacterial RNA sequencing, we demonstrated active transcription of both Fp2Cas9 and pre-crRNAs in strain CN46. Bioinformatics analysis further revealed that the transcription of Fp2Cas9 and pre-crRNAs was driven by a newly integrated promoter sequence and a promoter element embedded within each CRISPR repeat, respectively. To formally demonstrate that Fp2Cas9 and associated crRNAs yielded functional interference in strain CN46, a plasmid interference assay was performed, resulting in adaptive immunity to target DNA sequences in Flavobacterium bacteriophages. Phylogenetic analysis demonstrated that Fp2Cas9 was present only in several F. psychrophilum isolates. Phylogenetic analysis revealed that this novel endonuclease was probably acquired through horizontal gene transfer from the CRISPR-Cas9 system in an unidentified Flavobacterium species. Comparative genomics analysis further showed that the Fp2Cas9 was integrated into the type II-C CRISPR-Cas locus in strain CN38 instead of the original Fp1Cas9. Taken together, our results shed light on the origin and evolution of Fp2Cas9 gene and demonstrated that this novel endonuclease provided adaptive interference against bacteriophage infections.},
}
@article {pmid37395789,
year = {2023},
author = {Guo, Y and Zhao, G and Gao, X and Zhang, L and Zhang, Y and Cai, X and Yuan, X and Guo, X},
title = {CRISPR/Cas9 gene editing technology: a precise and efficient tool for crop quality improvement.},
journal = {Planta},
volume = {258},
number = {2},
pages = {36},
pmid = {37395789},
issn = {1432-2048},
support = {31971823//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Quality Improvement ; Plant Breeding ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; },
abstract = {This review provides a direction for crop quality improvement and ideas for further research on the application of CRISPR/Cas9 gene editing technology for crop improvement. Various important crops, such as wheat, rice, soybean and tomato, are among the main sources of food and energy for humans. Breeders have long attempted to improve crop yield and quality through traditional breeding methods such as crossbreeding. However, crop breeding progress has been slow due to the limitations of traditional breeding methods. In recent years, clustered regularly spaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has been continuously developed. And with the refinement of crop genome data, CRISPR/Cas9 technology has enabled significant breakthroughs in editing specific genes of crops due to its accuracy and efficiency. Precise editing of certain key genes in crops by means of CRISPR/Cas9 technology has improved crop quality and yield and has become a popular strategy for many breeders to focus on and adopt. In this paper, the present status and achievements of CRISPR/Cas9 gene technology as applied to the improvement of quality in several crops are reviewed. In addition, the shortcomings, challenges and development prospects of CRISPR/Cas9 gene editing technology are discussed.},
}
@article {pmid37395026,
year = {2023},
author = {Li, D and Ou, M and Zhang, W and Luo, Q and Cai, W and Mo, C and Liang, W and Dai, G and Yin, L and Zhu, P and Tang, D and Dai, Y},
title = {CRISPR/Cas9-Mediated Gene Correction in Osteopetrosis Patient-Derived iPSCs.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {28},
number = {6},
pages = {131},
doi = {10.31083/j.fbl2806131},
pmid = {37395026},
issn = {2768-6698},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; CRISPR-Cas Systems ; *Osteopetrosis/genetics/therapy/metabolism ; Mutation ; Chloride Channels/genetics/metabolism ; },
abstract = {BACKGROUND: Osteopetrosis represents a rare genetic disease with a wide range of clinical and genetic heterogeneity, which results from osteoclast failure. Although up to 10 genes have been identified to be related with osteopetrosis, the pathogenesis of osteopetrosis remains foggy. Disease-specific induced pluripotent stem cells (iPSCs) and gene-corrected disease specific iPSCs provide a platform to generate attractive in vitro disease cell models and isogenic control cellular models respectively. The purpose of this study is to rescue the disease causative mutation in osteopetrosis specific induced pluripotent stem cells and provide isogenic control cellular models.<br><br>METHODS: Based on our previously established osteopetrosis-specific iPSCs (ADO2-iPSCs), we repaired the point mutation R286W of the CLCN7 gene in ADO2-iPSCs by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated homologous recombination.<br><br>RESULTS: The obtained gene corrected ADO2-iPSCs (GC-ADO2-iPSCs) were characterized in terms of hESC-like morphology, a normal karyotype, expression of pluripotency markers, homozygous repaired sequence of CLCN7 gene, and the ability to differentiate into cells of three germ layers.<br><br>CONCLUSIONS: We successfully corrected the point mutation R286W of the CLCN7 gene in ADO2-iPSCs. This isogenic iPSC line is an ideal control cell model for deciphering the pathogenesis of osteopetrosis in future studies.},
}
@article {pmid37394947,
year = {2023},
author = {Campa, M and Miranda, S and Licciardello, C and Lashbrooke, JG and Dalla Costa, L and Guan, Q and Spök, A and Malnoy, M},
title = {Application of new breeding techniques in fruit trees.},
journal = {Plant physiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/plphys/kiad374},
pmid = {37394947},
issn = {1532-2548},
abstract = {Climate change and rapid adaption of invasive pathogens pose a constant pressure on fruit industry to develop improved varieties. Aiming to accelerate the development of better adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis and CRISPR/Cas genome editing hold significant potential for crop trait improvement, which have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance, tolerance to abiotic stress, and to improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. Regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.},
}
@article {pmid37302232,
year = {2023},
author = {Soprano, E and Migliavacca, M and López-Ferreiro, M and Pelaz, B and Polo, E and Del Pino, P},
title = {Fusogenic Cell-Derived nanocarriers for cytosolic delivery of cargo inside living cells.},
journal = {Journal of colloid and interface science},
volume = {648},
number = {},
pages = {488-496},
doi = {10.1016/j.jcis.2023.06.015},
pmid = {37302232},
issn = {1095-7103},
mesh = {*Drug Delivery Systems ; RNA, Guide, CRISPR-Cas Systems ; Cytosol/metabolism ; Lipids/chemistry ; *Nanoparticles/chemistry ; Drug Carriers/chemistry ; },
abstract = {A surface-engineered cell-derived nanocarrier was developed for efficient cytosolic delivery of encapsulated biologically active molecules inside living cells. Thus, a combination of aromatic-labeled and cationic lipids, instrumental in providing fusogenic properties, was intercalated into the biomimetic shell of self-assembled nanocarriers formed from cell membrane extracts. The nanocarriers were loaded, as a proof of concept, with either bisbenzimide molecules, a fluorescently labeled dextran polymer, the bicyclic heptapeptide phalloidin, fluorescently labeled polystyrene nanoparticles or a ribonucleoprotein complex (Cas9/sgRNA). The demonstrated nanocarrieŕs fusogenic behavior relies on the fusogen-like properties imparted by the intercalated exogenous lipids, which allows for circumventing lysosomal storage, thereby leading to efficient delivery into the cytosolic milieu where cargo regains function.},
}
@article {pmid37276914,
year = {2023},
author = {Singpant, P and Tubsuwan, A and Sakdee, S and Ketterman, AJ and Jearawiriyapaisarn, N and Kurita, R and Nakamura, Y and Songdej, D and Tangprasittipap, A and Bhukhai, K and Chiangjong, W and Hongeng, S and Saisawang, C},
title = {Recombinant Cas9 protein production in an endotoxin-free system and evaluation with editing the BCL11A gene in human cells.},
journal = {Protein expression and purification},
volume = {210},
number = {},
pages = {106313},
doi = {10.1016/j.pep.2023.106313},
pmid = {37276914},
issn = {1096-0279},
mesh = {Humans ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Endotoxins/genetics ; Gene Editing/methods ; Repressor Proteins ; },
abstract = {Many therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation. To mitigate this problem, we have employed the ClearColi BL21 (DE3) endotoxin-free cells as an expression host for Cas9 protein production. Cas9 is an endonuclease enzyme that plays a key role in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/Cas9) genome editing technique. This technology is very promising for use in diagnostics as well as treatment of diseases, especially for genetic diseases such as thalassemia. The potential uses for this technology thus generate a considerable interest for Cas9 utilization as a therapeutic protein in clinical treatment. Therefore, special care in protein production should be a major concern. Accordingly, we expressed the Cas9 protein in endotoxin-free bacterial cells achieving 99% purity with activity comparable to commercially available Cas9. Our protocol therefore yields a cost-effective product suitable for invitro experiments with stem cells.},
}
@article {pmid36481734,
year = {2023},
author = {Xu, X and Liao, T and Jiang, R and Ye, X and Yan, J},
title = {Increased Expression Levels of Soluble Fms-Like Tyrosine Kinase-1 Inhibit the Development of the Nervous System.},
journal = {Neuroendocrinology},
volume = {113},
number = {7},
pages = {692-704},
doi = {10.1159/000528571},
pmid = {36481734},
issn = {1423-0194},
mesh = {Humans ; Animals ; Female ; *Vascular Endothelial Growth Factor A ; Placenta Growth Factor ; Vascular Endothelial Growth Factor Receptor-1 ; RNA, Guide, CRISPR-Cas Systems ; Zebrafish ; *Pre-Eclampsia/metabolism ; Nervous System/metabolism ; Biomarkers ; },
abstract = {INTRODUCTION: Preeclampsia (PE) remains a leading cause of maternal and perinatal morbidity. At present, only limited options are available for the treatment of PE. Consequently, many patients need to terminate their pregnancies to relieve the disease. Soluble fms-like tyrosine kinase-1 (sFlt-1) is a decoy receptor of placental growth factor and vascular endothelial growth factor which can promote angiogenesis. Throughout pregnancy, the expression level of sFlt-1 continues to increase in both the mother with PE and her offspring.<br><br>MATERIAL AND METHODS: In this experiment, we generated a zebrafish line expressing high levels of sFlt-1 and investigated changes in behavior and development of the nervous system.<br><br>RESULTS: At 96 h post-fertilization (hpf), the brain volume area of zebrafish in the experimental group (zFLT1+CasRx) was significantly smaller after injection than in the WT group (p < 0.05) and the negative control group (CasRx) (p < 0.05). At 96 hpf, compared with the WT group, the cerebral blood vessels in the CasRx control group and experimental group (zFLT1-sgRNA+CasRx) were significantly lower after injection (p < 0.05). Compared with the CasRx control group, the track movement distance and the mean track speed of zebrafish in the experimental group (zFLT1-sgRNA+CasRx) after the 6th injection were significantly decreased (p < 0.05).<br><br>CONCLUSIONS: The increased expression levels of sFlt-1 in zebrafish inhibited the development of the cerebral blood vessels, influenced brain volumes, and inhibited behavioral activities. Our data suggest that the elevation of sFlt-1 in the pathological state of PE can inhibit the development of the nervous system in offspring.},
}
@article {pmid36042045,
year = {2023},
author = {Ghanbarnasab Behbahani, R and Danyaei, A and Teimoori, A and Tahmasbi, MJ and Neisi, N},
title = {CRISPR/Cas9 mediated knocking out of OPN gene enhances radiosensitivity in MDA-MB-231 breast cancer cell line.},
journal = {Journal of cancer research and clinical oncology},
volume = {149},
number = {7},
pages = {4117-4130},
pmid = {36042045},
issn = {1432-1335},
mesh = {Humans ; Female ; *Breast Neoplasms/genetics/radiotherapy/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; MDA-MB-231 Cells ; Osteopontin/genetics/metabolism ; Cell Proliferation/genetics ; Radiation Tolerance/genetics ; },
abstract = {PURPOSE: Although chemotherapy and radiotherapy in conjunction with surgery have been known as the standard methods for patients with breast cancer, they frequently face resistance due to the failure of cells to death. Accordingly, improving the results requires discovering novel therapeutic approaches based on the changes in the molecular biology of cancer cells. Osteopontin (OPN) is a secreted protein that previous studies have shown to be associated with progression, poor prognosis, and metastasis in breast cancer. The current study examined the synergistic effects of radiotherapy and knocking out of OPN gene, utilizing CRISPR/Cas9 technique in MDA-MB-231 breast cancer cells.<br><br>METHODS: We used to knock out the OPN gene by the two different gRNAs. The cells irradiated 24&#xa0;h after transfection. The mRNA expression, tumor cell proliferation, cell cycle distribution, growth, and apoptosis were measured. Moreover, activation of Chk1 and AKT were measured via western blot.<br><br>RESULTS: We demonstrated the OPN knocking out along with radiation led to the promotion of apoptosis, suppression of downstream genes, reduction of cell viability, and inhibition of cell-cycle progression. The western blot analysis has indicated that the knocking out of the OPN gene along with radiotherapy changes DNA damage responses substantially.<br><br>CONCLUSIONS: The OPN gene knocking out with radiotherapy might be an efficient approach to overcome the radioresistance in breast cancer.},
}
@article {pmid37394555,
year = {2023},
author = {Takahashi, H},
title = {[Accelerating drug discovery process with genome editing technology from Tokushima].},
journal = {Nihon yakurigaku zasshi. Folia pharmacologica Japonica},
volume = {158},
number = {4},
pages = {332-336},
doi = {10.1254/fpj.23015},
pmid = {37394555},
issn = {0015-5691},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Electroporation ; Electroporation Therapies ; },
abstract = {In a series of drug development processes from basic research to non-clinical and clinical trials, genome editing technologies have been making innovative breakthroughs. Genome editing using CRISPR/Cas9 system, which was awarded the Novel Prize in Chemistry in 2020, has greatly streamlined the production of genetically modified mice and cells, which have been used in a variety of drug discovery research and non-clinical trials. Setsuro Tech Inc. (Setsurotech) established in 2017 is a biotech startup originated in Tokushima University. In this paper, we will briefly review genome editing technology using CRISPR/Cas9 system, and then introduce our company, our fundamental technologies; GEEP method (Genome Editing by Electroporation of Cas9 Protein) developed by Takemoto et al., and VIKING method (Versatile NHEJ-based Knock-in using Genome Editing) established by Sawatsubashi et al. Also, we will introduce our contribution to the field of drug discovery research and industrial application of genome editing technology.},
}
@article {pmid37394299,
year = {2023},
author = {Chen, Z and Jin, W and Hoover, A and Chao, Y and Ma, Y},
title = {Decoding the microbiome: advances in genetic manipulation for gut bacteria.},
journal = {Trends in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tim.2023.05.007},
pmid = {37394299},
issn = {1878-4380},
abstract = {Studies of the gut microbiota have revealed associations between specific bacterial species or community compositions with health and disease, yet the causal mechanisms underlying microbiota gene-host interactions remain poorly understood. This is partly due to limited genetic manipulation (GM) tools for gut bacteria. Here, we review current advances and challenges in the development of GM approaches, including clustered regularly interspaced short palindromic repeats (CRISPR)-Cas and transposase-based systems in either model or non-model gut bacteria. By overcoming barriers to 'taming' the gut microbiome, GM tools allow molecular understanding of host-microbiome associations and accelerate microbiome engineering for clinical treatment of cancer and metabolic disorders. Finally, we provide perspectives on the future development of GM for gut microbiome species, where more effort should be placed on assembling a generalized GM pipeline to accelerate the application of groundbreaking GM tools in non-model gut bacteria towards both basic understanding and clinical translation.},
}
@article {pmid37393268,
year = {2023},
author = {Mikkelsen, NS and Bak, RO},
title = {Enrichment strategies to enhance genome editing.},
journal = {Journal of biomedical science},
volume = {30},
number = {1},
pages = {51},
pmid = {37393268},
issn = {1423-0127},
support = {4-1612-391/1//Sundhedsstyrelsen/ ; 101041231//HORIZON EUROPE European Research Council, ERC starting grant/ ; 101057438//HORIZON EUROPE Research and Innovation Actions/ ; R238-2016-3349//Lundbeckfonden/ ; 0134-00113B//Danmarks Frie Forskningsfond/ ; 0242-00009B//Danmarks Frie Forskningsfond/ ; 9144-00001B//Danmarks Frie Forskningsfond/ ; 609033//Aarhus Universitets Forskningsfond/ ; NNF19OC0058238//Novo Nordisk Fonden/ ; NNF17OC0028894//Novo Nordisk Fonden/ ; 8056-00010B//Innovationsfonden/ ; CF20-0424//Carlsbergfondet/ ; CF17-0129//Carlsbergfondet/ ; },
mesh = {*Gene Editing ; *Cell- and Tissue-Based Therapy ; },
abstract = {Genome editing technologies hold great promise for numerous applications including the understanding of cellular and disease mechanisms and the development of gene and cellular therapies. Achieving high editing frequencies is critical to these research areas and to achieve the overall goal of being able to manipulate any target with any desired genetic outcome. However, gene editing technologies sometimes suffer from low editing efficiencies due to several challenges. This is often the case for emerging gene editing technologies, which require assistance for translation into broader applications. Enrichment strategies can support this goal by selecting gene edited cells from non-edited cells. In this review, we elucidate the different enrichment strategies, their many applications in non-clinical and clinical settings, and the remaining need for novel strategies to further improve genome research and gene and cellular therapy studies.},
}
@article {pmid37390831,
year = {2023},
author = {Bush, K and Corsi, GI and Yan, AC and Haynes, K and Layzer, JM and Zhou, JH and Llanga, T and Gorodkin, J and Sullenger, BA},
title = {Utilizing directed evolution to interrogate and optimize CRISPR/Cas guide RNA scaffolds.},
journal = {Cell chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chembiol.2023.06.007},
pmid = {37390831},
issn = {2451-9448},
abstract = {CRISPR-based editing has revolutionized genome engineering despite the observation that many DNA sequences remain challenging to target. Unproductive interactions formed between the single guide RNA's (sgRNA) Cas9-binding scaffold domain and DNA-binding antisense domain are often responsible for such limited editing resolution. To bypass this limitation, we develop a functional SELEX (systematic evolution of ligands by exponential enrichment) approach, termed BLADE (binding and ligand activated directed evolution), to identify numerous, diverse sgRNA variants that bind Streptococcus pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in sgRNA sequence. We also observe that particular variants partner more effectively with specific DNA-binding antisense domains, yielding combinations with enhanced editing efficiencies at various target sites. Using molecular evolution, CRISPR-based systems could be created to efficiently edit even challenging DNA sequences making the genome more tractable to engineering. This selection approach will be valuable for generating sgRNAs with a range of useful activities.},
}
@article {pmid37389931,
year = {2023},
author = {Wei, N and Shang, L and Liu, J and Wang, M and Liu, Y and Zhu, C and Fei, C and Zhang, L and Yang, F and Gu, F},
title = {Engineered Staphylococcus auricularis Cas9 with high-fidelity.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {37},
number = {8},
pages = {e23060},
doi = {10.1096/fj.202202132RR},
pmid = {37389931},
issn = {1530-6860},
support = {32071443//National Natural Science Foundation of China (NSFC)/ ; 31871247//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Staphylococcus/genetics ; Staphylococcus aureus/genetics ; *Staphylococcal Infections ; },
abstract = {CRISPR-Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off-target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5'-NNGG-3' protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced-fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5'-NNGG-3' than SaCas9 of 5'-NNGRRT-3' PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9-HF1, harboring the N269D mutation; SauriCas9-HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE-seq. At certain sites, reduced off-target effects (approximately 61.6- and 111.9-fold improvements) of SauriCas9-HF2 compared with wild-type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9-HF1 and SauriCas9-HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications.},
}
@article {pmid36815235,
year = {2023},
author = {Giandomenico, SL and Schuman, EM},
title = {Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research.},
journal = {FEBS open bio},
volume = {13},
number = {7},
pages = {1164-1176},
pmid = {36815235},
issn = {2211-5463},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Proteolysis ; Gene Editing/methods ; Mutation ; *Pluripotent Stem Cells ; Mammals/genetics ; },
abstract = {Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise, and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e., knock-down, knock-out, mutation, and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination, and epigenetic silencing. The advent of CRISPR-Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often, the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA-based manipulation. Our aim is to present a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to providing the inexperienced reader with a conceptual framework on how to use these tools, and for more in-depth information, we will provide specific references throughout.},
}
@article {pmid37389430,
year = {2023},
author = {Rappleye, CA},
title = {Targeted gene deletions in the dimorphic fungal pathogen Histoplasma using an optimized episomal CRISPR/Cas9 system.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0017823},
doi = {10.1128/msphere.00178-23},
pmid = {37389430},
issn = {2379-5042},
abstract = {The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms with low rates of homologous recombination. Histoplasma is an important respiratory and systemic fungal pathogen that has few reverse genetic options. We describe an optimized CRISPR/Cas system for the efficient generation of mutations in desired genes. The limited requirements for CRISPR/Cas, namely a gene-targeting guide RNA (gRNA) and expression of a Cas endonuclease, enabled both the gRNA and the Streptococcus pyogenes Cas9 gene to be expressed from a single episomal vector. The gRNAs are expressed from a strong Pol(II) promoter, a critical parameter for increasing the recovery of mutated genes, and processed into the mature gRNA by ribozymes in the mRNA. Expression of dual-tandem gRNAs facilitates the generation of gene deletions at a good frequency which can be detected by PCR-based screening of pooled isolates resulting in the isolation of marker-less deletion mutants. The CRISPR/Cas system is encoded on an episomal telomeric vector facilitating curing strains of the CRISPR/Cas vector upon generation of the mutant. We demonstrate the successful application of this CRISPR/Cas system in diverse Histoplasma species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in Histoplasma spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen Histoplasma, methods to inactivate or deplete gene products are inefficient, which hampers progress in defining Histoplasma's virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in Histoplasma and show its validation on multiple genes with selectable and non-selectable phenotypes.},
}
@article {pmid37388410,
year = {2023},
author = {Sharma, A and Hill, KE and Schwarzbauer, JE},
title = {Extracellular matrix composition affects outgrowth of dendrites and dendritic spines on cortical neurons.},
journal = {Frontiers in cellular neuroscience},
volume = {17},
number = {},
pages = {1177663},
pmid = {37388410},
issn = {1662-5102},
abstract = {The composition of the extracellular matrix (ECM) in nervous tissue plays an important role in controlling neuronal outgrowth and synapse development. Changes in both protein and glycosaminoglycan components of the ECM occur with tissue injury and may affect neuron growth. To investigate neuron responses to alterations in fibronectin (FN), a major component of the wound ECM, we grew cortical neurons on cell-derived decellularized matrices composed of wild type FN (FN[+/+]) or of a mutant form of FN (FN[Δ/+]) from which the III13 heparin-binding site had been deleted by CRISPR-Cas 9 gene editing. The most significant effect of the mutant FN was a reduction in dendrite outgrowth. Not only were dendrites shorter on mutant FN[Δ/+]-collagen (COL) matrix than on wild type (FN[+/+]-COL) matrix, but the number of dendrites and dendritic spines per neuron and the spine densities were also dramatically reduced on FN[Δ/+]-COL matrices. Mass spectrometry and immunostaining identified a reduction in tenascin-C (TN-C) levels in the mutant matrix. TN-C is an ECM protein that binds to the III13 site of FN and modulates cell-matrix interactions and has been linked to dendrite development. We propose that TN-C binding to FN in the wound matrix supports dendrite and spine development during repair of damaged neural tissue. Overall, these results show that changes in ECM composition can dramatically affect elaboration of neurites and support the idea that the ECM microenvironment controls neuron morphology and connectivity.},
}
@article {pmid37388245,
year = {2023},
author = {Aouida, M and Saifaldeen, M and Al-Ansari, DE and Taleb, S and Hssain, AA and Ramotar, D},
title = {A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2.},
journal = {Frontiers in molecular biosciences},
volume = {10},
number = {},
pages = {1201347},
pmid = {37388245},
issn = {2296-889X},
abstract = {Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.},
}
@article {pmid37383425,
year = {2023},
author = {Khandia, R and Pandey, MK and Zaki, MEA and Al-Hussain, SA and Baklanov, I and Gurjar, P},
title = {Application of codon usage and context analysis in genes up- or down-regulated in neurodegeneration and cancer to combat comorbidities.},
journal = {Frontiers in molecular neuroscience},
volume = {16},
number = {},
pages = {1200523},
pmid = {37383425},
issn = {1662-5099},
abstract = {INTRODUCTION: Neurodegeneration and cancer present in comorbidities with inverse effects due to the expression of genes and pathways acting in opposition. Identifying and studying the genes simultaneously up or downregulated during morbidities helps curb both ailments together.<br><br>METHODS: This study examines four genes. Three of these (Amyloid Beta Precursor Protein (APP), Cyclin D1 (CCND1), and Cyclin E2 (CCNE2) are upregulated, and one protein phosphatase 2 phosphatase activator (PTPA) is simultaneously downregulated in both disorders. We investigated molecular patterns, codon usage, codon usage bias, nucleotide bias in the third codon position, preferred codons, preferred codon pairs, rare codons, and codon context.<br><br>RESULTS: Parity analysis revealed that T is preferred over A, and G is preferred over C in the third codon position, suggesting composition plays no role in nucleotide bias in both the upregulated and downregulated gene sets and that mutational forces are stronger in upregulated gene sets than in downregulated ones. Transcript length influenced the overall %A composition and codon bias, and the codon AGG exerted the strongest influence on codon usage in both the upregulated and downregulated gene sets. Codons ending in G/C were preferred for 16 amino acids, and glutamic acid-, aspartic acid-, leucine-, valine-, and phenylalanine-initiated codon pairs were preferred in all genes. Codons CTA (Leu), GTA (Val), CAA (Gln), and CGT (Arg) were underrepresented in all examined genes.<br><br>DISCUSSION: Using advanced gene editing tools such as CRISPR/Cas or any other gene augmentation technique, these recoded genes may be introduced into the human body to optimize gene expression levels to augment neurodegeneration and cancer therapeutic regimens simultaneously.},
}
@article {pmid37382533,
year = {2023},
author = {Arnold, J and Glazier, J and Mimee, M},
title = {Genetic Engineering of Resident Bacteria in the Gut Microbiome.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0012723},
doi = {10.1128/jb.00127-23},
pmid = {37382533},
issn = {1098-5530},
abstract = {Techniques by which to genetically manipulate members of the microbiota enable both the evaluation of host-microbe interactions and an avenue by which to monitor and modulate human physiology. Genetic engineering applications have traditionally focused on model gut residents, such as Escherichia coli and lactic acid bacteria. However, emerging efforts by which to develop synthetic biology toolsets for "nonmodel" resident gut microbes could provide an improved foundation for microbiome engineering. As genome engineering tools come online, so too have novel applications for engineered gut microbes. Engineered resident gut bacteria facilitate investigations of the roles of microbes and their metabolites on host health and allow for potential live microbial biotherapeutics. Due to the rapid pace of discovery in this burgeoning field, this minireview highlights advancements in the genetic engineering of all resident gut microbes.},
}
@article {pmid37381079,
year = {2023},
author = {Gehrke, F and Ruiz-Duarte, P and Schindele, A and Wolf, S and Puchta, H},
title = {An inducible CRISPR-Kill system for temporally controlled cell type-specific cell ablation in Arabidopsis thaliana.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.19102},
pmid = {37381079},
issn = {1469-8137},
support = {//Heidelberg Karlsruhe Strategic Partnership/ ; WO 1660/6-2//German Research Foundation (DFG)/ ; WO 1660/2-2//German Research Foundation (DFG)/ ; },
abstract = {The application of the CRISPR/Cas system as a biotechnological tool for genome editing has revolutionized plant biology. Recently, the repertoire was expanded by CRISPR-Kill, enabling CRISPR/Cas-mediated tissue engineering through genome elimination by tissue-specific expression. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), CRISPR-Kill relies on the induction of multiple double-strand breaks (DSBs) in conserved repetitive genome regions, such as the rDNA, causing cell death of the targeted cells. Here, we show that in addition to spatial control by tissue-specific expression, temporal control of CRISPR-mediated cell death is feasible in Arabidopsis thaliana. We established a chemically inducible tissue-specific CRISPR-Kill system that allows the simultaneous detection of targeted cells by fluorescence markers. As proof of concept, we were able to eliminate lateral roots and ablate root stem cells. Moreover, using a multi-tissue promoter, we induced targeted cell death at defined time points in different organs at select developmental stages. Thus, using this system makes it possible to gain new insights into the developmental plasticity of certain cell types. In addition to enabling tissue engineering in plants, our system provides an invaluable tool to study the response of developing plant tissue to cell elimination through positional signaling and cell-to-cell communication.},
}
@article {pmid37380027,
year = {2023},
author = {Saito, M and Xu, P and Faure, G and Maguire, S and Kannan, S and Altae-Tran, H and Vo, S and Desimone, A and Macrae, RK and Zhang, F},
title = {Fanzor is a eukaryotic programmable RNA-guided endonuclease.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-023-06356-2},
pmid = {37380027},
issn = {1476-4687},
abstract = {RNA-guided systems, which employ complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, play a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors, like Cas9 and Cas12, perform guide RNA-dependent DNA cleavage[1]. Although a few eukaryotic RNA-guided systems have been studied so far, such as RNA interference[2] and ribosomal RNA modification[3], it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided system (termed OMEGA) was reported[4,5]. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity[4,6]. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins[4,7], raising the possibility that eukaryotes are also equipped with CRISPR-Cas/OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolved the structure of Spizellomyces punctatus Fz (SpuFz) at 2.7Å using cryogenic-electron microscopy, revealing the conservation of the core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.},
}
@article {pmid37378659,
year = {2023},
author = {Zhang, H and Kong, X and Xue, M and Hu, J and Wang, Z and Wei, Y and Wang, H and Zhou, J and Zhang, W and Xu, M and Shen, X and Yin, F and Ai, Z and Huang, G and Xia, J and Song, X and Li, H and Yuan, Y and Li, J and Zhong, N and Zhang, M and Zhou, Y and Yang, H},
title = {An engineered xCas12i with high activity, high specificity, and broad PAM range.},
journal = {Protein &amp; cell},
volume = {14},
number = {7},
pages = {538-543},
pmid = {37378659},
issn = {1674-8018},
mesh = {*CRISPR-Associated Protein 9 ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37233755,
year = {2023},
author = {Bruder, MR and Aucoin, MG},
title = {A sensitive assay for scrutiny of Autographa californica multiple nucleopolyhedrovirus genes using CRISPR-Cas9.},
journal = {Applied microbiology and biotechnology},
volume = {107},
number = {13},
pages = {4323-4335},
pmid = {37233755},
issn = {1432-0614},
support = {RGPIN 355513-2017//Natural Sciences and Engineering Research Council of Canada/ ; STPGP 462995-14//Natural Sciences and Engineering Research Council of Canada/ ; PGS-D//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Spodoptera ; Baculoviridae/genetics ; Sf9 Cells ; Recombinant Proteins/genetics ; },
abstract = {Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.},
}
@article {pmid36992528,
year = {2023},
author = {Shannon, JG and Hinnebusch, BJ},
title = {Characterization and CRISPR/Cas9-mediated genetic manipulation of neutrophils derived from Hoxb8-ER-immortalized myeloid progenitors.},
journal = {Journal of leukocyte biology},
volume = {114},
number = {1},
pages = {42-52},
doi = {10.1093/jleuko/qiad036},
pmid = {36992528},
issn = {1938-3673},
support = {//NIAID/ ; //NIH/ ; },
mesh = {Mice ; Animals ; *Homeodomain Proteins/genetics/metabolism ; *Neutrophils/metabolism ; Receptors, Estrogen/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Myeloid Progenitor Cells ; },
abstract = {Neutrophils represent a first line of defense against a wide variety of microbial pathogens. Transduction with an estrogen receptor-Hoxb8 transcription factor fusion construct conditionally immortalizes myeloid progenitor cells (NeutPro) capable of differentiation into neutrophils. This system has been very useful for generating large numbers of murine neutrophils for in vitro and in vivo studies. However, some questions remain as to how closely neutrophils derived from these immortalized progenitors reflect primary neutrophils. Here we describe our experience with NeutPro-derived neutrophils as it relates to our studies of Yersinia pestis pathogenesis. NeutPro neutrophils have circular or multilobed nuclei, similar to primary bone marrow neutrophils. Differentiation of neutrophils from NeutPro cells leads to increased expression of CD11b, GR1, CD62L, and Ly6G. However, the NeutPro neutrophils expressed lower levels of Ly6G than bone marrow neutrophils. NeutPro neutrophils produced reactive oxygen species at slightly lower levels than bone marrow neutrophils, and the 2 cell types phagocytosed and killed Y. pestis in vitro to a similar degree. To further demonstrate their utility, we used a nonviral method for nuclear delivery of CRISPR/Cas9 guide RNA complexes to delete genes of interest in NeutPro cells. In summary, we have found these cells to be morphologically and functionally equivalent to primary neutrophils and useful for in vitro assays related to studies of bacterial pathogenesis.},
}
@article {pmid35997636,
year = {2023},
author = {Shnaider, Y and Elad, Y and Rav-David, D and Pashkovsky, E and Leibman, D and Kravchik, M and Shtarkman-Cohen, M and Gal-On, A and Spiegelman, Z},
title = {Development of Powdery Mildew Resistance in Cucumber Using CRISPR/Cas9-Mediated Mutagenesis of CsaMLO8.},
journal = {Phytopathology},
volume = {113},
number = {5},
pages = {786-790},
doi = {10.1094/PHYTO-06-22-0193-FI},
pmid = {35997636},
issn = {0031-949X},
mesh = {*Cucumis sativus/genetics/microbiology ; CRISPR-Cas Systems ; Plant Diseases/microbiology ; Plant Breeding ; Mutagenesis ; Erysiphe ; },
abstract = {Powdery mildew (PM) diseases may severely limit the production of various crops, including members of the family Cucurbitaceae. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility factors essential for fungus penetration into the host cell. In cucumber (Cucumis sativus), natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, we used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cultivar Ilan. Two transgene-free Csamlo8 CRISPR mutant lines (Csamlo-cr-1 and Csamlo-cr-2) were isolated, the first with a 5-bp deletion in exon 1, and the second harboring a 1,280-bp deletion and 10-bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semicommercial growth conditions in the summer growing seasons of 2019 and 2021. These results provide the basis for generating transgene-free powdery mildew resistance in cucumber in any genetic background. This method can directly be employed on commercial cultivars and hybrid parental lines, and thereby substantially shorten and simplify the breeding process for PM resistance in cucumber.},
}
@article {pmid37377914,
year = {2023},
author = {Tuffour, I and Amuzu, S and Bayoumi, H and Surtaj, I and Parrish, C and Willand-Charnley, R},
title = {Early in vitro evidence indicates that deacetylated sialic acids modulate multi-drug resistance in colon and lung cancers via breast cancer resistance protein.},
journal = {Frontiers in oncology},
volume = {13},
number = {},
pages = {1145333},
pmid = {37377914},
issn = {2234-943X},
abstract = {Cancers utilize sugar residues to engage in multidrug resistance. The underlying mechanism of action involving glycans, specifically the glycan sialic acid (Sia) and its various functional group alterations, has not been explored. ATP-binding cassette (ABC) transporter proteins, key proteins utilized by cancers to engage in multidrug resistant (MDR) pathways, contain Sias in their extracellular domains. The core structure of Sia can contain a variety of functional groups, including O-acetylation on the C6 tail. Modulating the expression of acetylated-Sias on Breast Cancer Resistance Protein (BCRP), a significant ABC transporter implicated in MDR, in lung and colon cancer cells directly impacted the ability of cancer cells to either retain or efflux chemotherapeutics. Via CRISPR-Cas-9 gene editing, acetylation was modulated by the removal of CAS1 Domain-containing protein (CASD1) and Sialate O-Acetyl esterase (SIAE) genes. Using western blot, immunofluorescence, gene expression, and drug sensitivity analysis, we confirmed that deacetylated Sias regulated a MDR pathway in colon and lung cancer in early in vitro models. When deacetylated Sias were expressed on BCRP, colon and lung cancer cells were able to export high levels of BCRP to the cell's surface, resulting in an increased BCRP efflux activity, reduced sensitivity to the anticancer drug Mitoxantrone, and high proliferation relative to control cells. These observations correlated with increased levels of cell survival proteins, BcL-2 and PARP1. Further studies also implicated the lysosomal pathway for the observed variation in BCRP levels among the cell variants. RNASeq data analysis of clinical samples revealed higher CASD1 expression as a favorable marker of survival in lung adenocarcinoma. Collectively, our findings indicate that deacetylated Sia is utilized by colon and lung cancers to engage in MDR via overexpression and efflux action of BCRP.},
}
@article {pmid37377126,
year = {2023},
author = {Zhang, H and Deng, M and Li, Z and Ren, Z and Zhang, L and Wang, M and Jiang, S and Yu, L and Wang, X and Li, J},
title = {Unamplified and Label-free Detection of HPV16 DNA Using CRISPR-Cas12a-functionalized Solution-Gated Graphene Transistors.},
journal = {Advanced healthcare materials},
volume = {},
number = {},
pages = {e2300563},
doi = {10.1002/adhm.202300563},
pmid = {37377126},
issn = {2192-2659},
abstract = {The persistent infection of high-risk-human papillomavirus type 16 (HPV16) is considered an essential element for suffering the cervical cancer. Despite polymerase chain reaction, loop-mediated amplification, and the microfluidic chips have been used to detect the HPV16, these methods still exist some drawbacks including time-consuming and false positive results. The CRISPR-Cas system has been widely used in the region of biological detection due to its precise targeted recognition capability. In this contribution, the novel solution-gated graphene transistor sensor is designed to realize the unamplified and label-free detection of HPV16 DNA. Using the precise recognition of the CRISPR-Cas12a system and the gate functionalization, HPV16 DNA can be precisely identified without need the amplification and the labeling. The limit of detection (LOD) of the sensor can be up to 8.3×10[-18 ] M and the detection can be within 20 min. Additionally, the heat-Inactivated clinical samples can be clearly distinguished by the sensor the diagnosis results have the high degree of agreement with q-PCR detection. This article is protected by copyright. All rights reserved.},
}
@article {pmid37376657,
year = {2023},
author = {Panda, K and Parashar, D and Viswanathan, R},
title = {An Update on Current Antiviral Strategies to Combat Human Cytomegalovirus Infection.},
journal = {Viruses},
volume = {15},
number = {6},
pages = {},
pmid = {37376657},
issn = {1999-4915},
mesh = {Humans ; *Cytomegalovirus Infections ; Antiviral Agents/pharmacology/therapeutic use ; Cytomegalovirus/genetics ; },
abstract = {Human cytomegalovirus (HCMV) remains an essential global concern due to its distinct life cycle, mutations and latency. As HCMV is a herpesvirus, it establishes a lifelong persistence in the host through a chronic state of infection. Immunocompromised individuals are at risk of significant morbidity and mortality from the virus. Until now, no effective vaccine has been developed to combat HCMV infection. Only a few antivirals targeting the different stages of the virus lifecycle and viral enzymes are licensed to manage the infection. Therefore, there is an urgent need to find alternate strategies to combat the infection and manage drug resistance. This review will provide an insight into the clinical and preclinical antiviral approaches, including HCMV antiviral drugs and nucleic acid-based therapeutics.},
}
@article {pmid37312453,
year = {2023},
author = {Peters, CW and Hanlon, KS and Ivanchenko, MV and Zinn, E and Linarte, EF and Li, Y and Levy, JM and Liu, DR and Kleinstiver, BP and Indzhykulian, AA and Corey, DP},
title = {Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2023.06.007},
pmid = {37312453},
issn = {1525-0024},
abstract = {Usher syndrome type 1F (USH1F), characterized by congenital lack of hearing and balance and progressive loss of vision, is caused by mutations in the PCDH15 gene. In the Ashkenazi population, a recessive truncation mutation accounts for a large proportion of USH1F cases. The truncation is caused by a single C→T mutation, which converts an arginine codon to a stop (R245X). To test the potential for base editors to revert this mutation, we developed a humanized Pcdh15[R245X] mouse model for USH1F. Mice homozygous for the R245X mutation were deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. Here we show that an adenine base editor (ABE) is capable of reversing the R245X mutation to restore the PCDH15 sequence and function. We packaged a split-intein ABE into dual adeno-associated virus (AAV) vectors and delivered them into cochleas of neonatal USH1F mice. Hearing was not restored in a Pcdh15 constitutive null mouse despite base editing, perhaps because of early disorganization of cochlear hair cells. However, injection of vectors encoding the split ABE into a late-deletion conditional Pcdh15 knockout rescued hearing. This study demonstrates the ability of an ABE to correct the PCDH15 R245X mutation in the cochlea and restore hearing.},
}
@article {pmid37376010,
year = {2023},
author = {Khan, A and Chen, S and Fatima, S and Ahamad, L and Siddiqui, MA},
title = {Biotechnological Tools to Elucidate the Mechanism of Plant and Nematode Interactions.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {12},
pages = {},
pmid = {37376010},
issn = {2223-7747},
support = {2020B0202090001//Key Realm R&amp;D Program of Guangdong Province, China/ ; },
abstract = {Plant-parasitic nematodes (PPNs) pose a threat to global food security in both the developed and developing worlds. PPNs cause crop losses worth a total of more than USD 150 billion worldwide. The sedentary root-knot nematodes (RKNs) also cause severe damage to various agricultural crops and establish compatible relationships with a broad range of host plants. This review aims to provide a broad overview of the strategies used to identify the morpho-physiological and molecular events that occur during RKN parasitism. It describes the most current developments in the transcriptomic, proteomic, and metabolomic strategies of nematodes, which are important for understanding compatible interactions of plants and nematodes, and several strategies for enhancing plant resistance against RKNs. We will highlight recent rapid advances in molecular strategies, such as gene-silencing technologies, RNA interference (RNAi), and small interfering RNA (siRNA) effector proteins, that are leading to considerable progress in understanding the mechanism of plant-nematode interactions. We also take into account genetic engineering strategies, such as targeted genome editing techniques, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system, and quantitative trait loci (QTL), to enhance the resistance of plants against nematodes.},
}
@article {pmid37375454,
year = {2023},
author = {Silva, AMA and Luz, ACO and Xavier, KVM and Barros, MPS and Alves, HB and Batista, MVA and Leal-Balbino, TC},
title = {Analysis of CRISPR/Cas Genetic Structure, Spacer Content and Molecular Epidemiology in Brazilian Acinetobacter baumannii Clinical Isolates.},
journal = {Pathogens (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
pmid = {37375454},
issn = {2076-0817},
abstract = {CRISPR/Cas is a molecular mechanism to prevent predatory viruses from invading bacteria via the insertion of small viral sequences (spacers) in its repetitive locus. The nature of spacer incorporation and the viral origins of spacers provide an overview of the genetic evolution of bacteria, their natural viral predators, and the mechanisms that prokaryotes may use to protect themselves, or to acquire mobile genetic elements such as plasmids. Here, we report on the CRISPR/Cas genetic structure, its spacer content, and strain epidemiology through MLST and CRISPR typing in Acinetobacter baumannii, an opportunistic pathogen intimately related to hospital infections and antimicrobial resistance. Results show distinct genetic characteristics, such as polymorphisms specific to ancestor direct repeats, a well-defined degenerate repeat, and a conserved leader sequence, as well as showing most spacers as targeting bacteriophages, and several self-targeting spacers, directed at prophages. There was a particular relationship between CRISPR/Cas and CC113 in the study of Brazilian isolates, and CRISPR-related typing techniques are interesting for subtyping strains with the same MLST profile. We want to emphasize the significance of descriptive genetic research on CRISPR loci, and we argue that spacer or CRISPR typing are helpful for small-scale investigations, preferably in conjunction with other molecular typing techniques such as MLST.},
}
@article {pmid37373413,
year = {2023},
author = {Cuevas-Ocaña, S and Yang, JY and Aushev, M and Schlossmacher, G and Bear, CE and Hannan, NRF and Perkins, ND and Rossant, J and Wong, AP and Gray, MA},
title = {A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells.},
journal = {International journal of molecular sciences},
volume = {24},
number = {12},
pages = {},
pmid = {37373413},
issn = {1422-0067},
support = {SRC003//Cystic Fibrosis Trust/ ; N/A//CFIT PROGRAM (SICKKIDS and CF-CANADA FOUNDATION)/ ; N/A//EMILY'S ENTOURAGE/ ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; Mutation ; Heterozygote ; },
abstract = {Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.},
}
@article {pmid37373404,
year = {2023},
author = {McBeath, E and Fujiwara, K and Hofmann, MC},
title = {Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice.},
journal = {International journal of molecular sciences},
volume = {24},
number = {12},
pages = {},
pmid = {37373404},
issn = {1422-0067},
support = {R03 HD101650/NH/NIH HHS/United States ; P30 CA016672/NH/NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; Introns/genetics ; Recombinases/genetics ; },
abstract = {Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene's protein after Cre-induced removal of the intron's branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.},
}
@article {pmid37365168,
year = {2023},
author = {Afzal, J and Kshitiz, },
title = {CRISPRing the hypertrophic cardiomyopathy: correcting one pathogenic variant at a time.},
journal = {Signal transduction and targeted therapy},
volume = {8},
number = {1},
pages = {254},
pmid = {37365168},
issn = {2059-3635},
mesh = {Humans ; *Cardiomyopathy, Hypertrophic/genetics/pathology ; *CRISPR-Cas Systems ; },
}
@article {pmid37309812,
year = {2023},
author = {Zhang, Y and Marshall-Phelps, K and de Almeida, RG},
title = {Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.},
journal = {Development (Cambridge, England)},
volume = {150},
number = {12},
pages = {},
doi = {10.1242/dev.201323},
pmid = {37309812},
issn = {1477-9129},
support = {Chancellor's Fellowship//University of Edinburgh/ ; ECG-1754-2021//Medical Research Scotland/ ; //The University of Edinburgh/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Zebrafish/genetics ; Gene Knock-In Techniques ; Genome ; Gene Editing ; },
abstract = {Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.},
}
@article {pmid37272803,
year = {2023},
author = {Liu, Y and Cheng, H and Li, H and Zhang, Y and Wang, M},
title = {A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis.},
journal = {Applied and environmental microbiology},
volume = {89},
number = {6},
pages = {e0023023},
pmid = {37272803},
issn = {1098-5336},
support = {NFSC-32100046//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Lycopene/metabolism ; *CRISPR-Cas Systems ; *Bacillus subtilis/genetics/metabolism ; Plasmids ; Metabolic Engineering ; },
abstract = {Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals. Thus, its metabolic engineering attracts significant attention. Nevertheless, the limited availability of selective markers makes this process difficult and time-consuming, especially in the case of multistep biosynthetic pathways. Here, we employ CRISPR/Cas9 technology to build an easy cloning toolkit that addresses commonly encountered obstacles in the metabolic engineering of B. subtilis, including the chromosomal integration locus, promoter, terminator, and guide RNA (gRNA) target. Six promoters were characterized, and the promoter strengths ranged from 0.9- to 23-fold that of the commonly used strong promoter P43. We characterized seven terminators in B. subtilis, and the termination efficiencies (TEs) of the seven terminators are all more than 90%. Six gRNA targets were designed upstream of the promoter and downstream of the terminator. Using a green fluorescent protein (GFP) reporter, we confirmed integration efficiency with the single-locus integration site is up to 100%. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important product, lycopene. By heterologous expression of the essential genes for lycopene synthesis on the B. subtilis genome, a total of 13 key genes involved in the lycopene biosynthetic pathway were manipulated. Moreover, our findings showed that the gene cluster ispG-idi-dxs-ispD could positively affect the production of lycopene, while the cluster dxr-ispE-ispF-ispH had a negative effect on lycopene production. Hence, our multilocus integration strategy can facilitate the pathway assembly for production of complex chemicals and pharmaceuticals in B. subtilis. IMPORTANCE We present a toolkit that allows for rapid cloning procedures and one-step subcloning to move from plasmid-based expression to stable chromosome integration and expression in a production strain in less than a week. The utility of the customized tool was demonstrated by integrating the MEP (2C-methyl-d-erythritol-4-phosphate) pathway, part of the pentose phosphate pathway (PPP), and the hetero-lycopene biosynthesis genes by stable expression in the genome. The tool could be useful to engineer B. subtilis strains through diverse recombination events and ultimately improve its potential and scope of industrial application as biological chassis.},
}
@article {pmid35636511,
year = {2022},
author = {Fischer, F and Benner, C and Goyala, A and Grigolon, G and Vitiello, D and Wu, J and Zarse, K and Ewald, CY and Ristow, M},
title = {Ingestion of single guide RNAs induces gene overexpression and extends lifespan in Caenorhabditis elegans via CRISPR activation.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {7},
pages = {102085},
pmid = {35636511},
issn = {1083-351X},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/metabolism ; *Caenorhabditis elegans Proteins/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Eating ; Longevity/genetics ; RNA, Small Untranslated ; CRISPR-Cas Systems ; },
abstract = {Inhibition of gene expression in Caenorhabditis elegans, a versatile model organism for studying the genetics of development and aging, is achievable by feeding nematodes with bacteria expressing specific dsRNAs. Overexpression of hypoxia-inducible factor 1 (hif-1) or heat-shock factor 1 (hsf-1) by conventional transgenesis has previously been shown to promote nematodal longevity. However, it is unclear whether other methods of gene overexpression are feasible, particularly with the advent of CRISPR-based techniques. Here, we show that feeding C. elegans engineered to stably express a Cas9-derived synthetic transcription factor with bacteria expressing promoter-specific single guide RNAs (sgRNAs) also allows activation of gene expression. We demonstrate that CRISPR activation via ingested sgRNAs specific for the respective promoter regions of hif-1 or hsf-1 increases gene expression and extends lifespan of C. elegans. Furthermore, and as an in silico resource for future studies aiming to use CRISPR activation in C. elegans, we provide predicted promoter-specific sgRNA target sequences for >13,000 C. elegans genes with experimentally defined transcription start sites. We anticipate that the approach and components described herein will help to facilitate genome-wide gene overexpression studies, for example, to identify modulators of aging or other phenotypes of interest, by enabling induction of transcription by feeding of sgRNA-expressing bacteria to nematodes.},
}
@article {pmid37372948,
year = {2023},
author = {Amberger, M and Grueso, E and Ivics, Z},
title = {CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch.},
journal = {International journal of molecular sciences},
volume = {24},
number = {12},
pages = {},
pmid = {37372948},
issn = {1422-0067},
support = {754658//European Union/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals, Genetically Modified ; },
abstract = {With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive Alu retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an Alu-specific single-guide RNA, were integrated into the genome of target cells via Sleeping-Beauty-mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future.},
}
@article {pmid37372396,
year = {2023},
author = {Guo, R and Wang, H and Meng, C and Gui, H and Li, Y and Chen, F and Zhang, C and Zhang, H and Ding, Q and Zhang, J and Zhang, J and Qian, Y and Zhong, J and Cao, S},
title = {Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR.},
journal = {Genes},
volume = {14},
number = {6},
pages = {},
pmid = {37372396},
issn = {2073-4425},
mesh = {Pregnancy ; Female ; Animals ; Sheep/genetics ; Mice ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Myostatin/genetics/metabolism ; Muscle, Skeletal/metabolism ; Mutation ; },
abstract = {Hu sheep, an indigenous breed in China known for its high fecundity, are being studied to improve their growth and carcass traits. MSTN is a negative regulator of muscle development, and its inactivation results in muscularity. The C-CRISPR system, utilizing multiple neighboring sgRNAs targeting a key exon, has been successfully used to generate genes for complete knockout (KO) monkeys and mice in one step. In this study, the C-CRISPR system was used to generate MSTN-edited Hu sheep; 70 embryos injected with Cas9 mRNA and four sgRNAs targeting exon 3 of sheep MSTN were transferred to 13 recipients. Out of 10 lambs born from five recipients after full-term pregnancies, nine had complete MSTN KO with various mutations. No off-target effects were found. These MSTN-KO Hu sheep showed a double-muscled (DM) phenotype, characterized by a higher body weight at 3 and 4 months old, prominent muscular protrusion, clearly visible intermuscular groves, and muscle hypertrophy. The molecular analysis indicated enhanced AKT and suppressed ERK1/2 signaling in the gluteus muscle of the edited Hu sheep. In conclusion, MSTN complete KO Hu sheep with a DM phenotype were efficiently and specifically generated using C-CRISPR, and the C-CRISPR method is a promising tool for farm animal breeding.},
}
@article {pmid37371069,
year = {2023},
author = {Stemerdink, M and Broekman, S and Peters, T and Kremer, H and de Vrieze, E and van Wijk, E},
title = {Generation and Characterization of a Zebrafish Model for ADGRV1-Associated Retinal Dysfunction Using CRISPR/Cas9 Genome Editing Technology.},
journal = {Cells},
volume = {12},
number = {12},
pages = {},
pmid = {37371069},
issn = {2073-4409},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Retina ; *Retinitis Pigmentosa/genetics ; },
abstract = {Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1[rmc22]). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1[rmc22] zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1[rmc22] zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1[rmc22] zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP.},
}
@article {pmid37371025,
year = {2023},
author = {Wang, G},
title = {Genome Editing for Cystic Fibrosis.},
journal = {Cells},
volume = {12},
number = {12},
pages = {},
pmid = {37371025},
issn = {2073-4409},
support = {HL150370/NH/NIH HHS/United States ; },
mesh = {Humans ; *Cystic Fibrosis/genetics/therapy/pathology ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Gene Editing ; Genetic Therapy ; Precision Medicine ; },
abstract = {Cystic fibrosis (CF) is a monogenic recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator gene (CFTR). Remarkable progress in basic research has led to the discovery of highly effective CFTR modulators. Now ~90% of CF patients are treatable. However, these modulator therapies are not curative and do not cover the full spectrum of CFTR mutations. Thus, there is a continued need to develop a complete and durable therapy that can treat all CF patients once and for all. As CF is a genetic disease, the ultimate therapy would be in-situ repair of the genetic lesions in the genome. Within the past few years, new technologies, such as CRISPR/Cas gene editing, have emerged as an appealing platform to revise the genome, ushering in a new era of genetic therapy. This review provided an update on this rapidly evolving field and the status of adapting the technology for CF therapy.},
}
@article {pmid37370394,
year = {2023},
author = {Javed, MU and Hayat, MT and Mukhtar, H and Imre, K},
title = {CRISPR-Cas9 System: A Prospective Pathway toward Combatting Antibiotic Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
pmid = {37370394},
issn = {2079-6382},
abstract = {Antibiotic resistance is rising to dangerously high levels throughout the world. To cope with this problem, scientists are working on CRISPR-based research so that antibiotic-resistant bacteria can be killed and attacked almost as quickly as antibiotic-sensitive bacteria. Nuclease activity is found in Cas9, which can be programmed with a specific target sequence. This mechanism will only attack pathogens in the microbiota while preserving commensal bacteria. This article portrays the delivery methods used in the CRISPR-Cas system, which are both viral and non-viral, along with its implications and challenges, such as microbial dysbiosis, off-target effects, and failure to counteract intracellular infections. CRISPR-based systems have a lot of applications, such as correcting mutations, developing diagnostics for infectious diseases, improving crops productions, improving breeding techniques, etc. In the future, CRISPR-based systems will revolutionize the world by curing diseases, improving agriculture, and repairing genetic disorders. Though all the drawbacks of the technology, CRISPR carries great potential; thus, the modification and consideration of some aspects could result in a mind-blowing technique to attain all the applications listed and present a game-changing potential.},
}
@article {pmid37370393,
year = {2023},
author = {Roson-Calero, N and Ballesté-Delpierre, C and Fernández, J and Vila, J},
title = {Insights on Current Strategies to Decolonize the Gut from Multidrug-Resistant Bacteria: Pros and Cons.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
pmid = {37370393},
issn = {2079-6382},
abstract = {In the last decades, we have witnessed a steady increase in infections caused by multidrug-resistant (MDR) bacteria. These infections are associated with higher morbidity and mortality. Several interventions should be taken to reduce the emergence and spread of MDR bacteria. The eradication of resistant pathogens colonizing specific human body sites that would likely cause further infection in other sites is one of the most conventional strategies. The objective of this narrative mini-review is to compile and discuss different strategies for the eradication of MDR bacteria from gut microbiota. Here, we analyse the prevalence of MDR bacteria in the community and the hospital and the clinical impact of gut microbiota colonisation with MDR bacteria. Then, several strategies to eliminate MDR bacteria from gut microbiota are described and include: (i) selective decontamination of the digestive tract (SDD) using a cocktail of antibiotics; (ii) the use of pre and probiotics; (iii) fecal microbiota transplantation; (iv) the use of specific phages; (v) engineered CRISPR-Cas Systems. This review intends to provide a state-of-the-art of the most relevant strategies to eradicate MDR bacteria from gut microbiota currently being investigated.},
}
@article {pmid37370299,
year = {2023},
author = {Jwair, NA and Al-Ouqaili, MTS and Al-Marzooq, F},
title = {Inverse Association between the Existence of CRISPR/Cas Systems with Antibiotic Resistance, Extended Spectrum β-Lactamase and Carbapenemase Production in Multidrug, Extensive Drug and Pandrug-Resistant Klebsiella pneumoniae.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
pmid = {37370299},
issn = {2079-6382},
abstract = {Antimicrobial resistance, with the production of extended-spectrum β-lactamases (ESBL) and carbapenemases, is common in the opportunistic pathogen, Klebsiella pneumoniae. This organism has a genome that can contain clustered regularly interspaced short palindromic repeats (CRISPRs), which operate as a defense mechanism against external invaders such as plasmids and viruses. This study aims to determine the association of the CRISPR/Cas systems with antibiotic resistance in K. pneumoniae isolates from Iraqi patients. A total of 100 K. pneumoniae isolates were collected and characterized according to their susceptibility to different antimicrobial agents. The CRISPR/Cas systems were detected via PCR. The phenotypic detection of ESBLs and carbapenemases was performed. The production of ESBL was detected in 71% of the isolates. Carbapenem-resistance was detected in 15% of the isolates, while only 14% were susceptible to all antimicrobial agents. Furthermore, the bacteria were classified into multidrug (77%), extensively drug-resistant (11.0%) and pandrug-resistant (4.0%). There was an inverse association between the presence of the CRISPR/Cas systems and antibiotic resistance, as resistance was higher in the absence of the CRISPR/Cas system. Multidrug resistance in ESBL-producing and carbapenem-resistant K. pneumoniae occurred more frequently in strains negative for the CRISPR/Cas system. Thus, we conclude that genes for exogenous antibiotic resistance can be acquired in the absence of the CRISPR/Cas modules that can protect the bacteria against acquiring foreign DNA.},
}
@article {pmid37367024,
year = {2023},
author = {Zhu, L and Liang, Z and Xu, Y and Chen, Z and Wang, J and Zhou, L},
title = {Ultrasensitive and Rapid Visual Detection of Escherichia coli O157:H7 Based on RAA-CRISPR/Cas12a System.},
journal = {Biosensors},
volume = {13},
number = {6},
pages = {},
pmid = {37367024},
issn = {2079-6374},
support = {2019ZT08L387//the introduced innovative R&amp;D team project under the "The Pearl River Talent Recruitment Program" of Guangdong Province/ ; 21906033//National Natural Science Foundation of China/ ; 22036002//National Natural Science Foundation of China/ ; 2021YFF0703300//National Key Research and Development Program of China/ ; 202102020570//Funding by Science and Technology Projects in Guangzhou/ ; 202201020392//Funding by Science and Technology Projects in Guangzhou/ ; },
mesh = {Humans ; Animals ; *Escherichia coli O157 ; CRISPR-Cas Systems ; Enzyme-Linked Immunosorbent Assay ; Milk ; Real-Time Polymerase Chain Reaction/methods ; Food Microbiology ; },
abstract = {Escherichia coli (E. coli) O157:H7 is a major foodborne and waterborne pathogen that can threaten human health. Due to its high toxicity at low concentrations, it is crucial to establish a time-saving and highly sensitive in situ detection method. Herein, we developed a rapid, ultrasensitive, and visualized method for detecting E. coli O157:H7 based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a technology. The CRISPR/Cas12a-based system was pre-amplified using the RAA method, which showed high sensitivity and enabled detecting as low as ~1 CFU/mL (fluorescence method) and 1 × 10[2] CFU/mL (lateral flow assay) of E. coli O157:H7, which was much lower than the detection limit of the traditional real-time PCR technology (10[3] CFU/mL) and ELISA (10[4]~10[7] CFU/mL). In addition, we demonstrated that this method still has good applicability in practical samples by simulating the detection in real milk and drinking water samples. Importantly, our RAA-CRISPR/Cas12a detection system could complete the overall process (including extraction, amplification, and detection) within 55 min under optimized conditions, which is faster than most other reported sensors, which take several hours to several days. The signal readout could also be visualized by fluorescence generated with a handheld UV lamp or a naked-eye-detected lateral flow assay depending on the DNA reporters used. Because of the advantages of being fast, having high sensitivity, and not requiring sophisticated equipment, this method has a promising application prospect for in situ detection of trace amounts of pathogens.},
}
@article {pmid37041404,
year = {2023},
author = {Lu, X and Jin, H},
title = {A Review of CRISPR-Based Advances in Dermatological Diseases.},
journal = {Molecular diagnosis &amp; therapy},
volume = {27},
number = {4},
pages = {445-456},
pmid = {37041404},
issn = {1179-2000},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) has revolutionized biomedical research by offering novel approaches to genetic and epigenetic manipulation. In dermatology, it has significantly promoted our understanding of complex diseases, and shown great potential in therapeutic applications. In this review, we introduce the adoption of CRISPR technology as a tool to study different types of skin disorders, including monogenic genodermatoses, inflammatory disorders, and cutaneous infections. We highlight the promising preclinical results of CRISPR-mediated treatment and important mechanic discoveries in investigative studies. Future opportunities and remaining challenges are also discussed. We predict that CRISPR will be more extensively used for dermatological research and even be accessible to patients in the future.},
}
@article {pmid36919203,
year = {2023},
author = {Tang, Y and Zhang, Z and Yang, Z and Wu, J},
title = {CRISPR/Cas9 and Agrobacterium tumefaciens virulence proteins synergistically increase efficiency of precise genome editing via homology directed repair in plants.},
journal = {Journal of experimental botany},
volume = {74},
number = {12},
pages = {3518-3530},
doi = {10.1093/jxb/erad096},
pmid = {36919203},
issn = {1460-2431},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Agrobacterium tumefaciens/genetics ; Virulence ; DNA ; },
abstract = {CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.},
}
@article {pmid37369103,
year = {2023},
author = {Giordano, A},
title = {From the lab to the field: CRISPR/Cas addressing challenges in agriculture.},
journal = {Journal of experimental botany},
volume = {74},
number = {12},
pages = {3399-3401},
doi = {10.1093/jxb/erad199},
pmid = {37369103},
issn = {1460-2431},
}
@article {pmid37368988,
year = {2023},
author = {Lim, SR and Lee, HJ and Kim, HJ and Lee, SJ},
title = {Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00323},
pmid = {37368988},
issn = {2161-5063},
abstract = {Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5'-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI[857] and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simultaneous and accurate editing of these two genes with an efficiency of 30%. This enabled the edited cells to retain their lysogenic state at 42 °C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNA method holds significant potential for widespread and practical use in synthetic biology.},
}
@article {pmid37367300,
year = {2023},
author = {Kang, DY and Kim, A and Kim, JN},
title = {CcpA and CodY Regulate CRISPR-Cas System of Streptococcus mutans.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0182623},
doi = {10.1128/spectrum.01826-23},
pmid = {37367300},
issn = {2165-0497},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are widely recognized as bacterial adaptive immune systems against invading viruses and bacteriophages. The oral pathogen Streptococcus mutans encodes two CRISPR-Cas loci (CRISPR1-Cas and CRISPR2-Cas), and their expression under environmental conditions is still under investigation. In this study, we investigated the transcriptional regulation of cas operons by CcpA and CodY, two global regulators that contribute to carbohydrate and (p)ppGpp metabolism. The possible promoter regions for cas operons and the binding sites for CcpA and CodY in the promoter regions of both CRISPR-Cas loci were predicted using computational algorithms. We found that CcpA could directly bind to the upstream region of both cas operons, and detected an allosteric interaction of CodY within the same region. The binding sequences of the two regulators were identified through footprinting analysis. Our results showed that the promoter activity of CRISPR1-Cas was enhanced under fructose-rich conditions, while deletion of the ccpA gene led to reduced activity of the CRISPR2-Cas promoter under the same conditions. Additionally, deletion of the CRISPR systems resulted in a significant decrease in fructose uptake ability compared to the parental strain. Interestingly, the accumulation of guanosine tetraphosphate (ppGpp) was reduced in the presence of mupirocin, which induces a stringent response, in the CRISPR1-Cas-deleted (ΔCR1cas) and both CRISPR-Cas-deleted (ΔCRDcas) mutant strains. Furthermore, the promoter activity of both CRISPRs was enhanced in response to oxidative or membrane stress, while the CRISPR1 promoter activity was reduced under low-pH conditions. Collectively, our findings demonstrate that the transcription of the CRISPR-Cas system is directly regulated by the binding of CcpA and CodY. These regulatory actions play a crucial role in modulating glycolytic processes and exerting effective CRISPR-mediated immunity in response to nutrient availability and environmental cues. IMPORTANCE An effective immune system has evolved not only in eukaryotic organisms but also in microorganisms, enabling them to rapidly detect and neutralize foreign invaders in the environment. Specifically, the CRISPR-Cas system in bacterial cells is established through a complex and sophisticated regulatory mechanism involving specific factors. In this study, we demonstrate that the expression of two CRISPR systems in S. mutans can be controlled by two global regulators, CcpA and CodY, which play critical roles in carbohydrate metabolism and amino acid biosynthesis. Importantly, our results show that the expression of the CRISPR-Cas system in S. mutans influences (p)ppGpp production during the stringent response, which is a gene expression regulatory response that aids in environmental stress adaptation. This transcriptional regulation by these regulators enables a CRISPR-mediated immune response in a host environment with limited availability of carbon sources or amino acids, while ensuring efficient carbon flux and energy expenditure to support multiple metabolic processes.},
}
@article {pmid37366949,
year = {2023},
author = {Mumtaz, Z and Rashid, Z and Ali, A and Arif, A and Ameen, F and AlTami, MS and Yousaf, MZ},
title = {Prospects of Microfluidic Technology in Nucleic Acid Detection Approaches.},
journal = {Biosensors},
volume = {13},
number = {6},
pages = {},
pmid = {37366949},
issn = {2079-6374},
abstract = {Conventional diagnostic techniques are based on the utilization of analyte sampling, sensing and signaling on separate platforms for detection purposes, which must be integrated to a single step procedure in point of care (POC) testing devices. Due to the expeditious nature of microfluidic platforms, the trend has been shifted toward the implementation of these systems for the detection of analytes in biochemical, clinical and food technology. Microfluidic systems molded with substances such as polymers or glass offer the specific and sensitive detection of infectious and noninfectious diseases by providing innumerable benefits, including less cost, good biological affinity, strong capillary action and simple process of fabrication. In the case of nanosensors for nucleic acid detection, some challenges need to be addressed, such as cellular lysis, isolation and amplification of nucleic acid before its detection. To avoid the utilization of laborious steps for executing these processes, advances have been deployed in this perspective for on-chip sample preparation, amplification and detection by the introduction of an emerging field of modular microfluidics that has multiple advantages over integrated microfluidics. This review emphasizes the significance of microfluidic technology for the nucleic acid detection of infectious and non-infectious diseases. The implementation of isothermal amplification in conjunction with the lateral flow assay greatly increases the binding efficiency of nanoparticles and biomolecules and improves the limit of detection and sensitivity. Most importantly, the deployment of paper-based material made of cellulose reduces the overall cost. Microfluidic technology in nucleic acid testing has been discussed by explicating its applications in different fields. Next-generation diagnostic methods can be improved by using CRISPR/Cas technology in microfluidic systems. This review concludes with the comparison and future prospects of various microfluidic systems, detection methods and plasma separation techniques used in microfluidic devices.},
}
@article {pmid37366636,
year = {2023},
author = {González de Aledo, M and Blasco, L and Lopez, M and Ortiz-Cartagena, C and Bleriot, I and Pacios, O and Hernández-García, M and Cantón, R and Tomas, M},
title = {Prophage identification and molecular analysis in the genomes of Pseudomonas aeruginosa strains isolated from critical care patients.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0012823},
doi = {10.1128/msphere.00128-23},
pmid = {37366636},
issn = {2379-5042},
abstract = {Prophages are bacteriophages integrated into the bacterial host's chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 Pseudomonas aeruginosa strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2% and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the P. aeruginosa strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host's quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense. IMPORTANCE Despite being known for decades, prophages remain understudied when compared to the lytic phages employed in phage therapy. This research aims to shed some light into the nature, composition, and role of prophages found within a set of circulating strains of Pseudomas aeruginosa, with special attention to high-risk clones. Given the fact that prophages can effectively influence bacterial pathogenesis, prophage basic research constitutes a topic of growing interest. Furthermore, the abundance of viral defense and regulatory proteins within prophage genomes detected in this study evidences the importance of characterizing the most frequent prophages in circulating clinical strains and in high-risk clones if phage therapy is to be used.},
}
@article {pmid37360717,
year = {2023},
author = {Zhang, Y and Wang, X and Wang, X and Wang, Y and Liu, J and Wang, S and Li, W and Jin, Y and Akhter, D and Chen, J and Hu, J and Pan, R},
title = {Bioinformatic analysis of short-chain dehydrogenase/reductase proteins in plant peroxisomes.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1180647},
pmid = {37360717},
issn = {1664-462X},
abstract = {Peroxisomes are ubiquitous eukaryotic organelles housing not only many important oxidative metabolic reactions, but also some reductive reactions that are less known. Members of the short-chain dehydrogenase/reductase (SDR) superfamily, which are NAD(P)(H)-dependent oxidoreductases, play important roles in plant peroxisomes, including the conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA), auxiliary β-oxidation of fatty acids, and benzaldehyde production. To further explore the function of this family of proteins in the plant peroxisome, we performed an in silico search for peroxisomal SDR proteins from Arabidopsis based on the presence of peroxisome targeting signal peptides. A total of 11 proteins were discovered, among which four were experimentally confirmed to be peroxisomal in this study. Phylogenetic analyses showed the presence of peroxisomal SDR proteins in diverse plant species, indicating the functional conservation of this protein family in peroxisomal metabolism. Knowledge about the known peroxisomal SDRs from other species also allowed us to predict the function of plant SDR proteins within the same subgroup. Furthermore, in silico gene expression profiling revealed strong expression of most SDR genes in floral tissues and during seed germination, suggesting their involvement in reproduction and seed development. Finally, we explored the function of SDRj, a member of a novel subgroup of peroxisomal SDR proteins, by generating and analyzing CRISPR/Cas mutant lines. This work provides a foundation for future research on the biological activities of peroxisomal SDRs to fully understand the redox control of peroxisome functions.},
}
@article {pmid37359892,
year = {2023},
author = {Joshi, T and Sehgal, H and Mandal, SK and Joshi, M and Deepa, PR and Sharma, PK},
title = {Nutraceutical and flavor profiles in underutilized desert legumes of India: gene editing strategies towards sustainable food development.},
journal = {Journal of plant biochemistry and biotechnology},
volume = {},
number = {},
pages = {1-8},
pmid = {37359892},
issn = {0971-7811},
abstract = {Climate change has posed a challenge for food security all over the world in the form of fluctuating crop yields and novel disease outbreaks in plants. Human society's overdependence on a few food crops does not seem a wise precedence. There are numerous underutilized/orphan/neglected legumes growing in the Indian desert regions that can come to the rescue and act as balanced and sustainable sources of nutrients and health-benefitting nutraceuticals. However, challenges such as low plant yield, unidentified metabolic pathways and off-flavor in the food products derived from them prevent the realization of their full potential. Conventional breeding techniques are too slow to achieve the desired modifications and cater to the sharply rising demand for functional foods. The novel gene editing tools like CRISPR-Cas provide more precise tool to manipulate the target genes with or without introduction of foreign DNA and therefore, have better chances to be accepted by governments and societies. The current article reports some of the relevant 'gene editing' success stories with respect to nutraceutical and flavor profiles in the popular legumes. It highlights gaps and future potential, along with areas requiring caution, in underutilized edible legumes of the Indian (semi) arid regions like Prosopis cineraria, Acacia senegal and Cyamopsis tetragonoloba.},
}
@article {pmid37359078,
year = {2023},
author = {Su, W and Li, J and Ji, C and Chen, C and Wang, Y and Dai, H and Li, F and Liu, P},
title = {CRISPR/Cas systems for the detection of nucleic acid and non-nucleic acid targets.},
journal = {Nano research},
volume = {},
number = {},
pages = {1-14},
pmid = {37359078},
issn = {1998-0124},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are becoming powerful tools for disease biomarkers detection. Due to the specific recognition, cis-cleavage and nonspecific trans-cleavage capabilities, CRISPR/Cas systems have implemented the detection of nucleic acid targets (DNA and RNA) as well as non-nucleic acid targets (e.g., proteins, exosomes, cells, and small molecules). In this review, we first summarize the principles and characteristics of various CRISPR/Cas systems, including CRISPR/Cas9, Cas12, Cas13 and Cas14 systems. Then, various types of applications of CRISPR/Cas systems used in detecting nucleic and non-nucleic acid targets are introduced emphatically. Finally, the prospects and challenges of their applications in biosensing are discussed.},
}
@article {pmid37357221,
year = {2023},
author = {Badenetti, L and Manzoli, R and Trevisan, M and D'Avanzo, F and Tomanin, R and Moro, E},
title = {A novel CRISPR/Cas9-based iduronate-2-sulfatase (IDS) knockout human neuronal cell line reveals earliest pathological changes.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {10289},
pmid = {37357221},
issn = {2045-2322},
mesh = {Humans ; Iduronic Acid ; CRISPR-Cas Systems ; *Iduronate Sulfatase/genetics/metabolism ; *Mucopolysaccharidosis II/genetics ; Cell Line ; },
abstract = {Multiple complex intracellular cascades contributing to Hunter syndrome (mucopolysaccharidosis type II) pathogenesis have been recognized and documented in the past years. However, the hierarchy of early cellular abnormalities leading to irreversible neuronal damage is far from being completely understood. To tackle this issue, we have generated two novel iduronate-2-sulfatase (IDS) loss of function human neuronal cell lines by means of genome editing. We show that both neuronal cell lines exhibit no enzymatic activity and increased GAG storage despite a completely different genotype. At a cellular level, they display reduced differentiation, significantly decreased LAMP1 and RAB7 protein levels, impaired lysosomal acidification and increased lipid storage. Moreover, one of the two clones is characterized by a marked decrease of the autophagic marker p62, while none of the two mutants exhibit marked oxidative stress and mitochondrial morphological changes. Based on our preliminary findings, we hypothesize that neuronal differentiation might be significantly affected by IDS functional impairment.},
}
@article {pmid37357000,
year = {2023},
author = {Hong, M and Wang, P and Shangguan, T and Li, GL and Bian, RP and He, W and Jiang, W and Chen, JP},
title = {[Correction of the pathogenic mutation in the G6PC3 gene by adenine base editing in mutant embryos].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {44},
number = {4},
pages = {308-315},
pmid = {37357000},
issn = {0253-2727},
support = {31901049//National Natural Science Foundation of China/ ; 2021MSXM100//Chongqing Science and Health Joint Medical Research Project/ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Adenine ; HEK293 Cells ; Mutation ; Glucose-6-Phosphatase/genetics/metabolism ; },
abstract = {Objective: To determine whether the adenine base editor (ABE7.10) can be used to fix harmful mutations in the human G6PC3 gene. Methods: To investigate the safety of base-edited embryos, off-target analysis by deep sequencing was used to examine the feasibility and editing efficiency of various sgRNA expression vectors. The human HEK293T mutation models and human embryos were also used to test the feasibility and editing efficiency of correction. Results: ①The G6PC3(C295T) mutant cell model was successfully created. ②In the G6PC3(C295T) mutant cell model, three distinct Re-sgRNAs were created and corrected, with base correction efficiency ranging from 8.79% to 19.56% . ③ ABE7.10 could successfully fix mutant bases in the human pathogenic embryo test; however, base editing events had also happened in other locations. ④ With the exception of one noncoding site, which had a high safety rate, deep sequencing analysis revealed that the detection of 32 probable off-target sites was <0.5% . Conclusion: This study proposes a new base correction strategy based on human pathogenic embryos; however, it also produces a certain nontarget site editing, which needs to be further analyzed on the PAM site or editor window.},
}
@article {pmid37353840,
year = {2023},
author = {Zhang, X and Wang, X and Lv, J and Huang, H and Wang, J and Zhuo, M and Tan, Z and Huang, G and Liu, J and Liu, Y and Li, M and Lin, Q and Li, L and Ma, S and Huang, T and Lin, Y and Zhao, X and Rong, Z},
title = {Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {145},
pmid = {37353840},
issn = {1474-760X},
support = {82070002//National Natural Science Foundation of China/ ; 82072329//National Natural Science Foundation of China/ ; 82102443//National Natural Science Foundation of China/ ; 82200072//National Natural Science Foundation of China/ ; 2021A1515110878//GuangDong Basic and Applied Basic Research Foundation/ ; 2021B1515140031//GuangDong Basic and Applied Basic Research Foundation/ ; 2021M701634//Fellowship of China Postdoctoral Science Foundation/ ; 2021M691473//Fellowship of China Postdoctoral Science Foundation/ ; 2022T150300//Fellowship of China Postdoctoral Science Foundation/ ; 2022YFA0806300//National Key R&amp;D Program of China/ ; 2022YFA0806300//National Key R&amp;D Program of China/ ; 2023A1515012269//GuangDong Basic and Applied Basic Research Foundation/ ; 2022A1515011091//GuangDong Basic and Applied Basic Research Foundation/ ; 2022A1515111046//GuangDong Basic and Applied Basic Research Foundation/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *RNA, Circular ; RNA Editing ; Gene Editing ; DNA/genetics ; },
abstract = {BACKGROUND: The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability.<br><br>RESULTS: Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA.<br><br>CONCLUSIONS: CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.},
}
@article {pmid37311609,
year = {2023},
author = {Cheng, M and Tan, C and Xiang, B and Lin, W and Cheng, B and Peng, X and Yang, Y and Lin, Y},
title = {Chain hybridization-based CRISPR-lateral flow assay enables accurate gene visual detection.},
journal = {Analytica chimica acta},
volume = {1270},
number = {},
pages = {341437},
doi = {10.1016/j.aca.2023.341437},
pmid = {37311609},
issn = {1873-4324},
mesh = {*Nucleic Acid Hybridization/methods ; CRISPR-Cas Systems ; SARS-CoV-2/genetics ; Humans ; Mutation ; COVID-19/virology ; },
abstract = {Visualized gene detection based on the CRISPR-Cas12/CRISPR-Cas13 technology and lateral flow assay device (CRISPR-LFA) has shown great potential in point-of-care testing sector. Current CRISPR-LFA methodology mainly utilizes conventional immuno-based LFA test strips, which could visualize whether the reporter probe is trans-cleaved by Cas protein, indicating the target positive detection. However, conventional CRISPR-LFA usually produces false-positive results in target negative assay. Herein, a nucleic acid Chain Hybridization-based Lateral Flow Assay platform, named CHLFA, has been developed to achieve the CRISPR-CHLFA concept. Different from the conventional CRISPR-LFA, the proposed CRISPR-CHLFA system was established based on the nucleic acid hybridization between the GNP-probe embedded in test strips and ssDNA (or ssRNA) reporter from CRISPR (LbaCas12a or LbuCas13a) reaction, which eliminated the requirement of immunoreaction in conventional immuno-based LFA. The assay realized the detection of 1-10 copy of target gene per reaction within 50 min. The CRISPR-CHLFA system achieved highly accurate visual detection of target negative samples, thus overcoming the false-positive problem that often produced in assays using conventional CRISPR-LFA. The CRISPR-CHLFA platform was further adopted for the visual detection of marker gene from SASR-CoV-2 Omicron variant and Mycobacterium tuberculosis (MTB), respectively, and 100% accuracy for the analysis of clinical specimens (45 SASR-CoV-2 specimens and 20 MTB specimens) was obtained. The proposed CRISPR-CHLFA system could provide an alternative platform for the development of POCT biosensors and can be widely adopted in accurate and visualized gene detection.},
}
@article {pmid37204239,
year = {2023},
author = {Márquez-Costa, R and Montagud-Martínez, R and Marqués, MC and Albert, E and Navarro, D and Daròs, JA and Ruiz, R and Rodrigo, G},
title = {Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.},
journal = {Analytical chemistry},
volume = {95},
number = {25},
pages = {9564-9574},
pmid = {37204239},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; DNA ; },
abstract = {Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.},
}
@article {pmid36480733,
year = {2023},
author = {Chen, Z and Yang, X and Xia, H and Wu, C and Yang, J and Dai, T},
title = {A Frontline, Rapid, Nucleic Acid-Based Fusarium circinatum Detection System Using CRISPR/Cas12a Combined with Recombinase Polymerase Amplification.},
journal = {Plant disease},
volume = {107},
number = {6},
pages = {1902-1910},
doi = {10.1094/PDIS-05-22-1234-RE},
pmid = {36480733},
issn = {0191-2917},
mesh = {*Recombinases/genetics ; CRISPR-Cas Systems ; *Fusarium/genetics ; DNA ; },
abstract = {Pitch canker caused by the fungus Fusarium circinatum is a damaging disease that affects pines in Europe, South Africa, and North America in both the southeast and west coast of the United States. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Timely detection, an important pillar of the quarantine effort, can efficiently prevent the introduction of F. circinatum into new areas or facilitate management and eradication strategies in already infested sites. In this study, we developed an F. circinatum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPA-CRISPR/Cas12a). After obtaining DNA, this novel method can be utilized for the molecular identification of F. circinatum using the naked eye and can specifically detect F. circinatum at DNA concentrations as low as 200 fg within 30 min at 37°C. The system is sensitive for both standard laboratory samples and samples from the field. In summary, we have developed a simple, rapid, sensitive, unaided-eye visualization, RPA-CRISPR/Cas12a-based detection system for the molecular identification of F. circinatum that does not require technical expertise or expensive ancillary equipment.},
}
@article {pmid36471470,
year = {2023},
author = {Wang, Q and Qin, M and Coleman, JJ and Shang, W and Hu, X},
title = {Rapid and Sensitive Detection of Verticillium dahliae from Complex Samples Using CRISPR/Cas12a Technology Combined with RPA.},
journal = {Plant disease},
volume = {107},
number = {6},
pages = {1664-1669},
doi = {10.1094/PDIS-08-22-1790-SC},
pmid = {36471470},
issn = {0191-2917},
mesh = {*CRISPR-Cas Systems ; Commerce ; Internationality ; *Ascomycota ; Soil ; Technology ; },
abstract = {Verticillium wilt is primarily caused by the fungus Verticillium dahliae and represents one of the most important worldwide soilborne plant diseases. The causal agent can be spread by microsclerotia and conidia attached to seeds during national/international trade or in soil between fields. Consequently, accurate, sensitive, and rapid detection of V. dahliae from complex samples is critical for restricting entry of the pathogen to a new region/environment and enforcing early management of Verticillium wilt. Based on CRISPR/Cas12a and recombinant polymerase amplification (RPA) technologies, we developed an accurate, sensitive, and rapid detection method for V. dahliae with paper-based lateral flow strips. A highly efficient and specific CRISPR RNA (crRNA) was designed for the GAPDH gene of V. dahliae and was validated using several closely related Verticillium spp. Excluding the time required for the DNA extraction from the complex samples, a minimum of 40 min was required for the detection time. The RPA-CRISPR/Cas12a detection system had a lower detection limit of ∼10 copies of genomic DNA per reaction and was able to successfully detect as little as one microsclerotium per gram of soil. In addition, field samples displaying symptoms suggestive of V. dahliae were able to be positively identified for the presence of V. dahliae. Taken together, this study broadens the applications of CRISPR/Cas12a nucleic acid detection to soilborne crop diseases and will contribute to the future development of field-deployable diagnostic tools.},
}
@article {pmid37353834,
year = {2023},
author = {Chen, H and Liu, X and Li, L and Tan, Q and Li, S and Li, L and Li, C and Fu, J and Lu, Y and Wang, Y and Sun, Y and Luo, ZG and Lu, Z and Sun, Q and Liu, Z},
title = {CATI: an efficient gene integration method for rodent and primate embryos by MMEJ suppression.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {146},
pmid = {37353834},
issn = {1474-760X},
support = {2018SHZDZX05//Shanghai Municipal Science and Technology Major Project/ ; XDB32060000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; ZDBS-LY-SM019//the Basic Frontier Scientific Research Program of CAS/ ; 31825018//National Natural Science Foundation of China Grant/ ; 82021001//National Natural Science Foundation of China Grant/ ; 2022YFF0710901//National Key Research and Development Program of China/ ; BX20200350//China National Postdoctoral Program for Innovative Talents/ ; },
mesh = {Animals ; Mice ; *Rodentia ; *Primates ; DNA Repair ; Gene Editing ; Haplorhini ; DNA End-Joining Repair ; CRISPR-Cas Systems ; },
abstract = {The efficiency of homology-directed repair (HDR) plays a crucial role in the development of animal models and gene therapy. We demonstrate that microhomology-mediated end-joining (MMEJ) constitutes a substantial proportion of DNA repair during CRISPR-mediated gene editing. Using CasRx to downregulate a key MMEJ factor, Polymerase Q (Polq), we improve the targeted integration efficiency of linearized DNA fragments and single-strand oligonucleotides (ssODN) in mouse embryos and offspring. CasRX-assisted targeted integration (CATI) also leads to substantial improvements in HDR efficiency during the CRISPR/Cas9 editing of monkey embryos. We present a promising tool for generating monkey models and developing gene therapies for clinical trials.},
}
@article {pmid37352230,
year = {2023},
author = {Paraan, M and Nasef, M and Chou-Zheng, L and Khweis, SA and Schoeffler, AJ and Hatoum-Aslan, A and Stagg, SM and Dunkle, JA},
title = {The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems.},
journal = {PloS one},
volume = {18},
number = {6},
pages = {e0287461},
pmid = {37352230},
issn = {1932-6203},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; RNA, Bacterial/metabolism ; Multiprotein Complexes/metabolism ; *CRISPR-Associated Proteins/genetics ; },
abstract = {Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.},
}
@article {pmid37351704,
year = {2023},
author = {Zhuo, C and Song, Z and Cui, J and Gong, Y and Tang, Q and Zhang, K and Song, X and Liao, X},
title = {Electrochemical biosensor strategy combining DNA entropy-driven technology to activate CRISPR-Cas13a activity and triple-stranded nucleic acids to detect SARS-CoV-2 RdRp gene.},
journal = {Mikrochimica acta},
volume = {190},
number = {7},
pages = {272},
pmid = {37351704},
issn = {1436-5073},
mesh = {Humans ; *Nucleic Acids ; SARS-CoV-2/genetics ; Entropy ; *COVID-19/diagnosis ; DNA/genetics ; Technology ; RNA-Dependent RNA Polymerase ; },
abstract = {By merging DNA entropy-driven technology with triple-stranded nucleic acids in an electrochemical biosensor to detect the SARS-CoV-2 RdRp gene, we tackled the challenges of false negatives and the high cost of SARS-CoV-2 detection. The approach generates a CRISPR-Cas 13a-activated RNA activator, which then stimulates CRISPR-Cas 13a activity using an entropy-driven mechanism. The activated CRISPR-Cas 13a can cleave Hoogsteen DNA due to the insertion of two uracil (-U-U-) in Hoogsteen DNA. The DNA tetrahedra changed on the electrode surface and can therefore not construct a three-stranded structure after cleaving Hoogsteen DNA. Significantly, this DNA tetrahedron/Hoogsteen DNA-based biosensor can regenerate at pH = 10.0, which keeps Hoogsteen DNA away from the electrode surface, allowing the biosensor to function at pH = 7.0. We could use this technique to detect the SARS-CoV-2 RdRp gene with a detection limit of 89.86 aM. Furthermore, the detection method is very stable and repeatable. This technique offers the prospect of detecting SARS-CoV-2 at a reasonable cost. This work has potential applications in the dynamic assessment of the diagnostic and therapeutic efficacy of SARS-CoV-2 infection and in the screening of environmental samples.},
}
@article {pmid37349508,
year = {2023},
author = {Patinios, C and de Vries, ST and Diallo, M and Lanza, L and Verbrugge, PLJVQ and López-Contreras, AM and van der Oost, J and Weusthuis, RA and Kengen, SWM},
title = {Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {10153},
pmid = {37349508},
issn = {2045-2322},
support = {642068//Marie Sklodowska Curie ITN/ ; },
mesh = {*Clostridium beijerinckii/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Clostridium/genetics ; Butanols/metabolism ; 1-Butanol/metabolism ; Gene Editing/methods ; },
abstract = {Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.},
}
@article {pmid37347230,
year = {2023},
author = {Mayorga-Ramos, A and Zúñiga-Miranda, J and Carrera-Pacheco, SE and Barba-Ostria, C and Guamán, LP},
title = {CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance.},
journal = {ACS infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsinfecdis.2c00649},
pmid = {37347230},
issn = {2373-8227},
abstract = {The emergence of antibiotic-resistant bacterial strains is a source of public health concern across the globe. As the discovery of new conventional antibiotics has stalled significantly over the past decade, there is an urgency to develop novel approaches to address drug resistance in infectious diseases. The use of a CRISPR-Cas-based system for the precise elimination of targeted bacterial populations holds promise as an innovative approach for new antimicrobial agent design. The CRISPR-Cas targeting system is celebrated for its high versatility and specificity, offering an excellent opportunity to fight antibiotic resistance in pathogens by selectively inactivating genes involved in antibiotic resistance, biofilm formation, pathogenicity, virulence, or bacterial viability. The CRISPR-Cas strategy can enact antimicrobial effects by two approaches: inactivation of chromosomal genes or curing of plasmids encoding antibiotic resistance. In this Review, we provide an overview of the main CRISPR-Cas systems utilized for the creation of these antimicrobials, as well as highlighting promising studies in the field. We also offer a detailed discussion about the most commonly used mechanisms for CRISPR-Cas delivery: bacteriophages, nanoparticles, and conjugative plasmids. Lastly, we address possible mechanisms of interference that should be considered during the intelligent design of these novel approaches.},
}
@article {pmid37347159,
year = {2023},
author = {Wu, Y and Zhang, J and Wang, B and Zhang, Y and Li, H and Liu, Y and Yin, J and He, D and Luo, H and Gan, F and Tang, B and Tang, XF},
title = {Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0028823},
doi = {10.1128/spectrum.00288-23},
pmid = {37347159},
issn = {2165-0497},
abstract = {The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.},
}
@article {pmid37347132,
year = {2023},
author = {Vardanyan, VH and Wang, Q and Kolomeisky, AB},
title = {Dynamics of single-base editing: Theoretical analysis.},
journal = {The Journal of chemical physics},
volume = {158},
number = {24},
pages = {},
doi = {10.1063/5.0157193},
pmid = {37347132},
issn = {1089-7690},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Mutation ; Cytosine ; DNA/genetics ; },
abstract = {Recent experimental advances led to the development of DNA base editors (BEs) with single-nucleotide precision, which is critical for future progress in various scientific and technological fields. The molecular mechanisms of single-base discrimination, however, remain poorly understood. Using a recently developed stochastic approach, we theoretically investigated the dynamics of single-base editing. More specifically, transient and mean times to edit "TC" motifs by cytosine BEs are explicitly evaluated for correct (target) and incorrect (bystander) locations on DNA. In addition, the effect of mutations on the dynamics of the single-base edition is also analyzed. It is found that for most ranges of parameters, it is possible to temporarily separate target and bystander products of base editing, supporting the idea of dynamic selectivity as a method of improving the precision of single-base editing. We conclude that to improve the efficiency of single-base editing, selecting the probability or selecting the time requires different strategies. Physical-chemical arguments to explain the observed dynamic properties are presented. The theoretical analysis clarifies some important aspects of the molecular mechanisms of selective base editing.},
}
@article {pmid37343690,
year = {2023},
author = {Yu, M and Hu, S and Tang, B and Yang, H and Sun, D},
title = {Engineering Escherichia coli Nissle 1917 as a microbial chassis for therapeutic and industrial applications.},
journal = {Biotechnology advances},
volume = {67},
number = {},
pages = {108202},
doi = {10.1016/j.biotechadv.2023.108202},
pmid = {37343690},
issn = {1873-1899},
abstract = {Genetically engineered microbes, especially Escherichia coli, have been widely used in the biosynthesis of proteins and metabolites for medical and industrial applications. As a traditional probiotic with a well-established safety record, E. coli Nissle 1917 (EcN) has recently emerged as a microbial chassis for generating living therapeutics, drug delivery vehicles, and microbial platforms for industrial production. Despite the availability of genetic tools for engineering laboratory E. coli K-12 and B strains, new genetic engineering systems are still greatly needed to expand the application range of EcN. In this review, we have summarized the latest progress in the development of genetic engineering systems in EcN, as well as their applications in the biosynthesis and delivery of valuable small molecules and biomacromolecules of medical and/or industrial interest, followed by a glimpse of how this rapidly growing field will evolve in the future.},
}
@article {pmid37343553,
year = {2023},
author = {Cui, N and Zhang, JT and Liu, Y and Liu, Y and Liu, XY and Wang, C and Huang, H and Jia, N},
title = {Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2023.05.036},
pmid = {37343553},
issn = {1097-4164},
abstract = {Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying Csf[crRNA] complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the Csf[crRNA] complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.},
}
@article {pmid37342929,
year = {2023},
author = {},
title = {Unleashing CRISPR on Cancer.},
journal = {The New England journal of medicine},
volume = {388},
number = {25},
pages = {e78},
doi = {10.1056/NEJMp2303851},
pmid = {37342929},
issn = {1533-4406},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *Neoplasms/genetics/therapy ; },
}
@article {pmid37341274,
year = {2023},
author = {Frâncio, L and Freitas, MV and Matte, U},
title = {CRISPR/Cas patents and health-related publications in South America.},
journal = {Anais da Academia Brasileira de Ciencias},
volume = {95},
number = {2},
pages = {e20220629},
doi = {10.1590/0001-3765202320220629},
pmid = {37341274},
issn = {1678-2690},
mesh = {*CRISPR-Cas Systems/genetics ; South America ; Databases, Factual ; },
abstract = {CRISPR/Cas is being increasingly used for various applications. However, different countries introduce new technologies at different paces and purposes. This study reviews research progress using the CRISPR/Cas system in South America, focusing on health-related applications. The PubMed database was used to identify relevant articles about gene editing with CRISPR/Cas, whereas patents were searched in the Patentscope database. In addition, ClinicalTrials.gov was used to find information on active and recruiting clinical trials. A total of 668 non-duplicated articles (extracted from PubMed) and 225 patents (not all health-related) were found. One hundred ninety-two articles on health-related applications of CRISPR/Cas were analyzed in detail. In 95 out of these, more than 50% of the authors were affiliated with South American institutions. Experimental CRISPR/Cas studies target different diseases, particularly cancer, neurological, and endocrine disorders. Most patents refer to generic applications, but those with clear disease indications are for inborn errors of metabolism, ophthalmological, hematological, and immunological disorders. No clinical trials were found involving Latin American countries. Although research on gene editing in South America is advancing, our data show the low number of national innovations protected by intellectual property in this field.},
}
@article {pmid37339984,
year = {2023},
author = {Aldag, P and Rutkauskas, M and Madariaga-Marcos, J and Songailiene, I and Sinkunas, T and Kemmerich, F and Kauert, D and Siksnys, V and Seidel, R},
title = {Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {3654},
pmid = {37339984},
issn = {2041-1723},
support = {GA 724863//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; SE 1646/9-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; },
abstract = {CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.},
}
@article {pmid37339909,
year = {2023},
author = {Lavelle, K and McDonnell, B and Fitzgerald, G and van Sinderen, D and Mahony, J},
title = {Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes.},
journal = {FEMS microbiology reviews},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsre/fuad032},
pmid = {37339909},
issn = {1574-6976},
abstract = {Bacteriophages (or phages) represent a persistent threat to the success and reliability of food fermentation processes. Recent reports of phages that infect Streptococcus thermophilus have highlighted the diversification of phages of this species. Phages of S. thermophilus typically exhibit a narrow range, a feature that is suggestive of diverse receptor moieties being presented on the cell surface of the host. Cell wall polysaccharides including rhamnose-glucose polysaccharides (RGP) and exopolysaccharides (EPS) have been implicated as being involved in the initial interactions with several phages of this species. Following internalisation of the phage genome, the host presents several defences including CRISPR-Cas and restriction and modification systems to limit phage proliferation. This review provides a current and holistic view of the interactions of phages and their S. thermophilus host cells and how this has influenced the diversity and evolution of both entities.},
}
@article {pmid37314680,
year = {2023},
author = {Testa, LC and Musunuru, K},
title = {Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {37},
number = {4},
pages = {453-462},
pmid = {37314680},
issn = {1179-190X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Genetic Therapy/methods ; Phenotype ; },
abstract = {Collectively, genetic disorders affect approximately 350 million individuals worldwide and are a major global health burden. Despite substantial progress in identification of new disease-causing genes, variants, and molecular etiologies, nearly all rare diseases have no targeted therapeutics that can address their underlying molecular causes. Base editing (BE) and prime editing (PE), two newly described iterations of CRISPR-Cas9 genome editing, represent potential therapeutic strategies that could be used to precisely, efficiently, permanently, and safely correct patients' pathogenic variants and ameliorate disease sequelae. Unlike "standard" CRISPR-Cas9 genome editing, these technologies do not rely on double-strand break (DSB) formation, thus improving safety by decreasing the likelihood of undesired insertions and deletions (indels) at the target site. Here, we provide an overview of BE and PE, including their structures, mechanisms, and differences from standard CRISPR-Cas9 genome editing. We describe several examples of the use of BE and PE to improve rare and common disease phenotypes in preclinical models and human patients, with an emphasis on in vivo editing efficacy, safety, and delivery method. We also discuss recently developed delivery methods for these technologies that may be used in future clinical settings.},
}
@article {pmid37244887,
year = {2023},
author = {Kheirandish, MH and Rahmani, B and Zarei Jaliani, H and Barkhordari, F and Mazlomi, MA and Davami, F},
title = {Efficient site-specific integration in CHO-K1 cells using CRISPR/Cas9-modified donors.},
journal = {Molecular biology reports},
volume = {50},
number = {7},
pages = {5889-5899},
pmid = {37244887},
issn = {1573-4978},
support = {99-1-148-47958//Tehran University of Medical Sciences and Health Services/ ; },
mesh = {Animals ; Cricetinae ; Humans ; *CRISPR-Cas Systems/genetics ; CHO Cells ; Clone Cells ; Cell Differentiation ; *Chromosomes, Human, Pair 3 ; Cricetulus ; },
abstract = {BACKGROUND: Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression.<br><br>METHODS AND RESULTS: In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line.<br><br>CONCLUSION: Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line&#xa0;development.},
}
@article {pmid37140059,
year = {2023},
author = {Huszár, K and Welker, Z and Györgypál, Z and Tóth, E and Ligeti, Z and Kulcsár, PI and Dancsó, J and Tálas, A and Krausz, SL and Varga, &#xc9; and Welker, E},
title = {Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs.},
journal = {Nucleic acids research},
volume = {51},
number = {11},
pages = {5847-5863},
pmid = {37140059},
issn = {1362-4962},
support = {GINOP-2.1.2-8.1.4-16-2018-00414//Ministry of National Economy/ ; K128188//National Research, Development and Innovation Office/ ; BO/764/20//J&#xe1;nos Bolyai Research Scholarship of the Hungarian Academy of Sciences/ ; },
mesh = {*Streptococcus pyogenes/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Nucleotides ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3' extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9.},
}
@article {pmid37339457,
year = {2023},
author = {Weller, J and Pallaseni, A and Koeppel, J and Parts, L},
title = {Predicting Mutations Generated by Cas9, Base Editing, and Prime Editing in Mammalian Cells.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2023.0016},
pmid = {37339457},
issn = {2573-1602},
abstract = {The first fruits of the CRISPR-Cas revolution are starting to enter the clinic, with gene editing therapies offering solutions to previously incurable genetic diseases. The success of such applications hinges on control over the mutations that are generated, which are known to vary depending on the targeted locus. In this review, we present the current state of understanding and predicting CRISPR-Cas cutting, base editing, and prime editing outcomes in mammalian cells. We first provide an introduction to the basics of DNA repair and machine learning that the models rely on. We then overview the datasets and methods created for characterizing edits at scale, as well as the insights that have been derived from them. The predictions generated from these models serve as a foundation for designing efficient experiments across the broad contexts where these tools are applied.},
}
@article {pmid37339299,
year = {2023},
author = {Afolabi, MOS and Sodeke, S},
title = {Social Inequality and Human Genome Editing: A Nuanced Analysis of the Ubuntuan Ethical Prism.},
journal = {The American journal of bioethics : AJOB},
volume = {23},
number = {7},
pages = {129-131},
doi = {10.1080/15265161.2023.2207526},
pmid = {37339299},
issn = {1536-0075},
mesh = {Humans ; *Gene Editing/ethics ; South Africa ; *CRISPR-Cas Systems ; Genome, Human ; Socioeconomic Factors ; },
}
@article {pmid37338599,
year = {2023},
author = {Bishnoi, R and Kaur, S and Sandhu, JS and Singla, D},
title = {Genome engineering of disease susceptibility genes for enhancing resistance in plants.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {3},
pages = {207},
pmid = {37338599},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Disease Susceptibility ; *Plant Diseases/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Disease Resistance/genetics ; Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Introgression of disease resistance genes (R-genes) to fight against an array of phytopathogens takes several years using conventional breeding approaches. Pathogens develop mechanism(s) to escape plants immune system by evolving new strains/races, thus making them susceptible to disease. Conversely, disruption of host susceptibility factors (or S-genes) provides opportunities for resistance breeding in crops. S-genes are often exploited by phytopathogens to promote their growth and infection. Therefore, identification and targeting of disease susceptibility genes (S-genes) are gaining more attention for the acquisition of resistance in plants. Genome engineering of S-genes results in targeted, transgene-free gene modification through CRISPR-Cas-mediated technology and has been reported in several agriculturally important crops. In this review, we discuss the defense mechanism in plants against phytopathogens, tug of war between R-genes and S-genes, in silico techniques for identification of host-target (S-) genes and pathogen effector molecule(s), CRISPR-Cas-mediated S-gene engineering, its applications, challenges, and future prospects.},
}
@article {pmid37336428,
year = {2023},
author = {Ali, Y and Inusa, I and Sanghvi, G and Mandaliya, VB and Bishoyi, AK},
title = {The current status of phage therapy and its advancement towards establishing standard antimicrobials for combating multi drug-resistant bacterial pathogens.},
journal = {Microbial pathogenesis},
volume = {181},
number = {},
pages = {106199},
doi = {10.1016/j.micpath.2023.106199},
pmid = {37336428},
issn = {1096-1208},
abstract = {Phage therapy; a revived antimicrobial weapon, has great therapeutic advantages with the main ones being its ability to eradicate multidrug-resistant pathogens as well as selective toxicity, which ensures that beneficial microbiota is not harmed, unlike antibiotics. These therapeutic properties make phage therapy a novel approach for combating resistant pathogens. Since millions of people across the globe succumb to multidrug-resistant infections, the implementation of phage therapy as a standard antimicrobial could transform global medicine as it offers greater therapeutic advantages than conventional antibiotics. Although phage therapy has incomplete clinical data, such as a lack of standard dosage and the ideal mode of administration, the conducted clinical studies report its safety and efficacy in some case studies, and therefore, this could lessen the concerns of its skeptics. Since its discovery, the development of phage therapeutics has been in a smooth progression. Concerns about phage resistance in populations of pathogenic bacteria are raised when bacteria are exposed to phages. Bacteria can use restriction-modification, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) defense, or mutations in the phage receptors to prevent phage invasion. Phage resistance, however, is often costly for the bacteria and may lead to a reduction in its virulence. The ongoing competition between bacteria and phage, on the other hand, ensures the emergence of phage strains that have evolved to infect resistant bacteria. A phage can quickly adapt by altering one or more aspects of its mode of infection, evading a resistance mechanism through genetic modifications, or directly thwarting the CRISPR-Cas defense. Using phage-bacterium coevolution as a technique could be crucial in the development of phage therapy as well. Through its recent advancement, gene-editing tools such as CRISPR-Cas allow the bioengineering of phages to produce phage cocktails that have broad spectrum activities, which could maximize the treatment's efficacy. This review presents the current state of phage therapy and its progression toward establishing standard medicine for combating antibiotic resistance. Recent clinical trials of phage therapy, some important case studies, and other ongoing clinical studies of phage therapy are all presented in this review. Furthermore, the recent advancement in the development of phage therapeutics, its application in various sectors, and concerns regarding its implementation are also highlighted here. Phage therapy has great potential and could help the fight against drug-resistant bacterial pathogens.},
}
@article {pmid37335501,
year = {2023},
author = {Chang, Y and Liu, B and Jiang, Y and Cao, D and Liu, Y and Li, Y},
title = {Induce male sterility by CRISPR/Cas9-mediated mitochondrial genome editing in tobacco.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {3},
pages = {205},
pmid = {37335501},
issn = {1438-7948},
mesh = {Animals ; Male ; Humans ; Gene Editing ; CRISPR-Cas Systems ; Tobacco/genetics ; *Genome, Mitochondrial ; DNA, Mitochondrial/genetics ; *Infertility, Male/genetics ; },
abstract = {Genome editing has become more and more popular in animal and plant systems following the emergence of CRISPR/Cas9 technology. However, target sequence modification by CRISPR/Cas9 has not been reported in the plant mitochondrial genome, mtDNA. In plants, a type of male sterility known as cytoplasmic male sterility (CMS) has been associated with certain mitochondrial genes, but few genes have been confirmed by direct mitochondrial gene-targeted modifications. Here, the CMS-associated gene (mtatp9) in tobacco was cleaved using mitoCRISPR/Cas9 with a mitochondrial localization signal. The male-sterile mutant, with aborted stamens, exhibited only 70% of the mtDNA copy number of the wild type and exhibited an altered percentage of heteroplasmic mtatp9 alleles; otherwise, the seed setting rate of the mutant flowers was zero. Transcriptomic analyses showed that glycolysis, tricarboxylic acid cycle metabolism and the oxidative phosphorylation pathway, which are all related to aerobic respiration, were inhibited in stamens of the male-sterile gene-edited mutant. In addition, overexpression of the synonymous mutations dsmtatp9 could restore fertility to the male-sterile mutant. Our results strongly suggest that mutation of mtatp9 causes CMS and that mitoCRISPR/Cas9 can be used to modify the mitochondrial genome of plants.},
}
@article {pmid37294549,
year = {2023},
author = {Feng, S and Chen, H and Hu, Z and Wu, T and Liu, Z},
title = {Ultrasensitive Detection of miRNA via CRISPR/Cas12a Coupled with Strand Displacement Amplification Reaction.},
journal = {ACS applied materials &amp; interfaces},
volume = {15},
number = {24},
pages = {28933-28940},
doi = {10.1021/acsami.3c03399},
pmid = {37294549},
issn = {1944-8252},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Gelatin ; Trypsin ; Food ; *MicroRNAs/genetics ; *Biosensing Techniques ; },
abstract = {MicroRNA (miRNA) is a promising biomarker for the diagnosis, monitoring, and prognostic evaluation of diseases, especially cancer. The existing miRNA detection methods usually need external instruments for quantitative signal output, limiting their practical applications in point-of-care (POC) settings. Here, we propose a distance-based biosensor through a responsive hydrogel, in combination with a CRISPR/Cas12a system and target-triggered strand displacement amplification (SDA) reaction for visual quantitative and sensitive measurement of miRNA. The target miRNA is first converted into plenty of double-stranded DNA (dsDNA) via target-triggered SDA reaction. Then, the dsDNA products trigger the collateral cleavage activity of CRISPR/Cas12a, leading to the release of trypsin from magnetic beads (MBs). The released trypsin can hydrolyze gelatin, and hence the permeability of gelatin-treated filter paper is increased, resulting in a visible distance signal on a cotton thread. Using this system, the concentration of the target miRNA can be quantified visually without any assistance of instruments, and a detection limit of 6.28 pM is obtained. In addition, the target miRNA in human serum samples and cell lysates can also be detected accurately. Owing to the characteristics of simplicity, sensitivity, specificity, and portability, the proposed biosensor provides a new tool for miRNA detection and holds great promise in POC applications.},
}
@article {pmid37261400,
year = {2023},
author = {Long, X and Li, J and Luo, T and Liu, H and Deng, Z and Ding, J and Gong, Z and Yang, Y and Zhong, S},
title = {CRISPR-Cas12a coupled with cyclic reverse transcription for amplified detection of miRNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {50},
pages = {7763-7766},
doi = {10.1039/d3cc01712e},
pmid = {37261400},
issn = {1364-548X},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Prospective Studies ; Reverse Transcription ; Biological Assay ; *MicroRNAs/genetics ; *Biosensing Techniques ; },
abstract = {In this work, we present a highly sensitive, specific, and versatile method to quantify miRNA expression by coupling CRISPR-Cas12a with cyclic reverse transcription (CRT), termed as CRISPR-CRT. Each miRNA target was first converted and amplified into multiple hairpin RT products via CRT. Afterward, the hairpin RT products could serve as activators to initiate the collateral cleavage activity of CRISPR-Cas12a. Due to the above two-stage amplification, this assay could detect miRNA at sub-femtomolar level (LOD, 0.201 fM). Since the sequence of target miRNA is double checked: first in the CRT and then in the CRISPR system, the proposed assay also shows an excellent specificity in detecting miR-21. Finally, with the usage of this assay, the sensitive assessment of miR-21 levels in human serum samples has been achieved and the disease human serum has been detected. Conclusively, CRISPR-CRT holds a great application prospective in the field of clinical molecular diagnosis.},
}
@article {pmid37173280,
year = {2023},
author = {Kamiya, A and Ueshima, H and Nishida, S and Honda, Y and Kamitsuji, H and Sato, T and Miyamoto, H and Sumita, T and Izumitsu, K and Irie, T},
title = {Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.},
journal = {FEMS microbiology letters},
volume = {370},
number = {},
pages = {},
doi = {10.1093/femsle/fnad042},
pmid = {37173280},
issn = {1574-6968},
support = {KAKENHI 22H00380//Grants-in-Aid for Scientific Research/ ; },
mesh = {*CRISPR-Cas Systems ; *Shiitake Mushrooms/genetics ; Carboxin ; Gene Editing/methods ; Gene Targeting ; },
abstract = {First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.},
}
@article {pmid37084283,
year = {2023},
author = {Li, H and Zhou, R and Liu, P and Yang, M and Xin, D and Liu, C and Zhang, Z and Wu, X and Chen, Q and Zhao, Y},
title = {Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.},
journal = {Plant biotechnology journal},
volume = {21},
number = {7},
pages = {1317-1319},
pmid = {37084283},
issn = {1467-7652},
mesh = {*Soybeans/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Fatty Acids, Monounsaturated ; Plant Proteins/genetics ; Plant Oils ; Seeds/metabolism ; Soybean Oil ; Fatty Acids ; },
}
@article {pmid37069831,
year = {2023},
author = {Jiao, J and Liu, Y and Yang, M and Zheng, J and Liu, C and Ye, W and Song, S and Bai, T and Song, C and Wang, M and Shi, J and Wan, R and Zhang, K and Hao, P and Feng, J and Zheng, X},
title = {The engineered CRISPR-Mb2Cas12a variant enables sensitive and fast nucleic acid-based pathogens diagnostics in the field.},
journal = {Plant biotechnology journal},
volume = {21},
number = {7},
pages = {1465-1478},
pmid = {37069831},
issn = {1467-7652},
mesh = {*Nucleic Acids ; Crops, Agricultural ; Plants, Genetically Modified ; RNA, Plant ; Recombinases/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.},
}
@article {pmid36964962,
year = {2023},
author = {Wolabu, TW and Mahmood, K and Jerez, IT and Cong, L and Yun, J and Udvardi, M and Tadege, M and Wang, Z and Wen, J},
title = {Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.},
journal = {Plant biotechnology journal},
volume = {21},
number = {7},
pages = {1383-1392},
pmid = {36964962},
issn = {1467-7652},
mesh = {*Medicago sativa/genetics ; *CRISPR-Cas Systems/genetics ; Biomass ; Detergents ; Plant Proteins/genetics/metabolism ; Plant Breeding ; Mutagenesis ; },
abstract = {Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.},
}
@article {pmid36951091,
year = {2023},
author = {Tian, S and Zhang, J and Zhao, H and Zong, M and Li, M and Gong, G and Wang, J and Zhang, J and Ren, Y and Zhang, H and Li, S and Wen, C and Xu, Y},
title = {Production of double haploid watermelon via maternal haploid induction.},
journal = {Plant biotechnology journal},
volume = {21},
number = {7},
pages = {1308-1310},
pmid = {36951091},
issn = {1467-7652},
mesh = {Haploidy ; *Citrullus/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plant Breeding ; },
}
@article {pmid37335486,
year = {2023},
author = {Potlapalli, BP and Ishii, T and Nagaki, K and Somasundaram, S and Houben, A},
title = {CRISPR-FISH: A CRISPR/Cas9-Based In Situ Labeling Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2672},
number = {},
pages = {315-335},
pmid = {37335486},
issn = {1940-6029},
mesh = {In Situ Hybridization, Fluorescence/methods ; *CRISPR-Cas Systems/genetics ; *Chromosomes ; DNA ; Repetitive Sequences, Nucleic Acid ; },
abstract = {Fluorescence in situ hybridization (FISH) has been widely used to visualize target DNA sequences in fixed chromosome samples by denaturing the dsDNA to allow complementary probe hybridization, thus damaging the chromatin structure by harsh treatments. To overcome this limitation, a CRISPR/Cas9-based in situ labeling method was developed, termed CRISPR-FISH. This method is also known as RNA-guided endonuclease-in situ labeling (RGEN-ISL). Here we present different protocols for the application of CRISPR-FISH on acetic acid: ethanol or formaldehyde-fixed nuclei and chromosomes as well as tissue sections for labeling repetitive sequences in a range of plant species. In addition, methods on how immunostaining can be combined with CRISPR-FISH are provided.},
}
@article {pmid37334268,
year = {2023},
author = {Li, Y and Liu, L and Qiao, L and Deng, F},
title = {Universal CRISPR/Cas12a-associated aptasensor suitable for rapid detection of small proteins with a plate reader.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1201175},
pmid = {37334268},
issn = {2296-4185},
abstract = {With the discovery of the collateral cleavage activity, CRISPR/Cas12a has recently been identified as a key enabling approach in novel DNA biosensor development. Despite its remarkable success in nucleic acid detection, realizing a universal CRISPR/Cas biosensing system for non-nucleic acid targets remains challenging, particularly at extremely high sensitivity ranges for analyte concentrations lower than the pM level. DNA aptamers can be designed to bind to a range of specific target molecules, such as proteins, small molecules, and cells, with high affinity and specificity through configuration changes. Here, by harnessing its diverse analyte-binding ability and also redirecting the specific DNA-cutting activity of Cas12a to selected aptamers, a simple, sensitive, and universal biosensing platform has been established, termed CRISPR/Cas and aptamer-mediated extra-sensitive assay (CAMERA). With simple modifications to the aptamer and guiding RNA of Cas12a RNP, CAMERA demonstrated 100 fM sensitivity for targeting small proteins, such as IFN-γ and insulin, with less than 1.5-h detection time. Compared with the gold-standard ELISA, CAMERA achieved higher sensitivity and a shorter detection time while retaining ELISA's simple setup. By replacing the antibody with an aptamer, CAMERA also achieved improved thermal stability, allowing to eliminate the requirement for cold storage. CAMERA shows potential to be used as a replacement for conventional ELISA for a variety of diagnostics but with no significant changes for the experimental setup.},
}
@article {pmid37333658,
year = {2023},
author = {Schultz, J and Modolon, F and Peixoto, RS and Rosado, AS},
title = {Shedding light on the composition of extreme microbial dark matter: alternative approaches for culturing extremophiles.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1167718},
pmid = {37333658},
issn = {1664-302X},
abstract = {More than 20,000 species of prokaryotes (less than 1% of the estimated number of Earth's microbial species) have been described thus far. However, the vast majority of microbes that inhabit extreme environments remain uncultured and this group is termed "microbial dark matter." Little is known regarding the ecological functions and biotechnological potential of these underexplored extremophiles, thus representing a vast untapped and uncharacterized biological resource. Advances in microbial cultivation approaches are key for a detailed and comprehensive characterization of the roles of these microbes in shaping the environment and, ultimately, for their biotechnological exploitation, such as for extremophile-derived bioproducts (extremozymes, secondary metabolites, CRISPR Cas systems, and pigments, among others), astrobiology, and space exploration. Additional efforts to enhance culturable diversity are required due to the challenges imposed by extreme culturing and plating conditions. In this review, we summarize methods and technologies used to recover the microbial diversity of extreme environments, while discussing the advantages and disadvantages associated with each of these approaches. Additionally, this review describes alternative culturing strategies to retrieve novel taxa with their unknown genes, metabolisms, and ecological roles, with the ultimate goal of increasing the yields of more efficient bio-based products. This review thus summarizes the strategies used to unveil the hidden diversity of the microbiome of extreme environments and discusses the directions for future studies of microbial dark matter and its potential applications in biotechnology and astrobiology.},
}
@article {pmid37333257,
year = {2023},
author = {Dhingra, Y and Sashital, DG},
title = {Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.06.05.543779},
pmid = {37333257},
abstract = {CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn [2+] dependent, Cas1 preferentially uses Mg [2+] over Mn [2+] . The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.},
}
@article {pmid37331842,
year = {2023},
author = {Cardi, T and Murovec, J and Bakhsh, A and Boniecka, J and Bruegmann, T and Bull, SE and Eeckhaut, T and Fladung, M and Galovic, V and Linkiewicz, A and Lukan, T and Mafra, I and Michalski, K and Kavas, M and Nicolia, A and Nowakowska, J and Sági, L and Sarmiento, C and Yıldırım, K and Zlatković, M and Hensel, G and Van Laere, K},
title = {CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2023.05.012},
pmid = {37331842},
issn = {1878-4372},
abstract = {The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.},
}
@article {pmid37292641,
year = {2023},
author = {Nemudryi, A and Nemudraia, A and Nichols, JE and Scherffius, AM and Zahl, T and Wiedenheft, B},
title = {CRISPR-based engineering of RNA viruses.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37292641},
abstract = {UNLABELLED: CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a new recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.<br><br>ONE-SENTENCE SUMMARY: Programmable CRISPR RNA-guided ribonucleases enable recombinant RNA technology.},
}
@article {pmid37329588,
year = {2023},
author = {Dos Santos-Neto, PC and Cuadro, F and Souza-Neves, M and Crispo, M and Menchaca, A},
title = {Refinements in embryo manipulation applied to CRISPR technology in livestock.},
journal = {Theriogenology},
volume = {208},
number = {},
pages = {142-148},
doi = {10.1016/j.theriogenology.2023.05.028},
pmid = {37329588},
issn = {1879-3231},
abstract = {The implementation of CRISPR technology in large animals requires further improvements in embryo manipulation and transfer to be applied with commercial purposes. In this study we report (a) developmental competence of CRISPR/Cas microinjected zygotes subjected to in vitro culture in large scale programs in sheep; (b) pregnancy outcomes after early-stage (2-8-cell) embryo transfer into the oviduct or the uterine horn; and (c) embryo survival and birth rate after vitrification/warming of CRISPR/Cas microinjected zygotes. Experiment 1 consisted of a retrospective analysis to evaluate embryo developmental rate of in vitro produced zygotes subjected to CRISPR/Cas microinjection (n = 7,819) compared with a subset of non-microinjected zygotes (n = 701). Development rates to blastocyst on Day 6 were 20.0% for microinjected zygotes and 44.9% for non-injected zygotes (P < 0.05). In Experiment 2, CRISPR/Cas microinjected zygotes were transferred on Day 2 after in vitro fertilization (2-8 cell embryos) into the oviductal ampulla (n = 262) or into the uterine horn (n = 276) in synchronized recipient ewes at prefixed time (i.e., approximately two days after ovulation). Pregnant/transferred recipients (24.0% vs. 25.0%), embryo survival/transferred embryos (6.9% vs. 6.2%), and born lambs/pregnant embryos (72.2% vs. 100.0%) did not differ significantly in the two groups. In Experiment 3, CRISPR/Cas microinjected zygotes were maintained under in vitro culture until blastocyst stage (Day 6), and subjected to vitrification/warming via the Cryotop method (n = 474), while a subset of embryos were left fresh as control group (n = 75). Embryos were transferred into the uterine horn of recipient females at prefixed time 8.5 days after the estrous synchronization treatment (i.e., approximately six days after ovulation). Pregnancy rate (30.8% vs. 48.0%), embryo survival rate (14.8% vs. 21.3%), and birth rate (85.7% vs. 75.0%) were not different (PNS) between vitrified and fresh embryos, respectively. In conclusion, the current study in sheep embryos reports (a) suitable developmental rate after CRISPR/Cas microinjection (i.e., 20%), even though it was lower than non-microinjected zygotes; (b) similar outcomes when Day 2-embryos were placed into the uterine horn instead of the oviduct, avoiding both time-consuming and invasive oviduct manipulation, and extended in vitro culture during one week; (c) promising pregnancy and birth rates obtained with vitrification of CRISPR/Cas microinjected embryos. This knowledge on in vitro embryo development, timing of embryo transfer, and cryopreservation of CRISPR/Cas microinjected zygotes have practical implications for the implementation of genome editing technology in large animals.},
}
@article {pmid37328571,
year = {2023},
author = {Booker, AE and D'Angelo, T and Adams-Beyea, A and Brown, JM and Nigro, O and Rappé, MS and Stepanauskas, R and Orcutt, BN},
title = {Life strategies for Aminicenantia in subseafloor oceanic crust.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
pmid = {37328571},
issn = {1751-7370},
abstract = {After decades studying the microbial "deep biosphere" in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use "primordial" siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.},
}
@article {pmid37326717,
year = {2023},
author = {Wu, W and Lei, H},
title = {Genome Editing Inhibits Retinal Angiogenesis in a Mouse Model of Oxygen-Induced Retinopathy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2678},
number = {},
pages = {207-217},
pmid = {37326717},
issn = {1940-6029},
mesh = {Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Endothelial Cells ; Vascular Endothelial Growth Factor A ; *Retinal Diseases ; Neovascularization, Pathologic/genetics ; Disease Models, Animal ; },
abstract = {This protocol describes a novel approach harnessing the technology of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9-based gene editing for treating retinal angiogenesis. In this system, adeno-associated virus (AAV)-mediated CRISPR/Cas9 was employed to edit the genome of vascular endothelial growth factor receptor (VEGFR)2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy. The results showed that genome editing of VEGFR2 suppressed pathological retinal angiogenesis. This mouse model mimics a critical aspect of abnormal retinal angiogenesis in patients with neovascular diabetic retinopathy and retinopathy of prematurity, indicating genome editing has high potential for treating angiogenesis-associated retinopathies.},
}
@article {pmid37326604,
year = {2023},
author = {Wu, Y and Liu, Y and Chang, Y and Liu, M},
title = {Integration of CRISPR/Cas13a and V-Shape PCR for Rapid, Sensitive, and Specific Genotyping of CYP2C19 Gene Polymorphisms.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c01968},
pmid = {37326604},
issn = {1520-6882},
abstract = {Rapid detection of single nucleotide polymorphisms (SNPs) in the CYP2C19 gene is of great significance for clopidogrel-accurate medicine. CRISPR/Cas systems have been increasingly used in SNP detection due to their single-nucleotide mismatch specificity. PCR, as a powerful amplification tool, has been incorporated into the CRISPR/Cas system to improve the sensitivity. However, the complicated three-step temperature control of the conventional PCR impeded rapid detection. The "V" shape PCR can shorten about 2/3 of the amplification time compared with conventional PCR. Herein, we present a new system termed the "V" shape PCR-coupled CRISPR/Cas13a (denoted as VPC) system, achieving the rapid, sensitive, and specific genotyping of CYP2C19 gene polymorphisms. The wild- and mutant-type alleles in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes can be discriminated by using the rationally programmed crRNA. A limit of detection (LOD) of 10[2] copies/μL was obtained within 45 min. In addition, the clinical applicability was demonstrated by genotyping SNPs in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes from clinical blood samples and buccal swabs within 1 h. Finally, we conducted the HPV16 and HPV18 detections to validate the generality of the VPC strategy.},
}
@article {pmid37315423,
year = {2023},
author = {Tracey, TJ and Jiang, L and Gill, MK and Ranie, SN and Ovchinnikov, DA and Wolvetang, EJ and Ngo, ST},
title = {Generation of a human induced pluripotent stem cell line (UQi001-A-1) edited with the CRISPR-Cas9 system to carry the heterozygous TARDBP c.1144G &gt; A (p.A382T) missense mutation.},
journal = {Stem cell research},
volume = {70},
number = {},
pages = {103137},
doi = {10.1016/j.scr.2023.103137},
pmid = {37315423},
issn = {1876-7753},
mesh = {Humans ; Mutation, Missense ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodegenerative Diseases/metabolism ; CRISPR-Cas Systems/genetics ; *Amyotrophic Lateral Sclerosis/genetics ; DNA-Binding Proteins/genetics/metabolism ; Mutation ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the TDP-43 protein is believed to play a central role in disease pathophysiology. Using the CRISPR-Cas9 system, we introduced the heterozygous c.1144G > A (p.A382T) missense mutation in exon 6 of the TARDBP gene into an iPSC line derived from a healthy individual. These edited iPSCs displayed normal cellular morphology, expressed major pluripotency markers, were capable of tri-lineage differentiation, and possessed a normal karyotype.},
}
@article {pmid36805562,
year = {2023},
author = {Cui, S and Shin, YJ and Fang, X and Lee, H and Eum, SH and Ko, EJ and Lim, SW and Shin, E and Lee, KI and Lee, JY and Lee, CB and Bae, SK and Yang, CW and Chung, BH},
title = {CRISPR/Cas9-mediated A4GALT suppression rescues Fabry disease phenotypes in a kidney organoid model.},
journal = {Translational research : the journal of laboratory and clinical medicine},
volume = {258},
number = {},
pages = {35-46},
doi = {10.1016/j.trsl.2023.02.005},
pmid = {36805562},
issn = {1878-1810},
mesh = {Humans ; *Fabry Disease/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Kidney/metabolism ; *Kidney Diseases/genetics ; Phenotype ; Organoids ; },
abstract = {The objective of this study was to investigate whether CRISPR/Cas9-mediated suppression of A4GALT could rescue phenotype of Fabry disease nephropathy (FDN) using human induced pluripotent stem cells (hiPSCs) derived kidney organoid system. We generated FDN patient-derived hiPSC (CMC-Fb-002) and FD-specific hiPSCs (GLA-KO) by knock-out (KO) of GLA in wild-type (WT) hiPSCs using CRISPR/Cas9. We then performed A4GALT KO in both CMC-Fb-002 and GLA-KO to make Fb-002-A4GALT-KO and GLA/A4GALT-KO, respectively. Using these hiPSCs, we generated kidney organoids and compared alpha-galactosidase-A enzyme (α-GalA) activity, globotriaosylceramide (Gb-3) deposition, and zebra body formation under electron microscopy (EM). We also compared mRNA expression levels using RNA-seq and qPCR. Generated hiPSCs showed typical pluripotency markers without chromosomal disruption. Expression levels of GLA in CMC-Fb-002 and GLA-KO and expression levels of A4GALT in Fb-002-A4GALT-KO and GLA/A4GALT-KO were successfully decreased compared to those in WT-hiPSCs, respectively. Generated kidney organoids using these hiPSCs expressed typical nephron markers. In CMC-Fb-002 and GLA-KO organoids, α-GalA activity was significantly decreased along with increased deposition of Gb-3 in comparison with WT organoids. Intralysosomal inclusion body was also detected under EM. However, these disease phenotypes were rescued by KO of A4GALT in both GLA/A4GALT-KO and Fb-002-A4GALT-KO kidney organoids. RNA-seq showed increased expression levels of genes related to FDN progression in both GLA-mutant organoids compared to those in WT. Such increases were rescued in GLA/A4GALT-KO or Fb-002-A4GALT-KO organoids. CRISPR/Cas9 mediated suppression of A4GALT could rescue FDN phenotype. Hence, it can be proposed as a therapeutic approach to treat FDN.},
}
@article {pmid36321718,
year = {2023},
author = {Kalaitzandonakes, N and Willig, C and Zahringer, K},
title = {The economics and policy of genome editing in crop improvement.},
journal = {The plant genome},
volume = {16},
number = {2},
pages = {e20248},
doi = {10.1002/tpg2.20248},
pmid = {36321718},
issn = {1940-3372},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; },
abstract = {In this review article we analyze the economics of genome editing and its potential long-term effect on crop improvement and agriculture. We describe the emergence of genome editing as a novel platform for crop improvement, distinct from the existing platforms of plant breeding and genetic engineering. We review key technical characteristics of genome editing and describe how it enables faster trait development, lower research and development costs, and the development of novel traits not possible through previous crop improvement methods. Given these fundamental technical and economic advantages, we describe how genome editing can greatly increase the productivity and broaden the scope of crop improvement with potential outsized economic effects. We further discuss how the global regulatory policy environment, which is still emerging, can shape the ultimate path of genome editing innovation, its effect on crop improvement, and its overall socioeconomic benefits to society.},
}
@article {pmid35713092,
year = {2023},
author = {Biswas, S and Ibarra, O and Shaphek, M and Molina-Risco, M and Faion-Molina, M and Bellinatti-Della Gracia, M and Thomson, MJ and Septiningsih, EM},
title = {Increasing the level of resistant starch in 'Presidio' rice through multiplex CRISPR-Cas9 gene editing of starch branching enzyme genes.},
journal = {The plant genome},
volume = {16},
number = {2},
pages = {e20225},
doi = {10.1002/tpg2.20225},
pmid = {35713092},
issn = {1940-3372},
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems ; *Oryza/genetics ; Resistant Starch ; *1,4-alpha-Glucan Branching Enzyme/genetics ; Plants, Genetically Modified/genetics ; Starch ; },
abstract = {Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR-Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.},
}
@article {pmid37326060,
year = {2023},
author = {Vasileva, AA and Aliukas, SA and Selkova, PA and Arseniev, AN and Chernova, VE and Musharova, OS and Klimuk, EI and Khodorkovskii, MA and Severinov, KV},
title = {[Type II CRISPR-Cas System Nucleases: A Pipeline for Prediction and In Vitro Characterization].},
journal = {Molekuliarnaia biologiia},
volume = {57},
number = {3},
pages = {546-560},
pmid = {37326060},
issn = {0026-8984},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing/methods ; Bacteria/genetics ; Recombinant Proteins/genetics ; DNA/metabolism ; },
abstract = {The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.},
}
@article {pmid37326047,
year = {2023},
author = {Sinyakov, AN and Kostina, EV},
title = {[DNA Fragment Enrichment for High-Throughput Sequencing].},
journal = {Molekuliarnaia biologiia},
volume = {57},
number = {3},
pages = {440-457},
pmid = {37326047},
issn = {0026-8984},
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; Polymerase Chain Reaction/methods ; Genome ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the enrichment of target genomic fragments. The methods of molecular hybridization, polymerase chain reaction, and CRISPR-Cas9 system for this purpose are considered. Examples of the practical use of the developed methods for research and diagnostic purposes are given.},
}
@article {pmid37326044,
year = {2023},
author = {Ukhatova, YV and Erastenkova, MV and Korshikova, ES and Krylova, EA and Mikhailova, AS and Semilet, TV and Tikhonova, NG and Shvachko, NA and Khlestkina, EK},
title = {[Improvement of Crops Using the CRISPR/Cas System: New Target Genes].},
journal = {Molekuliarnaia biologiia},
volume = {57},
number = {3},
pages = {387-410},
pmid = {37326044},
issn = {0026-8984},
mesh = {*CRISPR-Cas Systems ; *Genome, Plant ; Gene Editing/methods ; Crops, Agricultural/genetics ; Nucleotides ; Plants, Genetically Modified/genetics ; },
abstract = {The success of genome editing of crops using the CRISPR/Cas system largely depends on the correct choice of target genes, for which directed changes will increase yield and improve the quality of plant raw materials and resistance to biotic and abiotic stress factors. This work systematizes and catalogs data on target genes used to improve cultivated plants. The latest systematic review examined articles indexed in the Scopus database and published before August 17, 2019. Our work covers the period from August 18, 2019 to March 15, 2022. A search according to the given algorithm allowed us to identify 2090 articles, among which only 685 contain the results of gene editing of 28 species of cultivated plants (the search was carried out for 56 crops). A significant part of these papers considered either editing of target genes, which was previously carried out in similar works, or studies related to the field of reverse genetics, and only 136 articles contain data on editing of new target genes, whose modification is aimed at improving plant traits important for breeding. In total, 287 target genes of cultivated plants were subjected to editing in order to improve properties significant for breeding over the entire period of the CRISPR/Cas system application. This review presents a detailed analysis of the editing of new target genes. The studies were most often aimed at increasing productivity and disease resistance, as well as improving the properties of plant materials. It was noted whether it was possible to obtain stable transformants at the time of publication and whether editing was applied to non-model cultivars. The range of modified cultivars of a number of crops has been significantly expanded, in particular, for wheat, rice, soybean, tomato, potato, rapeseed, grape, and maize. In the vast majority of cases, editing constructs were delivered using agrobacterium-mediated transformation, less commonly, using biolistics, protoplast transfection, and haploinducers. The desired change in traits was most often achieved by gene knockout. In some cases, knockdown and nucleotide substitutions in the target gene were carried out. To obtain nucleotide substitutions in the genes of cultivated plants, base-editing and prime-editing technologies are increasingly used. The emergence of a convenient CRISPR/Cas editing system has contributed to the development of specific molecular genetics of many crop species.},
}
@article {pmid37326009,
year = {2023},
author = {Yosef, I and Mahata, T and Goren, MG and Degany, OJ and Ben-Shem, A and Qimron, U},
title = {Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad510},
pmid = {37326009},
issn = {1362-4962},
support = {818878/ERC_/European Research Council/International ; },
abstract = {Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.},
}
@article {pmid37323909,
year = {2023},
author = {Reva, O and Messina, E and La Cono, V and Crisafi, F and Smedile, F and La Spada, G and Marturano, L and Selivanova, EA and Rohde, M and Krupovic, M and Yakimov, MM},
title = {Functional diversity of nanohaloarchaea within xylan-degrading consortia.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1182464},
pmid = {37323909},
issn = {1664-302X},
abstract = {Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.},
}
@article {pmid37323580,
year = {2023},
author = {Abdullah, M and Greco, BM and Laurent, JM and Garge, RK and Boutz, DR and Vandeloo, M and Marcotte, EM and Kachroo, AH},
title = {Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast.},
journal = {Cell reports methods},
volume = {3},
number = {5},
pages = {100464},
pmid = {37323580},
issn = {2667-2375},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; Gene Editing ; Genetic Engineering ; Homologous Recombination ; },
abstract = {A major challenge to rationally building multi-gene processes in yeast arises due to the combinatorics of combining all of the individual edits into the same strain. Here, we present a precise and multi-site genome editing approach that combines all edits without selection markers using CRISPR-Cas9. We demonstrate a highly efficient gene drive that selectively eliminates specific loci by integrating CRISPR-Cas9-mediated double-strand break (DSB) generation and homology-directed recombination with yeast sexual assortment. The method enables marker-less enrichment and recombination of genetically engineered loci (MERGE). We show that MERGE converts single heterologous loci to homozygous loci at ∼100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at converting and combining multiple loci, thus identifying compatible genotypes. Finally, we establish MERGE proficiency by engineering a fungal carotenoid biosynthesis pathway and most of the human α-proteasome core into yeast. Therefore, MERGE lays the foundation for scalable, combinatorial genome editing in yeast.},
}
@article {pmid37295844,
year = {2023},
author = {Jiao, Y and Li, M and He, X and Wang, Y and Song, J and Hu, Y and Li, L and Zhou, L and Jiang, L and Qu, J and Xie, L and Chen, Q and Yao, S},
title = {Targeted, programmable, and precise tandem duplication in the mammalian genome.},
journal = {Genome research},
volume = {33},
number = {5},
pages = {779-786},
doi = {10.1101/gr.277261.122},
pmid = {37295844},
issn = {1549-5469},
mesh = {Animals ; *Genome ; *DNA/genetics ; Genomics ; CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {Tandem duplications are frequent structural variations of the genome and play important roles in genetic disease and cancer. However, interpreting the phenotypic consequences of tandem duplications remains challenging, in part owing to the lack of genetic tools to model such variations. Here, we developed a strategy, tandem duplication via prime editing (TD-PE), to create targeted, programmable, and precise tandem duplication in the mammalian genome. In this strategy, we design a pair of in trans prime editing guide RNAs (pegRNAs) for each targeted tandem duplication, which encode the same edits but prime the single-stranded DNA (ssDNA) extension in opposite directions. The reverse transcriptase (RT) template of each extension is designed homologous to the target region of the other single guide RNA (sgRNA) to promote the reannealing of the edited DNA strands and the duplication of the fragment in between. We showed that TD-PE produced robust and precise in situ tandem duplications of genomic fragments ranging from ∼50 bp to ∼10 kb, with a maximal efficiency up to 28.33%. By fine-tuning the pegRNAs, we achieved simultaneous targeted duplication and fragment insertion. Finally, we successfully produced multiple disease-relevant tandem duplications, showing the general utility of TD-PE in genetic research.},
}
@article {pmid37290935,
year = {2023},
author = {He, J and Zhang, K and Yan, S and Tang, M and Zhou, W and Yin, Y and Chen, K and Zhang, C and Li, M},
title = {Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library.},
journal = {Genome research},
volume = {33},
number = {5},
pages = {798-809},
doi = {10.1101/gr.277650.123},
pmid = {37290935},
issn = {1549-5469},
mesh = {*Brassica napus/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Plant Breeding ; Mutagenesis ; Plants, Genetically Modified/genetics ; Polyploidy ; },
abstract = {The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of Brassica napus A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.},
}
@article {pmid37276664,
year = {2023},
author = {Wang, C and Zhang, Y and Liu, S and Yin, Y and Fan, GC and Shen, Y and Han, H and Wang, W},
title = {Allosteric probe-triggered isothermal amplification to activate CRISPR/Cas12a for sensitive electrochemiluminescence detection of Salmonella.},
journal = {Food chemistry},
volume = {425},
number = {},
pages = {136382},
doi = {10.1016/j.foodchem.2023.136382},
pmid = {37276664},
issn = {1873-7072},
mesh = {CRISPR-Cas Systems ; DNA Primers ; DNA, Single-Stranded ; Electrodes ; *Metal-Organic Frameworks ; Salmonella/genetics ; *Biosensing Techniques ; },
abstract = {We report an electrochemiluminescence (ECL) sensor for Salmonella detection based on allosteric probe as a bio-recognition element and CRISPR/Cas12a as a signal amplification strategy. In the presence of Salmonella, the structure switching occurs on allosteric probes, resulting in their hybridization with primers to trigger isothermal amplification. Salmonella is then released to initiate the next reaction cycle accompanying by generating a large amount of dsDNA, which are subsequently recognized by CRISPR-gRNA for activating the trans-cleavage activity of Cas12a. Furthermore, the activated Cas12a can indiscriminately cut the ssDNA which is bound to the electrode, enabling the release of the ECL emitter porphyrinic Zr metal - organic framework (MOF, PCN-224) and exhibiting a decreased ECL signal accordingly. The linear range is 50 CFU·mL[-1]-5 × 10[6] CFU·mL[-1] and the detection limit is calculated to be 37 CFU·mL[-1]. This method sensitively detects Salmonella in different types of real samples, indicating it is a promising strategy for Salmonella detection.},
}
@article {pmid37266879,
year = {2023},
author = {Zhou, Y and Song, F and Yang, H and Li, D and Zhang, N and Huang, K and He, X and Wang, M and Tian, H and Li, C},
title = {Construction of a food-grade gene editing system based on CRISPR-Cas9 and its application in Lactococcus lactis NZ9000.},
journal = {Biotechnology letters},
volume = {45},
number = {8},
pages = {955-966},
pmid = {37266879},
issn = {1573-6776},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Lactococcus lactis/genetics/metabolism ; Plasmids/genetics ; Homologous Recombination ; },
abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system has been widely used in gene editing of various organisms. However, food-grade gene editing systems in lactic acid bacteria are still preliminary. Red/ET-dependent homologous recombination or CRISPR-based systems have been developed to gene editing in Lactococcus lactis, but these methods are overall inefficient. In the present study, a recombinant system based on CRISPR/Cas9 technology combined with Red/ET was developed using the plasmid pMG36e derived from Lactococcus lactis. Then, the developed recombinant system was applied to Lactococcus lactis. Knockout efficiency was significantly higher using the developed system (91%). In addition, this system showed the potential to be used as a high-throughput method for hierarchical screening. Finally, a gene-edited strain was obtained, and no antibiotics or exogenous genes were introduced using the developed gene editing system. Thus, the efficient system in lactic acid bacteria was constructed and optimized.},
}
@article {pmid37254851,
year = {2023},
author = {Xu, M and Zhu, Y and Zhang, Y and Gong, B and Kang, Y and Su, G and Yu, Y},
title = {Split G-quadruplex-programmed label-free CRISPR-Cas12a sensing system.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {49},
pages = {7615-7618},
doi = {10.1039/d3cc01239e},
pmid = {37254851},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; *G-Quadruplexes ; },
abstract = {A split G-quadruplex (G4)-programmed Cas12a platform was established, validated, and optimized. The split G4 motif was recruited as substrate for Cas12a, and the label-free sensing platform provided a concentration-dependent response towards the input target. Furthermore, exosomal surface proteins from cultured cancer cells and clinical samples were detected and profiled.},
}
@article {pmid37230419,
year = {2023},
author = {Shirani-Bidabadi, S and Mirian, M and Varshosaz, J and Tavazohi, N and Sadeghi, HMM and Shariati, L},
title = {Gene network analysis of oxaliplatin-resistant colorectal cancer to target a crucial gene using chitosan/hyaluronic acid/protamine polyplexes containing CRISPR-Cas9.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1867},
number = {8},
pages = {130385},
doi = {10.1016/j.bbagen.2023.130385},
pmid = {37230419},
issn = {1872-8006},
mesh = {Humans ; Oxaliplatin/pharmacology/therapeutic use ; Hyaluronic Acid ; CRISPR-Cas Systems/genetics ; *Chitosan ; Protamines/genetics/therapeutic use ; Gene Regulatory Networks ; *Colorectal Neoplasms/drug therapy/genetics ; },
abstract = {Colorectal cancer (CRC) treatment is dramatically hampered by resistance to oxaliplatin alone or in the combination of irinotecan or 5-fluorouracil and leucovorin. This study aims to design and assess Chitosan/Hyaluronic Acid/Protamine sulfate (CS/HA/PS) polyplexes loaded with CRISPR plasmid for targeting a key gene in cancer drug resistance. Here, recent findings were considered to validate oxaliplatin-resistant CRC-related genes and systems biology approaches employed to detect the critical gene. The polyplexes were characterized according to particle size, zeta potential, and stability. Moreover, carrier toxicity and transfection efficiency were assessed on oxaliplatin-resistant HT-29 cells. The post-transfection evaluations were performed to confirm gene disruption-mediated CRISPR. Eventually, excision cross complementation group 1(ERCC1), a crucial member of the nucleotide excision repair pathway, was selected to be targeted using CRISPR/Cas9 to reverse oxaliplatin resistance in HT-29 cells. CS/HA/PS polyplexes containing CRISPR/Cas9 plasmid exhibited negligible toxicity and comparable transfection efficiency with Lipofectamine™. Following the efficient gene delivery, sequences in CRISPR/Cas9 target sites were altered, ERCC1 was downregulated, and drug sensitivity was successfully restored in oxaliplatin-resistant cells. Findings indicate that CS/HA/PS/CRISPR polyplexes provide a potential strategy for delivering cargo and targeting oxaliplatin resistance-related gene to manipulate drug resistance as a rising concern in cancer therapeutic approaches.},
}
@article {pmid37224027,
year = {2023},
author = {Li, K and Qin, LY and Zhang, ZX and Yan, CX and Gu, Y and Sun, XM and Huang, H},
title = {Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications.},
journal = {ACS synthetic biology},
volume = {12},
number = {6},
pages = {1586-1598},
doi = {10.1021/acssynbio.3c00141},
pmid = {37224027},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.},
}
@article {pmid37217315,
year = {2023},
author = {Chen, KN and Ma, BG},
title = {OptoCRISPRi-HD: Engineering a Bacterial Green-Light-Activated CRISPRi System with a High Dynamic Range.},
journal = {ACS synthetic biology},
volume = {12},
number = {6},
pages = {1708-1715},
doi = {10.1021/acssynbio.3c00035},
pmid = {37217315},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; Metabolic Engineering/methods ; Reproducibility of Results ; Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/genetics ; },
abstract = {The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.},
}
@article {pmid37182624,
year = {2023},
author = {Wang, W and Zhang, F and Guo, K and Xu, J and Zhao, P and Xia, Q},
title = {CRISPR/Cas9-mediated gene editing of the let-7 seed sequence improves silk yield in the silkworm, Bombyx mori.},
journal = {International journal of biological macromolecules},
volume = {243},
number = {},
pages = {124793},
doi = {10.1016/j.ijbiomac.2023.124793},
pmid = {37182624},
issn = {1879-0003},
mesh = {Animals ; *Silk/genetics/metabolism ; *Bombyx/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Larva ; Insect Proteins/genetics/metabolism ; },
abstract = {Body size and silk protein synthesis ability are two crucial aspects of artificial selection in silkworm breeding; however, the role of genes in both pathways remains unknown. To determine whether let-7 microRNA could regulate larval development and silk gland growth simultaneously, we designed a guide RNA to edit let-7 using the CRISPR/Cas9 system. The indels predominantly appeared in the let-7 seed region, and the vast majority of the mutations were small-fragment deletions. Loss of let-7 function prolonged the fifth larval period, and substantially increased body weight during the wandering stage, but it resulted in developmental arrest during the pupal-moth transition. let-7 systemic knock down promoted silk gland growth and increased silk yield by >50 %, with efficiency significantly higher than in tissue-specific edited individuals. Hormone signaling and cell cycle pathway genes were activated in different patterns in the body and silk gland, implying that let-7 may regulate different target genes to play role in tissue growth. In summary, we first report that conditional knock down let-7 promoting the simultaneous growth of body and silk gland, greatly improve silk yield in the silkworm.},
}
@article {pmid37154683,
year = {2023},
author = {Li, F and Liu, S and Luo, B and Huang, M and Teng, Y and Wang, T},
title = {CRISPR/Cas12a Technology Combined with Recombinase Polymerase Amplification for Rapid and Portable Monkeypox Virus Detection.},
journal = {Microbiology spectrum},
volume = {11},
number = {3},
pages = {e0423322},
pmid = {37154683},
issn = {2165-0497},
mesh = {*Recombinases ; *Monkeypox virus ; CRISPR-Cas Systems ; },
}
@article {pmid37150989,
year = {2023},
author = {Li, S and Wu, Y and Wang, X},
title = {The Applications of CRISPR/Cas9 System for Urinary System Tumor.},
journal = {Current topics in medicinal chemistry},
volume = {23},
number = {10},
pages = {897-906},
doi = {10.2174/1568026623666230504100706},
pmid = {37150989},
issn = {1873-4294},
mesh = {Humans ; Male ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Prostatic Neoplasms ; },
abstract = {Tumors of the urinary system include those in the urinary and reproductive systems, of which tumors of the prostate, bladder, and kidney have the highest incidence. In recent years, due to changes in dietary structure, prostate cancer has become the most common type of male genitourinary system cancer. Furthermore, due to tobacco consumption, increases in industrialization, and the age of the population, the incidence of bladder cancer in both males and females in both urban and rural areas, has shown an increasing trend. The incidence and mortality of kidney cancer have also increased and negatively affected the lives and health of all residents. While surgery, radiotherapy, and chemotherapy have greatly improved the cure and survival rates of patients with urinary tumors, we lack methods for early detection and effective long-term treatment. New tools and methods for diagnosis and treatment are thus urgently needed. Recently, CRISPR/Cas9 has become an efficient method to alter the genome in many organisms. It can be used to activate or inhibit gene expression, which greatly facilitates the editing of targeted genes, both in vivo and in vitro. It provides a powerful scientific research tool to analyze the mechanisms of disease occurrence and development and to develop advanced targeted drug delivery. The diagnosis and treatment of human tumors will consequently be improved as this technology will surely accelerate cancer research. In this article, we discuss how CRISPR/Cas9 technology can be used to research and treat genitourinary system tumors will consequently be improved as this technology will surely accelerate cancer research. Here, we review the current applications of CRISPR/Cas9 technology for genitourinary system tumor research and therapy.},
}
@article {pmid37149929,
year = {2023},
author = {Xu, WW and Liao, L and Dai, W and Zheng, CC and Tan, XP and He, Y and Zhang, QH and Huang, ZH and Chen, WY and Qin, YR and Chen, KS and He, ML and Law, S and Lung, ML and He, QY and Li, B},
title = {Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis.},
journal = {EBioMedicine},
volume = {92},
number = {},
pages = {104587},
pmid = {37149929},
issn = {2352-3964},
mesh = {Humans ; *Esophageal Squamous Cell Carcinoma/pathology ; *Esophageal Neoplasms/genetics ; Molecular Docking Simulation ; CRISPR-Cas Systems ; Early Detection of Cancer ; *MicroRNAs/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Cell Proliferation ; Cell Movement/genetics ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; },
abstract = {BACKGROUND: Metastasis is one of the most lethal hallmarks of esophageal squamous cell carcinoma (ESCC), yet the mechanisms remain unclear due to a lack of reliable experimental models and systematic identification of key drivers. There is urgent need to develop useful therapies for this lethal disease.<br><br>METHODS: A genome-wide CRISPR/Cas9 screening, in combination with gene profiling of highly invasive and metastatic ESCC sublines, as well as PDX models, was performed to identify key regulators of cancer metastasis. The Gain- and loss-of-function experiments were taken to examine gene function. Protein interactome, RNA-seq, and whole genome methylation sequencing were used to investigate gene regulation and molecular mechanisms. Clinical significance was analyzed in tumor tissue microarray and TCGA databases. Homology modeling, modified ELISA, surface plasmon resonance and functional assays were performed to identify lead compound which targets MEST to suppress cancer metastasis.<br><br>FINDINGS: High MEST expression was associated with poor patient survival and promoted cancer invasion and metastasis in ESCC. Mechanistically, MEST activates SRCIN1/RASAL1-ERK-snail signaling by interacting with PURA. miR-449a was identified as a direct regulator of MEST, and hypermethylation of its promoter led to MEST upregulation, whereas systemically delivered miR-449a mimic could suppress tumor metastasis without overt toxicity. Furthermore, molecular docking and computational screening in a small-molecule library of 1,500,000 compounds and functional assays showed that G699-0288 targets the MEST-PURA interaction and significantly inhibits cancer metastasis.<br><br>INTERPRETATION: We identified the MEST-PURA-SRCIN1/RASAL1-ERK-snail signaling cascade as an important mechanism underlying cancer metastasis. Blockade of MEST-PURA interaction has therapeutic potential in management of cancer metastasis.<br><br>FUNDING: This work was supported by National Key Research and Development Program of China (2021YFC2501000, 2021YFC2501900, 2017YFA0505100); National Natural Science Foundation of China (31961160727, 82073196, 81973339, 81803551); NSFC/RGC Joint Research Scheme (N_HKU727/19); Natural Science Foundation of Guangdong Province (2021A1515011158, 2021A0505030035); Key Laboratory of Guangdong Higher Education Institutes of China (2021KSYS009).},
}
@article {pmid37127332,
year = {2023},
author = {Rossato, M and Marcolungo, L and De Antoni, L and Lopatriello, G and Bellucci, E and Cortinovis, G and Frascarelli, G and Nanni, L and Bitocchi, E and Di Vittori, V and Vincenzi, L and Lucchini, F and Bett, KE and Ramsay, L and Konkin, DJ and Delledonne, M and Papa, R},
title = {CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes.},
journal = {Genome research},
volume = {33},
number = {5},
pages = {787-797},
doi = {10.1101/gr.277628.122},
pmid = {37127332},
issn = {1549-5469},
mesh = {Genotype ; *Genome-Wide Association Study ; *CRISPR-Cas Systems ; Genome, Plant ; Genotyping Techniques ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; },
abstract = {High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods because of the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ∼40% of reads mapping to repeats, increasing those mapping to single-copy regions by ∼2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ∼10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ∼12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed because of low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely related species (L. orientalis). Our results showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.},
}
@article {pmid37042654,
year = {2023},
author = {Dua, S and Bansal, S and Gautam, D and Jose, B and Singh, P and Singh, MK and De, S and Kumar, D and Yadav, PS and Kues, W and Selokar, NL},
title = {Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.},
journal = {Cellular reprogramming},
volume = {25},
number = {3},
pages = {121-127},
doi = {10.1089/cell.2023.0003},
pmid = {37042654},
issn = {2152-4998},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Nuclear Transfer Techniques/veterinary ; Cloning, Organism ; Blastocyst ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.},
}
@article {pmid37028644,
year = {2023},
author = {Bennis, NX and Kostanjšek, M and van den Broek, M and Daran, JG},
title = {Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts.},
journal = {New biotechnology},
volume = {76},
number = {},
pages = {49-62},
doi = {10.1016/j.nbt.2023.04.001},
pmid = {37028644},
issn = {1876-4347},
mesh = {*CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; Beer ; Fermentation ; Genome, Fungal/genetics ; },
abstract = {Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.},
}
@article {pmid37323893,
year = {2023},
author = {Jia, HJ and Jia, PP and Yin, S and Bu, LK and Yang, G and Pei, DS},
title = {Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1172635},
pmid = {37323893},
issn = {1664-302X},
abstract = {Bacteriophages, the most abundant organisms on earth, have the potential to address the rise of multidrug-resistant bacteria resulting from the overuse of antibiotics. However, their high specificity and limited host range can hinder their effectiveness. Phage engineering, through the use of gene editing techniques, offers a means to enhance the host range of bacteria, improve phage efficacy, and facilitate efficient cell-free production of phage drugs. To engineer phages effectively, it is necessary to understand the interaction between phages and host bacteria. Understanding the interaction between the receptor recognition protein of bacteriophages and host receptors can serve as a valuable guide for modifying or replacing these proteins, thereby altering the receptor range of the bacteriophage. Research and development focused on the CRISPR-Cas bacterial immune system against bacteriophage nucleic acids can provide the necessary tools to promote recombination and counter-selection in engineered bacteriophage programs. Additionally, studying the transcription and assembly functions of bacteriophages in host bacteria can facilitate the engineered assembly of bacteriophage genomes in non-host environments. This review highlights a comprehensive summary of phage engineering methods, including in-host and out-of-host engineering, and the use of high-throughput methods to understand their role. The main aim of these techniques is to harness the intricate interactions between bacteriophages and hosts to inform and guide the engineering of bacteriophages, particularly in the context of studying and manipulating the host range of bacteriophages. By employing advanced high-throughput methods to identify specific bacteriophage receptor recognition genes, and subsequently introducing modifications or performing gene swapping through in-host recombination or out-of-host synthesis, it becomes possible to strategically alter the host range of bacteriophages. This capability holds immense significance for leveraging bacteriophages as a promising therapeutic approach against antibiotic-resistant bacteria.},
}
@article {pmid37323227,
year = {2023},
author = {Yao, T and Yuan, G and Lu, H and Liu, Y and Zhang, J and Tuskan, GA and Muchero, W and Chen, JG and Yang, X},
title = {CRISPR/Cas9-based gene activation and base editing in Populus.},
journal = {Horticulture research},
volume = {10},
number = {6},
pages = {uhad085},
pmid = {37323227},
issn = {2662-6810},
abstract = {The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone "717-1B4" (P. tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g. those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone "717-1B4" and poplar clone "WV94" (P. deltoides "WV94"), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%-14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone "717-1B4". Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.},
}
@article {pmid37323878,
year = {2022},
author = {Xu, Z and Wang, Q and Zhong, H and Jiang, Y and Shi, X and Yuan, B and Yu, N and Zhang, S and Yuan, X and Guo, S and Yang, Y},
title = {Carrier strategies boost the application of CRISPR/Cas system in gene therapy.},
journal = {Exploration (Beijing, China)},
volume = {2},
number = {2},
pages = {20210081},
pmid = {37323878},
issn = {2766-2098},
abstract = {Emerging clustered regularly interspaced short palindromic repeat/associated protein (CRISPR/Cas) genome editing technology shows great potential in gene therapy. However, proteins and nucleic acids suffer from enzymatic degradation in the physiological environment and low permeability into cells. Exploiting carriers to protect the CRISPR system from degradation, enhance its targeting of specific tissues and cells, and reduce its immunogenicity is essential to stimulate its clinical applications. Here, the authors review the state-of-the-art CRISPR delivery systems and their applications, and describe strategies to improve the safety and efficacy of CRISPR mediated genome editing, categorized by three types of cargo formats, that is, Cas: single-guide RNA ribonucleoprotein, Cas mRNA and single-guide RNA, and Cas plasmid expressing CRISPR/Cas systems. The authors hope this review will help develop safe and efficient nanomaterial-based carriers for CRISPR tools.},
}
@article {pmid37317864,
year = {2023},
author = {Devi, V and Harjai, K and Chhibber, S},
title = {Repurposing prokaryotic clustered regularly interspaced short palindromic repeats-Cas adaptive immune system to combat antimicrobial resistance.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {},
doi = {10.2217/fmb-2022-0222},
pmid = {37317864},
issn = {1746-0921},
abstract = {Despite achieving unparalleled progress in the field of science and technology, the global health community is still threatened by the looming pressure of infectious diseases. One of the greatest challenges is the rise in infections by antibiotic-resistant microorganisms. The misuse of antibiotics has led to the present circumstances, and there is seemingly no solution. There is imminent pressure to develop new antibacterial therapies to curb the rise and spread of multidrug resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas, having immense potential as a gene-editing tool, has gained considerable attention as an alternative antibacterial therapy. Strategies, aiming to either eliminate pathogenic strains or to restore sensitivity to antibiotics, are the main focus of research. This review deals with the development of CRISPR-Cas antimicrobials and their delivery challenges.},
}
@article {pmid37317115,
year = {2023},
author = {Tiwari, P and Dufossé, L},
title = {Focus and Insights into the Synthetic Biology-Mediated Chassis of Economically Important Fungi for the Production of High-Value Metabolites.},
journal = {Microorganisms},
volume = {11},
number = {5},
pages = {},
pmid = {37317115},
issn = {2076-2607},
abstract = {Substantial progress has been achieved and knowledge gaps addressed in synthetic biology-mediated engineering of biological organisms to produce high-value metabolites. Bio-based products from fungi are extensively explored in the present era, attributed to their emerging importance in the industrial sector, healthcare, and food applications. The edible group of fungi and multiple fungal strains defines attractive biological resources for high-value metabolites comprising food additives, pigments, dyes, industrial chemicals, and antibiotics, including other compounds. In this direction, synthetic biology-mediated genetic chassis of fungal strains to enhance/add value to novel chemical entities of biological origin is opening new avenues in fungal biotechnology. While substantial success has been achieved in the genetic manipulation of economically viable fungi (including Saccharomyces cerevisiae) in the production of metabolites of socio-economic relevance, knowledge gaps/obstacles in fungal biology and engineering need to be remedied for complete exploitation of valuable fungal strains. Herein, the thematic article discusses the novel attributes of bio-based products from fungi and the creation of high-value engineered fungal strains to promote yield, bio-functionality, and value-addition of the metabolites of socio-economic value. Efforts have been made to discuss the existing limitations in fungal chassis and how the advances in synthetic biology provide a plausible solution.},
}
@article {pmid37316664,
year = {2023},
author = {Wang, JY and Tuck, OT and Skopintsev, P and Soczek, KM and Li, G and Al-Shayeb, B and Zhou, J and Doudna, JA},
title = {Genome expansion by a CRISPR trimmer-integrase.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {37316664},
issn = {1476-4687},
abstract = {CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity[1]. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient[2-5]. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation[6,7], but many CRISPR-Cas systems lack Cas4[8]. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited[9,10] exonucleases for faithful acquisition of new CRISPR immune sequences.},
}
@article {pmid37316021,
year = {2023},
author = {Zheng, H and Huang, W and Li, X and Huang, H and Yuan, Q and Liu, R and Di, H and Liang, S and Wang, M and Li, M and Huang, Z and Tang, Y and Zheng, Y and Miao, H and Ma, J and Li, H and Wang, Q and Sun, B and Zhang, F},
title = {CRISPR/Cas9-mediated BoaAOP2s editing alters aliphatic glucosinolate side-chain metabolic flux and increases the glucoraphanin content in Chinese kale.},
journal = {Food research international (Ottawa, Ont.)},
volume = {170},
number = {},
pages = {112995},
doi = {10.1016/j.foodres.2023.112995},
pmid = {37316021},
issn = {1873-7145},
mesh = {*Brassica/genetics ; Glucosinolates ; CRISPR-Cas Systems ; },
abstract = {Glucoraphanin (GRA) is an aliphatic glucosinolate (GSL), and its hydrolysis product has powerful anticancer activity. ALKENYL HYDROXALKYL PRODUCING 2 (AOP2) gene, encodes a 2-oxoglutarate-dependent dioxygenase, which can catalyze GRA to form gluconapin (GNA). However, GRA only present in trace amounts in Chinese kale. To increase the content of GRA in Chinese kale, three copies of BoaAOP2 were isolated and edited using CRISPR/Cas9 system. The content of GRA was 11.71- to 41.29-fold (0.082-0.289 μmol g[-1] FW) higher in T1 generation of boaaop2 mutants than in wild-type plants, and this was accompanied by an increase in the GRA/GNA ratio and reductions in the content of GNA and total aliphatic GSLs. BoaAOP2.1 is an effective gene for the alkenylation of aliphatic GSLs in Chinese kale. Overall, targeted editing of CRISPR/Cas9-mediated BoaAOP2s altered aliphatic GSL side-chain metabolic flux and enhanced the GRA content in Chinese kale, suggesting that metabolic engineering of BoaAOP2s has huge potential in improving nutritional quality of Chinese kale.},
}
@article {pmid37315746,
year = {2023},
author = {Mesaki, K and Juvet, S and Yeung, J and Guan, Z and Wilson, GW and Hu, J and Davidson, AR and Kleinstiver, BP and Cypel, M and Liu, M and Keshavjee, S},
title = {Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression.},
journal = {The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.healun.2023.06.001},
pmid = {37315746},
issn = {1557-3117},
abstract = {BACKGROUND: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply CRISPR-Cas technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung.<br><br>METHODS: We explored the feasibility of CRISPR-mediated transcriptional upregulation of IL-10, a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting.<br><br>RESULTS: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, i.e. simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients.<br><br>CONCLUSIONS: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants.},
}
@article {pmid37313936,
year = {2023},
author = {Negi, R and Srivastava, A and Srivastava, AK and Pandeya, A and Vatsa, P and Ansari, UA and Pant, AB},
title = {Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders.},
journal = {Indian journal of pharmacology},
volume = {55},
number = {2},
pages = {108-118},
doi = {10.4103/ijp.ijp_56_23},
pmid = {37313936},
issn = {1998-3751},
mesh = {Humans ; Proteome ; *Amyotrophic Lateral Sclerosis/diagnosis/genetics ; *Induced Pluripotent Stem Cells ; Proteomics ; Early Diagnosis ; DNA-Binding Proteins ; Organoids ; },
abstract = {BACKGROUND AND OBJECTIVES: Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is emerging as a novel alternative to human diseased tissue to explore the disease etiology and potential drug discovery. In the interest of the same, we have generated a TDP-43-mutated human iPSCs (hiPSCs) derived 3D organoid model of ALS disease. The high-resolution mass spectrometry (MS)-based proteomic approach is used to explore the differential mechanism under disease conditions and the suitability of a 3D model to study the disease.<br><br>MATERIALS AND METHODS: The hiPSCs cell line was procured from a commercial source, grown, and characterized following standard protocols. The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA. The two groups of organoids were produced by normal and mutated hiPSCs and subjected to the whole proteomic profiling by high-resolution MS in two biological replicates with three technical replicas of each.<br><br>RESULTS: The proteomic analysis of normal and mutated organoids revealed the proteins associated with pathways of neurodegenerative disorders, proteasomes, autophagy, and hypoxia-inducible factor-1 signaling. Differential proteomic analysis revealed that the mutation in TDP-43 gene caused proteomic deregulation, which impaired protein quality mechanisms. Furthermore, this impairment may contribute to the generation of stress conditions that may ultimately lead to the development of ALS pathology.<br><br>CONCLUSION: The developed 3D model represents the majority of candidate proteins and associated biological mechanisms altered in ALS disease. The study also offers novel protein targets that may uncloud the precise disease pathological mechanism and be considered for future diagnostic and therapeutic purposes for various neurodegenerative disorders.},
}
@article {pmid37313752,
year = {2023},
author = {Dai, Y and Ignatyeva, N and Xu, H and Wali, R and Toischer, K and Brandenburg, S and Lenz, C and Pronto, J and Fakuade, FE and Sossalla, S and Zeisberg, EM and Janshoff, A and Kutschka, I and Voigt, N and Urlaub, H and Rasmussen, TB and Mogensen, J and Lehnart, SE and Hasenfuss, G and Ebert, A},
title = {An Alternative Mechanism of Subcellular Iron Uptake Deficiency in Cardiomyocytes.},
journal = {Circulation research},
volume = {},
number = {},
pages = {},
doi = {10.1161/CIRCRESAHA.122.321157},
pmid = {37313752},
issn = {1524-4571},
abstract = {BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis.<br><br>METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of MS-DIA-based proteomics and signaling pathway interrogation. We employed a genetic iPSC model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations.<br><br>RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a small compound, RhoA activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation.<br><br>CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.},
}
@article {pmid37308875,
year = {2023},
author = {Li, J and Song, X and Xiong, Z and Wang, G and Xia, Y and Yang, Y and Ai, L},
title = {Establishment of CRISPR-Cas9 system in Bifidobacteria animalis AR668.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {112},
pmid = {37308875},
issn = {1475-2859},
mesh = {*Bifidobacterium animalis ; CRISPR-Cas Systems ; Bifidobacterium ; Gene Editing ; *Probiotics ; },
abstract = {Bifidobacteria are representative intestinal probiotics that have extremely high application value in the food and medical fields. However, the lack of molecular biology tools limits the research on functional genes and mechanisms of bifidobacteria. The application of an accurate and efficient CRISPR system to genome engineering can fill the gap in efficient genetic tools for bifidobacteria. In this study, CRISPR system of B. animalis AR668 was established, which successfully knocked out gene 0348 and gene 0208. The influence of different homology arms and fragments on the knockout effect of the system was explored. In addition, the inducible plasmid curing system of bifidobacteria was innovatively established. This study contributes to the genetic modification and functional mechanism analysis of bifidobacteria.},
}
@article {pmid37307469,
year = {2023},
author = {Yadav, AK and Butler, C and Yamamoto, A and Patil, AA and Lloyd, AL and Scott, MJ},
title = {CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {25},
pages = {e2301525120},
doi = {10.1073/pnas.2301525120},
pmid = {37307469},
issn = {1091-6490},
mesh = {Female ; Animals ; *CRISPR-Cas Systems ; Fruit ; *Gene Drive Technology ; Gene Targeting ; Drosophila ; },
abstract = {Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. Here, we report the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). Our results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.},
}
@article {pmid37267883,
year = {2023},
author = {Zhao, Y and Zhu, L and Ding, Y and Ji, W and Liu, K and Liu, K and Gao, B and Tao, X and Dong, YG and Wang, FQ and Wei, D},
title = {Simple and cheap CRISPR/Cas12a biosensor based on plug-and-play of DNA aptamers for the detection of endocrine-disrupting compounds.},
journal = {Talanta},
volume = {263},
number = {},
pages = {124761},
doi = {10.1016/j.talanta.2023.124761},
pmid = {37267883},
issn = {1873-3573},
mesh = {*Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; Estradiol ; *Biosensing Techniques/methods ; },
abstract = {Endocrine-disrupting compounds (EDCs) are widely distributed in the environment. Here, we present a CRISPR/Cas12a (CAS) biosensor based on DNA aptamers for point-of-care detection of EDCs. Two typical EDCs, 17β-estradiol (E2) and bisphenol A (BPA), were selected to be detected by the CAS biosensors via the plug-and-play of their DNA aptamers. The results indicated that the performance of the CAS biosensors can be well regulated by controlling the trans-cleavage activity of Cas12a on a single-stranded DNA reporter and optimizing the sequence and ratio of DNA aptamer and activator DNA. Ultimately, two reliable and specific biosensors were developed, with the linear range and limit of detection of 0.2-25 nM and 0.08 nM for E2 and of 0.1-250 nM and 0.06 nM for BPA, respectively. Compared to the existing detection methods, the CAS biosensors showed higher reliability and sensitivity with simple operation, short detection time, and no costly equipment.},
}
@article {pmid37163250,
year = {2023},
author = {Reyes Gaido, OE and Anderson, ME},
title = {CRISPR Editing Takes Aim at Ischemia/Reperfusion Injury.},
journal = {JAMA cardiology},
volume = {8},
number = {6},
pages = {522-523},
doi = {10.1001/jamacardio.2023.0983},
pmid = {37163250},
issn = {2380-6591},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; CRISPR-Cas Systems ; *Reperfusion Injury/genetics/therapy ; Ischemia ; },
}
@article {pmid37309473,
year = {2021},
author = {Nuccio, ML and Claeys, H and Heyndrickx, KS},
title = {CRISPR-Cas technology in corn: a new key to unlock genetic knowledge and create novel products.},
journal = {Molecular breeding : new strategies in plant improvement},
volume = {41},
number = {2},
pages = {11},
pmid = {37309473},
issn = {1572-9788},
abstract = {UNLABELLED: Since its inception in 2012, CRISPR-Cas technologies have taken the life science community by storm. Maize genetics research is no exception. Investigators around the world have adapted CRISPR tools to advance maize genetics research in many ways. The principle application has been targeted mutagenesis to confirm candidate genes identified using map-based methods. Researchers are also developing tools to more effectively apply CRISPR-Cas technologies to maize because successful application of CRISPR-Cas relies on target gene identification, guide RNA development, vector design and construction, CRISPR-Cas reagent delivery to maize tissues, and plant characterization, each contributing unique challenges to CRISPR-Cas efficacy. Recent advances continue to chip away at major barriers that prevent more widespread use of CRISPR-Cas technologies in maize, including germplasm-independent delivery of CRISPR-Cas reagents and production of high-resolution genomic data in relevant germplasm to facilitate CRISPR-Cas experimental design. This has led to the development of novel breeding tools to advance maize genetics and demonstrations of how CRISPR-Cas technologies might be used to enhance maize germplasm.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-021-01200-9.},
}
@article {pmid37306585,
year = {2023},
author = {Wang, S and Sun, E and Liu, Y and Yin, B and Zhang, X and Li, M and Huang, Q and Tan, C and Qian, P and Rao, VB and Tao, P},
title = {Landscape of New Nuclease-Containing Antiphage Systems in Escherichia coli and the Counterdefense Roles of Bacteriophage T4 Genome Modifications.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0059923},
doi = {10.1128/jvi.00599-23},
pmid = {37306585},
issn = {1098-5514},
abstract = {Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. IMPORTANCE Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.},
}
@article {pmid37305433,
year = {2023},
author = {van der Does, C and Braun, F and Ren, H and Albers, SV},
title = {Putative nucleotide-based second messengers in archaea.},
journal = {microLife},
volume = {4},
number = {},
pages = {uqad027},
pmid = {37305433},
issn = {2633-6693},
abstract = {Second messengers transfer signals from changing intra- and extracellular conditions to a cellular response. Over the last few decades, several nucleotide-based second messengers have been identified and characterized in especially bacteria and eukaryotes. Also in archaea, several nucleotide-based second messengers have been identified. This review will summarize our understanding of nucleotide-based second messengers in archaea. For some of the nucleotide-based second messengers, like cyclic di-AMP and cyclic oligoadenylates, their roles in archaea have become clear. Cyclic di-AMP plays a similar role in osmoregulation in euryarchaea as in bacteria, and cyclic oligoadenylates are important in the Type III CRISPR-Cas response to activate CRISPR ancillary proteins involved in antiviral defense. Other putative nucleotide-based second messengers, like 3',5'- and 2',3'-cyclic mononucleotides and adenine dinucleotides, have been identified in archaea, but their synthesis and degradation pathways, as well as their functions as secondary messengers, still remain to be demonstrated. In contrast, 3'-3'-cGAMP has not yet been identified in archaea, but the enzymes required to synthesize 3'-3'-cGAMP have been found in several euryarchaeotes. Finally, the widely distributed bacterial second messengers, cyclic diguanosine monophosphate and guanosine (penta-)/tetraphosphate, do not appear to be present in archaea.},
}
@article {pmid37177893,
year = {2023},
author = {Lewald, KM and Song, W and Eweis-LaBolle, D and Truong, C and Godfrey, KE and Chiu, JC},
title = {Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest Phthorimaea absoluta (Lepidoptera: Gelechiidae).},
journal = {Journal of economic entomology},
volume = {116},
number = {3},
pages = {993-1001},
doi = {10.1093/jee/toad079},
pmid = {37177893},
issn = {1938-291X},
mesh = {Animals ; *Lepidoptera ; *Moths/genetics ; *Solanum lycopersicum/genetics ; CRISPR-Cas Systems ; Pathology, Molecular ; Polymerase Chain Reaction ; },
abstract = {The tomato pest Phthorimaea absoluta Meyrick is highly invasive but has not yet invaded North America. However, several morphologically similar species are already present, making detection of P. absoluta presence and invasion challenging. We designed a quantitative PCR molecular diagnostic to differentiate P. absoluta, P. operculella (Zeller), or Keiferia lycopersicella (Walsingham) (Lepidoptera: Gelechiidae) DNA. Additionally, we developed an RPA-Cas12a molecular diagnostic that allows for the isothermal detection of P. absoluta DNA, eliminating the need for a thermocycler. The results of the RPA-Cas12a diagnostic can be visualized simply using a UV light source and cell phone camera. We expect these diagnostics to improve quarantine and prevention measures against this serious agricultural threat.},
}
@article {pmid37161018,
year = {2023},
author = {Caccamo, M},
title = {New precision-breeding law unlocks gene editing in England.},
journal = {Nature biotechnology},
volume = {41},
number = {6},
pages = {752-753},
pmid = {37161018},
issn = {1546-1696},
mesh = {*Gene Editing ; *Genetic Engineering ; England ; Plant Breeding ; CRISPR-Cas Systems/genetics ; Genome, Plant ; },
}
@article {pmid37098587,
year = {2023},
author = {Wang, X and Zhang, R and Yang, D and Li, G and Fan, Z and Du, H and Wang, Z and Liu, Y and Lin, J and Wu, X and Shi, L and Yang, H and Zhou, Y},
title = {Develop a Compact RNA Base Editor by Fusing ADAR with Engineered EcCas6e.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {17},
pages = {e2206813},
doi = {10.1002/advs.202206813},
pmid = {37098587},
issn = {2198-3844},
mesh = {Animals ; Mice ; Humans ; *CRISPR-Cas Systems/genetics ; RNA/genetics ; HEK293 Cells ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Mutation ; },
abstract = {Catalytically inactive CRISPR-Cas13 (dCas13)-based base editors can achieve the conversion of adenine-to-inosine (A-to-I) or cytidine-to-uridine (C-to-U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199-amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre-crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A-to-I and C-to-U base editing with low transcriptome off-target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases.},
}
@article {pmid36967246,
year = {2023},
author = {Liu, Y and Cottle, WT and Ha, T},
title = {Mapping cellular responses to DNA double-strand breaks using CRISPR technologies.},
journal = {Trends in genetics : TIG},
volume = {39},
number = {7},
pages = {560-574},
doi = {10.1016/j.tig.2023.02.015},
pmid = {36967246},
issn = {0168-9525},
mesh = {*DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems/genetics ; DNA Repair/genetics ; DNA End-Joining Repair ; DNA/genetics ; Gene Editing/methods ; },
abstract = {DNA double-strand breaks (DSBs) are one of the most genotoxic DNA lesions, driving a range of pathological defects from cancers to immunodeficiencies. To combat genomic instability caused by DSBs, evolution has outfitted cells with an intricate protein network dedicated to the rapid and accurate repair of these lesions. Pioneering studies have identified and characterized many crucial repair factors in this network, while the advent of genome manipulation tools like clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has reinvigorated interest in DSB repair mechanisms. This review surveys the latest methodological advances and biological insights gained by utilizing Cas9 as a precise 'damage inducer' for the study of DSB repair. We highlight rapidly inducible Cas9 systems that enable synchronized and efficient break induction. When combined with sequencing and genome-specific imaging approaches, inducible Cas9 systems greatly expand our capability to spatiotemporally characterize cellular responses to DSB at specific genomic coordinates, providing mechanistic insights that were previously unobtainable.},
}
@article {pmid36377278,
year = {2023},
author = {Wang, D and Chen, J and Yuan, Y and Yu, L and Yang, G and Chen, W},
title = {CRISPR/Cas9-mediated knockout of period reveals its function in the circadian rhythms of the diamondback moth Plutella xylostella.},
journal = {Insect science},
volume = {30},
number = {3},
pages = {637-649},
doi = {10.1111/1744-7917.13139},
pmid = {36377278},
issn = {1744-7917},
mesh = {Animals ; Mice ; *Moths/genetics ; CRISPR-Cas Systems ; Phylogeny ; Circadian Rhythm/genetics ; Photoperiod ; },
abstract = {Circadian clocks control the rhythmicity of many behaviors and physiological features of insects. To study the circadian clock of the moth Plutella xylostella, we employed CRISPR/Cas9-mediated genome editing to investigate the effect of loss of the clock gene period on the circadian rhythms. P. xylostella harbors a single copy of period. Phylogenetic analysis showed that P. xylostella PERIOD is more homologous to mouse PERIOD than the PERIOD proteins from bees, flies, mosquitos, and many other Lepidoptera, such as Danaus plexippus and Bombyx mori. The circadian rhythms in adult locomotor activity were altered in the period knockout strain of P. xylostella under light-dark (LD) and continuous dark (DD) conditions. Under the LD cycle, the wild-type moths displayed nocturnal activity with activity peaking very early after lights off and quickly declining after lights on. In contrast, the period knockout strain had no peak in activity when the lights were turned off and exhibited steady activity throughout the hours of darkness. Interestingly, under DD conditions, our results showed that the locomotor rhythm can be maintained without period gene, but at a lower rhythmicity ratio than wild-type. In addition, knockout of period in P. xylostella changed circadian rhythms patterns related to pupal eclosion, mating, egg-laying, and egg hatching. Mechanistically, loss of PERIOD disrupted the molecular rhythm of period and changed the clock transcription rhythm in the heads of the moths under LD and DD conditions. Together, our study indicates that the PERIOD is required for normal expression of many behavioral rhythms in P. xylostella.},
}
@article {pmid36169087,
year = {2023},
author = {Han, WK and Tang, FX and Gao, HL and Wang, Y and Yu, N and Jiang, JJ and Liu, ZW},
title = {Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda.},
journal = {Insect science},
volume = {30},
number = {3},
pages = {625-636},
doi = {10.1111/1744-7917.13122},
pmid = {36169087},
issn = {1744-7917},
mesh = {Animals ; *Gene Editing/methods ; Spodoptera/genetics ; *CRISPR-Cas Systems ; Acetylcholinesterase/genetics ; Angiotensin-Converting Enzyme 2/genetics ; Mutation ; Larva/genetics ; },
abstract = {The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single-guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic-eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.},
}
@article {pmid37305413,
year = {2023},
author = {Guo, Y and Xia, H and Dai, T and Liu, T},
title = {RPA-CRISPR/Cas12a mediated isothermal amplification for visual detection of Phytophthora sojae.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1208837},
pmid = {37305413},
issn = {2235-2988},
mesh = {*Recombinases ; *Phytophthora/genetics ; CRISPR-Cas Systems ; Nucleotidyltransferases ; Fluorescent Dyes ; },
abstract = {INTRODUCTION: Phytophthora sojae is among the most devastating pathogens of soybean (Glycine max) and severely impacts soybean production in several countries. The resulting disease can be difficult to diagnose and other Phytophthora species can also infect soybean. Accurate diagnosis is important for management of the disease caused by P. sojae.<br><br>METHODS: In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system were used for detection of P. sojae. The assay was highly specific to P. sojae.<br><br>RESULTS: The test results were positive for 29 isolates of P. sojae, but negative for 64 isolates of 29 Phytophthora species, 7 Phytopythium and Pythium species, 32 fungal species, and 2 Bursaphelenchus species. The method was highly sensitive, detecting as little as 10 pg.&#xb5;L[-1] of P. sojae genomic DNA at 37&#xb0;C in 20 min. The test results were visible under UV light and readout coming from fluorophores. In addition, P. sojae was detected from natural inoculated hypocotyls of soybean seedlings using this novel assay. The rapidity and accuracy of the method were verified using 30 soybean rhizosphere samples.<br><br>DISCUSSION: In conclusion, the RPA-CRISPR/Cas12a detection assay developed here is sensitive, efficient, and convenient, and has potential for further development as a kit for monitoring root rot of soybean in the field.},
}
@article {pmid37304707,
year = {2023},
author = {Tripodi, P and Singh, NK and Abberton, M and Nankar, AN},
title = {Editorial: Enhancing allele mining for crop improvement amid the emerging challenge of climate change.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1197086},
doi = {10.3389/fpls.2023.1197086},
pmid = {37304707},
issn = {1664-462X},
}
@article {pmid37301980,
year = {2023},
author = {Wang, H and Qi, X and Zhu, J and Liu, C and Fan, H and Zhang, X and Li, X and Yang, Q and Xie, C},
title = {Pollen self-elimination CRISPR/Cas genome editing prevents transgenic pollen dispersal in maize.},
journal = {Plant communications},
volume = {},
number = {},
pages = {100637},
doi = {10.1016/j.xplc.2023.100637},
pmid = {37301980},
issn = {2590-3462},
}
@article {pmid37301944,
year = {2023},
author = {Peterson, KA and Khalouei, S and Hanafi, N and Wood, JA and Lanza, DG and Lintott, LG and Willis, BJ and Seavitt, JR and Braun, RE and Dickinson, ME and White, JK and Lloyd, KCK and Heaney, JD and Murray, SA and Ramani, A and Nutter, LMJ},
title = {Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {626},
pmid = {37301944},
issn = {2399-3642},
support = {UM1 OD023222/CD/ODCDC CDC HHS/United States ; UM1 OD023222/CD/ODCDC CDC HHS/United States ; UM1 OD023222/CD/ODCDC CDC HHS/United States ; UM1 HG006348/HG/NHGRI NIH HHS/United States ; UM1 HG006348/HG/NHGRI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genome ; Mutation ; Mutagenesis ; },
abstract = {Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.},
}
@article {pmid37300776,
year = {2023},
author = {Murray, L and Olson, MN and Barton, N and Dawes, P and Chan, Y and Lim, ET},
title = {FACS-Based Sequencing Approach to Evaluate Cell Type to Genotype Associations Using Cerebral Organoids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2683},
number = {},
pages = {193-199},
pmid = {37300776},
issn = {1940-6029},
mesh = {Flow Cytometry/methods ; Genotype ; *Organoids/metabolism ; *CRISPR-Cas Systems ; },
abstract = {Recent technological developments have led to widespread applications of large-scale transcriptomics-based sequencing methods to identify genotype-to-cell type associations. Here we describe a fluorescence-activated cell sorting (FACS)-based sequencing method to utilize CRISPR/Cas9 edited mosaic cerebral organoids to identify or validate genotype-to-cell type associations. Our approach is high-throughput and quantitative and uses internal controls to enable comparisons of the results across different antibody markers and experiments.},
}
@article {pmid37298481,
year = {2023},
author = {Paschoudi, K and Yannaki, E and Psatha, N},
title = {Precision Editing as a Therapeutic Approach for β-Hemoglobinopathies.},
journal = {International journal of molecular sciences},
volume = {24},
number = {11},
pages = {},
pmid = {37298481},
issn = {1422-0067},
mesh = {Humans ; CRISPR-Cas Systems ; Cell Line, Tumor ; Transcription Factors/metabolism ; DNA-Binding Proteins/metabolism ; *Hemoglobinopathies/genetics/therapy ; Gene Editing/methods ; *Anemia, Sickle Cell/genetics/therapy/metabolism ; gamma-Globins/genetics/metabolism ; *beta-Thalassemia/genetics/therapy/metabolism ; },
abstract = {Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.},
}
@article {pmid37296663,
year = {2023},
author = {Skryabin, EB and De Jong, KA and Subramanian, H and Bork, NI and Froese, A and Skryabin, BV and Nikolaev, VO},
title = {CRISPR/Cas9 Knock-Out in Primary Neonatal and Adult Cardiomyocytes Reveals Distinct cAMP Dynamics Regulation by Various PDE2A and PDE3A Isoforms.},
journal = {Cells},
volume = {12},
number = {11},
pages = {},
pmid = {37296663},
issn = {2073-4409},
mesh = {Mice ; Rats ; Animals ; *Myocytes, Cardiac/metabolism ; *CRISPR-Cas Systems/genetics ; Cyclic AMP/metabolism ; Diethylstilbestrol ; },
abstract = {Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.},
}
@article {pmid37296208,
year = {2023},
author = {Gopalakrishna, KP and Hillebrand, GH and Bhavana, VH and Elder, JL and D'Mello, A and Tettelin, H and Hooven, TA},
title = {Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {620},
pmid = {37296208},
issn = {2399-3642},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *RNA/metabolism ; Bacteria/genetics ; DNA/genetics ; Streptococcus/genetics ; },
abstract = {Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δcas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.},
}
@article {pmid37230190,
year = {2023},
author = {Mackon, E and Jeazet Dongho Epse Mackon, GC and Guo, Y and Ma, Y and Yao, Y and Liu, P},
title = {Development and application of CRISPR/Cas9 to improve anthocyanin pigmentation in plants: Opportunities and perspectives.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {333},
number = {},
pages = {111746},
doi = {10.1016/j.plantsci.2023.111746},
pmid = {37230190},
issn = {1873-2259},
mesh = {*Anthocyanins/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Plant Breeding ; Plants/genetics ; Vegetables/genetics ; Pigmentation/genetics ; Genome, Plant ; },
abstract = {Since its discovery in 2012, the novel technology of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has greatly contributed to revolutionizing molecular biology. It has been demonstrated to be an effective approach for identifying gene function and improving some important traits. Anthocyanins are secondary metabolites responsible for a wide spectrum of aesthetic coloration in various plant organs and are beneficial for health. As such, increasing anthocyanin content in plants, especially the edible tissue and organs, is always a main goal for plant breeding. Recently, CRISPR/Cas9 technology has been highly desired to enhance the amount of anthocyanin in vegetables, fruits, cereals, and other attractive plants with more precision. Here we reviewed the recent knowledge concerning CRISPR/Cas9-mediated anthocyanin enhancement in plants. In addition, we addressed the future avenues of promising potential target genes that could be helpful for achieving the same goal using CRISPR/Cas9 in several plants. Thus, molecular biologists, genetic engineers, agricultural scientists, plant geneticists, and physiologists may benefit from CRISPR technology to boost the biosynthesis and accumulation of anthocyanins in fresh fruits, vegetables, grains, roots, and ornamental plants.},
}
@article {pmid37226062,
year = {2023},
author = {Zhang, N and Zuo, Z},
title = {Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition.},
journal = {Journal of chemical information and modeling},
volume = {63},
number = {11},
pages = {3500-3509},
doi = {10.1021/acs.jcim.3c00256},
pmid = {37226062},
issn = {1549-960X},
mesh = {*CRISPR-Cas Systems ; Molecular Docking Simulation ; Ligands ; *Gene Editing/methods ; Binding Sites ; },
abstract = {The need for precision control of CRISPR-Cas9 genome editing has created a demand for anti-CRISPR molecules. Recently, the first class of small-molecule Cas9 inhibitors has been identified, verifying the feasibility of regulating CRISPR-Cas9 activity using direct-acting small molecules. However, it remains enigmatic as to the location of the ligand binding site(s) on CRISPR-Cas9 and how the ligand binding leads to Cas9 functional inhibition. Here, we established an integrative computational protocol, including massive binding site mapping, molecular docking, molecular dynamics simulations, and free energy calculations. Ultimately, a Cas9 ligand binding site was discovered from the dynamics trajectories that is hidden within its carboxyl-terminal domain (CTD), a domain recognizing the protospacer adjacent motif (PAM). Using the top inhibitor BRD0539 as a probe, we demonstrated that the ligand binding induces significant CTD structural rearrangements toward an incompetent conformation for PAM DNA engagement. The revealed molecular mechanism of BRD0539 inhibiting Cas9 is in well agreement with the experimental data. This study provides a structural and mechanistic basis for the potency improvement of existing ligands and the rational discovery of novel small-molecule brakes for developing safer CRISPR-Cas9 technologies.},
}
@article {pmid37207820,
year = {2023},
author = {Yang, X and Wang, J and Sun, X and Wang, P and Dou, H and Yang, Z and Wang, Y},
title = {A method for generating genome edited plant lines from CRISPR-transformed Shanxin poplar plants.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {333},
number = {},
pages = {111732},
doi = {10.1016/j.plantsci.2023.111732},
pmid = {37207820},
issn = {1873-2259},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Populus/genetics ; Genome, Plant/genetics ; Mutation ; Plants, Genetically Modified/genetics ; },
abstract = {Due to the reason of low efficiency of mutation in CRISPR-editing, a high frequency of CRISPR transformed plant lines failing in mutation had been generated and had to be discarded. In the present study, we built a method to increase the efficiency of CRISPR-editing. We used Shanxin poplar (Populus davidiana×P. bolleana) as the study material, and CRISPR-editing system was first built to generate the CRISPR-transformed lines. The line that failed in CRISPR-editing was used for improving the efficiency of mutation, which was treated with heat (37 °C) to improve the cleaving activity of Cas9, leading to increased frequency of the cleaved DNA. Our results indicated that 87-100% of cells in CRISPR-transformed plants whose DNA had been cleaved by heat treatment, and the heat treatment plants were then cut into explants to differentiate adventitious buds. Each differentiated bud can be considered as an independent line. Twenty independent lines were randomly selected for analysis, and all of them had been mutated by CRISPR editing, displaying 4 types of mutation. Our results indicated that heat treatment combined with re-differentiation can generate CRISPR-edited plants efficiently. This method could conquer the problem of low mutation efficiency of CRISPR-editing in Shanxin poplar, and will have a wide application in plant CRISPR-editing.},
}
@article {pmid37161298,
year = {2023},
author = {Tang, PZ and Ding, B and Reyes, C and Papp, D and Potter, J},
title = {Target-seq: single workflow for detection of genome integration site, DNA translocation and off-target events.},
journal = {BioTechniques},
volume = {74},
number = {5},
pages = {211-224},
doi = {10.2144/btn-2023-0013},
pmid = {37161298},
issn = {1940-9818},
mesh = {*CRISPR-Cas Systems/genetics ; Workflow ; *Gene Editing ; Genetic Therapy ; DNA/genetics ; },
abstract = {Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for in vivo and ex vivo delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.},
}
@article {pmid37296137,
year = {2023},
author = {Zimmermann, A and Prieto-Vivas, JE and Cautereels, C and Gorkovskiy, A and Steensels, J and Van de Peer, Y and Verstrepen, KJ},
title = {A Cas3-base editing tool for targetable in vivo mutagenesis.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {3389},
pmid = {37296137},
issn = {2041-1723},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; Mutation ; Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.},
}
@article {pmid37294164,
year = {2023},
author = {Wu, Y and Chang, D and Chang, Y and Zhang, Q and Liu, Y and Brennan, JD and Li, Y and Liu, M},
title = {Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2303007},
doi = {10.1002/smll.202303007},
pmid = {37294164},
issn = {1613-6829},
abstract = {clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.},
}
@article {pmid37293964,
year = {2023},
author = {Kenney, CT and Marraffini, LA},
title = {Rarely acquired type II-A CRISPR-Cas spacers mediate anti-viral immunity through the targeting of a non-canonical PAM sequence.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad501},
pmid = {37293964},
issn = {1362-4962},
support = {T32GM07739/GF/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
abstract = {The Streptococcus pyogenes type II-A CRISPR-Cas systems provides adaptive immunity through the acquisition of short DNA sequences from invading viral genomes, called spacers. Spacers are transcribed into short RNA guides that match regions of the viral genome followed by a conserved NGG DNA motif, known as the PAM. These RNA guides, in turn, are used by the Cas9 nuclease to find and destroy complementary DNA targets within the viral genome. While most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by NGG sequences, there is a small fraction that target non-canonical PAMs. Whether these spacers originate through accidental acquisition of phage sequences and/or provide efficient defense is unknown. Here we found that many of them match phage target regions flanked by an NAGG PAM. Despite being scarcely present in bacterial populations, NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro; with both activities comparable to spacers that target sequences followed by the canonical AGG PAM. In contrast, acquisition experiments showed that NAGG spacers are acquired at very low frequencies. We therefore conclude that discrimination against these sequences occurs during immunization of the host. Our results reveal unexpected differences in PAM recognition during the spacer acquisition and targeting stages of the type II-A CRISPR-Cas immune response.},
}
@article {pmid37293662,
year = {2023},
author = {Hu, Z and Sun, A and Yang, J and Naz, G and Sun, G and Li, Z and Gogo Liu, JJ and Zhang, S and Zhang, X},
title = {Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA.},
journal = {Chemical science},
volume = {14},
number = {22},
pages = {5945-5955},
pmid = {37293662},
issn = {2041-6520},
abstract = {Chemical modifications of CRISPR-Cas nucleases help decrease off-target editing and expand the biomedical applications of CRISPR-based gene manipulation tools. Here, we found that epigenetic modifications of guide RNA, such as m6A and m1A methylation, can effectively inhibit both the cis- and trans-DNA cleavage activities of CRISPR-Cas12a. The underlying mechanism is that methylations destabilize the secondary and tertiary structure of gRNA which prevents the assembly of the Cas12a-gRNA nuclease complex, leading to decreased DNA targeting ability. A minimum of three adenine methylated nucleotides are required to completely inhibit the nuclease activity. We also demonstrate that these effects are reversible through the demethylation of gRNA by demethylases. This strategy has been used in the regulation of gene expression, demethylase imaging in living cells and controllable gene editing. The results demonstrate that the methylation-deactivated and demethylase-activated strategy is a promising tool for regulation of the CRISPR-Cas12a system.},
}
@article {pmid37292749,
year = {2023},
author = {Gopalakrishna, KP and Hillebrand, GH and Bhavana, VH and Elder, JL and D'Mello, A and Tettelin, H and Hooven, TA},
title = {Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.05.24.542094},
pmid = {37292749},
abstract = {Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δ cas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.},
}
@article {pmid37291577,
year = {2023},
author = {Huang, X and Li, A and Xu, P and Yu, Y and Li, S and Hu, L and Feng, S},
title = {Current and prospective strategies for advancing the targeted delivery of CRISPR/Cas system via extracellular vesicles.},
journal = {Journal of nanobiotechnology},
volume = {21},
number = {1},
pages = {184},
pmid = {37291577},
issn = {1477-3155},
mesh = {*CRISPR-Cas Systems ; Prospective Studies ; Gene Editing/methods ; Gene Transfer Techniques ; *Extracellular Vesicles ; },
abstract = {Extracellular vesicles (EVs) have emerged as a promising platform for gene delivery owing to their natural properties and phenomenal functions, being able to circumvent the significant challenges associated with toxicity, problematic biocompatibility, and immunogenicity of the standard approaches. These features are of particularly interest for targeted delivery of the emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. However, the current efficiency of EV-meditated transport of CRISPR/Cas components remains insufficient due to numerous exogenous and endogenous barriers. Here, we comprehensively reviewed the current status of EV-based CRISPR/Cas delivery systems. In particular, we explored various strategies and methodologies available to potentially improve the loading capacity, safety, stability, targeting, and tracking for EV-based CRISPR/Cas system delivery. Additionally, we hypothesise the future avenues for the development of EV-based delivery systems that could pave the way for novel clinically valuable gene delivery approaches, and may potentially bridge the gap between gene editing technologies and the laboratory/clinical application of gene therapies.},
}
@article {pmid37291244,
year = {2023},
author = {Matsuyama, A and Hashimoto, A and Nishimura, S and Yoshida, M},
title = {A set of vectors and strains for chromosomal integration in fission yeast.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {9295},
pmid = {37291244},
issn = {2045-2322},
mesh = {*Schizosaccharomyces/genetics/metabolism ; Genetic Vectors/genetics ; Gene Editing/methods ; Mutation ; CRISPR-Cas Systems/genetics ; },
abstract = {The expression of heterologous genes is an important technique in yeast genetics. In fission yeast, the leu1 and ura4 genes have been used mainly as selectable markers for heterologous expression. To expand the repertoire of selection markers available for heterologous expression of genes, here we developed new host-vector systems employing lys1 and arg3. By employing genome editing with the CRISPR/Cas9 system, we isolated several alleles of lys1 and arg3, each having a critical mutation in the ORF region. In parallel, we developed a set of vectors that complement the amino acid auxotrophy of lys1 and arg3 mutants when integrated into each locus. Using these vectors in combination with the previously developed integration vector pDUAL, we successfully observed the localization of three proteins in a cell simultaneously by fusing them with different fluorescent proteins. Thus, these vectors enable combinatorial expression of heterologous genes, which addresses increasingly diverse experimental challenges.},
}
@article {pmid37291233,
year = {2023},
author = {Ramesh, A and Trivedi, V and Lee, S and Tafrishi, A and Schwartz, C and Mohseni, A and Li, M and Lonardi, S and Wheeldon, I},
title = {acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {617},
pmid = {37291233},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; Gene Library ; Genomics ; Genes, Essential ; *Yarrowia/genetics ; },
abstract = {High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.},
}
@article {pmid37291100,
year = {2023},
author = {Zheng, C and Liu, B and Dong, X and Gaston, N and Sontheimer, EJ and Xue, W},
title = {Template-jumping prime editing enables large insertion and exon rewriting in vivo.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {3369},
pmid = {37291100},
issn = {2041-1723},
mesh = {Mice ; Animals ; *Gene Editing ; *DNA/metabolism ; DNA Breaks, Double-Stranded ; Exons/genetics ; Genome ; CRISPR-Cas Systems/genetics ; },
abstract = {Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo.},
}
@article {pmid37289779,
year = {2023},
author = {Nakajima, I and Kawahigashi, H and Nishitani, C and Azuma, A and Haji, T and Toki, S and Endo, M},
title = {Targeted deletion of grape retrotransposon associated with fruit skin color via CRISPR/Cas9 in Vitis labrascana 'Shine Muscat'.},
journal = {PloS one},
volume = {18},
number = {6},
pages = {e0286698},
pmid = {37289779},
issn = {1932-6203},
mesh = {*Vitis/genetics/metabolism ; Skin Pigmentation ; Fruit/genetics/metabolism ; Retroelements/genetics ; CRISPR-Cas Systems ; Oman ; Anthocyanins/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal. PCR amplification and sequencing detected Gret1 eliminated cells in 19 of 45 transgenic plants. Although we have not yet confirmed any effects on grape berry skin color, we were successful in demonstrating that cleaving the long terminal repeat (LTR) present at both ends of Gret1 can efficiently eliminate the transposon.},
}
@article {pmid37288976,
year = {2023},
author = {Tariq, A and Mushtaq, M and Yaqoob, H and Bhat, BA and Zargar, SM and Raza, A and Ali, S and Charagh, S and Mubarik, MS and Zaman, QU and Prasad, PV and Mir, RA},
title = {Putting CRISPR-Cas system in action: a golden window for efficient and precise genome editing for crop improvement.},
journal = {GM crops &amp; food},
volume = {14},
number = {1},
pages = {1-27},
pmid = {37288976},
issn = {2164-5701},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; },
abstract = {The daunting task of feeding an ever-growing population is an immense challenge for the contemporary scientific community, especially in view of the rapidly changing climate throughout the world. Amidst these threatening crises, we witness rapid development in genome editing (GE) technologies, revolutionizing the field of applied genomics and molecular breeding. Various GE tools have been developed during the last two decades, but the CRISPR/Cas system has most recently made a significant impact on crop improvement. The major breakthroughs of this versatile toolbox are genomic modifications like single base-substitutions, multiplex GE, gene regulation, screening mutagenesis, and enhancing the breeding of wild crop plants. Previously, this toolbox was used to modify genes related to significant traits such as biotic/abiotic resistance/tolerance, post-harvest traits, nutritional regulation, and to address self-incompatibility analysis-related challenges. In the present review, we have demonstrated the functional dynamics of CRISPR-based GE and its applicability in targeting genes to accomplish novel editing of crops. The compiled knowledge will provide a solid foundation for highlighting the primary source for applying CRISPR/Cas as a toolbox for enhancing crops, to achieve food and nutritional security.},
}
@article {pmid37253059,
year = {2023},
author = {Sanvicente-García, M and García-Valiente, A and Jouide, S and Jaraba-Wallace, J and Bautista, E and Escobosa, M and Sánchez-Mejías, A and Güell, M},
title = {CRISPR-Analytics (CRISPR-A): A platform for precise analytics and simulations for gene editing.},
journal = {PLoS computational biology},
volume = {19},
number = {5},
pages = {e1011137},
pmid = {37253059},
issn = {1553-7358},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing characterization with currently available tools does not always give precise relative proportions among the different types of gene edits present in an edited bulk of cells. We have developed CRISPR-Analytics, CRISPR-A, which is a comprehensive and versatile genome editing web application tool and a nextflow pipeline to give support to gene editing experimental design and analysis. CRISPR-A provides a robust gene editing analysis pipeline composed of data analysis tools and simulation. It achieves higher accuracy than current tools and expands the functionality. The analysis includes mock-based noise correction, spike-in calibrated amplification bias reduction, and advanced interactive graphics. This expanded robustness makes this tool ideal for analyzing highly sensitive cases such as clinical samples or experiments with low editing efficiencies. It also provides an assessment of experimental design through the simulation of gene editing results. Therefore, CRISPR-A is ideal to support multiple kinds of experiments such as double-stranded DNA break-based engineering, base editing (BE), primer editing (PE), and homology-directed repair (HDR), without the need of specifying the used experimental approach.},
}
@article {pmid37156844,
year = {2023},
author = {},
title = {Capturing prime editor off-target sites within the genome.},
journal = {Nature methods},
volume = {20},
number = {6},
pages = {801-802},
pmid = {37156844},
issn = {1548-7105},
mesh = {*Gene Editing ; *Genome ; CRISPR-Cas Systems ; },
}
@article {pmid37156841,
year = {2023},
author = {Liang, SQ and Liu, P and Ponnienselvan, K and Suresh, S and Chen, Z and Kramme, C and Chatterjee, P and Zhu, LJ and Sontheimer, EJ and Xue, W and Wolfe, SA},
title = {Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag.},
journal = {Nature methods},
volume = {20},
number = {6},
pages = {898-907},
pmid = {37156841},
issn = {1548-7105},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *Genome ; DNA/genetics ; DNA Breaks, Double-Stranded ; Cell Line ; CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.},
}
@article {pmid37119891,
year = {2023},
author = {Zhang, M and Hu, Y and Liu, J and Guan, Z and Zhang, W},
title = {CRISPR/Cas9-mediated genome editing of gustatory receptor NlugGr23a causes male sterility in the brown planthopper Nilaparvata lugens.},
journal = {International journal of biological macromolecules},
volume = {241},
number = {},
pages = {124612},
doi = {10.1016/j.ijbiomac.2023.124612},
pmid = {37119891},
issn = {1879-0003},
mesh = {Male ; Female ; Animals ; Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; Seeds ; Receptors, Cell Surface/genetics ; *Drosophila Proteins ; *Hemiptera/genetics ; *Infertility, Male ; },
abstract = {Gustatory receptors (Grs) have an essential role in chemical recognition so as to evaluate food quality. Insect Grs also participate in non-gustatory functions, such as olfaction, temperature sensing, and mating. In this study, we knocked out NlugGr23a, a putative fecundity-related Gr, using the CRISPR/Cas9 system in the brown planthopper Nilaparvata lugens, a serious insect pest of rice. Surprisingly, homozygous NlugGr23a mutant (NlugGr23a[-/-]) males were sterile but their sperm were motile and morphologically normal. DAPI staining of mutant sperm inseminated eggs showed that most of NlugGr23a[-/-] sperm failed to fertilize eggs, even if they were capable of entering into the egg as a result of their arrested development prior to male pronucleus formation. Immunohistochemistry demonstrated the expression of NlugGr23a in testis. Moreover, prior mating by NlugGr23a[-/-] males suppressed female fertility. To our knowledge, it is the first report that a chemoreceptor is implicated in male sterility and provides a potential molecular target for genetic pest control alternatives.},
}
@article {pmid37094074,
year = {2023},
author = {Ma, S and Liao, K and Li, M and Wang, X and Lv, J and Zhang, X and Huang, H and Li, L and Huang, T and Guo, X and Lin, Y and Rong, Z},
title = {Phase-separated DropCRISPRa platform for efficient gene activation in mammalian cells and mice.},
journal = {Nucleic acids research},
volume = {51},
number = {10},
pages = {5271-5284},
pmid = {37094074},
issn = {1362-4962},
mesh = {Mice ; Animals ; Transcriptional Activation ; *Nuclear Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Transcription Factors/genetics ; Mammals ; },
abstract = {Liquid-liquid phase separation (LLPS) plays a critical role in regulating gene transcription via the formation of transcriptional condensates. However, LLPS has not been reported to be engineered as a tool to activate endogenous gene expression in mammalian cells or in vivo. Here, we developed a droplet-forming CRISPR (clustered regularly interspaced short palindromic repeats) gene activation system (DropCRISPRa) to activate transcription with high efficiency via combining the CRISPR-SunTag system with FETIDR-AD fusion proteins, which contain an N-terminal intrinsically disordered region (IDR) of a FET protein (FUS or TAF15) and a transcription activation domain (AD, VP64/P65/VPR). In this system, the FETIDR-AD fusion protein formed phase separation condensates at the target sites, which could recruit endogenous BRD4 and RNA polymerase II with an S2 phosphorylated C-terminal domain (CTD) to enhance transcription elongation. IDR-FUS9Y>S and IDR-FUSG156E, two mutants with deficient and aberrant phase separation respectively, confirmed that appropriate phase separation was required for efficient gene activation. Further, the DropCRISPRa system was compatible with a broad set of CRISPR-associated (Cas) proteins and ADs, including dLbCas12a, dAsCas12a, dSpCas9 and the miniature dUnCas12f1, and VP64, P65 and VPR. Finally, the DropCRISPRa system could activate target genes in mice. Therefore, this study provides a robust tool to activate gene expression for foundational research and potential therapeutics.},
}
@article {pmid37069311,
year = {2023},
author = {Parvez, S and Brandt, ZJ and Peterson, RT},
title = {Large-scale F0 CRISPR screens in vivo using MIC-Drop.},
journal = {Nature protocols},
volume = {18},
number = {6},
pages = {1841-1865},
pmid = {37069311},
issn = {1750-2799},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; Retrospective Studies ; Genetic Testing ; Phenotype ; },
abstract = {The zebrafish is a powerful model system for studying animal development, for modeling genetic diseases, and for large-scale in vivo functional genetics. Because of its ease of use and its high efficiency in targeted gene perturbation, CRISPR-Cas9 has recently gained prominence as the tool of choice for genetic manipulation in zebrafish. However, scaling up the technique for high-throughput in vivo functional genetics has been a challenge. We recently developed a method, Multiplexed Intermixed CRISPR Droplets (MIC-Drop), that makes large-scale CRISPR screening in zebrafish possible. Here, we outline the step-by-step protocol for performing functional genetic screens in zebrafish by using MIC-Drop. MIC-Drop uses multiplexed single-guide RNAs to generate biallelic mutations in injected zebrafish embryos, allowing genetic screens to be performed in F0 animals. Combining microfluidics and DNA barcoding enables simultaneous targeting of tens to hundreds of genes from a single injection needle, while also enabling retrospective and rapid identification of the genotype responsible for an observed phenotype. The primary target audiences for MIC-Drop are developmental biologists, zebrafish geneticists, and researchers interested in performing in vivo functional genetic screens in a vertebrate model system. MIC-Drop will also prove useful in the hands of chemical biologists seeking to identify targets of small molecules that cause phenotypic changes in zebrafish. By using MIC-Drop, a typical screen of 100 genes can be conducted within 2-3 weeks by a single user.},
}
@article {pmid36892416,
year = {2023},
author = {Sheng, X and Mahendra, RA and Wang, CT and Brunner, AM},
title = {CRISPR/Cas9 mutants delineate roles of Populus FT and TFL1/CEN/BFT family members in growth, dormancy release and flowering.},
journal = {Tree physiology},
volume = {43},
number = {6},
pages = {1042-1054},
doi = {10.1093/treephys/tpad027},
pmid = {36892416},
issn = {1758-4469},
mesh = {*Plant Proteins/metabolism ; *Populus/metabolism ; CRISPR-Cas Systems ; Flowers ; Meristem/genetics ; Gene Expression Regulation, Plant ; },
abstract = {Vegetative and reproductive phase change and phenology are economically and ecologically important traits. Trees typically require several years of growth before flowering and, once mature, seasonal control of the transition to flowering and flower development is necessary to maintain vegetative meristems and for reproductive success. Members of two related gene subfamilies, FLOWERING LOCUST (FT) and TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN)/BROTHER OF FT AND TFL1 (BFT), have antagonistic roles in flowering in diverse species and roles in vegetative phenology in trees, but many details of their functions in trees have yet to be resolved. Here, we used CRISPR/Cas9 to generate single and double mutants involving the five Populus FT and TFL1/CEN/BFT genes. The ft1 mutants exhibited wild-type-like phenotypes in long days and short days, but after chilling, to release dormancy, they showed delayed bud flush and GA3 could compensate for the ft1 mutation. After rooting and generating some phytomers in tissue culture, both cen1 and cen1ft1 mutants produced terminal as well as axillary flowers, indicating that the cen1 flowering phenotype is independent of FT1. The CEN1 showed distinct circannual expression patterns in vegetative and reproductive tissues and comparison with the expression patterns of FT1 and FT2 suggests that the relative levels of CEN1 compared with FT1 and FT2 regulate multiple phases of vegetative and reproductive seasonal development.},
}
@article {pmid37288365,
year = {2021},
author = {Zhang, Q and Chen, Z and Sun, B},
title = {Molecular mechanisms of Streptococcus pyogenes Cas9: a single-molecule perspective.},
journal = {Biophysics reports},
volume = {7},
number = {6},
pages = {475-489},
pmid = {37288365},
issn = {2364-3420},
abstract = {Cas9 is an RNA-guided endonuclease from the type II CRISPR-Cas system that employs RNA-DNA base pairing to target and cleave foreign DNA in bacteria. Due to its robust and programmable activity, Cas9 has been repurposed as a revolutionary technology for wide-ranging biological and medical applications. A comprehensive understanding of Cas9 mechanisms at the molecular level would aid in its better usage as a genome tool. Over the past few years, single-molecule techniques, such as fluorescence resonance energy transfer, DNA curtains, magnetic tweezers, and optical tweezers, have been extensively applied to characterize the detailed molecular mechanisms of Cas9 proteins. These techniques allow researchers to monitor molecular dynamics and conformational changes, probe essential DNA-protein interactions, detect intermediate states, and distinguish heterogeneity along the reaction pathway, thus providing enriched functional and mechanistic perspectives. This review outlines the single-molecule techniques that have been utilized for the investigation of Cas9 proteins and discusses insights into the mechanisms of the widely used Streptococcus pyogenes (Sp) Cas9 revealed through these techniques.},
}
@article {pmid37287467,
year = {2023},
author = {Jia, N and Wang, C and Liu, X and Huang, X and Xiao, F and Fu, J and Sun, C and Xu, Z and Wang, G and Zhou, J and Wang, Y},
title = {A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1192134},
pmid = {37287467},
issn = {2235-2988},
mesh = {Humans ; *Mycobacterium tuberculosis/genetics ; CRISPR-Cas Systems ; Pandemics ; Sensitivity and Specificity ; *COVID-19/genetics ; *Tuberculosis/microbiology ; },
abstract = {Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.},
}
@article {pmid37287312,
year = {2023},
author = {Armstrong, DA and Hudson, TR and Hodge, CA and Hampton, TH and Howell, AL and Hayden, MS},
title = {PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity.},
journal = {RNA biology},
volume = {20},
number = {1},
pages = {296-305},
pmid = {37287312},
issn = {1555-8584},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Mutation ; RNA/genetics ; Nucleotides ; Receptors, CCR5/genetics ; },
abstract = {Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.},
}
@article {pmid37287283,
year = {2023},
author = {Aoun Mehmood Sherazi, S and Rafique, F and Haris, M and Arshad, A and Qaiser, H and Uzair, M and Arshad, M},
title = {Applications of CRISPR Cas-9 in Ovarian Cancer Research.},
journal = {Protein and peptide letters},
volume = {},
number = {},
pages = {},
doi = {10.2174/0929866530666230607104453},
pmid = {37287283},
issn = {1875-5305},
abstract = {Ovarian cancer is a highly prevalent malignancy among women and affects a significant population worldwide. Different forms of hormonal treatments or chemotherapies are used to treat ovarian cancer, but the possible side effects, including menopausal symptoms, can be severe, forcing some patients to prematurely stop the treatment. The emerging genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9), has the potential to treat ovarian cancer via gene editing strategies. Studies have reported CRISPR knockouts of several oncogenes that are involved in the pathogenesis of ovarian cancer, such as BMI1, CXCR2, MTF1, miR-21, and BIRC5, and demonstrate the potential of the CRISPR-Cas9 genome editing technique to effectively treat ovarian cancer. However, there are limitations that restrict the biomedical applications of CRISPR-Cas9 and limit the implementation of Gene therapy for ovarian cancer. These include off-target DNA cleavage and the effects of CRISPR-Cas9 in non-target, normal cells. This article aims to review the current state of ovarian cancer research, highlight the significance of CRISPR-Cas9 in ovarian cancer treatment, and establish the groundwork for potential clinical research.},
}
@article {pmid37287132,
year = {2023},
author = {Giner, G and Ikram, S and Herold, MJ and Papenfuss, AT},
title = {A systematic review of computational methods for designing efficient guides for CRISPR DNA base editor systems.},
journal = {Briefings in bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1093/bib/bbad205},
pmid = {37287132},
issn = {1477-4054},
abstract = {In only a few years, as a breakthrough technology, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene-editing systems have ushered in the era of genome engineering with a plethora of applications. One of the most promising CRISPR tools, so-called base editors, opened an exciting avenue for exploring new therapeutic approaches through controlled mutagenesis. However, the efficiency of a base editor guide varies depending on several biological determinants, such as chromatin accessibility, DNA repair proteins, transcriptional activity, factors related to local sequence context and so on. Thus, the success of genetic perturbation directed by CRISPR/Cas base-editing systems relies on an optimal single guide RNA (sgRNA) design, taking those determinants into account. Although there is 11 commonly used software to design guides specifically for base editors, only three of them investigated and implemented those biological determinants into their models. This review presents the key features, capabilities and limitations of all currently available software with a particular focus on predictive model-based algorithms. Here, we summarize existing software for sgRNA design and provide a base for improving the efficiency of existing available software suites for precise target base editing.},
}
@article {pmid37286962,
year = {2023},
author = {Luo, J and Abid, M and Tu, J and Cai, X and Zhang, Y and Gao, P and Huang, H},
title = {Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.},
journal = {BMC plant biology},
volume = {23},
number = {1},
pages = {305},
pmid = {37286962},
issn = {1471-2229},
mesh = {*Gene Editing/methods ; *Tobacco/genetics/metabolism ; Cytosine/metabolism ; Plant Breeding ; DNA ; Plants/genetics ; Polyploidy ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration.<br><br>RESULTS: In the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs containing A3A, A3A (Y130F), and rAPOBEC1(R33A) to compare their base editing efficiency in allotetraploid N. benthamiana (n&#x2009;=&#x2009;4x). We used 14 target sites to compare their editing efficiency using transient transformation in tobacco plants. The sanger sequencing and deep sequencing results showed that A3A-CBE was the most efficient base editor. In addition, the results showed that A3A-CBE provided most comprehensive editing window (C1&#x2009;~&#x2009;C17 could be edited) and had a better editing efficiency under the base background of TC. The target sites (T2 and T6) analysis in transformed N. benthamiana showed that only A3A-CBE can have C-to-T editing events and the editing efficiency of T2 was higher than T6. Additionally, no off-target events were found in transformed N. benthamiana.<br><br>CONCLUSIONS: All in all, we conclude that A3A-CBE is the most suitable vector for specific C to T conversion in N. benthamiana. Current findings will provide valuable insights into selecting an appropriate base editor for breeding polyploid plants.},
}
@article {pmid37285261,
year = {2023},
author = {Wei, Y and Zhang, M and Hu, J and Zhou, Y and Xue, M and Yin, J and Liu, Y and Feng, H and Zhou, L and Li, Z and Wang, D and Zhang, Z and Zhou, Y and Liu, H and Yao, N and Zuo, E and Hu, J and Du, Y and Li, W and Xu, C and Yang, H},
title = {Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.},
journal = {Protein &amp; cell},
volume = {14},
number = {6},
pages = {416-432},
pmid = {37285261},
issn = {1674-8018},
mesh = {Humans ; *Apolipoprotein E4/genetics ; *Cytosine ; Mutation ; Blastocyst ; Heterozygote ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.},
}
@article {pmid37283391,
year = {2023},
author = {Suputri, NPAEO and Prasojo, IS and Prabowo, LAT and Purwestri, YA and Purnomo, and Semiarti, E},
title = {Identification of early flowering mutant gene in Phalaenopsis amabilis (L.) Blume for sgRNA construction in CRISPR/Cas9 genome editing system.},
journal = {Brazilian journal of biology = Revista brasleira de biologia},
volume = {84},
number = {},
pages = {e268133},
doi = {10.1590/1519-6984.268133},
pmid = {37283391},
issn = {1678-4375},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Phylogeny ; *Orchidaceae/genetics ; },
abstract = {Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented.},
}
@article {pmid37283088,
year = {2023},
author = {Shmakov, SA and Barth, ZK and Makarova, KS and Wolf, YI and Brover, V and Peters, JE and Koonin, EV},
title = {Widespread CRISPR-derived RNA regulatory elements in CRISPR-Cas systems.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad495},
pmid = {37283088},
issn = {1362-4962},
support = {R01 GM129118/NH/NIH HHS/United States ; },
abstract = {CRISPR-cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8, or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.},
}
@article {pmid37282821,
year = {2023},
author = {Wang, B and Yang, H},
title = {Progress of CRISPR-based programmable RNA manipulation and detection.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {},
number = {},
pages = {e1804},
doi = {10.1002/wrna.1804},
pmid = {37282821},
issn = {1757-7012},
abstract = {Prokaryotic clustered regularly interspaced short palindromic repeats and CRISPR associated (CRISPR-Cas) systems provide adaptive immunity by using RNA-guided endonucleases to recognize and eliminate invading foreign nucleic acids. Type II Cas9, type V Cas12, type VI Cas13, and type III Csm/Cmr complexes have been well characterized and developed as programmable platforms for selectively targeting and manipulating RNA molecules of interest in prokaryotic and eukaryotic cells. These Cas effectors exhibit remarkable diversity of ribonucleoprotein (RNP) composition, target recognition and cleavage mechanisms, and self discrimination mechanisms, which are leveraged for various RNA targeting applications. Here, we summarize the current understanding of mechanistic and functional characteristics of these Cas effectors, give an overview on RNA detection and manipulation toolbox established so far including knockdown, editing, imaging, modification, and mapping RNA-protein interactions, and discuss the future directions for CRISPR-based RNA targeting tools. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.},
}
@article {pmid37243666,
year = {2023},
author = {Yu, T and Liu, T and Wang, Y and Zhao, X and Zhang, W},
title = {Effect of Cas9 Protein on the Seed-Target Base Pair of the sgRNA/DNA Hybrid Duplex.},
journal = {The journal of physical chemistry. B},
volume = {127},
number = {22},
pages = {4989-4997},
doi = {10.1021/acs.jpcb.3c00997},
pmid = {37243666},
issn = {1520-5207},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Base Pairing ; Gene Editing/methods ; DNA/chemistry ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas9) has been widely used for gene editing. Not all guide RNAs can cleave the DNA efficiently remains a major challenge to CRISPR/Cas9-mediated genome engineering. Therefore, understanding how the Cas9 complex successfully and efficiently identifies specific functional targets through base-pairing has great implications for such applications. The 10-nt seed sequence at the 3' end of the guide RNA is critical to target recognition and cleavage. Here, through stretching molecular dynamics simulation, we studied the thermodynamics and kinetics of the binding-dissociation process of the seed base and the target DNA base with the Cas9 protein. The results showed that in the presence of Cas9 protein, the enthalpy change and entropy change in binding-dissociation of the seed base with the target are smaller than those without the Cas9 protein. The reduction of entropy penalty upon association with the protein resulted from the pre-organization of the seed base in an A-form helix, and the reduction of enthalpy change was due to the electrostatic attraction of the positively charged channel with the negative target DNA. The binding barrier coming from the entropy loss and the dissociation barrier resulting from the destruction of the base pair in the presence of Cas9 protein were lower than those without protein, which indicates that the seed region is crucial for efficiently searching the correct target by accelerating the binding rate and dissociating fast from the wrong target.},
}
@article {pmid37182559,
year = {2023},
author = {Ma, L and Yang, S and Peng, Q and Zhang, J and Zhang, J},
title = {CRISPR/Cas9-based gene-editing technology for sickle cell disease.},
journal = {Gene},
volume = {874},
number = {},
pages = {147480},
doi = {10.1016/j.gene.2023.147480},
pmid = {37182559},
issn = {1879-0038},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Hematopoietic Stem Cells ; *Anemia, Sickle Cell/genetics/therapy ; Genetic Therapy ; },
abstract = {Sickle cell disease (SCD) is the most common monogenic hematologic disorder and is essentially congenital hemolytic anemia caused by an inherited point mutation in the β-globin on chromosome 11. Although the genetic basis of SCD was revealed as early as 1957, treatment options for SCD have been very limited to date. Hematopoietic stem cell transplantation (HSCT) was thought to hold promise as a cure for SCD, but the available donors were still only 15% useful. Gene therapy has advanced rapidly into the 21st century with the promise of a cure for SCD, and gene editing strategies based on the cluster-based regularly interspaced short palindromic repeat sequence (CRISPR)/Cas9 system have revolutionized the field of gene therapy by precisely targeting genes. In this paper, we review the pathogenesis and therapeutic approaches of SCD, briefly summarize the delivery strategies of CRISPR/Cas9, and finally discuss in depth the current status, application barriers, and solution directions of CRISPR/Cas9 in SCD. Through the review in this paper, we hope to provide some references for gene therapy in SCD.},
}
@article {pmid36917984,
year = {2023},
author = {Tsuchiya, M and Tachibana, N and Nagao, K and Tamura, T and Hamachi, I},
title = {Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism.},
journal = {Cell metabolism},
volume = {35},
number = {6},
pages = {1072-1083.e9},
doi = {10.1016/j.cmet.2023.02.014},
pmid = {36917984},
issn = {1932-7420},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Flow Cytometry ; *Lipid Metabolism ; Phosphatidylcholines/metabolism ; Organelles/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.},
}
@article {pmid36908010,
year = {2023},
author = {Mircetic, J and Camgöz, A and Abohawya, M and Ding, L and Dietzel, J and Tobar, SG and Paszkowski-Rogacz, M and Seidlitz, T and Schmäche, T and Mehnert, MC and Sidorova, O and Weitz, J and Buchholz, F and Stange, DE},
title = {CRISPR/Cas9 Screen in Gastric Cancer Patient-Derived Organoids Reveals KDM1A-NDRG1 Axis as a Targetable Vulnerability.},
journal = {Small methods},
volume = {7},
number = {6},
pages = {e2201605},
doi = {10.1002/smtd.202201605},
pmid = {36908010},
issn = {2366-9608},
mesh = {Humans ; *Stomach Neoplasms/genetics/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Histone Demethylases/genetics/metabolism ; Organoids/metabolism/pathology ; },
abstract = {Viability CRISPR screens have proven indispensable in parsing genome function. However, their application in new, more physiologically relevant culturing systems like patient-derived organoids (PDOs) has been much slower. To probe epigenetic contribution to gastric cancer (GC), the third leading cause of cancer-related deaths worldwide, the first negative selection CRISPR screen in GC PDOs that faithfully preserve primary tumor characteristics is performed. Extensive quality control measurements showing feasibility of CRISPR screens in primary organoid culture are provided. The screen reveals the histone lysine demethylase-1A (KDM1A) to constitute a GC vulnerability. Both genetic and pharmacological inhibition of KDM1A cause organoid growth retardation. Further, it is shown that most of KDM1A cancer-supporting functions center on repression of N-myc downstream regulates gene-1 (NDRG1). De-repression of NDRG1 by KDM1A inhibitors (KDM1Ai) causes inhibition of Wnt signaling and a strong G1 cell cycle arrest. Finally, by profiling 20 GC PDOs, it is shown that NDRG1 upregulation predicts KDM1Ai response with 100% sensitivity and 82% specificity in the tested cohort. Thus, this work pioneers the use of negative selection CRISPR screens in patient-derived organoids, identifies a marker of KDM1Ai response, and accordingly a cohort of patients who may benefit from such therapy.},
}
@article {pmid37282668,
year = {2023},
author = {Tang, X and Zhang, Y},
title = {Beyond knockouts: fine-tuning regulation of gene expression in plants with CRISPR-Cas-based promoter editing.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.19020},
pmid = {37282668},
issn = {1469-8137},
abstract = {The CRISPR-Cas-based genome editing field in plants is expanding rapidly. Editing plant promoters to obtain cis-regulatory alleles with altered expression levels or patterns of target genes is a highly promising topic. However, primarily used CRISPR-Cas9 has significant limitations when editing noncoding sequences like promoters, which have unique structures and regulatory mechanisms, including A-T richness, repetitive redundancy, difficulty in identifying key regulatory regions, and a higher frequency of DNA structure, epigenetic modification, and protein binding accessibility issues. Researchers urgently require efficient and feasible editing tools and strategies to address these obstacles, enhance promoter editing efficiency, increase diversity in promoter polymorphism, and, most importantly, enable 'non-silent' editing events that achieve precise target gene expression regulation. This article provides insights into the key challenges and references for implementing promoter editing-based research in plants.},
}
@article {pmid37279911,
year = {2023},
author = {Zabrady, M and Zabrady, K and Li, AWH and Doherty, AJ},
title = {Reverse transcriptases prime DNA synthesis.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad478},
pmid = {37279911},
issn = {1362-4962},
support = {BB/S008691/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.},
}
@article {pmid37278656,
year = {2023},
author = {Huo, W and Price, VJ and Sharifi, A and Zhang, MQ and Palmer, KL},
title = {Enterococcus faecalis Strains with Compromised CRISPR-Cas Defense Emerge under Antibiotic Selection for a CRISPR-Targeted Plasmid.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0012423},
doi = {10.1128/aem.00124-23},
pmid = {37278656},
issn = {1098-5336},
abstract = {Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Non-MDR E. faecalis strains frequently possess CRISPR-Cas systems, which reduce the frequency of MGE acquisition. We demonstrated in previous studies that E. faecalis populations can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze these populations. In the presence of antibiotic selection for the plasmid, mutants with compromised CRISPR-Cas defense and enhanced ability to acquire a second antibiotic resistance plasmid emerged. Conversely, in the absence of selection, the plasmid was lost from wild-type E. faecalis populations but not E. faecalis populations that lacked the cas9 gene. Our results indicate that E. faecalis CRISPR-Cas can become compromised under antibiotic selection, generating populations with enhanced abilities to undergo horizontal gene transfer. IMPORTANCE Enterococcus faecalis is a leading cause of hospital-acquired infections and disseminator of antibiotic resistance plasmids among Gram-positive bacteria. We have previously shown that E. faecalis strains with an active CRISPR-Cas system can prevent plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. However, CRISPR-Cas is not a perfect barrier. In this study, we observed populations of E. faecalis with transient coexistence of CRISPR-Cas and one of its plasmid targets. Our experimental data demonstrate that antibiotic selection results in compromised E. faecalis CRISPR-Cas function, thereby facilitating the acquisition of additional resistance plasmids by E. faecalis.},
}
@article {pmid37278518,
year = {2023},
author = {Li, F and Li, J and Yang, W and Yang, S and Chen, C and Du, L and Mei, J and Tang, Q and Chen, X and Yao, C and Yang, D and Zuo, X and Liu, P},
title = {Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202305536},
doi = {10.1002/anie.202305536},
pmid = {37278518},
issn = {1521-3773},
abstract = {The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.},
}
@article {pmid37277632,
year = {2023},
author = {Meineke, B and Elsässer, SJ},
title = {Generation of Amber Suppression Cell Lines Using CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2676},
number = {},
pages = {169-180},
pmid = {37277632},
issn = {1940-6029},
mesh = {Animals ; Humans ; Codon, Terminator ; *CRISPR-Cas Systems/genetics ; *Amino Acids/chemistry ; Proteins/metabolism ; Cell Line ; Mammals/genetics ; },
abstract = {Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid.},
}
@article {pmid37277433,
year = {2023},
author = {Fraessle, SP and Tschulik, C and Effenberger, M and Cletiu, V and Gerget, M and Schober, K and Busch, DH and Germeroth, L and Stemberger, C and Poltorak, MP},
title = {Activation-inducible CAR expression enables precise control over engineered CAR T cell function.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {604},
pmid = {37277433},
issn = {2399-3642},
mesh = {*Receptors, Chimeric Antigen/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; Immunotherapy, Adoptive/methods ; T-Lymphocytes/metabolism ; },
abstract = {CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs.},
}
@article {pmid37275547,
year = {2023},
author = {Lučanský, V and Holubeková, V and Kolková, Z and Halašová, E and Samec, M and Golubnitschaja, O},
title = {Multi-faceted CRISPR/Cas technological innovation aspects in the framework of 3P medicine.},
journal = {The EPMA journal},
volume = {14},
number = {2},
pages = {201-217},
pmid = {37275547},
issn = {1878-5077},
abstract = {Since 2009, the European Association for Predictive, Preventive and Personalised Medicine (EPMA, Brussels) promotes the paradigm change from reactive approach to predictive, preventive, and personalized medicine (PPPM/3PM) to protect individuals in sub-optimal health conditions from the health-to-disease transition, to increase life-quality of the affected patient cohorts improving, therefore, ethical standards and cost-efficacy of healthcare to great benefits of the society at large. The gene-editing technology utilizing CRISPR/Cas gene-editing approach has demonstrated its enormous value as a powerful tool in a broad spectrum of bio/medical research areas. Further, CRISPR/Cas gene-editing system is considered applicable to primary and secondary healthcare, in order to prevent disease spread and to treat clinically manifested disorders, involving diagnostics of SARS-Cov-2 infection and experimental treatment of COVID-19. Although the principle of the proposed gene editing is simple and elegant, there are a lot of technological challenges and ethical considerations to be solved prior to its broadly scaled clinical implementation. This article highlights technological innovation beyond the state of the art, exemplifies current achievements, discusses unsolved technological and ethical problems, and provides clinically relevant outlook in the framework of 3PM.},
}
@article {pmid37275249,
year = {2023},
author = {Biswal, AK and Hernandez, LRB and Castillo, AIR and Debernardi, JM and Dhugga, KS},
title = {An efficient transformation method for genome editing of elite bread wheat cultivars.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1135047},
pmid = {37275249},
issn = {1664-462X},
abstract = {An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.},
}
@article {pmid37275127,
year = {2023},
author = {Zhao, G and Lu, D and Li, M and Wang, Y},
title = {Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1191812},
pmid = {37275127},
issn = {1664-302X},
abstract = {Mycoplasmas are successful pathogens that cause debilitating diseases in humans and various animal hosts. Despite the exceptionally streamlined genomes, mycoplasmas have evolved specific mechanisms to access essential nutrients from host cells. The paucity of genetic tools to manipulate mycoplasma genomes has impeded studies of the virulence factors of pathogenic species and mechanisms to access nutrients. This review summarizes several strategies for editing of mycoplasma genomes, including homologous recombination, transposons, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and synthetic biology. In addition, the mechanisms and features of different tools are discussed to provide references and future directions for efficient manipulation of mycoplasma genomes.},
}
@article {pmid37272863,
year = {2023},
author = {Sahakyan, H and Makarova, KS and Koonin, EV},
title = {Search for Origins of Anti-CRISPR Proteins by Structure Comparison.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {222-231},
doi = {10.1089/crispr.2023.0011},
pmid = {37272863},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics ; Gene Editing ; Bacteria/metabolism ; Viral Proteins/genetics/metabolism ; },
abstract = {Many bacterial and archaeal viruses encode anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas systems via various mechanisms. The majority of the Acrs are small, non-enzymatic proteins that abrogate CRISPR activity by binding to Cas effector proteins. The Acrs evolve fast, due to the arms race with the respective CRISPR-Cas systems, which hampers the elucidation of their evolutionary origins by sequence comparison. We performed comprehensive structural modeling using AlphaFold2 for 3693 experimentally characterized and predicted Acrs, followed by a comparison to the protein structures in the Protein Data Bank database. After clustering the Acrs by sequence similarity, 363 high-quality structural models were obtained that accounted for 102 Acr families. Structure comparisons allowed the identification of homologs for 13 of these families that could be ancestors of the Acrs. Despite the limited extent of structural conservation, the inferred origins of Acrs show distinct trends, in particular, recruitment of toxins and antitoxins and SOS repair system components for the Acr function.},
}
@article {pmid37272862,
year = {2023},
author = {Nety, SP and Altae-Tran, H and Kannan, S and Demircioglu, FE and Faure, G and Hirano, S and Mears, K and Zhang, Y and Macrae, RK and Zhang, F},
title = {The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {232-242},
doi = {10.1089/crispr.2023.0015},
pmid = {37272862},
issn = {2573-1602},
mesh = {*DNA Transposable Elements/genetics ; RNA, Messenger/genetics ; *Gene Editing ; CRISPR-Cas Systems ; RNA ; },
abstract = {TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5' processing. We also uncover a potential cis-regulatory mechanism whereby a region of the TnpB mRNA inhibits DNA cleavage by the RNP complex. We further expand the characterization of TnpB by examining ωRNA processing and RNA-guided nuclease activity in 59 orthologs spanning the natural diversity of the TnpB family. This work reveals a new functionality, ωRNA biogenesis, of TnpB, and characterizes additional members of this biotechnologically useful family of programmable enzymes.},
}
@article {pmid37272861,
year = {2023},
author = {Alexander, LM and Aliaga Goltsman, DS and Liu, J and Lin, JL and Temoche-Diaz, MM and Laperriere, SM and Neerincx, A and Bednarski, C and Knyphausen, P and Cohnen, A and Albers, J and Gonzalez-Osorio, L and Fregoso Ocampo, R and Oki, J and Devoto, AE and Castelle, CJ and Lamothe, RC and Cost, GJ and Butterfield, CN and Thomas, BC and Brown, CT},
title = {Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {261-277},
doi = {10.1089/crispr.2022.0090},
pmid = {37272861},
issn = {2573-1602},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Biotechnology ; RNA, Guide, CRISPR-Cas Systems ; Mammals/genetics/metabolism ; },
abstract = {Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.},
}
@article {pmid37272860,
year = {2023},
author = {Barrangou, R},
title = {CRISPR Conventions in a Polarized Era.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {183-184},
doi = {10.1089/crispr.2023.29160.editorial},
pmid = {37272860},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37272859,
year = {2023},
author = {Cress, B and Barrangou, R},
title = {Special Issue: Manipulating the Microbiome with CRISPR.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {185},
doi = {10.1089/crispr.2023.29159.cfp2},
pmid = {37272859},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; *Microbiota/genetics ; },
}
@article {pmid37271263,
year = {2023},
author = {Yee, BJ and Shafiqah, NF and Mohd-Naim, NF and Ahmed, MU},
title = {A CRISPR/Cas12a-based fluorescence aptasensor for the rapid and sensitive detection of ampicillin.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {125211},
doi = {10.1016/j.ijbiomac.2023.125211},
pmid = {37271263},
issn = {1879-0003},
abstract = {This study introduces CRISPR/Cas-based aptasensor for the highly sensitive and specific detection of the antibiotic, ampicillin. Ampicillin (AMPI) is a commonly used antibiotic for treating pathogenic bacteria and is additionally added to livestock feed in agriculture. This study can enable early detection of antibiotic residues, prevent their accumulation in the environment, and ensure compliance with food safety regulations. Herein, the aptasensor was developed with the CRISPR/Cas system by utilizing three different ampicillin-specific aptamers, each conjugated with a biotin at the 5'-end. The ssDNA activator was bound to the aptamers through complementary base pairings. The attraction of the aptamers to the ampicillin target released the bound ssDNA, causing the activation of the CRISPR/Cas system. The DNA reporter probe, labelled with Cy3 and a quencher, turns on the fluorescence signal when cleaved by the activated Cas12a through trans-cleavage measured using a fluorescence spectrophotometer at 590 nm. The fluorescence signal was linearly proportional to the ampicillin target concentration with a 0.01 nM limit of detection and a read-out time of 30 min. This aptasensor showed high sensitivity towards ampicillin even in the presence of other antibiotics. The method was also successfully implemented for ampicillin detection in spiked food samples.},
}
@article {pmid37219969,
year = {2023},
author = {Lamothe, RC and Storlie, MD and Espinosa, DA and Rudlaff, R and Browne, P and Liu, J and Rivas, A and Devoto, A and Oki, J and Khoubyari, A and Goltsman, DSA and Lin, JL and Butterfield, CN and Brown, CT and Thomas, BC and Cost, GJ},
title = {Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {243-260},
doi = {10.1089/crispr.2022.0089},
pmid = {37219969},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; T-Lymphocytes/metabolism ; Cell Differentiation ; Receptors, Antigen, T-Cell/genetics/metabolism ; },
abstract = {Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of β2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.},
}
@article {pmid37219623,
year = {2023},
author = {Wong, PK and Mohamad Zamberi, NN and Syafruddin, SE and Cheah, FC and Azmi, N and Law, JX and Chua, EW},
title = {The Promises and Pitfalls of CRISPR-Mediated Base Editing in Stem Cells.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {196-215},
doi = {10.1089/crispr.2023.0013},
pmid = {37219623},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Cell Line ; Embryonic Stem Cells/metabolism ; },
abstract = {Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic disorders. Precise base edits can be achieved using a variety of CRISPR-centric approaches, particularly homology-directed repair and the recently developed base editors and prime editors. Despite its much-touted potential, editing single DNA bases is technically challenging. In this review, we discuss the strategies for achieving exact base edits in the creation of various stem cell-based models for use in elucidating disease mechanisms and assessing drug efficacy, and the unique characteristics of stem cells that warrant special considerations.},
}
@article {pmid37200486,
year = {2023},
author = {Tschage, L and Kowarz, E and Marschalek, R},
title = {Model System to Analyze RNA-Mediated DNA Repair in Mammalian Cells.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {289-301},
doi = {10.1089/crispr.2022.0105},
pmid = {37200486},
issn = {2573-1602},
mesh = {Animals ; *DNA Breaks, Double-Stranded ; RNA Precursors ; CRISPR-Cas Systems/genetics ; Gene Editing ; DNA Repair/genetics ; DNA/genetics ; *RNA, Small Untranslated ; Mammals/genetics/metabolism ; },
abstract = {"RNA-templated/directed DNA repair" is a biological mechanism that has been experimentally demonstrated in bacteria, yeast, and mammalian cells. Recent study has shown that small noncoding RNAs (DDRNAs) and/or newly RNAPII transcribed RNAs (dilncRNAs) are orchestrating the initial steps of double-strand break (DSB) repair. In this study, we demonstrate that also pre-mRNA could be used as direct or indirect substrate for DSB repair. Our test system is based on (1) a stably integrated mutant reporter gene that produces constitutively a nonspliceable pre-mRNA, (2) a transiently expressed sgRNA-guided dCas13b::ADAR fusion protein to specifically RNA edit the nonspliceable pre-mRNA, and (3) transiently expressed I-SceI to create a DSB situation to study the effect of spliceable pre-mRNA on DNA repair. Based on our data, the RNA-edited pre-mRNA was used in cis for the DSB repair process, thereby converting the genomically encoded mutant reporter gene into an active reporter gene. Overexpression and knockdown of several cellular proteins were performed to delineate their role in this novel "RNA-mediated end joining" pathway.},
}
@article {pmid37163371,
year = {2023},
author = {Tornero-Écija, AR and Navas, MA and Muñoz-Braceras, S and Vincent, O and Escalante, R},
title = {Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency.},
journal = {Journal of cellular and molecular medicine},
volume = {27},
number = {11},
pages = {1557-1564},
pmid = {37163371},
issn = {1582-4934},
mesh = {Humans ; Vesicular Transport Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; HeLa Cells ; Sirolimus/pharmacology ; *Neurodegenerative Diseases/metabolism ; Lysosomes/metabolism ; *Neuroacanthocytosis/genetics/metabolism ; },
abstract = {VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.},
}
@article {pmid37134217,
year = {2023},
author = {Trasanidou, D and Barendse, P and Bouzetos, E and de Haan, L and Bouwmeester, H and Staals, RHJ and Mougiakos, I and van der Oost, J},
title = {Efficient Genome and Base Editing in Human Cells Using ThermoCas9.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {278-288},
doi = {10.1089/crispr.2023.0005},
pmid = {37134217},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Genetic Engineering ; Streptococcus pyogenes/genetics ; },
abstract = {Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.},
}
@article {pmid37083425,
year = {2023},
author = {Vasquez, CA and Evanoff, M and Ranzau, BL and Gu, S and Deters, E and Komor, AC},
title = {Curing "GFP-itis" in Bacteria with Base Editors: Development of a Genome Editing Science Program Implemented with High School Biology Students.},
journal = {The CRISPR journal},
volume = {6},
number = {3},
pages = {186-195},
doi = {10.1089/crispr.2023.0002},
pmid = {37083425},
issn = {2573-1602},
support = {T32 GM007240/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Students ; Escherichia coli/genetics ; Biology ; },
abstract = {The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research for the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in precollege biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics. To this end, we present a 3-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in Escherichia coli, accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools. We have included all requisite materials, including lecture slides, worksheets, experimental protocols, and suggestions on active learning strategies for others to reproduce our program with their local communities.},
}
@article {pmid37270558,
year = {2023},
author = {Haldrup, J and Weiss, S and Schmidt, L and Sørensen, KD},
title = {Investigation of enzalutamide, docetaxel, and cabazitaxel resistance in the castration resistant prostate cancer cell line C4 using genome-wide CRISPR/Cas9 screening.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {9043},
pmid = {37270558},
issn = {2045-2322},
mesh = {Male ; Humans ; Docetaxel/pharmacology/therapeutic use ; *Prostatic Neoplasms, Castration-Resistant/drug therapy/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Early Detection of Cancer ; Nitriles/therapeutic use ; Cell Line ; Treatment Outcome ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; GTPase-Activating Proteins/metabolism ; },
abstract = {Enzalutamide, docetaxel, and cabazitaxel treatment resistance is a major problem in metastatic castration resistant prostate cancer (mCRPC), but the underlying genetic determinants are poorly understood. To identify genes that modulate treatment response to these drugs, we performed three genome-wide CRISPR/Cas9 knockout screens in the mCRPC cell line C4. The screens identified seven candidates for enzalutamide (BCL2L13, CEP135, E2F4, IP6K2, KDM6A, SMS, and XPO4), four candidates for docetaxel (DRG1, LMO7, NCOA2, and ZNF268), and nine candidates for cabazitaxel (ARHGAP11B, DRG1, FKBP5, FRYL, PRKAB1, RP2, SMPD2, TCEA2, and ZNF585B). We generated single-gene C4 knockout clones/populations for all genes and could validate effect on treatment response for five genes (IP6K2, XPO4, DRG1, PRKAB1, and RP2). Altered enzalutamide response upon IP6K2 and XPO4 knockout was associated with deregulation of AR, mTORC1, and E2F signaling, and deregulated p53 signaling (IP6K2 only) in C4 mCRPC cells. Our study highlights the necessity of performing individual validation of candidate hits from genome-wide CRISPR screens. Further studies are needed to assess the generalizability and translational potential of these findings.},
}
@article {pmid37270532,
year = {2023},
author = {Noviello, G and Gjaltema, RAF and Schulz, EG},
title = {CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {3225},
pmid = {37270532},
issn = {2041-1723},
mesh = {Mice ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/metabolism ; Genes, Homeobox ; Gene Expression ; },
abstract = {Certain cellular processes are dose-dependent, requiring specific quantities or stoichiometries of gene products, as exemplified by haploinsufficiency and sex-chromosome dosage compensation. Understanding dosage-sensitive processes requires tools to quantitatively modulate protein abundance. Here we present CasTuner, a CRISPR-based toolkit for analog tuning of endogenous gene expression. The system exploits Cas-derived repressors that are quantitatively tuned by ligand titration through a FKBP12[F36V] degron domain. CasTuner can be applied at the transcriptional or post-transcriptional level using a histone deacetylase (hHDAC4) fused to dCas9, or the RNA-targeting CasRx, respectively. We demonstrate analog tuning of gene expression homogeneously across cells in mouse and human cells, as opposed to KRAB-dependent CRISPR-interference systems, which exhibit digital repression. Finally, we quantify the system's dynamics and use it to measure dose-response relationships of NANOG and OCT4 with their target genes and with the cellular phenotype. CasTuner thus provides an easy-to-implement tool to study dose-responsive processes in their physiological context.},
}
@article {pmid37269466,
year = {2023},
author = {Morshedzadeh, F and Ghanei, M and Lotfi, M and Ghasemi, M and Ahmadi, M and Najari-Hanjani, P and Sharif, S and Mozaffari-Jovin, S and Peymani, M and Abbaszadegan, MR},
title = {An Update on the Application of CRISPR Technology in Clinical Practice.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37269466},
issn = {1559-0305},
abstract = {The CRISPR/Cas system, an innovative gene-editing tool, is emerging as a promising technique for genome modifications. This straightforward technique was created based on the prokaryotic adaptive immune defense mechanism and employed in the studies on human diseases that proved enormous therapeutic potential. A genetically unique patient mutation in the process of gene therapy can be corrected by the CRISPR method to treat diseases that traditional methods were unable to cure. However, introduction of CRISPR/Cas9 into the clinic will be challenging because we still need to improve the technology's effectiveness, precision, and applications. In this review, we first describe the function and applications of the CRISPR-Cas9 system. We next delineate how this technology could be utilized for gene therapy of various human disorders, including cancer and infectious diseases and highlight the promising examples in the field. Finally, we document current challenges and the potential solutions to overcome these obstacles for the effective use of CRISPR-Cas9 in clinical practice.},
}
@article {pmid37268954,
year = {2023},
author = {Cairns, TC and de Kanter, T and Zheng, XZ and Zheng, P and Sun, J and Meyer, V},
title = {Regression modelling of conditional morphogene expression links and quantifies the impact of growth rate, fitness and macromorphology with protein secretion in Aspergillus niger.},
journal = {Biotechnology for biofuels and bioproducts},
volume = {16},
number = {1},
pages = {95},
pmid = {37268954},
issn = {2731-3654},
abstract = {BACKGROUND: Filamentous fungi are used as industrial cell factories to produce a diverse portfolio of proteins, organic acids, and secondary metabolites in submerged fermentation. Generating optimized strains for maximum product titres relies on a complex interplay of molecular, cellular, morphological, and macromorphological factors that are not yet fully understood.<br><br>RESULTS: In this study, we generate six conditional expression mutants in the protein producing ascomycete Aspergillus niger and use them as tools to reverse engineer factors which impact total secreted protein during submerged growth. By harnessing gene coexpression network data, we bioinformatically predicted six morphology and productivity associated 'morphogenes', and placed them under control of a conditional Tet-on gene switch using CRISPR-Cas genome editing. Strains were phenotypically screened on solid and liquid media following titration of morphogene expression, generating quantitative measurements of growth rate, filamentous morphology, response to various abiotic perturbations, Euclidean parameters of submerged macromorphologies, and total secreted protein. These data were built into a multiple linear regression model, which identified radial growth rate and fitness under heat stress as positively correlated with protein titres. In contrast, diameter of submerged pellets and cell wall integrity were negatively associated with productivity. Remarkably, our model predicts over 60% of variation in A. niger secreted protein titres is dependent on these four variables, suggesting that they play crucial roles in productivity and are high priority processes to be targeted in future engineering programs. Additionally, this study suggests A. niger dlpA and crzA genes are promising new leads for enhancing protein titres during fermentation.<br><br>CONCLUSIONS: Taken together this study has identified several potential genetic leads for maximizing protein titres, delivered a suite of chassis strains with user controllable macromorphologies during pilot fermentation studies, and has quantified four crucial factors which impact secreted protein titres in A. niger.},
}
@article {pmid37268340,
year = {2023},
author = {Lv, M and Zong, C and Chen, X and Lin, X and Kong, L and Li, C},
title = {A cathodic photoelectrochemical biosensor based on CRISPR/Cas12a trans-cleavage mediated p-n heterojunction quenching mode for microRNA determination.},
journal = {Analytica chimica acta},
volume = {1268},
number = {},
pages = {341399},
doi = {10.1016/j.aca.2023.341399},
pmid = {37268340},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis ; CRISPR-Cas Systems ; Electrochemical Techniques/methods ; Limit of Detection ; *Biosensing Techniques/methods ; },
abstract = {In this study, a cathodic photoelectrochemical (PEC) bioanalysis for sensitive determination of microRNA (miRNA) has been constructed based on CRISPR/Cas12a trans-cleavage mediated [(C6)2Ir(dcbpy)][+]PF6[-] (C6 represents coumarin-6 and dcbpy represents 4,4'-dicarboxyl-2,2'-bipyridine)-sensitized NiO photocathode and p-n heterojunction quenching mode. The [(C6)2Ir(dcbpy)][+]PF6[-]-sensitized NiO photocathode exhibits a stable and dramatically improved photocurrent signal due to highly effective photosensitization of [(C6)2Ir(dcbpy)][+] PF6[-]. Then Bi2S3 quantum dots (Bi2S3 QDs) is captured on the photocathode, resulting in markedly quenching of the photocurrent. When target miRNA is specifically recognized by the hairpin DNA to stimulate the trans-cleavage activity of CRISPR/Cas12a, leading to the leave of the Bi2S3 QDs. The photocurrent is gradually recovered with the increasing target concentration. Thus, the quantitative signal response to target is achieved. Benefiting from excellent performance of NiO photocathode, intense quenching effect of p-n heterojunction and accurate recognition ability of CRISPR/Cas12a, the cathodic PEC biosensor shows a wider linear range over 0.1 fM-10 nM, with a low detection limit of 36 aM. Also, the biosensor exhibits satisfying stability and selectivity.},
}
@article {pmid37267904,
year = {2023},
author = {Park, JU and Petassi, MT and Hsieh, SC and Mehrotra, E and Schuler, G and Budhathoki, J and Truong, VH and Thyme, SB and Ke, A and Kellogg, EH and Peters, JE},
title = {Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition.},
journal = {Molecular cell},
volume = {83},
number = {11},
pages = {1827-1838.e6},
doi = {10.1016/j.molcel.2023.05.005},
pmid = {37267904},
issn = {1097-4164},
mesh = {*RNA ; DNA/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; },
abstract = {CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.},
}
@article {pmid37267210,
year = {2023},
author = {Sharrar, A and Arake de Tacca, L and Collingwood, T and Meacham, Z and Rabuka, D and Staples-Ager, J and Schelle, M},
title = {Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2023.0006},
pmid = {37267210},
issn = {2573-1602},
abstract = {Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.},
}
@article {pmid37259535,
year = {2023},
author = {Matsumoto, D and Nomura, W},
title = {The history of genome editing: advances from the interface of chemistry &amp; biology.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3cc00559c},
pmid = {37259535},
issn = {1364-548X},
abstract = {Genome editing had a long history before the appearance of CRISPR. Although a decade has passed since the initial use of CRISPR with mammalian cells, the first attempts at gene editing occurred in the 1980's. Subsequently, many researchers tried to develop methods to edit specific genes. Here, we review the history of genome editing and improvements in genome editing tools. In the last two decades, genome editing tools have been applied in basic sciences, the bio-industry, and therapeutics. We provide examples in which genome editing tools have been applied to various tasks. Recently, new CRISPR-Cas techniques, such as base and prime editing and anti-CRISPR proteins, have attracted considerable interest. Accordingly, these topics are also reviewed.},
}
@article {pmid37257519,
year = {2023},
author = {Ko, KP and Huang, Y and Zhang, S and Zou, G and Kim, B and Zhang, J and Jun, S and Martin, C and Dunbar, KJ and Efe, G and Rustgi, AK and Nakagawa, H and Park, JI},
title = {Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion.},
journal = {Gastroenterology},
volume = {},
number = {},
pages = {},
doi = {10.1053/j.gastro.2023.05.030},
pmid = {37257519},
issn = {1528-0012},
abstract = {BACKGROUND AND AIMS: Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown.<br><br>METHODS: Using CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids (EOs). Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing (scRNA-seq). Tumorigenicity and immune evasion of organoids were monitored by allograft transplantation. Human ESCC scRNA-seq datasets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion.<br><br>RESULTS: We established 32 genetically engineered EOs and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple-knockout (KO) induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN KO also generates an immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via NF-&#x3ba;B. Moreover, comparative single-cell transcriptomic analyses stratified ESCC patients and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1.<br><br>CONCLUSIONS: Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable option for targeting PCN-type ESCC.},
}
@article {pmid37256960,
year = {2023},
author = {Boender, AJ and Boon, M and Albers, HE and Eck, SR and Fricker, BA and Kelly, AM and LeDoux, JE and Motta, SC and Shrestha, P and Taylor, JH and Trainor, BC and Triana-Del Rio, R and Young, LJ},
title = {An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept.},
journal = {Science advances},
volume = {9},
number = {22},
pages = {eadf4950},
doi = {10.1126/sciadv.adf4950},
pmid = {37256960},
issn = {2375-2548},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Receptors, Oxytocin/genetics ; Oxytocin/genetics ; },
abstract = {A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor (Oxtr) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species (n = 6) without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.},
}
@article {pmid37220689,
year = {2023},
author = {Cheng, X and Song, H and Ren, D and Gao, M and Xia, X and Yu, P and Bian, X},
title = {Rolling circle transcription and CRISPR/Cas12a-assisted versatile bicyclic cascade amplification assay for sensitive uracil-DNA glycosylase detection.},
journal = {Talanta},
volume = {262},
number = {},
pages = {124684},
doi = {10.1016/j.talanta.2023.124684},
pmid = {37220689},
issn = {1873-3573},
mesh = {*Uracil-DNA Glycosidase/metabolism ; CRISPR-Cas Systems ; Limit of Detection ; DNA/genetics ; DNA Probes ; *Biosensing Techniques/methods ; },
abstract = {Uracil-DNA glycosylase (UDG) is pivotal in maintaining genome integrity and aberrant expressed UDG is highly relevant to numerous diseases. Sensitive and accurate detecting UDG is critically significant for early clinical diagnosis. In this research, we demonstrated a sensitive UDG fluorescent assay based on rolling circle transcription (RCT)/CRISPR/Cas12a-assisted bicyclic cascade amplification strategy. Target UDG catalyzed to remove uracil base of DNA dumbbell-shape substrate probe (SubUDG) to produce an apurinic/apyrimidinic (AP) site, at which SubUDG was cleaved by apurinic/apyrimidinic endonuclease (APE1) subsequently. The exposed 5'-PO4 was ligated with the free 3'-OH terminus to form an enclosed DNA dumbbell-shape substrate probe (E-SubUDG). E-SubUDG functioned as a template can actuate T7 RNA polymerase-mediated RCT signal amplification, generating multitudes of crRNA repeats. The resultant Cas12a/crRNA/activator ternary complex activated the activity of Cas12a, causing a significantly enhanced fluorescence output. In this bicyclic cascade strategy, target UDG was amplified via RCT and CRISPR/Cas12a, and the whole reaction was completed without complex procedures. This method enabled sensitive and specific monitor UDG down to 0.0005 U/mL, screen corresponding inhibitors, and analyze endogenous UDG in A549 cells at single-cell level. Importantly, this assay can be extended to analyze other DNA glycosylase (hAAG and Fpg) by altering the recognition site in DNA substrates probe rationally, thereby offering a potent tool for DNA glycosylase-associated clinical diagnosis and biomedical research.},
}
@article {pmid37166482,
year = {2023},
author = {Fang, J and Liu, J and Cheng, N and Kang, X and Huang, Z and Wang, G and Xiong, X and Lu, T and Gong, Z and Huang, Z and Che, J and Xiang, T},
title = {Four thermostatic steps: A novel CRISPR-Cas12-based system for the rapid at-home detection of respiratory pathogens.},
journal = {Applied microbiology and biotechnology},
volume = {107},
number = {12},
pages = {3983-3996},
pmid = {37166482},
issn = {1432-0614},
mesh = {Humans ; Animals ; Cats ; *COVID-19/diagnosis ; SARS-CoV-2/genetics ; CRISPR-Cas Systems ; Real-Time Polymerase Chain Reaction ; Reverse Transcription ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has severely damaged the world's economy and public health and made people pay more attention to respiratory infectious diseases. However, traditional quantitative real-time polymerase chain reaction (qRT-PCR) nucleic acid detection kits require RNA extraction, reverse transcription, and amplification, as well as the support of large-scale equipment to enrich and purify nucleic acids and precise temperature control. Therefore, novel, fast, convenient, sensitive and specific detection methods are urgently being developed and moving to proof of concept test. In this study, we developed a new nucleic acid detection system, referred to as 4 Thermostatic steps (4TS), which innovatively allows all the detection processes to be completed in a constant temperature device, which performs extraction, amplification, cutting of targets, and detection within 40 min. The assay can specifically and sensitively detect five respiratory pathogens, namely SARS-CoV-2, Mycoplasma felis (MF), Chlamydia felis (CF), Feline calicivirus (FCV), and Feline herpes virus (FHV). In addition, a cost-effective and practical small-scale reaction device was designed and developed to maintain stable reaction conditions. The results of the detection of the five viruses show that the sensitivity of the system is greater than 94%, and specificity is 100%. The 4TS system does not require complex equipment, which makes it convenient and fast to operate, and allows immediate testing for suspected infectious agents at home or in small clinics. Therefore, the assay system has diagnostic value and significant potential for further reducing the cost of early screening of infectious diseases and expanding its application. KEY POINTS: • The 4TS system enables the accurate and specific detection of nucleic acid of pathogens at 37 °C in four simple steps, and the whole process only takes 40 min. •A simple alkali solution can be used to extract nucleic acid. • A small portable device simple to operate is developed for home diagnosis and detection of respiratory pathogens.},
}
@article {pmid37150408,
year = {2023},
author = {Monk, CH and Youngquist, BM and Brady, AD and Shaffer, JG and Hu, TY and Ning, B and Zwezdaryk, KJ},
title = {Development of a CRISPR-Cas12a rapid diagnostic for human cytomegalovirus.},
journal = {Antiviral research},
volume = {215},
number = {},
pages = {105624},
doi = {10.1016/j.antiviral.2023.105624},
pmid = {37150408},
issn = {1872-9096},
support = {R01 HD107790/HD/NICHD NIH HHS/United States ; },
mesh = {Infant, Newborn ; Humans ; *Cytomegalovirus/genetics ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Cytomegalovirus Infections ; Real-Time Polymerase Chain Reaction ; DNA, Viral/analysis ; },
abstract = {Despite decades of research, human cytomegalovirus (CMV) continues to contribute to significant morbidity and mortality in transplant settings and remains the leading cause of viral congenital infections. Clinical diagnosis of CMV infection and/or reactivation under these settings is completed using real time quantitative polymerase chain reaction (RT-qPCR). This assay performs well but is hampered by poor sensitivity and a lack of standardization among testing facilities. A point-of-care rapid diagnostic to determine CMV viremia could address these issues and improve patient care. In this manuscript, we introduce clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a technology to design and validate a rapid diagnostic for CMV. This system was tested using CMV spiked human saliva and urine samples. Sensitivity of the assay was ∼10 infectious units (IU)/mL. Specificity of the assay was robust and failed to detect other herpesviruses. Collectively, we have designed and validated a rapid diagnostic for CMV that overcomes limitations of the current standard diagnostic. This assay has the potential to be used as a point-of-care screening tool in transplant and neonatal settings.},
}
@article {pmid37142697,
year = {2023},
author = {Crunkhorn, S},
title = {Delivering genome editing tools to primary cells.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {6},
pages = {444},
doi = {10.1038/d41573-023-00069-x},
pmid = {37142697},
issn = {1474-1784},
mesh = {Humans ; *Gene Editing ; *Genetic Engineering ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37142191,
year = {2023},
author = {Yi, J and Lei, X and Guo, F and Chen, Q and Chen, X and Zhao, K and Zhu, C and Cheng, X and Lin, J and Yin, H and Xia, Y},
title = {Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models.},
journal = {Antiviral research},
volume = {215},
number = {},
pages = {105618},
doi = {10.1016/j.antiviral.2023.105618},
pmid = {37142191},
issn = {1872-9096},
mesh = {Mice ; Animals ; Hepatitis B virus ; Tupaia/genetics ; CRISPR-Cas Systems ; Tupaiidae/genetics ; RNA, Messenger ; Virus Replication ; *Hepatitis B ; DNA, Circular/genetics ; DNA, Viral/genetics ; *Hepatitis B, Chronic ; },
abstract = {With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.},
}
@article {pmid37255445,
year = {2023},
author = {Garrett, SC and Philippe, C and Kim, JG and Wei, Y and Johnson, KA and Olson, S and Graveley, BR and Terns, MP},
title = {Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0048222},
doi = {10.1128/jb.00482-22},
pmid = {37255445},
issn = {1098-5530},
abstract = {Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.},
}
@article {pmid37254817,
year = {2023},
author = {Gehlert, FO and Nickel, L and Vakirlis, N and Hammerschmidt, K and Vargas Gebauer, HI and Kießling, C and Kupczok, A and Schmitz, RA},
title = {Active in vivo translocation of the Methanosarcina mazei Gö1 Casposon.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad474},
pmid = {37254817},
issn = {1362-4962},
abstract = {Casposons are transposable elements containing the CRISPR associated gene Cas1solo. Identified in many archaeal genomes, casposons are discussed as the origin of CRISPR-Cas systems due to their proposed Cas1solo-dependent translocation. However, apart from bioinformatic approaches and the demonstration of Cas1solo integrase and endonuclease activity in vitro, casposon transposition has not yet been shown in vivo. Here, we report on active casposon translocations in Methanosarcina mazei Gö1 using two independent experimental approaches. First, mini-casposons, consisting of a R6Kγ origin and two antibiotic resistance cassettes, flanked by target site duplications (TSDs) and terminal inverted repeats (TIRs), were generated, and shown to actively translocate from a suicide plasmid and integrate into the chromosomal MetMaz-C1 TSD IS1a. Second, casposon excision activity was confirmed in a long-term evolution experiment using a Cas1solo overexpression strain in comparison to an empty vector control under four different treatments (native, high temperature, high salt, mitomycin C) to study stress-induced translocation. Analysis of genomic DNA using a nested qPCR approach provided clear evidence of casposon activity in single cells and revealed significantly different casposon excision frequencies between treatments and strains. Our results, providing the first experimental evidence for in vivo casposon activity are summarized in a modified hypothetical translocation model.},
}
@article {pmid37254802,
year = {2023},
author = {Casas-Mollano, JA and Zinselmeier, MH and Sychla, A and Smanski, MJ},
title = {Efficient gene activation in plants by the MoonTag programmable transcriptional activator.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad458},
pmid = {37254802},
issn = {1362-4962},
support = {NIHT32GM008347/GF/NIH HHS/United States ; },
abstract = {CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR/Cas-based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that works on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.},
}
@article {pmid37254060,
year = {2023},
author = {Fu, H and Shan, C and Kang, F and Yu, L and Li, Z and Yin, Y},
title = {CRISPR-GRANT: a cross-platform graphical analysis tool for high-throughput CRISPR-based genome editing evaluation.},
journal = {BMC bioinformatics},
volume = {24},
number = {1},
pages = {219},
pmid = {37254060},
issn = {1471-2105},
mesh = {*Gene Editing ; *High-Throughput Nucleotide Sequencing ; Software ; Sequence Analysis, DNA ; Computational Biology ; CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUD: CRISPR/Cas is an efficient genome editing system that has been widely used for functional genetic studies and exhibits high potential in biomedical translational applications. Indel analysis has thus become one of the most common practices in the lab to evaluate DNA editing events generated by CRISPR/Cas. Several indel analysis tools have been reported, however, it is often required that users have certain bioinformatics training and basic command-line processing capability.<br><br>RESULTS: Here, we developed CRISPR-GRANT, a stand-alone graphical CRISPR indel analysis tool, which could be easily installed for multi-platforms, including Linux, Windows, and macOS. CRISPR-GRANT offered a straightforward GUI by simple click-and-run for genome editing analysis of single or pooled amplicons and one-step analysis for whole-genome sequencing without the need of data pre-processing, making it ideal for novice lab scientists. Moreover, it also exhibited shorter run-time compared with tools currently available.<br><br>CONCLUSION: Therefore, CRISPR-GRANT is a valuable addition to the current CRISPR toolkits that significantly lower the barrier for wet-lab researchers to conduct indel analysis from large NGS datasets. CRISPR-GRANT binaries are freely available for Linux (above Ubuntu 16.04), macOS (above High Sierra 10.13) and Windows (above Windows 7) at https://github.com/fuhuancheng/CRISPR-GRANT . CRISPR-GRANT source code is licensed under the GPLv3 license and free to download and use.},
}
@article {pmid37253295,
year = {2023},
author = {Lahm, H and Dzilic, E and Neb, I and Doppler, SA and Schneider, S and Lange, R and Krane, M and Dreßen, M},
title = {Correction of a deleterious TBX5 mutation in an induced pluripotent stem cell line (DHMi004-A-1) using a completely plasmid-free CRISPR/Cas 9 approach.},
journal = {Stem cell research},
volume = {70},
number = {},
pages = {103126},
doi = {10.1016/j.scr.2023.103126},
pmid = {37253295},
issn = {1876-7753},
abstract = {TBX5 is a transcription factor which plays an essential role at different checkpoints during cardiac differentiation. However, regulatory pathways affected by TBX5 still remain ill-defined. We have applied the CRISPR/Cas9 technology using a completely plasmid-free approach to correct a heterozygous causative "loss-of function" TBX5 mutation in an iPSC line (DHMi004-A), that has been established from a patient suffering from Holt-Oram syndrome (HOS). This isogenic iPSC line, DHMi004-A-1, represents a powerful in vitro tool to dissect the regulatory pathways affected by TBX5 in HOS.},
}
@article {pmid37252785,
year = {2023},
author = {Li, Q and Song, ZL and Zhang, Y and Zhu, L and Yang, Q and Liu, X and Sun, X and Chen, X and Kong, R and Fan, GC and Luo, X},
title = {Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c00414},
pmid = {37252785},
issn = {1520-6882},
abstract = {CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.},
}
@article {pmid37250090,
year = {2023},
author = {Zhang, JX and Xu, JH and Yuan, B and Wang, XD and Mao, XH and Wang, JL and Zhang, XL and Yuan, Y},
title = {Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags.},
journal = {Frontiers in public health},
volume = {11},
number = {},
pages = {1153352},
pmid = {37250090},
issn = {2296-2565},
mesh = {Humans ; *Burkholderia pseudomallei/genetics ; *Melioidosis/diagnosis/genetics/microbiology ; CRISPR-Cas Systems ; },
abstract = {Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis.},
}
@article {pmid37210946,
year = {2023},
author = {Lahm, H and Stieglbauer, S and Neb, I and Doppler, S and Schneider, S and Dzilic, E and Lange, R and Krane, M and Dreßen, M},
title = {Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103123},
doi = {10.1016/j.scr.2023.103123},
pmid = {37210946},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; T-Box Domain Proteins/genetics/metabolism ; Mutation/genetics ; Transcription Factors/genetics/metabolism ; Phenotype ; Exons/genetics ; },
abstract = {TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding.},
}
@article {pmid37182381,
year = {2023},
author = {Luo, X and Yang, K and Jiang, M and Chen, T and Chi, Y and Ma, B and Lai, L and Zou, Q},
title = {Generation of a homozygous GRIN2A gene knockout human embryonic stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103121},
doi = {10.1016/j.scr.2023.103121},
pmid = {37182381},
issn = {1876-7753},
mesh = {Humans ; Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Embryonic Stem Cells/metabolism ; Cell Line ; },
abstract = {Schizophrenia is a group of common psychosis of unknown etiology and GRIN2A gene has been a risk gene for schizophrenia. In order to understand the relationship between the GRIN2A and schizophrenia, we generated a GRIN2A-KO human embryonic stem cell line by CRISPR/Cas9 system, which could provide a valuable resource for investing pathogenic mechanisms underlying schizophrenia and facilitating the development of targeted medicine.},
}
@article {pmid37148822,
year = {2023},
author = {Sun, R and Cui, Y and Liu, Z and Guo, J and Zhang, X and Zhu, P and Sha, J and Yang, X and Yuan, Y},
title = {A prime editor efficiently repaired human induced pluripotent stem cells with AR gene mutation (c.2710G &gt; A; p. V904M).},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103102},
doi = {10.1016/j.scr.2023.103102},
pmid = {37148822},
issn = {1876-7753},
mesh = {Male ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; Cell Line ; },
abstract = {Prime Editor (PE) is a precise genome manipulation technology based on the CRISPR-Cas9 system, while its application in human induced pluripotent stem cells (iPSCs) remains limited. Here, we established a repaired hiPS cell line (SKLRMi001-A-1) from hiPSCs with androgen receptor (AR) mutation (c.2710G > A; p.V904M). The repaired iPSC line expressed pluripotency markers, retained normal karyotype, showed the capability of differentiating into three germ layers and was absence of mycoplasma infection. The repaired iPSC line will help to elucidate the mechanism of androgen insensitivity syndrome (AIS) and benefit treatment for AIS in the future.},
}
@article {pmid37104932,
year = {2023},
author = {Merkert, S and Haase, A and Dahlmann, J and Göhring, G and Waqas, FH and Pessler, F and Martin, U and Olmer, R},
title = {Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103090},
doi = {10.1016/j.scr.2023.103090},
pmid = {37104932},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; NF-E2-Related Factor 2/genetics/metabolism ; Endothelial Cells/metabolism ; Clone Cells/metabolism ; },
abstract = {The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.},
}
@article {pmid37037372,
year = {2023},
author = {Yılmaz, VM and Ramnarine, TJS and Königer, A and Mussgnug, S and Grath, S},
title = {Tropical super flies: Integrating Cas9 into Drosophila ananassae and its phenotypic effects.},
journal = {Journal of insect physiology},
volume = {147},
number = {},
pages = {104516},
doi = {10.1016/j.jinsphys.2023.104516},
pmid = {37037372},
issn = {1879-1611},
mesh = {Animals ; *Drosophila/genetics ; *CRISPR-Cas Systems ; Temperature ; Cold Temperature ; Acclimatization ; },
abstract = {Ectotherms such as insects are animals whose body temperature largely depends on ambient temperature and temperature variations provide a selection pressure affecting the geographical distribution of these species. However, over the course of evolution, some insect species managed to colonize environments characterized by various temperature ranges. Therefore, insects provide an excellent study system to investigate the basis of adaptation to temperature changes and extremes. We are generally using the vinegar fly Drosophila ananassae as a model system to investigate the genetic basis of cold tolerance. This species has expanded from its tropical ancestral range to more temperate regions resulting in a cosmopolitan, domestic distribution. Previously, we identified candidate genes significantly associated with cold tolerance in this species. We now established molecular genetic tools to assess the function of these genes. Using CRISPR/Cas9 methodology for genome editing and the PiggyBac system, the Cas9 enzyme was successfully integrated into the genome of three fly strains with different levels of cold tolerance. We further report on preliminary findings that the Cas9 integration itself did not have a consistent effect on tolerance to cold. In conclusion, we offer with our study the molecular tools that allow studying stress-related candidate genes in D. ananassae in the future. In addition, we point out and provide guidance on the challenges that come with genome editing in a non-model species.},
}
@article {pmid37028180,
year = {2023},
author = {Rädecke, K and Gore, A and Burau, K and Laugsch, M and Köhler, K and Rappold, GA and Hoffmann, S},
title = {Generation of two homozygous SHOX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103089},
doi = {10.1016/j.scr.2023.103089},
pmid = {37028180},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Mutation ; Transcription Factors/genetics/metabolism ; *Atrial Fibrillation/genetics ; Homeodomain Proteins/genetics/metabolism ; },
abstract = {SHOX2 is a homeobox transcription factor associated with atrial fibrillation (AF) and sinus node dysfunction. Here, we generated two homozygous SHOX2 knock-out hiPSC lines from a healthy control line and a corrected AF patient line (disease-specific SHOX2 mutation corrected to WT) using CRISPR/Cas9. These cell lines maintained pluripotency, an ability to differentiate into all three germlayers and a normal karyotype, presenting a valuable tool to investigate the impact of a full SHOX2 knock-out with respect to arrhythmogenic diseases on a cellular level.},
}
@article {pmid37001365,
year = {2023},
author = {Jo, S and Kim, JW and Noh, H and Kim, H and Kim, JH and Park, HJ},
title = {Generation of a PDGFRB-mCherry knock-in reporter human induced pluripotent stem cell line (KITi001-A-1), using CRISPR/Cas9 nuclease.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103081},
doi = {10.1016/j.scr.2023.103081},
pmid = {37001365},
issn = {1876-7753},
mesh = {Humans ; *Receptor, Platelet-Derived Growth Factor beta/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Cell Line ; CRISPR-Cas Systems/genetics ; Platelet-Derived Growth Factor/metabolism ; Cell Differentiation ; },
abstract = {PDGFRB encodes platelet-derived growth factor receptor beta (PDGFR-β), a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. It is required for the normal development of the vascular and nervous systems and rearrangement of the actin cytoskeleton. PDGFR-β plays an essential role in early liver diseases, including liver fibrosis. Here, we generated a human induced pluripotent stem cell (iPSC) line, KITi001-A-1, using CRISPR/Cas9. This reporter iPSC line and its derivatives are useful for tracing PDGFR-β-expressing cells and for screening for liver fibrosis-inducing compounds.},
}
@article {pmid36965407,
year = {2023},
author = {Wang, X and Sun, J and Fu, C and Liu, C},
title = {Generation of a PPP1CA knockout human pluripotent stem cell line via CRISPR/Cas9.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103077},
doi = {10.1016/j.scr.2023.103077},
pmid = {36965407},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Protein Phosphatase 1/genetics/metabolism ; Cell Line ; Embryonic Stem Cells/metabolism ; },
abstract = {The Protein phosphatase 1 catalytic subunit alpha (PPP1CA) is a alpha subunit of the PP1 complex, which known to be involved in the regulation of a variety of cellular processes, such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and HIV-1 viral transcription. Increased PP1 activity has been observed in the end stage of heart failure. Here, a PPP1CA knockout human embryonic stem cell line, WAe009-A-B, was generated using the CRISPR/Cas9 system to further study the function of PPP1CA. The cell line confirmed with pluripotency, normal karyotype and differentiation potential.},
}
@article {pmid36963213,
year = {2023},
author = {Wu, X and Wang, X},
title = {Generation of a TBX20-knockout human embryonic stem line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103082},
doi = {10.1016/j.scr.2023.103082},
pmid = {36963213},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Embryonic Stem Cells/metabolism ; Cell Line ; T-Box Domain Proteins/genetics/metabolism ; },
abstract = {The TBX20 gene plays a crucial role in embryonic development and has been involved in various diseases, such as heart defects, intellectual disability, and cancer. Herein, we have established a TBX20-knockout human embryonic stem cell line (WAe009-A-84) that maintains stem cell-like features, pluripotency, a normal karyotype, and the ability to differentiate into all three germ layers in vivo. This cell line will be a valuable resource for exploring TBX20's role in human development and could have significant implications for regenerative medicine and disease modeling.},
}
@article {pmid36947995,
year = {2023},
author = {Schmitz, AS and Korneck, M and Raju, J and Lamsfus-Calle, A and Daniel-Moreno, A and Antony, JS and Mezger, M and Schöls, L and Hauser, S and Hayer, SN},
title = {Generation of a heterozygous and a homozygous CSF1R knockout line from iPSC using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103066},
doi = {10.1016/j.scr.2023.103066},
pmid = {36947995},
issn = {1876-7753},
mesh = {Adult ; Humans ; *Induced Pluripotent Stem Cells ; *Neurodegenerative Diseases/genetics ; CRISPR-Cas Systems/genetics ; Neuroglia ; *Leukoencephalopathies/genetics ; Mutation ; },
abstract = {Mutations in Colony-stimulating factor 1 receptor (CSF1R) lead to CSF1R-related leukoencephalopathy, also known as Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a rapidly progressing neurodegenerative disease with severe cognitive and motor impairment. In this study, a homozygous and a heterozygous CSF1R knockout induced pluripotent stem cell (iPSC) line were generated by CRISPR/Cas9-based gene editing. These in vitro models will provide a helpful tool for investigating the still largely unknown pathophysiology of CSF1R-related leukoencephalopathy.},
}
@article {pmid36947993,
year = {2023},
author = {Schuurmans, IME and Wu, KM and van Karnebeek, CDM and Nadif Kasri, N and Garanto, A},
title = {Generation of an induced pluripotent stem cell line carrying a biallelic deletion (SCTCi019-A) in GCDH using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103069},
doi = {10.1016/j.scr.2023.103069},
pmid = {36947993},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Amino Acid Metabolism, Inborn Errors/genetics ; *Brain Diseases, Metabolic/genetics/metabolism ; },
abstract = {GCDH encodes for the enzyme catalyzing the sixth step of the lysine catabolism pathway. Biallelic pathogenic variants in GCDH have been associated with glutaric aciduria type 1 (GA1). In this study CRISPR/Cas9 technology was used to create an isogenic GCDH knock-out human iPSC line. One clone with a biallelic deletion (SCTCi019-A) in GCDH was obtained and fully characterized, revealing a normal karyotype, no off-targets detected and expression of pluripotency markers. This iPSC line can contribute to gain insights in the molecular mechanism of disease.},
}
@article {pmid36913849,
year = {2023},
author = {Kim, AH and Lee, HM and Kim, HS and Jung, J and Seol, H and Choi, E and Lee, S and Min Choi, Y and Jun, JK and Kim, HS and Jang, J},
title = {Establishment of TUBB3-mCherry knock-in human pluripotent stem cell line using CRISPR/Cas9 (SNUe003-A-4).},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103064},
doi = {10.1016/j.scr.2023.103064},
pmid = {36913849},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Pluripotent Stem Cells ; Homologous Recombination ; Cell Differentiation/physiology ; Tubulin ; },
abstract = {TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.},
}
@article {pmid36481809,
year = {2023},
author = {Shi, H and Doench, JG and Chi, H},
title = {CRISPR screens for functional interrogation of immunity.},
journal = {Nature reviews. Immunology},
volume = {23},
number = {6},
pages = {363-380},
pmid = {36481809},
issn = {1474-1741},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics/therapy ; Phenotype ; Genomics ; Transcriptome ; },
abstract = {CRISPR-based technologies represent a major breakthrough in biomedical science as they offer a powerful platform for unbiased screening and functional genomics in various fields, including immunology. Pooled and arrayed CRISPR screens have uncovered previously unknown intracellular drivers in innate and adaptive immune cells for immune regulation as well as intercellular regulators mediating cell-cell interactions. Recent single-cell CRISPR screening platforms expand the readouts to the transcriptome and enable the inference of gene regulatory networks for better mechanistic insights. CRISPR screens also allow for mapping of genetic interactions to identify genes that synergize or alleviate complex immune phenotypes. Here, we review the progress in and emerging adaptation of CRISPR technologies to advance our fundamental immunological knowledge and identify novel disease targets for immunotherapy of infection, inflammation and cancer.},
}
@article {pmid36167511,
year = {2023},
author = {Shao, H and Jian, J and Peng, D and Yao, K and Abdulsalam, S and Huang, W and Kong, L and Li, C and Peng, H},
title = {Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a Technology for Rapid and Highly Sensitive Detection of Heterodera avenae and Heterodera filipjevi.},
journal = {Plant disease},
volume = {107},
number = {5},
pages = {1365-1376},
doi = {10.1094/PDIS-02-22-0386-RE},
pmid = {36167511},
issn = {0191-2917},
mesh = {Animals ; *Recombinases ; CRISPR-Cas Systems ; Edible Grain/parasitology ; *Tylenchoidea ; Soil ; },
abstract = {The cereal cyst nematodes Heterodera avenae and Heterodera filipjevi are recognized as cyst nematodes that infect cereal crops and cause severe economic losses worldwide. Rapid, visual detection of cyst nematodes is essential for more effective control of this pest. In this study, recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (formerly known as cpf1) was developed for the rapid detection of H. avenae and H. filipjevi from infested field samples. The RPA reaction was performed at a wide range of temperatures from 35 to 42°C within 15 min. There was no cross-reactivity between H. avenae, H. filipjevi, and the common closely related plant-parasitic nematodes, indicating the high specificity of this assay. The detection limit of RPA-Cas12a was as low as 10[-4] single second-stage juvenile (J2), 10[-5] single cyst, and 0.001 ng of genomic DNA, which is 10 times greater than that of RPA-lateral flow dipstick (LFD) detection. The RPA-Cas12a assay was able to detect 10[-1] single J2 of H. avenae and H. filipjevi in 10 g of soil. In addition, the RPA-LFD assay and RPA-Cas12a assays could both quickly detect H. avenae and H. filipjevi from naturally infested soil, and the entire detection process could be completed within 1 h. These results indicated that the RPA-Cas12a assay developed herein is a simple, rapid, specific, sensitive, and visual method that can be easily adapted for the quick detection of H. avenae and H. filipjevi in infested fields.},
}
@article {pmid37191624,
year = {2023},
author = {Qi, Q and Liu, X and Fu, F and Shen, W and Cui, S and Yan, S and Zhang, Y and Du, Y and Tian, T and Zhou, X},
title = {Utilizing Epigenetic Modification as a Reactive Handle To Regulate RNA Function and CRISPR-Based Gene Regulation.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {21},
pages = {11678-11689},
doi = {10.1021/jacs.3c01864},
pmid = {37191624},
issn = {1520-5126},
mesh = {*RNA/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Epigenesis, Genetic ; },
abstract = {The current methods to control RNA functions in living conditions are limited. The new RNA-controlling strategy presented in this study involves utilizing 5-formylcytidine (f5C)-directed base manipulation. This study shows that malononitrile and pyridine boranes can effectively manipulate the folding, small molecule binding, and enzyme recognition of f5C-bearing RNAs. We further demonstrate the efficiency of f5C-directed reactions in controlling two different clustered regularly interspaced short palindromic repeat (CRISPR) systems. Although further studies are needed to optimize the efficiency of these reactions in vivo, this small molecule-based approach presents exciting new opportunities for regulating CRISPR-based gene expression and other applications.},
}
@article {pmid37159025,
year = {2023},
author = {Chen, X and Huang, C and Hu, Q and Zhang, J and Wang, D and You, Q and Hu, M},
title = {Sensing platform for nucleic-acid detection based on a 2-aminopurine probe sheared by trans-cleavage activity of the CRISPR/Cas12a system.},
journal = {The Analyst},
volume = {148},
number = {11},
pages = {2482-2492},
doi = {10.1039/d3an00502j},
pmid = {37159025},
issn = {1364-5528},
mesh = {*2-Aminopurine ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; Coloring Agents ; Polymerase Chain Reaction ; *Biosensing Techniques ; },
abstract = {Target double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) can activate the trans-cleavage activity of the CRISPR/Cas12a, cutting the surrounding non-target ssDNA arbitrarily. In a typical CRISPR/Cas12a system, this non-target ssDNA, with a fluorescent tag and its quencher incorporated at both ends (ssDNA-FQ), is usually used as the reporter. Here, a 2-aminopurine probe (T-pro 4), made by inserting four 2-APs in non-target ssDNA, was screened for using as a reporter in the CRISPR/Cas12a system. Compared with ssDNA-FQ, each 2-AP probe is cleaved by the activated CRISPR/Cas12a system, multi-unit signals are generated. Therefore, the CRISPR/Cas12a system using the 2-AP probe as a reporter may be more sensitive than the CRISPR/Cas12a system which uses ssDNA-FQ as the reporter. We achieved ssDNA detection at as little as 10[-11] M using the 2-AP probe as the reporter in the CRISPR/Cas12a system. Compared to the CRISPR/Cas12a system using ssDNA-FQ as the reporter, its sensitivity increased by an order of magnitude. Furthermore, the method that combines PCR and the 2-AP-probe-mediated CRISPR/Cas12a system can detect goat pox virus (GTPV) down to 8.35 × 10[-2] copies per μL, 10 times lower than the method that combines PCR and the ssDNA-FQ-mediated CRISPR/Cas12a system. These results indicate that the CRISPR/Cas12a system using the screened 2-AP probe as a reporter has potential in highly sensitive detection of viruses.},
}
@article {pmid37159023,
year = {2023},
author = {Shen, P and Si, Z and Huang, D and Xu, Z and Wang, Z and Fang, M and Xu, Z},
title = {CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2.},
journal = {The Analyst},
volume = {148},
number = {11},
pages = {2573-2581},
doi = {10.1039/d3an00284e},
pmid = {37159023},
issn = {1364-5528},
mesh = {Female ; Pregnancy ; Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *Nucleic Acids ; Point-of-Care Testing ; *Pregnancy Tests ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; RNA, Viral/genetics ; },
abstract = {The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per μL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.},
}
@article {pmid37068691,
year = {2023},
author = {Deng, F and Pan, J and Chen, M and Liu, Z and Chen, J and Liu, C},
title = {Integrating CRISPR-Cas12a with catalytic hairpin assembly as a logic gate biosensing platform for the detection of polychlorinated biphenyls in water samples.},
journal = {The Science of the total environment},
volume = {881},
number = {},
pages = {163465},
doi = {10.1016/j.scitotenv.2023.163465},
pmid = {37068691},
issn = {1879-1026},
mesh = {Humans ; *Polychlorinated Biphenyls ; CRISPR-Cas Systems ; DNA ; Oligonucleotides ; *Biosensing Techniques/methods ; Water ; },
abstract = {Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that cause harmful effects on environmental safety and human health. There is an urgent need to develop an intelligent method for PCBs sensing. In this work, we proposed a logic gate biosensing platform for simultaneous detection of multiple PCBs. 2,3',5,5'-tetrachlorobiphenyl (PCB72) and 3,3',4,4'-tetrachlorobiphenyl (PCB77) were used as the two inputs to construct biocomputing logic gates. We used 0 and 1 to encode the inputs and outputs. The aptamer was used to recognize the inputs and release the trigger DNA. A catalytic hairpin assembly (CHA) module is designed to convert and amplify each trigger DNA into multiple programmable DNA duplexes, which initiate the trans-cleavage activity of CRISPR/Cas12a for the signal output. The activated Cas12 cleaves the BHQ-Cy5 modified single-stranded DNA (ssDNA) to yield the fluorescence reporting signals. In the YES logic gate, PCB72 was used as the only input to carry out the logic operation. In the OR, AND, and INHIBIT logic gates, PCB72 and PCB77 were used as the two inputs. The output signals can be visualized by the naked eye under UV light transilluminators or quantified by a microplate reader. Our constructed biosensing platform possesses the merits of multiple combinations of inputs, intuitive digital output, and high flexibility and scalability, which holds great promise for the intelligent detection of different PCBs.},
}
@article {pmid37251773,
year = {2023},
author = {Mipeshwaree Devi, A and Khedashwori Devi, K and Premi Devi, P and Lakshmipriyari Devi, M and Das, S},
title = {Metabolic engineering of plant secondary metabolites: prospects and its technological challenges.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1171154},
pmid = {37251773},
issn = {1664-462X},
abstract = {Plants produce a wide range of secondary metabolites that play vital roles for their primary functions such as growth, defence, adaptations or reproduction. Some of the plant secondary metabolites are beneficial to mankind as nutraceuticals and pharmaceuticals. Metabolic pathways and their regulatory mechanism are crucial for targeting metabolite engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated system has been widely applied in genome editing with high accuracy, efficiency, and multiplex targeting ability. Besides its vast application in genetic improvement, the technique also facilitates a comprehensive profiling approach to functional genomics related to gene discovery involved in various plant secondary metabolic pathways. Despite these wide applications, several challenges limit CRISPR/Cas system applicability in genome editing in plants. This review highlights updated applications of CRISPR/Cas system-mediated metabolic engineering of plants and its challenges.},
}
@article {pmid37251754,
year = {2023},
author = {Marín-Sanz, M and Barro, F and Sánchez-León, S},
title = {Unraveling the celiac disease-related immunogenic complexes in a set of wheat and tritordeum genotypes: implications for low-gluten precision breeding in cereal crops.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1171882},
pmid = {37251754},
issn = {1664-462X},
abstract = {The development of low-gluten immunogenic cereal varieties is a suitable way to fight the increment of pathologies associated with the consumption of cereals. Although RNAi and CRISPR/Cas technologies were effective in providing low-gluten wheat, the regulatory framework, particularly in the European Union, is an obstacle to the short- or medium-term implementation of such lines. In the present work, we carried out a high throughput amplicon sequencing of two highly immunogenic complexes of wheat gliadins in a set of bread and durum wheat, and tritordeum genotypes. Bread wheat genotypes harboring the 1BL/1RS translocation were included in the analysis and their amplicons successfully identified. The number of CD epitopes and their abundances were determined in the alpha- and gamma-gliadin amplicons, including 40k-γ-secalin ones. Bread wheat genotypes not containing the 1BL/1RS translocation showed a higher average number of both alpha- and gamma-gliadin epitopes than those containing such translocation. Interestingly, alpha-gliadin amplicons not containing CD epitopes accounted for the highest abundance (around 53%), and the alpha- and gamma-gliadin amplicons with the highest number of epitopes were present in the D-subgenome. The durum wheat and tritordeum genotypes showed the lowest number of alpha- and gamma-gliadin CD epitopes. Our results allow progress in unraveling the immunogenic complexes of alpha- and gamma-gliadins and can contribute to the development of low-immunogenic varieties within precision breeding programs, by crossing or by CRISPR/Cas gene editing.},
}
@article {pmid37249896,
year = {2023},
author = {Subedi, U and Burton Hughes, K and Chen, G and Hannoufa, A and Singer, SD},
title = {Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2659},
number = {},
pages = {219-239},
pmid = {37249896},
issn = {1940-6029},
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.},
}
@article {pmid37249290,
year = {2023},
author = {Cai, B and Chang, S and Tian, Y and Zhen, S},
title = {CRISPR/Cas9 for hepatitis B virus infection treatment.},
journal = {Immunity, inflammation and disease},
volume = {11},
number = {5},
pages = {e866},
pmid = {37249290},
issn = {2050-4527},
mesh = {Humans ; *Hepatitis B virus/genetics ; CRISPR-Cas Systems ; *Hepatitis B/drug therapy/genetics ; DNA, Circular/genetics/pharmacology ; },
abstract = {Hepatitis B virus (HBV) infection remains a global health challenge. Despite the availability of effective preventive vaccines, millions of people are at risk of cirrhosis and hepatocellular carcinoma. Current drug therapies inhibit viral replication, slow the progression of liver fibrosis and reduce infectivity, but they rarely remove the covalently sealed circular DNA (cccDNA) of the virus that causes HBV persistence. Alternative treatment strategies, including those based on CRISPR/cas9 knockout virus gene, can effectively inhibit HBV replication, so it has a good prospect. During chronic infection, some virus gene knockouts based on CRISPR/cas9 may even lead to cccDNA inactivation. This paper reviews the progress of different HBV CRISPR/cas9, vectors for delivering to the liver, and the current situation of preclinical and clinical research.},
}
@article {pmid37248464,
year = {2023},
author = {Mabuchi, A and Hata, S and Genova, M and Tei, C and Ito, KK and Hirota, M and Komori, T and Fukuyama, M and Chinen, T and Toyoda, A and Kitagawa, D},
title = {ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.},
journal = {BMC genomics},
volume = {24},
number = {1},
pages = {289},
pmid = {37248464},
issn = {1471-2164},
mesh = {Humans ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HCT116 Cells ; Diploidy ; DNA/metabolism ; DNA, Single-Stranded/genetics ; Gene Knock-In Techniques ; Gene Editing/methods ; },
abstract = {BACKGROUND: Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells.<br><br>RESULTS: In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration.<br><br>CONCLUSIONS: These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.},
}
@article {pmid37102700,
year = {2023},
author = {Iqbal, Z and Rehman, K and Xia, J and Shabbir, M and Zaman, M and Liang, Y and Duan, L},
title = {Biomaterial-assisted targeted and controlled delivery of CRISPR/Cas9 for precise gene editing.},
journal = {Biomaterials science},
volume = {11},
number = {11},
pages = {3762-3783},
doi = {10.1039/d2bm01636b},
pmid = {37102700},
issn = {2047-4849},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; Genetic Therapy/methods ; *Nanoparticles ; },
abstract = {RISPR-Cas9 has exhibited enormous potential in gene therapy. It can perform genome editing with single-nucleotide precision in various types of cell and tissue, providing a powerful breakthrough technology for genome editing in therapeutic development. But the limited delivery methods pose substantial challenges pertinent to safe and effective CRISPR/Cas9 delivery, thus hindering its application. These challenges should be tackled to develop next-generation genetic therapies. Biomaterial-based drug delivery systems can overcome these issues, for example using biomaterials as carriers for CRISPR/Cas9 targeted delivery, and conditional control of its function can improve precision, furnish on-demand and transient gene editing and reduce adverse consequences such as off-target events and immunogenicity, representing a promising direction for modern precision medicine. This review describes the application status and research progress of current CRISPR/Cas9 delivery approaches, including polymeric nanoparticles, liposomes, extracellular vesicles, inorganic nanoparticles and hydrogels. The unique properties of light-controlled and small-molecule drugs for spatially and temporally controlled genome editing are also illustrated. In addition, targetable delivery vehicles for the active delivery of CRISPR systems are also discussed. The perspectives to overcome the current limitations in the CRISPR/Cas9 delivery and their bench-to-bedside translation are also highlighted.},
}
@article {pmid37245014,
year = {2023},
author = {Zein-Eddine, R and Refrégier, G and Cervantes, J and Yokobori, NK},
title = {The future of CRISPR in Mycobacterium tuberculosis infection.},
journal = {Journal of biomedical science},
volume = {30},
number = {1},
pages = {34},
pmid = {37245014},
issn = {1423-0127},
mesh = {Humans ; CRISPR-Cas Systems ; Phylogeny ; *Tuberculosis/diagnosis/genetics ; *Mycobacterium tuberculosis/genetics ; Genes, Bacterial ; },
abstract = {Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis, the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments.},
}
@article {pmid37239967,
year = {2023},
author = {Zhang, F and Neik, TX and Thomas, WJW and Batley, J},
title = {CRISPR-Based Genome Editing Tools: An Accelerator in Crop Breeding for a Changing Future.},
journal = {International journal of molecular sciences},
volume = {24},
number = {10},
pages = {},
pmid = {37239967},
issn = {1422-0067},
mesh = {*Gene Editing ; *Plant Breeding ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Crops, Agricultural/genetics ; },
abstract = {Genome editing is an important strategy to maintain global food security and achieve sustainable agricultural development. Among all genome editing tools, CRISPR-Cas is currently the most prevalent and offers the most promise. In this review, we summarize the development of CRISPR-Cas systems, outline their classification and distinctive features, delineate their natural mechanisms in plant genome editing and exemplify the applications in plant research. Both classical and recently discovered CRISPR-Cas systems are included, detailing the class, type, structures and functions of each. We conclude by highlighting the challenges that come with CRISPR-Cas and offer suggestions on how to tackle them. We believe the gene editing toolbox will be greatly enriched, providing new avenues for a more efficient and precise breeding of climate-resilient crops.},
}
@article {pmid37239880,
year = {2023},
author = {Jiang, C and Geng, L and Wang, J and Liang, Y and Guo, X and Liu, C and Zhao, Y and Jin, J and Liu, Z and Mu, Y},
title = {Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript.},
journal = {International journal of molecular sciences},
volume = {24},
number = {10},
pages = {},
pmid = {37239880},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Gene Expression ; Transcriptional Activation ; RNA, Transfer/genetics ; Gene Editing ; },
abstract = {Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem-loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.},
}
@article {pmid37239814,
year = {2023},
author = {Feser, CJ and Williams, JM and Lammers, DT and Bingham, JR and Eckert, MJ and Tolar, J and Osborn, MJ},
title = {Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production.},
journal = {International journal of molecular sciences},
volume = {24},
number = {10},
pages = {},
pmid = {37239814},
issn = {1422-0067},
support = {R01 AR063070/GF/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Transcription Factors/genetics ; Transcriptional Activation ; Recombinant Proteins/genetics ; Gene Editing ; },
abstract = {Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a "dead" Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications.},
}
@article {pmid37239365,
year = {2023},
author = {Huang, S and Zhang, H and Chen, W and Su, N and Yuan, C and Zhang, J and Xiang, S and Hu, X},
title = {CRISPR/Cas9-Mediated Knockout of tnfaip1 in Zebrafish Plays a Role in Early Development.},
journal = {Genes},
volume = {14},
number = {5},
pages = {},
pmid = {37239365},
issn = {2073-4425},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; Adaptor Proteins, Signal Transducing/genetics ; CRISPR-Cas Systems ; *Neoplasms/genetics ; Human Umbilical Vein Endothelial Cells ; Receptors, Tumor Necrosis Factor/genetics ; Eye Proteins/genetics ; },
abstract = {TNF α-induced protein 1 (TNFAIP1) was first identified in human umbilical vein endothelial cells and can be induced by tumor necrosis factor α (TNFα). Early studies have found that TNFAIP1 is involved in the development of many tumors and is closely associated with the neurological disorder Alzheimer's disease. However, little is known about the expression pattern of TNFAIP1 under physiological conditions and its function during embryonic development. In this study, we used zebrafish as a model to illustrate the early developmental expression pattern of tnfaip1 and its role in early development. First, we examined the expression pattern of tnfaip1 during early zebrafish development using quantitative real-time PCR and whole mount in situ hybridization and found that tnfaip1 was highly expressed in early embryonic development and, subsequently, expression became localized to anterior embryonic structures. To investigate the function of tnfaip1 during early development, we constructed a model of a stably inherited tnfaip1 mutant using the CRISPR/Cas9 system. Tnfaip1 mutant embryos showed significant developmental delays as well as microcephaly and microphthalmia. At the same time, we found decreased expression of the neuronal marker genes tuba1b, neurod1, and ccnd1 in tnfaip1 mutants. Analysis of transcriptome sequencing data revealed altered expression of the embryonic development related genes dhx40, hspa13, tnfrsf19, nppa, lrp2b, hspb9, clul1, zbtb47a, cryba1a, and adgrg4a in the tnfaip1 mutants. These findings suggest an important role for tnfaip1 in the early development of zebrafish.},
}
@article {pmid37238739,
year = {2023},
author = {Zhao, X and Qu, K and Curci, B and Yang, H and Bolund, L and Lin, L and Luo, Y},
title = {Comparison of In-Frame Deletion, Homology-Directed Repair, and Prime Editing-Based Correction of Duchenne Muscular Dystrophy Mutations.},
journal = {Biomolecules},
volume = {13},
number = {5},
pages = {},
pmid = {37238739},
issn = {2218-273X},
mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy/metabolism ; Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; Genetic Therapy ; Mutation ; },
abstract = {Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74-77%), followed by HDR (21-24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.},
}
@article {pmid37201341,
year = {2023},
author = {Chen, K and Dai, L and Zhao, J and Deng, M and Song, L and Bai, D and Wu, Y and Zhou, X and Yang, Y and Yang, S and Zhao, L and Chen, X and Xie, G and Li, J},
title = {Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01.},
journal = {Talanta},
volume = {261},
number = {},
pages = {124674},
doi = {10.1016/j.talanta.2023.124674},
pmid = {37201341},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleotides ; Temperature ; Mutation ; Point Mutation ; },
abstract = {The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.},
}
@article {pmid37237769,
year = {2023},
author = {Gómez-Martínez, J and Rocha-Gracia, RDC and Bello-López, E and Cevallos, MA and Castañeda-Lucio, M and Sáenz, Y and Jiménez-Flores, G and Cortés-Cortés, G and López-García, A and Lozano-Zarain, P},
title = {Comparative Genomics of Pseudomonas aeruginosa Strains Isolated from Different Ecological Niches.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {12},
number = {5},
pages = {},
pmid = {37237769},
issn = {2079-6382},
abstract = {The Pseudomonas aeruginosa genome can change to adapt to different ecological niches. We compared four genomes from a Mexican hospital and 59 genomes from GenBank from different niches, such as urine, sputum, and environmental. The ST analysis showed that high-risk STs (ST235, ST773, and ST27) were present in the genomes of the three niches from GenBank, and the STs of Mexican genomes (ST167, ST2731, and ST549) differed from the GenBank genomes. Phylogenetic analysis showed that the genomes were clustering according to their ST and not their niche. When analyzing the genomic content, we observed that environmental genomes had genes involved in adapting to the environment not found in the clinics and that their mechanisms of resistance were mutations in antibiotic resistance-related genes. In contrast, clinical genomes from GenBank had resistance genes, in mobile/mobilizable genetic elements in the chromosome, except for the Mexican genomes that carried them mostly in plasmids. This was related to the presence of CRISPR-Cas and anti-CRISPR; however, Mexican strains only had plasmids and CRISPR-Cas. blaOXA-488 (a variant of blaOXA50) with higher activity against carbapenems was more prevalent in sputum genomes. The virulome analysis showed that exoS was most prevalent in the genomes of urinary samples and exoU and pldA in sputum samples. This study provides evidence regarding the genetic variability among P. aeruginosa isolated from different niches.},
}
@article {pmid37237494,
year = {2023},
author = {Jin, Y and Li, W and Zhang, H and Ba, X and Li, Z and Zhou, J},
title = {The Post-Antibiotic Era: A New Dawn for Bacteriophages.},
journal = {Biology},
volume = {12},
number = {5},
pages = {},
pmid = {37237494},
issn = {2079-7737},
abstract = {Phages are the most biologically diverse entities in the biosphere, infecting specific bacteria. Lytic phages quickly kill bacteria, while lysogenic phages integrate their genomes into bacteria and reproduce within the bacteria, participating in the evolution of natural populations. Thus, lytic phages are used to treat bacterial infections. However, due to the huge virus invasion, bacteria have also evolved a special immune mechanism (CRISPR-Cas systems, discovered in 1987). Therefore, it is necessary to develop phage cocktails and synthetic biology methods to infect bacteria, especially against multidrug-resistant bacteria infections, which are a major global threat. This review outlines the discovery and classification of phages and the associated achievements in the past century. The main applications of phages, including synthetic biology and PT, are also discussed, in addition to the effects of PT on immunity, intestinal microbes, and potential safety concerns. In the future, combining bioinformatics, synthetic biology, and classic phage research will be the way to deepen our understanding of phages. Overall, whether phages are an important element of the ecosystem or a carrier that mediates synthetic biology, they will greatly promote the progress of human society.},
}
@article {pmid37236860,
year = {2023},
author = {Awan, MJA and Akram, A and Amin, I and Mansoor, S},
title = {Viral vectors as carriers of genome-editing reagents.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2023.05.010},
pmid = {37236860},
issn = {1878-4372},
abstract = {The presence of a transgene in the genome of plants is a regulatory challenge. Recently, Liu et al. reported an engineered tomato spotted wilt virus (TSWV) that can carry large clustered regularly interspaced palindromic repeats (CRISPR)/Cas reagents for targeted genome editing in various crops without the integration of the transgene into the genome.},
}
@article {pmid37235011,
year = {2023},
author = {Jabran, M and Ali, MA and Zahoor, A and Muhae-Ud-Din, G and Liu, T and Chen, W and Gao, L},
title = {Intelligent reprogramming of wheat for enhancement of fungal and nematode disease resistance using advanced molecular techniques.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1132699},
pmid = {37235011},
issn = {1664-462X},
abstract = {Wheat (Triticum aestivum L.) diseases are major factors responsible for substantial yield losses worldwide, which affect global food security. For a long time, plant breeders have been struggling to improve wheat resistance against major diseases by selection and conventional breeding techniques. Therefore, this review was conducted to shed light on various gaps in the available literature and to reveal the most promising criteria for disease resistance in wheat. However, novel techniques for molecular breeding in the past few decades have been very fruitful for developing broad-spectrum disease resistance and other important traits in wheat. Many types of molecular markers such as SCAR, RAPD, SSR, SSLP, RFLP, SNP, and DArT, etc., have been reported for resistance against wheat pathogens. This article summarizes various insightful molecular markers involved in wheat improvement for resistance to major diseases through diverse breeding programs. Moreover, this review highlights the applications of marker assisted selection (MAS), quantitative trait loci (QTL), genome wide association studies (GWAS) and the CRISPR/Cas-9 system for developing disease resistance against most important wheat diseases. We also reviewed all reported mapped QTLs for bunts, rusts, smuts, and nematode diseases of wheat. Furthermore, we have also proposed how the CRISPR/Cas-9 system and GWAS can assist breeders in the future for the genetic improvement of wheat. If these molecular approaches are used successfully in the future, they can be a significant step toward expanding food production in wheat crops.},
}
@article {pmid37231285,
year = {2023},
author = {Yang, X and Zhang, B},
title = {A review on CRISPR/Cas: a versatile tool for cancer screening, diagnosis, and clinic treatment.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {2},
pages = {182},
pmid = {37231285},
issn = {1438-7948},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Early Detection of Cancer ; Genetic Therapy/methods ; *Neoplasms/diagnosis/genetics/therapy ; },
abstract = {Cancer is one of the leading causes of death worldwide and it has the trend of increase incidence. However, in the past decades, as quickly developed new technologies and modified old techniques for cancer screening, diagnosis, and treatment, the cancer-caused mortality rates dropped quickly, and the survival times of cancer patients are enhanced. However, the current death rate is still about 50% and the survival patients always suffer from the side effect of current cancer treatments. Recently developed Nobel Prize-winning CRISPR/Cas technology provides new hope on cancer screening, early diagnosis, and clinic treatment as well as new drug development. Currently, four major CRISPR/Cas9-derived genome editors, CRISPR/Cas9 nucleotide sequence editor, CRISPR/Cas base editor (BE), CRISPR prime editor (PE), and CRISPR interference (CRISPRi) (including both CRISPRa and CRISPRr), were well developed and used to various research and applications, including cancer biology study and cancer screening, diagnosis, and treatment. Additionally, CRISPR/Cas12 and CRISPR/Cas13 genome editors were also widely used in cancer-related basic and applied research as well as treatment. Cancer-associated SNPs and genetic mutations as well as both oncogenes and tumor suppressor genes are perfect targets for CRISPR/Cas-based gene therapy for cancer treatment. CRISPR/Cas is also employed to modify and generate new Chimeric antigen receptor (CAR) T-cells for improving its safety, efficiency, and longer-time last for treating various cancers. Currently, there are many clinic trails of CRISPR-based gene therapy for cancer treatments. Although all CRISPR/Cas-derived genome and epigenome tools are promising methods for cancer biology study and treatment, the efficiency and long term-safety are still the major concerns for CRISPR-based gene therapy. Developing new CRISPR/Cas delivery methods and reducing the potential side effects, including off-target impacts, will enhance CRISPR/Cas application in cancer-related research, diagnosis, and therapeutical treatment.},
}
@article {pmid37230569,
year = {2023},
author = {Zhou, S and Sun, H and Dong, J and Lu, P and Deng, L and Liu, Y and Yang, M and Huo, D and Hou, C},
title = {Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a.},
journal = {Analytica chimica acta},
volume = {1265},
number = {},
pages = {341278},
doi = {10.1016/j.aca.2023.341278},
pmid = {37230569},
issn = {1873-4324},
mesh = {*MicroRNAs/genetics/metabolism ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; DNA, Single-Stranded ; Biological Assay/methods ; *Biosensing Techniques/methods ; },
abstract = {MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.},
}
@article {pmid37228594,
year = {2023},
author = {Yang, L and Zhang, Y and Yi, W and Dong, X and Niu, M and Song, Y and Han, Y and Li, H and Sun, Y},
title = {A rapid and efficient platform for antiviral crRNA screening using CRISPR-Cas13a-based nucleic acid detection.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1116230},
pmid = {37228594},
issn = {1664-3224},
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Influenza A Virus, H1N1 Subtype/genetics ; RNA, Viral/genetics ; Mammals/genetics ; },
abstract = {INTRODUCTION: Rapid and high-throughput screening of antiviral clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) is urgently required for the CRISPR-Cas13a antiviral system. Based on the same principle, we established an efficient screening platform for antiviral crRNA through CRISPR-Cas13a nucleic acid detection.<br><br>METHOD: In this study, crRNAs targeting PA, PB1, NP, and PB2 of the influenza A virus (H1N1) were screened using CRISPR-Cas13a nucleic acid detection, and their antiviral effects were confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RNA secondary structures were predicted by bioinformatics methods.<br><br>RESULTS: The results showed that crRNAs screened by CRISPR-Cas13a nucleic acid detection could effectively inhibit viral RNA in mammalian cells. Besides, we found that this platform for antiviral crRNA screening was more accurate than RNA secondary structure prediction. In addition, we validated the feasibility of the platform by screening crRNAs targeting NS of the influenza A virus (H1N1).<br><br>DISCUSSION: This study provides a new approach for screening antiviral crRNAs and contributes to the rapid advancement of the CRISPR-Cas13a antiviral system.},
}
@article {pmid37228086,
year = {2023},
author = {Migunova, E and Rajamani, S and Bonanni, S and Wang, F and Zhou, C and Dubrovsky, EB},
title = {Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila.},
journal = {PloS one},
volume = {18},
number = {5},
pages = {e0286214},
pmid = {37228086},
issn = {1932-6203},
mesh = {Animals ; *Drosophila/genetics/metabolism ; *RNA Precursors/genetics ; CRISPR-Cas Systems ; Endoribonucleases/genetics/metabolism ; RNA, Transfer/genetics ; Cardiomegaly/genetics ; },
abstract = {Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3'-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. In this study, we used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. We found that this modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms.},
}
@article {pmid37201375,
year = {2023},
author = {Huang, DQ and Wu, Q and Yang, JH and Jiang, Y and Li, ZY and Fan, NS and Jin, RC},
title = {Deciphering endogenous and exogenous regulations of anammox consortia in responding to lincomycin by multiomics: quorum sensing and CRISPR system.},
journal = {Water research},
volume = {239},
number = {},
pages = {120061},
doi = {10.1016/j.watres.2023.120061},
pmid = {37201375},
issn = {1879-2448},
mesh = {Animals ; Humans ; *Quorum Sensing ; *CRISPR-Cas Systems ; Lincomycin/pharmacology ; Multiomics ; Anaerobic Ammonia Oxidation ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The widespread use of antibiotics has created an antibiotic resistance genes (ARGs)-enriched environment, which causes high risks on human and animal health. Although antibiotics can be partially adsorbed and degraded in wastewater treatment processes, striving for a complete understanding of the microbial adaptive mechanism to antibiotic stress remains urgent. Combined with metagenomics and metabolomics, this study revealed that anammox consortia could adapt to lincomycin by spontaneously changing the preference for metabolite utilization and establishing interactions with eukaryotes, such as Ascomycota and Basidiomycota. Specifically, quorum sensing (QS) based microbial regulation and the ARGs transfer mediated by clustered regularly interspaced short palindromic repeats (CRISPR) system and global regulatory genes were the principal adaptive strategies. Western blotting results validated that Cas9 and TrfA were mainly responsible for the alteration of ARGs transfer pathway. These findings highlight the potential adaptative mechanism of microbes to antibiotic stress and fill gaps in horizontal gene transfer pathways in the anammox process, further facilitating the ARGs control through molecular and synthetic biology techniques.},
}
@article {pmid37137305,
year = {2023},
author = {Martin-Rufino, JD and Castano, N and Pang, M and Grody, EI and Joubran, S and Caulier, A and Wahlster, L and Li, T and Qiu, X and Riera-Escandell, AM and Newby, GA and Al'Khafaji, A and Chaudhary, S and Black, S and Weng, C and Munson, G and Liu, DR and Wlodarski, MW and Sims, K and Oakley, JH and Fasano, RM and Xavier, RJ and Lander, ES and Klein, DE and Sankaran, VG},
title = {Massively parallel base editing to map variant effects in human hematopoiesis.},
journal = {Cell},
volume = {186},
number = {11},
pages = {2456-2474.e24},
pmid = {37137305},
issn = {1097-4172},
support = {R01 CA265726/CA/NCI NIH HHS/United States ; R01 DK103794/DK/NIDDK NIH HHS/United States ; R01 HL146500/HL/NHLBI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; *Hematopoietic Stem Cells/metabolism ; Hematopoiesis/genetics ; Cell Differentiation/genetics ; Genome ; CRISPR-Cas Systems ; },
abstract = {Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.},
}
@article {pmid37059184,
year = {2023},
author = {Sudhakar, S and Barkau, CL and Chilamkurthy, R and Barber, HM and Pater, AA and Moran, SD and Damha, MJ and Pradeepkumar, PI and Gagnon, KT},
title = {Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.},
journal = {The Journal of biological chemistry},
volume = {299},
number = {5},
pages = {104700},
pmid = {37059184},
issn = {1083-351X},
mesh = {*CRISPR-Cas Systems ; Phylogeny ; *RNA ; Ribonucleoproteins/genetics ; Gene Editing ; },
abstract = {Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.},
}
@article {pmid36779605,
year = {2023},
author = {Pantazica, AM and van Eerde, A and Dobrica, MO and Caras, I and Ionescu, I and Costache, A and Tucureanu, C and Steen, H and Lazar, C and Heldal, I and Haugslien, S and Onu, A and Stavaru, C and Branza-Nichita, N and Liu Clarke, J},
title = {The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.},
journal = {Plant biotechnology journal},
volume = {21},
number = {6},
pages = {1176-1190},
pmid = {36779605},
issn = {1467-7652},
mesh = {Humans ; Animals ; Mice ; *Hepatitis B virus/genetics ; Glycosylation ; Tobacco/genetics ; CRISPR-Cas Systems/genetics ; *COVID-19/genetics ; SARS-CoV-2 ; Hepatitis B Vaccines/genetics ; Antibodies, Neutralizing ; Hepatitis B Surface Antigens/genetics ; },
abstract = {The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of β-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.},
}
@article {pmid36759345,
year = {2023},
author = {Raffan, S and Oddy, J and Mead, A and Barker, G and Curtis, T and Usher, S and Burt, C and Halford, NG},
title = {Field assessment of genome-edited, low asparagine wheat: Europe's first CRISPR wheat field trial.},
journal = {Plant biotechnology journal},
volume = {21},
number = {6},
pages = {1097-1099},
pmid = {36759345},
issn = {1467-7652},
support = {BB/P016855/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T017007/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T50838X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Asparagine/metabolism ; *Triticum/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Europe ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37230046,
year = {2023},
author = {Li, W and Wang, Z and Jiang, Z and Yan, Y and Yao, X and Pan, Z and Chen, L and Wang, F and Wang, M and Qin, Z},
title = {MiR-3960 inhibits bladder cancer progression via targeting of DEXI.},
journal = {Biochemical and biophysical research communications},
volume = {668},
number = {},
pages = {8-18},
doi = {10.1016/j.bbrc.2023.05.055},
pmid = {37230046},
issn = {1090-2104},
abstract = {PURPOSE: MicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood.<br><br>MATERIALS AND METHODS: A microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression.<br><br>RESULTS: The results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49&#xa0;cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49&#xa0;cells in&#xa0;vivo.<br><br>CONCLUSION: Our results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.},
}
@article {pmid37228830,
year = {2023},
author = {Schmidt, M and Kircheis, W and Simons, A and Potthast, M and Stein, B},
title = {A diachronic perspective on citation latency in Wikipedia articles on CRISPR/Cas-9: an exploratory case study.},
journal = {Scientometrics},
volume = {128},
number = {6},
pages = {3649-3673},
pmid = {37228830},
issn = {0138-9130},
abstract = {This paper analyzes Wikipedia's representation of the Nobel Prize winning CRISPR/Cas9 technology, a method for gene editing. We propose and evaluate different heuristics to match publications from several publication corpora against Wikipedia's central article on CRISPR and against the complete Wikipedia revision history in order to retrieve further Wikipedia articles relevant to the topic and to analyze Wikipedia's referencing patterns. We explore to what extent the selection of referenced literature of Wikipedia's central article on CRISPR adheres to scientific standards and inner-scientific perspectives by assessing its overlap with (1) the Web of Science (WoS) database, (2) a WoS-based field-delineated corpus, (3) highly-cited publications within this corpus, and (4) publications referenced by field-specific reviews. We develop a diachronic perspective on citation latency and compare the delays with which publications are cited in relevant Wikipedia articles to the citation dynamics of these publications over time. Our results confirm that a combination of verbatim searches by title, DOI, and PMID is sufficient and cannot be improved significantly by more elaborate search heuristics. We show that Wikipedia references a substantial amount of publications that are recognized by experts and highly cited, but that Wikipedia also cites less visible literature, and, to a certain degree, even not strictly scientific literature. Delays in occurrence on Wikipedia compared to the publication years show (most pronounced in case of the central CRISPR article) a dependence on the dynamics of both the field and the editor's reaction to it in terms of activity.},
}
@article {pmid37226848,
year = {2023},
author = {Kayo, D and Kimura, S and Yamazaki, T and Naruse, K and Takeuchi, H and Ansai, S},
title = {Spatio-temporal control of targeted gene expression in combination with CRISPR/Cas and Tet-On systems in Medaka.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {},
number = {},
pages = {e23519},
doi = {10.1002/dvg.23519},
pmid = {37226848},
issn = {1526-968X},
abstract = {Spatial and temporal control of transgene expression is a powerful approach to understand gene functions in specific cells and tissues. The Tet-On system is a robust tool for controlling transgene expression spatially and temporally; however, few studies have examined whether this system can be applied to postembryonic stages of Medaka (Oryzias latipes) or other fishes. Here, we first improved a basal promoter sequence on the donor vector for a nonhomologous end joining (NHEJ)-based knock-in (KI) system. Next, using transgenic Medaka for establishing the Tet-On system by KI, we demonstrated that doxycycline administration for four or more days by feeding can be a stable and efficient method to achieve expression of the transduced reporter gene in adult fish. From these analyses, we propose an optimized approach for a spatio-temporal gene-expression system in the adult stage of Medaka and other small fishes.},
}
@article {pmid37226834,
year = {2023},
author = {Walker-Sünderhauf, D and Klümper, U and Pursey, E and Westra, ER and Gaze, WH and van Houte, S},
title = {Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.},
journal = {Microbiology (Reading, England)},
volume = {169},
number = {5},
pages = {},
doi = {10.1099/mic.0.001334},
pmid = {37226834},
issn = {1465-2080},
support = {MR/N0137941/1/MRC_/Medical Research Council/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S017674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Humans ; Animals ; Swine ; *CRISPR-Cas Systems ; Host Specificity ; *Bacteriophages ; Biological Transport ; Escherichia coli/genetics ; Plasmids/genetics ; },
abstract = {Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.},
}
@article {pmid37225346,
year = {2023},
author = {Yue, Y and Wang, S and Jin, Q and An, N and Wu, L and Huang, H},
title = {A triple amplification strategy using GR-5 DNAzyme as a signal medium for ultrasensitive detection of trace Pb[2+] based on CRISPR/Cas12a empowered electrochemical biosensor.},
journal = {Analytica chimica acta},
volume = {1263},
number = {},
pages = {341241},
doi = {10.1016/j.aca.2023.341241},
pmid = {37225346},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA, Catalytic ; Lead ; DNA, Single-Stranded ; },
abstract = {Lead ions (Pb[2+]) are a well-known toxic heavy metal that poses a significant threat to human health. Therefore, the development of a simple and ultrasensitive technique for detecting Pb[2+] is essential. With their trans-cleavage properties, the newly discovered CRISPR-V effectors have become a potential high-precision biometric tool. In this regard, a CRISPR/Cas12a-based electrochemical biosensor (E-CRISPR) has been developed, which is combined with the GR-5 DNAzyme that can specifically recognize Pb[2+]. In this strategy, the GR-5 DNAzyme acts as a signal-mediated intermediary, which can convert Pb[2+] into nucleic acid signals, thereby becoming single-stranded DNA that triggers strand displacement amplification (SDA) reaction. This is coupled with following activated CRISPR/Cas12a cleavage of the electrochemical signal probe, enabling cooperative signal amplification for ultrasensitive Pb[2+] detection. The proposed method has a detection limit as low as 0.02 pM. Therefore, we have developed an E-CRISPR detection platform with GR-5 DNAzyme as a signal medium (called SM-E-CRISPR biosensor). This provides a method for the CRISPR system to specifically detect non-nucleic substances by converting the signal using a medium.},
}
@article {pmid37223740,
year = {2023},
author = {Miezner, G and Turgeman-Grott, I and Zatopek, KM and Gardner, AF and Reshef, L and Choudhary, DK and Alstetter, M and Allers, T and Marchfelder, A and Gophna, U},
title = {An archaeal Cas3 protein facilitates rapid recovery from DNA damage.},
journal = {microLife},
volume = {4},
number = {},
pages = {uqad007},
pmid = {37223740},
issn = {2633-6693},
abstract = {CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.},
}
@article {pmid37222810,
year = {2023},
author = {Alinejad, T and Modarressi, S and Sadri, Z and Hao, Z and Chen, CS},
title = {Diagnostic applications and therapeutic option of Cascade CRISPR/Cas in the modulation of miRNA in diverse cancers: promises and obstacles.},
journal = {Journal of cancer research and clinical oncology},
volume = {},
number = {},
pages = {},
pmid = {37222810},
issn = {1432-1335},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology is a molecular tool specific to sequences for engineering genomes. Among diverse clusters of Cas proteins, the class 2/type II CRISPR/Cas9 system, despite several challenges, such as off-target effects, editing efficiency, and efficient delivery, has shown great promise for driver gene mutation discovery, high-throughput gene screening, epigenetic modulation, nucleic acid detection, disease modeling, and more importantly for therapeutic purposes. CRISPR-based clinical and experimental methods have applications across a wide range of areas, especially for cancer research and, possibly, anticancer therapy. On the other hand, given the influential role of microRNAs (miRNAs) in the regulations of cellular division, carcinogenicity, tumorigenesis, migration/invasion, and angiogenesis in diverse normal and pathogenic cellular processes, in different stages of cancer, miRNAs are either oncogenes or tumor suppressors, according to what type of cancer they are involved in. Hence, these noncoding RNA molecules are conceivable biomarkers for diagnosis and therapeutic targets. Moreover, they are suggested to be adequate predictors for cancer prediction. Conclusive evidence proves that CRISPR/Cas system can be applied to target small non-coding RNAs. However, the majority of studies have highlighted the application of the CRISPR/Cas system for targeting protein-coding regions. In this review, we specifically discuss diverse applications of CRISPR-based tools for probing miRNA gene function and miRNA-based therapeutic involvement in different types of cancers.},
}
@article {pmid37222701,
year = {2023},
author = {Engstler, M and Beneke, T},
title = {Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {37222701},
issn = {2050-084X},
mesh = {*Leishmania/genetics ; CRISPR-Cas Systems ; Gene Editing ; Genomics ; Cytosine ; },
abstract = {CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.},
}
@article {pmid37222511,
year = {2023},
author = {Lu, J and Wang, S},
title = {CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.},
journal = {Current protocols},
volume = {3},
number = {5},
pages = {e772},
doi = {10.1002/cpz1.772},
pmid = {37222511},
issn = {2691-1299},
support = {/NH/NIH HHS/United States ; /DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; Animals ; Mice ; Mice, Knockout ; *Lentivirus/genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Cloning, Molecular ; *Craniocerebral Trauma ; },
abstract = {CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.},
}
@article {pmid37129004,
year = {2023},
author = {Shi, Y and Kopparapu, N and Ohler, L and Dickinson, DJ},
title = {Efficient and rapid fluorescent protein knock-in with universal donors in mouse embryonic stem cells.},
journal = {Development (Cambridge, England)},
volume = {150},
number = {10},
pages = {},
doi = {10.1242/dev.201367},
pmid = {37129004},
issn = {1477-9129},
support = {R01 GM138443/GM/NIGMS NIH HHS/United States ; R01 GM138443/NH/NIH HHS/United States ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Mouse Embryonic Stem Cells ; Proteins/genetics ; Gene Knock-In Techniques ; Gene Editing/methods ; Mammals/genetics ; },
abstract = {Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.},
}
@article {pmid37101010,
year = {2023},
author = {Tanihara, F and Hirata, M and Namula, Z and Wittayarat, M and Do, LTK and Lin, Q and Takebayashi, K and Hara, H and Nagahara, M and Otoi, T},
title = {GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.},
journal = {Molecular biology reports},
volume = {50},
number = {6},
pages = {5049-5057},
pmid = {37101010},
issn = {1573-4978},
mesh = {Male ; Humans ; Swine/genetics ; Animals ; Female ; *Zygote ; *CRISPR-Cas Systems/genetics ; Receptors, Somatotropin/genetics ; Swine, Miniature ; Oocytes ; },
abstract = {BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa.<br><br>METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs.<br><br>CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.},
}
@article {pmid37016515,
year = {2023},
author = {Hu, M and Liu, R and Qiu, Z and Cao, F and Tian, T and Lu, Y and Jiang, Y and Zhou, X},
title = {Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {62},
number = {23},
pages = {e202300663},
doi = {10.1002/anie.202300663},
pmid = {37016515},
issn = {1521-3773},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Agriculture ; Biological Assay ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.},
}
@article {pmid36933474,
year = {2023},
author = {Davachi, ND and Bartlewski, PM and Masoudi, R and Fallahi, R},
title = {Suitability of a universal electroporation device for genome editing and production of transgenic rats.},
journal = {Reproductive biology},
volume = {23},
number = {2},
pages = {100755},
doi = {10.1016/j.repbio.2023.100755},
pmid = {36933474},
issn = {2300-732X},
mesh = {Animals ; Female ; Male ; Rats ; *Gene Editing/methods ; Rats, Transgenic ; *CRISPR-Cas Systems ; Rats, Sprague-Dawley ; Electroporation/methods ; RNA, Messenger/genetics ; Mammals/genetics ; },
abstract = {Mammalian genome editing has utilized expensive and highly specialized electroporator devices. The "Gene Pulser XCell," a modular electroporation system for transfecting all cell types, has not been used extensively in mammalian embryo genome editing. The present experiment was undertaken to determine the usefulness of the Gene Pulser XCell for inserting the CRISPR/Cas9 system into intact zygotes in order to obtain the enhanced green fluorescent protein reporter rats (eGFP-R). An electroporation pulse response test using mCherry mRNA was performed to optimize the settings of the electroporator. Forty-five combinations of five pulse voltages (15, 25, 30, 35 and 40 V), three pulse durations (5, 10 and 25 ms), and three pulse frequencies (2, 5 and 6 pulses) applied at a constant 100-ms pulse interval and temperature of 37.5 °C were evaluated. The test revealed that the 35 V was the only voltage suitable for insertion of mCherry mRNA into intact rat zygotes and the only one that resulted in the production of embryos attaining the blastocyst stage. The incorporation of mCherry mRNA increased but the survival of the electroporated embryos declined with an increment in the number of pulses. Subsequent transfer of 1112 surviving Sprague Dawley rat embryos (after 8 h of incubating 1800 zygotes electroporated with the CRISPR/Cas9) resulted in the production of 287 offspring (25.8%). Ensuing PCR and phenotypic evaluation confirmed that twenty animals (6.96%) expressed eGFP in all body organs/tissues except for blood and blood vessels. The mortality of males and females before the attainment of puberty was 2 and 3 pups, respectively, and the final number/ratio of male to female of offspring was 9:11. All the surviving rats mated naturally and successfully transmitted the GFP transgene to their progeny. The Gene Pulser XCell total system with the settings predetermined in the present experiment can effectively be used to produce transgenic rats through the CRISPR/Cas9-mediated genome editing of zygotes.},
}
@article {pmid36473602,
year = {2023},
author = {Li, X and Liu, X and Wei, J and Bu, S and Li, Z and Hao, Z and Zhang, W and Wan, J},
title = {Ultrasensitive detection of microRNAs based on click chemistry-terminal deoxynucleotidyl transferase combined with CRISPR/Cas12a.},
journal = {Biochimie},
volume = {208},
number = {},
pages = {38-45},
doi = {10.1016/j.biochi.2022.12.001},
pmid = {36473602},
issn = {1638-6183},
mesh = {*DNA Nucleotidylexotransferase ; CRISPR-Cas Systems ; Click Chemistry ; Coloring Agents ; DNA-Directed DNA Polymerase ; *MicroRNAs/genetics ; },
abstract = {The specificity and sensitivity of microRNA (miRNA) detection play a vital role in the early diagnosis of cancer and the treatment of various diseases. Here, we constructed a fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)12a cascade amplification system to achieve ultrasensitive miRNA-21 detection. Target miRNA-21 was employed as a template for click chemistry ligation of two nucleic acid probes, the product of which can be combined with magnetic microbeads (MBs). Then the 3'-end of the ligated nucleic acid and complementary strand miRNA-21 was extended by TdT. The extended poly-T tails activated the trans-cleavage ability of CRISPR/Cas12a, cleaving the reporter gene to generate the fluorescent signal. The proposed biosensor has a wide linear detection range, from 1 pM to 10[5] pM, with detection limits as low as 88 fM under optimal experimental conditions. Hence, this fluorescent biosensor enables simple, sensitive detection of miRNAs and offers a promising analytical platform for clinical diagnostics and biomedical research.},
}
@article {pmid37219967,
year = {2023},
author = {},
title = {Correction to: Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic by Panda et al. The CRISPR Journal, 2023;6(2):176-182; DOI: 10.1089/crispr.2022.0050.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0050.correx},
pmid = {37219967},
issn = {2573-1602},
}
@article {pmid37218113,
year = {2023},
author = {Chen, D and Huang, W and Zhang, Y and Chen, B and Tan, J and Yang, Y and Yuan, Q},
title = {CRISPR-Mediated Profiling of Viral RNA at Single Nucleotide Resolution.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202304298},
doi = {10.1002/anie.202304298},
pmid = {37218113},
issn = {1521-3773},
abstract = {Mass pathogen screening is critical to preventing the outbreaks and spread of infectious diseases. The large-scale epidemic of COVID-19 and the rapid mutation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have put forward new requirements for virus detection and identification techniques. Here, we report a CRISPR-based Amplification-free Viral RNA Electrical Detection platform (CAVRED) for the rapid detection and identification of SARS-CoV-2 variants. A series of CRISPR RNA assays were designed to amplify the CRISPR-Cas system's ability to discriminate between mutant and wild RNA genomes with a single-nucleotide difference. The identified viral RNA information was converted into readable electrical signals through field-effect transistor biosensors for the achievement of highly sensitive detection of single-base mutations. CAVRED can detect the SARS-CoV-2 virus genome as low as 1 cp/µL within 20 mins without amplification, and this value is comparable to the detection limit of real-time quantitative polymerase chain reaction. Based on the excellent RNA mutation detection ability, an 8-in-1 CAVRED array was constructed and realized the rapid identification of 40 simulated throat swab samples of SARS-CoV-2 variants with a 95.0% accuracy. The advantages of accuracy, sensitivity, and fast speed of CAVRED promise its application in rapid and large-scale epidemic screening.},
}
@article {pmid37218074,
year = {2023},
author = {Takebayashi, K and Wittayarat, M and Lin, Q and Torigoe, N and Liu, B and Hirata, M and Nagahara, M and Tanihara, F and Otoi, T},
title = {Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene.},
journal = {Animal science journal = Nihon chikusan Gakkaiho},
volume = {94},
number = {1},
pages = {e13842},
doi = {10.1111/asj.13842},
pmid = {37218074},
issn = {1740-0929},
mesh = {Male ; Animals ; Swine ; *Gene Editing/veterinary ; *Zygote ; CRISPR-Cas Systems ; Semen ; Electroporation/veterinary ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.},
}
@article {pmid37217056,
year = {2023},
author = {Lyu, M and Sun, Y and Yan, N and Chen, Q and Wang, X and Wei, Z and Zhang, Z and Xu, K},
title = {Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {124926},
doi = {10.1016/j.ijbiomac.2023.124926},
pmid = {37217056},
issn = {1879-0003},
abstract = {CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.},
}
@article {pmid37217031,
year = {2023},
author = {Hamdi, I and Boni, F and Shen, Q and Moukendza, L and Peibo, LI and Jianping, X},
title = {Characteristics of subtype III-A CRISPR-Cas system in Mycobacterium tuberculosis: An overview.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105445},
doi = {10.1016/j.meegid.2023.105445},
pmid = {37217031},
issn = {1567-7257},
abstract = {CRISPR-Cas systems are the only RNA- guided adaptive immunity pathways that trigger the detection and destruction of invasive phages and plasmids in bacteria and archaea. Due to its prevalence and mystery, the Class 1 CRISPR-Cas system has lately been the subject of several studies. This review highlights the specificity of CRISPR-Cas system III-A in Mycobacterium tuberculosis, the tuberculosis-causing pathogen, for over twenty years. We discuss the difference between the several subtypes of Type III and their defence mechanisms. The anti-CRISPRs (Acrs) recently described, the critical role of Reverse transcriptase (RT) and housekeeping nuclease for type III CRISPR-Cas systems, and the use of this cutting-edge technology, its impact on the search for novel anti-tuberculosis drugs.},
}
@article {pmid37119122,
year = {2023},
author = {Fatehi, S and Marks, RM and Rok, MJ and Perillat, L and Ivakine, EA and Cohn, RD},
title = {Advances in CRISPR/Cas9 Genome Editing for the Treatment of Muscular Dystrophies.},
journal = {Human gene therapy},
volume = {34},
number = {9-10},
pages = {388-403},
doi = {10.1089/hum.2023.059},
pmid = {37119122},
issn = {1557-7422},
support = {//CIHR/Canada ; },
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics ; Genetic Therapy/methods ; Dystrophin/genetics ; },
abstract = {Muscular dystrophies (MDs) comprise a diverse group of inherited disorders characterized by progressive muscle loss and weakness. Given the genetic etiology underlying MDs, researchers have explored the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing as a therapeutic intervention, resulting in significant advances. Here, we review recent progress on the use of CRISPR/Cas9 genome editing as a potential therapy for MDs. Significant strides have been made in this realm, made possible through innovative techniques such as precision genetic editing by modified forms of CRISPR/Cas9. These approaches have shown varying degrees of success in animal models of MD, including Duchenne MD, congenital muscular dystrophy type 1A, and myotonic dystrophy type 1. Even so, there are several challenges facing the development of CRISPR/Cas9-based MD therapies, including the targeting of satellite cells, improved editing efficiency in skeletal and cardiac muscle tissue, delivery vehicle enhancements, and the host immunogenic response. Although more work is needed to advance CRISPR/Cas9 genome editing past the preclinical stages, its therapeutic potential for MD is extremely promising and justifies concentrated efforts to move into clinical trials.},
}
@article {pmid37060194,
year = {2023},
author = {Chemello, F and Olson, EN and Bassel-Duby, R},
title = {CRISPR-Editing Therapy for Duchenne Muscular Dystrophy.},
journal = {Human gene therapy},
volume = {34},
number = {9-10},
pages = {379-387},
doi = {10.1089/hum.2023.053},
pmid = {37060194},
issn = {1557-7422},
mesh = {Mice ; Humans ; Animals ; *Muscular Dystrophy, Duchenne/genetics ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Exons ; Dystrophin/genetics ; Gene Editing/methods ; Disease Models, Animal ; },
abstract = {Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation.},
}
@article {pmid36355853,
year = {2023},
author = {Iyer, DN and Schimmer, AD and Chang, H},
title = {Applying CRISPR-Cas9 screens to dissect hematological malignancies.},
journal = {Blood advances},
volume = {7},
number = {10},
pages = {2252-2270},
doi = {10.1182/bloodadvances.2022008966},
pmid = {36355853},
issn = {2473-9537},
mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; *Neoplasms/therapy ; *Hematologic Neoplasms/genetics ; Regulatory Sequences, Nucleic Acid ; },
abstract = {Bit by bit, over the last few decades, functional genomic tools have been piecing together the molecular puzzle driving tumorigenesis in human patients. Nevertheless, our understanding of the role of several genes and regulatory elements that drive critical cancer-associated physiological processes from disease development to progression to spread is very limited, which significantly affects our ability of applying these insights in the context of improved disease management. The recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology and its application in cancer genomics has, however, allowed the generation of a wealth of knowledge that has helped decipher several critical questions associated with translational cancer research. Precisely, the high-throughput capability coupled with a high level of technological plasticity associated with the CRISPR-Cas9 screens have expanded our horizons from a mere struggle to appreciate cancer as a genetic disease to observing the integrated genomic/epigenomic network of numerous malignancies and correlating it with our present knowledge of drugging strategies to develop innovative approaches for next-generation precision cancer medicine. Specifically, within blood cancers, current CRISPR screens have specifically focused on improving our understanding of drug resistance mechanisms, disease biology, the development of novel therapeutic approaches, and identifying the molecular mechanisms of current therapies, with an underlying aim of improving disease outcomes. Here, we review the development of the CRISPR-Cas9 genome-editing strategy, explicitly focusing on the recent advances in the CRISPR-Cas9-based screening approaches, its current capabilities, limitations, and future applications in the context of hematological malignancies.},
}
@article {pmid37216595,
year = {2023},
author = {Zhou, F and Yu, X and Gan, R and Ren, K and Chen, C and Ren, C and Cui, M and Liu, Y and Gao, Y and Wang, S and Yin, M and Huang, T and Huang, Z and Zhang, F},
title = {CRISPRimmunity: an interactive web server for CRISPR-associated Important Molecular events and Modulators Used in geNome edIting Tool identifYing.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad425},
pmid = {37216595},
issn = {1362-4962},
abstract = {The CRISPR-Cas system is a highly adaptive and RNA-guided immune system found in bacteria and archaea, which has applications as a genome editing tool and is a valuable system for studying the co-evolutionary dynamics of bacteriophage interactions. Here introduces CRISPRimmunity, a new web server designed for Acr prediction, identification of novel class 2 CRISPR-Cas loci, and dissection of key CRISPR-associated molecular events. CRISPRimmunity is built on a suite of CRISPR-oriented databases providing a comprehensive co-evolutionary perspective of the CRISPR-Cas and anti-CRISPR systems. The platform achieved a high prediction accuracy of 0.997 for Acr prediction when tested on a dataset of 99 experimentally validated Acrs and 676 non-Acrs, outperforming other existing prediction tools. Some of the newly identified class 2 CRISPR-Cas loci using CRISPRimmunity have been experimentally validated for cleavage activity in vitro. CRISPRimmunity offers the catalogues of pre-identified CRISPR systems to browse and query, the collected resources or databases to download, a well-designed graphical interface, a detailed tutorial, multi-faceted information, and exportable results in machine-readable formats, making it easy to use and facilitating future experimental design and further data mining. The platform is available at http://www.microbiome-bigdata.com/CRISPRimmunity. Moreover, the source code for batch analysis are published on Github (https://github.com/HIT-ImmunologyLab/CRISPRimmunity).},
}
@article {pmid37216515,
year = {2023},
author = {Han, W and Li, Z and Guo, Y and He, K and Li, W and Xu, C and Ge, L and He, M and Yin, X and Zhou, J and Li, C and Yao, D and Bao, J and Liang, H},
title = {Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {22},
pages = {e2221127120},
doi = {10.1073/pnas.2221127120},
pmid = {37216515},
issn = {1091-6490},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Base Sequence ; *Gene Editing/methods ; DNA/genetics ; Homologous Recombination ; Mammals/genetics ; },
abstract = {CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.},
}
@article {pmid37215369,
year = {2023},
author = {Srivastava, P and Prasad, D},
title = {Isothermal nucleic acid amplification and its uses in modern diagnostic technologies.},
journal = {3 Biotech},
volume = {13},
number = {6},
pages = {200},
pmid = {37215369},
issn = {2190-572X},
abstract = {Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.},
}
@article {pmid37215153,
year = {2023},
author = {Fichter, KM and Setayesh, T and Malik, P},
title = {Strategies for precise gene edits in mammalian cells.},
journal = {Molecular therapy. Nucleic acids},
volume = {32},
number = {},
pages = {536-552},
pmid = {37215153},
issn = {2162-2531},
abstract = {CRISPR-Cas technologies have the potential to revolutionize genetic medicine. However, work is still needed to make this technology clinically efficient for gene correction. A barrier to making precise genetic edits in the human genome is controlling how CRISPR-Cas-induced DNA breaks are repaired by the cell. Since error-prone non-homologous end-joining is often the preferred cellular repair pathway, CRISPR-Cas-induced breaks often result in gene disruption. Homology-directed repair (HDR) makes precise genetic changes and is the clinically desired pathway, but this repair pathway requires a homology donor template and cycling cells. Newer editing strategies, such as base and prime editing, can affect precise repair for relatively small edits without requiring HDR and circumvent cell cycle dependence. However, these technologies have limitations in the extent of genetic editing and require the delivery of bulky cargo. Here, we discuss the pros and cons of precise gene correction using CRISPR-Cas-induced HDR, as well as base and prime editing for repairing small mutations. Finally, we consider emerging new technologies, such as recombination and transposases, which can circumvent both cell cycle and cellular DNA repair dependence for editing the genome.},
}
@article {pmid37212595,
year = {2023},
author = {Migliara, A and Cappelluti, MA and Giannese, F and Valsoni, S and Coglot, A and Merelli, I and Cittaro, D and Lombardo, A},
title = {In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {195},
pages = {},
doi = {10.3791/64403},
pmid = {37212595},
issn = {1940-087X},
mesh = {Humans ; *Gene Editing/methods ; *Epigenesis, Genetic ; Transcription Factors/metabolism ; Gene Silencing ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks. ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs' position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome. Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final evaluation in the therapeutically relevant setting of interest.},
}
@article {pmid37167072,
year = {2023},
author = {Mao, G and Luo, X and Ye, S and Wang, X and He, J and Kong, J and Dai, J and Yin, W and Ma, Y},
title = {Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.},
journal = {Analytical chemistry},
volume = {95},
number = {20},
pages = {8063-8069},
doi = {10.1021/acs.analchem.3c01033},
pmid = {37167072},
issn = {1520-6882},
mesh = {Animals ; Swine ; *Recombinases ; *African Swine Fever Virus/genetics ; CRISPR-Cas Systems/genetics ; Colorimetry ; Nucleotidyltransferases ; Alkaline Phosphatase ; Coloring Agents ; Nucleic Acid Amplification Techniques ; },
abstract = {It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.},
}
@article {pmid37156096,
year = {2023},
author = {Xiang, X and Song, M and Xu, X and Lu, J and Chen, Y and Chen, S and He, Y and Shang, Y},
title = {Microfluidic Biosensor Integrated with Signal Transduction and Enhancement Mechanism for Ultrasensitive Noncompetitive Assay of Multiple Mycotoxins.},
journal = {Analytical chemistry},
volume = {95},
number = {20},
pages = {7993-8001},
doi = {10.1021/acs.analchem.3c00813},
pmid = {37156096},
issn = {1520-6882},
mesh = {Microfluidics ; Biological Assay ; Chromatography, High Pressure Liquid ; *Mycotoxins ; Signal Transduction ; *Biosensing Techniques ; CRISPR-Cas Systems ; },
abstract = {To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10[-5]-10[1] ng·mL[-1]), low detection of limit (fg·mL[-1]), short detection period (∼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.},
}
@article {pmid37146364,
year = {2023},
author = {Gu, J and Sun, J and Tian, K and Bian, J and Peng, J and Xu, S and Zhao, L},
title = {Reversal of hepatic fibrosis by the co-delivery of drug and ribonucleoprotein-based genome editor.},
journal = {Biomaterials},
volume = {298},
number = {},
pages = {122133},
doi = {10.1016/j.biomaterials.2023.122133},
pmid = {37146364},
issn = {1878-5905},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Pharmaceutical Preparations ; Liver Cirrhosis/drug therapy ; Endonucleases/genetics/metabolism ; RNA, Messenger ; },
abstract = {Liver fibrosis is a chronic disease without effective treatment in the clinic. Gene editing systems such as the well-known CRISPR/Cas9 have shown great potential in the biomedical field. However, the delivery of the ribonucleoprotein is challenging due to the unstable RNA probe and the requirement for the entrance to the nucleus. Recently, a structure-guided endonuclease (SGN) has been reported as an effective gene-editing system composed of a nuclease and stable DNA probes, which can regulate the protein expression by targeting specific mRNA outside the nucleus. Here, we conjugated the SGN to a nanomicelle as the delivery system. In the resulting material, the chance of the collision between the endonuclease and the probe was raised due to the confinement of the two components within the 40-nm nanomicelle, thus the mRNA can be cleaved immediately after being captured by the probe, resulting in a space-induced nucleotide identification-cleavage acceleration effect. The delivery system was used to treat liver fibrosis via the co-delivery of SGN and a drug rosiglitazone to the hepatic stellate cells, which separately downregulated the expression of tissue inhibitor of metalloprotease-1 and inactivated the hepatic stellate cells. The system successfully reversed the liver fibrosis in mice through the bidirectional regulatory that simultaneously promoted the degradation and inhibited the production of the collagen, demonstrating the great potency of the SGN system as gene medicine.},
}
@article {pmid37214290,
year = {2023},
author = {Fang, L and Yang, L and Han, M and Xu, H and Ding, W and Dong, X},
title = {CRISPR-cas technology: A key approach for SARS-CoV-2 detection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1158672},
pmid = {37214290},
issn = {2296-4185},
abstract = {The CRISPR (Clustered Regularly Spaced Short Palindromic Repeats) system was first discovered in prokaryotes as a unique immune mechanism to clear foreign nucleic acids. It has been rapidly and extensively used in basic and applied research owing to its strong ability of gene editing, regulation and detection in eukaryotes. Hererin in this article, we reviewed the biology, mechanisms and relevance of CRISPR-Cas technology and its applications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. CRISPR-Cas nucleic acid detection tools include CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-Cas14, CRISPR nucleic acid amplification detection technology, and CRISPR colorimetric readout detection system. The above CRISPR technologies have been applied to the nucleic acid detection, including SARS-CoV-2 detection. Common nucleic acid detection based on CRISPR derivation technology include SHERLOCK, DETECTR, and STOPCovid. CRISPR-Cas biosensing technology has been widely applied to point-of-care testing (POCT) by targeting recognition of both DNA molecules and RNA Molecules.},
}
@article {pmid37212293,
year = {2023},
author = {Yang, X and Zeng, X and Chen, X and Huang, J and Wei, X and Ying, X and Tan, Q and Wang, Y and Li, S},
title = {Development of a CRISPR/Cas12a-recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus.},
journal = {Journal of medical virology},
volume = {95},
number = {5},
pages = {e28757},
doi = {10.1002/jmv.28757},
pmid = {37212293},
issn = {1096-9071},
mesh = {Humans ; *Recombinases/genetics ; *CRISPR-Cas Systems ; Monkeypox virus ; Congo ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; },
abstract = {Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L- and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.},
}
@article {pmid37210864,
year = {2023},
author = {Chirco, KR and Martinez, C and Lamba, DA},
title = {Advancements in pre-clinical development of gene editing-based therapies to treat inherited retinal diseases.},
journal = {Vision research},
volume = {209},
number = {},
pages = {108257},
doi = {10.1016/j.visres.2023.108257},
pmid = {37210864},
issn = {1878-5646},
abstract = {One of the major goals in the inherited retinal disease (IRD) field is to develop an effective therapy that can be applied to as many patients as possible. Significant progress has already been made toward this end, with gene editing at the forefront. The advancement of gene editing-based tools has been a recent focus of many research groups around the world. Here, we provide an update on the status of CRISPR/Cas-derived gene editors, promising options for delivery of these editing systems to the retina, and animal models that aid in pre-clinical testing of new IRD therapeutics.},
}
@article {pmid37094742,
year = {2023},
author = {Keppeke, GD and Diogenes, L and Gomes, K and Andrade, LEC},
title = {"Untargeting" autoantibodies using genome editing, a proof-of-concept study.},
journal = {Clinical immunology (Orlando, Fla.)},
volume = {251},
number = {},
pages = {109343},
doi = {10.1016/j.clim.2023.109343},
pmid = {37094742},
issn = {1521-7035},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Autoantibodies/genetics ; HEK293 Cells ; Genome ; },
abstract = {Autoantibodies (AAbs) are useful biomarkers and many have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. We apply CRISPR/Cas9 genome-editing to knockout V(D)J rearrangements that produce pathogenic AAbs in vitro. HEK293T cell-lines were established stably expressing a humanized anti-dsDNA Ab (clone 3H9) and a human-derived anti-nAChR-α1 Ab (clone B12L). For each clone, five CRISPR/Cas9 heavy-chain's CDR2/3-targeting guided-RNAs (T-gRNAs) were designed. Non-Target-gRNA (NT-gRNA) was control. After editing, levels of secreted Abs were evaluated, as well as 3H9 anti-dsDNA and B12L anti-AChR reactivities. T-gRNAs editing decreased expression of heavy-chain genes to ∼50-60%, compared to >90% in NT-gRNA, although secreted Abs levels and reactivity to their respective antigens in T-gRNAs decreased ∼90% and ∼ 95% compared with NT-gRNA for 3H9 and B12L, respectively. Sequencing indicated indels at Cas9 cut-site, which could lead to codon jam, and consequently, knockout. Additionally, remaining secreted 3H9-Abs presented variable dsDNA reactivity among the five T-gRNA, suggesting the exact Cas9 cut-site and indels further interfere with antibody-antigen interaction. CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting AAbs secretion and binding capacity, fostering application of this concept to in vivo models as a potential novel therapeutic approach for AAb-mediated diseases.},
}
@article {pmid37070192,
year = {2023},
author = {Carusillo, A and Haider, S and Schäfer, R and Rhiel, M and Türk, D and Chmielewski, KO and Klermund, J and Mosti, L and Andrieux, G and Schäfer, R and Cornu, TI and Cathomen, T and Mussolino, C},
title = {A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.},
journal = {Nucleic acids research},
volume = {51},
number = {9},
pages = {4660-4673},
pmid = {37070192},
issn = {1362-4962},
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; DNA Repair/genetics ; DNA Breaks, Double-Stranded ; Recombinational DNA Repair ; DNA End-Joining Repair/genetics ; Mammals/genetics ; },
abstract = {Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.},
}
@article {pmid36999618,
year = {2023},
author = {Byun, G and Yang, J and Seo, SW},
title = {CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli.},
journal = {Nucleic acids research},
volume = {51},
number = {9},
pages = {4650-4659},
pmid = {36999618},
issn = {1362-4962},
mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Gene Expression ; },
abstract = {Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.},
}
@article {pmid36938898,
year = {2023},
author = {Pan, C and Li, R and Shui, L and Xiao, Z and Wang, Y and Zhu, J and Wu, C and Zhang, L and Jia, J and Zheng, M},
title = {A new method to synthesize multiple gRNA libraries and functional mapping of mammalian H3K4me3 regions.},
journal = {Nucleic acids research},
volume = {51},
number = {9},
pages = {e50},
pmid = {36938898},
issn = {1362-4962},
mesh = {Animals ; *Genome ; *Histones/genetics ; Genomics ; DNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {Genetic screening based on the clustered regularly interspaced palindromic repeat (CRISPR) system has been indicated to be a powerful tool for identifying regulatory genes or cis-elements. However, when applying CRISPR screens to pinpoint functional elements at particular loci, a large number of guide RNA (gRNA) spacers may be required to achieve saturated coverage. Here, we present a controlled template-dependent elongation (CTDE) method relying on reversible terminators to synthesize gRNA libraries with genomic regions of interest. By applying this approach to H3K4me3 chromatin immunoprecipitation (ChIP)-derived DNA of mammalian cells, mega-sized gRNA libraries were synthesized in a tissue-specific manner, with which we conducted screening experiments to annotate essential sites for cell proliferation. Additionally, we confirmed that an essential site within the intron of LINC00339 regulates its own mRNA and that LINC00339 is a novel regulator of the cell cycle that maintains HepG2 proliferation. The CTDE method has the potential to be automated with high efficiency at low cost, and will be widely used to identify functional elements in mammalian genomes.},
}
@article {pmid34923124,
year = {2022},
author = {Rahman, K and Jamal, M and Chen, X and Zhou, W and Yang, B and Zou, Y and Xu, W and Lei, Y and Wu, C and Cao, X and Tyagi, R and Naeem, MA and Lin, D and Habib, Z and Peng, N and Fu, ZF and Cao, G},
title = {Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.},
journal = {Genomics, proteomics &amp; bioinformatics},
volume = {20},
number = {6},
pages = {1180-1196},
doi = {10.1016/j.gpb.2021.01.008},
pmid = {34923124},
issn = {2210-3244},
mesh = {Humans ; *Mycobacterium tuberculosis/genetics/metabolism ; Gene Editing ; RNA Interference ; *Tuberculosis/prevention &amp; control/genetics/microbiology ; Antitubercular Agents/metabolism ; CRISPR-Cas Systems ; },
abstract = {Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.},
}
@article {pmid37210856,
year = {2023},
author = {Wang, X and Jing, S and Wang, W and Wang, J},
title = {Direct and noninvasive fluorescence analysis of an RNA-protein interaction based on a CRISPR/Cas12a-powered assay.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {299},
number = {},
pages = {122884},
doi = {10.1016/j.saa.2023.122884},
pmid = {37210856},
issn = {1873-3557},
abstract = {RNA-protein interactions (RPIs) play critical roles in gene transcription and protein expression, but current analytical methods for RPIs are mainly performed in an invasive manner, involving special RNA/protein labeling, hampering access to intact and precise information on RPIs. In this work, we present the first CRISPR/Cas12a-based fluorescence assay for the direct analysis of RPIs without RNA/protein labeling steps. Select vascular endothelial growth factor 165 (VEGF165)/its RNA aptamer interaction as a model, the RNA sequence simultaneously serves as both the aptamer of VEGF165 and crRNA of CRISPR/Cas12a system, and the presence of VEGF165 facilitates VEGF165/its RNA aptamer interaction, thus prohibiting the formation of Cas12a-crRNA-DNA ternary complex along with low fluorescence signal. The assay showed a detection limit of 0.23 pg mL[-1], and good performance in serum-spiked samples with an RSD of 0.4 %-13.1 %. This simple and selective strategy opens the door for establishing CRISPR/Cas-based biosensors for gaining intact information on RPIs, and shows widespread potential for other RPIs analysis.},
}
@article {pmid37210555,
year = {2023},
author = {Wu, J and Tao, Y and Deng, D and Meng, Z and Zhao, Y},
title = {The applications of CRISPR/Cas-mediated genome editing in genetic hearing loss.},
journal = {Cell &amp; bioscience},
volume = {13},
number = {1},
pages = {93},
pmid = {37210555},
issn = {2045-3701},
abstract = {Hearing loss (HL) can be caused by a number of different genetic factors. Non-syndromic HL refers that HL occurs as an isolated symptom in an individual, whereas syndromic HL refers that HL is associated with other symptoms or abnormalities. To date, more than 140 genes have been identified as being associated with non-syndromic HL, and approximately 400 genetic syndromes can include HL as one of the clinical symptoms. However, no gene therapeutic approaches are currently available to restore or improve hearing. Therefore, there is an urgent necessity to elucidate the possible pathogenesis of specific mutations in HL-associated genes and to investigate the promising therapeutic strategies for genetic HL. The development of the CRISPR/Cas system has revolutionized the field of genome engineering, which has become an efficacious and cost-effective tool to foster genetic HL research. Moreover, several in vivo studies have demonstrated the therapeutic efficacy of the CRISPR/Cas-mediated treatments for specific genetic HL. In this review, we briefly introduce the progress in CRISPR/Cas technique as well as the understanding of genetic HL, and then we detail the recent achievements of CRISPR/Cas technique in disease modeling and therapeutic strategies for genetic HL. Furthermore, we discuss the challenges for the application of CRISPR/Cas technique in future clinical treatments.},
}
@article {pmid37209468,
year = {2023},
author = {Even-Zohar, N and Metin-Armagan, D and Ben-Shlomo, A and Sareen, D and Melmed, S},
title = {Generation of isogenic and homozygous MEN1 mutant cell lines from patient-derived iPSCs using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {69},
number = {},
pages = {103124},
doi = {10.1016/j.scr.2023.103124},
pmid = {37209468},
issn = {1876-7753},
abstract = {MEN1, an autosomal dominant disorder caused by mutations in the tumor suppressor gene MEN1, manifests with co-occurrence of multiple endocrine/neuroendocrine neoplasms. An iPSC line derived from an index patient carrying the mutation c.1273C>T (p.Arg465*) was edited using a single multiplex CRISPR/Cas approach to create an isogenic control non-mutated line and a homozygous double mutant line. These cell lines will be useful for elucidating subcellular MEN1 pathophysiology and for screening to identify potential MEN1 therapeutic targets.},
}
@article {pmid37208532,
year = {2023},
author = {Lee, K and Lee, C},
title = {Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {8109},
pmid = {37208532},
issn = {2045-2322},
support = {GM131283/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Fibroblasts/metabolism ; *Circadian Clocks/genetics ; Cell Culture Techniques ; Transcription Factors/metabolism ; Disease Models, Animal ; Circadian Rhythm/genetics ; },
abstract = {The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.},
}
@article {pmid37207121,
year = {2023},
author = {Wu, J and Xu, H and Hu, F and Jiang, Y and Fan, B and Khan, A and Sun, Y and Di, K and Gu, X and Shen, H and Li, Z},
title = {CRISPR-Cas and catalytic hairpin assembly technology for target-initiated amplification detection of pancreatic cancer specific tsRNAs.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1169424},
pmid = {37207121},
issn = {2296-4185},
abstract = {Transfer RNA-derived small RNAs (tsRNAs) tRF-LeuCAG-002 (ts3011a RNA) is a novel class of non-coding RNAs biomarker for pancreatic cancer (PC). Reverse transcription polymerase chain reaction (RT-qPCR) has been unfit for community hospitals that are short of specialized equipment or laboratory setups. It has not been reported whether isothermal technology can be used for detection, because the tsRNAs have rich modifications and secondary structures compared with other non-coding RNAs. Herein, we have employed a catalytic hairpin assembly (CHA) circuit and clustered regularly interspaced short palindromic repeats (CRISPR) to develop an isothermal and target-initiated amplification method for detecting ts3011a RNA. In the proposed assay, the presence of target tsRNA triggers the CHA circuit that transforms new DNA duplexes to activate collateral cleavage activity of CRISPR-associated proteins (CRISPR-Cas) 12a, achieving cascade signal amplification. This method showed a low detection limit of 88 aM at 37 °C within 2 h. Moreover, it was demonstrated for the first time that, this method is less likely to produce aerosol contamination than RT-qPCR by simulating aerosol leakage experiments. This method has good consistency with RT-qPCR in the detection of serum samples and showed great potential for PC-specific tsRNAs point-of-care testing (POCT).},
}
@article {pmid37204916,
year = {2023},
author = {Shi, L and Su, J and Cho, MJ and Song, H and Dong, X and Liang, Y and Zhang, Z},
title = {Promoter editing for the genetic improvement of crops.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erad175},
pmid = {37204916},
issn = {1460-2431},
abstract = {The proper gene expression plays a fundamental role in the regulation of agronomically important traits in crop plants. The genetic manipulation of plant promoters through genome editing has emerged as an effective strategy to create favorable traits in crops by altering the expression pattern of the pertinent genes. Promoter editing can be applied in a directed manner, where nucleotide sequences associated with favorable traits are precisely generated. Alternatively, promoter editing can also be exploited as a random mutagenic approach to generate novel genetic variations within a designated promoter, from which elite alleles are selected based on their phenotypic effects. Pioneering studies have demonstrated the potential of promoter editing in engineering agronomically important traits as well as in mining novel promoter alleles valuable for plant breeding. In this review article, we update the progress in the application of promoter editing in crops for increased yield, enhanced tolerance to biotic and abiotic stresses, and improved quality. We also discuss several remaining technical bottlenecks and how this strategy may be better employed for the genetic improvement of crops in the future.},
}
@article {pmid37204621,
year = {2023},
author = {Chaudhry, A and Hassan, AU and Khan, SH and Abbasi, A and Hina, A and Khan, MT and Abdelsalam, NR},
title = {The changing landscape of agriculture: role of precision breeding in developing smart crops.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {2},
pages = {167},
pmid = {37204621},
issn = {1438-7948},
mesh = {Humans ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; Crops, Agricultural/genetics ; Agriculture ; Genome, Plant ; },
abstract = {Food plants play a crucial role in human survival, providing them essential nutrients. However, traditional breeding methods have not been able to keep up with the demands of the growing population. The improvement of food plants aims to increase yield, quality, and resistance to biotic and abiotic stresses. With CRISPR/Cas9, researchers can identify and edit key genes conferring desirable qualities in agricultural plants, including increased yield, enhanced product quality attributes, and increased tolerance to biotic and abiotic challenges. These modifications have enabled the creation of "smart crops" that exhibit rapid climatic adaptation, resistance to extreme weather conditions and high yield and quality. The use of CRISPR/Cas9 combined with viral vectors or growth regulators has made it possible to produce more efficient modified plants with certain conventional breeding methods. However, ethical and regulatory aspects of this technology must be carefully considered. Proper regulation and application of genome editing technology can bring immense benefits to agriculture and food security. This article provides an overview of genetically modified genes and conventional as well as emerging tools, including CRISPR/Cas9, that have been utilized to enhance the quality of plants/fruits and their products. The review also discusses the challenges and prospects associated with these techniques.},
}
@article {pmid37204115,
year = {2023},
author = {Zhang, J and Zhu, A and Mei, M and Qu, J and Huang, Y and Shi, Y and Xue, M and Zhang, J and Zhang, R and Zhou, B and Tan, X and Zhao, J and Wang, Y},
title = {Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2300656},
doi = {10.1002/advs.202300656},
pmid = {37204115},
issn = {2198-3844},
abstract = {RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.},
}
@article {pmid37202186,
year = {2023},
author = {Ma, Y and Zou, L and Liang, Y and Liu, Q and Sun, Q and Pang, Y and Lin, H and Deng, X and Tang, S},
title = {[Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {43},
number = {4},
pages = {516-526},
doi = {10.12122/j.issn.1673-4254.2023.04.03},
pmid = {37202186},
issn = {1673-4254},
mesh = {Humans ; *COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics ; RNA ; COVID-19 Testing ; },
abstract = {OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.<br><br>METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.<br><br>RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/&#x3bc;L, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.<br><br>CONCLUSION: By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.},
}
@article {pmid37161551,
year = {2023},
author = {Liu, C and Yao, X and Liu, C and You, S and Qi, W and Wang, M},
title = {Development and evaluation of RPA-NFO-LFT and RPA-Cas12a-LFT systems for the detection of Candida albicans.},
journal = {Analytical methods : advancing methods and applications},
volume = {15},
number = {19},
pages = {2355-2365},
doi = {10.1039/d3ay00259d},
pmid = {37161551},
issn = {1759-9679},
mesh = {*Candida albicans/genetics ; Sensitivity and Specificity ; CRISPR-Cas Systems ; *Candidiasis, Invasive ; DNA Primers ; },
abstract = {Recently, the growing number of medical interventions has led to the risk of invasive candidiasis. Among them, Candida albicans (C. albicans) infection has the highest incidence, which has led to great demand for developing early diagnosis methods. In this study, two lateral flow device based molecular assay systems, RPA-NFO-LFT and RPA-Cas12a-LFT, were established and optimized to achieve the detection of C. albicans. Firstly, efficient and specific primers for C. albicans detection were designed and screened, and the purification of amplification products was also explored. Then, many important conditions and issues for each system were investigated and discussed to improve the performances of the test strip devices in C. albicans detection. An evaluation study revealed that both systems showed favorable specificity and sensitivity in the detection of C. albicans samples with a lower detection limit of 10[3] CFU ml[-1], while RPA-Cas12a-LFT is more accurate for visual interpretation and more stable toward samples that exhibit serum nucleic acid interference. Finally, the performances of RPA-NFO-LFT and RPA-Cas12a-LFT were compared with that of the conventional qPCR method. This work might provide a reference for the development of molecular assay devices in practical candidiasis diagnosis.},
}
@article {pmid37147433,
year = {2023},
author = {Foss, DV and Muldoon, JJ and Nguyen, DN and Carr, D and Sahu, SU and Hunsinger, JM and Wyman, SK and Krishnappa, N and Mendonsa, R and Schanzer, EV and Shy, BR and Vykunta, VS and Allain, V and Li, Z and Marson, A and Eyquem, J and Wilson, RC},
title = {Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes.},
journal = {Nature biomedical engineering},
volume = {7},
number = {5},
pages = {647-660},
pmid = {37147433},
issn = {2157-846X},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; L30 AI140341/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; T-Lymphocytes/metabolism ; Peptides/genetics ; Ribonucleoproteins ; },
abstract = {CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.},
}
@article {pmid37141888,
year = {2023},
author = {Guo, J and Yu, W and Li, M and Chen, H and Liu, J and Xue, X and Lin, J and Huang, S and Shu, W and Huang, X and Liu, Z and Wang, S and Qiao, Y},
title = {A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.},
journal = {Molecular cell},
volume = {83},
number = {10},
pages = {1710-1724.e7},
doi = {10.1016/j.molcel.2023.04.012},
pmid = {37141888},
issn = {1097-4164},
mesh = {Mice ; Animals ; *Gene Editing ; *Trans-Activators/metabolism ; Cytosine ; Mutation ; DNA, Mitochondrial/genetics ; CRISPR-Cas Systems ; },
abstract = {Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.},
}
@article {pmid37080758,
year = {2023},
author = {Sherkatghanad, Z and Abdar, M and Charlier, J and Makarenkov, V},
title = {Using traditional machine learning and deep learning methods for on- and off-target prediction in CRISPR/Cas9: a review.},
journal = {Briefings in bioinformatics},
volume = {24},
number = {3},
pages = {},
pmid = {37080758},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; *Deep Learning ; Gene Editing/methods ; Machine Learning ; },
abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) is a popular and effective two-component technology used for targeted genetic manipulation. It is currently the most versatile and accurate method of gene and genome editing, which benefits from a large variety of practical applications. For example, in biomedicine, it has been used in research related to cancer, virus infections, pathogen detection, and genetic diseases. Current CRISPR/Cas9 research is based on data-driven models for on- and off-target prediction as a cleavage may occur at non-target sequence locations. Nowadays, conventional machine learning and deep learning methods are applied on a regular basis to accurately predict on-target knockout efficacy and off-target profile of given single-guide RNAs (sgRNAs). In this paper, we present an overview and a comparative analysis of traditional machine learning and deep learning models used in CRISPR/Cas9. We highlight the key research challenges and directions associated with target activity prediction. We discuss recent advances in the sgRNA-DNA sequence encoding used in state-of-the-art on- and off-target prediction models. Furthermore, we present the most popular deep learning neural network architectures used in CRISPR/Cas9 prediction models. Finally, we summarize the existing challenges and discuss possible future investigations in the field of on- and off-target prediction. Our paper provides valuable support for academic and industrial researchers interested in the application of machine learning methods in the field of CRISPR/Cas9 genome editing.},
}
@article {pmid37069266,
year = {2023},
author = {Everette, KA and Newby, GA and Levine, RM and Mayberry, K and Jang, Y and Mayuranathan, T and Nimmagadda, N and Dempsey, E and Li, Y and Bhoopalan, SV and Liu, X and Davis, JR and Nelson, AT and Chen, PJ and Sousa, AA and Cheng, Y and Tisdale, JF and Weiss, MJ and Yen, JS and Liu, DR},
title = {Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.},
journal = {Nature biomedical engineering},
volume = {7},
number = {5},
pages = {616-628},
pmid = {37069266},
issn = {2157-846X},
support = {U01 AI142756/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 HL156647/HL/NHLBI NIH HHS/United States ; R01 HL136135/HL/NHLBI NIH HHS/United States ; },
mesh = {Adult ; Humans ; Mice ; Animals ; *Gene Editing ; CRISPR-Cas Systems ; beta-Globins/genetics ; *Anemia, Sickle Cell/therapy/genetics ; Hematopoietic Stem Cells ; Phenotype ; DNA ; },
abstract = {Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBB[S]) to wild type (HBB[A]) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBB[A] levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBB[A], exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBB[A]-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBB[S] to HBB[A], does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.},
}
@article {pmid37068307,
year = {2023},
author = {Guan, Z and Jiang, Z},
title = {Transformer-based anti-noise models for CRISPR-Cas9 off-target activities prediction.},
journal = {Briefings in bioinformatics},
volume = {24},
number = {3},
pages = {},
doi = {10.1093/bib/bbad127},
pmid = {37068307},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Base Sequence ; },
abstract = {The off-target effect occurring in the CRISPR-Cas9 system has been a challenging problem for the practical application of this gene editing technology. In recent years, various prediction models have been proposed to predict potential off-target activities. However, most of the existing prediction methods do not fully exploit guide RNA (gRNA) and DNA sequence pair information effectively. In addition, available prediction methods usually ignore the noise effect in original off-target datasets. To address these issues, we design a novel coding scheme, which considers the key features of mismatch type, mismatch location and the gRNA-DNA sequence pair information. Furthermore, a transformer-based anti-noise model called CrisprDNT is developed to solve the noise problem that exists in the off-target data. Experimental results of eight existing datasets demonstrate that the method with the inclusion of the anti-noise loss functions is superior to available state-of-the-art prediction methods. CrisprDNT is available at https://github.com/gzrgzx/CrisprDNT.},
}
@article {pmid37058502,
year = {2023},
author = {Maestroni, L and Butti, P and Milanesi, R and Pagliari, S and Campone, L and Serra, I and Branduardi, P},
title = {Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {12},
number = {5},
pages = {1508-1519},
doi = {10.1021/acssynbio.3c00015},
pmid = {37058502},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Fermentation ; *Metabolic Engineering ; CRISPR-Cas Systems ; },
abstract = {Nowadays, the yeast Saccharomyces cerevisiae is the platform of choice for demonstrating the proof of concept of the production of metabolites with a complex structure. However, introducing heterologous genes and rewiring the endogenous metabolism is still not standardized enough, affecting negatively the readiness-to-market of such metabolites. We developed the Easy Modular Integrative fuSion-ready Expression (Easy-MISE) toolkit, which is a novel combination of synthetic biology tools based on a single Golden Gate multiplasmid assembly meant to further ameliorate the rational predictability and flexibility of the process of yeast engineering. Thanks to an improved cloning screening strategy, double and independent transcription units are easily assembled and subsequently integrated into previously characterized loci. Moreover, the devices can be tagged for localization. This design allows for a higher degree of modularity and increases the flexibility of the engineering strategy. We show with a case study how the developed toolkit accelerates the construction and the analysis of the intermediate and the final engineered yeast strains, leaving space to better characterize the heterologous biosynthetic pathway in the final host and, overall, to improve the fermentation performances. Different S. cerevisiae strains were built harboring different versions of the biochemical pathway toward glucobrassicin (GLB) production, an indolyl-methyl glucosinolate. In the end, we could demonstrate that in the tested conditions the best-producing strain leads to a final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold higher than the best result previously reported in the literature.},
}
@article {pmid37058298,
year = {2023},
author = {Xiong, X and Tasan, I and Yang, C and Zhang, M and Hernandez Gonzalez, GA and Liu, S and Chaturvedi, P and Belmont, AS and Zhao, H},
title = {Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus.},
journal = {ACS synthetic biology},
volume = {12},
number = {5},
pages = {1424-1436},
doi = {10.1021/acssynbio.2c00433},
pmid = {37058298},
issn = {2161-5063},
mesh = {Animals ; *DNA Replication Timing ; *CRISPR-Cas Systems/genetics ; Chromosomes ; Genome ; DNA-Binding Proteins ; Molecular Chaperones ; Mammals ; },
abstract = {Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.},
}
@article {pmid37037965,
year = {2023},
author = {Kawamata, M and Suzuki, HI and Kimura, R and Suzuki, A},
title = {Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs.},
journal = {Nature biomedical engineering},
volume = {7},
number = {5},
pages = {672-691},
pmid = {37037965},
issn = {2157-846X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Cytosine ; Gene Editing ; Recombinational DNA Repair ; },
abstract = {The precise regulation of the activity of Cas9 is crucial for safe and efficient editing. Here we show that the genome-editing activity of Cas9 can be constrained by the addition of cytosine stretches to the 5'-end of conventional single-guide RNAs (sgRNAs). Such a 'safeguard sgRNA' strategy, which is compatible with Cas12a and with systems for gene activation and interference via CRISPR (clustered regularly interspaced short palindromic repeats), leads to the length-dependent inhibition of the formation of functional Cas9 complexes. Short cytosine extensions reduced p53 activation and cytotoxicity in human pluripotent stem cells, and enhanced homology-directed repair while maintaining bi-allelic editing. Longer extensions further decreased on-target activity yet improved the specificity and precision of mono-allelic editing. By monitoring indels through a fluorescence-based allele-specific system and computational simulations, we identified optimal windows of Cas9 activity for a number of genome-editing applications, including bi-allelic and mono-allelic editing, and the generation and correction of disease-associated single-nucleotide substitutions via homology-directed repair. The safeguard-sgRNA strategy may improve the safety and applicability of genome editing.},
}
@article {pmid36853024,
year = {2023},
author = {Meng, J and Qiu, Y and Zhang, Y and Zhao, H and Shi, S},
title = {CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {12},
number = {5},
pages = {1408-1414},
doi = {10.1021/acssynbio.2c00591},
pmid = {36853024},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; Genomics ; Gene Editing/methods ; },
abstract = {Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in Saccharomyces cerevisiae, which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of S. cerevisiae.},
}
@article {pmid36797418,
year = {2023},
author = {Yan, S and Zheng, X and Lin, Y and Li, C and Liu, Z and Li, J and Tu, Z and Zhao, Y and Huang, C and Chen, Y and Li, J and Song, X and Han, B and Wang, W and Liang, W and Lai, L and Li, XJ and Li, S},
title = {Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington's disease.},
journal = {Nature biomedical engineering},
volume = {7},
number = {5},
pages = {629-646},
pmid = {36797418},
issn = {2157-846X},
mesh = {Animals ; Swine ; *Huntington Disease/genetics/therapy/metabolism ; Trinucleotide Repeat Expansion ; CRISPR-Cas Systems/genetics ; Genetic Engineering ; },
abstract = {The monogenic nature of Huntington's disease (HD) and other neurodegenerative diseases caused by the expansion of glutamine-encoding CAG repeats makes them particularly amenable to gene therapy. Here we show the feasibility of replacing expanded CAG repeats in the mutant HTT allele with a normal CAG repeat in genetically engineered pigs mimicking the selective neurodegeneration seen in patients with HD. A single intracranial or intravenous injection of adeno-associated virus encoding for Cas9, a single-guide RNA targeting the HTT gene, and donor DNA containing the normal CAG repeat led to the depletion of mutant HTT in the animals and to substantial reductions in the dysregulated expression and neurotoxicity of mutant HTT and in neurological symptoms. Our findings support the further translational development of virally delivered Cas9-based gene therapies for the treatment of genetic neurodegenerative diseases.},
}
@article {pmid37202505,
year = {2023},
author = {Maier, CR and Hartmann, O and Prieto-Garcia, C and Al-Shami, KM and Schlicker, L and Vogel, FCE and Haid, S and Klann, K and Buck, V and Münch, C and Schmitz, W and Einig, E and Krenz, B and Calzado, MA and Eilers, M and Popov, N and Rosenfeldt, MT and Diefenbacher, ME and Schulze, A},
title = {USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.},
journal = {Cell death and differentiation},
volume = {},
number = {},
pages = {},
pmid = {37202505},
issn = {1476-5403},
abstract = {SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.},
}
@article {pmid37201524,
year = {2023},
author = {Kalamakis, G and Platt, RJ},
title = {CRISPR for neuroscientists.},
journal = {Neuron},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.neuron.2023.04.021},
pmid = {37201524},
issn = {1097-4199},
abstract = {Genome engineering technologies provide an entry point into understanding and controlling the function of genetic elements in health and disease. The discovery and development of the microbial defense system CRISPR-Cas yielded a treasure trove of genome engineering technologies and revolutionized the biomedical sciences. Comprising diverse RNA-guided enzymes and effector proteins that evolved or were engineered to manipulate nucleic acids and cellular processes, the CRISPR toolbox provides precise control over biology. Virtually all biological systems are amenable to genome engineering-from cancer cells to the brains of model organisms to human patients-galvanizing research and innovation and giving rise to fundamental insights into health and powerful strategies for detecting and correcting disease. In the field of neuroscience, these tools are being leveraged across a wide range of applications, including engineering traditional and non-traditional transgenic animal models, modeling disease, testing genomic therapies, unbiased screening, programming cell states, and recording cellular lineages and other biological processes. In this primer, we describe the development and applications of CRISPR technologies while highlighting outstanding limitations and opportunities.},
}
@article {pmid37200846,
year = {2023},
author = {Sheng, L and Madika, A and Lau, MSH and Zhang, Y and Minton, NP},
title = {Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius NCIMB 11955.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1191079},
pmid = {37200846},
issn = {2296-4185},
abstract = {The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.},
}
@article {pmid37199299,
year = {2022},
author = {Dholariya, S and Parchwani, D and Radadiya, M and Singh, RD and Sonagra, A and Patel, D and Sharma, G},
title = {CRISPR/Cas9: A Molecular Tool for Ovarian Cancer Management beyond Gene Editing.},
journal = {Critical reviews in oncogenesis},
volume = {27},
number = {4},
pages = {1-22},
doi = {10.1615/CritRevOncog.2022043814},
pmid = {37199299},
issn = {0893-9675},
mesh = {Female ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Ovarian Neoplasms/diagnosis/genetics/therapy ; Genetic Therapy/methods ; Carcinogenesis/genetics ; },
abstract = {Ovarian cancer manifests with early metastases and has an adverse outcome, impacting the health of women globally. Currently, this malignancy is often treated with cytoreductive surgery and platinum-based chemotherapy. This treatment option has a limited success rate due to tumor recurrence and chemoresistance. Consequently, the fundamental objective of ovarian cancer treatment is the development of novel treatment approaches. As a new robust tool, the CRISPR/Cas9 gene-editing system has shown immense promise in elucidating the molecular basis of all the facets of ovarian cancer. Due to the precise gene editing capabilities of CRISPR-Cas9, researchers have been able to conduct a more comprehensive investigation of the genesis of ovarian cancer. This gained knowledge can be translated into the development of novel diagnostic approaches and newer therapeutic targets for this dreadful malignancy. There is encouraging preclinical evidence that suggests that CRISPR/Cas9 is a powerful versatile tool for selectively targeting cancer cells and inhibiting tumor growth, establishing new signaling pathways involved in carcinogenesis, and verifying biomolecules as druggable targets. In this review, we analyzed the current research and progress made using CRISPR/Cas9-based engineering strategies in the diagnosis and treatment, as well as the challenges in bringing this method to clinics. This comprehensive analysis will lay the basis for subsequent research in the future for the treatment of ovarian cancer.},
}
@article {pmid37198545,
year = {2023},
author = {Li, J and Liang, Q and Zhou, H and Zhou, M and Huang, H},
title = {Profiling the impact of the promoters on CRISPR-Cas12a system in human cells.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {41},
pmid = {37198545},
issn = {1689-1392},
mesh = {Animals ; Humans ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Gene Editing/methods ; Genetic Vectors ; *Cytomegalovirus Infections/genetics ; Mammals/genetics ; },
abstract = {The plasmid vector platform is the most commonly used vector for the expression of the versatile CRISPR-Cas technique and the promoter is a crucial element for the expression vector, thus profiling the impact of the promoters on CRISPR editors provides the basic information for the gene-editing toolkits and can be a guideline for its design. Herein, we made a parallel comparison among four commonly used promoters (CAG, ~ 1700 bp; EF1a core, ~ 210 bp; CMV, ~ 500 bp; and PGK, ~ 500 bp) in CRISPR-Cas12a system in mammalian cells to explore the impact of promoters on this powerful tool. We found that without badly damaging targeting specificity, the CAG promoter-driving Cas12a editor exhibited the most active (efficiency takes as 100%, specificity index = ~ 75%) in genomic cleavage, multiplex editing, transcriptional activation, and base editing, followed by promoter CMV (efficiency = 70 ~ 90% (vs CAG), specificity index = ~ 78%), and then EF1a core and PGK (both efficiency = 40-60%, vs CAG) but with higher specificity (specificity index = ~ 84% and ~ 82%, respectively). Therefore, CAG is recommended in the CRISPR-Cas12a system for the applications that need a robust editing activity but without size limitation, CMV mostly can be an alternative for CAG when requiring a smaller space, EF1a is similar to PGK with relatively high specificity, but has a smaller size, thus is more suitable for in vivo therapeutic applications. The data outlined the properties of the widely used promoters in the CRISPR-Cas12a system, which can be a guide for its applications and can be a useful resource for the gene-editing field.},
}
@article {pmid37193354,
year = {2022},
author = {Qiu, HY and Ji, RJ and Zhang, Y},
title = {Current advances of CRISPR-Cas technology in cell therapy.},
journal = {Cell insight},
volume = {1},
number = {6},
pages = {100067},
pmid = {37193354},
issn = {2772-8927},
abstract = {CRISPR-Cas is a versatile genome editing technology that has been broadly applied in both basic research and translation medicine. Ever since its discovery, the bacterial derived endonucleases have been engineered to a collection of robust genome-editing tools for introducing frameshift mutations or base conversions at site-specific loci. Since the initiation of first-in-human trial in 2016, CRISPR-Cas has been tested in 57 cell therapy trials, 38 of which focusing on engineered CAR-T cells and TCR-T cells for cancer malignancies, 15 trials of engineered hematopoietic stem cells treating hemoglobinopathies, leukemia and AIDS, and 4 trials of engineered iPSCs for diabetes and cancer. Here, we aim to review the recent breakthroughs of CRISPR technology and highlight their applications in cell therapy.},
}
@article {pmid37192099,
year = {2023},
author = {Koonin, EV and Gootenberg, JS and Abudayyeh, OO},
title = {Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.3c00159},
pmid = {37192099},
issn = {1520-4995},
abstract = {CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.},
}
@article {pmid37128982,
year = {2023},
author = {Kang, Y and Zhang, J and Zhao, L and Yan, H},
title = {Colorimetric miRNA detection based on self-primer-initiated CRISPR-Cas12a-assisted amplification.},
journal = {BioTechniques},
volume = {74},
number = {4},
pages = {172-178},
doi = {10.2144/btn-2023-0008},
pmid = {37128982},
issn = {1940-9818},
mesh = {Female ; Pregnancy ; Humans ; CRISPR-Cas Systems/genetics ; Colorimetry ; Fetus ; *MicroRNAs/genetics ; *Biosensing Techniques ; Nucleic Acid Amplification Techniques ; },
abstract = {miRNAs alter significantly throughout pregnancy to support the development of the fetus. However, sensitive detection of miRNA remains a challenge. Herein, a reliable miRNA detection approach integrating self-assembly-triggered signal amplification and CRISPR-Cas12a-system cleavage-based color generation is described. The colorimetric approach contains three signal amplification processes. The first signal amplification is formed by the released miRNA in a chain extension process. The produced sequence that is similar to the target miRNA initiates the second signal recycle. Finally, CRISPR-Cas12a-based transcleavage on linker sequences induces the third signal amplification. The method exhibits high sensitivity and a low limit of detection of 254 aM, showing promising prospects in disease diagnosis.},
}
@article {pmid37191877,
year = {2023},
author = {Alalmaie, A and Diaf, S and Khashan, R},
title = {Insight into the molecular mechanism of the transposon-encoded type I-F CRISPR-Cas system.},
journal = {Journal, genetic engineering &amp; biotechnology},
volume = {21},
number = {1},
pages = {60},
pmid = {37191877},
issn = {2090-5920},
abstract = {CRISPR-Cas9 is a popular gene-editing tool that allows researchers to introduce double-strand breaks to edit parts of the genome. CRISPR-Cas9 system is used more than other gene-editing tools because it is simple and easy to customize. However, Cas9 may produce unintended double-strand breaks in DNA, leading to off-target effects. There have been many improvements in the CRISPR-Cas system to control the off-target effect and improve the efficiency. The presence of a nuclease-deficient CRISPR-Cas system in several bacterial Tn7-like transposons inspires researchers to repurpose to direct the insertion of Tn7-like transposons instead of cleaving the target DNA, which will eventually limit the risk of off-target effects. Two transposon-encoded CRISPR-Cas systems have been experimentally confirmed. The first system, found in Tn7 like-transposon (Tn6677), is associated with the variant type I-F CRISPR-Cas system. The second one, found in Tn7 like-transposon (Tn5053), is related to the variant type V-K CRISPR-Cas system. This review describes the molecular and structural mechanisms of DNA targeting by the transposon-encoded type I-F CRISPR-Cas system, from assembly around the CRISPR-RNA (crRNA) to the initiation of transposition.},
}
@article {pmid37191803,
year = {2023},
author = {Rivera-Torres, N and Bialk, P and Kmiec, EB},
title = {CRISPR-Directed Gene Editing as a Method to Reduce Chemoresistance in Lung Cancer Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2660},
number = {},
pages = {263-271},
pmid = {37191803},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/genetics ; *Lung Neoplasms/genetics/therapy ; Lung ; },
abstract = {We are advancing a novel strategy for the treatment of solid tumors by employing CRISPR-directed gene editing to reduce levels of standard of care required to halt or reverse the progression of tumor growth. We intend to do this by utilizing a combinatorial approach in which CRISPR-directed gene editing is used to eliminate or significantly reduce the acquired resistance emerging from chemotherapy, radiation therapy, or immunotherapy. We will utilize CRISPR/Cas as a biomolecular tool to disable specific genes involved in the sustainability of resistance to cancer therapy. We have also developed a CRISPR/Cas molecule that can distinguish between the genome of a tumor cell in the genome of a normal cell, thereby conferring target selectivity onto this therapeutic approach. We envision delivering these molecules by direct injection into solid tumors for the treatment of squamous cell carcinomas of the lung, esophageal cancer, and head and neck cancer. We provide experimental details and methodology for utilizing CRISPR/Cas as a supplement to chemotherapy to destroy lung cancer cells.},
}
@article {pmid37190012,
year = {2023},
author = {Bhokisham, N and Laudermilch, E and Traeger, LL and Bonilla, TD and Ruiz-Estevez, M and Becker, JR},
title = {CRISPR-Cas System: The Current and Emerging Translational Landscape.},
journal = {Cells},
volume = {12},
number = {8},
pages = {},
pmid = {37190012},
issn = {2073-4409},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Ecosystem ; Genetic Therapy ; Epigenome ; },
abstract = {CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics.},
}
@article {pmid36357719,
year = {2023},
author = {Neugebauer, ME and Hsu, A and Arbab, M and Krasnow, NA and McElroy, AN and Pandey, S and Doman, JL and Huang, TP and Raguram, A and Banskota, S and Newby, GA and Tolar, J and Osborn, MJ and Liu, DR},
title = {Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.},
journal = {Nature biotechnology},
volume = {41},
number = {5},
pages = {673-685},
pmid = {36357719},
issn = {1546-1696},
support = {R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Cytosine ; Adenine ; Gene Editing ; DNA/genetics ; Deoxyadenosines ; Cytidine/genetics ; },
abstract = {Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.},
}
@article {pmid36357717,
year = {2023},
author = {Chen, L and Zhu, B and Ru, G and Meng, H and Yan, Y and Hong, M and Zhang, D and Luan, C and Zhang, S and Wu, H and Gao, H and Bai, S and Li, C and Ding, R and Xue, N and Lei, Z and Chen, Y and Guan, Y and Siwko, S and Cheng, Y and Song, G and Wang, L and Yi, C and Liu, M and Li, D},
title = {Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.},
journal = {Nature biotechnology},
volume = {41},
number = {5},
pages = {663-672},
pmid = {36357717},
issn = {1546-1696},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *Cytosine/metabolism ; Aminohydrolases/metabolism ; RNA ; CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics/metabolism ; },
abstract = {Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.},
}
@article {pmid37189388,
year = {2023},
author = {Vora, DS and Yadav, S and Sundar, D},
title = {Hybrid Multitask Learning Reveals Sequence Features Driving Specificity in the CRISPR/Cas9 System.},
journal = {Biomolecules},
volume = {13},
number = {4},
pages = {},
pmid = {37189388},
issn = {2218-273X},
mesh = {*CRISPR-Cas Systems/genetics ; *Machine Learning ; Genome ; },
abstract = {CRISPR/Cas9 technology is capable of precisely editing genomes and is at the heart of various scientific and medical advances in recent times. The advances in biomedical research are hindered because of the inadvertent burden on the genome when genome editors are employed-the off-target effects. Although experimental screens to detect off-targets have allowed understanding the activity of Cas9, that knowledge remains incomplete as the rules do not extrapolate well to new target sequences. Off-target prediction tools developed recently have increasingly relied on machine learning and deep learning techniques to reliably understand the complete threat of likely off-targets because the rules that drive Cas9 activity are not fully understood. In this study, we present a count-based as well as deep-learning-based approach to derive sequence features that are important in deciding on Cas9 activity at a sequence. There are two major challenges in off-target determination-the identification of a likely site of Cas9 activity and the prediction of the extent of Cas9 activity at that site. The hybrid multitask CNN-biLSTM model developed, named CRISP-RCNN, simultaneously predicts off-targets and the extent of activity on off-targets. Employing methods of integrated gradients and weighting kernels for feature importance approximation, analysis of nucleotide and position preference, and mismatch tolerance have been performed.},
}
@article {pmid37189371,
year = {2023},
author = {Zuo, Y and Mei, X and Singson, A},
title = {CRISPR/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa.},
journal = {Biomolecules},
volume = {13},
number = {4},
pages = {},
pmid = {37189371},
issn = {2218-273X},
support = {R01 HD054681/NH/NIH HHS/United States ; },
mesh = {Animals ; Male ; *Caenorhabditis elegans/metabolism ; *Caenorhabditis elegans Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Membrane Proteins/metabolism ; Semen/metabolism ; Spermatozoa/metabolism ; },
abstract = {The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and/or fusion.},
}
@article {pmid37093234,
year = {2023},
author = {Chin, S and Goyon, A and Zhang, K and Kurita, KL},
title = {Middle-out sequence confirmation of CRISPR/Cas9 single guide RNA (sgRNA) using DNA primers and ribonuclease T1 digestion.},
journal = {Analytical and bioanalytical chemistry},
volume = {415},
number = {14},
pages = {2809-2818},
pmid = {37093234},
issn = {1618-2650},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Ribonuclease T1/genetics ; DNA Primers ; Oligonucleotides ; Digestion ; },
abstract = {Accurate sequencing of single guide RNAs (sgRNAs) for CRISPR/Cas9 genome editing is critical for patient safety, as the sgRNA guides the Cas9 nuclease to target site-specific cleavages in DNA. An approach to fully sequence sgRNA using protective DNA primers followed by ribonuclease (RNase) T1 digestion was developed to facilitate the analysis of these larger molecules by hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-HRMS). Without RNase digestion, top-down mass spectrometry alone struggles to properly fragment precursor ions in large RNA oligonucleotides to provide confidence in sequence coverage. With RNase T1 digestion of these larger oligonucleotides, however, bottom-up analysis cannot confirm full sequence coverage due to the presence of short, redundant digestion products. By combining primer protection with RNase T1 digestion, digestion products are large enough to prevent redundancy and small enough to provide base resolution by tandem mass spectrometry to allow for full sgRNA sequence coverage. An investigation into the general requirements for adequate primer protection of specific regions of the RNA was conducted, followed by the development of a generic protection and digestion strategy that may be applied to different sgRNA sequences. This middle-out technique has the potential to expedite accurate sequence confirmation of chemically modified sgRNA oligonucleotides.},
}
@article {pmid37040174,
year = {2023},
author = {Feng, ZY and Yun, YF and Li, X and Zhang, J},
title = {Impact of Divalent Metal Ions on Regulation of Trans-Cleavage Activity of CRISPR-Cas13a: A Combined Experimental and Computational Study.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {24},
number = {10},
pages = {e202300034},
doi = {10.1002/cbic.202300034},
pmid = {37040174},
issn = {1439-7633},
mesh = {*RNA ; *Manganese ; Calcium/metabolism ; Molecular Conformation ; Magnesium ; CRISPR-Cas Systems ; },
abstract = {CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg[2+] to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn[2+] and Ca[2+] could replace Mg[2+] as cofactors of LbuCas13a. In contrast, Ni[2+] , Zn[2+] , Cu[2+] , or Fe[2+] inhibits the cis- and trans-cleavage activity, while Pb[2+] does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg[2+] and Mn[2+] can further enhance the trans-cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.},
}
@article {pmid36823185,
year = {2023},
author = {Li, L and Liu, W and Zhang, H and Cai, Q and Wen, D and Du, J and Sun, J and Li, L and Gao, C and Lin, P and Wu, M and Jiang, J},
title = {A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.},
journal = {The Tohoku journal of experimental medicine},
volume = {260},
number = {1},
pages = {51-61},
doi = {10.1620/tjem.2023.J011},
pmid = {36823185},
issn = {1349-3329},
abstract = {Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.},
}
@article {pmid36656467,
year = {2023},
author = {Han, JL and Entcheva, E},
title = {Gene Modulation with CRISPR-based Tools in Human iPSC-Cardiomyocytes.},
journal = {Stem cell reviews and reports},
volume = {19},
number = {4},
pages = {886-905},
pmid = {36656467},
issn = {2629-3277},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac ; Gene Editing/methods ; Mammals ; },
abstract = {Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.},
}
@article {pmid37189522,
year = {2023},
author = {Altindiş, M and Kahraman Kilbaş, EP},
title = {Managing Viral Emerging Infectious Diseases via Current and Future Molecular Diagnostics.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {13},
number = {8},
pages = {},
doi = {10.3390/diagnostics13081421},
pmid = {37189522},
issn = {2075-4418},
abstract = {Emerging viral infectious diseases have been a constant threat to global public health in recent times. In managing these diseases, molecular diagnostics has played a critical role. Molecular diagnostics involves the use of various technologies to detect the genetic material of various pathogens, including viruses, in clinical samples. One of the most commonly used molecular diagnostics technologies for detecting viruses is polymerase chain reaction (PCR). PCR amplifies specific regions of the viral genetic material in a sample, making it easier to detect and identify viruses. PCR is particularly useful for detecting viruses that are present in low concentrations in clinical samples, such as blood or saliva. Another technology that is becoming increasingly popular for viral diagnostics is next-generation sequencing (NGS). NGS can sequence the entire genome of a virus present in a clinical sample, providing a wealth of information about the virus, including its genetic makeup, virulence factors, and potential to cause an outbreak. NGS can also help identify mutations and discover new pathogens that could affect the efficacy of antiviral drugs and vaccines. In addition to PCR and NGS, there are other molecular diagnostics technologies that are being developed to manage emerging viral infectious diseases. One of these is CRISPR-Cas, a genome editing technology that can be used to detect and cut specific regions of viral genetic material. CRISPR-Cas can be used to develop highly specific and sensitive viral diagnostic tests, as well as to develop new antiviral therapies. In conclusion, molecular diagnostics tools are critical for managing emerging viral infectious diseases. PCR and NGS are currently the most commonly used technologies for viral diagnostics, but new technologies such as CRISPR-Cas are emerging. These technologies can help identify viral outbreaks early, track the spread of viruses, and develop effective antiviral therapies and vaccines.},
}
@article {pmid37189462,
year = {2023},
author = {Ishikawa, K and Saitoh, S},
title = {Transcriptional Regulation Technology for Gene Perturbation in Fission Yeast.},
journal = {Biomolecules},
volume = {13},
number = {4},
pages = {},
doi = {10.3390/biom13040716},
pmid = {37189462},
issn = {2218-273X},
abstract = {Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3' untranslated region (3'UTR) disruption, are available for yeast systems, CRISPR-Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR-Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.},
}
@article {pmid37187674,
year = {2023},
author = {Liu, J and Carmichael, C and Hasturk, H and Shi, W and Bor, B},
title = {Rapid specific detection of oral bacteria using Cas13-based SHERLOCK.},
journal = {Journal of oral microbiology},
volume = {15},
number = {1},
pages = {2207336},
pmid = {37187674},
issn = {2000-2297},
abstract = {Decades of ongoing research has established that oral microbial communities play a role in oral diseases such as periodontitis and caries. Yet the detection of oral bacteria and the profiling of oral polymicrobial communities currently rely on methods that are costly, slow, and technically complex, such as qPCR or next-generation sequencing. For the widescale screening of oral microorganisms suitable for point-of-care settings, there exists the need for a low-cost, rapid detection technique. Here, we tailored the novel CRISPR-Cas-based assay SHERLOCK for the species-specific detection of oral bacteria. We developed a computational pipeline capable of generating constructs suitable for SHERLOCK and experimentally validated the detection of seven oral bacteria. We achieved detection within the single-molecule range that remained specific in the presence of off-target DNA found within saliva. Further, we adapted the assay for detecting target sequences directly from unprocessed saliva samples. The results of our detection, when tested on 30 healthy human saliva samples, fully aligned with 16S rRNA sequencing. Looking forward, this method of detecting oral bacteria is highly scalable and can be easily optimized for implementation at point-of-care settings.},
}
@article {pmid37186473,
year = {2023},
author = {Sahel, DK and Goswami, SG and Jatyan, R and Kumari, A and Mittal, A and Ramalingam, S and Chitkara, D},
title = {Lipopolymeric nanocarrier enables effective delivery of CRISPR/Cas9 expressing plasmid.},
journal = {Macromolecular rapid communications},
volume = {},
number = {},
pages = {e2300101},
doi = {10.1002/marc.202300101},
pmid = {37186473},
issn = {1521-3927},
abstract = {CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined nucleic acid sequence. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases in the biological fluid, etc. Viral vector has been vastly used to deliver CRISPR/Cas components but possesses ample drawbacks and is challenging to translate. We hereby explored a cationic lipopolymer, i.e, mPEG b-(CB-{g-cationic chain; g-Chol; g-Morph}
) for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The polymer was utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes were able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ∼98% and showed a size and zeta potential of ∼141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes showed a transfection efficiency of ∼69% in ARPE-19 cells and showed ∼22% of indel frequency indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they were found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes could be a potential carrier for CRISPR plasmids for genome editing applications. This article is protected by copyright. All rights reserved.},
}
@article {pmid37186287,
year = {2023},
author = {Luelf, UJ and Böhmer, LM and Li, S and Urlacher, VB},
title = {Effect of chromosomal integration on catalytic performance of a multi-component P450 system in Escherichia coli.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28404},
pmid = {37186287},
issn = {1097-0290},
abstract = {Cytochromes P450 are useful biocatalysts in synthetic chemistry and important bio-bricks in synthetic biology. Almost all bacterial P450s require separate redox partners for their activity, which are often expressed in recombinant Escherichia coli using multiple plasmids. However, the application of CRISPR/Cas recombineering facilitated chromosomal integration of heterologous genes which enables more stable and tunable expression of multi-component P450 systems for whole-cell biotransformations. Herein, we compared three E. coli strains W3110, JM109, and BL21(DE3) harboring three heterologous genes encoding a P450 and two redox partners either on plasmids or after chromosomal integration in two genomic loci. Both loci proved to be reliable and comparable for the model regio- and stereoselective two-step oxidation of (S)-ketamine. Furthermore, the CRISPR/Cas-assisted integration of the T7 RNA polymerase gene enabled an easy extension of T7 expression strains. Higher titers of soluble active P450 were achieved in E. coli harboring a single chromosomal copy of the P450 gene compared to E. coli carrying a medium copy pET plasmid. In addition, improved expression of both redox partners after chromosomal integration resulted in up to 80% higher (S)-ketamine conversion and more than fourfold increase in total turnover numbers.},
}
@article {pmid37186230,
year = {2023},
author = {Scrascia, M and Roberto, R and D'Addabbo, P and Ahmed, Y and Porcelli, F and Oliva, M and Calia, C and Marzella, A and Pazzani, C},
title = {Bioinformatic survey of CRISPR loci across 15 Serratia species.},
journal = {MicrobiologyOpen},
volume = {12},
number = {2},
pages = {e1339},
pmid = {37186230},
issn = {2045-8827},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) system of prokaryotes is an adaptative immune defense mechanism to protect themselves from invading genetic elements (e.g., phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order of Enterobacterales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotica, and ureilytica. Apart from subtypes I-E and I-F1 which had previously been identified in marcescens, we report that of I-C and the I-E unique locus 1, I-E*, and I-F1 unique locus 1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. The plasmid and/or phage origin of spacers was also established.},
}
@article {pmid37184565,
year = {2023},
author = {Ronai, I},
title = {How molecular techniques are developed from natural systems.},
journal = {Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/genetics/iyad067},
pmid = {37184565},
issn = {1943-2631},
abstract = {A striking characteristic of the molecular techniques of genetics is that they are derived from natural occurring systems. RNA interference, for example, utilizes a mechanism that evolved in eukaryotes to destroy foreign nucleic acid. Other case studies I highlight are restriction enzymes, DNA sequencing, polymerase chain reaction, gene targeting, fluorescent proteins (such as, green fluorescent protein), induced pluripotent stem cells, and clustered regularly interspaced short palindromic repeats-CRISPR associated 9. The natural systems' strategy for technique development means that biologists utilize the activity of a mechanism's effector (protein or RNA) and exploit biological specificity (protein or nucleic acid can cause precise reactions). I also argue that the developmental trajectory of novel molecular techniques, such as RNA interference, has 4 characteristic phases. The first phase is discovery of a biological phenomenon. The second phase is identification of the biological mechanism's trigger(s): the effector and biological specificity. The third phase is the application of the trigger(s) as a technique. The final phase is the maturation and refinement of the technique. Developing new molecular techniques from nature is crucial for future genetic research.},
}
@article {pmid37183261,
year = {2023},
author = {Mijiti, M and Maimaiti, A and Chen, X and Tuersun, M and Dilixiati, M and Dilixiati, Y and Zhu, G and Wu, H and Li, Y and Turhon, M and Abulaiti, A and Maimaitiaili, N and Yiming, N and Kasimu, M and Wang, Y},
title = {CRISPR-cas9 screening identified lethal genes enriched in Hippo kinase pathway and of predictive significance in primary low-grade glioma.},
journal = {Molecular medicine (Cambridge, Mass.)},
volume = {29},
number = {1},
pages = {64},
pmid = {37183261},
issn = {1528-3658},
mesh = {Humans ; Hippo Signaling Pathway ; CRISPR-Cas Systems/genetics ; Genes, Lethal ; *Glioma/genetics ; Oncogenes ; *Brain Neoplasms/genetics ; },
abstract = {BACKGROUND: Low-grade gliomas (LGG) are a type of brain tumor that can be lethal, and it is essential to identify genes that are correlated with patient prognosis. In this study, we aimed to use CRISPR-cas9 screening data to identify key signaling pathways and develop a genetic signature associated with high-risk, low-grade glioma patients.<br><br>METHODS: The study used CRISPR-cas9 screening data to identify essential genes correlated with cell survival in LGG. We used RNA-seq data to identify differentially expressed genes (DEGs) related to cell viability. Moreover, we used the least absolute shrinkage and selection operator (LASSO) method to construct a genetic signature for predicting overall survival in patients. We performed enrichment analysis to identify pathways mediated by DEGs, overlapping genes, and genes shared in the Weighted correlation network analysis (WGCNA). Finally, the study used western blot, qRT-PCR, and IHC to detect the expression of hub genes from signature in clinical samples.<br><br>RESULTS: The study identified 145 overexpressed oncogenes in low-grade gliomas using the TCGA database. These genes were intersected with lethal genes identified in the CRISPR-cas9 screening data from Depmap database, which are enriched in Hippo pathways. A total of 19 genes were used to construct a genetic signature, and the Hippo signaling pathway was found to be the predominantly enriched pathway. The signature effectively distinguished between low- and high-risk patients, with high-risk patients showing a shorter overall survival duration. Differences in hub gene expression were found in different clinical samples, with the protein and mRNA expression of REP65 being significantly up-regulated in tumor cells. The study suggests that the Hippo signaling pathway may be a critical regulator of viability and tumor proliferation and therefore is an innovative new target for treating cancerous brain tumors, including low-grade gliomas.<br><br>CONCLUSION: Our study identified a novel genetic signature associated with high-risk, LGG patients. We found that the Hippo signaling pathway was significantly enriched in this signature, indicating that it may be a critical regulator of tumor viability and proliferation in LGG. Targeting the Hippo pathway could be an innovative new strategy for treating LGG.},
}
@article {pmid36948241,
year = {2023},
author = {Pham, NN and Chang, CW and Chang, YH and Tu, Y and Chou, JY and Wang, HY and Hu, YC},
title = {Rational genome and metabolic engineering of Candida viswanathii by split CRISPR to produce hundred grams of dodecanedioic acid.},
journal = {Metabolic engineering},
volume = {77},
number = {},
pages = {76-88},
doi = {10.1016/j.ymben.2023.03.007},
pmid = {36948241},
issn = {1096-7184},
mesh = {*Metabolic Engineering ; *Candida/genetics/metabolism ; Dicarboxylic Acids/metabolism ; CRISPR-Cas Systems ; },
abstract = {Candida viswanathii is a promising cell factory for producing dodecanedioic acid (DDA) and other long chain dicarboxylic acids. However, metabolic engineering of C. viswanathii is difficult partly due to the lack of synthetic biology toolkits. Here we developed CRISPR-based approaches for rational genome and metabolic engineering of C. viswanathii. We first optimized the CRISPR system and protocol to promote the homozygous gene integration efficiency to >60%. We also designed a split CRISPR system for one-step integration of multiple genes into C. viswanathii. We uncovered that co-expression of CYP52A19, CPRb and FAO2 that catalyze different steps in the biotransformation enhances DDA production and abolishes accumulation of intermediates. We also unveiled that co-expression of additional enzyme POS5 further promotes DDA production and augments cell growth. We harnessed the split CRISPR system to co-integrate these 4 genes (13.6 kb) into C. viswanathii and generated a stable strain that doubles the DDA titer (224 g/L), molar conversion (83%) and productivity (1.87 g/L/h) when compared with the parent strain. This study altogether identifies appropriate enzymes/promoters to augment dodecane conversion to DDA and implicates the potential of split CRISPR system for metabolic engineering of C. viswanathii.},
}
@article {pmid37183363,
year = {2023},
author = {Comunanza, V and Gigliotti, C and Lamba, S and Doronzo, G and Vallariello, E and Martin, V and Isella, C and Medico, E and Bardelli, A and Sangiolo, D and Di Nicolantonio, F and Bussolino, F},
title = {Dual VEGFA/BRAF targeting boosts PD-1 blockade in melanoma through GM-CSF-mediated infiltration of M1 macrophages.},
journal = {Molecular oncology},
volume = {},
number = {},
pages = {},
doi = {10.1002/1878-0261.13450},
pmid = {37183363},
issn = {1878-0261},
abstract = {The introduction of targeted therapies represented one of the most significant advances in the treatment of BRAFV600E melanoma. However, the onset of acquired resistance remains a challenge. Previously, we showed in mouse xenografts that vascular endothelial growth factor (VEGFA) removal enhanced the antitumor effect of BRAF inhibition through the recruitment of M1 macrophages. In this work, we explored the strategy of VEGFA/BRAF inhibition in immunocompetent melanoma murine models. In BRAF mutant D4M melanoma tumors, VEGFA/BRAF targeting reshaped the tumor microenvironment, largely by stimulating infiltration of M1 macrophages and CD8[+] T cells, and sensitized tumors to immune checkpoint blockade (ICB). Further, we reported that the association of VEGFA/BRAF targeting with anti-PD-1 antibody (triple therapy) resulted in a durable response and enabled complete tumor eradication in 50% of the mice, establishing immunological memory. Neutralization and CRISPR-Cas-mediated editing of granulocyte-macrophage colony-stimulating factor (GM-CSF) abrogated anti-tumor response prompted by triple therapy and identified GM-CSF as the cytokine instrumental in M1-macrophage recruitment. Our data suggest that VEGFA/BRAF targeting in melanoma induces the activation of innate and adaptive immunity and prepares tumors for ICB. Our study contributes to understanding the tumor biology of BRAFV600E melanoma, and suggests VEGFA as therapeutic target.},
}
@article {pmid37182461,
year = {2023},
author = {Li, L and Hong, F and Pan, S and Ren, L and Xiao, R and Liu, P and Li, N and Wang, J and Chen, Y},
title = {"Lollipop" particle counting immunoassay based on antigen-powered CRISPR-Cas12a dual signal amplification for the sensitive detection of deoxynivalenol in the environment and food samples.},
journal = {Journal of hazardous materials},
volume = {455},
number = {},
pages = {131573},
doi = {10.1016/j.jhazmat.2023.131573},
pmid = {37182461},
issn = {1873-3336},
abstract = {Deoxynivalenol is one of the most widely distributed mycotoxins in cereals and poses tremendous threats to the agricultural environment and public health. Therefore, it is particularly important to develop sensitive and interference-resistant deoxynivalenol analysis methods. Here, we establish a "Lollipop" particle counting immunoassay (LPCI) based on antigen-powered CRISPR-Cas12a dual signal amplification. LPCI achieves high sensitivity and accuracy through antigen-powered CRISPR-Cas dual signal amplification combined with particle counting immunoassay. This strategy not only broadens the applicability of the CRISPR-Cas system in the field of non-nucleic acid target detection; it also improves the sensitivity of particle counting immunoassay. The introduction of a polystyrene "lollipop" immunoassay carrier further enables efficiently simultaneous pre-treatment of multiple samples and overcomes complex matrix interference in real samples. The linear detection range of LPCI for deoxynivalenol was 0.1-500 ng/mL with a detection limit of 0.061 ng/mL. The platform greatly broadens the scope of the CRISPR-Cas sensor for the detection of non-nucleic acid hazards in the environment and food samples.},
}
@article {pmid37179485,
year = {2023},
author = {Thevendran, R and Maheswaran, S},
title = {Recognizing CRISPR as the New Age Disease-Modifying Drug: Strategies to Bioengineer CRISPR/Cas for Direct in vivo Delivery.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2300077},
doi = {10.1002/biot.202300077},
pmid = {37179485},
issn = {1860-7314},
abstract = {CRISPR has established itself as a frontier technology in genetic engineering. Researchers have successfully used the CRISPR/Cas system as precise gene editing tools and have further expanded their scope beyond both imaging and diagnostic applications. The most prominent utility of CRISPR is its capacity for gene therapy, serving as the contemporary, disease-modifying drug at the genetic level of human medical disorders. Correcting these diseases using CRISPR-based gene editing has developed to the extent of preclinical trials and possible patient treatments. A major impediment in actualizing this is the complications associated with in vivo delivery of the CRISPR/Cas complex. Currently, only the viral vectors (e.g. lentivirus) and non-viral encapsulation (e.g. lipid particles, polymer-based and gold nanoparticles) techniques have been extensively reviewed, neglecting the efficiency of direct delivery. However, the direct delivery of CRISPR/Cas for in vivo gene editing therapies is an intricate process with numerous drawbacks. Hence, this paper discusses in detail both the need and the strategies that can potentially improve the direct delivery aspects of CRISPR/Cas biomolecules for gene therapy of human diseases. Here, we focus on enhancing the molecular and functional features of the CRISPR/Cas system for targeted in vivo delivery such as on-site localization, internalization, reduced immunogenicity, and better in vivo stability. We additionally emphasize the CRISPR/Cas complex as a multifaceted, biomolecular vehicle for co-delivery with therapeutic agents in targeted disease treatments. The delivery formats of efficient CRISPR/Cas systems for human gene editing are also briefly elaborated. This article is protected by copyright. All rights reserved.},
}
@article {pmid37176969,
year = {2023},
author = {Ijaz, M and Khan, F and Zaki, HEM and Khan, MM and Radwan, KSA and Jiang, Y and Qian, J and Ahmed, T and Shahid, MS and Luo, J and Li, B},
title = {Recent Trends and Advancements in CRISPR-Based Tools for Enhancing Resistance against Plant Pathogens.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {9},
pages = {},
pmid = {37176969},
issn = {2223-7747},
abstract = {Targeted genome editing technologies are becoming the most important and widely used genetic tools in studies of phytopathology. The "clustered regularly interspaced short palindromic repeats (CRISPR)" and its accompanying proteins (Cas) have been first identified as a natural system associated with the adaptive immunity of prokaryotes that have been successfully used in various genome-editing techniques because of its flexibility, simplicity, and high efficiency in recent years. In this review, we have provided a general idea about different CRISPR/Cas systems and their uses in phytopathology. This review focuses on the benefits of knock-down technologies for targeting important genes involved in the susceptibility and gaining resistance against viral, bacterial, and fungal pathogens by targeting the negative regulators of defense pathways of hosts in crop plants via different CRISPR/Cas systems. Moreover, the possible strategies to employ CRISPR/Cas system for improving pathogen resistance in plants and studying plant-pathogen interactions have been discussed.},
}
@article {pmid37176948,
year = {2023},
author = {Gajardo, HA and Gómez-Espinoza, O and Boscariol Ferreira, P and Carrer, H and Bravo, LA},
title = {The Potential of CRISPR/Cas Technology to Enhance Crop Performance on Adverse Soil Conditions.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {9},
pages = {},
pmid = {37176948},
issn = {2223-7747},
abstract = {Worldwide food security is under threat in the actual scenery of global climate change because the major staple food crops are not adapted to hostile climatic and soil conditions. Significant efforts have been performed to maintain the actual yield of crops, using traditional breeding and innovative molecular techniques to assist them. However, additional strategies are necessary to achieve the future food demand. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) technology, as well as its variants, have emerged as alternatives to transgenic plant breeding. This novelty has helped to accelerate the necessary modifications in major crops to confront the impact of abiotic stress on agriculture systems. This review summarizes the current advances in CRISPR/Cas applications in crops to deal with the main hostile soil conditions, such as drought, flooding and waterlogging, salinity, heavy metals, and nutrient deficiencies. In addition, the potential of extremophytes as a reservoir of new molecular mechanisms for abiotic stress tolerance, as well as their orthologue identification and edition in crops, is shown. Moreover, the future challenges and prospects related to CRISPR/Cas technology issues, legal regulations, and customer acceptance will be discussed.},
}
@article {pmid37176822,
year = {2023},
author = {Eckerstorfer, MF and Dolezel, M and Engelhard, M and Giovannelli, V and Grabowski, M and Heissenberger, A and Lener, M and Reichenbecher, W and Simon, S and Staiano, G and Wüst Saucy, AG and Zünd, J and Lüthi, C},
title = {Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {9},
pages = {},
pmid = {37176822},
issn = {2223-7747},
abstract = {The current initiative of the European Commission (EC) concerning plants produced using certain new genomic techniques, in particular, targeted mutagenesis and cisgenesis, underlines that a high level of protection for human and animal health and the environment needs to be maintained when using such applications. The current EU biosafety regulation framework ensures a high level of protection with a mandatory environmental risk assessment (ERA) of genetically modified (GM) products prior to the authorization of individual GMOs for environmental release or marketing. However, the guidance available from the European Food Safety Authority (EFSA) for conducting such an ERA is not specific enough regarding the techniques under discussion and needs to be further developed to support the policy goals towards ERA, i.e., a case-by-case assessment approach proportionate to the respective risks, currently put forward by the EC. This review identifies important elements for the case-by-case approach for the ERA that need to be taken into account in the framework for a risk-oriented regulatory approach. We also discuss that the comparison of genome-edited plants with plants developed using conventional breeding methods should be conducted at the level of a scientific case-by-case assessment of individual applications rather than at a general, technology-based level. Our considerations aim to support the development of further specific guidance for the ERA of genome-edited plants.},
}
@article {pmid37175878,
year = {2023},
author = {Karginov, AV and Tarutina, MG and Lapteva, AR and Pakhomova, MD and Galliamov, AA and Filkin, SY and Fedorov, AN and Agaphonov, MO},
title = {A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts.},
journal = {International journal of molecular sciences},
volume = {24},
number = {9},
pages = {},
pmid = {37175878},
issn = {1422-0067},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; },
abstract = {Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.},
}
@article {pmid37175730,
year = {2023},
author = {Sheng, X and Ai, Z and Tan, Y and Hu, Y and Guo, X and Liu, X and Sun, Z and Yu, D and Chen, J and Tang, N and Duan, M and Yuan, D},
title = {Novel Salinity-Tolerant Third-Generation Hybrid Rice Developed via CRISPR/Cas9-Mediated Gene Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {9},
pages = {},
pmid = {37175730},
issn = {1422-0067},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Salinity ; Plant Breeding/methods ; },
abstract = {Climate change has caused high salinity in many fields, particularly in the mud flats in coastal regions. The resulting salinity has become one of the most significant abiotic stresses affecting the world's rice crop productivity. Developing elite cultivars with novel salinity-tolerance traits is regarded as the most cost-effective and environmentally friendly approach for utilizing saline-alkali land. To develop a highly efficient green strategy and create novel rice germplasms for salt-tolerant rice breeding, this study aimed to improve rice salinity tolerance by combining targeted CRISPR/Cas9-mediated editing of the OsRR22 gene with heterosis utilization. The novel alleles of the genic male-sterility (GMS) and elite restorer line (733S[rr22]-T1447-1 and HZ[rr22]-T1349-3) produced 110 and 1 bp deletions at the third exon of OsRR22 and conferred a high level of salinity tolerance. Homozygous transgene-free progeny were identified via segregation in the T2 generation, with osrr22 showing similar agronomic performance to wild-type (733S and HZ). Furthermore, these two osrr22 lines were used to develop a new promising third-generation hybrid rice line with novel salinity tolerance. Overall, the results demonstrate that combining CRISPR/Cas9 targeted gene editing with the "third-generation hybrid rice system" approach allows for the efficient development of novel hybrid rice varieties that exhibit a high level of salinity tolerance, thereby ensuring improved cultivar stability and enhanced rice productivity.},
}
@article {pmid37175696,
year = {2023},
author = {Muto, V and Benigni, F and Magliocca, V and Borghi, R and Flex, E and Pallottini, V and Rosa, A and Compagnucci, C and Tartaglia, M},
title = {CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.},
journal = {International journal of molecular sciences},
volume = {24},
number = {9},
pages = {},
pmid = {37175696},
issn = {1422-0067},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells ; Endophenotypes ; Cell Differentiation/genetics ; Gene Editing ; Mutation ; Microtubule-Associated Proteins/metabolism ; },
abstract = {Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.},
}
@article {pmid37174639,
year = {2023},
author = {Lu, J and Ding, Y and Zhang, W and Qi, Y and Zhou, J and Xu, N and Zhang, Y and Xie, W},
title = {SQSTM1/p62 Knockout by Using the CRISPR/Cas9 System Inhibits Migration and Invasion of Hepatocellular Carcinoma.},
journal = {Cells},
volume = {12},
number = {9},
pages = {},
pmid = {37174639},
issn = {2073-4409},
mesh = {Animals ; Mice ; *Carcinoma, Hepatocellular/genetics/metabolism ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Sequestosome-1 Protein/genetics/metabolism ; NF-E2-Related Factor 2/genetics/metabolism ; Mice, Nude ; CRISPR-Cas Systems/genetics ; *Liver Neoplasms/genetics/metabolism ; Mice, Knockout ; Tumor Microenvironment ; },
abstract = {Migration and invasion play crucial roles in the progression of hepatocellular carcinoma (HCC), but the underlying mechanisms are not clear. Analysis of clinical samples indicates that SQSTM1/p62 is highly expressed in HCC and seriously affects the prognosis of patients. Subsequently, we showed that SQSTM1/p62 knockout using the CRISPR/Cas9 system led to impaired migration and invasion of HCC, upregulated Keap1, and promoted the inhibitory effect of Keap1 on Nrf2. Then, the inactivation of Nrf2 inhibited the expression of matrix metalloproteinases (MMPs), thus attenuating the migration and invasion of HCC. We also found that SQSTM1/p62 knockout significantly inhibited migration and invasion in a lung metastasis model of nude mice with HCC. Furthermore, we found that cisplatin not only significantly inhibited the expression of SQSTM1/p62 but also slowed down the migration and invasion of HCC, while the inflammatory microenvironment accelerated the migration and invasion of HCC. These results suggest for the first time that SQSTM1/p62 knockout inhibits the migration and invasion of HCC through the Keap1/Nrf2/MMP2 signaling pathway. SQSTM1/p62 may be developed into a key drug target to regulate the migration and invasion of HCC cells.},
}
@article {pmid37173533,
year = {2023},
author = {Perk, EA and Arruebarrena Di Palma, A and Colman, S and Mariani, O and Cerrudo, I and D'Ambrosio, JM and Robuschi, L and Pombo, MA and Rosli, HG and Villareal, F and Laxalt, AM},
title = {CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants are more resistant to Botrytis cinerea.},
journal = {Planta},
volume = {257},
number = {6},
pages = {117},
pmid = {37173533},
issn = {1432-2048},
mesh = {*Type C Phospholipases/metabolism ; *Solanum lycopersicum/genetics ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Oxylipins/metabolism ; Plant Breeding ; Botrytis/metabolism ; Phospholipases/genetics/metabolism ; Plant Diseases/genetics/microbiology ; Disease Resistance/genetics ; Gene Expression Regulation, Plant ; },
abstract = {CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.},
}
@article {pmid37172090,
year = {2023},
author = {Hany, D and Vafeiadou, V and Picard, D},
title = {CRISPR-Cas9 screen reveals a role of purine synthesis for estrogen receptor α activity and tamoxifen resistance of breast cancer cells.},
journal = {Science advances},
volume = {9},
number = {19},
pages = {eadd3685},
pmid = {37172090},
issn = {2375-2548},
mesh = {Humans ; Female ; *Tamoxifen/pharmacology ; *Breast Neoplasms/drug therapy/genetics/metabolism ; Estrogen Receptor alpha/genetics/metabolism ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Estrogens ; Purines/pharmacology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; },
abstract = {In breast cancer, resistance to endocrine therapies that target estrogen receptor α (ERα), such as tamoxifen and fulvestrant, remains a major clinical problem. Whether and how ERα[+] breast cancers switch from being estrogen-dependent to estrogen-independent remains unclear. With a genome-wide CRISPR-Cas9 knockout screen, we identified previously unknown biomarkers and potential therapeutic targets of endocrine resistance. We demonstrate that high levels of PAICS, an enzyme involved in the de novo biosynthesis of purines, can shift the balance of ERα activity to be more estrogen-independent and tamoxifen-resistant. We find that this may be due to elevated activities of cAMP-activated protein kinase A and mTOR, kinases known to phosphorylate ERα specifically and to stimulate its activity. Genetic or pharmacological targeting of PAICS sensitizes tamoxifen-resistant cells to tamoxifen. Addition of purines renders them more resistant. On the basis of these findings, we propose the combined targeting of PAICS and ERα as a new, effective, and potentially safe therapeutic regimen.},
}
@article {pmid37172086,
year = {2023},
author = {Bu, W and Creighton, CJ and Heavener, KS and Gutierrez, C and Dou, Y and Ku, AT and Zhang, Y and Jiang, W and Urrutia, J and Jiang, W and Yue, F and Jia, L and Ibrahim, AA and Zhang, B and Huang, S and Li, Y},
title = {Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice.},
journal = {Science advances},
volume = {9},
number = {19},
pages = {eade0059},
pmid = {37172086},
issn = {2375-2548},
mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; Recombinational DNA Repair ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.},
}
@article {pmid37170124,
year = {2023},
author = {Karapurkar, JK and Kim, MS and Colaco, JC and Suresh, B and Sarodaya, N and Kim, DH and Park, CH and Hong, SH and Kim, KS and Ramakrishna, S},
title = {CRISPR/Cas9-based genome-wide screening of the deubiquitinase subfamily identifies USP3 as a protein stabilizer of REST blocking neuronal differentiation and promotes neuroblastoma tumorigenesis.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {42},
number = {1},
pages = {121},
pmid = {37170124},
issn = {1756-9966},
mesh = {Humans ; Animals ; Mice ; *Transcription Factors/metabolism ; CRISPR-Cas Systems ; Ubiquitination ; Ubiquitin-Specific Proteases/metabolism ; Ubiquitin-Protein Ligases/metabolism ; *Neuroblastoma/genetics ; Cell Transformation, Neoplastic/genetics ; Cell Differentiation/genetics ; },
abstract = {BACKGROUND: The repressor element-1 silencing transcription factor (REST), a master transcriptional repressor, is essential for maintenance, self-renewal, and differentiation in neuroblastoma. An elevated expression of REST is associated with impaired neuronal differentiation, which results in aggressive neuroblastoma formation. E3 ligases are known to regulate REST protein abundance through the 26 S proteasomal degradation pathway in neuroblastoma. However, deubiquitinating enzymes (DUBs), which counteract the function of E3 ligase-mediated REST protein degradation and their impact on neuroblastoma tumorigenesis have remained unexplored.<br><br>METHODS: We employed a CRISPR/Cas9 system to perform a genome-wide knockout of ubiquitin-specific proteases (USPs) and used western blot analysis to screen for DUBs that regulate REST protein abundance. The interaction between USP3 and REST was confirmed by immunoprecipitation and Duolink in situ proximity assays. The deubiquitinating effect of USP3 on REST protein degradation, half-life, and neuronal differentiation was validated by immunoprecipitation, in vitro deubiquitination, protein-turnover, and immunostaining assays. The correlation between USP3 and REST expression was assessed using patient neuroblastoma datasets. The USP3 gene knockout in neuroblastoma cells was performed using CRISPR/Cas9, and the clinical relevance of USP3 regulating REST-mediated neuroblastoma tumorigenesis was confirmed by in vitro and in vivo oncogenic experiments.<br><br>RESULTS: We identified a deubiquitinase USP3 that interacts with, stabilizes, and increases the half-life of REST protein by counteracting its ubiquitination in neuroblastoma. An in silico analysis showed a correlation between USP3 and REST in multiple neuroblastoma cell lines and identified USP3 as a prognostic marker for overall survival in neuroblastoma patients. Silencing of USP3 led to a decreased self-renewal capacity and promoted retinoic acid-induced differentiation in neuroblastoma. A loss of USP3 led to attenuation of REST-mediated neuroblastoma tumorigenesis in a mouse xenograft model.<br><br>CONCLUSION: The findings of this study indicate that USP3 is a critical factor that blocks neuronal differentiation, which can lead to neuroblastoma. We envision that targeting USP3 in neuroblastoma tumors might provide an effective therapeutic differentiation strategy for improved survival rates of neuroblastoma patients.},
}
@article {pmid37166670,
year = {2023},
author = {Cheng, S and Park, M and Yong, J},
title = {RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of Genome Engineering and Stable Transfection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2666},
number = {},
pages = {247-263},
pmid = {37166670},
issn = {1940-6029},
mesh = {*RNA/genetics ; Epitopes/genetics ; *RNA-Binding Proteins/metabolism ; Transfection ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {To study the function of RNA-binding proteins (RBPs), an overexpression or knockout approach is generally used. However, as many RBPs are essential to cellular functions, the complete knockout of these proteins may be lethal to the cell. Overexpression of RBPs, on the other hand, may create an altered transcriptome and aberrant phenotypes that can mask their physiological function. Additionally, biochemical characterization of RBP often requires highly specific antibodies for efficient immunoprecipitation for downstream mass spectrometry or RNA footprinting profiling. To overcome these hurdles, we have developed a strategy to generate cellular systems either using a CRISPR-Cas9-mediated epitope tag knock-in approach or a two-step workflow to first stably express an exogenous Flag-tagged RBP and subsequently knockout the endogenous RBP using CRISPR-Cas9 gene editing. The generation of these cell lines will be beneficial for downstream RNA footprinting studies and mass spectrometry-mediated interactome studies.},
}
@article {pmid37166646,
year = {2023},
author = {Blanco-Fernandez, J and Jourdain, AA},
title = {Dead-Seq: Discovering Synthetic Lethal Interactions from Dead Cells Genomics.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2661},
number = {},
pages = {329-342},
pmid = {37166646},
issn = {1940-6029},
mesh = {*Genomics/methods ; *Genome ; Genetic Testing/methods ; CRISPR-Cas Systems ; },
abstract = {Pooled genetic screens have revolutionized the field of functional genomics, yet perturbations that decrease fitness, such as those leading to synthetic lethality, have remained difficult to quantify at the genomic level. We and colleagues previously developed "death screening," a protocol based on the purification of dead cells in genetic screens, and used it to identify a set of genes necessary for mitochondrial gene expression, translation, and oxidative phosphorylation (OXPHOS), thus offering new possibilities for the diagnosis of mitochondrial disorders. Here, we describe Dead-Seq, a refined protocol for death screening that is compatible with most pooled screening protocols, including genome-wide CRISPR/Cas9 screening. Dead-Seq converts negative-selection screens into positive-selection screens and generates high-quality data directly from dead cells, at limited sequencing costs.},
}
@article {pmid37141850,
year = {2023},
author = {Chen, X and Moran Torres, JP and Li, Y and Lugones, LG and Wösten, HAB},
title = {Inheritable CRISPR based epigenetic modification in a fungus.},
journal = {Microbiological research},
volume = {272},
number = {},
pages = {127397},
doi = {10.1016/j.micres.2023.127397},
pmid = {37141850},
issn = {1618-0623},
mesh = {Humans ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; DNA Methylation ; Gene Silencing ; Fungi/genetics ; Gene Editing/methods ; Mixed Function Oxygenases/genetics ; Proto-Oncogene Proteins/genetics ; },
abstract = {The CRISPRoff system was recently introduced as a programmable epigenetic memory writer that can be used to silence genes in human cells. The system makes use of a dead Cas9 protein (dCas9) that is fused with the ZNF10 KRAB, Dnmt3A, and Dnmt3L protein domains. The DNA methylation resulting from the CRISPRoff system can be removed by the CRISPRon system that consists of dCas9 fused to the catalytic domain of Tet1. Here, the CRISPRoff and CRISPRon systems were applied for the first time in a fungus. The CRISPRoff system resulted in an inactivation up to 100 % of the target genes flbA and GFP in Aspergillus niger. Phenotypes correlated with the degree of gene silencing in the transformants and were stable when going through a conidiation cycle, even when the CRISPRoff plasmid was removed from the flbA silenced strain. Introducing the CRISPRon system in a strain in which the CRISPRoff plasmid was removed fully reactivated flbA showing a phenotype similar to that of the wildtype. Together, the CRISPRoff and CRISPRon systems can be used to study gene function in A. niger.},
}
@article {pmid37119812,
year = {2023},
author = {Yu, G and Kim, HK and Park, J and Kwak, H and Cheong, Y and Kim, D and Kim, J and Kim, J and Kim, HH},
title = {Prediction of efficiencies for diverse prime editing systems in multiple cell types.},
journal = {Cell},
volume = {186},
number = {10},
pages = {2256-2272.e23},
doi = {10.1016/j.cell.2023.03.034},
pmid = {37119812},
issn = {1097-4172},
mesh = {*Knowledge ; *Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.},
}
@article {pmid37116617,
year = {2023},
author = {Wang, G and Wang, C and Chu, T and Wu, X and Anderson, CM and Huang, D and Li, J},
title = {Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency.},
journal = {Journal of biotechnology},
volume = {368},
number = {},
pages = {42-52},
doi = {10.1016/j.jbiotec.2023.04.008},
pmid = {37116617},
issn = {1873-4863},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; DNA/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an "enhanced specificity" SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.},
}
@article {pmid36864663,
year = {2023},
author = {Rojek, JB and Basavaraju, Y and Nallapareddy, S and Bulté, DB and Baumgartner, R and Schoffelen, S and Grav, LM and Goletz, S and Pedersen, LE},
title = {Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing.},
journal = {Biotechnology and bioengineering},
volume = {120},
number = {6},
pages = {1478-1491},
doi = {10.1002/bit.28367},
pmid = {36864663},
issn = {1097-0290},
mesh = {Cricetinae ; Animals ; *Gene Editing ; Cricetulus ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Endonucleases/genetics ; },
abstract = {The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.},
}
@article {pmid36762520,
year = {2023},
author = {Navaridas, R and Vidal-Sabanés, M and Ruiz-Mitjana, A and Perramon-Güell, A and Megino-Luque, C and Llobet-Navas, D and Matias-Guiu, X and Egea, J and Encinas, M and Bardia, L and Colombelli, J and Dolcet, X},
title = {Transient and DNA-free in vivo CRISPR/Cas9 genome editing for flexible modeling of endometrial carcinogenesis.},
journal = {Cancer communications (London, England)},
volume = {43},
number = {5},
pages = {620-624},
pmid = {36762520},
issn = {2523-3548},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; DNA ; },
}
@article {pmid36734182,
year = {2023},
author = {Cao, Y and Li, L and Ren, X and Mao, B and Yang, Y and Mi, H and Guan, Y and Li, S and Zhou, S and Guan, X and Yang, T and Zhao, X},
title = {CRISPR/Cas9 correction of a dominant cis-double-variant in COL1A1 isolated from a patient with osteogenesis imperfecta increases the osteogenic capacity of induced pluripotent stem cells.},
journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research},
volume = {38},
number = {5},
pages = {719-732},
doi = {10.1002/jbmr.4783},
pmid = {36734182},
issn = {1523-4681},
mesh = {Humans ; *Osteogenesis Imperfecta/genetics/therapy/pathology ; *Induced Pluripotent Stem Cells/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; Collagen Type I/genetics ; },
abstract = {Osteogenesis imperfecta (OI) is a hereditary skeletal disorder that is mainly caused by variants in COL1A1/2. So far, no specific treatment has been developed to correct its underlying etiology. We aimed to gain a better understanding of the pathological mechanisms of OI and develop gene therapies to correct OI-causing variants. A de novel cis-double-variant c.[175C>T; 187T>A] in COL1A1 was identified from a 5-year-old OI patient by whole-exome sequencing (WES). Three peptide nucleic acids (PNAs) were designed and then transfected patient-derived fibroblasts. PNA2 affected the translational strand and induced an optimal interfering effect at 0.25μM concentration, proved by Sanger sequencing, qPCR, Western blot, and immunostaining. Additionally, induced pluripotent stem cells (iPSCs) were cultured from patient-derived fibroblasts. Clones of iPSCs with c.187T>A variant and those with both variants largely restored their osteogenic capacities after CRISPR/Cas9 gene editing, which corrected the variants. Importantly, correcting c.187T>A variant alone in CRISPR-edited iPSCs was sufficient to alleviate OI phenotypes, as indicated by increased levels of COL1A1, COL1A2, ALP mRNAs, and COL1A1 protein. Our findings suggest that c.187T>A is the dominant variant of cis-double-variant in COL1A1 that led to OI, and PNA interference and CRISPR/Cas9 gene editing may be new therapeutic tools for OI treatment. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).},
}
@article {pmid36658005,
year = {2023},
author = {Strobbe, S and Wesana, J and Van Der Straeten, D and De Steur, H},
title = {Public acceptance and stakeholder views of gene edited foods: a global overview.},
journal = {Trends in biotechnology},
volume = {41},
number = {6},
pages = {736-740},
doi = {10.1016/j.tibtech.2022.12.011},
pmid = {36658005},
issn = {1879-3096},
mesh = {*Gene Editing ; Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {The increasing popularity of gene editing in plants has prompted research on stakeholder views. Gene edited foods are often more accepted than genetically modified foods, though differences occur within target groups, regions, and products. Nevertheless, marketing challenges related to a lack of familiarity with the technology, labeling, and risk perception remain.},
}
@article {pmid37175977,
year = {2023},
author = {Wettengel, JM and Hansen-Palmus, L and Yusova, S and Rust, L and Biswas, S and Carson, J and Ryu, J and Bimber, BN and Hennebold, JD and Burwitz, BJ},
title = {A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {9},
pages = {},
pmid = {37175977},
issn = {1422-0067},
support = {P51 OD011092/NH/NIH HHS/United States ; U24 OD026631-01/NH/NIH HHS/United States ; R21 AI108401-01/NH/NIH HHS/United States ; },
abstract = {CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.},
}
@article {pmid37175850,
year = {2023},
author = {Glinšek, K and Bozovičar, K and Bratkovič, T},
title = {CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering.},
journal = {International journal of molecular sciences},
volume = {24},
number = {9},
pages = {},
pmid = {37175850},
issn = {1422-0067},
abstract = {The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.},
}
@article {pmid37174743,
year = {2023},
author = {Melo-Silva, CR and Knudson, CJ and Tang, L and Kafle, S and Springer, LE and Choi, J and Snyder, CM and Wang, Y and Kim, SV and Sigal, LJ},
title = {Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.},
journal = {Cells},
volume = {12},
number = {9},
pages = {},
doi = {10.3390/cells12091343},
pmid = {37174743},
issn = {2073-4409},
abstract = {Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.},
}
@article {pmid37172271,
year = {2023},
author = {Fang, T and Zhang, L and Ding, W and Liu, Y and Li, P and Wang, W and Xiang, W and Wang, B and Sun, W},
title = {Point-of-Care Testing for Norovirus Typing Using CRISPR/Cas12a Combined with Reverse Transcription Recombinase Polymerase Amplification.},
journal = {Bioconjugate chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.bioconjchem.3c00181},
pmid = {37172271},
issn = {1520-4812},
abstract = {Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis in humans. This study combined reverse transcription recombinase polymerase amplification (RT-RPA) with a clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) nucleic acid detection system to develop a point-of-care testing (POCT) technology for typing NoVs. The detection can be completed within 35 min at 37 °C, covering each genotype of genogroup I (GI) and II (GII) NoVs. The sensitivity of this method is 10 copies/μL for GI and 1 copy/μL for GII NoV plasmids. For the detection of clinical samples, the detection results of this method for NoV infected samples are consistent with the RT-qPCR detection method in the laboratory, and this detection method has no cross-reactivity with rotavirus and adenovirus. Therefore, the detection method established in this study enables the diagnosis and screening of suspected patients and close contacts by POCT, which is important for the timely identification and control of NoV outbreaks. In addition, the typing detection of GI and GII NoVs can achieve a precise diagnosis and treatment of patients infected with NoVs.},
}
@article {pmid37170319,
year = {2023},
author = {Fu, L and Ma, E and Okada, M and Shibata, Y and Shi, YB},
title = {Competitive PCR with dual fluorescent primers enhances the specificity and reproducibility of genotyping animals generated from genome editing.},
journal = {Cell &amp; bioscience},
volume = {13},
number = {1},
pages = {83},
pmid = {37170319},
issn = {2045-3701},
abstract = {Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, leading to DNA repair through homologous recombination or non-homologous end joining (NHEJ). DNA repair through NHEJ mechanism is an error-prone process that often results in point mutations or stretches of indels (insertions and deletions) within the targeted region. Such mutations in embryos are germline transmissible, thus providing an easy means to generate organisms with gene mutations. However, point mutations and short indels present difficulty for genotyping, often requiring labor intensive sequencing to obtain reliable results. Here, we developed a single-tube competitive PCR assay with dual fluorescent primers that allowed simple and reliable genotyping. While we used Xenopus tropicalis as a model organism, the approach should be applicable to genotyping of any organisms.},
}
@article {pmid37169661,
year = {2023},
author = {Hołubowicz, R and Palczewski, K},
title = {Saving eyesight, one gene at a time.},
journal = {Trends in molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molmed.2023.04.008},
pmid = {37169661},
issn = {1471-499X},
abstract = {Kai Yao's group used prime editing to repair a blindness-causing mutation in the Pde6b gene in the mouse retina. This breakthrough was made possible by a Cas9 nickase that is not constrained by a protospacer adjacent motif (PAM) sequence requirement. This innovation brings prime editing technology one step closer to correcting disease-causing mutations at will.},
}
@article {pmid37168121,
year = {2023},
author = {Tao, S and Zhou, D and Chen, H and Li, N and Zheng, L and Fang, Y and Xu, Y and Jiang, Q and Liang, W},
title = {Analysis of genetic structure and function of clustered regularly interspaced short palindromic repeats loci in 110 Enterococcus strains.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1177841},
pmid = {37168121},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune system involved in specific defenses against the invasion of foreign mobile genetic elements, such as plasmids and phages. This study aims to analyze the gene structure and to explore the function of the CRISPR system in the Enterococcus genome, especially with regard to drug resistance. The whole genome information of 110 enterococci was downloaded from the NCBI database to analyze the distribution and the structure of the CRISPR-Cas system including the Cas gene, repeat sequences, and spacer sequence of the CRISPR-Cas system by bioinformatics methods, and to find drug resistance-related genes and analyze the relationship between them and the CRISPR-Cas system. Multilocus sequence typing (MLST) of enterococci was performed against the reference MLST database. Information on the drug resistance of Enterococcus was retrieved from the CARD database, and its relationship to the presence or absence of CRISPR was statistically analyzed. Among the 110 Enterococcus strains, 39 strains (35.45%) contained a complete CRISPR-Cas system, 87 CRISPR arrays were identified, and 62 strains contained Cas gene clusters. The CRISPR system in the Enterococcus genome was mainly type II-A (59.68%), followed by type II-C (33.87%). The phylogenetic analysis of the cas1 gene sequence was basically consistent with the typing of the CRISPR-Cas system. Of the 74 strains included in the study for MLST typing, only 19 (25.68%) were related to CRISPR-Cas typing, while the majority of the strains (74.32%) of MLST typing were associated with the untyped CRISPR system. Additionally, the CRISPR-Cas system may only be related to the carrying rate of some drug-resistant genes and the drug-resistant phenotype. In conclusion, the distribution of the enterococcus CRISPR-Cas system varies greatly among different species and the presence of CRISPR loci reduces the horizontal transfer of some drug resistance genes.},
}
@article {pmid37166088,
year = {2023},
author = {Ali, A and Zafar, MM and Farooq, Z and Ahmed, SR and Ijaz, A and Anwar, Z and Abbas, H and Tariq, MS and Tariq, H and Mustafa, M and Bajwa, MH and Shaukat, F and Razzaq, A and Maozhi, R},
title = {Breakthrough in CRISPR/Cas system: Current and future directions and challenges.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2200642},
doi = {10.1002/biot.202200642},
pmid = {37166088},
issn = {1860-7314},
abstract = {Targeted genome editing (GE) technology has brought a significant revolution in fictional genomic research and given hope to plant scientists to develop desirable varieties. This technology involves inducing site-specific DNA perturbations that can be repaired through DNA repair pathways. GE products currently include CRISPR-associated nuclease DNA breaks, prime editors generated DNA flaps, single nucleotide-modifications, transposases, and recombinases. The discovery of double-strand breaks, site-specific nucleases, and repair mechanisms paved the way for targeted genome editing, and the first-generation genome editing tools, ZFNs and TALENs, were successfully utilized in plant genome editing. However, CRISPR-Cas has now become the preferred tool for genome editing due to its speed, reliability, and cost-effectiveness. Plant functional genomics has benefited significantly from the widespread use of CRISPR technology for advancements and developments. This review highlights the progress made in CRISPR technology, including multiplex editing, base editing, and prime editing, as well as the challenges and potential delivery mechanisms. This article is protected by copyright. All rights reserved.},
}
@article {pmid37164010,
year = {2023},
author = {Fong, JHC and Chu, HY and Zhou, P and Wong, ASL},
title = {Parallel engineering and activity profiling of a base editor system.},
journal = {Cell systems},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cels.2023.03.007},
pmid = {37164010},
issn = {2405-4720},
abstract = {Selecting the most suitable existing base editors and engineering new variants for installing specific base conversions with maximal efficiency and minimal undesired edits are pivotal for precise genome editing applications. Here, we present a platform for creating and analyzing a library of engineered base editor variants to enable head-to-head evaluation of their editing performance at scale. Our comprehensive comparison provides quantitative measures on each variant's editing efficiency, purity, motif preference, and bias in generating single and multiple base conversions, while uncovering undesired higher indel generation rate and noncanonical base conversion for some of the existing base editors. In addition to engineering the base editor protein, we further applied this platform to investigate a hitherto underexplored engineering route and created guide RNA scaffold variants that augment the editor's base-editing activity. With the unknown performance and compatibility of the growing number of engineered parts including deaminase, CRISPR-Cas enzyme, and guide RNA scaffold variants for assembling the expanding collection of base editor systems, our platform addresses the unmet need for an unbiased, scalable method to benchmark their editing outcomes and accelerate the engineering of next-generation precise genome editors.},
}
@article {pmid37114454,
year = {2023},
author = {Wang, Y and Qi, Y and Guo, X and Zhang, K and Yu, B and Xu, FJ},
title = {Mannose-functionalized star polycation mediated CRISPR/Cas9 delivery for lung cancer therapy.},
journal = {Journal of materials chemistry. B},
volume = {11},
number = {18},
pages = {4121-4130},
doi = {10.1039/d2tb02274e},
pmid = {37114454},
issn = {2050-7518},
mesh = {Humans ; *Mannose ; Survivin ; CRISPR-Cas Systems/genetics ; DNA ; *Lung Neoplasms/genetics/therapy ; },
abstract = {The survivin gene, highly expressed in most cancer cells, is closely associated with inhibiting apoptosis. Therefore, gene editing for the survivin gene has great potential in tumor therapy. However, it is difficult for plasmid DNA (pDNA) to be taken up directly by cells, and thus the construction of gene vectors is a key step for successful gene editing. Ethanolamine-functionalized polyglycidyl methacrylate (PGEA) has been proved to facilitate the transfection of pDNA into cells in both in vivo and in vitro experiments. However, PGEA does not specifically recognize tumor cells. Some tumor cells express more mannose receptor (MR) than healthy cells. To achieve efficient target and transfection, we designed mannose-functionalized four-arm PGEA cationic polymers (P(GEA-co-ManMA), GM) with different molecular weights. GM was combined with pCas9-survivin. The mannose unit of GM/pCas9-survivin was identified by MR to selectively enter lung cancer cells. In vitro experiments showed that GM not only had excellent biocompatibility, gene transfection performance, and targeted ability, but also significantly inhibited the proliferation of tumor cells when used in combination with pCas9-survivin. At the same time, we also studied the relationship between the molecular weight and therapeutic effect.},
}
@article {pmid37163044,
year = {2023},
author = {Taylor, D and Schwartz, E and Bravo, J and Ahsan, M and Macias, L and McCafferty, C and Dangerfield, T and Walker, J and Brodbelt, J and Palermo, G and Fineran, P and Fagerlund, R},
title = {Type III CRISPR-Cas effectors act as protein-assisted ribozymes during RNA cleavage.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-2837968/v1},
pmid = {37163044},
abstract = {CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.},
}
@article {pmid37162202,
year = {2023},
author = {Huang, Z and Lyon, CJ and Wang, J and Lu, S and Hu, TY},
title = {CRISPR Assays for Disease Diagnosis: Progress to and Barriers Remaining for Clinical Applications.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2301697},
doi = {10.1002/advs.202301697},
pmid = {37162202},
issn = {2198-3844},
support = {U01CA252965/NH/NIH HHS/United States ; R01HD090927/NH/NIH HHS/United States ; R01AI144168/NH/NIH HHS/United States ; R01HD103511/NH/NIH HHS/United States ; R21NS130542/NH/NIH HHS/United States ; },
abstract = {Numerous groups have employed the special properties of CRISPR/Cas systems to develop platforms that have broad potential applications for sensitive and specific detection of nucleic acid (NA) targets. However, few of these approaches have progressed to commercial or clinical applications. This review summarizes the properties of known CRISPR/Cas systems and their applications, challenges associated with the development of such assays, and opportunities to improve their performance or address unmet assay needs using nano-/micro-technology platforms. These include rapid and efficient sample preparation, integrated single-tube, amplification-free, quantifiable, multiplex, and non-NA assays. Finally, this review discusses the current outlook for such assays, including remaining barriers for clinical or point-of-care applications and their commercial development.},
}
@article {pmid37162093,
year = {2023},
author = {Ishikawa, K and Soejima, S and Saitoh, S},
title = {Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe.},
journal = {Journal of cell science},
volume = {136},
number = {9},
pages = {},
doi = {10.1242/jcs.261186},
pmid = {37162093},
issn = {1477-9137},
mesh = {Animals ; *Schizosaccharomyces/genetics ; Cell Proliferation ; Gene Knockdown Techniques ; Phenotype ; Mammals ; },
abstract = {Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.},
}
@article {pmid37161535,
year = {2023},
author = {Wenger, A and Karlsson, I and Kling, T and Carén, H},
title = {CRISPR-Cas9 knockout screen identifies novel treatment targets in childhood high-grade glioma.},
journal = {Clinical epigenetics},
volume = {15},
number = {1},
pages = {80},
pmid = {37161535},
issn = {1868-7083},
mesh = {Humans ; Child ; *CRISPR-Cas Systems ; DNA Methylation ; *Glioma/genetics ; Genes, Regulator ; Histones/genetics ; },
abstract = {BACKGROUND: Brain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen.<br><br>RESULTS: The knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers.<br><br>CONCLUSIONS: The screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED) were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.},
}
@article {pmid37160974,
year = {2023},
author = {Du, S and Tong, X and Lai, ACK and Chan, CK and Mason, CE and Lee, PKH},
title = {Highly host-linked viromes in the built environment possess habitat-dependent diversity and functions for potential virus-host coevolution.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {2676},
pmid = {37160974},
issn = {2041-1723},
mesh = {Virome ; Built Environment ; *Microbiota/genetics ; *Alkanesulfonic Acids ; },
abstract = {Viruses in built environments (BEs) raise public health concerns, yet they are generally less studied than bacteria. To better understand viral dynamics in BEs, this study assesses viromes from 11 habitats across four types of BEs with low to high occupancy. The diversity, composition, metabolic functions, and lifestyles of the viromes are found to be habitat dependent. Caudoviricetes species are ubiquitous on surface habitats in the BEs, and some of them are distinct from those present in other environments. Antimicrobial resistance genes are identified in viruses inhabiting surfaces frequently touched by occupants and in viruses inhabiting occupants' skin. Diverse CRISPR/Cas immunity systems and anti-CRISPR proteins are found in bacterial hosts and viruses, respectively, consistent with the strongly coupled virus-host links. Evidence of viruses potentially aiding host adaptation in a specific-habitat manner is identified through a unique gene insertion. This work illustrates that virus-host interactions occur frequently in BEs and that viruses are integral members of BE microbiomes.},
}
@article {pmid37159673,
year = {2023},
author = {Chen, Y and Meng, L and Lang, B and Li, L and Liu, J and Wang, Y and Huang, Z and Tian, X and Tian, R and Hu, Z},
title = {A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18.},
journal = {Cell reports methods},
volume = {3},
number = {4},
pages = {100444},
pmid = {37159673},
issn = {2667-2375},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; *Papillomavirus Infections/diagnosis ; Biological Assay ; Capsid Proteins ; Papillomaviridae ; },
abstract = {CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.},
}
@article {pmid37160120,
year = {2023},
author = {Nguyen, LT and Rananaware, SR and Yang, LG and Macaluso, NC and Ocana-Ortiz, JE and Meister, KS and Pizzano, BLM and Sandoval, LSW and Hautamaki, RC and Fang, ZR and Joseph, SM and Shoemaker, GM and Carman, DR and Chang, L and Rakestraw, NR and Zachary, JF and Guerra, S and Perez, A and Jain, PK},
title = {Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.},
journal = {Cell reports. Medicine},
volume = {},
number = {},
pages = {101037},
doi = {10.1016/j.xcrm.2023.101037},
pmid = {37160120},
issn = {2666-3791},
abstract = {CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.},
}
@article {pmid37158910,
year = {2023},
author = {Bahrulolum, H and Tarrahimofrad, H and Rouzbahani, FN and Nooraei, S and Sameh, MM and Hajizade, A and Ahmadian, G},
title = {Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection.},
journal = {Virology journal},
volume = {20},
number = {1},
pages = {91},
pmid = {37158910},
issn = {1743-422X},
abstract = {Viral hepatitis, the most common cause of inflammatory liver disease, affects hundreds of millions of people worldwide. It is most commonly associated with one of the five nominal hepatitis viruses (hepatitis A-E viruses). HBV and HCV can cause acute infections and lifelong, persistent chronic infections, while HAV and HEV cause self-limiting acute infections. HAV and HEV are predominantly transmitted through the fecal-oral route, while diseases transmitted by the other forms are blood-borne diseases. Despite the success in the treatment of viral hepatitis and the development of HAV and HBV vaccines, there is still no accurate diagnosis at the genetic level for these diseases. Timely diagnosis of viral hepatitis is a prerequisite for efficient therapeutic intervention. Due to the specificity and sensitivity of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated sequences (Cas) technology, it has the potential to meet critical needs in the field of diagnosis of viral diseases and can be used in versatile point-of-care (POC) diagnostic applications to detect viruses with both DNA and RNA genomes. In this review, we discuss recent advances in CRISPR-Cas diagnostics tools and assess their potential and prospects in rapid and effective strategies for the diagnosis and control of viral hepatitis infection.},
}
@article {pmid37158576,
year = {2023},
author = {Yang, B and Khatri, M and Zheng, J and Deogun, J and Yin, Y},
title = {Genome mining for anti-CRISPR operons using machine learning.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btad309},
pmid = {37158576},
issn = {1367-4811},
abstract = {MOTIVATION: Encoded by (pro-)viruses, anti-CRISPR (Acr) proteins inhibit the CRISPR-Cas immune system of their prokaryotic hosts. As a result, Acr proteins can be employed to develop more controllable CRISPR-Cas genome editing tools. Recent studies revealed that known acr genes often coexist with other acr genes and with phage structural genes within the same operon. For example, we found that 47 of 98 known acr genes (or their homologs) co-exist in the same operons. None of the current Acr prediction tools have considered this important genomic context feature. We have developed a new software tool AOminer to facilitate the improved discovery of new Acrs by fully exploiting the genomic context of known acr genes and their homologs.<br><br>RESULTS: AOminer is the first machine learning based tool focused on the discovery of Acr operons (AOs). A two-state HMM (hidden Markov model) was trained to learn the conserved genomic context of operons that contain known acr genes or their homologs, and the learnt features could distinguish AOs and non-AOs. AOminer allows automated mining for potential AOs from query genomes or operons. AOminer outperformed all existing Acr prediction tools with an accuracy = 0.85. AOminer will facilitate the discovery of novel anti-CRISPR operons.<br><br>AVAILABILITY: The webserver is available at: http://aca.unl.edu/AOminer/AOminer_APP/. The python program is at: https://github.com/boweny920/AOminer.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid37154329,
year = {2023},
author = {Gao, S and Fang, G and Zhang, Y and DU, J},
title = {[Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {4},
pages = {1644-1654},
doi = {10.13345/j.cjb.221022},
pmid = {37154329},
issn = {1872-2075},
mesh = {Animals ; Mice ; Mice, Knockout ; *CRISPR-Cas Systems ; Blood Pressure ; Gene Knockout Techniques ; *Zygote ; },
abstract = {To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.},
}
@article {pmid37154311,
year = {2023},
author = {Hong, T and Luo, Q},
title = {[Advances in the RNA-targeting CRISPR-Cas systems].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {4},
pages = {1363-1373},
doi = {10.13345/j.cjb.220633},
pmid = {37154311},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA/genetics ; Bacteria/genetics ; Gene Editing ; Archaea ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.},
}
@article {pmid37153079,
year = {2023},
author = {Hendel, A and Bak, RO},
title = {Editorial: CRISPR and beyond: Cutting-edge technologies for gene correction in therapeutic applications.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1203864},
pmid = {37153079},
issn = {2673-3439},
}
@article {pmid37120835,
year = {2023},
author = {Xie, TJ and Xie, JL and Luo, YJ and Mao, K and Huang, CZ and Li, YF and Zhen, SJ},
title = {CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.},
journal = {Analytical chemistry},
volume = {95},
number = {18},
pages = {7237-7243},
doi = {10.1021/acs.analchem.3c00156},
pmid = {37120835},
issn = {1520-6882},
mesh = {Humans ; *COVID-19 ; SARS-CoV-2 ; DNA ; DNA, Single-Stranded ; *MicroRNAs ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; },
abstract = {DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.},
}
@article {pmid37119146,
year = {2023},
author = {Li, L and Yu, S and Wu, J and Ju, H},
title = {Regulation of Target-activated CRISPR/Cas12a on Surface Binding of Polymer Dots for Sensitive Electrochemiluminescence DNA Analysis.},
journal = {Analytical chemistry},
volume = {95},
number = {18},
pages = {7396-7402},
doi = {10.1021/acs.analchem.3c01521},
pmid = {37119146},
issn = {1520-6882},
mesh = {Humans ; *Quantum Dots ; Gold ; Polymers ; Reproducibility of Results ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; *Metal Nanoparticles ; DNA/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; },
abstract = {Polymer dots (Pdots) have emerged as a type of attractive electrochemiluminescence (ECL) emitter. However, the low ECL efficiency severely limits their practicability. In this work, we develop a sensitive ECL biosensing strategy for the detection of human papilloma virus subtype (HPV-16) DNA by using target-activated CRISPR/Cas12a to regulate the binding of Pdots-DNA to biosensor and local surface plasmon resonance (LSPR) effect of electrochemically deposited Au nanoparticles (depAuNPs) to enhance the ECL emission of Pdots bound on biosensor. The biosensor is prepared by simply assembling hairpin DNA on depAuNPs modified electrode. In the presence of target DNA, the designed specific CRISPR/Cas12a can be activated to digest single-stranded assistant DNA, which decreases the amount of hairpin DNA opened by assistant DNA to bind Pdots-DNA on the biosensor surface, thus reduces the ECL emission. The integration of target DNA-triggered catalysis and the LSPR effect of depAuNPs greatly improves the sensitivity of ECL analysis. Using HPV-16 DNA as a target model, the proposed method shows a limit of detection (LOD) of 3.2 fM at a signal-to-noise ratio of 3 and a detectable concentration range of 5.0 fM to 50 pM. The high sensitivity, excellent selectivity, good testing stability, and acceptable fabrication reproducibility of the designed ECL biosensing strategy demonstrate its potential application in DNA bioanalysis.},
}
@article {pmid37039522,
year = {2023},
author = {Hadi, J and Rapp, D and Dhawan, S and Gupta, SK and Gupta, TB and Brightwell, G},
title = {Molecular detection and characterization of foodborne bacteria: Recent progresses and remaining challenges.},
journal = {Comprehensive reviews in food science and food safety},
volume = {22},
number = {3},
pages = {2433-2464},
doi = {10.1111/1541-4337.13153},
pmid = {37039522},
issn = {1541-4337},
mesh = {*CRISPR-Cas Systems ; *Genome-Wide Association Study ; Bacteria/genetics ; Food Safety ; },
abstract = {The global food demand is expected to increase in the coming years, along with challenges around climate change and food security. Concomitantly, food safety risks, particularly those related to bacterial pathogens, may also increase. Thus, the food sector needs to innovate to rise to this challenge. Here, we discuss recent advancements in molecular techniques that can be deployed within various foodborne bacteria surveillance systems across food settings. To start with, we provide updates on nucleic acid-based detection, with a focus on polymerase chain reaction (PCR)-based technologies and loop-mediated isothermal amplification (LAMP). These include descriptions of novel genetic markers for several foodborne bacteria and progresses in multiplex PCR and droplet digital PCR. The next section provides an overview of the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems, such as CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a, as tools for enhanced sensitive and specific detection of foodborne pathogens. The final section describes utilizations of whole genome sequencing for accurate characterization of foodborne bacteria, ranging from epidemiological surveillance to model-based predictions of bacterial phenotypic traits through genome-wide association studies or machine learning.},
}
@article {pmid36800529,
year = {2023},
author = {Li, M and Zhao, Y and Xue, X and Zhong, J and Lin, J and Zhou, J and Yu, W and Chen, J and Qiao, Y},
title = {Cas9-orthologue-mediated cytosine and adenine base editors recognizing NNAAAA PAM sequences.},
journal = {Biotechnology journal},
volume = {18},
number = {5},
pages = {e2200533},
doi = {10.1002/biot.202200533},
pmid = {36800529},
issn = {1860-7314},
mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Adenine/metabolism ; Cytosine/metabolism ; Nucleotides ; },
abstract = {CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.},
}
@article {pmid37153078,
year = {2023},
author = {Pascher, K and Hainz-Renetzeder, C and Jagersberger, M and Kneissl, K and Gollmann, G and Schneeweiss, GM},
title = {Contamination of imported kernels by unapproved genome-edited varieties poses a major challenge for monitoring and traceability during transport and handling on a global scale: inferences from a study on feral oilseed rape in Austria.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1176290},
pmid = {37153078},
issn = {2673-3439},
abstract = {Novel techniques such as CRISPR/Cas are increasingly being applied for the development of modern crops. However, the regulatory framework for production, labelling and handling of genome-edited organisms varies worldwide. Currently, the European Commission is raising the question whether genome-edited organisms should still be regulated as genetically modified organisms in the future or whether a deregulation should be implemented. In our paper, based on the outcome of a 2-year case study on oilseed rape in Austria, we show that seed spillage during import and subsequent transport and handling activities is a key factor for the unintended dispersal of seeds into the environment, the subsequent emergence of feral oilseed rape populations and their establishment and long-term persistence in natural habitats. These facts must likewise be considered in case of genome-edited oilseed rape contaminants that might be accidentally introduced with conventional kernels. We provide evidence that in Austria a high diversity of oilseed rape genotypes, including some with alleles not known from cultivated oilseed rape in Austria, exists at sites with high seed spillage and low weed management, rendering these sites of primary concern with respect to possible escape of genome-edited oilseed rape varieties into the environment. Since appropriate detection methods for single genome-edited oilseed rape events have only recently started to be successfully developed and the adverse effects of these artificial punctate DNA exchanges remain largely unknown, tracing the transmission and spread of these genetic modifications places high requirements on their monitoring, identification, and traceability.},
}
@article {pmid37152166,
year = {2023},
author = {Ye, S and Ding, W and Bai, W and Lu, J and Zhou, L and Ma, X and Zhu, Q},
title = {Application of a novel strong promoter from Chinese fir (Cunninghamia lanceolate) in the CRISPR/Cas mediated genome editing of its protoplasts and transgenesis of rice and poplar.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1179394},
pmid = {37152166},
issn = {1664-462X},
abstract = {Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters (Cula11 and Cula08) from Chinese fir (C. lanceolate) by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.},
}
@article {pmid37149686,
year = {2023},
author = {Cetin, R and Wegner, M and Luwisch, L and Saud, S and Achmedov, T and Süsser, S and Vera-Guapi, A and Müller, K and Matthess, Y and Quandt, E and Schaubeck, S and Beisel, CL and Kaulich, M},
title = {Optimized metrics for orthogonal combinatorial CRISPR screens.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {7405},
pmid = {37149686},
issn = {2045-2322},
support = {865973/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems ; *Benchmarking ; Gene Editing ; Endonucleases/genetics ; },
abstract = {CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.},
}
@article {pmid37014013,
year = {2023},
author = {Pan, J and Mabuchi, M and Robb, GB},
title = {DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.},
journal = {Nucleic acids research},
volume = {51},
number = {8},
pages = {3903-3917},
pmid = {37014013},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Ribonucleoproteins/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism ; DNA/chemistry ; Streptococcus pyogenes ; DNA Cleavage ; },
abstract = {The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.},
}
@article {pmid36919598,
year = {2023},
author = {Lee, RRQ and Cher, WY and Wang, J and Chen, Y and Chae, E},
title = {Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg.},
journal = {Nucleic acids research},
volume = {51},
number = {8},
pages = {e43},
pmid = {36919598},
issn = {1362-4962},
mesh = {Humans ; Polymerase Chain Reaction ; Gene Knockout Techniques ; *Software ; *CRISPR-Cas Systems ; },
abstract = {MINORg is an offline gRNA design tool that generates the smallest possible combination of gRNA capable of covering all desired targets in multiple non-reference genomes. As interest in pangenomic research grows, so does the workload required for large screens in multiple individuals. MINORg aims to lessen this workload by capitalising on sequence homology to favour multi-target gRNA while simultaneously screening multiple genetic backgrounds in order to generate reusable gRNA panels. We demonstrated the practical application of MINORg by knocking out 11 homologous genes tandemly arrayed in a multi-gene cluster in two Arabidopsis thaliana lineages using three gRNA output by MINORg. We also described a new PCR-free modular cloning system for multiplexing gRNA, and used it to knockout three tandemly arrayed genes in another multi-gene cluster with gRNA designed by MINORg. Source code is freely available at https://github.com/rlrq/MINORg.},
}
@article {pmid36912089,
year = {2023},
author = {Deng, H and Xu, H and Wang, Y and Jia, R and Ma, X and Feng, Y and Chen, H},
title = {G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation.},
journal = {Nucleic acids research},
volume = {51},
number = {8},
pages = {4064-4077},
pmid = {36912089},
issn = {1362-4962},
mesh = {*Gene Editing/methods ; *Genomics ; Genome ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of CRISPR editing is essential to overcome its uncontrollable reaction process and excessive activity that leads to off-target editing. To overcome this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) for precisely controlled gene editing and expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated that rational design of the G-quadruplex onto crRNA conferred higher stability and sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization of AZD++ quickly transformed the on state of GqRNA, which facilitated rapid activation of ribonucleoprotein activity for genome editing of on-target sites in cells with excellent editing efficiency. In turn, AZD++-GqRNA promptly refolded to an off state to inhibit genomic cleavage, and limited the generation of off-target effects and by-products. Therefore, the proposed strategy of a photo-reversible modality presents a new opportunity for CRISPR-Cas9 modulation to improve its safety and applicability.},
}
@article {pmid36241547,
year = {2023},
author = {Jo, DH and Bae, S and Kim, HH and Kim, JS and Kim, JH},
title = {In vivo application of base and prime editing to treat inherited retinal diseases.},
journal = {Progress in retinal and eye research},
volume = {94},
number = {},
pages = {101132},
doi = {10.1016/j.preteyeres.2022.101132},
pmid = {36241547},
issn = {1873-1635},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; *Retinal Diseases/genetics/therapy ; },
abstract = {Inherited retinal diseases (IRDs) are vision-threatening retinal disorders caused by pathogenic variants of genes related to visual functions. Genomic analyses in patients with IRDs have revealed pathogenic variants which affect vision. However, treatment options for IRDs are limited to nutritional supplements regardless of genetic variants or gene-targeting approaches based on antisense oligonucleotides and adeno-associated virus vectors limited to targeting few genes. Genome editing, particularly that involving clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies, can correct pathogenic variants and provide additional treatment opportunities. Recently developed base and prime editing platforms based on CRISPR-Cas9 technologies are promising for therapeutic genome editing because they do not employ double-stranded breaks (DSBs), which are associated with P53 activation, large deletions, and chromosomal translocations. Instead, using attached deaminases and reverse transcriptases, base and prime editing efficiently induces specific base substitutions and intended genetic changes (substitutions, deletions, or insertions), respectively, without DSBs. In this review, we will discuss the recent in vivo application of CRISPR-Cas9 technologies, focusing on base and prime editing, in animal models of IRDs.},
}
@article {pmid37148798,
year = {2023},
author = {Kang, Y and Wang, J and Zhang, W and Xu, Y and Xu, B and Qu, G and Yu, Y and Yan, B and Su, G},
title = {RNA extraction-free workflow integrated with a single-tube CRISPR-Cas-based colorimetric assay for rapid SARS-CoV-2 detection in different environmental matrices.},
journal = {Journal of hazardous materials},
volume = {454},
number = {},
pages = {131487},
pmid = {37148798},
issn = {1873-3336},
abstract = {On-site environmental surveillance of viruses is increasingly important for infection prevention and pandemic control. Herein, we report a facile single-tube colorimetric assay for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from environmental compartments. Using glycerol as the phase separation additive, reverse transcription recombinase polymerase amplification (RT-RPA), CRISPR-Cas system activation, G-quadruplex (G4) cleavage, and G4-based colorimetric reaction were performed in a single tube. To further simplify the test, viral RNA genomes used for the one-tube assay were obtained via acid/base treatment without further purification. The whole assay from sampling to visual readout was completed within 30 min at a constant temperature without the need for sophisticated instruments. Coupling the RT-RPA to CRISPR-Cas improved the reliability by avoiding false positive results. Non-labeled cost-effective G4-based colorimetric systems are highly sensitive to CRISPR-Cas cleavage events, and the proposed assay reached the limit of detection of 0.84 copies/µL. Moreover, environmental samples from contaminated surfaces and wastewater were analyzed using this facile colorimetric assay. Given its simplicity, sensitivity, specificity, and cost-effectiveness, our proposed colorimetric assay is highly promising for applications in on-site environmental surveillance of viruses.},
}
@article {pmid37148478,
year = {2023},
author = {Liu, Y and Jing, P and Zhou, Y and Zhang, J and Shi, J and Zhang, M and Yang, H and Fei, J},
title = {The effects of length and sequence of gRNA on Cas13b and Cas13d activity in vitro and in vivo.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2300002},
doi = {10.1002/biot.202300002},
pmid = {37148478},
issn = {1860-7314},
abstract = {Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA. This article is protected by copyright. All rights reserved.},
}
@article {pmid37147507,
year = {2023},
author = {Sonnenburg, A and Stahlmann, R and Kreutz, R and Peiser, M},
title = {A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage.},
journal = {Archives of toxicology},
volume = {},
number = {},
pages = {},
pmid = {37147507},
issn = {1432-0738},
abstract = {Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation.},
}
@article {pmid37147050,
year = {2023},
author = {Wu, X and Ju, T and Li, Z and Li, J and Zhai, X and Han, K},
title = {Target-independent hybridization chain reaction-fluorescence resonance energy transfer for sensitive assay of ctDNA based on Cas12a.},
journal = {Analytica chimica acta},
volume = {1261},
number = {},
pages = {341170},
doi = {10.1016/j.aca.2023.341170},
pmid = {37147050},
issn = {1873-4324},
mesh = {*Fluorescence Resonance Energy Transfer ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; DNA/genetics ; *Biosensing Techniques/methods ; },
abstract = {Circulating tumor DNA (ctDNA) is a noninvasive biomarker which offer valuable information for cancer diagnosis and prognosis. In this study, a target-independent fluorescent signal system, Hybridization chain reaction-Fluorescence resonance energy transfer (HCR-FRET) system, is designed and optimized. Combined with CRISPR/Cas12a system, a fluorescent biosensing protocol was developed for sensing assay of T790 M. When the target is absent, the initiator remains intact, opens the fuel hairpins and triggers the following HCR-FRET. At presence of the target, the Cas12a/crRNA binary complex specifically recognizes the target, and the Cas12a trans-cleavage activity is activated. As a result, the initiator is cleaved and subsequent HCR responses and FRET processes are attenuated. This method showed detection range from 1 pM to 400 pM with a detection limit of 316 fM. The target independent property of the HCR-FRET system endows this protocol a promising potential to transplant to the assay of other DNA target in parallel.},
}
@article {pmid37146580,
year = {2023},
author = {Ferrari, S and Valeri, E and Conti, A and Scala, S and Aprile, A and Di Micco, R and Kajaste-Rudnitski, A and Montini, E and Ferrari, G and Aiuti, A and Naldini, L},
title = {Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.},
journal = {Cell stem cell},
volume = {30},
number = {5},
pages = {549-570},
doi = {10.1016/j.stem.2023.04.014},
pmid = {37146580},
issn = {1875-9777},
mesh = {*CRISPR-Cas Systems ; Prospective Studies ; *Hematopoietic Stem Cells ; Gene Editing ; Genetic Therapy ; Biology ; },
abstract = {The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.},
}
@article {pmid37144477,
year = {2023},
author = {Shariq, M and Khan, MF and Raj, R and Ahsan, N and Singh, R and Kumar, P},
title = {CRISPR‑based diagnostic approaches: Implications for rapid management of future pandemics (Review).},
journal = {Molecular medicine reports},
volume = {27},
number = {6},
pages = {},
doi = {10.3892/mmr.2023.13005},
pmid = {37144477},
issn = {1791-3004},
mesh = {Humans ; *COVID-19 ; SARS-CoV-2/genetics ; CRISPR-Cas Systems/genetics ; Pandemics ; Bacteria/genetics ; COVID-19 Testing ; },
abstract = {Sudden viral outbreaks have increased in the early part of the 21st century, such as those of severe acute respiratory syndrome coronavirus (SARS‑CoV), Middle East respiratory syndrome corona virus, and SARS‑CoV‑2, owing to increased human access to wildlife habitats. Therefore, the likelihood of zoonotic transmission of human‑associated viruses has increased. The emergence of severe acute respiratory syndrome coronavirus 2 in China and its spread worldwide within months have highlighted the need to be ready with advanced diagnostic and antiviral approaches to treat newly emerging diseases with minimal harm to human health. The gold‑standard molecular diagnostic approaches currently used are time‑consuming, require trained personnel and sophisticated equipment, and therefore cannot be used as point‑of‑care devices for widespread monitoring and surveillance. Clustered regularly interspaced short palindromic repeats (CRISPR)‑associated (Cas) systems are widespread and have been reported in bacteria, archaea and bacteriophages. CRISPR‑Cas systems are organized into CRISPR arrays and adjacent Cas proteins. The detection and in‑depth biochemical characterization of class 2 type V and VI CRISPR‑Cas systems and orthologous proteins such as Cas12 and Cas13 have led to the development of CRISPR‑based diagnostic approaches, which have been used to detect viral diseases and distinguish between serotypes and subtypes. CRISPR‑based diagnostic approaches detect human single nucleotide polymorphisms in samples from patients with cancer and are used as antiviral agents to detect and destroy viruses that contain RNA as a genome. CRISPR‑based diagnostic approaches are likely to improve disease detection methods in the 21st century owing to their ease of development, low cost, reduced turnaround time, multiplexing and ease of deployment. The present review discusses the biochemical properties of Cas12 and Cas13 orthologs in viral disease detection and other applications. The present review expands the scope of CRISPR‑based diagnostic approaches to detect diseases and fight viruses as antivirals.},
}
@article {pmid37066613,
year = {2023},
author = {Zhao, F and Wang, P and Wang, H and Liu, S and Sohail, M and Zhang, X and Li, B and Huang, H},
title = {CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing.},
journal = {Analytical methods : advancing methods and applications},
volume = {15},
number = {17},
pages = {2105-2113},
doi = {10.1039/d2ay01998a},
pmid = {37066613},
issn = {1759-9679},
mesh = {Humans ; *Monkeypox virus/genetics ; *Monkeypox/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; Recombinases/genetics ; Africa, Western ; },
abstract = {The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 10[1] copies per μL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 10[4] copies per μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.},
}
@article {pmid37024675,
year = {2023},
author = {Villanueva, MT},
title = {Base editing treats SMA in mice.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {5},
pages = {353},
doi = {10.1038/d41573-023-00057-1},
pmid = {37024675},
issn = {1474-1784},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Biotechnology ; },
}
@article {pmid37012339,
year = {2023},
author = {Kingwell, K},
title = {First CRISPR therapy seeks landmark approval.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {5},
pages = {339-341},
doi = {10.1038/d41573-023-00050-8},
pmid = {37012339},
issn = {1474-1784},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy ; },
}
@article {pmid37003192,
year = {2023},
author = {Yang, M and Jiao, Y and Yan, Y and Li, L and Hu, X and Jiao, Z and Li, M and Chen, Y and Shi, Y and Shen, Z and Peng, G},
title = {Safety and immunogenicity of a TK/ gI/gE gene-deleted feline herpesvirus-1 mutant constructed via CRISPR/Cas9 in feline.},
journal = {Veterinary microbiology},
volume = {281},
number = {},
pages = {109728},
doi = {10.1016/j.vetmic.2023.109728},
pmid = {37003192},
issn = {1873-2542},
mesh = {Cats ; Animals ; CRISPR-Cas Systems ; *Varicellovirus ; *Herpesviridae Infections/prevention &amp; control/veterinary ; Antibodies, Neutralizing/genetics ; *Viral Vaccines ; *Cat Diseases/prevention &amp; control ; },
abstract = {Feline herpesvirus-1 (FHV-1) is the aetiological agent of feline viral rhinotracheitis, which accounts for approximately 50 % of all viral upper respiratory diseases in cats. Commercially available modified live vaccines containing FHV-1 are generally safe and effective, but these FHV-1 vaccines retain full virulence genes and can establish latency and reactivate to cause infectious rhinotracheitis in vaccine recipients, raising safety concerns. To address this shortcoming, we constructed a novel TK/gI/gE -gene-deleted recombinant FHV-1 (WH2020-ΔTK/gI/gE) through CRISPR/Cas9-mediated homologous recombination. The growth kinetics of WH2020-ΔTK/gI/gE were slightly delayed compared to those of the parent strain WH2020. Recombinant FHV-1 had severely impaired pathogenicity in cats. Felines immunized with WH2020-ΔTK/gI/gE produced high levels of gB-specific antibodies, neutralizing antibodies and IFN-β. Additionally, WH2020-ΔTK/gI/gE provided greater protection against challenge with FHV-1 field strain WH2020 than did the commercial modified live vaccine. After challenge, the cats vaccinated with WH2020-ΔTK/gI/gE showed significantly fewer clinical signs, pathological changes, viral shedding, and viral loads in the lung and trigeminal ganglia than those vaccinated with the commercial vaccine or unvaccinated. Our results suggest that WH2020-ΔTK/gI/gE is a promising candidate as a safer and more efficacious live FHV-1 vaccine, with a decreased risk of vaccine-related complications, and could inform the design of other herpesvirus vaccines.},
}
@article {pmid36917276,
year = {2023},
author = {Zhang, C and Ren, Z and Gong, Z},
title = {Generation of Albino Phenotype in Ornamental Fish by CRISPR/Cas9-Mediated Genome Editing of slc45a2 Gene.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {25},
number = {2},
pages = {281-290},
pmid = {36917276},
issn = {1436-2236},
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Zebrafish/genetics ; Phylogeny ; *Albinism/genetics ; },
abstract = {Albinism is the most common color variation described in fish and is a fascinating trait of some ornamental fish species. Albino mutants can be generated by knocking out core genes affecting melanin synthesis like slc45a2 in several fish species. However, genetic mutation remains challenging for species with unknown genome information. In this study, we generated albino mutants in two selected ornamental fish species, royal farlowella (Sturisoma panamense), and redhead cichlid (Vieja melanura). For this purpose, we carried out phylogenetic analyses of fish slc45a2 sequences and identified a highly conserved region among different fish species. A pair of degenerate primers spanning this region was designed and used to amplify a conserved slc45a2 fragment of 340 bp from the two fish species. Based on the amplified sequences, a target site in the 6[th] exon was used for designing guide RNA and this targeted site was first verified by the CRISPR/Cas9 system in the zebrafish (Danio rerio) model for the effectiveness. Then, specific guide RNAs were designed for the two ornamental fish species and tested. Most of the injected larvae completely lost black pigment over the whole body and eyes. DNA sequencing confirmed a high degree of mutation at the targeted site. Overall, we described a fast and efficient method to generate albino phenotype in fish species by targeting the conserved 6[th] exon of slc45a2 gene for genome editing via CRISPR/Cas9 and this approach could be a new genetic tool to generate desirable albino ornamental fish.},
}
@article {pmid36896650,
year = {2023},
author = {Carneiro, SP and Greco, A and Chiesa, E and Genta, I and Merkel, OM},
title = {Shaping the future from the small scale: dry powder inhalation of CRISPR-Cas9 lipid nanoparticles for the treatment of lung diseases.},
journal = {Expert opinion on drug delivery},
volume = {20},
number = {4},
pages = {471-487},
doi = {10.1080/17425247.2023.2185220},
pmid = {36896650},
issn = {1744-7593},
mesh = {Humans ; Powders ; CRISPR-Cas Systems ; Administration, Inhalation ; *Nanoparticles ; *Lung Diseases/drug therapy ; Lung ; Dry Powder Inhalers ; Particle Size ; },
abstract = {INTRODUCTION: Most lung diseases are serious conditions resulting from genetic and environmental causes associated with high mortality and severe symptoms. Currently, treatments available have a palliative effect and many targets are still considered undruggable. Gene therapy stands as an attractive approach to offering innovative therapeutic solutions. CRISPRCas9 has established a remarkable potential for genome editing with high selectivity to targeted mutations. To ensure high efficacy with minimum systemic exposure, the delivery and administration route are key components that must be investigated.<br><br>AREAS COVERED: This review is focused on the delivery of CRISPRCas9 to the lungs, taking advantage of lipid nanoparticles (LNPs), the most clinically advanced nucleic acid carriers. We also aim to highlight the benefits of pulmonary administration as a local delivery route and the use of spray drying to prepare stable nucleic-acid-based dry powder formulations that can overcome multiple lung barriers.<br><br>EXPERT OPINION: Exploring the pulmonary administration to deliver CRISPRCas9 loaded in LNPs as a dry powder increases the chances to achieve high efficacy and reduced adverse effects. CRISPRCas9 loaded in LNP-embedded microparticles has not yet been reported in the literature but has the potential to reach and accumulate in target cells in the lung, thus, enhancing overall efficacy and safety.},
}
@article {pmid36861495,
year = {2023},
author = {Fujii, T and Inoue, N and Nobeyama, T and Inoue, J and Ogasawara, S and Otani, Y and Fujii, S and Ito, C and Fukunaga, R},
title = {A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {28},
number = {5},
pages = {390-397},
doi = {10.1111/gtc.13021},
pmid = {36861495},
issn = {1365-2443},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; Gene Knockout Techniques ; Plasmids ; Transfection ; *Gene Editing/methods ; },
abstract = {We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.},
}
@article {pmid36840757,
year = {2023},
author = {Galindo-Sotomonte, L and Jozefkowicz, C and Gómez, C and Stritzler, M and Frare, R and Bottero, E and Tajima, H and Blumwald, E and Ayub, N and Soto, G},
title = {CRISPR/Cas9-mediated knockout of a polyester synthase-like gene delays flowering time in alfalfa.},
journal = {Plant cell reports},
volume = {42},
number = {5},
pages = {953-956},
pmid = {36840757},
issn = {1432-203X},
mesh = {*Medicago sativa/genetics ; CRISPR-Cas Systems/genetics ; Flowers/genetics ; *Arabidopsis/genetics ; },
abstract = {T-DNA and CRISPR/Cas9-mediated knockout of polyester synthase-like genes delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Thus, we here present the first report of edited alfalfa with delayed flowering.},
}
@article {pmid36756818,
year = {2023},
author = {Zhang, X and Guo, B and Yang, L and Zhao, C and Wang, Y and Tang, Y and Yang, G and Wang, P and Gao, S},
title = {CRISPR/Cas12a combined with recombinase polymerase amplification for rapid and sensitive detection of Vibrio vulnificus in one tube.},
journal = {Acta biochimica et biophysica Sinica},
volume = {55},
number = {2},
pages = {322-326},
pmid = {36756818},
issn = {1745-7270},
mesh = {*Recombinases/genetics ; *Vibrio vulnificus/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; },
}
@article {pmid36734409,
year = {2023},
author = {Kurzhagen, JT and Noel, S and Lee, K and Sadasivam, M and Gharaie, S and Ankireddy, A and Lee, SA and Newman-Rivera, A and Gong, J and Arend, LJ and Hamad, ARA and Reddy, SP and Rabb, H},
title = {T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.},
journal = {Antioxidants &amp; redox signaling},
volume = {38},
number = {13-15},
pages = {959-973},
doi = {10.1089/ars.2022.0058},
pmid = {36734409},
issn = {1557-7716},
support = {R01 HL136946/HL/NHLBI NIH HHS/United States ; R01 DK111209/DK/NIDDK NIH HHS/United States ; },
mesh = {Mice ; Animals ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; *Antioxidants/metabolism ; NF-E2-Related Factor 2/genetics/metabolism ; CRISPR-Cas Systems ; Glutamate-Cysteine Ligase/genetics/metabolism ; Gene Editing ; Kidney/metabolism ; *Reperfusion Injury/genetics/therapy/metabolism ; Oxidative Stress ; },
abstract = {Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.},
}
@article {pmid37144131,
year = {2023},
author = {Singh, C and Kumar, R and Sehgal, H and Bhati, S and Singhal, T and Gayacharan, and Nimmy, MS and Yadav, R and Gupta, SK and Abdallah, NA and Hamwieh, A and Kumar, R},
title = {Unclasping potentials of genomics and gene editing in chickpea to fight climate change and global hunger threat.},
journal = {Frontiers in genetics},
volume = {14},
number = {},
pages = {1085024},
pmid = {37144131},
issn = {1664-8021},
abstract = {Genomics and genome editing promise enormous opportunities for crop improvement and elementary research. Precise modification in the specific targeted location of a genome has profited over the unplanned insertional events which are generally accomplished employing unadventurous means of genetic modifications. The advent of new genome editing procedures viz; zinc finger nucleases (ZFNs), homing endonucleases, transcription activator like effector nucleases (TALENs), Base Editors (BEs), and Primer Editors (PEs) enable molecular scientists to modulate gene expressions or create novel genes with high precision and efficiency. However, all these techniques are exorbitant and tedious since their prerequisites are difficult processes that necessitate protein engineering. Contrary to first generation genome modifying methods, CRISPR/Cas9 is simple to construct, and clones can hypothetically target several locations in the genome with different guide RNAs. Following the model of the application in crop with the help of the CRISPR/Cas9 module, various customized Cas9 cassettes have been cast off to advance mark discrimination and diminish random cuts. The present study discusses the progression in genome editing apparatuses, and their applications in chickpea crop development, scientific limitations, and future perspectives for biofortifying cytokinin dehydrogenase, nitrate reductase, superoxide dismutase to induce drought resistance, heat tolerance and higher yield in chickpea to encounter global climate change, hunger and nutritional threats.},
}
@article {pmid37143874,
year = {2023},
author = {Kumar, M and Prusty, MR and Pandey, MK and Singh, PK and Bohra, A and Guo, B and Varshney, RK},
title = {Application of CRISPR/Cas9-mediated gene editing for abiotic stress management in crop plants.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1157678},
pmid = {37143874},
issn = {1664-462X},
abstract = {Abiotic stresses, including drought, salinity, cold, heat, and heavy metals, extensively reducing global agricultural production. Traditional breeding approaches and transgenic technology have been widely used to mitigate the risks of these environmental stresses. The discovery of engineered nucleases as genetic scissors to carry out precise manipulation in crop stress-responsive genes and associated molecular network has paved the way for sustainable management of abiotic stress conditions. In this context, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas)-based gene-editing tool has revolutionized due to its simplicity, accessibility, adaptability, flexibility, and wide applicability. This system has great potential to build up crop varieties with enhanced tolerance against abiotic stresses. In this review, we summarize the latest findings on understanding the mechanism of abiotic stress response in plants and the application of CRISPR/Cas-mediated gene-editing system towards enhanced tolerance to a multitude of stresses including drought, salinity, cold, heat, and heavy metals. We provide mechanistic insights on the CRISPR/Cas9-based genome editing technology. We also discuss applications of evolving genome editing techniques such as prime editing and base editing, mutant library production, transgene free and multiplexing to rapidly deliver modern crop cultivars adapted to abiotic stress conditions.},
}
@article {pmid37142716,
year = {2023},
author = {Huang, D and Yuan, MM and Chen, J and Zheng, X and Wong, D and Alvarez, PJJ and Yu, P},
title = {The association of prokaryotic antiviral systems and symbiotic phage communities in drinking water microbiomes.},
journal = {ISME communications},
volume = {3},
number = {1},
pages = {46},
pmid = {37142716},
issn = {2730-6151},
abstract = {Prokaryotic antiviral systems are important mediators for prokaryote-phage interactions, which have significant implications for the survival of prokaryotic community. However, the prokaryotic antiviral systems under environmental stress are poorly understood, limiting the understanding of microbial adaptability. Here, we systematically investigated the profile of the prokaryotic antiviral systems at the community level and prokaryote-phage interactions in the drinking water microbiome. Chlorine disinfectant was revealed as the main ecological driver for the difference in prokaryotic antiviral systems and prokaryote-phage interactions. Specifically, the prokaryotic antiviral systems in the microbiome exhibited a higher abundance, broader antiviral spectrum, and lower metabolic burden under disinfectant stress. Moreover, significant positive correlations were observed between phage lysogenicity and enrichment of antiviral systems (e.g., Type IIG and IV restriction-modification (RM) systems, and Type II CRISPR-Cas system) in the presence of disinfection, indicating these antiviral systems might be more compatible with lysogenic phages and prophages. Accordingly, there was a stronger prokaryote-phage symbiosis in disinfected microbiome, and the symbiotic phages carried more auxiliary metabolic genes (AMGs) related to prokaryotic adaptability as well as antiviral systems, which might further enhance prokaryote survival in drinking water distribution systems. Overall, this study demonstrates that the prokaryotic antiviral systems had a close association with their symbiotic phages, which provides novel insights into prokaryote-phage interactions and microbial environmental adaptation.},
}
@article {pmid37142704,
year = {2023},
author = {Gencay, YE and Jasinskytė, D and Robert, C and Semsey, S and Martínez, V and Petersen, A&#xd8; and Brunner, K and de Santiago Torio, A and Salazar, A and Turcu, IC and Eriksen, MK and Koval, L and Takos, A and Pascal, R and Schou, TS and Bayer, L and Bryde, T and Johansen, KC and Bak, EG and Smrekar, F and Doyle, TB and Satlin, MJ and Gram, A and Carvalho, J and Jessen, L and Hallström, B and Hink, J and Damholt, B and Troy, A and Grove, M and Clube, J and Grøndahl, C and Haaber, JK and van der Helm, E and Zdravkovic, M and Sommer, MOA},
title = {Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37142704},
issn = {1546-1696},
abstract = {Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.},
}
@article {pmid37140042,
year = {2023},
author = {Johnson, MC and Laderman, E and Huiting, E and Zhang, C and Davidson, A and Bondy-Denomy, J},
title = {Core defense hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad317},
pmid = {37140042},
issn = {1362-4962},
support = {R01GM127489/NH/NIH HHS/United States ; },
abstract = {Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Recent advances in anti-phage system discovery and annotation tools have unearthed many unique systems, often encoded in horizontally transferred defense islands, which can be horizontally transferred. Here, we developed Hidden Markov Models (HMMs) for defense systems and queried microbial genomes on the NCBI database. Out of the 30 species with >200 completely sequenced genomes, our analysis found Pseudomonas aeruginosa exhibits the greatest diversity of anti-phage systems, as measured by Shannon entropy. Using network analysis to identify the common neighbors of anti-phage systems, we identified two core defense hotspot loci (cDHS1 and cDHS2). cDHS1 is up to 224 kb (median: 26 kb) with varied arrangements of more than 30 distinct immune systems across isolates, while cDHS2 has 24 distinct systems (median: 6 kb). Both cDHS regions are occupied in a majority of P. aeruginosa isolates. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identifying core genes flanking immune islands could simplify immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.},
}
@article {pmid37132114,
year = {2023},
author = {Gao, TT and Oh, TJ and Mehta, K and Huang, YA and Camp, T and Fan, H and Han, JW and Barnes, CM and Zhang, K},
title = {The clinical potential of optogenetic interrogation of pathogenesis.},
journal = {Clinical and translational medicine},
volume = {13},
number = {5},
pages = {e1243},
doi = {10.1002/ctm2.1243},
pmid = {37132114},
issn = {2001-1326},
support = {R01GM132438/GM/NIGMS NIH HHS/United States ; R01MH124827/MH/NIMH NIH HHS/United States ; },
mesh = {*Light ; *Optogenetics/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.<br><br>AIMS AND METHODS: This work summarizes the progress of current clinical trials and provides a brief overview of basic structures and photophysics of commonly used photoactivable proteins. We highlight recent achievements such as optogenetic control of the chimeric antigen receptor, CRISPR-Cas system, gene expression, and organelle dynamics. We discuss conceptual innovation and technical challenges faced by current optogenetic research.<br><br>CONCLUSION: In doing so, we provide a framework that showcases ever-growing applications of optogenetics in biomedical research and may inform novel precise medicine strategies based on this enabling technology.},
}
@article {pmid37127628,
year = {2023},
author = {Shabbir, S and Wang, W and Nawaz, M and Boruah, P and Kulyar, MF and Chen, M and Wu, B and Liu, P and Dai, Y and Sun, L and Gou, Q and Liu, R and Hu, G and Younis, T and He, M},
title = {Molecular mechanism of engineered Zymomonas mobilis to furfural and acetic acid stress.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {88},
pmid = {37127628},
issn = {1475-2859},
mesh = {*Acetic Acid/metabolism ; *Zymomonas/genetics ; Furaldehyde/metabolism ; DNA Shuffling ; Fermentation ; Ethanol/metabolism ; },
abstract = {Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.},
}
@article {pmid36930400,
year = {2023},
author = {Zhang, G and Wang, J and Li, Y and Shang, G},
title = {CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis and saturation mutagenesis of plasmid-encoded genes.},
journal = {Biotechnology letters},
volume = {45},
number = {5-6},
pages = {629-637},
pmid = {36930400},
issn = {1573-6776},
mesh = {*CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Mutagenesis, Site-Directed ; Mutagenesis ; Mutation ; *DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; Gene Editing/methods ; },
abstract = {Site-directed and saturation mutagenesis are critical DNA methodologies for studying protein structure and function. For plasmid-based gene mutation, PCR and overlap-extension PCR involve tedious cloning steps. When the plasmid size is large, PCR yield may be too low for cloning; and for saturation mutagenesis of a single codon, one experiment may not enough to generate all twenty coding variants. Oligo-mediated recombineering sidesteps the complicated cloning process by homologous recombination between a mutagenic oligo and its target site. However, the low recombineering efficiency and inability to select for the recombinant makes it necessary to screen a large number of clones. Herein, we describe two plasmid-based mutagenic strategies: CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis (CRM) and saturation mutagenesis (CRSM). CRM and CRSM involve co-electroporation of target plasmid, sgRNA expression plasmid and mutagenic oligonucleotide into Escherichia coli cells with induced expression of λ-Red recombinase and Cas9, followed by plasmid extraction and characterization. We established CRM and CRSM via ampicillin resistance gene repair and mutagenesis of N-acetyl‑D‑neuraminic acid aldolase. The mutational efficiency was between 80 and 100% and all twenty amino acid coding variants were obtained at a target site via a single CRSM strategy. CRM and CRSM have the potential to be general plasmid-based gene mutagenesis tools.},
}
@article {pmid36847422,
year = {2023},
author = {Wang, Z and Huang, C and Wei, S and Zhu, P and Li, Y and Fu, W and Zhang, Y},
title = {A CRISPR/Cas12a-Mediated Sensitive DNA Detection System for Gene-Edited Rice.},
journal = {Journal of AOAC International},
volume = {106},
number = {3},
pages = {558-567},
doi = {10.1093/jaoacint/qsad022},
pmid = {36847422},
issn = {1944-7922},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; DNA ; },
abstract = {BACKGROUND: In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing.<br><br>OBJECTIVE: We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products.<br><br>METHODS: In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods.<br><br>RESULTS: The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially&#xa0;for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products.<br><br>CONCLUSION: We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice.<br><br>HIGHLIGHTS: The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.},
}
@article {pmid37127441,
year = {2023},
author = {Vink, JNA and Hayhurst, M and Gerth, ML},
title = {Harnessing CRISPR-Cas for oomycete genome editing.},
journal = {Trends in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tim.2023.03.017},
pmid = {37127441},
issn = {1878-4380},
abstract = {Oomycetes are a group of microorganisms that include pathogens responsible for devastating diseases in plants and animals worldwide. Despite their importance, the development of genome editing techniques for oomycetes has progressed more slowly than for model microorganisms. Here, we review recent breakthroughs in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technologies that are expanding the genome editing toolbox for oomycetes - from the original Cas9 study to Cas12a editing, ribonucleoprotein (RNP) delivery, and complementation. We also discuss some of the challenges to applying CRISPR-Cas in oomycetes and potential ways to overcome them. Advances in CRISPR-Cas technologies are being used to illuminate the biology of oomycetes, which ultimately can guide the development of tools for managing oomycete diseases.},
}
@article {pmid37124148,
year = {2023},
author = {Balke-Want, H and Keerthi, V and Cadinanos-Garai, A and Fowler, C and Gkitsas, N and Brown, AK and Tunuguntla, R and Abou-El-Enein, M and Feldman, SA},
title = {Non-viral chimeric antigen receptor (CAR) T cells going viral.},
journal = {Immuno-oncology technology},
volume = {18},
number = {},
pages = {100375},
pmid = {37124148},
issn = {2590-0188},
abstract = {Chimeric antigen receptor (CAR) T cell therapy has made significant strides in the treatment of B-cell malignancies, but its application in treating solid tumors still poses significant challenges. Particularly, the widespread use of viral vectors to deliver CAR transgenes into T cells comes with limitations, including high costs and regulatory restrictions, which hinder the translation of novel genetic engineering concepts into clinical applications. Non-viral methods, such as transposon/transposase and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, offer promising alternatives for stable transgene insertion in CAR-T cells. These methods offer the potential to increase accessibility and efficiency in the development and delivery of CAR-T cell therapies. The main challenge in using non-viral methods, however, is their low knock-in efficiency, which leads to low transgene expression levels. In this review, we discuss recent developments in non-viral approaches for CAR-T cell production, the manufacturing requirements for clinical-grade production of non-viral CAR-T cells, and the adjustments needed in quality control for proper characterization of genomic features and evaluation of potential genotoxicity.},
}
@article {pmid37122009,
year = {2023},
author = {Zhang, L and Li, G and Zhang, Y and Cheng, Y and Roberts, N and Glenn, SE and DeZwaan-McCabe, D and Rube, HT and Manthey, J and Coleman, G and Vakulskas, CA and Qi, Y},
title = {Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {102},
pmid = {37122009},
issn = {1474-760X},
mesh = {Animals ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Mutagenesis ; Endonucleases/genetics/metabolism ; *Oryza/genetics/metabolism ; Genome, Plant ; Mammals/genetics ; },
abstract = {BACKGROUND: Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.<br><br>RESULTS: To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.<br><br>CONCLUSIONS: Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.},
}
@article {pmid36938935,
year = {2023},
author = {Park, J and Kang, SJ and Go, S and Lee, J and An, J and Chung, HS and Jeong, C and Ahn, DR},
title = {Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.},
journal = {Biomaterials science},
volume = {11},
number = {9},
pages = {3241-3251},
doi = {10.1039/d2bm01901a},
pmid = {36938935},
issn = {2047-4849},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Gene Editing ; RNA/genetics ; },
abstract = {Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).},
}
@article {pmid36780174,
year = {2023},
author = {Ma, T and Chen, X and Wang, M},
title = {Intracellular Delivery of mRNA for Cell-Selective CRISPR/Cas9 Genome Editing using Lipid Nanoparticles.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {24},
number = {9},
pages = {e202200801},
doi = {10.1002/cbic.202200801},
pmid = {36780174},
issn = {1439-7633},
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; RNA, Messenger/genetics ; *COVID-19/genetics ; *Nanoparticles ; },
abstract = {Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA-based vaccines to prevent COVID-19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue-targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP-based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.},
}
@article {pmid36757014,
year = {2023},
author = {Liu, Y and Yuan, G and Hyden, B and Tuskan, GA and Abraham, PE and Yang, X},
title = {Expanding the application of anti-CRISPR proteins in plants for tunable genome editing.},
journal = {Plant physiology},
volume = {192},
number = {1},
pages = {60-64},
pmid = {36757014},
issn = {1532-2548},
mesh = {*Gene Editing ; *Plants/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
abstract = {Anti-CRISPR proteins are very efficient for inhibiting CRISPR/Cas9-based genome editing tools in both herbaceous and woody plant species.},
}
@article {pmid36083715,
year = {2023},
author = {Nold, SP and Sych, K and Imre, G and Fuhrmann, DC and Pfeilschifter, J and Vutukuri, R and Schnutgen, F and Wittig, I and Meisterknecht, J and Frank, S and Goren, I},
title = {Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.},
journal = {The FEBS journal},
volume = {290},
number = {9},
pages = {2338-2365},
doi = {10.1111/febs.16625},
pmid = {36083715},
issn = {1742-4658},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Protein Isoforms/metabolism ; RNA Splicing ; Carrier Proteins/genetics/metabolism ; Pyruvate Kinase/genetics/metabolism ; },
abstract = {The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.},
}
@article {pmid37119838,
year = {2023},
author = {Pal, P and Anand, U and Saha, SC and Sundaramurthy, S and Okeke, ES and Kumar, M and Radha, and Bontempi, E and Albertini, E and Dey, A and Di Maria, F},
title = {Novel CRISPR/Cas technology in the realm of algal bloom biomonitoring: Recent trends and future perspectives.},
journal = {Environmental research},
volume = {},
number = {},
pages = {115989},
doi = {10.1016/j.envres.2023.115989},
pmid = {37119838},
issn = {1096-0953},
abstract = {In conjunction with global climate change, progressive ocean warming, and acclivity in pollution and anthropogenic eutrophication, the incidence of harmful algal blooms (HABs) and cyanobacterial harmful algal blooms (CHABs) continue to expand in distribution, frequency, and magnitude. Algal bloom-related toxins have been implicated in human health disorders and ecological dysfunction and are detrimental to the national and global economy. Biomonitoring programs based on traditional monitoring protocols were characterised by some limitations that can be efficiently overdone using the CRISPR/Cas technology. In the present review, the potential and challenges of exploiting the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology for early detection of HABs and CHABs-associated toxigenic species were analysed. Based on more than 30 scientific papers, the main results indicate the great potential of CRISPR/Cas technology for this issue, even if the high sensitivity detected for the Cas12 and Cas13 platforms represents a possible interference risk.},
}
@article {pmid37079720,
year = {2023},
author = {Zhu, F and Zhao, Q},
title = {Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution.},
journal = {Analytical chemistry},
volume = {95},
number = {17},
pages = {6769-6774},
doi = {10.1021/acs.analchem.3c00218},
pmid = {37079720},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Antibodies ; Biotin ; Catalysis ; Immunoassay ; },
abstract = {Sensitive detection of small molecules is crucial for many applications, like biomedical diagnosis, food safety, and environmental analysis. Here, we describe a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution. An active DNA (acDNA) modified with a specific small molecule serves as a competitor for antibody binding and an activator of CRISPR-Cas12a. Large-sized antibody binding with this acDNA probe inactivates the collateral cleavage activity of CRISPR-Cas12a due to a steric effect. When free small molecule target exists, it replaces the small molecule-modified acDNA from antibody, triggering catalytic cleavage of DNA reporters by CRISPR-Cas12a, and strong fluorescence is generated. With this strategy, we achieved detection of three important small molecules as models, biotin, digoxin, and folic acid, at picomolar levels by using streptavidin or antibody as recognition elements. With the progress of DNA-encoded small molecules and antibody, the proposed strategy provides a powerful toolbox for detection of small molecules in wide applications.},
}
@article {pmid36805659,
year = {2023},
author = {Malaiwong, N and Porta-de-la-Riva, M and Krieg, M},
title = {FLInt: single shot safe harbor transgene integration via Fluorescent Landmark Interference.},
journal = {G3 (Bethesda, Md.)},
volume = {13},
number = {5},
pages = {},
pmid = {36805659},
issn = {2160-1836},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; Transgenes ; Animals, Genetically Modified ; *Genome ; *Chromosomes ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {The stable incorporation of transgenes and recombinant DNA material into the host genome is a bottleneck in many bioengineering applications. Due to the low efficiency, identifying the transgenic animals is often a needle in the haystack. Thus, optimal conditions require efficient screening procedures, but also known and safe landing sites that do not interfere with host expression, low input material and strong expression from the new locus. Here, we leverage an existing library of ≈300 different loci coding for fluorescent markers that are distributed over all 6 chromosomes in Caenorhabditis elegans as safe harbors for versatile transgene integration sites using CRISPR/Cas9. We demonstrated that a single crRNA was sufficient for cleavage of the target region and integration of the transgene of interest, which can be easily followed by loss of the fluorescent marker. The same loci can also be used for extrachromosomal landing sites and as co-CRISPR markers without affecting body morphology or animal behavior. Thus, our method overcomes the uncertainty of transgene location during random mutagenesis, facilitates easy screening through fluorescence interference and can be used as co-CRISPR markers without further influence in phenotypes.},
}
@article {pmid37119416,
year = {2023},
author = {Jogam, P and Sandhya, D and Alok, A and Peddaboina, V and Singh, SP and Abbagani, S and Zhang, B and Allini, VR},
title = {Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {37119416},
issn = {1573-4978},
abstract = {BACKGROUND: Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species.<br><br>METHODS AND RESULTS: The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV.<br><br>CONCLUSIONS: CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.},
}
@article {pmid37030292,
year = {2023},
author = {Stokar-Avihail, A and Fedorenko, T and Hör, J and Garb, J and Leavitt, A and Millman, A and Shulman, G and Wojtania, N and Melamed, S and Amitai, G and Sorek, R},
title = {Discovery of phage determinants that confer sensitivity to bacterial immune systems.},
journal = {Cell},
volume = {186},
number = {9},
pages = {1863-1876.e16},
doi = {10.1016/j.cell.2023.02.029},
pmid = {37030292},
issn = {1097-4172},
mesh = {*Bacteria/genetics/virology ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Viral Proteins/metabolism ; Mutation ; Bacterial Physiological Phenomena ; },
abstract = {Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.},
}
@article {pmid37014765,
year = {2023},
author = {Wang, P and Guo, B and Zhang, X and Wang, Y and Yang, G and Shen, H and Gao, S and Zhang, L},
title = {One-Pot Molecular Diagnosis of Acute Hepatopancreatic Necrosis Disease by Recombinase Polymerase Amplification and CRISPR/Cas12a with Specially Designed crRNA.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {16},
pages = {6490-6498},
doi = {10.1021/acs.jafc.2c08689},
pmid = {37014765},
issn = {1520-5118},
mesh = {Humans ; *Recombinases ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Nucleotidyltransferases ; Acute Disease ; Necrosis ; Nucleic Acid Amplification Techniques ; },
abstract = {Acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases in aquaculture, causing significant economic losses in seafood supplies worldwide. Early detection is critical for its prevention, which requires reliable and fast-responding diagnosis tools with point-of-care testing (POCT) capacity. Recombinase polymerase amplification (RPA) has been combined with CRISPR/Cas12a for AHPND diagnosis with a two-step procedure, but the operation is inconvenient and has the risk of carryover contamination. Here, we develop an RPA-CRISPR one-pot assay that integrates RPA and CRISPR/Cas12a cleavage into simultaneous reactions. Using the special design of crRNA, which is based on suboptimal protospacer adjacent motifs (PAM), RPA and Cas12a are made compatible in one pot. The assay is highly specific with a good sensitivity of 10[2] copies/reaction. This study provides a new choice for AHPND diagnosis with a POCT facility and sets a good example for developing RPA-CRISPR one-pot molecular diagnosis assays.},
}
@article {pmid36848135,
year = {2023},
author = {Pathak, N and Patino, CA and Ramani, N and Mukherjee, P and Samanta, D and Ebrahimi, SB and Mirkin, CA and Espinosa, HD},
title = {Cellular Delivery of Large Functional Proteins and Protein-Nucleic Acid Constructs via Localized Electroporation.},
journal = {Nano letters},
volume = {23},
number = {8},
pages = {3653-3660},
doi = {10.1021/acs.nanolett.2c04374},
pmid = {36848135},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acids ; Gene Editing ; Electroporation ; Proteins/genetics ; },
abstract = {Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (β-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.},
}
@article {pmid36775840,
year = {2023},
author = {Zhang, YC and Gao, Y and Ye, WN and Peng, YX and Zhu, KY and Gao, CF},
title = {CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae).},
journal = {Pest management science},
volume = {79},
number = {6},
pages = {2239-2246},
doi = {10.1002/ps.7404},
pmid = {36775840},
issn = {1526-4998},
mesh = {Animals ; *Insecticides/pharmacology ; *Hemiptera/genetics ; CRISPR-Cas Systems ; Neonicotinoids/pharmacology ; Thiamethoxam ; Nitro Compounds/pharmacology ; *Chlorpyrifos/pharmacology ; Insecticide Resistance/genetics ; },
abstract = {BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens.<br><br>RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2- and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight.<br><br>CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. &#xa9; 2023 Society of Chemical Industry.},
}
@article {pmid35831613,
year = {2022},
author = {Li, S and Wu, H and Chen, LL},
title = {Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system.},
journal = {Nature protocols},
volume = {17},
number = {9},
pages = {2085-2107},
pmid = {35831613},
issn = {1750-2799},
support = {55008728/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Humans ; RNA/genetics/metabolism ; RNA Splicing ; *RNA, Circular ; RNA, Messenger/genetics/metabolism ; Sequence Analysis, RNA/methods ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3-4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.},
}
@article {pmid35023328,
year = {2022},
author = {Bozoglu, T and Lee, S and Ziegler, T and Jurisch, V and Maas, S and Baehr, A and Hinkel, R and Hoenig, A and Hariharan, A and Kim, CI and Decker, S and Sami, H and Koppara, T and Oellinger, R and Müller, OJ and Frank, D and Megens, R and Nelson, P and Weber, C and Schnieke, A and Sperandio, M and Santamaria, G and Rad, R and Moretti, A and Laugwitz, KL and Soehnlein, O and Ogris, M and Kupatt, C},
title = {Endothelial Retargeting of AAV9 In Vivo.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {9},
number = {7},
pages = {e2103867},
pmid = {35023328},
issn = {2198-3844},
support = {/ERC_/European Research Council/International ; },
mesh = {Animals ; Blood Pressure ; *Dependovirus/genetics ; *Endothelial Cells ; Mice ; Mice, Transgenic ; Swine ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2[CNN] AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2[CNN] AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE[-/-] mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2[CNN] AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2[CNN] AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2[CNN] AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2[CNN] AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2[CNN] AAV9-gRNA[eNOS] . Therefore, G2[CNN] AAV9 may enable gene transfer in vascular and atherosclerosis models.},
}
@article {pmid37118221,
year = {2023},
author = {Kushwaha, SK and Kumar, AA and Gupta, H and Marathe, SA},
title = {The Phylogenetic Study of the CRISPR-Cas System in Enterobacteriaceae.},
journal = {Current microbiology},
volume = {80},
number = {6},
pages = {196},
pmid = {37118221},
issn = {1432-0991},
mesh = {*Enterobacteriaceae/genetics ; Phylogeny ; *CRISPR-Cas Systems ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) system is a bacterial and archaeal adaptive immune system undergoing rapid multifaceted evolution. This evolution plausibly occurs due to the genetic exchanges of complete loci or individual entities. Here, we systematically investigate the evolutionary framework of the CRISPR-Cas system in six Enterobacteriaceae species and its evolutionary association with housekeeping genes as determined by the gyrB phenogram. The strains show high variability in the cas3 gene and the CRISPR1 locus among the closely related Enterobacteriaceae species, hinting at a series of genetic exchanges. The CRISPR leader is conserved, especially toward the distal end, and could be a core region of the leader. The spacers are conserved within the strains of most species, while some strains show unique sets of spacers. However, inter-species spacer conservation was rarely observed. For a considerable proportion of these spacers, protospacer sources were not detected. These results advance our understanding of the dynamics of the CRISPR-Cas system; however, the biological functions are yet to be characterised.},
}
@article {pmid37116358,
year = {2023},
author = {Zhao, D and Tang, J and Tan, Q and Xie, X and Zhao, X and Xing, D},
title = {CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection.},
journal = {Talanta},
volume = {260},
number = {},
pages = {124582},
doi = {10.1016/j.talanta.2023.124582},
pmid = {37116358},
issn = {1873-3573},
abstract = {Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application.},
}
@article {pmid37114789,
year = {2023},
author = {F Saad, J and A Saad, F},
title = {Gene Therapy for Alzheimer and Parkinson Diseases.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/1566523223666230419101023},
pmid = {37114789},
issn = {1875-5631},
abstract = {Alzheimer and Parkinson diseases are associated with cholinergic neuron loss and deterioration of bone mineral density. Gene therapy through either gene transfer, CRISPR gene editing, or CRISPR gene modulation holds the potential to cure Alzheimer and Parkinson diseases. The emerging role of weight-bearing exercise in the prevention of, and care for, osteoporosis, obesity, and diabetes has been previously recognized. Moreover, endurance exercise offers a viable alternative to reduce amyloid peptides deposits while increasing bone mineral density in Alzheimer and Parkinson patients. β-amyloid peptides, α-synuclein, and tau aggregates start building up two decades before the onset of Alzheimer and Parkinson diseases. Therefore, an early intervention program for the detection of these deposits is required to prevent or delay the onset of these diseases. This article spots light on the potential of gene therapy for Alzheimer and Parkinson diseases.},
}
@article {pmid37112797,
year = {2023},
author = {Teng, M and Liu, JL and Luo, Q and Zheng, LP and Yao, Y and Nair, V and Zhang, GP and Luo, J},
title = {Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses.},
journal = {Viruses},
volume = {15},
number = {4},
pages = {},
pmid = {37112797},
issn = {1999-4915},
support = {BBS/OS/NW/000007/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *Marek Disease ; Gene Editing ; CRISPR-Cas Systems ; Antibodies, Monoclonal/metabolism ; *Herpesvirus 2, Gallid/genetics ; Oncogene Proteins/metabolism ; Chickens ; *Poultry Diseases ; *Oncogene Proteins, Viral/genetics ; },
abstract = {Marek's disease (MD) caused by pathogenic Marek's disease virus type 1 (MDV-1) is one of the most important neoplastic diseases of poultry. MDV-1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV-1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV-1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.},
}
@article {pmid37108761,
year = {2023},
author = {Major, L and McClements, ME and MacLaren, RE},
title = {A Review of CRISPR Tools for Treating Usher Syndrome: Applicability, Safety, Efficiency, and In Vivo Delivery.},
journal = {International journal of molecular sciences},
volume = {24},
number = {8},
pages = {},
pmid = {37108761},
issn = {1422-0067},
support = {MR/V029924/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Usher Syndromes/genetics/therapy ; Genetic Therapy ; Epigenesis, Genetic ; },
abstract = {This review considers research into the treatment of Usher syndrome, a deaf-blindness syndrome inherited in an autosomal recessive manner. Usher syndrome mutations are markedly heterogeneous, involving many different genes, and research grants are limited due to minimal patient populations. Furthermore, gene augmentation therapies are impossible in all but three Usher syndromes as the cDNA sequence exceeds the 4.7 kb AAV packaging limit. It is, therefore, vital to focus research efforts on alternative tools with the broadest applicability. The CRISPR field took off in recent years following the discovery of the DNA editing activity of Cas9 in 2012. New generations of CRISPR tools have succeeded the original CRISPR/Cas9 model to enable more sophisticated genomic amendments such as epigenetic modification and precise sequence alterations. This review will evaluate the most popular CRISPR tools to date: CRISPR/Cas9, base editing, and prime editing. It will consider these tools in terms of applicability (in relation to the ten most prevalent USH2A mutations), safety, efficiency, and in vivo delivery potential with the intention of guiding future research investment.},
}
@article {pmid37108214,
year = {2023},
author = {Allemailem, KS and Almatroodi, SA and Almatroudi, A and Alrumaihi, F and Al Abdulmonem, W and Al-Megrin, WAI and Aljamaan, AN and Rahmani, AH and Khan, AA},
title = {Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management.},
journal = {International journal of molecular sciences},
volume = {24},
number = {8},
pages = {},
pmid = {37108214},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism ; Gene Editing ; *Neoplasms/genetics/therapy ; Technology ; },
abstract = {The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described.},
}
@article {pmid37107664,
year = {2023},
author = {Chapman, B and Han, JH and Lee, HJ and Ruud, I and Kim, TH},
title = {Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.},
journal = {Genes},
volume = {14},
number = {4},
pages = {},
pmid = {37107664},
issn = {2073-4425},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation ; Transcriptional Activation ; Cell Line ; },
abstract = {Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of IRF7 CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.},
}
@article {pmid37107564,
year = {2023},
author = {Yang, L and Li, H and Han, Y and Song, Y and Wei, M and Fang, M and Sun, Y},
title = {CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation.},
journal = {Genes},
volume = {14},
number = {4},
pages = {},
pmid = {37107564},
issn = {2073-4425},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Genome ; DNA Damage/genetics ; Gene Expression ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas) gene editing can induce P53 activation, large genome fragment deletions, and chromosomal structural variations. Here, gene expression was detected in host cells using transcriptome sequencing following CRISPR/Cas9 gene editing. We found that the gene editing reshaped the gene expression, and the number of differentially expressed genes was correlated with the gene editing efficiency. Moreover, we found that alternative splicing occurred at random sites and that targeting a single site for gene editing may not result in the formation of fusion genes. Further, gene ontology and KEGG enrichment analysis showed that gene editing altered the fundamental biological processes and pathways associated with diseases. Finally, we found that cell growth was not affected; however, the DNA damage response protein-γH2AX-was activated. This study revealed that CRISPR/Cas9 gene editing may induce cancer-related changes and provided basic data for research on the safety risks associated with the use of the CRISPR/Cas9 system.},
}
@article {pmid37106426,
year = {2023},
author = {Frank, E and Cailleret, M and Nelep, C and Fragner, P and Polentes, J and Herardot, E and El Kassar, L and Giraud-Triboult, K and Monville, C and Ben M'Barek, K},
title = {Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones.},
journal = {Stem cell research &amp; therapy},
volume = {14},
number = {1},
pages = {110},
pmid = {37106426},
issn = {1757-6512},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism ; Mutation ; Clone Cells ; },
abstract = {BACKGROUND: CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such "disease in a dish" models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious.<br><br>METHODS: Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones.<br><br>RESULTS: We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches.<br><br>CONCLUSIONS: This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening.},
}
@article {pmid37105976,
year = {2023},
author = {Yang, C and Ma, Z and Wang, K and Dong, X and Huang, M and Li, Y and Zhu, X and Li, J and Cheng, Z and Bi, C and Zhang, X},
title = {HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {2430},
pmid = {37105976},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *HMGN1 Protein/genetics ; Chromatin ; Genome ; Transcription Factors/genetics ; },
abstract = {C-to-G base editors have been successfully constructed recently, but limited work has been done on concurrent C-to-G and A-to-G base editing. In addition, there is also limited data on how chromatin-associated factors affect the base editing. Here, we test a series of chromatin-associated factors, and chromosomal protein HMGN1 was found to enhance the efficiency of both C-to-G and A-to-G base editing. By fusing HMGN1, GBE and ABE to Cas9, we develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE) which is capable of converting simultaneous A and C to G conversion with substantial editing efficiency. Accordingly, the HMGN1 role shown in this work and the resulting GGBE tool further broaden the genome manipulation capacity of CRISPR-directed base editors.},
}
@article {pmid37104211,
year = {2023},
author = {Xiong, L and Liu, Z and Li, J and Yao, S and Li, Z and Chen, X and Shen, L and Zhang, Z and Li, Y and Hou, Q and Zhang, Y and You, M and Yuchi, Z and You, S},
title = {Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.},
journal = {Toxins},
volume = {15},
number = {4},
pages = {},
pmid = {37104211},
issn = {2072-6651},
mesh = {Animals ; *Moths ; *Bacillus thuringiensis/genetics/metabolism ; Bacillus thuringiensis Toxins/metabolism ; CRISPR-Cas Systems ; Endotoxins/genetics/pharmacology/metabolism ; Larva ; Multidrug Resistance-Associated Protein 2 ; Hemolysin Proteins/genetics/pharmacology/metabolism ; Bacterial Proteins/genetics/pharmacology/metabolism ; Insecticide Resistance/genetics ; Insect Proteins/metabolism ; },
abstract = {Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.},
}
@article {pmid37088039,
year = {2023},
author = {Xiao, Y and Ren, H and Wang, H and Zou, D and Liu, Y and Li, H and Hu, P and Li, Y and Liu, Z and Lu, S},
title = {A rapid and inexpensive nucleic acid detection platform for Listeria monocytogenes based on the CRISPR/Cas12a system.},
journal = {Talanta},
volume = {259},
number = {},
pages = {124558},
doi = {10.1016/j.talanta.2023.124558},
pmid = {37088039},
issn = {1873-3573},
mesh = {Cattle ; Animals ; CRISPR-Cas Systems ; *Listeria monocytogenes/genetics ; Biological Assay ; Drug Contamination ; Recombinases ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; },
abstract = {Listeria monocytogenes (LM) is an important foodborne pathogen that is associated with a high mortality rate. Currently, there is an urgent need for an inexpensive and rapid assay for the large-scale diagnosis and monitoring of LM. To meet these requirements, we designed a one-step, low-cost platform for the simultaneous amplification and detection of LM based on the CRISPR/Cas12a system with a micro-amplification (named Cas12a-MA). This method utilizes a combination of CRISPR/Cas12a and recombinase polymerase amplification (RPA) in the same vessel to provide a contamination-free platform for rapid nucleic acid detection with high specificity and ultra-sensitivity. In this study, we screened for three specific genes and selected the hly gene in LM as the final target. Our data showed that the number of amplification products plays a crucial role in the function of the CRISPR/Cas12a system. Our method was then further optimized for the specific detection of target DNA on 4.4 CFU/g in 25min. These assays successfully detected LM in spiked pork samples and natural meat samples (pork, beef, and mutton). All results indicate that Cas12a-MA shows great promise for foodborne pathogen detection.},
}
@article {pmid37086563,
year = {2023},
author = {Tian, M and Zhang, R and Li, J},
title = {Emergence of CRISPR/Cas9-mediated bioimaging: A new dawn of in-situ detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {232},
number = {},
pages = {115302},
doi = {10.1016/j.bios.2023.115302},
pmid = {37086563},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; In Situ Hybridization, Fluorescence ; Gene Editing/methods ; },
abstract = {In-situ detection provides deep insights into the function of genes and their relationship with diseases by directly visualizing their spatiotemporal behavior. As an emerging in-situ imaging tool, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bioimaging can localize targets in living and fixed cells. CRISPR-mediated bioimaging has inherent advantages over the gold standard of fluorescent in-situ hybridization (FISH), including fast imaging, cost-effectiveness, and ease of preparation. Existing reviews have provided a detailed classification and overview of the principles of CRISPR-mediated bioimaging. However, the exploitation of potential clinical applicability of this bioimaging technique is still limited. Therefore, analyzing the potential value of CRISPR-mediated in-situ imaging is of great significance to the development of bioimaging. In this review, we initially discuss the available CRISPR-mediated imaging systems from the following aspects: summary of imaging substances, the design and optimization of bioimaging strategies, and factors influencing CRISPR-mediated in-situ detection. Subsequently, we highlight the potential of CRISPR-mediated bioimaging for application in biomedical research and clinical practice. Furthermore, we outline the current bottlenecks and future perspectives of CRISPR-based bioimaging. We believe that this review will facilitate the potential integration of bioimaging-related research with current clinical workflow.},
}
@article {pmid37027291,
year = {2023},
author = {Weng, Z and You, Z and Li, H and Wu, G and Song, Y and Sun, H and Fradlin, A and Neal-Harris, C and Lin, M and Gao, X and Zhang, Y},
title = {CRISPR-Cas12a Biosensor Array for Ultrasensitive Detection of Unamplified DNA with Single-Nucleotide Polymorphic Discrimination.},
journal = {ACS sensors},
volume = {8},
number = {4},
pages = {1489-1499},
doi = {10.1021/acssensors.2c02495},
pmid = {37027291},
issn = {2379-3694},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Reproducibility of Results ; DNA/genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; *Graphite ; Nucleotides ; },
abstract = {Quantitative polymerase chain reaction as a powerful tool for DNA detection has been pivotal to a vast range of applications, including disease screening, food safety assessment, environmental monitoring, and many others. However, the essential target amplification step in combination with fluorescence readout poses a significant challenge to rapid and streamlined analysis. The discovery and engineering of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) technology have recently paved the way for a novel approach to nucleic acid detection, but the majority of current CRISPR-mediated DNA detection platforms are limited by insufficient sensitivity and still require target preamplification. Herein, we report a CRISPR-Cas12a-mediated graphene field-effect transistor (gFET) array, named CRISPR Cas12a-gFET, for amplification-free, ultrasensitive, and reliable detection of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) targets. CRISPR Cas12a-gFET leverages the multiturnover trans-cleavage activity of CRISPR Cas12a for intrinsic signal amplification and ultrasensitivity of gFET. As demonstrated, CRISPR Cas12a-gFET achieves a limit of detection of 1 aM for the ssDNA human papillomavirus 16 synthetic target and 10 aM for the dsDNA Escherichia coli plasmid target without target preamplification. In addition, an array of 48 sensors on a single 1.5 cm × 1.5 cm chip is employed to improve data reliability. Finally, Cas12a-gFET demonstrates the capability to discriminate single-nucleotide polymorphisms. Together, the CRISPR Cas12a-gFET biosensor array provides a detection tool for amplification-free, ultrasensitive, reliable, and highly specific DNA detections.},
}
@article {pmid36800642,
year = {2023},
author = {Li, C and Georgakopoulou, A and Newby, GA and Chen, PJ and Everette, KA and Paschoudi, K and Vlachaki, E and Gil, S and Anderson, AK and Koob, T and Huang, L and Wang, H and Kiem, HP and Liu, DR and Yannaki, E and Lieber, A},
title = {In vivo HSC prime editing rescues sickle cell disease in a mouse model.},
journal = {Blood},
volume = {141},
number = {17},
pages = {2085-2099},
doi = {10.1182/blood.2022018252},
pmid = {36800642},
issn = {1528-0020},
mesh = {Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Anemia, Sickle Cell/genetics/therapy ; Hematopoietic Stem Cells ; Hemoglobin, Sickle/genetics ; },
abstract = {Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of βS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.},
}
@article {pmid36738269,
year = {2023},
author = {Brower-Toland, B and Shyu, C and Vega-Sanchez, ME and Slewinski, TL},
title = {Pedigree or identity? How genome editing can fundamentally change the path for crop development.},
journal = {Journal of experimental botany},
volume = {74},
number = {9},
pages = {2794-2798},
pmid = {36738269},
issn = {1460-2431},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genome, Plant/genetics ; Plant Breeding ; },
}
@article {pmid36111891,
year = {2023},
author = {Nguyen, NHK and Rafiee, R and Tagmount, A and Sobh, A and Loguinov, A and de Jesus Sosa, AK and Elsayed, AH and Gbadamosi, M and Seligson, N and Cogle, CR and Rubnitz, J and Ribeiro, R and Downing, J and Cao, X and Pounds, SB and Vulpe, CD and Lamba, JK},
title = {Genome-wide CRISPR/Cas9 screen identifies etoposide response modulators associated with clinical outcomes in pediatric AML.},
journal = {Blood advances},
volume = {7},
number = {9},
pages = {1769-1783},
doi = {10.1182/bloodadvances.2022007934},
pmid = {36111891},
issn = {2473-9537},
mesh = {Humans ; Child ; Etoposide/pharmacology/therapeutic use ; *CRISPR-Cas Systems ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology ; Cell Line ; DNA Helicases/genetics ; },
abstract = {Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.},
}
@article {pmid37111880,
year = {2023},
author = {Yuan, G and Liu, Y and Yao, T and Muchero, W and Chen, JG and Tuskan, GA and Yang, X},
title = {eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone '52-225'.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {8},
pages = {},
pmid = {37111880},
issn = {2223-7747},
abstract = {Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone '52-225' (P. trichocarpa × P. deltoides clone '52-225'). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.},
}
@article {pmid37111505,
year = {2023},
author = {Majumdar, A and Sharma, A and Belludi, R},
title = {Natural and Engineered Resistance Mechanisms in Plants against Phytoviruses.},
journal = {Pathogens (Basel, Switzerland)},
volume = {12},
number = {4},
pages = {},
pmid = {37111505},
issn = {2076-0817},
abstract = {Plant viruses, as obligate intracellular parasites, rely exclusively on host machinery to complete their life cycle. Whether a virus is pathogenic or not depends on the balance between the mechanisms used by both plants and viruses during the intense encounter. Antiviral defence mechanisms in plants can be of two types, i.e., natural resistance and engineered resistance. Innate immunity, RNA silencing, translational repression, autophagy-mediated degradation, and resistance to virus movement are the possible natural defence mechanisms against viruses in plants, whereas engineered resistance includes pathogen-derived resistance along with gene editing technologies. The incorporation of various resistance genes through breeding programmes, along with gene editing tools such as CRISPR/Cas technologies, holds great promise in developing virus-resistant plants. In this review, different resistance mechanisms against viruses in plants along with reported resistance genes in major vegetable crops are discussed.},
}
@article {pmid37110466,
year = {2023},
author = {Moreira, POL and Nogueira, PM and Monte-Neto, RL},
title = {Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania.},
journal = {Microorganisms},
volume = {11},
number = {4},
pages = {},
pmid = {37110466},
issn = {2076-2607},
abstract = {Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization-a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection-live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.},
}
@article {pmid37108063,
year = {2023},
author = {Gunitseva, N and Evteeva, M and Borisova, A and Patrushev, M and Subach, F},
title = {RNA-Dependent RNA Targeting by CRISPR-Cas Systems: Characterizations and Applications.},
journal = {International journal of molecular sciences},
volume = {24},
number = {8},
pages = {},
pmid = {37108063},
issn = {1422-0067},
abstract = {Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems.},
}
@article {pmid37107608,
year = {2023},
author = {He, Y and Yan, W and Long, L and Dong, L and Ma, Y and Li, C and Xie, Y and Liu, N and Xing, Z and Xia, W and Li, F},
title = {The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.},
journal = {Genes},
volume = {14},
number = {4},
pages = {},
doi = {10.3390/genes14040850},
pmid = {37107608},
issn = {2073-4425},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.},
}
@article {pmid37105997,
year = {2023},
author = {LaBauve, AE and Saada, EA and Jones, IKA and Mosesso, R and Noureddine, A and Techel, J and Gomez, A and Collette, N and Sherman, MB and Serda, RE and Butler, KS and Brinker, CJ and Schoeniger, JS and Sasaki, D and Negrete, OA},
title = {Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {6873},
pmid = {37105997},
issn = {2045-2322},
abstract = {Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann-Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.},
}
@article {pmid37105947,
year = {2023},
author = {Zhou, J and He, M and Yin, X and Yu, Y and Yao, D and Liang, H},
title = {Unexploited Performance of NLS in the dCas9-VPR-Mediated Transcriptional Activation.},
journal = {ACS chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acschembio.3c00195},
pmid = {37105947},
issn = {1554-8937},
abstract = {Nuclear localization signal (NLS) is a short peptide guiding the nuclear transport process, recognized as playing an important role in constructing clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) activators. Here, we investigate the effect of the position and number of the NLS on transcriptional activation based on the dCas9-VPR activator. Our results not only demonstrate that the position of the SV40 NLS could have different degrees of influence on activation efficiency but also, surprisingly, we find that the SV40 NLS plays a detrimental role. Complete deletion of the NLS from the system could increase the transcriptional activation efficiency by 2 to 4 times compared with the original dCas9-VPR. This finding is also supported by some typical first- and third-generation activators. Our work should be beneficial to the design of the NLS-based system.},
}
@article {pmid37104586,
year = {2023},
author = {VanderWal, AR and Park, JU and Polevoda, B and Nicosia, JK and Molina Vargas, AM and Kellogg, EH and O'Connell, MR},
title = {Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.},
journal = {Science (New York, N.Y.)},
volume = {380},
number = {6643},
pages = {410-415},
doi = {10.1126/science.abm1184},
pmid = {37104586},
issn = {1095-9203},
abstract = {Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.},
}
@article {pmid37100946,
year = {2023},
author = {},
title = {The gene-therapy revolution risks stalling if we don't talk about drug pricing.},
journal = {Nature},
volume = {616},
number = {7958},
pages = {629-630},
doi = {10.1038/d41586-023-01389-z},
pmid = {37100946},
issn = {1476-4687},
mesh = {*Drug Costs ; *Genetic Therapy ; Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid37100808,
year = {2023},
author = {Seda, M and Crespo, B and Corcelli, M and Osborn, DP and Jenkins, D},
title = {A CRISPR/Cas9-generated mutation in the zebrafish orthologue of PPP2R3B causes idiopathic scoliosis.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {6783},
pmid = {37100808},
issn = {2045-2322},
mesh = {Animals ; Adolescent ; Humans ; *Zebrafish/genetics/metabolism ; *Scoliosis/genetics ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics/metabolism ; Protein Phosphatase 2/genetics/metabolism ; Mutation ; },
abstract = {Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Here, we focus on PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a series of frameshift mutations in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres. In summary, we report a novel zebrafish model of IS and reduced bone mineral density. In future, it will be necessary to delineate the aetiology of these defects in relation to bone, muscle, neuronal and ependymal cilia function.},
}
@article {pmid37100476,
year = {2023},
author = {Deng, F and Li, Y and Hall, T and Vesey, G and Goldys, EM},
title = {Bi-functional antibody-CRISPR/Cas12a ribonucleoprotein conjugate for improved immunoassay performance.},
journal = {Analytica chimica acta},
volume = {1259},
number = {},
pages = {341211},
doi = {10.1016/j.aca.2023.341211},
pmid = {37100476},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems ; *Biosensing Techniques ; *Cryptosporidiosis ; *Cryptosporidium ; Immunoassay ; *Immunoconjugates ; Antibodies ; Ribonucleoproteins ; },
abstract = {Protein conjugates are commonly used in biochemistry, including diagnostic platforms such as antibody-based immunoassays. Antibodies can be bound to a variety of molecules creating conjugates with desirable functions, particularly for imaging and signal amplification. Cas12a is a recently discovered programable nuclease with the remarkable capability to amplify assay signals due to its trans-cleavage property. In this study, we directly conjugated antibody with Cas12a/gRNA ribonucleoprotein without the loss of function in either constituent. The conjugated antibody was suitable for immunoassays and the conjugated Cas12a was capable of amplifying the signal produced in an immunosensor without the need to change the original assay protocol. We applied the bi-functional antibody-Cas12a/gRNA conjugate to successfully detect two different types of targets, a whole pathogenic microorganism, Cryptosporidium, and a small protein, cytokine IFN-γ, with sensitivity reaching one single microorganism per sample and 10 fg/mL for IFN-γ, respectively. With simple substitution of the antibody conjugated with the Cas12a/gRNA RNP, this approach can potentially be applied to increase sensitivity of a variety of immunoassays for a broad range of different analytes.},
}
@article {pmid37100169,
year = {2023},
author = {Kraus, C and Sontheimer, EJ},
title = {Applications of Anti-CRISPR Proteins in Genome Editing and Biotechnology.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168120},
doi = {10.1016/j.jmb.2023.168120},
pmid = {37100169},
issn = {1089-8638},
abstract = {In the ten years since the discovery of the first anti-CRISPR (Acr) proteins, the number of validated Acrs has expanded rapidly, as has our understanding of the diverse mechanisms they employ to suppress natural CRISPR-Cas immunity. Many, though not all, function via direct, specific interaction with Cas protein effectors. The abilities of Acr proteins to modulate the activities and properties of CRISPR-Cas effectors have been exploited for an ever-increasing spectrum of biotechnological uses, most of which involve the establishment of control over genome editing systems. This control can be used to minimize off-target editing, restrict editing based on spatial, temporal, or conditional cues, limit the spread of gene drive systems, and select for genome-edited bacteriophages. Anti-CRISPRs have also been developed to overcome bacterial immunity, facilitate viral vector production, control synthetic gene circuits, and other purposes. The impressive and ever-growing diversity of Acr inhibitory mechanisms will continue to allow the tailored applications of Acrs.},
}
@article {pmid37098485,
year = {2023},
author = {Park, HM and Won, J and Park, Y and Anzaku, ET and Vankerschaver, J and Van Messem, A and De Neve, W and Shim, H},
title = {CRISPR-Cas-Docker: web-based in silico docking and machine learning-based classification of crRNAs with Cas proteins.},
journal = {BMC bioinformatics},
volume = {24},
number = {1},
pages = {167},
pmid = {37098485},
issn = {1471-2105},
mesh = {*CRISPR-Cas Systems ; *RNA/genetics ; Internet ; },
abstract = {BACKGROUND: CRISPR-Cas-Docker is a web server for in silico docking experiments with CRISPR RNAs (crRNAs) and Cas proteins. This web server aims at providing experimentalists with the optimal crRNA-Cas pair predicted computationally when prokaryotic genomes have multiple CRISPR arrays and Cas systems, as frequently observed in metagenomic data.<br><br>RESULTS: CRISPR-Cas-Docker provides two methods to predict the optimal Cas protein given a particular crRNA sequence: a structure-based method (in silico docking) and a sequence-based method (machine learning classification). For the structure-based method, users can either provide experimentally determined 3D structures of these macromolecules or use an integrated pipeline to generate 3D-predicted structures for in silico docking experiments.<br><br>CONCLUSION: CRISPR-Cas-Docker addresses the need of the CRISPR-Cas community to predict RNA-protein interactions in silico by optimizing multiple stages of computation and evaluation, specifically for CRISPR-Cas systems. CRISPR-Cas-Docker is available at www.crisprcasdocker.org as a web server, and at https://github.com/hshimlab/CRISPR-Cas-Docker as an open-source tool.},
}
@article {pmid36932303,
year = {2023},
author = {Lu, PJ and Zhang, P and Liu, YC and Jing, N and Guo, YN and Wang, PS and Su, LL and Guo, Q and Ma, Q and Xu, YM and Zhang, ST},
title = {Gene Repair of iPSC Line with GARS (G294R) Mutation of CMT2D Disease by CRISPR/Cas9.},
journal = {Current medical science},
volume = {43},
number = {2},
pages = {261-267},
pmid = {36932303},
issn = {2523-899X},
mesh = {Humans ; *Charcot-Marie-Tooth Disease/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; },
abstract = {OBJECTIVE: Charcot-Marie-Tooth disease (CMT) severely affects patient activity, and may cause disability. However, no clinical treatment is available to reverse the disease course. The combination of CRISPR/Cas9 and iPSCs may have therapeutic potential against nervous diseases, such as CMT.<br><br>METHODS: In the present study, the skin fibroblasts of CMT type 2D (CMT2D) patients with the c.880G>A heterozygous nucleotide mutation in the GARS gene were reprogrammed into iPSCs using three plasmids (pCXLE-hSK, pCXLE-hUL and pCXLE-hOCT3/4-shp5-F). Then, CRISPR/Cas9 technology was used to repair the mutated gene sites at the iPSC level.<br><br>RESULTS: An iPSC line derived from the GARS (G294R) family with fibular atrophy was successfully induced, and the mutated gene loci were repaired at the iPSC level using CRISPR/Cas9 technology. These findings lay the foundation for future research on drug screening and cell therapy.<br><br>CONCLUSION: iPSCs can differentiate into different cell types, and originate from autologous cells. Therefore, they are promising for the development of autologous cell therapies for degenerative diseases. The combination of CRISPR/Cas9 and iPSCs may open a new avenue for the treatment of nervous diseases, such as CMT.},
}
@article {pmid36693779,
year = {2023},
author = {Zhang, C and Wang, X and Liu, G and Ren, H and Liu, J and Jiang, Z and Zhang, Y},
title = {CRISPR/Cas9 and Chlorophyll Coordination Micelles for Cancer Treatment by Genome Editing and Photodynamic Therapy.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {19},
number = {17},
pages = {e2206981},
doi = {10.1002/smll.202206981},
pmid = {36693779},
issn = {1613-6829},
mesh = {Mice ; Animals ; Micelles ; Gene Editing ; Chlorophyll ; CRISPR-Cas Systems/genetics ; NF-E2-Related Factor 2 ; *Photochemotherapy ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR/Cas9-based gene therapy and photodynamic therapy both show promise for cancer treatment but still have their drawbacks limited by tumor microenvironment and long treatment duration. Herein, CRISPR/Cas9 genome editing and photodynamic strategy for a synergistic anti-tumor therapeutic modality is merged. Chlorophyll (Chl) extracted from natural green vegetables is encapsulated in Pluronic F127 (F127) micelles and Histidine-tagged Cas9 can be effectively chelated onto micelles via metal coordination by simple incubation, affording Cas9-Chl@F127 micelles. Mg[2+] acts as an enzyme cofactor to correlatively enhance Cas9 gene-editing activity. Upon laser irradiation, Chl as an effective photosensitizer generates reactive oxygen species (ROS) to kill tumor cells. Meanwhile, CRISPR/Cas9, mediated by dual deliberately designed gRNAs of APE1 and NRF2, can reprogram the tumor microenvironment by increasing the intracellular oxygen accumulation and impairing the oxidative defense system of tumor cells. Cas9-Chl@F127 micelles can responsively release Cas9 in the presence of abundant ATP or low pH in tumor cells. In a murine tumor model, Cas9-Chl@F127 complexed with dual gRNAs including APE1 and NRF2 significantly inhibits the tumor growth. Taken together, Cas9-Chl@F127 micelles, representing the first Chl-based green biomaterial for the delivery of Cas9, show great promise for the synergistic anti-tumor treatment by PDT and gene editing.},
}
@article {pmid37097167,
year = {2023},
author = {Wang, L and Huang, X and Jin, Q and Tang, J and Zhang, H and Zhang, JR and Wu, H},
title = {Two-Component Response Regulator OmpR Regulates Mucoviscosity through Energy Metabolism in Klebsiella pneumoniae.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0054423},
doi = {10.1128/spectrum.00544-23},
pmid = {37097167},
issn = {2165-0497},
abstract = {Hypermucoviscosity is a hallmark of hypervirulent Klebsiella pneumoniae (hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosity in vitro and virulence in vivo in a mouse pneumonia model. The ΔompR mutant lost its mucoidy but retained its capsule level and comparable rmpADC expression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in ΔompR mutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the ΔompR mutant, the atp operon encoding F-type ATP synthase and the gcvTHP encoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of the atp operon, not the gcvTHP, suggesting that OmpR regulates the expression of the atp operon directly and gcvTHP indirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of ΔompR cells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity. IMPORTANCE Hypermucoviscosity is a critical virulent factor for Klebsiella pneumoniae infections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity in K. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence of K. pneumoniae in a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the ΔompR mutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the ΔompR mutant phenotype. Our findings provide new insights into the regulation of K. pneumoniae hypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.},
}
@article {pmid37097160,
year = {2023},
author = {Esquerra-Ruvira, B and Baquedano, I and Ruiz, R and Fernandez, A and Montoliu, L and Mojica, FJM},
title = {Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering.},
journal = {Microbial biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1751-7915.14266},
pmid = {37097160},
issn = {1751-7915},
abstract = {Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5'-NGG-3' PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5'-NGG-3' PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.},
}
@article {pmid37096163,
year = {2023},
author = {McMahon, MA and Rahdar, M and Mukhopadhyay, S and Bui, HH and Hart, C and Damle, S and Courtney, M and Baughn, MW and Cleveland, DW and Bennett, CF},
title = {GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.},
journal = {Molecular therapy. Nucleic acids},
volume = {32},
number = {},
pages = {289-301},
pmid = {37096163},
issn = {2162-2531},
abstract = {Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.},
}
@article {pmid37010434,
year = {2023},
author = {Tian, X and Zhou, Y and Wang, S and Gao, M and Xia, Y and Li, Y and Zhong, Y and Xu, W and Bai, L and Fu, B and Zhou, Y and Lee, HR and Deng, H and Lan, K and Feng, P and Zhang, J},
title = {Genome-Wide CRISPR-Cas9 Screen Identifies SMCHD1 as a Restriction Factor for Herpesviruses.},
journal = {mBio},
volume = {14},
number = {2},
pages = {e0054923},
pmid = {37010434},
issn = {2150-7511},
mesh = {Mice ; Animals ; *Virus Replication/genetics ; DNA Replication ; CRISPR-Cas Systems ; DNA, Viral/genetics ; *Herpesvirus 8, Human/physiology ; Gene Expression Regulation, Viral ; Mammals/metabolism ; Chromosomal Proteins, Non-Histone/genetics ; },
abstract = {Intrinsic immunity is the frontline of host defense against invading pathogens. To combat viral infection, mammalian hosts deploy cell-intrinsic effectors to block viral replication prior to the onset of innate and adaptive immunity. In this study, SMCHD1 is identified as a pivotal cellular factor that restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation through a genome-wide CRISPR-Cas9 knockout screen. Genome-wide chromatin profiling revealed that SMCHD1 associates with the KSHV genome, most prominently the origin of lytic DNA replication (ORI-Lyt). SMCHD1 mutants defective in DNA binding could not bind ORI-Lyt and failed to restrict KSHV lytic replication. Moreover, SMCHD1 functioned as a pan-herpesvirus restriction factor that potently suppressed a wide range of herpesviruses, including alpha, beta, and gamma subfamilies. SMCHD1 deficiency facilitated the replication of a murine herpesvirus in vivo. These findings uncovered SMCHD1 as a restriction factor against herpesviruses, and this could be harnessed for the development of antiviral therapies to limit viral infection. IMPORTANCE Intrinsic immunity represents the frontline of host defense against invading pathogens. However, our understanding of cell-intrinsic antiviral effectors remains limited. In this study, we identified SMCHD1 as a cell-intrinsic restriction factor that controlled KSHV lytic reactivation. Moreover, SMCHD1 restricted the replication of a wide range of herpesviruses by targeting the origins of viral DNA replication (ORIs), and SMCHD1 deficiency facilitated the replication of a murine herpesvirus in vivo. This study helps us to better understand intrinsic antiviral immunity, which may be harnessed to develop new therapeutics for the treatment of herpesvirus infection and the related diseases.},
}
@article {pmid36929689,
year = {2023},
author = {Mund, M and Weber, W and Degreif, D and Schiklenk, C},
title = {A MAD7-based genome editing system for Escherichia coli.},
journal = {Microbial biotechnology},
volume = {16},
number = {5},
pages = {1000-1010},
pmid = {36929689},
issn = {1751-7915},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Genetic Engineering ; Plasmids ; },
abstract = {A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli. This system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of this approach.},
}
@article {pmid36880749,
year = {2023},
author = {Homberger, C and Hayward, RJ and Barquist, L and Vogel, J},
title = {Improved Bacterial Single-Cell RNA-Seq through Automated MATQ-Seq and Cas9-Based Removal of rRNA Reads.},
journal = {mBio},
volume = {14},
number = {2},
pages = {e0355722},
pmid = {36880749},
issn = {2150-7511},
mesh = {*CRISPR-Cas Systems ; *Single-Cell Gene Expression Analysis ; High-Throughput Nucleotide Sequencing/methods ; RNA/genetics ; RNA, Ribosomal ; Gene Expression Profiling/methods ; Bacteria/genetics ; Sequence Analysis, RNA/methods ; RNA-Directed DNA Polymerase/genetics ; Single-Cell Analysis/methods ; },
abstract = {Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria. IMPORTANCE Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.},
}
@article {pmid37095570,
year = {2023},
author = {Glaser, V and Flugel, C and Kath, J and Du, W and Drosdek, V and Franke, C and Stein, M and Pruß, A and Schmueck-Henneresse, M and Volk, HD and Reinke, P and Wagner, DL},
title = {Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {89},
pmid = {37095570},
issn = {1474-760X},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; T-Lymphocytes ; DNA Breaks, Double-Stranded ; Genome ; },
abstract = {BACKGROUND: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells.<br><br>RESULTS: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro.<br><br>CONCLUSIONS: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.},
}
@article {pmid35606905,
year = {2023},
author = {Kouhen, M and García-Caparrós, P and Twyman, RM and Abdelly, C and Mahmoudi, H and Schillberg, S and Debez, A},
title = {Improving environmental stress resilience in crops by genome editing: insights from extremophile plants.},
journal = {Critical reviews in biotechnology},
volume = {43},
number = {4},
pages = {559-574},
doi = {10.1080/07388551.2022.2042481},
pmid = {35606905},
issn = {1549-7801},
mesh = {*Gene Editing/methods ; *Extremophiles ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Crops, Agricultural/genetics ; Genome, Plant ; },
abstract = {In basic and applied sciences, genome editing has become an indispensable tool, especially the versatile and adaptable CRISPR/Cas9 system. Using CRISPR/Cas9 in plants has enabled modifications of many valuable traits, including environmental stress tolerance, an essential aspect when it comes to ensuring food security under climate change pressure. The CRISPR toolbox enables faster and more precise plant breeding by facilitating: multiplex gene editing, gene pyramiding, and de novo domestication. In this paper, we discuss the most recent advances in CRISPR/Cas9 and alternative CRISPR-based systems, along with the technical challenges that remain to be overcome. A revision of the latest proof-of-concept and functional characterization studies has indeed provided more insight into the quantitative traits affecting crop yield and stress tolerance. Additionally, we focus on the applications of CRISPR/Cas9 technology in regard to extremophile plants, due to their significance on: industrial, ecological and economic levels. These still unexplored genetic resources could provide the means to harden our crops against the threat of climate change, thus ensuring food security over the next century.},
}
@article {pmid37095348,
year = {2023},
author = {Zhang, Z and Baxter, AE and Ren, D and Qin, K and Chen, Z and Collins, SM and Huang, H and Komar, CA and Bailer, PF and Parker, JB and Blobel, GA and Kohli, RM and Wherry, EJ and Berger, SL and Shi, J},
title = {Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37095348},
issn = {1546-1696},
abstract = {Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.},
}
@article {pmid37095220,
year = {2023},
author = {Hao, L and Zhao, RT and Welch, NL and Tan, EKW and Zhong, Q and Harzallah, NS and Ngambenjawong, C and Ko, H and Fleming, HE and Sabeti, PC and Bhatia, SN},
title = {CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics.},
journal = {Nature nanotechnology},
volume = {},
number = {},
pages = {},
pmid = {37095220},
issn = {1748-3395},
abstract = {Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be 'read out' via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.},
}
@article {pmid37094126,
year = {2023},
author = {Zhang, M and Peng, R and Peng, Q and Liu, S and Li, Z and Zhang, Y and Song, H and Yang, J and Xing, X and Wang, P and Qi, J and Gao, GF},
title = {Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {18},
pages = {e2215098120},
doi = {10.1073/pnas.2215098120},
pmid = {37094126},
issn = {1091-6490},
abstract = {CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.},
}
@article {pmid37093294,
year = {2023},
author = {Hunt, JMT and Samson, CA and Rand, AD and Sheppard, HM},
title = {Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells.},
journal = {Human genetics},
volume = {},
number = {},
pages = {},
pmid = {37093294},
issn = {1432-1203},
abstract = {Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them.},
}
@article {pmid37085898,
year = {2023},
author = {Marinov, GK and Kim, SH and Bagdatli, ST and Higashino, SI and Trevino, AE and Tycko, J and Wu, T and Bintu, L and Bassik, MC and He, C and Kundaje, A and Greenleaf, WJ},
title = {CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {85},
pmid = {37085898},
issn = {1474-760X},
support = {P50 HG007735/HG/NHGRI NIH HHS/United States ; U19 AI057266/AI/NIAID NIH HHS/United States ; UM1 HG009442/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; U01 HG009431/HG/NHGRI NIH HHS/United States ; F99 DK126120/DK/NIDDK NIH HHS/United States ; T32 GM007365/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Single-Stranded/genetics ; Genome ; CRISPR-Associated Protein 9/genetics ; Epigenome ; Gene Editing/methods ; },
abstract = {Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.},
}
@article {pmid37084235,
year = {2023},
author = {Li, Y and Zhi, S and Wu, T and Chen, HX and Kang, R and Ma, DZ and Songyang, Z and He, C and Liang, P and Luo, GZ},
title = {Systematic identification of CRISPR off-target effects by CROss-seq.},
journal = {Protein &amp; cell},
volume = {14},
number = {4},
pages = {299-303},
pmid = {37084235},
issn = {1674-8018},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37083429,
year = {2023},
author = {Parker, J},
title = {How has CRISPR transformed therapeutic drug discovery?.},
journal = {BioTechniques},
volume = {74},
number = {3},
pages = {119-121},
doi = {10.2144/btn-2023-0020},
pmid = {37083429},
issn = {1940-9818},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neurodegenerative Diseases/drug therapy/genetics ; Drug Discovery ; },
abstract = {The genome is the blueprint for life, and over the past decade, CRISPR has become a very powerful method for editing our genetic makeup. In this article, we will explore the importance of CRISPR in developing breakthrough therapies for monogenic conditions and neurodegenerative diseases, and for enhancing the effectiveness of immuno-oncology.},
}
@article {pmid36928106,
year = {2023},
author = {Wang, Q and Liu, J and Janssen, JM and Gonçalves, MAFV},
title = {Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.},
journal = {Nucleic acids research},
volume = {51},
number = {7},
pages = {3465-3484},
pmid = {36928106},
issn = {1362-4962},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Induced Pluripotent Stem Cells ; Gene Editing/methods ; Genomics ; Mammals ; },
abstract = {Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.},
}
@article {pmid36718951,
year = {2023},
author = {Develtere, W and Waegneer, E and Debray, K and De Saeger, J and Van Glabeke, S and Maere, S and Ruttink, T and Jacobs, TB},
title = {SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.},
journal = {Nucleic acids research},
volume = {51},
number = {7},
pages = {e37},
pmid = {36718951},
issn = {1362-4962},
support = {833866/ERC_/European Research Council/International ; },
mesh = {*Multiplex Polymerase Chain Reaction ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome ; Genetic Variation ; CRISPR-Cas Systems ; },
abstract = {Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.},
}
@article {pmid37091050,
year = {2023},
author = {Waitkus, J and Chang, Y and Liu, L and Puttaswamy, SV and Chung, T and Vargas, AMM and Dollery, SJ and O'Connell, MR and Cai, H and Tobin, GJ and Bhalla, N and Du, K},
title = {Gold Nanoparticle Enabled Localized Surface Plasmon Resonance on Unique Gold Nanomushroom Structures for On-Chip CRISPR-Cas13a Sensing.},
journal = {Advanced materials interfaces},
volume = {10},
number = {1},
pages = {},
pmid = {37091050},
issn = {2196-7350},
abstract = {A novel localized surface plasmon resonance (LSPR) system based on the coupling of gold nanomushrooms (AuNMs) and gold nanoparticles (AuNPs) is developed to enable a significant plasmonic resonant shift. The AuNP size, surface chemistry, and concentration are characterized to maximize the LSPR effect. A 31 nm redshift is achieved when the AuNMs are saturated by the AuNPs. This giant redshift also increases the full width of the spectrum and is explained by the 3D finite-difference time-domain (FDTD) calculation. In addition, this LSPR substrate is packaged in a microfluidic cell and integrated with a CRISPR-Cas13a RNA detection assay for the detection of the SARS-CoV-2 RNA targets. Once activated by the target, the AuNPs are cleaved from linker probes and randomly deposited on the AuNM substrate, demonstrating a large redshift. The novel LSPR chip using AuNP as an indicator is simple, specific, isothermal, and label-free; and thus, provides a new opportunity to achieve the next generation multiplexing and sensitive molecular diagnostic system.},
}
@article {pmid37090614,
year = {2023},
author = {Shmakov, SA and Barth, ZK and Makarova, KS and Wolf, YI and Brover, V and Peters, JE and Koonin, EV},
title = {Widespread CRISPR repeat-like RNA regulatory elements in CRISPR-Cas systems.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.03.03.530964},
pmid = {37090614},
abstract = {CRISPR- cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR- cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR- cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8 , or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.},
}
@article {pmid37085666,
year = {2023},
author = {Pan, C and Qi, Y},
title = {CRISPR-Combo-mediated orthogonal genome editing and transcriptional activation for plant breeding.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {37085666},
issn = {1750-2799},
abstract = {CRISPR-Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break-mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.},
}
@article {pmid37084259,
year = {2023},
author = {Li, Y and Wei, Y and Xu, S and Tan, Q and Zong, L and Wang, J and Wang, Y and Chen, J and Hong, L and Li, Y},
title = {AcrNET: Predicting anti-CRISPR with Deep Learning.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btad259},
pmid = {37084259},
issn = {1367-4811},
abstract = {MOTIVATION: As an important group of proteins discovered in phages, anti-CRISPR inhibits the activity of the immune system of bacteria (i.e., CRISPR-Cas), offering promise for gene editing and phage therapy. However, the prediction and discovery of anti-CRISPR are challenging due to their high variability and fast evolution. Existing biological studies rely on known CRISPR and anti-CRISPR pairs, which may not be practical considering the huge number. Computational methods struggle with prediction performance. To address these issues, we propose a novel deep neural network for anti-CRISPR analysis (AcrNET), which achieves significant performance.<br><br>RESULTS: On both the cross-fold and cross-dataset validation, our method outperforms the state-of-the-art methods. Notably, AcrNET improves the prediction performance by at least 15% regarding the F1 score for the cross-dataset test problem comparing with state-of-art Deep Learning method. Moreover, AcrNET is the first computational method to predict the detailed anti-CRISPR classes, which may help illustrate the anti-CRISPR mechanism. Taking advantage of a Transformer protein language model ESM-1b, which was pre-trained on 250 million protein sequences, AcrNET overcomes the data scarcity problem. Extensive experiments and analysis suggest that the Transformer model feature, evolutionary feature, and local structure feature complement each other, which indicates the critical properties of anti-CRISPR proteins. AlphaFold prediction, further motif analysis, and docking experiments further demonstrate that AcrNET can capture the evolutionarily conserved pattern and the interaction between anti-CRISPR and the target implicitly.<br><br>Web server: https://proj.cse.cuhk.edu.hk/aihlab/AcrNET/. Training code and pre-trained model are available at https://github.com/banma12956/AcrNET.<br><br>&#x2022;: Supplementary Information.},
}
@article {pmid37083687,
year = {2023},
author = {Figueroa, W and Cazares, A and Cazares, D and Wu, Y and de la Cruz, A and Welch, M and Kameyama, L and Nobrega, FL and Guarneros, G},
title = {Distribution and molecular evolution of the anti-CRISPR family AcrIF7.},
journal = {PLoS biology},
volume = {21},
number = {4},
pages = {e3002072},
doi = {10.1371/journal.pbio.3002072},
pmid = {37083687},
issn = {1545-7885},
abstract = {Anti-clustered regularly interspaced short palindromic repeats (CRISPRs) are proteins capable of blocking CRISPR-Cas systems and typically their genes are located on mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPR's function and evolution. Here, we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered 5 subclusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological, and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.},
}
@article {pmid37083261,
year = {2023},
author = {Philippidis, A},
title = {PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime.},
journal = {Human gene therapy},
volume = {34},
number = {7-8},
pages = {255-258},
doi = {10.1089/hum.2023.29238.bfs},
pmid = {37083261},
issn = {1557-7422},
mesh = {*Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Genetic Engineering ; },
}
@article {pmid37083189,
year = {2023},
author = {Zhou, M and Li, X and Wen, H and Huang, B and Ren, J and Zhang, J},
title = {The construction of CRISPR/Cas9-mediated FRET 16S rDNA sensor for detection of Mycobacterium tuberculosis.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3an00462g},
pmid = {37083189},
issn = {1364-5528},
abstract = {The simple and efficient detection of nucleic acids is important in the diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis). However, base mismatch will lead to false positive and false negative nucleic acid test, which seriously interferes with the accuracy of the final results. Herein, we demonstrated a CRISPR/Cas-9-mediated fluorescent strategy utilizing fluorescence resonance energy transfer (FRET) for the detection of bacteria. High-variable region of M. tuberculosis 16S rDNA fragment was used as the target, and CRISPR/Cas9 was used as the recognition element. The binding of the P1 probe of upconversion nanoparticles (UCNPs) @SiO2-P1 and the P2 probe of Fe3O4@Au-P2 caused the fluorescence quenching of UCNPs. In the presence of the target, the P2 probe hybridized with the target to form double-stranded DNA (dsDNA), which was recognized and cleaved by CRISPR/Cas9, resulting in the breaking of the P1-P2 duplex linkage. UCNPs moved away from Fe3O4@Au under a magnetic field, and the fluorescence signal was restored; bacteria were detected under the excitation of a 980 nm laser source. Using the CRISPR/Cas-9-mediated system, the sensor could distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. The limit of detection (LOD) was 20 CFU mL[-1] and the detection time was 2 h. It developed a new way of accurate nucleic acid detection for disease diagnosis.},
}
@article {pmid37081092,
year = {2023},
author = {Leski, TA and Spangler, JR and Wang, Z and Schultzhaus, Z and Taitt, CR and Dean, SN and Stenger, DA},
title = {Machine learning for design of degenerate Cas13a crRNAs using lassa virus as a model of highly variable RNA target.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {6506},
pmid = {37081092},
issn = {2045-2322},
mesh = {*RNA/metabolism ; *Lassa virus/genetics ; RNA Processing, Post-Transcriptional ; CRISPR-Cas Systems/genetics ; },
abstract = {The design of minimum CRISPR RNA (crRNA) sets for detection of diverse RNA targets using sequence degeneracy has not been systematically addressed. We tested candidate degenerate Cas13a crRNA sets designed for detection of diverse RNA targets (Lassa virus). A decision tree machine learning (ML) algorithm (RuleFit) was applied to define the top attributes that determine the specificity of degenerate crRNAs to elicit collateral nuclease activity. Although the total number of mismatches (0-4) is important, the specificity depends as well on the spacing of mismatches, and their proximity to the 5' end of the spacer. We developed a predictive algorithm for design of candidate degenerate crRNA sets, allowing improved discrimination between "included" and "excluded" groups of related target sequences. A single degenerate crRNA set adhering to these rules detected representatives of all Lassa lineages. Our general ML approach may be applied to the design of degenerate crRNA sets for any CRISPR/Cas system.},
}
@article {pmid37031406,
year = {2023},
author = {Yin, W and Hu, H},
title = {CRISPR/Cas9-Mediated Genome Editing via Homologous Recombination in a Centric Diatom Chaetoceros muelleri.},
journal = {ACS synthetic biology},
volume = {12},
number = {4},
pages = {1287-1296},
doi = {10.1021/acssynbio.3c00051},
pmid = {37031406},
issn = {2161-5063},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Diatoms/genetics ; Chlorophyll A ; Homologous Recombination/genetics ; DNA ; },
abstract = {Chaetoceros, the most abundant genus of marine planktonic diatoms, can be used in mariculture. An effective genetic transformation system with a short transformation period was established in Chaetoceros muelleri by electroporation in our previous study. In this study, a sequence-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 vector applicable for C. muelleri was constructed, and the expressions of sgRNA, resistance gene, and Cas9 gene were driven by the endogenous promoters U6, acetyl-CoA acetyltransferase, and fucoxanthin chlorophyll a/c binding protein, respectively, in the vector. Nitrate reductase (NR) and urease (URE) genes were edited in C. muelleri, and the NR knockout and NR/URE double-knockout lines displayed the strict auxotrophic phenotype. In addition, the DNA double-strand break was repaired by homologous recombination when a donor DNA was introduced. CRISPR/Cas9 technology was successfully applied to C. muelleri with an editing efficiency of up to 86%, providing a molecular tool for the study of basic biology in C. muelleri and its synthetic biology applications.},
}
@article {pmid37024659,
year = {2023},
author = {Zhou, J and Liu, G and Zhao, Y and Zhang, R and Tang, X and Li, L and Jia, X and Guo, Y and Wu, Y and Han, Y and Bao, Y and He, Y and Han, Q and Yang, H and Zheng, X and Qi, Y and Zhang, T and Zhang, Y},
title = {An efficient CRISPR-Cas12a promoter editing system for crop improvement.},
journal = {Nature plants},
volume = {9},
number = {4},
pages = {588-604},
pmid = {37024659},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Crops, Agricultural/genetics ; Edible Grain ; Promoter Regions, Genetic ; },
abstract = {Promoter editing represents an innovative approach to introduce quantitative trait variation (QTV) in crops. However, an efficient promoter editing system for QTV needs to be established. Here we develop a CRISPR-Cas12a promoter editing (CAPE) system that combines a promoter key-region estimating model and an efficient CRISPR-Cas12a-based multiplexed or singular editing system. CAPE is benchmarked in rice to produce QTV continuums for grain starch content and size by targeting OsGBSS1 and OsGS3, respectively. We then apply CAPE for promoter editing of OsD18, a gene encoding GA3ox in the gibberellin biosynthesis pathway. The resulting lines carry a QTV continuum of semidwarfism without significantly compromising grain measures. Field trials demonstrated that the OsD18 promoter editing lines have the same yield performance and antilodging phenotype as the Green Revolution OsSD1 mutants in different genetic backgrounds. Hence, promoter editing of OsD18 generates a quantitative Green Revolution trait. Together, we demonstrate a CAPE-based promoter editing and tuning pipeline for efficient production of useful QTV continuum in crops.},
}
@article {pmid37017652,
year = {2023},
author = {Hao, X and Mu, T and Sharshar, MM and Jia, Y and Zhong, W and Chen, Z and Wen, Q and Yang, M and Wang, C and Xing, J},
title = {CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.},
journal = {ACS synthetic biology},
volume = {12},
number = {4},
pages = {1204-1215},
doi = {10.1021/acssynbio.2c00676},
pmid = {37017652},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Ectothiorhodospiraceae/genetics/metabolism ; Sulfur/metabolism ; },
abstract = {Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.},
}
@article {pmid37014634,
year = {2023},
author = {Machens, F and Ran, G and Ruehmkorff, C and Meyer Auf der Heyde, J and Mueller-Roeber, B and Hochrein, L},
title = {PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {12},
number = {4},
pages = {1046-1057},
doi = {10.1021/acssynbio.2c00517},
pmid = {37014634},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics ; Transcriptional Activation/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida/genetics ; Transcription Factors/genetics ; },
abstract = {Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.},
}
@article {pmid36973416,
year = {2023},
author = {},
title = {A genome-scale CRISPR tool for targeting multiple gene family members at once.},
journal = {Nature plants},
volume = {9},
number = {4},
pages = {511-512},
pmid = {36973416},
issn = {2055-0278},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; CRISPR-Cas Systems ; },
}
@article {pmid36973414,
year = {2023},
author = {Hu, Y and Patra, P and Pisanty, O and Shafir, A and Belew, ZM and Binenbaum, J and Ben Yaakov, S and Shi, B and Charrier, L and Hyams, G and Zhang, Y and Trabulsky, M and Caldararu, O and Weiss, D and Crocoll, C and Avni, A and Vernoux, T and Geisler, M and Nour-Eldin, HH and Mayrose, I and Shani, E},
title = {Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.},
journal = {Nature plants},
volume = {9},
number = {4},
pages = {572-587},
pmid = {36973414},
issn = {2055-0278},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Arabidopsis/genetics ; Plant Breeding ; Plants/genetics ; Genome, Plant ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Gene Editing ; },
abstract = {Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.},
}
@article {pmid36932144,
year = {2023},
author = {},
title = {Removing the toxic enantiomer from crop products with selective gene editing.},
journal = {Nature plants},
volume = {9},
number = {4},
pages = {513-514},
pmid = {36932144},
issn = {2055-0278},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Engineering ; Genome, Plant ; },
}
@article {pmid36914142,
year = {2023},
author = {Eslahi, A and Alizadeh, F and Avan, A and Ferns, GA and Moghbeli, M and Reza Abbaszadegan, M and Mojarrad, M},
title = {New advancements in CRISPR based gene therapy of Duchenne muscular dystrophy.},
journal = {Gene},
volume = {867},
number = {},
pages = {147358},
doi = {10.1016/j.gene.2023.147358},
pmid = {36914142},
issn = {1879-0038},
mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Dystrophin/genetics ; CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; },
abstract = {Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.},
}
@article {pmid36893454,
year = {2023},
author = {Dallo, T and Krishnakumar, R and Kolker, SD and Ruffing, AM},
title = {High-Density Guide RNA Tiling and Machine Learning for Designing CRISPR Interference in Synechococcus sp. PCC 7002.},
journal = {ACS synthetic biology},
volume = {12},
number = {4},
pages = {1175-1186},
doi = {10.1021/acssynbio.2c00653},
pmid = {36893454},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Synechococcus/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; RNA ; },
abstract = {While CRISPRi was previously established in Synechococcus sp. PCC 7002 (hereafter 7002), the design principles for guide RNA (gRNA) effectiveness remain largely unknown. Here, 76 strains of 7002 were constructed with gRNAs targeting three reporter systems to evaluate features that impact gRNA efficiency. Correlation analysis of the data revealed that important features of gRNA design include the position relative to the start codon, GC content, protospacer adjacent motif (PAM) site, minimum free energy, and targeted DNA strand. Unexpectedly, some gRNAs targeting upstream of the promoter region showed small but significant increases in reporter expression, and gRNAs targeting the terminator region showed greater repression than gRNAs targeting the 3' end of the coding sequence. Machine learning algorithms enabled prediction of gRNA effectiveness, with Random Forest having the best performance across all training sets. This study demonstrates that high-density gRNA data and machine learning can improve gRNA design for tuning gene expression in 7002.},
}
@article {pmid36732423,
year = {2023},
author = {Kodackattumannil, P and Lekshmi, G and Kottackal, M and Sasi, S and Krishnan, S and Al Senaani, S and Amiri, KMA},
title = {Hidden pleiotropy of agronomic traits uncovered by CRISPR-Cas9 mutagenesis of the tyrosinase CuA-binding domain of the polyphenol oxidase 2 of eggplant.},
journal = {Plant cell reports},
volume = {42},
number = {4},
pages = {825-828},
pmid = {36732423},
issn = {1432-203X},
mesh = {*Monophenol Monooxygenase/genetics ; *Solanum melongena/genetics ; CRISPR-Cas Systems/genetics ; Amino Acid Sequence ; Mutagenesis ; Gene Editing ; },
}
@article {pmid36645771,
year = {2023},
author = {Bonsma-Fisher, M and Goyal, S},
title = {Dynamics of immune memory and learning in bacterial communities.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {36645771},
issn = {2050-084X},
mesh = {Humans ; *Immunologic Memory ; Evolution, Molecular ; Bacteria/genetics ; *Bacteriophages/genetics ; Models, Theoretical ; CRISPR-Cas Systems ; },
abstract = {From bacteria to humans, adaptive immune systems provide learned memories of past infections. Despite their vast biological differences, adaptive immunity shares features from microbes to vertebrates such as emergent immune diversity, long-term coexistence of hosts and pathogens, and fitness pressures from evolving pathogens and adapting hosts, yet there is no conceptual model that addresses all of these together. To this end, we propose and solve a simple phenomenological model of CRISPR-based adaptive immunity in microbes. We show that in coexisting phage and bacteria populations, immune diversity in both populations is coupled and emerges spontaneously, that bacteria track phage evolution with a context-dependent lag, and that high levels of diversity are paradoxically linked to low overall CRISPR immunity. We define average immunity, an important summary parameter predicted by our model, and use it to perform synthetic time-shift analyses on available experimental data to reveal different modalities of coevolution. Finally, immune cross-reactivity in our model leads to qualitatively different states of evolutionary dynamics, including an influenza-like traveling wave regime that resembles a similar state in models of vertebrate adaptive immunity. Our results show that CRISPR immunity provides a tractable model, both theoretically and experimentally, to understand general features of adaptive immunity.},
}
@article {pmid37080908,
year = {2023},
author = {Du, H and Yin, T and Wang, J and Jie, G},
title = {Multifunctional Photoelectrochemical Biosensor Based on ZnIn2S4/ZnS QDs@Au-Ag-Reversed Photocurrent of Cu-Metal-Organic Framework Coupled with CRISPR/Cas-12a-Shearing for Assay of Dual Targets.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c00846},
pmid = {37080908},
issn = {1520-6882},
abstract = {False positives and negatives in bioanalytical assays remain a persistent problem. Herein, a multifunctional photoelectrochemical (PEC) biosensor based on ZnIn2S4 (ZIS)/ZnS quantum dots (QDs)@Au-Ag-reversed photocurrent of Cu-metal-organic framework (MOF) coupled with CRISPR/Cas-12a-shearing was innovatively developed for assay of dual targets. First, Cu-MOF as a good PEC material shows cathodic photocurrent. Then, numerous ZIS/ZnS QDs were assembled to the Au-Ag nanoparticles (NPs) to prepare a stable and highly amplified signal probe, which can just match the energy level of Cu-MOFs and realized the polarity-reversed photocurrent of Cu-MOF for the first time. As the empty-core nanostructure of Au-Ag NPs has a high specific surface area and low material density, the bimetallic nanocrystal can much increase the reaction rate and improve the redox efficiency. When target CEA-produced cDNA opened the hairpin DNA (HP1 DNA) on the electrode, the ZIS/ZnS QDs@Au-Ag signal probe was conjugated to the electrode via DNA hybridization, achieving a significantly reversed PEC current for CEA detection. Moreover, the specific binding of kanamycin/aptamer generated the acDNA (activator), which can activate the trans-cleavage activity of the CRISPR-CAS12a system on ssDNA, so the signal probe was sheared and caused the obvious decrease of PEC signal for kanamycin detection. The newly developed ZIS/ZnS QDs@Au-Ag NPs displayed excellent PEC properties and reversed photocurrent to MOF and were combined with the unique CRISPR-Cas12a system to achieve sensitive detection of dual targets, which can open a new polarity-reversed PEC sensing platform for rapid and accurate analysis of multiple targets and can effectively avoid false positives results in clinical testing.},
}
@article {pmid37079992,
year = {2023},
author = {Han, J and Shin, J and Lee, ES and Cha, BS and Kim, S and Jang, Y and Kim, S and Park, KS},
title = {Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection.},
journal = {Biosensors &amp; bioelectronics},
volume = {232},
number = {},
pages = {115323},
doi = {10.1016/j.bios.2023.115323},
pmid = {37079992},
issn = {1873-4235},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.},
}
@article {pmid37078688,
year = {2023},
author = {Huang, X and Zhou, J and Yang, D and Zhang, J and Xia, X and Chen, YE and Xu, J},
title = {Decoding CRISPR-Cas PAM recognition with UniDesign.},
journal = {Briefings in bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1093/bib/bbad133},
pmid = {37078688},
issn = {1477-4054},
support = {GM149016/NH/NIH HHS/United States ; },
abstract = {The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.},
}
@article {pmid37078593,
year = {2023},
author = {Walker, MWG and Klompe, SE and Zhang, DJ and Sternberg, SH},
title = {Novel molecular requirements for CRISPR RNA-guided transposition.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad270},
pmid = {37078593},
issn = {1362-4962},
support = {DP2HG011650/NH/NIH HHS/United States ; },
abstract = {CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.},
}
@article {pmid37078570,
year = {2023},
author = {Xia, B and Viswanatha, R and Hu, Y and Mohr, SE and Perrimon, N},
title = {Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {37078570},
issn = {2050-084X},
support = {GM132087/GM/NIGMS NIH HHS/United States ; CA120964/CA/NCI NIH HHS/United States ; P01CA120964/GF/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila/genetics ; *Sirolimus ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Genome ; },
abstract = {Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells.},
}
@article {pmid37076490,
year = {2023},
author = {Kawasaki, S and Ono, H and Hirosawa, M and Kuwabara, T and Sumi, S and Lee, S and Woltjen, K and Saito, H},
title = {Programmable mammalian translational modulators by CRISPR-associated proteins.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {2243},
pmid = {37076490},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics ; Gene Expression Regulation ; Gene Regulatory Networks ; RNA, Messenger ; Mammals/genetics ; },
abstract = {Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology.},
}
@article {pmid37013669,
year = {2023},
author = {Zhao, NN and Tian, X and Ma, F and Zhang, CY},
title = {CRISPR/Cas12a-enhanced single-molecule counting for sensitive detection of flap endonuclease 1 activity at the single-cell level.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {33},
pages = {4939-4942},
doi = {10.1039/d3cc00642e},
pmid = {37013669},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; Flap Endonucleases ; Kinetics ; Biological Assay ; Coloring Agents ; *Biosensing Techniques ; },
abstract = {We develop a new fluorescent biosensor for flap endonuclease 1 (FEN1) assay based on CRISPR/Cas12-enhanced single-molecule counting. This biosensor is simple, selective, and sensitive with a detection limit of 2.325 × 10[-5] U and it is applicable for inhibitor screening, kinetic parameter analysis, and quantifying cellular FEN1 with single-cell sensitivity.},
}
@article {pmid36952025,
year = {2023},
author = {Ma, X and Suo, T and Zhao, F and Shang, Z and Chen, Y and Wang, P and Li, B},
title = {Integrating CRISPR/Cas12a with strand displacement amplification for the ultrasensitive aptasensing of cadmium(II).},
journal = {Analytical and bioanalytical chemistry},
volume = {415},
number = {12},
pages = {2281-2289},
pmid = {36952025},
issn = {1618-2650},
mesh = {Humans ; CRISPR-Cas Systems ; Cadmium ; Agriculture ; DNA, Single-Stranded ; *Environmental Pollutants ; Oligonucleotides ; *Biosensing Techniques ; },
abstract = {Cadmium ion (Cd(II)) is a pernicious environmental pollutant that has been shown to contaminate agricultural lands, accumulate through the food chain, and seriously threaten human health. At present, Cd(II) monitoring is dependent on centralized instruments, necessitating the development of rapid and on-site detection platforms. Against this backdrop, the present study reports on the development of a fluorometric aptasensor designed to target Cd(II), which is achieved through the integration of strand displacement amplification (SDA) and CRISPR/Cas12a. In the absence of Cd(II), the aptamer initiates SDA, resulting in the generation of a profusion of ssDNA that activates Cas12a, leading to a substantial increase in fluorescence output. Conversely, the presence of Cd(II) curtails the SDA efficiency, culminating in a significant reduction in fluorescence output. The proposed approach has been demonstrated to enable the selective detection of Cd(II) at concentrations of 60 pM, with the performance of the aptasensor validated in real water and rice samples. The proposed platform based on aptamer-target interaction holds immense promise as a signal-amplified and precise method for the detection of Cd(II) and has the potential to transform current hazard detection practices in food samples.},
}
@article {pmid37077165,
year = {2023},
author = {Liu, DN and Wu, HP and Zhou, GH},
title = {Research progress of visual detection in rapid on-site detection of pathogen nucleic acid.},
journal = {Yi chuan = Hereditas},
volume = {45},
number = {4},
pages = {306-323},
doi = {10.16288/j.yczz.22-323},
pmid = {37077165},
issn = {0253-9772},
abstract = {Nucleic acid detection is widely used in pathogen screening and detection due to its high sensitivity and specificity. With the increase of detection requirements and the development of amplification technology, nucleic acid detection methods are gradually developing towards simple, fast and low-cost. Quantitative polymerase chain reaction (qPCR), as the "gold standard" for nucleic acid detection, relies on expensive equipment and professional operators, which is not suitable for rapid on-site detection of pathogens. The visual detection method without relying on excitation light source or complex equipment can present the detection results in a more intuitive and portable way after combining with rapid and efficient amplification technology, which has the potential of point-of-care testing (POCT). This paper focuses on the reported application of amplification technology and CRISPR/Cas technology in visual detection and compares their advantages and disadvantages, so as to provide reference for POCT strategy based on pathogen nucleic acid.},
}
@article {pmid37075815,
year = {2023},
author = {Farzanehpour, M and Miri, A and Ghorbani Alvanegh, A and Esmaeili Gouvarchinghaleh, H},
title = {Viral Vectors, Exosomes, and Vexosomes: Potential Armamentarium for Delivering CRISPR/Cas to Cancer Cells.},
journal = {Biochemical pharmacology},
volume = {},
number = {},
pages = {115555},
doi = {10.1016/j.bcp.2023.115555},
pmid = {37075815},
issn = {1873-2968},
abstract = {The underlying cause of cancer is genetic disruption, so gene editing technologies, particularly CRISPR/Cas systems can be used to go against cancer. The field of gene therapy has undergone many transitions over its 40-year history. Despite its many successes, it has also suffered many failures in the battle against malignancies, causing really adverse effects instead of therapeutic outcomes. At the tip of this double-edged sword are viral and non-viral-based vectors, which have profoundly transformed the way scientists and clinicians develop therapeutic platforms. Viruses such as lentivirus, adenovirus, and adeno-associated viruses are the most common viral vectors used for delivering the CRISPR/Cas system into human cells. In addition, among non-viral vectors, exosomes, especially tumor-derived exosomes (TDEs), have proven to be quite effective at delivering this gene editing tool. The combined use of viral vectors and exosomes, called vexosomes, seems to be a solution to overcoming the obstacles of both delivery systems.},
}
@article {pmid37071672,
year = {2023},
author = {Bryant, WB and Yang, A and Griffin, SH and Zhang, W and Rafiq, AM and Han, W and Deak, F and Mills, MK and Long, X and Miano, JM},
title = {CRISPR-Cas9 Long-Read Sequencing for Mapping Transgenes in the Mouse Genome.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {163-175},
doi = {10.1089/crispr.2022.0099},
pmid = {37071672},
issn = {2573-1602},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Transgenes ; Genome/genetics ; Mice, Transgenic ; },
abstract = {Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.},
}
@article {pmid37071671,
year = {2023},
author = {Barrangou, R},
title = {Amplifying CRISPR: Next-Generation Diagnostics.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {85},
doi = {10.1089/crispr.2023.0004.editorial},
pmid = {37071671},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
}
@article {pmid37071670,
year = {2023},
author = {Panda, A and Suvakov, M and Mariani, J and Drucker, KL and Park, Y and Jang, Y and Kollmeyer, TM and Sarkar, G and Bae, T and Kim, JJ and Yoon, WH and Jenkins, RB and Vaccarino, FM and Abyzov, A},
title = {Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {176-182},
doi = {10.1089/crispr.2022.0050},
pmid = {37071670},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; DNA ; },
abstract = {The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.},
}
@article {pmid37071669,
year = {2023},
author = {Cress, B and Barrangou, R},
title = {Special Issue: Manipulating the Microbiome with CRISPR.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {86},
doi = {10.1089/crispr.2023.0005.cfp},
pmid = {37071669},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; *Microbiota/genetics ; },
}
@article {pmid37069118,
year = {2023},
author = {Maloshenok, LG and Abushinova, GA and Ryazanova, AY and Bruskin, SA and Zherdeva, VV},
title = {Visualizing the Nucleome Using the CRISPR-Cas9 System: From in vitro to in vivo.},
journal = {Biochemistry. Biokhimiia},
volume = {88},
number = {Suppl 1},
pages = {S123-S149},
pmid = {37069118},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; Genomics ; Microscopy, Fluorescence ; },
abstract = {One of the latest methods in modern molecular biology is labeling genomic loci in living cells using fluorescently labeled Cas protein. The NIH Foundation has made the mapping of the 4D nucleome (the three-dimensional nucleome on a timescale) a priority in the studies aimed to improve our understanding of chromatin organization. Fluorescent methods based on CRISPR-Cas are a significant step forward in visualization of genomic loci in living cells. This approach can be used for studying epigenetics, cell cycle, cellular response to external stimuli, rearrangements during malignant cell transformation, such as chromosomal translocations or damage, as well as for genome editing. In this review, we focused on the application of CRISPR-Cas fluorescence technologies as components of multimodal imaging methods for in vivo mapping of chromosomal loci, in particular, attribution of fluorescence signal to morphological and anatomical structures in a living organism. The review discusses the approaches to the highly sensitive, high-precision labeling of CRISPR-Cas components, delivery of genetically engineered constructs into cells and tissues, and promising methods for molecular imaging.},
}
@article {pmid37062563,
year = {2023},
author = {Zhu, R and Jiang, H and Li, C and Li, Y and Peng, M and Wang, J and Wu, Q and Yan, C and Bo, Q and Wang, J and Shen, C and Qin, P},
title = {CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia.},
journal = {Analytica chimica acta},
volume = {1257},
number = {},
pages = {341175},
doi = {10.1016/j.aca.2023.341175},
pmid = {37062563},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems ; *Pneumonia, Mycoplasma/diagnosis/genetics ; Point-of-Care Systems ; *Biosensing Techniques/methods ; },
abstract = {Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.},
}
@article {pmid36944129,
year = {2023},
author = {Opstelten, R and Freen-van Heeren, JJ},
title = {CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {87-89},
doi = {10.1089/crispr.2023.0003},
pmid = {36944129},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; *Neoplasms/genetics/therapy ; T-Lymphocytes ; },
}
@article {pmid36944123,
year = {2023},
author = {Morales-Moreno, MD and Valdés-Galindo, EG and Reza, MM and Fiordelisio, T and Peon, J and Hernandez-Garcia, A},
title = {Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {116-126},
doi = {10.1089/crispr.2022.0074},
pmid = {36944123},
issn = {2573-1602},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; RNA, Viral/genetics ; Gene Editing ; RNA, Guide, Kinetoplastida/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.},
}
@article {pmid36912819,
year = {2023},
author = {Cullot, G and Amintas, S and Karembé, L and Prouzet-Mauléon, V and Rébillard, J and Boureau, L and Cappellen, D and Bedel, A and Moreau-Gaudry, F and Dulucq, S and Dabernat, S and Turcq, B},
title = {Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {140-151},
doi = {10.1089/crispr.2022.0070},
pmid = {36912819},
issn = {2573-1602},
mesh = {Humans ; Fusion Proteins, bcr-abl/genetics ; *Carcinoma, Non-Small-Cell Lung/diagnosis/genetics ; *Lung Neoplasms/diagnosis/genetics ; CRISPR-Cas Systems ; Gene Editing ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/genetics ; ErbB Receptors/genetics ; },
abstract = {Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.},
}
@article {pmid36683240,
year = {2023},
author = {Lv, Y and Sun, Y and Zhou, Y and Khan, IM and Niazi, S and Yue, L and Zhang, Y and Wang, Z},
title = {Cascade DNA Circuits Mediated CRISPR-Cas12a Fluorescent Aptasensor based on Multifunctional Fe3 O4 @hollow-TiO2 @MoS2 Nanochains for Tetracycline Determination.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {19},
number = {16},
pages = {e2206105},
doi = {10.1002/smll.202206105},
pmid = {36683240},
issn = {1613-6829},
mesh = {*CRISPR-Cas Systems/genetics ; Molybdenum ; Tetracycline ; Anti-Bacterial Agents ; DNA ; Oligonucleotides ; Coloring Agents ; *Biosensing Techniques ; },
abstract = {Herein, for the first time, the CRISPR-Cas12a system is combined with aptamer, cascaded dynamic DNA network circuits, and Fe3 O4 @hollow-TiO2 @MoS2 nanochains (Fe3 O4 @h-TiO2 @MoS2 NCs) to construct an efficient sensing platform for tetracycline (TC) analysis. In this strategy, specific recognition of the target is transduced and amplified into H1-H2 duplexes containing the specific sequence of Cas12a-crRNA through an aptamer recognition module and the dual amplification dynamic DNA network. Subsequently, the obtained activated Cas12a protein non-specifically cleaves the adjacent reporter gene ssDNA-FAM to dissociate the FAM molecule from the quencher Fe3 O4 @h-TiO2 @MoS2 NCs, resulting in the recovery of the fluorescence signal and further signal amplification. Particularly, the synthesized multifunctional Fe3 O4 @h-TiO2 @MoS2 NCs composites also exhibit superb magnetic separability and photocatalytic degradation ability. Under optimal conditions, the aptasensor displays a distinct linear relationship with the logarithm of TC concentration, and the limit of detection is as low as 0.384 pg mL[-1] . Furthermore, the results of spiked recovery confirm the viability of the proposed aptasensor for TC quantification in real samples. This study extends the application of the CRISPR-Cas12a system in the field of analytical sensing and contributes new insights into the exploration of reliable tools for monitoring and treating hazards in food and environment.},
}
@article {pmid36637878,
year = {2023},
author = {Kohabir, KAV and Nooi, LO and Brink, A and Brakenhoff, RH and Sistermans, EA and Wolthuis, RMF},
title = {In Vitro CRISPR-Cas12a-Based Detection of Cancer-Associated TP53 Hotspot Mutations Beyond the crRNA Seed Region.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {127-139},
doi = {10.1089/crispr.2022.0077},
pmid = {36637878},
issn = {2573-1602},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; Bacterial Proteins/genetics ; *CRISPR-Associated Proteins/genetics ; DNA/genetics ; Mutation ; Endonucleases/genetics ; *Neoplasms/diagnosis/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.},
}
@article {pmid36367987,
year = {2023},
author = {Patchsung, M and Homchan, A and Aphicho, K and Suraritdechachai, S and Wanitchanon, T and Pattama, A and Sappakhaw, K and Meesawat, P and Wongsatit, T and Athipanyasilp, A and Jantarug, K and Athipanyasilp, N and Buahom, J and Visanpattanasin, S and Niljianskul, N and Chaiyen, P and Tinikul, R and Wichukchinda, N and Mahasirimongkol, S and Sirijatuphat, R and Angkasekwinai, N and Crone, MA and Freemont, PS and Joung, J and Ladha, A and Abudayyeh, O and Gootenberg, J and Zhang, F and Chewapreecha, C and Chanarat, S and Horthongkham, N and Pakotiprapha, D and Uttamapinant, C},
title = {A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.},
journal = {The CRISPR journal},
volume = {6},
number = {2},
pages = {99-115},
pmid = {36367987},
issn = {2573-1602},
support = {216457/WT_/Wellcome Trust/United Kingdom ; BB/M025632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; COVID-19 Testing ; Pandemics ; Point-of-Care Systems ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.},
}
@article {pmid36258038,
year = {2023},
author = {Mondal, G and VanLith, CJ and Nicolas, CT and Thompson, WS and Cao, WS and Hillin, L and Haugo, BJ and Brien, DRO and Kocher, JP and Kaiser, RA and Lillegard, JB},
title = {Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction.},
journal = {Gene therapy},
volume = {30},
number = {3-4},
pages = {386-397},
pmid = {36258038},
issn = {1476-5462},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; DNA ; DNA Repair ; },
abstract = {Gene editing for the cure of inborn errors of metabolism (IEMs) has been limited by inefficiency of adult hepatocyte targeting. Here, we demonstrate that in utero CRISPR/Cas9-mediated gene editing in a mouse model of hereditary tyrosinemia type 1 provides stable cure of the disease. Following this, we performed an extensive gene expression analysis to explore the inherent characteristics of fetal/neonatal hepatocytes that make them more susceptible to efficient gene editing than adult hepatocytes. We showed that fetal and neonatal livers are comprised of proliferative hepatocytes with abundant expression of genes involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs), key for efficient gene editing by CRISPR/Cas9. We demonstrated the same is true of hepatocytes after undergoing a regenerative stimulus (partial hepatectomy), where post-hepatectomy cells show a higher efficiency of HDR and correction. Specifically, we demonstrated that HDR-related genome correction is most effective in the replicative phase, or S-phase, of an actively proliferating cell. In conclusion, this study shows that taking advantage of or triggering cell proliferation, specifically DNA replication in S-phase, may serve as an important tool to improve efficiency of CRISPR/Cas9-mediated genome editing in the liver and provide a curative therapy for IEMs in both children and adults.},
}
@article {pmid37073287,
year = {2021},
author = {Lu, J and Fang, W and Huang, J and Li, S},
title = {The application of genome editing technology in fish.},
journal = {Marine life science &amp; technology},
volume = {3},
number = {3},
pages = {326-346},
pmid = {37073287},
issn = {2662-1746},
abstract = {The advent and development of genome editing technology has opened up the possibility of directly targeting and modifying genomic sequences in the field of life sciences with rapid developments occurring in the last decade. As a powerful tool to decipher genome data at the molecular biology level, genome editing technology has made important contributions to elucidating many biological problems. Currently, the three most widely used genome editing technologies include: zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR). Researchers are still striving to create simpler, more efficient, and accurate techniques, such as engineered base editors and new CRISPR/Cas systems, to improve editing efficiency and reduce off-target rate, as well as a near-PAMless SpCas9 variants to expand the scope of genome editing. As one of the important animal protein sources, fish has significant economic value in aquaculture. In addition, fish is indispensable for research as it serves as the evolutionary link between invertebrates and higher vertebrates. Consequently, genome editing technologies were applied extensively in various fish species for basic functional studies as well as applied research in aquaculture. In this review, we focus on the application of genome editing technologies in fish species detailing growth, gender, and pigmentation traits. In addition, we have focused on the construction of a zebrafish (Danio rerio) disease model and high-throughput screening of functional genes. Finally, we provide some of the future perspectives of this technology.},
}
@article {pmid37077216,
year = {2021},
author = {Symeonidi, E and Regalado, J and Schwab, R and Weigel, D},
title = {CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals.},
journal = {Quantitative plant biology},
volume = {2},
number = {},
pages = {e1},
pmid = {37077216},
issn = {2632-8828},
abstract = {Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.},
}
@article {pmid37059915,
year = {2023},
author = {Lear, SK and Lopez, SC and González-Delgado, A and Bhattarai-Kline, S and Shipman, SL},
title = {Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {37059915},
issn = {1750-2799},
abstract = {Biological signals occur over time in living cells. Yet most current approaches to interrogate biology, particularly gene expression, use destructive techniques that quantify signals only at a single point in time. A recent technological advance, termed the Retro-Cascorder, overcomes this limitation by molecularly logging a record of gene expression events in a temporally organized genomic ledger. The Retro-Cascorder works by converting a transcriptional event into a DNA barcode using a retron reverse transcriptase and then storing that event in a unidirectionally expanding clustered regularly interspaced short palindromic repeats (CRISPR) array via acquisition by CRISPR-Cas integrases. This CRISPR array-based ledger of gene expression can be retrieved at a later point in time by sequencing. Here we describe an implementation of the Retro-Cascorder in which the relative timing of transcriptional events from multiple promoters of interest is recorded chronologically in Escherichia coli populations over multiple days. We detail the molecular components required for this technology, provide a step-by-step guide to generate the recording and retrieve the data by Illumina sequencing, and give instructions for how to use custom software to infer the relative transcriptional timing from the sequencing data. The example recording is generated in 2 d, preparation of sequencing libraries and sequencing can be accomplished in 2-3 d, and analysis of data takes up to several hours. This protocol can be implemented by someone familiar with basic bacterial culture, molecular biology and bioinformatics. Analysis can be minimally run on a personal computer.},
}
@article {pmid37059525,
year = {2023},
author = {Schilling, C and Klau, LJ and Aachmann, FL and Rühmann, B and Schmid, J and Sieber, V},
title = {CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.},
journal = {Carbohydrate polymers},
volume = {312},
number = {},
pages = {120763},
doi = {10.1016/j.carbpol.2023.120763},
pmid = {37059525},
issn = {1879-1344},
mesh = {Humans ; Carbohydrate Sequence ; *Paenibacillus polymyxa/genetics ; CRISPR-Cas Systems ; Polysaccharides/chemistry ; Magnetic Resonance Spectroscopy ; },
abstract = {Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-β-d-Glc, 1➔4-β-d-Man and a 1,3,4-branching β-d-Gal residue with a sidechain comprising of a terminal β-d-Gal[3,4-Pyr] and 1➔3-β-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-β-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric β-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.},
}
@article {pmid36424489,
year = {2023},
author = {Yarnall, MTN and Ioannidi, EI and Schmitt-Ulms, C and Krajeski, RN and Lim, J and Villiger, L and Zhou, W and Jiang, K and Garushyants, SK and Roberts, N and Zhang, L and Vakulskas, CA and Walker, JA and Kadina, AP and Zepeda, AE and Holden, K and Ma, H and Xie, J and Gao, G and Foquet, L and Bial, G and Donnelly, SK and Miyata, Y and Radiloff, DR and Henderson, JM and Ujita, A and Abudayyeh, OO and Gootenberg, JS},
title = {Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.},
journal = {Nature biotechnology},
volume = {41},
number = {4},
pages = {500-512},
pmid = {36424489},
issn = {1546-1696},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Integrases ; DNA Cleavage ; Gene Editing ; DNA/genetics ; DNA End-Joining Repair/genetics ; },
abstract = {Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.},
}
@article {pmid36008610,
year = {2023},
author = {Shy, BR and Vykunta, VS and Ha, A and Talbot, A and Roth, TL and Nguyen, DN and Pfeifer, WG and Chen, YY and Blaeschke, F and Shifrut, E and Vedova, S and Mamedov, MR and Chung, JJ and Li, H and Yu, R and Wu, D and Wolf, J and Martin, TG and Castro, CE and Ye, L and Esensten, JH and Eyquem, J and Marson, A},
title = {High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.},
journal = {Nature biotechnology},
volume = {41},
number = {4},
pages = {521-531},
pmid = {36008610},
issn = {1546-1696},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L30 AI140341/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; P01 AI138962/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *DNA, Single-Stranded/genetics ; Genome ; Recombinational DNA Repair ; Mutation ; DNA ; Gene Editing ; DNA End-Joining Repair ; },
abstract = {Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 10[9] modified cells) exceeding those of conventional approaches.},
}
@article {pmid37059363,
year = {2023},
author = {Kattner, AA},
title = {When it doesn't run in the blood(vessels) - events involved in vascular disorders.},
journal = {Biomedical journal},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bj.2023.03.004},
pmid = {37059363},
issn = {2320-2890},
abstract = {In the current issue of the Biomedical Journal the underlying pathology of hemodynamic compromise in acute small subcortical infarction are elucidated. A follow-up study in patients with childhood Kawasaki disease is presented, as well as an insight into the gradually decreasing antigen expression in cases of acute myeloid leukemia. Furthermore this issue provides an exciting update concerning COVID-19 and the use of CRISPR-Cas, a review about computational approaches in the research of kidney stone formation, factors connected to central precocious puberty, and why a rock star of paleogenetics recently received a Nobel Prize. Additionally, this issue contains an article proposing the repurposing of the lung cancer drug Capmatinib, a study of how the gut microbiome develops in neonates, an impulse about the role of the transmembrane protein TMED3 in esophageal carcinoma, and the revelation about how competing endogenous RNA influences ischemic stroke. Lastly, genetic reasons for male infertility are discussed, as well as the relation between non-alcoholic fatty liver disease and chronic kidney disease.},
}
@article {pmid37058530,
year = {2023},
author = {Schelling, MA and Nguyen, GT and Sashital, DG},
title = {CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.},
journal = {PLoS biology},
volume = {21},
number = {4},
pages = {e3002065},
doi = {10.1371/journal.pbio.3002065},
pmid = {37058530},
issn = {1545-7885},
abstract = {CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.},
}
@article {pmid37052300,
year = {2023},
author = {McBride, TM and Cameron, SC and Fineran, PC and Fagerlund, RD},
title = {The biology and type I/III hybrid nature of type I-D CRISPR-Cas systems.},
journal = {The Biochemical journal},
volume = {480},
number = {7},
pages = {471-488},
doi = {10.1042/BCJ20220073},
pmid = {37052300},
issn = {1470-8728},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; Biology ; },
abstract = {Prokaryotes have adaptive defence mechanisms that protect them from mobile genetic elements and viral infection. One defence mechanism is called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). There are six different types of CRISPR-Cas systems and multiple subtypes that vary in composition and mode of action. Type I and III CRISPR-Cas systems utilise multi-protein complexes, which differ in structure, nucleic acid binding and cleaving preference. The type I-D system is a chimera of type I and III systems. Recently, there has been a burst of research on the type I-D CRISPR-Cas system. Here, we review the mechanism, evolution and biotechnological applications of the type I-D CRISPR-Cas system.},
}
@article {pmid36628413,
year = {2023},
author = {Kan, J and Cai, Y and Cheng, C and Chen, S and Jiang, C and He, Z and Yang, P},
title = {CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.},
journal = {Plant biotechnology journal},
volume = {21},
number = {5},
pages = {893-895},
pmid = {36628413},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Triticum/genetics/metabolism ; Eukaryotic Initiation Factor-4E/genetics/metabolism ; *Potyviridae ; *Mosaic Viruses/metabolism ; *Hordeum/metabolism ; },
}
@article {pmid36591868,
year = {2023},
author = {Li, Y and Wu, P and Zhu, M and Liang, M and Zhang, L and Zong, Y and Wan, M},
title = {High-Performance Delivery of a CRISPR Interference System via Lipid-Polymer Hybrid Nanoparticles Combined with Ultrasound-Mediated Microbubble Destruction for Tumor-Specific Gene Repression.},
journal = {Advanced healthcare materials},
volume = {12},
number = {10},
pages = {e2203082},
doi = {10.1002/adhm.202203082},
pmid = {36591868},
issn = {2192-2659},
mesh = {Humans ; CRISPR-Cas Systems ; Polymers ; Clustered Regularly Interspaced Short Palindromic Repeats ; Microbubbles ; *Neoplasms/genetics ; *Nanoparticles ; Lipids ; *MicroRNAs ; },
abstract = {The dCas9-based CRISPR interference (CRISPRi) system efficiently silences genes without causing detectable off-target activity, thus showing great potential for the treatment of cancer at the transcriptional level. However, due to the large size of the commonly used CRISPRi system, effective delivery of the system has been a challenge that hinders its application in the clinic. Herein, a combination of pH-responsive lipid-polymer hybrid nanoparticles (PLPNs) and ultrasound-mediated microbubble destruction (UMMD) is used for the delivery of the CRISPRi system. The core-shell structure of PLPNs can effectively be loaded with the CRISPRi plasmid, and increases the time spent in the circulating in vivo, and "actively target" cancer cells. Moreover, the combination of PLPNs with UMMD achieves a higher cellular uptake of the CRISPRi plasmid in vitro and retention in vivo. Furthermore, when PLPNs loaded with a CRISPRi plasmid that targets microRNA-10b (miR-10b) are used in combination with UMMD, it results in the effective repression of miR-10b in breast cancer, simultaneous disturbance of multiple cell migration and invasion-related signaling pathways, and a significant inhibition of lung metastasis. Thus, the established system presents a versatile, highly efficient, and safe strategy for delivery of the CRISPRi system both in vitro and in vivo.},
}
@article {pmid37052221,
year = {2023},
author = {Wang, D and Wang, X and Ye, F and Zou, J and Qu, J and Jiang, X},
title = {An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.2c10143},
pmid = {37052221},
issn = {1936-086X},
abstract = {Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a "sample-in-answer-out" single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.},
}
@article {pmid36988873,
year = {2023},
author = {Sinclair, F and Begum, AA and Dai, CC and Toth, I and Moyle, PM},
title = {Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing.},
journal = {Drug delivery and translational research},
volume = {13},
number = {5},
pages = {1500-1519},
pmid = {36988873},
issn = {2190-3948},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Therapy ; Gene Transfer Techniques ; Genetic Vectors ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.},
}
@article {pmid36898908,
year = {2023},
author = {Awan, MJA and Mahmood, MA and Naqvi, RZ and Mansoor, S},
title = {PASTE: a high-throughput method for large DNA insertions.},
journal = {Trends in plant science},
volume = {28},
number = {5},
pages = {509-511},
doi = {10.1016/j.tplants.2023.02.013},
pmid = {36898908},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; Gene Editing/methods ; DNA ; },
abstract = {Prime editing (PE) enables precise genome editing at targeted locus without inducing double-stranded breaks (DSBs). Despite its precision, PE lacks the tendency to integrate large DNA fragments into the genome. Recently, Yarnall et al. reported clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and an integrase-based system that conducts targeted integration of large DNA sequences (~36 kb) into the genome more efficiently.},
}
@article {pmid36806179,
year = {2023},
author = {Gasser, T},
title = {[Introduction to Gene Therapy].},
journal = {Fortschritte der Neurologie-Psychiatrie},
volume = {91},
number = {4},
pages = {129-134},
doi = {10.1055/a-2002-5176},
pmid = {36806179},
issn = {1439-3522},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy ; RNA ; },
abstract = {After many years of research and numerous setbacks, there are now undeniable success stories of gene therapies, namely the direct modification of genetic information on the DNA or RNA level. Both "ex vivo" strategies, i. e. the genetic manipulation of patient cells in a dish, as well as "in vivo" approaches are being used successfully. In addition to the supplementation of defective genes, the use of the CRISPR-Cas9 system to alter nuclear DNA sequences and the sequence-specific interference with the transcriptional process on the RNA level can be designated as gene therapies in a broad sense.},
}
@article {pmid35993441,
year = {2023},
author = {Guo, W and Wang, H and Kumar Tharkeshwar, A and Couthouis, J and Braems, E and Masrori, P and Van Schoor, E and Fan, Y and Ahuja, K and Moisse, M and Jacquemyn, M and Furtado Madeiro da Costa, R and Gajjar, M and Balusu, S and Tricot, T and Fumagalli, L and Hersmus, N and Janky, R and Impens, F and Vanden Berghe, P and Ho, R and Thal, DR and Vandenberghe, R and Hegde, ML and Chandran, S and De Strooper, B and Daelemans, D and Van Damme, P and Van Den Bosch, L and Verfaillie, C},
title = {CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.},
journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association},
volume = {19},
number = {4},
pages = {1245-1259},
pmid = {35993441},
issn = {1552-5279},
support = {NC/N001419/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; R01 NS088645/NS/NINDS NIH HHS/United States ; RF1 NS112719/NS/NINDS NIH HHS/United States ; R01 NS094535/NS/NINDS NIH HHS/United States ; R03 AG064266/AG/NIA NIH HHS/United States ; MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; MR/N013255/1/MRC_/Medical Research Council/United Kingdom ; /NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Amyotrophic Lateral Sclerosis/genetics ; *Frontotemporal Dementia/genetics ; *Induced Pluripotent Stem Cells/metabolism ; C9orf72 Protein/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Zebrafish/genetics/metabolism ; Neurons/metabolism ; DNA Repeat Expansion/genetics ; NIMA-Related Kinases/genetics/metabolism ; },
abstract = {INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one.<br><br>METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies.<br><br>RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage.<br><br>DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.},
}
@article {pmid37051232,
year = {2023},
author = {Liu, X and Li, G and Liu, Y and Zhou, F and Huang, X and Li, K},
title = {Advances in CRISPR/Cas gene therapy for inborn errors of immunity.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1111777},
pmid = {37051232},
issn = {1664-3224},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Mutation ; Technology ; },
abstract = {Inborn errors of immunity (IEIs) are a group of inherited disorders caused by mutations in the protein-coding genes involved in innate and/or adaptive immunity. Hematopoietic stem cell transplantation (HSCT) is a mainstay definitive therapy for many severe IEIs. However, the lack of HLA-matched donors increases the risk of developing severe immunological complications. Gene therapy provides long-term clinical benefits and could be an attractive therapeutic strategy for IEIs. In this review, we describe the development and evolution of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene-editing systems, including double-strand break (DSB)-based gene editing and DSB-free base editing or prime editing systems. Here, we discuss the advances in and issues associated with CRISPR/Cas gene editing tools and their potential as therapeutic alternatives for IEIs. We also highlight the progress of preclinical studies for the treatment of human genetic diseases, including IEIs, using CRISR/Cas and ongoing clinical trials based on this versatile technology.},
}
@article {pmid37050170,
year = {2023},
author = {Tyagi, A and Ali, S and Park, S and Bae, H},
title = {Exploring the Potential of Multiomics and Other Integrative Approaches for Improving Waterlogging Tolerance in Plants.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {7},
pages = {},
pmid = {37050170},
issn = {2223-7747},
abstract = {Soil flooding has emerged as a serious threat to modern agriculture due to the rapid global warming and climate change, resulting in catastrophic crop damage and yield losses. The most detrimental effects of waterlogging in plants are hypoxia, decreased nutrient uptake, photosynthesis inhibition, energy crisis, and microbiome alterations, all of which result in plant death. Although significant advancement has been made in mitigating waterlogging stress, it remains largely enigmatic how plants perceive flood signals and translate them for their adaptive responses at a molecular level. With the advent of multiomics, there has been significant progress in understanding and decoding the intricacy of how plants respond to different stressors which have paved the way towards the development of climate-resistant smart crops. In this review, we have provided the overview of the effect of waterlogging in plants, signaling (calcium, reactive oxygen species, nitric oxide, hormones), and adaptive responses. Secondly, we discussed an insight into past, present, and future prospects of waterlogging tolerance focusing on conventional breeding, transgenic, multiomics, and gene-editing approaches. In addition, we have also highlighted the importance of panomics for developing waterlogging-tolerant cultivars. Furthermore, we have discussed the role of high-throughput phenotyping in the screening of complex waterlogging-tolerant traits. Finally, we addressed the current challenges and future perspectives of waterlogging signal perception and transduction in plants, which warrants future investigation.},
}
@article {pmid37047677,
year = {2023},
author = {Anuchina, AA and Zaynitdinova, MI and Demchenko, AG and Evtushenko, NA and Lavrov, AV and Smirnikhina, SA},
title = {Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.},
journal = {International journal of molecular sciences},
volume = {24},
number = {7},
pages = {},
pmid = {37047677},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Recombinational DNA Repair ; Gene Editing/methods ; DNA End-Joining Repair ; Ligases/genetics ; Mad2 Proteins/genetics ; },
abstract = {This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.},
}
@article {pmid37047489,
year = {2023},
author = {Yıldırım, K and Kavas, M and Küçük, &#x130;S and Seçgin, Z and Saraç, &#xc7;G},
title = {Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {7},
pages = {},
pmid = {37047489},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Beta vulgaris/genetics ; Iran ; Gene Editing/methods ; Vegetables ; Sugars ; },
abstract = {Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10-55%), disease symptoms and biomass reduction (13-45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53-88%), especially for those targeting BCTIV's movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.},
}
@article {pmid37043430,
year = {2023},
author = {Porto, EM and Komor, AC},
title = {In the business of base editors: Evolution from bench to bedside.},
journal = {PLoS biology},
volume = {21},
number = {4},
pages = {e3002071},
pmid = {37043430},
issn = {1545-7885},
mesh = {Humans ; Adolescent ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Engineering ; Genetic Therapy ; },
abstract = {With the advent of recombinant DNA technology in the 1970s, the idea of using gene therapies to treat human genetic diseases captured the interest and imagination of scientists around the world. Years later, enabled largely by the development of CRISPR-based genome editing tools, the field has exploded, with academic labs, startup biotechnology companies, and large pharmaceutical corporations working in concert to develop life-changing therapeutics. In this Essay, we highlight base editing technologies and their development from bench to bedside. Base editing, first reported in 2016, is capable of installing C•G to T•A and A•T to G•C point mutations, while largely circumventing some of the pitfalls of traditional CRISPR/Cas9 gene editing. Despite their youth, these technologies have been widely used by both academic labs and therapeutics-based companies. Here, we provide an overview of the mechanics of base editing and its use in clinical trials.},
}
@article {pmid37020015,
year = {2023},
author = {Sasnauskas, G and Tamulaitiene, G and Druteika, G and Carabias, A and Silanskas, A and Kazlauskas, D and Venclovas, &#x10c; and Montoya, G and Karvelis, T and Siksnys, V},
title = {TnpB structure reveals minimal functional core of Cas12 nuclease family.},
journal = {Nature},
volume = {616},
number = {7956},
pages = {384-389},
pmid = {37020015},
issn = {1476-4687},
mesh = {*Endonucleases/metabolism ; *DNA Transposable Elements ; Gene Editing ; RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells[1,2]. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases[3-5], which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally[6], the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.},
}
@article {pmid37047235,
year = {2023},
author = {Naeem, M and Alkhnbashi, OS},
title = {Current Bioinformatics Tools to Optimize CRISPR/Cas9 Experiments to Reduce Off-Target Effects.},
journal = {International journal of molecular sciences},
volume = {24},
number = {7},
pages = {},
doi = {10.3390/ijms24076261},
pmid = {37047235},
issn = {1422-0067},
abstract = {The CRISPR-Cas system has evolved into a cutting-edge technology that has transformed the field of biological sciences through precise genetic manipulation. CRISPR/Cas9 nuclease is evolving into a revolutionizing method to edit any gene of any species with desirable outcomes. The swift advancement of CRISPR-Cas technology is reflected in an ever-expanding ecosystem of bioinformatics tools designed to make CRISPR/Cas9 experiments easier. To assist researchers with efficient guide RNA designs with fewer off-target effects, nuclease target site selection, and experimental validation, bioinformaticians have built and developed a comprehensive set of tools. In this article, we will review the various computational tools available for the assessment of off-target effects, as well as the quantification of nuclease activity and specificity, including web-based search tools and experimental methods, and we will describe how these tools can be optimized for gene knock-out (KO) and gene knock-in (KI) for model organisms. We also discuss future directions in precision genome editing and its applications, as well as challenges in target selection, particularly in predicting off-target effects.},
}
@article {pmid37047101,
year = {2023},
author = {Tenea, GN},
title = {Metabiotics Signature through Genome Sequencing and In Vitro Inhibitory Assessment of a Novel Lactococcus lactis Strain UTNCys6-1 Isolated from Amazonian Camu-Camu Fruits.},
journal = {International journal of molecular sciences},
volume = {24},
number = {7},
pages = {},
doi = {10.3390/ijms24076127},
pmid = {37047101},
issn = {1422-0067},
abstract = {Metabiotics are the structural components of probiotic bacteria, functional metabolites, and/or signaling molecules with numerous beneficial properties. A novel Lactococcus lactis strain, UTNCys6-1, was isolated from wild Amazonian camu-camu fruits (Myrciaria dubia), and various functional metabolites with antibacterial capacity were found. The genome size is 2,226,248 base pairs, and it contains 2248 genes, 2191 protein-coding genes (CDSs), 50 tRNAs, 6 rRNAs, 1 16S rRNA, 1 23S rRNA, and 1 tmRNA. The average GC content is 34.88%. In total, 2148 proteins have been mapped to the EggNOG database. The specific annotation consisted of four incomplete prophage regions, one CRISPR-Cas array, six genomic islands (GIs), four insertion sequences (ISs), and four regions of interest (AOI regions) spanning three classes of bacteriocins (enterolysin_A, nisin_Z, and sactipeptides). Based on pangenome analysis, there were 6932 gene clusters, of which 751 (core genes) were commonly observed within the 11 lactococcal strains. Among them, 3883 were sample-specific genes (cloud genes) and 2298 were shell genes, indicating high genetic diversity. A sucrose transporter of the SemiSWEET family (PTS system: phosphoenolpyruvate-dependent transport system) was detected in the genome of UTNCys6-1 but not the other 11 lactococcal strains. In addition, the metabolic profile, antimicrobial susceptibility, and inhibitory activity of both protein-peptide extract (PPE) and exopolysaccharides (EPSs) against several foodborne pathogens were assessed in vitro. Furthermore, UTNCys6-1 was predicted to be a non-human pathogen that was unable to tolerate all tested antibiotics except gentamicin; metabolized several substrates; and lacks virulence factors (VFs), genes related to the production of biogenic amines, and acquired antibiotic resistance genes (ARGs). Overall, this study highlighted the potential of this strain for producing bioactive metabolites (PPE and EPSs) for agri-food and pharmaceutical industry use.},
}
@article {pmid37045931,
year = {2023},
author = {Wang, R and Shu, X and Zhao, H and Xue, Q and Liu, C and Wu, A and Cheng, F and Wang, L and Zhang, Y and Feng, J and Wu, N and Li, M},
title = {Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {2078},
pmid = {37045931},
issn = {2041-1723},
abstract = {CreTA, CRISPR-regulated toxin-antitoxin (TA), safeguards CRISPR-Cas immune systems by inducing cell dormancy/death upon their inactivation. Here, we characterize a bacterial CreTA associating with the I-F CRISPR-Cas in Acinetobacter. CreT is a distinct bactericidal small RNA likely targeting several essential RNA molecules that are required to initiate protein synthesis. CreA guides the CRISPR effector to transcriptionally repress CreT. We further demonstrate a proof-of-concept antimicrobial strategy named ATTACK, which AssociaTes TA and CRISPR-Cas to Kill multidrug resistant (MDR) pathogens. In this design, CRISPR-Cas is programed to target antibiotic resistance gene(s) to selectively kill MDR pathogens or cure their resistance, and when CRISPR-Cas is inactivated or suppressed by unwanted genetic or non-genetic events/factors, CreTA triggers cell death as the last resort. Our data highlight the diversity of RNA toxins coevolving with CRISPR-Cas, and illuminate a combined strategy of CRISPR and TA antimicrobials to 'ATTACK' MDR pathogens.},
}
@article {pmid37043060,
year = {2023},
author = {Achudhan, AB and Kannan, P and Saleena, LM},
title = {CRISPR detection in metagenome-assembled genomes (MAGs) of coal mine.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {2},
pages = {122},
pmid = {37043060},
issn = {1438-7948},
abstract = {Bacterial and archaeal CRISPR-Cas systems provide adaptive immune protection against foreign mobile genetic elements. When viruses infect bacteria, a small portion of the viral DNA is inserted into the bacterial DNA in a specific pattern to produce segments known as CRISPR arrays. Metagenome assembled genomes (MAGs) were used in our study to identify the CRISPR sequence for determining the interacted phage. Metagenomic data from a coal mine was used to perform a computational study. From raw reads, 206151 contigs were assembled. Then contigs were clustered into 150 Metagenome assembled genomes from which 78 non-redundant MAGs were selected. Using the CHECKM standard, seven MAGs were found to have >80 completeness and <20 contaminations. Those MAGs were analyzed for the presence of CRISPR elements. Out of seven MAGs, four MAGs have the CRISPR elements and are searched against the VIROblast database. CRISPR arrays have 4, 1, 3, and 7 spacer sequences in the MAGs of Burkholderia, Acinetobacter, Oxalobacteraceae, and Burkholderia multivorans respectively. The uncultured Caudovirales phage genomic regions were present in the genomes of Burkholderia, Oxalobacteriaceae, and Burkholderia multivorans. This study follows the unconventional metagenomics workflow to provide a better understanding of bacteria and phage interactions.},
}
@article {pmid37042651,
year = {2023},
author = {Buyukyoruk, M and Henriques, WS and Wiedenheft, B},
title = {Clarifying CRISPR: Why Repeats Identified in the Human Genome Should Not Be Considered CRISPRs.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0106},
pmid = {37042651},
issn = {2573-1602},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (cas) are essential components of adaptive immune systems that protect bacteria and archaea from viral infection. CRISPR-Cas systems are found in about 40% of bacterial and 85% of archaeal genomes, but not in eukaryotic genomes. Recently, an article published in Communications Biology reported the identification of 12,572 putative CRISPRs in the human genome, which they call "hCRISPR." In this study, we attempt to reproduce this analysis and show that repetitive elements identified as putative CRISPR loci in the human genome contain neither the repeat-spacer-repeat architecture nor the cas genes characteristic of functional CRISPR systems.},
}
@article {pmid37041450,
year = {2023},
author = {Li, L and Li, S and Gu, D and Xu, Y and Wang, J},
title = {CRISPR-Cas12-Based Diagnostic Applications in Infectious and Zoonotic Diseases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2621},
number = {},
pages = {267-278},
pmid = {37041450},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Zoonoses ; *Communicable Diseases/genetics ; RNA ; *Nucleic Acids/genetics ; },
abstract = {Rapid detection of infectious and zoonotic diseases is very important for pathogen identification and infection control. Molecular diagnostic assays are well-known for high accuracy and sensitivity; however, conventional methods such as real-time PCR may require professional instruments and operations, preventing their wide applications in scenarios including animal quarantine. The recently developed CRISPR diagnostic (CRISPR-Dx) methods, employing the trans-cleavage activities of either Cas12 (e.g., HOLMES) or Cas13 (e.g., SHERLOCK), have shown great potential in rapid and convenient nucleic acid detection. Guided by specially designed CRISPR RNA (crRNA), Cas12 binds target DNA sequences and trans-cleaves ssDNA reporters, generating detectable signals, while Cas13 recognizes target ssRNA and trans-cleaves ssRNA reporters. To achieve high detection sensitivity, both HOLMES and SHERLOCK systems can be combined with pre-amplification procedures including both PCR and isothermal amplifications. Here, we present the employment of the HOLMESv2 method for convenient detection of the infectious and zoonotic diseases. Specifically, target nucleic acid is first amplified by LAMP or RT-LAMP, and the products are then detected by the thermophilic Cas12b. In addition, Cas12b reaction can be combined with LAMP amplification to achieve one-pot reaction systems. In this chapter, we provide a step-by-step description of the HOLMESv2-mediated rapid and sensitive detection of Japanese encephalitis virus (JEV), an RNA pathogen as an example.},
}
@article {pmid37041195,
year = {2023},
author = {Kong, X and Zhang, H and Li, G and Wang, Z and Kong, X and Wang, L and Xue, M and Zhang, W and Wang, Y and Lin, J and Zhou, J and Shen, X and Wei, Y and Zhong, N and Bai, W and Yuan, Y and Shi, L and Zhou, Y and Yang, H},
title = {Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {2046},
pmid = {37041195},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9/metabolism ; Dependovirus/genetics ; Mammals/genetics ; },
abstract = {The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5' T-rich Protospacer Adjacent Motifs (PAMs) and 5' C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5'-TTN and 5'-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.},
}
@article {pmid37036877,
year = {2023},
author = {Bai, Y and He, Y and Shen, CZ and Li, K and Li, DL and He, ZQ},
title = {CRISPR/Cas9-Mediated genomic knock out of tyrosine hydroxylase and yellow genes in cricket Gryllus bimaculatus.},
journal = {PloS one},
volume = {18},
number = {4},
pages = {e0284124},
pmid = {37036877},
issn = {1932-6203},
mesh = {Animals ; *Tyrosine 3-Monooxygenase/genetics/metabolism ; *Melanins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; Genomics ; },
abstract = {Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn't been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.},
}
@article {pmid37036720,
year = {2022},
author = {Testa, G and Mainardi, M and Vannini, E and Pancrazi, L and Cattaneo, A and Costa, M},
title = {Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {36},
number = {11},
pages = {e22498},
doi = {10.1096/fj.202101760RR},
pmid = {37036720},
issn = {1530-6860},
mesh = {Rats ; Animals ; *Receptor, trkA/genetics/metabolism ; *Receptor, Nerve Growth Factor/genetics ; Nerve Growth Factor/genetics/metabolism ; CRISPR-Cas Systems ; Receptors, Nerve Growth Factor/genetics/metabolism ; },
abstract = {The binding of nerve growth factor (NGF) to the tropomyosin-related kinase A (TrkA) and p75[NTR] receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA-p75[NTR] interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma-derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75[NTR] , or both. We found that TrkA-null cells become unresponsive to NGF. Conversely, the absence of p75[NTR] enhances the phosphorylation of TrkA and its effectors. Using a patch-clamp, we demonstrated that the individual activation of TrkA and p75[NTR] by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75[NTR] in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing.},
}
@article {pmid37036212,
year = {2023},
author = {Tsuji, T and Zhang, Y and Tseng, YH},
title = {Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {193},
pages = {},
doi = {10.3791/65083},
pmid = {37036212},
issn = {1940-087X},
mesh = {Mice ; Animals ; Mice, Knockout ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Adipose Tissue, Brown ; Dependovirus/genetics ; },
abstract = {Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.},
}
@article {pmid37035707,
year = {2023},
author = {Sun, R and Zhao, Y and Wang, W and Liu, JG and Chen, C},
title = {Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage.},
journal = {Chemical science},
volume = {14},
number = {14},
pages = {3839-3851},
pmid = {37035707},
issn = {2041-6520},
abstract = {Cas12a is one of the most commonly used Cas proteins for genome editing and gene regulation. The first key step for Cas12a to fulfill its function is to search for its target among numerous nonspecific and off-target sites. Cas12a utilizes one-dimensional diffusion along the contour of dsDNA to efficiently search for its target. However, due to a lack of structural information of the transient diffusing complex, the residues mediating the one-dimensional diffusion of Cas12a are unknown. Here, combining single-molecule and ensemble assays, we found that nonspecific interactions between Cas12a and dsDNA at the PAM downstream cause asymmetric target search regions of Cas12a flanking the PAM site, which guided us to identify a positive-charge-enriched alpha helix in the REC2 domain serving as a conserved element to facilitate one-dimensional diffusion-driven target search of AsCas12a, LbCas12a and FnCas12a. In addition, this alpha helix assists the target cleavage process of AsCas12a via stabilizing the cleavage states. Thus, neutralizing positive charges within this helix not only significantly slows target search but also enhances the specificity of AsCas12a both in vitro and in living cells. Similar behaviors are detected when residues mediating diffusion of SpCas9 are mutated. Thus, engineering residues mediating diffusion on dsDNA is a new avenue to optimize and enrich the versatile CRISPR-Cas toolbox.},
}
@article {pmid37035482,
year = {2023},
author = {Pougnet, R and Derbez, B and Troadec, MB},
title = {Mapping the 'Ethical' Controversy of Human Heritable Genome Editing: a Multidisciplinary Approach.},
journal = {Asian bioethics review},
volume = {15},
number = {2},
pages = {189-204},
pmid = {37035482},
issn = {1793-9453},
abstract = {UNLABELLED: Genome editing, for instance by CRISPR-Cas, is a major advancement of the last 10 years in medicine but questions ethically our practices. In particular, human embryo heritable genome editing is a source of great controversy. We explored how this ethical question was debated in the literature from PubMed database, in a period of 4 years (2016-2020) around the announcement of the 'CRISPR babies' Chinese experiment in November 2018. We evaluated the weight of the arguments for and against this topic, through an analysis of reviews published on this question. The most important arguments come from the technical perspective: safety issues and benefits, putative long-term effects on the future generations and the need to assess this aspect. Next, foreseeable clinical benefits and the alternatives to these methods are discussed. The number of people that would benefit from such techniques is also considered. However, social and anthropological issues are addressed in a more disparate way. Parenthood and desire for children are sometimes overlooked. Few authors mention social justice, stigmatisation and equality of access. Consent and information are more clearly addressed, as well as the question of the relationship between generations. Finally, the effects on the nature of humankind or human species are far from being consensual; the risks of enhancement, eugenics and transhumanism are raised. We conclude that the risks associated with the immaturity of the technique were at the forefront of the ethical debate on human embryo heritable genome editing. Their consequences were seen as more immediate and easier to handle than those of sociological or anthropological projections, which are more speculative in nature.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s41649-022-00234-1.},
}
@article {pmid37034382,
year = {2022},
author = {Hasanzadeh, A and Hamblin, MR and Kiani, J and Noori, H and Hardie, JM and Karimi, M and Shafiee, H},
title = {Could artificial intelligence revolutionize the development of nanovectors for gene therapy and mRNA vaccines?.},
journal = {Nano today},
volume = {47},
number = {},
pages = {},
pmid = {37034382},
issn = {1748-0132},
support = {R01 AI138800/AI/NIAID NIH HHS/United States ; R01 EB033866/EB/NIBIB NIH HHS/United States ; R33 AI140489/AI/NIAID NIH HHS/United States ; R61 AI140489/AI/NIAID NIH HHS/United States ; },
abstract = {Gene therapy enables the introduction of nucleic acids like DNA and RNA into host cells, and is expected to revolutionize the treatment of a wide range of diseases. This growth has been further accelerated by the discovery of CRISPR/Cas technology, which allows accurate genomic editing in a broad range of cells and organisms in vitro and in vivo. Despite many advances in gene delivery and the development of various viral and non-viral gene delivery vectors, the lack of highly efficient non-viral systems with low cellular toxicity remains a challenge. The application of cutting-edge technologies such as artificial intelligence (AI) has great potential to find new paradigms to solve this issue. Herein, we review AI and its major subfields including machine learning (ML), neural networks (NNs), expert systems, deep learning (DL), computer vision and robotics. We discuss the potential of AI-based models and algorithms in the design of targeted gene delivery vehicles capable of crossing extracellular and intracellular barriers by viral mimicry strategies. We finally discuss the role of AI in improving the function of CRISPR/Cas systems, developing novel nanobots, and mRNA vaccine carriers.},
}
@article {pmid37033919,
year = {2023},
author = {Braun, T and Pruene, A and Darguzyte, M and Vom Stein, AF and Nguyen, PH and Wagner, DL and Kath, J and Roig-Merino, A and Heuser, M and Riehm, LL and Schneider, A and Awerkiew, S and Talbot, SR and Bleich, A and Figueiredo, C and Bornhäuser, M and Stripecke, R},
title = {Non-viral TRAC-knocked-in CD19[KI]CAR-T and gp350[KI]CAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1086433},
pmid = {37033919},
issn = {1664-3224},
mesh = {Humans ; Mice ; Animals ; *Epstein-Barr Virus Infections ; *Burkitt Lymphoma/therapy ; Herpesvirus 4, Human ; *Receptors, Chimeric Antigen ; Hepatitis A Virus Cellular Receptor 2 ; Programmed Cell Death 1 Receptor ; Prospective Studies ; Receptors, Antigen, T-Cell, alpha-beta ; },
abstract = {INTRODUCTION: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8[+] T cell exhaustion and dysregulates CD4[+] T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen.<br><br>METHODS: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus.<br><br>RESULTS: Applying upscaled methods with the ExPERT ATx[&#xae;] MaxCyte system, KI efficacy was ~20% of the total ~2 &#xd7; 10[8] TCR-knocked-out (KO) generated cells. [KO]TCR[KI]CAR-T cells were co-cultured in vitro with the gp350[+]CD19[+] BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8[+ KI]CAR-T cells showed higher persistency than CD4[+ KI]CAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 &#xd7; 10[5] Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19[KI]CAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4[+] CD19[KI]CAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3[+]CD4[+] T cells. Administration of gp350[KI]CAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8[+]PD-1[+]LAG-3[+] gp350[KI]CAR-T cells were predominant in the bone marrow.<br><br>DISCUSSION: The two types of [KO]TCR[KI]CAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.},
}
@article {pmid37032710,
year = {2023},
author = {Illa-Berenguer, E and LaFayette, PR and Parrott, WA},
title = {Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1074641},
pmid = {37032710},
issn = {2673-3439},
abstract = {The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.},
}
@article {pmid36876986,
year = {2023},
author = {Inotsume, M and Chiba, T and Matsushima, T and Kurimoto, R and Nakajima, M and Kato, T and Shishido, K and Liu, L and Kawakami, K and Asahara, H},
title = {One-step generation of mice with gene editing by Tol2 transposon-dependent gRNA delivery.},
journal = {FEBS letters},
volume = {597},
number = {7},
pages = {975-984},
doi = {10.1002/1873-3468.14605},
pmid = {36876986},
issn = {1873-3468},
mesh = {Mice ; Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Plasmids ; Mice, Knockout ; Transposases/genetics ; },
abstract = {Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.},
}
@article {pmid36864759,
year = {2023},
author = {Feng, YL and Liu, SC and Chen, RD and Sun, XN and Xiao, JJ and Xiang, JF and Xie, AY},
title = {Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.},
journal = {Nucleic acids research},
volume = {51},
number = {6},
pages = {2740-2758},
pmid = {36864759},
issn = {1362-4962},
mesh = {Animals ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Recombinational DNA Repair ; Gene Editing/methods ; DNA/genetics ; DNA Repair ; Mammals/genetics ; },
abstract = {In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.},
}
@article {pmid36705519,
year = {2023},
author = {Warsinger-Pepe, N and Chang, C and Desroberts, CR and Akbari, OS},
title = {Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {13},
number = {4},
pages = {},
pmid = {36705519},
issn = {2160-1836},
mesh = {Animals ; *Drosophila melanogaster/genetics ; Temperature ; CRISPR-Cas Systems ; *Drosophila Proteins/genetics ; Heat-Shock Proteins/genetics ; Response Elements ; Hot Temperature ; },
abstract = {Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for both basic and applied studies, to precisely control gene expression. Here we characterize the use of Polycomb response elements and the inducible Heat-shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat-shock durations and rearing temperatures. We show that Polycomb response elements can significantly reduce expression of Cas9 under Heat-shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. We further demonstrate that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat-shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance.},
}
@article {pmid37032058,
year = {2023},
author = {Wang, X and Jin, W and Yang, Y and Ma, H and Liu, H and Lei, J and Wu, Y and Zhang, L},
title = {CRISPR/Cas12a-mediated Enzymatic recombinase amplification for rapid visual quantitative authentication of halal food.},
journal = {Analytica chimica acta},
volume = {1255},
number = {},
pages = {341144},
doi = {10.1016/j.aca.2023.341144},
pmid = {37032058},
issn = {1873-4324},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Drug Contamination ; Fluorescence ; Food Safety ; Recombinases/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {Economically motivated adulteration (EMA) has become a concern in food safety. We propose a CRISPR/Cas12a Mediated Enzymatic Recombinase Amplification detection system (CAMERA) that integrates Enzymatic Recombinase Amplification (ERA) and Cas12a cleavage to detect halal food adulteration. We designed and screened crRNA targeting CLEC, a porcine-specific nuclear single-copy gene, and optimized the reagent concentrations and incubation times for the ERA and Cas12a cleavage steps. CAMERA was highly specific for pork ingredients detection. The DNA concentration and fluorescence signal intensity relationship was linear at DNA concentrations of 20-0.032 ng/μL. CAMERA detected as few as two CLEC copies and quantified samples with porcine DNA content as low as 5% within 25 min. The system could be operated in a miniaturized working mode that requires no technical expertise or professional equipment, making CAMERA a valuable tool in resource-limited areas for the qualitative and quantitative detection of pork ingredients in halal food.},
}
@article {pmid37029174,
year = {2023},
author = {Feng, Z and Kong, D and Jin, W and He, K and Zhao, J and Liu, B and Xu, H and Yu, X and Feng, S},
title = {Rapid detection of isocitrate dehydrogenase 1 mutation status in glioma based on Crispr-Cas12a.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {5748},
pmid = {37029174},
issn = {2045-2322},
mesh = {Adult ; Humans ; Isocitrate Dehydrogenase/genetics/metabolism ; *Brain Neoplasms/genetics/diagnosis ; CRISPR-Cas Systems/genetics ; *Glioma/diagnosis/genetics ; Mutation ; },
abstract = {The aim is to use Crispr-Cas12a for the rapid detection of the single nucleotide polymorphism (SNP) of isocitrate dehydrogenase 1 (IDH1)-R132H locus and explore the effectiveness and consistency of this method with direct sequencing method for detecting IDH1-R132H of glioma tissue samples. 58 previous frozen tissue and 46 recent fresh tissue samples of adult diffuse glioma were selected to detect IDH1-R132H using Crispr-Cas12a. The results of immunohistochemistry (IHC) and direct sequencing methods were analyzed. We calculated the efficiency index of Crispr-Cas12a and IHC, and analyzed the consistency among Crispr-Cas12a, IHC and direct sequencing method using paired Chi-sequare test and Kappa identity test. We accomplished the rapid detection of IDH1-R132H in 60 min using Crispr-Cas12a. Regarding direct sequencing method as the gold standard, the sensitivity, specificity and consistency rate of Crispr-Cas12a was 91.4%, 95.7% and 93.1% in the frozen sample group, while 96.1%, 89.7% and 92.0% in the fresh sample group, respectively. Kappa test showed good consistency between the two methods (k = 0.858). Crispr-Cas12a can quickly and accurately detect IDH1-R132H and has good stability. It is a promising method to detect IDH1 mutation status intraoperatively.},
}
@article {pmid36917981,
year = {2023},
author = {Wu, Q and Wu, J and Karim, K and Chen, X and Wang, T and Iwama, S and Carobbio, S and Keen, P and Vidal-Puig, A and Kotter, MR and Bassett, A},
title = {Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.},
journal = {Molecular cell},
volume = {83},
number = {7},
pages = {1125-1139.e8},
doi = {10.1016/j.molcel.2023.02.011},
pmid = {36917981},
issn = {1097-4164},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Neurons ; Transcriptional Activation ; Chromatin/genetics ; },
abstract = {CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.},
}
@article {pmid36804635,
year = {2023},
author = {Yan, AL and Du, SW and Palczewski, K},
title = {Genome editing, a superior therapy for inherited retinal diseases.},
journal = {Vision research},
volume = {206},
number = {},
pages = {108192},
doi = {10.1016/j.visres.2023.108192},
pmid = {36804635},
issn = {1878-5646},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Retinal Diseases/genetics/therapy ; Retina ; },
abstract = {Gene augmentation and genome editing are promising strategies for the treatment of monogenic inherited retinal diseases. Although gene augmentation treatments are commercially available for inherited retinal diseases, there are many shortcomings that need to be addressed, like progressive retinal degeneration and diminishing efficacy over time. Innovative CRISPR-Cas9-based genome editing technologies have broadened the proportion of treatable genetic disorders and can greatly improve or complement treatment outcomes from gene augmentation. Progress in this relatively new field involves the development of therapeutics including gene disruption, ablate-and-replace strategies, and precision gene correction techniques, such as base editing and prime editing. By making direct edits to endogenous DNA, genome editing theoretically guarantees permanent gene correction and long-lasting treatment effects. Improvements to delivery modalities aimed at limiting persistent gene editor activity have displayed an improved safety profile and minimal off-target editing. Continued progress to advance precise gene correction and associated delivery strategies will establish genome editing as the preferred treatment for genetic retinal disorders. This commentary describes the applications, strengths, and drawbacks of conventional gene augmentation approaches, recent advances in precise genome editing in the retina, and promising preclinical strategies to facilitate the use of robust genome editing therapies in human patients.},
}
@article {pmid36006888,
year = {2023},
author = {Wan, Y and Jiang, Z},
title = {TransCrispr: Transformer Based Hybrid Model for Predicting CRISPR/Cas9 Single Guide RNA Cleavage Efficiency.},
journal = {IEEE/ACM transactions on computational biology and bioinformatics},
volume = {20},
number = {2},
pages = {1518-1528},
doi = {10.1109/TCBB.2022.3201631},
pmid = {36006888},
issn = {1557-9964},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Guide, Kinetoplastida/genetics ; Gene Editing/methods ; Neural Networks, Computer ; },
abstract = {CRISPR/Cas9 is a widely used genome editing tool for site-directed modification of deoxyribonucleic acid (DNA) nucleotide sequences. However, how to accurately predict and evaluate the on- and off-target effects of single guide RNA (sgRNA) is one of the key problems for CRISPR/Cas9 system. Using computational methods to obtain high cell-specific sensitivity and specificity is a prerequisite for the optimal design of sgRNAs. Inspired by the work of predecessors, we found that sgRNA on-target knockout efficacy was not only related to the original sequence but also affected by important biological features. Hence, we introduce a novel approach called TransCrispr, which integrates Transformer and convolutional neural network (CNN) architecture to predict sgRNA knockout efficacy. Firstly, we encode the sequence data and send the transformed sgRNA sequence, positional information, and biological features into the network as input. Then, the convolutional neural network will automatically learn an appropriate feature representation for the sgRNA sequence and combine it with the positional information for self-attention learning of the Transformer. Finally, a regression score is generated by predicting biological features. Experiments on seven public datasets illustrate that TransCrispr outperforms state-of-the-art methods in terms of prediction accuracy and generalization ability.},
}
@article {pmid37031646,
year = {2023},
author = {Mukherjee, S and Perveen, S and Negi, A and Sharma, R},
title = {Evolution of tuberculosis diagnostics: From molecular strategies to nanodiagnostics.},
journal = {Tuberculosis (Edinburgh, Scotland)},
volume = {140},
number = {},
pages = {102340},
pmid = {37031646},
issn = {1873-281X},
abstract = {Tuberculosis has remained a global concern for public health affecting the lives of people for ages. Approximately 10 million people are affected by the disease and 1.5 million succumb to the disease worldwide annually. The COVID-19 pandemic has highlighted the role of early diagnosis to win the battle against such infectious diseases. Thus, advancement in the diagnostic approaches to provide early detection forms the foundation to eradicate and manage contagious diseases like tuberculosis. The conventional diagnostic strategies include microscopic examination, chest X-ray and tuberculin skin test. The limitations associated with sensitivity and specificity of these tests demands for exploring new techniques like probe-based assays, CRISPR-Cas and microRNA detection. The aim of the current review is to envisage the correlation between both the conventional and the newer approaches to enhance the specificity and sensitivity. A significant emphasis has been placed upon nanodiagnostic approaches manipulating quantum dots, magnetic nanoparticles, and biosensors for accurate diagnosis of latent, active and drug-resistant TB. Additionally, we would like to ponder upon a reliable method that is cost-effective, reproducible, require minimal infrastructure and provide point-of-care to the patients.},
}
@article {pmid37030198,
year = {2023},
author = {Mingarro, G and Del Olmo, ML},
title = {Corrigendum to "Improvements in the genetic editing technologies: CRISPR-Cas and beyond" [Gene 852 (2023) 147064].},
journal = {Gene},
volume = {870},
number = {},
pages = {147404},
doi = {10.1016/j.gene.2023.147404},
pmid = {37030198},
issn = {1879-0038},
}
@article {pmid37029313,
year = {2023},
author = {Hassan, S and Ganai, BA},
title = {Deciphering the recent trends in pesticide bioremediation using genome editing and multi-omics approaches: a review.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {39},
number = {6},
pages = {151},
pmid = {37029313},
issn = {1573-0972},
abstract = {Pesticide pollution in recent times has emerged as a grave environmental problem contaminating both aquatic and terrestrial ecosystems owing to their widespread use. Bioremediation using gene editing and system biology could be developed as an eco-friendly and proficient tool to remediate pesticide-contaminated sites due to its advantages and greater public acceptance over the physical and chemical methods. However, it is indispensable to understand the different aspects associated with microbial metabolism and their physiology for efficient pesticide remediation. Therefore, this review paper analyses the different gene editing tools and multi-omics methods in microbes to produce relevant evidence regarding genes, proteins and metabolites associated with pesticide remediation and the approaches to contend against pesticide-induced stress. We systematically discussed and analyzed the recent reports (2015-2022) on multi-omics methods for pesticide degradation to elucidate the mechanisms and the recent advances associated with the behaviour of microbes under diverse environmental conditions. This study envisages that CRISPR-Cas, ZFN and TALEN as gene editing tools utilizing Pseudomonas, Escherichia coli and Achromobacter sp. can be employed for remediation of chlorpyrifos, parathion-methyl, carbaryl, triphenyltin and triazophos by creating gRNA for expressing specific genes for the bioremediation. Similarly, systems biology accompanying multi-omics tactics revealed that microbial strains from Paenibacillus, Pseudomonas putida, Burkholderia cenocepacia, Rhodococcus sp. and Pencillium oxalicum are capable of degrading deltamethrin, p-nitrophenol, chlorimuron-ethyl and nicosulfuron. This review lends notable insights into the research gaps and provides potential solutions for pesticide remediation by using different microbe-assisted technologies. The inferences drawn from the current study will help researchers, ecologists, and decision-makers gain comprehensive knowledge of value and application of systems biology and gene editing in bioremediation assessments.},
}
@article {pmid37028423,
year = {2023},
author = {Usluer, S and Hallast, P and Crepaldi, L and Zhou, Y and Urgo, K and Dincer, C and Su, J and Noell, G and Alasoo, K and El Garwany, O and Gerety, SS and Newman, B and Dovey, OM and Parts, L},
title = {Optimized whole-genome CRISPR interference screens identify ARID1A-dependent growth regulators in human induced pluripotent stem cells.},
journal = {Stem cell reports},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.stemcr.2023.03.008},
pmid = {37028423},
issn = {2213-6711},
abstract = {Perturbing expression is a powerful way to understand the role of individual genes, but can be challenging in important models. CRISPR-Cas screens in human induced pluripotent stem cells (iPSCs) are of limited efficiency due to DNA break-induced stress, while the less stressful silencing with an inactive Cas9 has been considered less effective so far. Here, we developed the dCas9-KRAB-MeCP2 fusion protein for screening in iPSCs from multiple donors. We found silencing in a 200 bp window around the transcription start site in polyclonal pools to be as effective as using wild-type Cas9 for identifying essential genes, but with much reduced cell numbers. Whole-genome screens to identify ARID1A-dependent dosage sensitivity revealed the PSMB2 gene, and enrichment of proteasome genes among the hits. This selective dependency was replicated with a proteasome inhibitor, indicating a targetable drug-gene interaction. Many more plausible targets in challenging cell models can be efficiently identified with our approach.},
}
@article {pmid37028002,
year = {2023},
author = {Zhu, Y and Wu, J and Zhou, Q},
title = {Functional DNA sensors integrated with nucleic acid signal amplification strategies for non-nucleic acid targets detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {230},
number = {},
pages = {115282},
doi = {10.1016/j.bios.2023.115282},
pmid = {37028002},
issn = {1873-4235},
abstract = {In addition to carrying and transmitting genetic material, some DNA molecules have specific binding ability or catalytic function. DNA with this special function is collectively referred to as functional DNA (fDNA), such as aptamer, DNAzyme and so on. fDNA has the advantages of simple synthetic process, low cost and low toxicity. It also has high chemical stability, recognition specificity and biocompatibility. In recent years, fDNA biosensors have been widely investigated as signal recognition elements and signal transduction elements for the detection of non-nucleic acid targets. However, the main problem of fDNA sensors is their limited sensitivity to trace targets, especially when the affinity of fDNA to the targets is low. To further improve the sensitivity, various nucleic acid signal amplification strategies (NASAS) are explored to improve the limit of detection of fDNA. In this review, we will introduce four NASAS (hybridization chain reaction, entropy-driven catalysis, rolling circle amplification, CRISPR/Cas system) and the corresponding design principles. The principle and application of these fDNA sensors integrated with signal amplification strategies for detection of non-nucleic acid targets are summarized. Finally, the main challenges and application prospects of NASAS integrated fDNA biosensing system are discussed.},
}
@article {pmid37025992,
year = {2023},
author = {Bandara, G and Falduto, GH and Luker, A and Bai, Y and Pfeiffer, A and Lack, J and Metcalfe, DD and Olivera, A},
title = {CRISPR/Cas9-engineering of HMC-1.2 cells renders a human mast cell line with a single D816V-KIT mutation: An improved preclinical model for research on mastocytosis.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1078958},
pmid = {37025992},
issn = {1664-3224},
mesh = {Humans ; Animals ; Mice ; *Mastocytosis, Systemic/drug therapy/genetics/pathology ; CRISPR-Cas Systems ; Proto-Oncogene Proteins c-kit/genetics/metabolism ; *Mastocytosis/genetics ; Mutation ; Cell Line ; },
abstract = {The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells. Despite these similarities in liquid culture, survival of HMC-1.3 cells was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.},
}
@article {pmid37021953,
year = {2023},
author = {Wu, SZ and Ryken, SE and Bezanilla, M},
title = {CRISPR-Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella) patens.},
journal = {Current protocols},
volume = {3},
number = {4},
pages = {e725},
doi = {10.1002/cpz1.725},
pmid = {37021953},
issn = {2691-1299},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Bryopsida/genetics ; Homologous Recombination ; Plasmids ; },
abstract = {Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR-Cas9 mediated genome editing, P. patens was unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR-Cas9, molecular manipulations in P. patens have greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide-based homology-directed repair (HDR) template Alternate Protocol 2: Designing the plasmid-based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector into P. patens protoplasts Basic Protocol 4: Identifying edited plants.},
}
@article {pmid36793210,
year = {2023},
author = {Cromer, MK and Majeti, KR and Rettig, GR and Murugan, K and Kurgan, GL and Bode, NM and Hampton, JP and Vakulskas, CA and Behlke, MA and Porteus, MH},
title = {Comparative analysis of CRISPR off-target discovery tools following ex vivo editing of CD34[+] hematopoietic stem and progenitor cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {4},
pages = {1074-1087},
doi = {10.1016/j.ymthe.2023.02.011},
pmid = {36793210},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; RNA, Guide, Kinetoplastida/genetics ; Hematopoietic Stem Cells/metabolism ; },
abstract = {While a number of methods exist to investigate CRISPR off-target (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes. Therefore, we compared in silico tools (COSMID, CCTop, and Cas-OFFinder) and empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) after ex vivo hematopoietic stem and progenitor cell (HSPC) editing. We performed editing using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then performed targeted next-generation sequencing of nominated OT sites identified by in silico and empirical methods. We identified an average of less than one OT site per guide RNA (gRNA) and all OT sites generated using HiFi Cas9 and a 20-nt gRNA were identified by all OT detection methods with the exception of SITE-seq. This resulted in high sensitivity for the majority of OT nomination tools and COSMID, DISCOVER-Seq, and GUIDE-Seq attained the highest positive predictive value (PPV). We found that empirical methods did not identify OT sites that were not also identified by bioinformatic methods. This study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity and PPV, thereby enabling more efficient identification of potential OT sites without compromising a thorough examination for any given gRNA.},
}
@article {pmid36793209,
year = {2023},
author = {Yu, SY and Carlaw, T and Thomson, T and Birkenshaw, A and Basha, G and Kurek, D and Huang, C and Kulkarni, J and Zhang, LH and Ross, CJD},
title = {A luciferase reporter mouse model to optimize in vivo gene editing validated by lipid nanoparticle delivery of adenine base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {4},
pages = {1159-1166},
doi = {10.1016/j.ymthe.2023.02.009},
pmid = {36793209},
issn = {1525-0024},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Adenine ; Disease Models, Animal ; Luciferases/genetics ; },
abstract = {The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4 months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5% ± 17.5% and 8.4% ± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.},
}
@article {pmid36755493,
year = {2023},
author = {Tiukacheva, EA and Ulianov, SV and Karpukhina, A and Razin, SV and Vassetzky, Y},
title = {3D genome alterations and editing in pathology.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {4},
pages = {924-933},
doi = {10.1016/j.ymthe.2023.02.005},
pmid = {36755493},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing ; Genome, Human ; Cell Nucleus ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems ; },
abstract = {The human genome is folded into a multi-level 3D structure that controls many nuclear functions including gene expression. Recently, alterations in 3D genome organization were associated with several genetic diseases and cancer. As a consequence, experimental approaches are now being developed to modify the global 3D genome organization and that of specific loci. Here, we discuss emerging experimental approaches of 3D genome editing that may prove useful in biomedicine.},
}
@article {pmid36733252,
year = {2023},
author = {Li, B and Zhao, D and Li, Y and Yang, Y and Zhu, X and Li, J and Bi, C and Zhang, X},
title = {Obtaining the best igRNAs for bystander-less correction of all ABE-reversible pathogenic SNVs using high-throughput screening.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {4},
pages = {1167-1176},
doi = {10.1016/j.ymthe.2023.01.028},
pmid = {36733252},
issn = {1525-0024},
mesh = {*Gene Editing ; *Adenine Nucleotides ; High-Throughput Screening Assays ; CRISPR-Cas Systems ; },
abstract = {Imperfect -gRNA (igRNA) provides a simple strategy for single-base editing of a base editor. However, a significant number of igRNAs need to be generated and tested for each target locus to achieve efficient single-base reversion of pathogenic single nucleotide variations (SNVs), which hinders the direct application of this technology. To provide ready-to-use igRNAs for single-base and bystander-less correction of all the adenine base editor (ABE)-reversible pathogenic SNVs, we employed a high-throughput method to edit all 5,253 known ABE-reversible pathogenic SNVs, each with multiple systematically designed igRNAs, and two libraries of 96,000 igRNAs were tested. A total of 1,988 SNV loci could be single-base reversed by igRNA with a >30% efficiency. Among these 1,988 loci, 378 SNV loci exhibited an efficiency of more than 90%. At the same time, the bystander editing efficiency of 76.62% of the SNV loci was reduced to 0%, while remaining below 1% for another 18.93% of the loci. These ready-to-use igRNAs provided the best solutions for a substantial portion of the 4,657 pathogenic/likely pathogenic SNVs. In this work, we overcame one of the most significant obstacles of base editors and provide a ready-to-use platform for the genetic treatment of diseases caused by ABE-reversible SNVs.},
}
@article {pmid36733251,
year = {2023},
author = {Gao, S and Wang, Y and Qi, T and Wei, J and Hu, Z and Liu, J and Sun, S and Liu, H and Wang, Y},
title = {Genome editing with natural and engineered CjCas9 orthologs.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {4},
pages = {1177-1187},
doi = {10.1016/j.ymthe.2023.01.029},
pmid = {36733251},
issn = {1525-0024},
mesh = {Animals ; Swine ; *Gene Editing ; *CRISPR-Cas Systems ; Mutation ; DNA/genetics ; Genome ; Mammals ; },
abstract = {CjCas9 is one of the smallest CRISPR-associated (Cas9) nucleases for mammalian genome editing. However, it requires a long N4RYAC (R = A or G; Y = C or T) protospacer-adjacent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N4RAA and N4CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N4CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that can improve the specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. Hsp1-Hsp2Cas9-Y enables the knockout of B4GALNT2 and CMAH in porcine fetal fibroblasts (PFFs). Moreover, we developed a high-fidelity Hsp1-Hsp2Cas9-KY which displayed undetectable off-targets revealed by GUIDE-seq at four tested loci. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.},
}
@article {pmid37024653,
year = {2023},
author = {Zou, RS and Liu, Y and Gaido, OER and Konig, MF and Mog, BJ and Shen, LL and Aviles-Vazquez, F and Marin-Gonzalez, A and Ha, T},
title = {Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq.},
journal = {Nature methods},
volume = {},
number = {},
pages = {},
pmid = {37024653},
issn = {1548-7105},
abstract = {Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.},
}
@article {pmid37024652,
year = {2023},
author = {Jiménez, C and Crosetto, N},
title = {Discovering CRISPR-Cas off-target breaks.},
journal = {Nature methods},
volume = {},
number = {},
pages = {},
pmid = {37024652},
issn = {1548-7105},
}
@article {pmid37022538,
year = {2023},
author = {Sufyan, M and Daraz, U and Hyder, S and Zulfiqar, U and Iqbal, R and Eldin, SM and Rafiq, F and Mahmood, N and Shahzad, K and Uzair, M and Fiaz, S and Ali, I},
title = {An overview of genome engineering in plants, including its scope, technologies, progress and grand challenges.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {2},
pages = {119},
pmid = {37022538},
issn = {1438-7948},
abstract = {Genome editing is a useful, adaptable, and favored technique for both functional genomics and crop enhancement. Over the years, rapidly evolving genome editing technologies, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas), transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), have shown broad application prospects in gene function research and improvement of critical agronomic traits in many crops. These technologies have also opened up opportunities for plant breeding. These techniques provide excellent chances for the quick modification of crops and the advancement of plant science in the future. The current review describes various genome editing techniques and how they function, particularly CRISPR/Cas9 systems, which can contribute significantly to the most accurate characterization of genomic rearrangement and plant gene functions as well as the enhancement of critical traits in field crops. To accelerate the use of gene-editing technologies for crop enhancement, the speed editing strategy of gene-family members was designed. As it permits genome editing in numerous biological systems, the CRISPR technology provides a valuable edge in this regard that particularly captures the attention of scientists.},
}
@article {pmid37022199,
year = {2023},
author = {Liu, ZL and Hu, EZ and Niu, DK},
title = {Investigating the Relationship between CRISPR-Cas Content and Growth Rate in Bacteria.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0340922},
doi = {10.1128/spectrum.03409-22},
pmid = {37022199},
issn = {2165-0497},
abstract = {CRISPR-Cas systems provide adaptive immunity for prokaryotic cells by recognizing and eliminating the recurrent genetic invaders whose sequences had been captured in a prior infection and stored in the CRISPR arrays as spacers. However, the biological/environmental factors determining the efficiency of this immune system have yet to be fully characterized. Recent studies in cultured bacteria showed that slowing the growth rate of bacterial cells could promote their acquisition of novel spacers. This study examined the relationship between the CRISPR-Cas content and the minimal doubling time across the bacteria and the archaea domains. Every completely sequenced genome could be used to predict a minimal doubling time. With a large data set of 4,142 bacterial samples, we found that the predicted minimal doubling times are positively correlated with spacer number and other parameters of the CRISPR-Cas systems, like array number, Cas gene cluster number, and Cas gene number. Different data sets gave different results. Weak results were obtained in analyzing bacterial empirical minimal doubling times and the archaea domain. Still, the conclusion of more spacers in slowly grown prokaryotes was supported. In addition, we found that the minimal doubling times are negatively correlated with the occurrence of prophages, and the spacer numbers per array are negatively associated with the number of prophages. These observations support the existence of an evolutionary trade-off between bacterial growth and adaptive defense against virulent phages. IMPORTANCE Accumulating evidence indicates that slowing the growth of cultured bacteria could stimulate their CRISPR spacer acquisition. We observed a positive correlation between CRISPR-Cas content and cell cycle duration across the bacteria domain. This observation extends the physiological conclusion to an evolutionary one. In addition, the correlation provides evidence supporting the existence of a trade-off between bacterial growth/reproduction and antiviral resistance.},
}
@article {pmid35640601,
year = {2022},
author = {Nguyen, T and Ramachandran, H and Martins, S and Krutmann, J and Rossi, A},
title = {Identification of genome edited cells using CRISPRnano.},
journal = {Nucleic acids research},
volume = {50},
number = {W1},
pages = {W199-W203},
pmid = {35640601},
issn = {1362-4962},
mesh = {*Genome/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Nanopores ; *Sequence Analysis, DNA/methods ; *Software ; Whole Genome Sequencing ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Workflow ; Mutagenesis, Site-Directed ; Internet ; Computers ; Data Visualization ; Time Factors ; Cell Line ; },
abstract = {Genome engineering-induced cleavage sites can be resolved by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Identifying genetically modified clones at the target locus remains an intensive and laborious task. Different workflows and software that rely on deep sequencing data have been developed to detect and quantify targeted mutagenesis. Nevertheless, these pipelines require high-quality reads generated by Next Generation Sequencing (NGS) platforms. Here, we have developed a robust, versatile, and easy-to-use computational webserver named CRISPRnano (www.CRISPRnano.de) that enables the analysis of low-quality reads generated by affordable and portable sequencers including Oxford Nanopore Technologies (ONT) devices. CRISPRnano allows fast and accurate identification, quantification, and visualization of genetically modified cell lines, it is compatible with NGS and ONT sequencing reads, and it can be used without an internet connection.},
}
@article {pmid37020597,
year = {2023},
author = {Meng, H and Nan, M and Li, Y and Ding, Y and Yin, Y and Zhang, M},
title = {Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.},
journal = {Frontiers in endocrinology},
volume = {14},
number = {},
pages = {1148412},
pmid = {37020597},
issn = {1664-2392},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Gene Expression Regulation ; *Colonic Neoplasms ; Technology ; },
abstract = {Colon cancer is the fourth leading cause of cancer death worldwide, and its progression is accompanied by a complex array of genetic variations. CRISPR/Cas9 can identify new drug-resistant or sensitive mutations in colon cancer, and can use gene editing technology to develop new therapeutic targets and provide personalized treatments, thereby significantly improving the treatment of colon cancer patients. CRISPR/Cas9 systems are driving advances in biotechnology. RNA-directed Cas enzymes have accelerated the pace of basic research and led to clinical breakthroughs. This article reviews the rapid development of CRISPR/Cas in colon cancer, from gene editing to transcription regulation, gene knockout, genome-wide CRISPR tools, therapeutic targets, stem cell genomics, immunotherapy, metabolism-related genes and inflammatory bowel disease. In addition, the limitations and future development of CRISPR/Cas9 in colon cancer studies are reviewed. In conclusion, this article reviews the application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.},
}
@article {pmid37020072,
year = {2023},
author = {Liu, Z and Quan, L and Ma, F and Yang, M and Jiang, X and Chen, X},
title = {Determination of adenosine by CRISPR-Cas12a system based on duplexed aptamer and molecular beacon reporter linked to gold nanoparticles.},
journal = {Mikrochimica acta},
volume = {190},
number = {5},
pages = {173},
pmid = {37020072},
issn = {1436-5073},
mesh = {Adenosine ; Gold ; CRISPR-Cas Systems ; *Metal Nanoparticles ; *Aptamers, Nucleotide ; DNA, Single-Stranded ; },
abstract = {Adenosine as a potential tumor marker is of great value for clinical disease diagnosis. Since the CRISPR-cas12a system is only capable of recognizing nucleic acid targets we expanded the CRISPR-cas12a system to determine small molecules by designing a duplexed aptamer (DA) converting g-RNA recognition of adenosine to recognition of aptamer complementary DNA strands (ACD). To further improve the sensitivity of determination, we designed a molecule beacon (MB)/gold nanoparticle (AuNP)-based reporter, which has higher sensitivity than traditional ssDNA reporter. In addition, the AuNP-based reporter enables more efficient and fast determination. The determination of adenosine under 488-nm excitation can be realized within 7 min, which is more than 4 times faster than traditional ssDNA reporter. The linear determination range of the assay to adenosine was 0.5-100 μM with the determination limit of 15.67 nM. The assay was applied to recovery determination of adenosine in serum samples with satisfactory results. The recoveries were between 91 and 106% and the RSD values of different concertation were below 4.8%. This sensitive, highly selective, and stable sensing system is expected to play a role in the clinical determination of adenosine and other biomolecules.},
}
@article {pmid37018323,
year = {2023},
author = {Bridgeland, A and Biswas, S and Tsakirpaloglou, N and Thomson, MJ and Septiningsih, EM},
title = {Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.},
journal = {PloS one},
volume = {18},
number = {4},
pages = {e0283837},
pmid = {37018323},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; *Vigna/genetics ; CRISPR-Cas Systems ; Protoplasts/metabolism ; },
abstract = {Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.},
}
@article {pmid36964239,
year = {2023},
author = {},
title = {Gene editing holds promise for babies with deadly immune disease.},
journal = {Nature},
volume = {616},
number = {7955},
pages = {11},
pmid = {36964239},
issn = {1476-4687},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Therapy ; },
}
@article {pmid37020030,
year = {2023},
author = {Nakagawa, R and Hirano, H and Omura, SN and Nety, S and Kannan, S and Altae-Tran, H and Yao, X and Sakaguchi, Y and Ohira, T and Wu, WY and Nakayama, H and Shuto, Y and Tanaka, T and Sano, FK and Kusakizako, T and Kise, Y and Itoh, Y and Dohmae, N and van der Oost, J and Suzuki, T and Zhang, F and Nureki, O},
title = {Cryo-EM structure of the transposon-associated TnpB enzyme.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {37020030},
issn = {1476-4687},
abstract = {The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins[1]. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases[2,3]. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.},
}
@article {pmid37018843,
year = {2023},
author = {Rallapalli, KL and Komor, AC},
title = {The Design and Application of DNA-Editing Enzymes as Base Editors.},
journal = {Annual review of biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-biochem-052521-013938},
pmid = {37018843},
issn = {1545-4509},
abstract = {DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}
@article {pmid37018798,
year = {2023},
author = {Tian, T and Zhou, X},
title = {CRISPR-Based Biosensing Strategies: Technical Development and Application Prospects.},
journal = {Annual review of analytical chemistry (Palo Alto, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-anchem-090822-014725},
pmid = {37018798},
issn = {1936-1335},
abstract = {Biosensing based on CRISPR-Cas systems is a young but rapidly evolving technology. The unprecedented properties of the CRISPR-Cas system provide an innovative tool for developing new-generation biosensing strategies. To date, a series of nucleic acid and non-nucleic acid detection methods have been developed based on the CRISPR platform. In this review, we first introduce the core biochemical properties underpinning the development of CRISPR bioassays, such as diverse reaction temperatures, programmability in design, high reaction efficiency, and recognition specificity, and highlight recent efforts to improve these parameters. We then introduce the technical developments, including how to improve sensitivity and quantification capabilities, develop multiplex assays, achieve convenient one-pot assays, create advanced sensors, and extend the applications of detection. Finally, we analyze obstacles to the commercial application of CRISPR detection technology and explore development opportunities and directions. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}
@article {pmid37018035,
year = {2023},
author = {Muzahid, NH and Hussain, MH and Huët, MAL and Dwiyanto, J and Su, TT and Reidpath, D and Mustapha, F and Ayub, Q and Tan, HS and Rahman, S},
title = {Molecular characterization and comparative genomic analysis of Acinetobacter baumannii isolated from the community and the hospital: an epidemiological study in Segamat, Malaysia.},
journal = {Microbial genomics},
volume = {9},
number = {4},
pages = {},
doi = {10.1099/mgen.0.000977},
pmid = {37018035},
issn = {2057-5858},
abstract = {Acinetobacter baumannii is a common cause of multidrug-resistant (MDR) nosocomial infections around the world. However, little is known about the persistence and dynamics of A. baumannii in a healthy community. This study investigated the role of the community as a prospective reservoir for A. baumannii and explored possible links between hospital and community isolates. A total of 12 independent A. baumannii strains were isolated from human faecal samples from the community in Segamat, Malaysia, in 2018 and 2019. Another 15 were obtained in 2020 from patients at the co-located tertiary public hospital. The antimicrobial resistance profile and biofilm formation ability were analysed, and the relatedness of community and hospital isolates was determined using whole-genome sequencing (WGS). Antibiotic profile analysis revealed that 12 out of 15 hospital isolates were MDR, but none of the community isolates were MDR. However, phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) and a pangenome analysis of core genes showed clustering between four community and two hospital strains. Such clustering of strains from two different settings based on their genomes suggests that these strains could persist in both. WGS revealed 41 potential resistance genes on average in the hospital strains, but fewer (n=32) were detected in the community strains. In contrast, 68 virulence genes were commonly seen in strains from both sources. This study highlights the possible transmission threat to public health posed by virulent A. baumannii present in the gut of asymptomatic individuals in the community.},
}
@article {pmid37010073,
year = {2023},
author = {Liang, J and Tan, Y},
title = {Highly efficient CRISPR-mediated base editing for the gut Bacteroides spp. with pnCasBS-CBE.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2200504},
doi = {10.1002/biot.202200504},
pmid = {37010073},
issn = {1860-7314},
abstract = {Bacteroidales are the most abundant order of bacteria in the healthy human gut and have the potential as a therapeutic agent. We constructed a pnCasBS-CBE system for base editing in the Bacteroides thetaiotaomicron to expand their genetic toolkit, which is able to efficiently convert a C:G to a T:A in the genome. As a functional proof-of-concept, we used the pnCasBS-CBE system to successfully introduce nonsynonymous mutation and stop codons to the genes involved in carbohydrate metabolism. The system also allowed for multiplexed gene editing with a single plasmid, enabling efficient editing of up to four genes in a single experiment. Furthermore, the pnCasBS-CBE editing system was validated and successfully applied in four other non-model gut Bacteroides species for genome editing. An unbiased genome-wide SNPs analysis indicated that the pnCasBS-CBE system showed high fidelity and applicability. Thus, this study provides a powerful CRISPR-mediated genome editing toolbox for functional genomic analysis in Bacteroidales. This article is protected by copyright. All rights reserved.},
}
@article {pmid36993517,
year = {2023},
author = {Lampe, GD and King, RT and Halpin-Healy, TS and Klompe, SE and Hogan, MI and Vo, PLH and Tang, S and Chavez, A and Sternberg, SH},
title = {Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {36993517},
abstract = {Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.},
}
@article {pmid36737854,
year = {2023},
author = {Lei, H and Zeng, T and Ye, X and Fan, R and Xiong, W and Tian, T and Zhou, X},
title = {Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {10},
pages = {e2206433},
pmid = {36737854},
issn = {2198-3844},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida/genetics ; },
abstract = {Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.},
}
@article {pmid36378073,
year = {2023},
author = {Kobayashi, R and Kawabata-Iwakawa, R and Sugiyama, M and Oyama, T and Ohtsuka, M and Horii, T and Morita, S and Nishiyama, M and Hatada, I},
title = {Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice.},
journal = {International journal of cancer},
volume = {152},
number = {11},
pages = {2331-2337},
doi = {10.1002/ijc.34342},
pmid = {36378073},
issn = {1097-0215},
mesh = {Mice ; Female ; Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Ribonucleoproteins/genetics ; Electroporation/methods ; *Endometrial Neoplasms/genetics ; },
abstract = {Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.},
}
@article {pmid37009498,
year = {2023},
author = {Pan, Y and Xia, H and He, Y and Zeng, S and Shen, Z and Huang, W},
title = {The progress of molecules and strategies for the treatment of HBV infection.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1128807},
pmid = {37009498},
issn = {2235-2988},
mesh = {Humans ; *Hepatitis B virus/physiology ; Virus Replication ; *Hepatitis B/drug therapy ; Interferon-alpha/pharmacology ; Antiviral Agents/pharmacology/therapeutic use/metabolism ; DNA, Circular/metabolism/pharmacology/therapeutic use ; DNA, Viral/genetics ; },
abstract = {Hepatitis B virus infections have always been associated with high levels of mortality. In 2019, hepatitis B virus (HBV)-related diseases resulted in approximately 555,000 deaths globally. In view of its high lethality, the treatment of HBV infections has always presented a huge challenge. The World Health Organization (WHO) came up with ambitious targets for the elimination of hepatitis B as a major public health threat by 2030. To accomplish this goal, one of the WHO's strategies is to develop curative treatments for HBV infections. Current treatments in a clinical setting included 1 year of pegylated interferon alpha (PEG-IFNα) and long-term nucleoside analogues (NAs). Although both treatments have demonstrated outstanding antiviral effects, it has been difficult to develop a cure for HBV. The reason for this is that covalently closed circular DNA (cccDNA), integrated HBV DNA, the high viral burden, and the impaired host immune responses all hinder the development of a cure for HBV. To overcome these problems, there are clinical trials on a number of antiviral molecules being carried out, all -showing promising results so far. In this review, we summarize the functions and mechanisms of action of various synthetic molecules, natural products, traditional Chinese herbal medicines, as clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas)-based systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), all of which could destroy the stability of the HBV life cycle. In addition, we discuss the functions of immune modulators, which can enhance or activate the host immune system, as well some representative natural products with anti-HBV effects.},
}
@article {pmid37007603,
year = {2022},
author = {Gomez, MA and Berkoff, KC and Gill, BK and Iavarone, AT and Lieberman, SE and Ma, JM and Schultink, A and Karavolias, NG and Wyman, SK and Chauhan, RD and Taylor, NJ and Staskawicz, BJ and Cho, MJ and Rokhsar, DS and Lyons, JB},
title = {CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1079254},
pmid = {37007603},
issn = {1664-462X},
abstract = {Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.},
}
@article {pmid37005025,
year = {2023},
author = {Yi, M and Gong, Y and Zhan, Q and Dai, Y and Yang, T and Cheng, X and Ding, S and Gu, B and Cheng, W and Zhang, D},
title = {A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening.},
journal = {Analytica chimica acta},
volume = {1254},
number = {},
pages = {341115},
doi = {10.1016/j.aca.2023.341115},
pmid = {37005025},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems ; DNA Nucleotidylexotransferase ; Biological Assay ; *Biomedical Research ; DNA, Single-Stranded/genetics ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis/genetics ; *Biosensing Techniques ; },
abstract = {An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10[-5] U μL[-1] in the concentration scope from 1 × 10[-4] U μL[-1] to 1 × 10[-1] U μL[-1], and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.},
}
@article {pmid37004306,
year = {2023},
author = {Deng, X and Yuan, J and Chen, L and Chen, H and Wei, C and Nielsen, PH and Wuertz, S and Qiu, G},
title = {CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter.},
journal = {Water research},
volume = {235},
number = {},
pages = {119906},
doi = {10.1016/j.watres.2023.119906},
pmid = {37004306},
issn = {1879-2448},
abstract = {Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.},
}
@article {pmid36958604,
year = {2023},
author = {Bondy-Denomy, J and Maxwell, KL and Davidson, AR},
title = {Ten Years of Anti-CRISPR Research.},
journal = {Journal of molecular biology},
volume = {435},
number = {7},
pages = {168058},
doi = {10.1016/j.jmb.2023.168058},
pmid = {36958604},
issn = {1089-8638},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages ; },
}
@article {pmid36933297,
year = {2023},
author = {Yi, JY and Kim, M and Ahn, JH and Kim, BG and Son, J and Sung, C},
title = {CRISPR/deadCas9-based high-throughput gene doping analysis (HiGDA): A proof of concept for exogenous human erythropoietin gene doping detection.},
journal = {Talanta},
volume = {258},
number = {},
pages = {124455},
doi = {10.1016/j.talanta.2023.124455},
pmid = {36933297},
issn = {1873-3573},
mesh = {Humans ; *CRISPR-Cas Systems ; *Erythropoietin/genetics ; },
abstract = {A genetic approach targeted toward improving athletic performance is called gene doping and is prohibited by the World Anti-Doping Agency. Currently, the clustered regularly interspaced short palindromic repeats-associated protein (Cas)-related assays have been utilized to detect genetic deficiencies or mutations. Among the Cas proteins, deadCas9 (dCas9), a nuclease-deficient mutant of Cas9, acts as a DNA binding protein with a target-specific single guide RNA. On the basis of the principles, we developed a dCas9-based high-throughput gene doping analysis for exogenous gene detection. The assay comprises two distinctive dCas9s, a magnetic bead immobilized capture dCas9 for exogenous gene isolation and a biotinylated dCas9 with streptavidin-polyHRP that enables rapid signal amplification. For efficient biotin labeling via maleimide-thiol chemistry, two cysteine residues of dCas9 were structurally validated, and the Cys574 residue was identified as an essential labeling site. As a result, we succeeded in detecting the target gene in a concentration as low as 12.3 fM (7.41 × 10[5] copies) and up to 10 nM (6.07 × 10[11] copies) in a whole blood sample within 1 h with HiGDA. Assuming an exogenous gene transfer scenario, we added a direct blood amplification step to establish a rapid analytical procedure while detecting target genes with high sensitivity. Finally, we detected the exogenous human erythropoietin gene at concentrations as low as 2.5 copies within 90 min in 5 μL of the blood sample. Herein, we propose that HiGDA is a very fast, highly sensitive, and practical detection method for actual doping field in the future.},
}
@article {pmid36928506,
year = {2023},
author = {Trivedi, V and Ramesh, A and Wheeldon, I},
title = {Analyzing CRISPR screens in non-conventional microbes.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {50},
number = {1},
pages = {},
doi = {10.1093/jimb/kuad006},
pmid = {36928506},
issn = {1476-5535},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics ; Genome ; Computational Biology ; Mammals/genetics ; },
abstract = {UNLABELLED: The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction.<br><br>ONE-SENTENCE SUMMARY: This mini-review describes experimental and computational approaches for functional genomic screening using CRISPR technologies in non-conventional microbes.},
}
@article {pmid36924897,
year = {2023},
author = {Qiao, L and Gao, M and Yi, X and Peng, H and Zhang, R and Yao, W and Sun, G and He, X},
title = {Biomimetic gene editing system for precise tumor cell reprogramming and augmented tumor therapy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {356},
number = {},
pages = {663-677},
doi = {10.1016/j.jconrel.2023.03.020},
pmid = {36924897},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Biomimetics ; Cellular Reprogramming ; *Neoplasms/genetics/therapy/metabolism ; },
abstract = {The abnormal level of hypoxia-inducible factor-1 alpha (HIF-1α) is closely related to cancer metastasis and treatment resistance. CRISPR-Cas9-based gene editing technology has sparked profound hope to solve this issue by precise gene disruption, although the in vivo application remains hindered by the lack of a safe and efficient delivery strategy. Herein, we developed a cell membrane biomimetic core-shell system for light-controllable, precise gene editing. The inner core of the system comprises protamine for CRISPR-Cas9/sgRNA plasmid (pCas9) loading and calcium ions for efficient pCas9 transfection. The shell of the system is camouflaged by a cell membrane and modified with AS1411 aptamers for tumor targeting and photosensitizers to induce lysosomal escape and pCas9 release through reactive oxygen species production, thereby producing light-controllable enhanced gene editing. Neoplastic H1299 cells were reprogrammed using the biomimetic gene editing system upon laser irradiation with reduced VEGF and Vimentin expression, leading to enhanced antimetastatic effects. Genetic disruption of HIF-1α augmented the in vivo chemotherapeutic efficacy of paclitaxel. Our approach of using a membrane-camouflaged system combined with light augmentation provides a potential solution for the in vivo delivery of CRISPR-Cas9 as well as a feasible strategy for cancer therapy.},
}
@article {pmid36899271,
year = {2023},
author = {Kingwell, K},
title = {Testing the genome-editing toolkit in cardiomyopathy.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {4},
pages = {270},
pmid = {36899271},
issn = {1474-1784},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Cardiomyopathies/genetics ; },
}
@article {pmid36881874,
year = {2023},
author = {Dahlvang, JD and Dick, JK and Sangala, JA and Kennedy, PR and Pomeroy, EJ and Snyder, KM and Moushon, JM and Thefaine, CE and Wu, J and Hamilton, SE and Felices, M and Miller, JS and Walcheck, B and Webber, BR and Moriarity, BS and Hart, GT},
title = {Ablation of SYK Kinase from Expanded Primary Human NK Cells via CRISPR/Cas9 Enhances Cytotoxicity and Cytokine Production.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {210},
number = {8},
pages = {1108-1122},
pmid = {36881874},
issn = {1550-6606},
support = {R01 AI143828/AI/NIAID NIH HHS/United States ; R01 AI146031/AI/NIAID NIH HHS/United States ; R21 AI149659/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; Syk Kinase/genetics ; *Cytomegalovirus ; CRISPR-Cas Systems ; Killer Cells, Natural ; *Cytomegalovirus Infections ; Cytokines ; Antibody-Dependent Cell Cytotoxicity ; },
abstract = {CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.},
}
@article {pmid36871676,
year = {2023},
author = {Boubakri, H},
title = {Recent progress in CRISPR/Cas9-based genome editing for enhancing plant disease resistance.},
journal = {Gene},
volume = {866},
number = {},
pages = {147334},
doi = {10.1016/j.gene.2023.147334},
pmid = {36871676},
issn = {1879-0038},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Transcription Activator-Like Effector Nucleases/genetics ; Disease Resistance/genetics ; Plants/genetics ; Genome, Plant ; },
abstract = {Nowadays, agricultural production is strongly affected by both climate change and pathogen attacks which seriously threaten global food security. For a long time, researchers have been waiting for a tool allowing DNA/RNA manipulation to tailor genes and their expression. Some earlier genetic manipulation methods such as meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) allowed site directed modification but their successful rate was limited due to lack of flexibility when targeting a 'site-specific nucleic acid'. The discovery of clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome editing domain in different living organisms during the past 9 years. Based on RNA-guided DNA/RNA recognition, CRISPR/Cas9 optimizations have offered an unrecorded scientific opportunity to engineer plants resistant to diverse pathogens. In this report, we describe the main characteristics of the primary reported-genome editing tools ((MNs, ZFNs, TALENs) and evaluate the different CRISPR/Cas9 methods and achievements in developing crop plants resistant to viruses, fungi and bacteria.},
}
@article {pmid36868446,
year = {2023},
author = {Abe, T and Kaneko, M and Kiyonari, H},
title = {A reverse genetic approach in geckos with the CRISPR/Cas9 system by oocyte microinjection.},
journal = {Developmental biology},
volume = {497},
number = {},
pages = {26-32},
doi = {10.1016/j.ydbio.2023.02.005},
pmid = {36868446},
issn = {1095-564X},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Lizards/genetics ; Microinjections ; Reverse Genetics ; Gene Editing/methods ; Oocytes ; },
abstract = {Reptiles are important model organisms in developmental and evolutionary biology, but are used less widely than other amniotes such as mouse and chicken. One of the main reasons for this is that has proven difficult to conduct CRISPR/Cas9-mediated genome editing in many reptile species despite the widespread use of this technology in other taxa. Certain features of reptile reproductive systems make it difficult to access one-cell or early-stage zygotes, which represents a key impediment to gene editing techniques. Recently, Rasys and colleagues reported a genome editing method using oocyte microinjection that allowed them to produce genome-edited Anolis lizards. This method opened a new avenue to reverse genetics studies in reptiles. In the present article, we report the development of a related method for genome editing in the Madagascar ground gecko (Paroedura picta), a well-established experimental model, and describe the generation of Tyr and Fgf10 gene-knockout geckos in the F0 generation.},
}
@article {pmid36736009,
year = {2023},
author = {Han, J and Li, X and Li, W and Yang, Q and Li, Z and Cheng, Z and Lv, L and Zhang, L and Han, D},
title = {Isolation and preliminary functional analysis of FvICE1, involved in cold and drought tolerance in Fragaria vesca through overexpression and CRISPR/Cas9 technologies.},
journal = {Plant physiology and biochemistry : PPB},
volume = {196},
number = {},
pages = {270-280},
doi = {10.1016/j.plaphy.2023.01.048},
pmid = {36736009},
issn = {1873-2690},
mesh = {*Fragaria/metabolism ; Stress, Physiological ; Drought Resistance ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Droughts ; Plants, Genetically Modified/metabolism ; },
abstract = {Cold and drought stresses are serious problems of strawberry cultivation in temperate and subtropical regions. In the molecular regulation system of cold and drought stresses, ICE transcription factors (TFs) are crucial. In this research, the FvICE1 was isolated from Fragaria vesca 'Hawaii 4', a bioinformatics analysis was conducted, overexpression vector and CRISPR/cas9 vector were constructed. The results showed that FvICE1 was a member of the bHLH TF family, with a length of 1608 bp, encoding 535 amino acids, and its molecular formula was C2504H3987N745O811S22. By observing the fusion protein 35S-FvICE1-GFP, it was found that FvICE1 was a nuclear protein. The qRT-PCR results demonstrated that FvICE1 was significantly upregulated in different tissues of Fragaria vesca after cold, drought, salt and heat treatments. The wild type (WT) strawberry was selected as the control group, FvICE1-overexpression strawberries showed high tolerance to cold and drought treatments at the phenotypic and physiological levels. On the contrary, fvice1 mutant strawberries obtained by CRISPR/cas9 editing technology had lower tolerance to cold and drought treatments. Moreover, the expression of FvCBF1, FvCBF2, FvCBF3, FvCOR413, FvRD22 and FvKIN1 was positively regulated in the FvICE1-overexpression strawberries and inhibited in fvice1 mutant strawberries. Overall, the current results suggested that FvICE1 functioned as a positively regulator of cold and drought resistances.},
}
@article {pmid36478145,
year = {2023},
author = {Ge, X and Xu, J and Yang, Z and Yang, X and Wang, Y and Chen, Y and Wang, P and Li, F},
title = {Efficient genotype-independent cotton genetic transformation and genome editing.},
journal = {Journal of integrative plant biology},
volume = {65},
number = {4},
pages = {907-917},
doi = {10.1111/jipb.13427},
pmid = {36478145},
issn = {1744-7909},
mesh = {*Gene Editing/methods ; *Gossypium/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genotype ; Transformation, Genetic ; },
abstract = {Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem (SAM) cell-mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2-3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP- and RUBY-positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.},
}
@article {pmid36460630,
year = {2023},
author = {Wang, C and Li, Y and Wang, N and Yu, Q and Li, Y and Gao, J and Zhou, X and Ma, N},
title = {An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida).},
journal = {Journal of integrative plant biology},
volume = {65},
number = {4},
pages = {895-899},
doi = {10.1111/jipb.13421},
pmid = {36460630},
issn = {1744-7909},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Rosa/genetics ; Plants/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related nuclease 9 (Cas9) system enables precise, simple editing of genes in many animals and plants. However, this system has not been applied to rose (Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single-guide RNAs (sgRNAs) with high editing efficiency for CRISPR/Cas9-mediated gene editing in rose using suspension cells. We used the Arabidopsis thaliana U6-29 promoter, which showed high activity for driving sgRNA expression, to modify the CRISPR/Cas9 system. We used our highly efficient optimized CRISPR/Cas9 system to successfully edit RhEIN2, encoding an indispensable component of the ethylene signaling pathway, resulting in ethylene insensitivity in rose. Our optimized CRISPR/Cas9 system provides a powerful toolbox for functional genomics, molecular breeding, and synthetic biology in rose.},
}
@article {pmid36096547,
year = {2023},
author = {Ling, J and Jenny, LA and Zhou, A and Tsang, SH},
title = {Therapeutic Gene Editing in Inherited Retinal Disorders.},
journal = {Cold Spring Harbor perspectives in medicine},
volume = {13},
number = {4},
pages = {},
pmid = {36096547},
issn = {2157-1422},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; *Retinal Diseases/genetics/therapy ; },
abstract = {Since the development of CRISPR/Cas9 gene editing in 2012, therapeutic editing research has produced several phase 1-2a trials. Here we provide an overview of the mechanisms and applications of various gene-editing technologies including adeno-associated virus vectors, lentiviruses, CRISPR/Cas9 systems, base and prime editing, antisense oligonucleotides, short-hairpin RNAs, Cas13, and adenosine deaminase acting on RNA for the treatment of various inherited retinal diseases (IRDs). We outline the various stages of clinical trials using these technologies and the impacts they have made in advancing the practice of medicine.},
}
@article {pmid35670672,
year = {2022},
author = {Konstantakos, V and Nentidis, A and Krithara, A and Paliouras, G},
title = {CRISPRedict: a CRISPR-Cas9 web tool for interpretable efficiency predictions.},
journal = {Nucleic acids research},
volume = {50},
number = {W1},
pages = {W191-W198},
pmid = {35670672},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, Kinetoplastida/genetics ; },
abstract = {The development of the CRISPR-Cas9 technology has provided a simple yet powerful system for genome editing. Current gRNA design tools serve as an important platform for the efficient application of the CRISPR systems. However, most of the existing tools are black-box models that suffer from limitations, such as variable performance and unclear mechanism of decision making. Here, we introduce CRISPRedict, an interpretable gRNA efficiency prediction model for CRISPR-Cas9 gene editing. Its strength lies in the fact that it can accurately predict efficient guide RNAs-with equivalent performance to state-of-the-art tools-while being a simple linear model. Implemented as a user-friendly web server, CRISPRedict offers (i) quick and accurate predictions across various experimental conditions (e.g. U6/T7 transcription); (ii) regression and classification models for scoring gRNAs and (iii) multiple visualizations to explain the obtained results. Given its performance, interpretability, and versatility, we expect that it will assist researchers in the gRNA design process and facilitate genome editing research. CRISPRedict is available for use at http://www.crispredict.org/.},
}
@article {pmid37004283,
year = {2023},
author = {Zhang, Y and Chen, Y and Zhang, Q and Liu, Y and Zhang, X},
title = {An aM-level sensitive cascade CRISPR-Dx system (ASCas) for rapid detection of RNA without pre-amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {230},
number = {},
pages = {115248},
doi = {10.1016/j.bios.2023.115248},
pmid = {37004283},
issn = {1873-4235},
abstract = {The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus.},
}
@article {pmid37002219,
year = {2023},
author = {Thakku, SG and Lirette, J and Murugesan, K and Chen, J and Theron, G and Banaei, N and Blainey, PC and Gomez, J and Wong, SY and Hung, DT},
title = {Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {1803},
pmid = {37002219},
issn = {2041-1723},
mesh = {Cell-Free System ; DNA, Bacterial/analysis/genetics ; *Mycobacterium tuberculosis/genetics ; Genome, Bacterial ; CRISPR-Cas Systems ; Humans ; Tuberculosis/diagnosis/microbiology ; },
abstract = {Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.},
}
@article {pmid37001495,
year = {2023},
author = {Malech, HL and Notarangelo, LD},
title = {Gene therapy for inborn errors of immunity: Base editing comes into play.},
journal = {Cell},
volume = {186},
number = {7},
pages = {1302-1304},
doi = {10.1016/j.cell.2023.03.001},
pmid = {37001495},
issn = {1097-4172},
mesh = {Humans ; Adenine ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Mutation ; Genetic Diseases, Inborn/genetics/therapy ; *Immune System Diseases/genetics/therapy ; },
abstract = {CRISPR-Cas9-based base editing allows precise base editing to achieve conversion of adenosine to guanine or cytosine to thymidine. In this issue of Cell, McAuley et al. use adenine base editing to correct a single base-pair mutation causing human CD3δ deficiency, demonstrating superior efficiency of genetic correction with reduced undesired genetic alterations compared with standard CRISPR-Cas9 editing.},
}
@article {pmid37000655,
year = {2023},
author = {Kulishova, LM and Vokhtantsev, IP and Kim, DV and Zharkov, DO},
title = {[Mechanisms of the Specificity of the CRISPR/Cas9 System in Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {57},
number = {2},
pages = {269-284},
pmid = {37000655},
issn = {0026-8984},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Biotechnology ; RNA, Guide, Kinetoplastida/genetics ; Genome ; },
abstract = {The CRISPR/Cas9 system, which was discovered recently, utilizes nucleases targeted by sequence complementarity and is originally intended to protect bacteria from foreign genetic elements. The system provided a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology moved beyond the laboratory and already found application in biotechnology and agriculture. However, off-target activity of the CRISPR/Cas9 system can cause oncogenic mutations and thus limits its use for genome editing in human cells for medical purposes. Many studies are therefore aimed at developing variants of the CRISPR/Cas9 system with improved accuracy. The review considers the mechanisms of precise and erroneous actions of Cas9 RNA-guided nuclease, natural and artificial variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increasing the accuracy of the CRISPR/Cas9 system in genome editing.},
}
@article {pmid36958206,
year = {2023},
author = {Wang, Y and Chen, H and Gao, H and Wei, H and Wang, Y and Mu, K and Liu, L and Dai, E and Rong, Z and Wang, S},
title = {CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.},
journal = {Biosensors &amp; bioelectronics},
volume = {229},
number = {},
pages = {115238},
pmid = {36958206},
issn = {1873-4235},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Genotype ; SARS-CoV-2/genetics ; *Biosensing Techniques ; RNA, Viral/genetics ; },
abstract = {The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/μL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.},
}
@article {pmid37003351,
year = {2023},
author = {Huang, L and Wang, D and Chen, H and Hu, J and Dai, X and Liu, C and Li, A and Shen, X and Qi, C and Sun, H and Zhang, D and Chen, T and Jiang, Y},
title = {CRISPR-detector: fast and accurate detection, visualization, and annotation of genome-wide mutations induced by genome editing events.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2023.03.010},
pmid = {37003351},
issn = {1673-8527},
abstract = {The leading-edge CRISPR/Cas technology is revolutionizing biotechnologies through genome editing. To track on/off-target events with emerging new editing techniques, improved bioinformatic tools are indispensable. Existing tools suffer from limitations in speed and scalability, especially with whole-genome sequencing (WGS) data analysis. To address these limitations, we have developed a comprehensive tool called CRISPR-detector, a web-based and locally deployable pipeline for genome editing sequence analysis. The core analysis module of CRISPR-detector is based on the Sentieon TNscope pipeline, with additional novel annotation and visualization modules designed to fit CRISPR applications. Co-analysis of the treated and control samples is performed to remove background variants existing prior to genome editing. CRISPR-detector offers optimized scalability, enabling WGS data analysis beyond Browser Extensible Data (BED) file-defined regions, with improved accuracy due to haplotype-based variant calling to handle sequencing errors. In addition, the tool also provides integrated structural variation (SV) calling and included functional and clinical annotation of editing-induced mutations appreciated by users. These advantages facilitate rapid and efficient detection of mutations induced by genome editing events, especially for datasets generated from WGS. The web-based version of CRISPR-detector is available at https://db.cngb.org/crispr-detector, and the locally deployable version is available at https://github.com/hlcas/CRISPR-detector.},
}
@article {pmid37002157,
year = {2023},
author = {Zhao, Z and Shang, P and Mohanraju, P and Geijsen, N},
title = {Prime editing: advances and therapeutic applications.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.03.004},
pmid = {37002157},
issn = {1879-3096},
abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic and diagnostic landscape. However, CRISPR-Cas9, which edits DNA by activating DNA double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation repair is required. Prime editing, the latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without the requirement for DSBs. However, prime editing is still in its infancy, and further development is needed to improve editing efficiency and delivery strategies for therapeutic applications. We summarize latest developments in the optimization of prime editor (PE) variants with improved editing efficiency and precision. Moreover, we highlight some potential therapeutic applications.},
}
@article {pmid37001687,
year = {2023},
author = {Djermoun, S and Reuter, A and Derollez, E and Lesterlin, C and Bigot, S},
title = {Reprogramming Targeted-Antibacterial-Plasmids (TAPs) to achieve broad-host range antibacterial activity.},
journal = {Plasmid},
volume = {126},
number = {},
pages = {102680},
doi = {10.1016/j.plasmid.2023.102680},
pmid = {37001687},
issn = {1095-9890},
abstract = {The emergence and spread of antimicrobial resistance results in antibiotic inefficiency against multidrug resistant bacterial strains. Alternative treatment to antibiotics must be investigated to fight bacterial infections and limit this global public health problem. We recently developed an innovative strategy based on mobilizable Targeted-Antibacterial-Plasmids (TAPs) that deliver CRISPR/Cas systems with strain-specific antibacterial activity, using the F plasmid conjugation machinery for transfer into the targeted strains. These TAPs were shown to specifically kill a variety of Enterobacteriaceae strains, including E. coli K12 and the pathogen strains EPEC, Enterobacter cloacae and Citrobacter rodentium. Here, we extend the host-range of TAPs using the RP4 plasmid conjugation system for their mobilization, thus allowing the targeting of E. coli but also phylogenetically distant species, including Salmonella enterica Thyphimurium, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. This work demonstrates the versatility of the TAP strategy and represents a significant step toward the development of non-antibiotic strain-specific antimicrobial treatments.},
}
@article {pmid36997536,
year = {2023},
author = {Yan, N and Feng, H and Sun, Y and Xin, Y and Zhang, H and Lu, H and Zheng, J and He, C and Zuo, Z and Yuan, T and Li, N and Xie, L and Wei, W and Sun, Y and Zuo, E},
title = {Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {1784},
pmid = {36997536},
issn = {2041-1723},
mesh = {Animals ; Mice ; Mice, Transgenic ; *Cytosine/metabolism ; Mutation ; *Gene Editing/methods ; RNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Base editors have been reported to induce off-target mutations in cultured cells, mouse embryos and rice, but their long-term effects in vivo remain unknown. Here, we develop a Systematic evaluation Approach For gene Editing tools by Transgenic mIce (SAFETI), and evaluate the off-target effects of BE3, high fidelity version of CBE (YE1-BE3-FNLS) and ABE (ABE7.10[F148A]) in ~400 transgenic mice over 15 months. Whole-genome sequence analysis reveals BE3 expression generated de novo mutations in the offspring of transgenic mice. RNA-seq analysis reveals both BE3 and YE1-BE3-FNLS induce transcriptome-wide SNVs, and the numbers of RNA SNVs are positively correlated with CBE expression levels across various tissues. By contrast, ABE7.10[F148A] shows no detectable off-target DNA or RNA SNVs. Notably, we observe abnormal phenotypes including obesity and developmental delay in mice with permanent genomic BE3 overexpression during long-time monitoring, elucidating a potentially overlooked aspect of side effects of BE3 in vivo.},
}
@article {pmid36997524,
year = {2023},
author = {Lee, J and Lim, K and Kim, A and Mok, YG and Chung, E and Cho, SI and Lee, JM and Kim, JS},
title = {Prime editing with genuine Cas9 nickases minimizes unwanted indels.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {1786},
pmid = {36997524},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Deoxyribonuclease I/metabolism ; Mutation ; INDEL Mutation ; DNA ; },
abstract = {Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).},
}
@article {pmid36995680,
year = {2023},
author = {Preisinger, D and Winogrodzki, T and Klinger, B and Schnieke, A and Rieblinger, B},
title = {Genome Editing in Pigs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {393-417},
pmid = {36995680},
issn = {1940-6029},
mesh = {Swine/genetics ; Animals ; Female ; *Gene Editing ; *Genetic Engineering/methods ; Nuclear Transfer Techniques ; Oocytes ; Zygote ; CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; },
abstract = {The generation of genetically engineered (GE) pigs for disease modeling and xenotransplantation has been massively facilitated by the discovery of the CRISPR/Cas9 system. For livestock, genome editing is a powerful tool when used in combination with either somatic cell nuclear transfer (SCNT) or microinjection (MI) into fertilized oocytes. To generate either knockout or knock-in animals using SCNT, genome editing is carried out in vitro. This has the advantage that fully characterized cells are being employed to generate cloned pigs, predetermining their genetic makeups. However, this technique is labor-intensive and, hence, SCNT is better suited for more challenging projects such as the generation of multi-knockout- and knock-in pigs. Alternatively, CRISPR/Cas9 is introduced directly into fertilized zygotes via microinjection to produce knockout pigs more rapidly. Finally, the embryos are each transferred into recipient sows to deliver GE piglets.Both techniques, SCNT and MI, are technically challenging and therefore require skilled expertise, especially when applied for porcine embryos. Here, we present a detailed laboratory protocol for the generation of knockout and knock-in porcine somatic donor cells for SCNT and knockout pigs via microinjection. We describe the state-of-the-art method for isolation, cultivation, and manipulation of porcine somatic cells, which can then be used for SCNT. Moreover, we describe the isolation and maturation of porcine oocytes, their manipulation by microinjection, and the embryo transfer into surrogate sows.},
}
@article {pmid36995678,
year = {2023},
author = {Daniel, JG and Yu, X and Ferguson, AC and Shavit, JA},
title = {CRISPR/Cas9-Mediated Genome Editing in Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {371-380},
pmid = {36995678},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Zebrafish/genetics ; Genome ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.},
}
@article {pmid36995675,
year = {2023},
author = {Sato, M and Nakamura, A and Sekiguchi, M and Matsuwaki, T and Miura, H and Gurumurthy, CB and Kakuta, S and Ohtsuka, M},
title = {Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {325-340},
pmid = {36995675},
issn = {1940-6029},
mesh = {Humans ; Pregnancy ; Female ; Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Fallopian Tubes ; Oviducts ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Electroporation/methods ; Gonads ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.},
}
@article {pmid36995673,
year = {2023},
author = {Schaeffer, L and Lindner, L and Pavlovic, G and Hérault, Y and Birling, MC},
title = {CRISMERE Chromosome Engineering in Mouse and Rat.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {277-297},
pmid = {36995673},
issn = {1940-6029},
mesh = {Mice ; Rats ; Animals ; *DNA Copy Number Variations ; *Genome ; Genomics ; Mutation ; Chromosomes ; CRISPR-Cas Systems/genetics ; Genetic Engineering ; },
abstract = {CRISPR/Cas9 technology is a versatile tool for engineering biology that has dramatically transformed our ability to manipulate genomes. In this protocol, we use its capacity to generate two double-strand breaks simultaneously, at precise positions in the genome, to generate mouse or rat lines with deletion, inversion, and duplication of a specific genomic segment. The technic is called CRISMERE for CRISpr-MEdiated REarrangement. This protocol describes the different steps to generate and validate the different chromosomal rearrangements that can be obtained with the technology. These new genetic configurations can be useful to model rare diseases with copy number variation, understand the genomic organization, or provide genetic tools (like balancer chromosome) to keep lethal mutations.},
}
@article {pmid36995672,
year = {2023},
author = {Davis, DJ and Men, H and Bryda, EC},
title = {Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {267-276},
pmid = {36995672},
issn = {1940-6029},
mesh = {Rats ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Zygote ; Electroporation/methods ; Electroporation Therapies ; },
abstract = {Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.},
}
@article {pmid36995671,
year = {2023},
author = {Garza, S and Paik, R},
title = {CRISPR/Cas9 Endonuclease-Mediated Mouse Genome Editing of One-Cell and/or Two-Cell Embryos by Electroporation, and the Use of Rad51 to Enhance Knock-In Allele Homozygosity via Interhomolog Repair Mechanism.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {253-266},
pmid = {36995671},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; Electroporation/methods ; DNA ; Gene Knock-In Techniques ; },
abstract = {Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.},
}
@article {pmid36995669,
year = {2023},
author = {Wefers, B and Wurst, W and Kühn, R},
title = {Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {207-230},
pmid = {36995669},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Zygote/metabolism ; Gene Targeting/methods ; Mice, Knockout ; RNA, Guide, Kinetoplastida/genetics ; },
abstract = {Engineering of the mouse germline is a key technology in biomedical research for studying the function of genes in health and disease. Since the first knockout mouse was described in 1989, gene targeting was based on recombination of vector encoded sequences in mouse embryonic stem cell lines and their introduction into preimplantation embryos to obtain germline chimeric mice. This approach has been replaced in 2013 by the application of the RNA-guided CRISPR/Cas9 nuclease system, which is introduced into zygotes and directly creates targeted modifications in the mouse genome. Upon the introduction of Cas9 nuclease and guide RNAs into one-cell embryos, sequence-specific double-strand breaks are created that are highly recombinogenic and processed by DNA repair enzymes. Gene editing commonly refers to the diversity of DSB repair products that include imprecise deletions or precise sequence modifications copied from repair template molecules. Since gene editing can now be easily applied directly in mouse zygotes, it has rapidly become the standard procedure for generating genetically engineered mice. This article covers the design of guide RNAs, knockout and knockin alleles, options for donor delivery, preparation of reagents, microinjection or electroporation of zygotes, and the genotyping of pups derived from gene editing projects.},
}
@article {pmid36995667,
year = {2023},
author = {Narina, S and Connelly, JP and Pruett-Miller, SM},
title = {High-Throughput Analysis of CRISPR-Cas9 Editing Outcomes in Cell and Animal Models Using CRIS.py.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {155-182},
pmid = {36995667},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mutation ; Models, Animal ; },
abstract = {Genome editing using the CRISPR-Cas9 platform creates precise modifications in cells and whole organisms. Although knockout (KO) mutations can occur at high frequencies, determining the editing rates in a pool of cells or selecting clones that contain only KO alleles can be a challenge. User-defined knock-in (KI) modifications are achieved at much lower rates, making the identification of correctly modified clones even more challenging. The high-throughput format of targeted next-generation sequencing (NGS) provides a platform allowing sequence information to be gathered from a one to thousands of samples. However, it also poses a challenge in terms of analyzing the large amount of data that is generated. In this chapter, we present and discuss CRIS.py, a simple and highly versatile Python-based program for analyzing NGS data for genome-editing outcomes. CRIS.py can be used to analyze sequencing results for any kind of modification or multiplex modifications specified by the user. Moreover, CRIS.py runs on all fastq files found in a directory, thereby concurrently analyzing all uniquely indexed samples. CRIS.py results are consolidated into two summary files, which allows users to sort and filter results and quickly identify the clones (or animals) of greatest interest.},
}
@article {pmid36995665,
year = {2023},
author = {Mackenzie, M and Fower, A and Allan, AJ and Codner, GF and Bunton-Stasyshyn, RK and Teboul, L},
title = {Genotyping Genome-Edited Founders and Subsequent Generation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {103-134},
pmid = {36995665},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genotype ; Mutation ; Genome ; },
abstract = {Targeted nucleases allow the production of many types of genetic mutations directly in the early embryo. However, the outcome of their activity is a repair event of unpredictable nature, and the founder animals that are produced are generally of a mosaic nature. Here, we present the molecular assays and genotyping strategies that will support the screening of the first generation for potential founders and the validation of positive animals in the subsequent generation, according to the type of mutation generated.},
}
@article {pmid36995664,
year = {2023},
author = {Lintott, LG and Nutter, LMJ},
title = {Genetic and Molecular Quality Control of Genetically Engineered Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2631},
number = {},
pages = {53-101},
pmid = {36995664},
issn = {1940-6029},
mesh = {Mice ; Animals ; Humans ; *Gene Editing/methods ; *Genetic Engineering ; Embryonic Stem Cells ; Transgenes ; Quality Control ; CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {Genetically engineered mice are used as avatars to understand mammalian gene function and develop therapies for human disease. During genetic modification, unintended changes can occur, and these changes may result in misassigned gene-phenotype relationships leading to incorrect or incomplete experimental interpretations. The types of unintended changes that may occur depend on the allele type being made and the genetic engineering approach used. Here we broadly categorize allele types as deletions, insertions, base changes, and transgenes derived from engineered embryonic stem (ES) cells or edited mouse embryos. However, the methods we describe can be adapted to other allele types and engineering strategies. We describe the sources and consequ ences of common unintended changes and best practices for detecting both intended and unintended changes by screening and genetic and molecular quality control (QC) of chimeras, founders, and their progeny. Employing these practices, along with careful allele design and good colony management, will increase the chance that investigations using genetically engineered mice will produce high-quality reproducible results, to enable a robust understanding of gene function, human disease etiology, and therapeutic development.},
}
@article {pmid36995636,
year = {2023},
author = {González, MN and Massa, GA and Andersson, M and Storani, L and Olsson, N and Décima Oneto, CA and Hofvander, P and Feingold, SE},
title = {CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {333-361},
pmid = {36995636},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; Plant Breeding ; Gene Editing/methods ; Genomics ; },
abstract = {Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene(s). In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein (RNP) complex assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding.},
}
@article {pmid36995634,
year = {2023},
author = {Wu, FH and Hsu, CT and Lin, CS},
title = {Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {297-315},
pmid = {36995634},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Tobacco/genetics ; Protoplasts ; Oligodeoxyribonucleotides ; },
abstract = {Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simple protocol that eliminates the need for expensive equipment, chemicals, modifications of donor DNA, and complicated vector construction. The protocol uses polyethylene glycol (PEG)-calcium to deliver low-cost, unmodified single-stranded oligodeoxyribonucleotides (ssODNs) and CRISPR/Cas9 ribonucleoprotein (RNP) complexes into Nicotiana benthamiana protoplasts. Regenerated plants were obtained from edited protoplasts with an editing frequency of up to 50% at the target locus. The inserted sequence was inherited to the next generation; this method thus opens the possibility for the future exploration of genomes by targeted insertion in plants.},
}
@article {pmid36995633,
year = {2023},
author = {Jiang, Q and Yang, Q and Harwood, W and Tang, H and Wei, Y and Zheng, Y},
title = {A CRISPR/Cas9 Protocol for Target Gene Editing in Barley.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {287-296},
pmid = {36995633},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Hordeum/genetics ; CRISPR-Associated Protein 9/genetics ; Genome ; },
abstract = {Previous studies of gene function rely on the existing natural genetic variation or on induction of mutations by physical or chemical mutagenesis. The availability of alleles in nature, and random mutagenesis induced by physical or chemical means, limits the depth of research. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system provides the means to rapidly modify genomes in a precise and predictable way, making it possible to modulate gene expression and modify the epigenome. Barley is the most appropriate model species for functional genomic analysis of common wheat. Therefore, the genome editing system of barley is very important for the study of wheat gene function. Here we detail a protocol for barley gene editing. The effectiveness of this method has been confirmed in our previous published studies.},
}
@article {pmid36995632,
year = {2023},
author = {Quach, T and Nguyen, H and Meyer, O and Sato, SJ and Clemente, TE and Guo, M},
title = {Introduction of Genome Editing Reagents and Genotyping of Derived Edited Alleles in Soybean (Glycine max (L.) Merr.).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {273-285},
pmid = {36995632},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Soybeans/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; Genotype ; },
abstract = {Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome.},
}
@article {pmid36995630,
year = {2023},
author = {Lawrenson, T and Atkinson, N and Forner, M and Harwood, W},
title = {Highly Efficient Gene Knockout in Medicago truncatula Genotype R108 Using CRISPR-Cas9 System and an Optimized Agrobacterium Transformation Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {221-252},
pmid = {36995630},
issn = {1940-6029},
mesh = {*Agrobacterium/genetics ; CRISPR-Cas Systems/genetics ; *Medicago truncatula/genetics ; Gene Knockout Techniques ; Genotype ; },
abstract = {Medicago truncatula is the model plant species for studying symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizae, where edited mutants are invaluable for elucidating the contributions of known genes in these processes. Streptococcus pyogenes Cas9 (SpCas9)-based genome editing is a facile means of achieving loss of function, including where multiple gene knockouts are desired in a single generation. We describe how the user can customize our vector to target single or multiple genes, then how the vector is used to make M. truncatula transgenic plants containing target site mutations. Finally, obtaining transgene-free homozygous mutants is covered.},
}
@article {pmid36995629,
year = {2023},
author = {Dhokane, D and Kancharla, N and Savarimuthu, A and Bhadra, B and Bandyopadhyay, A and Dasgupta, S},
title = {Genome Editing in Chlamydomonas reinhardtii Using Cas9-gRNA Ribonucleoprotein Complex: A Step-by-Step Guide.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {207-217},
pmid = {36995629},
issn = {1940-6029},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Chlamydomonas reinhardtii/genetics/metabolism ; RNA, Guide, Kinetoplastida/genetics ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Genome editing technologies have provided opportunities to manipulate literally any genomic location, opening new avenues for reverse genetics-based improvements. Among them, CRISPR/Cas9 is the most versatile tool for genome editing applications in prokaryotes and eukaryotes. Here, we provide a guide to successfully carry out high-efficiency genome editing in Chlamydomonas reinhardtii using preassembled CRISPR/Cas9-gRNA ribonucleoprotein (RNP) complexes.},
}
@article {pmid36995627,
year = {2023},
author = {Becker, M and Hensel, G},
title = {Ribonucleoprotein (RNP)-Mediated Targeted Mutagenesis in Barley (Hordeum vulgare L.).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {187-197},
pmid = {36995627},
issn = {1940-6029},
mesh = {*Hordeum/genetics ; Ribonucleoproteins/genetics ; Genetic Engineering/methods ; Mutagenesis ; Edible Grain/genetics ; CRISPR-Cas Systems ; Genome, Plant ; },
abstract = {The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants.},
}
@article {pmid36995625,
year = {2023},
author = {Fang, H and Culver, JN and Niedz, RP and Qi, Y},
title = {Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {153-171},
pmid = {36995625},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants/genetics ; Ribonucleoproteins/genetics/metabolism ; Cell Line ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment.},
}
@article {pmid36995623,
year = {2023},
author = {Yuan, G and Tuskan, GA and Yang, X},
title = {Use of Fluorescent Protein Reporters for Assessing and Detecting Genome Editing Reagents and Transgene Expression in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {115-127},
pmid = {36995623},
issn = {1940-6029},
mesh = {*Gene Editing ; Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Indicators and Reagents ; Transgenes ; Genome, Plant/genetics ; },
abstract = {Fluorescent protein reporters have been widely used for monitoring the expression of target genes in various engineered organisms. Although a wide range of analytical approaches (e.g., genotyping PCR, digital PCR, DNA sequencing) have been utilized to detect and identify genome editing reagents and transgene expression in genetically modified plants, these methods are usually limited to use in the late stages of plant transformation and can only be used invasively. Here we describe GFP- and eYGFPuv-based strategies and methods for assessing and detecting genome editing reagents and transgene expression in plants, including protoplast transformation, leaf infiltration, and stable transformation. These methods and strategies enable easy, noninvasive screening of genome editing and transgenic events in plants.},
}
@article {pmid36995622,
year = {2023},
author = {Xiong, J and Wang, C and Wang, K},
title = {Construction of CRISPR/Cas9 Multiplex Genome Editing System in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {107-114},
pmid = {36995622},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Mutation ; },
abstract = {Multiplex genome editing (MGE) technologies constitute essential tools for rapid genome modification of multiple targets in one gene or multiple genes simultaneously. However, the vector construction process is complicated, and the number of mutation targets is constrained using the conventional binary vectors. Here, we describe a simple CRISPR/Cas9 MGE system based on classical isocaudomer technique in rice, which is comprised of only two simple vectors, and can theoretically be used to edit an unlimited number of genes simultaneously.},
}
@article {pmid36995619,
year = {2023},
author = {Li, G and Sretenovic, S and Coleman, G and Qi, Y},
title = {Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {53-71},
pmid = {36995619},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Agrobacterium/genetics ; Promoter Regions, Genetic ; },
abstract = {CRISPR-Cas9 systems have revolutionized genome editing in plants and facilitated gene knockout and functional genomic studies in woody plants, like poplar. However, in tree species, previous studies have only focused on targeting indel mutations through CRISPR-based nonhomologous end joining (NHEJ) pathway. Cytosine base editors (CBEs) and adenine base editors (ABEs) enable C-to-T and A-to-G base changes, respectively. These base editors can introduce premature stop codons and amino acid changes, alter RNA splicing sites, and edit cis-regulatory elements of promoters. Base editing systems have only been recently established in trees. In this chapter, we describe a detailed, robust, and thoroughly tested protocol for preparing T-DNA vectors with two highly efficient CBEs, PmCDA1-BE3 and A3A/Y130F-BE3, and the highly efficient ABE8e as well as delivering the T-DNA through an improved protocol for Agrobacterium-mediated transformation in poplar. This chapter will provide promising application potential for precise base editing in poplar and other trees.},
}
@article {pmid36995618,
year = {2023},
author = {Liang, D and Liu, Y and Li, C and Wen, Q and Xu, J and Geng, L and Liu, C and Jin, H and Gao, Y and Zhong, H and Dawson, J and Tian, B and Barco, B and Su, X and Dong, S and Li, C and Elumalai, S and Que, Q and Jepson, I and Shi, L},
title = {CRISPR/LbCas12a-Mediated Genome Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {39-52},
pmid = {36995618},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Soybeans/genetics/metabolism ; Endonucleases/genetics ; Plants, Genetically Modified/genetics/metabolism ; Genome, Plant/genetics ; },
abstract = {Currently methods for generating soybean edited lines are time-consuming, inefficient, and limited to certain genotypes. Here we describe a fast and highly efficient genome editing method based on CRISPR-Cas12a nuclease system in soybean. The method uses Agrobacterium-mediated transformation to deliver editing constructs and uses aadA or ALS genes as selectable marker. It only takes about 45 days to obtain greenhouse-ready edited plants at higher than 30% transformation efficiency and 50% editing rate. The method is applicable to other selectable markers including EPSPS and has low transgene chimera rate. The method is also genotype-flexible and has been applied to genome editing of several elite soybean varieties.},
}
@article {pmid36995616,
year = {2023},
author = {Sretenovic, S and Tang, X and Ren, Q and Zhang, Y and Qi, Y},
title = {PAM-Less CRISPR-SpRY Genome Editing in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {3-19},
pmid = {36995616},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Genome, Plant ; *Oryza/genetics/metabolism ; },
abstract = {Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.},
}
@article {pmid36995603,
year = {2023},
author = {He, L and He, Z and Li, Y and Sun, H and Wang, H},
title = {In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2640},
number = {},
pages = {287-311},
pmid = {36995603},
issn = {1940-6029},
mesh = {Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Satellite Cells, Skeletal Muscle ; Muscles ; },
abstract = {Skeletal muscle satellite cells (SCs) are adult stem cells responsible for muscle development and injury-induced muscle regeneration. Functional elucidation of intrinsic regulatory factors governing SC activity is constrained partially by the technological limitations in editing SCs in vivo. Although the power of CRISPR/Cas9 in genome manipulation has been widely documented, its application in endogenous SCs remains largely untested. Our recent study generates a muscle-specific genome editing system leveraging the Cre-dependent Cas9 knockin mice and AAV9-mediated sgRNAs delivery, which allows gene disruption in SCs in vivo. Here, we illustrate the step-by-step procedure for achieving efficient editing using the above system.},
}
@article {pmid36995602,
year = {2023},
author = {Nakamura, K and Tanaka, T and Yamanouchi, K},
title = {In Vivo Modeling of Skeletal Muscle Diseases Using the CRISPR/Cas9 System in Rats.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2640},
number = {},
pages = {277-285},
pmid = {36995602},
issn = {1940-6029},
mesh = {Animals ; Rats ; Mice ; Humans ; *Dystrophin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Muscle, Skeletal/metabolism ; Genetic Therapy/methods ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Disease Models, Animal ; },
abstract = {The CRISPR/Cas9 system is a powerful gene editing tool that can be used to modify a target gene in almost all species. It unlocks the possibility of generating knockout or knock-in genes in laboratory animals other than mice. The Dystrophin gene is implicated in human Duchenne muscular dystrophy; however, Dystrophin gene mutant mice do not show severe muscle degenerating phenotypes when compared to humans. On the other hand, Dystrophin gene mutant rats made with the CRISPR/Cas9 system show more severe phenotypes than those seen in mice. The phenotypes seen in dystrophin mutant rats are more representative of the features of human DMD. This implies that rats are better models of human skeletal muscle diseases than mice. In this chapter, we present a detailed protocol for the generation of gene-modified rats by microinjection into embryos using the CRISPR/Cas9 system.},
}
@article {pmid36997438,
year = {2023},
author = {Awan, MJA and Aslam, MQ and Naqvi, RZ and Amin, I and Mansoor, S},
title = {A graft that crafts nontransgenic and genome-edited plants.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2023.03.020},
pmid = {36997438},
issn = {1878-4372},
abstract = {Grafting in plants facilitates the transmission of biomolecules across the union formation. Recently, Yang et al. demonstrated that inter- and intraspecific grafting in plants can be exploited for trafficking tRNA-tagged mobile reagents of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system from the transgenic rootstock to wild-type scion for genetic improvement in plants through targeted mutagenesis.},
}
@article {pmid36995631,
year = {2023},
author = {Lawrenson, T and Youles, M and Chhetry, M and Clarke, M and Harwood, W and Hundleby, P},
title = {Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {253-271},
pmid = {36995631},
issn = {1940-6029},
abstract = {CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.},
}
@article {pmid36995628,
year = {2023},
author = {Becker, M and Hensel, G},
title = {Ribonucleoprotein (RNP)-Mediated Allele Replacement in Barley (Hordeum vulgare L.) Leaves.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {199-205},
pmid = {36995628},
issn = {1940-6029},
abstract = {Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes.},
}
@article {pmid36995626,
year = {2023},
author = {Ma, X and Li, X and Li, Z},
title = {Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {173-185},
pmid = {36995626},
issn = {1940-6029},
abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems have become the most widely adopted genome editing platform owing to their unprecedented simplicity, efficiency, and versatility. Typically, the genome editing enzyme is expressed in plant cells from an integrated transgene delivered by either Agrobacterium-mediated or biolistic transformation. Recently, plant virus vectors have emerged as promising tools for the in planta delivery of CRISPR/Cas reagent. Here, we provide a protocol for CRISPR/Cas9-mediated genome editing in the model tobacco plant Nicotiana benthamiana using a recombinant negative-stranded RNA rhabdovirus vector. The method is based on infection of N. benthamiana with a Sonchus yellow net virus (SYNV)-based vector that carries the Cas9 and guide RNA expression cassettes to target specific genome loci for mutagenesis. With this method, mutant plants free of foreign DNA can be obtained within 4-5 months.},
}
@article {pmid36995621,
year = {2023},
author = {Wu, Y and Zhang, T},
title = {Designing Guide-RNA for Generating Premature Stop Codons for Gene Knockout Using CRISPR-BETS.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {95-105},
pmid = {36995621},
issn = {1940-6029},
abstract = {Cytosine base editors (CBEs) accurately modify target sites by mediating a C to T change (or a G to A change on the opposite strand). This allows us to install premature stop codons for gene knockout. However, highly specific sgRNAs (single-guide RNAs) are necessary for the CRISPR-Cas nuclease to work efficiently. In this study, we introduce a method of designing highly specific gRNA to generate premature stop codons and knock out a gene using CRISPR-BETS software.},
}
@article {pmid36945419,
year = {2023},
author = {Koseki, S and Hong, L and Yudistyra, V and Stan, T and Tysinger, E and Silverstein, R and Kramme, C and Amrani, N and Savic, N and Pacesa, M and Rodriguez, TS and Ponnapati, M and Jacobson, J and Church, G and Truant, R and Jinek, M and Kleinstiver, B and Sontheimer, E and Chatterjee, P},
title = {PAM-Flexible Genome Editing with an Engineered Chimeric Cas9.},
journal = {Research square},
volume = {},
number = {},
pages = {},
pmid = {36945419},
abstract = {CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.},
}
@article {pmid36912653,
year = {2023},
author = {Li, J and Wang, X and Zou, J and Yang, K and Wang, X and Wang, Y and Zhang, H and Huang, H and Su, X and Yao, B and Luo, H and Qin, X},
title = {Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.},
journal = {Applied and environmental microbiology},
volume = {89},
number = {3},
pages = {e0210722},
pmid = {36912653},
issn = {1098-5336},
mesh = {Copper/pharmacology/metabolism ; CRISPR-Cas Systems ; Gene Editing ; *Ascomycota/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *P-type ATPases/genetics ; },
abstract = {Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.},
}
@article {pmid36890230,
year = {2023},
author = {Puig-Saus, C and Sennino, B and Peng, S and Wang, CL and Pan, Z and Yuen, B and Purandare, B and An, D and Quach, BB and Nguyen, D and Xia, H and Jilani, S and Shao, K and McHugh, C and Greer, J and Peabody, P and Nayak, S and Hoover, J and Said, S and Jacoby, K and Dalmas, O and Foy, SP and Conroy, A and Yi, MC and Shieh, C and Lu, W and Heeringa, K and Ma, Y and Chizari, S and Pilling, MJ and Ting, M and Tunuguntla, R and Sandoval, S and Moot, R and Hunter, T and Zhao, S and Saco, JD and Perez-Garcilazo, I and Medina, E and Vega-Crespo, A and Baselga-Carretero, I and Abril-Rodriguez, G and Cherry, G and Wong, DJ and Hundal, J and Chmielowski, B and Speiser, DE and Bethune, MT and Bao, XR and Gros, A and Griffith, OL and Griffith, M and Heath, JR and Franzusoff, A and Mandl, SJ and Ribas, A},
title = {Neoantigen-targeted CD8[+] T cell responses with PD-1 blockade therapy.},
journal = {Nature},
volume = {615},
number = {7953},
pages = {697-704},
pmid = {36890230},
issn = {1476-4687},
mesh = {Humans ; *Antigens, Neoplasm/immunology ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; *Immunotherapy ; *Melanoma/drug therapy/genetics/immunology/pathology ; Receptors, Antigen, T-Cell/immunology/metabolism ; *Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; HLA Antigens/immunology ; Neoplasm Metastasis ; Precision Medicine ; Gene Editing ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells[1-14]. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies[15-17] to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8[+] T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.},
}
@article {pmid36759244,
year = {2023},
author = {Ben Yacoub, T and Wohlschlegel, J and Sahel, JA and Zeitz, C and Audo, I},
title = {[CRISPR/Cas9: From research to therapeutic application].},
journal = {Journal francais d'ophtalmologie},
volume = {46},
number = {4},
pages = {398-407},
doi = {10.1016/j.jfo.2022.10.008},
pmid = {36759244},
issn = {1773-0597},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Neoplasms ; },
abstract = {For several decades, genome engineering has raised interest among many researchers and physicians in the study of genetic disorders and their treatments. Compared to its predecessors, zinc-finger nucleases (ZFN) and transcription activator-like effectors (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is currently the most efficient molecular tool for genome editing. This system, originally identified as a bacterial adaptive immune system, is capable of cutting and modifying any gene of a large number of living organisms. Numerous trials using this technology are being developed to provide effective treatment for several diseases, such as cancer, cardiovascular and ophthalmic disorders. In research, this technology is increasingly used for genetic disease modelling, providing meaningful models of relevant studies as well as a better understanding of underlying pathological mechanisms. Many molecular tools are now available to put this technique into practice in laboratories, and despite the technical and ethical issues raised by manipulation of the genome, CRIPSR/Cas9 offers a new breath of hope for therapeutic research around the world.},
}
@article {pmid36356599,
year = {2023},
author = {Foy, SP and Jacoby, K and Bota, DA and Hunter, T and Pan, Z and Stawiski, E and Ma, Y and Lu, W and Peng, S and Wang, CL and Yuen, B and Dalmas, O and Heeringa, K and Sennino, B and Conroy, A and Bethune, MT and Mende, I and White, W and Kukreja, M and Gunturu, S and Humphrey, E and Hussaini, A and An, D and Litterman, AJ and Quach, BB and Ng, AHC and Lu, Y and Smith, C and Campbell, KM and Anaya, D and Skrdlant, L and Huang, EY and Mendoza, V and Mathur, J and Dengler, L and Purandare, B and Moot, R and Yi, MC and Funke, R and Sibley, A and Stallings-Schmitt, T and Oh, DY and Chmielowski, B and Abedi, M and Yuan, Y and Sosman, JA and Lee, SM and Schoenfeld, AJ and Baltimore, D and Heath, JR and Franzusoff, A and Ribas, A and Rao, AV and Mandl, SJ},
title = {Non-viral precision T cell receptor replacement for personalized cell therapy.},
journal = {Nature},
volume = {615},
number = {7953},
pages = {687-696},
pmid = {36356599},
issn = {1476-4687},
support = {K08 AI139375/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; Antigens, Neoplasm/genetics/immunology ; Biopsy ; *Cell- and Tissue-Based Therapy/adverse effects/methods ; Cytokine Release Syndrome/complications ; Disease Progression ; Encephalitis/complications ; *Gene Editing ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, T-Cell Receptor alpha ; Genes, T-Cell Receptor beta ; Mutation ; *Neoplasms/complications/genetics/immunology/therapy ; Patient Safety ; *Precision Medicine/adverse effects/methods ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; *Transgenes/genetics ; HLA Antigens/immunology ; CRISPR-Cas Systems ; },
abstract = {T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells[1-3]. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.},
}
@article {pmid36995617,
year = {2023},
author = {Wada, N and Osakabe, K and Osakabe, Y},
title = {Type I-D CRISPR System-Mediated Genome Editing in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2653},
number = {},
pages = {21-38},
pmid = {36995617},
issn = {1940-6029},
abstract = {Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells.},
}
@article {pmid36995210,
year = {2023},
author = {Mahler, M and Malone, LM and van den Berg, DF and Smith, LM and Brouns, SJJ and Fineran, PC},
title = {An OmpW-dependent T4-like phage infects Serratia sp. ATCC 39006.},
journal = {Microbial genomics},
volume = {9},
number = {3},
pages = {},
doi = {10.1099/mgen.0.000968},
pmid = {36995210},
issn = {2057-5858},
abstract = {Serratia sp. ATCC 39006 is a Gram-negative bacterium that has been used to study the function of phage defences, such as CRISPR-Cas, and phage counter-defence mechanisms. To expand our phage collection to study the phage-host interaction with Serratia sp. ATCC 39006, we isolated the T4-like myovirus LC53 in Ōtepoti Dunedin, Aotearoa New Zealand. Morphological, phenotypic and genomic characterization revealed that LC53 is virulent and similar to other Serratia, Erwinia and Kosakonia phages belonging to the genus Winklervirus. Using a transposon mutant library, we identified the host ompW gene as essential for phage infection, suggesting that it encodes the phage receptor. The genome of LC53 encodes all the characteristic T4-like core proteins involved in phage DNA replication and generation of viral particles. Furthermore, our bioinformatic analysis suggests that the transcriptional organization of LC53 is similar to that of Escherichia coli phage T4. Importantly, LC53 encodes 18 tRNAs, which likely compensate for differences in GC content between phage and host genomes. Overall, this study describes a newly isolated phage infecting Serratia sp. ATCC 39006 that expands the diversity of phages available to study phage-host interactions.},
}
@article {pmid36993865,
year = {2023},
author = {Ahmad, M},
title = {Plant breeding advancements with "CRISPR-Cas" genome editing technologies will assist future food security.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1133036},
pmid = {36993865},
issn = {1664-462X},
abstract = {Genome editing techniques are being used to modify plant breeding, which might increase food production sustainably by 2050. A product made feasible by genome editing is becoming better known, because of looser regulation and widespread acceptance. The world's population and food supply would never have increased proportionally under current farming practices. The development of plants and food production has been greatly impacted by global warming and climate change. Therefore, minimizing these effects is crucial for agricultural production that is sustainable. Crops are becoming more resilient to abiotic stress because of sophisticated agricultural practices and a better understanding of the abiotic stress response mechanism. Both conventional and molecular breeding techniques have been used to create viable crop types both processes are time-consuming. Recently, plant breeders have shown an interest in genome editing approaches for genetic manipulation that use clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). To ensure the security of the food supply in the future, plant kinds with desired traits must be developed. A completely new era in plant breeding has begun because of the revolution in genome editing techniques based on the CRISPR/CRISPR-associated nuclease (Cas9) systems. All plants may effectively target a particular gene or group of loci using Cas9 and single-guide RNA (sgRNA). CRISPR/Cas9 can thereby save time and labor compared to conventional breeding methods. An easy, quick, and efficient method for directly altering the genetic sequences in cells is with the CRISPR and Cas9 systems. The CRISPR-Cas9 system, which was developed from components of the earliest known bacterial immune system, allows for targeted gene breakage and gene editing in a variety of cells/RNA sequences to guide endonuclease cleavage specificity in the CRISPR-Cas9 system. Editing can be directed to practically any genomic site by altering the guide RNA (gRNA) sequence and delivering it to a target cell along with the Cas9 endonuclease. We summarize recent CRISPR/Cas9 plant research findings, investigate potential applications in plant breeding, and make predictions about likely future breakthroughs and approaches to food security through 2050.},
}
@article {pmid36993599,
year = {2023},
author = {Meers, C and Le, H and Pesari, SR and Hoffmann, FT and Walker, MWG and Gezelle, J and Sternberg, SH},
title = {Transposon-encoded nucleases use guide RNAs to selfishly bias their inheritance.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.03.14.532601},
pmid = {36993599},
abstract = {Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS 200 /IS 605 elements undergo 'peel-and-paste' transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements from Geobacillus stearothermophilus that encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.},
}
@article {pmid36992681,
year = {2023},
author = {Sukegawa, S and Nureki, O and Toki, S and Saika, H},
title = {Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1138843},
pmid = {36992681},
issn = {2673-3439},
abstract = {Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4 h per day for 3 days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.},
}
@article {pmid36992399,
year = {2023},
author = {Mazurov, D and Ramadan, L and Kruglova, N},
title = {Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles.},
journal = {Viruses},
volume = {15},
number = {3},
pages = {},
doi = {10.3390/v15030690},
pmid = {36992399},
issn = {1999-4915},
abstract = {Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.},
}
@article {pmid36983530,
year = {2023},
author = {Wang, D and Jin, S and Lu, Q and Chen, Y},
title = {Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {9},
number = {3},
pages = {},
pmid = {36983530},
issn = {2309-608X},
abstract = {Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering.},
}
@article {pmid36983076,
year = {2023},
author = {Zhang, Y and Geng, Y and Li, S and Shi, T and Ma, X and Hua, R and Fang, L},
title = {Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1[T] via CRISPR/Cas9 with Red System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36983076},
issn = {1422-0067},
mesh = {*Insecticides/metabolism ; CRISPR-Cas Systems/genetics ; Organophosphorus Compounds/metabolism ; *Cupriavidus/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Cupriavidus nantongensis X1[T] is a type strain of the genus Cupriavidus, that can degrade eight kinds of organophosphorus insecticides (OPs). Conventional genetic manipulations in Cupriavidus species are time-consuming, difficult, and hard to control. The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) system has emerged as a powerful tool for genome editing applied in prokaryotes and eukaryotes due to its simplicity, efficiency, and accuracy. Here, we combined CRISPR/Cas9 with the Red system to perform seamless genetic manipulation in the X1[T] strain. Two plasmids, pACasN and pDCRH were constructed. The pACasN plasmid contained Cas9 nuclease and Red recombinase, and the pDCRH plasmid contained the dual single-guide RNA (sgRNA) of organophosphorus hydrolase (OpdB) in the X1[T] strain. For gene editing, two plasmids were transferred to the X1[T] strain and a mutant strain in which genetic recombination had taken place, resulting in the targeted deletion of opdB. The incidence of homologous recombination was over 30%. Biodegradation experiments suggested that the opdB gene was responsible for the catabolism of organophosphorus insecticides. This study was the first to use the CRISPR/Cas9 system for gene targeting in the genus Cupriavidus, and it furthered our understanding of the process of degradation of organophosphorus insecticides in the X1[T] strain.},
}
@article {pmid36982987,
year = {2023},
author = {Perdigão, PRL and Ollington, B and Sai, H and Leung, A and Sacristan-Reviriego, A and van der Spuy, J},
title = {Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982987},
issn = {1422-0067},
support = {MR/P02582X/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Child, Preschool ; Humans ; *Leber Congenital Amaurosis/pathology ; Carrier Proteins/metabolism ; Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Organoids/metabolism ; Eye Proteins/genetics/metabolism ; },
abstract = {Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and β, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.},
}
@article {pmid36982961,
year = {2023},
author = {Zhang, K and Zhang, W and Qin, M and Li, Y and Wang, H},
title = {Characterization and Application of the Sugar Transporter Zmo0293 from Zymomonas mobilis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982961},
issn = {1422-0067},
mesh = {*Zymomonas/genetics/metabolism ; Glucose/metabolism ; Escherichia coli/genetics/metabolism ; Biological Transport ; Ethanol/metabolism ; },
abstract = {Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for the commercial production of desirable bioproducts. Sugar transporters are responsible for the import of substrate sugars and the conversion of ethanol and other products. Glucose-facilitated diffusion protein Glf is responsible for facilitating the diffusion of glucose uptake in Z. mobilis. However, another sugar transporter-encoded gene, ZMO0293, is poorly characterized. We employed gene deletion and heterologous expression mediated by the CRISPR/Cas method to investigate the role of ZMO0293. The results showed that deletion of the ZMO0293 gene slowed growth and reduced ethanol production and the activities of key enzymes involved in glucose metabolism in the presence of high concentrations of glucose. Moreover, ZMO0293 deletion caused different transcriptional changes in some genes of the Entner Doudoroff (ED) pathway in the ZM4-ΔZM0293 strain but not in ZM4 cells. The integrated expression of ZMO0293 restored the growth of the glucose uptake-defective strain Escherichia coli BL21(DE3)-ΔptsG. This study reveals the function of the ZMO0293 gene in Z. mobilis in response to high concentrations of glucose and provides a new biological part for synthetic biology.},
}
@article {pmid36982733,
year = {2023},
author = {Moniruzzaman, M and Darwish, AG and Ismail, A and El-Kereamy, A and Tsolova, V and El-Sharkawy, I},
title = {Seedlessness Trait and Genome Editing-A Review.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982733},
issn = {1422-0067},
mesh = {*Gene Editing ; *Vitis/genetics ; Plant Breeding ; Seeds/genetics ; Fruit/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Parthenocarpy and stenospermocarpy are the two mechanisms underlying the seedless fruit set program. Seedless fruit occurs naturally and can be produced using hormone application, crossbreeding, or ploidy breeding. However, the two types of breeding are time-consuming and sometimes ineffective due to interspecies hybridization barriers or the absence of appropriate parental genotypes to use in the breeding process. The genetic engineering approach provides a better prospect, which can be explored based on an understanding of the genetic causes underlying the seedlessness trait. For instance, CRISPR/Cas is a comprehensive and precise technology. The prerequisite for using the strategy to induce seedlessness is identifying the crucial master gene or transcription factor liable for seed formation/development. In this review, we primarily explored the seedlessness mechanisms and identified the potential candidate genes underlying seed development. We also discussed the CRISPR/Cas-mediated genome editing approaches and their improvements.},
}
@article {pmid36982690,
year = {2023},
author = {Belalov, IS and Sokolov, AA and Letarov, AV},
title = {Diversity-Generating Retroelements in Prokaryotic Immunity.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982690},
issn = {1422-0067},
mesh = {*Bacteria/genetics ; *Retroelements/genetics ; Prokaryotic Cells ; Proteins/genetics ; CRISPR-Cas Systems ; },
abstract = {Adaptive immunity systems found in different organisms fall into two major types. Prokaryotes possess CRISPR-Cas systems that recognize former invaders using memorized (captured) pieces of their DNA as pathogen signatures. Mammals possess a vast repertoire of antibodies and T-cell receptor variants generated in advance. In this second type of adaptive immunity, a pathogen presentation to the immune system specifically activates the cells that express matching antibodies or receptors. These cells proliferate to fight the infection and form the immune memory. The principle of preemptive production of diverse defense proteins for future use can hypothetically take place in microbes too. We propose a hypothesis that prokaryotes employ diversity-generating retroelements to prepare defense proteins against yet-unknown invaders. In this study, we test this hypothesis with the methods of bioinformatics and identify several candidate defense systems based on diversity-generating retroelements.},
}
@article {pmid36982409,
year = {2023},
author = {Kim, MS and Ko, SR and Jung, YJ and Kang, KK and Lee, YJ and Cho, YG},
title = {Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982409},
issn = {1422-0067},
mesh = {*Oryza/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/metabolism ; Plant Breeding ; Stress, Physiological/genetics ; Plant Proteins/metabolism ; Gene Expression Regulation, Plant ; Droughts ; },
abstract = {Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein-protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.},
}
@article {pmid36982266,
year = {2023},
author = {Mohan, K and Dubey, SK and Jung, K and Dubey, R and Wang, QJ and Prajapati, S and Roney, J and Abney, J and Kleinman, ME},
title = {Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982266},
issn = {1422-0067},
support = {R01EY028206/NH/NIH HHS/United States ; K08EY021757/NH/NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Retina/metabolism ; Gene Editing/methods ; Electroretinography ; Genetic Vectors ; },
abstract = {The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.},
}
@article {pmid36982223,
year = {2023},
author = {Dyrkheeva, NS and Malakhova, AA and Zakharenko, AL and Okorokova, LS and Shtokalo, DN and Pavlova, SV and Medvedev, SP and Zakian, SM and Nushtaeva, AA and Tupikin, AE and Kabilov, MR and Khodyreva, SN and Luzina, OA and Salakhutdinov, NF and Lavrik, OI},
title = {Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.},
journal = {International journal of molecular sciences},
volume = {24},
number = {6},
pages = {},
pmid = {36982223},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; DNA ; DNA Repair ; DNA Topoisomerases, Type I/genetics/metabolism ; Esterases/metabolism ; *Phosphoric Diester Hydrolases/metabolism ; Proteasome Endopeptidase Complex/metabolism ; *Topotecan/pharmacology ; Transcriptome ; Poly (ADP-Ribose) Polymerase-1/metabolism ; },
abstract = {Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.},
}
@article {pmid36981028,
year = {2023},
author = {Mizushima, S and Sasanami, T and Ono, T and Kuroiwa, A},
title = {Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing.},
journal = {Genes},
volume = {14},
number = {3},
pages = {},
pmid = {36981028},
issn = {2073-4425},
mesh = {Animals ; Male ; Humans ; *Gene Editing ; *Sperm Injections, Intracytoplasmic ; CRISPR-Cas Systems/genetics ; Semen ; Chickens/genetics ; },
abstract = {Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.},
}
@article {pmid36980270,
year = {2023},
author = {Martínez-Fernández, C and Jha, S and Aliagas, E and Holmberg, CI and Nadal, E and Cerón, J},
title = {BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13.},
journal = {Cells},
volume = {12},
number = {6},
pages = {},
pmid = {36980270},
issn = {2073-4409},
mesh = {Animals ; Humans ; *Mesothelioma, Malignant ; Caenorhabditis elegans/genetics/metabolism ; *Mesothelioma/genetics/pathology ; Proteasome Endopeptidase Complex/genetics ; Synthetic Lethal Mutations ; Mutation/genetics ; Ubiquitin Thiolesterase/genetics/metabolism ; Tumor Suppressor Proteins/genetics ; },
abstract = {The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors.},
}
@article {pmid36977923,
year = {2023},
author = {Shi, P and Murphy, MR and Aparicio, AO and Kesner, JS and Fang, Z and Chen, Z and Trehan, A and Guo, Y and Wu, X},
title = {Collateral activity of the CRISPR/RfxCas13d system in human cells.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {334},
pmid = {36977923},
issn = {2399-3642},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA/genetics ; Transcriptome ; Bacteria/genetics ; },
abstract = {CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. While Cas13 nucleases are capable of degrading both target RNAs and bystander RNAs in vitro and in bacteria, initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells. Here we show that RfxCas13d, also known as CasRx, a widely used Cas13 system, can cause collateral transcriptome destruction when targeting abundant reporter RNA and endogenous RNAs, resulting in proliferation defect in target cells. While these results call for caution of using RfxCas13d for targeted RNA knockdown, we demonstrated that the collateral activity can be harnessed for selective depletion of a specific cell population defined by a marker RNA in an in vitro setting.},
}
@article {pmid36978193,
year = {2023},
author = {Ordon, J and Kiel, N and Becker, D and Kretschmer, C and Schulze-Lefert, P and Stuttmann, J},
title = {Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two.},
journal = {Plant methods},
volume = {19},
number = {1},
pages = {30},
pmid = {36978193},
issn = {1746-4811},
abstract = {BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.<br><br>RESULTS: We designed three pairs of guide RNAs for deletion of a&#x2009;~&#x2009;2.2&#xa0;kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.<br><br>CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.},
}
@article {pmid36977792,
year = {2023},
author = {Kolvenbach, CM and Dworschak, GC and Rieke, JM and Woolf, AS and Reutter, H and Odermatt, B and Hilger, AC},
title = {Modelling human lower urinary tract malformations in zebrafish.},
journal = {Molecular and cellular pediatrics},
volume = {10},
number = {1},
pages = {2},
pmid = {36977792},
issn = {2194-7791},
abstract = {Advances in molecular biology are improving our understanding of the genetic causes underlying human congenital lower urinary tract (i.e., bladder and urethral) malformations. This has recently led to the identification of the first disease-causing variants in the gene BNC2 for isolated lower urinary tract anatomical obstruction (LUTO), and of WNT3 and SLC20A1 as genes implicated in the pathogenesis of the group of conditions called bladder-exstrophy-epispadias complex (BEEC). Implicating candidate genes from human genetic data requires evidence of their influence on lower urinary tract development and evidence of the found genetic variants' pathogenicity. The zebrafish (Danio rerio) has many advantages for use as a vertebrate model organism for the lower urinary tract. Rapid reproduction with numerous offspring, comparable anatomical kidney and lower urinary tract homology, and easy genetic manipulability by Morpholino®-based knockdown or CRISPR/Cas editing are among its advantages. In addition, established marker staining for well-known molecules involved in urinary tract development using whole-mount in situ hybridization (WISH) and the usage of transgenic lines expressing fluorescent protein under a tissue-specific promoter allow easy visualization of phenotypic abnormalities of genetically modified zebrafish. Assays to examine the functionality of the excretory organs can also be modeled in vivo with the zebrafish. The approach of using these multiple techniques in zebrafish not only enables rapid and efficient investigation of candidate genes for lower urinary tract malformations derived from human data, but also cautiously allows transferability of causality from a non-mammalian vertebrate to humans.},
}
@article {pmid36976005,
year = {2023},
author = {Wang, C and Sun, Z and Hu, Y and Li, D and Guo, Q and Wang, M},
title = {A Novel Anti-CRISPR AcrIE9.2 Is Associated with Dissemination of blaKPC Plasmids in Klebsiella pneumoniae Sequence Type 15.},
journal = {Antimicrobial agents and chemotherapy},
volume = {},
number = {},
pages = {e0154722},
doi = {10.1128/aac.01547-22},
pmid = {36976005},
issn = {1098-6596},
abstract = {Sequence type (ST) 15 has become an emerging clone of carbapenem-resistant Klebsiella pneumoniae in which type I-E* CRISPR-Cas usually exists, indicating that the CRISPR-Cas system may not be able to block the transfer of blaKPC plasmids. The purpose of this study was to explore the mechanisms underlying dissemination of blaKPC plasmids in K. pneumoniae ST15. The type I-E* CRISPR-Cas system was present in 98.0% of 612 nonduplicate K. pneumoniae ST15 strains (88 clinical isolates and 524 from the NCBI database). Twelve ST15 clinical isolates were completely sequenced, and self-targeted protospacers were found on blaKPC plasmids flanked by a protospacer adjacent motif (PAM) of AAT in 11 isolates. The type I-E* CRISPR-Cas system was cloned from a clinical isolate and expressed in Escherichia coli BL21(DE3). In BL21(DE3) harboring the CRISPR system, the transformation efficiency of protospacer-bearing plasmids with a PAM of AAT was reduced by 96.2% compared to the empty vector, indicating that the type I-E* CRISPR-Cas system impeded blaKPC plasmid transfer. BLAST for known anti-CRISPR (Acr) amino acid sequences uncovered a novel AcrIE9-like protein with 40.5% to 44.6% sequence identity with AcrIE9 designated AcrIE9.2, which was present in 90.1% (146 of 162) of ST15 strains carrying both blaKPC and the CRISPR-Cas system. When AcrIE9.2 was cloned and expressed in a ST15 clinical isolate, the conjugation frequency of a CRISPR-targeted blaKPC plasmid was increased from 3.96 × 10[-6] to 2.01 × 10[-4] compared to the AcrIE9.2 absent strain. In conclusion, AcrIE9.2 may be associated with the dissemination of blaKPC in ST15 by repressing CRISPR-Cas activity.},
}
@article {pmid36975789,
year = {2023},
author = {Li, X and Bao, N and Yan, Z and Yuan, XZ and Wang, SG and Xia, PF},
title = {Degradation of Antibiotic Resistance Genes by VADER with CRISPR-Cas Immunity.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0005323},
doi = {10.1128/aem.00053-23},
pmid = {36975789},
issn = {1098-5336},
abstract = {The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes, e.g., biological wastewater treatment, are key barriers to prevent the spread of ARGs, they are often sources of ARGs at the same time, requiring upgraded biotechnology. Here, we present VADER, a synthetic biology system for the degradation of ARGs based on CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, to be implemented for wastewater treatment processes. Navigated by programmable guide RNAs, VADER targets and degrades ARGs depending on their DNA sequences, and by employing an artificial conjugation machinery, IncP, it can be delivered via conjugation. The system was evaluated by degrading plasmid-borne ARGs in Escherichia coli and further demonstrated via the elimination of ARGs on the environmentally relevant RP4 plasmid in Pseudomonas aeruginosa. Next, a prototype conjugation reactor at a 10-mL scale was devised, and 100% of the target ARG was eliminated in the transconjugants receiving VADER, giving a proof of principle for the implementation of VADER in bioprocesses. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. IMPORTANCE Antibiotic resistance has been causing severe health problems and has led to millions of deaths in recent years. Environmental processes, especially those of the wastewater treatment sector, are an important barrier to the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as a nonnegligible source of antibiotic resistance at the same time, as antibiotic resistance with its main cause, antibiotic resistance genes (ARGs), may accumulate in biological treatment units. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to tackle the antibiotic resistance problem raised in wastewater treatment processes, and we propose a new sector specialized in ARG removal with a conjugation reactor to implement the CRISPR-Cas system. Our study provides a new angle for resolving public health issues via the implementation of synthetic biology in environmental contexts at the process level.},
}
@article {pmid36974713,
year = {2023},
author = {Song, D and Wang, F and Ju, Y and He, Q and Sun, T and Deng, W and Ding, R and Zhang, C and Xu, Q and Qi, C and Bao, J},
title = {Application and development of noninvasive biomarkers for colorectal cancer screening: a systematic review.},
journal = {International journal of surgery (London, England)},
volume = {},
number = {},
pages = {},
doi = {10.1097/JS9.0000000000000260},
pmid = {36974713},
issn = {1743-9159},
abstract = {BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related death (9.4% of the 9.9 million cancer deaths). However, CRC develops slowly, and early detection and intervention can effectively improve the survival rate and quality of life. Although colonoscopy can detect and diagnose CRC, it is unsuitable for CRC screening in average-risk populations. Some commercial kits based on DNA mutation or methylation are approved for screening, but the low sensitivity for advanced adenoma or early-stage CRC would limit the applications.<br><br>MAIN RESULTS: Recently, researchers have focused on developing noninvasive or minimally invasive, easily accessible biomarkers with higher sensitivity and accuracy for CRC screening. Numerous reports describe advances in biomarkers, including DNA mutations and methylation, mRNA and miRNA, gut microbes, and metabolites, as well as low-throughput multiomics panels. In small cohorts, the specificity and sensitivity improved when fecal immunochemical testing combined with other biomarkers; further verification in large cohorts is expected. In addition, the continuous improvement of laboratory technology has also improved the sensitivity of detection technology, such as PCR, and the application of CRISPR/Cas technology. Besides, artificial intelligence has extensively promoted the mining of biomarkers. Machine learning was performed to construct a diagnosis model for CRC screening based on the cfDNA fragment features from whole-genome sequencing data. In another study, multiomics markers, including cfDNA, epigenetic, and protein signals, were also discovered by machine learning. Finally, advancements in sensor technology promote the applicability of volatile organic compounds in CRC early detection.<br><br>CONCLUSION: Here, the authors review advances in early detection and screening of CRC based on different biomarker types. Most studies reported optimistic findings based on preliminary research, and prospective clinical studies are ongoing. These promising biomarkers are expected to more accurately identify early-stage patients with CRC and be applied in the future.},
}
@article {pmid36974013,
year = {2023},
author = {Huang, W and Wei, H and Wang, C and Wang, J and Chen, L and Chen, W and Liu, Y and Zheng, Y and Lin, M},
title = {[Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum].},
journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control},
volume = {35},
number = {1},
pages = {38-43},
doi = {10.16250/j.32.1374.2022240},
pmid = {36974013},
issn = {1005-6661},
mesh = {Humans ; Plasmodium falciparum/genetics ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; CRISPR-Cas Systems ; *Malaria, Falciparum/genetics ; *Malaria/diagnosis ; DNA Primers ; *Malaria, Vivax ; RNA, Ribosomal, 18S/genetics ; },
abstract = {OBJECTIVE: To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.<br><br>METHODS: The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/&#x3bc;L for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/&#x3bc;L with healthy volunteers' O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested.<br><br>RESULTS: The Pf-F3/Pf-R3/crRNA2 combination, 2.5 &#x3bc;L as the addition amount of B buffer, 40 min as the RAA reaction time, 37 &#xb0;C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/&#x3bc;L, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/&#x3bc;L.<br><br>CONCLUSIONS: The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.},
}
@article {pmid36971438,
year = {2023},
author = {Sconocchia, T and Foßelteder, J and Köhnke, T and Majeti, R and Reinisch, A},
title = {Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {193},
pages = {},
doi = {10.3791/64558},
pmid = {36971438},
issn = {1940-087X},
mesh = {Humans ; *Gain of Function Mutation ; *Gene Editing/methods ; Hematopoietic Stem Cells ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.},
}
@article {pmid36746911,
year = {2023},
author = {Abdulmalek, HW and Yazgan-Karataş, A},
title = {Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon.},
journal = {Journal of microbiology and biotechnology},
volume = {33},
number = {3},
pages = {410-418},
doi = {10.4014/jmb.2209.09035},
pmid = {36746911},
issn = {1738-8872},
mesh = {*Bacillus subtilis/metabolism ; 5' Untranslated Regions ; *CRISPR-Cas Systems ; Operon ; Dipeptides/genetics ; },
abstract = {Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.},
}
@article {pmid36701041,
year = {2023},
author = {Mathew, SM},
title = {Strategies for generation of mice via CRISPR/HDR-mediated knock-in.},
journal = {Molecular biology reports},
volume = {50},
number = {4},
pages = {3189-3204},
pmid = {36701041},
issn = {1573-4978},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Recombinational DNA Repair ; Genetic Therapy ; DNA/genetics ; },
abstract = {CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. The clustered regularly interspaced short palindromic repeat gene editing technique, can efficiently generate knock-outs using the non-homologous end-joining repair pathway. Small knock-ins also work precisely using a repair template with help of homology-directed-repair (HDR) mechanism. However, when the fragment size is larger than 4-5 kb, the knock-in tends to be error prone and the efficiency decreases. Certain types of modifications, in particular insertions of very large DNA fragments (10-100 kb) or entire gene replacements, are still difficult. The HDR process needs further streamlining and improvement. Here in this review, we describe methods to enhance the efficiency of the knock-in through checking each step from the guide design to the microinjection and choice of the oocyte donors. This helps in understanding the parameters that can be modified to get improved knock-in efficiency via CRISPR targeting.},
}
@article {pmid36478032,
year = {2023},
author = {Beracochea, V and Stritzler, M and Radonic, L and Bottero, E and Jozefkowicz, C and Darqui, F and Ayub, N and Bilbao, ML and Soto, G},
title = {CRISPR/Cas9-mediated knockout of SPL13 radically increases lettuce yield.},
journal = {Plant cell reports},
volume = {42},
number = {3},
pages = {645-647},
pmid = {36478032},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems/genetics ; *Lettuce/genetics ; Gene Knockout Techniques ; Mutation ; Gene Editing ; },
}
@article {pmid36974023,
year = {2023},
author = {Li, M and Qin, Z and Yin, K and Zheng, B},
title = {[Application of CRISPR/Cas systems in the nucleic acid detection of pathogens: a review].},
journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control},
volume = {35},
number = {1},
pages = {98-103},
doi = {10.16250/j.32.1374.2022227},
pmid = {36974023},
issn = {1005-6661},
abstract = {Rapid, sensitive and specific detection tools are critical for the prevention and control of infectious diseases. The in vitro nucleic acid amplification assays, including polymerase chain reaction and isothermal amplification technology, have been widely used for the detection of pathogens. Recently, nucleic acid detection-based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) have been developed, which are rapid, highly sensitive, highly specific, and portable. This review describes the classification and principle of CRISPR/Cas systems and their applications in pathogen detection, and discusses the prospects of CRISPR/Cas systems.},
}
@article {pmid36970694,
year = {2023},
author = {Hu, Y and Jiang, J and Wang, D and Guo, Q and Wang, M},
title = {Coexistence of bla KPC-IncFII plasmids and type I-E[*] CRISPR-Cas systems in ST15 Klebsiella pneumoniae.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1125531},
pmid = {36970694},
issn = {1664-302X},
abstract = {The CRISPR-Cas system in Klebsiella pneumoniae can prevent the entry of bla KPC-IncF plasmids. However, some clinical isolates bear the KPC-2 plasmids despite carrying the CRISPR-Cas system. The purpose of this study was to characterize the molecular features of these isolates. A total of 697 clinical K. pneumoniae isolates were collected from 11 hospitals in China, and tested for the presence of CRISPR-Cas systems using polymerase chain reaction. Overall, 164 (23.5%) of 697 K. pneumoniae isolates had type I-E[*] (15.9%) or type I-E (7.7%) CRISPR-Cas systems. The most prevalent sequence type among isolates carrying type I-E[*] CRISPR was ST23 (45.9%), followed by ST15 (18.9%). Isolates with CRISPR-Cas system were more susceptible to ten antimicrobials tested, including carbapenems, compared with the CRISPR-negative isolates. However, there were still 21 CRISPR-Cas-carrying isolates that showed resistance to carbapenems, and these isolates were subjected to whole-genome sequencing. Thirteen of these 21 isolates carried bla KPC-2-bearing plasmids, of which nine had a new plasmid type, IncFIIK34, and two had IncFII(PHN7A8) plasmids. In addition, 12 of these 13 isolates belonged to ST15, while only eight (5.6%, 8/143) isolates belonged to ST15 in carbapenem-susceptible K. pneumoniae carrying CRISPR-Cas systems. In conclusion, we found that bla KPC-2-bearing IncFII plasmids could co-exist with the type I-E[*] CRISPR-Cas systems in ST15 K. pneumoniae.},
}
@article {pmid36969374,
year = {2023},
author = {Papaioannou, NY and Patsali, P and Naiisseh, B and Papasavva, PL and Koniali, L and Kurita, R and Nakamura, Y and Christou, S and Sitarou, M and Mussolino, C and Cathomen, T and Kleanthous, M and Lederer, CW},
title = {High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1141618},
pmid = {36969374},
issn = {2673-3439},
abstract = {Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34[+] cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34[+] cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34[+] cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.},
}
@article {pmid36968399,
year = {2023},
author = {Kumari, A and Sharma, D and Sharma, P and , and Wang, C and Verma, V and Patil, A and Imran, M and Singh, MP and Kumar, K and Paritosh, K and Caragea, D and Kapoor, S and Chandel, G and Grover, A and Jagadish, SVK and Katiyar-Agarwal, S and Agarwal, M},
title = {Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1133115},
pmid = {36968399},
issn = {1664-462X},
abstract = {Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.},
}
@article {pmid36967582,
year = {2023},
author = {Nieves-Cordones, M and Amo, J and Hurtado-Navarro, L and Martínez-Martínez, A and Martínez, V and Rubio, F},
title = {Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 uncovers CIPK-CBL-target network rewiring in land plants.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.18910},
pmid = {36967582},
issn = {1469-8137},
abstract = {Transport of K[+] to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK-CBL complexes have been widely shown to regulate K[+] uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage-gated K[+] channel SlSKOR and its regulation by the SlCIPK23-SlCBL1/9 complexes in tomato plants. We phenotyped gene-edited slskor and slcipk23 tomato knock-out mutants and carried out two-electrode voltage-clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches. SlSKOR was preferentially expressed in the root stele and was important not only for K[+] transport to shoots but also, indirectly, for that of Ca[2+] , Mg[2+] , Na[+] , NO3 [-] and Cl[-] . Surprisingly, the SlCIPK23-SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR-like channels by CIPK23-CBL1 complexes was also present in Medicago, grapevine and lettuce but not in Arabidopsis and saltwater cress. Our results provide a molecular framework for coordinating root K[+] uptake and its translocation to the shoot by SlCIPK23-SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK-CBL-target networks have evolved differently in land plants.},
}
@article {pmid36967147,
year = {2023},
author = {Hosoba, K and Morita, T and Zhang, Y and Kishi, H and Yamamoto, T and Miyamoto, T},
title = {High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.},
journal = {Methods in cell biology},
volume = {175},
number = {},
pages = {85-95},
doi = {10.1016/bs.mcb.2022.07.020},
pmid = {36967147},
issn = {0091-679X},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Targeting/methods ; Recombinational DNA Repair ; *Ciliopathies/genetics ; Mammals/genetics ; },
abstract = {Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.},
}
@article {pmid36967127,
year = {2023},
author = {June, CH},
title = {The Unlikely Development of CAR T Cells: a Brief History and Prospects for the Future.},
journal = {The Keio journal of medicine},
volume = {72},
number = {1},
pages = {26},
doi = {10.2302/kjm.ABSTRACT_72_1-1},
pmid = {36967127},
issn = {1880-1293},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Immunotherapy, Adoptive ; *Neoplasms/genetics/therapy ; T-Lymphocytes ; },
abstract = {The quest to cure cancer has been one of the Holy Grails of medicine. The discovery I am going to share with you, CAR T cells, is a promising new form of therapy of cancer that offers the prospect of curing cancer using the immune system. CAR T cells are the first form of synthetic biology to enter the practice of medicine. The notion of using the immune system to fight cancer is an old idea. Over a century ago, bacteria were ground up and injected into patients with late-stage cancers, and occasionally the cancer would disappear. However, we now have precise tools for genetic editing and gene insertion like CRISPR/Cas9 to rewrite the DNA code, offering the possibility to improve the immune system over what has evolved in a Darwinian fashion. In 2017 for the first time, CAR T cells were approved for the treatment of cancer. Today they are used worldwide in more than 15,000 patients and they offer the promise to move beyond cancer to other fields of medicine such as autoimmune disease and heart disease. Here I will discuss the promises and challenges faced by the evolving CAR T cell industry.},
}
@article {pmid36964659,
year = {2023},
author = {Bendixen, L and Jensen, TI and Bak, RO},
title = {CRISPR/Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2023.03.024},
pmid = {36964659},
issn = {1525-0024},
abstract = {The CRISPR/Cas system is commonly known for its ability to cleave DNA in a programmable manner, which has democratized gene editing and facilitated recent breakthroughs in gene therapy. However, newer iterations of the technology using nuclease-disabled Cas enzymes have spurred a variety of different types of genetic engineering platforms such as transcriptional modulation using the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems. This review introduces the creation of these programmable transcriptional modulators, various methods of delivery utilized for these systems, and recent technological developments. CRISPRa and CRISPRi have also been implemented in genetic screens for interrogating gene function and discovering genes involved in various biological pathways. We describe recent compelling examples of how these tools have become powerful means to unravel genetic networks and uncovering important information about devastating diseases. Finally, we provide an overview of preclinical studies in which transcriptional modulation has been used therapeutically, and we discuss potential future directions of these novel modalities.},
}
@article {pmid36924688,
year = {2023},
author = {Li, L and Tian, H and Wang, G and Ren, S and Ma, T and Wang, Y and Ge, S and Zhang, Y and Yu, J},
title = {Ready-to-use interactive dual-readout differential lateral flow biosensor for two genotypes of human papillomavirus.},
journal = {Biosensors &amp; bioelectronics},
volume = {228},
number = {},
pages = {115224},
doi = {10.1016/j.bios.2023.115224},
pmid = {36924688},
issn = {1873-4235},
mesh = {Humans ; Human Papillomavirus Viruses ; *Papillomavirus Infections/diagnosis ; Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Papillomaviridae/genetics ; Genotype ; CRISPR-Cas Systems ; },
abstract = {Ready-to-use in vitro diagnosis of multiple genotypes is vital for the prevention and treatment of cervical cancer. Herein, a paper-film-based interactive dual readout differential lateral flow biosensor is proposed to simultaneously assay two high-risk types of human papillomavirus (HPV) within the body-fluid. The CuCo2S4/ZnIn2S4 heterostructure is fabricated on the paper-film compound chip with high thermostability, and surface sulfur vacancy is introduced by mild annealing treatment to endow unexceptionable photoexcitation activity, such structure can be served as an initial energy harvester and converter. With the assistance of differential channels, the dual-target-propelled self-assembly of annular DNA and the cleavage activity of CRISPR-Cas12a are stepwise activated by sequential solution transfer. Accordingly, the input and release of polydopamine-coated gold nanoparticles with photothermal/photoelectric characteristic were implemented. The fabricated biosensor not only realized intelligent thermal-response without large instruments, but also actuated dynamic interfacial charge separation and transfer kinetics to further transmit photoelectric-signal, resulting in desirable interactive dual-signal with low limit-of-detection (0.21 pM for HPV-18 and 42.92 pM for HPV-16). Thanks to the sophisticated design of differential lateral flow paper-film compound chip and interactive dual-signal amplification strategy, sensitive detection of two HPV genotypes is realized, which provides a promising candidate for home medical intelligent diagnosis.},
}
@article {pmid36878066,
year = {2023},
author = {Zhu, Z and Guo, Y and Wang, C and Yang, Z and Li, R and Zeng, Z and Li, H and Zhang, D and Yang, L},
title = {An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.},
journal = {Biosensors &amp; bioelectronics},
volume = {228},
number = {},
pages = {115179},
pmid = {36878066},
issn = {1873-4235},
mesh = {Humans ; SARS-CoV-2/genetics ; *COVID-19/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; DNA ; RNA ; },
abstract = {Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.},
}
@article {pmid36812945,
year = {2023},
author = {Boontawon, T and Nakazawa, T and Choi, YJ and Ro, HS and Oh, M and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {370},
number = {},
pages = {},
doi = {10.1093/femsle/fnad015},
pmid = {36812945},
issn = {1574-6968},
mesh = {Gene Editing/methods ; *Pleurotus/genetics/metabolism ; *Agaricales/genetics ; CRISPR-Cas Systems ; Ribonucleoproteins/genetics/metabolism ; Gene Targeting ; },
abstract = {CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a preassembled Cas9 ribonucleoprotein (RNP) complex into the oyster mushroom Pleurotus ostreatus. However, the target gene was restricted to such a gene like pyrG, since screening of a genome-edited strain was indispensable and could be performed via examination of 5-fluoroorotic acid (5-FOA) resistance caused by the disruption of the target gene. In this study, we simultaneously introduced the Cas9 RNP complex targeting fcy1, a mutation that conferred P. ostreatus resistance to 5-fluorocytosine (5-FC), together with that targeting pyrG. A total of 76 5-FOA resistant strains were isolated during the first screening. Subsequently, a 5-FC resistance examination was conducted, and three strains exhibited resistance. Genomic PCR experiments followed by DNA sequencing revealed that mutations were successfully introduced into fcy1 and pyrG in the three strains. The results indicated that double gene-edited mutants could be obtained in one experiment employing 5-FOA resistance screening for strains with Cas9 RNP incorporation. This work may pave the way for safe CRISPR/Cas9 technology to isolate mutant strains in any gene of interest without an ectopic marker gene.},
}
@article {pmid36682390,
year = {2023},
author = {Sheikh Beig Goharrizi, MA and Ghodsi, S and Memarjafari, MR},
title = {Implications of CRISPR-Cas9 Genome Editing Methods in Atherosclerotic Cardiovascular Diseases.},
journal = {Current problems in cardiology},
volume = {48},
number = {5},
pages = {101603},
doi = {10.1016/j.cpcardiol.2023.101603},
pmid = {36682390},
issn = {1535-6280},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; CRISPR-Associated Protein 9/genetics/metabolism ; *Atherosclerosis/genetics/therapy ; },
abstract = {Today, new methods have been developed to treat or modify the natural course of cardiovascular diseases (CVDs), including atherosclerosis, by the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system. Genome-editing tools are CRISPR-related palindromic short iteration systems such as CRISPR-Cas9, a valuable technology for achieving somatic and germinal genomic manipulation in model cells and organisms for various applications, including the creation of deletion alleles. Mutations in genomic deoxyribonucleic acid and new genes' placement have emerged. Based on World Health Organization fact sheets, 17.9 million people die from CVDs each year, an estimated 32% of all deaths worldwide. 85% of all CVD deaths are due to acute coronary events and strokes. This review discusses the applications of CRISPR-Cas9 technology throughout atherosclerotic disease research and the prospects for future in vivo genome editing therapies. We also describe several limitations that must be considered to achieve the full scientific and therapeutic potential of cardiovascular genome editing in the treatment of atherosclerosis.},
}
@article {pmid36055523,
year = {2023},
author = {Liu, S and Duan, X and Peng, F and Wang, Y and Liu, Y and Wan, X and Zhang, J and Li, X and Sun, X},
title = {A tunable genome editing system of the prime editor mediated by dihydrofolate reductase.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {50},
number = {3},
pages = {204-207},
doi = {10.1016/j.jgg.2022.08.004},
pmid = {36055523},
issn = {1673-8527},
mesh = {*Gene Editing ; *Tetrahydrofolate Dehydrogenase/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid36963196,
year = {2023},
author = {Ma, L and Zhang, W and Yin, L and Li, Y and Zhuang, J and Shen, L and Man, S},
title = {A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel.},
journal = {Journal of hazardous materials},
volume = {452},
number = {},
pages = {131195},
pmid = {36963196},
issn = {1873-3336},
abstract = {The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.},
}
@article {pmid36961900,
year = {2023},
author = {Rubio, A and Sprang, M and Garzón, A and Moreno-Rodriguez, A and Pachón-Ibáñez, ME and Pachón, J and Andrade-Navarro, MA and Pérez-Pulido, AJ},
title = {Analysis of bacterial pangenomes reduces CRISPR dark matter and reveals strong association between membranome and CRISPR-Cas systems.},
journal = {Science advances},
volume = {9},
number = {12},
pages = {eadd8911},
pmid = {36961900},
issn = {2375-2548},
abstract = {CRISPR-Cas systems are prokaryotic acquired immunity mechanisms, which are found in 40% of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. However, the vast majority of these spacers have not yet been associated with the virus they recognize, and it has been named CRISPR dark matter. By analyzing the spacers of tens of thousands of genomes from six bacterial species, we have been able to reduce the CRISPR dark matter from 80% to as low as 15% in some of the species. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular sets of membrane proteins. Our results suggest that when bacteria present membrane proteins that make it compete better in its environment and these proteins are, in turn, receptors for specific phages, they would be forced to acquire CRISPR-Cas.},
}
@article {pmid36960537,
year = {2023},
author = {Jeon, W and Jung, SY and Lee, CY and Kim, WT and Kim, H and Jang, KW and Lim, H and Lee, M and Jeong, DH and Kim, SD and Kim, IA and Choi, SH and Son, TG and Kim, KS},
title = {Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.},
journal = {Technology in cancer research &amp; treatment},
volume = {22},
number = {},
pages = {15330338231165125},
doi = {10.1177/15330338231165125},
pmid = {36960537},
issn = {1533-0338},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; *Liver Neoplasms/genetics/radiotherapy/metabolism ; Mutation ; Organoids/metabolism/pathology ; Adenosine Triphosphate ; },
abstract = {BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1.<br><br>METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay.<br><br>RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations.<br><br>CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.},
}
@article {pmid36958270,
year = {2023},
author = {Botelho, J and Tüffers, L and Fuss, J and Buchholz, F and Utpatel, C and Klockgether, J and Niemann, S and Tümmler, B and Schulenburg, H},
title = {Phylogroup-specific variation shapes the clustering of antimicrobial resistance genes and defence systems across regions of genome plasticity in Pseudomonas aeruginosa.},
journal = {EBioMedicine},
volume = {90},
number = {},
pages = {104532},
doi = {10.1016/j.ebiom.2023.104532},
pmid = {36958270},
issn = {2352-3964},
abstract = {BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C). Here, we assessed phylogroup-specific evolutionary dynamics across available and also new P. aeruginosa genomes.<br><br>METHODS: In this genomic analysis, we first generated new genome assemblies for 18 strains of the major P.&#xa0;aeruginosa clone type (mPact) panel, comprising a phylogenetically diverse collection of clinical and environmental isolates for this species. Thereafter, we combined these new genomes with 1991 publicly available P.&#xa0;aeruginosa genomes for a phylogenomic and comparative analysis. We specifically explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups.<br><br>FINDINGS: We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of pangenome families, and show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these cargo genes is often significantly correlated. Moreover, inter- and intra-phylogroup interactions occur at the accessory genome level, suggesting frequent recombination events. Finally, we provide here the mPact panel of diverse P.&#xa0;aeruginosa strains that may serve as a valuable reference for functional analyses.<br><br>INTERPRETATION: Altogether, our results highlight distinct pangenome characteristics of the P.&#xa0;aeruginosa phylogroups, which are possibly influenced by variation in the abundance of CRISPR-Cas systems and are shaped by the differential distribution of other defence systems and AMR genes.<br><br>FUNDING: German Science Foundation, Max-Planck Society, Leibniz ScienceCampus Evolutionary Medicine of the Lung, BMBF program Medical Infection Genomics, Kiel Life Science Postdoc Award.},
}
@article {pmid36952190,
year = {2023},
author = {Chornyi, S and Koster, J and Waterham, HR},
title = {Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2643},
number = {},
pages = {233-245},
pmid = {36952190},
issn = {1940-6029},
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Peroxisomes/genetics ; Genome ; Base Sequence ; Mammals/genetics ; },
abstract = {The development and application of the CRISPR-Cas9 technology for genome editing of mammalian cells have opened up a wealth of possibilities for genetically modifying and manipulating human cells, and use in functional studies or therapeutic approaches.Here we describe the approach that we have been using successfully to generate multiple human cell lines with targeted (partial) gene deletions, i.e., knockout cells, or human cells with modified genomic nucleotide sequences, i.e., knock-in cells, in genes encoding known or putative proteins involved in peroxisome biogenesis or peroxisomal functions.},
}
@article {pmid36914213,
year = {2023},
author = {Zhao, J and Zuo, S and Huang, L and Lian, J and Xu, Z},
title = {CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.},
journal = {Journal of applied microbiology},
volume = {134},
number = {3},
pages = {},
doi = {10.1093/jambio/lxad049},
pmid = {36914213},
issn = {1365-2672},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Biotin/genetics/metabolism ; Plasmids ; },
abstract = {AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis.<br><br>MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5&#x2009;days. With the guidance of truncated crRNA containing 16&#x2009;bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously.<br><br>CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.},
}
@article {pmid36893808,
year = {2023},
author = {Peri, KVR and Faria-Oliveira, F and Larsson, A and Plovie, A and Papon, N and Geijer, C},
title = {Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.},
journal = {FEMS yeast research},
volume = {23},
number = {},
pages = {},
pmid = {36893808},
issn = {1567-1364},
mesh = {*Gene Editing ; *Saccharomycetales/genetics ; Homologous Recombination ; Candida albicans/genetics ; CRISPR-Cas Systems ; },
abstract = {Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.},
}
@article {pmid36623883,
year = {2023},
author = {Yang, W and Zhao, H and Dou, Y and Wang, P and Chang, Q and Qiao, X and Wang, X and Xu, C and Zhang, Z and Zhang, L},
title = {CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {51},
number = {4},
pages = {492-498},
doi = {10.1124/dmd.122.001111},
pmid = {36623883},
issn = {1521-009X},
mesh = {Humans ; *Cytochrome P-450 CYP3A/genetics/metabolism ; Hep G2 Cells ; *CRISPR-Cas Systems/genetics ; Rifampin/pharmacology ; RNA, Messenger/genetics ; Genotype ; },
abstract = {Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.},
}
@article {pmid36597709,
year = {2023},
author = {Zhang, R and Zhang, S and Li, J and Gao, J and Song, G and Li, W and Geng, S and Liu, C and Lin, Y and Li, Y and Li, G},
title = {CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.},
journal = {Plant biotechnology journal},
volume = {21},
number = {4},
pages = {839-853},
pmid = {36597709},
issn = {1467-7652},
mesh = {Male ; Humans ; *Triticum/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Plant Proteins/genetics/metabolism ; RNA, Small Interfering/genetics ; Mutagenesis/genetics ; Plants/genetics ; *Infertility, Male/genetics ; RNA, Plant/genetics ; Gene Expression Regulation, Plant ; },
abstract = {Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.},
}
@article {pmid36587283,
year = {2023},
author = {Cheng, J and Hill, C and Han, Y and He, T and Ye, X and Shabala, S and Guo, G and Zhou, M and Wang, K and Li, C},
title = {New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.).},
journal = {Plant biotechnology journal},
volume = {21},
number = {4},
pages = {806-818},
pmid = {36587283},
issn = {1467-7652},
mesh = {*Gibberellins ; Cotyledon ; *Hordeum/genetics ; CRISPR-Cas Systems/genetics ; Oxidoreductases/genetics ; Alleles ; Gene Editing ; Seedlings/genetics ; },
abstract = {The green revolution was based on genetic modification of the gibberellin (GA) hormone system with "dwarfing" gene mutations that reduces GA signals, conferring shorter stature, thus enabling plant adaptation to modern farming conditions. Strong GA-related mutants with shorter stature often have reduced coleoptile length, discounting yield gain due to their unsatisfactory seedling emergence under drought conditions. Here we present gibberellin (GA) 3-oxidase1 (GA3ox1) as an alternative semi-dwarfing gene in barley that combines an optimal reduction in plant height without restricting coleoptile and seedling growth. Using large-scale field trials with an extensive collection of barley accessions, we showed that a natural GA3ox1 haplotype moderately reduced plant height by 5-10 cm. We used CRISPR/Cas9 technology, generated several novel GA3ox1 mutants and validated the function of GA3ox1. We showed that altered GA3ox1 activities changed the level of active GA isoforms and consequently increased coleoptile length by an average of 8.2 mm, which could provide essential adaptation to maintain yield under climate change. We revealed that CRISPR/Cas9-induced GA3ox1 mutations increased seed dormancy to an ideal level that could benefit the malting industry. We conclude that selecting HvGA3ox1 alleles offers a new opportunity for developing barley varieties with optimal stature, longer coleoptile and additional agronomic traits.},
}
@article {pmid36529912,
year = {2023},
author = {Luo, W and Suzuki, R and Imai, R},
title = {Precise in planta genome editing via homology-directed repair in wheat.},
journal = {Plant biotechnology journal},
volume = {21},
number = {4},
pages = {668-670},
pmid = {36529912},
issn = {1467-7652},
mesh = {*Gene Editing ; *Triticum/genetics ; CRISPR-Cas Systems/genetics ; Recombinational DNA Repair ; },
}
@article {pmid36529879,
year = {2023},
author = {Zhu, Y and Lin, Y and Fan, Y and Wang, Y and Li, P and Xiong, J and He, Y and Cheng, S and Ye, X and Wang, F and Goodrich, J and Zhu, JK and Wang, K and Zhang, CJ},
title = {CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat.},
journal = {Plant biotechnology journal},
volume = {21},
number = {4},
pages = {665-667},
pmid = {36529879},
issn = {1467-7652},
mesh = {*Triticum/genetics ; CRISPR-Cas Systems/genetics ; Genes, Plant/genetics ; Seeds ; *Biological Phenomena ; },
}
@article {pmid36125667,
year = {2023},
author = {Zhang, Z and Wu, X and Yang, J and Liu, X and Liu, R and Song, Y},
title = {Highly efficient base editing in rabbit by using near-PAMless engineered CRISPR/Cas9 variants.},
journal = {Science China. Life sciences},
volume = {66},
number = {3},
pages = {635-638},
pmid = {36125667},
issn = {1869-1889},
mesh = {Animals ; Rabbits ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; RNA, Guide, Kinetoplastida ; },
}
@article {pmid36952053,
year = {2023},
author = {Huo, G and Shepherd, J and Pan, X},
title = {Craspase: A novel CRISPR/Cas dual gene editor.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {2},
pages = {98},
pmid = {36952053},
issn = {1438-7948},
}
@article {pmid36950734,
year = {2023},
author = {Daròs, JA and Pasin, F and Merwaiss, F},
title = {CRISPR-Cas-based plant genome engineering goes viral.},
journal = {Molecular plant},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molp.2023.03.010},
pmid = {36950734},
issn = {1752-9867},
}
@article {pmid36950281,
year = {2023},
author = {Didara, Z and Reithofer, F and Zöttl, K and Jürets, A and Kiss, I and Witte, A and Klein, R},
title = {Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene.},
journal = {Molecular therapy. Nucleic acids},
volume = {32},
number = {},
pages = {48-60},
pmid = {36950281},
issn = {2162-2531},
abstract = {DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results.},
}
@article {pmid36949234,
year = {2023},
author = {Ravikiran, KT and Thribhuvan, R and Sheoran, S and Kumar, S and Kushwaha, AK and Vineeth, TV and Saini, M},
title = {Tailoring crops with superior product quality through genome editing: an update.},
journal = {Planta},
volume = {257},
number = {5},
pages = {86},
pmid = {36949234},
issn = {1432-2048},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Plant Breeding/methods ; Crops, Agricultural/genetics ; },
abstract = {In this review, using genome editing, the quality trait alterations in important crops have been discussed, along with the challenges encountered to maintain the crop products' quality. The delivery of economic produce with superior quality is as important as high yield since it dictates consumer's acceptance and end use. Improving product quality of various agricultural and horticultural crops is one of the important targets of plant breeders across the globe. Significant achievements have been made in various crops using conventional plant breeding approaches, albeit, at a slower rate. To keep pace with ever-changing consumer tastes and preferences and industry demands, such efforts must be supplemented with biotechnological tools. Fortunately, many of the quality attributes are resultant of well-understood biochemical pathways with characterized genes encoding enzymes at each step. Targeted mutagenesis and transgene transfer have been instrumental in bringing out desired qualitative changes in crops but have suffered from various pitfalls. Genome editing, a technique for methodical and site-specific modification of genes, has revolutionized trait manipulation. With the evolution of versatile and cost effective CRISPR/Cas9 system, genome editing has gained significant traction and is being applied in several crops. The availability of whole genome sequences with the advent of next generation sequencing (NGS) technologies further enhanced the precision of these techniques. CRISPR/Cas9 system has also been utilized for desirable modifications in quality attributes of various crops such as rice, wheat, maize, barley, potato, tomato, etc. The present review summarizes salient findings and achievements of application of genome editing for improving product quality in various crops coupled with pointers for future research endeavors.},
}
@article {pmid36947920,
year = {2023},
author = {Zhan, X and Zhou, J and Jiang, Y and An, P and Luo, B and Lan, F and Ying, B and Wu, Y},
title = {DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging.},
journal = {Biosensors &amp; bioelectronics},
volume = {229},
number = {},
pages = {115229},
doi = {10.1016/j.bios.2023.115229},
pmid = {36947920},
issn = {1873-4235},
abstract = {Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes ([197]Au, [107]Ag, [195]Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218 fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.},
}
@article {pmid36914062,
year = {2023},
author = {Huang, H and Zhang, W and Zhang, J and Zhao, A and Jiang, H},
title = {Epigenome editing based on CRISPR/dCas9[p300] facilitates transdifferentiation of human fibroblasts into Leydig-like cells.},
journal = {Experimental cell research},
volume = {425},
number = {2},
pages = {113551},
doi = {10.1016/j.yexcr.2023.113551},
pmid = {36914062},
issn = {1090-2422},
mesh = {Humans ; Male ; *Cell Transdifferentiation/genetics ; *Epigenome ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cholesterol Side-Chain Cleavage Enzyme ; Testosterone ; Fibroblasts ; },
abstract = {Recently, Leydig cell (LCs) transplantation has a promising potential to treat male hypogonadism. However, the scarcity of seed cells is the actual barrier impeding the application of LCs transplantation. Utilizing the cutting-edge CRISPR/dCas9[VP64] technology, human foreskin fibroblasts (HFFs) were transdifferentiated into Leydig-like cells（iLCs） in previous study, but the efficiency of transdifferentiation is not very satisfactory. Therefore, this study was conducted to further optimize the CRISPR/dCas9 system for obtaining sufficient iLCs. First, the stable CYP11A1-Promoter-GFP-HFFs cell line was established by infecting HFFs with CYP11A1-Promoter-GFP lentiviral vectors, and then co-infected with dCas9[p300] and the combination of sgRNAs targeted to NR5A1, GATA4 and DMRT1. Next, this study adopted quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence to determine the efficiency of transdifferentiation, the generation of testosterone, the expression levels of steroidogenic biomarkers. Moreover, we utilized chromatin immuno-precipitation (ChIP) followed by quantitative polymerase chain reaction (ChIP-qPCR) to measure the levels of acetylation of targeted H3K27. The results revealed that advanced dCas9[p300] facilitated generation of iLCs. Moreover, the dCas9[p300]-mediated iLCs significantly expressed the steroidogenic biomarkers and produced more testosterone with or without LH treatment than the dCas9[VP64]-mediated. Additionally, preferred enrichment in H3K27ac at the promoters was detected only with dCas9[p300] treatment. The data provided here imply that the improved version of dCas9 can aid in the harvesting of iLCs, and will provide sufficient seed cells for cell transplantation treatment of androgen deficiency in the future.},
}
@article {pmid36945831,
year = {2023},
author = {Liang, F and Dong, Z and Ye, J and Hu, W and Bhandari, RK and Mai, K and Wang, X},
title = {In vivo DNA methylation editing in zebrafish.},
journal = {Epigenetics},
volume = {18},
number = {1},
pages = {2192326},
doi = {10.1080/15592294.2023.2192326},
pmid = {36945831},
issn = {1559-2308},
mesh = {Animals ; *DNA Methylation ; *CRISPR-Cas Systems ; Gene Editing/methods ; Zebrafish/genetics ; Epigenome ; },
abstract = {The CRISPR/dCas9-based epigenome editing technique has driven much attention. Fused with a catalytic domain from Dnmt or Tet protein, the CRISPR/dCas9-DnmtCD or -TetCD systems possess the targeted DNA methylation editing ability and have established a series of in vitro and in vivo disease models. However, no publication has been reported on zebrafish (Danio rerio), an important animal model in biomedicine. The present study demonstrated that CRISPR/dCas9-Dnmt7 and -Tet2 catalytic domain fusions could site-specifically edit genomic DNA methylation in vivo in zebrafish and may serve as an efficient toolkit for DNA methylation editing in the zebrafish model.},
}
@article {pmid36944919,
year = {2023},
author = {Chen, D and Liang, Y and Wang, H and Wang, H and Su, F and Zhang, P and Wang, S and Liu, W and Li, Z},
title = {CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.2c05767},
pmid = {36944919},
issn = {1520-6882},
abstract = {The target-dependent endonuclease activity (also known as the trans-cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.},
}
@article {pmid36940764,
year = {2023},
author = {Hussain, MS and Anand, V and Kumar, M},
title = {Functional PAM sequence for DNA interference by CRISPR-Cas I-B system of Leptospira interrogans and the role of LinCas11b encoded within lincas8b.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {124086},
doi = {10.1016/j.ijbiomac.2023.124086},
pmid = {36940764},
issn = {1879-0003},
abstract = {Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates LinCas11b. A mutant variant of LinCascade[-Cas11b] that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade[-Cas11b] rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.},
}
@article {pmid36940173,
year = {2023},
author = {Suliman Maashi, M},
title = {CRISPR/Cas-based Aptasensor as an Innovative Sensing Approaches for Food Safety Analysis: Recent Progresses and New Horizons.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10408347.2023.2188955},
pmid = {36940173},
issn = {1547-6510},
abstract = {Food safety is one of the greatest public problems occurring around the world. Chemical, physical, and microbiological hazards could lead to food safety problems, which might occur at all stages of the supply chain. To tackle food safety problems and protect consumer health, specific, accurate, and rapid diagnosis techniques meeting various requirements are the imperative measures to ensure food safety. CRISPR-Cas system, a novel emerging technology, is effectively repurposed in (bio)sensing and has shown a tremendous capability to develop on-site and portable diagnostic methods with high specificity and sensitivity. Among numerous existing CRISPR/Cas systems, CRISPR/Cas13a and CRISPR/Cas12a are extensively employed in the design of biosensors, owing to their ability to cleave both non-target and target sequences. However, the specificity limitation in CRISPR/Cas has hindered its progress. Nowadays, nucleic acid aptamers recognized for their specificity and high-affinity characteristics for their analytes are incorporated into CRISPR/Cas systems. With the benefits of reproducibility, high durability, portability, facile operation, and cost-effectiveness, CRISPR/Cas-based aptasensing approaches are an ideal choice for fabricating highly specific point-of-need analytical tools with enhanced response signals. In the current study, we explore some of the most recent progress in the CRISPR/Cas-mediated aptasensors for detecting food risk factors including veterinary drugs, pesticide residues, pathogens, mycotoxins, heavy metals, illegal additives, food additives, and other contaminants. The nanomaterial engineering support with CRISPR/Cas aptasensors is also signified to achieve a hopeful perspective to provide new straightforward test kits toward trace amounts of different contaminants encountered in food samples.},
}
@article {pmid36939849,
year = {2023},
author = {Kedlaya, MN and Puzhankara, L and Prasad, R and Raj, A},
title = {Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0094},
pmid = {36939849},
issn = {2573-1602},
abstract = {Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components during the infection stage and depend on defense mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas to counter them. Virulence genes regulated by the CRISPR-Cas system are often expressed by bacteria as part of the stress response to the presence of stress conditions and foreign elements. There is ever-growing evidence regarding the role of CRISPR-Cas in virulence of periodontal pathogens. The same CRISPR-Cas system may also be targeted to reduce bacterial virulence and it may also be utilized to develop diagnostic and therapeutic strategies for prevention and control of PD progression. This review article describes the CRISPR-Cas systems in the periodontal dysbiotic microbial communities, their role in the virulence of periodontal pathogens, and their potential role in understanding the pathogenesis of periodontitis and treatment of PD.},
}
@article {pmid36914863,
year = {2023},
author = {Ledford, H},
title = {Why CRISPR babies are still too risky - embryo studies highlight challenges.},
journal = {Nature},
volume = {615},
number = {7953},
pages = {568-569},
pmid = {36914863},
issn = {1476-4687},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/ethics ; Humans ; Infant, Newborn ; Risk Assessment ; *Embryo, Mammalian/metabolism ; Germ-Line Mutation ; },
}
@article {pmid36893726,
year = {2023},
author = {Liu, Y and Ma, L and Liu, W and Xie, L and Wu, Q and Wang, Y and Zhou, Y and Zhang, Y and Jiao, B and He, Y},
title = {RPA-CRISPR/Cas12a Combined with Rolling Circle Amplification-Enriched DNAzyme: A Homogeneous Photothermal Sensing Strategy for Plant Pathogens.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {11},
pages = {4736-4744},
doi = {10.1021/acs.jafc.2c07965},
pmid = {36893726},
issn = {1520-5118},
mesh = {Humans ; *DNA, Catalytic ; Recombinases ; CRISPR-Cas Systems ; DNA ; DNA Primers ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Alternaria is an endemic fungus associated with brown spot disease, which is one of the most serious citrus diseases. In addition, the mycotoxins metabolized by Alternaria threaten human health seriously. Herein, a novel homogeneous and portable qualitative photothermal method based on recombinase polymerase amplification (RPA), CRISPR/Cas12a, and rolling circle amplification (RCA) for the detection of Alternaria is described. Using RCA primers as substrates for CRISPR/Cas12a trans-cleavage, the two systems, RPA-CRISPR/Cas12a and RCA-enriched G-quadruplex/hemin DNAzyme, are intelligently combined. Target DNA at fg/μL levels can be detected with high specificity. Additionally, the practicability of the proposed method is demonstrated by analyzing cultured Alternaria from different fruit and vegetable samples, as well as citrus fruit samples collected in the field. Furthermore, the implementation of this method does not require any sophisticated equipment and complicated washing steps. Therefore, it has great potential to screen Alternaria in poor laboratories.},
}
@article {pmid36774242,
year = {2023},
author = {Papadopoulos, C and Albà, MM},
title = {Newly evolved genes in the human lineage are functional.},
journal = {Trends in genetics : TIG},
volume = {39},
number = {4},
pages = {235-236},
doi = {10.1016/j.tig.2023.02.001},
pmid = {36774242},
issn = {0168-9525},
mesh = {Animals ; Humans ; *Evolution, Molecular ; *Hominidae ; Genome ; Genomics ; CRISPR-Cas Systems ; },
abstract = {Genes restricted to a given species or lineage are mysterious. Many emerged de novo from ancestral noncoding genomic regions rather than from pre-existing genes. A new study by Vakirlis and colleagues shows that, in humans, many of these are associated with phenotypic effects, accelerating our understanding of their functional importance.},
}
@article {pmid36772799,
year = {2023},
author = {Bachtarzi, H},
title = {Genome Editing: Moving Toward a New Era of Innovation, Development, and Approval.},
journal = {Human gene therapy},
volume = {34},
number = {5-6},
pages = {171-176},
doi = {10.1089/hum.2022.230},
pmid = {36772799},
issn = {1557-7422},
mesh = {*Gene Editing ; *Genetic Therapy ; Endonucleases/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; CRISPR-Cas Systems ; },
abstract = {Therapeutic genome editing is currently reshaping and transforming the development of advanced therapies as more ex vivo and in vivo gene editing-based technologies are used to treat a broad range of debilitating and complex disorders. With first-generation gene editing modalities (notably those based on ZFNs, TALENs and CRISPR/Cas9), comes a new second-generation of gene editing-based therapeutics including base editing, prime editing and other nuclease-free genome editing modalities. Such ground-breaking innovative products warrant careful considerations from a product development and regulatory perspective, that take into account not only the common development considerations that apply to standard gene and cell therapy products, but also other specific considerations linked with the technology being used. This article sheds light into specific considerations for developing safe and effective in vivo and ex vivo genome editing medicines that will continue to push barriers even further for the cell and gene therapy field.},
}
@article {pmid36694939,
year = {2023},
author = {Nishimura, A and Tanahashi, R and Oi, T and Kan, K and Takagi, H},
title = {Plasmid-free CRISPR/Cas9 genome editing in Saccharomyces cerevisiae.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {87},
number = {4},
pages = {458-462},
doi = {10.1093/bbb/zbad008},
pmid = {36694939},
issn = {1347-6947},
mesh = {*Gene Editing ; *Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Transfection ; },
abstract = {The current CRISPR/Cas9 systems in the yeast Saccharomyces cerevisiae cannot be considered a non-genetic modification technology because it requires the introduction of Cas9 and sgRNA into yeast cells using plasmid expression systems. Our present study showed that the yeast genome can be edited without plasmid expression systems by using a commercially available protein transfection reagent and chemically modified sgRNAs.},
}
@article {pmid36937767,
year = {2023},
author = {Hu, M and Chen, S and Ni, Y and Wei, W and Mao, W and Ge, M and Qian, X},
title = {CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1141176},
pmid = {36937767},
issn = {2296-4185},
abstract = {Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%-97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%-40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.},
}
@article {pmid36937752,
year = {2023},
author = {Tang, Y and Wang, F and Wang, Y and Wang, Y and Liu, Y and Chen, Z and Li, W and Yang, S and Ma, L},
title = {In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1142637},
pmid = {36937752},
issn = {2296-4185},
abstract = {In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn[2+] or Mg[2+] and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.},
}
@article {pmid36935157,
year = {2023},
author = {Deng, L and He, X and Liu, K and Li, Y and Xia, H and Qian, H and Lu, X and Mao, X and Xiang, Y},
title = {One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.},
journal = {Analytica chimica acta},
volume = {1252},
number = {},
pages = {341059},
doi = {10.1016/j.aca.2023.341059},
pmid = {36935157},
issn = {1873-4324},
mesh = {Humans ; *Burkholderia pseudomallei/genetics ; *Melioidosis/diagnosis/genetics/microbiology ; CRISPR-Cas Systems ; },
abstract = {Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.},
}
@article {pmid36934130,
year = {2023},
author = {Schary, Y and Rotem, I and Caller, T and Lewis, N and Shaihov-Teper, O and Brzezinski, RY and Lendengolts, D and Raanani, E and Sternik, L and Naftali-Shani, N and Leor, J},
title = {CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {4481},
pmid = {36934130},
issn = {2045-2322},
mesh = {Humans ; Mice ; Animals ; *Toll-Like Receptor 4/genetics ; Cicatrix/pathology ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Myocardial Infarction/genetics/therapy/pathology ; Pericardium/pathology ; Cell- and Tissue-Based Therapy ; Inflammation/pathology ; },
abstract = {Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.},
}
@article {pmid36932065,
year = {2023},
author = {Karlikow, M and Amalfitano, E and Yang, X and Doucet, J and Chapman, A and Mousavi, PS and Homme, P and Sutyrina, P and Chan, W and Lemak, S and Yakunin, AF and Dolezal, AG and Kelley, S and Foster, LJ and Harpur, BA and Pardee, K},
title = {CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {1505},
pmid = {36932065},
issn = {2041-1723},
mesh = {Animals ; DNA/genetics ; *Nucleic Acids ; Agriculture ; Head ; RNA/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; },
abstract = {Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.},
}
@article {pmid36930418,
year = {2023},
author = {Thakur, N and Nigam, M and Awasthi, G and Shukla, A and Shah, AA and Negi, N and Khan, SA and Casini, R and Elansary, HO},
title = {Synergistic soil-less medium for enhanced yield of crops: a step towards incorporating genomic tools for attaining net zero hunger.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {2},
pages = {86},
pmid = {36930418},
issn = {1438-7948},
mesh = {Humans ; *CRISPR-Cas Systems ; *Soil ; Proteomics ; Hunger ; Phylogeny ; Genome, Plant ; Plant Breeding/methods ; Crops, Agricultural/genetics ; Genomics ; },
abstract = {Globally, industrial farming endangers crucial ecological mechanisms upon which food production relies, while 815 million people are undernourished and a significant number are malnourished. Zero Hunger aims to concurrently solve global ecological sustainability and food security concerns. Recent breakthroughs in molecular tools and approaches have allowed scientists to detect and comprehend the nature and structure of agro-biodiversity at the molecular and genetic levels, providing us an advantage over traditional methods of crop breeding. These bioinformatics techniques let us optimize our target plants for our soil-less medium and vice versa. Most of the soil-borne and seed-borne diseases are the outcomes of non-treated seed and growth media, which are important factors in low productivity. The farmers do not consider these issues, thereby facing problems growing healthy crops and suffering economic losses. This study is going to help the farmers increase their eco-friendly, chemical residue-free, quality yield of crops and their economic returns. The present invention discloses a synergistic soil-less medium that consists of only four ingredients mixed in optimal ratios by weight: vermicompost (70-80%), vermiculite (10-15%), coco peat (10-15%), and Rhizobium (0-1%). The medium exhibits better physical and chemical characteristics than existing conventional media. The vermiculite to coco peat ratio is reduced, while the vermicompost ratio is increased, with the goals of lowering toxicity, increasing plant and water holding capacity, avoiding drying of the media, and conserving water. The medium provides balanced nutrition and proper ventilation for seed germination and the growth of seedlings. Rhizobium is also used to treat the plastic bags and seeds. The results clearly show that the current synergistic soil-less environment is best for complete plant growth. Securing genetic advantages via sexual recombination, induced random mutations, and transgenic techniques have been essential for the development of improved agricultural varieties. The recent availability of targeted genome-editing technology provides a new path for integrating beneficial genetic modifications into the most significant agricultural species on the planet. Clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) has evolved into a potent genome-editing tool for imparting genetic modifications to crop species. In addition, the integration of analytical methods like population genomics, phylogenomics, and metagenomics addresses conservation problems, while whole genome sequencing has opened up a new dimension for explaining the genome architecture and its interactions with other species. The in silico genomic and proteomic investigation was also conducted to forecast future investigations for the growth of French beans on a synergistic soil-less medium with the purpose of studying how a blend of vermicompost, vermiculite, cocopeat, and Rhizobium secrete metal ions, and other chemical compounds into the soil-less medium and affect the development of our target plant as well as several other plants. This interaction was studied using functional and conserved region analysis, phylogenetic analysis, and docking tools.},
}
@article {pmid36930175,
year = {2023},
author = {Du, SW and Palczewski, K},
title = {Eye on genome editing.},
journal = {The Journal of experimental medicine},
volume = {220},
number = {5},
pages = {},
doi = {10.1084/jem.20230146},
pmid = {36930175},
issn = {1540-9538},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *Retinitis Pigmentosa/genetics/therapy ; Mutation/genetics ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR/Cas9 genome editing techniques have the potential to treat previously untreatable inherited genetic disorders of vision by correcting mutations that cause these afflictions. Using a prime editor, Qin et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20220776) restored visual functions in a mouse model (rd10) of retinitis pigmentosa.},
}
@article {pmid36876906,
year = {2023},
author = {Huang, M and Coral, D and Ardalani, H and Spegel, P and Saadat, A and Claussnitzer, M and Mulder, H and Franks, PW and Kalamajski, S},
title = {Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {36876906},
issn = {2050-084X},
support = {ERC-2015-CoG -681742 NASCENT/ERC_/European Research Council/International ; },
mesh = {Humans ; *Obesity/genetics/metabolism ; Causality ; Cell Line ; *Adipocytes/metabolism ; Weight Loss ; CRISPR-Cas Systems ; },
abstract = {Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r[2] > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.},
}
@article {pmid36825467,
year = {2023},
author = {Chen, Z and Zheng, S and Fu, C},
title = {Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast.},
journal = {Journal of cell science},
volume = {136},
number = {6},
pages = {},
doi = {10.1242/jcs.260769},
pmid = {36825467},
issn = {1477-9137},
mesh = {RNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Schizosaccharomyces/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Protein Serine-Threonine Kinases/metabolism ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; },
abstract = {The CRISPR-Cas13d system has a single small effector protein that targets RNA and does not require the presence of a protospacer flanking site in the targeted transcript. These features make CRISPR-Cas13d an attractive system for RNA manipulation. Here, we report the successful implementation of the CRISPR-Cas13d system in fission yeast for RNA knockdown. A high effectiveness of the CRISPR-Cas13d system was ensured by using an array of CRISPR RNAs (crRNAs) that are flanked by two self-cleaving ribozymes and are expressed from an RNA polymerase II promoter. Given the repressible nature of the promoter, RNA knockdown by the CRISPR-Cas13d system is reversible. Moreover, using the CRISPR-Cas13d system, we identified an effective crRNA array targeting the transcript of gfp and the effectiveness was demonstrated by successful knockdown of the transcripts of noc4-gfp, bub1-gfp and ade6-gfp. In principle, the effective GFP crRNA array allows knockdown of any transcript carrying the GFP sequences. This new CRISPR-Cas13d-based toolkit is expected to have a wide range of applications in many aspects of biology, including dissection of gene function and visualization of RNA.},
}
@article {pmid36651153,
year = {2023},
author = {Dubois, M},
title = {Phishing alert! A Cas9-based method reveals the identity of promoter-bound transcription factors.},
journal = {Plant physiology},
volume = {191},
number = {3},
pages = {1462-1464},
pmid = {36651153},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; },
}
@article {pmid36305686,
year = {2023},
author = {Wang, Z and He, Z and Liu, Z and Qu, M and Gao, C and Wang, C and Wang, Y},
title = {A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.},
journal = {Plant physiology},
volume = {191},
number = {3},
pages = {1505-1519},
pmid = {36305686},
issn = {1532-2548},
mesh = {Reproducibility of Results ; *DNA ; *Plants ; Chromatin Immunoprecipitation/methods ; RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {DNA-protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA-protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)-dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR-dCas9 system (R-ChIP-dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9-gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9-gRNA-DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP-dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.},
}
@article {pmid35388882,
year = {2023},
author = {Musunuru, K},
title = {CRISPR and cardiovascular diseases.},
journal = {Cardiovascular research},
volume = {119},
number = {1},
pages = {79-93},
doi = {10.1093/cvr/cvac048},
pmid = {35388882},
issn = {1755-3245},
mesh = {Animals ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Cardiovascular Diseases/diagnosis/genetics/therapy ; Genetic Therapy ; Gene Editing ; *Muscular Dystrophy, Duchenne/genetics ; CRISPR-Cas Systems ; },
abstract = {CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i) the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epigenome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, focusing on the examples of hypercholesterolaemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.},
}
@article {pmid36937283,
year = {2023},
author = {Gu, B and Zhuo, C and Xu, X and El Bissati, K},
title = {Editorial: Molecular diagnostics for infectious diseases: Novel approaches, clinical applications and future challenges.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1153827},
pmid = {36937283},
issn = {1664-302X},
}
@article {pmid36937152,
year = {2023},
author = {Xiao, H and Hu, J and Huang, C and Feng, W and Liu, Y and Kumblathan, T and Tao, J and Xu, J and Le, XC and Zhang, H},
title = {CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.},
journal = {Trends in analytical chemistry : TRAC},
volume = {161},
number = {},
pages = {117000},
pmid = {36937152},
issn = {0165-9936},
abstract = {The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.},
}
@article {pmid36934807,
year = {2023},
author = {Marino, ND},
title = {Phage against the machine: discovery and mechanism of type V anti-CRISPRs.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168054},
doi = {10.1016/j.jmb.2023.168054},
pmid = {36934807},
issn = {1089-8638},
abstract = {The discovery of diverse bacterial CRISPR-Cas systems has reignited interest in understanding bacterial defense pathways while yielding exciting new tools for genome editing. CRISPR-Cas systems are widely distributed in prokaryotes, found in 40% of bacteria and 90% of archaea, where they function as adaptive immune systems against bacterial viruses (phage) and other mobile genetic elements. In turn, phage have evolved inhibitors, called anti-CRISPR proteins, to prevent targeting. Type V CRISPR-Cas12 systems have emerged as a particularly exciting arena in this co-evolutionary arms race. Type V anti-CRISPRs have highly diverse and novel mechanisms of action, some of which appear to be unusually potent or widespread. In this review, we discuss the discovery and mechanism of these anti-CRISPRs as well as future areas for exploration.},
}
@article {pmid36933595,
year = {2023},
author = {Bhatia, S and Pooja, and Yadav, SK},
title = {CRISPR-Cas for genome editing: Classification, mechanism, designing and applications.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {124054},
doi = {10.1016/j.ijbiomac.2023.124054},
pmid = {36933595},
issn = {1879-0003},
abstract = {Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.},
}
@article {pmid36929199,
year = {2023},
author = {Rostain, W and Grebert, T and Vyhovskyi, D and Pizarro, PT and Tshinsele-Van Bellingen, G and Cui, L and Bikard, D},
title = {Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad170},
pmid = {36929199},
issn = {1362-4962},
support = {677823/ERC_/European Research Council/International ; 101044479/ERC_/European Research Council/International ; },
abstract = {Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9's application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR-Cas experiments in bacteria.},
}
@article {pmid36927663,
year = {2023},
author = {Wu, ZS and Gao, Y and Du, YT and Dang, S and He, KM},
title = {The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.},
journal = {Yi chuan = Hereditas},
volume = {45},
number = {2},
pages = {165-175},
doi = {10.16288/j.yczz.22-395},
pmid = {36927663},
issn = {0253-9772},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Recombinational DNA Repair ; DNA ; Mammals/genetics ; },
abstract = {The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.},
}
@article {pmid36927640,
year = {2023},
author = {Liu, MZ and Wang, LR and Li, YM and Ma, XY and Han, HH and Li, DL},
title = {Generation of genetically modified rat models via the CRISPR/Cas9 technology.},
journal = {Yi chuan = Hereditas},
volume = {45},
number = {1},
pages = {78-87},
doi = {10.16288/j.yczz.22-354},
pmid = {36927640},
issn = {0253-9772},
mesh = {Rats ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; Recombinational DNA Repair ; DNA End-Joining Repair/genetics ; Mammals/genetics ; },
abstract = {The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.},
}
@article {pmid36927528,
year = {2023},
author = {D'Antonio, L and Fieni, C and Ciummo, SL and Vespa, S and Lotti, L and Sorrentino, C and Di Carlo, E},
title = {Inactivation of interleukin-30 in colon cancer stem cells via CRISPR/Cas9 genome editing inhibits their oncogenicity and improves host survival.},
journal = {Journal for immunotherapy of cancer},
volume = {11},
number = {3},
pages = {},
doi = {10.1136/jitc-2022-006056},
pmid = {36927528},
issn = {2051-1426},
mesh = {Mice ; Animals ; *Colonic Neoplasms/pathology ; Cell Line, Tumor ; Gene Editing ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Neoplastic Stem Cells/pathology ; *Colorectal Neoplasms/pathology ; Cell Transformation, Neoplastic/pathology ; Carcinogenesis/genetics ; Interleukins/genetics ; },
abstract = {BACKGROUND: Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown.<br><br>METHODS: IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications.<br><br>RESULTS: We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival.<br><br>CONCLUSIONS: IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.},
}
@article {pmid36927151,
year = {2023},
author = {Wang, R and Zhang, R and Khodaverdian, A and Yosef, N},
title = {Theoretical guarantees for phylogeny inference from single-cell lineage tracing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {12},
pages = {e2203352120},
doi = {10.1073/pnas.2203352120},
pmid = {36927151},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems/genetics ; Phylogeny ; Cell Lineage/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Lineage-tracing technologies based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9) genome editing have emerged as a powerful tool for investigating development in single-cell contexts, but exact reconstruction of the underlying clonal relationships in experiment is complicated by features of the data. These complications are functions of the experimental parameters in these systems, such as the Cas9 cutting rate, the diversity of indel outcomes, and the rate of missing data. In this paper, we develop two theoretically grounded algorithms for the reconstruction of the underlying single-cell phylogenetic tree as well as asymptotic bounds for the number of recording sites necessary for exact recapitulation of the ground truth phylogeny at high probability. In doing so, we explore the relationship between the problem difficulty and the experimental parameters, with implications for experimental design. Lastly, we provide simulations showing the empirical performance of these algorithms and showing that the trends in the asymptotic bounds hold empirically. Overall, this work provides a theoretical analysis of phylogenetic reconstruction in single-cell CRISPR-Cas9 lineage-tracing technologies.},
}
@article {pmid36925702,
year = {2023},
author = {Meng, X and Wu, TG and Lou, QY and Niu, KY and Jiang, L and Xiao, QZ and Xu, T and Zhang, L},
title = {Optimization of CRISPR-Cas system for clinical cancer therapy.},
journal = {Bioengineering &amp; translational medicine},
volume = {8},
number = {2},
pages = {e10474},
pmid = {36925702},
issn = {2380-6761},
abstract = {Cancer is a genetic disease caused by alterations in genome and epigenome and is one of the leading causes for death worldwide. The exploration of disease development and therapeutic strategies at the genetic level have become the key to the treatment of cancer and other genetic diseases. The functional analysis of genes and mutations has been slow and laborious. Therefore, there is an urgent need for alternative approaches to improve the current status of cancer research. Gene editing technologies provide technical support for efficient gene disruption and modification in vivo and in vitro, in particular the use of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Currently, the applications of CRISPR-Cas systems in cancer rely on different Cas effector proteins and the design of guide RNAs. Furthermore, effective vector delivery must be met for the CRISPR-Cas systems to enter human clinical trials. In this review article, we describe the mechanism of the CRISPR-Cas systems and highlight the applications of class II Cas effector proteins. We also propose a synthetic biology approach to modify the CRISPR-Cas systems, and summarize various delivery approaches facilitating the clinical application of the CRISPR-Cas systems. By modifying the CRISPR-Cas system and optimizing its in vivo delivery, promising and effective treatments for cancers using the CRISPR-Cas system are emerging.},
}
@article {pmid36925687,
year = {2023},
author = {Khambhati, K and Bhattacharjee, G and Gohil, N and Dhanoa, GK and Sagona, AP and Mani, I and Bui, NL and Chu, DT and Karapurkar, JK and Jang, SH and Chung, HY and Maurya, R and Alzahrani, KJ and Ramakrishna, S and Singh, V},
title = {Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens.},
journal = {Bioengineering &amp; translational medicine},
volume = {8},
number = {2},
pages = {e10381},
pmid = {36925687},
issn = {2380-6761},
abstract = {Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.},
}
@article {pmid36925313,
year = {2023},
author = {Chen, H and Li, B and Shi, S and Zhou, T and Wang, X and Wang, Z and Zhou, X and Wang, M and Shi, W and Ren, L},
title = {Au-Fe3O4 nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing.},
journal = {Analytica chimica acta},
volume = {1251},
number = {},
pages = {341014},
doi = {10.1016/j.aca.2023.341014},
pmid = {36925313},
issn = {1873-4324},
mesh = {*Angiotensin Receptor Antagonists ; *Angiotensin-Converting Enzyme Inhibitors ; CRISPR-Cas Systems ; Ampicillin ; Drug Resistance, Microbial/genetics ; },
abstract = {The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au-Fe3O4 nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au-Fe3O4 nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL[-1]) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently.},
}
@article {pmid36925288,
year = {2023},
author = {Niu, C and Liu, J and Xing, X and Zhang, C},
title = {CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.},
journal = {Analytica chimica acta},
volume = {1251},
number = {},
pages = {340998},
doi = {10.1016/j.aca.2023.340998},
pmid = {36925288},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.},
}
@article {pmid36923939,
year = {2023},
author = {Yin, Z and Su, R and Ge, L and Wang, X and Yang, J and Huang, G and Li, C and Liu, Y and Zhang, K and Deng, L and Fei, J},
title = {Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.},
journal = {International journal of biological sciences},
volume = {19},
number = {4},
pages = {1211-1227},
pmid = {36923939},
issn = {1449-2288},
mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; GTP Phosphohydrolases/metabolism ; Gene Editing ; Hematopoietic Stem Cells/metabolism/pathology ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/drug therapy/pathology ; Carcinogenesis/genetics ; Neoplastic Stem Cells/metabolism ; ral GTP-Binding Proteins/genetics/metabolism ; },
abstract = {BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalA[Rosa26-Tg/+]) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.},
}
@article {pmid36922689,
year = {2023},
author = {Fudge, JB},
title = {Cardiac defect corrected in vivo with CRISPR.},
journal = {Nature biotechnology},
volume = {41},
number = {3},
pages = {323},
doi = {10.1038/s41587-023-01721-y},
pmid = {36922689},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid36842376,
year = {2023},
author = {Dyke, E and Bijnagte-Schoenmaker, C and Wu, KM and Oudakker, A and Roepman, R and Nadif Kasri, N},
title = {Generation of induced pluripotent stem cell line carrying frameshift variants in NPHP1 (UCSFi001-A-68) using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {68},
number = {},
pages = {103053},
doi = {10.1016/j.scr.2023.103053},
pmid = {36842376},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Frameshift Mutation ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; },
abstract = {NPHP1 (Nephrocystin 1) is a protein that localizes to the transition zone of the cilium, a small organelle that projects from the plasma membrane of most cells and allows for integration and coordination of signalling pathways during development and homeostasis. Loss of NPHP1 function due to biallelic NPHP1 gene mutations can lead to the development of ciliopathies - a heterogeneous spectra of disorders characterized by ciliary dysfunction. Here we report the generation of an NPHP1-null hiPSC line (UCSFi001-A-68) via CRISPR/Cas9-mediated non-homologous end joining in the UCSFi001-A background, for study of the role that this protein plays in different tissues.},
}
@article {pmid36825354,
year = {2023},
author = {Zhu, Y and Champer, J},
title = {Simulations Reveal High Efficiency and Confinement of a Population Suppression CRISPR Toxin-Antidote Gene Drive.},
journal = {ACS synthetic biology},
volume = {12},
number = {3},
pages = {809-819},
doi = {10.1021/acssynbio.2c00611},
pmid = {36825354},
issn = {2161-5063},
mesh = {Humans ; *Antidotes ; *Gene Drive Technology ; CRISPR-Cas Systems/genetics ; },
abstract = {Though engineered gene drives hold great promise for spreading through and suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called toxin-antidote dominant embryo (TADE) suppression drive could potentially address the issues of confinement and resistance. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in a continuous spatial landscape. We find that the drive is actually more confined than in simple models without space, even in its most efficient form with low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the population in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to spread, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.},
}
@article {pmid36807980,
year = {2023},
author = {Du, L and Zhang, D and Luo, Z and Lin, Z},
title = {Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081.},
journal = {Nucleic acids research},
volume = {51},
number = {5},
pages = {2485-2495},
pmid = {36807980},
issn = {1362-4962},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Second Messenger Systems ; Signal Transduction ; Endonucleases/metabolism ; Ions/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.},
}
@article {pmid36805322,
year = {2023},
author = {Ham, O and Kim, S and Lee, Y and Lee, MO},
title = {Generation of telomeric repeat binding factor 1 (TRF1)-knockout human embryonic stem cell lines, KRIBBe010-A-95, KRIBBe010-A-96, and KRIBBe010-A-97, using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {68},
number = {},
pages = {103045},
doi = {10.1016/j.scr.2023.103045},
pmid = {36805322},
issn = {1876-7753},
mesh = {Humans ; *Telomere/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Telomeric Repeat Binding Protein 1/genetics/metabolism ; Cell Line ; },
abstract = {Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.},
}
@article {pmid36802585,
year = {2023},
author = {Huan, YW and Torraca, V and Brown, R and Fa-Arun, J and Miles, SL and Oyarzún, DA and Mostowy, S and Wang, B},
title = {P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri.},
journal = {ACS synthetic biology},
volume = {12},
number = {3},
pages = {709-721},
doi = {10.1021/acssynbio.2c00465},
pmid = {36802585},
issn = {2161-5063},
support = {206444/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Anti-Infective Agents ; Gene Editing ; Bacteriophage P1/genetics ; Zebrafish/genetics ; Shigella flexneri/genetics ; Animals ; },
abstract = {The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.},
}
@article {pmid36801567,
year = {2023},
author = {Stępniewski, J and Jeż, M and Dulak, J},
title = {Generation of miR-15a/16-1 cluster-deficient human induced pluripotent stem cell line (DMBi001-A-2) using CRISPR/Cas9 gene editing.},
journal = {Stem cell research},
volume = {68},
number = {},
pages = {103046},
doi = {10.1016/j.scr.2023.103046},
pmid = {36801567},
issn = {1876-7753},
mesh = {Humans ; Animals ; Mice ; Gene Editing ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/metabolism ; Cardiomegaly ; },
abstract = {miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing. Obtained cells demonstrate expression of pluripotency markers, differentiation capacity into all three germ layers and normal karyotype.},
}
@article {pmid36787424,
year = {2023},
author = {Jeong, SH and Kim, HJ and Lee, SJ},
title = {New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.},
journal = {ACS synthetic biology},
volume = {12},
number = {3},
pages = {800-808},
doi = {10.1021/acssynbio.2c00595},
pmid = {36787424},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Xylose ; Nucleotides ; },
abstract = {CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10[12]). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N20) to 9 nucleotides (N9) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5'-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl[-] phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular l-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.},
}
@article {pmid36669880,
year = {2023},
author = {Nguyen, TTT and Tanaka, Y and Sanada, M and Hosaka, M and Tamai, M and Kagami, K and Komatsu, C and Somazu, S and Harama, D and Kasai, S and Watanabe, A and Akahane, K and Goi, K and Inukai, T},
title = {CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.},
journal = {Molecular pharmacology},
volume = {103},
number = {4},
pages = {199-210},
doi = {10.1124/molpharm.122.000546},
pmid = {36669880},
issn = {1521-0111},
mesh = {Humans ; *Mercaptopurine/pharmacology ; CRISPR-Cas Systems/genetics ; Mutation ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/genetics ; Recurrence ; 5'-Nucleotidase/genetics/metabolism/therapeutic use ; Ribose-Phosphate Pyrophosphokinase/genetics/metabolism ; },
abstract = {6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.},
}
@article {pmid36620901,
year = {2023},
author = {Lackner, M and Helmbrecht, N and Pääbo, S and Riesenberg, S},
title = {Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions.},
journal = {Nucleic acids research},
volume = {51},
number = {5},
pages = {e26},
pmid = {36620901},
issn = {1362-4962},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Nucleotides ; },
abstract = {CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our 'sequence-ascertained favorable editing' (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional 'diagnostic' substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with 'SAFE' donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with 'SAFE' donors and detect all copy number changes and almost all clones with gene conversion.},
}
@article {pmid36259148,
year = {2023},
author = {Zulhussnain, M and Zahoor, MK and Ranian, K and Ahmad, A and Jabeen, F},
title = {CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.},
journal = {Bulletin of entomological research},
volume = {113},
number = {2},
pages = {243-252},
doi = {10.1017/S0007485322000505},
pmid = {36259148},
issn = {1475-2670},
mesh = {Male ; Female ; Animals ; *Aedes/genetics ; CRISPR-Cas Systems ; Mosquito Vectors/genetics ; Protein Isoforms/genetics ; *Dengue ; },
abstract = {The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul[-1]) and gRNAs (each at 100 ng ul[-1]) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.},
}
@article {pmid36229610,
year = {2023},
author = {Lee, S and Lee, H and Baek, G and Kim, JS},
title = {Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors.},
journal = {Nature biotechnology},
volume = {41},
number = {3},
pages = {378-386},
pmid = {36229610},
issn = {1546-1696},
mesh = {Humans ; *DNA, Mitochondrial/genetics ; *Gene Editing/methods ; Mitochondria/metabolism ; Mutation ; Cytosine/metabolism ; CRISPR-Cas Systems ; },
abstract = {Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.},
}
@article {pmid36203014,
year = {2023},
author = {Christie, KA and Guo, JA and Silverstein, RA and Doll, RM and Mabuchi, M and Stutzman, HE and Lin, J and Ma, L and Walton, RT and Pinello, L and Robb, GB and Kleinstiver, BP},
title = {Precise DNA cleavage using CRISPR-SpRYgests.},
journal = {Nature biotechnology},
volume = {41},
number = {3},
pages = {409-416},
pmid = {36203014},
issn = {1546-1696},
support = {R35 HG010717/HG/NHGRI NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA Cleavage ; RNA, Guide, Kinetoplastida/genetics ; DNA/genetics ; Gene Editing/methods ; },
abstract = {Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.},
}
@article {pmid36163548,
year = {2023},
author = {Grünewald, J and Miller, BR and Szalay, RN and Cabeceiras, PK and Woodilla, CJ and Holtz, EJB and Petri, K and Joung, JK},
title = {Engineered CRISPR prime editors with compact, untethered reverse transcriptases.},
journal = {Nature biotechnology},
volume = {41},
number = {3},
pages = {337-343},
pmid = {36163548},
issn = {1546-1696},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Mice ; Humans ; *RNA-Directed DNA Polymerase/genetics ; *Moloney murine leukemia virus/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.},
}
@article {pmid36925473,
year = {2023},
author = {Zakrzewska, M and Burmistrz, M},
title = {Mechanisms regulating the CRISPR-Cas systems.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1060337},
pmid = {36925473},
issn = {1664-302X},
abstract = {The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated proteins) is a prokaryotic system that enables sequence specific recognition and cleavage of nucleic acids. This is possible due to cooperation between CRISPR array which contains short fragments of DNA called spacers that are complimentary to the targeted nucleic acid and Cas proteins, which take part in processes of: acquisition of new spacers, processing them into their functional form as well as recognition and cleavage of targeted nucleic acids. The primary role of CRISPR-Cas systems is to provide their host with an adaptive and hereditary immunity against exogenous nucleic acids. This system is present in many variants in both Bacteria and Archea. Due to its modular structure, and programmability CRISPR-Cas system become attractive tool for modern molecular biology. Since their discovery and implementation, the CRISPR-Cas systems revolutionized areas of gene editing and regulation of gene expression. Although our knowledge on how CRISPR-Cas systems work has increased rapidly in recent years, there is still little information on how these systems are controlled and how they interact with other cellular mechanisms. Such regulation can be the result of both auto-regulatory mechanisms as well as exogenous proteins of phage origin. Better understanding of these interaction networks would be beneficial for optimization of current and development of new CRISPR-Cas-based tools. In this review we summarize current knowledge on the various molecular mechanisms that affect activity of CRISPR-Cas systems.},
}
@article {pmid36925471,
year = {2023},
author = {Cerbino, GN and Traglia, GM and Ayala Nuñez, T and Parmeciano Di Noto, G and Ramírez, MS and Centrón, D and Iriarte, A and Quiroga, C},
title = {Comparative genome analysis of the genus Shewanella unravels the association of key genetic traits with known and potential pathogenic lineages.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1124225},
pmid = {36925471},
issn = {1664-302X},
abstract = {Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health.},
}
@article {pmid36905119,
year = {2023},
author = {Pedrazzoli, E and Bianchi, A and Umbach, A and Amistadi, S and Brusson, M and Frati, G and Ciciani, M and Badowska, KA and Arosio, D and Miccio, A and Cereseto, A and Casini, A},
title = {An optimized SpCas9 high-fidelity variant for direct protein delivery.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2023.03.007},
pmid = {36905119},
issn = {1525-0024},
abstract = {Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.},
}
@article {pmid36925314,
year = {2023},
author = {Wang, X and Mu, X and Li, J and Liu, G and Zhao, S and Tian, J},
title = {A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection.},
journal = {Analytica chimica acta},
volume = {1251},
number = {},
pages = {340950},
doi = {10.1016/j.aca.2023.340950},
pmid = {36925314},
issn = {1873-4324},
abstract = {The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.},
}
@article {pmid36922985,
year = {2023},
author = {Mikkelsen, NS and Hernandez, SS and Jensen, TI and Schneller, JL and Bak, RO},
title = {Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {29},
number = {},
pages = {1-16},
pmid = {36922985},
issn = {2329-0501},
abstract = {CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.},
}
@article {pmid36917574,
year = {2023},
author = {Jianwei, L and Jobichen, C and Machida, S and Meng, S and Read, RJ and Hongying, C and Jian, S and Yuan, YA and Sivaraman, J},
title = {Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.},
journal = {PLoS biology},
volume = {21},
number = {3},
pages = {e3002023},
pmid = {36917574},
issn = {1545-7885},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; DNA Cleavage ; RNA/chemistry ; DNA/chemistry ; Bacterial Proteins/metabolism ; },
abstract = {Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.},
}
@article {pmid36917274,
year = {2023},
author = {Spasskaya, DS and Davletshin, AI and Bachurin, SS and Tutyaeva, VV and Garbuz, DG and Karpov, DS},
title = {Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36917274},
issn = {1432-0614},
abstract = {Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.},
}
@article {pmid36914349,
year = {2023},
author = {Wang, D and Fletcher, GC and Gagic, D and On, SLW and Palmer, JS and Flint, SH},
title = {Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus.},
journal = {Food research international (Ottawa, Ont.)},
volume = {166},
number = {},
pages = {112605},
doi = {10.1016/j.foodres.2023.112605},
pmid = {36914349},
issn = {1873-7145},
mesh = {*Vibrio parahaemolyticus/genetics ; Biofilms ; Genomics ; Operon ; Cellulose ; },
abstract = {Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.},
}
@article {pmid36913066,
year = {2023},
author = {Adeyinka, OS and Tabassum, B and Koloko, BL and Ogungbe, IV},
title = {Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.},
journal = {Planta},
volume = {257},
number = {4},
pages = {78},
pmid = {36913066},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Breeding ; Gene Editing ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; Mutagenesis, Site-Directed ; Plants, Genetically Modified/genetics ; },
abstract = {The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.},
}
@article {pmid36801336,
year = {2023},
author = {Chen, J and Shi, G and Yan, C},
title = {Portable biosensor for on-site detection of kanamycin in water samples based on CRISPR-Cas12a and an off-the-shelf glucometer.},
journal = {The Science of the total environment},
volume = {872},
number = {},
pages = {162279},
doi = {10.1016/j.scitotenv.2023.162279},
pmid = {36801336},
issn = {1879-1026},
mesh = {*Kanamycin ; CRISPR-Cas Systems ; Reproducibility of Results ; beta-Fructofuranosidase ; Anti-Bacterial Agents ; Water ; *Biosensing Techniques ; },
abstract = {On-site and cost-effective monitoring of antibiotic residue in water samples using a ubiquitous device that is readily available to the general public is a big challenge. Herein, we developed a portable biosensor for kanamycin (KAN) detection based on a glucometer and CRISPR-Cas12a. The aptamer-KAN interactions liberate the trigger C strand, which can initiate the hairpin assembly to produce numerous double-stranded DNA. After recognition by CRISPR-Cas12a, Cas12a can cleave the magnetic bead and invertase-modified single-stranded DNA. After magnetic separation, the invertase can convert sucrose into glucose, which can be quantified by a glucometer. The linear range of the glucometer biosensor is from 1 pM to 100 nM and the detection limit is 1 pM. The biosensor also exhibited high selectivity and the nontarget antibiotics had no significant interference with KAN detection. The sensing system is robust and can work in complex samples with excellent accuracy and reliability. The recovery values were in the range of 89-107.2 % for water samples and 86-106.5 % for milk samples. The relative standard deviation (RSD) was below 5 %. With the advantages of simple operation, low cost, and easy accessibility to the public, this portable pocket-sized sensor can realize the on-site detection of antibiotic residue in resource-limited settings.},
}
@article {pmid36518081,
year = {2023},
author = {Abhinandan, K and Hickerson, NMN and Lan, X and Samuel, MA},
title = {Disabling of ARC1 through CRISPR-Cas9 leads to a complete breakdown of self-incompatibility responses in Brassica napus.},
journal = {Plant communications},
volume = {4},
number = {2},
pages = {100504},
doi = {10.1016/j.xplc.2022.100504},
pmid = {36518081},
issn = {2590-3462},
mesh = {*Brassica napus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Arabidopsis/metabolism ; Plant Proteins/genetics/metabolism ; },
}
@article {pmid36857724,
year = {2023},
author = {Ma, R and Jiang, J and Ya, Y and Lin, Y and Zhou, Y and Wu, Y and Tan, X and Huang, K and Du, F and Xu, J},
title = {A carbon dot-based nanoscale covalent organic framework as a new emitter combined with a CRISPR/Cas12a-mediated electrochemiluminescence biosensor for ultrasensitive detection of bisphenol A.},
journal = {The Analyst},
volume = {148},
number = {6},
pages = {1362-1370},
doi = {10.1039/d3an00024a},
pmid = {36857724},
issn = {1364-5528},
mesh = {Carbon ; *Metal-Organic Frameworks ; CRISPR-Cas Systems ; Luminescent Measurements ; *Biosensing Techniques ; Electrochemical Techniques ; Limit of Detection ; },
abstract = {Exploring new highly efficient electrochemiluminescence (ECL) luminophores is a necessary condition for developing ultrasensitive ECL biosensors. Therefore, a luminescent carbon dot-based covalent organic framework (CD-COF) was prepared using aldehyde-based carbon dots (CDs) and 1,3,5-tris (4-aminophenyl) benzene (TPB). Because the CD-COF made the regular arrangement of CDs conducive to improving the ECL response, CD-COF had a higher ECL intensity and efficiency than CDs. What's more, the ECL intensity of the CD-COF/S2O8[2-]/Bu4N[+] system was about 2.98, 7.50, and 28.08 times higher than those of the CD-COF/S2O8[2-], CDs/S2O8[2-] and S2O8[2-] systems, respectively. Considering the remarkable ECL performance, the CD-COF/S2O8[2-]/Bu4N[+] system was employed combined with the CRISPR/Cas12a trans-cutting strategy to construct an "off-on" ECL biosensor for BPA detection. The proposed ECL biosensor exhibited excellent performance with a wide linear range from 1.0 × 10[-14] mol L[-1] to 1.0 × 10[-5] mol L[-1] with a low detection limit of 2.21 fM (S/N = 3) under the optimized conditions. The biosensor demonstrated that CD-COF can be used as an efficient ECL emitter, thus expanding the application field of COFs. In addition, the good stability and specificity of the biosensor enabled the rapid detection of BPA, which will provide valuable insights into promising ultrasensitive ECL biosensors.},
}
@article {pmid36848598,
year = {2023},
author = {Puppulin, L and Ishikawa, J and Sumino, A and Marchesi, A and Flechsig, H and Umeda, K and Kodera, N and Nishimasu, H and Shibata, M},
title = {Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.},
journal = {ACS nano},
volume = {17},
number = {5},
pages = {4629-4641},
doi = {10.1021/acsnano.2c10709},
pmid = {36848598},
issn = {1936-086X},
mesh = {*CRISPR-Cas Systems ; *Staphylococcus aureus/metabolism ; Microscopy, Atomic Force ; Gene Editing/methods ; DNA/chemistry ; },
abstract = {Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.},
}
@article {pmid36641494,
year = {2023},
author = {Senthilnathan, R and Ilangovan, I and Kunale, M and Easwaran, N and Ramamoorthy, S and Veeramuthu, A and Kodiveri Muthukaliannan, G},
title = {An update on CRISPR-Cas12 as a versatile tool in genome editing.},
journal = {Molecular biology reports},
volume = {50},
number = {3},
pages = {2865-2881},
pmid = {36641494},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genetic Engineering ; Mutagenesis ; Mutation ; },
abstract = {Gene editing techniques, which help in modification of any DNA sequence at ease, have revolutionized the world of Genetic engineering. Although there are other gene-editing techniques, CRISPR has emerged as the chief and most preferred tool due to its simplicity and capacity to execute effective gene editing in a wide range of organisms. Although Cas9 has widely been employed for genetic modification over the years, Cas12 systems have lately emerged as a viable option. This review primarily focuses on assessing Cas12-mediated mutagenesis and elucidating the editing efficacy of both Cpf1 (Cas12a) and C2c1 (Cas12b) systems in microbes, plants, and other species. Also, we reviewed several genetic alterations that have been performed with these Cas12 systems to improve editing efficiency. Furthermore, the experimental benefits and applications of Cas12 systems are highlighted in this study.},
}
@article {pmid36550260,
year = {2023},
author = {Han, JY and Seo, J and Choi, Y and Im, W and Ban, JJ and Sung, JJ},
title = {CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.},
journal = {Molecular biology reports},
volume = {50},
number = {3},
pages = {2127-2136},
pmid = {36550260},
issn = {1573-4978},
mesh = {Mice ; Animals ; *Brain-Derived Neurotrophic Factor/genetics/metabolism ; *Huntington Disease/metabolism ; Mice, Transgenic ; Gene Editing ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; },
abstract = {BACKGROUND: Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed.<br><br>METHODS AND RESULTS: Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1&#x3b1;) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1&#x3b1; or BDNF.<br><br>CONCLUSION: Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1&#x3b1; and BDNF.&#xa0;Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.},
}
@article {pmid36428245,
year = {2023},
author = {Zhang, Y and Blahut-Beatty, L and Zheng, S and Clough, SJ and Simmonds, DH},
title = {The Role of a Soybean 14-3-3 Gene (Glyma05g29080) on White Mold Resistance and Nodulation Investigations Using CRISPR-Cas9 Editing and RNA Silencing.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {36},
number = {3},
pages = {159-164},
doi = {10.1094/MPMI-07-22-0157-R},
pmid = {36428245},
issn = {0894-0282},
mesh = {*CRISPR-Cas Systems/genetics ; RNA Interference ; *Soybeans/genetics ; },
abstract = {The role of a soybean 14-3-3 gene (Glyma05g29080) in defense against white mold and in nodulation was investigated by loss-of-gene-function with CRISPR-Cas9 editing and silencing of RNA interference (RNAi). Particle bombardment was used to introduce the CRISPR expression cassette to target the soybean 14-3-3 gene and an RNAi construct to silence gene transcription. Transmission of the edited 14-3-3 gene and the RNAi construct was confirmed in their respective progeny. The recovered transgenic plants and their progeny were significantly more susceptible to Sclerotinia sclerotiorum infection and showed a significant reduction in nodulation, thus confirming the role of the 14-3-3 gene (Glyma05g29080) in both nodulation and defense.},
}
@article {pmid36912935,
year = {2023},
author = {Timberlake, AT},
title = {Molecular scalpels: the future of pediatric craniofacial surgery?.},
journal = {Plastic and reconstructive surgery},
volume = {},
number = {},
pages = {},
doi = {10.1097/PRS.0000000000010402},
pmid = {36912935},
issn = {1529-4242},
abstract = {CRISPR-Cas genome editing tools are among the most substantial advances in the life sciences in modern history. Single dose gene therapies to correct pathogenic mutations have moved quickly from bench to bedside, with several therapeutics designed by CRISPR pioneers entering various stages of clinical investigation. Applications of these genetic technologies are poised to reshape the practice of both medicine and surgery. Many of the most morbid conditions treated by craniofacial surgeons are syndromic craniosynostoses caused by mutations in fibroblast growth factor receptor (FGFR) genes, including Apert, Pfeiffer, Crouzon, and Muenke syndromes. The fact that pathogenic mutations in these genes are recurrent in the majority of affected families presents a unique opportunity to develop "off the shelf" gene editing therapies to correct these mutations in affected children. The therapeutic potential of these interventions could reshape pediatric craniofacial surgery, potentially first eliminating the need for midface advancement procedures in affected children.},
}
@article {pmid36912817,
year = {2023},
author = {Wiegand, T and Wilkinson, R and Santiago-Frangos, A and Lynes, M and Hatzenpichler, R and Wiedenheft, B},
title = {Functional and Phylogenetic Diversity of Cas10 Proteins.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0085},
pmid = {36912817},
issn = {2573-1602},
abstract = {Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.},
}
@article {pmid36910714,
year = {2023},
author = {Jang, G and Shin, HR and Do, HS and Kweon, J and Hwang, S and Kim, S and Heo, SH and Kim, Y and Lee, BH},
title = {Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing.},
journal = {Molecular therapy. Nucleic acids},
volume = {31},
number = {},
pages = {586-595},
pmid = {36910714},
issn = {2162-2531},
abstract = {Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.},
}
@article {pmid36905519,
year = {2023},
author = {Freund, EC and Haag, SM and Haley, B and Murthy, A},
title = {Optimized Nonviral Gene Disruption in Primary Murine and Human Myeloid Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2618},
number = {},
pages = {201-217},
pmid = {36905519},
issn = {1940-6029},
mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Electroporation ; Genetic Engineering ; Macrophages ; },
abstract = {Genetically engineered myeloid cells such as monocytes, macrophages, and dendritic cells have broad applications in basic and translational research. Their central roles in innate and adaptive immunity make them attractive as putative therapeutic cell products. However, efficient gene editing of primary myeloid cells presents unique challenges owing to their sensitivity to foreign nucleic acids and poor editing efficiencies using current methodologies (Hornung et al., Science 314:994-997, 2006; Coch et al., PLoS One 8:e71057, 2013; Bartok and Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla et al., Gene Ther 20:514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva et al., BMC Biotechnol 11:13, 2011). This chapter describes nonviral CRISPR-mediated gene knockout in primary human and murine monocytes as well as monocyte-derived or bone marrow-derived macrophages and dendritic cells. Electroporation-mediated delivery of recombinant Cas9 complexed with synthetic guide RNAs can be applied for population-level disruption of single or multiple gene targets.},
}
@article {pmid36902207,
year = {2023},
author = {Ueda, J and Yamazaki, T and Funakoshi, H},
title = {Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges.},
journal = {International journal of molecular sciences},
volume = {24},
number = {5},
pages = {},
pmid = {36902207},
issn = {1422-0067},
mesh = {*Epigenome ; *Gene Editing ; Epigenesis, Genetic ; DNA Methylation ; Chromatin ; CRISPR-Cas Systems ; },
abstract = {The advancement in epigenetics research over the past several decades has led to the potential application of epigenome-editing technologies for the treatment of various diseases. In particular, epigenome editing is potentially useful in the treatment of genetic and other related diseases, including rare imprinted diseases, as it can regulate the expression of the epigenome of the target region, and thereby the causative gene, with minimal or no modification of the genomic DNA. Various efforts are underway to successfully apply epigenome editing in vivo, such as improving target specificity, enzymatic activity, and drug delivery for the development of reliable therapeutics. In this review, we introduce the latest findings, summarize the current limitations and future challenges in the practical application of epigenome editing for disease therapy, and introduce important factors to consider, such as chromatin plasticity, for a more effective epigenome editing-based therapy.},
}
@article {pmid36902202,
year = {2023},
author = {Zhou, J and Li, Z and Li, Y and Zhao, Q and Luan, X and Wang, L and Liu, Y and Liu, H and Zhang, J and Yao, D},
title = {Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family.},
journal = {International journal of molecular sciences},
volume = {24},
number = {5},
pages = {},
pmid = {36902202},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Soybeans/genetics ; CRISPR-Cas Systems ; Oleic Acid/metabolism ; Fatty Acid Desaturases/metabolism ; Plants, Genetically Modified/genetics ; Fatty Acids/metabolism ; },
abstract = {Δ[12]-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.},
}
@article {pmid36901914,
year = {2023},
author = {Ivanov, AV and Safenkova, IV and Biketov, SF and Zherdev, AV and Dzantiev, BB},
title = {Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.},
journal = {International journal of molecular sciences},
volume = {24},
number = {5},
pages = {},
pmid = {36901914},
issn = {1422-0067},
mesh = {*DNA/chemistry ; Endonucleases/metabolism ; Oligonucleotides ; *Biosensing Techniques ; Magnetic Phenomena ; CRISPR-Cas Systems ; },
abstract = {Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.},
}
@article {pmid36901844,
year = {2023},
author = {Wang, W and Huang, P and Dai, W and Tang, H and Qiu, Y and Chang, Y and Han, Z and Li, X and Du, L and Ye, X and Zou, C and Wang, K},
title = {Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid Wheat.},
journal = {International journal of molecular sciences},
volume = {24},
number = {5},
pages = {},
pmid = {36901844},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Triticum/genetics ; CRISPR-Cas Systems ; Histones/genetics ; Mutation ; },
abstract = {Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.},
}
@article {pmid36898814,
year = {2023},
author = {Dou, B and Zhang, Y and Gao, H and Zhang, S and Zheng, J and Lu, X and Liu, S and Zhou, H and Hun, X},
title = {CRISPR/Cas12a-based MUSCA-PEC strategy for HSV-1 assay.},
journal = {Analytica chimica acta},
volume = {1250},
number = {},
pages = {340955},
doi = {10.1016/j.aca.2023.340955},
pmid = {36898814},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems ; *Herpesvirus 1, Human ; Alkaline Phosphatase ; Biological Assay ; Coloring Agents ; *Biosensing Techniques ; },
abstract = {In the photoelectrochemical sensing, constant potential excitation to get the photoelectrochemical signal is the main excitation signal mode. Novel method for photoelectrochemical signal obtaining is needed. Inspired by this ideal, a photoelectrochemical strategy for Herpes simplex virus (HSV-1) detection with multiple potential step chronoamperometry (MUSCA) pattern was fabricated using CRISPR/Cas12a cleavage coupled with entropy-driven target recycling. In the presence of target, HSV-1, the Cas12a was activated by the H1-H2 complex obtained by entropy-driven, then digesting the circular fragment of csRNA to expose single-stranded crRNA2 and alkaline phosphatase (ALP). The inactive Cas12a was self-assembled with crRNA2 and activated again with the help of assistant dsDNA. After multiple rounds of CRISPR/Cas12a cleavage and magnetic separation, MUSCA, as a signal amplifier, collected the enhanced photocurrent responses generated by catalyzed p-Aminophenol (p-AP). Different from the reported signal enhancement strategies based on photoactive nanomaterials and sensing mechanisms, MUSCA technique endowed the strategy with unique advantages of direct, fast and ultrasensitive. A superior detection limit of 3 aM toward HSV-1 was achieved. This strategy was successfully applied for HSV-1 detection in Human serum samples. The combination of MUSCA technique and CRISPR/Cas12a assay brings broader potential prospect for the detection of nucleic acids.},
}
@article {pmid36858011,
year = {2023},
author = {Li, M and Luo, N and Liao, X and Zou, L},
title = {Proximity hybridization-regulated CRISPR/Cas12a-based dual signal amplification strategy for sensitive detection of circulating tumor DNA.},
journal = {Talanta},
volume = {257},
number = {},
pages = {124395},
doi = {10.1016/j.talanta.2023.124395},
pmid = {36858011},
issn = {1873-3573},
mesh = {Humans ; *Circulating Tumor DNA/genetics ; CRISPR-Cas Systems ; Nucleic Acid Hybridization/methods ; DNA ; *Neoplasms ; *Biosensing Techniques/methods ; },
abstract = {Circulating tumor DNA (ctDNA) is regarded as an ideal candidate biomarker for the non-invasive diagnosis of cancer. However, the lack of convenient and reliable detection methods for ctDNA restricts its clinical application. Herein, we developed a dual signal amplification strategy for sensitive detection of ctDNA based on hybridization chain reaction (HCR) and proximity hybridization-regulated CRISPR/Cas12a. The ctDNA initiates HCR through the continuous hybridization of two hairpin probes (H1 and H2), yielding long nicked double-stranded DNA nanowires composed of numerous split segments, which are successively connected to activate the trans-cleavage activity of CRISPR/Cas12a. In this case, the doubly labeled single-stranded DNA reporter can be cleaved to produce a strong fluorescent signal. Owing to the dual amplification of HCR and CRISPR/Cas12a, this strategy exhibits high sensitivity toward ctDNA with a low detection limit of 5.43 fM. Moreover, the proposed method was successfully applied for ctDNA detection in serum samples with satisfactory results, which has great potential in the clinical diagnosis of cancer.},
}
@article {pmid36841059,
year = {2023},
author = {Zhang, L and Bai, T and Wu, H},
title = {sgRNA-2wPSM: Identify sgRNAs on-target activity by combining two-window-based position specific mismatch and synthetic minority oversampling technique.},
journal = {Computers in biology and medicine},
volume = {155},
number = {},
pages = {106489},
doi = {10.1016/j.compbiomed.2022.106489},
pmid = {36841059},
issn = {1879-0534},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Algorithms ; Base Sequence ; Support Vector Machine ; },
abstract = {sgRNAs on-target activity prediction is a critical step in the CRISPR-Cas9 system. Due to its importance to RNA function research and genome editing application, some computational methods were introduced, treating it as a binary classification task or a regression task. Among these methods, sgRNA-PSM is a state-of-the-art method. In this work, we improved this method by proposing a new feature extraction method called two-window-based PSM, which divides the DNA sequences into two non-overlapping segments so as to extract different patterns in the two different segments. The two-window-based PSM were fed into Support Vector Machines (SVMs), and a new method called sgRNA-2wPSM was proposed. Furthermore, a new oversampling method called SCORE-SVM-SMOTE was proposed to solve the imbalanced training set problem based on the SVM-SMOTE algorithm. Results on the benchmark datasets indicated that sgRNA-2wPSM is superior to other methods.},
}
@article {pmid36827793,
year = {2023},
author = {Liu, D and Sun, XM and Zhu, L and Li, CY},
title = {Using time-shared scanning optical tweezers assisted two-photon fluorescence imaging to establish a versatile CRISPR/Cas12a-mediated biosensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {227},
number = {},
pages = {115158},
doi = {10.1016/j.bios.2023.115158},
pmid = {36827793},
issn = {1873-4235},
mesh = {Optical Tweezers ; CRISPR-Cas Systems ; *Biosensing Techniques ; Optical Imaging ; *DNA, Catalytic ; },
abstract = {Based on the admirable precision to identify target nucleic acids and the particular trans-cleavage feature, CRISPR/Cas12a system is a useful means to further improve the sensing accuracy and the design flexibility of fluorescence biosensors. However, the current construction concepts still suffer from insufficient sensitivity, unsuitable for complicated real samples and limited detection species. In this work, much efforts are achieved to address these obstacles. At first, we adopt a microsphere sustained signal enrichment, under which a home-made time-shared scanning optical tweezers assisted fluorescence imaging is employed to guarantee a stable excitation and also realize multiflux measurement. Furthermore, by taking advantage of the low background merit of the near-infrared light excited two-photon fluorescence, a commendable anti-interference capability is endowed to operate in complex media. After utilizing a functional DNA (e.g. aptamer and DNAzyme) regulated mediation pathway to respond non-nucleic acid analytes (alpha fetal protein and Pb[2+]), the newly-established CRISPR/Cas12a-mediated fluorescence biosensor is found to display favorable assay performance. More importantly, our analytical methodology can act as a versatile and reliable toolbox in various applications such as disease diagnosis and environmental analysis, propelling the development of CRISPR system in biosensing field.},
}
@article {pmid36809733,
year = {2023},
author = {Liu, W and Zhu, X and Li, L and Wang, S and Li, CZ and Liang, T},
title = {Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise gene interference.},
journal = {Biosensors &amp; bioelectronics},
volume = {227},
number = {},
pages = {115138},
doi = {10.1016/j.bios.2023.115138},
pmid = {36809733},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques ; RNA/genetics ; Peptide Hydrolases/genetics ; },
abstract = {Activation of Cas13 is exclusively dependent on crRNA-target RNA hybridization according to the canonical mode of Cas13 action. Upon activation Cas13 can cleave both target RNA and any surrounding RNA. The latter has been well adopted by therapeutic gene interference and biosensor development. This work for the first time, rationale designs and validates a multi-component controlled activation system of Cas13 by N-terminus tagging. A composite SUMO tag comprised of His, Twinstrep, and Smt3 tags fully suppresses target dependent activation of Cas13a by interfering with crRNA docking. The suppression releases upon proteases mediated proteolytic cleavage. The modular composition of the composite tag can be altered to fulfill customized response to alternative proteases. The biosensor SUMO-Cas13a is able to resolve a broad concentration range of protease Ulp1 with a calculated LOD of 48.8pg/μL in aqueous buffer. Further, in accordance with this finding Cas13a was successfully programmed to exert target gene knock down preferentially in SUMO protease high cell types. In summary the discovered regulatory component not only fulfills Cas13a based protease detection for the first time, but also delivers a novel strategy for multi-component controlled activation of Cas13a toward temporal and spacial precision.},
}
@article {pmid36801553,
year = {2023},
author = {Wang, X and Chen, Y and Ma, L and Han, Z and Liu, Y and Qiao, J},
title = {An amplification-free CRISPR/Cas12a-based fluorescence assay for ultrasensitive detection of nuclease activity.},
journal = {Talanta},
volume = {257},
number = {},
pages = {124329},
doi = {10.1016/j.talanta.2023.124329},
pmid = {36801553},
issn = {1873-3573},
mesh = {Humans ; *CRISPR-Cas Systems ; *Biological Assay ; DNA, Single-Stranded ; Deoxyribonuclease I ; Endonucleases ; RNA, Guide, CRISPR-Cas Systems ; Ribonuclease H ; },
abstract = {Nuclease, such as RNase H and DNase I, plays key roles in plenty of cellular processes and could be potential therapeutic target for drug development. It is necessary to establish rapid and simple-to-use methods to detect nuclease activity. Herein, we develop a Cas12a-based fluorescence assay without any nucleic acid amplification steps for ultrasensitive detection of RNase H or DNase I activity. By our design, the pre-assembled crRNA/ssDNA duplex triggered the cleavage of fluorescent probes in the presence of Cas12a enzymes. However, the crRNA/ssDNA duplex was selectively digested with the addition of RNase H or DNase I, which leaded to fluorescence intensity changes. Under optimized conditions, the method exhibited good analytical performance, achieving a limit of detection (LOD) as low as 0.0082 U/mL for RNase H and 0.13 U/mL for DNase I, respectively. The method was feasible for analysis of RNase H in human serum and cell lysates, as well as for screening of enzyme inhibitors. Moreover, it can be adopted to image RNase H activity in living cells. Together, this study provides a facile platform for nuclease detection and could be expanded for other biomedical research and clinical diagnostics.},
}
@article {pmid36371609,
year = {2023},
author = {Zhao, J and Tan, Y and Jiang, Y and Zhu-Salzman, K and Xiao, L},
title = {CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.},
journal = {Insect molecular biology},
volume = {32},
number = {2},
pages = {132-142},
doi = {10.1111/imb.12819},
pmid = {36371609},
issn = {1365-2583},
mesh = {Animals ; *Methoprene ; Larva ; Spodoptera/genetics ; *Beta vulgaris/genetics/metabolism ; CRISPR-Cas Systems ; Metamorphosis, Biological ; Juvenile Hormones/metabolism ; Insecta/genetics ; Pupa ; Insect Proteins/metabolism ; Gene Expression Regulation, Developmental ; },
abstract = {Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.},
}
@article {pmid36910196,
year = {2023},
author = {Kogawa, M and Nishikawa, Y and Saeki, T and Yoda, T and Arikawa, K and Takeyama, H and Hosokawa, M},
title = {Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1133917},
pmid = {36910196},
issn = {1664-302X},
abstract = {Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.},
}
@article {pmid36909407,
year = {2023},
author = {Majeed, Y and Zhu, X and Zhang, N and Ul-Ain, N and Raza, A and Haider, FU and Si, H},
title = {Harnessing the role of mitogen-activated protein kinases against abiotic stresses in plants.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {932923},
pmid = {36909407},
issn = {1664-462X},
abstract = {Crop plants are vulnerable to various biotic and abiotic stresses, whereas plants tend to retain their physiological mechanisms by evolving cellular regulation. To mitigate the adverse effects of abiotic stresses, many defense mechanisms are induced in plants. One of these mechanisms is the mitogen-activated protein kinase (MAPK) cascade, a signaling pathway used in the transduction of extracellular stimuli into intercellular responses. This stress signaling pathway is activated by a series of responses involving MAPKKKs→MAPKKs→MAPKs, consisting of interacting proteins, and their functions depend on the collaboration and activation of one another by phosphorylation. These proteins are key regulators of MAPK in various crop plants under abiotic stress conditions and also related to hormonal responses. It is revealed that in response to stress signaling, MAPKs are characterized as multigenic families and elaborate the specific stimuli transformation as well as the antioxidant regulation system. This pathway is directed by the framework of proteins and stopping domains confer the related associates with unique structure and functions. Early studies of plant MAPKs focused on their functions in model plants. Based on the results of whole-genome sequencing, many MAPKs have been identified in plants, such as Arbodiposis, tomato, potato, alfalfa, poplar, rice, wheat, maize, and apple. In this review, we summarized the recent work on MAPK response to abiotic stress and the classification of MAPK cascade in crop plants. Moreover, we highlighted the modern research methodologies such as transcriptomics, proteomics, CRISPR/Cas technology, and epigenetic studies, which proposed, identified, and characterized the novel genes associated with MAPKs and their role in plants under abiotic stress conditions. In-silico-based identification of novel MAPK genes also facilitates future research on MAPK cascade identification and function in crop plants under various stress conditions.},
}
@article {pmid36909403,
year = {2023},
author = {Tiwari, JK and Singh, AK and Behera, TK},
title = {CRISPR/Cas genome editing in tomato improvement: Advances and applications.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1121209},
pmid = {36909403},
issn = {1664-462X},
abstract = {The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters (e.g. leaf, stem, flower, male sterility, fruit, parthenocarpy), fruit ripening, quality and nutrition (e.g., lycopene, carotenoid, GABA, TSS, anthocyanin, shelf-life), disease resistance (e.g. TYLCV, powdery mildew, late blight), abiotic stress tolerance (e.g. heat, drought, salinity), C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of de novo domestication of elite traits from wild relatives to the cultivated tomato and vice versa. Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning (e.g. Golden Gate cloning, GoldenBraid, and BioBrick technology), robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.},
}
@article {pmid36907678,
year = {2023},
author = {Duan, W and Urani, E and Mattson, MP},
title = {The potential of gene editing for Huntington's disease.},
journal = {Trends in neurosciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tins.2023.02.005},
pmid = {36907678},
issn = {1878-108X},
abstract = {Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the huntingtin gene resulting in long stretches of polyglutamine repeats in the huntingtin protein. The disease involves progressive degeneration of neurons in the striatum and cerebral cortex resulting in loss of control of motor function, psychiatric problems, and cognitive deficits. There are as yet no treatments that can slow disease progression in HD. Recent advances in gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) systems and demonstrations of their ability to correct gene mutations in animal models of a range of diseases suggest that gene editing may prove effective in preventing or ameliorating HD. Here we describe (i) potential CRISPR-Cas designs and cellular delivery methods for the correction of mutant genes that cause inherited diseases, and (ii) recent preclinical findings demonstrating the efficacy of such gene-editing approaches in animal models, with a focus on HD.},
}
@article {pmid36905332,
year = {2023},
author = {Matsumoto, D and Kishi, K and Matsugi, E and Inoue, Y and Nigorikawa, K and Nomura, W},
title = {Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.14608},
pmid = {36905332},
issn = {1873-3468},
abstract = {Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.},
}
@article {pmid36903878,
year = {2023},
author = {Kong, Q and Li, J and Wang, S and Feng, X and Shou, H},
title = {Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {5},
pages = {},
pmid = {36903878},
issn = {2223-7747},
abstract = {The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.},
}
@article {pmid36902028,
year = {2023},
author = {Liu, B and Song, W and Wang, L and Wu, Y and Xu, X and Niu, X and Huang, S and Liu, Y and Tang, W},
title = {dCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae.},
journal = {International journal of molecular sciences},
volume = {24},
number = {5},
pages = {},
pmid = {36902028},
issn = {1422-0067},
abstract = {Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.},
}
@article {pmid36901793,
year = {2023},
author = {Wang, Y and Fan, H and Tong, Y},
title = {Unveil the Secret of the Bacteria and Phage Arms Race.},
journal = {International journal of molecular sciences},
volume = {24},
number = {5},
pages = {},
pmid = {36901793},
issn = {1422-0067},
abstract = {Bacteria have developed different mechanisms to defend against phages, such as preventing phages from being adsorbed on the surface of host bacteria; through the superinfection exclusion (Sie) block of phage's nucleic acid injection; by restricting modification (R-M) systems, CRISPR-Cas, aborting infection (Abi) and other defense systems to interfere with the replication of phage genes in the host; through the quorum sensing (QS) enhancement of phage's resistant effect. At the same time, phages have also evolved a variety of counter-defense strategies, such as degrading extracellular polymeric substances (EPS) that mask receptors or recognize new receptors, thereby regaining the ability to adsorb host cells; modifying its own genes to prevent the R-M systems from recognizing phage genes or evolving proteins that can inhibit the R-M complex; through the gene mutation itself, building nucleus-like compartments or evolving anti-CRISPR (Acr) proteins to resist CRISPR-Cas systems; and by producing antirepressors or blocking the combination of autoinducers (AIs) and its receptors to suppress the QS. The arms race between bacteria and phages is conducive to the coevolution between bacteria and phages. This review details bacterial anti-phage strategies and anti-defense strategies of phages and will provide basic theoretical support for phage therapy while deeply understanding the interaction mechanism between bacteria and phages.},
}
@article {pmid36894612,
year = {2023},
author = {Pavlou, S and Foskolou, S and Patikas, N and Field, SF and Papachristou, EK and Santos, C and Edwards, AR and Kishore, K and Ansari, R and Rajan, SS and Fernandes, HJR and Metzakopian, E},
title = {CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {3934},
pmid = {36894612},
issn = {2045-2322},
mesh = {Humans ; Tunicamycin/pharmacology ; *CRISPR-Cas Systems ; *Endoplasmic Reticulum/metabolism ; Cell Death ; Endoplasmic Reticulum Stress ; Dopaminergic Neurons/metabolism ; Apoptosis ; p300-CBP Transcription Factors/metabolism ; },
abstract = {Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.},
}
@article {pmid36762651,
year = {2023},
author = {Komori, T and Hata, S and Mabuchi, A and Genova, M and Harada, T and Fukuyama, M and Chinen, T and Kitagawa, D},
title = {A CRISPR-del-based pipeline for complete gene knockout in human diploid cells.},
journal = {Journal of cell science},
volume = {136},
number = {6},
pages = {},
doi = {10.1242/jcs.260000},
pmid = {36762651},
issn = {1477-9137},
mesh = {Humans ; Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Diploidy ; INDEL Mutation/genetics ; Cell Line ; Gene Editing/methods ; },
abstract = {The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion-deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked out by the indel-based gene disruption. Here, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform 'complete' gene knockout efficiently in human diploid cells. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enabled the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is an efficient and practical approach for producing 'complete' gene knockout cell lines in human diploid cells.},
}
@article {pmid36649793,
year = {2023},
author = {Dickson, A and Mullooly, N and Serrano, A and Escudero-Ibarz, L and Wiggins, C and Gianni, D},
title = {Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {28},
number = {2},
pages = {29-35},
doi = {10.1016/j.slasd.2023.01.003},
pmid = {36649793},
issn = {2472-5560},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Silencing ; Lung ; Epithelial Cells/metabolism ; },
abstract = {Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.},
}
@article {pmid36508706,
year = {2023},
author = {Hardouin, G and Antoniou, P and Martinucci, P and Felix, T and Manceau, S and Joseph, L and Masson, C and Scaramuzza, S and Ferrari, G and Cavazzana, M and Miccio, A},
title = {Adenine base editor-mediated correction of the common and severe IVS1-110 (G&gt;A) β-thalassemia mutation.},
journal = {Blood},
volume = {141},
number = {10},
pages = {1169-1179},
doi = {10.1182/blood.2022016629},
pmid = {36508706},
issn = {1528-0020},
mesh = {Humans ; *beta-Thalassemia/genetics/therapy ; Gene Editing ; CRISPR-Cas Systems ; Mutation ; beta-Globins/genetics ; },
abstract = {β-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients' HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.},
}
@article {pmid36434096,
year = {2023},
author = {Jin, S and Lin, Q and Gao, Q and Gao, C},
title = {Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs).},
journal = {Nature protocols},
volume = {18},
number = {3},
pages = {831-853},
pmid = {36434096},
issn = {1750-2799},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Genome, Plant ; Plants/genetics ; DNA ; RNA, Guide, Kinetoplastida ; },
abstract = {Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months.},
}
@article {pmid35915232,
year = {2023},
author = {Hu, X and Zhang, B and Li, X and Li, M and Wang, Y and Dan, H and Zhou, J and Wei, Y and Ge, K and Li, P and Song, Z},
title = {The application and progression of CRISPR/Cas9 technology in ophthalmological diseases.},
journal = {Eye (London, England)},
volume = {37},
number = {4},
pages = {607-617},
pmid = {35915232},
issn = {1476-5454},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; *Retinitis Pigmentosa/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is an adaptive immune defence system that has gradually evolved in bacteria and archaea to combat invading viruses and exogenous DNA. Advances in technology have enabled researchers to enhance their understanding of the immune process in vivo and its potential for use in genome editing. Thus far, applications of CRISPR/Cas9 genome editing technology in ophthalmology have included gene therapy for corneal dystrophy, glaucoma, congenital cataract, Leber's congenital amaurosis, retinitis pigmentosa, Usher syndrome, fundus neovascular disease, proliferative vitreoretinopathy, retinoblastoma and other eye diseases. Additionally, the combination of CRISPR/Cas9 genome editing technology with adeno-associated virus vector and inducible pluripotent stem cells provides further therapeutic avenues for the treatment of eye diseases. Nonetheless, many challenges remain in the development of clinically feasible retinal genome editing therapy. This review discusses the development, as well as mechanism of CRISPR/Cas9 and its applications and challenges in gene therapy for eye diseases.},
}
@article {pmid36897939,
year = {2023},
author = {Elmore, JR and Dexter, GN and Baldino, H and Huenemann, JD and Francis, R and Peabody, GL and Martinez-Baird, J and Riley, LA and Simmons, T and Coleman-Derr, D and Guss, AM and Egbert, RG},
title = {High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.},
journal = {Science advances},
volume = {9},
number = {10},
pages = {eade1285},
doi = {10.1126/sciadv.ade1285},
pmid = {36897939},
issn = {2375-2548},
abstract = {Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.},
}
@article {pmid36897609,
year = {2023},
author = {Yang, K and Qian, J and Zhang, C and Wang, Z and Huang, Q and Shi, G and Zhang, Z and Yang, Y and Han, X},
title = {Biogenic materials for CRISPR delivery and therapeutics.},
journal = {Biomaterials science},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2bm02169b},
pmid = {36897609},
issn = {2047-4849},
abstract = {CRISPR, as an emerging gene-editing technology, has been widely used in multidisciplinary fields, including genetic diseases and some cancers. However, it remains a challenge to efficiently deliver CRISPR for safe and efficient genome editing. Currently, biomimetic materials have become an attractive delivery strategy for CRISPR-mediated genome editing due to their low immunogenicity and application safety. The biomimetic materials delivery is involved in the improvement of cellular uptake of nanoparticle vectors, and the gene editing efficiency. In this review, we summarize the current delivery strategies of CRISPR/Cas systems based on biogenic materials such as viruses, bacteria, cells, bioactive substances, etc., focusing on the potential applications in disease research and therapy. Finally, the prospects and limitations of CRISPR-based systems in therapeutics are discussed.},
}
@article {pmid36896234,
year = {2023},
author = {Petersen, B},
title = {Editorial: Genetic engineering in farm animals.},
journal = {Frontiers in genetics},
volume = {14},
number = {},
pages = {1155201},
pmid = {36896234},
issn = {1664-8021},
}
@article {pmid36896016,
year = {2023},
author = {Hu, M and Bao, W and Peng, Q and Hu, W and Yang, X and Xiang, Y and Yan, X and Li, M and Xu, P and He, Q and Yang, S},
title = {Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1135484},
pmid = {36896016},
issn = {2296-4185},
abstract = {Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2 g/L lactate and 16.9 ± 0.3 g/L ethanol using 48 g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6 g/L lactate and 12.9 ± 0.8 g/L ethanol as well as 36.2 ± 1.0 g/L lactate and 40.3 ± 0.3 g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0 g/L/h and 2.2 ± 0.0 g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1 g/L D-lactate and 27.7 ± 0.2 g/L ethanol as well as 42.8 ± 0.0 g/L D-lactate and 53.1 ± 0.7 g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.},
}
@article {pmid36895064,
year = {2023},
author = {Li, ZH and Wang, J and Xu, JP and Wang, J and Yang, X},
title = {Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research.},
journal = {Military Medical Research},
volume = {10},
number = {1},
pages = {12},
pmid = {36895064},
issn = {2054-9369},
abstract = {The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein (Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases (CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.},
}
@article {pmid36893938,
year = {2023},
author = {Hwang, S and Maxwell, KL},
title = {Diverse mechanisms of CRISPR-Cas9 inhibition by type II anti-CRISPR proteins.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168041},
doi = {10.1016/j.jmb.2023.168041},
pmid = {36893938},
issn = {1089-8638},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.},
}
@article {pmid36893718,
year = {2023},
author = {Yang, H and Ledesma-Amaro, R and Gao, H and Ren, Y and Deng, R},
title = {CRISPR-based biosensors for pathogenic biosafety.},
journal = {Biosensors &amp; bioelectronics},
volume = {228},
number = {},
pages = {115189},
doi = {10.1016/j.bios.2023.115189},
pmid = {36893718},
issn = {1873-4235},
abstract = {Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis.},
}
@article {pmid36881658,
year = {2023},
author = {Yau, WW and Chen, GB and Zhou, J and Francisco, JC and Thimmukonda, NK and Li, S and Singh, BK and Yen, PM},
title = {Nicotinamide riboside rescues dysregulated glycolysis and fatty acid β-oxidation in a human hepatic cell model of Citrin deficiency.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddad018},
pmid = {36881658},
issn = {1460-2083},
abstract = {Citrin Deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using CRISPR/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic NADH/NAD+ ratio and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside (NR) increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.},
}
@article {pmid36881621,
year = {2023},
author = {Chauhan, VP and Sharp, PA and Langer, R},
title = {Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {11},
pages = {e2300605120},
doi = {10.1073/pnas.2300605120},
pmid = {36881621},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems/genetics ; *INDEL Mutation ; Mutation ; Culture ; DNA End-Joining Repair/genetics ; Endonucleases/genetics ; },
abstract = {CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.},
}
@article {pmid36881252,
year = {2023},
author = {Zha, MJ and Cai, CE and He, PM},
title = {Outlook on the Security and Potential Improvements of CRISPR-Cas9.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36881252},
issn = {1559-0305},
abstract = {Gene editing technology is regarded as a good news to save patients with genetic diseases because of its significant function of specifically changing genetic information. From zinc-finger proteins to transcription activator-like effector protein nucleases gene editing tools are constantly updated. At the same time, scientists are constantly developing a variety of new gene editing therapy strategies, in order to promote gene editing therapy from various aspects and realize the maturity of the technology as soon as possible. In 2016, CRISPR-Cas9-mediated CAR-T therapy was the first to enter the clinical trial stage, indicating that the use of CRISPR-Cas system as the blade of the genetic lancet to save patients is officially on the schedule. The first challenge to achieve this exciting goal is to improve the security of the technology. This review will introduce the gene security issues faced by the CRISPR system as a clinical treatment tool, the current safer delivery methods and the newly developed CRISPR editing tools with higher precision. Many reviews summarize the means of improving the security of gene editing therapy and the comprehensive delivery method, while few articles focus on the threat of gene editing to the genomic security of the treatment target. Therefore, this review focuses on the risks brought by gene editing therapy to the patient genome, which provides a broader perspective for exploring and improving the security of gene editing therapy from two aspects of delivery system and CRISPR editing tools.},
}
@article {pmid36880733,
year = {2023},
author = {Popescu, B and Shannon, K},
title = {Sidestepping SHP2 inhibition.},
journal = {The Journal of experimental medicine},
volume = {220},
number = {5},
pages = {},
doi = {10.1084/jem.20230082},
pmid = {36880733},
issn = {1540-9538},
mesh = {*CRISPR-Cas Systems/genetics ; *Mitogen-Activated Protein Kinases ; },
abstract = {Allosteric SHP2 inhibitors are a novel class of compounds that target hyperactive Ras/Mitogen Activated Protein Kinase (MAPK) signaling. In this issue of JEM, Wei et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221563) report a genome-wide CRISPR/Cas9 knockout screen that uncovered novel mechanisms of adaptive resistance to pharmacologic inhibition of SHP2.},
}
@article {pmid36880696,
year = {2023},
author = {Hernandes-Lopes, J and Yassitepe, JECT and Koltun, A and Pauwels, L and Silva, VCHD and Dante, RA and Gerhardt, IR and Arruda, P},
title = {Genome editing in maize: Toward improving complex traits in a global crop.},
journal = {Genetics and molecular biology},
volume = {46},
number = {1 Suppl 1},
pages = {e20220217},
pmid = {36880696},
issn = {1415-4757},
abstract = {Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.},
}
@article {pmid36880183,
year = {2023},
author = {Punetha, M and Saini, S and Chaudhary, S and S Yadav, P and Whitworth, K and Green, J and Kumar, D and Kues, W},
title = {Induced Pluripotent Stem Cells in the Era of Precise Genome Editing.},
journal = {Current stem cell research &amp; therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/1574888X18666230307115326},
pmid = {36880183},
issn = {2212-3946},
abstract = {Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.},
}
@article {pmid36878916,
year = {2023},
author = {Park, J and Cui, G and Lee, H and Jeong, H and Kwak, JJ and Lee, J and Byeon, SH},
title = {CRISPR/Cas9 mediated specific ablation of vegfa in retinal pigment epithelium efficiently regresses choroidal neovascularization.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {3715},
pmid = {36878916},
issn = {2045-2322},
mesh = {Humans ; Animals ; Mice ; Retinal Pigment Epithelium ; CRISPR-Cas Systems ; *Choroidal Neovascularization/genetics/therapy ; Retina ; *Craniocerebral Trauma ; Disease Models, Animal ; Vascular Endothelial Growth Factor A/genetics ; },
abstract = {The CRISPR/Cas9 system easily edits target genes in various organisms and is used to treat human diseases. In most therapeutic CRISPR studies, ubiquitously expressed promoters, such as CMV, CAG, and EF1α, are used; however, gene editing is sometimes necessary only in specific cell types relevant to the disease. Therefore, we aimed to develop a retinal pigment epithelium (RPE)-specific CRISPR/Cas9 system. We developed a CRISPR/Cas9 system that operates only in retinal pigment epithelium (RPE) by expressing Cas9 under the RPE-specific vitelliform macular dystrophy 2 promoter (pVMD2). This RPE-specific CRISPR/pVMD2-Cas9 system was tested in human retinal organoid and mouse model. We confirmed that this system works specifically in the RPE of human retinal organoids and mouse retina. In addition, the RPE-specific Vegfa ablation using the novel CRISPR-pVMD2-Cas9 system caused regression of choroidal neovascularization (CNV) without unwanted knock-out in the neural retina in laser-induced CNV mice, which is a widely used animal model of neovascular age-related macular degeneration. RPE-specific Vegfa knock-out (KO) and ubiquitous Vegfa KO were comparable in the efficient regression of CNV. The promoter substituted, cell type-specific CRISPR/Cas9 systems can be used in specific 'target cell' therapy, which edits genes while reducing unwanted off- 'target cell' effects.},
}
@article {pmid36812357,
year = {2023},
author = {Jiang, Y and Zheng, C and Jin, M and Zhou, R and Wu, Q and Huang, F and Lou, Y and Zheng, L},
title = {An Ultrasensitive Colorimetric Foodborne Pathogenic Detection Method Using a CRISPR/Cas12a Mediated Strand Displacement/Hybridization Chain Reaction.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {9},
pages = {4193-4200},
doi = {10.1021/acs.jafc.2c08888},
pmid = {36812357},
issn = {1520-5118},
mesh = {*Colorimetry/methods ; Hydrogen Peroxide ; CRISPR-Cas Systems ; DNA ; *DNA, Catalytic/genetics ; },
abstract = {Accurate, rapid, and sensitive pathogenic detections play an important role in food safety. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR) nucleic acid assay for foodborne pathogenic colorimetric detection. A biotinylated DNA toehold is coupled on avidin magnetic beads and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products to catalyze the TMB-H2O2 reaction. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no color change. Under optimal conditions, the CSDHCR has a satisfactory linear detection of DNA targets with a regression equation Y = 0.0531*X - 0.0091 (R[2] = 0.9903) in the range of 10 fM to 1 nM, and the limit of detection was determined as 4.54 fM. In addition, Vibrio vulnificus, one foodborne pathogen, was used to verify the practical application of the method, and it showed satisfactory specificity and sensitivity with a limit of detection at 1.0 × 10[0] CFU/mL coupling with recombinase polymerase amplification. Our proposed CSDHCR biosensor could be a promising alternative method for ultrasensitive and visual detection of nucleic acids and the practical application of foodborne pathogens.},
}
@article {pmid36811465,
year = {2023},
author = {Xu, Y and Tian, N and Shi, H and Zhou, C and Wang, Y and Liang, FS},
title = {A Split CRISPR/Cas13b System for Conditional RNA Regulation and Editing.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {9},
pages = {5561-5569},
doi = {10.1021/jacs.3c01087},
pmid = {36811465},
issn = {1520-5126},
mesh = {*RNA/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas13b system has been demonstrated as a robust tool for versatile RNA studies and relevant applications. New strategies enabling precise control of Cas13b/dCas13b activities and minimal interference with native RNA activities will further facilitate the understanding and regulation of RNA functions. Here, we engineered a split Cas13b system that can be conditionally activated and deactivated under the induction of abscisic acid (ABA), which achieved the downregulation of endogenous RNAs in dosage- and time-dependent manners. Furthermore, an ABA inducible split dCas13b system was generated to achieve temporally controlled deposition of m[6]A at specific sites on cellular RNAs through conditional assembly and disassembly of split dCas13b fusion proteins. We also showed that the activities of split Cas13b/dCas13b systems can be modulated by light via using a photoactivatable ABA derivative. Overall, these split Cas13b/dCas13b platforms expand the existing repertoire of the CRISPR and RNA regulation toolkit to achieve targeted manipulation of RNAs in native cellular environments with minimal functional disruption to these endogenous RNAs.},
}
@article {pmid36878037,
year = {2023},
author = {Zhu, Y and Yin, S and Li, Z},
title = {Mechanism of inhibition of CRISPR-Cas9 by anti-CRISPR protein AcrIIC1.},
journal = {Biochemical and biophysical research communications},
volume = {654},
number = {},
pages = {34-39},
doi = {10.1016/j.bbrc.2023.02.065},
pmid = {36878037},
issn = {1090-2104},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are bacterial and archaeal defense mechanisms against invading phages and viruses. To overcome these defenses, phages and other mobile genetic elements (MGEs) have evolved multiple anti-CRISPR proteins (Acrs) that can inhibit the function of CRISPR-Cas systems. The AcrIIC1 protein has been shown to be able to inhibit the activity of Neisseria meningitidis Cas9 (NmeCas9) in both bacteria and human cells. Here, we solve the structure of AcrIIC1 in complex with the HNH domain of NmeCas9 using X-ray crystallography. The structure shows that AcrIIC1 binds to the catalytic sites of the HNH domain, preventing it from accessing the DNA target. In addition, our biochemical data show that AcrIIC1 is a broad-spectrum inhibitor targeting Cas9 enzymes from different subtypes. Taken together, the structure and biochemical analysis reveal the molecular mechanism of AcrIIC1-mediated Cas9 inhibition and provide new insights into regulatory tools for Cas9-based applications.},
}
@article {pmid36802524,
year = {2023},
author = {Shen, B and Li, L and Liu, C and Li, X and Li, X and Cheng, X and Wu, H and Yang, T and Cheng, W and Ding, S},
title = {Mesoporous Nanozyme-Enhanced DNA Tetrahedron Electrochemiluminescent Biosensor with Three-Dimensional Walking Nanomotor-Mediated CRISPR/Cas12a for Ultrasensitive Detection of Exosomal microRNA.},
journal = {Analytical chemistry},
volume = {95},
number = {9},
pages = {4486-4495},
doi = {10.1021/acs.analchem.2c05217},
pmid = {36802524},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/analysis ; CRISPR-Cas Systems ; DNA/chemistry ; Photometry ; *Biosensing Techniques/methods ; },
abstract = {Exosomal microRNAs (exomiRNAs) have emerged as ideal biomarkers for early clinical diagnostics. The accurate detection of exomiRNAs plays a crucial role in facilitating clinical applications. Herein, an ultrasensitive electrochemiluminescent (ECL) biosensor was constructed using three-dimensional (3D) walking nanomotor-mediated CRISPR/Cas12a and tetrahedral DNA nanostructures (TDNs)-modified nanoemitters (TCPP-Fe@HMUiO@Au-ABEI) for exomiR-155 detection. Initially, the 3D walking nanomotor-mediated CRISPR/Cas12a strategy could effectively convert the target exomiR-155 into amplified biological signals for improving the sensitivity and specificity. Then, TCPP-Fe@HMUiO@Au nanozymes with excellent catalytic performance were used to amplify ECL signals because of the enhanced mass transfer and increased catalytic active sites, originating from its high surface areas (601.83 m[2]/g), average pore size (3.46 nm), and large pore volumes (0.52 cm[3]/g). Meanwhile, the TDNs as the scaffold to fabricate "bottom-up" anchor bioprobes could improve the trans-cleavage efficiency of Cas12a. Consequently, this biosensor achieved the limit of detection down to 273.20 aM ranging from 1.0 fM to 1.0 nM. Furthermore, the biosensor could discriminate breast cancer patients evidently by analyzing exomiR-155, and these results conformed to that of qRT-PCR. Thus, this work provides a promising tool for early clinical diagnostics.},
}
@article {pmid36877024,
year = {2023},
author = {Bleriot, I and Blasco, L and Pacios, O and Fernández-García, L and López, M and Ortiz-Cartagena, C and Barrio-Pujante, A and Fernández-Cuenca, F and Pascual, &#xc1; and Martínez-Martínez, L and Oteo-Iglesias, J and Tomás, M},
title = {Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0397422},
doi = {10.1128/spectrum.03974-22},
pmid = {36877024},
issn = {2165-0497},
abstract = {Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.},
}
@article {pmid36876071,
year = {2023},
author = {Kaltenbrunner, A and Reimann, V and Hoffmann, UA and Aoyagi, T and Sakata, M and Nimura-Matsune, K and Watanabe, S and Steglich, C and Wilde, A and Hess, WR},
title = {Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1112307},
pmid = {36876071},
issn = {1664-302X},
abstract = {Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.},
}
@article {pmid36874963,
year = {2023},
author = {Elston, KM and Maeda, GP and Perreau, J and Barrick, JE},
title = {Addressing the challenges of symbiont-mediated RNAi in aphids.},
journal = {PeerJ},
volume = {11},
number = {},
pages = {e14961},
pmid = {36874963},
issn = {2167-8359},
mesh = {Animals ; Bees ; *Aphids ; RNA Interference ; Agriculture ; Biological Assay ; Endonucleases ; Escherichia coli ; RNA, Double-Stranded ; },
abstract = {Because aphids are global agricultural pests and models for bacterial endosymbiosis, there is a need for reliable methods to study and control their gene function. However, current methods available for aphid gene knockout and knockdown of gene expression are often unreliable and time consuming. Techniques like CRISPR-Cas genome editing can take several months to achieve a single gene knockout because they rely on aphids going through a cycle of sexual reproduction, and aphids often lack strong, consistent levels of knockdown when fed or injected with molecules that induce an RNA interference (RNAi) response. In the hopes of addressing these challenges, we attempted to adapt a new method called symbiont-mediated RNAi (smRNAi) for use in aphids. smRNAi involves engineering a bacterial symbiont of the insect to continuously supply double-stranded RNA (dsRNA) inside the insect body. This approach has been successful in thrips, kissing bugs, and honeybees. We engineered the laboratory Escherichia coli strain HT115 and the native aphid symbiont Serratia symbiotica CWBI-2.3[T] to produce dsRNA inside the gut of the pea aphid (Acyrthosiphon pisum) targeting salivary effector protein (C002) or ecdysone receptor genes. For C002 assays, we also tested co-knockdown with an aphid nuclease (Nuc1) to reduce RNA degradation. However, we found that smRNAi was not a reliable method for aphid gene knockdown under our conditions. We were unable to consistently achieve the expected phenotypic changes with either target. However, we did see indications that elements of the RNAi pathway were modestly upregulated, and expression of some targeted genes appeared to be somewhat reduced in some trials. We conclude with a discussion of the possible avenues through which smRNAi, and aphid RNAi in general, could be improved in the future.},
}
@article {pmid36873375,
year = {2023},
author = {Ewaisha, R and Anderson, KS},
title = {Immunogenicity of CRISPR therapeutics-Critical considerations for clinical translation.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1138596},
pmid = {36873375},
issn = {2296-4185},
abstract = {CRISPR offers new hope for many patients and promises to transform the way we think of future therapies. Ensuring safety of CRISPR therapeutics is a top priority for clinical translation and specific recommendations have been recently released by the FDA. Rapid progress in the preclinical and clinical development of CRISPR therapeutics leverages years of experience with gene therapy successes and failures. Adverse events due to immunogenicity have been a major setback that has impacted the field of gene therapy. As several in vivo CRISPR clinical trials make progress, the challenge of immunogenicity remains a significant roadblock to the clinical availability and utility of CRISPR therapeutics. In this review, we examine what is currently known about the immunogenicity of CRISPR therapeutics and discuss several considerations to mitigate immunogenicity for the design of safe and clinically translatable CRISPR therapeutics.},
}
@article {pmid36869936,
year = {2023},
author = {Zhou, S and Deng, L and Dong, J and Lu, P and Qi, N and Huang, Z and Yang, M and Huo, D and Hou, C},
title = {Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.},
journal = {Mikrochimica acta},
volume = {190},
number = {4},
pages = {113},
pmid = {36869936},
issn = {1436-5073},
mesh = {Humans ; *CRISPR-Cas Systems ; *Genes, p53 ; DNA Primers ; Electrodes ; Fluorescence ; },
abstract = {An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.},
}
@article {pmid36821893,
year = {2023},
author = {Wang, Y and Peng, Y and Li, S and Han, D and Ren, S and Qin, K and Zhou, H and Han, T and Gao, Z},
title = {The development of a fluorescence/colorimetric biosensor based on the cleavage activity of CRISPR-Cas12a for the detection of non-nucleic acid targets.},
journal = {Journal of hazardous materials},
volume = {449},
number = {},
pages = {131044},
doi = {10.1016/j.jhazmat.2023.131044},
pmid = {36821893},
issn = {1873-3336},
mesh = {Male ; Humans ; CRISPR-Cas Systems ; Colorimetry ; Environmental Monitoring ; Hydrogels ; *Nucleic Acids ; Oligonucleotides ; DNA ; *Biosensing Techniques ; },
abstract = {Nano-biosensors are of great significance for the analysis and detection of important biological targets. Surprisingly, the CRISPR-Cas12a system not only provides us with excellent gene editing capabilities, it also plays an important role in biosensing due to its high base resolution and high levels of sensitivity. However, most CRISPR-Cas12a-based sensors are limited by their recognition and output modes, are therefore only utilized for the detection of nucleic acids using fluorescence as an output signal. In the present study, we further explored the potential application of CRISPR-Cas12a and developed a CRISPR-Cas12a-based fluorescence/colorimetric biosensor (UCNPs-Cas12a/hydrogel-MOF-Cas12a) that provides an efficient targeting system for small molecules and protein targets. These two sensors yield multiple types of signal outputs by converting the target molecule into a deoxyribonucleic acid (DNA) signal input system using aptamers, amplifying the DNA signal by catalyzed hairpin assembly (CHA), and then combining CRISPR-Cas12a with various nanomaterials. UCNPs-Cas12a/hydrogel-MOF-Cas12a exhibited prominent sensitivity and stability for the detection of estradiol (E2) and prostate-specific antigen (PSA), and was successfully applied for the detection of these targets in milk and serum samples. A major advantage of the hydrogel-MOF-Cas12a system is that the signal output can be observed directly. When combined with aptamers and nanomaterials, CRISPR-Cas12a can be used to target multiple targets, with a diverse array of signal outputs. Our findings create a foundation for the development of CRISPR-Cas12a-based technologies for application in the fields of food safety, environmental monitoring, and clinical diagnosis.},
}
@article {pmid36725926,
year = {2023},
author = {Eccleston, A},
title = {CRISPR base editing protects the heart.},
journal = {Nature reviews. Drug discovery},
volume = {22},
number = {3},
pages = {179},
pmid = {36725926},
issn = {1474-1784},
mesh = {Humans ; *Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
}
@article {pmid36696824,
year = {2023},
author = {Wang, W and Zhou, S and Cheng, Z and Ma, D and Liu, T},
title = {A glutathione-sensitive cationic polymer delivery of CRISPR-Cas9 RNA plasmid for targeting nasopharyngeal carcinoma gene therapy.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {223},
number = {},
pages = {113146},
doi = {10.1016/j.colsurfb.2023.113146},
pmid = {36696824},
issn = {1873-4367},
mesh = {Humans ; Nasopharyngeal Carcinoma/genetics/therapy ; *CRISPR-Cas Systems ; Polymers ; Genetic Therapy/methods ; Plasmids ; Glutathione ; Oligopeptides/chemistry ; RNA, Small Interfering/genetics ; Amines/chemistry ; *Nasopharyngeal Neoplasms/genetics/therapy ; Tumor Microenvironment ; },
abstract = {CRISPR-Cas9 technology has been proven to be the most straightforward and accurate tool for gene therapy, but some limitations, such as the inefficient transfection or inability to precisely target, prevent the gene therapy from achieving the desired therapeutic effect. To overcome these, a kind of glutathione-sensitive cationic vectors, hyperbranched polyamide amine (HPAA) was designed for Delivery of CRISPR-Cas9 RNA plasmid, and the cyclic RGD (Arg-Gly-Asp) was conjugated for the targeted treatment of nasopharyngeal carcinoma (NPC). Disulfide bonds in HPAA segments can specifically respond to the high glutathione concentration in the tumor microenvironment. Meanwhile, RGD could especially interact to the integrin αvβ3 receptors which are highly expressed on the surface of NPC tumor cells. The results showed that more HPAA-RGD/SGK3-gRNA complexes could be uptaken by NPC HNE-1 cells after RGD was conjugated, and then the plasmid could be accumulated in the NPC tumor as well, which may assure the satisfied NPC therapy effect in vivo. In transfection assays, this complex showed the acceptable gene transfection efficiency in vitro and the obvious tumor inhibition effect in vivo, suggested a potential application in gene therapy to NPC.},
}
@article {pmid36669556,
year = {2023},
author = {Yamada, K and Yamamoto, T and Uwasa, K and Osakabe, K and Takano, Y},
title = {The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {165},
number = {},
pages = {103777},
doi = {10.1016/j.fgb.2023.103777},
pmid = {36669556},
issn = {1096-0937},
mesh = {*CRISPR-Cas Systems ; Quinic Acid ; Integrases/genetics/metabolism ; *Colletotrichum/genetics ; Gene Editing/methods ; },
abstract = {Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.},
}
@article {pmid36868398,
year = {2023},
author = {Makarova, KS and I Wolf, Y and V Koonin, E},
title = {In silico approaches for prediction of anti-CRISPR proteins.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168036},
doi = {10.1016/j.jmb.2023.168036},
pmid = {36868398},
issn = {1089-8638},
abstract = {Numerous viruses infecting bacteria and archaea encode CRISPR-Cas system inhibitors, known as anti-CRISPR proteins (Acr). The Acrs typically are highly specific for particular CRISPR variants, resulting in remarkable sequence and structural diversity and complicating accurate prediction and identification of Acrs. In addition to their intrinsic interest for understanding the coevolution of defense and counter-defense systems in prokaryotes, Acrs could be natural, potent on-off switches for CRISPR-based biotechnological tools, so their discovery, characterization and application are of major importance. Here we discuss the computational approaches for Acr prediction. Due to the enormous diversity and likely multiple origins of the Acrs, sequence similarity searches are of limited use. However, multiple features of protein and gene organization have been successfully harnessed to this end including small protein size and distinct amino acid compositions of the Acrs, association of acr genes in virus genomes with genes encoding helix-turn-helix proteins that regulate Acr expression (Acr-associated proteins, Aca), and presence of self-targeting CRISPR spacers in bacterial and archaeal genomes containing Acr-encoding proviruses. Productive approaches for Acr prediction also involve genome comparison of closely related viruses, of which one is resistant and the other one is sensitive to a particular CRISPR variant, and "guilt by association" whereby genes adjacent to a homolog of a known Aca are identified as candidate Acrs. The distinctive features of Acrs are employed for Acr prediction both by developing dedicated search algorithms and through machine learning. New approaches will be needed to identify novel types of Acrs that are likely to exist.},
}
@article {pmid36867551,
year = {2023},
author = {Xia, L and Yin, J and Zhuang, J and Yin, W and Zou, Z and Mu, Y},
title = {Adsorption-Free Self-Priming Direct Digital Dual-crRNA CRISPR/Cas12a-Assisted Chip for Ultrasensitive Detection of Pathogens.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.2c05560},
pmid = {36867551},
issn = {1520-6882},
abstract = {Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 10[1] to 2.58 × 10[4] cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.},
}
@article {pmid36867054,
year = {2023},
author = {Li, Q and Sun, M and Lv, L and Zuo, Y and Zhang, S and Zhang, Y and Yang, S},
title = {Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00619},
pmid = {36867054},
issn = {2161-5063},
abstract = {Due to the high specificity in targeting DNA and highly convenient programmability, CRISPR-Cas-based antimicrobials applied for eliminating specific strains such as antibiotic-resistant bacteria in the microbiome were gradually developed. However, the generation of escapers makes the elimination efficiency far lower than the acceptable rate (10[-8]) recommended by the National Institutes of Health. Here, a systematic study was carried out providing insight into the escaping mechanisms in Escherichia coli, and strategies for reducing the escapers were devised accordingly. We first showed an escape rate of 10[-5]-10[-3] in E. coli MG1655 under the editing of pEcCas/pEcgRNA established previously. Detailed analysis of the escapers obtained at ligA site in E. coli MG1655 uncovered that the disruption of cas9 was the main cause of the generation of survivors, notably the frequent insertion of IS5. Hence, the sgRNA was next designed to target the "perpetrator" IS5, and subsequently the killing efficiency was improved 4-fold. Additionally, the escape rate in IS-free E. coli MDS42 was also tested at the ligA site, ∼10-fold decrease compared with MG1655, but the disruption of cas9 was still observed in all survivors manifested in the form of frameshifts or point mutations. Thus, we optimized the tool itself by increasing the copy number of cas9 to retain some cas9 that still has the correct DNA sequence. Fortunately, the escape rates dropped below 10[-8] at 9 of the 16 tested genes. Furthermore, the λ-Red recombination system was added to generate the pEcCas-2.0, and a 100% gene deletion efficiency was achieved at genes cadA, maeB, and gntT in MG1655, whereas those genes were edited with low efficiency previously. Last, the application of pEcCas-2.0 was then expanded to the E. coli B strain BL21(DE3) and W strain ATCC9637. This study reveals the mechanism of E. coli surviving Cas9-mediated death, and a highly efficient editing tool is established based on the mechanism, which will accelerate the further application of CRISPR-Cas.},
}
@article {pmid36861321,
year = {2023},
author = {Guzmán-Benito, I and Achkar, NP and Bologna, N and Ursache, R},
title = {CRISPR/Cas-mediated inplanta gene targeting: current advances and challenges.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erad072},
pmid = {36861321},
issn = {1460-2431},
abstract = {We can use gene targeting (GT) to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant GT. Several studies have recently demonstrated improvements in GT efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified GT-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated GT in plants and discuss potential efficiency improvements. Increasing the efficiency of GT technology will help us pave the way for increased crop yields and food safety in environmentally friendly agriculture.},
}
@article {pmid36861125,
year = {2023},
author = {Hou, X and Guo, X and Zhang, Y and Zhang, Q},
title = {CRISPR/Cas genome editing system and its application in potato.},
journal = {Frontiers in genetics},
volume = {14},
number = {},
pages = {1017388},
pmid = {36861125},
issn = {1664-8021},
abstract = {Potato is the largest non-cereal food crop worldwide and a vital substitute for cereal crops, considering its high yield and great nutritive value. It plays an important role in food security. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system has the advantages of easy operation, high efficiency, and low cost, which shows a potential in potato breeding. In this paper, the action mechanism and derivative types of the CRISPR/Cas system and the application of the CRISPR/Cas system in improving the quality and resistance of potatoes, as well as overcoming the self-incompatibility of potatoes, are reviewed in detail. At the same time, the application of the CRISPR/Cas system in the future development of the potato industry was analyzed and prospected.},
}
@article {pmid36803951,
year = {2023},
author = {Jiang, H},
title = {Genome editing coming of age for hemoglobinopathy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {3},
pages = {601-602},
doi = {10.1016/j.ymthe.2023.02.007},
pmid = {36803951},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing ; *Hemoglobinopathies/genetics/therapy ; CRISPR-Cas Systems ; },
}
@article {pmid36786127,
year = {2023},
author = {Feng, M and Liu, L and Qu, Z and Zhang, B and Wang, Y and Yan, L and Kong, L},
title = {CRISPR/Cas9 knockout of MTA1 enhanced RANKL-induced osteoclastogenesis in RAW264.7 cells partly via increasing ROS activities.},
journal = {Journal of cellular and molecular medicine},
volume = {27},
number = {5},
pages = {701-713},
pmid = {36786127},
issn = {1582-4934},
mesh = {Animals ; Mice ; *Osteogenesis ; Reactive Oxygen Species/metabolism ; Cell Differentiation/genetics ; *NF-E2-Related Factor 2/metabolism ; CRISPR-Cas Systems ; Osteoclasts/metabolism ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; RANK Ligand/metabolism ; RAW 264.7 Cells ; NFATC Transcription Factors/genetics/metabolism ; },
abstract = {Metastasis-associated protein 1 (MTA1), belonging to metastasis-associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. However, the effect of MTA1 on osteoclastogenesis is unknown. Currently, the regulation of MTA1 in osteoclastogenesis was reported for the first time. MTA1 knockout cells (KO) were established by CRISPR/Cas9 genome editing. RAW264.7 cells with WT and KO group were stimulated independently by RANKL to differentiate into mature osteoclasts. Further, western blotting and quantitative qRT-PCR were used to explore the effect of MTA1 on the expression of osteoclast-associated genes (including CTSK, MMP9, c-Fos and NFATc1) during osteoclastogenesis. Moreover, the effects of MTA1 on the expression of reactive oxygen species (ROS) in osteoclastogenesis was determined by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Nuclear translocation of Nrf2 was assessed by immunofluorescence staining and western blotting. Our results indicated that the MTA1 deletion group could differentiate into osteoclasts with larger volume and more TRAP positive. In addition, compared with WT group, KO group cells generated more actin rings. Mechanistically, the loss of MTA1 increased the expression of osteoclast-specific markers, including c-Fos, NFATc1, CTSK and MMP-9. Furthermore, the results of qRT-PCR and western blotting showed that MTA1 deficiency reduced basal Nrf2 expression and inhibited Nrf2-mediated expression of related antioxidant enzymes. Immunofluorescence staining demonstrated that MTA1 deficiency inhibited Nrf2 nuclear translocation. Taken together, the above increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclast formation.},
}
@article {pmid36782027,
year = {2023},
author = {Williams, MC and Reker, AE and Margolis, SR and Liao, J and Wiedmann, M and Rojas, ER and Meeske, AJ},
title = {Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy.},
journal = {Nature microbiology},
volume = {8},
number = {3},
pages = {400-409},
pmid = {36782027},
issn = {2058-5276},
mesh = {*Bacteriophages/genetics/metabolism ; DNA Restriction Enzymes/genetics/metabolism ; CRISPR-Cas Systems ; Bacteria/genetics ; DNA Restriction-Modification Enzymes/genetics ; RNA, Viral/genetics ; DNA ; },
abstract = {Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.},
}
@article {pmid36720917,
year = {2023},
author = {Kweon, J and Jang, AH and Kwon, E and Kim, U and Shin, HR and See, J and Jang, G and Lee, C and Koo, T and Kim, S and Kim, Y},
title = {Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery.},
journal = {Experimental &amp; molecular medicine},
volume = {55},
number = {2},
pages = {377-384},
pmid = {36720917},
issn = {2092-6413},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Campylobacter jejuni/genetics/metabolism ; HEK293 Cells ; RNA, Guide, Kinetoplastida ; },
abstract = {Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.},
}
@article {pmid36617193,
year = {2023},
author = {Simpson, BP and Yrigollen, CM and Izda, A and Davidson, BL},
title = {Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {3},
pages = {760-773},
doi = {10.1016/j.ymthe.2023.01.004},
pmid = {36617193},
issn = {1525-0024},
mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Mice, Transgenic ; Genome ; Brain ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.},
}
@article {pmid36584417,
year = {2023},
author = {Lee, BR and Yang, H and Byun, SJ and Park, TS},
title = {Research Note: Development of a chicken experimental model platform for induced pluripotent stem cells by using CRISPR/Cas9-mediated NANOG knock-in reporter DF1 cells.},
journal = {Poultry science},
volume = {102},
number = {3},
pages = {102425},
pmid = {36584417},
issn = {1525-3171},
mesh = {Animals ; *Induced Pluripotent Stem Cells/metabolism ; Chickens/genetics/metabolism ; CRISPR-Cas Systems ; Biological Specimen Banks ; Transcription Factors/genetics ; },
abstract = {NANOG, as a transcription factor, plays a key role in maintaining pluripotency in higher vertebrates. Thus, NANOG gene expression is a critical index for the transition from somatic cells to the pluripotent stage. Here, we established chicken knock-in DF1 cells in which the red fluorescent protein (RFP) gene was specifically inserted into the transcriptional start site of the NANOG gene through the CRISPR‒Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9) technical platform. Subsequently, 4 transcription factors (Pou5f3, Sox2, Nanog, and Lin28A) were introduced into the NANOG-RFP DF1 cells, and finally, the induced pluripotent cells were established and examined by endogenous NANOG promoter-controlled RFP gene expression. The development of induced pluripotent stem cells (iPSCs) in avians would be useful for practical applications in the field of avian biotechnology, including biobanking genetic materials and restoring endangered species. In this study, a reporter cell line system was established to efficiently identify the induced pluripotent stage, and it will facilitate potential use for various purposes in the field of avian experimental models.},
}
@article {pmid36457249,
year = {2023},
author = {Yin, S and Zhang, M and Liu, Y and Sun, X and Guan, Y and Chen, X and Yang, L and Huo, Y and Yang, J and Zhang, X and Han, H and Zhang, J and Xiao, MM and Liu, M and Hu, J and Wang, L and Li, D},
title = {Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {3},
pages = {744-759},
doi = {10.1016/j.ymthe.2022.11.014},
pmid = {36457249},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing ; Proprotein Convertase 9/genetics/metabolism ; gamma-Globins/genetics ; Genetic Therapy ; },
abstract = {Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of β-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.},
}
@article {pmid36859639,
year = {2023},
author = {Zeng, J and Zeng, XX},
title = {Systems Medicine for Precise Targeting of Glioblastoma Systems Medicine Against Glioblastoma.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {1-20},
pmid = {36859639},
issn = {1559-0305},
abstract = {Glioblastoma (GBM) is a malignant cancer that is fatal even after standard therapy and the effects of current available therapeutics are not promising due its complex and evolving epigenetic and genetic profile. The mysteries that lead to GBM intratumoral heterogeneity and subtype transitions are not entirely clear. Systems medicine is an approach to view the patient in a whole picture integrating systems biology and synthetic biology along with computational techniques. Since the GBM oncogenesis involves genetic mutations, various therapies including gene therapeutics based on CRISPR-Cas technique, MicroRNAs, and implanted synthetic cells endowed with synthetic circuits against GBM with neural stem cells and mesenchymal stem cells acting as potential vehicles carrying therapeutics via the intranasal route, avoiding the risks of invasive methods in order to reach the GBM cells in the brain are discussed and proposed in this review. Systems medicine approach is a rather novel strategy, and since the GBM of a patient is complex and unique, thus to devise an individualized treatment strategy to tailor personalized multimodal treatments for the individual patient taking into account the phenotype of the GBM, the unique body health profile of the patient and individual responses according to the systems medicine concept might show potential to achieve optimum effects.},
}
@article {pmid36858582,
year = {2023},
author = {Inoue, S and Nishimura, K and Gima, S and Nakano, M and Takata, K},
title = {CRISPR-Cas9-Edited SNCA Knockout Human Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons and Their Vulnerability to Neurotoxicity.},
journal = {Biological &amp; pharmaceutical bulletin},
volume = {46},
number = {3},
pages = {517-522},
doi = {10.1248/bpb.b22-00839},
pmid = {36858582},
issn = {1347-5215},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; alpha-Synuclein ; CRISPR-Cas Systems ; Dopaminergic Neurons ; *Parkinson Disease ; *Neurotoxicity Syndromes ; Dopamine ; Gene Expression ; },
abstract = {Parkinson's disease (PD) is an age-related disorder with selective dopaminergic (DA) neuronal degeneration in the substantia nigra pars compacta. The presence of mainly α-synuclein-composed Lewy bodies in DA neurons is among the disease hallmarks in the brain of patients with PD. Human induced pluripotent stem cells (hiPSCs) are powerful tools to investigate PD pathophysiology and understand its molecular and cellular mechanisms better. In this study, we generated an α-synuclein-null hiPSC line introducing a nonsense mutation in the α-synuclein-encoding SNCA alleles using clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9)-mediated gene editing. Our Western blotting analysis revealed the lack of α-synuclein protein expression in SNCA knockout hiPSC-derived cells. In addition, SNCA knockout hiPSCs retained healthy cell morphology, undifferentiated marker gene (e.g., NANOG, POU5F1, and SOX2) expression, and differentiation ability (based on the marker gene expression levels of the three germ layers). Finally, SNCA knockout hiPSC-derived DA neurons exhibited reduced vulnerability to the DA neurotoxin, 1-methyl-4-phenylpyridinium. In conclusion, the SNCA knockout hiPSC line we generated would provide a useful experimental tool for studying the physiological and pathological role of α-synuclein in PD.},
}
@article {pmid36856111,
year = {2023},
author = {Zheng, C and Wei, Y and Zhang, P and Xu, L and Zhang, Z and Lin, K and Hou, J and Lv, X and Ding, Y and Chiu, Y and Jain, A and Islam, N and Malovannaya, A and Wu, Y and Ding, F and Xu, H and Sun, M and Chen, X and Chen, Y},
title = {CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer.},
journal = {The Journal of clinical investigation},
volume = {133},
number = {5},
pages = {},
pmid = {36856111},
issn = {1558-8238},
mesh = {Humans ; Female ; *RNA, Long Noncoding/genetics ; Open Reading Frames ; CRISPR-Cas Systems ; *Breast Neoplasms/genetics ; Estrogens ; },
abstract = {Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α-positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the "hidden" lncRNA-encoded proteome might be a new space for therapeutic target discovery.},
}
@article {pmid36854897,
year = {2023},
author = {Ahmadi, SE and Soleymani, M and Shahriyary, F and Amirzargar, MR and Ofoghi, M and Fattahi, MD and Safa, M},
title = {Viral vectors and extracellular vesicles: innate delivery systems utilized in CRISPR/Cas-mediated cancer therapy.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {1-19},
pmid = {36854897},
issn = {1476-5500},
abstract = {Gene editing-based therapeutic strategies grant the power to override cell machinery and alter faulty genes contributing to disease development like cancer. Nowadays, the principal tool for gene editing is the clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR/Cas9) system. In order to bring this gene-editing system from the bench to the bedside, a significant hurdle remains, and that is the delivery of CRISPR/Cas to various target cells in vivo and in vitro. The CRISPR-Cas system can be delivered into mammalian cells using various strategies; among all, we have reviewed recent research around two natural gene delivery systems that have been proven to be compatible with human cells. Herein, we have discussed the advantages and limitations of viral vectors, and extracellular vesicles (EVs) in delivering the CRISPR/Cas system for cancer therapy purposes.},
}
@article {pmid36727483,
year = {2023},
author = {Dede, M and Hart, T},
title = {Recovering false negatives in CRISPR fitness screens with JLOE.},
journal = {Nucleic acids research},
volume = {51},
number = {4},
pages = {1637-1651},
pmid = {36727483},
issn = {1362-4962},
support = {R35GM130119/GM/NIGMS NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line ; Gene Library ; Genes, Essential ; *Neoplasms/genetics ; CRISPR-Cas Systems ; },
abstract = {It is widely accepted that pooled library CRISPR knockout screens offer greater sensitivity and specificity than prior technologies in detecting genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the assumption that CRISPR screens are saturating has been largely untested. Through integrated analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we show that a typical CRISPR screen has a ∼20% false negative rate, in addition to library-specific false negatives. Replicability falls sharply as gene expression decreases, while cancer subtype-specific genes within a tissue show distinct profiles compared to false negatives. Cumulative analyses across tissues improves our understanding of core essential genes and suggest only a small number of lineage-specific essential genes, enriched for transcription factors that define pathways of tissue differentiation. To recover false negatives, we introduce a method, Joint Log Odds of Essentiality (JLOE), which builds on our prior work with BAGEL to selectively rescue the false negatives without an increased false discovery rate.},
}
@article {pmid36611237,
year = {2023},
author = {Yang, H and Eremeeva, E and Abramov, M and Jacquemyn, M and Groaz, E and Daelemans, D and Herdewijn, P},
title = {CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.},
journal = {Nucleic acids research},
volume = {51},
number = {4},
pages = {1501-1511},
pmid = {36611237},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Nucleic Acids ; RNA, Guide, Kinetoplastida ; DNA ; },
abstract = {An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.},
}
@article {pmid36480796,
year = {2023},
author = {Arif, T and Farooq, A and Ahmad, FJ and Akhtar, M and Choudhery, MS},
title = {Prime editing: A potential treatment option for β-thalassemia.},
journal = {Cell biology international},
volume = {47},
number = {4},
pages = {699-713},
doi = {10.1002/cbin.11972},
pmid = {36480796},
issn = {1095-8355},
mesh = {Humans ; *CRISPR-Cas Systems ; *beta-Thalassemia/genetics ; Gene Editing ; Genome ; },
abstract = {The potential to therapeutically alter the genome is one of the remarkable scientific developments in recent years. Genome editing technologies have provided an opportunity to precisely alter genomic sequence(s) in eukaryotic cells as a treatment option for various genetic disorders. These technologies allow the correction of harmful mutations in patients by precise nucleotide editing. Genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeat) and base editors have greatly contributed to the practical applications of gene editing. However, these technologies have certain limitations, including imperfect editing, undesirable mutations, off-target effects, and lack of potential to simultaneously edit multiple loci. Recently, prime editing (PE) has emerged as a new gene editing technology with the potential to overcome the above-mentioned limitations. Interestingly, PE not only has higher specificity but also does not require double-strand breaks. In addition, a minimum possibility of potential off-target mutant sites makes PE a preferred choice for therapeutic gene editing. Furthermore, PE has the potential to introduce insertion and deletions of all 12 single-base mutations at target sequences. Considering its potential, PE has been applied as a treatment option for genetic diseases including hemoglobinopathies. β-Thalassemia, for example, one of the most significant blood disorders characterized by reduced levels of functional hemoglobin, could potentially be treated using PE. Therapeutic reactivation of the γ-globin gene in adult β-thalassemia patients through PE technology is considered a promising therapeutic strategy. The current review aims to briefly discuss the genome editing strategies and potential applications of PE for the treatment of β-thalassemia. In addition, the review will also focus on challenges associated with the use of PE.},
}
@article {pmid36854069,
year = {2023},
author = {Ahumada-Ayala, M and Aguilar-López, R and González-Stoylov, N and Palacio-Sosa, E and Cervantes-Barragán, DE and Fernández-Hernández, L},
title = {Editing the Human Genome with CRISPR/Cas: A Review of its Molecular Basis, Current Clinical Applications, and Bioethical Implications.},
journal = {Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion},
volume = {75},
number = {1},
pages = {13-28},
doi = {10.24875/RIC.22000252},
pmid = {36854069},
issn = {0034-8376},
mesh = {Humans ; *Genome, Human ; *CRISPR-Cas Systems ; Gene Editing ; Molecular Biology ; },
abstract = {CRISPR/Cas genes evolved in prokaryotic organisms as a mechanism of defense designed to identify and destroy genetic material from threatening viruses. A breakthrough discovery is that CRISPR/Cas system can be used in eukaryotic cells to edit almost any desired gene. This comprehensive review addresses the most relevant work in the CRISPR/Cas field, including its history, molecular biology, gene editing capability, ongoing clinical trials, and bioethics. Although the science involved is complex, we intended to describe it in a concise manner that could be of interest to diverse readers, including anyone dedicated to the treatment of patients who could potentially benefit from gene editing, molecular biologists, and bioethicists. CRISPR/Cas has the potential to correct inherited diseases caused by single point mutations, to knock-in the promoter of a gene whose expression is highly desirable or knockout the gene coding for a deleterious protein. CRISPR/Cas technique can also be used to edit ex vivo immune cells and reinsert them in patients, improving their efficiency in attacking malignant cells, limiting the infectious potential of viruses or modulating xenotransplant rejection. Very important bioethical considerations on this topic include the need to internationally regulate its use by ad hoc expert committees and to limit its use until safety and bioethical issues are satisfactorily resolved.},
}
@article {pmid36853759,
year = {2023},
author = {Wang, Z and Wei, L and Ruan, S and Chen, Y},
title = {CRISPR/Cas12a-Assisted Chemiluminescence Sensor for Aflatoxin B1 Detection in Cereal Based on Functional Nucleic Acid and In-Pipet Rolling Circle Amplification.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c00341},
pmid = {36853759},
issn = {1520-5118},
abstract = {Herein, we report a CRISPR/Cas12a-assisted chemiluminescence sensor for aflatoxin B1 (AFB1) detection based on functional nucleic-acid-mediated target recognition and in-pipet rolling circle amplification-mediated signal amplification. In this sensor, we performed rolling circle amplification on the inside of the pipet to enrich horseradish peroxidase (pipet-poly-HRP). When AFB1 is present, it interacts with functional nucleic acids and results in the release of the activator. The activator is designed to activate the CRISPR/Cas12a system, which cleaves the pipet-poly-HRP to liberate HRP. The freed HRP can then be measured by chemiluminescence to quantify AFB1. This CRISPR/Cas12a-assisted chemiluminescence sensor enables facile, highly sensitive, and specific detection of AFB1, with a linear range from 50 pg/mL to 100 ng/mL and a detection limit of 5.2 pg/mL. Furthermore, it exhibits satisfactory recovery and has successfully challenged AFB1 detection in cereal samples. The proposed sensor offers a novel rapid screening approach that holds great promise for food security monitoring.},
}
@article {pmid36853698,
year = {2023},
author = {Uranga, M and Aragonés, V and Daròs, JA and Pasin, F},
title = {Heritable CRISPR-Cas9 editing of plant genomes using RNA virus vectors.},
journal = {STAR protocols},
volume = {4},
number = {1},
pages = {102091},
pmid = {36853698},
issn = {2666-1667},
abstract = {Viral vectors hold enormous potential for genome editing in plants as transient delivery vehicles of CRISPR-Cas components. Here, we describe a protocol to assemble plant viral vectors for single-guide RNA (sgRNA) delivery. The obtained viral constructs are based on compact T-DNA binary vectors of the pLX series and are delivered into Cas9-expressing plants through agroinoculation. This approach allows rapidly assessing sgRNA design for plant genome targeting, as well as the recovery of progeny with heritable mutations at targeted loci. For complete details on the use and execution of this protocol, please refer to Uranga et al. (2021)[1] and Aragonés et al. (2022).[2].},
}
@article {pmid36853461,
year = {2023},
author = {Abu-Daya, A and Godwin, A},
title = {CRISPR/Cas9 Gene Disruption Studies in F0 Xenopus Tadpoles: Understanding Development and Disease in the Frog.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2633},
number = {},
pages = {111-130},
pmid = {36853461},
issn = {1940-6029},
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; Xenopus laevis ; *Anura ; CRISPR-Associated Protein 9/genetics ; DNA End-Joining Repair ; },
abstract = {CRISPR/Cas9 has become the favorite method for gene knockouts in a range of vertebrate model organisms due to its ease of use and versatility. Gene-specific guide RNAs can be designed to a unique genomic sequence and used to target the Cas9 endonuclease, which causes a double-stranded break at the desired locus. Repair of the breaks through non-homologous end joining often results in the deletion or insertion of several nucleotides, which frequently result in nonsense mutations. Xenopus frogs have long been an excellent model organism in which to study gene function, and they have proven to be useful in gene-editing experiments, especially the diploid species, X. tropicalis. In this chapter, we present our protocols for gene disruption in Xenopus, which we regularly use to investigate developmental processes and model human genetic disease.},
}
@article {pmid36797405,
year = {2023},
author = {Yoon, PH and Adler, BA and Doudna, JA},
title = {To TnpB or not TnpB? Cas12 is the answer.},
journal = {Nature chemical biology},
volume = {19},
number = {3},
pages = {263-264},
pmid = {36797405},
issn = {1552-4469},
mesh = {*DNA Transposable Elements ; *Bacterial Proteins/metabolism ; CRISPR-Cas Systems ; },
}
@article {pmid36625577,
year = {2023},
author = {Shelake, RM and Pramanik, D and Kim, JY},
title = {Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli.},
journal = {mBio},
volume = {14},
number = {1},
pages = {e0229622},
pmid = {36625577},
issn = {2150-7511},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; Adenine ; Cytosine ; Gene Editing/methods ; },
abstract = {Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.},
}
@article {pmid36539178,
year = {2023},
author = {Shirk, BD and Shirk, PD and Furlong, RB and Scully, ED and Wu, K and Siegfried, BD},
title = {Gene editing of the ABC Transporter/White locus using CRISPR/Cas9-mediated mutagenesis in the Indian Meal Moth.},
journal = {Journal of insect physiology},
volume = {145},
number = {},
pages = {104471},
doi = {10.1016/j.jinsphys.2022.104471},
pmid = {36539178},
issn = {1879-1611},
mesh = {Male ; Female ; Animals ; *Gene Editing ; CRISPR-Cas Systems ; ATP-Binding Cassette Transporters/genetics ; Ovum/metabolism ; Mutagenesis ; *Moths/genetics/metabolism ; },
abstract = {ATP binding cassette (ABC) proteins are involved in transport of substrates across membranes including eye pigments. Mutations of ABC transporter white, brown and scarlet genes of Drosophila and other insects result in visible eye color phenotypes. White locus was identified in a genome assembly of Plodia interpunctella and was found to extend for 16,670 bp comprising 13 exons. We report here recovery of heritable mutants in white in the Indian meal moth, P. interpunctella, using CRISPR/Cas9-mediated mutagenesis. A white eye strain of P. interpunctella c.737delC (Piw[-/-]) was previously isolated in 1986. Guide RNA (sgRNA) was designed for exon 1 (sgRNA242). Microinjection of Cas9/sgRNA242 complex into Plodia wild type eggs (≤20 min post oviposition) produced 156 viable larvae of which 81 eclosed as adults. Forty-five (56 %) adults displayed wild type phenotype, while 26 females (32 %) and 10 males (12 %) showed full or partial white eye phenotype. The 26 white eye females were mated with Piw[-/-] males and 21 matings resulted in F1 white eye progeny. Thirteen of the Piw[-242] lines were established and sequencing showed indels at the CRISPR/Cas9 242AM site. Based on RT-PCR analysis, most white mutations resulted in suppressed levels of transcript. These results demonstrate the utility of CRISPR/Cas9 gene editing in Plodia which suggests this technology can be used to characterize the role of various genetic elements including those that encode novel targets or confer insecticide resistance mechanisms.},
}
@article {pmid36251123,
year = {2023},
author = {Sen, D and Sarkar, S and Mukhopadhyay, P},
title = {Prime Editing: An Emerging Tool in Cancer Treatment.},
journal = {Molecular biotechnology},
volume = {65},
number = {4},
pages = {509-520},
pmid = {36251123},
issn = {1559-0305},
mesh = {Humans ; *Gene Editing/methods ; Mutation ; CRISPR-Cas Systems ; *Neoplasms/genetics/therapy ; },
abstract = {Prime Editing is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technique having promising potential in terms of reducing off target activity. It introduces fragments of DNA sequences into the target site using a guide RNA (gRNA) molecule, composed of both the sequence that is to be inserted into the target site along with an inactive Cas9 nickase and a reverse transcriptase. Prime Editing can cause insertions, deletions, and various point mutations for reverting the phenetic characteristics of a disease specially tested in human adult stem cells and cancer cell lines. The main aim of our review is to explore how Prime Editing and its various forms are being utilized as an emerging tool to cure deleterious diseases like cancer, also as a delivery strategy of the tool into cells. There are almost five generations of Prime Editors (PE) with increasing levels of efficiency from one level to another that have huge clinical potential in correcting mutations; however, the necessity for a pegRNA design is extremely significant. But besides having such advantages, the limitations of this technology particularly include generation of double nicks while optimizing the efficiency of PE3. So, it is important to consider all such consequences and customize PE as per requirements.},
}
@article {pmid36853462,
year = {2023},
author = {Martin, SA},
title = {A CRISPR/Cas-Based Method for Precise DNA Integration in Xenopus laevis Oocytes Followed by Intracytoplasmic Sperm Injection (ICSI) Fertilization.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2633},
number = {},
pages = {131-143},
pmid = {36853462},
issn = {1940-6029},
abstract = {Xenopus has long had a reputation for being a powerful model organism for use in developmental cell and biochemistry research. With the advent of gene-editing technologies, and the full genome sequencing of Xenopus genomes revealing the extent of the genetic conservation between Xenopus and humans, Xenopus has the potential to become an ideal model for human genetic disease. However, the inability to produce non-mosaic, precise DNA insertions through homology directed repair has limited the strength of Xenopus this field. Furthermore, it has prevented researchers from taking full advantage of fusion tagging, a method for directly tagging genes with either epitope or fluorescent tags, allowing the visualization, quantification, and tracking of proteins without the use of protein-specific antibodies. Here, we describe a method for precise DNA insertion into oocytes using CRISPR/Cas9, followed by in vitro maturation and fertilization by intracytoplasmic sperm injection (ICSI), culminating in the production of embryos carrying a non-mosaic, heterozygous insertion.},
}
@article {pmid36851743,
year = {2023},
author = {Mahmood, MA and Naqvi, RZ and Rahman, SU and Amin, I and Mansoor, S},
title = {Plant Virus-Derived Vectors for Plant Genome Engineering.},
journal = {Viruses},
volume = {15},
number = {2},
pages = {},
pmid = {36851743},
issn = {1999-4915},
mesh = {*Genome, Plant ; Agrobacterium ; Climate Change ; Crops, Agricultural/genetics ; *Plant Viruses/genetics ; },
abstract = {Advances in genome engineering (GE) tools based on sequence-specific programmable nucleases have revolutionized precise genome editing in plants. However, only the traditional approaches are used to deliver these GE reagents, which mostly rely on Agrobacterium-mediated transformation or particle bombardment. These techniques have been successfully used for the past decades for the genetic engineering of plants with some limitations relating to lengthy time-taking protocols and transgenes integration-related regulatory concerns. Nevertheless, in the era of climate change, we require certain faster protocols for developing climate-smart resilient crops through GE to deal with global food security. Therefore, some alternative approaches are needed to robustly deliver the GE reagents. In this case, the plant viral vectors could be an excellent option for the delivery of GE reagents because they are efficient, effective, and precise. Additionally, these are autonomously replicating and considered as natural specialists for transient delivery. In the present review, we have discussed the potential use of these plant viral vectors for the efficient delivery of GE reagents. We have further described the different plant viral vectors, such as DNA and RNA viruses, which have been used as efficient gene targeting systems in model plants, and in other important crops including potato, tomato, wheat, and rice. The achievements gained so far in the use of viral vectors as a carrier for GE reagent delivery are depicted along with the benefits and limitations of each viral vector. Moreover, recent advances have been explored in employing viral vectors for GE and adapting this technology for future research.},
}
@article {pmid36851656,
year = {2023},
author = {Evseev, P and Tikhonova, I and Krasnopeev, A and Sorokovikova, E and Gladkikh, A and Timoshkin, O and Miroshnikov, K and Belykh, O},
title = {Tychonema sp. BBK16 Characterisation: Lifestyle, Phylogeny and Related Phages.},
journal = {Viruses},
volume = {15},
number = {2},
pages = {},
pmid = {36851656},
issn = {1999-4915},
abstract = {Cyanobacterial expansion is harmful to the environment, the ecology of Lake Baikal and the economy of nearby regions and can be dangerous to people and animals. Since 2011, the process of colonisation of the lake with potentially toxic cyanobacteria belonging to the genus Tychonema has continued. An understanding of the mechanism of successful expansion of Tychonema requires scrutiny of biological and genomic features. Tychonema sp. BBK16 was isolated from the coastal zone of Lake Baikal. The morphology of BBK16 biofilm was studied with light, scanning electron and confocal microscopy. The biofilm is based on filaments of cyanobacteria, which are intertwined like felt; there are also dense fascicles of rope-like twisted filaments that impart heterogeneity to the surface of the biofilm. Genome sequencing, intergenomic comparisons and phylogenetic analyses indicated that Tychonema sp. BBK16 represent a new species related to planktic cyanobacterium Tychonema bourrellyi, isolated from Alpine lentic freshwater. Genome investigation revealed the genes possibly responsible for the mixotrophic lifestyle. The presence of CRISPR-Cas and restriction modification defence mechanisms allowed to suggest the existence of phages infecting Tychonema sp. BBK16. Analysis of CRISPR spacers and prophage-derived regions allowed to suggest related cyanophages. Genomic analysis supported the assumption that mobile elements and horizontal transfer participate in shaping the Tychonema sp. BBK16 genome. The findings of the current research suggest that the aptitude of Tychonema sp. BBK16 for biofilm formation and, possibly, its mixotrophic lifestyle provide adaptation advantages that lead to the successful expansion of this cyanobacterium in the Baikal's conditions of freshwater lake environments.},
}
@article {pmid36849628,
year = {2023},
author = {Goh, YX and Wang, M and Hou, XP and He, Y and Ou, HY},
title = {Analysis of CRISPR-Cas Loci and their Targets in Levilactobacillus brevis.},
journal = {Interdisciplinary sciences, computational life sciences},
volume = {},
number = {},
pages = {},
pmid = {36849628},
issn = {1867-1462},
abstract = {The CRISPR‒Cas system acts as a bacterial defense mechanism by conferring adaptive immunity and limiting genetic reshuffling. However, under adverse environmental hazards, bacteria can employ their CRISPR‒Cas system to exchange genes that are vital for adaptation and survival. Levilactobacillus brevis is a lactic acid bacterium with great potential for commercial purposes because it can be genetically manipulated to enhance its functionality and nutritional value. Nevertheless, the CRISPR‒Cas system might interfere with the genetic modification process. Additionally, little is known about the CRISPR‒Cas system in this industrially important microorganism. Here, we investigate the prevalence, diversity, and targets of CRISPR‒Cas systems in the genus Levilactobacillus, further focusing on complete genomes of L. brevis. Using the CRISPRCasFinder webserver, we identified 801 putative CRISPR-Cas systems in the genus Levilactobacillus. Further investigation focusing on the complete genomes of L. brevis revealed 54 putative CRISPR-Cas systems. Of these, 46 were orphan CRISPRs, and eight were CRISPR‒Cas systems. The type II-A CRISPR‒Cas system is the most common in Levilactobacillus and L. brevis complete genomes. Analysis of the spacer's target showed that the CRISPR‒Cas systems of L. brevis mainly target the enterococcal plasmids. Comparative analysis of putative CRISPR-Cas loci in Levilactobacillus brevis.},
}
@article {pmid36847375,
year = {2023},
author = {Hartweger, H and Gautam, R and Nishimura, Y and Schmidt, F and Yao, KH and Escolano, A and Jankovic, M and Martin, MA and Nussenzweig, MC},
title = {Gene Editing of Primary Rhesus Macaque B Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {192},
pages = {},
doi = {10.3791/64858},
pmid = {36847375},
issn = {1940-087X},
mesh = {Animals ; Humans ; *Gene Editing/methods ; Macaca mulatta/genetics ; *Leukocytes, Mononuclear ; Prospective Studies ; B-Lymphocytes ; CRISPR-Cas Systems ; },
abstract = {B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.},
}
@article {pmid36847371,
year = {2023},
author = {Voigt, B and Minowa, R and Gray, RS},
title = {Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {192},
pages = {},
doi = {10.3791/64686},
pmid = {36847371},
issn = {1940-087X},
mesh = {Animals ; Male ; *Zebrafish/genetics/metabolism ; *Semen/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems ; Endonucleases/genetics ; Germ Cells/metabolism ; Spermatozoa/metabolism ; },
abstract = {The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.},
}
@article {pmid36847161,
year = {2023},
author = {Richardson, C and Kelsh, RN and J Richardson, R},
title = {New advances in CRISPR/Cas-mediated precise gene-editing techniques.},
journal = {Disease models &amp; mechanisms},
volume = {16},
number = {2},
pages = {},
doi = {10.1242/dmm.049874},
pmid = {36847161},
issn = {1754-8411},
support = {BB/T008741/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Zebrafish/genetics ; CRISPR-Associated Protein 9/genetics ; Mutation/genetics ; Mammals ; },
abstract = {Over the past decade, CRISPR/Cas-based gene editing has become a powerful tool for generating mutations in a variety of model organisms, from Escherichia coli to zebrafish, rodents and large mammals. CRISPR/Cas-based gene editing effectively generates insertions or deletions (indels), which allow for rapid gene disruption. However, a large proportion of human genetic diseases are caused by single-base-pair substitutions, which result in more subtle alterations to protein function, and which require more complex and precise editing to recreate in model systems. Precise genome editing (PGE) methods, however, typically have efficiencies of less than a tenth of those that generate less-specific indels, and so there has been a great deal of effort to improve PGE efficiency. Such optimisations include optimal guide RNA and mutation-bearing donor DNA template design, modulation of DNA repair pathways that underpin how edits result from Cas-induced cuts, and the development of Cas9 fusion proteins that introduce edits via alternative mechanisms. In this Review, we provide an overview of the recent progress in optimising PGE methods and their potential for generating models of human genetic disease.},
}
@article {pmid36847080,
year = {2023},
author = {Yin, W and Chen, Z and Huang, J and Ye, H and Lu, T and Lu, M and Rao, Y},
title = {[Application of CRISPR-Cas9 gene editing technology in crop breeding].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {2},
pages = {399-424},
doi = {10.13345/j.cjb.220664},
pmid = {36847080},
issn = {1872-2075},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Technology ; },
abstract = {The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.},
}
@article {pmid36846807,
year = {2023},
author = {Wang, S and Wang, S and Tang, Y and Peng, G and Hao, T and Wu, X and Wei, J and Qiu, X and Zhou, D and Zhu, S and Li, Y and Wu, S},
title = {Detection of Klebsiella pneumonia DNA and ESBL positive strains by PCR-based CRISPR-LbCas12a system.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1128261},
pmid = {36846807},
issn = {1664-302X},
abstract = {INTRODUCTION: Klebsiella pneumonia (K. pneumonia) is a Gram-negative bacterium that opportunistically causes nosocomial infections in the lung, bloodstream, and urinary tract. Extended-spectrum β-Lactamases (ESBLs)-expressed K. pneumonia strains are widely reported to cause antibiotic resistance and therapy failure. Therefore, early identification of K. pneumonia, especially ESBL-positive strains, is essential in preventing severe infections. However, clinical detection of K. pneumonia requires a time-consuming process in agar disk diffusion. Nucleic acid detection, like qPCR, is precise but requires expensive equipment. Recent research reveals that collateral cleavage activity of CRISPR-LbCas12a has been applied in nucleic acid detection, and the unique testing model can accommodate various testing models.<br><br>METHODS: This study established a system that combined PCR with CRISPR-LbCas12a targeting the K. pneumoniae system. Additionally, this study summarized the antibiotic-resistant information of the past five years' K. pneumoniae clinic cases in Luohu Hospital and found that the ESBL-positive strains were growing. This study then designs a crRNA that targets SHV to detect ESBL-resistant K. pneumoniae. This work is to detect K. pneumoniae and ESBL-positive strains' nucleic acid using CRISPR-Cas12 technology. We compared PCR-LbCas12 workflow with PCR and qPCR techniques.<br><br>RESULTS AND DISCUSSION: This system showed excellent detection specificity and sensitivity in both bench work and clinical samples. Due to its advantages, its application can meet different detection requirements in health centers where qPCR is not accessible. The antibiotic-resistant information is valuable for further research.},
}
@article {pmid36846759,
year = {2023},
author = {Wei, TS and Gao, ZM and Gong, L and Li, QM and Zhou, YL and Chen, HG and He, LS and Wang, Y},
title = {Genome-centric view of the microbiome in a new deep-sea glass sponge species Bathydorus sp.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1078171},
pmid = {36846759},
issn = {1664-302X},
abstract = {Sponges are widely distributed in the global ocean and harbor diverse symbiotic microbes with mutualistic relationships. However, sponge symbionts in the deep sea remain poorly studied at the genome level. Here, we report a new glass sponge species of the genus Bathydorus and provide a genome-centric view of its microbiome. We obtained 14 high-quality prokaryotic metagenome-assembled genomes (MAGs) affiliated with the phyla Nitrososphaerota, Pseudomonadota, Nitrospirota, Bdellovibrionota, SAR324, Bacteroidota, and Patescibacteria. In total, 13 of these MAGs probably represent new species, suggesting the high novelty of the deep-sea glass sponge microbiome. An ammonia-oxidizing Nitrososphaerota MAG B01, which accounted for up to 70% of the metagenome reads, dominated the sponge microbiomes. The B01 genome had a highly complex CRISPR array, which likely represents an advantageous evolution toward a symbiotic lifestyle and forceful ability to defend against phages. A sulfur-oxidizing Gammaproteobacteria species was the second most dominant symbiont, and a nitrite-oxidizing Nitrospirota species could also be detected, but with lower relative abundance. Bdellovibrio species represented by two MAGs, B11 and B12, were first reported as potential predatory symbionts in deep-sea glass sponges and have undergone dramatic genome reduction. Comprehensive functional analysis indicated that most of the sponge symbionts encoded CRISPR-Cas systems and eukaryotic-like proteins for symbiotic interactions with the host. Metabolic reconstruction further illustrated their essential roles in carbon, nitrogen, and sulfur cycles. In addition, diverse putative phages were identified from the sponge metagenomes. Our study expands the knowledge of microbial diversity, evolutionary adaption, and metabolic complementarity in deep-sea glass sponges.},
}
@article {pmid36840774,
year = {2023},
author = {Errum, A and Rehman, N and Uzair, M and Inam, S and Ali, GM and Khan, MR},
title = {CRISPR/Cas9 editing of wheat Ppd-1 gene homoeologs alters spike architecture and grain morphometric traits.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {1},
pages = {66},
pmid = {36840774},
issn = {1438-7948},
mesh = {*Triticum/genetics ; *Photoperiod ; CRISPR-Cas Systems ; Phenotype ; Edible Grain/genetics ; },
abstract = {Mutations in Photoperiod-1 (Ppd-1) gene are known to modify flowering time and yield in wheat. We cloned TaPpd-1 from wheat and found high similarity among the three homoeologs of TaPpd-1. To clarify the characteristics of TaPpd-1 homoeologs in different photoperiod conditions for inflorescence architecture and yield, we used CRISPR/Cas9 system to generate Tappd-1 mutant plants by simultaneous modification of the three homoeologs of wheat Ppd-1. Tappd-1 mutant plants showed no off-target mutations. Four T0-edited lines under short-day length and three lines under long-day length conditions with the mutation frequency of 25% and 21%, respectively. These putative transgenic plants of all the lines were self-fertilized and generated T1 and T2 progenies and were evaluated by phenotypic and expression analysis. Results demonstrated that simultaneously edited TaPpd-1- A1, B1, and D1 homoeologs gene copies in T2_SDL-8-4, T2_SDL-4-5, T2_SDL-3-9, and T2_LDL-10-9 showed similar spike inflorescence, flowering time, and significantly increase in 1000-grain weight, grain area, grain width, grain length, plant height, and spikelets per spike due to mutation in both alleles of Ppd-B1 and Ppd-D1 homoeologs but only spike length was decreased in T2_SDL-8-4, T2_SDL-4-5, and T2_LDL-13-3 mutant lines due to mutation in both alleles of Ppd-A1 homoeolog under both conditions. Our results indicate that all TaPpd1 gene homoeologs influence wheat spike development by affecting both late flowering and earlier flowering but single mutant TaPpd-A1 homoeolog affect lowest as compared to the combination with double mutants of TaPpd-B1 and TaPpd-D1, TaPpd-A1 and TaPpd-B1, and TaPpd-A1 and TaPpd-D1 homoeologs for yield enhancement. Our findings further raised the idea that the relative expression of the various genomic copies of TaPpd-1 homoeologs may have an impact on the spike inflorescence architecture and grain morphometric features in wheat cultivars.},
}
@article {pmid36835061,
year = {2023},
author = {Siles, L and Gaudó, P and Pomares, E},
title = {High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs.},
journal = {International journal of molecular sciences},
volume = {24},
number = {4},
pages = {},
pmid = {36835061},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems ; *Color Vision Defects/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Mutation ; Gene Editing/methods ; },
abstract = {Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies that currently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.},
}
@article {pmid36834878,
year = {2023},
author = {Guo, H and Chen, F and Zhou, M and Lan, W and Zhang, W and Shen, G and Lin, P and Xia, Q and Zhao, P and Li, Z},
title = {CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori.},
journal = {International journal of molecular sciences},
volume = {24},
number = {4},
pages = {},
pmid = {36834878},
issn = {1422-0067},
mesh = {Animals ; *Methyltransferases/metabolism ; *Bombyx/genetics ; CRISPR-Cas Systems ; Mutation ; Methylation ; Insect Proteins/genetics ; },
abstract = {DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.},
}
@article {pmid36834852,
year = {2023},
author = {Qi, Q and Hu, B and Jiang, W and Wang, Y and Yan, J and Ma, F and Guan, Q and Xu, J},
title = {Advances in Plant Epigenome Editing Research and Its Application in Plants.},
journal = {International journal of molecular sciences},
volume = {24},
number = {4},
pages = {},
pmid = {36834852},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Epigenome ; Genome, Plant ; Plant Breeding/methods ; Plants/genetics ; Gene Editing/methods ; },
abstract = {Plant epistatic regulation is the DNA methylation, non-coding RNA regulation, and histone modification of gene sequences without altering the genome sequence, thus regulating gene expression patterns and the growth process of plants to produce heritable changes. Epistatic regulation in plants can regulate plant responses to different environmental stresses, regulate fruit growth and development, etc. Genome editing can effectively improve plant genetic efficiency by targeting the design and efficient editing of genome-specific loci with specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9). As research progresses, the CRISPR/Cas9 system has been widely used in crop breeding, gene expression, and epistatic modification due to its high editing efficiency and rapid translation of results. In this review, we summarize the recent progress of CRISPR/Cas9 in epigenome editing and look forward to the future development direction of this system in plant epigenetic modification to provide a reference for the application of CRISPR/Cas9 in genome editing.},
}
@article {pmid36834483,
year = {2023},
author = {Zhang, Y and Li, P and Niu, Y and Zhang, Y and Wen, G and Zhao, C and Jiang, M},
title = {Evolution of the WRKY66 Gene Family and Its Mutations Generated by the CRISPR/Cas9 System Increase the Sensitivity to Salt Stress in Arabidopsis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {4},
pages = {},
pmid = {36834483},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics ; Abscisic Acid/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Plant Proteins/genetics ; Salt Tolerance/genetics ; Plants, Genetically Modified/genetics ; Salt Stress ; Transcription Factors/metabolism ; Stress, Physiological/physiology ; Antioxidants/metabolism ; Mutation ; Gene Expression Regulation, Plant ; },
abstract = {Group Ⅲ WRKY transcription factors (TFs) play pivotal roles in responding to the diverse abiotic stress and secondary metabolism of plants. However, the evolution and function of WRKY66 remains unclear. Here, WRKY66 homologs were traced back to the origin of terrestrial plants and found to have been subjected to both motifs' gain and loss, and purifying selection. A phylogenetic analysis showed that 145 WRKY66 genes could be divided into three main clades (Clade A-C). The substitution rate tests indicated that the WRKY66 lineage was significantly different from others. A sequence analysis displayed that the WRKY66 homologs had conserved WRKY and C2HC motifs with higher proportions of crucial amino acid residues in the average abundance. The AtWRKY66 is a nuclear protein, salt- and ABA- inducible transcription activator. Simultaneously, under salt stress and ABA treatments, the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, as well as the seed germination rates of Atwrky66-knockdown plants generated by the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system, were all lower than those of wild type (WT) plants, but the relative electrolyte leakage (REL) was higher, indicating the increased sensitivities of the knockdown plants to the salt stress and ABA treatments. Moreover, RNA-seq and qRT-PCR analyses revealed that several regulatory genes in the ABA-mediated signaling pathway involved in stress response of the knockdown plants were significantly regulated, being evidenced by the more moderate expressions of the genes. Therefore, the AtWRKY66 likely acts as a positive regulator in the salt stress response, which may be involved in an ABA-mediated signaling pathway.},
}
@article {pmid36833415,
year = {2023},
author = {Debbarma, J and Saikia, B and Singha, DL and Das, D and Keot, AK and Maharana, J and Velmurugan, N and Arunkumar, KP and Reddy, PS and Chikkaputtaiah, C},
title = {CRISPR/Cas9-Mediated Mutation in XSP10 and SlSAMT Genes Impart Genetic Tolerance to Fusarium Wilt Disease of Tomato (Solanum lycopersicum L.).},
journal = {Genes},
volume = {14},
number = {2},
pages = {},
pmid = {36833415},
issn = {2073-4425},
mesh = {*Solanum lycopersicum ; *Fusarium/genetics ; CRISPR-Cas Systems ; Salicylic Acid/metabolism ; Mutation ; Xylem/metabolism ; },
abstract = {Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.},
}
@article {pmid36833328,
year = {2023},
author = {Erbs, V and Lorentz, R and Eisenman, B and Schaeffer, L and Luppi, L and Lindner, L and Hérault, Y and Pavlovic, G and Wattenhofer-Donzé, M and Birling, MC},
title = {Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells.},
journal = {Genes},
volume = {14},
number = {2},
pages = {},
pmid = {36833328},
issn = {2073-4425},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; Mice, Inbred C57BL ; *Embryonic Stem Cells/metabolism ; Homologous Recombination ; Mutagenesis ; },
abstract = {The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for 'à la carte' mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5' and 3' PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes.},
}
@article {pmid36833200,
year = {2023},
author = {Mary, L and Leclerc, D and Labalme, A and Bellaud, P and Mazaud-Guittot, S and Dréano, S and Evrard, B and Bigand, A and Cauchoix, A and Loget, P and Lokchine, A and Cluzeau, L and Gilot, D and Belaud-Rotureau, MA and Jaillard, S},
title = {Functional Assessment of a New PBX1 Variant in a 46,XY Fetus with Severe Syndromic Difference of Sexual Development through CRISPR-Cas9 Gene Editing.},
journal = {Genes},
volume = {14},
number = {2},
pages = {},
pmid = {36833200},
issn = {2073-4425},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; HEK293 Cells ; Fetus ; Sexual Development ; Pre-B-Cell Leukemia Transcription Factor 1/genetics ; },
abstract = {Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1, encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.},
}
@article {pmid36831203,
year = {2023},
author = {Godbout, K and Tremblay, JP},
title = {Prime Editing for Human Gene Therapy: Where Are We Now?.},
journal = {Cells},
volume = {12},
number = {4},
pages = {},
pmid = {36831203},
issn = {2073-4409},
support = {418059/CAPMC/CIHR/Canada ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Mutation ; Genetic Therapy ; *Cystic Fibrosis/genetics ; },
abstract = {Gene therapy holds tremendous potential in the treatment of inherited diseases. Unlike traditional medicines, which only treat the symptoms, gene therapy has the potential to cure the disease by addressing the root of the problem: genetic mutations. The discovery of CRISPR/Cas9 in 2012 paved the way for the development of those therapies. Improvement of this system led to the recent development of an outstanding technology called prime editing. This system can introduce targeted insertions, deletions, and all 12 possible base-to-base conversions in the human genome. Since the first publication on prime editing in 2019, groups all around the world have worked on this promising technology to develop a treatment for genetic diseases. To date, prime editing has been attempted in preclinical studies for liver, eye, skin, muscular, and neurodegenerative hereditary diseases, in addition to cystic fibrosis, beta-thalassemia, X-linked severe combined immunodeficiency, and cancer. In this review, we portrayed where we are now on prime editing for human gene therapy and outlined the best strategies for correcting pathogenic mutations by prime editing.},
}
@article {pmid36830693,
year = {2023},
author = {Sellaththurai, S and Jung, S and Kim, MJ and Nadarajapillai, K and Ganeshalingam, S and Jeong, JB and Lee, J},
title = {CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection.},
journal = {Biomolecules},
volume = {13},
number = {2},
pages = {},
pmid = {36830693},
issn = {2218-273X},
mesh = {Animals ; *Zebrafish/metabolism ; NF-kappa B/metabolism ; CRISPR-Cas Systems ; Lipopolysaccharides ; Nucleotidyltransferases/genetics/metabolism ; *Bacterial Infections/genetics ; Immunity, Innate ; },
abstract = {Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting[(-/-)] with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting[(-/-)] embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting[(-/-)] than in healthy WT fish. The late response was observed in sting[(-/-)] larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS-STING pathway. Furthermore, adult sting[(-/-)] fish had a high mortality rate and significantly downregulated cGAS-STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.},
}
@article {pmid36734062,
year = {2023},
author = {Zhen, S and Chen, H and Lu, J and Yang, X and Tuo, X and Chang, S and Tian, Y and Li, X},
title = {Intravaginal delivery for CRISPR-Cas9 technology: For example, the treatment of HPV infection.},
journal = {Journal of medical virology},
volume = {95},
number = {2},
pages = {e28552},
doi = {10.1002/jmv.28552},
pmid = {36734062},
issn = {1096-9071},
mesh = {Humans ; Female ; CRISPR-Cas Systems ; Gene Editing/methods ; *Papillomavirus Infections/genetics ; Gene Silencing ; *Nanoparticles ; },
abstract = {The increasing incidence of sexually transmitted diseases in women, including human papillomavirus (HPV) infection, has led to the need to develop user-friendly potential prevention methods. At present, although there are several therapeutic parts, none of them has a preventive effect, but they are only limited to providing patients with symptom relief. Researchers have now recognized the need to find effective local preventive agents. One of the potential undiscovered local fungicides is the vaginal delivery of CRISPR/Cas9. CRISPR/Cas9 delivery involves silencing gene expression in a sequence-specific manner in the pathogenic agent, thus showing microbicidal activity. However, vaginal mucosal barrier and physiological changes (such as pH value and variable epithelial thickness in the menstrual cycle) are the main obstacles to effective delivery and cell uptake of CRISPR/Cas9. To enhance the vaginal delivery of CRISPR/Cas9, so far, nano-carrier systems such as lipid delivery systems, macromolecular systems, polymer nanoparticles, aptamers, and cell-penetrating peptides have been extensively studied. In this paper, various nano-carriers and their prospects in the preclinical stage are described, as well as the future significance of CRISPR/Cas9 vaginal delivery based on nano-carriers.},
}
@article {pmid36623445,
year = {2023},
author = {Zhang, N and Huang, D and Ruan, X and Ng, AT and Tsu, JH and Jiang, G and Huang, J and Zhan, Y and Na, R},
title = {CRISPR screening reveals gleason score and castration resistance related oncodriver ring finger protein 19 A (RNF19A) in prostate cancer.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {67},
number = {},
pages = {100912},
doi = {10.1016/j.drup.2022.100912},
pmid = {36623445},
issn = {1532-2084},
mesh = {Humans ; Male ; ATPases Associated with Diverse Cellular Activities/genetics/metabolism/therapeutic use ; Cell Cycle Proteins/genetics/metabolism ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Early Detection of Cancer ; High-Throughput Screening Assays ; Neoplasm Grading ; Prostatic Neoplasms/genetics ; *Prostatic Neoplasms, Castration-Resistant/drug therapy/genetics/pathology ; Proteomics ; *Ubiquitin-Protein Ligases/genetics ; Ubiquitins/genetics/metabolism/therapeutic use ; },
abstract = {Prostate cancer (PCa) is one of the most lethal causes of cancer-related death in male. It is characterized by chromosomal instability and disturbed signaling transduction. E3 ubiquitin ligases are well-recognized as mediators leading to genomic alterations and malignant phenotypes. There is a lack of systematic study on novel oncodrivers with genomic and clinical significance in PCa. In this study we used clustered regularly interspaced short palindromic repeats (CRISPR) system to screen 656 E3 ubiquitin ligases as oncodrivers or tumor repressors in PCa cells. We identified 51 significantly changed genes, and conducted genomic and clinical analysis on these genes. It was found that the Ring Finger Protein 19 A (RNF19A) was a novel oncodriver in PCa. RNF19A was frequently amplified and highly expressed in PCa and other cancer types. Clinically, higher RNF19A expression correlated with advanced Gleason Score and predicted castration resistance. Mechanistically, transcriptomics, quantitative and ubiquitination proteomic analysis showed that RNF19A ubiquitylated Thyroid Hormone Receptor Interactor 13 (TRIP13) and was transcriptionally activated by androgen receptor (AR) and Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A). This study uncovers the genomic and clinical significance of a oncodriver RNF19A in PCa. The results of this study indicate that targeting AR/HIF1A-RNF19A-TRIP13 signaling axis could be an alternative option for PCa diagnosis and therapy.},
}
@article {pmid36596342,
year = {2023},
author = {Sellers, DL and Lee, K and Murthy, N and Pun, SH},
title = {TAxI-peptide targeted Cas12a ribonuclease protein nanoformulations increase genome editing in hippocampal neurons.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {354},
number = {},
pages = {188-195},
doi = {10.1016/j.jconrel.2022.12.057},
pmid = {36596342},
issn = {1873-4995},
support = {R01 NS118247/NS/NINDS NIH HHS/United States ; },
mesh = {Mice ; Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Ribonucleases ; Peptides ; Neurons ; },
abstract = {Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3 nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease.},
}
@article {pmid36845106,
year = {2023},
author = {Ashley, CL and McSharry, BP and McWilliam, HEG and Stanton, RJ and Fielding, CA and Mathias, RA and Fairlie, DP and McCluskey, J and Villadangos, JA and Rossjohn, J and Abendroth, A and Slobedman, B},
title = {Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1107497},
pmid = {36845106},
issn = {1664-3224},
abstract = {INTRODUCTION: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells).<br><br>METHODS: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout.<br><br>RESULTS: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells.<br><br>DISCUSSION: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.},
}
@article {pmid36843874,
year = {2023},
author = {Kadam, US and Cho, Y and Park, TY and Hong, JC},
title = {Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets.},
journal = {Applied biological chemistry},
volume = {66},
number = {1},
pages = {13},
pmid = {36843874},
issn = {2468-0834},
abstract = {CRISPR-Cas systems have been widely used in genome editing and transcriptional regulation. Recently, CRISPR-Cas effectors are adopted for biosensor construction due to its adjustable properties, such as simplicity of design, easy operation, collateral cleavage activity, and high biocompatibility. Aptamers' excellent sensitivity, specificity, in vitro synthesis, base-pairing, labeling, modification, and programmability has made them an attractive molecular recognition element for inclusion in CRISPR-Cas systems. Here, we review current advances in aptamer-based CRISPR-Cas sensors. We briefly discuss aptamers and the knowledge of Cas effector proteins, crRNA, reporter probes, analytes, and applications of target-specific aptamers. Next, we provide fabrication strategies, molecular binding, and detection using fluorescence, electrochemical, colorimetric, nanomaterials, Rayleigh, and Raman scattering. The application of CRISPR-Cas systems in aptamer-based sensing of a wide range of biomarkers (disease and pathogens) and toxic contaminants is growing. This review provides an update and offers novel insights into developing CRISPR-Cas-based sensors using ssDNA aptamers with high efficiency and specificity for point-of-care setting diagnostics.},
}
@article {pmid36843655,
year = {2023},
author = {Cannone, G and Kompaniiets, D and Graham, S and White, MF and Spagnolo, L},
title = {Structure of the Saccharolobus solfataricus type III-D CRISPR effector.},
journal = {Current research in structural biology},
volume = {5},
number = {},
pages = {100098},
pmid = {36843655},
issn = {2665-928X},
abstract = {CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.},
}
@article {pmid36842543,
year = {2023},
author = {Nourani, L and Mehrizi, AA and Pirahamdi, S and Pourhashem, Z and Asadollahi, E and Jahangiri, B},
title = {CRISPR/Cas advancements for genome editing, diagnosis, therapeutics, and vaccine development for Plasmodium parasites, and genetic engineering of Anopheles mosquito vector.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105419},
doi = {10.1016/j.meegid.2023.105419},
pmid = {36842543},
issn = {1567-7257},
abstract = {Malaria as vector-borne disease remains important health concern with over 200 million cases globally. Novel antimalarial medicines and more effective vaccines must be developed to eliminate and eradicate malaria. Appraisal of preceding genome editing approaches confirmed the CRISPR-Cas nuclease system as a novel proficient genome editing system and a tool for species-specific diagnosis, and drug resistance researches for Plasmodium species, and gene drive to control Anopheles population. CRISPR-Cas technology, as a handy tool for genome editing can be justified for the production of transgenic malaria parasites like Plasmodium transgenic lines expressing Cas9, chimeric Plasmodium transgenic lines, knockdown and knockout transgenic parasites, and transgenic parasites expressing alternative alleles, and also mutant strains of Anopheles such as only male mosquito populations, generation of wingless mosquitoes, and creation of knock-out/ knock-in mutants. Though, the incorporation of traditional methods and novel molecular techniques could noticeably enhance the quality of results. The striking development of a CRISPR/Cas-based diagnostic kit that can specifically diagnose the Plasmodium species or drug resistance markers is highly required in malaria settings with affordable cost and high-speed detection. Furthermore, the advancement of genome modifications by CRISPR/Cas technologies resolves contemporary restrictions to culturing, maintaining, and analyzing these parasites, and the aptitude to investigate parasite genome functions opens up new vistas in the better understanding of pathogenesis.},
}
@article {pmid36842456,
year = {2023},
author = {Sen, D and Mukhopadhyay, P},
title = {Application of CRISPR Cas systems in DNA recorders and writers.},
journal = {Bio Systems},
volume = {},
number = {},
pages = {104870},
doi = {10.1016/j.biosystems.2023.104870},
pmid = {36842456},
issn = {1872-8324},
abstract = {The necessity to record and store biological data is increasing in due course of time. However, it is quite difficult to understand biological mechanisms and keep a track of these events in some storage mediums. DNA (deoxyribonucleic acid) is the best candidate for the storage of cellular events in the biological system. It is energy efficient as well as stable at the same time. DNA-based writers and memory devices are continually evolving and finding new avenues in terms of their wide range of applications. Among all the DNA-based storage devices that employ enzymes like recombinases, nucleases, integrases, and polymerases, one of the most popular tools used for these devices is the emerging and versatile CRISPR Cas technology. CRISPR Cas is a prokaryotic immune system that keeps a memory of viral attacks and protects prokaryotes from potential future infections. The main aim of this short review is to study such molecular recorders and writers that employ CRISPR Cas technologies and obtain an in-depth overview of the mechanisms involved and the applications of these molecular devices.},
}
@article {pmid36841425,
year = {2023},
author = {Malekshoar, M and Ataei Azimi, S and Kaki, A and Mousazadeh, L and Motaei, J and Vatankhah, M},
title = {CRISPR-Cas9 Targeted Enrichment and Next Generation Sequencing for Mutations Detection.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmoldx.2023.01.010},
pmid = {36841425},
issn = {1943-7811},
abstract = {Despite the rapid application of next-generation sequencing (NGS) technologies, target sequencing in regions of the genome is often required to diagnose many genetic diseases. Target enrichment can be a very effective factor in reducing the cost of sequencing and the duration of sequencing. Recently, several CRISPR-based methods (amplification-free sequencing) have been developed to target enrichment in combination with one of the NGS platforms. CRISPR-based target enrichment strategies act as an auxiliary tool to improve NGS analytical performance, thereby indirectly facilitating nucleic acid detection. The direct DNA cleavage approach by CRISPR-Cas at genome-specific sites, enhances the possibility of separating native large fragments from disease-related genomic regions. The CRISPR-Cas can isolate the target region without any amplification and subsequently long-read sequencing technologies were also implemented . These methods, as promising tools, have the ability to assess genetic and epigenetic composition for clinical application and treatment responses in cancer precision medicine. By modifying CRISPR-based enrichment protocols, it was possible to identify different types of mutations, including structural variants, short tandem repeats (STRs), fusion genes, and mobile elements. The Cas9 can specifically eliminate wild-type sequences, and also enables the enrichment and detection of very small amounts of tumor DNA fragments among the highly heterogeneous fragments of wild-type DNA.},
}
@article {pmid36840794,
year = {2023},
author = {Bhattacharjee, S and Bhowmick, R and Kant, L and Paul, K},
title = {Strategic transgene-free approaches of CRISPR-based genome editing in plants.},
journal = {Molecular genetics and genomics : MGG},
volume = {},
number = {},
pages = {},
pmid = {36840794},
issn = {1617-4623},
abstract = {Genome editing through the alteration of nucleotide sequence has already revolutionized the field of site-directed mutagenesis for a decade. However, research in terms of precision and efficacy in targeting the loci and reduction in off-target mutation has always been a priority when DNA is involved. Therefore, recent research interest lies in utilizing the same precision technology but results in non-transgenic. In this review article, different technological advancements have been explained, which may provide a holistic concept of and need for transgene-free genome editing. The advantage and lacunas of each technology have been critically discussed to deliver a transparent view to the readers. A systematic analysis and evaluation of published research articles implied that researchers across the globe are putting continuous efforts in this direction to eliminate the hindrance of transgenic regulation. Nevertheless, this approach has severe implications legitimate for mitigating the conflict of acceptance, reliability, and generosity of gene-editing technology and sustainably retorting to the expanding global population feeding challenges.},
}
@article {pmid36840698,
year = {2023},
author = {},
title = {Correction to 'Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences'.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad147},
pmid = {36840698},
issn = {1362-4962},
}
@article {pmid36823429,
year = {2023},
author = {Lu, F and Leach, LL and Gross, JM},
title = {A CRISPR-Cas9-mediated F0 screen to identify pro-regenerative genes in the zebrafish retinal pigment epithelium.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {3142},
pmid = {36823429},
issn = {2045-2322},
support = {R01 EY29410/NH/NIH HHS/United States ; },
mesh = {Animals ; *Retinal Pigment Epithelium/physiology ; *Zebrafish/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Ocular diseases resulting in death of the retinal pigment epithelium (RPE) lead to vision loss and blindness. There are currently no FDA-approved strategies to restore damaged RPE cells. Stimulating intrinsic regenerative responses within damaged tissues has gained traction as a possible mechanism for tissue repair. Zebrafish possess remarkable regenerative abilities, including within the RPE; however, our understanding of the underlying mechanisms remains limited. Here, we conducted an F0 in vivo CRISPR-Cas9-mediated screen of 27 candidate RPE regeneration genes. The screen involved injection of a ribonucleoprotein complex containing three highly mutagenic guide RNAs per target gene followed by PCR-based genotyping to identify large intragenic deletions and MATLAB-based automated quantification of RPE regeneration. Through this F0 screening pipeline, eight positive and seven negative regulators of RPE regeneration were identified. Further characterization of one candidate, cldn7b, revealed novel roles in regulating macrophage/microglia infiltration after RPE injury and in clearing RPE/pigment debris during late-phase RPE regeneration. Taken together, these data support the utility of targeted F0 screens for validating pro-regenerative factors and reveal novel factors that could regulate regenerative responses within the zebrafish RPE.},
}
@article {pmid36820830,
year = {2023},
author = {Wei, W and Geer, MJ and Guo, X and Dolgalev, I and Sanjana, NE and Neel, BG},
title = {Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition.},
journal = {The Journal of experimental medicine},
volume = {220},
number = {5},
pages = {},
doi = {10.1084/jem.20221563},
pmid = {36820830},
issn = {1540-9538},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Signal Transduction ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics ; },
abstract = {SHP2 (PTPN11) acts upstream of SOS1/2 to enable RAS activation. Allosteric SHP2 inhibitors (SHP2i) in the clinic prevent SHP2 activation, block proliferation of RTK- or cycling RAS mutant-driven cancers, and overcome "adaptive resistance." To identify SHP2i resistance mechanisms, we performed genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive cell lines, recovering genes expected to cause resistance (NF1, PTEN, CDKN1B, LZTR1, and RASA2) and novel targets (INPPL1, MAP4K5, epigenetic modifiers). We screened 14 additional lines with a focused CRISPR library targeting common "hits" from the genome-wide screens. LZTR1 deletion conferred resistance in 12/14 lines, followed by MAP4K5 (8/14), SPRED2/STK40 (6/14), and INPPL1 (5/14). INPPL1, MAP4K5, or LZTR1 deletion reactivated ERK signaling. INPPL1-mediated sensitization to SHP2i required its NPXY motif but not lipid phosphatase activity. MAP4K5 acted upstream of MEK through a kinase-dependent target(s); LZTR1 had cell-dependent effects on RIT and RAS stability. INPPL1, MAP4K5, or LZTR1 deletion also conferred SHP2i resistance in vivo. Defining the SHP2i resistance landscape could suggest effective combination approaches.},
}
@article {pmid36796252,
year = {2023},
author = {Bai, J and Zhang, J and Fu, W and Li, S and Tian, X and Li, X and Zhao, X and Dong, J},
title = {Generation of a TRPM8 knockout hESC line (WAe009-A-A) derived from H9 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103040},
doi = {10.1016/j.scr.2023.103040},
pmid = {36796252},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; Karyotype ; Cell Line ; },
abstract = {The transient receptor potential cation channel subfamily M member 8 (TRPM8) is a kind of non-selective cation channel which controls Ca[2+] homeostasis. Mutations in TRPM8 were related to dry eye diseases (DED). Here we constructed a TRPM8 knockout cell line WAe009-A-A from the original embryonic stem cell line H9 using CRISPR/Cas9 technology, which maybe helpful for exploring the pathogenesis of DED. WAe009-A-A cells possess stem cell morphology and pluripotency as well as normal karyotype, and have the ability of differentiating into three germ layers in vitro.},
}
@article {pmid36753833,
year = {2023},
author = {Srisook, P and Laowtammathron, C and Lorthongpanich, C and Klaihmon, P and Terbto, P and Waeteekul, S and U-Pratya, Y and Issaragrisil, S},
title = {Generation of RUNX1c-eGFP induced pluripotent stem cell, MUSIi012-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103035},
doi = {10.1016/j.scr.2023.103035},
pmid = {36753833},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Cell Differentiation ; },
abstract = {Runt-Related Transcription Factor 1c (RUNX1c) plays an important role in regulating the development of hematopoietic stem cells (HSC). Using CRISPR/Cas9 gene editing technology, we established a RUNX1c-eGFP reporter cell line from the MUSIi012-A cell line. The MUSIi012-A-4 cell line has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro and in vivo. This cell line serves as a valuable model to observe the expression of RUNX1c via eGFP tracking during human hematopoietic development.},
}
@article {pmid36746102,
year = {2023},
author = {Diouf, D and Vitale, MR and Zöller, JEM and Pineau, AM and Klopocki, E and Hamann, C and Ziegler, GC and Vanmierlo, T and Van den Hove, D and Lesch, KP},
title = {Generation of a ST3GAL3 null mutant induced pluripotent stem cell (iPSC) line (UKWMPi002-A-3) by CRISPR/Cas9 genome editing.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103038},
doi = {10.1016/j.scr.2023.103038},
pmid = {36746102},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; },
abstract = {Fibroblasts isolated from a skin biopsy of a healthy individual were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate an isogenic cell line carrying an inactivation of ST3GAL3, a risk gene associated with neurodevelopmental and psychiatric disorders. This ST3GAL3 null mutant (ST3GAL3-/-) iPSC line, which displays the expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal karyotype, is a powerful tool to investigate the impact of deficient sialylation of glycoproteins in neural development and plasticity.},
}
@article {pmid36724553,
year = {2023},
author = {Ding, T and Gao, Z and Liu, H and Li, Y and Liu, S and Jian, L},
title = {Establishment of a PPP2CA homozygous knockout human embryonic stem cell line via CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103029},
doi = {10.1016/j.scr.2023.103029},
pmid = {36724553},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; Protein Phosphatase 2/genetics/metabolism ; Cell Line ; Embryonic Stem Cells/metabolism ; },
abstract = {Protein phosphatase 2A (PPP2CA) is one of the four main Ser/Thr phosphatase enzymes, which involved in the negative control of cell growth and division. PPP2CA is the main protein phosphatase in the heart, which regulates Ca (2+) through a series of ion channels and transporters. In this study, we generated a PPP2CA homozygous knockout human embryonic stem cell line WAe009-A-25 based on the transient expression CRISPR/Cas9 system to investigate functional effect of PP1 deficiency. This cell line has multidirectional differentiation potential, normal karyotypic and trilineage differentiation potential in vivo.},
}
@article {pmid36724552,
year = {2023},
author = {Aalders, J and Léger, L and Demolder, A and Muiño Mosquera, L and Coucke, P and Menten, B and De Backer, J and van Hengel, J},
title = {Generation of human induced pluripotent stem cell line UGENTi001-A from a patient with Marfan syndrome carrying a heterozygous c.7754 T &gt; C variant in FBN1 and the isogenic control UGENT001-A-1 using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103036},
doi = {10.1016/j.scr.2023.103036},
pmid = {36724552},
issn = {1876-7753},
mesh = {Humans ; Fibrillin-1/genetics ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; *Marfan Syndrome/genetics ; Heterozygote ; Mutation ; },
abstract = {Marfan syndrome is an autosomal dominant genetic disorder resulting from pathogenic variants in FBN1 gene. FBN1 encodes for fibrillin-1, an important extracellular matrix protein. Impaired fibrillin-1 affects multiple organ systems, including the cardiovascular system. We generated an iPSC line carrying a heterozygous variant c.7754 T > C (p.Ile2585Thr, missense) in FBN1 from a patient with Marfan syndrome. Also, an isogenic control is generated, where the pathogenic variant is repaired using CRISPR-Cas9. This isogenic pair provides a valuable resource for in vitro disease modelling.},
}
@article {pmid36719060,
year = {2023},
author = {Oulhen, N and Morita, S and Warner, JF and Wessel, G},
title = {CRISPR/Cas9 knockin methodology for the sea urchin embryo.},
journal = {Molecular reproduction and development},
volume = {90},
number = {2},
pages = {69-72},
doi = {10.1002/mrd.23672},
pmid = {36719060},
issn = {1098-2795},
support = {/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Sea Urchins/genetics ; Embryo, Nonmammalian ; },
}
@article {pmid36716678,
year = {2023},
author = {Yi, T and Liu, J and Zhang, S and He, Y and Han, J},
title = {Generation of a TIMP3 knockout stem cell line via CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103034},
doi = {10.1016/j.scr.2023.103034},
pmid = {36716678},
issn = {1876-7753},
mesh = {Humans ; CRISPR-Cas Systems ; Cell Line ; Embryonic Stem Cells/metabolism ; *Macular Degeneration/genetics ; *Human Embryonic Stem Cells/metabolism ; Tissue Inhibitor of Metalloproteinase-3/genetics/metabolism ; },
abstract = {The tissue inhibitors of metalloproteinases 3 (TIMP3) play an essential role in the tumorigenesis of human pancreatic endocrine tumors and Sorsby fundus dystrophy. To further investigate the significance of TIMP3 in disease, we used CRISPR/Cas9 to create a TIMP3 knock out human embryonic stem cell line (WAe009-A-89) that can differentiate into any desired cell type. Our results show that the WAe009-A-89 cell line retains the typical colony form and normal karyotype of stem cells. The cells strongly expressed pluripotency markers and could differentiate into tissues of all three germ layers in vivo. This cell line allowed exploring the role of the TIMP3 gene in related diseases.},
}
@article {pmid36682125,
year = {2023},
author = {Yammine, KM and Mirda Abularach, S and Sampurno, L and Bateman, JF and Lamandé, SR and Shoulders, MD},
title = {Using CRISPR/Cas9 to generate a heterozygous COL2A1 p.R719C iPSC line (MCRIi019-A-6) model of human precocious osteoarthritis.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103020},
doi = {10.1016/j.scr.2023.103020},
pmid = {36682125},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Heterozygote ; Mutation ; *Osteoarthritis/metabolism ; Collagen Type II/genetics ; },
abstract = {The human iPSC line MCRIi019-A-6 was generated using CRISPR/Cas9-mediated gene editing to introduce a heterozygous COL2A1 exon 33 c.2155C>T (p.R719C) mutation into the control human iPSC line MCRIi019-A. Both the edited and parental lines display typical iPSC characteristics, including the expression of pluripotency markers, the ability to be differentiated into the three germ lines, and a normal karyotype. This cell line, along with the isogenic control line, can be used to study the molecular pathology of precocious osteoarthritis in a human model, more broadly understand type II collagenopathies, and explore novel therapeutic targets for this class of diseases.},
}
@article {pmid36650342,
year = {2023},
author = {Tian, L and Yan, B and Huo, D and Sun, W and Cui, S and Li, X and Zhang, X and Dong, H},
title = {Establishment of a visual gene knockout system based on CRISPR/Cas9 for the rare actinomycete Nonomuraea gerenzanensis.},
journal = {Biotechnology letters},
volume = {45},
number = {3},
pages = {401-410},
pmid = {36650342},
issn = {1573-6776},
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; *Actinobacteria/genetics ; },
abstract = {OBJECTIVES: To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis (N. gerenzanensis).<br><br>RESULTS: With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis. Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500&#xa0;bp in this system could still reach the relatively higher conjugation transfer frequency.<br><br>CONCLUSIONS: The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis.},
}
@article {pmid36642056,
year = {2023},
author = {Kamat, K and Inamdar, MS},
title = {Generation of OCIAD2 homozygous knockout (BJNhem20-OCIAD2-CRISPR-33) and heterozygous knockout (BJNhem20-OCIAD2-CRISPR-40) human embryonic stem cell lines using CRISPR-Cas9 mediated targeting.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103026},
doi = {10.1016/j.scr.2023.103026},
pmid = {36642056},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Karyotype ; Embryonic Stem Cells/metabolism ; Neoplasm Proteins/metabolism ; },
abstract = {Ovarian Carcinoma Immunoreactive Antigen domain containing 2 (OCIAD2) was knocked out by targeting its exon 4 through CRISPR-Cas9 paired nickase strategy to generate two OCIAD2 knockout human embryonic stem cell lines- one homozygous (BJNhem20-OCIAD2-CRISPR-33) and one heterozygous (BJNhem20-OCIAD2-CRISPR-40) for mutant ociad2. Both lines maintain pluripotency, normal karyotype, and trilineage differentiation potential.},
}
@article {pmid36630838,
year = {2023},
author = {Guo, R and Yuan, S and Li, B and Wang, J and Sun, C},
title = {Generation of NANOS3-mCherry reporter human embryonic stem cell line SYSUe-009-A using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103022},
doi = {10.1016/j.scr.2023.103022},
pmid = {36630838},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; Embryonic Stem Cells/metabolism ; Cell Line ; Germ Cells/metabolism ; Cell Differentiation ; RNA-Binding Proteins/genetics ; },
abstract = {NANOS3 is a zinc-finger containing RNA-binding protein that has been demonstrated to be highly expressed in human primordial germ cell(hPGC), thus NANOS3 can serve as a marker for hPGC development. Due to the ethical and technical restrictions, it is difficult to get primary human germline cells. Human primordial germ cell-like cells (hPGCLCs) generated from pluripotent stem cells is an excellent alternatives in human germ cell-related studies. This hESC line with an mCherry knock-in at the site before NANOS3's stop codon serves as a useful tool to learn human PGC specification.},
}
@article {pmid36610307,
year = {2023},
author = {Akdaş, EY and Turan, S and Guhathakurta, D and Ekici, A and Salar, S and Lie, DC and Winner, B and Fejtova, A},
title = {CRISPR/Cas9-mediated generation of hESC lines with homozygote and heterozygote p.R331W mutation in CTBP1 to model HADDTS syndrome.},
journal = {Stem cell research},
volume = {67},
number = {},
pages = {103012},
doi = {10.1016/j.scr.2022.103012},
pmid = {36610307},
issn = {1876-7753},
mesh = {Humans ; Homozygote ; *Human Embryonic Stem Cells ; Heterozygote ; CRISPR-Cas Systems ; Muscle Hypotonia/genetics ; Mutation ; Transcription Factors/genetics ; Ataxia/genetics ; },
abstract = {C-terminal Binding Protein 1 (CTBP1) is a ubiquitously expressed transcriptional co-repressor and membrane trafficking regulator. A recurrent de novo c.991C>T mutation in CTBP1 leads to expression of p.R331W CTBP1 and causes hypotonia, ataxia, developmental delay, and tooth enamel defects syndrome (HADDTS), a rare early onset neurodevelopmental disorder. We generated hESCs lines with heterozygote and homozygote c.991C>T in CTBP1 using CRISPR/Cas9 genome editing and validated them for genetic integrity, off-target mutations, and pluripotency. They will be useful for investigation of HADDTS pathophysiology and for screening for potential therapeutics.},
}
@article {pmid36840654,
year = {2023},
author = {Fu, R and Xianyu, Y},
title = {Gold Nanomaterials-Implemented CRISPR-Cas Systems for Biosensing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2300057},
doi = {10.1002/smll.202300057},
pmid = {36840654},
issn = {1613-6829},
abstract = {Due to their superiority in the simple design and precise targeting, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have attracted significant interest for biosensing. On the one hand, CRISPR-Cas systems have the capacity to precisely recognize and cleave specific DNA and RNA sequences. On the other hand, CRISPR-Cas systems such as orthologs of Cas9, Cas12, and Cas13 exhibit cis-cleavage or trans-cleavage activities after recognizing the target sequence. Owing to the cleavage activities, CRISPR-Cas systems can be designed for biosensing by degrading tagged nucleic acids to produce detectable signals. To meet the requirements of point-of-care detection and versatile signal readouts, gold nanomaterials with excellent properties such as high extinction coefficients, easy surface functionalization, and biocompatibility are implemented in CRISPR-Cas-based biosensors. In combination with gold nanomaterials such as gold nanoparticles, gold nanorods, and gold nanostars, great efforts are devoted to fabricating CRISPR-Cas-based biosensors for the detection of diverse targets. This review focuses on the current advances in gold nanomaterials-implemented CRISPR-Cas-based biosensors, particularly the working mechanism and the performance of these biosensors. CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a are discussed and highlighted. Meanwhile, prospects and challenges are also discussed in the design of biosensing strategies based on gold nanomaterials and CRISPR-Cas systems.},
}
@article {pmid36838970,
year = {2023},
author = {Mollashahi, B and Latifi-Navid, H and Owliaee, I and Shamdani, S and Uzan, G and Jamehdor, S and Naserian, S},
title = {Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit.},
journal = {Molecules (Basel, Switzerland)},
volume = {28},
number = {4},
pages = {},
pmid = {36838970},
issn = {1420-3049},
abstract = {The most widely used genome editing toolkit is CRISPR (clustered regularly interspaced short palindromic repeats). It provides the possibility of replacing and modifying DNA and RNA nucleotides. Furthermore, with advancements in biological technology, inhibition and activation of the transcription of specific gene(s) has become possible. Bioinformatics tools that target the evolution of CRISPR-associated protein 9 (Cas9) turn this protein into a vehicle that is specific for a DNA or RNA region with single guide RNA (sgRNA). This toolkit could be used by researchers to investigate the function of stem cell gene(s). Here, in this review article, we cover recent developments and applications of this technique in stem cells for research and clinical purposes and discuss different CRISPR/Cas technologies for knock-out, knock-in, activation, or inhibition of gene expression. Additionally, a comparison of several deliveries and off-target detecting strategies is discussed.},
}
@article {pmid36835087,
year = {2023},
author = {Yuan, Q and Zhai, Y and Zhou, L and Ai, X and Chen, J and Yan, F},
title = {A Novel miRNA in Rice Associated with the Low Seed Setting Rate Symptom of Rice Stripe Virus.},
journal = {International journal of molecular sciences},
volume = {24},
number = {4},
pages = {},
doi = {10.3390/ijms24043675},
pmid = {36835087},
issn = {1422-0067},
abstract = {MicroRNAs play key regulatory roles in plant development. The changed pattern of miRNA expression is involved in the production of viral symptoms. Here, we showed that a small RNA, Seq119, a putative novel microRNA, is associated with the low seed setting rate, a viral symptom of rice stripe virus (RSV)-infected rice. The expression of Seq 119 was downregulated in RSV-infected rice. The overexpression of Seq119 in transgenic rice plants did not cause any obvious phenotypic changes in plant development. When the expression of Seq119 was suppressed in rice plants either by expressing a mimic target or by CRISPR/Cas editing, seed setting rates were extremely low, similar to the effects of RSV infection. The putative targets of Seq119 were then predicted. The overexpression of the target of Seq119 in rice caused a low seed setting rate, similar to that in Seq119-suppressed or edited rice plants. Consistently, the expression of the target was upregulated in Seq119-suppressed and edited rice plants. These results suggest that downregulated Seq119 is associated with the low seed setting rate symptom of the RSV in rice.},
}
@article {pmid36833410,
year = {2023},
author = {Phan, HTL and Kim, K and Lee, H and Seong, JK},
title = {Progress in and Prospects of Genome Editing Tools for Human Disease Model Development and Therapeutic Applications.},
journal = {Genes},
volume = {14},
number = {2},
pages = {},
doi = {10.3390/genes14020483},
pmid = {36833410},
issn = {2073-4425},
abstract = {Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are widely accepted because of their diversity and enormous potential for targeted genomic modifications in eukaryotes and other animals. Moreover, rapid advances in genome editing tools have accelerated the ability to produce various genetically modified animal models for studying human diseases. Given the advances in gene editing tools, these animal models are gradually evolving toward mimicking human diseases through the introduction of human pathogenic mutations in their genome rather than the conventional gene knockout. In the present review, we summarize the current progress in and discuss the prospects for developing mouse models of human diseases and their therapeutic applications based on advances in the study of programmable nucleases.},
}
@article {pmid36833321,
year = {2023},
author = {Yang, W and Tao, D and Xu, B and Zheng, Y and Zhao, S},
title = {Detecting Melanocortin 1 Receptor Gene's SNPs by CRISPR/enAsCas12a.},
journal = {Genes},
volume = {14},
number = {2},
pages = {},
doi = {10.3390/genes14020394},
pmid = {36833321},
issn = {2073-4425},
abstract = {Beyond its powerful genome-editing capabilities, the CRISPR/Cas system has opened up a new era of molecular diagnostics due to its highly specific base recognition and trans-cleavage activity. However, most CRISPR/Cas detection systems are mainly used to detect nucleic acids of bacteria or viruses, while the application of single nucleotide polymorphism (SNP) detection is limited. The MC1R SNPs were investigated by CRISPR/enAsCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enAsCas12a has a preference for divalent magnesium ion (Mg[2+]) and can effectively distinguish the genes with a single base difference in the presence of Mg[2+], and the Melanocortin l receptor (MC1R) gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enAsCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enAsCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.},
}
@article {pmid36832059,
year = {2023},
author = {Tang, H and Peng, J and Jiang, X and Peng, S and Wang, F and Weng, X and Zhou, X},
title = {A CRISPR-Cas and Tat Peptide with Fluorescent RNA Aptamer System for Signal Amplification in RNA Imaging.},
journal = {Biosensors},
volume = {13},
number = {2},
pages = {},
doi = {10.3390/bios13020293},
pmid = {36832059},
issn = {2079-6374},
abstract = {We reported on an efficient RNA imaging strategy based on a CRISPR-Cas and Tat peptide with a fluorescent RNA aptamer (TRAP-tag). Using modified CRISPR-Cas RNA hairpin binding proteins fused with a Tat peptide array that recruits modified RNA aptamers, this simple and sensitive strategy is capable of visualizing endogenous RNA in cells with high precision and efficiency. In addition, the modular design of the CRISPR-TRAP-tag facilitates the substitution of sgRNAs, RNA hairpin binding proteins, and aptamers in order to optimize imaging quality and live cell affinity. With CRISPR-TRAP-tag, exogenous GCN4, endogenous mRNA MUC4, and lncRNA SatIII were distinctly visualized in single live cells.},
}
@article {pmid36831968,
year = {2023},
author = {Kumaran, A and Jude Serpes, N and Gupta, T and James, A and Sharma, A and Kumar, D and Nagraik, R and Kumar, V and Pandey, S},
title = {Advancements in CRISPR-Based Biosensing for Next-Gen Point of Care Diagnostic Application.},
journal = {Biosensors},
volume = {13},
number = {2},
pages = {},
doi = {10.3390/bios13020202},
pmid = {36831968},
issn = {2079-6374},
abstract = {With the move of molecular tests from diagnostic labs to on-site testing becoming more common, there is a sudden rise in demand for nucleic acid-based diagnostic tools that are selective, sensitive, flexible to terrain changes, and cost-effective to assist in point-of-care systems for large-scale screening and to be used in remote locations in cases of outbreaks and pandemics. CRISPR-based biosensors comprise a promising new approach to nucleic acid detection, which uses Cas effector proteins (Cas9, Cas12, and Cas13) as extremely specialized identification components that may be used in conjunction with a variety of readout approaches (such as fluorescence, colorimetry, potentiometry, lateral flow assay, etc.) for onsite analysis. In this review, we cover some technical aspects of integrating the CRISPR Cas system with traditional biosensing readout methods and amplification technologies such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) and continue to elaborate on the prospects of the developed biosensor in the detection of some major viral and bacterial diseases. Within the scope of this article, we also discuss the recent COVID pandemic and the numerous CRISPR biosensors that have undergone development since its advent. Finally, we discuss some challenges and future prospects of CRISPR Cas systems in point-of-care testing.},
}
@article {pmid36831926,
year = {2023},
author = {Wang, M and Liu, H and Ren, J and Huang, Y and Deng, Y and Liu, Y and Chen, Z and Chow, FW and Leung, PH and Li, S},
title = {Enzyme-Assisted Nucleic Acid Amplification in Molecular Diagnosis: A Review.},
journal = {Biosensors},
volume = {13},
number = {2},
pages = {},
doi = {10.3390/bios13020160},
pmid = {36831926},
issn = {2079-6374},
abstract = {Infectious diseases and tumors have become the biggest medical challenges in the 21st century. They are driven by multiple factors such as population growth, aging, climate change, genetic predispositions and more. Nucleic acid amplification technologies (NAATs) are used for rapid and accurate diagnostic testing, providing critical information in order to facilitate better follow-up treatment and prognosis. NAATs are widely used due their high sensitivity, specificity, rapid amplification and detection. It should be noted that different NAATs can be selected according to different environments and research fields; for example, isothermal amplification with a simple operation can be preferred in developing countries or resource-poor areas. In the field of translational medicine, CRISPR has shown great prospects. The core component of NAAT lies in the activity of different enzymes. As the most critical material of nucleic acid amplification, the key role of the enzyme is self-evident, playing the upmost important role in molecular diagnosis. In this review, several common enzymes used in NAATs are compared and described in detail. Furthermore, we summarize both the advances and common issues of NAATs in clinical application.},
}
@article {pmid36830769,
year = {2023},
author = {Wei, W and Jiang, X and Zhang, L and Yan, Y and Yan, J and Xu, L and Gao, CH and Yang, M},
title = {Regulation of CRISPR-Associated Genes by Rv1776c (CasR) in Mycobacterium tuberculosis.},
journal = {Biomolecules},
volume = {13},
number = {2},
pages = {},
doi = {10.3390/biom13020400},
pmid = {36830769},
issn = {2218-273X},
abstract = {The CRISPR-Cas system is an adaptive immune system for many bacteria and archaea to defend against foreign nucleic acid invasion, and this system is conserved in the genome of M. tuberculosis (Mtb). Although the CRISPR-Cas system-mediated immune defense mechanism has been revealed in Mtb, the regulation of cas gene expression is poorly understood. In this study, we identified a transcription factor, CasR (CRISPR-associated protein repressor, encoded by Rv1776c), and it could bind to the upstream DNA sequence of the CRISPR-Cas gene cluster and regulate the expression of cas genes. EMSA and ChIP assays confirmed that CasR could interact with the upstream sequence of the csm6 promoter, both in vivo and in vitro. Furthermore, DNA footprinting assay revealed that CasR recognized a 20 bp palindromic sequence motif and negatively regulated the expression of csm6. In conclusion, our research elucidates the regulatory effect of CasR on the expression of CRISPR-associated genes in mycobacteria, thus providing insight into gene expression regulation of the CRISPR-Cas system.},
}
@article {pmid36830633,
year = {2023},
author = {Belato, HB and Lisi, GP},
title = {The Many (Inter)faces of Anti-CRISPRs: Modulation of CRISPR-Cas Structure and Dynamics by Mechanistically Diverse Inhibitors.},
journal = {Biomolecules},
volume = {13},
number = {2},
pages = {},
doi = {10.3390/biom13020264},
pmid = {36830633},
issn = {2218-273X},
support = {R01 GM136815/NH/NIH HHS/United States ; T32 GM077995/NH/NIH HHS/United States ; },
abstract = {The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs), has enabled the development of highly controllable and precise CRISPR-Cas tools. Anti-CRISPRs share very little structural or sequential resemblance to each other or to other proteins, which raises intriguing questions regarding their modes of action. Many structure-function studies have shed light on the mechanism(s) of Acrs, which can act as orthosteric or allosteric inhibitors of CRISPR-Cas machinery, as well as enzymes that irreversibly modify CRISPR-Cas components. Only recently has the breadth of diversity of Acr structures and functions come to light, and this remains a rapidly evolving field. Here, we draw attention to a plethora of Acr mechanisms, with particular focus on how their action toward Cas proteins modulates conformation, dynamic (allosteric) signaling, nucleic acid binding, and cleavage ability.},
}
@article {pmid36830114,
year = {2023},
author = {Shim, H},
title = {Three Innovations of Next-Generation Antibiotics: Evolvability, Specificity, and Non-Immunogenicity.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {12},
number = {2},
pages = {},
doi = {10.3390/antibiotics12020204},
pmid = {36830114},
issn = {2079-6382},
abstract = {Antimicrobial resistance is a silent pandemic exacerbated by the uncontrolled use of antibiotics. Since the discovery of penicillin, we have been largely dependent on microbe-derived small molecules to treat bacterial infections. However, the golden era of antibiotics is coming to an end, as the emergence and spread of antimicrobial resistance against these antibacterial compounds are outpacing the discovery and development of new antibiotics. The current antibiotic market suffers from various shortcomings, including the absence of profitability and investment. The most important underlying issue of traditional antibiotics arises from the inherent properties of these small molecules being mostly broad-spectrum and non-programmable. As the scientific knowledge of microbes progresses, the scientific community is starting to explore entirely novel approaches to tackling antimicrobial resistance. One of the most prominent approaches is to develop next-generation antibiotics. In this review, we discuss three innovations of next-generation antibiotics compared to traditional antibiotics as specificity, evolvability, and non-immunogenicity. We present a number of potential antimicrobial agents, including bacteriophage-based therapy, CRISPR-Cas-based antimicrobials, and microbiome-derived antimicrobial agents. These alternative antimicrobial agents possess innovative properties that may overcome the inherent shortcomings of traditional antibiotics, and some of these next-generation antibiotics are not merely far-fetched ideas but are currently in clinical development. We further discuss some related issues and challenges such as infection diagnostics and regulatory frameworks that still need to be addressed to bring these next-generation antibiotics to the antibiotic market as viable products to combat antimicrobial resistance using a diversified set of strategies.},
}
@article {pmid36829149,
year = {2023},
author = {Park, BS and Jeon, H and Chi, SG and Kim, T},
title = {Efficient prioritization of CRISPR screen hits by accounting for targeting efficiency of guide RNA.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {45},
pmid = {36829149},
issn = {1741-7007},
abstract = {BACKGROUND: CRISPR-based screens are revolutionizing drug discovery as tools to identify genes whose ablation induces a phenotype of interest. For instance, CRISPR-Cas9 screening has been successfully used to identify novel therapeutic targets in cancer where disruption of genes leads to decreased viability of malignant cells. However, low-activity guide RNAs may give rise to variable changes in phenotype, preventing easy identification of hits and leading to false negative results. Therefore, correcting the effects of bias due to differences in guide RNA efficiency in CRISPR screening data can improve the efficiency of prioritizing hits for further validation. Here, we developed an approach to identify hits from negative CRISPR screens by correcting the fold changes (FC) in gRNA frequency by the actual, observed frequency of indel mutations generated by gRNA.<br><br>RESULTS: Each gRNA was coupled with the "reporter sequence" that can be targeted by the same gRNA so that the frequency of mutations in the reporter sequence can be used as a proxy for the endogenous target gene. The measured gRNA activity was used to correct the FC. We identified indel generation efficiency as the dominant factor contributing significant bias to screening results, and our method significantly removed such bias and was better at identifying essential genes when compared to conventional fold change analysis. We successfully applied our gRNA activity data to previously published gRNA screening data, and identified novel genes whose ablation could synergize with vemurafenib in the A375 melanoma cell line. Our method identified nicotinamide N-methyltransferase, lactate dehydrogenase B, and polypyrimidine tract-binding protein 1 as synergistic targets whose ablation sensitized A375 cells to vemurafenib.<br><br>CONCLUSIONS: We identified the variations in target cleavage efficiency, even in optimized sgRNA libraries, that pose a strong bias in phenotype and developed an analysis method that corrects phenotype score by the measured differences in the targeting efficiency among sgRNAs. Collectively, we expect that our new analysis method will more accurately identify genes that confer the phenotype of interest.},
}
@article {pmid36826896,
year = {2023},
author = {Chen, H and Zhou, X and Wang, M and Ren, L},
title = {Towards Point of Care CRISPR-Based Diagnostics: From Method to Device.},
journal = {Journal of functional biomaterials},
volume = {14},
number = {2},
pages = {},
doi = {10.3390/jfb14020097},
pmid = {36826896},
issn = {2079-4983},
abstract = {Rapid, accurate, and portable on-site detection is critical in the face of public health emergencies. Infectious disease control and public health emergency policymaking can both be aided by effective and trustworthy point of care tests (POCT). A very promising POCT method appears to be the clustered regularly interspaced short palindromic repeats and associated protein (CRISPR/Cas)-based molecular diagnosis. For on-site detection, CRISPR/Cas-based detection can be combined with multiple signal sensing methods and integrated into smart devices. In this review, sensing methods for CRISPR/Cas-based diagnostics are introduced and the advanced strategies and recent advances in CRISPR/Cas-based POCT are reviewed. Finally, the future perspectives of CRISPR and POCT are summarized and prospected.},
}
@article {pmid36826113,
year = {2023},
author = {Rabaan, AA and AlSaihati, H and Bukhamsin, R and Bakhrebah, MA and Nassar, MS and Alsaleh, AA and Alhashem, YN and Bukhamseen, AY and Al-Ruhimy, K and Alotaibi, M and Alsubki, RA and Alahmed, HE and Al-Abdulhadi, S and Alhashem, FA and Alqatari, AA and Alsayyah, A and Farahat, RA and Abdulal, RH and Al-Ahmed, AH and Imran, M and Mohapatra, RK},
title = {Application of CRISPR/Cas9 Technology in Cancer Treatment: A Future Direction.},
journal = {Current oncology (Toronto, Ont.)},
volume = {30},
number = {2},
pages = {1954-1976},
pmid = {36826113},
issn = {1718-7729},
abstract = {Gene editing, especially with clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9), has advanced gene function science. Gene editing's rapid advancement has increased its medical/clinical value. Due to its great specificity and efficiency, CRISPR/Cas9 can accurately and swiftly screen the whole genome. This simplifies disease-specific gene therapy. To study tumor origins, development, and metastasis, CRISPR/Cas9 can change genomes. In recent years, tumor treatment research has increasingly employed this method. CRISPR/Cas9 can treat cancer by removing genes or correcting mutations. Numerous preliminary tumor treatment studies have been conducted in relevant fields. CRISPR/Cas9 may treat gene-level tumors. CRISPR/Cas9-based personalized and targeted medicines may shape tumor treatment. This review examines CRISPR/Cas9 for tumor therapy research, which will be helpful in providing references for future studies on the pathogenesis of malignancy and its treatment.},
}
@article {pmid36824842,
year = {2023},
author = {Jain, P and Rananaware, S and Vesco, E and Shoemaker, G and Anekar, S and Sandoval, LS and Meister, K and Macaluso, N and Nguyen, L},
title = {Programmable RNA detection with CRISPR-Cas12a.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-2549171/v1},
pmid = {36824842},
abstract = {CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or 'SAHARA' to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.},
}
@article {pmid36824723,
year = {2023},
author = {Casas-Mollano, JA and Zinselmeier, M and Sychla, A and Smanski, MJ},
title = {Efficient gene activation in plants by the MoonTag programmable transcriptional activator.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.02.15.528671},
pmid = {36824723},
abstract = {CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR-Cas based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that worked on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.},
}
@article {pmid36824201,
year = {2023},
author = {Sustek-Sánchez, F and Rognli, OA and Rostoks, N and Sõmera, M and Jaškūnė, K and Kovi, MR and Statkevičiūtė, G and Sarmiento, C},
title = {Improving abiotic stress tolerance of forage grasses - prospects of using genome editing.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1127532},
pmid = {36824201},
issn = {1664-462X},
abstract = {Due to an increase in the consumption of food, feed, and fuel and to meet global food security needs for the rapidly growing human population, there is a necessity to obtain high-yielding crops that can adapt to future climate changes. Currently, the main feed source used for ruminant livestock production is forage grasses. In temperate climate zones, perennial grasses grown for feed are widely distributed and tend to suffer under unfavorable environmental conditions. Genome editing has been shown to be an effective tool for the development of abiotic stress-resistant plants. The highly versatile CRISPR-Cas system enables increasingly complex modifications in genomes while maintaining precision and low off-target frequency mutations. In this review, we provide an overview of forage grass species that have been subjected to genome editing. We offer a perspective view on the generation of plants resilient to abiotic stresses. Due to the broad factors contributing to these stresses the review focuses on drought, salt, heat, and cold stresses. The application of new genomic techniques (e.g., CRISPR-Cas) allows addressing several challenges caused by climate change and abiotic stresses for developing forage grass cultivars with improved adaptation to the future climatic conditions. Genome editing will contribute towards developing safe and sustainable food systems.},
}
@article {pmid36603730,
year = {2023},
author = {Lu, W and Ma, S and Sun, L and Zhang, T and Wang, X and Feng, M and Wang, A and Shi, R and Jia, L and Xia, Q},
title = {Combined CRISPR toolkits reveal the domestication landscape and function of the ultra-long and highly repetitive silk genes.},
journal = {Acta biomaterialia},
volume = {158},
number = {},
pages = {190-202},
doi = {10.1016/j.actbio.2022.12.063},
pmid = {36603730},
issn = {1878-7568},
mesh = {Animals ; *Bombyx/genetics ; Domestication ; *Fibroins/genetics ; Silk/genetics ; CRISPR-Cas Systems ; },
abstract = {Highly repetitive sequences play a major structural and function role in the genome. In the present study, we developed Cas9-assisted cloning and SMRT sequencing of long repetitive sequences (CACS) to sequence and manipulate highly repetitive genes from eukaryotic genomes. CACS combined Cas9-mediated cleavage of a target segment from an intact genome, Gibson assembly cloning, and PacBio SMRT sequencing. Applying CACS, we directly cloned and sequenced the complete sequences of fibroin heavy chain (FibH) genes from 17 domesticated (Bombyx mori) and 7 wild (Bombyx mandarina) silkworms. Our analysis revealed the unique fine structure organization, genetic variations, and domestication dynamics of FibH. We also demonstrated that the length of the repetitive regions determined the mechanical properties of silk fiber, which was further confirmed by Cas9 editing of FibH. CACS is a simple, robust, and efficient approach, providing affordable accessibility to highly repetitive regions of a genome. STATEMENT OF SIGNIFICANCE: Silkworm silk is the earliest and most widely used animal fiber, and its excellent performance mainly depends on the fibroin heavy chain (FibH) protein. The FibH gene is the main breakthrough in understanding the formation mechanism and improvement of silk fiber. In the study, we developed a CACS method for characterizing the fine structure and domestication landscape of 24 silkworm FibH genes. We used CRISPR/Cas9 to edit the repetitive sequence of FibH genes, revealing the relationship between FibH genes and mechanical properties of silkworm silk. Our study is helpful in modifying silk genes to manipulate other valuable highly repetitive sequences, and provides insight for silkworm breeding.},
}
@article {pmid36824195,
year = {2023},
author = {Verma, V and Kumar, A and Partap, M and Thakur, M and Bhargava, B},
title = {CRISPR-Cas: A robust technology for enhancing consumer-preferred commercial traits in crops.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1122940},
doi = {10.3389/fpls.2023.1122940},
pmid = {36824195},
issn = {1664-462X},
abstract = {The acceptance of new crop varieties by consumers is contingent on the presence of consumer-preferred traits, which include sensory attributes, nutritional value, industrial products and bioactive compounds production. Recent developments in genome editing technologies provide novel insight to identify gene functions and improve the various qualitative and quantitative traits of commercial importance in plants. Various conventional as well as advanced gene-mutagenesis techniques such as physical and chemical mutagenesis, CRISPR-Cas9, Cas12 and base editors are used for the trait improvement in crops. To meet consumer demand, breakthrough biotechnologies, especially CRISPR-Cas have received a fair share of scientific and industrial interest, particularly in plant genome editing. CRISPR-Cas is a versatile tool that can be used to knock out, replace and knock-in the desired gene fragments at targeted locations in the genome, resulting in heritable mutations of interest. This review highlights the existing literature and recent developments in CRISPR-Cas technologies (base editing, prime editing, multiplex gene editing, epigenome editing, gene delivery methods) for reliable and precise gene editing in plants. This review also discusses the potential of gene editing exhibited in crops for the improvement of consumer-demanded traits such as higher nutritional value, colour, texture, aroma/flavour, and production of industrial products such as biofuel, fibre, rubber and pharmaceuticals. In addition, the bottlenecks and challenges associated with gene editing system, such as off targeting, ploidy level and the ability to edit organelle genome have also been discussed.},
}
@article {pmid36823453,
year = {2023},
author = {Webb, EA and Held, NA and Zhao, Y and Graham, ED and Conover, AE and Semones, J and Lee, MD and Feng, Y and Fu, FX and Saito, MA and Hutchins, DA},
title = {Importance of mobile genetic element immunity in numerically abundant Trichodesmium clades.},
journal = {ISME communications},
volume = {3},
number = {1},
pages = {15},
pmid = {36823453},
issn = {2730-6151},
abstract = {The colony-forming cyanobacteria Trichodesmium spp. are considered one of the most important nitrogen-fixing genera in the warm, low nutrient ocean. Despite this central biogeochemical role, many questions about their evolution, physiology, and trophic interactions remain unanswered. To address these questions, we describe Trichodesmium pangenomic potential via significantly improved genomic assemblies from two isolates and 15 new >50% complete Trichodesmium metagenome-assembled genomes from hand-picked, Trichodesmium colonies spanning the Atlantic Ocean. Phylogenomics identified ~four N2 fixing clades of Trichodesmium across the transect, with T. thiebautii dominating the colony-specific reads. Pangenomic analyses showed that all T. thiebautii MAGs are enriched in COG defense mechanisms and encode a vertically inherited Type III-B Clustered Regularly Interspaced Short Palindromic Repeats and associated protein-based immunity system (CRISPR-Cas). Surprisingly, this CRISPR-Cas system was absent in all T. erythraeum genomes, vertically inherited by T. thiebautii, and correlated with increased signatures of horizontal gene transfer. Additionally, the system was expressed in metaproteomic and transcriptomic datasets and CRISPR spacer sequences with 100% identical hits to field-assembled, putative phage genome fragments were identified. While the currently CO2-limited T. erythraeum is expected to be a 'winner' of anthropogenic climate change, their genomic dearth of known phage resistance mechanisms, compared to T. thiebautii, could put this outcome in question. Thus, the clear demarcation of T. thiebautii maintaining CRISPR-Cas systems, while T. erythraeum does not, identifies Trichodesmium as an ecologically important CRISPR-Cas model system, and highlights the need for more research on phage-Trichodesmium interactions.},
}
@article {pmid36818884,
year = {2023},
author = {Ercolano, MR and Wang, K},
title = {Editorial: Targeted genome editing for crop improvement.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1106996},
pmid = {36818884},
issn = {1664-462X},
}
@article {pmid36818498,
year = {2023},
author = {Yang, H and Zhang, Y and Teng, X and Hou, H and Deng, R and Li, J},
title = {CRISPR-based nucleic acid diagnostics for pathogens.},
journal = {Trends in analytical chemistry : TRAC},
volume = {160},
number = {},
pages = {116980},
pmid = {36818498},
issn = {0165-9936},
abstract = {Pathogenic infection remains the primary threat to human health, such as the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutated variants, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we firstly describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges involved in on-site diagnostic assays and present emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.},
}
@article {pmid36814855,
year = {2023},
author = {Smaruj, P and Kieliszek, M},
title = {Casposons - silent heroes of the CRISPR-Cas systems evolutionary history.},
journal = {EXCLI journal},
volume = {22},
number = {},
pages = {70-83},
pmid = {36814855},
issn = {1611-2156},
abstract = {Many archaeal and bacterial organisms possess an adaptive immunity system known as CRISPR-Cas. Its role is to recognize and degrade foreign DNA showing high similarity to repeats within the CRISPR array. In recent years computational techniques have been used to identify cas1 genes that are not associated with CRISPR systems, named cas1-solo. Often, cas1-solo genes are present in a conserved neighborhood of PolB-like polymerase genes, which is a characteristic feature of self-synthesizing, eukaryotic transposons of the Polinton class. Nearly all cas1-polB genomic islands are flanked by terminal inverted repeats and direct repeats which correspond to target site duplications. Considering the patchy taxonomic distribution of the identified islands in archaeal and bacterial genomes, they were characterized as a new superfamily of mobile genetic elements and called casposons. Here, we review recent experiments on casposons' mobility and discuss their discovery, classification, and evolutionary relationship with the CRISPR-Cas systems.},
}
@article {pmid36813818,
year = {2023},
author = {Rahmani, B and Kheirandish, MH and Ghanbari, S and Raz, A and Shamsi, F and Davami, F},
title = {Targeting DNA repair pathways with B02 and Nocodazole small molecules to improve CRIS-PITCh mediated cassette integration in CHO-K1 cells.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {3116},
pmid = {36813818},
issn = {2045-2322},
mesh = {Cricetinae ; Animals ; Nocodazole ; *CRISPR-Cas Systems ; CHO Cells ; Cricetulus ; *DNA Repair ; },
abstract = {CRISPR-mediated integration could be used to develop the recombinant CHO (rCHO) cells by knock-in into the hotspot loci. However, low HDR efficiency besides the complex donor design is the main barrier for achieving so. The recently introduced MMEJ-mediated CRISPR system (CRIS-PITCh) uses a donor with short homology arms, being linearized in the cells via two sgRNAs. In this paper, a new approach to improve CRIS-PITCh knock-in efficiency by employing small molecules was investigated. Two small molecules, B02, a Rad51 inhibitor, and Nocodazole, a G2/M cell cycle synchronizer, were used to target the S100A hotspot site using a bxb1 recombinase comprised landing pad in CHO-K1 cells. Following transfection, the CHO-K1 cells were treated with the optimum concentration of one or combination of small molecules, being determined by the cell viability or flow cytometric cell cycle assay. Stable cell lines were generated and the single-cell clones were achieved by the clonal selection procedure. The finding showed that B02 improved the PITCh-mediated integration approximately twofold. In the case of Nocodazole treatment, the improvement was even more significant, up to 2.4-fold. However, the combinatorial effects of both molecules were not substantial. Moreover, according to the copy number and out-out PCR analyses, 5 and 6 of 20 clonal cells exhibited mono-allelic integration in Nocodazole and B02 groups, respectively. The results of the present study as the first attempt to enhance the CHO platform generation by exploiting two small molecules in the CRIS-PITCh system could be used in future researches to establish rCHO clones.},
}
@article {pmid36813454,
year = {2023},
author = {Zhou, C and Li, W and Zhao, Y and Gu, K and Liao, Z and Guo, B and Huang, Z and Yang, M and Wei, H and Ma, P and Li, C and Li, H and Tang, Y and Lei, C and Wang, H},
title = {Sensitive detection of viable salmonella bacteria based on tertiary cascade signal amplification via splintR ligase ligation-PCR amplification-CRISPR/Cas12a cleavage.},
journal = {Analytica chimica acta},
volume = {1248},
number = {},
pages = {340885},
doi = {10.1016/j.aca.2023.340885},
pmid = {36813454},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *Food Microbiology ; *Ligases/genetics ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; RNA ; *Salmonella/isolation &amp; purification ; },
abstract = {Several viable Salmonella bacteria are capable of causing severe human diseases and huge economic losses. In this regard, viable Salmonella bacteria detection techniques that can identify small numbers of microbial cells are highly valuable. Here, we present a detection method (referred to as SPC) based on the amplification of tertiary signals using splintR ligase ligation, PCR amplification and CRISPR/Cas12a cleavage. The detection limit of the SPC assay was 6 copies (HilA RNA) and 10 CFU (cell). Based on Intracellular HilA RNA detection, this assay can be used to distinguish between viable and dead Salmonella. In addition, it is able to detect multiple serotypes of Salmonella and has been successfully used to detect Salmonella in milk or isolated from farms. Overall, this assay is a promising test for viable pathogens detection and biosafety control.},
}
@article {pmid36813257,
year = {2023},
author = {Shen, Y and Hu, K and Yuan, M and Duan, G and Guo, Y and Chen, S},
title = {Progress and bioapplication of CRISPR-based one-step, quantitative and multiplexed infectious disease diagnostics.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxad035},
pmid = {36813257},
issn = {1365-2672},
abstract = {In Vitro Diagnosis (IVD) technology is able to accurately detect pathogens or biomarkers at an initial stage of disease, which works as an important toolbox for disease diagnosis. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system, as an emerging IVD method, plays a crucial role in the field of infectious disease detection due to its superior sensitivity and specificity. Recently, an increasing number of scientists have been devoted to improving the performance of CRISPR-based detection and on-site point-of-care testing (POCT) from extraction-free detection, amplification-free, modified Cas/crRNA complexes, quantitative assays, one-pot detection and multiplexed platform. In this review, we describe the potential roles of these novel approaches and platforms in one-pot methods, quantitative molecular diagnostics as well as multiplexed detection. This review will not only help guide the full use of the CRISPR-Cas tools for quantification, multiplexed detection, POCT and as next-generation diagnostic biosensing platforms but also inspire new ideas, technological advances and engineering strategies to address real-world challenges like the ongoing COVID-19 pandemic.},
}
@article {pmid36812799,
year = {2023},
author = {Antony Ceasar, S and Ignacimuthu, S},
title = {CRISPR/Cas genome editing in plants: Dawn of Agrobacterium transformation for recalcitrant and transgene-free plants for future crop breeding.},
journal = {Plant physiology and biochemistry : PPB},
volume = {196},
number = {},
pages = {724-730},
doi = {10.1016/j.plaphy.2023.02.030},
pmid = {36812799},
issn = {1873-2690},
abstract = {Genome editing tools based on CRISPR/Cas system have been posed to solve many issues in agriculture and improve food production. Genetic engineering by Agrobacterium-mediated transformation has helped to impart specific traits straightaway in many crops. Many GM crops have also reached the field for commercial cultivation. Genetic engineering requires mostly a transformation protocol often mediated by Agrobacterium to insert a specific gene at a random locus. Genome editing with CRISPR/Cas system is a more precise technique for the targeted modification of genes/bases in the host plant genome. Unlike the conventional transformation system, wherein elimination of marker/foreign gene was possible only post-transformation, CRISPR/Cas system could generate transgene-free plants by delivering CRISPR/Cas reagents such as the Cas protein and guide RNAs gRNA(s) preassembled to form ribonucleoproteins (RNPs) into plant cells. CRISPR reagent delivery might be helpful to overcome issues with plants that are recalcitrant to Agrobacterium transformation and the legal hurdles due to the presence of the foreign gene. More recently, the grafting of wild-type shoots to transgenic donor rootstocks developed by the CRISPR/Cas system has reported transgene-free genome editing. CRISPR/Cas system also requires only a small piece of gRNA besides Cas9 or other effectors to target a specific region in the genome. So this system has been projected to be a key contributor to future crop breeding. In this article, we recap the main events of plant transformation, compare the difference between genetic transformation and CRISPR/Cas-mediated genome editing, and draw insights into the future application of the CRISPR/Cas system.},
}
@article {pmid36806764,
year = {2023},
author = {Dong, K and Shu, W and Zhang, J and Cheng, S and Zhang, J and Zhao, R and Hua, T and Zhang, W and Wang, H},
title = {Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging.},
journal = {Biosensors &amp; bioelectronics},
volume = {226},
number = {},
pages = {115118},
doi = {10.1016/j.bios.2023.115118},
pmid = {36806764},
issn = {1873-4235},
mesh = {*Uracil-DNA Glycosidase ; CRISPR-Cas Systems ; *Biosensing Techniques ; Limit of Detection ; DNA, Single-Stranded ; },
abstract = {As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a "double-edged sword" for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10[-6] U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a.},
}
@article {pmid36797479,
year = {2023},
author = {Strong, A},
title = {CRISPR gene-editing therapies for hypertrophic cardiomyopathy.},
journal = {Nature medicine},
volume = {29},
number = {2},
pages = {305-306},
pmid = {36797479},
issn = {1546-170X},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Cardiomyopathy, Hypertrophic ; CRISPR-Cas Systems ; },
}
@article {pmid36775826,
year = {2023},
author = {Mallapaty, S},
title = {Disgraced CRISPR-baby scientist's 'publicity stunt' frustrates researchers.},
journal = {Nature},
volume = {614},
number = {7949},
pages = {599-600},
pmid = {36775826},
issn = {1476-4687},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; CRISPR-Cas Systems ; Research Personnel ; },
}
@article {pmid36774734,
year = {2023},
author = {Wang, S and Li, H and Dong, K and Shu, W and Zhang, J and Zhang, J and Zhao, R and Wei, S and Feng, D and Xiao, X and Zhang, W},
title = {A universal and specific RNA biosensor via DNA circuit-mediated PAM-independent CRISPR/Cas12a and PolyA-rolling circle amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {226},
number = {},
pages = {115139},
doi = {10.1016/j.bios.2023.115139},
pmid = {36774734},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Clinical Relevance ; DNA ; Piwi-Interacting RNA ; Poly A ; RNA ; },
abstract = {Point of care testing (POCT) has important clinical significance for the diagnosis and prognosis evaluation of diseases. At present, the biosensor based on CRISPR/Cas12a has become a powerful diagnostic tool due to its high sensitivity. However, CRISPR/Cas12a requires PAM sequence to recognize target double strand and only can recognize specific sequence, so it is not universal. The current RNA detection techniques either lack consideration for specificity and universality, are expensive and difficult, or both. Therefore, it is crucial to create a CRISPR/Cas12a-based RNA detection system that is easy to use, cheap, specific, and universal in order to further its use in molecular diagnostics. Here, we established a DNA circuit-mediated PAM-independent CRISPR/Cas12a coupled PolyA-rolling circle amplification for RNA detection biosensor, namely DCPRBiosensor. The DCPRBiosensor not only functions as a simple, inexpensive, and highly sensitive RNA detection sensor, but it also boasts innovative specificity and universality features. More importantly, DCPRBiosensor removes the PAM restriction of CRISPR/Cas12a. The DCPRBiosensor's detection limit reached 100 aM and it had a linear relationship between 100 aM and 10 pM. We detected four piRNAs to verify the universality and stability of DCPRBiosensor. Then, we verified that DCPRBiosensor has good discrimination ability for single-base mismatch. Finally, we successfully detected piRNA in DLD-1 and HCT-116 cells and urine mixed samples within 4.5 h. In conclusion, we believe that DCPRBiosensor will have a substantial impact on both the development of CRISPR/as12a's applications and the investigation of the clinical value of piRNA.},
}
@article {pmid36759580,
year = {2023},
author = {Wang, N and Ryan, L and Sardesai, N and Wu, E and Lenderts, B and Lowe, K and Che, P and Anand, A and Worden, A and van Dyk, D and Barone, P and Svitashev, S and Jones, T and Gordon-Kamm, W},
title = {Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum.},
journal = {Nature plants},
volume = {9},
number = {2},
pages = {255-270},
pmid = {36759580},
issn = {2055-0278},
mesh = {*Zea mays/genetics ; *Sorghum/genetics ; Plants, Genetically Modified/genetics ; Edible Grain/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.},
}
@article {pmid36748269,
year = {2023},
author = {Imran, M and Shafiq, S and Tang, X},
title = {CRISPR-Cas9-mediated editing of BADH2 gene triggered fragrance revolution in rice.},
journal = {Physiologia plantarum},
volume = {175},
number = {1},
pages = {e13871},
doi = {10.1111/ppl.13871},
pmid = {36748269},
issn = {1399-3054},
mesh = {*Oryza/genetics ; Odorants ; CRISPR-Cas Systems ; Phenotype ; Genes, Plant ; },
abstract = {Fragrance is one of the most important quality traits for breeding in rice. The natural aroma substance 2-acetyl-1-pyrroline (2-AP) is a key fragrance compound among over 200 volatiles identified in fragrant rice. In addition to rice, there are other plant species that contain a germplasm that naturally produces a fragrant aroma. These other plant species all have lower activity levels of the enzyme BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2). Therefore, improving fragrance efficiency has been a focus of intensive research. Recent studies have engineered BADH2 gene, which is responsible for fragrance trait in non-fragrant cultivars of rice, using CRISPR-Cas9. Although engineering rice BADH2 can be useful for upregulating 2-AP, there are still a lot of restrictions on how it can be applied in practice. In this review article, we discuss the recent developments in BADH2 editing and propose potential future strategies to effectively target BADH2 for transcriptional regulation, with the goal of producing a better fragrance.},
}
@article {pmid36690788,
year = {2023},
author = {Lyu, J},
title = {Cas9-gRNA delivery via grafting.},
journal = {Nature plants},
volume = {9},
number = {2},
pages = {196},
doi = {10.1038/s41477-023-01353-9},
pmid = {36690788},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; RNA, Guide, Kinetoplastida ; },
}
@article {pmid36669950,
year = {2023},
author = {Khirallah, J and Eimbinder, M and Li, Y and Xu, Q},
title = {Clinical progress in genome-editing technology and in vivo delivery techniques.},
journal = {Trends in genetics : TIG},
volume = {39},
number = {3},
pages = {208-216},
doi = {10.1016/j.tig.2022.12.001},
pmid = {36669950},
issn = {0168-9525},
support = {UG3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Treatment Outcome ; },
abstract = {There is wide interest in applying genome-editing tools to prevent, treat, and cure a variety of diseases. Since the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, these techniques have been used in combination with different delivery systems to create highly efficacious treatment options. Each delivery system has its own advantages and disadvantages and is being used for various applications. With the large number of gene-editing applications being studied but very few being brought into the clinic, we review current progress in the field, specifically where genome editing has been applied in vivo and in the clinic, and identify current challenges and areas of future growth.},
}
@article {pmid36651298,
year = {2023},
author = {Yu, L and Marchisio, MA},
title = {CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits.},
journal = {Nucleic acids research},
volume = {51},
number = {3},
pages = {1473-1487},
pmid = {36651298},
issn = {1362-4962},
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; Genes, Synthetic ; Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger ; RNA Stability ; },
abstract = {Type V-A CRISPR-(d)Cas system has been used in multiplex genome editing and transcription regulation in both eukaryotes and prokaryotes. However, mRNA degradation through the endonuclease activity of Cas12a has never been studied. In this work, we present an efficient and powerful tool to induce mRNA degradation in the yeast Saccharomyces cerevisiae via the catalytic activity of (d)Cas12a on pre-crRNA structure. Our results point out that dFnCas12a, (d)LbCas12a, denAsCas12a and two variants (which carry either NLSs or NESs) perform significant mRNA degradation upon insertion of pre-crRNA fragments into the 5'- or 3' UTR of the target mRNA. The tool worked well with two more Cas12 proteins-(d)MbCas12a and Casϕ2-whereas failed by using type VI LwaCas13a, which further highlights the great potential of type V-A Cas proteins in yeast. We applied our tool to the construction of Boolean NOT, NAND, and IMPLY gates, whose logic operations are fully based on the control of the degradation of the mRNA encoding for a reporter protein. Compared to other methods for the regulation of mRNA stability in yeast synthetic gene circuits (such as RNAi and riboswitches/ribozymes), our system is far easier to engineer and ensure very high performance.},
}
@article {pmid36513383,
year = {2023},
author = {Park, SJ and Yoon, S and Choi, EH and Hyeon, H and Lee, K and Kim, KP},
title = {Elevated expression of exogenous RAD51 enhances the CRISPR/Cas9-mediated genome editing efficiency.},
journal = {BMB reports},
volume = {56},
number = {2},
pages = {102-107},
pmid = {36513383},
issn = {1976-670X},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; DNA Breaks, Double-Stranded ; Genome ; },
abstract = {Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to singlestranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology. [BMB Reports 2023; 56(2): 102-107].},
}
@article {pmid36382936,
year = {2023},
author = {Schindele, P and Merker, L and Schreiber, T and Prange, A and Tissier, A and Puchta, H},
title = {Enhancing gene editing and gene targeting efficiencies in Arabidopsis thaliana by using an intron-containing version of ttLbCas12a.},
journal = {Plant biotechnology journal},
volume = {21},
number = {3},
pages = {457-459},
pmid = {36382936},
issn = {1467-7652},
mesh = {*Gene Editing ; *Arabidopsis/genetics ; Introns ; CRISPR-Cas Systems ; Gene Targeting ; },
}
@article {pmid35909208,
year = {2023},
author = {Jayachandran, M and Fei, Z and Qu, S},
title = {Genetic advancements in obesity management and CRISPR-Cas9-based gene editing system.},
journal = {Molecular and cellular biochemistry},
volume = {478},
number = {3},
pages = {491-501},
pmid = {35909208},
issn = {1573-4919},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Obesity Management ; Genetic Therapy/methods ; Body Weight ; },
abstract = {Human genome research has reached new heights in the recent decade thanks to a major advance in genome editing. Genome editing enables scientists to understand better the functions of a single gene and its impact on a wide range of diseases. In brief, genome editing is a technique for introducing alterations into specific DNA sequences, such as insertions, deletions, or base substitutions. Several methods are adopted to perform genome editing and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) systems. Unfortunately, despite substantial progress in understanding the molecular pathways behind obesity, anti-obesity medications are now ineffective. If you are obese, a 10% weight decrease would be preferable to healthy body weight for most people. CRISPR-Cas9, on the other hand, has been shown to reduce body weight by an astonishing 20%. Hence, this updated review elaborates on the molecular basis of obesity, risk factors, types of gene therapy, possible mechanisms, and advantages of the CRISPR-Cas9 system over other methods.},
}
@article {pmid36809461,
year = {2023},
author = {Mitic, D and Bolt, EL and Ivancic-Bace, I},
title = {CRISPR-Cas adaptation in Escherichia coli.},
journal = {Bioscience reports},
volume = {},
number = {},
pages = {},
doi = {10.1042/BSR20221198},
pmid = {36809461},
issn = {1573-4935},
abstract = {Prokaryotes use the adaptive immunity mediated via the Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR associated (CRISPR-Cas) system for protection against invading elements such as phages and plasmids. The immunity is achieved by capturing small DNA fragments or spacers from foreign nucleic acids (protospacers) and integrating them into the host CRISPR locus. This step of CRISPR-Cas immunity called 'naïve CRISPR adaptation' requires the conserved Cas1-Cas2 complex and is often supported by variable host proteins that assist in spacer processing and integration. Bacteria that have acquired new spacers become immune to the same invading elements when reinfected. CRISPR-Cas immunity can also be updated by integrating new spacers from the same invading elements, a process called 'primed adaptation'. Only properly selected and integrated spacers are functional in the next steps of CRISPR immunity when their processed transcripts are used for RNA-guided target recognition and interference (target degradation). Capturing, trimming, and integrating new spacers in the correct orientation are universal steps of adaptation to all CRISPR-Cas systems, but some details are CRISPR-Cas type-specific and species-specific. In this review, we provide an overview of the mechanisms of CRISPR-Cas class 1 type I-E adaptation in Escherichia coli as a general model of adaptation processes (DNA capture and integration) that have been studied in detail. We focus on the role of many host non-Cas proteins involved in adaptation, particularly on the role of homologous recombination.},
}
@article {pmid36807264,
year = {2023},
author = {Nieweglowska, ES and Brilot, AF and Méndez-Moran, M and Kokontis, C and Baek, M and Li, J and Cheng, Y and Baker, D and Bondy-Denomy, J and Agard, DA},
title = {The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {927},
pmid = {36807264},
issn = {2041-1723},
abstract = {To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry. We further demonstrate that recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into similar 2D sheets with p2 and p4 symmetry. We resolve the dominant p2 symmetric state to 3.9 Å by cryo-EM. Our structure reveals a two-domain core, organized into quasi-symmetric tetramers. Flexible loops and termini mediate adaptable inter-tetramer contacts that drive subunit assembly into a lattice and enable the adoption of different symmetric states. While the interfaces between subunits are mostly well packed, two are open, forming channels that likely have functional implications for the transport of proteins, mRNA, and small molecules.},
}
@article {pmid36804928,
year = {2023},
author = {Heidersbach, AJ and Dorighi, KM and Gomez, JA and Jacobi, AM and Haley, B},
title = {A versatile, high-efficiency platform for CRISPR-based gene activation.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {902},
pmid = {36804928},
issn = {2041-1723},
mesh = {Transcriptional Activation ; Cell Line ; *Genome ; *RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with an optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts.},
}
@article {pmid36804154,
year = {2023},
author = {Farhadi, L and Soleimani, F and Fakhari, S and Jalili, A},
title = {The Disruption of Mage-11 Gene via CRISPR/Cas9 Method Induced Apoptosis in the in vitro Model of Prostate Cancer.},
journal = {The Gulf journal of oncology},
volume = {1},
number = {41},
pages = {7-16},
pmid = {36804154},
issn = {2078-2101},
mesh = {Humans ; Male ; Apoptosis/genetics ; *CRISPR-Cas Systems ; *Prostatic Neoplasms/genetics/pathology ; Survivin/genetics ; *Antigens, Neoplasm/genetics ; },
abstract = {BACKGROUNDS AND AIMS: Prostate cancer is the most common malignant cancer among men and is the second deadliest cancer in men after lung cancer. Understanding the molecular mechanisms involved in development and progression of prostate cancer is essential to improve both diagnostic and therapeutic strategies in this regard. In addition, using novel gene therapy-based methods for treatment of cancers has gotten increasing attention during the recent years. Accordingly, this study was aimed to evaluate the inhibitory effect of MAGE-A11 gene, as an important oncogene involved in the pathophysiology of prostate cancer invitro model. The study was also aimed to evaluate the downstream genes related to MAGE-A11.<br><br>MATERIALS AND METHODS: First, MAGE-A11 gene was knocked out in PC-3 cell line using "Clustered regularly interspaced short palindromic repeats" (CRISPR)/ "CRISPR-associated genes 9" (CRISPR/Cas9) method. Next, the expression levels of MAGE-A11, survivin and Ribonucleotide Reductase Small Subunit M2 (RRM2) genes were determined by quantitative polymerase chain reaction (qPCR) technique. The levels of proliferation and apoptosis were also analyzed in PC-3 cells using CCK-8 and Annexin V-PE/7-AAD assays.<br><br>RESULTS: The results showed that the disruption of MAGE-A11 by CRISPR/Cas9 method significantly decreased proliferation (P< 0.0001) and enhanced apoptosis (P< 0.05) in PC-3 cells compared to control group. Moreover, the disruption of MAGE-A11 significantly down regulated the expression levels of survivin and RRM2 genes (P< 0.05).<br><br>CONCLUSION: Our results demonstrated that knocking out MAGE-11 gene by CRISPR/CAS9 technique could efficiently inhibit cell proliferation and induce apoptosis in PC3 cells. Survivin and RRM2 genes might also participated in these processes.},
}
@article {pmid36752591,
year = {2023},
author = {Ansari, AH and Kumar, M and Sarkar, S and Maiti, S and Chakraborty, D},
title = {CriSNPr, a single interface for the curated and de novo design of gRNAs for CRISPR diagnostics using diverse Cas systems.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {36752591},
issn = {2050-084X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Pandemics ; *COVID-19/diagnosis/genetics ; SARS-CoV-2/genetics ; RNA, Guide, Kinetoplastida/genetics ; COVID-19 Testing ; },
abstract = {CRISPR-based diagnostics (CRISPRDx) have improved clinical decision-making, especially during the COVID-19 pandemic, by detecting nucleic acids and identifying variants. This has been accelerated by the discovery of new and engineered CRISPR effectors, which have expanded the portfolio of diagnostic applications to include a broad range of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline necessitates customized detection schemes based on the fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is especially relevant for variant detection, a low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPRDx currently exists. In this manuscript, we fill this lacuna using a unified web server, CriSNPr (CRISPR-based SNP recognition), which provides the user with the opportunity to de novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. Furthermore, we provide a database of curated pre-designed gRNAs as well as target/off-target for all human and SARS-CoV-2 variants reported thus far. CriSNPr has been validated on multiple Cas proteins, demonstrating its broad and immediate applicability across multiple detection platforms. CriSNPr can be found at http://crisnpr.igib.res.in/.},
}
@article {pmid36720378,
year = {2023},
author = {Sasagawa, T and Nagamatsu, T and Shibuya, M},
title = {CRISPR/Cas9-mediated mutations in both a cAMP response element and an ETS-binding site suppress FLT1 gene expression.},
journal = {Experimental cell research},
volume = {424},
number = {1},
pages = {113500},
doi = {10.1016/j.yexcr.2023.113500},
pmid = {36720378},
issn = {1090-2422},
mesh = {Female ; Pregnancy ; Humans ; *Vascular Endothelial Growth Factor Receptor-1/genetics/metabolism ; *Placenta/metabolism ; CRISPR-Cas Systems/genetics ; Endothelial Cells/metabolism ; HEK293 Cells ; Binding Sites/genetics ; Mutation/genetics ; Response Elements ; Gene Expression ; },
abstract = {The Fms-like tyrosine kinase-1 (FLT1) gene is expressed in various types of cells, including vascular endothelial cells and placental trophoblasts, and regulates angiogenesis, inflammation, and pregnancy. However, the basal transcriptional machinery of FLT1 is still not well understood. In this study, we first examined FLT1 promoter activity in three different types of cells, that is, trophoblast-derived cells, vascular endothelial-related cells, and HEK293 cells, using plasmid-based luciferase reporter assays, and showed that a cAMP-response element (CRE) and an ETS-binding site (EBS) are important for FLT1 expression in all cell types. To further examine the importance of these sites at the chromosomal level using HEK293 cells, we introduced CRISPR/Cas9-mediated mutations in these sites on the genomic DNA. HEK293 cells carrying these mutations clearly showed a significant decrease in endogenous FLT1 gene expression. These results suggest that CRE and EBS transcription regulatory elements are crucial for FLT1 gene expression in human tissues.},
}
@article {pmid36805026,
year = {2023},
author = {O'Brien, RE and Bravo, JPK and Ramos, D and Hibshman, GN and Wright, JT and Taylor, DW},
title = {Structural snapshots of R-loop formation by a type I-C CRISPR Cascade.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2023.01.024},
pmid = {36805026},
issn = {1097-4164},
abstract = {Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the "belly" subunits, securing the NTS in place. This "molecular seatbelt" mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.},
}
@article {pmid36801863,
year = {2023},
author = {Ziemann, M and Reimann, V and Liang, Y and Shi, Y and Ma, H and Xie, Y and Li, H and Zhu, T and Lu, X and Hess, WR},
title = {CvkR is a MerR-type transcriptional repressor of class 2 type V-K CRISPR-associated transposase systems.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {924},
pmid = {36801863},
issn = {2041-1723},
abstract = {Certain CRISPR-Cas elements integrate into Tn7-like transposons, forming CRISPR-associated transposon (CAST) systems. How the activity of these systems is controlled in situ has remained largely unknown. Here we characterize the MerR-type transcriptional regulator Alr3614 that is encoded by one of the CAST (AnCAST) system genes in the genome of cyanobacterium Anabaena sp. PCC 7120. We identify a number of Alr3614 homologs across cyanobacteria and suggest naming these regulators CvkR for Cas V-K repressors. Alr3614/CvkR is translated from leaderless mRNA and represses the AnCAST core modules cas12k and tnsB directly, and indirectly the abundance of the tracr-CRISPR RNA. We identify a widely conserved CvkR binding motif 5'-AnnACATnATGTnnT-3'. Crystal structure of CvkR at 1.6 Å resolution reveals that it comprises distinct dimerization and potential effector-binding domains and that it assembles into a homodimer, representing a discrete structural subfamily of MerR regulators. CvkR repressors are at the core of a widely conserved regulatory mechanism that controls type V-K CAST systems.},
}
@article {pmid36800641,
year = {2023},
author = {Zhu, M and Wang, X and Xie, R and Wang, Y and Xu, X and Burger, J and Gong, S},
title = {Guanidinium-Rich Lipopeptide-Based Nanoparticle Enables Efficient Gene Editing in Skeletal Muscles.},
journal = {ACS applied materials &amp; interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.2c21683},
pmid = {36800641},
issn = {1944-8252},
abstract = {Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both in vitro and in vivo. An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail via a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (i.e., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.},
}
@article {pmid36799285,
year = {2023},
author = {Selma García, S and Ntelkis, N and Hieu Nguyen, T and Goossens, A},
title = {Engineering the plant metabolic system by exploiting metabolic regulation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.16157},
pmid = {36799285},
issn = {1365-313X},
abstract = {Plants are the most sophisticated biofactories and sources of food and biofuels present in nature. Engineering the plant metabolism can increase the production of desired compounds and improve the nutritional or commercial value of the plant species. However, this can be challenging because of the complexity of the regulation of multiple genes and the involvement of different protein interactions. To improve metabolic engineering (ME) capabilities, different tools and strategies for rerouting the metabolic pathways have been developed, including genome editing and transcriptional regulation approaches. In addition, cutting-edge technologies have provided new methods for understanding uncharacterized biosynthetic pathways, protein degradation mechanisms, protein-protein interactions, or allosteric feedback, enabling the design of novel ME approaches.},
}
@article {pmid36797756,
year = {2023},
author = {Liu, Z and Shi, M and Ren, Y and Xu, H and Weng, S and Ning, W and Ge, X and Liu, L and Guo, C and Duo, M and Li, L and Li, J and Han, X},
title = {Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy.},
journal = {Molecular cancer},
volume = {22},
number = {1},
pages = {35},
pmid = {36797756},
issn = {1476-4598},
mesh = {Humans ; *CRISPR-Cas Systems ; Genetic Therapy ; Gene Editing ; Immunotherapy ; *Neoplasms/genetics/therapy ; },
abstract = {The incidence and mortality of cancer are the major health issue worldwide. Apart from the treatments developed to date, the unsatisfactory therapeutic effects of cancers have not been addressed by broadening the toolbox. The advent of immunotherapy has ushered in a new era in the treatments of solid tumors, but remains limited and requires breaking adverse effects. Meanwhile, the development of advanced technologies can be further boosted by gene analysis and manipulation at the molecular level. The advent of cutting-edge genome editing technology, especially clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), has demonstrated its potential to break the limits of immunotherapy in cancers. In this review, the mechanism of CRISPR-Cas9-mediated genome editing and a powerful CRISPR toolbox are introduced. Furthermore, we focus on reviewing the impact of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy (knockout or knockin). Finally, we discuss the CRISPR-Cas9-based genome-wide screening for target identification, emphasis the potential of spatial CRISPR genomics, and present the comprehensive application and challenges in basic research, translational medicine and clinics of CRISPR-Cas9.},
}
@article {pmid36637817,
year = {2023},
author = {Paradise, J},
title = {The CRISPR Patent Ruling and Implications for Medicine.},
journal = {JAMA},
volume = {329},
number = {6},
pages = {461-462},
doi = {10.1001/jama.2022.24986},
pmid = {36637817},
issn = {1538-3598},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/legislation &amp; jurisprudence ; *CRISPR-Cas Systems ; *Patents as Topic/legislation &amp; jurisprudence ; United States ; Legislation, Medical ; },
}
@article {pmid36314243,
year = {2023},
author = {Lee, RG and Mazzola, AM and Braun, MC and Platt, C and Vafai, SB and Kathiresan, S and Rohde, E and Bellinger, AM and Khera, AV},
title = {Efficacy and Safety of an Investigational Single-Course CRISPR Base-Editing Therapy Targeting PCSK9 in Nonhuman Primate and Mouse Models.},
journal = {Circulation},
volume = {147},
number = {3},
pages = {242-253},
doi = {10.1161/CIRCULATIONAHA.122.062132},
pmid = {36314243},
issn = {1524-4539},
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Cholesterol, LDL/genetics/metabolism ; Primates/genetics/metabolism ; *Proprotein Convertase 9/genetics/therapeutic use ; Semen/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Hyperlipoproteinemia Type II/genetics/therapy ; Atherosclerosis/genetics/therapy ; },
abstract = {BACKGROUND: VERVE-101 is an investigational in vivo CRISPR base-editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower low-density lipoprotein cholesterol. We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of nonhuman primates and a murine F1 progeny study.<br><br>METHODS: Cynomolgus monkeys were given a single intravenous infusion of a vehicle control (n=10) or VERVE-101 at a dose of 0.75 mg/kg (n=4) or 1.5 mg/kg (n=22) with subsequent follow-up up to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male nonhuman primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu).<br><br>RESULTS: Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 (proprotein convertase subtilisin/kexin type 9) of 67% and 83% and reductions of low-density lipoprotein cholesterol of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days after dosing. Liver safety monitoring noted a transient rise in alanine aminotransferase and aspartate aminotransferase concentrations after infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male nonhuman primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals.<br><br>CONCLUSIONS: VERVE-101 was well tolerated in nonhuman primates and led to 83% lower blood PCSK9 protein and 69% lower low-density lipoprotein cholesterol with durable effects up to 476 days after dosing. These results have supported the initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.},
}
@article {pmid36797253,
year = {2023},
author = {Mi, L and Shi, M and Li, YX and Xie, G and Rao, X and Wu, D and Cheng, A and Niu, M and Xu, F and Yu, Y and Gao, N and Wei, W and Wang, X and Wang, Y},
title = {DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {874},
pmid = {36797253},
issn = {2041-1723},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Mitochondria/genetics ; DNA, Mitochondrial/genetics ; Cytosine ; },
abstract = {Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.},
}
@article {pmid36795971,
year = {2023},
author = {Liu, G and Wang, H and Tong, B and Cui, Y and Vonesch, SC and Dong, H and Zhang, D},
title = {An Efficient CRISPR/Cas12e System for Genome Editing in Sinorhizobium meliloti.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00629},
pmid = {36795971},
issn = {2161-5063},
abstract = {CRISPR/Cas systems have been widely used in the precise and traceless genetic engineering of bacteria. Sinorhizobium meliloti 320 (SM320) is a Gram-negative bacterium with a low efficiency of homologous recombination but a strong ability to produce vitamin B12. Here, a CRISPR/Cas12e-based genome engineering toolkit, CRISPR/Cas12eGET, was constructed in SM320. The expression level of CRISPR/Cas12e was tuned through promoter optimization and the use of a low copy plasmid to adjust Cas12e cutting activity to the low homologous recombination efficiency of SM320, resulting in improved transformation and precision editing efficiencies. Furthermore, the accuracy of CRISPR/Cas12eGET was improved by deleting the ku gene involved in NHEJ repair in SM320. This advance will be useful for metabolic engineering and basic research on SM320, and it further provides a platform to develop the CRISPR/Cas system in strains where the efficiency of homologous recombination is low.},
}
@article {pmid36795789,
year = {2023},
author = {Mohamed, F and Ruiz Rodriguez, LG and Zorzoli, A and Dorfmueller, HC and Raya, RR and Mozzi, F},
title = {Genomic diversity in Fructobacillus spp. isolated from fructose-rich niches.},
journal = {PloS one},
volume = {18},
number = {2},
pages = {e0281839},
pmid = {36795789},
issn = {1932-6203},
mesh = {Fructose/metabolism ; Phylogeny ; *Leuconostocaceae/genetics/metabolism ; *Lactobacillales/genetics ; Genomics ; },
abstract = {The Fructobacillus genus is a group of obligately fructophilic lactic acid bacteria (FLAB) that requires the use of fructose or another electron acceptor for their growth. In this work, we performed a comparative genomic analysis within the genus Fructobacillus by using 24 available genomes to evaluate genomic and metabolic differences among these organisms. In the genome of these strains, which varies between 1.15- and 1.75-Mbp, nineteen intact prophage regions, and seven complete CRISPR-Cas type II systems were found. Phylogenetic analyses located the studied genomes in two different clades. A pangenome analysis and a functional classification of their genes revealed that genomes of the first clade presented fewer genes involved in the synthesis of amino acids and other nitrogen compounds. Moreover, the presence of genes strictly related to the use of fructose and electron acceptors was variable within the genus, although these variations were not always related to the phylogeny.},
}
@article {pmid36795787,
year = {2023},
author = {Yanagawa, Y and Suenaga, Y and Iijima, Y and Endo, M and Sanada, N and Katoh, E and Toki, S and Okino, A and Mitsuhara, I},
title = {Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.},
journal = {PloS one},
volume = {18},
number = {2},
pages = {e0281767},
pmid = {36795787},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plant Cells ; Temperature ; Plant Breeding ; Plants, Genetically Modified/genetics ; Genome, Plant ; },
abstract = {Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.},
}
@article {pmid36795295,
year = {2023},
author = {Nguyen, NH and Bui, TP and Le, NT and Nguyen, CX and Le, MTT and Dao, NT and Phan, Q and Van Le, T and To, HMT and Pham, NB and Chu, HH and Do, PT},
title = {Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum).},
journal = {Planta},
volume = {257},
number = {3},
pages = {57},
pmid = {36795295},
issn = {1432-2048},
mesh = {*Transcription Factors/genetics ; Amino Acids/metabolism ; Sugars/metabolism ; *Solanum lycopersicum/genetics ; CRISPR-Cas Systems ; Fruit/genetics/metabolism ; },
abstract = {Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.},
}
@article {pmid36794977,
year = {2023},
author = {Philippidis, A},
title = {Graphite Bio Pauses Lead Gene Editing Program in Sickle Cell Disease.},
journal = {Human gene therapy},
volume = {34},
number = {3-4},
pages = {90-93},
doi = {10.1089/hum.2023.29234.bfs},
pmid = {36794977},
issn = {1557-7422},
mesh = {Humans ; Gene Editing ; *Graphite ; *Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; },
}
@article {pmid36750230,
year = {2023},
author = {Mohr, M and Damas, N and Gudmand-Høyer, J and Zeeberg, K and Jedrzejczyk, D and Vlassis, A and Morera-Gómez, M and Pereira-Schoning, S and Puš, U and Oliver-Almirall, A and Lyholm Jensen, T and Baumgartner, R and Tate Weinert, B and Gill, RT and Warnecke, T},
title = {The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells.},
journal = {ACS synthetic biology},
volume = {12},
number = {2},
pages = {375-389},
pmid = {36750230},
issn = {2161-5063},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Transgenes/genetics ; Endonucleases/genetics ; DNA End-Joining Repair ; },
abstract = {CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.},
}
@article {pmid36731114,
year = {2023},
author = {Lei, Z and Meng, H and Zhuang, Y and Zhu, Q and Yi, C},
title = {Chemical and Biological Approaches to Interrogate off-Target Effects of Genome Editing Tools.},
journal = {ACS chemical biology},
volume = {18},
number = {2},
pages = {205-217},
doi = {10.1021/acschembio.2c00836},
pmid = {36731114},
issn = {1554-8937},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome ; Genomics ; },
abstract = {Various genome editing tools have been developed for programmable genome manipulation at specified genomic loci. However, it is crucial to comprehensively interrogate the off-target effect induced by these genome editing tools, especially when apply them onto the therapeutic applications. Here, we outlined the off-target effect that has been observed for various genome editing tools. We also reviewed detection methods to determine or evaluate the off-target editing, and we have discussed their advantages and limitations. Additionally, we have summarized current RNA editing tools for RNA therapy and medicine that may serve as alternative approaches for genome editing tools in both research and clinical applications.},
}
@article {pmid36701857,
year = {2023},
author = {Zhang, J and Xiang, J and Liao, L and Jiang, B and Yuan, R and Xiang, Y},
title = {Proximity binding-initiated DNA walker and CRISPR/Cas12a reaction for dual signal amplification detection of thrombin.},
journal = {Talanta},
volume = {256},
number = {},
pages = {124286},
doi = {10.1016/j.talanta.2023.124286},
pmid = {36701857},
issn = {1873-3573},
mesh = {CRISPR-Cas Systems ; Gold ; *Metal Nanoparticles ; Thrombin ; DNA/genetics ; *Biosensing Techniques ; },
abstract = {We report here a highly sensitive fluorescent thrombin biomarker sensing method by integrating the DNA walker and CRISPR/Cas12a system. The presence of thrombin causes the localization of DNA moving arms on AuNP tracks via their proximity bindings with the dye-labeled probes immobilized on AuNPs. With the assistance of the primer and DNA polymerase, the arm sequences move continuously on the AuNP tracks to generate many CRISPR/Cas12a-responsive dsDNAs, which push the dye to move away from AuNPs to restore its fluorescence. Moreover, the dsDNAs can be recognized and cut by the CRISPR/Cas12a to trigger its trans-cleavage activity for cyclically cleaving the fluorescently quenched signal probes on the AuNP tracks, which liberates the dye from AuNPs to further enhance the fluorescence restoration to achieve highly sensitive thrombin assay with detection limit of 29.5 fM. Selectively detecting thrombin against other interference proteins and in diluted serums by such sensing method has also been verified, making it an attractive approach for monitoring other protein biomarkers at low levels for the diagnosis of diseases.},
}
@article {pmid36650116,
year = {2023},
author = {Shively, CA and Dong, F and Mitra, RD},
title = {A Suite of New Strain Construction Vectors for Gene Expression Knockdown in Budding Yeast.},
journal = {ACS synthetic biology},
volume = {12},
number = {2},
pages = {624-633},
doi = {10.1021/acssynbio.2c00547},
pmid = {36650116},
issn = {2161-5063},
mesh = {Animals ; *Saccharomycetales/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Indoleacetic Acids/metabolism ; Gene Expression ; CRISPR-Cas Systems/genetics ; Gene Editing ; Mammals/genetics ; },
abstract = {Numerous tools for gene expression knockdown have been developed and characterized in the model organism Saccharomyces cerevisiae and extended to facilitate studies in multicellular models. To comparatively evaluate the efficacy of these approaches, we systematically applied seven such published constitutive and inducible knockdown strategies to a panel of essential genes encoding nuclear-localized proteins. In this effort, we created the CEAS (C-SWAT for Essential Allele Strains) collection, a suite of tagging vectors for improved utility and ease of strain construction. Of particular note, we adapted an improved auxin inducible degron (AID) protein degradation strategy previously available only in mammalian tissue culture for one-step strain construction in budding yeast by leveraging both the C-SWAT system and CRISPR/Cas9 editing. Taken together, this work presents a toolbox for endogenous gene expression knockdown and allows us to make recommendations on the efficacy and applicability of these tools for the perturbation of essential genes.},
}
@article {pmid36572137,
year = {2023},
author = {Liu, H and Wang, J and Hu, X and Tang, X and Zhang, C},
title = {A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {540},
number = {},
pages = {117201},
doi = {10.1016/j.cca.2022.12.013},
pmid = {36572137},
issn = {1873-3492},
mesh = {Humans ; Recombinases ; *Helicobacter pylori/genetics ; Sensitivity and Specificity ; CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Nucleotidyltransferases ; *Nucleic Acids ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Helicobacter pylori lives in the human stomach and causes gastric cancer and other gastric diseases. The development of molecular technology has facilitated low-cost, rapid, and high-throughput detection of H. pylori.<br><br>MATERIALS AND METHODS: The combination of isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a was used for early diagnosis and monitoring of H. pylori in clinical settings. The UreB genes from 242 H. pylori strains were subjected to cluster analysis, and we designed corresponding RPA primers and screened 2 sets of CRISPR-derived RNAs (crRNAs) for accurate H. pylori recognition. We then performed specificity and sensitivity validation of seven strains using this RPA-CRISPR/Cas12a method. In addition, the cut-off values of this RPA-CRISPR/Cas12a method based on fluorescence values (i.e., RPA-CRISPR/Cas12a-FT) were determined by comparison with quantitative PCR (qPCR), and further experiments comparing different methods were performed using clinical samples.<br><br>RESULTS: We developed a rapid detection system based on the combination of RPA and CRISPR-Cas12a, which was applied to the early diagnosis and monitoring of H. pylori in clinical settings. The RPA-CRISPR/Cas12a system was used to detect the UreB gene. We found that the limit of detection (LOD) for the CRISPR/Cas12a method based on the lateral flow dipstick result (i.e., CRISPR/Cas12a-LFD) was 100 copies, the cut-off value was 1.4; and for CRISPR/Cas12a-FT the LOD was 50 copies. This system was used to assess clinical samples and showed high reproducibility with proof-of-concept sensitivity, and the whole detection process was completed within 40&#x202f;min.<br><br>CONCLUSION: As a diagnostic method that can detect the UreB gene of H. pylori in gastric tissue samples rapidly, sensitively, visually, and in a high throughput manner, our method provides a new diagnostic option for clinicians. This system is ideal for hospitals or testing sites with limited medical resources.},
}
@article {pmid36538017,
year = {2023},
author = {Liu, L and Li, W and Li, J and Zhao, D and Li, S and Jiang, G and Wang, J and Chen, X and Bi, C and Zhang, X},
title = {Circular Guide RNA for Improved Stability and CRISPR-Cas9 Editing Efficiency in Vitro and in Bacteria.},
journal = {ACS synthetic biology},
volume = {12},
number = {2},
pages = {350-359},
doi = {10.1021/acssynbio.2c00381},
pmid = {36538017},
issn = {2161-5063},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Circular ; Escherichia coli/genetics ; Bacteria/genetics ; },
abstract = {Due to its intrinsic RNA properties, guide RNA (gRNA) is the least stable component of the CRISPR-Cas9 complex and is a major target for modification and engineering to increase the stability of the system. While most strategies involve chemical modification and special processes, we created a more stable gRNA with an easy-to-use biological technique. Since circular RNAs are theoretically immune to all RNA exonucleases, we attempted to construct a circular gRNA (cgRNA) employing the autocatalytic splicing mechanism of the RNA cyclase ribozyme. First, the formation of the cgRNA, which has a length requirement, was optimized in vivo in E. coli cells. It was found that a cgRNA with an insert length of 251 bp, designated 251cgRNA, was functional. More importantly, cgRNA increased the editing efficiency of the tested base editors relative to normal linear gRNA. The cgRNAs were more stable in vitro under all tested temperature conditions and maintained their function for 24 h at 37 °C, while linear gRNAs completely lost their activity within 8 h. Enzymatically purified 251cgRNA demonstrated even higher stability, which was obviously presented on gels after 48 h at 37 °C, and maintained partial function. By inserting a homologous arm into the 251cgRNA to 251HAcgRNA cassette, the circularization efficiency reached 88.2%, and the half-life of 251HAcgRNA was 30 h, very similar to that of purified 251cgRNA. This work provides a simple innovative strategy to greatly increase the stability of gRNA both in vivo in E. coli and in vitro, with no additional cost or labor. We think this work is very interesting and might revolutionize the form of gRNAs people are using in research and therapeutic applications.},
}
@article {pmid36471056,
year = {2023},
author = {Ekundayo, B and Torre, D and Beckert, B and Nazarov, S and Myasnikov, A and Stahlberg, H and Ni, D},
title = {Structural insights into the regulation of Cas7-11 by TPR-CHAT.},
journal = {Nature structural &amp; molecular biology},
volume = {30},
number = {2},
pages = {135-139},
pmid = {36471056},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *Tetratricopeptide Repeat ; Protein Conformation ; Nucleic Acid Conformation ; RNA, Bacterial/chemistry ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT (tetratricopeptide repeats (TPR) fused with a CHAT domain). The Craspase complex holds promise as a tool for gene therapy and biomedical research, but its regulation is poorly understood. TPR-CHAT regulates Cas7-11 nuclease activity via an unknown mechanism. In the present study, we use cryoelectron microscopy to determine structures of the Desulfonema magnum (Dm) Craspase complex to gain mechanistic insights into its regulation. We show that DmTPR-CHAT stabilizes crRNA-bound DmCas7-11 in a closed conformation via a network of interactions mediated by the DmTPR-CHAT N-terminal domain, the DmCas7-11 insertion finger and Cas11-like domain, resulting in reduced target RNA accessibility and cleavage.},
}
@article {pmid35581402,
year = {2023},
author = {Leal, AF and Alméciga-Díaz, CJ},
title = {Efficient CRISPR/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA.},
journal = {Gene therapy},
volume = {30},
number = {1-2},
pages = {107-114},
pmid = {35581402},
issn = {1476-5462},
mesh = {Humans ; *Mucopolysaccharidosis IV/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing ; *Chondroitinsulfatases/genetics/metabolism/therapeutic use ; Keratan Sulfate/metabolism/therapeutic use ; Glycosaminoglycans/genetics/metabolism ; },
abstract = {Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.},
}
@article {pmid36795578,
year = {2023},
author = {Nguyen, T and Urrutia-Cabrera, D and Wang, L and Lees, JG and Wang, JH and Hung, SSC and Hewitt, AW and Edwards, TL and McLenachan, S and Chen, FK and Lim, SY and Luu, CD and Guymer, R and Wong, RCB},
title = {Knockout of AMD-associated gene POLDIP2 reduces mitochondrial superoxide in human retinal pigment epithelial cells.},
journal = {Aging},
volume = {15},
number = {},
pages = {},
doi = {10.18632/aging.204522},
pmid = {36795578},
issn = {1945-4589},
abstract = {Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line ARPE-19 with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that it retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 caused a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2 in ARPE-19, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.},
}
@article {pmid36792705,
year = {2023},
author = {Nordling, L},
title = {Putting CRISPR into African hands to future-proof crops.},
journal = {Nature biotechnology},
volume = {41},
number = {2},
pages = {165-166},
doi = {10.1038/s41587-023-01668-0},
pmid = {36792705},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; },
}
@article {pmid36792696,
year = {2023},
author = {},
title = {Base editors.},
journal = {Nature biotechnology},
volume = {41},
number = {2},
pages = {193},
doi = {10.1038/s41587-023-01672-4},
pmid = {36792696},
issn = {1546-1696},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid36792645,
year = {2023},
author = {Eghbalsaied, S and Kues, WA},
title = {CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {2690},
pmid = {36792645},
issn = {2045-2322},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Nocodazole ; Animals, Genetically Modified ; *Ribonuclease H/genetics ; DNA/genetics ; Recombinational DNA Repair ; DNA End-Joining Repair ; Gene Editing/methods ; Gene Knock-In Techniques ; Mammals/genetics ; },
abstract = {On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1-13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3-7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome.},
}
@article {pmid36508310,
year = {2023},
author = {Huang, A and Cui, T and Zhang, Y and Ren, X and Wang, M and Jia, L and Zhang, Y and Wang, G},
title = {CRISPR/Cas9-Engineered Large Fragment Deletion Mutations in Arabidopsis CEP Peptide-Encoding Genes Reveal Their Role in Primary and Lateral Root Formation.},
journal = {Plant &amp; cell physiology},
volume = {64},
number = {1},
pages = {19-26},
doi = {10.1093/pcp/pcac171},
pmid = {36508310},
issn = {1471-9053},
mesh = {*Arabidopsis/metabolism ; CRISPR-Cas Systems/genetics ; Plant Roots/metabolism ; Peptides/genetics/metabolism ; Gene Editing ; Sequence Deletion ; },
abstract = {C-TERMINALLY ENCODED PEPTIDEs (CEPs) are post-translationally modified peptides that play essential roles in root and shoot development, nitrogen absorption, nodule formation and stress resilience. However, it has proven challenging to determine biological activities of CEPs because of difficulties in obtaining loss-of-function mutants for these small genes. To overcome this challenge, we thus assembled a collection of easily detectable large fragment deletion mutants of Arabidopsis CEP genes through the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9-engineered genome editing. This collection was then evaluated for the usability by functionally analyzing the Arabidopsis growth and development with a focus on the root. Most cep mutants displayed developmental defects in primary and lateral roots showing an increased primary root length and an enhanced lateral root number, demonstrating that the genetic resource provides a useful tool for further investigations into the roles of CEPs.},
}
@article {pmid36163549,
year = {2023},
author = {Pihlajamaa, P and Kauko, O and Sahu, B and Kivioja, T and Taipale, J},
title = {A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites.},
journal = {Nature biotechnology},
volume = {41},
number = {2},
pages = {197-203},
pmid = {36163549},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Promoter Regions, Genetic/genetics ; *Gene Editing/methods ; Transcription Factors/genetics/metabolism ; Binding Sites ; CRISPR-Cas Systems/genetics ; },
abstract = {Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness.},
}
@article {pmid36794770,
year = {2023},
author = {Cornaro, L and Banfi, C and Cucinotta, M and Colombo, L and van Dijk, PJ},
title = {Asexual reproduction through seeds: the complex case of diplosporous apomixis.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erad054},
pmid = {36794770},
issn = {1460-2431},
abstract = {Apomixis is considered a potentially revolutionary tool to generate high-quality food at a lower cost and shorter developmental time due to clonal seed production through apomeiosis and parthenogenesis. In the diplosporous type of apomixis, meiotic recombination and reduction are circumvented either by avoiding or failing meiosis or by a mitotic-like division. Here, we review the literature about diplospory, from early cytological studies dating back to the late 19th century to recent genetic findings. We discuss diplosporous developmental mechanisms, including their inheritance. Furthermore, we compare the strategies adopted to isolate the genes controlling diplospory with those to produce mutants forming unreduced gametes. Nowadays, the dramatically improved technologies of long-read sequencing and targeted CRISPR-Cas mutagenesis justify the expectation that natural diplospory genes will soon be identified. Their identification will answer questions such as how the apomictic phenotype can be superimposed upon the sexual pathway and how diplospory genes have evolved. This knowledge will contribute to the application of apomixis in agriculture.},
}
@article {pmid36791369,
year = {2023},
author = {Lembacher, MJ and Arnoldner, C and Landegger, LD},
title = {Patient Acceptance of Novel Therapeutic Options for Sensorineural Hearing Loss-A Pilot Study.},
journal = {Otology &amp; neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology},
volume = {},
number = {},
pages = {},
doi = {10.1097/MAO.0000000000003828},
pmid = {36791369},
issn = {1537-4505},
abstract = {OBJECTIVES: Numerous preclinical experiments over the past years have shown the potential of novel therapeutic approaches for sensorineural hearing loss (SNHL) that are now awaiting clinical translation. In this pilot study, we aimed to evaluate the patient acceptance of these future innovative therapies in individuals with SNHL.<br><br>STUDY DESIGN: Cross-sectional exploratory pilot study.<br><br>SETTING: Tertiary care academic hospital.<br><br>PATIENTS: In total, 72 individuals (43 female and 29 male, 59 affected subjects and 13 parents) with different types of SNHL were surveyed between May 2020 and November 2020.<br><br>INTERVENTION: The interest/willingness to consider new therapeutic options (viral vectors, stem cells, CRISPR/Cas) for themselves or their children was assessed with the help of a questionnaire, and the answers were matched with a quality-of-life score and sociodemographic as well as clinical characteristics.<br><br>MAIN OUTCOME MEASURE: Acceptance of new therapeutic strategies for SNHL in a representative population.<br><br>RESULTS: Even with the currently associated treatment uncertainties, 48 patients (66.7%) suffering from SNHL stated that new therapies could be a potential future option for them. Half of these (24 individuals; 33.3%) expressed high acceptance toward the novel strategies. Subjects with a positive attitude toward new therapies in general and viral vectors specifically were significantly older.<br><br>CONCLUSION: With two-thirds of patients affected by SNHL expressing acceptance toward novel therapies, this pilot study highlights the importance of investigating such attitudes and motivates further translational research to offer additional treatment strategies to this patient population.},
}
@article {pmid36788695,
year = {2023},
author = {Mishra, G and Srivastava, K and Rais, J and Dixit, M and Kumari Singh, V and Chandra Mishra, L},
title = {CRISPR-Cas9: A Potent Gene-editing Tool for the Treatment of Cancer.},
journal = {Current molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.2174/1566524023666230213094308},
pmid = {36788695},
issn = {1875-5666},
abstract = {The prokaryotic adaptive immune system has clustered regularly interspaced short palindromic repeat. CRISPR-associated protein (CRISPR-Cas) genome editing systems have been harnessed. A robust programmed technique for efficient and accurate genome editing and gene targeting has been developed. Engineered cell therapy, in vivo gene therapy, animal modeling, and cancer diagnosis and treatment are all possible applications of this ground-breaking approach. Multiple genetic and epigenetic changes in cancer cells induce malignant cell growth and provide chemoresistance. The capacity to repair or ablate such mutations has enormous potential in the fight against cancer. The CRISPR-Cas9 genome editing method has recently become popular in cancer treatment research due to its excellent efficiency and accuracy. The preceding study has shown therapeutic potential in expanding our anticancer treatments by using CRISPR-Cas9 to directly target cancer cell genomic DNA in cellular and animal cancer models. In addition, CRISPR-Cas9 can combat oncogenic infections and test anticancer medicines. It may design immune cells and oncolytic viruses for cancer immunotherapeutic applications. In this review, these preclinical CRISPRCas9-based cancer therapeutic techniques are summarised, along with the hurdles and advancements in converting therapeutic CRISPR-Cas9 into clinical use. It will increase their applicability in cancer research.},
}
@article {pmid36788244,
year = {2023},
author = {Jin, DY and Chen, X and Liu, Y and Williams, CM and Pedersen, LC and Stafford, DW and Tie, JK},
title = {A genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {828},
pmid = {36788244},
issn = {2041-1723},
mesh = {Humans ; Anticoagulants/pharmacology ; CRISPR-Cas Systems ; *NAD(P)H Dehydrogenase (Quinone)/metabolism ; Ubiquinone/pharmacology/metabolism ; Vitamin K/metabolism ; Vitamin K Epoxide Reductases/genetics/metabolism ; *Warfarin/pharmacology ; *Apoptosis Regulatory Proteins/genetics ; },
abstract = {Vitamin K is a vital micronutrient implicated in a variety of human diseases. Warfarin, a vitamin K antagonist, is the most commonly prescribed oral anticoagulant. Patients overdosed on warfarin can be rescued by administering high doses of vitamin K because of the existence of a warfarin-resistant vitamin K reductase. Despite the functional discovery of vitamin K reductase over eight decades ago, its identity remained elusive. Here, we report the identification of warfarin-resistant vitamin K reductase using a genome-wide CRISPR-Cas9 knockout screen with a vitamin K-dependent apoptotic reporter cell line. We find that ferroptosis suppressor protein 1 (FSP1), a ubiquinone oxidoreductase, is the enzyme responsible for vitamin K reduction in a warfarin-resistant manner, consistent with a recent discovery by Mishima et al. FSP1 inhibitor that inhibited ubiquinone reduction and thus triggered cancer cell ferroptosis, displays strong inhibition of vitamin K-dependent carboxylation. Intriguingly, dihydroorotate dehydrogenase, another ubiquinone-associated ferroptosis suppressor protein parallel to the function of FSP1, does not support vitamin K-dependent carboxylation. These findings provide new insights into selectively controlling the physiological and pathological processes involving electron transfers mediated by vitamin K and ubiquinone.},
}
@article {pmid36787302,
year = {2023},
author = {Blažević, T and Ciotu, CI and Gold-Binder, M and Heiss, EH and Fischer, MJM and Dirsch, VM},
title = {Cultured rat aortic vascular smooth muscle cells do not express a functional TRPV1.},
journal = {PloS one},
volume = {18},
number = {2},
pages = {e0281191},
pmid = {36787302},
issn = {1932-6203},
mesh = {Rats ; Humans ; Animals ; *Capsaicin/pharmacology/metabolism ; *Muscle, Smooth, Vascular/metabolism ; HEK293 Cells ; Aorta/metabolism ; TRPV Cation Channels/metabolism ; Cells, Cultured ; Calcium/metabolism ; },
abstract = {We showed previously that capsaicin, an active compound of chili peppers, can inhibit platelet-derived growth factor-induced proliferation in primary rat vascular smooth muscle cells (VSMCs). The inhibition of BrdU incorporation by capsaicin in these cells was revoked by BCTC, which might be explained by a role of TRPV1 in VSMCs proliferation. To further pursue the hypothesis of a TRPV1-dependent effect of capsaicin, we investigated TRPV1 expression and function. Commercially available antibodies against two different TRPV1 epitopes (N-terminus and C-terminus) were rendered invalid in detecting TRPV1, as shown: i) in western blot experiments using control lysates of TRPV1-expressing (PC-12 and hTRPV1 transfected HEK293T) and TRPV1-downregulated (CRISPR/Cas gene edited A10) cells, and ii) by substantial differences in staining patterns between the applied antibodies using fluorescence confocal microscopy. The TRPV1 agonists capsaicin, resiniferatoxin, piperine and evodiamine did not increase intracellular calcium levels in primary VSMCs and in A10 cells. Using RT qPCR, we could detect a rather low TRPV1 expression in VSMCs at the mRNA level (Cp value around 30), after validating the primer pair in NGF-stimulated PC-12 cells. We conclude that rat vascular smooth muscle cells do not possess canonical TRPV1 channel activity, which could explain the observed antiproliferative effect of capsaicin.},
}
@article {pmid36787118,
year = {2023},
author = {Barrangou, R},
title = {CRISPR Crops and Sustainable Agriculture.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {1},
doi = {10.1089/crispr.2023.0002.editorial},
pmid = {36787118},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Agriculture ; Crops, Agricultural/genetics ; },
}
@article {pmid36787117,
year = {2023},
author = {Petiwala, S and Modi, A and Anton, T and Murphy, E and Kadri, S and Hu, H and Lu, C and Flister, MJ and Verduzco, D},
title = {Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {75-82},
doi = {10.1089/crispr.2022.0093},
pmid = {36787117},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Gene Library ; },
abstract = {Genomewide loss-of-function (LOF) screening using clustered regularly interspaced short palindromic repeats (CRISPR) has facilitated the discovery of novel gene functions across diverse physiological and pathophysiological systems. A challenge with conventional genomewide CRISPR-Cas9 libraries is the unwieldy size (60,000-120,000 constructs), which is resource intensive and prohibitive in some experimental contexts. One solution to streamlining CRISPR screening is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. In this regard, AsCas12a (Cpf1) and its derivatives, for example, enhanced AsCas12a (enAsCas12a), have enabled multiplexed guide arrays to be specifically and efficiently processed for genome editing. Prior studies have established that multiplexed CRISPR-Cas12a libraries perform comparably to the larger equivalent CRISPR-Cas9 libraries, yet the most efficient CRISPR-Cas12a library design remains unresolved. In this study, we demonstrate that CRISPR-Cas12a genomewide LOF screening performed optimally with three guides arrayed per gene construct and could be adapted to robotic cell culture without noticeable differences in screen performance. Thus, the conclusions from this study provide novel insight to streamlining genomewide LOF screening using CRISPR-Cas12a and robotic cell culture.},
}
@article {pmid36787116,
year = {2023},
author = {},
title = {Acknowledgment of Reviewers 2022.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {83-84},
doi = {10.1089/crispr.2023.29158.ack},
pmid = {36787116},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid36716261,
year = {2023},
author = {Mahmood, MA and Mansoor, S},
title = {PASTE: The Way Forward for Large DNA Insertions.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {2-4},
doi = {10.1089/crispr.2023.0001},
pmid = {36716261},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; DNA/genetics ; },
}
@article {pmid36669486,
year = {2023},
author = {Coelho, MA and Cooper, S and Strauss, ME and Karakoc, E and Bhosle, S and Gonçalves, E and Picco, G and Burgold, T and Cattaneo, CM and Veninga, V and Consonni, S and Dinçer, C and Vieira, SF and Gibson, F and Barthorpe, S and Hardy, C and Rein, J and Thomas, M and Marioni, J and Voest, EE and Bassett, A and Garnett, MJ},
title = {Base editing screens map mutations affecting interferon-γ signaling in cancer.},
journal = {Cancer cell},
volume = {41},
number = {2},
pages = {288-303.e6},
doi = {10.1016/j.ccell.2022.12.009},
pmid = {36669486},
issn = {1878-3686},
mesh = {Humans ; Interferon-gamma/genetics/metabolism ; Gene Editing ; *Neoplasms/genetics ; Mutation ; Signal Transduction/genetics ; *Hematologic Neoplasms ; CRISPR-Cas Systems ; },
abstract = {Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive T cells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.},
}
@article {pmid36662546,
year = {2023},
author = {Ureña-Bailén, G and Block, M and Grandi, T and Aivazidou, F and Quednau, J and Krenz, D and Daniel-Moreno, A and Lamsfus-Calle, A and Epting, T and Handgretinger, R and Wild, S and Mezger, M},
title = {Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {5-16},
doi = {10.1089/crispr.2022.0086},
pmid = {36662546},
issn = {2573-1602},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Hemoglobinopathies/genetics/therapy ; Hematopoietic Stem Cells ; *Hematopoietic Stem Cell Transplantation ; },
abstract = {Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy[®] system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for β-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.},
}
@article {pmid36651760,
year = {2023},
author = {Chang, PK},
title = {A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger.},
journal = {Microbiology spectrum},
volume = {11},
number = {1},
pages = {e0464822},
pmid = {36651760},
issn = {2165-0497},
mesh = {*Aspergillus fumigatus ; Aspergillus niger ; CRISPR-Cas Systems ; *Aspergillus nidulans ; },
abstract = {For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.},
}
@article {pmid36629845,
year = {2023},
author = {Leclerc, D and Goujon, L and Jaillard, S and Nouyou, B and Cluzeau, L and Damaj, L and Dubourg, C and Etcheverry, A and Levade, T and Froissart, R and Dréano, S and Guillory, X and Eriksson, LA and Launay, E and Mouriaux, F and Belaud-Rotureau, MA and Odent, S and Gilot, D},
title = {Gene Editing Corrects In Vitro a G &gt; A GLB1 Transition from a GM1 Gangliosidosis Patient.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {17-31},
doi = {10.1089/crispr.2022.0045},
pmid = {36629845},
issn = {2573-1602},
mesh = {Humans ; *Gangliosidosis, GM1/therapy/drug therapy ; beta-Galactosidase/genetics/chemistry/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Alleles ; },
abstract = {Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of β-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on β-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.},
}
@article {pmid36629744,
year = {2023},
author = {Li, S and Sun, Y and Du, M and Shangguan, A and Liu, Z and Li, W and Lina, L and Liu, W and Zhang, S and Han, H},
title = {Graphene Oxide Nanoparticles Combined with CRISPR/Cas9 System Enable Efficient Inhibition of Pseudorabies Virus.},
journal = {Bioconjugate chemistry},
volume = {34},
number = {2},
pages = {326-332},
doi = {10.1021/acs.bioconjchem.2c00570},
pmid = {36629744},
issn = {1520-4812},
mesh = {Animals ; *Herpesvirus 1, Suid/genetics ; CRISPR-Cas Systems/genetics ; Virus Replication ; *Nanoparticles ; Antiviral Agents/pharmacology ; },
abstract = {We describe an application where graphene oxide nanoparticles (GONs) enable combined inhibition of Pseudorabies Virus (PRV) through delivery of a CRISPR/Cas9 system for targeted cleaving of a PRV genome and direct interaction with viral particles. The sheeted GONs could load CRISPR plasmid DNA (pDNA) to form a small sized, near-spheroidal GONs-CRISPR complex, which enables CRISPR pDNA efficient intracellular delivery and transient expression under serum conditions. Cell studies showed that GONs-CRISPR could allow rapid cellular uptake, endolysosomes escape, and nucleus transport within 3 h. Virus studies demonstrated that the pure GONs have antiviral activity and GONs-CRISPR could significantly inhibit PRV replication and result in progeny PRV decreasing by approximately 4000 times in infected host cells. Transmission electron microscopy (TEM) imaging showed that GONs-CRISPR could destroy the PRV structures by directly interacting with viral particles. This GONs-based strategy may extend the advanced application of the CRISPR system for antiviral action.},
}
@article {pmid36622174,
year = {2023},
author = {Qiu, X and Liu, X and Wang, R and Ma, X and Han, L and Yao, J and Li, Z},
title = {Accurate, Sensitive, and Rapid Detection of Pseudomonas aeruginosa Based on CRISPR/Cas12b with One Fluid-Handling Step.},
journal = {Microbiology spectrum},
volume = {11},
number = {1},
pages = {e0352322},
pmid = {36622174},
issn = {2165-0497},
mesh = {Humans ; *Pseudomonas aeruginosa/genetics ; CRISPR-Cas Systems ; Real-Time Polymerase Chain Reaction/methods ; *Cystic Fibrosis/microbiology ; DNA, Bacterial/analysis ; },
abstract = {Pseudomonas aeruginosa is a major bacterial pathogen causing nosocomial infections and accounts for morbidity and mortality among patients with cystic fibrosis. An accurate, sensitive, and rapid method to detect P. aeruginosa is critical for the early control of infection and patient management. In this study, we established a P. aeruginosa clustered regularly interspaced short palindromic repeats testing in one pot (CRISPR-top) assay which detected P. aeruginosa with one fluid-handling step in one tube. The reaction was performed isothermally within 1 h; thus, specific instruments were not required. The optimal reaction conditions of this assay were determined to be a temperature of 55°C; working concentrations of 1 μM for the forward inner primer and backward inner primer, 0.5 μM for the loop forward primer and loop backward primer, and 0.25 μM for the forward outer primer and backward outer primer; as well as a 2 μM concentration single-stranded DNA reporter molecules. In terms of specificity, our assay showed 100% inclusivity and exclusivity among 48 strains, including 15 P. aeruginosa clinical isolates and 33 non-P. aeruginosa strains. The limit of detection of our method was 10 copies per reaction mixture. Forty-six human sputum specimens from patients with respiratory symptoms were tested. Using the results of quantitative real-time PCR as the gold standard, our method showed 85.3% (29/34) sensitivity, 100% (12/12) specificity, a positive predictive value of 100% (29/29), and a negative predictive value of 70.6% (12/17). In summary, the P. aeruginosa CRISPR-top assay developed in the present study is a high-efficiency alternative tool for the accurate and rapid detection of P. aeruginosa, especially in resource-limited settings. IMPORTANCE This study reports a P. aeruginosa CRISPR-top assay which can precisely identify P. aeruginosa using nucleic acids from pure cultures or clinical samples in one pot with one fluid-handling step. The P. aeruginosa CRISPR-top reaction is suitable for on-site testing, and its diagnostic performance can be compared with that of qPCR.},
}
@article {pmid36515555,
year = {2023},
author = {Zheng, XN and Wang, JL and Elsheikha, HM and Wang, M and Zhang, ZW and Sun, LX and Wang, XC and Zhu, XQ and Li, TT},
title = {Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System.},
journal = {Microbiology spectrum},
volume = {11},
number = {1},
pages = {e0307822},
pmid = {36515555},
issn = {2165-0497},
mesh = {Animals ; Mice ; *Toxoplasma/genetics ; Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Persistent Infection ; *Toxoplasmosis ; },
abstract = {The analysis of the subcellular localization and function of dense granule proteins (GRAs) is of central importance for the understanding of host-parasite interaction and pathogenesis of Toxoplasma gondii infection. Here, we identified 15 novel GRAs and used C-terminal endogenous gene tagging to determine their localization at the intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in the tachyzoites and at the periphery of the bradyzoites-containing cysts. The functions of the 15 gra genes were examined in type I RH strain and 5 of these gra genes were also evaluated in the cyst-forming type II Pru strain. The 15 novel gra genes were successfully disrupted by using CRISPR-Cas9 mediated homologous recombination and the results showed that 13 gra genes were not individually essential for T. gondii replication in vitro or virulence in mice during acute and chronic infection. Intriguingly, deletion of TGME49_266410 and TGME49_315910 in both RH and Pru strains decreased the parasite replication in vitro and attenuated its virulence, and also reduced the cyst-forming ability of the Pru strain in mice during chronic infection. Comparison of the transcriptomic profiles of the 15 gra genes suggests that they may play roles in other life cycle stages and genotypes of T. gondii. Taken together, our findings improve the understanding of T. gondii pathogenesis and demonstrate the involvement of two novel GRAs, TGME49_266410 and TGME49_315910, in the parasite replication and virulence. IMPORTANCE Dense granule proteins (GRAs) play important roles in Toxoplasma gondii pathogenicity. However, the functions of many putative GRAs have not been elucidated. Here, we found that 15 novel GRAs are secreted into intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in tachyzoites and are located at the periphery of the bradyzoite-containing cysts. TGME49_266410 and TGME49_315910 were crucial to the growth of RH and Pru strains in vitro. Deletion of TGME49_266410 and TGME49_315910 attenuated the parasite virulence in mice. However, disruption of other 13 gra genes did not have a significant impact on the proliferation and pathogenicity of T. gondii in vitro or in vivo. The marked effects of the two novel GRAs (TGME49_266410 and TGME49_315910) on the in vitro growth and virulence of T. gondii are notable and warrant further elucidation of the temporal and spatial dynamics of translocation of these two novel GRAs and how do they interfere with host cell functions.},
}
@article {pmid36493370,
year = {2023},
author = {Hauk, P and Weeks, R and Ostermeier, M},
title = {A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo in Escherichia coli.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {43-51},
doi = {10.1089/crispr.2022.0041},
pmid = {36493370},
issn = {2573-1602},
mesh = {Humans ; *Escherichia coli/genetics ; *Ribonuclease III/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; RNA ; },
abstract = {Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range of important functions and are found in all organisms, yet a simple and high-throughput in vivo method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on in vitro RNA analysis or in vivo methods that are not suitable for high-throughput analysis. In this study, we describe our development of a deactivated Cas9 (dCas9)-based in vivo assay for RNase III activity that utilizes RNase III's cleavage of the 5'-untranslated region (UTR) of its own messenger RNA. The key molecule in the system is a hybrid guide RNA (gRNA) between the 5'-UTR of RNase III and gGFP, a gRNA that works with dCas9 to repress GFP expression. This fusion must be cleaved by RNase III for full GFP repression. Our system uses GFP fluorescence to report on Escherichia coli RNase III activity in culture and on an individual cell basis, making it effective for selecting individual cells through fluorescence-activated cell sorting. Homology between enzymes within the RNase III family suggests this assay might be adapted to measure the activity of other enzymes in the RNase III family such as human Dicer or Drosha.},
}
@article {pmid36453226,
year = {2023},
author = {Wang, H and Wang, Y and Luo, Z and Lin, X and Liu, M and Wu, F and Shao, H and Zhang, W},
title = {Advances in Off-Target Detection for CRISPR-Based Genome Editing.},
journal = {Human gene therapy},
volume = {34},
number = {3-4},
pages = {112-128},
doi = {10.1089/hum.2022.198},
pmid = {36453226},
issn = {1557-7422},
abstract = {The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome editing system exhibits marked potential for both gene editing and gene therapy, and its continuous improvement contributes to its great clinical potential. However, the largest hindrance to its application in clinical practice is the presence of off-target effects (OTEs). Thus, in addition to continuous optimization of the CRISPR system to reduce and eventually eliminate OTEs, further development of unbiased genome-wide detection of OTEs is key for its successful clinical application. This article summarizes detection strategies for OTEs of different CRISPR systems, to provide detailed guidance for the detection of OTEs in CRISPR-based genome editing.},
}
@article {pmid36342783,
year = {2023},
author = {Gurel, F and Wu, Y and Pan, C and Cheng, Y and Li, G and Zhang, T and Qi, Y},
title = {On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice.},
journal = {The CRISPR journal},
volume = {6},
number = {1},
pages = {62-74},
doi = {10.1089/crispr.2022.0072},
pmid = {36342783},
issn = {2573-1602},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Genome, Plant ; Mutation ; },
abstract = {The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.},
}
@article {pmid36781248,
year = {2023},
author = {Ling, C and Chang, Y and Wang, X and Cao, X and Tu, Q and Liu, B and Huang, S},
title = {Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations.},
journal = {Analytica chimica acta},
volume = {1247},
number = {},
pages = {340881},
doi = {10.1016/j.aca.2023.340881},
pmid = {36781248},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Proteolysis ; Mutation ; *Recombinases ; DNA Primers ; },
abstract = {Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. This highlights the critical need for speedy and accurate technology capable of detecting single-base alterations. Using clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a), two fundamental approaches for getting 100% differentiation of single-base mutations have been established, by which fluorescence signals could be detected for variants but not for wild strains. The first method required both polymerase chain reaction (PCR) and CRISPR/Cas12a cleavage: By introducing a mismatched base at the 3' end of the primers and adjusting the PCR settings, the wild strain strand amplifications were completely blocked prior to CRISPR/Cas12a cleavage. The parameters for Method 1 (PCR + CRISPR/Cas12a) could be easily controlled and adjusted to attain a sensitivity of one copy (about 6 copies μL-1). The second method included isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12a cleavage: By introducing an extra mismatched base adjacent to the single-base mutant site by RPA (IMAS-RPA), the RPA products from the wild strains were rendered incapable of triggering the cleavage activity of CRISPR/Cas12a. Method 2 (IMAS-RPA) was rapid and easy to implement (can be finished within 1 h). Because each method has its own set of advantages, the laboratory environment-appropriate methods can be selected independently. Both approaches are expected to aid in clinical diagnosis to some extent in the near future.},
}
@article {pmid36735954,
year = {2023},
author = {Zhou, H and Xu, Z and He, L and Wang, Z and Zhang, T and Hu, T and Huang, F and Chen, D and Li, Y and Yang, Y and Huang, X},
title = {Coupling CRISPR/Cas12a and Recombinase Polymerase Amplification on a Stand-Alone Microfluidics Platform for Fast and Parallel Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {95},
number = {6},
pages = {3379-3389},
doi = {10.1021/acs.analchem.2c04713},
pmid = {36735954},
issn = {1520-6882},
mesh = {Humans ; *Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Microfluidics ; Sensitivity and Specificity ; *Papillomavirus Infections/diagnosis ; CRISPR-Cas Systems/genetics ; Nucleotidyltransferases/genetics ; DNA, Viral/genetics ; },
abstract = {Timely identification of human papillomavirus (HPV) infection is crucial for the prevention of cervical cancer. Current HPV detection methods mainly rely on polymerase chain reaction (PCR), which often requires bulky equipment and a long assay time. In this work, we report a heating-membrane-assisted multiplexed microfluidics platform that couples recombinase polymerase amplification (RPA) and CRISPR technology (termed M3-CRISPR) for fast and low-cost detection of multiple HPV subtypes. The heating membrane can provide convenient temperature control for the on-chip RPA and CRISPR assays. This stand-alone system allows simultaneous detection of HPV16 and HPV18 with high specificity and detection sensitivity (0.5 nM and 1 × 10[-18] M for unamplified and amplified plasmids, respectively) in 30 min with a fluorescence-based readout. Furthermore, we introduced an optimized lateral flow dipstick (LFD) into the portable system to allow visualized detection of HPV DNA. The LFD-based readout also reached a detection sensitivity of 1 × 10[-18] M for amplified plasmids and realized successful detection of HPV subtypes in the clinical samples. Finally, we established an automatic microfluidic system that enables the sample-in-answer-out detection of HPV subtypes. We believe that this fast, convenient, and affordable molecular diagnostic platform can serve as a useful tool in point-of-care testing of HPV or other pathogens.},
}
@article {pmid36724385,
year = {2023},
author = {Zhao, Y and Chen, D and Xu, Z and Li, T and Zhu, J and Hu, R and Xu, G and Li, Y and Yang, Y and Liu, M},
title = {Integrating CRISPR-Cas12a into a Microfluidic Dual-Droplet Device Enables Simultaneous Detection of HPV16 and HPV18.},
journal = {Analytical chemistry},
volume = {95},
number = {6},
pages = {3476-3485},
doi = {10.1021/acs.analchem.2c05320},
pmid = {36724385},
issn = {1520-6882},
mesh = {Humans ; *Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; CRISPR-Cas Systems ; *Papillomavirus Infections/diagnosis ; Microfluidics ; Human Papillomavirus Viruses ; Nucleotidyltransferases ; Recombinases ; Nucleic Acid Amplification Techniques ; },
abstract = {Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10[-18] M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.},
}
@article {pmid36716431,
year = {2023},
author = {Zhou, Y and Xie, S and Liu, B and Wang, C and Huang, Y and Zhang, X and Zhang, S},
title = {Chemiluminescence Sensor for miRNA-21 Detection Based on CRISPR-Cas12a and Cation Exchange Reaction.},
journal = {Analytical chemistry},
volume = {95},
number = {6},
pages = {3332-3339},
doi = {10.1021/acs.analchem.2c04484},
pmid = {36716431},
issn = {1520-6882},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Luminescence ; Hydrogen Peroxide/chemistry ; DNA/genetics ; *MicroRNAs/genetics/chemistry ; *Biosensing Techniques/methods ; },
abstract = {Herein, a chemiluminescence (CL) biosensor based on CRISPR-Cas12a and cation exchange reaction was constructed to detect the biomarker microRNA-21 (miRNA-21). The rolling circle amplification (RCA) reaction was introduced to convert each target RNA strand into a long single-stranded DNA with repeated sequences, which acted as triggers to initiate the transcleavage activity of CRISPR-Cas12a. The activated Cas12a could cleave the biotinylated linker DNA of CuS nanoparticles (NPs) to inhibit the binding of CuS NPs to streptavidin immobilized on the surface of the microplate, which strongly reduced the generation of Cu[2+] from a cation exchange between CuS NPs and AgNO3, and thus efficiently suppressed the CL of Cu[2+]-luminol-H2O2 system, giving a "signal off" biosensor. With the multiple amplification, the detection limit of the developed sensor for miRNA-21 reached 16 aM. In addition, this biosensor is not only suitable for a professional chemiluminescence instrument but also for a smartphone used as a detection tool for the purpose of portable and low-cost assay. This method could be used to specifically detect quite a low level of miRNA-21 in human serum samples and various cancer cells, indicating its potential in ultrasensitive molecular diagnostics.},
}
@article {pmid36781955,
year = {2023},
author = {Awwad, SW and Serrano-Benitez, A and Thomas, JC and Gupta, V and Jackson, SP},
title = {Revolutionizing DNA repair research and cancer therapy with CRISPR-Cas screens.},
journal = {Nature reviews. Molecular cell biology},
volume = {},
number = {},
pages = {},
pmid = {36781955},
issn = {1471-0080},
abstract = {All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most notably cancer, these mechanisms have been studied extensively, yet new DNA repair and/or DDR factors and functional interactions between them are still being uncovered. The emergence of CRISPR technologies and CRISPR-based genetic screens has enabled genome-scale analyses of gene-gene and gene-drug interactions, thereby providing new insights into cellular processes in distinct DDR-deficiency genetic backgrounds and conditions. In this Review, we discuss the mechanistic basis of CRISPR-Cas genetic screening approaches and describe how they have contributed to our understanding of DNA repair and DDR pathways. We discuss how DNA repair pathways are regulated, and identify and characterize crosstalk between them. We also highlight the impacts of CRISPR-based studies in identifying novel strategies for cancer therapy, and in understanding, overcoming and even exploiting cancer-drug resistance, for example in the contexts of PARP inhibition, homologous recombination deficiencies and/or replication stress. Lastly, we present the DDR CRISPR screen (DDRcs) portal , in which we have collected and reanalysed data from CRISPR screen studies and provide a tool for systematically exploring them.},
}
@article {pmid36780840,
year = {2023},
author = {Abavisani, M and Khayami, R and Hoseinzadeh, M and Kodori, M and Kesharwani, P and Sahebkar, A},
title = {CRISPR-Cas system as a promising player against bacterial infection and antibiotic resistance.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {68},
number = {},
pages = {100948},
doi = {10.1016/j.drup.2023.100948},
pmid = {36780840},
issn = {1532-2084},
abstract = {The phenomenon of antibiotic resistance (AR) and its increasing global trends and destructive waves concerns patients and the healthcare system. In order to combat AR, it is necessary to explore new strategies when the current antibiotics fail to be effective. Thus, knowing the resistance mechanisms and appropriate diagnosis of bacterial infections may help enhance the sensitivity and specificity of novel strategies. On the other hand, resistance to antimicrobial compounds can spread from resistant populations to susceptible ones. Antimicrobial resistance genes (ARGs) significantly disseminate AR via horizontal and vertical gene transfer. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is a member of the bacterial immune system with the ability to remove the ARGs; therefore, it can be introduced as an effective and innovative strategy in the battle against AR. Here, we reviewed CRISPR-based bacterial diagnosis technologies. Moreover, the strategies to battle AR based on targeting bacterial chromosomes and resistance plasmids using the CRISPR-Cas system have been explained. Besides, we have presented the limitations of CRISPR delivery and potential solutions to help improve the future development of CRISPR-based platforms.},
}
@article {pmid36780784,
year = {2023},
author = {Sodani, M and Misra, CS and Rath, D and Kulkarni, S},
title = {Harnessing CRISRP-Cas9 as an anti-mycobacterial system.},
journal = {Microbiological research},
volume = {270},
number = {},
pages = {127319},
doi = {10.1016/j.micres.2023.127319},
pmid = {36780784},
issn = {1618-0623},
abstract = {Rapid emergence of drug resistance has posed new challenges to the treatment of mycobacterial infections. As the pace of development of new drugs is slow, alternate treatment approaches are required. Recently, CRISPR-Cas systems have emerged as potential antimicrobials. These sequence-specific nucleases introduce double strand cuts in the target DNA, which if left unrepaired, prove fatal to the host. For most bacteria, homologous recombination repair (HRR) is the only pathway for repair and survival. Mycobacteria is one of the few bacteria which possesses the non-homologous end joining (NHEJ) system in addition to HRR for double strand break repair. To assess the antimicrobial potential of CRISPR-system, Cas9-induced breaks were introduced in the genome of Mycobacterium smegmatis and the survival was studied. While the single strand breaks were efficiently repaired, the organism was unable to repair the double strand breaks efficiently. In a mixed population of antibiotic-resistant and sensitive mycobacterial cells, selectively targeting a factor that confers hygromycin resistance, turned the entire population sensitive to the drug. Further, we demonstrate that the sequence-specific targeting could also be used for curing plasmids from mycobacterium cells. Considering the growing interest in nucleic acid-based therapy to curtail infections and combat antimicrobial resistance, our data shows that CRISPR-systems hold promise for future use as an antimicrobial against drug-resistant mycobacterial infections.},
}
@article {pmid36773162,
year = {2023},
author = {Sugi, T},
title = {Genome Editing of C. elegans.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {389-396},
pmid = {36773162},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; *Caenorhabditis elegans/genetics ; CRISPR-Cas Systems/genetics ; Genome ; Eukaryota/genetics ; },
abstract = {Caenorhabditis elegans, a 1 mm long free-living nematode, is a traditional model animal for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle, well-annotated genome, simple morphology, and transparency. Recently, genome editing technologies have been increasingly used in C. elegans, thereby facilitating their genetic analyses. Here, I introduce a protocol frequently used in C. elegans genome editing.},
}
@article {pmid36773161,
year = {2023},
author = {Sasakura, Y and Horie, T},
title = {Improved Genome Editing in the Ascidian Ciona with CRISPR/Cas9 and TALEN.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {375-388},
doi = {10.1007/978-1-0716-3016-7_28},
pmid = {36773161},
issn = {1940-6029},
mesh = {Animals ; Gene Editing/methods ; *Ciona/metabolism ; *Ciona intestinalis/genetics/metabolism ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Transcription Activator-Like Effectors/genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; },
abstract = {The ascidian Ciona intestinalis type A (or Ciona robusta) is an important organism for elucidating the mechanisms that make the chordate body plan. CRISPR/Cas9 and TAL effector nuclease (TALEN) are widely used to quickly address genetic functions in Ciona. Our previously reported method of CRISPR/Cas9-mediated mutagenesis in this animal has inferior mutation rates compared to those of TALENs. We here describe an updated way to effectively mutate genes with CRISPR/Cas9 in Ciona. Although the construction of TALENs is much more laborious than that of CRISPR/Cas9, this technique is useful for tissue-specific knockouts that are not easy even by the optimized CRISPR/Cas9 method.},
}
@article {pmid36773160,
year = {2023},
author = {Tsubota, T and Sakai, H and Sezutsu, H},
title = {Genome Editing of Silkworms.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {359-374},
pmid = {36773160},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Bombyx/genetics/metabolism ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {Silkworm is a lepidopteran insect that has been used as a model for a wide variety of biological studies. The microinjection technique is available, and it is possible to cause transgenesis as well as target gene disruption via the genome editing technique. TALEN-mediated knockout is especially effective in this species. We also succeeded in the precise and efficient integration of a donor vector using the precise integration into target chromosome (PITCh) method. Here we describe protocols for ZFN (zinc finger nuclease)-, TALEN (transcription activator-like effector nuclease)-, and CRISPR/Cas9-mediated genome editing as well as the PITCh technique in the silkworm. We consider that all of these techniques can contribute to the further promotion of various biological studies in the silkworm and other insect species.},
}
@article {pmid36773157,
year = {2023},
author = {Kamachi, Y and Kawahara, A},
title = {CRISPR-Cas9-Mediated Genome Modifications in Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {313-324},
pmid = {36773157},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome/genetics ; DNA Repair ; DNA End-Joining Repair/genetics ; },
abstract = {CRISPR-Cas9 genome editing technology has been successfully applied to generate various genetic modifications in zebrafish. The CRISPR-Cas9 system, which originally consisted of three components, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9, efficiently induces DNA double-strand breaks (DSBs) at targeted genomic loci, often resulting in frameshift-mediated target gene disruption (knockout). However, it remains difficult to perform the targeted integration of exogenous DNA fragments (knock-in) with CRISPR-Cas9. DSBs can be restored through DNA repair mechanisms, such as nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR). One of our two research groups established a method for the precise MMEJ-mediated targeted integrations of exogenous genes containing homologous microhomology sequences flanking a targeted genomic locus in zebrafish. The other group recently developed a method for knocking in ~200 nt sequences encoding composite tags using long single-stranded DNA (ssDNA) donors. This chapter summarizes the CRISPR-Cas9-mediated genome modification strategy in zebrafish.},
}
@article {pmid36773156,
year = {2023},
author = {Han, JY and Lee, HJ},
title = {Genome Editing Mediated by Primordial Germ Cell in Chicken.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {301-312},
pmid = {36773156},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Germ Cells ; },
abstract = {Genome editing technology has facilitated the studies on exploring specific gene functions in diverse living organisms. The technology has also contributed to creating high-value livestock in industry fields in terms of enhancing productivity or acquiring disease resistance. Particularly, applying genome editing technologies in avian species has been emphasized in both academic and industrial fields due to their unique developmental patterns as well as application possibilities. To accomplish genome editing in avian species, gene integration into chicken primordial germ cell (PGC) genome using a virus or transposition systems has been widely used, and recently developed programmable genome editing technologies including clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) systems enable to edit the genetic information precisely for maximizing the application potentials of avian species. In these regards, this chapter will cover the methods for producing genome-edited chickens, particularly by CRISPR/Cas9 technologies allowing targeted gene insertion, gene knockout, and gene tagging.},
}
@article {pmid36773155,
year = {2023},
author = {Tanihara, F and Hirata, M and Otoi, T},
title = {GEEP Method: An Optimized Electroporation-Mediated Gene Editing Approach for Establishment of Knockout Pig Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {293-300},
pmid = {36773155},
issn = {1940-6029},
mesh = {Humans ; Animals ; Swine/genetics ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Mutation ; Zygote/metabolism ; Electroporation/methods ; },
abstract = {Pigs are excellent large animal models owing to their several physiological and anatomical similarities to humans. Somatic cell nuclear transfer using gene-modified cells is the mainstream approach for generating genetically modified pigs. Recent advances in improving gene editors such as the CRISPR/Cas9 system have enabled direct gene modification in zygotes/embryos. Here, we describe the gene editing by electroporation of Cas9 protein (GEEP) method, an optimized electroporation-mediated method for the introduction of CRISPR/Cas9 into porcine zygotes/embryos. The simplicity and micromanipulation-free procedures are the major advantages of this method.},
}
@article {pmid36773154,
year = {2023},
author = {Watanabe, M and Nagashima, H},
title = {Genome Editing of Pig.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {269-292},
doi = {10.1007/978-1-0716-3016-7_21},
pmid = {36773154},
issn = {1940-6029},
mesh = {Humans ; Animals ; Swine/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; Nuclear Transfer Techniques ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Pigs have anatomical and physiological characteristics similar to humans; therefore, genetically modified pigs have the potential to become a valuable bioresource in biomedical research. In fact, considering the increasing need for translational research, pigs are useful for studying intractable diseases, organ transplantation, and regenerative medicine as large-scale experimental animals with excellent potential for extrapolation to humans. With the advent of zinc finger nucleases (ZFNs), breakthroughs in genome editing tools such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) have facilitated the efficient generation of genetically modified pigs. Genome editing has been used in pigs for more than 10 years; now, along with knockout pigs, knock-in pigs are also gaining increasing importance. In this chapter, we describe the establishment of gene-modified cells (nuclear donor cells), which are necessary for gene knockout and production of knock-in pigs via somatic cell nuclear transplantation, as well as the production of gene knockout pigs using a simple cytoplasmic injection method.},
}
@article {pmid36773153,
year = {2023},
author = {Honda, A},
title = {Gene Targeting in Rabbits: Single-Step Generation of Knockout Rabbits by Microinjection of CRISPR/Cas9 Plasmids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {255-267},
pmid = {36773153},
issn = {1940-6029},
mesh = {Male ; Animals ; Rabbits ; Female ; *CRISPR-Cas Systems ; Microinjections ; Plasmids/genetics ; *Gene Targeting/methods ; DNA ; Mammals/genetics ; },
abstract = {The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This chapter describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into a pseudopregnant rabbits, and offspring are born within approximately 29-30 days. The genomic DNA of the offspring is then examined to check what type of genetic modifications has occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This chapter summarizes specifically how to carry out gene disruptions in rabbits.},
}
@article {pmid36773150,
year = {2023},
author = {Kaneko, T},
title = {Genome Editing of Rat.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {223-231},
pmid = {36773150},
issn = {1940-6029},
mesh = {Rats ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {Many genetically engineered rat strains have been produced by the development of genome editing technology, although it used to be technical difficulty and low production efficiency. Knockout and knock-in strains can be simple and quick produced using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Presently, genome edited strains have been produced by microinjection and a new electroporation method named technique for animal knockout system by electroporation (TAKE). This chapter presents the latest protocols for producing genome edited rats.},
}
@article {pmid36773149,
year = {2023},
author = {Hara, H and Munkh-Erdene, N and Byambaa, S and Hanazono, Y},
title = {Nonviral Ex Vivo Genome Editing in Mouse Bona Fide Hematopoietic Stem Cells with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {213-221},
pmid = {36773149},
issn = {1940-6029},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Genetic Therapy/methods ; Lentivirus/genetics ; },
abstract = {Knock-in therapy, in which an insertion site can be controlled, would be more suitable for the treatment of genetic blood disorders as compared to conventional gene therapy with lentivirus vectors that introduce genes into the genome randomly. Recent advancements in genome editing technology have substantially improved the knock-in efficiency, making it a reality. We present the details of a virus-free CRISPR/Cas9-based genome editing method for bona fide mouse hematopoietic stem cells.},
}
@article {pmid36773148,
year = {2023},
author = {Kashiwakura, Y and Ohmori, T},
title = {Genome Editing of Murine Liver Hepatocytes by AAV Vector-Mediated Expression of Cas9 In Vivo.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {195-211},
pmid = {36773148},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Liver/metabolism ; Hepatocytes ; Promoter Regions, Genetic ; Dependovirus/genetics ; Genetic Vectors/genetics ; },
abstract = {Adeno-associated virus (AAV) vectors are attractive tools for gene transfer to the liver and are used as gene therapeutic drugs for inherited disorders. The intravenous injection of an AAV vector harboring the gene of interest driven by the hepatocyte-specific promoter could efficiently express the target gene in liver hepatocytes. The delivery of genome editing tools including Cas9 and gRNA, by the AAV vector, can efficiently disrupt the target gene expression in the liver in vivo by intravenous administration in mice. We can quickly obtain mice lacking specific gene expression in the liver only by administering the AAV vector. The method could be suitable for developing genome editing treatments for inherited disorders and basic research exploring the physiological role of the target gene produced from liver hepatocytes.},
}
@article {pmid36773147,
year = {2023},
author = {Abe, T and Inoue, KI and Kiyonari, H},
title = {Efficient CRISPR/Cas9-Assisted Knockin of Large DNA Donors by Pronuclear Microinjection During S-Phase in Mouse Zygotes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {181-194},
pmid = {36773147},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Zygote/metabolism ; *CRISPR-Cas Systems/genetics ; Microinjections/methods ; Homologous Recombination ; Gene Knock-In Techniques ; DNA/genetics/metabolism ; },
abstract = {In the CRISPR/Cas9-mediated gene cassette knockin (KI) strategy, a gene cassette is integrated into a target locus through a proper DNA repair pathway after the Cas9-induced double-strand DNA breaks; the activation of the DNA repair pathway is known to be correlated with the cell cycle. Recently, we have reported a new KI approach named SPRINT (S-phase pronuclear injection for targeting)-CRISPR, focusing on the correlation between the cell cycle and the KI efficiency in the mouse zygote microinjection. Our results suggest that the CRISPR-mediated KI with a homologous recombination-based donor vector during S-phase enhances the KI efficiency. For SPRINT-CRISPR, the uniformity of the zygotes in the cell cycle is achieved by in vitro fertilization, and the zygotes are cryopreserved until use. These reproductive techniques are necessary for efficient KI. Furthermore, Piezo-assisted microinjection has been successful in improving the survival rate of the injected embryos. In this chapter, we describe the protocols that focus on the zygote preparation and Piezo-assisted microinjection of the SPRINT-CRISPR method.},
}
@article {pmid36773145,
year = {2023},
author = {Kobayashi, R and Horii, T and Hatada, I},
title = {Efficient Detection of Flox Mice Using In Vitro Cre Recombination.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {149-159},
pmid = {36773145},
issn = {1940-6029},
mesh = {Mice ; Animals ; Mice, Knockout ; *Integrases/genetics/metabolism ; *Gene Editing ; Recombination, Genetic ; CRISPR-Cas Systems ; },
abstract = {Advances in CRISPR/Cas9 genome editing technologies have allowed for the rapid generation of Cre-loxP conditional knockout mice. However, current strategies for genotyping flox mice, typically based on Sanger sequencing following cloning of target sequences from dozens of pups, are time-consuming. Here, we describe a rapid screening method for flox mice, using in vitro Cre recombination that can be performed using simple enzymatic reactions and enables detection of functional flox mouse within 1 day. In addition, we introduce an efficient strategy for subsequent sequence analysis by cloning of floxed regions using the In-Fusion system. Our genotyping pipeline reduces laborious tasks and thus contributes to the rapid selection of accurately edited flox mice.},
}
@article {pmid36773144,
year = {2023},
author = {Horii, T and Kobayashi, R and Hatada, I},
title = {Generation of Floxed Mice by Sequential Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {135-147},
pmid = {36773144},
issn = {1940-6029},
mesh = {Mice ; Animals ; Mice, Knockout ; *Electroporation ; *Electroporation Therapies ; Integrases/genetics ; CRISPR-Cas Systems ; Mice, Transgenic ; },
abstract = {Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.},
}
@article {pmid36773143,
year = {2023},
author = {Kaneko, T},
title = {Genome Editing in Mouse and Rat by Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {125-134},
pmid = {36773143},
issn = {1940-6029},
mesh = {Rats ; Mice ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Mice, Knockout ; Electroporation/methods ; Electroporation Therapies ; Endonucleases/genetics/metabolism ; *Craniocerebral Trauma ; },
abstract = {Many genome-edited mouse and rat strains have been produced using engineered endonucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Especially, CRISPR-Cas9 is powerful tool that can be easy, rapid, and high-efficiency-produced new genome-edited strains. Furthermore, new technique, Technique for Animal Knockout system by Electroporation (TAKE), efficiently accelerate production of new strains by direct nuclease introduction into intact embryos using electroporation. This chapter presents a latest technical information in the production of genome-edited mouse and rat by TAKE method.},
}
@article {pmid36773141,
year = {2023},
author = {Fujii, W},
title = {Generation of Knock-In Mouse by Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {99-109},
doi = {10.1007/978-1-0716-3016-7_8},
pmid = {36773141},
issn = {1940-6029},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *Gene Targeting/methods ; Embryonic Stem Cells ; Oligodeoxyribonucleotides/genetics ; CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques ; Zygote/metabolism ; },
abstract = {Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome-editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-strand oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.},
}
@article {pmid36773140,
year = {2023},
author = {Ozawa, M and Emori, C and Ikawa, M},
title = {Gene Targeting in Mouse Embryonic Stem Cells via CRISPR/Cas9 Ribonucleoprotein (RNP)-Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {87-97},
pmid = {36773140},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells ; Ribonucleoproteins/genetics ; Gene Targeting ; DNA ; },
abstract = {The CRISPR/Cas9-mediated genome-editing system enables the development of gene-modified mice using fertilized eggs. However, while the efficiency in developing gene knockout mice by inducing small indel mutations would be good enough, the successful ratio to create large side DNA knock-in (KI) by embryonic genome editing is still low. In contrast to the direct embryo KI method, gene targeting using embryonic stem cells (ESC) followed by chimeric mouse development by blastocyst injection still has several advantages, e.g., high-throughput in vitro targeting/screening or large-size DNA KI such as Cre, CreERT, TetON, and reporter fluorescent protein, or their fusion proteins can be carried out without serving animal lives. The ESC targeting can also be applied to strains such as BALB/c, of which embryos are known to be difficult to handle in vitro. This text describes the optimized method for either short- or large-size DNA KI in ESC by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.},
}
@article {pmid36773139,
year = {2023},
author = {Horii, T and Hatada, I},
title = {Generation of Genome-Edited Mice by Cytoplasmic Injection of CRISPR-Cas9 RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {75-86},
pmid = {36773139},
issn = {1940-6029},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; Cytoplasm ; Mice, Knockout ; RNA, Guide, Kinetoplastida/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) gene editing enables rapid production of genome-edited animals. The Cas9/guide RNA (gRNA) component can be introduced into zygotes in several ways. Here, we provide an instructional guide for the generation of knockout mice using cytoplasmic injection of in vitro transcribed Cas9 RNA and gRNA.},
}
@article {pmid36773137,
year = {2023},
author = {Maruyama, S and Kusakabe, T and Zou, X and Kobayashi, Y and Asano, Y and Wang, QS and Ui-Tei, K},
title = {SNPD-CRISPR: Single Nucleotide Polymorphism-Distinguishable Repression or Enhancement of a Target Gene Expression by CRISPR System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {49-62},
pmid = {36773137},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; RNA/genetics ; Nucleotides ; Gene Expression ; },
abstract = {A wide range of diseases, including cancer, autoimmune diseases, or neurodegenerative diseases, have been associated with single nucleotide mutations in their causative genes. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a flexible and efficient genome engineering technology widely used for researches and therapeutic applications which offers immense opportunity to treat genetic diseases. The complex of Cas9 and the guide RNA acts as an RNA-guided endonuclease. Cas9 recognizes a sequence motif known as a protospacer adjacent motif (PAM), and then the guide RNA base pairs with its proximal target region of 20 nucleotides with sequence complementarity. Here we describe the procedure named single nucleotide polymorphism-distinguishable (SNPD)-CRISPR system which can suppress or enhance the expression of disease-causative gene with single nucleotide mutation distinguished from its wild-type. In this study, we used HRAS, one of most famous cancer-causative genes, as an example of a target gene.},
}
@article {pmid36773136,
year = {2023},
author = {Hatada, I and Morita, S and Horii, T},
title = {CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {41-47},
pmid = {36773136},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
abstract = {CRISPR/Cas9 is the genome-editing technology that is most widely used around the world. Its widespread adoption is largely due to its simplicity and ease of use. Here, we introduce the construction of vectors and genome editing of the target gene in cells using the CRISPR/Cas9 system.},
}
@article {pmid36769335,
year = {2023},
author = {Lim, SW and Fang, X and Cui, S and Lee, H and Shin, YJ and Ko, EJ and Lee, KI and Lee, JY and Chung, BH and Yang, CW},
title = {CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {3},
pages = {},
pmid = {36769335},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Solute Carrier Family 12, Member 3 ; Mutation ; Kidney ; Phenotype ; Organoids ; *Induced Pluripotent Stem Cells ; },
abstract = {The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001[corr]. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001[corr] group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm[2] vs. 3.7 ± 0.2/cm[2], p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001[corr] group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001[corr] group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001[corr] group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.},
}
@article {pmid36769177,
year = {2023},
author = {Yu, Z and Xu, J and She, Q},
title = {Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection.},
journal = {International journal of molecular sciences},
volume = {24},
number = {3},
pages = {},
pmid = {36769177},
issn = {1422-0067},
mesh = {Humans ; *MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics ; },
abstract = {In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5'-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.},
}
@article {pmid36768765,
year = {2023},
author = {Nikolić, I and Samardžić, J and Stevanović, S and Miljuš-Đukić, J and Milisavljević, M and Timotijević, G},
title = {CRISPR/Cas9-Targeted Disruption of Two Highly Homologous Arabidopsis thaliana DSS1 Genes with Roles in Development and the Oxidative Stress Response.},
journal = {International journal of molecular sciences},
volume = {24},
number = {3},
pages = {},
pmid = {36768765},
issn = {1422-0067},
mesh = {*Arabidopsis/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Arabidopsis Proteins/genetics/metabolism ; Oxidative Stress/genetics ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Global climate change has a detrimental effect on plant growth and health, causing serious losses in agriculture. Investigation of the molecular mechanisms of plant responses to various environmental pressures and the generation of plants tolerant to abiotic stress are imperative to modern plant science. In this paper, we focus on the application of the well-established technology CRISPR/Cas9 genome editing to better understand the functioning of the intrinsically disordered protein DSS1 in plant response to oxidative stress. The Arabidopsis genome contains two highly homologous DSS1 genes, AtDSS1(I) and AtDSS1(V). This study was designed to identify the functional differences between AtDSS1s, focusing on their potential roles in oxidative stress. We generated single dss1(I) and dss1(V) mutant lines of both Arabidopsis DSS1 genes using CRISPR/Cas9 technology. The homozygous mutant lines with large indels (dss1(I)del25 and dss1(V)ins18) were phenotypically characterized during plant development and their sensitivity to oxidative stress was analyzed. The characterization of mutant lines revealed differences in root and stem lengths, and rosette area size. Plants with a disrupted AtDSS1(V) gene exhibited lower survival rates and increased levels of oxidized proteins in comparison to WT plants exposed to oxidative stress induced by hydrogen peroxide. In this work, the dss1 double mutant was not obtained due to embryonic lethality. These results suggest that the DSS1(V) protein could be an important molecular component in plant abiotic stress response.},
}
@article {pmid36768619,
year = {2023},
author = {Brooks, IR and Sheriff, A and Moran, D and Wang, J and Jacków, J},
title = {Challenges of Gene Editing Therapies for Genodermatoses.},
journal = {International journal of molecular sciences},
volume = {24},
number = {3},
pages = {},
pmid = {36768619},
issn = {1422-0067},
support = {218452/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Genetic Therapy ; Mutation ; Recombinational DNA Repair ; },
abstract = {Genodermatoses encompass a wide range of inherited skin diseases, many of which are monogenic. Genodermatoses range in severity and result in early-onset cancers or life-threatening damage to the skin, and there are few curative options. As such, there is a clinical need for single-intervention treatments with curative potential. Here, we discuss the nascent field of gene editing for the treatment of genodermatoses, exploring CRISPR-Cas9 and homology-directed repair, base editing, and prime editing tools for correcting pathogenic mutations. We specifically focus on the optimisation of editing efficiency, the minimisation off-targets edits, and the tools for delivery for potential future therapies. Honing each of these factors is essential for translating gene editing therapies into the clinical setting. Therefore, the aim of this review article is to raise important considerations for investigators aiming to develop gene editing approaches for genodermatoses.},
}
@article {pmid36766782,
year = {2023},
author = {Hansen, S and McClements, ME and Corydon, TJ and MacLaren, RE},
title = {Future Perspectives of Prime Editing for the Treatment of Inherited Retinal Diseases.},
journal = {Cells},
volume = {12},
number = {3},
pages = {},
pmid = {36766782},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Retinal Diseases/genetics/therapy ; Genetic Therapy/methods ; Mutation/genetics ; },
abstract = {Inherited retinal diseases (IRD) are a clinically and genetically heterogenous group of diseases and a leading cause of blindness in the working-age population. Even though gene augmentation therapies have shown promising results, they are only feasible to treat a small number of autosomal recessive IRDs, because the size of the gene is limited by the vector used. DNA editing however could potentially correct errors regardless of the overall size of the gene and might also be used to correct dominant mutations. Prime editing is a novel CRISPR/Cas9 based gene editing tool that enables precise correction of point mutations, insertions, and deletions without causing double strand DNA breaks. Due to its versatility and precision this technology may be a potential treatment option for virtually all genetic causes of IRD. Since its initial description, the prime editing technology has been further improved, resulting in higher efficacy and a larger target scope. Additionally, progress has been achieved concerning the size-related delivery issue of the prime editor components. This review aims to give an overview of these recent advancements and discusses prime editing as a potential treatment for IRDs.},
}
@article {pmid36765063,
year = {2023},
author = {Cheng, X and Li, Z and Shan, R and Li, Z and Wang, S and Zhao, W and Zhang, H and Chao, L and Peng, J and Fei, T and Li, W},
title = {Modeling CRISPR-Cas13d on-target and off-target effects using machine learning approaches.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {752},
pmid = {36765063},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *RNA, Long Noncoding ; RNA, Guide, Kinetoplastida/genetics ; Machine Learning ; },
abstract = {A major challenge in the application of the CRISPR-Cas13d system is to accurately predict its guide-dependent on-target and off-target effect. Here, we perform CRISPR-Cas13d proliferation screens and design a deep learning model, named DeepCas13, to predict the on-target activity from guide sequences and secondary structures. DeepCas13 outperforms existing methods to predict the efficiency of guides targeting both protein-coding and non-coding RNAs. Guides targeting non-essential genes display off-target viability effects, which are closely related to their on-target efficiencies. Choosing proper negative control guides during normalization mitigates the associated false positives in proliferation screens. We apply DeepCas13 to the guides targeting lncRNAs, and identify lncRNAs that affect cell viability and proliferation in multiple cell lines. The higher prediction accuracy of DeepCas13 over existing methods is extensively confirmed via a secondary CRISPR-Cas13d screen and quantitative RT-PCR experiments. DeepCas13 is freely accessible via http://deepcas13.weililab.org .},
}
@article {pmid36764778,
year = {2023},
author = {Chen, H and Zhuang, Z and Chen, Y and Qiu, C and Qin, Y and Tan, C and Tan, Y and Jiang, Y},
title = {A universal platform for one-pot detection of circulating non-coding RNA combining CRISPR-Cas12a and branched rolling circle amplification.},
journal = {Analytica chimica acta},
volume = {1246},
number = {},
pages = {340896},
doi = {10.1016/j.aca.2023.340896},
pmid = {36764778},
issn = {1873-4324},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Untranslated ; *MicroRNAs/genetics ; Piwi-Interacting RNA ; Biological Assay ; *Cell-Free Nucleic Acids ; Nucleic Acid Amplification Techniques ; },
abstract = {Multiple circulating non-coding RNAs (ncRNAs) in serum may serve as vital biomarkers for use in diagnosing early-stage colorectal cancer (CRC). Herein, a universal platform for one-pot detection of CRC-related ncRNAs was developed based on branched rolling circle amplification and CRISPR-Cas12a (BRCACas). For the implementation of the method, primers incorporating ncRNA sequences of circulating CRC-associated RNAs (piRNA or miRNA) were designed that could specifically hybridize with circular probes to initiate the BRCA process. Thereafter, the generation of dendritic DNA products triggered Cas12a trans-cleavage activity to elicit a fluorescent signal. The proposed method, combining high BRCA reaction efficiency with powerful Cas12a trans-cleavage activity, provided greatly enhanced detection sensitivity, as reflected by limits of detection (LODs) for model piRNA (piR-54265) and model miRNA (miR21) of 0.76 fM and 0.87 fM, respectively. Notably, the proposed BRCACas platform, assaying two different types of CRC-associated ncRNAs in patient samples, produced consistent results with the conventional reverse transcription-quantitative PCR (RT-qPCR) method. Therefore, the one-pot, isothermal, and specific BRCACas platform provided excellent performance, thus demonstrating its promise as a rapid, adaptable, and practical diagnostic/prognostic cancer screening method.},
}
@article {pmid36764772,
year = {2023},
author = {Wang, H and Liu, R and Dong, K and Zhang, L and Zhang, J and Zhang, X and Zhang, J and Xiao, X and Zhang, W and Wang, X},
title = {A universal and sensitive gene mutation detection method based on CRISPR-Cas12a.},
journal = {Analytica chimica acta},
volume = {1246},
number = {},
pages = {340886},
doi = {10.1016/j.aca.2023.340886},
pmid = {36764772},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Clinical Relevance ; Mutation ; Nucleotides ; Polymerase Chain Reaction ; Nucleic Acid Amplification Techniques ; },
abstract = {Single nucleotide mutations are highly related to the occurrence and development of cancer. The development of simple single nucleotide mutation detection methods with high sensitivity and specificity has great clinical significance for the prevention, diagnosis, treatment and prognosis evaluation of cancer. In recent years, CRISPR/Cas12a has been developed as a highly sensitive, simple and fast tool for nucleic acid detection. However, the specificity and universality of current detection methods based on it are still insufficient, so their clinical applications are limited. Herein, we developed a simple and rapid single nucleotide mutation detection method based on CRISPR/Cas12a system. This method not only solves the problem of PAM sequence restriction of CRISPR/Cas12a, but also significantly improves the specificity of CRISPR/Cas12a for single nucleotide mutation and greatly improves the sensitivity. We detected three clinically significant mutations, PTEN R130Q, BRAF V600E, and TP53 R248W, with a detection limit of 0.1%. Finally, we further verified the clinical practicability of this method. We selected TP53 R248W mutation site for testing. The accuracy of testing results for 10 clinical samples was as high as 100%. In conclusion, the detection method of specific PCR combined with CRISPR/Cas12a is simple, rapid, universal and highly sensitive. We believe that this method has promising application prospects in clinical diagnosis of cancer.},
}
@article {pmid36764753,
year = {2023},
author = {Mehta, S and Buyanbat, A and Orkin, S and Nabet, B},
title = {High-efficiency knock-in of degradable tags (dTAG) at endogenous loci in cell lines.},
journal = {Methods in enzymology},
volume = {681},
number = {},
pages = {1-22},
doi = {10.1016/bs.mie.2022.08.045},
pmid = {36764753},
issn = {1557-7988},
mesh = {Animals ; Mice ; Humans ; *CRISPR-Cas Systems ; *Gene Editing ; DNA ; DNA, Single-Stranded ; Recombinational DNA Repair ; },
abstract = {The dTAG system is a versatile strategy for tunable control of protein abundance and facilitates the time-resolved assessment of disease-associated protein function. A "co-opted" fusion-based degron peptide, the "dTAG" facilitates the study of endogenous protein function when knocked-in at the endogenous genetic loci of proteins of interest. We combine CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with the delivery of a single-stranded DNA HDR-donor-template via crude preparations of recombinant adeno-associated virus (rAAV). Our approach to knock-in of large (1-2kb) DNA fragments via crude-rAAV mediated HDR donor delivery is rapid and inexpensive. It facilitates genetic modification of a variety of human as well as mouse cell lines at high efficiency and precision.},
}
@article {pmid36731397,
year = {2023},
author = {Song, D and Xu, W and Han, X and Wang, H and Zhuo, Y and Liu, J and Zhu, A and Long, F},
title = {CRISPR/Cas12a-powered evanescent wave fluorescence nanobiosensing platform for nucleic acid amplification-free detection of Staphylococcus aureus with multiple signal enhancements.},
journal = {Biosensors &amp; bioelectronics},
volume = {225},
number = {},
pages = {115109},
doi = {10.1016/j.bios.2023.115109},
pmid = {36731397},
issn = {1873-4235},
mesh = {Humans ; Staphylococcus aureus/genetics ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Staphylococcal Infections/diagnosis ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; },
abstract = {Although CRISPR-based biosensors for pathogenic detection are highly specific, they not sensitive enough and nucleic acid amplification is generally required to improve their sensitivity. However, this allows only binary operations and significantly limits practical applications. Here, a CRISPR/Cas12a-powered Evanescent wAve fluorescence nanobiosensing plaTform (CREAT) was developed for ultrasensitive nucleic acid amplification-free quantitative detection of pathogens with multiple signal enhancements. In addition to collateral cleavage amplification of the CRISPR/Cas12a system, we constructed nanophotonic structure-based evanescent wave fluorescence enhancement, Mg[2+] or DNA-mediated fluorescence enhancement, and air-displacement fluorescence enhancement strategies for ultrasensitive detection of Staphylococcus aureus (S. aureus). Especially, the fluorescence signal detected by CREAT can be significantly enhanced by adding a simple air displacement step, thus improving detection sensitivity. This nanobiosensor detected real samples containing S. aureus, with a detection limit of 592 CFU/mL and 13.2 CFU/mL in 45 min and 90 min, respectively, which are comparable to those of RT-qPCR. This paves a new way for simple, rapid, sensitive, robust, and flexible on-site detection of S. aureus as well as other pathogens.},
}
@article {pmid36724657,
year = {2023},
author = {Zhao, Y and Wu, W and Tang, X and Zhang, Q and Mao, J and Yu, L and Li, P and Zhang, Z},
title = {A universal CRISPR/Cas12a-powered intelligent point-of-care testing platform for multiple small molecules in the healthcare, environment, and food.},
journal = {Biosensors &amp; bioelectronics},
volume = {225},
number = {},
pages = {115102},
doi = {10.1016/j.bios.2023.115102},
pmid = {36724657},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; Tandem Mass Spectrometry ; *Biosensing Techniques ; Aflatoxin B1 ; Capsaicin ; Coloring Agents ; Point-of-Care Testing ; Delivery of Health Care ; },
abstract = {Growing studies focusing on nuclear acid detection via the emerging CRISPR technique demonstrate its promising application. However, limited works solve the identification of non-nucleic acid targets, especially multiple small molecules. To address challenges for point-of-care testing (POCT) in complex matrices for healthcare, environment, and food safety, we developed CRISPR Cas12a-powered highly sensitive, high throughput, intelligent POCT (iPOCT) for multiple small molecules based on a smartphone-controlled reader. As a proof of concept, aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), and capsaicin (CAP) were chosen as multiple targets. First, three antigens were preloaded in independent microwells. Then, the antibody/antigen-induced fluorescent signals were consecutively transferred from the biotin-streptavidin to CRISPR/Cas12a system. Third, the fluorescent signals were recorded by a smartphone-controlled handheld dark-box readout. Under optimization, detection limits in AFB1, BaP, and CAP were 0.00257, 4.971, and 794.6 fg/mL with wide linear ranges up to four orders of magnitude. Using urine, water, soybean oil, wheat, and peanuts as the complex matrix, we recorded high selectivity, considerable recovery, repeatability, and high consistency comparison to HPLC-MS/MS methods. This work promises a practical intelligent POCT platform for multiple targets in lipid-soluble and water-soluble matrices and could be extensively applied for healthcare, environment, and food safety.},
}
@article {pmid36696850,
year = {2023},
author = {Song, J and Song, Y and Jang, H and Moon, J and Kang, H and Huh, YM and Son, HY and Rho, HW and Park, M and Lim, EK and Jung, J and Jung, Y and Park, HG and Lee, KG and Im, SG and Kang, T},
title = {Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.},
journal = {Biosensors &amp; bioelectronics},
volume = {225},
number = {},
pages = {115085},
doi = {10.1016/j.bios.2023.115085},
pmid = {36696850},
issn = {1873-4235},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; DNA/analysis ; Oligonucleotides ; },
abstract = {Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.},
}
@article {pmid36681183,
year = {2023},
author = {Naeem, M and Hazafa, A and Bano, N and Ali, R and Farooq, M and Razak, SIA and Lee, TY and Devaraj, S},
title = {Explorations of CRISPR/Cas9 for improving the long-term efficacy of universal CAR-T cells in tumor immunotherapy.},
journal = {Life sciences},
volume = {316},
number = {},
pages = {121409},
doi = {10.1016/j.lfs.2023.121409},
pmid = {36681183},
issn = {1879-0631},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/metabolism ; Immunotherapy ; *Neoplasms/genetics/therapy ; T-Lymphocytes ; Tumor Microenvironment ; },
abstract = {Chimeric antigen receptor (CAR) T therapy has shown remarkable success in discovering novel CAR-T cell products for treating malignancies. Despite of successful results from clinical trials, CAR-T cell therapy is ineffective for long-term disease progression. Numerous challenges of CAR-T cell immunotherapy such as cell dysfunction, cytokine-related toxicities, TGF-β resistance, GvHD risks, antigen escape, restricted trafficking, and tumor cell infiltration still exist that hamper the safety and efficacy of CAR-T cells for malignancies. The accumulated data revealed that these challenges could be overcome with the advanced CRISPR genome editing technology, which is the most promising tool to knockout TRAC and HLA genes, inhibiting the effects of dominant negative receptors (PD-1, TGF-β, and B2M), lowering the risks of cytokine release syndrome (CRS), and regulating CAR-T cell function in the tumor microenvironment (TME). CRISPR technology employs DSB-free genome editing methods that robustly allow efficient and controllable genetic modification. The present review explored the innovative aspects of CRISPR/Cas9 technology for developing next-generation/universal allogeneic CAR-T cells. The present manuscript addressed the ongoing status of clinical trials of CRISPR/Cas9-engineered CAR-T cells against cancer and pointed out the off-target effects associated with CRISPR/Cas9 genome editing. It is concluded that CAR-T cells modified by CRISPR/Cas9 significantly improved antitumor efficacy in a cost-effective manner that provides opportunities for novel cancer immunotherapies.},
}
@article {pmid36436172,
year = {2023},
author = {Iwasa, K and Yamagishi, A and Yamamoto, S and Haruta, C and Maruyama, K and Yoshikawa, K},
title = {GPR137 Inhibits Cell Proliferation and Promotes Neuronal Differentiation in the Neuro2a Cells.},
journal = {Neurochemical research},
volume = {48},
number = {3},
pages = {996-1008},
pmid = {36436172},
issn = {1573-6903},
mesh = {Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Cycle ; Cell Differentiation ; *CRISPR-Cas Systems ; },
abstract = {The orphan receptor, G protein-coupled receptor 137 (GPR137), is an integral membrane protein involved in several types of cancer. GPR137 is expressed ubiquitously, including in the central nervous system (CNS). We established a GPR137 knockout (KO) neuro2A cell line to analyze GPR137 function in neuronal cells. KO cells were generated by genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and cultured as single cells by limited dilution. Rescue cells were then constructed to re-express GPR137 in GPR137 KO neuro2A cells using an expression vector with an EF1-alpha promoter. GPR137 KO cells increased cellular proliferation and decreased neurite outgrowth (i.e., a lower level of neuronal differentiation). Furthermore, GPR137 KO cells exhibited increased expression of a cell cycle regulator, cyclin D1, and decreased expression of a neuronal differentiation marker, NeuroD1. Additionally, GPR137 KO cells exhibited lower expression levels of the neurite outgrowth markers STAT3 and GAP43. These phenotypes were all abrogated in the rescue cells. In conclusion, GPR137 deletion increased cellular proliferation and decreased neuronal differentiation, suggesting that GPR137 promotes cell cycle exit and neuronal differentiation in neuro2A cells. Regulation of neuronal differentiation by GPR137 could be vital to constructing neuronal structure during brain development.},
}
@article {pmid36779782,
year = {2023},
author = {Thomason, LC and Costantino, N and Li, X and Court, DL},
title = {Recombineering: Genetic Engineering in Escherichia coli Using Homologous Recombination.},
journal = {Current protocols},
volume = {3},
number = {2},
pages = {e656},
doi = {10.1002/cpz1.656},
pmid = {36779782},
issn = {2691-1299},
abstract = {The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using either PCR products or synthetic double-stranded DNA (dsDNA) or single-stranded DNA as substrates. Multiple linear dsDNA molecules can be assembled into an intact plasmid. The technology of recombineering is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to the location of restriction sites and can also be used in combination with CRISPR/Cas targeting systems. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: Making electrocompetent cells and transforming with linear DNA Support Protocol 1: Selection/counter-selections for genome engineering Support Protocol 2: Creating and screening for oligo recombinants by PCR Support Protocol 3: Other methods of screening for unselected recombinants Support Protocol 4: Curing recombineering plasmids containing a temperature-sensitive replication function Support Protocol 5: Removal of the prophage by recombineering Alternate Protocol 1: Using CRISPR/Cas9 as a counter-selection following recombineering Alternate Protocol 2: Assembly of linear dsDNA fragments into functional plasmids Alternate Protocol 3: Retrieval of alleles onto a plasmid by gap repair Alternate Protocol 4: Modifying multicopy plasmids with recombineering Support Protocol 6: Screening for unselected plasmid recombinants Alternate Protocol 5: Recombineering with an intact λ prophage Alternate Protocol 6: Targeting an infecting λ phage with the defective prophage strains.},
}
@article {pmid36779580,
year = {2023},
author = {Ghani, MW and Iqbal, A and Ghani, H and Bibi, S and Wang, Z and Pei, R},
title = {Recent advances in nanocomposite-based delivery systems for targeted CRISPR/Cas delivery and therapeutic genetic manipulation.},
journal = {Journal of materials chemistry. B},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2tb02610d},
pmid = {36779580},
issn = {2050-7518},
abstract = {CRISPR/Cas systems are novel gene editing tools with tremendous capacity and accuracy for gene editing and hold great potential for therapeutic genetic manipulation. However, the lack of safe and efficient delivery methods for CRISPR/Cas and its guide RNA hinders their wide adoption for therapeutic applications. To this end, there is an increasing demand for safe, efficient, precise, and non-pathogenic delivery approaches, both in vitro and in vivo. With the convergence of nanotechnology and biomedicine, functional nanocomposites have demonstrated unparalleled sophistication to overcome the limits of CRISPR/Cas delivery. The tunability of the physicochemical properties of nanocomposites makes it very easy to conjugate them with different functional substances. The combinatorial application of diverse functional materials in the form of nanocomposites has shown excellent properties for CRISPR/Cas delivery at the target site with therapeutic potential. The recent highlights of selective organ targeting and phase I clinical trials for gene manipulation by CRISPR/Cas after delivery through LNPs are at the brink of making it to routine clinical practice. Here we summarize the recent advances in delivering CRISPR/Cas systems through nanocomposites for targeted delivery and therapeutic genome editing.},
}
@article {pmid36778248,
year = {2023},
author = {Rananaware, SR and Vesco, EK and Shoemaker, GM and Anekar, SS and Sandoval, LSW and Meister, KS and Macaluso, NC and Nguyen, LT and Jain, PK},
title = {Programmable RNA detection with CRISPR-Cas12a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.01.29.525716},
pmid = {36778248},
abstract = {CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans- cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or 'SAHARA' to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg [2+] concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.},
}
@article {pmid36775572,
year = {2022},
author = {Zhou, Y and Zhang, L and Xie, YH and Wu, J},
title = {Advancements in detection of SARS-CoV-2 infection for confronting COVID-19 pandemics.},
journal = {Laboratory investigation; a journal of technical methods and pathology},
volume = {102},
number = {1},
pages = {4-13},
doi = {10.1038/s41374-021-00663-w},
pmid = {36775572},
issn = {1530-0307},
abstract = {As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.},
}
@article {pmid36773957,
year = {2023},
author = {Onuma, H and Sato, Y and Harashima, H},
title = {Lipid nanoparticle-based ribonucleoprotein delivery for in vivo genome editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {355},
number = {},
pages = {406-416},
doi = {10.1016/j.jconrel.2023.02.008},
pmid = {36773957},
issn = {1873-4995},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system is a technology that is used to perform site-specific gene disruption, repair, and the modification of genomic DNA via DNA repair mechanisms, and is expected to be a fundamental therapeutic strategy for the treatment of infectious diseases and genetic disorders. For clinical applications, the non-viral vector-based delivery of the CRISPR/Cas ribonucleoprotein (RNP) is important, but the poor efficiency of delivery and the lack of a practical method for its manufacture remains as an issue. We report herein on the development of a lipid nanoparticle (LNP)-based Cas RNP delivery system based on optimally designed single stranded oligonucleotides (ssODNs) that allow efficient in vivo genome editing. The formation of sequence-specific RNP-ssODN complexes was found to be important for the functional delivery of RNP. Furthermore, the melting temperature (Tm) between sgRNA and ssODN had a significant effect on in vivo gene knockout efficiency. An ssODN with a high Tm resulted in limited knockout (KO) activity while that at near room temperature showed the highest KO activity, indicating the importance of the cytosolic release of RNPs. Two consecutive intravenous injections of the Tm optimized formulation achieved approximately 70% and 80% transthyretin KO at the DNA and protein level, respectively, without any obvious toxicity. These findings represent a significant contribution to the development of safe in vivo CRISPR/Cas RNP delivery technology and its practical application in genome editing therapies.},
}
@article {pmid36725187,
year = {2023},
author = {Wang, D and Zhang, Y and Li, R and Li, J and Zhang, R},
title = {Consistency and reproducibility of large panel next-generation sequencing: Multi-laboratory assessment of somatic mutation detection on reference materials with mismatch repair and proofreading deficiency.},
journal = {Journal of advanced research},
volume = {44},
number = {},
pages = {161-172},
doi = {10.1016/j.jare.2022.03.016},
pmid = {36725187},
issn = {2090-1224},
mesh = {Humans ; DNA Mismatch Repair/genetics ; *High-Throughput Nucleotide Sequencing/methods ; Mutation ; *Neoplasms/genetics ; *Precision Medicine ; Reproducibility of Results ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Clinical precision oncology increasingly relies on accurate genome-wide profiling using large panel next generation sequencing; however, difficulties in accurate and consistent detection of somatic mutation from individual platforms and pipelines remain an open question.<br><br>OBJECTIVES: To obtain paired tumor-normal reference materials that can be effectively constructed and interchangeable with clinical samples, and evaluate the performance of 56 panels under routine testing conditions based on the reference samples.<br><br>METHODS: Genes involved in mismatch repair and DNA proofreading were knocked down using the CRISPR-Cas9 technology to accumulate somatic mutations in a defined GM12878 cell line. They were used as reference materials to comprehensively evaluate the reproducibility and accuracy of detection results of oncopanels and explore the potential influencing factors.<br><br>RESULTS: In total, 14 paired tumor-normal reference DNA samples from engineered cell lines were prepared, and a reference dataset comprising 168 somatic mutations in a high-confidence region of 1.8&#xa0;Mb were generated. For mutations with an allele frequency (AF) of more than 5% in reference samples, 56 panels collectively reported 1306 errors, including 729 false negatives (FNs), 179 false positives (FPs) and 398 reproducibility errors. The performance metric varied among panels with precision and recall ranging from 0.773 to 1 and 0.683 to 1, respectively. Incorrect and inadequate filtering accounted for a large proportion of false discovery (including FNs and FPs), while low-quality detection, cross-contamination and other sequencing errors during the wet bench process were other sources of FNs and FPs. In addition, low AF (<5%) considerably influenced the reproducibility and comparability among panels.<br><br>CONCLUSIONS: This study provided an integrated practice for developing reference standard to assess oncopanels in detecting somatic mutations and quantitatively revealed the source of detection errors. It will promote optimization, validation, and quality control among laboratories with potential applicability in clinical use.},
}
@article {pmid36773159,
year = {2023},
author = {Suzuki, M and Iida, M and Hayashi, T and Suzuki, KT},
title = {CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {341-357},
doi = {10.1007/978-1-0716-3016-7_26},
pmid = {36773159},
issn = {1940-6029},
abstract = {Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.},
}
@article {pmid36773158,
year = {2023},
author = {Hara, R and Ansai, S and Kinoshita, M},
title = {Genome Editing of Medaka.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {325-339},
pmid = {36773158},
issn = {1940-6029},
abstract = {Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knockout strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knockin using a plasmid carrying a sequence that does not cause off-target effects in medaka. Additionally, knockin method without plasmid cloning is described.},
}
@article {pmid36773142,
year = {2023},
author = {Sasaguri, H},
title = {Introduction of Genetic Mutations Into Mice by Base Editor and Target-AID.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {111-124},
pmid = {36773142},
issn = {1940-6029},
abstract = {Generating genetically modified animal models that precisely recapitulate disease characteristics forms an integral and indispensable tool to understanding disease pathophysiology. Recently, important advances in genome editing technologies have enabled us to efficiently create sophisticated animal models in short periods of time. Base editing is a modified CRISPR/Cas system that induces base substitution at targeted genomic regions. Here I describe a basic protocol to introduce disease-relevant pathogenic mutations into mice utilizing two representative base editing tools, Base Editor and Target-AID.},
}
@article {pmid36773138,
year = {2023},
author = {Wang, QS and Ui-Tei, K},
title = {Utilizing Large Functional and Population Genomics Resources for CRISPR/Cas Perturbation Experiment Design.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {63-73},
pmid = {36773138},
issn = {1940-6029},
abstract = {Genome sequencing technologies have rapidly evolved in the past decades, enabling us to interpret the human genome through multiple perspectives, ranging from cross-species comparisons, naturally occurring variation in health and disease state to regulatory mechanisms.Although such perspectives are all informative to narrow down the list of genes or variants for perturbation experiments based on specific biological aims, utilizing multiple sources of information is often challenging in practice.In this chapter, we provide an overview of major large-scale functional and population genomics resources, followed by a practical example of selecting target variants for genetic perturbation experiments involving genome engineering techniques such as CRISPR/Cas.},
}
@article {pmid36773134,
year = {2023},
author = {Ochiai, H and Yamamoto, T},
title = {Construction and Evaluation of Zinc Finger Nucleases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2637},
number = {},
pages = {1-25},
pmid = {36773134},
issn = {1940-6029},
abstract = {Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences. However, fundamental ZFN patents have expired, enabling a wide range of their distribution for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-strand annealing (SSA) assay.},
}
@article {pmid36768486,
year = {2023},
author = {Michalski, K and Ziąbska, P and Sowa, S and Zimny, J and Linkiewicz, AM},
title = {Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures.},
journal = {International journal of molecular sciences},
volume = {24},
number = {3},
pages = {},
doi = {10.3390/ijms24032162},
pmid = {36768486},
issn = {1422-0067},
abstract = {Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8'-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches.},
}
@article {pmid36766007,
year = {2023},
author = {Wang, M and Wang, H and Li, K and Li, X and Wang, X and Wang, Z},
title = {Review of CRISPR/Cas Systems on Detection of Nucleotide Sequences.},
journal = {Foods (Basel, Switzerland)},
volume = {12},
number = {3},
pages = {},
doi = {10.3390/foods12030477},
pmid = {36766007},
issn = {2304-8158},
abstract = {Nowadays, with the rapid development of biotechnology, the CRISPR/Cas technology in particular has produced many new traits and products. Therefore, rapid and high-resolution detection methods for biotechnology products are urgently needed, which is extremely important for safety regulation. Recently, in addition to being gene editing tools, CRISPR/Cas systems have also been used in detection of various targets. CRISPR/Cas systems can be successfully used to detect nucleic acids, proteins, metal ions and others in combination with a variety of technologies, with great application prospects in the future. However, there are still some challenges need to be addressed. In this review, we will list some detection methods of genetically modified (GM) crops, gene-edited crops and single-nucleotide polymorphisms (SNPs) based on CRISPR/Cas systems, hoping to bring some inspiration or ideas to readers.},
}
@article {pmid36763662,
year = {2023},
author = {Wang, Y and Qi, T and Liu, J and Yang, Y and Wang, Z and Wang, Y and Wang, T and Li, M and Li, M and Lu, D and Chang, ACY and Yang, L and Gao, S and Wang, Y and Lan, F},
title = {A highly specific CRISPR-Cas12j nuclease enables allele-specific genome editing.},
journal = {Science advances},
volume = {9},
number = {6},
pages = {eabo6405},
doi = {10.1126/sciadv.abo6405},
pmid = {36763662},
issn = {2375-2548},
abstract = {The CRISPR-Cas system can treat autosomal dominant diseases by nonhomologous end joining (NHEJ) gene disruption of mutant alleles. However, many single-nucleotide mutations cannot be discriminated from wild-type alleles by current CRISPR-Cas systems. Here, we functionally screened six Cas12j nucleases and determined Cas12j-8 as an ideal genome editor with a hypercompact size. Cas12j-8 displayed comparable activity to AsCas12a and Un1Cas12f1. Cas12j-8 is a highly specific nuclease sensitive to single-nucleotide mismatches in the protospacer adjacent motif (PAM)-proximal region. We experimentally proved that Cas12j-8 enabled allele-specific disruption of genes with a single-nucleotide polymorphism (SNP). Cas12j-8 recognizes a simple TTN PAM that provides for high target site density. In silico analysis reveals that Cas12j-8 enables allele-specific disruption of 25,931 clinically relevant variants in the ClinVar database, and 485,130,147 SNPs in the dbSNP database. Therefore, Cas12j-8 would be particularly suitable for therapeutic applications.},
}
@article {pmid36760507,
year = {2023},
author = {Quansah, E and Chen, Y and Yang, S and Wang, J and Sun, D and Zhao, Y and Chen, M and Yu, L and Zhang, C},
title = {CRISPR-Cas13 in malaria parasite: Diagnosis and prospective gene function identification.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1076947},
pmid = {36760507},
issn = {1664-302X},
abstract = {Malaria caused by Plasmodium is still a serious public health problem. Genomic editing is essential to understand parasite biology, elucidate mechanical pathways, uncover gene functions, identify novel therapeutic targets, and develop clinical diagnostic tools. Recent advances have seen the development of genomic diagnostic technologies and the emergence of genetic manipulation toolbox comprising a host of several systems for editing the genome of Plasmodium at the DNA, RNA, and protein level. Genomic manipulation at the RNA level is critical as it allows for the functional characterization of several transcripts. Of notice, some developed artificial RNA genome editing tools hinge on the endogenous RNA interference system of Plasmodium. However, Plasmodium lacks a robust RNAi machinery, hampering the progress of these editing tools. CRISPR-Cas13, which belongs to the VI type of the CRISPR system, can specifically bind and cut RNA under the guidance of crRNA, with no or minimal permanent genetic scar on genes. This review summarizes CRISPR-Cas13 system from its discovery, classification, principle of action, and diagnostic platforms. Further, it discusses the application prospects of Cas13-based systems in Plasmodium and highlights its advantages and drawbacks.},
}
@article {pmid36759683,
year = {2023},
author = {Sapozhnikov, DM and Szyf, M},
title = {The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {2280},
doi = {10.1038/s41598-023-29332-2},
pmid = {36759683},
issn = {2045-2322},
support = {PJT159583/CAPMC/CIHR/Canada ; PJT159583/CAPMC/CIHR/Canada ; },
abstract = {Off-target mutagenesis of CRISPR/Cas systems must be solved to facilitate safe gene therapy. Here, we report a novel approach, termed "PROTECTOR", to shield known off-target sites by directing the binding of an orthologous nuclease-dead Cas protein to the off-target site to sterically interfere with Cas activity. We show that this method reduces off-target mutation rates of two well-studied guide RNAs without compromising on-target activity and that it can be used in combination with high-fidelity Cas enzymes to further reduce off-target editing. This expands the suite of off-target mitigation strategies and offers an ability to protect off-target sites even when their sequences are fully identical to target sites.},
}
@article {pmid36758652,
year = {2023},
author = {Kalkan, AK and Palaz, F and Sofija, S and Elmousa, N and Ledezma, Y and Cachat, E and Rios-Solis, L},
title = {Improving recombinant protein production in CHO cells using the CRISPR-Cas system.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108115},
doi = {10.1016/j.biotechadv.2023.108115},
pmid = {36758652},
issn = {1873-1899},
abstract = {Chinese hamster ovary (CHO) cells are among the most widely used mammalian cell lines in the biopharmaceutical industry. Therefore, it is not surprising that significant efforts have been made around the engineering of CHO cells using genetic engineering methods such as the CRISPR-Cas system. In this review, we summarize key recent studies that have used different CRISPR-Cas systems such as Cas9, Cas13 or dCas9 fused with effector domains to improve recombinant protein (r-protein) production in CHO cells. Here, every relevant stage of production was considered, underscoring the advantages and limitations of these systems, as well as discussing their bottlenecks and probable solutions. A special emphasis was given on how these systems could disrupt and/or regulate genes related to glycan composition, which has relevant effects over r-protein properties and in vivo activity. Furthermore, the related promising future applications of CRISPR to achieve a tunable, reversible, or highly stable editing of CHO cells are discussed. Overall, the studies covered in this review show that despite the complexity of mammalian cells, the synthetic biology community has developed many mature strategies to improve r-protein production using CHO cells. In this regard, CRISPR-Cas technology clearly provides efficient and flexible genetic manipulation and allows for the generation of more productive CHO cell lines, leading to more cost-efficient production of biopharmaceuticals, however, there is still a need for many emerging techniques in CRISPR to be reported in CHO cells; therefore, more research in these cells is needed to realize the full potential of this technology.},
}
@article {pmid36754958,
year = {2023},
author = {Collias, D and Vialetto, E and Yu, J and Co, K and Almási, &#xc9;DH and Rüttiger, AS and Achmedov, T and Strowig, T and Beisel, CL},
title = {Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {680},
pmid = {36754958},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; DNA/genetics ; Genome, Bacterial/genetics ; Bacteria/genetics ; },
abstract = {Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.},
}
@article {pmid36757178,
year = {2023},
author = {Montagud-Martínez, R and Márquez-Costa, R and Rodrigo, G},
title = {Programmable regulation of translation by harnessing the CRISPR-Cas13 system.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3cc00058c},
pmid = {36757178},
issn = {1364-548X},
abstract = {The ability to control protein expression at both the transcriptional and post-transcriptional levels is instrumental for the cell to integrate multiple molecular signals and then reach high operational sophistication. Although challenging, fully artificial regulations at different levels are required for boosting systems and synthetic biology. Here, we report the development of a novel framework to regulate translation by repurposing the CRISPR-Cas13 immune system, which uses an RNA-guided ribonuclease. By exploiting a cell-free expression system for prototyping gene regulatory structures, our results demonstrate that CRISPR-dCas13a ribonucleoproteins (d means catalytically dead) can be programmed to repress or activate translation initiation. The performance assessment of the engineered systems also revealed guide RNA design principles. Moreover, we show that the system can work in vivo. This development complements the ability to regulate transcription with other CRISPR-Cas systems and offers potential applications.},
}
@article {pmid36755494,
year = {2023},
author = {Jiang, L and Wang, D and He, Y and Shu, Y},
title = {Advances in gene therapy hold promise for treating hereditary hearing loss.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2023.02.001},
pmid = {36755494},
issn = {1525-0024},
abstract = {Gene therapy focuses on genetic modification to produce therapeutic effects or treat diseases by repairing or reconstructing genetic material, thus being expected to be the most promising therapeutic strategy for genetic disorders. Due to the growing attention to hearing impairment, an increasing amount of research is attempting to utilize gene therapy for hereditary hearing loss (HHL)-an important monogenic disease and the most common type of congenital deafness. Several gene therapy clinical trials for HHL have recently been approved, and additionally, CRISPR/Cas tools have been attempted for HHL treatment. Therefore, in order to further advance the development of inner ear gene therapy and promote its broad application in other forms of genetic disease, it is imperative to review the progress of gene therapy for HHL. Herein, we address three main gene therapy strategies-gene replacement, gene suppression, and gene editing, summarizing which strategy is most appropriate for particular monogenic diseases based on different pathogenic mechanisms and then focusing on their successful applications for HHL in preclinical trials. Finally, we elaborate on the challenges and outlooks of gene therapy for HHL.},
}
@article {pmid36755261,
year = {2023},
author = {Shaigani, P and Fuchs, T and Graban, P and Prem, S and Haack, M and Masri, M and Mehlmer, N and Brueck, T},
title = {Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {25},
pmid = {36755261},
issn = {1475-2859},
abstract = {BACKGROUND: Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported.<br><br>RESULTS: The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions.<br><br>CONCLUSION: This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.},
}
@article {pmid36755180,
year = {2023},
author = {Abe, M and Nakatsukasa, E and Natsume, R and Hamada, S and Sakimura, K and Watabe, AM and Ohtsuka, T},
title = {A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {2245},
doi = {10.1038/s41598-023-29468-1},
pmid = {36755180},
issn = {2045-2322},
abstract = {CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.},
}
@article {pmid36754343,
year = {2023},
author = {Yin, P and Zhang, Y and Yang, L and Feng, Y},
title = {Non-canonical inhibition strategies and structural basis of anti-CRISPR proteins targeting type I CRISPR-Cas systems.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {167996},
doi = {10.1016/j.jmb.2023.167996},
pmid = {36754343},
issn = {1089-8638},
abstract = {Mobile genetic elements (MGEs) such as bacteriophages and their host prokaryotes are trapped in an eternal battle against each other. To cope with foreign infection, bacteria and archaea have evolved multiple immune strategies, out of which CRISPR-Cas system is up to now the only discovered adaptive system in prokaryotes. Despite the fact that CRISPR-Cas system provides powerful and delicate protection against MGEs, MGEs have also evolved anti-CRISPR proteins (Acrs) to counteract the CRISPR-Cas immune defenses. To date, 46 families of Acrs targeting type I CRISPR-Cas system have been characterized, out of which structure information of 21 families have provided insights on their inhibition strategies. Here, we review the non-canonical inhibition strategies adopted by Acrs targeting type I CRISPR-Cas systems based on their structure information by incorporating the most recent advances in this field, and discuss our current understanding and future perspectives. The delicate interplay between type I CRISPR-Cas systems and their Acrs provides us with important insights into the ongoing fierce arms race between prokaryotic hosts and their predators.},
}
@article {pmid36754153,
year = {2023},
author = {Fu, L and Li, Z and Ren, Y and Yu, H and Liu, B and Qiu, Y},
title = {CRISPR/Cas genome editing in triple negative breast cancer: Current situation and future directions.},
journal = {Biochemical pharmacology},
volume = {},
number = {},
pages = {115449},
doi = {10.1016/j.bcp.2023.115449},
pmid = {36754153},
issn = {1873-2968},
abstract = {Triple negative breast cancer (TNBC) has been well-known to be closely associated with the abnormal expression of both oncogenes and tumor suppressors. Although several pathogenic mutations in TNBC have been identified, the current therapeutic strategy is usually aimed at symptom relief rather than correcting mutations in the DNA sequence. Of note, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has been gradually regarded as a breakthrough gene-editing tool with potential therapeutic applications in human cancers, including TNBC. Thus, in this review, we focus on summarizing the molecular subtypes of TNBC, as well as the CRISPR system and its potential applications in TNBC treatment. Moreover, we further discuss several emerging strategies for utilizing the CRISPR/Cas system to aid in the precise diagnosis of TNBC, as well as the limitations of the CRISPR/Cas system. Taken together, these findings would demonstrate that CRISPR/Cas system is not only an effective genome editing tool in TNBC, but a promising strategy for the future therapeutic purposes.},
}
@article {pmid36703533,
year = {2023},
author = {Li, Y and Huang, D and Pei, Y and Wu, Y and Xu, R and Quan, F and Gao, H and Zhang, J and Hou, H and Zhang, K and Li, J},
title = {CasSABER for Programmable In Situ Visualization of Low and Nonrepetitive Gene Loci.},
journal = {Analytical chemistry},
volume = {95},
number = {5},
pages = {2992-3001},
doi = {10.1021/acs.analchem.2c04867},
pmid = {36703533},
issn = {1520-6882},
mesh = {Reproducibility of Results ; *Genetic Loci ; Molecular Probes ; Fluorescence ; *RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Site-specific imaging of target genes using CRISPR probes is essential for understanding the molecular mechanisms of gene function and engineering tools to modulate its downstream pathways. Herein, we develop CRISPR/Cas9-mediated signal amplification by exchange reaction (CasSABER) for programmable in situ imaging of low and nonrepetitive regions of the target gene in the cell nucleus. The presynthesized primer-exchange reaction (PER) probe is able to hybridize multiple fluorophore-bearing imager strands to specifically light up dCas9/sgRNA target-bound gene loci, enabling in situ imaging of fixed cellular gene loci with high specificity and signal-to-noise ratio. In combination with a multiround branching strategy, we successfully detected nonrepetitive gene regions using a single sgRNA. As an intensity-codable and orthogonal probe system, CasSABER enables the adjustable amplification of local signals in fixed cells, resulting in the simultaneous visualization of multicopy and single-copy gene loci with similar fluorescence intensity. Owing to avoiding the complexity of controlling in situ mutistep enzymatic reactions, CasSABER shows good reliability, sensitivity, and ease of implementation, providing a rapid and cost-effective molecular toolkit for studying multigene interaction in fundamental research and gene diagnosis.},
}
@article {pmid36652615,
year = {2023},
author = {Larkin, HD},
title = {Gene Edited Tumor Cells Kill and Prevent Cancer in Mice.},
journal = {JAMA},
volume = {329},
number = {5},
pages = {363},
doi = {10.1001/jama.2022.24492},
pmid = {36652615},
issn = {1538-3598},
mesh = {Mice ; Animals ; *Gene Editing ; *Neoplasms ; CRISPR-Cas Systems ; },
}
@article {pmid36640150,
year = {2023},
author = {Maestroni, L and Butti, P and Senatore, VG and Branduardi, P},
title = {pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {23},
number = {},
pages = {},
doi = {10.1093/femsyr/foad002},
pmid = {36640150},
issn = {1567-1364},
mesh = {*Gene Editing/methods ; *Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; Plasmids ; RNA, Guide, Kinetoplastida ; DNA/metabolism ; },
abstract = {CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted double-strand breaks are controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming, and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome editing in S. cerevisiae, pCEC-red. This tool allows to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and (iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.},
}
@article {pmid35994168,
year = {2023},
author = {Yu, J and Li, T and Chen, K and Tang, Q and Zhu, J},
title = {Stereopure AIMer: A Promising RNA Base-editing Tool for Monogenic Neurological Diseases.},
journal = {Neuroscience bulletin},
volume = {39},
number = {2},
pages = {353-355},
pmid = {35994168},
issn = {1995-8218},
mesh = {*RNA ; *CRISPR-Cas Systems ; },
}
@article {pmid36753844,
year = {2023},
author = {Hemalatha, P and Abda, EM and Shah, S and Venkatesa Prabhu, S and Jayakumar, M and Karmegam, N and Kim, W and Govarthanan, M},
title = {Multi-faceted CRISPR-Cas9 strategy to reduce plant based food loss and waste for sustainable bio-economy - A review.},
journal = {Journal of environmental management},
volume = {332},
number = {},
pages = {117382},
doi = {10.1016/j.jenvman.2023.117382},
pmid = {36753844},
issn = {1095-8630},
abstract = {Currently, international development requires innovative solutions to address imminent challenges like climate change, unsustainable food system, food waste, energy crisis, and environmental degradation. All the same, addressing these concerns with conventional technologies is time-consuming, causes harmful environmental impacts, and is not cost-effective. Thus, biotechnological tools become imperative for enhancing food and energy resilience through eco-friendly bio-based products by valorisation of plant and food waste to meet the goals of circular bioeconomy in conjunction with Sustainable Developmental Goals (SDGs). Genome editing can be accomplished using a revolutionary DNA modification tool, CRISPR-Cas9, through its uncomplicated guided mechanism, with great efficiency in various organisms targeting different traits. This review's main objective is to examine how the CRISPR-Cas system, which has positive features, could improve the bioeconomy by reducing food loss and waste with all-inclusive food supply chain both at on-farm and off-farm level; utilising food loss and waste by genome edited microorganisms through food valorisation; efficient microbial conversion of low-cost substrates as biofuel; valorisation of agro-industrial wastes; mitigating greenhouse gas emissions through forestry plantation crops; and protecting the ecosystem and environment. Finally, the ethical implications and regulatory issues that are related to CRISPR-Cas edited products in the international markets have also been taken into consideration.},
}
@article {pmid36753615,
year = {2023},
author = {Gutiérrez-Rodríguez, A and Cruz-Fuentes, C and Genis-Mendoza, AD and Nicolini, H},
title = {CRISPR/Cas9 Genome Editing Approaches for Psychiatric Research.},
journal = {Revista brasileira de psiquiatria (Sao Paulo, Brazil : 1999)},
volume = {},
number = {},
pages = {},
doi = {10.47626/1516-4446-2022-2913},
pmid = {36753615},
issn = {1809-452X},
abstract = {Currently, genome editing technologies, such as the Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), are predominantly used to model genetic diseases. This genome editing system can correct point or frameshift mutations of risk genes. Here, we analyze and discuss the genome editing advantages, current applications, and the feasibility of the CRISPR/Cas9 system in psychiatric disorders research. These disorders produce cognitive and behavioral alterations. Besides, their etiology is associated with polygenetic and environmental factors. CRISPR/Cas9 may allow the understanding of the biological mechanisms of psychiatric disorders at a basic research level. We propose that the knowledge of these post-editing mechanisms in the psychiatric field may ease the translation of a suitable clinical approach in genetic diagnosis and treatment of psychiatric disorders. Currently, the diagnosis and treatment in psychiatry are not fully established because of the limited understanding of the heterogeneity and polygenicity of these disorders. Here, we discussed the challenges and ethical issues that could lead to the CRISPR/Cas9 application as a diagnosis or gene therapy tool in the future.},
}
@article {pmid36751129,
year = {2023},
author = {Liu, Q and Zhao, C and Sun, K and Deng, Y and Li, Z},
title = {Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes.},
journal = {Molecular plant},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molp.2023.02.003},
pmid = {36751129},
issn = {1752-9867},
abstract = {CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding. However, the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science. Here, we report an efficient, non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus, an RNA virus with a host range of over 1,000 plant species. We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts. The resulting non-insect-transmissible viral vectors attained effective and stable in planta delivery of CRISPR/Cas12a and Cas9 nucleases as well as adenine and cytosine base editors. In systemically infected plant tissues, the deconstructed virus vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency. Plants with heritable, bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection. Moreover, we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100% of regenerated plants and further augmented the recovery of heritable mutations. Because many plants are recalcitrant to stable transformation, the viral delivery system developed in this work should provide a promising tool to overcome gene delivery bottlenecks for genome-editing various crop species and elite varieties.},
}
@article {pmid36748042,
year = {2023},
author = {Froehlich, JJ and Rajewsky, N},
title = {C. elegans Gene Regulatory Alleles and Reporter Bashing Studies.},
journal = {microPublication biology},
volume = {2023},
number = {},
pages = {},
pmid = {36748042},
issn = {2578-9430},
abstract = {Gene regulation has been studied in C. elegans for over 30 years. In this analysis of 102 publications, we find that most transcriptional cis-regulatory elements are located within 5,000 bp of the transcription start site. Over 75% of studies conclude that transcriptional elements and 5'UTRs activate-, while 3'UTRs repress gene expression. While gene regulatory mutations make up less than 0.8% of alleles in forward genetics screens, recent CRISPR-Cas approaches are increasing the number of tested mutations. This work provides a resource of known gene regulatory sequences in C.elegans .},
}
@article {pmid36746216,
year = {2023},
author = {Jeong, BR and Jang, J and Jin, E},
title = {Genome engineering via gene editing technologies in microalgae.},
journal = {Bioresource technology},
volume = {},
number = {},
pages = {128701},
doi = {10.1016/j.biortech.2023.128701},
pmid = {36746216},
issn = {1873-2976},
abstract = {CRISPR-Cas has revolutionized genetic modification with its comparative simplicity and accuracy, and it can be used even at the genomic level. Microalgae are excellent feedstocks for biofuels and nutraceuticals because they contain high levels of fatty acids, carotenoids, and other metabolites; however, genome engineering for microalgae is not yet as developed as for other model organisms. Microalgal engineering at the genetic and metabolic levels is relatively well established, and a few genomic resources are available. Their genomic information was used for a "safe harbor" site for stable transgene expression in microalgae. This review proposes further genome engineering schemes including the construction of sgRNA libraries, pan-genomic and epigenomic resources, and mini-genomes, which can together be developed into synthetic biology for carbon-based engineering in microalgae. Acetyl-CoA is at the center of carbon metabolic pathways and is further reviewed for the production of molecules including terpenoids in microalgae.},
}
@article {pmid36056851,
year = {2023},
author = {Dashtaki, ME and Ghasemi, S},
title = {CRISPR/Cas9-based Gene Therapies for Fighting Drug Resistance Mediated by Cancer Stem Cells.},
journal = {Current gene therapy},
volume = {23},
number = {1},
pages = {41-50},
doi = {10.2174/1566523222666220831161225},
pmid = {36056851},
issn = {1875-5631},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; *Neoplasms/genetics/therapy/pathology ; Neoplastic Stem Cells/pathology ; },
abstract = {Cancer stem cells (CSCs) are cancer-initiating cells found in most tumors and hematological cancers. CSCs are involved in cells progression, recurrence of tumors, and drug resistance. Current therapies have been focused on treating the mass of tumor cells and cannot eradicate the CSCs. CSCs drug-specific targeting is considered as an approach to precisely target these cells. Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing systems are making progress and showing promise in the cancer research field. One of the attractive applications of CRISPR/Cas9 as one approach of gene therapy is targeting the critical genes involved in drug resistance and maintenance of CSCs. The synergistic effects of gene editing as a novel gene therapy approach and traditional therapeutic methods, including chemotherapy, can resolve drug resistance challenges and regression of the cancers. This review article considers different aspects of CRISPR/Cas9 ability in the study and targeting of CSCs with the intention to investigate their application in drug resistance.},
}
@article {pmid35899957,
year = {2023},
author = {Lasemi, MV and Sahebkar, A},
title = {Non-surgical In Vivo Germ Cell-mediated Gene Editing by CRISPR Mutagenic Chain Reaction with the Aid of Magnetic Nanoparticles.},
journal = {Current medicinal chemistry},
volume = {30},
number = {7},
pages = {809-819},
doi = {10.2174/0929867329666220722113832},
pmid = {35899957},
issn = {1875-533X},
mesh = {Animals ; Male ; Female ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mutagens ; *Magnetite Nanoparticles ; Germ Cells ; },
abstract = {Gene therapy via germline cells leads to a permanent genetic modification. The promise of this method is due to its potential for providing a stable therapeutic effect for all who inherit the gene of interest. If germinal therapy is successfully performed, it can eliminate certain diseases from the family and the population. The feasibility of genetic modification in the human germline raises several controversial and bioethical issues. However, gene transfer via male and female germinal cells has been recently explored in animal models. Previous studies have shown that delivering DNA to the testes followed by electroporation is relatively successful in producing germline-mediated alterations. Since this method includes surgical procedures, non-surgical, safer, and less timeconsuming methods would be ideal. Herein, we discuss a potential approach for nonsurgical in vivo germ cell-mediated gene editing by CRISPR mutagenic chain reaction with the aid of magnetic nanoparticles.},
}
@article {pmid35345999,
year = {2023},
author = {Mani, S and Jindal, D and Singh, M},
title = {Gene Therapy, A Potential Therapeutic Tool for Neurological and Neuropsychiatric Disorders: Applications, Challenges and Future Perspective.},
journal = {Current gene therapy},
volume = {23},
number = {1},
pages = {20-40},
doi = {10.2174/1566523222666220328142427},
pmid = {35345999},
issn = {1875-5631},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Autism Spectrum Disorder/genetics ; Genetic Therapy/methods ; Gene Transfer Techniques ; },
abstract = {Neurological and neuropsychiatric disorders are the main risks for the health care system, exhibiting a huge socioeconomic load. The available range of pharmacotherapeutics mostly provides palliative consequences and fails to treat such conditions. The molecular etiology of various neurological and neuropsychiatric disorders is mostly associated with a change in genetic background, which can be inherited/triggered by other environmental factors. To address such conditions, gene therapy is considered a potential approach claiming a permanent cure of the disease primarily by deletion, silencing, or edition of faulty genes and by insertion of healthier genes. In gene therapy, vectors (viral/nonvial) play an important role in delivering the desired gene to a specific region of the brain. Targeted gene therapy has unraveled opportunities for the treatment of many neurological and neuropsychiatric disorders. For improved gene delivery, the current techniques mainly focus on designing a precise viral vector, plasmid transfection, nanotechnology, microRNA, and in vivo clustered regulatory interspaced short palindromic repeats (CRISPR)-based therapy. These latest techniques have great benefits in treating predominant neurological and neurodevelopmental disorders, including Parkinson's disease, Alzheimer's disease, and autism spectrum disorder, as well as rarer diseases. Nevertheless, all these delivery methods have their limitations, including immunogenic reactions, off-target effects, and a deficiency of effective biomarkers to appreciate the effectiveness of therapy. In this review, we present a summary of the current methods in targeted gene delivery, followed by the limitations and future direction of gene therapy for the cure of neurological and neuropsychiatric disorders.},
}
@article {pmid36746179,
year = {2023},
author = {Sha, Y and Liu, W and Tang, S and Zhang, X and Xiao, Z and Xiao, Y and Deng, H and Zhou, H and Wei, X},
title = {TENT5D disruption causes oligoasthenoteratozoospermia and male infertility.},
journal = {Andrology},
volume = {},
number = {},
pages = {},
doi = {10.1111/andr.13407},
pmid = {36746179},
issn = {2047-2927},
abstract = {BACKGROUND: Oligoasthenoteratozoospermia (OAT) is one of the most complex aggregators of male gametic problems. However, the genetic etiology of OAT is still largely unknown.<br><br>OBJECTIVES: To reveal the new genetic factors responsible for male infertility owning to OAT and reveal the outcomes of the affected patients from intracytoplasmic sperm injection (ICSI).<br><br>MATERIALS AND METHODS: Two infertile men with typical OAT were recruited in 2018 and retrospected a cohort that included 47 patients with OAT from 2013 to 2021. Fifty healthy men with proven fertility served as control subjects. Whole-exome sequencing and Sanger sequencing to identify the novel pathogenic variants. In silico analysis revealed the affecting of the variants. Field emission scanning electron microscopy was employed to observe the morphological defects of the spermatozoa. Immunofluorescence was used to analyze the expression and localization of the related protein. CRISPR/Cas 9 was used to generate the mouse model. ICSI was used as a treatment for the patients and to assess the effects of the pathogenic variant on fertilization and embryo development.<br><br>RESULTS: We identified a loss-of-function mutation NM_001170574.2:c.823G>T (p.Glu275*) in X-linked TENT5D from two patients with OAT. This variant is highly deleterious and has not been found in the human population. The count of patients' spermatozoa is dramatically decreased and displays multiple morphologic abnormalities with poor motility. Tent5d knockout mice are infertile and exhibit parallel defects. ICSI could rescue the infertility of the Tent5d knockout male mice. Moreover, the proband was treated with ICSI and achieved a successful pregnancy outcome for the first time. Subsequent mutation screening identified no TENT5D mutations among 47 additional patients with OAT and fifty control subjects.<br><br>CONCLUSION: Mutation in TENT5D results in OAT and male infertility, and this terrible situation could be rescued by ICSI. This article is protected by copyright. All rights reserved.},
}
@article {pmid36736311,
year = {2023},
author = {Chang, HY and Qi, LS},
title = {Reversing the Central Dogma: RNA-guided control of DNA in epigenetics and genome editing.},
journal = {Molecular cell},
volume = {83},
number = {3},
pages = {442-451},
doi = {10.1016/j.molcel.2023.01.010},
pmid = {36736311},
issn = {1097-4164},
mesh = {Animals ; *Gene Editing ; DNA/genetics ; Epigenesis, Genetic ; Chromatin/genetics ; *RNA, Long Noncoding/genetics ; CRISPR-Cas Systems/genetics ; Mammals/metabolism ; },
abstract = {The Central Dogma of the flow of genetic information is arguably the crowning achievement of 20[th] century molecular biology. Reversing the flow of information from RNA to DNA or chromatin has come to the fore in recent years, from the convergence of fundamental discoveries and synthetic biology. Inspired by the example of long noncoding RNAs (lncRNAs) in mammalian genomes that direct chromatin modifications and gene expression, synthetic biologists have repurposed prokaryotic RNA-guided genome defense systems such as CRISPR to edit eukaryotic genomes and epigenomes. Here we explore the parallels of these two fields and highlight opportunities for synergy and future breakthroughs.},
}
@article {pmid36656895,
year = {2023},
author = {Bui, M and Dalla Benetta, E and Dong, Y and Zhao, Y and Yang, T and Li, M and Antoshechkin, IA and Buchman, A and Bottino-Rojas, V and James, AA and Perry, MW and Dimopoulos, G and Akbari, OS},
title = {CRISPR mediated transactivation in the human disease vector Aedes aegypti.},
journal = {PLoS pathogens},
volume = {19},
number = {1},
pages = {e1010842},
pmid = {36656895},
issn = {1553-7374},
mesh = {Animals ; Humans ; *Aedes ; Hedgehog Proteins/metabolism ; Mosquito Vectors/genetics ; RNA/metabolism ; Transcriptional Activation ; CRISPR-Cas Systems ; },
abstract = {As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.},
}
@article {pmid36635153,
year = {2023},
author = {Lahr, WS and Sipe, CJ and Skeate, JG and Webber, BR and Moriarity, BS},
title = {CRISPR-Cas9 base editors and their current role in human therapeutics.},
journal = {Cytotherapy},
volume = {25},
number = {3},
pages = {270-276},
doi = {10.1016/j.jcyt.2022.11.013},
pmid = {36635153},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; DNA ; },
abstract = {BACKGROUND: Consistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification.<br><br>METHODS: This review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes.<br><br>RESULTS: CRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction.<br><br>CONCLUSION: As such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.},
}
@article {pmid36627091,
year = {2023},
author = {Augustin, L and Agarwal, N},
title = {Designing a Cas9/gRNA-assisted quantitative Real-Time PCR (CARP) assay for identification of point mutations leading to rifampicin resistance in the human pathogen Mycobacterium tuberculosis.},
journal = {Gene},
volume = {857},
number = {},
pages = {147173},
doi = {10.1016/j.gene.2023.147173},
pmid = {36627091},
issn = {1879-0038},
mesh = {Humans ; Animals ; Rifampin/pharmacology ; *Mycobacterium tuberculosis ; Point Mutation ; Real-Time Polymerase Chain Reaction ; CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; *Tuberculosis, Multidrug-Resistant/diagnosis/drug therapy/genetics ; Mutation ; DNA-Directed RNA Polymerases/genetics ; *Carps ; Microbial Sensitivity Tests ; Bacterial Proteins/genetics/pharmacology ; },
abstract = {A simple, rapid and low-cost diagnostic test, which can detect both the drug-sensitive and the drug-resistant tuberculosis (TB) cases is the need of the hour. Here, we developed a Cas9/gRNA-assisted quantitative Real-Time PCR (qRT-PCR) (CARP) assay to detect single nucleotide mutations causing drug resistance in the TB pathogen, Mycobacterium tuberculosis (Mtb). Guide RNAs (gRNAs) were designed against S531 and H526 positions in the rifampicin (RIF)-resistance-determining region (RRDR) of the Mtb rpoB gene that exhibit frequent mutations in the RR clinical isolates of Mtb. Conditions were optimised for in vitro Cas9 cleavage such that single nucleotide changes at these positions can be recognised by Cas9/gRNA complex with high sensitivity and 100% specificity. Further estimation of Cas9/gRNA-based cleavage of target DNA by qRT-PCR led to rapid detection of drug-resistant sequences. The newly designed CARP assay holds a great deal of promise in the diagnosis and prognosis of patients suffering from TB, in a cost-effective manner.},
}
@article {pmid36610813,
year = {2023},
author = {Ugalde, L and Fañanas, S and Torres, R and Quintana-Bustamante, O and Río, P},
title = {CRISPR/Cas9-mediated gene editing. A promising strategy in hematological disorders.},
journal = {Cytotherapy},
volume = {25},
number = {3},
pages = {277-285},
doi = {10.1016/j.jcyt.2022.11.014},
pmid = {36610813},
issn = {1477-2566},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; Mutation ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the gene editing field, making it possible to interrupt, insert or replace a sequence of interest with high precision in the human genome. Its easy design and wide applicability open up a variety of therapeutic alternatives for the treatment of genetic diseases. Indeed, very promising approaches for the correction of hematological disorders have been developed in the recent years, based on the self-renewal and multipotent differentiation properties of hematopoietic stem and progenitor cells, which make this cell subset the ideal target for gene therapy purposes. This technology has been applied in different congenital blood disorders, such as primary immunodeficiencies, X-linked severe combined immunodeficiency, X-linked chronic granulomatous disease or Wiskott-Aldrich syndrome, and inherited bone marrow failure syndromes, such as Fanconi anemia, congenital amegakaryocytic thrombocytopenia or severe congenital neutropenia. Furthermore, CRISPR/Cas9-based gene editing has been implemented successfully as a novel therapy for cancer immunotherapy, by the development of promising strategies such as the use of oncolytic viruses or adoptive cellular therapy to the chimeric antigen receptor-T-cell therapy. Therefore, considering the variety of genes and mutations affected, we can take advantage of the different DNA repair mechanisms by CRISPR/Cas9 in different manners, from homology-directed repair to non-homologous-end-joining to the latest emerging technologies such as base and prime editing. Although the delivery systems into hematopoietic stem and progenitor cells are still the bottleneck of this technology, some of the advances in genome editing shown in this review have already reached a clinical stage and show very promising preliminary results.},
}
@article {pmid36571980,
year = {2023},
author = {Yang, T and Curtis, S and Bai, A and Young, A and Derosier, D and Ripley, S and Bai, S},
title = {CRISPR/Cas9 targeting liposomes knocked down multidrug resistance proteins in brain endothelial cells as a model to predict potential pharmacoresistance.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {222},
number = {},
pages = {113103},
pmid = {36571980},
issn = {1873-4367},
support = {R03 EB028572/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Liposomes ; *Endothelial Cells/metabolism ; CRISPR-Cas Systems/genetics ; Rhodamine 123 ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; Brain/metabolism ; Doxorubicin/pharmacology ; },
abstract = {This investigation aimed to use CRISPR-Cas9 gene-editing to knock down P-glycoprotein (P-gp) expression and then establish a feasible cell line to evaluate the potential pharmacoresistance of therapeutic agents mediated by efflux. A cationic liposome was prepared as a "smart bomb" by conjugating with a peptide-based targeting ligand (THRPPMWSPVWP), specifically binding to transferrin receptors at the blood-brain barrier (BBB), and then formed a nanocomplex with P-gp knockdown CRISPR/Cas9 plasmid. Higher uptakes of targeted and stable liposomes in bEND.3 cells were observed compared to non-peptide conjugated ones (p < 0.05). The P-gp transporters were successfully knocked down by the cell-nontoxic CRISPR/Cas9 targeted liposomes and P-gp associated ATP activities were higher in the transfected cells (p < 0.05). Functional studies of knocked down cells were evaluated by using prototypical P-gp substrates rhodamine 123 and doxorubicin. More accumulation of rhodamine 123 and higher cytotoxic sensitivity of doxorubicin was observed in the transfected cells as compared with those in the wild-type cells.},
}
@article {pmid36565936,
year = {2023},
author = {Okita, TW and Delseny, M},
title = {Genome editing in plants: New advances and applications in plant biology and agriculture.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {328},
number = {},
pages = {111577},
doi = {10.1016/j.plantsci.2022.111577},
pmid = {36565936},
issn = {1873-2259},
mesh = {*Gene Editing ; *Plants/genetics ; CRISPR-Cas Systems ; Agriculture ; Biology ; Genome, Plant/genetics ; Plant Breeding ; },
}
@article {pmid36482283,
year = {2023},
author = {Alkanli, SS and Alkanli, N and Ay, A and Albeniz, I},
title = {CRISPR/Cas9 Mediated Therapeutic Approach in Huntington's Disease.},
journal = {Molecular neurobiology},
volume = {60},
number = {3},
pages = {1486-1498},
pmid = {36482283},
issn = {1559-1182},
mesh = {Mice ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; *Huntington Disease/genetics/therapy/metabolism ; *Neurodegenerative Diseases/genetics ; Gene Editing ; Neurons/metabolism ; },
abstract = {The pathogenic mechanisms of these diseases must be well understood for the treatment of neurological disorders such as Huntington's disease. Huntington's Disease (HD), a dominant and neurodegenerative disease, is characterized by the CAG re-expansion that occurs in the gene encoding the polyglutamine-expanded mutant Huntingtin (mHTT) protein. Genome editing approaches include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats/Caspase 9 (CRISPR/Cas9) systems. CRISPR/Cas9 technology allows effective gene editing in different cell types and organisms. Through these systems are created isogenic control of human origin induced pluripotent stem cells (iPSCs). In human and mouse models, HD-iPSC lines can be continuously corrected using these systems. HD-iPSCs can be corrected through the CRISPR/Cas9 system and the cut-and-paste mechanism using isogenic control iPSCs. This mechanism is a piggyBac transposon-based selection system that can effectively switch between vectors and chromosomes. In studies conducted, it has been determined that in neural cells derived from HD-iPSC, there are isogenic controls as corrected lines recovered from phenotypic abnormalities and gene expression changes. It has been determined that trinucleotide repeat disorders occurring in HD can be cured by single-guide RNA (sgRNA) and normal exogenous DNA restoration, known as the single guideline RNA specific to Cas9. The purpose of this review in addition to give general information about HD, a neurodegenerative disorder is to explained the role of CRISPR/Cas9 system with iPSCs in HD treatment.},
}
@article {pmid36481341,
year = {2023},
author = {Lear, SK and Shipman, SL},
title = {Molecular recording: transcriptional data collection into the genome.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102855},
doi = {10.1016/j.copbio.2022.102855},
pmid = {36481341},
issn = {1879-0429},
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; Genome/genetics ; Genomics ; Data Collection ; Gene Editing ; },
abstract = {Advances in regenerative medicine depend upon understanding the complex transcriptional choreography that guides cellular development. Transcriptional molecular recorders, tools that record different transcriptional events into the genome of cells, hold promise to elucidate both the intensity and timing of transcriptional activity at single-cell resolution without requiring destructive multitime point assays. These technologies are dependent on DNA writers, which translate transcriptional signals into stable genomic mutations that encode the duration, intensity, and order of transcriptional events. In this review, we highlight recent progress toward more informative and multiplexable transcriptional recording through the use of three different types of DNA writing - recombineering, Cas1-Cas2 acquisition, and prime editing - and the architecture of the genomic data generated.},
}
@article {pmid36326169,
year = {2023},
author = {Kim, S and Jeong, YK and Cho, CS and Lee, S and Sohn, CH and Kim, JH and Jeong, Y and Jo, DH and Bae, S and Lee, H},
title = {Enhancement of Gene Editing and Base Editing with Therapeutic Ribonucleoproteins through In Vivo Delivery Based on Absorptive Silica Nanoconstruct.},
journal = {Advanced healthcare materials},
volume = {12},
number = {4},
pages = {e2201825},
doi = {10.1002/adhm.202201825},
pmid = {36326169},
issn = {2192-2659},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Ribonucleoproteins/genetics/metabolism ; Genetic Therapy ; },
abstract = {Key to the widespread and secure application of genome editing tools is the safe and effective delivery of multiple components of ribonucleoproteins (RNPs) into single cells, which remains a biological barrier to their clinical application. To overcome this issue, a robust RNP delivery platform based on a biocompatible sponge-like silica nanoconstruct (SN) for storing and directly delivering therapeutic RNPs, including Cas9 nuclease RNP (Cas9-RNP) and base editor RNP (BE-RNP) is designed. Compared with commercialized material such as lipid-based methods, up to 50-fold gene deletion and 10-fold base substitution efficiency is obtained with a low off-target efficiency by targeting various cells and genes. In particular, gene correction is successfully induced by SN-based delivery through intravenous injection in an in vivo solid-tumor model and through subretinal injection in mouse eye. Moreover, because of its low toxicity and high biodegradability, SN has negligible effect on cellular function of organs. As the engineered SN can overcome practical challenges associated with therapeutic RNP application, it is strongly expected this platform to be a modular RNPs delivery system, facilitating in vivo gene deletion and editing.},
}
@article {pmid36123234,
year = {2023},
author = {Bhoopalan, SV and Yen, JS and Levine, RM and Sharma, A},
title = {Editing human hematopoietic stem cells: advances and challenges.},
journal = {Cytotherapy},
volume = {25},
number = {3},
pages = {261-269},
doi = {10.1016/j.jcyt.2022.08.003},
pmid = {36123234},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Hematopoietic Stem Cells ; Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {Genome editing of hematopoietic stem and progenitor cells is being developed for the treatment of several inherited disorders of the hematopoietic system. The adaptation of CRISPR-Cas9-based technologies to make precise changes to the genome, and developments in altering the specificity and efficiency, and improving the delivery of nucleases to target cells have led to several breakthroughs. Many clinical trials are ongoing, and several pre-clinical models have been reported that would allow these genetic therapies to one day offer a potential cure to patients with diseases where limited options currently exist. However, there remain several challenges with respect to establishing safety, expanding accessibility and improving the manufacturing processes of these therapeutic products. This review focuses on some of the recent advances in the field of genome editing of hematopoietic stem and progenitor cells and illustrates the ongoing challenges.},
}
@article {pmid36745596,
year = {2023},
author = {Shao, F and Park, JS and Zhao, G and Hsieh, K and Wang, TH},
title = {Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.2c05632},
pmid = {36745596},
issn = {1520-6882},
abstract = {Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.},
}
@article {pmid36744495,
year = {2023},
author = {Sun, W and Zhao, X and Wang, J and Yang, X and Cheng, Z and Liu, S and Wang, J and Sheng, G and Wang, Y},
title = {Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad052},
pmid = {36744495},
issn = {1362-4962},
abstract = {Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH domains. We employ biochemical and mutational analyses to build a model for AcrIIC5's mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.},
}
@article {pmid36744219,
year = {2023},
author = {Liu, Z and Gao, X and Kan, C and Li, L and Zhang, Y and Gao, Y and Zhang, S and Zhou, L and Zhao, H and Li, M and Zhang, Z and Sun, Y},
title = {CRISPR-Cas13d effectively targets SARS-CoV-2 variants, including Delta and Omicron, and inhibits viral infection.},
journal = {MedComm},
volume = {4},
number = {1},
pages = {e208},
pmid = {36744219},
issn = {2688-2663},
abstract = {The recent pandemic of variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need for innovative anti-SARS-CoV-2 approaches in addition to vaccines and antiviral therapeutics. Here, we demonstrate that a CRISPR-Cas13-based strategy against SARS-CoV-2 can effectively degrade viral RNA. First, we conducted a cytological infection experiment, screened CRISPR-associated RNAs (crRNAs) targeting conserved regions of viruses, and used an in vitro system to validate functional crRNAs. Reprogrammed Cas13d effectors targeting NSP13, NSP14, and nucleocapsid transcripts achieved >99% silencing efficiency in human cells which are infected with coronavirus 2, including the emerging variants in the last 2 years, B.1, B.1.1.7 (Alpha), D614G B.1.351 (Beta), and B.1.617 (Delta). Furthermore, we conducted bioinformatics data analysis. We collected the sequence information of COVID-19 and its variants from China, and phylogenetic analysis revealed that these crRNA oligos could target almost 100% of the SARS-CoV family, including the emerging new variant, Omicron. The reprogrammed Cas13d exhibited high specificity, efficiency, and rapid deployment properties; therefore, it is promising for antiviral drug development. This system could possibly be used to protect against unexpected SARS-CoV-2 variants carrying multiple mutations.},
}
@article {pmid36744155,
year = {2023},
author = {Zhang, Y and Zhang, C and Huo, W and Wang, X and Zhang, M and Palmer, K and Chen, M},
title = {An expectation-maximization algorithm for estimating proportions of deletions among bacterial populations with application to study antibiotic resistance gene transfer in Enterococcus faecalis.},
journal = {Marine life science &amp; technology},
volume = {},
number = {},
pages = {1-16},
pmid = {36744155},
issn = {2662-1746},
abstract = {UNLABELLED: The emergence of antibiotic resistance in bacteria limits the availability of antibiotic choices for treatment and infection control, thereby representing a major threat to human health. The de novo mutation of bacterial genomes is an essential mechanism by which bacteria acquire antibiotic resistance. Previously, deletion mutations within bacterial immune systems, ranging from dozens to thousands of base pairs (bps) in length, have been associated with the spread of antibiotic resistance. Most current methods for evaluating genomic structural variations (SVs) have concentrated on detecting them, rather than estimating the proportions of populations that carry distinct SVs. A better understanding of the distribution of mutations and subpopulations dynamics in bacterial populations is needed to appreciate antibiotic resistance evolution and movement of resistance genes through populations. Here, we propose a statistical model to estimate the proportions of genomic deletions in a mixed population based on Expectation-Maximization (EM) algorithms and next-generation sequencing (NGS) data. The method integrates both insert size and split-read mapping information to iteratively update estimated distributions. The proposed method was evaluated with three simulations that demonstrated the production of accurate estimations. The proposed method was then applied to investigate the horizontal transfers of antibiotic resistance genes in concert with changes in the CRISPR-Cas system of E. faecalis.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42995-022-00144-z.},
}
@article {pmid36743551,
year = {2022},
author = {Anand, A and Subramanian, M and Kar, D},
title = {Breeding techniques to dispense higher genetic gains.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1076094},
pmid = {36743551},
issn = {1664-462X},
abstract = {Plant breeding techniques encompass all the processes aimed at improving the genetic characteristics of a crop. It helps in achieving desirable characteristics like resistance to diseases and pests, tolerance to environmental stresses, higher yield and improved quality of the crop. This review article aims to describe and evaluate the current plant breeding techniques and novel methods. This qualitative review employs a comparative approach in exploring the different plant breeding techniques. Conventional plant breeding techniques were compared with modern ones to understand the advancements in plant biotechnology. Backcross breeding, mass selection, and pure-line selection were all discussed in conventional plant breeding for self-pollination and recurrent selection and hybridisation were employed for cross-pollinated crops. Modern techniques comprise of CRISPR Cas-9, high-throughput phenotyping, marker-assisted selection and genomic selection. Further, novel techniques were reviewed to gain more insight. An in-depth analysis of conventional and modern plant breeding has helped gain insight on the advantages and disadvantages of the two. Modern breeding techniques have an upper hand as they are more reliable and less time consuming. It is also more accurate as it is a genotype-based method. However, conventional breeding techniques are cost effective and require less expertise. Modern plant breeding has an upper hand as it uses genomics techniques. Techniques like QTL mapping, marker assisted breeding aid in selection of superior plants right at the seedling stage, which is impossible with conventional breeding. Unlike the conventional method, modern methods are capable of selecting recessive alleles by using different markers. Modern plant breeding is a science and therefore more reliable and accurate.},
}
@article {pmid36741753,
year = {2023},
author = {Jiang, W and Aman, R and Ali, Z and Mahfouz, M},
title = {Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1118684},
pmid = {36741753},
issn = {2296-4185},
abstract = {Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2 μM in a short time, requiring only 15 min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.},
}
@article {pmid36740587,
year = {2023},
author = {Das, T and Anand, U and Pal, T and Mandal, S and Kumar, M and Radha, and Gopalakrishnan, AV and Lastra, JMP and Dey, A},
title = {Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28344},
pmid = {36740587},
issn = {1097-0290},
abstract = {Vegetables provide many nutrients in the form of fiber, vitamins and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement. This article is protected by copyright. All rights reserved.},
}
@article {pmid36738621,
year = {2023},
author = {Xing, W and Li, Q and Han, C and Sun, D and Zhang, Z and Fang, X and Guo, Y and Ge, F and Ding, W and Luo, Z and Zhang, L},
title = {Customization of aptamer to develop CRISPR/Cas12a-derived ultrasensitive biosensor.},
journal = {Talanta},
volume = {256},
number = {},
pages = {124312},
doi = {10.1016/j.talanta.2023.124312},
pmid = {36738621},
issn = {1873-3573},
abstract = {The CRISPR/Cas systems have provided wide biosensing applications. Particularly, the aptamer-involved CRISPR/Cas sensor system powerfully expanded to non-nucleic-acid targets. However, tailoring the sequence of the aptamer to explore the relationship between affinity and the activation of CRISPR/Cas12a trans-cleavage activity has not been reported yet. Herein, we developed a series of new aptamers toward the spike protein 1(S1) of SARS-CoV-2. Surface plasmon resonance measurements showed that the affinity of these aptamers to S1 was at the nM level. Subsequently, a "SET" effect (Sequence Essential Trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, an aptamer, as the activator, sequence needs to be tailored to activate CRISPR/Cas12a efficiently. A balance should be reached between affinity and activation ability. On the one hand, high affinity ensures target recognition performance, and on the other hand, activation can achieve adequate amplification and output of recognition signals. The optimized sequence (with 27 nucleotides, for short 27-nt) not only recognizes the target with a high affinity and specificity but also can trigger the CRISPR/Cas12a trans-cleavage activity efficiently, showing an excellent detection performance in electrochemical biosensors. The detection limit for SARS-CoV-2 S1 can be low at 1.5 pg mL[-1]. The new CRISPR/Cas12a-derived aptasensor also displays a remarkable ability to detect Beta, Delta, and Omicron variants but is selective toward other kinds of proteins. Above all, it is robust for point-of-care testing (POCT) in complex biological fluids, such as saliva, urine, and serum, and provides a universal and scalable detecting platform. Our results provide new insights into aptamer development and a different strategy for COVID-19 antigen detection and biosensor development.},
}
@article {pmid36732720,
year = {2023},
author = {Tissier, RLM and Schie, JJMV and Wolthuis, RMF and Lange, J and Menezes, R},
title = {ShrinkCRISPR: a flexible method for differential fitness analysis of CRISPR-Cas9 screen data.},
journal = {BMC bioinformatics},
volume = {24},
number = {1},
pages = {36},
pmid = {36732720},
issn = {1471-2105},
mesh = {*CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Bayes Theorem ; Gene Knockout Techniques ; },
abstract = {BACKGROUND: CRISPR screens provide large-scale assessment of cellular gene functions. Pooled libraries typically consist of several single guide RNAs (sgRNAs) per gene, for a large number of genes, which are transduced in such a way that every cell receives at most one sgRNA, resulting in the disruption of a single gene in that cell. This approach is often used to investigate effects on cellular fitness, by measuring sgRNA abundance at different time points. Comparing gene knockout effects between different cell populations is challenging due to variable cell-type specific parameters and between replicates variation. Failure to take those into account can lead to inflated or false discoveries.<br><br>RESULTS: We propose a new, flexible approach called ShrinkCRISPR that can take into account multiple sources of variation. Impact on cellular fitness between conditions is inferred by using a mixed-effects model, which allows to test for gene-knockout effects while taking into account sgRNA-specific variation. Estimates are obtained using an empirical Bayesian approach. ShrinkCRISPR can be applied to a variety of experimental designs, including multiple factors. In simulation studies, we compared ShrinkCRISPR results with those of drugZ and MAGeCK, common methods used to detect differential effect on cell fitness. ShrinkCRISPR yielded as many true discoveries as drugZ using a paired screen design, and outperformed both drugZ and MAGeCK for an independent screen design. Although conservative, ShrinkCRISPR was the only approach that kept false discoveries under control at the desired level, for both designs. Using data from several publicly available screens, we showed that ShrinkCRISPR can take data for several time points into account simultaneously, helping to detect early and late differential effects.<br><br>CONCLUSIONS: ShrinkCRISPR is a robust and flexible approach, able to incorporate different sources of variations and to test for differential effect on cell fitness at the gene level. These improve power to find effects on cell fitness, while keeping multiple testing under the correct control level and helping to improve reproducibility. ShrinkCrispr can be applied to different study designs and incorporate multiple time points, making it a complete and reliable tool to analyze CRISPR screen data.},
}
@article {pmid36713455,
year = {2022},
author = {Du, X and McManus, DP and French, JD and Collinson, N and Sivakumaran, H and MacGregor, SR and Fogarty, CE and Jones, MK and You, H},
title = {CRISPR interference for sequence-specific regulation of fibroblast growth factor receptor A in Schistosoma mansoni.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {1105719},
pmid = {36713455},
issn = {1664-3224},
mesh = {Animals ; Mice ; Gene Expression ; *Parasites ; Receptors, Fibroblast Growth Factor ; *Schistosoma mansoni ; Stem Cells ; CRISPR-Cas Systems ; },
abstract = {Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2'-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.},
}
@article {pmid36738203,
year = {2023},
author = {Yao, X and Hu, X and Wang, X and Ge, J},
title = {[Application of cell-free transcription and translation system in CRISPR technologies and the associated biosensors].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {1},
pages = {86-102},
doi = {10.13345/j.cjb.220347},
pmid = {36738203},
issn = {1872-2075},
abstract = {Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.},
}
@article {pmid36711797,
year = {2023},
author = {Alves, CRR and Ha, LL and Yaworski, R and Lazzarotto, CR and Christie, KA and Reilly, A and Beauvais, A and Doll, RM and de la Cruz, D and Maguire, CA and Swoboda, KJ and Tsai, SQ and Kothary, R and Kleinstiver, BP},
title = {Base editing as a genetic treatment for spinal muscular atrophy.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {36711797},
abstract = {Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C•G-to-T•A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2 , reverting the exon 7 mutation via an A•T-to-G•C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.},
}
@article {pmid36738201,
year = {2023},
author = {Sun, W and Huang, X and Wang, X},
title = {[CRISPR-based molecular diagnostics: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {1},
pages = {60-73},
doi = {10.13345/j.cjb.220317},
pmid = {36738201},
issn = {1872-2075},
abstract = {Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.},
}
@article {pmid36738198,
year = {2023},
author = {Zhang, Y and Zhang, C and Wu, Y and Yu, R and Su, J},
title = {[Principle and development of single base editing technology and its application in livestock breeding].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {1},
pages = {19-33},
doi = {10.13345/j.cjb.220339},
pmid = {36738198},
issn = {1872-2075},
abstract = {CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is widely used in the field of livestock breeding. However, its low efficiency, untargeted cutting and low safety have greatly hampered its use for introducing single base mutations in livestock breeding. Single base editing, as a new gene editing tool, can directly replace bases without introducing double strand breaks. Single base editing shows high efficiency and strong specificity, and provides a simpler and more effective method for precise gene modification in livestock breeding. This paper introduces the principle and development of single base editing technology and its application in livestock breeding.},
}
@article {pmid36738178,
year = {2023},
author = {Ayswaria, R and Vijayan, J and Nathan, VK},
title = {Antimicrobial peptides derived from microalgae for combating antibiotic resistance: Current status and prospects.},
journal = {Cell biochemistry and function},
volume = {},
number = {},
pages = {},
doi = {10.1002/cbf.3779},
pmid = {36738178},
issn = {1099-0844},
abstract = {Microalgae are photosynthetic cell factories that produce a spectrum of bioactive compounds extensively used for various applications. Owing to the increase in antibiotic resistance among microbial pathogens, there is a significant thrust for identifying new treatment strategies, and antimicrobial peptides (AMPs) generation is one such method. These AMPs have multiple roles and are active against bacteria, fungi, and viruses. Such peptides synthesized in microalgae have a significant role in medical application, managing aquaculture-associated diseases, and the food industry. To increase their effectiveness and novel peptides, genetically modified microalgae are used as cell factories. With the advancement of new technologies like the CRISPR-Cas system, new avenues are opened for developing novel AMPs using microalgae. This review gives us insight into the various AMPs produced by microalgae and multiple technologies involved in creating such therapeutically essential molecules.},
}
@article {pmid36737140,
year = {2023},
author = {Du, Y and Ji, S and Dong, Q and Wang, J and Han, D and Gao, Z},
title = {Amplification-free detection of HBV DNA mediated by CRISPR-Cas12a using surface-enhanced Raman spectroscopy.},
journal = {Analytica chimica acta},
volume = {1245},
number = {},
pages = {340864},
doi = {10.1016/j.aca.2023.340864},
pmid = {36737140},
issn = {1873-4324},
abstract = {Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans-activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy. This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.},
}
@article {pmid36736884,
year = {2023},
author = {Hwang, S and Shah, M and Garcia, B and Hashem, N and Davidson, AR and Moraes, TF and Maxwell, KL},
title = {Anti-CRISPR protein AcrIIC5 inhibits CRISPR-Cas9 by occupying the target DNA binding pocket.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {167991},
doi = {10.1016/j.jmb.2023.167991},
pmid = {36736884},
issn = {1089-8638},
abstract = {Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5Nch) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5Smu, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.},
}
@article {pmid36735185,
year = {2023},
author = {Aslan, A and Yuka, SA},
title = {Stem Cell-Based Therapeutic Approaches in Genetic Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {},
number = {},
pages = {},
pmid = {36735185},
issn = {0065-2598},
abstract = {Stem cells, which can self-renew and differentiate into different cell types, have become the keystone of regenerative medicine due to these properties. With the achievement of superior clinical results in the therapeutic approaches of different diseases, the applications of these cells in the treatment of genetic diseases have also come to the fore. Foremost, conventional approaches of stem cells to genetic diseases are the first approaches in this manner, and they have brought safety issues due to immune reactions caused by allogeneic transplantation. To eliminate these safety issues and phenotypic abnormalities caused by genetic defects, firstly, basic genetic engineering practices such as vectors or RNA modulators were combined with stem cell-based therapeutic approaches. However, due to challenges such as immune reactions and inability to target cells effectively in these applications, advanced molecular methods have been adopted in ZFN, TALEN, and CRISPR/Cas genome editing nucleases, which allow modular designs in stem cell-based genetic diseases' therapeutic approaches. Current studies in genetic diseases are in the direction of creating permanent treatment regimens by genomic manipulation of stem cells with differentiation potential through genome editing tools. In this chapter, the stem cell-based therapeutic approaches of various vital genetic diseases were addressed wide range from conventional applications to genome editing tools.},
}
@article {pmid36734119,
year = {2023},
author = {Luo, X and Yang, Z and Zeng, J and Chen, J and Chen, N and Jiang, X and Wei, Q and Yi, P and Xu, J},
title = {Mutation of FLNA attenuating the migration of abdominal muscles contributed to Melnick-Needles syndrome (MNS) in a family with recurrent miscarriage.},
journal = {Molecular genetics &amp; genomic medicine},
volume = {},
number = {},
pages = {e2145},
doi = {10.1002/mgg3.2145},
pmid = {36734119},
issn = {2324-9269},
abstract = {BACKGROUND: Filamin A, encoded by the X-linked gene FLNA, links the cell membrane with the cytoskeleton and acts as a regulator of the actin cytoskeleton. Mutations in FLNA cause a large spectrum of congenital malformations during embryonic development, including Melnick-Needles syndrome (MNS). However, reports of MNS, especially in males, are rare, and the pathogenesis molecular mechanisms are not well understood.<br><br>METHODS: We found a family with two consecutive miscarriages of similar fetuses with multiple malformations. DNA was extracted from peripheral blood and tissues, and whole exome sequencing was performed for genetic analysis. Then, we created a C57BL/6 mouse with a point mutation by CRISPR/Cas-mediated genome engineering. The migration of primary abdominal muscle cell was detected by wound healing assay.<br><br>RESULTS: The first fetus showed congenital hygroma colli and omphalocele identified by ultrasound at 12 wks; the second fetus showed hygroma colli and thoraco abdominoschisis at 12 wks, with a new hemizygous mutation c.4420G>A in exon 26 of the FLNA gene, which is predicted to cause an amino acid substitution (p.Asp1474Asn). The mother and grandmother were both present in the c.4420G>A heterozygous state, and the mother's healthy brother had wild-type FLNA. These FLNA-mutated mice exhibited a broader central gap between the rectus abdominis than the wild type (WT), similar to the midline structure dysplasia of the abdominal wall in the two fetuses. Wound healing assays showed the attenuated migration capacity of abdominal muscle cells in mice with mutated FLNA. Finally, we summarized the cases of MNS with FLNA mutation from the accessible published literature thus far.<br><br>CONCLUSION: Our research revealed a mutation site of the FLNA for MNS and explored the mechanism of midline structure dysplasia in the abdominal wall of male patients, which could provide more evidence for the clinical diagnosis and genetic counseling of families with these disorders.},
}
@article {pmid36733604,
year = {2022},
author = {Al-Khayri, JM and Banadka, A and Rashmi, R and Nagella, P and Alessa, FM and Almaghasla, MI},
title = {Cadmium toxicity in medicinal plants: An overview of the tolerance strategies, biotechnological and omics approaches to alleviate metal stress.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1047410},
pmid = {36733604},
issn = {1664-462X},
abstract = {Medicinal plants, an important source of herbal medicine, are gaining more demand with the growing human needs in recent times. However, these medicinal plants have been recognized as one of the possible sources of heavy metal toxicity in humans as these medicinal plants are exposed to cadmium-rich soil and water because of extensive industrial and agricultural operations. Cadmium (Cd) is an extremely hazardous metal that has a deleterious impact on plant development and productivity. These plants uptake Cd by symplastic, apoplastic, or via specialized transporters such as HMA, MTPs, NRAMP, ZIP, and ZRT-IRT-like proteins. Cd exerts its effect by producing reactive oxygen species (ROS) and interfere with a range of metabolic and physiological pathways. Studies have shown that it has detrimental effects on various plant growth stages like germination, vegetative and reproductive stages by analyzing the anatomical, morphological and biochemical changes (changes in photosynthetic machinery and membrane permeability). Also, plants respond to Cd toxicity by using various enzymatic and non-enzymatic antioxidant systems. Furthermore, the ROS generated due to the heavy metal stress alters the genes that are actively involved in signal transduction. Thus, the biosynthetic pathway of the important secondary metabolite is altered thereby affecting the synthesis of secondary metabolites either by enhancing or suppressing the metabolite production. The present review discusses the abundance of Cd and its incorporation, accumulation and translocation by plants, phytotoxic implications, and morphological, physiological, biochemical and molecular responses of medicinal plants to Cd toxicity. It explains the Cd detoxification mechanisms exhibited by the medicinal plants and further discusses the omics and biotechnological strategies such as genetic engineering and gene editing CRISPR- Cas 9 approach to ameliorate the Cd stress.},
}
@article {pmid36732857,
year = {2023},
author = {Kuninobu, KI and Takemura, T and Takizawa, Y and Hasebe, F and Yamashiro, T},
title = {Whole-genome analysis of a Vibrio cholerae O1 biotype classical strain isolated in 1946 in Sasebo city, Nagasaki prefecture, from a returnee from the northeast part of China.},
journal = {Tropical medicine and health},
volume = {51},
number = {1},
pages = {5},
pmid = {36732857},
issn = {1348-8945},
abstract = {BACKGROUND: Cholera is a water-borne disease caused by toxigenic Vibrio cholerae serogroups O1 and O139. Not a few studies on the whole-genome analyses of V. cholerae O1 biotype El Tor have been published; however, the number of analyses for biotype classical is limited. The whole-genome analysis was made on a V. cholerae biotype classical strain, Man9, isolated in 1946 in Sasebo city, Nagasaki prefecture, from a returnee from the northeast part of China.<br><br>METHODS: PacBio RSII was used to determine the whole-genome of Man9. De novo assemblies were made with CLC Genomics Workbench 8.5.1 and Canu. 2.0 and annotated by Prokka version 1.12. Upon determining the configuration of the CTX prophage region, combined procedures of PCR, RFLP with Southern blotting, and Sanger sequencing method were used. The phylogenetic tree was constructed by RaxML and visualized by Phandango. The identification of Cas genes and spacer sequences was made by CRISPR-finder and NCBI Blast search. These data were compared with those of V. cholerae serogroup O1 biotype classical O395.<br><br>RESULTS: The Man9 carried the 2.9&#xa0;Mb (Chr1) and 1.1&#xa0;Mb (Chr2) chromosomes with 2683 and 1198 CDSs, respectively. The genome similarity between Man9 and O395 was 97.0% when the total genomes were compared. Man9 carried a 380-kb inversion on the Chr1, and 95-kb and 35-kb fragments were not present on the Chr1 and on the Chr2, respectively. Man9 monophyletically clustered with 23 other biotype classical strains on the core gene phylogenetic tree analyses. Man9 carries "CTX[cla]" and a stretch of "truncated CTX[cla]-CTX[cla]" on the Chr1 and the Chr2, respectively, which is the opposite arrangement of O395. Man9 carries CRISPR-Cas system subtype I-E with 33 spacers, 64% of which were identical to those of O395.<br><br>CONCLUSIONS: Man9 differs from O395 by 3% on the total genome comparison; however, genomic analysis of a strain having circulated in the interpandemic period between the 6th and the 7th cholera pandemic is valuable and contributes to understanding the evolution of pathogenic V. cholerae.},
}
@article {pmid36731748,
year = {2023},
author = {García-Murillo, L and Valencia-Lozano, E and Priego-Ranero, NA and Cabrera-Ponce, JL and Duarte-Aké, FP and Vizuet-de-Rueda, JC and Rivera-Toro, DM and Herrera-Ubaldo, H and de Folter, S and Alvarez-Venegas, R},
title = {CRISPRa-mediated transcriptional activation of the SlPR-1 gene in edited tomato plants.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {111617},
doi = {10.1016/j.plantsci.2023.111617},
pmid = {36731748},
issn = {1873-2259},
abstract = {With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.},
}
@article {pmid36726758,
year = {2023},
author = {Feng, F and Zhu, Y and Ma, Y and Wang, Y and Yu, Y and Sun, X and Song, Y and Shao, Z and Huang, X and Liao, Y and Ma, J and He, Y and Wang, M and Tang, L and Huang, Y and Zhao, J and Ding, Q and Xie, Y and Cai, Q and Xiao, H and Li, C and Yuan, Z and Zhang, R},
title = {A CRISPR activation screen identifies genes that enhance SARS-CoV-2 infection.},
journal = {Protein &amp; cell},
volume = {14},
number = {1},
pages = {64-68},
pmid = {36726758},
issn = {1674-8018},
mesh = {Humans ; *COVID-19/genetics ; SARS-CoV-2/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; RNA, Viral/genetics ; },
}
@article {pmid36726140,
year = {2023},
author = {Li, Y and Xu, J and Guo, X and Li, Z and Cao, L and Liu, S and Guo, Y and Wang, G and Luo, Y and Zhang, Z and Wei, X and Zhao, Y and Liu, T and Wang, X and Xia, H and Kuang, M and Guo, Q and Li, J and Chen, L and Wang, Y and Li, Q and Wang, F and Liu, Q and You, F},
title = {The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {20},
pmid = {36726140},
issn = {1474-760X},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *RNA/genetics ; Transcriptome ; MAP Kinase Signaling System ; Mammals/genetics ; },
abstract = {BACKGROUND: The CRISPR-Cas13 system is an RNA-guided RNA-targeting system and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells.<br><br>RESULTS: Here, we find that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which may result from the collateral activity of RfxCas13d rather than the loss of target gene function or off-target effects. Mechanistically, we show that RfxCas13d exhibits collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAK&#x3b1;-JNK/p38-immediate early gene pathway.<br><br>CONCLUSIONS: These findings provide new mechanistic insights into the collateral activity of RfxCas13d in mammalian cells and warn that the biosafety of the CRISPR-Cas13 system needs further evaluation before application to clinical treatments.},
}
@article {pmid36587640,
year = {2023},
author = {Cheng, FP and Hu, XF and Pan, LX and Gong, ZX and Qin, KX and Li, Z and Wang, ZL},
title = {Transcriptome changes of Apis mellifera female embryos with fem gene knockout by CRISPR/Cas9.},
journal = {International journal of biological macromolecules},
volume = {229},
number = {},
pages = {260-267},
doi = {10.1016/j.ijbiomac.2022.12.229},
pmid = {36587640},
issn = {1879-0003},
mesh = {Bees/genetics ; Female ; Animals ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Alternative Splicing/genetics ; RNA-Seq ; },
abstract = {The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees.},
}
@article {pmid36423657,
year = {2023},
author = {Rouillon, C and Schneberger, N and Chi, H and Blumenstock, K and Da Vela, S and Ackermann, K and Moecking, J and Peter, MF and Boenigk, W and Seifert, R and Bode, BE and Schmid-Burgk, JL and Svergun, D and Geyer, M and White, MF and Hagelueken, G},
title = {Antiviral signalling by a cyclic nucleotide activated CRISPR protease.},
journal = {Nature},
volume = {614},
number = {7946},
pages = {168-174},
doi = {10.1038/s41586-022-05571-7},
pmid = {36423657},
issn = {1476-4687},
mesh = {Nucleotides, Cyclic ; Peptide Hydrolases/metabolism ; Antiviral Agents ; *Protease La/metabolism ; RNA ; Endopeptidases/metabolism ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes[1,2]. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA[3-5]. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates[6,7], a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family[7] fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.},
}
@article {pmid36413603,
year = {2023},
author = {Iwagawa, T and Masumoto, H and Tabuchi, H and Tani, K and Conklin, BR and Watanabe, S},
title = {Evaluation of CRISPR/Cas9 exon-skipping vector for choroideremia using human induced pluripotent stem cell-derived RPE.},
journal = {The journal of gene medicine},
volume = {25},
number = {2},
pages = {e3464},
doi = {10.1002/jgm.3464},
pmid = {36413603},
issn = {1521-2254},
support = {P01-HL146366/NH/NIH HHS/United States ; R01-HL13535801/NH/NIH HHS/United States ; R01-HL130533/NH/NIH HHS/United States ; R01-EY027789/NH/NIH HHS/United States ; R01-EY028249/NH/NIH HHS/United States ; P01-HL146366/NH/NIH HHS/United States ; R01-HL13535801/NH/NIH HHS/United States ; R01-HL130533/NH/NIH HHS/United States ; R01-EY027789/NH/NIH HHS/United States ; R01-EY028249/NH/NIH HHS/United States ; },
mesh = {Humans ; Retinal Pigment Epithelium/metabolism ; *Choroideremia/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Artificial Intelligence ; Exons/genetics ; },
abstract = {BACKGROUND: Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene.<br><br>METHODS: We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting.<br><br>RESULTS: Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM.<br><br>CONCLUSIONS: CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.},
}
@article {pmid36223984,
year = {2023},
author = {Rozenberg, I and Moses, E and Harel, I},
title = {CRISPR-Cas9 Genome Editing in Nothobranchius furzeri for Gene Knockout and Knock-In.},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {2},
pages = {pdb.prot107742},
doi = {10.1101/pdb.prot107742},
pmid = {36223984},
issn = {1559-6095},
mesh = {Animals ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; Gene Editing ; Longevity/genetics ; Aging/genetics ; *Cyprinodontiformes/genetics ; },
abstract = {The African turquoise killifish Nothobranchius furzeri has recently gained interest as an emerging vertebrate model system for the study of aging, owing to its naturally short life span and generation time. Here, we provide a step-by-step guide for effective genome engineering using the CRISPR-Cas9 system to generate loss-of-function (i.e., knockout) alleles and for precise editing (i.e., knock-in) of short sequences into the genome. Using this approach, a new stable line can be created within several months. The killifish's tough chorion, rapid growth, and short life span are considered in this protocol and account for the key deviations from similar protocols in other fish models.},
}
@article {pmid35294257,
year = {2022},
author = {Böck, D and Rothgangl, T and Villiger, L and Schmidheini, L and Matsushita, M and Mathis, N and Ioannidi, E and Rimann, N and Grisch-Chan, HM and Kreutzer, S and Kontarakis, Z and Kopf, M and Thöny, B and Schwank, G},
title = {In vivo prime editing of a metabolic liver disease in mice.},
journal = {Science translational medicine},
volume = {14},
number = {636},
pages = {eabl9238},
pmid = {35294257},
issn = {1946-6242},
mesh = {Animals ; Dependovirus/genetics/metabolism ; Gene Editing ; *Liver Diseases/genetics/therapy ; Mice ; *Phenylketonurias/genetics/therapy ; },
abstract = {Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2[ΔRnH]) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2[ΔRnH] via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah[enu2] mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10[14] vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.},
}
@article {pmid36727449,
year = {2023},
author = {Handelmann, CR and Tsompana, M and Samudrala, R and Buck, MJ},
title = {The impact of nucleosome structure on CRISPR/Cas9 fidelity.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad021},
pmid = {36727449},
issn = {1362-4962},
support = {R01GM132199/GM/NIGMS NIH HHS/United States ; T15LM012495/LM/NLM NIH HHS/United States ; /NH/NIH HHS/United States ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) Cas system is a powerful tool that has the potential to become a therapeutic gene editor in the near future. Cas9 is the best studied CRISPR system and has been shown to have problems that restrict its use in therapeutic applications. Chromatin structure is a known impactor of Cas9 targeting and there is a gap in knowledge on Cas9's efficacy when targeting such locations. To quantify at a single base pair resolution how chromatin inhibits on-target gene editing relative to off-target editing of exposed mismatching targets, we developed the gene editor mismatch nucleosome inhibition assay (GEMiNI-seq). GEMiNI-seq utilizes a library of nucleosome sequences to examine all target locations throughout nucleosomes in a single assay. The results from GEMiNI-seq revealed that the location of the protospacer-adjacent motif (PAM) sequence on the nucleosome edge drives the ability for Cas9 to access its target sequence. In addition, Cas9 had a higher affinity for exposed mismatched targets than on-target sequences within a nucleosome. Overall, our results show how chromatin structure impacts the fidelity of Cas9 to potential targets and highlight how targeting sequences with exposed PAMs could limit off-target gene editing, with such considerations improving Cas9 efficacy and resolving current limitations.},
}
@article {pmid36727270,
year = {2023},
author = {van der Gulik, PTS and Egas, M and Kraaijeveld, K and Dombrowski, N and Groot, AT and Spang, A and Hoff, WD and Gallie, J},
title = {On distinguishing between canonical tRNA genes and tRNA gene fragments in prokaryotes.},
journal = {RNA biology},
volume = {20},
number = {1},
pages = {48-58},
doi = {10.1080/15476286.2023.2172370},
pmid = {36727270},
issn = {1555-8584},
abstract = {Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.},
}
@article {pmid36725417,
year = {2023},
author = {Stankov, S and Cuchel, M},
title = {Gene editing for dyslipidemias: New tools to "cut" lipids.},
journal = {Atherosclerosis},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.atherosclerosis.2023.01.010},
pmid = {36725417},
issn = {1879-1484},
abstract = {Effective lipid lowering therapies are essential for the prevention of atherosclerosis and cardiovascular disease. Available treatments have evolved in both their efficacy and their frequency of administration, and currently include monoclonal antibodies, antisense oligonucleotides and siRNA approaches. However, an unmet need remains for more effective and long-lasting therapeutics. Gene editing permanently alters endogenous gene expression and has the potential to revolutionize disease treatment. Despite the existence of several gene editing approaches, the CRISPR/Cas9 system has emerged as the preferred technology because of its high efficiency and relative simplicity. This review provides a general overview of this promising technology and an update on the progress made towards the development of treatments of dyslipidemia. The recently started phase 1b gene editing clinical trial targeting PCSK9 in patients with heterozygous familial hypercholesterolemia and cardiovascular disease highlights how gene editing may become available to treat not only patients affected by rare disorders of lipid metabolism, but also patients that are difficult-to-treat or at high risk. Other targets like ANGPTL3, LDLR, and APOC3 are on track for further pre-clinical development. The identification of novel targets using electronic health record-linked biobanks and human sequencing studies will continue to expand the potential target pool, and clinical assessment of treated patients will provide essential efficacy and safety information on current strategies. Gene editing of genes regulating lipid metabolism holds promise as an exciting new therapeutic approach. However, since gene editing permanently alters a patient's genome, its therapeutic application in humans will require careful safety assessment and ethical considerations.},
}
@article {pmid36725305,
year = {2023},
author = {Roh, YH and Lee, CY and Lee, S and Kim, H and Ly, A and Castro, CM and Cheon, J and Lee, JH and Lee, H},
title = {CRISPR-Enhanced Hydrogel Microparticles for Multiplexed Detection of Nucleic Acids.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2206872},
doi = {10.1002/advs.202206872},
pmid = {36725305},
issn = {2198-3844},
support = {R21DA049577/GF/NIH HHS/United States ; R01CA229777/GF/NIH HHS/United States ; R01CA239078/GF/NIH HHS/United States ; R01CA237500/GF/NIH HHS/United States ; R21CA267222/GF/NIH HHS/United States ; U01CA233360/GF/NIH HHS/United States ; R01CA264363/GF/NIH HHS/United States ; },
abstract = {CRISPR/Cas systems offer a powerful sensing mechanism to transduce sequence-specific information into amplified analytical signals. However, performing multiplexed CRISPR/Cas assays remains challenging and often requires complex approaches for multiplexed assays. Here, a hydrogel-based CRISPR/Cas12 system termed CLAMP (Cas-Loaded Annotated Micro-Particles) is described. The approach compartmentalizes the CRISPR/Cas reaction in spatially-encoded hydrogel microparticles (HMPs). Each HMP is identifiable by its face code and becomes fluorescent when target DNA is present. The assay is further streamlined by capturing HMPs inside a microfluidic device; the captured particles are then automatically recognized by a machine-learning algorithm. The CLAMP assay is fast, highly sensitive (attomolar detection limits with preamplification), and capable of multiplexing in a single-pot assay. As a proof-of-concept clinical application, CLAMP is applied to detect nucleic acid targets of human papillomavirus in cervical brushing samples.},
}
@article {pmid36722224,
year = {2023},
author = {Wysocka, E and Gonicka, A and Anbalagan, S},
title = {CRISPR-Cas9 F0 knockout approach using predesigned in vitro transcribed guide RNAs partially recapitulates Rx3 function in eye morphogenesis.},
journal = {Journal of genetics},
volume = {102},
number = {},
pages = {},
pmid = {36722224},
issn = {0973-7731},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Homozygote ; Larva ; Phenotype ; },
abstract = {CRISPR-Cas9-based F0 knockout (KO) approach permits relatively simple and rapid generation of homozygous KOs and allows quick investigation of gene functions in zebrafish. However, F0 KO studies are largely performed using commercial synthetic guide RNAs (gRNAs) which are unaffordable by majority of the researchers. We tested (i) how effective is the CRISPR-Cas9-based F0 KO approach using in vitro transcribed gRNAs; (ii) how penetrant are the resulting phenotype at the later developmental stages and (iii) whether Coughlin's group pre-designed gRNAs are functional even without validating the gRNAs or testing for lack of SNP's in target loci. We targeted the rx3 gene that is required for the formation of the eye, a structure that exhibits robustness and can quickly recover from early phenotypes. Our results indicate that, in the majority of the samples, injection of Cas9 protein complex with four different in vitro transcribed gRNAs; targeting rx3 results in lack of eyes or disrupted eye development. Thus, the CRISPR-Cas9-based F0 KO approach using pre-designed, quadruple in vitro transcribed gRNAs can recapitulate the function of a gene at least until 5-dpf stage of larval zebrafish.},
}
@article {pmid36721198,
year = {2023},
author = {Ferrando, J and Filluelo, O and Zeigler, DR and Picart, P},
title = {Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {21},
pmid = {36721198},
issn = {1475-2859},
mesh = {*Bacillus subtilis/genetics ; *CRISPR-Cas Systems ; Colorimetry ; Mutagenesis, Insertional ; Operon ; },
abstract = {BACKGROUND: Despite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming.<br><br>RESULTS: In this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes.<br><br>CONCLUSIONS: The novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.},
}
@article {pmid36720908,
year = {2023},
author = {Singh, S and Banerjee, A and Vanden Broeck, A and Klinge, S},
title = {Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {1719},
pmid = {36720908},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Cell Membrane ; Clone Cells ; Epitopes ; *Gene Editing ; },
abstract = {One of the challenges faced by current CRISPR/Cas9 editing strategies is the difficulty in rapidly selecting clonal populations of biallelically edited cells. Here we present Surface engiNeered fluorEscence Assisted Kit with Protein Epitope Enhanced Capture (SNEAK PEEC), a platform that combines human genome editing with cell-surface display, which enables the direct identification of biallelically edited clones with minimal screening.},
}
@article {pmid36719476,
year = {2023},
author = {Hajirnis, N and Pandey, S and Mishra, RK},
title = {CRISPR/Cas9 and FLP-FRT mediated regulatory dissection of the BX-C of Drosophila melanogaster.},
journal = {Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology},
volume = {31},
number = {1},
pages = {7},
pmid = {36719476},
issn = {1573-6849},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Drosophila Proteins/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/genetics/metabolism ; Regulatory Sequences, Nucleic Acid ; },
abstract = {The homeotic genes or Hox define the anterior-posterior (AP) body axis formation in bilaterians and are often present on the chromosome in an order collinear to their function across the AP axis. However, there are many cases wherein the Hox are not collinear, but their expression pattern is conserved across the AP axis. The expression pattern of Hox is attributed to the cis-regulatory modules (CRMs) consisting of enhancers, initiators, or repressor elements that regulate the genes in a segment-specific manner. In the Drosophila melanogaster Hox complex, the bithorax complex (BX-C) and even the CRMs are organized in an order that is collinear to their function in the thoracic and abdominal segments. In the present study, the regulatorily inert regions were targeted using CRISPR/Cas9 to generate a series of transgenic lines with the insertion of FRT sequences. These FRT lines are repurposed to shuffle the CRMs associated with Abd-B to generate modular deletion, duplication, or inversion of multiple CRMs. The rearrangements yielded entirely novel phenotypes in the fly suggesting the requirement of such complex manipulations to address the significance of higher order arrangement of the CRMs. The functional map and the transgenic flies generated in this study are important resources to decipher the collective ability of multiple regulatory elements in the eukaryotic genome to function as complex modules.},
}
@article {pmid36625087,
year = {2023},
author = {Roy, SK and Srivastava, S and Hancock, A and Shrivastava, A and Morvant, J and Shankar, S and Srivastava, RK},
title = {Inhibition of ribosome assembly factor PNO1 by CRISPR/Cas9 technique suppresses lung adenocarcinoma and Notch pathway: Clinical application.},
journal = {Journal of cellular and molecular medicine},
volume = {27},
number = {3},
pages = {365-378},
doi = {10.1111/jcmm.17657},
pmid = {36625087},
issn = {1582-4934},
mesh = {Male ; Female ; Humans ; CRISPR-Cas Systems/genetics ; *Adenocarcinoma of Lung/genetics ; *Adenocarcinoma/pathology ; Receptors, Notch/genetics/metabolism ; *Lung Neoplasms/pathology ; Ribosomes/metabolism/pathology ; Epithelial-Mesenchymal Transition/genetics ; Cell Line, Tumor ; RNA-Binding Proteins/genetics/metabolism ; },
abstract = {Growth is crucially controlled by the functional ribosomes available in cells. To meet the enhanced energy demand, cancer cells re-wire and increase their ribosome biogenesis. The RNA-binding protein PNO1, a ribosome assembly factor, plays an essential role in ribosome biogenesis. The purpose of this study was to examine whether PNO1 can be used as a biomarker for lung adenocarcinoma and also examine the molecular mechanisms by which PNO1 knockdown by CRISPR/Cas9 inhibited growth and epithelial-mesenchymal transition (EMT). The expression of PNO1 was significantly higher in lung adenocarcinoma compared to normal lung tissues. PNO1 expression in lung adenocarcinoma patients increased with stage, nodal metastasis, and smoking. Lung adenocarcinoma tissues from males expressed higher PNO1 than those from females. Furthermore, lung adenocarcinoma tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53, suggesting the influence of Tp53 status on PNO1 expression. PNO1 knockdown inhibited cell viability, colony formation, and EMT, and induced apoptosis. Since dysregulated signalling through the Notch receptors promotes lung adenocarcinoma, we measured the effects of PNO1 inhibition on the Notch pathway. PNO1 knockdown inhibited Notch signalling by suppressing the expression of Notch receptors, their ligands, and downstream targets. PNO1 knockdown also suppressed CCND1, p21, PTGS-2, IL-1α, IL-8, and CXCL-8 genes. Overall, our data suggest that PNO1 can be used as a diagnostic biomarker, and also can be an attractive therapeutic target for the treatment of lung adenocarcinoma.},
}
@article {pmid36512423,
year = {2023},
author = {Li, G and Jin, M and Li, Z and Xiao, Q and Lin, J and Yang, D and Liu, Y and Wang, X and Xie, L and Ying, W and Wang, H and Zuo, E and Shi, L and Wang, N and Chen, W and Xu, C and Yang, H},
title = {Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy.},
journal = {The Journal of clinical investigation},
volume = {133},
number = {3},
pages = {},
doi = {10.1172/JCI162809},
pmid = {36512423},
issn = {1558-8238},
mesh = {Mice ; Animals ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Dystrophin/genetics ; RNA Editing ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Muscle, Skeletal/metabolism ; Gene Editing/methods ; Disease Models, Animal ; },
abstract = {Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, mxABE packaged in a single adeno-associated virus (AAV) reached A-to-G editing rates up to 84% in vivo, at least 20-fold greater than rates reported in previous studies using other RNA editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of WT levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases.},
}
@article {pmid36507966,
year = {2023},
author = {Dhuriya, YK and Naik, AA},
title = {CRISPR: a tool with potential for genomic reprogramming in neurological disorders.},
journal = {Molecular biology reports},
volume = {50},
number = {2},
pages = {1845-1856},
doi = {10.1007/s11033-022-08136-z},
pmid = {36507966},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; RNA Interference ; Genomics ; },
abstract = {The intricate neural circuitry of the brain necessitates precise and synchronized transcriptional programs. Any disturbance during embryonic or adult development, whether caused by genetic or environmental factors, may result in refractory and recurrent neurological disorders. Inadequate knowledge of the pathogenic mechanisms underlying neurological disorders is the primary obstacle to the development of effective treatments, necessitating the development of alternative therapeutic approaches to identify rational molecular targets. Recently, with the evolution of CRISPR-Cas9 technology, an engineered RNA system provides precise and highly effective correction or silencing of disease-causing mutations by modulating expression and thereby avoiding the limitations of the RNA interference strategy. This article discusses the CRISPR-Cas9 technology, its mechanisms, and the limitations of the new technology. We provide a glimpse of how the far-reaching implications of CRISPR can open new avenues for the development of tools to combat neurological disorders, as well as a review of recent attempts by neuroscientists to launch therapeutic correction.},
}
@article {pmid36441374,
year = {2023},
author = {Goto, T and Yogo, K and Hochi, S and Hirabayashi, M},
title = {Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease.},
journal = {Molecular biology reports},
volume = {50},
number = {2},
pages = {1231-1239},
pmid = {36441374},
issn = {1573-4978},
mesh = {Animals ; Rats ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; DNA ; RNA, Messenger/genetics ; },
abstract = {BACKGROUND: The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA.<br><br>METHODS AND RESULTS: We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60&#xa0;ng/&#xb5;l was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60&#xa0;ng/&#xb5;l protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction.<br><br>CONCLUSIONS: The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.},
}
@article {pmid36723794,
year = {2023},
author = {Jeong, SH and Lee, HJ and Lee, SJ},
title = {Recent Advances in CRISPR-Cas Technologies for Synthetic Biology.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {},
number = {},
pages = {},
doi = {10.1007/s12275-022-00005-5},
pmid = {36723794},
issn = {1976-3794},
abstract = {With developments in synthetic biology, "engineering biology" has emerged through standardization and platformization based on hierarchical, orthogonal, and modularized biological systems. Genome engineering is necessary to manufacture and design synthetic cells with desired functions by using bioparts obtained from sequence databases. Among various tools, the CRISPR-Cas system is modularly composed of guide RNA and Cas nuclease; therefore, it is convenient for editing the genome freely. Recently, various strategies have been developed to accurately edit the genome at a single nucleotide level. Furthermore, CRISPR-Cas technology has been extended to molecular diagnostics for nucleic acids and detection of pathogens, including disease-causing viruses. Moreover, CRISPR technology, which can precisely control the expression of specific genes in cells, is evolving to find the target of metabolic biotechnology. In this review, we summarize the status of various CRISPR technologies that can be applied to synthetic biology and discuss the development of synthetic biology combined with CRISPR technology in microbiology.},
}
@article {pmid36721057,
year = {2023},
author = {Rish, AD and Fu, TM},
title = {CRISPR-Cas has a new juggling act: interplay between nuclease and protease.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {36721057},
issn = {1545-9985},
}
@article {pmid36655772,
year = {2023},
author = {Cao, H and Xie, J and Cheng, J and Xu, Y and Lu, X and Tang, J and Zhang, X and Wang, H},
title = {CRISPR Cas12a-Powered Silicon Surface-Enhanced Raman Spectroscopy Ratiometric Chip for Sensitive and Reliable Quantification.},
journal = {Analytical chemistry},
volume = {95},
number = {4},
pages = {2303-2311},
doi = {10.1021/acs.analchem.2c03990},
pmid = {36655772},
issn = {1520-6882},
mesh = {*Metal Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; Silicon/chemistry ; Spectrum Analysis, Raman/methods ; Becaplermin ; Reproducibility of Results ; Silver/chemistry ; DNA/chemistry ; DNA, Single-Stranded ; *Biosensing Techniques ; },
abstract = {Sensitive and reliable clustered regularly interspaced short palindromic repeats (CRISPR) quantification without preamplification of the sample remains a challenge. Herein, we report a CRISPR Cas12a-powered silicon surface-enhanced Raman spectroscopy (SERS) ratiometric chip for sensitive and reliable quantification. As a proof-of-concept application, we select the platelet-derived growth factor-BB (PDGF-BB) as the target. We first develop a microfluidic synthetic strategy to prepare homogeneous silicon SERS substrates, in which uniform silver nanoparticles (AgNPs) are in situ grown on a silicon wafer (AgNPs@Si) by microfluidic galvanic deposition reactions. Next, one 5'-SH-3'-ROX-labeled single-stranded DNA (ssDNA) is modified on AgNPs via Ag-S bonds. In our design, such ssDNA has two fragments: one fragment hybridizes to its complementary DNA (5'-Cy3-labeled ssDNA) to form double-stranded DNA (dsDNA) and the other fragment labeled with 6'-carboxy-X-rhodmine (ROX) extends out as a substrate for Cas12a. The cleavage of the ROX-tagged fragment by Cas12a is controlled by the presence or not of PDGF-BB. Meanwhile, Cy3 molecules serving as internal standard molecules still stay at the end of the rigid dsDNA, and their signals remain constant. Thereby, the ratio of ROX signal intensity to Cy3 intensity can be employed for the reliable quantification of PDGF-BB concentration. The developed chip features an ultrahigh sensitivity (e.g., the limit of detection is as low as 3.2 pM, approximately 50 times more sensitive than the fluorescence counterpart) and good reproducibility (e.g., the relative standard deviation is less than 5%) in the detection of PDGF-BB.},
}
@article {pmid36442503,
year = {2023},
author = {Park, JU and Tsai, AW and Rizo, AN and Truong, VH and Wellner, TX and Schargel, RD and Kellogg, EH},
title = {Structures of the holo CRISPR RNA-guided transposon integration complex.},
journal = {Nature},
volume = {613},
number = {7945},
pages = {775-782},
pmid = {36442503},
issn = {1476-4687},
support = {R01 GM144566/GM/NIGMS NIH HHS/United States ; S10 OD030470/OD/NIH HHS/United States ; U24 GM129539/GM/NIGMS NIH HHS/United States ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA Transposable Elements/genetics ; DNA-Binding Proteins/chemistry/metabolism/ultrastructure ; *Gene Editing/methods ; *Transposases/chemistry/metabolism/ultrastructure ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Holoenzymes/chemistry/metabolism/ultrastructure ; *Multiprotein Complexes/chemistry/metabolism/ultrastructure ; Cryoelectron Microscopy ; Ribosome Subunits/chemistry/metabolism/ultrastructure ; Bacterial Proteins/chemistry/metabolism/ultrastructure ; },
abstract = {CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism[1-3]. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.},
}
@article {pmid35568962,
year = {2022},
author = {Brown, EA and Eikenbary, SR and Landis, WG},
title = {Bayesian network-based risk assessment of synthetic biology: Simulating CRISPR-Cas9 gene drive dynamics in invasive rodent management.},
journal = {Risk analysis : an official publication of the Society for Risk Analysis},
volume = {42},
number = {12},
pages = {2835-2846},
doi = {10.1111/risa.13948},
pmid = {35568962},
issn = {1539-6924},
mesh = {Animals ; Mice ; CRISPR-Cas Systems ; *Gene Drive Technology/methods ; Rodentia/genetics ; Synthetic Biology ; Bayes Theorem ; *Rodenticides ; Risk Assessment ; },
abstract = {Gene drive technology has been proposed to control invasive rodent populations as an alternative to rodenticides. However, this approach has not undergone risk assessment that meets criteria established by Gene Drives on the Horizon, a 2016 report by the National Academies of Sciences, Engineering, and Medicine. To conduct a risk assessment of gene drives, we employed the Bayesian network-relative risk model to calculate the risk of mouse eradication on Southeast Farallon Island using a CRISPR-Cas9 homing gene drive construct. We modified and implemented the R-based model "MGDrivE" to simulate and compare 60 management strategies for gene drive rodent management. These scenarios spanned four gene drive mouse release schemes, three gene drive homing rates, three levels of supplemental rodenticide dose, and two timings of rodenticide application relative to gene drive release. Simulation results showed that applying a supplemental rodenticide simultaneously with gene drive mouse deployment resulted in faster eradication of the island mouse population. Gene drive homing rate had the highest influence on the overall probability of successful eradication, as increased gene drive accuracy reduces the likelihood of mice developing resistance to the CRISPR-Cas9 homing mechanism.},
}
@article {pmid36720828,
year = {2023},
author = {Sánchez-Gómez, C and Posé, D and Martín-Pizarro, C},
title = {Genome Editing by CRISPR/Cas9 in Polyploids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2545},
number = {},
pages = {459-473},
pmid = {36720828},
issn = {1940-6029},
abstract = {CRISPR/Cas system has been widely used for genome editing in the past few years. Even though it has been performed in many polyploid species to date, its efficient accomplishment in these organisms is still a challenge. The presence of multiple homoeologous genes as targets for their editing requires more rigorous work and specific needs to assess successful genome editing. Here, we describe a general stepwise protocol to select target sites, design sgRNAs, indicate vector requirements, and screen CRISPR/Cas9-mediated genome editing in polyploid species.},
}
@article {pmid36718242,
year = {2022},
author = {Tarek, N and El-Gendy, AO and Khairalla, AS and Abdel-Fattah, M and Tawfik, E and Azmy, AF},
title = {Genomic analysis of Enterococcus durans NT21, a putative bacteriocin-producing isolate.},
journal = {Molecular biology research communications},
volume = {11},
number = {3},
pages = {143-153},
pmid = {36718242},
issn = {2345-2005},
abstract = {Enterococcus species are a long-standing and non-pathogenic commensal bacterium, representing an important part of the normal. Enterococcus durans is a rarely isolated species from animals and humans, and it was a tiny constituent of human oral cavity and animal intestinal flora, as well as animal-derived foods, particularly dairy products. This study evaluated the security of our strain E. durans NT21 by using whole-genome sequencing (WGS), physicochemical features, and antimicrobial activity. The complete genomic of our strain Enterococcus durans NT21was sequenced and analyzed by using several bioinformatics tools to identify bacteriocin genes, virulence genes, antibiotic resistance genes, Crispr-Cas and pathogenicity islands. The results showed that our strain NT21 lacks the presence of virulence genes, pathogenicity islands, plasmids and has only two antibiotic resistance genes. On the other hand, it produces three bacteriocin-like inhibitory substances (Enterolysin A, P and L50a). It has six gene-encoded Crisper-Cas and one cluster Crispr-Cas gene. According to our findings, E. durans NT21 is a possible probiotic strain that is safe for both human and animal use.},
}
@article {pmid36716555,
year = {2023},
author = {Liao, X and Xia, X and Yang, H and Zhu, Y and Deng, R and Ding, T},
title = {Bacterial drug-resistance and viability phenotyping upon disinfectant exposure revealed by single-nucleotide resolved-allele specific isothermal RNA amplification.},
journal = {Journal of hazardous materials},
volume = {448},
number = {},
pages = {130800},
pmid = {36716555},
issn = {1873-3336},
abstract = {Disinfectant abuse poses a risk of bacterial evolution against stresses, especially during the coronavirus disease 2019 (COVID-19) pandemic. However, bacterial phenotypes, such as drug resistance and viability, are hard to access quickly. Here, we reported an allele specific isothermal RNA amplification (termed AlleRNA) assay, using an isothermal RNA amplification technique, i.e., nucleic acid sequence-based amplification (NASBA), integrated the amplification refractory mutation system (ARMS), involving the use of sequence-specific primers to allow the amplification of the targets with complete complementary sequences. AlleRNA assay enables rapid and simultaneous detection of the single nucleotide polymorphism (SNP) (a detection limit, a LOD of 0.5 % SNP) and the viability (a LOD of 80 CFU) of the quinolone resistant Salmonella enterica. With the use of AlleRNA assay, we found that the quinolone resistant S. enterica exhibited higher survival ability during exposure toquaternary ammonium salt, 75 % ethanol and peracetic acid, which might be attributed to the upregulation of stress response-associated genescompared with the susceptible counterparts. Additionally, the AlleRNA assay indicated the potential risk in a high-frequency occurrence of viable but nonculturable (VBNC) quinolone resistant S. enterica induced by disinfectants due to the depression of ATP biosynthesis. The excessive usage of disinfectants during the COVID-19 pandemic should be carefully evaluated due to the latent threat to ecological and human health.},
}
@article {pmid36714073,
year = {2023},
author = {Chen, J and Li, Y and Liu, Z},
title = {Functional nucleic acids as potent therapeutics against SARS-CoV-2 infection.},
journal = {Cell reports. Physical science},
volume = {},
number = {},
pages = {101249},
pmid = {36714073},
issn = {2666-3864},
abstract = {The COVID-19 pandemic has posed a severe threat to human life and the global economy. Although conventional treatments, including vaccines, antibodies, and small-molecule inhibitors, have been broadly developed, they usually fall behind the constant mutation of SARS-CoV-2, due to the long screening process and high production cost. Functional nucleic acid (FNA)-based therapeutics are a newly emerging promising means against COVID-19, considering their timely adaption to different mutants and easy design for broad-spectrum virus inhibition. In this review, we survey typical FNA-related therapeutics against SARS-CoV-2 infection, including aptamers, aptamer-integrated DNA frameworks, functional RNA, and CRISPR-Cas technology. We first introduce the pathogenesis, transmission, and evolution of SARS-CoV-2, then analyze the existing therapeutic and prophylactic strategies, including their pros and cons. Subsequently, the FNAs are recommended as potent alternative therapeutics from their screening process and controllable engineering to effective neutralization. Finally, we put forward the remaining challenges of the existing field and sketch out the future development directions.},
}
@article {pmid36707549,
year = {2023},
author = {Sieliwonczyk, E and Vandendriessche, B and Claes, C and Mayeur, E and Alaerts, M and Holmgren, P and Canter Cremers, T and Snyders, D and Loeys, B and Schepers, D},
title = {Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {1491},
pmid = {36707549},
issn = {2045-2322},
support = {Genomia - ERC-COG- 2017-771945/ERC_/European Research Council/International ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Zebrafish/genetics ; Genotype ; High-Throughput Nucleotide Sequencing ; },
abstract = {Despite numerous prior attempts to improve knock-in (KI) efficiency, the introduction of precise base pair substitutions by the CRISPR-Cas9 technique in zebrafish remains challenging. In our efforts to generate KI zebrafish models of human CACNA1C mutations, we have tested the effect of several CRISPR determinants on KI efficiency across two sites in a single gene and developed a novel method for early selection to ameliorate KI efficiency. We identified optimal KI conditions for Cas9 protein and non-target asymmetric PAM-distal single stranded deoxynucleotide repair templates at both cacna1c sites. An effect of distance to the cut site on the KI efficiency was only observed for a single repair template conformation at one of the two sites. By combining minimally invasive early genotyping with the zebrafish embryo genotyper (ZEG) device and next-generation sequencing, we were able to obtain an almost 17-fold increase in somatic editing efficiency. The added benefit of the early selection procedure was particularly evident for alleles with lower somatic editing efficiencies. We further explored the potential of the ZEG selection procedure for the improvement of germline transmission by demonstrating germline transmission events in three groups of pre-selected embryos.},
}
@article {pmid36373224,
year = {2023},
author = {Hyde, L and Osman, K and Winfield, M and Sanchez-Moran, E and Higgins, JD and Henderson, IR and Sparks, C and Franklin, FCH and Edwards, KJ},
title = {Identification, characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants.},
journal = {Plant biotechnology journal},
volume = {21},
number = {2},
pages = {405-418},
pmid = {36373224},
issn = {1467-7652},
support = {BB/M014908/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/W003317/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Triticum/genetics ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; Chromosomes ; Meiosis/genetics ; },
abstract = {Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.},
}
@article {pmid36301462,
year = {2023},
author = {Kim, K and Shin, J and Kang, TA and Kim, B and Kim, WC},
title = {CRISPR/Cas9-mediated AtGATA25 mutant represents a novel model for regulating hypocotyl elongation in Arabidopsis thaliana.},
journal = {Molecular biology reports},
volume = {50},
number = {1},
pages = {31-41},
pmid = {36301462},
issn = {1573-4978},
mesh = {*Arabidopsis/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; Hypocotyl/genetics ; CRISPR-Cas Systems/genetics ; Transcription Factors/genetics/metabolism ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; *Phytochrome ; Gene Expression Regulation, Plant/genetics ; },
abstract = {BACKGROUND: Plants have evolved to adapt to the ever-changing environments through various morphological changes. An organism anticipates and responds to changes in its environment via the circadian clock, an endogenous oscillator lasting approximately 24 h. The circadian clock regulates various physiological processes, such as hypocotyl elongation in Arabidopsis thaliana. Phytochrome interacting factor 4 (PIF4), a member of the bHLH protein family, plays a vital hub role in light signaling pathways and temperature-mediated growth response mechanisms. PIF4 is controlled by the circadian clock and interacts with several factors. However, the components that regulate PIF4 transcription and activity are not clearly understood.<br><br>METHODS AND RESULTS: Here, we showed that the Arabidopsis thaliana GATA25 (AtGATA25) transcription factor plays a fundamental role in promoting hypocotyl elongation by positively regulating the expression of PIF4. This was confirmed to in the loss-of-function mutant of AtGATA25 via CRISPR/Cas9-mediated gene editing, which inhibits hypocotyl elongation and decreases the expression of PIF4. In contrast, the overexpression of AtGATA25 in transgenic plants resulted in increased expression of PIF4 and enhanced hypocotyl elongation. To better understand AtGATA25-mediated PIF4 transcriptional regulation, we analyzed the promoter region of the target gene PIF4 and characterized the role of GATA25 through transcriptional activation analysis.<br><br>CONCLUSION: Our findings suggest a novel role of the AtGATA25 transcription factor in hypocotyl elongation.},
}
@article {pmid36713205,
year = {2022},
author = {Gumustop, I and Ortakci, F},
title = {Analyzing the genetic diversity and biotechnological potential of Leuconostoc pseudomesenteroides by comparative genomics.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {1074366},
pmid = {36713205},
issn = {1664-302X},
abstract = {Leuconostoc pseudomesenteroides is a lactic acid bacteria species widely exist in fermented dairy foods, cane juice, sourdough, kimchi, apple dumpster, caecum, and human adenoid. In the dairy industry, Ln. pseudomesenteroides strains are usually found in mesophilic starter cultures with lactococci. This species plays a crucial role in the production of aroma compounds such as acetoin, acetaldehyde, and diacetyl, thus beneficially affecting dairy technology. We performed genomic characterization of 38 Ln. pseudomesenteroides from diverse ecological niches to evaluate this species' genetic diversity and biotechnological potential. A mere ~12% of genes conserved across 38 Ln. pseudomesenteroides genomes indicate that accessory genes are the driving force for genotypic distinction in this species. Seven main clades were formed with variable content surrounding mobile genetic elements, namely plasmids, transposable elements, IS elements, prophages, and CRISPR-Cas. All but three genomes carried CRISPR-Cas system. Furthermore, a type IIA CRISPR-Cas system was found in 80% of the CRISPR-Cas positive strains. AMBR10, CBA3630, and MGBC116435 were predicted to encode bacteriocins. Genes responsible for citrate metabolism were found in all but five strains belonging to cane juice, sourdough, and unknown origin. On the contrary, arabinose metabolism genes were only available in nine strains isolated from plant-related systems. We found that Ln. pseudomesenteroides genomes show evolutionary adaptation to their ecological environment due to niche-specific carbon metabolism and forming closely related phylogenetic clades based on their isolation source. This species was found to be a reservoir of type IIA CRISPR-Cas system. The outcomes of this study provide a framework for uncovering the biotechnological potential of Ln. pseudomesenteroides and its future development as starter or adjunct culture for dairy industry.},
}
@article {pmid36712915,
year = {2023},
author = {Xu, J and Ma, Y and Song, Z and Sun, W and Liu, Y and Shu, C and Hua, H and Yang, M and Liang, Q},
title = {Evaluation of an automated CRISPR-based diagnostic tool for rapid detection of COVID-19.},
journal = {Heliyon},
volume = {},
number = {},
pages = {e13190},
pmid = {36712915},
issn = {2405-8440},
abstract = {The performance of an automated commercial CRISPR/Cas based technology was evaluated and compared with routine RT-PCR testing to diagnose COVID-19. Suspected and discharged COVID-19 cases were included and tested with CRISPR-based SARS-CoV-2 test and RT-PCR assay using throat swab and sputum specimens. The diagnostic yield was calculated and compared using the McNemar test. A total of 437 patients were included for analysis, including COVID-19 (n = 171), discharged cases (n = 155), and others (n = 111). For the diagnosis of COVID-19, the CRISPR-SARS-CoV-2 test had a sensitivity and specificity of 98.2% (168/171) and 100.0% (266/266), respectively; the RT-PCR test had a sensitivity and specificity of 100.0% (171/171) and 100.0% (266/266), respectively. No significant difference was found in the sensitivity of CRISPR-SARS-CoV-2 and RT-PCR. In conclusion, the CRISPR-SARS-CoV-2 test had a comparable performance with RT-PCR and showed several advantages, such as short assay time, low cost, and no requirement for expensive equipment.},
}
@article {pmid36712576,
year = {2022},
author = {Zhang, X and Yang, Y and Cao, J and Qi, Z and Li, G},
title = {Point-of-care CRISPR/Cas biosensing technology: A promising tool for preventing the possible COVID-19 resurgence caused by contaminated cold-chain food and packaging.},
journal = {Food frontiers},
volume = {},
number = {},
pages = {},
pmid = {36712576},
issn = {2643-8429},
abstract = {The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused great public health concern and has been a global threat due to its high transmissibility and morbidity. Although the SARS-CoV-2 transmission mainly relies on the person-to-person route through the respiratory droplets, the possible transmission through the contaminated cold-chain food and packaging to humans has raised widespread concerns. This review discussed the possibility of SARS-CoV-2 transmission via the contaminated cold-chain food and packaging by tracing the occurrence, the survival of SARS-CoV-2 in the contaminated cold-chain food and packaging, as well as the transmission and outbreaks related to the contaminated cold-chain food and packaging. Rapid, accurate, and reliable diagnostics of SARS-CoV-2 is of great importance for preventing and controlling the COVID-19 resurgence. Therefore, we summarized the recent advances on the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system-based biosensing technology that is promising and powerful for preventing the possible COVID-19 resurgence caused by the contaminated cold-chain food and packaging during the COVID-19 pandemic, including CRISPR/Cas system-based biosensors and their integration with portable devices (e.g., smartphone, lateral flow assays, microfluidic chips, and nanopores). Impressively, this review not only provided an insight on the possibility of SARS-CoV-2 transmission through the food supply chain, but also proposed the future opportunities and challenges on the development of CRISPR/Cas system-based detection methods for the diagnosis of SARS-CoV-2.},
}
@article {pmid36712324,
year = {2022},
author = {Zhao, L and You, D and Wang, T and Zou, ZP and Yin, BC and Zhou, Y and Ye, BC},
title = {Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.},
journal = {PNAS nexus},
volume = {1},
number = {5},
pages = {pgac277},
pmid = {36712324},
issn = {2752-6542},
abstract = {CRISPR-Cas, the immune system of bacteria and archaea, has been widely harnessed for genome editing, including gene knockouts and knockins, single-base editing, gene activation, and silencing. However, the molecular mechanisms underlying fluctuations in the genome editing efficiency of crispr in various cells under different conditions remain poorly understood. In this work, we found that Cas9 can be ac(et)ylated by acetyl-phosphate or acyl-CoA metabolites both in vitro and in vivo. Several modifications are associated with the DNA or sgRNA binding sites. Notably, ac(et)ylation of Cas9 driven by these metabolites in host cells potently inhibited its binding and cleavage activity with the target DNA, thereby decreasing Crispr genome editing efficiency. This study provides more insights into understanding the effect of the intracellular environment on genome editing application of crispr with varying efficiency in hosts.},
}
@article {pmid36712129,
year = {2023},
author = {Esmaeili Anvar, N and Lin, C and Wilson, LL and Sangree, AK and Ma, X and Colic, M and Doench, JG and Hart, T},
title = {Combined genome-scale fitness and paralog synthetic lethality screens with just 44k clones: the IN4MER CRISPR/Cas12a multiplex knockout platform.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.01.03.522655},
pmid = {36712129},
abstract = {Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for multiplex genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different CRISPR/Cas multiplexing technologies and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the CRISPR/enCas12a platform provides superior sensitivity and assay replicability. We demonstrate that enCas12a can independently target up to four genes from a single guide array, and build on this knowledge by constructing a one-component library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 44k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than two thousand paralog pairs, triples, and quads. Proof of concept screens in two cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state of the art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking (also known as buffering) genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.},
}
@article {pmid36711804,
year = {2023},
author = {Walker, MWG and Klompe, SE and Zhang, DJ and Sternberg, SH},
title = {Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.01.19.524723},
pmid = {36711804},
abstract = {CRISPR-associated transposons (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system. On the donor DNA, large mutagenic libraries identified core binding sites recognized by the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications of CAST systems.},
}
@article {pmid36711562,
year = {2023},
author = {Armstrong, DA and Hudson, TR and Hodge, CA and Hampton, TH and Howell, AL and Hayden, MS},
title = {CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.01.02.522476},
pmid = {36711562},
abstract = {CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.},
}
@article {pmid36710911,
year = {2022},
author = {Alipanahi, R and Safari, L and Khanteymoori, A},
title = {CRISPR genome editing using computational approaches: A survey.},
journal = {Frontiers in bioinformatics},
volume = {2},
number = {},
pages = {1001131},
pmid = {36710911},
issn = {2673-7647},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has been widely used in various cell types and organisms. To make genome editing with Clustered regularly interspaced short palindromic repeats far more precise and practical, we must concentrate on the design of optimal gRNA and the selection of appropriate Cas enzymes. Numerous computational tools have been created in recent years to help researchers design the best gRNA for Clustered regularly interspaced short palindromic repeats researches. There are two approaches for designing an appropriate gRNA sequence (which targets our desired sites with high precision): experimental and predicting-based approaches. It is essential to reduce off-target sites when designing an optimal gRNA. Here we review both traditional and machine learning-based approaches for designing an appropriate gRNA sequence and predicting off-target sites. In this review, we summarize the key characteristics of all available tools (as far as possible) and compare them together. Machine learning-based tools and web servers are believed to become the most effective and reliable methods for predicting on-target and off-target activities of Clustered regularly interspaced short palindromic repeats in the future. However, these predictions are not so precise now and the performance of these algorithms -especially deep learning one's-depends on the amount of data used during training phase. So, as more features are discovered and incorporated into these models, predictions become more in line with experimental observations. We must concentrate on the creation of ideal gRNA and the choice of suitable Cas enzymes in order to make genome editing with Clustered regularly interspaced short palindromic repeats far more accurate and feasible.},
}
@article {pmid36710268,
year = {2023},
author = {Weng, Z and You, Z and Yang, J and Mohammad, N and Lin, M and Wei, Q and Gao, X and Zhang, Y},
title = {CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {},
doi = {10.1002/anie.202214987},
pmid = {36710268},
issn = {1521-3773},
abstract = {Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) detection, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.},
}
@article {pmid36702845,
year = {2023},
author = {Zhang, S and Song, L and Yuan, B and Zhang, C and Cao, J and Chen, J and Qiu, J and Tai, Y and Chen, J and Qiu, Z and Zhao, XM and Cheng, TL},
title = {TadA reprogramming to generate potent miniature base editors with high precision.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {413},
pmid = {36702845},
issn = {2041-1723},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Mutation ; Cytidine Deaminase/genetics/metabolism ; Cytosine/metabolism ; },
abstract = {Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.},
}
@article {pmid36702837,
year = {2023},
author = {Zhang, S and Yuan, B and Cao, J and Song, L and Chen, J and Qiu, J and Qiu, Z and Zhao, XM and Chen, J and Cheng, TL},
title = {TadA orthologs enable both cytosine and adenine editing of base editors.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {414},
pmid = {36702837},
issn = {2041-1723},
mesh = {*Gene Editing ; *Cytosine ; Adenine ; DNA ; RNA ; Adenosine/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Cytidine and adenosine deaminases are required for cytosine and adenine editing of base editors respectively, and no single deaminase could enable concurrent and comparable cytosine and adenine editing. Additionally, distinct properties of cytidine and adenosine deaminases lead to various types of off-target effects, including Cas9-indendepent DNA off-target effects for cytosine base editors (CBEs) and RNA off-target effects particularly severe for adenine base editors (ABEs). Here we demonstrate that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single or double mutations, which display minimized Cas9-independent DNA off-target effects and genotoxicity, with orthologs B5ZCW4, Q57LE3, E8WVH3, Q13XZ4 and B3PCY2 as promising candidates for further engineering. Furthermore, RNA off-target effects of TadA ortholog-derived base editors could be further reduced or even eliminated by additional single mutation. Taken together, our work expands the base editing toolkits, and also provides important clues for the potential evolutionary process of deaminases.},
}
@article {pmid36702819,
year = {2023},
author = {Bisio, H and Legendre, M and Giry, C and Philippe, N and Alempic, JM and Jeudy, S and Abergel, C},
title = {Evolution of giant pandoravirus revealed by CRISPR/Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {428},
pmid = {36702819},
issn = {2041-1723},
mesh = {DNA Viruses/genetics ; CRISPR-Cas Systems/genetics ; *Acanthamoeba castellanii/genetics ; *Giant Viruses/genetics ; *Viruses/genetics ; Genome, Viral/genetics ; Phylogeny ; Evolution, Molecular ; },
abstract = {Giant viruses (GVs) are a hotspot of unresolved controversies since their discovery, including the definition of "Virus" and their origin. While increasing knowledge of genome diversity has accumulated, GV functional genomics was largely neglected. Here, we describe an experimental framework to genetically modify nuclear GVs and their host Acanthamoeba castellanii using CRISPR/Cas9, shedding light on the evolution from small icosahedral viruses to amphora-shaped GVs. Ablation of the icosahedral major capsid protein in the phylogenetically-related mollivirus highlights a transition in virion shape and size. We additionally demonstrate the existence of a reduced core essential genome in pandoravirus, reminiscent of their proposed smaller ancestors. This proposed genetic expansion led to increased genome robustness, indicating selective pressures for adaptation to uncertain environments. Overall, we introduce new tools for manipulation of the unexplored genome of nuclear GVs and provide experimental evidence suggesting that viral gigantism has aroused as an emerging trait.},
}
@article {pmid36701275,
year = {2023},
author = {Qi, L and Sui, Y and Tang, XX and McGinty, RJ and Liang, XZ and Dominska, M and Zhang, K and Mirkin, SM and Zheng, DQ and Petes, TD},
title = {Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements.},
journal = {PLoS genetics},
volume = {19},
number = {1},
pages = {e1010590},
pmid = {36701275},
issn = {1553-7404},
mesh = {Humans ; *Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Retroelements/genetics ; Chromosome Aberrations ; Homologous Recombination/genetics ; },
abstract = {Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution.},
}
@article {pmid36700990,
year = {2023},
author = {Yi, P and Luo, D and Gao, Z and Chen, Q and Zhou, Y},
title = {Fluorescent aptasensor based on the MNPs-CRISPR/Cas12a-TdT for the determination of nasopharyngeal carcinoma-derived exosomes.},
journal = {Mikrochimica acta},
volume = {190},
number = {2},
pages = {74},
pmid = {36700990},
issn = {1436-5073},
mesh = {Humans ; *Exosomes/metabolism ; DNA Nucleotidylexotransferase/metabolism ; CRISPR-Cas Systems ; Nasopharyngeal Carcinoma/diagnosis/genetics/metabolism ; DNA/genetics ; Fluorescent Dyes/metabolism ; DNA-Directed DNA Polymerase/metabolism ; *Nasopharyngeal Neoplasms/diagnosis/genetics/metabolism ; },
abstract = {A fluorescence aptasensor based on taking the advantage of the combination of magnetic nanoparticles (MNPs), terminal deoxynucleotidyl transferase (TdT), and CRISPR/Cas12a was developed for the determination of nasopharyngeal carcinoma (NPC)-derived exosomes. The MNPs can eliminate background interference due to their magnetic separation capability. TdT can form an ultra-long polynucleotide tail which can bind with multiple crRNA, generating a signal amplification effect. The trans-cleavage activity of CRISPR/Cas12a can be specifically triggered via the crRNA binding with DNA, resulting in the bi-labeled DNA reporter with fluorophore and quencher being cleaved. The excitation wavelength of the fluorescence spectra was 490 nm. Fluorescence spectra with emission wavelengths ranging from 511 to 600 nm were collected. Under the optimization condition, the fabricated fluorescence aptasensor for NPC-derived exosome determination exhibited excellent sensitivity and specificity, with the linear range between 500 to 5 × 10[4] particles mL[-1] and the limit of detection of 100 particles mL[-1]. It can be used for the determination of NPC-derived exosomes in clinical samples, which has a considerable clinical potential and prospect.},
}
@article {pmid36629257,
year = {2023},
author = {Pellegrino, GM and Browne, TS and Sharath, K and Bari, KA and Vancuren, SJ and Allen-Vercoe, E and Gloor, GB and Edgell, DR},
title = {Metabolically-targeted dCas9 expression in bacteria.},
journal = {Nucleic acids research},
volume = {51},
number = {2},
pages = {982-996},
doi = {10.1093/nar/gkac1248},
pmid = {36629257},
issn = {1362-4962},
support = {PJT-159708/CAPMC/CIHR/Canada ; PJT-159708/CAPMC/CIHR/Canada ; },
mesh = {Humans ; *Escherichia coli/genetics ; *Glucuronides ; Bacteria ; Gene Expression Regulation ; Transcription Factors/genetics ; CRISPR-Cas Systems ; },
abstract = {The ability to restrict gene expression to a relevant bacterial species in a complex microbiome is an unsolved problem. In the context of the human microbiome, one desirable target metabolic activity are glucuronide-utilization enzymes (GUS) that are implicated in the toxic re-activation of glucuronidated compounds in the human gastrointestinal (GI) tract, including the chemotherapeutic drug irinotecan. Here, we take advantage of the variable distribution of GUS enzymes in bacteria as a means to distinguish between bacteria with GUS activity, and re-purpose the glucuronide-responsive GusR transcription factor as a biosensor to regulate dCas9 expression in response to glucuronide inducers. We fused the Escherichia coli gusA regulatory region to the dCas9 gene to create pGreg-dCas9, and showed that dCas9 expression is induced by glucuronides, but not other carbon sources. When conjugated from E. coli to Gammaproteobacteria derived from human stool, dCas9 expression from pGreg-dCas9 was restricted to GUS-positive bacteria. dCas9-sgRNAs targeted to gusA specifically down-regulated gus operon transcription in Gammaproteobacteria, with a resulting ∼100-fold decrease in GusA activity. Our data outline a general strategy to re-purpose bacterial transcription factors responsive to exogenous metabolites for precise ligand-dependent expression of genetic tools such as dCas9 in diverse bacterial species.},
}
@article {pmid36709094,
year = {2023},
author = {van der Oost, J and Patinios, C},
title = {The genome editing revolution.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.12.022},
pmid = {36709094},
issn = {1879-3096},
abstract = {A series of spectacular scientific discoveries and technological advances in the second half of the 20th century have provided the basis for the ongoing genome editing revolution. The elucidation of structural and functional features of DNA and RNA was followed by pioneering studies on genome editing: Molecular biotechnology was born. Since then, four decades followed during which progress of scientific insights and technological methods continued at an overwhelming pace. Fundamental insights into microbial host-virus interactions led to the development of tools for genome editing using restriction enzymes or the revolutionary CRISPR-Cas technology. In this review, we provide a historical overview of milestones that led to the genome editing revolution and speculate about future trends in biotechnology.},
}
@article {pmid36708962,
year = {2023},
author = {Sun, SX and Liu, YC and Limbu, SM and Li, DL and Chen, LQ and Zhang, ML and Yin, Z and Du, ZY},
title = {Vitellogenin 1 is essential for fish reproduction by transporting DHA-containing phosphatidylcholine from liver to ovary.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {},
number = {},
pages = {159289},
doi = {10.1016/j.bbalip.2023.159289},
pmid = {36708962},
issn = {1879-2618},
abstract = {Vitellogenins (Vtgs) are essential for female reproduction in oviparous animals, yet the exact roles and mechanisms remain unknown. In the present study, we knocked out vtg1, which is the most abundant Vtg in zebrafish, Danio rerio via the CRISPR/Cas 9 technology. We aimed to identify the roles of Vtg1 and related mechanisms in reproduction and development. We found that, the Vtg1-deficient female zebrafish reduced gonadosomatic index, egg production, yolk granules and mature follicles in ovary compared to the wide type (WT). Moreover, the Vtg1-deficient zebrafish diminished hatching rates, cumulative survival rate, swimming capacity and food intake, but increased malformation rate, and delayed swim bladder development during embryo and early-larval phases. The Vtg1-deficiency in female broodstock inhibited docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) transportation from liver to ovary, which lowered DHA-PC content in ovary and offspring during larval stage. However, the Vtg1-deficient zebrafish increased gradually the total DHA-PC content via exogeneous food intake, and the differences in swimming capacity and food intake returned to normal as they matured. Furthermore, supplementing Vtg1-deficient zebrafish with dietary PC and DHA partly ameliorated the impaired female reproductive capacity and larval development during early phases. This study indicates that, DHA and PC carried by Vtg1 are crucial for female fecundity, and affect embryo and larval development through maternal-nutrition effects. This is the first study elucidating the nutrient and physiological functions of Vtg1 and the underlying biochemical mechanisms in fish reproduction and development.},
}
@article {pmid36707422,
year = {2023},
author = {Aman Mohammadi, M and Maximiano, MR and Hosseini, SM and Franco, OL},
title = {CRISPR-Cas engineering in food science and sustainable agriculture: recent advancements and applications.},
journal = {Bioprocess and biosystems engineering},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00449-022-02842-5},
pmid = {36707422},
issn = {1615-7605},
abstract = {The developments in the food supply chain to support the growing population of the world is one of today's most pressing issues, and to achieve this goal improvements should be performed in both crops and microbes. For this purpose, novel approaches such as genome editing (GE) methods have upgraded the biological sciences for genome manipulation and, among such methods, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are the main exciting innovations since the Green Revolution. CRISPR/Cas systems can be a potent tool for the food industry, improvement of agricultural crops and even for protecting food-grade bacteria from foreign genetic invasive elements. This review introduces the history and mechanism of the CRISPR-Cas system as a genome editing tool and its applications in the vaccination of starter cultures, production of antimicrobials and bioactive compounds, and genome editing of microorganisms.},
}
@article {pmid36704579,
year = {2022},
author = {Heinemann, JA and Clark, K and Hiscox, TC and McCabe, AW and Agapito-Tenfen, SZ},
title = {Are null segregants new combinations of heritable material and should they be regulated?.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {1064103},
pmid = {36704579},
issn = {2673-3439},
abstract = {Through genome editing and other techniques of gene technology, it is possible to create a class of organism called null segregants. These genetically modified organisms (GMOs) are products of gene technology but are argued to have no lingering vestige of the technology after the segregation of chromosomes or deletion of insertions. From that viewpoint regulations are redundant because any unique potential for the use of gene technology to cause harm has also been removed. We tackle this question of international interest by reviewing the early history of the purpose of gene technology regulation. The active ingredients of techniques used for guided mutagenesis, e.g., site-directed nucleases, such as CRISPR/Cas, are promoted for having a lower potential per reaction to create a hazard. However, others see this as a desirable industrial property of the reagents that will lead to genome editing being used more and nullifying the promised hazard mitigation. The contest between views revolves around whether regulations could alter the risks in the responsible use of gene technology. We conclude that gene technology, even when used to make null segregants, has characteristics that make regulation a reasonable option for mitigating potential harm. Those characteristics are that it allows people to create more harm faster, even if it creates benefits as well; the potential for harm increases with increased use of the technique, but safety does not; and regulations can control harm scaling.},
}
@article {pmid36702942,
year = {2023},
author = {Huo, Y and Zhao, H and Dong, Q and Jiang, T},
title = {Cryo-EM structure and protease activity of the type III-E CRISPR-Cas effector.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {36702942},
issn = {2058-5276},
abstract = {The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here we present four cryo-EM structures of Desulfonema ishimotonii Cas7-11-crRNA complex in pre-target and target RNA-bound states, and the cryo-EM structure of DiCas7-11-crRNA bound to its accessory protein DiCsx29. These data reveal structural elements for pre-crRNA processing, target RNA cleavage and regulation. Moreover, a 3' seed region of crRNA is involved in regulating RNA cleavage activity of DiCas7-11-crRNA-Csx29. Our analysis also shows that both the minimal mismatch of 4 nt to the 5' handle of crRNA and the minimal matching of the first 12 nt of the spacer by the target RNA are essential for triggering the protease activity of DiCas7-11-crRNA-Csx29 towards DiCsx30. Taken together, we propose that target RNA recognition and cleavage regulate and fine-tune the protease activity of DiCas7-11-crRNA-Csx29, thus preventing auto-immune responses.},
}
@article {pmid36700476,
year = {2022},
author = {Wu, WH and Ma, XM and Huang, JQ and Lai, Q and Jiang, FN and Zou, CY and Chen, LT and Yu, L},
title = {CRISPR/Cas9 (D10A) nickase-mediated Hb CS gene editing and genetically modified fibroblast identification.},
journal = {Bioengineered},
volume = {13},
number = {5},
pages = {13398-13406},
doi = {10.1080/21655979.2022.2069940},
pmid = {36700476},
issn = {2165-5987},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/metabolism ; Mutation ; Fibroblasts/metabolism ; },
abstract = {This study investigated whether CRISPR/Cas9 (D10A) nickase-mediated gene editing can correct the aberrant Hb Constant Spring mutation (Hb CS or HBA2: c.427 T > C) in fibroblasts. Vectors for repairing the α-globin-encoding gene, HBA2:c.427 T > C mutation, includingthe CRISPR/Cas9(D10A)-sg plasmid and donor with homology arms, were constructed and used to perform gene editing in patient-derived fibroblasts. We subsequently analyzed the genetic correction, the gene editing efficiency and off-target effect. Sequencing analysis and the BamHI assay showed that HB CS mutant cells were repaired with Hb CS point mutations, the editing efficiency was 4.18%~9.34% and no off-target effects were detected. The results indicate that the HB CS mutant gene is effectively repaired by the CRISPR/Cas9 (D10A)system, which may enable truly personalized therapy for precise repair of α-thalassemia.},
}
@article {pmid36697180,
year = {2023},
author = {Xu, S and Wang, S and Guo, L and Tong, Y and Wu, L and Huang, X},
title = {Nanozyme-catalysed CRISPR-Cas12a system for the preamplification-free colorimetric detection of lead ion.},
journal = {Analytica chimica acta},
volume = {1243},
number = {},
pages = {340827},
doi = {10.1016/j.aca.2023.340827},
pmid = {36697180},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems ; Colorimetry ; *DNA, Catalytic ; Lead ; Manganese Compounds ; Oxides ; *Biosensing Techniques ; },
abstract = {CRISPR-based detection was often based on the target preamplification to realize the high sensitivity. Here, we prepared a CRISPR-Cas12a system for the colorimetric detection of lead ion (Pb[2+]) based on the assistance of DNAzyme and nanozyme instead of preamplification. The recognition between GR-5 DNAzyme and Pb[2+] could trigger the CRISPR-Cas12a system. MnO2 nanozymes connected with magnetic beads through single stranded DNA were prepared as the colorimetric signal probes and catalyst of CRISPR-Cas12a system for the strong oxidase-like activity inducing the color change of 3,3',5,5'-tetramethylbenzidine. The nanozyme-catalysed CRISPR-Cas12a system could be used to detect Pb[2+] through the color change with high specificity and sensitivity. The linear range of this approach was 0.8 nM-2500 nM, with a limit of detection of 0.54 nM. This method was applied for the detection of the Pb[2+] in food samples indicating good accuracy and anti-interference ability.},
}
@article {pmid36641031,
year = {2023},
author = {Shi, K and Yi, Z and Han, Y and Chen, J and Hu, Y and Cheng, Y and Liu, S and Wang, W and Song, J},
title = {PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA.},
journal = {Analytical biochemistry},
volume = {664},
number = {},
pages = {115046},
pmid = {36641031},
issn = {1096-0309},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; RNA, Viral/genetics ; Reproducibility of Results ; SARS-CoV-2/genetics ; RNA, Guide, Kinetoplastida/genetics ; *Biosensing Techniques ; },
abstract = {The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDR, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a's trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDR, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.},
}
@article {pmid36525790,
year = {2023},
author = {Leal, AF and Fnu, N and Benincore-Flórez, E and Herreño-Pachón, AM and Echeverri-Peña, OY and Alméciga-Díaz, CJ and Tomatsu, S},
title = {The landscape of CRISPR/Cas9 for inborn errors of metabolism.},
journal = {Molecular genetics and metabolism},
volume = {138},
number = {1},
pages = {106968},
doi = {10.1016/j.ymgme.2022.106968},
pmid = {36525790},
issn = {1096-7206},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy ; Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Since its discovery as a genome editing tool, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system has opened new horizons in the diagnosis, research, and treatment of genetic diseases. CRISPR/Cas9 can rewrite the genome at any region with outstanding precision to modify it and further instructions for gene expression. Inborn Errors of Metabolism (IEM) are a group of more than 1500 diseases produced by mutations in genes encoding for proteins that participate in metabolic pathways. IEM involves small molecules, energetic deficits, or complex molecules diseases, which may be susceptible to be treated with this novel tool. In recent years, potential therapeutic approaches have been attempted, and new models have been developed using CRISPR/Cas9. In this review, we summarize the most relevant findings in the scientific literature about the implementation of CRISPR/Cas9 in IEM and discuss the future use of CRISPR/Cas9 to modify epigenetic markers, which seem to play a critical role in the context of IEM. The current delivery strategies of CRISPR/Cas9 are also discussed.},
}
@article {pmid35708078,
year = {2022},
author = {Wang, W and Yuan, D and Jiang, K and Li, R and Qu, H and Jiang, FN and Zhong, WD and Sun, F and Jia, Z and Zhu, J},
title = {Genome-Wide CRISPR-Cas9 Screening and Identification of Potential Genes Promoting Prostate Cancer Growth and Metastasis.},
journal = {Current cancer drug targets},
volume = {23},
number = {1},
pages = {71-86},
doi = {10.2174/1568009622666220615154137},
pmid = {35708078},
issn = {1873-5576},
mesh = {Humans ; Male ; Mice ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Early Detection of Cancer ; *Prostatic Neoplasms/genetics/pathology ; *MicroRNAs/genetics ; GTP-Binding Protein Regulators/genetics ; },
abstract = {OBJECTIVE: Identification and validation of genes that functionally account for the growth and metastasis of prostate cancer.<br><br>METHODS: DU145-KO cell line was constructed by transfecting DU145 cells with lentivirus packaged with the genome-wide knock-out library. The DU145-KO cells were transplanted into the armpits of immunocompromised Nu/Nu mice, followed by the tissue collection from the lung at week 3 (early lung tissue) or week 7 (late lung tissue with micro-metastasis), as well as from primary tumor site at week 7 (late primary tumor) after inoculation. Lung metastasis was retrieved at various time points for DNA sequencing analysis to identify enriched sgRNAs, thus candidate genes/miRNAs. Further bioinformatics analysis and limited functional validation studies were carried out.<br><br>RESULTS: DU145-KO cells promoted the formation of transplanted tumors in mice and promoted the growth and metastasis of primary tumors, compared to the controls (DU145-NC cells). The analysis of sequence data showed that the abundance of sgRNAs significantly changed in the primary tumor and micro-metastasis site. Fifteen target genes (C1QTNF9B, FAM229A, hsa-mir-3929, KRT23, TARS2, CRADD, GRIK4, PLA2G15, LOXL1, SLITRK6, CDC42EP5, SLC2A4, PTGDS, MYL9 and ACOX2 for the enriched sgRNAs) have been selected for experimental validation, which showed that knock-out of any of these genes led to the enhanced potential of invasion and metastasis of DU145 cells.<br><br>CONCLUSION: Genome-wide CRISPR-Cas9 knock-out screening technology combined with highthroughput sequencing analysis identified genes that potentially relate to prostate tumor invasion and metastasis. Analysis of these genes provided insights into biological pathways relevant to the disease and disclosed innovative markers for diagnosis or prognosis as well as potential targets for therapy.},
}
@article {pmid36698803,
year = {2022},
author = {Zarghamian, P and Klermund, J and Cathomen, T},
title = {Clinical genome editing to treat sickle cell disease-A brief update.},
journal = {Frontiers in medicine},
volume = {9},
number = {},
pages = {1065377},
pmid = {36698803},
issn = {2296-858X},
abstract = {Sickle cell disease (SCD) is one of the most common hemoglobinopathies. Due to its high prevalence, with about 20 million affected individuals worldwide, the development of novel effective treatments is highly warranted. While transplantation of allogeneic hematopoietic stem cells (HSC) is the standard curative treatment approach, a variety of gene transfer and genome editing strategies have demonstrated their potential to provide a prospective cure for SCD patients. Several stratagems employing CRISPR-Cas nucleases or base editors aim at reactivation of γ-globin expression to replace the faulty β-globin chain. The fetal hemoglobin (HbF), consisting of two α-globin and two γ-globin chains, can compensate for defective adult hemoglobin (HbA) and reverse the sickling of hemoglobin-S (HbS). Both disruption of cis-regulatory elements that are involved in inhibiting γ-globin expression, such as BCL11A or LRF binding sites in the γ-globin gene promoters (HBG1/2), or the lineage-specific disruption of BCL11A to reduce its expression in human erythroblasts, have been demonstrated to reestablish HbF expression. Alternatively, the point mutation in the HBB gene has been corrected using homology-directed repair (HDR)-based methodologies. In general, genome editing has shown promising results not only in preclinical animal models but also in clinical trials, both in terms of efficacy and safety. This review provides a brief update on the recent clinical advances in the genome editing space to offer cure for SCD patients, discusses open questions with regard to off-target effects induced by the employed genome editors, and gives an outlook of forthcoming developments.},
}
@article {pmid36698226,
year = {2023},
author = {Wu, D and Liu, J and Liu, Y and Qiu, Y and Cao, Z and Pan, Y and Shi, J and Yuan, X},
title = {Temperature-dependent affinity changes in substrate binding affect the cleavage activity of BthC2c1.},
journal = {Protein and peptide letters},
volume = {},
number = {},
pages = {},
doi = {10.2174/0929866530666230125100320},
pmid = {36698226},
issn = {1875-5305},
abstract = {BACKGROUND: The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.<br><br>OBJECTIVES: Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.<br><br>METHODS: The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.<br><br>RESULTS: BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37&#xb0;C to 67&#xb0;C. The cleavage ability of BthC2c1 at 42&#x2103; was stronger than that at 37&#x2103;. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42&#x2103; was stronger than that at 37&#x2103;.<br><br>CONCLUSION: In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37&#x2103; to 67&#x2103;. The affinity of BthC2c1-sgRNA to DNA at 42&#xb0;C was significantly enhanced than that at 37&#xb0;C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42&#x2103;. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.},
}
@article {pmid36696817,
year = {2023},
author = {Zhou, Y and Yang, Y and Li, X and Tian, D and Ai, W and Wang, W and Wang, B and Kreiswirth, BN and Yu, F and Chen, L and Jiang, X},
title = {Exploiting a conjugative endogenous CRISPR-Cas3 system to tackle multidrug-resistant Klebsiella pneumoniae.},
journal = {EBioMedicine},
volume = {88},
number = {},
pages = {104445},
doi = {10.1016/j.ebiom.2023.104445},
pmid = {36696817},
issn = {2352-3964},
abstract = {BACKGROUND: Mobile plasmids play a key role in spurring the global dissemination of multidrug-resistant (MDR) K. pneumoniae, while plasmid curing has been recognized as a promising strategy to combat antimicrobial resistance. Here we exploited a K. pneumoniae native CRISPR system to cure the high-risk IncFII plasmids.<br><br>METHODS: We examined matched protospacers in 725 completely sequenced IncFII plasmids from K.&#xa0;pneumoniae genomes. Then, we re-engineered a native CRISPR-Cas3 system and deliver the CRISPR-Cas3 system via conjugation. Plasmid killing efficiency and G.&#xa0;mellonella infection model were applied to evaluate the CRISPR-Cas3 immunity in&#xa0;vitro and in&#xa0;vivo.<br><br>FINDINGS: Genomic analysis revealed that most IncFII plasmids could be targeted by the native CRISPR-Cas3 system with multiple matched protospacers, and the targeting regions were highly conserved across different IncFII plasmids. This conjugative endogenous CRISPR-Cas3 system demonstrated high plasmid curing efficiency in vitro (8-log decrease) and in&#xa0;vivo (&#x223c;100% curing) in a Galleria mellonella infection model, as well as provided immunization against the invasion of IncFII plasmids once the system entering a susceptible bacterial host.<br><br>INTERPRETATION: Overall, our work demonstrated the applicability of using native CRISPR-mediated plasmid curing to re-sensitize drug-resistant K.&#xa0;pneumoniae to multiple antibiotics. This work provided strong support for the idea of utilizing native CRISPR-Cas systems to tackle AMR in K.&#xa0;pneumoniae.<br><br>FUNDING: This work was supported by research grants National Natural Science Foundation of China [grant numbers 81871692, 82172315, 82102439, and 82202564], the Shanghai Science and Technology Commission [grant number 19JC1413002], and Shanghai Sailing Program [grant number 22YF1437500].},
}
@article {pmid36693839,
year = {2023},
author = {Gao, Y and Guitton-Sert, L and Dessapt, J and Coulombe, Y and Rodrigue, A and Milano, L and Blondeau, A and Larsen, NB and Duxin, JP and Hussein, S and Fradet-Turcotte, A and Masson, JY},
title = {A CRISPR-Cas9 screen identifies EXO1 as a formaldehyde resistance gene.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {381},
pmid = {36693839},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems ; DNA Repair/genetics ; DNA Damage ; DNA Replication ; Genomic Instability/genetics ; DNA ; Formaldehyde/toxicity ; *Fanconi Anemia/genetics ; Exodeoxyribonucleases/genetics/metabolism ; DNA Repair Enzymes/metabolism ; },
abstract = {Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.},
}
@article {pmid36626587,
year = {2023},
author = {Ma, D and Yuan, Q and Peng, F and Paredes, V and Zeng, H and Osikpa, EC and Yang, Q and Peddi, A and Patel, A and Liu, MS and Sun, Z and Gao, X},
title = {Engineered PROTAC-CID Systems for Mammalian Inducible Gene Regulation.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {3},
pages = {1593-1606},
doi = {10.1021/jacs.2c09129},
pmid = {36626587},
issn = {1520-5126},
mesh = {Humans ; Mice ; Animals ; Dimerization ; *DNA/metabolism ; *Recombinases/metabolism ; Gene Editing ; Genome ; CRISPR-Cas Systems ; Mammals/genetics/metabolism ; },
abstract = {Gene regulation via chemically induced dimerization (CID) is useful for biomedical research. However, the number, type, versatility, and in vivo applications of CID tools remain limited. Here, we demonstrate the development of proteolysis-targeting chimera-based scalable CID (PROTAC-CID) platforms by systematically engineering the available PROTAC systems for inducible gene regulation and gene editing. Further, we show orthogonal PROTAC-CIDs that can fine-tune gene expression at gradient levels or multiplex biological signals with different logic gating operations. Coupling the PROTAC-CID platform with genetic circuits, we achieve digitally inducible expression of DNA recombinases, base- and prime-editors for transient genome manipulation. Finally, we package a compact PROTAC-CID system into adeno-associated viral vectors for inducible and reversible gene activation in vivo. This work provides a versatile molecular toolbox that expands the scope of chemically inducible gene regulation in human cells and mice.},
}
@article {pmid36626407,
year = {2023},
author = {Yan, F and Wang, J and Zhang, S and Lu, Z and Li, S and Ji, Z and Song, C and Chen, G and Xu, J and Feng, J and Zhou, X and Zhou, H},
title = {CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.},
journal = {PLoS pathogens},
volume = {19},
number = {1},
pages = {e1010961},
pmid = {36626407},
issn = {1553-7374},
mesh = {Bacterial Proteins/metabolism ; Gene Editing/methods ; Genome, Bacterial ; *Oryza/microbiology ; Plasmids ; *Xanthomonas/genetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.},
}
@article {pmid36624936,
year = {2023},
author = {Hu, JJ and Liu, D and Cai, MZ and Zhou, Y and Yin, WX and Luo, CX},
title = {One-Pot Assay for Rapid Detection of Benzimidazole Resistance in Venturia carpophila by Combining RPA and CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {3},
pages = {1381-1390},
doi = {10.1021/acs.jafc.2c06549},
pmid = {36624936},
issn = {1520-5118},
mesh = {Humans ; *Recombinases ; CRISPR-Cas Systems ; Nucleotidyltransferases ; Benzimidazoles/pharmacology ; *Fungicides, Industrial ; Nucleic Acid Amplification Techniques ; },
abstract = {High resistance to benzimidazole fungicides in Venturia carpophila is caused by the point mutation E198K of the β-tubulin (TUB2) gene. Traditional methods for detection of fungicide resistance are time-consuming, which are routinely based on tedious operation, reliance on expensive equipment, and specially trained people. Therefore, it is important to establish efficient methods for field detection of benzimidazole resistance in V. carpophila to make suitable management strategies and ensure food safety. Based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a, a rapid one-pot assay ORCas12a-BRVc (one-pot RPA-CRISPR/Cas12 platform) was established for the detection of benzimidazole resistance in V. carpophila. The ORCas12a-BRVc assay enabled one-pot detection by adding components at the bottom and wall of the tube separately, solving the problems of aerosol contamination and decreased sensitivity caused by competing DNA substrates between Cas12a cleavage and RPA amplification. The ORCas12a-BRVc assay could accomplish the detection with a minimum of 7.82 × 10[3] fg μL[-1] V. carpophila genomic DNA in 45 min at 37 °C. Meanwhile, this assay showed excellent specificity due to the specific recognition ability of the Cas12a-crRNA complex. Further, we combined a method that could rapidly extract DNA from V. carpophila within 2 min with the ORCas12a-BRVc to achieve more rapid and simple detection of V. carpophila with benzimidazole resistance in fields. The ORCas12a-BRVc assay has the advantages of simplicity, rapidity, high sensitivity, high specificity, and ease of operation without the need for precision instruments and the need to isolate and culture pathogens. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in fungicide resistance detection and can be used for monitoring of resistant populations in fields, providing guidance on making suitable management strategies for peach scab.},
}
@article {pmid36693761,
year = {2023},
author = {Morelli, KH and Smargon, AA and Yeo, GW},
title = {Programmable Macromolecule-based RNA-targeting Therapies To Treat Human Neurological Disorders.},
journal = {RNA (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1261/rna.079519.122},
pmid = {36693761},
issn = {1469-9001},
abstract = {Disruptions in RNA processing play critical roles in the pathogenesis of neurological diseases. In this perspective, we discuss recent progress in the development of RNA-targeting therapeutic modalities. We focus on progress, limitations, and opportunities in a new generation of therapies engineered from RNA binding proteins and other endogenous RNA regulatory macromolecules to treat human neurological disorders.},
}
@article {pmid36689187,
year = {2023},
author = {Wu, Q and Michaels, YS and Fulga, TA},
title = {Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2630},
number = {},
pages = {243-264},
pmid = {36689187},
issn = {1940-6029},
mesh = {Animals ; *MicroRNAs/genetics ; CRISPR-Cas Systems ; Cell Line ; Genome ; Response Elements ; },
abstract = {Posttranscriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22 nt) noncoding RNAs capable of specifically targeting the miRNA-induced silencing complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct in situ interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering (Bassett G et al., Nat Commun 5, 4640, 2014). In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).},
}
@article {pmid36689186,
year = {2023},
author = {Godden, AM and Antonaci, M and Wheeler, GN},
title = {An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2630},
number = {},
pages = {231-241},
pmid = {36689186},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing ; CRISPR-Cas Systems ; *MicroRNAs/genetics ; Xenopus/genetics ; RNA, Guide, Kinetoplastida ; },
abstract = {In recent years CRISPR-Cas9 knockouts (KO) have become increasingly utilized to study gene function. MicroRNAs (miRNAs) are short noncoding RNAs, 20-25 nucleotides long, which affect gene expression through posttranscriptional repression. As miRNAs are so small and due to the limitations of known PAM sequences, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore developed a novel approach using two guide RNAs to effectively "drop out" a miRNA. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of q-RT-PCR and Sanger sequencing. To show specificity of the phenotype, we provide a protocol to use miRNA mimics to rescue the KO phenotype.},
}
@article {pmid36631579,
year = {2023},
author = {Barabas, O and Rice, PA},
title = {First full views of a CRISPR-guided system for gene insertion.},
journal = {Nature},
volume = {613},
number = {7945},
pages = {634-635},
pmid = {36631579},
issn = {1476-4687},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Mutagenesis, Insertional ; *CRISPR-Cas Systems ; Gene Editing ; },
}
@article {pmid36519469,
year = {2023},
author = {Huang, T and Cheng, J and Feng, H and Zhou, W and Qiu, P and Zhou, D and Yang, D and Zhang, J and Willer, C and Chen, YE and Mizrak, D and Yang, B},
title = {Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {43},
number = {2},
pages = {312-322},
doi = {10.1161/ATVBAHA.122.318566},
pmid = {36519469},
issn = {1524-4636},
abstract = {BACKGROUND: The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT.<br><br>METHODS: A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction.<br><br>RESULTS: The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032.<br><br>CONCLUSIONS: BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.},
}
@article {pmid36690762,
year = {2023},
author = {Colognori, D and Trinidad, M and Doudna, JA},
title = {Precise transcript targeting by CRISPR-Csm complexes.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36690762},
issn = {1546-1696},
abstract = {Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90-99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.},
}
@article {pmid36690071,
year = {2023},
author = {Pons Conceptualisation, BJ and van Houte Conceptualisation, S and Westra Conceptualisation, ER and Chevallereau Conceptualisation, A},
title = {Ecology and evolution of phages encoding anti-CRISPR proteins.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {167974},
doi = {10.1016/j.jmb.2023.167974},
pmid = {36690071},
issn = {1089-8638},
abstract = {CRISPR-Cas are prokaryotic defence systems that provide protection against invasion by mobile genetic elements (MGE), including bacteriophages. MGE can overcome CRISPR-Cas defences by encoding anti-CRISPR (Acr) proteins. These proteins are produced in the early stages of the infection and inhibit the CRISPR-Cas machinery to allow phage replication. While research on Acr has mainly focused on their discovery, structure and mode of action, and their application in biotechnology, the impact of Acr on the ecology of MGE as well as on the coevolution with their bacterial hosts only begins to be unravelled. In this review, we summarise our current understanding on the distribution of anti-CRISPR genes in MGE, the ecology of phages encoding Acr, and their coevolution with bacterial defence mechanisms. We highlight the need to use more diverse and complex experimental models to better understand the impact of anti-CRISPR in MGE-host interactions.},
}
@article {pmid36685559,
year = {2022},
author = {Brault, J and Liu, T and Liu, S and Lawson, A and Choi, U and Kozhushko, N and Bzhilyanskaya, V and Pavel-Dinu, M and Meis, RJ and Eckhaus, MA and Burkett, SS and Bosticardo, M and Kleinstiver, BP and Notarangelo, LD and Lazzarotto, CR and Tsai, SQ and Wu, X and Dahl, GA and Porteus, MH and Malech, HL and De Ravin, SS},
title = {CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {1067417},
pmid = {36685559},
issn = {1664-3224},
mesh = {Animals ; Mice ; *X-Linked Combined Immunodeficiency Diseases/genetics/therapy ; Dependovirus ; CRISPR-Cas Systems ; Mice, SCID ; Hematopoietic Stem Cells ; },
abstract = {INTRODUCTION: Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs.<br><br>METHODS: We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus.<br><br>RESULTS AND DISCUSSION: In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.},
}
@article {pmid36685499,
year = {2022},
author = {Xiong, E and Popp, O and Salomon, C and Mertins, P and Kocks, C and Rajewsky, K and Chu, VT},
title = {A CRISPR/Cas9-mediated screen identifies determinants of early plasma cell differentiation.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {1083119},
pmid = {36685499},
issn = {1664-3224},
mesh = {*CRISPR-Cas Systems ; *B-Lymphocytes ; Plasma Cells ; Cell Differentiation/genetics ; Antibodies ; },
abstract = {INTRODUCTION: The differentiation of B cells into antibody-secreting plasma cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood.<br><br>METHODS: We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells.<br><br>RESULTS: Among known genes whose deletion preferentially or mostly affected plasmablast formation were the transcription factors Prdm1 (BLIMP1), Irf4 and Pou2af1 (OBF-1), and the Ern1 gene encoding IRE1a, while deletion of XBP1, the transcriptional master regulator that specifies the expansion of the secretory program in plasma cells, had no effect. Defective plasmablast formation caused by Ern1 deletion could not be rescued by the active, spliced form of XBP1 whose processing is dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1. Moreover, we newly identified several genes involved in NF-kB signaling (Nfkbia), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complex (Hdac1, Mta2, Mbd2) to be required for plasmablast formation. Deletion of ARF4, a small GTPase required for COPI vesicle formation, impaired plasmablast formation and blocked antibody secretion. After Hdac1 deletion plasmablast differentiation was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that govern plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5).<br><br>DISCUSSION: Taken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation.},
}
@article {pmid36683434,
year = {2023},
author = {Pakari, K and Wittbrodt, J and Thumberger, T},
title = {De novo PAM generation to reach initially inaccessible target sites for base editing.},
journal = {Development (Cambridge, England)},
volume = {150},
number = {2},
pages = {},
doi = {10.1242/dev.201115},
pmid = {36683434},
issn = {1477-9129},
support = {ERC-SyG H2020 810172/ERC_/European Research Council/International ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.},
}
@article {pmid36674715,
year = {2023},
author = {Du, W and Zhu, H and Qian, J and Xue, D and Zheng, S and Huang, Q},
title = {Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.},
journal = {International journal of molecular sciences},
volume = {24},
number = {2},
pages = {},
pmid = {36674715},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *RNA, Guide, Kinetoplastida ; Gene Editing ; DNA/chemistry ; },
abstract = {Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.},
}
@article {pmid36674592,
year = {2023},
author = {Lopatriello, G and Maestri, S and Alfano, M and Papa, R and Di Vittori, V and De Antoni, L and Bellucci, E and Pieri, A and Bitocchi, E and Delledonne, M and Rossato, M},
title = {CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions.},
journal = {International journal of molecular sciences},
volume = {24},
number = {2},
pages = {},
pmid = {36674592},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Sequence Analysis, DNA ; *Nanopore Sequencing ; High-Throughput Nucleotide Sequencing ; Genomics ; *Nanopores ; },
abstract = {Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore sequencing to perform a targeted de novo assembly and accurately reconstruct a genomic region of interest. This approach was used to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype-phenotype association analysis.},
}
@article {pmid36671975,
year = {2023},
author = {Qian, S and Chen, Y and Wang, X and Wang, T and Che, Y and Wu, J and Ye, Z and Xu, J},
title = {CRISPR/Cas12a-Assisted Dual Visualized Detection of SARS-CoV-2 on Frozen Shrimps.},
journal = {Biosensors},
volume = {13},
number = {1},
pages = {},
pmid = {36671975},
issn = {2079-6374},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Given the possibility that food contaminated with SARS-CoV-2 might become an infection source, there is an urgent need for us to develop a rapid and accurate nucleic acid detection method for SARS-CoV-2 in food to ensure food safety. Here, we propose a sensitive, specific, and reliable molecular detection method for SARS-CoV-2. It has a mechanism to control amplicon contamination. Swabs from spiked frozen shrimps were used as detection samples, which were processed by heating at 95 °C for 30 s. These preprocessed samples served as the templates for subsequent amplification. A colorimetric LAMP reaction was carried out to amplify both the SARS-CoV-2 target and the MS2 phage simultaneously in one tube. MS2 phage was detected by colorimetric LAMP as the internal control, while SARS-CoV-2 was detected with a CRISPR/Cas12a system. The fluorescence results could be visually detected with an ultraviolet lamp. Meanwhile, uracil was incorporated during the LAMP reaction to provide an amplicon contamination proof mechanism. This test could detect as low as 20 copies of SARS-CoV-2 in one reaction. Additionally, the detection could be finished in 45 min. The test only needs a heating block and an ultraviolet lamp, which shows the potential for field detection.},
}
@article {pmid36671946,
year = {2023},
author = {Yang, T and Chen, Y and He, J and Wu, J and Wang, M and Zhong, X},
title = {A Designed Vessel Using Dissolvable Polyvinyl Alcohol Membrane as Automatic Valve to Couple LAMP with CRISPR/Cas12a System for Visual Detection.},
journal = {Biosensors},
volume = {13},
number = {1},
pages = {},
pmid = {36671946},
issn = {2079-6374},
mesh = {*Polyvinyl Alcohol ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; *Vibrio parahaemolyticus/genetics ; Catheters ; },
abstract = {A rapid and intuitive method for detecting Vibrio parahaemolyticus (VP) was established by a designed reaction vessel which coupled CRISPR/Cas12a with loop-mediated isothermal nucleic acid amplification (LAMP). There were two spaces in the vessel-holding LAMP reaction solution and CRISPR reaction solution, respectively, which were separated with a polyvinyl alcohol (PVA) membrane. The PVA membrane could be dissolved with a water solution. The thermolabile hemolysin (TLH) gene of VP was employed as the detection target. After the target sequence of the TLH gene was amplified with LAMP, the PVA membrane would be dissolved and the CRISPR reaction solution mixed with the LAMP reaction solution. In this way, amplicons could be detected with CRISPR/Cas12a in the reaction vessel. The fluorescent signals produced by the positive samples were clearly identified by the naked eye under a UV light, while the negative samples were dark. The whole detection procedure could be finished within 35 min with a detection limit of 100 copies/µL. The designed reaction vessel is easy to produce and can effectively prevent contamination due to the opening of the reaction vessel after the LAMP reaction. Thus, it will have the potential to provide a new solution for rapid detection in the field.},
}
@article {pmid36641878,
year = {2023},
author = {You, J and Park, H and Lee, H and Jang, K and Park, J and Na, S},
title = {Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {224},
number = {},
pages = {115078},
doi = {10.1016/j.bios.2023.115078},
pmid = {36641878},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Polymerization ; *Biosensing Techniques/methods ; DNA/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.},
}
@article {pmid36638795,
year = {2023},
author = {Nyberg, WA and Ark, J and To, A and Clouden, S and Reeder, G and Muldoon, JJ and Chung, JY and Xie, WH and Allain, V and Steinhart, Z and Chang, C and Talbot, A and Kim, S and Rosales, A and Havlik, LP and Pimentel, H and Asokan, A and Eyquem, J},
title = {An evolved AAV variant enables efficient genetic engineering of murine T cells.},
journal = {Cell},
volume = {186},
number = {2},
pages = {446-460.e19},
doi = {10.1016/j.cell.2022.12.022},
pmid = {36638795},
issn = {1097-4172},
mesh = {Mice ; Humans ; Animals ; *T-Lymphocytes ; *Dependovirus/genetics ; Genetic Engineering ; Gene Targeting ; Genome ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Vectors ; },
abstract = {Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.},
}
@article {pmid36621165,
year = {2023},
author = {Hao, J and Xie, L and Yang, T and Huo, Z and Liu, G and Liu, Y and Xiong, W and Zeng, Z},
title = {Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with recombinase polymerase amplification.},
journal = {Talanta},
volume = {255},
number = {},
pages = {124220},
doi = {10.1016/j.talanta.2022.124220},
pmid = {36621165},
issn = {1873-3573},
mesh = {Humans ; *Recombinases ; CRISPR-Cas Systems ; *Pasteurella multocida/genetics ; Biological Assay ; Cross Reactions ; Nucleic Acid Amplification Techniques ; },
abstract = {Pasteurella multocida (P. multocida) is an important pathogenic bacterium that poses a serious threat to the development of the livestock economy and human health. Currently, the existing methods for P. multocida detection are time-consuming and require complex professional operations, limiting the application of field detection. In the study, we presented a single-pot naked-eye CRISPR-Cas12a platform (Cas12a-NEye) for the detection of P. multocida. The round tube cover allowed more Cas12a detection solution to be temporarily stored than the flat cap, enabling single-pot assays and avoiding aerosol contamination. The positive samples generated obvious red using naked eye using no excitation light and the negative samples generated blue. The limit of detection (LOD) was a single copy, without cross-reactivity with other closely related bacteria. Furthermore, we validated this platform using 16 P. multocida clinical lung samples and obtained consistent results with the real-time quantitative polymerase chain reaction (qPCR) method. The entire experimental process included rapid DNA extraction (<1 h) and Cas12a-NEye assay (25 min), which was accomplished within 1.5 h. Thus, this "sample-to-answer" platform has significant potential for P. multocida detection.},
}
@article {pmid36596263,
year = {2023},
author = {Yuan, H and Kang, Q and Li, Z and Bai, X and Jia, J and Han, D and Wu, X and Li, M},
title = {Crispr-Cas9 mediated complete deletion of glucagon receptor in mice display hyperglucagonemia and α-cell hyperplasia.},
journal = {Biochemical and biophysical research communications},
volume = {643},
number = {},
pages = {121-128},
doi = {10.1016/j.bbrc.2022.12.079},
pmid = {36596263},
issn = {1090-2104},
mesh = {Animals ; Mice ; *Receptors, Glucagon/genetics ; Hyperplasia/genetics ; *CRISPR-Cas Systems ; Glucagon/genetics/metabolism ; Mice, Knockout ; },
abstract = {Glucagon receptor plays an important role in the regulation of glucose metabolism. Studies have revealed that glucagon receptor antagonism is a potential effective treatment for diabetes. However, the functions of GCGR have not been fully illustrated. Although two Gcgr truncation knockout mice models have been widely used for GCGR function studies, truncated gene may remain neomorphic and/or dominant-negative function. In this study, we took the advantages of Crispr-Cas9 technique and generated a novel allele of GCGR in the mouse that yields complete loss of GCGR protein. Our studies reveal that complete deletion of Gcgr results in hyperglucagonemia, α-cell hyperplasia, improvement of glucose tolerance. These results are similar to the Gcgr-truncated mutation in mice. Hence, we provide a novel strain of GCGR knockout mice for the GCGR function studies.},
}
@article {pmid36566557,
year = {2023},
author = {Pan, J and Deng, F and Liu, Z and Zeng, L and Chen, J},
title = {Construction of molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly and CRISPR-Cas12a.},
journal = {Talanta},
volume = {255},
number = {},
pages = {124210},
doi = {10.1016/j.talanta.2022.124210},
pmid = {36566557},
issn = {1873-3573},
mesh = {*DNA, Catalytic ; CRISPR-Cas Systems ; Cadmium ; Lead ; *Metals, Heavy ; *Mercury ; DNA ; Ions ; DNA, Single-Stranded ; },
abstract = {We successfully constructed several molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly (CHA) and CRISPR-Cas12a. The corresponding DNAzymes were used to recognize heavy metal ions (Hg[2+], Cd[2+], Pb[2+], and Mn[2+]). The specific cleavage between heavy metal ions and DNAzymes leads to the release of the trigger DNA, which can be used to activate CHA through logic computation. The CHA-generated DNA duplexes contain the protospacer adjacent motifs (PAM) sequence, which can be distinguished by CRISPR-Cas12a. The hybridization interactions between the duplexes and gRNA will activate the trans-cleavage capability of Cas12a, which can cleave the single-stranded DNA (ssDNA) reporter. The separation of the fluorescence group and quench group in ssDNA will generate a high fluorescence signal for readout. Using Hg[2+] and Cd[2+] as the two inputs, several basic logic gates were constructed, including OR, AND, and INHIBT. Using Hg[2+], Cd[2+], Pb[2+], and Mn[2+] as the four inputs, cascaded logic gates were further fabricated. With the advantages of scalability, versatility, and logic computing capability, our proposed molecular logic gates can provide an intelligent sensing system for heavy metal ions monitoring.},
}
@article {pmid36326850,
year = {2023},
author = {Lao, K and Xiao, Y and Huang, Q and Mo, B and Dong, X and Wang, X},
title = {Establishment of an efficient early flowering-assisted CRISPR/Cas9 gene-editing system in Arabidopsis.},
journal = {Plant cell reports},
volume = {42},
number = {1},
pages = {211-214},
pmid = {36326850},
issn = {1432-203X},
mesh = {*Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Phenotype ; Plants, Genetically Modified/genetics ; Reproduction ; },
abstract = {Ectopic expression of the florigen FT gene along with the Cas9 cassette promotes fast reproduction of the T1 transgenic plants, and the late-flowering phenotype serves as a convenient marker for transgene-free plants screening in T2 segregation population.},
}
@article {pmid35282729,
year = {2023},
author = {Dong, BR and Jiang, R and Chen, JF and Xiao, Y and Lv, ZY and Chen, WS},
title = {Strategic nanoparticle-mediated plant disease resistance.},
journal = {Critical reviews in biotechnology},
volume = {43},
number = {1},
pages = {22-37},
doi = {10.1080/07388551.2021.2007842},
pmid = {35282729},
issn = {1549-7801},
mesh = {*Gene Editing ; Disease Resistance/genetics ; Plants, Genetically Modified/genetics ; *Nanoparticles ; Plant Diseases/prevention &amp; control ; CRISPR-Cas Systems ; Genome, Plant ; },
abstract = {Nanotechnology is a promising means for development of sustainable agriculture while the study of nanoparticle-mediated plant disease resistance is still in its primary stage. Nanotechnology has shown great promise in regulating: the content of secondary metabolites, inducing disease resistance genes, delivering hormones, delivering biomolecules (such as: nucleotides, proteins, and activators), and obtaining transgenic plants to resist plant diseases. In this review, we conclude its versatility and applicability in disease management strategies and diagnostics and as molecular tools. With the advent of new biotechnologies (e.g. de novo regeneration, CRISPR/Cas9, and GRF4-GIF1 fusion protein), we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering. In order to ensure the safe use and social acceptance of plant nanoparticle technology, its adverse effects are discussed, including the risk of transferring nanoparticles through the food chain.},
}
@article {pmid36689174,
year = {2023},
author = {Qiu, X and Liu, C and Zhu, C and Zhu, L},
title = {MicroRNA Detection with CRISPR/Cas.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2630},
number = {},
pages = {25-45},
pmid = {36689174},
issn = {1940-6029},
abstract = {Low-cost detection of miRNAs has caught broad attention in recent years due to the potential application of these small noncoding RNAs for diagnostics and therapeutic purposes. Their small size and low abundance, however, derive challenges in engineering robust detection tools. To date, multiple detection assays have been developed to achieve highly specific recognition of trace amount of miRNA with state-of-the-art nucleic acid detection and signal amplification techniques. In this chapter we describe how isothermal amplification techniques and CRISPR/Cas-based techniques can be integrated to generate rationally designed miRNA detection systems for specific miRNA.},
}
@article {pmid36688751,
year = {2023},
author = {Karmakar, K and Kumar, S and Roy, D and Singh, M and Kolte, V and Choudhury, A and Sharma, G},
title = {Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.},
journal = {Letters in applied microbiology},
volume = {76},
number = {1},
pages = {},
doi = {10.1093/lambio/ovac004},
pmid = {36688751},
issn = {1472-765X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.},
}
@article {pmid36688528,
year = {2023},
author = {Rumah, BL and Claxton Stevens, BH and Yeboah, JE and Stead, CE and Harding, EL and Minton, NP and Zhang, Y},
title = {In Vivo Genome Editing in Type I and II Methanotrophs Using a CRISPR/Cas9 System.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00554},
pmid = {36688528},
issn = {2161-5063},
abstract = {Methanotrophic bacteria are Gram-negative, aerobic organisms that use methane as their sole source of carbon and energy. In this study, we constructed and exemplified a CRISPR/Cas9 genome editing system and used it to successfully make gene deletions and insertions in the type I methanotroph Methylococcus capsulatus Bath and the type II methanotroph Methylocystis parvus OBBP. High frequencies of gene deletions and insertions were achieved in combination with homology-directed repair. In M. parvus OBBP, we also investigated the impact of several parameters on the CRISPR/Cas9 genome editing, where the ligD gene was targeted with various PAM sequences and guide RNA spacer sequences, homology arms of variable length, differences in the duration of mating during conjugation, and exploiting promoters of different strengths to control the expression of cas9 and sgRNA. Although not the first attempt to develop a CRISPR/Cas system in methanotrophs, this work demonstrated for the first time an efficient CRISPR/Cas9 system generating scarless clean gene deletions and insertions in methanotroph genomes.},
}
@article {pmid36687779,
year = {2022},
author = {Mohammadian Gol, T and Ureña-Bailén, G and Hou, Y and Sinn, R and Antony, JS and Handgretinger, R and Mezger, M},
title = {CRISPR medicine for blood disorders: Progress and challenges in delivery.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {1037290},
pmid = {36687779},
issn = {2673-3439},
abstract = {Blood disorders are a group of diseases including hematological neoplasms, clotting disorders and orphan immune deficiency diseases that affects human health. Current improvements in genome editing based therapeutics demonstrated preclinical and clinical proof to treat different blood disorders. Genome editing components such as Cas nucleases, guide RNAs and base editors are supplied in the form of either a plasmid, an mRNA, or a ribonucleoprotein complex. The most common delivery vehicles for such components include viral vectors (e.g., AAVs and RV), non-viral vectors (e.g., LNPs and polymers) and physical delivery methods (e.g., electroporation and microinjection). Each of the delivery vehicles specified above has its own advantages and disadvantages and the development of a safe transferring method for ex vivo and in vivo application of genome editing components is still a big challenge. Moreover, the delivery of genome editing payload to the target blood cells possess key challenges to provide a possible cure for patients with inherited monogenic blood diseases and hematological neoplastic tumors. Here, we critically review and summarize the progress and challenges related to the delivery of genome editing elements to relevant blood cells in an ex vivo or in vivo setting. In addition, we have attempted to provide a future clinical perspective of genome editing to treat blood disorders with possible clinical grade improvements in delivery methods.},
}
@article {pmid36687471,
year = {2023},
author = {Joseph, RC and Sandoval, NR},
title = {Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference.},
journal = {Synthetic and systems biotechnology},
volume = {8},
number = {1},
pages = {148-156},
pmid = {36687471},
issn = {2405-805X},
abstract = {The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.},
}
@article {pmid36686235,
year = {2022},
author = {Xu, H and Peng, L and Wu, J and Khan, A and Sun, Y and Shen, H and Li, Z},
title = {Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins13a combined with magnetic beads, chemiluminescence and reverse transcription-recombinase aided amplification for detection of avian influenza a (H7N9) virus.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {1094028},
pmid = {36686235},
issn = {2296-4185},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins (CRISPR-Cas) have promising prospects in the field of nucleic acid molecular diagnostics. However, Clustered Regularly Interspaced Short Palindromic Repeats-based fluorescence detection technology is mainly hindered by proteins with conjugated double bonds and autofluorescence, resulting in high fluorescence background, low sensitivity and incompatible reaction systems, which are not conducive to automatic clinical testing. Chemiluminescence (CL) detection technology has been applied mainly owing to its greatly high sensitivity, as well as low background and rapid response. Therefore, we developed a rapid, ultrasensitive and economical detection system based on Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 13a combined with magnetic beads (MBs) and chemiluminescence (CL) (Cas13a-MB-CL) to detect Influenza A (H7N9), an acute respiratory tract infectious disease. The carboxyl functionalized magnetic beads (MBs-COOH) were covalently coupled with aminated RNA probe while the other end of the RNA probe was modified with biotin. Alkaline phosphatase labeled streptavidin (SA-ALP) binds with biotin to form magnetic beads composites. In presence of target RNA, the collateral cleavage activity of Cas13a was activated to degrade the RNA probes on MBs and released Alkaline phosphatase from the composites. The composites were then magnetically separated followed by addition of ALP substrate Disodium 2-chloro-5-{4-methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo (3.3.1.13,7) decan]-4-yl}
-1-phenyl phosphate (CDP-star), to generate the chemiluminescence signal. The activity of Associated Proteins 13a and presence of target RNA was quantified by measuring the chemiluminescence intensity. The proposed method accomplished the detection of H7N9 within 30 min at 25°C. When combined with Reverse Transcription- Recombinase Aides Amplification (RT-RAA), the low detection limit limit of detection was as low as 19.7 fM (3S/N). Our proposed MB-Associated Proteins 13a-chemiluminescence was further evaluated to test H7N9 clinical samples, showing superior sensitivity and specificity.},
}
@article {pmid36685866,
year = {2022},
author = {Kor, SD and Chowdhury, N and Keot, AK and Yogendra, K and Chikkaputtaiah, C and Sudhakar Reddy, P},
title = {RNA Pol III promoters-key players in precisely targeted plant genome editing.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {989199},
pmid = {36685866},
issn = {1664-8021},
abstract = {The clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein Cas) system is a powerful and highly precise gene-editing tool in basic and applied research for crop improvement programs. CRISPR/Cas tool is being extensively used in plants to improve crop yield, quality, and nutritional value and make them tolerant to environmental stresses. CRISPR/Cas system consists of a Cas protein with DNA endonuclease activity and one CRISPR RNA transcript that is processed to form one or several short guide RNAs that direct Cas9 to the target DNA sequence. The expression levels of Cas proteins and gRNAs significantly influence the editing efficiency of CRISPR/Cas-mediated genome editing. This review focuses on insights into RNA Pol III promoters and their types that govern the expression levels of sgRNA in the CRISPR/Cas system. We discussed Pol III promoters structural and functional characteristics and their comparison with Pol II promoters. Further, the use of synthetic promoters to increase the targeting efficiency and overcome the structural, functional, and expressional limitations of RNA Pol III promoters has been discussed. Our review reports various studies that illustrate the use of endogenous U6/U3 promoters for improving editing efficiency in plants and the applicative approach of species-specific RNA pol III promoters for genome editing in model crops like Arabidopsis and tobacco, cereals, legumes, oilseed, and horticultural crops. We further highlight the significance of optimizing these species-specific promoters' systematic identification and validation for crop improvement and biotic and abiotic stress tolerance through CRISPR/Cas mediated genome editing.},
}
@article {pmid36685816,
year = {2022},
author = {Basu, U and Riaz Ahmed, S and Bhat, BA and Anwar, Z and Ali, A and Ijaz, A and Gulzar, A and Bibi, A and Tyagi, A and Nebapure, SM and Goud, CA and Ahanger, SA and Ali, S and Mushtaq, M},
title = {A CRISPR way for accelerating cereal crop improvement: Progress and challenges.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {866976},
pmid = {36685816},
issn = {1664-8021},
abstract = {Humans rely heavily on cereal grains as a key source of nutrients, hence regular improvement of cereal crops is essential for ensuring food security. The current food crisis at the global level is due to the rising population and harsh climatic conditions which prompts scientists to develop smart resilient cereal crops to attain food security. Cereal crop improvement in the past generally depended on imprecise methods like random mutagenesis and conventional genetic recombination which results in high off targeting risks. In this context, we have witnessed the application of targeted mutagenesis using versatile CRISPR-Cas systems for cereal crop improvement in sustainable agriculture. Accelerated crop improvement using molecular breeding methods based on CRISPR-Cas genome editing (GE) is an unprecedented tool for plant biotechnology and agriculture. The last decade has shown the fidelity, accuracy, low levels of off-target effects, and the high efficacy of CRISPR technology to induce targeted mutagenesis for the improvement of cereal crops such as wheat, rice, maize, barley, and millets. Since the genomic databases of these cereal crops are available, several modifications using GE technologies have been performed to attain desirable results. This review provides a brief overview of GE technologies and includes an elaborate account of the mechanisms and applications of CRISPR-Cas editing systems to induce targeted mutagenesis in cereal crops for improving the desired traits. Further, we describe recent developments in CRISPR-Cas-based targeted mutagenesis through base editing and prime editing to develop resilient cereal crop plants, possibly providing new dimensions in the field of cereal crop genome editing.},
}
@article {pmid36685074,
year = {2023},
author = {Chen, K and Li, A and Wang, J and Li, D and Wang, X and Liu, C and Wang, Z},
title = {Arenobufagin causes ferroptosis in human gastric cancer cells by increasing rev-erbα expression.},
journal = {Journal of traditional and complementary medicine},
volume = {13},
number = {1},
pages = {72-80},
pmid = {36685074},
issn = {2225-4110},
abstract = {BACKGROUND AND AIMS: Gastric cancer is the fifth most diagnosed malignant tumor worldwide with limited effective chemotherapy. Ferroptosis is a new type of programmed cell death, which is becoming as a novel therapeutic target for tumors. Arenobufagin (ArBu) is a bufadienolide isolated from toad skin and venom, which exhibits broad-spectrum anti-tumor activity. It is unclear whether ArBu causes ferroptosis, thereby exhibiting anti-tumor activity in gastric cancer. We aimed to determine whether ArBu causes ferroptosis in cultured human gastric cancer cells.<br><br>EXPERIMENTAL PROCEDURE: Different human gastric cancer cells were treated with ArBu (5-20&#xa0;&#x3bc;M, 48&#xa0;h). Indicators of apoptosis and ferroptosis were measured. CRISPR/Cas-9 system was employed to delete Nr1d1 gene.<br><br>RESULTS: ArBu incubation reduced cell viability in a concentration-dependent manner. ArBu caused ferroptosis but not apoptosis at a lower concentration (10&#xa0;&#x3bc;M), despite it caused both of them at a higher concentration (20&#xa0;&#x3bc;M). Cotreatment with a selective ferroptosis inhibitor ferrostatin-1 protected against ArBu (10&#xa0;&#x3bc;M)-induced reduction in cell viability. ArBu-mediated ferroptosis was associated with abnormal expression of genes involved in iron uptake, lipid peroxidation, and antioxidants. Particularly, Nr1d1 gene expression was most significantly increased after ArBu treatment. Furthermore, activating Rev-erb&#x3b1; encoded by Nr1d1 by a selective agonist GSK4112 (1 and 2&#xa0;&#x3bc;M, 48&#xa0;h) caused ferroptosis. In contrast, Rev-erb&#x3b1; knockout using the CRISPR/Cas-9 system diminished ArBu-induced ferroptosis in cultured human gastric cancer cells.<br><br>CONCLUSION: ArBu causes ferroptosis by increasing Rev-erb&#x3b1; expression in human gastric cancer cells. This has implications of ArBu as a promising therapy for gastric cancer.<br><br>SECTION: 1. Natural Products.<br><br>Traditional medicine, pharmacology, gastric cancer, signal pathway.},
}
@article {pmid36683073,
year = {2023},
author = {Sharma, A and Gupta, AK and Devi, B},
title = {Current trends in management of bacterial pathogens infecting plants.},
journal = {Antonie van Leeuwenhoek},
volume = {},
number = {},
pages = {},
pmid = {36683073},
issn = {1572-9699},
abstract = {Plants are continuously challenged by different pathogenic microbes that reduce the quality and quantity of produce and therefore pose a serious threat to food security. Among them bacterial pathogens are known to cause disease outbreaks with devastating economic losses in temperate, tropical and subtropical regions throughout the world. Bacteria are structurally simple prokaryotic microorganisms and are diverse from a metabolic standpoint. Bacterial infection process mainly involves successful attachment or penetration by using extracellular enzymes, type secretion systems, toxins, growth regulators and by exploiting different molecules that modulate plant defence resulting in successful colonization. Theses bacterial pathogens are extremely difficult to control as they develop resistance to antibiotics. Therefore, attempts are made to search for innovative methods of disease management by the targeting bacterial virulence and manipulating the genes in host plants by exploiting genome editing methods. Here, we review the recent developments in bacterial disease management including the bioactive antimicrobial compounds, bacteriophage therapy, quorum-quenching mediated control, nanoparticles and CRISPR/Cas based genome editing techniques for bacterial disease management. Future research should focus on implementation of smart delivery systems and consumer acceptance of these innovative methods for sustainable disease management.},
}
@article {pmid36682874,
year = {2023},
author = {Martin, L and Rostami, S and Rajan, R},
title = {Optimized protocols for the characterization of Cas12a activities.},
journal = {Methods in enzymology},
volume = {679},
number = {},
pages = {97-129},
doi = {10.1016/bs.mie.2022.08.048},
pmid = {36682874},
issn = {1557-7988},
abstract = {The CRISPR-associated (Cas) Cas12a is the effector protein for type V-A CRISPR systems. Cas12a is a sequence-specific endonuclease that targets and cleaves DNA containing a cognate short signature motif, called the protospacer adjacent motif (PAM), flanked by a 20 nucleotide (nt) segment that is complementary to the "guide" region of its CRISPR RNA (crRNA). The guide sequence of the crRNA can be programmed to target any DNA with a cognate PAM and is the basis for Cas12a's current use for gene editing in numerous organisms and for medical diagnostics. While Cas9 (type II effector protein) is widely used for gene editing, Cas12a possesses favorable features such as its smaller size and creation of staggered double-stranded DNA ends after cleavage that enhances cellular recombination events. Collected here are protocols for the recombinant purification of Cas12a and the transcription of its corresponding programmable crRNA that are used in a variety of Cas12a-specific in vitro activity assays such as the cis, the trans and the guide-RNA independent DNA cleavage activities with multiple substrates. Correspondingly, protocols are included for the quantification of the activity assay data using ImageJ and the use of MATLAB for rate constant calculations. These procedures can be used for further structural and mechanistic studies of Cas12a orthologs and other Cas proteins.},
}
@article {pmid36658599,
year = {2023},
author = {Yu, D and Liang, B and Xu, H and Chen, L and Ye, Z and Wu, Z and Wang, X},
title = {CRISPR/Cas12a-based assay for the rapid and high-sensitivity detection of Streptococcus agalactiae colonization in pregnant women with premature rupture of membrane.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {22},
number = {1},
pages = {8},
pmid = {36658599},
issn = {1476-0711},
mesh = {Infant, Newborn ; Pregnancy ; Female ; Humans ; Pregnant Women ; Streptococcus agalactiae/genetics ; *Pregnancy Complications, Infectious/diagnosis ; CRISPR-Cas Systems ; *Streptococcal Infections/diagnosis ; Vagina ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Streptococcus agalactiae or group B Streptococcus (GBS) is a leading infectious cause of neonatal morbidity and mortality. It is essential to establish a robust method for the rapid and ultra-sensitive detection of GBS in pregnant women with premature rupture of membrane (PROM).<br><br>METHODS: This study developed a CRISPR-GBS assay that combined the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for GBS detection. The clinical performance of the CRISPR-GBS assay was assessed using vaginal or cervical swabs that were collected from 179 pregnant women with PROM, compared in parallel to culture-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (culture-MS) method and real-time quantitative polymerase chain reaction (qPCR) assay.<br><br>RESULTS: The CRISPR-GBS assay can be completed within 35&#xa0;min and the limit of detection was as low as 5 copies &#x3bc;L[-1]. Compared with the culture-MS, the CRISPR-GBS assay demonstrated a sensitivity of 96.64% (144/149, 95% confidence interval [CI] 92.39-98.56%) and a specificity of 100% (30/30, 95% CI&#x2009; 88.65-100%). It also had a high concordance rate of 98.88% with the qPCR assay.<br><br>CONCLUSIONS: The established CRISPR-GBS platform can detect GBS in a rapid, accurate, easy-to-operate, and cost-efficient manner. It offered a promising tool for the intrapartum screening of GBS colonization.},
}
@article {pmid36658146,
year = {2023},
author = {Li, X and Zhang, G and Huang, S and Liu, Y and Tang, J and Zhong, M and Wang, X and Sun, W and Yao, Y and Ji, Q and Wang, X and Liu, J and Zhu, S and Huang, X},
title = {Development of a versatile nuclease prime editor with upgraded precision.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {305},
pmid = {36658146},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing ; *DNA Breaks, Double-Stranded ; HEK293 Cells ; DNA End-Joining Repair ; CRISPR-Cas Systems ; },
abstract = {The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential.},
}
@article {pmid36656942,
year = {2023},
author = {Wang, JY and Doudna, JA},
title = {CRISPR technology: A decade of genome editing is only the beginning.},
journal = {Science (New York, N.Y.)},
volume = {379},
number = {6629},
pages = {eadd8643},
doi = {10.1126/science.add8643},
pmid = {36656942},
issn = {1095-9203},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Plant Breeding ; Plants/genetics ; Genome, Plant ; },
abstract = {The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing, coupled with advances in computing and imaging capabilities, has initiated a new era in which genetic diseases and individual disease susceptibilities are both predictable and actionable. Likewise, genes responsible for plant traits can be identified and altered quickly, transforming the pace of agricultural research and plant breeding. In this Review, we discuss the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants along with relevant successes and challenges and present a roadmap for the future of this technology.},
}
@article {pmid36543986,
year = {2023},
author = {Burgess, DJ},
title = {New cuts for CRISPR effectors.},
journal = {Nature reviews. Genetics},
volume = {24},
number = {2},
pages = {71},
pmid = {36543986},
issn = {1471-0064},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Gene Editing ; },
}
@article {pmid36516042,
year = {2023},
author = {Je, HW and Ji, CH and Kim, JY and Kang, HS},
title = {CaExTun: Mitigating Cas9-Related Toxicity in Streptomyces through Species-Specific Expression Tuning with Randomized Constitutive Promoters.},
journal = {ACS synthetic biology},
volume = {12},
number = {1},
pages = {61-70},
doi = {10.1021/acssynbio.2c00539},
pmid = {36516042},
issn = {2161-5063},
mesh = {*Streptomyces/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Gene Editing ; },
abstract = {The CRISPR/Cas9 system provides an efficient tool for engineering genomes. However, its application to Streptomyces genome engineering has been hampered by excessive toxicity associated with overexpression of Cas9 protein. As the level of Cas9 toxicity varies significantly between Streptomyces species, species-specific optimization of Cas9 expression is a strategy to mitigate its toxicity while maintaining sufficient double-strand break (DSB) activity for genome engineering. Using a pool of randomized constitutive promoters and a blue pigment indigoidine biosynthetic gene (IndC) as a reporter, we developed the CaExTun (Cas9 Expression Tuning) platform, which enables rapid screening of a large pool of promoter-Cas9 constructs to quickly recover the one with high DSB activity and no apparent toxicity. We demonstrate the utility of CaExTun using four model Streptomyces species. We also show that CaExTun can be applied to the CRISPRi system by allowing the construction of a library of promoter-dCas9 constructs that confer a wide range of gene repression levels. As demonstrated here, CaExTun is a versatile tool for the rapid optimization of the CRISPR/Cas9 system in a species-specific manner and thus will facilitate CRISPR/Cas9-based genome engineering efforts in Streptomyces.},
}
@article {pmid36357212,
year = {2023},
author = {Hummel, A},
title = {Editing a healthier future in plants.},
journal = {Trends in biotechnology},
volume = {41},
number = {2},
pages = {255-256},
doi = {10.1016/j.tibtech.2022.09.015},
pmid = {36357212},
issn = {1879-3096},
mesh = {*Plants/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Genome, Plant ; },
}
@article {pmid36682394,
year = {2023},
author = {Seo, PW and Gu, DH and Kim, JW and Kim, JH and Park, SY and Kim, JS},
title = {Structural characterization of the type IB CRISPR Cas7 from Thermobaculum terrenum.},
journal = {Biochimica et biophysica acta. Proteins and proteomics},
volume = {},
number = {},
pages = {140900},
doi = {10.1016/j.bbapap.2023.140900},
pmid = {36682394},
issn = {1878-1454},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex. In this study, we structurally characterized type IB Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems.},
}
@article {pmid36680641,
year = {2023},
author = {Campos-Madueno, EI and Moradi, M and Eddoubaji, Y and Shahi, F and Moradi, S and Bernasconi, OJ and Moser, AI and Endimiani, A},
title = {Intestinal colonization with multidrug-resistant Enterobacterales: screening, epidemiology, clinical impact, and strategies to decolonize carriers.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {36680641},
issn = {1435-4373},
abstract = {The clinical impact of infections due to extended-spectrum β-lactamase (ESBL)- and/or carbapenemase-producing Enterobacterales (Ent) has reached dramatic levels worldwide. Infections due to these multidrug-resistant (MDR) pathogens-especially Escherichia coli and Klebsiella pneumoniae-may originate from a prior asymptomatic intestinal colonization that could also favor transmission to other subjects. It is therefore desirable that gut carriers are rapidly identified to try preventing both the occurrence of serious endogenous infections and potential transmission. Together with the infection prevention and control countermeasures, any strategy capable of effectively eradicating the MDR-Ent from the intestinal tract would be desirable. In this narrative review, we present a summary of the different aspects linked to the intestinal colonization due to MDR-Ent. In particular, culture- and molecular-based screening techniques to identify carriers, data on prevalence and risk factors in different populations, clinical impact, length of colonization, and contribution to transmission in various settings will be overviewed. We will also discuss the standard strategies (selective digestive decontamination, fecal microbiota transplant) and those still in development (bacteriophages, probiotics, microcins, and CRISPR-Cas-based) that might be used to decolonize MDR-Ent carriers.},
}
@article {pmid36680242,
year = {2023},
author = {Rothemejer, FH and Lauritsen, NP and Juhl, AK and Schleimann, MH and König, S and Søgaard, OS and Bak, RO and Tolstrup, M},
title = {Development of HIV-Resistant CAR T Cells by CRISPR/Cas-Mediated CAR Integration into the CCR5 Locus.},
journal = {Viruses},
volume = {15},
number = {1},
pages = {},
doi = {10.3390/v15010202},
pmid = {36680242},
issn = {1999-4915},
abstract = {Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has been highly successful in treating B cell malignancies and holds great potential as a curative strategy for HIV infection. Recent advances in the use of anti-HIV broadly neutralizing antibodies (bNAbs) have provided vital information for optimal antigen targeting of CAR T cells. However, CD4+ CAR T cells are susceptible to HIV infection, limiting their therapeutic potential. In the current study, we engineered HIV-resistant CAR T cells using CRISPR/Cas9-mediated integration of a CAR cassette into the CCR5 locus. We used a single chain variable fragment (scFv) of the clinically potent bNAb 10-1074 as the antigen-targeting domain in our anti-HIV CAR T cells. Our anti-HIV CAR T cells showed specific lysis of HIV-infected cells in vitro. In a PBMC humanized mouse model of HIV infection, the anti-HIV CAR T cells expanded and transiently limited HIV infection. In conclusion, this study provides proof-of-concept for developing HIV-resistant CAR T cells using CRISPR/Cas9 targeted integration.},
}
@article {pmid36680093,
year = {2022},
author = {Sari-Ak, D and Alomari, O and Shomali, RA and Lim, J and Govinda Raj, DBT},
title = {Advances in CRISPR-Cas9 for the Baculovirus Vector System: A Systematic Review.},
journal = {Viruses},
volume = {15},
number = {1},
pages = {},
doi = {10.3390/v15010054},
pmid = {36680093},
issn = {1999-4915},
abstract = {The baculovirus expression vector systems (BEVS) have been widely used for the recombinant production of proteins in insect cells and with high insert capacity. However, baculovirus does not replicate in mammalian cells; thus, the BacMam system, a heterogenous expression system that can infect certain mammalian cells, was developed. Since then, the BacMam system has enabled transgene expression via mammalian-specific promoters in human cells, and later, the MultiBacMam system enabled multi-protein expression in mammalian cells. In this review, we will cover the continual development of the BEVS in combination with CRPISPR-Cas technologies to drive genome-editing in mammalian cells. Additionally, we highlight the use of CRISPR-Cas in glycoengineering to potentially produce a new class of glycoprotein medicines in insect cells. Moreover, we anticipate CRISPR-Cas9 to play a crucial role in the development of protein expression systems, gene therapy, and advancing genome engineering applications in the future.},
}
@article {pmid36680056,
year = {2022},
author = {Barchi, Y and Philippe, C and Chaïb, A and Oviedo-Hernandez, F and Claisse, O and Le Marrec, C},
title = {Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus.},
journal = {Viruses},
volume = {15},
number = {1},
pages = {},
doi = {10.3390/v15010015},
pmid = {36680056},
issn = {1999-4915},
abstract = {The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species.},
}
@article {pmid36679018,
year = {2023},
author = {Nascimento, FDS and Rocha, AJ and Soares, JMDS and Mascarenhas, MS and Ferreira, MDS and Morais Lino, LS and Ramos, APS and Diniz, LEC and Mendes, TAO and Ferreira, CF and Santos-Serejo, JAD and Amorim, EP},
title = {Gene Editing for Plant Resistance to Abiotic Factors: A Systematic Review.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {2},
pages = {},
doi = {10.3390/plants12020305},
pmid = {36679018},
issn = {2223-7747},
abstract = {Agricultural crops are exposed to various abiotic stresses, such as salinity, water deficits, temperature extremes, floods, radiation, and metal toxicity. To overcome these challenges, breeding programs seek to improve methods and techniques. Gene editing by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR/Cas-is a versatile tool for editing in all layers of the central dogma with focus on the development of cultivars of plants resistant or tolerant to multiple biotic or abiotic stresses. This systematic review (SR) brings new contributions to the study of the use of CRISPR/Cas in gene editing for tolerance to abiotic stress in plants. Articles deposited in different electronic databases, using a search string and predefined inclusion and exclusion criteria, were evaluated. This SR demonstrates that the CRISPR/Cas system has been applied to several plant species to promote tolerance to the main abiotic stresses. Among the most studied crops are rice and Arabidopsis thaliana, an important staple food for the population, and a model plant in genetics/biotechnology, respectively, and more recently tomato, whose number of studies has increased since 2021. Most studies were conducted in Asia, specifically in China. The Cas9 enzyme is used in most articles, and only Cas12a is used as an additional gene editing tool in plants. Ribonucleoproteins (RNPs) have emerged as a DNA-free strategy for genome editing without exogenous DNA. This SR also identifies several genes edited by CRISPR/Cas, and it also shows that plant responses to stress factors are mediated by many complex-signaling pathways. In addition, the quality of the articles included in this SR was validated by a risk of bias analysis. The information gathered in this SR helps to understand the current state of CRISPR/Cas in the editing of genes and noncoding sequences, which plays a key role in the regulation of various biological processes and the tolerance to multiple abiotic stresses, with potential for use in plant genetic improvement programs.},
}
@article {pmid36678404,
year = {2022},
author = {Tripathi, S and Khatri, P and Fatima, Z and Pandey, RP and Hameed, S},
title = {A Landscape of CRISPR/Cas Technique for Emerging Viral Disease Diagnostics and Therapeutics: Progress and Prospects.},
journal = {Pathogens (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/pathogens12010056},
pmid = {36678404},
issn = {2076-0817},
abstract = {Viral diseases have emerged as a serious threat to humanity and as a leading cause of morbidity worldwide. Many viral diagnostic methods and antiviral therapies have been developed over time, but we are still a long way from treating certain infections caused by viruses. Acquired immunodeficiency syndrome (AIDS) is one of the challenges where current medical science advancements fall short. As a result, new diagnostic and treatment options are desperately needed. The CRISPR/Cas9 system has recently been proposed as a potential therapeutic approach for viral disease treatment. CRISPR/Cas9 is a specialised, effective, and adaptive gene-editing technique that can be used to modify, delete, or correct specific DNA sequences. It has evolved into an advanced, configurable nuclease-based single or multiple gene-editing tool with a wide range of applications. It is widely preferred simply because its operational procedures are simple, inexpensive, and extremely efficient. Exploration of infectious virus genomes is required for a comprehensive study of infectious viruses. Herein, we have discussed the historical timeline-based advancement of CRISPR, CRISPR/Cas9 as a gene-editing technology, the structure of CRISPR, and CRISPR as a diagnostic tool for studying emerging viral infections. Additionally, utilizing CRISPR/Cas9 technology to fight viral infections in plants, CRISPR-based diagnostics of viruses, pros, and cons, and bioethical issues of CRISPR/Cas9-based genomic modification are discussed.},
}
@article {pmid36678392,
year = {2022},
author = {Morgan, T and Rezende, RR and Lima, TTM and Souza, FO and Alfenas-Zerbini, P},
title = {Genomic Analysis Unveils the Pervasiveness and Diversity of Prophages Infecting Erwinia Species.},
journal = {Pathogens (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/pathogens12010044},
pmid = {36678392},
issn = {2076-0817},
abstract = {Prophages are abundant elements integrated into bacterial genomes and contribute to inter-strain genetic variability and, in some cases, modulate the environmental behavior of bacteria, such as pathogen virulence. Here, we described prophage occurrence and diversity in publicly available Erwinia genome assemblies, a genus containing plant pathogens. Prophage-like sequences were identified and taxonomically classified. Sequence diversity was analyzed through intergenomic similarities. Furthermore, we searched for anti-phage defense systems in Erwinia spp., such as DISARM, BREX, and CRISPR-Cas systems, and identified the putative targets of CRISPR spacers. We identified 939 prophage-like sequences in 221 Erwinia spp. genome assemblies. Only 243 prophage-like sequences were classified, all belonging to the Caudoviricetes class. The set of putative Erwinia prophages was mostly unique since only three sequences showed more than 70% intergenomic similarities to known Erwinia phages. Overall, the number and type of CRISPR-Cas systems were conserved within Erwinia species, with many spacers directed to the putative prophages identified. This study increased the knowledge of the diversity and distribution of Erwinia prophages, contributing to the characterization of genetic and ecological factors influencing Erwinia spp. environmental fitness.},
}
@article {pmid36677927,
year = {2023},
author = {Zhang, P and Zhu, H},
title = {Anthocyanins in Plant Food: Current Status, Genetic Modification, and Future Perspectives.},
journal = {Molecules (Basel, Switzerland)},
volume = {28},
number = {2},
pages = {},
doi = {10.3390/molecules28020866},
pmid = {36677927},
issn = {1420-3049},
abstract = {Anthocyanins are naturally occurring polyphenolic pigments that give food varied colors. Because of their high antioxidant activities, the consumption of anthocyanins has been associated with the benefit of preventing various chronic diseases. However, due to natural evolution or human selection, anthocyanins are found only in certain species. Additionally, the insufficient levels of anthocyanins in the most common foods also limit the optimal benefits. To solve this problem, considerable work has been done on germplasm improvement of common species using novel gene editing or transgenic techniques. This review summarized the recent advances in the molecular mechanism of anthocyanin biosynthesis and focused on the progress in using the CRISPR/Cas gene editing or multigene overexpression methods to improve plant food anthocyanins content. In response to the concerns of genome modified food, the future trends in developing anthocyanin-enriched plant food by using novel transgene or marker-free genome modified technologies are discussed. We hope to provide new insights and ideas for better using natural products like anthocyanins to promote human health.},
}
@article {pmid36677496,
year = {2023},
author = {Xu, PX and Ren, HY and Li, R and Jin, XJ and Gao, ZC and Qin, T},
title = {Legionnaires' Disease in China Caused by Legionella pneumophila Corby.},
journal = {Microorganisms},
volume = {11},
number = {1},
pages = {},
doi = {10.3390/microorganisms11010204},
pmid = {36677496},
issn = {2076-2607},
abstract = {Legionella pneumophila is an intracellular pathogen causing pneumonia in humans. In February 2022, Legionnaires' disease caused by L. pneumophila strain Corby in a patient with lung adenocarcinoma was identified for the first time in China. This paper includes the case report and phenotypic and genomic analysis of the Corby (ICDC) strain. Its biological characteristics were evaluated by antibiotic sensitivity testing and cytology experiments, and genomic analysis was performed to understand its genetic evolution. The patient's clinical manifestations included cough, fever, pulmonary infiltration, and significantly decreased activity endurance. After empirical antimicrobial therapy, infection indicators decreased. The Corby (ICDC) strain was susceptible to nine antibiotics and exhibited strong intracellular proliferation ability. A phylogenetic tree showed that the Corby (ICDC) strain was closely related to the Corby strain, but under the pressure of a complex environment, its genome had undergone more rearrangement and inversion. The type IF CRISPR-Cas system was identified in its genome, and spacer analysis indicated that it had been invaded by several foreign plasmids, bacteria, and viruses during evolution. Legionnaires' disease caused by L. pneumophila strain Corby may be ignored in China, and it is urgent to improve long-term monitoring and investigation of aquatic environments and patients with respiratory infections to prevent a large-scale outbreak of Legionnaires' disease.},
}
@article {pmid36675077,
year = {2023},
author = {Hussein, M and Molina, MA and Berkhout, B and Herrera-Carrillo, E},
title = {A CRISPR-Cas Cure for HIV/AIDS.},
journal = {International journal of molecular sciences},
volume = {24},
number = {2},
pages = {},
doi = {10.3390/ijms24021563},
pmid = {36675077},
issn = {1422-0067},
abstract = {Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure.},
}
@article {pmid36672870,
year = {2023},
author = {Boti, MA and Athanasopoulou, K and Adamopoulos, PG and Sideris, DC and Scorilas, A},
title = {Recent Advances in Genome-Engineering Strategies.},
journal = {Genes},
volume = {14},
number = {1},
pages = {},
doi = {10.3390/genes14010129},
pmid = {36672870},
issn = {2073-4425},
abstract = {In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients' quality of life.},
}
@article {pmid36671408,
year = {2022},
author = {Brandt, CB and Fonager, SV and Haskó, J and Helmig, RB and Degn, S and Bolund, L and Jessen, N and Lin, L and Luo, Y},
title = {HIF1A Knockout by Biallelic and Selection-Free CRISPR Gene Editing in Human Primary Endothelial Cells with Ribonucleoprotein Complexes.},
journal = {Biomolecules},
volume = {13},
number = {1},
pages = {},
doi = {10.3390/biom13010023},
pmid = {36671408},
issn = {2218-273X},
abstract = {Primary endothelial cells (ECs), especially human umbilical vein endothelial cells (HUVECs), are broadly used in vascular biology. Gene editing of primary endothelial cells is known to be challenging, due to the low DNA transfection efficiency and the limited proliferation capacity of ECs. We report the establishment of a highly efficient and selection-free CRISPR gene editing approach for primary endothelial cells (HUVECs) with ribonucleoprotein (RNP) complex. We first optimized an efficient and cost-effective protocol for messenger RNA (mRNA) delivery into primary HUVECs by nucleofection. Nearly 100% transfection efficiency of HUVECs was achieved with EGFP mRNA. Using this optimized DNA-free approach, we tested RNP-mediated CRISPR gene editing of primary HUVECs with three different gRNAs targeting the HIF1A gene. We achieved highly efficient (98%) and biallelic HIF1A knockout in HUVECs without selection. The effects of HIF1A knockout on ECs' angiogenic characteristics and response to hypoxia were validated by functional assays. Our work provides a simple method for highly efficient gene editing of primary endothelial cells (HUVECs) in studies and manipulations of ECs functions.},
}
@article {pmid36671367,
year = {2023},
author = {Chukamnerd, A and Pomwised, R and Chusri, S and Singkhamanan, K and Chumtong, S and Jeenkeawpiam, K and Sakunrang, C and Saroeng, K and Saengsuwan, P and Wonglapsuwan, M and Surachat, K},
title = {Antimicrobial Susceptibility and Molecular Features of Colonizing Isolates of Pseudomonas aeruginosa and the Report of a Novel Sequence Type (ST) 3910 from Thailand.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/antibiotics12010165},
pmid = {36671367},
issn = {2079-6382},
abstract = {Pseudomonas aeruginosa is an important pathogen as it can cause hospital-acquired infections. Additionally, it can also colonize in patients and in other various environments. Hence, this study aimed to investigate the antimicrobial susceptibility, and to study the molecular features, of colonizing isolates of P. aeruginosa from Songklanagarind Hospital, Thailand. Genomic DNA extraction, whole-genome sequencing (WGS), and bioinformatics analysis were performed in all studied isolates. The findings demonstrated that the majority of isolates were non-susceptible to colistin and carbapenem. For in silico study, multilocus sequence typing (MLST) revealed one novel sequence type (ST) 3910 and multiple defined STs. The isolates carried several antimicrobial resistance genes (blaOXA-50, aph(3')-IIb, etc.) and virulence-associated genes (fleN, waaA, etc.). CRISPR-Cas sequences with different spacers and integrated bacteriophage sequences were also identified in these isolates. Very high SNPs were found in the alignments of the novel ST-3910 isolate with other isolates. A comparative genomic analysis exhibited phylogenetic clustering of our colonizing isolates with clinical isolates from many countries. Interestingly, ST-3981, ST-3982, ST-3983, ST-3984, ST-3985, ST-3986, ST-3986, ST-3986, ST-3987, and ST-3988, the new STs from published genomes, were assigned in this study. In conclusion, this WGS data might be useful for tracking the spread of P. aeruginosa colonizing isolates.},
}
@article {pmid36669890,
year = {2023},
author = {Diaz Quiroz, JF and Siskel, LD and Rosenthal, JJ},
title = {Site-directed A→I RNA editing as a therapeutic tool: moving beyond genetic mutations.},
journal = {RNA (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1261/rna.079518.122},
pmid = {36669890},
issn = {1469-9001},
abstract = {Adenosine deamination by the ADAR family of enzymes is a natural process that edits genetic information as it passes through messenger RNA. Adenosine is converted to inosine in mRNAs, and this base is interpreted as guanosine during translation. Realizing the potential of this activity for therapeutics, a number of researchers have developed systems that redirect ADAR activity to new targets, ones that aren't normally edited. These site-directed RNA editing (SDRE) systems can be broadly classified into two categories: ones that deliver an antisense RNA oligonucleotide to bind opposite a target adenosine, creating an editable structure that endogenously expressed ADARs recognize, and ones that tether the catalytic domain of recombinant ADAR to an antisense RNA oligonucleotide that serves as a targeting mechanism, much like with CRISPR-Cas or RNAi. To date, SDRE has been used mostly to try and correct genetic mutations. Here we argue that these applications are not ideal SDRE, mostly because RNA edits are transient and genetic mutations are not. Instead, we suggest that SDRE could be used to tune cell physiology to achieve temporary outcomes that are therapeutically advantageous, particularly in the nervous system. These include manipulating excitability in nociceptive neural circuits, abolishing specific phosphorylation events to reduce protein aggregation related to neurodegeneration or reduce the glial scarring that inhibits nerve regeneration, or enhancing G protein-coupled receptor signaling to increase nerve proliferation for the treatment of sensory disorders like blindness and deafness.},
}
@article {pmid36669116,
year = {2023},
author = {Pons, BJ and Dimitriu, T and Westra, ER and van Houte, S},
title = {Antibiotics that affect translation can antagonize phage infectivity by interfering with the deployment of counter-defenses.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {4},
pages = {e2216084120},
doi = {10.1073/pnas.2216084120},
pmid = {36669116},
issn = {1091-6490},
abstract = {It is becoming increasingly clear that antibiotics can both positively and negatively impact the infectivity of bacteriophages (phage), but the underlying mechanisms often remain unclear. Here we demonstrate that antibiotics that target the protein translation machinery can fundamentally alter the outcome of bacteria-phage interactions by interfering with the production of phage-encoded counter-defense proteins. Specifically, using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model, we show that bacteria with Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR associated (CRISPR-Cas) immune systems have elevated levels of immunity against phage that encode anti-CRISPR (acr) genes when translation inhibitors are present in the environment. CRISPR-Cas are highly prevalent defense systems that enable bacteria to detect and destroy phage genomes in a sequence-specific manner. In response, many phages encode acr genes that are expressed immediately following the infection to inhibit key steps of the CRISPR-Cas immune response. Our data show that while phage-carrying acr genes can amplify efficiently on bacteria with CRISPR-Cas immune systems in the absence of antibiotics, the presence of antibiotics that act on protein translation prevents phage amplification, while protecting bacteria from lysis.},
}
@article {pmid36661529,
year = {2023},
author = {Antropov, DN and Stepanov, GA},
title = {Molecular Mechanisms Underlying CRISPR/Cas-Based Assays for Nucleic Acid Detection.},
journal = {Current issues in molecular biology},
volume = {45},
number = {1},
pages = {649-662},
doi = {10.3390/cimb45010043},
pmid = {36661529},
issn = {1467-3045},
abstract = {Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.},
}
@article {pmid36660867,
year = {2023},
author = {Zheng, X and Zhang, S and Liang, Y and Zhang, R and Liu, L and Qin, P and Zhang, Z and Wang, Y and Zhou, J and Tang, X and Zhang, Y},
title = {Loss-function mutants of OsCKX gene family based on CRISPR-Cas systems revealed their diversified roles in rice.},
journal = {The plant genome},
volume = {},
number = {},
pages = {e20283},
doi = {10.1002/tpg2.20283},
pmid = {36660867},
issn = {1940-3372},
abstract = {Cytokinin (CTK) is an important plant hormone that promotes cell division, controls cell differentiation, and regulates a variety of plant growth and development processes. Cytokinin oxidase/dehydrogenase (CKX) is an irreversible cytokinin-degrading enzyme that affects plant growth and development by regulating the dynamic balance of CTKs synthesis and degradation. There are presumed 11 members of the CKX gene family in rice (Oryza sativa L.), but limited members have been reported. In this study, based on CRISPR-Cas9 and CRISPR-Cas12a genome-editing technology, we established a complete set of OsCKX1-OsCKX11 single-gene mutants, as well as double-gene and triple-gene mutants of different OsCKXs gene combinations with high similarity. The results revealed that CRISPR-Cas12a outperformed Cas9 to generate biallelic mutations, multi-gene mutants, and more diverse genotypes. And then, we found, except the reported OsCKX2, OsCKX4, OsCKX9 and OsCKX11, OsCKX5, OsCKX6, OsCKX7, and OsCKX8 also had significant effects on agronomic traits such as plant height, panicle size, grain size, and grain number per panicle in rice. In addition, the different loss-of-function of the OsCKX genes also changed the seed appearance quality and starch composition. Interestingly, by comparing different combinations of multi-gene mutants, we found significant functional redundancy among OsCKX gene members in the same phylogenetic clade. These data collectively reveal the diversified regulating capabilities of OsCKX genes in rice, and also provide the valuable reference for further rice molecular breeding.},
}
@article {pmid36657729,
year = {2023},
author = {Meyer, MB and Pike, JW},
title = {Genomic Mechanisms Controlling Renal Vitamin D Metabolism.},
journal = {The Journal of steroid biochemistry and molecular biology},
volume = {},
number = {},
pages = {106252},
doi = {10.1016/j.jsbmb.2023.106252},
pmid = {36657729},
issn = {1879-1220},
abstract = {Vitamin D metabolism centers on regulation in the kidney of CYP27B1 induction by PTH, suppression by FGF23 and 1,25(OH)2D3, and reciprocal CYP24A1 suppression by PTH, and induction by FGF23 and 1,25(OH)2D3. This coordinated genomic regulation through enhancer modules results in the production and dynamic maintenance of circulating endocrine 1,25(OH)2D3 which, together with PTH and FGF23, controls mineral homeostasis. We discovered enhancers near Cyp27b1 in the mouse kidney located within intronic regions of Mettl1 and Mettl21b genes. These kidney-specific enhancers ("M1", "M21") control Cyp27b1. Through CRISPR/Cas deletion, we found that PTH activation of Cyp27b1 is lost with deletion of M1, whereas FGF23 suppression is lost with deletion of M21. The combination of both deletions (M1/M21-DIKO) eliminated the suppression by 1,25(OH)2D3. Cyp24a1 activation by 1,25(OH)2D3 is controlled by a promoter proximal pair of VDREs as well as a distal region -35 to -37 kb (DS2). We also found that FGF23 activation and PTH suppression of Cyp24a1 was located in a region -21 to -37 kb downstream (DS1). More recently, using in vivo ChIP-seq in mouse kidney, we demonstrate that PTH activation rapidly induces increased recruitment of pCREB and its coactivators, CBP and CRTC2, to the M1 and M21 enhancers near the Cyp27b1 gene. At distal enhancers of the Cyp24a1 gene, PTH suppression promotes dismisses CBP with only minor changes in pCREB and CRTC2 occupancy, all of which correlate with a suppression of basal histone acetylation across this locus and reduced transcripts. Surprisingly, we find that 1,25(OH)2D3 suppression increases the occupancy of CRTC2 in the M1 enhancer, a novel observation for CRTC2 and/or 1,25(OH)2D3 action. The suppressive actions of 1,25(OH)2D3 and FGF23 at the Cyp27b1 gene are associated with a reduction in CBP recruitment at these enhancers. Although FGF23-regulated transcription factors remain unknown, we hypothesize that VDR occupancy induced at the M1 and M21 enhancers by 1,25(OH)2D3 likely disrupts or competes with the active conformation of these CREB modules thereby preventing full induction by PTH. Our findings show coactivators such as CRTC2 and CBP contribute to Cyp27b1 and Cyp24a1 transcription and provide molecular insight into the coordinated mechanistic actions of PTH, FGF23, and 1,25(OH)2D3 in the kidney that regulate mineral homeostasis.},
}
@article {pmid36652148,
year = {2023},
author = {Zhou, X and Fu, Q and Yang, T and Sun, M},
title = {CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {1},
pages = {42},
pmid = {36652148},
issn = {1438-7948},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Mice, Inbred C57BL ; Mice, Transgenic ; *Gene Editing/methods ; Insulin/genetics ; },
abstract = {Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic β cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.},
}
@article {pmid36599980,
year = {2023},
author = {Bravo, JPK and Hallmark, T and Naegle, B and Beisel, CL and Jackson, RN and Taylor, DW},
title = {RNA targeting unleashes indiscriminate nuclease activity of CRISPR-Cas12a2.},
journal = {Nature},
volume = {613},
number = {7944},
pages = {582-587},
pmid = {36599980},
issn = {1476-4687},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; RNA/genetics ; *CRISPR-Associated Proteins/metabolism ; DNA/genetics/metabolism ; DNA, Single-Stranded ; RNA, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection[1]. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.},
}
@article {pmid36563996,
year = {2023},
author = {Carroll, MS and Giacca, M},
title = {CRISPR activation and interference as investigative tools in the cardiovascular system.},
journal = {The international journal of biochemistry &amp; cell biology},
volume = {155},
number = {},
pages = {106348},
doi = {10.1016/j.biocel.2022.106348},
pmid = {36563996},
issn = {1878-5875},
mesh = {*CRISPR-Cas Systems/genetics ; Transcriptional Activation ; RNA, Guide, Kinetoplastida ; Transcription Factors/genetics ; *Cardiovascular System ; },
abstract = {CRISPR activation and interference (CRISPRa/i) technology offers the unprecedented possibility of achieving regulated gene expression both in vitro and in vivo. The DNA pairing specificity of a nuclease dead Cas9 (dCas9) is exploited to precisely target a transcriptional activator or repressor in proximity to a gene promoter. This permits both the study of phenotypes arising from gene modulation for investigative purposes, and the development of potential therapeutics. As with virtually all other organ systems, the cardiovascular system can deeply benefit from a broader utilisation of CRISPRa/i. However, application of this technology is still in its infancy. Significant areas for improvement include the identification of novel and more effective transcriptional regulators that can be docked to dCas9, and the development of more efficient methods for their delivery and expression in vivo.},
}
@article {pmid36448318,
year = {2023},
author = {Miyata, M and Yoshida, J and Takagishi, I and Horie, K},
title = {Comparison of CRISPR-Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells.},
journal = {DNA research : an international journal for rapid publication of reports on genes and genomes},
volume = {30},
number = {1},
pages = {},
doi = {10.1093/dnares/dsac045},
pmid = {36448318},
issn = {1756-1663},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Mouse Embryonic Stem Cells ; Gene Editing/methods ; Genome ; Recombinational DNA Repair ; DNA End-Joining Repair ; },
abstract = {The genome contains large functional units ranging in size from hundreds of kilobases to megabases, such as gene clusters and topologically associating domains. To analyse these large functional units, the technique of deleting the entire functional unit is effective. However, deletion of such large regions is less efficient than conventional genome editing, especially in cultured cells, and a method that can ensure success is anticipated. Here, we compared methods to delete the 2.5-Mb Krüppel-associated box zinc finger protein (KRAB-ZFP) gene cluster in mouse embryonic stem cells using CRISPR-Cas9. Three methods were used: first, deletion by non-homologous end joining (NHEJ); second, homology-directed repair (HDR) using a single-stranded oligodeoxynucleotide (ssODN); and third, HDR employing targeting vectors with a selectable marker and 1-kb homology arms. NHEJ-mediated deletion was achieved in 9% of the transfected cells. Inversion was also detected at similar efficiency. The deletion frequency of NHEJ and HDR was found to be comparable when the ssODN was transfected. Deletion frequency was highest when targeting vectors were introduced, with deletions occurring in 31-63% of the drug-resistant clones. Biallelic deletion was observed when targeting vectors were used. This study will serve as a benchmark for the introduction of large deletions into the genome.},
}
@article {pmid36076084,
year = {2023},
author = {Huang, TP and Heins, ZJ and Miller, SM and Wong, BG and Balivada, PA and Wang, T and Khalil, AS and Liu, DR},
title = {High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.},
journal = {Nature biotechnology},
volume = {41},
number = {1},
pages = {96-107},
pmid = {36076084},
issn = {1546-1696},
support = {R01 EB031172/EB/NIBIB NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Human/genetics ; Pyrimidines ; },
abstract = {Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci-especially those with pyrimidine-rich PAM sequences-remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to N4TN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at N4CN PAM sequences.},
}
@article {pmid35962198,
year = {2023},
author = {},
title = {Engineered Cas13 variants with minimal collateral RNA targeting.},
journal = {Nature biotechnology},
volume = {41},
number = {1},
pages = {29-30},
pmid = {35962198},
issn = {1546-1696},
mesh = {*RNA/genetics ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism ; },
}
@article {pmid35953673,
year = {2023},
author = {Tong, H and Huang, J and Xiao, Q and He, B and Dong, X and Liu, Y and Yang, X and Han, D and Wang, Z and Wang, X and Ying, W and Zhang, R and Wei, Y and Xu, C and Zhou, Y and Li, Y and Cai, M and Wang, Q and Xue, M and Li, G and Fang, K and Zhang, H and Yang, H},
title = {High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.},
journal = {Nature biotechnology},
volume = {41},
number = {1},
pages = {108-119},
pmid = {35953673},
issn = {1546-1696},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *RNA/genetics ; RNA Stability/genetics ; Mice, Transgenic ; Transcriptome ; Mammals/genetics ; },
abstract = {CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.},
}
@article {pmid36659571,
year = {2019},
author = {Liu, Y and Li, J and Zhou, C and Meng, B and Wei, Y and Yang, G and Lu, Z and Shen, Q and Zhang, Y and Yang, H and Qiao, Y},
title = {Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9).},
journal = {Science bulletin},
volume = {64},
number = {21},
pages = {1592-1600},
doi = {10.1016/j.scib.2019.08.023},
pmid = {36659571},
issn = {2095-9281},
abstract = {Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions (DMRs), and aberrant genomic imprinted DNA methylation is associated with some human diseases, including Prader-Willi syndrome and cancer. Thus, the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases. To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system, which is an efficient gene-targeting technique in various organisms, we examined the targeting efficiency of Staphylococcus aureus Cas9 (SaCas9) and Streptococcus pyogenes Cas9 (SpCas9) in response to DNA methylation interference. We found that the targeting efficiency of SaCas9, but not SpCas9, was enhanced by targeted DNA demethylation using the dCas9-Tet1 catalytic domain (CD) but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-dCas9. An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine (5mC) in a synthesized CpG-containing context. Further analysis with ChIP-Q-PCR demonstrated that the non-methylated sequence targeting of SaCas9 depends on the binding preference of SaCas9 to non-methylated sequences. Taking advantage of this feature of SaCas9, we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes, H19 and Snrpn, with relatively high efficiencies of 28.6% and 47.4%, respectively. These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation. By using SaCas9, we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.},
}
@article {pmid36658818,
year = {2018},
author = {Zhao, Y and Wang, J and Sun, Q and Dou, C and Gu, Y and Nie, C and Zhu, X and Wei, Y and Cheng, W},
title = {Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6.},
journal = {Science bulletin},
volume = {63},
number = {11},
pages = {691-699},
doi = {10.1016/j.scib.2018.03.017},
pmid = {36658818},
issn = {2095-9281},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) system is an adaptive immune system in bacteria and archaea that resists exogenous invasion through nucleic acid-mediated cleavage. In the type III-A system, the Csm complex contains five effectors and a CRISPR RNA, which edits both single stranded RNA and double stranded DNA. It has recently been demonstrated that cyclic oligoadenylates (cOAs), which are synthesized by the Csm complex, act as second messengers that bind and activate Csm6. Here, we report the crystal structures of Staphylococcus epidermidis Csm3 (SeCsm3) and an N-terminally truncated Csm6 (SeCsm6ΔN) at 2.26 and 2.0 Å, respectively. The structure of SeCsm3 highly resembled previously reported Csm3 structures from other species; however, it provided novel observations allowing further enzyme characterization. The homodimeric SeCsm6ΔN folds into a compact structure. The dimerization of the HEPN domain leads to the formation of the ribonuclease active site, which is consistent with the reported Csm6 structures. Altogether, our studies provide a structural view of the ribonuclease activity mediated by Csm3 and Csm6 of the type III-A CRISPR-Cas system.},
}
@article {pmid36658815,
year = {2018},
author = {Gu, F},
title = {Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6: a step for the development of novel genome editing tool.},
journal = {Science bulletin},
volume = {63},
number = {11},
pages = {672-674},
doi = {10.1016/j.scib.2018.05.012},
pmid = {36658815},
issn = {2095-9281},
}
@article {pmid36657010,
year = {2023},
author = {Yin, L and Li, Y and Zhang, W and Han, X and Wu, Q and Xie, Y and Fan, J and Ma, L},
title = {Detection Methods for Foodborne Viruses: Current State-of-Art and Future Perspectives.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.2c06537},
pmid = {36657010},
issn = {1520-5118},
abstract = {Foodborne viruses have been recognized as important threats to food safety and human health. Rapid and accurate detection is one of the most crucial measures for food safety control. With the development of biology, chemistry, nanoscience, and related interdisciplines, detection strategies have been devised and advanced continuously. This review mainly focuses on the progress of detection methods for foodborne viruses. The current detection methods for foodborne viruses are summarized, including traditional electron microscopy and cultural isolation, immunoassay, molecular technology, biosensors, and newly emerging CRISPR/Cas-based detection technology. Furthermore, a comparison of the detection methods was objectively discussed. This review provides a comprehensive account of foodborne virus detection methods from fundamentals to state-of-the-art and illustrates the advantages and disadvantages of the current methods and proposes the future trends and directions for foodborne virus detection. It is hoped that this review can update current knowledge and present blueprints in order to accelerate futuristic development.},
}
@article {pmid36656806,
year = {2023},
author = {Yaméogo, P and Majeau, N and Mbakam, CH and Tremblay, JP},
title = {Small-molecule inhibitors of proteasome increase CjCas9 protein stability.},
journal = {PloS one},
volume = {18},
number = {1},
pages = {e0280353},
doi = {10.1371/journal.pone.0280353},
pmid = {36656806},
issn = {1932-6203},
abstract = {The small size of CjCas9 can make easier its vectorization for in vivo gene therapy. However, compared to the SpCas9, the CjCas9 is, in general, less efficient to generate indels in target genes. The factors that affect its efficacity are not yet determined. We observed that the CjCas9 protein expressed in HEK293T cells after transfection of this transgene under a CMV promoter was much lower than the SpCas9 protein in the same conditions. We thus evaluated the effect of proteasome inhibitors on CjCas9 protein stability and its efficiency on FXN gene editing. Western blotting showed that the addition of MG132 or bortezomib, significantly increased CjCas9 protein levels in HEK293T and HeLa cells. Moreover, bortezomib increased the level of CjCas9 protein expressed under promoters weaker than CMV such as CBH or EFS but which are specific for certain tissues. Finally, ddPCR quantification showed that bortezomib treatment enhanced CjCas9 efficiency to delete GAA repeat region of FXN gene in HEK293T cells. The improvement of CjCas9 protein stability would facilitate its used in CRISPR/Cas system.},
}
@article {pmid36655998,
year = {2023},
author = {Zhang, YM and Zheng, L and Xie, K},
title = {CRISPR/dCas9-Mediated Gene Silencing in Two Plant Fungal Pathogens.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0059422},
doi = {10.1128/msphere.00594-22},
pmid = {36655998},
issn = {2379-5042},
abstract = {Magnaporthe oryzae and Ustilaginoidea virens are two filamentous fungal pathogens that threaten rice production worldwide. Genetic tools that permit fast gene deletion and silencing are of great interest for functional genomics of fungal pathogens. As a revolutionary genome editing tool, clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) enable many innovative applications. Here, we developed a CRISPR interference (CRISPRi) toolkit using nuclease activity dead Cas9 (dCas9) to silence genes of interest in M. oryzae and U. virens. We optimized the components of CRISPRi vectors, including transcriptional repression domains, dCas9 promoters, and guide RNA (gRNA) promoters. The CRISPRi tool was tested using nine gRNAs to target the promoters of MoATG3, MoATG7, and UvPal1. The results indicated that a single gRNA could direct the dCas9-fused transcriptional repression domain to efficiently silence the target gene in M. oryzae and U. virens. In both fungi, the target genes were repressed >100-fold, and desired phenotypes were observed in CRISPRi strains. Importantly, we showed that multiple genes could be easily silenced using polycistronic tRNA-gRNA in CRISPRi. Furthermore, gRNAs that bind different promoter regions displayed variable repression levels of target genes, highlighting the importance of gRNA design for CRISPRi efficiency. Together, this study provides an efficient and robust CRISPRi tool for targeted gene silencing in M. oryzae and U. virens. Owing to its simplicity and multiplexity, CRISPRi will be a useful tool for gene function discovery in fungal pathogens. IMPORTANCE Many devastating plant diseases are caused by fungal pathogens that evolve rapidly to adapt to host resistance and environmental changes. Therefore, genetic tools that enable fast gene function discovery are needed to study the pathogenicity and stress adaptation of fungal pathogens. In this study, we adopted the CRISPR/Cas9 system to silence genes in Magnaporthe oryzae and Ustilaginoidea virens, which are two dominant fungal pathogens that threaten rice production worldwide. We present a versatile and robust CRISPRi toolkit that represses target gene expression >100-fold using a single gRNA. We also demonstrated that CRISPRi could simultaneously silence multiple genes using the tRNA-gRNA strategy. The CRISPRi technologies described in this study would accelerate the functional genomics of fungal pathogens.},
}
@article {pmid36655267,
year = {2023},
author = {Tian, BS and Wu, YJ and Cui, XX and Lyu, JW and Chen, MH and Zhu, C and Gu, B},
title = {[Research progress of CRISPR/Cas biosensors based on different signal amplification strategies].},
journal = {Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]},
volume = {57},
number = {1},
pages = {112-119},
doi = {10.3760/cma.j.cn112150-20220220-00158},
pmid = {36655267},
issn = {0253-9624},
abstract = {CRISPR/Cas(the clustered regularly interspaced short palindromic repeats-CRISPR associated)system exists in most bacteria and all archaea. It is an important strategy for bacteria and archaea to resist foreign nucleic acid invasion and use for self-defense. The CRISPR/Cas system is a simple, fast, and specific diagnostic tool, which is widely used in agriculture, industry, animal husbandry, and medicine. This article mainly introduces and discusses recently advantages and limitations of biosensors combining CRISPR/Cas system with fluorescence, visualization and surface enhanced raman related technologies, as well as future research directions.},
}
@article {pmid36654480,
year = {2023},
author = {Liu, X and Cai, L and Zhu, L and Tian, Z and Shen, Z and Cheng, J and Zhang, S and Li, Z and Liu, X},
title = {Mutation of the clock gene timeless disturbs diapause induction and adult emergence rhythm in Helicoverpa armigera.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.7363},
pmid = {36654480},
issn = {1526-4998},
abstract = {BACKGROUND: Circadian rhythms are physical and behavioral changes that follow the 24-h cycle of Earth's light and temperature and are regulated by clock genes. Timeless (Tim) has been identified as a canonical clock gene in some insects, however, its functions have been little studied in lepidopteran pests.<br><br>RESULTS: To investigate Tim (HaTim) gene function in Helicoverpa armigera, an important lepidopteran pest, we obtained the HaTim mutant using the CRISPR/Cas 9 gene editing system. Our results showed that the transcript levels of HaTim rhythmically peaked at night in heads of the wild larvae and adult, and the diel expression of HaTim was sensitive to photoperiod and temperature. The expression rhythms of other clock genes, such as HaPer, HaCry1, HaCry2 and HaCwo, were disturbed in the HaTim mutant larvae, as that stage is a sensitivity period for diapause induction. Fifth-instar wild-type larvae could be induced to pupate in diapause under a short-day photoperiod and low temperature, however, fifth-instar HaTim mutant larvae could not be induced under the same conditions. In addition, the emergence of wild-type adults peaked early at night, but the rhythm was disturbed in the HaTim mutant with arrhythmic expression of some clock genes, such as HaPer, HaCry1 and HaCwo in adults.<br><br>CONCLUSION: Our results suggest that the clock gene Tim is involved in diapause induction and adult emergence in H. armigera, and is a potential target gene for controlling pest.},
}
@article {pmid36654470,
year = {2023},
author = {Li, J and Liang, X and Wang, F and Wang, J and Ding, F},
title = {The Current Status of Antisense Gene Therapies for Bacteria-caused Diseases-Challenges and Opportunities.},
journal = {Current pharmaceutical design},
volume = {},
number = {},
pages = {},
doi = {10.2174/1381612829666230118152428},
pmid = {36654470},
issn = {1873-4286},
abstract = {Bacteria-caused diseases continue to pose a serious threat to human health. The current situation of overused antibiotics against those diseases further spurs and exacerbates the ever-increasing drug resistance problems, which really leaves us very few options to combat those nasty bugs. Gene therapies based on the antisense oligonucleotide, though developed more than 40 years ago, did not reform the current treatments as originally expected. Along with the advances of new delivery technologies, this old field thrives again. In addition, newly evolving gene-editing tools based on the CRISPR-Cas system shed new light on this old field, bringing a breeze of hope to gene therapies for bacteria-caused diseases. As a fast-growing field, we strive to summarize in this review the recent progress in using gene therapies in those areas, analyze the potential challenges or problems from using antisense or gene-editing tools for targeting bacterial diseases and seek to explore any potential solutions to the current dilemmas. As a short review, we will focus our discussion mainly on antisense oligonucleotide-based gene therapies while briefly touching on the CRISPR-Cas based ones as the latter is just beginning to get more attention for application in the prokaryotic kingdom.},
}
@article {pmid36652483,
year = {2023},
author = {Li, Z and Zhong, Z and Wu, Z and Pausch, P and Al-Shayeb, B and Amerasekera, J and Doudna, JA and Jacobsen, SE},
title = {Genome editing in plants using the compact editor CasΦ.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {4},
pages = {e2216822120},
doi = {10.1073/pnas.2216822120},
pmid = {36652483},
issn = {1091-6490},
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.},
}
@article {pmid36650600,
year = {2023},
author = {Zhan, Y and Cao, C and Li, A and Mei, H and Liu, Y},
title = {Enhanced RNA knockdown efficiency with engineered fusion guide RNAs that function with both CRISPR-CasRx and hammerhead ribozyme.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {9},
pmid = {36650600},
issn = {1474-760X},
mesh = {Humans ; *RNA ; *RNA, Catalytic/genetics/metabolism ; RNA, Messenger/metabolism ; RNA Interference ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: CRISPR-Cas13 is a newly emerging RNA knockdown technology that is comparable to RNAi. Among all members of Cas13, CasRx degrades RNA in human cells with high precision and effectiveness. However, it remains unclear whether the efficiency of this technology can be further improved and applied to gene therapy.<br><br>RESULTS: In this study, we fuse CasRx crRNA with an antisense ribozyme to construct a synthetic fusion guide RNA that can interact with both CasRx protein and ribozyme and tested the ability of this approach in RNA knockdown and cancer gene therapy. We show that the CasRx-crRNA-ribozyme system (CCRS) is more efficient for RNA knockdown of mRNAs and non-coding RNAs than conventional methods, including CasRx, shRNA, and ribozyme. In particular, CCRS is more effective than wild-type CasRx when targeting multiple transcripts simultaneously. We next use bladder cancer as a model to evaluate the anticancer effects of CCRS targeting multiple genes in vitro and in vivo. CCRS shows a higher anticancer effect than conventional methods, consistent with the gene knockdown results.<br><br>CONCLUSIONS: Thus, our study demonstrates that CCRS expands the design ideas and RNA knockdown capabilities of Cas13 technology and has the potential to be used in disease treatment.},
}
@article {pmid36650523,
year = {2023},
author = {Jin, Q and Liu, X and Zhuang, Z and Huang, J and Gou, S and Shi, H and Zhao, Y and Ouyang, Z and Liu, Z and Li, L and Mao, J and Ge, W and Chen, F and Yu, M and Guan, Y and Ye, Y and Tang, C and Huang, R and Wang, K and Lai, L},
title = {Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {8},
pmid = {36650523},
issn = {1474-760X},
mesh = {Humans ; Animals ; Swine ; *CRISPR-Cas Systems ; *Doxycycline/pharmacology ; RNA, Guide, Kinetoplastida/genetics ; Mutation ; Genome ; Gene Editing/methods ; },
abstract = {BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals.<br><br>RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases.<br><br>CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.},
}
@article {pmid36650384,
year = {2023},
author = {Rodrigo, S and Senasinghe, K and Quazi, S},
title = {Molecular and therapeutic effect of CRISPR in treating cancer.},
journal = {Medical oncology (Northwood, London, England)},
volume = {40},
number = {2},
pages = {81},
pmid = {36650384},
issn = {1559-131X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Immunotherapy, Adoptive/methods ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer has become one of the common causes of mortality around the globe due to mutations in the genome which allows rapid growth of cells uncontrollably without repairing DNA errors. Cancers could arise due alterations in DNA repair mechanisms (errors in mismatch repair genes), activation of oncogenes and inactivation of tumor suppressor genes. Each cancer type is different and each individual has a unique genetic change which leads them to cancer. Studying genetic and epigenetic alterations in the genome leads to understanding the underlying features. CAR T therapy over other immunotherapies such as monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines and adoptive cell therapies has been widely used to treat cancer in recent days and gene editing has now become one of the promising treatments for many genetic diseases. This tool allows scientists to change the genome by adding, removing or altering genetic material of an organism. Due to advance in genetics and novel molecular techniques such as CRISPR, TALEN these genes can be edited in such a way that their original function could be replaced which in turn improved the treatment possibilities and can be used against malignancies and even cure cancer in future along with CAR T cell therapy due to the specific recognition and attacking of tumor.},
}
@article {pmid36650182,
year = {2023},
author = {Chen, R and Cao, Y and Liu, Y and Zhao, D and Li, J and Cheng, Z and Bi, C and Zhang, X},
title = {Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {257},
pmid = {36650182},
issn = {2041-1723},
mesh = {*Transcription Factor RelA ; *Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Prime editing is a versatile gene editing tool that enables precise sequence changes of all types in the genome, but its application is rather limited by the editing efficiency. Here, we first apply the Suntag system to recruit the transcription factor P65 and enhance the desired editing outcomes in the prime editing system. Next, MS2 hairpins are used to recruit MS2-fused P65 and confirmed that the recruitment of the P65 protein could effectively improve the prime editing efficiency in both the PE3 and PE5 systems. Moreover, this suggests the increased editing efficiency is most likely associated with the induction of chromatin accessibility change by P65. In conclusion, we apply different systems to recruit P65 and enhance the prime editing efficiency of various PE systems. Furthermore, our work provides a variety of methods to work as protein scaffolds for screening target factors and thus supports further optimization of prime editing systems.},
}
@article {pmid36541937,
year = {2023},
author = {Long, W and Yang, J and Zhao, Q and Pan, Y and Luan, X and He, B and Han, X and Wang, Y and Song, Y},
title = {Metal-Organic Framework-DNA Bio-Barcodes Amplified CRISPR/Cas12a Assay for Ultrasensitive Detection of Protein Biomarkers.},
journal = {Analytical chemistry},
volume = {95},
number = {2},
pages = {1618-1626},
doi = {10.1021/acs.analchem.2c04737},
pmid = {36541937},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; *Metal-Organic Frameworks ; Biomarkers, Tumor/genetics ; DNA ; Antibodies ; *Biosensing Techniques ; },
abstract = {CRISPR/Cas12a shows excellent potential in disease diagnostics. However, insensitive signal conversion strategies hindered its application in detecting protein biomarkers. Here, we report a metal-organic framework (MOF)-based DNA bio-barcode integrated with the CRISPR/Cas12a system for ultrasensitive detection of protein biomarkers. In this work, zirconium-based MOF nanoparticles were comodified with antibodies and bio-barcode phosphorylated DNA as an efficient signal converter, which not only recognized the protein biomarker to form the sandwich complex but also released the bio-barcode DNA activators after MOF dissociation to activate the trans-cleavage activity of Cas12a. Due to the obvious advantages, including numerous loaded oligonucleotides, a convenient release process, and the nontoxic release reagent, this MOF-DNA bio-barcode strategy could amplify the CRISPR/Cas12a system to achieve simple and highly sensitive detection of tumor protein biomarkers with detection limits of 0.03 pg/mL (PSA) and 0.1 pg/mL (CEA), respectively. Furthermore, this platform could detect PSA directly in clinical serum samples, offering a powerful tool for early disease diagnosis.},
}
@article {pmid36654316,
year = {2021},
author = {Zhao, X and Li, S and Liu, G and Wang, Z and Yang, Z and Zhang, Q and Liang, M and Liu, J and Li, Z and Tong, Y and Zhu, G and Wang, X and Jiang, L and Wang, W and Tan, GY and Zhang, L},
title = {A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes.},
journal = {Science bulletin},
volume = {66},
number = {1},
pages = {69-77},
doi = {10.1016/j.scib.2020.09.004},
pmid = {36654316},
issn = {2095-9281},
abstract = {Rapid and sensitive detection of various analytes is in high demand. Apart from its application in genome editing, CRISPR-Cas also shows promises in nucleic acid detection applications. To further exploit the potential of CRISPR-Cas for detection of diverse analytes, we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12a. We demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background, i.e., the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 μmol/L, respectively, highlighting the advantages of simplicity, sensitivity, short detection time, and low cost compared with the state-of-the-art biosensing approaches. Altogether, this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes.},
}
@article {pmid36651835,
year = {2023},
author = {Ma, L and Liao, D and Zhao, Z and Kou, J and Guo, H and Xiong, X and Man, S},
title = {Sensitive Small Molecule Aptasensing based on Hybridization Chain Reaction and CRISPR/Cas12a Using a Portable 3D-Printed Visualizer.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.2c02097},
pmid = {36651835},
issn = {2379-3694},
abstract = {Next-generation biosensing tools based on CRISPR/Cas have revolutionized the molecular detection. A number of CRISPR/Cas-based biosensors have been reported for the detection of nucleic acid targets. The establishment of efficient methods for non-nucleic acid target detection would further broaden the scope of this technique, but up to now, the concerning research is limited. In the current study, we reported a versatile biosensing platform for non-nucleic acid small-molecule detection called SMART-Cas12a (small-molecule aptamer regulated test using CRISPR/Cas12a). Simply, hybridization chain reaction cascade signal amplification was first trigged by functional nucleic acid (aptamer) through target binding. Then, the CRISPR/Cas system was integrated to recognize the amplified products followed by activation of the trans-cleavage. As such, the target can be ingeniously converted to nucleic acid signals and then fluorescent signals that can be readily visualized and analyzed by a customized 3D-printed visualizer with the help of a home-made App-enabled smartphone. Adenosine triphosphate was selected as a model target, and under the optimized conditions, we achieved fine analytical performance with a linear range from 0.1 to 750 μM and a detection limit of 1.0 nM. The satisfactory selectivity and recoveries that we have obtained further demonstrated this method to be suitable for a complex sample environment. The sample-to-answer time was less than 100 min. Our work not only expanded the reach of the CRISPR-Cas system in biosensing but also provided a prototype method that can be generalized for detecting a wider range of analytes with desirable adaptability, sensitivity, specificity, and on-site capability.},
}
@article {pmid36650285,
year = {2023},
author = {Sun, A and Li, CP and Chen, Z and Zhang, S and Li, DY and Yang, Y and Li, LQ and Zhao, Y and Wang, K and Li, Z and Liu, J and Liu, S and Wang, J and Liu, JG},
title = {The compact Casπ (Cas12l) 'bracelet' provides a unique structural platform for DNA manipulation.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
pmid = {36650285},
issn = {1748-7838},
abstract = {CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.},
}
@article {pmid36646768,
year = {2023},
author = {Li, C and Wang, L and Cseke, LJ and Vasconcelos, F and Huguet-Tapia, JC and Gassmann, W and Pauwels, L and White, FF and Dong, H and Yang, B},
title = {Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {56},
pmid = {36646768},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; *Cytosine/metabolism ; Gene Editing/methods ; Bacteria/genetics/metabolism ; RNA ; },
abstract = {Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.},
}
@article {pmid36646687,
year = {2023},
author = {Li, T and Yang, Y and Qi, H and Cui, W and Zhang, L and Fu, X and He, X and Liu, M and Li, PF and Yu, T},
title = {CRISPR/Cas9 therapeutics: progress and prospects.},
journal = {Signal transduction and targeted therapy},
volume = {8},
number = {1},
pages = {36},
pmid = {36646687},
issn = {2059-3635},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications.},
}
@article {pmid36587560,
year = {2023},
author = {Lorincz, R and Alvarez, AB and Walkey, CJ and Mendonça, SA and Lu, ZH and Martinez, AE and Ljungberg, C and Heaney, JD and Lagor, WR and Curiel, DT},
title = {In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {158},
number = {},
pages = {114189},
doi = {10.1016/j.biopha.2022.114189},
pmid = {36587560},
issn = {1950-6007},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Vectors/genetics ; Genetic Therapy/methods ; Adenoviridae/genetics ; Capsid Proteins/genetics ; Endothelium ; },
abstract = {Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.},
}
@article {pmid36164603,
year = {2022},
author = {Mayuramart, O and Poomipak, W and Rattanaburi, S and Khongnomnan, K and Anuntakarun, S and Saengchoowong, S and Chavalit, T and Chantaravisoot, N and Payungporn, S},
title = {IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production.},
journal = {PeerJ},
volume = {10},
number = {},
pages = {e13989},
pmid = {36164603},
issn = {2167-8359},
mesh = {Animals ; Dogs ; Humans ; *Influenza Vaccines/genetics ; Madin Darby Canine Kidney Cells ; *Influenza, Human/genetics ; Factor VII/genetics ; CRISPR-Cas Systems/genetics ; *Orthomyxoviridae ; Virus Replication/genetics ; *Interferon Type I/genetics ; Technology ; },
abstract = {The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7[-/ -] MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7[-/ -] MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7[-/ -] MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7[-/ -] MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7[-/ -] MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7[-/ -] MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7[-/ -] MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency.},
}
@article {pmid36124130,
year = {2022},
author = {Tian, H and Niu, H and Luo, J and Yao, W and Gao, W and Wen, Y and Cheng, M and Lei, A and Hua, J},
title = {Effects of CRISPR/Cas9-mediated stearoyl-Coenzyme A desaturase 1 knockout on mouse embryo development and lipid synthesis.},
journal = {PeerJ},
volume = {10},
number = {},
pages = {e13945},
pmid = {36124130},
issn = {2167-8359},
mesh = {Animals ; Female ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Mice, Knockout ; *Embryonic Development/genetics ; Fatty Acids, Monounsaturated/metabolism ; Triglycerides/metabolism ; Fatty Acid Desaturases/metabolism ; Water/metabolism ; },
abstract = {BACKGROUND: Lipid synthesis is an indispensable process during embryo and growth development. Abnormal lipid synthesis metabolism can cause multiple metabolic diseases including obesity and hyperlipidemia. Stearoyl-Coenzyme A desaturase 1 (SCD1) is responsible for catalyzing the synthesis of monounsaturated fatty acids (MUFA) and plays an essential role in lipid metabolism. The aim of our study was to evaluate the effects of SCD1 on embryo development and lipid synthesis in a knockout mice model.<br><br>METHODS: We used the CRISPR/Cas9 system together with microinjection for the knockout mouse model generation. Ten-week-old female C57BL/6 mice were used for zygote collection. RNase-free water was injected into mouse zygotes at different cell phases in order to select the optimal time for microinjection. Five sgRNAs were designed and in vitro transcription was performed to obtain sgRNAs and Cas9 mRNA. RNase-free water, NC sgRNA/Cas9 mRNA, and Scd1 sgRNA/Cas9 mRNA were injected into zygotes to observe the morula and blastocyst formation rates. Embryos that were injected with Scd1 sgRNA/Cas9 mRNA and developed to the two-cell stage were used for embryo transfer. Body weight, triacylglycerol (TAG), and cholesterol in Scd1 knockout mice serum were analyzed to determine the effects of SCD1 on lipid metabolism.<br><br>RESULTS: Microinjection performed during the S phase presented with the highest zygote survival rate (P < 0.05). Of the five sgRNAs targeted to Scd1, two sgRNAs with relatively higher gene editing efficiency were used for Scd1 knockout embryos and mice generation. Genome sequence modification was observed at Scd1 exons in embryos, and Scd1 knockout reduced blastocyst formation rates (P < 0.05). Three Scd1 monoallelic knockout mice were obtained. In mice, the protein level of SCD1 decreased (P < 0.05), and the body weight and serum TAG and cholesterol contents were all reduced (P < 0.01).},
}
@article {pmid35999358,
year = {2023},
author = {Tamai, M and Fujisawa, S and Nguyen, TTT and Komatsu, C and Kagami, K and Kamimoto, K and Omachi, K and Kasai, S and Harama, D and Watanabe, A and Akahane, K and Goi, K and Naka, K and Kaname, T and Teshima, T and Inukai, T},
title = {Creation of Philadelphia chromosome by CRISPR/Cas9-mediated double cleavages on BCR and ABL1 genes as a model for initial event in leukemogenesis.},
journal = {Cancer gene therapy},
volume = {30},
number = {1},
pages = {38-50},
pmid = {35999358},
issn = {1476-5500},
mesh = {Humans ; *Philadelphia Chromosome ; Fusion Proteins, bcr-abl/genetics ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; CRISPR-Cas Systems ; Translocation, Genetic ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics ; Carcinogenesis/genetics ; },
abstract = {The Philadelphia (Ph) chromosome was the first translocation identified in leukemia. It is supposed to be generated by aberrant ligation between two DNA double-strand breaks (DSBs) at the BCR gene located on chromosome 9q34 and the ABL1 gene located on chromosome 22q11. Thus, mimicking the initiation process of translocation, we induced CRISPR/Cas9-mediated DSBs simultaneously at the breakpoints of the BCR and ABL1 genes in a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent human leukemia cell line. After transfection of two single guide RNAs (sgRNAs) targeting intron 13 of the BCR gene and intron 1 of the ABL1 gene, a factor-independent subline was obtained. In the subline, p210 BCR::ABL1 and its reciprocal ABL1::BCR fusions were generated as a result of balanced translocation corresponding to the Ph chromosome. Another set of sgRNAs targeting intron 1 of the BCR gene and intron 1 of the ABL1 gene induced a factor-independent subline expressing p190 BCR::ABL1. Both p210 and p190 BCR::ABL1 induced factor-independent growth by constitutively activating intracellular signaling pathways for transcriptional regulation of cell cycle progression and cell survival that are usually regulated by GM-CSF. These observations suggested that simultaneous DSBs at the BCR and ABL1 gene breakpoints are initiation events for oncogenesis in Ph+ leukemia. (200/200 words).},
}
@article {pmid36648696,
year = {2023},
author = {Yang, Y and Wang, D and Lü, P and Ma, S and Chen, K},
title = {Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {1-16},
pmid = {36648696},
issn = {1573-4978},
abstract = {PURPOSE: This work characterizes the applications of CRISPR/Cas12 system, including nucleic acid detection, animal, plant and microbial genome editing.<br><br>METHODS: The literature on CRISPR/Cas12 system was collected and reviewed.<br><br>RESULTS: CRISPR/Cas system is an acquired immune system derived from bacteria and archaea, which has become the most popular technology around the world because of its outstanding contribution in genome editing. Type V CRISPR/Cas systems are distinguished by a single RNA-guided RuvC nuclease domain with single effector molecule. Cas12a, the first reported type V CRISPR/Cas system, targets double-stranded DNA (dsDNA) adjacent to PAM sequences and trans-cleaves single-stranded DNA (ssDNA). We present the applications of CRISPR/Cas12 system for nucleic acid detection and genome editing in animals, plants and microorganisms. Furthermore, this review also summarizes the applications of other Cas12 proteins, such as Cas12b, Cas12c, Cas12d, and so on, which further widen the application prospects of CRISPR/Cas12 system.<br><br>CONCLUSIONS: Knowledge of the applications of CRISPR/Cas12 system is necessary for improving the understanding of the functional diversity of CRISPR/Cas12 system and also provides significant references for further research and utilization of CRISPR/Cas12 in other new fields.},
}
@article {pmid36647130,
year = {2023},
author = {Huang, H and Lv, W and Li, J and Huang, G and Tan, Z and Hu, Y and Ma, S and Zhang, X and Huang, L and Lin, Y},
title = {Comparison of DNA targeting CRISPR editors in human cells.},
journal = {Cell &amp; bioscience},
volume = {13},
number = {1},
pages = {11},
pmid = {36647130},
issn = {2045-3701},
abstract = {BACKGROUND: Profiling and comparing the performance of current widely used DNA targeting CRISPR systems provide the basic information for the gene-editing toolkit and can be a useful resource for this field. In the current study, we made a parallel comparison between the recently reported miniature Cas12f1 (Un1Cas12f1 and AsCas12f1) and the widely used Cas12a and Cas9 nucleases in mammalian cells.<br><br>RESULTS: We found that as a CRISPRa activator, Un1Cas12f1 could induce gene expression with a comparable level to that of Cas12a and Cas9, while as a DNA cleavage editor, Cas12f1 exhibited similar properties to Cas12a, like high specificity and dominantly induced deletions over insertions, but with less activity. In contrast, wild-type SpCas9 showed the highest activity, lowest specificity, and induced balanced deletions and insertions. Thus, Cas12f1 is recommended for gene-activation-based applications, Cas12a is for therapy applications, and wild-type Cas9 is for in vitro and animal investigations.<br><br>CONCLUSION: The comparison provided the editing properties of the widely used DNA-targeting CRISPR systems in the gene-editing field.},
}
@article {pmid36641157,
year = {2023},
author = {Cheng, X and Xia, X and Ren, D and Chen, Q and Xu, G and Wei, F and Yang, J and Wang, L and Hu, Q and Zou, J and Cen, Y},
title = {Programmable CRISPR-Cas12a and self-recruiting crRNA assisted dual biosensing platform for simultaneous detection of lung cancer biomarkers hOGG1 and FEN1.},
journal = {Analytica chimica acta},
volume = {1240},
number = {},
pages = {340748},
doi = {10.1016/j.aca.2022.340748},
pmid = {36641157},
issn = {1873-4324},
mesh = {Humans ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Flap Endonucleases ; *Lung Neoplasms/diagnosis/genetics ; Lung ; *Biosensing Techniques ; },
abstract = {Human 8-oxoguanine DNA glycosylase (hOGG1) and flap endonuclease 1 (FEN1) are recognized as potential biomarkers in lung cancer investigations. Developing analytical platforms of simultaneously targeting hOGG1 and FEN1 with high selectivity, sensitivity, especially programmability and universality is highly valuable for clinical research. Herein, we established a signal-amplified platform for simultaneously detecting hOGG1 and FEN1 on the basis of cleavage-induced ligation of DNA dumbbell probes, rolling circle transcription (RCT) and CRISPR-Cas12a. A hOGG1 cleavable site and FEN1 cleavable flap were dexterously designed at the 5' end of DNA flapped dumbbell probes (FDP) for hOGG1 and FEN1. After cleavage, the resulting nick sites with juxtaposition of 5' phosphate and 3' hydroxyl terminus could be linked to closed DNA dumbbell probes (CDP) by DNA ligase. The CDP served as a template for RCT, producing plentiful crRNA repeats to activate the trans-cleavage activity of CRISPR-Cas12a which could cleave fluorophores (TAMRA and FAM) and quenchers (BHQ2 and BHQ1) double-labeled ssDNA reporters. Then, hOGG1 and FEN1 could be detected by the recovered fluorescence signal, allowing for the highly sensitive calculated detection limits of 0.0013 and 0.0052 U/mL, respectively. Additionally, this method made it possible to evaluate the inhibitory effects, even to measure hOGG1 and FEN1 activities at the single-cell level. This novel target enzyme-initiated, circles-transcription without promoters, real-time generation, and self-assembly features of FDP-RCT-Cas12a system suppressed nonspecific background remarkably and relieved rigorous requirement of protospacer adjacent motif site. Hence, the universality of FDP-RCT-Cas12a system toward various disease-related non-nucleic acid targets which are tested without using aptamers was extremely improved.},
}
@article {pmid36640045,
year = {2023},
author = {Chen, H and Sun, H and Xie, J and Yao, Z and Zheng, W and Li, Z and Deng, Z and Li, X and Zhang, H},
title = {CRISPR/Cas9-induced Mutation of Sex Peptide Receptor Gene Bdspr Affects Ovary, Egg Laying, and Female Fecundity in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).},
journal = {Journal of insect science (Online)},
volume = {23},
number = {1},
pages = {},
doi = {10.1093/jisesa/ieac078},
pmid = {36640045},
issn = {1536-2442},
mesh = {Female ; Animals ; *Tephritidae/physiology ; Ovary ; CRISPR-Cas Systems ; Ovum ; Drosophila ; Fertility/genetics ; Mutation ; Receptors, Peptide/genetics ; },
abstract = {The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an invasive and polyphagous pest of horticultural crops, and it can cause huge economic losses in agricultural production. The rapid development of CRISPR/Cas9 gene editing technology has provided new opportunities for the scientific control of agricultural pests. Here, we explore the applicability of the B. dorsalis sex peptide receptor (Bdspr) as a target gene for the CRISPR/Cas9-based sterile insect technique (SIT) in B. dorsalis. We screened two high-efficient single guide RNAs (sgRNAs) for gene editing. The results showed that both mutation efficiency and germline transmission rate were 100% in the surviving G0 females (8/8) from injected embryos, and that 75% of mosaically mutated G0 females (6/8) were sterile. The 50% of heterozygous G1 females (4/8) could not lay eggs; 100% of eggs laid by them could not survive; and 62.5% of individual females (5/8) had abnormal ovaries. These results indicate that Bdspr plays an important role in regulating fertility, egg viability, and ovary development in female B. dorsalis, suggesting that the spr gene can be used for CRISPR/Cas9-based SIT in B. dorsalis.},
}
@article {pmid36639800,
year = {2023},
author = {Gaillochet, C and Peña Fernández, A and Goossens, V and D'Halluin, K and Drozdzecki, A and Shafie, M and Van Duyse, J and Van Isterdael, G and Gonzalez, C and Vermeersch, M and De Saeger, J and Develtere, W and Audenaert, D and De Vleesschauwer, D and Meulewaeter, F and Jacobs, TB},
title = {Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {6},
pmid = {36639800},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Zea mays/genetics ; Triticum/genetics ; Gene Editing/methods ; Mutagenesis ; },
abstract = {BACKGROUND: Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging.<br><br>RESULTS: We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle - from design to results - within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components - NLS, crRNA, LbCas12a, adenine deaminase, and linker - leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox.<br><br>CONCLUSION: Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.},
}
@article {pmid36639729,
year = {2023},
author = {Liao, J and Chen, S and Hsiao, S and Jiang, Y and Yang, Y and Zhang, Y and Wang, X and Lai, Y and Bauer, DE and Wu, Y},
title = {Therapeutic adenine base editing of human hematopoietic stem cells.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {207},
pmid = {36639729},
issn = {2041-1723},
mesh = {Adult ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; gamma-Globins/genetics ; *beta-Thalassemia/genetics ; Adenine/metabolism ; Hematopoietic Stem Cells/metabolism ; Antigens, CD34/genetics/metabolism ; Fetal Hemoglobin/genetics ; },
abstract = {In β-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or β-globin repair to restore adult hemoglobin (α2β2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of β-thalassemia patient CD34[+] hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34[+] HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders.},
}
@article {pmid36639508,
year = {2023},
author = {Wagner, BK},
title = {Identifying type 2 diabetes risk genes by β-cell CRISPR screening.},
journal = {Nature genetics},
volume = {55},
number = {1},
pages = {4-5},
pmid = {36639508},
issn = {1546-1718},
mesh = {Humans ; *Diabetes Mellitus, Type 2/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid36593304,
year = {2023},
author = {Saha, K},
title = {Accounting for diversity in the design of CRISPR-based therapeutic genome editing.},
journal = {Nature genetics},
volume = {55},
number = {1},
pages = {6-7},
pmid = {36593304},
issn = {1546-1718},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid36572334,
year = {2023},
author = {Yu, X and Li, S and Feng, H and Liao, X and Xing, XH and Bai, Z and Liu, X and Zhang, C},
title = {CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion.},
journal = {Metabolic engineering},
volume = {75},
number = {},
pages = {192-204},
doi = {10.1016/j.ymben.2022.12.004},
pmid = {36572334},
issn = {1096-7184},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Microfluidics ; Recombinant Proteins/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression. Meanwhile, we developed a highly efficient droplet-based microfluidics system integrating the FlAsH-tetracysteine assay that enables screening of millions of strains to identify potential knockdowns conducive to nanobody VHH secretion. Among our highest-ranking candidates are a slew of previously unknown targets involved in transmembrane transport, amino-acid metabolism and redox regulation. Guided by these findings, we eventually constructed a hyperproducer for multiple proteins via combinatorial engineering of redox-response transcription factors. As the near-universal applicability of CRISPRi technology and the FlAsH-based screening platform, this procedure might be expanded to include a varied variety of microbial species and recombinant proteins.},
}
@article {pmid36536407,
year = {2023},
author = {Žun, G and Doberšek, K and Petrovič, U},
title = {Construction and evaluation of gRNA arrays for multiplex CRISPR-Cas9.},
journal = {Yeast (Chichester, England)},
volume = {40},
number = {1},
pages = {32-41},
doi = {10.1002/yea.3833},
pmid = {36536407},
issn = {1097-0061},
mesh = {*CRISPR-Cas Systems ; *Saccharomyces cerevisiae/genetics ; Gene Editing/methods ; RNA, Guide, Kinetoplastida/genetics ; Plasmids/genetics ; },
abstract = {Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiae genome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressing guide RNAs (gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.},
}
@article {pmid36522432,
year = {2023},
author = {Cancellieri, S and Zeng, J and Lin, LY and Tognon, M and Nguyen, MA and Lin, J and Bombieri, N and Maitland, SA and Ciuculescu, MF and Katta, V and Tsai, SQ and Armant, M and Wolfe, SA and Giugno, R and Bauer, DE and Pinello, L},
title = {Human genetic diversity alters off-target outcomes of therapeutic gene editing.},
journal = {Nature genetics},
volume = {55},
number = {1},
pages = {34-43},
pmid = {36522432},
issn = {1546-1718},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida/genetics ; Hematopoietic Stem Cells ; INDEL Mutation ; },
abstract = {CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34[+] hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination.},
}
@article {pmid36463563,
year = {2023},
author = {Zhang, B},
title = {Genome Editing.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {137},
doi = {10.1007/s12033-022-00622-w},
pmid = {36463563},
issn = {1559-0305},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid36455778,
year = {2023},
author = {Kwak, JS and Kim, KH},
title = {Effect of CRISPR/Cas9-mediated knockout of either IRF-3 or IRF-5 gene in Epithelioma papulosum cyprini cells on type I interferon response and NF-κB activity.},
journal = {Fish &amp; shellfish immunology},
volume = {132},
number = {},
pages = {108463},
doi = {10.1016/j.fsi.2022.108463},
pmid = {36455778},
issn = {1095-9947},
mesh = {Animals ; *Interferon Type I/genetics/metabolism ; NF-kappa B/genetics/metabolism ; CRISPR-Cas Systems ; Interferon Regulatory Factors/metabolism ; Poly I-C ; *Virus Diseases ; *Carcinoma ; },
abstract = {Transcription factors related to the activation of type I interferons (IFNs) and nuclear factor-kappa B (NF-κB) are known to be critical in innate immune responses. Interferon regulatory factors (IRFs) are a family of transcription factors. IRF-3 is known to act as the primary regulator in type I IFN signaling in response to viral infections, and the upregulation of IRF5 by virus infection has been reported in various fish species. One of the ways to know the functional role of certain genes is the production of target gene(s) knockout cells or organisms. In the present study, we produced either IRF3 or IRF5 gene knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 system, and investigated the effect of IRF3 gene and IRF5 gene knockout on polyinosinic:polycytidylic acid (ploly (I:C))-mediated and viral hemorrhagic septicemia virus (VHSV) infection-mediated type I IFN response and NF-κB activation. Both IRF3 knockout and IRF5 knockout EPC cells showed severely decreased type I IFN responses measured by ISRE activity and the expression of Mx1 and ISG15 genes when stimulated with poly (I:C), while the decreased level of type I IFN responses was not high as by poly (I:C) stimulation when infected with VHSV. Different from type I IFN response, NF-κB activities in IRF3 and IRF5 knockout cells were not highly different between poly (I:C) stimulated cells and VHSV-infected cells. Further studies are needed to elucidate pathways responsible for the type I IFN responses and NF-κB activation by VHSV infection.},
}
@article {pmid36404524,
year = {2023},
author = {Wang, J and Teng, Y and Gong, X and Zhang, J and Wu, Y and Lou, L and Li, M and Xie, ZR and Yan, Y},
title = {Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.},
journal = {Metabolic engineering},
volume = {75},
number = {},
pages = {68-77},
doi = {10.1016/j.ymben.2022.10.005},
pmid = {36404524},
issn = {1096-7184},
support = {R35 GM128620/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Mutagenesis ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.},
}
@article {pmid36375746,
year = {2023},
author = {Wang, J and Li, C and Jiang, T and Yan, Y},
title = {Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes.},
journal = {Metabolic engineering},
volume = {75},
number = {},
pages = {58-67},
doi = {10.1016/j.ymben.2022.11.004},
pmid = {36375746},
issn = {1096-7184},
mesh = {*Escherichia coli/genetics/metabolism ; *High-Throughput Screening Assays ; Coumaric Acids ; Phenotype ; CRISPR-Cas Systems/genetics ; Metabolic Engineering ; },
abstract = {With rapid advances in the development of metabolic pathways and synthetic biology toolkits, a persisting challenge in microbial bioproduction is how to optimally rewire metabolic fluxes and accelerate the concomitant high-throughput phenotype screening. Here we developed a biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening approach that combines a titratable mismatch CRISPR interference and a biosensor mediated screening for high-production phenotypes in Escherichia coli. We first developed a programmable mismatch CRISPRi that could afford multiple levels of interference efficacy with a one-pot sgRNA pool (a total of 16 variants for each target gene) harboring two consecutive random mismatches in the seed region of sgRNA spacers. The mismatch CRISPRi was demonstrated to enable almost a full range of gene knockdown when targeting different positions on genes. As a proof-of-principle demonstration of the BATCH screening system, we designed doubly mismatched sgRNA pools targeting 20 relevant genes in E. coli and optimized a PadR-based p-coumaric acid biosensor with broad dynamic range for the eGFP fluorescence guided high-production screening. Using sgRNA variants for the combinatorial knockdown of pfkA and ptsI, the p-coumaric acid titer was increased by 40.6% to o 1308.6 mg/l from glycerol in shake flasks. To further demonstrate the general applicability of the BATCH screening system, we recruited a HpdR-based butyrate biosensor that facilitated the screening of E. coli strains achieving 19.0% and 25.2% increase of butyrate titer in shake flasks with sgRNA variants targeting sucA and ldhA, respectively. This work reported the establishment of a plug-and-play approach that enables multilevel modulation of metabolic fluxes and high-throughput screening of high-production phenotypes.},
}
@article {pmid36264628,
year = {2023},
author = {Sun, X and Hua, W and Zhu, B and Liang, P and Gao, X},
title = {CRISPR/Cas9-mediated D472N substitution in the Rdl1 of Plutella xylostella confers low resistance to abamectin.},
journal = {Pest management science},
volume = {79},
number = {2},
pages = {741-747},
doi = {10.1002/ps.7252},
pmid = {36264628},
issn = {1526-4998},
mesh = {Animals ; *Moths/genetics ; CRISPR-Cas Systems ; Ivermectin/pharmacology ; *Insecticides/pharmacology ; Insecticide Resistance/genetics ; },
abstract = {BACKGROUND: Abamectin is one of the main insecticides used for the control of Plutella xylostella, a destructive pest of cruciferous crops. Target-site mutation plays an important role in insecticide resistance. A point mutation (D472N) has been reported in the Rdl1 γ-aminobutyric acid receptor (GABAR) in P. xylostella, but its roles in insecticide resistance remain unknown.<br><br>RESULTS: In this study, the D472N mutation of the Rdl1 GABAR was detected in several field populations of P. xylostella and showed a positive correlation with abamectin resistance. A knock-in homozygous mutation strain (D472N-KI) of P. xylostella was successfully constructed using CRISPR/Cas9 coupled with homology-directed repair, and the bioassay results demonstrated that compared with the susceptible strain, the D472N-KI strain had 11.1- and 3.7-fold increased resistance to abamectin and endosulfan, respectively. There was no difference in resistance to fipronil, broflanilide or isocycloseram, which also target the GABAR. In addition, the total fecundity of the D472N-KI strain was significantly reduced by 50.0%.<br><br>CONCLUSION: Our results suggest that the homozygous D472N mutation in Rdl1 confers a low level of resistance to abamectin in P. xylostella but causes significant fecundity disadvantages, which may delay the development of resistance to some extent. These results lay a foundation for further understanding the mechanisms of abamectin resistance in insect pests. &#xa9; 2022 Society of Chemical Industry.},
}
@article {pmid36030521,
year = {2023},
author = {Konu, M and Kulmuni, J and Viljakainen, L},
title = {Genetic modification of the ant Lasius niger using CRISPR-Cas9 technology.},
journal = {Insect molecular biology},
volume = {32},
number = {1},
pages = {11-25},
doi = {10.1111/imb.12809},
pmid = {36030521},
issn = {1365-2583},
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Ants/genetics ; Niger ; Pigmentation ; },
abstract = {CRISPR-Cas9 has become one of the most prominent gene editing tools available and it has been utilized in various organisms from bacteria to fungi, plants, and animals. In this study, we developed a CRISPR-Cas9 protocol for the black garden ant Lasius niger, a common and easily available study species for lab and field experiments. To create indel mutations using CRISPR-Cas9 in L. niger, we targeted three different locations in a well-studied eye pigmentation gene cinnabar, generating several mutations that disrupt the ommochrome biosynthesis pathway and result in the lack of the pigment and therefore, abnormal eye coloration in adult workers. We also developed a protocol to collect L. niger eggs, inject them with CRISPR-Cas9 construct, and rear the eggs into mature adult workers with the assistance of nursing workers. We demonstrated for the first time in L. niger that CRISPR-Cas9 is an excellent tool to create targeted mutations for this species. Our protocol can be referred to when developing similar studies for other species of ants and eusocial insects.},
}
@article {pmid35999480,
year = {2023},
author = {Maity, S and Mukherjee, R and Banerjee, S},
title = {Recent Advances and Therapeutic Strategies Using CRISPR Genome Editing Technique for the Treatment of Cancer.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {206-226},
pmid = {35999480},
issn = {1559-0305},
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Neoplasms/genetics/therapy ; Genetic Therapy/methods ; },
abstract = {CRISPR genome editing technique has the potential to target cancer cells in a precise manner. The latest advancements have helped to address one of the prominent concerns about this strategy which is the off-target integrations observed with dsDNA and have resulted in more studies being carried out for potentially safer and more targeted gene therapy, so as to make it available for the clinical trials in order to effectively treat cancer. CRISPR screens offer great potential for the high throughput investigation of the gene functionality in various tumors. It extends its capability to identify the tumor growth essential genes, therapeutic resistant genes, and immunotherapeutic responses. CRISPR screens are mostly performed in in vitro models, but latest advancements focus on developing in vivo models to view cancer progression in animal models. It also allows the detection of factors responsible for tumorigenesis. In CRISPR screens key parameters are optimized in order to meet proficient gene targeting efficiencies. It also detects various molecular effectors required for gene regulation in different cancers, essential pathways which modulate cytotoxicity to immunotherapy in cancer cells, important genes which contribute to cancer cell survival in hypoxic states and modulate cancer long non-coding RNAs. The current review focuses on the recent developments in the therapeutic application of CRISPR technology for cancer therapy. Furthermore, the associated challenges and safety concerns along with the various strategies that can be implemented to overcome these drawbacks has been discussed.},
}
@article {pmid35840848,
year = {2023},
author = {Tao, D and Liu, J and Li, Q and Jiang, Y and Xu, B and Khazalwa, EM and Gong, P and Xu, J and Ma, Y and Ruan, J and Niu, L and Xie, S},
title = {A Simple, Affordable, and Rapid Visual CRISPR-Based Field Test for Sex Determination of Earlier Porcine Embryos and Pork Products.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {263-272},
pmid = {35840848},
issn = {1559-0305},
mesh = {Swine/genetics ; Animals ; *Meat Products ; *Red Meat ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; DNA Primers/genetics ; CRISPR-Cas Systems ; Sensitivity and Specificity ; },
abstract = {Sex selection technologies have immensely impacted swine production globally. Conventional earlier embryo sex identification methods require professional technicians and sophisticated laboratory instruments. Rapid on-site gender identification of porcine embryos and pork products remains challenging. In this study, we developed a CRISPR/Cas12a-based fluorescence visualization point-of-care sex determination test that is rapid, accurate and easy to implement on-site. The CRISPR/Cas12a assay coupled with either the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) employs precisely designed primers and single-guide RNAs targeting the sex-determining region Y (SRY) and the zinc finger protein X-linked (ZFX) genes. PCR and LAMP amplicons were cleaved with the subsequent generation of fluorescing products detectable with portable blue and ultraviolet light transilluminators. Approximately two copies per microliter of the ZFX and SRY genes were detected using the RApid VIsual CRISPR (RAVI-CRISPR) assay. This method is a sensitive, inexpensive, versatile, and point-of-care test. The technology has other potential applications like determining the sex of diverse livestock species, detecting livestock disease-causing pathogens and evaluating the quality of meat products.},
}
@article {pmid35587334,
year = {2023},
author = {Jia, Z and Yang, M and Zhao, Y and Li, X and Yang, C and Qiao, L and Li, H and Du, J and Lin, J and Guan, L},
title = {CRISPR-Cas9-Mediated NPC1 Gene Deletion Enhances HEK 293 T Cell Adhesion by Regulating E-Cadherin.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {252-262},
pmid = {35587334},
issn = {1559-0305},
mesh = {Humans ; Cell Adhesion/genetics ; *CRISPR-Cas Systems ; Gene Deletion ; HEK293 Cells ; *Cadherins ; Niemann-Pick C1 Protein ; },
abstract = {NPC1 gene encodes a transmembrane glycoprotein on the late endosome/lysosomal membrane. Its mutation leads to a rare and aggravated autosomal recessive neurovisceral condition, termed Niemann-Pick disease type C1 (NPC1), which is characterized by progressive neurodegeneration, visceral symptoms, and premature death. To investigate the influence of NPC1 gene deletion on cell morphology, adhesion, proliferation, and apoptosis, CRISPR-Cas9 technology was used to knockout the NPC1 gene in HEK 293 T cells. Sanger sequencing, western blotting, and immunofluorescence were used to confirm successful NPC1 ablation. Filipin staining results indicated that deletion of NPC1 gene led to accumulation of unesterified cholesterol in HEK 293 T cells. Phalloidin staining results revealed cell aggregation, synapse shortening, nuclear enlargement, and cytoskeleton filamentous actin thinning in HEK 293 T cells with NPC1 gene mutation. Furthermore, NPC1 gene mutated HEK 293 T cell showed enhanced cell adhesion, inhibited cell proliferation, and increased cell apoptosis. In addition, NPC1 gene mutations significantly increased the protein expression levels of E-cadherin and γ-catenin and significantly decreased the protein expression levels of Wnt 3a, c-Myc, and cyclin D1. These results suggest that NPC1 may regulate cell adhesion by affecting the cadherin-catenin complex through E-cadherin, and that the classical Wnt signaling pathway may be inhibited by restricting β-catenin from entering the nucleus to inhibit cell proliferation.},
}
@article {pmid35474409,
year = {2023},
author = {Akram, F and Sahreen, S and Aamir, F and Haq, IU and Malik, K and Imtiaz, M and Naseem, W and Nasir, N and Waheed, HM},
title = {An Insight into Modern Targeted Genome-Editing Technologies with a Special Focus on CRISPR/Cas9 and its Applications.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {227-242},
pmid = {35474409},
issn = {1559-0305},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *COVID-19 ; SARS-CoV-2/genetics ; Gene Editing/methods ; Plants/genetics ; Technology ; },
abstract = {Genome-editing technology has enabled scientists to make changes in model organisms' DNA at the genomic level to get biotechnologically important products from them. Most commonly employed technologies for this purpose are transcription activator like effector nucleases (TALENs), homing-endonucleases or meganucleases, zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9). Among these tools, CRISPR/Cas9 is most preferred because it's easy to use, has a small mutation rate, has great effectiveness, low cost of development, and decreased rate of advancement. CRISPR/Cas9 has a lot of applications in plants, animals, humans, and microbes. It also has applications in many fields such as horticulture, cancer, food biotechnology, and targeted human genome treatments. CRISPR technology has shown great potential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic to provide early and easy detection methods, possible treatment, and vaccine development. In the present review, genome-editing tools with their basic assembly and features have been discussed. Exceptional notice has been paid to CRISPR technology on basis of its structure and significant applications in humans, plants, animals, and microbes such as bacteria, viruses, and fungi. The review has also shed a little light on current CRISPR challenges and future perspectives.},
}
@article {pmid35467255,
year = {2023},
author = {Hashemabadi, M and Sasan, H and Amandadi, M and Esmaeilzadeh-Salestani, K and Esmaeili-Mahani, S and Ravan, H},
title = {CRISPR/Cas9-Mediated Disruption of ZNF543 Gene: An Approach Toward Discovering Its Relation to TRIM28 Gene in Parkinson's Disease.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {243-251},
pmid = {35467255},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems ; *Parkinson Disease/genetics ; Cell Line ; Gene Expression Regulation ; Mutation ; Tripartite Motif-Containing Protein 28 ; },
abstract = {Genetic studies of familial forms of Parkinson's disease (PD) have shown that the ZNF543 gene is a candidate gene that operates relevant to this disease. However, until now, there is no evidence for ZNF543 gene function in PD, and mechanisms resulting from its mutation have not been elucidated. Given the same genetic location of the ZNF543 gene with TRIM28 and their effects on PD pathogenesis, we surmised that ZNF543 might act as a transcription factor for TRIM28 gene expression. By knocking out the ZNF543 gene via the CRISPR/Cas9 editing platform, we assessed the functional effect of loss of expression of this gene on TRIM28 gene expression. Four sgRNAs with different PAM sequences were designed against two parts of the regulatory region of ZNF543 gene, and highly efficient disruption of ZNF543 expression in human neuroblastoma cell line was evaluated by polymerase chain reaction and T7 endonuclease assay. Moreover, evaluation of TRIM28 gene expression in ZNF543-knocked-out cells indicated a significant increase in TRIM28 gene expression, suggesting that ZNF543 probably regulates the expression of TRIM28. This approach offers a window into pinpointing the mechanism by which ZNF543 gene mutations mediate PD pathogenicity.},
}
@article {pmid35119645,
year = {2023},
author = {Rahman, SU and McCoy, E and Raza, G and Ali, Z and Mansoor, S and Amin, I},
title = {Improvement of Soybean; A Way Forward Transition from Genetic Engineering to New Plant Breeding Technologies.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {162-180},
pmid = {35119645},
issn = {1559-0305},
mesh = {Animals ; *Soybeans/genetics ; Plants, Genetically Modified/genetics ; *Plant Breeding/methods ; Chickens/genetics ; Genetic Engineering/methods ; CRISPR-Cas Systems ; Genome, Plant ; },
abstract = {Soybean is considered one of the important crops among legumes. Due to high nutritional contents in seed (proteins, sugars, oil, fatty acids, and amino acids), soybean is used globally for food, feed, and fuel. The primary consumption of soybean is vegetable oil and feed for chickens and livestock. Apart from this, soybean benefits soil fertility by fixing atmospheric nitrogen through root nodular bacteria. While conventional breeding is practiced for soybean improvement, with the advent of new biotechnological methods scientists have also engineered soybean to improve different traits (herbicide, insect, and disease resistance) to fulfill consumer requirements and to meet the global food deficiency. Genetic engineering (GE) techniques such as transgenesis and gene silencing help to minimize the risks and increase the adaptability of soybean. Recently, new plant breeding technologies (NPBTs) emerged such as zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), which paved the way for enhanced genetic modification of soybean. These NPBTs have the potential to improve soybean via gene functional characterization precision genome engineering for trait improvement. Importantly, these NPBTs address the ethical and public acceptance issues related to genetic modifications and transgenesis in soybean. In the present review, we summarized the improvement of soybean through GE and NPBTs. The valuable traits that have been improved through GE for different constraints have been discussed. Moreover, the traits that have been improved through NPBTs and potential targets for soybean improvements via NPBTs and solutions for ethical and public acceptance are also presented.},
}
@article {pmid34586618,
year = {2023},
author = {Kesavan, G},
title = {Innovations in CRISPR-Based Therapies.},
journal = {Molecular biotechnology},
volume = {65},
number = {2},
pages = {138-145},
pmid = {34586618},
issn = {1559-0305},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Genetic Therapy ; *Anemia, Sickle Cell/genetics/therapy ; Cell Separation ; },
abstract = {Gene and cell therapies have shown tremendous advancement in the last 5 years. Prominent examples include the successful use of CRISPR-edited stem cells for treating blood disorders like sickle cell anemia and beta-thalassemia, and ongoing clinical trials for treating blindness. This mini-review assesses the status of CRISPR-based therapies, both in vivo and ex vivo, and the challenges associated with clinical translation. In vivo CRISPR therapies have been used to treat eye and liver diseases due to the practicality of delivering editing components to the target tissue. In contrast, even though ex vivo CRISPR therapy involves cell isolation, expansion, and infusion, its advantages include characterizing the gene edits before infusion and restricting off-target effects in other tissues. Further, the safety, affordability, and feasibility of CRISPR therapies, especially for treating large number of patients, are discussed.},
}
@article {pmid36646742,
year = {2023},
author = {Figueiredo, D and Cascalheira, A and Goncalves, J},
title = {Rapid, multiplex detection of SARS-CoV-2 using isothermal amplification coupled with CRISPR-Cas12a.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {849},
pmid = {36646742},
issn = {2045-2322},
abstract = {In December 2019 an outbreak erupted due to the beta coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 in Wuhan, China. The disease caused by this virus (COVID-19) rapidly spread to all parts of the globe leading to a global pandemic. Efforts to combat the pandemic rely on RT-qPCR diagnostic tests that have high turnaround times (~ 24 h), are easily contaminated, need specialized equipment, facilities, and personnel that end up increasing the overall costs of this method. Loop-mediated isothermal amplification (LAMP) coupled with a reverse transcription step (RT-LAMP) is an alternative diagnostic method that can easily overcome these obstacles, when coupled with CRISPR/Cas it can eliminate false positives. Here we report a fast (~ 40 min), highly sensitive, point-of-care multiplex RT-LAMP and CRISPR/Cas12a assay to detect SARS-CoV-2. This fluorescence-based test achieved 100% specificity and 93% sensitivity using 25 positives and 50 negative patient samples for Ct < 35. Our reported LoD of 3 copies/µL will enable the robust, fast detection of the virus in a dedicated equipment which is a major step towards population-wide accessible testing.},
}
@article {pmid36639728,
year = {2023},
author = {Tao, J and Bauer, DE and Chiarle, R},
title = {Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {212},
pmid = {36639728},
issn = {2041-1723},
abstract = {CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients.},
}
@article {pmid36635554,
year = {2023},
author = {Hua, H},
title = {CRISPR inspirations from nature.},
journal = {Nature methods},
volume = {20},
number = {1},
pages = {37},
doi = {10.1038/s41592-022-01756-0},
pmid = {36635554},
issn = {1548-7105},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; },
}
@article {pmid36635291,
year = {2023},
author = {Terradas, G and Bennett, JB and Li, Z and Marshall, JM and Bier, E},
title = {Genetic conversion of a split-drive into a full-drive element.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {191},
pmid = {36635291},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Transgenes ; Phenotype ; *Fertility ; },
abstract = {The core components of CRISPR-based gene drives, Cas9 and guide RNA (gRNA), either can be linked within a self-contained single cassette (full gene-drive, fGD) or be provided in two separate elements (split gene-drive, sGD), the latter offering greater control options. We previously engineered split systems that could be converted genetically into autonomous full drives. Here, we examine such dual systems inserted at the spo11 locus that are recoded to restore gene function and thus organismic fertility. Despite minimal differences in transmission efficiency of the sGD or fGD drive elements in single generation crosses, the reconstituted spo11 fGD cassette surprisingly exhibits slower initial drive kinetics than the unlinked sGD element in multigenerational cage studies, but then eventually catches up to achieve a similar level of final introduction. These unexpected kinetic behaviors most likely reflect differing transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.},
}
@article {pmid36634166,
year = {2023},
author = {Lebek, S and Chemello, F and Caravia, XM and Tan, W and Li, H and Chen, K and Xu, L and Liu, N and Bassel-Duby, R and Olson, EN},
title = {Ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease.},
journal = {Science (New York, N.Y.)},
volume = {379},
number = {6628},
pages = {179-185},
doi = {10.1126/science.ade1105},
pmid = {36634166},
issn = {1095-9203},
mesh = {Humans ; Mice ; Animals ; Gene Editing ; CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac/physiology ; *Heart Diseases/genetics/therapy ; },
abstract = {CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.},
}
@article {pmid36565681,
year = {2023},
author = {Polak, K and Stępniewski, J and Ścieżyńska, A and Podgórska, A and Dulak, J and Florczyk-Soluch, U},
title = {Generation of human induced pluripotent stem cell lines with HMOX1 promoter polymorphism and CRISPR/Cas9-mediated deletion of exon 50 of DMD gene.},
journal = {Stem cell research},
volume = {66},
number = {},
pages = {103004},
doi = {10.1016/j.scr.2022.103004},
pmid = {36565681},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Heme Oxygenase-1/genetics/metabolism ; Leukocytes, Mononuclear/metabolism ; Dystrophin/genetics/metabolism ; *Muscular Dystrophy, Duchenne/metabolism ; Exons/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD), originating from the lack of functional dystrophin, clinically manifests as devastating disease of skeletal muscles with progressive cardiac involvement. HMOX1 promoter polymorphism may reflect different activity of heme oxygenase-1 (HO-1) that may be critical for DMD progression. Here we generated human induced pluripotent stem cell (hiPSC) lines from healthy donors-derived peripheral blood mononuclear cells with different variants of HMOX1 promoter (GT repeats), and engineered by CRISPR/Cas9-mediated deletion of exon 50 of DMD gene. Such in vitro model could add to molecular understanding of DMD and verify the prognostic value of HMOX1 promoter polymorphism.},
}
@article {pmid36528979,
year = {2023},
author = {Khampang, S and Jiamvoraphong, N and Laowtammathron, C and Lorthongpanich, C and Klaihmon, P and Srisook, P and Kang, X and Luanpitpong, S and U-Pratya, Y and Issaragrisil, S},
title = {CRISPR/Cas9 mediated approach to generate YAP-depleted human embryonic stem cell line (MUSIe002-A-1).},
journal = {Stem cell research},
volume = {66},
number = {},
pages = {102990},
doi = {10.1016/j.scr.2022.102990},
pmid = {36528979},
issn = {1876-7753},
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Signal Transduction/physiology ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; YAP-Signaling Proteins ; Cell Line ; },
abstract = {Yes-associated protein (YAP), an important effector protein of the Hippo signaling pathway, acts as a molecular switch in controlling cell proliferation and apoptosis. In this study, a YAP-targeted isogenic sub-clone of the MUSIe002-A was generated, designated as MUSIe002-A-1. The MUSIe002-1 cell line had normal pluripotent stem cell characteristics and karyotype. Its ability to differentiate into three germ layers was confirmed. As reduction of YAP does not disturb the pluripotency of hESCs, this cell line serves as a valuable model to extrapolate the functional role of YAP in stem cell biology and its applications.},
}
@article {pmid36521383,
year = {2023},
author = {Guan, M and Chai, Y and Yi, R and Chen, Y and Ip, JPK and Ye, T and Chen, Y},
title = {Generation of a homozygous ABCA7-knockout human iPSC line using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {66},
number = {},
pages = {103000},
doi = {10.1016/j.scr.2022.103000},
pmid = {36521383},
issn = {1876-7753},
mesh = {Humans ; *Alzheimer Disease/genetics ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Homozygote ; ATP-Binding Cassette Transporters/genetics ; },
abstract = {ABCA7 encodes an ATP-binding cassette transporter, and its loss-of-function variants are associated with Alzheimer's disease. To investigate the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, we generated a homozygous ABCA7-knockout induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-mediated gene editing. This ABCA7-deficient iPSC line maintains a normal karyotype, expression of pluripotency markers, and trilineage differentiation capacity.},
}
@article {pmid36516658,
year = {2023},
author = {Choi, JB and Seo, D and Do, HS and Han, YM},
title = {Generation of a CRISPR/Cas9-corrected-hiPSC line (DDLABi001-A) from Fabry disease (FD)-derived iPSCs having α-galactosidase (GLA) gene mutation (c.803_806del).},
journal = {Stem cell research},
volume = {66},
number = {},
pages = {103001},
doi = {10.1016/j.scr.2022.103001},
pmid = {36516658},
issn = {1876-7753},
mesh = {Humans ; alpha-Galactosidase/genetics/metabolism ; *Fabry Disease/genetics ; CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; },
abstract = {Fabry disease (FD) is a lysosomal storage disorder caused by mutations in GLA gene. Here, GLA mutation (1268fs*1 (c.803_806del)) of FD iPSCs was corrected using the CRISPR-Cas9 gene editing system. The corrected (cor) FD-iPSCs retained normal morphology, karyotype, expression of pluripotency-associated markers, trilineage differentiation potential, and GLA activity. Thus, FD(cor)-iPSCs can be used as valuable tools to study the mechanism how GLA mutation[1268fs*1] induces various pathophysiologic phenotypes in FD patients.},
}
@article {pmid36493708,
year = {2023},
author = {Kang, EH and Kim, JW and Noh, H and Park, SH and Park, HJ and Yoon, S and Kim, H},
title = {Establishment of a human embryonic stem cell line, WAe009-A-99, with constitutive expression of the dCas9-p300 fusion protein.},
journal = {Stem cell research},
volume = {66},
number = {},
pages = {102986},
doi = {10.1016/j.scr.2022.102986},
pmid = {36493708},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Associated Protein 9/genetics ; *Human Embryonic Stem Cells ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Transcription Factors/genetics ; Cell Line ; Transcriptional Activation ; },
abstract = {CRISPR/Cas9-based transcriptional regulation systems can induce the site-specific activation or repression of endogenous genes. p300 is a transcriptional co-activator that functions as a histone acetyltransferase that regulates gene transcription via chromatin remodeling. Here, we generated a human embryonic stem cell line stably expressing catalytically dead Cas9 (dCas9) fused to the catalytic core domain of human p300 via lentiviral transduction. This cell line can be used for locus-specific histone acetylation in combination with guide RNAs, and is a valuable tool for gene regulation in stem cell research.},
}
@article {pmid36470413,
year = {2023},
author = {Okshevsky, M and Xu, Y and Masson, L and Arbour, M},
title = {Efficiency of CRISPR-Cas9 genetic engineering in Escherichia coli BL21 is impaired by lack of Lon protease.},
journal = {Journal of microbiological methods},
volume = {204},
number = {},
pages = {106648},
doi = {10.1016/j.mimet.2022.106648},
pmid = {36470413},
issn = {1872-8359},
mesh = {Escherichia coli/genetics/metabolism ; *Protease La/genetics ; *Escherichia coli Proteins/genetics/metabolism ; ATP-Dependent Proteases/genetics ; CRISPR-Cas Systems/genetics ; Serine Endopeptidases/genetics/metabolism ; Genetic Engineering ; },
abstract = {The efficiency with which E.coli BL21 can be modified using CRISPR-Cas9 genetic engineering is several orders of magnitude lower than that of E. coli W3110. We show that the lack of Lon protease is responsible, and demonstrate that restoration of the Lon protease or knock-out of sulA improves CRISPR-Cas9 engineering efficiency of BL21 to levels comparable to E. coli W3110.},
}
@article {pmid35909075,
year = {2023},
author = {Chey, YCJ and Arudkumar, J and Aartsma-Rus, A and Adikusuma, F and Thomas, PQ},
title = {CRISPR applications for Duchenne muscular dystrophy: From animal models to potential therapies.},
journal = {WIREs mechanisms of disease},
volume = {15},
number = {1},
pages = {e1580},
doi = {10.1002/wsbm.1580},
pmid = {35909075},
issn = {2692-9368},
mesh = {Animals ; *Muscular Dystrophy, Duchenne/genetics ; Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; },
abstract = {CRISPR gene-editing technology creates precise and permanent modifications to DNA. It has significantly advanced our ability to generate animal disease models for use in biomedical research and also has potential to revolutionize the treatment of genetic disorders. Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disease that could potentially benefit from the development of CRISPR therapy. It is commonly associated with mutations that disrupt the reading frame of the DMD gene that encodes dystrophin, an essential scaffolding protein that stabilizes striated muscles and protects them from contractile-induced damage. CRISPR enables the rapid generation of various animal models harboring mutations that closely simulates the wide variety of mutations observed in DMD patients. These models provide a platform for the testing of sequence-specific interventions like CRISPR therapy that aim to reframe or skip DMD mutations to restore functional dystrophin expression. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics.},
}
@article {pmid35393779,
year = {2023},
author = {Hu, LF and Li, YX and Wang, JZ and Zhao, YT and Wang, Y},
title = {Controlling CRISPR-Cas9 by guide RNA engineering.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {14},
number = {1},
pages = {e1731},
doi = {10.1002/wrna.1731},
pmid = {35393779},
issn = {1757-7012},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, Kinetoplastida/genetics ; RNA/genetics ; RNA Interference ; Genetic Engineering ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is a product of million years of evolution by microbes to fight against invading genetic materials. Around 10 years ago, scientists started to repurpose the CRISPR as genetic tools by molecular engineering approaches. The guide RNA provides a versatile and unique platform for the innovation to improve and expand the application of CRISPR-Cas9 system. In this review, we will first introduce the basic sequence and structure of guide RNA and its role during the function of CRISPR-Cas9. We will then summarize recent progress on the development of various guide RNA engineering strategies. These strategies have been dedicated to improve the performance of CRISPR-Cas9, to achieve precise spatiotemporal control of CRISPR-Cas9, and to broaden the application of CRISPR-Cas9. Finally, we will briefly discuss the uniqueness and advantage of guide RNA-engineering based systems versus those with engineered Cas9 proteins and speculate potential future directions in guide RNA engineering. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Methods > RNA Nanotechnology Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.},
}
@article {pmid36638909,
year = {2023},
author = {Forsberg, KJ},
title = {Anti-CRISPR discovery: using magnets to find needles in haystacks.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {167952},
doi = {10.1016/j.jmb.2023.167952},
pmid = {36638909},
issn = {1089-8638},
abstract = {CRISPR-Cas immune systems in bacteria and archaea protect against viral infection, which has spurred viruses to develop dedicated inhibitors of these systems called anti-CRISPRs (Acrs). Like most host-virus arms races, many diverse examples of these immune and counter-immune proteins are encoded by the genomes of bacteria, archaea, and their viruses. For the case of Acrs, it is almost certain that just a small minority of nature's true diversity has been described. In this review, I discuss the various approaches used to identify these Acrs and speculate on the future for Acr discovery. Because Acrs can determine infection outcomes in nature and regulate CRISPR-Cas activities in applied settings, they have a dual importance to both host-virus conflicts and emerging biotechnologies. Thus, revealing the largely hidden world of Acrs should provide important lessons in microbiology that have the potential to ripple far beyond the field.},
}
@article {pmid36636380,
year = {2023},
author = {Mohamad, F and Alzahrani, RR and Alsaadi, A and Alrfaei, BM and Yassin, AEB and Alkhulaifi, MM and Halwani, M},
title = {An Explorative Review on Advanced Approaches to Overcome Bacterial Resistance by Curbing Bacterial Biofilm Formation.},
journal = {Infection and drug resistance},
volume = {16},
number = {},
pages = {19-49},
pmid = {36636380},
issn = {1178-6973},
abstract = {The continuous emergence of multidrug-resistant pathogens evoked the development of innovative approaches targeting virulence factors unique to their pathogenic cascade. These approaches aimed to explore anti-virulence or anti-infective therapies. There are evident concerns regarding the bacterial ability to create a superstructure, the biofilm. Biofilm formation is a crucial virulence factor causing difficult-to-treat, localized, and systemic infections. The microenvironments of bacterial biofilm reduce the efficacy of antibiotics and evade the host's immunity. Producing a biofilm is not limited to a specific group of bacteria; however, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus biofilms are exemplary models. This review discusses biofilm formation as a virulence factor and the link to antimicrobial resistance. In addition, it explores insights into innovative multi-targeted approaches and their physiological mechanisms to combat biofilms, including natural compounds, phages, antimicrobial photodynamic therapy (aPDT), CRISPR-Cas gene editing, and nano-mediated techniques.},
}
@article {pmid36631688,
year = {2023},
author = {Rasmussen, TS and Koefoed, AK and Deng, L and Muhammed, MK and Rousseau, GM and Kot, W and Sprotte, S and Neve, H and Franz, CMAP and Hansen, AK and Vogensen, FK and Moineau, S and Nielsen, DS},
title = {CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
pmid = {36631688},
issn = {1751-7370},
abstract = {Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.},
}
@article {pmid36563314,
year = {2023},
author = {Okafor, IC and Ha, T},
title = {Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.},
journal = {The journal of physical chemistry. B},
volume = {127},
number = {1},
pages = {45-51},
doi = {10.1021/acs.jpcb.2c05428},
pmid = {36563314},
issn = {1520-5207},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer ; DNA/metabolism ; },
abstract = {CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.},
}
@article {pmid36550277,
year = {2023},
author = {Wessels, HH and Méndez-Mancilla, A and Hao, Y and Papalexi, E and Mauck, WM and Lu, L and Morris, JA and Mimitou, EP and Smibert, P and Sanjana, NE and Satija, R},
title = {Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq.},
journal = {Nature methods},
volume = {20},
number = {1},
pages = {86-94},
pmid = {36550277},
issn = {1548-7105},
mesh = {*RNA/genetics ; *Chromatin ; CRISPR-Cas Systems/genetics ; },
abstract = {Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq), which leverages the RNA-targeting CRISPR-Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable CRISPR array that is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.},
}
@article {pmid36537539,
year = {2023},
author = {Liu, Y and Wang, F and Ge, S and Zhang, L and Zhang, Z and Liu, Y and Zhang, Y and Ge, S and Yu, J},
title = {Programmable T-Junction Structure-Assisted CRISPR/Cas12a Electrochemiluminescence Biosensor for Detection of Sa-16S rDNA.},
journal = {ACS applied materials &amp; interfaces},
volume = {15},
number = {1},
pages = {617-625},
doi = {10.1021/acsami.2c18930},
pmid = {36537539},
issn = {1944-8252},
mesh = {DNA, Ribosomal ; *Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; },
abstract = {Herein, a strand displacement amplification (SDA)-assisted CRISPR/Cas12a (LbCpf1) electrochemiluminescence (ECL) biosensor was fabricated for ultrasensitive identification of Staphylococcus aureus (Sa)-16S rDNA. A porphyrinic Zr metal-organic framework (MOF) (PCN-224) nanomaterial was prepared as the coreactant accelerator, which promoted the conversion of S2O8[2-] and SO4[*-], thus enhancing the reaction with CdS quantum dots (QDs) and amplifying the ECL emission signal. Meanwhile, with the presence of Sa-16S rDNA, the auxiliary probes and primers stimulated the SDA reaction under the action of Klenow fragment (3'-5' exo-) and Nt. BbvCI specifically recognized Sa-16S rDNA to form a defective T-junction structure and generated second primers to initiate the cycles. Such a structure transformed the input signal (Sa-16S rDNA) into substantial single-stranded DNA products (SP) through SDA. SP acted as activators and activated arbitrary side chain cleavage of CRISPR/Cas12a (trans-cleavage) and further realized effective annihilation of ECL signals. This ECL platform demonstrated desirable assay performance for Sa-16S rDNA with a wide response range of 1 fM to 10 nM, and the limit of detection was 0.437 fM (S/N = 3), showing good sensitivity and specificity. Therefore, the method not only expanded the applications of CRISPR/Cas12a but also opened up a novel strategy for clinical diagnosis.},
}
@article {pmid36525967,
year = {2023},
author = {Lee, J and Wang, J and Ally, R and Trzaska, S and Hickey, J and Mujica, A and Miloscio, L and Mastaitis, J and Morse, B and Smith, J and Atanasio, A and Chiao, E and Chen, H and Latuszek, A and Hu, Y and Valenzuela, D and Romano, C and Zambrowicz, B and Auerbach, W},
title = {Production of large, defined genome modifications in rats by targeting rat embryonic stem cells.},
journal = {Stem cell reports},
volume = {18},
number = {1},
pages = {394-409},
doi = {10.1016/j.stemcr.2022.11.012},
pmid = {36525967},
issn = {2213-6711},
mesh = {Humans ; Rats ; Animals ; Mice ; *Embryonic Stem Cells ; Gene Editing ; Genetic Engineering ; *Retinal Degeneration ; CRISPR-Cas Systems/genetics ; },
abstract = {Rats were more frequently used than mice to model human disease before mouse embryonic stem cells (mESCs) revolutionized genetic engineering in mice. Rat ESCs (rESCs) were first reported over 10 years ago, yet they are not as frequently used as mESCs. CRISPR-based gene editing in zygotes is widely used in rats but is limited by the difficulty of inserting or replacing DNA sequences larger than about 10 kb. We report here the generation of germline-competent rESC lines from several rat strains. These rESC lines maintain their potential for germline transmission after serial targeting with bacterial artificial chromosome (BAC)-based targeting vectors, and CRISPR-Cas9 cutting can increase targeting efficiency. Using these methods, we have successfully replaced entire rat genes spanning up to 101 kb with the human ortholog.},
}
@article {pmid36399647,
year = {2023},
author = {Lin, Y and Wilk, U and Pöhmerer, J and Hörterer, E and Höhn, M and Luo, X and Mai, H and Wagner, E and Lächelt, U},
title = {Folate Receptor-Mediated Delivery of Cas9 RNP for Enhanced Immune Checkpoint Disruption in Cancer Cells.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {19},
number = {2},
pages = {e2205318},
doi = {10.1002/smll.202205318},
pmid = {36399647},
issn = {1613-6829},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; B7-H1 Antigen/metabolism ; Ribonucleoproteins/genetics ; Gene Editing ; DNA ; *Neoplasms/therapy/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.},
}
@article {pmid36253612,
year = {2023},
author = {Cody, JP and Maher, MF and Nasti, RA and Starker, CG and Chamness, JC and Voytas, DF},
title = {Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana.},
journal = {Nature protocols},
volume = {18},
number = {1},
pages = {81-107},
pmid = {36253612},
issn = {1750-2799},
support = {T32 GM008347/GM/NIGMS NIH HHS/United States ; },
mesh = {*Agrobacterium/genetics ; *Tobacco/genetics ; Plants, Genetically Modified/genetics ; Coculture Techniques ; Gene Editing/methods ; Genome, Plant ; Soil ; CRISPR-Cas Systems ; Transformation, Genetic ; },
abstract = {There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.},
}
@article {pmid36628731,
year = {2023},
author = {Zhang, X and Qiu, H and Zhong, X and Yi, S and Jia, Z and Chen, L and Hu, S},
title = {A CRISPR/Cas12a-assisted array for Helicobacter pylori DNA analysis in saliva.},
journal = {Analytica chimica acta},
volume = {1239},
number = {},
pages = {340736},
doi = {10.1016/j.aca.2022.340736},
pmid = {36628731},
issn = {1873-4324},
mesh = {Humans ; Saliva ; *Helicobacter pylori/genetics ; *Helicobacter Infections/diagnosis/genetics ; CRISPR-Cas Systems ; DNA ; *Biosensing Techniques ; },
abstract = {Helicobacter pylori infection has become a threat to the world populations. This leads to an urgent need of an efficient and convenient approach to accurately diagnose H. pylori infection. Saliva-based diagnoses are particularly welcomed for their efficiency and convenience. Aiming at saliva sample analysis, we proposed a CRISPR/Cas12a-assisted array, which had integrated H. pylori concentration detection and genotype screening functions. Single-nucleotide variations (SNVs) could be distinguished using the screening array with different probes, and an isothermal cycling strategy was combined with the trans-cleavage activity of Cas12a for signal amplification to improve accuracy of the diagnosis. As a demonstration, the SNV screening array was fabricated by utilizing the hybridization efficiency difference caused by mismatched bases. The array was able to successfully distinguish between ten H. pylori genotypes, and combined with the successful SDA biosensing, it had a LOD of as low as 60 fM. It was also able to diagnose H. pylori infection in saliva samples from infected patients. Together, the developed array has a potential in large-scale clinical screening and is a promising tool for the diagnosis and prevention of H. pylori infection-related diseases.},
}
@article {pmid36628729,
year = {2023},
author = {Sun, H and Zhou, S and Liu, Y and Lu, P and Qi, N and Wang, G and Yang, M and Huo, D and Hou, C},
title = {A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection.},
journal = {Analytica chimica acta},
volume = {1239},
number = {},
pages = {340732},
doi = {10.1016/j.aca.2022.340732},
pmid = {36628729},
issn = {1873-4324},
mesh = {*Site-Specific DNA-Methyltransferase (Adenine-Specific) ; CRISPR-Cas Systems ; DNA Modification Methylases/metabolism ; Coloring Agents ; *Biosensing Techniques/methods ; DNA Methylation ; DNA/genetics/metabolism ; },
abstract = {DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3' end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10[-4] U/mL and a broad detection range from 2 × 10[-4] to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.},
}
@article {pmid36628706,
year = {2023},
author = {Kim, U and Lee, SY and Oh, SW},
title = {Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers.},
journal = {Analytica chimica acta},
volume = {1239},
number = {},
pages = {340679},
doi = {10.1016/j.aca.2022.340679},
pmid = {36628706},
issn = {1873-4324},
mesh = {*Escherichia coli O157/genetics ; CRISPR-Cas Systems ; Polymerase Chain Reaction/methods ; Food Microbiology ; },
abstract = {BACKGROUND: s: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required.<br><br>RESULTS: In this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 10[1]&#xa0;CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 10[3]&#xa0;CFU/g E. coli O157:H7.<br><br>CONCLUSION: This study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.},
}
@article {pmid36628703,
year = {2023},
author = {Qian, S and Chen, Y and Peng, C and Wang, X and Che, Y and Wang, T and Wu, J and Xu, J},
title = {Integrated slip valve-assisted fluidic chip coupling with CRISPR/Cas12a system for nucleic acid analysis.},
journal = {Analytica chimica acta},
volume = {1239},
number = {},
pages = {340670},
doi = {10.1016/j.aca.2022.340670},
pmid = {36628703},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Salmonella typhimurium ; },
abstract = {Currently, some on-site nucleic acid detection platforms have been developed. However, these platforms still need to be improved in device integration and multiple detection capability. In this work, an integrated dual nucleic acid analysis platform was developed by slip valve-assisted fluidic chip coupled with CRISPR/Cas12a system. All the reagents, including nucleic acid extraction, air-dried loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a detection reagents, were preloaded on the fluidic chip. Liquids transfer and stirring could be controlled by a slip valve and a syringe. By combining duplex LAMP reaction with two CRISPR detection units, CRISPR/Cas12a-based dual nucleic acid analysis was successfully constructed. Benefiting from high-quality nucleic acid extraction on the chip, as low as 30 copies/reaction of Vibrio parahaemolyticus (V. parahaemolyticus) and 20 copies/reaction of Salmonella typhimurium (S. typhimurium) could be simultaneously detected. Detection results could be observed by the naked eye under a portable ultraviolet lamp. The whole detection procedure was finished within 60 min. This method with integrated nucleic acid analysis, dual detection capability and fluorescence visualized results provides a new solution for on-site nucleic acid analysis.},
}
@article {pmid36627653,
year = {2023},
author = {Kwon, J and Kim, M and Hwang, W and Jo, A and Hwang, GH and Jung, M and Kim, UG and Cui, G and Kim, H and Eom, JH and Hur, JK and Lee, J and Kim, Y and Kim, JS and Bae, S and Lee, JK},
title = {Extru-seq: a method for predicting genome-wide Cas9 off-target sites with advantages of both cell-based and in vitro approaches.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {4},
pmid = {36627653},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Genome ; RNA, Guide, Kinetoplastida/genetics ; Gene Editing ; },
abstract = {We present a novel genome-wide off-target prediction method named Extru-seq and compare it with cell-based (GUIDE-seq), in vitro (Digenome-seq), and in silico methods using promiscuous guide RNAs with large numbers of valid off-target sites. Extru-seq demonstrates a high validation rate and retention of information about the intracellular environment, both beneficial characteristics of cell-based methods. Extru-seq also shows a low miss rate and could easily be performed in clinically relevant cell types with little optimization, which are major positive features of the in vitro methods. In summary, Extru-seq shows beneficial features of cell-based and in vitro methods.},
}
@article {pmid36626564,
year = {2023},
author = {Ferreira, SS and Reis, RS},
title = {CRISPR/Cas to enhance gene expression for crop trait improvement by editing miRNAs targets.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erad003},
pmid = {36626564},
issn = {1460-2431},
}
@article {pmid36576240,
year = {2022},
author = {Replogle, JM and Bonnar, JL and Pogson, AN and Liem, CR and Maier, NK and Ding, Y and Russell, BJ and Wang, X and Leng, K and Guna, A and Norman, TM and Pak, RA and Ramos, DM and Ward, ME and Gilbert, LA and Kampmann, M and Weissman, JS and Jost, M},
title = {Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {36576240},
issn = {2050-084X},
support = {R00GM130964/NH/NIH HHS/United States ; T32AI132120/NH/NIH HHS/United States ; RM1HG009490-01/NH/NIH HHS/United States ; F31NS115380/NH/NIH HHS/United States ; F30AG066418/NH/NIH HHS/United States ; Investigator//Howard Hughes Medical Institute/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.},
}
@article {pmid36350677,
year = {2023},
author = {Novak, LC and Chou, J and Colic, M and Bristow, CA and Hart, T},
title = {PICKLES v3: the updated database of pooled in vitro CRISPR knockout library essentiality screens.},
journal = {Nucleic acids research},
volume = {51},
number = {D1},
pages = {D1117-D1121},
pmid = {36350677},
issn = {1362-4962},
support = {R35GM130119/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Gene Library ; Genome ; Gene Knockout Techniques ; },
abstract = {PICKLES (https://pickles.hart-lab.org) is an updated web interface to a freely available database of genome-scale CRISPR knockout fitness screens in human cell lines. Using a completely rewritten interface, researchers can explore gene knockout fitness phenotypes across cell lines and tissue types and compare fitness profiles with fitness, expression, or mutation profiles of other genes. The database has been updated to include data from three CRISPR libraries (Avana, Score, and TKOv3), and includes information from 1162 whole-genome screens probing the knockout fitness phenotype of 18 959 genes. Source code for the interface and the integrated database are available for download.},
}
@article {pmid34695289,
year = {2023},
author = {Niu, Q and Wu, S and Xie, H and Wu, Q and Liu, P and Xu, Y and Lang, Z},
title = {Efficient A·T to G·C base conversions in dicots using adenine base editors expressed under the tomato EF1α promoter.},
journal = {Plant biotechnology journal},
volume = {21},
number = {1},
pages = {5-7},
pmid = {34695289},
issn = {1467-7652},
mesh = {*Solanum lycopersicum/genetics ; Adenine ; Gene Editing ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; },
}
@article {pmid36625124,
year = {2023},
author = {Feng, W and Zhang, H and Le, XC},
title = {Signal Amplification by the trans-Cleavage Activity of CRISPR-Cas Systems: Kinetics and Performance.},
journal = {Analytical chemistry},
volume = {95},
number = {1},
pages = {206-217},
doi = {10.1021/acs.analchem.2c04555},
pmid = {36625124},
issn = {1520-6882},
}
@article {pmid36624149,
year = {2023},
author = {Lam, DK and Feliciano, PR and Arif, A and Bohnuud, T and Fernandez, TP and Gehrke, JM and Grayson, P and Lee, KD and Ortega, MA and Sawyer, C and Schwaegerle, ND and Peraro, L and Young, L and Lee, SJ and Ciaramella, G and Gaudelli, NM},
title = {Improved cytosine base editors generated from TadA variants.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36624149},
issn = {1546-1696},
abstract = {Cytosine base editors (CBEs) enable programmable genomic C·G-to-T·A transition mutations and typically comprise a modified CRISPR-Cas enzyme, a naturally occurring cytidine deaminase, and an inhibitor of uracil repair. Previous studies have shown that CBEs utilizing naturally occurring cytidine deaminases may cause unguided, genome-wide cytosine deamination. While improved CBEs that decrease stochastic genome-wide off-targets have subsequently been reported, these editors can suffer from suboptimal on-target performance. Here, we report the generation and characterization of CBEs that use engineered variants of TadA (CBE-T) that enable high on-target C·G to T·A across a sequence-diverse set of genomic loci, demonstrate robust activity in primary cells and cause no detectable elevation in genome-wide mutation. Additionally, we report cytosine and adenine base editors (CABEs) catalyzing both A-to-I and C-to-U editing (CABE-Ts). Together with ABEs, CBE-Ts and CABE-Ts enable the programmable installation of all transition mutations using laboratory-evolved TadA variants with improved properties relative to previously reported CBEs.},
}
@article {pmid36622025,
year = {2022},
author = {Li, M and Qin, H and Long, Y and Cheng, M and Li, L and Huang, A and Wang, N and Duan, S},
title = {Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {190},
pages = {},
doi = {10.3791/64070},
pmid = {36622025},
issn = {1940-087X},
mesh = {*Liberibacter ; *Rhizobiaceae/genetics ; Recombinases/genetics ; CRISPR-Cas Systems ; Plant Diseases ; },
abstract = {The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.},
}
@article {pmid36614293,
year = {2023},
author = {Tian, Y and Zhou, Y and Gao, G and Zhang, Q and Li, Y and Lou, G and He, Y},
title = {Creation of Two-Line Fragrant Glutinous Hybrid Rice by Editing the Wx and OsBADH2 Genes via the CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36614293},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Odorants ; Plant Breeding ; Genes, Plant ; Gene Editing ; },
abstract = {Global food security has benefited from the development and promotion of the two-line hybrid rice system. Excellent eating quality determines the market competitiveness of hybrid rice varieties based on achieving the fundamental requirements of high yield and good adaptability. Developing sterile and restorer lines with improved quality for two-line hybrid breeding by editing quality genes with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is an efficient and practical alternative to the lengthy and laborious process of conventional breeding to improve rice quality. We edited Wx and OsBADH2 using CRISPR/Cas9 technology to produce both homozygous male sterile mutant lines and homozygous restorer mutant lines with Cas9-free. These mutants have a much lower amylose content while having a significantly higher 2-acetyl-1-pyrroline aroma content. Based on this, a fragrant glutinous hybrid rice was developed without too much effect on most agronomic traits. This study demonstrates the use of CRISPR/Cas9 in creating two-line fragrant glutinous hybrid rice by editing the components of the male sterile and the restorative lines.},
}
@article {pmid36613993,
year = {2022},
author = {Shin, YH and Park, YD},
title = {CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36613993},
issn = {1422-0067},
mesh = {*Brassica rapa/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Mutagenesis ; *Brassica/genetics ; },
abstract = {Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the 'CT001' pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, 'CT001'. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E1LFY-edited lines bolted later than the inbred line, 'CT001'. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.},
}
@article {pmid36613947,
year = {2022},
author = {Hamada, T and Yokoyama, S and Akahane, T and Matsuo, K and Tanimoto, A},
title = {Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36613947},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Glioblastoma/genetics ; Ribonucleoproteins/genetics/metabolism ; Cell Line ; Nucleotides/metabolism ; },
abstract = {Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.},
}
@article {pmid36613871,
year = {2022},
author = {Zhen, C and Hua, X and Jiang, X and Tong, G and Li, C and Yang, C and Cheng, Y},
title = {Cas9/gRNA-Mediated Mutations in PtrFLA40 and PtrFLA45 Reveal Redundant Roles in Modulating Wood Cell Size and SCW Synthesis in Poplar.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36613871},
issn = {1422-0067},
mesh = {*Wood/metabolism ; Lignin/metabolism ; CRISPR-Cas Systems ; Xylem/metabolism ; Plant Proteins/genetics/metabolism ; Mutation ; *Populus/metabolism ; Cell Wall/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Fasciclin-like arabinogalactan proteins (FLAs) play an important role in plant development and adaptation to the environment. However, the roles of FLAs in wood formation remain poorly understood. Here, we identified a total of 50 PtrFLA genes in poplar. They were classified into four groups: A to D, among which group A was the largest group with 28 members clustered into four branches. Most PtrFLAs of group A were dominantly expressed in developing xylem based on microarray and RT-qPCR data. The roles of PtrFLA40 and PtrFLA45 in group A were investigated via the Cas9/gRNA-induced mutation lines. Loss of PtrFLA40 and PtrFLA45 increased stem length and diameter in ptrfla40ptrfla45 double mutants, but not in ptrfla40 or ptrfla45 single mutants. Further, our findings indicated that the ptrfla40ptrfla45 mutants enlarged the cell size of xylem fibers and vessels, suggesting a negative modulation in stem xylem cell size. In addition, wood lignin content in the ptrfla40fla45 mutants was increased by nearly 9%, and the lignin biosynthesis-related genes were significantly up-regulated in the ptrfla40fla45 mutants, in agreement with the increase in wood lignin content. Overall, Cas9/gRNA-mediated mutations in PtrFLA40 and PtrFLA45 reveal redundant roles in modulating wood cell size and secondary cell wall (SCW) synthesis in poplar.},
}
@article {pmid36613839,
year = {2022},
author = {Kuzmin, AA and Tomilin, AN},
title = {Building Blocks of Artificial CRISPR-Based Systems beyond Nucleases.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36613839},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Genome ; Tissue Engineering ; Gene Regulatory Networks ; Gene Editing ; },
abstract = {Tools developed in the fields of genome engineering, precise gene regulation, and synthetic gene networks have an increasing number of applications. When shared with the scientific community, these tools can be used to further unlock the potential of precision medicine and tissue engineering. A large number of different genetic elements, as well as modifications, have been used to create many different systems and to validate some technical concepts. New studies have tended to optimize or improve existing elements or approaches to create complex synthetic systems, especially those based on the relatively new CRISPR technology. In order to maximize the output of newly developed approaches and to move from proof-of-principle experiments to applications in regenerative medicine, it is important to navigate efficiently through the vast number of genetic elements to choose those most suitable for specific needs. In this review, we have collected information regarding the main genetic elements and their modifications, which can be useful in different synthetic systems with an emphasis of those based on CRISPR technology. We have indicated the most suitable elements and approaches to choose or combine in planning experiments, while providing their deeper understanding, and have also stated some pitfalls that should be avoided.},
}
@article {pmid36613611,
year = {2022},
author = {Gabellini, C and Pucci, C and De Cesari, C and Martini, D and Di Lauro, C and Digregorio, M and Norton, W and Zippo, A and Sessa, A and Broccoli, V and Andreazzoli, M},
title = {CRISPR/Cas9-Induced Inactivation of the Autism-Risk Gene setd5 Leads to Social Impairments in Zebrafish.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36613611},
issn = {1422-0067},
mesh = {Animals ; Humans ; *Autistic Disorder/genetics ; Zebrafish/genetics/metabolism ; *Autism Spectrum Disorder/genetics/metabolism ; CRISPR-Cas Systems ; Brain/metabolism ; Methyltransferases/metabolism ; },
abstract = {Haploinsufficiency of the SETD5 gene, encoding a SET domain-containing histone methyltransferase, has been identified as a cause of intellectual disability and Autism Spectrum Disorder (ASD). Recently, the zebrafish has emerged as a valuable model to study neurodevelopmental disorders because of its genetic tractability, robust behavioral traits and amenability to high-throughput drug screening. To model human SETD5 haploinsufficiency, we generated zebrafish setd5 mutants using the CRISPR/Cas9 technology and characterized their morphological, behavioral and molecular phenotypes. According to our observation that setd5 is expressed in adult zebrafish brain, including those areas controlling social behavior, we found that setd5 heterozygous mutants exhibit defective aggregation and coordination abilities required for shoaling interactions, as well as indifference to social stimuli. Interestingly, impairment in social interest is rescued by risperidone, an antipsychotic drug used to treat behavioral traits in ASD individuals. The molecular analysis underscored the downregulation of genes encoding proteins involved in the synaptic structure and function in the adult brain, thus suggesting that brain hypo-connectivity could be responsible for the social impairments of setd5 mutant fishes. The zebrafish setd5 mutants display ASD-like features and are a promising setd5 haploinsufficiency model for drug screening aimed at reversing the behavioral phenotypes.},
}
@article {pmid36613549,
year = {2022},
author = {Kim, JY and Kim, JH and Jang, YH and Yu, J and Bae, S and Kim, MS and Cho, YG and Jung, YJ and Kang, KK},
title = {Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
pmid = {36613549},
issn = {1422-0067},
mesh = {*Solanum lycopersicum/genetics ; Transcriptome ; CRISPR-Cas Systems/genetics ; Gas Chromatography-Mass Spectrometry ; Carotenoids/metabolism ; Chlorophyll/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography-tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.},
}
@article {pmid36611948,
year = {2022},
author = {Xiu, K and Saunders, L and Wen, L and Ruan, J and Dong, R and Song, J and Yang, D and Zhang, J and Xu, J and Chen, YE and Ma, PX},
title = {Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.},
journal = {Cells},
volume = {12},
number = {1},
pages = {},
pmid = {36611948},
issn = {2073-4409},
support = {R01 HL136231/HL/NHLBI NIH HHS/United States ; R42 TR001711/TR/NCATS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; Plasmids/genetics ; *Nanoparticles ; DNA ; Polymers ; },
abstract = {Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.},
}
@article {pmid36611199,
year = {2023},
author = {Xie, X and Yang, J and Du, H and Chen, J and Sanganyado, E and Gong, Y and Du, H and Chen, W and Liu, Z and Liu, X},
title = {Golgi fucosyltransferase 1 reveals its important role in α-1,4-fucose modification of N-glycan in CRISPR/Cas9 diatom Phaeodactylum tricornutum.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {6},
pmid = {36611199},
issn = {1475-2859},
mesh = {*Fucosyltransferases/genetics/metabolism ; *Diatoms/genetics/metabolism ; Fucose/metabolism ; CRISPR-Cas Systems ; Recombinant Proteins/metabolism ; Polysaccharides/metabolism ; Golgi Apparatus/genetics/metabolism ; },
abstract = {Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum, PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.},
}
@article {pmid36609377,
year = {2023},
author = {Su, R and Wang, T and Bo, T and Cai, N and Yuan, M and Wu, C and Jiang, H and Peng, H and Chen, N and Li, Y},
title = {Enhanced production of D-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR-Cpf1 genome editing method.},
journal = {Microbial cell factories},
volume = {22},
number = {1},
pages = {3},
pmid = {36609377},
issn = {1475-2859},
mesh = {Humans ; *Gene Editing/methods ; Pantothenic Acid/genetics/metabolism ; *Corynebacterium glutamicum/genetics/metabolism ; Plasmids/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum.<br><br>RESULTS: In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62&#xa0;g/L from glucose only.<br><br>CONCLUSIONS: We developed a RecET-assisted CRISPR-Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.},
}
@article {pmid36484104,
year = {2022},
author = {Zhang, W and Mu, Y and Dong, K and Zhang, L and Yan, B and Hu, H and Liao, Y and Zhao, R and Shu, W and Ye, Z and Lu, Y and Wan, C and Sun, Q and Li, L and Wang, H and Xiao, X},
title = {PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA.},
journal = {Nucleic acids research},
volume = {50},
number = {22},
pages = {12674-12688},
pmid = {36484104},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins/metabolism ; DNA/chemistry ; DNA, Single-Stranded/genetics ; },
abstract = {Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combining pre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM-SE+ dsDNA). We discovered that CRISPR-Cas12a had special enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.},
}
@article {pmid36383017,
year = {2023},
author = {},
title = {Personalized TCR T Cells Enabled by CRISPR.},
journal = {Cancer discovery},
volume = {13},
number = {1},
pages = {6-7},
doi = {10.1158/2159-8290.CD-NB2022-0071},
pmid = {36383017},
issn = {2159-8290},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Receptors, Antigen, T-Cell/genetics/metabolism ; Gene Editing ; T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; },
abstract = {A 16-person trial demonstrated the feasibility and safety of using CRISPR gene editing to create personalized T-cell therapies equipped with neoantigen-specific T-cell receptors. However, the elaborate manufacturing process and limited efficacy of the engineered cells have prompted the therapy's sponsor to pursue other strategies.},
}
@article {pmid36215032,
year = {2022},
author = {Piñero-Lambea, C and Garcia-Ramallo, E and Miravet-Verde, S and Burgos, R and Scarpa, M and Serrano, L and Lluch-Senar, M},
title = {SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome.},
journal = {Nucleic acids research},
volume = {50},
number = {22},
pages = {e127},
pmid = {36215032},
issn = {1362-4962},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Mycoplasma pneumoniae/genetics ; Plasmids/genetics ; Bacteria/genetics ; },
abstract = {The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.},
}
@article {pmid36121194,
year = {2023},
author = {Zhen, S and Qiang, R and Lu, J and Tuo, X and Yang, X and Li, X},
title = {CRISPR/Cas9-HPV-liposome enhances antitumor immunity and treatment of HPV infection-associated cervical cancer.},
journal = {Journal of medical virology},
volume = {95},
number = {1},
pages = {e28144},
doi = {10.1002/jmv.28144},
pmid = {36121194},
issn = {1096-9071},
mesh = {Female ; Humans ; *Uterine Cervical Neoplasms/therapy/pathology ; Liposomes ; *Oncogene Proteins, Viral/genetics ; *Papillomavirus Infections/complications/therapy ; CRISPR-Cas Systems ; Repressor Proteins/genetics ; RNA ; Papillomavirus E7 Proteins/genetics ; Tumor Microenvironment ; },
abstract = {Increasing evidence shows that human papillomavirus (HPV) E6/E7 deletion in cervical cancer cells may be related to the immunosuppressive tumor microenvironment and adverse reactions or resistance to immune checkpoint blockade. Here, we demonstrate that liposome delivery of CRISPR/cas9 can effectively knock out HPV, which, in turn, induces autophagy and triggers cell death-related immune activation by releasing damage-related molecular patterns. The results of in vivo experiments showed that HPV-targeting guide RNA-liposomes could promote CD8+ T cell infiltration in tumor tissues; enhance the expression of proinflammatory cytokines, such as interleukin-12, tumor necrosis factor-α, and interferon-γ, and reduce regulatory T cells and myeloid suppressor cells. The combination of HPV-targeting guide RNA-liposomes with immune checkpoint inhibitors and antiprogrammed death-1 antibodies produced highly effective antitumor effects. In addition, combination therapy induced immune memory in the cervical cancer model.},
}
@article {pmid35980592,
year = {2023},
author = {Ashwath, P and Somanath, D and Sannejal, AD},
title = {CRISPR and Antisense RNA Technology: Exploiting Nature's Tool to Restrain Virulence in Tenacious Pathogens.},
journal = {Molecular biotechnology},
volume = {65},
number = {1},
pages = {17-27},
pmid = {35980592},
issn = {1559-0305},
mesh = {Virulence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; Virulence Factors/genetics ; CRISPR-Cas Systems ; },
abstract = {Pathogenic bacteria constitute a significant threat to mankind and at the same time represent a huge reservoir of abeyant therapeutics to prevent and treat various diseases. The concept of virulence determinants has been a compelling tool in driving research in the field of bacterial pathogenesis and infectious diseases. In this review, we highlight a few virulence elements forged by the pathogens from the viewpoint of the damage-response scaffold, vandalizing the susceptible host. Seeking an alternative to target the virulence determinants heads a road map toward the exemplary molecular approach. Hence, here we explore some of the exceptional applications of the clustered regulatory interspaced short palindromic repeat (CRISPR)- based therapy and antisense RNA (asRNA) approach, which could be exploited to selectively dismantle adamant components of the pathogen's virulence machinery. To the best of our knowledge, this is the first review paper involving both CRISPR and antisense RNA technology, as an alternative strategy to evade virulence mechanisms in bacterial pathogens.},
}
@article {pmid35691554,
year = {2022},
author = {Chen, X and Niu, X and Liu, Y and Zheng, R and Yang, L and Lu, J and Yin, S and Wei, Y and Pan, J and Sayed, A and Ma, X and Liu, M and Jing, F and Liu, M and Hu, J and Wang, L and Li, D},
title = {Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {49},
number = {12},
pages = {1114-1126},
doi = {10.1016/j.jgg.2022.06.001},
pmid = {35691554},
issn = {1673-8527},
mesh = {Rats ; Animals ; *Hemophilia B/therapy/drug therapy ; Factor IX/genetics/metabolism/therapeutic use ; CRISPR-Cas Systems/genetics ; Genetic Therapy ; },
abstract = {CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.},
}
@article {pmid36621223,
year = {2023},
author = {Alamillo, JM and López, CM and Martínez Rivas, FJ and Torralbo, F and Bulut, M and Alseekh, S},
title = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein and hairy roots: a perfect match for gene functional analysis and crop improvement.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102876},
doi = {10.1016/j.copbio.2022.102876},
pmid = {36621223},
issn = {1879-0429},
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene editing has become a powerful tool in genome manipulation for crop improvement. Advances in omics technologies, including genomics, transcriptomics, and metabolomics, allow the identification of causal genes that can be used to improve crops. However, the functional validation of these genetic components remains a challenge due to the lack of efficient protocols for crop engineering. Hairy roots gene editing using CRISPR/Cas, coupled with omics analyses, provide a platform for rapid, precise, and cost-effective functional analysis of genes. Here, we describe common requirements for efficient crop genome editing, focused on the transformation of recalcitrant legumes, and highlight the great opportunities that gene editing in hairy roots offers for future crop improvement.},
}
@article {pmid36619397,
year = {2022},
author = {Geng, B and Liu, S and Chen, Y and Wu, Y and Wang, Y and Zhou, X and Li, H and Li, M and Yang, S},
title = {A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {1110513},
pmid = {36619397},
issn = {2296-4185},
abstract = {Genome minimization is an effective way for industrial chassis development. In this study, Zymomonas mobilis ZMNP, a plasmid-free mutant strain of Z. mobilis ZM4 with four native plasmids deleted, was constructed using native type I-F CRISPR-Cas system. Cell growth of ZMNP under different temperatures and industrial effluent of xylose mother liquor were examined to investigate the impact of native plasmid removal. Despite ZMNP grew similarly as ZM4 under different temperatures, ZMNP had better xylose mother liquor utilization than ZM4. In addition, genomic, transcriptomic, and proteomic analyses were applied to unravel the molecular changes between ZM4 and ZMNP. Whole-genome resequencing result indicated that an S267P mutation in the C-terminal of OxyR, a peroxide-sensing transcriptional regulator, probably alters the transcription initiation of antioxidant genes for stress responses. Transcriptomic and proteomic studies illustrated that the reason that ZMNP utilized the toxic xylose mother liquor better than ZM4 was probably due to the upregulation of genes in ZMNP involving in stress responses as well as cysteine biosynthesis to accelerate the intracellular ROS detoxification and nucleic acid damage repair. This was further confirmed by lower ROS levels in ZMNP compared to ZM4 in different media supplemented with furfural or ethanol. The upregulation of stress response genes due to the OxyR mutation to accelerate ROS detoxification and DNA/RNA repair not only illustrates the underlying mechanism of the robustness of ZMNP in the toxic xylose mother liquor, but also provides an idea for the rational design of synthetic inhibitor-tolerant microorganisms for economic lignocellulosic biochemical production.},
}
@article {pmid36619383,
year = {2022},
author = {Dong, H and Wang, H and Fu, S and Zhang, D},
title = {CRISPR/Cas tools for enhancing the biopreservation ability of lactic acid bacteria in aquatic products.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {1114588},
pmid = {36619383},
issn = {2296-4185},
abstract = {Lactic acid bacteria (LAB) plays a crucial role in aquatic products biopreservation as it can inhibit many bacteria, in particular the specific spoilage organisms (SSOs) of aquatic products, by competing for nutrients or producing one or more metabolites which have antimicrobial activity, such as bacteriocins. Lactobacillus spp. and Lactococcus spp. are the most commonly used Lactic acid bacterias in aquatic products preservation. The improvement of gene editing tools is particularly important for developing new lactic acid bacteria strains with superior properties for aquatic products biopreservation. This review summarized the research progress of the most widely used CRISPR/Cas-based genome editing tools in Lactobacillus spp. and Lactococcus spp. The genome editing tools based on homologous recombination and base editor were described. Then, the research status of CRISPRi in transcriptional regulation was reviewed briefly. This review may provide a reference for the application of CRISPR/Cas-based genome editing tools to other lactic acid bacteria species.},
}
@article {pmid36614195,
year = {2023},
author = {Jia, Y and Guo, Z and Zhu, J and Qin, G and Sun, W and Yin, Y and Wang, H and Guo, R},
title = {Snap29 Is Dispensable for Self-Renewal Maintenance but Required for Proper Differentiation of Mouse Embryonic Stem Cells.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
doi = {10.3390/ijms24010750},
pmid = {36614195},
issn = {1422-0067},
abstract = {Pluripotent embryonic stem cells (ESCs) can self-renew indefinitely and are able to differentiate into all three embryonic germ layers. Synaptosomal-associated protein 29 (Snap29) is implicated in numerous intracellular membrane trafficking pathways, including autophagy, which is involved in the maintenance of ESC pluripotency. However, the function of Snap29 in the self-renewal and differentiation of ESCs remains elusive. Here, we show that Snap29 depletion via CRISPR/Cas does not impair the self-renewal and expression of pluripotency-associated factors in mouse ESCs. However, Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Furthermore, transcriptome analysis reveals that Snap29 depletion significantly decreased the expression of numerous genes required for germ layer differentiation. Interestingly, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our data show that Snap29 is dispensable for self-renewal maintenance, but required for the proper differentiation of mouse ESCs.},
}
@article {pmid36606637,
year = {2023},
author = {Yaqoob, H and Tariq, A and Bhat, BA and Bhat, KA and Nehvi, IB and Raza, A and Djalovic, I and Prasad, PV and Mir, RA},
title = {Integrating genomics and genome editing for orphan crop improvement: a bridge between orphan crops and modern agriculture system.},
journal = {GM crops &amp; food},
volume = {14},
number = {1},
pages = {1-20},
doi = {10.1080/21645698.2022.2146952},
pmid = {36606637},
issn = {2164-5701},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Plant Breeding ; Agriculture ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; Genomics ; },
abstract = {Domestication of orphan crops could be explored by editing their genomes. Genome editing has a lot of promise for enhancing agricultural output, and there is a lot of interest in furthering breeding in orphan crops, which are sometimes plagued with unwanted traits that resemble wild cousins. Consequently, applying model crop knowledge to orphan crops allows for the rapid generation of targeted allelic diversity and innovative breeding germplasm. We explain how plant breeders could employ genome editing as a novel platform to accelerate the domestication of semi-domesticated or wild plants, resulting in a more diversified base for future food and fodder supplies. This review emphasizes both the practicality of the strategy and the need to invest in research that advances our understanding of plant genomes, genes, and cellular systems. Planting more of these abandoned orphan crops could help alleviate food scarcities in the challenge of future climate crises.},
}
@article {pmid36604145,
year = {2022},
author = {Zhang, H and Yang, F and Yang, M and Liu, J and Wang, M and Fei, C and Zhang, L and Xue, F and Zhu, C and Liu, Y and Gu, F},
title = {An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips.},
journal = {Acta biochimica et biophysica Sinica},
volume = {54},
number = {11},
pages = {1630-1636},
doi = {10.3724/abbs.2022163},
pmid = {36604145},
issn = {1745-7270},
mesh = {*Nucleic Acid Amplification Techniques/methods ; Oseltamivir/pharmacology ; CRISPR-Cas Systems ; Recombinases/metabolism ; *Viruses/metabolism ; },
abstract = {Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.},
}
@article {pmid36593295,
year = {2023},
author = {Alonso-Lerma, B and Jabalera, Y and Samperio, S and Morin, M and Fernandez, A and Hille, LT and Silverstein, RA and Quesada-Ganuza, A and Reifs, A and Fernández-Peñalver, S and Benitez, Y and Soletto, L and Gavira, JA and Diaz, A and Vranken, W and Sanchez-Mejias, A and Güell, M and Mojica, FJM and Kleinstiver, BP and Moreno-Pelayo, MA and Montoliu, L and Perez-Jimenez, R},
title = {Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.},
journal = {Nature microbiology},
volume = {8},
number = {1},
pages = {77-90},
pmid = {36593295},
issn = {2058-5276},
mesh = {Humans ; *CRISPR-Cas Systems ; *Endonucleases/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing ; RNA, Guide, Kinetoplastida ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.},
}
@article {pmid36525857,
year = {2023},
author = {Xu, X and Zhang, X and Peng, X and Liu, C and Li, W and Liu, M},
title = {Introduction of the FecG[F] mutation in GDF9 gene via CRISPR/Cas9 system with single-stranded oligodeoxynucleotide.},
journal = {Theriogenology},
volume = {197},
number = {},
pages = {177-185},
doi = {10.1016/j.theriogenology.2022.11.044},
pmid = {36525857},
issn = {1879-3231},
mesh = {Pregnancy ; Female ; Animals ; Sheep/genetics ; *CRISPR-Cas Systems ; *Oligodeoxyribonucleotides ; Mutation ; Zygote ; Embryonic Development ; Gene Editing/veterinary ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has been a recent focus of breeders owing to its potential to improve economically significant traits of livestock. The introduction of defined point mutations into the ovine genome via CRISPR/Cas9-mediated homology-directed repair has been reported; however, indel and mosaic events observed in genetically modified animals limit the practical application of this system in sheep breeding. The FecG[F] mutation (g. G1111A, p. V371 M) in the growth differentiation factor 9 (GDF9) gene is strongly associated with litter size in Belclare and Norwegian White Sheep. In the present study, we introduced the FecG[F] mutation in GDF9 by co-injecting the CRISPR/Cas9 system, single-stranded oligodeoxynucleotide (ssODN), and Scr7 into ovine zygotes. Scr7 at various concentrations (0 μM, 1 μM, and 2 μM) had no adverse effects on embryonic development in vitro. No significant differences in total mutation, point mutation, and indel rates in embryos were observed among groups treated with different concentrations of Scr7. However, the mosaicism rates of embryos from zygotes microinjected with 1 and 2 μM Scr7 were significantly lower than that for 0 μM Scr7 (7.7% and 7.5% vs. 19.7%). We successfully obtained lambs with defined nucleotide substitutions by the coinjection of Cas9 mRNA, sgRNA, ssODN, and 1 μM Scr7 into Altay sheep zygotes. The single nucleotide mutation efficiency was 7.69% (3/39) in newborn lambs, with one mosaic. Our findings provide evidence that Scr7 could improve the specificity of the CRISPR/Cas9 system for the introduction of a defined point mutation in livestock to some extent.},
}
@article {pmid36403178,
year = {2023},
author = {Khiabani, A and Kohansal, MH and Keshavarzi, A and Shahraki, H and Kooshesh, M and Karimzade, M and Gholizadeh Navashenaq, J},
title = {CRISPR/Cas9, a promising approach for the treatment of β-thalassemia: a systematic review.},
journal = {Molecular genetics and genomics : MGG},
volume = {298},
number = {1},
pages = {1-11},
doi = {10.1007/s00438-022-01978-z},
pmid = {36403178},
issn = {1617-4623},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *beta-Thalassemia/genetics/therapy ; Mutation ; },
abstract = {The CRISPR/Cas9 technique is easily programmable, fast, more powerful, and efficient at generating a mutation compared to previous gene therapy methods. β-thalassemia is the most common autosomal recessive disorder worldwide. Appropriate genomic changes in the β gene can be modified to alleviate the symptoms of the disease using the CRISPR/Cas9 system. PubMed/Medline, Scopus, Web of Science, and SID databases were searched in Persian and English from February 2000 to September 2022. Finally, 39 articles had inclusion criteria which were reviewed by two separate individuals. Among the reviewed articles, articles were divided into three categories. In the first group, studies attemped to increase the expression of γ-globin and production of hemoglobin F. The strategy of second group of studies were the reduction of the α-globin chain to prevent hemolysis of RBCs by accumulation of excessive α-globins. The third group corrected the mutations causing β-thalassemia. Studies have shown that the genome of β-thalassemia patients can be modified using the CRISPR/Cas9 technique, and this approach might be promising for the treatment of β-thalassemia.},
}
@article {pmid36379415,
year = {2023},
author = {Cassidy, A and Onal, M and Pelletier, S},
title = {Novel methods for the generation of genetically engineered animal models.},
journal = {Bone},
volume = {167},
number = {},
pages = {116612},
doi = {10.1016/j.bone.2022.116612},
pmid = {36379415},
issn = {1873-2763},
mesh = {Animals ; Mice ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Genetic Engineering/methods ; },
abstract = {Genetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology.},
}
@article {pmid36245127,
year = {2023},
author = {Huang, X and Wang, M and Wu, X and Zou, Y and Xu, J and Cao, C and Ma, Q and Yu, B and Liu, Y and Gui, Y},
title = {Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {1},
pages = {260-268},
doi = {10.1016/j.ymthe.2022.10.009},
pmid = {36245127},
issn = {1525-0024},
mesh = {*CRISPR-Associated Protein 9/genetics ; Recombinational DNA Repair ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/genetics ; DNA Cleavage ; Gene Editing/methods ; },
abstract = {Accurate genome editing based on various molecular tools has always been the focus of gene-editing research and the primary goal for therapeutic application. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a well-established gene-editing method that is preferred due to its simplicity and high efficiency. In this study, a group of single-stranded DNA aptamers with high affinity and high specificity for the Cas9 protein were obtained by the systematic evolution of ligands through the exponential enrichment method. Their binding affinity and possible binding domains to the Cas9 protein were analyzed. In addition, we demonstrated the effectiveness of aptamers in regulating dCas9-modulated gene transcription, in terms of both transcriptional activation and repression. Additionally, the aptamers successfully reduced the off-target effect and improved the efficiency of gene homologous recombination repair mediated by CRISPR-Cas9. The findings suggest a potential method to better control precise gene editing and enrich the diversity of modulating tools for the CRISPR-Cas9 system.},
}
@article {pmid36114670,
year = {2023},
author = {Kweon, J and Hwang, HY and Ryu, H and Jang, AH and Kim, D and Kim, Y},
title = {Targeted genomic translocations and inversions generated using a paired prime editing strategy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {1},
pages = {249-259},
doi = {10.1016/j.ymthe.2022.09.008},
pmid = {36114670},
issn = {1525-0024},
mesh = {Humans ; *Translocation, Genetic ; Gene Rearrangement ; Genome ; *Neoplasms ; Endonucleases ; Genomics ; Receptor Protein-Tyrosine Kinases ; Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.},
}
@article {pmid35982622,
year = {2023},
author = {Crippa, S and Conti, A and Vavassori, V and Ferrari, S and Beretta, S and Rivis, S and Bosotti, R and Scala, S and Pirroni, S and Jofra-Hernandez, R and Santi, L and Basso-Ricci, L and Merelli, I and Genovese, P and Aiuti, A and Naldini, L and Di Micco, R and Bernardo, ME},
title = {Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {1},
pages = {230-248},
doi = {10.1016/j.ymthe.2022.08.011},
pmid = {35982622},
issn = {1525-0024},
mesh = {Humans ; Animals ; Mice ; Gene Editing ; CRISPR-Cas Systems ; Hematopoietic Stem Cells ; *Mesenchymal Stem Cells ; *Hematopoietic Stem Cell Transplantation/methods ; },
abstract = {Mesenchymal stromal cells (MSCs) have been employed in vitro to support hematopoietic stem and progenitor cell (HSPC) expansion and in vivo to promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.},
}
@article {pmid35622155,
year = {2023},
author = {Kang, H and Naing, AH and Park, SK and Chung, MY and Kim, CK},
title = {Protoplast isolation and transient gene expression in different petunia cultivars.},
journal = {Protoplasma},
volume = {260},
number = {1},
pages = {271-280},
pmid = {35622155},
issn = {1615-6102},
mesh = {*CRISPR-Cas Systems ; *Petunia/genetics ; Protoplasts ; Gene Editing/methods ; Gene Expression ; },
abstract = {The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 10[5] protoplast density, 40% polyethylene glycol (PEG) concentration, 10 µg plasmid DNA, and 15 min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein-protein interactions, and functional gene expression in the petunias.},
}
@article {pmid36613684,
year = {2022},
author = {Kurishev, AO and Karpov, DS and Nadolinskaia, NI and Goncharenko, AV and Golimbet, VE},
title = {CRISPR/Cas-Based Approaches to Study Schizophrenia and Other Neurodevelopmental Disorders.},
journal = {International journal of molecular sciences},
volume = {24},
number = {1},
pages = {},
doi = {10.3390/ijms24010241},
pmid = {36613684},
issn = {1422-0067},
abstract = {The study of diseases of the central nervous system (CNS) at the molecular level is challenging because of the complexity of neural circuits and the huge number of specialized cell types. Moreover, genomic association studies have revealed the complex genetic architecture of schizophrenia and other genetically determined mental disorders. Investigating such complex genetic architecture to decipher the molecular basis of CNS pathologies requires the use of high-throughput models such as cells and their derivatives. The time is coming for high-throughput genetic technologies based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas systems to manipulate multiple genomic targets. CRISPR/Cas systems provide the desired complexity, versatility, and flexibility to create novel genetic tools capable of both altering the DNA sequence and affecting its function at higher levels of genetic information flow. CRISPR/Cas tools make it possible to find and investigate the intricate relationship between the genotype and phenotype of neuronal cells. The purpose of this review is to discuss innovative CRISPR-based approaches for studying the molecular mechanisms of CNS pathologies using cellular models.},
}
@article {pmid36610752,
year = {2023},
author = {Botelho, J and Cazares, A and Schulenburg, H},
title = {The ESKAPE mobilome contributes to the spread of antimicrobial resistance and CRISPR-mediated conflict between mobile genetic elements.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac1220},
pmid = {36610752},
issn = {1362-4962},
abstract = {Mobile genetic elements (MGEs) mediate the shuffling of genes among organisms. They contribute to the spread of virulence and antibiotic resistance (AMR) genes in human pathogens, such as the particularly problematic group of ESKAPE pathogens. Here, we performed the first systematic analysis of MGEs, including plasmids, prophages, and integrative and conjugative/mobilizable elements (ICEs/IMEs), across all ESKAPE pathogens. We found that different MGE types are asymmetrically distributed across these pathogens, and that most horizontal gene transfer (HGT) events are restricted by phylum or genus. We show that the MGEs proteome is involved in diverse functional processes and distinguish widespread proteins within the ESKAPE context. Moreover, anti-CRISPRs and AMR genes are overrepresented in the ESKAPE mobilome. Our results also underscore species-specific trends shaping the number of MGEs, AMR, and virulence genes across pairs of conspecific ESKAPE genomes with and without CRISPR-Cas systems. Finally, we observed that CRISPR spacers found on prophages, ICEs/IMEs, and plasmids have different targeting biases: while plasmid and prophage CRISPRs almost exclusively target other plasmids and prophages, respectively, ICEs/IMEs CRISPRs preferentially target prophages. Overall, our study highlights the general importance of the ESKAPE mobilome in contributing to the spread of AMR and mediating conflict among MGEs.},
}
@article {pmid36610369,
year = {2023},
author = {Liu, T and Zhang, X and Li, K and Yao, Q and Zhong, D and Deng, Q and Lu, Y},
title = {Large-scale genome editing in plants: approaches, applications, and future perspectives.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102875},
doi = {10.1016/j.copbio.2022.102875},
pmid = {36610369},
issn = {1879-0429},
abstract = {As a powerful genome editing technology, CRISPR/Cas is revolutionizing both fundamental research and crop breeding, and has now evolved into large-scale editing tools that are efficient, simple, and programmable. With such CRISPR screening technologies, the numbers of genome-edited crops are rapidly increasing. Here, we describe the general workflow of a CRISPR screen in plants, including the selection of appropriate editors, genome-wide guide RNA design, pooled library construction, massive transformation, and high-throughput genotyping. We also discuss applications for the screening of candidate genes, the optimization of spatiotemporal expression, the evolution of protein activities, and the establishment of genome-wide libraries of knockout mutant. After considering the current challenges and limitations, we finally envision a virus-mediated strategy to improve CRISPR screens.},
}
@article {pmid36610368,
year = {2023},
author = {Han, YH and Kim, G and Seo, SW},
title = {Programmable synthetic biology tools for developing microbial cell factories.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102874},
doi = {10.1016/j.copbio.2022.102874},
pmid = {36610368},
issn = {1879-0429},
abstract = {Microbial conversion to generate value-added chemicals from diverse biomass is one of the keystones of energy biotechnology. Programmable synthetic biology tools offer versatile, standardized options for developing microbial cell factories. These tools thus can be reprogrammed in a user-defined manner for flexible wiring of stimuli and response, highly efficient genome engineering, and extensive perturbation of metabolic flux and genetic circuits. They also can be modularly assembled to construct elaborate and unprecedented biological systems with unique features. This review highlights recent advances in programmable synthetic biology tools based on biosensors, CRISPR-Cas, and RNA devices for developing microbial cell factories that have the potential to be utilized for energy biotechnology.},
}
@article {pmid36610218,
year = {2022},
author = {Lahm, H and Stieglbauer, S and Neb, I and Doppler, SA and Schneider, S and Dzilic, E and Lange, R and Krane, M and Dreßen, M},
title = {Generation of two CRISPR/Cas edited human induced pluripotent stem cell lines (DHMi005-A-3 and DHMi005-A-4) carrying a FLAG-tag after exon 9 of the TBX5 gene.},
journal = {Stem cell research},
volume = {66},
number = {},
pages = {103011},
doi = {10.1016/j.scr.2022.103011},
pmid = {36610218},
issn = {1876-7753},
abstract = {Although TBX5 plays a major role during human cardiogenesis and initiates and controls limb development, many of its interactions with genomic DNA and the resulting biological consequences are not well known. Existing anti-TBX5-antibodies work very inefficiently in certain applications such as ChIP-Seq analysis. To circumvent this drawback, we introduced a FLAG-tag sequence into the TBX5 locus at the end of exon 9 prior to the stop codon by CRISPR/Cas9. The expressed TBX5-FLAG fusion protein can effectively be precipitated by anti-FLAG antibodies. Therefore, these gene-edited iPSC lines represent powerful cellular in vitro tools to unravel TBX5:DNA interactions in detail.},
}
@article {pmid36610156,
year = {2022},
author = {Shen, B and Wang, Y and Cheng, J and Peng, Y and Zhang, Q and Li, Z and Zhao, L and Deng, X and Feng, H},
title = {Pterostilbene alleviated NAFLD via AMPK/mTOR signaling pathways and autophagy by promoting Nrf2.},
journal = {Phytomedicine : international journal of phytotherapy and phytopharmacology},
volume = {109},
number = {},
pages = {154561},
doi = {10.1016/j.phymed.2022.154561},
pmid = {36610156},
issn = {1618-095X},
abstract = {BACKGROUND: NAFLD is a liver disease that is caused by liver damage or extreme lipid deposition but not alcohol. Nrf2 could mediate resistance to oxidative stress injury. Autophagy can degrade metabolic waste and accumulated toxic endogenous substances. Pterostilbene (PTE) is an active compound extracted from blueberry, and grape, that exhibits many biological effects, such as antiinflammation and antitumor.<br><br>PURPOSE: This study provides a mechanism of PTE affecting on oxidative stress and autophagy in NAFLD mice. Tyloxapol, oil acid (OA) and palmitic acid (PA) were used to induce lipid accumulation in mice and HepG2 cells.<br><br>METHODS: Western blotting, CRISPR/Cas 9 and other molecular biological approaches were applied to explore the mechanisms of PTE effected on NAFLD.<br><br>RESULTS: PTE pretreatment effectively reduced the lipid accumulation in OA and PA induced HepG2 cells and tyloxapol induced mice, and significantly promoted the expression of nNrf2, PPAR-&#x3b1; and HO-1, and AMPK activity, but inhibited the expression of mTORC 1 and SREBP-1c. PTE activated phosphatidylinositide 3-kinase (PI3K) and proteins in the autophagy-related gene (ATG) family, and promoted the transformation of LC3&#x2160; to LC3&#x2161; which indicated the activation of autophagy, however, these effects were abolished after Nrf2 knockout.<br><br>CONCLUSION: PTE effectively alleviated oxidative stress damage induced by excessive lipid accumulation in hepatocytes, thus promoting the metabolism and decomposition of fatty acids to improve NAFLD.},
}
@article {pmid36609885,
year = {2023},
author = {Yu, S and Cao, S and He, S and Zhang, K},
title = {Locus-Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR-Cas Assisted Biosensors.},
journal = {Small methods},
volume = {},
number = {},
pages = {e2201624},
doi = {10.1002/smtd.202201624},
pmid = {36609885},
issn = {2366-9608},
abstract = {Deoxyribonucleic acid (DNA) methylation is one of the epigenetic characteristics that result in heritable and revisable phenotype changes but without sequence changes in DNA. Aberrant methylation occurring at a specific locus was reported to be associated with cancers, insulin resistance, obesity, Alzheimer's disease, Parkinson's disease, etc. Therefore, locus-specific DNA methylation can serve as a valuable biomarker for disease diagnosis and therapy. Recently, Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are applied to develop biosensors for DNA, ribonucleic acid, proteins, and small molecules detection. Because of their highly specific binding ability and signal amplification capacity, CRISPR-Cas assisted biosensor also serve as a potential tool for locus-specific detection of DNA methylation. In this perspective, based on the detection principle, a detailed classification and comprehensive discussion of recent works about the latest advances in locus-specific detection of DNA methylation using CRISPR-Cas systems are provided. Furthermore, current challenges and future perspectives of CRISPR-based locus-specific detection of DNA methylation are outlined.},
}
@article {pmid36605051,
year = {2022},
author = {George, A and Ravi, NS and Prasad, K and Panigrahi, L and Koikkara, S and Rajendiran, V and Devaraju, N and Paul, J and Pai, AA and Nakamura, Y and Kurita, R and Balasubramanian, P and Thangavel, S and Marepally, S and Velayudhan, SR and Srivastava, A and Mohankumar, KM},
title = {Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {1085111},
pmid = {36605051},
issn = {2673-3439},
abstract = {Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.},
}
@article {pmid36603502,
year = {2023},
author = {Pan, C and Li, G and Bandyopadhyay, A and Qi, Y},
title = {Guide RNA library-based CRISPR screens in plants: opportunities and challenges.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102883},
doi = {10.1016/j.copbio.2022.102883},
pmid = {36603502},
issn = {1879-0429},
abstract = {Next-generation sequencing technologies have revolutionized our ability to read sequence information at the genome and transcriptome levels in a high-throughput manner. However, genetic screening at a large or genomic scale remains challenging in plants. Recently, the RNA-guided CRISPR-Cas nucleases have been optimized for high-throughput functional genomic screens combined with guide RNA (gRNA) libraries in plants. This approach has shown great promise in facilitating genetic screening, directed evolution, and quantitative trait engineering. However, this technology is still in its infancy. In this short review, we describe the recent progress in gRNA library-based CRISPR screens in plants. We provide a critical assessment of the current approaches and emerging delivery methods for CRISPR screens. We also highlight the challenges and present future perspectives on CRISPR screens in plants.},
}
@article {pmid36602175,
year = {2022},
author = {Singh, R and Chandel, S and Ghosh, A and Gautam, A and Huson, DH and Ravichandiran, V and Ghosh, D},
title = {Easy efficient HDR-based targeted knock-in in Saccharomyces cerevisiae genome using CRISPR-Cas9 system.},
journal = {Bioengineered},
volume = {13},
number = {6},
pages = {14857-14871},
doi = {10.1080/21655979.2022.2162667},
pmid = {36602175},
issn = {2165-5987},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Genome ; Metabolic Engineering/methods ; Homologous Recombination ; Gene Editing/methods ; },
abstract = {During the last two decades, yeast has been used as a biological tool to produce various small molecules, biofuels, etc., using an inexpensive bioprocess. The application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) techniques in yeast genetic and metabolic engineering has made a paradigm shift, particularly with a significant improvement in targeted chromosomal integration using synthetic donor constructs, which was previously a challenge. This study reports the CRISPR-Cas9-based highly efficient strategy for targeted chromosomal integration and in-frame expression of a foreign gene in the genome of Saccharomyces cerevisiae (S. cerevisiae) by homology-dependent recombination (HDR); our optimized methods show that CRISPR-Cas9-based chromosomal targeted integration of small constructs at multiple target sites of the yeast genome can be achieved with an efficiency of 74%. Our study also suggests that 15 bp microhomology flanked arms are sufficient for 50% targeted knock-in at minimal knock-in construct concentration. Whole-genome sequencing confirmed that there is no off-target effect. This study provides a comprehensive and streamlined protocol that will support the targeted integration of essential genes into the yeast genome for synthetic biology and other industrial purposes.Highlights• CRISPR-Cas9 based in-frame expression of foreign protein in Saccharomyces cerevisiae using Homology arm without a promoter.• As low as 15 base pairs of microhomology (HDR) are sufficient for targeted integration in Saccharomyces cerevisiae.• The methodology is highly efficient and very specific as no off-targeted effects were shown by the whole-genome sequence.},
}
@article {pmid36598341,
year = {2023},
author = {Schjeide, BM and Püschel, GP},
title = {Determining On-Target, Off-Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe-Harbor Modification of iPSCs Using Double-Control Quantitative Copy Number PCR.},
journal = {Current protocols},
volume = {3},
number = {1},
pages = {e635},
doi = {10.1002/cpz1.635},
pmid = {36598341},
issn = {2691-1299},
mesh = {Animals ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; DNA Copy Number Variations ; Animals, Genetically Modified ; Polymerase Chain Reaction ; },
abstract = {Double-control quantitative copy number PCR (dc-qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert-confirmation PCR, the technique can quickly and easily identify clones containing on-target heterozygous or homozygous donor DNA integrations and exclude off-target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription-activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost-prohibitive or create dependency on manufacturers for assay design and production. The dc-qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step-by-step instructions to prepare and perform the dc-qcnPCR, and its companion insert-confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90-4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert-confirmation PCR Basic Protocol 3: Design and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells.},
}
@article {pmid35036088,
year = {2022},
author = {Zhang, S and Wu, S and Hu, C and Yang, Q and Dong, T and Sheng, O and Deng, G and He, W and Dou, T and Li, C and Sun, C and Yi, G and Bi, F},
title = {Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct.},
journal = {PeerJ},
volume = {10},
number = {},
pages = {e12664},
pmid = {35036088},
issn = {2167-8359},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Musa/genetics ; Mutation ; Mutagenesis, Site-Directed ; },
abstract = {The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species' triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.},
}
@article {pmid36601854,
year = {2023},
author = {Avaro, AS and Santiago, JG},
title = {A critical review of microfluidic systems for CRISPR assays.},
journal = {Lab on a chip},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2lc00852a},
pmid = {36601854},
issn = {1473-0189},
abstract = {Reviewed are nucleic acid detection assays that incorporate clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics and microfluidic devices and techniques. The review serves as a reference for researchers who wish to use CRISPR-Cas systems for diagnostics in microfluidic devices. The review is organized in sections reflecting a basic five-step workflow common to most CRISPR-based assays. These steps are analyte extraction, pre-amplification, target recognition, transduction, and detection. The systems described include custom microfluidic chips and custom (benchtop) chip control devices for automated assays steps. Also included are partition formats for digital assays and lateral flow biosensors as a readout modality. CRISPR-based, microfluidics-driven assays offer highly specific detection and are compatible with parallel, combinatorial implementation. They are highly reconfigurable, and assays are compatible with isothermal and even room temperature operation. A major drawback of these assays is the fact that reports of kinetic rates of these enzymes have been highly inconsistent (many demonstrably erroneous), and the low kinetic rate activity of these enzymes limits achievable sensitivity without pre-amplification. Further, the current state-of-the-art of CRISPR assays is such that nearly all systems rely on off-chip assays steps, particularly off-chip sample preparation.},
}
@article {pmid36599979,
year = {2023},
author = {Dmytrenko, O and Neumann, GC and Hallmark, T and Keiser, DJ and Crowley, VM and Vialetto, E and Mougiakos, I and Wandera, KG and Domgaard, H and Weber, J and Gaudin, T and Metcalf, J and Gray, BN and Begemann, MB and Jackson, RN and Beisel, CL},
title = {Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {36599979},
issn = {1476-4687},
abstract = {Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate[1,2]. Several RNA-targeting CRISPR-Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases[3-5]. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.},
}
@article {pmid36596996,
year = {2023},
author = {Kong, W and Wang, M and Huang, L and Wu, F and Tao, J and Mo, B and Yu, Y},
title = {A high-efficient and naked-eye visible CRISPR/Cas9 system in Arabidopsis.},
journal = {Planta},
volume = {257},
number = {2},
pages = {30},
pmid = {36596996},
issn = {1432-2048},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; },
abstract = {Introducing 35S-dsRED2 into the Cas9 vector which expresses naked-eye visible dsRED2 greatly facilitates the genetic screening, and the WUS promoter driving the Cas9 expression can improve editing efficiency in Arabidopsis. CRISPR/Cas9-dependent genome editing has been applied to generate random insertions and deletions, targeted insertions or replacements, and precise base changes for both fundamental studies in many plant species and crop improvement. To simplify the screening procedure for target gene-edited transformants, we introduced a CaMV 35S-driven dsRED2 cassette (35S-dsRED2) into the Cas9 vector to express the naked-eye visible protein dsRED2, which can be observed under white light, greatly facilitated the genetic screening and reduced labor intensity without using any instrument. In addition, the WUS promoter was used to drive the expression of Cas9, which successfully improved the target genes editing efficiency and enabled the homozygous mutagenesis of two genes in T1 generation in Arabidopsis. Considering the conserved function and expression pattern of WUS across the plant species, this dsRED2-WUS/Cas9 system could also be used in many crops.},
}
@article {pmid36596482,
year = {2023},
author = {Tomizawa, I and Chiu, YW and Hori, Y and Tomita, T},
title = {[Identification of novel regulators involved in AD pathogenesis using the CRISPR-Cas9 system].},
journal = {Nihon yakurigaku zasshi. Folia pharmacologica Japonica},
volume = {158},
number = {1},
pages = {21-25},
doi = {10.1254/fpj.22081},
pmid = {36596482},
issn = {0015-5691},
mesh = {Humans ; *Alzheimer Disease/genetics/metabolism ; Amyloid beta-Peptides/genetics/metabolism ; Amyloid Precursor Protein Secretases/genetics/metabolism ; CRISPR-Cas Systems ; Amyloid beta-Protein Precursor/genetics/metabolism ; Neurons/metabolism ; },
abstract = {The production of amyloid β peptide (Aβ) is an important process relating to the pathogenesis of Alzheimer disease (AD). It is widely known that the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases lead to the production of Aβ. However, the precise regulatory mechanism for Aβ production remains unclear. We have established a CRISPR-Cas9 based screening system to identify the novel regulators of Aβ production. Calcium and integrin-binding protein 1 (CIB1) was identified as a novel potential negative regulator of Aβ production. The knockdown and knockout of Cib1 significantly increased Aβ levels. In addition, immunoprecipitation showed that CIB1 interacts with the γ-secretase complex but did not alter its enzymatic activity. Moreover, Cib1 disruption specifically reduced the cell-surface localization of the γ-secretase complex. Finally, the single-cell RNA-seq analysis in the human brain demonstrated that early-stage AD patients have lower neuronal CIB1 mRNA levels compared to healthy controls. Taken together, we have shown that CIB1 controls the subcellular localization of γ-secretase, resulting in the regulation of Aβ production, suggesting the involvement of CIB1 in the development of AD pathogenesis.},
}
@article {pmid36595926,
year = {2022},
author = {Zheng, X and Cairns, T and Zheng, P and Meyer, V and Sun, J},
title = {Protocol for gene characterization in Aspergillus niger using 5S rRNA-CRISPR-Cas9-mediated Tet-on inducible promoter exchange.},
journal = {STAR protocols},
volume = {3},
number = {4},
pages = {101838},
pmid = {36595926},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Aspergillus niger/genetics ; RNA, Ribosomal, 5S ; Promoter Regions, Genetic/genetics ; },
abstract = {This protocol presents an efficient genetic strategy to investigate gene function in the fungus Aspergillus niger. We combined 5S rRNA-CRISPR-Cas9 technology with Tet-on gene switch to generate conditional-expression mutants via precisely replacing native promoter with inducible promoter. We describe the design and DNA preparation for sgRNAs and donor DNA. We then detail the steps for DNA co-transformation into A. niger protoplasts by PEG-mediated transformation, followed by homozygote isolation. Finally, we describe the genome verification and strain validation of the isolates. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2019).[1].},
}
@article {pmid36414074,
year = {2023},
author = {Ma, P and Guo, H and Ye, H and Zhang, Y and Wang, Z},
title = {Structural insights into the AFB1 aptamer coupled with a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB1 detection.},
journal = {International journal of biological macromolecules},
volume = {225},
number = {},
pages = {1164-1171},
doi = {10.1016/j.ijbiomac.2022.11.177},
pmid = {36414074},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; *Aptamers, Nucleotide/chemistry ; Food Contamination/analysis ; Aflatoxin B1/chemistry ; Limit of Detection ; },
abstract = {Aflatoxin B1 (AFB1) is a typical food contaminant. A truncated DNA aptamer of AFB1 was reported by our team in previous work. However, the recognition mechanism between aptamer and AFB1 was lacking, which was crucial for the design of related aptasensor. Herein, the binding of aptamer to AFB1 was systematically studied and found that it was an exothermic process and the conformation of aptamer changed during the recognition process. Loop bases in the secondary structure of aptamer formed a special binding pocket to recognize AFB1. Van der Waals and electrostatic interaction were the main driving forces. By blocking the stem bases guided by the structural investigation, a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB1 detection was constructed, and the sensitivity was improved by target recycling. Under optimal conditions, the linear detection range for AFB1 was 0.01-20 ng/mL, and AFB1 was accurately determined in corn and wheat samples. This work laid a theoretical foundation for the design of AFB1 aptasensor, and the developed detection model came up with new ideas for the development of CRISPR/Cas12a-based aptasensor.},
}
@article {pmid36598279,
year = {2023},
author = {Xu, C and Rao, J and Xie, Y and Lu, J and Li, Z and Dong, C and Wang, L and Jiang, J and Chen, C and Chen, S},
title = {The DNA Phosphorothioation Restriction-Modification System Influences the Antimicrobial Resistance of Pathogenic Bacteria.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0350922},
doi = {10.1128/spectrum.03509-22},
pmid = {36598279},
issn = {2165-0497},
abstract = {Bacterial defense barriers, such as DNA methylation-associated restriction-modification (R-M) and the CRISPR-Cas system, play an important role in bacterial antimicrobial resistance (AMR). Recently, a novel R-M system based on DNA phosphorothioate (PT) modification has been shown to be widespread in the kingdom of Bacteria as well as Archaea. However, the potential role of the PT R-M system in bacterial AMR remains unclear. In this study, we explored the role of PT R-Ms in AMR with a series of common clinical pathogenic bacteria. By analyzing the distribution of AMR genes related to mobile genetic elements (MGEs), it was shown that the presence of PT R-M effectively reduced the distribution of horizontal gene transfer (HGT)-derived AMR genes in the genome, even in the bacteria that did not tend to acquire AMR genes by HGT. In addition, unique gene variation analysis based on pangenome analysis and MGE prediction revealed that the presence of PT R-M could suppress HGT frequency. Thus, this is the first report showing that the PT R-M system has the potential to repress HGT-derived AMR gene acquisition by reducing the HGT frequency. IMPORTANCE In this study, we demonstrated the effect of DNA PT modification-based R-M systems on horizontal gene transfer of AMR genes in pathogenic bacteria. We show that there is no apparent association between the genetic background of the strains harboring PT R-Ms and the number of AMR genes or the kinds of gene families. The strains equipped with PT R-M harbor fewer plasmid-derived, prophage-derived, or integrating mobile genetic element (iMGE)-related AMR genes and have a lower HGT frequency, but the degree of inhibition varies among different bacteria. In addition, compared with Salmonella enterica and Escherichia coli, Klebsiella pneumoniae prefers to acquire MGE-derived AMR genes, and there is no coevolution between PT R-M clusters and bacterial core genes.},
}
@article {pmid36597180,
year = {2023},
author = {Amiri, S and Adibzadeh, S and Ghanbari, S and Rahmani, B and Kheirandish, MH and Farokhi-Fard, A and Dastjerdeh, MS and Davami, F},
title = {CRISPR-interceded CHO Cell Line Development approaches.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28329},
pmid = {36597180},
issn = {1097-0290},
abstract = {For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high-quality biologics with human-like post-translational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long-term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR-Cas systems, the use of CRISPR-Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast-track product development process with consistent, improved product yield and quality. This article is protected by copyright. All rights reserved.},
}
@article {pmid36593207,
year = {2022},
author = {Shen, Y and Chen, Z and Chen, J and Zhao, B and Lü, J and Gui, L and Lu, F and Li, M},
title = {[An efficient marker-free genome editing method for Aspergillus niger].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {12},
pages = {4744-4755},
doi = {10.13345/j.cjb.220162},
pmid = {36593207},
issn = {1872-2075},
mesh = {*Gene Editing ; *Aspergillus niger/genetics ; CRISPR-Cas Systems/genetics ; Plasmids/genetics ; },
abstract = {Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.},
}
@article {pmid36592319,
year = {2023},
author = {Liu, Y and Liu, Y and Zheng, P and Wang, Y and Wang, M},
title = {Cytosine Base Editing in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {219-231},
pmid = {36592319},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Cytosine ; Mutation ; DNA ; Bacteria/genetics ; },
abstract = {Base editing is a new genome editing technology that enables DNA base mutations without requiring double-stranded DNA backbone cleavage or a donor template. It has been widely used for genome engineering of eukaryotic and prokaryotic microorganisms. In this chapter, we describe a routine protocol for cytosine base editing in two model bacteria Corynebacterium glutamicum and Bacillus subtilis. The protocol can be adapted to base editing in other bacteria with modifications.},
}
@article {pmid36592318,
year = {2023},
author = {Kim, H and Oh, Y and Park, E and Kang, M and Choi, Y and Kim, SG},
title = {Heritable Virus-Induced Genome Editing (VIGE) in Nicotiana attenuata.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {203-218},
pmid = {36592318},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Tobacco/genetics ; CRISPR-Cas Systems/genetics ; Plants/genetics ; Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {The CRISPR/Cas9 system is an extremely powerful tool for targeted mutagenesis in plants. However, plant genome editing relies on the labor-intensive plant regeneration method for generating gene-edited plants. To overcome this bottleneck, several virus-induced genome editing (VIGE) techniques have been developed. The VIGE system aims to induce targeted mutations in germ cells without plant regeneration. However, due to the delivery issues of a large Cas9 protein, scientists focus on developing a virus-mediated delivery system for guide RNA into Cas9-overproducing plants. Here, we describe how to induce heritable targeted mutations in a non-model plant, Nicotiana attenuata, using VIGE system. This method will be applied for manipulating the target genes in any plants that scientists are interested in.},
}
@article {pmid36592316,
year = {2023},
author = {Wu, J and Cho, CS and Jo, DH and Kim, JH},
title = {Application of Base Editor-Mediated Genome Editing in Mouse Retina.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {179-188},
pmid = {36592316},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Retina ; Genome ; DNA Breaks, Double-Stranded ; },
abstract = {Base editor is a newly developed genome editing technology that enables conversion of single nucleotides without DNA double-strand breaks (DSB) and maintains a low rate of insertion-deletion (INDEL) errors. With these flexibility and safety, base editor has been widely used in many fields, including inherited retinal disease. The majority of retinal genome editing requires intravitreal and subretinal injection delivery of the therapeutic vector in order to transduce the target cells. Here, we provide an application guide of base editor as performed in the mouse retina.},
}
@article {pmid36592313,
year = {2023},
author = {Lim, CKW and Miskalis, AJ and Perez-Pinera, P and Gaj, T},
title = {Delivering Base Editors In Vivo by Adeno-Associated Virus Vectors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {135-158},
pmid = {36592313},
issn = {1940-6029},
mesh = {*Dependovirus/genetics ; *Genetic Vectors/genetics ; Gene Transfer Techniques ; DNA ; Genome ; CRISPR-Cas Systems ; },
abstract = {CRISPR base editors are genome-modifying proteins capable of creating single-base substitutions in DNA but without the requirement for a DNA double-strand break. Given their ability to precisely edit DNA, they hold tremendous therapeutic potential. Here, we describe procedures for delivering base editors in vivo via adeno-associated virus (AAV) vectors, a promising engineered gene delivery vehicle capable of transducing a range of cell types and tissues. We provide step by step protocols for (i) designing and validating base editing systems, (ii) packaging base editors into recombinant AAV vector particles, (iii) delivering AAV to the central nervous system via intrathecal injection, and (iv) quantifying base editing frequencies by next-generation sequencing.},
}
@article {pmid36592312,
year = {2023},
author = {Lee, SN and Jang, HS and Woo, JS},
title = {Heterologous Expression and Purification of a CRISPR-Cas9-Based Adenine Base Editor.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {123-133},
pmid = {36592312},
issn = {1940-6029},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Adenine/metabolism ; Gene Editing/methods ; Plasmids/genetics ; RNA, Guide, Kinetoplastida/genetics ; },
abstract = {CRISPR-cas9-guided adenine base editors (ABEs) site-specifically convert the A-T base pair to G-C base pair in genomic DNA. The intracellular delivery of ABE proteins preassembled with guide RNAs (gRNAs) has shown greatly reduced off-target effects compared with that of plasmids or viral vectors containing ABE and gRNA-encoding sequences. For efficient gene editing by the ribonucleoprotein delivery method, the ABE-gRNA complexes need to be prepared in high purity and quantity. Here we describe the expression and purification procedure of ABEmax, one of high-efficiency ABE versions.},
}
@article {pmid36592311,
year = {2023},
author = {Jeong, TY and Lim, SY and Seong, JK and Kim, K},
title = {Targeted Mutagenesis in Mice Using a Base Editor.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {99-119},
pmid = {36592311},
issn = {1940-6029},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mutagenesis ; Point Mutation ; Nucleotides ; },
abstract = {Base editors, such as cytosine and adenine base editors, are composed of nickase Cas9 (nCas9) and deaminase and serve as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based enzymatic tools for specific nucleotide substitutions. They are mainly the most effective genome editing tools for introducing point mutations, such as C-to-T and A-to-G conversions. The enhanced base editor, a C-to-G base editor (CGBE), can perform other nucleotide substitutions, such as C-to-G conversions. Here, we introduce a method for generating mouse models with point mutations using a base editing system.},
}
@article {pmid36592309,
year = {2023},
author = {See, JE and Kim, Y},
title = {Functional Analysis of Variants in BRCA1 Using CRISPR Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {73-85},
pmid = {36592309},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cytosine ; Nucleotides ; Genes, Tumor Suppressor ; },
abstract = {To date, methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays have been used to evaluate the function of the variants of uncertain significance (VUS) of the BRCA genes. However, these methods have limitations as they are associated with overexpression and do not apply to post-transcriptional regulation. Therefore, there are several VUS whose functions are unclear. Recently, we devised a new way to assess the functionality of variants in BRCA1 via a CRISPR-mediated base editor to overcome these limitations. We precisely introduced the target nucleotide substitution in living cells and identified variants whose functions were not defined. Here, we describe the methods for the functional appraisal of BRCA1 variants using CRISPR-based base editors.},
}
@article {pmid36592308,
year = {2023},
author = {Zhang, X and Guan, Y and Li, D},
title = {A/C Simultaneous Conversion Using the Dual Base Editor in Human Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {63-72},
pmid = {36592308},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; Mutation ; Cytosine ; Adenine ; },
abstract = {Base editors, mostly cytidine base editors (CBEs) and adenine base editors (ABEs), are powerful tools for precise base editing. However, current base editors can only edit either adenines or cytosines. Thus, our lab has developed a dual base editor (A&amp;C-BEmax) through the fusion of cytidine and adenosine deaminases to Cas9n to achieve both C•G to T•A and A•T to G•C mutations, which enables A/C simultaneous conversion in the same allele (up to 30%) in human cells. Here, we described a protocol for the usage of A&amp;C-BEmax in human cells. This protocol includes standard dual base editing experiments in HEK293T cells and data analysis of dual base editing outcomes using BE-analyzer. All the workflow of experiments can be completed within 2-3 weeks.},
}
@article {pmid36592306,
year = {2023},
author = {Kim, D},
title = {Profiling Genome-Wide Specificity of dCpf1 Cytidine Base Editors Using Digenome-Seq.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {33-40},
pmid = {36592306},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Cytidine/genetics ; Escherichia coli/genetics ; RNA, Guide, Kinetoplastida/genetics ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Digenome-seq is a powerful approach for determining the genome-wide specificity of programmable nuclease including CRISPR-Cas9 and CRISPR-Cpf1 (also known as Cas12a) and programmable deaminase including cytosine base editors (CBEs) and adenine base editors (ABEs). To define the genome-wide specificity of dLbCpf1-BE (also known as dLbCas12a-BE), genomic DNA is first incubated with dLbCpf1-BE, which induces C-to-U conversion at on-target and off-target sites, and then treated with a mixture of E. coli uracil DNA glycosylase (UDG) and Endonuclease VIII, which creates single-strand breaks (SSBs) by removing uracil in vitro. Digested genomic DNA is subjected to WGS, and then sequencing reads are aligned to the reference genome, resulting in straight alignments at on-target and off-target sites. The in vitro cleavage sites related to the straight alignments can be identified using the Digenome-seq computer tool.},
}
@article {pmid36592305,
year = {2023},
author = {Park, J and Kim, HK},
title = {Prediction of Base Editing Efficiencies and Outcomes Using DeepABE and DeepCBE.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {23-32},
pmid = {36592305},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Point Mutation ; Cytosine ; Nucleotides ; DNA ; },
abstract = {Adenine base editors (ABEs) and cytosine base editors (CBEs) have been widely used to introduce disease-relevant point mutations at target DNA sites of interest. However, the introduction of point mutations using base editors can be difficult due to low editing efficiencies and/or the existence of multiple target nucleotides within the base editing window at the target site. Thus, previous works have relied heavily on experimentally evaluating the base editing efficiencies and outcomes using time-consuming and labor-intensive multi-step experimental processes. DeepABE and DeepCBE are deep learning-based computational models to predict the efficiencies and outcome frequencies of ABE and CBE at given target DNA sites, in silico. Here, we describe the step-by-step procedure for the accurate determination of specific target nucleotides for ABE or CBE editing on the online available web tool, (DeepBaseEditor, https://deepcrispr.info/DeepBaseEditor).},
}
@article {pmid36592304,
year = {2023},
author = {Hwang, GH and Bae, S},
title = {Web-Based Computational Tools for Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {13-22},
pmid = {36592304},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Internet ; },
abstract = {CRISPR-based base editors are efficient genome editing tools for use in base correction. Currently, there are various versions and types of base editors with different substitution patterns, editing windows, and protospacer adjacent motif (PAM) sequences. For the design of target sequences, consideration of off-target sequences is required. In addition, for assessment of base editing outcomes in bulk populations, the analysis of high-throughput sequencing data is required. Several web browser-based computation programs have been developed for the purpose of target design and NGS data analysis, especially for users with less computational knowledge. In this manuscript, depending on the purpose of each program, we provide an explanation of useful tools including BE-Designer for design of targets and BE-Analyzer for analysis of NGS data that were developed by our group, CRISPResso2 for analysis of NGS data developed by Luca Pinello group, DeepBaseEditor for prediction of target efficiency developed by Hyongbum Henry Kim group, and BE-Hive for prediction of target outcome developed by David Liu group.},
}
@article {pmid36592303,
year = {2023},
author = {Song, B and Bae, S},
title = {Introduction and Perspectives of DNA Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {3-11},
pmid = {36592303},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; Point Mutation ; DNA/genetics ; },
abstract = {DNA base editors, one of the CRISPR-based genome editing tools, can induce targeted point mutations at desired sites. Their superiority is based on the fact that they can perform efficient and precise gene editing without generating a DNA double-strand break (DSB) or requiring a donor DNA template. Since they were first developed, significant efforts have been made to improve DNA base editors in order to overcome problems such as off-target edits on DNA/RNA and bystander mutations in editing windows. Here, we provide an overview of DNA base editors with a summary about the history of development of DNA base editors and report on efforts to improve them.},
}
@article {pmid36593183,
year = {2022},
author = {Ning, S and Wu, X and Luo, Y},
title = {[Recent advances in CRISPR-related transposable elements].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {12},
pages = {4371-4384},
doi = {10.13345/j.cjb.220197},
pmid = {36593183},
issn = {1872-2075},
abstract = {A new wave of research has been inspired by the CRISPR-Cas system with respect to their application in genome editing. The CRISPR-Cas system can not only be applied in gene knockout and insertion, but also be used in base editing, transcriptional regulation and recombination of gene clusters. However, the low efficiency of homology-directed repair (HDR) limits its application. Unlike the CRISPR-Cas system, mobile genetic elements (MGE) can insert DNA fragments into cell chromosomes without the aid of HDR. Recently, it is reported that CRISPR-related transposable elements can guide targeted DNA insertion. Their transposition mechanisms and reprogramming abilities have brought novel opportunities to the development of this field. This review summarized the research progress and application development of natural CRISPR-related transposable elements in recent years, as well as the applications of fused dCas9-transposase. It proposed the application prospects and potential challenges of CRISPR-related transposable elements in the future, which provided a reference for the development direction of gene editing tools.},
}
@article {pmid36591999,
year = {2022},
author = {Diallo, CK and Modzelewski, AJ},
title = {Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {190},
pages = {},
doi = {10.3791/64302},
pmid = {36591999},
issn = {1940-087X},
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Zygote ; Mice, Inbred C57BL ; Electroporation/methods ; Ribonucleoproteins/genetics ; },
abstract = {With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.},
}
@article {pmid36587138,
year = {2023},
author = {Maranda, V and Zhang, Y and Vizeacoumar, FS and Freywald, A and Vizeacoumar, FJ},
title = {A CRISPR Platform for Targeted In Vivo Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2614},
number = {},
pages = {397-409},
pmid = {36587138},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome ; *Neoplasms/genetics ; Tumor Microenvironment/genetics ; },
abstract = {Large-scale genetic screens are becoming increasingly used as powerful tools to query the genome to identify therapeutic targets in cancer. The advent of the CRISPR technology has revolutionized the effectiveness of these screens and has made it possible to carry out loss-of-function screens to identify cancer-specific genetic interactions. Such loss-of-function screens can be performed in silico, in vitro, and in vivo, depending on the scale of the screen, as well as research questions to be answered. Performing screens in vivo has its challenges but also advantages, providing opportunities to study the tumor microenvironment and cancer immunity. In this chapter, we present a procedural framework and associated notes for conducting in vivo CRISPR knockout screens in cancer models to study cancer biology, anti-tumor immune responses, tumor microenvironment, and predicting treatment responses.},
}
@article {pmid36587075,
year = {2023},
author = {Yamaji, T and Homma, Y},
title = {Construction of Sphingolipid Remodeled Cells by Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2613},
number = {},
pages = {111-125},
pmid = {36587075},
issn = {1940-6029},
mesh = {Animals ; Humans ; *Sphingolipids/metabolism ; *Gene Editing/methods ; HeLa Cells ; Ceramides/metabolism ; Sphingomyelins/metabolism ; Glycosphingolipids ; CRISPR-Cas Systems ; Mammals/metabolism ; },
abstract = {Sphingolipids are ubiquitously expressed in eukaryotes and play various functional roles. The key characteristic of sphingolipids is their diversity of molecular species. Sphingomyelin (SM) and glycosphingolipids (GSLs) are the major components of sphingolipids in the plasma membrane, which are composed of ceramide and a polar head-group. SM is the most abundant sphingolipid species in mammalian cells, while GSLs have a wide variety of glycans as head groups. Various fatty acids in ceramide also contribute to the diversity of sphingolipid species. To analyze the cellular function of each sphingolipid species, precise gene manipulation is essential. Recent developments in genome editing technologies have facilitated complete gene disruption in cultured cells. This chapter describes protocols for the construction of various sphingolipid-related gene knockout HeLa cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and for confirmation of changes in their lipid composition using radioisotopes and thin layer chromatography. This sphingolipid-remodeled cell panel is a useful tool for analyzing the cellular functions of sphingolipid species and as a reference for lipid analysis.},
}
@article {pmid36585106,
year = {2023},
author = {Leuillier, M and Duflot, T and Ménoret, S and Messaoudi, H and Djerada, Z and Groussard, D and Denis, RGP and Chevalier, L and Karoui, A and Panthu, B and Thiébaut, PA and Schmitz-Afonso, I and Nobis, S and Campart, C and Henry, T and Sautreuil, C and Luquet, SH and Beseme, O and Féliu, C and Peyret, H and Nicol, L and Henry, JP and Renet, S and Mulder, P and Wan, D and Tesson, L and Heslan, JM and Duché, A and Jacques, S and Ziegler, F and Brunel, V and Rautureau, GJP and Monteil, C and do Rego, JL and do Rego, JC and Afonso, C and Hammock, B and Madec, AM and Pinet, F and Richard, V and Anegon, I and Guignabert, C and Morisseau, C and Bellien, J},
title = {CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury.},
journal = {Journal of advanced research},
volume = {43},
number = {},
pages = {163-174},
doi = {10.1016/j.jare.2022.03.004},
pmid = {36585106},
issn = {2090-1224},
support = {R35 ES030443/ES/NIEHS NIH HHS/United States ; R15 DK114790/DK/NIDDK NIH HHS/United States ; P42 ES004699/ES/NIEHS NIH HHS/United States ; },
mesh = {Male ; Female ; Rats ; Animals ; Epoxide Hydrolases/genetics/metabolism ; CRISPR-Cas Systems ; Obesity/genetics ; *Insulin Resistance/genetics ; *Heart Diseases ; *Heart Injuries/genetics ; Phosphoric Monoester Hydrolases/genetics/metabolism ; Lysophospholipids ; },
abstract = {INTRODUCTION: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown.<br><br>OBJECTIVES: This study aimed to assess in vivo the physiological role of sEH-P.<br><br>METHODS: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity.<br><br>RESULTS: The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPAR&#x3b3;, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury.<br><br>CONCLUSION: Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.},
}
@article {pmid36584049,
year = {2022},
author = {Söllner, JH and Sake, HJ and Frenzel, A and Lechler, R and Herrmann, D and Fuchs, W and Petersen, B},
title = {In vitro genome editing activity of Cas9 in somatic cells after random and transposon-based genomic Cas9 integration.},
journal = {PloS one},
volume = {17},
number = {12},
pages = {e0279123},
pmid = {36584049},
issn = {1932-6203},
mesh = {Animals ; Swine ; Male ; Humans ; *Gene Editing/methods ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; Genetic Engineering/methods ; Genomics ; },
abstract = {Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2-60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.},
}
@article {pmid36463799,
year = {2023},
author = {Zhou, B and Yang, R and Sohail, M and Kong, X and Zhang, X and Fu, N and Li, B},
title = {CRISPR/Cas14 provides a promising platform in facile and versatile aptasensing with improved sensitivity.},
journal = {Talanta},
volume = {254},
number = {},
pages = {124120},
doi = {10.1016/j.talanta.2022.124120},
pmid = {36463799},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; Oligonucleotides ; DNA, Single-Stranded/genetics ; *Biosensing Techniques/methods ; Aflatoxin B1/analysis ; },
abstract = {CRISPR is reshaping biosensing technology due to its programmability, sensitivity, and specificity. Most current CRISPR-based biosensors are developed based on Cas12 and Cas13, while the biosensing potentials of the newly discovered Cas14 have not been fully elucidated yet. Herein, a fluorometric biosensor named HARRY (highly sensitive aptamer-regulated Cas14 R-loop for bioanalysis) was developed. The diblock ssDNA is designed to contain the activator sequence of Cas14 and the aptamer sequence of specific targets. In the absence of targets, the ssDNA activates Cas14a, then the Cas14a trans-cleavages the fluorescent reporter, causing fluorescence enhancement. In the presence of the targets, ssDNA-target assembly is formed via aptamer interaction, resulting in the inhibition of Cas14a activation. HARRY can detect ATP, Cd[2+], histamine, aflatoxin B1, and thrombin with detection limits at the low-nanomolar level, which shows improvement compared with Cas12a-based aptasensors in sensitivity and versatility. We reasoned that the improvement is derived from the ssDNA specificity of Cas14a and found that the detection limit of HARRY is correlated to the binding affinities of aptamers. This study unlocks the potential of Cas14a in versatile aptasensing, which may inspire the development of CRISPR-based biosensors from the Cas14a branch.},
}
@article {pmid36423223,
year = {2023},
author = {Negin, B and Aharoni, A},
title = {Let's connect nature with hypothesis-based experimentation and explore life in context.},
journal = {The Plant journal : for cell and molecular biology},
volume = {113},
number = {1},
pages = {23-25},
doi = {10.1111/tpj.16030},
pmid = {36423223},
issn = {1365-313X},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome ; Gene Editing/methods ; },
abstract = {In a recent paper in Nature, Edith Heard from the European Molecular Biology Laboratory (EMBL) suggested that molecular biologists should 'reconnect with nature' by diversifying sampling locations. Although this approach has its own benefits, we suggest that advanced methods should rather be used to take hypothesis-based experiments to nature, thereby supplying a much-needed context for experimentation under controlled conditions. Following the CRISPR (clustered regularly interspaced short palindromic repeats) revolution, this approach has become accessible to many research groups. For the past several years we have developed the groundwork and initiated such experimentation. This included the assembly of a mobile laboratory on a four-wheel drive truck and examining genome-edited metabolic mutants in wild tree tobacco (Nicotiana glauca), grown in nature. Our findings included both targeted answers to focused questions, but also surprising results that could only be reached while working in natural settings.},
}
@article {pmid36401738,
year = {2023},
author = {Miranda, S and Piazza, S and Nuzzo, F and Li, M and Lagrèze, J and Mithöfer, A and Cestaro, A and Tarkowska, D and Espley, R and Dare, A and Malnoy, M and Martens, S},
title = {CRISPR/Cas9 genome-editing applied to MdPGT1 in apple results in reduced foliar phloridzin without impacting plant growth.},
journal = {The Plant journal : for cell and molecular biology},
volume = {113},
number = {1},
pages = {92-105},
doi = {10.1111/tpj.16036},
pmid = {36401738},
issn = {1365-313X},
mesh = {*Malus/genetics/metabolism ; CRISPR-Cas Systems ; Phlorhizin/metabolism ; Plant Growth Regulators/metabolism ; Gibberellins/metabolism ; Gene Editing/methods ; },
abstract = {Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin-specific UDP-2'-O-glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome-edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome-edited lines exhibited normal growth despite reduced foliar phloridzin. RNA-sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome-edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up-regulated, whereas gibberellin GA4 was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase-quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.},
}
@article {pmid36224265,
year = {2022},
author = {Carvalho, T},
title = {CRISPR-Cas9 hits its target in amyloidosis.},
journal = {Nature medicine},
volume = {28},
number = {12},
pages = {2438},
doi = {10.1038/d41591-022-00101-4},
pmid = {36224265},
issn = {1546-170X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism ; Gene Editing ; *Amyloidosis/genetics ; },
}
@article {pmid36066192,
year = {2023},
author = {Lorenzo, CD and Debray, K and Herwegh, D and Develtere, W and Impens, L and Schaumont, D and Vandeputte, W and Aesaert, S and Coussens, G and De Boe, Y and Demuynck, K and Van Hautegem, T and Pauwels, L and Jacobs, TB and Ruttink, T and Nelissen, H and Inzé, D},
title = {BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize.},
journal = {The Plant cell},
volume = {35},
number = {1},
pages = {218-238},
doi = {10.1093/plcell/koac243},
pmid = {36066192},
issn = {1532-298X},
support = {833866/ERC_/European Research Council/International ; },
mesh = {*Gene Editing ; *Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Multifactorial Inheritance ; Plant Breeding ; Genome, Plant/genetics ; },
abstract = {Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs.},
}
@article {pmid36592317,
year = {2023},
author = {Yan, F and Yu, M and Wang, M and Zhou, H},
title = {High-Throughput Base Editing-Mediated Artificial Evolution Streamlines Trait Gene Identification in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {191-202},
pmid = {36592317},
issn = {1940-6029},
abstract = {Base Editing-Mediated Gene Evolution (BEMGE) method develops novel rice germplasms with mutations in any endogenous gene through employing both Cas9n-based cytosine and adenine base editors combined with an sgRNA library tiling the full-length coding region, for obtaining unknown functional SNPs. Here we describe the process of artificial evolution of OsALS1 in rice cells using BEMGE through Agrobacterium-mediated transformation. BEMGE method has the potential to generate numerous nucleotide changes to screen pivotal amino acids (AAs) and to accelerate crop improvement.},
}
@article {pmid36592310,
year = {2023},
author = {Sasaguri, H},
title = {Use of the Representative Base Editing Tool Target-AID to Introduce Pathogenic Mutations into Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2606},
number = {},
pages = {87-97},
pmid = {36592310},
issn = {1940-6029},
abstract = {Base editing technology is a modified CRISPR/Cas system that directly introduces point mutations into targeted genomic regions without causing double-stranded DNA breaks. Target-AID (activation-induced cytidine deaminase) is a representative base editing tool and may serve as a potent option to create genetically modified animals that harbor disease-causing pathogenic point mutations. In this chapter, I describe the basic protocol used to introduce disease-relevant pathogenic mutations into mice by Target-AID.},
}
@article {pmid36591257,
year = {2022},
author = {Ramamurthy, RM and Rodriguez, M and Ainsworth, HC and Shields, J and Meares, D and Bishop, C and Farland, A and Langefeld, CD and Atala, A and Doering, CB and Spencer, HT and Porada, CD and Almeida-Porada, G},
title = {Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {954984},
pmid = {36591257},
issn = {1664-3224},
abstract = {INTRODUCTION: Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing.<br><br>METHODS: We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs).<br><br>RESULTS: PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness.<br><br>DISCUSSION: Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.},
}
@article {pmid36589238,
year = {2022},
author = {Shaytan, AK and Novikov, RV and Vinnikov, RS and Gribkova, AK and Glukhov, GS},
title = {From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems.},
journal = {Frontiers in molecular biosciences},
volume = {9},
number = {},
pages = {1070526},
pmid = {36589238},
issn = {2296-889X},
abstract = {In the last decade, the CRISPR-Cas technology has gained widespread popularity in different fields from genome editing and detecting specific DNA/RNA sequences to gene expression control. At the heart of this technology is the ability of CRISPR-Cas complexes to be programmed for targeting particular DNA loci, even when using catalytically inactive dCas-proteins. The repertoire of naturally derived and engineered dCas-proteins including fusion proteins presents a promising toolbox that can be used to construct functional synthetic genetic circuits. Rational genetic circuit design, apart from having practical relevance, is an important step towards a deeper quantitative understanding of the basic principles governing gene expression regulation and functioning of living organisms. In this minireview, we provide a succinct overview of the application of CRISPR-dCas-based systems in the emerging field of synthetic genetic circuit design. We discuss the diversity of dCas-based tools, their properties, and their application in different types of genetic circuits and outline challenges and further research directions in the field.},
}
@article {pmid36589095,
year = {2022},
author = {Wang, Y and Tang, Q and Pu, L and Zhang, H and Li, X},
title = {CRISPR-Cas technology opens a new era for the creation of novel maize germplasms.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1049803},
pmid = {36589095},
issn = {1664-462X},
abstract = {Maize (Zea mays) is one of the most important food crops in the world with the greatest global production, and contributes to satiating the demands for human food, animal feed, and biofuels. With population growth and deteriorating environment, efficient and innovative breeding strategies to develop maize varieties with high yield and stress resistance are urgently needed to augment global food security and sustainable agriculture. CRISPR-Cas-mediated genome-editing technology (clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated)) has emerged as an effective and powerful tool for plant science and crop improvement, and is likely to accelerate crop breeding in ways dissimilar to crossbreeding and transgenic technologies. In this review, we summarize the current applications and prospects of CRISPR-Cas technology in maize gene-function studies and the generation of new germplasm for increased yield, specialty corns, plant architecture, stress response, haploid induction, and male sterility. Optimization of gene editing and genetic transformation systems for maize is also briefly reviewed. Lastly, the challenges and new opportunities that arise with the use of the CRISPR-Cas technology for maize genetic improvement are discussed.},
}
@article {pmid36586428,
year = {2022},
author = {Madhavan, A and Arun, KB and Sindhu, R and Nair, BG and Pandey, A and Awasthi, MK and Szakacs, G and Binod, P},
title = {Design and genome engineering of microbial cell factories for efficient conversion of lignocellulose to fuel.},
journal = {Bioresource technology},
volume = {370},
number = {},
pages = {128555},
doi = {10.1016/j.biortech.2022.128555},
pmid = {36586428},
issn = {1873-2976},
abstract = {The gradually increasing need for fossil fuels demands renewable biofuel substitutes. This has fascinated an increasing investigation to design innovative energy fuels that have comparable Physico-chemical and combustion characteristics with fossil-derived fuels. The efficient microbes for bioenergy synthesis desire the proficiency to consume a large quantity of carbon substrate, transfer various carbohydrates through efficient metabolic pathways, capability to withstand inhibitory components and other degradation compounds, and improve metabolic fluxes to synthesize target compounds. Metabolically engineered microbes could be an efficient methodology for synthesizing biofuel from cellulosic biomass by cautiously manipulating enzymes and metabolic pathways. This review offers a comprehensive perspective on the trends and advances in metabolic and genetic engineering technologies for advanced biofuel synthesis by applying various heterologous hosts. Probable technologies include enzyme engineering, heterologous expression of multiple genes, CRISPR-Cas technologies for genome editing, and cell surface display.},
}
@article {pmid36581624,
year = {2022},
author = {Kwon, J and Kim, M and Bae, S and Jo, A and Kim, Y and Lee, JK},
title = {TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7975},
pmid = {36581624},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *Genome ; High-Throughput Nucleotide Sequencing ; Deoxyribonuclease I/genetics ; CRISPR-Cas Systems ; },
abstract = {Prime editors (PEs) are powerful tools that widen the possibilities for sequence modifications during genome editing. Although methods based on the analysis of Cas9 nuclease or nickase activity have been used to predict genome-wide off-target activities of PEs, no tool that directly uses PEs for this purpose has been reported yet. In this study, we present a cell-based assay, named TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs. TAPE-seq analyses are successfully performed using many different versions of PEs. The TAPE-seq predictions are compared with results from two other off-site prediction methods, Cas9 nuclease-based GUIDE-seq and Cas9 nickase-based Digenome-seq (nDigenome-seq). TAPE-seq shows a lower miss rate, and a higher area under the receiver operating characteristic curve compared to the other methods. TAPE-seq also identified valid off-target sites that were missed by the other methods.},
}
@article {pmid36585738,
year = {2022},
author = {Escalona, RM and Chu, S and Kadife, E and Kelly, JK and Kannourakis, G and Findlay, JK and Ahmed, N},
title = {Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer.},
journal = {Cancer cell international},
volume = {22},
number = {1},
pages = {422},
pmid = {36585738},
issn = {1475-2867},
abstract = {BACKGROUND: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9.<br><br>METHODS: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model.<br><br>RESULTS: Approximately 70-90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model.<br><br>CONCLUSIONS: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.},
}
@article {pmid36584777,
year = {2022},
author = {Dao, FY and Liu, ML and Su, W and Lv, H and Zhang, ZY and Lin, H and Liu, L},
title = {AcrPred: A hybrid optimization with enumerated machine learning algorithm to predict Anti-CRISPR proteins.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ijbiomac.2022.12.250},
pmid = {36584777},
issn = {1879-0003},
abstract = {CRISPR-Cas, as a tool for gene editing, has received extensive attention in recent years. Anti-CRISPR (Acr) proteins can inactivate the CRISPR-Cas defense system during interference phase, and can be used as a potential tool for the regulation of gene editing. In-depth study of Anti-CRISPR proteins is of great significance for the implementation of gene editing. In this study, we developed a high-accuracy prediction model based on two-step model fusion strategy, called AcrPred, which could produce an AUC of 0.952 with independent dataset validation. To further validate the proposed model, we compared with published tools and correctly identified 9 of 10 new Acr proteins, indicating the strong generalization ability of our model. Finally, for the convenience of related wet-experimental researchers, a user-friendly web-server AcrPred (Anti-CRISPR proteins Prediction) was established at http://lin-group.cn/server/AcrPred, by which users can easily identify potential Anti-CRISPR proteins.},
}
@article {pmid36584292,
year = {2022},
author = {Xue, Y and Luo, X and Xu, W and Wang, K and Wu, M and Chen, L and Yang, G and Ma, K and Yao, M and Zhou, Q and Lv, Q and Li, X and Zhou, J and Wang, J},
title = {PddCas: A Polydisperse Droplet Digital CRISPR/Cas-Based Assay for the Rapid and Ultrasensitive Amplification-Free Detection of Viral DNA/RNA.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.2c03590},
pmid = {36584292},
issn = {1520-6882},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays have been an emerging diagnostic technology for pathogen diagnosis. In this work, we developed a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) for the rapid and ultrasensitive amplification-free detection of viral DNA/RNA with minimum instruments. LbaCas12a and LbuCas13a were used for the direct detection of viral DNA and RNA, respectively. The reaction mixtures were partitioned with a common vortex mixer to generate picoliter-scale polydisperse droplets in several seconds. The limit of detection (LoD) for the target DNA and RNA is approximately 100 aM and 10 aM, respectively, which is about 3 × 10[4]-10[5] fold more sensitive than corresponding bulk CRISPR assays. We applied the PddCas to successfully detect severe acute respiratory syndrome coronavirus (SARS-CoV-2) and human papillomavirus type 18 (HPV 18) in clinical samples. For the 23 HPV 18-suspected cervical epithelial cell samples and 32 nasopharyngeal swabs for SARS-CoV-2, 100% sensitivity and 100% specificity were demonstrated. The dual-gene virus detection with PddCas was also established and verified. Therefore, PddCas has potential for point-of-care application and is envisioned to be readily deployed for frequent testing as part of an integrated public health surveillance program.},
}
@article {pmid36493528,
year = {2023},
author = {Yu, Y and Zhang, F and Duan, X and Yang, C and Cui, Y and Yu, L},
title = {ORFV can carry TRAP gene expression via intracellular CRISPR/Cas9 gene editing technology.},
journal = {Journal of virological methods},
volume = {312},
number = {},
pages = {114652},
doi = {10.1016/j.jviromet.2022.114652},
pmid = {36493528},
issn = {1879-0984},
mesh = {Animals ; Humans ; Sheep ; *Orf virus/genetics ; CRISPR-Cas Systems ; Gene Editing ; Staphylococcus aureus/genetics/metabolism ; Vascular Endothelial Growth Factor A/genetics/metabolism ; *Ecthyma, Contagious ; Gene Expression ; },
abstract = {Orf is an acute and highly contracted human and animal infection caused by orf virus (ORFV), which mainly affects sheep, goats, and other species. Clinically, opportunistic or conditional pathogens such as Staphylococcus aureus (S. aureus) are often detected in cases of orf, which greatly increases the risk of disease progression and clinical death. It has been reported that TRAP gene products of S. aureus can broadly influence bacterial life and pathogenicity in vivo, and introduction of exogenous TRAP genes may help to inhibit the proliferation of bacteria. In order to achieve the combined control of ORFV and S. aureus, a novel approach to design a S. aureus TRAP gene vaccine using a live attenuated ORFV vector is proposed. In this study, CRISPR/Cas9 gene editing technology was used to disable vascular endothelial growth factor E of ORFV (VEGF-v) and introduced TRAP gene into this position. TRAP gene expression was detected in keratinocytes infected with recombinant virus. The construction and experimental verification of recombinant ORFV (ORFV-v/TRAP) will provide a reference for in-depth studies on the prevention and control of mixed infectious disease.},
}
@article {pmid36374008,
year = {2022},
author = {Atsuta, Y and Suzuki, K and Iikawa, H and Yaguchi, H and Saito, D},
title = {Prime editing in chicken fibroblasts and primordial germ cells.},
journal = {Development, growth &amp; differentiation},
volume = {64},
number = {9},
pages = {548-557},
doi = {10.1111/dgd.12823},
pmid = {36374008},
issn = {1440-169X},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; Gene Editing/methods ; Germ Cells/metabolism ; },
abstract = {CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.},
}
@article {pmid36229683,
year = {2023},
author = {Chen, L and Zhang, S and Xue, N and Hong, M and Zhang, X and Zhang, D and Yang, J and Bai, S and Huang, Y and Meng, H and Wu, H and Luan, C and Zhu, B and Ru, G and Gao, H and Zhong, L and Liu, M and Liu, M and Cheng, Y and Yi, C and Wang, L and Zhao, Y and Song, G and Li, D},
title = {Engineering a precise adenine base editor with minimal bystander editing.},
journal = {Nature chemical biology},
volume = {19},
number = {1},
pages = {101-110},
pmid = {36229683},
issn = {1552-4469},
mesh = {Animals ; Mice ; Rats ; *Gene Editing ; *Adenine ; Mutation ; Cytosine ; RNA, Guide, Kinetoplastida/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.},
}
@article {pmid36138140,
year = {2023},
author = {Yang, J and Song, Y and Deng, X and Vanegas, JA and You, Z and Zhang, Y and Weng, Z and Avery, L and Dieckhaus, KD and Peddi, A and Gao, Y and Zhang, Y and Gao, X},
title = {Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection.},
journal = {Nature chemical biology},
volume = {19},
number = {1},
pages = {45-54},
pmid = {36138140},
issn = {1552-4469},
mesh = {Humans ; SARS-CoV-2/genetics ; *COVID-19 ; *Nucleic Acids/genetics ; Genome ; CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.},
}
@article {pmid36574681,
year = {2023},
author = {Rottinghaus, AG and Vo, S and Moon, TS},
title = {Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {1},
pages = {e2213154120},
doi = {10.1073/pnas.2213154120},
pmid = {36574681},
issn = {1091-6490},
support = {R01 AT009741/AT/NCCIH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Microbial Consortia ; RNA, Guide, Kinetoplastida/genetics ; Plasmids/genetics ; Gene Editing ; },
abstract = {Microbes naturally coexist in complex, multistrain communities. However, extracting individual microbes from and specifically manipulating the composition of these consortia remain challenging. The sequence-specific nature of CRISPR guide RNAs can be leveraged to accurately differentiate microorganisms and facilitate the creation of tools that can achieve these tasks. We developed a computational program, ssCRISPR, which designs strain-specific CRISPR guide RNA sequences with user-specified target strains, protected strains, and guide RNA properties. We experimentally verify the accuracy of the strain specificity predictions in both Escherichia coli and Pseudomonas spp. and show that up to three nucleotide mismatches are often required to ensure perfect specificity. To demonstrate the functionality of ssCRISPR, we apply computationally designed CRISPR-Cas9 guide RNAs to two applications: the purification of specific microbes through one- and two-plasmid transformation workflows and the targeted removal of specific microbes using DNA-loaded liposomes. For strain purification, we utilize gRNAs designed to target and kill all microbes in a consortium except the specific microbe to be isolated. For strain elimination, we utilize gRNAs designed to target only the unwanted microbe while protecting all other strains in the community. ssCRISPR will be of use in diverse microbiota engineering applications.},
}
@article {pmid36512680,
year = {2022},
author = {Cai, Q and Shi, H and Sun, M and Ma, N and Wang, R and Yang, W and Qiao, Z},
title = {Sensitive Detection of Salmonella Based on CRISPR-Cas12a and the Tetrahedral DNA Nanostructure-Mediated Hyperbranched Hybridization Chain Reaction.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {51},
pages = {16382-16389},
doi = {10.1021/acs.jafc.2c05831},
pmid = {36512680},
issn = {1520-5118},
mesh = {Humans ; Gold ; CRISPR-Cas Systems ; *Metal Nanoparticles ; *Nanostructures ; Salmonella/genetics ; *Biosensing Techniques ; },
abstract = {Salmonella severely threatens global human health and causes financial burden. The ability to sensitively detect Salmonella in food samples is highly valuable but remains a challenge. Herein, a sensitive detection method for Salmonella was developed by coupling immunomagnetic separation with the CRISPR-Cas12a system and the tetrahedral DNA nanostructure-mediated hyperbranched hybridization chain reaction (TDN-hHCR). In the detection system, the target Salmonella was immunomagnetically separated and labeled with bio-barcode DNA-modified gold nanoparticles (AuNPs), which could transfer and magnify the signal of a bacterial cell into numerous bio-barcode DNA molecules. Afterward, the bio-barcode DNA can trigger the trans-cleavage activity of CRISPR-Cas12a to inhibit the process of the TDN-hHCR to generate a fluorescence readout. Due to the high immunomagnetic separation efficiency and the effective signal amplification of CRISPR-Cas12a and the TDN-hHCR, Salmonella as low as 8 CFU/mL could be easily detected. Meanwhile, this has been applied for practical use and showed the capability to detect 17 and 25 CFU/mL in spiked milk and egg white, respectively, indicating its potential application in real samples.},
}
@article {pmid36494589,
year = {2022},
author = {Wu, SS and Lee, H and Szép-Bakonyi, R and Colozza, G and Boese, A and Gert, KR and Hallay, N and Lee, JH and Kim, J and Zhu, Y and Linssen, MM and Pilat-Carotta, S and Hohenstein, P and Theussl, HC and Pauli, A and Koo, BK},
title = {SCON-a Short Conditional intrON for conditional knockout with one-step zygote injection.},
journal = {Experimental &amp; molecular medicine},
volume = {54},
number = {12},
pages = {2188-2199},
pmid = {36494589},
issn = {2092-6413},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Introns/genetics ; *Zygote ; Gene Knockout Techniques ; },
abstract = {The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.},
}
@article {pmid36577887,
year = {2022},
author = {Li, X and Wang, J and Geng, J and Xiao, L and Wang, H},
title = {Emerging Landscape of SARS-CoV-2 Variants and Detection Technologies.},
journal = {Molecular diagnosis &amp; therapy},
volume = {},
number = {},
pages = {},
pmid = {36577887},
issn = {1179-2000},
abstract = {In 2019, a new coronavirus was identified that has caused significant morbidity and mortality worldwide. Like all RNA viruses, severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) evolves over time through random mutation resulting in genetic variations in the population. Although the currently approved coronavirus disease 2019 vaccines can be given to those over 5 years of age and older in most countries, strikingly, the number of people diagnosed positive for SARS-Cov-2 is still increasing. Therefore, to prevent and control this epidemic, early diagnosis of infected individuals is of great importance. The current detection of SARS-Cov-2 coronavirus variants are mainly based on reverse transcription-polymerase chain reaction. Although the sensitivity of reverse transcription-polymerase chain reaction is high, it has some disadvantages, for example, multiple temperature changes, long detection time, complicated operation, expensive instruments, and the need for professional personnel, which brings considerable inconvenience to the early diagnosis of this virus. This review comprehensively summarizes the development and application of various current detection technologies for novel coronaviruses, including isothermal amplification, CRISPR-Cas detection, serological detection, biosensor, ensemble, and microfluidic technology, along with next-generation sequencing. Those findings offer us a great potential to replace or combine with reverse transcription-polymerase chain reaction detection to achieve the purpose of allowing predictive diagnostics and targeted prevention of SARS-Cov-2 in the future.},
}
@article {pmid36576897,
year = {2022},
author = {Lee, HJ and Kim, HJ and Lee, SJ},
title = {Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3'-Truncated sgRNA.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0071},
pmid = {36576897},
issn = {2573-1602},
abstract = {The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3'-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3'-truncated sgRNA-Cas12f1 complex in vivo. Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.},
}
@article {pmid36576859,
year = {2022},
author = {Zhang, W and Bhoobalan-Chitty, Y and Zhai, X and Hui, Y and Hansen, LH and Deng, L and Peng, X},
title = {Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0037},
pmid = {36576859},
issn = {2573-1602},
abstract = {Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δgp16) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold less virus in a CRISPR-null host, suggesting that Rep is the major replication initiator. In addition to Rep-dependent replication initiation from the viral genomic termini, we detected Rep-independent replication initiation from nonterminal sites. Intriguingly, despite the presence of Acrs, lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the Δgp16 mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.},
}
@article {pmid36575525,
year = {2022},
author = {Holland, K and Blazeck, J},
title = {High throughput mutagenesis and screening for yeast engineering.},
journal = {Journal of biological engineering},
volume = {16},
number = {1},
pages = {37},
pmid = {36575525},
issn = {1754-1611},
abstract = {The eukaryotic yeast Saccharomyces cerevisiae is a model host utilized for whole cell biocatalytic conversions, protein evolution, and scientific inquiries into the pathogenesis of human disease. Over the past decade, the scale and pace of such studies has drastically increased alongside the advent of novel tools for both genome-wide studies and targeted genetic mutagenesis. In this review, we will detail past and present (e.g., CRISPR/Cas) genome-scale screening platforms, typically employed in the context of growth-based selections for improved whole cell phenotype or for mechanistic interrogations. We will further highlight recent advances that enable the rapid and often continuous evolution of biomolecules with improved function. Additionally, we will detail the corresponding advances in high throughput selection and screening strategies that are essential for assessing or isolating cellular and protein improvements. Finally, we will describe how future developments can continue to advance yeast high throughput engineering.},
}
@article {pmid36566209,
year = {2022},
author = {Peng, Q and Huang, Z and Sun, K and Liu, Y and Yoon, CW and Harrison, RES and Schmitt, DL and Zhu, L and Wu, Y and Tasan, I and Zhao, H and Zhang, J and Zhong, S and Chien, S and Wang, Y},
title = {Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7933},
pmid = {36566209},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems ; *Genome ; Transcription Factors ; Genomics ; },
abstract = {Genome architecture and organization play critical roles in cell life. However, it remains largely unknown how genomic loci are dynamically coordinated to regulate gene expression and determine cell fate at the single cell level. We have developed an inducible system which allows Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies (SIMBA) in living cells. In SIMBA, the human heterochromatin protein 1α (HP1α) is fused to mCherry and FRB, which can be induced to form biomolecular assemblies (BAs) with FKBP-scFv, guided to specific genomic loci by a nuclease-defective Cas9 (dCas9) or a transcriptional factor (TF) carrying tandem repeats of SunTag. The induced BAs can not only enhance the imaging signals at target genomic loci using a single sgRNA, either at repetitive or non-repetitive sequences, but also recruit epigenetic modulators such as histone methyltransferase SUV39H1 to locally repress transcription. As such, SIMBA can be applied to simultaneously visualize and manipulate, in principle, any genomic locus with controllable timing in living cells.},
}
@article {pmid36525708,
year = {2023},
author = {Zhang, C and Cai, Z and Zhou, Z and Li, M and Hong, W and Zhou, W and Yu, D and Wei, P and He, J and Wang, Y and Huang, C and Wang, X and Wu, J},
title = {CASMART, a one-step CRISPR Cas12a-mediated isothermal amplification for rapid and high-resolution digital detection of rare mutant alleles.},
journal = {Biosensors &amp; bioelectronics},
volume = {222},
number = {},
pages = {114956},
doi = {10.1016/j.bios.2022.114956},
pmid = {36525708},
issn = {1873-4235},
mesh = {Humans ; Alleles ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; Mutation ; ErbB Receptors/genetics ; },
abstract = {Convenient, ultrasensitive, and accurate detection of rare variants is essential for early cancer diagnosis and precision medicine, however, despite years of efforts, tools that have all these qualities remain elusive. Here, we developed a one-step CRISPR/Cas12a-based digital diagnostic platform for accurately quantifying mutant alleles, referred to as the CRISPR ASsoaciated Mutation Allele Rapid Test (CASMART). The platform accurately quantifies the variant allele frequency of EGFR L858R within 1 h at 42 °C and can detect mutant targets as low as 0.3 copies/μL (0.498 aM) in mock multiplex cfDNA samples. We further investigated the applicability of CASMART using human genomic samples with confirmed EGFR L858R mutations previously measured variant allele frequency by next-generation sequencing. Comparison across platforms revealed equivalent detection performance (Pearson's correlation coefficient, R[2] = 0.9208) and high quantification accuracy for mutation allele frequency (intraclass correlation coefficient = 0.959). Our one-step approach enables easy and accurate variant allele frequency measurement of rare mutant alleles without PCR instrumentation, while the assay time was reduced by approximately half compared to the digital PCR with the shortest turnaround. The CASMART is an alternative to conventional single nucleotide polymorphism detection methods with great potential as a next-generation biosensor for rapidly quantifying the variant allele fraction, especially in resource-limited settings.},
}
@article {pmid36493720,
year = {2023},
author = {Wei, L and Wang, Z and Wu, L and Chen, Y},
title = {CRISPR/Cas12a-based magnetic relaxation switching biosensor for nucleic acid amplification-free and ultrasensitive detection of methicillin-resistant Staphylococcus aureus.},
journal = {Biosensors &amp; bioelectronics},
volume = {222},
number = {},
pages = {114984},
doi = {10.1016/j.bios.2022.114984},
pmid = {36493720},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *Methicillin-Resistant Staphylococcus aureus/genetics ; CRISPR-Cas Systems/genetics ; Alkaline Phosphatase ; Coloring Agents ; Eggs ; *Nucleic Acids ; Magnetic Phenomena ; Nucleic Acid Amplification Techniques ; },
abstract = {Herein, we develop a CRISPR/Cas12a-based magnetic relaxation switching (C-MRS) biosensor for ultrasensitive and nucleic acid amplification-free detection of methicillin-resistant Staphylococcus aureus (MRSA) in food. In this biosensor, mecA gene in MRSA was recognized by CRISPR-RNA, which will activate the trans-cleavage activity of Cas12a and release the fastened alkaline phosphatase (ALP) on the particle. The freed ALP can then use to hydrolyze substrate to produce ascorbic acid that trigger the click reaction between magnetic probe. The transverse relaxation time of the unbound magnetic probe can be measured for signal readout. By incorporating collateral activity of CRISPR/Cas12a, on-particle rolling circle amplification, and ALP-triggered click chemistry into background-free MRS, as low as 16 CFU/mL MRSA can be detected without any nucleic acid pre-amplification, which avoids carryover contamination, but without compromising sensitivity. Moreover, this C-MRS biosensor could distinguish 0.01% target DNA from the single-base mutant. Recovery in eggs, milk and pork ranged from 75% to 112%, 82%-104%, and 81%-91%, respectively, revealing its satisfactory accuracy and applicability in the complex food matrix. The developed C-MRS biosensor fleshes out the CRISPR toolbox for food safety and provides a new approach for the sensitive and accurate detection of foodborne drug-resistant bacteria.},
}
@article {pmid36493719,
year = {2023},
author = {Cheng, L and Yang, F and Zhao, Y and Liu, Z and Yao, X and Zhang, J},
title = {Tetrahedron supported CRISPR/Cas13a cleavage for electrochemical detection of circular RNA in bladder cancer.},
journal = {Biosensors &amp; bioelectronics},
volume = {222},
number = {},
pages = {114982},
doi = {10.1016/j.bios.2022.114982},
pmid = {36493719},
issn = {1873-4235},
mesh = {Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Circular ; *Biosensing Techniques/methods ; DNA ; *Urinary Bladder Neoplasms/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {As a diagnostic biomarker, the detection of circular RNA (circRNA) is vital for the early screening of bladder cancer. Usually, the low abundance of circRNA in clinic samples results in the necessarily complicated extraction before detection. In this work, a tetrahedron supported CRISPR/Cas13a cleavage has been explored for direct electrochemical detection of circRNA in urine from bladder cancer. CRISPR/Cas13a system has been reasonably designed to recognize the characteristic back-splice junction site of circRNA. The activated CRISPR/Cas13a by circRNA can cleave uracil bases composed of DNA tetrahedron immobilized on the surface of gold electrode, resulting in the breakage of DNA tetrahedron and the release of electrochemical active molecule methylene blue. By virtue of highly catalytic efficiency of CRISPR/Cas13a and rigid structure of tetrahedron, the developed electrochemical biosensor can directly detect circRNA in 25 μL urine sample with the lowest detection limit of 0.089 fM and linear detection range from 10 fM to 50 nM in less than 10 min, so as to avoid complicated extraction process and benefit for on-site detection.},
}
@article {pmid36463654,
year = {2023},
author = {Sun, Y and Yang, C and Jiang, X and Zhang, P and Chen, S and Su, F and Wang, H and Liu, W and He, X and Chen, L and Man, B and Li, Z},
title = {High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple with stabilized graphene field-effect transistor.},
journal = {Biosensors &amp; bioelectronics},
volume = {222},
number = {},
pages = {114979},
pmid = {36463654},
issn = {1873-4235},
mesh = {Humans ; *COVID-19/diagnosis ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Graphite ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; },
abstract = {False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a "big subtraction" signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/μL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.},
}
@article {pmid36459819,
year = {2023},
author = {Habimana, JD and Mukama, O and Chen, G and Chen, M and Amissah, OB and Wang, L and Liu, Y and Sun, Y and Li, AL and Deng, S and Huang, J and Yan, XX and Rutaganda, T and Mutangana, D and Wu, LP and Huang, R and Li, Z},
title = {Harnessing enhanced CRISPR/Cas12a trans-cleavage activity with extended reporters and reductants for early diagnosis of Helicobacter pylori, the causative agent of peptic ulcers and stomach cancer.},
journal = {Biosensors &amp; bioelectronics},
volume = {222},
number = {},
pages = {114939},
doi = {10.1016/j.bios.2022.114939},
pmid = {36459819},
issn = {1873-4235},
mesh = {Humans ; Antigens, Bacterial/metabolism ; Bacterial Proteins/genetics ; *Helicobacter pylori/genetics/metabolism ; *Stomach Neoplasms/diagnosis/genetics ; Reducing Agents ; CRISPR-Cas Systems ; *Biosensing Techniques ; Early Detection of Cancer ; *Peptic Ulcer/diagnosis/genetics ; Genotype ; Cytotoxins/genetics ; *Helicobacter Infections/diagnosis/genetics/metabolism ; },
abstract = {Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the [13]C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.},
}
@article {pmid36456803,
year = {2022},
author = {Mok, YG and Hong, S and Bae, SJ and Cho, SI and Kim, JS},
title = {Targeted A-to-G base editing of chloroplast DNA in plants.},
journal = {Nature plants},
volume = {8},
number = {12},
pages = {1378-1384},
pmid = {36456803},
issn = {2055-0278},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; DNA, Chloroplast/genetics ; DNA, Mitochondrial ; Crops, Agricultural/genetics ; Mammals/genetics ; },
abstract = {Chloroplast DNA (cpDNA) encodes up to 315 (typically, 120-130) genes[1], including those for essential components in photosystems I and II and the large subunit of RuBisCo, which catalyses CO2 fixation in plants. Targeted mutagenesis in cpDNA will be broadly useful for studying the functions of these genes in molecular detail and for developing crops and other plants with desired traits. Unfortunately, CRISPR-Cas9 and CRISPR-derived base editors, which enable targeted genetic modifications in nuclear DNA, are not suitable for organellar DNA editing[2], owing to the difficulty of delivering guide RNA into organelles. CRISPR-free, protein-only base editors (including DddA-derived cytosine base editors[3-8] and zinc finger deaminases[9]), originally developed for mitochondrial DNA editing in mammalian cells, can be used for C-to-T, rather than A-to-G, editing in cpDNA[10-12]. Here we show that heritable homoplasmic A-to-G edits can be induced in cpDNA, leading to phenotypic changes, using transcription activator-like effector-linked deaminases[13].},
}
@article {pmid36374037,
year = {2022},
author = {Heo, YB and Hwang, GH and Kang, SW and Bae, S and Woo, HM},
title = {High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects.},
journal = {Microbiology spectrum},
volume = {10},
number = {6},
pages = {e0376022},
pmid = {36374037},
issn = {2165-0497},
mesh = {Animals ; Rats ; *Gene Editing/methods ; *Corynebacterium glutamicum/genetics ; Cytosine ; Mutation ; DNA/genetics ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; APOBEC-1 Deaminase/genetics ; },
abstract = {Genome editing technology is a powerful tool for programming microbial cell factories. However, rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of the bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we demonstrate the genome engineering of Corynebacterium glutamicum as a GC-rich model industrial bacterium by generating premature termination codons (PTCs) in desired genes using high-fidelity cytosine base editors (CBEs). Through this CBE-STOP approach of introducing specific cytosine conversions, we constructed several single-gene-inactivated strains for three genes (ldh, idsA, and pyc) with high base editing efficiencies of average 95.6% (n = 45, C6 position) and the highest success rate of up to 100% for PTCs and ultimately developed a strain with five genes (ldh, actA, ackA, pqo, and pta) that were inactivated sequentially for enhancing succinate production. Although these mutant strains showed the desired phenotypes, whole-genome sequencing (WGS) data revealed that genome-wide point mutations occurred in each strain and further accumulated according to the duration of CBE plasmids. To lower the undesirable mutations, high-fidelity CBEs (pCoryne-YE1-BE3 and pCoryne-BE3-R132E) was employed for single or multiplexed genome editing in C. glutamicum, resulting in drastically reduced sgRNA-independent off-targets. Thus, we provide a CRISPR-assisted bacterial genome engineering tool with an average high efficiency of 90.5% (n = 76, C5 or C6 position) at the desired targets. IMPORTANCE Rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we identified the DNA off-targets for single and multiple genome engineering of the industrial bacterium Corynebacterium glutamicum using whole-genome sequencing. Further, we developed the high-fidelity (HF)-CBE with significantly reduced off-targets with comparable efficiency and precision. We believe that our DNA off-target analysis and the HF-CBE can promote CRISPR-assisted genome engineering over conventional gene manipulation tools by providing a markerless genetic tool without need for a foreign DNA donor.},
}
@article {pmid36342324,
year = {2022},
author = {Yang, J and Barua, N and Rahman, MN and Li, C and Lo, N and Yeong, KY and Tsang, TF and Yang, X and Cheung, YY and Tsang, AKL and Chan, RCW and Leung, EC and Chan, PKS and Ip, M},
title = {Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.},
journal = {Microbiology spectrum},
volume = {10},
number = {6},
pages = {e0326022},
pmid = {36342324},
issn = {2165-0497},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; SARS-CoV-2/genetics ; *Expeditions ; Nucleotides ; RNA ; },
abstract = {The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.},
}
@article {pmid36100522,
year = {2023},
author = {Hou, Z and Tan, R and Zhang, Y},
title = {Snapshots of a tiny ancestral nuclease of Cas9.},
journal = {Trends in biochemical sciences},
volume = {48},
number = {1},
pages = {9-10},
doi = {10.1016/j.tibs.2022.08.008},
pmid = {36100522},
issn = {0968-0004},
mesh = {*CRISPR-Cas Systems ; *RNA ; Endonucleases/genetics ; },
abstract = {High-resolution structures solved by Schuler et al. shed light on how Cas9's evolutionary ancestor IscB operates as an RNA-guided nuclease. With only two-fifths the size of Cas9, IscB holds great promise for alleviating the cargo size constraint of in vivo CRISPR delivery.},
}
@article {pmid36572753,
year = {2022},
author = {Tarverdizadeh, Y and Khalili, M and Esmaeili, S and Ahmadian, G and Golchin, M and Hajizade, A},
title = {Targeted gene inactivation in Salmonella Typhi by CRISPR/Cas9-assisted homologous recombination.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {39},
number = {2},
pages = {58},
pmid = {36572753},
issn = {1573-0972},
abstract = {BACKGROUND: Targeted gene inactivation (TGI) is a widely used technique for the study of genes' functions. There are many different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineering tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed repair (HDR).<br><br>METHODS: For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombination was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme. pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed bacteria were screened for recombination using PCR, restriction digestion and sequencing.<br><br>RESULTS: The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which the gidA gene is disrupted.<br><br>CONCLUSION: In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria's genes.},
}
@article {pmid36569102,
year = {2022},
author = {Zhao, L and Qiu, M and Li, X and Yang, J and Li, J},
title = {CRISPR-Cas13a system: A novel tool for molecular diagnostics.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {1060947},
pmid = {36569102},
issn = {1664-302X},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system.},
}
@article {pmid36558152,
year = {2022},
author = {Yuan, X and Yuan, H and Liu, B and Liu, Y},
title = {Self-Supplying Guide RNA-Mediated CRISPR/Cas12a Fluorescence System for Sensitive Detection of T4 PNKP.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {24},
pages = {},
pmid = {36558152},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics/chemistry ; Polynucleotide 5'-Hydroxyl-Kinase/genetics/metabolism ; DNA Probes/chemistry ; Fluorescent Dyes/chemistry ; RNA ; },
abstract = {Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better guidance in PNKP-related diagnostics and drug screening. Although the CRISPR/Cas12a system shows great promise in DNA-based signal amplification protocols, its guide RNAs with small molecular weight often suffer nuclease degradation during storage and utilization, resulting in reduced activation efficiency. Herein, we proposed a self-supplying guide RNA-mediated CRISPR/Cas12a system for the sensitive detection of T4 PNKP in cancer cells, in which multiple copies of guide RNA were generated by in situ transcription. In this assay, T4 PNKP was chosen as a model, and a dsDNA probe with T7 promoter region and the transcription region of guide RNA were involved. Under the action of T4 PNKP, the 5'-hydroxyl group of the dsDNA probe was converted to a phosphate group, which can be recognized and digested by Lambda Exo, resulting in dsDNA hydrolysis. The transcription template was destroyed, which resulted in the failure to generate guide RNA by the transcription pathway. Therefore, the CRISPR/Cas12a system could not be activated to effectively cleavage the F-Q-reporter, and the fluorescence signal was turned off. In the absence of T4 PNKP, the 5'-hydroxyl group of the substrate DNA cannot be digested by Lambda Exo. The intact dsDNA acts as the transcription template to generate a large amount of guide RNA. Finally, the formed Cas12a/gRNA complex triggered the reverse cleavage of Cas12a on the F-Q-reporter, resulting in a "turn-on" fluorescence signal. This strategy displayed sharp sensitivity of T4 PNKP with the limit of detection (LOD) down to 0.0017 mU/mL, which was mainly due to the multiple regulation effect of transcription amplification. In our system, the dsDNA simultaneously serves as the T4 PNKP substrate, transcription template, and Lambda Exo substrate, avoiding the need for multiple probe designs and saving costs. By integrating the target recognition, Lambda Exo activity, and trans-cleavage activity of Cas12a, CRISPR/Cas12a catalyzed the cleavage of fluorescent-labeled short-stranded DNA probes and enabled synergetic signal amplification for sensitive T4 PNKP detection. Furthermore, the T4 PNKP in cancer cells has been evaluated as a powerful tool for biomedical research and clinical diagnosis, proving a good practical application capacity.},
}
@article {pmid36555815,
year = {2022},
author = {Zhang, Z and Wang, W and Ali, S and Luo, X and Xie, L},
title = {CRISPR/Cas9-Mediated Multiple Knockouts in Abscisic Acid Receptor Genes Reduced the Sensitivity to ABA during Soybean Seed Germination.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
pmid = {36555815},
issn = {1422-0067},
mesh = {Abscisic Acid/pharmacology/metabolism ; Germination ; *Arabidopsis Proteins/genetics ; Soybeans/genetics/metabolism ; *Arabidopsis/genetics ; CRISPR-Cas Systems ; Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Abscisic acid (ABA) is an important plant hormone that regulates numerous functions in plant growth, development, and stress responses. Several proteins regulate the ABA signal transduction mechanism in response to environmental stress. Among them, the PYR1/PYL/RCAR family act as ABA receptors. This study used the CRISPR/Cas9 gene-editing system with a single gRNA to knock out three soybean PYL genes: GmPYL17, GmPYL18, and GmPYL19. The gRNA may efficiently cause varying degrees of deletion of GmPYL17, GmPYL18, and GmPYL19 gene target sequences, according to the genotyping results of T0 plants. A subset of induced alleles was successfully transferred to progeny. In the T2 generation, we obtained double and triple mutant genotypes. At the seed germination stage, CRISPR/Cas9-created GmPYL gene knockout mutants, particularly gmpyl17/19 double mutants, are less susceptible to ABA than the wild type. RNA-Seq was used to investigate the differentially expressed genes related to the ABA response from germinated seedlings under diverse treatments using three biological replicates. The gmpyl17/19-1 double mutant was less susceptible to ABA during seed germination, and mutant plant height and branch number were higher than the wild type. Under ABA stress, the GO enrichment analysis showed that certain positive germination regulators were activated, which reduced ABA sensitivity and enhanced seed germination. This research gives a theoretical basis for a better understanding of the ABA signaling pathway and the participation of the key component at their molecular level, which helps enhance soybean abiotic stress tolerance. Furthermore, this research will aid breeders in regulating and improving soybean production and quality under various stress conditions.},
}
@article {pmid36555723,
year = {2022},
author = {Li, Z and Rao, MJ and Li, J and Wang, Y and Chen, P and Yu, H and Ma, C and Wang, L},
title = {CRISPR/Cas9 Mutant Rice Ospmei12 Involved in Growth, Cell Wall Development, and Response to Phytohormone and Heavy Metal Stress.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
pmid = {36555723},
issn = {1422-0067},
mesh = {*Oryza/genetics/metabolism ; Plant Growth Regulators/metabolism ; Cadmium/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Cell Wall/genetics/metabolism ; Pectins/metabolism ; Plants/metabolism ; Seedlings/genetics/metabolism ; },
abstract = {Pectin is one of the constituents of the cell wall, distributed in the primary cell wall and middle lamella, affecting the rheological properties and the cell wall stickiness. Pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) are the most important factors for modifying methyl esterification. In this study, 45 PMEI genes from rice (Oryza sativa L.) were screened by bioinformatics tools, and their structure, motifs, cis-acting elements in the promoter region, chromosomal distribution, gene duplication, and phylogenetic relationship were analyzed. Furthermore, CRISPR/Cas9 was used to edit the OsPMEI12 (LOC_Os03G01020) and two mutant pmei12 lines were obtained to explore the functions of OsPMEI in plant growth and development, and under cadmium (Cd) stress. Compared to wild type (WT) Nipponbare, the second inverted internodes of the mutant plants shortened significantly, resulting in the reduction in plant height at mature stage. The seed setting rate, and fresh and dry weights of the mutants were also decreased in mutant plants. In addition, the pectin methylation of pmei12 lines is decreased as expected, and the pectin content of the cell wall increased at both seedling and maturity stages; however, the cellulose and hemicellulose increased only at seedling stage. Interestingly, the growth of the pmei12 lines was better than the WT in both normal conditions and under two phytohormone (GA3 and NAA) treatments at seedling stage. Under Cd stress, the fresh and dry weights were increased in pmei12 lines. These results indicated that OsPMEI12 was involved in the regulation of methyl esterification during growth, affected cell wall composition and agronomic traits, and might play an important role in responses to phytohormones and stress.},
}
@article {pmid36555398,
year = {2022},
author = {Yan, B and Liang, Y},
title = {New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
pmid = {36555398},
issn = {1422-0067},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Therapy/methods ; *Neoplasms/genetics/therapy ; *Extracellular Vesicles/genetics ; },
abstract = {Cancers are defined by genetic defects, which underlines the prospect of using gene therapy in patient care. During the past decade, CRISPR technology has rapidly evolved into a powerful gene editing tool with high fidelity and precision. However, one of the impediments slowing down the clinical translation of CRISPR-based gene therapy concerns the lack of ideal delivery vectors. Extracellular vesicles (EVs) are nano-sized membrane sacs naturally released from nearly all types of cells. Although EVs are secreted for bio-information conveyance among cells or tissues, they have been recognized as superior vectors for drug or gene delivery. Recently, emerging evidence has spotlighted EVs in CRISPR delivery towards cancer treatment. In this review, we briefly introduce the biology and function of the CRISPR system and follow this with a summary of current delivery methods for CRISPR applications. We emphasize the recent progress in EV-mediated CRISPR editing for various cancer types and target genes. The reported strategies for constructing EV-CRISPR vectors, as well as their limitations, are discussed in detail. The review aims to throw light on the clinical potential of engineered EVs and encourage the expansion of our available toolkit to defeat cancer.},
}
@article {pmid36555281,
year = {2022},
author = {Skowronek, D and Pilz, RA and Bonde, L and Schamuhn, OJ and Feldmann, JL and Hoffjan, S and Much, CD and Felbor, U and Rath, M},
title = {Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM Genes.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
pmid = {36555281},
issn = {1422-0067},
mesh = {Humans ; *Proto-Oncogene Proteins/genetics ; *Nanopore Sequencing ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Multiplex Polymerase Chain Reaction ; },
abstract = {Deletions in the CCM1, CCM2, and CCM3 genes are a common cause of familial cerebral cavernous malformations (CCMs). In current molecular genetic laboratories, targeted next-generation sequencing or multiplex ligation-dependent probe amplification are mostly used to identify copy number variants (CNVs). However, both techniques are limited in their ability to specify the breakpoints of CNVs and identify complex structural variants (SVs). To overcome these constraints, we established a targeted Cas9-mediated nanopore sequencing approach for CNV detection with single nucleotide resolution. Using a MinION device, we achieved complete coverage for the CCM genes and determined the exact size of CNVs in positive controls. Long-read sequencing for a CCM1 and CCM2 CNV revealed that the adjacent ANKIB1 and NACAD genes were also partially or completely deleted. In addition, an interchromosomal insertion and an inversion in CCM2 were reliably re-identified by long-read sequencing. The refinement of CNV breakpoints by long-read sequencing enabled fast and inexpensive PCR-based variant confirmation, which is highly desirable to reduce costs in subsequent family analyses. In conclusion, Cas9-mediated nanopore sequencing is a cost-effective and flexible tool for molecular genetic diagnostics which can be easily adapted to various target regions.},
}
@article {pmid36553630,
year = {2022},
author = {Jurlina, SL and Jones, MK and Agarwal, D and De La Toba, DV and Kambli, N and Su, F and Martin, HM and Anderson, R and Wong, RM and Seid, J and Attaluri, SV and Chow, M and Wahlin, KJ},
title = {A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.},
journal = {Genes},
volume = {13},
number = {12},
pages = {},
pmid = {36553630},
issn = {2073-4425},
support = {K99/R00 EY024648/EY/NEI NIH HHS/United States ; EY026590/EY/NEI NIH HHS/United States ; R01EY031318/EY/NEI NIH HHS/United States ; P30EY022589/EY/NEI NIH HHS/United States ; Investigator Award/FFB/Foundation Fighting Blindness/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Pluripotent Stem Cells ; DNA End-Joining Repair ; Mutagenesis ; },
abstract = {Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.},
}
@article {pmid36553615,
year = {2022},
author = {Hou, Y and Ureña-Bailén, G and Mohammadian Gol, T and Gratz, PG and Gratz, HP and Roig-Merino, A and Antony, JS and Lamsfus-Calle, A and Daniel-Moreno, A and Handgretinger, R and Mezger, M},
title = {Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing.},
journal = {Genes},
volume = {13},
number = {12},
pages = {},
pmid = {36553615},
issn = {2073-4425},
mesh = {Humans ; *X-Linked Combined Immunodeficiency Diseases/genetics/therapy ; CRISPR-Cas Systems/genetics ; Mosaicism ; Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.},
}
@article {pmid36553489,
year = {2022},
author = {Psatha, N and Paschoudi, K and Papadopoulou, A and Yannaki, E},
title = {In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations.},
journal = {Genes},
volume = {13},
number = {12},
pages = {},
pmid = {36553489},
issn = {2073-4425},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Hematopoietic Stem Cells ; Genetic Therapy ; Transcription Activator-Like Effector Nucleases ; },
abstract = {The tremendous evolution of genome-editing tools in the last two decades has provided innovative and effective approaches for gene therapy of congenital and acquired diseases. Zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR-Cas9 have been already applied by ex vivo hematopoietic stem cell (HSC) gene therapy in genetic diseases (i.e., Hemoglobinopathies, Fanconi anemia and hereditary Immunodeficiencies) as well as infectious diseases (i.e., HIV), and the recent development of CRISPR-Cas9-based systems using base and prime editors as well as epigenome editors has provided safer tools for gene therapy. The ex vivo approach for gene addition or editing of HSCs, however, is complex, invasive, technically challenging, costly and not free of toxicity. In vivo gene addition or editing promise to transform gene therapy from a highly sophisticated strategy to a "user-friendly' approach to eventually become a broadly available, highly accessible and potentially affordable treatment modality. In the present review article, based on the lessons gained by more than 3 decades of ex vivo HSC gene therapy, we discuss the concept, the tools, the progress made and the challenges to clinical translation of in vivo HSC gene editing.},
}
@article {pmid36550159,
year = {2022},
author = {Yu, J and Zhou, B and Zhang, S and Yin, H and Sun, L and Pu, Y and Zhou, B and Sun, Y and Li, X and Fang, Y and Wang, L and Zhao, C and Du, D and Zhang, Y and Xu, H},
title = {Design of a self-driven probiotic-CRISPR/Cas9 nanosystem for sono-immunometabolic cancer therapy.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7903},
pmid = {36550159},
issn = {2041-1723},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Gene Editing/methods ; Reactive Oxygen Species ; Immunotherapy ; *Lung Neoplasms/genetics ; Tumor Microenvironment ; },
abstract = {Reprogramming the tumor immunosuppressive microenvironment is a promising strategy for improving tumor immunotherapy efficacy. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system can be used to knockdown tumor immunosuppression-related genes. Therefore, here, a self-driven multifunctional delivery vector is constructed to efficiently deliver the CRISPR-Cas9 nanosystem for indoleamine 2,3-dioxygenase-1 (IDO1) knockdown in order to amplify immunogenic cell death (ICD) and then reverse tumor immunosuppression. Lactobacillus rhamnosus GG (LGG) is a self-driven safety probiotic that can penetrate the hypoxia tumor center, allowing efficient delivery of the CRISPR/Cas9 system to the tumor region. While LGG efficiently colonizes the tumor area, it also stimulates the organism to activate the immune system. The CRISPR/Cas9 nanosystem can generate abundant reactive oxygen species (ROS) under the ultrasound irradiation, resulting in ICD, while the produced ROS can induce endosomal/lysosomal rupture and then releasing Cas9/sgRNA to knock down the IDO1 gene to lift immunosuppression. The system generates immune responses that effectively attack tumor cells in mice, contributing to the inhibition of tumor re-challenge in vivo. In addition, this strategy provides an immunological memory effect which offers protection against lung metastasis.},
}
@article {pmid36550137,
year = {2022},
author = {Yin, J and Fang, K and Gao, Y and Ou, L and Yuan, S and Xin, C and Wu, W and Wu, WW and Hong, J and Yang, H and Hu, J},
title = {Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7867},
pmid = {36550137},
issn = {2041-1723},
mesh = {Mice ; Animals ; *Gene Editing ; Translocation, Genetic ; Genetic Therapy ; *Macular Degeneration/genetics/therapy ; CRISPR-Cas Systems/genetics ; },
abstract = {Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly reduced from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.},
}
@article {pmid36399094,
year = {2022},
author = {Yang, T and Li, J and Zhang, D and Cheng, X and Li, J and Huang, X and Ding, S and Tang, BZ and Cheng, W},
title = {Pre-Folded G-Quadruplex as a Tunable Reporter to Facilitate CRISPR/Cas12a-Based Visual Nucleic Acid Diagnosis.},
journal = {ACS sensors},
volume = {7},
number = {12},
pages = {3710-3719},
doi = {10.1021/acssensors.2c01391},
pmid = {36399094},
issn = {2379-3694},
mesh = {Female ; Humans ; CRISPR-Cas Systems ; Gold ; *Metal Nanoparticles ; Fluorescent Dyes ; *Nucleic Acids ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or "signal-on" colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection.},
}
@article {pmid36216710,
year = {2023},
author = {Ahmad, N and Awan, MJA and Mansoor, S},
title = {Improving editing efficiency of prime editor in plants.},
journal = {Trends in plant science},
volume = {28},
number = {1},
pages = {1-3},
doi = {10.1016/j.tplants.2022.09.001},
pmid = {36216710},
issn = {1878-4372},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Genome ; Plants, Genetically Modified/genetics ; },
abstract = {Prime editing creates targeted insertions or deletions in the genome without double-stranded breaks (DSBs). However, prime-editing efficiency in plants is low, thereby limiting its utilization. A recent study by Zong et al. used a simple strategy that led to substantially improvements in the editing efficiency in both protoplasts and transgenic plants.},
}
@article {pmid36005831,
year = {2022},
author = {Fang, T and Shen, J and Xue, J and Jiang, Y and Guo, D and Yang, J and Kong, X and Xu, X and Wang, X},
title = {Sensitive and Rapid Detection of Escherichia coli O157:H7 From Beef Samples Based on Recombinase Aided Amplification Assisted CRISPR/Cas12a System.},
journal = {Journal of AOAC International},
volume = {106},
number = {1},
pages = {156-164},
doi = {10.1093/jaoacint/qsac101},
pmid = {36005831},
issn = {1944-7922},
mesh = {Animals ; Cattle ; Humans ; *Escherichia coli O157/genetics ; Food Microbiology ; CRISPR-Cas Systems/genetics ; Real-Time Polymerase Chain Reaction/methods ; Limit of Detection ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs to be developed since the traditional detection methods are complex, costly, and time-consuming.<br><br>OBJECTIVE: In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive, and rapid nucleic acid detection of E. coli O157:H7 was introduced.<br><br>METHODS: First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, a real-time PCR assay for detection of the specific wzy gene of E. coli O157:H7 (FDA's Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out.<br><br>RESULTS: The developed RAA-CRISPR/Cas12a method showed high specificity, and the detection could be completed within 30&#x2009;min (after 4&#x2009;h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4&#x2009;&#xd7;&#x2009;102 CFU/mL and 7.5&#x2009;&#xd7;&#x2009;10-4&#x2009;ng/&#x3bc;L, respectively, which exhibited higher sensitivity than the RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157:H7 in ground beef samples could be positively detected after 4&#x2009;h enrichment when the initial bacterial inoculum was 9.0&#x2009; CFU/25 g. The detection results of the RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157:H7 from 93 local collected ground beef samples.<br><br>CONCLUSIONS: The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples.<br><br>HIGHLIGHTS: The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive, and accurate detection of E. coli O157:H7 in foods.},
}
@article {pmid36567185,
year = {2022},
author = {Kim, ER and Joe, C and Mitchell, RJ and Gu, MB},
title = {Biosensors for healthcare: current and future perspectives.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.12.005},
pmid = {36567185},
issn = {1879-3096},
abstract = {Biosensors are utilized in several different fields, including medicine, food, and the environment; in this review, we examine recent developments in biosensors for healthcare. These involve three distinct types of biosensor: biosensors for in vitro diagnosis with blood, saliva, or urine samples; continuous monitoring biosensors (CMBs); and wearable biosensors. Biosensors for in vitro diagnosis have seen a significant expansion recently, with newly reported clustered regularly interspaced short palindromic repeats (CRISPR)/Cas methodologies and improvements to many established integrated biosensor devices, including lateral flow assays (LFAs) and microfluidic/electrochemical paper-based analytical devices (μPADs/ePADs). We conclude with a discussion of two novel groups of biosensors that have drawn great attention recently, continuous monitoring and wearable biosensors, as well as with perspectives on the commercialization and future of biosensors.},
}
@article {pmid36562862,
year = {2022},
author = {Chincinska, IA and Miklaszewska, M and Sołtys-Kalina, D},
title = {Recent advances and challenges in potato improvement using CRISPR/Cas genome editing.},
journal = {Planta},
volume = {257},
number = {1},
pages = {25},
pmid = {36562862},
issn = {1432-2048},
abstract = {Genome editing using CRISPR/Cas technology improves the quality of potato as a food crop and enables its use as both a model plant in fundamental research and as a potential biofactory for producing valuable compounds for industrial applications. Potato (Solanum tuberosum L.) plays a significant role in ensuring global food and nutritional security. Tuber yield is negatively affected by biotic and abiotic stresses, and enzymatic browning and cold-induced sweetening significantly contribute to post-harvest quality losses. With the dual challenges of a growing population and a changing climate, potato enhancement is essential for its sustainable production. However, due to several characteristics of potato, including high levels of heterozygosity, tetrasomic inheritance, inbreeding depression, and self-incompatibility of diploid potato, conventional breeding practices are insufficient to achieve substantial trait improvement in tetraploid potato cultivars within a relatively short time. CRISPR/Cas-mediated genome editing has opened new possibilities to develop novel potato varieties with high commercialization potential. In this review, we summarize recent developments in optimizing CRISPR/Cas-based methods for potato genome editing, focusing on approaches addressing the challenging biology of this species. We also discuss the feasibility of obtaining transgene-free genome-edited potato varieties and explore different strategies to improve potato stress resistance, nutritional value, starch composition, and storage and processing characteristics. Altogether, this review provides insight into recent advances, possible bottlenecks, and future research directions in potato genome editing using CRISPR/Cas technology.},
}
@article {pmid36560658,
year = {2022},
author = {Martinez, MG and Smekalova, E and Combe, E and Gregoire, F and Zoulim, F and Testoni, B},
title = {Gene Editing Technologies to Target HBV cccDNA.},
journal = {Viruses},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/v14122654},
pmid = {36560658},
issn = {1999-4915},
abstract = {Hepatitis B virus (HBV) remains a significant cause of mortality and morbidity worldwide, since chronic HBV infection is associated with elevated risk of cirrhosis and hepatocellular carcinoma. Current licensed therapies against HBV efficiently suppress viral replication; however, they do not have significant effects on the intrahepatic covalently closed circular DNA (cccDNA) of the viral minichromosome responsible for viral persistence. Thus, life-long treatment is required to avoid viral rebound. There is a significant need for novel therapies that can reduce, silence or eradicate cccDNA, thus preventing HBV reemergence after treatment withdrawal. In this review, we discuss the latest developments and applications of gene editing and related approaches for directly targeting HBV DNA and, more specifically, cccDNA in infected hepatocytes.},
}
@article {pmid36558949,
year = {2022},
author = {Kundar, R and Gokarn, K},
title = {CRISPR-Cas System: A Tool to Eliminate Drug-Resistant Gram-Negative Bacteria.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {15},
number = {12},
pages = {},
doi = {10.3390/ph15121498},
pmid = {36558949},
issn = {1424-8247},
abstract = {Rapidly emerging drug-resistant superbugs, especially Gram-negative bacteria, pose a serious threat to healthcare systems all over the globe. Newer strategies are being developed to detect and overcome the arsenal of weapons that these bacteria possess. The development of antibiotics is time-consuming and may not provide full proof of action on evolving drug-resistant pathogens. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are promising in curbing drug-resistant bacteria. This review focuses on the pathogenesis of Gram-negative bacteria, emergence of antimicrobial drug resistance, and their treatment failures. It also draws attention to the present status of the CRISPR-Cas system in diagnosisand treatment of Gram-negative bacterial infections.},
}
@article {pmid36556333,
year = {2022},
author = {Li, Q and Gao, Y and Wang, H},
title = {CRISPR-Based Tools for Fighting Rare Diseases.},
journal = {Life (Basel, Switzerland)},
volume = {12},
number = {12},
pages = {},
doi = {10.3390/life12121968},
pmid = {36556333},
issn = {2075-1729},
abstract = {Rare diseases affect the life of a tremendous number of people globally. The CRISPR-Cas system emerged as a powerful genome engineering tool and has facilitated the comprehension of the mechanism and development of therapies for rare diseases. This review focuses on current efforts to develop the CRISPR-based toolbox for various rare disease therapy applications and compares the pros and cons of different tools and delivery methods. We further discuss the therapeutic applications of CRISPR-based tools for fighting different rare diseases.},
}
@article {pmid36555862,
year = {2022},
author = {Strømsnes, TAH and Schmidke, SE and Azad, M and Singstad, &#xd8; and Grønsberg, IM and Dalmo, RA and Okoli, AS},
title = {CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
doi = {10.3390/ijms232416218},
pmid = {36555862},
issn = {1422-0067},
abstract = {Finfish production has seen over three-fold increase in the past 30 years (1990-2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci-cr2 and mmp9 of A. salmon-were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells.},
}
@article {pmid36555835,
year = {2022},
author = {Song, S and Semenova, E and Severinov, K and Fernández-García, L and Benedik, MJ and Maeda, T and Wood, TK},
title = {CRISPR-Cas Controls Cryptic Prophages.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
doi = {10.3390/ijms232416195},
pmid = {36555835},
issn = {1422-0067},
abstract = {The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.},
}
@article {pmid36555505,
year = {2022},
author = {Kim, HM and Hong, Y and Chen, J},
title = {A Decade of CRISPR-Cas Gnome Editing in C. elegans.},
journal = {International journal of molecular sciences},
volume = {23},
number = {24},
pages = {},
doi = {10.3390/ijms232415863},
pmid = {36555505},
issn = {1422-0067},
abstract = {CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions, with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields, with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly established over the past decade. Ironically, the emergence of numerous methods makes the choice of the correct method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans, illustrating key strategies. Our overview of significant advances in CRISPR-Cas will help readers understand the current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.},
}
@article {pmid36552850,
year = {2022},
author = {Viushkov, VS and Lomov, NA and Rubtsov, MA and Vassetzky, YS},
title = {Visualizing the Genome: Experimental Approaches for Live-Cell Chromatin Imaging.},
journal = {Cells},
volume = {11},
number = {24},
pages = {},
doi = {10.3390/cells11244086},
pmid = {36552850},
issn = {2073-4409},
abstract = {Over the years, our vision of the genome has changed from a linear molecule to that of a complex 3D structure that follows specific patterns and possesses a hierarchical organization. Currently, genomics is becoming "four-dimensional": our attention is increasingly focused on the study of chromatin dynamics over time, in the fourth dimension. Recent methods for visualizing the movements of chromatin loci in living cells by targeting fluorescent proteins can be divided into two groups. The first group requires the insertion of a special sequence into the locus of interest, to which proteins that recognize the sequence are recruited (e.g., FROS and ParB-INT methods). In the methods of the second approach, "programmed" proteins are targeted to the locus of interest (i.e., systems based on CRISPR/Cas, TALE, and zinc finger proteins). In the present review, we discuss these approaches, examine their strengths and weaknesses, and identify the key scientific problems that can be studied using these methods.},
}
@article {pmid36551141,
year = {2022},
author = {Ivanov, AV and Safenkova, IV and Drenova, NV and Zherdev, AV and Dzantiev, BB},
title = {Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora.},
journal = {Biosensors},
volume = {12},
number = {12},
pages = {},
doi = {10.3390/bios12121174},
pmid = {36551141},
issn = {2079-6374},
abstract = {Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5' ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 10[4] CFU/mL for LAMP-LFT, 10[3] CFU/mL for LAMP-CRISPR/Cas, and 10[2] CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30-55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.},
}
@article {pmid36546784,
year = {2022},
author = {Belato, HB and Norbrun, C and Luo, J and Pindi, C and Sinha, S and D'Ordine, AM and Jogl, G and Palermo, G and Lisi, GP},
title = {Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.},
journal = {The Journal of chemical physics},
volume = {157},
number = {22},
pages = {225103},
pmid = {36546784},
issn = {1089-7690},
mesh = {*CRISPR-Associated Protein 9/chemistry/metabolism ; *CRISPR-Cas Systems ; DNA Cleavage ; Static Electricity ; Temperature ; },
abstract = {Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.},
}
@article {pmid36546144,
year = {2022},
author = {Chen, Q and Chuai, G and Zhang, C and Zhang, Q and Liu, Q},
title = {Toward a molecular mechanism-based prediction of CRISPR-Cas9 targeting effects.},
journal = {Science bulletin},
volume = {67},
number = {12},
pages = {1201-1204},
doi = {10.1016/j.scib.2022.04.023},
pmid = {36546144},
issn = {2095-9281},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Targeting ; },
}
@article {pmid36546116,
year = {2022},
author = {Ma, L and Dong, C and Yu, M and Song, X and Yu, Y and Chen, Y},
title = {Nanomedicine enables efficient CRISPR-Cas9 genome editing for disease treatment.},
journal = {Science bulletin},
volume = {67},
number = {6},
pages = {572-576},
doi = {10.1016/j.scib.2021.11.016},
pmid = {36546116},
issn = {2095-9281},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Nanomedicine ; Palliative Care ; },
}
@article {pmid36541895,
year = {2022},
author = {Ngan, KC and Lue, NZ and Lee, C and Liau, BB},
title = {CRISPR-Suppressor Scanning for Systematic Discovery of Drug-Resistance Mutations.},
journal = {Current protocols},
volume = {2},
number = {12},
pages = {e614},
doi = {10.1002/cpz1.614},
pmid = {36541895},
issn = {2691-1299},
support = {DDR 60S-20/DRCRF/Damon Runyon Cancer Research Foundation/United States ; /GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; Gene Library ; Lentivirus/genetics ; },
abstract = {CRISPR-Cas9 genome editing technologies have enabled complex genetic manipulations in situ, including large-scale, pooled screening approaches to probe and uncover mechanistic insights across various biological processes. The RNA-programmable nature of CRISPR-Cas9 greatly empowers tiling mutagenesis approaches to elucidate molecular details of protein function, in particular the interrogation of mechanisms of resistance to small molecules, an approach termed CRISPR-suppressor scanning. In a typical CRISPR-suppressor scanning experiment, a pooled library of single-guide RNAs is designed to target across the coding sequence(s) of one or more genes, enabling the Cas9 nuclease to systematically mutate the targeted proteins and generate large numbers of diverse protein variants in situ. This cellular pool of protein variants is then challenged with drug treatment to identify mutations conferring a fitness advantage. Drug-resistance mutations identified with this approach can not only elucidate drug mechanism of action but also reveal deeper mechanistic insights into protein structure-function relationships. In this article, we outline the framework for a standard CRISPR-suppressor scanning experiment. Specifically, we provide instructions for the design and construction of a pooled sgRNA library, execution of a CRISPR-suppressor scanning screen, and basic computational analysis of the resulting data. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of a pooled sgRNA library Support Protocol 1: sgRNA library design using command-line CRISPOR Support Protocol 2: Production and titering of pooled sgRNA library lentivirus Basic Protocol 2: Execution and analysis of a CRISPR-suppressor scanning experiment.},
}
@article {pmid36453883,
year = {2022},
author = {Wang, X and Jin, Q and Xiao, W and Fang, P and Lai, L and Xiao, S and Wang, K and Fang, L},
title = {Genome-Wide CRISPR/Cas9 Screen Reveals a Role for SLC35A1 in the Adsorption of Porcine Deltacoronavirus.},
journal = {Journal of virology},
volume = {96},
number = {24},
pages = {e0162622},
pmid = {36453883},
issn = {1098-5514},
mesh = {Humans ; Animals ; Swine ; Adsorption ; CRISPR-Cas Systems ; N-Acetylneuraminic Acid ; Trypsin ; *Coronavirus/genetics ; *Coronavirus Infections/genetics ; *Swine Diseases ; *Nucleotide Transport Proteins/genetics ; },
abstract = {Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. IMPORTANCE Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.},
}
@article {pmid36416551,
year = {2022},
author = {Lombardi, L and Bergin, SA and Ryan, A and Zuniga-Soto, E and Butler, G},
title = {CRISPR-Cas9 Editing Induces Loss of Heterozygosity in the Pathogenic Yeast Candida parapsilosis.},
journal = {mSphere},
volume = {7},
number = {6},
pages = {e0039322},
pmid = {36416551},
issn = {2379-5042},
mesh = {Humans ; *CRISPR-Cas Systems ; *Candida parapsilosis/genetics ; Saccharomyces cerevisiae/genetics ; Gene Editing/methods ; Loss of Heterozygosity ; },
abstract = {Genetic manipulation is often used to study gene function. However, unplanned genome changes (including single nucleotide polymorphisms [SNPs], aneuploidy, and loss of heterozygosity [LOH]) can affect the phenotypic traits of the engineered strains. Here, we compared the effect of classical deletion methods (replacing target alleles with selectable markers by homologous recombination) with CRISPR-Cas9 editing in the diploid human-pathogenic yeast Candida parapsilosis. We sequenced the genomes of 9 isolates that were modified using classic recombination methods and 12 that were edited using CRISPR-Cas9. As a control, the genomes of eight isolates that were transformed with a Cas9-expressing plasmid in the absence of a guide RNA were also sequenced. Following gene manipulation using classic homologous recombination, only one strain exhibited extensive LOH near the targeted gene (8.9 kb), whereas another contained multiple LOH events not associated with the intended modification. In contrast, large regions of LOH (up to >1,100 kb) were observed in most CRISPR-Cas9-edited strains. LOH most commonly occurred adjacent to the Cas9 cut site and extended to the telomere in four isolates. In two isolates, we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Among the CRISPR-edited isolates, two exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is required when using CRISPR-Cas9. IMPORTANCE CRISPR-Cas9 has greatly streamlined gene editing and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species, extra care should be taken in confirming the cause of any phenotypic changes observed. We show that the Cas9-induced double-strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g., stop gain in one allele) becoming homozygous, resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.},
}
@article {pmid36547823,
year = {2022},
author = {Saber Sichani, A and Ranjbar, M and Baneshi, M and Torabi Zadeh, F and Fallahi, J},
title = {A Review on Advanced CRISPR-Based Genome-Editing Tools: Base Editing and Prime Editing.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36547823},
issn = {1559-0305},
abstract = {In the field of medicine, it is axiomatic that the need of a precise gene-editing tool is critical to employ therapeutic approaches toward pathogenic mutations, occurring in human genome. Today we know that most of genetic defects are caused by single-base pair substitutions in genomic DNA. The ability to make practically any targeted substitutions of DNA sequences at specified regions in the human genome gives us the chance to employ gene therapy in most known diseases associated with genetic variants. In this regard, CRISPR/Cas9 applications is becoming more and more popular along with the significant advancements of life sciences, by employing this technology in genome-editing and high-throughput screenings. Several CRISPR/Cas-based mammalian cell gene-editing techniques have been developed during the last decade, including nucleases, base editors, and prime editors, all of which have the exact mechanism at first glance. However, they address a subset of known pathogenic sequence mutations using different methods. First, we highlight the development of CRISPR-based gene-editing tools. Then we describe their functions and summarize the conducted research studies, which are increasing the reliability of these strategies to better efficiencies for prospective gene therapies in the near future. Lastly, we compare the capabilities of all these platforms together besides their probable limitations.},
}
@article {pmid36546891,
year = {2022},
author = {Corts, A and Thomason, LC and Costantino, N and Court, DL},
title = {Recombineering in Non-Model Bacteria.},
journal = {Current protocols},
volume = {2},
number = {12},
pages = {e605},
doi = {10.1002/cpz1.605},
pmid = {36546891},
issn = {2691-1299},
abstract = {The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.},
}
@article {pmid36546759,
year = {2022},
author = {Stoyko, D and O, T and Hernandez, A and Konstantinidou, P and Meng, Q and Haase, AD},
title = {CRISPR-Cas9 Genome Editing and Rapid Selection of Cell Pools.},
journal = {Current protocols},
volume = {2},
number = {12},
pages = {e624},
doi = {10.1002/cpz1.624},
pmid = {36546759},
issn = {2691-1299},
support = {/DK/NIDDK NIH HHS/United States ; ZIA DK075111/NH/NIH HHS/United States ; },
abstract = {The harnessing of the CRISPR-Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR-Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome for nontransformed cell culture models such as Drosophila ovarian somatic sheath cells (OSCs). OSCs are a unique ex vivo model for epigenetic regulation by PIWI-interacting RNAs (piRNAs) that establish restriction of mobile genetic elements in germ cells to protect genome integrity. Here, we provide a protocol to generate endogenously tagged proteins and gene knockouts without the need for clonal selection. We combine CRISPR-Cas genome editing and knockin of antibiotic selection markers to generate edited cell pools. At the example of Drosophila piwi in OSCs, we demonstrate a strategy that relies on the insertion of an artificial intron to accommodate a selection marker with minimal disturbance of the resulting mRNA. In brief, our donor cassette contains a peptide tag and an optimized intron that accommodates a selection marker driven by an independent promoter on the other genomic strand. The selection marker is transcribed as an independent mRNA, and the intron is efficiently removed from the mRNA encoding the endogenously tagged (endo-tagged) piwi gene. The endo-tagged Piwi protein is expressed at wild-type levels and appropriately localizes to the nucleus of OSCs. We also describe strategies for C-terminal tagging and generation of knockout alleles in OSCs and in human embryonic kidney cells, discuss different design strategies, and provide a plasmid toolkit (available at Addgene). Our protocol enables robust genome editing in OSCs for the first time and provides a simple and time-saving alternative for other cell culture systems. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Design and cloning of single-guide RNA plasmids Basic Protocol 2: Design and cloning of donor template plasmids for epitope tagging Alternate Protocol: Design and cloning of donor template plasmids for gene knockout Basic Protocol 3: Transfection and selection of edited cell pools.},
}
@article {pmid36542213,
year = {2022},
author = {Chen, Y and Hu, Y and Luo, S and Wang, X and Mao, B and Chen, Y and Xu, J and Li, Z and Zhou, Q and Li, W},
title = {Computer-aided engineering of CRISPR-Cas proteins for enhanced human genome editing.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {36542213},
issn = {1869-1889},
}
@article {pmid36541764,
year = {2022},
author = {Sharma, A and Omer Aden, R and Puhar, A and Cisneros, DA},
title = {CRISPR-Cas-Guided Mutagenesis of Chromosome and Virulence Plasmid in Shigella flexneri by Cytosine Base Editing.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0104522},
doi = {10.1128/msystems.01045-22},
pmid = {36541764},
issn = {2379-5077},
abstract = {Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large-scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas-dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by single guide RNA (sgRNA) to introduce targeted C→T transitions, resulting in internal stop codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both Escherichia coli and Shigella flexneri with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion was in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Furthermore, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella flexneri genes and found the mutant phenotype to be similar to those of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well-plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. IMPORTANCE Loss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches systems biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen-Shigella. This pathogen causes high morbidity and mortality in children, and antibiotic-resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of the utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generate multiple mutants in a single, massively parallel reaction. By virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.},
}
@article {pmid36427699,
year = {2023},
author = {Ustiugova, AS and Ekaterina, DM and Nataliya, MV and Alexey, DA and Dmitry, KV and Marina, AA},
title = {CRISPR/Cas9 genome editing demonstrates functionality of the autoimmunity-associated SNP rs12946510.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1869},
number = {2},
pages = {166599},
doi = {10.1016/j.bbadis.2022.166599},
pmid = {36427699},
issn = {1879-260X},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Genome-Wide Association Study ; Autoimmunity ; Polymorphism, Single Nucleotide ; },
abstract = {Genome-wide association studies (GWAS) map genetic associations of complex traits with precision limited to a linkage disequilibrium group. To translate GWAS results into new understanding of disease mechanisms, individual causative polymorphisms and their target genes should be identified. CRISPR/Cas9 genome editing can be used to create isogenic cell lines bearing alternative genotypes of candidate single-nucleotide polymorphisms to test their causality and to reveal gene targets. An intergenic polymorphism rs12946510 is associated with multiple sclerosis, inflammatory bowel disease and asthma. We created sublines of the T-helper cell line bearing alternative genotypes of rs12946510 and showed that its risk ("T") allele is associated with lower expression of IKZF3 and ORMDL3 genes and reduced cell activation. Our editing procedure can become an effective tool for discovering new genes involved in pathogenesis of complex diseases.},
}
@article {pmid36385254,
year = {2023},
author = {Schimmel, J and van Wezel, MD and van Schendel, R and Tijsterman, M},
title = {Chromosomal breaks at the origin of small tandem DNA duplications.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {45},
number = {1},
pages = {e2200168},
doi = {10.1002/bies.202200168},
pmid = {36385254},
issn = {1521-1878},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA End-Joining Repair ; Chromosome Breakage ; DNA/genetics ; DNA Repair ; Genome, Human ; },
abstract = {Small tandem DNA duplications in the range of 15 to 300 base-pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9-based experimental data from different model systems. Recent work points to fill-in synthesis at double-stranded DNA breaks with complementary sequences, regulated by end-joining mechanisms, to account for small tandem duplications. We will review the prevalence of small tandem duplications in the population, and we will speculate on the potential sources of DNA damage that could give rise to this mutational signature. With the development of novel algorithms to analyse sequencing data, small tandem duplications are now more frequently detected in the human genome and identified as oncogenic gain-of-function mutations. Understanding their origin could lead to optimized treatment regimens to prevent therapy-induced activation of oncogenes and might expose novel vulnerabilities in cancer.},
}
@article {pmid36115027,
year = {2022},
author = {Caillaud, A and Lévêque, A and Thédrez, A and Girardeau, A and Canac, R and Bray, L and Baudic, M and Barc, J and Gaborit, N and Lamirault, G and Gardie, B and Idriss, S and Rimbert, A and Le May, C and Cariou, B and Si-Tayeb, K},
title = {FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.},
journal = {STAR protocols},
volume = {3},
number = {4},
pages = {101680},
pmid = {36115027},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells ; Clone Cells ; Cell Culture Techniques ; },
abstract = {This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).},
}
@article {pmid36115024,
year = {2022},
author = {Qi, S and Sivakumar, S and Yu, H},
title = {CRISPR-Cas9 screen in human embryonic stem cells to identify genes required for neural differentiation.},
journal = {STAR protocols},
volume = {3},
number = {4},
pages = {101682},
pmid = {36115024},
issn = {2666-1667},
mesh = {Humans ; *Human Embryonic Stem Cells ; CRISPR-Cas Systems ; *Neural Stem Cells ; Cell Differentiation ; },
abstract = {Human embryonic stem cells (hESCs) continuously self-renew in culture and can be induced to differentiate into multiple cell types, including neural progenitor cells (NPCs). Here, we present a protocol to perform a CRISPR-Cas9 screen in hESCs to identify regulators that promote SOX1 expression during NPC formation. This screening protocol can be adapted with other endpoint reporters for the identification of genes involved in the commitment of hESCs to other cell lineages. For complete details on the use and execution of this protocol, please refer to Sivakumar et al. (2022).},
}
@article {pmid36107744,
year = {2022},
author = {Lin, K and Chang, YC and Marron Fernandez de Velasco, E and Wickman, K and Myers, CL and Bielinsky, AK},
title = {Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells.},
journal = {STAR protocols},
volume = {3},
number = {4},
pages = {101675},
pmid = {36107744},
issn = {2666-1667},
support = {F30 CA257227/CA/NCI NIH HHS/United States ; R35 GM141805/GM/NIGMS NIH HHS/United States ; UL1 TR002494/TR/NCATS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Genetic Testing ; Gene Library ; Genome ; Gene Knockout Techniques ; },
abstract = {Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53[-/-] cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.},
}
@article {pmid36538648,
year = {2022},
author = {Perillo, G and Shibata, K and Wu, PH},
title = {piRNAs in sperm function and embryo viability.},
journal = {Reproduction (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1530/REP-22-0312},
pmid = {36538648},
issn = {1741-7899},
abstract = {Unique to animals, PIWI-interacting RNAs (piRNAs) defend organisms against threats to germline integrity evoked by transposons, retroviruses, and inappropriate expression of protein-coding genes. Characterization of mouse piRNAs and studies of more than a dozen piRNA pathway protein mutants detailed in the past 15 years have firmly established an essential role for piRNAs in male fertility. Despite their vital function in spermatogenesis, mammalian piRNAs were thought to be dispensable beyond gamete formation because all piRNA pathway protein mouse mutants are invariably sterile and do not produce sperm. In contrast to the specialized purpose of piRNAs in gamete formation, tRNA-derived fragments (tRFs) and microRNAs (miRNAs) have been the focus of research in RNA-mediated paternal contribution, providing additional examples of the versatility of non-coding RNAs. In recent years, direct elimination of mouse piRNAs using CRISPR/Cas revealed their extended function in post-testicular sperm maturation. An intergenerational contribution from paternal piRNAs has also been proposed. Together with insights into piRNAs in oocytes and early embryos in mice and other mammals, these newly proposed functions of mammalian piRNAs invite further investigations of piRNA dynamics during sperm maturation and fertilization as well as their roles in reproduction beyond gametogenesis.},
}
@article {pmid36537251,
year = {2022},
author = {Ma, E and Chen, K and Shi, H and Stahl, EC and Adler, B and Trinidad, M and Liu, J and Zhou, K and Ye, J and Doudna, JA},
title = {Improved genome editing by an engineered CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac1192},
pmid = {36537251},
issn = {1362-4962},
support = {RM1HG009490/NH/NIH HHS/United States ; F32GM140637-01/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.},
}
@article {pmid36537206,
year = {2022},
author = {Hsieh, SC and Peters, JE},
title = {Discovery and characterization of novel type I-D CRISPR-guided transposons identified among diverse Tn7-like elements in cyanobacteria.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac1216},
pmid = {36537206},
issn = {1362-4962},
abstract = {CRISPR-Cas defense systems have been naturally coopted for guide RNA-directed transposition by Tn7 family bacterial transposons. We find cyanobacterial genomes are rich in Tn7-like elements, including most of the known guide RNA-directed transposons, the type V-K, I-B1, and I-B2 CRISPR-Cas based systems. We discovered and characterized an example of a type I-D CRISPR-Cas system which was naturally coopted for guide RNA-directed transposition. Multiple novel adaptations were found specific to the I-D subtype, including natural inactivation of the Cas10 nuclease. The type I-D CRISPR-Cas transposition system showed flexibility in guide RNA length requirements and could be engineered to function with ribozyme-based self-processing guide RNAs removing the requirement for Cas6 in the heterologous system. The type I-D CRISPR-Cas transposon also has naturally fused transposase proteins that are functional for cut-and-paste transposition. Multiple attributes of the type I-D system offer unique possibilities for future work in gene editing. Our bioinformatic analysis also revealed a broader understanding of the evolution of Tn7-like elements. Extensive swapping of targeting systems was identified among Tn7-like elements in cyanobacteria and multiple examples of convergent evolution, including systems targeting integration into genes required for natural transformation.},
}
@article {pmid36526651,
year = {2022},
author = {Yamazaki, K and Matsuo, K and Okada, A and Uno, N and Suzuki, T and Abe, S and Hamamichi, S and Kishima, N and Togai, S and Tomizuka, K and Kazuki, Y},
title = {Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {21790},
pmid = {36526651},
issn = {2045-2322},
mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Recombinational DNA Repair ; Chromosomes, Artificial ; Genome ; Polymerase Chain Reaction ; Gene Editing ; Mammals/genetics ; },
abstract = {Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor containing homology arm by PCR could bypass the cloning steps of target sequence and selection markers. To confirm the functionality of the loaded HDR donors, we constructed a MAC with human leukocyte antigen A (HLA-A) gene in the DT40 cells, and verified the expression of this genomic region by reverse transcription PCR (RT-PCR) and western blotting. Collectively, the simHDR offers a rapid and convenient approach to generate genetically modified models for investigating gene functions, as well as understanding disease mechanisms and therapeutic interventions.},
}
@article {pmid36482251,
year = {2022},
author = {Thomas, T},
title = {Off-target Cas9 crystallized.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {12},
pages = {1147},
doi = {10.1038/s41594-022-00900-w},
pmid = {36482251},
issn = {1545-9985},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, Kinetoplastida ; CRISPR-Associated Protein 9/genetics ; Gene Editing ; },
}
@article {pmid36471058,
year = {2022},
author = {Hall, PM and Inman, JT and Fulbright, RM and Le, TT and Brewer, JJ and Lambert, G and Darst, SA and Wang, MD},
title = {Polarity of the CRISPR roadblock to transcription.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {12},
pages = {1217-1227},
pmid = {36471058},
issn = {1545-9985},
mesh = {*CRISPR-Associated Proteins/metabolism ; RNA, Guide, Kinetoplastida/genetics ; Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/chemistry ; DNA/chemistry ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) utility relies on a stable Cas effector complex binding to its target site. However, a Cas complex bound to DNA may be removed by motor proteins carrying out host processes and the mechanism governing this removal remains unclear. Intriguingly, during CRISPR interference, RNA polymerase (RNAP) progression is only fully blocked by a bound endonuclease-deficient Cas (dCas) from the protospacer adjacent motif (PAM)-proximal side. By mapping dCas-DNA interactions at high resolution, we discovered that the collapse of the dCas R-loop allows Escherichia coli RNAP read-through from the PAM-distal side for both Sp-dCas9 and As-dCas12a. This finding is not unique to RNAP and holds for the Mfd translocase. This mechanistic understanding allowed us to modulate the dCas R-loop stability by modifying the guide RNAs. This work highlights the importance of the R-loop in dCas-binding stability and provides valuable mechanistic insights for broad applications of CRISPR technology.},
}
@article {pmid36460008,
year = {2022},
author = {Patel, DJ and Yu, Y and Jia, N},
title = {Bacterial origins of cyclic nucleotide-activated antiviral immune signaling.},
journal = {Molecular cell},
volume = {82},
number = {24},
pages = {4591-4610},
doi = {10.1016/j.molcel.2022.11.006},
pmid = {36460008},
issn = {1097-4164},
mesh = {*Nucleotides, Cyclic/metabolism ; CRISPR-Cas Systems ; Antiviral Agents ; Archaea/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteriophages/genetics/metabolism ; Immunity, Innate ; },
abstract = {Second-messenger-mediated signaling by cyclic oligonucleotides (cOs) composed of distinct base, ring size, and 3'-5'/2'-5' linkage combinations constitutes the initial trigger resulting in activation of signaling pathways that have an impact on immune-mediated antiviral defense against invading viruses and phages. Bacteria and archaea have evolved CRISPR, CBASS, Pycsar, and Thoeris surveillance complexes that involve cO-mediated activation of effectors resulting in antiviral defense through either targeted nuclease activity, effector oligomerization-mediated depletion of essential cellular metabolites or disruption of host cell membrane functions. Notably, antiviral defense capitalizes on an abortive infection mechanism, whereby infected cells die prior to completion of the phage replication cycle. In turn, phages have evolved small proteins that target and degrade/sequester cOs, thereby suppressing host immunity. This review presents a structure-based mechanistic perspective of recent advances in the field of cO-mediated antiviral defense, in particular highlighting the ancient evolutionary adaptation by metazoans of bacterial cell-autonomous innate immune mechanisms.},
}
@article {pmid36209487,
year = {2022},
author = {Allemailem, KS and Alsahli, MA and Almatroudi, A and Alrumaihi, F and Alkhaleefah, FK and Rahmani, AH and Khan, AA},
title = {Current updates of CRISPR/Cas9-mediated genome editing and targeting within tumor cells: an innovative strategy of cancer management.},
journal = {Cancer communications (London, England)},
volume = {42},
number = {12},
pages = {1257-1287},
doi = {10.1002/cac2.12366},
pmid = {36209487},
issn = {2523-3548},
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics/metabolism ; Endonucleases/genetics/metabolism ; DNA ; *Neoplasms/genetics/therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9), an adaptive microbial immune system, has been exploited as a robust, accurate, efficient and programmable method for genome targeting and editing. This innovative and revolutionary technique can play a significant role in animal modeling, in vivo genome therapy, engineered cell therapy, cancer diagnosis and treatment. The CRISPR/Cas9 endonuclease system targets a specific genomic locus by single guide RNA (sgRNA), forming a heteroduplex with target DNA. The Streptococcus pyogenes Cas9/sgRNA:DNA complex reveals a bilobed architecture with target recognition and nuclease lobes. CRISPR/Cas9 assembly can be hijacked, and its nanoformulation can be engineered as a delivery system for different clinical utilizations. However, the efficient and safe delivery of the CRISPR/Cas9 system to target tissues and cancer cells is very challenging, limiting its clinical utilization. Viral delivery strategies of this system may have many advantages, but disadvantages such as immune system stimulation, tumor promotion risk and small insertion size outweigh these advantages. Thus, there is a desperate need to develop an efficient non-viral physical delivery system based on simple nanoformulations. The delivery strategies of CRISPR/Cas9 by a nanoparticle-based system have shown tremendous potential, such as easy and large-scale production, combination therapy, large insertion size and efficient in vivo applications. This review aims to provide in-depth updates on Streptococcus pyogenic CRISPR/Cas9 structure and its mechanistic understanding. In addition, the advances in its nanoformulation-based delivery systems, including lipid-based, polymeric structures and rigid NPs coupled to special ligands such as aptamers, TAT peptides and cell-penetrating peptides, are discussed. Furthermore, the clinical applications in different cancers, clinical trials and future prospects of CRISPR/Cas9 delivery and genome targeting are also discussed.},
}
@article {pmid36535150,
year = {2022},
author = {Wang, T and Hellmer, H and Simmel, FC},
title = {Genetic switches based on nucleic acid strand displacement.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102867},
doi = {10.1016/j.copbio.2022.102867},
pmid = {36535150},
issn = {1879-0429},
abstract = {Toehold-mediated strand displacement (TMSD) is an isothermal switching process that enables the sequence-programmable and reversible conversion of DNA or RNA strands between single- and double-stranded conformations or other secondary structures. TMSD processes have already found widespread application in DNA nanotechnology, where they are used to drive DNA-based molecular devices or for the realization of synthetic biochemical computing circuits. Recently, researchers have started to employ TMSD also for the control of RNA-based gene regulatory processes in vivo, in particular in the context of synthetic riboregulators and conditional guide RNAs for CRISPR/Cas. Here, we provide a review over recent developments in this emerging field and discuss the opportunities and challenges for such systems in in vivo applications.},
}
@article {pmid36533445,
year = {2022},
author = {Medert, R and Thumberger, T and Tavhelidse-Suck, T and Hub, T and Kellner, T and Oguchi, Y and Dlugosz, S and Zimmermann, F and Wittbrodt, J and Freichel, M},
title = {Efficient single copy integration via homology-directed repair (scHDR) by 5'modification of large DNA donor fragments in mice.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac1150},
pmid = {36533445},
issn = {1362-4962},
abstract = {CRISPR/Cas-based approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging given that donor constructs preferentially integrate as concatemers. Here, we showed that injecting 5' biotinylated donor DNA into mouse embryos at the two-cell stage led to efficient single-copy HDR (scHDR) allele generation. Our dedicated genotyping strategy showed that these alleles occurred with frequencies of 19%, 20%, and 26% at three independent gene loci, indicating that scHDR was dramatically increased by 5' biotinylation. Thus, we suggest that the combination of a 5' biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins in the mouse genome.},
}
@article {pmid36533412,
year = {2022},
author = {Li, H and Xie, Y and Chen, F and Bai, H and Xiu, L and Zhou, X and Guo, X and Hu, Q and Yin, K},
title = {Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives.},
journal = {Chemical Society reviews},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2cs00594h},
pmid = {36533412},
issn = {1460-4744},
abstract = {Rapid and accurate molecular diagnosis is a prerequisite for precision medicine, food safety, and environmental monitoring. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based detection, as a cutting-edged technique, has become an immensely effective tool for molecular diagnosis because of its outstanding advantages including attomolar level sensitivity, sequence-targeted single-base specificity, and rapid turnover time. However, the CRISPR/Cas-based detection methods typically require a pre-amplification step to elevate the concentration of the analyte, which may produce non-specific amplicons, prolong the detection time, and raise the risk of carryover contamination. Hence, various strategies for target amplification-free CRISPR/Cas-based detection have been developed, aiming to minimize the sensitivity loss due to lack of pre-amplification, enable detection for non-nucleic acid targets, and facilitate integration in portable devices. In this review, the current status and challenges of target amplification-free CRISPR/Cas-based detection are first summarized, followed by highlighting the four main strategies to promote the performance of target amplification-free CRISPR/Cas-based technology. Furthermore, we discuss future perspectives that will contribute to developing more efficient amplification-free CRISPR/Cas detection systems.},
}
@article {pmid36532627,
year = {2022},
author = {Ulyanov, AV and Karlov, AV and Khatefov, EB},
title = {The use of maize haploidy inducers as a tool in agricultural plant biotechnology.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {26},
number = {7},
pages = {704-713},
doi = {10.18699/VJGB-22-85},
pmid = {36532627},
issn = {2500-0462},
abstract = {The discovery of the ability of some mutations to stimulate haploidy during hybridization made it possible to create one of the most promising and sought-after trends in the f ield of reproductive biology. Haploid inducers created on their basis are capable of increasing the frequency of haploidy up to 15 %. The improvement of the existing haploid inducer lines and the search for new genes that contribute to a high frequency of haploidy are underway. Along with these studies, the f ield of application of haploid inducers in genetics and plant breeding is expanding. Haploid inducers carrying R1-nj genes for anthocyanin pigmentation of the seed and embryo are able not only to mark the hybrid embryo and identify haploid genotypes, but also to detect genes that suppress the anthocyanin color of the grain, like C1-I, C2-Idf, and In1-D. Depending on their quantity, the phenotypic manifestation of the gene in the seed varies. Haploidy is widely used for accelerating hybrid breeding and obtaining both new maize lines with improved traits and their sterile counterparts. By introducing certain genes into the genome of the improved line, breeders can use the doubled haploid (DH) breeding technology to accelerate the creation of pure lines carrying the desired gene. Haploid inducer maize lines and their tetraploid analogs are used in the selection of rediploid maize lines by their resynthesis from tetraploid genotypes. In 2019, Syngenta Company synthesized a haploid inducer maize line carrying a CRISPR/cas construct capable of simultaneously stimulating haploidy and editing the genome at a specif ied DNA site. Thanks to this technology, it became possible to improve haploid inducers by introducing various CRISPR/cas constructs into the haploid inducer genome for editing any DNA site. Maize haploid inducers are widely used in doubled haploid wheat breeding. The f irst experiments showed that the most effective haploid inducer for stimulating haploidy in wheat is maize pollen. Researchers are intensively searching for other ways of using maize haploid inducers in plant breeding.},
}
@article {pmid36532491,
year = {2022},
author = {Salgado, O and Guajardo-Leiva, S and Moya-Beltrán, A and Barbosa, C and Ridley, C and Tamayo-Leiva, J and Quatrini, R and Mojica, FJM and Díez, B},
title = {Global phylogenomic novelty of the Cas1 gene from hot spring microbial communities.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {1069452},
pmid = {36532491},
issn = {1664-302X},
abstract = {The Cas1 protein is essential for the functioning of CRISPR-Cas adaptive systems. However, despite the high prevalence of CRISPR-Cas systems in thermophilic microorganisms, few studies have investigated the occurrence and diversity of Cas1 across hot spring microbial communities. Phylogenomic analysis of 2,150 Cas1 sequences recovered from 48 metagenomes representing hot springs (42-80°C, pH 6-9) from three continents, revealed similar ecological diversity of Cas1 and 16S rRNA associated with geographic location. Furthermore, phylogenetic analysis of the Cas1 sequences exposed a broad taxonomic distribution in thermophilic bacteria, with new clades of Cas1 homologs branching at the root of the tree or at the root of known clades harboring reference Cas1 types. Additionally, a new family of casposases was identified from hot springs, which further completes the evolutionary landscape of the Cas1 superfamily. This ecological study contributes new Cas1 sequences from known and novel locations worldwide, mainly focusing on under-sampled hot spring microbial mat taxa. Results herein show that circumneutral hot springs are environments harboring high diversity and novelty related to adaptive immunity systems.},
}
@article {pmid36531381,
year = {2022},
author = {Scintilla, S and Salvagnin, U and Giacomelli, L and Zeilmaker, T and Malnoy, MA and Rouppe van der Voort, J and Moser, C},
title = {Regeneration of non-chimeric plants from DNA-free edited grapevine protoplasts.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1078931},
pmid = {36531381},
issn = {1664-462X},
abstract = {The application of New Breeding Techniques (NBTs) in Vitis vinifera is highly desirable to introduce valuable traits while preserving the genotype of the elite cultivars. However, a broad application of NBTs through standard DNA-based transformation is poorly accepted by public opinion and law regulations in Europe and other countries due to the stable integration of exogenous DNA, which leads to transgenic plants possibly affected by chimerism. A single-cell based approach, coupled with a DNA-free transfection of the CRISPR/Cas editing machinery, constitutes a powerful tool to overcome these problems and maintain the original genetic make-up in the whole organism. We here describe a successful single-cell based, DNA-free methodology to obtain edited grapevine plants, regenerated from protoplasts isolated from embryogenic callus of two table grapevine varieties (V. vinifera cv. Crimson seedless and Sugraone). The regenerated, non-chimeric plants were edited on the downy- and powdery-mildew susceptibility genes, VviDMR6 and VviMlo6 respectively, either as single or double mutants.},
}
@article {pmid36530849,
year = {2023},
author = {Huang, X and Fu, R and Qiao, S and Zhang, J and Xianyu, Y},
title = {Nanotechnology-based diagnostic methods for coronavirus: From nucleic acid extraction to amplification.},
journal = {Biosensors &amp; bioelectronics: X},
volume = {13},
number = {},
pages = {100289},
pmid = {36530849},
issn = {2590-1370},
abstract = {The recent emergence of human coronaviruses (CoVs) causing severe acute respiratory syndrome (SARS) is posing a great threat to global public health. Therefore, the rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people's lives and preventing epidemics. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are natural biopolymers composed of nucleotides that store, transmit, and express genetic information. Applications of nucleic acid detection range from genotyping and genetic prognostics, to expression profiling and detection of infectious disease. The nucleic acid detection for infectious diseases is widely used, as evidenced by the widespread use of COVID-19 tests for the containment of the pandemic. Nanotechnology influences all medical disciplines and has been considered as an essential tool for novel diagnostics, nanotherapeutics, vaccines, medical imaging, and the utilization of biomaterials for regenerative medicine. In this review, the recent advances in the development of nanotechnology-based diagnostic methods for coronavirus, and their applications in nucleic acid detection are discussed in detail. The techniques for the amplification of nucleic acids are summarized, as well as the use of magnetic nanoparticles for nucleic acid extraction. Besides, current challenges and future prospects are proposed, along with the great potential of nanotechnology for the effective diagnosis of coronavirus.},
}
@article {pmid36522342,
year = {2022},
author = {Aliaga Goltsman, DS and Alexander, LM and Lin, JL and Fregoso Ocampo, R and Freeman, B and Lamothe, RC and Perez Rivas, A and Temoche-Diaz, MM and Chadha, S and Nordenfelt, N and Janson, OP and Barr, I and Devoto, AE and Cost, GJ and Butterfield, CN and Thomas, BC and Brown, CT},
title = {Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7602},
pmid = {36522342},
issn = {2041-1723},
mesh = {Animals ; Humans ; *Gene Editing ; *Escherichia coli/genetics/metabolism ; Endonucleases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Ribonucleases/genetics ; RNA ; CRISPR-Cas Systems/genetics ; Mammals/genetics ; },
abstract = {Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.},
}
@article {pmid36449008,
year = {2022},
author = {Zheng, X and Li, Y and Yuan, M and Shen, Y and Chen, S and Duan, G},
title = {Rapid detection of HPV16/18 based on a CRISPR-Cas13a/Cas12a dual-channel system.},
journal = {Analytical methods : advancing methods and applications},
volume = {14},
number = {48},
pages = {5065-5075},
doi = {10.1039/d2ay01536f},
pmid = {36449008},
issn = {1759-9679},
mesh = {Female ; Humans ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; *Uterine Cervical Neoplasms/diagnosis ; Early Detection of Cancer ; CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis ; },
abstract = {PURPOSE: cervical cancer is the leading cause of cancer deaths in women in the developing world, with high-risk HPV16 and HPV18 accounting for approximately 70% of all cervical cancers. Early detection of HPV, especially high-risk HPV types, is essential to prevent disease progression.<br><br>METHODS: in this study, we established a highly sensitive and specific nucleic acid assay based on a CRISPR-Cas13a/Cas12a dual-channel system combined with multiplex RAA for rapid detection and typing of HPV16/18, which provides a new idea for cervical cancer screening. To meet the application of field testing, we designed a portable fluorescence imaging assay that can judge the test results directly with the naked eye or through cell phone imaging.<br><br>RESULTS: the lower limit of detection for both HPV16 and HPV18 based on the CRISPR-Cas12a/Cas13a dual-channel assay was 10[0] copies per &#x3bc;L. The dual-channel assay was validated with 55 clinical samples, showing 97.06% sensitivity, 100% specificity, 100% positive predictive value, and 96.55% negative predictive value. The results of the portable fluorescence imaging assay were fully comparable to those of the real-time fluorescent RAA-based CRISPR-Cas12a/Cas13a dual-channel assay.<br><br>CONCLUSIONS: this developed portable dual gene assay platform may provide new technical support for cervical cancer screening in resource-limited settings.},
}
@article {pmid36332126,
year = {2022},
author = {Xing, D and Li, S and Shang, M and Wang, W and Zhang, Q and Wang, J and Hasin, T and Hettiarachchi, D and Alston, V and Bern, L and Parrales, AP and Lu, C and Coogan, M and Johnson, A and Qin, Z and Su, B and Dunham, R},
title = {A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences.},
journal = {ACS synthetic biology},
volume = {11},
number = {12},
pages = {4210-4219},
doi = {10.1021/acssynbio.2c00252},
pmid = {36332126},
issn = {2161-5063},
mesh = {Animals ; Fatty Acid Elongases/genetics ; *Flavin-Adenine Dinucleotide ; Transgenes/genetics ; Animals, Genetically Modified/genetics ; *Fatty Acid Desaturases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon (Oncorhynchus masou) elovl2 gene and rabbitfish (Siganus canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust target KI of elovl2 and Δ6 fad genes at multiple sites of LRS1 and LRS3, respectively, in the initial generation. This demonstrated that donor plasmid homology arms, which were nearly identical but not completely the same as the genome sequence, still led to on-target KI. Although the target KI efficiencies at LRS1, LRS2, and LRS3 sites were still relatively low in the current study, it is very promising that 100% KI efficiency in the future could be realized and perfected by selection of better LRSs and optimization of sgRNAs.},
}
@article {pmid36309322,
year = {2022},
author = {Shanaka, KASN and Jung, S and Madushani, KP and Wijerathna, HMSM and Neranjan Tharuka, MD and Kim, MJ and Lee, J},
title = {Generation of viperin-knockout zebrafish by CRISPR/Cas9-mediated genome engineering and the effect of this mutation under VHSV infection.},
journal = {Fish &amp; shellfish immunology},
volume = {131},
number = {},
pages = {672-681},
doi = {10.1016/j.fsi.2022.10.040},
pmid = {36309322},
issn = {1095-9947},
mesh = {Animals ; *Hemorrhagic Septicemia, Viral ; Zebrafish/genetics/metabolism ; CRISPR-Cas Systems ; *Fish Diseases ; *Novirhabdovirus/physiology ; Viral Proteins/genetics ; Mutation ; Interferons/genetics ; },
abstract = {Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin[-/-] compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin[-/-] fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin[-/-] fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.},
}
@article {pmid36256845,
year = {2022},
author = {Hu, L and Zhai, Y and Xu, L and Wang, J and Yang, S and Sun, Y and Yu, K and He, H and Fan, C},
title = {Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome.},
journal = {Journal of cellular physiology},
volume = {237},
number = {12},
pages = {4544-4550},
doi = {10.1002/jcp.30904},
pmid = {36256845},
issn = {1097-4652},
mesh = {*Gene Editing/methods ; *Brassica napus/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida ; Adenine ; },
abstract = {Rapeseed is an important source of oilseed crop in the world. Achieving genetic improvement has always been the major goal in rapeseed production. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Nowadays, Cas-base editing acts as an efficient tool to mediate single-base substitution at the target site. In this study, four adenine base editors (ABE) were modified to achieve adenosine base editing at different genome sites in allotetraploid Brassica napus. We designed 18 small guide RNAs to target phytoene desaturase (PDS), acetolactate synthase (ALS), CLAVATA3 (CLV3), CLV2, TRANSPARENT TESTA12 (TT12), carotenoid isomerase (CRTISO), designated de-etiolated-2 (DET2), BRANCHED1 (BRC1), zeaxanthin epoxidase (ZEP) genes, respectively. Among the four ABE systems, pBGE17 had the highest base-editing efficiency, with an average editing efficiency of 3.51%. Target sequencing results revealed that the editing window ranged from A5 to A8 of the protospacer-adjacent motif (PAM) sequence. Moreover, the ABEmax-nCas9NG system with NG PAM was developed, with a base-editing efficiency of 1.22%. These results revealed that ABE system developed in this study could efficiently induce A to G substitution and the ABE-nCas9NG system could broaden editing window in oilseed rape.},
}
@article {pmid36043456,
year = {2022},
author = {Asad, M and Liu, D and Chen, J and Yang, G},
title = {Applications of gene drive systems for population suppression of insect pests.},
journal = {Bulletin of entomological research},
volume = {112},
number = {6},
pages = {724-733},
doi = {10.1017/S0007485322000268},
pmid = {36043456},
issn = {1475-2670},
mesh = {Humans ; Animals ; *Gene Drive Technology ; CRISPR-Cas Systems ; Insecta/genetics ; Disease Vectors ; },
abstract = {Population suppression is an effective way for controlling insect pests and disease vectors, which cause significant damage to crop and spread contagious diseases to plants, animals and humans. Gene drive systems provide innovative opportunities for the insect pests population suppression by driving genes that impart fitness costs on populations of pests or disease vectors. Different gene-drive systems have been developed in insects and applied for their population suppression. Here, different categories of gene drives such as meiotic drive (MD), under-dominance (UD), homing endonuclease-based gene drive (HEGD) and especially the CRISPR/Cas9-based gene drive (CCGD) were reviewed, including the history, types, process and mechanisms. Furthermore, the advantages and limitations of applying different gene-drive systems to suppress the insect population were also summarized. This review provides a foundation for developing a specific gene-drive system for insect population suppression.},
}
@article {pmid36527830,
year = {2022},
author = {Aggarwal, N and Liang, Y and Foo, JL and Ling, H and Hwang, IY and Chang, MW},
title = {FELICX: A robust nucleic acid detection method using flap endonuclease and CRISPR-Cas12.},
journal = {Biosensors &amp; bioelectronics},
volume = {222},
number = {},
pages = {115002},
doi = {10.1016/j.bios.2022.115002},
pmid = {36527830},
issn = {1873-4235},
abstract = {Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/μL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics.},
}
@article {pmid36527789,
year = {2022},
author = {Metzger, JM and Wang, Y and Neuman, SS and Snow, KJ and Murray, SA and Lutz, CM and Bondarenko, V and Felton, J and Gimse, K and Xie, R and Li, D and Zhao, Y and Flowers, MT and Simmons, HA and Roy, S and Saha, K and Levine, JE and Emborg, ME and Gong, S},
title = {Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.},
journal = {Biomaterials},
volume = {293},
number = {},
pages = {121959},
doi = {10.1016/j.biomaterials.2022.121959},
pmid = {36527789},
issn = {1878-5905},
abstract = {Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.},
}
@article {pmid36517844,
year = {2022},
author = {Kim, MS and Jeong, DE and Choi, SK},
title = {Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {259},
pmid = {36517844},
issn = {1475-2859},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Bacillus/genetics ; Genes, Synthetic ; Plasmids/genetics ; },
abstract = {BACKGROUND: Owing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems.<br><br>RESULTS: Here, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains.<br><br>CONCLUSION: The Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.},
}
@article {pmid36515846,
year = {2023},
author = {Karpova, Y and Tulin, AV},
title = {Generating PARP Knockout D. melanogaster with CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2609},
number = {},
pages = {353-362},
pmid = {36515846},
issn = {1940-6029},
mesh = {Animals ; *Drosophila melanogaster/genetics/metabolism ; *Poly(ADP-ribose) Polymerases/metabolism ; Poly(ADP-ribose) Polymerase Inhibitors ; CRISPR-Cas Systems ; Nuclear Proteins/metabolism ; },
abstract = {Long-branched negatively charged poly(ADP-ribose) (pADPr) is a posttranslation modification of nuclear proteins that play a key role in many chromatin remodeling events. While several enzymes of PARP family could synthesize it across all multicellular organisms, Drosophila melanogaster is very suitable model to study pADPr-regulated processes because only one PARP gene is present. Despite the fact that PARP is an intensively studied protein with multiple important functions, no total knockout PARP flies were obtained in mobile element mutagenesis-based projects, mainly because PARP gene localizes in heterochromatic region. Here, we describe all steps of generating PARP mutated D. melanogaster with CRISPR/Cas9 system from the gRNA design, plasmid cloning to fly crosses and mutation detection. Provided gRNAs sequences target the region with high efficiency and results in more than 90% mutant stocks. This method could also be modified to generate PARP mutations in other gene locus, knockins with donor sequences for homology recombination or to be adjusted for other pADPr turnover-regulating enzymes.},
}
@article {pmid36418198,
year = {2022},
author = {Morera, C and Kim, J and Paredes-Redondo, A and Nobles, M and Rybin, D and Moccia, R and Kowala, A and Meng, J and Garren, S and Liu, P and Morgan, JE and Muntoni, F and Christoforou, N and Owens, J and Tinker, A and Lin, YY},
title = {CRISPR-mediated correction of skeletal muscle Ca[2+] handling in a novel DMD patient-derived pluripotent stem cell model.},
journal = {Neuromuscular disorders : NMD},
volume = {32},
number = {11-12},
pages = {908-922},
doi = {10.1016/j.nmd.2022.10.007},
pmid = {36418198},
issn = {1873-2364},
mesh = {Humans ; Dystrophin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; Muscle, Skeletal/pathology ; Muscle Fibers, Skeletal/pathology ; *Pluripotent Stem Cells/metabolism/pathology ; },
abstract = {Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca[2+] handling is a pathological feature of DMD, mechanisms underlying defective Ca[2+] homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca[2+] handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca[2+] transients and mathematical modeling of Ca[2+] dynamics reveal significantly reduced cytosolic Ca[2+] clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca[2+] flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca[2+] homeostasis in DMD and suggests that Ca[2+] signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.},
}
@article {pmid36271848,
year = {2022},
author = {Anthon, C and Corsi, GI and Gorodkin, J},
title = {CRISPRon/off: CRISPR/Cas9 on- and off-target gRNA design.},
journal = {Bioinformatics (Oxford, England)},
volume = {38},
number = {24},
pages = {5437-5439},
pmid = {36271848},
issn = {1367-4811},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; RNA, Guide, Kinetoplastida/genetics ; Gene Editing ; },
abstract = {SUMMARY: The effectiveness of CRISPR/Cas9-mediated genome editing experiments largely depends on the guide RNA (gRNA) used by the CRISPR/Cas9 system for target recognition and cleavage activation. Careful design is necessary to select a gRNA with high editing efficiency at the on-target site and with minimum off-target potential. Here, we present our webserver for gRNA design with a user-friendly graphical interface, which provides interoperability between our on- and off-target prediction tools, CRISPRon and CRISPRoff, for a complete and streamlined gRNA selection.<br><br>The graphical interface uses the Integrative Genomic Viewer (IGV) JavaScript plugin. The backend tools are implemented in Python and C. The CRISPRon and CRISPRoff webservers and command-line tools are freely available at https://rth.dk/resources/crispr.},
}
@article {pmid35773341,
year = {2022},
author = {Nahmad, AD and Reuveni, E and Goldschmidt, E and Tenne, T and Liberman, M and Horovitz-Fried, M and Khosravi, R and Kobo, H and Reinstein, E and Madi, A and Ben-David, U and Barzel, A},
title = {Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage.},
journal = {Nature biotechnology},
volume = {40},
number = {12},
pages = {1807-1813},
pmid = {35773341},
issn = {1546-1696},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; In Situ Hybridization, Fluorescence ; *T-Lymphocytes ; Gene Editing/methods ; Receptors, Antigen, T-Cell/genetics ; Aneuploidy ; },
abstract = {Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes[1-6]. In this study, using single-cell RNA sequencing, we investigated genome editing outcomes in primary human T cells transfected with CRISPR-Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1. Four days after transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells and a chromosome 14 gain in up to 1.4% of the cells. Chromosome 7, harboring the TCRβ locus, was truncated in 9.9% of the cells. Aberrations were validated using fluorescence in situ hybridization and digital droplet PCR. Aneuploidy was associated with reduced proliferation, induced p53 activation and cell death. However, at 11 days after transfection, 0.9% of T cells still had a chromosome 14 loss. Aneuploidy and chromosomal truncations are, thus, frequent outcomes of CRISPR-Cas9 cleavage that should be monitored and minimized in clinical protocols.},
}
@article {pmid35534612,
year = {2022},
author = {Wang, DN and Wang, ZQ and Jin, M and Lin, MT and Wang, N},
title = {CRISPR/Cas9-based genome editing for the modification of multiple duplications that cause Duchenne muscular dystrophy.},
journal = {Gene therapy},
volume = {29},
number = {12},
pages = {730-737},
pmid = {35534612},
issn = {1476-5462},
mesh = {Humans ; *Dystrophin/genetics/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; DNA Copy Number Variations ; },
abstract = {With the development of basic research, some genetic-based methods have been found to treat Duchenne muscular dystrophy (DMD) with large deletion mutations and nonsense mutations. Appropriate therapeutic approaches for repairing multiple duplications are limited. We used the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system with patient-derived primary myoblasts to correct multiple duplications of the dystrophin gene. Muscle tissues from a patient carrying duplications of dystrophin were obtained, and tissue-derived primary cells were cultured. Myoblasts were purified with an immunomagnetic sorting system using CD56 microbeads. After transduction by lentivirus with a designed single guide RNA (sgRNA) targeting a duplicated region, myoblasts were allowed to differentiate for 7 days. Copy number variations in the exons of the patient's myotubes were quantified by real-time PCR before and after genetic editing. Western blot analysis was performed to detect the full-length dystrophin protein before and after genetic editing. The ten sequences predicted to be the most likely off-targets were determined by Sanger sequencing. The patient carried duplications of exon 18-25, dystrophin protein expression was completely abrogated. Real-time PCR showed that the copy number of exon 25 in the patient's myotubes was 2.015 ± 0.079 compared with that of the healthy controls. After editing, the copy number of exon 25 in the patient's modified myotubes was 1.308 ± 0.083 compared with that of the healthy controls (P < 0.001). Western blot analysis revealed no expression of the dystrophin protein in the patient's myotubes before editing. After editing, the patient's myotubes expressed the full-length dystrophin protein at a level that was ~6.12% of that in the healthy control samples. Off-target analysis revealed no abnormal editing at the ten sites predicted to be the most likely off-target sites. The excision of multiple duplications by the CRISPR/Cas9 system restored the expression of full-length dystrophin. This study provides proof of evidence for future genome-editing therapy in patients with DMD caused by multiple duplication mutations.},
}
@article {pmid36522348,
year = {2022},
author = {Nemudraia, A and Nemudryi, A and Buyukyoruk, M and Scherffius, AM and Zahl, T and Wiegand, T and Pandey, S and Nichols, JE and Hall, LN and McVey, A and Lee, HH and Wilkinson, RA and Snyder, LR and Jones, JD and Koutmou, KS and Santiago-Frangos, A and Wiedenheft, B},
title = {Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7762},
pmid = {36522348},
issn = {2041-1723},
abstract = {Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.},
}
@article {pmid36521436,
year = {2022},
author = {Nemudryi, A and Nemudraia, A and Wiegand, T and Sternberg, SH and Wiedenheft, B},
title = {A viral "codebreaker" intercepts a host alarm.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {12},
pages = {1647-1648},
doi = {10.1016/j.chom.2022.11.005},
pmid = {36521436},
issn = {1934-6069},
abstract = {Immune systems generate diverse alarm signals in response to invading pathogens. In a recent Nature paper, Leavitt et al. identified a family of phage-encoded "codebreakers" that intercept nucleotide-derived immune signals and render the cell defenseless to viral infection.},
}
@article {pmid36513736,
year = {2022},
author = {Ozawa, M and Taguchi, J and Katsuma, K and Ishikawa-Yamauchi, Y and Kikuchi, M and Sakamoto, R and Yamada, Y and Ikawa, M},
title = {Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {21558},
pmid = {36513736},
issn = {2045-2322},
support = {INV-001902/GATES/Bill &amp; Melinda Gates Foundation/United States ; },
mesh = {Mice ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Gene Editing/methods ; Plasmids/genetics ; Embryonic Stem Cells/metabolism ; DNA/metabolism ; Gene Knock-In Techniques ; Mammals/metabolism ; },
abstract = {Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.},
}
@article {pmid36513282,
year = {2022},
author = {Wang, C and Zhang, M and Yan, J and Wang, R and Wang, Z and Sun, X and Dong, S},
title = {Chemokine-like receptor 1 deficiency impedes macrophage phenotypic transformation and cardiac repair after myocardial infarction.},
journal = {International journal of cardiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ijcard.2022.12.015},
pmid = {36513282},
issn = {1874-1754},
abstract = {BACKGROUND: Timely and appropriate transformation of macrophage phenotypes from proinflammatory to anti-inflammatory is essential for cardiac repair after myocardial infarction (MI). Chemokine-like receptor 1 (CMKLR1), which is expressed on macrophages, is regulated by proinflammatory and anti-inflammatory stimuli. However, the contribution of CMKLR1 to macrophage phenotypic transformation and the role it plays in modulating cardiac repair after MI remain unclear.<br><br>METHODS: CMKLR1 knockout (CMKLR1[-/-]) mice were generated by CRISPR/Cas-mediated genome engineering. A model of murine MI was induced by permanent ligation along the left anterior descending artery. Cardiac function was evaluated by echocardiography. Infarct size and collagen deposition were detected by Masson's trichrome staining. Cardiac macrophages were obtained by fluorescence-activated cell sorting. The protein and mRNA expression of associated molecules was determined by Western blotting and qRT-PCR.<br><br>RESULTS: We demonstrated that macrophages highly expressed CMKLR1 and accumulated in murine infarcted hearts during the anti-inflammatory reparative phase of MI. CMKLR1 deficiency impaired cardiac function, increased infarct size, induced maladaptive cardiac remodeling, and decreased long-term survival after MI. Furthermore, CMKLR1 deficiency impeded macrophage phenotypic transformation from M1 to M2 in vivo and in vitro. In addition, we demonstrated that CMKLR1 signaling through the PI3K/Akt/mTOR pathway stimulated C/EBP&#x3b2; activation while simultaneously limiting NF-&#x3ba;B activation, thereby promoting anti-inflammatory and prohibiting proinflammatory macrophage polarization.<br><br>CONCLUSIONS: Our results reveal that CMKLR1 deficiency impedes macrophage phenotypic transformation and cardiac repair after MI involving the PI3K/AKT/mTOR pathway. CMKLR1 may thus represent a potential therapeutic target for MI.},
}
@article {pmid36512351,
year = {2022},
author = {Wang, D and Fan, X and Li, M and Liu, T and Lu, P and Wang, G and Li, Y and Han, J and Zhao, J},
title = {Prime Editing in Mammals: The Next Generation of Precision Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {746-768},
doi = {10.1089/crispr.2022.0084},
pmid = {36512351},
issn = {2573-1602},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, Kinetoplastida/genetics ; Genome/genetics ; Mammals/genetics ; },
abstract = {The recently established prime editor (PE) system is regarded as next-generation gene-editing technology. This methodology can install any base-to-base change as well as insertions and deletions without the requirement for double-stranded break formation or donor DNA templates; thus, it offers more targeting flexibility and greater editing precision than conventional CRISPR-Cas systems or base editors. In this study, we introduce the basic principles of PE and then review its most recent progress in terms of editing versatility, specificity, and efficiency in mammals. Next, we summarize key considerations regarding the selection of PE variants, prime editing guide RNA (pegRNA) design rules, and the efficiency and accuracy evaluation of PE. Finally, we highlight and discuss how PE can assist in a wide range of biological studies and how it can be applied to make precise genomic corrections in animal models, which paves the way for curing human diseases.},
}
@article {pmid36512350,
year = {2022},
author = {Davies, K},
title = {Breaking the Rules.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {742-745},
doi = {10.1089/crispr.2022.29157.kd},
pmid = {36512350},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid36512349,
year = {2022},
author = {Barrangou, R},
title = {Better Guidance for CRISPR.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {741},
doi = {10.1089/crispr.2022.29156.editorial},
pmid = {36512349},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid36511936,
year = {2022},
author = {Larkin, H},
title = {Researchers Use Plasmid Gene Editing to Personalize Solid Tumor T-Cell Therapy.},
journal = {JAMA},
volume = {328},
number = {22},
pages = {2201-2202},
doi = {10.1001/jama.2022.20034},
pmid = {36511936},
issn = {1538-3598},
mesh = {Humans ; *Gene Editing ; Plasmids/genetics ; CRISPR-Cas Systems ; *Neoplasms/genetics/therapy ; Cell- and Tissue-Based Therapy ; },
}
@article {pmid36509783,
year = {2022},
author = {Xi, Z and Vats, A and Sahel, JA and Chen, Y and Byrne, LC},
title = {Gene augmentation prevents retinal degeneration in a CRISPR/Cas9-based mouse model of PRPF31 retinitis pigmentosa.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7695},
pmid = {36509783},
issn = {2041-1723},
mesh = {Mice ; Animals ; Humans ; Eye Proteins/metabolism ; *Retinal Degeneration/genetics ; CRISPR-Cas Systems/genetics ; *Retinitis Pigmentosa/genetics/therapy ; Genes, Dominant ; Mutation ; Disease Models, Animal ; },
abstract = {Mutations in PRPF31 cause autosomal dominant retinitis pigmentosa, an untreatable form of blindness. Gene therapy is a promising treatment for PRPF31-retinitis pigmentosa, however, there are currently no suitable animal models in which to develop AAV-mediated gene augmentation. Here we establish Prpf31 mutant mouse models using AAV-mediated CRISPR/Cas9 knockout, and characterize the resulting retinal degeneration phenotype. Mouse models with early-onset morphological and functional impairments like those in patients were established, providing new platforms in which to investigate pathogenetic mechanisms and develop therapeutic methods. AAV-mediated PRPF31 gene augmentation restored the retinal structure and function in a rapidly degenerating mouse model, demonstrating the first in vivo proof-of-concept for AAV-mediated gene therapy to treat PRPF31-retinitis pigmentosa. AAV-CRISPR/Cas9-PRPF31 knockout constructs also mediated efficient PRPF31 knockout in human and non-human primate retinal explants, laying a foundation for establishing non-human primate models using the method developed here.},
}
@article {pmid36509752,
year = {2022},
author = {Yu, Z and Lu, Z and Li, J and Wang, Y and Wu, P and Li, Y and Zhou, Y and Li, B and Zhang, H and Liu, Y and Ma, L},
title = {PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7545},
pmid = {36509752},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Endonucleases/genetics ; High-Throughput Nucleotide Sequencing ; Mammals/genetics ; },
abstract = {CRISPR technology holds significant promise for biological studies and gene therapies because of its high flexibility and efficiency when applied in mammalian cells. But endonuclease (e.g., Cas9) potentially generates undesired edits; thus, there is an urgent need to comprehensively identify off-target sites so that the genotoxicities can be accurately assessed. To date, it is still challenging to streamline the entire process to specifically label and efficiently enrich the cleavage sites from unknown genomic locations. Here we develop PEAC-seq, in which we adopt the Prime Editor to insert a sequence-optimized tag to the editing sites and enrich the tagged regions with site-specific primers for high throughput sequencing. Moreover, we demonstrate that PEAC-seq could identify DNA translocations, which are more genotoxic but usually overlooked by other off-target detection methods. As PEAC-seq does not rely on exogenous oligodeoxynucleotides to label the editing site, we also conduct in vivo off-target identification as proof of concept. In summary, PEAC-seq provides a comprehensive and streamlined strategy to identify CRISPR off-targeting sites in vitro and in vivo, as well as DNA translocation events. This technique further diversified the toolkit to evaluate the genotoxicity of CRISPR applications in research and clinics.},
}
@article {pmid36412588,
year = {2022},
author = {Zhao, Q and Pan, B and Long, W and Pan, Y and Zhou, D and Luan, X and He, B and Wang, Y and Song, Y},
title = {Metal Organic Framework-Based Bio-Barcode CRISPR/Cas12a Assay for Ultrasensitive Detection of MicroRNAs.},
journal = {Nano letters},
volume = {22},
number = {23},
pages = {9714-9722},
doi = {10.1021/acs.nanolett.2c04022},
pmid = {36412588},
issn = {1530-6992},
mesh = {Humans ; Female ; *MicroRNAs/genetics ; *Metal-Organic Frameworks ; CRISPR-Cas Systems/genetics ; *Breast Neoplasms/diagnosis/genetics ; Cell Differentiation ; *Biosensing Techniques ; },
abstract = {CRISPR/Cas12a has shown great potential in molecular diagnostics, but its application in sensing of microRNAs (miRNAs) was limited by sensitivity and complexity. Here, we have sensitively and conveniently detected microRNAs by reasonably integrating metal-organic frameworks (MOFs) based biobarcodes with CRISPR/Cas12a assay (designated as MBCA). In this work, DNA-functionalized Zr-MOFs were designed as the converter to convert and amplify each miRNA target into activators that can initiate the trans-cleavage activity of CRISPR/Cas12a to further amplify the signal. Such integration provides a universal strategy for sensitive detection of miRNAs. By tuning the complementary sequences modified on nanoprobes, this assay achieves subattomolar sensitivity for different miRNAs and was selective to single-based mismatches. With the proposed method, the expression of miR-21 in different cancer cells can be assessed, and breast cancer patients and healthy individuals can be differentiated by analyzing the target miRNAs extracted from serum samples, holding great potential in clinical diagnosis.},
}
@article {pmid36378258,
year = {2022},
author = {Morianou, I and Crisanti, A and Nolan, T and Hammond, AM},
title = {CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {868-876},
doi = {10.1089/crispr.2022.0026},
pmid = {36378258},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Recombinational DNA Repair ; Genome ; },
abstract = {The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5-41 × higher rates of editing than homology-directed repair or prime editing.},
}
@article {pmid36378256,
year = {2022},
author = {Hoy, A and Zheng, YY and Sheng, J and Royzen, M},
title = {Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {787-798},
doi = {10.1089/crispr.2022.0065},
pmid = {36378256},
issn = {2573-1602},
support = {R21 HG012257/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *Thiouridine ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; RNA, Guide, Kinetoplastida/genetics ; Oligonucleotides ; },
abstract = {The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N[1]-methyladenosine (m[1]A), N[6]-methyladenosine (m[6]A), 2-thiouridine (s[2]U), and 4-thiouridine (s[4]U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.},
}
@article {pmid36374965,
year = {2022},
author = {Zhu, GH and Gaddelapati, SC and Jiao, Y and Koo, J and Palli, SR},
title = {CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {813-824},
doi = {10.1089/crispr.2022.0066},
pmid = {36374965},
issn = {2573-1602},
mesh = {Animals ; Methoprene/pharmacology/metabolism ; *Aedes/genetics/metabolism ; *Yellow Fever/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; Metamorphosis, Biological/physiology ; Insect Proteins/genetics/metabolism ; Juvenile Hormones/genetics/pharmacology/metabolism ; Pupa/genetics/metabolism ; Larva/genetics/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 (Kr-h1), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1, which suppresses the expression of E93, resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.},
}
@article {pmid36374245,
year = {2022},
author = {Liu, J and Li, B and Yang, L and Ren, N and Xu, M and Huang, Q},
title = {Increasing Genome Editing Efficiency of Cas9 Nucleases by the Simultaneous Use of Transcriptional Activators and Histone Acetyltransferase Activator.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {854-867},
doi = {10.1089/crispr.2022.0001},
pmid = {36374245},
issn = {2573-1602},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Histones/genetics ; Histone Acetyltransferases/genetics ; Endonucleases/genetics ; Chromatin ; Transcription Factors/genetics ; Mammals/genetics ; },
abstract = {The CRISPR-Cas9 system shows diverse levels of genome editing activities on eukaryotic chromatin, and high-efficiency sgRNA targets are usually desired in application. In this study, we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing. However, the strategy that increases chromatin openness by fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR-Cas9 targets in Lenti-X 293T cells. Under the enlightenment that histone acetylation increases eukaryotic chromatin accessibility, we developed a composite strategy to further improve genome editing by activating histone acetylation. We demonstrate that promoting histone acetylation using the histone acetyltransferase activator YF-2 can improve the genome editing by Cas9 and, more robustly, by the Cas9 transcriptional activator (Cas9-AD). This strategy holds great potential to enhance CRISPR-Cas9 genome editing and to enable broader CRISPR gRNA target choices for experiments in eukaryotes.},
}
@article {pmid36374019,
year = {2022},
author = {Pareek, M and Hegedüs, B and Hou, Z and Csernetics, &#xc1; and Wu, H and Virágh, M and Sahu, N and Liu, XB and Nagy, L},
title = {Preassembled Cas9 Ribonucleoprotein-Mediated Gene Deletion Identifies the Carbon Catabolite Repressor and Its Target Genes in Coprinopsis cinerea.},
journal = {Applied and environmental microbiology},
volume = {88},
number = {23},
pages = {e0094022},
pmid = {36374019},
issn = {1098-5336},
mesh = {Lignin/metabolism ; Gene Deletion ; Carbon/metabolism ; Fungal Proteins/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; CRISPR-Cas Systems ; *Catabolite Repression ; *Agaricales/metabolism ; *Basidiomycota/metabolism ; *Ascomycota/metabolism ; Transcription Factors/genetics/metabolism ; Membrane Transport Proteins/genetics ; Gene Expression Regulation, Fungal ; },
abstract = {Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. The cre1 gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota species remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9 RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor-encoding genes, among others. Our results support the notion that, like reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs, and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented here will expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. IMPORTANCE Mushroom-forming fungi include some of the most efficient lignocellulosic plant biomass degraders. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose-, and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulations of these metabolic enzymes is known as carbon catabolite repression, which is orchestrated by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g., glucose) are not available. Here, we identified the Cre1 ortholog in a litter decomposer Agaricomycete, Coprinopsis cinerea, knocked it out, and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes of C. cinerea cre1. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.},
}
@article {pmid36350691,
year = {2022},
author = {Prokhorova, DV and Vokhtantsev, IP and Tolstova, PO and Zhuravlev, ES and Kulishova, LM and Zharkov, DO and Stepanov, GA},
title = {Natural Nucleoside Modifications in Guide RNAs Can Modulate the Activity of the CRISPR-Cas9 System In Vitro.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {799-812},
doi = {10.1089/crispr.2022.0069},
pmid = {36350691},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Nucleosides ; RNA, Guide, Kinetoplastida/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {At the present time, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has been widely adopted as an efficient genomic editing tool. However, there are some actual problems such as the off-target effects, cytotoxicity, and immunogenicity. The incorporation of modifications into guide RNAs permits enhancing both the efficiency and the specificity of the CRISPR-Cas9 system. In this study, we demonstrate that the inclusion of N[6]-methyladenosine, 5-methylcytidine, and pseudouridine in trans-activating RNA (tracrRNA) or in single guide RNA (sgRNA) enables efficient gene editing in vitro. We found that the complexes of modified guide RNAs with Cas9 protein promoted cleavage of the target short/long duplexes and plasmid substrates. In addition, the modified monomers in guide RNAs allow increasing the specificity of CRISPR-Cas9 system in vitro and promote diminishing both the immunostimulating and the cytotoxic effects of sgRNAs.},
}
@article {pmid36315201,
year = {2022},
author = {Liang, X and Lan, J and Xu, M and Qin, K and Liu, H and Sun, G and Liu, X and Chen, Y and He, Z},
title = {Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {825-842},
doi = {10.1089/crispr.2022.0039},
pmid = {36315201},
issn = {2573-1602},
mesh = {Swine/genetics ; Mice ; Animals ; *Hair Color/genetics ; *Proto-Oncogene Proteins c-kit/genetics ; Monophenol Monooxygenase/genetics ; Phosphatidylinositol 3-Kinases/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; Pigmentation/genetics ; Meat ; },
abstract = {The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice mutations is expected to affect Yorkshire pig pigmentation. In this study, we describe the use of a Yorkshire pig kidney cell strain with the I[?]/I[Be-ed] genotype, previously created by CRISPR-Cas9, as donor cells for somatic cell nuclear transfer to generate gene-edited Yorkshire pigs. The removal of the 450 kb duplications harboring the KIT copy with splice mutation did not alter the white coat color of Yorkshire pigs, which was confirmed by the absence of fully mature melanocytes and melanin accumulation in the hair follicles. Except for the improved transcription of tyrosinase, and slight increase in microphthalmia transcription factor and tyrosinase-related protein 1 protein expression, there was no significant impact of the removal of splice mutations on genes and signaling pathways (PI3K/AKT) involved in melanogenesis. However, the removal of the 450 kb duplications harboring the KIT copy with splice mutation substantially improved fresh meat color accompanied by significantly increased red blood cell number, which merits further investigation. Our study provides new insights into the role of structural mutations of the KIT gene in the formation of white coat color and erythropoiesis in Yorkshire pigs.},
}
@article {pmid36263914,
year = {2022},
author = {Paulo, DF and Cha, AY and Kauwe, AN and Curbelo, K and Corpuz, RL and Simmonds, TJ and Sim, SB and Geib, SM},
title = {A Unified Protocol for CRISPR/Cas9-Mediated Gene Knockout in Tephritid Fruit Flies Led to the Recreation of White Eye and White Puparium Phenotypes in the Melon Fly.},
journal = {Journal of economic entomology},
volume = {115},
number = {6},
pages = {2110-2115},
doi = {10.1093/jee/toac166},
pmid = {36263914},
issn = {1938-291X},
mesh = {Animals ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Cucurbitaceae ; Reproducibility of Results ; *Tephritidae/genetics ; Drosophila/genetics ; Phenotype ; Recreation ; },
abstract = {Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare diverse strategies for delivering CRISPR/Cas9 components into tephritid embryos, optimize procedures, and advance the technology to systems outside the most thoroughly studied species within the family. Here, we revisit five years of CRISPR research in Tephritidae and propose a unified protocol for candidate gene knockout in fruit flies using CRISPR/Cas9. We demonstrated the efficiency of our protocol by disrupting the eye pigmentation gene white eye (we) in the melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae). High rates of somatic and germline mutagenesis were induced by microinjecting pre-assembled Cas9-sgRNA complexes through the chorion of embryos at early embryogenesis, leading to the rapid development of new mutant lines. We achieved comparable results when targeting the we orthologue in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), illustrating the reliability of our methods when transferred to other related species. Finally, we functionally validated the recently discovered white pupae (wp) loci in the melon fly, successfully recreating the white puparium phenotype used in suppression programs of this and other major economically important tephritids. This is the first demonstration of CRISPR-based genome-editing in the genus Zeugodacus, and we anticipate that the procedures described here will contribute to advancing genome-editing in other non-model tephritid fruit flies.},
}
@article {pmid36257604,
year = {2022},
author = {Mills, C and Riching, A and Keller, A and Stombaugh, J and Haupt, A and Maksimova, E and Dickerson, SM and Anderson, E and Hemphill, K and Ebmeier, C and Schiel, JA and Levenga, J and Perkett, M and Smith, AVB and Strezoska, Z},
title = {A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs.},
journal = {The CRISPR journal},
volume = {5},
number = {6},
pages = {769-786},
doi = {10.1089/crispr.2022.0056},
pmid = {36257604},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Induced Pluripotent Stem Cells ; RNA, Guide, Kinetoplastida/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with in vitro-transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.},
}
@article {pmid36505425,
year = {2022},
author = {Dang, W and Li, T and Xu, F and Wang, Y and Yang, F and Zheng, H},
title = {Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {1016545},
pmid = {36505425},
issn = {1664-3224},
mesh = {Humans ; Swine ; Animals ; Mice ; *Interferon Type I/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; Gene Library ; Vesiculovirus ; Antiviral Restriction Factors ; Membrane Proteins ; RNA-Binding Proteins ; },
abstract = {Diseases caused by emerging swine viruses had a great economic impact, constituting a new challenge for researchers and practicing veterinarians. Innate immune control of viral pathogen invasion is mediated by interferons (IFNs), resulting in transcriptional elevation of hundreds of IFN-stimulated genes (ISGs). However, the ISG family is vast and species-specific, and despite remarkable advancements in uncovering the breadth of IFN-induced gene expression in mouse and human, it is less characterized with respect to the repertoire of porcine ISGs and their functional annotation. Herein, with the application of RNA-sequencing (RNA-Seq) gene profiling, the breadth of IFN-induced gene expression in the context of type I IFN stimulation was explored by using IBRS-2 cell, a commonly used high-efficient cultivation system for porcine picornaviruses. By establishing inclusion criteria, a total of 359 ISGs were selected. Aiming to identify key effectors mediating type I IFN inhibition of swine viruses, a CRISPR/Cas9 knockout library of 1908 sgRNAs targeting 5' constitutive exons of 359 ISGs with an average of 5 to 6 sgRNAs per gene was constructed. Using VSV-eGFP (vesicular stomatitis virus, fused with GFP) as a model virus, a subset of highest-ranking candidates were identified, including previously validated anti-VSV genes IRF9, IFITM3, LOC100519082 and REC8, as well as several novel hits. This approach attains a high level of feasibility and reliability, and a high rate of hit identification, providing a forward-looking platform to systematically profile the effectors of type I IFN antiviral response against porcine viruses.},
}
@article {pmid36503743,
year = {2023},
author = {Chen, H and Ye, R and Liang, Y and Zhang, S and Liu, X and Sun, C and Li, F and Yi, J},
title = {Generation of low-cadmium rice germplasms via knockout of OsLCD using CRISPR/Cas9.},
journal = {Journal of environmental sciences (China)},
volume = {126},
number = {},
pages = {138-152},
doi = {10.1016/j.jes.2022.05.047},
pmid = {36503743},
issn = {1001-0742},
mesh = {*Cadmium/toxicity ; *Oryza/genetics ; CRISPR-Cas Systems ; Seedlings ; Hydroponics ; },
abstract = {The OsLCD gene, which has been implicated in cadmium (Cd) accumulation in rice, might be a useful target for CRISPR/Cas9 editing. However, the effects of OsLCD gene editing on Cd accumulation, plant growth, and yield traits remain unknown. Here, we used CRISPR/Cas9 to generate oslcd single mutants from indica and japonica rice cultivars. We also generated osnramp5 single mutants and oslcd osnramp5 double mutants in the indica background. When grown in Cd-contaminated paddy soils, all oslcd single mutants accumulated less Cd than the wild types (WTs). Consistent with this, oslcd single mutants grown in Cd-contaminated hydroponic culture accumulated significantly less Cd in the shoots as compared to WTs. This decrease in accumulation probably resulted from the reduction of Cd translocation under Cd stress. Oxidative damage also decreased, and plant growth increased in all oslcd single mutant seedlings as compared to WTs in the presence of Cd. Plant growth and most yield traits, as well essential element concentrations in rice seedling shoots, brown rice, and rice straw, were similar between oslcd single mutants and WTs. In the presence of Cd, Cd concentrations in the brown rice and shoots of oslcd osnramp5 double mutants were significantly decreased compared with WTs as well as osnramp single mutants. Our results suggested that OsLCD knockout may reduce Cd accumulation alone or in combination with other knockout mutations in a variety of rice genotypes; unlike OsNramp5 mutations, OsLCD knockout did not reduce essential element contents. Therefore, OsLCD knockout might be used to generate low-Cd rice germplasms.},
}
@article {pmid36499605,
year = {2022},
author = {Li, G and Ma, Y and Wang, X and Cheng, N and Meng, D and Chen, S and Wang, W and Wang, X and Hu, X and Yan, L and Wang, S},
title = {CRISPR/Cas9 Gene Editing of NtAITRs, a Family of Transcription Repressor Genes, Leads to Enhanced Drought Tolerance in Tobacco.},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
pmid = {36499605},
issn = {1422-0067},
mesh = {*Tobacco/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Drought Resistance ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Abscisic Acid/pharmacology/metabolism ; Droughts ; Stress, Physiological/genetics ; Transcription Factors/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Tobacco is a cash crop throughout the world, and its growth and development are affected by abiotic stresses including drought stress; therefore, drought-tolerant breeding may help to improve tobacco yield and quality under drought stress conditions. Considering that the plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via activating ABA response genes, the characterization of ABA response genes may enable the identification of genes that can be used for molecular breeding to improve drought tolerance in tobacco. We report here the identification of NtAITRs (Nicotiana tabacum ABA-induced transcription repressors) as a family of novel regulators of drought tolerance in tobacco. Bioinformatics analysis shows that there are a total of eight NtAITR genes in tobacco, and all the NtAITRs have a partially conserved LxLxL motif at their C-terminus. RT-PCR results show that the expression levels of at least some NtAITRs were increased in response to ABA and drought treatments, and NtAITRs, when recruited to the Gal4 promoter via a fused GD (Gal4 DNA-binding domain), were able to repress transcription activator LD-VP activated expression of the LexA-Gal4-GUS reporter gene. Roles of NtAITRs in regulating drought tolerance in tobacco were analyzed by generating CRISPR/Cas9 gene-edited mutants. A total of three Cas9-free ntaitr12356 quintuple mutants were obtained, and drought treatment assays show that drought tolerance was increased in the ntaitr12356 quintuple mutants. On the other hand, results of seed germination and seedling greening assays show that ABA sensitivity was increased in the ntaitr12356 quintuple mutants, and the expression levels of some ABA signaling key regulator genes were altered in the ntaitr12356-c3 mutant. Taken together, our results suggest that NtAITRs are ABA-responsive genes, and that NtAITRs function as transcription repressors and negatively regulate drought tolerance in tobacco, possibly by affecting plant ABA response via affecting the expression of ABA signaling key regulator genes.},
}
@article {pmid36499376,
year = {2022},
author = {Chupradit, K and Sornsuwan, K and Saiprayong, K and Wattanapanitch, M and Tayapiwatana, C},
title = {Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication.},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
pmid = {36499376},
issn = {1422-0067},
mesh = {Humans ; Jurkat Cells ; *CRISPR-Cas Systems ; HEK293 Cells ; *HIV-1/genetics ; Gene Editing ; Virus Replication/genetics ; },
abstract = {Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.},
}
@article {pmid36499334,
year = {2022},
author = {Shin, YH and Lee, HM and Park, YD},
title = {CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis).},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
pmid = {36499334},
issn = {1422-0067},
mesh = {Humans ; *Brassica rapa/metabolism ; CRISPR-Cas Systems/genetics ; East Asian People ; *Brassica/genetics ; Phenotype ; Gene Editing ; },
abstract = {Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting-the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E0&amp;nbsp;AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line 'CT001.' Generational progression using bud pollination obtained T-DNA-free E1&amp;nbsp;AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis).},
}
@article {pmid36499273,
year = {2022},
author = {Guo, J and Zeng, L and Chen, H and Ma, C and Tu, J and Shen, J and Wen, J and Fu, T and Yi, B},
title = {CRISPR/Cas9-Mediated Targeted Mutagenesis of BnaCOL9 Advances the Flowering Time of Brassica napus L.},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
pmid = {36499273},
issn = {1422-0067},
mesh = {*Brassica napus/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Mutagenesis ; Flowers/genetics ; *Brassica rapa ; Gene Expression Regulation, Plant ; },
abstract = {Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. The planting area and output of rapeseed are affected by the flowering time, which is a critical agronomic feature. COL9 controls growth and development in many different plant species as a member of the zinc finger transcription factor family. However, BnaCOL9 in rapeseed has not been documented. The aim of this study was to apply CRISPR/Cas9 technology to create an early-flowering germplasm resource to provide useful material for improving the early-maturing breeding of rapeseed. We identified four COL9 homologs in rapeseed that were distributed on chromosomes A05, C05, A03, and C03. We successfully created quadruple BnaCOL9 mutations in rapeseed using the CRISPR/Cas9 platform. The quadruple mutants of BnaCOL9 flowered earlier than the wild-type. On the other hand, the flowering time of the BnaCOL9 overexpression lines was delayed. An analysis of the expression patterns revealed that these genes were substantially expressed in the leaves and flowers. A subcellular localization experiment demonstrated that BnaCOL9 was in the nucleus. Furthermore, we discovered that two key flowering-related genes, BnaCO and BnaFT, were highly elevated in the BnaCOL9 mutants, but dramatically downregulated in the BnaCOL9 overexpression lines. Our findings demonstrate that BnaCOL9 is a significant flowering inhibitor in rapeseed and may be employed as a crucial gene for early-maturing breeding.},
}
@article {pmid36498869,
year = {2022},
author = {Li, R and Maioli, A and Yan, Z and Bai, Y and Valentino, D and Milani, AM and Pompili, V and Comino, C and Lanteri, S and Moglia, A and Acquadro, A},
title = {CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars.},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
pmid = {36498869},
issn = {1422-0067},
mesh = {*Solanum lycopersicum/genetics ; CRISPR-Cas Systems/genetics ; Plant Diseases/genetics ; *Phytophthora infestans/genetics ; *Solanum tuberosum/genetics ; *Arabidopsis/genetics ; Glucosyltransferases/genetics ; *Arabidopsis Proteins/genetics ; },
abstract = {Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR-Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: 'San Marzano' (SM) and 'Oxheart' (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a -7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (-7 bp and -2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.},
}
@article {pmid36497134,
year = {2022},
author = {Zhao, L and Li, M and Yin, Z and Lv, L and Zhou, M and Wang, Y and Zhang, M and Guo, T and Guo, X and Liu, H and Cheng, L and Liang, X and Duo, S and Li, R},
title = {Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2.},
journal = {Cells},
volume = {11},
number = {23},
pages = {},
pmid = {36497134},
issn = {2073-4409},
mesh = {Pregnancy ; Female ; Mice ; Animals ; Humans ; Thyroid Nuclear Factor 1/genetics ; *CRISPR-Cas Systems/genetics ; *Tracheoesophageal Fistula ; Lung ; Disease Models, Animal ; Mice, Knockout ; },
abstract = {A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.},
}
@article {pmid36490276,
year = {2022},
author = {Hou, Y and Cheng, X and Witman, GB},
title = {Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis.},
journal = {PloS one},
volume = {17},
number = {12},
pages = {e0278972},
pmid = {36490276},
issn = {1932-6203},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Mutagenesis, Insertional ; Flagella/genetics ; Genome ; },
abstract = {Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin (HA) tag with an efficiency of 7.4%. Sequencing confirmed that these strains are correctly edited. Western blotting confirmed the expression of HA-tagged LF5, and immunofluorescence microscopy showed that LF5-HA is localized normally. These strains have normal length flagella and appear wild type. We successfully tagged the N-terminus of Cre09.g416350 with mNeonGreen-3xFLAG with an efficiency of 9%. Sequencing showed that the tag region in these strains is as expected. Western blotting confirmed the expression of tagged protein of the expected size in these strains, which appeared to have normal cell size, growth rate, and swimming speed. This is the first time that C. reinhardtii endogenous genes have been edited in situ to express a wild-type tagged protein. This effective, efficient, and convenient TIM-tagging strategy promises to be a useful tool for the study of nuclear genes, including essential genes, in C. reinhardtii.},
}
@article {pmid36372102,
year = {2022},
author = {Hosseini, SA and Salehifard Jouneghani, A and Ghatrehsamani, M and Yaghoobi, H and Elahian, F and Mirzaei, SA},
title = {CRISPR/Cas9 as precision and high-throughput genetic engineering tools in gastrointestinal cancer research and therapy.},
journal = {International journal of biological macromolecules},
volume = {223},
number = {Pt A},
pages = {732-754},
doi = {10.1016/j.ijbiomac.2022.11.018},
pmid = {36372102},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; *Gastrointestinal Neoplasms/diagnosis/genetics/therapy ; },
abstract = {Gastrointestinal cancer (GI) is one of the most serious and health-threatening diseases worldwide. Many countries have encountered an escalating prevalence of shock. Therefore, there is a pressing need to clarify the molecular pathogenesis of these cancers. The use of high-throughput technologies that allow the precise and simultaneous investigation of thousands of genes, proteins, and metabolites is a critical step in disease diagnosis and cure. Recent innovations have provided easy and reliable methods for genome investigation, including TALENs, ZFNs, and the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats system). Among these, CRISPR/Cas9 has been revolutionary tool in genetic research. Recent years were prosperous years for CRISPR by the discovery of novel Cas enzymes, the Nobel Prize, and the development of critical clinical trials. This technology utilizes comprehensive information on genes associated with tumor development, provides high-throughput libraries for tumor therapy by developing screening platforms, and generates rapid tools for cancer therapy. This review discusses the various applications of CRISPR/Cas9 in genome editing, with a particular focus on genome manipulation, including infection-related genes, RNAi targets, pooled library screening for identification of unknown driver mutations, and molecular targets for gastrointestinal cancer modeling. Finally, it provides an overview of CRISPR/Cas9 clinical trials, as well as the challenges associated with its use.},
}
@article {pmid36334622,
year = {2023},
author = {Zhang, J and Xing, J and Mi, Q and Yang, W and Xiang, H and Xu, L and Zeng, W and Wang, J and Deng, L and Jiang, J and Yang, G and Gao, Q and Li, X},
title = {Highly efficient transgene-free genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {326},
number = {},
pages = {111523},
doi = {10.1016/j.plantsci.2022.111523},
pmid = {36334622},
issn = {1873-2259},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Tobacco/genetics ; Protein-Tyrosine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; Genome, Plant/genetics ; Plants/genetics ; RNA, Transfer ; },
abstract = {CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T1 generation as a result of editing the NtLHT1 gene in tobacco, and the plant height and the contents of most free amino acids in the leaves of the T2 mutant plants were significantly different from those in the leaves of WT plants, demonstrating the high efficiency of the new editing system. This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements for increasing the efficiency of plant genome editing.},
}
@article {pmid36243370,
year = {2022},
author = {Li, S and Du, M and Deng, J and Deng, G and Li, J and Song, Z and Han, H},
title = {Gene editing of Duchenne muscular dystrophy using biomineralization-based spCas9 variant nanoparticles.},
journal = {Acta biomaterialia},
volume = {154},
number = {},
pages = {597-607},
doi = {10.1016/j.actbio.2022.10.015},
pmid = {36243370},
issn = {1878-7568},
mesh = {Animals ; *Gene Editing/methods ; *Muscular Dystrophy, Duchenne/genetics/therapy/metabolism ; CRISPR-Associated Protein 9/metabolism ; Dystrophin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA Splice Sites ; Mammals/metabolism ; },
abstract = {The CRISPR/Cas9 mediated genome editing have provided a promising strategy to correct multiple mutations of Duchenne muscular dystrophy (DMD). However, the delivery of CRISPR/Cas9 system into mammalian cell for DMD gene editing mainly relies on adeno associated virus (AAV)-mediated transport. Meanwhile, the protospacer adjacent motif (PAM) requirement of wild-typed Cas9 protein causing the target sites for exon splice acceptor site are restricted to limited regions. Here, we developed a biomineralized PAMLess Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This method described a facile synthesis of biomineralized NPs with high SpRY pDNA encapsulation efficiency. In vitro results show that the Bm-SpRY NPs have the obvious advantages of well biocompatibility and protecting SpRY pDNA from enzyme degradation and efficient delivery under high serum condition. Cell studies demonstrated that Bm-SpRY NPs enable rapid cellular uptake, endo-lysosomes escape and nucleus transport. Meanwhiles, the DMD gene editing via Bm-SpRY NPs pathway is transient process without genomic integration. We evaluated multiple target regions with different PAMs for the DMD exon 51 splice acceptor site through Bm-SpRY NPs method and found that the target region with TAG PAM has the highest editing efficiency and significant preferential mutation. In vivo results show that intramuscular injection of Bm-SpRY NPs enable DMD gene mutation in muscle tissue without tissue damage. This study may extend the advanced application of CRISPR system for DMD therapy. STATEMENT OF SIGNIFICANCE: The gene editing technology of CRISPR/Cas9 provides an effective treatment strategy for the Duchenne muscular dystrophy (DMD) therapy. However, the delivery of CRISPR system in mammalian cell mainly relies on viral mediated transport and the NGG or NAG requirement of wild-typed Cas9 protein limits the target region in DMD gene. Here, the present study provides a biomineralized PAM Less Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This study may extend the application of CRISPR system for DMD gene therapy.},
}
@article {pmid36170778,
year = {2022},
author = {Nguyen, GT and Dhingra, Y and Sashital, DG},
title = {Miniature CRISPR-Cas12 endonucleases - Programmed DNA targeting in a smaller package.},
journal = {Current opinion in structural biology},
volume = {77},
number = {},
pages = {102466},
doi = {10.1016/j.sbi.2022.102466},
pmid = {36170778},
issn = {1879-033X},
mesh = {*Endonucleases/chemistry ; *CRISPR-Cas Systems ; DNA/genetics ; RNA/metabolism ; Amino Acids/metabolism ; },
abstract = {CRISPR-associated (Cas) endonucleases specifically target and cleave RNA or DNA based on complementarity to a guide RNA. Cas endonucleases - including Cas9, Cas12a, and Cas13 - have been adopted for a wide array of biotechnological tools, including gene editing, transcriptional modulation, and diagnostics. These tools are facilitated by ready reprogramming of guide RNA sequences and the varied nucleic acid binding and cleavage activities observed across diverse Cas endonucleases. However, the large size of most Cas endonucleases (950-1400 amino acids) can restrict applications. The recent discovery of miniature Cas endonucleases (400-800 amino acids) provides the potential to overcome this limitation. Here, we review recent advances in understanding the structural mechanisms of two miniature Cas endonucleases, Cas12f and Cas12j.},
}
@article {pmid36134724,
year = {2022},
author = {Chen, BX and Xue, LN and Wei, T and Wang, N and Zhong, JR and Ye, ZW and Guo, LQ and Lin, JF},
title = {Multiplex gene precise editing and large DNA fragment deletion by the CRISPR-Cas9-TRAMA system in edible mushroom Cordyceps militaris.},
journal = {Microbial biotechnology},
volume = {15},
number = {12},
pages = {2982-2991},
pmid = {36134724},
issn = {1751-7915},
mesh = {CRISPR-Cas Systems ; *Cordyceps/genetics/metabolism ; *Agaricales/genetics ; Gene Editing/methods ; DNA/metabolism ; Gene Deletion ; },
abstract = {The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA-processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker-free CRISPR-Cas9-TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9-TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9-TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.},
}
@article {pmid36508352,
year = {2022},
author = {Ghouneimy, A and Mahas, A and Marsic, T and Aman, R and Mahfouz, M},
title = {CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00496},
pmid = {36508352},
issn = {2161-5063},
abstract = {The COVID-19 pandemic has challenged the conventional diagnostic field and revealed the need for decentralized Point of Care (POC) solutions. Although nucleic acid testing is considered to be the most sensitive and specific disease detection method, conventional testing platforms are expensive, confined to central laboratories, and are not deployable in low-resource settings. CRISPR-based diagnostics have emerged as promising tools capable of revolutionizing the field of molecular diagnostics. These platforms are inexpensive, simple, and do not require the use of special instrumentation, suggesting they could democratize access to disease diagnostics. However, there are several obstacles to the use of the current platforms for POC applications, including difficulties in sample processing and stability. In this review, we discuss key advancements in the field, with an emphasis on the challenges of sample processing, stability, multiplexing, amplification-free detection, signal interpretation, and process automation. We also discuss potential solutions for revolutionizing CRISPR-based diagnostics toward sample-to-answer diagnostic solutions for POC and home use.},
}
@article {pmid36508307,
year = {2023},
author = {Evans, CH and Ghivizzani, SC and Robbins, PD},
title = {Osteoarthritis gene therapy in 2022.},
journal = {Current opinion in rheumatology},
volume = {35},
number = {1},
pages = {37-43},
pmid = {36508307},
issn = {1531-6963},
abstract = {PURPOSE OF REVIEW: To assess the present status of gene therapy for osteoarthritis (OA).<br><br>RECENT FINDINGS: An expanding list of cDNAs show therapeutic activity when introduced into the joints of animals with experimental models of OA. In vivo delivery with adenovirus or adeno-associated virus is most commonly used for this purpose. The list of encoded products includes cytokines, cytokine antagonists, enzymes, enzyme inhibitors, growth factors and noncoding RNA. Elements of CRISPR-Cas have also been delivered to mouse knees to ablate key genes. Several human trials have been initiated, using transgenes encoding transforming growth factor-&#x3b2;1, interleukin-1 receptor antagonist, interferon-&#x3b2;, the NKX3.2 transcription factor or variant interleukin-10. The first of these, using ex vivo delivery with allogeneic chondrocytes, gained approval in Korea which was subsequently retracted. However, it is undergoing Phase III clinical trials in the United States. The other trials are in Phase I or II. No gene therapy for OA has current marketing approval in any jurisdiction.<br><br>SUMMARY: Extensive preclinical data support the use of intra-articular gene therapy for treating OA. Translation is beginning to accelerate and six gene therapeutics are in clinical trials. Importantly, venture capital has begun to flow and at least seven companies are developing products. Significant progress in the future can be expected.},
}
@article {pmid36506027,
year = {2022},
author = {Ramamurthy, T and Ghosh, A and Chowdhury, G and Mukhopadhyay, AK and Dutta, S and Miyoshi, SI},
title = {Deciphering the genetic network and programmed regulation of antimicrobial resistance in bacterial pathogens.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {952491},
pmid = {36506027},
issn = {2235-2988},
abstract = {Antimicrobial resistance (AMR) in bacteria is an important global health problem affecting humans, animals, and the environment. AMR is considered as one of the major components in the "global one health". Misuse/overuse of antibiotics in any one of the segments can impact the integrity of the others. In the presence of antibiotic selective pressure, bacteria tend to develop several defense mechanisms, which include structural changes of the bacterial outer membrane, enzymatic processes, gene upregulation, mutations, adaptive resistance, and biofilm formation. Several components of mobile genetic elements (MGEs) play an important role in the dissemination of AMR. Each one of these components has a specific function that lasts long, irrespective of any antibiotic pressure. Integrative and conjugative elements (ICEs), insertion sequence elements (ISs), and transposons carry the antimicrobial resistance genes (ARGs) on different genetic backbones. Successful transfer of ARGs depends on the class of plasmids, regulons, ISs proximity, and type of recombination systems. Additionally, phage-bacterial networks play a major role in the transmission of ARGs, especially in bacteria from the environment and foods of animal origin. Several other functional attributes of bacteria also get successfully modified to acquire ARGs. These include efflux pumps, toxin-antitoxin systems, regulatory small RNAs, guanosine pentaphosphate signaling, quorum sensing, two-component system, and clustered regularly interspaced short palindromic repeats (CRISPR) systems. The metabolic and virulence state of bacteria is also associated with a range of genetic and phenotypic resistance mechanisms. In spite of the availability of a considerable information on AMR, the network associations between selection pressures and several of the components mentioned above are poorly understood. Understanding how a pathogen resists and regulates the ARGs in response to antimicrobials can help in controlling the development of resistance. Here, we provide an overview of the importance of genetic network and regulation of AMR in bacterial pathogens.},
}
@article {pmid36504499,
year = {2023},
author = {Pons, BJ and Westra, ER and van Houte, S},
title = {Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa.},
journal = {MethodsX},
volume = {10},
number = {},
pages = {101941},
pmid = {36504499},
issn = {2215-0161},
abstract = {Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the 'CRISPR-Cas' (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated) system keeps record of previous infections to prevent re-infection and thus provides acquired immunity. However, phages are not defenceless against CRISPR-based bacterial immunity. Indeed, they can escape CRISPR systems by encoding one or several anti-CRISPR (Acr) proteins. Acr proteins are among the earliest proteins produced upon phage infection, as they need to quickly inhibit CRISPR-Cas system before it can destroy phage genetic material. As a result, Acrs do not perfectly protect phage from the CRISPR-Cas system, and infection often fails. However, even if the infection fails, Acr can induce a lasting inactivation of the CRISPR-Cas system. The method presented here aims to assess the lasting CRISPR-Cas inhibition in Pseudomonas aeruginosa induced by Acr proteins by:•Infecting the P. aeruginosa strain with a phage carrying an acr gene.•Making the cell electrocompetent while eliminating the phage•Transforming the cells with a plasmid targeted by the CRISPR-Cas system and a non-targeted one to measure the relative transformation efficiency of the plasmids. This method can be adapted to measure which parameters influence Acr-induced immunosuppression in different culture conditions.},
}
@article {pmid36504444,
year = {2022},
author = {Cheng, J and Lin, G and Wang, T and Wang, Y and Guo, W and Liao, J and Yang, P and Chen, J and Shao, X and Lu, X and Zhu, L and Wang, Y and Fan, X},
title = {Massively Parallel CRISPR-Based Genetic Perturbation Screening at Single-Cell Resolution.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2204484},
doi = {10.1002/advs.202204484},
pmid = {36504444},
issn = {2198-3844},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-based genetic screening has been demonstrated as a powerful approach for unbiased functional genomics research. Single-cell CRISPR screening (scCRISPR) techniques, which result from the combination of single-cell toolkits and CRISPR screening, allow dissecting regulatory networks in complex biological systems at unprecedented resolution. These methods allow cells to be perturbed en masse using a pooled CRISPR library, followed by high-content phenotyping. This is technically accomplished by annotating cells with sgRNA-specific barcodes or directly detectable sgRNAs. According to the integration of distinct single-cell technologies, these methods principally fall into four categories: scCRISPR with RNA-seq, scCRISPR with ATAC-seq, scCRISPR with proteome probing, and imaging-based scCRISPR. scCRISPR has deciphered genotype-phenotype relationships, genetic regulations, tumor biological issues, and neuropathological mechanisms. This review provides insight into the technical breakthrough of scCRISPR by systematically summarizing the advancements of various scCRISPR methodologies and analyzing their merits and limitations. In addition, an application-oriented approach guide is offered to meet researchers' individualized demands.},
}
@article {pmid36504030,
year = {2023},
author = {Hijjawi, N and Zahedi, A and Ryan, U},
title = {Point of care diagnostics for Cryptosporidium: new and emerging technologies.},
journal = {Current opinion in gastroenterology},
volume = {39},
number = {1},
pages = {3-8},
doi = {10.1097/MOG.0000000000000895},
pmid = {36504030},
issn = {1531-7056},
abstract = {PURPOSE OF REVIEW: Although Cryptosporidium detection and typing techniques have improved dramatically in recent years, relatively little research has been conducted on point of care (POC) detection and typing tools. Therefore, the main purpose of the present review is to summarize and evaluate recent and emerging POC diagnostic methods for Cryptosporidium spp.<br><br>RECENT FINDINGS: Microscopy techniques such as light-emitting diode fluorescence microscopy with auramine-phenol staining (LED-AP), still have utility for (POC) diagnostics but require fluorescent microscopes and along with immunological-based techniques, suffer from lack of specificity and sensitivity. Molecular detection and typing tools offer higher sensitivity, specificity and speciation, but are currently too expensive for routine POC diagnostics. Isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) including a commercially available LAMP kit have been developed for Cryptosporidium but are prone to false positives. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas diagnostic technologies (CRISPRDx) have recently been combined with isothermal amplification to increase its specificity and sensitivity for detection and typing. Other emerging technologies including amplification-free CRISPR detection methods are currently being developed for Cryptosporidium using a smartphone to read the results.<br><br>SUMMARY: Many challenges are still exist in the development of POC diagnostics for Cryptosporidium. The ideal POC tool would be able to concentrate the pathogen prior to detection and typing, which is complicated and research in this area is still very limited. In the short-term, CRISPR-powered isothermal amplification lateral flow tools offer the best opportunity for POC Cryptosporidium species and subtype detection, with a fully integrated autonomous biosensor for the long-term goal.},
}
@article {pmid36479105,
year = {2022},
author = {Wei, Y and Zhao, Z and Ma, X},
title = {Description of CRISPR-Cas9 development and its prospects in human papillomavirus-driven cancer treatment.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {1037124},
pmid = {36479105},
issn = {1664-3224},
mesh = {Humans ; Human Papillomavirus Viruses ; CRISPR-Cas Systems ; *Papillomavirus Infections/genetics/therapy ; *Neoplasms/genetics/therapy ; },
abstract = {Human papillomaviruses (HPVs) have been recognized as the etiologic agents of various cancers and are called HPV-driven cancers. Concerning HPV-mediated carcinogenic action, gene therapy can cure cancer at the molecular level by means of the correction of specific genes or sites. CRISPR-Cas9, as a novel genetic editing technique, can correct errors in the genome and change the gene expression and function in cells efficiently, quickly, and with relative ease. Herein, we overviewed studies of CRISPR-mediated gene remedies for HPV-driven cancers and summarized the potential applications of CRISPR-Cas9 in gene therapy for cancer.},
}
@article {pmid36475478,
year = {2022},
author = {Sharaev, N and Chacon-Machado, L and Musharova, O and Savitskaya, E and Severinov, K},
title = {[Repair of Double-Stranded DNA Breaks Generated by CRISPR-Cas9 in Pseudomonas putida KT2440].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {914},
doi = {10.31857/S0026898422060180},
pmid = {36475478},
issn = {0026-8984},
mesh = {*DNA Breaks, Double-Stranded ; *Pseudomonas putida/genetics ; CRISPR-Cas Systems ; },
abstract = {Pseudomonas putida KT2440 is a metabolically versatile bacterium with considerable promise as a chassis strain for production and degradation of complex organic compounds. Unlike most bacteria, P. putida KT2440 encodes the Ku and LigD proteins involved in Non-Homologous End Joining (NHEJ). This pathway of repair of double-strand breaks (DSBs) in DNA has an intrinsic mutagenic potential that could be exploited in combination with currently available genome editing tools that generate programmable DSBs. Here, we investigated the effect of removal or overproduction of NHEJ-associated P. putida KT2440 enzymes on mutations generated upon repair of Cas9-mediated DSBs with the double purpose of characterizing the NHEJ pathway and investigating how it functionally interacts with the current gold standard tool for gene editing. The results of our work shed light on non-templated mechanisms of DSB repair in P. putida KT2440, an information that will serve as foundation to expand the gene engineering toolbox for this important microorganism.},
}
@article {pmid36475475,
year = {2022},
author = {Kostyusheva, AP and Brezgin, SA and Ponomareva, NI and Goptar, IA and Nikiforova, AV and Gegechkori, VI and Poluektova, VB and Turkadze, KA and Sudina, AE and Chulanov, VP and Kostyushev, DS},
title = {[Antiviral Activity of CRISPR/Cas9 Ribonucleoprotein Complexes on a Hepatitis B Virus Model In Vivo].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {884-891},
doi = {10.31857/S002689842206012X},
pmid = {36475475},
issn = {0026-8984},
mesh = {Animals ; Mice ; *Hepatitis B virus/genetics ; *Antiviral Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Ribonucleoproteins/genetics ; },
abstract = {Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. This disease is a key issue for global health. Modern methods of therapy do not completely eliminate HBV from infected cells and do not cure chronic infection. The CRISPR/Cas9 systems of site-specific nucleases can effectively cleave do not target DNA including viral genomes. The cleavage of the major form of the HBV genome, i.e., covalently closed circular DNA (cccDNA), leads to a robust reduction in viral replication and degradation or mutational inactivation of cccDNA. CRISPR/Cas9-based approaches are one of the most promising ways to achieve a 'sterilizing' cure of CHB, i.e., complete elimination of the virus from the body. Here, the HBV mouse model in vivo has been used to analyze the antiviral activity of the high-specific Cas9 protein and sgRNA targeting HBV genome. We have found that a single injection of short-lived ribonucleoprotein complexes of CRISPR/Cas9 results in a ~10-fold reduction in HBV DNA levels in the serum and liver of mice as early as 48 h after the start of the experiment. The remaining HBV DNAs have been found to harbor rare indel mutations. Developing new antivirals for treating CHB based on CRISPR/Cas9 ribonucleoprotein complexes could substantially reduce the duration of CHB therapy and, potentially, achieve complete elimination of viral infection.},
}
@article {pmid36474180,
year = {2022},
author = {Mak, JK and Störtz, F and Minary, P},
title = {Comprehensive computational analysis of epigenetic descriptors affecting CRISPR-Cas9 off-target activity.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {805},
pmid = {36474180},
issn = {1471-2164},
support = {BB/S507593/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S507593/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S507593/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Epigenomics ; },
abstract = {BACKGROUND: A common issue in CRISPR-Cas9 genome editing is off-target activity, which prevents the widespread use of CRISPR-Cas9 in medical applications. Among other factors, primary chromatin structure and epigenetics may influence off-target activity.<br><br>METHODS: In this work, we utilize crisprSQL, an off-target database, to analyze the effect of 19 epigenetic descriptors on CRISPR-Cas9 off-target activity. Termed as 19 epigenetic features/scores, they consist of 6 experimental epigenetic and 13 computed nucleosome organization-related features. In terms of novel features, 15 of the epigenetic scores are newly considered. The 15 newly considered scores consist of 13 freshly computed nucleosome occupancy/positioning scores and 2 experimental features (MNase and DRIP). The other 4 existing scores are experimental features (CTCF, DNase I, H3K4me3, RRBS) commonly used in deep learning models for off-target activity prediction. For data curation, MNase was aggregated from existing experimental nucleosome occupancy data. Based on the sequence context information available in crisprSQL, we also computed nucleosome occupancy/positioning scores for off-target sites.<br><br>RESULTS: To investigate the relationship between the 19 epigenetic features and off-target activity, we first conducted Spearman and Pearson correlation analysis. Such analysis shows that some computed scores derived from training-based models and training-free algorithms outperform all experimental epigenetic features. Next, we evaluated the contribution of all epigenetic features in two successful machine/deep learning models which predict off-target activity. We found that some computed scores, unlike all 6 experimental features, significantly contribute to the predictions of both models. As a practical research contribution, we make the off-target dataset containing all 19 epigenetic features available to the research community.<br><br>CONCLUSIONS: Our comprehensive computational analysis helps the CRISPR-Cas9 community better understand the relationship between epigenetic features and CRISPR-Cas9 off-target activity.},
}
@article {pmid36436397,
year = {2022},
author = {Wang, M and Tang, J and Zhou, T and Mai, Y and Zhu, Y},
title = {Generation of a homozygous FIS1 knockout human embryonic stem cell line GIBHe015-B by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {65},
number = {},
pages = {102980},
doi = {10.1016/j.scr.2022.102980},
pmid = {36436397},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells ; CRISPR-Cas Systems/genetics ; Cell Line ; Membrane Proteins ; Mitochondrial Proteins/genetics ; },
abstract = {The mitochondrial fission protein 1 (FIS1) is essential for mitochondrial division or fission and has been determined to mediate mitochondrial and peroxisomal fission. Other studies also found that FIS1 functions as an essential component of the mitophagy and apoptosis pathways in mammalian cells, suggesting that FIS1 has multiple important roles. Here, we generated homozygous FIS1 knockout human embryonic stem cells (hESCs) using the CRISPR/Cas9 system. This cell line exhibits normal karyotype, pluripotency, and trilineage differentiation potential, which could provide a useful cellular resource for exploring the functions of FIS1 and their implications in human health and diseases.},
}
@article {pmid36367552,
year = {2022},
author = {Cai, W and Liu, J and Chen, X and Mao, L and Wang, M},
title = {Orthogonal Chemical Activation of Enzyme-Inducible CRISPR/Cas9 for Cell-Selective Genome Editing.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {48},
pages = {22272-22280},
doi = {10.1021/jacs.2c10545},
pmid = {36367552},
issn = {1520-5126},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; },
abstract = {The precision and therapeutic potential of CRISPR/Cas9 genome editing are greatly challenged by the less control over Cas9-mediated DNA cleavage. Herein, we introduce a conditional and cell-selective genome editing system controlled by disease-associated enzymes, termed enzyme-inducible CRISPR (eiCRISPR). eiCRISPR comprises Cas9 protein, a self-blocked inactive single-guide RNA (bsgRNA), and a chemically caged deoxyribozyme (DNAzyme) that activates bsgRNA and eiCRISPR in a controllable manner. We design chemical modifications of DNAzyme to suppress its ability to cleave the blocking region of bsgRNA, while the decaging of DNAzyme triggered by the tumor cell-overexpressed enzyme, for instance, NAD(P)H:quinone oxidoreductase (NQO1), restores the activity of bsgRNA and switches on eiCRISPR selectively for genome editing in cancer cells. Moreover, using a biodegradable lipid nanoparticle to deliver eiCRISPR in a tumor-bearing xenograft, we show that the in vivo activation of eiCRISPR enables the editing of human papillomavirus 18 E6 for potential cancer therapy. The strategy of postsynthetic and site-specific modification of DNAzyme is compatible with endogenous chemistries for regulating eiCRISPR for cell-selective genome editing and targeted gene therapy.},
}
@article {pmid36310121,
year = {2022},
author = {Yang, C and Lee, D and Zhang, MS and Tse, AP and Wei, L and Bao, MH and Wong, BP and Chan, CY and Yuen, VW and Chen, Y and Wong, CC},
title = {Genome-Wide CRISPR/Cas9 Library Screening Revealed Dietary Restriction of Glutamine in Combination with Inhibition of Pyruvate Metabolism as Effective Liver Cancer Treatment.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {9},
number = {34},
pages = {e2202104},
doi = {10.1002/advs.202202104},
pmid = {36310121},
issn = {2198-3844},
mesh = {Mice ; Animals ; *Liver Neoplasms/genetics/therapy ; Early Detection of Cancer ; Mice, Knockout ; *Carcinoma, Hepatocellular/genetics/therapy ; Glutamine ; Pyruvic Acid ; CRISPR-Cas Systems/genetics ; Oxidoreductases ; },
abstract = {Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Glutamine is an essential, extracellular nutrient which supports HCC growth. Dietary glutamine deficiency may be a potential therapeutic approach for HCC. HCC cells overcome metabolic challenges by rewiring their metabolic pathways for rapid adaptations. The efficiency of dietary glutamine deficiency as HCC treatment is examined and the adaptation machinery under glutamine depletion in HCC cells is unraveled. Using genome-wide CRISPR/Cas9 knockout library screening, this study identifies that pyruvate dehydrogenase α (PDHA), pyruvate dehydrogenase β (PDHB), and pyruvate carboxylase (PC) in pyruvate metabolism are crucial to the adaptation of glutamine depletion in HCC cells. Knockout of either PDHA, PDHB or PC induced metabolic reprogramming of the tricarboxylic acid (TCA) cycle, disrupts mitochondrial function, leading to the suppression of HCC cell proliferation under glutamine depletion. Surprisingly, dietary glutamine restriction improves therapeutic responses of HCC to PDH or PC inhibitor in mouse HCC models. Stable isotope carbon tracing confirms that PDH or PC inhibitors further disrupt the metabolic rewiring of the TCA cycle induced by dietary glutamine depletion in HCC. In summary, the results demonstrate that pyruvate metabolism acts as novel targetable metabolic vulnerabilities for HCC treatment in combination with a glutamine-deficient diet.},
}
@article {pmid36283273,
year = {2022},
author = {Schneider, Y and Turan, S and Koller, A and Krumbiegel, M and Farrell, M and Plötz, S and Winkler, J and Xiang, W},
title = {Generation of a homozygous and a heterozygous SNCA gene knockout human-induced pluripotent stem cell line by CRISPR/Cas9 mediated allele-specific tuning of SNCA expression.},
journal = {Stem cell research},
volume = {65},
number = {},
pages = {102952},
doi = {10.1016/j.scr.2022.102952},
pmid = {36283273},
issn = {1876-7753},
mesh = {Humans ; *alpha-Synuclein/genetics ; Gene Knockout Techniques ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; },
abstract = {Aggregation of alpha-synuclein (aSyn) is closely linked to Parkinson's disease, probably due to the loss of physiological functions and/or gain of toxic functions of aggregated aSyn. Significant efforts have been made elucidating the physiological structure and function of aSyn, however, with limited success thus far in human-derived cells, partly because of restricted resources. Here, we developed two human-induced pluripotent stem cell lines using CRISPR/Cas9-mediated allele-specific frame-shift deletion of the aSyn encoding gene SNCA, resulting in homo- and heterozygous SNCA knockout. The generated cell lines are promising cellular tools for studying aSyn dosage-dependent functions and structural alterations in human neural cells.},
}
@article {pmid36260954,
year = {2022},
author = {Mun, D and Kang, JY and Chun, Y and Park, DS and Kim, H and Yun, N and Lee, SH and Joung, B},
title = {Generation of two PITX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {65},
number = {},
pages = {102940},
doi = {10.1016/j.scr.2022.102940},
pmid = {36260954},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; *Atrial Fibrillation ; CRISPR-Cas Systems/genetics ; },
abstract = {PITX2 is a homeobox gene located in the human 4q25 locus and is commonly associated with atrial fibrillation (AF). Here, we generated two PITX2 knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 genome editing. The edited iPSCs maintained fullpluripotency, normal karyotype and spontaneousdifferentiation capability. This cell line provides a suitable model for investigating the physiopathologyof PITX2 mutation in atrial fibrillation.},
}
@article {pmid36257094,
year = {2022},
author = {Li, Q and Shao, L and Li, L and Shen, H and Yu, Y and Shen, Z},
title = {Generation of a human embryonic stem cell line targeted homozygous deletion of BMP10 (WAe007-A-2) by CRISPR/Cas9-dgRNA.},
journal = {Stem cell research},
volume = {65},
number = {},
pages = {102942},
doi = {10.1016/j.scr.2022.102942},
pmid = {36257094},
issn = {1876-7753},
mesh = {Humans ; Homozygote ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells ; Sequence Deletion ; Bone Morphogenetic Proteins ; },
abstract = {BMP10 signaling has been implicated in regulation of cardiovascular cell fate determination and diseases, while the underlying molecular mechanism still remains uncertain. Here, the human embryonic stem cell line (H7-BMP10[del]) with homozygous deletion of BMP10 was generated by CRISPR/Cas9 method. Thus, the crosstalk related to BMP10 signaling could be investigated in cell fate determination and the molecular pathogenesis of cardiovascular disease.},
}
@article {pmid36073293,
year = {2023},
author = {Alon, DM and Partosh, T and Burstein, D and Pines, G},
title = {Rapid and sensitive on-site genetic diagnostics of pest fruit flies using CRISPR-Cas12a.},
journal = {Pest management science},
volume = {79},
number = {1},
pages = {68-75},
doi = {10.1002/ps.7173},
pmid = {36073293},
issn = {1526-4998},
mesh = {*CRISPR-Cas Systems ; Europe ; Asia, Eastern ; Australia ; *Nuclear Family ; },
abstract = {BACKGROUND: Bactrocera zonata, a major fruit pest species, is gradually spreading west from its native habitat in East Asia. In recent years it has become a significant threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent it spreading, monitoring efforts in cultivation sites and border controls are carried out. Despite these efforts, and due to morphological similarities between B. zonata and other pests in relevant developmental stages, the monitoring process is challenging, time-consuming, and requires external assistance from professional laboratories. CRISPR-Cas12a genetic diagnostics has been rapidly developing in recent years and provides an efficient tool for the genetic identification of pathogens, viruses, and other genetic targets. Here we design a CRISPR-Cas12a detection assay that differentially detects two major pest species, B. zonata and Ceratitis capitata.<br><br>RESULTS: We demonstrate the specificity and high sensitivity of this method. Identification of target pests was done using specific and universal primers on pooled samples, enabling differentiation of pests with high certainty. We also demonstrate reaction stability over time for future on-site applications.<br><br>DISCUSSION: Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification and Cas12a-based detection. This method is highly modular, and the presented target design method can be applied to a wide array of pests. This approach can be easily adapted to fit local threats and requires minimal training of operators in border controls and other relevant locations, reshaping pest control and making state-of-the-art technologies available worldwide, including in developing countries. &#xa9; 2022 The Authors. Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.},
}
@article {pmid35713529,
year = {2022},
author = {Zhang, Q and Wang, S and Li, W and Yau, E and Hui, H and Singh, PK and Achuthan, V and Young Karris, MA and Engelman, AN and Rana, TM},
title = {Genome-wide CRISPR/Cas9 transcriptional activation screen identifies a histone acetyltransferase inhibitor complex as a regulator of HIV-1 integration.},
journal = {Nucleic acids research},
volume = {50},
number = {12},
pages = {6687-6701},
pmid = {35713529},
issn = {1362-4962},
support = {CA177322/NH/NIH HHS/United States ; CA177322/NH/NIH HHS/United States ; },
mesh = {Humans ; *HIV-1/genetics ; Transcriptional Activation ; Leukocytes, Mononuclear ; CRISPR-Cas Systems ; Histone Acetyltransferases ; Nuclear Proteins ; RNA-Binding Proteins ; },
abstract = {The retrovirus human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS. Although treatment of HIV/AIDS with antiretroviral therapy provides suppression of viremia, latent reservoirs of integrated proviruses preclude cure by current antiviral treatments. Understanding the mechanisms of host-viral interactions may elucidate new treatment strategies. Here, we performed a CRISPR/Cas9 transcriptional activation screen using a high-complexity, genome-wide sgRNA library to identify cellular factors that inhibit HIV-1 infection of human CD4+ T cells. MT4 cells were transduced with a CRISPR/Cas9 sgRNA library and infected with nef-deficient HIV-1NL4-3 expressing ganciclovir-sensitive thymidine kinase, thus enabling selection of HIV-1-resistant cells for analysis of enriched sgRNAs. After validation of screen hits, multiple host factors essential for HIV-1 infection were identified, including SET (SET nuclear proto-oncogene) and ANP32A (acidic nuclear phosphoprotein 32A, PP32A), which together form a histone acetylase inhibitor complex. Using multiple human cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-1-infected individuals, we demonstrate that SET depletion increased HIV-1 infectivity by augmenting DNA integration without significantly changing sites of integration. Conversely, SET overexpression decreased HIV-1 integration and infectivity. SET protein expression was significantly reduced in PBMCs from HIV-1-infected individuals and was downregulated by HIV-1 infection of healthy donor cells in vitro. Notably, HIV-1-induced downregulation of SET could be alleviated by inhibition of the protease granzyme A. Altogether, we have identified cellular inhibitors of HIV-1 infection on a genome-wide scale, which affords new insight into host-virus interactions and may provide new strategies for HIV-1 treatment.},
}
@article {pmid36500347,
year = {2022},
author = {Zhou, Y and Wang, H and Yang, L and Wang, Q},
title = {Progress on COVID-19 Chemotherapeutics Discovery and Novel Technology.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {23},
pages = {},
doi = {10.3390/molecules27238257},
pmid = {36500347},
issn = {1420-3049},
abstract = {COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel highly contagious and pathogenic coronavirus that emerged in late 2019. SARS-CoV-2 spreads primarily through virus-containing droplets and small particles of air pollution, which greatly increases the risk of inhaling these virus particles when people are in close proximity. COVID-19 is spreading across the world, and the COVID-19 pandemic poses a threat to human health and public safety. To date, there are no specific vaccines or effective drugs against SARS-CoV-2. In this review, we focus on the enzyme targets of the virus and host that may be critical for the discovery of chemical compounds and natural products as antiviral drugs, and describe the development of potential antiviral drugs in the preclinical and clinical stages. At the same time, we summarize novel emerging technologies applied to the research on new drug development and the pathological mechanisms of COVID-19.},
}
@article {pmid36499601,
year = {2022},
author = {Benati, D and Leung, A and Perdigao, P and Toulis, V and van der Spuy, J and Recchia, A},
title = {Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders.},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
doi = {10.3390/ijms232315276},
pmid = {36499601},
issn = {1422-0067},
abstract = {Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.},
}
@article {pmid36499174,
year = {2022},
author = {Karpov, DS and Demidova, NA and Kulagin, KA and Shuvalova, AI and Kovalev, MA and Simonov, RA and Karpov, VL and Snezhkina, AV and Kudryavtseva, AV and Klimova, RR and Kushch, AA},
title = {Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems.},
journal = {International journal of molecular sciences},
volume = {23},
number = {23},
pages = {},
doi = {10.3390/ijms232314847},
pmid = {36499174},
issn = {1422-0067},
abstract = {Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.},
}
@article {pmid36497186,
year = {2022},
author = {Wang, Y and Zafar, N and Ali, Q and Manghwar, H and Wang, G and Yu, L and Ding, X and Ding, F and Hong, N and Wang, G and Jin, S},
title = {CRISPR/Cas Genome Editing Technologies for Plant Improvement against Biotic and Abiotic Stresses: Advances, Limitations, and Future Perspectives.},
journal = {Cells},
volume = {11},
number = {23},
pages = {},
doi = {10.3390/cells11233928},
pmid = {36497186},
issn = {2073-4409},
abstract = {Crossbreeding, mutation breeding, and traditional transgenic breeding take much time to improve desirable characters/traits. CRISPR/Cas-mediated genome editing (GE) is a game-changing tool that can create variation in desired traits, such as biotic and abiotic resistance, increase quality and yield in less time with easy applications, high efficiency, and low cost in producing the targeted edits for rapid improvement of crop plants. Plant pathogens and the severe environment cause considerable crop losses worldwide. GE approaches have emerged and opened new doors for breeding multiple-resistance crop varieties. Here, we have summarized recent advances in CRISPR/Cas-mediated GE for resistance against biotic and abiotic stresses in a crop molecular breeding program that includes the modification and improvement of genes response to biotic stresses induced by fungus, virus, and bacterial pathogens. We also discussed in depth the application of CRISPR/Cas for abiotic stresses (herbicide, drought, heat, and cold) in plants. In addition, we discussed the limitations and future challenges faced by breeders using GE tools for crop improvement and suggested directions for future improvements in GE for agricultural applications, providing novel ideas to create super cultivars with broad resistance to biotic and abiotic stress.},
}
@article {pmid36495467,
year = {2022},
author = {Kazemizadeh, H and Kashefizadeh, A},
title = {CRISPR-Cas9-mediated gene therapy in lung cancer.},
journal = {Clinical &amp; translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico},
volume = {},
number = {},
pages = {},
pmid = {36495467},
issn = {1699-3055},
abstract = {As the largest cause of cancer-related deaths worldwide, pulmonary cancer is the most common form of the disease. Several genetic, epigenetic, and environmental factors come into play during the multi-step mechanism of tumorigenesis. The heterogeneity that makes discovering successful therapeutics for pulmonary cancer problematic is significantly influenced by the epigenetic landscape, including DNA methylation, chromatin architecture, histone modifications, and noncoding RNA control. Clinical activity of epigenetic-targeted medicines has been reported in hematological tumors, and these compounds may also have therapeutic effects in solid tumors. Over the course of the past few years, some researchers have successfully modified the expression of genes in cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) technique. The utilization of this technology allows for the induction of site-specific mutagenesis, epigenetic alterations, and the regulation of gene expression. This study will present an overview of the primary epigenetic alterations seen in pulmonary cancer, as well as a summary of therapeutic implications for targeting epigenetics in the management of pulmonary cancer, with a particular emphasis on the technique known as CRISPR/Cas9.},
}
@article {pmid36494593,
year = {2022},
author = {Zahra, A and Shahid, A and Shamim, A and Khan, SH and Arshad, MI},
title = {The SHERLOCK Platform: An Insight into Advances in Viral Disease Diagnosis.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36494593},
issn = {1559-0305},
abstract = {Persistence and prevalence of microbial diseases (pandemics, epidemics) is the most alarming threats to the human resulting in huge health and economic losses. Rapid detection and understanding of the disease dynamics by molecular biotechnology tools allow for robust reporting, treatment and control of diseases. As per WHO, the optimal diagnostic approach should be quick, specific, sensitive, without a stringed instrument, and low cost. The drawbacks of traditional detection techniques promote the use of CRISPR-mediated nucleic acid detection methods such as SHERLOCK as detection method. It takes advantage of the unexpected in vitro features of CRISPR-Cas system to develop field-deployable sensitive detection tools. Previously, CRISPR-mediated diagnostic methods have extensively been reviewed particularly for SARS-COV-2 detection, but it fails to provide the insight into advances of this technique. This study is the first attempt to review the advances of SHERLOCK approach as diagnostic tool for viral diseases detection. Variations of SHERLOCK mechanism for improved efficiency are discussed. Particularly integrated SHERLOCK approaches in terms of extraction-free assay and Bluetooth-enabled detection are reviewed to access their feasibility for the development of simpler and cost-effective diagnostic toolkits. Insight in to perks and limitations of diagnostic methods indicates its potential as ultimate diagnostic instrument for disease management.},
}
@article {pmid36494336,
year = {2022},
author = {Dong, X and Guthrie, BGH and Alexander, M and Noecker, C and Ramirez, L and Glasser, NR and Turnbaugh, PJ and Balskus, EP},
title = {Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7624},
pmid = {36494336},
issn = {2041-1723},
abstract = {Eggerthella lenta is a prevalent human gut Actinobacterium implicated in drug, dietary phytochemical, and bile acid metabolism and associated with multiple human diseases. No genetic tools are currently available for the direct manipulation of E. lenta. Here, we construct shuttle vectors and develop methods to transform E. lenta and other Coriobacteriia. With these tools, we characterize endogenous E. lenta constitutive and inducible promoters using a reporter system and construct inducible expression systems, enabling tunable gene regulation. We also achieve genome editing by harnessing an endogenous type I-C CRISPR-Cas system. Using these tools to perform genetic knockout and complementation, we dissect the functions of regulatory proteins and enzymes involved in catechol metabolism, revealing a previously unappreciated family of membrane-spanning LuxR-type transcriptional regulators. Finally, we employ our genetic toolbox to study the effects of E. lenta genes on mammalian host biology. By greatly expanding our ability to study and engineer gut Coriobacteriia, these tools will reveal mechanistic details of host-microbe interactions and provide a roadmap for genetic manipulation of other understudied human gut bacteria.},
}
@article {pmid36494308,
year = {2022},
author = {Li, Y and Liao, D and Kou, J and Tong, Y and Daniels, LC and Man, S and Ma, L},
title = {Comparison of CRISPR/Cas and Argonaute for nucleic acid tests.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.11.002},
pmid = {36494308},
issn = {1879-3096},
abstract = {Guided, programmable, and target-activated nucleases, exemplified by Cas in the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system and Argonaute (Ago), are emerging as a new generation of nucleic acid tests (NATs). A specific approach for comparison of these two nucleases side by side in terms of similarities, differences, and complementarities is instrumental for the sensible design of novel NATs.},
}
@article {pmid36484100,
year = {2022},
author = {Wang, X and Yu, G and Wen, Y and An, Q and Li, X and Liao, F and Lian, C and Zhang, K and Yin, H and Wei, Y and Deng, Z and Zhang, H},
title = {Target RNA-guided protease activity in type III-E CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac1151},
pmid = {36484100},
issn = {1362-4962},
abstract = {The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.},
}
@article {pmid36483767,
year = {2022},
author = {Huang, J and Zhou, Y and Li, J and Lu, A and Liang, C},
title = {CRISPR/Cas systems: Delivery and application in gene therapy.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {942325},
doi = {10.3389/fbioe.2022.942325},
pmid = {36483767},
issn = {2296-4185},
abstract = {The CRISPR/Cas systems in prokaryotes such as bacteria and archaea are the adaptive immune system to prevent infection from viruses, phages, or other foreign substances. When viruses or phages first invade the bacteria, Cas proteins recognize and cut the DNA from viruses or phages into short fragments that will be integrated into the CRISPR array. Once bacteria are invaded again, the modified CRISPR and Cas proteins react quickly to cut DNA at the specified target location, protecting the host. Due to its high efficiency, versatility, and simplicity, the CRISPR/Cas system has become one of the most popular gene editing technologies. In this review, we briefly introduce the CRISPR/Cas systems, focus on several delivery methods including physical delivery, viral vector delivery, and non-viral vector delivery, and the applications of disease therapy. Finally, some problems in CRISPR/Cas9 technology have been proposed, such as the off-target effects, the efficiency of DNA repair mechanisms, and delivery of CRISPR/Cas system safely and efficiently to the target location.},
}
@article {pmid36482900,
year = {2022},
author = {Devi, R and Chauhan, S and Dhillon, TS},
title = {Genome editing for vegetable crop improvement: Challenges and future prospects.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {1037091},
doi = {10.3389/fgene.2022.1037091},
pmid = {36482900},
issn = {1664-8021},
abstract = {Vegetable crops are known as protective foods due to their potential role in a balanced human diet, especially for vegetarians as they are a rich source of vitamins and minerals along with dietary fibers. Many biotic and abiotic stresses threaten the crop growth, yield and quality of these crops. These crops are annual, biennial and perennial in breeding behavior. Traditional breeding strategies pose many challenges in improving economic crop traits. As in most of the cases the large number of backcrosses and stringent selection pressure is required for the introgression of the useful traits into the germplasm, which is time and labour-intensive process. Plant scientists have improved economic traits like yield, quality, biotic stress resistance, abiotic stress tolerance, and improved nutritional quality of crops more precisely and accurately through the use of the revolutionary breeding method known as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 (Cas9). The high mutation efficiency, less off-target consequences and simplicity of this technique has made it possible to attain novel germplasm resources through gene-directed mutation. It facilitates mutagenic response even in complicated genomes which are difficult to breed using traditional approaches. The revelation of functions of important genes with the advancement of whole-genome sequencing has facilitated the CRISPR-Cas9 editing to mutate the desired target genes. This technology speeds up the creation of new germplasm resources having better agro-economical traits. This review entails a detailed description of CRISPR-Cas9 gene editing technology along with its potential applications in olericulture, challenges faced and future prospects.},
}
@article {pmid36482771,
year = {2022},
author = {Carroll, D},
title = {RNA in Therapeutics: CRISPR in the Clinic.},
journal = {Molecules and cells},
volume = {},
number = {},
pages = {},
pmid = {36482771},
issn = {0219-1032},
abstract = {The advent of the CRISPR-Cas genome editing platform has greatly enhanced the capabilities of researchers in many areas of biology. Its use has also been turned to the development of therapies for genetic diseases and to the enhancement of cell therapies. This review describes some recent advances in these areas.},
}
@article {pmid36481907,
year = {2023},
author = {Tsai, YT and da Costa, BL and Nolan, ND and Caruso, SM and Jenny, LA and Levi, SR and Tsang, SH and Quinn, PMJ},
title = {Prime Editing for the Installation and Correction of Mutations Causing Inherited Retinal Disease: A Brief Methodology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2560},
number = {},
pages = {313-331},
pmid = {36481907},
issn = {1940-6029},
abstract = {Inherited retinal diseases (IRDs) encompass a large heterogeneous group of rare blinding disorders whose etiology originates from mutations in the 280 genes identified to date. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems represent a promising avenue for the treatment of IRDs, as exemplified by FDA clinical trial approval of EDIT-101 (AGN-151587), which removes a deep intronic variant in the CEP290 gene that causes Leber congenital amaurosis (LCA) type 10. Prime editing is a novel double-strand break (DSB) independent CRISPR/Cas system which has the potential to correct all 12 possible transition and transversion mutations in addition to small deletions and insertions. Here, as a proof-of-concept study, we describe a methodology using prime editing for the in vitro installation and correction of the classical Pde6b[rd10] c.1678C > T (p.Arg560Cys) mutation which causes autosomal recessive retinitis pigmentosa (RP) in mice.},
}
@article {pmid36479971,
year = {2022},
author = {Chou-Zheng, L and Hatoum-Aslan, A},
title = {Critical roles for 'housekeeping' nucleases in Type III CRISPR-Cas immunity.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
doi = {10.7554/eLife.81897},
pmid = {36479971},
issn = {2050-084X},
abstract = {CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e. phages). Type III systems launch a multi-layered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the Type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third 'housekeeping' nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation, and identify specific interactions with Csm5, a member of the Type III effector complex, which facilitate nuclease recruitment/stimulation. Further, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for Type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.},
}
@article {pmid36479615,
year = {2022},
author = {Li, J and Zhang, C and He, Y and Li, S and Yan, L and Li, Y and Zhu, Z and Xia, L},
title = {Plant base editing and prime editing: The current status and future perspectives.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13425},
pmid = {36479615},
issn = {1744-7909},
abstract = {Precise replacement of an allele with an elite allele controlling important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement. Base editing and prime editing are two newly developed precision gene editing systems that can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks (DSBs) and donor repair templates (DRTs), respectively. Since their discoveries, various strategies have been attempted to optimize both base editor (BE) and prime editor (PE) in order to improve the precise editing efficacy, specificity, and expand the targeting scopes. Here, we summarize the latest development of various BEs and PEs, as well as their applications in plants. Based on these progresses, we recommend the appropriate BEs and PEs for both basic plant research and crop improvement. Moreover, we propose the perspectives for further optimization of these two editors. We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future. This article is protected by copyright. All rights reserved.},
}
@article {pmid36478403,
year = {2022},
author = {Qiao, D and Wang, J and Lu, MH and Xin, C and Chai, Y and Jiang, Y and Sun, W and Cao, Z and Guo, S and Wang, XC and Chen, QJ},
title = {Optimized prime editing efficiently generates heritable mutations in maize.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13428},
pmid = {36478403},
issn = {1744-7909},
abstract = {Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed. This article is protected by copyright. All rights reserved.},
}
@article {pmid36478250,
year = {2022},
author = {Chen, Q and Gul, I and Liu, C and Lei, Z and Li, X and Raheem, MA and He, Q and Haihui, Z and Leeansyah, E and Zhang, CY and Pandey, V and Du, K and Qin, P},
title = {CRISPR-Cas12-based field-deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome.},
journal = {Journal of medical virology},
volume = {},
number = {},
pages = {},
doi = {10.1002/jmv.28385},
pmid = {36478250},
issn = {1096-9071},
abstract = {The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost-effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system harnesses the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification (RPA). It can detect both the current circulating MPXV clade and the original clades. We reached an LoD of 22.4 aM (13.5 copies/µL) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µL) in a two-step assay, which is further reduced to 25 aM (15 copies/µL) in a one-pot system. We compared our results with quantitative (q) PCR and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample-to-answer time of 35 minutes. This article is protected by copyright. All rights reserved.},
}
@article {pmid36477884,
year = {2022},
author = {Zhang, Q and Zhang, X and Zou, X and Ma, F and Zhang, CY},
title = {CRISPR/Cas-Based MicroRNA Biosensors.},
journal = {Chemistry (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {},
doi = {10.1002/chem.202203412},
pmid = {36477884},
issn = {1521-3765},
abstract = {As important post-transcriptional regulators, microRNAs (miRNAs) play irreplaceable roles in diverse cellular functions. Dysregulated miRNA expression is implicated in various diseases including cancers, and thus miRNAs have become the valuable biomarkers for disease monitoring. Recently, clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system has shown great promise for the development of next-generation bio-sensors because of its precise localization capability, good fidelity, and high cleavage activity. Herein, we review recent advance in development of CRISPR/Cas-based biosensors for miRNA detection. We summarize the principles, features, and performance of these miRNA biosensors, and further highlight the remaining challenges and future directions.},
}
@article {pmid36477756,
year = {2022},
author = {Ramos, JN and Baio, PVP and Veras, JFC and Vieira, &#xc9;MD and Mattos-Guaraldi, AL and Vieira, VV},
title = {Novel configurations of type I-E CRISPR-Cas system in Corynebacterium striatum clinical isolates.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {},
number = {},
pages = {},
pmid = {36477756},
issn = {1678-4405},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) are a prokaryotic adaptive immune system that, through Cas proteins, promote the degradation of foreign nucleic acids such as phages and plasmids. We analyzed 10 genomes of Corynebacterium striatum clinical isolates from a public hospital in Rio de Janeiro, Brazil, the most emergent multidrug-resistant Corynebacterium species. All isolates were submitted to antimicrobial susceptibility testing. The occurrence and diversity of the CRISPR system were investigated by bioinformatics tools. Our analysis revealed that the isolates exhibited type I-E gene arrangements, and 3 more multidrug-resistant isolates, alternative type I-E gene arrangements, showing a divergent gene arrangement within the cas operon. Phylogenetic analysis of the cas1 gene of this type I-E CRISPR-Cas system alternative arrangement, termed here type I-E', showed a cluster in a distinct clade of the type I-E CRISPR-Cas system. The systems' guanine-cytosine (GC) content is lower than the genomic DNA's GC content, and mobile genetic elements were found in some isolates near the CRISPR-Cas system. Most CRISPR spacers are unknown indicating that there is a reservoir of unexplored corynebacteriophages and plasmids. Some spacers showed perfect homologies with phage and plasmid sequences. Intact phage regions were found in 3 of our isolates, ranging from 9.1 to 43.8 kb, with regions showing similarity to Rhodococcus and Corynebacterium phages. Our results may contribute to research about the CRISPR-Cas system diversity in C. striatum, where there are no published data to date.},
}
@article {pmid36477448,
year = {2022},
author = {Cui, N and Zhang, JT and Li, Z and Liu, XY and Wang, C and Huang, H and Jia, N},
title = {Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7549},
pmid = {36477448},
issn = {2041-1723},
abstract = {The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMP[crRNA] and gRAMP[crRNA]-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions -1 and -2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.},
}
@article {pmid36476798,
year = {2022},
author = {Hiramoto, T and Ohmori, T},
title = {[Application of genome editing technology in gene therapy].},
journal = {[Rinsho ketsueki] The Japanese journal of clinical hematology},
volume = {63},
number = {11},
pages = {1558-1565},
doi = {10.11406/rinketsu.63.1558},
pmid = {36476798},
issn = {0485-1439},
abstract = {Genome editing has been attracting increasing attention as a new treatment for several refractory diseases since the CRISPR-Cas discovery has facilitated easy modification of target chromosomal DNA. The concept of treating refractory diseases by genome editing has been achieved in various animal models, and genome editing has been applied to human clinical trials for β-thalassemia, sickle cell disease, mucopolysaccharidosis, transthyretin amyloidosis, HIV infection, and CAR-T therapy. The genome editing technology targets the germline in industrial applications in animals and plants and is directed at the chromosomal DNA of the somatic cells in human therapeutic applications. Genome editing therapy for germline cells is currently forbidden due to ethical and safety concerns. Concerns regarding genome editing technology include safety (off-target effects) as well as technical aspects (low homologous recombination). Various technological innovations for genome editing are expected to expand its clinical application to various diseases in the future.},
}
@article {pmid36476134,
year = {2022},
author = {Lu, Y and Yang, H and Bai, J and He, Q and Deng, R},
title = {CRISPR-Cas based molecular diagnostics for foodborne pathogens.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/10408398.2022.2153792},
pmid = {36476134},
issn = {1549-7852},
abstract = {Foodborne pathogenic infection has brought multifaceted issues to human life, leading to an urgent demand for advanced detection technologies. CRISPR/Cas-based biosensors have the potential to address various challenges that exist in conventional assays such as insensitivity, long turnaround time and complex pretreatments. In this perspective, we review the relevant strategies of CRISPR/Cas-assisted diagnostics on foodborne pathogens, focusing on biosensing platforms for foodborne pathogens based on fluorescence, colorimetric, (electro)chemiluminescence, electrochemical, and surface-enhanced Raman scattering detection. It summarizes their detection principles by the clarification of foodborne pathogenic bacteria, fungi, and viruses. Finally, we discuss the current challenges or technical barriers of these methods against broad application, and put forward alternative solutions to improve CRISPR/Cas potential for food safety.},
}
@article {pmid36475481,
year = {2022},
author = {Kuluev, BR and Mikhailova, EV and Kuluev, AR and Galimova, AA and Zaikina, EA and Khlestkina, EK},
title = {[Genome Editing in Species of the Tribe Triticeae with the CRISPR/Cas System].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {949-968},
doi = {10.31857/S0026898422060155},
pmid = {36475481},
issn = {0026-8984},
abstract = {The tribe Triticeae includes important agricultural crops, such as bread wheat, durum wheat, barley, rye, and triticale. Research in the field of reverse genetics and genetic engineering of Triticeae received a new impetus as the CRISPR/Cas genome editing system came into broad use. The review describes and analyzes the data on recent advances in genomic editing of cultivated plants of the tribe Triticeae and tools used in the field. The tools most commonly used for genome editing in Triticeae include the codon-optimized Cas9 gene under the control of the maize ubiquitin gene promoter and guide RNAs under the control of Pol III promoters U6 and U3 in one or more binary vectors. Phosphinothricin and hygromycin resistance genes are used as selectable genes. Agrobacterium-mediated transformation and biolistics are performed to obtain genome-edited plants, and immature embryos are used as explants. Approaches developed to overcome the problem of low regenerative capacity of Triticeae include in planta transformation of shoot apical meristems, transformation of microspores and pollen grains, and the use of haploinductors. Bread wheat and barley were subject to genomic editing in the majority of studies published to date, and durum wheat and triticale were recently used in CRISPR/Cas knockout studies of target genes. Further progress in the development of genome editing of cultivated plants of the tribe Triticeae should be aimed at expanding the range of species and varieties involved and overcoming the problems of low regenerative capacity. This will allow genetic modification of elite varieties, which will be in demand in agricultural production.},
}
@article {pmid36475480,
year = {2022},
author = {Spasskaya, DS and Davletshin, AI and Tutyaeva, VV and Kulagin, KA and Garbuz, DG and Karpov, DS},
title = {[A Test System for Assessment of the Activity of Mutant Cas9 Variants in Saccharomyces cerevisiae].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {937-948},
doi = {10.31857/S0026898422060209},
pmid = {36475480},
issn = {0026-8984},
abstract = {The key component of the revolutionary Streptococcus pyogenes CRISPR/Cas genome editing technology is the multidomain protein Cas9. However, the specificity of wild type Cas9 is not sufficiently high for editing large genomes of higher eukaryotes, which limits the realization of the potential of genomic editing both in fundamental investigations and in the therapy of genetic diseases. The main way to obtain more specific variants of Cas9 is through mutagenesis followed by characterization of mutant proteins in in vitro or in vivo test systems. The in vitro and some in vivo test systems described in the literature are often labor-intensive and have scaling limitations, which makes it challenging to screen SpCas9 mutant variant libraries. In order to develop a simple method for high-throughput screening of Cas9 mutants in vivo, we characterized three test systems using CRISPR/Cas9-mediated inactivation of the reporter genes, tsPurple, ADE2, and URA3, in the Saccharomyces cerevisiae yeast as a model subject. We measured the activities of high-precision forms of Cas9, evoCas9, and HiFiCas9, and compared them with the wild-type form. ADE2 gene inactivation was found to be the most valid method for the evaluation of Cas9 activity. In the test-system developed, the sensitivity to chromatin structure was demonstrated for the high-fidelity variant of Cas9, HiFiCas9. The proposed test-system can be used for the development of new generation genome editors.},
}
@article {pmid36475477,
year = {2022},
author = {Armianinova, DK and Karpov, DS and Kotliarova, MS and Goncharenko, AV},
title = {[Genetic Engineering in Mycobacteria].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {900-913},
doi = {10.31857/S0026898422060040},
pmid = {36475477},
issn = {0026-8984},
abstract = {Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.},
}
@article {pmid36475476,
year = {2022},
author = {Nadolinskaia, NI and Goncharenko, AV},
title = {[CRISPR Interference in Regulation of Bacterial Gene Expression].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {892-899},
doi = {10.31857/S0026898422060167},
pmid = {36475476},
issn = {0026-8984},
abstract = {The review is devoted to the use of the CRISPR/Cas system for obtaining knockdowns of target bacterial genes by CRISPR-mediated interference (CRISPRi). CRISPRi is based on the preservation of the ability of the inactivated dCas nuclease in complex with guide RNA to bind a target, which leads to reversible repression of the selected genes. The review describes the principle of operation of CRISPR/Cas and CRIS-PRi/dCas and provides examples of various approaches to the use of CRISPRi with the most popular inactivated nucleases dCas9 and dCas12a. Also, attention is paid to the use of CRISPRi screening for genome-wide studies and the modular system for identifying many important patterns at the Mobile-CRISPRi genome level. In addition, we discuss the use of CRISPRi to optimize biotechnological production, such as the synthesis of malonyl-CoA, L-lysine, L-glutamate, and other significant products.},
}
@article {pmid36475474,
year = {2022},
author = {Isaev, A and Andriianov, A and Znobishcheva, E and Zorin, E and Morozova, N and Severinov, K},
title = {[Editing of Phage Genomes - Recombineering-Assisted SpCas9 Modification of Model Coliphages T7, T5, and T3].},
journal = {Molekuliarnaia biologiia},
volume = {56},
number = {6},
pages = {883},
doi = {10.31857/S002689842206009X},
pmid = {36475474},
issn = {0026-8984},
abstract = {Bacteriophages-viruses that infect bacterial cells - are the most abundant biological entities on Earth. The use of phages in fundamental research and industry requires tools for precise manipulation of their genomes. Yet, compared to bacterial genome engineering, modification of phage genomes is challenging because of the lack of selective markers and thus requires laborious screenings of recombinant/mutated phage variants. The development of the CRISPR-Cas technologies allowed to solve this issue by the implementation of negative selection that eliminates the parental phage genomes. In this manuscript, we summarize current methods of phage genome engineering and their coupling with CRISPR-Cas technologies. We also provide examples of our successful application of these methods for introduction of specific insertions, deletions, and point mutations in the genomes of model Escherichia coli lytic phages T7, T5, and T3.},
}
@article {pmid36474907,
year = {2022},
author = {Tao, S and Chen, H and Li, N and Fang, Y and Xu, Y and Liang, W},
title = {Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus.},
journal = {Infection and drug resistance},
volume = {15},
number = {},
pages = {6939-6949},
pmid = {36474907},
issn = {1178-6973},
abstract = {PURPOSE: This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes.<br><br>MATERIALS AND METHODS: Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests.<br><br>RESULTS: A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene.<br><br>CONCLUSION: In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.},
}
@article {pmid36473330,
year = {2022},
author = {Selma, S and Ceulemans, E and Goossens, A and Lacchini, E},
title = {Clustered regularly interspaced short palindromic repeats tools for plant metabolic engineering: achievements and perspectives.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102856},
doi = {10.1016/j.copbio.2022.102856},
pmid = {36473330},
issn = {1879-0429},
abstract = {The plant kingdom represents the biggest source of feedstock, food, and added-value compounds. Engineering plant metabolic pathways to increase the phytochemical production or improve the nutraceutical value of crops is challenging because of the intricate interaction networks that link multiple genes, enzymatic steps, and metabolites, even when pathways are fully elucidated. The development of clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) technologies has helped to overcome limitations in metabolic engineering, providing efficient and versatile tools for multigene editing. CRISPR approaches in plants were shown to have a remarkable efficiency in genome editing of different species to improve agronomic and metabolic traits. Here, we give an overview of the different achievements and perspectives of CRISPR technology in plant metabolic engineering.},
}
@article {pmid36471243,
year = {2022},
author = {Gumustop, I and Ortakci, F},
title = {Comparative Genomics of Lentilactobacillus parabuchneri isolated from dairy, KEM complex, Makgeolli, and Saliva Microbiomes.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {803},
pmid = {36471243},
issn = {1471-2164},
mesh = {Phylogeny ; *Genome, Bacterial ; Histamine ; Saliva ; Genomics ; Prophages ; *Microbiota ; },
abstract = {BACKGROUND: Lentilactobacillus parabuchneri is of particular concern in fermented food bioprocessing due to causing unwanted gas formation, cracks, and off-flavor in fermented dairy foods. This species is also a known culprit of histamine poisonings because of decarboxylating histidine to histamine in ripening cheese. Twenty-eight genomes in NCBI GenBank were evaluated via comparative analysis to determine genomic diversity within this species and identify potential avenues for reducing health associated risks and economic losses in the food industry caused by these organisms.<br><br>RESULT: Core genome-based phylogenetic analysis revealed four distinct major clades. Eight dairy isolates, two strains from an unknown source, and a saliva isolate formed the first clade. Three out of five strains clustered on clade 2 belonged to dairy, and the remaining two strains were isolated from the makgeolli and Korean effective microorganisms (KEM) complex. The third and fourth clade members were isolated from Tete de Moine and dairy-associated niches, respectively. Whole genome analysis on twenty-eight genomes showed&#x2009;~&#x2009;40% of all CDS were conserved across entire strains proposing a considerable diversity among L. parabuchneri strains analyzed. After assigning CDS to their corresponding function, ~&#x2009;79% of all strains were predicted to carry putative intact prophages, and ~&#x2009;43% of the strains harbored at least one plasmid; however, all the strains were predicted to encode genomic island, insertion sequence, and CRISPR-Cas system. A type I-E CRISPR-Cas subgroup was identified in all the strains, with the exception of DSM15352, which carried a type II-A CRISPR-Cas system. Twenty strains were predicted to encode histidine decarboxylase gene cluster that belongs to not only dairy but also saliva, KEM complex, and unknown source. No bacteriocin-encoding gene(s) or antibiotic resistome was found in any of the L. parabuchneri strains screened.<br><br>CONCLUSION: The findings of the present work provide in-depth knowledge of the genomics of L. parabuchneri by comparing twenty-eight genomes available to date. For example, the hdc gene cluster was generally reported in cheese isolates; however, our findings in the current work indicated that it could also be encoded in those strains isolated from saliva, KEM complex, and unknown source. We think prophages are critical mobile elements of L. parabuchneri genomes that could pave the way for developing novel tools to reduce the occurrence of this unwanted species in the food industry.},
}
@article {pmid36469612,
year = {2022},
author = {Blomme, J and Ribera, JA and Develtere, W and Jacobs, TB},
title = {A Simple and Low-Tech Heat-Shock Method to Increase Genome Editing Efficiency in Plants.},
journal = {Current protocols},
volume = {2},
number = {12},
pages = {e608},
doi = {10.1002/cpz1.608},
pmid = {36469612},
issn = {2691-1299},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants/genetics ; Mutagenesis ; Heat-Shock Response/genetics ; },
abstract = {CRISPR/Cas is now the standard technique to generate novel plant genotypes. However, optimizing the efficiency of the system continues to be an aspect of research and development. One of the improvements for increasing mutagenesis efficiency in different species is the application of heat stress. However, many experimental setups are limited by the requirement of using dedicated climate chambers to impose heat stress and by difficulties in the phenotyping of soil-grown plants. Here, we describe a simplified heat stress assay for in vitro-grown plants that can be completed in 6 days using commonly available laboratory equipment. We show that three 24-hr heat shocks (3×HS) at 37°C alternated with 24 hr of recovery at 21°C efficiently increases indel rates of LbCas12a and Cas9. We illustrate how visual mutant phenotypes (pds3 and gl1) can assist in quantifying genome editing efficiency, and describe how to quantify genome editing efficiency using genotyping by Sanger sequencing. We also provide a support protocol to efficiently clone a CRISPR expression vector in a single step. Together, our methods allow researchers to increase CRISPR-induced mutations using a low-tech setup in plants. © 2022 Wiley Periodicals LLC. Basic Protocol 1: 3×HS protocol Basic Protocol 2: Genotyping by Sanger sequencing Support Protocol: One-step cloning of a CRISPR expression vector.},
}
@article {pmid36463195,
year = {2022},
author = {Chien, Y and Hsiao, YJ and Chou, SJ and Lin, TY and Yarmishyn, AA and Lai, WY and Lee, MS and Lin, YY and Lin, TW and Hwang, DK and Lin, TC and Chiou, SH and Chen, SJ and Yang, YP},
title = {Nanoparticles-mediated CRISPR-Cas9 gene therapy in inherited retinal diseases: applications, challenges, and emerging opportunities.},
journal = {Journal of nanobiotechnology},
volume = {20},
number = {1},
pages = {511},
pmid = {36463195},
issn = {1477-3155},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Prospective Studies ; *Retinal Diseases/genetics/therapy ; Retina ; Genetic Therapy ; *Nanoparticles ; },
abstract = {Inherited Retinal Diseases (IRDs) are considered one of the leading causes of blindness worldwide. However, the majority of them still lack a safe and effective treatment due to their complexity and genetic heterogeneity. Recently, gene therapy is gaining importance as an efficient strategy to address IRDs which were previously considered incurable. The development of the clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has strongly empowered the field of gene therapy. However, successful gene modifications rely on the efficient delivery of CRISPR-Cas9 components into the complex three-dimensional (3D) architecture of the human retinal tissue. Intriguing findings in the field of nanoparticles (NPs) meet all the criteria required for CRISPR-Cas9 delivery and have made a great contribution toward its therapeutic applications. In addition, exploiting induced pluripotent stem cell (iPSC) technology and in vitro 3D retinal organoids paved the way for prospective clinical trials of the CRISPR-Cas9 system in treating IRDs. This review highlights important advances in NP-based gene therapy, the CRISPR-Cas9 system, and iPSC-derived retinal organoids with a focus on IRDs. Collectively, these studies establish a multidisciplinary approach by integrating nanomedicine and stem cell technologies and demonstrate the utility of retina organoids in developing effective therapies for IRDs.},
}
@article {pmid36463018,
year = {2022},
author = {Chen, Y and Zeng, Z and She, Q and Han, W},
title = {The abortive infection functions of CRISPR-Cas and Argonaute.},
journal = {Trends in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tim.2022.11.005},
pmid = {36463018},
issn = {1878-4380},
abstract = {CRISPR-Cas and prokaryotic Argonaute (pAgo) are nucleic acid (NA)-guided defense systems that protect prokaryotes against the invasion of mobile genetic elements. Previous studies established that they are directed by NA fragments (guides) to recognize invading complementary NA (targets), and that they cleave the targets to silence the invaders. Nevertheless, growing evidence indicates that many CRISPR-Cas and pAgo systems exploit the abortive infection (Abi) strategy to confer immunity. The CRISPR-Cas and pAgo Abi systems typically sense invaders using the NA recognition ability and activate various toxic effectors to kill the infected cells to prevent the invaders from spreading. This review summarizes the diverse mechanisms of these CRISPR-Cas and pAgo systems, and highlights their critical roles in the arms race between microbes and invaders.},
}
@article {pmid36460652,
year = {2022},
author = {Rutkauskas, M and Songailiene, I and Irmisch, P and Kemmerich, FE and Sinkunas, T and Siksnys, V and Seidel, R},
title = {A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7460},
pmid = {36460652},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Recognition, Psychology ; *Nucleic Acids ; Cognition ; Engineering ; },
abstract = {CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.},
}
@article {pmid36459983,
year = {2022},
author = {Steens, JA and van der Oost, J and Staals, RHJ},
title = {Compact but mighty: Biology and applications of type III-E CRISPR-Cas systems.},
journal = {Molecular cell},
volume = {82},
number = {23},
pages = {4405-4406},
doi = {10.1016/j.molcel.2022.11.007},
pmid = {36459983},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Endopeptidases ; Peptide Hydrolases ; RNA ; Biology ; },
abstract = {In this issue, Liu et al. present an in-depth study aiming to unravel the structural, biochemical, and physiological aspects of how type III-E CRISPR-Cas systems trigger abortive infection by activating a protease upon target RNA recognition.[1].},
}
@article {pmid36459982,
year = {2022},
author = {Mahata, T and Qimron, U},
title = {Thou shalt not cleave DNA-only repress transcription: A compact Cas protein representing a new CRISPR-Cas subtype.},
journal = {Molecular cell},
volume = {82},
number = {23},
pages = {4403-4404},
doi = {10.1016/j.molcel.2022.10.021},
pmid = {36459982},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; },
abstract = {Wu et al.[1] characterize Cas12m, a compact Cas protein that silences transcription without cleaving DNA and is a prototype protein of the novel CRISPR-Cas subtype V-M.},
}
@article {pmid36459645,
year = {2022},
author = {Chen, E and Lin-Shiao, E and Trinidad, M and Saffari Doost, M and Colognori, D and Doudna, JA},
title = {Decorating chromatin for enhanced genome editing using CRISPR-Cas9.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {49},
pages = {e2204259119},
doi = {10.1073/pnas.2204259119},
pmid = {36459645},
issn = {1091-6490},
mesh = {Humans ; *Chromatin/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Recombinational DNA Repair ; Homologous Recombination ; Histone-Lysine N-Methyltransferase ; },
abstract = {CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.},
}
@article {pmid36457126,
year = {2022},
author = {Guo, X and Steinman, RA and Sheng, Y and Cao, G and Wiley, CA and Wang, Q},
title = {An AGS-associated mutation in ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the brain.},
journal = {Journal of neuroinflammation},
volume = {19},
number = {1},
pages = {285},
pmid = {36457126},
issn = {1742-2094},
support = {I01 RX001455/RX/RRD VA/United States ; R01 AI139544/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; Catalytic Domain ; *Neurodegenerative Diseases ; Brain ; Mutation/genetics ; Disease Models, Animal ; *Brain Injuries ; RNA ; Adenosine Deaminase/genetics ; },
abstract = {BACKGROUND: Aicardi-Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous.<br><br>METHODS: Via CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms.<br><br>RESULTS: We established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5.<br><br>CONCLUSIONS: The Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.},
}
@article {pmid36457057,
year = {2022},
author = {Bhowmik, R and Chaubey, B},
title = {CRISPR/Cas9: a tool to eradicate HIV-1.},
journal = {AIDS research and therapy},
volume = {19},
number = {1},
pages = {58},
pmid = {36457057},
issn = {1742-6405},
mesh = {Humans ; Animals ; *HIV-1/genetics ; CRISPR-Cas Systems ; *HIV Infections/drug therapy ; Virus Latency ; *HIV Seropositivity ; Proviruses/genetics ; },
abstract = {The development of antiretroviral therapy (ART) has been effective in suppressing HIV replication. However, severe drug toxicities due to the therapy and its failure in targeting the integrated proviral genome have led to the introduction of a new paradigm of gene-based therapies. With its effective inhibition and high precision, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) or CRISPR/Cas9 has emerged as an effective genome editing tool in the last decade. Mediated by guide RNAs (gRNAs), Cas9 endonuclease acts like genetic scissors that can modify specific target sites. With this concept, CRISPR/Cas9 has been used to target the integrated proviral HIV-1 genome both in in vitro as well as in vivo studies including non-human primates. The CRISPR has also been tested for targeting latent HIV-1 by modulating the proviral transcription with the help of a specialized Cas9 mutant. Overcoming the limitations of the current therapy, CRISPR has the potential to become the primary genome editing tool for eradicating HIV-1 infection. In this review, we summarize the recent advancements of CRISPR to target the proviral HIV-1 genome, the challenges and future prospects.},
}
@article {pmid36456580,
year = {2022},
author = {Arias, CF and Acosta, FJ and Bertocchini, F and Herrero, MA and Fernández-Arias, C},
title = {The coordination of anti-phage immunity mechanisms in bacterial cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7412},
pmid = {36456580},
issn = {2041-1723},
mesh = {*Bacteriophages/genetics ; DNA Replication ; DNA Restriction-Modification Enzymes ; DNA, Viral ; Virus Replication ; *Bacteria/virology ; },
abstract = {Bacterial cells are equipped with a variety of immune strategies to fight bacteriophage infections. Such strategies include unspecific mechanisms directed against any phage infecting the cell, ranging from the identification and cleavage of the viral DNA by restriction nucleases (restriction-modification systems) to the suicidal death of infected host cells (abortive infection, Abi). In addition, CRISPR-Cas systems generate an immune memory that targets specific phages in case of reinfection. However, the timing and coordination of different antiviral systems in bacterial cells are poorly understood. Here, we use simple mathematical models of immune responses in individual bacterial cells to propose that the intracellular dynamics of phage infections are key to addressing these questions. Our models suggest that the rates of viral DNA replication and cleavage inside host cells define functional categories of phages that differ in their susceptibility to bacterial anti-phage mechanisms, which could give raise to alternative phage strategies to escape bacterial immunity. From this viewpoint, the combined action of diverse bacterial defenses would be necessary to reduce the chances of phage immune evasion. The decision of individual infected cells to undergo suicidal cell death or to incorporate new phage sequences into their immune memory would be determined by dynamic interactions between the host's immune mechanisms and the phage DNA. Our work highlights the importance of within-cell dynamics to understand bacterial immunity, and formulates hypotheses that may inspire future research in this area.},
}
@article {pmid36455451,
year = {2022},
author = {Capdeville, N and Schindele, P and Puchta, H},
title = {Getting better all the time - recent progress in the development of CRISPR/Cas-based tools for plant genome engineering.},
journal = {Current opinion in biotechnology},
volume = {79},
number = {},
pages = {102854},
doi = {10.1016/j.copbio.2022.102854},
pmid = {36455451},
issn = {1879-0429},
abstract = {Since their first adaptation for plant genome editing, clustered regularly interspaced short palindromic repeats/CRISPR-associated system nucleases and tools have revolutionized the field. While early approaches focused on targeted mutagenesis that relies on mutagenic repair of induced double-strand breaks, newly developed tools now enable the precise induction of predefined modifications. Constant efforts to optimize these tools have led to the generation of more efficient base editors with enlarged editing windows and have enabled previously unachievable C-G transversions. Prime editors were also optimized for the application in plants and now allow to accurately induce substitutions, insertions, and deletions. Recently, great progress was made through precise restructuring of chromosomes, which enables not only the breakage or formation of genetic linkages but also the swapping of promoters.},
}
@article {pmid36454836,
year = {2022},
author = {Leslie, M},
title = {CRISPR is so popular even viruses may use it.},
journal = {Science (New York, N.Y.)},
volume = {378},
number = {6623},
pages = {935-936},
doi = {10.1126/science.adg0519},
pmid = {36454836},
issn = {1095-9203},
mesh = {*Bacteriophages/genetics ; *Gene Editing ; *CRISPR-Cas Systems ; },
abstract = {Thousands of phages appear to have stolen the gene-cutting mechanism.},
}
@article {pmid36454475,
year = {2023},
author = {Ilahibaks, NF and Hulsbos, MJ and Lei, Z and Vader, P and Sluijter, JPG},
title = {Enabling Precision Medicine with CRISPR-Cas Genome Editing Technology: A Translational Perspective.},
journal = {Advances in experimental medicine and biology},
volume = {1396},
number = {},
pages = {315-339},
pmid = {36454475},
issn = {0065-2598},
mesh = {Humans ; *Gene Editing ; *Precision Medicine ; CRISPR-Cas Systems/genetics ; Genetic Therapy ; Endonucleases ; },
abstract = {Genome editing technologies, particularly CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleases), are redefining the boundaries of therapeutic gene therapy. CRISPR-Cas is a robust, straightforward, and programmable genome editing tool capable of mediating site-specific DNA modifications. The rapid advancements from discovery to clinical adaptation have expanded the therapeutic landscape to treat genetically defined diseases. Together with the technical developments in human DNA and RNA sequencing, CRISPR-directed gene therapy enables a new era to realize precision medicine where pathogenic mutations underlying monogenic disorders can potentially be corrected. Also, protective or therapeutic genomic alterations can be introduced as preventative or curative therapy. Despite its high therapeutic potential, CRISPR-Cas´ clinical translation is still in its infancy and is highly dependent on its efficiency, specificity in gene corrections, and cell-specific delivery. Therefore, this chapter focuses on the challenges and opportunities the CRISPR-Cas toolbox offers together with delivery vehicles to realize its use for therapeutic gene editing. Furthermore, we discuss the obstacles the CRISPR-Cas system faces for successful clinical translation and summarize its current clinical progress.},
}
@article {pmid36454473,
year = {2023},
author = {Giallongo, S and Lo Re, O and Resnick, I and Raffaele, M and Vinciguerra, M},
title = {Gene Editing and Human iPSCs in Cardiovascular and Metabolic Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1396},
number = {},
pages = {275-298},
pmid = {36454473},
issn = {0065-2598},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; Gene Editing ; *Diabetes Mellitus, Type 2/genetics/therapy ; *Cardiovascular System ; *Metabolic Diseases/genetics/therapy ; },
abstract = {The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.},
}
@article {pmid36454471,
year = {2023},
author = {Lu, D and Cushman, S and Thum, T and Bär, C},
title = {Gene Therapy and Cardiovascular Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1396},
number = {},
pages = {235-254},
pmid = {36454471},
issn = {0065-2598},
mesh = {Humans ; *Cardiovascular Diseases/genetics/therapy ; Genetic Therapy ; Gene Editing ; Cloning, Molecular ; Erythrocyte Count ; },
abstract = {Cardiovascular diseases (CVDs) are the leading causes of death globally and urgently require new novel therapeutic strategies. Gene therapy is the application of gene modulation technology to treat abnormal gene expression under disease conditions. Viral- and nonviral-based gene delivery systems are the foundation of gene modulation in target cells. Moreover, plasmid- or oligo-based gene modulation tools as well as new advancements in gene editing using CRISPR/Cas technology are currently being tested in a variety of clinical trials. Here, we summarized state-of-the-art gene therapy technologies as well as recent clinical trials and discuss the applications and lessons of gene therapy in CVDs.},
}
@article {pmid36454465,
year = {2023},
author = {Chen, Z and Lehertshuber, C and Schunkert, H},
title = {Genome Editing in Dyslipidemia and Atherosclerosis.},
journal = {Advances in experimental medicine and biology},
volume = {1396},
number = {},
pages = {139-156},
pmid = {36454465},
issn = {0065-2598},
mesh = {Humans ; Gene Editing ; *Dyslipidemias/genetics/therapy ; *Atherosclerosis/genetics/therapy ; Transcription Activator-Like Effector Nucleases ; *Cardiovascular Diseases ; },
abstract = {Despite successive advancement of genome editing technology with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the recent breakthrough in the field has been related to clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas). The high efficiency and convenience of CRIPSR/Cas systems dramatically accelerate pre- and clinical experimentations of dyslipidemia and atherosclerosis. In this chapter, we review the latest state of genome editing in translational research of dyslipidemia and atherosclerosis. We highlight recent progress in therapeutic development for familial dyslipidemia by genome editing. We point to the challenges in maximizing efficacy and minimizing safety issues related to the once-and-done therapy focusing on CRISPR/Cas systems. We give an outlook on the potential gene targets prioritized by large-scale genetic studies of cardiovascular diseases and genome editing in precision medicine of dyslipidemia and atherosclerosis.},
}
@article {pmid36454461,
year = {2023},
author = {Kato, T},
title = {Genome Editing and Pathological Cardiac Hypertrophy.},
journal = {Advances in experimental medicine and biology},
volume = {1396},
number = {},
pages = {87-101},
pmid = {36454461},
issn = {0065-2598},
mesh = {Humans ; *Gene Editing ; Transcription Activator-Like Effector Nucleases/genetics ; *Cardiomyopathy, Hypertrophic ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cardiomegaly/genetics/therapy ; },
abstract = {Three major genome editing tools, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeat (CRISPR) systems, are increasingly important technologies used in the study and treatment of hereditary myocardial diseases. Germ cell genome editing and modification can permanently eliminate monogenic cardiovascular disease from the offspring of affected families and the next generation, although ethically controversial. Somatic genome editing may be a promising method for the treatment of hereditary cardiomyopathy various diseases for which gene knockout is favorable and can also treat people who are already ill, although there are currently some technical challenges. This chapter describes the application of genome editing in the experimental studies and treatment of hypertrophic cardiomyopathy as well as other cardiomyopathies.},
}
@article {pmid36454457,
year = {2023},
author = {Carrillo-Rodriguez, P and Martinez-Lopez, J and Gómez-Hernández, L and Boraita-Morales, L and Uceda-Molina, M and Cuenca-Bermejo, I and Valencia-Aguirre, JP and Mora-López, M and Plaza-Diaz, J},
title = {Online Databases of Genome Editing in Cardiovascular and Metabolic Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1396},
number = {},
pages = {19-33},
pmid = {36454457},
issn = {0065-2598},
mesh = {Humans ; Gene Editing ; *Cardiovascular System ; *Metabolic Diseases/genetics/therapy ; CRISPR-Cas Systems/genetics ; Databases, Factual ; },
abstract = {Metabolic and cardiovascular diseases are world-concerning pathologies that affect an important percentage of the population. Nowadays, advances in the genetic background of these diseases allow new approaches to models and therapies, as well as different gene edition trials. Furthermore, technological improvements in gene editing go along with the development of new online and biocomputational tools that provide us alternative ways to explore pathologies. In this chapter, historical gene editing methods are discussed but focusing on CRISPR-Cas system in detail and also online resources available to perform these types of experiments. Here, the different strategies for gene editing and their online tools are gathered, putting the light on its application in the study and treatment of cardiovascular and metabolic diseases.},
}
@article {pmid36453982,
year = {2022},
author = {Sun, YJ and Chen, WD and Liu, J and Li, JJ and Zhang, Y and Cai, WQ and Liu, L and Tang, XJ and Hou, J and Wang, M and Cheng, L},
title = {A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {},
doi = {10.1002/anie.202212413},
pmid = {36453982},
issn = {1521-3773},
abstract = {The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report a spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformational restricted gRNAs. This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs which often required extensive screening and time-consuming optimization. The tactic could direct precise cleavage of GFP and VEGFA within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated MSTN gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significantly new strategy in CRISPR/Cas editing with modified circular guide RNAs to precisely manipulate where and when genes are edited.},
}
@article {pmid36452329,
year = {2022},
author = {Forte-Gomez, HF and Gioia, R and Tonelli, F and Kobbe, B and Koch, P and Bloch, W and Paulsson, M and Zaucke, F and Forlino, A and Wagener, R},
title = {Structure, evolution and expression of zebrafish cartilage oligomeric matrix protein (COMP, TSP5). CRISPR-Cas mutants show a dominant phenotype in myosepta.},
journal = {Frontiers in endocrinology},
volume = {13},
number = {},
pages = {1000662},
pmid = {36452329},
issn = {1664-2392},
mesh = {Adult ; Humans ; Mice ; Animals ; Cartilage Oligomeric Matrix Protein/genetics ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Phenotype ; Thrombospondins/genetics ; Mammals ; },
abstract = {COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish comp is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.},
}
@article {pmid36449402,
year = {2022},
author = {Yao, Y and Cao, J and Wang, W and Liu, B and Pei, X and Zhang, L and Rao, S},
title = {Highly Efficient One-Step Tagging of Endogenous Genes in Primary Cells Using CRISPR-Cas Ribonucleoproteins.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0046},
pmid = {36449402},
issn = {2573-1602},
abstract = {Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein complexes and chemically modified donor DNA into cells. Upon CRISPR-Cas9 blunt-end double-strand breaks, highly efficient site-specific insertion of genetic materials (3 × FLAG or eGFP) was achieved in both cell lines and primary cells. We further optimized the gene-tagging efficiency and precision by using CRISPR-Cas12a, which produces a staggered cut with a 5' overhang and thus enables precise ligation of DNA donors with a complementary 3' overhang. With high efficiency and flexibility, this platform would be extremely useful for multiplex endogenous genes tagging and further exploration of protein functions in various cell types.},
}
@article {pmid36449171,
year = {2023},
author = {Simpson, DM and Chuong, EB},
title = {Genetic Knockout of TE Insertions by CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2607},
number = {},
pages = {369-379},
pmid = {36449171},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Gene Editing ; Cell Line ; DNA Transposable Elements/genetics ; Mammals ; },
abstract = {Transposable elements (TEs) are abundant in the genome, and specific insertions may be co-opted to act as coding or noncoding functional elements. CRISPR-based genome editing technologies enable functional studies of TE insertions in cell lines. Here, we describe the use of CRISPR-Cas9 to create and validate genetic knockouts of TEs in mammalian cell lines.},
}
@article {pmid36449169,
year = {2023},
author = {Weber, VM and Doucet, AJ and Cristofari, G},
title = {Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2607},
number = {},
pages = {329-353},
pmid = {36449169},
issn = {1940-6029},
mesh = {Humans ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems/genetics ; Cell Line ; Cicatrix ; Genes, Reporter ; },
abstract = {Transposable element insertions can have broad effects on gene expression, ranging from new regulatory functions to pathogenic consequences by transplanting new cis-regulating elements or perturbing existing ones. Genetic manipulation of such DNA sequences can help decipher their mechanism of action. Here, we describe a CRISPR-Cas9-mediated two-step approach to precisely insert transposable elements into into the genome of cultured human cells, without scar or reporter gene. First, a double-selection cassette is inserted into the desired target locus. Once a clone containing a single copy of this cassette has been isolated, a second editing step is performed to exchange the double-selection cassette with a markerless transposable element sequence. More generally, this method can be used for knocking in any large insert without genetic markers.},
}
@article {pmid36445095,
year = {2022},
author = {Yip, CC and Sridhar, S and Chan, WM and Ip, JD and Chu, AW and Leung, KH and Cheng, VC and Yuen, KY and To, KK},
title = {Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0196222},
doi = {10.1128/spectrum.01962-22},
pmid = {36445095},
issn = {2165-0497},
abstract = {Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings. IMPORTANCE Accurate tests for the diagnosis of SARS-CoV-2, the virus causing coronavirus disease 2019 (COVID-19), are important for timely treatment and infection control decisions. Conventional tests such as real-time reverse transcription-PCR (RT-PCR) require specialized equipment and are expensive. On the other hand, rapid antigen tests suffer from a lack of sensitivity. In this study, we describe a novel assay format for the diagnosis of COVID-19 that is based on principles of loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas chemistry. A major advantage of this assay format is that it does not require expensive equipment to perform, and results can be read visually. This method proved to be fast, easy to perform, and inexpensive. The test compared well against an RT-PCR assay in terms of the ability to detect SARS-CoV-2 RNA in clinical samples. No false-positive test results were observed. The new assay format is ideal for SARS-CoV-2 diagnosis in resource-limited settings.},
}
@article {pmid36443399,
year = {2022},
author = {McGrail, M and Sakuma, T and Bleris, L},
title = {Genome editing.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {20497},
pmid = {36443399},
issn = {2045-2322},
mesh = {*Gene Editing ; },
abstract = {Recent advances in genome editing technologies have redefined our ability to probe and precisely edit the human genome and epigenome in vitro and in vivo. More specifically, RNA-guided CRISPR/Cas systems have revolutionized the field due to their simplicity in design and adaptability across biological systems. This Collection highlights results in CRISPR/Cas technology that increase the efficiency of precision genome editing, and allow genetic manipulation in model systems traditionally intractable to site-directed gene modification.},
}
@article {pmid36443301,
year = {2022},
author = {Huang, Z and You, X and Chen, L and Du, Y and Brodeur, K and Jee, H and Wang, Q and Linder, G and Darbousset, R and Cunin, P and Chang, MH and Wactor, A and Wauford, BM and Todd, MJC and Wei, K and Li, Y and Levescot, A and Iwakura, Y and Pascual, V and Baldwin, NE and Quartier, P and Li, T and Gianatasio, MT and Hasserjian, RP and Henderson, LA and Sykes, DB and Mellins, ED and Canna, SW and Charles, JF and Nigrovic, PA and Lee, PY},
title = {mTORC1 links pathology in experimental models of Still's disease and macrophage activation syndrome.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6915},
pmid = {36443301},
issn = {2041-1723},
mesh = {Adult ; Child ; Humans ; Mice ; Animals ; *Macrophage Activation Syndrome/genetics ; Mechanistic Target of Rapamycin Complex 1/genetics ; *Arthritis, Juvenile ; *Lymphohistiocytosis, Hemophagocytic/genetics ; Models, Theoretical ; },
abstract = {Still's disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still's disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still's disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still's disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still's disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still's disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still's disease and macrophage activation syndrome.},
}
@article {pmid36442853,
year = {2022},
author = {Hu, T and Ke, X and Li, W and Lin, Y and Liang, A and Ou, Y and Chen, C},
title = {CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2204689},
doi = {10.1002/advs.202204689},
pmid = {36442853},
issn = {2198-3844},
abstract = {Most multiplex nucleic acids detection methods require numerous reagents and high-priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point-of-care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR-RDB (CRISPR-based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost-effectiveness, instrument-free, ease to use, and visualization is reported. CRISPR-RDB integrates the trans-cleavage activity of CRISPR-Cas12a with a commercial RDB technique. It utilizes different Cas12a-crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific-oligonucleotide probes. It has demonstrated that the versatility of CRISPR-RDB by constructing a four-channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2. With a simple modification of crRNAs, the CRISPR-RDB can be modified to detect human papillomavirus, saving two-thirds of the time compared to a commercial PCR-RDB kit. Further, a user-friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR-RDB represents a desirable option for multiplexed biosensing and on-site diagnosis.},
}
@article {pmid36442697,
year = {2022},
author = {Patra, P and B R, D and Kundu, P and Das, M and Ghosh, A},
title = {Recent advances in machine learning applications in metabolic engineering.},
journal = {Biotechnology advances},
volume = {62},
number = {},
pages = {108069},
doi = {10.1016/j.biotechadv.2022.108069},
pmid = {36442697},
issn = {1873-1899},
abstract = {Metabolic engineering encompasses several widely-used strategies, which currently hold a high seat in the field of biotechnology when its potential is manifesting through a plethora of research and commercial products with a strong societal impact. The genomic revolution that occurred almost three decades ago has initiated the generation of large omics-datasets which has helped in gaining a better understanding of cellular behavior. The itinerary of metabolic engineering that has occurred based on these large datasets has allowed researchers to gain detailed insights and a reasonable understanding of the intricacies of biosystems. However, the existing trail-and-error approaches for metabolic engineering are laborious and time-intensive when it comes to the production of target compounds with high yields through genetic manipulations in host organisms. Machine learning (ML) coupled with the available metabolic engineering test instances and omics data brings a comprehensive and multidisciplinary approach that enables scientists to evaluate various parameters for effective strain design. This vast amount of biological data should be standardized through knowledge engineering to train different ML models for providing accurate predictions in gene circuits designing, modification of proteins, optimization of bioprocess parameters for scaling up, and screening of hyper-producing robust cell factories. This review briefs on the premise of ML, followed by mentioning various ML methods and algorithms alongside the numerous omics datasets available to train ML models for predicting metabolic outcomes with high-accuracy. The combinative interplay between the ML algorithms and biological datasets through knowledge engineering have guided the recent advancements in applications such as CRISPR/Cas systems, gene circuits, protein engineering, metabolic pathway reconstruction, and bioprocess engineering. Finally, this review addresses the probable challenges of applying ML in metabolic engineering which will guide the researchers toward novel techniques to overcome the limitations.},
}
@article {pmid36441794,
year = {2022},
author = {Takacs, CN and Nakajima, Y and Haber, JE and Jacobs-Wagner, C},
title = {Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.},
journal = {PloS one},
volume = {17},
number = {11},
pages = {e0278151},
doi = {10.1371/journal.pone.0278151},
pmid = {36441794},
issn = {1932-6203},
mesh = {Humans ; *Borrelia burgdorferi/genetics ; CRISPR-Cas Systems/genetics ; *Lyme Disease ; Plasmids/genetics ; *Borrelia ; },
abstract = {The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.},
}
@article {pmid36441701,
year = {2022},
author = {Xie, Y and Wang, M and Gu, L and Wang, Y},
title = {CRISPR/Cas9-mediated knock-in strategy at the Rosa26 locus in cattle fetal fibroblasts.},
journal = {PloS one},
volume = {17},
number = {11},
pages = {e0276811},
doi = {10.1371/journal.pone.0276811},
pmid = {36441701},
issn = {1932-6203},
mesh = {Cattle/genetics ; Animals ; Humans ; Female ; Pregnancy ; *CRISPR-Cas Systems ; *Fetus ; Prenatal Care ; Fibroblasts ; Research ; },
abstract = {The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often leads to the unstable or differentially expression of the exogenous genes, which limit the application and development of transgenic technologies. Finding a safe locus suitable for site-specific insertion and efficient expression of exogenous genes is a good way to overcome these hurdles. In this study, we efficiently integrated three targeted vector into the cattle Rosa26 (cRosa26) by CRISPR/Cas9 technology in which EGFP was driven by CAG, EF1a, PGK and cRosa26 endogenous promoter respectively. The CRISPR/Cas9 knock-in system allows highly efficient gene insertion of different expression units at the cRosa26 locus. We also find that in the four cell lines, EGFP was stable expressed at different times, and the CAG promoter has the highest activity to activate the expression of EGFP, when compared with the cRosa26, EF1a and PGK promoter. Our results proved that cRosa26 was a locus that could integrate different expression units efficiently, and supported the friendly expression of different expression units. Our findings described here will be useful for a variety of studies using cattle.},
}
@article {pmid36439395,
year = {2022},
author = {Bhoobalan-Chitty, Y and Duan, X and Peng, X},
title = {High-MOI induces rapid CRISPR spacer acquisition in Sulfolobus from an acr deficient virus.},
journal = {microPublication biology},
volume = {2022},
number = {},
pages = {},
pmid = {36439395},
issn = {2578-9430},
abstract = {Spacer acquisition, the first step in CRISPR-Cas adaptive immunity, plays a critical role in establishing and strengthening host defense against mobile genetic elements (MGEs). Here we present a host-virus system, where an increase in the multiplicity of infection (MOI), of a CRISPR-Cas susceptible virus, forces rapid spacer acquisition in the Sulfolobus islandicus LAL14/1 CRISPR arrays. Spacer acquisition was observed as early as 30 minutes post infection, with the newly acquired spacers uniformly distributed across the genome of the virus. Although the newly acquired spacers were predominantly effective only against the CRISPR-Cas susceptible mutant virus, we were able to isolate a host mutant with a novel spacer which provides immunity against the multiple Acr encoding wildtype virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2).},
}
@article {pmid36439054,
year = {2023},
author = {Liu, S and Xie, T and Pei, X and Li, S and He, Y and Tong, Y and Liu, G},
title = {CRISPR-Cas12a coupled with universal gold nanoparticle strand-displacement probe for rapid and sensitive visual SARS-CoV-2 detection.},
journal = {Sensors and actuators. B, Chemical},
volume = {377},
number = {},
pages = {133009},
pmid = {36439054},
issn = {0925-4005},
abstract = {Point of care (POC) diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are particularly significant for preventing transmission of coronavirus disease 2019 (COVID-19) by any user at any given time and place. CRISPR/Cas-assisted SARS-CoV-2 assays are viewed as supplemental to RT-PCR due to simple operation, convenient use and low cost. However, most current CRISPR molecular diagnostics based on fluorescence measurement increased the difficulty of POC test with need of the additional light sources. Some instrument-free visual detection with the naked eye has limitations in probe universality. Herein, we developed a universal, rapid, sensitive and specific SARS-CoV-2 POC test that combines the outstanding DNase activity of Cas12a with universal AuNPs strand-displacement probe. The oligo trigger, which is the switch the AuNPs of the strand-displacement probe, is declined as a result of Cas12a recognition and digestion. The amount of released AuNPs produced color change which can be visual with the naked eye and assessed by UV-Vis spectrometer for quantitative detection. Furthermore, a low-cost hand warmer is used as an incubator for the visual assay, enabling an instrument-free, visual SARS-CoV-2 detection within 20 min. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. After rapid virus RNA extraction and RT-PCR amplification, a minimum of 2.7 × 10[2] copies/mL was obtained successfully. The modular design can be applied to many nucleic acid detection applications, such as viruses, bacteria, species, etc., by simply modifying the crRNA, showing great potential in POC diagnosis.},
}
@article {pmid36438568,
year = {2022},
author = {Park, CH and Jeoung, YH and Zhang, L and Yeddula, SGR and Park, KE and Waters, J and Telugu, BP},
title = {Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification.},
journal = {Frontiers in cell and developmental biology},
volume = {10},
number = {},
pages = {1059710},
pmid = {36438568},
issn = {2296-634X},
abstract = {Fibroblasts are the common cell type in the connective tissue-the most abundant tissue type in the body. Fibroblasts are widely used for cell culture, for the generation of induced pluripotent stem cells (iPSCs), and as nuclear donors for somatic cell nuclear transfer (SCNT). We report for the first time, the derivation of embryonic fibroblasts (EFs) from porcine embryonic outgrowths, which share similarities in morphology, culture characteristics, molecular markers, and transcriptional profile to fetal fibroblasts (FFs). We demonstrated the efficient use of EFs as nuclear donors in SCNT, for enhanced post-blastocyst development, implantation, and pregnancy outcomes. We further validated EFs as a source for CRISPR/Cas genome editing with overall editing frequencies comparable to that of FFs. Taken together, we established an alternative and efficient pipeline for genome editing and for the generation of genetically engineered animals.},
}
@article {pmid36437912,
year = {2022},
author = {Cobos, M and Condori, RC and Grandez, MA and Estela, SL and Del Aguila, MT and Castro, CG and Rodríguez, HN and Vargas, JA and Tresierra, AB and Barriga, LA and Marapara, JL and Adrianzén, PM and Ruiz, R and Castro, JC},
title = {Genomic analysis and biochemical profiling of an unaxenic strain of Synechococcus sp. isolated from the Peruvian Amazon Basin region.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {973324},
pmid = {36437912},
issn = {1664-8021},
abstract = {Cyanobacteria are diverse photosynthetic microorganisms able to produce a myriad of bioactive chemicals. To make possible the rational exploitation of these microorganisms, it is fundamental to know their metabolic capabilities and to have genomic resources. In this context, the main objective of this research was to determine the genome features and the biochemical profile of Synechococcus sp. UCP002. The cyanobacterium was isolated from the Peruvian Amazon Basin region and cultured in BG-11 medium. Growth parameters, genome features, and the biochemical profile of the cyanobacterium were determined using standardized methods. Synechococcus sp. UCP002 had a specific growth rate of 0.086 ± 0.008 μ and a doubling time of 8.08 ± 0.78 h. The complete genome of Synechococcus sp. UCP002 had a size of ∼3.53 Mb with a high coverage (∼200x), and its quality parameters were acceptable (completeness = 99.29%, complete and single-copy genes = 97.5%, and contamination = 0.35%). Additionally, the cyanobacterium had six plasmids ranging from 24 to 200 kbp. The annotated genome revealed ∼3,422 genes, ∼ 3,374 protein-coding genes (with ∼41.31% hypothetical protein-coding genes), two CRISPR Cas systems, and 61 non-coding RNAs. Both the genome and plasmids had the genes for prokaryotic defense systems. Additionally, the genome had genes coding the transcription factors of the metalloregulator ArsR/SmtB family, involved in sensing heavy metal pollution. The biochemical profile showed primary nutrients, essential amino acids, some essential fatty acids, pigments (e.g., all-trans-β-carotene, chlorophyll a, and phycocyanin), and phenolic compounds. In conclusion, Synechococcus sp. UCP002 shows biotechnological potential to produce human and animal nutrients and raw materials for biofuels and could be a new source of genes for synthetic biological applications.},
}
@article {pmid36437283,
year = {2022},
author = {Yoshida, M and Saito, T and Takayanagi, Y and Totsuka, Y and Onaka, T},
title = {Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {20390},
pmid = {36437283},
issn = {2045-2322},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; *Genomics ; Base Sequence ; Mutation ; Alleles ; },
abstract = {The CRISPR-Cas9 method for generation of knock-in mutations in rodent embryos yields many F0 generation candidates that may have the designed mutations. The first task for selection of promising F0 generations is to analyze genomic DNA which likely contains a mixture of designed and unexpected mutations. In our study, while generating Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopies at a target allele locus, and mosaic genotypes for two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism nor discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In the F1 and F2 generations, droplet digital PCR assisted in establishing the strain, although a multicopy was falsely detected as one copy by analysis of the F0 generation. Thus, the combination of these methods allowed us to select promising F0 generations and facilitated establishment of the designed strain. We emphasize that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.},
}
@article {pmid36436108,
year = {2022},
author = {Ianevski, A and Ahmad, S and Anunnitipat, K and Oksenych, V and Zusinaite, E and Tenson, T and Bjørås, M and Kainov, DE},
title = {Seven classes of antiviral agents.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {12},
pages = {605},
pmid = {36436108},
issn = {1420-9071},
mesh = {Humans ; *Antiviral Agents/pharmacology/therapeutic use/chemistry ; Interferons ; *Virus Diseases/drug therapy ; },
abstract = {The viral epidemics and pandemics have stimulated the development of known and the discovery of novel antiviral agents. About a hundred mono- and combination antiviral drugs have been already approved, whereas thousands are in development. Here, we briefly reviewed 7 classes of antiviral agents: neutralizing antibodies, neutralizing recombinant soluble human receptors, antiviral CRISPR/Cas systems, interferons, antiviral peptides, antiviral nucleic acid polymers, and antiviral small molecules. Interferons and some small molecules alone or in combinations possess broad-spectrum antiviral activity, which could be beneficial for treatment of emerging and re-emerging viral infections.},
}
@article {pmid36435853,
year = {2022},
author = {Liu, Z and Chen, S and Xie, W and Yu, H and Lai, L and Li, Z},
title = {Versatile and efficient genome editing with Neisseria cinerea Cas9.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {1296},
pmid = {36435853},
issn = {2399-3642},
mesh = {Mice ; Humans ; Animals ; *Gene Editing ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics/metabolism ; *Neisseria cinerea/genetics/metabolism ; Genome ; Mammals/genetics ; },
abstract = {The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.},
}
@article {pmid36435506,
year = {2022},
author = {Mingarro, G and Del Olmo, ML},
title = {Improvements in the genetic editing technologies: CRISPR-Cas and beyond.},
journal = {Gene},
volume = {852},
number = {},
pages = {147064},
doi = {10.1016/j.gene.2022.147064},
pmid = {36435506},
issn = {1879-0038},
abstract = {Gene editing is a great hope not only for the scientific community, but also for society in general. This is due to its potential therapeutic applications that would allow curing diseases of genetic origin. The first realistic approach to achieve this goal was the development of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) tools. This review deals with some of the improvements that have been designed to obtain more efficient and safer genome editing. Initial CRISPR-Cas (CRISPR associated) editing systems yield low efficiency and undesired editing products. To solve these problems, new approaches emerged, such as the creation of base editors. Recent discoveries have led to the development of many interesting alternatives, such as the CRISPR-associated transposable systems, which open the range by generating guided insertions, or the discovery of other programmable nucleases like the IscB family, which greatly increase the range of proteins available for editing uses. Also, to address the limitations of base editors, prime editors were created; this novel system, despite having some disadvantages compared to base editor systems, has the potential to generate all the possible point mutations. On the other hand, dual prime editing systems (like twin and homologous 3' extension-mediated prime editors) have been developed to create targeted insertions and enhance the editing outcomes, respectively. Furthermore, advances in gene editing do not reside solely in CRISPR-dependent systems, as we will discuss when treating the Replication Interrupted Template-Driven DNA Modification technique.},
}
@article {pmid36435179,
year = {2022},
author = {Schmitz, M and Querques, I and Oberli, S and Chanez, C and Jinek, M},
title = {Structural basis for the assembly of the type V CRISPR-associated transposon complex.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2022.11.009},
pmid = {36435179},
issn = {1097-4172},
abstract = {CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.},
}
@article {pmid36434686,
year = {2022},
author = {Zhuo, C and Ruan, Q and Zhao, X and Shen, Y and Lin, R},
title = {CXCL1 promotes colon cancer progression through activation of NF-κB/P300 signaling pathway.},
journal = {Biology direct},
volume = {17},
number = {1},
pages = {34},
pmid = {36434686},
issn = {1745-6150},
mesh = {Humans ; NF-kappa B/metabolism/pharmacology ; Signal Transduction ; *Colonic Neoplasms/genetics ; *Colorectal Neoplasms/metabolism ; Chemokine CXCL1/genetics/metabolism/pharmacology ; },
abstract = {BACKGROUND: The upregulated expression of CXCL1 has been validated in colorectal cancer patients. As a potential biotherapeutic target for colorectal cancer, the mechanism by which CXCL1 affects the development of colorectal cancer is not clear.<br><br>METHODS: Expression data of CXCL1 in colorectal cancer were obtained from the GEO database and verified using the GEPIA database and the TIMER 2.0 database. Knockout and overexpression of CXCL1 in colorectal cancer cells by CRISPR/Cas and "Sleeping Beauty" transposon-mediated gene editing techniques. Cell biological function was demonstrated by CCK-8, transwell chamber and Colony formation assay. RT-qPCR and Western Blot assays measured RNA and protein expression. Protein localization and expression were measured by immunohistochemistry and immunofluorescence.<br><br>RESULTS: Bioinformatics analysis showed significant overexpression of CXCL1 in the colorectal cancer tissues compared to normal human tissues, and identified CXCL1 as a potential therapeutic target for colorectal cancer. We demonstrate that CXCL1 promotes the proliferation and migration of colon cancer cells and has a facilitative effect on tumor angiogenesis. Furthermore, CXCL1 elevation promoted the migration of M2-tumor associated macrophages (TAMs) while disrupting the aggregation of CD4+ and CD8+ T cells at tumor sites. Mechanistic studies suggested that CXCL1 activates the NF-&#x3ba;B pathway. In the in vivo colon cancer transplantation tumor model, treatment with the P300 inhibitor C646 significantly inhibited the growth of CXCL1-overexpressing colon cancer.<br><br>CONCLUSION: CXCL1 promotes colon cancer development through activation of NF-&#x3ba;B/P300, and that CXCL1-based therapy is a potential novel strategy to prevent colon cancer development.},
}
@article {pmid36434031,
year = {2022},
author = {Pappadà, M and Bonuccelli, O and Buratto, M and Fontana, R and Sicurella, M and Caproni, A and Fuselli, S and Benazzo, A and Bertorelli, R and De Sanctis, V and Cavallerio, P and Simioni, V and Tugnoli, V and Salvatori, F and Marconi, P},
title = {Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {20285},
pmid = {36434031},
issn = {2045-2322},
mesh = {Animals ; *Spinocerebellar Ataxias/genetics/therapy/metabolism ; Gain of Function Mutation ; CRISPR-Cas Systems ; Ataxin-1/genetics/metabolism ; Italy ; },
abstract = {SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.},
}
@article {pmid36434000,
year = {2022},
author = {He, X and Zhang, Z and Xue, J and Wang, Y and Zhang, S and Wei, J and Zhang, C and Wang, J and Urip, BA and Ngan, CC and Sun, J and Li, Y and Lu, Z and Zhao, H and Pei, D and Li, CK and Feng, B},
title = {Low-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7275},
pmid = {36434000},
issn = {2041-1723},
mesh = {Mice ; Animals ; Humans ; *Hemophilia B/genetics/therapy ; Gene Editing ; CRISPR-Cas Systems/genetics ; Antibody Formation ; Genetic Vectors/genetics ; Hemostasis ; Liver ; },
abstract = {AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose (2 × 10[9] vg/neonate and 1 × 10[10] vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9[R338L] variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.},
}
@article {pmid36431249,
year = {2022},
author = {Fu, Q and Hu, L and Shen, T and Yang, R and Jiang, L},
title = {Recent Advances in Gene Therapy for Familial Hypercholesterolemia: An Update Review.},
journal = {Journal of clinical medicine},
volume = {11},
number = {22},
pages = {},
doi = {10.3390/jcm11226773},
pmid = {36431249},
issn = {2077-0383},
abstract = {(1) Background: Existing lipid-lowering therapies have difficulty in achieving lipid target levels in patients with familial hypercholesterolemia (FH), especially in the treatment of patients with homozygous familial hypercholesterolemia. (2) Method: All of the literature data containing "Familial hypercholesterolemia" and "Gene Therapy" in PubMed and Clinical Trials from 2018 to 2022 were selected. (3) Results: The rapid development of gene therapy technology in recent years is expected to change the treatment status of FH patients. As emerging gene therapy vectors, the optimized adeno-associated viruses, exosomes, and lipid nanoparticles have demonstrated an improved safety and higher transfection efficiency. Various RNA-targeted therapies are in phase 1-3 clinical trials, such as small interfering RNA-based drugs inclisiran, ARO-ANG3, ARO-APOC3, olpasiran, SLN360, and antisense oligonucleotide-based drugs AZD8233, vupanorsen, volanesorsen, IONIS-APO(a)Rx, etc., all of which have demonstrated excellent lipid-lowering effects. With gene editing technologies, such as CRISPR-Cas 9 and meganuclease, completing animal experiments in mice or cynomolgus monkeys and demonstrating lasting lipid-lowering effects, patients with FH are expected to reach a permanent cure in the future. (4) Conclusion: Gene therapy is being widely used for the lipid-lowering treatment of FH patients and has shown excellent therapeutic promise, but the current delivery efficiency, economic burden, immunogenicity and the precision of gene therapy can be further optimized.},
}
@article {pmid36430971,
year = {2022},
author = {Kandasamy, S and Yoo, J and Yun, J and Lee, KH and Kang, HB and Kim, JE and Oh, MH and Ham, JS},
title = {Probiogenomic In-Silico Analysis and Safety Assessment of Lactiplantibacillus plantarum DJF10 Strain Isolated from Korean Raw Milk.},
journal = {International journal of molecular sciences},
volume = {23},
number = {22},
pages = {},
doi = {10.3390/ijms232214494},
pmid = {36430971},
issn = {1422-0067},
mesh = {Animals ; *Lactobacillus plantarum/metabolism ; Genome, Bacterial ; Phylogeny ; Milk ; *Bacteriocins/metabolism ; Anti-Bacterial Agents/metabolism ; Republic of Korea ; },
abstract = {The whole genome sequence of Lactiplantibacillus plantarum DJF10, isolated from Korean raw milk, is reported, along with its genomic analysis of probiotics and safety features. The genome consists of 29 contigs with a total length of 3,385,113 bp and a GC content of 44.3%. The average nucleotide identity and whole genome phylogenetic analysis showed the strain belongs to Lactiplantibacillus plantarum with 99% identity. Genome annotation using Prokka predicted a total of 3235 genes, including 3168 protein-coding sequences (CDS), 59 tRNAs, 7 rRNAs and 1 tmRNA. The functional annotation results by EggNOG and KEGG showed a high number of genes associated with genetic information and processing, transport and metabolism, suggesting the strain's ability to adapt to several environments. Various genes conferring probiotic characteristics, including genes related to stress adaptation to the gastrointestinal tract, biosynthesis of vitamins, cell adhesion and production of bacteriocins, were identified. The CAZyme analysis detected 98 genes distributed under five CAZymes classes. In addition, several genes encoding carbohydrate transport and metabolism were identified. The genome also revealed the presence of insertion sequences, genomic islands, phage regions, CRISPR-cas regions, and the absence of virulence and toxin genes. However, the presence of hemolysin and antibiotic-resistance-related genes detected in the KEGG search needs further experimental validation to confirm the safety of the strain. The presence of two bacteriocin clusters, sactipeptide and plantaricin J, as detected by the BAGEL 4 webserver, confer the higher antimicrobial potential of DJF10. Altogether, the analyses in this study performed highlight this strain's functional characteristics. However, further in vitro and in vivo studies are required on the safety assurance and potential application of L. plantarum DJF10 as a probiotic agent.},
}
@article {pmid36430848,
year = {2022},
author = {Yu, H and Wang, Y and Fu, F and Li, W},
title = {Transgenic Improvement for Biotic Resistance of Crops.},
journal = {International journal of molecular sciences},
volume = {23},
number = {22},
pages = {},
doi = {10.3390/ijms232214370},
pmid = {36430848},
issn = {1422-0067},
mesh = {Animals ; Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; RNA Interference ; *Nematoda/genetics ; },
abstract = {Biotic constraints, including pathogenic fungi, viruses and bacteria, herbivory insects, as well as parasitic nematodes, cause significant yield loss and quality deterioration of crops. The effect of conventional management of these biotic constraints is limited. The advances in transgenic technologies provide a direct and directional approach to improve crops for biotic resistance. More than a hundred transgenic events and hundreds of cultivars resistant to herbivory insects, pathogenic viruses, and fungi have been developed by the heterologous expression of exogenous genes and RNAi, authorized for cultivation and market, and resulted in a significant reduction in yield loss and quality deterioration. However, the exploration of transgenic improvement for resistance to bacteria and nematodes by overexpression of endogenous genes and RNAi remains at the testing stage. Recent advances in RNAi and CRISPR/Cas technologies open up possibilities to improve the resistance of crops to pathogenic bacteria and plant parasitic nematodes, as well as other biotic constraints.},
}
@article {pmid36430696,
year = {2022},
author = {Wang, Q and Bai, H and Zada, A and Jiao, Q},
title = {DORN1 Is Involved in Drought Stress Tolerance through a Ca[2+]-Dependent Pathway.},
journal = {International journal of molecular sciences},
volume = {23},
number = {22},
pages = {},
doi = {10.3390/ijms232214213},
pmid = {36430696},
issn = {1422-0067},
mesh = {*Droughts ; *Calcium ; Calcium Chloride ; Photosynthesis/physiology ; Water ; },
abstract = {Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants. DORN1 was the first identified purinoceptor for the plant response to extracellular ATP. It has been established that DORN1 could play key roles in a series of biological activities in plants. However, the biological roles of DORN1 and the mechanism remain unclear under drought stress conditions in plants. Here, DORN1 was targeted for knockout by using the CRISPR/Cas 9 system. It was found that the loss function of DORN1 resulted in a significant decrease in the effective quantum yield of PSII [Y(II)], the photochemical quenching coefficient (qP), and the rate of photosynthetic electron transport through PSII (ETR), which reflected plants' photochemical efficiency. Whereas Y(NO) values showed obvious enhancement under drought stress conditions. Further experimental results showed that the Y(II), qP, and ETR, which reflect plants' photochemical efficiency, increased significantly with CaCl2 treatment. These results indicated that the drought tolerance of the mutant was decreased, and the exogenous application of calcium ions could effectively promote the drought tolerance of the dorn1 mutant. Transpiration loss controlled by stomata is closely related to drought tolerance, further, we examined the transpirational water loss in dorn1 and found that it was greater than wild-type (WT). Besides, the dorn1 mutant's stomatal aperture significantly increased compared with the WT and the stomata of dorn1 mutant plants tend to close after CaCl2 treatment. Taken together, our results show that DORN1 plays a key role in drought stress tolerance in plants, which may depend on calcium and calcium-related signaling pathways.},
}
@article {pmid36430643,
year = {2022},
author = {Li, C and Gong, C and Wu, J and Yang, L and Zhou, L and Wu, B and Gao, L and Ling, F and You, A and Li, C and Lin, Y},
title = {Improvement of Rice Agronomic Traits by Editing Type-B Response Regulators.},
journal = {International journal of molecular sciences},
volume = {23},
number = {22},
pages = {},
doi = {10.3390/ijms232214165},
pmid = {36430643},
issn = {1422-0067},
mesh = {*Oryza/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Gene Editing/methods ; Phenotype ; Plants/genetics ; },
abstract = {Type-B response regulator proteins in rice contain a conserved receiver domain, followed by a GARP DNA binding domain and a longer C-terminus. Some type-B response regulators such as RR21, RR22 and RR23 are involved in the development of rice leaf, root, flower and trichome. In this study, to evaluate the application potential of type-B response regulators in rice genetic improvement, thirteen type-B response regulator genes in rice were respectively knocked out by using CRISPR/Cas9 genome editing technology. Two guide RNAs (gRNAs) were simultaneously expressed on a knockout vector to mutate one gene. T0 transformed plants were used to screen the plants with deletion of large DNA fragments through PCR with specific primers. The mutants of CRISPR/Cas9 gene editing were detected by Cas9 specific primer in the T1 generation, and homozygous mutants without Cas9 were screened, whose target regions were confirmed by sequencing. Mutant materials of 12 OsRRs were obtained, except for RR24. Preliminary phenotypic observation revealed variations of various important traits in different mutant materials, including plant height, tiller number, tillering angle, heading date, panicle length and yield. The osrr30 mutant in the T2 generation was then further examined. As a result, the heading date of the osrr30 mutant was delayed by about 18 d, while the yield was increased by about 30%, and the chalkiness was significantly reduced compared with those of the wild-type under field high temperature stress. These results indicated that osrr30 has great application value in rice breeding. Our findings suggest that it is feasible to perform genetic improvement of rice by editing the type-B response regulators.},
}
@article {pmid36430465,
year = {2022},
author = {Pechenov, PY and Garagulya, DA and Stanovov, DS and Letarov, AV},
title = {New Effective Method of Lactococcus Genome Editing Using Guide RNA-Directed Transposition.},
journal = {International journal of molecular sciences},
volume = {23},
number = {22},
pages = {},
doi = {10.3390/ijms232213978},
pmid = {36430465},
issn = {1422-0067},
mesh = {RNA, Guide/genetics ; Gene Editing ; *Lactococcus lactis/genetics ; *Bacteriophages/genetics ; *Lactobacillales/genetics ; },
abstract = {Lactococcus lactis is an important industrial microorganism and a widely used model object for research in the field of lactic acid bacteria (LAB) biology. The development of new L. lactis and related LAB strains with improved properties, including phage-resistant strains for dairy fermentation, LAB-based vaccines or strains with altered genotypes for research purposes, are hindered by the lack of genome-editing tools that allow for the easy and straightforward incorporation of a significant amount of the novel genetic material, such as large genes or operons, into the chromosomes of these bacteria. We recently employed a suggested system based on the CRISPR-Cas-associated transposon for the editing of the L. lactis genome. After the in-depth redesign of the system, we were able to achieve the stable incorporation of the fragments that were sized up to 10 kbp into the L. lactis beta-galactosidase gene. The efficiency of editing under the optimized conditions were 2 × 10[-4] and 4 × 10[-5] for 1 kbp and 10 kbp, respectively, which are sufficient for fast and easy modifications if a positive selection marker can be used.},
}
@article {pmid36430368,
year = {2022},
author = {Baranova, SV and Zhdanova, PV and Lomzov, AA and Koval, VV and Chernonosov, AA},
title = {Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems.},
journal = {International journal of molecular sciences},
volume = {23},
number = {22},
pages = {},
doi = {10.3390/ijms232213889},
pmid = {36430368},
issn = {1422-0067},
mesh = {*DNA Cleavage ; *CRISPR-Cas Systems ; Endonucleases/metabolism ; Gene Editing/methods ; DNA/chemistry ; },
abstract = {Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson-Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the "seed" region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the "unwinding" of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.},
}
@article {pmid36429042,
year = {2022},
author = {Nasrallah, A and Sulpice, E and Kobaisi, F and Gidrol, X and Rachidi, W},
title = {CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements.},
journal = {Cells},
volume = {11},
number = {22},
pages = {},
doi = {10.3390/cells11223615},
pmid = {36429042},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Zinc Finger Nucleases ; Transcription Activator-Like Effector Nucleases/genetics ; Technology ; },
abstract = {This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.},
}
@article {pmid36429003,
year = {2022},
author = {Yang, Y and Li, D and Wan, F and Chen, B and Wu, G and Li, F and Ren, Y and Liang, P and Wan, J and Songyang, Z},
title = {Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells.},
journal = {Cells},
volume = {11},
number = {22},
pages = {},
doi = {10.3390/cells11223574},
pmid = {36429003},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; CRISPR-Associated Protein 9/metabolism ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.},
}
@article {pmid36428986,
year = {2022},
author = {Gu, J and Wang, J and Bi, H and Li, X and Merchant, A and Zhang, P and Zhang, Q and Zhou, X},
title = {CRISPR/Cas9-Mediated Mutagenesis of Sex-Specific Doublesex Splicing Variants Leads to Sterility in Spodoptera frugiperda, a Global Invasive Pest.},
journal = {Cells},
volume = {11},
number = {22},
pages = {},
doi = {10.3390/cells11223557},
pmid = {36428986},
issn = {2073-4409},
mesh = {Animals ; Female ; Male ; Spodoptera/genetics ; *Introduced Species ; CRISPR-Cas Systems/genetics ; *Infertility/genetics ; Mutagenesis ; },
abstract = {Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, we cloned and functionally characterized Doublesex (dsx), a "molecular switch" modulating sexual dimorphism in S. frugiperda using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct S. frugiperda mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of S. frugiperda dsx (Sfdsx), respectively. As expected, abnormal development of both the external and internal genitalia was observed during the pupal and adult stages. Interestingly, knocking out sex-specific dsx variants in S. frugiperda led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of dsx in S. frugiperda sex differentiation but also provide empirical evidence for dsx as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way.},
}
@article {pmid36428958,
year = {2022},
author = {Yagi, Y and Teramoto, T and Kaieda, S and Imai, T and Sasaki, T and Yagi, M and Maekawa, N and Nakamura, T},
title = {Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.},
journal = {Cells},
volume = {11},
number = {22},
pages = {},
doi = {10.3390/cells11223529},
pmid = {36428958},
issn = {2073-4409},
mesh = {Animals ; Humans ; *RNA Splicing ; *RNA-Binding Proteins/genetics/metabolism ; RNA/metabolism ; RNA, Messenger/metabolism ; Mammals/metabolism ; },
abstract = {RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.},
}
@article {pmid36427491,
year = {2022},
author = {Wu, WY and Mohanraju, P and Liao, C and Adiego-Pérez, B and Creutzburg, SCA and Makarova, KS and Keessen, K and Lindeboom, TA and Khan, TS and Prinsen, S and Joosten, R and Yan, WX and Migur, A and Laffeber, C and Scott, DA and Lebbink, JHG and Koonin, EV and Beisel, CL and van der Oost, J},
title = {The miniature CRISPR-Cas12m effector binds DNA to block transcription.},
journal = {Molecular cell},
volume = {82},
number = {23},
pages = {4487-4502.e7},
doi = {10.1016/j.molcel.2022.11.003},
pmid = {36427491},
issn = {1097-4164},
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/genetics ; Plasmids ; RNA ; RNA, Guide/metabolism ; },
abstract = {CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.},
}
@article {pmid36427302,
year = {2022},
author = {Shiriaeva, AA and Kuznedelov, K and Fedorov, I and Musharova, O and Khvostikov, T and Tsoy, Y and Kurilovich, E and Smith, GR and Semenova, E and Severinov, K},
title = {Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system.},
journal = {Science advances},
volume = {8},
number = {47},
pages = {eabn8650},
doi = {10.1126/sciadv.abn8650},
pmid = {36427302},
issn = {2375-2548},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids. In Escherichia coli, immunity is acquired upon integration of 33-bp spacers into CRISPR arrays. DNA targets complementary to spacers get degraded and serve as a source of new spacers during a process called primed adaptation. Precursors of such spacers, prespacers, are ~33-bp double-stranded DNA fragments with a ~4-nt 3' overhang. The mechanism of prespacer generation is not clear. Here, we use FragSeq and biochemical approaches to determine enzymes involved in generation of defined prespacer ends. We demonstrate that RecJ is the main exonuclease trimming 5' ends of prespacer precursors, although its activity can be partially substituted by ExoVII. The RecBCD complex allows single strand-specific RecJ to process double-stranded regions flanking prespacers. Our results reveal intricate functional interactions of genome maintenance proteins with CRISPR interference and adaptation machineries during generation of prespacers capable of integration into CRISPR arrays.},
}
@article {pmid36426905,
year = {2022},
author = {Maguire, JA and Gadue, P and French, DL},
title = {Highly Efficient CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.},
journal = {Current protocols},
volume = {2},
number = {11},
pages = {e590},
pmid = {36426905},
issn = {2691-1299},
support = {U01 HL134696/HL/NHLBI NIH HHS/United States ; /NH/NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells ; Cell- and Tissue-Based Therapy ; Oligonucleotides ; },
abstract = {Human pluripotent stem cells hold tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR/Cas9 technology has expanded this potential by providing a valuable tool to engineer or correct disease-associated mutations. Because of the high efficiency with which CRISPR/Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele without indel formation on the non-targeted allele. To overcome this obstacle, we describe use of two oligonucleotide repair templates: one expressing the sequence change and the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodologies to make the protocols efficient, with small numbers of cells and a limited amount of labor-intensive clone passaging. This article provides a technically simple approach for generating valuable tools to model human disease in stem cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Application and optimization of CRISPR-based genome editing in human pluripotent stem cells Basic Protocol 2: Genetic modification of human pluripotent stem cells using a double-oligonucleotide CRISPR/Cas9 recombination system.},
}
@article {pmid36425434,
year = {2022},
author = {de Oliveira, HL and Dias, GM and Neves, BC},
title = {Genome sequence of Pseudomonas aeruginosa PA1-Petro-A role model of environmental adaptation and a potential biotechnological tool.},
journal = {Heliyon},
volume = {8},
number = {11},
pages = {e11566},
pmid = {36425434},
issn = {2405-8440},
abstract = {Pseudomonas aeruginosa is a ubiquitous microorganism, capable of colonizing a wide range of habitats due to its metabolic versatility and wide adaptability to different conditions. Industrial and environmental research involving petroleum microbiology play a pivotal role in controlling many technical, operational, and environmental issues. P. aeruginosa PA1-Petro strain was isolated from oil production water in Northeastern Brazil. Herein we report the genomic sequencing and annotation of PA1-Petro, and a comparative genomics study against two widely used reference P. aeruginosa strains (PAO1 and PA14). PA1-Petro has a genome of 6,893,650 bp, the largest among the three analyzed in this study, with a 65.87% GC content. The analyzes resulted in a wide repertoire of 544 unique genes in PA1-Petro, and the highest copy numbers of common genes among the three strains (PA1-Petro, PAO1 and PA14). Unique sequences are hypothetical proteins, prophage sequences, mobile genetic elements, transcriptional regulators, metal resistance genes to copper, tellurium and arsenic, type IE CRISPR-Cas, Type VI Secretion System (T6SS)-associated proteins, and a toxin-antitoxin system. Taken together, these results provide intriguing insights on adaptive evolution within PA1-Petro genome, adding unprecedented information to the species' plasticity and ubiquitous characteristics.},
}
@article {pmid36424814,
year = {2022},
author = {Nasef, M and Khweis, SA and Dunkle, JA},
title = {The effect of crRNA-target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system.},
journal = {RNA biology},
volume = {19},
number = {1},
pages = {1293-1304},
pmid = {36424814},
issn = {1555-8584},
support = {R35 GM142966/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Oligoribonucleotides ; *Nucleic Acids ; Coenzyme A ; },
abstract = {CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target-crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target-crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target-crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA-target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.},
}
@article {pmid36423792,
year = {2022},
author = {Prasad, D and Mani, NK and Pandey, DM},
title = {CRISPR/Cas technology: Opportunities for phytopathogenic viruses detection.},
journal = {Journal of biotechnology},
volume = {360},
number = {},
pages = {211-217},
doi = {10.1016/j.jbiotec.2022.11.010},
pmid = {36423792},
issn = {1873-4863},
abstract = {Detection and monitoring of viruses are essential for healthy plants and prosperity. Recent development in CRISPR/Cas system in diagnosis has open an avenue well suited for pathogen detection. Variety of CRISPR associated proteins are being discovered, suggesting array of application and detection strategies in diagnosis. Phytopathogenic viruses are diverse with respect to their nucleic acid compositions, which presents a challenge in developing a single device applicable for almost all viruses. The review describes about the efficient use of CRISPR/Cas Technology in diagnosis, such as SHERLOCK, DETECTR and SATORI. These methods are different in their characteristic to identify specific nucleic acids and processing the detectable signals. These technologies are in their infancy and lot of scope is there to develop commercial kits. Plant tissue culture-based industries, climate control green houses, indoor cultivation facilities etc. has been considered as few examples. This review will be beneficial for researchers seeking to develop detection mechanism based on CRISPR/Cas technology. The outcome in the form of cost-effective detection of viruses will be boon for agro-based industries, which are facing challenges through virus contamination.},
}
@article {pmid36423580,
year = {2022},
author = {Al-Shayeb, B and Skopintsev, P and Soczek, KM and Stahl, EC and Li, Z and Groover, E and Smock, D and Eggers, AR and Pausch, P and Cress, BF and Huang, CJ and Staskawicz, B and Savage, DF and Jacobsen, SE and Banfield, JF and Doudna, JA},
title = {Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors.},
journal = {Cell},
volume = {185},
number = {24},
pages = {4574-4586.e16},
doi = {10.1016/j.cell.2022.10.020},
pmid = {36423580},
issn = {1097-4172},
support = {U01 AI142817/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; Gene Editing ; Genome ; *Bacteriophages/genetics ; DNA ; RNA ; Mammals/genetics ; },
abstract = {CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.},
}
@article {pmid36423327,
year = {2022},
author = {Yang, H and Li, F and Xue, T and Khan, MR and Xia, X and Busquets, R and Gao, H and Dong, Y and Zhou, W and Deng, R},
title = {Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice.},
journal = {Analytical chemistry},
volume = {94},
number = {48},
pages = {16953-16959},
doi = {10.1021/acs.analchem.2c04589},
pmid = {36423327},
issn = {1520-6882},
mesh = {Mice ; Humans ; Animals ; *DNA, Catalytic/metabolism ; Lead ; Bioaccumulation ; *Biosensing Techniques ; Ions ; Limit of Detection ; },
abstract = {Lead contamination in the environment tends to enter the food chain and further into the human body, causing serious health issues. Herein, we proposed a Csm6-DNAzyme tandem assay (termed cDNAzyme) using CRISPR/Cas III-A Csm6 and GR-5 DNAzyme, enabling one-pot and sensitive detection of lead contamination. We found that Pb[2+]-activated GR-5 DNAzyme produced cleaved substrates that can serve as the activator of Csm6, and the Csm6-DNAzyme tandem improved the sensitivity for detecting Pb[2+] by 6.1 times compared to the original GR-5 DNAzyme. Due to the high specificity of DNAzyme, the cDNAzyme assay can discriminate Pb[2+] from other bivalent and trivalent interfering ions and allowed precise detection of Pb[2+] in water and food samples. Particularly, the assay can achieve one-step, mix-and-read detection of Pb[2+] at room temperature. We used the cDNAzyme assay to investigate the accumulation of lead in mice, and found that lead accumulated at higher levels in the colon and kidney compared to the liver, and most of the lead was excreted. The cDNAzyme assay is promising to serve as analytical tools for lead-associated environmental and biosafety issues.},
}
@article {pmid36423304,
year = {2022},
author = {Kato, K and Okazaki, S and Schmitt-Ulms, C and Jiang, K and Zhou, W and Ishikawa, J and Isayama, Y and Adachi, S and Nishizawa, T and Makarova, KS and Koonin, EV and Abudayyeh, OO and Gootenberg, JS and Nishimasu, H},
title = {RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.},
journal = {Science (New York, N.Y.)},
volume = {378},
number = {6622},
pages = {882-889},
doi = {10.1126/science.add7347},
pmid = {36423304},
issn = {1095-9203},
support = {R21 AI149694/AI/NIAID NIH HHS/United States ; R01 EB031957/EB/NIBIB NIH HHS/United States ; R56 HG011857/HG/NHGRI NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Endonucleases/chemistry/metabolism ; *Proteolysis ; *CRISPR-Associated Proteins/chemistry/metabolism ; *RNA, Guide/chemistry/metabolism ; *Deltaproteobacteria/enzymology ; Protein Conformation ; HEK293 Cells ; },
abstract = {The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.},
}
@article {pmid36423276,
year = {2022},
author = {Strecker, J and Demircioglu, FE and Li, D and Faure, G and Wilkinson, ME and Gootenberg, JS and Abudayyeh, OO and Nishimasu, H and Macrae, RK and Zhang, F},
title = {RNA-activated protein cleavage with a CRISPR-associated endopeptidase.},
journal = {Science (New York, N.Y.)},
volume = {378},
number = {6622},
pages = {874-881},
doi = {10.1126/science.add7450},
pmid = {36423276},
issn = {1095-9203},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Endopeptidases/chemistry/metabolism ; RNA, Bacterial/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/metabolism ; *Proteolysis ; *Deltaproteobacteria/enzymology/genetics ; *RNA, Guide/chemistry/metabolism ; Sigma Factor/metabolism ; Transcription, Genetic ; Substrate Specificity ; Allosteric Regulation ; *Bacterial Proteins/chemistry/metabolism ; Enzyme Activation ; },
abstract = {In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.},
}
@article {pmid36421156,
year = {2022},
author = {Atçeken, N and Yigci, D and Ozdalgic, B and Tasoglu, S},
title = {CRISPR-Cas-Integrated LAMP.},
journal = {Biosensors},
volume = {12},
number = {11},
pages = {},
pmid = {36421156},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Molecular Diagnostic Techniques ; Point-of-Care Testing ; },
abstract = {Pathogen-specific point-of-care (PoC) diagnostic tests have become an important need in the fight against infectious diseases and epidemics in recent years. PoC diagnostic tests are designed with the following parameters in mind: rapidity, accuracy, sensitivity, specificity, and ease of use. Molecular techniques are the gold standard for pathogen detection due to their accuracy and specificity. There are various limitations in adapting molecular diagnostic methods to PoC diagnostic tests. Efforts to overcome limitations are focused on the development of integrated molecular diagnostics by utilizing the latest technologies available to create the most successful PoC diagnostic platforms. With this point of view, a new generation technology was developed by combining loop-mediated isothermal amplification (LAMP) technology with clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) technology. This integrated approach benefits from the properties of LAMP technology, namely its high efficiency, short turnaround time, and the lack of need for a complex device. It also makes use of the programmable function of CRISPR-Cas technology and the collateral cleavage activity of certain Cas proteins that allow for convenient reporter detection. Thus, this combined technology enables the development of PoC diagnostic tests with high sensitivity, specificity, and ease of use without the need for complicated devices. In this review, we discuss the advantages and limitations of the CRISPR/Cas combined LAMP technology. We review current limitations to convert CRISPR combined LAMP into pathogen-specific PoC platforms. Furthermore, we point out the need to design more useful PoC platforms using microfabrication technologies by developing strategies that overcome the limitations of this new technology, reduce its complexity, and reduce the risk of contamination.},
}
@article {pmid36420593,
year = {2022},
author = {Zhang, Y and Lei, J and Liu, W and Jin, Y and Li, B},
title = {Chemiluminescence resonance energy transfer as a simple and sensitive readout mode for a CRISPR/Cas12a-based biosensing platform.},
journal = {The Analyst},
volume = {147},
number = {24},
pages = {5687-5693},
doi = {10.1039/d2an01735k},
pmid = {36420593},
issn = {1364-5528},
mesh = {*Luminescence ; *CRISPR-Cas Systems/genetics ; Hydrogen Peroxide ; Vibration ; Fluorescence Resonance Energy Transfer ; },
abstract = {Various CRISPR/Cas12a-based biosensing systems have been developed in the past few years, and most of these systems used Taqman probes to report signals through fluorescence resonance energy transfer (FRET). In this study, we explored chemiluminescence resonance energy transfer (CRET) as the readout mode for CRISPR/Cas12a-based biosensing. The chemiluminescence (CL) reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) and H2O2 was used to excite the fluorophore dye of the Taqman probe. Different from FRET, CRET does not need external excitation light, which can effectively avoid autofluorescence and photobleaching. The detection limit of this CRET readout mode was estimated to be 10 pM for target DNA, which was about 8 times lower than that of the widely used FRET readout mode. These results suggest that CRET can serve as a rapid, sensitive and simple readout mode of CRISPR/Cas12-based biosensing, and can further enrich the toolbox of CRISPR/Cas12-based biosensing.},
}
@article {pmid36419420,
year = {2022},
author = {Ding, J and Schuergers, N and Baehre, H and Wilde, A},
title = {Enzymatic properties of CARF-domain proteins in Synechocystis sp. PCC 6803.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {1046388},
pmid = {36419420},
issn = {1664-302X},
abstract = {Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.},
}
@article {pmid36418881,
year = {2022},
author = {Ledford, H},
title = {CRISPR tools found in thousands of viruses could boost gene editing.},
journal = {Nature},
volume = {612},
number = {7938},
pages = {21},
pmid = {36418881},
issn = {1476-4687},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; *Viruses/genetics ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid36418866,
year = {2022},
author = {Huang, J and Rowe, D and Subedi, P and Zhang, W and Suelter, T and Valent, B and Cook, DE},
title = {CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7168},
pmid = {36418866},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; Mutation ; DNA/genetics ; },
abstract = {CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, understanding of DNA repair following Cas-mediated DNA cleavage remains incomplete. Using Cas12a ribonucleoprotein genome editing in the fungal pathogen, Magnaporthe oryzae, we detail non-canonical DNA repair outcomes from hundreds of transformants. Sanger and nanopore sequencing analysis reveals significant variation in DNA repair profiles, ranging from small INDELs to kilobase size deletions and insertions. Furthermore, we find the frequency of DNA repair outcomes varies between loci. The results are not specific to the Cas-nuclease or selection procedure. Through Ku80 deletion analysis, a key protein required for canonical non-homologous end joining, we demonstrate activity of an alternative end joining mechanism that creates larger DNA deletions, and uses longer microhomology compared to C-NHEJ. Together, our results suggest preferential DNA repair pathway activity in the genome that can create different mutation profiles following repair, which could create biased genome variation and impact genome engineering and genome evolution.},
}
@article {pmid36418691,
year = {2023},
author = {Niklander, SE and Hunter, KD},
title = {A Protocol to Produce Genetically Edited Primary Oral Keratinocytes Using the CRISPR-Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2588},
number = {},
pages = {217-229},
pmid = {36418691},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Keratinocytes ; Gene Editing ; Genomics ; RNA ; },
abstract = {The Nobel Prize awarded gene editing system, CRISPR-Cas9, is probably one of the greatest achievements of the last decades. CRISPR-Cas9 can introduce irreversible genomic changes in its target DNA by simple specifying a 20-nucleotide sequence within its RNA guide. Due to its simplicity, efficacy, and relative low cost in comparison with other genome editing systems, it has become the most common gene editing system used in research laboratories. Here we describe a step-by-step protocol to produce genetically edited primary oral keratinocytes using the CRISPR-Cas9 system.},
}
@article {pmid36418298,
year = {2022},
author = {Willis, JCW and Silva-Pinheiro, P and Widdup, L and Minczuk, M and Liu, DR},
title = {Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7204},
pmid = {36418298},
issn = {2041-1723},
mesh = {Mice ; Animals ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; Zinc Fingers/genetics ; Cytosine/metabolism ; Mitochondria/genetics/metabolism ; },
abstract = {DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase (DddA), and a uracil glycosylase inhibitor to mediate C•G-to-T•A editing in nuclear and organelle DNA. Here we report the development of zinc finger DdCBEs (ZF-DdCBEs) and the improvement of their editing performance through engineering their architectures, defining improved ZF scaffolds, and installing DddA activity-enhancing mutations. We engineer variants with improved DNA specificity by integrating four strategies to reduce off-target editing. We use optimized ZF-DdCBEs to install or correct disease-associated mutations in mitochondria and in the nucleus. Leveraging their small size, we use a single AAV9 to deliver into heart, liver, and skeletal muscle in post-natal mice ZF-DdCBEs that efficiently install disease-associated mutations. While off-target editing of ZF-DdCBEs is likely too high for therapeutic applications, these findings demonstrate a compact, all-protein base editing research tool for precise editing of organelle or nuclear DNA without double-strand DNA breaks.},
}
@article {pmid36417506,
year = {2022},
author = {Yan, MY and Zheng, D and Li, SS and Ding, XY and Wang, CL and Guo, XP and Zhan, L and Jin, Q and Yang, J and Sun, YC},
title = {Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.},
journal = {Science advances},
volume = {8},
number = {47},
pages = {eadd5907},
doi = {10.1126/sciadv.add5907},
pmid = {36417506},
issn = {2375-2548},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Mycobacterium tuberculosis/genetics ; CRISPR-Cas Systems ; Genomics/methods ; Genome ; },
abstract = {CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.},
}
@article {pmid36414618,
year = {2022},
author = {Verkuijl, SAN and Gonzalez, E and Li, M and Ang, JXD and Kandul, NP and Anderson, MAE and Akbari, OS and Bonsall, MB and Alphey, L},
title = {A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7145},
pmid = {36414618},
issn = {2041-1723},
mesh = {Male ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Drive Technology ; Germ Cells ; Inheritance Patterns/genetics ; *Aedes/genetics ; Animals ; Transgenes ; },
abstract = {CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (w[GDe]). Here, through an analysis using this linkage we show that in males inheritance bias of w[GDe] did not occur by homing, rather through increased propagation of the donor drive element. We test the same w[GDe] drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.},
}
@article {pmid36414383,
year = {2022},
author = {Gu, S and Zhang, J and Li, L and Zhong, J},
title = {Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00374},
pmid = {36414383},
issn = {2161-5063},
abstract = {Lactobacilli such as Lacticaseibacillus (Lcb) paracasei are generally regarded as safe and health-promoting microbes, and have been widely applied in food and pharmaceutical industries. However, the genetic bases of their beneficial properties were mostly uncertain because of the lack of effective genetic manipulation tools. The type II CRISPR-Cas9 system is the largest family present in lactobacilli, but none of them yet have been developed for genetic modifications. Here, we establish the first endogenous CRISPR-Cas9 genome-editing system in lactobacilli. With a validated protospacer adjacent motif (PAM) and customized single guide RNA (sgRNA) expression cassette, the native CRISPR-Cas9 system was reprogrammed to achieve gene deletion and chromosomal insertion at over 90% efficiency, as well as nucleotide substitution at ≥50% efficiency. We also effectively accomplished deletions of large genomic fragments (5-10 kb) and simultaneous deletion of multiple genes at distal loci, both of which are the first cases in lactobacilli when either endogenous or exogenous CRISPR-Cas systems were employed. In addition, we designed a controllable plasmid-targeting sgRNA expression module and integrated it into the editing plasmid. The all-in-one vector realized gene deletion and plasmid curing at high efficiency (>90%). Collectively, the present study develops a convenient and precise genetic tool in Lcb. paracasei and contributes to the genetics and engineering of lactobacilli.},
}
@article {pmid36413017,
year = {2022},
author = {Yang, B and Zheng, J and Yin, Y},
title = {AcaFinder: Genome Mining for Anti-CRISPR-Associated Genes.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0081722},
doi = {10.1128/msystems.00817-22},
pmid = {36413017},
issn = {2379-5077},
abstract = {Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize to form acr-aca operons. Here, we present AcaFinder as the first Aca genome mining tool. AcaFinder can (i) predict Acas and their associated acr-aca operons using guilt-by-association (GBA); (ii) identify homologs of known Acas using an HMM (Hidden Markov model) database; (iii) take input genomes for potential prophages, CRISPR-Cas systems, and self-targeting spacers (STSs); and (iv) provide a standalone program (https://github.com/boweny920/AcaFinder) and a web server (http://aca.unl.edu/Aca). AcaFinder was applied to mining over 16,000 prokaryotic and 142,000 gut phage genomes. After a multistep filtering, 36 high-confident new Aca families were identified, which is three times that of the 12 known Aca families. Seven new Aca families were from major human gut bacteria (Bacteroidota, Actinobacteria, and Fusobacteria) and their phages, while most known Aca families were from Proteobacteria and Firmicutes. A complex association network between Acrs and Acas was revealed by analyzing their operonic colocalizations. It appears very common in evolution that the same aca genes can recombine with different acr genes and vice versa to form diverse acr-aca operon combinations. IMPORTANCE At least four bioinformatics programs have been published for genome mining of Acrs since 2020. In contrast, no bioinformatics tools are available for automated Aca discovery. As the self-transcriptional repressor of acr-aca operons, Aca can be viewed as anti-anti-CRISPRs, with great potential in the improvement of CRISPR-Cas technology. Although all the 12 known Aca proteins contain a conserved helix-turn-helix (HTH) domain, not all HTH-containing proteins are Acas. However, HTH-containing proteins with adjacent Acr homologs encoded in the same genetic operon are likely Aca proteins. AcaFinder implements this guilt-by-association idea and the idea of using HMMs of known Acas for homologs into one software package. Applying AcaFinder in screening prokaryotic and gut phage genomes reveals a complex acr-aca operonic colocalization network between different families of Acrs and Acas.},
}
@article {pmid36411629,
year = {2022},
author = {Hazrati, A and Malekpour, K and Soudi, S and Hashemi, SM},
title = {CRISPR/Cas9-engineered mesenchymal stromal/stem cells and their extracellular vesicles: A new approach to overcoming cell therapy limitations.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {156},
number = {},
pages = {113943},
doi = {10.1016/j.biopha.2022.113943},
pmid = {36411629},
issn = {1950-6007},
mesh = {CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; *Mesenchymal Stem Cells/metabolism ; Cell- and Tissue-Based Therapy ; },
abstract = {Cell therapy is one of the newest therapeutic approaches for treating tissue destruction diseases and replacing damaged parts in defective tissues. Among different cells, mesenchymal stem cells (MSCs) have received a lot of attention due to their advantages and desirable properties. Also, MSCs-derived secretome, which includes various growth factors, cytokines, and extracellular vesicles (EVs), is used in the treatment of different types of diseases. However, the application of MSCs in an intact form brings their functionality with limitations. For this reason, different methods are recommended to increase their efficiency and the extracellular vesicles derived from them. One of these methods is gene editing of these cells. Among the different techniques for MSCs gene editing, CRISPR/Cas9 can increase the therapeutic potential of MSCs in a targeted manner due to its advantages. In order to achieve the desired result, various genes have been manipulated in MSCs, including genes involved in stemness, aging, migration, proliferation, survival, and inflammatory responses. Engineering MSCs with this method affects the cells' characteristics, changes their cytokine and different growth factors secretions, and increases their therapeutic efficiency.},
}
@article {pmid36411557,
year = {2022},
author = {Saad, FA and Saad, JF and Siciliano, G and Merlini, L and Angelini, C},
title = {Duchenne Muscular Dystrophy Gene therapy.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/1566523223666221118160932},
pmid = {36411557},
issn = {1875-5631},
abstract = {Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular diseases affecting both skeletal and cardiac muscles. Therefore, owing to their single X chromosome, the affected boys receive the pathogenic gene mutations from their unknowing carrier mothers. Current pharmacological drugs are palliative that address the symptoms of the disease rather than the genetic cause imbedded in the Dystrophin gene DNA sequence. Therefore, alternative therapies like gene drugs that can address the genetic cause of the disease at its root is crucial, which include gene transfer/implantation, exon skipping, and gene editing. Presently, it is possible through genetic reprogramming to engineer AAV vectors to deliver certain therapeutic cargos specifically to muscle or other organs regardless of their serotype. Similarly, it is possible to direct the biogenesis of exosomes to carry gene editing constituents or certain therapeutic cargos to specific tissue or cell type like brain and muscle. While autologous exosomes are immunologically inert, it is possible to camouflage AAV capsids, gold and lipid nanoparticles to evade the immune system recognition. In this review, we highlight current opportunities for Duchenne muscular dystrophy gene therapy, which has been known thus far as an incurable genetic disease. This article is a part of Gene Therapy of Rare Genetic Diseases thematic issue.},
}
@article {pmid36410158,
year = {2023},
author = {Xing, G and Shang, Y and Wang, X and Lin, H and Chen, S and Pu, Q and Lin, L},
title = {Multiplexed detection of foodborne pathogens using one-pot CRISPR/Cas12a combined with recombinase aided amplification on a finger-actuated microfluidic biosensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {220},
number = {},
pages = {114885},
doi = {10.1016/j.bios.2022.114885},
pmid = {36410158},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Hydrolases ; *Microfluidics ; Recombinases/genetics ; *Food Contamination/analysis ; Food Microbiology ; Bacillus cereus/isolation &amp; purification ; },
abstract = {Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings.},
}
@article {pmid36409894,
year = {2022},
author = {Zhao, H and Yang, M and Bishop, J and Teng, Y and Cao, Y and Beall, BD and Li, S and Liu, T and Fang, Q and Fang, C and Xin, H and Nützmann, HW and Osbourn, A and Meng, F and Jiang, J},
title = {Identification and functional validation of super-enhancers in Arabidopsis thaliana.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {48},
pages = {e2215328119},
doi = {10.1073/pnas.2215328119},
pmid = {36409894},
issn = {1091-6490},
mesh = {Animals ; *Arabidopsis/genetics ; Regulatory Sequences, Nucleic Acid ; *Triterpenes ; Chromatin/genetics ; Mammals/genetics ; },
abstract = {Super-enhancers (SEs) are exceptionally large enhancers and are recognized to play prominent roles in cell identity in mammalian species. We surveyed the genomic regions containing large clusters of accessible chromatin regions (ACRs) marked by deoxyribonuclease (DNase) I hypersensitivity in Arabidopsis thaliana. We identified a set of 749 putative SEs, which have a minimum length of 1.5 kilobases and represent the top 2.5% of the largest ACR clusters. We demonstrate that the genomic regions associating with these SEs were more sensitive to DNase I than other nonpromoter ACRs. The SEs were preferentially associated with topologically associating domains. Furthermore, the SEs and their predicted cognate genes were frequently associated with organ development and tissue identity in A. thaliana. Therefore, the A. thaliana SEs and their cognate genes mirror the functional characteristics of those reported in mammalian species. We developed CRISPR/Cas-mediated deletion lines of a 3,578-bp SE associated with the thalianol biosynthetic gene cluster (BGC). Small deletions (131-157 bp) within the SE resulted in distinct phenotypic changes and transcriptional repression of all five thalianol genes. In addition, T-DNA insertions in the SE region resulted in transcriptional alteration of all five thalianol genes. Thus, this SE appears to play a central role in coordinating the operon-like expression pattern of the thalianol BGC.},
}
@article {pmid36409126,
year = {2022},
author = {Maslać, N and Sidhu, C and Teeling, H and Wagner, T},
title = {Comparative Transcriptomics Sheds Light on Remodeling of Gene Expression during Diazotrophy in the Thermophilic Methanogen Methanothermococcus thermolithotrophicus.},
journal = {mBio},
volume = {},
number = {},
pages = {e0244322},
doi = {10.1128/mbio.02443-22},
pmid = {36409126},
issn = {2150-7511},
abstract = {Some marine thermophilic methanogens are able to perform energy-consuming nitrogen fixation despite deriving only little energy from hydrogenotrophic methanogenesis. We studied this process in Methanothermococcus thermolithotrophicus DSM 2095, a methanogenic archaeon of the order Methanococcales that contributes to the nitrogen pool in some marine environments. We successfully grew this archaeon under diazotrophic conditions in both batch and fermenter cultures, reaching the highest cell density reported so far. Diazotrophic growth depended strictly on molybdenum and, in contrast to other diazotrophs, was not inhibited by tungstate or vanadium. This suggests an elaborate control of metal uptake and a specific metal recognition system for the insertion into the nitrogenase cofactor. Differential transcriptomics of M. thermolithotrophicus grown under diazotrophic conditions with ammonium-fed cultures as controls revealed upregulation of the nitrogenase machinery, including chaperones, regulators, and molybdate importers, as well as simultaneous upregulation of an ammonium transporter and a putative pathway for nitrate and nitrite utilization. The organism thus employs multiple synergistic strategies for uptake of nitrogen nutrients during the early exponential growth phase without altering transcription levels for genes involved in methanogenesis. As a counterpart, genes coding for transcription and translation processes were downregulated, highlighting the maintenance of an intricate metabolic balance to deal with energy constraints and nutrient limitations imposed by diazotrophy. This switch in the metabolic balance included unexpected processes, such as upregulation of the CRISPR-Cas system, probably caused by drastic changes in transcription levels of putative mobile and virus-like elements. IMPORTANCE The thermophilic anaerobic archaeon M. thermolithotrophicus is a particularly suitable model organism to study the coupling of methanogenesis to diazotrophy. Likewise, its capability of simultaneously reducing N2 and CO2 into NH3 and CH4 with H2 makes it a viable target for biofuel production. We optimized M. thermolithotrophicus cultivation, resulting in considerably higher cell yields and enabling the successful establishment of N2-fixing bioreactors. Improved understanding of the N2 fixation process would provide novel insights into metabolic adaptations that allow this energy-limited extremophile to thrive under diazotrophy, for instance, by investigating its physiology and uncharacterized nitrogenase. We demonstrated that diazotrophic growth of M. thermolithotrophicus is exclusively dependent on molybdenum, and complementary transcriptomics corroborated the expression of the molybdenum nitrogenase system. Further analyses of differentially expressed genes during diazotrophy across three cultivation time points revealed insights into the response to nitrogen limitation and the coordination of core metabolic processes.},
}
@article {pmid36407602,
year = {2022},
author = {An, Y and Wang, Y and Wang, X and Xiao, J},
title = {Development of chloroplast transformation and gene expression regulation technology in land plants.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1037038},
pmid = {36407602},
issn = {1664-462X},
abstract = {Chloroplasts in land plants have their own small circular DNA that is presumed to have originated from cyanobacteria-related endosymbionts, and the chloroplast genome is an attractive target to improve photosynthetic ability and crop yield. However, to date, most transgenic or genetic engineering technologies for plants are restricted to manipulations of the nuclear genome. In this review, we provide a comprehensive overview of chloroplast genetic engineering and regulation of gene expression from the perspective of history and biology, focusing on current and latest methods. In addition, we suggest techniques that may regulate the chloroplast gene expression at the transcriptional or post-transcriptional level.},
}
@article {pmid36406953,
year = {2022},
author = {Ngamsom, B and Iles, A and Kamita, M and Kimani, R and Wakaba, P and Rodriguez-Mateos, P and Mungai, M and Dyer, CE and Walter, C and Gitaka, J and Pamme, N},
title = {A sample-to-answer COVID-19 diagnostic device based on immiscible filtration and CRISPR-Cas12a-assisted detection.},
journal = {Talanta open},
volume = {6},
number = {},
pages = {100166},
pmid = {36406953},
issn = {2666-8319},
abstract = {In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip device, namely 'IFAST-LAMP-CRISPR', as an affordable, rapid and high-precision molecular diagnostic means for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The herein proposed 'sample-to-answer' platform integrates RNA extraction, amplification and molecular detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and guanidine hydrochloride, streamline sample preparation (including RNA extraction, concentration and purification) in 15 min with minimal hands-on steps. The pre-amplification in combination with CRISPR-Cas12a detection assays targeting the nucleoprotein (N) gene achieved visual identification of ≥ 470 copies mL[-1] genomic SARS-CoV-2 samples in 45 min. On-chip assays showed the ability to isolate and detect SARS-CoV-2 RNA from 100 genome copies mL[-1] of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity- and sensitivity-comparable alternative to the costly gold-standard reverse transcription-polymerase chain reaction (RT-PCR) assay, requiring only a simple heating source. Initial testing illustrates the platform viability both on nasopharyngeal swab and saliva samples collected using the easily accessible Swan-brand cigarette filter, providing a complete workflow for COVID-19 diagnostics in low-resource settings.},
}
@article {pmid36406407,
year = {2022},
author = {Zhu, Y and Xing, C and Yang, L and Li, Q and Wang, X and Zhou, J and Zhang, C and Ren, C and Liu, F and He, J and Shen, B and Du, Y and Liu, Y},
title = {Dual-gene detection in a single-tube system based on CRISPR-Cas12a/Cas13a for severe fever thrombocytopenia syndrome virus.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {977382},
pmid = {36406407},
issn = {1664-302X},
abstract = {Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease, which is caused by severe fever with thrombocytopenia syndrome virus (SFTSV). The disease results in high mortality and increased morbidity and threatens global public health. Rapid detection of SFTSV is crucial for epidemic prevention in low-resource settings. Here we developed deployable, sensitive and rapid detection methods based on CRISPR/Cas12a or Cas13a technologies. The CRISPR/Cas12a-based detection assay could stably detect the SFTSV L or M genes at 10 cp/μl. The Cas13a-based method could detect the L gene as low as 0.75 cp/μl. For point-of-care testing, we combined fluorescence visualization and lateral flow detection with CRISPR/Cas-based assays. Furthermore, using the orthogonal DNA/RNA collateral activity of the Cas12a/Cas13a system, we present the dual-gene detection platform for SFTSV, which can simultaneously detect the L and M genes in a single tube. Based on the dual-gene detection, we designed multiplexed test strips to detect SFTSV. All our methods were initially validated using 52 clinical samples, showing 100% sensitivity and specificity. These new CRISPR/Cas-based detection methods are promising candidates for on-site detection of SFTSV.},
}
@article {pmid36406402,
year = {2022},
author = {Abriouel, H and Manetsberger, J and Caballero Gómez, N and Benomar, N},
title = {In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {989824},
pmid = {36406402},
issn = {1664-302X},
abstract = {Lactiplantibacillus pentosus CF2-10 N, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. High throughput sequencing and annotation of genome sequences underline the potential of L. pentosus CF2-10 N as excellent probiotic candidate of vegetable origin. In a previous study we could show the probiotic potential of CF2-10 N in vitro, while in this study in silico analysis of its genome revealed new insights into its safety and functionality. Our findings highlight the microorganism's ecological flexibility and adaptability to a broad range of environmental niches, food matrices and the gastrointestinal tract. These features are shared by both phylogenetically very close L. pentosus strains (CF2-10 N and MP-10) isolated from the same ecological niche with respect to their genome size (≅ 3.6 Mbp), the presence of plasmids (4-5) and several other properties. Nonetheless, additional and unique features are reported in the present study for L. pentosus CF2-10 N. Notably, the safety of L. pentosus CF2-10 N was shown by the absence of virulence determinants and the determination of acquired antibiotic resistance genes, i.e., resistome, which is mostly represented by efflux-pump resistance genes responsible for the intrinsic resistance. On the other hand, defense mechanisms of L. pentosus CF2-10 N include eight prophage regions and a CRISPR/cas system (CRISPR-I and CRISPR-II) as acquired immune system against mobile elements. Finally, the probiotic potential of this strain was further demonstrated by the presence of genes coding for proteins involved in adhesion, exopolysaccharide biosynthesis, tolerance to low pH and bile salts, immunomodulation, and vitamin and enzyme production. Taken together these results, we propose the use of L. pentosus CF2-10 N as a potential and promising probiotic candidate able to colonize several niches and adapt to different lifestyles. The strain can provide attractive functional and probiotic features necessary for its application as starter culture and probiotic.},
}
@article {pmid36405602,
year = {2022},
author = {Álvarez-Rodríguez, A and Jin, BK and Radwanska, M and Magez, S},
title = {Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030.},
journal = {Frontiers in medicine},
volume = {9},
number = {},
pages = {1037094},
pmid = {36405602},
issn = {2296-858X},
abstract = {Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei. The subspecies T. b. gambiense is mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting for roughly 95% of all HAT cases. Trypanosoma b. rhodesiense results in more acute zoonotic infections in East-Africa. Because HAT has a two-stage pathogenesis, treatment depends on clinical assessment of patients and the determination whether or not parasites have crossed the blood brain barrier. Today, ultimate confirmation of parasitemia is still done by microscopy analysis. However, the introduction of diagnostic lateral flow devices has been a major contributor to the recent dramatic drop in T. b. gambiense HAT. Other techniques such as loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA)-based tests have been published but are still not widely used in the field. Most recently, CRISPR-Cas technology has been proposed to improve the intrinsic diagnostic characteristics of molecular approaches. This will become crucial in the near future, as preventing the resurgence of HAT will be a priority and will require tools with extreme high positive and negative predicted values, as well as excellent sensitivity and specificity. As for treatment, pentamidine and suramin have historically been the drugs of choice for the treatment of blood-stage gambiense-HAT and rhodesiense-HAT, respectively. For treatment of second-stage infections, drugs that pass the blood brain barrier are needed, and melarsoprol has been effectively used for both forms of HAT in the past. However, due to the high occurrence of post-treatment encephalopathy, the drug is not recommended for use in T. b. gambiense HAT. Here, a combination therapy of eflornithine and nifurtimox (NECT) has been the choice of treatment since 2009. As this treatment requires IV perfusion of eflornithine, efforts were launched in 2003 by the drugs for neglected disease initiative (DNDi) to find an oral-only therapy solution, suitable for rural sub-Saharan Africa treatment conditions. In 2019 this resulted in the introduction of fexinidazole, with a treatment regimen suitable for both the blood-stage and non-severe second-stage T. b. gambiense infections. Experimental treatment of T. b. rhodesiense HAT has now been initiated as well.},
}
@article {pmid36405570,
year = {2022},
author = {Johnston, M and Ceren Ates, H and Glatz, RT and Mohsenin, H and Schmachtenberg, R and Göppert, N and Huzly, D and Urban, GA and Weber, W and Dincer, C},
title = {Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management.},
journal = {Materials today (Kidlington, England)},
volume = {},
number = {},
pages = {},
pmid = {36405570},
issn = {1369-7021},
abstract = {In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: ∼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.},
}
@article {pmid36403709,
year = {2022},
author = {Wang, SY and Li, X and Wang, SG and Xia, PF},
title = {Base editing for reprogramming cyanobacterium Synechococcus elongatus.},
journal = {Metabolic engineering},
volume = {75},
number = {},
pages = {91-99},
doi = {10.1016/j.ymben.2022.11.005},
pmid = {36403709},
issn = {1096-7184},
abstract = {Cyanobacteria can directly convert carbon dioxide (CO2) at the atmospheric level to biofuels, value-added chemicals and food products, making them ideal candidates to alleviate global climate change. Despite decades-long pioneering successes, the development of genome-editing tools, especially the CRISPR-Cas-based approaches, seems to lag behind other microbial chassis, slowing down the innovations of cyanobacteria. Here, we adapted and tailored base editing for cyanobacteria based on the CRISPR-Cas system and deamination. We achieved precise and efficient genome editing at a single-nucleotide resolution and demonstrated multiplex base editing in the model cyanobacterium Synechococcus elongatus. By using the base-editing tool, we successfully manipulated the glycogen metabolic pathway via the introduction of premature STOP codons in the relevant genes, building engineered strains with elevated potentials to produce chemicals and food from CO2. We present here the first report of base editing in the phylum of cyanobacteria, and a paradigm for applying CRISPR-Cas systems in bacteria. We believe that our work will accelerate the metabolic engineering and synthetic biology of cyanobacteria and drive more innovations to alleviate global climate change.},
}
@article {pmid36403665,
year = {2022},
author = {Perveen, S and Negi, A and Gopalakrishnan, V and Panda, S and Sharma, V and Sharma, R},
title = {COVID-19 diagnostics: Molecular biology to nanomaterials.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {538},
number = {},
pages = {139-156},
doi = {10.1016/j.cca.2022.11.017},
pmid = {36403665},
issn = {1873-3492},
abstract = {The SARS-CoV-2 pandemic has claimed around 6.4 million lives worldwide. The disease symptoms range from mild flu-like infection to life-threatening complications. The widespread infection demands rapid, simple, and accurate diagnosis. Currently used methods include molecular biology-based approaches that consist of conventional amplification by RT-PCR, isothermal amplification-based techniques such as RT-LAMP, and gene editing tools like CRISPR-Cas. Other methods include immunological detection including ELISA, lateral flow immunoassay, chemiluminescence, etc. Radiological-based approaches are also being used. Despite good analytical performance of these current methods, there is an unmet need for less costly and simpler tests that may be performed at point of care. Accordingly, nanomaterial-based testing has been extensively pursued. In this review, we discuss the currently used diagnostic techniques for SARS-CoV-2, their usefulness, and limitations. In addition, nanoparticle-based approaches have been highlighted as another potential means of detection. The review provides a deep insight into the current diagnostic methods and future trends to combat this deadly menace.},
}
@article {pmid36403643,
year = {2022},
author = {Çerçi, B and Uzay, IA and Kara, MK and Dinçer, P},
title = {Clinical trials and promising preclinical applications of CRISPR/Cas gene editing.},
journal = {Life sciences},
volume = {312},
number = {},
pages = {121204},
doi = {10.1016/j.lfs.2022.121204},
pmid = {36403643},
issn = {1879-0631},
abstract = {Treatment of genetic disorders by genomic manipulation has been the unreachable goal of researchers for many decades. Although our understanding of the genetic basis of genetic diseases has advanced tremendously in the last few decades, the tools developed for genomic editing were not efficient and practical for their use in the clinical setting until now. The recent advancements in the research of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems offered an easy and efficient way to edit the genome and accelerated the research on their potential use in the treatment of genetic disorders. In this review, we summarize the clinical trials that evaluate the CRISPR/Cas systems for treating different genetic diseases and highlight promising preclinical research on CRISPR/Cas mediated treatment of a great diversity of genetic disorders. Ultimately, we discuss the future of CRISPR/Cas mediated genome editing in genetic diseases.},
}
@article {pmid36401300,
year = {2022},
author = {Leng, K and Kampmann, M},
title = {Towards elucidating disease-relevant states of neurons and glia by CRISPR-based functional genomics.},
journal = {Genome medicine},
volume = {14},
number = {1},
pages = {130},
pmid = {36401300},
issn = {1756-994X},
support = {R01 AG062359/AG/NIA NIH HHS/United States ; U01 AG072464/AG/NIA NIH HHS/United States ; F30 AG066418/AG/NIA NIH HHS/United States ; U54 NS123746/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Genome-Wide Association Study ; Neuroglia ; Neurons ; Genomics ; *Nervous System Diseases/genetics ; },
abstract = {Our understanding of neurological diseases has been tremendously enhanced over the past decade by the application of new technologies. Genome-wide association studies have highlighted glial cells as important players in diseases. Single-cell profiling technologies are providing descriptions of disease states of neurons and glia at unprecedented molecular resolution. However, significant gaps remain in our understanding of the mechanisms driving disease-associated cell states, and how these states contribute to disease. These gaps in our understanding can be bridged by CRISPR-based functional genomics, a powerful approach to systematically interrogate gene function. In this review, we will briefly review the current literature on neurological disease-associated cell states and introduce CRISPR-based functional genomics. We discuss how advances in CRISPR-based screens, especially when implemented in the relevant brain cell types or cellular environments, have paved the way towards uncovering mechanisms underlying neurological disease-associated cell states. Finally, we will delineate current challenges and future directions for CRISPR-based functional genomics to further our understanding of neurological diseases and potential therapeutic strategies.},
}
@article {pmid36401282,
year = {2022},
author = {Bhat, AA and Nisar, S and Mukherjee, S and Saha, N and Yarravarapu, N and Lone, SN and Masoodi, T and Chauhan, R and Maacha, S and Bagga, P and Dhawan, P and Akil, AA and El-Rifai, W and Uddin, S and Reddy, R and Singh, M and Macha, MA and Haris, M},
title = {Integration of CRISPR/Cas9 with artificial intelligence for improved cancer therapeutics.},
journal = {Journal of translational medicine},
volume = {20},
number = {1},
pages = {534},
pmid = {36401282},
issn = {1479-5876},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Artificial Intelligence ; Gene Editing/methods ; Immunotherapy ; *Neoplasms/genetics/therapy ; },
abstract = {Gene editing has great potential in treating diseases caused by well-characterized molecular alterations. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene-editing tools has substantially improved the precision and efficiency of gene editing. The CRISPR/Cas9 system offers several advantages over the existing gene-editing approaches, such as its ability to target practically any genomic sequence, enabling the rapid development and deployment of novel CRISPR-mediated knock-out/knock-in methods. CRISPR/Cas9 has been widely used to develop cancer models, validate essential genes as druggable targets, study drug-resistance mechanisms, explore gene non-coding areas, and develop biomarkers. CRISPR gene editing can create more-effective chimeric antigen receptor (CAR)-T cells that are durable, cost-effective, and more readily available. However, further research is needed to define the CRISPR/Cas9 system's pros and cons, establish best practices, and determine social and ethical implications. This review summarizes recent CRISPR/Cas9 developments, particularly in cancer research and immunotherapy, and the potential of CRISPR/Cas9-based screening in developing cancer precision medicine and engineering models for targeted cancer therapy, highlighting the existing challenges and future directions. Lastly, we highlight the role of artificial intelligence in refining the CRISPR system's on-target and off-target effects, a critical factor for the broader application in cancer therapeutics.},
}
@article {pmid36401043,
year = {2023},
author = {Jin, Y and Shen, Y and Kim, IM and Weintraub, NL and Hamrick, M and Tang, Y},
title = {Restoration of Dystrophin Expression in Mdx-Derived Muscle Progenitor Cells Using CRISPR/Cas9 System and Homology-Directed Repair Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2587},
number = {},
pages = {455-464},
pmid = {36401043},
issn = {1940-6029},
support = {R01 HL146481/HL/NHLBI NIH HHS/United States ; R01 HL134354/HL/NHLBI NIH HHS/United States ; R01 HL086555/HL/NHLBI NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Dystrophin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mice, Inbred mdx ; Myoblasts/metabolism ; Muscles/metabolism ; Technology ; },
abstract = {Duchenne muscular dystrophy (DMD) is a progressive myopathy caused by mutations in genes encoding dystrophin proteins that ultimately lead to depletion of myogenic progenitor cells (MPCs). Several approaches have been used to correctly express the dystrophin gene in induced pluripotent stem cells (iPSCs), including deletion of mutated exon 23 (ΔEx23) by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated gene 9 (Cas9)-mediated gene editing technology. However, this approach is labor-intensive due to individual colony picking and genotyping to verify allelic modification. Here, we present a protocol to restore the function of the dystrophin gene by using homology-directed repair (HDR)-based CRISPR/Cas9 and inducing myogenic program of reprogrammed iPSCs from Mdx mice by inducible muscle-specific transcription factor MyoD.},
}
@article {pmid36401042,
year = {2023},
author = {Watanabe, K and Gee, P and Hotta, A},
title = {Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2587},
number = {},
pages = {427-453},
pmid = {36401042},
issn = {1940-6029},
mesh = {Animals ; RNA, Guide/genetics ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Ribonucleoproteins/genetics ; Exons/genetics ; Genomics ; *Extracellular Vesicles/genetics ; *HIV Infections/genetics ; },
abstract = {The CRISPR-Cas9 system has quickly become the standard tool for genome editing. To deliver this system to target cells, adeno-associated virus (AAV) vectors are commonly used. In fact, AAV vectors have been utilized to deliver the CRISPR-Cas9 system to induce genomic exon skipping and restore the dystrophin protein in various Duchenne muscular dystrophy model animals. Despite the high transduction efficiency, AAV vector-mediated delivery has several limitations, such as the packaging size, prolonged overexpression of Cas9, immunogenicity against the AAV capsid, and the risk of integrating a part of the AAV genomic sequence into the host cell. To overcome these issues, we have recently engineered a transient delivery system utilizing VSV-G pseudotyped extracellular vesicles (EVs) termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). NanoMEDIC utilizes an HIV-derived Gag protein to package Cas9 protein and gRNA into EVs. The Cas9 and Gag proteins are fused to a heterodimerizer and conditionally dimerized by the addition of an inducible chemical ligand to recruit Cas9 protein into EVs. sgRNA is packaged into EVs through an HIV-derived RNA packaging signal and is subsequently released by two self-cleaving ribozymes. Utilizing these features, NanoMEDIC can achieve highly efficient packaging of the Cas9 protein and gRNA for genome editing into a variety of target cells and in vivo. Here, we describe a step-by-step protocol, including the gRNA-expressing vector construction and large-scale NanoMEDIC production, for in vivo genome editing.},
}
@article {pmid36401041,
year = {2023},
author = {Zhang, Y and Bassel-Duby, R and Olson, EN},
title = {CRISPR-Cas9 Correction of Duchenne Muscular Dystrophy in Mice by a Self-Complementary AAV Delivery System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2587},
number = {},
pages = {411-425},
pmid = {36401041},
issn = {1940-6029},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Dystrophin/genetics/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy/metabolism ; Dependovirus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Exons ; Muscle, Skeletal/metabolism ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder, caused by mutations in the DMD gene coding dystrophin. Applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) for therapeutic gene editing represents a promising technology to correct this devastating disease through elimination of underlying genetic mutations. Adeno-associated virus (AAV) has been widely used for gene therapy due to its low immunogenicity and high tissue tropism. In particular, CRISPR-Cas9 gene editing components packaged by self-complementary AAV (scAAV) demonstrate robust viral transduction and efficient gene editing, enabling restoration of dystrophin expression throughout skeletal and cardiac muscle in animal models of DMD. Here, we describe protocols for cloning CRISPR single guide RNAs (sgRNAs) into a scAAV plasmid and procedures for systemic delivery of AAVs into a DMD mouse model. We also provide methodologies for quantification of dystrophin restoration after systemic CRISPR-Cas9-mediated correction of DMD.},
}
@article {pmid36401035,
year = {2023},
author = {Hakim, CH and Pérez-López, D and Burke, MJ and Teixeira, J and Duan, D},
title = {Molecular and Biochemical Assessment of Gene Therapy in the Canine Model of Duchenne Muscular Dystrophy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2587},
number = {},
pages = {255-301},
pmid = {36401035},
issn = {1940-6029},
mesh = {Animals ; Dogs ; *Muscular Dystrophy, Duchenne/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; Gene Editing/methods ; Dependovirus/genetics ; },
abstract = {Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease. Adeno-associated virus (AAV) mediated gene replacement, and CRISPR/Cas9-mediated genome editing hold the potential to treat DMD. Molecular and biochemical analyses are essential to determine gene transfer efficiency and therapeutic efficacy. In this chapter, we present a series of methods routinely used in our laboratory to extract and quantify DNA, RNA, and protein in gene therapy studies performed in the canine DMD model.},
}
@article {pmid36400778,
year = {2022},
author = {Zhao, Y and Hu, J and Yang, SS and Zhong, J and Liu, J and Wang, S and Jiao, Y and Jiang, F and Zhai, R and Ren, B and Cong, H and Zhu, Y and Han, F and Zhang, J and Xu, Y and Huang, Z and Zhang, S and Yang, F},
title = {A redox switch regulates the assembly and anti-CRISPR activity of AcrIIC1.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {7071},
pmid = {36400778},
issn = {2041-1723},
mesh = {*CRISPR-Associated Protein 9/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Oxidation-Reduction ; },
abstract = {Anti-CRISPRs (Acrs) are natural inhibitors of bacteria's CRISPR-Cas systems, and have been developed as a safeguard to reduce the off-target effects of CRISPR gene-editing technology. Acrs can directly bind to CRISPR-Cas complexes and inhibit their activities. However, whether this process is under regulation in diverse eukaryotic cellular environments is poorly understood. In this work, we report the discovery of a redox switch for NmeAcrIIC1, which regulates NmeAcrIIC1's monomer-dimer interconversion and inhibitory activity on Cas9. Further structural studies reveal that a pair of conserved cysteines mediates the formation of inactive NmeAcrIIC1 dimer and directs the redox cycle. The redox switch also applies to the other two AcrIIC1 orthologs. Moreover, by replacing the redox-sensitive cysteines, we generated a robust AcrIIC1 variant that maintains potent inhibitory activity under various redox conditions. Our results reveal a redox-dependent regulation mechanism of Acr, and shed light on the design of superior Acr for CRISPR-Cas systems.},
}
@article {pmid36400115,
year = {2022},
author = {Liu, X and Qiu, X and Han, L and Yue, Y and Xu, S and Li, F and Yao, J and Sun, L and Li, Z},
title = {Three novel Cas12a orthologs with robust DNA cleavage activity suitable for nucleic acid detection.},
journal = {Gene},
volume = {852},
number = {},
pages = {147055},
doi = {10.1016/j.gene.2022.147055},
pmid = {36400115},
issn = {1879-0038},
abstract = {Developing rapid and accurate pathogen detection methods is increasingly important, and CRISPR-Cas system can be optimized for this purpose. CRISPR-Cas12a is a single RNA-guided endonuclease system with the potential for nucleic acid detection. There is a broad diversity among Cas12a nucleases with robust detection capability. Herein, we characterised three Cas12a orthologs (ObCas12a, MbCas12a, and ScCas12a), including cis- and trans-cleavage activities, the identification of PAM, single-base resolution ability, and the application for nucleic acid detection. These Cas12a orthologs displayed robust cis- and trans-cleavage activities, and performed well in terms of specificity and sensitivity for nucleic acid detection. Furthermore, they have subtle differences in single-base resolution and recognised PAM sites in vitro. Therefore, these Cas12a nucleases are candidate proteins for CRISPR-based diagnostic methods. Addition of these enzymes to the nucleic acid detection toolbox will further expand the utility of this powerful technology.},
}
@article {pmid36399393,
year = {2022},
author = {Ki, J and Na, HK and Yoon, SW and Le, VP and Lee, TG and Lim, EK},
title = {CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever Virus.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.2c02007},
pmid = {36399393},
issn = {2379-3694},
abstract = {African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymatic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically separating the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the solution. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clinical specimens collected from porcine clinical tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective analytical technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools.},
}
@article {pmid36395770,
year = {2022},
author = {Mayo-Muñoz, D and Smith, LM and Garcia-Doval, C and Malone, LM and Harding, KR and Jackson, SA and Hampton, HG and Fagerlund, RD and Gumy, LF and Fineran, PC},
title = {Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection.},
journal = {Molecular cell},
volume = {82},
number = {23},
pages = {4471-4486.e9},
doi = {10.1016/j.molcel.2022.10.028},
pmid = {36395770},
issn = {1097-4164},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Cell Nucleus ; Chromosomes, Bacterial ; Endonucleases ; RNA, Messenger ; },
abstract = {Bacteria have diverse defenses against phages. In response, jumbo phages evade multiple DNA-targeting defenses by protecting their DNA inside a nucleus-like structure. We previously demonstrated that RNA-targeting type III CRISPR-Cas systems provide jumbo phage immunity by recognizing viral mRNA exported from the nucleus for translation. Here, we demonstrate that recognition of phage mRNA by the type III system activates a cyclic triadenylate-dependent accessory nuclease, NucC. Although unable to access phage DNA in the nucleus, NucC degrades the bacterial chromosome, triggers cell death, and disrupts phage replication and maturation. Hence, type-III-mediated jumbo phage immunity occurs via abortive infection, with suppression of the viral epidemic protecting the population. We further show that type III systems targeting jumbo phages have diverse accessory nucleases, including RNases that provide immunity. Our study demonstrates how type III CRISPR-Cas systems overcome the inaccessibility of jumbo phage DNA to provide robust immunity.},
}
@article {pmid36394005,
year = {2022},
author = {Ma, L and Li, Y},
title = {Editorial: CRISPR-based biosensors.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {1060249},
pmid = {36394005},
issn = {2296-4185},
}
@article {pmid36388977,
year = {2022},
author = {Kotta-Loizou, I and Giuliano, MG and Jovanovic, M and Schaefer, J and Ye, F and Zhang, N and Irakleidi, DA and Liu, X and Zhang, X and Buck, M and Engl, C},
title = {The RNA repair proteins RtcAB regulate transcription activator RtcR via its CRISPR-associated Rossmann fold domain.},
journal = {iScience},
volume = {25},
number = {11},
pages = {105425},
pmid = {36388977},
issn = {2589-0042},
abstract = {CRISPR-associated Rossmann fold (CARF) domain signaling underpins modulation of CRISPR-Cas nucleases; however, the RtcR CARF domain controls expression of two conserved RNA repair enzymes, cyclase RtcA and ligase RtcB. Here, we demonstrate that RtcAB are required for RtcR-dependent transcription activation and directly bind to RtcR CARF. RtcAB catalytic activity is not required for complex formation with CARF, but is essential yet not sufficient for RtcRAB-dependent transcription activation, implying the need for an additional RNA repair-dependent activating signal. This signal differs from oligoadenylates, a known ligand of CARF domains, and instead appears to originate from the translation apparatus: RtcB repairs a tmRNA that rescues stalled ribosomes and increases translation elongation speed. Taken together, our data provide evidence for an expanded range for CARF domain signaling, including the first evidence of its control via in trans protein-protein interactions, and a feed-forward mechanism to regulate RNA repair required for a functioning translation apparatus.},
}
@article {pmid36388608,
year = {2022},
author = {Juma, BS and Mukami, A and Mweu, C and Ngugi, MP and Mbinda, W},
title = {Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1009860},
pmid = {36388608},
issn = {1664-462X},
abstract = {Cassava is the world's most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content.},
}
@article {pmid36388532,
year = {2022},
author = {Singh, AK and Mishra, P and Kashyap, SP and Karkute, SG and Singh, PM and Rai, N and Bahadur, A and Behera, TK},
title = {Molecular insights into mechanisms underlying thermo-tolerance in tomato.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {1040532},
pmid = {36388532},
issn = {1664-462X},
abstract = {Plant productivity is being seriously compromised by climate-change-induced temperature extremities. Agriculture and food safety are threatened due to global warming, and in many cases the negative impacts have already begun. Heat stress leads to significant losses in yield due to changes in growth pattern, plant phonologies, sensitivity to pests, flowering, grain filling, maturity period shrinkage, and senescence. Tomato is the second most important vegetable crop. It is very sensitive to heat stress and thus, yield losses in tomato due to heat stress could affect food and nutritional security. Tomato plants respond to heat stress with a variety of cellular, physiological, and molecular responses, beginning with the early heat sensing, followed by signal transduction, antioxidant defense, osmolyte synthesis and regulated gene expression. Recent findings suggest that specific plant organs are extremely sensitive to heat compared to the entire plant, redirecting the research more towards generative tissues. This is because, during sexual reproduction, developing pollens are the most sensitive to heat. Often, just a few degrees of temperature elevation during pollen development can have a negative effect on crop production. Furthermore, recent research has discovered certain genetic and epigenetic mechanisms playing key role in thermo-tolerance and have defined new directions for tomato heat stress response (HSR). Present challenges are to increase the understanding of molecular mechanisms underlying HS, and to identify superior genotypes with more tolerance to extreme temperatures. Several metabolites, genes, heat shock factors (HSFs) and microRNAs work together to regulate the plant HSR. The present review provides an insight into molecular mechanisms of heat tolerance and current knowledge of genetic and epigenetic control of heat-tolerance in tomato for sustainable agriculture in the future. The information will significantly contribute to improve breeding programs for development of heat tolerant cultivars.},
}
@article {pmid36387576,
year = {2022},
author = {Hossain, TJ},
title = {Functional genomics of the lactic acid bacterium Limosilactobacillus fermentum LAB-1: metabolic, probiotic and biotechnological perspectives.},
journal = {Heliyon},
volume = {8},
number = {11},
pages = {e11412},
pmid = {36387576},
issn = {2405-8440},
abstract = {A genome-based systematic analysis was conducted to characterize the metabolic, probiotic, fitness, and safety properties of Limosilactobacillus fermentum LAB-1, a lactic acid bacterium demonstrating strong antimicrobial effects against clinical pathogens. Gene functional characterization revealed a large number of genes for carbohydrate metabolism and a heterofermentative system for carbon dissimilation. Genes for intact pyruvate oxidation, pentose phosphate, and PRPP biosynthetic pathways were identified. Substantial carbohydrate-active enzymes and transporters were also predicted. Metabolic reconstruction revealed complete sets of enzymes for arginine, lysine, methionine, threonine, proline, and ornithine biosynthesis. The bacterium harbors a diverse range of peptidases, and a large variety of peptide and amino acid uptake systems. It encodes restriction-modification and CRISPR-Cas systems for protection against phage infections and carries a wide spectrum of stress proteins for adaptation in the gut and industrial conditions. Genes related to the biosynthesis of B-group and K vitamins were identified allowing its application for novel bio-enriched food production. Other beneficial traits of probiotic and industrial importance such as production of flavor compounds, exopolysaccharide, acetoin, and butanediol were identified. Three antimicrobial peptides were predicted which showed >98% sequence-identity to experimentally validated bacteriocins. Negative traits such as transmissible antibiotic resistance, pathogenicity or virulence appeared to be absent suggesting the strain to be considered safe. The genome analysis will allow precisely targeted laboratory research and full exploitation of the probiotic potentials towards functional-food, biotechnology and health-related applications.},
}
@article {pmid36386633,
year = {2022},
author = {Chen, W and Wang, W and Wang, X and Li, Z and Wu, K and Li, X and Li, Y and Yi, L and Zhao, M and Ding, H and Fan, S and Chen, J},
title = {Advances in the differential molecular diagnosis of vesicular disease pathogens in swine.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {1019876},
pmid = {36386633},
issn = {1664-302X},
abstract = {Foot-and-mouth disease virus (FMDV), Senecavirus A (SVA) and swine vesicular disease virus (SVDV) are members of the family Picornaviridae, which can cause similar symptoms - vesicular lesions in the tissues of the mouth, nose, feet, skin and mucous membrane of animals. Rapid and accurate diagnosis of these viruses allows for control measures to prevent the spread of these diseases. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR are traditional and reliable methods for pathogen detection, while their amplification reaction requires a thermocycler. Isothermal amplification methods including loop-mediated isothermal amplification and recombinase polymerase amplification developed in recent years are simple, rapid and do not require specialized equipment, allowing for point of care diagnostics. Luminex technology allows for simultaneous detection of multiple pathogens. CRISPR-Cas diagnostic systems also emerging nucleic acid detection technologies which are very sensitivity and specificity. In this paper, various nucleic acid detection methods aimed at vesicular disease pathogens in swine (including FMDV, SVA and SVDV) are summarized.},
}
@article {pmid36385843,
year = {2022},
author = {Chakraborty, J and Chaudhary, AA and Khan, SU and Rudayni, HA and Rahaman, SM and Sarkar, H},
title = {CRISPR/Cas-Based Biosensor As a New Age Detection Method for Pathogenic Bacteria.},
journal = {ACS omega},
volume = {7},
number = {44},
pages = {39562-39573},
pmid = {36385843},
issn = {2470-1343},
abstract = {Methods enabling rapid and on-site detection of pathogenic bacteria are a prerequisite for public health assurance, medical diagnostics, ensuring food safety and security, and research. Many current bacteria detection technologies are inconvenient and time-consuming, making them unsuitable for field detection. New technology based on the CRISPR/Cas system has the potential to fill the existing gaps in detection. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a part of the bacterial adaptive immune system to protect them from intruding bacteriophages. The immunological memory is saved by the CRISPR array of bacteria in the form of short DNA sequences (spacers) from invading viruses and incorporated with the CRISPR DNA repeats. Cas proteins are responsible for triggering and initiating the adaptive immune function of CRISPR/Cas systems. In advanced biological research, the CRISPR/Cas system has emerged as a significant tool from genome editing to pathogen detection. By considering its sensitivity and specificity, this system can become one of the leading detection methods for targeting DNA/RNA. This technique is well applied in virus detection like Dengue, ZIKA, SARS-CoV-2, etc., but for bacterial detection, this CRISPR/Cas system is limited to only a few organisms to date. In this review, we have discussed the different techniques based on the CRISPR/Cas system that have been developed for the detection of various pathogenic bacteria like L. monocytogenes, M. tuberculosis, Methicillin-resistant S. aureus, Salmonella, E. coli, P. aeruginosa, and A. baumannii.},
}
@article {pmid36385310,
year = {2022},
author = {Watts, EA and Garrett, SC and Catchpole, RJ and Clark, LM and Graveley, BR and Terns, MP},
title = {Hyper-stimulation of Pyrococcus furiosus CRISPR DNA uptake by a self-transmissible plasmid.},
journal = {Extremophiles : life under extreme conditions},
volume = {26},
number = {3},
pages = {36},
pmid = {36385310},
issn = {1433-4909},
support = {R35GM118140/NH/NIH HHS/United States ; R35GM118160/NH/NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Pyrococcus furiosus/genetics ; CRISPR-Cas Systems ; Plasmids/genetics ; DNA/genetics ; },
abstract = {Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation.},
}
@article {pmid36384993,
year = {2022},
author = {Liu, Y and Zhou, XH and Huang, SH and Wang, XL},
title = {Prime editing: a search and replace tool with versatile base changes.},
journal = {Yi chuan = Hereditas},
volume = {44},
number = {11},
pages = {993-1008},
doi = {10.16288/j.yczz.22-156},
pmid = {36384993},
issn = {0253-9772},
mesh = {Animals ; *RNA, Guide/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants/genetics ; Point Mutation ; },
abstract = {Prime editing is a newly developed CRISPR/Cas system-based genome editing technique. The effector of prime editor (PE) is termed PE2, which is generated by fusing a reverse transcriptase (RT) with a Cas9 H840A nickase. The guide RNA of PE is termed prime editing guide RNA (pegRNA), which consists of a single guide RNA (sgRNA) with a 3' extension containing the RT template (RTT) and primer binding site (PBS). PE can install all 12 types of point mutations, small insertions and deletions and combinations thereof. Since its emergence in 2019, with the high versatility and specificity, PE has been applied to many living organisms, including animals, plants and bacteria. This led to many explorations of PE on gene therapy and genetic improvement in agriculture. In this review, we systematically describe the development, characteristics, optimizations, applications and security of PE. In addition, we discuss the future applications of PE. We expect that this review will help researchers to grasp and better use PE.},
}
@article {pmid36384669,
year = {2022},
author = {Zhang, XJ and Xu, K and Shen, JC and Mu, L and Qian, HR and Cui, JY and Ma, BX and Chen, ZL and Zhang, ZY and Wei, ZH},
title = {A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair.},
journal = {Yi chuan = Hereditas},
volume = {44},
number = {8},
pages = {708-719},
doi = {10.16288/j.yczz.22-118},
pmid = {36384669},
issn = {0253-9772},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Recombinational DNA Repair ; DNA ; },
abstract = {The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5'-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.},
}
@article {pmid36384163,
year = {2022},
author = {Roberts, A and Nethery, MA and Barrangou, R},
title = {Functional characterization of diverse type I-F CRISPR-associated transposons.},
journal = {Nucleic acids research},
volume = {50},
number = {20},
pages = {11670-11681},
doi = {10.1093/nar/gkac985},
pmid = {36384163},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Plasmids ; Endonucleases/metabolism ; Escherichia coli/genetics/metabolism ; DNA ; RNA ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.},
}
@article {pmid36383999,
year = {2022},
author = {Tang, C and Wu, J and Chen, Q and Wang, Y},
title = {CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus.},
journal = {Plant disease},
volume = {},
number = {},
pages = {},
doi = {10.1094/PDIS-07-22-1648-SR},
pmid = {36383999},
issn = {0191-2917},
abstract = {The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes significant damage to pine trees and thus poses a serious threat to pine forests worldwide, particularly in China, Korea, and Japan. A fast, affordable, and ultrasensitive detection of B. xylophilus is urgently needed for disease diagnosis. Recently clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have reshaped molecular diagnosis, with high speed, precision, specificity, strength, efficiency, and versatility. Herein, we established two isothermal diagnostics methods based on CRISPR-based platforms (CRISPR/Cas12a and CRISPR/Cas13a) for B. xylophilus-specific detection via fluorescence or lateral flow strip readout. The guide RNA (gRNA) and CRISPR RNA (crRNA) were designed to target the 5S rDNA intergenic spacer sequences (IGS) region of B. xylophilus. Recombinase-aided amplification (RAA) was used for pre-amplification whose reaction condition was 37 oC for 15 min. The sensitivity of CRISPR/Cas12a could reach 94 copies/μl of plasmid DNA, or 2.33 copies/μl of purified genomic DNA (gDNA) within 45 min at 37 oC, while the sensitivity of CRISPR/Cas13a was 1000 times higher than that of CRISPR/Cas12a of plasmid DNA in 15 min or 100 times higher of purified gDNA at the minimum reaction time of 4 min via fluorescence measurement. The CRISPR/Cas12a assay enabled the detection of 0.01 PWNs/100 mg of pine wood, 10-times higher than that of the CRISPR/Cas13a assay. This work enriches molecular detection approaches of B. xylophilus and provides huge potential for ultrasensitive and rapid methods to detect B. xylophilus in pine wood, facilitating point-of-sample diagnostic processing for pine wilt disease management.},
}
@article {pmid36380794,
year = {2022},
author = {Cantone, A and Sanguettoli, F and Dal Passo, B and Serenelli, M and Rapezzi, C},
title = {The treatment of amyloidosis is being refined.},
journal = {European heart journal supplements : journal of the European Society of Cardiology},
volume = {24},
number = {Suppl I},
pages = {I131-I138},
pmid = {36380794},
issn = {1520-765X},
abstract = {The therapy of transthyretin (TTR)-related cardiac amyloidosis consists, on the one hand, of the prevention and management of complications (supportive therapy) and on the other of treatments aimed at interrupting or slowing down the production and deposition of fibrils (disease-modifying therapy). This definition includes drugs that act on different phases of amyloidogenesis: (i) silencing of the gene encoding TTR (small interfering RNA: patisiran, vutrisiran; antisense oligonucleotides: inotersen, eplontersen; new CRISPR Cas-9 drug technology for editing in vivo DNA); (ii) stabilization of circulating TTR to inhibit its dissociation and subsequent assembly of the resulting monomers in amyloidotic fibrils (tafamidis, acoramidis, and tolcapone); (iii) destruction and re-absorption of already formed amyloid tissue deposits. Drugs related to the latter strategy (antibodies) are still the subject of Phase 1 or 2 studies.},
}
@article {pmid36379609,
year = {2022},
author = {Gao, H and Shang, Z and Chan, SY and Ma, D},
title = {Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {11},
pages = {881-898},
pmid = {36379609},
issn = {1862-1783},
mesh = {Humans ; *CRISPR-Cas Systems ; *Communicable Diseases ; Gene Editing/methods ; },
abstract = {Infectious diseases cause great economic loss and individual and even social anguish. Existing detection methods lack sensitivity and specificity, have a poor turnaround time, and are dependent on expensive equipment. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)‍-CRISPR-associated protein (Cas) system has been widely used in the detection of pathogens that cause infectious diseases owing to its high specificity, sensitivity, and speed, and good accessibility. In this review, we discuss the discovery and development of the CRISPR-Cas system, summarize related analysis and interpretation methods, and discuss the existing applications of CRISPR-based detection of infectious pathogens using Cas proteins. We conclude the challenges and prospects of the CRISPR-Cas system in the detection of pathogens.},
}
@article {pmid36378874,
year = {2022},
author = {Kiattisewee, C and Karanjia, AV and Legut, M and Daniloski, Z and Koplik, SE and Nelson, J and Kleinstiver, BP and Sanjana, NE and Carothers, JM and Zalatan, JG},
title = {Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.2c00405},
pmid = {36378874},
issn = {2161-5063},
abstract = {CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the trade-off that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.},
}
@article {pmid36378103,
year = {2022},
author = {Green, CM and Spangler, J and Susumu, K and Stenger, DA and Medintz, IL and Díaz, SA},
title = {Quantum Dot-Based Molecular Beacons for Quantitative Detection of Nucleic Acids with CRISPR/Cas(N) Nucleases.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.2c07749},
pmid = {36378103},
issn = {1936-086X},
abstract = {Strategies utilizing the CRISPR/Cas nucleases Cas13 and Cas12 have shown great promise in the development of highly sensitive and rapid diagnostic assays for the detection of pathogenic nucleic acids. The most common approaches utilizing fluorophore-quencher molecular beacons require strand amplification strategies or highly sensitive optical setups to overcome the limitations of the readout. Here, we demonstrate a flexible strategy for assembling highly luminescent and colorimetric quantum dot-nucleic acid hairpin (QD-HP) molecular beacons for use in CRISPR/Cas diagnostics. This strategy utilizes a chimeric peptide-peptide nucleic acid (peptide-PNA) to conjugate fluorescently labeled DNA or RNA hairpins to ZnS-coated QDs. QDs are particularly promising alternatives for molecular beacons due to their greater brightness, strong UV absorbance with large emission offset, exceptional photostability, and potential for multiplexing due to their sharp emission peaks. Using Förster resonance energy transfer (FRET), we have developed ratiometric reporters capable of pM target detection (without nucleotide amplification) for both target DNA and RNA, and we further demonstrated their capabilities for multiplexing and camera-phone detection. The flexibility of this system is imparted by the dual functionality of the QD as both a FRET donor and a central nanoscaffold for arranging nucleic acids and fluorescent acceptors on its surface. This method also provides a generalized approach that could be applied for use in other CRISPR/Cas nuclease systems.},
}
@article {pmid36374358,
year = {2022},
author = {Lebedeva, M and Komakhin, R and Konovalova, L and Ivanova, L and Taranov, V and Monakhova, Y and Babakov, A and Klepikova, A and Zlobin, N},
title = {Development of potato (Solanum tuberosum L.) plants with StLEAFY knockout.},
journal = {Planta},
volume = {256},
number = {6},
pages = {116},
pmid = {36374358},
issn = {1432-2048},
mesh = {*Solanum tuberosum/genetics ; Tetraploidy ; Meristem ; Inflorescence ; Flowers ; },
abstract = {StLFY-knockout potato plants were developed using CRISPR/Cas9 system. Inflorescences of edited plants transited to flowering, but inflorescence structures lacked flowers and were indeterminate, producing multiple shoot meristems. The tetraploid potato (Solanum tuberosum L.) is an important agricultural crop worldwide. In this study, we used CRISPR/Cas9 to inactivate the potato homolog (StLFY) of the LEAFY gene-a key regulator of the transition to flowering and floral meristem identity-in a tetraploid potato cultivar. We achieved high rates of all-allelic knockouts. Frameshift indels led to phenotypic alterations, including indeterminate inflorescence development and the replacement of flowers with the leafy-like structures.},
}
@article {pmid36372024,
year = {2023},
author = {Ma, L and Wang, J and Li, Y and Liao, D and Zhang, W and Han, X and Man, S},
title = {A ratiometric fluorescent biosensing platform for ultrasensitive detection of Salmonella typhimurium via CRISPR/Cas12a and silver nanoclusters.},
journal = {Journal of hazardous materials},
volume = {443},
number = {Pt B},
pages = {130234},
doi = {10.1016/j.jhazmat.2022.130234},
pmid = {36372024},
issn = {1873-3336},
mesh = {*Silver ; Salmonella typhimurium/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; *Biosensing Techniques/methods ; },
abstract = {Rapid, sensitive and specific detection of bacteria is of great importance. Herein, we developed a versatile biosensing platform for ultrasensitive detection of pathogenic bacteria, termed as SCENT-Cas (Silver nanoCluster Empowered Nucleic acids Test using CRISPR/Cas12a). Simply, the species-specific invA gene of Salmonella typhimurium (S. typhi) was isothermally amplified using LAMP, which subsequently triggered the trans-cleavage of CRISPR/Cas12a. The trans-cleavage degraded any single-stranded DNA (ssDNA) non-specifically. A DNA-templated AgNCs probe was then employed, in which green fluorescence emissive AgNCs effectively converted to red fluorescence emissive AgNCs when placed in close vicinity to a pre-designed converter ssDNA. As such, the trans-cleavage was utilized for shredding converter ssDNA, enabling the green-to-red fluorescent change to form a ratiometric biosensing platform. With this strategy, target nucleic acid was dexterously converted into ratiometric fluorescence that was recorded to detect as low as 1 CFU/mL S. typhi with a dynamic range from 1 to 10[8] CFU/mL. To our knowledge, this is the first report regarding the use of ratiometric fluorescence in CRISPR/Cas-based detection, which minimizes interference and improves reliability. Lastly, this proposed strategy was challenged by detecting S. typhi contamination in real food samples. Our work enriches CRISPR/Cas toolbox in biosensing by providing a desirable method for bacterial detection.},
}
@article {pmid36371895,
year = {2022},
author = {Bravo, JP and Hibshman, GN and Taylor, DW},
title = {Constructing next-generation CRISPR-Cas tools from structural blueprints.},
journal = {Current opinion in biotechnology},
volume = {78},
number = {},
pages = {102839},
doi = {10.1016/j.copbio.2022.102839},
pmid = {36371895},
issn = {1879-0429},
abstract = {Clustered regularly interspaced short palindromic repeats - CRISPR-associated protein (CRISPR-Cas) systems are a critical component of the bacterial adaptive immune response. Since the discovery that they can be reengineered as programmable RNA-guided nucleases, there has been significant interest in using these systems to perform diverse and precise genetic manipulations. Here, we outline recent advances in the mechanistic understanding of CRISPR-Cas9, how these findings have been leveraged in the rational redesign of Cas9 variants with altered activities, and how these novel tools can be exploited for biotechnology and therapeutics. We also discuss the potential of the ubiquitous, yet often-overlooked, multisubunit CRISPR effector complexes for large-scale genomic deletions. Furthermore, we highlight how future structural studies will bolster these technologies.},
}
@article {pmid36371870,
year = {2022},
author = {Ul Haq, SI and Zheng, D and Feng, N and Jiang, X and Qiao, F and He, JS and Qiu, QS},
title = {Progresses of CRISPR/Cas9 genome editing in forage crops.},
journal = {Journal of plant physiology},
volume = {279},
number = {},
pages = {153860},
doi = {10.1016/j.jplph.2022.153860},
pmid = {36371870},
issn = {1618-1328},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated-genome editing has evolved into a powerful tool that is widely used in plant species to induce editing in the genome for analyzing gene function and crop improvement. CRISPR/Cas9 is an RNA-guided genome editing tool consisting of a Cas9 nuclease and a single-guide RNA (sgRNA). The CRISPR/Cas9 system enables more accurate and efficient genome editing in crops. In this review, we summarized the advances of the CRISPR/Cas9 technology in plant genome editing and its applications in forage crops. We described briefly about the development of CRISPR/Cas9 technology in plant genome editing. We assessed the progress of CRISPR/Cas9-mediated targeted-mutagenesis in various forage crops, including alfalfa, Medicago truncatula, Hordeum vulgare, Sorghum bicolor, Setaria italica and Panicum virgatum. The potentials and challenges of CRISPR/Cas9 in forage breeding were discussed.},
}
@article {pmid36371486,
year = {2022},
author = {Siegner, SM and Ugalde, L and Clemens, A and Garcia-Garcia, L and Bueren, JA and Rio, P and Karasu, ME and Corn, JE},
title = {Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6900},
pmid = {36371486},
issn = {2041-1723},
mesh = {Humans ; *Fanconi Anemia/genetics/therapy/metabolism ; Adenine/metabolism ; Hematopoietic Stem Cells/metabolism ; Gene Editing ; DNA Repair ; },
abstract = {Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused by deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions leads to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from FA patients was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.},
}
@article {pmid36370369,
year = {2022},
author = {Pinto, DS and Prithvisagar, KS and Rohit, A and Karunasagar, I and Karunasagar, I and Kumar, BK},
title = {Genome analysis of clinical isolate of Campylobacter fetus subspecies fetus MMM01 from India reveals genetic determinants of pathogenesis and adaptation.},
journal = {Acta microbiologica et immunologica Hungarica},
volume = {69},
number = {4},
pages = {332-344},
doi = {10.1556/030.2022.01900},
pmid = {36370369},
issn = {1588-2640},
abstract = {In this study we report the whole genome sequencing (WGS) based analysis of blood-borne Campylobacter fetus subsp. fetus MMM01 isolated from a diabetic patient to obtain deeper insights in to the virulence and host adaptability. The sequenced genome of C. fetus subsp. fetus MMM01 along with reference genomes retrieved from NCBI was subjected to various in-silico analysis including JSpecies, MLST server, PATRIC server, VFanalyzer, CARD, PHASTER to understand their phylogenetic relation, virulence and antimicrobial resistance profile. The genome had a size of 1,788,790 bp, with a GC content of 33.09%, nearly identical to the reference strain C. fetus subsp. fetus 82-40. The MLST based phylogenetic tree constructed revealed the polyphyletic branching and MMM01 (ST25) was found to be closely related to ST11, both belong to the sap-A serotype which are more common in human infections. VFanalyzer identified 88 protein-coding genes coding for several virulence factors including Campylobacter adhesion to fibronectin, flagellar apparatus, cytolethal distending toxin operons and Campylobacter invasion antigen proteins which enhance the virulence of bacteria along with resistance genes against antibiotics including fluoroquinolone, chloramphenicol, tetracycline, and aminoglycoside in MMM01, which points to enhanced survival and pathogenicity of this zoonotic pathogen. It was interesting to find that MMM01 lacked FGI-II island found in most of the clinical isolates, which encoded CRISPR Cas and prophage II regions. More details about the complexity and evolution of this zoonotic pathogen could be learned from future studies that concentrate on comparative genome analysis using larger genome datasets.},
}
@article {pmid36370349,
year = {2023},
author = {Okusha, Y and Eguchi, T},
title = {Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2582},
number = {},
pages = {157-167},
pmid = {36370349},
issn = {1940-6029},
mesh = {*Matrix Metalloproteinase 3/genetics/metabolism ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Cell Communication/genetics ; Connective Tissue Growth Factor/metabolism ; },
abstract = {The Cellular Communication Network Factor (CCN) family is composed of six members: CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3. The second member, CCN2/CTGF is a matricellular protein that promotes extracellular matrix (ECM) synthesis and controls angiogenesis. On the other hand, moonlighting/matrix metalloproteinase 3 (MMP3) is an ECM-degrading enzyme that also functions as an intracellular transcription factor. Importantly, extracellular MMP3 is uptaken into cells, translocating into nuclei, and transcriptionally activating CCN2/CTGF gene in cancer and chondrocytes. Thus, the MMP3-CTGF axis balances the matrix metabolism and turnover in the tissue and tumor microenvironments. We established an MMP3 knockout cell line using the CRISPR/Cas9 system, demonstrating the sequential regulatory events of the MMP3-CCN2 axis in the microenvironment. Notably, our protocol is useful for generation of CCN knockout cells as well. Here we serve a protocol of the CRISPR/Cas9-based gene targeting in cultured cells for investigating cellular communication network.},
}
@article {pmid36370236,
year = {2022},
author = {Kerber-Diaz, JC and Leos-Ramírez, MA and Flores-Ceron, AA and Ponce-Mendoza, A and Estrada-de Los Santos, P and Ibarra, JA},
title = {Distribution of CRISPR-Cas systems in the Burkholderiaceae family and its biological implications.},
journal = {Archives of microbiology},
volume = {204},
number = {12},
pages = {703},
pmid = {36370236},
issn = {1432-072X},
mesh = {CRISPR-Cas Systems ; *Burkholderiaceae/genetics ; Plasmids ; Computational Biology ; *Bacteriophages/genetics ; Bacteria/genetics ; },
abstract = {CRISPR-Cas systems are composed of repeated sequences separated by non-repeated sequences that are near genes coding for Cas proteins, which are involved in the function of these systems. Their function has been mostly related to "genetic immunity" against foreign genetic material, among other roles. Interest in them increased after their use in genetic manipulation was uncovered and surveys to find and classify them have been done in several bacterial groups. To determine the presence of these genetic elements in the Burkholderiaceae family members, a bioinformatic approach was followed. Attention in this family comes as it is formed by a great diversity of microorganisms that include opportunistic and true pathogens, and symbiotic and saprophytic organisms, among others. Results show that, in contrast to other bacterial groups, only 8.4% of family members harbor complete CRISPR-Cas systems and the rest either do not have one or have remains or sections of one. Analyses of the spacer sequences indicated that most of them have identity to sections of the same genomes they were found, while a few had identities with either plasmids or phages. The genus with the higher proportion of self-directed spacers is Ralstonia, and their possible roles are discussed. Most of the systems (60%) belong to the class I subtype I-E and a few to subtypes I-C (13.3%), I-F (18.3%), II-C (5%), IV-A (1.7%) and V-C (1.7%). To the best of our knowledge, this is the first study to uncover the CRISPR-Cas system for the whole Burkholderiaceae family.},
}
@article {pmid36369279,
year = {2022},
author = {Kulcsár, PI and Tálas, A and Ligeti, Z and Krausz, SL and Welker, E},
title = {SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6858},
pmid = {36369279},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Endonucleases/genetics/metabolism ; DNA/genetics ; Cytosine ; },
abstract = {Several advancements have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased-fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high-fidelity SpCas9 variant, free from the drawbacks of first-generation IFNs. In this study, we characterize the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells, comparing it to first-generation IFNs. SuperFi-Cas9 demonstrates strongly reduced activity but high fidelity features that are in many aspects similar to those of some first-generation variants, such as evo- and HeFSpCas9. SuperFi-cytosine (CBE3) and -adenine (ABE7.10) base editors, as well as SuperFi-prime editor show no meaningful activity. When combined with ABE8e, SuperFi-Cas9, similarly to HeFSpCas9, executes DNA editing with high activity as well as high specificity reducing both bystander and SpCas9-dependent off-target base editing.},
}
@article {pmid36369085,
year = {2022},
author = {Su, J and Wang, P and Li, J and Zhao, D and Li, S and Fan, F and Dai, Z and Liao, X and Mao, Z and Zhang, C and Bi, C and Zhang, X},
title = {A CRISPR-based chromosomal-separation technique for Escherichia coli.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {235},
pmid = {36369085},
issn = {1475-2859},
mesh = {Humans ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Plasmids/genetics ; Chromosomes ; Synthetic Biology ; },
abstract = {BACKGROUND: Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains.<br><br>RESULTS: The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10&#xa0;Mb and 4.54&#xa0;Mb, and 2.28&#xa0;Mb and 2.36&#xa0;Mb were created respectively, designated as E. coli[0.10/4.54] and E. coli[2.28/2.36]. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli[0.10/4.54] was quite stable, the two chromosomes of E. coli[2.28/2.36] population recombined into a new chromosome (Chr.4.64M[Mut]), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated.<br><br>CONCLUSION: Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.},
}
@article {pmid36369053,
year = {2022},
author = {Van Tricht, C and Voet, T and Lammertyn, J and Spasic, D},
title = {Imaging the unimaginable: leveraging signal generation of CRISPR-Cas for sensitive genome imaging.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.10.003},
pmid = {36369053},
issn = {1879-3096},
abstract = {Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique.},
}
@article {pmid36368142,
year = {2023},
author = {Cui, C and Lau, CH and Chu, LT and Kwong, HK and Tin, C and Chen, TH},
title = {Multimodal detection of flap endonuclease 1 activity through CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides.},
journal = {Biosensors &amp; bioelectronics},
volume = {220},
number = {},
pages = {114859},
doi = {10.1016/j.bios.2022.114859},
pmid = {36368142},
issn = {1873-4235},
mesh = {*Flap Endonucleases/genetics/metabolism ; Oligonucleotides ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; DNA, Single-Stranded ; DNA/chemistry ; },
abstract = {Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.},
}
@article {pmid36367481,
year = {2022},
author = {Huang, T and Zhang, R and Li, J},
title = {CRISPR-Cas-based techniques for pathogen detection: Retrospect, recent advances, and future perspectives.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2022.10.011},
pmid = {36367481},
issn = {2090-1224},
abstract = {BACKGROUND: Early detection of pathogen-associated diseases are critical for effective treatment. Rapid, specific, sensitive, and cost-effective diagnostic technologies continue to be challenging to develop. The current gold standard for pathogen detection, polymerase chain reaction technology, has limitations such as long operational cycles, high cost, and high technician and instrumentation requirements.<br><br>AIM OF REVIEW: This review examines and highlights the technical advancements of CRISPR-Cas in pathogen detection and provides an outlook for future development, multi-application scenarios, and clinical translation.<br><br>Approaches enabling clinical detection of pathogen nucleic acids that are highly sensitive, specific, cheap, and portable are necessary. CRISPR-Cas9 specificity in targeting nucleic acids and "collateral cleavage" activity of CRISPR-Cas12/Cas13/Cas14 show significant promise in nucleic acid detection technology. These methods have a high specificity, versatility, and rapid detection cycle. In this paper, CRISPR-Cas-based detection methods are discussed in depth. Although CRISPR-Cas-mediated pathogen diagnostic solutions face challenges, their powerful capabilities will pave the way for ideal diagnostic tools.},
}
@article {pmid36367403,
year = {2022},
author = {Yan, J and Xu, Z and Zhou, H and Li, T and Du, X and Hu, R and Zhu, J and Ou, G and Li, Y and Yang, Y},
title = {Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping.},
journal = {Analytical chemistry},
volume = {94},
number = {47},
pages = {16481-16490},
doi = {10.1021/acs.analchem.2c04079},
pmid = {36367403},
issn = {1520-6882},
mesh = {Humans ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Nucleotidyltransferases/metabolism ; },
abstract = {Fast and on-site detection is important for an effective antigene-doping strategy. However, the current gene doping (GD) evaluation methods require sophisticated instruments and laborious procedures, limiting their field applications. This study proposes a CRISPR/Cas12a-based detection platform (termed CasGDP) combining CRISPR/Cas12a and multiplexed Recombinase Polymerase Amplification (RPA) for rapid evaluation of GD. CasGDP showed high specificity for identifying the putative target genes such as EPO, IGF-1, and GH-1. By using fluorescence as the readout, the method achieved a limit-of-detection of 0.1 nM and 1 aM for unamplified and amplified target plasmids, respectively. Additionally, an in vitro GD cell model was successfully established with the human EPO gene (hEPO). The results indicated that the hEPO gene transfection promoted the hEPO protein expression. Furthermore, trace amounts of EPO transgene spiked in human serum were efficiently measured by CasGDP with fluorescence- and lateral flow device (LFD)-based readouts in 40 min. Finally, we designed a multiplexed microfluidic device and realized simultaneous detection of the three transgenes via LFD embedded in the device. To our knowledge, this is the first work that combines the CRISPR-based system and multiplexed RPA for GD detection. We anticipate CasGDP to be widely used as a rapid, sensitive, and robust tool for GD evaluation.},
}
@article {pmid36365450,
year = {2022},
author = {Chu, P and Agapito-Tenfen, SZ},
title = {Unintended Genomic Outcomes in Current and Next Generation GM Techniques: A Systematic Review.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {21},
pages = {},
pmid = {36365450},
issn = {2223-7747},
abstract = {Classical genetic engineering and new genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities for modifying the genetic material in organisms. These technologies have the potential to provide novel agricultural traits, including modified microorganisms and environmental applications. However, legitimate safety concerns arise from the unintended genetic modifications (GM) that have been reported as side-effects of such techniques. Here, we systematically review the scientific literature for studies that have investigated unintended genomic alterations in plants modified by the following GM techniques: Agrobacterium tumefaciens-mediated gene transfer, biolistic bombardment, and CRISPR-Cas9 delivered via Agrobacterium-mediated gene transfer (DNA-based), biolistic bombardment (DNA-based) and as ribonucleoprotein complexes (RNPs). The results of our literature review show that the impact of such techniques in host genomes varies from small nucleotide polymorphisms to large genomic variation, such as segmental duplication, chromosome truncation, trisomy, chromothripsis, breakage fusion bridge, including large rearrangements of DNA vector-backbone sequences. We have also reviewed the type of analytical method applied to investigate the genomic alterations and found that only five articles used whole genome sequencing in their analysis methods. In addition, larger structural variations detected in some studies would not be possible without long-read sequencing strategies, which shows a potential underestimation of such effects in the literature. As new technologies are constantly evolving, a more thorough examination of prospective analytical methods should be conducted in the future. This will provide regulators working in the field of genetically modified and gene-edited organisms with valuable information on the ability to detect and identify genomic interventions.},
}
@article {pmid36365188,
year = {2022},
author = {Srivastava, P and Kim, KS},
title = {Membrane Vesicles Derived from Gut Microbiota and Probiotics: Cutting-Edge Therapeutic Approaches for Multidrug-Resistant Superbugs Linked to Neurological Anomalies.},
journal = {Pharmaceutics},
volume = {14},
number = {11},
pages = {},
pmid = {36365188},
issn = {1999-4923},
abstract = {Multidrug-resistant (MDR) superbugs can breach the blood-brain barrier (BBB), leading to a continuous barrage of pro-inflammatory modulators and induction of severe infection-related pathologies, including meningitis and brain abscess. Both broad-spectrum or species-specific antibiotics (β-lactamase inhibitors, polymyxins, vancomycin, meropenem, plazomicin, and sarecycline) and biocompatible poly (lactic-co-glycolic acid) (PLGA) nanoparticles have been used to treat these infections. However, new therapeutic platforms with a broad impact that do not exert off-target deleterious effects are needed. Membrane vesicles or extracellular vesicles (EVs) are lipid bilayer-enclosed particles with therapeutic potential owing to their ability to circumvent BBB constraints. Bacteria-derived EVs (bEVs) from gut microbiota are efficient transporters that can penetrate the central nervous system. In fact, bEVs can be remodeled via surface modification and CRISPR/Cas editing and, thus, represent a novel platform for conferring protection against infections breaching the BBB. Here, we discuss the latest scientific research related to gut microbiota- and probiotic-derived bEVs, and their therapeutic modifications, in terms of regulating neurotransmitters and inhibiting quorum sensing, for the treatment of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases. We also emphasize the benefits of probiotic-derived bEVs to human health and propose a novel direction for the development of innovative heterologous expression systems to combat BBB-crossing pathogens.},
}
@article {pmid36363691,
year = {2022},
author = {González-Vázquez, R and Zúñiga-León, E and Torres-Maravilla, E and Leyte-Lugo, M and Mendoza-Pérez, F and Hernández-Delgado, NC and Pérez-Pastén-Borja, R and Azaola-Espinosa, A and Mayorga-Reyes, L},
title = {Genomic and Biochemical Characterization of Bifidobacterium pseudocatenulatum JCLA3 Isolated from Human Intestine.},
journal = {Microorganisms},
volume = {10},
number = {11},
pages = {},
pmid = {36363691},
issn = {2076-2607},
abstract = {Bifidobacteria have been investigated due to their mutualistic microbe-host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5-5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3.},
}
@article {pmid36362256,
year = {2022},
author = {Sakovina, L and Vokhtantsev, I and Vorobyeva, M and Vorobyev, P and Novopashina, D},
title = {Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.},
journal = {International journal of molecular sciences},
volume = {23},
number = {21},
pages = {},
pmid = {36362256},
issn = {1422-0067},
mesh = {*RNA, Guide/genetics ; *CRISPR-Cas Systems/genetics ; Nucleotides ; Gene Editing/methods ; Endonucleases/genetics ; },
abstract = {The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2'-fluoro modified crRNA. The 2'-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.},
}
@article {pmid36362061,
year = {2022},
author = {Prudencio, AS and Devin, SR and Mahdavi, SME and Martínez-García, PJ and Salazar, JA and Martínez-Gómez, P},
title = {Spontaneous, Artificial, and Genome Editing-Mediated Mutations in Prunus.},
journal = {International journal of molecular sciences},
volume = {23},
number = {21},
pages = {},
pmid = {36362061},
issn = {1422-0067},
mesh = {Animals ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; *Prunus/genetics/metabolism ; Plant Breeding ; Mutation ; Endonucleases/metabolism ; Genome, Plant ; },
abstract = {Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the Prunus species, as well as in the rest of crop species. Mutation can occur in nature at a very low frequency or can be induced artificially. Spontaneous or bud sport mutations in somatic cells can be vegetatively propagated to get an individual with the mutant phenotype. Unlike animals, plants have unlimited growth and totipotent cells that let somatic mutations to be transmitted to the progeny. On the other hand, in vitro tissue culture makes it possible to induce mutation in plant material and perform large screenings for mutant's selection and cleaning of chimeras. Finally, targeted mutagenesis has been boosted by the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 and Transcription activator-like effector nuclease (TALEN) editing technologies. Over the last few decades, environmental stressors such as global warming have been threatening the supply of global demand for food based on population growth in the near future. For this purpose, the release of new varieties adapted to such changes is a requisite, and selected or generated Prunus mutants by properly regulated mechanisms could be helpful to this task. In this work, we reviewed the most relevant mutations for breeding traits in Prunus species such as flowering time, self-compatibility, fruit quality, and disease tolerance, including new molecular perspectives in the present postgenomic era including CRISPR/Cas9 and TALEN editing technologies.},
}
@article {pmid36361828,
year = {2022},
author = {Zhang, Z and Xiong, L and Xie, C and Shen, L and Chen, X and Ye, M and Sun, L and Yang, X and Yao, S and Yue, Z and Liang, Z and You, M and You, S},
title = {Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth.},
journal = {International journal of molecular sciences},
volume = {23},
number = {21},
pages = {},
pmid = {36361828},
issn = {1422-0067},
mesh = {Animals ; *Gene Editing ; *Moths/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; Promoter Regions, Genetic ; },
abstract = {The CRISPR/Cas9 system is an efficient tool for reverse genetics validation, and the application of this system in the cell lines provides a new perspective on target gene analysis for the development of biotechnology tools. However, in the cell lines of diamondback moth, Plutella xylostella, the integrity of the CRISPR/Cas9 system and the utilization of this cell lines still need to be improved to ensure the application of the system. Here, we stabilize the transfection efficiency of the P. xylostella cell lines at different passages at about 60% by trying different transfection reagents and adjusting the transfection method. For Cas9 expression in the CRIPSPR/Cas9 system, we identified a strong endogenous promoter: the 217-2 promoter. The dual-luciferase and EGFP reporter assay demonstrated that it has a driving efficiency close to that of the IE1 promoter. We constructed pB-Cas9-Neo plasmid and pU6-sgRNA plasmid for CRISPR/Cas9 system and subsequent cell screening. The feasibility of the CRISPR/Cas9 system in P. xylostella cell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line of P. xylostella that would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in the P. xylostella cell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.},
}
@article {pmid36361647,
year = {2022},
author = {Mu, Y and Zhang, C and Li, T and Jin, FJ and Sung, YJ and Oh, HM and Lee, HG and Jin, L},
title = {Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus.},
journal = {International journal of molecular sciences},
volume = {23},
number = {21},
pages = {},
pmid = {36361647},
issn = {1422-0067},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Lactobacillus/genetics ; Mammals/genetics ; },
abstract = {Lactobacillus, a genus of lactic acid bacteria, plays a crucial function in food production preservation, and probiotics. It is particularly important to develop new Lactobacillus strains with superior performance by gene editing. Currently, the identification of its functional genes and the mining of excellent functional genes mainly rely on the traditional gene homologous recombination technology. CRISPR/Cas9-based genome editing is a rapidly developing technology in recent years. It has been widely applied in mammalian cells, plants, yeast, and other eukaryotes, but less in prokaryotes, especially Lactobacillus. Compared with the traditional strain improvement methods, CRISPR/Cas9-based genome editing can greatly improve the accuracy of Lactobacillus target sites and achieve traceless genome modification. The strains obtained by this technology may even be more efficient than the traditional random mutation methods. This review examines the application and current issues of CRISPR/Cas9-based genome editing in Lactobacillus, as well as the development trend of CRISPR/Cas9-based genome editing in Lactobacillus. In addition, the fundamental mechanisms of CRISPR/Cas9-based genome editing are also presented and summarized.},
}
@article {pmid36361580,
year = {2022},
author = {Zhang, Y and Cai, Y and Sun, S and Han, T and Chen, L and Hou, W},
title = {Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean.},
journal = {International journal of molecular sciences},
volume = {23},
number = {21},
pages = {},
pmid = {36361580},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Staphylococcus aureus/metabolism ; Soybeans/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean (Glycinemax). The Streptococcuspyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5'-NGG-3' (SpCas9) or 5'-TTTN-3' (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5'-NNGRRT-3' (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5'-AAGGGT-3', 5'-GGGGAT-3', 5'-TTGAAT-3', and 5'-TAGGGT-3' in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.},
}
@article {pmid36361556,
year = {2022},
author = {Iordache, D and Baci, GM and Căpriță, O and Farkas, A and Lup, A and Butiuc-Keul, A},
title = {Correlation between CRISPR Loci Diversity in Three Enterobacterial Taxa.},
journal = {International journal of molecular sciences},
volume = {23},
number = {21},
pages = {},
pmid = {36361556},
issn = {1422-0067},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; *Enterobacteriaceae/genetics ; Escherichia coli/genetics ; Klebsiella pneumoniae/genetics ; Animals ; },
abstract = {CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains).},
}
@article {pmid36360266,
year = {2022},
author = {Almeida, RS and Wisnieski, F and Takao Real Karia, B and Smith, MAC},
title = {CRISPR/Cas9 Genome-Editing Technology and Potential Clinical Application in Gastric Cancer.},
journal = {Genes},
volume = {13},
number = {11},
pages = {},
pmid = {36360266},
issn = {2073-4425},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Stomach Neoplasms/genetics/therapy ; Quality of Life ; Technology ; },
abstract = {Gastric cancer is the subject of clinical and basic studies due to its high incidence and mortality rates worldwide. Due to the diagnosis occurring in advanced stages and the classic treatment methodologies such as gastrectomy and chemotherapy, they are extremely aggressive and limit the quality of life of these patients. CRISPR/Cas9 is a tool that allows gene editing and has been used to explore the functions of genes related to gastric cancer, in addition to being used in the treatment of this neoplasm, greatly increasing our understanding of cancer genomics. In this mini-review, we seek the current status of the CRISPR/Cas9 gene-editing technology in gastric cancer research and clinical research.},
}
@article {pmid36360244,
year = {2022},
author = {Zhang, X},
title = {Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry.},
journal = {Genes},
volume = {13},
number = {11},
pages = {},
pmid = {36360244},
issn = {2073-4425},
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Livestock/genetics ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids/genetics ; },
abstract = {The rapid rate of virus transmission and pathogen mutation and evolution highlight the necessity for innovative approaches to the diagnosis and prevention of infectious diseases. Traditional technologies for pathogen detection, mostly PCR-based, involve costly/advanced equipment and skilled personnel and are therefore not feasible in resource-limited areas. Over the years, many promising methods based on clustered regularly interspaced short palindromic repeats and the associated protein systems (CRISPR/Cas), i.e., orthologues of Cas9, Cas12, Cas13 and Cas14, have been reported for nucleic acid detection. CRISPR/Cas effectors can provide one-tube reaction systems, amplification-free strategies, simultaneous multiplex pathogen detection, visual colorimetric detection, and quantitative identification as alternatives to quantitative PCR (qPCR). This review summarizes the current development of CRISPR/Cas-mediated molecular diagnostics, as well as their design software and readout methods, highlighting technical improvements for integrating CRISPR/Cas technologies into on-site applications. It further highlights recent applications of CRISPR/Cas-based nucleic acid detection in livestock industry, including emerging infectious diseases, authenticity and composition of meat/milk products, as well as sex determination of early embryos.},
}
@article {pmid36359866,
year = {2022},
author = {Wang, Y and Tang, Q and Kang, Y and Wang, X and Zhang, H and Li, X},
title = {Analysis of the Utilization and Prospects of CRISPR-Cas Technology in the Annotation of Gene Function and Creation New Germplasm in Maize Based on Patent Data.},
journal = {Cells},
volume = {11},
number = {21},
pages = {},
pmid = {36359866},
issn = {2073-4409},
mesh = {*Zea mays/genetics ; *CRISPR-Cas Systems/genetics ; Edible Grain ; Phenotype ; Technology ; },
abstract = {Maize (Zea mays L.) is a food crop with the largest planting area and the highest yield in the world, and it plays a vital role in ensuring global food security. Conventional breeding methods are costly, time-consuming, and ineffective in maize breeding. In recent years, CRISPR-Cas editing technology has been used to quickly generate new varieties with high yield and improved grain quality and stress resistance by precisely modifying key genes involved in specific traits, thus becoming a new engine for promoting crop breeding and the competitiveness of seed industries. Using CRISPR-Cas, a range of new maize materials with high yield, improved grain quality, ideal plant type and flowering period, male sterility, and stress resistance have been created. Moreover, many patents have been filed worldwide, reflecting the huge practical application prospects and commercial value. Based on the existing patent data, we analyzed the development process, current status, and prospects of CRISPR-Cas technology in dissecting gene function and creating new germplasm in maize, providing information for future basic research and commercial production.},
}
@article {pmid36358971,
year = {2022},
author = {Aiba, W and Amai, T and Ueda, M and Kuroda, K},
title = {Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases.},
journal = {Biomolecules},
volume = {12},
number = {11},
pages = {},
pmid = {36358971},
issn = {2218-273X},
mesh = {*Gene Editing/methods ; *RNA, Guide/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA ; },
abstract = {In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing.},
}
@article {pmid36357583,
year = {2022},
author = {Ledford, H},
title = {CRISPR cancer trial success paves the way for personalized treatments.},
journal = {Nature},
volume = {611},
number = {7936},
pages = {433-434},
pmid = {36357583},
issn = {1476-4687},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; *Precision Medicine/trends ; },
}
@article {pmid36356930,
year = {2022},
author = {Fachel, FNS and Frâncio, L and Poletto, &#xc9; and Schuh, RS and Teixeira, HF and Giugliani, R and Baldo, G and Matte, U},
title = {Gene editing strategies to treat lysosomal disorders: The example of mucopolysaccharidoses.},
journal = {Advanced drug delivery reviews},
volume = {191},
number = {},
pages = {114616},
doi = {10.1016/j.addr.2022.114616},
pmid = {36356930},
issn = {1872-8294},
mesh = {Animals ; Gene Editing ; *Mucopolysaccharidoses/genetics/therapy/diagnosis ; *Lysosomal Storage Diseases/therapy/drug therapy ; Enzyme Replacement Therapy/methods ; Lysosomes ; },
abstract = {Lysosomal storage disorders are a group of progressive multisystemic hereditary diseases with a combined incidence of 1:4,800. Here we review the clinical and molecular characteristics of these diseases, with a special focus on Mucopolysaccharidoses, caused primarily by the lysosomal storage of glycosaminoglycans. Different gene editing techniques can be used to ameliorate their symptoms, using both viral and nonviral delivery methods. Whereas these are still being tested in animal models, early results of phase I/II clinical trials of gene therapy show how this technology may impact the future treatment of these diseases. Hurdles related to specific hard-to-reach organs, such as the central nervous system, heart, joints, and the eye must be tackled. Finally, the regulatory framework necessary to advance into clinical practice is also discussed.},
}
@article {pmid36355258,
year = {2022},
author = {Quijano-Rubio, C and Silginer, M and Weller, M},
title = {CRISPR/Cas9-mediated abrogation of CD95L/CD95 signaling-induced glioma cell growth and immunosuppression increases survival in murine glioma models.},
journal = {Journal of neuro-oncology},
volume = {160},
number = {2},
pages = {299-310},
pmid = {36355258},
issn = {1573-7373},
mesh = {Mice ; Animals ; fas Receptor ; CRISPR-Cas Systems ; *Glioma/pathology ; *Brain Neoplasms/pathology ; *Glioblastoma/pathology ; Apoptosis/physiology ; Immunosuppression Therapy ; },
abstract = {PURPOSE: Glioblastoma is the most common brain tumor in adults and is virtually incurable. Therefore, new therapeutic strategies are urgently needed. Over the last decade, multiple growth-promoting functions have been attributed to CD95, a prototypic death receptor well characterized as an apoptosis mediator upon CD95L engagement. Strategic targeting of non-apoptotic or apoptotic CD95 signaling may hold anti-glioblastoma potential. Due to its antithetic nature, understanding the constitutive role of CD95 signaling in glioblastoma is indispensable.<br><br>METHODS: We abrogated constitutive Cd95 and Cd95l gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo.<br><br>RESULTS: Expression of canonical CD95 but not CD95L was identified in mouse glioma cells in vitro. Instead, a soluble isoform-encoding non-canonical Cd95l transcript variant was detected. In vivo, an upregulation of the membrane-bound canonical CD95L form was revealed. Cd95 or Cd95l gene deletion decreased cell growth in vitro. The growth-supporting role of constitutive CD95 signaling was validated by Cd95 re-transfection, which rescued growth. In vivo, Cd95 or Cd95l gene deletion prolonged survival involving tumor-intrinsic and immunological mechanisms in the SMA-497 model. In the GL-261 model, that expresses no CD95, only CD95L gene deletion prolonged survival, involving a tumor-intrinsic mechanism.<br><br>CONCLUSION: Non-canonical CD95L/CD95 interactions are growth-promoting in murine glioma models, and glioma growth and immunosuppression may be simultaneously counteracted by Cd95l gene silencing.},
}
@article {pmid36354495,
year = {2022},
author = {Janghorban, M and Aradanas, I and Kazemi, S and Ngaju, P and Pandey, R},
title = {Recent Advances, Opportunities, and Challenges in Developing Nucleic Acid Integrated Wearable Biosensors for Expanding the Capabilities of Wearable Technologies in Health Monitoring.},
journal = {Biosensors},
volume = {12},
number = {11},
pages = {},
pmid = {36354495},
issn = {2079-6374},
mesh = {*Nucleic Acids ; *Wearable Electronic Devices ; *Biosensing Techniques ; Point-of-Care Testing ; },
abstract = {Wearable biosensors are becoming increasingly popular due to the rise in demand for non-invasive, real-time monitoring of health and personalized medicine. Traditionally, wearable biosensors have explored protein-based enzymatic and affinity-based detection strategies. However, in the past decade, with the success of nucleic acid-based point-of-care diagnostics, a paradigm shift has been observed in integrating nucleic acid-based assays into wearable sensors, offering better stability, enhanced analytical performance, and better clinical applicability. This narrative review builds upon the current state and advances in utilizing nucleic acid-based assays, including oligonucleotides, nucleic acid, aptamers, and CRISPR-Cas, in wearable biosensing. The review also discusses the three fundamental blocks, i.e., fabrication requirements, biomolecule integration, and transduction mechanism, for creating nucleic acid integrated wearable biosensors.},
}
@article {pmid36351608,
year = {2022},
author = {Kiga, K},
title = {[RNA functions in bacterial infections and its application to antimicrobial therapy].},
journal = {Nihon saikingaku zasshi. Japanese journal of bacteriology},
volume = {77},
number = {3},
pages = {139-144},
doi = {10.3412/jsb.77.139},
pmid = {36351608},
issn = {1882-4110},
mesh = {Humans ; RNA/genetics ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Anti-Bacterial Agents/therapeutic use ; *Anti-Infective Agents ; *Staphylococcal Infections/genetics ; },
abstract = {In the concept of central dogma (RNA is transcribed from DNA to produce proteins), RNA was thought to be merely an intermediary for genetic information to synthesize proteins from DNA. Since the discovery of RNA interference in 2000, research on RNA has progressed remarkably, especially in mammals. On the other hand, the role of RNA in bacterial infections was largely unknown. At that time, we started research on RNA and bacterial infection and revealed that miR-210, a small RNA in the gastric epithelial cells, is involved in gastric diseases caused by Helicobacter pylori in-fection. Furthermore, we have successfully developed sequence-specific antimicrobials by loading CRISPR-Cas13, an RNA-targeting CRISPR-Cas, on bacteriophage. The constructed antimicrobials were effective against at least Escherichia coli and Staphylococcus aureus. In this paper, we would like to introduce the importance of RNA in bacteriology.},
}
@article {pmid36350608,
year = {2022},
author = {Fan, J and Shi, L and Liu, Q and Zhu, Z and Wang, F and Song, R and Su, J and Zhou, D and Chen, X and Li, K and Xue, L and Sun, L and Mao, F},
title = {Annotation and evaluation of base editing outcomes in multiple cell types using CRISPRbase.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkac967},
pmid = {36350608},
issn = {1362-4962},
abstract = {CRISPR-Cas base editing (BE) system is a powerful tool to expand the scope and efficiency of genome editing with single-nucleotide resolution. The editing efficiency, product purity, and off-target effect differ among various BE systems. Herein, we developed CRISPRbase (http://crisprbase.maolab.org), by integrating 1 252 935 records of base editing outcomes in more than 50 cell types from 17 species. CRISPRbase helps to evaluate the putative editing precision of different BE systems by integrating multiple annotations, functional predictions and a blasting system for single-guide RNA sequences. We systematically assessed the editing window, editing efficiency and product purity of various BE systems. Intensive efforts were focused on increasing the editing efficiency and product purity of base editors since the byproduct could be detrimental in certain applications. Remarkably, more than half of cancer-related off-target mutations were non-synonymous and extremely damaging to protein functions in most common tumor types. Luckily, most of these cancer-related mutations were passenger mutations (4840/5703, 84.87%) rather than cancer driver mutations (863/5703, 15.13%), indicating a weak effect of off-target mutations on carcinogenesis. In summary, CRISPRbase is a powerful and convenient tool to study the outcomes of different base editors and help researchers choose appropriate BE designs for functional studies.},
}
@article {pmid36350521,
year = {2022},
author = {Cortés-Mancera, FM and Sarno, F and Goubert, D and Rots, MG},
title = {Gene-Targeted DNA Methylation: Towards Long-Lasting Reprogramming of Gene Expression?.},
journal = {Advances in experimental medicine and biology},
volume = {1389},
number = {},
pages = {515-533},
pmid = {36350521},
issn = {0065-2598},
mesh = {Humans ; *DNA Methylation/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Gene Expression ; },
abstract = {DNA methylation is an essential epigenetic mark, strongly associated with gene expression regulation. Aberrant DNA methylation patterns underlie various diseases and efforts to intervene with DNA methylation signatures are of great clinical interest. Technological developments to target writers or erasers of DNA methylation to specific genomic loci by epigenetic editing resulted in successful gene expression modulation, also in in vivo models. Application of epigenetic editing in human health could have a huge impact, but clinical translation is still challenging. Despite successes for a wide variety of genes, not all genes mitotically maintain their (de)methylation signatures after editing, and reprogramming requires further understanding of chromatin context-dependency. In addition, difficulties of current delivery systems and off-target effects are hurdles to be tackled. The present review describes findings towards effective and sustained DNA (de)methylation by epigenetic editing and discusses the need for multi-effector approaches to achieve highly efficient long-lasting reprogramming.},
}
@article {pmid36347819,
year = {2022},
author = {Dai, P and Hu, D},
title = {The making of hypervirulent Klebsiella pneumoniae.},
journal = {Journal of clinical laboratory analysis},
volume = {},
number = {},
pages = {e24743},
doi = {10.1002/jcla.24743},
pmid = {36347819},
issn = {1098-2825},
abstract = {Klebsiella pneumoniae is a notorious bacterium in clinical practice. Virulence, carbapenem-resistance and their convergence among K. pneumoniae are extensively discussed in this article. Hypervirulent K. pneumoniae (HvKP) has spread from the Asian Pacific Rim to the world, inducing various invasive infections, such as pyogenic liver abscess, endophthalmitis, and meningitis. Furthermore, HvKP has acquired more and more drug resistance. Among multidrug-resistant HvKP, hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP), and carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) are both devastating for their extreme drug resistance and virulence. The hypervirulence of HvKP is primarily attributed to hypercapsule, macromolecular exopolysaccharides, or excessive siderophores, although it has many other factors, for example, lipopolysaccharides, fimbriae, and porins. In contrast with classical determination of HvKP, that is, animal lethality test, molecular determination could be an optional and practical method after improvement. HvKP, including Hv-CRKP and CR-HvKP, has been progressing. R-M and CRISPR-Cas systems may play pivotal roles in such evolutions. Hv-CRKP and CR-HvKP, in particular the former, should be of severe concern due to their being more and more prevalent.},
}
@article {pmid36347254,
year = {2022},
author = {Funk, L and Su, KC and Ly, J and Feldman, D and Singh, A and Moodie, B and Blainey, PC and Cheeseman, IM},
title = {The phenotypic landscape of essential human genes.},
journal = {Cell},
volume = {185},
number = {24},
pages = {4634-4653.e22},
doi = {10.1016/j.cell.2022.10.017},
pmid = {36347254},
issn = {1097-4172},
support = {R01 HG009283/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Genes, Essential ; HeLa Cells ; Gene Knockout Techniques ; Phenotype ; },
abstract = {Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.},
}
@article {pmid36346319,
year = {2022},
author = {Zhang, L and Ma, J and Jin, X and Zhang, L and Zhang, M and Li, PZ and Li, J and Zhang, L},
title = {Human IFNAR2 Mutant Generated by CRISPR/Cas9-Induced Exon Skipping Upregulates a Subset of Tonic-Like Interferon-Stimulated Genes Upon IFNβ Stimulation.},
journal = {Journal of interferon &amp; cytokine research : the official journal of the International Society for Interferon and Cytokine Research},
volume = {42},
number = {11},
pages = {580-589},
doi = {10.1089/jir.2022.0158},
pmid = {36346319},
issn = {1557-7465},
mesh = {Humans ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; Exons/genetics ; *Interferon Type I/metabolism ; Interferon-beta/genetics/metabolism ; *Receptor, Interferon alpha-beta/genetics/metabolism ; },
abstract = {Type I interferons (IFN-Is) play central roles in regulating immune responses. The role of IFNAR2 in IFN-I signaling is an open question since a previous report showed that IFNβ was still functional in the absence of IFNAR2 in mice. In this study, we report that IFN-I signaling in human monocyte-derived THP1 cells absolutely depends on IFNAR2, as determined by using a knockout mutant made by CRISPR/Cas9. Additionally, we demonstrated that a 7-bp deletion mutant (Δ7) of IFNAR2 remains responsive to IFNβ stimulation and upregulates a subset of interferon-stimulated genes (s-ISGs). The s-ISGs largely overlap with tonic ISGs, which depend on the basal expression level of IFN-I. We also showed that IFN signaling in Δ7 still depends on IFNAR2. Then, we found that the 7-bp deletion in the genome results in the loss of the entire third exon (42 bp) from the mRNA and in the expression of a functionally impaired IFNAR2. These findings clarified the requirement of IFNAR2 for human IFN-I signaling and highlighted that caution should be used with CRISPR/Cas9 technology to prevent misleading interpretations caused by residual protein expression due to exon skipping or other mechanisms.},
}
@article {pmid36344749,
year = {2022},
author = {Chen, PJ and Liu, DR},
title = {Prime editing for precise and highly versatile genome manipulation.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {36344749},
issn = {1471-0064},
abstract = {Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR-Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.},
}
@article {pmid36344504,
year = {2022},
author = {Kato, K and Okazaki, S and Kannan, S and Altae-Tran, H and Esra Demircioglu, F and Isayama, Y and Ishikawa, J and Fukuda, M and Macrae, RK and Nishizawa, T and Makarova, KS and Koonin, EV and Zhang, F and Nishimasu, H},
title = {Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6719},
pmid = {36344504},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *RNA, Guide/metabolism ; Endonucleases/metabolism ; RNA/metabolism ; DNA/metabolism ; Ribonucleoproteins/metabolism ; },
abstract = {Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.},
}
@article {pmid36343261,
year = {2022},
author = {Nethery, MA and Hidalgo-Cantabrana, C and Roberts, A and Barrangou, R},
title = {CRISPR-based engineering of phages for in situ bacterial base editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {46},
pages = {e2206744119},
pmid = {36343261},
issn = {1091-6490},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Ecosystem ; Soil ; },
abstract = {Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal (lacZ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.},
}
@article {pmid36342558,
year = {2022},
author = {Yasmeen, A and Shakoor, S and Azam, S and Bakhsh, A and Shahid, N and Latif, A and Shahid, AA and Husnain, T and Rao, AQ},
title = {CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.},
journal = {Planta},
volume = {256},
number = {6},
pages = {107},
pmid = {36342558},
issn = {1432-2048},
mesh = {*beta-Fructofuranosidase/genetics/metabolism ; *Solanum tuberosum/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Gene Expression ; Sugars/metabolism ; },
abstract = {VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.},
}
@article {pmid36342156,
year = {2022},
author = {Yu, L and Zhang, Y and Marchisio, MA},
title = {Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {188},
pages = {},
doi = {10.3791/64539},
pmid = {36342156},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Regulatory Networks ; Promoter Regions, Genetic ; Transcription Factors/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; RNA, Guide/genetics ; },
abstract = {Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to β-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.},
}
@article {pmid36341480,
year = {2022},
author = {Paßreiter, A and Naumann, N and Thomas, A and Grogna, N and Delahaut, P and Thevis, M},
title = {How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes.},
journal = {The Analyst},
volume = {147},
number = {23},
pages = {5528-5536},
doi = {10.1039/d2an01318e},
pmid = {36341480},
issn = {1364-5528},
mesh = {Mice ; Animals ; CRISPR-Cas Systems/genetics ; *Doping in Sports ; Gene Editing/methods ; Streptococcus pyogenes ; *Nucleic Acids ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.},
}
@article {pmid36340792,
year = {2022},
author = {Altay, HY and Ozdemir, F and Afghah, F and Kilinc, Z and Ahmadian, M and Tschopp, M and Agca, C},
title = {Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.},
journal = {Frontiers in neuroscience},
volume = {16},
number = {},
pages = {924917},
pmid = {36340792},
issn = {1662-4548},
abstract = {Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.},
}
@article {pmid36338968,
year = {2022},
author = {Garrood, WT and Cuber, P and Willis, K and Bernardini, F and Page, NM and Haghighat-Khah, RE},
title = {Driving down malaria transmission with engineered gene drives.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {891218},
pmid = {36338968},
issn = {1664-8021},
abstract = {The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.},
}
@article {pmid36338309,
year = {2022},
author = {Liu, L and Xu, Z and Awayda, K and Dollery, SJ and Bao, M and Fan, J and Cormier, D and O'Connell, M and Tobin, GJ and Du, K},
title = {Gold Nanoparticle-Labeled CRISPR-Cas13a Assay for the Sensitive Solid-State Nanopore Molecular Counting.},
journal = {Advanced materials technologies},
volume = {7},
number = {3},
pages = {},
pmid = {36338309},
issn = {2365-709X},
support = {R35 GM133462/GM/NIGMS NIH HHS/United States ; R35 GM142763/GM/NIGMS NIH HHS/United States ; },
abstract = {A gold nanoparticle (AuNP) labeled CRISPR-Cas13a nucleic acid assay has been developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, our system utilizes non-covalent conjugates of AuNPs and RNA targets. Upon CRISPR activation, the AuNPs are liberated from the RNA, isolated, and passed through a nanopore sensor. Detection of the AuNPs can be observed as increasing ionic current in the chip. Each AuNP that is detected is enumerated as an event, leading to quantitative of molecular targets. Leveraging the high signal-to-noise ratio enabled by the AuNPs, a detection limit of 50 fM before front-end target amplification is achieved using SARS-CoV-2 RNA segments as a Cas13 target. Furthermore, a dynamic range of six orders of magnitude is demonstrated for quantitative RNA sensing. This simplified AuNP-based CRISPR assay is performed at the physiological temperature without relying on thermal cyclers. In addition, the nanopore reader is similar in size to a smartphone, making the assay system suitable for rapid and portable nucleic acid biomarker detection in either low-resource settings or hospitals.},
}
@article {pmid36336952,
year = {2022},
author = {Ubiparipovic, S and Christ, D and Rouet, R},
title = {Antibody-mediated delivery of CRISPR-Cas9 ribonucleoproteins in human cells.},
journal = {Protein engineering, design &amp; selection : PEDS},
volume = {35},
number = {},
pages = {},
doi = {10.1093/protein/gzac011},
pmid = {36336952},
issn = {1741-0134},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; Gene Editing ; Antibodies, Monoclonal/genetics/metabolism ; },
abstract = {The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies.},
}
@article {pmid36334915,
year = {2022},
author = {Subramaniam, S and Smith, GR},
title = {RecBCD enzyme and Chi recombination hotspots as determinants of self vs. non-self: Myths and mechanisms.},
journal = {Advances in genetics},
volume = {109},
number = {},
pages = {1-37},
doi = {10.1016/bs.adgen.2022.06.001},
pmid = {36334915},
issn = {0065-2660},
mesh = {Exodeoxyribonuclease V/genetics ; *Escherichia coli/genetics ; *Recombination, Genetic ; DNA Helicases/genetics ; DNA/genetics ; },
abstract = {Bacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5' GCTGGTGG 3'), a hotspot of homologous genetic recombination recognized by Escherichia coli's RecBCD helicase-nuclease, allows E. coli to distinguish its DNA (self) from any other DNA (non-self) and to destroy non-self DNA, and that Chi is "over-represented" in the E. coli genome. We show here that these latter statements (dogmas) are not supported by available evidence. We note Chi's wide-spread occurrence and activity in distantly related bacterial species and phages. We illustrate multiple, highly non-random features of the genomes of E. coli and coliphage P1 that account for Chi's high frequency and genomic position, leading us to propose that P1 selects for Chi's enhancement of recombination, whereas E. coli selects for the preferred codons in Chi. We discuss other, previously described mechanisms for self vs. non-self determination involving RecBCD and for RecBCD's destruction of DNA that cannot recombine, whether foreign or domestic, with or without Chi.},
}
@article {pmid36334630,
year = {2022},
author = {Beyers, WC and Detry, AM and Di Pietro, SM},
title = {OCA7 is a melanosome membrane protein that defines pigmentation by regulating early stages of melanosome biogenesis.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {12},
pages = {102669},
doi = {10.1016/j.jbc.2022.102669},
pmid = {36334630},
issn = {1083-351X},
abstract = {Mutations in C10orf11 (oculocutaneous albinism type 7 [OCA7]) cause OCA, a disorder that presents with hypopigmentation in skin, eyes, and hair. The OCA7 pathophysiology is unknown, and there is virtually no information on the OCA7 protein and its cellular function. Here, we discover that OCA7 localizes to the limiting membrane of melanosomes, the specialized pigment cell organelles where melanin is synthesized. We demonstrate that OCA7 is recruited through interaction with a canonical effector-binding surface of melanosome proteins Rab32 and Rab38. Using newly generated OCA7-KO MNT1 cells, we show OCA7 regulates overall melanin levels in a melanocyte autonomous manner by controlling melanosome maturation. Importantly, we found that OCA7 regulates premelanosome protein (PMEL) processing, impacting fibrillation and the striations that define transition from melanosome stage I to stage II. Furthermore, the melanosome lumen of OCA7-KO cells displays lower pH than control cells. Together, our results reveal that OCA7 regulates pigmentation through two well-established determinants of melanosome biogenesis and function, PMEL processing, and organelle pH.},
}
@article {pmid36334619,
year = {2023},
author = {Liu, Z and Zhang, M and Huang, P and Ji, Z and Qi, C and Jiao, S and Zhao, D and Jiang, Y and Chen, X and Lv, D and Pang, D and Zhang, X and Feng, L and Xie, Z and Ouyang, H},
title = {Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy.},
journal = {Gene},
volume = {851},
number = {},
pages = {147007},
doi = {10.1016/j.gene.2022.147007},
pmid = {36334619},
issn = {1879-0038},
mesh = {Swine/genetics ; Animals ; Gene Editing ; CRISPR-Cas Systems ; *Porcine epidemic diarrhea virus/genetics ; *Transmissible gastroenteritis virus/genetics ; *Coronavirus Infections/genetics/veterinary ; Antiviral Agents ; *Swine Diseases/genetics ; },
abstract = {The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.},
}
@article {pmid36334354,
year = {2022},
author = {Huang, K and Zapata, D and Tang, Y and Teng, Y and Li, Y},
title = {In vivo delivery of CRISPR-Cas9 genome editing components for therapeutic applications.},
journal = {Biomaterials},
volume = {291},
number = {},
pages = {121876},
doi = {10.1016/j.biomaterials.2022.121876},
pmid = {36334354},
issn = {1878-5905},
support = {R01 DE028351/DE/NIDCR NIH HHS/United States ; R03 DE032084/DE/NIDCR NIH HHS/United States ; R03 DE028387/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics ; Gene Transfer Techniques ; *Lung Neoplasms/genetics ; Mammals/genetics ; },
abstract = {Since its mechanism discovery in 2012 and the first application for mammalian genome editing in 2013, CRISPR-Cas9 has revolutionized the genome engineering field and created countless opportunities in both basic science and translational medicine. The first clinical trial of CRISPR therapeutics was initiated in 2016, which employed ex vivo CRISPR-Cas9 edited PD-1 knockout T cells for the treatment of non-small cell lung cancer. So far there have been dozens of clinical trials registered on ClinicalTrials.gov in regard to using the CRISPR-Cas9 genome editing as the main intervention for therapeutic applications; however, most of these studies use ex vivo genome editing approach, and only a few apply the in vivo editing strategy. Compared to ex vivo editing, in vivo genome editing bypasses tedious procedures related to cell isolation, maintenance, selection, and transplantation. It is also applicable to a wide range of diseases and disorders. The main obstacles to the successful translation of in vivo therapeutic genome editing include the lack of safe and efficient delivery system and safety concerns resulting from the off-target effects. In this review, we highlight the therapeutic applications of in vivo genome editing mediated by the CRISPR-Cas9 system. Following a brief introduction of the history, biology, and functionality of CRISPR-Cas9, we showcase a series of exemplary studies in regard to the design and implementation of in vivo genome editing systems that target the brain, inner ear, eye, heart, liver, lung, muscle, skin, immune system, and tumor. Current challenges and opportunities in the field of CRISPR-enabled therapeutic in vivo genome editing are also discussed.},
}
@article {pmid36333584,
year = {2022},
author = {Wirth, AK and Wange, L and Vosberg, S and Henrich, KO and Rausch, C and Özdemir, E and Zeller, CM and Richter, D and Feuchtinger, T and Kaller, M and Hermeking, H and Greif, PA and Senft, D and Jurinovic, V and Bahrami, E and Jayavelu, AK and Westermann, F and Mann, M and Enard, W and Herold, T and Jeremias, I},
title = {In vivo PDX CRISPR/Cas9 screens reveal mutual therapeutic targets to overcome heterogeneous acquired chemo-resistance.},
journal = {Leukemia},
volume = {36},
number = {12},
pages = {2863-2874},
pmid = {36333584},
issn = {1476-5551},
mesh = {Humans ; Mice ; Animals ; CRISPR-Cas Systems ; *Antineoplastic Agents/therapeutic use ; *Neoplasms/genetics ; Disease Models, Animal ; Transcriptome ; Xenograft Model Antitumor Assays ; },
abstract = {Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.},
}
@article {pmid36333335,
year = {2022},
author = {Schmiderer, L and Yudovich, D and Oburoglu, L and Hjort, M and Larsson, J},
title = {Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18687},
pmid = {36333335},
issn = {2045-2322},
mesh = {Humans ; *RNA, Guide/genetics/metabolism ; *DNA, Mitochondrial/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Mitochondria/genetics/metabolism ; },
abstract = {Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C → T mutations) on mtDNA. Unlike regular Cas9-cutting, this leaves a permanent mark on mtDNA that can be detected with amplicon sequencing, even if the efficiency is low. We detected low levels of C → T mutations in cells that were exposed to mitochondrially targeted Cas9-BE3, but, surprisingly, these occurred regardless of whether a guide RNA (gRNA) specific to the targeted site, or non-targeting gRNA was used. This unspecific off-target activity shows that Cas9-BE3 can technically edit mtDNA, but also strongly indicates that gRNA import to mitochondria was not successful. Going forward mitochondria-targeted Cas9 base editors will be a useful tool for validating successful gRNA delivery to mitochondria without the ambiguity of approaches that rely on quantifying mtDNA copy numbers.},
}
@article {pmid36332793,
year = {2022},
author = {Wang, Y and Du, T and Li, A and Qiao, L and Zhang, Z and Sun, W},
title = {Establishment and application of a silkworm CRISPR/Cas9 tool for conditionally manipulating gene disruption in the epidermis.},
journal = {Insect biochemistry and molecular biology},
volume = {151},
number = {},
pages = {103861},
doi = {10.1016/j.ibmb.2022.103861},
pmid = {36332793},
issn = {1879-0240},
mesh = {Animals ; *Bombyx/genetics ; CRISPR-Cas Systems ; Gene Editing ; Epidermis ; Epidermal Cells ; },
abstract = {Spatial or temporal specific gene knockout system is a valuable tool for studying the molecular mechanisms underlying developmental processes. The integument is essential for insect fitness and survival, but tools for dissecting function of genes in this tissue are lacking. In this study, we firstly identified an epidermis specifically expressed gene of the domesticated silkworm, BmCPG25, by comparative transcriptomic analysis. Furthermore, a transgenic silkworm expressing the RNA dependent CRISPR-Cas9 protein driven by the regulatory region of the BmCPG25 was established. Immunochemistry analysis showed the endonuclease was specifically expressed in the nuclear of epidermal cells. We also validated the efficiency of this system by disrupting the function of an epidermis specifically expressed cuticular protein gene (Cpr21) and a ubiquitously expressed cuticular gene (Cph18), respectively. In summary, we successfully constructed a conditional knockout toolkit to manipulate the gene editing in epidermal cells, which provides a valuable approach to study the molecular mechanism of integument development.},
}
@article {pmid36327561,
year = {2022},
author = {Guk, K and Yi, S and Kim, H and Bae, Y and Yong, D and Kim, S and Lee, KS and Lim, EK and Kang, T and Jung, J},
title = {Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {219},
number = {},
pages = {114819},
doi = {10.1016/j.bios.2022.114819},
pmid = {36327561},
issn = {1873-4235},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.},
}
@article {pmid36327313,
year = {2022},
author = {Qin, P and Chen, P and Deng, N and Tan, L and Yin, BC and Ye, BC},
title = {Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing.},
journal = {Analytical chemistry},
volume = {94},
number = {45},
pages = {15908-15914},
doi = {10.1021/acs.analchem.2c04315},
pmid = {36327313},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; DNA/genetics ; Oligonucleotides ; *Biosensing Techniques ; },
abstract = {The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.},
}
@article {pmid36326904,
year = {2022},
author = {Maharajan, T and Krishna, TPA and Rakkammal, K and Ceasar, SA and Ramesh, M},
title = {Application of CRISPR/Cas system in cereal improvement for biotic and abiotic stress tolerance.},
journal = {Planta},
volume = {256},
number = {6},
pages = {106},
pmid = {36326904},
issn = {1432-2048},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Edible Grain/genetics ; Plant Breeding ; Stress, Physiological/genetics ; Genome, Plant ; },
abstract = {Application of the recently developed CRISPR/Cas tools might help enhance cereals' growth and yield under biotic and abiotic stresses. Cereals are the most important food crops for human life and an essential source of nutrients for people in developed and developing countries. The growth and yield of all major cereals are affected by both biotic and abiotic stresses. To date, molecular breeding and functional genomic studies have contributed to the understanding and improving cereals' growth and yield under biotic and abiotic stresses. Clustered, regularly inter-spaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been predicted to play a major role in precision plant breeding and developing non-transgenic cereals that can tolerate adverse effects of climate change. Variants of next-generation CRISPR/Cas tools, such as prime editor, base editor, CRISPR activator and repressor, chromatin imager, Cas12a, and Cas12b, are currently used in various fields, including plant science. However, few studies have been reported on applying the CRISPR/Cas system to understand the mechanism of biotic and abiotic stress tolerance in cereals. Rice is the only plant used frequently for such studies. Genes responsible for biotic and abiotic stress tolerance have not yet been studied by CRISPR/Cas system in other major cereals (sorghum, barley, maize and small millets). Examining the role of genes that respond to biotic and abiotic stresses using the CRISPR/Cas system may help enhance cereals' growth and yield under biotic and abiotic stresses. It will help to develop new and improved cultivars with biotic- and abiotic-tolerant traits for better yields to strengthen food security. This review provides information for cereal researchers on the current status of the CRISPR/Cas system for improving biotic and abiotic stress tolerance in cereals.},
}
@article {pmid36326816,
year = {2022},
author = {Schubert, OT and Bloom, JS and Sadhu, MJ and Kruglyak, L},
title = {Genome-wide base editor screen identifies regulators of protein abundance in yeast.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {36326816},
issn = {2050-084X},
support = {R01 GM102308/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *RNA, Guide/genetics ; Genetic Testing ; CRISPR-Cas Systems ; },
abstract = {Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.},
}
@article {pmid36324900,
year = {2022},
author = {Rittiner, J and Cumaran, M and Malhotra, S and Kantor, B},
title = {Therapeutic modulation of gene expression in the disease state: Treatment strategies and approaches for the development of next-generation of the epigenetic drugs.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {1035543},
pmid = {36324900},
issn = {2296-4185},
abstract = {Epigenetic dysregulation is an important determinant of many pathological conditions and diseases. Designer molecules that can specifically target endogenous DNA sequences provide a means to therapeutically modulate gene function. The prokaryote-derived CRISPR/Cas editing systems have transformed our ability to manipulate the expression program of genes through specific DNA and RNA targeting in living cells and tissues. The simplicity, utility, and robustness of this technology have revolutionized epigenome editing for research and translational medicine. Initial success has inspired efforts to discover new systems for targeting and manipulating nucleic acids on the epigenetic level. The evolution of nuclease-inactive and RNA-targeting Cas proteins fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions opened up an unprecedented level of possibilities for the development of "next-generation" gene therapy therapeutics. The rational design and construction of different types of designer molecules paired with viral-mediated gene-to-cell transfers, specifically using lentiviral vectors (LVs) and adeno-associated vectors (AAVs) are reviewed in this paper. Furthermore, we explore and discuss the potential of these molecules as therapeutic modulators of endogenous gene function, focusing on modulation by stable gene modification and by regulation of gene transcription. Notwithstanding the speedy progress of CRISPR/Cas-based gene therapy products, multiple challenges outlined by undesirable off-target effects, oncogenicity and other virus-induced toxicities could derail the successful translation of these new modalities. Here, we review how CRISPR/Cas-based gene therapy is translated from research-grade technological system to therapeutic modality, paying particular attention to the therapeutic flow from engineering sophisticated genome and epigenome-editing transgenes to delivery vehicles throughout efficient and safe manufacturing and administration of the gene therapy regimens. In addition, the potential solutions to some of the obstacles facing successful CRISPR/Cas utility in the clinical research are discussed in this review. We believe, that circumventing these challenges will be essential for advancing CRISPR/Cas-based tools towards clinical use in gene and cell therapies.},
}
@article {pmid36323848,
year = {2022},
author = {Chen, F and Lian, M and Ma, B and Gou, S and Luo, X and Yang, K and Shi, H and Xie, J and Ge, W and Ouyang, Z and Lai, C and Li, N and Zhang, Q and Jin, Q and Liang, Y and Chen, T and Wang, J and Zhao, X and Li, L and Yu, M and Ye, Y and Wang, K and Wu, H and Lai, L},
title = {Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {1163},
pmid = {36323848},
issn = {2399-3642},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Cytosine ; Adenine ; Point Mutation ; },
abstract = {Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.},
}
@article {pmid36323688,
year = {2022},
author = {Hoberecht, L and Perampalam, P and Lun, A and Fortin, JP},
title = {A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6568},
pmid = {36323688},
issn = {2041-1723},
mesh = {*RNA, Guide/genetics ; *CRISPR-Cas Systems/genetics ; Ecosystem ; Endonucleases/genetics/metabolism ; Deoxyribonucleases/genetics ; },
abstract = {The success of CRISPR-mediated gene perturbation studies is highly dependent on the quality of gRNAs, and several tools have been developed to enable optimal gRNA design. However, these tools are not all adaptable to the latest CRISPR modalities or nucleases, nor do they offer comprehensive annotation methods for advanced CRISPR applications. Here, we present a new ecosystem of R packages, called crisprVerse, that enables efficient gRNA design and annotation for a multitude of CRISPR technologies. This includes CRISPR knockout (CRISPRko), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR base editing (CRISPRbe) and CRISPR knockdown (CRISPRkd). The core package, crisprDesign, offers a user-friendly and unified interface to add off-target annotations, rich gene and SNP annotations, and on- and off-target activity scores. These functionalities are enabled for any RNA- or DNA-targeting nucleases, including Cas9, Cas12, and Cas13. The crisprVerse ecosystem is open-source and deployed through the Bioconductor project (https://github.com/crisprVerse).},
}
@article {pmid36323073,
year = {2022},
author = {Lu, Q and Chen, R and Du, S and Chen, C and Pan, Y and Luan, X and Yang, J and Zeng, F and He, B and Han, X and Song, Y},
title = {Activation of the cGAS-STING pathway combined with CRISPR-Cas9 gene editing triggering long-term immunotherapy.},
journal = {Biomaterials},
volume = {291},
number = {},
pages = {121871},
doi = {10.1016/j.biomaterials.2022.121871},
pmid = {36323073},
issn = {1878-5905},
mesh = {Humans ; B7-H1 Antigen/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Immunotherapy ; *Neoplasms/genetics ; Nucleotidyltransferases/genetics/metabolism ; Tumor Microenvironment ; },
abstract = {Effective activation of cGAS-STING pathway combined with immune checkpoint blockade (ICB) within the immunosuppressive tumor microenvironment to induce stronger immune responsiveness yet remains challenging. CRISPR-Cas9 gene editing technology, which offers the benefits of permanence and irreversibility, could recognize the target genome sequence with sgRNA (Guide RNA) and guide the Cas9 protease to knock down the target gene. Herein, a nanoplatform (HMnMPH) for dual activation of cGAS-STING pathway in combination with CRISPR-Cas9 gene editing to silence programmed death ligand 1 (PD-L1) to trigger long-term immunotherapy was reported. The HMnMPH consists of hollow manganese dioxide (HMn) loaded with STING agonist (MSA-2) and CRISPR-Cas9/sg-PD-L1 plasmid with further modification of hyaluronic acid (HA). In acidic and GSH overexpressed tumor environment, HMnPMH was degraded to release large amounts of Mn ions and STING agonists, strongly and persistently activating the cGAS-STING pathway to promote the release of type I interferon and pro-inflammatory factors. Meanwhile, the released CRISPR-Cas9 plasmid could knockdown the PD-L1 immune checkpoint and restart immunosuppressive T cells to differentiate into cytotoxic T lymphocytes significantly, which reduced the activity of primary and distal tumors and demonstrated a long-term immune memory effect on distal tumors.},
}
@article {pmid36322747,
year = {2022},
author = {Lee, J and Kim, DH and Karolak, MC and Shin, S and Lee, K},
title = {Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {45},
pages = {e2214344119},
pmid = {36322747},
issn = {1091-6490},
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; Ducks/genetics ; CRISPR-Cas Systems ; Adenoviridae/genetics ; Germ Cells ; },
abstract = {Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.},
}
@article {pmid36321657,
year = {2022},
author = {Zhang, B and Lin, J and Perčulija, V and Li, Y and Lu, Q and Chen, J and Ouyang, S},
title = {Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system.},
journal = {Nucleic acids research},
volume = {50},
number = {20},
pages = {11820-11833},
doi = {10.1093/nar/gkac987},
pmid = {36321657},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; RNA, Guide/metabolism ; Gene Editing ; DNA/chemistry ; Deoxyribonucleases/metabolism ; DNA Cleavage ; },
abstract = {Cas12c is the recently characterized dual RNA-guided DNase effector of type V-C CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems. Due to minimal requirements for a protospacer adjacent motif (PAM), Cas12c is an attractive candidate for genome editing. Here we report the crystal structure of Cas12c1 in complex with single guide RNA (sgRNA) and target double-stranded DNA (dsDNA) containing the 5'-TG-3' PAM. Supported by biochemical and mutation assays, this study reveals distinct structural features of Cas12c1 and the associated sgRNA, as well as the molecular basis for PAM recognition, target dsDNA unwinding, heteroduplex formation and recognition, and cleavage of non-target and target DNA strands. Cas12c1 recognizes the PAM through a mechanism that is interdependent on sequence identity and Cas12c1-induced conformational distortion of the PAM region. Another special feature of Cas12c1 is the cleavage of both non-target and target DNA strands at a single, uniform site with indistinguishable cleavage capacity and order. Location of the sgRNA seed region and minimal length of target DNA required for triggering Cas12c1 DNase activity were also determined. Our findings provide valuable information for developing the CRISPR-Cas12c1 system into an efficient, high-fidelity genome editing tool.},
}
@article {pmid36320584,
year = {2022},
author = {Zhao, X and Na, N and Ouyang, J},
title = {CRISPR/Cas9-based coronal nanostructures for targeted mitochondria single molecule imaging.},
journal = {Chemical science},
volume = {13},
number = {38},
pages = {11433-11441},
pmid = {36320584},
issn = {2041-6520},
abstract = {The biological state at the subcellular level is highly relevant to many diseases, and the monitoring of organelles such as mitochondria is crucial based on this. However, most DNA and protein based nanoprobes used for the detection of mitochondrial RNAs (mitomiRs) lack spatial selectivity, which leads to inefficiencies in probe delivery and signal turn-on. Herein, we constructed a novel DNA nanoprobe named protein delivery nano-corona (PDNC) to improve the delivery efficiency of Cas protein, for spatially selective imaging of mitomiRs in living cells switched on by a CRISPR/Cas system. Combined with a single-molecule counting method, this strategy enables highly sensitive detection of low-abundance mitomiR. Therefore, the strategy in this work opens up new opportunities for cell identification, early clinical diagnosis, and research in biological behaviour at the subcellular level.},
}
@article {pmid36320315,
year = {2022},
author = {Lohia, A and Sahel, DK and Salman, M and Singh, V and Mariappan, I and Mittal, A and Chitkara, D},
title = {Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies: challenges and opportunities.},
journal = {Asian journal of pharmaceutical sciences},
volume = {17},
number = {2},
pages = {153-176},
pmid = {36320315},
issn = {2221-285X},
abstract = {CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc. Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the gene mutations. CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions. Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight, negative charge, and in vivo stability of CRISPR components. Recently, non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid (miRNA/siRNA/CRISPR) delivery. This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.},
}
@article {pmid36318504,
year = {2022},
author = {Gong, S and Wang, X and Zhou, P and Pan, W and Li, N and Tang, B},
title = {AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs.},
journal = {Analytical chemistry},
volume = {94},
number = {45},
pages = {15839-15846},
doi = {10.1021/acs.analchem.2c03666},
pmid = {36318504},
issn = {1520-6882},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Colorimetry ; *MicroRNAs/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Single-Stranded ; Logic ; *Biosensing Techniques/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still challenging. In this study, we develop an AND logic-gate-based CRISPR-Cas12a biosensing platform to achieve the sensitive colorimetric detection of dual miRNAs. Specifically, the DNA probe was designed to recognize the binary input of miRNAs and to output trigger DNA, which activated the CRISPR-Cas12a system to cut single-stranded DNA (ssDNA). The ssDNA on magnetic beads (MBs) was cleaved by the activated CRISPR-Cas12a, causing the separation of glucose oxidase (GOx) from MB and the subsequent generation of a colorimetric signal. The color change induced by 1 pM of target miRNAs can be directly distinguished by the naked eye and the instrumental limit of detection reaches 36.4 fM. The overexpressed miR-205 and miR-944 in the real human serum can be detected, allowing us to differentiate between lung cancer patients and healthy people. Furthermore, the developed strategy achieves simultaneous detection of dual miRNAs using CRISPR-Cas12a with one kind of crRNA, avoiding sophisticated nucleic acid amplifications and the use of bulky instruments. The current method can broaden the CRISPR-Cas12a-based applications for multiple biomarkers detection and precise disease diagnosis.},
}
@article {pmid36318259,
year = {2022},
author = {Wu, Y and Luo, W and Weng, Z and Guo, Y and Yu, H and Zhao, R and Zhang, L and Zhao, J and Bai, D and Zhou, X and Song, L and Chen, K and Li, J and Yang, Y and Xie, G},
title = {A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration.},
journal = {Nucleic acids research},
volume = {50},
number = {20},
pages = {11727-11737},
doi = {10.1093/nar/gkac886},
pmid = {36318259},
issn = {1362-4962},
mesh = {Humans ; *CRISPR-Cas Systems ; ErbB Receptors/genetics ; Mutation ; *Lung Neoplasms/genetics ; Protein Kinase Inhibitors ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.},
}
@article {pmid36317989,
year = {2022},
author = {Blicharska, D and Szućko-Kociuba, I and Filip, E and Skuza, L},
title = {[CRISPR/Cas as the intelligent immune system of bacteria and archea].},
journal = {Postepy biochemii},
volume = {68},
number = {3},
pages = {235-245},
doi = {10.18388/pb.2021_453},
pmid = {36317989},
issn = {0032-5422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Quality of Life ; Bacteria/genetics ; Immune System ; },
abstract = {The invention of CRISPR is considered to be one of the most breakthrough discoveries in recent years in the history of biology, biotechnology, medicine, as well as the pharmaceutical and agricultural industries. The methods developed using CRISPR create new, previously unattainable possibilities that can significantly improve the quality of life. From the invention of this intelligent immune system to the present day, much research has been done using the CRISPR/Cas systems. The result of these studies was the development of a modern tool for genetic manipulation, which allows for the introduction of many modifications within the DNA, which may contribute to the silencing of the expression of given genes or their overexpression through e.g. mutations or deletions. The paper describes the application of the method for genetic manipulation with the use of the second class system – CRISPR/Cas9 and the advantages of this method and its advantage over the previously used genetic engineering tools, as well as its limitations and disadvantages, which significantly limit the possibility of its application. The potential use of the method was also presented as well as the research carried out with the use of CRSPR/Cas9.},
}
@article {pmid36317180,
year = {2022},
author = {Medishetti, R and Balamurugan, K and Yadavalli, K and Rani, R and Sevilimedu, A and Challa, AK and Parsa, K and Chatti, K},
title = {CRISPR-Cas9-induced gene knockout in zebrafish.},
journal = {STAR protocols},
volume = {3},
number = {4},
pages = {101779},
pmid = {36317180},
issn = {2666-1667},
mesh = {Animals ; Humans ; Gene Knockout Techniques ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Germ Cells ; },
abstract = {The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).},
}
@article {pmid36317129,
year = {2022},
author = {Robinson, EL and Port, JD},
title = {Utilization and Potential of RNA-Based Therapies in Cardiovascular Disease.},
journal = {JACC. Basic to translational science},
volume = {7},
number = {9},
pages = {956-969},
pmid = {36317129},
issn = {2452-302X},
abstract = {Cardiovascular disease (CVD) remains the largest cause of mortality worldwide. The development of new effective therapeutics is a major unmet need. The current review focuses broadly on the concept of nucleic acid (NA)-based therapies, considering the use of various forms of NAs, including mRNAs, miRNAs, siRNA, and guide RNAs, the latter specifically for the purpose of CRISPR-Cas directed gene editing. We describe the current state-of-the-art of RNA target discovery and development, the status of RNA therapeutics in the context of CVD, and some of the challenges and hurdles to be overcome.},
}
@article {pmid36316452,
year = {2022},
author = {Guan, J and Oromí-Bosch, A and Mendoza, SD and Karambelkar, S and Berry, JD and Bondy-Denomy, J},
title = {Bacteriophage genome engineering with CRISPR-Cas13a.},
journal = {Nature microbiology},
volume = {7},
number = {12},
pages = {1956-1966},
pmid = {36316452},
issn = {2058-5276},
support = {R01 AI171041/AI/NIAID NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; RNA ; },
abstract = {Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.},
}
@article {pmid36316451,
year = {2022},
author = {Adler, BA and Hessler, T and Cress, BF and Lahiri, A and Mutalik, VK and Barrangou, R and Banfield, J and Doudna, JA},
title = {Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing.},
journal = {Nature microbiology},
volume = {7},
number = {12},
pages = {1967-1979},
pmid = {36316451},
issn = {2058-5276},
mesh = {*Gene Editing ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Phylogeny ; RNA/genetics ; Antiviral Agents ; },
abstract = {CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.},
}
@article {pmid36315306,
year = {2022},
author = {Singh, S and Chaudhary, R and Deshmukh, R and Tiwari, S},
title = {Opportunities and challenges with CRISPR-Cas mediated homologous recombination based precise editing in plants and animals.},
journal = {Plant molecular biology},
volume = {},
number = {},
pages = {},
pmid = {36315306},
issn = {1573-5028},
abstract = {We summarise recent advancements to achieve higher homologous recombination based gene targeting efficiency in different animals and plants. The genome editing has revolutionized the agriculture and human therapeutic sectors by its ability to create precise, stable and predictable mutations in the genome. It depends upon targeted double-strand breaks induction by the engineered endonucleases, which then gets repaired by highly conserved endogenous DNA repair mechanisms. The repairing could be done either through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The HDR-based editing can be applied for precise gene targeting such as insertion of a new gene, gene replacement and altering of the regulatory sequence of a gene to control the existing protein expression. However, HDR-mediated editing is considered challenging because of lower efficiency in higher eukaryotes, thus, preventing its widespread application. This article reviews the recent progress of HDR-mediated editing and discusses novel strategies such as cell cycle synchronization, modulation of DNA damage repair factors, engineering of Cas protein favoring HDR and CRISPR-Cas reagents delivery methods to improve efficiency for generating knock-in events in both plants and animals. Further, multiplexing of described methods may be promising towards achieving higher donor template-assisted homologous recombination efficiency at the target locus.},
}
@article {pmid36312941,
year = {2022},
author = {Adade, NE and Aniweh, Y and Mosi, L and Valvano, MA and Duodu, S and Ahator, SD},
title = {Comparative analysis of Vibrio cholerae isolates from Ghana reveals variations in genome architecture and adaptation of outbreak and environmental strains.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {998182},
pmid = {36312941},
issn = {1664-302X},
abstract = {Recurrent epidemics of cholera denote robust adaptive mechanisms of Vibrio cholerae for ecological shifting and persistence despite variable stress conditions. Tracking the evolution of pathobiological traits requires comparative genomic studies of isolates from endemic areas. Here, we investigated the genetic differentiation among V. cholerae clinical and environmental isolates by highlighting the genomic divergence associated with gene decay, genome plasticity, and the acquisition of virulence and adaptive traits. The clinical isolates showed high phylogenetic relatedness due to a higher frequency of shared orthologs and fewer gene variants in contrast to the evolutionarily divergent environmental strains. Divergence of the environmental isolates is linked to extensive genomic rearrangements in regions containing mobile genetic elements resulting in numerous breakpoints, relocations, and insertions coupled with the loss of virulence determinants acf, zot, tcp, and ctx in the genomic islands. Also, four isolates possessed the CRISPR-Cas systems with spacers specific for Vibrio phages and plasmids. Genome synteny and homology analysis of the CRISPR-Cas systems suggest horizontal acquisition. The marked differences in the distribution of other phage and plasmid defense systems such as Zorya, DdmABC, DdmDE, and type-I Restriction Modification systems among the isolates indicated a higher propensity for plasmid or phage disseminated traits in the environmental isolates. Our results reveal that V. cholerae strains undergo extensive genomic rearrangements coupled with gene acquisition, reflecting their adaptation during ecological shifts and pathogenicity.},
}
@article {pmid36309986,
year = {2022},
author = {Huang, S and Xue, Y and Zhou, C and Ma, Y},
title = {An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production.},
journal = {Microbial biotechnology},
volume = {15},
number = {11},
pages = {2730-2743},
pmid = {36309986},
issn = {1751-7915},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; Xylose ; *Bacillus/genetics ; Lactic Acid ; },
abstract = {Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D-lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D-lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis.},
}
@article {pmid36309609,
year = {2022},
author = {Das, N and Ghosh Dhar, D and Dhar, P},
title = {Editing the genome of common cereals (Rice and Wheat): techniques, applications, and industrial aspects.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {36309609},
issn = {1573-4978},
abstract = {Gene editing techniques have made a significant contribution to the development of better crops. Gene editing enables precise changes in the genome of crops, which can introduce new possibilities for altering the crops' traits. Since the last three decades, various gene editing techniques such as meganucleases, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspersed short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins) have been discovered. In this review, we discuss various gene editing techniques and their applications to common cereals. Further, we elucidate the future of gene-edited crops, their regulatory features, and industrial aspects globally. To achieve this, we perform a comprehensive literature survey using databases such as PubMed, Web of Science, SCOPUS, Google Scholar etc. For the literature search, we used keywords such as gene editing, crop genome modification, CRISPR/Cas, ZFN, TALEN, meganucleases etc. With the advent of the CRISPR/Cas technology in the last decade, the future of gene editing has transitioned into a new dimension. The functionality of CRISPR/Cas in both DNA and RNA has increased through the use of various Cas enzymes and their orthologs. Constant research efforts in this direction have improved the gene editing process for crops by minimizing its off-target effects. Scientists also use computational tools, which help them to design experiments and analyze the results of gene editing experiments in advance. Gene editing has diverse potential applications. In the future, gene editing will open new avenues for solving more agricultural issues and boosting crop production, which may have great industrial prospects.},
}
@article {pmid36309521,
year = {2022},
author = {Xu, Z and Chen, D and Li, T and Yan, J and Zhu, J and He, T and Hu, R and Li, Y and Yang, Y and Liu, M},
title = {Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6480},
pmid = {36309521},
issn = {2041-1723},
mesh = {Humans ; *Recombinases ; CRISPR-Cas Systems/genetics ; Microfluidics ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Nucleotidyltransferases ; },
abstract = {Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a microfluidic device with CRISPR-Cas12a and multiplex recombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.},
}
@article {pmid36309502,
year = {2022},
author = {Ciciani, M and Demozzi, M and Pedrazzoli, E and Visentin, E and Pezzè, L and Signorini, LF and Blanco-Miguez, A and Zolfo, M and Asnicar, F and Casini, A and Cereseto, A and Segata, N},
title = {Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6474},
pmid = {36309502},
issn = {2041-1723},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; RNA, Guide/genetics ; Metagenome ; Gene Editing/methods ; Endonucleases/metabolism ; },
abstract = {The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation in genome editing. Here we develop a computational pipeline that was used to interrogate a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions. This procedure allows the identification and isolation of sequence-tailored Cas9 nucleases by using the target sequence as bait. As proof of concept, starting from the disease-causing mutation P23H in the RHO gene, we find, isolate and experimentally validate a Cas9 which uses the mutated sequence as PAM. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement.},
}
@article {pmid36309062,
year = {2023},
author = {Phulpoto, IA and Yu, Z and Qazi, MA and Ndayisenga, F and Yang, J},
title = {A comprehensive study on microbial-surfactants from bioproduction scale-up toward electrokinetics remediation of environmental pollutants: Challenges and perspectives.},
journal = {Chemosphere},
volume = {311},
number = {Pt 1},
pages = {136979},
doi = {10.1016/j.chemosphere.2022.136979},
pmid = {36309062},
issn = {1879-1298},
mesh = {Surface-Active Agents ; *Soil Pollutants/analysis ; *Environmental Pollutants ; Biodegradation, Environmental ; Bacteria ; *Environmental Restoration and Remediation ; },
abstract = {Currently, researchers have focused on electrokinetic (EK) bioremediation due to its potential to remove a wide-range of pollutants. Further, to improve their performance, synthetic surfactants are employed as effective additives because of their excellent solubility and mobility. Synthetic surfactants have an excessive position in industries since they are well-established, cheap, and easily available. Nevertheless, these surfactants have adverse environmental effects and could be detrimental to aquatic and terrestrial life. Owing to social and environmental awareness, there is a rising demand for bio-based surfactants in the global market, from environmental sustainability to public health, because of their excellent surface and interfacial activity, higher and stable emulsifying property, biodegradability, non- or low toxicity, better selectivity and specificity at extreme environmental conditions. Unfortunately, challenges to biosurfactants, like expensive raw materials, low yields, and purification processes, hinder their applicability to large-scale. To date, extensive research has already been conducted for production scale-up using multidisciplinary approaches. However, it is still essential to research and develop high-yielding bacteria for bioproduction through traditional and biotechnological advances to reduce production costs. Herein, this review evaluates the recent progress made on microbial-surfactants for bioproduction scale-up and provides detailed information on traditional and advanced genetic engineering approaches for cost-effective bioproduction. Furthermore, this study emphasized the role of electrokinetic (EK) bioremediation and discussed the application of BioS-mediated EK for various pollutants remediation.},
}
@article {pmid36308835,
year = {2022},
author = {Ding, L and Wu, Y and Liu, LE and He, L and Yu, S and Effah, CY and Liu, X and Qu, L and Wu, Y},
title = {Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer.},
journal = {Biosensors &amp; bioelectronics},
volume = {219},
number = {},
pages = {114827},
doi = {10.1016/j.bios.2022.114827},
pmid = {36308835},
issn = {1873-4235},
abstract = {Exosomal proteins are considered to be promising indicators of cancer. Herein, a novel DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a strategy was proposed for the synchronous determination of exosomal proteins: serum amyloid A-1 protein (SAA1) and coagulation factor V (FV). In this design, the paired antibodies were used to recognize targets, thereby ensuring the specificity. DNAzyme walkers were employed to convert the contents of SAA1 and FV into activators (P1 and P2), and one target can produce abundant activators, thus achieving an initial amplification of signal. Furthermore, the P1 and P2 can activate CRISPR-Cas12a/Cas13a system, which in turn trans-cleaves the reporters, enabling a second amplification and generating two fluorescent signals. The assay is highly sensitive (limits of detection as low as 30.00 pg/mL for SAA1 and 200.00 pg/mL for FV), highly specific and ideally accurate. More importantly, it is universal and can be used to detect both non-membrane and membrane proteins in exosome. Besides, the method can be successfully applied to detect SAA1 and FV in plasma exosomes to differentiate between lung cancer patients and healthy individuals. To explore the application of the developed method in tumor diagnosis, a deep learning model based on the expressions of SAA1 and FV was developed. The accuracy of this model can achieve 86.96%, which proves that it has a promising practical application capacity. Thus, this study does not only provide a new tool for the detection of exosomal proteins and cancer diagnosis, but also propose a new strategy to detect non-nucleic acid analytes for CRISPR-Cas system.},
}
@article {pmid36308834,
year = {2022},
author = {Duan, M and Li, B and Zhao, Y and Liu, Y and Liu, Y and Dai, R and Li, X and Jia, F},
title = {A CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor for Salmonella Typhimurium.},
journal = {Biosensors &amp; bioelectronics},
volume = {219},
number = {},
pages = {114823},
doi = {10.1016/j.bios.2022.114823},
pmid = {36308834},
issn = {1873-4235},
abstract = {CRISPR/Cas-based biosensors were typically used for nucleic-acid targets detection and complex DNA extraction and amplification procedures were usually inevitable. Here, we report a CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor (abbreviated as "CATCHER") for Salmonella Typhimurium (S. Typhimurium) with a simple (3 steps) and fast (∼2 h) sensing workflow. Magnetic nanoparticle immobilized anti-S. Typhimurium antibody was worked as capture probe to capture the target and provide movable reaction interface. Colloidal gold labeled with anti-S. Typhimurium antibody and DNase I was used as detection probe to bridge the input target and output signal. First, in the presence of S. Typhimurium, an immuno-sandwich structure was formed. Second, DNase I in sandwich structure degraded the valid, complete activator DNA to invalid DNA fragments which can't trigger the trans-cleavage activity of Cas12a. Finally, the integrity of reporter DNA was preserved presenting a low fluorescence signal. Conversely, in the absence of S. Typhimurium, strong fluorescence recovery appeared owing to the cutting of reporter by activated Cas12a. Significantly, the proposed "CATCHER" showed satisfactory detection performance for S. Typhimurium with the limit of detection (LOD) of 7.9 × 10[1] CFU/mL in 0.01 M PBS and 6.31 × 10[3] CFU/mL in spiked chicken samples, providing a general platform for non-nucleic acid targets.},
}
@article {pmid36307966,
year = {2022},
author = {Belshaw, N and Grouneva, I and Aram, L and Gal, A and Hopes, A and Mock, T},
title = {Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.18587},
pmid = {36307966},
issn = {1469-8137},
abstract = {CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. HR was induced in T. pseudonana using sequence specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to approximately 85% of NAT resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.},
}
@article {pmid36307542,
year = {2022},
author = {Bäckström, A and Yudovich, D and Žemaitis, K and Nilsén Falck, L and Subramaniam, A and Larsson, J},
title = {Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18169},
pmid = {36307542},
issn = {2045-2322},
support = {648894/ERC_/European Research Council/International ; },
mesh = {Humans ; Mice ; Animals ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing ; Gene Targeting ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.},
}
@article {pmid36307508,
year = {2022},
author = {Westermann, L and Li, Y and Göcmen, B and Niedermoser, M and Rhein, K and Jahn, J and Cascante, I and Schöler, F and Moser, N and Neubauer, B and Hofherr, A and Behrens, YL and Göhring, G and Köttgen, A and Köttgen, M and Busch, T},
title = {Wildtype heterogeneity contributes to clonal variability in genome edited cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18211},
pmid = {36307508},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems/genetics ; Reproducibility of Results ; *Gene Editing/methods ; Cell Line ; Cells, Cultured ; },
abstract = {Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.},
}
@article {pmid36307477,
year = {2022},
author = {Bellinvia, E and García-González, J and Cifrová, P and Martinek, J and Sikorová, L and Havelková, L and Schwarzerová, K},
title = {CRISPR-Cas9 Arabidopsis mutants of genes for ARPC1 and ARPC3 subunits of ARP2/3 complex reveal differential roles of complex subunits.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {18205},
pmid = {36307477},
issn = {2045-2322},
mesh = {*Actin-Related Protein 2-3 Complex/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; Actins/metabolism ; CRISPR-Cas Systems ; Actin-Related Protein 2/genetics ; Actin-Related Protein 3/metabolism ; },
abstract = {Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.},
}
@article {pmid36307400,
year = {2022},
author = {Coelho, R and Tozzi, A and Disler, M and Lombardo, F and Fedier, A and López, MN and Freuler, F and Jacob, F and Heinzelmann-Schwarz, V},
title = {Overlapping gene dependencies for PARP inhibitors and carboplatin response identified by functional CRISPR-Cas9 screening in ovarian cancer.},
journal = {Cell death &amp; disease},
volume = {13},
number = {10},
pages = {909},
pmid = {36307400},
issn = {2041-4889},
mesh = {Humans ; Female ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Carcinoma, Ovarian Epithelial/drug therapy/genetics ; Carboplatin/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Early Detection of Cancer ; Phthalazines/pharmacology/therapeutic use ; *Ovarian Neoplasms/drug therapy/genetics/pathology ; Genes, Overlapping ; },
abstract = {PARP inhibitors (PARPi) have revolutionized the therapeutic landscape of epithelial ovarian cancer (EOC) treatment with outstanding benefits in regard to progression-free survival, especially in patients either carrying BRCA1/2 mutations or harboring defects in the homologous recombination repair system. Yet, it remains uncertain which PARPi to apply and how to predict responders when platinum sensitivity is unknown. To shed light on the predictive power of genes previously suggested to be associated with PARPi response, we systematically reviewed the literature and identified 79 publications investigating a total of 93 genes. The top candidate genes were further tested using a comprehensive CRISPR-Cas9 mutagenesis screening in combination with olaparib treatment. Therefore, we generated six constitutive Cas9[+] EOC cell lines and profiled 33 genes in a CRISPR-Cas9 cell competition assay using non-essential (AAVS1) and essential (RPA3 and PCNA) genes for cell fitness as negative and positive controls, respectively. We identified only ATM, MUS81, NBN, BRCA2, and RAD51B as predictive markers for olaparib response. As the major survival benefit of PARPi treatment was reported in platinum-sensitive tumors, we next assessed nine top candidate genes in combination with three PARPi and carboplatin. Interestingly, we observed similar dropout rates in a gene and compound independent manner, supporting the strong correlation of cancer cell response to compounds that rely on DNA repair for their effectiveness. In addition, we report on CDK12 as a common vulnerability for EOC cell survival and proliferation without altering the olaparib response, highlighting its potential as a therapeutic target in EOC.},
}
@article {pmid36306795,
year = {2022},
author = {Liu, X and Zhang, L and Wang, H and Xiu, Y and Huang, L and Gao, Z and Li, N and Li, F and Xiong, W and Gao, T and Zhang, Y and Yang, M and Feng, Y},
title = {Target RNA activates the protease activity of Craspase to confer antiviral defense.},
journal = {Molecular cell},
volume = {82},
number = {23},
pages = {4503-4518.e8},
doi = {10.1016/j.molcel.2022.10.007},
pmid = {36306795},
issn = {1097-4164},
mesh = {*CRISPR-Associated Proteins/genetics ; RNA/metabolism ; Antiviral Agents ; CRISPR-Cas Systems ; Peptide Hydrolases/genetics/metabolism ; },
abstract = {In the type III-E CRISPR-Cas system, a Cas effector (gRAMP) is associated with a TPR-CHAT to form Craspase (CRISPR-guided caspase). However, both the structural features of gRAMP and the immunity mechanism remain unknown for this system. Here, we report structures of gRAMP-crRNA and gRAMP:cRNA:target RNA as well as structures of Craspase and Craspase complexed with cognate target RNA (CTR) or non-cognate target RNA (NTR). Importantly, the 3' anti-tag region of NTR and CTR binds at two distinct channels in Craspase, and CTR with a non-complementary 3' anti-tag induces a marked conformational change of the TPR-CHAT, which allosterically activates its protease activity to cleave an ancillary protein Csx30. This cleavage then triggers an abortive infection as the antiviral strategy of the type III-E system. Together, our study provides crucial insights into both the catalytic mechanism of the gRAMP and the immunity mechanism of the type III-E system.},
}
@article {pmid36306733,
year = {2022},
author = {Pacesa, M and Lin, CH and Cléry, A and Saha, A and Arantes, PR and Bargsten, K and Irby, MJ and Allain, FH and Palermo, G and Cameron, P and Donohoue, PD and Jinek, M},
title = {Structural basis for Cas9 off-target activity.},
journal = {Cell},
volume = {185},
number = {22},
pages = {4067-4081.e21},
doi = {10.1016/j.cell.2022.09.026},
pmid = {36306733},
issn = {1097-4172},
mesh = {*RNA, Guide/metabolism ; *CRISPR-Cas Systems ; Endonucleases/metabolism ; Base Pairing ; Nucleotides ; Gene Editing ; },
abstract = {The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.},
}
@article {pmid36306216,
year = {2022},
author = {Das, A and Doss, K and Mandal, J},
title = {CRISPR-cas heterogeneity and plasmid incompatibility types in relation to virulence determinants of Shigella.},
journal = {Journal of medical microbiology},
volume = {71},
number = {10},
pages = {},
doi = {10.1099/jmm.0.001607},
pmid = {36306216},
issn = {1473-5644},
mesh = {Virulence/genetics ; *CRISPR-Cas Systems ; *Shigella/genetics ; Virulence Factors/genetics ; Plasmids/genetics ; },
abstract = {Introduction. Virulence factors (VFs) are the most potent weapon in the molecular armoury of Shigella. In bacteria, the mobile genetic elements (MGEs) are contributors to the evolution of different types of clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-cas) variants and plasmid incompatibility types. The present study explored the virulence potential of Shigella in relation to the CRISPR-cas pattern and incompatibility types among the isolates.Hypothesis/Gap Statement. The profile of the CRISPR-cas systems among clinical isolates of Shigella in India has not been reported earlier. Limited knowledge is available on the pattern of plasmid incompatibility groups among clinical isolates Shigella. The bias is always towards studying the genetic elements associated with AMR, but the present study highlights CRISPR-cas and incompatibility types among Shigella in association with virulence.Aim. We aimed to investigate the distribution of virulence factors, CRISPR-cas pattern followed by plasmid incompatibility types among Shigella isolates.Methodology. Between 2012-2017, a total of 187 isolates of Shigella were included in the study. The virulence genes' distribution was carried out. CRISPR-cas profiling followed by analysis of the repeats and spacers was carried out. PCR-based replicon typing was used to determine the incompatibility types. The interplay was statistically determined using STATA.Results. The distribution of virulence genes showed varied pattern with ipaH present in all the isolates followed by ompA (93.6 %), virF (66.8 %), ial and sen (60.4 %), set1A (39.6 %) and set1B (39 %). CRISPR 1, CRISPR 3 and Cas6-Cas5 region were dominantly conserved. Twenty-two types of spacers were identified. The CRISPR3 repeat appeared to have a highly conserved sequence. CRISPR2 being the least common CRISPR type showed a strong association with an array of virulence genes (ial-set1A-set1B-virF) while CRISPR1 being the most dominant showed the least association with virulence genes (sen-virF). The dominant plasmids were found to be belonging to the inc FII group. The incompatibility groups FII, IncIγ, U, FIIS, FIIK, K, A/C, I1alpha was found to be associated with a greater number of virulence genes.Conclusion. The isolates showed increasing diversity in their gene content that contributes to increasing heterogeneity among the isolates, which is a known virulence strategy among pathogens.},
}
@article {pmid36306089,
year = {2023},
author = {Kohm, K and Lutz, VT and Friedrich, I and Hertel, R},
title = {CRISPR-Cas9 Shaped Viral Metagenomes Associated with Bacillus subtilis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2555},
number = {},
pages = {205-212},
pmid = {36306089},
issn = {1940-6029},
mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems/genetics ; Metagenome ; *Bacteriophages ; },
abstract = {Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.},
}
@article {pmid36305833,
year = {2022},
author = {Shangguan, Q and Graham, S and Sundaramoorthy, R and White, MF},
title = {Structure and mechanism of the type I-G CRISPR effector.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11214-11228},
pmid = {36305833},
issn = {1362-4962},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/S021647/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Associated Proteins/chemistry ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/chemistry ; *Ectothiorhodospiraceae ; *Bacterial Proteins/chemistry ; },
abstract = {Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.},
}
@article {pmid36305591,
year = {2022},
author = {Vos, PD and Filipovska, A and Rackham, O},
title = {Frankenstein Cas9: engineering improved gene editing systems.},
journal = {Biochemical Society transactions},
volume = {50},
number = {5},
pages = {1505-1516},
doi = {10.1042/BST20220873},
pmid = {36305591},
issn = {1470-8752},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; RNA, Guide/genetics/metabolism ; Endonucleases/genetics/metabolism ; },
abstract = {The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an adaptable gene editing system, it has many characteristics that could be improved for use in biotechnology. Cas9 exhibits significant off-target activity and low on-target nuclease activity in certain contexts. Scientists have undertaken ambitious protein engineering campaigns to bypass these limitations, producing several promising variants of Cas9. Cas9 variants with improved and alternative activities provide exciting new tools to expand the scope and fidelity of future CRISPR applications.},
}
@article {pmid36304520,
year = {2022},
author = {Li, J and Yu, X and Zhang, C and Li, N and Zhao, J},
title = {The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives.},
journal = {aBIOTECH},
volume = {3},
number = {2},
pages = {146-161},
pmid = {36304520},
issn = {2662-1738},
abstract = {Brassica species are a global source of nutrients and edible vegetable oil for humans. However, all commercially important Brassica crops underwent a whole-genome triplication event, hindering the development of functional genomics and breeding programs. Fortunately, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies, by allowing multiplex and precise genome engineering, have become valuable genome-editing tools and opened up new avenues for biotechnology. Here, we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing. We also summarize the application of CRISPR/Cas technologies to Brassica crops for trait improvements. Finally, we discuss the challenges and future directions of these technologies for comprehensive application in Brassica crops. Ongoing advancement in CRISPR/Cas technologies, in combination with other achievements, will play a significant role in the genetic improvement and molecular breeding of Brassica crops.},
}
@article {pmid36302881,
year = {2022},
author = {Yu, G and Wang, X and Zhang, Y and An, Q and Wen, Y and Li, X and Yin, H and Deng, Z and Zhang, H},
title = {Structure and function of a bacterial type III-E CRISPR-Cas7-11 complex.},
journal = {Nature microbiology},
volume = {7},
number = {12},
pages = {2078-2088},
pmid = {36302881},
issn = {2058-5276},
mesh = {Cryoelectron Microscopy ; *CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; Catalytic Domain ; RNA ; },
abstract = {The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR-Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA-target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.},
}
@article {pmid36302757,
year = {2022},
author = {Li, M and Zhong, A and Wu, Y and Sidharta, M and Beaury, M and Zhao, X and Studer, L and Zhou, T},
title = {Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6354},
pmid = {36302757},
issn = {2041-1723},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R35 GM145260/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Tumor Suppressor Protein p53/genetics ; Cytosine ; Gene Editing/methods ; *Pluripotent Stem Cells/physiology ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems ; },
abstract = {Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.},
}
@article {pmid36301754,
year = {2022},
author = {Guo, JZ and Su, J and Dai, H and Wang, XY and Wu, WB and Chen, T and Zhang, J and Wang, WH},
title = {Establishment of a mouse model of Netherton syndrome based on CRISPR/Cas9 technology.},
journal = {European journal of dermatology : EJD},
volume = {32},
number = {4},
pages = {459-463},
doi = {10.1684/ejd.2022.4287},
pmid = {36301754},
issn = {1952-4013},
mesh = {Animals ; Humans ; Mice ; CRISPR-Cas Systems ; *Dermatitis/genetics ; Disease Models, Animal ; Mice, Inbred C57BL ; Mice, Knockout ; *Netherton Syndrome/genetics/pathology ; Proteinase Inhibitory Proteins, Secretory/genetics ; Serine Peptidase Inhibitor Kazal-Type 5/genetics ; },
abstract = {BACKGROUND: Netherton syndrome is a rare but severe autosomal recessive disorder with dominant impaired skin barrier function, caused by mutations in the SPINK5 (serine protease inhibitor Kazal-type 5) gene, which encodes LEKTI (lymphoepithelial Kazal-type-related inhibitor).<br><br>OBJECTIVES: To establish a murine model of Netherton syndrome based on CRISPR/Cas9 gene editing technology.<br><br>MATERIALS &amp; METHODS: Spink5-sgRNA was designed to target exon 3 of the mouse Spink5 gene. Cas9 mRNA and sgRNA were microinjected into the zygotes of C57BL/6J mice. Spink5 homozygous knockout mice were born from a heterozygous intercross, and the phenotype of these mice was compared with wild-type regarding gross morphology, histopathology and immunofluorescent detection of LEKTI.<br><br>RESULTS: Following microinjection of zygotes using the CRISPR/Cas9 system, sequencing demonstrated a 22-bp deletion at exon 3 of the mouse Spink5 gene. Histological investigation revealed complete detachment of the stratum corneum from the underlying granular layer and an absence of LEKTI in skin from Spink5 homozygous knockout mice.<br><br>CONCLUSION: The 22-bp deleted Spink5 transgenic mouse model demonstrates the clinical phenotype and genotype of human Netherton syndrome, representing a useful tool for future gene correction and skin barrier/inflammation studies.},
}
@article {pmid36301532,
year = {2022},
author = {Matsuo, M and Matsuyama, M and Kobayashi, T and Kanda, S and Ansai, S and Kawakami, T and Hosokawa, E and Daido, Y and Kusakabe, TG and Naruse, K and Fukamachi, S},
title = {Retinal Cone Mosaic in sws1-Mutant Medaka (Oryzias latipes), A Teleost.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {63},
number = {11},
pages = {21},
pmid = {36301532},
issn = {1552-5783},
mesh = {Animals ; *Opsins/genetics/metabolism ; *Oryzias/genetics/metabolism ; Retina/metabolism ; *Retinal Cone Photoreceptor Cells/metabolism ; Vision, Ocular ; },
abstract = {PURPOSE: Ablation of short single cones (SSCs) expressing short-wavelength-sensitive opsin (SWS1) is well analyzed in the field of regenerative retinal cells. In contrast with ablation studies, the phenomena caused by the complete deletion of SWS1 are less well-understood. To assess the effects of SWS1 deficiency on retinal structure, we established and analyzed sws1-mutant medaka.<br><br>METHODS: To visualize SWS1, a monoclonal anti-SWS1 antibody and transgenic reporter fish (Tg(sws1:mem-egfp)) were generated. We also developed a CRISPR/Cas-driven sws1-mutant line. Retinal structure of sws1 mutant was visualized using anti-SWS1, 1D4, and ZPR1 antibodies and coumarin derivatives and compared with wild type, Tg(sws1:mem-egfp), and another opsin (lws) mutant.<br><br>RESULTS: Our rat monoclonal antibody specifically recognized medaka SWS1. Sws1 mutant retained regularly arranged cone mosaic as lws mutant and its SSCs had neither SWS1 nor long wavelength sensitive opsin. Depletion of sws1 did not affect the expression of long wavelength sensitive opsin, and vice versa. ZPR1 antibody recognized arrestin spread throughout double cones and long single cones in wild-type, transgenic, and sws1-mutant lines.<br><br>CONCLUSIONS: Comparative observation of sws1-mutant and wild-type retinas revealed that ZPR1 negativity is not a marker for SSCs with SWS1, but SSCs themselves. Loss of functional sws1 did not cause retinal degeneration, indicating that sws1 is not essential for cone mosaic development in medaka. Our two fish lines, one with visualized SWS1 and the other lacking functional SWS1, offer an opportunity to study neural network synapsing with SSCs and to clarify the role of SWS1 in vision.},
}
@article {pmid36301479,
year = {2023},
author = {Tiwari, K and Kumar, R and Saudagar, P},
title = {Design of SaCas9-HF for In Vivo Gene Therapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2575},
number = {},
pages = {261-268},
pmid = {36301479},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Staphylococcus aureus/metabolism ; Gene Editing/methods ; Genetic Therapy ; Endonucleases/genetics ; },
abstract = {Genome alteration results in several diseases for which therapeutics are limited. Gene editing provides a strong and potential alternative for the treatment of rare and genetic diseases. CRISPR-Cas9-based system is now being envisaged as a potential tool for the cure of genetic diseases. The RNA-guided nuclease, SaCas9 enzyme, along with its HF versions is widely employed for in vivo gene editing because of its small size and high efficiency. The current work summarizes the widely used and improved methods for in vivo manipulation of genes. The potential of CRISPR-Cas9-based systems can be harnessed to treat genetic diseases and holds great promise for therapeutic interventions in gene therapy. The in vivo gene editing poses a caveat in the form of delivery systems, the tissue in question, and several other factors. This work describes the methods which have been optimized to offer high efficiency, delivery, and gene editing in vivo.},
}
@article {pmid36301478,
year = {2023},
author = {Kumar, R and Tiwari, K and Saudagar, P},
title = {Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2575},
number = {},
pages = {241-260},
pmid = {36301478},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Eukaryota/genetics ; RNA, Guide/genetics/metabolism ; Gene Editing/methods ; DNA/genetics ; },
abstract = {The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.},
}
@article {pmid36301469,
year = {2023},
author = {Pereira, GC},
title = {Latest Trends in Nucleic Acids' Engineering Techniques Applied to Precision Medicine.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2575},
number = {},
pages = {25-38},
pmid = {36301469},
issn = {1940-6029},
mesh = {Humans ; *Nucleic Acids ; Precision Medicine ; *COVID-19 ; Genetic Engineering/methods ; DNA ; CRISPR-Cas Systems ; },
abstract = {Nucleic acids are paving the way for advanced therapeutics. Unveiling the genome enabled a better understanding of unique genotype-phenotype profiling. Methods for engineering and analysis of nucleic acids, from polymerase chain reaction to Cre-Lox recombination, are contributing greatly to biomarkers' discovery, mapping of cellular signaling cascades, and smart design of therapeutics in precision medicine. Investigating the different subtypes of DNA and RNA via sequencing and profiling is empowering the scientific community with valuable information, to be used in advanced therapeutics, tracking epigenetics linked to disease. Recent results from the application of nucleic acids in novel therapeutics and precision medicine are very encouraging, demonstrating great potential to treat cancer, viral infections via inoculation (e.g., SAR-COV-2 mRNA vaccines), along with metabolic and genetic disorders. Limitations posed by challenges in delivery mode are being addressed to enable efficient guided-gene-programmed precision therapies. With the focus on genetic engineering and novel therapeutics, more precisely, in precision medicine, this chapter discusses the advance enabled by knowledge derived from these innovative branches of biotechnology.},
}
@article {pmid36301087,
year = {2022},
author = {Wu, L and Wang, X and Wu, X and Xu, S and Liu, M and Cao, X and Tang, T and Huang, X and Huang, H},
title = {MnO2 Nanozyme-Mediated CRISPR-Cas12a System for the Detection of SARS-CoV-2.},
journal = {ACS applied materials &amp; interfaces},
volume = {14},
number = {45},
pages = {50534-50542},
pmid = {36301087},
issn = {1944-8252},
mesh = {Humans ; *SARS-CoV-2/genetics ; CRISPR-Cas Systems/genetics ; Manganese Compounds ; *COVID-19/diagnosis ; Nucleic Acid Amplification Techniques/methods ; Oxides ; DNA, Single-Stranded ; },
abstract = {The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3',5,5'-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.},
}
@article {pmid36300761,
year = {2022},
author = {Zhang, Y and Yang, J and Yao, H and Zhang, Z and Song, Y},
title = {CRISPR/Cas9-mediated deletion of Fam83h induces defective tooth mineralization and hair development in rabbits.},
journal = {Journal of cellular and molecular medicine},
volume = {26},
number = {22},
pages = {5670-5679},
pmid = {36300761},
issn = {1582-4934},
mesh = {Humans ; Mice ; Animals ; Rabbits ; *CRISPR-Cas Systems/genetics ; Proteins/genetics ; *Amelogenesis Imperfecta/genetics/pathology ; Tooth Calcification ; Hair/pathology ; },
abstract = {Family with sequence similarity 83 members H (Fam83h) is essential for dental enamel formation. Fam83h mutations cause human amelogenesis imperfecta (AI), an inherited disorder characterized by severe hardness defects in dental enamel. Nevertheless, previous studies showed no enamel defects in Fam83h-knockout/lacZ-knockin mice. In this study, a large deletion of the Fam83h gene (900 bp) was generated via a dual sgRNA-directed CRISPR/Cas9 system in rabbits. Abnormal tooth mineralization and loose dentine were found in homozygous Fam83h knockout (Fam83h[-/-]) rabbits compared with WT rabbits. In addition, reduced hair follicle counts in dorsal skin, hair cycling dysfunction and hair shaft differentiation deficiency were observed in Fam83h[-/-] rabbits. Moreover, X-rays and staining of bone sections showed abnormal bending of the ulna and radius and an ulnar articular surface with insufficient trabecular bone in Fam83h[-/-] rabbits. Taken together, these data are the first report of defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius in Fam83h[-/-] rabbits. This novel Fam83h[-/-] rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of the Fam83h mutation.},
}
@article {pmid36298718,
year = {2022},
author = {Kanojia, A and Sharma, M and Shiraz, R and Tripathi, S},
title = {Flavivirus-Host Interaction Landscape Visualized through Genome-Wide CRISPR Screens.},
journal = {Viruses},
volume = {14},
number = {10},
pages = {},
pmid = {36298718},
issn = {1999-4915},
mesh = {Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Flavivirus/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; *Viruses/genetics ; Antiviral Agents/pharmacology ; },
abstract = {Flaviviruses comprise several important human pathogens which cause significant morbidity and mortality worldwide. Like any other virus, they are obligate intracellular parasites. Therefore, studying the host cellular factors that promote or restrict their replication and pathogenesis becomes vital. Since inhibiting the host dependency factors or activating the host restriction factors can suppress the viral replication and propagation in the cell, identifying them reveals potential targets for antiviral therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has provided an effective means of producing customizable genetic modifications and performing forward genetic screens in a broad spectrum of cell types and organisms. The ease, rapidity, and high reproducibility of CRISPR technology have made it an excellent tool for carrying out genome-wide screens to identify and characterize viral host dependency factors systematically. Here, we review the insights from various Genome-wide CRISPR screens that have advanced our understanding of Flavivirus-Host interactions.},
}
@article {pmid36298520,
year = {2022},
author = {Rabaan, AA and Mutair, AA and Hajissa, K and Alfaraj, AH and Al-Jishi, JM and Alhajri, M and Alwarthan, S and Alsuliman, SA and Al-Najjar, AH and Al Zaydani, IA and Al-Absi, GH and Alshaikh, SA and Alkathlan, MS and Almuthree, SA and Alawfi, A and Alshengeti, A and Almubarak, FZ and Qashgari, MS and Abdalla, ANK and Alhumaid, S},
title = {A Comprehensive Review on the Current Vaccines and Their Efficacies to Combat SARS-CoV-2 Variants.},
journal = {Vaccines},
volume = {10},
number = {10},
pages = {},
pmid = {36298520},
issn = {2076-393X},
abstract = {Since the first case of Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, SARS-CoV-2 infection has affected many individuals worldwide. Eventually, some highly infectious mutants-caused by frequent genetic recombination-have been reported for SARS-CoV-2 that can potentially escape from the immune responses and induce long-term immunity, linked with a high mortality rate. In addition, several reports stated that vaccines designed for the SARS-CoV-2 wild-type variant have mixed responses against the variants of concern (VOCs) and variants of interest (VOIs) in the human population. These results advocate the designing and development of a panvaccine with the potential to neutralize all the possible emerging variants of SARS-CoV-2. In this context, recent discoveries suggest the design of SARS-CoV-2 panvaccines using nanotechnology, siRNA, antibodies or CRISPR-Cas platforms. Thereof, the present comprehensive review summarizes the current vaccine design approaches against SARS-CoV-2 infection, the role of genetic mutations in the emergence of new viral variants, the efficacy of existing vaccines in limiting the infection of emerging SARS-CoV-2 variants, and efforts or challenges in designing SARS panvaccines.},
}
@article {pmid36297672,
year = {2022},
author = {Du, R and Wang, C and Zhu, L and Yang, Y},
title = {Extracellular Vesicles as Delivery Vehicles for Therapeutic Nucleic Acids in Cancer Gene Therapy: Progress and Challenges.},
journal = {Pharmaceutics},
volume = {14},
number = {10},
pages = {},
pmid = {36297672},
issn = {1999-4923},
abstract = {Extracellular vesicles (EVs) are nanoscale vesicles secreted by most types of cells as natural vehicles to transfer molecular information between cells. Due to their low toxicity and high biocompatibility, EVs have attracted increasing attention as drug delivery systems. Many studies have demonstrated that EV-loaded nucleic acids, including RNA-based nucleic acid drugs and CRISPR/Cas gene-editing systems, can alter gene expressions and functions of recipient cells for cancer gene therapy. Here in this review, we discuss the advantages and challenges of EV-based nucleic acid delivery systems in cancer therapy. We summarize the techniques and methods to increase EV yield, enhance nucleic acid loading efficiency, extend circulation time, and improve targeted delivery, as well as their applications in gene therapy and combination with other cancer therapies. Finally, we discuss the current status, challenges, and prospects of EVs as a therapeutic tool for the clinical application of nucleic acid drugs.},
}
@article {pmid36297131,
year = {2022},
author = {Olech, M},
title = {Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus.},
journal = {Pathogens (Basel, Switzerland)},
volume = {11},
number = {10},
pages = {},
pmid = {36297131},
issn = {2076-0817},
abstract = {Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of an acute and devastating enteric disease that causes moderate-to-high mortality in suckling piglets. The accurate and early detection of PEDV infection is essential for the prevention and control of the spread of the disease. Many molecular assays have been developed for the detection of PEDV, including reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR) and loop-mediated isothermal amplification assays. Additionally, several serological methods have been developed and are widely used for the detection of antibodies against PEDV. Some of them, such as the immunochromatography assay, can generate results very quickly and in field conditions. Molecular assays detect viral RNA in clinical samples rapidly, and with high sensitivity and specificity. Serological assays can determine prior immune exposure to PEDV, can be used to monitor the efficacy of vaccination strategies and may help to predict the duration of immunity in piglets. However, they are less sensitive than nucleic acid-based detection methods. Sanger and next-generation sequencing (NGS) allow the analysis of PEDV cDNA or RNA sequences, and thus, provide highly specific results. Furthermore, NGS based on nonspecific DNA cleavage in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems promise major advances in the diagnosis of PEDV infection. The objective of this paper was to summarize the current serological and molecular PEDV assays, highlight their diagnostic performance and emphasize the advantages and drawbacks of the application of individual tests.},
}
@article {pmid36296588,
year = {2022},
author = {Yuan, B and Yuan, C and Li, L and Long, M and Chen, Z},
title = {Application of the CRISPR/Cas System in Pathogen Detection: A Review.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {20},
pages = {},
pmid = {36296588},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Polymerase Chain Reaction ; *CRISPR-Associated Proteins/genetics ; },
abstract = {Early and rapid diagnosis of pathogens is important for the prevention and control of epidemic disease. The polymerase chain reaction (PCR) technique requires expensive instrument control, a special test site, complex solution treatment steps and professional operation, which can limit its application in practice. The pathogen detection method based on the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated protein (CRISPR/Cas) system is characterized by strong specificity, high sensitivity and convenience for detection, which is more suitable for practical applications. This article first reviews the CRISPR/Cas system, and then introduces the application of the two types of systems represented by Type II (cas9), Type V (cas12a, cas12b, cas14a) and Type VI (cas13a) in pathogen detection. Finally, challenges and prospects are proposed.},
}
@article {pmid36296288,
year = {2022},
author = {Ferdous, MA and Islam, SI and Habib, N and Almehmadi, M and Allahyani, M and Alsaiari, AA and Shafie, A},
title = {CRISPR-Cas Genome Editing Technique for Fish Disease Management: Current Study and Future Perspective.},
journal = {Microorganisms},
volume = {10},
number = {10},
pages = {},
pmid = {36296288},
issn = {2076-2607},
abstract = {Scientists have discovered many ways to treat bacteria, viruses, and parasites in aquaculture; however, there is still an impossibility in finding a permanent solution for all types of diseases. In that case, the CRISPR-Cas genome-editing technique can be the potential solution to preventing diseases for aquaculture sustainability. CRISPR-Cas is cheaper, easier, and more precise than the other existing genome-editing technologies and can be used as a new disease treatment tool to solve the far-reaching challenges in aquaculture. This technique may now be employed in novel ways, such as modifying a single nucleotide base or tagging a location in the DNA with a fluorescent protein. This review paper provides an informative discussion on adopting CRISPR technology in aquaculture disease management. Starting with the basic knowledge of CRISPR technology and phages, this study highlights the development of RNA-guided immunity to combat the Chilodonella protozoan group and nervous necrosis virus (NNV) in marine finfish. Additionally, we highlight the immunological application of CRISPR-Cas against bacterial diseases in channel catfish and the white spot syndrome virus (WSSV) in shrimp. In addition, the review summarizes a synthesis of bioinformatics tools used for CRISPR-Cas sgRNA design, and acceptable solutions are discussed, considering the limitations.},
}
@article {pmid36294556,
year = {2022},
author = {Schusterbauer, V and Fischer, JE and Gangl, S and Schenzle, L and Rinnofner, C and Geier, M and Sailer, C and Glieder, A and Thallinger, GG},
title = {Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in Komagataella phaffii: A Budding Yeast in Distress.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {8},
number = {10},
pages = {},
pmid = {36294556},
issn = {2309-608X},
abstract = {The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies. We compared the outcomes of single target CRISPR transformations to double target experiments. Furthermore, we examined the extent of possible large deletions by targeting a large genomic region, which is likely to be non-essential. The analysis of on-target mutations showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target loci. We also observed an increase of on-target structural variants in double target experiments as compared to single target experiments. Targeting of two loci within a putatively non-essential region led to a truncation of chromosome 3 at the target locus in multiple cases, causing the deletion of 20 genes and several ribosomal DNA repeats. The identified de novo off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites.},
}
@article {pmid36293232,
year = {2022},
author = {Yee, T and Wert, KJ},
title = {Base and Prime Editing in the Retina-From Preclinical Research toward Human Clinical Trials.},
journal = {International journal of molecular sciences},
volume = {23},
number = {20},
pages = {},
pmid = {36293232},
issn = {1422-0067},
support = {5T32GM131945/NH/NIH HHS/United States ; P30EY030413/EY/NEI NIH HHS/United States ; },
mesh = {Humans ; Aged ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/metabolism ; Gene Editing ; Retina/metabolism ; *Retinal Diseases/metabolism ; DNA/metabolism ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases that are one of the leading causes of vision loss in young and aged individuals. IRDs are mainly caused by a loss of the post-mitotic photoreceptor neurons of the retina, or by the degeneration of the retinal pigment epithelium. Unfortunately, once these cells are damaged, it is irreversible and leads to permanent vision impairment. Thought to be previously incurable, gene therapy has been rapidly evolving to be a potential treatment to prevent further degeneration of the retina and preserve visual function. The development of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) base and prime editors have increased the capabilities of the genome editing toolbox in recent years. Both base and prime editors evade the creation of double-stranded breaks in deoxyribonucleic acid (DNA) and the requirement of donor template of DNA for repair, which make them advantageous methods in developing clinical therapies. In addition, establishing a permanent edit within the genome could be better suited for patients with progressive degeneration. In this review, we will summarize published uses of successful base and prime editing in treating IRDs.},
}
@article {pmid36292796,
year = {2022},
author = {König, S and Fliegauf, M and Rhiel, M and Grimbacher, B and Cornu, TI and Cathomen, T and Mussolino, C},
title = {Allele-Specific Disruption of a Common STAT3 Autosomal Dominant Allele Is Not Sufficient to Restore Downstream Signaling in Patient-Derived T Cells.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
pmid = {36292796},
issn = {2073-4425},
mesh = {Humans ; Alleles ; *Immunoglobulin E/genetics ; T-Lymphocytes/metabolism ; *Job Syndrome/genetics/therapy/diagnosis ; Signal Transduction/genetics ; STAT3 Transcription Factor/genetics/metabolism ; },
abstract = {Dominant negative mutations in the STAT3 gene account for autosomal dominant hyper-IgE syndrome (AD-HIES). Patients typically present high IgE serum levels, recurrent infections, and soft tissue abnormalities. While current therapies focus on alleviating the symptoms, hematopoietic stem cell transplantation (HSCT) has recently been proposed as a strategy to treat the immunological defect and stabilize the disease, especially in cases with severe lung infections. However, because of the potentially severe side effects associated with allogeneic HSCT, this has been considered only for a few patients. Autologous HSCT represents a safer alternative but it requires the removal of the dominant negative mutation in the patients' cells prior to transplantation. Here, we developed allele-specific CRISPR-Cas9 nucleases to selectively disrupt five of the most common STAT3 dominant negative alleles. When tested ex vivo in patient-derived hematopoietic cells, allele-specific disruption frequencies varied in an allele-dependent fashion and reached up to 62% of alleles harboring the V637M mutation without detectable alterations in the healthy STAT3 allele. However, assessment of the gene expression profiles of the STAT3 downstream target genes revealed that, upon activation of those edited patient cells, mono-allelic STAT3 expression (functional haploinsufficiency) is not able to sufficiently restore STAT3-dependent signaling in edited T cells cultured in vitro. Moreover, the stochastic mutagenesis induced by the repair of the nuclease-induced DNA break could further contribute to dominant negative effects. In summary, our results advocate for precise genome editing strategies rather than allele-specific gene disruption to correct the underlying mutations in AD-HIES.},
}
@article {pmid36292795,
year = {2022},
author = {Wang, L and Gao, Y and Wang, J and Huang, N and Jiang, Q and Ju, Z and Yang, C and Wei, X and Xiao, Y and Zhang, Y and Yang, L and Huang, J},
title = {Selection Signature and CRISPR/Cas9-Mediated Gene Knockout Analyses Reveal ZC3H10 Involved in Cold Adaptation in Chinese Native Cattle.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
pmid = {36292795},
issn = {2073-4425},
mesh = {Animals ; Cattle/genetics ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Thermogenesis ; Glucose ; Lipids ; },
abstract = {Cold stress is an important factor affecting cattle health, production performance, and reproductive efficiency. Understanding of the potential mechanism underlying genetic adaptation to local environments, particularly extreme cold environment, is limited. Here, by using FLK and hapFLK methods, we found that the Zinc finger CCCH-type containing 10 (ZC3H10) gene underwent positive selection in the Menggu, Fuzhou, Anxi, and Shigatse humped cattle breeds that are distributed in the cold areas of China. Furthermore, ZC3H10 expression significantly increased in bovine fetal fibroblast (BFF) cells at 28 °C for 4 h. ZC3H10 knockout BFFs were generated using CRISPR/Cas9. Wild and ZC3H10-deleted BFFs were treated at two temperatures and were divided into four groups (WT, wild and cultured at 38 °C; KO, ZC3H10[-/-] and 38 °C; WT_LT, wild, and 28 °C for 4 h; and KO_LT, ZC3H10[-/-] and 28 °C for 4 h. A total of 466, 598, 519, and 650 differently expressed genes (two-fold or more than two-fold changes) were identified by determining transcriptomic difference (KO_LT vs. KO, WT_LT vs. WT, KO vs. WT, and KO_LT vs. WT_LT, respectively). Loss of ZC3H10 dysregulated pathways involved in thermogenesis and immunity, and ZC3H10 participated in immunity-related pathways induced by cold stress and regulated genes involved in glucose and lipid metabolism and lipid transport (PLTP and APOA1), thereby facilitating adaptability to cold stress. Our findings provide a foundation for further studies on the function of ZC3H10 in cold stress and development of bovine breeding strategies for combatting the influences of cold climate.},
}
@article {pmid36292757,
year = {2022},
author = {Kreppel, F and Hagedorn, C},
title = {Episomes and Transposases-Utilities to Maintain Transgene Expression from Nonviral Vectors.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
pmid = {36292757},
issn = {2073-4425},
mesh = {*Transposases/genetics ; *Epigenesis, Genetic ; DNA Transposable Elements ; Transgenes ; Plasmids/genetics ; Chromatin ; },
abstract = {The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.},
}
@article {pmid36292612,
year = {2022},
author = {Cosenza, LC and Zuccato, C and Zurlo, M and Gambari, R and Finotti, A},
title = {Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β[0]39-Globin Gene Editing and Induction of Fetal Hemoglobin.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
pmid = {36292612},
issn = {2073-4425},
support = {208872/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adult ; Humans ; Fetal Hemoglobin/genetics/metabolism ; Gene Editing/methods ; *beta-Thalassemia/genetics/therapy ; CRISPR-Cas Systems/genetics ; Erythroid Cells/metabolism ; *Thalassemia/genetics ; Sirolimus ; },
abstract = {Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β[0]39-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β[0]39-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β[0]39-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.},
}
@article {pmid36292566,
year = {2022},
author = {Shi, J and Ni, X and Huang, J and Fu, Y and Wang, T and Yu, H and Zhang, Y},
title = {CRISPR/Cas9-Mediated Gene Editing of BnFAD2 and BnFAE1 Modifies Fatty Acid Profiles in Brassica napus.},
journal = {Genes},
volume = {13},
number = {10},
pages = {},
pmid = {36292566},
issn = {2073-4425},
mesh = {*Brassica napus/genetics ; Gene Editing/methods ; Erucic Acids ; Fatty Acids/genetics ; Fatty Acid Elongases/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Fatty Acids, Unsaturated ; Oleic Acid ; Oxidoreductases/genetics ; },
abstract = {Fatty acid (FA) composition determines the quality of oil from oilseed crops, and thus is a major target for genetic improvement. FAD2 (Fatty acid dehydrogenase 2) and FAE1 (fatty acid elongase 1) are critical FA synthetic genes, and have been the focus of genetic manipulation to alter fatty acid composition in oilseed plants. In this study, to improve the nutritional quality of rapeseed cultivar CY2 (about 50% oil content; of which 40% erucic acid), we generated novel knockout plants by CRISPR/Cas9 mediated genome editing of BnFAD2 and BnFAE1 genes. Two guide RNAs were designed to target one copy of the BnFAD2 gene and two copies of the BnFAE1 gene, respectively. A number of lines with mutations at three target sites of BnFAD2 and BnFAE1 genes were identified by sequence analysis. Three of these lines showed mutations in all three target sites of the BnFAD2 and BnFAE1 genes. Fatty acid composition analysis of seeds revealed that mutations at all three sites resulted in significantly increased oleic acid (70-80%) content compared with that of CY2 (20%), greatly reduced erucic acid levels and slightly decreased polyunsaturated fatty acids content. Our results confirmed that the CRISPR/Cas9 system is an effective tool for improving this important trait.},
}
@article {pmid36292145,
year = {2022},
author = {Li, J and Wang, Y and Wang, B and Lou, J and Ni, P and Jin, Y and Chen, S and Duan, G and Zhang, R},
title = {Application of CRISPR/Cas Systems in the Nucleic Acid Detection of Infectious Diseases.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {10},
pages = {},
pmid = {36292145},
issn = {2075-4418},
abstract = {The CRISPR/Cas system is a protective adaptive immune system against attacks from foreign mobile genetic elements. Since the discovery of the excellent target-specific sequence recognition ability of the CRISPR/Cas system, the CRISPR/Cas system has shown excellent performance in the development of pathogen nucleic-acid-detection technology. In combination with various biosensing technologies, researchers have made many rapid, convenient, and feasible innovations in pathogen nucleic-acid-detection technology. With an in-depth understanding and development of the CRISPR/Cas system, it is no longer limited to CRISPR/Cas9, CRISPR/Cas12, and other systems that had been widely used in the past; other CRISPR/Cas families are designed for nucleic acid detection. We summarized the application of CRISPR/Cas-related technology in infectious-disease detection and its development in SARS-CoV-2 detection.},
}
@article {pmid36291798,
year = {2022},
author = {Thomsen, MK},
title = {Application of CRISPR for In Vivo Mouse Cancer Studies.},
journal = {Cancers},
volume = {14},
number = {20},
pages = {},
pmid = {36291798},
issn = {2072-6694},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) are widely used in cancer research to edit specific genes and study their functions. This applies both to in vitro and in vivo studies where CRISPR technology has accelerated the generation of specific loss- or gain-of-function mutations. This review focuses on CRISPR for generating in vivo models of cancer by editing somatic cells in specific organs. The delivery of CRISPR/Cas to designated tissues and specific cell compartments is discussed with a focus on different methods and their advantages. One advantage of CRISPR/Cas is the possibility to target multiple genes simultaneously in the same cell and therefore generate complex mutation profiles. This complexity challenges the interpretation of results and different methods to analyze the samples discussed herein. CRISPR-induced tumors are also different from classical tumors in pre-clinical models. Especially the clonal evolution of CRISPR-induced tumors adds new insight into cancer biology. Finally, the review discusses future perspectives for CRISPR technology in pre-clinical models with a focus on in vivo screening, CRISPR activation/inhibition, and the development of prime/ base-editing for the introduction of specific gene editing.},
}
@article {pmid36291103,
year = {2022},
author = {Karagyaur, M and Primak, A and Efimenko, A and Skryabina, M and Tkachuk, V},
title = {The Power of Gene Technologies: 1001 Ways to Create a Cell Model.},
journal = {Cells},
volume = {11},
number = {20},
pages = {},
pmid = {36291103},
issn = {2073-4409},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genome ; Cloning, Molecular ; },
abstract = {Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies.},
}
@article {pmid36290917,
year = {2022},
author = {Huang, Z and Liu, S and Pei, X and Li, S and He, Y and Tong, Y and Liu, G},
title = {Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection.},
journal = {Biosensors},
volume = {12},
number = {10},
pages = {},
pmid = {36290917},
issn = {2079-6374},
mesh = {CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Nucleic Acids ; },
abstract = {The CRISPR/Cas system is now being used extensively in nucleic acid detection applications, particularly after the trans-cleavage activity of several Cas effectors was found. A CRISPR/Cas system combined with multiple signal-readout techniques has been developed for various molecular diagnostics applications. Fluorescence is now a widely utilized dominant read-out technique in CRISPR biosensors. An in-depth understanding of various fluorescence readout types and variables affecting the fluorescence signals can facilitate better experimental designs to effectively improve the analytical performance. There are the following two commonly used types of CRISPR/Cas detection modes: the first is based on binding activity, such as Cas9 and dCas9; the second is based on cleavage activity, such as Cas12a, Cas12b, Cas13, and Cas14. In this review, fluorescence signal-readout strategies from the last 5 years based on the binding activity and cleavage activity of the CRISPR/Cas system with fundamentals and examples are fully discussed. A detailed comparison of the available fluorescent reporter sequences and design principles is summarized. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.},
}
@article {pmid36290480,
year = {2022},
author = {Wang, J and Yang, X and Wang, X and Wang, W},
title = {Recent Advances in CRISPR/Cas-Based Biosensors for Protein Detection.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {9},
number = {10},
pages = {},
pmid = {36290480},
issn = {2306-5354},
abstract = {CRISPR is an acquired immune system found in prokaryotes that can accurately recognize and cleave foreign nucleic acids, and has been widely explored for gene editing and biosensing. In the past, CRISPR/Cas-based biosensors were mainly applied to detect nucleic acids in the field of biosensing, and their applications for the detection of other types of analytes were usually overlooked such as small molecules and disease-related proteins. The recent work shows that CRISPR/Cas biosensors not only provide a new tool for protein analysis, but also improve the sensitivity and specificity of protein detections. However, it lacks the latest review to summarize CRISPR/Cas-based biosensors for protein detection and elucidate their mechanisms of action, hindering the development of superior biosensors for proteins. In this review, we summarized CRISPR/Cas-based biosensors for protein detection based on their mechanism of action in three aspects: antibody-assisted CRISPR/Cas-based protein detection, aptamer-assisted CRISPR/Cas-based protein detection, and miscellaneous CRISPR/Cas-based methods for protein detection, respectively. Moreover, the prospects and challenges for CRISPR/Cas-based biosensors for protein detection are also discussed.},
}
@article {pmid36289480,
year = {2022},
author = {Zarate, OA and Yang, Y and Wang, X and Wang, JP},
title = {BoostMEC: predicting CRISPR-Cas9 cleavage efficiency through boosting models.},
journal = {BMC bioinformatics},
volume = {23},
number = {1},
pages = {446},
pmid = {36289480},
issn = {1471-2105},
mesh = {*RNA, Guide/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Machine Learning ; },
abstract = {BACKGROUND: In the CRISPR-Cas9 system, the efficiency of genetic modifications has been found to vary depending on the single guide RNA (sgRNA) used. A variety of sgRNA properties have been found to be predictive of CRISPR cleavage efficiency, including the position-specific sequence composition of sgRNAs, global sgRNA sequence properties, and thermodynamic features. While prevalent existing deep learning-based approaches provide competitive prediction accuracy, a more interpretable model is desirable to help understand how different features may contribute to CRISPR-Cas9 cleavage efficiency.<br><br>RESULTS: We propose a gradient boosting approach, utilizing LightGBM to develop an integrated tool, BoostMEC (Boosting Model for Efficient CRISPR), for the prediction of wild-type CRISPR-Cas9 editing efficiency. We benchmark BoostMEC against 10 popular models on 13 external datasets and show its competitive performance.<br><br>CONCLUSIONS: BoostMEC can provide state-of-the-art predictions of CRISPR-Cas9 cleavage efficiency for sgRNA design and selection. Relying on direct and derived sequence features of sgRNA sequences and based on conventional machine learning, BoostMEC maintains an advantage over other state-of-the-art CRISPR efficiency prediction models that are based on deep learning through its ability to produce more interpretable feature insights and predictions.},
}
@article {pmid36289424,
year = {2022},
author = {O'Leary, K},
title = {A new frontier in CRISPR technology.},
journal = {Nature medicine},
volume = {},
number = {},
pages = {},
doi = {10.1038/d41591-022-00103-2},
pmid = {36289424},
issn = {1546-170X},
}
@article {pmid36289338,
year = {2022},
author = {Xu, J and Song, F and Lyu, H and Kobayashi, M and Zhang, B and Zhao, Z and Hou, Y and Wang, X and Luan, Y and Jia, B and Stasiak, L and Wong, JH and Wang, Q and Jin, Q and Jin, Q and Fu, Y and Yang, H and Hardison, RC and Dovat, S and Platanias, LC and Diao, Y and Yang, Y and Yamada, T and Viny, AD and Levine, RL and Claxton, D and Broach, JR and Zheng, H and Yue, F},
title = {Subtype-specific 3D genome alteration in acute myeloid leukaemia.},
journal = {Nature},
volume = {611},
number = {7935},
pages = {387-398},
pmid = {36289338},
issn = {1476-4687},
support = {R35 GM124820/GM/NIGMS NIH HHS/United States ; R01 HG009906/HG/NHGRI NIH HHS/United States ; R01 HG011207/HG/NHGRI NIH HHS/United States ; U01 CA200060/CA/NCI NIH HHS/United States ; R24 DK106766/DK/NIDDK NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA216421/CA/NCI NIH HHS/United States ; K08 CA215317/CA/NCI NIH HHS/United States ; U01 DA053691/DA/NIDA NIH HHS/United States ; },
mesh = {Humans ; Chromatin/genetics ; DNA Methylation ; *Leukemia, Myeloid, Acute/genetics ; *Genome, Human/genetics ; Promoter Regions, Genetic ; Enhancer Elements, Genetic ; Gene Silencing ; Reproducibility of Results ; CRISPR-Cas Systems ; Sequence Analysis ; DNA (Cytosine-5-)-Methyltransferases ; Gene Expression Regulation, Leukemic ; },
abstract = {Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases[1-5]. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&amp;Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.},
}
@article {pmid36288511,
year = {2022},
author = {Chen, J and Liu, J and Wu, D and Pan, R and Chen, J and Wu, Y and Huang, M and Li, G},
title = {CRISPR/Cas Precisely Regulated DNA-Templated Silver Nanocluster Fluorescence Sensor for Meat Adulteration Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {44},
pages = {14296-14303},
doi = {10.1021/acs.jafc.2c04500},
pmid = {36288511},
issn = {1520-5118},
mesh = {*Silver/chemistry ; CRISPR-Cas Systems ; Limit of Detection ; DNA/chemistry ; Fluorescent Dyes/chemistry ; Meat/analysis ; *Biosensing Techniques/methods ; },
abstract = {Meat adulteration can cause consumer fraud, food allergies, and religious issues. Rapid and sensitive detection methods are urgently demanded to supervise meat authenticity. Herein, a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas precisely regulated DNA-templated silver nanocluster (DNA-AgNC) sensor was ingeniously designed to detect meat adulteration. Specific sequence recognition of CRISPR/Cas12a allowed accurate identification of target DNA. The emerging label-free fluorescent probes, DNA-AgNCs, a class of promising fluorophores in biochemical analysis with attractive photostability and remarkably enhanced fluorescence properties, were first introduced as the substrates of CRISPR/Cas12a system, allowing a sensitive output of amplified signals through the precise regulation of the unique target DNA-activated trans-cleavage activity of Cas12a. Based on this specific recognition, efficient signal transduction of CRISPR/Cas12a, and the outstanding fluorescence properties of DNA-AgNCs, the proposed strategy achieved a satisfactory linear range from 10 pM to 1 μM with a limit of detection (LOD) as low as 1.9 pM, which can achieve sensitive detection of meat adulteration.},
}
@article {pmid36288366,
year = {2022},
author = {Zhang, X and Ge, X and Shen, F and Qiao, J and Zhang, Y and Li, H},
title = {Diagnostic efficiency of RPA/RAA integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis.},
journal = {PloS one},
volume = {17},
number = {10},
pages = {e0276728},
pmid = {36288366},
issn = {1932-6203},
mesh = {Humans ; *COVID-19/diagnosis ; SARS-CoV-2/genetics ; COVID-19 Testing ; CRISPR-Cas Systems/genetics ; Recombinases ; Sensitivity and Specificity ; },
abstract = {OBJECTIVE: To evaluate the diagnostic value of recombinase polymerase/ aided amplification (RPA/RAA) integrated clustered regularly interspaced short palindromic repeats (CRISPR) in the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).<br><br>METHODS: We searched relevant literature on CRISPR technology for COVID-19 diagnosis using "novel coronavirus", "clustered regularly interspaced short palindromic repeats" and "RPA/RAA" as subject terms in PubMed, Cochrane, Web of Science, and Embase databases. Further, we performed a meta-analysis after screening the literature, quality assessment, and data extraction.<br><br>RESULTS: The pooled sensitivity, specificity and a rea under the summary receiver operator characteristic curve (AUC) were 0.98 [95% confidence interval (CI):0.97-0.99], 0.99 (95% CI: 0.97-1.00) and 1.00 (95% CI: 0.98-1.00), respectively. For CRISPR-associated (Cas) proteins-12, the sensitivity, specificity was 0.98 (95% CI: 0.96-1.00), 1.00 (95% CI: 0.99-1.00), respectively. For Cas13, the sensitivity and specificity were 0.99 (95% CI: 0.97-1.00) and 0.95 (95% CI: 0.91-1.00). The positive likelihood ratio (PLR) was 183.2 (95% CI: 28.8, 1166.8); the negative likelihood ratio (NLR) was 0.02 (95% CI: 0.01, 0.03).<br><br>CONCLUSION: RPA/RAA integrated with CRISPR technology is used to diagnose coronavirus disease-19 (COVID-19) with high accuracy and can be used for large-scale population screening.},
}
@article {pmid36288050,
year = {2022},
author = {Zakiyyah, SN and Ibrahim, AU and Babiker, MS and Gaffar, S and Ozsoz, M and Zein, MIHL and Hartati, YW},
title = {Detection of Tropical Diseases Caused by Mosquitoes Using CRISPR-Based Biosensors.},
journal = {Tropical medicine and infectious disease},
volume = {7},
number = {10},
pages = {},
pmid = {36288050},
issn = {2414-6366},
abstract = {Tropical diseases (TDs) are among the leading cause of mortality and fatality globally. The emergence and reemergence of TDs continue to challenge healthcare system. Several tropical diseases such as yellow fever, tuberculosis, cholera, Ebola, HIV, rotavirus, dengue, and malaria outbreaks have led to endemics and epidemics around the world, resulting in millions of deaths. The increase in climate change, migration and urbanization, overcrowding, and other factors continue to increase the spread of TDs. More cases of TDs are recorded as a result of substandard health care systems and lack of access to clean water and food. Early diagnosis of these diseases is crucial for treatment and control. Despite the advancement and development of numerous diagnosis assays, the healthcare system is still hindered by many challenges which include low sensitivity, specificity, the need of trained pathologists, the use of chemicals and a lack of point of care (POC) diagnostic. In order to address these issues, scientists have adopted the use of CRISPR/Cas systems which are gene editing technologies that mimic bacterial immune pathways. Recent advances in CRISPR-based biotechnology have significantly expanded the development of biomolecular sensors for diagnosing diseases and understanding cellular signaling pathways. The CRISPR/Cas strategy plays an excellent role in the field of biosensors. The latest developments are evolving with the specific use of CRISPR, which aims for a fast and accurate sensor system. Thus, the aim of this review is to provide concise knowledge on TDs associated with mosquitoes in terms of pathology and epidemiology as well as background knowledge on CRISPR in prokaryotes and eukaryotes. Moreover, the study overviews the application of the CRISPR/Cas system for detection of TDs associated with mosquitoes.},
}
@article {pmid36284357,
year = {2022},
author = {Ferre-Fernández, JJ and Muheisen, S and Thompson, S and Semina, EV},
title = {CRISPR-Cas9-mediated functional dissection of the foxc1 genomic region in zebrafish identifies critical conserved cis-regulatory elements.},
journal = {Human genomics},
volume = {16},
number = {1},
pages = {49},
pmid = {36284357},
issn = {1479-7364},
support = {R01EY025718/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Zebrafish/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Developmental ; Forkhead Transcription Factors/genetics/metabolism ; *Glaucoma/genetics ; Genomics ; DNA, Intergenic/genetics/metabolism ; Nucleotides/metabolism ; },
abstract = {FOXC1 encodes a forkhead-domain transcription factor associated with several ocular disorders. Correct FOXC1 dosage is critical to normal development, yet the mechanisms controlling its expression remain unknown. Together with FOXQ1 and FOXF2, FOXC1 is part of a cluster of FOX genes conserved in vertebrates. CRISPR-Cas9-mediated dissection of genomic sequences surrounding two zebrafish orthologs of FOXC1 was performed. This included five zebrafish-human conserved regions, three downstream of foxc1a and two remotely upstream of foxf2a/foxc1a or foxf2b/foxc1b clusters, as well as two intergenic regions between foxc1a/b and foxf2a/b lacking sequence conservation but positionally corresponding to the area encompassing a previously reported glaucoma-associated SNP in humans. Removal of downstream sequences altered foxc1a expression; moreover, zebrafish carrying deletions of two or three downstream elements demonstrated abnormal phenotypes including enlargement of the anterior chamber of the eye reminiscent of human congenital glaucoma. Deletions of distant upstream conserved elements influenced the expression of foxf2a/b or foxq1a/b but not foxc1a/b within each cluster. Removal of either intergenic sequence reduced foxc1a or foxc1b expression during late development, suggesting a role in transcriptional regulation despite the lack of conservation at the nucleotide level. Further studies of the identified regions in human patients may explain additional individuals with developmental ocular disorders.},
}
@article {pmid36284311,
year = {2022},
author = {Tanaka, M and Yokoyama, K and Hayashi, H and Isaki, S and Kitatani, K and Wang, T and Kawata, H and Matsuzawa, H and Gurumurthy, CB and Miura, H and Ohtsuka, M},
title = {CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {228},
pmid = {36284311},
issn = {1474-760X},
support = {R35HG010719/HG/NHGRI NIH HHS/United States ; R21GM129559/GM/NIGMS NIH HHS/United States ; },
mesh = {Mice ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knock-In Techniques ; *CRISPR-Cas Systems ; DNA/genetics ; Genome ; Gene Editing/methods ; },
abstract = {CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.},
}
@article {pmid36284264,
year = {2022},
author = {Zhu, L and Wang, X and Li, F and Song, J},
title = {PreAcrs: a machine learning framework for identifying anti-CRISPR proteins.},
journal = {BMC bioinformatics},
volume = {23},
number = {1},
pages = {444},
pmid = {36284264},
issn = {1471-2105},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Machine Learning ; Algorithms ; RNA ; },
abstract = {BACKGROUND: Anti-CRISPR proteins are potent modulators that inhibit the CRISPR-Cas immunity system and have huge potential in gene editing and gene therapy as a genome-editing tool. Extensive studies have shown that anti-CRISPR proteins are essential for modifying endogenous genes, promoting the RNA-guided binding and cleavage of DNA or RNA substrates. In recent years, identifying and characterizing anti-CRISPR proteins has become a hot and significant research topic in bioinformatics. However, as most anti-CRISPR proteins fall short in sharing similarities to those currently known, traditional screening methods are time-consuming and inefficient. Machine learning methods could fill this gap with powerful predictive capability and provide a new perspective for anti-CRISPR protein identification.<br><br>RESULTS: Here, we present a novel machine learning ensemble predictor, called PreAcrs, to identify anti-CRISPR proteins from protein sequences directly. Three features and eight different machine learning algorithms were used to train PreAcrs. PreAcrs outperformed other existing methods and significantly improved the prediction accuracy for identifying anti-CRISPR proteins.<br><br>CONCLUSIONS: In summary, the PreAcrs predictor achieved a competitive performance for predicting new anti-CRISPR proteins in terms of accuracy and robustness. We anticipate PreAcrs will be a valuable tool for researchers to speed up the research process. The source code is available at: https://github.com/Lyn-666/anti_CRISPR.git .},
}
@article {pmid36283791,
year = {2022},
author = {Lai, Y and Li, M and Liao, X and Zou, L},
title = {DNAzyme-regulated CRISPR/Cas12a based fluorescent biosensor for sensitive detection of alkaline phosphatase activity and inhibition.},
journal = {Analytica chimica acta},
volume = {1233},
number = {},
pages = {340518},
doi = {10.1016/j.aca.2022.340518},
pmid = {36283791},
issn = {1873-4324},
mesh = {*DNA, Catalytic/chemistry ; Diphosphates/metabolism ; Alkaline Phosphatase/chemistry ; CRISPR-Cas Systems ; DNA, Single-Stranded ; Spectrometry, Fluorescence/methods ; *Biosensing Techniques/methods ; DNA/chemistry ; },
abstract = {Alkaline phosphatase (ALP) is regarded as an emerging biomarker and therapeutic target for various diseases. Herein, we developed a DNAzyme-regulated CRISPR/Cas12a cascade signal amplification strategy for sensitive and selective detection of ALP activity and inhibition. In this assay, the substrate strand of Cu[2+]-specific DNAzyme was designed as the DNA activator for Cas12a. The substrate strand would be cleaved into two fragments when ALP-catalyzed hydrolysis reaction disabled the complexation between Cu[2+] and pyrophosphate (PPi). In this case, the cleaved sequences could not further bind to the Cas12a-crRNA complex to activate the trans-cleavage activity of CRISPR/Cas12a, which would hamper the cleavage of doubly labeled single-stranded DNA (ssDNA) fluorescence reporter, and thus a turn-off fluorescence signal. The cascade signal amplification strategy greatly improved the detection sensitivity for ALP activity, with a detection limit as low as 0.04 U/L. Moreover, the proposed method was successfully applied for ALP activity detection in serum samples and ALP inhibitory capability evaluation. This method overcomes the shortcoming of conventional methods that show unsatisfactory sensitivity and are susceptible to interfering substances, which might hold great promise in clinical diagnosis and biomedical research.},
}
@article {pmid36283790,
year = {2022},
author = {Cai, X and Zhao, D and Li, X and Zheng, Q and Shu, X and Ding, S and Zhang, D and Yan, Y},
title = {An ultrasensitive biosensing platform for FEN1 activity detection based on target-induced primer extension to trigger the collateral cleavage of CRISPR/Cas12a.},
journal = {Analytica chimica acta},
volume = {1233},
number = {},
pages = {340519},
doi = {10.1016/j.aca.2022.340519},
pmid = {36283790},
issn = {1873-4324},
mesh = {*Flap Endonucleases ; CRISPR-Cas Systems ; DNA/genetics ; DNA Primers ; *Biosensing Techniques ; },
abstract = {Flap endonuclease 1 (FEN1), a structure-selective endonuclease essential for DNA replication and repair, has been considered as a new promising marker for early cancer diagnosis. However, reliable, sensitive and convenient biosensors for FEN1 detection are still technically challenging. Herein, a fluorometric biosensor based on target-induced primer extension to initiate the collateral cleavage of CRISPR/Cas12a has been established for ultrasensitive and specific detection of FEN1 activity. Using branched DNA to probe FEN1 activity, the cleaved 5' flap initiated DNA polymerase-mediated primer extension to produce plenty of DNA duplexes containing protospacer adjacent motif (PAM) which act as activators to initiate the collateral cleavage activity of Cas12a protein, producing an significantly amplified fluorescence response for ultrasensitive determination of FEN1 activity. The developed biosensing platform displays excellent analytical performance, with a limit of detection (LOD) down to 8.9 × 10[-5] U μL[-1], and a wide linear range from 1.0 × 10[-4] to 5.0 × 10[-1] U μL[-1]. Moreover, the proposed strategy was successfully used for FEN1 detection in serums and cell lysates and suggests potential clinical applications, which may provide a reliable approach for FEN1 that will allow effective diagnosis in the early stages of related cancer.},
}
@article {pmid36282000,
year = {2022},
author = {Steens, JA and Salazar, CRP and Staals, RHJ},
title = {The diverse arsenal of type III CRISPR-Cas-associated CARF and SAVED effectors.},
journal = {Biochemical Society transactions},
volume = {50},
number = {5},
pages = {1353-1364},
pmid = {36282000},
issn = {1470-8752},
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; RNA, Guide ; Second Messenger Systems ; Catalytic Domain ; },
abstract = {Type III CRISPR-Cas systems make use of a multi-subunit effector complex to target foreign (m)RNA transcripts complementary to the guide/CRISPR RNA (crRNA). Base-pairing of the target RNA with specialized regions in the crRNA not only triggers target RNA cleavage, but also activates the characteristic Cas10 subunit and sets in motion a variety of catalytic activities that starts with the production of cyclic oligoadenylate (cOA) second messenger molecules. These messenger molecules can activate an extensive arsenal of ancillary effector proteins carrying the appropriate sensory domain. Notably, the CARF and SAVED effector proteins have been responsible for renewed interest in type III CRISPR-Cas due to the extraordinary diversity of defenses against invading genetic elements. Whereas only a handful of CARF and SAVED proteins have been studied so far, many of them seem to provoke abortive infection, aimed to kill the host and provide population-wide immunity. A defining feature of these effector proteins is the variety of in silico-predicted catalytic domains they are fused to. In this mini-review, we discuss all currently characterized type III-associated CARF and SAVED effector proteins, highlight a few examples of predicted CARF and SAVED proteins with interesting predicted catalytic activities, and speculate how they could contribute to type III immunity.},
}
@article {pmid36280712,
year = {2022},
author = {Wang, S and Guo, M and Zhu, Y and Lin, Z and Huang, Z},
title = {Cryo-EM structure of the type III-E CRISPR-Cas effector gRAMP in complex with TPR-CHAT.},
journal = {Cell research},
volume = {32},
number = {12},
pages = {1128-1131},
pmid = {36280712},
issn = {1748-7838},
mesh = {*CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *CRISPR-Associated Proteins/chemistry ; RNA, Bacterial/chemistry ; },
}
@article {pmid36280711,
year = {2022},
author = {Yang, H and Patel, DJ},
title = {A type III-E CRISPR Craspase exhibiting RNase and protease activities.},
journal = {Cell research},
volume = {32},
number = {12},
pages = {1044-1046},
pmid = {36280711},
issn = {1748-7838},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Ribonucleases ; Endoribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Peptide Hydrolases ; Ribonuclease III ; CRISPR-Cas Systems ; },
}
@article {pmid36280707,
year = {2022},
author = {Chavez, M and Chen, X and Finn, PB and Qi, LS},
title = {Advances in CRISPR therapeutics.},
journal = {Nature reviews. Nephrology},
volume = {},
number = {},
pages = {1-14},
pmid = {36280707},
issn = {1759-507X},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR-Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.},
}
@article {pmid36280360,
year = {2022},
author = {Sengupta, A and Liu, D and Pakrasi, HB},
title = {CRISPR-Cas mediated genome engineering of cyanobacteria.},
journal = {Methods in enzymology},
volume = {676},
number = {},
pages = {403-432},
doi = {10.1016/bs.mie.2022.07.023},
pmid = {36280360},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Cyanobacteria/genetics ; Synthetic Biology ; Photosynthesis/genetics ; Metabolic Engineering ; },
abstract = {Over the past decade, several cyanobacterial strains have emerged as exciting model systems for unraveling important biological process like photosynthesis and nitrogen fixation. In parallel, novel strains are being developed as platforms for production of various value-added products. To meet either of these goals, synthetic biology tool development has been prioritized, and among many such tools, CRISPR-mediated genome editing tools distinctly hold the potential to revolutionize cyanobacterial research. This chapter reviews our current understanding of the existing and emerging CRISPR-based technologies and their potential application for advanced genome editing in cyanobacterial strains of interest. CRISPR-based tools have gained traction in cyanobacterial research for their ability to target the polyploid genomes in these organisms and generate fully segregated mutants in a remarkably short time. We discuss the native cyanobacterial CRISPR system and the promise they hold for use as precision tools for cyanobacterial genome editing. We elaborate the methodologies for the development of CRISPR-based markerless mutants in cyanobacteria as well as discuss strategies for large scale, regulated genome silencing with CRISPRi. We also highlight some of the emerging CRISPR tools that have shown promise in other prokaryotic and eukaryotic systems but are yet to be adapted for cyanobacterial research.},
}
@article {pmid36280324,
year = {2022},
author = {Gupta, A and Singh, V and Mani, I},
title = {Dysbiosis of human microbiome and infectious diseases.},
journal = {Progress in molecular biology and translational science},
volume = {192},
number = {1},
pages = {33-51},
doi = {10.1016/bs.pmbts.2022.06.016},
pmid = {36280324},
issn = {1878-0814},
mesh = {Humans ; Dysbiosis ; Prebiotics ; *Gastrointestinal Microbiome ; *Microbiota ; *Probiotics ; Bacteria ; *Communicable Diseases ; },
abstract = {Since birth, the human body gets colonized by various communities of symbiotic or commensal microorganisms and they persist till the death of an individual. The human microbiome is comprised of the genomes of microorganisms such as viruses, archaea, eukaryotes, protozoa, and, most remarkably, bacteria. The development of "omics" technologies gave way to the Human Microbiome Project (HMP) which aimed at exploring the collection of microbial genes and genomes inhabiting the human body. Eubiosis, i.e., a healthy and balanced composition of such microbes contributes to the metabolic function, protection against pathogens and provides nutrients and energy to the host. Whereas, an imbalance in the diversity of microorganisms, termed dysbiosis, greatly influences the state of health and disease. This chapter summarizes the impact of gut bacteria on the well-being of humans and highlights the protective role played by the human microbiota during bacterial and viral infections. The condition of dysbiosis and how it plays a role in the establishment of various infections and metabolic disorders such as Clostridioides difficile infection (CFI), inflammatory bowel disease (IBD), cancer, periodontitis, and obesity are described in detail. Further, treatments such as fecal transplantation, probiotics, prebiotics, phage therapy, and CRISPR/Cas system, which target gut microbiota during digestive diseases are also discussed.},
}
@article {pmid36279605,
year = {2022},
author = {Yang, K and Zhang, W and Xu, L and Liu, Q and Song, X and Shao, Y and Tu, J and Qi, K},
title = {Facile, ultrasensitive, and highly specific diagnosis of goose astrovirus via reverse transcription-enzymatic recombinase amplification coupled with a CRISPR-Cas12a system detection.},
journal = {Poultry science},
volume = {101},
number = {12},
pages = {102208},
pmid = {36279605},
issn = {1525-3171},
mesh = {Animals ; *Reverse Transcription ; Recombinases ; Geese/genetics ; CRISPR-Cas Systems ; Chickens ; *Avastrovirus/genetics ; },
abstract = {Fatal gout in geese caused by goose astrovirus (GAstV) has been spreading rapidly in China since 2018, causing serious economic losses in the goose breeding industry. To achieve simple, convenient and sensitive detection of GAstV, a novel diagnostic test was developed by combining reverse transcription-enzymatic recombinase amplification (RT-ERA) and CRISPR-Cas12a technologies. RT-ERA primers were designed to pre-amplify the conserved region of the ORF2 gene of GAstV and the predefined target sequence detected using the Cas12a/crRNA complex at 37℃ for 30 min. Specific detection of GAstV was achieved with no cross-reaction with non-GAstV templates and a sensitivity detection limit of 2 copies. The experimental procedure could be completed within 1 h, including RNA extraction (15 min), RT-ERA reaction (20 min), CRISPR-Cas12a/crRNA detection (5 min) and result readout (within 2 min) steps. In conclusion, the combination of RT-ETA and CRISPR-Cas12a provides a rapid and specific method that should be effective for the control and surveillance of GAstV infections in farms from remote locations.},
}
@article {pmid36279540,
year = {2022},
author = {Yuan, J and Zhang, J and Zhang, Y and QiQiGe, W and Liu, W and Yan, S and Wang, G},
title = {Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {187},
pages = {},
doi = {10.3791/64195},
pmid = {36279540},
issn = {1940-087X},
mesh = {Animals ; Female ; CRISPR-Cas Systems ; *Insecticides ; *Tephritidae/genetics ; Drosophila ; Mutagenesis ; Insecta ; },
abstract = {The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.},
}
@article {pmid36278890,
year = {2022},
author = {Yin, X and Yang, H and Piao, Y and Zhu, Y and Zheng, Q and Khan, MR and Zhang, Y and Busquets, R and Hu, B and Deng, R and Cao, J},
title = {CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {43},
pages = {14052-14060},
doi = {10.1021/acs.jafc.2c05974},
pmid = {36278890},
issn = {1520-5118},
mesh = {Animals ; *Colorimetry/methods ; DNA Barcoding, Taxonomic ; *DNA, Catalytic ; DNA ; Takifugu ; },
abstract = {Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3',5,5'-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.},
}
@article {pmid36278822,
year = {2022},
author = {Turco, F and Garavaglia, M and Van Houdt, R and Hill, P and Rawson, FJ and Kovacs, K},
title = {Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.},
journal = {ACS synthetic biology},
volume = {11},
number = {11},
pages = {3617-3628},
pmid = {36278822},
issn = {2161-5063},
mesh = {*Synthetic Biology ; CRISPR-Cas Systems/genetics ; *Cupriavidus/genetics/metabolism ; Plasmids/genetics ; Metals/metabolism ; },
abstract = {Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.},
}
@article {pmid36278560,
year = {2022},
author = {Krishna, R and Ansari, WA and Soumia, PS and Yadav, A and Jaiswal, DK and Kumar, S and Singh, AK and Singh, M and Verma, JP},
title = {Biotechnological Interventions in Tomato (Solanum lycopersicum) for Drought Stress Tolerance: Achievements and Future Prospects.},
journal = {Biotech (Basel (Switzerland))},
volume = {11},
number = {4},
pages = {},
pmid = {36278560},
issn = {2673-6284},
abstract = {Tomato production is severely affected by abiotic stresses (drought, flood, heat, and salt) and causes approximately 70% loss in yield depending on severity and duration of the stress. Drought is the most destructive abiotic stress and tomato is very sensitive to the drought stress, as cultivated tomato lack novel gene(s) for drought stress tolerance. Only 20% of agricultural land worldwide is irrigated, and only 14.51% of that is well-irrigated, while the rest is rain fed. This scenario makes drought very frequent, which restricts the genetically predetermined yield. Primarily, drought disturbs tomato plant physiology by altering plant-water relation and reactive oxygen species (ROS) generation. Many wild tomato species have drought tolerance gene(s); however, their exploitation is very difficult because of high genetic distance and pre- and post-transcriptional barriers for embryo development. To overcome these issues, biotechnological methods, including transgenic technology and CRISPR-Cas, are used to enhance drought tolerance in tomato. Transgenic technology permitted the exploitation of non-host gene/s. On the other hand, CRISPR-Cas9 technology facilitated the editing of host tomato gene(s) for drought stress tolerance. The present review provides updated information on biotechnological intervention in tomato for drought stress management and sustainable agriculture.},
}
@article {pmid36276961,
year = {2022},
author = {Sharma, KK and Palakolanu, SR and Bhattacharya, J and Shankhapal, AR and Bhatnagar-Mathur, P},
title = {CRISPR for accelerating genetic gains in under-utilized crops of the drylands: Progress and prospects.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {999207},
pmid = {36276961},
issn = {1664-8021},
abstract = {Technologies and innovations are critical for addressing the future food system needs where genetic resources are an essential component of the change process. Advanced breeding tools like "genome editing" are vital for modernizing crop breeding to provide game-changing solutions to some of the "must needed" traits in agriculture. CRISPR/Cas-based tools have been rapidly repurposed for editing applications based on their improved efficiency, specificity and reduced off-target effects. Additionally, precise gene-editing tools such as base editing, prime editing, and multiplexing provide precision in stacking of multiple traits in an elite variety, and facilitating specific and targeted crop improvement. This has helped in advancing research and delivery of products in a short time span, thereby enhancing the rate of genetic gains. A special focus has been on food security in the drylands through crops including millets, teff, fonio, quinoa, Bambara groundnut, pigeonpea and cassava. While these crops contribute significantly to the agricultural economy and resilience of the dryland, improvement of several traits including increased stress tolerance, nutritional value, and yields are urgently required. Although CRISPR has potential to deliver disruptive innovations, prioritization of traits should consider breeding product profiles and market segments for designing and accelerating delivery of locally adapted and preferred crop varieties for the drylands. In this context, the scope of regulatory environment has been stated, implying the dire impacts of unreasonable scrutiny of genome-edited plants on the evolution and progress of much-needed technological advances.},
}
@article {pmid36276652,
year = {2022},
author = {Nedorezova, DD and Dubovichenko, MV and Belyaeva, EP and Grigorieva, ED and Peresadina, AV and Kolpashchikov, DM},
title = {Specificity of oligonucleotide gene therapy (OGT) agents.},
journal = {Theranostics},
volume = {12},
number = {16},
pages = {7132-7157},
pmid = {36276652},
issn = {1838-7640},
mesh = {Animals ; RNA, Guide/genetics ; Oligonucleotides ; CRISPR-Cas Systems/genetics ; *Peptide Nucleic Acids ; RNA, Small Interfering/genetics ; *DNA, Catalytic ; Genetic Therapy ; RNA, Messenger ; Untranslated Regions ; },
abstract = {Oligonucleotide gene therapy (OGT) agents (e. g. antisense, deoxyribozymes, siRNA and CRISPR/Cas) are promising therapeutic tools. Despite extensive efforts, only few OGT drugs have been approved for clinical use. Besides the problem of efficient delivery to targeted cells, hybridization specificity is a potential limitation of OGT agents. To ensure tight binding, a typical OGT agent hybridizes to the stretch of 15-25 nucleotides of a unique targeted sequence. However, hybrids of such lengths tolerate one or more mismatches under physiological conditions, the problem known as the affinity/specificity dilemma. Here, we assess the scale of this problem by analyzing OGT hybridization-dependent off-target effects (HD OTE) in vitro, in animal models and clinical studies. All OGT agents except deoxyribozymes exhibit HD OTE in vitro, with most thorough evidence of poor specificity reported for siRNA and CRISPR/Cas9. Notably, siRNA suppress non-targeted genes due to (1) the partial complementarity to mRNA 3'-untranslated regions (3'-UTR), and (2) the antisense activity of the sense strand. CRISPR/Cas9 system can cause hundreds of non-intended dsDNA breaks due to low specificity of the guide RNA, which can limit therapeutic applications of CRISPR/Cas9 by ex-vivo formats. Contribution of this effects to the observed in vivo toxicity of OGT agents is unclear and requires further investigation. Locked or peptide nucleic acids improve OGT nuclease resistance but not specificity. Approaches that use RNA marker dependent (conditional) activation of OGT agents may improve specificity but require additional validation in cell culture and in vivo.},
}
@article {pmid36273308,
year = {2022},
author = {Zhang, L and Wang, L and Xiang, S and Hu, Y and Zhao, S and Liao, Y and Zhu, Z and Wu, X},
title = {CRISPR/Cas9-mediated gene knockout of Sj16 in Schistosoma japonicum eggs upregulates the host-to-egg immune response.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {36},
number = {11},
pages = {e22615},
doi = {10.1096/fj.202200600RR},
pmid = {36273308},
issn = {1530-6860},
mesh = {Mice ; Animals ; *Schistosoma japonicum/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Anti-Inflammatory Agents/metabolism ; Immunity ; },
abstract = {Schistosomiasis is an important, neglected tropical disease. Schistosoma japonicum can evade host attacks by regulating the host's immunity, causing continuous infection. However, interactions between the host's immune system and S. japonicum are unclear. Our previous research found that the Sj16 protein isolated from S. japonicum has an anti-inflammatory effect in the host. However, the role of Sj16 in the regulation of host immunity in S. japonicum infection is not clear. Here, we applied the CRISPR/Cas9 technique to knockout Sj16 in S. japonicum eggs and investigated the effect of Sj16 in regulating host immunity. We found egg viability decreased after Sj16 knockout. In addition, we found granulomatous inflammation increased, the T-cell immune response enhanced and the immune microenvironment changed in mice model injected with Sj16-knockout eggs by tail vein. These findings suggested that S. japonicum could regulate host immunity through Sj16 to evade the host immune attack and cause continuous infection. In addition, we confirmed the application of CRISPR/Cas9-mediated gene reprogramming for functional genomics in S. japonicum.},
}
@article {pmid36272994,
year = {2022},
author = {Ishibashi, R and Maki, R and Kitano, S and Miyachi, H and Toyoshima, F},
title = {Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {17775},
pmid = {36272994},
issn = {2045-2322},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Endonucleases/genetics ; Plasmids/genetics ; RNA/genetics ; DNA ; Transgenes ; },
abstract = {The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.},
}
@article {pmid36272820,
year = {2022},
author = {Islam, T and Kasfy, SH},
title = {CRISPR-based point-of-care plant disease diagnostics.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.10.002},
pmid = {36272820},
issn = {1879-3096},
abstract = {The coupling of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas RNA-programmable nucleases with nucleic acid detection platforms has brought radical changes to the field of disease diagnosis. Recently, Sánchez et al. developed a simple, rapid, highly sensitive, precise, and in-field deployable point-of-care (POC) and point-of-need (PON) molecular disease detection tool that can be used in diverse agricultural applications.},
}
@article {pmid36272411,
year = {2022},
author = {Dhingra, Y and Suresh, SK and Juneja, P and Sashital, DG},
title = {PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.},
journal = {Molecular cell},
volume = {82},
number = {22},
pages = {4353-4367.e6},
pmid = {36272411},
issn = {1097-4164},
support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; R35 GM140876/GM/NIGMS NIH HHS/United States ; U24 GM129539/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; },
abstract = {Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.},
}
@article {pmid36272409,
year = {2022},
author = {Shi, YJ and Duan, M and Ding, JM and Wang, FQ and Bi, LL and Zhang, CX and Zhang, YZ and Duan, JY and Huang, AH and Lei, XL and Yin, H and Zhang, Y},
title = {DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.},
journal = {Molecular cell},
volume = {82},
number = {21},
pages = {4160-4175.e6},
doi = {10.1016/j.molcel.2022.09.032},
pmid = {36272409},
issn = {1097-4164},
mesh = {Animals ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; Gene Editing ; DNA/genetics ; Plasmids ; Mammals/metabolism ; },
abstract = {CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.},
}
@article {pmid36272087,
year = {2023},
author = {Wang, L and Zeng, YY and Liu, Y and Fei, JF},
title = {Applying a Knock-In Strategy to Create Reporter-Tagged Knockout Alleles in Axolotls.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2562},
number = {},
pages = {351-368},
pmid = {36272087},
issn = {1940-6029},
mesh = {Animals ; *Ambystoma mexicanum/genetics ; *CRISPR-Cas Systems/genetics ; Alleles ; Gene Knock-In Techniques ; Gene Knockout Techniques ; },
abstract = {Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the "knocking out" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.},
}
@article {pmid36272086,
year = {2023},
author = {Sousounis, K and Courtemanche, K and Whited, JL},
title = {A Practical Guide for CRISPR-Cas9-Induced Mutations in Axolotls.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2562},
number = {},
pages = {335-349},
pmid = {36272086},
issn = {1940-6029},
support = {K99 EY029361/EY/NEI NIH HHS/United States ; DP2 HD087953/HD/NICHD NIH HHS/United States ; R01 HD095494/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Ambystoma mexicanum/genetics ; Genome ; Mutation ; RNA, Guide/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool that enables editing of the axolotl genome. In this chapter, we will cover how to retrieve gene sequences, confirm annotation, design CRISPR targets, analyze indels, and screen for Crispant axolotls. This is a comprehensive guide on how to use CRISPR on your favorite gene and gain insights into its function.},
}
@article {pmid36271789,
year = {2022},
author = {Molina, R and Garcia-Martin, R and López-Méndez, B and Jensen, ALG and Ciges-Tomas, JR and Marchena-Hurtado, J and Stella, S and Montoya, G},
title = {Molecular basis of cyclic tetra-oligoadenylate processing by small standalone CRISPR-Cas ring nucleases.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11199-11213},
pmid = {36271789},
issn = {1362-4962},
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Oligoribonucleotides/chemistry ; Adenine Nucleotides/metabolism ; Endonucleases/metabolism ; },
abstract = {Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.},
}
@article {pmid36271076,
year = {2022},
author = {Meijboom, KE and Abdallah, A and Fordham, NP and Nagase, H and Rodriguez, T and Kraus, C and Gendron, TF and Krishnan, G and Esanov, R and Andrade, NS and Rybin, MJ and Ramic, M and Stephens, ZD and Edraki, A and Blackwood, MT and Kahriman, A and Henninger, N and Kocher, JA and Benatar, M and Brodsky, MH and Petrucelli, L and Gao, FB and Sontheimer, EJ and Brown, RH and Zeier, Z and Mueller, C},
title = {CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {6286},
pmid = {36271076},
issn = {2041-1723},
mesh = {Animals ; Mice ; *Frontotemporal Dementia/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/metabolism ; C9orf72 Protein/genetics/metabolism ; DNA Repeat Expansion/genetics ; CRISPR-Cas Systems ; Motor Neurons/metabolism ; Dipeptides/metabolism ; RNA/metabolism ; },
abstract = {A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.},
}
@article {pmid36270083,
year = {2022},
author = {Kang, X and Lei, C and Shi, J and Liu, X and Ren, W and Liu, C},
title = {A versatile CRISPR/Cas12a-based biosensing platform coupled with a target-protected transcription strategy.},
journal = {Biosensors &amp; bioelectronics},
volume = {219},
number = {},
pages = {114801},
doi = {10.1016/j.bios.2022.114801},
pmid = {36270083},
issn = {1873-4235},
abstract = {Besides the critical role in gene editing, CRISPR/Cas system also brings a new signal amplification mechanism to the development of next generation biosensing technologies. Herein, we have developed a versatile CRISPR/Cas12a sensing platform by combining a target protection-based transcription amplification strategy with the Cas12a-based signal amplification mechanism, which allows for the sensitive detection of both nucleic acid and non-nucleic acid targets. In this design, a rationally designed transcription template sequence is able to avoid Exonuclease I (Exo I) degradation only in the existence of the target-mediated binding events including either nucleic acid hybridization or protein-based affinity interactions. This target binding-induced protection effect can facilitate the subsequent transcription amplification to generate crRNA and activate the subsequent Cas12a trans-cleavage signal amplification mechanism to yield target dosage-responsive fluorescence signal. In contrast, if the target is absent, the protection-free transcription template will be completely digested by Exo I, thus no fluorescence response is produced. This new strategy well eliminates the T7 polymerase-associated non-specific transcription background and realizes the sensitive detection of various kinds of biomolecules including microRNA, protein, as well as exosome, broadening the application scenarios of CRISPR/Cas system in the field of bioanalysis and biosensing.},
}
@article {pmid36269834,
year = {2022},
author = {Park, SH and Cao, M and Pan, Y and Davis, TH and Saxena, L and Deshmukh, H and Fu, Y and Treangen, T and Sheehan, VA and Bao, G},
title = {Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing.},
journal = {Science advances},
volume = {8},
number = {42},
pages = {eabo7676},
pmid = {36269834},
issn = {2375-2548},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Programmed Cell Death 1 Receptor/metabolism ; Hematopoietic Stem Cells/metabolism ; Cell Line ; },
abstract = {Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.},
}
@article {pmid36268581,
year = {2022},
author = {Urbaitis, T and Gasiunas, G and Young, JK and Hou, Z and Paulraj, S and Godliauskaite, E and Juskeviciene, MM and Stitilyte, M and Jasnauskaite, M and Mabuchi, M and Robb, GB and Siksnys, V},
title = {A new family of CRISPR-type V nucleases with C-rich PAM recognition.},
journal = {EMBO reports},
volume = {23},
number = {12},
pages = {e55481},
doi = {10.15252/embr.202255481},
pmid = {36268581},
issn = {1469-3178},
abstract = {Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.},
}
@article {pmid36267139,
year = {2022},
author = {Farheen, J and Hosmane, NS and Zhao, R and Zhao, Q and Iqbal, MZ and Kong, X},
title = {Nanomaterial-assisted CRISPR gene-engineering - A hallmark for triple-negative breast cancer therapeutics advancement.},
journal = {Materials today. Bio},
volume = {16},
number = {},
pages = {100450},
pmid = {36267139},
issn = {2590-0064},
abstract = {Triple-negative breast cancer (TNBC) is the most violent class of tumor and accounts for 20-24% of total breast carcinoma, in which frequently rare mutation occurs in high frequency. The poor prognosis, recurrence, and metastasis in the brain, heart, liver and lungs decline the lifespan of patients by about 21 months, emphasizing the need for advanced treatment. Recently, the adaptive immunity mechanism of archaea and bacteria, called clustered regularly interspaced short palindromic repeats (CRISPR) combined with nanotechnology, has been utilized as a potent gene manipulating tool with an extensive clinical application in cancer genomics due to its easeful usage and cost-effectiveness. However, CRISPR/Cas are arguably the efficient technology that can be made efficient via organic material-assisted approaches. Despite the efficacy of the CRISPR/Cas@nano complex, problems regarding successful delivery, biodegradability, and toxicity remain to render its medical implications. Therefore, this review is different in focus from past reviews by (i) detailing all possible genetic mechanisms of TNBC occurrence; (ii) available treatments and gene therapies for TNBC; (iii) overview of the delivery system and utilization of CRISPR-nano complex in TNBC, and (iv) recent advances and related toxicity of CRISPR-nano complex towards clinical trials for TNBC.},
}
@article {pmid36266475,
year = {2022},
author = {Zhai, R and Zheng, C and Yang, Z and Li, T and Chen, J and Shen, X},
title = {Contribution of CRISPRable DNA to human complex traits.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {1111},
pmid = {36266475},
issn = {2399-3642},
mesh = {Humans ; *CRISPR-Cas Systems ; DNA/genetics ; Gene Editing/methods ; Genomics ; *Multifactorial Inheritance ; },
abstract = {CRISPR-Cas is a powerful genome editing tool for various species and human cell lines, widely used in many research areas including studying the mechanisms, targets, and gene therapies of human diseases. Recent developments have even allowed high-throughput genetic screening using the CRISPR system. However, due to the practical and ethical limitations in human gene editing research, little is known about whether CRISPR-editable DNA segments could influence human complex traits or diseases. Here, we investigated the human genomic regions condensed with different CRISPR Cas enzymes' protospacer-adjacent motifs (PAMs). We found that Cas enzymes with GC-rich PAMs could interfere more with the genomic regions that harbor enriched heritability for human complex traits and diseases. The results linked GC content across the genome to the functional genomic elements in the heritability enrichment of human complex traits. We provide a genetic overview of the effects of high-throughput genome editing on human complex traits.},
}
@article {pmid36264489,
year = {2023},
author = {Salafranca, J and Ai, Z and Wang, L and Udalova, IA and van Grinsven, E},
title = {Analysis of Neutrophil Morphology and Function Under Genetic Perturbation of Transcription Factors In Vitro.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2594},
number = {},
pages = {69-86},
pmid = {36264489},
issn = {1940-6029},
support = {/VAC_/Versus Arthritis/United Kingdom ; 209422/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Neutrophils/metabolism ; *Homeodomain Proteins/genetics ; Transcription Factors/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Cell Differentiation/genetics ; },
abstract = {Hoxb8 cells are immortalized myeloid progenitors that maintain their multipotent potential and can be differentiated into neutrophils. Genetic modification of Hoxb8 cells can be used as a model system for the functional analysis of regulators of neutrophil maturation and effector functions, such as transcription factors. Here we describe the generation of transcription factor (TF) knockout Hoxb8 cell lines in vitro with the lentivirus (lenti)CRISPR-Cas 9 technique. After their differentiation into neutrophils, the study of their maturation profile, morphology, and effector functions, including NETosis, phagocytosis, and ROS production, is described.},
}
@article {pmid36264488,
year = {2023},
author = {Yeo, NC and Church, GM},
title = {Perturbation of Gene Regulation by Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2594},
number = {},
pages = {59-68},
pmid = {36264488},
issn = {1940-6029},
support = {RM1 HG008525/HG/NHGRI NIH HHS/United States ; P50 HG005550/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Gene Expression Regulation ; Endonucleases/genetics ; RNA ; Mammals/genetics ; },
abstract = {The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor. Here we describe a set of protocols for using the catalytically inactive dead Cas9 (dCas9)-based tools, including the bipartite super repressor consisting of the KRAB and MeCP2 domains, to achieve efficient and scalable gene silencing in mammalian cells.},
}
@article {pmid36263801,
year = {2022},
author = {Liu, X and Cui, S and Qi, Q and Lei, H and Zhang, Y and Shen, W and Fu, F and Tian, T and Zhou, X},
title = {G-quadruplex-guided RNA engineering to modulate CRISPR-based genomic regulation.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11387-11400},
pmid = {36263801},
issn = {1362-4962},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genomics ; RNA/genetics ; *RNA, Guide/genetics/metabolism ; G-Quadruplexes ; },
abstract = {It is important to develop small moelcule-based methods to modulate gene editing and expression in human cells. The roles of the G-quadruplex (G4) in biological systems have been widely studied. Here, G4-guided RNA engineering is performed to generate guide RNA with G4-forming units (G4-gRNA). We further demonstrate that chemical targeting of G4-gRNAs holds promise as a general approach for modulating gene editing and expression in human cells. The rich structural diversity of RNAs offers a reservoir of targets for small molecules to bind, thus creating the potential to modulate RNA biology.},
}
@article {pmid36263429,
year = {2022},
author = {Imai, T and Hippo, Y},
title = {Editorial: Development of in vitro toxicology methods using organoid systems and toxicogenomic approaches.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {1030580},
doi = {10.3389/fgene.2022.1030580},
pmid = {36263429},
issn = {1664-8021},
}
@article {pmid36261968,
year = {2022},
author = {Naz, M and Benavides-Mendoza, A and Tariq, M and Zhou, J and Wang, J and Qi, S and Dai, Z and Du, D},
title = {CRISPR/Cas9 technology as an innovative approach to enhancing the phytoremediation: Concepts and implications.},
journal = {Journal of environmental management},
volume = {323},
number = {},
pages = {116296},
doi = {10.1016/j.jenvman.2022.116296},
pmid = {36261968},
issn = {1095-8630},
mesh = {*CRISPR-Cas Systems ; Biodegradation, Environmental ; Ecosystem ; Plants/genetics ; Metals ; Technology ; *Environmental Pollutants ; },
abstract = {Phytoremediation is currently an active field of research focusing chiefly on identifying and characterizing novel and high chelation action super-accumulators. In the last few years, molecular tools have been widely exploited to understand better metal absorption, translocation, cation, and tolerance mechanisms in plants. Recently more advanced CRISPR-Cas9 genome engineering technology is also employed to enhance detoxification efficiency. Further, advances in molecular science will trigger the understanding of adaptive phytoremediation ability plant production in current global warming conditions. The enhanced abilities of nucleases for genome modification can improve plant repair capabilities by modifying the genome, thereby achieving a sustainable ecosystem. The purpose of this manuscript focuses on biotechnology's fundamental principles and application to promote climate-resistant metal plants, especially the CRISPR-Cas9 genome editing system for enhancing the phytoremediation of harmful contamination and pollutants.},
}
@article {pmid36261092,
year = {2023},
author = {Xu, L and Sun, B and Liu, S and Gao, X and Zhou, H and Li, F and Li, Y},
title = {The evaluation of active transcriptional repressor domain for CRISPRi in plants.},
journal = {Gene},
volume = {851},
number = {},
pages = {146967},
doi = {10.1016/j.gene.2022.146967},
pmid = {36261092},
issn = {1879-0038},
mesh = {*Transcription, Genetic ; *RNA, Guide/genetics ; Gene Silencing ; Plants/genetics ; CRISPR-Cas Systems ; },
abstract = {With the development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system in gene editing, the catalytic site in Cas9 has been mutated to dead Cas9 (dCas9) to regulate target gene's expression with the guidance of single guide RNA (sgRNA) in many organisms. When dCas9 was navigated to the region close to the transcription start site, duo to sterical hindrance, it could downregulate the expression level of target gene specifically without genomic alteration. Furthermore, the fusion of synthetic transcriptional repressor domain (TRD) to dCas9 could improve the gene silencing efficiency dramatically, the above all was also known as CRISPR interference system (CRISPRi). Till now, SRDX repressor domain was the most frequently used TRD in plant. Nevertheless, its incomplete repression limited the application of CRISPRi system. Hereafter, in this study, we identified three more effective TRDs, DLN144, DLS and MIX in plant. To dissect the transcriptional repressing activity of DLN144, DLS and MIX in plant, first and foremost, we proved their transcriptional repression efficiency in transient transformed Nicotiana benthamiana leaves. Then, their intrinsic transcriptional repressing activity was corroborated in stable transgenic wheat and N. benthamiana. These three functional TRDs, DLN144, DLS and MIX, provide more options for the application of CRISPRi in plant and shed new light on the advancement of more robust TRDs by combining different individual effective repressor domain in plant which will facilitate the application of CRISPRi when higher repression efficiency is required.},
}
@article {pmid36260301,
year = {2022},
author = {Demirci, S and Essawi, K and Germino-Watnick, P and Liu, X and Hakami, W and Tisdale, JF},
title = {Advances in CRISPR Delivery Methods: Perspectives and Challenges.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {660-676},
doi = {10.1089/crispr.2022.0051},
pmid = {36260301},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics ; },
abstract = {With the advent of new genome editing technologies and the emphasis placed on their optimization, the genetic and phenotypic correction of a plethora of diseases sit on the horizon. Ideally, genome editing approaches would provide long-term solutions through permanent disease correction instead of simply treating patients symptomatically. Although various editing machinery options exist, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein) editing technique has emerged as the most popular due to its high editing efficiency, simplicity, and affordability. However, while CRISPR technology is gradually being perfected, optimization is futile without accessible, effective, and safe delivery to the desired cell or tissue. Therefore, it is important that scientists simultaneously focus on inventing and improving delivery modalities for editing machinery as well. In this review, we will discuss the critical details of viral and nonviral delivery systems, including payload, immunogenicity, efficacy in delivery, clinical application, and future directions.},
}
@article {pmid36260300,
year = {2022},
author = {Barrangou, R},
title = {Extreme Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {629-630},
doi = {10.1089/crispr.2022.29155.editorial},
pmid = {36260300},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid36260299,
year = {2022},
author = {Montesinos-Rongen, M and Sanchez-Ruiz, M and Siebert, S and Winter, C and Siebert, R and Brunn, A and Deckert, M},
title = {Expression of Cas9 in a Syngeneic Model of Primary Central Nervous System Lymphoma Induces Intracerebral NK and CD8 T Cell-Mediated Lymphoma Cell Lysis Via Perforin.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {726-739},
doi = {10.1089/crispr.2022.0049},
pmid = {36260299},
issn = {2573-1602},
mesh = {Animals ; Mice ; Perforin/genetics ; *CRISPR-Associated Protein 9/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; CD8-Positive T-Lymphocytes ; Central Nervous System ; },
abstract = {The development of clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene modification has opened an exciting avenue of targeting genes to study the pathogenesis of diseases and to develop novel therapeutic concepts. However, as the effector protein Cas9 is of bacterial origin, unwanted side effects due to a host immune response against Cas9 need to be considered. Here, we used the syngeneic model of BAL17[CNS]-induced primary lymphoma of the central nervous system (PCNSL, CNS) in BALB/c mice to address this issue. Surprisingly, stable expression of Cas9 in BAL17[CNS] (BAL17[CNS]/Cas9) cells rendered them unable to establish PCNSL on intracerebral transplantation. Instead, they induced a prominent intracerebral immune response mediated by CD8 T cells, which lysed BAL17[CNS]/Cas9 cells via perforin. In addition, B cells contributed to the immune response as evidenced by serum anti-Cas9 antibodies in BALB/c mice as early as day 8 after transplantation of BAL17[CNS]/Cas9 cells. In athymic BALB/c[nu/nu] mice, NK cells mounted a vigorous intracerebral immune response with perforin-mediated destruction of BAL17[CNS]/Cas9 cells. Thus, in the CNS, perforin produced by NK and CD8 T cells was identified as a mediator of cytotoxicity against BAL17[CNS]/Cas9 cells. These observations should be taken into account when considering therapeutic CRISPR-Cas9-mediated tumor cell manipulation for PCNSL.},
}
@article {pmid36260269,
year = {2022},
author = {Hu, YF and Fang, YH and Lai, YR and Feng, XQ and Xu, SQ},
title = {Application of Gene Therapy in Hemophilia.},
journal = {Current medical science},
volume = {42},
number = {5},
pages = {925-931},
pmid = {36260269},
issn = {2523-899X},
mesh = {Humans ; *Hemophilia A/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Therapy/methods ; Antibodies, Neutralizing ; },
abstract = {Gene therapy refers to introducing normal exogenous genes into target cells to correct or compensate for the diseases caused by defective and abnormal genes for the purpose of therapy. It holds out hope of a cure for single-gene genetic diseases such as thalassemia, hemophilia, etc. At present, gene therapy is performed in two ways: introducing exogenous genes, and gene editing. A great number of clinical trials of gene therapy in hemophilia have been carried out using viral vectors to introduce foreign genes into target cells. However, the production of neutralizing antibodies following injection and the inability to prepare viral vectors in large quantities limit their application. Although gene-editing methods like CRISPR avoid the above problems, the potential risks of off-target effects are still unknown. More trials and evidence are needed to elucidate the safety and accuracy of gene therapy. This paper will review the bench and clinical work of gene therapy in hemophilia in recent years, and summarize the challenges and prospects of gene therapy, so as to provide directions for future scientific research in this field.},
}
@article {pmid36260063,
year = {2022},
author = {López-Muñoz, AD and Rastrojo, A and Martín, R and Alcami, A},
title = {High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9.},
journal = {The Journal of general virology},
volume = {103},
number = {10},
pages = {},
doi = {10.1099/jgv.0.001797},
pmid = {36260063},
issn = {1465-2099},
support = {BB/L024209/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; Genome, Viral ; *Herpesvirus 1, Human/genetics ; *Viruses/genetics ; },
abstract = {The application of CRISPR/Cas9 to improve genome engineering efficiency for large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desired locus has not yet been reported. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiencies, depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex virus (HSV) type 1 and 2 to optimize a CRISPR/Cas9 method that can be used versatilely for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and colour-based selection of recombinants. This application of CRISPR/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2 weeks.},
}
@article {pmid36259649,
year = {2022},
author = {Nguyen, MHT and Netter, Z and Angermeyer, A and Seed, KD},
title = {A phage weaponizes a satellite recombinase to subvert viral restriction.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11138-11153},
pmid = {36259649},
issn = {1362-4962},
support = {R01 AI153303/AI/NIAID NIH HHS/United States ; R01 AI127652/AI/NIAID NIH HHS/United States ; },
mesh = {*Bacteriophages/metabolism ; Recombinases/genetics/metabolism ; *Vibrio cholerae/metabolism ; CRISPR-Cas Systems ; },
abstract = {Bacteria can acquire mobile genetic elements (MGEs) to combat infection by viruses (phages). Satellite viruses, including the PLEs (phage-inducible chromosomal island-like elements) in epidemic Vibrio cholerae, are MGEs that restrict phage replication to the benefit of their host bacterium. PLEs parasitize the lytic phage ICP1, unleashing multiple mechanisms to restrict phage replication and promote their own spread. In the arms race against PLE, ICP1 uses nucleases, including CRISPR-Cas, to destroy PLE's genome during infection. However, through an unknown CRISPR-independent mechanism, specific ICP1 isolates subvert restriction by PLE. Here, we discover ICP1-encoded Adi that counteracts PLE by exploiting the PLE's large serine recombinase (LSR), which normally mobilizes PLE in response to ICP1 infection. Unlike previously characterized ICP1-encoded anti-PLE mechanisms, Adi is not a nuclease itself but instead appears to modulate the activity of the LSR to promote destructive nuclease activity at the LSR's specific attachment site, attP. The PLE LSR, its catalytic activity, and attP are additionally sufficient to sensitize a PLE encoding a resistant variant of the recombination module to Adi activity. This work highlights a unique type of adaptation arising from inter-genome conflicts, in which the intended activity of a protein can be weaponized to overcome the antagonizing genome.},
}
@article {pmid36259008,
year = {2022},
author = {Lohiya, DV and Pathak, SS},
title = {Role of Technology in Detection of COVID-19.},
journal = {Cureus},
volume = {14},
number = {9},
pages = {e29138},
pmid = {36259008},
issn = {2168-8184},
abstract = {The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus caused coronavirus infection termed as COVID-19, an illness that has spread devastation all over the world. It was developed first in China and had swiftly spread throughout the world. COVID has created imposed burden on health in the lives of all individuals around the globe. This article provides a number of unprecedented detection technologies used in the detection of infection. COVID has created a large number of symptoms in the young, adolescent as well as elderly population. Old age people are susceptible to fatal serious symptoms because of low immunity. With these goals in mind, this article includes substantial condemning descriptions of the majority of initiatives in order to create diagnostic tools for easy diagnosis. It also provides the reader with a multidisciplinary viewpoint on how traditional approaches such as serology and reverse transcriptase polymerase chain reaction (RT-PCR) along with the frontline techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas and artificial intelligence/machine learning have been utilized to gather information. The story will inspire creative new ways for successful detection therapy and to prevent this pandemic among a wide audience of operating and aspiring biomedical scientists and engineers.},
}
@article {pmid36257831,
year = {2022},
author = {Zhang, JQ and Guo, JX and Wu, XJ and Wang, Y and Yue, W and Li, ZH and Wang, W and Wei, XF and Wu, XL and Shen, QY and Lei, AM and Wei, HJ and Hua, JL},
title = {Optimization of sgRNA expression strategy to generate multiplex gene-edited pigs.},
journal = {Zoological research},
volume = {43},
number = {6},
pages = {1005-1008},
pmid = {36257831},
issn = {2095-8137},
mesh = {Swine/genetics ; Animals ; *Gene Editing/veterinary ; *RNA, Guide/genetics/metabolism ; CRISPR-Cas Systems ; },
}
@article {pmid36257813,
year = {2022},
author = {Xie, Y and Li, H and Chen, F and Udayakumar, S and Arora, K and Chen, H and Lan, Y and Hu, Q and Zhou, X and Guo, X and Xiu, L and Yin, K},
title = {Clustered Regularly Interspaced short palindromic repeats-Based Microfluidic System in Infectious Diseases Diagnosis: Current Status, Challenges, and Perspectives.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2204172},
doi = {10.1002/advs.202204172},
pmid = {36257813},
issn = {2198-3844},
abstract = {Mitigating the spread of global infectious diseases requires rapid and accurate diagnostic tools. Conventional diagnostic techniques for infectious diseases typically require sophisticated equipment and are time consuming. Emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) detection systems have shown remarkable potential as next-generation diagnostic tools to achieve rapid, sensitive, specific, and field-deployable diagnoses of infectious diseases, based on state-of-the-art microfluidic platforms. Therefore, a review of recent advances in CRISPR-based microfluidic systems for infectious diseases diagnosis is urgently required. This review highlights the mechanisms of CRISPR/Cas biosensing and cutting-edge microfluidic devices including paper, digital, and integrated wearable platforms. Strategies to simplify sample pretreatment, improve diagnostic performance, and achieve integrated detection are discussed. Current challenges and future perspectives contributing to the development of more effective CRISPR-based microfluidic diagnostic systems are also proposed.},
}
@article {pmid36257021,
year = {2022},
author = {Jacobus, AP and Barreto, JA and de Bem, LS and Menegon, YA and Fier, &#xcd; and Bueno, JGR and Dos Santos, LV and Gross, J},
title = {EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9 Applications in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {11},
number = {11},
pages = {3886-3891},
doi = {10.1021/acssynbio.2c00348},
pmid = {36257021},
issn = {2161-5063},
mesh = {*RNA, Guide/genetics/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3-5 days for its precloning in Escherichia coli, purification, validation, and cotransformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/Cas9 applications in Saccharomyces cerevisiae. The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing precloning in E. coli, yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished through a quick protocol.},
}
@article {pmid36255694,
year = {2023},
author = {Shahbazi, R and Lipson, P and Gottimukkala, KSV and Lane, DD and Adair, JE},
title = {CRISPR Gene Editing of Hematopoietic Stem and Progenitor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2567},
number = {},
pages = {39-62},
pmid = {36255694},
issn = {1940-6029},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Oligonucleotides ; Stem Cells ; RNA ; DNA, Mitochondrial ; },
abstract = {Genetic editing of hematopoietic stem and progenitor cells can be employed to understand gene-function relationships underlying hematopoietic cell biology, leading to new therapeutic approaches to treat disease. The ability to collect, purify, and manipulate primary cells outside the body permits testing of many different gene editing approaches. RNA-guided nucleases, such as CRISPR, have revolutionized gene editing based simply on Watson-Crick base-pairing, employed to direct activity to specific genomic loci. Given the ease and affordability of synthetic, custom RNA guides, testing of precision edits or large random pools in high-throughput screening studies is now widely available. With the ever-growing number of CRISPR nucleases being discovered or engineered, researchers now have a plethora of options for directed genomic change, including single base edits, nicks or double-stranded DNA cuts with blunt or staggered ends, as well as the ability to target CRISPR to other cellular oligonucleotides such as RNA or mitochondrial DNA. Except for single base editing strategies, precise rewriting of larger segments of the genetic code requires delivery of an additional component, templated DNA oligonucleotide(s) encoding the desired changes flanked by homologous sequences that permit recombination at or near the site of CRISPR activity. Altogether, the ever-growing CRISPR gene editing toolkit is an invaluable resource. This chapter outlines available technologies and the strategies for applying CRISPR-based editing in hematopoietic stem and progenitor cells.},
}
@article {pmid36255641,
year = {2023},
author = {Jung, P and Schmalbrock, L and Wirth, M},
title = {CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2589},
number = {},
pages = {429-454},
pmid = {36255641},
issn = {1940-6029},
mesh = {*Histone Deacetylase Inhibitors/pharmacology ; *Gene Regulatory Networks ; CRISPR-Associated Protein 9 ; Gene Expression ; Histone Deacetylases/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.},
}
@article {pmid36255638,
year = {2023},
author = {Muscolini, M and Hiscott, J and Tassone, E},
title = {A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic Virotherapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2589},
number = {},
pages = {379-399},
pmid = {36255638},
issn = {1940-6029},
mesh = {Humans ; *Oncolytic Virotherapy ; CRISPR-Cas Systems/genetics ; RNA, Guide/genetics ; *Oncolytic Viruses/genetics ; *Neoplasms/genetics/therapy ; High-Throughput Nucleotide Sequencing ; },
abstract = {Oncolytic virotherapy represents an efficient immunotherapeutic approach for cancer treatment. Oncolytic viruses (OVs) promote antitumor responses through tumor-selective cell lysis and immune system activation. However, some tumor cell lines and primary tumors display resistance to therapy. Here we describe a protocol to identify novel host factors responsible for tumor resistance to oncolysis using an unbiased genome-wide CRISPR-Cas9 loss-of-function screening. Cas9-expressing tumor cells are transduced with a library of pooled single-guide RNA (sgRNA)-expressing lentiviruses that target all human genes to obtain a cell population where each cell is knocked out for a single gene. Upon OV infection, resistant cells survive, while sensitive cells die. The relative abundance of each genome-integrated sgRNA is measured by next-generation sequencing (NGS) in resistant and control cells. This protocol is amenable to uncover host factors involved in the resistance to different OVs in multiple tumor models.},
}
@article {pmid36255392,
year = {2022},
author = {Xu, X and Liu, C and Wang, S and Mäkilä, E and Wang, J and Koivisto, O and Zhou, J and Rosenholm, JM and Shu, Y and Zhang, H},
title = {Microfluidic-assisted biomineralization of CRISPR/Cas9 in near-infrared responsive metal-organic frameworks for programmable gene-editing.},
journal = {Nanoscale},
volume = {14},
number = {42},
pages = {15832-15844},
doi = {10.1039/d2nr04095f},
pmid = {36255392},
issn = {2040-3372},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Metal-Organic Frameworks/metabolism ; Microfluidics ; Biomineralization ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Ribonucleoprotein (RNP) based CRISPR/Cas9 gene-editing system shows great potential in biomedical applications. However, due to the large size, charged surface and high biological sensitivity of RNP, its efficient delivery with precise control remains highly challenging. Herein, a microfluidic-assisted metal-organic framework (MOF) based biomineralization strategy is designed and utilized for the efficient delivery and remote regulation of CRISPR/Cas9 RNP gene editing. The strategy is realized by biomimetic growing of thermo-responsive EuMOFs onto photothermal template Prussian blue (PB). The RNP is loaded during MOFs crystallization in microfluidic channels. By adjusting different microfluidic parameters, well-defined and comparable RNP encapsulated nanocarrier (PB@RNP-EuMOFs) are obtained with high loading efficiency (60%), remarkable RNP protection and NIR-stimulated release capacity. Upon laser exposure, the nanocarrier induces effective endosomal escape (4 h) and precise gene knockout of green fluorescent protein by 40% over 2 days. Moreover, the gene-editing activity can be programmed by tuning exposure times (42% for three times and 47% for four times), proving more controllable and inducible editing modality compared to control group without laser irradiation. This novel microfluidic-assisted MOFs biomineralization strategy thus offers an attractive route to optimize delivery systems and reduce off-target side effects by NIR-triggered remote control of CRISPR/Cas9 RNP, improving the potential for its highly efficient and precise therapeutic application.},
}
@article {pmid36255384,
year = {2022},
author = {Ceballos-Garzon, A and Muñoz, AB and Plata, JD and Sanchez-Quitian, ZA and Ramos-Vivas, J},
title = {Phages, anti-CRISPR proteins, and drug-resistant bacteria: what do we know about this triad?.},
journal = {Pathogens and disease},
volume = {80},
number = {1},
pages = {},
doi = {10.1093/femspd/ftac039},
pmid = {36255384},
issn = {2049-632X},
mesh = {Humans ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Bacteria/genetics ; Viral Proteins/genetics/metabolism ; },
abstract = {Phages are viruses that infect bacteria, relying on their genetic machinery to replicate. To survive the constant attack of phages, bacteria have developed diverse defense strategies to act against them. Nevertheless, phages rapidly co-evolve to overcome these barriers, resulting in a constant, and often surprising, molecular arms race. Thus, some phages have evolved protein inhibitors known as anti-CRISPRs (∼50-150 amino acids), which antagonize the bacterial CRISPR-Cas immune response. To date, around 45 anti-CRISPRs proteins with different mechanisms and structures have been discovered against the CRISPR-Cas type I and type II present in important animal and human pathogens such as Escherichia, Morganella, Klebsiella, Enterococcus, Pseudomonas, Staphylococcus, and Salmonella. Considering the alarming growth of antibiotic resistance, phage therapy, either alone or in combination with antibiotics, appears to be a promising alternative for the treatment of many bacterial infections. In this review, we illustrated the biological and clinical aspects of using phage therapy; furthermore, the CRISPR-Cas mechanism, and the interesting activity of anti-CRISPR proteins as a possible weapon to combat bacteria.},
}
@article {pmid36254536,
year = {2022},
author = {Akram, F and Haq, IU and Sahreen, S and Nasir, N and Naseem, W and Imitaz, M and Aqeel, A},
title = {CRISPR/Cas9: A revolutionary genome editing tool for human cancers treatment.},
journal = {Technology in cancer research &amp; treatment},
volume = {21},
number = {},
pages = {15330338221132078},
pmid = {36254536},
issn = {1533-0338},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Humans ; Immunotherapy ; Mice ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer is a genetic disease stemming from genetic and epigenetic mutations and is the second most common cause of death across the globe. Clustered regularly interspaced short palindromic repeats (CRISPR) is an emerging gene-editing tool, acting as a defense system in bacteria and archaea. CRISPR/Cas9 technology holds immense potential in cancer diagnosis and treatment and has been utilized to develop cancer disease models such as medulloblastoma and glioblastoma mice models. In diagnostics, CRISPR can be used to quickly and efficiently detect genes involved in various cancer development, proliferation, metastasis, and drug resistance. CRISPR/Cas9 mediated cancer immunotherapy is a well-known treatment option after surgery, chemotherapy, and radiation therapy. It has marked a turning point in cancer treatment. However, despite its advantages and tremendous potential, there are many challenges such as off-target effects, editing efficiency of CRISPR/Cas9, efficient delivery of CRISPR/Cas9 components into the target cells and tissues, and low efficiency of HDR, which are some of the main issues and need further research and development for completely clinical application of this novel gene editing tool. Here, we present a CRISPR/Cas9 mediated cancer treatment method, its role and applications in various cancer treatments, its challenges, and possible solution to counter these challenges.},
}
@article {pmid36252775,
year = {2022},
author = {Radtke, L and Majchrzak-Celińska, A and Awortwe, C and Vater, I and Nagel, I and Sebens, S and Cascorbi, I and Kaehler, M},
title = {CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme.},
journal = {Pharmacological research},
volume = {185},
number = {},
pages = {106510},
doi = {10.1016/j.phrs.2022.106510},
pmid = {36252775},
issn = {1096-1186},
mesh = {Humans ; Temozolomide/pharmacology/therapeutic use ; *Glioblastoma/drug therapy/genetics/pathology ; Carmustine/pharmacology/therapeutic use ; ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism ; Lomustine/therapeutic use/pharmacology ; CRISPR-Cas Systems ; ATP-Binding Cassette Transporters/metabolism ; Neoplasm Proteins/metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; },
abstract = {Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.},
}
@article {pmid36252276,
year = {2022},
author = {Malcı, K and Jonguitud-Borrego, N and van der Straten Waillet, H and Puodžiu Naitė, U and Johnston, EJ and Rosser, SJ and Rios-Solis, L},
title = {ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.},
journal = {ACS synthetic biology},
volume = {11},
number = {11},
pages = {3629-3643},
pmid = {36252276},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Indicators and Reagents/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; },
abstract = {Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.},
}
@article {pmid36252206,
year = {2022},
author = {Kua, JM and Azizi, MMF and Abdul Talib, MA and Lau, HY},
title = {Adoption of analytical technologies for verification of authenticity of halal foods - a review.},
journal = {Food additives &amp; contaminants. Part A, Chemistry, analysis, control, exposure &amp; risk assessment},
volume = {},
number = {},
pages = {1-27},
doi = {10.1080/19440049.2022.2134591},
pmid = {36252206},
issn = {1944-0057},
abstract = {Halal authentication has become essential in the food industry to ensure food is free from any prohibited ingredients according to Islamic law. Diversification of food origin and adulteration issues have raised concerns among Muslim consumers. Therefore, verification of food constituents and their quality is paramount. From conventional methods based on physical and chemical properties, various diagnostic methods have emerged relying on protein or DNA measurements. Protein-based methods that have been used in halal detection including electrophoresis, chromatographic-based methods, molecular spectroscopy and immunoassays. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are DNA-based techniques that possess better accuracy and sensitivity. Biosensors are miniatured devices that operate by converting biochemical signals into a measurable quantity. CRISPR-Cas is one of the latest novel emerging nucleic acid detection tools in halal food analysis as well as quantification of stable isotopes method for identification of animal species. Within this context, this review provides an overview of the various techniques in halal detection along with their advantages and limitations. The future trend and growth of detection technologies are also discussed in this review.},
}
@article {pmid36251847,
year = {2022},
author = {Blanluet, C and Huyke, DA and Ramachandran, A and Avaro, AS and Santiago, JG},
title = {Detection and Discrimination of Single Nucleotide Polymorphisms by Quantification of CRISPR-Cas Catalytic Efficiency.},
journal = {Analytical chemistry},
volume = {94},
number = {43},
pages = {15117-15123},
doi = {10.1021/acs.analchem.2c03338},
pmid = {36251847},
issn = {1520-6882},
mesh = {Humans ; *SARS-CoV-2/genetics ; CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; *COVID-19/diagnosis ; },
abstract = {The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., cancer and SARS-CoV-2 variants. Such assays often employ endpoint measurements of SNP or wild type (WT) activated Cas12 trans-cleavage activity; however, the fundamental kinetic effects of SNP versus WT activation remain unknown. We here show that endpoint-based assays are limited by arbitrary experimental choices (like used reporter concentration and assay duration) and work best for known target concentrations. More importantly, we show that SNP (versus WT) activation results in measurable kinetic shifts in the Cas12 trans-cleavage substrate affinity (KM) and apparent catalytic efficiency (kcat[*]/KM). To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michaelis-Menten parameters and apply this assay to a 20 base pair WT target of the SARS-CoV-2 E gene. We find that the kcat[*]/KM measured for WT is 130-fold greater than the lowest kcat[*]/KM among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). KM also offers a strong ability to distinguish SNPs, varies 27-fold over all the cases, and, importantly, is insensitive to the target concentration. Last, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.},
}
@article {pmid36250792,
year = {2022},
author = {Zhao, Y and Yu, L and Wu, X and Li, H and Coombes, KR and Au, KF and Cheng, L and Li, L},
title = {CEDA: integrating gene expression data with CRISPR-pooled screen data identifies essential genes with higher expression.},
journal = {Bioinformatics (Oxford, England)},
volume = {38},
number = {23},
pages = {5245-5252},
pmid = {36250792},
issn = {1367-4811},
support = {P30CA016058/GF/NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Genes, Essential ; Bayes Theorem ; Reproducibility of Results ; RNA, Guide/genetics ; Gene Expression ; CRISPR-Cas Systems ; },
abstract = {MOTIVATION: Clustered regularly interspaced short palindromic repeats (CRISPR)-based genetic perturbation screen is a powerful tool to probe gene function. However, experimental noises, especially for the lowly expressed genes, need to be accounted for to maintain proper control of false positive rate.<br><br>METHODS: We develop a statistical method, named CRISPR screen with Expression Data Analysis (CEDA), to integrate gene expression profiles and CRISPR screen data for identifying essential genes. CEDA stratifies genes based on expression level and adopts a three-component mixture model for the log-fold change of single-guide RNAs (sgRNAs). Empirical Bayesian prior and expectation-maximization algorithm are used for parameter estimation and false discovery rate inference.<br><br>RESULTS: Taking advantage of gene expression data, CEDA identifies essential genes with higher expression. Compared to existing methods, CEDA shows comparable reliability but higher sensitivity in detecting essential genes with moderate sgRNA fold change. Therefore, using the same CRISPR data, CEDA generates an additional hit gene list.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid36250791,
year = {2022},
author = {Reid, W and Williams, AE and Sanchez-Vargas, I and Lin, J and Juncu, R and Olson, KE and Franz, AWE},
title = {Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {12},
pages = {},
pmid = {36250791},
issn = {2160-1836},
support = {R01 AI130085/AI/NIAID NIH HHS/United States ; R56 AI167980/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Aedes/genetics ; *Gene Drive Technology ; CRISPR-Cas Systems/genetics ; Mosquito Vectors/genetics ; *Zika Virus/genetics ; *Zika Virus Infection/genetics ; },
abstract = {The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, β2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.},
}
@article {pmid36250060,
year = {2022},
author = {Monshizadeh, M and Zomorodi, S and Mortensen, K and Ye, Y},
title = {Revealing bacteria-phage interactions in human microbiome through the CRISPR-Cas immune systems.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {933516},
pmid = {36250060},
issn = {2235-2988},
support = {R01 AI143254/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Humans ; Immune System ; *Microbiota/genetics ; },
abstract = {The human gut microbiome is composed of a diverse consortium of microorganisms. Relatively little is known about the diversity of the bacteriophage population and their interactions with microbial organisms in the human microbiome. Due to the persistent rivalry between microbial organisms (hosts) and phages (invaders), genetic traces of phages are found in the hosts' CRISPR-Cas adaptive immune system. Mobile genetic elements (MGEs) found in bacteria include genetic material from phage and plasmids, often resultant from invasion events. We developed a computational pipeline (BacMGEnet), which can be used for inference and exploratory analysis of putative interactions between microbial organisms and MGEs (phages and plasmids) and their interaction network. Given a collection of genomes as the input, BacMGEnet utilizes computational tools we have previously developed to characterize CRISPR-Cas systems in the genomes, which are then used to identify putative invaders from publicly available collections of phage/prophage sequences. In addition, BacMGEnet uses a greedy algorithm to summarize identified putative interactions to produce a bacteria-MGE network in a standard network format. Inferred networks can be utilized to assist further examination of the putative interactions and for discovery of interaction patterns. Here we apply the BacMGEnet pipeline to a few collections of genomic/metagenomic datasets to demonstrate its utilities. BacMGEnet revealed a complex interaction network of the Phocaeicola vulgatus pangenome with its phage invaders, and the modularity analysis of the resulted network suggested differential activities of the different P. vulgatus' CRISPR-Cas systems (Type I-C and Type II-C) against some phages. Analysis of the phage-bacteria interaction network of human gut microbiome revealed a mixture of phages with a broad host range (resulting in large modules with many bacteria and phages), and phages with narrow host range. We also showed that BacMGEnet can be used to infer phages that invade bacteria and their interactions in wound microbiome. We anticipate that BacMGEnet will become an important tool for studying the interactions between bacteria and their invaders for microbiome research.},
}
@article {pmid36248738,
year = {2022},
author = {Chen, L and Vedula, P and Tang, HY and Dong, DW and Kashina, AS},
title = {Differential N-terminal processing of beta and gamma actin.},
journal = {iScience},
volume = {25},
number = {10},
pages = {105186},
pmid = {36248738},
issn = {2589-0042},
abstract = {Cytoplasmic beta- and gamma-actin are ubiquitously expressed in every eukaryotic cell. They are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-termini, making it difficult to dissect their individual regulation. Here, we analyzed actin from cultured cells and tissues by mass spectrometry and found that beta, unlike gamma actin, undergoes sequential removal of N-terminal Asp residues, leading to truncated actin species found in both F- and G-actin preparations. This processing affects up to ∼3% of beta actin in different cell types. We used CRISPR/Cas-9 in cultured cells to delete two candidate enzymes capable of mediating this type of processing. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration. Our results demonstrate previously unknown isoform-specific actin regulation that can potentially affect actin functions in cells.},
}
@article {pmid36246877,
year = {2022},
author = {Gao, Y and Zhang, X and Yuan, J and Zhang, C and Li, S and Li, F},
title = {CRISPR/Cas9-mediated mutation on an insulin-like peptide encoding gene affects the growth of the ridgetail white prawn Exopalaemon carinicauda.},
journal = {Frontiers in endocrinology},
volume = {13},
number = {},
pages = {986491},
pmid = {36246877},
issn = {1664-2392},
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila melanogaster ; Hormones ; *Insulins ; Mutation ; *Palaemonidae/genetics ; *Penaeidae ; Peptides/genetics ; },
abstract = {Insulin-like peptides (ILPs) play key roles in animal growth, metabolism and reproduction in vertebrates. In crustaceans, one type of ILPs, insulin-like androgenic gland hormone (IAG) had been reported to be related to the sex differentiations. However, the function of other types of ILPs is rarely reported. Here, we identified another type of ILPs in the ridgetail white prawn Exopalaemon carinicauda (EcILP), which is an ortholog of Drosophila melanogaster ILP7. Sequence characterization and expression analyses showed that EcILP is similar to vertebrate insulin/IGFs and insect ILPs in its heterodimeric structure and expression profile. Using CRISPR/Cas9 genome editing technology, we generated EcILP knockout (KO) prawns. EcILP-KO individuals have a significant higher growth-inhibitory trait and mortality than those in the normal group. In addition, knockdown of EcILP by RNA interference (RNAi) resulted in slower growth rate and higher mortality. These results indicated that EcILP was an important growth regulator in E. carinicauda.},
}
@article {pmid36246582,
year = {2022},
author = {Tyagi, S and Jha, SK and Kumar, A and Saripalli, G and Bhurta, R and Hurali, DT and Sathee, L and Mallick, N and Mir, RR and Chinnusamy, V and Vinod, },
title = {Genome-wide characterization and identification of cyclophilin genes associated with leaf rust resistance in bread wheat (Triticum aestivum L.).},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {972474},
pmid = {36246582},
issn = {1664-8021},
abstract = {Cyclophilins (CYPs) are a group of highly conserved proteins involved in host-pathogen interactions in diverse plant species. However, the role of CYPs during disease resistance in wheat remains largely elusive. In the present study, the systematic genome-wide survey revealed a set of 81 TaCYP genes from three subfamilies (GI, GII, and GIII) distributed on all 21 wheat chromosomes. The gene structures of TaCYP members were found to be highly variable, with 1-14 exons/introns and 15 conserved motifs. A network of miRNA targets with TaCYPs demonstrated that TaCYPs were targeted by multiple miRNAs and vice versa. Expression profiling was done in leaf rust susceptible Chinese spring (CS) and the CS-Ae. Umbellulata derived resistant IL "Transfer (TR). Three homoeologous TaCYP genes (TaCYP24, TaCYP31, and TaCYP36) showed high expression and three homoeologous TaCYP genes (TaCYP44, TaCYP49, and TaCYP54) showed low expression in TR relative to Chinese Spring. Most of the other TaCYPs showed comparable expression changes (down- or upregulation) in both contrasting TR and CS. Expression of 16 TaCYPs showed significant association (p < 0.05) with superoxide radical and hydrogen peroxide abundance, suggesting the role of TaCYPs in downstream signaling processes during wheat-leaf rust interaction. The differentially expressing TaCYPs may be potential targets for future validation using transgenic (overexpression, RNAi or CRISPR-CAS) approaches and for the development of leaf rust-resistant wheat genotypes.},
}
@article {pmid36246261,
year = {2022},
author = {Dela Ahator, S and Liu, Y and Wang, J and Zhang, LH},
title = {The virulence factor regulator and quorum sensing regulate the type I-F CRISPR-Cas mediated horizontal gene transfer in Pseudomonas aeruginosa.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {987656},
pmid = {36246261},
issn = {1664-302X},
abstract = {Pseudomonas aeruginosa is capable of thriving in diverse environments due to its network of regulatory components for effective response to stress factors. The survival of the bacteria is also dependent on the ability to discriminate between the acquisition of beneficial and non-beneficial genetic materials via horizontal gene transfer (HGT). Thus, bacteria have evolved the CRISPR-Cas adaptive immune system for defense against the deleterious effect of phage infection and HGT. By using the transposon mutagenesis approach, we identified the virulence factor regulator (Vfr) as a key regulator of the type I-F CRISPR-Cas system in P. aeruginosa. We showed that Vfr influences the expression of the CRISPR-Cas system through two signaling pathways in response to changes in calcium levels. Under calcium-rich conditions, Vfr indirectly regulates the CRISPR-Cas system via modulation of the AHL-QS gene expression, which could be vital for defense against phage infection at high cell density. When encountering calcium deficiency, however, Vfr can directly regulate the CRISPR-Cas system via a cAMP-dependent pathway. Furthermore, we provide evidence that mutation of vfr reduces the CRISPR-Cas spacer acquisition and interference of HGT. The results from this study add to the regulatory network of factors controlling the CRISPR-Cas system in response to abiotic factors in the environment. The findings may facilitate the design of effective and reliable phage therapies against P. aeruginosa infections, as targeting Vfr could prevent the development of the CRISPR-Cas mediated phage resistance.},
}
@article {pmid36243978,
year = {2022},
author = {Wu, J and Zou, Z and Liu, Y and Liu, X and Zhangding, Z and Xu, M and Hu, J},
title = {CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11128-11137},
pmid = {36243978},
issn = {1362-4962},
mesh = {Animals ; Mice ; *T-Lymphocytes ; *Gene Editing ; CRISPR-Cas Systems/genetics ; DNA, Viral ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.},
}
@article {pmid36243977,
year = {2022},
author = {Kim, GE and Lee, SY and Birkholz, N and Kamata, K and Jeong, JH and Kim, YG and Fineran, PC and Park, HH},
title = {Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11344-11358},
pmid = {36243977},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacteriophages/genetics ; Pseudomonas aeruginosa/metabolism ; Operon/genetics ; },
abstract = {CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.},
}
@article {pmid36243247,
year = {2022},
author = {Valcárcel-Hernández, V and Guillén-Yunta, M and Bueno-Arribas, M and Montero-Pedrazuela, A and Grijota-Martínez, C and Markossian, S and García-Aldea, &#xc1; and Flamant, F and Bárez-López, S and Guadaño-Ferraz, A},
title = {A CRISPR/Cas9-engineered avatar mouse model of monocarboxylate transporter 8 deficiency displays distinct neurological alterations.},
journal = {Neurobiology of disease},
volume = {174},
number = {},
pages = {105896},
doi = {10.1016/j.nbd.2022.105896},
pmid = {36243247},
issn = {1095-953X},
mesh = {Animals ; Mice ; *Monocarboxylic Acid Transporters/genetics/metabolism ; *Symporters/genetics/metabolism ; CRISPR-Cas Systems ; Thyroid Hormones/metabolism ; Disease Models, Animal ; Mammals/metabolism ; },
abstract = {Inactivating mutations in the specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to an X-linked rare disease named MCT8 deficiency or Allan-Herndon-Dudley Syndrome. Patients exhibit a plethora of severe endocrine and neurological alterations, with no effective treatment for the neurological symptoms. An optimal mammalian model is essential to explore the pathological mechanisms and potential therapeutic approaches. Here we have generated by CRISPR/Cas9 an avatar mouse model for MCT8 deficiency with a point mutation found in two MCT8-deficient patients (P253L mice). We have predicted by in silico studies that this mutation alters the substrate binding pocket being the probable cause for impairing thyroid hormone transport. We have characterized the phenotype of MCT8-P253L mice and found endocrine alterations similar to those described in patients and in MCT8-deficient mice. Importantly, we detected brain hypothyroidism, structural and functional neurological alterations resembling the patient's neurological impairments. Thus, the P253L mouse provides a valuable model for studying the pathophysiology of MCT8 deficiency and in the future will allow to test therapeutic alternatives such as in vivo gene therapy and pharmacological chaperone therapy to improve the neurological impairments in MCT8 deficiency.},
}
@article {pmid36242902,
year = {2022},
author = {Chen, M and Zhang, J and Peng, Y and Bai, J and Li, S and Han, D and Ren, S and Qin, K and Zhou, H and Han, T and Wang, Y and Gao, Z},
title = {Design and synthesis of DNA hydrogel based on EXPAR and CRISPR/Cas14a for ultrasensitive detection of creatine kinase MB.},
journal = {Biosensors &amp; bioelectronics},
volume = {218},
number = {},
pages = {114792},
doi = {10.1016/j.bios.2022.114792},
pmid = {36242902},
issn = {1873-4235},
mesh = {DNA, Complementary ; *Hydrogels ; *Biosensing Techniques ; Isoenzymes/genetics ; DNA ; Creatine Kinase, MB Form ; Biomarkers ; CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems exhibit significant potential in developing biosensing technology due to their collateral cleavage capabilities. Herein, we introduced the collateral cleavage activity of CRISPR/Cas14a to activate DNA hydrogel for ultrasensitive detection of the myocardial infarction biomarker creatine kinase MB (CK-MB). In this strategy, the designed CRISPR/Cas14a system can be activated by introducing complementary DNA (cDNA) derived from competitive dissociation and exponential amplification (EXPAR), which is positively correlated with creatine kinase isoenzyme (CK-MB) concentration. Then the activated Cas14a protein can be utilized to indiscriminately cleave the DNA hydrogel cross-linker strand, leading to the degradation of the gel matrix and thus releasing the pre-encapsulated PtNPs/Cu-TCPP(Fe). PtNPs/Cu-TCPP(Fe) can trigger the TMB reaction, leading to an increase in absorbance value at 450 nm, thus enabling the quantitative detection of CK-MB. The proposed strategy combines CRISPR/Cas14a with DNA hydrogel for the first time, improving the programmability of DNA hydrogel and providing a reliable, sensitive, and versatile detection platform for trace non-nucleic acid targets.},
}
@article {pmid36242898,
year = {2022},
author = {Tetard, M and Peterson, NA and Egan, ES},
title = {Erythrocyte-Plasmodium interactions: genetic manipulation of the erythroid lineage.},
journal = {Current opinion in microbiology},
volume = {70},
number = {},
pages = {102221},
doi = {10.1016/j.mib.2022.102221},
pmid = {36242898},
issn = {1879-0364},
mesh = {Humans ; Cell Lineage ; Erythrocytes/parasitology ; Plasmodium falciparum/genetics ; *Plasmodium/genetics ; *Malaria/parasitology ; CRISPR-Cas Systems ; },
abstract = {Targeting critical host factors is an emerging concept in the treatment of infectious diseases. As obligate pathogens of erythrocytes, the Plasmodium spp. parasites that cause malaria must exploit erythroid host factors for their survival. However, our understanding of this important aspect of the malaria lifecycle is limited, in part because erythrocytes are enucleated cells that lack a nucleus and DNA, rendering them genetically intractable. Recent advances in genetic analysis of the erythroid lineage using small-hairpin RNAs and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) in red-blood cells derived from stem cells have generated new insights into the functions of several candidate host factors for Plasmodium parasites. Along with efforts in other hematopoietic cells, these advances have also laid a strong foundation for genetic screens to identify novel erythrocyte host factors for malaria.},
}
@article {pmid36242861,
year = {2023},
author = {Figueredo, EF and Cruz, TAD and Almeida, JR and Batista, BD and Marcon, J and Andrade, PAM and Hayashibara, CAA and Rosa, MS and Azevedo, JL and Quecine, MC},
title = {The key role of indole-3-acetic acid biosynthesis by Bacillus thuringiensis RZ2MS9 in promoting maize growth revealed by the ipdC gene knockout mediated by the CRISPR-Cas9 system.},
journal = {Microbiological research},
volume = {266},
number = {},
pages = {127218},
doi = {10.1016/j.micres.2022.127218},
pmid = {36242861},
issn = {1618-0623},
mesh = {*Zea mays/genetics/metabolism ; Plant Growth Regulators/metabolism ; *Bacillus thuringiensis/genetics/metabolism ; Tryptophan/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Indoleacetic Acids/metabolism ; },
abstract = {The bacterial biosynthesis of indole-3-acetic acid (IAA) is often related to the beneficial effects of plant growth-promoting rhizobacteria (PGPR) on plant development. In PGPR belonging to the Bacillus genus, the synthesis of IAA may occur through different metabolic pathways that are still poorly understood. B. thuringiensis (Bt) is well known for its insecticidal properties; however, its beneficial features are not limited to pest control. Our group has been studed the beneficial effects of Bt strain RZ2MS9 as growth promoter in a range of plant crops, including soybean, tomato, and maize. We recently demonstrated that bacterial IAA biosynthesis plays an important role in the ability of RZ2MS9 to benefit plant development. However, the molecular involved mechanisms in the IAA biosynthesis by this bacterium in the beneficial interaction with plants remain unclear. Here, we investigated the genetic basis of IAA biosynthesis by RZ2MS9. We knocked out the ipdC gene, involved in IAA biosynthesis via the tryptophan-dependent IPyA pathway, using the CRISPR-Cas9 system. Our results showed that, by disrupting the IPyA pathway, the amount of IAA synthesized by the mutant RZ2MS9 (ΔipdC) in the presence of tryptophan drops 57%. The gene knockout did not affect the bacterial growth, but it did affect its ability to colonize maize. Moreover, deactivating the ipdC gene in RZ2MS9 significantly reduces its ability to promote maize growth. ΔipdC performed worse than RZ2MS9 in almost all evaluated plant parameters, including total root length, projected root area, lateral roots, aerial part dry matter, and germination speed index. Therefore, we demonstrated that tryptophan-dependent IAA biosynthesis via the IPyA pathway by RZ2MS9 is strongly influenced by the ipdC gene. Furthermore, IAA biosynthesis by RZ2MS9 is a major mechanism used by this PGPR to promote maize growth.},
}
@article {pmid36241974,
year = {2022},
author = {Mann, JG and Pitts, RJ},
title = {PrimedSherlock: a tool for rapid design of highly specific CRISPR-Cas12 crRNAs.},
journal = {BMC bioinformatics},
volume = {23},
number = {1},
pages = {428},
pmid = {36241974},
issn = {1471-2105},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acids ; RNA ; SARS-CoV-2/genetics ; *Zika Virus/genetics ; *Zika Virus Infection ; },
abstract = {BACKGROUND: CRISPR-Cas based diagnostic assays provide a portable solution which bridges the benefits of qRT-PCR and serological assays in terms of portability, specificity and ease of use. CRISPR-Cas assays are rapidly fieldable, specific and have been rigorously validated against a number of targets, including HIV and vector-borne pathogens. Recently, CRISPR-Cas12 and CRISPR-Cas13 diagnostic assays have been granted FDA approval for the detection of SARS-CoV-2. A critical step in utilizing this technology requires the design of highly-specific and efficient CRISPR RNAs (crRNAs) and isothermal primers. This process involves intensive manual curation and stringent parameters for design in order to minimize off-target detection while also preserving detection across divergent strains. As such, a single, streamlined bioinformatics platform for rapidly designing crRNAs for use with the CRISPR-Cas12 platform is needed. Here we offer PrimedSherlock, an automated, computer guided process for selecting highly-specific crRNAs and primers for targets of interest.<br><br>RESULTS: Utilizing PrimedSherlock and publicly available databases, crRNAs were designed against a selection of Flavivirus genomes, including West Nile, Zika and all four serotypes of Dengue. Using outputs from PrimedSherlock in concert with both wildtype A.s Cas12a and Alt-R Cas12a Ultra nucleases, we demonstrated sensitive detection of nucleic acids of each respective arbovirus in in-vitro fluorescence assays. Moreover, primer and crRNA combinations facilitated the detection of their intended targets with minimal off-target background noise.<br><br>CONCLUSIONS: PrimedSherlock is a novel crRNA design tool, specific for CRISPR-Cas12 diagnostic platforms. It allows for the rapid identification of highly conserved crRNA targets from user-provided primer pairs or PrimedRPA output files. Initial testing of crRNAs against arboviruses of medical importance demonstrated a robust ability to distinguish multiple strains by exploiting polymorphisms within otherwise highly conserved genomic regions. As a freely-accessible software package, PrimedSherlock could significantly increase the efficiency of CRISPR-Cas12 diagnostics. Conceptually, the portability of detection kits could also be enhanced when coupled with isothermal amplification technologies.},
}
@article {pmid36241739,
year = {2022},
author = {Varshney, P and Varshney, GK},
title = {Expanded precision genome-editing toolbox for human disease modeling in zebrafish.},
journal = {Lab animal},
volume = {51},
number = {11},
pages = {287-289},
pmid = {36241739},
issn = {1548-4475},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid36241651,
year = {2022},
author = {Casarin, T and Freitas, NC and Pinto, RT and Breitler, JC and Rodrigues, LAZ and Marraccini, P and Etienne, H and Diniz, LEC and Andrade, AC and Paiva, LV},
title = {Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {17270},
pmid = {36241651},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; *Coffea/genetics ; Coffee ; Gene Editing ; Oxidoreductases ; Plant Breeding ; beta Carotene ; },
abstract = {Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in β-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.},
}
@article {pmid36241097,
year = {2022},
author = {Edelstein, J and Fritz, M and Lai, SK},
title = {Challenges and opportunities in gene editing of B cells.},
journal = {Biochemical pharmacology},
volume = {206},
number = {},
pages = {115285},
doi = {10.1016/j.bcp.2022.115285},
pmid = {36241097},
issn = {1873-2968},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
abstract = {B cells have long been an underutilized target in immune cell engineering, despite a number of unique attributes that could address longstanding challenges in medicine. Notably, B cells evolved to secrete large quantities of antibodies for prolonged periods, making them suitable platforms for long-term protein delivery. Recent advances in gene editing technologies, such as CRISPR-Cas, have improved the precision and efficiency of engineering and expanded potential applications of engineered B cells. While most work on B cell editing has focused on ex vivo modification, a body of recent work has also advanced the possibility of in vivo editing applications. In this review, we will discuss both past and current approaches to B cell engineering, and its promising applications in immunology research and therapeutic gene editing.},
}
@article {pmid36240636,
year = {2022},
author = {Xu, T and Zhong, J and Huang, Z and Yu, L and Zheng, J and Xie, L and Sun, L and Liu, X and Lu, Y},
title = {Optimization of the base editor BE4max in chicken somatic cells.},
journal = {Poultry science},
volume = {101},
number = {12},
pages = {102174},
pmid = {36240636},
issn = {1525-3171},
mesh = {Animals ; *CRISPR-Cas Systems ; *Chickens/genetics ; Gene Editing/veterinary/methods ; Mutation ; },
abstract = {Advanced animal reproductive and breeding biotechnology has made it possible to alter traits or create new genetic resources by the direct knock-in or knock-out of target genes. Base editing technology can achieve single-base mutations without double-stranded DNA breaks, and is a promising tool for use in the genetic modification and breeding of livestock. However, the application of base editors (BEs) in chicken has not been optimized. We evaluated the efficacy of BE4max in chicken somatic cells (DF-1). The key element of BE4max, cytosine deaminase (APOBEC), was optimized for chicken. The base editing efficiency of the optimized chBE4max editor, compared with the original BE4max editor, was improved by 10.4% ± 4.6. By inhibiting the expression of the uracil DNA glycosylase-related gene methyl binding domain protein 4 (MBD4) by siRNA in chicken DF-1 cells, the editing efficiency was enhanced by 4.43% ± 1.4 compared to the control. These results suggest that this editor may have applications in poultry breeding studies.},
}
@article {pmid36240131,
year = {2022},
author = {Song, Y and Gao, K and Cai, X and Cheng, W and Ding, S and Zhang, D and Deng, S},
title = {Controllable crRNA Self-Transcription Aided Dual-Amplified CRISPR-Cas12a Strategy for Highly Sensitive Biosensing of FEN1 Activity.},
journal = {ACS synthetic biology},
volume = {11},
number = {11},
pages = {3847-3854},
doi = {10.1021/acssynbio.2c00420},
pmid = {36240131},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; Endonucleases/genetics ; DNA ; *Biosensing Techniques ; },
abstract = {A controllable crRNA self-transcription aided dual-amplified CRISPR-Cas12a strategy (termed CST-Cas12a) was developed for highly sensitive and specific biosensing of flap endonuclease 1 (FEN1), a structure-selective nuclease in eukaryotic cells. In this strategy, a branched DNA probe with a 5' overhanging flap was designed to serve as a hydrolysis substrate of FEN1. The flap cut by FEN1 was annealed with a template probe and functioned as a primer for an extension reaction to produce a double-stranded DNA (dsDNA) containing a T7 promoter and crRNA transcription template. Assisting the T7 RNA polymerase, abundant crRNA was generated and assembled with Cas12a to form a Cas12a/crRNA complex, which can be activated by a dsDNA trigger and unlock the indiscriminate fluorophore-quencher reporter cleavage. The highly efficient dual signal amplification and near-zero background enabled CST-Cas12a with extraordinarily high sensitivity. Under optimized conditions, this method allowed highly sensitive biosensing of FEN1 activity in the range of 1 × 10[-5] U μL[-1] to 5 × 10[-2] U μL[-1] with a detection limit of 5.2 × 10[-6] U μL[-1] and achieved excellent specificity for FEN1 in the presence of other interfering enzymes. The inhibitory capabilities of chemicals on FEN1 were also investigated. Further, the newly established CST-Cas12a strategy was successfully applied to FEN1 biosensing in complex biological samples, which might be a reliable biosensing platform for highly sensitive and specific detection of FEN1 activity in clinical applications.},
}
@article {pmid36239984,
year = {2022},
author = {Mohammad, N and Katkam, SS and Wei, Q},
title = {A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double-Stranded λ DNA Reporter.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {50},
pages = {e202213920},
doi = {10.1002/anie.202213920},
pmid = {36239984},
issn = {1521-3773},
mesh = {*CRISPR-Cas Systems ; DNA ; DNA Cleavage ; DNA, Single-Stranded ; *Biosensing Techniques ; },
abstract = {CRISPR-based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR-Cas12a-based sensing platform to detect ssDNA targets by sizing double-stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massive trans-nuclease activity of Cas12a toward λ DNA is due to the presence of single-stranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes the trans-cleavage activity and helps achieve sub-picomolar detection sensitivity, ≈100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters.},
}
@article {pmid36239875,
year = {2022},
author = {Li, R and Wang, Q and She, K and Lu, F and Yang, Y},
title = {CRISPR/Cas systems usher in a new era of disease treatment and diagnosis.},
journal = {Molecular biomedicine},
volume = {3},
number = {1},
pages = {31},
pmid = {36239875},
issn = {2662-8651},
abstract = {The discovery and development of the CRISPR/Cas system is a milestone in precise medicine. CRISPR/Cas nucleases, base-editing (BE) and prime-editing (PE) are three genome editing technologies derived from CRISPR/Cas. In recent years, CRISPR-based genome editing technologies have created immense therapeutic potential with safe and efficient viral or non-viral delivery systems. Significant progress has been made in applying genome editing strategies to modify T cells and hematopoietic stem cells (HSCs) ex vivo and to treat a wide variety of diseases and disorders in vivo. Nevertheless, the clinical translation of this unique technology still faces many challenges, especially targeting, safety and delivery issues, which require further improvement and optimization. In addition, with the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), CRISPR-based molecular diagnosis has attracted extensive attention. Growing from the specific set of molecular biological discoveries to several active clinical trials, CRISPR/Cas systems offer the opportunity to create a cost-effective, portable and point-of-care diagnosis through nucleic acid screening of diseases. In this review, we describe the development, mechanisms and delivery systems of CRISPR-based genome editing and focus on clinical and preclinical studies of therapeutic CRISPR genome editing in disease treatment as well as its application prospects in therapeutics and molecular detection.},
}
@article {pmid36235491,
year = {2022},
author = {Hamdan, MF and Karlson, CKS and Teoh, EY and Lau, SE and Tan, BC},
title = {Genome Editing for Sustainable Crop Improvement and Mitigation of Biotic and Abiotic Stresses.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {19},
pages = {},
pmid = {36235491},
issn = {2223-7747},
abstract = {Climate change poses a serious threat to global agricultural activity and food production. Plant genome editing technologies have been widely used to develop crop varieties with superior qualities or can tolerate adverse environmental conditions. Unlike conventional breeding techniques (e.g., selective breeding and mutation breeding), modern genome editing tools offer more targeted and specific alterations of the plant genome and could significantly speed up the progress of developing crops with desired traits, such as higher yield and/or stronger resilience to the changing environment. In this review, we discuss the current development and future applications of genome editing technologies in mitigating the impacts of biotic and abiotic stresses on agriculture. We focus specifically on the CRISPR/Cas system, which has been the center of attention in the last few years as a revolutionary genome-editing tool in various species. We also conducted a bibliographic analysis on CRISPR-related papers published from 2012 to 2021 (10 years) to identify trends and potential in the CRISPR/Cas-related plant research. In addition, this review article outlines the current shortcomings and challenges of employing genome editing technologies in agriculture with notes on future prospective. We believe combining conventional and more innovative technologies in agriculture would be the key to optimizing crop improvement beyond the limitations of traditional agricultural practices.},
}
@article {pmid36235360,
year = {2022},
author = {Karlson, D and Mojica, JP and Poorten, TJ and Lawit, SJ and Jali, S and Chauhan, RD and Pham, GM and Marri, P and Guffy, SL and Fear, JM and Ochsenfeld, CA and Lincoln Chapman, TA and Casamali, B and Venegas, JP and Kim, HJ and Call, A and Sublett, WL and Mathew, LG and Shariff, A and Watts, JM and Mann, M and Hummel, A and Rapp, R},
title = {Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {19},
pages = {},
pmid = {36235360},
issn = {2223-7747},
abstract = {Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the 'mustard bomb' reaction in reducing pungency upon the mastication of fresh tissue-a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.},
}
@article {pmid36235340,
year = {2022},
author = {Antonova, EV and Shimalina, NS and Korotkova, AM and Kolosovskaya, EV and Gerasimova, SV and Khlestkina, EK},
title = {Seedling Biometry of nud Knockout and win1 Knockout Barley Lines under Ionizing Radiation.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {19},
pages = {},
pmid = {36235340},
issn = {2223-7747},
abstract = {The genes NUD and WIN1 play a regulatory role in cuticle organization in barley. A knockout (KO) of each gene may alter plant mechanisms of adaptation to adverse environmental conditions. A putative pleiotropic effect of NUD or WIN1 gene mutations in barley can be assessed in a series of experiments in the presence or absence of a provoking factor. Ionizing radiation is widely used in research as a provoking factor for quantifying adaptive potential of living organisms. Our aim was to evaluate initial stages of growth and development of barley lines with a KO of NUD or WIN1 under radiation stress. Air-dried barley grains with different KOs and wild-type control (WT) were exposed to γ-radiation at 50, 100, or 200 Gy at a dose rate of 0.74 R/min. Approximately 30 physiological parameters were evaluated, combined into groups: (1) viability, (2) radiosensitivity, and (3) mutability of barley seed progeny. Seed germination, seedling survival, and shoot length were similar among all barley lines. Naked nud KO lines showed lower weights of seeds, roots, and seedlings and shorter root length as compared to win1 KO lines. The shoot-to-root length ratio of nud KO lines' seedlings exceeded that of win1 KO and WT lines. In terms of the number of seedlings with leaves, all the KO lines were more sensitive to pre-sowing γ-irradiation. Meanwhile, the radioresistance of nud KO lines (50% growth reduction dose [RD50] = 318-356 Gy) and WT plants (RD50 = 414 Gy) judging by seedling weight was higher than that of win1 KO lines (RD50 = 201-300 Gy). Resistance of nud KO lines to radiation was also demonstrated by means of root length (RD50 = 202-254 Gy) and the shoot-to-root length ratio. WT seedlings had the fewest morphological anomalies. In nud KO lines, mainly alterations of root shape were found, whereas in win1 KO lines, changes in the color and shape of leaves were noted. Thus, seedlings of nud KO lines are characterized mainly by changes in the root system (root length, root number, and root anomalies). For win1 KO lines, other parameters are sensitive (shoot length and alterations of leaf shape and color). These data may indicate a pleiotropic effect of genes NUD and WIN1 in barley.},
}
@article {pmid36234804,
year = {2022},
author = {Shin, S and Jang, S and Lim, D},
title = {Small Molecules for Enhancing the Precision and Safety of Genome Editing.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {19},
pages = {},
pmid = {36234804},
issn = {1420-3049},
support = {HV22C0235//Korea Health Industry Development Institute/Republic of Korea ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Endonucleases/genetics/metabolism ; *Gene Editing ; RNA, Guide/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing technologies have revolutionized biology, biotechnology, and medicine, and have spurred the development of new therapeutic modalities. However, there remain several barriers to the safe use of CRISPR technologies, such as unintended off-target DNA cleavages. Small molecules are important resources to solve these problems, given their facile delivery and fast action to enable temporal control of the CRISPR systems. Here, we provide a comprehensive overview of small molecules that can precisely modulate CRISPR-associated (Cas) nucleases and guide RNAs (gRNAs). We also discuss the small-molecule control of emerging genome editors (e.g., base editors) and anti-CRISPR proteins. These molecules could be used for the precise investigation of biological systems and the development of safer therapeutic modalities.},
}
@article {pmid36233222,
year = {2022},
author = {Reuven, N and Shaul, Y},
title = {Selecting for CRISPR-Edited Knock-In Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36233222},
issn = {1422-0067},
support = {1/CX/CSRD VA/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA Repair ; *Gene Editing ; Genome ; Mammals ; },
abstract = {CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, "scarless" selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.},
}
@article {pmid36233218,
year = {2022},
author = {Aoto, K and Takabayashi, S and Mutoh, H and Saitsu, H},
title = {Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36233218},
issn = {1422-0067},
mesh = {Animals ; Antibodies/metabolism ; Blotting, Western ; CRISPR-Cas Systems/genetics ; *Electroporation ; Epitopes/genetics/metabolism ; *Gene Editing ; Gonads/metabolism ; Mice ; },
abstract = {Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable.},
}
@article {pmid36233166,
year = {2022},
author = {Khusnutdinov, E and Artyukhin, A and Sharifyanova, Y and Mikhaylova, EV},
title = {A Mutation in the MYBL2-1 Gene Is Associated with Purple Pigmentation in Brassica oleracea.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36233166},
issn = {1422-0067},
mesh = {*Anthocyanins/metabolism ; *Brassica/genetics/metabolism ; DNA/metabolism ; Gene Expression Regulation, Plant ; Mutation ; Nucleotides/metabolism ; Pigmentation/genetics ; Plant Proteins/genetics/metabolism ; RNA/metabolism ; Transcription Factors/metabolism ; },
abstract = {Anthocyanins are well-known antioxidants that are beneficial for plants and consumers. Dihydroflavonol-4-reductase (DFR) is a key gene of anthocyanin biosynthesis, controlled by multiple transcription factors. Its expression can be enhanced by mutations in the negative regulator of anthocyanin biosynthesis myeloblastosis family transcription factor-like 2 (MYBL2). The expression profiles of the DFR gene were examined in 43 purple and green varieties of Brassica oleracea L., Brassica napus L., Brassica juncea L., and Brassica rapa L. MYBL2 gene expression was significantly reduced in purple varieties of B. oleracea, and green varieties of B. juncea. The MYBL2 gene sequences were screened for mutations that can affect pigmentation. Expression of the DFR gene was cultivar-specific, but in general it correlated with anthocyanin content and was higher in purple plants. Two single nucleotide polymorphysms (SNPs) were found at the beginning of the DNA-binding domain of MYBL2 gene in all purple varieties of B. oleracea. This mutation, leading to an amino acid substitution and the formation of a mononucleotide repeat (A)8, significantly affects RNA structure. No other noteworthy mutations were found in the MYBL2 gene in green varieties of B. oleracea and other studied species. These results bring new insights into the regulation of anthocyanin biosynthesis in genus Brassica and provide opportunities for generation of new purple varieties with precise mutations introduced via genetic engineering and CRISPR/Cas.},
}
@article {pmid36232946,
year = {2022},
author = {Banicka, V and Martens, MC and Panzer, R and Schrama, D and Emmert, S and Boeckmann, L and Thiem, A},
title = {Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36232946},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; DNA Damage ; DNA Repair/genetics ; Exons/genetics ; Humans ; Immune Checkpoint Inhibitors ; *Melanoma/genetics ; Programmed Cell Death 1 Receptor/metabolism ; Ultraviolet Rays ; *Xeroderma Pigmentosum/genetics ; *Xeroderma Pigmentosum Group A Protein/genetics/metabolism ; },
abstract = {Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.},
}
@article {pmid36232782,
year = {2022},
author = {Ahmad, I},
title = {CRISPR/Cas9-A Promising Therapeutic Tool to Cure Blindness: Current Scenario and Future Prospects.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36232782},
issn = {1422-0067},
mesh = {Animals ; Blindness/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Mice ; },
abstract = {CRISPR-based targeted genome editing is bringing revolutionary changes in the research arena of biological sciences. CRISPR/Cas9 has been explored as an efficient therapeutic tool for the treatment of genetic diseases. It has been widely used in ophthalmology research by using mouse models to correct pathogenic mutations in the eye stem cells. In recent studies, CRISPR/Cas9 has been used to correct a large number of mutations related to inherited retinal disorders. In vivo therapeutic advantages for retinal diseases have been successfully achieved in some rodents. Current advances in the CRISPR-based gene-editing domain, such as modified Cas variants and delivery approaches have optimized its application to treat blindness. In this review, recent progress and challenges of the CRISPR-Cas system have been discussed to cure blindness and its prospects.},
}
@article {pmid36232699,
year = {2022},
author = {Liu, L and Pei, DS},
title = {Insights Gained from RNA Editing Targeted by the CRISPR-Cas13 Family.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36232699},
issn = {1422-0067},
mesh = {Amino Acids/metabolism ; *CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; Gene Editing ; *Nucleic Acids/metabolism ; RNA/genetics/metabolism ; RNA Editing/genetics ; RNA, Guide/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely developed for DNA targeting and formed a set of mature precision gene-editing systems. However, the basic research and application of the CRISPR-Cas system in RNA is still in its early stages. Recently, the discovery of the CRISPR-Cas13 type VI system has provided the possibility for the expansion of RNA targeting technology, which has broad application prospects. Most type VI Cas13 effectors have dinuclease activity that catalyzes pre-crRNA into mature crRNA and produces strong RNA cleavage activity. Cas13 can specifically recognize targeted RNA fragments to activate the Cas13/crRNA complex for collateral cleavage activity. To date, the Cas13X protein is the smallest effector of the Cas13 family, with 775 amino acids, which is a promising platform for RNA targeting due to its lack of protospacer flanking sequence (PFS) restrictions, ease of packaging, and absence of permanent damage. This study highlighted the latest progress in RNA editing targeted by the CRISPR-Cas13 family, and discussed the application of Cas13 in basic research, nucleic acid diagnosis, nucleic acid tracking, and genetic disease treatment. Furthermore, we clarified the structure of the Cas13 protein family and their molecular mechanism, and proposed a future vision of RNA editing targeted by the CRISPR-Cas13 family.},
}
@article {pmid36232425,
year = {2022},
author = {Revathidevi, S and Hosomichi, K and Natsume, T and Nakaoka, H and Fujito, NT and Akatsuka, H and Sato, T and Munirajan, AK and Inoue, I},
title = {AMBRA1 p.Gln30Arg Mutation, Identified in a Cowden Syndrome Family, Exhibits Hyperproliferative Potential in hTERT-RPE1 Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36232425},
issn = {1422-0067},
mesh = {Animals ; Beclin-1/genetics ; Germ-Line Mutation ; *Hamartoma Syndrome, Multiple/complications/genetics/pathology ; Mutation ; PTEN Phosphohydrolase/genetics ; RNA-Directed DNA Polymerase/genetics ; Tensins/genetics ; Zebrafish/genetics ; },
abstract = {Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1 (humanTelomerase Reverse Transcriptase-immortalized Retinal Pigmented Epithelial cells) using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.},
}
@article {pmid36232363,
year = {2022},
author = {Arriaga-Canon, C and Contreras-Espinosa, L and Rebollar-Vega, R and Montiel-Manríquez, R and Cedro-Tanda, A and García-Gordillo, JA and Álvarez-Gómez, RM and Jiménez-Trejo, F and Castro-Hernández, C and Herrera, LA},
title = {Transcriptomics and RNA-Based Therapeutics as Potential Approaches to Manage SARS-CoV-2 Infection.},
journal = {International journal of molecular sciences},
volume = {23},
number = {19},
pages = {},
pmid = {36232363},
issn = {1422-0067},
mesh = {*COVID-19/genetics/therapy ; Humans ; Pandemics ; RNA, Small Interfering ; SARS-CoV-2/genetics ; Transcriptome ; },
abstract = {SARS-CoV-2 is a coronavirus family member that appeared in China in December 2019 and caused the disease called COVID-19, which was declared a pandemic in 2020 by the World Health Organization. In recent months, great efforts have been made in the field of basic and clinical research to understand the biology and infection processes of SARS-CoV-2. In particular, transcriptome analysis has contributed to generating new knowledge of the viral sequences and intracellular signaling pathways that regulate the infection and pathogenesis of SARS-CoV-2, generating new information about its biology. Furthermore, transcriptomics approaches including spatial transcriptomics, single-cell transcriptomics and direct RNA sequencing have been used for clinical applications in monitoring, detection, diagnosis, and treatment to generate new clinical predictive models for SARS-CoV-2. Consequently, RNA-based therapeutics and their relationship with SARS-CoV-2 have emerged as promising strategies to battle the SARS-CoV-2 pandemic with the assistance of novel approaches such as CRISPR-CAS, ASOs, and siRNA systems. Lastly, we discuss the importance of precision public health in the management of patients infected with SARS-CoV-2 and establish that the fusion of transcriptomics, RNA-based therapeutics, and precision public health will allow a linkage for developing health systems that facilitate the acquisition of relevant clinical strategies for rapid decision making to assist in the management and treatment of the SARS-CoV-2-infected population to combat this global public health problem.},
}
@article {pmid36231081,
year = {2022},
author = {Zayat, V and Szlendak, R and Hoffman-Zacharska, D},
title = {Concise Review: Stem Cell Models of SCN1A-Related Encephalopathies-Current Perspective and Future Therapies.},
journal = {Cells},
volume = {11},
number = {19},
pages = {},
pmid = {36231081},
issn = {2073-4409},
mesh = {Animals ; *Brain Diseases/genetics/therapy ; *Epilepsies, Myoclonic/genetics/therapy ; *Induced Pluripotent Stem Cells ; NAV1.1 Voltage-Gated Sodium Channel/genetics ; *Seizures, Febrile/genetics ; },
abstract = {Mutations in the SCN1A gene can cause a variety of phenotypes, ranging from mild forms, such as febrile seizures and generalized epilepsy with febrile seizures plus, to severe, such as Dravet and non-Dravet developmental epileptic encephalopathies. Until now, more than two thousand pathogenic variants of the SCN1A gene have been identified and different pathogenic mechanisms (loss vs. gain of function) described, but the precise molecular mechanisms responsible for the deficits exhibited by patients are not fully elucidated. Additionally, the phenotypic variability proves the involvement of other genetic factors in its final expression. This is the reason why animal models and cell line models used to explore the molecular pathology of SCN1A-related disorders are only of limited use. The results of studies based on such models cannot be directly translated to affected individuals because they do not address each patient's unique genetic background. The generation of functional neurons and glia for patient-derived iPSCs, together with the generation of isogenic controls using CRISPR/Cas technology, and finally, the 3D brain organoid models, seem to be a good way to solve this problem. Here, we review SCN1A-related encephalopathies, as well as the stem cell models used to explore their molecular basis.},
}
@article {pmid36231007,
year = {2022},
author = {Ding, X and Yu, L and Chen, L and Li, Y and Zhang, J and Sheng, H and Ren, Z and Li, Y and Yu, X and Jin, S and Cao, J},
title = {Recent Progress and Future Prospect of CRISPR/Cas-Derived Transcription Activation (CRISPRa) System in Plants.},
journal = {Cells},
volume = {11},
number = {19},
pages = {},
pmid = {36231007},
issn = {2073-4409},
mesh = {*CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Plants/genetics ; RNA, Guide ; Transcriptional Activation/genetics ; },
abstract = {Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed.},
}
@article {pmid36230926,
year = {2022},
author = {Chen, G and Wei, T and Yang, H and Li, G and Li, H},
title = {CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives.},
journal = {Cells},
volume = {11},
number = {19},
pages = {},
pmid = {36230926},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dogs ; *Dystrophin/genetics/metabolism ; Gene Editing ; Genetic Therapy ; Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Swine ; },
abstract = {Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation.},
}
@article {pmid36228744,
year = {2022},
author = {Abe, T and Horisawa, Y and Kikuchi, O and Ozawa-Umeta, H and Kishimoto, A and Katsuura, Y and Imaizumi, A and Hashimoto, T and Shirakawa, K and Takaori-Kondo, A and Yusa, K and Asakura, T and Kakeya, H and Kanai, M},
title = {Pharmacologic characterization of TBP1901, a prodrug form of aglycone curcumin, and CRISPR-Cas9 screen for therapeutic targets of aglycone curcumin.},
journal = {European journal of pharmacology},
volume = {935},
number = {},
pages = {175321},
doi = {10.1016/j.ejphar.2022.175321},
pmid = {36228744},
issn = {1879-0712},
mesh = {Mice ; Animals ; *Curcumin/pharmacology ; *Prodrugs/pharmacology/therapeutic use ; Glucuronides/metabolism/pharmacology/therapeutic use ; Reactive Oxygen Species/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; NF-kappa B/metabolism ; Glucuronidase/metabolism ; },
abstract = {Curcumin (aglycone curcumin) has antitumor properties in a variety of malignancies via the alteration of multiple cancer-related biological pathways; however, its clinical application has been hampered due to its poor bioavailability. To overcome this limitation, we have developed a synthesized curcumin β-D-glucuronide sodium salt (TBP1901), a prodrug form of aglycone curcumin. In this study, we aimed to clarify the pharmacologic characteristics of TBP1901. In β-glucuronidase (GUSB)-proficient mice, both curcumin β-D-glucuronide and its active metabolite, aglycone curcumin, were detected in the blood after TBP1901 injection, whereas only curcumin β-D-glucuronide was detected in GUSB-impaired mice, suggesting that GUSB plays a pivotal role in the conversion of TBP1901 into aglycone curcumin in vivo. TBP1901 itself had minimal antitumor effects in vitro, whereas it demonstrated significant antitumor effects in vivo. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen disclosed the genes associated with NF-κB signaling pathway and mitochondria were among the highest hit. In vitro, aglycone curcumin inhibited NF-kappa B signaling pathways whereas it caused production of reactive oxygen species (ROS). ROS scavenger, N-acetyl-L-cysteine, partially reversed antitumor effects of aglycone curcumin. In summary, TBP1901 can exert antitumor effects as a prodrug of aglycone curcumin through GUSB-dependent activation.},
}
@article {pmid36228392,
year = {2022},
author = {Wang, Y and Zhang, D and Zeng, Y and Sun, Y and Qi, P},
title = {Simultaneous ultrasensitive ADP and ATP quantification based on CRISPR/Cas12a integrated ZIF-90@Ag3AuS2@Fe3O4 nanocomposites.},
journal = {Biosensors &amp; bioelectronics},
volume = {218},
number = {},
pages = {114784},
doi = {10.1016/j.bios.2022.114784},
pmid = {36228392},
issn = {1873-4235},
mesh = {CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Zeolites ; *Nanocomposites ; Adenosine Triphosphate ; Adenosine Diphosphate ; },
abstract = {ADP/ATP ratio is a sensitive indicator of changes in cellular energy status and is important for regulating cell signaling activities. Ultrasensitive quantification of ADP and ATP concentration in a single system is in great demand for bioanalysis and early disease diagnosis. Hence, a target-regulated luminous nanoplatform based on clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a integrated zeolite imidazolate framework (ZIF-90)@Ag3AuS2@Fe3O4 nanocomposites was established for the simultaneous detection of ADP and ATP. This simultaneous and ultrasensitive quantification nanoplatform (dsDNA-ZIF-90@Ag3AuS2@Fe3O4) composed an ADP sensitive module based on the aptamer-activated CRISPR/Cas12a and an ATP responsive module based on ATP-triggered ZIF-90 decomposition and quencher loading release. The binding and signaling processes of the different nucleotides were independent, and there was no interference between the two modules. Utilizing the high specific binding and strong signal amplification of this method, limits of detection as low as 0.022 and 0.079 nM were obtained within 30 min for ADP and ATP, respectively. Moreover, the proposed biosensor exhibited high accuracy, specificity, and excellent recovery in serum samples and bacterial biofilms. Therefore, the dsDNA-ZIF-90@Ag3AuS2@Fe3O4-based nanoplatform provides a promising method for ultrasensitive dual-mode quantification of ADP and ATP in the same system, possessing great potential for bioanalysis and early disease diagnosis.},
}
@article {pmid36227031,
year = {2022},
author = {Chen, Y and Wang, X},
title = {Evaluation of efficiency prediction algorithms and development of ensemble model for CRISPR/Cas9 gRNA selection.},
journal = {Bioinformatics (Oxford, England)},
volume = {38},
number = {23},
pages = {5175-5181},
pmid = {36227031},
issn = {1367-4811},
support = {R35GM141535/NH/NIH HHS/United States ; },
mesh = {*RNA, Guide/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Algorithms ; Software ; },
abstract = {MOTIVATION: The CRISPR/Cas9 system is widely used for genome editing. The editing efficiency of CRISPR/Cas9 is mainly determined by the guide RNA (gRNA). Although many computational algorithms have been developed in recent years, it is still a challenge to select optimal bioinformatics tools for gRNA design in different experimental settings.<br><br>RESULTS: We performed a comprehensive comparison analysis of 15 public algorithms for gRNA design, using 16 experimental gRNA datasets. Based on this analysis, we identified the top-performing algorithms, with which we further implemented various computational strategies to build ensemble models for performance improvement. Validation analysis indicates that the new ensemble model had improved performance over any individual algorithm alone at predicting gRNA efficacy under various experimental conditions.<br><br>The new sgRNA design tool is freely accessible as a web application via https://crisprdb.org. The source code and stand-alone version is available at Figshare (https://doi.org/10.6084/m9.figshare.21295863) and Github (https://github.com/wang-lab/CRISPRDB).<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid36226578,
year = {2022},
author = {Wang, J and Luo, L and Li, Y and Chen, L and Gui, C and Zhu, J and Li, H and Wang, W and Chen, D},
title = {A signal on-off strategy based on the digestion of DNA cubes assisted by the CRISPR-Cas12a system for ultrasensitive HBV detection in solid-state nanopores.},
journal = {The Analyst},
volume = {147},
number = {24},
pages = {5623-5632},
doi = {10.1039/d2an01402e},
pmid = {36226578},
issn = {1364-5528},
mesh = {Humans ; *Nanopores ; Hepatitis B virus/genetics ; CRISPR-Cas Systems ; DNA/chemistry ; Digestion ; },
abstract = {Solid-state nanopores have been proven as a powerful platform for label-free single-molecule analysis. However, due to its relatively low resolution and selectivity, developing biosensors with good translocation signals faces two significant challenges: (1) small-sized chemical or biological targets show difficulty in producing recognizable translocation signals because of their weak interaction with the nanopore and (2) protein interferents that widely exist in biological samples or buffers would considerably deteriorate the noise level of the nanopore, submerging the translocation signal. Herein, we demonstrate an effective way to overcome both the challenges. DNA cubes were used as signal transducers that can achieve an ultra-high (>50 : 1) signal-to-noise ratio (SNR) translocation signal, which is maintained even in protein interferent-rich buffers. A sensing strategy was constructed via hepatitis B virus (HBV) target-triggered cleavage of the component elements of the DNA cube with the assistance of the CRISPR-Cas12a technology, which caused a great drop in the translocation rate. The elements to cleave were optimized, and the sensor performance was tested in different protein stabilizer-rich buffers and human serum. Coupling with the polymerase chain reaction (PCR) pre-amplification technology, HBV-positive or -negative classification was achieved with the detection limit reaching 5 aM. It is worth noting that in our method, all reaction buffers were directly used without further optimization, which is of great help for the practical application of solid-state nanopores.},
}
@article {pmid36225138,
year = {2022},
author = {Crowther, LM and Poms, M and Zandl-Lang, M and Abela, L and Hartmann, H and Seiler, M and Mathis, D and Plecko, B},
title = {Metabolomics analysis of antiquitin deficiency in cultured human cells and plasma: Relevance to pyridoxine-dependent epilepsy.},
journal = {Journal of inherited metabolic disease},
volume = {},
number = {},
pages = {},
doi = {10.1002/jimd.12569},
pmid = {36225138},
issn = {1573-2665},
abstract = {Deficiency of antiquitin (α-aminoadipic semialdehyde dehydrogenase), an enzyme involved in lysine degradation and encoded by ALDH7A1, is the major cause of vitamin B6 -dependent epilepsy (PDE-ALDH7A1). Despite seizure control with high dose pyridoxine (PN), developmental delay still occurs in approximately 70% of patients. We aimed to investigate metabolic perturbations due to possible previously unidentified roles of antiquitin, which may contribute to developmental delay, as well as metabolic effects of high dose pyridoxine supplementation reflecting the high doses used for seizure control in patients with PDE-ALDH7A1. Untargeted metabolomics by high resolution mass spectrometry (HRMS) was used to analyze plasma of patients with PDE-ALDH7A1 and two independently generated lines of cultured ReNcell CX human neuronal progenitor cells (NPCs) with CRISPR/Cas mediated antiquitin deficiency. Accumulation of lysine pathway metabolites in antiquitin-deficient NPCs and western-blot analysis confirmed knockdown of ALDH7A1. Metabolomics analysis of antiquitin-deficient NPCs in conditions of lysine restriction and PN supplementation identified changes in metabolites related to the transmethylation and transsulfuration pathways and osmolytes, indicating a possible unrecognized role of antiquitin outside the lysine degradation pathway. Analysis of plasma samples of PN treated patients with PDE-ALDH7A1 and antiquitin-deficient NPCs cultured in conditions comparable to the patient plasma samples demonstrated perturbation of metabolites of the gamma-glutamyl cycle, suggesting potential oxidative stress-related effects in PN-treated patients with PDE-ALDH7A1. We postulate that a model of human NPCs with CRISPR/Cas mediated antiquitin deficiency is well suited to characterize previously unreported roles of antiquitin, relevant to this most prevalent form of pyridoxine-dependent epilepsy.},
}
@article {pmid36224758,
year = {2023},
author = {Mitra, S and Anand, U and Ghorai, M and Kant, N and Kumar, M and Radha, and Jha, NK and Swamy, MK and Proćków, J and de la Lastra, JMP and Dey, A},
title = {Genome editing technologies, mechanisms and improved production of therapeutic phytochemicals: Opportunities and prospects.},
journal = {Biotechnology and bioengineering},
volume = {120},
number = {1},
pages = {82-94},
doi = {10.1002/bit.28260},
pmid = {36224758},
issn = {1097-0290},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plants/genetics ; Metabolic Engineering ; Phytochemicals ; },
abstract = {Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.},
}
@article {pmid36224639,
year = {2022},
author = {Sorrentino, C and D'Antonio, L and Ciummo, SL and Fieni, C and Landuzzi, L and Ruzzi, F and Vespa, S and Lanuti, P and Lotti, LV and Lollini, PL and Di Carlo, E},
title = {CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality.},
journal = {Journal of hematology &amp; oncology},
volume = {15},
number = {1},
pages = {145},
pmid = {36224639},
issn = {1756-8722},
mesh = {Animals ; *B7-H1 Antigen/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Chemokine CXCL5/genetics/metabolism ; Cyclooxygenase 2/genetics/metabolism ; Humans ; Insulin-Like Growth Factor I ; Interleukins/metabolism ; Male ; Mice ; *Prostatic Neoplasms/genetics/pathology ; Proto-Oncogene Proteins c-bcl-2/genetics ; Receptors, Chemokine ; Suppressor of Cytokine Signaling 3 Protein/genetics ; Toll-Like Receptor 4/genetics/metabolism ; },
abstract = {BACKGROUND: Metastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30.<br><br>METHODS: PC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings.<br><br>RESULTS: Human membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30's ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test, p&#x2009;=&#x2009;0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30[Neg]SOCS3[Pos]PC, when compared to patients with IL30[Pos]SOCS3[Neg]PC.<br><br>CONCLUSIONS: Membrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use.},
}
@article {pmid36224386,
year = {2022},
author = {Hirano, S and Kappel, K and Altae-Tran, H and Faure, G and Wilkinson, ME and Kannan, S and Demircioglu, FE and Yan, R and Shiozaki, M and Yu, Z and Makarova, KS and Koonin, EV and Macrae, RK and Zhang, F},
title = {Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.},
journal = {Nature},
volume = {610},
number = {7932},
pages = {575-581},
pmid = {36224386},
issn = {1476-4687},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Deoxyribonuclease I/chemistry/metabolism/ultrastructure ; *DNA/chemistry/metabolism/ultrastructure ; *RNA, Guide/chemistry/metabolism/ultrastructure ; Cryoelectron Microscopy ; CRISPR-Associated Proteins/chemistry ; },
abstract = {RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies[1,2]. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development[3]. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain[4]. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.},
}
@article {pmid36224252,
year = {2022},
author = {Novakova, Z and Milosevic, M and Kutil, Z and Ondrakova, M and Havlinova, B and Kasparek, P and Sandoval-Acuña, C and Korandova, Z and Truksa, J and Vrbacky, M and Rohlena, J and Barinka, C},
title = {Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {17081},
pmid = {36224252},
issn = {2045-2322},
mesh = {Aminolevulinic Acid/metabolism ; CRISPR-Cas Systems ; Cell Line ; Heme ; Humans ; *Oxidoreductases/genetics/metabolism ; *Porphyria, Variegate/genetics ; Protoporphyrinogen Oxidase/genetics/metabolism ; Protoporphyrins ; },
abstract = {In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.},
}
@article {pmid36222238,
year = {2022},
author = {Coakley-Youngs, E and Ranatunga, M and Richardson, S and Getti, G and Shorter, S and Fivaz, M},
title = {Autism-associated CHD8 keeps proliferation of human neural progenitors in check by lengthening the G1 phase of the cell cycle.},
journal = {Biology open},
volume = {11},
number = {9},
pages = {},
pmid = {36222238},
issn = {2046-6390},
mesh = {*Autism Spectrum Disorder/genetics/metabolism ; *Autistic Disorder/genetics/metabolism ; Cell Cycle/genetics ; Cell Division ; Chromatin/metabolism ; Cyclins/genetics/metabolism ; DNA-Binding Proteins/genetics/metabolism ; G1 Phase ; Humans ; *Neural Stem Cells/metabolism ; Transcription Factors/metabolism ; },
abstract = {De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/- NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells - two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/- developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.},
}
@article {pmid36220450,
year = {2023},
author = {Zhang, H and Lin, Y and Zhuang, M and Zhu, L and Dai, Y and Lin, M},
title = {Screening and identification of CNIH4 gene associated with cell proliferation in gastric cancer based on a large-scale CRISPR-Cas9 screening database DepMap.},
journal = {Gene},
volume = {850},
number = {},
pages = {146961},
doi = {10.1016/j.gene.2022.146961},
pmid = {36220450},
issn = {1879-0038},
mesh = {Humans ; Cell Cycle ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; Early Detection of Cancer/methods ; *Receptors, Cytoplasmic and Nuclear/genetics ; *Stomach Neoplasms/diagnosis/drug therapy/genetics ; Databases, Genetic ; },
abstract = {BACKGROUND: Gastric cancer is a common malignant tumor of digestive tract. The study aimed to identify candidate genes associated with the proliferation and survival of gastric cancer cell through CRISPR-cas9 screening data, which may provide new therapeutic targets for gastric cancer patients.<br><br>METHODS: Candidate genes related to gastric cancer cell viability by CRISPR-cas9 screening from Depmap and genes differentially expressed between gastric cancer tissues and normal gastric tissues from TCGA were overlapped. WGCNA and KEGG analysis was conducted to performed to identify key pathways and genes. Using CMap, we identified small molecules that might reverse candidate gene expression of gastric cancer. LASSO regression was used to construct a signature to predict overall survival of gastric cancer patients. CCK8 assay was performed to assess the effects of candidate gene on gastric cancer cell proliferation.<br><br>RESULTS: A total of 710 candidate genes related to gastric cancer cell viability in the DepMap were identified and overlapped with differentially expressed genes in TCGA database, which were enriched in the cell cycle pathway. CMap analysis suggested that molecule drug LY294002 might be a novel choice for gastric cancer treatment. Using Cox univariate analysis and Lasso analysis, we developed a prognostic model including 12 candidate genes, and conducted subgroup analysis and external validation. Moreover, knockdown of the key candidate gene CNIH4 inhibited the proliferation of gastric cancer cells.<br><br>CONCLUSION: Cell cycle pathway and CNIH4, identified by CRISPR-cas9 screening, were a key pathway and gene that regulate cell viability in gastric cancer. CNIH4 has significant prognostic values and can serve as a new target for gastric cancer patient treatment.},
}
@article {pmid36220409,
year = {2022},
author = {Liu, Y and Wang, Y and Ma, L and Fu, R and Liu, H and Cui, Y and Zhao, Q and Zhang, Y and Jiao, B and He, Y},
title = {A CRISPR/Cas12a-based photothermal platform for the portable detection of citrus-associated Alternaria genes using a thermometer.},
journal = {International journal of biological macromolecules},
volume = {222},
number = {Pt B},
pages = {2661-2669},
doi = {10.1016/j.ijbiomac.2022.10.048},
pmid = {36220409},
issn = {1879-0003},
mesh = {*Alternaria/genetics ; *Citrus/microbiology ; Thermometers ; CRISPR-Cas Systems/genetics ; Plant Diseases/microbiology ; },
abstract = {The outbreak of citrus brown spot because of Alternaria is one of the most destructive citrus diseases. Additionally, Alternaria species produce highly toxic mycotoxins. Mass screening is a valid method to control the spread of Alternaria. Morphological analysis and polymerase chain reaction combined with gene-sequencing technique are the most commonly used techniques for detecting Alternaria. However, they are limited by either low convenience and accuracy or low instrument accessibility and high cost. To balance the convenience, accuracy, test availability, and low cost, we develop a CRISPR/Cas12a-based photothermal platform for the portable detection of Alternaria genes using a thermometer. Using this platform, the Alternaria genes from the synthetic sequences and cultured fungus of citrus, tomato, and apple can be detected using a thermometer with a detection limit of 1.5 pM. With the aid of the CRISPR/Cas12a system, citrus-associated Alternaria can be specifically differentiated from other citrus disease-associated microorganisms. When the photothermal platform is applied to analyze the citrus fruit samples collected in the field, good-consistency results are obtained with the gene-sequencing technology. The excellent performance of this portable method shows that it can be applied to screen for Alternaria in resource-poor settings.},
}
@article {pmid36220300,
year = {2022},
author = {Ma, W and Liu, M and Xie, S and Liu, B and Jiang, L and Zhang, X and Yuan, X},
title = {CRISPR/Cas12a system responsive DNA hydrogel for label-free detection of non-glucose targets with a portable personal glucose meter.},
journal = {Analytica chimica acta},
volume = {1231},
number = {},
pages = {340439},
doi = {10.1016/j.aca.2022.340439},
pmid = {36220300},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; Blood Glucose Self-Monitoring ; CRISPR-Cas Systems ; DNA/genetics ; DNA, Single-Stranded ; *Glucose/analysis ; Hydrogels ; beta-Fructofuranosidase/genetics ; },
abstract = {In this work, personal glucose meter (PGM) as a portable electrochemical device was utilized for sensitive detection of non-glucose targets: N-gene and PCB77, respectively. DNA hydrogel, which can respond to CRISPR/Cas system, was prepared for label-free encapsulating invertase. In the presence of targets, the repeated sequence for the activation of Cas12a was obtained due to the performance of RCA. Unlike "one-to-one" recognition, activated Cas12a can efficiently cleave multiple single-stranded linker DNAs on DNA hydrogels, thus releasing many invertase that can be used for PGM detection. With the amplification of RCA and CRISPR/Cas system, high detection sensitivity can be obtained even using portable PGM. The detection limits for N-gene and PCB77 were 2.6 fM and 3.2 × 10[-5] μg/L, respectively, with high specificity and good practical application performance. The developed biosensor can be used for online monitoring with the merit of low cost, easy operation and can be used for various targets analysis.},
}
@article {pmid36220290,
year = {2022},
author = {Chen, Y and Yang, T and Qian, S and Peng, C and Wang, X and Wang, T and Che, Y and Ji, F and Wu, J and Xu, J},
title = {Multiple crRNAs-assisted CRISPR/Cas12a assay targeting cytochrome b gene for amplification-free detection of meat adulteration.},
journal = {Analytica chimica acta},
volume = {1231},
number = {},
pages = {340417},
doi = {10.1016/j.aca.2022.340417},
pmid = {36220290},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems/genetics ; *Cytochromes b/genetics ; Food Contamination/analysis ; Meat/analysis ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have been widely applied in nucleic acid analysis for the high specificity. Coupled with pre-amplification steps, the sensitivity of CRISPR-based detection is greatly improved. However, an extra pre-amplification step not only complicates the detection procedures but may also cause aerosol contaminations in the process of transferring amplified solution into CRISPR system. In this study, we demonstrate that combination of multiple crRNAs in CRISPR/Cas12a system can enhance the detection sensitivity. Based on it, we establish a multiple crRNAs enhanced CRISPR (meCRISPR) method and apply it to meat adulteration identification. Take cytochrome b (Cyt b) gene as a target, meCRISPR method can directly detect as low as 1.13 ng/μL extracted pork DNA and 5% (w/w) pork contamination in pork and beef meat mixtures. There is no cross-reaction with extracted chicken, beef, duck and fish DNA. meCRISPR reaction is incubated at an isothermal temperature, and the detection process can be completed in a designed portable apparatus with a heat block, a light emitting diode and filters. For the simplicity, specificity and sufficient sensitivity of meCRISPR method, it will have great prospects in species identification, food adulteration, and genetically modified food detection.},
}
@article {pmid36219983,
year = {2022},
author = {Wu, S and Deng, H and Lu, B and Yuan, L and Wang, X},
title = {Generation of CD16A gene knockout human embryonic stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102935},
doi = {10.1016/j.scr.2022.102935},
pmid = {36219983},
issn = {1876-7753},
mesh = {Humans ; Gene Knockout Techniques ; *Human Embryonic Stem Cells/metabolism ; Receptors, IgG/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Immunoglobulin G/genetics/metabolism ; },
abstract = {CD16A is a receptor for the Fc portion of immunoglobulin G, and is involved in the antibody dependent cellular cytotoxicity (ADCC) of nature killer cells(Zhu et al., 2020) and antibody dependent enhancement (ADE) of virus infections(Wan et al., 2020). However, the role of CD16A in human embryonic stem cell modeled development has been merely documented. Hence, to illustrate the role of CD16A in the human cell development, we reported a CD16A knockout human embryonic stem(hESC) cell line via CRISPR/Cas9 mediated gene knockout. The CD16A mutated cell line displayed normal karyotype, pluripotent stem cell marker gene expression and differentiation potential.},
}
@article {pmid36219982,
year = {2022},
author = {Zhou, M and Huang, P and Bai, R and Liu, X},
title = {Generation of a homozygous TAZ knockout hESCs line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102923},
doi = {10.1016/j.scr.2022.102923},
pmid = {36219982},
issn = {1876-7753},
mesh = {Male ; Female ; Humans ; *Barth Syndrome/genetics/metabolism/pathology ; Cardiolipins/metabolism ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Transcription Factors/genetics/metabolism ; *Cardiomyopathies/genetics ; },
abstract = {Tafazzin (TAZ), a mitochondrial transacylase located on chromosome X, is required for the production of the mitochondrial phospholipid cardiolipin. Mutations occurring in the TAZ gene will lead to Barth syndrome, an X-linked recessive disease generally presenting as cardiomyopathy affecting males. Disease modeling strategies based on pluripotent stem cells (PSCs) provide an unprecedented and powerful platform to study Barth Syndrome. However, current studies were mostly based on male PSCs, the results and conclusions of which neglected the potential distinctions existing in disease phenotypes and mechanisms between gender. In this study, based on the H9 cell line (Female), we generated a homozygous TAZ knockout (TAZ-KO) human embryonic stem cell (hESC) line by employing CRISPR/Cas9 genome editing tools. This female TAZ-KO cell line, with normal karyotype, robust pluripotency and remarkably reduced TAZ expression, would be a useful tool for further deeply studying the pathogenesis of Barth syndrome cardiomyopathy in females.},
}
@article {pmid36219232,
year = {2022},
author = {Liu, M and Ma, W and Zhou, Y and Liu, B and Zhang, X and Zhang, S},
title = {A Label-Free Photoelectrochemical Biosensor Based on CRISPR/Cas12a System Responsive Deoxyribonucleic Acid Hydrogel and "Click" Chemistry.},
journal = {ACS sensors},
volume = {7},
number = {10},
pages = {3153-3160},
doi = {10.1021/acssensors.2c01636},
pmid = {36219232},
issn = {2379-3694},
mesh = {*Cadmium Compounds ; Hydrogels ; CRISPR-Cas Systems ; *Biosensing Techniques ; DNA/genetics ; },
abstract = {A novel label-free photoelectrochemical (PEC) biosensor is presented in this work. As a barrier, the DNA hydrogel could block the coupling between g-C3N4 and CdS quantum dots (QDs). Therefore, extremely low photocurrent signals were obtained. The presence of target microRNA-21 can initiate the rolling circle amplification (RCA) reaction, which in turn produces many repeated sequences to activate the CRISPR/Cas12a system. The trans-cleavage activity of the CRISPR/Cas12a system led to the degradation of DNA hydrogels efficiently. As a result, the g-C3N4/CdS QDs heterojunction was formed through "click" chemistry. Through the amplification of the RCA and CRISPR/Cas12a system, the sensitivity of the PEC biosensor was improved significantly with the detection limit of 3.2 aM. The proposed sensor also showed excellent selectivity and could be used to detect actual samples. In addition, the modular design could facilitate the detection of different objects. Thus, the proposed CRISPR/Cas12a system responsive DNA hydrogel provides a simple, sensitive, and flexible way for label-free PEC analysis.},
}
@article {pmid36218268,
year = {2022},
author = {Tian, Y and Zhong, D and Li, X and Shen, R and Han, H and Dai, Y and Yao, Q and Zhang, X and Deng, Q and Cao, X and Zhu, JK and Lu, Y},
title = {High-throughput genome editing in rice with a virus-based surrogate system.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13381},
pmid = {36218268},
issn = {1744-7909},
abstract = {With the widespread use of CRISPR/Cas technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called WDV-Gate (Wheat Dwarf Virus-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two sgRNAs targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in. This article is protected by copyright. All rights reserved.},
}
@article {pmid36218189,
year = {2022},
author = {Liu, J and Wu, D and Chen, J and Jia, S and Chen, J and Wu, Y and Li, G},
title = {CRISPR-Cas systems mediated biosensing and applications in food safety detection.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-26},
doi = {10.1080/10408398.2022.2128300},
pmid = {36218189},
issn = {1549-7852},
abstract = {Food safety, closely related to economic development of food industry and public health, has become a global concern and gained increasing attention worldwide. Effective detection technology is of great importance to guarantee food safety. Although several classical detection methods have been developed, they have some limitations in portability, selectivity, and sensitivity. The emerging CRISPR-Cas systems, uniquely integrating target recognition specificity, signal transduction, and efficient signal amplification abilities, possess superior specificity and sensitivity, showing huge potential to address aforementioned challenges and develop next-generation techniques for food safety detection. In this review, we focus on recent progress of CRISPR-Cas mediated biosensing and their applications in food safety monitoring. The properties and principles of commonly used CRISPR-Cas systems are highlighted. Notably, the frequently coupled nucleic acid amplification strategies to enhance their selectivity and sensitivity, especially isothermal amplification methods, as well as various signal output modes are also systematically summarized. Meanwhile, the application of CRISPR-Cas systems-based biosensors in food safety detection including foodborne virus, foodborne bacteria, food fraud, genetically modified organisms (GMOs), toxins, heavy metal ions, antibiotic residues, and pesticide residues is comprehensively described. Furthermore, the current challenges and future prospects in this field are tentatively discussed.},
}
@article {pmid36216550,
year = {2022},
author = {Yoshimura, Y and Nakamura, K and Seno, M and Mochizuki, M and Kawai, K and Koba, S and Watanabe, T},
title = {Generation of c-Fos knockout rats, and observation of their phenotype.},
journal = {Experimental animals},
volume = {},
number = {},
pages = {},
doi = {10.1538/expanim.22-0077},
pmid = {36216550},
issn = {1881-7122},
abstract = {c-Fos is a useful marker gene of neuron activation for neuroscience and physiology research. The mechanism and function of neural networks have been elucidated using c-Fos reporter knock-in (KI) mice, but the small size of the mice makes it difficult to perform surgical procedures on specific brain regions. On the other hand, there is a large amount of accumulated data on behavioral studies using rats. Thus, the generation of c-Fos reporter rat is expected, but it is difficult to generate gene-modified rats. Furthermore, c-Fos gene abnormality is expected to be severe in rats, as shown in homozygous of c-Fos knockout (KO) mouse, but such analysis has rarely been performed and is not certain. This study generated c-Fos-deficient rats using CRISPR/Cas, with 1067 bp deletion including exon 1 of the c-Fos gene. Homozygous c-Fos KO rats had growth latency and the same tooth and bone abnormality as homozygous c-Fos KO mice but not heterozygous c-Fos KO rats. Therefore, the c-Fos gene in rats is expected to have the same function as that in mice, and the generation of c-Fos reporter KI rats is further anticipated.},
}
@article {pmid36216296,
year = {2022},
author = {Guo, G and Li, MJ and Lai, JL and Du, ZY and Liao, QS},
title = {Development of tobacco rattle virus-based platform for dual heterologous gene expression and CRISPR/Cas reagent delivery.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {325},
number = {},
pages = {111491},
doi = {10.1016/j.plantsci.2022.111491},
pmid = {36216296},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems ; Indicators and Reagents/metabolism ; *Plant Viruses/genetics ; Tobacco/metabolism ; Recombinant Proteins/metabolism ; Gene Expression ; Genetic Vectors/genetics ; },
abstract = {A large number of viral delivery systems have been developed for characterizing functional genes and producing heterologous recombinant proteins in plants, and but most of them are unable to co-express two fusion-free foreign proteins in the whole plant for extended periods of time. In this study, we modified tobacco rattle virus (TRV) as a TRVe dual delivery vector, using the strategy of gene substitution. The reconstructed TRVe had the capability to simultaneously produce two fusion-free foreign proteins at the whole level of Nicotiana benthamiana, and maintained the genetic stability for the insert of double foreign genes. Moreover, TRVe allowed systemic expression of two foreign proteins with the total lengths up to ∼900 aa residues. In addition, Cas12a protein and crRNA were delivered by the TRVe expression system for site-directed editing of genomic DNA in N. benthamiana 16c line constitutively expressing green fluorescent protein (GFP). Taker together, the TRV-based delivery system will be a simple and powerful means to rapidly co-express two non-fused foreign proteins at the whole level and facilitate functional genomics studies in plants.},
}
@article {pmid36215034,
year = {2022},
author = {Patterson, A and White, A and Waymire, E and Fleck, S and Golden, S and Wilkinson, RA and Wiedenheft, B and Bothner, B},
title = {Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {11243-11254},
pmid = {36215034},
issn = {1362-4962},
support = {S10 OD028650/OD/NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; P20 GM103474/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; Allosteric Regulation ; Models, Molecular ; Proteins/genetics ; Thermodynamics ; RNA ; CRISPR-Cas Systems ; },
abstract = {CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here, we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.},
}
@article {pmid36214815,
year = {2022},
author = {Zhou, S and Dong, J and Deng, L and Wang, G and Yang, M and Wang, Y and Huo, D and Hou, C},
title = {Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation.},
journal = {ACS sensors},
volume = {7},
number = {10},
pages = {3032-3040},
doi = {10.1021/acssensors.2c01330},
pmid = {36214815},
issn = {2379-3694},
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Recombinases/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; DNA Methylation ; Bacterial Proteins/genetics ; DNA, Single-Stranded/genetics ; DNA/analysis ; Phosphates ; },
abstract = {DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5'-phosphate terminuses. After treating with Lambda, the sequence with 5'-phosphate modification will be degraded, leaving the single-stranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.},
}
@article {pmid36214677,
year = {2022},
author = {Opazo-Capurro, A and Morales-León, F and Jerez, C and Olivares-Pacheco, J and Alcalde-Rico, M and González-Muñoz, P and Bello-Toledo, H and Cardenas-Arias, A and Esposito, F and Lincopán, N and Illesca, V and González-Rocha, G},
title = {Isolation of an Extensively Drug-Resistant Pseudomonas aeruginosa exoS[+]/O4 Strain Belonging to the "High-Risk" Clone ST654 and Coproducer of NDM-1 and the Novel VIM-80.},
journal = {Microbiology spectrum},
volume = {10},
number = {5},
pages = {e0143922},
pmid = {36214677},
issn = {2165-0497},
mesh = {Humans ; *Pseudomonas aeruginosa/genetics ; Colistin ; *Pseudomonas Infections/epidemiology ; Microbial Sensitivity Tests ; Agar ; Anti-Bacterial Agents/pharmacology ; beta-Lactamases/genetics ; Clone Cells ; },
abstract = {The aim of this study was to investigate the genomic features of an extensively drug-resistant (XDR) Pseudomonas aeruginosa isolate (P-469) emerging in Chile. Antibiotic susceptibility was determined by disk diffusion and "colistin agar" test. Whole-genome sequencing (WGS) was performed by the Illumina NextSeq 2000 platform, and epidemiologically and clinically relevant data (i.e., sequence-type, serotype, mobile genetic elements, virulome, resistome, plasmidome, prophages, and CRISPR-Cas systems) were retrieved using multiple bioinformatic tools. The P-469 strain displayed an XDR profile, remaining susceptible to colistin. Genomic analysis revealed that this isolate belonged to the "high-risk" clone ST654 (CC654), serotype O4, and genotype exoS[+]. Strikingly, two CRISPR-Cas systems, five intact prophages sequences, and a broad resistome that included blaNDM-1 and the novel blaVIM-80 carbapenemase genes were predicted. Our results revealed the genomic characteristics of P. aeruginosa belonging to the high-risk clone ST654/O4 coproducing NDM-1 and VIM-80 in Chile, supporting that genomic surveillance is necessary to track the emergence and spread of epidemiologically successful WHO's critical priority pathogens in order to prevent their rapid dissemination.},
}
@article {pmid36212909,
year = {2022},
author = {García, M and Bonafont, J and Martínez-Palacios, J and Xu, R and Turchiano, G and Svensson, S and Thrasher, AJ and Larcher, F and Del Rio, M and Hernández-Alcoceba, R and Garín, MI and Mencía, &#xc1; and Murillas, R},
title = {Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {27},
number = {},
pages = {96-108},
pmid = {36212909},
issn = {2329-0501},
abstract = {Recessive dystrophic epidermolysis bullosa, a devastating skin fragility disease characterized by recurrent skin blistering, scarring, and a high risk of developing squamous cell carcinoma is caused by mutations in COL7A1, the gene encoding type VII collagen, which is the major component of the anchoring fibrils that bind the dermis and epidermis. Ex vivo correction of COL7A1 by gene editing in patients' cells has been achieved before. However, in vivo editing approaches are necessary to address the direct treatment of the blistering lesions characteristic of this disease. We have now generated adenoviral vectors for CRISPR-Cas9 delivery to remove exon 80 of COL7A1, which contains a highly prevalent frameshift mutation in Spanish patients. For in vivo testing, a humanized skin mouse model was used. Efficient viral transduction of skin was observed after excisional wounds generated with a surgical punch on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. Type VII collagen deposition in the basement membrane zone of the wounded areas treated with the vectors correlated with restoration of dermal-epidermal adhesion, demonstrating that recessive dystrophic epidermolysis bullosa (RDEB) patient skin lesions can be directly treated by CRISPR-Cas9 delivery in vivo.},
}
@article {pmid36212739,
year = {2022},
author = {Liu, S and Xie, T and Huang, Z and Pei, X and Li, S and He, Y and Tong, Y and Liu, G},
title = {Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection.},
journal = {Sensors and actuators. B, Chemical},
volume = {373},
number = {},
pages = {132746},
pmid = {36212739},
issn = {0925-4005},
abstract = {The CRISPR/Cas system is widely used for molecular diagnostics after the discovery of trans-cleavage activity, especially now with the COVID-19 outbreak. However, the majority of contemporary trans-cleavage activity-based CRISPR/Cas biosensors exploited standard single-strand DNA (ssDNA) reporters, which were based on the FRET principle from pioneering research. An in-depth comparison and understanding of various fluorescent readout types are essential to facilitate the outstanding analytical performance of CRISPR probes. We investigated various types of fluorescent reporters of Cas12a comprehensively. Results show that trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MB). And MB reporters can achieve superior analytical performance compared with ssDNA and ds DNA reporters at the same conditions. Accordingly, we developed a highly-sensitive SARS-CoV-2 detection with the sensitivity as low as 100 fM were successfully achieved without amplification strategy. The model target of ORF1a could robustly identify the current widespread emerging SARS-CoV-2 variants. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. And a minimum of 27 copies/mL was achieved successfully. This inspiration can also be applied to other Cas proteins with trans-cleavage activity, which provides new perspectives for simple, highly-sensitive and universal molecular diagnosis in various applications.},
}
@article {pmid36209895,
year = {2022},
author = {Zhang, B and Li, M and Wei, Y and Wang, J and Wang, Y and Shi, P and Tang, H and Song, Z},
title = {Detection of target DNA with a visual CRISPR-associated hyperbranched rolling circle amplification technique.},
journal = {Analytical biochemistry},
volume = {658},
number = {},
pages = {114940},
doi = {10.1016/j.ab.2022.114940},
pmid = {36209895},
issn = {1096-0309},
mesh = {Humans ; *Nucleic Acid Amplification Techniques/methods ; *DNA/genetics ; DNA, Circular/genetics ; Papillomaviridae ; CRISPR-Cas Systems/genetics ; },
abstract = {This paper presents a novel clustered regularly interspaced short palindromic repeat (CRISPR)-associated HRCA technique (CART). During the entire detection process of CART, the target DNA is first specifically recognized and cleaved by a pair of Cas9/sgRNA complexes; then, the cleaved product is ligated into circular DNA as the template of HRCA, and the circular DNA is efficiently amplified by HRCA. Therefore, CART has the advantages of Cas9/sgRNA (single-base mismatch specificity) and HRCA (isothermal reaction temperature and high sensitivity). This technique has been verified by detecting various human papillomavirus (HPV) genes with numerous subtypes. In summary, this study provides a new and effective method for the detection of nucleic acids.},
}
@article {pmid36209819,
year = {2022},
author = {Gao, Z and Zhang, L and Ge, Z and Wang, H and Yue, Y and Jiang, Z and Wang, X and Xu, C and Zhang, Y and Yang, M and Feng, Y},
title = {Anti-CRISPR protein AcrIF4 inhibits the type I-F CRISPR-Cas surveillance complex by blocking nuclease recruitment and DNA cleavage.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {11},
pages = {102575},
pmid = {36209819},
issn = {1083-351X},
mesh = {*CRISPR-Associated Proteins/metabolism ; DNA Cleavage ; CRISPR-Cas Systems ; Pseudomonas aeruginosa/metabolism ; *Bacteriophages/metabolism ; Endonucleases/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti-CRISPR (Acr) proteins to evade this immunity. AcrIF4, an Acr targeting the type I-F CRISPR-Cas system, has been reported to bind the crRNA-guided surveillance (Csy) complex. However, it remains controversial whether AcrIF4 inhibits target DNA binding to the Csy complex. Here, we present structural and mechanistic studies into AcrIF4, exploring its unique anti-CRISPR mechanism. While the Csy-AcrIF4 complex displays decreased affinity for target DNA, it is still able to bind the DNA. Our structural and functional analyses of the Csy-AcrIF4-dsDNA complex revealed that AcrIF4 binding prevents rotation of the helical bundle of the Cas8f subunit induced by dsDNA binding, therefore resulting in failure of nuclease Cas2/3 recruitment and DNA cleavage. Overall, our study provides an interesting example of attack on the nuclease recruitment event by an Acr, but not conventional mechanisms of blocking binding of target DNA.},
}
@article {pmid36208845,
year = {2022},
author = {Wu, S and Tian, P and Tan, T},
title = {CRISPR-Cas13 technology portfolio and alliance with other genetic tools.},
journal = {Biotechnology advances},
volume = {61},
number = {},
pages = {108047},
doi = {10.1016/j.biotechadv.2022.108047},
pmid = {36208845},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA/genetics ; Synthetic Biology ; Technology ; },
abstract = {The exploitation of CRISPR-Cas systems especially CRISPR-Cas9 has led to radical advances in genome editing, gene activation, gene repression, protein imaging, and beyond. However, these applications are limited to targeting of DNA rather than RNA. CRISPR-Cas13 is the first reported CRISPR system targeting RNA and thus opens a new avenue for transcription regulation. While a plethora of reviews have systematically documented CRISPR-Cas9 toolbox, this review focuses on CRISPR-Cas13 family, covering aspects of classification, structures, response to foreign invaders, and genetic toolbox. In particular, we compare CRISPR-Cas13 with other RNA regulation tools such as RNA interference and antisense RNA technology to ponder the possibility of combining them to engineer hierarchical regulatory networks fulfilling novel functions. Lastly, we summarize the wide applications of CRISPR-Cas13 toolbox and the status quo that requires amelioration. Overall, this review charts a landscape of CRISPR-Cas13 technology portfolio to provide insights for gene regulation, metabolic engineering and synthetic biology.},
}
@article {pmid36208239,
year = {2022},
author = {Li, QC and Huang, JB and Xue, HM and Yang, M and Zhu, CM and Li, CK and Dong, JC and Chen, C},
title = {[Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {5},
pages = {1384-1390},
doi = {10.19746/j.cnki.issn.1009-2137.2022.05.013},
pmid = {36208239},
issn = {1009-2137},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Plasmids ; },
abstract = {OBJECTIVE: In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction.<br><br>METHODS: Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-&#x394;cas9, LentiCRISPRV2GFP-&#x394;cas9-AF4, LentiCRISPRV2GFP-&#x394; cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus.<br><br>RESULTS: Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning.<br><br>CONCLUSION: Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.},
}
@article {pmid36207827,
year = {2022},
author = {Sun, P and Zou, W},
title = {Research progress of live-cell RNA imaging techniques.},
journal = {Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences},
volume = {51},
number = {3},
pages = {362-372},
pmid = {36207827},
issn = {1008-9292},
mesh = {*Fluorescent Dyes/chemistry ; *RNA/chemistry/genetics/metabolism ; },
abstract = {RNA molecules play diverse roles in many physiological and pathological processes as they interact with various nucleic acids and proteins. The various biological processes of RNA are highly dynamic. Tracking RNA dynamics in living cells is crucial for a better understanding of the spatiotemporal control of gene expression and the regulatory roles of RNA. Genetically encoded RNA-tagging systems include MS2/MCP, PP7/PCP, boxB/λN22 and CRISPR-Cas. The MS2/MCP system is the most widely applied, and it has the advantages of stable binding and high signal-to-noise ratio, while the realization of RNA imaging requires gene editing of the target RNA, which may change the characteristics of the target RNA. Recently developed CRISPR-dCas13 system does not require RNA modification, but the uncertainty in CRISPR RNA (crRNA) efficiency and low signal-to-noise ratio are its limitations. Fluorescent dye-based RNA-tagging systems include molecular beacons and fluorophore-binding aptamers. The molecular beacons have high specificity and high signal-to-noise ratio; Mango and Peppers outperform the other RNA-tagging system in signal-to-noise, but they also need gene editing. Live-cell RNA imaging allows us to visualize critical steps of RNA activities, including transcription, splicing, transport, translation (for message RNA only) and subcellular localization. It will contribute to studying biological processes such as cell differentiation and the transcriptional regulation mechanism when cells adapt to the external environment, and it improves our understanding of the pathogenic mechanism of various diseases caused by abnormal RNA behavior and helps to find potential therapeutic targets. This review provides an overview of current progress of live-cell RNA imaging techniques and highlights their major strengths and limitations.},
}
@article {pmid36207756,
year = {2022},
author = {Park, HM and Park, Y and Berani, U and Bang, E and Vankerschaver, J and Van Messem, A and De Neve, W and Shim, H},
title = {In silico optimization of RNA-protein interactions for CRISPR-Cas13-based antimicrobials.},
journal = {Biology direct},
volume = {17},
number = {1},
pages = {27},
pmid = {36207756},
issn = {1745-6150},
mesh = {Anti-Bacterial Agents ; Bacteria/genetics ; *CRISPR-Cas Systems ; Humans ; *RNA, Bacterial ; Ribonucleases/genetics/metabolism ; },
abstract = {RNA-protein interactions are crucial for diverse biological processes. In prokaryotes, RNA-protein interactions enable adaptive immunity through CRISPR-Cas systems. These defence systems utilize CRISPR RNA (crRNA) templates acquired from past infections to destroy foreign genetic elements through crRNA-mediated nuclease activities of Cas proteins. Thanks to the programmability and specificity of CRISPR-Cas systems, CRISPR-based antimicrobials have the potential to be repurposed as new types of antibiotics. Unlike traditional antibiotics, these CRISPR-based antimicrobials can be designed to target specific bacteria and minimize detrimental effects on the human microbiome during antibacterial therapy. In this study, we explore the potential of CRISPR-based antimicrobials by optimizing the RNA-protein interactions of crRNAs and Cas13 proteins. CRISPR-Cas13 systems are unique as they degrade specific foreign RNAs using the crRNA template, which leads to non-specific RNase activities and cell cycle arrest. We show that a high proportion of the Cas13 systems have no colocalized CRISPR arrays, and the lack of direct association between crRNAs and Cas proteins may result in suboptimal RNA-protein interactions in the current tools. Here, we investigate the RNA-protein interactions of the Cas13-based systems by curating the validation dataset of Cas13 protein and CRISPR repeat pairs that are experimentally validated to interact, and the candidate dataset of CRISPR repeats that reside on the same genome as the currently known Cas13 proteins. To find optimal CRISPR-Cas13 interactions, we first validate the 3-D structure prediction of crRNAs based on their experimental structures. Next, we test a number of RNA-protein interaction programs to optimize the in silico docking of crRNAs with the Cas13 proteins. From this optimized pipeline, we find a number of candidate crRNAs that have comparable or better in silico docking with the Cas13 proteins of the current tools. This study fully automatizes the in silico optimization of RNA-protein interactions as an efficient preliminary step for designing effective CRISPR-Cas13-based antimicrobials.},
}
@article {pmid36207463,
year = {2022},
author = {Sapozhnikov, DM and Szyf, M},
title = {Enzyme-free targeted DNA demethylation using CRISPR-dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression.},
journal = {Nature protocols},
volume = {17},
number = {12},
pages = {2840-2881},
pmid = {36207463},
issn = {1750-2799},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *RNA, Guide/genetics ; DNA Methylation ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; DNA Demethylation ; Gene Expression ; },
abstract = {DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally confounded DNA demethylation of specific sites in the genome of cultured cells by clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and without the involvement of an epigenetic-modifying enzyme, the purpose of which is the evaluation of the functional (i.e., gene expression or phenotypic) consequences of DNA demethylation of specific sites that have been previously implicated in particular pathological or physiological contexts. This protocol maximizes the ability of the easily reprogrammable CRISPR-dCas9 system to assess the impact of DNA methylation from a causal rather than correlational perspective: alternative protocols for CRISPR-dCas9-based site-specific DNA methylation or demethylation rely on the recruitment of epigenetic enzymes that exhibit additional nonspecific activities at both the targeted site and throughout the genome, confounding conclusions of causality of DNA methylation. Inhibition or loss of DNA methylation is accomplished by three consecutive lentiviral transductions. The first two lentiviruses establish stable expression of dCas9 and a guide RNA, which will physically obstruct either maintenance or de novo DNA methyltransferase activity at the guide RNA target site. A third lentivirus introduces Cre recombinase to delete the dCas9 transgene, which leads to loss of dCas9 from the target site, allowing transcription factors and/or the transcription machinery to interact with the demethylated target site. This protocol requires 3-8 months to complete owing to prolonged cell passaging times, but there is little hands-on time, and no specific skills beyond basic molecular biology techniques are necessary.},
}
@article {pmid36207338,
year = {2022},
author = {Levesque, S and Mayorga, D and Fiset, JP and Goupil, C and Duringer, A and Loiselle, A and Bouchard, E and Agudelo, D and Doyon, Y},
title = {Marker-free co-selection for successive rounds of prime editing in human cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5909},
pmid = {36207338},
issn = {2041-1723},
support = {//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; Gene Editing ; Humans ; Sodium ; *Sodium-Potassium-Exchanging ATPase/genetics ; },
abstract = {Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na[+]/K[+] ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.},
}
@article {pmid36206765,
year = {2022},
author = {Deep, A and Gu, Y and Gao, YQ and Ego, KM and Herzik, MA and Zhou, H and Corbett, KD},
title = {The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.},
journal = {Molecular cell},
volume = {82},
number = {21},
pages = {4145-4159.e7},
pmid = {36206765},
issn = {1097-4164},
support = {R01 GM116897/GM/NIGMS NIH HHS/United States ; R35 GM138206/GM/NIGMS NIH HHS/United States ; R35 GM144121/GM/NIGMS NIH HHS/United States ; S10 OD023498/OD/NIH HHS/United States ; },
mesh = {*DNA, Circular ; *Multiprotein Complexes/genetics/chemistry ; Cryoelectron Microscopy ; DNA-Binding Proteins/metabolism ; Chromosomes/metabolism ; Plasmids/genetics ; DNA/genetics ; Bacteria/genetics ; },
abstract = {Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.},
}
@article {pmid36206730,
year = {2022},
author = {Ferrari, S and Jacob, A and Cesana, D and Laugel, M and Beretta, S and Varesi, A and Unali, G and Conti, A and Canarutto, D and Albano, L and Calabria, A and Vavassori, V and Cipriani, C and Castiello, MC and Esposito, S and Brombin, C and Cugnata, F and Adjali, O and Ayuso, E and Merelli, I and Villa, A and Di Micco, R and Kajaste-Rudnitski, A and Montini, E and Penaud-Budloo, M and Naldini, L},
title = {Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells.},
journal = {Cell stem cell},
volume = {29},
number = {10},
pages = {1428-1444.e9},
pmid = {36206730},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; DNA Damage ; *DNA, Viral ; Gene Editing ; Hematopoietic Stem Cells ; Humans ; Integrases ; *Tumor Suppressor Protein p53/genetics ; },
abstract = {Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.},
}
@article {pmid36206729,
year = {2022},
author = {Bauer, DE},
title = {Pervasive donor DNA integration defies precision gene editing of hematopoietic stem cells.},
journal = {Cell stem cell},
volume = {29},
number = {10},
pages = {1426-1427},
doi = {10.1016/j.stem.2022.09.003},
pmid = {36206729},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; DNA ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *Gene Editing ; Hematopoietic Stem Cells ; Recombinational DNA Repair ; },
abstract = {Therapeutic gene editing based on homology-directed repair (HDR) promises to precisely recombine donor template sequences at programmed double-strand break sites. In this issue, Ferrari et al. report that commonly used AAV donors blunt hematopoietic repopulation and result in frequent genomic integrations that could interfere with normal gene expression.},
}
@article {pmid36206672,
year = {2022},
author = {You, J and Ma, S and Zhang, H and Chen, L},
title = {Generation of a homozygous P4HA2 knockout human embryonic stem cell line (FDCHDPe012-A) by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102930},
doi = {10.1016/j.scr.2022.102930},
pmid = {36206672},
issn = {1876-7753},
mesh = {Humans ; Cell Line ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; *Myopia/genetics/metabolism ; *Prolyl Hydroxylases/genetics/metabolism ; },
abstract = {Prolyl 4-hydroxylase subunit alpha-2(P4HA2) is associated with autosomal dominant high myopia. A significant reduction of P4HA2 protein expression has been observed in fibroblast cells of high myopia patients with inherited P4HA2 mutations. To explore the function of P4HA2 in cases of high myopia, we generated a P4HA2-KO hES cell line (FDCHDPe012-A) efficiently through CRISPR/Cas9 system. We confirmed the presence of a 5-bp biallelic deletion, causing a frameshift and premature P4HA2 translation termination. The FDCHDPe012-A presented morphology of typical stem cells expressed pluripotent genes, possessed a normal parental karyotype, and could differentiate into three germ layers.},
}
@article {pmid36206523,
year = {2022},
author = {Presnell, KV and Melhem, O and Morse, NJ and Alper, HS},
title = {Modular, Synthetic Boolean Logic Gates Enabled in Saccharomyces cerevisiae through T7 Polymerases/CRISPR dCas9 Designs.},
journal = {ACS synthetic biology},
volume = {11},
number = {10},
pages = {3414-3425},
doi = {10.1021/acssynbio.2c00327},
pmid = {36206523},
issn = {2161-5063},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Saccharomyces cerevisiae/genetics ; Galactose ; Copper ; Synthetic Biology ; CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic control of gene expression, whether simply promoter selection or higher-order Boolean-style logic, is an important tool for metabolic engineering and synthetic biology. This work develops a suite of orthogonal T7 RNA polymerase systems capable of exerting AND/OR switchlike control over transcription in the yeastSaccharomyces cerevisiae. When linked with CRISPR dCas9-based regulation systems, more complex circuitry is possible including AND/OR/NAND/NOR style control in response to combinations of extracellular copper and galactose. Additionally, we demonstrate that these T7 system designs are modular and can accommodate alternative stimuli sensing as demonstrated through blue light induction. These designs should greatly reduce the time and labor necessary for developing Boolean gene circuits in yeast with novel applications including metabolic pathway control in the future.},
}
@article {pmid36206316,
year = {2022},
author = {Gao, H and Feng, M and Li, F and Zhang, K and Zhang, T and Zhang, Z and Yang, C and Deng, R and Zhang, J and Jiang, P},
title = {G-Quadruplex DNAzyme-Substrated CRISPR/Cas12 Assay for Label-Free Detection of Single-Celled Parasitic Infection.},
journal = {ACS sensors},
volume = {7},
number = {10},
pages = {2968-2977},
doi = {10.1021/acssensors.2c01104},
pmid = {36206316},
issn = {2379-3694},
mesh = {Mice ; Animals ; *DNA, Catalytic/genetics ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *G-Quadruplexes ; *Parasitic Diseases/genetics ; },
abstract = {Early diagnosis of parasitic diseases can dramatically alleviate medical, economic, and social burdens. Herein, we report a sensitive and label-free assay for diagnosing single-celled parasitic infections using G-quadruplex (G4) DNAzyme as a reporter for CRISPR/Cas12. The substitution of a fluorescent DNA reporter with G4 DNAzyme increased the sensitivity for detecting Leishmania donovani (L. donovani) by 5 times and obviated the need for using chemically labeled DNA probes. The G4 DNAzyme-substrated CRISPR/Cas12 (GsubCas12) assay yielded a limit of detection of 3.1 parasites in the detection of cultured L. donovani and was further applied to analyze L. donovani in infected mice. The results showed that the GsubCas12 assay could positively detect L. donovani in spleen samples from infected mice about 2 weeks after low-dose inoculation, nearly 2 weeks earlier than that of parasitological analysis. GsubCas12 assay is promising as a diagnostic tool for parasitic infection in resource-limited regions.},
}
@article {pmid36206027,
year = {2022},
author = {Pelea, O and Fulga, TA and Sauka-Spengler, T},
title = {RNA-Responsive gRNAs for Controlling CRISPR Activity: Current Advances, Future Directions, and Potential Applications.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {642-659},
pmid = {36206027},
issn = {2573-1602},
mesh = {*RNA, Guide/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA ; Streptococcus pyogenes/genetics ; },
abstract = {CRISPR-Cas9 has emerged as a major genome manipulation tool. As Cas9 can cause off-target effects, several methods for controlling the expression of CRISPR systems were developed. Recent studies have shown that CRISPR activity could be controlled by sensing expression levels of endogenous transcripts. This is particularly interesting, as endogenous RNAs could harbor important information about the cell type, disease state, and environmental challenges cells are facing. Single-guide RNA (sgRNA) engineering played a major role in the development of RNA-responsive CRISPR systems. Following further optimizations, RNA-responsive sgRNAs could enable the development of novel therapeutic and research applications. This review introduces engineering strategies that could be employed to modify Streptococcus pyogenes sgRNAs with a focus on recent advances made toward the development of RNA-responsive sgRNAs. Future directions and potential applications of these technologies are also discussed.},
}
@article {pmid36206022,
year = {2022},
author = {Hatoum-Aslan, A},
title = {A Protein-Cutting CRISPR Complex Caught in Action.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {631-633},
doi = {10.1089/crispr.2022.29154.aha},
pmid = {36206022},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; RNA, Guide ; },
}
@article {pmid36206017,
year = {2022},
author = {Cui, N and Faure, G and Singh, A and Macrae, R and Zhang, F},
title = {Microfluidic Enrichment and Computational Analysis of Rare Sequences from Mixed Genomic Samples for Metagenomic Mining.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {677-684},
pmid = {36206017},
issn = {2573-1602},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Microfluidics ; Metagenome/genetics ; Genomics ; },
abstract = {Many powerful molecular biology tools have their origins in natural systems, including restriction modification enzymes and the CRISPR effectors, Cas9, Cas12, and Cas13. Heightened interest in these systems has led to mining of genomic and metagenomic data to identify new orthologs of these proteins, new types of CRISPR systems, and uncharacterized natural systems with novel mechanisms. To accelerate metagenomic mining, we developed a high-throughput, low-cost droplet microfluidic-based method for enrichment of rare sequences in a mixed starting population. Using a computational pipeline, we then searched in the enriched data for the presence of CRISPR-Cas systems, identifying a previously unknown CRISPR-Cas system. Our approach enables researchers to efficiently mine metagenomic samples for sequences of interest, greatly accelerating the search for nature's treasures.},
}
@article {pmid36205282,
year = {2022},
author = {Khan, SH},
title = {Cancer: Can CRISPR Become a Game Changer?.},
journal = {Journal of the College of Physicians and Surgeons--Pakistan : JCPSP},
volume = {32},
number = {10},
pages = {1339-1340},
doi = {10.29271/jcpsp.2022.10.1339},
pmid = {36205282},
issn = {1681-7168},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Humans ; Mutation ; *Neoplasms/genetics/therapy ; },
abstract = {Recent development in CRISPR technologies has emerged as a powerful tool for genome editing, which can become a powerful diagnostic and therapeutic option for cancer patients. While literature search demonstrates some successes in this dimension in animal models and phase-I human trials, still the promised panacea has shortcomings in terms of off-target mutations (OTMs), minimal efficiency, and tailoring individualised interventions. While there is a promise for the future, although the CRISPR technology still has to climb up the ladder in terms of improvements with more enhanced efficiency tools for delivering the payload into cell and reducing the off-target mutations. Key Words: CRISPR, Oncology, Off-target mutations (OTMs), Genome editing, 3-D cell cultures.},
}
@article {pmid36205232,
year = {2022},
author = {Woodcraft, C and Chooi, YH and Roux, I},
title = {The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi.},
journal = {Natural product reports},
volume = {},
number = {},
pages = {},
doi = {10.1039/d2np00055e},
pmid = {36205232},
issn = {1460-4752},
abstract = {Covering: up to May 2022Fungal genetics has transformed natural product research by enabling the elucidation of cryptic metabolites and biosynthetic steps. The enhanced capability to add, subtract, modulate, and rewrite genes via CRISPR/Cas technologies has opened up avenues for the manipulation of biosynthetic gene clusters across diverse filamentous fungi. This review discusses the innovative and diverse strategies for fungal natural product discovery and engineering made possible by CRISPR/Cas-based tools. We also provide a guide into multiple angles of CRISPR/Cas experiment design, and discuss current gaps in genetic tool development for filamentous fungi and the promising opportunities for natural product research.},
}
@article {pmid36203248,
year = {2022},
author = {Sánchez-Popoca, D and Serrano-Fujarte, I and Fernández-Mora, M and Calva, E},
title = {The LeuO regulator and quiescence: About transcriptional roadblocks, multiple promoters, and crispr-cas.},
journal = {Molecular microbiology},
volume = {118},
number = {5},
pages = {503-509},
doi = {10.1111/mmi.14990},
pmid = {36203248},
issn = {1365-2958},
mesh = {*Gene Expression Regulation, Bacterial/genetics ; Transcription Factors/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics/metabolism ; },
abstract = {LeuO is a LysR-type transcriptional regulator in bacteria. It determines the regulation of numerous genes related to stress response and virulence. Thus, four exciting areas of research are discussed herein. One pertains the leuO gene, which in S. Typhi and in E. coli contains multiple forward promoters as well as reverse promoters, even though it is expressed at very low levels, that is, it is quiescent. Such multiplicity might allow for a greater plasticity in regulation, or even aid in maintaining the quiescence, in processes that appear to involve many nucleoid-associated proteins in a second area of opportunity. A third one relates to the effector-binding domain of the LeuO regulator, which is highly conserved in S. enterica and in E. coli and determines its activity as a regulator of transcription. A fourth area regards the role of the CRISPR-Cas system in gene regulation in S. Typhi; a system that is regulated by LeuO.},
}
@article {pmid36201634,
year = {2022},
author = {Huang, C and Han, Z and Evangelopoulos, M and Mirkin, CA},
title = {CRISPR Spherical Nucleic Acids.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {41},
pages = {18756-18760},
doi = {10.1021/jacs.2c07913},
pmid = {36201634},
issn = {1520-5126},
support = {U54 CA199091/CA/NCI NIH HHS/United States ; },
mesh = {*RNA, Guide/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Nuclear Localization Signals/genetics/metabolism ; *Nucleic Acids ; Peptide Hydrolases/metabolism ; },
abstract = {The use of CRISPR/Cas9 systems in genome editing has been limited by the inability to efficiently deliver the key editing components to and across tissues and cell membranes, respectively. Spherical nucleic acids (SNAs) are nanostructures that provide privileged access to both but have yet to be explored as a means of facilitating gene editing. Herein, a new class of CRISPR SNAs are designed and evaluated in the context of genome editing. Specifically, Cas9 ProSNAs comprised of Cas9 cores densely modified with DNA on their exteriors and preloaded with single-guide RNA were synthesized and evaluated for their genome editing capabilities in the context of multiple cell lines. The radial orientation of the DNA on the Cas9 protein surface enhances cellular uptake, without the need for electroporation or transfection agents. In addition, the Cas9 proteins defining the cores of the ProSNAs were fused with GALA peptides on their N-termini and nuclear localization signals on their C-termini to facilitate endosomal escape and maximize nuclear localization and editing efficiency, respectively. These constructs were stable against protease digestion under conditions that fully degrade the Cas9 protein, when not transformed into an SNA, and used to achieve genome editing efficiency between 32 and 47%. Taken together, these novel constructs and advances point toward a way of significantly broadening the scope of use and impact of CRISPR-Cas9 genome editing systems.},
}
@article {pmid36201342,
year = {2022},
author = {King, S and Quick, A and King, K and Walker, AR and Shields, RC},
title = {Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans.},
journal = {Microbiology (Reading, England)},
volume = {168},
number = {10},
pages = {},
doi = {10.1099/mic.0.001254},
pmid = {36201342},
issn = {1465-2080},
mesh = {Amino Acids/metabolism ; Bacterial Proteins/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; Mitomycin ; *Streptococcus mutans/metabolism ; },
abstract = {Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.},
}
@article {pmid36201269,
year = {2022},
author = {Dilnawaz, F and Acharya, S},
title = {Nanoparticle-based CRISPR/Cas delivery: An emerging tactic for cancer therapy.},
journal = {Current medicinal chemistry},
volume = {},
number = {},
pages = {},
doi = {10.2174/0929867329666221006112615},
pmid = {36201269},
issn = {1875-533X},
abstract = {Genome editing arose as a new promising approach for the treatment of innumerable intricate ailments including cancer. Over the past couple of decades, delivery technologies that have serendipitously been developed using viral vectors are successful to some extent in protein and nucleic acid delivery but their effectiveness still lags due to their efficiency, tissue targeting capabilities, and toxicity which must be further improved. With the infiltration of nanotechnology into every sphere of life, nano-vehicles can be implemented as an ideal modality that can overcome challenges, also can be introspective as new genome editing tools for cancer therapy owing to the safety and efficiency in clinical settings. Such projected substitution can help in developing highly efficacious therapy regimes which are successful in clinical settings. This emerging approach of incorporation of genome editors (CRISPR/Cas) in different nano vehicles and their utility in targeting various aspects of cancer therapy like treatment, diagnostics, modelling has been comprehensively done in this review.},
}
@article {pmid36199433,
year = {2022},
author = {Rahman, MR and Majumder, TR and Apu, MAI and Paul, AK and Afrose, A and Dash, BK},
title = {CRISPR-Based Programmable Nucleic Acid-Binding Protein Technology Can Specifically Detect Fatal Tropical Disease-Causing Pathogens.},
journal = {Journal of tropical medicine},
volume = {2022},
number = {},
pages = {5390685},
pmid = {36199433},
issn = {1687-9686},
abstract = {Diagnostic approaches capable of ultrasensitive pathogen detection from low-volume clinical samples, running without any sophisticated instrument and laboratory setup, are easily field-deployable, inexpensive, and rapid, and are considered ideal for monitoring disease progression and surveillance. However, standard pathogen detection methods, including culture and microscopic observation, antibody-based serologic tests, and primarily polymerase chain reaction (PCR)-oriented nucleic acid screening techniques, have shortcomings that limit their widespread use in responding to outbreaks and regular diagnosis, especially in remote resource-limited settings (RLSs). Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based programmable technology has emerged to challenge the unmet criteria of conventional methods. It consists of CRISPR-associated proteins (Cas) capable of targeting virtually any specific RNA or DNA genome based on the guide RNA (gRNA) sequence. Furthermore, the discovery of programmable trans-cleavage Cas proteins like Cas12a and Cas13 that can collaterally damage reporter-containing single-stranded DNA or RNA upon formation of target Cas-gRNA complex has strengthened this technology with enhanced sensitivity. Current advances, including automated multiplexing, ultrasensitive single nucleotide polymorphism (SNP)-based screening, inexpensive paper-based lateral flow readouts, and ease of use in remote global settings, have attracted the scientific community to introduce this technology in nucleic acid-based precise detection of bacterial and viral pathogens at the point of care (POC). This review highlights CRISPR-Cas-based molecular technologies in diagnosing several tropical diseases, namely malaria, zika, chikungunya, human immunodeficiency virus and acquired immunodeficiency syndrome (HIV-AIDS), tuberculosis (TB), and rabies.},
}
@article {pmid36199268,
year = {2022},
author = {Guo, Y and Guo, L and Su, Y and Xiong, Y},
title = {CRISPR-Cas system manipulating nanoparticles signal transduction for cancer diagnosis.},
journal = {Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology},
volume = {},
number = {},
pages = {e1851},
doi = {10.1002/wnan.1851},
pmid = {36199268},
issn = {1939-0041},
abstract = {Early diagnosis of cancer is important to improve the survival rate and relieve patient pain. Sensitive detection of cancer related biomarkers in body fluids is a critical approach for the early diagnosis of cancer. The clustered regularly interspaced short palindromic repeat-associated protein (CRISPR-Cas) system has emerged as a molecular manipulation technology because of its simple detection procedure, high base resolution, and isothermal signal amplification. Recently, various nanomaterials with unique optical and electrical characteristics have been introduced as the novel signal transducers to enhance the detection performance of CRISPR-Cas-based nanosensors. This review summarizes the working mechanisms of the CRISPR-Cas system for biosensing. It also enumerates the strategies of CRISPR-manipulated nanosensors based on various signal models for cancer diagnosis, including colorimetric, fluorescence, electrochemical, electrochemiluminescence, pressure, and other signals. Finally, the prospects and challenges of CRISPR-Cas-based nanosensors for cancer diagnostic are also discussed. This article is categorized under: Diagnostic Tools > Biosensing.},
}
@article {pmid36199015,
year = {2022},
author = {Costigan, R and Stoakes, E and Floto, RA and Parkhill, J and Grant, AJ},
title = {Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni.},
journal = {BMC microbiology},
volume = {22},
number = {1},
pages = {238},
pmid = {36199015},
issn = {1471-2180},
support = {/WT_/Wellcome Trust/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Arylsulfatases/genetics/metabolism ; CRISPR-Cas Systems ; *Campylobacter jejuni/metabolism ; Flagella/genetics ; Gene Expression Regulation ; Humans ; Streptococcus pyogenes/genetics ; },
abstract = {BACKGROUND: Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176.<br><br>RESULTS: The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM).<br><br>CONCLUSIONS: This is the first report of a CRISPRi-based tool in&#xa0;C. jejuni, which will provide a valuable resource to the&#xa0;Campylobacter&#xa0;community.},
}
@article {pmid36197945,
year = {2022},
author = {Yuan, B and Keller, NP and Oakley, BR and Stajich, JE and Wang, CCC},
title = {Manipulation of the Global Regulator mcrA Upregulates Secondary Metabolite Production in Aspergillus wentii Using CRISPR-Cas9 with In Vitro Assembled Ribonucleoproteins.},
journal = {ACS chemical biology},
volume = {17},
number = {10},
pages = {2828-2835},
pmid = {36197945},
issn = {1554-8937},
support = {R21 AI156320/AI/NIAID NIH HHS/United States ; },
mesh = {*Emodin ; Polyketide Synthases/genetics ; CRISPR-Cas Systems/genetics ; Ribonucleoproteins ; Aspergillus/genetics ; Multigene Family ; Glucose ; },
abstract = {Genome sequencing of filamentous fungi has demonstrated that most secondary metabolite biosynthetic gene clusters (BGCs) are silent under standard laboratory conditions. In this work, we have established an in vitro CRISPR-Cas9 system in Aspergillus wentii. To activate otherwise silent BGCs, we deleted the negative transcriptional regulator mcrA. Deletion of mcrA (mcrAΔ) resulted in differential production of 17 SMs in total when the strain was cultivated on potato dextrose media (PDA). Nine out of fifteen of these SMs were fully characterized, including emodin (1), physcion (2), sulochrin (3), physcion bianthrone (4), 14-O-demethylsulochrin (5), (trans/cis)-emodin bianthrone (6 and 7), and (trans/cis)-emodin physcion bianthrone (8 and 9). These compounds were all found to be produced by the same polyketide synthase (PKS) BGC. We then performed a secondary knockout targeting this PKS cluster in the mcrAΔ background. The metabolite profile of the dual-knockout strain revealed new metabolites that were not previously detected in the mcrAΔ parent strain. Two additional SMs were purified from the dual-knockout strain and were characterized as aspergillus acid B (16) and a structurally related but previously unidentified compound (17). For the first time, this work presents a facile genetic system capable of targeted gene editing in A. wentii. This work also illustrates the utility of performing a dual knockout to eliminate major metabolic products, enabling additional SM discovery.},
}
@article {pmid36197590,
year = {2022},
author = {Gagaletsios, LA and Papagiannitsis, CC and Petinaki, E},
title = {Prevalence and analysis of CRISPR/Cas systems in Pseudomonas aeruginosa isolates from Greece.},
journal = {Molecular genetics and genomics : MGG},
volume = {297},
number = {6},
pages = {1767-1776},
pmid = {36197590},
issn = {1617-4623},
mesh = {*Pseudomonas aeruginosa/genetics ; Greece/epidemiology ; *CRISPR-Cas Systems/genetics ; Multilocus Sequence Typing ; Prevalence ; Carbapenems/pharmacology ; },
abstract = {The objective of this study was to investigate the prevalence of CRISPR/Cas systems in P. aeruginosa, isolated from a Greek hospital. Additionally, we aimed to determine the origin of the sequenced spacers. A collection of 100 nonrepetitive P. aeruginosa was analyzed. Isolates were typed by MLST. The presence of CRISPR/Cas systems, as well as amplification of CRISPR arrays, was examined by PCR using specific primers. CRISPR/Cas systems were detected in 36 isolates, of which 27 isolates exhibited resistance to carbapenems, with 10 of the later isolates producing a VIM-type MβL. The majority (n = 19) of CRISPR/Cas-positive isolates harbored a type I-F system, while I-C and I-E systems were found in 9 and 8 isolates, respectively. Based on MLST, isolates carrying I-E and I-F systems belonged to different STs and included CRISPR arrays with diverse number of spacers. Isolates with I-C systems belonged to clonal complex 235 and exhibited identical CRISPR arrays. Among 425 unique spacers, identified during this study, BLASTn search showed that they matched with P. aeruginosa chromosomal sequences (47.0%), phages (31.9%), plasmids, PAGIs, and an ICE. 16.3% of the spacers exhibited no significant similarity with sequences submitted to GenBank database. In conclusion, we observed the presence of type I-C, I-E and I-F CRISPR/Cas systems in P. aeruginosa of clinical origin. CRISPR/Cas were also observed among isolates carrying the carbapenemase-encoding blaVIM gene, which is usually associated with integrons, questioning the defense role against mobile elements. Therefore, further experimental characterization is needed to clarify their functional role.},
}
@article {pmid36194543,
year = {2022},
author = {Liyanagedera, SBW and Williams, J and Wheatley, JP and Biketova, AY and Hasan, M and Sagona, AP and Purdy, KJ and Puxty, RJ and Feher, T and Kulkarni, V},
title = {SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies.},
journal = {ACS synthetic biology},
volume = {11},
number = {10},
pages = {3330-3342},
doi = {10.1021/acssynbio.2c00244},
pmid = {36194543},
issn = {2161-5063},
support = {BB/M017982/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Phage Therapy ; Epidermal Growth Factor/genetics ; *Bacteriophages/genetics ; Genetic Engineering ; Escherichia coli/genetics ; },
abstract = {The past decade has seen the emergence of multidrug resistant pathogens as a leading cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including host range alterations, constitutive lytic growth, and control over phage replication. However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to this problem, we propose the SpyPhage system where a "scaffold" bacteriophage is engineered to incorporate a SpyTag moiety on its capsid head to enable rapid postsynthetic modification of their surfaces with SpyCatcher-fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and whole genome assembly, we targeted a SpyTag capsid fusion to K1F, a phage targeting the pathogenic strain Escherichia coli K1. We demonstrate for the first time the cell-free assembly and decoration of the phage surface with two alternative fusion proteins, SpyCatcher-mCherry-EGF and SpyCatcher-mCherry-Rck, both of which facilitate the endocytotic uptake of the phages by a urinary bladder epithelial cell line. Overall, our work presents a cell-free phage production pipeline for the generation of multiple phenotypically distinct phages with a single underlying "scaffold" genotype. These phages could become the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations.},
}
@article {pmid36193597,
year = {2022},
author = {Khan, A and Sarkar, E},
title = {CRISPR/Cas9 encouraged CAR-T cell immunotherapy reporting efficient and safe clinical results towards cancer.},
journal = {Cancer treatment and research communications},
volume = {33},
number = {},
pages = {100641},
doi = {10.1016/j.ctarc.2022.100641},
pmid = {36193597},
issn = {2468-2942},
abstract = {CRISPR is a customized genome-editing tool that snips DNA in a simpler, cheaper and more precise way than any other gene editing tool. In recent years CRISPR/Cas has completely transformed an existing discipline of genetic engineering. This 'review' focuses on the generations and modifications in CAR-T as an advanced cancer therapeutic tool and CAR-T-approved products. It also highlights three path-breaking successful autologous and allogenic ex vivo CAR-T clinical trials in treating cancer using CRISPR/Cas9 which reported successful results despite the controversies regarding the safety of this technique. Outcomes from the first successful clinical trial showed the beneficial long-term effect on genetically modified T-cells in targeting cancer cells which opens the door for CRISPR to be the most preferred technique to help treat cancer and other diseases in the future. We searched the MEDLINE, EMBASE and PUBMED databases for original studies and meta-analysis on the use of CRISPR/Cas9 to edit T-cells until 2021. We finally selected 15 pre-clinical and 26 clinical studies for the review.},
}
@article {pmid36193328,
year = {2022},
author = {Zhu, Y},
title = {Advances in CRISPR/Cas9.},
journal = {BioMed research international},
volume = {2022},
number = {},
pages = {9978571},
pmid = {36193328},
issn = {2314-6141},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Humans ; Plasmids ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas9 technology has become the most examined gene editing technology in recent years due to its simple design, yet low cost, high efficiency, and simple operation, which can also achieve simultaneous editing of multiple loci. It can also be carried out without using plasmids, saving lots of troubles caused by plasmids. CRISPR/Cas9 has shown great potential in the study of genes or genomic functions in microorganisms, plants, animals, and human beings. In this review, we will examine the history, structure, and basic mechanisms of the CRISPR/Cas9 system, describe its great value in precision medicine and sgRNA library screening, and dig its great potential in a new field: DNA information storage.},
}
@article {pmid36192057,
year = {2022},
author = {Wei, L and Wang, Z and Wang, J and Wang, X and Chen, Y},
title = {Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system and recombinase polymerase amplification.},
journal = {Analytica chimica acta},
volume = {1230},
number = {},
pages = {340357},
doi = {10.1016/j.aca.2022.340357},
pmid = {36192057},
issn = {1873-4324},
mesh = {Anti-Bacterial Agents ; *Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; Colorimetry ; Cytosine ; *Methicillin-Resistant Staphylococcus aureus/genetics ; Recombinases ; Silver ; },
abstract = {Detection of methicillin-resistant Staphylococcus aureus (MRSA) with superior accuracy, timeliness, and simplicity is highly valuable in clinical diagnosis and food safety. In this study, an aptamer-based colorimetric biosensor was developed to detect MRSA by using a CRISPR/Cas12a system and recombinase polymerase amplification (RPA). The aptamer of silver ion (Ag[+]) pre-coupled to magnetic nanoparticles was employed not only as the substrate of trans-cleavage in the CRISPR/Cas12a system, but also as the modulator of Ag[+]-3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction, innovatively integrating the powerful CRISPR/Cas12a system with convenient colorimetry. The utilized aptamer containing consecutive and interrupted cytosine: cytosine mismatched base pairs also served as a signal amplifier because of the one-to-multiple binding of the aptamer to Ag[+]. Using triple amplification of RPA, multiple-turnover nuclease activity of Cas12a, and cytosine-Ag[+]-cytosine coordination chemistry, MRSA was detected as low as 8 CFU mL[-1]. Moreover, its satisfactory accuracy in the analysis of real samples, together with visualization and simplicity, revealed the great potential of the proposed biosensor as a robust antibiotic-resistant bacteria detection platform.},
}
@article {pmid36192055,
year = {2022},
author = {Zhou, M and Wang, H and Li, C and Yan, C and Qin, P and Huang, L},
title = {CRISPR/Cas9 mediated triple signal amplification platform for high selective and sensitive detection of single base mutations.},
journal = {Analytica chimica acta},
volume = {1230},
number = {},
pages = {340421},
doi = {10.1016/j.aca.2022.340421},
pmid = {36192055},
issn = {1873-4324},
mesh = {Biotin ; CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gold ; *Metal Nanoparticles ; Mutation ; Nucleic Acid Amplification Techniques/methods ; Proto-Oncogene Proteins p21(ras)/genetics ; Recombinases/genetics ; },
abstract = {Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.},
}
@article {pmid36191879,
year = {2022},
author = {Dong, M and Liu, J and Liu, C and Wang, H and Sun, W and Liu, B},
title = {CRISPR/CAS9: A promising approach for the research and treatment of cardiovascular diseases.},
journal = {Pharmacological research},
volume = {185},
number = {},
pages = {106480},
doi = {10.1016/j.phrs.2022.106480},
pmid = {36191879},
issn = {1096-1186},
mesh = {Humans ; *CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing/methods ; Genetic Engineering/methods ; },
abstract = {The development of gene-editing technology has been one of the biggest advances in biomedicine over the past two decades. Not only can it be used as a research tool to build a variety of disease models for the exploration of disease pathogenesis at the genetic level, it can also be used for prevention and treatment. This is done by intervening with the expression of target genes and carrying out precise molecular targeted therapy for diseases. The simple and flexible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technology overcomes the limitations of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). For this reason, it has rapidly become a preferred method for gene editing. As a new gene intervention method, CRISPR/Cas9 has been widely used in the clinical treatment of tumours and rare diseases; however, its application in the field of cardiovascular diseases is currently limited. This article reviews the application of the CRISPR/Cas9 editing technology in cardiovascular disease research and treatment, and discusses the limitations and prospects of this technology.},
}
@article {pmid36191156,
year = {2022},
author = {Miller, KJ and Thorpe, C and Eggenberger, AL and Lee, K and Kang, M and Liu, F and Wang, K and Jiang, S},
title = {Identifying Factors that Determine Effectiveness of Delivery Agents in Biolistic Delivery Using a Library of Amine-Containing Molecules.},
journal = {ACS applied bio materials},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsabm.2c00689},
pmid = {36191156},
issn = {2576-6422},
abstract = {Biolistic transfection is a popular and versatile tool for plant transformation. A key step in the biolistic process is the binding of DNA to the heavy microprojectile using a delivery agent, usually a positively charged molecule containing amine groups. Currently, the choice of the commercial delivery agent is mostly limited to spermidine. In addition, the detailed delivery mechanism has not been reported. To help broaden the selection of the delivery agent and reveal the fundamental mechanisms that lead to high delivery performance, a library of amine-containing molecules was investigated. A double-barrel biolistic delivery device was utilized for testing hundreds of samples with much-improved consistency. The performance was evaluated on onion epidermis. The binding and release of DNA were measured via direct high-performance liquid chromatography analysis. This study shows that the overwhelming majority of the amine library performed at the same level as spermidine. To further interpret these results, correlations were performed with thousands of molecular descriptors generated by chemical modeling. It was discovered that the overall charge is most likely the key factor to a successful binding and delivery. Furthermore, even after increasing the amount of the DNA concentration 50-fold to stress the binding capacity of the molecules, the amines in the library continued to deliver at a near identical level while binding all the DNA. The increased DNA was also demonstrated with a Cas9 editing test that required a large amount of DNA to be delivered, and the result was consistent with the previously determined amine performance. This study greatly expands the delivery agent selection for biolistic delivery, allowing alternatives to a commercial reagent that are more shelf-stable and cheaper. The library also offers an approach to investigate more challenging delivery of protein and CRISPR-Cas via the biolistic process in the future.},
}
@article {pmid36190280,
year = {2022},
author = {Simpson, KE and Faizi, S and Venkateshappa, R and Yip, M and Johal, R and Poburko, D and Cheng, YM and Hunter, D and Lin, E and Tibbits, GF and Claydon, TW},
title = {CRISPR-Cas9-mediated Precise Knock-in Edits in Zebrafish Hearts.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {187},
pages = {},
doi = {10.3791/64209},
pmid = {36190280},
issn = {1940-087X},
support = {//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Genome ; *RNA, Guide/genetics ; *Zebrafish/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.},
}
@article {pmid36190192,
year = {2022},
author = {Goswami, HN and Rai, J and Das, A and Li, H},
title = {Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {36190192},
issn = {2050-084X},
support = {R01 GM100143/GM/NIGMS NIH HHS/United States ; S10 RR025080/RR/NCRR NIH HHS/United States ; U24 GM116788/GM/NIGMS NIH HHS/United States ; S10 RR024564/RR/NCRR NIH HHS/United States ; },
mesh = {*RNA/genetics ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; RNA Processing, Post-Transcriptional ; Repetitive Sequences, Nucleic Acid ; RNA, Guide/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.},
}
@article {pmid36189259,
year = {2022},
author = {Campos, JS and Henrickson, SE},
title = {Defining and targeting patterns of T cell dysfunction in inborn errors of immunity.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {932715},
pmid = {36189259},
issn = {1664-3224},
support = {K08 AI135091/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cytokines/genetics ; Gene Editing ; Humans ; Mice ; *Neoplasms/therapy ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {Inborn errors of immunity (IEIs) are a group of more than 450 monogenic disorders that impair immune development and function. A subset of IEIs blend increased susceptibility to infection, autoimmunity, and malignancy and are known collectively as primary immune regulatory disorders (PIRDs). While many aspects of immune function are altered in PIRDs, one key impact is on T-cell function. By their nature, PIRDs provide unique insights into human T-cell signaling; alterations in individual signaling molecules tune downstream signaling pathways and effector function. Quantifying T-cell dysfunction in PIRDs and the underlying causative mechanisms is critical to identifying existing therapies and potential novel therapeutic targets to treat our rare patients and gain deeper insight into the basic mechanisms of T-cell function. Though there are many types of T-cell dysfunction, here we will focus on T-cell exhaustion, a key pathophysiological state. Exhaustion has been described in both human and mouse models of disease, where the chronic presence of antigen and inflammation (e.g., chronic infection or malignancy) induces a state of altered immune profile, transcriptional and epigenetic states, as well as impaired T-cell function. Since a subset of PIRDs amplify T-cell receptor (TCR) signaling and/or inflammatory cytokine signaling cascades, it is possible that they could induce T-cell exhaustion by genetically mimicking chronic infection. Here, we review the fundamentals of T-cell exhaustion and its possible role in IEIs in which genetic mutations mimic prolonged or amplified T-cell receptor and/or cytokine signaling. Given the potential insight from the many forms of PIRDs in understanding T-cell function and the challenges in obtaining primary cells from these rare disorders, we also discuss advances in CRISPR-Cas9 genome-editing technologies and potential applications to edit healthy donor T cells that could facilitate further study of mechanisms of immune dysfunctions in PIRDs. Editing T cells to match PIRD patient genetic variants will allow investigations into the mechanisms underpinning states of dysregulated T-cell function, including T-cell exhaustion.},
}
@article {pmid36189249,
year = {2022},
author = {Pinter, T and Fischer, M and Schäfer, M and Fellner, M and Jude, J and Zuber, J and Busslinger, M and Wöhner, M},
title = {Comprehensive CRISPR-Cas9 screen identifies factors which are important for plasmablast development.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {979606},
pmid = {36189249},
issn = {1664-3224},
mesh = {Animals ; B-Lymphocytes ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Germinal Center ; Mice ; *Plasma Cells ; },
abstract = {Plasma cells (PCs) and their progenitors plasmablasts (PBs) are essential for the acute and long-term protection of the host against infections by providing vast levels of highly specific antibodies. Several transcription factors, like Blimp1 and Irf4, are already known to be essential for PC and PB differentiation and survival. We set out to identify additional genes, that are essential for PB development by CRISPR-Cas9 screening of 3,000 genes for the loss of PBs by employing the in vitro-inducible germinal center B cell (iGB) culture system and Rosa26[Cas9/+] mice. Identified hits in the screen were Mau2 and Nipbl, which are known to contribute to the loop extrusion function of the cohesin complex. Other examples of promising hits were Taf6, Stat3, Ppp6c and Pgs1. We thus provide a new set of genes, which are important for PB development.},
}
@article {pmid36188128,
year = {2022},
author = {Li, Y and Wu, X and Zhang, Y and Zhang, Q},
title = {CRISPR/Cas genome editing improves abiotic and biotic stress tolerance of crops.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {987817},
pmid = {36188128},
issn = {2673-3439},
abstract = {Abiotic stress such as cold, drought, saline-alkali stress and biotic stress including disease and insect pest are the main factors that affect plant growth and limit agricultural productivity. In recent years, with the rapid development of molecular biology, genome editing techniques have been widely used in botany and agronomy due to their characteristics of high efficiency, controllable and directional editing. Genome editing techniques have great application potential in breeding resistant varieties. These techniques have achieved remarkable results in resistance breeding of important cereal crops (such as maize, rice, wheat, etc.), vegetable and fruit crops. Among them, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) provides a guarantee for the stability of crop yield worldwide. In this paper, the development of CRISRR/Cas and its application in different resistance breeding of important crops are reviewed, the advantages and importance of CRISRR/Cas technology in breeding are emphasized, and the possible problems are pointed out.},
}
@article {pmid36187950,
year = {2022},
author = {Dueñas, E and Nakamoto, JA and Cabrera-Sosa, L and Huaihua, P and Cruz, M and Arévalo, J and Milón, P and Adaui, V},
title = {Novel CRISPR-based detection of Leishmania species.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {958693},
pmid = {36187950},
issn = {1664-302X},
abstract = {Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10[-2] (kDNA) or 5 × 10[0] (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.},
}
@article {pmid36186424,
year = {2022},
author = {Walker, AR and Shields, RC},
title = {Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {997341},
pmid = {36186424},
issn = {1664-8021},
abstract = {CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted ∼8,000 CRISPR spacers from a collection of over three hundred Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. S. mutans strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and 'openness' of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome.},
}
@article {pmid36185740,
year = {2023},
author = {Breier, D and Peer, D},
title = {Genome editing in cancer: Challenges and potential opportunities.},
journal = {Bioactive materials},
volume = {21},
number = {},
pages = {394-402},
pmid = {36185740},
issn = {2452-199X},
abstract = {Ever since its mechanism was discovered back in 2012, the CRISPR/Cas9 system have revolutionized the field of genome editing. While at first it was seen as a therapeutic tool mostly relevant for curing genetic diseases, it has been recently shown to also hold the potential to become a clinically relevant therapy for cancer. However, there are multiple challenges that must be addressed prior to clinical testing. Predominantly, the safety of the system when used for in-vivo therapies, including off-target activity and the effects of the double strand break induction on genomic stability. Here, we will focus on the inherent challenges in the CRISPR/Cas9 system and discuss various opportunities to overcoming these challenges.},
}
@article {pmid36185592,
year = {2022},
author = {Naeem, M and Alkhodairy, HF and Ashraf, I and Khalil, AB},
title = {CRISPR/Cas System Toward the Development of Next-Generation Recombinant Vaccines: Current Scenario and Future Prospects.},
journal = {Arabian journal for science and engineering},
volume = {},
number = {},
pages = {1-11},
pmid = {36185592},
issn = {2193-567X},
abstract = {The initially developed vaccines were relying mostly on attenuation and inactivation of pathogens. The use of recombinant DNA technology allows the targeting of immune responses focused against a few protective antigens. The conventional recombination methods for generating vaccines are time-consuming, laborious, and less efficient. To overcome these limitations, a new precise CRISPR/Cas9 with high efficacy, specificity, and low-cost properties has solved a lot of current problems of recombinant vaccines that intrigued the inspiration for novel recombinant vaccine development. CRISPR/Cas9 system was discovered as a bacterial adaptive immune system. In the domain of virology, CRISPR/Cas9 is used to engineer the virus genome to understand the fundamentals of viral pathogenesis, gene therapy, and virus-host interactions. One step ahead CRISPR/Cass9 bypassed the vaccine to precisely engineer the B-cells to secrete the specific antibodies against deadly viral pathogens. There is a critical literature review gap especially in the use of CRISPR/Cas9 to generate recombinant vaccines against viral diseases and its prospective application to engineering the B-cells in immunocompromised people. This review heights the application of CRISPR/Cas9 compared to conventional approaches for the development of recombinant vaccine vectors, editing the genes of B-cells, and challenges that need to be overcome. The factors affecting CRISPR/Cas9-edited recombinant vaccines and prospects in the context of viral genome editing for the development of vaccines will be discussed.},
}
@article {pmid36185462,
year = {2022},
author = {Chen, K and Shen, Z and Wang, G and Gu, W and Zhao, S and Lin, Z and Liu, W and Cai, Y and Mushtaq, G and Jia, J and Wan, CC and Yan, T},
title = {Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {986233},
pmid = {36185462},
issn = {2296-4185},
abstract = {CRISPR/Cas technology originated from the immune mechanism of archaea and bacteria and was awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Molecular diagnostics is highly valued globally for its development as a new generation of diagnostic technology. An increasing number of studies have shown that CRISPR/Cas technology can be integrated with biosensors and bioassays for molecular diagnostics. CRISPR-based detection has attracted much attention as highly specific and sensitive sensors with easily programmable and device-independent capabilities. The nucleic acid-based detection approach is one of the most sensitive and specific diagnostic methods. With further research, it holds promise for detecting other biomarkers such as small molecules and proteins. Therefore, it is worthwhile to explore the prospects of CRISPR technology in biosensing and summarize its application strategies in molecular diagnostics. This review provides a synopsis of CRISPR biosensing strategies and recent advances from nucleic acids to other non-nucleic small molecules or analytes such as proteins and presents the challenges and perspectives of CRISPR biosensors and bioassays.},
}
@article {pmid36184667,
year = {2022},
author = {Tenjo-Castaño, F and Sofos, N and López-Méndez, B and Stutzke, LS and Fuglsang, A and Stella, S and Montoya, G},
title = {Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5792},
pmid = {36184667},
issn = {2041-1723},
mesh = {DNA/genetics ; DNA Transposable Elements/genetics ; *Dichlorodiphenyl Dichloroethylene ; Protein Subunits/genetics ; RNA ; *Transposases/genetics/metabolism ; },
abstract = {CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition.},
}
@article {pmid36184032,
year = {2022},
author = {Wu, Q and Glitscher, M and Tonnemacher, S and Schollmeier, A and Raupach, J and Zahn, T and Eberle, R and Krijnse-Locker, J and Basic, M and Hildt, E},
title = {Presence of Intact Hepatitis B Virions in Exosomes.},
journal = {Cellular and molecular gastroenterology and hepatology},
volume = {15},
number = {1},
pages = {237-259},
pmid = {36184032},
issn = {2352-345X},
abstract = {BACKGROUND &amp; AIMS: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells.<br><br>METHODS: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells.<br><br>RESULTS: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na[+]-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells.<br><br>CONCLUSION: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.},
}
@article {pmid36183557,
year = {2022},
author = {Cheng, P and Wu, Y and Guo, S and Ma, X and Fei, C and Xue, F and Zhu, C and Wang, M and Gu, F},
title = {RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples.},
journal = {Veterinary parasitology},
volume = {311},
number = {},
pages = {109810},
doi = {10.1016/j.vetpar.2022.109810},
pmid = {36183557},
issn = {1873-2550},
mesh = {Animals ; Chickens/parasitology ; *Coccidiosis/diagnosis/veterinary/genetics ; CRISPR-Cas Systems ; DNA ; *Eimeria/genetics ; Nucleic Acid Amplification Techniques/methods/veterinary ; Recombinases/genetics ; },
abstract = {Chicken coccidiosis is one of the most common and economically important diseases in the global poultry industry, and it is caused by at least one of the seven Eimeria species. A simple and reliable way to distinguish Eimeria species in infected chicken is critical for the surveillance, control, and eradication of chicken coccidiosis. In this study, a recombinase polymerase amplification (RPA) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system (RPA-CRISPR/Cas12a) was developed for the detection of Eimeria species in chicken fecal samples. This assay is highly specific to the seven Eimeria species and it does not cross react between species. Assessment of analytical sensitivity revealed that a single copy of plasmid DNA could be detected. Comparative analysis revealed strong agreement between RPA-CRISPR/Cas12a assays and real-time qPCR to reliably detect all seven Eimeria species in fecal chicken samples. Importantly, the cleavage products could be visualized under a blue light instrument, making it possible for the rapid detection of Eimeria species for on-site testing. Collectively, our study demonstrated that RPA-CRISPR/Cas12a assays offer a simple and reliable diagnostic method for Eimeria species.},
}
@article {pmid36183037,
year = {2022},
author = {Nafian, F and Nafian, S and Kamali Doust Azad, B and Hashemi, M},
title = {CRISPR-Based Diagnostics and Microfluidics for COVID-19 Point-of-Care Testing: A Review of Main Applications.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {1-12},
pmid = {36183037},
issn = {1559-0305},
abstract = {An ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). So far, there have been various approaches for SARS-CoV-2 detection, each having its pros and cons. The current gold-standard method for SARS-CoV-2 detection, which offers acceptable specificity and sensitivity, is the quantitative reverse transcription-PCR (qRT-PCR). However, this method requires considerable cost and time to transport samples to specialized laboratories and extract, amplify, and detect the viral genome. On the other hand, antigen and antibody testing approaches that bring rapidity and affordability into play have lower sensitivity and specificity during the early stages of COVID-19. Moreover, the immune response is variable depending on the individual. Methods based on clustered regularly interspaced short palindromic repeats (CRISPR) can be used as an alternative approach to controlling the spread of disease by a high-sensitive, specific, and low-cost molecular diagnostic system. CRISPR-based detection systems (CRISPR-Dx) target the desired sequences by specific CRISPR-RNA (crRNA)-pairing on a pre-amplified sample and a subsequent collateral cleavage. In the present article, we have reviewed different CRISPR-Dx methods and presented their benefits and drawbacks for point-of-care testing (POCT) of suspected SARS-CoV-2 infections at home or in small clinics.},
}
@article {pmid36182708,
year = {2022},
author = {Zhang, P and Wang, H and Li, S and Guo, T and Zhang, R and Zhou, P and Ding, S},
title = {Generation of an S100B homozygous knockout pluripotent stem cell line (WAe009-A-94) by the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102924},
doi = {10.1016/j.scr.2022.102924},
pmid = {36182708},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Calcium/metabolism ; Homozygote ; Calcium-Binding Proteins ; S100 Calcium Binding Protein beta Subunit/genetics/metabolism ; },
abstract = {S100 calcium binding protein beta (S100B) is an S-100 low molecular weight binding protein that regulates intracellular processes. This protein is involved in myocardial contractility and calcium handling capacity. In this study, a human embryonic stem cell (hESC) line with homozygous S100B knockout (S100B-KO) was generated using the CRISPR/Cas9 editing system. This S100B-KO hESC line maintained normal cell morphology and karyotype, expressed pluripotency markers, and could differentiate into cells of all three germ layers.},
}
@article {pmid36181622,
year = {2022},
author = {Wang, X and Zhao, Y and Wang, Z and Liao, Z and Zhang, Y},
title = {Construction of TSC2 knockout cell line using CRISPR/Cas9 system and demonstration of its effects on NIH-3T3 cells.},
journal = {Cell biochemistry and biophysics},
volume = {80},
number = {4},
pages = {681-687},
pmid = {36181622},
issn = {1559-0283},
mesh = {*Angiomyolipoma/genetics/metabolism/pathology ; Animals ; CRISPR-Cas Systems ; Carotenoids ; Humans ; *Kidney Neoplasms/pathology ; Mice ; NIH 3T3 Cells ; TOR Serine-Threonine Kinases/metabolism ; *Tuberous Sclerosis/genetics/metabolism/pathology ; Tuberous Sclerosis Complex 2 Protein/genetics/metabolism ; Vitamin A/analogs &amp; derivatives ; },
abstract = {Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder involving multiple organ systems. TSC2 gene plays an important role in the development of TSC. The most common kidney manifestation of TSC is renal angiomyolipoma (RAML). TSC-RAML is more likely to be bilateral multiple tumors and tends to destroy the renal structure and damages renal function severely. As a result, patients with TSC-RAML often miss the opportunity for surgical treatment when TSC-RAML is diagnosed, causing difficulty in obtaining tumor specimens through surgery. Due to this difficulty, model cell lines must be constructed for scientific research. In this paper, TSC2 was knocked out in NIH-3T3 cell lines by CRISPR/Cas9 system. PCR, WB and mTOR inhibitor drug sensitivity test showed that the TSC2 knockout NIH-3T3 cells were successfully constructed. The ability of proliferation and invasion in TSC2 KO NIH-3T3 cells were higher than those in wild type group. The constructed KO cell line lay the foundation for further study of TSC.},
}
@article {pmid36180879,
year = {2022},
author = {Li, S and Wang, X and Yu, Y and Cao, S and Liu, J and Zhao, P and Li, J and Zhang, X and Li, X and Zhang, N and Sun, M and Cao, L and Gong, P},
title = {Establishment and application of a CRISPR-Cas12a-based RPA-LFS and fluorescence for the detection of Trichomonas vaginalis.},
journal = {Parasites &amp; vectors},
volume = {15},
number = {1},
pages = {350},
pmid = {36180879},
issn = {1756-3305},
mesh = {Actins/genetics ; CRISPR-Cas Systems ; *Cryptosporidiosis/genetics ; *Cryptosporidium/genetics ; Female ; Humans ; Male ; Nucleic Acid Amplification Techniques/methods ; Pregnancy ; Sensitivity and Specificity ; *Trichomonas vaginalis/genetics ; },
abstract = {BACKGROUND: Infection with Trichomonas vaginalis can lead to cervicitis, urethritis, pelvic inflammatory disease, prostatitis and perinatal complications and increased risk of HIV transmission. Here, we used an RPA-based CRISPR-Cas12a assay system in combination with a lateral flow strip (LFS) (referred to as RPA-CRISPR-Cas12a) to establish a highly sensitive and field-ready assay and evaluated its ability to detect clinical samples.<br><br>METHODS: We developed a one-pot CRISPR-Cas12a combined with RPA-based field detection technology for T. vaginalis, chose actin as the target gene to design crRNA and designed RPA primers based on the crRNA binding site. The specificity of the method was demonstrated by detecting genomes from nine pathogens. To improve the usability and visualize the RPA-CRISPR-Cas12a assay results, both fluorescence detection and LFS readouts were devised.<br><br>RESULTS: The RPA-CRISPR-Cas12a assay platform was completed within 60&#xa0;min and had a maximum detection limit of 1 copy/&#xb5;l and no cross-reactivity with Candida albicans, Mycoplasma hominis, Neisseria gonorrhoeae, Escherichia coli, Cryptosporidium parvum, G. duodenalis or Toxoplasma gondii after specificity validation. Thirty human vaginal secretions were tested by RPA-CRISPR-Cas12a assays, and the results were read by a fluorescent reporter and LFS biosensors and then compared to the results from nested PCR detection of these samples. Both RPA-CRISPR-Cas12a assays showed 26.7% (8/30) T. vaginalis-positive samples and a consistency of 100% (8/8). The RPA-CRISPR-Cas12a assays had a higher sensitivity than nested PCR (only seven T. vaginalis-positive samples were detected).<br><br>CONCLUSIONS: The T. vaginalis RPA-CRISPR-Cas12a assay platform in this study can be used for large-scale field testing and on-site tests without the need for trained technicians or costly ancillary equipment.},
}
@article {pmid36180232,
year = {2022},
author = {Geng, K and Merino, LG and Wedemann, L and Martens, A and Sobota, M and Sanchez, YP and Søndergaard, JN and White, RJ and Kutter, C},
title = {Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.},
journal = {Genome research},
volume = {32},
number = {10},
pages = {1876-1891},
doi = {10.1101/gr.276901.122},
pmid = {36180232},
issn = {1549-5469},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanopore Sequencing ; Genomics ; RNA, Guide/genetics ; DNA/genetics ; Alleles ; },
abstract = {The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.},
}
@article {pmid36180213,
year = {2022},
author = {Weber, D and Richter, V and Rohwedder, A and Großjohann, A and Thum, AS},
title = {Learning and Memory in Drosophila Larvae.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top107863},
pmid = {36180213},
issn = {1559-6095},
abstract = {The Drosophila larva has become an attractive model system for studying fundamental questions in neuroscience. Although the focus was initially on topics such as the structure of genes, mechanisms of inheritance, genetic regulation of development, and the function and physiology of ion channels, today it is often on the cellular and molecular principles of naive and learned behavior. Drosophila larvae have developed different mechanisms, often widespread in similar manifestations in the animal kingdom, to orient themselves toward olfactory, gustatory, mechanosensory, thermal, and visual stimuli to coordinate their locomotion appropriately. To adapt to changes in the environment, larvae are able to learn to categorize some of these sensory impressions as "good" or "bad." Depending on their relevance and reliability, the larva learns them and constantly updates these memories. Laboratory experiments allow us to parametrically study and describe many of these processes (e.g., olfactory appetitive and aversive memory or visual appetitive and aversive memory). Combining behavioral tests with various neurogenetic techniques allows us to thermally or optogenetically activate or inhibit individual cells during learning, memory consolidation, and memory retrieval. The molecular and genetic bases of larval learning can be analyzed by using specific mutants. The CRISPR-Cas method has established extensive new directions in this area, in addition to the already wide-ranging traditional approaches, like the GAL4/UAS system. The combination of these genetic methods with the simplicity and cost-effectiveness of the introduced behavioral assay provides a platform for discovering the fundamental mechanisms underlying learning and memory formation in the rather simple larval brain.},
}
@article {pmid36179433,
year = {2022},
author = {Li, X and Chen, X and Mao, M and Peng, C and Wang, Z},
title = {Accelerated CRISPR/Cas12a-based small molecule detection using bivalent aptamer.},
journal = {Biosensors &amp; bioelectronics},
volume = {217},
number = {},
pages = {114725},
doi = {10.1016/j.bios.2022.114725},
pmid = {36179433},
issn = {1873-4235},
mesh = {Adenosine ; Adenosine Triphosphate/analysis ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Kanamycin ; *Nucleic Acids ; },
abstract = {CRISPR/Cas holds great promise for biosensing applications, however, restricted to nucleic acid targets. Here, we broaden the sensing target of CRISPR/Cas to small molecules via integrating a bivalent aptamer as a recognition component. Using adenosine 5'-triphosphate (ATP) as a model molecule, we found that a bivalent aptamer we selected could shorten the binding time between the aptamer and ATP from 30 min to 3 min, thus dramatically accelerating the detection of ATP. The accelerated bivalent aptamer binding to ATP was mainly ascribed to the extended conformation of the aptamer, which was stabilized through linking with a 5 T bases connector on specific loops of the monovalent aptamer. To facilitate on-site detection, we integrated lateral flow assay (LFA) with the CRISPR/Cas sensing strategy (termed BA-CASLFA) to serve as a visual readout of the presence of ATP. In addition, in the CASLFA platform, due to the unique characteristics of LFA, the thermal step of Cas12a inactivation can be omitted. The BA-CASLFA could output a colorimetric "TURN ON" signal for ATP within 26 min, which could be easily discriminated by the naked eye and sensitively quantified by the portable reader. Furthermore, we showed the versatility of BA-CASLFA for detecting kanamycin using a kanamycin bivalent aptamer obtained through the same design as the ATP bivalent aptamer. Therefore, this strategy is amenable to serve as a general sensing strategy for small molecular targets. The above work opened a new way in developing CRISPR-based on-site sensors for clinic diagnosis, food safety, and environmental analysis.},
}
@article {pmid36178561,
year = {2022},
author = {Razzaq, HA and Ijaz, S and Haq, IU and Khan, IA},
title = {Functional inhibition of the StERF3 gene by dual targeting through CRISPR/Cas9 enhances resistance to the late blight disease in Solanum tuberosum L.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11675-11684},
pmid = {36178561},
issn = {1573-4978},
mesh = {Humans ; *Solanum tuberosum/genetics ; CRISPR-Cas Systems/genetics ; Plant Diseases/genetics/microbiology ; *Phytophthora infestans/genetics ; Genes, Plant ; Disease Resistance/genetics ; },
abstract = {BACKGROUND: Disease-resistant cultivars are the best solution to get their maximum yield potential and avoid fungicide application. There is no doubt about the contribution, and use of R genes (resistance genes) in resistance development in plants, while S genes (susceptibility genes) also hold a strong position in pathogenesis by resistance repression, and their loss of function contributes to enhanced resistance. Hence, we attempted to knock out the function of the StERF3 gene in potatoes through CRISPR/Cas9-based genome editing and investigated the CRISPR/Cas9 approach as strategic control against late blight disease in potato plants.<br><br>METHODS AND RESULTS: The StERF3 gene was edited in late blight susceptible cv. Lady Rosetta. Full allelic edited plants were identified through DnpI, and N1aIV mediated restriction digestion and then further analyzed through Indel Detection by Amplicon Analysis. Sequence analysis of targeted plants for indel identification showed full allelic editing. The detached leaf assay of full allelic edited plants demonstrated the role of the StERF3 gene in susceptibility to late blight in potatoes. In planta disease assay also showed reduced, slowed, and delayed disease progression in StERF3-loss-of-function mutants compared to wild-type (control) plants. Less fungal biomass was quantified in knockouts through Real-time qPCR that supported less susceptibility of edited plants to late blight. Besides, relatively high expression of pathogens-related genes, StPR1, and StNPR1, were also observed in StERF3-loss-of-function mutants compared to the corresponding control.<br><br>CONCLUSION: The results showed the functional inhibition of StERF3 genes using the CRISPR/Cas9 approach. The functional knockouts (StERF3 gene-edited potato plants) revealed enhanced resistance against Phytophthora infestans, thereby demonstrating the best strategic control for late blight disease in potato plants.},
}
@article {pmid36178514,
year = {2022},
author = {Soliman, M and Said, HS and El-Mowafy, M and Barwa, R},
title = {Novel PCR detection of CRISPR/Cas systems in Pseudomonas aeruginosa and its correlation with antibiotic resistance.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {21},
pages = {7223-7234},
pmid = {36178514},
issn = {1432-0614},
mesh = {Humans ; Pseudomonas aeruginosa/genetics ; CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; *CRISPR-Associated Proteins/genetics ; *Bacteriophages/genetics ; Drug Resistance, Microbial ; Polymerase Chain Reaction ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) systems are considered as acquired immune mechanisms in Gram-positive and Gram-negative bacteria and also in archaea. They provide resistance/immunity to attacking bacteriophages or mobile genetic elements as integrative conjugative elements (ICE) as well as plasmid transformation. As an opportunistic pathogen, Pseudomonas aeruginosa has been held responsible for serious infections especially in hospitalized and immunocompromised patients. Three subtypes of type I CRISPR system (I-C, I-E, &amp; I-F1) have been detected in P. aeruginosa genomes. In this work, P. aeruginosa isolates were collected from different clinical sources, and the three CRISPR/Cas subtypes (I-C, I-E, &amp; I-F1) were detected via singleplex and multiplex PCR techniques using novel universal primers that were designed specifically in this study. CRISPR subtypes I-C, I-E, and I-F1 were detected in 10, 9, and 13 isolates, respectively. Furthermore, antimicrobial susceptibility of CRISPR/Cas-positive and negative isolates to different antibiotics and the capacity of biofilm formation were detected using disc diffusion method and tissue culture plate method, respectively. There was a significant correlation between the presence/absence of CRISPR/Cas system and both antimicrobial susceptibility to some antibiotics and biofilm-forming capacity among P. aeruginosa clinical isolates. KEY POINTS: • A novel multiplex-PCR for detection of CRISPR/Cas-positive strains of P. aeruginosa. • Understand the correlation between CRISPR/Cas systems and other characters of P. aeruginosa. • Correlation between antimicrobial susceptibility and CRISPR systems in P. aeruginosa.},
}
@article {pmid36177842,
year = {2022},
author = {Zhang, Y and Wu, Y and Li, G and Qi, A and Zhang, Y and Zhang, T and Qi, Y},
title = {Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice.},
journal = {The plant genome},
volume = {},
number = {},
pages = {e20266},
doi = {10.1002/tpg2.20266},
pmid = {36177842},
issn = {1940-3372},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) nucleases like Cas9 and Cas12a are revolutionizing plant basic research and crop breeding. A major advantage of CRISPR over earlier nucleases systems is its capability of multiplexed genome editing. However, it remains unknown about the potential off-target effects when multiple concurrent DNA double-strand breaks (DSBs) are induced in a crop genome. Here, we investigated this important question in rice (Oryza sativa) using a highly multiplexed CRISPR-Cas12a system. With whole-genome sequencing, we first revealed high genome editing specificity of Mb2Cas12a and protospacer adjacent motif promiscuity of LbCas12a. We discovered large chromosomal rearrangement events in edited rice plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured lower order DSBs (e.g., <10). Our results shed important light on the analysis and regulation of engineered crops derived from CRISPR-Cas mediated multiplexed genome editing.},
}
@article {pmid36175516,
year = {2022},
author = {Guo, X and Sanchez-Londono, M and Gomes-Filho, JV and Hernandez-Tamayo, R and Rust, S and Immelmann, LM and Schäfer, P and Wiegel, J and Graumann, PL and Randau, L},
title = {Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans.},
journal = {Nature microbiology},
volume = {7},
number = {11},
pages = {1870-1878},
pmid = {36175516},
issn = {2058-5276},
mesh = {*Pseudomonas oleovorans/genetics ; CRISPR-Cas Systems ; Bacteria/genetics ; DNA ; Deoxyribonucleases ; },
abstract = {Bacterial Type IV CRISPR-Cas systems are thought to rely on multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements, but the substrate requirements and potential DNA nuclease activities for many systems within this type are uncharacterized. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in a PAM-dependent manner and elicits interference without showing DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. Deletion of the native Type IV CRISPR array resulted in upregulation of pilN operon transcription in the absence of genome cleavage, indicating that Type IV-A CRISPR-Cas systems can function in host gene regulation. These systems resemble CRISPR interference (CRISPRi) methodology but represent a natural CRISPRi-like system that is found in many Pseudomonas and Klebsiella species and allows for gene silencing using engineered crRNAs.},
}
@article {pmid36174966,
year = {2022},
author = {Schmidt, TJN and Berarducci, B and Konstantinidou, S and Raffa, V},
title = {CRISPR/Cas9 in the era of nanomedicine and synthetic biology.},
journal = {Drug discovery today},
volume = {28},
number = {1},
pages = {103375},
doi = {10.1016/j.drudis.2022.103375},
pmid = {36174966},
issn = {1878-5832},
abstract = {The CRISPR/Cas system was first discovered as a defense mechanism in bacteria and is now used as a tool for precise gene-editing applications. Rapidly evolving, it is increasingly applied in therapeutics. However, concerns about safety, specificity, and delivery still limit its potential. In this context, we introduce the concept of nanogenetics and speculate how the rational engineering of the CRISPR/Cas machinery could advance the biomedical field. In nanogenetics, the advantages of traditional approaches of synthetic biology could be expanded by nanotechnology approaches, enabling the design of a new generation of intrinsically safe and specific genome-editing platforms.},
}
@article {pmid36174845,
year = {2022},
author = {Luo, C and Wang, Q and Guo, R and Zhang, J and Zhang, J and Zhang, R and Ma, X and Wang, P and Adam, FEA and Zeshan, B and Yang, Z and Zhou, Y and Wang, X},
title = {A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.},
journal = {Virus research},
volume = {322},
number = {},
pages = {198937},
doi = {10.1016/j.virusres.2022.198937},
pmid = {36174845},
issn = {1872-7492},
mesh = {Swine ; Mice ; Animals ; Pseudorabies Vaccines/genetics ; *Herpesvirus 1, Suid ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; CRISPR-Cas Systems ; *Swine Diseases ; Antibodies, Viral ; *Pseudorabies/prevention &amp; control ; Immunity, Cellular ; Viral Envelope Proteins/genetics ; },
abstract = {Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9[D10A] targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.},
}
@article {pmid36174804,
year = {2022},
author = {Ruan, W and Jiao, M and Xu, S and Ismail, M and Xie, X and An, Y and Guo, H and Qian, R and Shi, B and Zheng, M},
title = {Brain-targeted CRISPR/Cas9 nanomedicine for effective glioblastoma therapy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {351},
number = {},
pages = {739-751},
doi = {10.1016/j.jconrel.2022.09.046},
pmid = {36174804},
issn = {1873-4995},
mesh = {Animals ; Humans ; Mice ; *RNA, Guide ; CRISPR-Cas Systems ; *Glioblastoma/genetics/therapy/pathology ; Nanomedicine ; Guanidine ; Fluorine ; Gene Editing/methods ; Brain/metabolism ; },
abstract = {CRISPR/Cas9 gene-editing technology shows great potential for treating a variety of diseases, such as glioblastoma multiforme (GBM). However, CRISPR components suffer from inherent delivery challenges, such as poor in vivo stability of Cas9 protein and gRNA, low blood-brain barrier (BBB) permeability and non-specific tissue or cell targeting. These defects have limited the application of Cas9/gRNA ribonucleoprotein (RNP) complexes for GBM therapy. Here, we developed a brain-targeted CRISPR/Cas9 based nanomedicine by fabricating an angiopep-2 decorated, guanidinium and fluorine functionalized polymeric nanoparticle with loading Cas9/gRNA RNP for the treatment of GBM. The guanidinium and fluorine domains of our polymeric nanoparticles were both capable of interacting with Cas9/gRNA RNP to stabilize it in blood circulation, without impairing its activity. Moreover, by leveraging angiopep-2 peptide functionality, the RNP nanoparticles efficiently crossed the BBB and accumulated in brain tumors. In U87MG cells, we achieved approximately 32% gene knockout and 67% protein reduction in the targeted proto-oncogene polo-like kinase 1 (PLK1). This was sufficient to suppress tumor growth and significantly improved the median survival time of mice bearing orthotopic glioblastoma to 40 days, while inducing negligible side or off-target effects. These results suggest that the developed brain-targeted CRISPR/Cas9 based nanomedicine shows promise for effective human glioblastoma gene therapy.},
}
@article {pmid36174801,
year = {2022},
author = {Tuncel, A and Qi, Y},
title = {CRISPR/Cas mediated genome editing in potato: Past achievements and future directions.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {325},
number = {},
pages = {111474},
doi = {10.1016/j.plantsci.2022.111474},
pmid = {36174801},
issn = {1873-2259},
mesh = {*Gene Editing/methods ; *Solanum tuberosum/genetics ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Plants/genetics ; Genome, Plant/genetics ; },
abstract = {Genome engineering has been re-shaping plant biotechnology and agriculture. Crop improvement using the recently developed gene editing techniques is now easier, faster, and more precise than ever. Although considered to be a global food security crop, potato has not benefitted enough from diverse collection of these techniques. Unique genetic features of cultivated potatoes such as tetrasomic inheritance, high genomic heterozygosity, and inbreeding depression hamper conventional breeding of this important crop. Therefore, genome editing provides an excellent arsenal of tools for trait improvement in potato. Moreover, using specific transformation protocols, it is possible to engineer transgene free commercial varieties. In this review we first describe the past achievements in potato genome editing and highlight some of the missing aspects of these efforts. Then, we discuss about technical challenges of genome editing in potato and present approaches to overcome these difficulties. Finally, we talk about genome editing applications that have not been explored in potato and point out some of the missing venues in literature.},
}
@article {pmid36174769,
year = {2022},
author = {Yang, Z and Wu, H and Lin, Q and Wang, X and Kang, S},
title = {Lymphopenic condition enhanced the antitumor immunity of PD-1-knockout T cells mediated by CRISPR/Cas9 system in malignant melanoma.},
journal = {Immunology letters},
volume = {250},
number = {},
pages = {15-22},
doi = {10.1016/j.imlet.2022.09.004},
pmid = {36174769},
issn = {1879-0542},
mesh = {Animals ; Antigens, Neoplasm ; B7-H1 Antigen/genetics ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Lymphopenia/genetics ; Melanoma ; *Melanoma, Experimental/therapy ; Mice ; Mice, Inbred C57BL ; Programmed Cell Death 1 Receptor/genetics ; Skin Neoplasms ; T-Lymphocytes ; },
abstract = {BACKGROUND: Adoptive transfer of PD-1 knockout T cells mediated by CRISPR/Cas9 technology has been used in various cancers and got satisfactory treatment effectiveness. However, the effectiveness was limited due to the low proliferation ability, antigen recognition ability and short lifetime of T cells in vivo.<br><br>METHOD: In this study, PD-1 knockout T cells mediated by CRISPR/Cas9 system were transferred into lymphopenic mice after sub-lethal dose of total body irradiation. The antitumor effects of PD-1 knockout T cells were comprehensively analyzed by flow cytometry. Moreover, PD-L1 knockout B16 cells were inoculated subcutaneously in lymphopenic mice receiving infusion of na&#xef;ve T cells to value the role of PD-1/PD-L1 axis on lymphopenia-induced antitumor immunity RESULT: In this study, we found that the PD-1-knockout T cells underwent several rounds of homeostatic proliferation in vivo when they were transferred into lymphopenic mice. The number of IFN-&#x3b3;-releasing CTL was significantly increased and the tumor growth was remarkably inhibited in lymphopenic mice receiving infusion of PD-1 knockout T cells. The expression of PD-L1 on tumor cells rose smartly in lymphopenic mice undergoing homeostatic proliferation. PD-L1 gene knockout on B16 melanoma cells could effectively enhance the antitumor immunity mediated by the homeostatic proliferation of T cells and significantly inhibited the growth of tumor CONCLUSION: These findings suggested that lymphopenic condition after total body irradiation might be able to create an environment to promote the PD-1 knockout T cells to recognize tumor antigen and undergo homeostatic proliferation, thus induced a more powerful antitumor immunity than adoptively transferring into immunocompetent hosts.},
}
@article {pmid36173579,
year = {2023},
author = {Horii, T and Morita, S and Hatada, I},
title = {Generation of Epigenetic Disease Model Mice by Targeted Demethylation of the Epigenome.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2577},
number = {},
pages = {255-268},
pmid = {36173579},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Demethylation ; DNA Methylation ; Epigenesis, Genetic ; Epigenome ; *Gene Editing/methods ; Mice ; },
abstract = {Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation.},
}
@article {pmid36173577,
year = {2023},
author = {Otabe, T and Nihongaki, Y and Sato, M},
title = {A Split CRISPR-Cpf1 Platform for Inducible Gene Activation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2577},
number = {},
pages = {229-240},
pmid = {36173577},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Genome ; Mammals/metabolism ; Mice ; RNA ; Transcriptional Activation ; },
abstract = {The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.},
}
@article {pmid36173576,
year = {2023},
author = {Kunii, A and Yamamoto, T and Sakuma, T},
title = {Design, Construction, and Validation of Targeted Gene Activation with TREE System in Human Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2577},
number = {},
pages = {211-226},
pmid = {36173576},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics ; Transcription Factors/genetics ; Transcriptional Activation ; },
abstract = {Genome editing technologies can be diverted into artificial transcription activators. In particular, researchers have improved dCas9-based technologies by tandem-fusing or trans-accumulating effector domains. Previously, we developed a hierarchical effector accumulation system named "TREE," enabling robust activation of target genes even when strongly silenced. In this chapter, we describe our protocol to design, construct, and validate the TREE-mediated target gene activation in cultured human cells.},
}
@article {pmid36173575,
year = {2023},
author = {Lebar, T and Jerala, R},
title = {Concatenated Coiled-Coil Tag for Highly Efficient, Small Molecule-Inducible Upregulation of Endogenous Mammalian Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2577},
number = {},
pages = {197-209},
pmid = {36173575},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; *DNA ; Mammals/genetics ; Peptides/genetics ; *Transcription Factors/genetics ; Transcriptional Activation ; Up-Regulation ; },
abstract = {Regulation of epigenomic functions requires controlled site-specific alteration of epigenetic information. This can be achieved by using designed DNA-binding domains, associated with effector domains, that function as targeted transcription factors or epigenetic modifiers. These effectors have been employed to study the implications of epigenetic modifications, and sequence-specific targeting has been instrumental in understanding the effect of these modification on gene regulation. Ultimately, these tools could be used for therapeutic applications to revert the epigenetic aberrations that have been linked to various diseases. The ability to spatiotemporally control gene expression is especially important for precise regulation of the epigenomic state. In this chapter, we describe the protocol for achieving highly efficient small molecule-inducible transcriptional activation of endogenous mammalian genes, mediated by a dCas9-based system that recruits transcriptional activation domains binding to a chain of concatenated coiled-coil peptides.},
}
@article {pmid36173574,
year = {2023},
author = {Morita, S and Horii, T and Hatada, I},
title = {Regulation of Gene Expression Using dCas9-SunTag Platforms.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2577},
number = {},
pages = {189-195},
pmid = {36173574},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; DNA Methylation ; Epigenesis, Genetic ; *Gene Editing/methods ; Gene Expression Regulation ; Transcriptional Activation ; },
abstract = {Regulating gene expression is important for basic research and therapeutic applications. The epigenome is a record of genetic modifications such as DNA methylation and histone modifications, and epigenetic changes can play a key role in modifying gene expression. With the advent of genome editing technologies, it has become possible to manipulate the epigenome of specific genomic regions to control gene expression. In particular, CRISPR-Cas9 systems have been used widely for epigenome editing due to their high efficiency, versatility, specificity, and ease of use. Here, we describe a protocol for the upregulation of specific genes using the dCas9-SunTag system.},
}
@article {pmid36173573,
year = {2023},
author = {Bashtrykov, P and Rajaram, N and Jeltsch, A},
title = {Efficient Targeted DNA Methylation with dCas9-Coupled DNMT3A-DNMT3L Methyltransferase.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2577},
number = {},
pages = {177-188},
pmid = {36173573},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Chromatin ; DNA/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; *DNA Methylation ; DNA Modification Methylases/genetics ; Epigenesis, Genetic ; Gene Editing ; Methyltransferases/metabolism ; *RNA, Guide/genetics/metabolism ; Transcription Factors/metabolism ; },
abstract = {Epigenome editing is a powerful approach for the establishment of a chromatin environment with desired properties at a selected genomic locus, which is used to influence the transcription of target genes and to study properties and functions of gene regulatory elements. Targeted DNA methylation is one of the most often used types of epigenome editing, which typically aims for gene silencing by methylation of gene promoters. Here, we describe the design principles of EpiEditors for targeted DNA methylation and provide step-by-step guidelines for the realization of this approach. We focus on the dCas9 protein as the state-of-the-art DNA targeting module fused to 10×SunTag as the most frequently used system for editing enhancement. Further, we discuss different flavors of DNA methyltransferase modules used for this purpose including the most specific variants developed recently. Finally, we explain the principles of gRNA selection, outline the setup of the cell culture experiments, and briefly introduce the available options for the downstream DNA methylation data analysis.},
}
@article {pmid36172044,
year = {2022},
author = {Anand, V and Prabhakaran, HS and Gogoi, P and Kanaujia, SP and Kumar, M},
title = {Structural and functional characterization of Cas2 of CRISPR-Cas subtype I-C lacking the CRISPR component.},
journal = {Frontiers in molecular biosciences},
volume = {9},
number = {},
pages = {988569},
pmid = {36172044},
issn = {2296-889X},
abstract = {The genome of pathogenic Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni (rLinCas2C, 12 kDa) and Lai (rLinCas2C_Lai, 8.6 kDa) were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1-71 amino acids) and C-terminal (72-90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues (Tyr7, Asp8, Arg33, and Phe39) with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.},
}
@article {pmid36171658,
year = {2022},
author = {Gostimskaya, I},
title = {CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing.},
journal = {Biochemistry. Biokhimiia},
volume = {87},
number = {8},
pages = {777-788},
pmid = {36171658},
issn = {1608-3040},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; *Gene Editing/methods ; Genomics ; Humans ; *Viruses ; },
abstract = {The development of a method for genome editing based on CRISPR-Cas9 technology was awarded The Nobel Prize in Chemistry in 2020, less than a decade after the discovery of all principal molecular components of the system. For the first time in history a Nobel prize was awarded to two women, Emmanuelle Charpentier and Jennifer Doudna, who made key discoveries in the field of DNA manipulation with the CRISPR-Cas9 system, so-called "genetic scissors". It is difficult to overestimate the importance of the technique as it enables one not only to manipulate genomes of model organisms in scientific experiments, and modify characteristics of important crops and animals, but also has the potential of introducing revolutionary changes in medicine, especially in treatment of genetic diseases. The original biological function of CRISPR-Cas9 system is the protection of prokaryotes from mobile genetic elements, in particular viruses. Currently, CRISPR-Cas9 and related technologies have been successfully used to cure life-threatening diseases, make coronavirus detection tests, and even to modify human embryo cells with the consequent birth of babies carrying the introduced modifications. This intervention with human germplasm cells resulted in wide disapproval in the scientific community due to ethical concerns, and calls for a moratorium on inheritable genomic manipulations. This review focuses on the history of the discovery of the CRISPR-Cas9 system with some aspects of its current applications, including ethical concerns about its use in humans.},
}
@article {pmid36171473,
year = {2022},
author = {Shinkado, S and Saito, H and Yamazaki, M and Kotera, S and Arazoe, T and Arie, T and Kamakura, T},
title = {Genome editing using a versatile vector-based CRISPR/Cas9 system in Fusarium species.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {16243},
pmid = {36171473},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; *Fusarium/genetics ; *Gene Editing ; Humans ; Nuclear Localization Signals/genetics ; RNA, Guide/genetics ; },
abstract = {Fusarium species include important filamentous fungal pathogens that can infect plants, animals, and humans. Meanwhile, some nonpathogenic Fusarium species are promising biocontrol agents against plant pathogens. Here, we developed a genome editing technology using a vector-based CRISPR/Cas9 system for Fusarium oxysporum f. sp. lycopersici (Fol). This optimized CRISPR/Cas9 system, harboring an endogenous U6 small nuclear RNA promoter for the expression of single-guide RNA and an endogenous H2B nuclear localization signal for the localization of Cas9, enabled efficient targeted gene knock-out, including in the accessory chromosomal regions in Fol. We further demonstrated single crossover-mediated targeted base editing and endogenous gene tagging. This system was also applicable for genome editing in F. oxysporum f. sp. spinaciae and F. commune without any modifications, suggesting that this CRISPR/Cas9 vector has a potential application for a broad range of researches on other Fusarium species.},
}
@article {pmid36171290,
year = {2022},
author = {Xu, Y and Viswanatha, R and Sitsel, O and Roderer, D and Zhao, H and Ashwood, C and Voelcker, C and Tian, S and Raunser, S and Perrimon, N and Dong, M},
title = {CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.},
journal = {Nature},
volume = {610},
number = {7931},
pages = {349-355},
pmid = {36171290},
issn = {1476-4687},
support = {R01 NS080833/NS/NINDS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AI170835/AI/NIAID NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; P41 GM132087/GM/NIGMS NIH HHS/United States ; R01 NS117626/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Bacterial Toxins/metabolism ; Biological Control Agents ; *CRISPR-Cas Systems ; Culicidae ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/cytology/genetics/metabolism/microbiology ; Fat Body/cytology ; *Gene Editing ; Gene Knockdown Techniques ; Hemocytes ; Humans ; Moths ; Mucins ; Pest Control, Biological ; Phagocytosis ; Photorhabdus/metabolism ; Repetitive Sequences, Amino Acid ; Transgenes ; *Virulence Factors/metabolism ; },
abstract = {Entomopathogenic nematodes are widely used as biopesticides[1,2]. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors[3-6]. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.},
}
@article {pmid36171275,
year = {2022},
author = {van Beljouw, SPB and Sanders, J and Rodríguez-Molina, A and Brouns, SJJ},
title = {RNA-targeting CRISPR-Cas systems.},
journal = {Nature reviews. Microbiology},
volume = {},
number = {},
pages = {},
pmid = {36171275},
issn = {1740-1534},
abstract = {CRISPR-Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR-Cas systems sense and cleave viral DNA, type III and type VI CRISPR-Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR-Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.},
}
@article {pmid36170834,
year = {2022},
author = {Wang, Y and Wang, Y and Pan, D and Yu, H and Zhang, Y and Chen, W and Li, F and Wu, Z and Ji, Q},
title = {Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease.},
journal = {Cell reports},
volume = {40},
number = {13},
pages = {111418},
doi = {10.1016/j.celrep.2022.111418},
pmid = {36170834},
issn = {2211-1247},
mesh = {Amino Acids/metabolism ; *CRISPR-Cas Systems/genetics ; Clostridiales ; Endonucleases/genetics/metabolism ; Gene Editing ; Humans ; RNA/metabolism ; *RNA, Guide/metabolism ; },
abstract = {Gene therapy is limited by inefficient delivery of large clustered regularly interspaced short palindromic repeat (CRISPR) effectors, such as Cas9 and Cas12a nucleases. Cas12f nucleases are currently one of the most compact CRISPR genome editors. However, the available toolkit of efficient Cas12f editors is limited. Here, we report the characterization and engineering of a miniature CRISPR-Cas12f system from Syntrophomonas palmitatica (SpaCas12f1, 497 amino acids). We show that CRISPR-SpaCas12f1 cleaves double-stranded DNA (dsDNA) with 5' T-rich PAM specificity and is naturally active for genome editing in bacteria. We identify that CRISPR-SpaCas12f1 trans-activating CRISPR RNA (tracrRNA) harbors a unique head-to-toe hairpin structure, and the natural hairpin structure is a key factor in restricting genome editing by SpaCas12f1 in human cells. Systematical engineering of SpaCas12f1 guide RNA transforms CRISPR-SpaCas12f1 into an efficient genome editor comparable to Francisella novicida CRISPR-Cas12a. Our findings expand the mini CRISPR toolbox, paving the way for therapeutic applications of CRISPR-SpaCas12f1 and engineering compact genome manipulation technologies.},
}
@article {pmid36169633,
year = {2022},
author = {Verhagen, HJMP and Kuijk, C and Rutgers, L and Kokke, AM and van der Meulen, SA and van Mierlo, G and Voermans, C and van den Akker, E},
title = {Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {702-716},
doi = {10.1089/crispr.2021.0112},
pmid = {36169633},
issn = {2573-1602},
mesh = {Humans ; *RNA, Guide/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics ; Ribonucleoproteins/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34[+] HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.},
}
@article {pmid36169448,
year = {2022},
author = {Ortiz-Cartagena, C and Fernández-García, L and Blasco, L and Pacios, O and Bleriot, I and López, M and Cantón, R and Tomás, M},
title = {Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2.},
journal = {Microbiology spectrum},
volume = {10},
number = {5},
pages = {e0239822},
pmid = {36169448},
issn = {2165-0497},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; COVID-19 Testing ; Clinical Laboratory Techniques/methods ; Molecular Diagnostic Techniques/methods ; CRISPR-Cas Systems ; Technology ; Endonucleases ; RNA ; Nucleotides ; },
abstract = {At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (CT) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples.},
}
@article {pmid36165075,
year = {2022},
author = {Zhai, LM and Zhao, Y and Xiao, RL and Zhang, SQ and Tian, BH and Li, XX and Zhang, R and Ma, RS and Liang, HX},
title = {Nuclear-targeted carbon quantum dot mediated CRISPR/Cas9 delivery for fluorescence visualization and efficient editing.},
journal = {Nanoscale},
volume = {14},
number = {39},
pages = {14645-14660},
doi = {10.1039/d2nr04281a},
pmid = {36165075},
issn = {2040-3372},
mesh = {*CRISPR-Cas Systems ; Carbon ; HeLa Cells ; Humans ; Polyethylene Glycols ; Polyethyleneimine ; *Quantum Dots ; },
abstract = {Nuclear targeted delivery has great potential in improving the efficiency of non-viral carrier mediated genome editing. However, direct and efficient delivery of CRISPR/Cas9 plasmid into the nucleus remains a challenge. In this study, a nuclear targeted gene delivery platform based on fluorescent carbon quantum dots (CQDs) was developed. Polyethylenimine (PEI) and polyethylene glycol (PEG) synergistically passivated the surface of CQDs, providing an excitation-independent green-emitting fluorescent CQDs-PEI-PEG conjugate (CQDs-PP) with an ultra-small size and positive surface charge. Here we show that CQDs-PP could bind CRISPR/Cas9 plasmid to form a nano-complex by electrostatic attraction, which can bypass lysosomes and enter the nucleus by passive diffusion, and thereby improve the transfection efficiency. Also, CQDs-PP could deliver CRISPR/Cas9 plasmid into HeLa cells, resulting in the insertion/deletion mutation of the target EFHD1 gene. More importantly, CQDs-PP exhibited a considerably higher gene editing efficiency as well as comparable or lower cytotoxicity relative to Lipo2000 and PEI-passivated CQDs-PEI (CQDs-P). Thus, the nuclear-targeted CQDs-PP is expected to constitute an efficient CRISPR/Cas9 delivery carrier in vitro with imaging-trackable ability.},
}
@article {pmid36163606,
year = {2022},
author = {Hillary, VE and Ceasar, SA},
title = {A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {1-15},
pmid = {36163606},
issn = {1559-0305},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA). The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9, Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins and their applications, which have the potential to be used in next-generation precise genome engineering.},
}
@article {pmid36163402,
year = {2022},
author = {Saleem, A and Abbas, MK and Wang, Y and Lan, F},
title = {hPSC gene editing for cardiac disease therapy.},
journal = {Pflugers Archiv : European journal of physiology},
volume = {474},
number = {11},
pages = {1123-1132},
pmid = {36163402},
issn = {1432-2013},
mesh = {CRISPR-Cas Systems/genetics ; *Cardiovascular Diseases/genetics/metabolism/therapy ; Gene Editing/methods ; *Heart Diseases/genetics/metabolism/therapy ; Humans ; *Pluripotent Stem Cells/metabolism ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. However, the lack of human cardiomyocytes with proper genetic backgrounds limits the study of disease mechanisms. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have significantly advanced the study of these conditions. Moreover, hPSC-CMs made it easy to study CVDs using genome-editing techniques. This article discusses the applications of these techniques in hPSC for studying CVDs. Recently, several genome-editing systems have been used to modify hPSCs, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9). We focused on the recent advancement of genome editing in hPSCs, which dramatically improved the efficiency of the cell-based mechanism study and therapy for cardiac diseases.},
}
@article {pmid36163386,
year = {2022},
author = {Mukherjee, IA and Gabel, C and Noinaj, N and Bondy-Denomy, J and Chang, L},
title = {Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor.},
journal = {Nature chemical biology},
volume = {18},
number = {12},
pages = {1417-1424},
pmid = {36163386},
issn = {1552-4469},
support = {R01 GM138675/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/genetics ; Viral Proteins/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; *Bacteriophages/genetics/metabolism ; Pseudomonas aeruginosa/genetics/metabolism ; },
abstract = {Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy-AcrIF24-promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.},
}
@article {pmid36162812,
year = {2022},
author = {Escobar, M and Li, J and Patel, A and Liu, S and Xu, Q and Hilton, IB},
title = {Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells.},
journal = {ACS synthetic biology},
volume = {11},
number = {10},
pages = {3239-3250},
pmid = {36162812},
issn = {2161-5063},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome ; Genomics ; },
abstract = {CRISPR/Cas technologies have revolutionized the ability to redesign genomic information and tailor endogenous gene expression. Nevertheless, the discovery and development of new CRISPR/Cas systems has resulted in a lack of clarity surrounding the relative efficacies among these technologies in human cells. This deficit makes the optimal selection of CRISPR/Cas technologies in human cells unnecessarily challenging, which in turn hampers their adoption, and thus ultimately limits their utility. Here, we designed a series of endogenous testbed systems to methodically quantify and compare the genome editing, CRISPRi, and CRISPRa capabilities among 10 different natural and engineered Cas protein variants spanning Type II and Type V CRISPR/Cas families. We show that although all Cas protein variants are capable of genome editing and transcriptional control in human cells, hierarchies exist, particularly for genome editing and CRISPRa applications, wherein Cas9 ≥ Cas12a > Cas12e/Cas12j. Our findings also highlight the utility of our modular testbed platforms to rapidly and systematically quantify the functionality of practically any natural or engineered genomic-targeting Cas protein in human cells.},
}
@article {pmid36162766,
year = {2022},
author = {Wang, Y and Tang, Y and Zhao, XM and Huang, G and Gong, JH and Yang, SD and Li, H and Wan, WJ and Jia, CH and Chen, G and Zhang, XN},
title = {A multifunctional non-viral vector for the delivery of MTH1-targeted CRISPR/Cas9 system for non-small cell lung cancer therapy.},
journal = {Acta biomaterialia},
volume = {153},
number = {},
pages = {481-493},
doi = {10.1016/j.actbio.2022.09.046},
pmid = {36162766},
issn = {1878-7568},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Genetic Vectors ; *Lung Neoplasms/genetics/therapy ; Gene Editing/methods ; DNA ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.},
}
@article {pmid36162731,
year = {2022},
author = {Ghosh, D and Nilavar, NM and Raghavan, SC},
title = {A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1866},
number = {12},
pages = {130246},
doi = {10.1016/j.bbagen.2022.130246},
pmid = {36162731},
issn = {1872-8006},
mesh = {Humans ; Mice ; Animals ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; DNA/metabolism ; Cell Line ; },
abstract = {KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70-2-DG). We observed that the N6-KU70-2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.},
}
@article {pmid36162509,
year = {2022},
author = {Chang, C and Ma, G and Cheung, E and Hutchins, AP},
title = {A programmable system to methylate and demethylate N[6]-methyladenosine (m[6]A) on specific RNA transcripts in mammalian cells.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {11},
pages = {102525},
pmid = {36162509},
issn = {1083-351X},
mesh = {Animals ; Humans ; Mice ; *Adenosine/metabolism ; Gene Expression Regulation ; *RNA/genetics/metabolism ; *RNA, Long Noncoding/genetics ; RNA-Binding Proteins/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Mouse Embryonic Stem Cells ; RNA, Circular/genetics ; },
abstract = {RNA N[6]-methyladenosine (m[6]A) is the most abundant internal mRNA modification and forms part of an epitranscriptomic system that modulates RNA function. m[6]A is reversibly catalyzed by specific enzymes, and those modifications can be recognized by RNA-binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m[6]A participation in critical biological functions, this exploration has mainly been conducted through the global KO or knockdown of the writers, erasers, or readers of m[6]A. Consequently, there is a lack of information about the role of m[6]A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m[6]A. Here, we demonstrate a CRISPR/dCas13a-based RNA m[6]A editors, which can target RNAs using a single or multiple CRISPR RNA array to methylate or demethylate m[6]A in human 293T cells and mouse embryonic stem cells. We systematically assay its capabilities to enable the targeted rewriting of m[6]A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we use the system to specifically modulate m[6]A levels on the noncoding XIST (X-inactive specific transcript) to modulate X chromosome silencing and activation. The editors described here can be used to explore the roles of m[6]A in biological processes.},
}
@article {pmid36161926,
year = {2022},
author = {Marciano, S and Ionescu, TM and Saw, RS and Cheong, RY and Kirik, D and Maurer, A and Pichler, BJ and Herfert, K},
title = {Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {40},
pages = {e2122552119},
pmid = {36161926},
issn = {1091-6490},
mesh = {Animals ; *Brain/metabolism ; *CRISPR-Cas Systems ; *Dopamine/metabolism ; *Dopaminergic Neurons/metabolism ; Gene Editing ; *Neuroimaging ; Rats ; *Vesicular Monoamine Transport Proteins/genetics ; },
abstract = {Receptors, transporters, and ion channels are important targets for therapy development in neurological diseases, but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on the whole-brain level. We combine CRISPR-Cas9 gene editing with in vivo positron emission tomography (PET) and functional MRI (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene network relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR-Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a tool to investigate the impact of specific genes on brain molecular and functional dynamics, which will help to develop tailored therapies for normalizing brain function.},
}
@article {pmid36161449,
year = {2022},
author = {Zhao, X and Yuan, K and Liu, Y and Zhang, N and Yang, L and Zhang, Y and Wang, Y and Ji, J and Fang, Z and Han, F and Lv, H},
title = {In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea.},
journal = {Plant biotechnology journal},
volume = {20},
number = {12},
pages = {2242-2244},
pmid = {36161449},
issn = {1467-7652},
mesh = {*Gene Editing ; Haploidy ; CRISPR-Cas Systems ; Genome, Plant/genetics ; *Brassica/genetics ; },
}
@article {pmid36160971,
year = {2022},
author = {Pandey, PK and Bhowmik, P and Kagale, S},
title = {Optimized methods for random and targeted mutagenesis in field pea (Pisum sativum L.).},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {995542},
pmid = {36160971},
issn = {1664-462X},
abstract = {Field pea is an important pulse crop for its dense nutritional profile and contribution to sustainable agricultural practices. Recently, it has received extensive attention as a potential leading source of plant-based proteins. However, the adoption of peas as a mainstream source of proteins is affected by a relatively moderate protein content, anti-nutritional factors and high levels of off-flavor components that reduce protein quality. Availability of genetic variation for desirable seed quality traits is the foundation for the sustainable development of pea varieties with improved protein content and quality. Mutagenesis has been an important tool in gene functional characterization studies and creating genetic variability for crop breeding. Large-scale mutagenesis of a crop using physical and chemical agents requires diligent selection of the mutagen and optimization of its dose to increase the frequency of mutations. In this study, we present detailed optimized protocols for physical and chemical mutagenesis of pea using gamma irradiation and ethyl methanesulfonate (EMS), respectively. Gamma radiation and EMS titration kill curves were established to identify optimal doses of the two mutagenic agents. Based on germination, survival rate and growth phenotypes, a gamma radiation dose of 225 Gy and EMS concentration of 5 mm were selected as optimal dosages for mutagenesis in field pea. The presented protocol has been modified from previously established mutagenesis protocols in other crop plants. Our results indicate that the optimal mutagen dosage is genotype dependent. CRISPR/Cas-based gene editing provides a precise and rapid method for targeted genetic manipulation in plants. With the recent success of gene editing in pea using CRISPR/Cas, this innovative technology is expected to become an integral component of the gene discovery and crop improvement toolkit in pea. Here, we describe an optimized methods for targeted mutagenesis of pea protoplasts, including mesophyll protoplast extraction, PEG-mediated transformation and gene editing of a LOX gene using CRISPR/Cas system. The general strategies and methods of mutagenesis described here provide an essential resource for mutation breeding and functional genomics studies in pea. These methods also provide a foundation for similar studies in other crops.},
}
@article {pmid36160203,
year = {2022},
author = {Hossain, MM},
title = {Wheat blast: A review from a genetic and genomic perspective.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {983243},
pmid = {36160203},
issn = {1664-302X},
abstract = {The newly emerged wheat blast fungus Magnaporthe oryzae Triticum (MoT) is a severe threat to global wheat production. The fungus is a distinct, exceptionally diverse lineage of the M. oryzae, causing rice blast disease. Genome-based approaches employing MoT-specific markers are used to detect MoT field isolates. Sequencing the whole genome indicates the presence of core chromosome and mini-chromosome sequences that harbor effector genes and undergo divergent evolutionary routes. Significant genetic and pathotype diversity within the fungus population gives ample potential for evolutionary change. Identifying and refining genetic markers allows for tracking genomic regions with stable blast resistance. Introgression of quantitative and R gene resistance into popular cultivars is crucial to controlling disease in areas where the pathogen population is diverse and well established. Novel approaches such as CRISPR/Cas-9 genome editing could generate resistant varieties in wheat within a short time. This chapter provides an extensive summary of the genetic and genomic aspects of the wheat blast fungus MoT and offers an essential resource for wheat blast research in the affected areas.},
}
@article {pmid36157513,
year = {2022},
author = {Li, CZ and Hu, TY},
title = {Nanotechnology Powered CRISPR-Cas Systems for Point of Care Diagnosis and Therapeutic.},
journal = {Research (Washington, D.C.)},
volume = {2022},
number = {},
pages = {9810237},
pmid = {36157513},
issn = {2639-5274},
}
@article {pmid36156805,
year = {2022},
author = {Lau, RK and Enustun, E and Gu, Y and Nguyen, JV and Corbett, KD},
title = {A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage.},
journal = {The EMBO journal},
volume = {41},
number = {22},
pages = {e111540},
pmid = {36156805},
issn = {1460-2075},
support = {F31 GM137600/GM/NIGMS NIH HHS/United States ; R35 GM144121/GM/NIGMS NIH HHS/United States ; T32 GM127235/GM/NIGMS NIH HHS/United States ; T32 GM127235/GM/NIGMS NIH HHS/United States ; F31 GM137600/GM/NIGMS NIH HHS/United States ; R35 GM144121/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages/genetics ; Bacteria ; Signal Transduction ; DNA Damage ; },
abstract = {To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction-modification, CRISPR-Cas, and many others. Here, we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS immune systems and demonstrate that this module drives the expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is activated in vitro by single-stranded DNA, and in cells by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. We identify CapH- and CapP-related proteins associated with diverse known and putative bacterial immune systems including DISARM and Pycsar antiphage operons. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal signal of cell stress, potentially enabling multiple immune systems to mount a coordinated defensive response against an invading pathogen.},
}
@article {pmid36156623,
year = {2022},
author = {Zhou, W and Yang, K and Zhang, L and Liang, Z and Zhang, Y},
title = {[Recent advances in clustered regularly interspaced short palindromic repeats-based detection of severe acute respiratory syndrome coronavirus 2].},
journal = {Se pu = Chinese journal of chromatography},
volume = {40},
number = {9},
pages = {773-781},
pmid = {36156623},
issn = {1000-8713},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; SARS-CoV-2/genetics ; },
abstract = {The rapid global spread of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has introduced various challenges in global public health systems. The poor applicability and sensitivity of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and antigen-based tests, as well as the persistent emergence of SARS-CoV-2 variants with different mutations hinder satisfactory epidemic prevention and control. Therefore, there is an urgent need for diagnostic technologies capable of distinguishing SARS-CoV-2 variants with high sensitivity and low (or no) equipment dependence. Diagnosis based on clustered regularly interspaced short palindromic repeats (CRISPR) has low equipment requirements and is programmable, sensitive, and easy to use. Various nucleic acid detection tools with great clinical potential have been developed for the diagnosis of infectious diseases. Therefore, this review focuses on the reported state-of-the-art CRISPR diagnostic technologies developed for the detection and differentiation of SARS-CoV-2 variants, summarizes their characteristics and provides an outlook for their development.},
}
@article {pmid36156138,
year = {2022},
author = {Sheng, A and Yang, J and Tang, L and Niu, L and Cheng, L and Zeng, Y and Chen, X and Zhang, J and Li, G},
title = {Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis.},
journal = {Nucleic acids research},
volume = {50},
number = {18},
pages = {10562-10570},
pmid = {36156138},
issn = {1362-4962},
mesh = {*CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; DNA, Ribosomal ; Gene Editing/methods ; Hydrazones/pharmacology ; },
abstract = {In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.},
}
@article {pmid36155952,
year = {2022},
author = {Kumar, M and Maiti, S and Chakraborty, D},
title = {Capturing nucleic acid variants with precision using CRISPR diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {217},
number = {},
pages = {114712},
doi = {10.1016/j.bios.2022.114712},
pmid = {36155952},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Humans ; *Nucleic Acids/genetics ; Nucleotides ; RNA ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas systems have the ability to precisely target nucleotide sequences and enable their rapid identification and modification. While nucleotide modification has enabled the therapeutic correction of diseases, the process of identifying the target DNA or RNA has greatly expanded the field of molecular diagnostics in recent times. CRISPR-based DNA/RNA detection through programmable nucleic acid binding or cleavage has been demonstrated for a large number of pathogenic and non-pathogenic targets. Combining CRISPR detection with nucleic acid amplification and a terminal signal readout step allowed the development of numerous rapid and robust nucleic acid platforms. Wherever the Cas effector can faithfully distinguish nucleobase variants in the target, the platform can also be extended for sequencing-free rapid variant detection. Some initial PAM disruption-based SNV detection reports were limited to finding or integrating mutated/mismatched nucleotides within the PAM sequences. In this review, we try to summarize the developments made in CRISPR diagnostics (CRISPRDx) to date emphasizing CRISPR-based SNV detection. We also discuss the applications where such diagnostic modalities can be put to use, covering various fields of clinical research, SNV screens, disease genotyping, primary surveillance during microbial infections, agriculture, food safety, and industrial biotechnology. The ease of rapid design and implementation of such multiplexable assays can potentially expand the applications of CRISPRDx in the domain of affinity-based target sequencing, with immense possibilities for low-cost, quick, and widespread usage. In the end, in combination with proximity assays and a suicidal gene approach, CRISPR-based in vivo SNV detection and cancer cell targeting can be formulated as personalized gene therapy.},
}
@article {pmid36155858,
year = {2022},
author = {Konwar, AN and Hazarika, SN and Bharadwaj, P and Thakur, D},
title = {Emerging Non-Traditional Approaches to Combat Antibiotic Resistance.},
journal = {Current microbiology},
volume = {79},
number = {11},
pages = {330},
pmid = {36155858},
issn = {1432-0991},
mesh = {Anti-Bacterial Agents/pharmacology/therapeutic use ; *Anti-Infective Agents/pharmacology ; Bacteria/genetics ; Drug Resistance, Microbial ; Humans ; *Peptide Nucleic Acids/pharmacology ; },
abstract = {An increasing number of bacterial pathogens are acquiring resistance to the commonly used antibiotics. This has spurred a global threat leading to a resistance era and has penetrated the consciousness of the common people and the clinicians alike. The delay in discovering new antibiotics has exacerbated the resistance problem, forcing researchers to focus on unconventional antimicrobial therapeutics that differ from conventional antibiotics. Alternative therapies have emerged in recent years, including antimicrobial peptides, phage therapy, efflux pump inhibitors, antibodies, and immunomodulatory agents, which have produced impressive results in both laboratory and in clinical trials. Additionally, ultra-narrow-spectrum therapeutics such as CRISPR-Cas system and peptide nucleic acids aided in the development of sequence-specific antimicrobials. Moreover, combinatorial therapies that combine these new approaches have been efficient enough to get approval for clinical use and have accelerated the discovery of novel combination approaches that enhance the performance of already in-use antibiotics. In this review, we provide an overview of these approaches along with studies that focus on the uncharted microbial territories that have been able to deliver some of the important new antibiotics of recent times. It is hoped that the information gathered in this article will provide an update on the current antibiotic resistance threat and encourage profound research.},
}
@article {pmid36155855,
year = {2022},
author = {Du, Y and Han, D and An, Z and Wang, J and Gao, Z},
title = {CRISPR/dCas9-surface-enhanced Raman scattering for the detection of drug resistance gene macB.},
journal = {Mikrochimica acta},
volume = {189},
number = {10},
pages = {394},
pmid = {36155855},
issn = {1436-5073},
mesh = {Animals ; Anti-Bacterial Agents ; CRISPR-Cas Systems ; Cattle ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance ; *Nucleic Acids ; Spectrum Analysis, Raman ; },
abstract = {Antibiotics have brought many benefits to public health systems worldwide since their first use in the last century, yet with their overuse in clinical treatment and livestock farming, new public health issues have arisen. Previously, we found in our experiments that the levels of macB genes in bovine raw milk ranked among the top of many drug resistance genes. In this paper, we present an analysis of regularly interspaced clustered short palindromic repeats (CRISPR) combined with surface-enhanced Raman scattering (SERS) technology for the detection of the drug resistance gene macB. The analysis was accomplished through the collaboration of the CRISPR system's ability to specifically identify genes and the more sensitive performance of the SERS. The analysis detects the drug resistance gene macB and does not yet require complex steps such as nucleic acid amplification. This method may prove to be an effective method for accurate detection of the drug-resistant gene macB, thus enabling more effective prevention of contamination of drug-resistant genes in food hygiene.},
}
@article {pmid36154917,
year = {2022},
author = {Heider, J and Sperlich, D and Vogel, S and Breitmeyer, R and Volkmer, H},
title = {Generation of two induced pluripotent stem cell lines (TMOi001-A-5, TMOi001-A-6) carrying variants in DISC1 exon 2 using CRISPR/Cas9 gene editing.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102925},
doi = {10.1016/j.scr.2022.102925},
pmid = {36154917},
issn = {1876-7753},
mesh = {*Gene Editing ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Mutation ; Exons/genetics ; },
abstract = {DISC1 is a scaffold protein involved in key developmental processes such as neuronal migration, differentiation and neurogenesis. Genetic variants of the DISC1 gene have been linked to neuropsychiatric disorders like schizophrenia, bipolar disorder and major depression. Here, we generated two isogenic iPSC lines carrying mutations in DISC1 exon 2 using CRISPR/Cas9 gene editing. Both lines express pluripotency markers, can be differentiated into the three germ layers and present a normal karyotype. The generated iPSC lines can be used to study the implications of DISC1 mutations in the context of neuropsychiatric diseases in vitro.},
}
@article {pmid36154577,
year = {2022},
author = {Abkhooie, L and Saberianpour, S},
title = {CRISPR/Cas9 Tool for MicroRNAs Editing in Cardiac Development, Function, and Disease.},
journal = {MicroRNA (Shariqah, United Arab Emirates)},
volume = {},
number = {},
pages = {},
doi = {10.2174/2211536611666220922092601},
pmid = {36154577},
issn = {2211-5374},
abstract = {CRISPR/Cas9 is a powerful gene-editing technology. Extensive scientific data exist that the CRISPR/Cas9 system can target small, non-coding, active RNA molecules including microRNAs [miRNAs]. miRNAs have been recognized as key regulators of different cell biological processes, such as modulation of fibrosis and cardiac hypertrophy, as well as the regulation of cardiomyocytes. Also, it has been demonstrated that miRNAs strongly affect organ evolution and the concentration of miRNAs can involve in the differentiation, development, and function of different organs. In addition, the current findings clearly indicate that miRNAs can select and control their targets based on their own concentrations. CRISPR/Cas9 genome-editing technology is a stronger system for stopping miRNAs than previous methods including antisense inhibitors. CRISPR/Cas9 tools can be used to eliminate small areas of DNA and determine miRNA in cases where similar groups of miRNAs are in the same strand. Herein, besides other emerging strategies we critically summarize the recent investigations linking miRNA-targeted therapeutics and CRISPR/Cas9 system, to precisely clarify and combine different delivery platforms and cell-fate engineering of miRNAs function and miRNA-based therapeutic intervention in cardiac development, function, and disease. Based on our findings from the literature, it appears that the use of the CRISPR/Cas technology provides new perspectives for understanding the molecular mechanism of cardiovascular disease and can be effective in the treatment and control of cardiac development, function, and disease in the future.},
}
@article {pmid36153319,
year = {2022},
author = {Xin, C and Yin, J and Yuan, S and Ou, L and Liu, M and Zhang, W and Hu, J},
title = {Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5623},
pmid = {36153319},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Dependovirus/genetics/metabolism ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Genetic Therapy ; },
abstract = {Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.},
}
@article {pmid36151929,
year = {2022},
author = {Zönnchen, J and Gantner, J and Lapin, D and Barthel, K and Eschen-Lippold, L and Erickson, JL and Villanueva, SL and Zantop, S and Kretschmer, C and Joosten, MHAJ and Parker, JE and Guerois, R and Stuttmann, J},
title = {EDS1 complexes are not required for PRR responses and execute TNL-ETI from the nucleus in Nicotiana benthamiana.},
journal = {The New phytologist},
volume = {236},
number = {6},
pages = {2249-2264},
doi = {10.1111/nph.18511},
pmid = {36151929},
issn = {1469-8137},
mesh = {Tobacco/genetics/metabolism ; *Arabidopsis Proteins/metabolism ; DNA-Binding Proteins/metabolism ; Plant Immunity/genetics ; *Arabidopsis/metabolism ; Receptors, Cell Surface/metabolism ; Plant Diseases ; Carboxylic Ester Hydrolases/metabolism ; },
abstract = {Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown.},
}
@article {pmid36151800,
year = {2022},
author = {Hai, Y and Wang, X and Xie, J},
title = {[The role of bacterial toxin-antitoxin systems in phage abortive infections].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {9},
pages = {3291-3300},
doi = {10.13345/j.cjb.220140},
pmid = {36151800},
issn = {1872-2075},
mesh = {Anti-Bacterial Agents ; *Antitoxins/chemistry ; Bacteria/genetics ; Bacterial Proteins/chemistry ; *Bacterial Toxins/genetics ; *Bacteriophages/genetics ; *Toxin-Antitoxin Systems/genetics ; },
abstract = {Bacteria are often infected by large numbers of phages, and host bacteria have evolved diverse molecular strategies in the race with phages, with abortive infection (Abi) being one of them. The toxin-antitoxin system (TA) is expressed in response to bacterial stress, mediating hypometabolism and even dormancy, as well as directly reducing the formation of offspring phages. In addition, some of the toxins' sequences and structures are highly homologous to Cas, and phages even encode antitoxin analogs to block the activity of the corresponding toxins. This suggests that the failure of phage infection due to bacterial death in abortive infections is highly compatible with TA function, whereas TA may be one of the main resistance and defense forces for phage infestation of the host. This review summarized the TA systems involved in phage abortive infections based on classification and function. Moreover, TA systems with abortive functions and future use in antibiotic development and disease treatment were predicted. This will facilitate the understanding of bacterial-phage interactions as well as phage therapy and related synthetic biology research.},
}
@article {pmid36151310,
year = {2022},
author = {},
title = {Redirecting meiotic recombination by CRISPR-Cas-mediated chromosome engineering.},
journal = {Nature plants},
volume = {8},
number = {10},
pages = {1144-1145},
pmid = {36151310},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; *Homologous Recombination ; Genetic Engineering ; Meiosis/genetics ; Chromosomes ; Gene Editing ; },
}
@article {pmid36151095,
year = {2022},
author = {Chan, WF and Coughlan, HD and Chen, Y and Keenan, CR and Smyth, GK and Perkins, AC and Johanson, TM and Allan, RS},
title = {Activation of stably silenced genes by recruitment of a synthetic de-methylating module.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5582},
pmid = {36151095},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Methylation ; Epigenesis, Genetic ; Promoter Regions, Genetic/genetics ; Transcription Factors/metabolism ; Transcriptional Activation ; },
abstract = {Stably silenced genes that display a high level of CpG dinucleotide methylation are refractory to the current generation of dCas9-based activation systems. To counter this, we create an improved activation system by coupling the catalytic domain of DNA demethylating enzyme TET1 with transcriptional activators (TETact). We show that TETact demethylation-coupled activation is able to induce transcription of suppressed genes, both individually and simultaneously in cells, and has utility across a number of cell types. Furthermore, we show that TETact can effectively reactivate embryonic haemoglobin genes in non-erythroid cells. We anticipate that TETact will expand the existing CRISPR toolbox and be valuable for functional studies, genetic screens and potential therapeutics.},
}
@article {pmid36149915,
year = {2022},
author = {Rubben, K and Tilleman, L and Deserranno, K and Tytgat, O and Deforce, D and Van Nieuwerburgh, F},
title = {Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping.},
journal = {PLoS genetics},
volume = {18},
number = {9},
pages = {e1010176},
pmid = {36149915},
issn = {1553-7404},
mesh = {Alleles ; CRISPR-Cas Systems ; *Cytochrome P-450 CYP2D6/genetics ; DNA ; Genotype ; *Nanopore Sequencing ; Nucleotides ; },
abstract = {CYP2D6 is a very important pharmacogene as it is responsible for the metabolization or bioactivation of 20 to 30% of the clinically used drugs. However, despite its relatively small length of only 4.4 kb, it is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.},
}
@article {pmid36149578,
year = {2022},
author = {Wienmeister, A},
title = {Rereading Habermas in Times of CRISPR-cas: A Critique of and an Alternative to the Instrumentalist Interpretation of the Human Nature Argument.},
journal = {Journal of bioethical inquiry},
volume = {},
number = {},
pages = {},
pmid = {36149578},
issn = {1176-7529},
abstract = {Habermas's argument from human nature, which speaks in favour of holding back the use of human germline editing for purposes of enhancement, has lately received criticism anew. Prominent are objections to its supposedly genetic essentialist and determinist framework, which underestimates social impacts on human development. I argue that this criticism originates from an instrumentalist reading of Habermas's argument, which wrongly focuses on empirical conditions and means-ends-relations. Drawing on Habermas's distinction of a threefold use of practical reason, I show how an alternative-the ethical-reading avoids essentialist and determinist objections by addressing an existential level of sense making. I present three reasons that speak in favour of the ethical reading and I demonstrate how it incorporates social aspects of character formation. Habermas's account therefore offers exactly what the critics claim is missing. The paper concludes with a conceptual challenge that the ethical reading has to face within Habermas's overall approach to genetic engineering.},
}
@article {pmid36149314,
year = {2022},
author = {Salvi, P and Varshney, V and Majee, M},
title = {Raffinose family oligosaccharides (RFOs): role in seed vigor and longevity.},
journal = {Bioscience reports},
volume = {42},
number = {10},
pages = {},
pmid = {36149314},
issn = {1573-4935},
mesh = {*Longevity/genetics ; Oligosaccharides/metabolism ; Raffinose/metabolism ; *Seeds/genetics ; Sugars/metabolism ; },
abstract = {Seed vigor and longevity are important agronomic attributes, as they are essentially associated with crop yield and thus the global economy. Seed longevity is a measure of seed viability and the most essential property in gene bank management since it affects regeneration of seed recycling. Reduced seed life or storability is a serious issue in seed storage since germplasm conservation and agricultural enhancement initiatives rely on it. The irreversible and ongoing process of seed deterioration comprises a complex gene regulatory network and altered metabolism that results in membrane damage, DNA integrity loss, mitochondrial dysregulation, protein damage, and disrupted antioxidative machinery. Carbohydrates and/or sugars, primarily raffinose family oligosaccharides (RFOs), have emerged as feasible components for boosting or increasing seed vigor and longevity in recent years. RFOs are known to perform diverse functions in plants, including abiotic and biotic stress tolerance, besides being involved in regulating seed germination, desiccation tolerance, vigor, and longevity. We emphasized and analyzed the potential impact of RFOs on seed vigor and longevity in this review. Here, we comprehensively reviewed the molecular mechanisms involved in seed longevity, RFO metabolism, and how RFO content is critical and linked with seed vigor and longevity. Further molecular basis, biotechnological approaches, and CRISPR/Cas applications have been discussed briefly for the improvement of seed attributes and ultimately crop production. Likewise, we suggest advancements, challenges, and future possibilities in this area.},
}
@article {pmid36146851,
year = {2022},
author = {Teng, M and Zhou, ZY and Yao, Y and Nair, V and Zhang, GP and Luo, J},
title = {A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology.},
journal = {Viruses},
volume = {14},
number = {9},
pages = {},
pmid = {36146851},
issn = {1999-4915},
support = {BBS/OS/NW/000007/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Antibodies, Monoclonal ; Antigens, Viral ; CRISPR-Cas Systems ; Chickens ; Chromatography, Liquid ; Epitopes/genetics ; *Herpesvirus 2, Gallid/genetics ; *Marek Disease ; Tandem Mass Spectrometry ; Technology ; Viral Proteins/genetics ; },
abstract = {Marek's disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek's disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant-GX0101Δpp38-and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas-namely, 4F9, 31G7, 34F2, and 35G9-were demonstrated to secrete specific antibodies against MDV-1's pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses-especially for large DNA viruses such as herpesviruses.},
}
@article {pmid36146815,
year = {2022},
author = {Wiegand, T and Nemudryi, A and Nemudraia, A and McVey, A and Little, A and Taylor, DN and Walk, ST and Wiedenheft, B},
title = {The Rise and Fall of SARS-CoV-2 Variants and Ongoing Diversification of Omicron.},
journal = {Viruses},
volume = {14},
number = {9},
pages = {},
pmid = {36146815},
issn = {1999-4915},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; K99 AI171893/AI/NIAID NIH HHS/United States ; },
mesh = {*COVID-19 ; Humans ; Nucleocapsid Proteins ; Retrospective Studies ; *SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus/genetics ; Viral Proteins ; },
abstract = {In late December of 2019, high-throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we provide a short retrospective analysis of SARS-CoV-2 variants by analyzing a subset (n = 97,437) of all publicly available SARS-CoV-2 genomes (n = ~11.9 million) that were randomly selected but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron (BA.1) and Omicron sub-lineages (BA.2-BA.5) are significantly elevated compared to previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike and nucleocapsid proteins, while 24 other viral proteins-including those involved in SARS-CoV-2 replication-are generally conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered.},
}
@article {pmid36146732,
year = {2022},
author = {Denner, J},
title = {Virus Safety of Xenotransplantation.},
journal = {Viruses},
volume = {14},
number = {9},
pages = {},
pmid = {36146732},
issn = {1999-4915},
mesh = {Animals ; *Endogenous Retroviruses/genetics ; Heterografts ; Humans ; Macaca fascicularis ; Papio ; Pharmaceutical Preparations ; Primates ; Transplantation, Heterologous/adverse effects/methods ; },
abstract = {The practice of xenotransplantation using pig islet cells or organs is under development to alleviate the shortage of human donor islet cells or organs for the treatment of diabetes or organ failure. Multiple genetically modified pigs were generated to prevent rejection. Xenotransplantation may be associated with the transmission of potentially zoonotic porcine viruses. In order to prevent this, we developed highly sensitive PCR-based, immunologicals and other methods for the detection of numerous xenotransplantation-relevant viruses. These methods were used for the screening of donor pigs and xenotransplant recipients. Of special interest are the porcine endogenous retroviruses (PERVs) that are integrated in the genome of all pigs, which are able to infect human cells, and that cannot be eliminated by methods that other viruses can. We showed, using droplet digital PCR, that the number of PERV proviruses is different in different pigs (usually around 60). Furthermore, the copy number is different in different organs of a single pig, indicating that PERVs are active in the living animals. We showed that in the first clinical trials treating diabetic patients with pig islet cells, no porcine viruses were transmitted. However, in preclinical trials transplanting pig hearts orthotopically into baboons, porcine cytomegalovirus (PCMV), a porcine roseolovirus (PCMV/PRV), and porcine circovirus 3 (PCV3), but no PERVs, were transmitted. PCMV/PRV transmission resulted in a significant reduction of the survival time of the xenotransplant. PCMV/PRV was also transmitted in the first pig heart transplantation to a human patient and possibly contributed to the death of the patient. Transmission means that the virus was detected in the recipient, however it remains unclear whether it can infect primate cells, including human cells. We showed previously that PCMV/PRV can be eliminated from donor pigs by early weaning. PERVs were also not transmitted by inoculation of human cell-adapted PERV into small animals, rhesus monkey, baboons and cynomolgus monkeys, even when pharmaceutical immunosuppression was applied. Since PERVs were not transmitted in clinical, preclinical, or infection experiments, it remains unclear whether they should be inactivated in the pig genome by CRISPR/Cas. In summary, by using our sensitive methods, the safety of xenotransplantation can be ensured.},
}
@article {pmid36146709,
year = {2022},
author = {Khanal, S and Cao, D and Zhang, J and Zhang, Y and Schank, M and Dang, X and Nguyen, LNT and Wu, XY and Jiang, Y and Ning, S and Zhao, J and Wang, L and Gazzar, ME and Moorman, JP and Yao, ZQ},
title = {Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.},
journal = {Viruses},
volume = {14},
number = {9},
pages = {},
pmid = {36146709},
issn = {1999-4915},
support = {R15 AG069544/AG/NIA NIH HHS/United States ; I01 BX004281/BX/BLRD VA/United States ; I01 BX005428/BX/BLRD VA/United States ; R21AI138598/NH/NIH HHS/United States ; 1I01BX002670/VA/VA/United States ; R15AG069544/NH/NIH HHS/United States ; R21 AI138598/AI/NIAID NIH HHS/United States ; R21AI157909/NH/NIH HHS/United States ; R21 AI157909/AI/NIAID NIH HHS/United States ; 1I01BX004281/VA/VA/United States ; I01 BX002670/BX/BLRD VA/United States ; 5I01BX005428-02/VA/VA/United States ; },
mesh = {Antiviral Agents ; CRISPR-Cas Systems ; DNA ; *HIV Infections ; *HIV-1/genetics/metabolism ; Humans ; Nucleotides/metabolism ; Proviruses/genetics ; RNA, Guide/genetics ; Ribonucleoproteins/genetics/metabolism ; Virus Latency ; },
abstract = {The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.},
}
@article {pmid36145593,
year = {2022},
author = {Salman, A and Kantor, A and McClements, ME and Marfany, G and Trigueros, S and MacLaren, RE},
title = {Non-Viral Delivery of CRISPR/Cas Cargo to the Retina Using Nanoparticles: Current Possibilities, Challenges, and Limitations.},
journal = {Pharmaceutics},
volume = {14},
number = {9},
pages = {},
pmid = {36145593},
issn = {1999-4923},
abstract = {The discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool have revolutionized the field of molecular biology and generated excitement for its potential to treat a wide range of human diseases. As a gene therapy target, the retina offers many advantages over other tissues because of its surgical accessibility and relative immunity privilege due to its blood-retinal barrier. These features explain the large advances made in ocular gene therapy over the past decade, including the first in vivo clinical trial using CRISPR gene-editing reagents. Although viral vector-mediated therapeutic approaches have been successful, they have several shortcomings, including packaging constraints, pre-existing anti-capsid immunity and vector-induced immunogenicity, therapeutic potency and persistence, and potential genotoxicity. The use of nanomaterials in the delivery of therapeutic agents has revolutionized the way genetic materials are delivered to cells, tissues, and organs, and presents an appealing alternative to bypass the limitations of viral delivery systems. In this review, we explore the potential use of non-viral vectors as tools for gene therapy, exploring the latest advancements in nanotechnology in medicine and focusing on the nanoparticle-mediated delivery of CRIPSR genetic cargo to the retina.},
}
@article {pmid36145585,
year = {2022},
author = {Schüller, A and Studt-Reinhold, L and Strauss, J},
title = {How to Completely Squeeze a Fungus-Advanced Genome Mining Tools for Novel Bioactive Substances.},
journal = {Pharmaceutics},
volume = {14},
number = {9},
pages = {},
pmid = {36145585},
issn = {1999-4923},
abstract = {Fungal species have the capability of producing an overwhelming diversity of bioactive substances that can have beneficial but also detrimental effects on human health. These so-called secondary metabolites naturally serve as antimicrobial "weapon systems", signaling molecules or developmental effectors for fungi and hence are produced only under very specific environmental conditions or stages in their life cycle. However, as these complex conditions are difficult or even impossible to mimic in laboratory settings, only a small fraction of the true chemical diversity of fungi is known so far. This also implies that a large space for potentially new pharmaceuticals remains unexplored. We here present an overview on current developments in advanced methods that can be used to explore this chemical space. We focus on genetic and genomic methods, how to detect genes that harbor the blueprints for the production of these compounds (i.e., biosynthetic gene clusters, BGCs), and ways to activate these silent chromosomal regions. We provide an in-depth view of the chromatin-level regulation of BGCs and of the potential to use the CRISPR/Cas technology as an activation tool.},
}
@article {pmid36145402,
year = {2022},
author = {Kaewsapsak, P and Chantaravisoot, N and Nimsamer, P and Mayuramart, O and Mankhong, S and Payungporn, S},
title = {In Silico Evaluation of CRISPR-Based Assays for Effective Detection of SARS-CoV-2.},
journal = {Pathogens (Basel, Switzerland)},
volume = {11},
number = {9},
pages = {},
pmid = {36145402},
issn = {2076-0817},
abstract = {Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.},
}
@article {pmid36144436,
year = {2022},
author = {Mori, H and Kataoka, M and Yang, X},
title = {Past, Present, and Future of Genome Modification in Escherichia coli.},
journal = {Microorganisms},
volume = {10},
number = {9},
pages = {},
pmid = {36144436},
issn = {2076-2607},
abstract = {Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design.},
}
@article {pmid36142589,
year = {2022},
author = {Takabayashi, S and Iijima, K and Tsujimura, M and Aoshima, T and Takagi, H and Aoto, K and Sato, M},
title = {Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142589},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Electroporation/methods ; Female ; *Gene Editing/methods ; Gonads ; Humans ; Hyaluronoglucosaminidase/genetics ; Mammals/genetics ; Mice ; *Nucleic Acids ; Oviducts ; Pregnancy ; Rats ; Ribonucleoproteins/genetics ; Zygote ; },
abstract = {Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.},
}
@article {pmid36142495,
year = {2022},
author = {Song, JH and Shin, G and Kim, HJ and Lee, SB and Moon, JY and Jeong, JC and Choi, HK and Kim, IA and Song, HJ and Kim, CY and Chung, YS},
title = {Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142495},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; Iron ; Micronutrients ; Mutation ; Nucleotides ; *Phytic Acid ; Seeds/genetics ; *Soybeans/genetics ; Zinc ; },
abstract = {Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1-84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1-9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development.},
}
@article {pmid36142407,
year = {2022},
author = {Wang, M and He, L and Chen, B and Wang, Y and Wang, L and Zhou, W and Zhang, T and Cao, L and Zhang, P and Xie, L and Zhang, Q},
title = {Transgenerationally Transmitted DNA Demethylation of a Spontaneous Epialleles Using CRISPR/dCas9-TET1cd Targeted Epigenetic Editing in Arabidopsis.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142407},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Catalytic Domain ; DNA Demethylation ; DNA Methylation/genetics ; Epigenesis, Genetic ; Gene Editing/methods ; Pheophytins ; },
abstract = {CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.},
}
@article {pmid36142353,
year = {2022},
author = {Liu, H and Chen, W and Li, Y and Sun, L and Chai, Y and Chen, H and Nie, H and Huang, C},
title = {CRISPR/Cas9 Technology and Its Utility for Crop Improvement.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142353},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems/genetics ; Crops, Agricultural ; Gene Editing/methods ; Genome, Plant ; Plant Breeding ; RNA, Guide ; Technology ; },
abstract = {The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most widely used of which is CRISPR/Cas9, may enable the rapid improvement of crop traits. Specifically, CRISPR/Cas9 genome-editing technology involves the use of a guide RNA and a Cas9 protein that can cleave the genome at specific loci. Due to its simplicity and efficiency, the CRISPR/Cas9 system has rapidly become the most widely used tool for editing animal and plant genomes. It is ideal for modifying the traits of many plants, including food crops, and for creating new germplasm materials. In this review, the development of the CRISPR/Cas9 system, the underlying mechanism, and examples of its use for editing genes in important crops are discussed. Furthermore, certain limitations of the CRISPR/Cas9 system and potential solutions are described. This article will provide researchers with important information regarding the use of CRISPR/Cas9 gene-editing technology for crop improvement, plant breeding, and gene functional analyses.},
}
@article {pmid36142330,
year = {2022},
author = {Yoon, S and Min, Y and Park, C and Kim, D and Heo, Y and Kim, M and Son, E and Ghosh, M and Son, YO and Hur, CG},
title = {Innate Immune Response Analysis in Meniscus Xenotransplantation Using Normal and Triple Knockout Jeju Native Pigs.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142330},
issn = {1422-0067},
mesh = {Animals ; Humans ; Mice ; Animals, Genetically Modified ; Cytokines/genetics ; Epitopes ; Galactosyltransferases/genetics ; Gene Knockout Techniques ; Graft Rejection ; *Immunity, Innate ; *Meniscus/transplantation ; Mice, Knockout ; Swine ; Transplantation, Heterologous ; },
abstract = {Although allogenic meniscus grafting can be immunologically safe, it causes immune rejection due to an imbalanced tissue supply between donor and recipient. Pigs are anatomically and physiologically similar to adult humans and are, therefore, considered to be advantageous xenotransplantation models. However, immune rejection caused by genetic difference damages the donor tissue and can sometimes cause sudden death. Immune rejection is caused by genes; porcine GGTA1, CMAH, and B4GLANT2 are the most common. In this study, we evaluated immune cells infiltrating the pig meniscus transplanted subcutaneously into BALB/c mice bred for three weeks. We compared the biocompatibility of normal Jeju native black pig (JNP) meniscus with that of triple knockout (TKO) JNP meniscus (α-gal epitope, N-glycolylneuraminic acid (Neu5Gc), and Sd (a) epitope knockout using CRISPR-Cas 9). Mast cells, eosinophils, neutrophils, and macrophages were found to have infiltrated the transplant boundary in the sham (without transplantation), normal (normal JNP), and test (TKO JNP) samples after immunohistochemical analysis. When compared to normal and sham groups, TKO was lower. Cytokine levels did not differ significantly between normal and test groups. Because chronic rejection can occur after meniscus transplantation associated with immune cell infiltration, we propose studies with multiple genetic editing to prevent immune rejection.},
}
@article {pmid36142294,
year = {2022},
author = {Kim, JH and Yu, J and Kim, HK and Kim, JY and Kim, MS and Cho, YG and Bae, S and Kang, KK and Jung, YJ},
title = {Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142294},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Carotenoids ; DNA ; *Gene Editing/methods ; Intramolecular Lyases ; *Oryza/genetics ; Reactive Oxygen Species ; Replicon/genetics ; },
abstract = {Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.},
}
@article {pmid36142132,
year = {2022},
author = {Mahdi, AK and Medrano, JF and Ross, PJ},
title = {Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142132},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Electroporation ; *Gene Editing ; Goats/genetics ; *RNA, Guide/genetics ; Ruminants ; Sheep/genetics ; Zygote ; },
abstract = {We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.},
}
@article {pmid36142116,
year = {2022},
author = {Zhang, C and Liu, S and Li, X and Zhang, R and Li, J},
title = {Virus-Induced Gene Editing and Its Applications in Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {18},
pages = {},
pmid = {36142116},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant ; Plants/genetics ; Plants, Genetically Modified/genetics ; *Viruses/genetics ; },
abstract = {CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generating gene-edited DNA-free plants. Here, we briefly describe CRISPR/Cas-based genome editing. We then focus on VIGE systems and the types of viruses used currently for CRISPR/Cas9 cassette delivery and genome editing. We also highlight recent applications of and advances in VIGE in plants. Finally, we discuss the challenges and potential for VIGE in plants.},
}
@article {pmid36139477,
year = {2022},
author = {Li, B and Fu, C and Zhou, J and Hui, F and Wang, Q and Wang, F and Wang, G and Xu, Z and Che, L and Yuan, D and Wang, Y and Zhang, X and Jin, S},
title = {Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.},
journal = {Cells},
volume = {11},
number = {18},
pages = {},
pmid = {36139477},
issn = {2073-4409},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Geminiviridae/genetics/metabolism ; *Gene Editing/methods ; Gossypium/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Ubiquitins/metabolism ; },
abstract = {Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes' function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1-10 bp in length. The main editing sites are located at positions 11-17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.},
}
@article {pmid36139356,
year = {2022},
author = {Chira, S and Nutu, A and Isacescu, E and Bica, C and Pop, L and Ciocan, C and Berindan-Neagoe, I},
title = {Genome Editing Approaches with CRISPR/Cas9 for Cancer Treatment: Critical Appraisal of Preclinical and Clinical Utility, Challenges, and Future Research.},
journal = {Cells},
volume = {11},
number = {18},
pages = {},
pmid = {36139356},
issn = {2073-4409},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Epigenesis, Genetic ; *Gene Editing ; Humans ; Neoplasm Recurrence, Local/genetics ; },
abstract = {The increasing burden on human malignant diseases became a major concern for healthcare practitioners, that must deal with tumor relapse and the inability to efficiently treat metastasis, in addition to side effects. Throughout the decades, many therapeutic strategies have been employed to improve the clinical outcomes of cancer patients and great efforts have been made to develop more efficient and targeted medicines. The malignant cell is characterized by genetic and epigenetic modifications, therefore targeting those specific drivers of carcinogenesis is highly desirable. Among the genome editing technologies, CRISPR/Cas9 stood as a promising candidate for cancer treatment alternatives, due to its low complexity design. First described as a defense mechanism of bacteria against invading foreign DNA, later it was shown that CRISPR components can be engineered to target specific DNA sequences in a test tube, a discovery that was awarded later with the Nobel Prize in chemistry for its rapid expansion as a reliable genome editing tool in many fields of research, including medicine. The present paper aims of describing CRISPR/Cas9 potential targets for malignant disorders, and the approaches used for achieving this goal. Aside from preclinical studies, we also present the clinical trials that use CRISPR-based technology for therapeutic purposes of cancer. Finally, a summary of the presented studies adds a more focused view of the therapeutic value CRISPR/Cas9 holds and the associated shortcomings.},
}
@article {pmid36139078,
year = {2022},
author = {Nie, D and Guo, T and Yue, M and Li, W and Zong, X and Zhu, Y and Huang, J and Lin, M},
title = {Research Progress on Nanoparticles-Based CRISPR/Cas9 System for Targeted Therapy of Tumors.},
journal = {Biomolecules},
volume = {12},
number = {9},
pages = {},
pmid = {36139078},
issn = {2218-273X},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; *Nanoparticles ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer is a genetic mutation disease that seriously endangers the health and life of all human beings. As one of the most amazing academic achievements in the past decade, CRISPR/Cas9 technology has been sought after by many researchers due to its powerful gene editing capability. CRISPR/Cas9 technology shows great potential in oncology, and has become one of the most promising technologies for cancer genome-editing therapeutics. However, its efficiency and the safety issues of in vivo gene editing severely limit its widespread application. Therefore, developing a suitable delivery method for the CRISPR/Cas9 system is an urgent problem to be solved at present. Rapid advances in nanomedicine suggest nanoparticles could be a viable option. In this review, we summarize the latest research on the potential use of nanoparticle-based CRISPR/Cas9 systems in cancer therapeutics, in order to further their clinical application. We hope that this review will provide a novel insight into the CRISPR/Cas9 system and offer guidance for nanocarrier designs that will enable its use in cancer clinical applications.},
}
@article {pmid36137027,
year = {2022},
author = {Kaiser, J},
title = {CRISPR infusion eases symptoms in genetic disease.},
journal = {Science (New York, N.Y.)},
volume = {377},
number = {6613},
pages = {1367},
doi = {10.1126/science.ade9805},
pmid = {36137027},
issn = {1095-9203},
mesh = {*Angioedemas, Hereditary/therapy ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; },
abstract = {Some patients no longer need drugs to prevent swelling.},
}
@article {pmid36136587,
year = {2022},
author = {Zhang, W and Xiao, D and Li, X and Zhang, Y and Rasouli, J and Casella, G and Boehm, A and Hwang, D and Ishikawa, LL and Thome, R and Ciric, B and Curtis, MT and Rostami, A and Zhang, GX},
title = {SIRT1 inactivation switches reactive astrocytes to an antiinflammatory phenotype in CNS autoimmunity.},
journal = {The Journal of clinical investigation},
volume = {132},
number = {22},
pages = {},
pmid = {36136587},
issn = {1558-8238},
mesh = {Animals ; Mice ; *Astrocytes/enzymology/pathology ; Autoimmunity ; *Encephalomyelitis, Autoimmune, Experimental/genetics/pathology ; Mice, Inbred C57BL ; Phenotype ; *Sirtuin 1/genetics/metabolism ; Mice, Knockout ; },
abstract = {Astrocytes are highly heterogeneous in their phenotype and function, which contributes to CNS disease, repair, and aging; however, the molecular mechanism of their functional states remains largely unknown. Here, we show that activation of sirtuin 1 (SIRT1), a protein deacetylase, played an important role in the detrimental actions of reactive astrocytes, whereas its inactivation conferred these cells with antiinflammatory functions that inhibited the production of proinflammatory mediators by myeloid cells and microglia and promoted the differentiation of oligodendrocyte progenitor cells. Mice with astrocyte-specific Sirt1 knockout (Sirt1-/-) had suppressed progression of experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammatory demyelinating disease. Ongoing EAE was also suppressed when Sirt1 expression in astrocytes was diminished by a CRISPR/Cas vector, resulting in reduced demyelination, decreased numbers of T cells, and an increased rate of IL-10-producing macrophages and microglia in the CNS, whereas the peripheral immune response remained unaffected. Mechanistically, Sirt1-/- astrocytes expressed a range of nuclear factor erythroid-derived 2-like 2 (Nfe2l2) target genes, and Nfe2l2 deficiency shifted the beneficial action of Sirt1-/- astrocytes to a detrimental one. These findings identify an approach for switching the functional state of reactive astrocytes that will facilitate the development of astrocyte-targeting therapies for inflammatory neurodegenerative diseases such as multiple sclerosis.},
}
@article {pmid36135925,
year = {2022},
author = {Langmüller, AM and Champer, J and Lapinska, S and Xie, L and Metzloff, M and Champer, SE and Liu, J and Xu, Y and Du, J and Clark, AG and Messer, PW},
title = {Fitness effects of CRISPR endonucleases in Drosophila melanogaster populations.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {36135925},
issn = {2050-084X},
support = {R21 AI130635/AI/NIAID NIH HHS/United States ; F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics/metabolism ; *Endonucleases/genetics/metabolism ; *Gene Drive Technology ; Gene Editing/methods ; RNA, Guide/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 provides a highly efficient and flexible genome editing technology with numerous potential applications ranging from gene therapy to population control. Some proposed applications involve the integration of CRISPR/Cas9 endonucleases into an organism's genome, which raises questions about potentially harmful effects to the transgenic individuals. One example for which this is particularly relevant are CRISPR-based gene drives conceived for the genetic alteration of entire populations. The performance of such drives can strongly depend on fitness costs experienced by drive carriers, yet relatively little is known about the magnitude and causes of these costs. Here, we assess the fitness effects of genomic CRISPR/Cas9 expression in Drosophila melanogaster cage populations by tracking allele frequencies of four different transgenic constructs that allow us to disentangle 'direct' fitness costs due to the integration, expression, and target-site activity of Cas9, from fitness costs due to potential off-target cleavage. Using a maximum likelihood framework, we find that a model with no direct fitness costs but moderate costs due to off-target effects fits our cage data best. Consistent with this, we do not observe fitness costs for a construct with Cas9HF1, a high-fidelity version of Cas9. We further demonstrate that using Cas9HF1 instead of standard Cas9 in a homing drive achieves similar drive conversion efficiency. These results suggest that gene drives should be designed with high-fidelity endonucleases and may have implications for other applications that involve genomic integration of CRISPR endonucleases.},
}
@article {pmid36135023,
year = {2022},
author = {Kang, K and Song, Y and Kim, I and Kim, TJ},
title = {Therapeutic Applications of the CRISPR-Cas System.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {9},
number = {9},
pages = {},
pmid = {36135023},
issn = {2306-5354},
abstract = {The clustered regularly interspaced palindromic repeat (CRISPR)-Cas system has revolutionized genetic engineering due to its simplicity, stability, and precision since its discovery. This technology is utilized in a variety of fields, from basic research in medicine and biology to medical diagnosis and treatment, and its potential is unbounded as new methods are developed. The review focused on medical applications and discussed the most recent treatment trends and limitations, with an emphasis on CRISPR-based therapeutics for infectious disease, oncology, and genetic disease, as well as CRISPR-based diagnostics, screening, immunotherapy, and cell therapy. Given its promising results, the successful implementation of the CRISPR-Cas system in clinical practice will require further investigation into its therapeutic applications.},
}
@article {pmid36134715,
year = {2022},
author = {Klanschnig, M and Cserjan-Puschmann, M and Striedner, G and Grabherr, R},
title = {CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli.},
journal = {Nucleic acids research},
volume = {50},
number = {18},
pages = {10772-10784},
pmid = {36134715},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; Gene Editing ; RNA, Guide/genetics ; Recombinant Proteins/metabolism ; Up-Regulation/genetics ; },
abstract = {Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide-ranging prokaryotic CRISPRa studies might be attributed to intrinsic limitations of bacterial activators and Cas9 proteins. While bacterial activators need accurate spatial orientation and distancing towards the target promoter to be functional, Cas9-based CRISPR tools only bind sites adjacent to NGG PAM sequences. These circumstances hampered Cas9-guided activators from mediating the up-regulation of endogenous genes at precise positions in bacteria. We could overcome this limitation by combining the PAM independent Cas9 variant SpRY and a CRISPRa construct using phage protein MCP fused to transcriptional activator SoxS. This CRISPRa construct, referred to as SMS, was compared with previously reported CRISPRa constructs and showed up-regulation of a reporter gene library independent of its PAM sequence in Escherichia coli. We also demonstrated down-regulation and multi-gene expression control with SMS at non-NGG PAM sites. Furthermore, we successfully applied SMS to up-regulate endogenous genes, and transgenes at non-NGG PAM sites, which was impossible with the previous CRISPRa construct.},
}
@article {pmid36134452,
year = {2022},
author = {Lalonde, ME and Sasseville, M and Gélinas, AM and Milanese, JS and Béland, K and Drouin, S and Haddad, E and Marcotte, R},
title = {Genome-wide CRISPR screens identify ferroptosis as a novel therapeutic vulnerability in acute lymphoblastic leukemia.},
journal = {Haematologica},
volume = {},
number = {},
pages = {},
doi = {10.3324/haematol.2022.280786},
pmid = {36134452},
issn = {1592-8721},
abstract = {Acute lymphoblastic leukemia (ALL) is the most frequent cancer diagnosed in children. Despite the great progress achieved over the last 40 years, with cure rates now exceeding 85%, refractory or relapsed ALL still exhibit a dismal prognosis. This poor outcome reflects the lack of treatment options specifically targeting relapsed or refractory ALL. To address this gap, we have performed whole-genome CRISPR/Cas drop-out screens on a panel of seven B-ALL cell lines. Our results demonstrate that while there was a significant overlap in gene essentiality between ALL cell lines and other cancer types survival of ALL cell lines was dependent on several unique metabolic pathways, including an exquisite sensitivity to GPX4 depletion and ferroptosis induction. Detailed molecular analysis of B-ALL cells suggest that they are primed to undergo ferroptosis as they exhibit high steady-state oxidative stress potential, a low buffering capacity, and a disabled GPX4-independent secondary lipid peroxidation detoxification pathway. Finally, we validated the sensitivity of B-ALL to ferroptosis induction using patient-derived B-ALL samples.},
}
@article {pmid36130724,
year = {2022},
author = {Tenjo-Castaño, F and Montoya, G and Carabias, A},
title = {Transposons and CRISPR: Rewiring Gene Editing.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.2c00379},
pmid = {36130724},
issn = {1520-4995},
abstract = {CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.},
}
@article {pmid36130423,
year = {2022},
author = {Okamoto, N and Maeda, M and Yamamoto, C and Kodama, R and Sugimoto, K and Shinozaki, Y and Ezura, H and Kimura, Y},
title = {Construction of tomato plants with suppressed endo-β-N-acetylglucosaminidase activity using CRISPR-Cas9 mediated genome editing.},
journal = {Plant physiology and biochemistry : PPB},
volume = {190},
number = {},
pages = {203-211},
doi = {10.1016/j.plaphy.2022.08.009},
pmid = {36130423},
issn = {1873-2690},
mesh = {Acetylglucosamine ; Acetylglucosaminidase/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Lycopersicon esculentum/genetics ; Mannose/chemistry ; Polysaccharides/chemistry ; },
abstract = {High mannose-type free N-glycans with a single N-acetyl-D-glucosamine (GlcNAc) residue at the reducing end (GN1-HMT-FNGs) are produced by cytosolic endo-β-N-acetylglucosaminidase (EC:3.2.1.96) (ENGase) and are ubiquitous in differentiating and growing plant cells. To elucidate the physiological functions of HMT-FNGs in plants, we identified the ENGase gene in tomato (Solyc06g050930) and detected ENGase activity and increased production of GN1-HMT-FNGs during tomato fruit maturation. However, the precise role of GN1-HMT-FNGs in fruit maturation remains unclear. In this study, we established tomato ENGase mutants with suppressed ENGase activity via CRISPR/Cas9 genome editing technology. DNA sequencing of the Δeng mutants (T0 and T1 generations) revealed that they had the same mutations in the genomic DNA around the target sequences. Three null CRISPR/Cas9 segregant plants of the T1 generation (Δeng1-2, -22, and -26) were used to measure ENGase activity and analyze the structural features of HMT-FNGs in the leaves. The Δeng mutants did not exhibit ENGase activity and produced GN2-HMT-FNGs bearing tow GlcNAc residues at the reducing end side instead of GN1-HMT-FNGs. The Δeng mutants lack the N-terminal region of ENGase, indicating that the N-terminal region is important for full ENGase activity. The fruits of Δeng mutants (T2 generation) also showed loss of ENGase activity and similar structural features of HMT-FNGs of the T1 generation. However, there was no significant difference in fruit maturation between the T2 generation of the Δeng mutants and the wild type. The Δeng mutants rich in GN2-HMT-FNGs could be offered as a new tomato that is different from wild type containing GN1-HMT-FNGs.},
}
@article {pmid36130036,
year = {2022},
author = {Senger, K and Akhmetzyanova, I and Haley, B and Rutz, S and Oh, SA},
title = {Plasmid-Based Donor Templates for Nonviral CRISPR/Cas9-Mediated Gene Knock-In in Human T Cells.},
journal = {Current protocols},
volume = {2},
number = {9},
pages = {e538},
doi = {10.1002/cpz1.538},
pmid = {36130036},
issn = {2691-1299},
mesh = {CD28 Antigens/genetics ; *CD8-Positive T-Lymphocytes/metabolism ; *CRISPR-Cas Systems/genetics ; DNA ; Gene Knock-In Techniques ; Humans ; Plasmids/genetics ; Receptors, Antigen, T-Cell/genetics ; Ribonucleoproteins/genetics ; },
abstract = {Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next-generation cellular therapies. Although methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9-based gene knock-in utilizing plasmid-based donor DNA templates. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Purification of human CD4[+] or CD8[+] T cells from blood Basic Protocol 2: Activation of purified CD4[+] or CD8[+] T cells using TransAct CD3/CD28 agonist-conjugated nanomatrix Basic Protocol 3: Preparation of Cas9/sgRNA RNPs Basic Protocol 4: Transfection of CAS9-RNP and knock-in template into human T cells Support Protocol 1: Purity check following magnetic T cell isolation Support Protocol 2: Dextramer staining of TCR-edited T cells Support Protocol 3: Functional characterization of TCR knock-in T cells Support Protocol 4: Detection of knock-in reporter activity in CRISPR/CAS9-edited T cells.},
}
@article {pmid36128975,
year = {2022},
author = {Yan, C and Shi, G and Chen, J},
title = {Fluorescent Detection of Two Pesticides Based on CRISPR-Cas12a and Its Application for the Construction of Four Molecular Logic Gates.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {39},
pages = {12700-12707},
doi = {10.1021/acs.jafc.2c04548},
pmid = {36128975},
issn = {1520-5118},
mesh = {*Atrazine ; *Biosensing Techniques ; CRISPR-Cas Systems ; DNA ; Oligonucleotides ; *Pesticides ; },
abstract = {An intelligent detection platform was developed through molecular logic gate operation based on CRISPR-Cas12a and signal amplification circuits using two kinds of pesticides [acetamiprid (ACE) and atrazine (ATR)] as inputs. The pesticide-aptamer bindings activate the signal amplification process to produce numerous double-stranded DNA, which can be identified by CRISPR-Cas12a. Under the optimal assay conditions, the sensor exhibits excellent analytical performance, with the detection limits for ACE and ATR of 2.5 and 0.2 pM, respectively. The practicality of the platform was verified by testing pesticide concentrations in food samples. Several molecular logic gates (OR, AND, XOR, and INHIBIT) were constructed using "0" and "1" to encode the target pesticides and the fluorescence readout. The logic detection platform with simple operation, high sensitivity, and multiple logic functions is promising to become a powerful sensing system for the intelligent assay of different pesticides in food samples.},
}
@article {pmid36128934,
year = {2022},
author = {Khurana, A and Sayed, N and Singh, V and Khurana, I and Allawadhi, P and Rawat, PS and Navik, U and Pasumarthi, SK and Bharani, KK and Weiskirchen, R},
title = {A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery.},
journal = {Journal of cellular biochemistry},
volume = {123},
number = {10},
pages = {1674-1698},
doi = {10.1002/jcb.30329},
pmid = {36128934},
issn = {1097-4644},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Engineering/methods ; Drug Discovery ; Technology ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas technology possesses revolutionary potential to positively affect various domains of drug discovery. It has initiated a rise in the area of genetic engineering and its advantages range from classical science to translational medicine. These genome editing systems have given a new dimension to our capabilities to alter, detect and annotate specified gene sequences. Moreover, the ease, robustness and adaptability of the CRISPR/Cas9 technology have led to its extensive utilization in research areas in such a short period of time. The applications include the development of model cell lines, understanding disease mechanisms, discovering disease targets, developing transgenic animals and plants, and transcriptional modulation. Further, the technology is rapidly growing; hence, an overlook of progressive success is crucial. This review presents the current status of the CRISPR-Cas technology in a tailor-made format from its discovery to several advancements for drug discovery alongwith future trends associated with possibilities and hurdles including ethical concerns.},
}
@article {pmid36127876,
year = {2022},
author = {Liu, S and Sretenovic, S and Fan, T and Cheng, Y and Li, G and Qi, A and Tang, X and Xu, Y and Guo, W and Zhong, Z and He, Y and Liang, Y and Han, Q and Zheng, X and Gu, X and Qi, Y and Zhang, Y},
title = {Hypercompact CRISPR-Cas12j2 (CasΦ) enables genome editing, gene activation, and epigenome editing in plants.},
journal = {Plant communications},
volume = {3},
number = {6},
pages = {100453},
doi = {10.1016/j.xplc.2022.100453},
pmid = {36127876},
issn = {2590-3462},
mesh = {*Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenome/genetics ; Transcriptional Activation ; CRISPR-Cas Systems/genetics ; Plants/genetics ; },
}
@article {pmid36127475,
year = {2022},
author = {Armat, M and Vaz, PK and Browning, GF and Noormohammadi, AH and Hartley, CA and Devlin, JM},
title = {Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques.},
journal = {Virus genes},
volume = {58},
number = {6},
pages = {540-549},
pmid = {36127475},
issn = {1572-994X},
mesh = {Animals ; CRISPR-Cas Systems ; Australia ; *Herpesvirus 1, Gallid/genetics ; Chickens ; Glycoproteins/genetics ; Green Fluorescent Proteins/genetics ; Homologous Recombination ; *Herpesviridae Infections ; *Poultry Diseases ; },
abstract = {In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.},
}
@article {pmid36125750,
year = {2022},
author = {Wimmer, E and Zink, IA and Schleper, C},
title = {Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2522},
number = {},
pages = {177-201},
pmid = {36125750},
issn = {1940-6029},
mesh = {DNA ; Endonucleases/genetics ; *Genes, Essential ; RNA/genetics ; RNA Interference ; RNA, Messenger ; *Sulfolobales/genetics ; },
abstract = {The manipulation of gene expression levels in vivo is often key to elucidating gene function and regulatory network interactions, especially when it comes to the investigation of essential genes that cannot be deleted from the model organism's genome. Several techniques have been developed for prokaryotes that allow to interfere with transcription initiation of specific genes by blocking or modifying promoter regions. However, a tool functionally similar to RNAi used in eukaryotes to efficiently degrade mRNA posttranscriptionally did not exist until recently. Type III CRISPR-Cas systems use small RNAs (crRNAs) that guide effector complexes (encoded by cas genes) which act as site-specific RNA endonuclease and can thus be harnessed for targeted posttranscriptional gene silencing. Guide RNAs complementary to the desired target mRNA that, in addition, exhibit complementarity to repeat sequences found in the CRISPR arrays, effectively suppress unspecific DNA and RNA activities of the CRISPR-Cas complexes. Here we describe the use of endogenous type III CRISPR-Cas systems in two model organisms of Crenarchaeota, Saccharolobus solfataricus and Sulfolobus acidocaldarius.},
}
@article {pmid36125743,
year = {2022},
author = {Schwarz, TS and Schreiber, SS and Marchfelder, A},
title = {CRISPR Interference as a Tool to Repress Gene Expression in Haloferax volcanii.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2522},
number = {},
pages = {57-85},
pmid = {36125743},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression ; *Haloferax volcanii/genetics ; Molecular Biology ; RNA, Guide ; },
abstract = {To date, a plethora of tools for molecular biology have been developed on the basis of the CRISPR-Cas system. Almost all use the class 2 systems since here the setup is the simplest with only one protein and one guide RNA, allowing for easy transfer to and expression in other organisms. However, the CRISPR-Cas components harnessed for applications are derived from mesophilic bacteria and are not optimal for use in extremophilic archaea.Here, we describe the application of an endogenous CRISPR-Cas system as a tool for silencing gene expression in a halophilic archaeon. Haloferax volcanii has a CRISPR-Cas system of subtype I-B, which can be easily used to repress the transcription of endogenous genes, allowing to study the effects of their depletion. This article gives a step-by-step introduction on how to use the implemented system for any gene of interest in Haloferax volcanii. The concept of CRISPRi described here for Haloferax can be transferred to any other archaeon, that is genetically tractable and has an endogenous CRISPR-Cas I systems.},
}
@article {pmid36125723,
year = {2022},
author = {Shortt, K and Heruth, DP},
title = {Identification of Genes Regulating Hepatocyte Injury by a Genome-Wide CRISPR-Cas9 Screen.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2544},
number = {},
pages = {227-251},
pmid = {36125723},
issn = {1940-6029},
mesh = {*Acetaminophen/adverse effects ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome ; Hepatocytes ; },
abstract = {Gene editing introduces stable mutations into the genome and has powerful applications extending from research to clinical gene therapy. CRISPR-Cas9 gene editing can be employed to study directly the functional impact of stable gene knockout, activation, and knockdown. Here, we describe the end-to-end methodology by which we employ genome-wide CRISPR-Cas9 knockout to study drug toxicity using acetaminophen (APAP) in a hepatocellular carcinoma liver model as an example. This methodology can be extended to other proliferative cell types and chemical metabolic and toxicity models. By employing a massively parallelized genome-wide knockout model, the genes responsible for cellular toxicity and proliferation may be assayed concurrently. Resultant data are interrogated in the context of existing gene expression data, pathway analysis, drug-gene interactions, and orthogonal confirmatory assays to better understand the metabolic mechanisms.},
}
@article {pmid36124799,
year = {2022},
author = {Mitrofanov, A and Ziemann, M and Alkhnbashi, OS and Hess, WR and Backofen, R},
title = {CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection.},
journal = {Bioinformatics (Oxford, England)},
volume = {38},
number = {Supplement_2},
pages = {ii42-ii48},
pmid = {36124799},
issn = {1367-4811},
mesh = {Genome ; Humans ; *RNA/genetics ; *RNA, Guide/genetics ; Sequence Alignment ; },
abstract = {MOTIVATION: The CRISPR-Cas9 system is a Type II CRISPR system that has rapidly become the most versatile and widespread tool for genome engineering. It consists of two components, the Cas9 effector protein, and a single guide RNA that combines the spacer (for identifying the target) with the tracrRNA, a trans-activating small RNA required for both crRNA maturation and interference. While there are well-established methods for screening Cas effector proteins and CRISPR arrays, the detection of tracrRNA remains the bottleneck in detecting Class 2 CRISPR systems.<br><br>RESULTS: We introduce a new pipeline CRISPRtracrRNA for screening and evaluation of tracrRNA candidates in genomes. This pipeline combines evidence from different components of the Cas9-sgRNA complex. The core is a newly developed structural model via covariance models from a sequence-structure alignment of experimentally validated tracrRNAs. As additional evidence, we determine the terminator signal (required for the tracrRNA transcription) and the RNA-RNA interaction between the CRISPR array repeat and the 5'-part of the tracrRNA. Repeats are detected via an ML-based approach (CRISPRidenify). Providing further evidence, we detect the cassette containing the Cas9 (Type II CRISPR systems) and Cas12 (Type V CRISPR systems) effector protein. Our tool is the first for detecting tracrRNA for Type V systems.<br><br>The implementation of the CRISPRtracrRNA is available on GitHub upon requesting the access permission, (https://github.com/BackofenLab/CRISPRtracrRNA). Data generated in this study can be obtained upon request to the corresponding person: Rolf Backofen (backofen@informatik.uni-freiburg.de).<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid36123438,
year = {2022},
author = {Vassallo, CN and Doering, CR and Littlehale, ML and Teodoro, GIC and Laub, MT},
title = {A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome.},
journal = {Nature microbiology},
volume = {7},
number = {10},
pages = {1568-1579},
pmid = {36123438},
issn = {2058-5276},
support = {F32 GM139231/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Antiviral Agents ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Prophages/genetics ; },
abstract = {The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species.},
}
@article {pmid36122469,
year = {2022},
author = {Xu, Y and Wang, C and Liu, G and Zhao, X and Qian, Q and Li, S and Mi, X},
title = {Tetrahedral DNA framework based CRISPR electrochemical biosensor for amplification-free miRNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {217},
number = {},
pages = {114671},
doi = {10.1016/j.bios.2022.114671},
pmid = {36122469},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *MicroRNAs ; },
abstract = {microRNA (miRNA) is a kind of small non-coding RNA that has been regarded as potential biomarkers for cancers. Sensitive and specific detection of miRNA at low expression levels is highly desirable but remains challenging, especially for amplification-free and portable point of care (POC) diagnostics. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a has been recently discovered and used in the field of RNA detection. Nonetheless, most CRISPR/Cas13a-based methods were burdened with expensive equipment, time-consuming procedures, and complicated operations which were not suitable for POC analysis. In this work, we constructed a three-dimensional tetrahedral DNA framework based CRISPR-electrochemical biosensor (CRISPR-E). By combining tetrahedral DNA framework, CRISPR, and electrochemical biosensor, the process of activation, cleavage of Cas13a, and signal readout were all finished on the chip, and a simple, amplification-free and sensitive detection of miRNA-19b was realized. Under the optimal experimental conditions, a linear range from 10 pM to 10[4] pM with detection limit of 10 pM for miRNA-19b in buffer solution was achieved. Selectivity analysis indicated that our CRISPR-E had good distinguishing ability between miRNA-19b and miRNA-197. The results of miRNA-19b detection in mimic serum samples were consistent with that of the buffer solution. This all-on-chip strategy of our CRISPR-E is very suitable for POC testing.},
}
@article {pmid36122230,
year = {2022},
author = {Suh, S and Choi, EH and Raguram, A and Liu, DR and Palczewski, K},
title = {Precision genome editing in the eye.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {39},
pages = {e2210104119},
pmid = {36122230},
issn = {1091-6490},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing ; Mutation ; *Vision Disorders/genetics/therapy ; },
abstract = {CRISPR-Cas-based genome editing technologies could, in principle, be used to treat a wide variety of inherited diseases, including genetic disorders of vision. Programmable CRISPR-Cas nucleases are effective tools for gene disruption, but they are poorly suited for precisely correcting pathogenic mutations in most therapeutic settings. Recently developed precision genome editing agents, including base editors and prime editors, have enabled precise gene correction and disease rescue in multiple preclinical models of genetic disorders. Additionally, new delivery technologies that transiently deliver precision genome editing agents in vivo offer minimized off-target editing and improved safety profiles. These improvements to precision genome editing and delivery technologies are expected to revolutionize the treatment of genetic disorders of vision and other diseases. In this Perspective, we describe current preclinical and clinical genome editing approaches for treating inherited retinal degenerative diseases, and we discuss important considerations that should be addressed as these approaches are translated into clinical practice.},
}
@article {pmid36121880,
year = {2022},
author = {Verkuijl, SAN and Anderson, MAE and Alphey, L and Bonsall, MB},
title = {Daisy-chain gene drives: The role of low cut-rate, resistance mutations, and maternal deposition.},
journal = {PLoS genetics},
volume = {18},
number = {9},
pages = {e1010370},
pmid = {36121880},
issn = {1553-7404},
support = {BB/H01814X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L00948X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/V008110/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Alleles ; CRISPR-Cas Systems ; *Gene Drive Technology/methods ; Mutation ; },
abstract = {The introgression of genetic traits through gene drive may serve as a powerful and widely applicable method of biological control. However, for many applications, a self-perpetuating gene drive that can spread beyond the specific target population may be undesirable and preclude use. Daisy-chain gene drives have been proposed as a means of tuning the invasiveness of a gene drive, allowing it to spread efficiently into the target population, but be self-limiting beyond that. Daisy-chain gene drives are made up of multiple independent drive elements, where each element, except one, biases the inheritance of another, forming a chain. Under ideal inheritance biasing conditions, the released drive elements remain linked in the same configuration, generating copies of most of their elements except for the last remaining link in the chain. Through mathematical modelling of populations connected by migration, we have evaluated the effect of resistance alleles, different fitness costs, reduction in the cut-rate, and maternal deposition on two alternative daisy-chain gene drive designs. We find that the self-limiting nature of daisy-chain gene drives makes their spread highly dependent on the efficiency and fidelity of the inheritance biasing mechanism. In particular, reductions in the cut-rate and the formation of non-lethal resistance alleles can cause drive elements to lose their linked configuration. This severely reduces the invasiveness of the drives and allows for phantom cutting, where an upstream drive element cuts a downstream target locus despite the corresponding drive element being absent, creating and biasing the inheritance of additional resistance alleles. This phantom cutting can be mitigated by an alternative indirect daisy-chain design. We further find that while dominant fitness costs and maternal deposition reduce daisy-chain invasiveness, if overcome with an increased release frequency, they can reduce the spread of the drive into a neighbouring population.},
}
@article {pmid36121799,
year = {2022},
author = {Ding, S and Wei, Y and Chen, G and Du, F and Cui, X and Huang, X and Yuan, Y and Dong, J and Tang, Z},
title = {Detection of Cancer Marker Flap Endonuclease 1 Using One-Pot Transcription-Powered Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Signal Expansion.},
journal = {Analytical chemistry},
volume = {94},
number = {39},
pages = {13549-13555},
doi = {10.1021/acs.analchem.2c03054},
pmid = {36121799},
issn = {1520-6882},
mesh = {Biomarkers ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; DNA, Single-Stranded ; Deoxyribonucleases ; Flap Endonucleases/*analysis/genetics ; Humans ; *Neoplasms/genetics ; RNA, Guide/genetics ; },
abstract = {As a critical functional protein in DNA replication and genome stability, flap endonuclease 1 (FEN1) has been considered a promising biomarker and druggable target for multiple cancers. We report here a transcription-powered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a signal expansion platform for rapid and sensitive detection of FEN1. In this method, the probe cleavage by FEN1 generated a free 5' flap single-stranded DNA which could hybridize with the single-stranded T7 promoter-bearing template and trigger the extension. Then, the CRISPR guide RNA (crRNA) transcribed from the extended template activated the collateral DNase activity of Cas12a, releasing the fluorophore from the quenched DNA signal probe to report the FEN1 detection result. The high specificity for FEN1 was validated by comparing with other repair-relevant proteins. The limit of detection (LOD) could be as low as 0.03 mU, which is sensitive enough to detect the FEN1 activity in biological samples. In addition, the inhibition assay of FEN1 was also successfully achieved with this platform, proving its potential in inhibitor screening. In summary, this study provides a novel biosensor for FEN1 activity analysis and provides new insights into the development of CRISPR-based biosensors for non-nucleic acid targets.},
}
@article {pmid36121484,
year = {2022},
author = {Sheikh, BA and Bhat, BA and Mir, MA},
title = {Antimicrobial resistance: new insights and therapeutic implications.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {19-20},
pages = {6427-6440},
pmid = {36121484},
issn = {1432-0614},
mesh = {Animals ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Bacteria ; *Drug Resistance, Bacterial ; Humans ; },
abstract = {Antimicrobial resistance has not been a new phenomenon. Still, the number of resistant organisms, the geographic areas affected by emerging drug resistance, and the magnitude of resistance in a single organism are enormous and mounting. Disease and disease-causing agents formerly thought to be contained by antibiotics are now returning in new forms resistant to existing therapies. Antimicrobial resistance is one of the most severe and complicated health issues globally, driven by interrelated dynamics in humans, animals, and environmental health sectors. Coupled with various epidemiological factors and a limited pipeline for new antimicrobials, all these misappropriations allow the transmission of drug-resistant organisms. The problem is likely to worsen soon. Antimicrobial resistance in general and antibiotic resistance in particular is a shared global problem. Actions taken by any single country can adversely or positively affect the other country. Targeted coordination and prevention strategies are critical in stopping the spread of antibiotic-resistant organisms and hence its overall management. This article has provided in-depth knowledge about various methods that can help mitigate the emergence and spread of antimicrobial resistance globally. KEY POINTS: • Overview of antimicrobial resistance as a global challenge and explain various reasons for its rapid progression. • Brief about the intrinsic and acquired resistance to antimicrobials and development of antibiotic resistance in bacteria. • Systematically organized information is provided on different strategies for tackling antimicrobial resistance for the welfare of human health.},
}
@article {pmid36120975,
year = {2022},
author = {Mayran, C and Henry, S and Pinchon, E and Fournier-Wirth, C and Cantaloube, JF and Foulongne, V},
title = {[CRISPR-Cas: the bacterial immunity that supports diagnostic in virology].},
journal = {Virologie (Montrouge, France)},
volume = {26},
number = {4},
pages = {303-313},
doi = {10.1684/vir.2022.0966},
pmid = {36120975},
issn = {1267-8694},
mesh = {*Bacteria/genetics ; *CRISPR-Cas Systems ; DNA ; Endonucleases/genetics ; RNA ; },
abstract = {CRISPR-Cas is an adaptive immune system that prevents bacteria and archea from nucleic acids invasion such as viral genomes. The ability of the CRISPR-Cas technology to effectively and precisely cut a targeted genomic DNA region was exploited to develop powerful genome editing tools that were adapted for a wide range of applications, revolutionizing biological sciences. The CRISPR-Cas system consists of a Cas endonuclease triggered by a RNA guide for highly specific cleavage of targeted DNA or RNA sequences. In addition to the target specific cleavage, some Cas enzymes, including Cas12a and Cas13a, display a collateral trans-cleavage activity that allows the cleavage of all surrounding single-stranded nucleic acids. These biosensing activities of CRISPR-Cas systems, based on target specific binding and cleavage, are promising tools to develop accurate diagnostic methods to detect specific nucleic acids. CRISPRCas could therefore be used to diagnose a wide variety of diseases. In the current review we propose to describe the more significant advances for virus detection based on CRISPR-Cas systems.},
}
@article {pmid36120556,
year = {2022},
author = {Barragán-Álvarez, CP and Flores-Fernandez, JM and Hernández-Pérez, OR and Ávila-Gónzalez, D and Díaz, NF and Padilla-Camberos, E and Dublan-García, O and Gómez-Oliván, LM and Diaz-Martinez, NE},
title = {Recent advances in the use of CRISPR/Cas for understanding the early development of molecular gaps in glial cells.},
journal = {Frontiers in cell and developmental biology},
volume = {10},
number = {},
pages = {947769},
pmid = {36120556},
issn = {2296-634X},
abstract = {Glial cells are non-neuronal elements of the nervous system (NS) and play a central role in its development, maturation, and homeostasis. Glial cell interest has increased, leading to the discovery of novel study fields. The CRISPR/Cas system has been widely employed for NS understanding. Its use to study glial cells gives crucial information about their mechanisms and role in the central nervous system (CNS) and neurodegenerative disorders. Furthermore, the increasingly accelerated discovery of genes associated with the multiple implications of glial cells could be studied and complemented with the novel screening methods of high-content and single-cell screens at the genome-scale as Perturb-Seq, CRISP-seq, and CROPseq. Besides, the emerging methods, GESTALT, and LINNAEUS, employed to generate large-scale cell lineage maps have yielded invaluable information about processes involved in neurogenesis. These advances offer new therapeutic approaches to finding critical unanswered questions about glial cells and their fundamental role in the nervous system. Furthermore, they help to better understanding the significance of glial cells and their role in developmental biology.},
}
@article {pmid36117747,
year = {2022},
author = {Lee, S and Nam, D and Park, JS and Kim, S and Lee, ES and Cha, BS and Park, KS},
title = {Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor.},
journal = {Biochip journal},
volume = {},
number = {},
pages = {1-8},
pmid = {36117747},
issn = {1976-0280},
abstract = {UNLABELLED: In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5'-TTATT-CCCCC-3'; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5'-TTATT-3') used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin (stn) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13206-022-00081-0.},
}
@article {pmid36117459,
year = {2022},
author = {Avaro, AS and Santiago, JG},
title = {Uncertainty Quantification of Michaelis-Menten Kinetic Rates and Its Application to the Analysis of CRISPR-Based Diagnostics.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {45},
pages = {e202209527},
doi = {10.1002/anie.202209527},
pmid = {36117459},
issn = {1521-3773},
mesh = {*Kinetics ; Uncertainty ; Monte Carlo Method ; Catalysis ; },
abstract = {Michaelis-Menten kinetics is an essential model to rationalize enzyme reactions. The quantification of Michaelis-Menten parameters can be very challenging as it is sensitive to even small experimental errors. We here present a quantification of the uncertainty inherent to the experimental determination of kinetic rate parameters for enzymatic reactions. We study the influence of several sources of uncertainty and bias, including the inner filter effect, pipetting errors, number of points in the Michaelis-Menten curve, and flat-field correction. Using Monte Carlo simulations and analyses of experimental data, we compute typical uncertainties of k c a t ${{k}
_{cat}
}
$ , K M ${{K}
_{M}
}
$ , and catalytic efficiency k c a t / K M ${{k}
_{cat}
/{K}
_{M}
}
$ . As a salient example, we analyze the extraction of such parameters for CRISPR-Cas systems. CRISPR diagnostics have recently attracted much interest and yet reports of these enzymatic kinetic rates have been highly unreliable and inconsistent.},
}
@article {pmid36117025,
year = {2022},
author = {Mahler, M and Costa, AR and van Beljouw, SPB and Fineran, PC and Brouns, SJJ},
title = {Approaches for bacteriophage genome engineering.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.08.008},
pmid = {36117025},
issn = {1879-3096},
abstract = {In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and control gene functions, or alter phage properties such as host range, has remained challenging due to a lack of universal selectable markers. Here, we discuss the state-of-the-art techniques to engineer and select desired phage genomes using advances in cell-free methodologies and clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) counter-selection approaches.},
}
@article {pmid36115975,
year = {2022},
author = {Qian, W and Huang, J and Wang, T and Fan, C and Kang, J and Zhang, Q and Li, Y and Chen, S},
title = {Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay.},
journal = {Virology journal},
volume = {19},
number = {1},
pages = {150},
pmid = {36115975},
issn = {1743-422X},
mesh = {CRISPR-Cas Systems ; *Caliciviridae Infections/diagnosis ; Genotype ; Humans ; *Norovirus/genetics ; Sensitivity and Specificity ; Viral Proteins/genetics ; },
abstract = {BACKGROUND: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens.<br><br>METHODS: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored.<br><br>RESULTS: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/&#x3bc;L within approximately 30-40&#xa0;min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement.<br><br>CONCLUSIONS: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.},
}
@article {pmid36115160,
year = {2022},
author = {Bot, JF and van der Oost, J and Geijsen, N},
title = {The double life of CRISPR-Cas13.},
journal = {Current opinion in biotechnology},
volume = {78},
number = {},
pages = {102789},
doi = {10.1016/j.copbio.2022.102789},
pmid = {36115160},
issn = {1879-0429},
abstract = {Since the discovery of RNA-programmable nucleases from the prokaryotic adaptive immune system CRISPR-Cas, these proteins have seen rapid and widespread adoption for biotechnological and clinical research. A recently discovered system, CRISPR-Cas13, uses CRISPR RNA guides to target RNA. Interestingly, RNA targeting by Cas13 results in cleavage of both target RNA and bystander RNA. This feature has been used to develop innovative diagnostic tools for the detection of specific RNAs. Unlike in vitro detection of RNA using collateral RNA cleavage, however, initial studies of mammalian cells only revealed highly specific target RNA-knockdown activity. Although these findings have been confirmed subsequently, several recent publications do report Cas13-mediated toxicity and collateral RNA cleavage when using Cas13 in eukaryotes. Here, we review these conflicting observations and discuss its potential molecular basis.},
}
@article {pmid36114353,
year = {2022},
author = {Trakarnsanga, K and Thongsin, N and Metheetrairut, C and Tipgomut, C and Poldee, S and Wattanapanitch, M},
title = {Genetic correction of haemoglobin E in an immortalised haemoglobin E/beta-thalassaemia cell line using the CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {15551},
pmid = {36114353},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Hemoglobin E/genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells ; beta-Globins/genetics/metabolism ; *beta-Thalassemia/genetics/metabolism/therapy ; },
abstract = {β-thalassaemia is one of the most common genetic blood diseases worldwide with over 300 mutations in the HBB gene affecting red blood cell functions. Recently, advances in genome editing technology have provided a powerful tool for precise genetic correction. Generation of patient-derived induced pluripotent stem cells (iPSCs) followed by genetic correction of HBB mutations and differentiation into haematopoietic stem/progenitor cells (HSPCs) offers a potential therapy to cure the disease. However, the biggest challenge is to generate functional HSPCs that are capable of self-renewal and transplantable. In addition, functional analyses of iPSC-derived erythroid cells are hampered by poor erythroid expansion and incomplete erythroid differentiation. Previously, we generated an immortalised erythroid cell line (SiBBE) with unique properties, including unlimited expansion and the ability to differentiate into mature erythrocytes. In this study, we report a highly efficient genetic correction of HbE mutation in the SiBBE cells using the CRISPR/Cas9 system. The HbE-corrected clones restored β-globin production with reduced levels of HbE upon erythroid differentiation. Our approach provides a sustainable supply of corrected erythroid cells and represents a valuable model for validating the therapeutic efficacy of gene editing systems.},
}
@article {pmid36114266,
year = {2022},
author = {Altgilbers, S and Dierks, C and Klein, S and Weigend, S and Kues, WA},
title = {Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {15587},
pmid = {36114266},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Chickens/genetics ; Germ Cells ; Homozygote ; Male ; Polymerase Chain Reaction ; *Proviruses/genetics ; *RNA, Guide/genetics ; Semen ; Sequence Deletion ; },
abstract = {Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5'-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.},
}
@article {pmid36114193,
year = {2022},
author = {Crone, MA and MacDonald, JT and Freemont, PS and Siciliano, V},
title = {gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells.},
journal = {NPJ systems biology and applications},
volume = {8},
number = {1},
pages = {34},
pmid = {36114193},
issn = {2056-7189},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Expression ; Mammals/genetics ; Promoter Regions, Genetic/genetics ; *RNA, Guide/genetics ; Transcription Factors/genetics ; },
abstract = {Synthetic networks require complex intertwined genetic regulation often relying on transcriptional activation or repression of target genes. CRISPRi-based transcription factors facilitate the programmable modulation of endogenous or synthetic promoter activity and the process can be optimised by using software to select appropriate gRNAs and limit non-specific gene modulation. Here, we develop a computational software pipeline, gDesigner, that enables the automated selection of orthogonal gRNAs with minimized off-target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. Our system expands the toolkit of mammalian synthetic promoters with a new complementary and orthogonal CRISPRi-based system, ultimately enabling the design of synthetic promoter libraries for multiplex gene perturbation that facilitate the understanding of complex cellular phenotypes.},
}
@article {pmid36114150,
year = {2022},
author = {Zhang, X and Shi, Y and Chen, G and Wu, D and Wu, Y and Li, G},
title = {CRISPR/Cas Systems-Inspired Nano/Biosensors for Detecting Infectious Viruses and Pathogenic Bacteria.},
journal = {Small methods},
volume = {6},
number = {10},
pages = {e2200794},
doi = {10.1002/smtd.202200794},
pmid = {36114150},
issn = {2366-9608},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Bacteria/genetics ; *CRISPR-Associated Proteins ; *Biosensing Techniques ; *Communicable Diseases ; *Virus Diseases/diagnosis ; *Viruses/genetics ; },
abstract = {Infectious pathogens cause severe human illnesses and great deaths per year worldwide. Rapid, sensitive, and accurate detection of pathogens is of great importance for preventing infectious diseases caused by pathogens and optimizing medical healthcare systems. Inspired by a microbial defense system (i.e., CRISPR/ CRISPR-associated proteins (Cas) system, an adaptive immune system for protecting microorganisms from being attacked by invading species), a great many new biosensors have been successfully developed and widely applied in the detection of infectious viruses and pathogenic bacteria. Moreover, advanced nanotechnologies have also been integrated into these biosensors to improve their detection stability, sensitivity, and accuracy. In this review, the recent advance in CRISPR/Cas systems-based nano/biosensors and their applications in the detection of infectious viruses and pathogenic bacteria are comprehensively reviewed. First of all, the categories and working principles of CRISPR/Cas systems for establishing the nano/biosensors are simply introduced. Then, the design and construction of CRISPR/Cas systems-based nano/biosensors are comprehensively discussed. In the end, attentions are focused on the applications of CRISPR/Cas systems-based nano/biosensors in the detection of infectious viruses and pathogenic bacteria. Impressively, the remaining opportunities and challenges for the further design and development of CRISPR/Cas system-based nano/biosensors and their promising applications are proposed.},
}
@article {pmid36113404,
year = {2022},
author = {Chirgwin, ME and Schroeder, EA and Derbyshire, ER},
title = {Cut it out! A CRISPR-Cas9 screen identifies host regulators of the Plasmodium liver stage.},
journal = {Cell chemical biology},
volume = {29},
number = {9},
pages = {1365-1367},
doi = {10.1016/j.chembiol.2022.08.005},
pmid = {36113404},
issn = {2451-9448},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Liver/metabolism ; *Malaria/metabolism ; *Parasites ; *Plasmodium ; },
abstract = {In this issue of Cell Chemical Biology, Vijayan and colleagues identify host factors integral for Plasmodium liver-stage infection using a whole-genome CRISPR-Cas9 knockout screen. Their efforts reveal that liver-stage parasites redistribute host microtubules to the parasite membrane in a process dependent on the host Golgi.},
}
@article {pmid36112587,
year = {2022},
author = {Hänggeli, KPA and Hemphill, A and Müller, N and Schimanski, B and Olias, P and Müller, J and Boubaker, G},
title = {Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.},
journal = {PloS one},
volume = {17},
number = {9},
pages = {e0271011},
pmid = {36112587},
issn = {1932-6203},
mesh = {Antigens, Surface/pharmacology ; CRISPR-Cas Systems/genetics ; DNA/pharmacology ; DNA Copy Number Variations ; Mutagenesis, Site-Directed ; Polymerase Chain Reaction ; Pyrimethamine/pharmacology ; Tetrahydrofolate Dehydrogenase/genetics/metabolism ; *Thymidylate Synthase/genetics/metabolism ; *Toxoplasma ; },
abstract = {Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.},
}
@article {pmid36112496,
year = {2022},
author = {Shen, B and Schmidt, MA and Collet, KH and Liu, ZB and Coy, M and Abbitt, S and Molloy, L and Frank, M and Everard, JD and Booth, R and Samadar, PP and He, Y and Kinney, A and Herman, EM},
title = {RNAi and CRISPR-Cas silencing E3-ring ubiquitin ligase AIP2 enhances soybean seed protein content.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erac376},
pmid = {36112496},
issn = {1460-2431},
abstract = {The majority of plant protein in the world's food supply is derived from soybean. Soybean (Glycine max) is a key protein source for global animal feed and in its incorporation into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. AIP2 (ABI3-interacting protein 2) is an E3-ring ubiquitin ligase that targets the seed-specific transcription factor, ABI3. Silencing both soybean AIP2 genes (AIP2a/AIP2b) by RNAi enhanced seed protein content by up to seven percentage-points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR Cas 9-mediated mutation increased seed protein content, this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean which may also be applicable to other protein-source crops.},
}
@article {pmid36110841,
year = {2022},
author = {Shi, H and Guo, J and Yu, Q and Hou, X and Liu, L and Gao, M and Wei, L and Zhang, L and Huang, W and Wang, Y and Liu, G and Tontonoz, P and Xian, X},
title = {CRISPR/Cas9 based blockade of IL-10 signaling impairs lipid and tissue homeostasis to accelerate atherosclerosis.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {999470},
pmid = {36110841},
issn = {1664-3224},
mesh = {Animals ; *Atherosclerosis/genetics/metabolism ; CRISPR-Cas Systems ; Cholesterol/metabolism ; Cricetinae ; Diet, High-Fat/adverse effects ; Homeostasis ; Humans ; *Interleukin-10/genetics/metabolism ; Lipopolysaccharides ; Lipoproteins/metabolism ; Triglycerides ; },
abstract = {Interleukin-10 (IL-10) is a widely recognized immunosuppressive factor. Although the concept that IL-10 executes an anti-inflammatory role is accepted, the relationship between IL-10 and atherosclerosis is still unclear, thus limiting the application of IL-10-based therapies for this disease. Emerging evidence suggests that IL-10 also plays a key role in energy metabolism and regulation of gut microbiota; however, whether IL-10 can affect atherosclerotic lesion development by integrating lipid and tissue homeostasis has not been investigated. In the present study, we developed a human-like hamster model deficient in IL-10 using CRISPR/Cas9 technology. Our results showed that loss of IL-10 changed the gut microbiota in hamsters on chow diet, leading to an increase in lipopolysaccharide (LPS) production and elevated concentration of LPS in plasma. These changes were associated with systemic inflammation, lipodystrophy, and dyslipidemia. Upon high cholesterol/high fat diet feeding, IL-10-deficient hamsters exhibited abnormal distribution of triglyceride and cholesterol in lipoprotein particles, impaired lipid transport in macrophages and aggravated atherosclerosis. These findings show that silencing IL-10 signaling in hamsters promotes atherosclerosis by affecting lipid and tissue homeostasis through a gut microbiota/adipose tissue/liver axis.},
}
@article {pmid36109610,
year = {2022},
author = {Rönspies, M and Schmidt, C and Schindele, P and Lieberman-Lazarovich, M and Houben, A and Puchta, H},
title = {Massive crossover suppression by CRISPR-Cas-mediated plant chromosome engineering.},
journal = {Nature plants},
volume = {8},
number = {10},
pages = {1153-1159},
pmid = {36109610},
issn = {2055-0278},
mesh = {*Arabidopsis/genetics/metabolism ; *Chromosomes, Plant/genetics ; CRISPR-Cas Systems ; Plant Breeding ; },
abstract = {Recent studies have demonstrated that not only genes but also entire chromosomes can be engineered using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPER-associated protein 9 (Cas9)[1-5]. A major objective of applying chromosome restructuring in plant breeding is the manipulation of genetic exchange[6]. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using chromosome restructuring. We were able to induce a heritable inversion of a >17 Mb-long chromosome fragment that contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In single-nucleotide polymorphism marker analysis of the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers within the inverted chromosome region, coupled with a shift of crossovers to the telomeric ends. The few genetic exchanges detected within the inversion all originated from double crossovers. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny.},
}
@article {pmid36109551,
year = {2022},
author = {Kupcinskaite, E and Tutkus, M and Kopūstas, A and Ašmontas, S and Jankunec, M and Zaremba, M and Tamulaitiene, G and Sinkunas, T},
title = {Disarming of type I-F CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {15548},
pmid = {36109551},
issn = {2045-2322},
mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Crystallography, X-Ray ; DNA/metabolism ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.},
}
@article {pmid36109427,
year = {2022},
author = {Nidhi, S and Tripathi, P and Tripathi, V},
title = {Phylogenetic Analysis of Anti-CRISPR and Member Addition in the Families.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36109427},
issn = {1559-0305},
abstract = {CRISPR-Cas is a widespread anti-viral adaptive immune system in the microorganisms. Viruses living in bacteria or some phages carry anti-CRISPR proteins to evade immunity by CRISPR-Cas. The anti-CRISPR proteins are prevalent in phages capable of lying dormant in a CRISPR-carrying host, while their orthologs frequently found in virulent phages. Here, we propose a probabilistic strategy of ancestral sequence reconstruction (ASR) and Hidden Markov Model (HMM) profile search to fish out sequences of anti-CRISPR proteins from environmental metagenomic, human microbiome metagenomic, human microbiome reference genome, and NCBI's non-redundant databases. Our results revealed that the metagenome database dark matter might contain anti-CRISPR encoding genes.},
}
@article {pmid36109386,
year = {2022},
author = {Tong, S and An, K and Chen, W and Zhou, W and Sun, Y and Wang, Q and Li, D},
title = {Evasion of Cas9 toxicity to develop an efficient genome editing system and its application to increase ethanol yield in Fusarium venenatum TB01.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {19-20},
pages = {6583-6593},
pmid = {36109386},
issn = {1432-0614},
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Ethanol/toxicity ; Fusarium ; *Gene Editing/methods ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40-50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene-edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future.Key points• Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss• Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374•Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield.},
}
@article {pmid36107780,
year = {2022},
author = {Friskes, A and Koob, L and Krenning, L and Severson, TM and Koeleman, ES and Vergara, X and Schubert, M and van den Berg, J and Evers, B and Manjón, AG and Joosten, S and Kim, Y and Zwart, W and Medema, RH},
title = {Double-strand break toxicity is chromatin context independent.},
journal = {Nucleic acids research},
volume = {50},
number = {17},
pages = {9930-9947},
pmid = {36107780},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Chromatin/genetics ; *DNA Breaks, Double-Stranded ; DNA Repair ; Humans ; Lamins ; RNA ; },
abstract = {Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.},
}
@article {pmid36107767,
year = {2022},
author = {Wörle, E and Newman, A and D'Silva, J and Burgio, G and Grohmann, D},
title = {Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.},
journal = {Nucleic acids research},
volume = {50},
number = {17},
pages = {10153-10168},
pmid = {36107767},
issn = {1362-4962},
mesh = {Allosteric Regulation ; Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; DNA/genetics ; Endonucleases/metabolism ; Gene Editing ; RNA, Guide/genetics ; },
abstract = {Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.},
}
@article {pmid36107373,
year = {2022},
author = {Aksoy, E and Yildirim, K and Kavas, M and Kayihan, C and Yerlikaya, BA and Çalik, I and Sevgen, &#x130; and Demirel, U},
title = {General guidelines for CRISPR/Cas-based genome editing in plants.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {12151-12164},
pmid = {36107373},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; RNA, Guide/genetics ; Genetic Engineering ; Plants/genetics ; Genome, Plant/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.},
}
@article {pmid36107342,
year = {2023},
author = {Tamura, R and Kamiyama, D},
title = {CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2564},
number = {},
pages = {185-201},
pmid = {36107342},
issn = {1940-6029},
support = {R01 NS107558/NS/NINDS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; *DNA, Single-Stranded ; Green Fluorescent Proteins/genetics ; Humans ; Nucleotides ; Ribonucleoproteins/genetics ; },
abstract = {The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration of GFP11 into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use the GFP11 sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration of GFP11 is performed in cells stably expressing GFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.},
}
@article {pmid36107141,
year = {2022},
author = {Feng, Y and Guo, Z and Chen, J and Zhang, S and Wu, J and Tian, H and Chen, X},
title = {Cationic polymer synergizing with a disulfide-containing enhancer achieved efficient nucleic acid and protein delivery.},
journal = {Biomaterials science},
volume = {10},
number = {21},
pages = {6230-6243},
doi = {10.1039/d2bm01211a},
pmid = {36107141},
issn = {2047-4849},
mesh = {*Polymers/chemistry ; *Disulfides ; Cations ; DNA ; RNA, Messenger ; Sulfur ; Gene Transfer Techniques ; },
abstract = {To improve the efficiency of nucleic acid and protein delivery by cationic polymers, there is a trade-off between increasing the positive charge density of cationic polymers and decreasing cytotoxicity. In this work, a strategy to introduce multiple interactions between the cell membrane and a delivery system based on cationic polymers was proposed. A novel delivery system consisting of PEI1.8k and an enhancer (LA-RT) was fabricated. The introduction of LA-RT contributed to multiple interactions between the delivery system and the cell membrane including electrostatic interactions, hydrogen bonding, hydrophobic interaction, and dynamic sulfur exchange reactions, which enabled efficient intracellular delivery of nucleic acids and proteins. For nucleic acid delivery, plasmid DNA and mRNA were loaded to realize CRISPR/Cas 9 gene editing in vivo and protein expression in vivo, respectively. For protein delivery, the delivery system carrying OVA protein and CpG formed a nano-vaccine, which induced enhanced humoral and cellular immunity in vivo. In addition, the delivery system based on PEI1.8k revealed negligible cytotoxicity. This work provided a novel strategy to prepare efficient delivery systems based on cationic polymers via the introduction of a multifunctional enhancer.},
}
@article {pmid36106983,
year = {2022},
author = {Zhang, Z and Zeng, W and Zhang, W and Li, J and Kong, D and Zhang, L and Wang, R and Peng, F and Kong, Z and Ke, Y and Zhang, H and Kim, C and Zhang, H and Botella, JR and Zhu, JK and Miki, D},
title = {Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis.},
journal = {Plant physiology},
volume = {190},
number = {4},
pages = {2203-2216},
pmid = {36106983},
issn = {1532-2548},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Gene Targeting/methods ; Homologous Recombination/genetics ; Agrobacterium tumefaciens/genetics ; Gene Editing ; },
abstract = {Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.},
}
@article {pmid36106530,
year = {2022},
author = {Yang, X and Li, J and Zhang, S and Li, C and Ma, J},
title = {Amplification-Free, Single-Microbead-Based Cas12a Assay for One-Step DNA Detection at the Single-Molecule Level.},
journal = {Analytical chemistry},
volume = {94},
number = {38},
pages = {13076-13083},
doi = {10.1021/acs.analchem.2c02283},
pmid = {36106530},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Microspheres ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {CRISPR/Cas-based systems are highly attractive for developing next-generation diagnostic technologies because of their intrinsic merits such as simplicity, sensitivity, and specificity. However, currently, nucleic acid amplification procedures are still needed to achieve attomolar sensitivity in most CRISPR/Cas-based assays, which causes high cost, operation difficulty, and low efficiency. Herein, we combine the CRISPR/Cas12a-based assay and a single-microbead detection platform for one-step and amplification-free detection of DNA at the single-molecule level. By modifying DNA reporters on a biomimetic membrane-coated microbead, the activated Cas12a by targets will cleave these reporters and lighten the bead within 10 min. The method allows the detection of the target down to three copies in a 5 μL sample. Furthermore, we successfully apply this method for the specific identification of viral infection, foodborne bacteria, and DNA mutation in real samples without extra nucleic acid amplification. We believe that this approach offers new insights for developing CRISPR/Cas-based DNA assays in biomedical applications.},
}
@article {pmid36106387,
year = {2022},
author = {Chatterjee, S and Mukhopadhyay, S},
title = {Recent advances of lateral flow immunoassay components as "point of need".},
journal = {Journal of immunoassay &amp; immunochemistry},
volume = {43},
number = {6},
pages = {579-604},
doi = {10.1080/15321819.2022.2122063},
pmid = {36106387},
issn = {1532-4230},
mesh = {*COVID-19/diagnosis ; Humans ; Immunoassay/methods ; Pandemics ; Recombinases ; SARS-CoV-2 ; Sensitivity and Specificity ; *Zika Virus ; *Zika Virus Infection ; },
abstract = {Lateral flow immunoassay is the leading Point of Care test and is becoming increasingly essential for its versatile properties. The attraction of lateral flow assay (LFA) has reached its prime position during recent SARS-CoV-2 pandemic and Ebola, Zika epidemics in third world countries where primary screening of the disease and financial issues are very important. During the last decade traditional methodology of LFA was limited to visual detection and qualitative assessment only. However, recently researchers are focusing on the development and improvement of this tool to enhance its specificity, assessment power (quantitative) to make it an alternative to traditional lab-based technology. Modifying working principle and instrumentation, combination of different modern molecular techniques such as Reverse transcription loop mediated isothermal amplification (RT-LAMP), Clustered regularly inter-spaced short palindromic repeat (CRISPR-Cas), Recombinase amplification polymerase (RPA), also association of image-based software, involvement of nanotechnology, implementation of LFA ruler have established authenticity and ultra-specific detection level. These leading immunochromatographic techniques offer simultaneous detection of different analytes from a single sample unit into one multiplex strip and provide the necessary information. This review is a foremost attempt to encompass recent advances of lateral flow assays in combination with molecular biology techniques along with improvements of assay components for improved diagnostic sensitivity and specificity. Some infectious disease diagnosis by LFA with its reporter and low detection limit have also been mentioned in this review.},
}
@article {pmid36104588,
year = {2022},
author = {Kumari, D and Prasad, BD and Dwivedi, P and Hidangmayum, A and Sahni, S},
title = {CRISPR/Cas9 mediated genome editing tools and their possible role in disease resistance mechanism.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11587-11600},
pmid = {36104588},
issn = {1573-4978},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Plant Breeding ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; },
abstract = {Several phytopathogens have detrimental effects on crop production and productivity potentially threatening global food security. Studying the genetic mechanisms of virulence in phytopathogens is vital to assist in their management. Genome editing tools are paving their fascinating roles from the first-generation site-specific nucleases ZNF and TALEN to the current generation clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein9. The discovery of CRISPR/Cas9 has revolutionised the understanding of resistance as well as the susceptibility mechanism against phytopathogens in crop plants. This emerging tool allows researchers to perform precise genome manipulation, genetic screening, regulation, and correction to develop resistance in crop plants with fewer off-target effects. It provides a new opportunity for disease improvement and strengthens the resistant breeding programme. CRISPR/Cas9-based targeted gene manipulation and its enormous application potential as well as the challenges for developing transgene-free disease-resistant crop plants have been discussed in this review.},
}
@article {pmid36104507,
year = {2022},
author = {Lyu, XY and Deng, Y and Huang, XY and Li, ZZ and Fang, GQ and Yang, D and Wang, FL and Kang, W and Shen, EZ and Song, CQ},
title = {CRISPR FISHer enables high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification.},
journal = {Cell research},
volume = {32},
number = {11},
pages = {969-981},
pmid = {36104507},
issn = {1748-7838},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; In Situ Hybridization, Fluorescence ; *DNA/metabolism ; Chromatin ; Genome ; CRISPR-Cas Systems/genetics ; },
abstract = {The dynamic three-dimensional structures of chromatin and extrachromosomal DNA molecules regulate fundamental cellular processes and beyond. However, the visualization of specific DNA sequences in live cells, especially nonrepetitive sequences accounting for most of the genome, is still vastly challenging. Here, we introduce a robust CRISPR-mediated fluorescence in situ hybridization amplifier (CRISPR FISHer) system, which exploits engineered sgRNA and protein trimerization domain-mediated, phase separation-based exponential assembly of fluorescent proteins in the CRISPR-targeting locus, conferring enhancements in both local brightness and signal-to-background ratio and thus achieving single sgRNA-directed visualization of native nonrepetitive DNA loci in live cells. In one application, by labeling and tracking the broken ends of chromosomal fragments, CRISPR FISHer enables real-time visualization of the entire process of chromosome breakage, separation, and subsequent intra- or inter-chromosomal ends rejoining in a single live cell. Furthermore, CRISPR FISHer allows the movement of small extrachromosomal circular DNAs (eccDNAs) and invading DNAs to be recorded, revealing substantial differences in dynamic behaviors between chromosomal and extrachromosomal loci. With the potential to track any specified self or non-self DNA sequences, CRISPR FISHer dramatically broadens the scope of live-cell imaging in biological events and for biomedical diagnoses.},
}
@article {pmid36103687,
year = {2022},
author = {Lee, YG and Kim, C and Kuanyshev, N and Kang, NK and Fatma, Z and Wu, ZY and Cheng, MH and Singh, V and Yoshikuni, Y and Zhao, H and Jin, YS},
title = {Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {38},
pages = {12085-12094},
doi = {10.1021/acs.jafc.2c04251},
pmid = {36103687},
issn = {1520-5118},
mesh = {Aldehyde Reductase/genetics ; CRISPR-Cas Systems ; D-Xylulose Reductase/genetics ; Ethanol/metabolism ; Fermentation ; *Metabolic Engineering/methods ; Nitrogen/metabolism ; Pichia ; Saccharomyces cerevisiae/metabolism ; *Xylose/metabolism ; },
abstract = {Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.},
}
@article {pmid36103526,
year = {2022},
author = {Wan, T and Zhong, J and Pan, Q and Zhou, T and Ping, Y and Liu, X},
title = {Exosome-mediated delivery of Cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases.},
journal = {Science advances},
volume = {8},
number = {37},
pages = {eabp9435},
pmid = {36103526},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems ; *Exosomes/genetics/metabolism ; Genetic Therapy ; *Liver Diseases/genetics/therapy ; Mice ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR-Cas9 gene editing has emerged as a powerful therapeutic technology, but the lack of safe and efficient in vivo delivery systems, especially for tissue-specific vectors, limits its broad clinical applications. Delivery of Cas9 ribonucleoprotein (RNP) owns competitive advantages over other options; however, the large size of RNPs exceeds the loading capacity of currently available delivery vectors. Here, we report a previously unidentified genome editing delivery system, named exosome[RNP], in which Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells through electroporation. Exosome[RNP] facilitated effective cytosolic delivery of RNP in vitro while specifically accumulated in the liver tissue in vivo. Exosome[RNP] showed vigorous therapeutic potential in acute liver injury, chronic liver fibrosis, and hepatocellular carcinoma mouse models via targeting p53 up-regulated modulator of apoptosis (PUMA), cyclin E1 (CcnE1), and K (lysine) acetyltransferase 5 (KAT5), respectively. The developed exosome[RNP] provides a feasible platform for precise and tissue-specific gene therapies of liver diseases.},
}
@article {pmid36100328,
year = {2022},
author = {Bhardwaj, S and Kesari, KK and Rachamalla, M and Mani, S and Ashraf, GM and Jha, SK and Kumar, P and Ambasta, RK and Dureja, H and Devkota, HP and Gupta, G and Chellappan, DK and Singh, SK and Dua, K and Ruokolainen, J and Kamal, MA and Ojha, S and Jha, NK},
title = {CRISPR/Cas9 gene editing: New hope for Alzheimer's disease therapeutics.},
journal = {Journal of advanced research},
volume = {40},
number = {},
pages = {207-221},
pmid = {36100328},
issn = {2090-1224},
mesh = {*Alzheimer Disease/genetics/therapy ; Amyloid beta-Peptides/genetics ; Amyloid beta-Protein Precursor/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
abstract = {BACKGROUND: Alzheimer's disease (AD) is an insidious, irreversible, and progressive neurodegenerative health condition manifesting as cognitive deficits and amyloid beta (Aβ) plaques and neurofibrillary tangles. Approximately 50 million individuals are affected by AD, and the number is rapidly increasing globally. This review explores the role of CRISPR/Cas9 gene editing in the management of AD and its clinical manifestations.<br><br>AIM OF REVIEW: This review aims to provide a deep insight into the recent progress in CRISPR/Cas9-mediated genome editing and its use against neurodegenerative disorders, specifically AD. However, we have referred to its use against parkinsons's disease (PD), Huntington's disease (HD), and other human diseases, as is one of the most promising and emerging technologies for disease treatment.<br><br>The pathophysiology of AD is known to be linked with gene mutations, that is, presenilin (PSEN) and amyloid beta precursor protein (APP). However, clinical trials focused at the genetic level could not meet the desired efficiency. The CRISPR/Cas9 genome editing tool is one of the most powerful technologies for correcting inconsistent genetic signatures and now extensively used for AD management. It has significant potential for the correction of undesired gene mutations associated with AD. This technology has allowed the development of empirical AD models, therapeutic lines, and diagnostic approaches for better understanding the nervous system, from in vitro to in vivo models.},
}
@article {pmid36100322,
year = {2022},
author = {Guo, N and Liu, JB and Li, W and Ma, YS and Fu, D},
title = {The power and the promise of CRISPR/Cas9 genome editing for clinical application with gene therapy.},
journal = {Journal of advanced research},
volume = {40},
number = {},
pages = {135-152},
pmid = {36100322},
issn = {2090-1224},
mesh = {CRISPR-Cas Systems/genetics ; *Cardiovascular Diseases/genetics ; Gene Editing/methods ; Genetic Therapy ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is derived from the bacterial innate immune system and engineered as a robust gene-editing tool. Due to the higher specificity and efficiency of CRISPR/Cas9, it has been widely applied to many genetic and non-genetic disease, including cancers, genetic hemolytic diseases, acquired immunodeficiency syndrome, cardiovascular diseases, ocular diseases, and neurodegenerative diseases, and some X-linked diseases. Furthermore, in terms of the therapeutic strategy of cancers, many researchers used the CRISPR/Cas9 technique to cure or alleviate cancers through different approaches, such as gene therapy and immune therapy.<br><br>AIM OF REVIEW: Here, we conclude the recent application and clinical trials of CRISPR/Cas9 in non-cancerous diseases and cancers and pointed out some of the problems to be solved.<br><br>CRISPR/Cas9, derived from the microbial innate immune system, is developed as a robust gene-editing tool and has been applied widely. Due to its high accuracy and efficiency, CRISPR/Cas9 techniques may provide a great chance to treat some gene-related diseases by disrupting, inserting, correcting, replacing, or blocking genes for clinical application with gene therapy.},
}
@article {pmid36099191,
year = {2022},
author = {Wang, Y and Zhao, D and Sun, L and Wang, J and Fan, L and Cheng, G and Zhang, Z and Ni, X and Feng, J and Wang, M and Zheng, P and Bi, C and Zhang, X and Sun, J},
title = {Engineering of the Translesion DNA Synthesis Pathway Enables Controllable C-to-G and C-to-A Base Editing in Corynebacterium glutamicum.},
journal = {ACS synthetic biology},
volume = {11},
number = {10},
pages = {3368-3378},
doi = {10.1021/acssynbio.2c00265},
pmid = {36099191},
issn = {2161-5063},
mesh = {*Corynebacterium glutamicum/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; DNA-Directed DNA Polymerase/genetics ; DNA/metabolism ; },
abstract = {Expanding the base conversion type is expected to largely broaden the application of base editing, whereas it requires decipherment of the machinery controlling the editing outcome. Here, we discovered that the DNA polymerase V-mediated translesion DNA synthesis (TLS) pathway controlled the C-to-A editing by a glycosylase base editor (GBE) in Escherichia coli. However, C-to-G conversion was surprisingly found to be the main product of the GBE in Corynebacterium glutamicum and subsequent gene inactivation identified the decisive TLS enzymes. Introduction of the E. coli TLS pathway into a TLS-deficient C. glutamicum mutant completely changed the GBE outcome from C-to-G to C-to-A. Combining the canonical C-to-T editor, a pioneering C-to-N base editing toolbox was established in C. glutamicum. The expanded base conversion capability produces greater genetic diversity and promotes the application of base editing in gene inactivation and protein evolution. This study demonstrates the possibility of engineering TLS systems to develop advanced genome editing tools.},
}
@article {pmid36098733,
year = {2022},
author = {Kan, MJ and Doudna, JA},
title = {Treatment of Genetic Diseases With CRISPR Genome Editing.},
journal = {JAMA},
volume = {328},
number = {10},
pages = {980-981},
doi = {10.1001/jama.2022.13468},
pmid = {36098733},
issn = {1538-3598},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Genetic Diseases, Inborn/genetics/therapy ; },
}
@article {pmid36097111,
year = {2022},
author = {Chaudhary, M and Sharma, P and Mukherjee, TK},
title = {Applications of CRISPR/Cas technology against drug-resistant lung cancers: an update.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11491-11502},
pmid = {36097111},
issn = {1573-4978},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; *Lung Neoplasms/genetics ; Neoplasm Recurrence, Local/genetics ; Technology ; },
abstract = {Out of all the cancer types, the most prevalent one is lung cancer. Multiple genes and signaling pathways play role in the progression of lung cancer. Considering the wider prevalence and fatality of lung cancer it has become the focus of current cancer research. Though currently used approaches have shown positive results against lung cancer but success against non-small cell lung cancer (NSCLC) still looms as an enigma for the entire research fraternity. The development of resistance against inhibitors within a short span is one of the reasons responsible for the failure and relapse of lung cancer. Under these prevailing conditions genome/gene-editing technology using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated proteins (Cas), popularly known as CRISPR/Cas technology offers a convenient and flexible method for inducing precise changes within the lung cancer cell. Additionally, CRISPR-barcoding and CRISPR knockout screens at the genome-wide level can help in the functional investigation of specific mutations and identification of novel cancer drivers respectively. Several variants of the CRISPR/Cas system are being developed to limit off-targeting with enhanced precision. The present review article updates the usefulness of CRISPR/Cas technology against various types of lung cancers.},
}
@article {pmid36094255,
year = {2022},
author = {Ozawa, M and Emori, C and Ikawa, M},
title = {CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {186},
pages = {},
doi = {10.3791/64385},
pmid = {36094255},
issn = {1940-087X},
support = {R01 HD088412/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA ; Embryonic Stem Cells ; Gene Editing/methods ; *Gene Targeting/methods ; Mice ; },
abstract = {The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.},
}
@article {pmid36092658,
year = {2022},
author = {Hejabi, F and Abbaszadeh, MS and Taji, S and O'Neill, A and Farjadian, F and Doroudian, M},
title = {Nanocarriers: A novel strategy for the delivery of CRISPR/Cas systems.},
journal = {Frontiers in chemistry},
volume = {10},
number = {},
pages = {957572},
pmid = {36092658},
issn = {2296-2646},
abstract = {In recent decades, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) has become one of the most promising genome-editing tools for therapeutic purposes in biomedical and medical applications. Although the CRISPR/Cas system has truly revolutionized the era of genome editing, the safe and effective delivery of CRISPR/Cas systems represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In addition, there are some challenges in the in vivo delivery to the targeted cells/tissues. Nanotechnology-based drug delivery systems can be employed to overcome this issue. This review discusses different types and forms of CRISPR/Cas systems and the current CRISPR/Cas delivery systems, including non-viral carriers such as liposomes, polymeric, and gold particles. The focus then turns to the viral nanocarriers which have been recently used as a nanocarrier for CRISPR/Cas delivery.},
}
@article {pmid36091445,
year = {2022},
author = {Li, W and Huang, C and Chen, J},
title = {The application of CRISPR /Cas mediated gene editing in synthetic biology: Challenges and optimizations.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {890155},
pmid = {36091445},
issn = {2296-4185},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated enzymes (Cas) is a simple and convenient genome editing tool that has been used in various cell factories and emerging synthetic biology in the recent past. However, several problems, including off-target effects, cytotoxicity, and low efficiency of multi-gene editing, are associated with the CRISPR/Cas system, which have limited its application in new species. In this review, we briefly describe the mechanisms of CRISPR/Cas engineering and propose strategies to optimize the system based on its defects, including, but not limited to, enhancing targeted specificity, reducing toxicity related to Cas protein, and improving multi-point editing efficiency. In addition, some examples of improvements in synthetic biology are also highlighted. Finally, future perspectives of system optimization are discussed, providing a reference for developing safe genome-editing tools for new species.},
}
@article {pmid36089769,
year = {2022},
author = {Qi, X and Gao, H and Lv, R and Mao, W and Zhu, J and Liu, C and Mao, L and Li, X and Xie, C},
title = {CRISPR/dCas-mediated gene activation toolkit development and its application for parthenogenesis induction in maize.},
journal = {Plant communications},
volume = {},
number = {},
pages = {100449},
doi = {10.1016/j.xplc.2022.100449},
pmid = {36089769},
issn = {2590-3462},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate (CRISPRa) or inhibit transcription. Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds. We report here a CRISPR/dCas9-based toolkit equipped with dCas9-VP64 and MS2-p65-HSF1 effectors that may specifically target genes with high activation capability. We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2 (ZmBBM2), acting as a fertilization checkpoint, as a means to engineer parthenogenesis. We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells. Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds. Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.},
}
@article {pmid36089315,
year = {2022},
author = {Zhao, X and Tian, X and Wang, Y and Li, L and Yu, Y and Zhao, S and Zhang, J},
title = {CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA.},
journal = {Analytica chimica acta},
volume = {1227},
number = {},
pages = {340303},
doi = {10.1016/j.aca.2022.340303},
pmid = {36089315},
issn = {1873-4324},
mesh = {*Acquired Immunodeficiency Syndrome/genetics ; CRISPR-Cas Systems ; DNA/chemistry ; *HIV Infections/diagnosis ; *HIV-1/genetics ; Humans ; RNA ; },
abstract = {Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA. After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment, direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.},
}
@article {pmid36088673,
year = {2022},
author = {Hang, XM and Wang, HY and Liu, PF and Zhao, KR and Wang, L},
title = {Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {216},
number = {},
pages = {114683},
pmid = {36088673},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA, Viral/analysis/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/μL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/μL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.},
}
@article {pmid36087841,
year = {2022},
author = {van Senten, JR and Møller, TC and Moo, EV and Seiersen, SD and Bräuner-Osborne, H},
title = {Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {10},
pages = {102466},
pmid = {36087841},
issn = {1083-351X},
mesh = {Animals ; Humans ; Rats ; CRISPR-Cas Systems ; GTP-Binding Protein alpha Subunits, Gq-G11/metabolism ; HEK293 Cells ; *Isoenzymes/genetics/metabolism ; Phosphorylation ; *Protein Kinase C/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {The internalization of G protein-coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser[901], a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.},
}
@article {pmid36087523,
year = {2022},
author = {Pera, J and Castaño, J and Casamitjana, J and Giorgetti, A and Romero-Moya, D},
title = {Generation of heterozygous SAMD9 CRISPR/Cas9-edited iPSC line (ESi086-A-3), carrying p.I1567M mutation.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102906},
doi = {10.1016/j.scr.2022.102906},
pmid = {36087523},
issn = {1876-7753},
mesh = {Humans ; Child ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Myelodysplastic Syndromes/genetics/metabolism ; Heterozygote ; Mutation/genetics ; *Leukemia, Myeloid, Acute/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/genetics ; },
abstract = {Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9[mut] (p.I1567M), taking advantage of CRISPR/Cas9 system. As a result, the genetic engineered hiPSC line represent a new in vitro disease model to understand the impact of SAMD9 mutation at molecular and cellular level during hematopoiesis.},
}
@article {pmid36087195,
year = {2022},
author = {Crowther, MD and Legut, M and Sewell, AK},
title = {Ligand Identification for Orphan MHC-Agnostic T-Cell Receptors by Whole Genome CRISPR-Cas9 Screening.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2574},
number = {},
pages = {3-14},
pmid = {36087195},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Histocompatibility Antigens ; Ligands ; Major Histocompatibility Complex ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {Killer T-cells play important roles in immunity to infection and cancer by detecting intracellular anomalies at the cell surface and destroying the cells that bear them. Conventional killer T-cells scan the intracellular proteome by sampling peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. It is becoming apparent that some T-cells can also respond to pathogens and neoplasms by sensing intracellular changes through molecules other than MHC. We describe an unbiased methodology for T-cell receptor ligand discovery that requires no a priori knowledge regarding the nature of the antigen.},
}
@article {pmid36084670,
year = {2022},
author = {Sui, Y and Xu, Q and Liu, M and Zuo, K and Liu, X and Liu, J},
title = {CRISPR-Cas12a-based detection of monkeypox virus.},
journal = {The Journal of infection},
volume = {85},
number = {6},
pages = {702-769},
pmid = {36084670},
issn = {1532-2742},
mesh = {Humans ; *CRISPR-Cas Systems ; *Monkeypox virus/genetics ; },
}
@article {pmid36083788,
year = {2022},
author = {Aziz, A and Rehman, U and Sheikh, A and Abourehab, MAS and Kesharwani, P},
title = {Lipid-based nanocarrier mediated CRISPR/Cas9 delivery for cancer therapy.},
journal = {Journal of biomaterials science. Polymer edition},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/09205063.2022.2121592},
pmid = {36083788},
issn = {1568-5624},
abstract = {CRISPR/Cas mediated gene-editing has opened new avenues for therapies that show great potential for treating or curing cancers, genetic disorders, and microbial infections such as HIV. CRISPR/Cas9 tool is highly efficacious in revolutionizing the advent of genome editing; however, its efficient and safe delivery is a major hurdle due to its cellular impermeability and instability. Nano vectors could be explored to scale up the safe and effective delivery of CRISPR/Cas9. This review highlights the importance of CRISPR/Cas9 genome editing system in cancer treatment along with the effect of lipid-based nanoparticles in its safe delivery to cancer cells. The solid-lipid nanoparticles, nanostructured lipid carrier, lipid nanoparticles and niosomes have shown great effect in the delivery of CRISPR compounds to the cancer cells. The design and genome editing application in cancer therapy has been discussed along with the future concern and prospects of lipid nanoparticle based CRISPR/Cas9 has been focused toward the end.},
}
@article {pmid36082852,
year = {2022},
author = {Lakhawat, SS and Malik, N and Kumar, V and Kumar, S and Sharma, PK},
title = {Implications of CRISPR-Cas9 in Developing Next Generation Biofuel: A Mini-review.},
journal = {Current protein &amp; peptide science},
volume = {23},
number = {9},
pages = {574-584},
doi = {10.2174/1389203723666220907110310},
pmid = {36082852},
issn = {1875-5550},
mesh = {*Biofuels ; *CRISPR-Cas Systems ; Lignin/metabolism ; Gene Editing/methods ; Saccharomyces cerevisiae/genetics/metabolism ; Ethanol/metabolism ; },
abstract = {The major drawbacks of biofuel production at the commercial level are its low yield, nonavailability of feedstock, feedback inhibition, presence of inhibitory pathways in various organisms, and biofuel intolerance of organisms. The present review focuses on the implications of the CRISPRCas9 mediated gene editing tool to alter the genome of bacteria, algae, fungi, and higher plants for efficient biofuel production. Gene knockout and gene cassette insertions employing CRISPR-Cas9 in Saccharomyces cerevisiae and Kluyveromyces marxianus have resulted in enhanced production of bioethanol and 2-Phenyl ethanol in these organisms, respectively. Genomes of several bacterial strains were also modified to enhance ethanol and butanol production in them. CRISPR-Cas9 modification of microalgae has demonstrated improved total lipid content, a prerequisite for biofuel production. All over, CRISPR-Cas9 has emerged as a tool of choice for engineering the genome and metabolic pathways of organisms for producing industrial biofuel. In plant-based biofuel production, the biosynthetic pathways of lignin interfere with the satisfactory release of fermentable sugars thus hampering efficient biofuel production. CRISPR-Cas9 has shown a promising role in reducing lignin content in various plants including barley, switchgrass, and rice straw.},
}
@article {pmid36082675,
year = {2022},
author = {Wu, MM and Chen, X and Xu, QX and Zang, LS and Wang, S and Li, M and Xiao, D},
title = {Melanin Synthesis Pathway Interruption: CRISPR/Cas9-mediated Knockout of dopa decarboxylase (DDC) in Harmonia axyridis (Coleoptera: Coccinellidae).},
journal = {Journal of insect science (Online)},
volume = {22},
number = {5},
pages = {},
pmid = {36082675},
issn = {1536-2442},
mesh = {Animals ; CRISPR-Cas Systems ; *Coleoptera/genetics ; Dopa Decarboxylase ; Larva ; Melanins ; Ovum ; },
abstract = {CRISPR/Cas9 technology is a very powerful genome editing tool and has been used in many insect species for functional genomics studies through targeted gene mutagenesis. Here, we successfully established CRISPR/Cas9 research platform in Asian multi-colored ladybird beetle, Harmonia axyridis, an important natural enemy in biological control. In this study, one pivotal gene dopa decarboxylase (DDC) in melanin synthesis was targeted by CRISPR/Cas9 to generate mutants in H. axyridis by CRISPR/Cas9 technology. Our results showed that injection of single guide RNA of the DDC and Cas9 protein into preblastoderm eggs induced one insertion and four deletion (indels) mutant H. axyridis. Mutations of HaDDC gene generated 25% mutant rate with melanin missing phenotype in larva, pupa,l and adult stage. The predation ability of the fourth instar larvae has no significant difference between wild (control) and mutant H. axyridis (G0), while these mutant fourth instar larvae had longer developmental period than that of the wild type. Consequently, the total predation of the fourth instar larvae was significantly increased in H. axyridis mutants comparing with the wild type. These results indicated that the success of CRISPR/Cas9 gene editing in H. axyridis. The gene editing platform in H. axyridis would facilitate the gene function research and promote special strain of predatory ladybird beetle generation.},
}
@article {pmid36082579,
year = {2022},
author = {Zhao, J and Cao, S and Zhang, Y and Guo, Y and Li, B and Ma, J and Ma, X},
title = {[Research advances in photoactivatable CRISPR gene editing technology].},
journal = {Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics},
volume = {39},
number = {9},
pages = {1025-1029},
doi = {10.3760/cma.j.cn511374-20211027-00851},
pmid = {36082579},
issn = {1003-9406},
mesh = {*CRISPR-Cas Systems ; DNA ; *Gene Editing ; Humans ; Technology ; },
abstract = {Since the emergence of CRISPR/Cas9, gene editing technologies have attracted increasing attention, in particular type II systems, in which nucleases consist of only a single protein. The effectors include type II Cas9, type V Cas12 and type VI Cas13, which allow precise genomic DNA or RNA editing. Catalytically inactive CRISPR/Cas9 can also be used as a platform to recruit effectors such as transcription factors, epigenetic factors, and/or base modification enzymes to target gene loci. On the other hand, optogenetics offers spatial, temporal, and reversible control of biological processes. CRISPR and optogenetics can enable precise gene editing in vitro and in vivo at the spatiotemporal level, which has a broad applicability in biology and medicine. This article has provided a review for the research advance in photoactivatable CRISPR systems, with details for the design and application of such tools and a discussion over the limitations of the current methods, which may shed light on this emerging field.},
}
@article {pmid36082000,
year = {2022},
author = {Dhakate, P and Sehgal, D and Vaishnavi, S and Chandra, A and Singh, A and Raina, SN and Rajpal, VR},
title = {Comprehending the evolution of gene editing platforms for crop trait improvement.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {876987},
pmid = {36082000},
issn = {1664-8021},
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base editing uses base editors to engineer precise changes for trait improvement. Newer technologies such as prime editing have now been developed as a "search-and-replace" tool to engineer all possible single-base changes. Owing to the availability of these, the field of genome editing has evolved rapidly to develop crop plants with improved traits. In this review, we present the evolution of the CRISPR/Cas system into new-age methods of genome engineering across various plant species and the impact they have had on tweaking plant genomes and associated outcomes on crop improvement initiatives.},
}
@article {pmid36081307,
year = {2023},
author = {Wu, Z and Sun, DW and Pu, H and Wei, Q},
title = {A novel fluorescence biosensor based on CRISPR/Cas12a integrated MXenes for detecting Aflatoxin B1.},
journal = {Talanta},
volume = {252},
number = {},
pages = {123773},
doi = {10.1016/j.talanta.2022.123773},
pmid = {36081307},
issn = {1873-3573},
mesh = {Aflatoxin B1/analysis ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/genetics/chemistry ; Limit of Detection ; *Biosensing Techniques ; },
abstract = {Aflatoxin B1 (AFB1) contamination in food threatens global food safety, and rapid quantitative detection of AFB1 remains a challenge. Herein, a novel fluorescence biosensor was developed for AFB1 detection based on CRISPR/Cas12a and MXenes. Specifically, the well-designed activator was locked by dual-AFB1 aptamers, Cas12a was directly linked to crRNA to form inactivated complexes, and MXenes efficiently adsorbed FAM fluorophore-modified single-stranded DNA (ssDNA-FAM), quenching its fluorescence. In the presence of AFB1, the activator was released due to the preferential binding of the aptamer to AFB1, and the released activator then activated the trans-cleavage activity of Cas12a to indiscriminately cleave ssDNA on MXenes, leading to the recovery of the fluorescence signal. The fluorescent biosensor had a wide detection range from 0.001 to 80 ng mL[-1], a detection limit of 0.92 pg mL[-1], and the ability to detect within 80 min. More importantly, the platform demonstrates excellent detection performance in real peanut samples.},
}
@article {pmid36081278,
year = {2022},
author = {Rong, L and Chen, D and Huang, X and Sun, L},
title = {Delivery of Cas9-guided ABE8e into stem cells using poly(l-lysine) polypeptides for correction of the hemophilia-associated FIX missense mutation.},
journal = {Biochemical and biophysical research communications},
volume = {628},
number = {},
pages = {49-56},
doi = {10.1016/j.bbrc.2022.08.076},
pmid = {36081278},
issn = {1090-2104},
mesh = {Aminohydrolases/genetics ; Blood Coagulation Factors/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/metabolism ; *Gene Editing/methods ; HEK293 Cells ; *Hemophilia A/genetics/metabolism ; *Hemophilia B/genetics/therapy ; Humans ; Mutation ; Mutation, Missense ; Polylysine/chemistry ; Stem Cells/metabolism ; },
abstract = {The coagulation factor 9 gene (FIX) point mutation contributes to most hemophilia B cases, providing ideal gene correction models. Here we identified the frequent mutation G20519A (R226Q) in FIX, which resulted in many severe and moderate hemophilia B patients. This study aimed to investigate the effect of HDR and base editing in correcting FIX mutant. We first constructed HEK293 and liver-derived cell lines Huh7 cells stabling carrying mutated FIX containing G20519A (HEK293-FIXmut and Huh7-FIXmut). Then, CRISPR/Cas9-based homology-directed repair (HDR) and base editing were used for the correction of this mutated point. We used Cas9 nickase (nCas9) mediated HDR and the advanced base editor ABE8e to correct G20519A and then measured the concentration and activity of FIX. Furthermore, we used the star-shaped poly(lysine) gene nanocarriers to deliver the ABE8e correction systems into HEK293-FIXmut and Huh7-FIXmut stem cells to correct mutated FIX. As a result, we found that gRNAs directed inefficient HDR in correcting G20519A. The ABE8e corrected the mutation efficiently in both HEK293-FIXmut and Huh7-FIXmut stem cells. In addition, the star-shaped poly(lysine) carriers delivered non-viral vectors into stem cells efficiently. The nanocarriers-delivered ABE8e system corrected mutated FIX in stem cells, and the stem cells secreted active FIX in high concentration. In conclusion, our study provides a potential alternative for correcting mutated FIX in hemophilia B patients.},
}
@article {pmid36080431,
year = {2022},
author = {Guo, T and Yang, J and Sun, X and Wang, Y and Yang, L and Kong, G and Jiao, H and Bao, G and Li, G},
title = {Whole-Genome Analysis of Acinetobacter baumannii Strain AB43 Containing a Type I-Fb CRISPR-Cas System: Insights into the Relationship with Drug Resistance.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {17},
pages = {},
pmid = {36080431},
issn = {1420-3049},
mesh = {*Acinetobacter Infections/microbiology ; *Acinetobacter baumannii ; Anti-Bacterial Agents/pharmacology ; Bacteria ; Drug Resistance ; Drug Resistance, Multiple, Bacterial ; Genome, Bacterial ; Humans ; },
abstract = {The CRISPR-Cas system is a bacterial and archaea adaptive immune system and is a newly recognized mechanism for controlling antibiotic resistance gene transfer. Acinetobacter baumannii (A. baumannii) is an important organism responsible for a variety of nosocomial infections. A. baumannii infections have become problematic worldwide because of the resistance of A. baumannii to multiple antibiotics. Thus, it is clinically significant to explore the relationship between the CRISPR-Cas system and drug resistance in A. baumannii. This study aimed to analyze the genomic characteristics of the A. baumannii strain AB3 containing the type I-Fb CRISPR-Cas system, which was isolated from a tertiary care hospital in China, and to investigate the relationship between the CRISPR-Cas system and antibiotic resistance in this strain. The whole-genome sequencing (WGS) of the AB43 strain was performed using Illumina and PacBio sequencing. The complete genome of AB43 consisted of a 3,854,806 bp chromosome and a 104,309 bp plasmid. The specific characteristics of the CRISPR-Cas system in AB43 are described as follows: (1) The strain AB43 carries a complete type I-Fb CRISPR-Cas system; (2) Homology analysis confirmed that the cas genes in AB43 share high sequence similarity with the same subtype cas genes; (3) A total of 28 of 105 A. baumannii AB43 CRISPR spacers matched genes in the bacteriophage genome database and the plasmid database, implying that the CRISPR-Cas system in AB43 provides immunity against invasive bacteriophage and plasmids; (4) None of the CRISPR spacers in A. baumannii AB43 were matched with antimicrobial resistance genes in the NCBI database. In addition, we analyzed the presence of antibiotic resistance genes and insertion sequences in the AB43 strain and found that the number of antibiotic resistance genes was not lower than in the "no CRISPR-Cas system" strain. This study supports the idea that the CRISPR-Cas system may inhibit drug-resistance gene expression via endogenous gene regulation, except to the published mechanism that the CRISPR-Cas system efficiently limits the acquisition of antibiotic resistance genes that make bacteria sensitive to antibiotics.},
}
@article {pmid36079653,
year = {2022},
author = {Wijerathna-Yapa, A and Ramtekey, V and Ranawaka, B and Basnet, BR},
title = {Applications of In Vitro Tissue Culture Technologies in Breeding and Genetic Improvement of Wheat.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {17},
pages = {},
pmid = {36079653},
issn = {2223-7747},
abstract = {Sources of new genetic variability have been limited to existing germplasm in the past. Wheat has been studied extensively for various agronomic traits located throughout the genome. The large size of the chromosomes and the ability of its polyploid genome to tolerate the addition or loss of chromosomes facilitated rapid progress in the early study of wheat genetics using cytogenetic techniques. At the same time, its large genome size has limited the progress in genetic characterization studies focused on diploid species, with a small genome and genetic engineering procedures already developed. Today, the genetic transformation and gene editing procedures offer attractive alternatives to conventional techniques for breeding wheat because they allow one or more of the genes to be introduced or altered into an elite cultivar without affecting its genetic background. Recently, significant advances have been made in regenerating various plant tissues, providing the essential basis for regenerating transgenic plants. In addition, Agrobacterium-mediated, biolistic, and in planta particle bombardment (iPB) gene delivery procedures have been developed for wheat transformation and advanced transgenic wheat development. As a result, several useful genes are now available that have been transferred or would be helpful to be transferred to wheat in addition to the current traditional effort to improve trait values, such as resistance to abiotic and biotic factors, grain quality, and plant architecture. Furthermore, the in planta genome editing method will significantly contribute to the social implementation of genome-edited crops to innovate the breeding pipeline and leverage unique climate adaptations.},
}
@article {pmid36078090,
year = {2022},
author = {Wei, X and Pu, A and Liu, Q and Hou, Q and Zhang, Y and An, X and Long, Y and Jiang, Y and Dong, Z and Wu, S and Wan, X},
title = {The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide.},
journal = {Cells},
volume = {11},
number = {17},
pages = {},
pmid = {36078090},
issn = {2073-4409},
mesh = {*Bibliometrics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Publications ; },
abstract = {Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology.},
}
@article {pmid36078073,
year = {2022},
author = {Kavuri, NR and Ramasamy, M and Qi, Y and Mandadi, K},
title = {Applications of CRISPR/Cas13-Based RNA Editing in Plants.},
journal = {Cells},
volume = {11},
number = {17},
pages = {},
pmid = {36078073},
issn = {2073-4409},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Plants/genetics ; RNA/genetics ; *RNA Editing/genetics ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants.},
}
@article {pmid36077578,
year = {2022},
author = {Liu, T and Xu, Y and Wang, X and Ye, Q and Liu, Z and Zhang, Z and Liu, J and Yang, Y and Peng, X and Peng, N},
title = {DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus.},
journal = {International journal of molecular sciences},
volume = {23},
number = {17},
pages = {},
pmid = {36077578},
issn = {1422-0067},
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Integrases/metabolism ; Nucleotide Motifs ; *Sulfolobus/genetics/metabolism ; },
abstract = {CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.},
}
@article {pmid36077571,
year = {2022},
author = {Min, T and Hwarari, D and Li, D and Movahedi, A and Yang, L},
title = {CRISPR-Based Genome Editing and Its Applications in Woody Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {17},
pages = {},
pmid = {36077571},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Genome, Plant/*genetics ; Trees/*genetics ; Wood/genetics ; },
abstract = {CRISPR/Cas-based genome editing technology provides straightforward, proficient, and multifunctional ways for the site-directed modification of organism genomes and genes. The application of CRISPR-based technology in plants has a vast potential value in gene function research, germplasm innovation, and genetic improvement. The complexity of woody plants genome may pose significant challenges in the application and expansion of various new editing techniques, such as Cas9, 12, 13, and 14 effectors, base editing, particularly for timberland species with a long life span, huge genome, and ploidy. Therefore, many novel optimisms have been drawn to molecular breeding research based on woody plants. This review summarizes the recent development of CRISPR/Cas applications for essential traits, including wood properties, flowering, biological stress, abiotic stress, growth, and development in woody plants. We outlined the current problems and future development trends of this technology in germplasm and the improvement of products in woody plants.},
}
@article {pmid36077252,
year = {2022},
author = {Lu, C and Kuang, J and Shao, T and Xie, S and Li, M and Zhu, L and Zhu, L},
title = {Prime Editing: An All-Rounder for Genome Editing.},
journal = {International journal of molecular sciences},
volume = {23},
number = {17},
pages = {},
pmid = {36077252},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {Prime editing (PE), as a "search-and-replace" genome editing technology, has shown the attractive potential of versatile genome editing ability, which is, in principle, currently superior to other well-established genome-editing technologies in the all-in-one operation scope. However, essential technological solutions of PE technology, such as the improvement of genome editing efficiency, the inhibition of potential off-targets and intended edits accounting for unexpected side-effects, and the development of effective delivery systems, are necessary to broaden its application. Since the advent of PE, many optimizations have been performed on PE systems to improve their performance, resulting in bright prospects for application in many fields. This review briefly discusses the development of PE technology, including its functional principle, noteworthy barriers restraining its application, current efforts in technical optimization, and its application directions and potential risks. This review may provide a concise and informative insight into the burgeoning field of PE, highlight the exciting prospects for this powerful tool, and provide clues for questions that may propel the field forward.},
}
@article {pmid36077206,
year = {2022},
author = {Biswas, S and Bridgeland, A and Irum, S and Thomson, MJ and Septiningsih, EM},
title = {Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity.},
journal = {International journal of molecular sciences},
volume = {23},
number = {17},
pages = {},
pmid = {36077206},
issn = {1422-0067},
mesh = {Arachis/genetics ; CRISPR-Cas Systems/genetics ; *Cicer/genetics ; Crops, Agricultural/genetics ; Gene Editing/methods ; Genome, Plant ; Humans ; *Oryza/genetics ; Plant Breeding ; Protoplasts ; RNA, Guide/genetics ; *Vigna/genetics ; },
abstract = {Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement.},
}
@article {pmid36077152,
year = {2022},
author = {Laoharawee, K and Johnson, MJ and Lahr, WS and Sipe, CJ and Kleinboehl, E and Peterson, JJ and Lonetree, CL and Bell, JB and Slipek, NJ and Crane, AT and Webber, BR and Moriarity, BS},
title = {A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.},
journal = {International journal of molecular sciences},
volume = {23},
number = {17},
pages = {},
pmid = {36077152},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; Endoribonucleases/genetics ; Gene Editing ; Gene Knockout Techniques ; Genetic Engineering ; Humans ; Monocytes ; RNA, Messenger/genetics ; *Ribonucleases/genetics ; },
abstract = {Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.},
}
@article {pmid36076934,
year = {2022},
author = {Zhai, YL and Dong, SJ and Zou, MM and Qin, YD and Liu, LL and Cao, MH and Huang, MQ and Vasseur, L and You, MS and Peng, L},
title = {Vitelline Membrane Protein 26 Mutagenesis, Using CRISPR/Cas9, Results in Egg Collapse in Plutella xylostella.},
journal = {International journal of molecular sciences},
volume = {23},
number = {17},
pages = {},
pmid = {36076934},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Egg Proteins ; Female ; *Moths/metabolism ; Mutagenesis ; Vitelline Membrane ; },
abstract = {Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation and embryo development. Here, we characterized the molecular structure and expression patterns of the VMP26 gene and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a worldwide migratory pest of cruciferous plants. The PxVMP26 gene was shown to be a single exon gene that contained an open reading frame of 852 base pairs (bp) encoding 283 amino acids. Both qPCR and western blot analyses showed that PxVMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level of PxVMP26 in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-induced PxVMP26 knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of the PxVMP26 protein was detected in the mutant strains using immunofluorescence and western blot. No significant difference was found in the number of eggs laid within three days between wild and mutant individuals, but there was a lower egg hatchability. The loss of the PxVMP26 gene changed the mean egg size, damaged the structure of the vitelline membrane, and increased the proportion of abnormal eggs due to water loss, resulting in egg collapse. This first analysis of the roles of the VMP gene in the oocyte formation and embryonic development of P. xylostella, using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets of P. xylostella.},
}
@article {pmid36076759,
year = {2022},
author = {Mikhaylova, Y and Shelenkov, A and Chernyshkov, A and Tyumentseva, M and Saenko, S and Egorova, A and Manzeniuk, I and Akimkin, V},
title = {Whole-Genome Analysis of Staphylococcus aureus Isolates from Ready-to-Eat Food in Russia.},
journal = {Foods (Basel, Switzerland)},
volume = {11},
number = {17},
pages = {},
pmid = {36076759},
issn = {2304-8158},
abstract = {This study provides a thorough investigation of a diverse set of antimicrobial resistant (AMR) Staphylococcus aureus isolates collected from a broad range of ready-to-eat (RTE) food in various geographic regions of Russia ranging from Pskov to Kamchatka. Thirty-five isolates were characterized using the whole genome sequencing (WGS) analysis in terms of clonal structure, the presence of resistance and virulence determinants, as well as plasmid replicon sequences and CRISPR/Cas systems. To the best of our knowledge, this is the first WGS-based surveillance of Russian RTE food-associated S. aureus isolates. The isolates belonged to fifteen different multilocus sequence typing (MLST)-based types with a predominant being the ones of clonal complex (CC) 22. The isolates studied can pose a threat to public health since about 40% of the isolates carried at least one enterotoxin gene, and 70% of methicillin-resistant (MRSA) isolates carried a tsst1 gene encoding a toxin that may cause severe acute disease. In addition, plasmid analysis revealed some important characteristics, e.g., Rep5 and Rep20 plasmid replicons were a "signature" of MRSA CC22. By analyzing the isolates belonging to the same/single strain based on cgMLST analysis, we were able to identify the differences in their accessory genomes marking their dynamics and plasticity. This data is very important since S. aureus isolates studied and RTE food, in general, represent an important route of transmission and dissemination of multiple pathogenic determinants. We believe that the results obtained will facilitate performing epidemiological surveillance and developing protection measures against this important pathogen in community settings.},
}
@article {pmid36076376,
year = {2022},
author = {Yu, L and Zang, X and Chen, Y and Gao, Y and Pei, Z and Yang, B and Zhang, H and Narbad, A and Tian, F and Zhai, Q and Chen, W},
title = {Phenotype-genotype analysis of Latilactobacills curvatus from different niches: Carbohydrate metabolism, antibiotic resistance, bacteriocin, phage fragments and linkages with CRISPR-Cas systems.},
journal = {Food research international (Ottawa, Ont.)},
volume = {160},
number = {},
pages = {111640},
doi = {10.1016/j.foodres.2022.111640},
pmid = {36076376},
issn = {1873-7145},
mesh = {*Bacteriocins/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Carbohydrate Metabolism/genetics ; Drug Resistance, Microbial ; Genome, Bacterial/genetics ; Genotype ; Phenotype ; },
abstract = {The potential probiotic function of Latilactobacills curvatus has attracted the attention of researchers. To explore the differences in the genomes of L. curvatus, nine strains were isolated from various sources, including feces and fermented vegetables and compared with 25 strains from the NCBI database. The findings indicated that the average genome size, GC content, and CDS of L. curvatus were 1.94 MB, 41.9%, and 1825, respectively. Its core genome is associated with transcription, translation, carbohydrate transport and metabolism, and defense functions. The pan-genome of L. curvatus was in a closed state. The genetic diversity of L. curatus is mainly manifested in its ability to use carbohydrates, antibiotic resistance, bacteriocin operon, and polymeric regularly interspaced short palindromic repeats (CRISPR)-Cas for bacterial immunity. The CRISPR system of 34 strains of L. curvatus was predominantly found to be of the IIA type with a few IIC and IE types. These findings will contribute to a better understanding of this species.},
}
@article {pmid36075911,
year = {2022},
author = {van de Kooij, B and Kruswick, A and van Attikum, H and Yaffe, MB},
title = {Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5295},
pmid = {36075911},
issn = {2041-1723},
support = {R01 ES015339/ES/NIEHS NIH HHS/United States ; R35 ES028374/ES/NIEHS NIH HHS/United States ; R01 CA226898/CA/NCI NIH HHS/United States ; P30 ES002109/ES/NIEHS NIH HHS/United States ; C42454/A28596/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; Homologous Recombination ; },
abstract = {DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.},
}
@article {pmid36073935,
year = {2022},
author = {Campbell, JA and Cianciotto, NP},
title = {Legionella pneumophila Cas2 Promotes the Expression of Small Heat Shock Protein C2 That Is Required for Thermal Tolerance and Optimal Intracellular Infection.},
journal = {Infection and immunity},
volume = {90},
number = {10},
pages = {e0036922},
pmid = {36073935},
issn = {1098-5522},
support = {R01 AI139054/AI/NIAID NIH HHS/United States ; R21 AI123462/AI/NIAID NIH HHS/United States ; P30 CA060553/CA/NCI NIH HHS/United States ; P41 GM108569/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Legionella pneumophila/physiology ; *Heat-Shock Proteins, Small/metabolism ; Bacterial Proteins/genetics/metabolism ; *Acanthamoeba castellanii ; Ribonucleases/metabolism ; },
abstract = {Previously, we demonstrated that Cas2 encoded within the CRISPR-Cas locus of Legionella pneumophila strain 130b promotes the ability of the Legionella pathogen to infect amoebal hosts. Given that L. pneumophila Cas2 has RNase activity, we posited that the cytoplasmic protein is regulating the expression of another Legionella gene(s) that fosters intracellular infection. Proteomics revealed 10 proteins at diminished levels in the cas2 mutant, and reverse transcription-quantitative (qRT-PCR) confirmed the reduced expression of a gene encoding putative small heat shock protein C2 (HspC2), among several others. As predicted, the gene was expressed more highly at 37°C to 50°C than that at 30°C, and an hspC2 mutant, but not its complemented derivative, displayed ~100-fold reduced CFU following heat shock at 55°C. Compatible with the effect of Cas2 on hspC2 expression, strains lacking Cas2 also had impaired thermal tolerance. The hspC2 mutant, like the cas2 mutant before it, was greatly impaired for infection of Acanthamoeba castellanii, a frequent host for legionellae in waters. HspC2 and Cas2 were not required for entry into these host cells but promoted the replicative phase of intracellular infection. Finally, the hspC2 mutant exhibited an additional defect during the infection of macrophages, which are the primary host for legionellae during lung infection. In summary, hspC2 is upregulated by the presence of Cas2, and HspC2 uniquely promotes both L. pneumophila extracellular survival at high temperatures and infection of amoebal and human host cells. To our knowledge, these findings also represent the first genetic proof linking Cas2 to thermotolerance, expanding the repertoire of noncanonical functions associated with CRISPR-Cas proteins.},
}
@article {pmid36073895,
year = {2022},
author = {Li, J and Tang, L and Li, T and Li, K and Zhang, Y and Ni, W and Xiao, MM and Zhao, Y and Zhang, ZY and Zhang, GJ},
title = {Tandem Cas13a/crRNA-Mediated CRISPR-FET Biosensor: A One-for-All Check Station for Virus without Amplification.},
journal = {ACS sensors},
volume = {7},
number = {9},
pages = {2680-2690},
doi = {10.1021/acssensors.2c01200},
pmid = {36073895},
issn = {2379-3694},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Hepatitis C/genetics ; Humans ; RNA, Viral/genetics ; },
abstract = {The path toward field-effect transistor (FET) application from laboratory to clinic has delivered a compelling push in the biomedical domain, yet ultrasensitive and timely pathogen identification without PCR remains a long-lasting challenge. Herein, we create a generic check station termed "CRISPR-FET", first incorporating the CRISPR/Cas13a system within the FET modality, for accelerated and unamplified detection of viral RNA. Unlike conventional FETs bearing target-specific receptors, this sensor holds three unique advancements: (i) an ingenious sensing mechanism is used, which converts the signal of a large-sized analyte into an on-chip cleavage response of an immobilized CRISPR reporter, enabling signal generation events to occur all within the Debye length; (ii) the multipurpose inspection of the CoV ORF1ab, CoV N gene, and HCV RNA unveils the potential for "one-for-all" scalable FET-based molecular diagnostics; and (iii) it is shown that Cas13a-crRNAs targeting different sites of the viral genome can be deployed in tandem to amplify the FET response, empowering the detection limit down to 1.56 aM, which is a world-record level of sensitivity in the FET for direct viral gene sensing. Notably, a brilliant clinical applicability was made in distinguishing HCV-infected patients from normal controls. Overall, this study sheds new insights into FET-based nucleic acid sensing technology and invokes a vision for its possible future roles in diagnosis of various viral diseases.},
}
@article {pmid36073082,
year = {2022},
author = {Kretzmer, C and Narasimhan, RL and Lal, RD and Balassi, V and Ravellette, J and Kotekar Manjunath, AK and Koshy, JJ and Viano, M and Torre, S and Zanda, VM and Kumravat, M and Saldanha, KMR and Chandranpillai, H and Nihad, I and Zhong, F and Sun, Y and Gustin, J and Borgschulte, T and Liu, J and Razafsky, D},
title = {De novo assembly and annotation of the CHOZN® GS[-/-] genome supports high-throughput genome-scale screening.},
journal = {Biotechnology and bioengineering},
volume = {119},
number = {12},
pages = {3632-3646},
doi = {10.1002/bit.28226},
pmid = {36073082},
issn = {1097-0290},
mesh = {Cricetinae ; Animals ; Cricetulus ; CHO Cells ; *CRISPR-Cas Systems/genetics ; *Genome/genetics ; RNA, Guide/genetics ; },
abstract = {Chinese hamster ovary (CHO) cells have been used as the industry standard for the production of therapeutic monoclonal antibodies for several decades. Despite significant improvements in commercial-scale production processes and media, the CHO cell has remained largely unchanged. Due to the cost and complexity of whole-genome sequencing and gene-editing it has been difficult to obtain the tools necessary to improve the CHO cell line. With the advent of next-generation sequencing and the discovery of the CRISPR/Cas9 system it has become more cost effective to sequence and manipulate the CHO genome. Here, we provide a comprehensive de novo assembly and annotation of the CHO-K1 based CHOZN® GS[-/-] genome. Using this platform, we designed, built, and confirmed the functionality of a whole genome CRISPR guide RNA library that will allow the bioprocessing community to design a more robust CHO cell line leading to the production of life saving medications in a more cost-effective manner.},
}
@article {pmid36072833,
year = {2022},
author = {Ming, M and Long, H and Ye, Z and Pan, C and Chen, J and Tian, R and Sun, C and Xue, Y and Zhang, Y and Li, J and Qi, Y and Wu, J},
title = {Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli.},
journal = {Horticulture research},
volume = {9},
number = {},
pages = {uhac148},
pmid = {36072833},
issn = {2662-6810},
abstract = {CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.},
}
@article {pmid36071435,
year = {2022},
author = {Zhang, K and Duan, X and Cai, P and Gao, L and Wu, X and Yao, L and Zhou, YJ},
title = {Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {182},
pmid = {36071435},
issn = {1475-2859},
mesh = {*CRISPR-Cas Systems ; Coenzyme A Ligases ; *DNA End-Joining Repair ; Homologous Recombination ; Saccharomycetales ; },
abstract = {BACKGROUND: The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts.<br><br>RESULTS: The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number.<br><br>CONCLUSIONS: Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.},
}
@article {pmid36071253,
year = {2022},
author = {Rakshit, S and Shanmugam, G and Sarkar, K},
title = {Coronary artery disease and cancer: a significant resemblance.},
journal = {Medical oncology (Northwood, London, England)},
volume = {39},
number = {12},
pages = {187},
pmid = {36071253},
issn = {1559-131X},
mesh = {CRISPR-Cas Systems ; *Coronary Artery Disease/genetics ; Gene Editing ; Genetic Therapy ; Humans ; *Neoplasms/genetics ; },
abstract = {Cancer and coronary artery disease (CAD) are two of the most common causes of death, and they frequently coexist, especially as the world's population ages. CAD can develop prior to or following cancer diagnosis, as well as a side effect of cancer treatment. CAD develops as complex interactions of lifestyle and hereditary variables, just like the development of the most complex and non-communicable diseases. Cancer is caused by both external/acquired factors (tobacco, food, physical activity, alcohol consumption, epigenetic alterations) and internal/inherited factors (genetic mutations, hormones, and immunological diseases). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system has recently emerged as a strong tool for gene therapy for both cancer as well as CAD treatment due to its great accuracy and efficiency. A deeper understanding of the complex link between CAD and cancer should lead to better prevention, faster detection, and safer treatment strategies.},
}
@article {pmid36070819,
year = {2022},
author = {Antoniêto, ACC and Nogueira, KMV and Mendes, V and Maués, DB and Oshiquiri, LH and Zenaide-Neto, H and de Paula, RG and Gaffey, J and Tabatabaei, M and Gupta, VK and Silva, RN},
title = {Use of carbohydrate-directed enzymes for the potential exploitation of sugarcane bagasse to obtain value-added biotechnological products.},
journal = {International journal of biological macromolecules},
volume = {221},
number = {},
pages = {456-471},
doi = {10.1016/j.ijbiomac.2022.08.186},
pmid = {36070819},
issn = {1879-0003},
mesh = {*Saccharum/chemistry ; Cellulose/chemistry ; Biotechnology ; Biomass ; Hydrolysis ; Lignin/chemistry ; },
abstract = {Microorganisms, such as fungi and bacteria, are crucial players in the production of enzymatic cocktails for biomass hydrolysis or the bioconversion of plant biomass into products with industrial relevance. The biotechnology industry can exploit lignocellulosic biomass for the production of high-value chemicals. The generation of biotechnological products from lignocellulosic feedstock presents several bottlenecks, including low efficiency of enzymatic hydrolysis, high cost of enzymes, and limitations on microbe metabolic performance. Genetic engineering offers a route for developing improved microbial strains for biotechnological applications in high-value product biosynthesis. Sugarcane bagasse, for example, is an agro-industrial waste that is abundantly produced in sugar and first-generation processing plants. Here, we review the potential conversion of its feedstock into relevant industrial products via microbial production and discuss the advances that have been made in improving strains for biotechnological applications.},
}
@article {pmid36070530,
year = {2022},
author = {Iyer, S and Mir, A and Vega-Badillo, J and Roscoe, BP and Ibraheim, R and Zhu, LJ and Lee, J and Liu, P and Luk, K and Mintzer, E and Guo, D and Soares de Brito, J and Emerson, CP and Zamore, PD and Sontheimer, EJ and Wolfe, SA},
title = {Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {685-701},
pmid = {36070530},
issn = {2573-1602},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; *DNA, Single-Stranded/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; HEK293 Cells ; K562 Cells ; },
abstract = {While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.},
}
@article {pmid36070109,
year = {2022},
author = {Wang, W and Yu, Z and He, F and Bai, G and Trick, HN and Akhunova, A and Akhunov, E},
title = {Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system.},
journal = {Plant biotechnology journal},
volume = {20},
number = {12},
pages = {2332-2341},
pmid = {36070109},
issn = {1467-7652},
mesh = {*RNA, Guide/genetics ; Gene Editing ; Triticum/genetics ; *RNA Viruses ; RNA, Viral ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {The low efficiency of genetic transformation and gene editing across diverse cultivars hinder the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we perform direct inoculation of wheat leaves with the barley stripe mosaic virus (BSMV) transcripts to deliver guide RNAs (sgRNA) into the Cas9-expressing wheat. We demonstrate that wheat inoculation with the pool of BSMV-sgRNAs could be used to generate heritable precise deletions in the promoter region of a transcription factor and to perform multiplexed editing of agronomic genes. We transfer the high-expressing locus of Cas9 into adapted spring and winter cultivars by marker-assisted introgression and use of the BSMV-sgRNAs to edit two agronomic genes. A strategy presented in our study could be applied to any adapted cultivar for creating new cis-regulatory diversity or large-scale editing of multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes, and accelerating gene discovery and trait improvement efforts.},
}
@article {pmid36069759,
year = {2022},
author = {Li, H and Busquets, O and Verma, Y and Syed, KM and Kutnowski, N and Pangilinan, GR and Gilbert, LA and Bateup, HS and Rio, DC and Hockemeyer, D and Soldner, F},
title = {Highly efficient generation of isogenic pluripotent stem cell models using prime editing.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {36069759},
issn = {2050-084X},
support = {DP2 CA239597/CA/NCI NIH HHS/United States ; R01 HG012227/HG/NHGRI NIH HHS/United States ; P30 CA013330/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide/genetics/metabolism ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism ; Deoxyribonuclease I/genetics/metabolism ; RNA, Messenger/metabolism ; RNA-Directed DNA Polymerase ; Ribonucleoproteins/metabolism ; CRISPR-Cas Systems ; },
abstract = {The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.},
}
@article {pmid36069378,
year = {2022},
author = {Shin, W and Jeong, S and Lee, JU and Jeong, SY and Shin, J and Kim, HH and Cheon, J and Lee, JH},
title = {Magnetogenetics with Piezo1 Mechanosensitive Ion Channel for CRISPR Gene Editing.},
journal = {Nano letters},
volume = {22},
number = {18},
pages = {7415-7422},
doi = {10.1021/acs.nanolett.2c02314},
pmid = {36069378},
issn = {1530-6992},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Ion Channels/genetics ; },
abstract = {Regulation of genetic activity in single cells and tissues is pivotal to determine key cellular functions in current biomedicine, yet the conventional biochemical activators lack spatiotemporal precision due to the diffusion-mediated slow kinetics and nonselectivity. Here, we describe a magnetogenetic method for target-specific activation of a clustered regularly interspaced short palindromic repeats (CRISPR) system for the regulation of intracellular proteins. We used magnetomechanical force generated by the magnetic nanostructure to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca[2+] signaling pathway, inducing the pre-encoded genes to express genes of interest (GOIs), which is Cas9 protein for the CRISPR regulation of the target proteins. We demonstrated that this magnetogenetic CRISPR system successfully edits the target genome for both in vitro and pseudo-in vivo environments, providing a versatile magnetic platform for remote gene editing of animals with various size scales.},
}
@article {pmid36068235,
year = {2022},
author = {DeWeirdt, PC and McGee, AV and Zheng, F and Nwolah, I and Hegde, M and Doench, JG},
title = {Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5255},
pmid = {36068235},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome ; *RNA, Guide/genetics ; Transcription, Genetic ; },
abstract = {CRISPR technology is a powerful tool for studying genome function. To aid in picking sgRNAs that have maximal efficacy against a target of interest from many possible options, several groups have developed models that predict sgRNA on-target activity. Although multiple tracrRNA variants are commonly used for screening, no existing models account for this feature when nominating sgRNAs. Here we develop an on-target model, Rule Set 3, that makes optimal predictions for multiple tracrRNA variants. We validate Rule Set 3 on a new dataset of sgRNAs tiling essential and non-essential genes, demonstrating substantial improvement over prior prediction models. By analyzing the differences in sgRNA activity between tracrRNA variants, we show that Pol III transcription termination is a strong determinant of sgRNA activity. We expect these results to improve the performance of CRISPR screening and inform future research on tracrRNA engineering and sgRNA modeling.},
}
@article {pmid36068216,
year = {2022},
author = {Nishijima, S and Nagata, N and Kiguchi, Y and Kojima, Y and Miyoshi-Akiyama, T and Kimura, M and Ohsugi, M and Ueki, K and Oka, S and Mizokami, M and Itoi, T and Kawai, T and Uemura, N and Hattori, M},
title = {Extensive gut virome variation and its associations with host and environmental factors in a population-level cohort.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {5252},
pmid = {36068216},
issn = {2041-1723},
mesh = {Bacteria ; *Bacteriophages/genetics ; Humans ; Metagenome ; Metagenomics ; *Virome/genetics ; },
abstract = {Indigenous bacteriophage communities (virome) in the human gut have a huge impact on the structure and function of gut bacterial communities (bacteriome), but virome variation at a population scale is not fully investigated yet. Here, we analyse the gut dsDNA virome in the Japanese 4D cohort of 4198 deeply phenotyped individuals. By assembling metagenomic reads, we discover thousands of high-quality phage genomes including previously uncharacterised phage clades with different bacterial hosts than known major ones. The distribution of host bacteria is a strong determinant for the distribution of phages in the gut, and virome diversity is highly correlated with anti-viral defence mechanisms of the bacteriome, such as CRISPR-Cas and restriction-modification systems. We identify 97 various intrinsic/extrinsic factors that significantly affect the virome structure, including age, sex, lifestyle, and diet, most of which showed consistent associations with both phages and their predicted bacterial hosts. Among the metadata categories, disease and medication have the strongest effects on the virome structure. Overall, these results present a basis to understand the symbiotic communities of bacteria and their viruses in the human gut, which will facilitate the medical and industrial applications of indigenous viruses.},
}
@article {pmid36067720,
year = {2022},
author = {Qiu, F and Gan, X and Yao, J and Jiang, B and Yuan, R and Xiang, Y},
title = {CRISPR/Cas12a-derived sensitive electrochemical biosensing of NF-κB p50 based on hybridization chain reaction and DNA hydrogel.},
journal = {Biosensors &amp; bioelectronics},
volume = {216},
number = {},
pages = {114665},
doi = {10.1016/j.bios.2022.114665},
pmid = {36067720},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/chemistry ; HeLa Cells ; Humans ; Hydrogels ; *NF-kappa B/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA ; Transcription Factors/genetics ; },
abstract = {Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.},
}
@article {pmid36066674,
year = {2022},
author = {Katta, M and Mathew, BA and Chaturvedi, P and Ludhiadch, A and Munshi, A},
title = {Advanced molecular therapies for neurological diseases: focus on stroke, alzheimer's disease, and parkinson's disease.},
journal = {Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology},
volume = {},
number = {},
pages = {},
pmid = {36066674},
issn = {1590-3478},
abstract = {Neurological diseases (NDs) are one of the leading causes of disability and the second leading cause of death globally. Among these stroke, Alzheimer's disease (AD), and Parkinson's disease (PD) are the most common NDs. A rise in the absolute number of individuals affected with these diseases indicates that the current treatment strategies in management and prevention of these debilitating diseases are not effective sufficiently. Therefore, novel treatment strategies are being explored to cure these diseases by addressing the causative mechanisms at the molecular level. Advanced therapies like gene therapy (gene editing and gene silencing) and stem cell therapies aim to cure diseases by gene editing, gene silencing and tissue regeneration, respectively. Gene editing results in the deletion of the aberrant gene or insertion of the corrected gene which can be executed using the CRISPR/Cas gene editing tool a promising treatment strategy being explored for many other prevalent diseases. Gene silencing using siRNA silences the gene by inhibiting protein translation, thereby silencing its expression. Stem cell therapy aims to regenerate damaged cells or tissues because of their ability to divide into any type of cell in the human body. Among these approaches, gene editing and gene silencing have currently been applied in vitro and to animal models, while stem cell therapy has reached the clinical trial stage for the treatment of NDs. The current status of these strategies suggests a promising outcome in their clinical translation.},
}
@article {pmid36066245,
year = {2022},
author = {Velázquez, E and Al-Ramahi, Y and de Lorenzo, V},
title = {CRISPR/Cas9-enhanced Targetron Insertion for Delivery of Heterologous Sequences into the Genome of Gram-Negative Bacteria.},
journal = {Current protocols},
volume = {2},
number = {9},
pages = {e532},
doi = {10.1002/cpz1.532},
pmid = {36066245},
issn = {2691-1299},
mesh = {Bacteria/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; Escherichia coli/genetics ; *Lactococcus lactis/genetics ; },
abstract = {Targetron technology, a gene-editing approach based on the use of mobile group II introns, is particularly useful for bacterial strains deficient in homologous recombination. Specifically, the Ll.LtrB intron from Lactococcus lactis can be used in a wide range of species and can be easily retargeted, that is, modified for integration into any locus of interest. Targetron technology is thus a powerful tool for generating genomic insertions in a broad range of genetic backgrounds, mainly when no other techniques can be efficiently employed. Notably, the approach can be coupled to CRISPR/Cas9 counterselection of wildtype DNA sequences to decrease the population of unmodified cells and ultimately improve Ll.LtrB insertion efficiency. Here, we describe a step-by-step protocol for delivering exogenous sequences into the genome of Gram-negative bacteria by means of targetron technology and CRISPR/Cas9 counterselection using Pseudomonas putida as a model. We describe the retargeting of the Ll.LtrB intron to the locus selected for insertion, the design of specific spacers for eliminating unmutated cells through CRISPR/Cas9 counterselection, and the cloning of exogenous sequences into Ll.LtrB. We also provide a protocol for delivering a specific cargo to the locus of choice once all necessary components of the system are ready. Lastly, we describe a general protocol for curing the engineered strain of all plasmids. CRISPR/Cas9-enhanced Ll.LtrB insertion can be an efficient alternative for overcoming low recombination-based editing efficiency and can be used in numerous bacterial species. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retargeting the Ll.LtrB intron to the target locus Support Protocol 1: Preparation of competent E. coli Basic Protocol 2: Design and cloning of CRISPR spacers to counterselect Ll.LtrB insertions Support Protocol 2: Interference assay to check efficiency of selected spacers Basic Protocol 3: Cloning cargos into Ll.LtrB Basic Protocol 4: Ll.LtrB/CRISPR/Cas9-mediated insertion Basic Protocol 5: Curing the engineered strain of plasmids.},
}
@article {pmid36066120,
year = {2022},
author = {Ader, F and Russi, M and Tixier-Cardoso, L and Jullian, E and Martin, E and Richard, P and Villard, E and Monnier, V},
title = {Drosophila CRISPR/Cas9 mutants as tools to analyse cardiac filamin function and pathogenicity of human FLNC variants.},
journal = {Biology open},
volume = {11},
number = {9},
pages = {},
pmid = {36066120},
issn = {2046-6390},
mesh = {Actins/metabolism ; Animals ; CRISPR-Cas Systems ; *Cardiomyopathies/genetics/metabolism ; *Drosophila/metabolism ; Drosophila Proteins ; Drosophila melanogaster/genetics/metabolism ; Filamins/chemistry/genetics/metabolism ; Humans ; Virulence ; },
abstract = {Filamins are large proteins with actin-binding properties. Mutations in FLNC, one of the three filamin genes in humans, have recently been implicated in dominant cardiomyopathies, but the underlying mechanisms are not well understood. Here, we aimed to use Drosophila melanogaster as a new in vivo model to study these diseases. First, we show that adult-specific cardiac RNAi-induced depletion of Drosophila Filamin (dFil) induced cardiac dilatation, impaired systolic function and sarcomeric alterations, highlighting its requirement for cardiac function and maintenance of sarcomere integrity in the adult stage. Next, we introduced in the cheerio gene, using CRISPR/Cas9 gene editing, three missense variants, previously identified in patients with hypertrophic cardiomyopathy. Flies carrying these variants did not exhibit cardiac defects or increased propensity to form filamin aggregates, arguing against their pathogenicity. Finally, we show that deletions of the C-term part of dFil carrying the last four Ig-like domains are dispensable for cardiac function. Collectively, these results highlight the relevance of this model to explore the cardiac function of filamins and increase our understanding of physio-pathological mechanisms involved in FLNC-related cardiomyopathies. This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid36065829,
year = {2022},
author = {Tian, K and Hong, X and Guo, M and Li, Y and Wu, H and Caiyin, Q and Qiao, J},
title = {Development of Base Editors for Simultaneously Editing Multiple Loci in Lactococcus lactis.},
journal = {ACS synthetic biology},
volume = {11},
number = {11},
pages = {3644-3656},
doi = {10.1021/acssynbio.1c00561},
pmid = {36065829},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Lactococcus lactis/genetics ; Gene Editing ; Plasmids/genetics ; Cytidine ; },
abstract = {Lactococcus lactis serves as the most extensively studied model organism and an important dairy species. Though CRISPR-Cas9 systems have been developed for robust genetic manipulations, simultaneously editing multiple endogenous loci in L. lactis is still challenging. Herein, we first report the development of a double-strand break-free, robust, multiloci editing system CRISPR-deaminase-assisted base editor (CRISPR-DBE), which comprises a cytidine (CRISPR-cDBE) and an adenosine deaminase-assisted base editor (CRISPR-aDBE). Specifically targeted by a sgRNA, CRISPR-cDBE can efficiently introduce a cytidine-to-thymidine mutation and CRISPR-aDBE can high-efficiently convert adenosine to guanosine within a 5 nt editing window. CRISPR-cDBE was validated and successfully applied to simultaneously inactivate multiple genes using a single plasmid in L. lactis strain NZ9000. Meanwhile, the temperature-sensitive plasmid of CRISPR-DBE can be cured quickly, and the continuous gene editing of L. lactis has been achieved. Furthermore, CRISPR-cDBE can also efficiently convert the targeted C to T in a nisin-producing, industrial L. lactis strain F44. Finally, we applied genome-wide bioinformatics analysis to determine the scope of gene inactivation for these base editors using different Cas9 variants and evaluated the preference of SpGn and SpRYn variants for the protospacer adjacent motif in L. lactis NZ9000. Taken together, our study provides a powerful tool for simultaneously editing multiple loci in L. lactis, which may have a wide range of industrial applications in the future.},
}
@article {pmid36064968,
year = {2022},
author = {Zou, RS and Marin-Gonzalez, A and Liu, Y and Liu, HB and Shen, L and Dveirin, RK and Luo, JXJ and Kalhor, R and Ha, T},
title = {Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites.},
journal = {Nature cell biology},
volume = {24},
number = {9},
pages = {1433-1444},
pmid = {36064968},
issn = {1476-4679},
support = {U01 HL156056/HL/NHLBI NIH HHS/United States ; T32 GM007445/GM/NIGMS NIH HHS/United States ; U01 DK127432/DK/NIDDK NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; F30 CA254160/CA/NCI NIH HHS/United States ; T32 GM007057/GM/NIGMS NIH HHS/United States ; T32 GM136577/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; Chromatin/genetics ; DNA/metabolism ; DNA Repair/genetics ; Genomics ; *RNA, Guide/genetics/metabolism ; },
abstract = {Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.},
}
@article {pmid36064264,
year = {2022},
author = {Han, RC and MacLaren, RE},
title = {RNA gene editing in the eye and beyond: The neglected tool of the gene editing armatorium?.},
journal = {International review of cell and molecular biology},
volume = {372},
number = {},
pages = {175-205},
doi = {10.1016/bs.ircmb.2022.04.009},
pmid = {36064264},
issn = {1937-6448},
mesh = {CRISPR-Cas Systems ; DNA ; *Gene Editing/methods ; RNA ; *RNA Editing ; RNA, Guide/genetics/metabolism ; },
abstract = {RNA editing allows correction of pathological point mutations without permanently altering genomic DNA. Theoretically targetable to any RNA type and site, its flexibility and reversibility makes it a potentially powerful gene editing tool. RNA editing offers a host of potential advantages in specific niches when compared to currently available alternative gene manipulation techniques. Unlike DNA editors, which are currently too large to be delivered in vivo using a viral vector, smaller RNA editors fit easily within the capabilities of an adeno-associated virus (AAV). Unlike gene augmentation, which is limited by gene size and viral packaging constraints, RNA editing may correct transcripts too long to fit within a viral vector. In this article we examine the development of RNA editing and discuss potential applications and pitfalls. We argue that, although in its infancy, an RNA editing approach can offer unique advantages for selected retinal diseases.},
}
@article {pmid36063702,
year = {2022},
author = {Liu, JX and Sun, XM and Liu, D and Liu, YH and Li, CY},
title = {Smart NIR light-gated CRISPR/Cas12a fluorescent biosensor with boosted biological delivery and trans-cleavage activity for high-performance in vivo operation.},
journal = {Biosensors &amp; bioelectronics},
volume = {216},
number = {},
pages = {114646},
doi = {10.1016/j.bios.2022.114646},
pmid = {36063702},
issn = {1873-4235},
mesh = {Animals ; Biomarkers, Tumor ; *Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Manganese Compounds ; Mice ; Oxides ; RNA, Messenger ; },
abstract = {Despite the in vitro usage of CRISPR/Cas12a system in fluorescent biosensors has made remarkable achievements, many challenges such as poor biological delivery, insufficient sensitivity, and uncontrollable initiation compel them hard to conduct in vivo analysis. Thus, we propose here some fruitful sensing concepts. First, the multiple biomolecular components of CRISPR/Cas12a system are collectively carried by MnO2 nanosheets via a simple physical absorption to achieve a highly-efficient biological uptake. Under the reduction of widespread biothiols, not only the sensing frame is easily released but also sufficient Mn[2+] is produced to serve as an effective trans-cleavage accelerator. Furthermore, a photocleavge-linker induced smart near-infrared (NIR) light-gated manner is designed to offer a spatiotemporal target recognition, for which a 808 nm NIR light transduced ultraviolet upconversion luminescence with weak thermal effect is employed to completely prevent the sensing flow from pre-initiating during the biological delivery. As a conceptual validation, this biosensor has satisfactory sensitivity and specificity to survivn messenger RNA (a broad-spectrum cancer biomarker). More importantly, it can work as a reliable imaging platform for differentiating cancers in live cellular level and also presents a high-performance operation ability for analyzing live mice, greatly promoting the CRISPR technology in biosensing field.},
}
@article {pmid36063701,
year = {2022},
author = {Elumalai, P and Ezhilarasan, D},
title = {Emerging applications of CRISPR/Cas9 gene editing technology in reversing drug resistance in oral squamous cell carcinoma.},
journal = {Oral oncology},
volume = {134},
number = {},
pages = {106100},
doi = {10.1016/j.oraloncology.2022.106100},
pmid = {36063701},
issn = {1879-0593},
mesh = {CRISPR-Cas Systems/genetics ; *Carcinoma, Squamous Cell/drug therapy/genetics ; Drug Resistance ; Gene Editing ; *Head and Neck Neoplasms/genetics ; Humans ; *Mouth Neoplasms/drug therapy/genetics ; Squamous Cell Carcinoma of Head and Neck/genetics ; Technology ; },
}
@article {pmid36063327,
year = {2022},
author = {Newton, MD and Taylor, BJ and Cuomo, ME and Rueda, DS},
title = {CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2478},
number = {},
pages = {349-378},
pmid = {36063327},
issn = {1940-6029},
support = {206292/C/17/Z/WT_/Wellcome Trust/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; UKRI MC-A658-5TY10/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Single Molecule Imaging ; },
abstract = {The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.},
}
@article {pmid36063051,
year = {2022},
author = {Dvir, E and Shohat, S and Flint, J and Shifman, S},
title = {Identification of genetic mechanisms for tissue-specific genetic effects based on CRISPR screens.},
journal = {Genetics},
volume = {222},
number = {3},
pages = {},
pmid = {36063051},
issn = {1943-2631},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cell Line ; *Neoplasms/genetics ; Gene Amplification ; },
abstract = {A major challenge in genetic studies of complex diseases is to determine how the action of risk genes is restricted to a tissue or cell type. Here, we investigate tissue specificity of gene action using CRISPR screens from 786 cancer cell lines originating from 24 tissues. We find that the expression pattern of the gene across tissues explains only a minority of cases of tissue-specificity (9%), while gene amplification and the expression levels of paralogs account for 39.5% and 15.5%, respectively. In addition, the transfer of small molecules to mutant cells explains tissue-specific gene action in blood. The tissue-specific genes we found are not specific just for human cancer cell lines: we found that the tissue-specific genes are intolerant to functional mutations in the human population and are associated with human diseases more than genes that are essential across all cell types. Our findings offer important insights into genetic mechanisms for tissue specificity of human diseases.},
}
@article {pmid36063049,
year = {2022},
author = {Sottolano, CJ and Revaitis, NT and Geneva, AJ and Yakoby, N},
title = {Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {11},
pages = {},
pmid = {36063049},
issn = {2160-1836},
support = {R15 GM101597/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *Gene Editing ; Drosophila/genetics ; Drosophila melanogaster/genetics ; Animals, Genetically Modified ; },
abstract = {The diversity among Drosophila species presents an opportunity to study the molecular mechanisms underlying the evolution of biological phenomena. A challenge to investigating these species is that, unlike the plethora of molecular and genetics tools available for D. melanogaster research, many other species do not have sequenced genomes; a requirement for employing these tools. Selecting transgenic flies through white (w) complementation has been commonly practiced in numerous Drosophila species. While tolerated, the disruption of w is associated with impaired vision, among other effects in D. melanogaster. The D. nebulosa fly has a unique mating behavior which requires vision, and is thus unable to successfully mate in dark conditions. Here, we hypothesized that the disruption of w will impede mating success. As a first step, using PacBio long-read sequencing, we assembled a high-quality annotated genome of D. nebulosa. Using these data, we employed CRISPR/Cas9 to successfully disrupt the w gene. As expected, D. nebulosa males null for w did not court females, unlike several other mutant strains of Drosophila species whose w gene has been disrupted. In the absence of mating, no females became homozygous null for w. We conclude that gene disruption via CRISPR/Cas9 genome engineering is a successful tool in D. nebulosa, and that the w gene is necessary for mating. Thus, an alternative selectable marker unrelated to vision is desirable.},
}
@article {pmid36060733,
year = {2022},
author = {Yadav, G and Singh, R},
title = {In silico analysis reveals the co-existence of CRISPR-Cas type I-F1 and type I-F2 systems and its association with restricted phage invasion in Acinetobacter baumannii.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {909886},
pmid = {36060733},
issn = {1664-302X},
abstract = {INTRODUCTION: Acinetobacter baumannii, an opportunistic pathogen, rapidly acquires antibiotic resistance, thus compelling researchers to develop alternative treatments at utmost priority. Phage-based therapies are of appreciable benefit; however, CRISPR-Cas systems are a major constraint in this approach. Hence for effective implementation and a promising future of phage-based therapies, a multifaceted understanding of the CRISPR-Cas systems is necessary.<br><br>METHODS: This study investigated 4,977 RefSeq genomes of A. baumannii from the NCBI database to comprehend the distribution and association of CRISPR-Cas systems with genomic determinants.<br><br>RESULTS: Approximately 13.84% (n = 689/4,977) isolates were found to carry the CRSIPR-Cas system, and a small fraction of isolates, 1.49% (n = 74/4,977), exhibited degenerated CRISPR-Cas systems. Of these CRISPR-Cas positive (+) isolates, 67.48% (465/689) isolates harbored type I-F1, 28.59% (197/689) had type I-F2, and 3.7% (26/689) had co-existence of both type I-F1 and type I-F2 systems. Co-existing type I-F1 and type I-F2 systems are located distantly (&#x223c;1.733 Mb). We found a strong association of CRISPR-Cas systems within STs for type I-F1 and type I-F2, whereas the type I-F1 + F2 was not confined to any particular ST. Isolates with type I-F1 + F2 exhibited a significantly high number of mean spacers (n = 164.58 &#xb1; 46.41) per isolate as compared to isolates with type I-F2 (n = 82.87 &#xb1; 36.14) and type I-F1 (n = 54.51 &#xb1; 26.27) with majority targeting the phages. Isolates with type I-F1 (p < 0.0001) and type I-F2 (p < 0.0115) displayed significantly larger genome sizes than type I-F1 + F2. A significantly reduced number of integrated phages in isolates with co-existence of type I-F1 + F2 compared with other counterparts was observed (p = 0.0041). In addition, the isolates carrying type I-F1 + F2 did not exhibit reduced resistance and virulence genes compared to CRISPR-Cas(-) and CRISPR-Cas (+) type I-F1 and type I-F2, except for bap, abaI, and abaR.<br><br>CONCLUSION: Our observation suggests that the co-existence of type I-F1 and F2 is more effective in constraining the horizontal gene transfer and phage invasion in A. baumannii than the isolates exhibiting only type I-F1 and only type I-F2 systems.},
}
@article {pmid36060092,
year = {2022},
author = {Tang, S and Wu, X and Liu, J and Zhang, Q and Wang, X and Shao, S and Gokbag, B and Fan, K and Liu, X and Li, F and Cheng, L and Li, L},
title = {Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101556},
pmid = {36060092},
issn = {2666-1667},
support = {R01 CA157429/CA/NCI NIH HHS/United States ; R01 CA196634/CA/NCI NIH HHS/United States ; R01 CA264652/CA/NCI NIH HHS/United States ; R01 CA256893/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Library ; Genes, Lethal ; *RNA, Guide/genetics ; Synthetic Lethal Mutations ; },
abstract = {Combinatorial CRISPR screening is useful for investigating synthetic lethality (SL) gene pairs. Here, we detail the steps for dual-gRNA library construction, with the introduction of two backbones, LentiGuide_DKO and LentiCRISPR_DKO. We describe steps for in vitro screening with 22Rv1-Cas9 and SaOS2-Cas9 cells followed by sequencing and data analysis. By introducing two backbones, we optimized the library construction process, facilitated standard pair-end sequencing, and provided options of screening on cells with or without modification of Cas9 expression.},
}
@article {pmid36058644,
year = {2022},
author = {Ye, H and Jiang, C and Li, L and Li, H and Rong, Z and Lin, Y},
title = {Live-cell imaging of genomic loci with Cas9 variants.},
journal = {Biotechnology journal},
volume = {17},
number = {12},
pages = {e2100381},
doi = {10.1002/biot.202100381},
pmid = {36058644},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; *Endonucleases/genetics ; Genomics ; Genetic Loci ; RNA ; },
abstract = {BACKGROUND: Endonuclease-deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (dCas9) has been repurposed for live-cell imaging of genomic loci. Engineered or evolved dCas9 variants have been developed to increase the applicability of the CRISPR/dCas9 system. However, there have been no systematic comparisons of these dCas9 variants in terms of their performance in the visualization of genomic loci.<br><br>Here we demonstrate that dSpCas9 and its variants deSpCas9(1.1), dSpCas9-HF1, devoCas9, and dxCas9(3.7) can be used for CRISPR-based live-cell genomic imaging. dSpCas9 had the greatest utility, with a high labeling efficiency of repetitive sequences-including those with a low number of repeats-and good compatibility with target RNA sequences at the MUC4 locus that varied in length from 13 to 23 nucleotides. We combined CRISPR-Tag with the dSpCas9 imaging system to observe the dynamics of the Tet promoter and found that its movement was restricted when it was active.<br><br>CONCLUSIONS AND IMPLICATIONS: These novel Cas9 variants provide a new set of tools for investigating the spatiotemporal regulation of gene expression through live imaging of genomic sites.},
}
@article {pmid36058302,
year = {2022},
author = {Chen, Z and Du, H and Tao, Y and Xu, Y and Wang, F and Li, B and Zhu, QH and Niu, H and Yang, J},
title = {Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {324},
number = {},
pages = {111449},
doi = {10.1016/j.plantsci.2022.111449},
pmid = {36058302},
issn = {1873-2259},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Glutens/genetics ; *Oryza/genetics/metabolism ; Phenotype ; Plant Breeding ; },
abstract = {Chronic kidney disease (CKD) and phenylketonuria (PKU) patients need to eat rice with low glutelin content. Therefore, breeding low glutelin content rice varieties with high yield and delicious taste is one of the major goals of rice breeders due to the high demand for the product. In this study, we designed three sgRNAs targeting nine glutelin genes and generated nine T-DNA-free homozygous editing lines with reduced glutelin content compared with the wild-type due to simultaneous mutation(s) in 5-7 glutelin genes. The glutelin content of two lines is even significantly lower than that of the low glutelin content cultivar, LGC-1. Compared to the wild-type, these low glutelin lines showed similar agronomic traits, including yield components and viscosity properties, and can be used as new varieties or parental materials for further breeding.},
}
@article {pmid36057300,
year = {2022},
author = {Jogam, P and Sandhya, D and Alok, A and Peddaboina, V and Allini, VR and Zhang, B},
title = {A review on CRISPR/Cas-based epigenetic regulation in plants.},
journal = {International journal of biological macromolecules},
volume = {219},
number = {},
pages = {1261-1271},
doi = {10.1016/j.ijbiomac.2022.08.182},
pmid = {36057300},
issn = {1879-0003},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Epigenesis, Genetic/genetics ; *Gene Editing ; RNA, Untranslated ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {Epigenetic changes are the heritable modifications in genes without altering DNA sequences. The epigenetic changes occur in the plant genomes to regulate gene expression patterns, which were used to regulate different biological processes, including coping various environmental stresses. These changes, including DNA methylation, non-coding RNA regulation, and histone modification, play a vital role in the transcription and translation processes to regulate gene expression. Gene engineering for the development of stress-tolerant crops via the DNA methylation pathway initially needs a proper selection of genes and its promoter. Manipulating epigenetics requires genetic engineering tools such as Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas). However, CRISPR/Cas9 mediated epigenetic editing refers to transcriptional reprogramming at the targeted sites using epigenetic enzymes fused with decatalytical Cas9 (dCas9). This review focused on the different epigenetic mechanisms in plants and their potential contribution to developing epigenetic tools. The dCas9 endonuclease tethered with transcriptional repressor or activator domain leads to CRISPR inhibitor (CRISPRi) or activator (CRISPRa) for regulating gene expression. The dCas9 has been successfully fused with other various effector domains for constructing epigenetic tools, including the DNA methyltransferase 3A (DNMT3A), or the DNA demethylase TET. Multiple efforts have been made to improve epigenome editing in plants. Initially, incorporating SunTag into the dCas9-EpiEffector complex was used as an epigenetic tool; demethylation of target loci with dCas9-SunTag-TET1 futher increased its efficiency. Additionally, SunTag could also be fused with the dCas9-DNMT3A complex to augment CpG methylation at a targeted loci.},
}
@article {pmid36057005,
year = {2022},
author = {Zhao, Z and Qi, Y and Yang, Z and Cheng, L and Sharif, R and Raza, A and Chen, P and Hou, D and Li, Y},
title = {Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.).},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11481-11490},
pmid = {36057005},
issn = {1573-4978},
mesh = {*Agrobacterium/genetics ; *Cucumis sativus/genetics ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Mutagenesis, Site-Directed ; Transformation, Genetic ; },
abstract = {BACKGROUNDS: The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristic of simple design, low cost and high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber.<br><br>METHODS AND RESULTS: In this study, a Histochemical &#x3b2;-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1&#xa0;day, Agrobacterium bacterial solution containing AS, and 20&#xa0;min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines.<br><br>CONCLUSION: In this study, 2.4&#x2030; of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further&#xa0;optimize and improve the efficiency of the genetic transformation of cucumber.},
}
@article {pmid36056433,
year = {2022},
author = {Umbach, A and Maule, G and Kheir, E and Cutarelli, A and Foglia, M and Guarrera, L and Fava, LL and Conti, L and Garattini, E and Terao, M and Cereseto, A},
title = {Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology.},
journal = {Stem cell research &amp; therapy},
volume = {13},
number = {1},
pages = {440},
pmid = {36056433},
issn = {1757-6512},
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics ; Clone Cells/metabolism ; *De Lange Syndrome/genetics/therapy ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Phenotype ; Technology ; },
abstract = {BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60-70%). Currently, there are no specific cures available for CdLS and clinical management is needed for life. Disease models are highly needed to find a cure. Among therapeutic possibilities are genome editing strategies based on CRISPR-Cas technology.<br><br>METHODS: A comparative analysis was performed to test the most recent CRISPR-Cas technologies comprising base- and prime-editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed repair (HDR) induced by Cas9 nuclease activity. The HDR method that was found more efficient was applied to repair a CdLS-causing mutation in the NIPBL gene. Human-induced pluripotent stem cells (hiPSCs) derived from a CdLS patient carrying the c.5483G&#x2009;>&#x2009;A mutation in the NIPBL were modified through HDR to generate isogenic corrected clones.<br><br>RESULTS: This study reports an efficient method to repair the NIPBL gene through HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed the generation of isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency.<br><br>CONCLUSIONS: CdLS cellular models were generated which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular, the hiPSC-based cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that were generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions.},
}
@article {pmid36050244,
year = {2022},
author = {Gurney, M and O'Reilly, E and Corcoran, S and Brophy, S and Krawczyk, J and Otto, NM and Hermanson, DL and Childs, RW and Szegezdi, E and O'Dwyer, ME},
title = {Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.},
journal = {Cytotherapy},
volume = {24},
number = {11},
pages = {1087-1094},
doi = {10.1016/j.jcyt.2022.07.008},
pmid = {36050244},
issn = {1477-2566},
support = {203930/B/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cytokines/metabolism ; Cytotoxicity, Immunologic ; DNA Transposable Elements/genetics ; *Epstein-Barr Virus Infections ; Gene Editing ; Herpesvirus 4, Human/genetics ; Humans ; Immunotherapy, Adoptive/methods ; Killer Cells, Natural ; *Leukemia, Lymphocytic, Chronic, B-Cell/genetics/therapy ; *Leukemia, Myeloid, Acute ; *Receptors, Chimeric Antigen/genetics/metabolism ; },
abstract = {BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development.<br><br>AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion.<br><br>RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype.<br><br>CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.},
}
@article {pmid36049732,
year = {2022},
author = {El Zowalaty, ME and Falgenhauer, L and Forsythe, S},
title = {Draft genome sequence of Cronobacter sakazakii strain MEZCS99 sequence type 3 isolated from chicken in South Africa.},
journal = {Journal of global antimicrobial resistance},
volume = {31},
number = {},
pages = {292-294},
doi = {10.1016/j.jgar.2022.08.016},
pmid = {36049732},
issn = {2213-7173},
abstract = {OBJECTIVES: Cronobacter sakazakii is an emerging opportunistic foodborne pathogen that is frequently associated with life-threatening infections such as infantile septicemia, meningitis, and necrotizing enterocolitis. The emergence of antimicrobial-resistant, livestock-associated C. sakazakii is a great public health concern. Here, we report on the first draft genome sequence of C. sakazakii strain MEZCS99 sequence type 3 (ST3) isolated from feces from a healthy chicken in KwaZulu-Natal Province, South Africa.<br><br>METHODS: The genomic DNA of C. sakazakii was sequenced using an Illumina MiSeq platform (Illumina Inc., San Diego, CA). Generated reads were trimmed and de novo assembled. The assembled contigs were analyzed for virulence and antimicrobial resistance genes, extra-chromosomal plasmids, and multilocus sequence type (MLST). To compare the sequenced strains to other previously sequenced C. sakazakii strains, available raw read sequences of C. sakazakii were downloaded and all sequence files were treated identically to generate a core genome phylogenetic tree.<br><br>RESULTS: Intrinsic beta-lactam resistance gene blaCSA-1 was detected in MEZCS99. No colistin or other antibiotic resistance genes were detected. MEZCS99 belonged to ST3 and harbored an extra-chromosomal plasmid (IncFIB (pCTU3)). The genome of MEZCS99 strain showed two CRISPR/Cas cluster arrays of I-E (n&#xa0;=&#xa0;1) and I-F (n&#xa0;=&#xa0;1) type.<br><br>CONCLUSION: The genome sequence of strain MEZCS99 will serve as a reference point for molecular epidemiological studies of livestock-associated C. sakazakii in Africa. In addition, this study allows in-depth analysis of the genomic structure and will provide valuable information that helps understand the pathogenesis and antimicrobial resistance of livestock-associated C. sakazakii.},
}
@article {pmid36048424,
year = {2022},
author = {Chen, Y and Cheng, M and Feng, X and Niu, X and Song, H and Cao, Y},
title = {Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1.},
journal = {ACS synthetic biology},
volume = {11},
number = {9},
pages = {2947-2955},
doi = {10.1021/acssynbio.2c00208},
pmid = {36048424},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Homologous Recombination ; *Shewanella/genetics ; },
abstract = {Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of genome-wide editing in Shewanella oneidensis. Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9. AsCpf1 and BhCas12b show low cell toxicity and superior ability to target sequences and are thus screened out in S. oneidensis MR-1. The PAMs of AsCpf1 and BhCas12b are 5'-TTTV-3' and 5'-ATTN-3'. For gene deletion, ∼1-kb gene is knocked out and the editing efficiency is 41.67% by BhCas12b-mediated system. For gene replacement, endogenous promoter of nagK was substituted to a constitutive promoter with the efficiency of 25% through BhCas12b system. For gene insertion, the integration efficiency was up to 94.4% and 83.9% via CRISPR-BhCas12b and AsCpf1 tools. This study implies a great potential of CRISPR-BhCas12b/AsCpf1 systems recognizing AT-rich PAMs for genomic editing in S. oneidensis to facilitate multifaceted gene manipulation.},
}
@article {pmid36048170,
year = {2022},
author = {Amendola, M and Brusson, M and Miccio, A},
title = {CRISPRthripsis: The Risk of CRISPR/Cas9-induced Chromothripsis in Gene Therapy.},
journal = {Stem cells translational medicine},
volume = {11},
number = {10},
pages = {1003-1009},
pmid = {36048170},
issn = {2157-6580},
support = {865797 DITSB/ERC_/European Research Council/International ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; *Chromothripsis ; Genetic Therapy/adverse effects ; DNA/genetics ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system has allowed the generation of disease models and the development of therapeutic approaches for many genetic and non-genetic disorders. However, the generation of large genomic rearrangements has raised safety concerns for the clinical application of CRISPR/Cas9 nuclease approaches. Among these events, the formation of micronuclei and chromosome bridges due to chromosomal truncations can lead to massive genomic rearrangements localized to one or few chromosomes. This phenomenon, known as chromothripsis, was originally described in cancer cells, where it is believed to be caused by defective chromosome segregation during mitosis or DNA double-strand breaks. Here, we will discuss the factors influencing CRISPR/Cas9-induced chromothripsis, hereafter termed CRISPRthripsis, and its outcomes, the tools to characterize these events and strategies to minimize them.},
}
@article {pmid36047802,
year = {2022},
author = {Wu, S and Xu, R and Su, M and Gao, C and Liu, Y and Chen, Y and Luan, G and Jia, X and Wang, R},
title = {A pyrF-Based Efficient Genetic Manipulation Platform in Acinetobacter baumannii To Explore the Vital DNA Components of Adaptive Immunity for I-F CRISPR-Cas.},
journal = {Microbiology spectrum},
volume = {10},
number = {5},
pages = {e0195722},
pmid = {36047802},
issn = {2165-0497},
mesh = {Humans ; *CRISPR-Cas Systems ; *Acinetobacter baumannii/genetics ; Plasmids/genetics ; DNA ; Sucrose ; },
abstract = {Acinetobacter baumannii is an important pathogenic bacterium with multidrug resistance which causes infections with high mortality rates. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. In this study, we utilized the conserved pyrF-flanking fragment to rapidly generate uracil auxotrophy hosts with pyrF deleted in model and clinical A. baumannii strains and then introduced the pyrF gene as the selectable and counterselectable marker to establish a series of gene manipulation vectors. For gene deletion with the suicide pyrF-based plasmid, the second-crossover colonies screened with the pyrF/5-fluoroorotic acid (5-FOA) system were obtained more quickly and efficiently than those screened with the sacB/sucrose system. By using the replicative plasmid, the recognized protospacer-adjacent motif (PAM) bias for type I-F CRISPR was experimentally revealed in A. baumannii AYE. Interestingly, interference recognized only the PAM-CC sequence, whereas adaptation priming tolerates 4 PAM sequences. Furthermore, we also performed a rapid and extensive modification of the I-F CRISPR-Cas elements and revealed that the role of double-nucleotide sequence mutants at the end of the repeat could be critical during both CRISPR interference and priming; we also found strong biases for A and demonstrated that adaptation could tolerate certain sequence and size variations of the leader in A. baumannii. In conclusion, this pyrF-based genetic manipulation system was readily applicable and efficient for exploring the genetic characteristics of A. baumannii. IMPORTANCE In this study, we developed the widely applicable and efficient pyrF-based selection and counterselection system in A. baumannii for gene manipulation. In most cases, this pyrF/5-FOA genetic manipulation system was very effective and enabled us to obtain marker-free mutants in a very short period of time. Utilizing this system and the separate mechanism of interference and/or primed adaptation, our experiments revealed some recognition mechanism differences for the key DNA elements of PAM, leader, and repeat in the priming adaptation process of the I-F CRISPR-Cas systems of A. baumannii, which provided some new and original insights for the study of the molecular mechanisms of these processes and laid a foundation for further studies.},
}
@article {pmid36046896,
year = {2022},
author = {Tang, H and Cheng, YT and Guo, J and Bao, JC and Huang, LQ},
title = {[Effects of Cas9 expression on cell growth and production of natural products in Saccharomyces cerevisiae and optimization of CRISPR-Cas9 editing system].},
journal = {Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica},
volume = {47},
number = {15},
pages = {4066-4073},
doi = {10.19540/j.cnki.cjcmm.20220417.101},
pmid = {36046896},
issn = {1001-5302},
mesh = {*Biological Products ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Gene Editing/methods ; *Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.},
}
@article {pmid36046671,
year = {2022},
author = {Shi, S and Wang, J and Ling, H},
title = {Editorial: Synthetic biology for the sustainable production of biochemicals in engineered microbes.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {984875},
doi = {10.3389/fbioe.2022.984875},
pmid = {36046671},
issn = {2296-4185},
}
@article {pmid36044769,
year = {2022},
author = {Wang, T and Wang, Y and Chen, P and Yin, BC and Ye, BC},
title = {An Ultrasensitive, One-Pot RNA Detection Method Based on Rationally Engineered Cas9 Nickase-Assisted Isothermal Amplification Reaction.},
journal = {Analytical chemistry},
volume = {94},
number = {36},
pages = {12461-12471},
doi = {10.1021/acs.analchem.2c02617},
pmid = {36044769},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/analysis ; *Deoxyribonuclease I/chemistry ; Escherichia coli ; HIV ; RNA, Ribosomal, 16S/analysis ; RNA, Viral/analysis ; SARS-CoV-2 ; Salmonella typhimurium ; },
abstract = {RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized molecular diagnostics by offering versatile Cas effectors. We previously developed an isothermal amplification reaction method using Cas9 nickase (Cas9 nAR) to detect genomic DNA. However, slow dissociation of Cas9n from nicked double-stranded DNA (dsDNA) substrates dramatically hampers the cooperation between Cas9n and DNA polymerase, leading to low amplification efficiency. Here, we use structure-guided protein engineering to generate a Cas9n variant with faster kinetics and enhanced targeting specificity, and apply it to develop Cas9 nAR version 2 (Cas9 nAR-v2) by deftly merging reverse transcription with nicking-extension-displacement-based amplification for isothermal, one-pot RNA detection. This assay is validated by detecting Salmonella typhimurium 16S rRNA, Escherichia coli O157:H7 16S rRNA, synthetic SARS-CoV-2 genes, and HIV virus RNA, showing a quantitative analysis over a wide, linear range and a detection limit as low as fewer than ten copies of RNA molecules per reaction (20 μL volume). It also shows an excellent nucleotide-mutation discrimination capability in detecting SARS-CoV-2 variants. Furthermore, Cas9 nAR-v2 is compatible with low-cost point-of-care (POC) tests based on fluorescence and lateral-flow readouts. In summary, this method provides a new paradigm for sensitive, direct RNA detection and would spur the exploration of engineered Cas effectors with improved properties for a wide range of biological applications.},
}
@article {pmid36043880,
year = {2022},
author = {Xu, L and Li, B and Liu, H and Zhang, H and Liu, R and Yu, H and Li, D},
title = {CRISPR/Cas9-Mediated Knockout Reveals the Involvement of CYP304F1 in β-Cypermethrin and Chlorpyrifos Resistance in Spodoptera litura.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {36},
pages = {11192-11200},
doi = {10.1021/acs.jafc.2c04352},
pmid = {36043880},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; *Chlorpyrifos/metabolism/pharmacology ; Insecticide Resistance/genetics ; *Insecticides/metabolism/pharmacology ; Larva/genetics ; *Pyrethrins/metabolism/pharmacology ; Spodoptera ; },
abstract = {Functions of insect CYP2 clan P450s in insecticide resistance are relatively less reported. In Spodoptera litura, a gene from the CYP2 clan (CYP304F1) was validated to be up-regulated significantly in a pyrethroid- and organophosphate-resistant population (QJ) than a susceptible population by RNA-Seq and qRT-PCR. Spatial-temporal expression indicated the high expression of CYP304F1 in the fourth, fifth, and sixth instar larvae and the metabolism-related tissue fat body and malpighian tubules. CYP304F1 was knocked out by CRISPR/Cas9, and a homozygous population (QJ-CYP304F1) with a G-base deletion at exon 2 was obtained after selection. Bioassay results showed that the LD50 values to β-cypermethrin and chlorpyrifos in the QJ-CYP304F1 population decreased significantly, and the resistance ratio was both 1.81-fold in the QJ population compared with that in the QJ-CYP304F1 population. The toxicity of fenvalerate, cyhalothrin, or phoxim showed no significant change. These results suggested that CYP304F1 is involved in β-cypermethrin and chlorpyrifos resistance in S. litura.},
}
@article {pmid36043443,
year = {2022},
author = {Yang, Y and Zhang, C and Song, Y and Li, Y and Li, P and Huang, M and Meng, F and Zhang, M},
title = {Small-molecule activators specific to adenine base editors through blocking the canonical TGF-β pathway.},
journal = {Nucleic acids research},
volume = {50},
number = {17},
pages = {9632-9646},
pmid = {36043443},
issn = {1362-4962},
mesh = {*Adenine/chemistry ; CRISPR-Cas Systems ; Gene Editing ; Genome ; Humans ; *Transforming Growth Factor beta/genetics/metabolism ; Transforming Growth Factors/metabolism ; },
abstract = {Adenine base editors (ABEs) catalyze A-to-G conversions, offering therapeutic options to treat the major class of human pathogenic single nucleotide polymorphisms (SNPs). However, robust and precise editing at diverse genome loci remains challenging. Here, using high-throughput chemical screening, we identified and validated SB505124, a selective ALK5 inhibitor, as an ABE activator. Treating cells with SB505124 enhanced on-target editing at multiple genome loci, including epigenetically refractory regions, and showed little effect on off-target conversion on the genome. Furthermore, SB505124 facilitated the editing of disease-associated genes in vitro and in vivo. Intriguingly, SB505124 served as a specific activator by selectively promoting ABE activity. Mechanistically, SB505124 promotes ABE editing, at least in part, by enhancing ABE expression and modulating DNA repair-associated genes. Our findings reveal the role of the canonical transforming growth factor-β pathway in gene editing and equip ABEs with precise chemical control.},
}
@article {pmid36042887,
year = {2022},
author = {Elango, R and Panday, A and Willis, NA and Scully, R},
title = {Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101551},
pmid = {36042887},
issn = {2666-1667},
support = {R01 CA095175/CA/NCI NIH HHS/United States ; R01 GM073894/GM/NIGMS NIH HHS/United States ; R35 CA263813/CA/NCI NIH HHS/United States ; R01 CA217991/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Mouse Embryonic Stem Cells ; Sequence Deletion ; },
abstract = {In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Panday et al. (2021).},
}
@article {pmid36042883,
year = {2022},
author = {Sinha, T and Yazdani, SS},
title = {Genome editing in Penicillium funiculosum using in vitro assembled CRISPR-Cas9 ribonucleoprotein complexes.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101629},
pmid = {36042883},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Reproducibility of Results ; Ribonucleoproteins/genetics ; Talaromyces ; },
abstract = {The plasmid-free CRISPR-Cas9-based genome editing in fungi is a precise and time-saving approach. Here, we present a detailed protocol for genetic manipulation in Penicillium funiculosum, which includes design and synthesis of sgRNA, high-quality protoplast preparation, and PEG-mediated protoplast transformation of linear donor DNA along with in vitro synthesized RNP complex composed of sgRNA and host-specific Cas9. This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment. For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021).},
}
@article {pmid36042877,
year = {2022},
author = {Martinez-Silgado, A and Yousef Yengej, FA and Puschhof, J and Geurts, V and Boot, C and Geurts, MH and Rookmaaker, MB and Verhaar, MC and Beumer, J and Clevers, H},
title = {Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101639},
pmid = {36042877},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Genetic Engineering ; Humans ; Intestines ; *Organoids ; },
abstract = {Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells. For complete details on the use and execution of this protocol, please refer to Beumer et al. (2022).},
}
@article {pmid36042688,
year = {2022},
author = {Lupish, B and Hall, J and Schwartz, C and Ramesh, A and Morrison, C and Wheeldon, I},
title = {Genome-wide CRISPR-Cas9 screen reveals a persistent null-hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica.},
journal = {Biotechnology and bioengineering},
volume = {119},
number = {12},
pages = {3623-3631},
doi = {10.1002/bit.28219},
pmid = {36042688},
issn = {1097-0290},
mesh = {*Yarrowia/genetics/metabolism ; Hyphae ; Lycopene/metabolism ; CRISPR-Cas Systems ; Metabolic Engineering ; Carotenoids/metabolism ; Phenotype ; },
abstract = {Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress-induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome-wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null-hyphal phenotype-ΔRAS2, ΔRHO5, ΔSFL1, ΔSNF2, and ΔPAXIP1. Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L[-1] OD[-1] from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.},
}
@article {pmid36042215,
year = {2022},
author = {Yoshimi, K and Takeshita, K and Kodera, N and Shibumura, S and Yamauchi, Y and Omatsu, M and Umeda, K and Kunihiro, Y and Yamamoto, M and Mashimo, T},
title = {Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4917},
pmid = {36042215},
issn = {2041-1723},
mesh = {CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; DNA Helicases/*metabolism ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/*metabolism ; },
abstract = {Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific single-stranded DNA (ssDNA) cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target double-stranded DNA (dsDNA) substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.},
}
@article {pmid36042173,
year = {2022},
author = {Dobner, J and Ramachandran, H and Rossi, A},
title = {Genome Editing in Translational Medicine: An Inventory.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {27},
number = {8},
pages = {241},
doi = {10.31083/j.fbl2708241},
pmid = {36042173},
issn = {2768-6698},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome ; Humans ; Transcription Activator-Like Effector Nucleases ; Translational Science, Biomedical ; },
abstract = {Genomic mutations are the driving force of biological diversity but they are also the cause of a plethora of human diseases ranging from heritable disorders to neurological pathologies and cancer. For most genetic disorders, there is no curative treatment available to date. The demand for precise, preferably patient-specific, treatment regimen offering cure is naturally high. Genome editing by Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas enables targeted manipulation of genomes, thereby offering the opportunity to treat such diseases. While ethical and regulatory guidelines need to be developed and considered, the prospect of genome editing for curative treatment is certainly exciting. Here, we review the current state of therapeutics based on genome editing techniques. We highlight recent breakthroughs, describe clinical trials employing genome editing-based medicine, discuss the benefits and pitfalls, and take a look into the future of genome editing.},
}
@article {pmid36041149,
year = {2022},
author = {Salman, M and Verma, A and Singh, VK and Jaffet, J and Chaurasia, S and Sahel, DK and Ramappa, M and Singh, V},
title = {New Frontier in the Management of Corneal Dystrophies: Basics, Development, and Challenges in Corneal Gene Therapy and Gene Editing.},
journal = {Asia-Pacific journal of ophthalmology (Philadelphia, Pa.)},
volume = {11},
number = {4},
pages = {346-359},
doi = {10.1097/APO.0000000000000443},
pmid = {36041149},
issn = {2162-0989},
mesh = {Cornea/pathology ; *Corneal Dystrophies, Hereditary/genetics/pathology/therapy ; *Corneal Transplantation ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; },
abstract = {Corneal dystrophies represent a group of heterogeneous hereditary disorders causing progressive corneal opacification and blindness. Current corneal transplant management for corneal dystrophies faces the challenges of repeated treatments, complex surgical procedures, shortage of appropriate donor cornea, and, more importantly, graft rejection. Genetic medicine could be an alternative treatment regime to overcome such challenges. Cornea carries promising scope for a gene-based therapy involving gene supplementation, gene silencing, and gene editing in both ex vivo and in vivo platforms. In the cornea, ex vivo gene therapeutic strategies were attempted for corneal graft survival, and in vivo gene augmentation therapies aimed to prevent herpes stromal keratitis, neovascularization, corneal clouding, and wound healing. However, none of these studies followed a clinical trial-based successful outcome. CRISPR/Cas system offers a broad scope of gene editing and engineering to correct underlying genetic causes in corneal dystrophies. Corneal tissue--specific gene correction in vitro with minimal off-target effects and optimal gene correction efficiency followed by their successful surgical implantation, or in vivo CRISPR administration targeting pathogenic genes finds a way to explore therapeutic intervention for corneal dystrophies. However, there are many limitations associated with such CRISPR-based corneal treatment management. This review will look into the development of corneal gene therapy and CRISPR-based study in corneal dystrophies, associated challenges, potential approaches, and future directions.},
}
@article {pmid36040863,
year = {2022},
author = {Cao, H and Mao, K and Ran, F and Xu, P and Zhao, Y and Zhang, X and Zhou, H and Yang, Z and Zhang, H and Jiang, G},
title = {Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater.},
journal = {Environmental science &amp; technology},
volume = {56},
number = {18},
pages = {13245-13253},
pmid = {36040863},
issn = {1520-5851},
mesh = {Biotin/genetics ; CRISPR-Cas Systems ; Fluoresceins ; Nucleic Acid Amplification Techniques ; Pandemics ; RNA, Viral/genetics ; *SARS-CoV-2/isolation &amp; purification ; Sensitivity and Specificity ; *Waste Water/virology ; },
abstract = {Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.},
}
@article {pmid36040815,
year = {2022},
author = {Shin, JW and Hong, EP and Park, SS and Choi, DE and Seong, IS and Whittaker, MN and Kleinstiver, BP and Chen, RZ and Lee, JM},
title = {Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR/Cas9.},
journal = {JCI insight},
volume = {7},
number = {19},
pages = {},
pmid = {36040815},
issn = {2379-3708},
support = {R01 NS105709/NS/NINDS NIH HHS/United States ; R01 NS119471/NS/NINDS NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems ; Gain of Function Mutation ; Humans ; Huntingtin Protein/genetics/metabolism ; *Huntington Disease/genetics/therapy ; Mutant Proteins/genetics/metabolism ; RNA, Messenger ; },
abstract = {Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic protospacer adjacent motif-altering (PAM-altering) SNP (PAS), we developed an allele-specific CRISPR/Cas9 strategy to permanently inactivate mutant HTT through nonsense-mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant-specific CRISPR/Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR/Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR/Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR/Cas9 strategy inactivates mutant HTT through NMD. In addition, GUIDE-Seq analysis and subsequent validation experiments support high levels of on-target gene specificity. Our data demonstrate a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR/Cas9 and supporting its therapeutic potential in HD.},
}
@article {pmid36040587,
year = {2022},
author = {Ziegler, T and Bozoglu, T and Kupatt, C},
title = {AAV-Mediated Somatic Gene Editing for Cardiac and Skeletal Muscle in a Large Animal Model.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2573},
number = {},
pages = {63-74},
pmid = {36040587},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics/metabolism ; Disease Models, Animal ; Dystrophin/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Genetic Vectors/genetics ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics ; RNA, Guide/genetics ; Swine ; },
abstract = {Here we describe a protocol to produce a recombinant adeno-associated viral vector (rAAV)-based system to deliver the CRISPR-Cas9 complex into porcine skeletal muscle and myocardial cells. We initially describe the genomic composition of the rAAV-CRISPR vectors used in our lab. Furthermore, we give a step-by-step instruction into the production of recombinant viral vectors with high yields and purity. Lastly we describe the minimally invasive injection regimes to target the myocardium in a pig.},
}
@article {pmid36040586,
year = {2022},
author = {Schoger, E and Zelarayán, LC},
title = {Enhancing Cardiomyocyte Transcription Using In Vivo CRISPR/Cas9 Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2573},
number = {},
pages = {53-61},
pmid = {36040586},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Myocytes, Cardiac ; RNA, Guide/genetics ; Transcription Factors/genetics ; Transcriptional Activation ; },
abstract = {Endogenous gene activation by programmable transcription factors offers gene-dose-dependent phenotyping of target cells embedded in their in vivo natural tissue environment. Modified CRISPR/Cas9 systems were developed to be used as guide (g) RNA programmable transcriptional activation platforms (CRISPRa) in vitro and in vivo allowing targeted or multiplexed gene activation studies. We specifically developed these tools to be applied in cardiomyocytes providing dCas9VPR expressing mice under the control of the Myosin heavy chain 6 (Myh6) promoter. Here, we describe a protocol for the efficient design and validation of newly identified gRNA for enhancing transcriptional activity of a selected gene of interest. Additionally, we are providing insights into a downstream application in a dCas9VPR expressing mouse model specifically for cardiomyocyte biology.},
}
@article {pmid36040585,
year = {2022},
author = {Yamaguchi, N and Zhang, XH and Morad, M},
title = {CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca[2+] Signaling Aberrancies of CPVT Arrhythmogenesis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2573},
number = {},
pages = {41-52},
pmid = {36040585},
issn = {1940-6029},
mesh = {Arrhythmias, Cardiac/genetics/pathology ; CRISPR-Cas Systems ; Calcium/metabolism ; Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Myocytes, Cardiac/metabolism ; *Ryanodine Receptor Calcium Release Channel/genetics/metabolism ; Tachycardia, Ventricular ; },
abstract = {Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca[2+] signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca[2+] release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations in RYR2 gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca[2+] signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.},
}
@article {pmid36040369,
year = {2022},
author = {Sheng, A and Yang, J and Cheng, L and Zhang, J},
title = {Boronic Ester-Mediated Dual Recognition Coupled with a CRISPR/Cas12a System for Lipopolysaccharide Analysis.},
journal = {Analytical chemistry},
volume = {94},
number = {36},
pages = {12523-12530},
doi = {10.1021/acs.analchem.2c02776},
pmid = {36040369},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Boron ; Boronic Acids/chemistry ; CRISPR-Cas Systems/genetics ; Esters ; *Lipopolysaccharides ; },
abstract = {In this work, boronic ester-mediated dual recognition has been coupled with a CRISPR/Cas12a system; thus, a new method for highly specific and sensitive detection of lipopolysaccharide (LPS) is proposed via the simultaneous recognition of boronic acid and an LPS aptamer (LPSA) as well as signal amplification by CRISPR/Cas12a. Specifically, boronic acid-modified magnetic beads (MB@APBA) and aptamers are employed for the simultaneous dual recognition of LPS, while polymerase isotherm amplification is further utilized to induce LPS cycling and form a double strand, which can activate the CRISPR/Cas12a system so as to amplify the signal. Consequently, a linear detection range can be obtained from 0.05 to 5000 ng/mL, with the lowest detection limit of 44.86 pg/mL. The capturing of MB@APBA on 1, 2- and 1, 3-cis dihydroxyl-containing substances can not only eliminate the interference of other molecules but also enhance the highly specific recognition of LPSA on LPS. Moreover, MB@APBA can be reused by adjusting the pH value of the reaction system. The method can be developed as a universal platform for the analytical detection of other carbohydrates.},
}
@article {pmid36040193,
year = {2022},
author = {Samanta, D and Ebrahimi, SB and Ramani, N and Mirkin, CA},
title = {Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {36},
pages = {16310-16315},
doi = {10.1021/jacs.2c07625},
pmid = {36040193},
issn = {1520-5126},
mesh = {Adenosine Triphosphate/analysis ; *Biosensing Techniques ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; Horseradish Peroxidase ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; SARS-CoV-2/genetics ; },
abstract = {We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.},
}
@article {pmid36038232,
year = {2022},
author = {Liang, J and Teng, P and Hu, L and He, G and Song, Q and Zhang, Y and Peng, B and Li, G and Xiao, W and Cao, D and Tang, Y},
title = {Platinum nanoparticles (PtNPs)-based CRISPR/Cas12a platform for detection of nucleic acid and protein in clinical samples.},
journal = {Analytica chimica acta},
volume = {1225},
number = {},
pages = {340203},
pmid = {36038232},
issn = {1873-4324},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Platinum ; SARS-CoV-2 ; },
abstract = {Early rapid screening diagnostic assay is essential for the identification, prevention, and evaluation of many contagious or refractory diseases. The optical density transducer created by platinum nanoparticles (PtNPs) (OD-CRISPR) is reported in the present research as a cheap and easy-to-execute CRISPR/Cas12a-based diagnostic platform. The OD-CRISPR uses PtNPs, with ultra-high peroxidase-mimicking activity, to increase the detection sensitivity, thereby enabling the reduction of detection time and cost. The OD-CRISPR can be utilized to identify nucleic acid or protein biomarkers within an incubation time of 30-40min in clinical specimens. In the case of taking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene as an instance, when compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR), the OD-CRISPR test attains a sensitivity of 79.17% and a specificity of 100%. In terms of detecting prostate-specific antigen (PSA), aptamer-based OD-CRISPR assay achieves the least discoverable concentration of 0.01 ng mL[-1]. In general, the OD-CRISPR can detect nucleic acid and protein biomarkers, and is a potential strategy for early rapid screening diagnostic tools.},
}
@article {pmid36038144,
year = {2022},
author = {Singh, G and Sharma, S and Rawat, S and Sharma, RK},
title = {Plant Specialised Glycosides (PSGs): their biosynthetic enzymatic machinery, physiological functions and commercial potential.},
journal = {Functional plant biology : FPB},
volume = {49},
number = {12},
pages = {1009-1028},
doi = {10.1071/FP21294},
pmid = {36038144},
issn = {1445-4416},
mesh = {*Plants/genetics ; Glycosides/metabolism ; Plant Extracts/metabolism ; *Alkaloids/metabolism ; Terpenes/metabolism ; },
abstract = {Plants, the primary producers of our planet, have evolved from simple aquatic life to very complex terrestrial habitat. This habitat transition coincides with evolution of enormous chemical diversity, collectively termed as 'Plant Specialised Metabolisms (PSMs)', to cope the environmental challenges. Plant glycosylation is an important process of metabolic diversification of PSMs to govern their in planta stability, solubility and inter/intra-cellular transport. Although, individual category of PSMs (terpenoids, phenylpropanoids, flavonoids, saponins, alkaloids, phytohormones, glucosinolates and cyanogenic glycosides) have been well studied; nevertheless, deeper insights of physiological functioning and genomic aspects of plant glycosylation/deglycosylation processes including enzymatic machinery (CYPs, GTs, and GHs) and regulatory elements are still elusive. Therefore, this review discussed the paradigm shift on genomic background of enzymatic machinery, transporters and regulatory mechanism of 'Plant Specialised Glycosides (PSGs)'. Current efforts also update the fundamental understanding about physiological, evolutionary and adaptive role of glycosylation/deglycosylation processes during the metabolic diversification of PSGs. Additionally, futuristic considerations and recommendations for employing integrated next-generation multi-omics (genomics, transcriptomics, proteomics and metabolomics), including gene/genome editing (CRISPR-Cas) approaches are also proposed to explore commercial potential of PSGs.},
}
@article {pmid36037982,
year = {2022},
author = {Wu, H and Zhang, K and Zhang, Z and Wang, J and Jia, P and Cong, L and Li, J and Duan, Y and Ke, F and Zhang, F and Liu, Z and Lu, F and Wang, Y and Li, Z and Chang, M and Zou, J and Zhu, K},
title = {Cell-penetrating peptide: A powerful delivery tool for DNA-free crop genome editing.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {324},
number = {},
pages = {111436},
doi = {10.1016/j.plantsci.2022.111436},
pmid = {36037982},
issn = {1873-2259},
mesh = {CRISPR-Cas Systems ; *Cell-Penetrating Peptides/genetics ; DNA ; *Gene Editing ; Genome, Plant/genetics ; Transcription Factors/genetics ; },
abstract = {Genome editing system based on the CRISPR/Cas (clustered regularly interspaced short palindromic repeats) technology is a milestone for biology. However, public concerns regarding genetically modified organisms (GMOs) and recalcitrance in the crop of choice for regeneration have limited its application. Cell-penetrating peptides (CPPs) are derived from protein transduction domains (PTDs) that can take on various cargoes across the plant wall, and membrane of target cells. Selected CPPs show mild cytotoxicity and are a suitable delivery tool for DNA-free genome editing. Moreover, CPPs may also be applied for the transient delivery of morphogenic transcription factors, also known as developmental regulators (DRs), to overcome the bottleneck of the crop of choice regeneration. In this review, we introduce a brief history of cell-penetrating peptides and discuss the practice of CPP-mediated DNA-free transfection and the prospects of this potential delivery tool for improving crop genome editing.},
}
@article {pmid36037573,
year = {2022},
author = {Gim, GM and Uhm, KH and Kwon, DH and Kim, MJ and Jung, DJ and Kim, DH and Yi, JK and Ha, JJ and Yum, SY and Son, WJ and Lee, JH and Park, JH and Song, KY and Lee, WW and Jang, G},
title = {Germline transmission of MSTN knockout cattle via CRISPR-Cas9.},
journal = {Theriogenology},
volume = {192},
number = {},
pages = {22-27},
doi = {10.1016/j.theriogenology.2022.08.021},
pmid = {36037573},
issn = {1879-3231},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle/genetics ; Electroporation/veterinary ; Female ; Gene Editing/veterinary ; Male ; Mutation ; Myostatin/genetics ; Oocytes ; *Semen ; },
abstract = {Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.},
}
@article {pmid36037555,
year = {2022},
author = {Kang, JY and Mun, D and Chun, Y and Park, DS and Kim, H and Yun, N and Lee, SH and Joung, B},
title = {Generation of three TTN knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102901},
doi = {10.1016/j.scr.2022.102901},
pmid = {36037555},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Cardiomyopathy, Dilated/genetics ; *Cardiomyopathies/metabolism ; *Arrhythmogenic Right Ventricular Dysplasia/genetics ; Mutation ; Connectin/genetics/metabolism ; },
abstract = {TTN mutations are the common genetic cause for various types of cardiomyopathies (e.g., dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy) and skeletal myopathies. Here, we generated three TTN knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 system. These cell lines, which exhibit normal karyotype, typical morphology and pluripotency, could provide useful platform for investigating the role of TTN in associated disorders.},
}
@article {pmid36037444,
year = {2022},
author = {Fang, TT and Zou, ZP and Zhou, Y and Ye, BC},
title = {Prebiotics-Controlled Disposable Engineered Bacteria for Intestinal Diseases.},
journal = {ACS synthetic biology},
volume = {11},
number = {9},
pages = {3004-3014},
doi = {10.1021/acssynbio.2c00182},
pmid = {36037444},
issn = {2161-5063},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/genetics ; Codon, Initiator ; *Gene Editing/methods ; Humans ; *Intestinal Diseases/genetics ; Prebiotics ; Thiosulfates ; Xylose ; },
abstract = {As a new method of diagnosis and treatment for intestinal diseases, intelligent engineered bacteria based on synthetic biology have been developed vigorously in recent years. However, how to deal with the engineered bacteria in vivo after completing the tasks is an urgent problem to be resolved. In this study, we constructed a thiosulfate (a biomarker of inflammatory bowel disease)-responsive engineered bacteria to generate two signals, sfGFP (monitoring) and gain-of-function (translation activation) mutation (ACG to ATG), in the initiation codon of lysisE (recording) via the CRISPR/Cas9-mediated base editing system. Once these two signals were detected, xylose could be added to induce lysis E expression, resulting in the destruction of the edited bacteria and the release of AvCystain simultaneously. Overall, our innovative engineered bacteria can record instant and historical information of the disease, and especially, the edited bacteria can be artificially attenuated and release drug in situ when needed, ultimately serving as a disposable and recyclable candidate for more types of diseases.},
}
@article {pmid36037185,
year = {2022},
author = {Wongpalee, SP and Thananchai, H and Chewapreecha, C and Roslund, HB and Chomkatekaew, C and Tananupak, W and Boonklang, P and Pakdeerat, S and Seng, R and Chantratita, N and Takarn, P and Khamnoi, P},
title = {Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a.},
journal = {PLoS neglected tropical diseases},
volume = {16},
number = {8},
pages = {e0010659},
pmid = {36037185},
issn = {1935-2735},
support = {/WT_/Wellcome Trust/United Kingdom ; 221418/Z/20/Z/WT_/Wellcome Trust/United Kingdom ; 216457/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Burkholderia pseudomallei/genetics ; CRISPR-Cas Systems ; DNA ; Genomics ; Humans ; *Melioidosis/microbiology ; Sensitivity and Specificity ; },
abstract = {Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.},
}
@article {pmid36036862,
year = {2022},
author = {Vuong, UT and Iswanto, ABB and Nguyen, QM and Kang, H and Lee, J and Moon, J and Kim, SH},
title = {Engineering plant immune circuit: walking to the bright future with a novel toolbox.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13916},
pmid = {36036862},
issn = {1467-7652},
abstract = {Plant pathogens destroy crops and cause severe yield losses, leading to an insufficient food supply to sustain the human population. Apart from relying on natural plant immune systems to combat biological agents or waiting for the appropriate evolutionary steps to occur over time, researchers are currently seeking new breakthrough methods to boost disease resistance in plants through genetic engineering. Here, we summarize the past two decades of research in disease resistance engineering against an assortment of pathogens through modifying the plant immune components (internal and external) with several biotechnological techniques. We also discuss potential strategies and provide perspectives on engineering plant immune systems for enhanced pathogen resistance and plant fitness.},
}
@article {pmid36036597,
year = {2022},
author = {Venkatesan, V and Christopher, AC and Karuppusamy, KV and Babu, P and Alagiri, MKK and Thangavel, S},
title = {CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {186},
pages = {},
doi = {10.3791/64064},
pmid = {36036597},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Genetic Therapy/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Mice ; },
abstract = {CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.},
}
@article {pmid36036427,
year = {2022},
author = {Poli, FE and Yusuf, IH and Clouston, P and Shanks, M and Whitfield, J and Charbel Issa, P and MacLaren, RE},
title = {MERTK missense variants in three patients with retinitis pigmentosa.},
journal = {Ophthalmic genetics},
volume = {},
number = {},
pages = {1-9},
pmid = {36036427},
issn = {1744-5094},
abstract = {BACKGROUND: MERTK (MER proto-oncogene, tyrosine kinase) is a transmembrane protein essential in regulating photoreceptor outer segment phagocytosis. Biallelic mutations in MERTK cause retinal degeneration. Here we present the retinal phenotype of three patients with missense variants in MERTK.<br><br>MATERIALS AND METHODS: All patients underwent a full clinical examination, fundus photography, short-wavelength fundus autofluorescence and optical coherence tomography imaging. Two patients also underwent Goldmann visual field testing and electroretinography was undertaken for the third patient. Molecular genetic testing was undertaken using next generation or whole-exome sequencing with all variants confirmed by Sanger sequencing.<br><br>RESULTS: The first patient was a 29-year-old female heterozygous for a missense variant (c.1133C>T, p.Thr378&#x2009;Met) and a nonsense variant (c.1744_1751delinsT, p.Ile582Ter) in MERTK. The second patient was a 26-year-old male homozygous for a c.2163T>A, p.His721Gln variant in MERTK. The third patient was an 11-year-old female heterozygous for a deletion of exons 5-19 and a missense variant (c.1866&#x2009;G>C, p.Lys622Asn) in MERTK. Reduced night vision was the initial symptom in all patients. Fundoscopy revealed typical signs of retinitis pigmentosa (RP) with early-onset macular atrophy. All three MERTK missense variants affect highly conserved residues within functional domains, have low population frequencies and are predicted to be pathogenic in silico.<br><br>CONCLUSIONS: We report three missense variants in MERTK and present the associated phenotypic data, which are supportive of non-syndromic RP. MERTK is a promising candidate for viral-mediated gene replacement therapy. Moreover, one variant represents a single nucleotide transition, which is theoretically targetable with CRISPR-Cas9 base-editing.},
}
@article {pmid36036114,
year = {2022},
author = {Chen, Z and Zhu, J and Chen, Z and Du, M and Yao, R and Fu, W and Lei, A and Wang, J},
title = {High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2048-2050},
pmid = {36036114},
issn = {1467-7652},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Euglena gracilis ; Microinjections ; Indicators and Reagents ; Electroporation ; Ribonucleoproteins/genetics ; High-Throughput Nucleotide Sequencing ; RNA, Guide/genetics ; },
}
@article {pmid36035749,
year = {2022},
author = {Zhang, Y and Li, H and Nishiyama, T and McAnally, JR and Sanchez-Ortiz, E and Huang, J and Mammen, PPA and Bassel-Duby, R and Olson, EN},
title = {A humanized knockin mouse model of Duchenne muscular dystrophy and its correction by CRISPR-Cas9 therapeutic gene editing.},
journal = {Molecular therapy. Nucleic acids},
volume = {29},
number = {},
pages = {525-537},
pmid = {36035749},
issn = {2162-2531},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; },
abstract = {Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the X-linked dystrophin (DMD) gene. Exon deletions flanking exon 51, which disrupt the dystrophin open reading frame (ORF), represent one of the most common types of human DMD mutations. Previously, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) gene editing to restore the reading frame of exon 51 in mice and dogs with exon 50 deletions. Due to genomic sequence variations between species, the single guide RNAs (sgRNAs) used for DMD gene editing are often not conserved, impeding direct clinical translation of CRISPR-Cas therapeutic gene-editing strategies. To circumvent this potential obstacle, we generated a humanized DMD mouse model by replacing mouse exon 51 with human exon 51, followed by deletion of mouse exon 50, which disrupted the dystrophin ORF. Systemic CRISPR-Cas9 gene editing using an sgRNA that targets human exon 51 efficiently restored dystrophin expression and ameliorated pathologic hallmarks of DMD, including histopathology and grip strength in this mouse model. This unique DMD mouse model with the human genomic sequence allows in vivo assessment of clinically relevant gene editing strategies as well as other therapeutic approaches and represents a significant step toward therapeutic translation of CRISPR-Cas9 gene editing for correction of DMD.},
}
@article {pmid36032355,
year = {2022},
author = {Su, G and Zhu, M and Li, D and Xu, M and Zhu, Y and Zhang, Y and Zhu, H and Li, F and Yu, Y},
title = {Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection.},
journal = {Sensors and actuators. B, Chemical},
volume = {371},
number = {},
pages = {132537},
pmid = {36032355},
issn = {0925-4005},
abstract = {The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.},
}
@article {pmid36032098,
year = {2022},
author = {Lee, KY and Choi, HJ and Park, KJ and Woo, SJ and Kim, YM and Han, JY},
title = {Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {892476},
pmid = {36032098},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Cas Systems ; Chickens ; *Genes, RAG-1 ; Homeodomain Proteins ; Immunoglobulin Heavy Chains ; *Immunologic Deficiency Syndromes ; T-Lymphocytes ; },
abstract = {Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 [+/-] and RAG1 [-/-] embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 [+/-] chickens, lymphatic organs in 3-week old RAG1 [-/-] chickens were severely damaged. Furthermore, relative to WT chickens, RAG1[+/-] and RAG1[-/-] birds had reduced serum Igs, fewer mature CD4[+] and CD8[+] T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 [+/-] chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.},
}
@article {pmid36031021,
year = {2022},
author = {Saeed, S and Usman, B and Shim, SH and Khan, SU and Nizamuddin, S and Saeed, S and Shoaib, Y and Jeon, JS and Jung, KH},
title = {CRISPR/Cas-mediated editing of cis-regulatory elements for crop improvement.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {324},
number = {},
pages = {111435},
doi = {10.1016/j.plantsci.2022.111435},
pmid = {36031021},
issn = {1873-2259},
mesh = {*CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Genome, Plant/genetics ; Genome-Wide Association Study ; Plants, Genetically Modified/genetics ; },
abstract = {To improve future agricultural production, major technological advances are required to increase crop production and yield. Targeting the coding region of genes via the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has been well established and has enabled the rapid generation of transgene-free plants, which can lead to crop improvement. The emergence of the CRISPR/Cas system has also enabled scientists to achieve cis-regulatory element (CRE) editing and, consequently, engineering endogenous critical CREs to modulate the expression of target genes. Recent genome-wide association studies have identified the domestication of natural CRE variants to regulate complex agronomic quantitative traits and have allowed for their engineering via the CRISPR/Cas system. Although engineering plant CREs can be advantageous to drive gene expression, there are still many limitations to its practical application. Here, we review the current progress in CRE editing and propose future strategies to effectively target CREs for transcriptional regulation for crop improvement.},
}
@article {pmid36030476,
year = {2022},
author = {Javaid, D and Ganie, SY and Hajam, YA and Reshi, MS},
title = {CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {12133-12150},
pmid = {36030476},
issn = {1573-4978},
mesh = {Swine ; Animals ; Dogs ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome/genetics ; Biology ; },
abstract = {Genome engineering has always been a versatile technique in biological research and medicine, with several applications. In the last several years, the discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 technology has swept the scientific community and revolutionised the speed of modern biology, heralding a new era of disease detection and rapid biotechnology discoveries. It enables successful gene editing by producing targeted double-strand breaks in virtually any organism or cell type. So, this review presents a comprehensive knowledge about the mechanism and structure of Cas9-mediated RNA-guided DNA targeting and cleavage. In addition, genome editing via CRISPR-Cas9 technology in various animals which are being used as models in scientific research including Non-Human Primates Pigs, Dogs, Zebra, fish and Drosophila has been discussed in this review. This review also aims to understand the applications, serious concerns and future perspective of CRISPR/Cas9-mediated genome editing.},
}
@article {pmid36029893,
year = {2022},
author = {Mirjalili Mohanna, SZ and Djaksigulova, D and Hill, AM and Wagner, PK and Simpson, EM and Leavitt, BR},
title = {LNP-mediated delivery of CRISPR RNP for wide-spread in vivo genome editing in mouse cornea.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {350},
number = {},
pages = {401-413},
doi = {10.1016/j.jconrel.2022.08.042},
pmid = {36029893},
issn = {1873-4995},
mesh = {Animals ; CRISPR-Cas Systems ; Cornea/metabolism ; DNA ; *Gene Editing ; Liposomes ; Mice ; Nanoparticles ; *RNA, Guide/genetics ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR/Cas9-based genome-editing therapies are poised to change the clinical outcome for many diseases with validated therapeutic targets awaiting an appropriate delivery system. Recent advances in lipid nanoparticle (LNP) technology make them an attractive platform for the delivery of various forms of CRISPR/Cas9, including the efficient and transient Cas9/gRNA ribonucleoprotein (RNP) complexes. In this study, we initially tested our novel LNP platform by delivering pre-complexed RNPs and template DNA to cultured mouse cortical neurons, and obtained successful ex vivo genome editing. We then directly injected LNP-packaged RNPs and DNA template into the mouse cornea to evaluate in vivo delivery. For the first time, we demonstrated wide-spread genome editing in the cornea using our LNP-RNPs. The ability of our LNPs to transfect the cornea highlights the potential of our novel delivery platform to be used in CRISPR/Cas9-based genome editing therapies of corneal diseases.},
}
@article {pmid36029686,
year = {2023},
author = {Pu, Q and Ye, Y and Hu, J and Xie, C and Zhou, X and Yu, H and Liao, F and Jiang, S and Jiang, L and Xie, G and Chen, W},
title = {XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification.},
journal = {Talanta},
volume = {252},
number = {},
pages = {123754},
doi = {10.1016/j.talanta.2022.123754},
pmid = {36029686},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques ; Nucleic Acid Probes ; DNA/genetics/chemistry ; RNA/genetics/chemistry ; },
abstract = {N6-methyladenosine (m6A) in RNAs is closely related to various biological progresses, but the specific regulatory mechanisms are still unclear. The existing m6A single-base resolution analysis techniques have problems of specificity and sensitivity to be improved, which can hardly meet the urgent needs of basic research and clinical applications. This work proposes a new strategy based on xeno nucleic acid (XNA) probe and CRISPR/Cas12a signal amplification for the sensitive detection of site-specific m6A modifications. According to the difference in the thermodynamic stability of hybridization between XNA probe with m6A-RNA and A-RNA, XNA was designed as a block probe to mediate m6A-RNA specific reverse transcription polymerase chain reaction (MsRT-PCR). Therefore, m6A can be specifically distinguished by converting difficult-to-test m6A modifications into easily detectable dsDNA fragments. Integration of CRISPR/Cas12a technology, skilfully designed sequences of crRNAs targeting m6A site-specific amplification dsDNA. The specificity was significantly improved through dual specific recognition of XNA probe and crRNA. Furthermore, the sensitivity of the assay was also greatly increased by the combined signal amplification of PCR and CRISPR/Cas12a. Additionally, we extend the application of CRISPR/Cas12a to flexible fluorescent and electrochemical biosensing system, which can accurately detect m6A modifications with different ranges of methylation fractions. The analysis results of m6A sites in MALAT1, ACTB and TPT1 further demonstrated the feasibility of the constructed biosensor for the accurate detection of hypomethylated samples in cells. The implementation of this work will provide strong technical support to promote the in-depth research on m6A in disease regulation mechanisms and in vitro molecular diagnosis.},
}
@article {pmid36029350,
year = {2022},
author = {Opel, F and Axmann, IM and Klähn, S},
title = {The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories.},
journal = {Advances in biochemical engineering/biotechnology},
volume = {},
number = {},
pages = {},
pmid = {36029350},
issn = {0724-6145},
abstract = {Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, a solar-driven process which allows them to obtain electrons from water to reduce and finally assimilate carbon dioxide. Consequently, they are in the spotlight of biotechnology as photoautotrophic cell factories to generate a large variety of chemicals and biofuels in a sustainable way. Recent progress in synthetic biology has enlarged the molecular toolset to genetically engineer the metabolism of cyanobacteria, mainly targeting common model strains, such as Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, Synechococcus sp. PCC 7002, or Anabaena sp. PCC 7120. Nevertheless, the accessibility and flexibility of engineering cyanobacteria is still somewhat limited and less predictable compared to other biotechnologically employed microorganisms.This chapter gives a broad overview of currently available methods for the genetic modification of cyanobacterial model strains as well as more recently discovered and promising species, such as Synechococcus elongatus PCC 11801. It comprises approaches based on homologous recombination, replicative broad-host-range or strain-specific plasmids, CRISPR/Cas, as well as markerless selection. Furthermore, common and newly introduced molecular tools for gene expression regulation are presented, comprising promoters, regulatory RNAs, genetic insulators like transcription terminators, ribosome binding sites, CRISPR interference, and the utilization of heterologous RNA polymerases. Additionally, potential DNA assembly strategies, like modular cloning, are described. Finally, considerations about post-translational control via protein degradation tags and heterologous proteases, as well as small proteins working as enzyme effectors are briefly discussed.},
}
@article {pmid36029324,
year = {2022},
author = {Feng, T and Liu, J and Chen, G and Wu, L and Ren, F and Yang, Y and Zhu, J and Shen, F and Wang, L and Chen, Q},
title = {The fluorescent aptasensor based on CRISPR-Cas12a combined with TdT for highly sensitive detection of cocaine.},
journal = {Analytical and bioanalytical chemistry},
volume = {414},
number = {24},
pages = {7291-7297},
pmid = {36029324},
issn = {1618-2650},
mesh = {*Aptamers, Nucleotide/genetics ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; *Cocaine ; DNA ; DNA Nucleotidylexotransferase ; DNA-Directed DNA Polymerase ; Fluorescent Dyes ; Humans ; },
abstract = {Ultrasensitive and specific detection of cocaine is of great significance for monitoring cocaine abuse. Herein, a fluorescent aptasensor via coupling CRISPR-Cas12a, with magnetic nanoparticles (MNPs), split-aptamer, and terminal deoxynucleotidyl transferase (TdT), was developed for the detection of cocaine. In short, the complete cocaine aptamer is split into two parts, one is modified on magnetic nanoparticles (MNPs) and the other is free. The presence of cocaine will mediate the binding of these two segments. Then TdT will mediate the extension to form an ultra-long sequence that can bind with multiple CRISPR-Cas12a resulting in the trans-cleavage activity of CRISPR-Cas12a being triggered. Thence, the DNA reporter which is bi-labeled with fluorophore and quencher is cleaved resulting in the generation of a fluorescence signal. The developed fluorescent aptasensor realizes the detection of cocaine with excellent sensitivity and specificity. The detection limit is low down to 33 pM, and the linear range is from 330 to 1.65 × 10[5] pM. Most importantly, this fluorescent aptasensor can be successfully applied to the determination of cocaine in human plasma samples.},
}
@article {pmid36029126,
year = {2022},
author = {Han, W and Huang, W and Wei, T and Ye, Y and Mao, M and Wang, Z},
title = {Programmable RNA base editing with a single gRNA-free enzyme.},
journal = {Nucleic acids research},
volume = {50},
number = {16},
pages = {9580-9595},
pmid = {36029126},
issn = {1362-4962},
mesh = {Humans ; Mice ; Animals ; *RNA, Guide/genetics/chemistry ; *RNA Editing/genetics ; RNA/genetics ; Genome ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Programmable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60-80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.},
}
@article {pmid36028909,
year = {2022},
author = {Somerville, V and Schowing, T and Chabas, H and Schmidt, RS and von Ah, U and Bruggmann, R and Engel, P},
title = {Extensive diversity and rapid turnover of phage defense repertoires in cheese-associated bacterial communities.},
journal = {Microbiome},
volume = {10},
number = {1},
pages = {137},
pmid = {36028909},
issn = {2049-2618},
mesh = {Bacteria ; *Bacteriophages ; *Cheese ; Genome, Bacterial ; Metagenome ; },
abstract = {BACKGROUND: Phages are key drivers of genomic diversity in bacterial populations as they impose strong selective pressure on the evolution of bacterial defense mechanisms across closely related strains. The pan-immunity model suggests that such diversity is maintained because the effective immune system of a bacterial species is the one distributed across all strains present in the community. However, only few studies have analyzed the distribution of bacterial defense systems at the community-level, mostly focusing on CRISPR and comparing samples from complex environments. Here, we studied 2778 bacterial genomes and 188 metagenomes from cheese-associated communities, which are dominated by a few bacterial taxa and occur in relatively stable environments.<br><br>RESULTS: We corroborate previous laboratory findings that in cheese-associated communities nearly identical strains contain diverse and highly variable arsenals of innate and adaptive (i.e., CRISPR-Cas) immunity systems suggesting rapid turnover. CRISPR spacer abundance correlated with the abundance of matching target sequences across the metagenomes providing evidence that the identified defense repertoires are functional and under selection. While these characteristics align with the pan-immunity model, the detected CRISPR spacers only covered a subset of the phages previously identified in cheese, providing evidence that CRISPR does not enable complete immunity against all phages, and that the innate immune mechanisms may have complementary roles.<br><br>CONCLUSIONS: Our findings show that the evolution of bacterial defense mechanisms is a highly dynamic process and highlight that experimentally tractable, low complexity communities such as those found in cheese, can help to understand ecological and molecular processes underlying phage-defense system relationships. These findings can have implications for the design of robust synthetic communities used in biotechnology and the food industry. Video Abstract.},
}
@article {pmid36028578,
year = {2022},
author = {Le, NT and Tran, HT and Bui, TP and Nguyen, GT and Van Nguyen, D and Ta, DT and Trinh, DD and Molnar, A and Pham, NB and Chu, HH and Do, PT},
title = {Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {14627},
pmid = {36028578},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Mutation ; Plant Diseases ; *Potyvirus ; *Solanum tuberosum ; Tobacco ; },
abstract = {Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVY[O] accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVY[O] confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVY[O] in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.},
}
@article {pmid36027912,
year = {2022},
author = {Nakagawa, R and Kannan, S and Altae-Tran, H and Takeda, SN and Tomita, A and Hirano, H and Kusakizako, T and Nishizawa, T and Yamashita, K and Zhang, F and Nishimasu, H and Nureki, O},
title = {Structure and engineering of the minimal type VI CRISPR-Cas13bt3.},
journal = {Molecular cell},
volume = {82},
number = {17},
pages = {3178-3192.e5},
pmid = {36027912},
issn = {1097-4164},
support = {MC_UP_A025_1012/MRC_/Medical Research Council/United Kingdom ; R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Mammals/genetics ; RNA/genetics ; *RNA, Guide/genetics ; Transcriptome ; },
abstract = {Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.},
}
@article {pmid36027801,
year = {2022},
author = {Li, Y and Pei, Y and Huang, D and Xu, R and Wu, Y and Li, Y and Zhou, R and Wei, Y and Tan, X and Gao, H and Shi, J and Zhang, Z and Zhang, K and Li, J},
title = {Direct visualization of living bacterial genotypes using CRISPR/Cas12a-circular reporter nanoprobes.},
journal = {Biosensors &amp; bioelectronics},
volume = {216},
number = {},
pages = {114641},
doi = {10.1016/j.bios.2022.114641},
pmid = {36027801},
issn = {1873-4235},
mesh = {Animals ; Bacteria/genetics/metabolism ; Bacterial Proteins/genetics ; *Biosensing Techniques ; *CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Genotype ; In Situ Hybridization, Fluorescence ; Mice ; Polyethyleneimine ; },
abstract = {Bacterial genotyping is important for understanding the complex microbiota. Although fluorescence in situ hybridization (FISH) has enabled bacterial community identification with high spatial resolution, its unavoidable cell fixation steps and signal generation by multi-probe stacking greatly limit its application in living bacterial genotyping. Here, we designed polyethyleneimine-encapsulated CRISPR/Cas12a-circular reporter nanoprobes (CasCLR) for rapid and sensitive visualization of gene information in living bacteria. We found that, nanoprobe-based sequential delivery of Cas12a/crRNA and circular reporter into bacteria allowed single genomic loci to initiate trans-cleavage activity of Cas12a, thereby cleaving CLR to generate amplified fluorescent signals for imaging of target gene. Using CasCLR, we can sensitively analyze the percentage of target bacteria in co-culture experiments and directly detect pathogenic bacteria in uncultured mouse gut microbe. In addition, CasCLR has the ability to sensitively analyze specific genotype of microbial communities in vivo. This nanobiotechnology-based bacterial gene analysis is expected to advance understanding of in vivo bacterial cytogenetic information.},
}
@article {pmid36027620,
year = {2023},
author = {Huang, L and Yuan, N and Guo, W and Zhang, Y and Zhang, W},
title = {An electrochemical biosensor for the highly sensitive detection of Staphylococcus aureus based on SRCA-CRISPR/Cas12a.},
journal = {Talanta},
volume = {252},
number = {},
pages = {123821},
doi = {10.1016/j.talanta.2022.123821},
pmid = {36027620},
issn = {1873-3573},
mesh = {Humans ; Staphylococcus aureus/genetics ; CRISPR-Cas Systems ; Gold/chemistry ; Limit of Detection ; Reproducibility of Results ; *Metal Nanoparticles ; *Biosensing Techniques/methods ; *Staphylococcal Infections/diagnosis ; DNA/chemistry ; DNA, Single-Stranded ; },
abstract = {As one of the major foodborne pathogens, Staphylococcus aureus (S. aureus) can cause infectious diseases. In the current study, a novel electrochemical biosensor based on saltatory rolling circle amplification (SRCA) combined with CRISPR/Cas12a system was developed for the accurate detection of S. aureus. The thio-modified reporter probes (SH-ssDNA-MB) was immobilized on the surface of gold nanoparticle-modified electrode through the Au-S bond. In the presence of S. aureus, the target DNA double strands obtained by SRCA can be specifically recognized with Cas12a/crRNA complex. The trans-cleavage activity of Cas12a induces SH-ssDNA-MB to be cleaved from the electrode surface, resulting in a decrease in the current signal. Subsequently, the ratio of the current can be calculated as the detection result. Under optimal conditions, the detection limits were 2.51 fg/μL for genomic DNA and 3 CFU/mL for S. aureus in pure cultures, respectively. Moreover, the method demonstrated satisfactory specificity, acceptable stability and reproducibility. In comparison with ISO methods, the sensitivity, specificity and accuracy of the developed method were 100%, 97.8% and 98%, respectively. In conclusion, the developed novel electrochemical biosensor provides a potential powerful platform for the accurate detection of S. aureus.},
}
@article {pmid36018812,
year = {2022},
author = {Cheng, F and Wu, A and Liu, C and Cao, X and Wang, R and Shu, X and Wang, L and Zhang, Y and Xiang, H and Li, M},
title = {The toxin-antitoxin RNA guards of CRISPR-Cas evolved high specificity through repeat degeneration.},
journal = {Nucleic acids research},
volume = {50},
number = {16},
pages = {9442-9452},
pmid = {36018812},
issn = {1362-4962},
mesh = {*RNA ; CRISPR-Cas Systems ; *Antitoxins/genetics ; },
abstract = {Recent discovery of ectopic repeats (outside CRISPR arrays) provided unprecedented insights into the nondefense roles of CRISPR-Cas. A striking example is the addiction module CreTA (CRISPR-regulated toxin-antitoxins), where one or two (in most cases) ectopic repeats produce CRISPR-resembling antitoxic (CreA) RNAs that direct the CRISPR effector Cascade to transcriptionally repress a toxic RNA (CreT). Here, we demonstrated that CreTA repeats are extensively degenerated in sequence, with the first repeat (ψR1) being more diverged than the second one (ψR2). As a result, such addiction modules become highly specific to their physically-linked CRISPR-Cas loci, and in most cases, CreA could not harness a heterologous CRISPR-Cas to suppress its cognate toxin. We further disclosed that this specificity primarily derives from the degeneration of ψR1, and could generally be altered by modifying this repeat element. We also showed that the degenerated repeats of CreTA were insusceptible to recombination and thus more stable compared to a typical CRISPR array, which could be exploited to develop highly stable CRISPR-based tools. These data illustrated that repeat degeneration (a common feature of ectopic repeats) improves the stability and specificity of CreTA in protecting CRISPR-Cas, which could have contributed to the widespread occurrence and deep diversification of CRISPR systems.},
}
@article {pmid36018009,
year = {2022},
author = {Alfano, M and De Antoni, L and Centofanti, F and Visconti, VV and Maestri, S and Degli Esposti, C and Massa, R and D'Apice, MR and Novelli, G and Delledonne, M and Botta, A and Rossato, M},
title = {Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {36018009},
issn = {2050-084X},
mesh = {Alleles ; CRISPR-Cas Systems ; Genetic Association Studies ; Humans ; *Myotonic Dystrophy/genetics ; *Nanopore Sequencing ; RNA-Binding Proteins/genetics ; },
abstract = {Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.},
}
@article {pmid36017994,
year = {2022},
author = {Specht, DA and Cortes, LB and Lambert, G},
title = {Overcoming Leak Sensitivity in CRISPRi Circuits Using Antisense RNA Sequestration and Regulatory Feedback.},
journal = {ACS synthetic biology},
volume = {11},
number = {9},
pages = {2927-2937},
pmid = {36017994},
issn = {2161-5063},
support = {R35 GM133759/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/metabolism ; Escherichia coli/genetics/metabolism ; Feedback ; RNA, Antisense/genetics/metabolism ; *RNA, Guide/genetics/metabolism ; },
abstract = {The controlled binding of the catalytically dead CRISPR nuclease (dCas) to DNA can be used to create complex, programmable transcriptional genetic circuits, a fundamental goal of synthetic biology. This approach, called CRISPR interference (CRISPRi), is advantageous over existing methods because the programmable nature of CRISPR proteins in principle enables the simultaneous regulation of many different targets without crosstalk. However, the performance of dCas-based genetic circuits is limited by both the sensitivity to leaky repression within CRISPRi logic gates and retroactive effects due to a shared pool of dCas proteins. By utilizing antisense RNAs (asRNAs) to sequester gRNA transcripts as well as CRISPRi feedback to self-regulate asRNA production, we demonstrate a mechanism that suppresses unwanted repression by CRISPRi and improves logical gene circuit function in Escherichia coli. This improvement is particularly pronounced during stationary expression when CRISPRi circuits do not achieve the expected regulatory dynamics. Furthermore, the use of dual CRISPRi/asRNA inverters restores the logical performance of layered circuits such as a double inverter. By studying circuit induction at the single-cell level in microfluidic channels, we provide insight into the dynamics of antisense sequestration of gRNA and regulatory feedback on dCas-based repression and derepression. These results demonstrate how CRISPRi inverters can be improved for use in more complex genetic circuitry without sacrificing the programmability and orthogonality of dCas proteins.},
}
@article {pmid36017348,
year = {2022},
author = {Asadbeigi, A and Norouzi, M and Vafaei Sadi, MS and Saffari, M and Bakhtiarizadeh, MR},
title = {CaSilico: A versatile CRISPR package for in silico CRISPR RNA designing for Cas12, Cas13, and Cas14.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {957131},
pmid = {36017348},
issn = {2296-4185},
abstract = {The efficiency of the CRISPR-Cas system is highly dependent on well-designed CRISPR RNA (crRNA). To facilitate the use of various types of CRISPR-Cas systems, there is a need for the development of computational tools to design crRNAs which cover different CRISPR-Cas systems with off-target analysis capability. Numerous crRNA design tools have been developed, but nearly all of them are dedicated to design crRNA for genome editing. Hence, we developed a tool matching the needs of both beginners and experts, named CaSilico, which was inspired by the limitations of the current crRNA design tools for designing crRNAs for Cas12, Cas13, and Cas14 CRISPR-Cas systems. This tool considers a comprehensive list of the principal rules that are not yet well described to design crRNA for these types. Using a list of important features such as mismatch tolerance rules, self-complementarity, GC content, frequency of cleaving base around the target site, target accessibility, and PFS (protospacer flanking site) or PAM (protospacer adjacent motif) requirement, CaSilico searches all potential crRNAs in a user-input sequence. Considering these features help users to rank all crRNAs for a sequence and make an informed decision about whether a crRNA is suited for an experiment or not. Our tool is sufficiently flexible to tune some key parameters governing the design of crRNA and identification of off-targets, which can lead to an increase in the chances of successful CRISPR-Cas experiments. CaSilico outperforms previous crRNA design tools in the following aspects: 1) supporting any reference genome/gene/transcriptome for which an FASTA file is available; 2) designing crRNAs that simultaneously target multiple sequences through conserved region detection among a set of sequences; 3) considering new CRISPR-Cas subtypes; and 4) reporting a list of different features for each candidate crRNA, which can help the user to select the best one. Given these capabilities, CaSilico addresses end-user concerns arising from the use of sophisticated bioinformatics algorithms and has a wide range of potential research applications in different areas, especially in the design of crRNA for pathogen diagnosis. CaSilico was successfully applied to design crRNAs for different genes in the SARS-CoV-2 genome, as some of the crRNAs have been experimentally tested in the previous studies.},
}
@article {pmid36016778,
year = {2022},
author = {Ghosh, S and Lahiri, D and Nag, M and Sarkar, T and Pati, S and Edinur, HA and Kumar, M and Mohd Zain, MRA and Ray, RR},
title = {Precision targeting of food biofilm-forming genes by microbial scissors: CRISPR-Cas as an effective modulator.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {964848},
pmid = {36016778},
issn = {1664-302X},
abstract = {The abrupt emergence of antimicrobial resistant (AMR) bacterial strains has been recognized as one of the biggest public health threats affecting the human race and food processing industries. One of the causes for the emergence of AMR is the ability of the microorganisms to form biofilm as a defense strategy that restricts the penetration of antimicrobial agents into bacterial cells. About 80% of human diseases are caused by biofilm-associated sessile microbes. Bacterial biofilm formation involves a cascade of genes that are regulated via the mechanism of quorum sensing (QS) and signaling pathways that control the production of the extracellular polymeric matrix (EPS), responsible for the three-dimensional architecture of the biofilm. Another defense strategy utilized commonly by various bacteria includes clustered regularly interspaced short palindromic repeats interference (CRISPRi) system that prevents the bacterial cell from viral invasion. Since multigenic signaling pathways and controlling systems are involved in each and every step of biofilm formation, the CRISPRi system can be adopted as an effective strategy to target the genomic system involved in biofilm formation. Overall, this technology enables site-specific integration of genes into the host enabling the development of paratransgenic control strategies to interfere with pathogenic bacterial strains. CRISPR-RNA-guided Cas9 endonuclease, being a promising genome editing tool, can be effectively programmed to re-sensitize the bacteria by targeting AMR-encoding plasmid genes involved in biofilm formation and virulence to revert bacterial resistance to antibiotics. CRISPRi-facilitated silencing of genes encoding regulatory proteins associated with biofilm production is considered by researchers as a dependable approach for editing gene networks in various biofilm-forming bacteria either by inactivating biofilm-forming genes or by integrating genes corresponding to antibiotic resistance or fluorescent markers into the host genome for better analysis of its functions both in vitro and in vivo or by editing genes to stop the secretion of toxins as harmful metabolites in food industries, thereby upgrading the human health status.},
}
@article {pmid36016245,
year = {2022},
author = {Apinda, N and Muenthaisong, A and Chomjit, P and Sangkakam, K and Nambooppha, B and Rittipornlertrak, A and Koonyosying, P and Yao, Y and Nair, V and Sthitmatee, N},
title = {Simultaneous Protective Immune Responses of Ducks against Duck Plague and Fowl Cholera by Recombinant Duck Enteritis Virus Vector Expressing Pasteurella multocida OmpH Gene.},
journal = {Vaccines},
volume = {10},
number = {8},
pages = {},
pmid = {36016245},
issn = {2076-393X},
support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Duck enteritis virus and Pasteurella multocida are major duck pathogens that induce duck plague and fowl cholera, respectively, in ducks and other waterfowl populations, leading to high levels of morbidity and mortality. Immunization with live attenuated DEV vaccine containing P. multocida outer membrane protein H (OmpH) can provide the most effective protection against these two infectious diseases in ducks. We have recently reported the construction of recombinant DEV expressing P. multocida ompH gene using the CRISPR/Cas9 gene editing strategy with the goal of using it as a bivalent vaccine that can simultaneously protect against both infections. Here we describe the findings of our investigation into the systemic immune responses, potency and clinical protection induced by the two recombinant DEV-ompH vaccine constructs, where one copy each of the ompH gene was inserted into the DEV genome at the UL55-LORF11 and UL44-44.5 intergenic regions, respectively. Our study demonstrated that the insertion of the ompH gene exerted no adverse effect on the DEV parental virus. Moreover, ducklings immunized with the rDEV-ompH-UL55 and rDEV-ompH-UL44 vaccines induced promising levels of P. multocida OmpH-specific as well as DEV-specific antibodies and were completely protected from both diseases. Analysis of the humoral and cellular immunity confirmed the immunogenicity of both recombinant vaccines, which provided strong immune responses against DEV and P. multocida. This study not only provides insights into understanding the immune responses of ducks to recombinant DEV-ompH vaccines but also demonstrates the potential for simultaneous prevention of viral and bacterial infections using viral vectors expressing bacterial immunogens.},
}
@article {pmid36014577,
year = {2022},
author = {Liu, Q and Liu, J and He, N and Zhang, M and Wu, L and Chen, X and Zhu, J and Ran, F and Chen, Q and Zhang, H},
title = {CRISPR/Cas12a Coupling with Magnetic Nanoparticles and Cascaded Strand Displacement Reaction for Ultrasensitive Fluorescence Determination of Exosomal miR-21.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {16},
pages = {},
pmid = {36014577},
issn = {1420-3049},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; Humans ; *Lung Neoplasms/diagnosis/genetics ; *Magnetite Nanoparticles ; *MicroRNAs/genetics ; },
abstract = {Exosomal MicroRNA-21 (miRNA-21, miR-21) is significantly up-regulated in blood samples of patients with lung cancer. Exosomal-derived miR-21 can be used as a promising biomarker for the early diagnosis of lung cancer. This paper develops a fluorescent biosensor based on the combination of magnetic nanoparticles (MNPs), cascade strand displacement reaction (CSDR) and CRISPR/Cas12a to detect the exosomal miR-21 from lung cancer. The powerful separation performance of MNPs can eliminate the potential interference of matrix and reduce the background signal, which is very beneficial for the improvement of specificity and sensitivity. The CSDR can specifically transform one miR-21 into plenty of DNA which can specifically trigger the trans-cleavage nuclease activity of Cas12a, resulting in the cleavage of ssDNA bi-labeled with fluorescent and a quencher. Under the optimized experimental conditions, the developed fluorescence biosensor exhibited high sensitivity and specificity towards the determination of exosomal-derived miR-21 with a linear range from 10 to 1 × 10[5] fM and a low detection limit of about 0.89 fM. Most importantly, this method can be successfully applied to distinguish the exosomal miR-21 from the lung cancer patients and the healthy people.},
}
@article {pmid36014003,
year = {2022},
author = {Mateos, G and Bonilla, AM and de Francisco de Polanco, S and Martínez, JM and Escudero, C and Rodríguez, N and Sánchez-Andrea, I and Amils, R},
title = {Shewanella sp. T2.3D-1.1 a Novel Microorganism Sustaining the Iron Cycle in the Deep Subsurface of the Iberian Pyrite Belt.},
journal = {Microorganisms},
volume = {10},
number = {8},
pages = {},
pmid = {36014003},
issn = {2076-2607},
abstract = {The Iberian Pyrite Belt (IPB) is one of the largest deposits of sulphidic minerals on Earth. Río Tinto raises from its core, presenting low a pH and high metal concentration. Several drilling cores were extracted from the IPB's subsurface, and strain T2.3D-1.1 was isolated from a core at 121.8 m depth. We aimed to characterize this subterranean microorganism, revealing its phylogenomic affiliation (Average Nucleotide Identity, digital DNA-DNA Hybridization) and inferring its physiology through genome annotation, backed with physiological experiments to explore its relationship with the Fe biogeochemical cycle. Results determined that the isolate belongs to the Shewanella putrefaciens (with ANI 99.25 with S. putrefaciens CN-32). Its genome harbours the necessary genes, including omcA mtrCAB, to perform the Extracellular Electron Transfer (EET) and reduce acceptors such as Fe[3+], napAB to reduce NO3[-] to NO2[-], hydAB to produce H2 and genes sirA, phsABC and ttrABC to reduce SO3[2-], S2O3[2-] and S4O6[2-], respectively. A full CRISPR-Cas 1F type system was found as well. S. putrefaciens T2.3D-1.1 can reduce Fe[3+] and promote the oxidation of Fe[2+] in the presence of NO3[-] under anaerobic conditions. Production of H2 has been observed under anaerobic conditions with lactate or pyruvate as the electron donor and fumarate as the electron acceptor. Besides Fe[3+] and NO3[-], the isolate also grows with Dimethyl Sulfoxide and Trimethyl N-oxide, S4O6[2-] and S2O3[2-] as electron acceptors. It tolerates different concentrations of heavy metals such as 7.5 mM of Pb, 5 mM of Cr and Cu and 1 mM of Cd, Co, Ni and Zn. This array of traits suggests that S. putrefaciens T2.3D-1.1 could have an important role within the Iberian Pyrite Belt subsurface participating in the iron cycle, through the dissolution of iron minerals and therefore contributing to generate the extreme conditions detected in the Río Tinto basin.},
}
@article {pmid36013970,
year = {2022},
author = {Hoang, PT and Luong, QXT and Ayun, RQ and Lee, Y and Vo, TTB and Kim, T and Lee, S},
title = {A Novel Approach of Antiviral Drugs Targeting Viral Genomes.},
journal = {Microorganisms},
volume = {10},
number = {8},
pages = {},
pmid = {36013970},
issn = {2076-2607},
abstract = {Outbreaks of viral diseases, which cause morbidity and mortality in animals and humans, are increasing annually worldwide. Vaccines, antiviral drugs, and antibody therapeutics are the most effective tools for combating viral infection. The ongoing coronavirus disease 2019 pandemic, in particular, raises an urgent need for the development of rapid and broad-spectrum therapeutics. Current antiviral drugs and antiviral antibodies, which are mostly specific at protein levels, have encountered difficulties because the rapid evolution of mutant viral strains resulted in drug resistance. Therefore, degrading viral genomes is considered a novel approach for developing antiviral drugs. The current article highlights all potent candidates that exhibit antiviral activity by digesting viral genomes such as RNases, RNA interference, interferon-stimulated genes 20, and CRISPR/Cas systems. Besides that, we introduce a potential single-chain variable fragment (scFv) that presents antiviral activity against various DNA and RNA viruses due to its unique nucleic acid hydrolyzing characteristic, promoting it as a promising candidate for broad-spectrum antiviral therapeutics.},
}
@article {pmid36012737,
year = {2022},
author = {Gao, Q and Liu, J and Weng, H and Yuan, X and Xiao, W and Wang, H},
title = {A Long Noncoding RNA Derived from lncRNA-mRNA Networks Modulates Seed Vigor.},
journal = {International journal of molecular sciences},
volume = {23},
number = {16},
pages = {},
pmid = {36012737},
issn = {1422-0067},
mesh = {Gene Expression Profiling ; Gene Regulatory Networks ; Indoleacetic Acids/metabolism ; *Oryza/genetics/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Seeds/genetics/metabolism ; },
abstract = {The discovery of long noncoding RNAs (lncRNAs) has filled a great gap in our understanding of posttranscriptional gene regulation in a variety of biological processes related to plant stress responses. However, systematic analyses of the lncRNAs expressed in rice seeds that germinate under cold stress have been elusive. In this study, we performed strand-specific whole transcriptome sequencing in germinated rice seeds under cold stress and normal temperature. A total of 6258 putative lncRNAs were identified and expressed in a stage-specific manner compared to mRNA. By investigating the targets of differentially expressed (DE) lncRNAs of LT-I (phase I of low temperature)/NT-I (phase I of normal temperature), it was shown that the auxin-activated signaling pathway was significantly enriched, and twenty-three protein-coding genes with most of the members of the SAUR family located in chromosome 9 were identified as the candidate target genes that may interact with five lncRNAs. A seed vigor-related lncRNA, SVR, which interplays with the members of the SAUR gene family in cis was eventually identified. The CRISPR/Cas 9 engineered mutations in SVR cause delay of germination. The findings provided new insights into the connection between lncRNAs and the auxin-activated signaling pathway in the regulation of rice seed vigor.},
}
@article {pmid36012553,
year = {2022},
author = {Vasilev, R and Gunitseva, N and Shebanova, R and Korzhenkov, A and Vlaskina, A and Evteeva, M and Polushkina, I and Nikitchina, N and Toshchakov, S and Kamenski, P and Patrushev, M and Mazunin, I},
title = {Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii.},
journal = {International journal of molecular sciences},
volume = {23},
number = {16},
pages = {},
pmid = {36012553},
issn = {1422-0067},
mesh = {Acidaminococcus/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; DNA/metabolism ; *Endonucleases/metabolism ; Gene Editing ; Humans ; Mammals/metabolism ; Ruminococcus ; },
abstract = {Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.},
}
@article {pmid36012245,
year = {2022},
author = {Wang, H and Ouyang, Q and Yang, C and Zhang, Z and Hou, D and Liu, H and Xu, H},
title = {Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism.},
journal = {International journal of molecular sciences},
volume = {23},
number = {16},
pages = {},
pmid = {36012245},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; *Gravitropism/genetics ; Indoleacetic Acids/metabolism ; Mutation ; *Oryza/metabolism ; Plant Proteins/genetics/metabolism ; Plant Roots/metabolism ; Seedlings/genetics/metabolism ; },
abstract = {The distribution and content of auxin within plant tissues affect a variety of important growth and developmental processes. Polar auxin transport (PAT), mainly mediated by auxin influx and efflux transporters, plays a vital role in determining auxin maxima and gradients in plants. The auxin efflux carrier PIN-FORMED (PIN) family is one of the major protein families involved in PAT. Rice (Oryza sativa L.) genome possesses 12 OsPIN genes. However, the detailed functions of OsPIN genes involved in regulating the rice architecture and gravity response are less well understood. In the present study, OsPIN1b was disrupted by CRISPR/Cas9 technology, and its roles in modulating rice architecture and root gravitropism were investigated. Tissue-specific analysis showed that OsPIN1b was mainly expressed in roots, stems and sheaths at the seedling stage, and the transcript abundance was progressively decreased during the seedling stages. Expression of OsPIN1b could be quickly and greatly induced by NAA, indicating that OsPIN1b played a vital role in PAT. IAA homeostasis was disturbed in ospin1b mutants, as evidenced by the changed sensitivity of shoot and root to NAA and NPA treatment, respectively. Mutation of OsPIN1b resulted in pleiotropic phenotypes, including decreased growth of shoots and primary roots, reduced adventitious root number in rice seedlings, as well as shorter and narrower leaves, increased leaf angle, more tiller number and decreased plant height and panicle length at the late developmental stage. Moreover, ospin1b mutants displayed a curly root phenotype cultured with tap water regardless of lighting conditions, while nutrient solution culture could partially rescue the curly root phenotype in light and almost completely abolish this phenotype in darkness, indicating the involvement of the integration of light and nutrient signals in root gravitropism regulation. Additionally, amyloplast sedimentation was impaired in the peripheral tiers of the ospin1b root cap columella cell, while it was not the main contributor to the abnormal root gravitropism. These data suggest that OsPIN1b not only plays a vital role in regulating rice architecture but also functions in regulating root gravitropism by the integration of light and nutrient signals.},
}
@article {pmid36012157,
year = {2022},
author = {Zhdanova, PV and Lomzov, AA and Prokhorova, DV and Stepanov, GA and Chernonosov, AA and Koval, VV},
title = {Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms.},
journal = {International journal of molecular sciences},
volume = {23},
number = {16},
pages = {},
pmid = {36012157},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Computer Simulation ; DNA/chemistry ; Gene Editing/methods ; *RNA/genetics ; RNA, Guide/metabolism ; Thermodynamics ; },
abstract = {Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR-Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR-Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex's components of Cas9-RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9-RNA/DNA formation from the Cas9-RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR-Cas9 system.},
}
@article {pmid36011351,
year = {2022},
author = {Yan, Q and Li, W and Gong, X and Hu, R and Chen, L},
title = {Transcriptomic and Phenotypic Analysis of CRISPR/Cas9-Mediated gluk2 Knockout in Zebrafish.},
journal = {Genes},
volume = {13},
number = {8},
pages = {},
pmid = {36011351},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Larva/genetics ; Period Circadian Proteins ; *Transcriptome/genetics ; *Zebrafish/genetics ; Zebrafish Proteins/genetics ; },
abstract = {As a subtype of kainite receptors (KARs), GluK2 plays a role in the perception of cold in the periphery sensory neuron. However, the molecular mechanism for gluk2 on the cold stress in fish has not been reported. In this article, real-time PCR assays showed that gluk2 was highly expressed in the brain and eyes of adult zebrafish. To study the functions of gluk2, gene knockout was carried out using the CRISPR/Cas9 system. According to RNA-seq analysis, we selected the differentially expressed genes (DEGs) that had significant differences in at least three tissues of the liver, gill, intestine, skin, brain, and eyes. Gene Ontology (GO) enrichment analysis revealed that cry1ba, cry2, per1b, per2, hsp70.1, hsp70.2, hsp70l, hsp90aa1.1, hsp90aa1.2, hspb1, trpv1, slc27a1b, park2, ucp3, and METRNL were significantly enriched in the 'Response to temperature stimulus' pathway. Through behavioral phenotyping assay, the gluk2[-/-] larval mutant displayed obvious deficiency in cold stress. Furthermore, TUNEL (TdT-mediated dUTP Nick-End Labeling) staining proved that the gill apoptosis of gluk2[-/-] mutant was increased approximately 60 times compared with the wild-type after gradient cooling to 8 °C for 15 h. Overall, our data suggested that gluk2 was necessary for cold tolerance in zebrafish.},
}
@article {pmid36010544,
year = {2022},
author = {Yuan, G and Martin, S and Hassan, MM and Tuskan, GA and Yang, X},
title = {PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies.},
journal = {Cells},
volume = {11},
number = {16},
pages = {},
pmid = {36010544},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Polymerase Chain Reaction ; *RNA, Guide/genetics ; },
abstract = {Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.},
}
@article {pmid36009017,
year = {2022},
author = {Vora, DS and Verma, Y and Sundar, D},
title = {A Machine Learning Approach to Identify the Importance of Novel Features for CRISPR/Cas9 Activity Prediction.},
journal = {Biomolecules},
volume = {12},
number = {8},
pages = {},
pmid = {36009017},
issn = {2218-273X},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Gene Editing ; Machine Learning ; *RNA, Guide/genetics ; },
abstract = {The reprogrammable CRISPR/Cas9 genome editing tool's growing popularity is hindered by unwanted off-target effects. Efforts have been directed toward designing efficient guide RNAs as well as identifying potential off-target threats, yet factors that determine efficiency and off-target activity remain obscure. Based on sequence features, previous machine learning models performed poorly on new datasets, thus there is a need for the incorporation of novel features. The binding energy estimation of the gRNA-DNA hybrid as well as the Cas9-gRNA-DNA hybrid allowed generating better performing machine learning models for the prediction of Cas9 activity. The analysis of feature contribution towards the model output on a limited dataset indicated that energy features played a determining role along with the sequence features. The binding energy features proved essential for the prediction of on-target activity and off-target sites. The plateau, in the performance on unseen datasets, of current machine learning models could be overcome by incorporating novel features, such as binding energy, among others. The models are provided on GitHub (GitHub Inc., San Francisco, CA, USA).},
}
@article {pmid36008654,
year = {2022},
author = {Lombardi, L and Butler, G},
title = {Plasmid-Based CRISPR-Cas9 Editing in Multiple Candida Species.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2542},
number = {},
pages = {13-40},
pmid = {36008654},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Candida/genetics ; Gene Deletion ; Gene Editing ; Plasmids/genetics ; },
abstract = {CRISPR-Cas9 technology radically changed the approach to genetic manipulation of both medically and industrially relevant Candida species, as attested by the ever-increasing number of applications to the study of pathogenesis, drug resistance, gene expression, and host pathogen interaction and drug discovery. Here, we describe the use of plasmid-based systems for high efficiency CRISPR-Cas9 gene editing into any strain of four non-albicans Candida species, namely, Candida parapsilosis, Candida orthopsilosis, Candida metapsilosis, and Candida tropicalis. The plasmids pCP-tRNA and pCT-tRNA contain all the elements necessary for expressing the CRISPR-Cas9 machinery, and they can be used in combination with a repair template for disrupting gene function by insertion of a premature stop codon or by gene deletion. The plasmids are easily lost in the absence of selection, allowing scarless gene editing and minimizing detrimental effects of prolonged Cas9 expression.},
}
@article {pmid36008653,
year = {2022},
author = {Evans, BA and Bernstein, DA},
title = {CRISPR-Mediated Genome Editing in the Human Fungal Pathogen C. albicans.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2542},
number = {},
pages = {3-12},
pmid = {36008653},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Candida albicans/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Genome, Fungal ; Humans ; *RNA, Guide/genetics ; },
abstract = {Cas9-mediated genome editing is one tool investigators can use to study fungal pathogens. Such methodologies allow the investigator to examine how fungal cells differ from human cells and thus potentially identify novel therapeutic targets. In this chapter, we describe how CRISPR-mediated genome editing can be used to edit the genome of the most prevalent human fungal pathogen C. albicans. A cassette encoding a fungal optimized Cas9 nuclease and guide RNA is integrated into the C. albicans genome. The guide RNA targets Cas9 to the complementary genome sequence, and Cas9 cleaves the DNA. A repair template encoding whatever changes the investigator wished to make to the genome is co-transformed with the cassette and repairs the break via homologous recombination, thus introducing the change to the genome. The method we describe enables the researcher to edit the C. albicans genome and then efficiently remove the editing machinery and antibiotic resistance markers. This allows one to sequentially edit the C. albicans genome when multiple changes are desired. In addition, we provide notes that provide guidance on how the described protocols can be altered to meet the demands of the researcher. In these notes, we also describe the recent development of a more flexible CRISPR system that has a relaxed PAM site specificity. These and other advancements make CRISPR-mediated genome editing a practical approach when one needs to genetically alter C. albicans.},
}
@article {pmid36008567,
year = {2022},
author = {Rajan, A and Shrivastava, S and Janhawi, and Kumar, A and Singh, AK and Arora, PK},
title = {CRISPR-Cas system: from diagnostic tool to potential antiviral treatment.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {18},
pages = {5863-5877},
pmid = {36008567},
issn = {1432-0614},
mesh = {Antiviral Agents/therapeutic use ; *COVID-19/diagnosis/drug therapy ; COVID-19 Testing ; CRISPR-Cas Systems ; *HIV Infections/diagnosis/drug therapy ; Humans ; *Influenza, Human/diagnosis/drug therapy ; SARS-CoV-2/genetics ; *Virus Diseases/diagnosis/drug therapy ; },
abstract = {This mini review focuses on the diagnosis and treatment of virus diseases using Crisper-Cas technology. The present paper describes various strategies involved in diagnosing diseases using Crispr-Cas-based assays. Additionally, CRISPR-Cas systems offer great potential as new therapeutic tools for treating viral infections including HIV, Influenza, and SARS-CoV-2. There are several major challenges to be overcome before this technology can be applied routinely in clinical settings, such as finding a suitable delivery tool, toxicity, and immunogenicity, as well as off-target effects. This review also discusses ways to deal with the challenges associated with Crisper-Cas technology. KEY POINTS: • Crisper technology is being applied to diagnose infectious and non-infectious diseases. • A new generation of CRISPR-Cas-based assays has been developed which detect pathogens within minutes, providing rapid diagnosis of diseases. • Crispr-Cas tools can be used to combat viral infections, specifically HIV, influenza, and SARS-CoV-2.},
}
@article {pmid36008396,
year = {2022},
author = {Shaw, WM and Studená, L and Roy, K and Hapeta, P and McCarty, NS and Graham, AE and Ellis, T and Ledesma-Amaro, R},
title = {Inducible expression of large gRNA arrays for multiplexed CRISPRai applications.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4984},
pmid = {36008396},
issn = {2041-1723},
support = {BB/R01602X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T011408/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T013176/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide/genetics/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Transcriptional Activation ; },
abstract = {CRISPR gene activation and inhibition (CRISPRai) has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here we develop a method for near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems to increase CRISPRai multiplexing capacity and target gene flexibility. To achieve strong inducibility, we create a technology to silence gRNA expression within the array in the absence of the inducer, since we found that long gRNA arrays for CRISPRai can express themselves even without promoter. Using this method, we create a highly tuned and easy-to-use CRISPRai toolkit in the industrially relevant yeast, Saccharomyces cerevisiae, establishing the first system to combine simultaneous activation and repression, large multiplexing capacity, and inducibility. We demonstrate this toolkit by targeting 11 genes in central metabolism in a single transformation, achieving a 45-fold increase in succinic acid, which could be precisely controlled in an inducible manner. Our method offers a highly effective way to regulate genes and rewire metabolism in yeast, with principles of gRNA array construction and inducibility that should extend to other chassis organisms.},
}
@article {pmid36008137,
year = {2022},
author = {Duff, K},
title = {A Recipe for ORANGE-CAKE-This Time with Two Layers!.},
journal = {eNeuro},
volume = {9},
number = {4},
pages = {},
pmid = {36008137},
issn = {2373-2822},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid36007683,
year = {2022},
author = {Herbert, DR and Stoltzfus, JDC and Rossi, HL and Abraham, D},
title = {Is Strongyloides stercoralis hyperinfection induced by glucocorticoids a result of both suppressed host immunity and altered parasite genetics?.},
journal = {Molecular and biochemical parasitology},
volume = {251},
number = {},
pages = {111511},
doi = {10.1016/j.molbiopara.2022.111511},
pmid = {36007683},
issn = {1872-9428},
support = {U01 AI163062/AI/NIAID NIH HHS/United States ; R01 AI164715/AI/NIAID NIH HHS/United States ; R01 AI123173/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Glucocorticoids/adverse effects ; Humans ; Larva ; Mice ; Mice, Inbred NOD ; *Parasites ; *Strongyloides stercoralis/genetics ; *Strongyloidiasis ; },
abstract = {The gastrointestinal (GI) nematode Strongyloides stercoralis (S.s.) causes human strongyloidiasis, a potentially life-threatening disease that currently affects over 600 million people globally. The uniquely pernicious aspect of S.s. infection, as compared to all other GI nematodes, is its autoinfective larval stage (L3a) that maintains a low-grade chronic infection, allowing undetectable persistence for decades. Infected individuals who are administered glucocorticoid therapy can develop a rapid and often lethal hyperinfection syndrome within days. Hyperinfection patients often present with dramatic increases in first- and second-stage larvae and L3a in their GI tract, with L3a widely disseminating throughout host organs leading to sepsis. How glucocorticoid administration drives hyperinfection remains a critical unanswered question; specifically, it is unknown whether these steroids promote hyperinfection through eliminating essential host protective mechanisms and/or through dysregulating parasite development. This current deficiency in understanding is largely due to the previous absence of a genetically defined mouse model that would support all S.s. life-cycle stages and the lack of successful approaches for S.s. genetic manipulation. However, there are currently new possibilities through the recent demonstration that immunodeficient NOD.Cg-Prkdc[scid]Il2rg[tm1Wjl]/SzJ (NSG) mice support sub-clinical infections that can be transformed to lethal hyperinfection syndrome following glucocorticoid administration. This is coupled with advances in transcriptomics, transgenesis, and gene inactivation strategies that now allow rigorous scientific inquiry into S.s. biology. We propose that combining in vivo manipulation of host immunity and deep immunoprofiling strategies with the latest advances in S.s. transcriptomics, piggyBac transposon-mediated transgene insertion, and CRISPR/Cas-9-mediated gene inactivation will facilitate new insights into the mechanisms that could be targeted to block lethality in humans with S.s. hyperinfection.},
}
@article {pmid36007061,
year = {2022},
author = {Hu, C and van Beljouw, SPB and Nam, KH and Schuler, G and Ding, F and Cui, Y and Rodríguez-Molina, A and Haagsma, AC and Valk, M and Pabst, M and Brouns, SJJ and Ke, A},
title = {Craspase is a CRISPR RNA-guided, RNA-activated protease.},
journal = {Science (New York, N.Y.)},
volume = {377},
number = {6612},
pages = {1278-1285},
doi = {10.1126/science.add5064},
pmid = {36007061},
issn = {1095-9203},
support = {R35 GM118174/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacterial Proteins/chemistry ; *CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; *Caspases/chemistry ; Cryoelectron Microscopy ; *Planctomycetes/enzymology ; Protein Conformation ; *RNA, Guide/chemistry ; },
abstract = {The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.},
}
@article {pmid36006707,
year = {2022},
author = {Li, C and Georgakopoulou, A and Newby, GA and Everette, KA and Nizamis, E and Paschoudi, K and Vlachaki, E and Gil, S and Anderson, AK and Koob, T and Huang, L and Wang, H and Kiem, HP and Liu, DR and Yannaki, E and Lieber, A},
title = {In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies.},
journal = {JCI insight},
volume = {7},
number = {19},
pages = {},
pmid = {36006707},
issn = {2379-3708},
mesh = {Adenine ; *Anemia, Sickle Cell/genetics/therapy ; Animals ; CRISPR-Cas Systems ; Fetal Hemoglobin/genetics/metabolism ; Gene Editing/methods ; *Hemoglobinopathies/genetics/therapy ; Humans ; Mice ; beta-Globins/genetics ; *beta-Thalassemia/genetics/therapy ; gamma-Globins/genetics ; },
abstract = {Individuals with β-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in > 60% -113 A>G conversion, with 30% γ-globin of β-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from β-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.},
}
@article {pmid36006039,
year = {2022},
author = {Kushwaha, SK and Narasimhan, LP and Chithananthan, C and Marathe, SA},
title = {Clustered regularly interspaced short palindromic repeats-Cas system: diversity and regulation in Enterobacteriaceae.},
journal = {Future microbiology},
volume = {17},
number = {},
pages = {1249-1267},
doi = {10.2217/fmb-2022-0081},
pmid = {36006039},
issn = {1746-0921},
mesh = {Bacteria ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; *Enterobacteriaceae/genetics ; Quorum Sensing ; Virulence ; },
abstract = {Insights into the arms race between bacteria and invading mobile genetic elements have revealed the intricacies of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system and the counter-defenses of bacteriophages. Incredible spacer diversity but significant spacer conservation among species/subspecies dictates the specificity of the CRISPR-Cas system. Researchers have exploited this feature to type/subtype the bacterial strains, devise targeted antimicrobials and regulate gene expression. This review focuses on the nuances of the CRISPR-Cas systems in Enterobacteriaceae that predominantly harbor type I-E and I-F CRISPR systems. We discuss the systems' regulation by the global regulators, H-NS, LeuO, LRP, cAMP receptor protein and other regulators in response to environmental stress. We further discuss the regulation of noncanonical functions like DNA repair pathways, biofilm formation, quorum sensing and virulence by the CRISPR-Cas system. The review comprehends multiple facets of the CRISPR-Cas system in Enterobacteriaceae including its diverse attributes, association with genetic features, regulation and gene regulatory mechanisms.},
}
@article {pmid36005038,
year = {2022},
author = {Li, X and Jiang, W and Qi, Q and Liang, Q},
title = {A Gene Circuit Combining the Endogenous I-E Type CRISPR-Cas System and a Light Sensor to Produce Poly-β-Hydroxybutyric Acid Efficiently.},
journal = {Biosensors},
volume = {12},
number = {8},
pages = {},
pmid = {36005038},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli ; Gene Regulatory Networks ; Hydroxybutyrates/metabolism ; Polyesters ; },
abstract = {'Metabolic burden,' which arises when introducing exogenic synthesizing pathways into a host strain, remains a challenging issue in metabolic engineering. Redirecting metabolic flux from cell growth to product synthesis at an appropriate culture timepoint is ideal for resolving this issue. In this report, we introduce optogenetics-which is capable of precise temporal and spatial control-as a genetic switch, accompanied by the endogenous type I-E CRISPRi system in Escherichia coli (E. coli) to generate a metabolic platform that redirects metabolic flux. Poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of this platform. A two-to-three-fold increase in PHB content was observed under green light when compared with the production of PHB under red light, confirming the regulatory activity of this platform and its potential to redirect metabolic flux to synthesize target products.},
}
@article {pmid36005032,
year = {2022},
author = {Wang, L and Fu, J and Cai, G and Cheng, X and Zhang, D and Shi, S and Zhang, Y},
title = {Rapid and Visual RPA-Cas12a Fluorescence Assay for Accurate Detection of Dermatophytes in Cats and Dogs.},
journal = {Biosensors},
volume = {12},
number = {8},
pages = {},
pmid = {36005032},
issn = {2079-6374},
mesh = {Animals ; *Arthrodermataceae ; CRISPR-Cas Systems ; Cats ; *Dermatomycoses/diagnosis/microbiology/veterinary ; Dogs ; Hair/microbiology ; Recombinases ; },
abstract = {Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes. Wood's lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.},
}
@article {pmid36002653,
year = {2022},
author = {Singh, J and Sharma, D and Brar, GS and Sandhu, KS and Wani, SH and Kashyap, R and Kour, A and Singh, S},
title = {CRISPR/Cas tool designs for multiplex genome editing and its applications in developing biotic and abiotic stress-resistant crop plants.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11443-11467},
pmid = {36002653},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Crops, Agricultural/genetics ; Stress, Physiological/genetics ; },
abstract = {Crop plants are prone to several yield-reducing biotic and abiotic stresses. The crop yield reductions due to these stresses need addressing to maintain an adequate balance between the increasing world population and food production to avoid food scarcities in the future. It is impossible to increase the area under food crops proportionately to meet the rising food demand. In such an adverse scenario overcoming the biotic and abiotic stresses through biotechnological interventions may serve as a boon to help meet the globe's food requirements. Under the current genomic era, the wide availability of genomic resources and genome editing technologies such as Transcription Activator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered-Regularly Interspaced Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) has widened the scope of overcoming these stresses for several food crops. These techniques have made gene editing more manageable and accessible with changes at the embryo level by adding or deleting DNA sequences of the target gene(s) from the genome. The CRISPR construct consists of a single guide RNA having complementarity with the nucleotide fragments of the target gene sequence, accompanied by a protospacer adjacent motif. The target sequence in the organism's genome is then cleaved by the Cas9 endonuclease for obtaining a desired trait of interest. The current review describes the components, mechanisms, and types of CRISPR/Cas techniques and how this technology has helped to functionally characterize genes associated with various biotic and abiotic stresses in a target organism. This review also summarizes the application of CRISPR/Cas technology targeting these stresses in crops through knocking down/out of associated genes.},
}
@article {pmid36002574,
year = {2022},
author = {Carnevale, J and Shifrut, E and Kale, N and Nyberg, WA and Blaeschke, F and Chen, YY and Li, Z and Bapat, SP and Diolaiti, ME and O'Leary, P and Vedova, S and Belk, J and Daniel, B and Roth, TL and Bachl, S and Anido, AA and Prinzing, B and Ibañez-Vega, J and Lange, S and Haydar, D and Luetke-Eversloh, M and Born-Bony, M and Hegde, B and Kogan, S and Feuchtinger, T and Okada, H and Satpathy, AT and Shannon, K and Gottschalk, S and Eyquem, J and Krenciute, G and Ashworth, A and Marson, A},
title = {RASA2 ablation in T cells boosts antigen sensitivity and long-term function.},
journal = {Nature},
volume = {609},
number = {7925},
pages = {174-182},
pmid = {36002574},
issn = {1476-4687},
support = {K99 CA256262/CA/NCI NIH HHS/United States ; R35 NS105068/NS/NINDS NIH HHS/United States ; R01 CA173750/CA/NCI NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R01 NS106379/NS/NINDS NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; K08 CA252605/CA/NCI NIH HHS/United States ; S10 OD010786/OD/NIH HHS/United States ; R01 NS121249/NS/NINDS NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Antigens, Neoplasm/immunology ; Bone Marrow ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Knockdown Techniques ; Humans ; Immunotherapy, Adoptive ; Leukemia/immunology/pathology/therapy ; Mice ; *Neoplasms/immunology/pathology/therapy ; Receptors, Antigen, T-Cell/immunology ; Receptors, Chimeric Antigen/immunology ; *T-Lymphocytes/immunology/metabolism ; Time Factors ; Xenograft Model Antitumor Assays ; *ras GTPase-Activating Proteins/deficiency/genetics ; },
abstract = {The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints[1,2]. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function[3-10]. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.},
}
@article {pmid36002571,
year = {2022},
author = {Pacesa, M and Loeff, L and Querques, I and Muckenfuss, LM and Sawicka, M and Jinek, M},
title = {R-loop formation and conformational activation mechanisms of Cas9.},
journal = {Nature},
volume = {609},
number = {7925},
pages = {191-196},
pmid = {36002571},
issn = {1476-4687},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/metabolism/ultrastructure ; *CRISPR-Cas Systems ; Catalysis ; *Cryoelectron Microscopy ; DNA/metabolism ; DNA Cleavage ; Enzyme Activation ; Gene Editing ; *Protein Domains ; *R-Loop Structures ; RNA, Guide/metabolism ; *Streptococcus pyogenes/enzymology ; Substrate Specificity ; },
abstract = {Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage[1-3]. The programmable activity of Cas9 has been widely utilized for genome editing applications[4-6], yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.},
}
@article {pmid36002405,
year = {2022},
author = {Ding, X and Pan, Z and Yang, L and Luo, X and Jiang, N and Zhu, M and Wu, C and Lan, G and Li, P},
title = {[Advances of CRISPR/Cas9 activation system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {8},
pages = {2713-2724},
doi = {10.13345/j.cjb.220040},
pmid = {36002405},
issn = {1872-2075},
mesh = {Biotechnology ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Phenotype ; RNA, Guide/genetics/metabolism ; },
abstract = {Gene editing technology has been a hotspot in the field of biotechnology. CRISPR/Cas systems are efficient gene editing tools because of its specificity, simplicity and flexibility, these features enabled the rapid application of CRISPR/Cas systems in a variety of organisms. Moreover, the combination of transcriptional activator with dead Cas protein can achieve specific regulation of gene expression at the transcription level, which has made important contributions to the development of biotechnology in medical and agriculture. Overexpression of foreign genes is a common method to verify gene function and regulation. However, due to the limitation of vector capacity, it is difficult to achieve overexpression of multiple genes. CRISPR/Cas9 activation system can regulate the expression of multiple genes under the guidance of different guide RNAs to verify gene functions at the regulatory level. This review summarizes the composition of the CRISPR/Cas9 activation system and different activation strategies, and summarizes solutions for excessive activation. It may facilitate the application of CRISPR/Cas9 activation system in genetic improvement of cotton and herbicide resistance research.},
}
@article {pmid36001999,
year = {2022},
author = {Yamasaki, F and Nakazawa, T and Oh, M and Bao, D and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {Gene targeting of dikaryotic Pleurotus ostreatus nuclei using the CRISPR/Cas9 system.},
journal = {FEMS microbiology letters},
volume = {369},
number = {1},
pages = {},
doi = {10.1093/femsle/fnac083},
pmid = {36001999},
issn = {1574-6968},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; Gene Targeting ; Plant Breeding ; *Pleurotus/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene targeting is a promising method used in molecular breeding. We recently reported the successful introduction of this method in the monokaryotic Pleurotus ostreatus (oyster mushroom), PC9. However, considering their application in mushroom breeding, dikaryotic strains (with targeted gene mutations in both nuclei) need to be generated. This is laborious and time-consuming because a classical crossing technique is used. Herein, we report a technique that targets both nuclei of dikaryotic P. ostreatus, PC9×#64 in a transformation experiment using plasmid-based CRISPR/Cas9, with the aim of developing a method for efficient and rapid molecular breeding. As an example, we targeted strains with low basidiospore production ability through the meiosis-related genes mer3 or msh4. Four different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting mer3 or msh4 were constructed and separately introduced into PC9×#64. Eight of the 38 dikaryotic transformants analyzed produced no basidiospores. Genomic PCR suggested that msh4 or mer3 mutations were introduced into both nuclei of seven out of eight strains. Thus, in this study, we demonstrated simultaneous gene targeting using our CRISPR/Cas9 system, which may be useful for the molecular breeding of cultivated agaricomycetes.},
}
@article {pmid36001082,
year = {2022},
author = {Wang, Z and Wang, Y and Wang, Y and Chen, W and Ji, Q},
title = {CRISPR/Cpf1-Mediated Multiplex and Large-Fragment Gene Editing in Staphylococcus aureus.},
journal = {ACS synthetic biology},
volume = {11},
number = {9},
pages = {3049-3057},
doi = {10.1021/acssynbio.2c00248},
pmid = {36001082},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Staphylococcus aureus/genetics ; },
abstract = {Staphylococcus aureus is a major human pathogen that causes a variety of infections, including life-threatening diseases. Research on S. aureus is constrained by complex and limited genetic manipulation methods. Here, we report a CRISPR/Cpf1-mediated system, pCpfSA, for rapid and versatile genome editing in S. aureus. In direct comparison with the existing CRISPR/Cas9-mediated genome-editing system, the pCpfSA system exhibits enhanced colony-forming units (CFUs) after editing and an expanded targetable range with comparable editing efficiency. Given the precursor crRNA (pre-crRNA) processing activity of Cpf1, the pCpfSA system also allows multiplex gene editing and large-fragment DNA knockout simply by introducing two crRNAs and the corresponding donor templates, which is difficult to achieve using the CRISPR/Cas9 system, thereby greatly expanding the genome editor toolbox for S. aureus.},
}
@article {pmid36000854,
year = {2022},
author = {Ipoutcha, T and Rideau, F and Gourgues, G and Arfi, Y and Lartigue, C and Blanchard, A and Sirand-Pugnet, P},
title = {Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System.},
journal = {Applied and environmental microbiology},
volume = {88},
number = {17},
pages = {e0099622},
pmid = {36000854},
issn = {1098-5336},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; *Gene Editing ; Humans ; Mycoplasma ; Phylogeny ; Virulence Factors/genetics ; },
abstract = {Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically relevant livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools for most species, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is limited. Here, we demonstrate the adaptation of an efficient Cas9-Base Editor system to introduce targeted mutations into three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible SpdCas9-pmcDA1 cytosine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and likely the entire class Mollicutes. IMPORTANCE Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies of many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. Here, we demonstrate the adaptation of a CRISPR-derived base editor for three major pathogenic species: Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted, and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies of these economically important bacteria.},
}
@article {pmid35999638,
year = {2022},
author = {Zou, Y and Qiu, L and Xie, A and Han, W and Zhang, S and Li, J and Zhao, S and Li, Y and Liang, Y and Hu, Y},
title = {Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {173},
pmid = {35999638},
issn = {1475-2859},
mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Knockout Techniques ; Plasmids/genetics ; },
abstract = {BACKGROUND: Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability.<br><br>RESULTS: To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5&#xa0;days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL.<br><br>CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.},
}
@article {pmid35999261,
year = {2022},
author = {Adriaanse, FRS and Kamens, JL and Vogel, P and Sakurada, SM and Pruett-Miller, SM and Stam, RW and Michel Zwaan, C and Gruber, TA},
title = {A CRISPR/Cas9 engineered Mpl[S504N] mouse model recapitulates human myelofibrosis.},
journal = {Leukemia},
volume = {36},
number = {10},
pages = {2535-2538},
pmid = {35999261},
issn = {1476-5551},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Humans ; Mice ; *Primary Myelofibrosis/genetics ; Receptors, Thrombopoietin/genetics ; },
}
@article {pmid35998924,
year = {2022},
author = {Zhang, T and Jia, Y and Li, H and Xu, D and Zhou, J and Wang, G},
title = {CRISPRCasStack: a stacking strategy-based ensemble learning framework for accurate identification of Cas proteins.},
journal = {Briefings in bioinformatics},
volume = {23},
number = {5},
pages = {},
doi = {10.1093/bib/bbac335},
pmid = {35998924},
issn = {1477-4054},
mesh = {*Archaea/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Machine Learning ; *Proteomics ; },
abstract = {CRISPR-Cas system is an adaptive immune system widely found in most bacteria and archaea to defend against exogenous gene invasion. One of the most critical steps in the study of exploring and classifying novel CRISPR-Cas systems and their functional diversity is the identification of Cas proteins in CRISPR-Cas systems. The discovery of novel Cas proteins has also laid the foundation for technologies such as CRISPR-Cas-based gene editing and gene therapy. Currently, accurate and efficient screening of Cas proteins from metagenomic sequences and proteomic sequences remains a challenge. For Cas proteins with low sequence conservation, existing tools for Cas protein identification based on homology cannot guarantee identification accuracy and efficiency. In this paper, we have developed a novel stacking-based ensemble learning framework for Cas protein identification, called CRISPRCasStack. In particular, we applied the SHAP (SHapley Additive exPlanations) method to analyze the features used in CRISPRCasStack. Sufficient experimental validation and independent testing have demonstrated that CRISPRCasStack can address the accuracy deficiencies and inefficiencies of the existing state-of-the-art tools. We also provide a toolkit to accurately identify and analyze potential Cas proteins, Cas operons, CRISPR arrays and CRISPR-Cas locus in prokaryotic sequences. The CRISPRCasStack toolkit is available at https://github.com/yrjia1015/CRISPRCasStack.},
}
@article {pmid35998847,
year = {2022},
author = {Wang, Z and Wei, H and Bu, S and Li, X and Zhou, H and Zhang, W and Wan, J},
title = {Ultrasensitive, rapid, and highly specific detection of microRNAs based on PER-CRISPR/CAS.},
journal = {Bioorganic &amp; medicinal chemistry letters},
volume = {74},
number = {},
pages = {128949},
doi = {10.1016/j.bmcl.2022.128949},
pmid = {35998847},
issn = {1464-3405},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded ; Fluorescent Dyes/chemistry ; *MicroRNAs/genetics ; },
abstract = {Abnormal microRNA (miRNA) expression levels are confirmed as diagnostic biomarkers of the emergence and development of diseases. In this study, we developed a fluorescence biosensor for detecting miRNAs based on double amplification reactions with the primer exchange reaction (PER) and CRISPR/Cas12a. In the absence of target miRNA-21, PER hairpins remained locked by the protector strands and the primers did not extend. In the presence of target miRNA-21, the miRNA-21 bound to the guard sequence and exposed primer binding sites. Also, the closed PER hairpin was unlocked to specifically extend primers into single-stranded DNA (ssDNA) of unequal lengths. These ssDNAs of unequal lengths could activate the cleavage of a reporter by Cas12a, leading to an increase in detectable fluorescence signals. A large number of short nucleic acid fragments were amplified by PER-CRISPR multiple cycle cleavage fluorescent probes. Based on PER-combined CRISPR/Cas12a established dual signal amplification method was characterized by a low limit of detection of 10fM. The fluorescent biosensor for miRNA detection had the advantages of low detection cost, simple operation, and mobility, providing a very promising platform for the point-of-care testing of miRNA-21.},
}
@article {pmid35998200,
year = {2022},
author = {McKindles, KM and McKay, RM and Bullerjahn, GS},
title = {Genomic comparison of Planktothrix agardhii isolates from a Lake Erie embayment.},
journal = {PloS one},
volume = {17},
number = {8},
pages = {e0273454},
pmid = {35998200},
issn = {1932-6203},
support = {P01 ES028939/ES/NIEHS NIH HHS/United States ; },
mesh = {*Cyanobacteria/metabolism ; Genome, Bacterial ; Genomics ; *Lakes/microbiology ; Microcystins/genetics ; Phylogeny ; Planktothrix ; },
abstract = {Planktothrix agardhii is a filamentous cyanobacterial species that dominates harmful algal blooms in Sandusky Bay, Lake Erie and other freshwater basins across the world. P. agardhii isolates were obtained from early (June) blooms via single filament isolation; eight have been characterized from 2016, and 12 additional isolates have been characterized from 2018 for a total of 20 new cultures. These novel isolates were processed for genomic sequencing, where reads were used to generate scaffolds and contigs which were annotated with DIAMOND BLAST hit, Pfam, and GO. Analyses include whole genome alignment to generate phylogenetic trees and comparison of genetic rearrangements between isolates. Nitrogen acquisition and metabolism was compared across isolates. Secondary metabolite production was genetically explored including microcystins, two types of aeruginosin clusters, anabaenopeptins, cyanopeptolins, microviridins, and prenylagaramides. Two common and 4 unique CRISPR-cas islands were analyzed for similar sequences across all isolates and against the known Planktothrix-specific cyanophage, PaV-LD. Overall, the uniqueness of each genome from Planktothrix blooms sampled from the same site and at similar times belies the unexplored diversity of this genus.},
}
@article {pmid35994211,
year = {2022},
author = {Janthabut, T and Tristianto, C and Sakulkoo, J and Sunvittayakul, P and Suttangkakul, A and Gomez, LD and Vuttipongchaikij, S and Sakulsingharoj, C},
title = {Effects of CRISPR/Cas9 generated drooping leaf (dl) alleles on midrib and carpel formations in Oryza sativa Nipponbare.},
journal = {Planta},
volume = {256},
number = {3},
pages = {61},
pmid = {35994211},
issn = {1432-2048},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Leucine/metabolism ; *Oryza/metabolism ; Plant Proteins/genetics/metabolism ; Poaceae/genetics ; Serine/genetics/metabolism ; },
abstract = {We generated drooping leaf rice mutants by CRISPR/Cas and identified two novel alleles with specific editing that allow underpinning of the function of the DL protein domain towards midrib and carpel formations. The DROOPING LEAF (DL) gene plays an essential role in regulating midrib formation and carpel specification in rice and other grass species, but the specific function of DL protein domains in different developmental processes is unclear. Analysis of different dl mutant alleles will allow dissecting the function of DL. Here, we generated Nipponbare rice dl mutants using CRISPR/Cas gene editing and identified two novel dl alleles with different effects on midrib formation and carpel development. Phenotypic and genotypic analysis of T0 and segregated T1 edited lines showed that while dl-51S allele (a 3 bp deletion and a serine deletion at position 51) reduces midrib sizes and produces normal carpels, the dl-50LS allele (a 6 bp deletion and a leucine-serine deletion at position 50-51) causes the lack of midribs and abnormal stigma. This result indicates that the 51-serine is important for midrib formation and the 50-leucine is essential for midrib and carpel development. These dl mutant alleles contribute to the DL gene functional analysis and to gain insights into possible modifications of leaf architecture of rice and other grass species.},
}
@article {pmid35994136,
year = {2022},
author = {Jindal, I and Wang, X},
title = {Programmable Genome-Editing Technologies as Single-Course Therapeutics for Atherosclerotic Cardiovascular Disease.},
journal = {Current atherosclerosis reports},
volume = {24},
number = {11},
pages = {861-866},
pmid = {35994136},
issn = {1534-6242},
mesh = {Adenine ; Animals ; *Atherosclerosis/genetics/therapy ; CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Cholesterol, LDL ; Gene Editing ; Humans ; Liposomes ; Nanoparticles ; Proprotein Convertase 9/genetics ; },
abstract = {PURPOSE OF REVIEW: To establish genome editing as a promising therapeutic approach for the treatment and prevention of atherosclerotic cardiovascular disease.<br><br>RECENT FINDINGS: Systemic delivery of a CRISPR adenine base editor using lipid nanoparticles demonstrated a near 90% reduction in circulating PCSK9 and over 60% reduction in blood LDL-C in nonhuman primates with the effects remaining durable at least 8&#xa0;months following a single course. Preclinical proof-of-concept studies have elucidated the superior therapeutic potential of genome-editing approaches for the treatment of hyperlipidemia, thus substantiating their progression to clinical studies.},
}
@article {pmid35993350,
year = {2022},
author = {Zhao, L and Li, D and Zhang, Y and Huang, Q and Zhang, Z and Chen, C and Xu, CF and Chu, X and Zhang, Y and Yang, X},
title = {HSP70-Promoter-Driven CRISPR/Cas9 System Activated by Reactive Oxygen Species for Multifaceted Anticancer Immune Response and Potentiated Immunotherapy.},
journal = {ACS nano},
volume = {16},
number = {9},
pages = {13821-13833},
doi = {10.1021/acsnano.2c01885},
pmid = {35993350},
issn = {1936-086X},
mesh = {*B7-H1 Antigen/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Dendrimers ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Immunity ; Immunologic Factors ; Immunotherapy ; Reactive Oxygen Species/metabolism ; Tumor Microenvironment/genetics ; },
abstract = {To address the low response rate to immune checkpoint blockade (ICB) therapy, we propose a specific promoter-driven CRISPR/Cas9 system, F-PC/pHCP, that achieves permanent genomic disruption of PD-L1 and elicits a multifaceted anticancer immune response to potentiate immunotherapy. This system consists of a chlorin e6-encapsulated fluorinated dendrimer and HSP70-promoter-driven CRISPR/Cas9. F-PC/pHCP under 660 nm laser activated the HSP70 promoter and enabled the specific expression of the Cas9 protein to disrupt the PD-L1 gene, preventing immune escape. Moreover, F-PC/pHCP also induced immunogenic cell death (ICD) of tumor cells and reprogrammed the immunosuppressive tumor microenvironment. Overall, this specific promoter-driven CRISPR/Cas9 system showed great anticancer efficacy and, more importantly, stimulated an immune memory response to inhibit distant tumor growth and lung metastasis. This CRISPR/Cas9 system represents an alternative strategy for ICB therapy as well as enhanced cancer immunotherapy.},
}
@article {pmid35993342,
year = {2022},
author = {Meyerink, BL and Kc, P and Tiwari, NK and Kittock, CM and Klein, A and Evans, CM and Pilaz, LJ},
title = {Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.},
journal = {Development (Cambridge, England)},
volume = {149},
number = {18},
pages = {},
pmid = {35993342},
issn = {1477-9129},
support = {P20 GM103620/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Electroporation/methods ; Epitopes ; *Gene Editing/methods ; Mice ; },
abstract = {In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.},
}
@article {pmid35989704,
year = {2022},
author = {Berghuis, NF and Mars-Groenendijk, R and Busker, RW and Paauw, A and van Leeuwen, HC},
title = {Combining CRISPR-Cas12a with terminal deoxynucleotidyl transferase dependent reporter elongation for pathogen detection using lateral flow test strips.},
journal = {Biology methods &amp; protocols},
volume = {7},
number = {1},
pages = {bpac015},
pmid = {35989704},
issn = {2396-8923},
abstract = {CRISPR-Cas (CC)-based detection technologies have some exceptional features, which hold the promise of developing into the next-generation diagnostic platforms. One of these features is the ability to trigger non-specific single-stranded DNA/RNA cleavage activity after specific target recognition and Cas enzyme activation. This cleavage activity can be visualized either by single-stranded DNA/RNA fluorescence resonance energy transfer quenching reporters or via lateral flow strips, which separate and detect the cleaved reporters. In a previous study, we reported coupling CC-cleavage activity with the enzyme terminal deoxynucleotidyl transferase (TdT) that elongates cleaved ssDNA reporter fragments with dTTP nucleotides. These elongated poly(thymine) tails then act as scaffolds for the formation of copper nanoparticles which generate a bright fluorescent signal upon UV excitation. In the current study, we visualize the poly(thymine) tails on lateral flow strips, using different combinations of biotinylated or fluorescein-labeled nucleotides, various reporters, and capture oligos. One particular approach, using a fluorescein reporter, reached a target sensitivity of <1 pM and was named Cas activity assay on a strip and was tested using Bacillus anthracis genomic DNA.},
}
@article {pmid35989112,
year = {2022},
author = {Chen, Y and Qian, S and Yu, X and Wu, J and Xu, J},
title = {Microfluidics: the propellant of CRISPR-based nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2022.07.015},
pmid = {35989112},
issn = {1879-3096},
abstract = {Since the discovery of collateral cleavage activity, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have become the new generation of nucleic acid detection tools. However, their widespread application remains limited. A pre-amplification step is required to improve the sensitivity of CRISPR systems, complicating the operating procedure and limiting quantitative precision. In addition, nonspecific collateral cleavage activity makes it difficult to realize multiplex detection in a one-pot CRISPR reaction with a single Cas protein. Microfluidics, which can transfer nucleic acid analysis process to a chip, has the advantages of miniaturization, integration, and automation. Microfluidics coupled with CRISPR systems improves the detection ability of CRISPR, enabling fast, high-throughput, integrated, multiplex, and digital detection, which results in the further popularization of CRISPR for a range of scenarios.},
}
@article {pmid35988346,
year = {2023},
author = {Jiang, W and Chen, Z and Lu, J and Ren, X and Ma, Y},
title = {Ultrasensitive visual detection of miRNA-143 using a CRISPR/Cas12a-based platform coupled with hyperbranched rolling circle amplification.},
journal = {Talanta},
volume = {251},
number = {},
pages = {123784},
doi = {10.1016/j.talanta.2022.123784},
pmid = {35988346},
issn = {1873-3573},
mesh = {CRISPR-Cas Systems ; Gold ; Humans ; Male ; *Metal Nanoparticles ; *MicroRNAs/genetics ; Nucleic Acid Amplification Techniques/methods ; *Prostatic Neoplasms/diagnosis/genetics ; },
abstract = {MicroRNAs are proposed novel biomarker for noninvasive diagnosis of cancer. miRNA-143 is reported to be associated with the development of prostate cancer. However, detection of miRNAs is still challenging due to their unique characteristics, such as small size and high sequence homology among family members. We here developed a gold nanoparticle (AuNP)-based visual assay that combines with CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (HRCA), which is called HRCA enhanced CRISPR/Cas12a-based assay (HECA) for sensitive detection of miRNA-143. The sequence-specific recognition character of CRISPR/Cas12a and HRCA signal amplification strategy enables the HECA outstanding specificity and sensitivity. In optimal condition, 1 fM miRNA-143 could be detected by naked eyes, and down to aM level with the aid of UV-Vis instrument. The diagnostic performance of the HECA for clinical samples was also evaluated based on the receiving operating characteristic algorithm (ROC), and our results suggest the miR-143 is a promising biomarker for noninvasive diagnosis of prostate cancer. This method is simple in operation and requires minimum instrument. We expect it to be widely applied in clinical diagnostics, especially in low-resource settings.},
}
@article {pmid35988299,
year = {2023},
author = {Camperi, J and Console, G and Zheng, L and Stephens, N and Montti, M and Roper, B and Zheng, M and Moshref, M and Dagdas, Y and Holder, P and Stella, C},
title = {Comprehensive UHPLC- and CE-based methods for engineered Cas9 characterization.},
journal = {Talanta},
volume = {252},
number = {},
pages = {123780},
doi = {10.1016/j.talanta.2022.123780},
pmid = {35988299},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; Chromatography, High Pressure Liquid ; *Tandem Mass Spectrometry ; CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; Electrophoresis, Capillary ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). This work describes for the first time the development of ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9. The potential of using a low-resolution MS detector, especially for a GMP environment, as a low-cost and simple method to identify SpyFi Cas9 is discussed.},
}
@article {pmid35987443,
year = {2022},
author = {Burris, BJD and Molina Vargas, AM and Park, BJ and O'Connell, MR},
title = {Optimization of specific RNA knockdown in mammalian cells with CRISPR-Cas13.},
journal = {Methods (San Diego, Calif.)},
volume = {206},
number = {},
pages = {58-68},
pmid = {35987443},
issn = {1095-9130},
support = {R35 GM133462/GM/NIGMS NIH HHS/United States ; T32 GM068411/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA ; Humans ; Mammals/genetics ; RNA/genetics ; *RNA, Guide/genetics ; Ribonucleases ; },
abstract = {Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR Associated (Cas) proteins to target and cleave foreign genetic elements in an RNA-guided manner [1-3]. Type VI CRISPR-Cas systems contain a single effector ribonuclease, Cas13, that binds and processes a CRISPR-RNA (crRNA; also known as a guide-RNA), forming an RNA-guided RNA-targeting effector complex [4,5]. Previous studies have shown that Cas13 can be engineered to target and modulate RNA processes in human cells, illustrating the versatility and specificity of Cas13 as an RNA knockdown (KD), splicing, editing, or imaging tool [6-8]. While Cas13 has been successfully used by several groups, our lab has observed significant variability in Cas13 KD ability depending which protocol is being followed [9-12]. To further understand this variability and generate a robust Cas13 KD protocol we thoroughly tested which Cas13 ortholog to use, the duration of KD experiments, the amount of plasmid DNA transfected, methods for analyzing KD efficiency, and report an optimized method for carrying out and analyzing Cas13 mediated RNA KD experiments. The method outlined in this paper illustrates a faster and more reliable protocol to iteratively test gRNA performance and target gene KD.},
}
@article {pmid35987123,
year = {2023},
author = {Guo, Z and Tan, X and Yuan, H and Zhang, L and Wu, J and Yang, Z and Qu, K and Wan, Y},
title = {Bis-enzyme cascade CRISPR-Cas12a platform for miRNA detection.},
journal = {Talanta},
volume = {252},
number = {},
pages = {123837},
doi = {10.1016/j.talanta.2022.123837},
pmid = {35987123},
issn = {1873-3573},
mesh = {CRISPR-Cas Systems ; *MicroRNAs/genetics ; DNA Cleavage ; DNA, Single-Stranded ; DNA ; *Biosensing Techniques ; },
abstract = {MicroRNA (miRNA) play a vital role in the pathological development of many diseases. It is considered to be the diagnosis and potential biomarkers of prognosis. Herein, we proposed Bis-enzyme cascade Platform by combining T7 RNA polymerase and CRISPR-Cas12a (BPTC) for a miRNA detection. In the proposed BPTC, the RNA to DNA conversion ability of phi29 amplification and trans-cleavage of CRISPR-Cas12a are combined. The target miRNA can be amplified after binding to the recognizer ssDNA, and then transcribed the CRISPR-derived RNA (crRNA) by T7 RNA polymerase. The produced crRNA can thereby be assembled by CRISPR-Cas12a and recognized with its target dsDNA, thus triggered its trans-cleavage towards surrounding fluorescent reporters, labeled with a fluorophore and a corresponding quenching group. Based on the bis-enzyme cascade system, the biosensor shows highly sensitivity and excellent specificity. Moreover, this study provided a novel all-in-one detect strategy for miRNA and may open a new idea for the design of CRISR-Cas-based miRNA biosensing platforms.},
}
@article {pmid35986652,
year = {2022},
author = {Zhou, C and Huang, D and Wang, Z and Shen, P and Wang, P and Xu, Z},
title = {CRISPR Cas12a-based "sweet" biosensor coupled with personal glucose meter readout for the point-of-care testing of Salmonella.},
journal = {Journal of food science},
volume = {87},
number = {9},
pages = {4137-4147},
doi = {10.1111/1750-3841.16287},
pmid = {35986652},
issn = {1750-3841},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; *Glucose/analysis ; Humans ; Point-of-Care Testing ; Salmonella/genetics ; },
abstract = {Salmonella is a pathogen that comes from different animal-originated foods and poses a significant threat to human health. The present detection methods for Salmonella are time-consuming and labor-intensive and requires skilled workers and specialized instruments. In this study, the conservative invA was selected as the target gene, and a quantitative detection method for Salmonella with wide availability and user-friendliness was established based on CRISPR Cas12a and a personal glucose meter (PGM). The indirect signal transformation from the original target DNA to the final glucose signal was achieved through RAA, CRISPR Cas12a reaction, enzymic reaction, and glucose signal reading by a PGM (accu-chek type from Roche). This PGMs-CRISPR assay showed a detection sensitivity of Salmonella as low as 5 colony-forming units (CFU)/reaction in either pure culture or artificially contaminated food samples and exhibited specificity between Salmonella isolates and non-Salmonella isolates. Furthermore, quantitative detection of Salmonella in spiked milk samples was also achieved within the range from 1 to 1 × 10[3] CFU/reaction. Subsequently, the correlation and consistency between the PGMs-CRISPR assay and quantitative polymerase chain reaction (qPCR) in detection of Salmonella in spiked milk samples were achieved. Therefore, a highly sensitive, portable, quantitative, and user-friendly detection method based on CRISPR Cas12a and PGMs was developed in this study for Salmonella detection and identification. PRACTICAL APPLICATION: A sensitive, rapid, user-friendly, and quantitative detection method based on CRISPR Cas12a for Salmonella in food has been developed in this study, which is of great significance to food safety supervision and management.},
}
@article {pmid35986182,
year = {2022},
author = {Ghouneimy, A and Mahfouz, M},
title = {Streamlined detection of SARS-CoV-2 via Cas13.},
journal = {Nature biomedical engineering},
volume = {6},
number = {8},
pages = {925-927},
pmid = {35986182},
issn = {2157-846X},
mesh = {*COVID-19 ; *CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Humans ; SARS-CoV-2 ; },
}
@article {pmid35983948,
year = {2022},
author = {Raza, A and Charagh, S and García-Caparrós, P and Rahman, MA and Ogwugwa, VH and Saeed, F and Jin, W},
title = {Melatonin-mediated temperature stress tolerance in plants.},
journal = {GM crops &amp; food},
volume = {13},
number = {1},
pages = {196-217},
pmid = {35983948},
issn = {2164-5701},
mesh = {Gene Expression Regulation, Plant ; *Melatonin ; Plants, Genetically Modified/genetics ; Stress, Physiological/genetics ; Temperature ; },
abstract = {Global climate changes cause extreme temperatures and a significant reduction in crop production, leading to food insecurity worldwide. Temperature extremes (including both heat and cold stresses) is one of the most limiting factors in plant growth and development and severely affect plant physiology, biochemical, and molecular processes. Biostimulants like melatonin (MET) have a multifunctional role that acts as a "defense molecule" to safeguard plants against the noxious effects of temperature stress. MET treatment improves plant growth and temperature tolerance by improving several defense mechanisms. Current research also suggests that MET interacts with other molecules, like phytohormones and gaseous molecules, which greatly supports plant adaptation to temperature stress. Genetic engineering via overexpression or CRISPR/Cas system of MET biosynthetic genes uplifts the MET levels in transgenic plants and enhances temperature stress tolerance. This review highlights the critical role of MET in plant production and tolerance against temperature stress. We have documented how MET interacts with other molecules to alleviate temperature stress. MET-mediated molecular breeding would be great potential in helping the adverse effects of temperature stress by creating transgenic plants.},
}
@article {pmid35983328,
year = {2022},
author = {Qian, C and Ma, J and Liang, J and Zhang, L and Liang, X},
title = {Comprehensive deciphering prophages in genus Acetobacter on the ecology, genomic features, toxin-antitoxin system, and linkage with CRISPR-Cas system.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {951030},
pmid = {35983328},
issn = {1664-302X},
abstract = {Acetobacter is the predominant microbe in vinegar production, particularly in those natural fermentations that are achieved by complex microbial communities. Co-evolution of prophages with Acetobacter, including integration, release, and dissemination, heavily affects the genome stability and production performance of industrial strains. However, little has been discussed yet about prophages in Acetobacter. Here, prophage prediction analysis using 148 available genomes from 34 Acetobacter species was carried out. In addition, the type II toxin-antitoxin systems (TAs) and CRISPR-Cas systems encoded by prophages or the chromosome were analyzed. Totally, 12,000 prophage fragments were found, of which 350 putatively active prophages were identified in 86.5% of the selected genomes. Most of the active prophages (83.4%) belonged to the order Caudovirales dominated by the families Siphoviridae and Myroviridae prophages (71.4%). Notably, Acetobacter strains survived in complex environments that frequently carried multiple prophages compared with that in restricted habits. Acetobacter prophages showed high genome diversity and horizontal gene transfer across different bacterial species by genomic feature characterization, average nucleotide identity (ANI), and gene structure visualization analyses. About 31.14% of prophages carry type II TAS, suggesting its important role in addiction, bacterial defense, and growth-associated bioprocesses to prophages and hosts. Intriguingly, the genes coding for Cse1, Cse2, Cse3, Cse4, and Cas5e involved in type I-E and Csy4 involved in type I-F CRISPR arrays were firstly found in two prophages. Type II-C CRISPR-Cas system existed only in Acetobacter aceti, while the other Acetobacter species harbored the intact or eroded type I CRISPR-Cas systems. Totally, the results of this study provide fundamental clues for future studies on the role of prophages in the cell physiology and environmental behavior of Acetobacter.},
}
@article {pmid35983086,
year = {2022},
author = {Getahun, YA and Ali, DA and Taye, BW and Alemayehu, YA},
title = {Multidrug-Resistant Microbial Therapy Using Antimicrobial Peptides and the CRISPR/Cas9 System.},
journal = {Veterinary medicine (Auckland, N.Z.)},
volume = {13},
number = {},
pages = {173-190},
pmid = {35983086},
issn = {2230-2034},
abstract = {The emergence and spread of multidrug-resistant microbes become a serious threat to animal and human health globally because of their less responsiveness to conventional antimicrobial therapy. Multidrug-resistant microbial infection poses higher morbidity and mortality rate with significant economic losses. Currently, antimicrobial peptides and the CRISPR/Cas9 system are explored as alternative therapy to circumvent the challenges of multidrug-resistant organisms. Antimicrobial peptides are small molecular weight, cationic peptides extracted from all living organisms. It is a promising drug candidate for the treatment of multidrug-resistant microbes by direct microbial killing or indirectly modulating the innate immune system. The CRISPR/Cas9 system is another novel antimicrobial alternative used to manage multidrug-resistant microbial infection. It is a versatile gene-editing tool that uses engineered single guide RNA for targeted gene recognition and the Cas9 enzyme for the destruction of target nucleic acids. Both the CRISPR/Cas9 system and antimicrobial peptides were used to successfully treat nosocomial infections caused by ESKAPE pathogens, which developed resistance to various antimicrobials. Despite, their valuable roles in multidrug-resistant microbial treatments, both the antimicrobial peptides and the CRISPR/Cas systems have various limitations like toxicity, instability, and incurring high manufacturing costs. Thus, this review paper gives detailed explanations of the roles of the CRISPR/Cas9 system and antimicrobial peptides in circumventing the challenges of multidrug-resistant microbial infections, its limitation and prospects in clinical applications.},
}
@article {pmid35982368,
year = {2022},
author = {Jiang, Y and Li, J and Liu, B and Cao, D and Zong, Y and Chang, Y and Li, Y},
title = {Novel Hina alleles created by genome editing increase grain hardness and reduce grain width in barley.},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {35982368},
issn = {1573-9368},
abstract = {The hordoindolina genes (Hina and Hinb) are believed to play critical roles in barley (Hordeum vulgare L.) grain texture. In this study, we created novel alleles of the Hina gene using CRISPR/Cas9 (Clustered regularly inter spaced short palindromic repeat-associated protein, CRISPR-Cas) genome editing. Mutagenesis of single bases in these novel alleles led to loss of Hina protein function in edited lines. The grain hardness index of hina mutants was 95.5 on average, while that of the wild type was only 53.7, indicating successful conversion of soft barley into hard barley. Observation of cross-sectional grain structure using scanning electron microscopy revealed different adhesion levels between starch granules and protein matrix. Starch granules were loose and separated from the protein matrix in the wild type, but deeply trapped and tightly integrated with the protein matrix in hina02 mutants. In addition, the grain width and thousand-grain weight of the hina02 mutant were significantly lower than those of the wild type.},
}
@article {pmid35981997,
year = {2022},
author = {Liang, WJ and Hu, JZ and Long, JQ and Zhu, AC and Duan, G},
title = {[Evaluation of effect based on different typing methods in Escherichia coli].},
journal = {Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi},
volume = {43},
number = {8},
pages = {1321-1325},
doi = {10.3760/cma.j.cn112338-20220303-00167},
pmid = {35981997},
issn = {0254-6450},
mesh = {*Escherichia coli/genetics ; *Escherichia coli Infections ; Humans ; Multilocus Sequence Typing ; },
abstract = {Objective: To evaluate the typing and clinical application effect based on clustered regularly interspaced short palindromic repeats (CRISPRs), serotype, and Multilocus Sequence Typing (MLST). Methods: The spacers, serotype and sequence type (ST) were obtained with CRISPRsFinder, SeroTypeFinder and MLST. PCR was used to amplify the CRISPRs, and the spacers were used to predict serotype and ST, then comparing with the serotype and ST. Results: We defined the I-E CRISPR/Cas as CT-Ⅰ, I-F CRISPR/Cas as CT-Ⅱ, and only CRISPR3-4 as CT-Ⅲ. We designated each unique arrangement spacer profile as a unique CRISPRs type. A total of 79 CT types, 76 serotypes, and 66 STs were identified. The CRISPRs typing was the most discriminating, with the Simpson index of 0.936, having the highest correlation with serology with the adjusted Rand index of 0.908. The CRISPRs type could divide the same serotype (ST) into two subtypes [O157∶H7(ST11), O104∶H4(ST678), and O26∶H11(ST21)]. The detection rates of CRISPR1, CRISPR2, CRISPR3, CRISPR4, and CRISPR3-4 were 81.1%, 94.5%, 1.4%, 1.4%, and 4.6%, with the accuracy rate of 95.0% and 100.0% according to the spacers to forecast O157∶H7 (ST11) and ST131. Conclusion: Based on the CRISPRs spacer, this method can be used as an essential molecular typing for E.coli, as it presents a good typing and clinical application effect.},
}
@article {pmid35981819,
year = {2022},
author = {Kong, S and Yu, W and Gao, N and Zhai, X and Zhou, YJ},
title = {Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae.},
journal = {FEMS microbiology letters},
volume = {369},
number = {1},
pages = {},
doi = {10.1093/femsle/fnac081},
pmid = {35981819},
issn = {1574-6968},
mesh = {CRISPR-Cas Systems ; Gene Expression ; Metabolic Engineering/methods ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {Construction of efficient microbial cell factories always requires assembling biosynthetic pathways and rewiring cellular metabolism with overexpression of multiple genes. Genomic integration is considered to be helpful for stable gene expression in compared with the episomal plasmids. However, the limited availability of suitable loci hinders the extensive metabolic engineering. We here characterized 30 neutral sites in Saccharomyces cerevisiae genome that did not affect cellular fitness by using expression cassettes of green fluorescent protein (eGFP) and fatty acyl-CoA reductase (MaFAR1) with the aid of efficient CRISPR-Cas9 technique. We found that integration of gene expression cassettes to different genome loci resulted a varied GFP signal and fatty alcohol production, which showed that genomic loci could be used for tuning gene expression. The characterized set of neutral sites should be helpful for extensively metabolic engineering of S. cerevisiae for chemical production and other purposes.},
}
@article {pmid35981511,
year = {2022},
author = {Kellogg, EH and Gootenberg, J and Abudayyeh, O and Wong, ASL and Dahlman, JE and Lapinaite, A and Myhrvold, C and Liu, CC and Hsu, PD and Mali, P and Qi, LS},
title = {What are the current bottlenecks in developing and applying CRISPR technologies?.},
journal = {Cell systems},
volume = {13},
number = {8},
pages = {589-593},
doi = {10.1016/j.cels.2022.07.004},
pmid = {35981511},
issn = {2405-4720},
support = {R01 GM144566/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35980582,
year = {2022},
author = {Avellaneda, J and Schnorrer, F},
title = {Tagging Drosophila Proteins with Genetically Encoded Fluorophores.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2540},
number = {},
pages = {251-268},
pmid = {35980582},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Fluorescent Dyes ; },
abstract = {Proteins are typically not expressed homogeneously in all cells of a complex organism. Within cells, proteins can dynamically change locations, be transported to their destinations, or be degraded upon external signals. Thus, revealing the cellular and subcellular localizations as well as the temporal dynamics of a protein provides important insights into the possible function of the studied protein. Tagging a protein of interest with a genetically encoded fluorophore enables us to follow its expression dynamics in the living organism. Here, we summarize the genetic resources available for tagged Drosophila proteins that assist in studying protein expression and dynamics. We also review the various techniques used in the past and at present to tag a protein of interest with a genetically encoded fluorophore. Comparing the pros and cons of the various techniques guides the reader to judge the suitable applications possible with these tagged proteins in Drosophila.},
}
@article {pmid35980578,
year = {2022},
author = {Han, Y and Lu, X and Li, Y and Qiu, Y and Dong, X and Li, X and Si, X and Liu, Q and Ni, JQ},
title = {CRISPR-Based Transcriptional Activation in Drosophila.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2540},
number = {},
pages = {177-199},
pmid = {35980578},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Drosophila/genetics/metabolism ; Transcription Factors/metabolism ; Transcriptional Activation ; },
abstract = {Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors or by using the CRISPR/dCas9-based transcriptional activation system. To date, the most efficient CRISPR/dCas9-based transcriptional activator is the Synergistic Activation Mediator (SAM) system whereby three different transcriptional activation domains are directly fused to dCas9 and MS2 phage Coat Protein (MCP), respectively, and the system in Drosophila is named flySAM. Here we describe the effective and convenient transcriptional activation system, flySAM, starting from vector construction, microinjection, and transgenic fly selection to the phenotypic analysis.},
}
@article {pmid35980577,
year = {2022},
author = {Port, F and Boutros, M},
title = {Tissue-Specific CRISPR-Cas9 Screening in Drosophila.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2540},
number = {},
pages = {157-176},
pmid = {35980577},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila/genetics ; Genetic Testing/methods ; Genomics/methods ; Mutagenesis ; RNA, Guide/genetics ; },
abstract = {Over the last century research in Drosophila has resulted in many fundamental contributions to our understanding of the biology of multicellular organisms. Many of these breakthroughs have been based on the identification of novel gene functions in large-scale genetic screens. However, conventional forward-genetic screens have been limited by the random nature of mutagenesis and difficulties in mapping causal mutations, while reverse-genetic RNAi screens suffer from incomplete knockdown of gene expression. Recently developed large-scale CRISPR-Cas9 libraries promise to address these limitations by allowing the induction of targeted mutations in genes with spatial and temporal control. Here, we provide a guide for tissue-specific CRISPR screening in Drosophila, including the characterization of Gal4 UAS-Cas9 lines, selection of sgRNA libraries, and various quality control measures. We also discuss confounding factors that can give rise to false-positive and false-negative results in such experiments and suggest strategies on how to detect and avoid them. Conditional CRISPR screening represents an exciting new approach for functional genomics in vivo and is set to further expand our knowledge of the molecular underpinning of development, homeostasis, and disease.},
}
@article {pmid35980576,
year = {2022},
author = {Nyberg, KG and Carthew, RW},
title = {CRISPR-/Cas9-Mediated Precise and Efficient Genome Editing in Drosophila.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2540},
number = {},
pages = {135-156},
pmid = {35980576},
issn = {1940-6029},
support = {F32 GM122349/GM/NIGMS NIH HHS/United States ; R35 GM118144/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Drosophila/genetics ; *Gene Editing/methods ; Recombinational DNA Repair ; },
abstract = {The CRISPR/Cas9 system provides the means to make precise and purposeful modifications to the genome via homology-directed repair (HDR). In Drosophila, a wide variety of tools provide flexibility to achieve these ends. Here, we detail a method to generate precise genome edits via HDR that is efficient and broadly applicable to any Drosophila stock or species. sgRNAs are first tested for their cleavage efficiency by injecting embryos with Cas9/sgRNA ribonucleoproteins using commercially available Cas9 protein. Using an empirically validated sgRNA, HDR is performed using a donor repair plasmid that carries two transformation markers. A fluorescent eye marker that can be seamlessly removed using PiggyBac transposase marks integration of the repair sequence. A counter-selection marker that produces small rough eyes via RNAi against eyes absent is used to screen against imprecise HDR events. Altogether, the enhancements implemented in this method expand the ease and scope of achieving precise CRISPR/Cas9 genome edits in Drosophila.},
}
@article {pmid35980575,
year = {2022},
author = {Bosch, JA and Perrimon, N},
title = {Prime Editing for Precise Genome Engineering in Drosophila.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2540},
number = {},
pages = {113-134},
pmid = {35980575},
issn = {1940-6029},
support = {T32 GM007748/GM/NIGMS NIH HHS/United States ; P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Drosophila/genetics ; Gene Editing/methods ; Genome, Insect ; Mammals/genetics ; },
abstract = {Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months.},
}
@article {pmid35980530,
year = {2022},
author = {Yan, X and Yang, J and Xiao, L and Gao, J and Xu, G and Wang, L and Qi, Y and Song, G},
title = {Construction of Fzd6[Q152E ]mice through CRISPR/Cas9 technology and their reproduction and identification.},
journal = {Molecular biology reports},
volume = {49},
number = {10},
pages = {9575-9584},
pmid = {35980530},
issn = {1573-4978},
mesh = {Animals ; *Brain-Derived Neurotrophic Factor/genetics ; *CRISPR-Cas Systems/genetics ; Frizzled Receptors/genetics ; Glycogen Synthase Kinase 3 beta/genetics ; Humans ; Mice ; Norepinephrine ; RNA, Messenger ; Rats ; Reproduction ; Serotonin ; Technology ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system is widely used for genome editing in human, rat and mouse cells. In this study, we established Fzd6 mutant mice using CRISPR/Cas9 technology, and obtained Fzd6 homozygous mutant (Fzd6[Q152E]) mice through breeding. Fzd6 plays a role in depression, but there are few related reports. We used this model to investigate the mechanism of Fzd6 involved in depression, and build a solid foundation for subsequent in-depth studies.<br><br>METHODS AND RESULTS: The target of Fzd6 mutation was obtained by CRISPR/Cas9 technology and hippocampal tissue was collected for Nissl staining and histological analysis. Blood was collected for enzyme linked immunosorbent assay (ELISA); The gene expression of Fzd6 and the related genes expression in wnt pathway was quantified by quantitative real-time PCR (qRT-PCR), and then expression of Fzd6 and proteins in the Wnt pathway were identified by western blotting. ELISA results showed that the expression levels of brain derived neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT), and Noradrenaline (NE) in serum were significantly decreased in Fzd6[Q152E] mice, whereas the mRNA expression of Lrp5, Lrp6, and Dkk2 is increased. The western blotting revealed that the expression of Fzd6 and Lrp6 is decreased, although the expression of Dkk2 and Gsk-3&#x3b2; increased.<br><br>CONCLUSION: Our study successfully established homozygous Fzd6 mutant mice model. The relationship between Fzd6-Wnt and depression was preliminarily clarified, which provides an ideal animal model for subsequent research on diseases induced by the Fzd6 mutation.},
}
@article {pmid35979687,
year = {2022},
author = {Raffan, S and Kaur, N and Halford, NG},
title = {Epigenetic switch reveals CRISPR/Cas9 response to cytosine methylation in plants.},
journal = {The New phytologist},
volume = {235},
number = {6},
pages = {2146-2148},
pmid = {35979687},
issn = {1469-8137},
support = {BB/P016855/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/W007134/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T017007/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cytosine ; DNA Methylation/genetics ; *Gene Editing ; Plants/genetics ; },
abstract = {This article is a Commentary on Přibylová et al. (2022) 235: 2285–2299.},
}
@article {pmid35978051,
year = {2022},
author = {Palazzo, A and Piccolo, I and Minervini, CF and Purgato, S and Capozzi, O and D'Addabbo, P and Cumbo, C and Albano, F and Rocchi, M and Catacchio, CR},
title = {Genome characterization and CRISPR-Cas9 editing of a human neocentromere.},
journal = {Chromosoma},
volume = {131},
number = {4},
pages = {239-251},
pmid = {35978051},
issn = {1432-0886},
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; *Centromere/genetics ; Kinetochores ; Chromosome Segregation ; Chromosome Breakage ; Mammals ; },
abstract = {The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.},
}
@article {pmid35977955,
year = {2022},
author = {Spangler, JR and Leski, TA and Schultzhaus, Z and Wang, Z and Stenger, DA},
title = {Large scale screening of CRISPR guide RNAs using an optimized high throughput robotics system.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {13953},
pmid = {35977955},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; RNA/genetics ; RNA Cleavage ; *RNA, Guide/metabolism ; },
abstract = {All CRISPR/CAS systems utilize CRISPR guide RNAs (crRNAs), the design of which depend on the type of CAS protein, genetic target and the environment/matrix. While machine learning approaches have recently been developed to optimize some crRNA designs, candidate crRNAs must still be screened for efficacy under relevant conditions. Here, we demonstrate a high-throughput method to screen hundreds of candidate crRNAs for activation of Cas13a collateral RNA cleavage. Entire regions of a model gene transcript (Y. pestis lcrV gene) were tiled to produce overlapping crRNA sets. We tested for possible effects that included crRNA/target sequence, size and secondary structures, and the commercial source of DNA oligomers used to generate crRNAs. Detection of a 981 nt target RNA was initially successful with 271 out of 296 tested guide RNAs, and that was improved to 287 out of 296 (97%) after protocol optimizations. For this specific example, we determined that crRNA efficacy did not strongly depend on the target region or crRNA physical properties, but was dependent on the source of DNA oligomers used for RNA preparation. Our high-throughput methods for screening crRNAs has general applicability to the optimization of Cas12 and Cas13 guide RNA designs.},
}
@article {pmid35977479,
year = {2022},
author = {Kelley, CP and Haerle, MC and Wang, ET},
title = {Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells.},
journal = {Cell reports},
volume = {40},
number = {7},
pages = {111226},
doi = {10.1016/j.celrep.2022.111226},
pmid = {35977479},
issn = {2211-1247},
support = {P50 NS048843/NS/NINDS NIH HHS/United States ; R01 AG058636/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Homeostasis ; Humans ; Mammals/genetics ; *Myotonic Dystrophy/genetics ; RNA/genetics ; RNA, Guide/genetics ; Ribonucleases/genetics ; },
abstract = {CRISPR-Cas13 RNA endonucleases show promise for programmable RNA knockdown. However, sequence-specific binding of Cas13 unleashes non-specific bystander RNA cleavage, or collateral activity, raising concerns for experiments and therapeutic applications. Although robust in cell-free and bacterial environments, collateral activity in mammalian cells remains disputed. We investigate Cas13d collateral activity in a therapeutic context for myotonic dystrophy type 1, caused by a transcribed CTG repeat expansion. We find that, when targeting CUGn RNA in mammalian cells, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis. Collateral effects also occur when targeting abundant endogenous transcripts. To minimize collateral activity for repeat-targeting approaches, we introduce GENO, an adeno-associated virus-compatible strategy that leverages guide RNA processing to control Cas13d expression. We argue that thorough assessment of collateral activity is necessary when applying Cas13 in mammalian cells and that GENO illustrates advantages of compact regulatory systems for Cas-based gene therapies.},
}
@article {pmid35976402,
year = {2022},
author = {Fu, K and Song, W and Chen, C and Mou, C and Huang, Y and Zhang, F and Hao, Q and Wang, P and Ma, T and Chen, Y and Zhu, Z and Zhang, M and Tong, Q and Liu, X and Jiang, L and Wan, J},
title = {Improving pre-harvest sprouting resistance in rice by editing OsABA8ox using CRISPR/Cas9.},
journal = {Plant cell reports},
volume = {41},
number = {10},
pages = {2107-2110},
pmid = {35976402},
issn = {1432-203X},
mesh = {Abscisic Acid/metabolism/pharmacology ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Germination/genetics ; Mixed Function Oxygenases/genetics ; *Oryza/genetics/metabolism ; Plant Dormancy/genetics ; Plant Growth Regulators/metabolism ; Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; },
abstract = {Knock out OsABA8ox helps improve pre-harvest spouting resistance and do not affect rice yield. Pre-harvest sprouting(PHS) is a phenomenon that the seeds of crops germinate preharvest, which reduces the yield and quality of rice. Abscisic acid(ABA) is one of the phytohormones that promotes seed dormancy. ABA8' hydroxylase is the main enzyme that can catabolism ABA in plant. There are three genes that encode ABA8' hydroxylase in rice, named OsABA8ox1, OsABA8ox2 and OsABA8ox3. In this study, we use CRISPR/Cas9 gene editing technology to target these three genes in Ningjing6 and find that the knockout transgenic lines are all significantly strengthen in seed dormancy and have no effect on the yield. By a series of quantitative experiments, we consider that after knock out OsABA8ox, the high endogenous ABA level will influence the ABA signal which suppress the substantial and energy metabolism in the seeds, and finally led to higher dormancy.},
}
@article {pmid35976141,
year = {2022},
author = {Sprink, T and Wilhelm, R and Hartung, F},
title = {Genome editing around the globe: An update on policies and perceptions.},
journal = {Plant physiology},
volume = {190},
number = {3},
pages = {1579-1587},
pmid = {35976141},
issn = {1532-2548},
mesh = {*Gene Editing ; *Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Policy ; },
abstract = {A decade ago, the CRISPR/Cas system has been adapted for genome editing. Since then, hundreds of organisms have been altered using genome editing and discussions were raised on the regulatory status of genome edited organisms esp. crops. To date, many countries have made decisions on the regulatory status of products of genome editing, by exempting some kinds of edits from the classical GMO regulation. However, the guidance differs between countries even in the same region. Several countries are still debating the issue or are in the progress of updating guidance and regulatory systems to cover products of genome editing. The current global situation of different regulatory systems is putting a harmonized framework on genome-edited crops in the far future. In this update, we summarize the current developments in the field of regulation concerning edited crops and present a short insight into perception of genome editing in the society.},
}
@article {pmid35975201,
year = {2022},
author = {Shams, F and Bayat, H and Mohammadian, O and Mahboudi, S and Vahidnezhad, H and Soosanabadi, M and Rahimpour, A},
title = {Advance trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified CRISPR-Cas9 systems.},
journal = {BioImpacts : BI},
volume = {12},
number = {4},
pages = {371-391},
pmid = {35975201},
issn = {2228-5652},
abstract = {Introduction: Clustered regularly interspaced short palindromic repeat and its associated protein (CRISPR-Cas)-based technologies generate targeted modifications in host genome by inducing site-specific double-strand breaks (DSBs) that can serve as a substrate for homology-directed repair (HDR) in both in vitro and in vivo models. HDR pathway could enhance incorporation of exogenous DNA templates into the CRISPR-Cas9-mediated DSB site. Owing to low rate of HDR pathway, the efficiency of accurate genome editing is diminished. Enhancing the efficiency of HDR can provide fast, easy, and accurate technologies based on CRISPR-Cas9 technologies. Methods: The current study presents an overview of attempts conducted on the precise genome editing strategies based on small molecules and modified CRISPR-Cas9 systems. Results: In order to increase HDR rate in targeted cells, several logical strategies have been introduced such as generating CRISPR effector chimeric proteins, anti-CRISPR proteins, modified Cas9 with donor template, and using validated synthetic or natural small molecules for either inhibiting non-homologous end joining (NHEJ), stimulating HDR, or synchronizing cell cycle. Recently, high-throughput screening methods have been applied for identification of small molecules which along with the CRISPR system can regulate precise genome editing through HDR. Conclusion: The stimulation of HDR components or inhibiting NHEJ can increase the accuracy of CRISPR-Cas-mediated engineering systems. Generating chimeric programmable endonucleases provide this opportunity to direct DNA template close proximity of CRISPR-Cas-mediated DSB. Small molecules and their derivatives can also proficiently block or activate certain DNA repair pathways and bring up novel perspectives for increasing HDR efficiency, especially in human cells. Further, high throughput screening of small molecule libraries could result in more discoveries of promising chemicals that improve HDR efficiency and CRISPR-Cas9 systems.},
}
@article {pmid35974394,
year = {2022},
author = {Jiang, J and Chen, Y and Zhang, L and Jin, Q and Wang, L and Xu, S and Chen, K and Li, L and Zeng, T and Fan, X and Liu, T and Li, J and Wang, J and Han, C and Gao, F and Yang, Y and Wang, Y},
title = {i-CRISPR: a personalized cancer therapy strategy through cutting cancer-specific mutations.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {164},
pmid = {35974394},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; Gene Editing ; Humans ; Mutation ; *Neoplasms/genetics/therapy ; },
abstract = {Developing a strategy to specifically kill cancer cells without inducing obvious damage to normal cells may be of great clinical significance for cancer treatment. In the present study, we developed a new precise personalized strategy named "i-CRISPR" for cancer treatment through adding DNA damage repair inhibitors(i) and inducing cancer cell-specific DNA double strand breaks by CRISPR. Through in vitro and in vivo experiments, we confirmed the efficacy of this strategy in multiple cancer models and revealed the mechanism of cell death. Our strategy might provide a novel concept for precise cancer therapy.},
}
@article {pmid35973998,
year = {2022},
author = {Yedier-Bayram, O and Gokbayrak, B and Kayabolen, A and Aksu, AC and Cavga, AD and Cingöz, A and Kala, EY and Karabiyik, G and Günsay, R and Esin, B and Morova, T and Uyulur, F and Syed, H and Philpott, M and Cribbs, AP and Kung, SHY and Lack, NA and Onder, TT and Bagci-Onder, T},
title = {EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities.},
journal = {Cell death &amp; disease},
volume = {13},
number = {8},
pages = {710},
pmid = {35973998},
issn = {2041-4889},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromatin ; Early Detection of Cancer ; Humans ; Male ; *Triple Negative Breast Neoplasms/genetics ; },
abstract = {Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.},
}
@article {pmid35973902,
year = {2022},
author = {Tsee Dawson, A and Tonkin, CJ},
title = {A CRISPR upgrade unlocks Toxoplasma gene function.},
journal = {Trends in parasitology},
volume = {38},
number = {10},
pages = {826-828},
doi = {10.1016/j.pt.2022.07.007},
pmid = {35973902},
issn = {1471-5007},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Testing ; *Toxoplasma/genetics ; },
abstract = {Forward genetic screens are invaluable in describing gene function. CRISPR has reinvigorated phenotypic screens in Toxoplasma - a model apicomplexan parasite. Two recent papers by Smith et al. and Li et al. take the next big leap in performing forward genetic screens in Toxoplasma by combining conditional gene regulation with CRISPR.},
}
@article {pmid35972825,
year = {2022},
author = {Jones, IR and Ren, X and Shen, Y},
title = {High-throughput CRISPRi and CRISPRa technologies in 3D genome regulation for neuropsychiatric diseases.},
journal = {Human molecular genetics},
volume = {31},
number = {R1},
pages = {R47-R53},
pmid = {35972825},
issn = {1460-2083},
support = {R01 AG057497/AG/NIA NIH HHS/United States ; R01 EY027789/EY/NEI NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; U01 DA052713/DA/NIDA NIH HHS/United States ; T32 GM007175/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genomics ; Alleles ; DNA ; },
abstract = {Advances in genomics have led to the identification of many risk loci with hundreds of genes and thousands of DNA variants associated with neuropsychiatric disorders. A significant barrier to understanding the genetic underpinnings of complex diseases is the lack of functional characterization of risk genes and variants in biological systems relevant to human health and connecting disease-associated variants to pathological phenotypes. Characterizing gene and DNA variant functions requires genetic perturbations followed by molecular and cellular assays of neurobiological phenotypes. However, generating null or mutant alleles is low throughput, making it impossible to characterize disease-associated variants in large quantities efficiently. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens can be leveraged to dissect the biological consequences of the tested genes and variants in their native context. Nevertheless, testing non-coding variants associated with complex diseases remains non-trivial. In this review, we first discuss the current challenges of interpreting the function of the non-coding genome and approaches to prioritizing disease-associated variants in the context of the 3D epigenome. Second, we provide a brief overview of high-throughput CRISPRi and CRISPRa screening strategies applicable for characterizing non-coding sequences in appropriate biological systems. Lastly, we discuss the promising prospects of using CRISPR-based technologies to dissect DNA sequences associated with neuropsychiatric diseases.},
}
@article {pmid35972707,
year = {2022},
author = {Evazi Bakhshi, S and Mohammadi Roushandeh, A and Habibi Roudkenar, M and Shekarchi, S and Bahadori, MH},
title = {CRISPR/Cas9-mediated knockout of HO-1 decreased the proliferation and migration of T47D cells and increased cisplatin-induced apoptosis: an in vitro study.},
journal = {Medical oncology (Northwood, London, England)},
volume = {39},
number = {11},
pages = {175},
pmid = {35972707},
issn = {1559-131X},
mesh = {Apoptosis ; *Breast Neoplasms/drug therapy/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; *Cisplatin/pharmacology/therapeutic use ; Female ; Heme Oxygenase-1/genetics/metabolism ; Humans ; },
abstract = {Breast cancer is the most common type of neoplasm and the second cause of cancer-related death in women. Despite the development of novel therapeutic strategies and improved the clinical outcomes, the mortality rate for breast cancer is still high. Therefore, development of a new modality, particularly based on knocking out key genes, is under focus of investigation. Heme oxygenase-1 (HO-1) deregulation has been associated with various neoplasms-related behaviors of many types of tumor cells including breast cancer. In the current study, in order to evaluate the role of the HO-1 gene in breast cancer, we utilized the CRISPR/Cas9 technology to knock out HO-1 gene in T47D breast cancer cell line and studied its potential therapeutic effects in vitro. The cell proliferation and their sensitivity to Cisplatin were determined by CCK-8 kit. In addition, the apoptosis and the migratory potential of the cells were evaluated using Hoechst staining, and Transwell/Scratch methods, respectively. Our findings revealed that HO-1 suppression significantly reduced the proliferation ability of T47D cells (P < 0.001). Moreover, sensitivity to Cisplatin-induced toxicity increased significantly in KO-T47D cells compared to the control T47D cells. Furthermore, our findings indicated that Cisplatin-induced apoptosis increased in the KO-T47D cells. Moreover, the migratory capability of KO-T47D cells was abolished significantly (P < 0.001) as determined by Transwell migration assay. In a nutshell, our findings strongly suggest that HO-1 involved in breast cancer progression and metastasis and chemotherapy resistance. However, further comprehensive studies are required to clarify the precise role of the HO-1 gene on breast cancer cells.},
}
@article {pmid35972568,
year = {2022},
author = {Dai, Z and Zhang, Z and Zhu, L and Zhu, Z and Jiang, L},
title = {Complete Genome Sequencing Analysis of Deinococcus wulumuqiensis R12, an Extremely Radiation-Resistant Strain.},
journal = {Current microbiology},
volume = {79},
number = {10},
pages = {292},
pmid = {35972568},
issn = {1432-0991},
mesh = {*Deinococcus/genetics ; *Gene Editing ; Genome, Bacterial/genetics ; Plasmids ; Whole Genome Sequencing ; },
abstract = {Genome sequencing was performed by the PacBio RS II platform and Illumina HiSeq 4000 platform to discover the metabolic profile of the Deinococcus wulumuqiensis R12, which was isolated from radiation-contaminated soils in Xinjiang Uygur Autonomous Region of northwest China. The genome of 3.5 Mbp comprises one circular chromosome and four circular plasmids with 3679 genes and a GC content of 66.97%. A total of 41 new transcriptional factors were identified using the DeepTFactor tool. Genomic analysis revealed the presence of genes for homologous recombination repair, which suggested high recombination efficiency in R12. Three Type I and one Type II RM systems, two CRISPR arrays, and one Cas-Type IC protein were found, allowing the development of endogenous CRISPR-Cas gene-editing tools. Additionally, we found that R12 has a broad spectrum of substrate utilization, which was validated by physiological experiments. Genes involved in the carotenoid biosynthesis pathway and the antioxidative system were also identified. Overall, the comprehensive description of the genome of R12 will facilitate the additional exploitation of this strain as a versatile cell factory for biotechnological applications.},
}
@article {pmid35972367,
year = {2022},
author = {Becklin, KL and Draper, GM and Madden, RA and Kluesner, MG and Koga, T and Huang, M and Weiss, WA and Spector, LG and Largaespada, DA and Moriarity, BS and Webber, BR},
title = {Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {517-535},
pmid = {35972367},
issn = {2573-1602},
support = {R21 CA237789/CA/NCI NIH HHS/United States ; R21 AI163731/AI/NIAID NIH HHS/United States ; P01 CA254849/CA/NCI NIH HHS/United States ; P50 CA136393/CA/NCI NIH HHS/United States ; R01 AI161017/AI/NIAID NIH HHS/United States ; R01 AI146009/AI/NIAID NIH HHS/United States ; R01 NS115438/NS/NINDS NIH HHS/United States ; R00 CA197484/CA/NCI NIH HHS/United States ; R01 CA255369/CA/NCI NIH HHS/United States ; R01 NS106155/NS/NINDS NIH HHS/United States ; R01 CA221969/CA/NCI NIH HHS/United States ; P01 CA217959/CA/NCI NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; P50 CA097257/CA/NCI NIH HHS/United States ; U01 CA217864/CA/NCI NIH HHS/United States ; U54 CA243125/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; *Neoplasms/genetics/therapy ; },
abstract = {Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into "bottom-up" cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.},
}
@article {pmid35972366,
year = {2022},
author = {Barrangou, R},
title = {Navigating Viral Space with CRISPR Technologies.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {487},
doi = {10.1089/crispr.2022.29153.editorial},
pmid = {35972366},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35972365,
year = {2022},
author = {Maire, A and Bikard, D},
title = {What if Bacteria Could Tell Us What They Have Seen?.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {488-489},
doi = {10.1089/crispr.2022.29151.bik},
pmid = {35972365},
issn = {2573-1602},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid35972327,
year = {2022},
author = {Philippidis, A},
title = {Precision BioSciences, Novartis Launch Up-to-$1.5B In Vivo Genome Editing Collaboration.},
journal = {Human gene therapy},
volume = {33},
number = {15-16},
pages = {777-779},
doi = {10.1089/hum.2022.29214.bfs},
pmid = {35972327},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid35972246,
year = {2022},
author = {Qin, S and Liu, Y and Chen, Y and Hu, J and Xiao, W and Tang, X and Li, G and Lin, P and Pu, Q and Wu, Q and Zhou, C and Wang, B and Gao, P and Wang, Z and Yan, A and Nadeem, K and Xia, Z and Wu, M},
title = {Engineered Bacteriophages Containing Anti-CRISPR Suppress Infection of Antibiotic-Resistant P. aeruginosa.},
journal = {Microbiology spectrum},
volume = {10},
number = {5},
pages = {e0160222},
pmid = {35972246},
issn = {2165-0497},
mesh = {Humans ; *Bacteriophages/genetics ; Pseudomonas aeruginosa/genetics ; Anti-Bacterial Agents/pharmacology/therapeutic use ; *Phage Therapy ; Drug Resistance, Multiple, Bacterial ; },
abstract = {The therapeutic use of bacteriophages (phages) provides great promise for treating multidrug-resistant (MDR) bacterial infections. However, an incomplete understanding of the interactions between phages and bacteria has negatively impacted the application of phage therapy. Here, we explored engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) by introducing Type I anti-CRISPR (AcrIF1, AcrIF2, and AcrIF3) genes into the P. aeruginosa bacteriophage DMS3/DMS3m to render the potential for blocking P. aeruginosa replication and infection. In order to achieve effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo, the inhibitory concentration for EATPs was 1 × 10[8] PFU/mL with a multiplicity of infection value of 0.2. In addition, the EATPs significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection. Collectively, these findings provide evidence that engineered phages may be an alternative, viable approach by which to treat patients with an intractable bacterial infection, especially an infection by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy. IMPORTANCE Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic Gram-negative bacterium that causes severe infection in immune-weakened individuals, especially patients with cystic fibrosis, burn wounds, cancer, or chronic obstructive pulmonary disease (COPD). Treating P. aeruginosa infection with conventional antibiotics is difficult due to its intrinsic multidrug resistance. Engineered bacteriophage therapeutics, acting as highly viable alternative treatments of multidrug-resistant (MDR) bacterial infections, have great potential to break through the evolutionary constraints of bacteriophages to create next-generation antimicrobials. Here, we found that engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) display effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo. EATPs also significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection, which may provide novel insight toward developing bacteriophages to treat patients with intractable bacterial infections, especially infections by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy.},
}
@article {pmid35970947,
year = {2022},
author = {Imai, Y and Tanave, A and Matsuyama, M and Koide, T},
title = {Efficient genome editing in wild strains of mice using the i-GONAD method.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {13821},
pmid = {35970947},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Electroporation/methods ; *Gene Editing/methods ; Genetic Engineering/methods ; Gonads ; Mice ; },
abstract = {Wild mouse strains have been used for many research studies, because of the high level of inter-strain genetic and phenotypic variations in them, in addition to the characteristic phenotype maintained from wild mice. However, since application of the current genetic engineering method on wild strains is not easy, there are limited studies that have attempted to apply gene modification techniques in wild strains. Recently, i-GONAD, a new method for genome editing that does not involve any ex vivo manipulation of unfertilized or fertilized eggs has been reported. We applied i-GONAD method for genome editing on a series of wild strains and showed that genome editing is efficiently possible using this method. We successfully made genetically engineered mice in seven out of the nine wild strains. Moreover, we believe that it is still possible to apply milder conditions and improve the efficiencies for the remaining two strains. These results will open avenues for studying the genetic basis of various phenotypes that are characteristic to wild strains. Furthermore, applying i-GONAD will be also useful for other mouse resources in which genetic manipulation is difficult using the method of microinjection into fertilized eggs.},
}
@article {pmid35969224,
year = {2022},
author = {Cengic, I and Cañadas, IC and Minton, NP and Hudson, EP},
title = {Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803.},
journal = {ACS synthetic biology},
volume = {11},
number = {9},
pages = {3100-3113},
pmid = {35969224},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; DNA-Binding Proteins/genetics ; Endoribonucleases/genetics ; Escherichia coli/genetics ; *Escherichia coli Proteins/genetics ; Gene Editing ; Nickel ; *Riboswitch ; *Synechocystis/genetics ; Theophylline ; },
abstract = {Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here, we describe a riboswitch-inducible CRISPR/Cas9 system, contained on a single replicative vector, for the model cyanobacterium Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector intoSynechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits performed better, reaching, e.g., 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system mediated multiplexed editing of up to three targets in parallel inSynechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction. Lastly, a vector curing system based on the nickel-inducible expression of the toxic mazF (from Escherichia coli) was added to the CRISPR/Cas9 vector. This inducible system allowed for curing of the vector in 25-75% of screened colonies, enabling edited mutants to become markerless.},
}
@article {pmid35968922,
year = {2022},
author = {Raza, A and Tabassum, J and Fakhar, AZ and Sharif, R and Chen, H and Zhang, C and Ju, L and Fotopoulos, V and Siddique, KHM and Singh, RK and Zhuang, W and Varshney, RK},
title = {Smart reprograming of plants against salinity stress using modern biotechnological tools.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-28},
doi = {10.1080/07388551.2022.2093695},
pmid = {35968922},
issn = {1549-7801},
abstract = {Climate change gives rise to numerous environmental stresses, including soil salinity. Salinity/salt stress is the second biggest abiotic factor affecting agricultural productivity worldwide by damaging numerous physiological, biochemical, and molecular processes. In particular, salinity affects plant growth, development, and productivity. Salinity responses include modulation of ion homeostasis, antioxidant defense system induction, and biosynthesis of numerous phytohormones and osmoprotectants to protect plants from osmotic stress by decreasing ion toxicity and augmented reactive oxygen species scavenging. As most crop plants are sensitive to salinity, improving salt tolerance is crucial in sustaining global agricultural productivity. In response to salinity, plants trigger stress-related genes, proteins, and the accumulation of metabolites to cope with the adverse consequence of salinity. Therefore, this review presents an overview of salinity stress in crop plants. We highlight advances in modern biotechnological tools, such as omics (genomics, transcriptomics, proteomics, and metabolomics) approaches and different genome editing tools (ZFN, TALEN, and CRISPR/Cas system) for improving salinity tolerance in plants and accomplish the goal of "zero hunger," a worldwide sustainable development goal proposed by the FAO.},
}
@article {pmid35968912,
year = {2022},
author = {Thapliyal, G and Bhandari, MS and Vemanna, RS and Pandey, S and Meena, RK and Barthwal, S},
title = {Engineering traits through CRISPR/cas genome editing in woody species to improve forest diversity and yield.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/07388551.2022.2092714},
pmid = {35968912},
issn = {1549-7801},
abstract = {Dangers confronting forest ecosystems are many and the strength of these biological systems is deteriorating, thus substantially affecting tree physiology, phenology, and growth. The establishment of genetically engineered trees into degraded woodlands, which would be adaptive to changing climate, could help in subsiding ecological threats and bring new prospects. This should not be resisted due to the apprehension of transgene dispersal in forests. Consequently, it is important to have a deep insight into the genetic structure and phenotypic limits of the reproductive capability of tree stands/population(s) to endure tolerance and survival. Importantly, for a better understanding of genes and their functional mechanisms, gene editing (GeEd) technology is an excellent molecular tool to unravel adaptation progressions. Therefore, GeEd could be harnessed for resolving the allelic interactions for the creation of gene diversity, and transgene dispersal may be alleviated among the population or species in different bioclimatic zones around the globe. This review highlights the potential of the CRISPR/Cas tools in genomic, transcriptomic, and epigenomic-based assorted and programmable alterations of genes in trees that might be able to fix the trait-specific gene function. Also, we have discussed the application of diverse forms of GeEd to genetically improve several traits, such as wood density, phytochemical constituents, biotic and abiotic stress tolerance, and photosynthetic efficiency in trees. We believe that the technology encourages fundamental research in the forestry sector besides addressing key aspects, which might fasten tree breeding and germplasm improvement programs worldwide.},
}
@article {pmid35968255,
year = {2022},
author = {Kim, S and Lee, JH},
title = {Current Advances in Paper-Based Biosensor Technologies for Rapid COVID-19 Diagnosis.},
journal = {Biochip journal},
volume = {},
number = {},
pages = {1-21},
pmid = {35968255},
issn = {1976-0280},
abstract = {The global coronavirus disease 2019 (COVID-19) pandemic has had significant economic and social impacts on billions of people worldwide since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in November 2019. Although polymerase chain reaction (PCR)-based technology serves as a robust test to detect SARS-CoV-2 in patients with COVID-19, there is a high demand for cost-effective, rapid, comfortable, and accurate point-of-care diagnostic tests in medical facilities. This review introduces the SARS-CoV-2 viral structure and diagnostic biomarkers derived from viral components. A comprehensive introduction of a paper-based diagnostic platform, including detection mechanisms for various target biomarkers and a COVID-19 commercial kit is presented. Intrinsic limitations related to the poor performance of currently developed paper-based devices and unresolved issues are discussed. Furthermore, we provide insight into novel paper-based diagnostic platforms integrated with advanced technologies such as nanotechnology, aptamers, surface-enhanced Raman spectroscopy (SERS), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Finally, we discuss the prospects for the development of highly sensitive, accurate, cost-effective, and easy-to-use point-of-care COVID-19 diagnostic methods.},
}
@article {pmid35966677,
year = {2022},
author = {Prakash, A and Kumar, M},
title = {Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {960559},
pmid = {35966677},
issn = {1664-302X},
abstract = {In the genome of various Leptospira interrogans serovars, the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas-operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.},
}
@article {pmid35966591,
year = {2022},
author = {Tyagi, A and Kaushal, K and Chandrasekaran, AP and Sarodaya, N and Das, S and Park, CH and Hong, SH and Kim, KS and Ramakrishna, S},
title = {CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.},
journal = {Theranostics},
volume = {12},
number = {13},
pages = {5949-5970},
pmid = {35966591},
issn = {1838-7640},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Cisplatin/pharmacology/therapeutic use ; Early Detection of Cancer ; Humans ; *Lung Neoplasms ; Mice ; Microtubule-Associated Proteins/metabolism ; Microtubules/metabolism ; Protein Serine-Threonine Kinases/genetics ; Ubiquitin-Specific Proteases/genetics/metabolism ; },
abstract = {Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown. Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48- and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1-mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice. Results: The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model. Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.},
}
@article {pmid35966585,
year = {2022},
author = {Yin, B and Zhang, Q and Xia, X and Li, C and Ho, WKH and Yan, J and Huang, Y and Wu, H and Wang, P and Yi, C and Hao, J and Wang, J and Chen, H and Wong, SHD and Yang, M},
title = {A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection.},
journal = {Theranostics},
volume = {12},
number = {13},
pages = {5914-5930},
pmid = {35966585},
issn = {1838-7640},
mesh = {CRISPR-Cas Systems/genetics ; DNA/chemistry ; Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Nucleic Acids ; RNA ; },
abstract = {Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising "on-off" nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity. Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.},
}
@article {pmid35965331,
year = {2022},
author = {Ni, L and Li, Y and Wu, K and Deng, F and Wang, H and Ning, YJ},
title = {Antitumor efficacy of CRISPR/Cas9-engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma.},
journal = {Journal of medical virology},
volume = {94},
number = {12},
pages = {6000-6015},
doi = {10.1002/jmv.28069},
pmid = {35965331},
issn = {1096-9071},
mesh = {*Adenocarcinoma of Lung/genetics/therapy ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; *Herpesvirus 1, Human/genetics ; Heterografts ; Humans ; *Lung Neoplasms/genetics/therapy ; Mice ; *Ribonucleotide Reductases/genetics ; Tumor Microenvironment ; },
abstract = {Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6-mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR] domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV-1 causing severe illness or death in the mouse model, the ICP6-mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti-adenocarcinoma potential of the CRISPR/Cas9-engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV-based therapeutics against lung adenocarcinoma or other types of tumors.},
}
@article {pmid35965014,
year = {2022},
author = {Hu, C and Ke, A},
title = {Reconstitution and biochemical characterization of the RNA-guided helicase-nuclease protein Cas3 from type I-A CRISPR-Cas system.},
journal = {Methods in enzymology},
volume = {673},
number = {},
pages = {405-424},
doi = {10.1016/bs.mie.2022.03.059},
pmid = {35965014},
issn = {1557-7988},
mesh = {*CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; DNA Helicases/chemistry ; Endonucleases/genetics/metabolism ; Humans ; RNA ; RNA Helicases ; },
abstract = {Type I is the most prevalent CRISPR system found in nature. It can be further defined into six subtypes, from I-A to I-G. Among them, the Type I-A CRISPR-Cas systems are almost exclusively found in hyperthermophilic archaeal organisms. The system achieves RNA-guided DNA degradation through the concerted action of a CRISPR RNA containing complex Cascade and a helicase-nuclease fusion enzyme Cas3. Here, we summarize assays to characterize the biochemical behavior of Cas3. A steep temperature-dependency was found for the helicase component of Cas3HEL, but not the nuclease component HD. This finding enabled us to establish the correct experimental condition to carry out I-A CRISPR-Cas based genome editing in human cells with extremely high efficiency.},
}
@article {pmid35964771,
year = {2022},
author = {Gatticchi, L and Grottelli, S and Ambrosini, G and Pampalone, G and Gualtieri, O and Dando, I and Bellezza, I and Cellini, B},
title = {CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1.},
journal = {Biochimie},
volume = {202},
number = {},
pages = {110-122},
doi = {10.1016/j.biochi.2022.08.005},
pmid = {35964771},
issn = {1638-6183},
mesh = {Humans ; Hep G2 Cells ; *CRISPR-Cas Systems ; *Pyridoxine/pharmacology ; Transaminases/genetics ; Oxalates ; Pyridoxal Phosphate ; },
abstract = {AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5'-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates. Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.},
}
@article {pmid35962717,
year = {2022},
author = {Duan, Z and Yang, X and Ji, X and Chen, Y and Niu, X and Guo, A and Zhu, JK and Li, F and Lang, Z and Zhao, H},
title = {Cas12a-based on-site, rapid detection of genetically modified crops.},
journal = {Journal of integrative plant biology},
volume = {64},
number = {10},
pages = {1856-1859},
doi = {10.1111/jipb.13342},
pmid = {35962717},
issn = {1744-7909},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; *Nucleic Acids ; Plant Extracts ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {A CRISPR/LbCas12a-based nucleic acid detection method that uses crude leaf extracts as samples and is rapid (≤40 min for a full run) and highly sensitive (0.01%) can be used to monitor genetically modified organisms in the field.},
}
@article {pmid35962451,
year = {2022},
author = {Chen, W and Lin, Y and Jiang, M and Wang, Q and Shu, Q},
title = {Identification of LARS as an essential gene for osteosarcoma proliferation through large-Scale CRISPR-Cas9 screening database and experimental verification.},
journal = {Journal of translational medicine},
volume = {20},
number = {1},
pages = {355},
pmid = {35962451},
issn = {1479-5876},
mesh = {Adolescent ; *Bone Neoplasms/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Child ; Genes, Essential ; Humans ; Leucine-tRNA Ligase/*genetics ; *Osteosarcoma/genetics/pathology ; },
abstract = {BACKGROUND: Osteosarcoma is one of the most malignant tumors, and it occurs mostly in children and adolescents. Currently, surgery and chemotherapy are the main treatments. The recurrence rate is high and the prognosis is often poor. Finding an effective target gene therapy for osteosarcoma may effectively improve its prognosis.<br><br>METHOD: In this study, genes essential for the survival of osteosarcoma cells were identified by genome-wide screening of CRISPR-Cas9 based on the DepMap database. The expression of these essential genes in osteosarcoma patients' tissues and normal tissues was identified in the GSE19276 database. Functional pathway enrichment analysis, protein interaction network construction, and LASSO were performed to construct a prognostic risk model based on these essential genes. CCK8 assay was used to detect the effect of essential gene-LARS (Leucyl-TRNA Synthetase 1) on the proliferation of osteosarcoma.<br><br>RESULTS: In this study, 785 genes critical for osteosarcoma cell proliferation were identified from the DepMap. Among these 785 essential genes, 59 DEGs were identified in osteosarcoma tissues. In the functional enrichment analysis, these 59 essential genes were mainly enriched in cell cycle-related signaling pathways. Furthermore, we established a risk score module, including LARS and DNAJC17, screened from these 59 genes, and this module could divide osteosarcoma patients into the low-risk and high-risk groups. In addition, knockdown of LARS expression inhibited the proliferative ability of osteosarcoma cells. A significant correlation was found between LARS expression and Monocytic lineage, T cells, and Fibroblasts.<br><br>CONCLUSION: In conclusion, LARS was identified as an essential gene for survival in osteosarcoma based on the DepMap database. Knockdown of LARS expression significantly inhibited the proliferation of osteosarcoma cells, suggesting that it is involved in the formation and development of osteosarcoma. The results are useful as a foundation for further studies to elucidate a potential osteosarcoma diagnostic index and therapeutic targets.},
}
@article {pmid35961953,
year = {2022},
author = {Zhuo, C and Tao, Y and Li, M},
title = {Engineered virus-like particles: paving the way for effective somatic genome editing.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {279},
pmid = {35961953},
issn = {2059-3635},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
}
@article {pmid35960410,
year = {2022},
author = {Luo, N and Zhong, W and Li, J and Lu, J and Dong, R},
title = {CRISPR/dCas9 for hepatic fibrosis therapy: implications and challenges.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11403-11408},
pmid = {35960410},
issn = {1573-4978},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Transcriptional Activation ; Liver Cirrhosis/genetics/therapy ; },
abstract = {Hepatic fibrosis is a pathological reaction of tissue damage and repair caused by various pathogenic factors acting on liver. At present, there is no effective anti-fibrotic specific therapy. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (dCas9) system is a new generation of gene editing technology. The CRISPR/dCas9 system provides a platform for studying site-specific transcriptional regulation, which has high efficiency in gene transcriptional activation for achieving robust. This system holds promise for hepatic fibrosis therapy via acting on liver fibrosis effector cells. However, there are some challenges associated with this novel technology, such as large structural variants at on-target, off-target sites, and targeted delivery efficiency. In this review, we present the potential implications and describe the challenges of CRISPR/dCas9 system that might be encountered in hepatic fibrosis therapy.},
}
@article {pmid35960248,
year = {2022},
author = {Alp, A},
title = {[Recent Advances in Molecular Point of Care Tests].},
journal = {Mikrobiyoloji bulteni},
volume = {56},
number = {3},
pages = {580-591},
doi = {10.5578/mb.20229717},
pmid = {35960248},
issn = {0374-9096},
mesh = {*Coronavirus Infections/diagnosis ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Point-of-Care Systems ; Point-of-Care Testing ; },
abstract = {Molecular point of care tests has become promising methods for the global control of infectious diseases in recent years. Different kinds of point of care testing devices have been introduced into the market in the last decade. They are mainly based on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. These devices must offer a low-cost, sensitive, specific, and practical analysis to be used especially in low-resource settings. Microfluidics has high potential for handling very small volumes of fluids on a single platform. The key design features for molecular point of care tests in resource-limited settings include isothermal nucleic acid amplification methods to eliminate the need for a thermocycler, lyophilized reagents for long-term stability at high temperature and relatively simple test procedures. CRISPR-Cas-based new generation molecular point of care tests have been developed in recent years. In these tests Cas enzymes are used as highly specific target sequence recognition elements. Collateral cleavage activity of these enzymes cleaves both target sequence and labeled RNA in the mixture and a signal is generated. The ongoing Coronavirus 2019 pandemic has shown the importance of rapid diagnostic tests for the prevention of further spread. Although real-time polymerase chain reaction method was used successfully for the rapid diagnosis during the pandemic, additional rapid and practical tests that could be performed outside the laboratories would provide even faster diagnosis and lighten the burden of test load in large central laboratories. Molecular point of care tests are considered to be one of the best candidates to fill the this gap in the near future. The future technology challenges will be the standardization of the methods and further miniaturization of the system components. In this review article, point-of-care tests adapted from nucleic acid amplification methods with a large number of studies in the literature and tests based on the CRISPR-Cas system which constitutes the newest group and which is among the point-of-care molecular tests based on new nextgeneration sequencing have been discussed.},
}
@article {pmid35959889,
year = {2022},
author = {Wei, J and Hou, L and Liu, J and Wang, Z and Gao, S and Qi, T and Gao, S and Sun, S and Wang, Y},
title = {Closely related type II-C Cas9 orthologs recognize diverse PAMs.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35959889},
issn = {2050-084X},
mesh = {*CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Endonucleases/genetics ; Gene Editing ; Humans ; Purines ; Pyrimidines ; },
abstract = {The RNA-guided CRISPR/Cas9 system is a powerful tool for genome editing, but its targeting scope is limited by the protospacer-adjacent motif (PAM). To expand the target scope, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N4C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.},
}
@article {pmid35959589,
year = {2022},
author = {Bhujbal, S and Bhujbal, R and Giram, P},
title = {An overview: CRISPR/Cas-based gene editing for viral vaccine development.},
journal = {Expert review of vaccines},
volume = {21},
number = {11},
pages = {1581-1593},
doi = {10.1080/14760584.2022.2112952},
pmid = {35959589},
issn = {1744-8395},
mesh = {Animals ; Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; *Viral Vaccines/genetics ; Vaccine Development ; *Viruses/genetics ; RNA ; Antiviral Agents ; },
abstract = {INTRODUCTION: Gene-editing technology revolutionized vaccine manufacturing and offers a variety of benefits over traditional vaccinations, such as improved immune response, higher production rate, stability, precise immunogenic activity, and fewer adverse effects. The more recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated protein 9 (Cas9) system has become the most widely utilized technology based on its efficiency, utility, flexibility, versatility, ease of use, and cheaper compared to other gene-editing techniques. Considering its wider scope for genomic modification, CRISPR/Cas9-based technology's potential is explored for vaccine development.<br><br>AREAS COVERED: In this review, we will address the recent advances in the CRISPR/Cas system for the development of vaccines and viral vectors for delivery. In addition, we will discuss strategies for the development of the vaccine, as well as the limitations and future prospects of the CRISPR/Cas system.<br><br>EXPERT OPINION: Human and animal viruses have been exposed to antiviral CRISPR/Cas9-based engineering to prevent infection, which uses knockout, knock-in, gene activation/deactivation, RNA targeting, and editing cell lines strategies for gene editing of viruses. Because of that CRISPR/Cas system is used to boost the vaccine production yield by removing unwanted genes that cause disease or are required for viral infection.},
}
@article {pmid35959108,
year = {2022},
author = {Goel, K and Ploski, JE},
title = {RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives.},
journal = {Frontiers in molecular neuroscience},
volume = {15},
number = {},
pages = {914430},
pmid = {35959108},
issn = {1662-5099},
abstract = {Manipulating gene expression within and outside the nervous system is useful for interrogating gene function and developing therapeutic interventions for a variety of diseases. Several approaches exist which enable gene manipulation in preclinical models, and some of these have been approved to treat human diseases. For the last couple of decades, RNA interference (RNAi) has been a leading technique to knockdown (i.e., suppress) specific RNA expression. This has been partly due to the technology's simplicity, which has promoted its adoption throughout biomedical science. However, accumulating evidence indicates that this technology can possess significant shortcomings. This review highlights the overwhelming evidence that RNAi can be prone to off-target effects and is capable of inducing cytotoxicity in some cases. With this in mind, we consider alternative CRISPR/Cas-based approaches, which may be safer and more reliable for gene knockdown. We also discuss the pros and cons of each approach.},
}
@article {pmid35958050,
year = {2022},
author = {Lu, Y and Happi Mbakam, C and Song, B and Bendavid, E and Tremblay, JP},
title = {Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {892769},
pmid = {35958050},
issn = {2673-3439},
abstract = {Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.},
}
@article {pmid35958049,
year = {2022},
author = {Liu, S and Rao, X and Zhao, R and Han, W},
title = {The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {929929},
pmid = {35958049},
issn = {2673-3439},
abstract = {Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans in vitro. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in Escherichia coli, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity in vivo.},
}
@article {pmid35955908,
year = {2022},
author = {Tamaddondoust, RN and Wong, A and Chandrashekhar, M and Azzam, EI and Alain, T and Wang, Y},
title = {Identification of Novel Regulators of Radiosensitivity Using High-Throughput Genetic Screening.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955908},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; DNA ; *Gene Editing/methods ; Genetic Testing ; Humans ; Radiation Tolerance/genetics ; },
abstract = {The biological impact of ionizing radiation (IR) on humans depends not only on the physical properties and absorbed dose of radiation but also on the unique susceptibility of the exposed individual. A critical target of IR is DNA, and the DNA damage response is a safeguard mechanism for maintaining genomic integrity in response to the induced cellular stress. Unrepaired DNA lesions lead to various mutations, contributing to adverse health effects. Cellular sensitivity to IR is highly correlated with the ability of cells to repair DNA lesions, in particular coding sequences of genes that affect that process and of others that contribute to preserving genomic integrity. However, accurate profiling of the molecular events underlying individual sensitivity requires techniques with sensitive readouts. Here we summarize recent studies that have used whole-genome analysis and identified genes that impact individual radiosensitivity. Whereas microarray and RNA-seq provide a snapshot of the transcriptome, RNA interference (RNAi) and CRISPR-Cas9 techniques are powerful tools that enable modulation of gene expression and characterizing the function of specific genes involved in radiosensitivity or radioresistance. Notably, CRISPR-Cas9 has altered the landscape of genome-editing technology with its increased readiness, precision, and sensitivity. Identifying critical regulators of cellular radiosensitivity would help tailor regimens that enhance the efficacy of therapeutic treatments and fast-track prediction of clinical outcomes. It would also contribute to occupational protection based on average individual sensitivity, as well as the formulation of countermeasures to the harmful effects of radiation.},
}
@article {pmid35955895,
year = {2022},
author = {Lyu, P and Lu, B},
title = {New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955895},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Eukaryota/genetics ; *Gene Editing/methods ; Zinc Finger Nucleases/genetics ; },
abstract = {The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions.},
}
@article {pmid35955894,
year = {2022},
author = {Leong, SK and Hsiao, JC and Shie, JJ},
title = {A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955894},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Cluster Analysis ; *Epidermal Growth Factor/metabolism/pharmacology ; ErbB Receptors/metabolism ; HeLa Cells ; Humans ; Ligands ; *Molecular Dynamics Simulation ; Neuraminidase/genetics/metabolism ; Phosphorylation ; },
abstract = {Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&amp;B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.},
}
@article {pmid35955847,
year = {2022},
author = {De Plano, LM and Calabrese, G and Conoci, S and Guglielmino, SPP and Oddo, S and Caccamo, A},
title = {Applications of CRISPR-Cas9 in Alzheimer's Disease and Related Disorders.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955847},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/therapy ; Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; *Neurodegenerative Diseases/drug therapy ; },
abstract = {Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease represent some of the most prevalent neurodegenerative disorders afflicting millions of people worldwide. Unfortunately, there is a lack of efficacious treatments to cure or stop the progression of these disorders. While the causes of such a lack of therapies can be attributed to various reasons, the disappointing results of recent clinical trials suggest the need for novel and innovative approaches. Since its discovery, there has been a growing excitement around the potential for CRISPR-Cas9 mediated gene editing to identify novel mechanistic insights into disease pathogenesis and to mediate accurate gene therapy. To this end, the literature is rich with experiments aimed at generating novel models of these disorders and offering proof-of-concept studies in preclinical animal models validating the great potential and versatility of this gene-editing system. In this review, we provide an overview of how the CRISPR-Cas9 systems have been used in these neurodegenerative disorders.},
}
@article {pmid35955778,
year = {2022},
author = {Permyakova, NV and Marenkova, TV and Belavin, PA and Zagorskaya, AA and Sidorchuk, YV and Deineko, EV},
title = {CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955778},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Gene Rearrangement ; Plants/genetics ; Plasmids ; },
abstract = {Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.},
}
@article {pmid35955649,
year = {2022},
author = {Zhang, Y and Lin, J and Tian, X and Wang, Y and Zhao, R and Wu, C and Wang, X and Zhao, P and Bi, X and Yu, Z and Han, W and Peng, N and Liang, YX and She, Q},
title = {Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955649},
issn = {1422-0067},
mesh = {Autoimmunity/genetics ; *CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; RNA/genetics ; RNA Cleavage ; *Sulfolobus/genetics ; },
abstract = {Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2α[HD] and Cmr2α[Palm] mutants, but the inhibition was diminished in Cmr2α[HD,Palm]. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.},
}
@article {pmid35955636,
year = {2022},
author = {Zhi, H and Zhou, S and Pan, W and Shang, Y and Zeng, Z and Zhang, H},
title = {The Promising Nanovectors for Gene Delivery in Plant Genome Engineering.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955636},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering/methods ; Genome, Plant ; Gold ; *Metal Nanoparticles ; *Nanotubes, Carbon ; Plant Breeding/methods ; Plants/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Highly efficient gene delivery systems are essential for genetic engineering in plants. Traditional delivery methods have been widely used, such as Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated delivery, biolistic particle bombardment, and viral transfection. However, genotype dependence and other drawbacks of these techniques limit the application of genetic engineering, particularly genome editing in many crop plants. There is a great need to develop newer gene delivery vectors or methods. Recently, nanomaterials such as mesoporous silica particles (MSNs), AuNPs, carbon nanotubes (CNTs), and layer double hydroxides (LDHs), have emerged as promising vectors for the delivery of genome engineering tools (DNA, RNA, proteins, and RNPs) to plants in a species-independent manner with high efficiency. Some exciting results have been reported, such as the successful delivery of cargo genes into plants and the generation of genome stable transgenic cotton and maize plants, which have provided some new routines for genome engineering in plants. Thus, in this review, we summarized recent progress in the utilization of nanomaterials for plant genetic transformation and discussed the advantages and limitations of different methods. Furthermore, we emphasized the advantages and potential broad applications of nanomaterials in plant genome editing, which provides guidance for future applications of nanomaterials in plant genetic engineering and crop breeding.},
}
@article {pmid35955484,
year = {2022},
author = {Fang, Y and Yang, J and Guo, X and Qin, Y and Zhou, H and Liao, S and Liu, F and Qin, B and Zhuang, C and Li, R},
title = {CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.).},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35955484},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Infertility, Male/genetics ; Male ; Mutagenesis ; *Oryza/genetics ; Plant Breeding ; Plant Infertility/genetics ; Proteomics ; Temperature ; },
abstract = {The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.},
}
@article {pmid35953824,
year = {2022},
author = {Lam, TJ and Mortensen, K and Ye, Y},
title = {Diversity and dynamics of the CRISPR-Cas systems associated with Bacteroides fragilis in human population.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {573},
pmid = {35953824},
issn = {1471-2164},
support = {R01 AI108888/AI/NIAID NIH HHS/United States ; R01 AI143254/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteria/genetics ; Bacteroides fragilis/genetics ; *CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Genomics ; Humans ; },
abstract = {BACKGROUND: CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems are adaptive immune systems commonly found in prokaryotes that provide sequence-specific defense against invading mobile genetic elements (MGEs). The memory of these immunological encounters are stored in CRISPR arrays, where spacer sequences record the identity and history of past invaders. Analyzing such CRISPR arrays provide insights into the dynamics of CRISPR-Cas systems and the adaptation of their host bacteria to rapidly changing environments such as the human gut.<br><br>RESULTS: In this study, we utilized 601 publicly available Bacteroides fragilis genome isolates from 12 healthy individuals, 6 of which include longitudinal observations, and 222 available B. fragilis reference genomes to update the understanding of B. fragilis CRISPR-Cas dynamics and their differential activities. Analysis of longitudinal genomic data showed that some CRISPR array structures remained relatively stable over time whereas others involved radical spacer acquisition during some periods, and diverse CRISPR arrays (associated with multiple isolates) co-existed in the same individuals with some persisted over time. Furthermore, features of CRISPR adaptation, evolution, and microdynamics were highlighted through an analysis of host-MGE network, such as modules of multiple MGEs and hosts, reflecting complex interactions between B. fragilis and its invaders mediated through the CRISPR-Cas systems.<br><br>CONCLUSIONS: We made available of all annotated CRISPR-Cas systems and their target MGEs, and their interaction network as a web resource at https://omics.informatics.indiana.edu/CRISPRone/Bfragilis . We anticipate it will become an important resource for studying of B. fragilis, its CRISPR-Cas systems, and its interaction with mobile genetic elements providing insights into evolutionary dynamics that may shape the species virulence and lead to its pathogenicity.},
}
@article {pmid35953477,
year = {2022},
author = {Cromer, MK and Barsan, VV and Jaeger, E and Wang, M and Hampton, JP and Chen, F and Kennedy, D and Xiao, J and Khrebtukova, I and Granat, A and Truong, T and Porteus, MH},
title = {Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4724},
pmid = {35953477},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Hematopoietic Stem Cells/metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; RNA, Guide/genetics ; },
abstract = {As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.},
}
@article {pmid35953473,
year = {2022},
author = {Xu, X and Tang, H and Guo, J and Xin, H and Ping, Y},
title = {A dual-specific CRISPR-Cas nanosystem for precision therapeutic editing of liver disorders.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {269},
pmid = {35953473},
issn = {2059-3635},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Liver Diseases/genetics/therapy ; },
}
@article {pmid35953242,
year = {2022},
author = {Nakayama, T and Grainger, RM and Cha, SW},
title = {Homology-Directed Repair by CRISPR-Cas9 Mutagenesis in Xenopus Using Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos.},
journal = {Cold Spring Harbor protocols},
volume = {2022},
number = {12},
pages = {pdb.prot107599},
doi = {10.1101/pdb.prot107599},
pmid = {35953242},
issn = {1559-6095},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA, Single-Stranded/genetics ; Xenopus laevis/genetics ; Xenopus/genetics ; Microinjections ; Gene Editing/methods ; Mutagenesis ; },
abstract = {We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing in Xenopus embryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR-Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilized Xenopus eggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F1 generation. Therefore, we can avoid the more challenging "oocyte host transfer" technique, which is particularly difficult for Xenopus tropicalis, that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5'-end phosphorothioate-modification to obtain higher-efficiency HDR.},
}
@article {pmid35952606,
year = {2022},
author = {Kang, YJ and Park, HH},
title = {High-resolution crystal structure of the anti-CRISPR protein AcrIC5.},
journal = {Biochemical and biophysical research communications},
volume = {625},
number = {},
pages = {102-108},
doi = {10.1016/j.bbrc.2022.08.005},
pmid = {35952606},
issn = {1090-2104},
mesh = {Archaea ; Bacteria/genetics ; *CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; Pseudomonas aeruginosa/metabolism ; },
abstract = {As a result of the long-term battle of bacteria and archaea against invaders such as viruses and genetic mobile elements, they have developed CRISPR-Cas systems for self-defense, which allows them to remove the viral genetic material introduced into host cells via infection. To fight against this bacterial immune system, however, viruses have also evolved to produce multiple anti-CRISPR proteins that can inhibit the bacterial CRISPR-Cas system. In this study, we introduced a tentative inhibitory activity against a type I-C CRISPR-Cas system by determining the crystal structure of AcrIC5 from Pseudomonas delhiensis. Structural analysis revealed that AcrIC5 was composed of noble folds comprising two antiparallel sheets and three helices. Although AcrIC5 did not directly interact with either the type I-C cascade from Neisseria lactamia or the type I-F cascade from Pseudomonas aeruginosa in our analysis, a highly acidic surface feature indicated that AcrIC5 may be DNA mimic Acrs that directly binds to the target DNA binding site in type I-C cascade and inhibits the recruitment of the target DNA to this cascade.},
}
@article {pmid35952502,
year = {2023},
author = {Mu, X and Wang, X and Qin, Y and Huang, Y and Tian, J and Zhao, S},
title = {A novel label-free universal biosensing platform based on CRISPR/Cas12a for biomarker detection.},
journal = {Talanta},
volume = {251},
number = {},
pages = {123795},
doi = {10.1016/j.talanta.2022.123795},
pmid = {35952502},
issn = {1873-3573},
mesh = {Biomarkers ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/genetics ; DNA, Single-Stranded/genetics ; Limit of Detection ; *MicroRNAs ; Silver ; },
abstract = {The development of a biosensing platform with high sensitivity, high specificity, and low cost for the detection of biomarkers, especially one that is programmable and universal, is critical for disease surveillance and diagnosis, yet it remains a difficulty. Herein, we combined the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system with a fluorescent label-free biosensor platform for sensitive and specific detection of disease-related protein, small molecule and nucleic acid. In this strategy, we designed an exonuclease III-mediated target cycle and released a universal trigger chain to stimulate the enzyme activity of CRISPR/Cas12a for additional signal amplification. The hydrolysis of ssDNA-templated silver nanoclusters (ssDNA-Ag NCs) as the reporter probe resulted in a significant decrease of fluorescence intensity. This biosensing platform can be flexibly used to the sensitive and specific determination of protein, small molecule, or microRNA in biological samples by simply transforming the target recognized sequences in the DNA hairpin. In this work, a new label-free sensing system used the fluorescent ssDNA-Ag NCs as the signal output does not need to be marked in advance and has no background signal. In addition, the method has the advantages of low cost, simple operation and high speed, and provides an innovative idea for the development of a powerful clinical diagnosis tool.},
}
@article {pmid35951752,
year = {2022},
author = {Wu, J and Liu, H and Ren, S and Li, P and Li, X and Lin, L and Sun, Q and Zhang, L and Lin, C and Wang, Y},
title = {Generating an oilseed rape mutant with non-abscising floral organs using CRISPR/Cas9 technology.},
journal = {Plant physiology},
volume = {190},
number = {3},
pages = {1562-1565},
pmid = {35951752},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Brassica napus/genetics ; Technology ; },
abstract = {Oilseed rape plants with abscission-defective floral organs acquired through genome editing show less susceptibility to Sclerotinia sclerotiorum infection and longer flowering-period for flower tourism.},
}
@article {pmid35950852,
year = {2022},
author = {Patel, A and Iannello, G and Diaz, AG and Sirabella, D and Thaker, V and Corneo, B},
title = {Efficient Cas9-based Genome Editing Using CRISPR Analysis Webtools in Severe Early-onset-obesity Patient-derived iPSCs.},
journal = {Current protocols},
volume = {2},
number = {8},
pages = {e519},
pmid = {35950852},
issn = {2691-1299},
support = {P30 DK026687/DK/NIDDK NIH HHS/United States ; },
mesh = {Adolescent ; Animals ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; *Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells ; Mice ; Obesity/genetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR system is an adaptive defense mechanism used by bacteria and archaea against viruses and plasmids. The discovery of the CRISPR-associated protein Cas9 and its RNA-guided cleavage mechanism marked the beginning of a new era in genomic engineering by enabling the editing of a target region in the genome. Gene-edited cells or mice can be used as models for understanding human diseases. Given its high impact in functional genomic experiments on different model systems, several CRISPR/Cas9 protocols have been generated in the past years. The technique uses a straightforward "cut and stitch" mechanism, but requires an accurate step-by-step design. One of the key points is the use of an efficient programmable guide RNA to increase the rate of success in obtaining gene-specific edited clones. Here, we describe an efficient editing protocol using a ribonucleotide protein (RNP) complex for homology-directed repair (HDR)-based correction of a point mutation in an induced pluripotent stem cell (iPSC) line generated from a 14-year-old patient with severe early-onset obesity carrying a de novo variant of ARNT2. The resulting isogenic iPSC line, named CUIMCi003-A-1, has a normal karyotype, expresses stemness markers, and can be differentiated into progenies from all three germ layers. We provide a detailed workflow for designing a single guide RNA and donor DNA, and for isolating clonal human iPSCs edited with the desired modification. This article also focuses on parameters to consider when selecting reagents for CRISPR/Cas9 gene editing after testing their efficiency with in silico tools. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of sgRNAs and PCR primers Basic Protocol 2: Testing the efficiency of sgRNAs Basic Protocol 3: Design of template or donor DNA Basic Protocol 4: Targeted gene editing Basic Protocol 5: Selection of positive clones Basic Protocol 6: Freezing, thawing, and expansion of cells Basic Protocol 7: Characterization of edited cell lines.},
}
@article {pmid35949837,
year = {2022},
author = {Huang, YY and Zhang, XY and Zhu, P and Ji, L},
title = {Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications.},
journal = {World journal of clinical cases},
volume = {10},
number = {18},
pages = {5934-5945},
pmid = {35949837},
issn = {2307-8960},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins constitute the innate adaptive immune system in several bacteria and archaea. This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity. Owing to the numerous advantages such as ease of use, low cost, high efficiency, good accuracy, and a diverse range of applications, the CRISPR-Cas system has become the most widely used genome editing technology. Hence, the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine. This study reviews the recently reported application platforms for screening, diagnosis, and treatment of different diseases based on CRISPR/Cas systems. The limitations, current challenges, and future prospectus are summarized; this article would be a valuable reference for future genome-editing practices.},
}
@article {pmid35948929,
year = {2022},
author = {Schmidt, JK and Reynolds, MR and Golos, TG and Slukvin, II},
title = {CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection.},
journal = {Retrovirology},
volume = {19},
number = {1},
pages = {17},
pmid = {35948929},
issn = {1742-4690},
support = {P51 OD011106/OD/NIH HHS/United States ; R01HL132891/HL/NHLBI NIH HHS/United States ; K99 HD099154/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; *HIV Infections ; Humans ; Primates ; Stem Cells ; },
abstract = {Nonhuman primates (NHPs) are well-established basic and translational research models for human immunodeficiency virus (HIV) infections and pathophysiology, hematopoietic stem cell (HSC) transplantation, and assisted reproductive technologies. Recent advances in CRISPR/Cas9 gene editing technologies present opportunities to refine NHP HIV models for investigating genetic factors that affect HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 and conferring resistance to HIV/simian immunodeficiency virus (SIV) infections. In this report, we provide an overview of recent advances and challenges in gene editing NHP embryos and discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV.},
}
@article {pmid35948771,
year = {2022},
author = {Perdigoto, CN},
title = {Whole-body CRISPR editing.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {8},
pages = {729},
doi = {10.1038/s41594-022-00822-7},
pmid = {35948771},
issn = {1545-9985},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35948641,
year = {2022},
author = {Bartonicek, N and Rouet, R and Warren, J and Loetsch, C and Rodriguez, GS and Walters, S and Lin, F and Zahra, D and Blackburn, J and Hammond, JM and Reis, ALM and Deveson, IW and Zammit, N and Zeraati, M and Grey, S and Christ, D and Mattick, JS and Chtanova, T and Brink, R and Dinger, ME and Weatheritt, RJ and Sprent, J and King, C},
title = {The retroelement Lx9 puts a brake on the immune response to virus infection.},
journal = {Nature},
volume = {608},
number = {7924},
pages = {757-765},
pmid = {35948641},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics/immunology ; Evolution, Molecular ; *Host Microbial Interactions/genetics/immunology ; *Immunity/genetics ; Mice ; RNA, Untranslated/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; *Retroelements/genetics/immunology ; *Virus Diseases/genetics/immunology ; },
abstract = {The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago[1,2]. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes[3,4]. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements[5-8] has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype[8-14]. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11[-/-] mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.},
}
@article {pmid35947755,
year = {2022},
author = {de Bruijn, S},
title = {Eight (histone H4 genes) is too much: A CRISPR/Cas9 strategy to replace highly duplicated genes with a single modified version for mutational analysis.},
journal = {The Plant cell},
volume = {34},
number = {10},
pages = {3487-3488},
pmid = {35947755},
issn = {1532-298X},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genes, Duplicate ; *Histones/metabolism ; RNA, Guide/genetics ; },
}
@article {pmid35947749,
year = {2022},
author = {Shehreen, S and Birkholz, N and Fineran, PC and Brown, CM},
title = {Widespread repression of anti-CRISPR production by anti-CRISPR-associated proteins.},
journal = {Nucleic acids research},
volume = {50},
number = {15},
pages = {8615-8625},
pmid = {35947749},
issn = {1362-4962},
mesh = {*CRISPR-Associated Proteins/genetics ; Escherichia coli/genetics ; CRISPR-Cas Systems ; Operon/genetics ; Helix-Turn-Helix Motifs ; Bacteria/genetics ; Bacterial Proteins/genetics ; },
abstract = {Many bacteria use CRISPR-Cas systems to defend against invasive mobile genetic elements (MGEs). In response, MGEs have developed strategies to resist CRISPR-Cas, including the use of anti-CRISPR (Acr) proteins. Known acr genes may be followed in an operon by a putative regulatory Acr-associated gene (aca), suggesting the importance of regulation. Although ten families of helix-turn-helix (HTH) motif containing Aca proteins have been identified (Aca1-10), only three have been tested and shown to be transcriptional repressors of acr-aca expression. The AcrIIA1 protein (a Cas9 inhibitor) also contains a functionally similar HTH containing repressor domain. Here, we identified and analysed Aca and AcrIIA1 homologs across all bacterial genomes. Using HMM models we found aca-like genes are widely distributed in bacteria, both with and without known acr genes. The putative promoter regions of acr-aca operons were analysed and members of each family of bacterial Aca tested for regulatory function. For each Aca family, we predicted a conserved inverted repeat binding site within a core promoter. Promoters containing these sites directed reporter expression in E. coli and were repressed by the cognate Aca protein. These data demonstrate that acr repression by Aca proteins is widely conserved in nature.},
}
@article {pmid35946585,
year = {2022},
author = {Naranitus, P and Aiamsa-At, P and Sukonta, T and Hannanta-Anan, P and Chaijarasphong, T},
title = {Smartphone-compatible, CRISPR-based platforms for sensitive detection of acute hepatopancreatic necrosis disease in shrimp.},
journal = {Journal of fish diseases},
volume = {45},
number = {12},
pages = {1805-1816},
doi = {10.1111/jfd.13702},
pmid = {35946585},
issn = {1365-2761},
mesh = {Animals ; *Smartphone ; *Fish Diseases ; Nucleic Acid Amplification Techniques/veterinary/methods ; DNA ; Necrosis ; },
abstract = {Acute Hepatopancreatic Necrosis Disease (AHPND), caused by bacterial isolates expressing PirAB binary toxins, represents the severest and most economically destructive disease affecting penaeid shrimp. Its rapid disease progression and associated massive mortalities call for vigilant monitoring and early diagnosis, but molecular detection methods that simultaneously satisfy the requirements of sensitivity, specificity, and portability are still scarce. In this work, the CRISPR-Cas12a technology was harnessed for the development of two fluorescent assays compatible with naked-eye visualization. The first assay, AP4-Cas12a, was based on the OIE-recommended AP4 two-tubed nested PCR method and was designed to bypass the time-consuming and potentially hazardous agarose gel electrophoresis step. Using AP4-Cas12a, the detection limit of 10 copies per reaction could be achieved within less than 30 minutes post-PCR. The second assay, RPA-Cas12a, utilized recombinase polymerase amplification (RPA) to rapidly and isothermally amplify the target DNA, followed by amplicon detection by Cas12a, resulting in a protocol that can be completed in less than an hour at a constant temperature of 37°C. The detection limit of RPA-Cas12a is 100 copies of plasmid DNA or 100 fg of bacterial genomic DNA per reaction. Importantly, we validated that both assays are compatible with a previously reported smartphone-based device for facile visualization of fluorescence, thereby providing an affordable option that requires less consumables than lateral flow detection. Using this portable device for readouts, the AP4-Cas12a and RPA-Cas12a methods showed excellent concordance with the AP4-agarose gel electrophoresis approach in the evaluation of clinical samples. Therefore, the developed Cas12a assays have the potential to streamline both in-laboratory and onsite diagnosis of AHPND.},
}
@article {pmid35946380,
year = {2022},
author = {Sahel, DK and Salman, M and Azhar, M and Goswami, SG and Singh, V and Dalela, M and Mohanty, S and Mittal, A and Ramalingam, S and Chitkara, D},
title = {Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery.},
journal = {Journal of materials chemistry. B},
volume = {10},
number = {37},
pages = {7634-7649},
doi = {10.1039/d2tb00645f},
pmid = {35946380},
issn = {2050-7518},
mesh = {*CRISPR-Cas Systems/genetics ; Clathrin/genetics/metabolism ; Endonucleases/genetics/metabolism ; HEK293 Cells ; Humans ; *Ribonucleoproteins/genetics/metabolism ; Serum Albumin, Bovine/metabolism ; },
abstract = {sgRNA/Cas9 ribonucleoproteins (RNPs) provide a site-specific robust gene-editing approach avoiding the mutagenesis and unwanted off-target effects. However, the high molecular weight (∼165 kDa), hydrophilicity and net supranegative charge (∼-20 mV) hinder the intracellular delivery of these RNPs. In the present study, we have prepared cationic RNPs lipopolymeric nanoplexes that showed a size of 117.3 ± 7.64 nm with +6.17 ± 1.04 mV zeta potential and >90% entrapment efficiency of RNPs. Further, these RNPs lipopolymeric nanoplexes showed good complexation efficiency and were found to be stable for 12 h with fetal bovine serum. These RNPs lipopolymeric nanoplexes did not induce any significant cytotoxicity in HEK293T cells, and were efficiently uptaken via a clathrin-mediated pathway with optimal transfection efficiency and nuclear localization after 48 h. Further, HEK293T cells having the mGFP insert were used as a cell line model for gene editing, wherein the loss of the mGFP signal was observed as a function of gene editing after transfection with mGFP targeting RNPs lipopolymeric nanoplexes. Further, the T7 endonuclease and TIDE assay data showed a decent gene editing efficiency. Additionally, the lipopolymeric nanoplexes were able to transfect muscle cells in vivo, when injected intra-muscularly. Collectively, this study explored the potential of cationic lipopolymeric nanoplexes for delivering gene-editing endonucleases.},
}
@article {pmid35946354,
year = {2022},
author = {Shu, X and Zhang, D and Li, X and Zheng, Q and Cai, X and Ding, S and Yan, Y},
title = {Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor.},
journal = {ACS synthetic biology},
volume = {11},
number = {8},
pages = {2829-2836},
doi = {10.1021/acssynbio.2c00224},
pmid = {35946354},
issn = {2161-5063},
mesh = {Adenosine Triphosphate ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA Cleavage ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 μM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 μM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics.},
}
@article {pmid35945437,
year = {2022},
author = {},
title = {Verve takes base editors into humans.},
journal = {Nature biotechnology},
volume = {40},
number = {8},
pages = {1159},
doi = {10.1038/s41587-022-01445-5},
pmid = {35945437},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid35943638,
year = {2022},
author = {Coogan, M and Alston, V and Su, B and Khalil, K and Elaswad, A and Khan, M and Johnson, A and Xing, D and Li, S and Wang, J and Simora, RMC and Lu, C and Page-McCaw, P and Chen, W and Michel, M and Wang, W and Hettiarachchi, D and Hasin, T and Butts, IAE and Cone, RD and Dunham, RA},
title = {Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {24},
number = {5},
pages = {843-855},
pmid = {35943638},
issn = {1436-2236},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Humans ; *Ictaluridae/genetics/metabolism ; Mutation ; Receptor, Melanocortin, Type 4/genetics/metabolism ; },
abstract = {Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F1 homozygous/bi-allelic mutants reaching market size 30% faster than F1 heterozygotes in earthen ponds (p = 0.022). At the stocker stage (~ 50 g), MC4R × MC4R mutants generated in 2019 were 40% larger than the mean of combined CNTRL × CNTRL families (p = 0.005) and 54% larger than F1 MC4R × CNTRL mutants (p = 0.001) indicating mutation may be recessive. With a high mutation rate and inheritance of the mutation as well as improved growth, the use of gene-edited MC4R channel catfish appears to be beneficial for application on commercial farms.},
}
@article {pmid35942309,
year = {2022},
author = {Tao, S and Chen, H and Li, N and Liang, W},
title = {The Application of the CRISPR-Cas System in Antibiotic Resistance.},
journal = {Infection and drug resistance},
volume = {15},
number = {},
pages = {4155-4168},
pmid = {35942309},
issn = {1178-6973},
abstract = {The emergence and global epidemic of antimicrobial resistance (AMR) poses a serious threat to global public health in recent years. AMR genes are shared between bacterial pathogens mainly via horizontal gene transfer (HGT) on mobile genetic elements (MGEs), thereby accelerating the spread of antimicrobial resistance (AMR) and increasing the burden of drug resistance. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are an RNA-guided adaptive immune system in prokaryotes that recognizes and defends against invasive genetic elements such as phages and plasmids. Because of its specifically target and cleave DNA sequences encoding antibiotic resistance genes, CRISPR/Cas system has been developed into a new gene-editing tool for the prevention and control of bacterial drug resistance. CRISPR-Cas plays a potentially important role in controlling horizontal gene transfer and limiting the spread of antibiotic resistance. In this review, we will introduce the structure and working mechanism of CRISPR-Cas systems, followed by delivery strategies, and then focus on the relationship between antimicrobial resistance and CRISPR-Cas. Moreover, the challenges and prospects of this research field are discussed, thereby providing a reference for the prevention and control of the spread of antibiotic resistance.},
}
@article {pmid35941224,
year = {2022},
author = {Doman, JL and Sousa, AA and Randolph, PB and Chen, PJ and Liu, DR},
title = {Designing and executing prime editing experiments in mammalian cells.},
journal = {Nature protocols},
volume = {17},
number = {11},
pages = {2431-2468},
pmid = {35941224},
issn = {1750-2799},
support = {R01 EB031172/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA/genetics ; RNA, Guide/genetics ; Mammals/genetics ; },
abstract = {Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3' ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2-4 weeks.},
}
@article {pmid35941130,
year = {2022},
author = {Li, A and Mitsunobu, H and Yoshioka, S and Suzuki, T and Kondo, A and Nishida, K},
title = {Cytosine base editing systems with minimized off-target effect and molecular size.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4531},
pmid = {35941130},
issn = {2041-1723},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Cytosine ; DNA/genetics ; Gene Editing/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Cytosine base editing enables the installation of specific point mutations without double-strand breaks in DNA and is advantageous for various applications such as gene therapy, but further reduction of off-target risk and development of efficient delivery methods are desired. Here we show structure-based rational engineering of the cytosine base editing system Target-AID to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 is eliminated and additional mutations are introduced to restore enzyme function. The resulting tCDA1EQ is effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized RNA-mediated editing and gRNA-dependent/independent DNA off-targets, as assessed in human cells. Combining with the smaller Cas9 ortholog system (SaCas9), a cytosine base editing system is created that is within the size limit of AAV vector.},
}
@article {pmid35940609,
year = {2022},
author = {Pourrajab, F and Zare-Khormizi, MR},
title = {Extrachromosomal Circular DNAs, Amplified Oncogenes, and CRISPR-Cas9 System.},
journal = {Molecular pharmacology},
volume = {102},
number = {4},
pages = {209-215},
doi = {10.1124/molpharm.122.000553},
pmid = {35940609},
issn = {1521-0111},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; DNA ; DNA, Circular/genetics ; ErbB Receptors/genetics ; Humans ; Oncogenes/genetics ; },
abstract = {Structurally rearranged extrachromosomal circular DNAs (eccDNAs) have been identified in tumor cells, many of which carry regions related to recurrent cancer driver oncogenes (e.g., CCND1, EGFR, and MYC). In a tumor cell, eccDNAs are carrying regions associated with oncogene amplification (>10-fold amplified-copy numbers in human tumors) and poor outcome across multiple cancers. Even though dual-delivery of pairs of CRISPR and CRISPR-associated protein 9 (Cas9) guiding RNAs into normal human cells was reported to induce circularization of genes and chromosomes, in bacteria, the CRISPR-Cas9 system primarily targets extrachromosomal rearranged elements. Likewise, in cancer cells, it is expected that a designed CRISPR-Cas9 system would be able to target extrachromosomal copy number amplifications and produce double strand breaks detrimental to cellular fitness by dictating gene-independent copy number loss-of-fitness effects and antiproliferative responses. A system designed against amplified amplicons may provide a novel approach for cancer therapy and propose a practical implication for CRISPR-Cas9 pairs as a pathway in therapeutic strategies of cancer. SIGNIFICANCE STATEMENT: Structurally rearranged extrachromosomal circular DNAs (eccDNAs) have been identified in tumor cells. Many eccDNAs are carrying regions related to recurrent cancer driver oncogenes (e.g. CCND1, EGFR and MYC). It is expected that a designed CRISPR-Cas9 system would able to target extrachromosomal recurrent oncogenes.},
}
@article {pmid35940236,
year = {2022},
author = {Jena, R and Vishwas, S and Kumar, R and Kaur, J and Khursheed, R and Gulati, M and Singh, TG and Vanathi, BM and Alam, A and Kumar, B and Chaitanya, MVNL and Gupta, S and Negi, P and Pandey, NK and Bhatt, S and Gupta, G and Chellappan, DK and Oliver, BG and Dua, K and Singh, SK},
title = {Treatment strategies for HIV infection with emphasis on role of CRISPR/Cas9 gene: Success so far and road ahead.},
journal = {European journal of pharmacology},
volume = {931},
number = {},
pages = {175173},
doi = {10.1016/j.ejphar.2022.175173},
pmid = {35940236},
issn = {1879-0712},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; *HIV Infections/drug therapy/genetics ; Humans ; Receptors, Chemokine/genetics ; },
abstract = {Advances in biotechnology have led to improving human health with number of novel approaches to mitigate life-threatening diseases such as human immunodeficiency virus (HIV) infection, cancer, and neurodegenerative diseases. In the case of HIV, the damage caused by the retrovirus to the immune system leads to opportunistic infection as well as an elevated risk of autoimmune disease and cancer. Furthermore, clinical symptoms associated with the virus itself may arise. Antiretroviral drug therapy using reverse transcriptase inhibitors, protease inhibitors, fusion inhibitor, chemokine receptor 5 antagonist and integrase strand transfer inhibitors have shown promising results in treating HIV infection and available in market in the form of various dosage forms. However, they are unable to completely cure the disease because of complexity in pathogenesis of HIV. In addition, these drugs have some limitations of poor solubility, permeability or, poor receptor binding capacity. To overcome these drawbacks, many novel drug delivery systems for the drugs belonging to above mentioned categories have been developed. The possibility of treating HIV infection using CRISPR-Cas9 gene editing has been found in 2015. This provided a new area of research to the scientists who are working towards alternative treatment strategies for HIV infections. The present article describes about various treatment strategies used to treat HIV infections with special emphasis on the role of CRISPR/Cas9 gene-based technology. The potential benefits of specific epigenetic modification in the c-c chemokine receptor 5 gene (CCR5) via various delivery methods are also highlighted.},
}
@article {pmid35939208,
year = {2022},
author = {Khan, S and Sallard, E},
title = {Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {35939208},
issn = {1559-0305},
abstract = {CRISPR-Cas systems play a critical role in the prokaryotic adaptive immunity against mobile genetic elements, such as phages and foreign plasmids. In the last decade, Cas9 has been established as a powerful and versatile gene editing tool. In its wake, the novel RNA-guided endonuclease system CRISPR-Cas12a is transforming biological research due to its unique properties, such as its high specificity or its ability to target T-rich motifs, to induce staggered double-strand breaks and to process RNA arrays. Meanwhile, there is an increasing need for efficient and safe gene activation, repression or editing in pluricellular organisms for crop improvement, gene therapy, research model development, and other goals. In this article, we review CRISPR-Cas12a applications in pluricellular organisms and discuss how the challenges characteristic of these complex models, such as vectorization or temperature variations in ectothermic species, can be overcome.},
}
@article {pmid35939207,
year = {2022},
author = {Saha, U and Gondi, R and Patil, A and Saroj, SD},
title = {CRISPR in Modulating Antibiotic Resistance of ESKAPE Pathogens.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {35939207},
issn = {1559-0305},
abstract = {The ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) isolates both from the clinical settings and food products are demonstrated to gain resistance to multiple antimicrobials. Therefore, the ESKAPE pathogens pose a serious threat to public health, which warrants specific attention to developing alternative novel therapeutics. The clustered regularly interspaced short palindromic repeats associated (CRISPR-Cas) system is one of the novel methods for managing antibiotic-resistant strains. Specific Cas nucleases can be programmed against bacterial genomic sequences to decrease bacterial resistance to antibiotics. Moreover, a few CRISPR-Cas nucleases have the ability to the sequence-specific killing of bacterial strains. However, some pathogens acquire antibiotic resistance due to the presence of the CRISPR-Cas system. In brief, there is a wide range of functional diversity of CRISPR-Cas systems in bacterial pathogens. Hence, to be an effective and safe infection treatment strategy, a comprehensive understanding of the role of CRISPR-Cas systems in modulating antibiotic resistance in ESKAPE pathogens is essential. The present review summarizes all the mechanisms by which CRISPR confers and prevents antibiotic resistance in ESKAPE. The review also emphasizes the relationship between CRISPR-Cas systems, biofilm formation, and antibiotic resistance in ESKAPE.},
}
@article {pmid35938813,
year = {2022},
author = {Wang, Y and Yang, J and Sun, X and Li, M and Zhang, P and Zhu, Z and Jiao, H and Guo, T and Li, G},
title = {CRISPR-Cas in Acinetobacter baumannii Contributes to Antibiotic Susceptibility by Targeting Endogenous AbaI.},
journal = {Microbiology spectrum},
volume = {10},
number = {4},
pages = {e0082922},
pmid = {35938813},
issn = {2165-0497},
mesh = {*Acinetobacter baumannii/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Bacteria/metabolism ; Bacterial Proteins/genetics/metabolism ; Biofilms ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Drug Resistance, Multiple, Bacterial/genetics ; Humans ; Reactive Oxygen Species/metabolism ; },
abstract = {Acinetobacter baumannii is a well-known human opportunistic pathogen in nosocomial infections, and the emergence of multidrug-resistant Acinetobacter baumannii has become a complex problem for clinical anti-infective treatments. The ways this organism obtains multidrug resistance phenotype include horizontal gene transfer and other mechanisms, such as altered targets, decreased permeability, increased enzyme production, overexpression of efflux pumps, metabolic changes, and biofilm formation. A CRISPR-Cas system generally consists of a CRISPR array and one or more operons of cas genes, which can restrict horizontal gene transfer in bacteria. Nevertheless, it is unclear how CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii. Thus, we sought to assess how CRISPR-Cas affects biofilm formation, membrane permeability, efflux pump, reactive oxygen species, and quorum sensing to clarify further the mechanism of CRISPR-Cas regulation of Acinetobacter baumannii antibiotic resistance. In the clinical isolate AB43, which has a complete I-Fb CRISPR-Cas system, we discovered that the Cas3 nuclease of this type I-F CRISPR-Cas system regulates Acinetobacter baumannii quorum sensing and has a unique function in changing drug resistance. As a result of quorum sensing, synthase abaI is reduced, allowing efflux pumps to decrease, biofilm formation to become weaker, reactive oxygen species to generate, and drug resistance to decrease in response to CRISPR-Cas activity. These observations suggest that the CRISPR-Cas system targeting endogenous abaI may boost bacterial antibiotic sensitivity. IMPORTANCE CRISPR-Cas systems are vital for genome editing, bacterial virulence, and antibiotic resistance. How CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii is almost wholly unknown. In this study, we reveal that the quorum sensing regulator abaI mRNA was a primary target of the I-Fb CRISPR-Cas system and the cleavage activity of Cas3 was the most critical factor in regulating abaI mRNA degradation. These results advance our understanding of how CRISPR-Cas systems inhibit drug resistance. However, the mechanism of endogenous targeting of abaI by CRISPR-Cas needs to be further explored.},
}
@article {pmid35937379,
year = {2022},
author = {Yu, H and Yang, Q and Fu, F and Li, W},
title = {Three strategies of transgenic manipulation for crop improvement.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {948518},
pmid = {35937379},
issn = {1664-462X},
abstract = {Heterologous expression of exogenous genes, overexpression of endogenous genes, and suppressed expression of undesirable genes are the three strategies of transgenic manipulation for crop improvement. Up to 2020, most (227) of the singular transgenic events (265) of crops approved for commercial release worldwide have been developed by the first strategy. Thirty-eight of them have been transformed by synthetic sequences transcribing antisense or double-stranded RNAs and three by mutated copies for suppressed expression of undesirable genes (the third strategy). By the first and the third strategies, hundreds of transgenic events and thousands of varieties with significant improvement of resistance to herbicides and pesticides, as well as nutritional quality, have been developed and approved for commercial release. Their application has significantly decreased the use of synthetic pesticides and the cost of crop production and increased the yield of crops and the benefits to farmers. However, almost all the events overexpressing endogenous genes remain at the testing stage, except one for fertility restoration and another for pyramiding herbicide tolerance. The novel functions conferred by the heterologously expressing exogenous genes under the control of constitutive promoters are usually absent in the recipient crops themselves or perform in different pathways. However, the endogenous proteins encoded by the overexpressing endogenous genes are regulated in complex networks with functionally redundant and replaceable pathways and are difficult to confer the desirable phenotypes significantly. It is concluded that heterologous expression of exogenous genes and suppressed expression by RNA interference and clustered regularly interspaced short palindromic repeats-cas (CRISPR/Cas) of undesirable genes are superior to the overexpression of endogenous genes for transgenic improvement of crops.},
}
@article {pmid35934339,
year = {2022},
author = {Liao, K and Peng, W and Qian, B and Nan, W and Shan, Y and Zeng, D and Tang, F and Wu, X and Chen, Y and Xue, F and Dai, J},
title = {A highly adaptable platform powered by CRISPR-Cas12a to diagnose lumpy skin disease in cattle.},
journal = {Analytica chimica acta},
volume = {1221},
number = {},
pages = {340079},
doi = {10.1016/j.aca.2022.340079},
pmid = {35934339},
issn = {1873-4324},
mesh = {Animals ; CRISPR-Cas Systems ; *Capripoxvirus/genetics ; Cattle ; *Lumpy Skin Disease/diagnosis/genetics/prevention &amp; control ; Lysergic Acid Diethylamide ; Real-Time Polymerase Chain Reaction/methods ; Reproducibility of Results ; Sheep/genetics ; },
abstract = {Lumpy skin disease (LSD) in cattle, a transboundary viral disease of cattle once restricted to Africa, has been spreading to many European and Asian countries in the past decade with huge economic losses. This emerging worldwide threat to cattle warrants the development of diagnostic methods for accurate disease screening of suspected samples to effectively control the spread of LSD. In this study, we integrated pre-amplification and three kinds of sensor systems with CRISPR and therefore established an LSD diagnosis platform with highly adaptable and ultra-sensitive advantages. It was the first CRISPR-powered platform that could identify lumpy skin disease virus from vaccine strains of goat pox virus and sheep pox virus. Its limit of detection (LOD) was one copy/reaction after introducing PCR or recombinase-aided amplification (RAA). Moreover, this platform achieved a satisfactory overall agreement in clinical diagnoses of 50 samples and its reproducibility and accuracy were superior to other qPCR methods we tested. The whole diagnostic procedure, from DNA extraction to the results, could complete in 5 h with a total cost of 1.7-9.6 $/test. Overall, this CRISPR-powered platform provided a novel diagnostic tool for portable, ultra-sensitive, rapid, and highly adaptable disease screening of LSD and may be an effective method to control this transboundary disease's spread.},
}
@article {pmid35933957,
year = {2022},
author = {Liu, J and Black, GC and Kimber, SJ and Sergouniotis, PI},
title = {Generation of a human induced pluripotent stem cell line carrying the TYR c.575C&gt;A (p.Ser192Tyr) and c.1205G&gt;A (p.Arg402Gln) variants in homozygous state using CRISPR-Cas9 genome editing.},
journal = {Stem cell research},
volume = {64},
number = {},
pages = {102880},
doi = {10.1016/j.scr.2022.102880},
pmid = {35933957},
issn = {1876-7753},
support = {224643/Z/21/Z/WT_/Wellcome Trust/United Kingdom ; CL-2017-06-001/DH_/Department of Health/United Kingdom ; },
mesh = {Humans ; Gene Editing ; *Induced Pluripotent Stem Cells/metabolism ; Monophenol Monooxygenase/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Melanins/genetics/metabolism ; *Albinism/genetics ; Retinal Pigments ; },
abstract = {TYR encodes tyrosinase, the enzyme catalysing the first steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). The TYR c.575C>A (p.Ser192Tyr) [rs1042602] and c.1205G>A (p.Arg402Gln) [rs1126809] variants are prevalent genetic changes that have been associated with multiple pigmentation traits. Notably, individuals who are homozygous for these two missense variants are predisposed to having albinism. Here we used CRISPR-Cas9 technology to generate an induced pluripotent stem cell (iPSC) line (WTSIi253-A-2) that carries both c.575C>A and c.1205G>A in homozygous state. The line expresses pluripotency markers and exhibits multi-lineage differentiation potential, providing a useful in vitro model for investigating albinism pathogenesis.},
}
@article {pmid35933519,
year = {2022},
author = {Ipsen, MB and Sørensen, EMG and Thomsen, EA and Weiss, S and Haldrup, J and Dalby, A and Palmfeldt, J and Bross, P and Rasmussen, M and Fredsøe, J and Klingenberg, S and Jochumsen, MR and Bouchelouche, K and Ulhøi, BP and Borre, M and Mikkelsen, JG and Sørensen, KD},
title = {A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.},
journal = {Oncogene},
volume = {41},
number = {37},
pages = {4271-4281},
pmid = {35933519},
issn = {1476-5594},
mesh = {*Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Humans ; Male ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; *Prostatic Neoplasms, Castration-Resistant/drug therapy/genetics/pathology ; },
abstract = {DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.},
}
@article {pmid35933268,
year = {2022},
author = {Zhang, P and Chan, MM},
title = {A multifaceted signal recorder of cellular experiences using Cas12a base-editing.},
journal = {Trends in biotechnology},
volume = {40},
number = {11},
pages = {1279-1281},
doi = {10.1016/j.tibtech.2022.07.009},
pmid = {35933268},
issn = {1879-3096},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mammals ; },
abstract = {Technological advances have led to the emergence of lineage tracers, but signal recorders for mammalian systems have remained elusive. Kempton et al. have developed a Cas12a base-editing signal recorder capable of capturing diverse signals and operating in various experimental designs. The recorder enables new opportunities to chronicle cellular history.},
}
@article {pmid35931820,
year = {2022},
author = {Jia, N and Patel, DJ},
title = {Structure-based evolutionary relationship between IscB and Cas9.},
journal = {Cell research},
volume = {32},
number = {10},
pages = {875-877},
pmid = {35931820},
issn = {1748-7838},
mesh = {*Awards and Prizes ; Biological Evolution ; *CRISPR-Cas Systems ; Computational Biology ; },
}
@article {pmid35931681,
year = {2022},
author = {Möller, L and Aird, EJ and Schröder, MS and Kobel, L and Kissling, L and van de Venn, L and Corn, JE},
title = {Recursive Editing improves homology-directed repair through retargeting of undesired outcomes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4550},
pmid = {35931681},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *Gene Editing ; Humans ; RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {CRISPR-Cas induced homology-directed repair (HDR) enables the installation of a broad range of precise genomic modifications from an exogenous donor template. However, applications of HDR in human cells are often hampered by poor efficiency, stemming from a preference for error-prone end joining pathways that yield short insertions and deletions. Here, we describe Recursive Editing, an HDR improvement strategy that selectively retargets undesired indel outcomes to create additional opportunities to produce the desired HDR allele. We introduce a software tool, named REtarget, that enables the rational design of Recursive Editing experiments. Using REtarget-designed guide RNAs in single editing reactions, Recursive Editing can simultaneously boost HDR efficiencies and reduce undesired indels. We also harness REtarget to generate databases for particularly effective Recursive Editing sites across the genome, to endogenously tag proteins, and to target pathogenic mutations. Recursive Editing constitutes an easy-to-use approach without potentially deleterious cell manipulations and little added experimental burden.},
}
@article {pmid35930710,
year = {2022},
author = {Huang, J and Liang, Z and Liu, Y and Zhou, J and He, F},
title = {Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis.},
journal = {Analytical chemistry},
volume = {94},
number = {32},
pages = {11409-11415},
doi = {10.1021/acs.analchem.2c02538},
pmid = {35930710},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; DNA/genetics ; *Mycobacterium tuberculosis/genetics ; *Nucleic Acids ; RNA, Ribosomal, 16S ; },
abstract = {Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by Mycobacterium tuberculosis (M.TB). However, false-negative and false-positive results caused by base mismatches could affect the detection accuracy. Inspired by the unique property of CRISPR/Cas9, we proposed a new MSPQC M.TB sensor based on the CRISPR/Cas9 system, which can distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. In the proposed sensor, single-stranded DNA on Au interdigital electrodes was used as a capture probe for the target and an initiator for hybridization chain reaction (HCR). HCR was used to generate long double-stranded DNA (dsDNA), which could span the Au interdigital electrodes. CRISPR/Cas9 was used as recognition components to recognize capture/target dsDNA. When the target existed, the capture probe hybridized with the target to form dsDNA, which could be recognized and cut by CRISPR/Cas9. Thus, the DNA connection between electrodes was cut off and resulted in the MSPQC response. When no target existed, the capture probe remained single-stranded and could not be recognized and cut by CRISPR/Cas9. Therefore, DNA connection between electrodes was reserved. Moreover, silver staining technology was utilized to improve the sensitivity of detection. M.TB was detected by the proposed sensor using specific sequence fragments of 16S rRNA of M.TB as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.},
}
@article {pmid35930700,
year = {2022},
author = {Park, JJ and Lee, KAV and Lam, SZ and Tang, K and Chen, S},
title = {Genome Engineering for Next-Generation Cellular Immunotherapies.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.2c00340},
pmid = {35930700},
issn = {1520-4995},
support = {DP2 CA238295/CA/NCI NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; },
abstract = {Over the past decade, cellular immunotherapies such as CAR-T, TCR-T, and NK cell therapies have achieved tremendous success in cancer treatment. However, various challenges and obstacles remain, including antigen escape, immunosuppression in the tumor microenvironment, toxicities, and on-target off-tumor effects. Recent strategies for overcoming these roadblocks have included the use of genome engineering. Multiplexed CRISPR-Cas and synthetic biology approaches facilitate the development of cell therapies with higher potency and sophisticated modular control; they also offer a toolkit for allogeneic therapy development. Engineering approaches have targeted genetic modifications to enhance long-term persistence through cytokine modulation, knockout of genes mediating immunosuppressive signals, and genes such as the endogenous TCR and MHC-I that elicit adverse host-graft interactions in an allogeneic context. Genome engineering approaches for other immune cell types are also being explored, such as CAR macrophages and CAR-NK cells. Future therapeutic development of cellular immunotherapies may also be guided by novel target discovery through unbiased CRISPR genetic screening approaches.},
}
@article {pmid35930037,
year = {2022},
author = {Wen, Z and Al Makishah, NH},
title = {Recent advances in genetic technology development of oleaginous yeasts.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {17},
pages = {5385-5397},
pmid = {35930037},
issn = {1432-0614},
mesh = {Biofuels ; Gene Editing ; Genetic Engineering ; *Industrial Development ; *Yeasts ; },
abstract = {As important chemical raw materials and potential nutritional supplements, microbial lipids play an important role in ensuring economic development, food security, and energy security. Compared with non-natural hosts, oleaginous yeasts exhibit obvious advantages in lipid yield and productivity and have great potential to be genetically engineered into an oil cell factory. The main bottleneck in the current oleaginous yeasts engineering is the lack of genetic manipulation tools. Fortunately, the rapid development of synthetic biology has provided numerous new approaches, resources, and ideas for the field. Most importantly, gene editing technology mediated by CRISPR/Cas systems has been successfully applied to some oleaginous yeasts, almost completely rewriting the development pattern of genetic manipulation technology applicable. This paper reviews recent progress in genetic technology with regard to oleaginous yeasts, with a special focus on transformation methods and genome editing tools, discussing the effects of some important genetic parts. KEY POINTS: •Contribution of microbiotechnology in food safety and biofuel by oleaginous yeasts. •Advancement of genetic manipulation and transformation for oleaginous yeasts.},
}
@article {pmid35929067,
year = {2022},
author = {Gao, K and Zhang, X and Zhang, Z and Wu, X and Guo, Y and Fu, P and Sun, A and Peng, J and Zheng, J and Yu, P and Wang, T and Ye, Q and Jiang, J and Wang, H and Lin, CP and Gao, G},
title = {Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing.},
journal = {Nucleic acids research},
volume = {50},
number = {19},
pages = {e109},
pmid = {35929067},
issn = {1362-4962},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Homologous Recombination/genetics ; DNA Breaks, Double-Stranded ; DNA/genetics ; Mammals/genetics ; },
abstract = {Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.},
}
@article {pmid35928165,
year = {2022},
author = {Anniballi, F and Smith, TJ and Austin, JW},
title = {Editorial: New insights on botulism, botulinum neurotoxins, and botulinum toxin-producing clostridia.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {979653},
doi = {10.3389/fmicb.2022.979653},
pmid = {35928165},
issn = {1664-302X},
}
@article {pmid35928004,
year = {2022},
author = {Yang, Q and Zhou, Y and He, L and Zhang, Y and Tao, L},
title = {Protocol for genome-wide CRISPR knockout screens of bacterial cytotoxins in HeLa cells.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101595},
pmid = {35928004},
issn = {2666-1667},
mesh = {*Bacterial Toxins/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytotoxins ; HeLa Cells ; Humans ; },
abstract = {CRISPR screening is a powerful tool to identify host factors for pathogenic agents including viruses and bacterial toxins. Here, we present a protocol to conduct a genome-scale CRISPR screen on HeLa cells for host factors involved in the toxin action of Clostridioides difficile TcdB4. We describe in detail how to prepare the library, set up the screen, obtain the gene sequences, and analyze the results. This protocol can also be modified for other genome-scale libraries, cell lines, and cytotoxins. For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).},
}
@article {pmid35927945,
year = {2022},
author = {Shelake, RM and Kadam, US and Kumar, R and Pramanik, D and Singh, AK and Kim, JY},
title = {Engineering drought and salinity tolerance traits in crops through CRISPR-mediated genome editing: Targets, tools, challenges, and perspectives.},
journal = {Plant communications},
volume = {3},
number = {6},
pages = {100417},
doi = {10.1016/j.xplc.2022.100417},
pmid = {35927945},
issn = {2590-3462},
mesh = {*Gene Editing ; *Droughts ; Salt Tolerance/genetics ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Genome, Plant/genetics ; Crops, Agricultural/genetics ; },
abstract = {Prolonged periods of drought triggered by climate change hamper plant growth and cause substantial agricultural yield losses every year. In addition to drought, salinity is one of the major abiotic stresses that severely affect crop health and agricultural production. Plant responses to drought and salinity involve multiple processes that operate in a spatiotemporal manner, such as stress sensing, perception, epigenetic modifications, transcription, post-transcriptional processing, translation, and post-translational changes. Consequently, drought and salinity stress tolerance are polygenic traits influenced by genome-environment interactions. One of the ideal solutions to these challenges is the development of high-yielding crop varieties with enhanced stress tolerance, together with improved agricultural practices. Recently, genome-editing technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR) tools, have been effectively applied to elucidate how plants deal with drought and saline environments. In this work, we aim to portray that the combined use of CRISPR-based genome engineering tools and modern genomic-assisted breeding approaches are gaining momentum in identifying genetic determinants of complex traits for crop improvement. This review provides a synopsis of plant responses to drought and salinity stresses at the morphological, physiological, and molecular levels. We also highlight recent advances in CRISPR-based tools and their use in understanding the multi-level nature of plant adaptations to drought and salinity stress. Integrating CRISPR tools with modern breeding approaches is ideal for identifying genetic factors that regulate plant stress-response pathways and for the introgression of beneficial traits to develop stress-resilient crops.},
}
@article {pmid35927321,
year = {2022},
author = {Broto, M and Kaminski, MM and Adrianus, C and Kim, N and Greensmith, R and Dissanayake-Perera, S and Schubert, AJ and Tan, X and Kim, H and Dighe, AS and Collins, JJ and Stevens, MM},
title = {Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs.},
journal = {Nature nanotechnology},
volume = {17},
number = {10},
pages = {1120-1126},
pmid = {35927321},
issn = {1748-3395},
support = {RE/18/4/34215/BHF_/British Heart Foundation/United Kingdom ; },
mesh = {Biomarkers ; *CRISPR-Cas Systems/genetics ; DNA ; Humans ; Immunosorbents ; *MicroRNAs/genetics ; RNA, Circular ; },
abstract = {CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence that activates Cas enzymes to cleave reporter molecules. Currently, most CRISPR-based diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here we show the combination of a CRISPR-Cas-based reaction with a nanozyme-linked immunosorbent assay, which allows for the quantitative and colorimetric readout of Cas13-mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate that CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilize this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme will serve as a universally applicable signal catalyst for CRISPR-based diagnostics, which will expand the spectrum of targets for preamplification-free, quantitative detection.},
}
@article {pmid35927263,
year = {2022},
author = {Álvarez, MM and Biayna, J and Supek, F},
title = {TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4520},
pmid = {35927263},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Chromatin ; *Gene Editing ; Genetic Testing ; Genomics ; },
abstract = {CRISPR/Cas9 gene editing can inactivate genes in a precise manner. This process involves DNA double-strand breaks (DSB), which may incur a loss of cell fitness. We hypothesize that DSB toxicity may be variable depending on the chromatin environment in the targeted locus. Here, by analyzing isogenic cell line pair CRISPR experiments jointly with previous screening data from across ~900 cell lines, we show that TP53-associated break toxicity is higher in genomic regions that harbor active chromatin, such as gene regulatory elements or transcription elongation histone marks. DSB repair pathway choice and DNA sequence context also associate with toxicity. We also show that, due to noise introduced by differential toxicity of sgRNA-targeted sites, the power of genetic screens to detect conditional essentiality is reduced in TP53 wild-type cells. Understanding the determinants of Cas9 cut toxicity will help improve design of CRISPR reagents to avoid incidental selection of TP53-deficient and/or DNA repair deficient cells.},
}
@article {pmid35927241,
year = {2022},
author = {Tang, H and Wang, D and Shu, Y},
title = {Structural insights into Cas9 mismatch: promising for development of high-fidelity Cas9 variants.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {271},
pmid = {35927241},
issn = {2059-3635},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
}
@article {pmid35926679,
year = {2022},
author = {Pšeničnik, A and Reberšek, R and Slemc, L and Godec, T and Kranjc, L and Petković, H},
title = {Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces species.},
journal = {Journal of microbiological methods},
volume = {200},
number = {},
pages = {106545},
doi = {10.1016/j.mimet.2022.106545},
pmid = {35926679},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Gene Editing/methods ; Genetic Engineering ; *Streptomyces/genetics/metabolism ; },
abstract = {CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to "escaper colonies" carrying unedited genotype. Our approach allows a simple detection of "escaper" phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.},
}
@article {pmid35926468,
year = {2022},
author = {Kishton, RJ and Patel, SJ and Decker, AE and Vodnala, SK and Cam, M and Yamamoto, TN and Patel, Y and Sukumar, M and Yu, Z and Ji, M and Henning, AN and Gurusamy, D and Palmer, DC and Stefanescu, RA and Girvin, AT and Lo, W and Pasetto, A and Malekzadeh, P and Deniger, DC and Wood, KC and Sanjana, NE and Restifo, NP},
title = {Cancer genes disfavoring T cell immunity identified via integrated systems approach.},
journal = {Cell reports},
volume = {40},
number = {5},
pages = {111153},
pmid = {35926468},
issn = {2211-1247},
support = {DP2 HG010099/HG/NHGRI NIH HHS/United States ; R00 HG008171/HG/NHGRI NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; ZIA BC010763/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Antigen Presentation ; CRISPR-Cas Systems/genetics ; Humans ; *Neoplasms/genetics ; Oncogenes ; Systems Analysis ; *T-Lymphocytes ; },
abstract = {Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.},
}
@article {pmid35926276,
year = {2022},
author = {Kumagai, H and Kondo, K and Kunieda, T},
title = {Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades.},
journal = {Biochemical and biophysical research communications},
volume = {623},
number = {},
pages = {196-201},
doi = {10.1016/j.bbrc.2022.07.060},
pmid = {35926276},
issn = {1090-2104},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; INDEL Mutation ; RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; *Tardigrada/genetics/metabolism ; },
abstract = {Tardigrades are small aquatic animals known for the tolerant ability against various extreme stresses. Recent studies identified several tardigrade-unique proteins as protective factors of biomolecules from extreme stresses. Due to the limitation of the technique available in tardigrades, the function of these protective molecules has largely been studied utilizing the systems of in vitro and the heterologous expression in other organisms. Although RNAi is feasible in tardigrades, their effects are variable and not always sufficient. To analyze the functions of the tardigrade protective proteins, in vivo genetic manipulations have been desired. In this study, we used a tardigrade Hypsibius exemplaris as a model whose genome is available, and developed the delivery method of Cas9 ribonucleoproteins (RNPs) to adult tardigrade cells. Cas9 RNPs containing two kinds of crRNAs were injected to the body cavity of adult tardigrades and subjected to the subsequent electroporation to facilitate the incorporation of RNPs to the cells. Using this delivery method, we detected the deletion of the intervening region between two crRNAs from the genome. Intriguingly, all examined joining sites exhibited no incorporation of insertions/deletions (indels), suggesting that no-indel end-joining is dominant repair system in this tardigrade. We also detected similar removal of the intervening region even in the tardigrades injected with Cas9 RNPs without electroporation and in this case the no-indel end-joining is detected in still dominant but not all examined joining sites. This study provides the development of the delivery method of Cas9 RNPs to tardigrade cells and our data also suggested that simultaneous application of more than two crRNAs/gRNAs are recommended to disrupt the target gene by CRISPR/Cas9 system to avoid scarless repair in the tardigrade.},
}
@article {pmid35925472,
year = {2022},
author = {Wu, C and Chen, Z and Li, C and Hao, Y and Tang, Y and Yuan, Y and Chai, L and Fan, T and Yu, J and Ma, X and Al-Hartomy, OA and Wageh, S and Al-Sehemi, AG and Luo, Z and He, Y and Li, J and Xie, Z and Zhang, H},
title = {CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant.},
journal = {Nano-micro letters},
volume = {14},
number = {1},
pages = {159},
pmid = {35925472},
issn = {2150-5551},
abstract = {Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which is time-consuming and requires expensive instrumentation, and the confirmation of variants relies on further sequencing techniques. Herein, we first proposed a robust technique-methodology of electrochemical CRISPR sensing with the advantages of rapid, highly sensitivity and specificity for the detection of SARS-CoV-2 variant. To enhance the sensing capability, gold electrodes are uniformly decorated with electro-deposited gold nanoparticles. Using DNA template identical to SARS-CoV-2 Delta spike gene sequence as model, our biosensor exhibits excellent analytical detection limit (50 fM) and high linearity (R[2] = 0.987) over six orders of magnitude dynamic range from 100 fM to 10 nM without any nucleic-acid-amplification assays. The detection can be completed within 1 h with high stability and specificity which benefits from the CRISPR-Cas system. Furthermore, based on the wireless micro-electrochemical platform, the proposed biosensor reveals promising application ability in point-of-care testing.},
}
@article {pmid35923689,
year = {2022},
author = {Riaz, A and Kanwal, F and Ahmad, I and Ahmad, S and Farooq, A and Madsen, CK and Brinch-Pedersen, H and Bekalu, ZE and Dai, F and Zhang, G and Alqudah, AM},
title = {New Hope for Genome Editing in Cultivated Grasses: CRISPR Variants and Application.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {866121},
pmid = {35923689},
issn = {1664-8021},
abstract = {With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) mediated genome editing, crop improvement has progressed significantly in recent years. In this genome editing tool, CRISPR-associated Cas nucleases are restricted to their target of DNA by their preferred protospacer adjacent motifs (PAMs). A number of CRISPR-Cas variants have been developed e.g. CRISPR-Cas9, -Cas12a and -Cas12b, with different PAM requirements. In this mini-review, we briefly explain the components of the CRISPR-based genome editing tool for crop improvement. Moreover, we intend to highlight the information on the latest development and breakthrough in CRISPR technology, with a focus on a comparison of major variants (CRISPR-Cas9, -Cas12a, and -Cas12b) to the newly developed CRISPR-SpRY that have nearly PAM-less genome editing ability. Additionally, we briefly explain the application of CRISPR technology in the improvement of cultivated grasses with regard to biotic and abiotic stress tolerance as well as improving the quality and yield.},
}
@article {pmid35922667,
year = {2022},
author = {Fu, B and Liao, J and Chen, S and Li, W and Wang, Q and Hu, J and Yang, F and Hsiao, S and Jiang, Y and Wang, L and Chen, F and Zhang, Y and Wang, X and Li, D and Liu, M and Wu, Y},
title = {CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric β[0]/β[0] transfusion-dependent β-thalassemia.},
journal = {Nature medicine},
volume = {28},
number = {8},
pages = {1573-1580},
pmid = {35922667},
issn = {1546-170X},
mesh = {CRISPR-Cas Systems/genetics ; Child ; *Gene Editing/methods ; Humans ; Leukocytes, Mononuclear/metabolism ; Repressor Proteins/genetics ; beta-Globins/genetics ; *beta-Thalassemia/genetics/therapy ; gamma-Globins/genetics ; },
abstract = {Gene editing to disrupt the GATA1-binding site at the +58 BCL11A erythroid enhancer could induce γ-globin expression, which is a promising therapeutic strategy to alleviate β-hemoglobinopathy caused by HBB gene mutation. In the present study, we report the preliminary results of an ongoing phase 1/2 trial (NCT04211480) evaluating safety and efficacy of gene editing therapy in children with blood transfusion-dependent β-thalassemia (TDT). We transplanted BCL11A enhancer-edited, autologous, hematopoietic stem and progenitor cells into two children, one carrying the β[0]/β[0] genotype, classified as the most severe type of TDT. Primary endpoints included engraftment, overall survival and incidence of adverse events (AEs). Both patients were clinically well with multilineage engraftment, and all AEs to date were considered unrelated to gene editing and resolved after treatment. Secondary endpoints included achieving transfusion independence, editing rate in bone marrow cells and change in hemoglobin (Hb) concentration. Both patients achieved transfusion independence for >18 months after treatment, and their Hb increased from 8.2 and 10.8 g dl[-1] at screening to 15.0 and 14.0 g dl[-1] at the last visit, respectively, with 85.46% and 89.48% editing persistence in bone marrow cells. Exploratory analysis of single-cell transcriptome and indel patterns in edited peripheral blood mononuclear cells showed no notable side effects of the therapy.},
}
@article {pmid35922579,
year = {2022},
author = {Gurumurthy, CB and Quadros, RM and Ohtsuka, M},
title = {Prototype mouse models for researching SEND-based mRNA delivery and gene therapy.},
journal = {Nature protocols},
volume = {17},
number = {10},
pages = {2129-2138},
pmid = {35922579},
issn = {1750-2799},
support = {R35 HG010719/HG/NHGRI NIH HHS/United States ; R21 AI143394/AI/NIAID NIH HHS/United States ; R21 DA046831/DA/NIDA NIH HHS/United States ; R21 GM129559/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; Genetic Therapy ; Lipids ; Mice ; RNA, Messenger/genetics ; },
abstract = {One of the major challenges of gene therapy-an approach to treat diseases caused by faulty genes-is a lack of technologies that deliver healthy gene copies to target tissues and cells. Some commonly used approaches include viral vectors or coating therapeutic nucleic acids with lipid-based nanoparticles to pass through cell membranes, but these technologies have had limited success. A revolutionary tool, the CRISPR-Cas gene-editing system, offers tremendous promise, but it too suffers from problems with delivery. Another tool, called 'SEND' (for 'selective endogenous encapsidation for cellular delivery'), seems to offer a better solution. The SEND system uses endogenous genetic components to package mRNA cargoes to deliver them to other cells via virus-like particles (VLPs). The SEND-VLP tool has enormous potential as a gene-therapy tool, if the endogenous components of SEND can be repurposed to produce VLPs containing therapeutic cargoes. However, several aspects of this newly identified phenomenon are not yet fully understood. Genetically engineered mouse (GEM) models, expressing different combinations of SEND components in a controllable and inducible fashion, could serve as valuable tools to understand more about this tool and to repurpose it for gene-therapy applications. In this Perspective, we discuss how GEM models and mouse molecular genetics tools could be used for SEND-VLP research.},
}
@article {pmid35922510,
year = {2022},
author = {Laughlin, TG and Deep, A and Prichard, AM and Seitz, C and Gu, Y and Enustun, E and Suslov, S and Khanna, K and Birkholz, EA and Armbruster, E and McCammon, JA and Amaro, RE and Pogliano, J and Corbett, KD and Villa, E},
title = {Architecture and self-assembly of the jumbo bacteriophage nuclear shell.},
journal = {Nature},
volume = {608},
number = {7922},
pages = {429-435},
pmid = {35922510},
issn = {1476-4687},
support = {R35 GM144121/GM/NIGMS NIH HHS/United States ; T32 GM133351/GM/NIGMS NIH HHS/United States ; R01 GM129325/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM129245/GM/NIGMS NIH HHS/United States ; R01 GM031749/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteria/cytology/immunology/metabolism/virology ; *Bacteriophages/chemistry/immunology/physiology/ultrastructure ; *Cell Compartmentation ; Cryoelectron Microscopy ; *Viral Proteins/chemistry/metabolism/ultrastructure ; *Virus Assembly ; },
abstract = {Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems[1]. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors[2-4]. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.},
}
@article {pmid35921985,
year = {2022},
author = {Wang, J and Li, D and Yang, J and Chang, L and Zhang, R and Li, J},
title = {CRISPR/Cas9-mediated epigenetic editing tool: An optimized strategy for targeting de novo DNA methylation with stable status via homology directed repair pathway.},
journal = {Biochimie},
volume = {202},
number = {},
pages = {190-205},
doi = {10.1016/j.biochi.2022.07.013},
pmid = {35921985},
issn = {1638-6183},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; Epigenomics ; DNA Methylation ; HeLa Cells ; },
abstract = {With the development of epigenetic engineering tools for manipulating DNA methylation at the desired locus, the stable maintenance of targeted methylation status in edited cells after division poses a major obstacle. Transient methylation levels, along with the reversible inhibition of the desired genes, by some epigenetic editing techniques, limit their further application in functional and therapeutic studies of critical regulators in the cancer epigenome. In this study, we utilized CRISPR/Cas9-mediated the homology-directed repair (HDR) pathway following double-strand breaks (DSBs) information to target de novo methylation of the vital epigenetic biomarker, O[6]-methylguanine-DNA methyltransferase (MGMT). Based on synthetic single and double stranded methylated repair templates, DNA methylation patterns can be incorporated into the endogenous MGMT gene. In addition, we observed upregulation of DNA methyltransferases (DNMTs) in edited HeLa cells. In particular, we employed an optimized method of using the DNA ligase IV inhibitor SCR7 to markedly enhance the insertion efficiency induced by the long methylated repair template, contributing to the induction of stable methylation alterations through epigenetic recombination after cell division. Additionally, our study confirmed that CRISPR/Cas9-mediated target methylation in a site-specific manner enabled the maintenance of gene silencing in vitro and in vivo. Collectively, we show that a combination of CRISPR/Cas9 components, SCR7 treatment, and the long methylated HDR template can enhance CRISPR/Cas9-directed epigenomic editing efficiency and further induce stable effects on methylation modifications and transcriptional suppression.},
}
@article {pmid35921742,
year = {2023},
author = {Lin, C and Huang, Q and Tian, M and Luo, F and Wang, J and Qiu, B and Yang, S and Lin, Z},
title = {Electrochemiluminescence biosensor for DNA adenine methylation methyltransferase based on CRISPR/Cas12a trans-cleavage-induced dual signal enhancement.},
journal = {Talanta},
volume = {251},
number = {},
pages = {123748},
doi = {10.1016/j.talanta.2022.123748},
pmid = {35921742},
issn = {1873-3573},
mesh = {Adenine ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; DNA ; DNA Methylation ; DNA Restriction Enzymes ; Indium ; Metallocenes ; Methyltransferases ; Oxides ; Tin ; },
abstract = {In this work, an electrochemiluminescence (ECL) biosensor with dual signal enhancement was constructed and used for DNA adenine methylation methyltransferase (Dam MTase) detection. At present of Dam MTase, restriction endonuclease (DPnI) cleaves hairpin DNA (HP) and releases the HP stem end as a single strand that can activate CRISPR/Cas12a trans-cleavage activity. Assisted by trans-cleavage, the distance between the signal quenching factor ferrocene (Fc) and the ECL signal unit increased, and the repulsion between the signal unit and the Indium Tin Oxides (ITO) electrode decreased. The above results resulted in an enhanced ECL signal. ECL intensity has a good linear relationship with the logarithm of Dam MTase concentration in the range of 5-70 U/mL with a detection limit of 23.4 mU/mL. The proposed biosensor was successfully utilized to detect of Dam MTase in serum samples.},
}
@article {pmid35920723,
year = {2022},
author = {Li, X and Zhang, Y and Liu, Q and Jin, Y and Li, B},
title = {CRISPR/Cas12a-based fluorescence immunoassay: combination of efficient signal generation with specific molecule recognition.},
journal = {The Analyst},
volume = {147},
number = {17},
pages = {3833-3837},
doi = {10.1039/d2an01048h},
pmid = {35920723},
issn = {1364-5528},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; DNA/genetics ; Immunoassay ; },
abstract = {Using the DNA activator of Cas to label the secondary antibody of traditional ELISA, the CRISPR/Cas12a-based fluorescence immunoassay was presented. The sensing strategy ingeniously combines the efficient signal generation of the CRISPR/Cas12a system with antigen-antibody-specific recognition. This work provides an alternative strategy to expand the application of the CRISPR/Cas system for the detection of non-nucleic acid targets.},
}
@article {pmid35920441,
year = {2022},
author = {Guhathakurta, S and Adams, L and Jeong, I and Sivakumar, A and Cha, M and Bernardo Fiadeiro, M and Hu, HN and Kim, YS},
title = {Precise epigenomic editing with a SunTag-based modular epigenetic toolkit.},
journal = {Epigenetics},
volume = {17},
number = {13},
pages = {2075-2081},
pmid = {35920441},
issn = {1559-2308},
mesh = {*Epigenomics ; *Epigenesis, Genetic ; Gene Editing ; CRISPR-Cas Systems ; DNA Methylation ; Promoter Regions, Genetic ; },
abstract = {Epigenetic regulation is a crucial factor controlling gene expression. Here, we report our CRISPR/dCas9-based modular epigenetic toolkit that enables gene-specific modulation of epigenetic architecture. By modifying the SunTag framework of dCas9 tagged with five GCN4 moieties, each epigenetic writer is bound to scFv and target-specific sgRNA, and this system is able to modify multiple epigenetic marks in a target-specific manner. We successfully demonstrated that this system is efficient in modifying individual histone post-translational modifications. We display its utility as a tool to understand the contributions of specific histone marks on gene expression by screening a large promoter region and identifying differential outcomes with high base-pair resolution. This epigenetic toolkit can be easily altered with a large variety of epigenetic effectors and is a useful tool for researchers to use in understanding gene-specific epigenetic changes and their relation to gene expression.},
}
@article {pmid35920325,
year = {2022},
author = {Lee, SY and Birkholz, N and Fineran, PC and Park, HH},
title = {Molecular basis of anti-CRISPR operon repression by Aca10.},
journal = {Nucleic acids research},
volume = {50},
number = {15},
pages = {8919-8928},
pmid = {35920325},
issn = {1362-4962},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; *CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Operon/genetics ; Transcription Factors/genetics ; },
abstract = {CRISPR-Cas systems are bacterial defense systems for fighting against invaders such as bacteriophages and mobile genetic elements. To escape destruction by these bacterial immune systems, phages have co-evolved multiple anti-CRISPR (Acr) proteins, which inhibit CRISPR-Cas function. Many acr genes form an operon with genes encoding transcriptional regulators, called anti-CRISPR-associated (Aca) proteins. Aca10 is the most recently discovered Aca family that is encoded within an operon containing acrIC7 and acrIC6 in Pseudomonas citronellolis. Here, we report the high-resolution crystal structure of an Aca10 protein to unveil the molecular basis of transcriptional repressor role of Aca10 in the acrIC7-acrIC6-aca10 operon. We identified that Aca10 forms a dimer in solution, which is critical for binding specific DNA. We also showed that Aca10 directly recognizes a 21 bp palindromic sequence in the promoter of the acr operon. Finally, we revealed that R44 of Aca10 is a critical residue involved in the DNA binding, which likely results in a high degree of DNA bending.},
}
@article {pmid35920322,
year = {2022},
author = {Cui, Y and Dong, H and Tong, B and Wang, H and Chen, X and Liu, G and Zhang, D},
title = {A versatile Cas12k-based genetic engineering toolkit (C12KGET) for metabolic engineering in genetic manipulation-deprived strains.},
journal = {Nucleic acids research},
volume = {50},
number = {15},
pages = {8961-8973},
pmid = {35920322},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Escherichia coli/genetics/metabolism ; Gene Editing ; Genetic Engineering ; *Metabolic Engineering ; *Sinorhizobium meliloti/genetics ; },
abstract = {The genetic modification of microorganisms is conducive to the selection of high-yield producers of high-value-added chemicals, but a lack of genetic tools hinders the industrialization of most wild species. Therefore, it is crucial to develop host-independent gene editing tools that can be used for genetic manipulation-deprived strains. The Tn7-like transposon from Scytonema hofmanni has been shown to mediate homologous recombination-independent genomic integration after heterologous expression in Escherichia coli, but the integration efficiency of heterologous sequences larger than 5 kb remains suboptimal. Here, we constructed a versatile Cas12k-based genetic engineering toolkit (C12KGET) that can achieve genomic integration of fragments up to 10 kb in size with up to 100% efficiency in challenging strains. Using C12KGET, we achieved the first example of highly efficient genome editing in Sinorhizobium meliloti, which successfully solved the problem that industrial strains are difficult to genetically modify, and increased vitamin B12 production by 25%. In addition, Cas12k can be directly used for transcriptional regulation of genes with up to 92% efficiency due to its naturally inactivated nuclease domain. The C12KGET established in this study is a versatile and efficient marker-free tool for gene integration as well as transcriptional regulation that can be used for challenging strains with underdeveloped genetic toolkits.},
}
@article {pmid35920132,
year = {2022},
author = {Robertson, G and Burger, J and Campa, M},
title = {CRISPR/Cas-based tools for the targeted control of plant viruses.},
journal = {Molecular plant pathology},
volume = {23},
number = {11},
pages = {1701-1718},
pmid = {35920132},
issn = {1364-3703},
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Crops, Agricultural ; Gene Editing ; Genome, Plant ; Plant Breeding ; *Plant Viruses/genetics ; RNA ; },
abstract = {Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the "Genetic modification" issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas-based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research.},
}
@article {pmid35918694,
year = {2022},
author = {Fang, T and Cao, X and Ibnat, M and Chen, G},
title = {Stimuli-responsive nanoformulations for CRISPR-Cas9 genome editing.},
journal = {Journal of nanobiotechnology},
volume = {20},
number = {1},
pages = {354},
pmid = {35918694},
issn = {1477-3155},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; },
abstract = {The CRISPR-Cas9 technology has changed the landscape of genome editing and has demonstrated extraordinary potential for treating otherwise incurable diseases. Engineering strategies to enable efficient intracellular delivery of CRISPR-Cas9 components has been a central theme for broadening the impact of the CRISPR-Cas9 technology. Various non-viral delivery systems for CRISPR-Cas9 have been investigated given their favorable safety profiles over viral systems. Many recent efforts have been focused on the development of stimuli-responsive non-viral CRISPR-Cas9 delivery systems, with the goal of achieving efficient and precise genome editing. Stimuli-responsive nanoplatforms are capable of sensing and responding to particular triggers, such as innate biological cues and external stimuli, for controlled CRISPR-Cas9 genome editing. In this Review, we overview the recent advances in stimuli-responsive nanoformulations for CRISPR-Cas9 delivery, highlight the rationale of stimuli and formulation designs, and summarize their biomedical applications.},
}
@article {pmid35918643,
year = {2022},
author = {Gumustop, I and Ortakci, F},
title = {Comparative genomics of Leuconostoc lactis strains isolated from human gastrointestinal system and fermented foods microbiomes.},
journal = {BMC genomic data},
volume = {23},
number = {1},
pages = {61},
pmid = {35918643},
issn = {2730-6844},
mesh = {*Fermented Foods ; Genomics ; Humans ; Leuconostoc/genetics ; *Microbiota/genetics ; },
abstract = {BACKGROUND: Leuconostoc lactis forms a crucial member of the genus Leuconostoc and has been widely used in the fermentation industry to convert raw material into acidified and flavored products in dairy and plant-based food systems. Since the ecological niches that strains of Ln. lactis being isolated from were truly diverse such as the human gut, dairy, and plant environments, comparative genome analysis studies are needed to better understand the strain differences from a metabolic adaptation point of view across diverse sources of origin. We compared eight Ln. lactis strains of 1.2.28, aa_0143, BIOML-A1, CBA3625, LN19, LN24, WIKIM21, and WiKim40 using bioinformatics to elucidate genomic level characteristics of each strain for better utilization of this species in a broad range of applications in food industry.<br><br>RESULTS: Phylogenomic analysis of twenty-nine Ln. lactis strains resulted in nine clades. Whole-genome sequence analysis was performed on eight Ln. lactis strains representing human gastrointestinal tract and fermented foods microbiomes. The findings of the present study are based on comparative genome analysis against the reference Ln. lactis CBA3625 genome. Overall, a&#x2009;~&#x2009;41% of all CDS were conserved between all strains. When the coding sequences were assigned to a function, mobile genetic elements, mainly insertion sequences were carried by all eight strains. All strains except LN24 and WiKim40 harbor at least one intact putative prophage region, and two of the strains contained CRISPR-Cas system. All strains encoded Lactococcin 972 bacteriocin biosynthesis gene clusters except for CBA3625.<br><br>CONCLUSIONS: The findings in the present study put forth new perspectives on genomics of Ln. lactis via complete genome sequence&#xa0;based comparative analysis and further determination of genomic characteristics. The outcomes of this work could potentially pave the way for developing elements for future strain engineering applications.},
}
@article {pmid35918621,
year = {2022},
author = {Wang, C and Sun, Z and Wang, M and Jiang, Z and Zhang, M and Cao, H and Luo, L and Qiao, C and Xiao, H and Chen, G and Li, X and Liu, J and Wei, Z and Shen, B and Wang, J and Feng, J},
title = {Novel CRISPR/Cas9-mediated knockout of LIG4 increases efficiency of site-specific integration in Chinese hamster ovary cell line.},
journal = {Biotechnology letters},
volume = {44},
number = {9},
pages = {1063-1072},
pmid = {35918621},
issn = {1573-6776},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; DNA End-Joining Repair/genetics ; DNA Ligase ATP/genetics ; *Gene Editing ; },
abstract = {AIM: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells.<br><br>RESULTS: CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4[-/-]-CHO cell lines was estimated by a green fluorescence protein promoterless reporter system. Notably, the RI efficiency, most likely mediated by NHEJ in CHO, was inhibited by LIG4 knockout, whereas SSI efficiency strongly increased 9.2-fold under the precise control of the promoter in the ROSA26 site in LIG4[-/-]-CHO cells. Moreover, deletion of LIG4 had no obvious side effects on CHO cell proliferation.<br><br>CONCLUSIONS: Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.},
}
@article {pmid35918499,
year = {2022},
author = {McAleese, CE and Butcher, NJ and Minchin, RF},
title = {Arylamine N-acetyltransferase 1 deficiency inhibits drug-induced cell death in breast cancer cells: switch from cytochrome C-dependent apoptosis to necroptosis.},
journal = {Breast cancer research and treatment},
volume = {195},
number = {3},
pages = {223-236},
pmid = {35918499},
issn = {1573-7217},
mesh = {*Antineoplastic Agents/pharmacology/therapeutic use ; Apoptosis ; *Arylamine N-Acetyltransferase/deficiency/genetics ; *Breast Neoplasms/drug therapy/genetics/metabolism ; Caspase 8/genetics/metabolism/therapeutic use ; Cell Death ; Cytochromes c/metabolism/therapeutic use ; Female ; Humans ; Isoenzymes/deficiency/genetics ; Necroptosis ; },
abstract = {PURPOSE: Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response.<br><br>METHODS: The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide.<br><br>RESULTS: Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin.<br><br>CONCLUSIONS: NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.},
}
@article {pmid35918324,
year = {2022},
author = {Chen, Y and Hysolli, E and Chen, A and Casper, S and Liu, S and Yang, K and Liu, C and Church, G},
title = {Multiplex base editing to convert TAG into TAA codons in the human genome.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4482},
pmid = {35918324},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Codon, Terminator ; Exons ; *Gene Editing ; Genes, Essential ; *Genome, Human/genetics ; Humans ; Mammals/genetics ; },
abstract = {Whole-genome recoding has been shown to enable nonstandard amino acids, biocontainment and viral resistance in bacteria. Here we take the first steps to extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and highly multiplex editing in mammalian cells.},
}
@article {pmid35917936,
year = {2022},
author = {Oulhen, N and Pieplow, C and Perillo, M and Gregory, P and Wessel, GM},
title = {Optimizing CRISPR/Cas9-based gene manipulation in echinoderms.},
journal = {Developmental biology},
volume = {490},
number = {},
pages = {117-124},
doi = {10.1016/j.ydbio.2022.07.008},
pmid = {35917936},
issn = {1095-564X},
support = {R35 GM140897/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Morpholinos/genetics ; RNA, Guide/genetics ; *Sea Urchins/genetics ; },
abstract = {The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.},
}
@article {pmid35917610,
year = {2022},
author = {Cheng, X and Li, Y and Kou, J and Liao, D and Zhang, W and Yin, L and Man, S and Ma, L},
title = {Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review.},
journal = {Biosensors &amp; bioelectronics},
volume = {215},
number = {},
pages = {114559},
doi = {10.1016/j.bios.2022.114559},
pmid = {35917610},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Transcription Factors/genetics ; },
abstract = {Various non-nucleic acid targets (ions, small molecules, polysaccharides, peptides/proteins/enzymes, cells, transcription factors) are important biomarkers. They play important roles in environmental protection, food safety and medical diagnosis. Therefore, it is necessary to detect non-nucleic acid targets from various samples before the situation deteriorates. Derived from prokaryotic immune systems, CRISPR/Cas tools have exhibited great promise in the field of biosensing, in addition to the well-known gene-editing function. However, most reported CRISPR/Cas-based biosensors are for nucleic acid detection and the application of non-nucleic acid targets is still in its infancy. To fully explore the potential of CRISPR/Cas-based biosensing systems, it is of great significance to summarize the strategies and prospects of CRISPR/Cas toolboxes in non-nucleic acid targets recognition. In this review, we introduced CRISPR/Cas systems and their characteristics in the field of detection. The progress of detecting six non-nucleic acid targets was outlined and reviewed based on CRISPR/Cas systems coupled with biotransduction elements, including aptamers, DNAzymes, riboswitches, enzymatic reactions, transcription factors, antigen-antibody interactions, allosteric probes, in vitro transcription processes, steric hindrance effectors, etc. The development challenges and prospects in this field were also put forward. As such, this comprehensive review would provide valuable information for the expansion of the powerful CRISPR/Cas toolboxes into multiple detection fields.},
}
@article {pmid35917600,
year = {2022},
author = {Kang, JY and Mun, D and Chun, Y and Kim, H and Yun, N and Lee, SH and Joung, B},
title = {Generation of a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {63},
number = {},
pages = {102878},
doi = {10.1016/j.scr.2022.102878},
pmid = {35917600},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Cardiomyopathies/metabolism ; Gene Editing ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Tropomyosin/genetics ; },
abstract = {E192K missense mutation of TPM1 has been found in different types of cardiomyopathies (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, and left ventricular non-compaction), leading to systolic dysfunction, diastolic dysfunction, and/or tachyarrhythmias. Here, we generated a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-based genome editing system. The cells exhibit normal karyotype, typical stem cell morphology, expression of pluripotency markers and differentiation ability into three germ layers. Accordingly, this cell line could provide a useful cell resource for exploring the pathogenic role of TPM1-E192K mutation in different types of cardiomyopathies.},
}
@article {pmid35917561,
year = {2022},
author = {Lattanzi, A and Maddalo, D},
title = {The CRISPR Revolution in the Drug Discovery Workflow: An Industry Perspective.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {634-641},
doi = {10.1089/crispr.2022.0002},
pmid = {35917561},
issn = {2573-1602},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Workflow ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Drug Discovery ; },
abstract = {In a relatively short time, the pharmaceutical industry has witnessed a rapid integration of the CRISPR technology in multiple areas of research, development, therapy, and diagnostics. A unique feature to this system compared with other technologies is the exceptional versatility in adapting to the broad range of needs across the drug discovery pipeline, such as target identification, cell engineering, and in vivo modeling. As a consequence, the CRISPR toolbox has been evolving to address key questions around preclinical and clinical drug development. In this review, we provide a high-level perspective of how CRISPR has impacted several aspects of the drug discovery workflow and the future ahead for this exciting technology.},
}
@article {pmid35917293,
year = {2022},
author = {Park, BJ and Yoo, JR and Heo, ST and Kim, M and Lee, KH and Song, YJ},
title = {A CRISPR-Cas12a-based diagnostic method for multiple genotypes of severe fever with thrombocytopenia syndrome virus.},
journal = {PLoS neglected tropical diseases},
volume = {16},
number = {8},
pages = {e0010666},
pmid = {35917293},
issn = {1935-2735},
mesh = {Aged ; *Bunyaviridae Infections ; CRISPR-Cas Systems ; Genotype ; Humans ; *Phlebovirus/genetics ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) infection is commonly reported in countries of Northeast Asia including China, Japan and South Korea. The majority of the SFTS patients are elderly and the average fatality rate is more than 10%. A rapid and sensitive diagnostic method to monitor and prevent SFTSV transmission remains an urgent clinical challenge. In this study, we developed a molecular diagnostic technique for detection of SFTSV using the CRISPR-Cas12a system combined with reverse transcription recombinase polymerase amplification (RT-RPA). Using this method, we successfully diagnosed SFTSV infections with the reaction time of 50 min from blood plasma without cross-reactivity to other viruses, supporting its application for rapid and sensitive diagnosis of SFTS.},
}
@article {pmid35917009,
year = {2022},
author = {Perera, BPU and Morgan, RK and Polemi, KM and Sala-Hamrick, KE and Svoboda, LK and Dolinoy, DC},
title = {PIWI-Interacting RNA (piRNA) and Epigenetic Editing in Environmental Health Sciences.},
journal = {Current environmental health reports},
volume = {},
number = {},
pages = {},
pmid = {35917009},
issn = {2196-5412},
support = {ES017885/ES/NIEHS NIH HHS/United States ; ES032048/ES/NIEHS NIH HHS/United States ; ES031686/ES/NIEHS NIH HHS/United States ; P30 ES017885/ES/NIEHS NIH HHS/United States ; ES0077062/ES/NIEHS NIH HHS/United States ; ES026697/ES/NIEHS NIH HHS/United States ; },
abstract = {PURPOSE OF REVIEW: The epigenome modulates gene expression in response to environmental stimuli. Modifications to the epigenome are potentially reversible, making them a promising therapeutic approach to mitigate environmental exposure effects on human health. This review details currently available genome and epigenome editing technologies and highlights ncRNA, including piRNA, as potential tools for targeted epigenome editing. RECENT FINDINGS: Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease (CRISPR/Cas) research has significantly advanced genome editing technology, with broad promise in genetic research and targeted therapies. Initial epigenome-directed therapies relied on global modification and suffered from limited specificity. Adapted from current genome editing tools, zinc finger protein (ZFP), TALE, and CRISPR/nuclease-deactivated Cas (dCas) systems now confer locus-specific epigenome editing, with promising applicability in the field of environmental health sciences. However, high incidence of off-target effects and time taken for screening limit their use. FUTURE DEVELOPMENT: ncRNA serve as a versatile biomarker with well-characterized regulatory mechanisms that can easily be adapted to edit the epigenome. For instance, the transposon silencing mechanism of germline PIWI-interacting RNAs (piRNA) could be engineered to specifically methylate a given gene, overcoming pitfalls of current global modifiers. Future developments in epigenome editing technologies will inform risk assessment through mechanistic investigation and serve as potential modes of intervention to mitigate environmentally induced adverse health outcomes later in life.},
}
@article {pmid35916620,
year = {2022},
author = {Wang, ZY and Li, DL and Tian, X and Li, Y and Zhang, CY},
title = {Single-Molecule Counting of FTO in Human Breast Tissues Based on a Rolling Circle Transcription Amplification-Driven Clustered Regularly Interspaced Short Palindromic Repeat─Cas12a.},
journal = {Analytical chemistry},
volume = {94},
number = {32},
pages = {11425-11432},
doi = {10.1021/acs.analchem.2c02578},
pmid = {35916620},
issn = {1520-6882},
mesh = {Adenosine/metabolism ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Humans ; Mammals/metabolism ; *Neoplasms/genetics ; Obesity/genetics ; },
abstract = {N6-methyladenosine modification as an mRNA modification in mammalian cells is dynamically reversible, regulated by RNA demethylase [e.g., fat mass and obesity-associated protein (FTO)]. The abnormal expression of FTO is closely related to numerous diseases (e.g., various cancers and obesity). Herein, we demonstrate the single-molecule counting of FTO in human cancer cells and breast tissues based on a T7 RNA polymerase-mediated rolling circle transcription (RCT) amplification-driven clustered regularly interspaced short palindromic repeat (CRISPR)─Cas12a. When FTO is present, it demethylates the DNA substrate, initiating the DpnII-mediated cleavage reaction. After magnetic separation, the cleaved DNA fragments trigger the T7 RNA polymerase-mediated RCT amplification, activating CRISPR-/Cas12a-mediated cleavage of signal probes and releasing abundant FAM molecules that are simply counted via single-molecule detection. In this assay, only target FTO can generate CRISPR RNAs, efficiently improving detection specificity. Moreover, the integration of single-molecule detection with magnetic separation achieves zero background and effectively enhances detection sensitivity. This method can specifically and sensitively monitor FTO activity with a limit of detection of 1.20 × 10[-13] M, and it may measure FTO at the single-cell level. Furthermore, it may accurately discriminate the FTO expression level in breast tissues between healthy persons and breast cancer patients and screen the FTO inhibitors as well, with great potential in clinical diagnosis and drug discovery.},
}
@article {pmid35916139,
year = {2022},
author = {Ishikawa, M and Nomura, T and Tamaki, S and Ozasa, K and Suzuki, T and Toyooka, K and Hirota, K and Yamada, K and Suzuki, K and Mochida, K},
title = {CRISPR/Cas9-mediated generation of non-motile mutants to improve the harvesting efficiency of mass-cultivated Euglena gracilis.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2042-2044},
pmid = {35916139},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; *Euglena gracilis/genetics ; Gene Editing ; Ribonucleoproteins/genetics ; },
}
@article {pmid35915475,
year = {2022},
author = {Liu, SC and Feng, YL and Sun, XN and Chen, RD and Liu, Q and Xiao, JJ and Zhang, JN and Huang, ZC and Xiang, JF and Chen, GQ and Yang, Y and Lou, C and Li, HD and Cai, Z and Xu, SM and Lin, H and Xie, AY},
title = {Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {165},
pmid = {35915475},
issn = {1474-760X},
mesh = {Animals ; CRISPR-Cas Systems ; DNA ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; *Gene Editing/methods ; Mice ; },
abstract = {BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood.<br><br>RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements.<br><br>CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.},
}
@article {pmid35915300,
year = {2022},
author = {Pfromm, JK and Bonillo, M and Dauletbekov, D and Bucher, K and Fischer, MD},
title = {Plasmid-mediated gene transfer of Cas9 induces vector-related but not SpCas9-related immune responses in human retinal pigment epithelial cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {13202},
pmid = {35915300},
issn = {2045-2322},
mesh = {*Bacterial Proteins/metabolism ; *CRISPR-Cas Systems ; Epithelial Cells/metabolism ; Gene Editing/methods ; Humans ; Immunity ; Plasmids/genetics ; Retinal Pigments ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system represents a powerful gene-editing tool and could enable treatment of blinding diseases of the retina. As a peptide of bacterial origin, we investigated the immunogenic potential of Cas9 in models of retinal immunocompetent cells: human microglia (IMhu) and ARPE-19 cells. Transfection with Streptococcus pyogenes-Cas9 expression plasmids (SpCas9 plasmid) induced Cas9 protein expression in both cell lines. However, only ARPE-19 cells, not IMhu cells, responded with pro-inflammatory immune responses as evidenced by the upregulation of IL-8, IL-6, and the cellular activation markers HLA-ABC and CD54 (ICAM). These pro-inflammatory responses were also induced through transfection with equally sized non-coding control plasmids. Moreover, viability rates of ARPE-19 cells were reduced after transfection with both the SpCas9 plasmids and the control plasmids. Although these results demonstrate cell type-specific responses to the DNA plasmid vector, they show no evidence of an immunogenic effect due to the presence of Cas9 in models of human retinal pigment epithelial and microglia cells. These findings add another layer of confidence in the immunological safety of potential future Cas9-mediated retinal gene therapies.},
}
@article {pmid35915259,
year = {2022},
author = {Kim, DY and Chung, Y and Lee, Y and Jeong, D and Park, KH and Chin, HJ and Lee, JM and Park, S and Ko, S and Ko, JH and Kim, YS},
title = {Hypercompact adenine base editors based on transposase B guided by engineered RNA.},
journal = {Nature chemical biology},
volume = {18},
number = {9},
pages = {1005-1013},
pmid = {35915259},
issn = {1552-4469},
mesh = {*Adenine/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Mammals/genetics ; *RNA/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Transposases/genetics/metabolism ; },
abstract = {Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1. Previously, we reported a set of engineered guide RNAs supporting high indel efficiency for Cas12f1 in human cells. Here we suggest a new technology whereby the engineered guide RNAs also manifest high-efficiency programmable endonuclease activity for TnpB. We have termed this technology TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors (ABEs). A Tad-Tad mutant (V106W, D108Q) dimer fused to the C terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded with engineered variants of TnpB or optimized deaminases. Delivery of TaRGET-ABE also ensured potent A-to-G conversion rates in mammalian genomes. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by adeno-associated viruses, thereby harnessing the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene therapy.},
}
@article {pmid35914977,
year = {2022},
author = {Awan, MJA and Amin, I and Mansoor, S},
title = {CRISPR-Cas12c: a noncleaving DNA binder with minimal PAM requirement.},
journal = {Trends in biotechnology},
volume = {40},
number = {10},
pages = {1141-1143},
doi = {10.1016/j.tibtech.2022.07.005},
pmid = {35914977},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; Nucleotides ; RNA ; },
abstract = {The CRISPR-Cas toolbox is expanding swiftly. Every discovery of a novel and unique variant opens new frontiers in the field of synthetic and applied biology. Recently, Huang et al. revealed the CRISPR-Cas12c system that requires a single-nucleotide protospacer adjacent motif (PAM) to perform RNA-guided DNA targeting without DNA cleavage.},
}
@article {pmid35914975,
year = {2022},
author = {Gudmunds, E and Wheat, CW and Khila, A and Husby, A},
title = {Functional genomic tools for emerging model species.},
journal = {Trends in ecology &amp; evolution},
volume = {37},
number = {12},
pages = {1104-1115},
doi = {10.1016/j.tree.2022.07.004},
pmid = {35914975},
issn = {1872-8383},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genomics/methods ; Phenotype ; },
abstract = {Most studies in the field of ecology and evolution aiming to connect genotype to phenotype rarely validate identified loci using functional tools. Recent developments in RNA interference (RNAi) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas genome editing have dramatically increased the feasibility of functional validation. However, these methods come with specific challenges when applied to emerging model organisms, including limited spatial control of gene silencing, low knock-in efficiencies, and low throughput of functional validation. Moreover, many functional studies to date do not recapitulate ecologically relevant variation, and this limits their scope for deeper insights into evolutionary processes. We therefore argue that increased use of gene editing by allelic replacement through homology-directed repair (HDR) would greatly benefit the field of ecology and evolution.},
}
@article {pmid35914146,
year = {2022},
author = {Park, JU and Tsai, AW and Chen, TH and Peters, JE and Kellogg, EH},
title = {Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {32},
pages = {e2202590119},
pmid = {35914146},
issn = {1091-6490},
mesh = {Bacterial Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy ; *Cyanobacteria/enzymology/genetics ; *DNA Transposable Elements ; DNA-Binding Proteins/metabolism ; *Transposases/genetics/metabolism ; },
abstract = {CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.},
}
@article {pmid35913596,
year = {2022},
author = {Zhang, DF and Yao, YF and Xue, HP and Fu, ZY and Zhang, XM and Shao, Z},
title = {Characterization of Marinilongibacter aquaticus gen. nov., sp. nov., a unique marine bacterium harboring four CRISPR-Cas systems in the phylum Bacteroidota.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {60},
number = {9},
pages = {905-915},
pmid = {35913596},
issn = {1976-3794},
mesh = {Bacterial Typing Techniques ; *Bacteroidetes/genetics ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Fatty Acids/chemistry ; Phospholipids/chemistry ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; Ubiquinone/chemistry ; },
abstract = {A novel bacterium, designated YYF0007[T], was isolated from an agar-degrading co-culture. The strain was found harboring four CRISPR-Cas systems of two classes in the chromosome and subsequently subjected to a study on polyphasic taxonomy. Pairwise analyses of the 16S rRNA gene sequences indicated that strain YYF0007[T] had highest 16S rRNA gene sequence similarity (92.2%) to Jiulongibacter sediminis JN-14-9[T]. The phylogenomic trees based on the 16S rRNA gene and 269 single-copy orthologous gene clusters (OCs) indicated that strain YYF0007[T] should be recognized as a novel genus of the family Spirosomaceae. The cells were Gramstain-negative, nonmotile, strictly aerobic, and straight long rods with no flagellum. Optimum growth occurred at 28°C and pH 7.0 with the presence of NaCl concentration 1.0-3.0% (w/v). The strain showed oxidase and catalase activities. The major fatty acids were C16:1ω5c, iso-C15:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The predominant isoprenoid quinone was MK-7. The complete genome size was 4.64 Mb with a DNA G + C content of 44.4%. Further typing of CRISPR-Cas systems in the family Spirosomaceae and the phylum Bacteroidota indicated that it was remarkable for strain YYF0007[T] featured by such a set of CRISPR-Cas systems. This trait highlights the applications of strain YYF-0007[T] in studies on the evolutionary dynamics and bacterial autoimmunity of CRISPR-Cas system as a potential model. The name Marinilongibacter aquaticus gen. nov., sp. nov. is proposed, and the type strain is YYF0007[T] (= MCCC 1K06017[T] = GDMCC 1.2428[T] = JCM 34683[T]).},
}
@article {pmid35910203,
year = {2022},
author = {Kumar, D and Yadav, A and Ahmad, R and Dwivedi, UN and Yadav, K},
title = {CRISPR-Based Genome Editing for Nutrient Enrichment in Crops: A Promising Approach Toward Global Food Security.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {932859},
pmid = {35910203},
issn = {1664-8021},
abstract = {The global malnutrition burden imparts long-term developmental, economic, social, and medical consequences to individuals, communities, and countries. The current developments in biotechnology have infused biofortification in several food crops to fight malnutrition. However, these methods are not sustainable and suffer from several limitations, which are being solved by the CRISPR-Cas-based system of genome editing. The pin-pointed approach of CRISPR-based genome editing has made it a top-notch method due to targeted gene editing, thus making it free from ethical issues faced by transgenic crops. The CRISPR-Cas genome-editing tool has been extensively used in crop improvement programs due to its more straightforward design, low methodology cost, high efficiency, good reproducibility, and quick cycle. The system is now being utilized in the biofortification of cereal crops such as rice, wheat, barley, and maize, including vegetable crops such as potato and tomato. The CRISPR-Cas-based crop genome editing has been utilized in imparting/producing qualitative enhancement in aroma, shelf life, sweetness, and quantitative improvement in starch, protein, gamma-aminobutyric acid (GABA), oleic acid, anthocyanin, phytic acid, gluten, and steroidal glycoalkaloid contents. Some varieties have even been modified to become disease and stress-resistant. Thus, the present review critically discusses CRISPR-Cas genome editing-based biofortification of crops for imparting nutraceutical properties.},
}
@article {pmid35909968,
year = {2022},
author = {Lyimo, BM and Popkin-Hall, ZR and Giesbrecht, DJ and Mandara, CI and Madebe, RA and Bakari, C and Pereus, D and Seth, MD and Ngamba, RM and Mbwambo, RB and MacInnis, B and Mbwambo, D and Garimo, I and Chacky, F and Aaron, S and Lusasi, A and Molteni, F and Njau, R and Cunningham, JA and Lazaro, S and Mohamed, A and Juliano, JJ and Bailey, JA and Ishengoma, DS},
title = {Potential Opportunities and Challenges of Deploying Next Generation Sequencing and CRISPR-Cas Systems to Support Diagnostics and Surveillance Towards Malaria Control and Elimination in Africa.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {757844},
pmid = {35909968},
issn = {2235-2988},
support = {001/WHO_/World Health Organization/International ; K24 AI134990/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Communicable Diseases ; High-Throughput Nucleotide Sequencing ; Humans ; *Malaria/diagnosis/epidemiology/prevention &amp; control ; Tanzania ; },
abstract = {Recent developments in molecular biology and genomics have revolutionized biology and medicine mainly in the developed world. The application of next generation sequencing (NGS) and CRISPR-Cas tools is now poised to support endemic countries in the detection, monitoring and control of endemic diseases and future epidemics, as well as with emerging and re-emerging pathogens. Most low and middle income countries (LMICs) with the highest burden of infectious diseases still largely lack the capacity to generate and perform bioinformatic analysis of genomic data. These countries have also not deployed tools based on CRISPR-Cas technologies. For LMICs including Tanzania, it is critical to focus not only on the process of generation and analysis of data generated using such tools, but also on the utilization of the findings for policy and decision making. Here we discuss the promise and challenges of NGS and CRISPR-Cas in the context of malaria as Africa moves towards malaria elimination. These innovative tools are urgently needed to strengthen the current diagnostic and surveillance systems. We discuss ongoing efforts to deploy these tools for malaria detection and molecular surveillance highlighting potential opportunities presented by these innovative technologies as well as challenges in adopting them. Their deployment will also offer an opportunity to broadly build in-country capacity in pathogen genomics and bioinformatics, and to effectively engage with multiple stakeholders as well as policy makers, overcoming current workforce and infrastructure challenges. Overall, these ongoing initiatives will build the malaria molecular surveillance capacity of African researchers and their institutions, and allow them to generate genomics data and perform bioinformatics analysis in-country in order to provide critical information that will be used for real-time policy and decision-making to support malaria elimination on the continent.},
}
@article {pmid35908288,
year = {2022},
author = {Wang, Y and Chen, H and Ma, L and Gong, M and Wu, Y and Bao, D and Zou, G},
title = {Use of CRISPR-Cas tools to engineer Trichoderma species.},
journal = {Microbial biotechnology},
volume = {15},
number = {10},
pages = {2521-2532},
pmid = {35908288},
issn = {1751-7915},
mesh = {Biotechnology ; *CRISPR-Cas Systems ; Gene Editing ; *Trichoderma/genetics ; },
abstract = {Given their lignocellulose degradability and biocontrol activities, fungi of the ubiquitously distributed genus Trichoderma have multiple industrial and agricultural applications. Genetic manipulation plays a valuable role in tailoring novel engineered strains with enhanced target traits. Nevertheless, as applied to fungi, the classic tools of genetic manipulation tend to be time-consuming and tedious. However, the recent development of the CRISPR-Cas system for gene editing has enabled researchers to achieve genome-wide gene disruptions, gene replacements, and precise editing, and this technology has emerged as a primary focus for novel developments in engineered strains of Trichoderma. Here, we provide a brief overview of the traditional approaches to genetic manipulation, the different strategies employed in establishing CRSIPR-Cas systems, the utilization of these systems to develop engineered strains of Trichoderma for desired applications, and the future trends in biotechnology.},
}
@article {pmid35908059,
year = {2022},
author = {Finney, M and Romanowski, J and Adelman, ZN},
title = {Strategies to improve homology-based repair outcomes following CRISPR-based gene editing in mosquitoes: lessons in how to keep any repair disruptions local.},
journal = {Virology journal},
volume = {19},
number = {1},
pages = {128},
pmid = {35908059},
issn = {1743-422X},
support = {R01 AI137112/AI/NIAID NIH HHS/United States ; R01AI148787/AI/NIAID NIH HHS/United States ; R01AI137112/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Culicidae/genetics ; DNA ; *Gene Editing/methods ; Mammals ; Mosquito Vectors/genetics ; },
abstract = {Programmable gene editing systems such as CRISPR-Cas have made mosquito genome engineering more practical and accessible, catalyzing the development of cutting-edge genetic methods of disease vector control. This progress, however, has been limited by the low efficiency of homology-directed repair (HDR)-based sequence integration at DNA double-strand breaks (DSBs) and a lack of understanding about DSB repair in mosquitoes. Innovative efforts to optimize HDR sequence integration by inhibiting non-homologous end joining or promoting HDR have been performed in mammalian systems, however many of these approaches have not been applied to mosquitoes. Here, we review some of the most relevant steps of DNA DSB repair choice and highlight promising approaches that influence this choice to enhance HDR in the context of mosquito gene editing.},
}
@article {pmid35906195,
year = {2022},
author = {Zhukova, M and Sapountzis, P and Schiøtt, M and Boomsma, JJ},
title = {Phylogenomic analysis and metabolic role reconstruction of mutualistic Rhizobiales hindgut symbionts of Acromyrmex leaf-cutting ants.},
journal = {FEMS microbiology ecology},
volume = {98},
number = {9},
pages = {},
doi = {10.1093/femsec/fiac084},
pmid = {35906195},
issn = {1574-6941},
mesh = {*Alphaproteobacteria ; Animals ; *Ants ; Arginine ; Fungi ; Nitrogen ; Phylogeny ; Symbiosis ; },
abstract = {Rhizobiales are well-known plant-root nitrogen-fixing symbionts, but the functions of insect-associated Rhizobiales are poorly understood. We obtained genomes of three strains associated with Acromyrmex leaf-cutting ants and show that, in spite of being extracellular gut symbionts, they lost all pathways for essential amino acid biosynthesis, making them fully dependent on their hosts. Comparison with 54 Rhizobiales genomes showed that all insect-associated Rhizobiales lost the ability to fix nitrogen and that the Acromyrmex symbionts had exceptionally also lost the urease genes. However, the Acromyrmex strains share biosynthesis pathways for riboflavin vitamin, queuosine and a wide range of antioxidant enzymes likely to be beneficial for the ant fungus-farming symbiosis. We infer that the Rhizobiales symbionts catabolize excess of fungus-garden-derived arginine to urea, supplementing complementary Mollicutes symbionts that turn arginine into ammonia and infer that these combined symbiont activities stabilize the fungus-farming mutualism. Similar to the Mollicutes symbionts, the Rhizobiales species have fully functional CRISPR/Cas and R-M phage defenses, suggesting that these symbionts are important enough for the ant hosts to have precluded the evolution of metabolically cheaper defenseless strains.},
}
@article {pmid35905895,
year = {2022},
author = {Li, Z and Ma, R and Liu, D and Wang, M and Zhu, T and Deng, Y},
title = {A straightforward plant prime editing system enabled highly efficient precise editing of rice Waxy gene.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {323},
number = {},
pages = {111400},
doi = {10.1016/j.plantsci.2022.111400},
pmid = {35905895},
issn = {1873-2259},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Oryza/genetics ; Plants/genetics ; Recombinational DNA Repair ; Waxes ; },
abstract = {CRISPR Cas9-mediated genome editing is highly efficient at targeted site-specific gene knock-out through NHEJ (Non-Homology End Joining), but ineffective for specific DNA integration through HDR (Homology Directed Repair) for precise gene editing. Base editors can make limited base substitutions but only within restricted small windows of the protospacer. Prime editing has been applied in plants with various degrees of success. However, several questions such as low and inconsistent editing efficiencies across different target sites need to be addressed. We compared two prime editing approaches PE3 and PE2 at two neighboring target sites within rice Waxy gene to partially address those questions. A straightforward PE2 plant prime editing system retrofitted from a regular CRISPR-Cas9 editing system can deliver highly efficient up to 66.7% precise gene editing. Various forms of precise editing including base substitutions, small deletions and insertions can be accurately achieved. The secondary structure variations of different pegRNAs may be the primary reason for inconsistent editing across different target sites and should be the optimization focus to further improve plant prime editing.},
}
@article {pmid35905712,
year = {2022},
author = {Vinceti, A and Perron, U and Trastulla, L and Iorio, F},
title = {Reduced gene templates for supervised analysis of scale-limited CRISPR-Cas9 fitness screens.},
journal = {Cell reports},
volume = {40},
number = {4},
pages = {111145},
doi = {10.1016/j.celrep.2022.111145},
pmid = {35905712},
issn = {2211-1247},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Library ; Genome ; Humans ; *RNA, Guide/genetics ; },
abstract = {Pooled genome-wide CRISPR-Cas9 screens are furthering our mechanistic understanding of human biology and have allowed us to identify new oncology therapeutic targets. Scale-limited CRISPR-Cas9 screens-typically employing guide RNA libraries targeting subsets of functionally related genes, biological pathways, or portions of the druggable genome-constitute an optimal setting for investigating narrow hypotheses and are easier to execute on complex models, such as organoids and in vivo models. Different supervised methods are used for computational analysis of genome-wide CRISPR-Cas9 screens; most are not well suited for scale-limited screens, as they require large sets of positive/negative control genes (gene templates) to be included among the screened ones. Here, we develop a computational framework identifying optimal subsets of known essential and nonessential genes (at different subsampling percentages) that can be used as templates for supervised analyses of scale-limited CRISPR-Cas9 screens, while having a reduced impact on the size of the employed library.},
}
@article {pmid35902672,
year = {2022},
author = {Kim, DE and Lee, JH and Ji, KB and Lee, EJ and Li, C and Oh, HJ and Park, KS and Lee, SH and Koo, O and Kim, MK},
title = {Prime editor-mediated correction of a pathogenic mutation in purebred dogs.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {12905},
pmid = {35902672},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Dogs ; Gene Editing ; *Hip Dysplasia, Canine/diagnosis/genetics/pathology ; Mice ; Mutation ; },
abstract = {Canine hip dysplasia (HD) is a multifactorial disease caused by interactions between genetic and environmental factors. HD, which mainly occurs in medium- to large-sized dogs, is a disease that causes severe pain and requires surgical intervention. However, the procedure is not straight-forward, and the only way to ameliorate the situation is to exclude individual dogs with HD from breeding programs. Recently, prime editing (PE), a novel genome editing tool based on the CRISPR-Cas9 system, has been developed and validated in plants and mice. In this study, we successfully corrected a mutation related to HD in Labrador retriever dogs for the first time. We collected cells from a dog diagnosed with HD, corrected the mutation using PE, and generated mutation-corrected dogs by somatic cell nuclear transfer. The results indicate that PE technology can potentially be used as a platform to correct genetic defects in dogs.},
}
@article {pmid35902408,
year = {2022},
author = {Nakazawa, T and Inoue, C and Nguyen, DX and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {17},
pages = {5575-5585},
pmid = {35902408},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Plasmids ; Polyporales ; *RNA, Guide ; },
abstract = {Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.},
}
@article {pmid35902079,
year = {2022},
author = {Li, X and Zhang, H and Zhang, J and Song, Y and Shi, X and Zhao, C and Wang, J},
title = {Diagnostic accuracy of CRISPR technology for detecting SARS-CoV-2: a systematic review and meta-analysis.},
journal = {Expert review of molecular diagnostics},
volume = {22},
number = {6},
pages = {655-663},
doi = {10.1080/14737159.2022.2107425},
pmid = {35902079},
issn = {1744-8352},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; Humans ; Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/genetics ; Sensitivity and Specificity ; Technology ; },
abstract = {OBJECTIVE: To evaluate the diagnostic accuracy of CRISPR-Cas technology for SARS-CoV-2.<br><br>METHODS: RT-qPCR is defined as the reference standard. Data was collected and assessed by Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 tool. A bivariate model for pooling was employed and subgroups analysis was used to explore heterogeneity.<br><br>RESULTS: 2264 samples from 28 articles were extracted for evaluating the accuracy of CRISPR technology for diagnosing SARS-CoV-2. The pooled sensitivity and specificity of CRISPR technology were 0.98 (95% CI: 0.95-0.99) and 1.0 (95% CI: 0.98-1.00), respectively. High risks in patient selection bias and unclear risk of index test bias may affect accuracy. Subgroup analysis showed that CRISPR-Cas12 is applicable for molecular diagnostics for its active editing characteristics. RT-LAMP and RT-RPA are usually used for pre-amplification and fluorescence detection to output results quantitatively. Nasopharyngeal swabs and dual-genes perform greatly in our study.<br><br>CONCLUSION: The results concluded from all studies showed that CRISPR technology is a promising molecular method for detecting SARS-CoV-2. Standard methods including comparable sample material, patient selection, operating procedure and operators should be established.},
}
@article {pmid35900273,
year = {2022},
author = {Lin, G and Zhang, K and Han, Y and Peng, R and Zhang, J and Li, D and Li, J},
title = {Reprogramming of Human B Cells from Secreting IgG to IgM by Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {5},
pages = {717-725},
doi = {10.1089/crispr.2021.0093},
pmid = {35900273},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing ; *RNA, Guide ; CRISPR-Cas Systems/genetics ; Immunoglobulin M/genetics ; Immunoglobulin A/genetics ; Immunoglobulin G/genetics ; Immunoglobulin E ; },
abstract = {B lymphocytes are activated and regulated by their interactions with T cells, a process that results in one-way class switching of immunoglobulins (ig) from IgM to IgG, IgE, or IgA. In this study, we show the application of clustered regularly interspaced short palindromic repeat-Cas9-induced nonhomologous end joining in B cells to achieve reverse-directional Ig class switching. By electroporating Cas9 and guide RNA and a Cμ encoding donor into cells, we engineered IgG-secreting human B cell lines to switch to express IgM antibody. This approach offers a new potential path for the production of IgM antibodies.},
}
@article {pmid35900171,
year = {2022},
author = {El Mouridi, S and Alkhaldi, F and Frøkjær-Jensen, C},
title = {Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {9},
pages = {},
pmid = {35900171},
issn = {2160-1836},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; *Caenorhabditis elegans/genetics ; *Caenorhabditis elegans Proteins/genetics ; Chromosomes ; Gene Transfer Techniques ; Nerve Tissue Proteins/genetics ; Transgenes ; },
abstract = {Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here, we describe a modular safe-harbor transgene insertion (MosTI) for use in Caenorhabditis elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by nonhomologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25-75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7-44%) at modular safe-harbor transgene insertion landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for 8 integrated arrays (6-37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of 1 array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.},
}
@article {pmid35897807,
year = {2022},
author = {Kim, S and Nowakowska, A and Kim, YB and Shin, HY},
title = {Integrated CRISPR-Cas9 System-Mediated Knockout of IFN-γ and IFN-γ Receptor 1 in the Vero Cell Line Promotes Viral Susceptibility.},
journal = {International journal of molecular sciences},
volume = {23},
number = {15},
pages = {},
pmid = {35897807},
issn = {1422-0067},
mesh = {Animals ; Antiviral Agents/pharmacology ; *CRISPR-Cas Systems ; Cell Line ; Chlorocebus aethiops ; Interferon-beta/pharmacology ; *Interferon-gamma/genetics/pharmacology ; Receptors, Interferon ; Vero Cells ; Virus Replication/genetics ; },
abstract = {The current pandemic and the possible emergence of new viruses urgently require the rapid development of antiviral vaccines and therapeutics. However, some viruses or newly generated variants are difficult to culture in common cell types or exhibit low viral susceptibility in vivo, making it difficult to manufacture viral vector-based vaccines and understand host-virus interactions. To address these issues, we established new cell lines deficient in both type I and type II interferon responses, which are essential for host immunity and interference with virus replication. These cell lines were generated by developing an integrated CRISPR-Cas9 system that simultaneously expresses dual-guide RNA cassettes and Cas9 nuclease in a single plasmid. Using this highly efficient gene-editing system, we successfully established three cell lines starting from IFN-α/β-deficient Vero cells, deleting the single interferon-gamma (IFNG) gene, the IFNG receptor 1 (IFNGR1) gene, or both genes. All cell lines clearly showed a decrease in IFN-γ-responsive antiviral gene expression and cytokine production. Moreover, production of IFN-γ-induced cytokines remained low, even after HSV-1 or HCoV-OC43 infection, while expression of the receptor responsible for viral entry increased. Ultimately, knockout of IFN-signaling genes in these cell lines promoted cytopathic effects and increased apoptosis after viral infection up to three-fold. These results indicate that our integrated CRISPR-Cas9-mediated IFNG- and IFNGR1-knockout cell lines promote virus replication and will be useful in viral studies used to design novel vaccines and therapies.},
}
@article {pmid35896844,
year = {2022},
author = {Yan, M and Li, B and Wang, J and Bai, Y and Ke, Q and Zhou, T and Xu, P},
title = {Disruption of mstn Gene by CRISPR/Cas9 in Large Yellow Croaker (Larimichthys crocea).},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {24},
number = {4},
pages = {681-689},
pmid = {35896844},
issn = {1436-2236},
mesh = {Animals ; Aquaculture ; *CRISPR-Cas Systems ; Gene Editing/methods ; Mammals/genetics ; *Perciformes/genetics ; Phylogeny ; },
abstract = {The large yellow croaker (Larimichthys crocea) plays an economically vital role in the marine aquaculture in China. Suffering from infection of bacteria and protozoon, effect of extreme weather and stress from high-density farming, genome editing is thought to be an important tool applied to L. croea for enhancing commercial traits such as growth rate, disease resistance, and nutrition component. In this study, we identified two mstn genes in L. croea and investigated the different phylogenetic clades, gene structures, and conserved syntenic relationships. To obtain fast-growing large yellow croaker, we specially selected two validated targets for mstnb knockout, which was homologous to mammalian myostatin gene (MSTN) and downregulated skeletal muscle growth and development. Five significant mutation types were generated in two mosaic mutants by transferring specific CRISPR/Cas9 RNPs (ribonucleoprotein) into the one-cell fertilized embryos based on CRISPR/Cas9 technology. Subsequently, we also elucidated the obstacles and possible measures to improve the success rate of inducing modified large yellow croaker. Our results would provide valuable method and reference for facilitating genome editing programs of the large yellow croaker in the future.},
}
@article {pmid35896746,
year = {2022},
author = {Bhattarai-Kline, S and Lear, SK and Fishman, CB and Lopez, SC and Lockshin, ER and Schubert, MG and Nivala, J and Church, GM and Shipman, SL},
title = {Recording gene expression order in DNA by CRISPR addition of retron barcodes.},
journal = {Nature},
volume = {608},
number = {7921},
pages = {217-225},
pmid = {35896746},
issn = {1476-4687},
support = {DP2 GM140917/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/biosynthesis/genetics ; *Gene Editing/methods ; *Gene Expression ; Genome/genetics ; *Information Storage and Retrieval/methods ; Integrases/metabolism ; Prokaryotic Cells/metabolism ; *RNA/genetics ; *Reverse Transcription ; Time Factors ; },
abstract = {Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons[1], that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system[2]. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.},
}
@article {pmid35894926,
year = {2022},
author = {McVey, SL and Olson, MA and Pawlowski, WP and Nannas, NJ},
title = {Beyond editing, CRISPR/Cas9 for protein localization: an educational primer for use with "A dCas9-based system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination".},
journal = {Genetics},
volume = {222},
number = {1},
pages = {},
pmid = {35894926},
issn = {1943-2631},
mesh = {*CRISPR-Cas Systems ; Centromere ; Gene Editing/methods ; Homologous Recombination ; Humans ; *Kinetochores ; Meiosis/genetics ; },
abstract = {CRISPR/Cas9 has dramatically changed how we conduct genetic research, providing a tool for precise sequence editing. However, new applications of CRISPR/Cas9 have emerged that do not involve nuclease activity. In the accompanying article "A dCas9-based system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination," Kuhl et al. utilize a catalytically dead Cas9 to localize proteins at specific genomic locations. The authors seek to understand the role of kinetochore proteins in the suppression of meiotic recombination, a phenomenon that has been observed in centromere regions. By harnessing the power of CRISPR/Cas9 to bind specific genomic sequences, Kuhl et al. localized individual kinetochore proteins to areas of high meiotic recombination and assessed their role in suppression. This primer article provides undergraduate students with background information on chromosomes, meiosis, recombination and CRISPR/Cas9 to support their reading of the Kuhl et al. study. This primer is intended to help students and instructors navigate the study's experimental design, interpret the results, and appreciate the broader scope of meiotic recombination and CRISPR/Cas9. Questions are included to facilitate discussion of the study.},
}
@article {pmid35894125,
year = {2022},
author = {Mayta, ML and Dotto, M and Orellano, EG and Krapp, AR},
title = {An experimental protocol for teaching CRISPR/Cas9 in a post-graduate plant laboratory course: An analysis of mutant-edited plants without sequencing.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {50},
number = {5},
pages = {537-546},
doi = {10.1002/bmb.21659},
pmid = {35894125},
issn = {1539-3429},
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded ; Gene Editing/methods ; Humans ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system is widely used for editing genes in various organisms and is a very useful tool due to its versatility, simplicity, and efficiency. To teach its principles to post-graduate students we designed a laboratory activity to obtain and analyze PDS3 mutants in Arabidopsis thaliana plants consisting of: 1) Design of guide RNAs using bioinformatics tools; 2) plant transformation (which is optional depending on the length of the course); 3) observation and evaluation of the mutant's phenotypes in the Phytoene desaturase (PDS3) gene, which exhibit an albino phenotype and different degrees of mosaicism in the editing events we evaluated; 4) PCR amplification of a fragment that includes the mutated region followed by analysis of single-stranded DNA conformation polymorphisms (SSCP) using native polyacrylamide gel electrophoresis and silver nitrate staining to detect changes in the amplicon sequence due to gene editing. Through SSCP, the students were able to distinguish between homozygous and heterozygous edited plants. A highlight feature of this protocol is the visualization and detection of the mutation/edition without sequencing the edited fragment.},
}
@article {pmid35892930,
year = {2022},
author = {Freen-van Heeren, JJ},
title = {Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV.},
journal = {Biotech (Basel (Switzerland))},
volume = {11},
number = {3},
pages = {},
pmid = {35892930},
issn = {2673-6284},
abstract = {Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.},
}
@article {pmid35892709,
year = {2022},
author = {Böldicke, T},
title = {Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions.},
journal = {Antibodies (Basel, Switzerland)},
volume = {11},
number = {3},
pages = {},
pmid = {35892709},
issn = {2073-4468},
abstract = {Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies with activated autologous T cells from cancer patients transduced with novel recombinant TCRs or chimeric antigen receptors have been successfully applied. Many TAAs and most neoantigens are expressed in the cytoplasm or nucleus of tumor cells. As alternative to adoptive T-cell therapy, the mRNA of intracellular tumor antigens can be depleted by RNAi, the corresponding genes or proteins deleted by CRISPR-Cas or inactivated by kinase inhibitors or by intrabodies, respectively. Intrabodies are suitable to knockdown TAAs and neoantigens without off-target effects. RNA sequencing and proteome analysis of single tumor cells combined with computational methods is bringing forward the identification of new neoantigens for the selection of anti-cancer intrabodies, which can be easily performed using phage display antibody repertoires. For specifically delivering intrabodies into tumor cells, the usage of new capsid-modified adeno-associated viruses and lipid nanoparticles coupled with specific ligands to cell surface receptors can be used and might bring cancer intrabodies into the clinic.},
}
@article {pmid35891620,
year = {2022},
author = {Barka, GD and Lee, J},
title = {Advances in S gene targeted genome-editing and its applicability to disease resistance breeding in selected Solanaceae crop plants.},
journal = {Bioengineered},
volume = {13},
number = {6},
pages = {14646-14666},
pmid = {35891620},
issn = {2165-5987},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Disease Resistance/genetics ; Gene Editing/methods ; Genome, Plant/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; *Solanaceae/genetics ; Vegetables ; },
abstract = {Genome-editing tools for the development of traits to tolerate abiotic and biotic adversaries are the recently devised breeding techniques revolutionizing molecular breeding by addressing the issues of rapidness and precision. To that end, disease resistance development by disrupting disease susceptibility genes (S genes) to intervene in the biological mechanism of pathogenicity has significantly improved the techniques of molecular breeding. Despite the achievements in genome-editing aimed at the intervention of the function of susceptibility determinants or gene regulatory elements, off-target effects associated with yield-related traits are still the main setbacks. The challenges are attributed to the complexity of the inheritance of traits controlled by pleiotropic genes. Therefore, a more rigorous genome-editing tool with ultra-precision and efficiency for the development of broad-spectrum and durable disease resistance applied to staple crop plants is of critical importance in molecular breeding programs. The main objective of this article is to review the most impressive progresses achieved in resistance breeding against the main diseases of three Solanaceae crops (potato, Solanum tuberosum; tomato, Solanum lycopersicum and pepper, Capsicum annuum) using genome-editing by disrupting the sequences of S genes, their promoters, or pathogen genes. In this paper, we discussed the complexity and applicability of genome-editing tools, summarized the main disease of Solanaceae crops, and compiled the recent reports on disease resistance developed by S-gene silencing and their off-target effects. Moreover, GO count and gene annotation were made for pooled S-genes from biological databases. Achievements and prospects of S-gene-based next-generation breeding technologies are also discussed.},
}
@article {pmid35891539,
year = {2022},
author = {Boutin, L and Mosca, E and Iseni, F},
title = {Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9.},
journal = {Viruses},
volume = {14},
number = {7},
pages = {},
pmid = {35891539},
issn = {1999-4915},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Gene Editing/methods ; Point Mutation ; *Vaccinia virus/genetics ; },
abstract = {The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.},
}
@article {pmid35887335,
year = {2022},
author = {Zeng, D and Zheng, Z and Liu, Y and Liu, T and Li, T and Liu, J and Luo, Q and Xue, Y and Li, S and Chai, N and Yu, S and Xie, X and Liu, YG and Zhu, Q},
title = {Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools.},
journal = {International journal of molecular sciences},
volume = {23},
number = {14},
pages = {},
pmid = {35887335},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/genetics ; Escherichia coli/genetics ; *Gene Editing ; Guanine/analogs &amp; derivatives ; Humans ; *Oryza/genetics ; },
abstract = {CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5'-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.},
}
@article {pmid35886856,
year = {2022},
author = {Lu, S and Tao, T and Su, Y and Hu, J and Zhang, L and Wang, G and Li, X and Guo, X},
title = {Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses.},
journal = {International journal of molecular sciences},
volume = {23},
number = {14},
pages = {},
pmid = {35886856},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Enterotoxigenic Escherichia coli/genetics ; Enterotoxins/chemistry/genetics ; *Escherichia coli Infections/genetics ; *Escherichia coli Proteins/metabolism ; Gene Editing/methods ; Optical Imaging/methods ; Rats ; Rats, Sprague-Dawley ; Whole Genome Sequencing ; },
abstract = {Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311-mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311-mCherry challenge in Sprague-Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-ST[KO] was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-ST[KO] than with E. coli BE311. Therefore, the E. coli BE311-mCherry and E. coli BE311-ST[KO] strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.},
}
@article {pmid35885963,
year = {2022},
author = {Tanaka, J and Minkenberg, B and Poddar, S and Staskawicz, B and Cho, MJ},
title = {Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics.},
journal = {Genes},
volume = {13},
number = {7},
pages = {},
pmid = {35885963},
issn = {2073-4425},
mesh = {*Biolistics/methods ; CRISPR-Cas Systems/genetics ; Genomics ; Gold ; Mutation ; *Triticum/genetics ; },
abstract = {Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in the ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation efficiency bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 μm gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34 °C for 24 h resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34 °C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.},
}
@article {pmid35885915,
year = {2022},
author = {Zou, H and Yu, D and Yao, S and Ding, F and Li, J and Li, L and Li, X and Zhao, S and Pang, Y and Hao, H and Du, W and Zhao, X and Dai, Y and Zhu, H},
title = {Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System.},
journal = {Genes},
volume = {13},
number = {7},
pages = {},
pmid = {35885915},
issn = {2073-4425},
mesh = {Animals ; Binding Sites ; *CRISPR-Cas Systems ; Cattle ; Female ; Introns/genetics ; *Mammals/genetics ; Mutation ; Promoter Regions, Genetic ; Swine ; },
abstract = {BACKGROUND: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2-which disrupts the ZBED6-binding site-affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle.<br><br>METHODS: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression.<br><br>RESULTS: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming.<br><br>CONCLUSIONS: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding.},
}
@article {pmid35884169,
year = {2022},
author = {Hasan, M and Ahn, J},
title = {Evolutionary Dynamics between Phages and Bacteria as a Possible Approach for Designing Effective Phage Therapies against Antibiotic-Resistant Bacteria.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {11},
number = {7},
pages = {},
pmid = {35884169},
issn = {2079-6382},
abstract = {With the increasing global threat of antibiotic resistance, there is an urgent need to develop new effective therapies to tackle antibiotic-resistant bacterial infections. Bacteriophage therapy is considered as a possible alternative over antibiotics to treat antibiotic-resistant bacteria. However, bacteria can evolve resistance towards bacteriophages through antiphage defense mechanisms, which is a major limitation of phage therapy. The antiphage mechanisms target the phage life cycle, including adsorption, the injection of DNA, synthesis, the assembly of phage particles, and the release of progeny virions. The non-specific bacterial defense mechanisms include adsorption inhibition, superinfection exclusion, restriction-modification, and abortive infection systems. The antiphage defense mechanism includes a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system. At the same time, phages can execute a counterstrategy against antiphage defense mechanisms. However, the antibiotic susceptibility and antibiotic resistance in bacteriophage-resistant bacteria still remain unclear in terms of evolutionary trade-offs and trade-ups between phages and bacteria. Since phage resistance has been a major barrier in phage therapy, the trade-offs can be a possible approach to design effective bacteriophage-mediated intervention strategies. Specifically, the trade-offs between phage resistance and antibiotic resistance can be used as therapeutic models for promoting antibiotic susceptibility and reducing virulence traits, known as bacteriophage steering or evolutionary medicine. Therefore, this review highlights the synergistic application of bacteriophages and antibiotics in association with the pleiotropic trade-offs of bacteriophage resistance.},
}
@article {pmid35883629,
year = {2022},
author = {Huang, X and Yang, D and Zhang, J and Xu, J and Chen, YE},
title = {Recent Advances in Improving Gene-Editing Specificity through CRISPR-Cas9 Nuclease Engineering.},
journal = {Cells},
volume = {11},
number = {14},
pages = {},
pmid = {35883629},
issn = {2073-4409},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing ; Protein Engineering ; },
abstract = {CRISPR-Cas9 is the state-of-the-art programmable genome-editing tool widely used in many areas. For safe therapeutic applications in clinical medicine, its off-target effect must be dramatically minimized. In recent years, extensive studies have been conducted to improve the gene-editing specificity of the most popular CRISPR-Cas9 nucleases using different strategies. In this review, we summarize and discuss these strategies and achievements, with a major focus on improving the gene-editing specificity through Cas9 protein engineering.},
}
@article {pmid35883217,
year = {2022},
author = {Krause, AL and Stinear, TP and Monk, IR},
title = {Barriers to genetic manipulation of Enterococci: Current Approaches and Future Directions.},
journal = {FEMS microbiology reviews},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsre/fuac036},
pmid = {35883217},
issn = {1574-6976},
abstract = {Enterococcus faecalis and Enterococcus faecium are Gram-positive commensal gut bacteria that can also cause fatal infections. To study clinically relevant multi-drug resistant E. faecalis and E. faecium strains, methods are needed to overcome physical (thick cell wall) and enzymatic barriers that limit the transfer of foreign DNA and thus prevent facile genetic manipulation. Enzymatic barriers to DNA uptake identified in E. faecalis and E. faecium include type I, II and IV restriction modification systems and CRISPR-Cas. This review examines E. faecalis and E. faecium DNA defence systems and the methods with potential to overcome these barriers. DNA defence system bypass will allow the application of innovative genetic techniques to expedite molecular-level understanding of these important, but somewhat neglected, pathogens.},
}
@article {pmid35883203,
year = {2022},
author = {Ma, Y and Sun, W and Zhao, L and Yao, M and Wu, C and Su, P and Yang, L and Wang, G},
title = {Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology.},
journal = {Stem cell research &amp; therapy},
volume = {13},
number = {1},
pages = {353},
pmid = {35883203},
issn = {1757-6512},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Codon, Nonsense/genetics ; Factor IX/genetics/metabolism ; *Hemophilia A/genetics ; *Hemophilia B/genetics/therapy ; Humans ; Mice ; Mouse Embryonic Stem Cells/metabolism ; Mutation ; Technology ; },
abstract = {BACKGROUND: Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B.<br><br>METHODS: First, a donor plasmid with a mutation (F9 c.223 C&#x2009;>&#x2009;T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system.<br><br>RESULTS: The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system.<br><br>CONCLUSIONS: The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level.},
}
@article {pmid35882427,
year = {2022},
author = {Misra, CS and Rangu, SS and Phulsundar, RD and Bindal, G and Singh, M and Shashidhar, R and Saha, TK and Rao, AVSSN and Rath, D},
title = {An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.},
journal = {Journal of applied microbiology},
volume = {133},
number = {4},
pages = {2668-2677},
doi = {10.1111/jam.15737},
pmid = {35882427},
issn = {1365-2672},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/methods ; Ribonuclease P/genetics ; *SARS-CoV-2/genetics ; },
abstract = {AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost.<br><br>METHODS AND RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples.<br><br>CONCLUSIONS: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE.<br><br>Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.},
}
@article {pmid35882411,
year = {2022},
author = {Jiang, HJ and Tan, R and Jin, M and Yin, J and Gao, ZX and Li, HB and Shi, DY and Zhou, SQ and Chen, TJ and Yang, D and Li, JW},
title = {Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification.},
journal = {Biomedical and environmental sciences : BES},
volume = {35},
number = {6},
pages = {518-527},
doi = {10.3967/bes2022.069},
pmid = {35882411},
issn = {2214-0190},
mesh = {CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics ; *Vibrio parahaemolyticus/genetics ; },
abstract = {OBJECTIVE: To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .<br><br>METHODS: We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).<br><br>RESULTS: CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 [-18] M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 [2] CFU/g.<br><br>CONCLUSION: The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.},
}
@article {pmid35881862,
year = {2022},
author = {Coorey, B and Haase, F and Ellaway, C and Clarke, A and Lisowski, L and Gold, WA},
title = {Gene Editing and Rett Syndrome: Does It Make the Cut?.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {490-499},
doi = {10.1089/crispr.2022.0020},
pmid = {35881862},
issn = {2573-1602},
mesh = {Brain/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Rett Syndrome/genetics/metabolism/therapy ; },
abstract = {Rett syndrome (RTT) is a rare neurogenetic disorder caused by pathogenic variants of the Methyl CpG binding protein 2 (MECP2) gene. The RTT is characterized by apparent normal early development followed by regression of communicative and fine motor skills. Comorbidities include epilepsy, severe cognitive impairment, and autonomic and motor dysfunction. Despite almost 60 clinical trials and the promise of a gene therapy, no cure has yet emerged with treatment remaining symptomatic. Advances in understanding RTT has provided insight into the complexity and exquisite control of MECP2 expression, where loss of expression leads to RTT and overexpression leads to MECP2 duplication syndrome. Therapy development requires regulated expression that matches the spatiotemporal endogenous expression of MECP2 in the brain. Gene editing has revolutionized gene therapy and promises an exciting strategy for many incurable monogenic disorders, including RTT, by editing the native locus and retaining endogenous gene expression. Here, we review the literature on the currently available editing technologies and discuss their limitations and applicability to the treatment of RTT.},
}
@article {pmid35881633,
year = {2022},
author = {Chabas, H and Müller, V and Bonhoeffer, S and Regoes, RR},
title = {Epidemiological and evolutionary consequences of different types of CRISPR-Cas systems.},
journal = {PLoS computational biology},
volume = {18},
number = {7},
pages = {e1010329},
pmid = {35881633},
issn = {1553-7358},
mesh = {Bacteria ; *Bacteriophages/genetics ; Biological Evolution ; *CRISPR-Cas Systems/genetics ; },
abstract = {Bacteria have adaptive immunity against viruses (phages) in the form of CRISPR-Cas immune systems. Currently, 6 types of CRISPR-Cas systems are known and the molecular study of three of these has revealed important molecular differences. It is unknown if and how these molecular differences change the outcome of phage infection and the evolutionary pressure the CRISPR-Cas systems faces. To determine the importance of these molecular differences, we model a phage outbreak entering a population defending exclusively with a type I/II or a type III CRISPR-Cas system. We show that for type III CRISPR-Cas systems, rapid phage extinction is driven by the probability to acquire at least one resistance spacer. However, for type I/II CRISPR-Cas systems, rapid phage extinction is characterized by an a threshold-like behaviour: any acquisition probability below this threshold leads to phage survival whereas any acquisition probability above it, results in phage extinction. We also show that in the absence of autoimmunity, high acquisition rates evolve. However, when CRISPR-Cas systems are prone to autoimmunity, intermediate levels of acquisition are optimal during a phage outbreak. As we predict an optimal probability of spacer acquisition 2 factors of magnitude above the one that has been measured, we discuss the origin of such a discrepancy. Finally, we show that in a biologically relevant parameter range, a type III CRISPR-Cas system can outcompete a type I/II CRISPR-Cas system with a slightly higher probability of acquisition.},
}
@article {pmid35881350,
year = {2022},
author = {Zhang, X and Deitsch, KW and Dzikowski, R},
title = {CRISPR-Cas9 Editing of the Plasmodium falciparum Genome: Special Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2470},
number = {},
pages = {241-253},
pmid = {35881350},
issn = {1940-6029},
mesh = {Animals ; Antigenic Variation ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; *Malaria, Falciparum/genetics/parasitology ; *Parasites/metabolism ; Plasmodium falciparum/metabolism ; Protozoan Proteins/metabolism ; },
abstract = {The virulence of Plasmodium falciparum has been attributed in large part to the expression on the surface of infected red blood cells of the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Different forms of this protein are encoded by individual members of the multicopy gene family called var. Two attributes of the var gene family are key to the pathogenesis of malaria caused by P. falciparum; the hyperrecombinogenic nature of the var gene family that continuously generates antigenic diversity within parasite populations, and the ability of parasites to express only a single var gene at a time and to switch which gene is expressed over the course of an infection. The unique attributes of CRISPR-Cas9 have been applied to help decipher the molecular mechanisms underlying these unusual properties of the var gene family, both as a source of the DNA double strand breaks that initiate var gene recombination and as a way to recruit molecular probes to specific regions of the genome. In this chapter, we describe these somewhat unusual applications of the CRISPR-Cas9 system.},
}
@article {pmid35881349,
year = {2022},
author = {Adjalley, S and Lee, MCS},
title = {CRISPR/Cas9 Editing of the Plasmodium falciparum Genome.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2470},
number = {},
pages = {221-239},
pmid = {35881349},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Protozoan ; *Plasmodium ; *Plasmodium falciparum/genetics ; },
abstract = {The ability to interrogate gene function in Plasmodium parasites has been greatly enhanced by the advent of CRISPR/Cas9 systems. The breadth of genome manipulations ranges from single point mutations to large multigene deletions, however many of the technical considerations for designing CRISPR-based experiments are common to any editing approach. This review will discuss protocols for vector construction and donor design for genome editing P. falciparum, including pitfalls, variables, and validation methods.},
}
@article {pmid35880867,
year = {2022},
author = {Zhang, X and Zhang, C and Liang, C and Li, B and Meng, F and Ai, Y},
title = {CRISPR-Cas9 Based Bacteriophage Genome Editing.},
journal = {Microbiology spectrum},
volume = {10},
number = {4},
pages = {e0082022},
pmid = {35880867},
issn = {2165-0497},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Escherichia coli/genetics ; *Gene Editing/methods ; Vibrio ; },
abstract = {Bacteriophages are the most abundant entities in the biosphere, and many genomes of rare and novel bacteriophages have been sequenced to date. However, bacteriophage functional genomics has been limited by a lack of effective research methods. Clustered regularly interspaced short palindromic repeat/CRISPR-associated gene (CRISPR-Cas) systems provide bacteriophages with a new mechanism for attacking host bacteria as well as new tools for study bacteriophage functional genomics. It has been reported that bacteriophages are not only the driving elements of the evolution of prokaryote CRISPR arrays but also the targets of CRISPR-Cas systems. In this study, a phage genome editing platform based on the heterologous CRISPR-Cas9 system was theoretically designed, and a Vibrio natriegens phage TT4P2 genome editing experiment was carried out in vivo in the host bacterium Vibrio natriegens TT4 to achieve phage gene deletion and replacement. The construction of this phage genome editing platform is expected to solve the problem of insufficient research on phage gene diversity, promote the development of phage synthetic biology and nanotechnology, and even accelerate the discovery of new molecular biology tools. IMPORTANCE Bacteriophages are the most numerous organisms on earth and are known for their diverse lifestyles. Since the discovery of bacteriophages, our knowledge of the wider biological world has undergone immense and unforeseen changes. A variety of V. natriegens phages have been detected, but few have been well characterized. CRISPR was first documented in Escherichia coli in 1987. It has been reported that the CRISPR-Cas system can target and cleave invaders, including bacteriophages, in a sequence-specific manner. Here, we show that the construction of a phage genome editing platform based on the heterologous CRISPR-Cas9 system can achieve V. natriegens phage TT4P2 gene editing and can also improve the efficiency and accuracy of phage TT4P2 gene editing.},
}
@article {pmid35880832,
year = {2022},
author = {Dubois, M},
title = {And … cut! Identifying chromatin features affecting CRISPR-Cas9 activity in plants.},
journal = {Plant physiology},
volume = {190},
number = {2},
pages = {1074-1076},
pmid = {35880832},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Chromatin/genetics ; Gene Editing ; Plants/genetics ; },
}
@article {pmid35880637,
year = {2022},
author = {Shalaby, KE and Aouida, M and Gupta, V and Abdesselem, H and El-Agnaf, OMA},
title = {Development of non-viral vectors for neuronal-targeted delivery of CRISPR-Cas9 RNA-proteins as a therapeutic strategy for neurological disorders.},
journal = {Biomaterials science},
volume = {10},
number = {17},
pages = {4959-4977},
doi = {10.1039/d2bm00368f},
pmid = {35880637},
issn = {2047-4849},
mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Parkinson Disease/genetics/therapy ; Peptides/genetics ; RNA ; },
abstract = {The aging population contributes to an increase in the prevalence of neurodegenerative diseases, such as Parkinson's disease (PD). Due to the progressive nature of these diseases and an incomplete understanding of their pathophysiology, current drugs are inefficient, with a limited efficacy and major side effects. In this study, CRISPR-Cas9 RNA-proteins (RNP) composed of a Cas9 nuclease and single-guide RNA were delivered with a non-viral targeted delivery system to rescue the PD-associated phenotype in neuronal cells. Here, we fused the cell-penetrating amphipathic peptide, PepFect14 (PF14), with a short fragment of the rabies virus glycoprotein (C2) previously shown to have an affinity towards nicotinic acetylcholine receptors expressed on neuronal cells and on the blood-brain barrier. The resultant peptide, C2-PF14, was used to complex with and deliver RNPs to neuronal cells. We observed that RNP/C2-PF14 complexes formed nanosized, monodispersed, and nontoxic nanoparticles that led to a specific delivery into neuronal cells. α-Synuclein (α-syn) plays a major role in the pathology of PD and is considered to be a target for therapy. We demonstrated that CRISPR/Cas9 RNP delivered by C2-PF14 achieved α-syn gene (SNCA) editing in neuronal cells as determined by T7EI assay and western blotting. Furthermore, RNP/C2-PF14 relieved PD-associated toxicity in neuronal cells in vitro. This is a proof-of-concept towards simple and safe targeted genome-editing for treating PD and other neurological disorders.},
}
@article {pmid35880456,
year = {2022},
author = {Bharucha, N and Arias, A and Karakikes, I},
title = {The potential of CRISPR-Cas9 prime editing for cardiovascular disease research and therapy.},
journal = {Current opinion in cardiology},
volume = {37},
number = {5},
pages = {413-418},
doi = {10.1097/HCO.0000000000000985},
pmid = {35880456},
issn = {1531-7080},
support = {R01 HL139679/HL/NHLBI NIH HHS/United States ; R01 HL150414/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing/methods ; Humans ; },
abstract = {PURPOSE OF REVIEW: The ability to edit any genomic sequence has led to a better understanding of gene function and holds promise for the development of therapies for genetic diseases. This review describes prime editing - the latest CRISPR-Cas9 genome editing technology. Prime editing enables precise and accurate genome editing in terminally differentiated, postmitotic cells like cardiomyocytes, paving the way for therapeutic applications for genetic cardiomyopathies.<br><br>RECENT FINDINGS: Prime editing has been used to precisely insert up to 40 bases, create deletions up to 80 base pairs, and can perform all 12 possible transition and transversion base mutations with lower indels and off-target effects than other genome editing methods. The development of several software tools has simplified the experimental design and led to increased efficiency of the process. Improvements in methods for in-vivo delivery of the prime editing components should enable this technology to be used to edit the genome in patients.<br><br>SUMMARY: Prime editing has the potential to revolutionize the future of biomedical research and transform cardiovascular medicine. Improved understanding of the prime editing process and developments in agent design, efficacy and delivery will benefit scientists and patients and could be an effective way to cure cardiovascular diseases.},
}
@article {pmid35880295,
year = {2022},
author = {Wu, L and Xiao, H and Zhao, L and Cheng, Q},
title = {CRISPR/Cas9-mediated generation of fls2 mutant in Nicotiana benthamiana for investigating the flagellin recognition spectrum of diverse FLS2 receptors.},
journal = {Plant biotechnology journal},
volume = {20},
number = {10},
pages = {1853-1855},
pmid = {35880295},
issn = {1467-7652},
mesh = {*Arabidopsis/metabolism ; *Arabidopsis Proteins/genetics ; CRISPR-Cas Systems/genetics ; Flagellin/genetics ; Protein Kinases/metabolism ; Tobacco/genetics/metabolism ; },
}
@article {pmid35879771,
year = {2022},
author = {Li, J and Chen, L and Liang, J and Xu, R and Jiang, Y and Li, Y and Ding, J and Li, M and Qin, R and Wei, P},
title = {Development of a highly efficient prime editor 2 system in plants.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {161},
pmid = {35879771},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Plant ; *Oryza/genetics ; Plants/genetics ; },
abstract = {Low efficiency has seriously restricted the application of prime editing (PE) systems in plants. In this study, we develop an enhanced plant prime editor 2 system, enpPE2, by stacking various optimization strategies, including updating the PE architecture to PEmax and expressing engineered pegRNA with a structured motif under the control of a composite promoter. In T0 rice plants, enpPE2 exhibits editing frequencies of 64.58% to 77.08%, which are much higher than the frequencies with unmodified pPE2. Our results indicate that the enpPE2 system provides a robust and powerful tool for the precise modification of plant genomes.},
}
@article {pmid35878992,
year = {2022},
author = {Zhu, C and Bai, C and Gomez-Gomez, L and Sandmann, G and Baysal, C and Capell, T and Christou, P},
title = {Rice callus as a high-throughput platform for synthetic biology and metabolic engineering of carotenoids.},
journal = {Methods in enzymology},
volume = {671},
number = {},
pages = {511-526},
doi = {10.1016/bs.mie.2021.09.016},
pmid = {35878992},
issn = {1557-7988},
mesh = {Carotenoids/metabolism ; Humans ; Metabolic Engineering ; *Oryza/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Synthetic Biology ; beta Carotene/metabolism ; },
abstract = {Carotenoids are a large class of important lipid-soluble phytonutrients that are widely used as nutritional supplements due to their health-promoting activities. For example, β-carotene is the precursor for vitamin A synthesis, and astaxanthin is a powerful antioxidant. However, these carotenoids cannot be synthesized de novo by humans. These properties of β-carotene and astaxanthin make them attractive targets for metabolic engineering in rice (Oryza sativa) endosperm because rice is an important staple food in developing countries, and rice endosperm is devoid of carotenoids. In this chapter, we introduce an assay based on rice embryogenic callus for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation. The system is also an ideal platform to characterize cereal endosperm specific promoters. Four diverse cereal endosperm specific promoters were demonstrated to be active in rice callus despite their restricted activity in mature plants. The use of endosperm specific promoters that are expressed in rice callus, but remain silent in regenerated vegetative tissue, directs accumulation of carotenoids in the endosperm without interfering with plant growth. Rice callus is a useful platform for improving gene editing methods and for further optimizing pathway engineering. Thus, the rice callus platform provides a unique opportunity to test strategies for metabolic engineering of synthetic carotenoid pathways, leading to novel carotenoid-biofortified crops.},
}
@article {pmid35877342,
year = {2022},
author = {Xue, Y and Chen, Z and Zhang, W and Zhang, J},
title = {Engineering CRISPR/Cas13 System against RNA Viruses: From Diagnostics to Therapeutics.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {9},
number = {7},
pages = {},
pmid = {35877342},
issn = {2306-5354},
abstract = {Over the past decades, RNA viruses have been threatened people's health and led to global health emergencies. Significant progress has been made in diagnostic methods and antiviral therapeutics for combating RNA viruses. ELISA and RT-qPCR are reliable methods to detect RNA viruses, but they suffer from time-consuming procedures and limited sensitivities. Vaccines are effective to prevent virus infection and drugs are useful for antiviral treatment, while both need a relatively long research and development cycle. In recent years, CRISPR-based gene editing and modifying tools have been expanded rapidly. In particular, the CRISPR-Cas13 system stands out from the CRISPR-Cas family due to its accurate RNA-targeting ability, which makes it a promising tool for RNA virus diagnosis and therapy. Here, we review the current applications of the CRISPR-Cas13 system against RNA viruses, from diagnostics to therapeutics, and use some medically important RNA viruses such as SARS-CoV-2, dengue virus, and HIV-1 as examples to demonstrate the great potential of the CRISPR-Cas13 system.},
}
@article {pmid35877119,
year = {2022},
author = {Jin, P and Jin, Q and Wang, X and Zhao, M and Dong, F and Jiang, G and Li, Z and Shen, J and Zhang, W and Wu, S and Li, R and Zhang, Y and Li, X and Li, J},
title = {Large-Scale In Vitro and In Vivo CRISPR-Cas9 Knockout Screens Identify a 16-Gene Fitness Score for Improved Risk Assessment in Acute Myeloid Leukemia.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {28},
number = {18},
pages = {4033-4044},
doi = {10.1158/1078-0432.CCR-22-1618},
pmid = {35877119},
issn = {1557-3265},
mesh = {*CRISPR-Cas Systems ; Cohort Studies ; Humans ; *Leukemia, Myeloid, Acute/diagnosis/drug therapy/genetics ; Prognosis ; Risk Assessment ; },
abstract = {PURPOSE: The molecular complexity of acute myeloid leukemia (AML) presents a considerable challenge to implementation of clinical genetic testing for accurate risk stratification. Identification of better biomarkers therefore remains a high priority to enable improving established stratification and guiding risk-adapted therapy decisions.<br><br>EXPERIMENTAL DESIGN: We systematically integrated and analyzed the genome-wide CRISPR-Cas9 data from more than 1,000 in vitro and in vivo knockout screens to identify the AML-specific fitness genes. A prognostic fitness score was developed using the sparse regression analysis in a training cohort of 618 cases and validated in five publicly available independent cohorts (n = 1,570) and our RJAML cohort (n = 157) with matched RNA sequencing and targeted gene sequencing performed.<br><br>RESULTS: A total of 280 genes were identified as AML fitness genes and a 16-gene AML fitness (AFG16) score was further generated and displayed highly prognostic power in more than 2,300 patients with AML. The AFG16 score was able to distill downstream consequences of several genetic abnormalities and can substantially improve the European LeukemiaNet classification. The multi-omics data from the RJAML cohort further demonstrated its clinical applicability. Patients with high AFG16 scores had significantly poor response to induction chemotherapy. Ex vivo drug screening indicated that patients with high AFG16 scores were more sensitive to the cell-cycle inhibitors flavopiridol and SNS-032, and exhibited strongly activated cell-cycle signaling.<br><br>CONCLUSIONS: Our findings demonstrated the utility of the AFG16 score as a powerful tool for better risk stratification and selecting patients most likely to benefit from chemotherapy and alternative experimental therapies.},
}
@article {pmid35873151,
year = {2022},
author = {Dai, H and Wu, J and Yang, H and Guo, Y and Di, H and Gao, M and Wang, J},
title = {Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {942987},
pmid = {35873151},
issn = {2235-2988},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Defective Viruses/genetics/metabolism ; RNA, Messenger/metabolism ; *Viral Proteins/genetics/metabolism ; Virus Replication ; },
abstract = {Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenger RNA (mRNA) to interrupt host defense and facilitate the rapid proliferation of BHV-1. However, studies on the BHV-1 UL41 gene are limited, and BHV-1 defective virus construction using the CRISPR/Cas9 system is somewhat challenging. In this study, we rapidly constructed a BHV-1 UL41-deficient strain using the CRISPR/Cas9 system in BL primary bovine-derived cells. BHV-1 UL41-defective mutants were screened by Western blot analysis using specific polyclonal antibodies as the primary antibodies. During the isolation and purification of the defective strain, a mixed virus pool edited by an efficient single-guide RNA (sgRNA) showed a plaque number reduction. Viral growth property assessment showed that BHV-1 UL41 was dispensable for replication, but the UL41-defective strain exhibited early and slowed viral replication. Furthermore, the BHV-1 UL41-deficient strain exhibited enhanced sensitivity to temperature and acidic environments. The BHV-1 UL41-deficient strain regulated viral and host mRNA levels to affect viral replication.},
}
@article {pmid35871983,
year = {2022},
author = {Soh, JH and Balleza, E and Abdul Rahim, MN and Chan, HM and Mohd Ali, S and Chuah, JKC and Edris, S and Atef, A and Bahieldin, A and Ying, JY and Sabir, JSM},
title = {CRISPR-based systems for sensitive and rapid on-site COVID-19 diagnostics.},
journal = {Trends in biotechnology},
volume = {40},
number = {11},
pages = {1346-1360},
pmid = {35871983},
issn = {1879-3096},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques ; Pandemics ; Point-of-Care Systems ; SARS-CoV-2/genetics ; },
abstract = {The COVID-19 pandemic has strained healthcare systems. Sensitive, specific, and timely COVID-19 diagnosis is crucial for effective medical intervention and transmission control. RT-PCR is the most sensitive/specific, but requires costly equipment and trained personnel in centralized laboratories, which are inaccessible to resource-limited areas. Antigen rapid tests enable point-of-care (POC) detection but are significantly less sensitive/specific. CRISPR-Cas systems are compatible with isothermal amplification and dipstick readout, enabling sensitive/specific on-site testing. However, improvements in sensitivity and workflow complexity are needed to spur clinical adoption. We outline the mechanisms/strategies of major CRISPR-Cas systems, evaluate their on-site diagnostic capabilities, and discuss future research directions.},
}
@article {pmid35871616,
year = {2022},
author = {Zhu, Q and Xu, C and Zhang, S and Xie, N and Pang, X and Lü, J},
title = {[Advances in utilizing the endogenous CRISPR-Cas system for genome editing of lactic acid bacteria].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {7},
pages = {2447-2458},
doi = {10.13345/j.cjb.210958},
pmid = {35871616},
issn = {1872-2075},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Homologous Recombination ; *Lactobacillales/genetics ; Mammals/genetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is an efficient and precise gene editing tool. The development of this technology has promoted genome editing into a rapid development stage. The most widely used Cas9 protein is SpyCas9 from Streptococcus pyogenes. As a "gene scissors", the SpyCas9 protein is widely used in eukaryotes including mammals and plants. However, the application of this protein in some lactic acid bacteria (LAB) is still hampered by many factors. It has been identified that LAB genomes possess many types of CRISPR system and are rich in uncharacterized Cas proteins. Genome editing of LAB is possible by repurposing the endogenous CRISPR-Cas systems in LAB combined with exogenous single guide RNA (sgRNA) and homologous recombination template. This method employs its endogenous CRISPR-Cas system for gene editing, which has the advantages including easiness for transformation due to the relatively small targeting vector, and no concern about the toxicity of heterologous Cas9 to host cells. Compared to CRISPR-SpyCas9, the endogenous CRISPR-Cas system is more suitable for genome editing of LAB, and it may become the main genome editing tool for some LAB in the future. This article summarizes the advances in this field.},
}
@article {pmid35870723,
year = {2022},
author = {Pant, S and Ritika, and Nag, P and Ghati, A and Chakraborty, D and Maximiano, MR and Franco, OL and Mandal, AK and Kuila, A},
title = {Employment of the CRISPR/Cas9 system to improve cellulase production in Trichoderma reesei.},
journal = {Biotechnology advances},
volume = {60},
number = {},
pages = {108022},
doi = {10.1016/j.biotechadv.2022.108022},
pmid = {35870723},
issn = {1873-1899},
mesh = {CRISPR-Cas Systems/genetics ; *Cellulase/genetics ; Employment ; Hypocreales ; *Trichoderma/genetics/metabolism ; },
abstract = {Trichoderma reesei has been explored intensively in the laboratory and on an industrial scale for its highly potent cellulase secretion machinery since its characterization over 70 years ago. Emergence of new genetic tools over the past decade has strengthened the understanding of mechanism involved in transcription of cellulase genes in fungi and provided a boost to edit them at molecular level. Since several transcriptional factors work synergistically for cellulase expression in fungi; engineering of cellulase secretome for enhanced cellulase titer require combined manipulation of these factors. In the same context, CRISPR/Cas9 has emerged as a powerful, versatile genetic engineering tool for multiplex gene editing in fungi. It is true that considerable efforts with CRISPR technologies have largely developed fungal genetic engineering, but its application in fungi is still challenging and limited. The present review illustrates the precision, strengths and challenges of using CRISPR/Cas9 technology for cellulase engineering in T. reesei, highlighting key strategies that could be employed for strain improvement.},
}
@article {pmid35870449,
year = {2022},
author = {Liu, B and Jing, Z and Zhang, X and Chen, Y and Mao, S and Kaundal, R and Zou, Y and Wei, G and Zang, Y and Wang, X and Lin, W and Di, M and Sun, Y and Chen, Q and Li, Y and Xia, J and Sun, J and Lin, CP and Huang, X and Chi, T},
title = {Large-scale multiplexed mosaic CRISPR perturbation in the whole organism.},
journal = {Cell},
volume = {185},
number = {16},
pages = {3008-3024.e16},
doi = {10.1016/j.cell.2022.06.039},
pmid = {35870449},
issn = {1097-4172},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; Mice ; *RNA, Guide/genetics/metabolism ; Transgenes ; },
abstract = {Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.},
}
@article {pmid35870342,
year = {2022},
author = {Chukamnerd, A and Pomwised, R and Jeenkeawpiam, K and Sakunrang, C and Chusri, S and Surachat, K},
title = {Genomic insights into blaNDM-carrying carbapenem-resistant Klebsiella pneumoniae clinical isolates from a university hospital in Thailand.},
journal = {Microbiological research},
volume = {263},
number = {},
pages = {127136},
doi = {10.1016/j.micres.2022.127136},
pmid = {35870342},
issn = {1618-0623},
mesh = {Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Carbapenems/pharmacology ; Genomics ; Hospitals ; Humans ; *Klebsiella Infections/drug therapy/microbiology ; *Klebsiella pneumoniae/genetics ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Plasmids/genetics ; Thailand ; Virulence Factors ; beta-Lactamases/genetics ; },
abstract = {The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates is a serious threat to global health. Here, we elucidate the genetic features of blaNDM-carrying CRKP clinical isolates from a university hospital in Thailand. The entire genomes of 19 CRKP isolates were extracted and then sequenced using the MGISEQ200 platform. Using various bioinformatics tools, we analyzed the antimicrobial resistance (AMR), virulence factors, gene transfer, bacterial defense mechanisms, and genomic diversity of the CRKP isolates. The sequence type (ST) 16 was found in most of the isolates, along with carriages of the blaNDM-1, blaOXA-232, and blaCTX-M-15 genes. The IncFIB(pQil), Col440II, and ColKP3 plasmids were identified with high frequency. The CRKP isolates harbored genes encoding for virulence factors such as adherence, biofilm formation, immune evasion, and iron uptake. The CRISPR-Cas region in the CRKP9 isolate consisted of 28 distinct spacer sequences. The genomes of the CRKP isolates presented restriction-modification (R-M) sites (M.Kpn34618Dcm and M.Kpn928I) and integrated bacteriophage genomes (Klebsiella phage ST16-OXA48phi5.4 and Enterobacteria phage mEp390). Bottromycin and sactipeptides were also identified. The isolates could be separated into three clades according to STs and pairwise single nucleotide polymorphism (SNP) distance. Pairwise average nucleotide identity (ANI) values revealed intra-species. These findings support the importance of whole-genome sequencing (WGS) to the rapid and accurate genomic analysis of clinical isolates of CRKP.},
}
@article {pmid35870335,
year = {2022},
author = {Li, Q and Li, X and Zhou, P and Chen, R and Xiao, R and Pang, Y},
title = {Split aptamer regulated CRISPR/Cas12a biosensor for 17β-estradiol through a gap-enhanced Raman tags based lateral flow strategy.},
journal = {Biosensors &amp; bioelectronics},
volume = {215},
number = {},
pages = {114548},
doi = {10.1016/j.bios.2022.114548},
pmid = {35870335},
issn = {1873-4235},
mesh = {*Aptamers, Nucleotide/chemistry ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Estradiol/chemistry ; },
abstract = {It is significant to exploit the full potential of CRISPR/Cas based biosensor for non-nucleic-acid targets. Here, we developed a split aptamer regulated CRISPR/Cas12a and gap-enhanced Raman tags based lateral flow biosensor for small-molecule target, 17β-estradiol. In this assay, one split aptamer of 17β-estradiol was designed to complement with crRNA of Cas12a so that the trans-cleavage ability of CRISPR/Cas12a can be regulated by the competitive binding of 17β-estradiol and split aptamers. Through integration of the signal amplification ability of CRISPR/Cas12a and the ultra-sensitive gap-enhanced Raman tags based lateral flow assay, a visible-SERS dual mode determination of 17β-estradiol can be established. 17β-estradiol can be visibly recognized as low as 10 pM and accurately quantified with a detection limit of 180 fM by SERS signals, which is at least 10[3]-fold lower than that of the previous immunoassay lateral flow strategies. Our assay provides a novel perspective to develop split aptamer regulated CRISPR/Cas12a coupling with SERS lateral flow strips for ultrasensitive and easy-to-use non-nucleic-acid targets detection.},
}
@article {pmid35870248,
year = {2022},
author = {Sun, Y and Fu, J and Yang, J and Zhao, J and Rong, J},
title = {Generation of a RRAGA knockout human iPSC line GIBHi002-A-5 using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {63},
number = {},
pages = {102859},
doi = {10.1016/j.scr.2022.102859},
pmid = {35870248},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems ; *Cell Line ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mechanistic Target of Rapamycin Complex 1/genetics/metabolism ; *Monomeric GTP-Binding Proteins/deficiency/genetics/metabolism ; },
abstract = {Ras-related GTP-binding protein A (RagA), encoded by RRAGA gene, initially senses the availability of cellular amino acids (e.g., leucine) and controls the translocation of mTORC1 to the lysosomal membrane. RagA overexpression appears to be associated with the onset of depression. To discover the biological roles of RagA, we employed the CRISPR/Cas9 system to generate a RRAGA gene knockout stem cell line from human induced pluripotent stem cell (iPSC) iPSN0003. Such RRAGA knockout iPSC cell line may help the development of new therapeutics for depression.},
}
@article {pmid35868558,
year = {2022},
author = {Chaya, T and Maeda, Y and Sugimura, R and Okuzaki, D and Watanabe, S and Varner, LR and Motooka, D and Gyoten, D and Yamamoto, H and Kato, H and Furukawa, T},
title = {Multiple knockout mouse and embryonic stem cell models reveal the role of miR-124a in neuronal maturation.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {9},
pages = {102293},
pmid = {35868558},
issn = {1083-351X},
mesh = {Animals ; *DNA-Binding Proteins/genetics/metabolism ; Mice ; Mice, Knockout ; *MicroRNAs/genetics/metabolism ; Mouse Embryonic Stem Cells ; *Neurogenesis/genetics ; *Neurons/cytology/metabolism ; },
abstract = {MicroRNA-124a (miR-124a) is one of the most abundantly expressed microRNAs in the central nervous system and is encoded in mammals by the three genomic loci miR-124a-1/2/3; however, its in vivo roles in neuronal development and function remain ambiguous. In the present study, we investigated the effect of miR-124a loss on neuronal differentiation in mice and in embryonic stem (ES) cells. Since miR-124a-3 exhibits only background expression levels in the brain and we were unable to obtain miR-124a-1/2/3 triple knockout (TKO) mice by mating, we generated and analyzed miR-124a-1/2 double knockout (DKO) mice. We found that these DKO mice exhibit perinatal lethality. RNA-seq analysis demonstrated that the expression levels of proneural and neuronal marker genes were almost unchanged between the control and miR-124a-1/2 DKO brains; however, genes related to neuronal synaptic formation and function were enriched among downregulated genes in the miR-124a-1/2 DKO brain. In addition, we found the transcription regulator Tardbp/TDP-43, loss of which leads to defects in neuronal maturation and function, was inactivated in the miR-124a-1/2 DKO brain. Furthermore, Tardbp knockdown suppressed neurite extension in cultured neuronal cells. We also generated miR-124a-1/2/3 TKO ES cells using CRISPR-Cas9 as an alternative to TKO mice. Phase-contrast microscopic, immunocytochemical, and gene expression analyses showed that miR-124a-1/2/3 TKO ES cell lines were able to differentiate into neurons. Collectively, these results suggest that miR-124a plays a role in neuronal maturation rather than neurogenesis in vivo and advance our understanding of the functional roles of microRNAs in central nervous system development.},
}
@article {pmid35868252,
year = {2022},
author = {},
title = {Meet the authors: Joseph E. Peters and Marcin Nowotny.},
journal = {Molecular cell},
volume = {82},
number = {14},
pages = {2533-2535},
doi = {10.1016/j.molcel.2022.07.001},
pmid = {35868252},
issn = {1097-4164},
mesh = {*DNA Transposable Elements ; },
abstract = {We talk to Joseph Peters and Marcin Nowotny about their paper, "Structural basis of transposon end recognition explains central features of Tn7 transposition systems," their love for transposons, and how their collaboration was established and led to the success of the project.},
}
@article {pmid35867255,
year = {2022},
author = {Seow, WQ and Agarwal, P and Wang, KC},
title = {CLOuD9: CRISPR-Cas9-Mediated Technique for Reversible Manipulation of Chromatin Architecture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2532},
number = {},
pages = {293-309},
pmid = {35867255},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Chromatin/genetics ; Epigenomics ; Gene Expression Regulation ; Genome ; },
abstract = {The spatial organization of the genome plays a critical role in cell-specific biological functions such as gene expression. Existing genome-wide technologies reveal a dynamic interplay between chromatin looping and gene regulation, but the mechanisms by which regulatory interactions between genetic elements are established or maintained remain unclear. Here, we present CLOuD9, a CRISPR-based technology that can create de novo, pairwise chromatin interactions in cells. This technique for chromatin loop reorganization employs dCas9-targeting and ABI1-PYL heterodimerization. It is reversible, but can also establish epigenetic memory under certain conditions, which provides a way to dissect gene regulation mechanisms.},
}
@article {pmid35867229,
year = {2022},
author = {MacLeod, G and Rajakulendran, N and Angers, S},
title = {Identification of Drug Resistance Mechanisms Using Genome-Wide CRISPR-Cas9 Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2535},
number = {},
pages = {141-156},
pmid = {35867229},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Drug Resistance ; Gene Editing/methods ; Gene Knockout Techniques ; Humans ; Mammals/genetics ; *RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 genome editing provides a means for simple and scalable production of gene knockouts in mammalian cell lines. The development of guide RNA (gRNA) libraries targeting tens of thousands of genes has allowed researchers to produce pools of cells, each containing a single gene knockout for use in genetic screens. In addition to assessing the effect of gene knockout on cell proliferation, CRISPR-Cas9 genetic screens can be used to assess gene-drug interactions. Here, we outline a protocol for performing positive and negative selection genome-wide CRISPR-Cas9 screens for identifying gene knockouts that cause drug resistance and hypersensitivity. This protocol is designed for the use of the TKOv3 library in human cell lines, but can be readily adapted for different libraries.},
}
@article {pmid35867109,
year = {2022},
author = {Li, X and Li, R and Yuan, Z and Zhu, Z and Xu, W and Wang, Y and Zhang, D and Yang, L},
title = {One Versatile Cas9-Integrated Single-Tube Duplex Quantitative Real-Time PCR System for Rapid Analysis of CRISPR/Cas-Induced Mutants.},
journal = {Analytical chemistry},
volume = {94},
number = {30},
pages = {10832-10840},
doi = {10.1021/acs.analchem.2c01837},
pmid = {35867109},
issn = {1520-6882},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Oryza/genetics ; RNA, Guide ; Real-Time Polymerase Chain Reaction ; },
abstract = {Clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 gene editing has become a common tool for rapid crop and animal breeding, but efficiently screening out and genotyping for the CRISPR/Cas9-induced mutant lines at a low cost remains challenging. Using rice (Oryza sativa L.) samples genetically edited at the Waxy locus as an example, we developed a single-tube duplex quantitative real-time PCR assisted by an in vitro CRISPR/Cas9 cleavage (Cc-qPCR) method to screen for expected genetically edited lines, identify genotypes, and evaluate gene-editing frequency. In Cc-qPCR, genomic DNA is first cleaved at the target site by the single-guide RNA (sgRNA)/Cas9 complex and then quantified with qPCR to assess for the presence of a mutant and identify sample genotypes. Our findings suggest that Cc-qPCR can successfully identify mutants with small insertions or deletions (indels), even in mutant lines with single-base indels or substitutions. Cc-qPCR was also able to successfully identify heterozygous and homozygous mutants. The sensitivity of Cc-qPCR was determined to be as low as 0.5%, indicating that the method could be used to evaluate the editing efficiency of gene-editing systems. After testing our novel method on Waxy locus-edited rice offspring, our results show that Cc-qPCR is an accurate and effective approach to rapidly identify expected mutants and their genotypes and to evaluate editing efficiency. This method will prove useful for increasing the efficiency and range of molecular breeding techniques.},
}
@article {pmid35865929,
year = {2022},
author = {Li, Y and Wang, Y and Liu, J},
title = {Genomic Insights Into the Interspecific Diversity and Evolution of Mobiluncus, a Pathogen Associated With Bacterial Vaginosis.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {939406},
pmid = {35865929},
issn = {1664-302X},
abstract = {Bacterial vaginosis (BV) is a common vaginal infection and has been associated with increased risk for a wide array of health issues. BV is linked with a variety of heterogeneous pathogenic anaerobic bacteria, among which Mobiluncus is strongly associated with BV diagnosis. However, their genetic features, pathogenicity, interspecific diversity, and evolutionary characters have not been illustrated at genomic level. The current study performed phylogenomic and comparative genomic analyses of Mobiluncus. Phylogenomic analyses revealed remarkable phylogenetic distinctions among different species. Compared with M. curtisii, M. mulieris had a larger genome and pangenome size with more insertion sequences but less CRISPR-Cas systems. In addition, these two species were diverse in profile of virulence factors, but harbored similar antibiotic resistance genes. Statistically different functional genome profiles between strains from the two species were determined, as well as correlations of some functional genes/pathways with putative pathogenicity. We also showed that high levels of horizontal gene transfer might be an important strategy for species diversification and pathogenicity. Collectively, this study provides the first genome sequence level description of Mobiluncus, and may shed light on its virulence/pathogenicity, functional diversification, and evolutionary dynamics. Our study could facilitate the further investigations of this important pathogen, and might improve the future treatment of BV.},
}
@article {pmid35864956,
year = {2022},
author = {Huang, S and Ma, Z and Zhou, Q and Wang, A and Gong, Y and Li, Z and Wang, S and Yan, Q and Wang, D and Hou, B and Zhang, C},
title = {Genome-Wide CRISPR/Cas9 Library Screening Identified that DUSP4 Deficiency Induces Lenvatinib Resistance in Hepatocellular Carcinoma.},
journal = {International journal of biological sciences},
volume = {18},
number = {11},
pages = {4357-4371},
pmid = {35864956},
issn = {1449-2288},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Carcinoma, Hepatocellular/drug therapy/genetics/metabolism ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/genetics ; *Dual-Specificity Phosphatases/genetics/metabolism ; Humans ; *Liver Neoplasms/drug therapy/genetics/metabolism ; Mice ; Mitogen-Activated Protein Kinase Kinases/genetics/metabolism ; Phenylurea Compounds ; Protein Kinase Inhibitors/pharmacology ; *Quinolines/pharmacology/therapeutic use ; },
abstract = {Background: Lenvatinib is in a first-line therapy for advanced hepatocellular carcinoma (HCC). However, drug resistance is one of the principal obstacles for treatment failure. The molecular mechanism of Lenvatinib resistance has not been well investigated. Materials and methods: A genome-wide CRISPR/Cas9 knockout screening system was established and bioinformatic analysis was used to identify critical genes associated with Lenvatinib resistance. Cell proliferation assays, colony formation assays and cell migration assays were performed to investigate the effect of drug resistance associated genes, particularly DUSP4, on cancer cell malignant behavior during Lenvatinib treatment. In vivo experiments were conducted by using a xenograft mouse model. Results: We identified six genes that were associated with Lenvatinib resistance in HCC, including DUSP4, CCBL1, DHDH, CNTN2, NOS3 and TNF. DUSP4 was found to be significantly decreased at the mRNA and protein levels in Lenvatinib resistant HCC cells. DUSP4 knockout enhanced HCC cell survival, cell proliferation and migration during Lenvatinib treatment in vitro and in vivo, accompanied by regulation of p-ERK and p-MEK levels. This finding implied that DUSP4 deficiency induced Lenvatinib resistance. Interestingly, DUSP4 deficiency induced Lenvatinib resistance was abrogated by the MEK inhibitor Selumetinib, implying that MEK phosphorylation and DUSP4-inhibition dependent ERK activation were required for drug resistance. Finally, we found that DUSP4 deficiency was associated with HCC prognosis and response to Lenvatinib based on clinical data. Conclusions: DUSP4 deficiency mediates Lenvatinib resistance by activating MAPK/ERK signaling and combination therapy using Lenvatinib and MEK inhibitors may be a promising therapeutic strategy for overcoming Lenvatinib resistance.},
}
@article {pmid35862996,
year = {2022},
author = {Domazetovska, A and Jensen, SO and Gray, M and Radzieta, M and Maley, M},
title = {Culture-Free Phylogenetic Analysis of Legionella pneumophila Using Targeted CRISPR/Cas9 Next-Generation Sequencing.},
journal = {Microbiology spectrum},
volume = {10},
number = {4},
pages = {e0035922},
pmid = {35862996},
issn = {2165-0497},
mesh = {CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; *Legionella ; *Legionella pneumophila/genetics ; *Legionnaires' Disease/epidemiology ; Phylogeny ; },
abstract = {Currently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method's utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms. IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires' disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.},
}
@article {pmid35862665,
year = {2022},
author = {Dai, K and Fu, H and Guo, X and Qu, C and Lan, Y and Wang, J},
title = {Exploiting the Type I-B CRISPR Genome Editing System in Thermoanaerobacterium aotearoense SCUT27 and Engineering the Strain for Enhanced Ethanol Production.},
journal = {Applied and environmental microbiology},
volume = {88},
number = {15},
pages = {e0075122},
pmid = {35862665},
issn = {1098-5336},
mesh = {Biofuels ; CRISPR-Cas Systems ; Ethanol/metabolism ; *Gene Editing/methods ; *Thermoanaerobacterium/genetics/metabolism ; Xylose/metabolism ; },
abstract = {Thermoanaerobacterium aotearoense strain SCUT27 is a potential industrial biofuel-producing strain because of its broad substrate spectrum, especially the ability to co-use glucose and xylose. The bottleneck hindering the development of strain SCUT27 is the lack of selective markers for polygene manipulation in this thermophilic bacterium. In this study, the endogenous type I-B clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was developed for multiplex genome editing of strain SCUT27. The protospacer-adjacent motif was identified by in silico analysis and verified with orotidine-5'-phosphate decarboxylase (pyrF) or lactate dehydrogenase (ldh) as the editing target. The type I-B CRISPR/Cas system was functional in strain SCUT27 with 58.3% to 100% editing efficiency. A multiplex genome editing method based on thymidine kinase (tdk) as a negative selection marker was developed, and strain SCUT27/Δtdk/Δldh/ΔargR, in which ldh and the arginine repressor (argR) were knocked out successively, was successfully obtained. Strain SCUT27/Δtdk/Δldh/ΔargR exhibited prominent advantages over wild-type SCUT27 in ethanol production, with significantly improved ability to metabolize xylose. IMPORTANCE Thermophilic microbes have attracted great attention as potential candidates for production of biofuels and chemicals from lignocellulose because of their thermal tolerance and wide substrate spectra. The ability to edit multiple genes using the native type I-B CRISPR/Cas system would speed up engineering of Thermoanaerobacterium aotearoense strain SCUT27 for higher ethanol production from lignocellulosic hydrolysates. Here, we produced a mutant strain, T. aotearoense SCUT27/Δtdk/Δldh/ΔargR, using the native CRISPR/Cas system. The engineered strain showed satisfactory performance with improved ethanol productivity from various lignocellulosic hydrolysates. Our data lay the foundations for development of this thermophilic microbe into an excellent ethanol producer using lignocellulosic hydrolysates. The methods described here may also provide a reference to develop multigene editing methods for other microorganisms.},
}
@article {pmid35862015,
year = {2022},
author = {Bchetnia, M and Dionne Gagné, R and Powell, J and Morin, C and McCuaig, C and Dupérée, A and Germain, L and Tremblay, JP and Laprise, C},
title = {Allele-Specific Inactivation of an Autosomal Dominant Epidermolysis Bullosa Simplex Mutation Using CRISPR-Cas9.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {586-597},
doi = {10.1089/crispr.2021.0132},
pmid = {35862015},
issn = {2573-1602},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; *Epidermolysis Bullosa Simplex/genetics/metabolism/pathology ; Gene Editing ; Humans ; Mutation ; Phenotype ; },
abstract = {Epidermolysis bullosa simplex (EBS) is a rare mechanobullous disease caused by dominant-negative mutations in either keratin 5 (KRT5) or keratin 14 (KRT14) genes. Until now, there is no cure for EBS and the care is primarily palliative. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system raised hope for the treatment of EBS and many other autosomal dominant diseases by mutant allele-specific gene disruption. In this study, we aim to disrupt the mutant allele for the heterozygous EBS pathogenic variation c.449T>C (p.Leu150Pro) within KRT5. This mutation generates, naturally, a novel protospacer-adjacent motif for the endonuclease Streptococcus pyogenes Cas9. Thus, we designed a single-guide RNA that guides the Cas9 to introduce a DNA cleavage of the mutant allele in patient's keratinocytes. Then, transfected cells were single-cell cloned and analyzed by deep sequencing. The expression of KRT5 and KRT14 was quantified, and the keratin intermediate filament stability was assessed. Results showed successful stringent mutant allele-specific knockout. An absence of synthesis of mutant transcript was further confirmed indicating permanent mutant allele-specific inactivation. Edited EBS patient keratinocytes produced a lower amount of K5 and K14 proteins compared with nonedited EBS cells, and no disturbance of cellular properties was observed.},
}
@article {pmid35861526,
year = {2022},
author = {Nie, M and Dong, Y and Cao, Q and Zhao, D and Ji, S and Huang, H and Jiang, M and Liu, G and Liu, Y},
title = {CRISPR Contributes to Adhesion, Invasion, and Biofilm Formation in Streptococcus agalactiae by Repressing Capsular Polysaccharide Production.},
journal = {Microbiology spectrum},
volume = {10},
number = {4},
pages = {e0211321},
pmid = {35861526},
issn = {2165-0497},
mesh = {Biofilms ; CRISPR-Cas Systems ; *Operon ; Polysaccharides ; *Streptococcus agalactiae/genetics ; Virulence ; },
abstract = {The clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) system functions classically as a prokaryotic defense system against invading mobile genetic elements, such as phages, plasmids, and viruses. Our previous study revealed that CRISPR deletion caused increased transcription of capsular polysaccharide (CPS) synthesis-related genes and severely attenuated virulence in the hypervirulent piscine Streptococcus agalactiae strain GD201008-001. Here, we found that CRISPR deficiency resulted in reduced adhesion, invasion, and biofilm formation abilities in this strain by upregulating the production of CPS. However, enhanced CPS production was not responsible for the attenuated phenotype of the ΔCRISPR mutant. RNA degradation assays indicated that inhibited transcription of the cps operon by CRISPR RNA (crRNA) was not due to the base pairing of the crRNA with the cps mRNA but to the repression of the promoter activity of cpsA, which is a putative transcriptional regulator of the capsule locus. IMPORTANCE Beyond protection from invading nucleic acids, CRISPR-Cas systems have been shown to have an important role in regulating bacterial endogenous genes. In this study, we demonstrate that crRNA inhibits the transcription of the cps operon by repressing the activity of promoter PcpsA, leading to increases in the abilities of adhesion, invasion, and biofilm formation in S. agalactiae. This study highlights the regulatory role of crRNA in bacterial physiology and provides a new explanation for the mechanism of crRNA-mediated endogenous gene regulation in S. agalactiae.},
}
@article {pmid35861371,
year = {2022},
author = {Yang, C and Dong, X and Ma, Z and Li, B and Bi, C and Zhang, X},
title = {Pioneer Factor Improves CRISPR-Based C-To-G and C-To-T Base Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {9},
number = {26},
pages = {e2202957},
pmid = {35861371},
issn = {2198-3844},
mesh = {Alkanesulfonic Acids ; *CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Cytosine ; *Gene Editing ; },
abstract = {Base editing events in eukaryote require a compatible chromatin environment, but there is little research on how chromatin factors contribute to the editing efficiency or window. By engineering BEs (base editors) fused with various pioneer factors, the authors found that SOX2 substantially increased the editing efficiency for GBE and CBE. While SoxN-GBE (SOX2-NH3-GBE) improved the editing efficiency at overall cytosines of the protospacer, SoxM-GBE/CBE (SOX2-Middle-GBE/CBE) enabled the higher base editing at PAM-proximal cytosines. By separating functional domains of SOX2, the SadN-GBE (SOX2 activation domain-NH3-GBE) is constructed for higher editing efficiency and SadM-CBE for broader editing window to date. With the DNase I assay, it is also proved the increased editing efficiency is most likely associated with the induction of chromatin accessibility by SAD. Finally, SadM-CBE is employed to introduce a stop codon in the proto-oncogene MYC, at a locus rarely edited by previous editors with high efficiency. In this work, a new class of pioneer-BEs is constructed by fusion of pioneer factor or its functional domains, which exhibits higher editing efficiency or broader editing window in eukaryote.},
}
@article {pmid35859738,
year = {2022},
author = {Wang, P and Li, M and Dong, L and Zhang, C and Xie, W},
title = {Comparative Genomics of Thaumarchaeota From Deep-Sea Sponges Reveal Their Niche Adaptation.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {869834},
pmid = {35859738},
issn = {1664-302X},
abstract = {Thaumarchaeota account for a large portion of microbial symbionts in deep-sea sponges and are even dominant in some cases. In this study, we investigated three new sponge-associated Thaumarchaeota from the deep West Pacific Ocean. Thaumarchaeota were found to be the most dominant phylum in this sponge by both prokaryotic 16S rRNA amplicons and metagenomic sequencing. Fifty-seven published Thaumarchaeota genomes from sponges and other habitats were included for genomic comparison. Similar to shallow sponge-associated Thaumarchaeota, those Thaumarchaeota in deep-sea sponges have extended genome sizes and lower coding density compared with their free-living lineages. Thaumarchaeota in deep-sea sponges were specifically enriched in genes related to stress adapting, symbiotic adhesion and stability, host-microbe interaction and protein transportation. The genes involved in defense mechanisms, such as the restriction-modification system, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and toxin-antitoxin system were commonly enriched in both shallow and deep sponge-associated Thaumarchaeota. Our study demonstrates the significant effects of both depth and symbiosis on forming genomic characteristics of Thaumarchaeota, and provides novel insights into their niche adaptation in deep-sea sponges.},
}
@article {pmid35858770,
year = {2022},
author = {Zhang, C and Wei, ZX and Wang, M and Chen, YS and He, ZY},
title = {Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis.},
journal = {Yi chuan = Hereditas},
volume = {44},
number = {7},
pages = {581-590},
doi = {10.16288/j.yczz.22-037},
pmid = {35858770},
issn = {0253-9772},
mesh = {Animals ; CRISPR-Cas Systems ; Humans ; Mammals/metabolism ; Melanins/genetics ; *Melanoma/genetics/metabolism ; Monophenol Monooxygenase/metabolism ; *Receptor, Melanocortin, Type 1/genetics/metabolism ; Swine ; },
abstract = {MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color.},
}
@article {pmid35858572,
year = {2022},
author = {Dong, X and Yang, C and Ma, Z and Chen, M and Zhang, X and Bi, C},
title = {Enhancing glycosylase base-editor activity by fusion to transactivation modules.},
journal = {Cell reports},
volume = {40},
number = {3},
pages = {111090},
doi = {10.1016/j.celrep.2022.111090},
pmid = {35858572},
issn = {2211-1247},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; HEK293 Cells ; Humans ; Mutation/genetics ; Transcriptional Activation/genetics ; },
abstract = {Base editors (BEs) are a group of genetic tools with potential in both scientific and medical research. Recently, a glycosylase BE (GBE), which converts C to G, has been constructed. However, the editing efficiency and targeting scope remains to be further exploited. Here, we renovate the GBE by first fusing it to various transactivation modules including Vp64, leading to a higher conversion of C to G relative to GBE in HEK293T cells. Further, higher editing efficiency, enhanced editing purity, and an enlarged editing window are acquired by the combination of SunTag system, GBE, and VP64. Finally, a SpRY-Cas9 variant is used to expand the targeting scope for Vp64-GBE. Vp64-SpRY-GBE and SpRY-GBE target genomic sites with non-NGG PAM, and Vp64-SpRY-GBE demonstrates better performance compared with SpRY-GBE. The construction of GBE variants with superior performance and versatile editing scope broadens the toolbox of BEs and may contribute to genetic therapies with C-to-G mutation.},
}
@article {pmid35858470,
year = {2022},
author = {Shannon, SR and Ben-Akiva, E and Green, JJ},
title = {Approaches towards biomaterial-mediated gene editing for cancer immunotherapy.},
journal = {Biomaterials science},
volume = {10},
number = {23},
pages = {6675-6687},
doi = {10.1039/d2bm00806h},
pmid = {35858470},
issn = {2047-4849},
support = {R01 EY031097/EY/NEI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Biocompatible Materials ; Gene Transfer Techniques ; Immunotherapy ; *Neoplasms/genetics/therapy ; },
abstract = {Gene therapies are transforming treatment modalities for many human diseases and disorders, including those in ophthalmology, oncology, and nephrology. To maximize the clinical efficacy and safety of these treatments, consideration of both delivery materials and cargos is critical. In consideration of the former, a large effort has been placed on transitioning away from potentially immunoreactive and toxic viral delivery mechanisms towards safer and highly tunable nonviral delivery mechanisms, including polymeric, lipid-based, and inorganic carriers. This change of paradigm does not come without obstacles, as efficient non-viral delivery is challenging, particularly to immune cells, and has yet to see clinical translation breakthroughs for gene editing. This mini-review describes notable examples of biomaterial-based gene delivery to immune cells, with emphasis on recent in vivo successes. In consideration of delivery cargos, clustered regularly interspaced palindromic repeat (CRISPR) technology is reviewed and its great promise in the field of immune cell gene editing is described. This mini-review describes how leading non-viral delivery materials and CRISPR technology can be integrated together to advance its clinical potential for therapeutic gene transfer to immune cells to treat cancer.},
}
@article {pmid35858375,
year = {2022},
author = {Zhao, J and Makhija, S and Zhou, C and Zhang, H and Wang, Y and Muralidharan, M and Huang, B and Cheng, Y},
title = {Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {33},
pages = {e2207200119},
pmid = {35858375},
issn = {1091-6490},
support = {S10 OD021741/OD/NIH HHS/United States ; R35 GM140847/GM/NIGMS NIH HHS/United States ; S10 OD020054/OD/NIH HHS/United States ; P30 CA030199/CA/NCI NIH HHS/United States ; R01 GM131641/GM/NIGMS NIH HHS/United States ; S10 OD026926/OD/NIH HHS/United States ; S10 OD026881/OD/NIH HHS/United States ; R01 GM124334/GM/NIGMS NIH HHS/United States ; P50 AI150476/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Flow Cytometry/methods ; *Gene Editing/methods ; HEK293 Cells ; Humans ; *Muscle Proteins/chemistry/genetics/isolation &amp; purification ; *Proteasome Endopeptidase Complex/chemistry/genetics/isolation &amp; purification ; Proteolysis ; },
abstract = {The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.},
}
@article {pmid35857900,
year = {2022},
author = {Sanchez-Álvarez, NT and Bautista-Niño, PK and Trejos-Suárez, J and Serrano-Díaz, NC},
title = {A model of metformin mitochondrial metabolism in metachromatic leukodystrophy: first description of human Schwann cells transfected with CRISPR-Cas9.},
journal = {Open biology},
volume = {12},
number = {7},
pages = {210371},
pmid = {35857900},
issn = {2046-2441},
mesh = {CRISPR-Cas Systems ; Humans ; *Leukodystrophy, Metachromatic/genetics/metabolism ; *Metformin/pharmacology ; Reactive Oxygen Species/metabolism ; Schwann Cells/metabolism ; Sulfoglycosphingolipids/metabolism ; },
abstract = {Metachromatic leukodystrophy is a neurological lysosomal deposit disease that affects public health despite its low incidence in the population. Currently, few reports are available on pathophysiological events related to enzyme deficiencies and subsequent sulfatide accumulation. This research aims to examine the use of metformin as an alternative treatment to counteract these effects. This was evaluated in human Schwann cells (HSCs) transfected or non-transfected with CRISPR-Cas9, and later treated with sulfatides and metformin. This resulted in transfected HSCs showing a significant increase in cell reactive oxygen species (ROS) production when exposed to 100 µM sulfatides (p = 0.0007), compared to non-transfected HSCs. Sulfatides at concentrations of 10 to 100 µM affected mitochondrial bioenergetics in transfected HSCs. Moreover, these analyses showed that transfected cells showed a decrease in basal and maximal respiration rates after exposure to 100 µM sulfatide. However, maximal and normal mitochondrial respiratory capacity decreased in cells treated with both sulfatide and metformin. This study has provided valuable insights into bioenergetic and mitochondrial effects of sulfatides in HSCs for the first time. Treatment with metformin (500 µM) restored the metabolic activity of these cells and decreased ROS production.},
}
@article {pmid35857897,
year = {2022},
author = {Zeng, M and Ke, Y and Zhuang, Z and Qin, C and Li, LY and Sheng, G and Li, Z and Meng, H and Ding, X},
title = {Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection.},
journal = {Analytical chemistry},
volume = {94},
number = {30},
pages = {10805-10812},
doi = {10.1021/acs.analchem.2c01588},
pmid = {35857897},
issn = {1520-6882},
mesh = {*African Swine Fever Virus/genetics ; Animals ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA, Single-Stranded ; Nucleic Acid Amplification Techniques ; Swine ; },
abstract = {CRISPR-associated (Cas) protein systems have been increasingly incorporated in nucleic-acid diagnosis. CRISPR/Cas12a can cleave single-stranded DNA (ssDNA) after being guided to the target double-stranded DNA (dsDNA) with crRNA, making it a specific tool for dsDNA detection. Assisted by nucleic acid preamplification, CRISPR/Cas12a enables dsDNA detection at the attomolar level. However, such mandatory preamplification in CRISPR/Cas12a also accompanies the extra step of transferring preamplification products into the CRISPR/Cas12a system, which is not only cumbersome and time-consuming but also induces the risk of cross-contamination. Herein, we demonstrate a multiplex-crRNA strategy to enhance the sensitivity of the CRISPR/Cas12a system without any preamplification. This multiplex-crRNA strategy harnesses multiple sequences of crRNA which target different regions of the same dsDNA substrate in the same CRISPR/Cas12a system. Therefore, detection signals are accumulated without amplification, which augments the conventional detection limit. For application demonstration, the B646L gene from the African swine fever virus (ASFV), which is a dsDNA virus, is exemplified. The detection limit of the multiplex-crRNA system can be improved to ∼1 picomolar (pM) without amplification, which is ∼64 times stronger than the conventional single-crRNA system. The multiplex-crRNA system presented in this study, with slight modifications, can be generalized to other biosensing settings where preamplification is not readily available.},
}
@article {pmid35857876,
year = {2022},
author = {Pan, M and Morovic, W and Hidalgo-Cantabrana, C and Roberts, A and Walden, KKO and Goh, YJ and Barrangou, R},
title = {Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {30},
pages = {e2205068119},
pmid = {35857876},
issn = {1091-6490},
mesh = {*Bifidobacterium/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Bacterial/genetics ; Genomics ; Humans ; *Probiotics ; },
abstract = {Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.},
}
@article {pmid35857372,
year = {2022},
author = {Wait, A and Srivastava, A and Diaz-Lara, A and Al Rwahnih, M and Villamor, DEV and Tzanetakis, IE},
title = {A novel, divergent member of the Rhabdoviridae infects strawberry.},
journal = {Plant disease},
volume = {},
number = {},
pages = {},
doi = {10.1094/PDIS-05-22-1078-SC},
pmid = {35857372},
issn = {0191-2917},
abstract = {Strawberry (Fragaria x ananassa) is the most important berry crop worldwide and viruses pose a constant threat to the industry. In this communication, we describe a novel virus in the family Rhabdoviridae referred to as strawberry virus 3 (StrV-3). The virus does not show significant homology when compared to recognized rhabdoviruses and therefore the establishment of a new genus should be considered. A triplex RT-PCR test was developed and successfully employed in a survey of the National Clonal Germplasm Repository Fragaria collection. A CRISPR-Cas based protocol was also developed and shown to detect the virus in as little as 1fg of total RNA, a protocol to be used in the detection of the virus in candidate G1 plants. The strawberry aphid (Chaetosiphon fragaefolii) was evaluated, alas unsuccessfully as a potential vector of the virus. This work broadens our understanding of the Rhabdoviridae and assists in the quest of releasing plant material free of viruses.},
}
@article {pmid35857318,
year = {2022},
author = {Zhi, S and Shen, J and Li, X and Jiang, Y and Xue, J and Fang, T and Xu, J and Wang, X and Cao, Y and Yang, D and Yao, Z and Yu, D},
title = {Development of Recombinase-Aided Amplification (RAA)-Exo-Probe and RAA-CRISPR/Cas12a Assays for Rapid Detection of Campylobacter jejuni in Food Samples.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {30},
pages = {9557-9566},
doi = {10.1021/acs.jafc.2c02581},
pmid = {35857318},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; *Campylobacter jejuni/genetics ; Cattle ; Chickens ; *Food Analysis ; Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics ; },
abstract = {Campylobacter jejuni is the major cause of campylobacteriosis, one of the most common foodborne illnesses worldwide. Here, we report the development of RAA-exo-probe and RAA-CRIPSR/Cas12a assays for the detection of C. jejuni in food samples. The two assays were found to be highly specific to C. jejuni and highly sensitive, as they were one log more sensitive compared to the traditional culture method, with detection thresholds of 9 and 5 copies per reaction, respectively. These assays successfully detected C. jejuni in spiked chicken samples and natural meat samples (chicken, beef, mutton, etc.) and were overall less dependent on expensive equipment, only requiring a fluorescent reader. Their ease of use compared to other nucleic acid amplification-based methods indicates that these assays could be adapted for the rapid, routine surveillance of C. jejuni contamination in food samples, particularly for work done in the field or poorly equipped labs.},
}
@article {pmid35857175,
year = {2022},
author = {Lou, J and Wang, B and Li, J and Ni, P and Jin, Y and Chen, S and Xi, Y and Zhang, R and Duan, G},
title = {The CRISPR-Cas system as a tool for diagnosing and treating infectious diseases.},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {11301-11311},
pmid = {35857175},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Communicable Diseases/diagnosis/genetics/therapy ; },
abstract = {Emerging and relapsing infectious diseases pose a huge health threat to human health and a new challenge to global public health. Rapid, sensitive and simple diagnostic tools are keys to successful management of infectious patients and containment of disease transmission. In recent years, international research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related proteins (Cas) has revolutionized our understanding of biology. The CRISPR-Cas system has the advantages of high specificity, high sensitivity, simple, rapid, low cost, and has begun to be used for molecular diagnosis and treatment of infectious diseases. In this paper, we described the biological principles, application fields and prospects of CRISPR-Cas system in the molecular diagnosis and treatment of infectious diseases, and compared it with existing molecular diagnosis methods, the advantages and disadvantages were summarized.},
}
@article {pmid35856644,
year = {2022},
author = {Mohammad, N and Katkam, SS and Wei, Q},
title = {Recent Advances in CRISPR-Based Biosensors for Point-of-Care Pathogen Detection.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {500-516},
doi = {10.1089/crispr.2021.0146},
pmid = {35856644},
issn = {2573-1602},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plants ; Point-of-Care Systems ; },
abstract = {Infectious pathogens are pressing concerns due to their heavy toll on global health and socioeconomic infrastructure. Rapid, sensitive, and specific pathogen detection methods are needed more than ever to control disease spreading. The fast evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics (CRISPR-Dx) has opened a new horizon in the field of molecular diagnostics. This review highlights recent efforts in configuring CRISPR technology as an efficient diagnostic tool for pathogen detection. It starts with a brief introduction of different CRISPR-Cas effectors and their working principles for disease diagnosis. It then focuses on the evolution of laboratory-based CRISPR technology toward a potential point-of-care test, including the development of new signaling mechanisms, elimination of preamplification and sample pretreatment steps, and miniaturization of CRISPR reactions on digital assay chips and lateral flow devices. In addition, promising examples of CRISPR-Dx for pathogen detection in various real samples, such as blood, saliva, nasal swab, plant, and food samples, are highlighted. Finally, the challenges and perspectives of future development of CRISPR-Dx for infectious disease monitoring are discussed.},
}
@article {pmid35856642,
year = {2022},
author = {Swartjes, T and Shang, P and van den Berg, DTM and Künne, T and Geijsen, N and Brouns, SJJ and van der Oost, J and Staals, RHJ and Notebaart, RA},
title = {Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {571-585},
pmid = {35856642},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; DNA, Complementary ; *Gene Editing ; Humans ; Oligonucleotides/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.},
}
@article {pmid35854181,
year = {2022},
author = {Devi, V and Harjai, K and Chhibber, S},
title = {CRISPR-Cas systems: role in cellular processes beyond adaptive immunity.},
journal = {Folia microbiologica},
volume = {67},
number = {6},
pages = {837-850},
pmid = {35854181},
issn = {1874-9356},
mesh = {*CRISPR-Cas Systems ; Archaea ; Bacteria/genetics ; Virulence ; *Biological Phenomena ; },
abstract = {Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) are the only known adaptive immune system in prokaryotes. CRISPR-Cas system provides sequence-specific immunity against invasion by foreign genetic elements. It carries out its functions by incorporating a small part of the invading DNA sequence, termed as spacer into the CRISPR array. Although the CRISPR-Cas systems are mainly responsible for adaptive immune functions, their alternative role in the gene regulation, bacterial pathophysiology, virulence, and evolution has started to unravel. In several species, these systems are revealed to regulate the processes beyond adaptive immunity by employing various components of CRISPR-Cas machinery, independently or in combination. The molecular mechanisms entailing the regulatory processes are not clear in most of the instances. In this review, we have discussed some well-known and some recently established noncanonical functions of CRISPR-Cas system and its fast-extending applications in other biological processes.},
}
@article {pmid35854143,
year = {2022},
author = {Géry, C and Téoulé, E},
title = {Cold acclimation diversity in Arabidopsis thaliana: CRISPR/Cas9 as a tool to fine analysis of Tandem Gene Arrays, application to CBF genes.},
journal = {Development genes and evolution},
volume = {232},
number = {5-6},
pages = {147-154},
pmid = {35854143},
issn = {1432-041X},
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Freezing ; Acclimatization/genetics ; },
abstract = {In this period of climate change, it is of major importance to increase knowledge about the mechanisms by whose plants adapt to their environment. Tandem gene arrays (TAG) are overrepresented in the pool of tandem duplicates involved in stress response and are consequently of special interest. Nevertheless, until recently, addressing questions about individual genes or fine regulations in such structures was very difficult due to the close arrangement of the genome, almost preventing the production of targeted simple or multiple mutants. The CRISPR/Cas9 new tool offers new opportunities as the setting of gene deletion strategies in various genetic backgrounds. Here, we used this technology on the cold acclimation CBF pathway in different accessions of Arabidopsis thaliana with the same set of guide RNAs. Deleted lines free of T-DNA have been produced for simple or multiple copies of CBF genes and evaluated for cold tolerance after acclimation. Expression levels of CBF genes and five COR genes have also been analyzed. Our data suggest first that two or three missing CBF genes are necessary to induce a strong reduction in cold tolerance and secondly that most deletions have a low impact on the expression of remaining CBF copies which contradicts the previous hypothesis in the literature. Our results thus show that the CRISPR/Cas9 deletion strategy is a useful performance tool to investigate how tandem gene arrays work.},
}
@article {pmid35854035,
year = {2022},
author = {Wu, Q and Cui, L and Liu, Y and Li, R and Dai, M and Xia, Z and Wu, M},
title = {CRISPR-Cas systems target endogenous genes to impact bacterial physiology and alter mammalian immune responses.},
journal = {Molecular biomedicine},
volume = {3},
number = {1},
pages = {22},
pmid = {35854035},
issn = {2662-8651},
support = {5R01AI138203-01/NH/NIH HHS/United States ; },
abstract = {CRISPR-Cas systems are an immune defense mechanism that is widespread in archaea and bacteria against invasive phages or foreign genetic elements. In the last decade, CRISPR-Cas systems have been a leading gene-editing tool for agriculture (plant engineering), biotechnology, and human health (e.g., diagnosis and treatment of cancers and genetic diseases), benefitted from unprecedented discoveries of basic bacterial research. However, the functional complexity of CRISPR systems is far beyond the original scope of immune defense. CRISPR-Cas systems are implicated in influencing the expression of physiology and virulence genes and subsequently altering the formation of bacterial biofilm, drug resistance, invasive potency as well as bacterial own physiological characteristics. Moreover, increasing evidence supports that bacterial CRISPR-Cas systems might intriguingly influence mammalian immune responses through targeting endogenous genes, especially those relating to virulence; however, unfortunately, their underlying mechanisms are largely unclear. Nevertheless, the interaction between bacterial CRISPR-Cas systems and eukaryotic cells is complex with numerous mysteries that necessitate further investigation efforts. Here, we summarize the non-canonical functions of CRISPR-Cas that potentially impact bacterial physiology, pathogenicity, antimicrobial resistance, and thereby altering the courses of mammalian immune responses.},
}
@article {pmid35853769,
year = {2022},
author = {Tan, J and Forner, J and Karcher, D and Bock, R},
title = {DNA base editing in nuclear and organellar genomes.},
journal = {Trends in genetics : TIG},
volume = {38},
number = {11},
pages = {1147-1169},
doi = {10.1016/j.tig.2022.06.015},
pmid = {35853769},
issn = {0168-9525},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA Breaks, Double-Stranded ; Gene Editing/methods ; *Nucleoside Deaminases/genetics/metabolism ; Nucleotides ; RNA-Directed DNA Polymerase/genetics/metabolism ; Transcription Activator-Like Effectors/genetics/metabolism ; },
abstract = {Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.},
}
@article {pmid35853277,
year = {2022},
author = {Safarzadeh Kozani, P and Shokrgozar, MA and Evazalipour, M and Roudkenar, MH},
title = {CRISPR/Cas9-medaited knockout of endogenous T-cell receptor in Jurkat cells and generation of NY-ESO-1-specific T cells: An in vitro study.},
journal = {International immunopharmacology},
volume = {110},
number = {},
pages = {109055},
doi = {10.1016/j.intimp.2022.109055},
pmid = {35853277},
issn = {1878-1705},
mesh = {Antigens, Neoplasm ; CRISPR-Cas Systems ; *Esophageal Neoplasms ; *Esophageal Squamous Cell Carcinoma ; Humans ; Jurkat Cells ; Peptides/metabolism ; Receptors, Antigen, T-Cell/genetics/metabolism ; },
abstract = {Adoptive transfer of T-cell receptor (TCR)-engineered T cells has been successful in mediating favorable clinical outcomes. TCR-engineered T cells can be applied for targeting cancers whose associated antigens are intracellular and presented through major histocompatibility complexes (MHC). The mispairing of the exogenous TCR chains with the endogenous TCR chains leads to functionally impaired TCR-engineered T cells. The CRISPR/Cas9 genome-editing system can be utilized for the knockout of the endogenous TCR in T cells before introducing the exogenous TCR chains. In this study, we used the lentiviral delivery of CRISPR/Cas9 for disrupting the expression of the endogenous TCR in the Jurkat cell line. Next, an exogenous TCR targeting human leukocyte antigen (HLA)-A*0201-restricted New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide was transduced into the TCR-knockout (KO) Jurkat cells. Further, we assessed lentiviral transduction efficacy using tetramer assay and evaluated the functionality of the NY-ESO-1-specific TCR-engineered T cells by quantifying the cell surface expression of CD69 upon co-cultivation with peptide-pulsed T2 cells. We successfully knocked out the endogenous TCR in ∼40% of the Jurkat cells. TCR-KO cells were selected and subjected to express NY-ESO-1-specific TCRs using lentiviral vectors. Flow cytometry analysis confirmed that up to 55% of the cells expressed the transgenic TCR on their surface. The functionality assay demonstrated that >90% of the engineered cells expressed CD69 when co-cultured with peptide-pulsed T2 cells. Conclusively, we developed a pipeline to engineer Jurkat cells using the state-of-the-art technique CRISPR/Cas9 and generated TCR-engineered cells that can become activated by a tumor-specific antigen.},
}
@article {pmid35852729,
year = {2022},
author = {Flusche, T and Rajan, R},
title = {Molecular Details of DNA Integration by CRISPR-Associated Proteins During Adaptation in Bacteria and Archaea.},
journal = {Advances in experimental medicine and biology},
volume = {},
number = {},
pages = {},
pmid = {35852729},
issn = {0065-2598},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in bacteria and archaea, where immunological memory is retained in the CRISPR locus as short pieces of the intruding nucleic acid, termed spacers. The adaptation to new infections occurs through the integration of a new spacer into the CRISPR array. For immune protection, spacers are transcribed into CRISPR RNAs (crRNA) that are used to guide the effector nuclease of the system in sequence-dependent target cleavage. Spacers originate as a prespacer from either DNA or RNA depending on the CRISPR-Cas system being observed, and the nearly universal Cas proteins, Cas1 and Cas2, insert the prespacer into the CRISPR locus during adaptation in all systems that contain them. The mechanism of site-specific prespacer integration varies across CRISPR classes and types, and distinct differences can even be found within the same subtype. In this review, the current knowledge on the mechanisms of prespacer integration in type II-A CRISPR-Cas systems will be described. Comparisons of the currently characterized type II-A systems show that distinct mechanisms exist within different members of this subtype and are correlated to sequence-specific interactions of Cas proteins and the DNA elements present in the CRISPR array. These observations indicate that nature has fine-tuned the mechanistic details while performing the basic step of DNA integration by Cas proteins, which offers unique advantages to develop Cas1-Cas2-based biotechnology.},
}
@article {pmid35849921,
year = {2022},
author = {Padmanaban, V and Ranganathan, UDK},
title = {CRISPR-Cas system and its use in the diagnosis of infectious diseases.},
journal = {Microbiological research},
volume = {263},
number = {},
pages = {127100},
doi = {10.1016/j.micres.2022.127100},
pmid = {35849921},
issn = {1618-0623},
mesh = {*CRISPR-Cas Systems ; *Communicable Diseases/diagnosis ; Humans ; Pathology, Molecular ; },
abstract = {Rapid and accurate diagnostic methods for detecting pathogens are needed for effective management and treatment of infectious diseases. The conventional pathogen detection approach based on culture is considered the gold standard method, but needs several days to corroborate its results. Using nucleic acids from pathogens as detection targets has a considerable advantage in overcoming these time-consuming issues. The development of several molecular techniques has started to change the landscape of infectious disease diagnosis. However, these require expensive reagents, equipment, and sophisticated infrastructure, as well as highly trained workers. In this context, it is necessary to identify new diagnostic strategies to overcome these issues. Recently, CRISPR/Cas based diagnosis has revolutionized the area of molecular diagnostics of pathogenic diseases. In this review, we have discussed the different classes of CRISPR-Cas systems and their functions, and then focused on recent advances in CRISPR-based diagnosis technologies and the perspective of using this as a potential biosensing platform to detect infectious disease.},
}
@article {pmid35849532,
year = {2022},
author = {Zaayman, M and Wheatley, RM},
title = {Fitness costs of CRISPR-Cas systems in bacteria.},
journal = {Microbiology (Reading, England)},
volume = {168},
number = {7},
pages = {},
doi = {10.1099/mic.0.001209},
pmid = {35849532},
issn = {1465-2080},
mesh = {*Bacteria/genetics ; *CRISPR-Cas Systems ; Genome, Bacterial/genetics ; },
abstract = {CRISPR-Cas systems provide bacteria with both specificity and adaptability in defence against invading genetic elements. From a theoretical perspective, CRISPR-Cas systems confer many benefits. However, they are observed at an unexpectedly low prevalence across the bacterial domain. While these defence systems can be gained horizontally, fitness costs may lead to selection against their carriage. Understanding the source of CRISPR-related fitness costs will help us to understand the evolutionary dynamics of CRISPR-Cas systems and their role in shaping bacterial genome evolution. Here, we review our current understanding of the potential fitness costs associated with CRISPR-Cas systems. In addition to potentially restricting the acquisition of genetic material that could confer fitness benefits, we explore five alternative biological factors that from a theoretical perspective may influence the fitness costs associated with CRISPR-Cas system carriage: (1) the repertoire of defence mechanisms a bacterium has available to it, (2) the potential for a metabolic burden, (3) larger-scale population and environmental factors, (4) the phenomenon of self-targeting spacers, and (5) alternative non-defence roles for CRISPR-Cas.},
}
@article {pmid35849129,
year = {2022},
author = {Wang, K and Escobar, M and Li, J and Mahata, B and Goell, J and Shah, S and Cluck, M and Hilton, IB},
title = {Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer-promoter interactions.},
journal = {Nucleic acids research},
volume = {50},
number = {14},
pages = {7842-7855},
pmid = {35849129},
issn = {1362-4962},
support = {R35 GM143532/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Enhancer Elements, Genetic ; Epigenomics ; Gene Expression Regulation ; Humans ; Peptide Fragments ; *Promoter Regions, Genetic ; RNA ; Sialoglycoproteins ; Transcription Factors/genetics ; *Transcriptional Activation ; },
abstract = {Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.},
}
@article {pmid35848536,
year = {2022},
author = {Wan, L and Ma, J and Yi, J and Dong, Y and Niu, R and Su, Y and Li, Q and Dan Zhu, and Chao, J and Su, S and Fan, C and Wang, L and Wan, Y},
title = {CRISPR-empowered hybridization chain reaction amplification for an attomolar electrochemical sensor.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {63},
pages = {8826-8829},
doi = {10.1039/d2cc01155g},
pmid = {35848536},
issn = {1364-548X},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques ; Humans ; *Influenza A Virus, H7N9 Subtype/genetics ; *Influenza, Human ; Limit of Detection ; Nucleic Acid Hybridization ; },
abstract = {Rapid pathogen screening holds the key against certain viral infections, especially in an overwhelming pandemic. Herein, a CRISPR-empowered electrochemical biosensor was designed for the ultrasensitive detection of the avian influenza A (H7N9) virus gene sequence. Combining the CRISPR/Cas system, a signal-amplification strategy and a high-conductivity sensing substrate, the developed biosensor showed an ultrawide dynamic range, an ultralow detection limit, and excellent selectivity for H7N9 detection, providing a potential sensing platform for the simple, fast, sensitive, and on-site detection of infectious diseases.},
}
@article {pmid35847087,
year = {2022},
author = {Shingote, PR and Wasule, DL and Parma, VS and Holkar, SK and Karkute, SG and Parlawar, ND and Senanayake, DMJB},
title = {An Overview of Chili Leaf Curl Disease: Molecular Mechanisms, Impact, Challenges, and Disease Management Strategies in Indian Subcontinent.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {899512},
pmid = {35847087},
issn = {1664-302X},
abstract = {Leaf curl disease in a chili plant is caused mainly by Chili leaf curl virus (ChiLCV) (Family: Geminiviridae, Genus: Begomovirus). ChiLCV shows a widespread occurrence in most of the chili (Capsicum spp.) growing regions. ChiLCV has a limited host range and infects tomatoes (Solanum lycopersicum), potatoes (S. tuberosum), and amaranth (Amaranthus tricolor). The virus genome is a monopartite circular single-stranded DNA molecule of 2.7 kb and associated with α and β-satellites of 1.3 and 1.4 kb, respectively. The virus genome is encapsulated in distinct twinned icosahedral particles of around 18-30 nm in size and transmitted by Bemisia tabaci (Family: Aleyrodidae, Order: Hemiptera). Recently, bipartite begomovirus has been found to be associated with leaf curl disease. The leaf curl disease has a widespread distribution in the major equatorial regions viz., Australia, Asia, Africa, Europe, and America. Besides the PCR, qPCR, and LAMP-based detection systems, recently, localized surface-plasmon-resonance (LPSR) based optical platform is used for ChiLCV detection in a 20-40 μl of sample volume using aluminum nanoparticles. Management of ChiLCV is more challenging due to the vector-borne nature of the virus, therefore integrated disease management strategies need to be followed to contain the spread and heavy crop loss. CRISPR/Cas-mediated virus resistance has gained importance in disease management of DNA and RNA viruses due to certain advantages over the conventional approaches. Therefore, CRISPR/Cas system-mediated resistance needs to be explored in chili against ChiLCV.},
}
@article {pmid35847068,
year = {2022},
author = {Kayesh, MEH and Hashem, MA and Kohara, M and Tsukiyama-Kohara, K},
title = {In vivo Delivery Tools for Clustered Regularly Interspaced Short Palindromic Repeat/Associated Protein 9-Mediated Inhibition of Hepatitis B Virus Infection: An Update.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {953218},
pmid = {35847068},
issn = {1664-302X},
abstract = {Chronic hepatitis B virus (HBV) infection remains a major global health problem despite the availability of an effective prophylactic HBV vaccine. Current antiviral therapies are unable to fully cure chronic hepatitis B (CHB) because of the persistent nature of covalently closed circular DNA (cccDNA), a replicative template for HBV, which necessitates the development of alternative therapeutic approaches. The CRISPR/Cas system, a newly emerging genome editing tool, holds great promise for genome editing and gene therapy. Several in vitro and/or in vivo studies have demonstrated the effectiveness of HBV-specific clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) systems in cleaving HBV DNA and cccDNA. Although recent advances in CRISPR/Cas technology enhance its prospects for clinical application against HBV infection, in vivo delivery of the CRISPR/Cas9 system at targets sites remains a major challenge that needs to be resolved before its clinical application in gene therapy for CHB. In the present review, we discuss CRISPR/Cas9 delivery tools for targeting HBV infection, with a focus on the development of adeno-associated virus vectors and lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery to treat CHB. In addition, we discuss the importance of delivery tools in the enhancement of the antiviral efficacy of CRISPR/Cas9 against HBV infection.},
}
@article {pmid35844563,
year = {2022},
author = {Pfenninger, P and Yerly, L and Abe, J},
title = {Naïve Primary Mouse CD8[+] T Cells Retain In Vivo Immune Responsiveness After Electroporation-Based CRISPR/Cas9 Genetic Engineering.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {777113},
pmid = {35844563},
issn = {1664-3224},
mesh = {Animals ; *CD8-Positive T-Lymphocytes ; *CRISPR-Cas Systems ; Electroporation ; Gene Editing ; Genetic Engineering ; Mice ; },
abstract = {CRISPR/Cas9 technology has revolutionized genetic engineering of primary cells. Although its use is gaining momentum in studies on CD8[+] T cell biology, it remains elusive to what extent CRISPR/Cas9 affects in vivo function of CD8[+] T cells. Here, we optimized nucleofection-based CRISPR/Cas9 genetic engineering of naïve and in vitro-activated primary mouse CD8[+] T cells and tested their in vivo immune responses. Nucleofection of naïve CD8[+] T cells preserved their in vivo antiviral immune responsiveness to an extent that is indistinguishable from non-nucleofected cells, whereas nucleofection of in vitro-activated CD8[+] T cells led to slightly impaired expansion/survival at early time point after adoptive transfer and more pronounced contraction. Of note, different target proteins displayed distinct decay rates after gene editing. This is in stark contrast to a comparable period of time required to complete gene inactivation. Thus, for optimal experimental design, it is crucial to determine the kinetics of the loss of target gene product to adapt incubation period after gene editing. In sum, nucleofection-based CRISPR/Cas9 genome editing achieves efficient and rapid generation of mutant CD8[+] T cells without imposing detrimental constraints on their in vivo functions.},
}
@article {pmid35842430,
year = {2022},
author = {Jedrzejczyk, DJ and Poulsen, LD and Mohr, M and Damas, ND and Schoffelen, S and Barghetti, A and Baumgartner, R and Weinert, BT and Warnecke, T and Gill, RT},
title = {CRISPR-Cas12a nucleases function with structurally engineered crRNAs: SynThetic trAcrRNA.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {12193},
pmid = {35842430},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Gene Editing ; *RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate sixteen types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo. We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region led to comparable genome editing efficiencies. In conclusion, we show that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications.},
}
@article {pmid35840676,
year = {2022},
author = {Ledford, H},
title = {CRISPR 'cousin' put to the test in landmark heart-disease trial.},
journal = {Nature},
volume = {607},
number = {7920},
pages = {647},
pmid = {35840676},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/standards ; *Heart Diseases/genetics/therapy ; Humans ; },
}
@article {pmid35840661,
year = {2022},
author = {Braun, CJ and Adames, AC and Saur, D and Rad, R},
title = {Tutorial: design and execution of CRISPR in vivo screens.},
journal = {Nature protocols},
volume = {17},
number = {9},
pages = {1903-1925},
pmid = {35840661},
issn = {1750-2799},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; Genetic Testing ; Mice ; Phenotype ; },
abstract = {Here we provide a detailed tutorial on CRISPR in vivo screening. Using the mouse as the model organism, we introduce a range of CRISPR tools and applications, delineate general considerations for 'transplantation-based' or 'direct in vivo' screening design, and provide details on technical execution, sequencing readouts, computational analyses and data interpretation. In vivo screens face unique pitfalls and limitations, such as delivery issues or library bottlenecking, which must be counteracted to avoid screening failure or flawed conclusions. A broad variety of in vivo phenotypes can be interrogated such as organ development, hematopoietic lineage decision and evolutionary licensing in oncogenesis. We describe experimental strategies to address various biological questions and provide an outlook on emerging CRISPR applications, such as genetic interaction screening. These technological advances create potent new opportunities to dissect the molecular underpinnings of complex organismal phenotypes.},
}
@article {pmid35840489,
year = {2022},
author = {Kumar, S and Fry, LE and Wang, JH and Martin, KR and Hewitt, AW and Chen, FK and Liu, GS},
title = {RNA-targeting strategies as a platform for ocular gene therapy.},
journal = {Progress in retinal and eye research},
volume = {},
number = {},
pages = {101110},
doi = {10.1016/j.preteyeres.2022.101110},
pmid = {35840489},
issn = {1873-1635},
abstract = {Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.},
}
@article {pmid35838958,
year = {2022},
author = {Phutela, R and Gulati, S and Kumar, M and Maiti, S and Chakraborty, D},
title = {FnCas9 Editor Linked Uniform Detection Assay for COVID-19.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2511},
number = {},
pages = {149-159},
pmid = {35838958},
issn = {1940-6029},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; Pandemics ; Point-of-Care Testing ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {The recent COVID-19 outbreak and pandemic of 2020 and its surveillance were implemented by quickly adapting the existing diagnostic methods to detect the SARS-CoV-2 RNA. While traditional methods for detecting pathogenic DNA and RNA have relied heavily on gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and sequencing-based methods, their shortcomings under resource-limited settings have emphasized the need of developing point-of-care (POC) diagnostics. Clustered regularly interspaced short palindromic repeats (CRISPR)-based detection systems provide a rapid and accurate alternative. Here, we describe a CRISPR-Cas9-based detection system FnCas9 Editor Linked Uniform Detection Assay (FELUDA) using a lateral flow test that can detect nucleobase and nucleotide sequences depending upon the stoichiometric-based binding of FnCas9 ribonucleoprotein complex (RNP)-target sequences. The assay has been optimized to be conducted within 1 h and shows 100% sensitivity and 97% specificity in clinical samples across a range of viral loads. The lateral strip results are read using the True Outcome Predicted via Strip Evaluation (TOPSE) smartphone application. This assay is versatile and can be optimized and adjusted to target various diseases.},
}
@article {pmid35838756,
year = {2022},
author = {Ksiezarek, M and Grosso, F and Ribeiro, TG and Peixe, L},
title = {Genomic diversity of genus Limosilactobacillus.},
journal = {Microbial genomics},
volume = {8},
number = {7},
pages = {},
pmid = {35838756},
issn = {2057-5858},
mesh = {*Genome ; *Genomics ; Lactobacillaceae ; },
abstract = {The genus Limosilactobacillus (formerly Lactobacillus) contains multiple species considered to be adapted to vertebrates, yet their genomic diversity has not been explored. In this study, we performed comparative genomic analysis of Limosilactobacillus (22 species; 332 genomes) isolated from different niches, further focusing on human strains (11 species; 74 genomes) and their adaptation features to specific body sites. Phylogenomic analysis of Limosilactobacillus showed misidentification of some strains deposited in public databases and existence of putative novel Limosilactobacillus species. The pangenome analysis revealed a remarkable genomic diversity (only 1.3 % of gene clusters are shared), and we did not observe a strong association of the accessory genome with different niches. The pangenome of Limosilactobacillus reuteri and Limosilactobacillus fermentum was open, suggesting that acquisition of genes is still occurring. Although most Limosilactobacillus were predicted as antibiotic susceptible (83%), acquired antibiotic-resistance genes were common in L. reuteri from food-producing animals. Genes related to lactic acid isoform production (>95 %) and putative bacteriocins (70.2%) were identified in most Limosilactobacillus strains, while prophages (55.4%) and CRISPR-Cas systems (32.0%) were less prevalent. Among strains from human sources, several metabolic pathways were predicted as conserved and completed. Their accessory genome was highly variable and did not cluster according to different human body sites, with some exceptions (urogenital Limosilactobacillus vaginalis , Limosilactobacillus portuensis , Limosilactobacillus urinaemulieris and Limosilactobacillus coleohominis or gastrointestinal Limosilactobacillus mucosae). Moreover, we identified 12 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologues that were significantly enriched in strains from particular body sites. We concluded that evolution of the highly diverse Limosilactobacillus is complex and not always related to niche or human body site origin.},
}
@article {pmid35837551,
year = {2022},
author = {Touzdjian Pinheiro Kohlrausch Távora, F and de Assis Dos Santos Diniz, F and de Moraes Rêgo-Machado, C and Chagas Freitas, N and Barbosa Monteiro Arraes, F and Chumbinho de Andrade, E and Furtado, LL and Osiro, KO and Lima de Sousa, N and Cardoso, TB and Márcia Mertz Henning, L and Abrão de Oliveira Molinari, P and Feingold, SE and Hunter, WB and Fátima Grossi de Sá, M and Kobayashi, AK and Lima Nepomuceno, A and Santiago, TR and Correa Molinari, HB},
title = {CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {913728},
pmid = {35837551},
issn = {2296-4185},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe.},
}
@article {pmid35836018,
year = {2022},
author = {Naqvi, MM and Lee, L and Montaguth, OET and Diffin, FM and Szczelkun, MD},
title = {CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage.},
journal = {Nature chemical biology},
volume = {18},
number = {9},
pages = {1014-1022},
pmid = {35836018},
issn = {1552-4469},
support = {BB/S001239/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; Constriction ; DNA/genetics ; DNA Cleavage ; Gene Editing ; Nucleic Acid Conformation ; RNA ; *RNA, Guide/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable 'clamped' Cas12a-DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3' CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3' CRISPR RNA to downstream DNA.},
}
@article {pmid35835393,
year = {2022},
author = {Karmakar, S and Das, P and Panda, D and Xie, K and Baig, MJ and Molla, KA},
title = {A detailed landscape of CRISPR-Cas-mediated plant disease and pest management.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {323},
number = {},
pages = {111376},
doi = {10.1016/j.plantsci.2022.111376},
pmid = {35835393},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Pest Control ; Plant Diseases/genetics ; Plants/genetics ; },
abstract = {Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection.},
}
@article {pmid35835111,
year = {2022},
author = {Hu, C and Ni, D and Nam, KH and Majumdar, S and McLean, J and Stahlberg, H and Terns, MP and Ke, A},
title = {Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.},
journal = {Molecular cell},
volume = {82},
number = {15},
pages = {2754-2768.e5},
pmid = {35835111},
issn = {1097-4164},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; R35 GM118174/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/genetics/metabolism ; Endonucleases/genetics ; Gene Editing ; Humans ; RNA ; },
abstract = {Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.},
}
@article {pmid35833801,
year = {2022},
author = {Mighell, TL and Nishida, A and O'Connell, BL and Miller, CV and Grindstaff, S and Thornton, CA and Adey, AC and Doherty, D and O'Roak, BJ},
title = {Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {548-557},
pmid = {35833801},
issn = {2573-1602},
support = {U54 HD083091/HD/NICHD NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Nucleotides ; RNA, Guide/genetics ; },
abstract = {Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.},
}
@article {pmid35833800,
year = {2022},
author = {Wu, WY and Jackson, SA and Almendros, C and Haagsma, AC and Yilmaz, S and Gort, G and van der Oost, J and Brouns, SJJ and Staals, RHJ},
title = {Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {536-547},
pmid = {35833800},
issn = {2573-1602},
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; DNA ; Endonucleases/genetics ; Gene Editing ; },
abstract = {Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements.},
}
@article {pmid35833799,
year = {2022},
author = {Mendoza, B and Fry, T and Dooley, D and Herman, J and Trinh, CT},
title = {CASPER: An Integrated Software Platform for Rapid Development of CRISPR Tools.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {609-617},
doi = {10.1089/crispr.2022.0025},
pmid = {35833799},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Gene Editing ; *RNA, Guide/genetics ; Software ; },
abstract = {Both academic and enterprise software solutions exist for designing CRISPR targets. They offer advantages when designing guide RNAs (gRNAs) but often focus on a select number of model organisms. Those that offer a wide variety of organisms can be limited in support of alternative endonucleases and downstream analyses such as multitargeting and population analyses to interrogate a microbiome. To accommodate broad CRISPR utilization, we developed a flexible platform software CRISPR Associated Software for Pathway Engineering and Research (CASPER) for gRNA generation and analysis in any organism and with any CRISPR-Cas system. CASPER combines traditional gRNA design tools with unique functions such as multiple Cas-type gRNA generation and evaluation of spacer redundancy in a single species or microbiome. The analyses have implications for strain-, species-, or genus-specific CRISPR diagnostic probe design and microbiome manipulation. The novel features of CASPER are packaged in a user-friendly interface to create a computational environment for researchers to streamline the utility of CRISPR-Cas systems.},
}
@article {pmid35832085,
year = {2022},
author = {Jiang, L and Long, J and Yang, Y and Zhou, L and Su, J and Qin, F and Tang, W and Tao, R and Chen, Q and Yao, S},
title = {Internally inlaid SaCas9 base editors enable window specific base editing.},
journal = {Theranostics},
volume = {12},
number = {10},
pages = {4767-4778},
pmid = {35832085},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems ; Cytosine/metabolism ; Cytosine Deaminase/metabolism ; DNA ; *Gene Editing/methods ; },
abstract = {Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.},
}
@article {pmid35831865,
year = {2022},
author = {Graffeuil, A and Guerrero-Castro, J and Assefa, A and Uhlin, BE and Cisneros, DA},
title = {Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {139},
pmid = {35831865},
issn = {1475-2859},
mesh = {Adenosine Triphosphate ; *CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Editing ; Mutagenesis ; Operon ; },
abstract = {BACKGROUND: Functionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.<br><br>RESULTS: Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator's sequence, whilst other types of mutations, such as a 97&#xa0;bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (&#x394;atpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.<br><br>CONCLUSIONS: We conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate&#xa0;polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless &#x394;atpIBEFHAGDC mutant.},
}
@article {pmid35831828,
year = {2022},
author = {Kim, DE and Lee, JH and Ji, KB and Park, KS and Kil, TY and Koo, O and Kim, MK},
title = {Generation of genome-edited dogs by somatic cell nuclear transfer.},
journal = {BMC biotechnology},
volume = {22},
number = {1},
pages = {19},
pmid = {35831828},
issn = {1472-6750},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; Cloning, Organism ; Dogs ; Gene Editing ; *Nuclear Transfer Techniques ; },
abstract = {BACKGROUND: Canine cloning technology based on somatic cell nuclear transfer (SCNT) combined with genome-editing tools such as CRISPR-Cas9 can be used to correct pathogenic mutations in purebred dogs or to generate animal models of disease.<br><br>RESULTS: We constructed a CRISPR-Cas9 vector targeting canine DJ-1. Genome-edited canine fibroblasts were established using vector transfection and antibiotic selection. We performed canine SCNT using genome-edited fibroblasts and successfully generated two genome-edited dogs. Both genome-edited dogs had insertion-deletion mutations at the target locus, and DJ-1 expression was either downregulated or completely repressed.<br><br>CONCLUSION: SCNT successfully produced genome-edited dogs by using the CRISPR-Cas9 system for the first time.},
}
@article {pmid35831515,
year = {2022},
author = {Kingwell, K},
title = {Base editors hit the clinic.},
journal = {Nature reviews. Drug discovery},
volume = {21},
number = {8},
pages = {545-547},
doi = {10.1038/d41573-022-00124-z},
pmid = {35831515},
issn = {1474-1784},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid35831384,
year = {2022},
author = {Feuer, KL and Wahbeh, MH and Yovo, C and Rabie, E and Lam, AN and Abdollahi, S and Young, LJ and Rike, B and Umamageswaran, A and Avramopoulos, D},
title = {CRISPR Del/Rei: a simple, flexible, and efficient pipeline for scarless genome editing.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {11928},
pmid = {35831384},
issn = {2045-2322},
support = {MH122936/MH/NIMH NIH HHS/United States ; P50 MH094268/MH/NIMH NIH HHS/United States ; R01 MH113215/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome, Human ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Scarless genome editing of induced pluripotent stem cells (iPSCs) is crucial for the precise modeling of genetic disease. Here we present CRISPR Del/Rei, a two-step deletion-reinsertion strategy with high editing efficiency and simple PCR-based screening that generates isogenic clones in ~ 2 months. We apply our strategy to edit iPSCs at 3 loci with only rare off target editing.},
}
@article {pmid35831290,
year = {2022},
author = {Pan, X and Qu, K and Yuan, H and Xiang, X and Anthon, C and Pashkova, L and Liang, X and Han, P and Corsi, GI and Xu, F and Liu, P and Zhong, J and Zhou, Y and Ma, T and Jiang, H and Liu, J and Wang, J and Jessen, N and Bolund, L and Yang, H and Xu, X and Church, GM and Gorodkin, J and Lin, L and Luo, Y},
title = {Massively targeted evaluation of therapeutic CRISPR off-targets in cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {4049},
pmid = {35831290},
issn = {2041-1723},
support = {RM1 HG008525/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/genetics/metabolism ; High-Throughput Nucleotide Sequencing/methods ; Humans ; *RNA, Guide/genetics ; Ribonucleases/metabolism ; },
abstract = {Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.},
}
@article {pmid35831117,
year = {2022},
author = {Wake, Y and Vakulskas, CA and Glenn, SE and Kaneko, T},
title = {Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate.},
journal = {The Journal of reproduction and development},
volume = {68},
number = {5},
pages = {307-311},
pmid = {35831117},
issn = {1348-4400},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Electroporation/methods ; *Gene Editing/methods ; Mice ; Microinjections ; },
abstract = {Genetically engineered animals can be produced quickly using genome editing technology. A new electroporation technique, technique for animal knockout system by electroporation (TAKE), aids in the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of microinjection. It is difficult to confirm nuclease delivery into embryos after electroporation using the conventional TAKE method. We previously reported the successful visualization of fluorescently-labeled tracrRNA in embryos after electroporation Cas9 paired with the crRNA:tracrRNA-ATTO550 duplex. However, the amount of fluorescence signal from labeled tracrRNA in embryos did not correlate with the genome editing rate of the offspring. This study examined the visualization of Cas9 protein in embryos after electroporation and its correlation with the genome editing rate of the offspring using a fluorescent Cas9 fusion protein. The fluorescent Cas9 protein was observed in all embryos that survived following electroporation. We found that the efficiency of Cas9 protein delivery into embryos via electroporation depended on the pulse length. Furthermore, we demonstrated that the amount of fluorescent Cas9 protein detected in the embryos correlated with the genome editing efficiency of the embryos. These data indicate that the TAKE method using fluorescently-labeled nucleases can be used to optimize the delivery conditions and verify nuclease delivery into individual embryos prior to embryo transfer for the efficient production of genome-edited animals.},
}
@article {pmid35830793,
year = {2022},
author = {Liu, L and Zou, L and Li, K and Hou, H and Hu, Q and Liu, S and Li, J and Song, C and Chen, J and Wang, S and Wang, Y and Li, C and Du, H and Li, JL and Chen, F and Xu, Z and Sun, W and Sun, Q and Xiong, W},
title = {Template-independent genome editing in the Pcdh15[av-3j] mouse, a model of human DFNB23 nonsyndromic deafness.},
journal = {Cell reports},
volume = {40},
number = {2},
pages = {111061},
doi = {10.1016/j.celrep.2022.111061},
pmid = {35830793},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems ; *Cadherin Related Proteins/genetics ; Disease Models, Animal ; Gene Editing ; *Hearing Loss, Sensorineural/genetics/pathology ; Humans ; Mechanotransduction, Cellular ; Mice ; *Protein Precursors/genetics ; },
abstract = {Although frameshift mutations lead to 22% of inherited Mendelian disorders in humans, there is no efficient in vivo gene therapy strategy available to date, particularly in nondividing cells. Here, we show that nonhomologous end-joining (NHEJ)-mediated nonrandom editing profiles compensate the frameshift mutation in the Pcdh15 gene and restore the lost mechanotransduction function in postmitotic hair cells of Pcdh15[av-3J] mice, an animal model of human nonsyndromic deafness DFNB23. Identified by an ex vivo evaluation system in cultured cochlear explants, the selected guide RNA restores reading frame in approximately 50% of indel products and recovers mechanotransduction in more than 70% of targeted hair cells. In vivo treatment shows that half of the animals gain improvements in auditory responses, and balance function is restored in the majority of injected mutant mice. These results demonstrate that NHEJ-mediated reading-frame restoration is a simple and efficient strategy in postmitotic systems.},
}
@article {pmid35830604,
year = {2022},
author = {Zhu, JJ and Cheng, AW},
title = {JACKIE: Fast Enumeration of Genome-Wide Single- and Multicopy CRISPR Target Sites and Their Off-Target Numbers.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {618-628},
pmid = {35830604},
issn = {2573-1602},
support = {R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {Algorithms ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome/genetics ; Software ; },
abstract = {Zinc finger protein-, transcription activator like effector-, and CRISPR-based methods for genome and epigenome editing and imaging have provided powerful tools to investigate functions of genomes. Targeting sequence design is vital to the success of these experiments. Although existing design software mainly focus on designing target sequence for specific elements, we report here the implementation of Jackie and Albert's Comprehensive K-mer Instances Enumerator (JACKIE), a suite of software for enumerating all single- and multicopy sites in the genome that can be incorporated for genome-scale designs as well as loaded onto genome browsers alongside other tracks for convenient web-based graphic-user-interface-enabled design. We also implement fast algorithms to identify sequence neighborhoods or off-target counts of targeting sequences so that designs with low probability of off-target can be identified among millions of design sequences in reasonable time. We demonstrate the application of JACKIE-designed CRISPR site clusters for genome imaging.},
}
@article {pmid35830256,
year = {2022},
author = {Xie, S and Qin, C and Zhao, F and Shang, Z and Wang, P and Sohail, M and Zhang, X and Li, B},
title = {A DNA-Cu nanocluster and exonuclease I integrated label-free reporting system for CRISPR/Cas12a-based SARS-CoV-2 detection with minimized background signals.},
journal = {Journal of materials chemistry. B},
volume = {10},
number = {32},
pages = {6107-6117},
doi = {10.1039/d2tb00857b},
pmid = {35830256},
issn = {2050-7518},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Copper ; DNA/genetics ; Exodeoxyribonucleases ; Humans ; RNA, Viral ; *SARS-CoV-2/genetics ; },
abstract = {CRISPR-driven biosensing is developing rapidly, but current studies mostly adopt dye-labeled ssDNA as the signal reporter, which is costly and unstable. Herein, we developed a label-free and low-background reporter for CRISPR/Cas12a signaling by integrating DNA-templated copper nanoclusters (DNA-CuNCs) and exonuclease I (EXO I). The template of the DNA-CuNCs was rationally designed as a ds-/ss-DNA hybrid, ensuring that after a quick and nonpersistent cut of Cas12a, a majority of the template can be digested by EXO I. Based on this novel reporter, a biosensor termed CRISPR-CNS (cost-effective, nimble, and sensitive copper nanocluster sensor integrating CRISPR) was developed. Due to the high signal-to-background ratio of our proposed reporter, CRISPR-CNS shows excellent performances for nucleic acid detection, yielding a detection limit of 20 copies for SARS-CoV-2 RNA. Considering its facile synthesis, robust fluorescence, effective cost, and good sensitivity, this combination shall serve as a highly potential output for CRISPR-based point-of-care testing.},
}
@article {pmid35829671,
year = {2022},
author = {Meng, G and Wang, X and Liu, M and Wang, F and Liu, Q and Dong, C},
title = {Efficient CRISPR/Cas9 system based on autonomously replicating plasmid with an AMA1 sequence and precisely targeted gene deletion in the edible fungus, Cordyceps militaris.},
journal = {Microbial biotechnology},
volume = {15},
number = {10},
pages = {2594-2606},
pmid = {35829671},
issn = {1751-7915},
mesh = {CRISPR-Cas Systems ; *Cordyceps/genetics ; Gene Deletion ; Plasmids/genetics ; Polyethylene Glycols ; RNA, Guide ; RNA, Transfer, Gly ; RNA, Transfer, Pro ; },
abstract = {Cordyceps militaris is a popular edible fungus with important economic value worldwide. In this study, an efficient CRISPR/Cas9 genome-editing system based on an autonomously replicating plasmid with an AMA1 sequence was constructed. Further, a precisely targeted gene deletion via homology-directed repair was effectively introduced in C. militaris. Gene editing was successful, with efficiencies of 55.1% and 89% for Cmwc-1 and Cmvvd, respectively. Precisely targeted gene deletion was achieved at an efficiency of 73.9% by a single guide RNA supplementation with donor DNAs. Double genes, Cmwc-1 and Cmvvd, were edited simultaneously with an efficiency of 10%. Plasmid loss was observed under non-selective culture conditions, which could permit recycling of the selectable marker and avoid the adverse effects of the CRISPR/Cas9 system on the fungus, which is beneficial for the generation of new cultivars. RNA Pol III promoters, endogenous tRNAPro of C. militaris, and chimeric AfU6-tRNAGly can be used to improve the efficiency. Polyethylene glycol-mediated protoplast transformation was markedly more efficient than Agrobacterium tumefaciens-mediated transformation of C. militaris. To our knowledge, this is the first description of genome editing and precisely targeted gene deletion in mushrooms based on AMA1 plasmids. Our findings will enable the modification of multiple genes in both functional genomics research and strain breeding.},
}
@article {pmid35822842,
year = {2022},
author = {Rossetti, M and Merlo, R and Bagheri, N and Moscone, D and Valenti, A and Saha, A and Arantes, PR and Ippodrino, R and Ricci, F and Treglia, I and Delibato, E and van der Oost, J and Palermo, G and Perugino, G and Porchetta, A},
title = {Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.},
journal = {Nucleic acids research},
volume = {50},
number = {14},
pages = {8377-8391},
pmid = {35822842},
issn = {1362-4962},
support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/genetics ; DNA Cleavage ; DNA, Single-Stranded/genetics ; },
abstract = {The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.},
}
@article {pmid35822764,
year = {2021},
author = {Eckerstorfer, MF and Grabowski, M and Lener, M and Engelhard, M and Simon, S and Dolezel, M and Heissenberger, A and Lüthi, C},
title = {Biosafety of Genome Editing Applications in Plant Breeding: Considerations for a Focused Case-Specific Risk Assessment in the EU.},
journal = {Biotech (Basel (Switzerland))},
volume = {10},
number = {3},
pages = {},
pmid = {35822764},
issn = {2673-6284},
abstract = {An intensely debated question is whether or how a mandatory environmental risk assessment (ERA) should be conducted for plants obtained through novel genomic techniques, including genome editing (GE). Some countries have already exempted certain types of GE applications from their regulations addressing genetically modified organisms (GMOs). In the European Union, the European Court of Justice confirmed in 2018 that plants developed by novel genomic techniques for directed mutagenesis are regulated as GMOs. Thus, they have to undergo an ERA prior to deliberate release or being placed on the market. Recently, the European Food Safety Authority (EFSA) published two opinions on the relevance of the current EU ERA framework for GM plants obtained through novel genomic techniques (NGTs). Regarding GE plants, the opinions confirmed that the existing ERA framework is suitable in general and that the current ERA requirements need to be applied in a case specific manner. Since EFSA did not provide further guidance, this review addresses a couple of issues relevant for the case-specific assessment of GE plants. We discuss the suitability of general denominators of risk/safety and address characteristics of GE plants which require particular assessment approaches. We suggest integrating the following two sets of considerations into the ERA: considerations related to the traits developed by GE and considerations addressing the assessment of method-related unintended effects, e.g., due to off-target modifications. In conclusion, we recommend that further specific guidance for the ERA and monitoring should be developed to facilitate a focused assessment approach for GE plants.},
}
@article {pmid35822533,
year = {2022},
author = {Selma, S and Gianoglio, S and Uranga, M and Vázquez-Vilar, M and Espinosa-Ruiz, A and Drapal, M and Fraser, PD and Daròs, JA and Orzáez, D},
title = {Potato virus X-delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.},
journal = {The Plant journal : for cell and molecular biology},
volume = {111},
number = {6},
pages = {1550-1564},
pmid = {35822533},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; *Potexvirus/genetics/metabolism ; RNA, Guide/genetics ; Tobacco/metabolism ; Transcription Factors/metabolism ; },
abstract = {Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X-derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus-induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show that gRNA-mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X-based virus-induced gene reprogramming strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.},
}
@article {pmid35822391,
year = {2022},
author = {Wei, TY and Zheng, Y and Wan, M and Yang, S and Tang, J and Wu, Y and Li, J and Chen, SX},
title = {Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation.},
journal = {ACS chemical biology},
volume = {17},
number = {8},
pages = {2130-2141},
doi = {10.1021/acschembio.2c00250},
pmid = {35822391},
issn = {1554-8937},
mesh = {*Antifungal Agents/chemistry ; Ascomycota ; *CRISPR-Cas Systems/genetics ; Echinocandins/chemistry ; Genomics ; Peptides, Cyclic ; Tyrosine/analogs &amp; derivatives ; },
abstract = {The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.},
}
@article {pmid35821638,
year = {2022},
author = {Tu, T and Song, Z and Liu, X and Wang, S and He, X and Xi, H and Wang, J and Yan, T and Chen, H and Zhang, Z and Lv, X and Lv, J and Huang, XF and Zhao, J and Lin, CP and Gao, C and Zhang, J and Gu, F},
title = {A precise and efficient adenine base editor.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {9},
pages = {2933-2941},
pmid = {35821638},
issn = {1525-0024},
support = {ZIA DK075136/ImNIH/Intramural NIH HHS/United States ; },
mesh = {*Adenine ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Mutation ; },
abstract = {Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.},
}
@article {pmid35820824,
year = {2022},
author = {Wei, L and Mu, Y and Deng, J and Wu, Y and Qiao, Y and Zhang, K and Wang, X and Huang, W and Shao, A and Chen, L and Zhang, Y and Li, Z and Lai, L and Qu, S and Xu, L},
title = {α-Gal antigen-deficient rabbits with GGTA1 gene disruption via CRISPR/Cas9.},
journal = {BMC genomic data},
volume = {23},
number = {1},
pages = {54},
pmid = {35820824},
issn = {2730-6844},
mesh = {Animals ; *Antigens, Heterophile ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Infant ; Mice ; Rabbits ; },
abstract = {BACKGROUND: Previous studies have identified the carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system.<br><br>RESULTS: The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the &#x3b1;-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6&#x2009;months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits.<br><br>CONCLUSIONS: &#x3b1;-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these &#x3b1;-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.},
}
@article {pmid35819774,
year = {2022},
author = {Gulbranson, DR},
title = {Generating Custom Pooled CRISPR Libraries for Genetic Dissection of Biological Pathways.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2473},
number = {},
pages = {333-347},
pmid = {35819774},
issn = {1940-6029},
support = {F32 AG062039/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Genetic Testing/methods ; Genomic Library ; Mammals/genetics ; },
abstract = {Genetic screens are a classic approach to dissecting biological pathways including membrane trafficking. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have enabled the utility of this approach in diploid models, including cultured mammalian cells. Here, we present detailed protocols for generating custom CRISPR libraries. These methods are useful for generating genome-wide libraries for new model organisms that lack an existing genome-wide library, and for generating smaller focused libraries.},
}
@article {pmid35819374,
year = {2022},
author = {Nicol, D and Rudge, C and Paxton, R and Niemeyer, S},
title = {How Should We Regulate Heritable Human Genome Editing in Australia?.},
journal = {Journal of law and medicine},
volume = {29},
number = {2},
pages = {322-336},
pmid = {35819374},
issn = {1320-159X},
mesh = {Australia ; CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human ; Humans ; },
abstract = {Heritable human genome editing is a form of modification of the human genome that will be inherited by progeny of the person whose DNA has been edited. Editing human genomes in ways that are heritable is currently prohibited in many countries throughout the world, including in Australia. This section starts with an examination of the historical backdrop to Australia's current laws relating to heritable human genome editing, with particular focus on how technological advances and community responses have shaped our legislative environment for innovative artificial reproductive technologies. The section then examines how community responses to current developments in heritable human genome editing might shape future law reform. The aim is to provide a foundation for examining how the future regulatory environment for heritable human genome editing in Australia might be shaped in ways that are responsive both to technological developments and to contemporary ethical norms and social values.},
}
@article {pmid35819243,
year = {2022},
author = {Talluri, S},
title = {Engineering and Design of Programmable Genome Editors.},
journal = {The journal of physical chemistry. B},
volume = {126},
number = {28},
pages = {5140-5150},
doi = {10.1021/acs.jpcb.2c03761},
pmid = {35819243},
issn = {1520-5207},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; Humans ; },
abstract = {Programmable genome editors are enzymes that can be targeted to a specific location in the genome for making site-specific alterations or deletions. The engineering, design, and development of sequence-specific editors has resulted in a dramatic increase in the precision of editing for nucleotide sequences. These editors can target specific locations in a genome, in vivo. The genome editors are being deployed for the development of genetically modified organisms for agriculture and industry, and for gene therapy of inherited human genetic disorders, cancer, and immunotherapy. Experimental and computational studies of structure, binding, activity, dynamics, and folding, reviewed here, have provided valuable insights that have the potential for increasing the functional efficiency of these gene/genome editors. Biochemical and biophysical studies of the specificities of natural and engineered genome editors reveal that increased binding affinity can be detrimental because of the increase of off-target effects and that the engineering and design of genome editors with higher specificity may require modulation and control of the conformational dynamics.},
}
@article {pmid35819195,
year = {2022},
author = {Hanscom, T and Woodward, N and Batorsky, R and Brown, AJ and Roberts, SA and McVey, M},
title = {Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks.},
journal = {Nucleic acids research},
volume = {50},
number = {13},
pages = {7465-7478},
pmid = {35819195},
issn = {1362-4962},
support = {R01 CA218112/CA/NCI NIH HHS/United States ; T32 GM008336/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/chemistry/genetics ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA Repair ; Drosophila melanogaster ; },
abstract = {Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.},
}
@article {pmid35818792,
year = {2022},
author = {Yang, W and Yan, J and Zhuang, P and Ding, T and Chen, Y and Zhang, Y and Zhang, H and Cui, W},
title = {Progress of delivery methods for CRISPR-Cas9.},
journal = {Expert opinion on drug delivery},
volume = {19},
number = {8},
pages = {913-926},
doi = {10.1080/17425247.2022.2100342},
pmid = {35818792},
issn = {1744-7593},
mesh = {*CRISPR-Cas Systems ; Endonucleases/genetics ; Gene Editing/methods ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Humans ; },
abstract = {INTRODUCTION: Gene therapy is becoming increasingly common in clinical practice, giving hope for the correction of a wide range of human diseases and defects. The CRISPR/Cas9 system, consisting of the Cas9 nuclease and single-guide RNA (sgRNA), has revolutionized the field of gene editing. However, efficiently delivering the CRISPR-Cas9 to the target organ or cell remains a significant challenge. In recent years, with rapid advances in nanoscience, materials science, and medicine, researchers have developed various technologies that can deliver CRISPR-Cas9 in different forms for in vitro and in vivo gene editing. Here, we review the development of the CRISPR-Cas9 and describe the delivery forms and the vectors that have emerged in CRISPR-Cas9 delivery, summarizing the key barriers and the promising strategies that vectors currently face in delivering the CRISPR-Cas9.<br><br>AREAS COVERED: With the rapid development of CRISPR-Cas9, delivery methods are becoming increasingly important in the in vivo delivery of CRISPR-Cas9.<br><br>EXPERT OPINION: CRISPR-Cas9 is becoming increasingly common in clinical trials. However, the complex nuclease and protease environment is a tremendous challenge for in vivo clinical applications. Therefore, the development of delivery methods is highly likely to take the application of CRISPR-Cas9 technology to another level.},
}
@article {pmid35817985,
year = {2022},
author = {Davies, K},
title = {30 years of progress from positional cloning to precision genome editing.},
journal = {Nature genetics},
volume = {54},
number = {7},
pages = {908-910},
pmid = {35817985},
issn = {1546-1718},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Techniques ; },
}
@article {pmid35815994,
year = {2022},
author = {Tanimoto, Y and Mikami, N and Ishida, M and Iki, N and Kato, K and Sugiyama, F and Takahashi, S and Mizuno, S},
title = {Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {184},
pages = {},
doi = {10.3791/64161},
pmid = {35815994},
issn = {1940-087X},
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; DNA/genetics ; Female ; Gene Editing/methods ; Gene Knock-In Techniques ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Microinjections ; *Zygote ; },
abstract = {CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.},
}
@article {pmid35814004,
year = {2022},
author = {Yu, HY and Wang, SG and Xia, PF},
title = {Reprogramming Microbial CO2-Metabolizing Chassis With CRISPR-Cas Systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {897204},
pmid = {35814004},
issn = {2296-4185},
abstract = {Global warming is approaching an alarming level due to the anthropogenic emission of carbon dioxide (CO2). To overcome the challenge, the reliance on fossil fuels needs to be alleviated, and a significant amount of CO2 needs to be sequestrated from the atmosphere. In this endeavor, carbon-neutral and carbon-negative biotechnologies are promising ways. Especially, carbon-negative bioprocesses, based on the microbial CO2-metabolizing chassis, possess unique advantages in fixing CO2 directly for the production of fuels and value-added chemicals. In order to fully uncover the potential of CO2-metabolizing chassis, synthetic biology tools, such as CRISPR-Cas systems, have been developed and applied to engineer these microorganisms, revolutionizing carbon-negative biotechnology. Herein, we review the recent advances in the adaption of CRISPR-Cas systems, including CRISPR-Cas based genome editing and CRISPR interference/activation, in cyanobacteria, acetogens, and methanogens. We also envision future innovations via the implementation of rising CRISPR-Cas systems, such as base editing, prime editing, and transposon-mediated genome editing.},
}
@article {pmid35811100,
year = {2022},
author = {Qin, Y and Geng, F and Wen, D},
title = {Generation of sex-reversed female clonal mice via CRISPR/Cas9-mediated Y chromosome deletion in male embryonic stem cells.},
journal = {Methods in cell biology},
volume = {170},
number = {},
pages = {203-210},
pmid = {35811100},
issn = {0091-679X},
support = {R01 GM129380/GM/NIGMS NIH HHS/United States ; R21 OD031973/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Chromosome Deletion ; Chromosomes, Human, Y ; Embryonic Stem Cells ; Female ; Infertility, Male ; Male ; Mice ; Sex Chromosome Aberrations ; Sex Chromosome Disorders of Sex Development ; *Tetraploidy ; },
abstract = {Mice derived entirely from embryonic stem (ES) cells can be generated through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR/Cas9-mediated deletion of the Y chromosome allowing generation of female clonal mice by tetraploid complementation. The monosomic XO female mice are viable and able to produce normal male and female offspring. Direct generation of clonal mice in both sexes can significantly accelerate the production of complex genetically modified mouse models.},
}
@article {pmid35810160,
year = {2022},
author = {Yang, B and Kong, J and Fang, X},
title = {Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNA.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3999},
pmid = {35810160},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Cell-Free Nucleic Acids/genetics ; *Epstein-Barr Virus Infections ; Herpesvirus 4, Human/genetics ; Mice ; Needles ; },
abstract = {Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles can extract analytes of interest from interstitial fluid in a minimally invasive fashion. However, some microneedles are limited by their ability to perform highly effective extraction and real-time monitoring for macromolecule biomarkers simultaneously. Here we show the synergetic effect of CRISPR-activated graphene biointerfaces, and report an on-line wearable microneedle patch for extraction and in vivo long-term monitoring of universal cell-free DNA. In this study, this wearable system enables real-time monitoring of Epstein-Barr virus, sepsis, and kidney transplantation cell-free DNA, with anti-interference ability of 60% fetal bovine serum, and has satisfactory stable sensitivity for 10 days in vivo. The experimental results of immunodeficient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring of cell-free DNA and could potentially be used for early disease screening and prognosis.},
}
@article {pmid35810156,
year = {2022},
author = {Dobbs, FM and van Eijk, P and Fellows, MD and Loiacono, L and Nitsch, R and Reed, SH},
title = {Precision digital mapping of endogenous and induced genomic DNA breaks by INDUCE-seq.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3989},
pmid = {35810156},
issn = {2041-1723},
support = {BB/P504841/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R00756X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA/genetics ; *DNA Breaks, Double-Stranded ; DNA Repair/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Genomics ; },
abstract = {Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemical's DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We further discuss how the method can be adapted to detect other genomic features.},
}
@article {pmid35810003,
year = {2022},
author = {Huang, J and Cook, DE},
title = {The contribution of DNA repair pathways to genome editing and evolution in filamentous pathogens.},
journal = {FEMS microbiology reviews},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsre/fuac035},
pmid = {35810003},
issn = {1574-6976},
abstract = {DNA double-strand breaks require repair or risk corrupting the language of life. To ensure genome integrity and viability, multiple DNA double-strand break repair pathways function in eukaryotes. Two such repair pathways, canonical non-homologous end joining and homologous recombination, have been extensively studied, while other pathways such as microhomology-mediated end joint and single-strand annealing, once thought to serve as back-ups, now appear to play a fundamental role in DNA repair. Here, we review the molecular details and hierarchy of these four DNA repair pathways, and where possible, a comparison for what is known between animal and fungal models. We address the factors contributing to break repair pathway choice, and aim to explore our understanding and knowledge gaps regarding mechanisms and regulation in filamentous pathogens. We additionally discuss how DNA double-strand break repair pathways influence genome engineering results, including unexpected mutation outcomes. Finally, we review the concept of biased genome evolution in filamentous pathogens, and provide a model, termed Biased Variation, that links DNA double-strand break repair pathways with properties of genome evolution. Despite our extensive knowledge for this universal process, there remain many unanswered questions, for which the answers may improve genome engineering and our understanding of genome evolution.},
}
@article {pmid35809388,
year = {2022},
author = {Zeng, Y and Hong, Y and Azi, F and Liu, Y and Chen, Y and Guo, C and Lin, D and Wu, Z and Chen, W and Xu, P},
title = {Advanced genome-editing technologies enable rapid and large-scale generation of genetic variants for strain engineering and synthetic biology.},
journal = {Current opinion in microbiology},
volume = {69},
number = {},
pages = {102175},
doi = {10.1016/j.mib.2022.102175},
pmid = {35809388},
issn = {1879-0364},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering ; Synthetic Biology ; },
abstract = {Targeted genome editing not only improves our understanding of fundamental rules in life sciences but also affords us versatile toolkits to improve industrially relevant phenotypes in various host cells. In this review, we summarize the recent endeavor to develop efficient genome-editing tools, and emphasize the utility of these tools to generate massive scale of genetic variants. We categorize these tools into traditional recombination-based tools, and more advanced CRISPR as well as RNA-based genome-editing tools. This diverse panel of sophisticated tools has been applied to accelerate strain engineering, upgrade biomanufacturing, and customize biosensing. In parallel with high-throughput phenotyping and AI-based optimization algorithms, we envision that genome-editing technologies will become a driving force to automate and streamline biological engineering, and empower us to address critical challenges in health, environment, energy, and sustainability.},
}
@article {pmid35808824,
year = {2022},
author = {Gao, Z and Ravendran, S and Mikkelsen, NS and Haldrup, J and Cai, H and Ding, X and Paludan, SR and Thomsen, MK and Mikkelsen, JG and Bak, RO},
title = {A truncated reverse transcriptase enhances prime editing by split AAV vectors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {9},
pages = {2942-2951},
pmid = {35808824},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Gene Editing ; Genetic Vectors/genetics ; Mice ; *Proprotein Convertase 9/genetics ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.},
}
@article {pmid35807704,
year = {2022},
author = {Schlegel, R},
title = {100 Years of Chromosome Research in Rye, Secale L.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {13},
pages = {},
pmid = {35807704},
issn = {2223-7747},
abstract = {Although microscopy and genetics were still in their infancy, there are cytological results produced a hundred years ago that are still relevant today. Since the 1920s, rye has been a subject of chromosome research. It started by plotting its mitotic and meiotic chromosomes to determine genome size. After controversial evidence, it became clear that the base number is n = 7. However, structural differences exist between species within the genus Secale. Some rye populations even carry accessory chromosomes evolutionary derived from the A genome. The development of tetraploid strains significantly promoted chromosome analysis. Various techniques have tried to stabilize the disturbed chromosome pairing of the induced tetraploids. Although slight improvements could be achieved, they did not lead to a breakthrough. However, the various aneuploid derivatives of the polyploids found major advances in the genetic analysis of rye. Trisomics, telo-trisomics, and reciprocal translocation have served as important tools for gene mapping. Since the 1970s, various chromosome banding techniques have stimulated scientific progress. The seven haploid chromosomes could be diagnosed unequivocally, not only in S. cereale but also in related species. These findings led to a clear homoeologous assignment to the genomes of related grass species such as wheat, barley, rice, etc. Current applications of in situ fluorescence staining methods, such as GISH and FISH, allow even more precise results, depending on the specificity of the DNA samples. Advanced preparation techniques are supplemented by the variety of innovations in the field of molecular genome analysis. They replace complex cytological examinations. In this way, introgressions can be safely detected by DNA markers and be much more detailed. In addition, CRISPR/CAS-mediated chromosome engineering will become an important method of the future.},
}
@article {pmid35806334,
year = {2022},
author = {Liu, Z and Wu, T and Xiang, G and Wang, H and Wang, B and Feng, Z and Mu, Y and Li, K},
title = {Enhancing Animal Disease Resistance, Production Efficiency, and Welfare through Precise Genome Editing.},
journal = {International journal of molecular sciences},
volume = {23},
number = {13},
pages = {},
pmid = {35806334},
issn = {1422-0067},
mesh = {*Animal Diseases/genetics ; Animals ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Endonucleases/genetics ; *Gene Editing ; Genetic Engineering ; Mammals/genetics ; },
abstract = {The major goal of animal breeding is the genetic enhancement of economic traits. The CRISPR/Cas system, which includes nuclease-mediated and base editor mediated genome editing tools, provides an unprecedented approach to modify the mammalian genome. Thus, farm animal genetic engineering and genetic manipulation have been fundamentally revolutionized. Agricultural animals with traits of interest can be obtained in just one generation (and without long time selection). Here, we reviewed the advancements of the CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) genome editing tools and their applications in animal breeding, especially in improving disease resistance, production performance, and animal welfare. Additionally, we covered the regulations on genome-edited animals (GEAs) and ways to accelerate their use. Recommendations for how to produce GEAs were also discussed. Despite the current challenges, we believe that genome editing breeding and GEAs will be available in the near future.},
}
@article {pmid35806003,
year = {2022},
author = {Kim, NS and Yu, J and Bae, S and Kim, HS and Park, S and Lee, K and Lee, SI and Kim, JA},
title = {Identification and Characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage (Brassica rapa L.).},
journal = {International journal of molecular sciences},
volume = {23},
number = {13},
pages = {},
pmid = {35806003},
issn = {1422-0067},
mesh = {*Brassica/genetics ; *Brassica rapa/genetics ; CRISPR-Cas Systems ; China ; Circadian Rhythm/physiology ; Gene Expression Regulation, Plant ; Mutagenesis ; Plant Breeding ; RNA, Messenger ; },
abstract = {The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9-16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2-4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.},
}
@article {pmid35805149,
year = {2022},
author = {Fang, T and Je, G and Pacut, P and Keyhanian, K and Gao, J and Ghasemi, M},
title = {Gene Therapy in Amyotrophic Lateral Sclerosis.},
journal = {Cells},
volume = {11},
number = {13},
pages = {},
pmid = {35805149},
issn = {2073-4409},
support = {P50 HD060848/HD/NICHD NIH HHS/United States ; },
mesh = {*Amyotrophic Lateral Sclerosis/genetics/metabolism/therapy ; C9orf72 Protein/genetics/metabolism ; *Frontotemporal Dementia/genetics ; Genetic Therapy ; Humans ; RNA-Binding Protein FUS/genetics/metabolism ; Superoxide Dismutase/metabolism ; Superoxide Dismutase-1/genetics/metabolism ; },
abstract = {Since the discovery of Cu/Zn superoxide dismutase (SOD1) gene mutation, in 1993, as the first genetic abnormality in amyotrophic lateral sclerosis (ALS), over 50 genes have been identified as either cause or modifier in ALS and ALS/frontotemporal dementia (FTD) spectrum disease. Mutations in C9orf72, SOD1, TAR DNA binding protein 43 (TARDBP), and fused in sarcoma (FUS) genes are the four most common ones. During the last three decades, tremendous effort has been made worldwide to reveal biological pathways underlying the pathogenesis of these gene mutations in ALS/FTD. Accordingly, targeting etiologic genes (i.e., gene therapies) to suppress their toxic effects have been investigated widely. It includes four major strategies: (i) removal or inhibition of abnormal transcribed RNA using microRNA or antisense oligonucleotides (ASOs), (ii) degradation of abnormal mRNA using RNA interference (RNAi), (iii) decrease or inhibition of mutant proteins (e.g., using antibodies against misfolded proteins), and (iv) DNA genome editing with methods such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas). The promising results of these studies have led to the application of some of these strategies into ALS clinical trials, especially for C9orf72 and SOD1. In this paper, we will overview advances in gene therapy in ALS/FTD, focusing on C9orf72, SOD1, TARDBP, and FUS genes.},
}
@article {pmid36304720,
year = {2020},
author = {Hsieh-Feng, V and Yang, Y},
title = {Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing.},
journal = {aBIOTECH},
volume = {1},
number = {2},
pages = {123-134},
pmid = {36304720},
issn = {2662-1738},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized transgene-free and multiplex genome editing. In this review, we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.},
}
@article {pmid36305010,
year = {2020},
author = {Gürel, F and Zhang, Y and Sretenovic, S and Qi, Y},
title = {CRISPR-Cas nucleases and base editors for plant genome editing.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {74-87},
pmid = {36305010},
issn = {2662-1738},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) and base editors are fundamental tools in plant genome editing. Cas9 from Streptococcus pyogenes (SpCas9), recognizing an NGG protospacer adjacent motif (PAM), is a widely used nuclease for genome editing in living cells. Cas12a nucleases, targeting T-rich PAMs, have also been recently demonstrated in several plant species. Furthermore, multiple Cas9 and Cas12a engineered variants and orthologs, with different PAM recognition sites, editing efficiencies and fidelity, have been explored in plants. These RNA-guided sequence-specific nucleases (SSN) generate double-stranded breaks (DSBs) in DNA, which trigger non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR), resulting in insertion and deletion (indel) mutations or precise gene replacement, respectively. Alternatively, genome editing can be achieved by base editors without introducing DSBs. So far, several base editors have been applied in plants to introduce C-to-T or A-to-G transitions, but they are still undergoing improvement in editing window size, targeting scope, off-target effects in DNA and RNA, product purity and overall activity. Here, we summarize recent progress on the application of Cas nucleases, engineered Cas variants and base editors in plants.},
}
@article {pmid36305005,
year = {2020},
author = {Li, S and Xia, L},
title = {Precise gene replacement in plants through CRISPR/Cas genome editing technology: current status and future perspectives.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {58-73},
pmid = {36305005},
issn = {2662-1738},
abstract = {CRISPR/Cas, as a simple, versatile, robust and cost-effective system for genome manipulation, has dominated the genome editing field over the past few years. The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change, as well as diverse agricultural, environmental and ecological challenges. Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci, transcriptome regulation and epigenome editing, base editing, and precise targeted gene/allele replacement or tagging in plants. In particular, precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair (HDR) is a holy grail in genome editing for crop improvement as it has been very difficult, laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice. However, it still remains very challenging in crop plants. This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology, with a focus on achievements, potential mechanisms and future perspectives in plant biological science as well as crop improvement.},
}
@article {pmid36305004,
year = {2020},
author = {Char, SN and Yang, B},
title = {Genome editing in grass plants.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {41-57},
pmid = {36305004},
issn = {2662-1738},
abstract = {Cereal crops including maize, rice, wheat, sorghum, barley, millet, oats and rye are the major calorie sources in our daily life and also important bioenergy sources of the world. The rapidly advancing and state-of-the-art genome-editing tools such as zinc finger nucleases, TAL effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (CRISPR-Cas9-, CRISPR-Cas12a- and CRISPR/Cas-derived base editors) have accelerated the functional genomics and have promising potential for precision breeding of grass crops. With the availability of annotated genomes of the major cereal crops, application of these established genome-editing toolkits to grass plants holds promise to increase the nutritional value and productivity. Furthermore, these easy-to-use and robust genome-editing toolkits have advanced the reverse genetics for discovery of novel gene functions in crop plants. In this review, we document some of important progress in development and utilization of genome-editing tool sets in grass plants. We also highlight present and future uses of genome-editing toolkits that can sustain and improve the quality of cereal grain for food consumption.},
}
@article {pmid36305002,
year = {2020},
author = {Schmidt, C and Schindele, P and Puchta, H},
title = {From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas.},
journal = {aBIOTECH},
volume = {1},
number = {1},
pages = {21-31},
pmid = {36305002},
issn = {2662-1738},
abstract = {In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.},
}
@article {pmid36249839,
year = {2018},
author = {Menchaca, A and Dos Santos-Neto, PC and Cuadro, F and Souza-Neves, M and Crispo, M},
title = {From reproductive technologies to genome editing in small ruminants: an embryo's journey.},
journal = {Animal reproduction},
volume = {15},
number = {Suppl 1},
pages = {984-995},
pmid = {36249839},
issn = {1984-3143},
abstract = {The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.},
}
@article {pmid35804405,
year = {2022},
author = {Shi, Z and Jiang, H and Liu, G and Shi, S and Zhang, X and Chen, Y},
title = {Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis.},
journal = {Cell &amp; bioscience},
volume = {12},
number = {1},
pages = {104},
pmid = {35804405},
issn = {2045-3701},
abstract = {BACKGROUND: The true diploid frog, Xenopus tropicalis (X. tropicalis) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation.<br><br>RESULTS: Through one-cell stage injection of Cas/gRNAs into X. tropicalis embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in X. tropicalis embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in X. tropicalis embryos.<br><br>CONCLUSION: SaCas9 and KKH SaCas9 are robust genome editing tools in X. tropicalis embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in frogs.},
}
@article {pmid35804017,
year = {2022},
author = {Ijaz, F and Nakazato, R and Setou, M and Ikegami, K},
title = {A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {11681},
pmid = {35804017},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; DNA Primers ; Gene Editing/methods ; Genotyping Techniques ; *INDEL Mutation ; Polymerase Chain Reaction/methods ; },
abstract = {The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using "face-to-face" primers where the 3' ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We present actual cases of screening of CRISPR/Cas9-engineered knockout (KO) cells for six genes, where we screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR is a simple and fast method to screen KO candidates generated by the CRISPR/Cas9 system before the final selection of clones with sequencing.},
}
@article {pmid35803994,
year = {2022},
author = {Fujikura, Y and Kimura, K and Yamanouchi, K and Sugihara, H and Hatakeyama, M and Zhuang, H and Abe, T and Daimon, M and Morita, H and Komuro, I and Oishi, K},
title = {A medium-chain triglyceride containing ketogenic diet exacerbates cardiomyopathy in a CRISPR/Cas9 gene-edited rat model with Duchenne muscular dystrophy.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {11580},
pmid = {35803994},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; *Cardiomyopathies/pathology ; *Diet, Ketogenic ; Disease Models, Animal ; Dystrophin/genetics/metabolism ; Gene Editing ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/pathology ; Rats ; Triglycerides ; },
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy caused by dystrophin mutations. Although respiratory management has improved the prognosis of patients with DMD, inevitable progressive cardiomyopathy is a current leading cause of premature death. Recently, we showed that a medium-chain triglyceride containing ketogenic diet (MCTKD) improves skeletal muscle function and pathology in a CRISPR/Cas9 gene-edited rat model with DMD. In this study, we sought to clarify whether MCTKD also improves the cardiomyopathy in these rats. DMD rats were fed either the MCTKD or normal diet (ND) from ages of 3 weeks to 9 months old. Compared with the ND-fed rats, MCTKD-fed rats showed significantly prolonged QRS duration, decreased left ventricular fractional shortening, an increased heart weight/body weight ratio, and progression of cardiac fibrosis. In contrast to our previous study which found that MCTKD improved skeletal myopathy, the current study showed unexpected exacerbation of the cardiomyopathy. Further studies are needed to explore the underlying mechanisms for these differences and to explore modified dietary options that improve skeletal and cardiac muscles simultaneously.},
}
@article {pmid35802645,
year = {2022},
author = {Sayed, S and Sidorova, OA and Hennig, A and Augsburg, M and Cortés Vesga, CP and Abohawya, M and Schmitt, LT and Sürün, D and Stange, DE and Mircetic, J and Buchholz, F},
title = {Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing.},
journal = {Cancer research},
volume = {82},
number = {17},
pages = {3002-3015},
doi = {10.1158/0008-5472.CAN-21-2519},
pmid = {35802645},
issn = {1538-7445},
mesh = {CRISPR-Cas Systems ; Carcinogenesis/genetics ; Gene Editing ; Humans ; Mutation ; *Neoplasms/genetics ; Oncogenes ; Precision Medicine ; *Proto-Oncogene Proteins p21(ras)/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {UNLABELLED: KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications.<br><br>SIGNIFICANCE: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.},
}
@article {pmid35801912,
year = {2022},
author = {Aulicino, F and Pelosse, M and Toelzer, C and Capin, J and Ilegems, E and Meysami, P and Rollarson, R and Berggren, PO and Dillingham, MS and Schaffitzel, C and Saleem, MA and Welsh, GI and Berger, I},
title = {Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus.},
journal = {Nucleic acids research},
volume = {50},
number = {13},
pages = {7783-7799},
pmid = {35801912},
issn = {1362-4962},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/R013942/1/MRC_/Medical Research Council/United Kingdom ; MR/R003017/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*Baculoviridae/genetics ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Gene Editing ; Genetic Vectors ; Humans ; },
abstract = {CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.},
}
@article {pmid35801861,
year = {2022},
author = {Ameruoso, A and Villegas Kcam, MC and Cohen, KP and Chappell, J},
title = {Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces.},
journal = {Nucleic acids research},
volume = {50},
number = {13},
pages = {7751-7760},
pmid = {35801861},
issn = {1362-4962},
mesh = {Anti-Bacterial Agents ; *Biological Products ; CRISPR-Cas Systems/genetics ; Multigene Family ; *Streptomyces/genetics ; },
abstract = {The rise of antibiotic-resistant bacteria represents a major threat to global health, creating an urgent need to discover new antibiotics. Natural products derived from the genus Streptomyces represent a rich and diverse repertoire of chemical molecules from which new antibiotics are likely to be found. However, a major challenge is that the biosynthetic gene clusters (BGCs) responsible for natural product synthesis are often poorly expressed under laboratory culturing conditions, thus preventing the isolation and screening of novel chemicals. To address this, we describe a novel approach to activate silent BGCs through rewiring endogenous regulation using synthetic gene regulators based upon CRISPR-Cas. First, we refine CRISPR interference (CRISPRi) and create CRISPR activation (CRISPRa) systems that allow for highly programmable and effective gene repression and activation in Streptomyces. We then harness these tools to activate a silent BGC by perturbing its endogenous regulatory network. Together, this work advances the synthetic regulatory toolbox for Streptomyces and facilitates the programmable activation of silent BGCs for novel chemical discovery.},
}
@article {pmid35799444,
year = {2022},
author = {Huang, S and Zhang, Z and Tao, W and Liu, Y and Li, X and Wang, X and Harati, J and Wang, PY and Huang, X and Lin, CP},
title = {Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {9},
pages = {2923-2932},
pmid = {35799444},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; Promoter Regions, Genetic ; *RNA Polymerase II/genetics ; RNA Polymerase III/genetics ; RNA, Guide/genetics ; },
abstract = {The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.},
}
@article {pmid35798852,
year = {2022},
author = {Mitschka, S and Mayr, C},
title = {Context-specific regulation and function of mRNA alternative polyadenylation.},
journal = {Nature reviews. Molecular cell biology},
volume = {23},
number = {12},
pages = {779-796},
pmid = {35798852},
issn = {1471-0080},
mesh = {*Polyadenylation/genetics ; 3' Untranslated Regions/genetics ; RNA, Messenger/genetics/metabolism ; *Gene Expression Regulation/genetics ; Protein Biosynthesis ; },
abstract = {Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions (UTRs). The expression of alternative 3' UTR isoforms is highly cell type specific and is further controlled in a gene-specific manner by environmental cues. In this Review, we discuss how the dynamic, fine-grained regulation of APA is accomplished by several mechanisms, including cis-regulatory elements in RNA and DNA and factors that control transcription, pre-mRNA cleavage and post-transcriptional processes. Furthermore, signalling pathways modulate the activity of these factors and integrate APA into gene regulatory programmes. Dysregulation of APA can reprogramme the outcome of signalling pathways and thus can control cellular responses to environmental changes. In addition to the regulation of protein abundance, APA has emerged as a major regulator of mRNA localization and the spatial organization of protein synthesis. This role enables the regulation of protein function through the addition of post-translational modifications or the formation of protein-protein interactions. We further discuss recent transformative advances in single-cell RNA sequencing and CRISPR-Cas technologies, which enable the mapping and functional characterization of alternative 3' UTRs in any biological context. Finally, we discuss new APA-based RNA therapeutics, including compounds that target APA in cancer and therapeutic genome editing of degenerative diseases.},
}
@article {pmid35798006,
year = {2022},
author = {Raguram, A and Banskota, S and Liu, DR},
title = {Therapeutic in vivo delivery of gene editing agents.},
journal = {Cell},
volume = {185},
number = {15},
pages = {2806-2827},
pmid = {35798006},
issn = {1097-4172},
support = {R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; UG3 AI150551/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Genetic Vectors ; Liposomes ; *Nanoparticles ; },
abstract = {In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.},
}
@article {pmid35797934,
year = {2022},
author = {Zhong, M and Chen, K and Sun, W and Li, X and Huang, S and Meng, Q and Sun, B and Huang, X and Wang, X and Ma, X and Ma, P},
title = {PCDetection: PolyA-CRISPR/Cas12a-based miRNA detection without PAM restriction.},
journal = {Biosensors &amp; bioelectronics},
volume = {214},
number = {},
pages = {114497},
doi = {10.1016/j.bios.2022.114497},
pmid = {35797934},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/analysis ; Nucleic Acid Amplification Techniques/methods ; Poly A/genetics ; },
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.},
}
@article {pmid35797389,
year = {2022},
author = {Chupradit, K and Thongsin, N and Tayapiwatana, C and Wattanapanitch, M},
title = {A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors.},
journal = {PloS one},
volume = {17},
number = {7},
pages = {e0270963},
pmid = {35797389},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Gene Editing/methods ; Genetic Vectors/genetics ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs' low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.},
}
@article {pmid35797344,
year = {2022},
author = {Song, Y and He, S and Jopkiewicz, A and Setroikromo, R and van Merkerk, R and Quax, WJ},
title = {Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages.},
journal = {Journal of applied microbiology},
volume = {133},
number = {4},
pages = {2280-2298},
doi = {10.1111/jam.15704},
pmid = {35797344},
issn = {1365-2672},
mesh = {*Bacillus/genetics ; *Bacillus Phages/genetics ; *Bacillus anthracis/genetics ; Bacillus subtilis/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.},
}
@article {pmid35796955,
year = {2022},
author = {Sun, R and Li, M and McMeniman, CJ and Akbari, OS},
title = {CRISPR-Mediated Genome Engineering in Aedes aegypti.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2509},
number = {},
pages = {23-51},
pmid = {35796955},
issn = {1940-6029},
support = {R21 AI146450/AI/NIAID NIH HHS/United States ; R01 AI151004/AI/NIAID NIH HHS/United States ; },
mesh = {*Aedes/genetics ; Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Chikungunya Fever ; Gene Editing/methods ; RNA, Guide ; *Yellow Fever ; *Zika Virus/genetics ; *Zika Virus Infection/genetics ; },
abstract = {CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegypti , is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya. The key to successful CRISPR-mediated gene editing applications is the delivery of both Cas9 ribonuclease and single-guide RNA (sgRNA) to the nucleus of desired cells. Various methods have been developed for supplying the Cas9 endonuclease, sgRNA , and donor DNA to Ae. aegypti. In this chapter, we focus on methods of direct embryo delivery of editing components, presenting detailed step-by-step CRISPR/Cas9-based genome-editing protocols for inducing desired heritable edits in mosquitoes as well as insights into successful application of these protocols. We also highlight potential opportunities for customizing these protocols to manipulate the mosquito genome for innovative in vivo gene function studies.},
}
@article {pmid35796760,
year = {2022},
author = {Rozners, E},
title = {Chemical Modifications of CRISPR RNAs to Improve Gene-Editing Activity and Specificity.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {28},
pages = {12584-12594},
pmid = {35796760},
issn = {1520-5126},
support = {R35 GM130207/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; RNA/genetics ; },
abstract = {CRISPR (clustered, regularly interspaced, short palindromic repeats) has become a cutting-edge research method and holds great potential to revolutionize biotechnology and medicine. However, like other nucleic acid technologies, CRISPR will greatly benefit from chemical innovation to improve activity and specificity for critical in vivo applications. Chemists have started optimizing various components of the CRISPR system; the present Perspective focuses on chemical modifications of CRISPR RNAs (crRNAs). As with other nucleic acid-based technologies, early efforts focused on well-established sugar and backbone modifications (2'-deoxy, 2'-F, 2'-OMe, and phosphorothioates). Some more significant alterations of crRNAs have been done using bicyclic (locked) riboses and phosphate backbone replacements (phosphonoacetates and amides); however, the range of chemical innovation applied to crRNAs remains limited to modifications that have been successful in RNA interference and antisense technologies. The encouraging results given by these tried-and-true modifications suggest that, going forward, chemists should take a bolder approach─research must aim to investigate what chemistry will have the most impact on maturing CRISPR as therapeutic and other in vivo technologies. With an eye to the future, this Perspective argues that the complexity of CRISPR presents rich unprecedented opportunities for chemists to synergize advances in synthetic methodology and structural biochemistry to rationally optimize crRNA-protein interactions.},
}
@article {pmid35796629,
year = {2022},
author = {Herath, D and Voogd, C and Mayo-Smith, M and Yang, B and Allan, AC and Putterill, J and Varkonyi-Gasic, E},
title = {CRISPR-Cas9-mediated mutagenesis of kiwifruit BFT genes results in an evergrowing but not early flowering phenotype.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2064-2076},
pmid = {35796629},
issn = {1467-7652},
mesh = {*Actinidia/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Flowers/genetics/metabolism ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Amino Acid Sequence ; Phenotype ; Mutagenesis ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Phosphatidylethanolamine-binding protein (PEBP) genes regulate flowering and architecture in many plant species. Here, we study kiwifruit (Actinidia chinensis, Ac) PEBP genes with homology to BROTHER OF FT AND TFL1 (BFT). CRISPR-Cas9 was used to target AcBFT genes in wild-type and fast-flowering kiwifruit backgrounds. The editing construct was designed to preferentially target AcBFT2, whose expression is elevated in dormant buds. Acbft lines displayed an evergrowing phenotype and increased branching, while control plants established winter dormancy. The evergrowing phenotype, encompassing delayed budset and advanced budbreak after defoliation, was identified in multiple independent lines with edits in both alleles of AcBFT2. RNA-seq analyses conducted using buds from gene-edited and control lines indicated that Acbft evergrowing plants had a transcriptome similar to that of actively growing wild-type plants, rather than dormant controls. Mutations in both alleles of AcBFT2 did not promote flowering in wild-type or affect flowering time, morphology and fertility in fast-flowering transgenic kiwifruit. In summary, editing of AcBFT2 has the potential to reduce plant dormancy with no adverse effect on flowering, giving rise to cultivars better suited for a changing climate.},
}
@article {pmid35796408,
year = {2022},
author = {Li, Y and Man, S and Ye, S and Liu, G and Ma, L},
title = {CRISPR-Cas-based detection for food safety problems: Current status, challenges, and opportunities.},
journal = {Comprehensive reviews in food science and food safety},
volume = {21},
number = {4},
pages = {3770-3798},
doi = {10.1111/1541-4337.13000},
pmid = {35796408},
issn = {1541-4337},
mesh = {*CRISPR-Cas Systems ; Food Safety ; *Gene Editing/methods ; },
abstract = {Food safety is one of the biggest public issues occurring around the world. Microbiological, chemical, and physical hazards can lead to food safety issues, which may occur at all stages of the supply chain. In order to tackle food safety issues and safeguard consumer health, rapid, accurate, specific, and field-deployable detection methods meeting diverse requirements are one of the imperative measures for food safety assurance. CRISPR-Cas system, a newly emerging technology, has been successfully repurposed in biosensing and has demonstrated huge potential to establish conceptually novel detection methods with high sensitivity and specificity. This review focuses on CRISPR-Cas-based detection and its current status and huge potential specifically for food safety inspection. We firstly illustrate the pending problems in food safety and summarize the popular detection methods. We then describe the potential applications of CRISPR-Cas-based detection in food safety inspection. Finally, the challenges and futuristic opportunities are proposed and discussed. Generally speaking, the current food safety detection methods are still unsatisfactory in some ways such as being time-consuming, displaying unmet sensitivity and specificity standards, and there is a comparative paucity of multiplexed testing and POCT. Recent studies have shown that CRISPR-Cas-based biosensing is an innovative and fast-expanding technology, which could make up for the shortcomings of the existing methods or even replace them. To sum up, the implementation of CRISPR-Cas and the integration of CRISPR-Cas with other techniques is promising and desirable, which is expected to provide "customized" and "smart" detection methods for food safety inspection in the coming future.},
}
@article {pmid35795354,
year = {2022},
author = {Van Huffel, K and Stock, M and Ruttink, T and De Baets, B},
title = {Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {907095},
pmid = {35795354},
issn = {1664-462X},
abstract = {Over the past years, CRISPR/Cas-mediated genome editing has revolutionized plant genetic studies and crop breeding. Specifically, due to its ability to simultaneously target multiple genes, the multiplex CRISPR/Cas system has emerged as a powerful technology for functional analysis of genetic pathways. As such, it holds great potential for application in plant systems to discover genetic interactions and to improve polygenic agronomic traits in crop breeding. However, optimal experimental design regarding coverage of the combinatorial design space in multiplex CRISPR/Cas screens remains largely unexplored. To contribute to well-informed experimental design of such screens in plants, we first establish a representation of the design space at different stages of a multiplex CRISPR/Cas experiment. We provide two independent computational approaches yielding insights into the plant library size guaranteeing full coverage of all relevant multiplex combinations of gene knockouts in a specific multiplex CRISPR/Cas screen. These frameworks take into account several design parameters (e.g., the number of target genes, the number of gRNAs designed per gene, and the number of elements in the combinatorial array) and efficiencies at subsequent stages of a multiplex CRISPR/Cas experiment (e.g., the distribution of gRNA/Cas delivery, gRNA-specific mutation efficiency, and knockout efficiency). With this work, we intend to raise awareness about the limitations regarding the number of target genes and order of genetic interaction that can be realistically analyzed in multiplex CRISPR/Cas experiments with a given number of plants. Finally, we establish guidelines for designing multiplex CRISPR/Cas experiments with an optimal coverage of the combinatorial design space at minimal plant library size.},
}
@article {pmid35794474,
year = {2022},
author = {Choi, J and Chen, W and Minkina, A and Chardon, FM and Suiter, CC and Regalado, SG and Domcke, S and Hamazaki, N and Lee, C and Martin, B and Daza, RM and Shendure, J},
title = {A time-resolved, multi-symbol molecular recorder via sequential genome editing.},
journal = {Nature},
volume = {608},
number = {7921},
pages = {98-107},
pmid = {35794474},
issn = {1476-4687},
support = {R01 HG010632/HG/NHGRI NIH HHS/United States ; UM1 HG011586/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *DNA/genetics ; *Gene Editing/methods ; *Genome/genetics ; RNA, Guide/genetics ; RNA-Seq ; Single-Cell Analysis ; Time Factors ; },
abstract = {DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained in terms of the number of distinct 'symbols' that can be concurrently recorded and/or by a failure to capture the order in which events occur[1]. Here we describe DNA Typewriter, a general system for in vivo molecular recording that overcomes these and other limitations. For DNA Typewriter, the blank recording medium ('DNA Tape') consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5' ends and therefore inactive. Short insertional edits serve as symbols that record the identity of the prime editing guide RNA[2] mediating the edit while also shifting the position of the 'type guide' by one unit along the DNA Tape, that is, sequential genome editing. In this proof of concept of DNA Typewriter, we demonstrate recording and decoding of thousands of symbols, complex event histories and short text messages; evaluate the performance of dozens of orthogonal tapes; and construct 'long tape' potentially capable of recording as many as 20 serial events. Finally, we leverage DNA Typewriter in conjunction with single-cell RNA-seq to reconstruct a monophyletic lineage of 3,257 cells and find that the Poisson-like accumulation of sequential edits to multicopy DNA tape can be maintained across at least 20 generations and 25 days of in vitro clonal expansion.},
}
@article {pmid35793201,
year = {2022},
author = {Marqués, MC and Sánchez-Vicente, J and Ruiz, R and Montagud-Martínez, R and Márquez-Costa, R and Gómez, G and Carbonell, A and Daròs, JA and Rodrigo, G},
title = {Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.},
journal = {ACS synthetic biology},
volume = {11},
number = {7},
pages = {2384-2393},
pmid = {35793201},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Genome, Viral ; *Plant Viruses/genetics ; Plants/genetics ; RNA, Viral/genetics ; Tobacco/genetics ; },
abstract = {Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.},
}
@article {pmid35792375,
year = {2022},
author = {He, M and Zhou, X and Li, Z and Yin, X and Han, W and Zhou, J and Sun, X and Liu, X and Yao, D and Liang, H},
title = {Programmable Transcriptional Modulation with a Structured RNA-Mediated CRISPR-dCas9 Complex.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {28},
pages = {12690-12697},
doi = {10.1021/jacs.2c02271},
pmid = {35792375},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA/genetics ; Transcriptional Activation ; },
abstract = {Multi-module dCas9 engineering systems have been developed for controllable transcriptional manipulation such as chemical- or light-induced systems. However, there is still a need for a separate module that can be used for internal control over the CRISPR-dCas9 system. Here, we describe a multi-module CRISPR-dCas9 system in which a separate structured RNA was applied as a programmable component that could control dCas9-based gene regulation and achieved a higher activation efficiency than dCas9-VPR that is traditionally used. By introducing a microRNA sensor, we generated a dCas9-based transcriptional regulation platform that responded to endogenous microRNAs and allowed controllable activation of endogenous genes. Moreover, we applied the platform to selectively identify HCT116 cells in a cell mixture. This work provides a flexible platform for efficient and controllable gene regulation based on CRISPR-dCas9.},
}
@article {pmid35789697,
year = {2022},
author = {Madireddy, I and Pierson Smela, M},
title = {Stably Integrating an Inducible CRISPR-Cas9 to Protect Against Viral Infections in Vitro.},
journal = {microPublication biology},
volume = {2022},
number = {},
pages = {},
pmid = {35789697},
issn = {2578-9430},
abstract = {CRISPR-Cas systems protect bacteria from viral nucleic acids. The Cas9 enzyme cleaves bacteriophage DNA preventing viral genes from being expressed in the bacterial host. In this work, the Cas9 protein is repurposed to function as an intracellular mammalian defense mechanism that protects human cells from cytomegaloviral DNA. The A549 lung adenocarcinoma cell line was genetically modified to express a doxycycline-inducible Cas9, and a guide RNA targeting a luciferase reporter plasmid. This investigation revealed a robust inducible Cas9 system that successfully reduced the expression of the luciferase viral reporter by up to 98% and by 75% on average.},
}
@article {pmid35788357,
year = {2022},
author = {Abdulrachman, D and Champreda, V and Eurwilaichitr, L and Chantasingh, D and Pootanakit, K},
title = {Efficient multiplex CRISPR/Cpf1 (Cas12a) genome editing system in Aspergillus aculeatus TBRC 277.},
journal = {Journal of biotechnology},
volume = {355},
number = {},
pages = {53-64},
doi = {10.1016/j.jbiotec.2022.06.011},
pmid = {35788357},
issn = {1873-4863},
mesh = {Aspergillus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; Francisella ; *Gene Editing/methods ; RNA ; },
abstract = {CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93 %) among the three Cpf1s. It was further investigated for its ability to delete a 1.7 kb and a 0.5 kb from pksP and pyrG genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA, which resulted in combined editing efficiency of 40 %. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts.},
}
@article {pmid35787470,
year = {2022},
author = {Vandiver, AR and Pielstick, B and Gilpatrick, T and Hoang, AN and Vernon, HJ and Wanagat, J and Timp, W},
title = {Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation.},
journal = {Mitochondrion},
volume = {65},
number = {},
pages = {176-183},
pmid = {35787470},
issn = {1872-8278},
support = {T32 GM136577/GM/NIGMS NIH HHS/United States ; T32 GM007445/GM/NIGMS NIH HHS/United States ; U01 CA253481/CA/NCI NIH HHS/United States ; T32 AR071307/AR/NIAMS NIH HHS/United States ; K02 AG059847/AG/NIA NIH HHS/United States ; R01 HG009190/HG/NHGRI NIH HHS/United States ; R01 AG055518/AG/NIA NIH HHS/United States ; R56 AG060880/AG/NIA NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA, Mitochondrial/genetics ; *Genome, Mitochondrial ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Mitochondria/genetics ; Sequence Analysis, DNA/methods ; },
abstract = {The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.},
}
@article {pmid35786883,
year = {2022},
author = {Xie, S and Xu, B and Tang, R and Chen, S and Lei, C and Nie, Z},
title = {Kinetics Accelerated CRISPR-Cas12a Enabling Live-Cell Monitoring of Mn[2+] Homeostasis.},
journal = {Analytical chemistry},
volume = {94},
number = {28},
pages = {10159-10167},
doi = {10.1021/acs.analchem.2c01461},
pmid = {35786883},
issn = {1520-6882},
mesh = {*CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Homeostasis ; Kinetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas12a system has been repurposed as a versatile nuclei acid bio-imaging tool, but its utility in sensing non-nucleic acid analytes in living cells has been less exploited. Herein, we demonstrated the ability of Mn[2+] to accelerate cleavage kinetics of Cas12a and deployed for live-cell Mn[2+] sensing by leveraging the accelerated trans-cleavage for signal reporting. In this work, we found that Mn[2+] could significantly boost both the cis-cleavage and trans-cleavage activities of Cas12a. On the basis of this phenomenon, we harnessed CRISPR-Cas12a as a direct sensing system for Mn[2+], which achieved robust Mn[2+] detection in the concentration range of 0.5-700 μM within 15 min in complex biological samples. Furthermore, we also demonstrated the versatility of this system to sense Mn[2+] in the cytoplasm of living cells. With the usage of a conditional guide RNA, this Cas12a-based sensing method was applied to study the cytotoxicity of Mn[2+] in living nerve cells, offering a valuable tool to reveal the cellular response of nerve cells to Mn[2+] disorder and homeostasis.},
}
@article {pmid35786464,
year = {2022},
author = {Ma, Y and Deng, L and Li, S},
title = {[Application of nanoparticles in CRISPR/Cas9-based gene therapy].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {6},
pages = {2087-2104},
doi = {10.13345/j.cjb.210739},
pmid = {35786464},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Gold ; *Metal Nanoparticles ; },
abstract = {CRISPR/Cas9 is a cutting-edge gene-editing technology that has emerged as a promising tool for gene therapy. Nevertheless, the safe and efficient delivery of CRISPR/Cas9 is still an urgent issue in clinical application. Nanoparticles, such as lipid-based nanoparticles, polymer nanoparticles, gold nanoparticles, and biofilm nanoparticles, are expected to bring new opportunities for CRISPR/Cas9-based gene therapy because of their biocompatibility, safety and designability. This review briefly introduced the characteristics of nanoparticles and the development of CRISPR/Cas9 in gene therapy. Moreover, the application of nanoparticles in the delivery of different forms of CRISPR/Cas9 were elaborated. Finally, the challenges and safety of nanoparticle-based gene therapy were discussed.},
}
@article {pmid35786463,
year = {2022},
author = {Zhang, J and Luo, J and Zhao, Y and Huang, Y and Chen, J and Hao, W},
title = {[Advances of using CRISPR-Cas13a system for tumor diagnosis and treatment].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {6},
pages = {2079-2086},
doi = {10.13345/j.cjb.210814},
pmid = {35786463},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Gene Editing ; Humans ; *Neoplasms/diagnosis/genetics/therapy ; RNA ; },
abstract = {CRISPR-Cas systems are well known gene editing tools, among which CRISPR-Cas9 system targeting DNA is the most well developed. Compared with CRISPR-Cas9 system, CRISPR-C2c2/ Cas13a system derived from TYPE VI of CRISPR family that can target RNA has attracted increasingly intense investigations in recent years. The CRISPR-Cas13a system is featured by specific recognition and binding of single stranded RNA sequences, thus playing a role in non-specific cleavage of RNA. This feature could be potentially applied to detect free nucleic acid in tumors or peripheral blood as a diagnostic approach. Since Cas13a specifically targets RNA, it can directly edit mRNA transcripts of genomic DNA to achieve the downregulation of target proteins without involving DNA editing. Therefore, Cas13a system could be used in tumor treatment. This review summarized the advances of using CRISPR-Cas13a for RNA targeting in tumor diagnosis and treatment, and prospected future applications.},
}
@article {pmid35784280,
year = {2022},
author = {Wagner, DL and Koehl, U and Chmielewski, M and Scheid, C and Stripecke, R},
title = {Review: Sustainable Clinical Development of CAR-T Cells - Switching From Viral Transduction Towards CRISPR-Cas Gene Editing.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {865424},
pmid = {35784280},
issn = {1664-3224},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Immunotherapy ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; },
abstract = {T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as "off-the-shelf" products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.},
}
@article {pmid35783286,
year = {2022},
author = {Taj, M and Sajjad, M and Li, M and Yasmeen, A and Mubarik, MS and Kaniganti, S and He, C},
title = {Potential Targets for CRISPR/Cas Knockdowns to Enhance Genetic Resistance Against Some Diseases in Wheat (Triticum aestivum L.).},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {926955},
pmid = {35783286},
issn = {1664-8021},
abstract = {Wheat is one of the most important food crops worldwide. Even though wheat yields have increased considerably in recent years, future wheat production is predicted to face enormous challenges due to global climate change and new versions of diseases. CRISPR/Cas technology is a clean gene technology and can be efficiently used to target genes prone to biotic stress in wheat genome. Herein, the published research papers reporting the genetic factors corresponding to stripe rust, leaf rust, stem rust, powdery mildew, fusarium head blight and some insect pests were critically reviewed to identify negative genetic factors (Susceptible, S genes) in bread wheat. Out of all reported genetic factors related to these disease, 33 genetic factors (S genes) were found as negative regulators implying that their down-regulation, deletion or silencing improved disease tolerance/resistance. The results of the published studies provided the concept of proof that these 33 genetic factors are potential targets for CRISPR/Cas knockdowns to improve genetic tolerance/resistance against these diseases in wheat. The sequences of the 33 genes were retrieved and re-mapped on the latest wheat reference genome IWGSC RefSeq v2.1. Phylogenetic analysis revealed that pathogens causing the same type of disease had some common conserved motifs and were closely related. Considering the significance of these disease on wheat yield, the S genes identified in this study are suggested to be disrupted using CRISPR/Cas system in wheat. The knockdown mutants of these S genes will add to genetic resources for improving biotic stress resistance in wheat crop.},
}
@article {pmid35782122,
year = {2022},
author = {Hawsawi, YM and Shams, A and Theyab, A and Siddiqui, J and Barnawee, M and Abdali, WA and Marghalani, NA and Alshelali, NH and Al-Sayed, R and Alzahrani, O and Alqahtani, A and Alsulaiman, AM},
title = {The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {869889},
pmid = {35782122},
issn = {2235-2988},
mesh = {*COVID-19/therapy ; Gene Editing ; Humans ; Pandemics ; SARS-CoV-2/genetics ; *Virus Diseases ; },
abstract = {Gene therapy delivers a promising hope to cure many diseases and defects. The discovery of gene-editing technology fueled the world with valuable tools that have been employed in various domains of science, medicine, and biotechnology. Multiple means of gene editing have been established, including CRISPR/Cas, ZFNs, and TALENs. These strategies are believed to help understand the biological mechanisms of disease progression. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been designated the causative virus for coronavirus disease 2019 (COVID-19) that emerged at the end of 2019. This viral infection is a highly pathogenic and transmissible disease that caused a public health pandemic. As gene editing tools have shown great success in multiple scientific and medical areas, they could eventually contribute to discovering novel therapeutic and diagnostic strategies to battle the COVID-19 pandemic disease. This review aims to briefly highlight the history and some of the recent advancements of gene editing technologies. After that, we will describe various biological features of the CRISPR-Cas9 system and its diverse implications in treating different infectious diseases, both viral and non-viral. Finally, we will present current and future advancements in combating COVID-19 with a potential contribution of the CRISPR system as an antiviral modality in this battle.},
}
@article {pmid35782118,
year = {2022},
author = {Zhan, Y and Gao, X and Li, S and Si, Y and Li, Y and Han, X and Sun, W and Li, Z and Ye, F},
title = {Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for Pneumocystis jirovecii Pneumonia.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {904485},
pmid = {35782118},
issn = {2235-2988},
mesh = {CRISPR-Cas Systems ; Humans ; *Pneumocystis carinii/genetics ; *Pneumonia, Pneumocystis/diagnosis/genetics/microbiology ; RNA ; Real-Time Polymerase Chain Reaction ; },
abstract = {BACKGROUND: Pneumocystis jirovecii can result in a serious pulmonary infection, Pneumocystis jirovecii pneumonia, in immunocompetent hosts. The diagnosis of Pneumocystis jirovecii pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies (e.g., insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific transcription mediated amplification and CRISPR/Cas13a-based diagnostics targeted P. jirovecii-mitochondrial large subunit ribosomal RNA.<br><br>METHODS: The procedure of the diagnostics included amplification of the extracted RNA samples by transcription mediated amplification, followed by CRISPR/Cas13 detection, and ultimately, the judgment of the results after 30 minutes of fluorescence signal. Later, the diagnostic performance of the CRISPR/Cas13-based diagnostics were tested on the 62 surplus clinical samples.<br><br>RESULTS: This CRISPR/Cas13-based diagnostics achieved limits of detection of approximately 2 copies/&#xb5;L transcribed RNA templates, with no cross reaction to other respiratory pathogens, including bacteria and fungi. Similar to in-house quantitative real-time polymerase chain reaction, CRISPR/Cas13-based diagnostics was still positive in 243-fold diluted bronchial alveolar lavage fluid. A preliminary evaluation of 62 surplus bronchial alveolar lavage fluid samples from patients suspected of Pneumocystis jirovecii pneumonia showed that CRISPR/Cas13-based diagnostics achieved a 78.9% sensitivity and a 97.7% specificity in the diagnosis of Pneumocystis jirovecii pneumonia.<br><br>CONCLUSION: Our study demonstrates that the CRISPR/Cas13-based diagnostics technique has good performance for the accurate and specific diagnosis of Pneumocystis jirovecii pneumonia.},
}
@article {pmid35781470,
year = {2022},
author = {Beaufils, M and Tourel, A and Petiot, A and Halmai, NB and Segal, DJ and Rendu, J and Marty, I},
title = {Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {184},
pages = {},
doi = {10.3791/64114},
pmid = {35781470},
issn = {1940-087X},
mesh = {CRISPR-Cas Systems ; Cell Line ; *Gene Editing/methods ; Humans ; Lentivirus/genetics ; Muscle Cells ; Muscles ; *RNA, Guide/genetics ; },
abstract = {One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.},
}
@article {pmid35781203,
year = {2022},
author = {Fischer, JE and Glieder, A},
title = {CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2513},
number = {},
pages = {121-133},
pmid = {35781203},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Mutagenesis ; *RNA, Guide/genetics ; Saccharomycetales ; },
abstract = {Efficient targeted genome engineering of Komagataella phaffii requires balanced expression of Cas9 nuclease and a target-specific guide RNA (gRNA). In addition, correct processing of the transcribed RNA to provide the designed gRNA as a target selective partner of targeted Cas9 protein for binding to genomic DNA is essential for efficient genome engineering. This method describes a step-by-step procedure and recommended tools for simple and efficient design of gRNAs to introduce insertions or deletions at targeted sites by CRISPR/Cas9-directed double-strand breaks, followed by error-prone nonhomologous end-joining repair.},
}
@article {pmid35781198,
year = {2022},
author = {Jakočiūnas, T and Jensen, MK and Keasling, JD},
title = {CasPER: A CRISPR/Cas9-Based Method for Directed Evolution in Genomic Loci in Saccharomyces cerevisiae.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2513},
number = {},
pages = {23-37},
pmid = {35781198},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; Genomics ; Polymerase Chain Reaction ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Here, in this chapter, we describe a detailed protocol for the method named Cas9-mediated protein evolution reaction or short CasPER. CasPER is based on the generation of large 300-600-bp mutagenized linear DNA fragments by error-prone PCR which are used as a donor for repair of double-strand break mediated by Cas9 and subsequently integrated to the genome. This method can be efficiently used for directed evolution of desired essential or nonessential genes in the genome and most importantly can be multiplexed. Altogether, the described method allows for heterogeneous DNA integration with successful transformation efficiencies of 98-100% for both single and multiplex targeting.},
}
@article {pmid35781172,
year = {2022},
author = {Wani, AK and Akhtar, N and Singh, R and Prakash, A and Raza, SHA and Cavalu, S and Chopra, C and Madkour, M and Elolimy, A and Hashem, NM},
title = {Genome centric engineering using ZFNs, TALENs and CRISPR-Cas9 systems for trait improvement and disease control in Animals.},
journal = {Veterinary research communications},
volume = {},
number = {},
pages = {},
pmid = {35781172},
issn = {1573-7446},
abstract = {Livestock is an essential life commodity in modern agriculture involving breeding and maintenance. The farming practices have evolved mainly over the last century for commercial outputs, animal welfare, environment friendliness, and public health. Modifying genetic makeup of livestock has been proposed as an effective tool to create farmed animals with characteristics meeting modern farming system goals. The first technique used to produce transgenic farmed animals resulted in random transgene insertion and a low gene transfection rate. Therefore, genome manipulation technologies have been developed to enable efficient gene targeting with a higher accuracy and gene stability. Genome editing (GE) with engineered nucleases-Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) regulates the targeted genetic alterations to facilitate multiple genomic modifications through protein-DNA binding. The application of genome editors indicates usefulness in reproduction, animal models, transgenic animals, and cell lines. Recently, CRISPR/Cas system, an RNA-dependent genome editing tool (GET), is considered one of the most advanced and precise GE techniques for on-target modifications in the mammalian genome by mediating knock-in (KI) and knock-out (KO) of several genes. Lately, CRISPR/Cas9 tool has become the method of choice for genome alterations in livestock species due to its efficiency and specificity. The aim of this review is to discuss the evolution of engineered nucleases and GETs as a powerful tool for genome manipulation with special emphasis on its applications in improving economic traits and conferring resistance to infectious diseases of animals used for food production, by highlighting the recent trends for maintaining sustainable livestock production.},
}
@article {pmid35780949,
year = {2022},
author = {Xia, K and Zhang, D and Xu, X and Liu, G and Yang, Y and Chen, Z and Wang, X and Zhang, GQ and Sun, HX and Gu, Y},
title = {Protoplast technology enables the identification of efficient multiplex genome editing tools in Phalaenopsis.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {322},
number = {},
pages = {111368},
doi = {10.1016/j.plantsci.2022.111368},
pmid = {35780949},
issn = {1873-2259},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Orchidaceae/genetics ; Plant Breeding ; Protoplasts ; Technology ; },
abstract = {Phalaenopsis orchids are popular ornamental plants worldwide. The application and optimization of efficient CRISPR-Cas genome editing toolkits in Phalaenopsis greatly accelerate the development of orchid gene function and breeding research. However, these methods are greatly hindered by the deficiency of a rapid screening system. In this study, we established a fast and convenient Phalaenopsis protoplast technology for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9-HPG (PTG, polycistronic tRNA-gRNA) system and RMC-Cpf1-HPG (RMC, ribozyme-based multi-crRNA) system, were developed for Phalaenopsis genome editing and further evaluated by established protoplast technology. We successfully detected various editing events comprising substitution and indel at designed target sites of the PDS gene and MADS gene, showing that both PTG-Cas9-HPG and RMC-Cpf1-HPG multiplex genome editing systems are functional in Phalaenopsis. Additionally, by optimizing the promoter that drives Cpf1 expression, we found that Super promoter can significantly improve the editing efficiency of the RMC-Cpf1-HPG system. Altogether, we successfully developed two efficient multiplex genome editing systems, PTG-Cas9-HPG and RMC-Cpf1-HPG, for Phalaenopsis, and the established protoplast-based screening technology provides a valuable foundation for developing more diverse and efficient genome editing toolkits and facilitating the development of orchid precision breeding.},
}
@article {pmid35780537,
year = {2022},
author = {Mei-Ling, L and Yi, L and Mei-Ling, Z and Ying, Z and Xiao-Jing, H},
title = {Y-shaped DNA nanostructures assembled-spherical nucleic acids as target converters to activate CRISPR-Cas12a enabling sensitive ECL biosensing.},
journal = {Biosensors &amp; bioelectronics},
volume = {214},
number = {},
pages = {114512},
doi = {10.1016/j.bios.2022.114512},
pmid = {35780537},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/chemistry ; Gold/chemistry ; *Metal Nanoparticles ; *Nanostructures ; *Nucleic Acids ; },
abstract = {Considering the trans-cleavage capabilities, high-specificity and programmability, the CRISPR-Cas system has been recognized as a valuable platform to develop the next-generation diagnostic biosensors. However, due to the natural interaction with nucleic acids, current CRISPR-Cas-based detection mostly applies in nucleic acid analysis rather than non-nucleic acid analysis. By virtue of spherical nucleic acids (SNAs) with programmability and specificity, the Y-shaped DNA nanostructures assembled-SNAs (Y-SNAs) were rationally designed as target converters to achieve the quantitative activation of CRISPR-Cas12a, enabling a highly specific and sensitive electrochemiluminescence (ECL) determination of alpha-methylacyl-CoA racemase (AMACR), a high specific protein biomarker of prostate cancer. Significantly, the Y-shaped DNA nanostructures comprised of assisted DNA (A1), AMACR aptamer and DNA activator of CRISPR-Cas12a were loaded on Au nanoparticles modified Fe3O4 magnetic beads (Au@Fe3O4 MBs) to construct the robust Y-SNAs. In the presence of the target AMACR, the Y-SNAs as target converters could achieve quantitative activation of CRISPR-Cas12a by outputting the DNA activators with a linear relationship to the target. The amplified ECL signals were triggered by the release of the ferrocene-labeled quenching probes (QPs) on the electrode surface due to the trans-cleavage activity of CRISPR-Cas12a, thereby realizing the sensitive ECL determination of AMACR from 10 ng/mL to 100 μg/mL with the detection limit of 1.25 ng/mL. In general, this approach provides novel perspectives on how to design a universal ECL platform of the CRISPR-Cas system to detect the non-nucleic acid targets beyond the traditional methods.},
}
@article {pmid35780186,
year = {2022},
author = {Ogasawara, T and Watanabe, J and Adachi, R and Ono, Y and Kamimura, Y and Muramoto, T},
title = {CRISPR/Cas9-based genome-wide screening of Dictyostelium.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {11215},
pmid = {35780186},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; *Dictyostelium/genetics ; Gene Library ; Genome ; RNA, Guide/genetics ; },
abstract = {Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a unique life cycle and is often used as a crucial research model for a wide range of biological processes and rare metabolites. To address the inadequacies of conventional genetic screening approaches, we developed a highly efficient CRISPR/Cas9-based genome-wide screening system for Dictyostelium. A genome-wide library of 27,405 gRNAs and a kinase library of 4,582 gRNAs were compiled and mutant pools were generated. The resulting mutants were screened for defects in cell growth and more than 10 candidate genes were identified. Six of these were validated and five recreated mutants presented with growth abnormalities. Finally, the genes implicated in developmental defects were screened to identify the unknown genes associated with a phenotype of interest. These findings demonstrate the potential of the CRISPR/Cas9 system as an efficient genome-wide screening method.},
}
@article {pmid35779647,
year = {2022},
author = {Alfaro, T and Elmore, JR and Stromberg, ZR and Hutchison, JR and Hess, BM},
title = {Engineering Citrobacter freundii using CRISPR/Cas9 system.},
journal = {Journal of microbiological methods},
volume = {200},
number = {},
pages = {106533},
doi = {10.1016/j.mimet.2022.106533},
pmid = {35779647},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems ; *Citrobacter freundii/genetics ; Gene Editing/methods ; Genome, Bacterial ; },
abstract = {The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) system is a useful tool to edit genomes quickly and efficiently. However, the use of CRISPR/Cas9 to edit bacterial genomes has been limited to select microbial chassis primarily used for bioproduction of high value products. Thus, expansion of CRISPR/Cas9 tools to other microbial organisms is needed. Here, our aim was to assess the suitability of CRISPR/Cas9 for genome editing of the Citrobacter freundii type strain ATCC 8090. We evaluated the commonly used two plasmid pCas/pTargetF system to enable gene deletions and insertions in C. freundii and determined editing efficiency. The CRISPR/Cas9 based method enabled high editing efficiency (~91%) for deletion of galactokinase (galk) and enabled deletion with various single guide RNA (sgRNA) sequences. To assess the ability of CRISPR/Cas9 tools to insert genes, we used the fluorescent reporter mNeonGreen, an endopeptidase (yebA), and a transcriptional regulator (xylS) and found successful insertion with high efficiency (81-100%) of each gene individually. These results strengthen and expand the use of CRISPR/Cas9 genome editing to C. freundii as an additional microbial chassis.},
}
@article {pmid35778883,
year = {2022},
author = {Pechar, GS and Donaire, L and Gosalvez, B and García-Almodovar, C and Sánchez-Pina, MA and Truniger, V and Aranda, MA},
title = {Editing melon eIF4E associates with virus resistance and male sterility.},
journal = {Plant biotechnology journal},
volume = {20},
number = {10},
pages = {2006-2022},
pmid = {35778883},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; *Cucurbitaceae/genetics/virology ; *Eukaryotic Initiation Factor-4E/genetics ; Eukaryotic Initiation Factor-4F/metabolism ; Eukaryotic Initiation Factor-4G/metabolism ; *Gametogenesis, Plant/genetics ; Gene Editing ; *Plant Diseases/genetics/virology ; *Plant Infertility/genetics ; *Plant Proteins/genetics ; *Pollen/genetics/growth &amp; development ; *Potyvirus ; },
abstract = {The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.},
}
@article {pmid35777218,
year = {2022},
author = {Zavvar, TS and Khoshbin, Z and Ramezani, M and Alibolandi, M and Abnous, K and Taghdisi, SM},
title = {CRISPR/Cas-engineered technology: Innovative approach for biosensor development.},
journal = {Biosensors &amp; bioelectronics},
volume = {214},
number = {},
pages = {114501},
doi = {10.1016/j.bios.2022.114501},
pmid = {35777218},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Reproducibility of Results ; Ribonucleases/metabolism ; },
abstract = {On-site and real-time clinical monitoring have been progressed dramatically by integrating biosensor science with portable digital electronic technology. Clustered regularly interspaced short palindromic repeats (CRISPR) with association of RNA-guided nucleases (CrRNA-Cas enzymes) have achieved novel CRISPR/Cas biosensing science as a promising revolutionized diagnostic technology for portable and on-site healthcare monitoring and diagnostics. Among several available CRISPR/Cas systems, CRISPR/Cas12a and CRISPR/Cas13a conjugates are utilized broadly in biosensor design, because of their capability to cleave both target and non-target sequences. With the advantages of portability, cost-effectiveness, facile operation, high durability, and reproducibility, CRISPR/Cas-based biosensing techniques are a perfect choice for designing ultra-sensitive point-of-care diagnostic devices with amplified response signals. In the present review, we summarize the advances in the CRISPR/Cas-based biosensors with the focus on healthcare and diagnostic purposes. The cooperation of nanomaterial engineering with CRISPR/Cas biosensors is also represented to attain a promising viewpoint for offering novel user-friendly test kits for announcing ultra-low levels of diverse targets in the future.},
}
@article {pmid35776981,
year = {2022},
author = {Wang, Y and Li, X and Chen, X and Siewers, V},
title = {CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {22},
number = {1},
pages = {},
pmid = {35776981},
issn = {1567-1364},
support = {EP-D-16-017/EPA/EPA/United States ; },
mesh = {Amylases/genetics ; CRISPR-Cas Systems ; Point Mutation ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; alpha-Amylases/genetics/metabolism ; },
abstract = {The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were reintroduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation), and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. A total of 11 point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication, and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.},
}
@article {pmid35776792,
year = {2022},
author = {Roy, S and Juste, SS and Sneider, M and Auradkar, A and Klanseck, C and Li, Z and Julio, AHF and Lopez del Amo, V and Bier, E and Guichard, A},
title = {Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells.},
journal = {Science advances},
volume = {8},
number = {26},
pages = {eabo0721},
doi = {10.1126/sciadv.abo0721},
pmid = {35776792},
issn = {2375-2548},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Chromatids ; *Deoxyribonuclease I ; *Drosophila ; },
abstract = {Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. Here, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process we refer to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.},
}
@article {pmid35776127,
year = {2022},
author = {Tasca, F and Brescia, M and Wang, Q and Liu, J and Janssen, JM and Szuhai, K and Gonçalves, MAFV},
title = {Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells.},
journal = {Nucleic acids research},
volume = {50},
number = {13},
pages = {7761-7782},
pmid = {35776127},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; *Dystrophin/genetics/metabolism ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Humans ; Muscle Cells/metabolism ; Muscular Dystrophy, Duchenne/*genetics/pathology/*therapy ; Tumor Suppressor Protein p53/metabolism ; },
abstract = {Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.},
}
@article {pmid35776123,
year = {2022},
author = {Cheng, ZH and Wu, J and Liu, JQ and Min, D and Liu, DF and Li, WW and Yu, HQ},
title = {Repurposing CRISPR RNA-guided integrases system for one-step, efficient genomic integration of ultra-long DNA sequences.},
journal = {Nucleic acids research},
volume = {50},
number = {13},
pages = {7739-7750},
pmid = {35776123},
issn = {1362-4962},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genomics ; *Integrases/metabolism ; RNA ; },
abstract = {Genomic integration techniques offer opportunities for generation of engineered microorganisms with improved or even entirely new functions but are currently limited by inability for efficient insertion of long genetic payloads due to multiplexing. Herein, using Shewanella oneidensis MR-1 as a model, we developed an optimized CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST system), which enables programmable, RNA-guided transposition of ultra-long DNA sequences (30 kb) onto bacterial chromosomes at ∼100% efficiency in a single orientation. In this system, a crRNA (CRISPR RNA) was used to target multicopy loci like insertion-sequence elements or combining I-SceI endonuclease, thereby allowing efficient single-step multiplexed or iterative DNA insertions. The engineered strain exhibited drastically improved substrate diversity and extracellular electron transfer ability, verifying the success of this system. Our work greatly expands the application range and flexibility of genetic engineering techniques and may be readily extended to other bacteria for better controlling various microbial processes.},
}
@article {pmid35775831,
year = {2022},
author = {Chen, Y and Zong, N and Ye, F and Mei, Y and Qu, J and Jiang, X},
title = {Dual-CRISPR/Cas12a-Assisted RT-RAA for Ultrasensitive SARS-CoV-2 Detection on Automated Centrifugal Microfluidics.},
journal = {Analytical chemistry},
volume = {94},
number = {27},
pages = {9603-9609},
doi = {10.1021/acs.analchem.2c00638},
pmid = {35775831},
issn = {1520-6882},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; Microfluidics ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Recombinases ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection can be combined with recombinase-aided amplification (RAA) to enable rapid, accurate, and early detection of SARS-CoV-2. Current CRISPR-based approaches to detecting viral nucleic acid typically require immense manual operations to transfer RPA amplicons for CRISPR detection or suffer from compromised sensitivity by mixing the competing RPA amplification and CRISPR detection. Here, we develop dual-CRISPR/Cas12a-assisted RT-RAA assay and a ″sample-to-answer″ centrifugal microfluidic platform that can automatically detect 1 copy/μL of the SARS-CoV-2 within 30 min. This chip separates the amplification (RAA) from detection (CRISPR), such that sensitivity is maximized and the time consumption is decreased by a factor of 3. For the 26 positive and 8 negative clinical SARS-CoV-2 samples, this automated centrifugal microfluidics achieved 100% accuracy compared to the gold-standard RT-PCR technique. This point-of-care test, with the advantages of being one-step, automated, rapid, and sensitive, will have a significant potential for clinical diagnosis and disease prevention.},
}
@article {pmid35775700,
year = {2022},
author = {Quan, M and Oh, Y and Cho, SY and Kim, JH and Moon, HG},
title = {Polo-Like Kinase 1 Regulates Chromosomal Instability and Paclitaxel Resistance in Breast Cancer Cells.},
journal = {Journal of breast cancer},
volume = {25},
number = {3},
pages = {178-192},
pmid = {35775700},
issn = {1738-6756},
support = {2019R1A2C2005277//National Research Foundation of Korea/Korea ; 2019R1C1C1006898//National Research Foundation of Korea/Korea ; //Seoul National University Hospital/Korea ; //Bertis Inc/Korea ; },
abstract = {PURPOSE: Chromosomal instability (CIN) contributes to intercellular genetic heterogeneity and has been implicated in paclitaxel (PTX) resistance in breast cancer. In this study, we explored polo-like kinase 1 (PLK1) as an important regulator of mitotic integrity and as a useful predictive biomarker for PTX resistance in breast cancer.<br><br>METHODS: We performed PTX resistance screening using the human kinome CRISPR/Cas9 library in breast cancer cells. In vitro cell proliferation and apoptosis assays and in vivo xenograft experiments were performed to determine the effects of PLK1 on breast cancer cells. Immunofluorescence microscopy was used to measure the degree of multipolar cell division.<br><br>RESULTS: Kinome-wide CRISPR/Cas9 screening identified various kinases involved in PTX resistance in breast cancer cells; among these, PLK1 was chosen for further experiments. PLK1 knockdown inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro and in vivo. Moreover, PLK1 silencing sensitized breast cancer cells and mouse xenograft tumor models to PTX cytotoxicity. Silencing of PLK1 induced the formation of multipolar spindles and increased the percentage of multipolar cells. In addition, PLK1 silencing resulted in the downregulation of BubR1 and Mad2 in breast cancer cells. Furthermore, PLK1 upregulation in primary breast cancer was associated with decreased overall patient survival based on the analysis of The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium databases.<br><br>CONCLUSION: PLK1 plays an important role in PTX resistance by regulating CIN in breast cancer cells. Targeting PLK1 may be an effective treatment strategy for PTX-resistant breast cancers.},
}
@article {pmid35774509,
year = {2022},
author = {Sathee, L and Jagadhesan, B and Pandesha, PH and Barman, D and Adavi B, S and Nagar, S and Krishna, GK and Tripathi, S and Jha, SK and Chinnusamy, V},
title = {Genome Editing Targets for Improving Nutrient Use Efficiency and Nutrient Stress Adaptation.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {900897},
pmid = {35774509},
issn = {1664-8021},
abstract = {In recent years, the development of RNA-guided genome editing (CRISPR-Cas9 technology) has revolutionized plant genome editing. Under nutrient deficiency conditions, different transcription factors and regulatory gene networks work together to maintain nutrient homeostasis. Improvement in the use efficiency of nitrogen (N), phosphorus (P) and potassium (K) is essential to ensure sustainable yield with enhanced quality and tolerance to stresses. This review outlines potential targets suitable for genome editing for understanding and improving nutrient use (NtUE) efficiency and nutrient stress tolerance. The different genome editing strategies for employing crucial negative and positive regulators are also described. Negative regulators of nutrient signalling are the potential targets for genome editing, that may improve nutrient uptake and stress signalling under resource-poor conditions. The promoter engineering by CRISPR/dead (d) Cas9 (dCas9) cytosine and adenine base editing and prime editing is a successful strategy to generate precise changes. CRISPR/dCas9 system also offers the added advantage of exploiting transcriptional activators/repressors for overexpression of genes of interest in a targeted manner. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) are variants of CRISPR in which a dCas9 dependent transcription activation or interference is achieved. dCas9-SunTag system can be employed to engineer targeted gene activation and DNA methylation in plants. The development of nutrient use efficient plants through CRISPR-Cas technology will enhance the pace of genetic improvement for nutrient stress tolerance of crops and improve the sustainability of agriculture.},
}
@article {pmid35774227,
year = {2022},
author = {Obermeier, M and Vadolas, J and Verhulst, S and Goossens, E and Baert, Y},
title = {Lipofection of Non-integrative CRISPR/Cas9 Ribonucleoproteins in Male Germline Stem Cells: A Simple and Effective Knockout Tool for Germline Genome Engineering.},
journal = {Frontiers in cell and developmental biology},
volume = {10},
number = {},
pages = {891173},
pmid = {35774227},
issn = {2296-634X},
abstract = {Gene editing in male germline stem (GS) cells is a potent tool to study spermatogenesis and to create transgenic mice. Various engineered nucleases already demonstrated the ability to modify the genome of GS cells. However, current systems are limited by technical complexity diminishing application options. To establish an easier method to mediate gene editing, we tested the lipofection of site-specific Cas9:gRNA ribonucleoprotein (RNP) complexes to knockout the enhanced green fluorescent protein (Egfp) in mouse EGFP-GS cells via non-homologous end joining. To monitor whether gene conversion through homology-directed repair events occurred, single-stranded oligodeoxynucleotides were co-lipofected to deliver a Bfp donor sequence. Results showed Egfp knockout in up to 22% of GS cells, which retained their undifferentiated status following transfection, while only less than 0.7% EGFP to BFP conversion was detected in gated GS cells. These data show that CRISPR/Cas9 RNP-based lipofection is a promising system to simply and effectively knock out genes in mouse GS cells. Understanding the genes involved in spermatogenesis could expand therapeutic opportunities for men suffering from infertility.},
}
@article {pmid35773498,
year = {2022},
author = {Liu, Y and Andersson, M and Granell, A and Cardi, T and Hofvander, P and Nicolia, A},
title = {Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).},
journal = {Plant cell reports},
volume = {41},
number = {9},
pages = {1843-1852},
pmid = {35773498},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Lycopersicon esculentum/genetics ; Plant Breeding ; Protoplasts ; Ribonucleoproteins/genetics ; },
abstract = {We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M0 plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.},
}
@article {pmid35773055,
year = {2022},
author = {Croteau, SE},
title = {Hemophilia A/B.},
journal = {Hematology/oncology clinics of North America},
volume = {36},
number = {4},
pages = {797-812},
doi = {10.1016/j.hoc.2022.03.009},
pmid = {35773055},
issn = {1558-1977},
mesh = {Adult ; Dependovirus/genetics ; Factor IX/genetics ; Factor VIII/genetics ; Genetic Vectors/genetics ; *Hemophilia A/genetics/therapy ; *Hemophilia B/genetics ; Humans ; Quality of Life ; },
abstract = {Adeno-associated virus (AAV)-mediated gene transfer has successfully raised, and in some cases transiently normalized, FVIII or FIX activity levels in adults with severe hemophilia. Raising FVIII/IX levels, particularly greater than ∼15 IU/dL (mild deficiency), corresponds to a marked decrease in spontaneous and provoked bleeding, dramatic reduction in factor concentrate use, and improved quality of life (QoL). Limited understanding of innate and adaptive immune system responses and hepatocyte transgene expression and stress responses to AAV-mediated gene transfer contribute to the variability in initial and long-term factor protein expression. Lentiviral (LV) and CRISPR/Cas-9 gene therapy approaches may further bolster the range of eligible participants and improve transgene expression and durability.},
}
@article {pmid35773054,
year = {2022},
author = {Porteus, MH and Pavel-Dinu, M and Pai, SY},
title = {A Curative DNA Code for Hematopoietic Defects: Novel Cell Therapies for Monogenic Diseases of the Blood and Immune System.},
journal = {Hematology/oncology clinics of North America},
volume = {36},
number = {4},
pages = {647-665},
pmid = {35773054},
issn = {1558-1977},
support = {R01 AI097320/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA ; *Gene Editing ; *Genetic Vectors ; Hematopoietic Stem Cells ; Humans ; Immune System ; },
abstract = {Innovations in programmable nucleases have expanded genetic engineering capabilities, raising the possibility of a new approach to curing monogenic hematological diseases. Feasibility studies using ex vivo targeted genome-editing, and nonintegrating viral vectors show outstanding potential for correcting genetic conditions at their root cause. This article reviews the latest technological advances in the CRISPR/Cas9 system alone and combined with engineered viruses as editing tools for human hematopoietic stem and progenitor cells (HSPCs). We discuss the early phase in human trials of genome editing-based therapies for hemoglobinopathies.},
}
@article {pmid35773047,
year = {2022},
author = {Ottaviano, G and Qasim, W},
title = {Genome-Edited T Cell Therapies.},
journal = {Hematology/oncology clinics of North America},
volume = {36},
number = {4},
pages = {729-744},
doi = {10.1016/j.hoc.2022.03.006},
pmid = {35773047},
issn = {1558-1977},
mesh = {*CRISPR-Cas Systems ; Child ; Gene Editing/methods ; Humans ; Immunotherapy, Adoptive/methods ; *Neoplasms/therapy ; T-Lymphocytes ; },
abstract = {Chimeric antigen receptor (CAR) T-cells are widely being investigated against malignancies, and allogeneic 'universal donor' CAR-T cells offer the possibility of widened access to pre-manufactured, off-the-shelf therapies. Different genome-editing platforms have been used to address human leukocyte antigen (HLA) barriers to generate universal CAR-T cell therapy and early applications have been reported in children and adults against B cell malignancies. Recently developed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based systems and related technologies offer the prospect of enhanced cellular immunotherapies for a wider range of hematological malignancies.},
}
@article {pmid35771483,
year = {2022},
author = {Zou, J and Meng, X and Liu, Q and Shang, M and Wang, K and Li, J and Yu, H and Wang, C},
title = {Improving the efficiency of prime editing with epegRNAs and high-temperature treatment in rice.},
journal = {Science China. Life sciences},
volume = {65},
number = {11},
pages = {2328-2331},
pmid = {35771483},
issn = {1869-1889},
mesh = {*Oryza/genetics ; Temperature ; Gene Editing ; CRISPR-Cas Systems ; *Hyperthermia, Induced ; },
}
@article {pmid35768165,
year = {2022},
author = {Zhao, S and Wang, Y and Yang, N and Mu, M and Wu, Z and Li, H and Tang, X and Zhong, K and Zhang, Z and Huang, C and Cao, T and Zheng, M and Wang, G and Nie, C and Yang, H and Guo, G and Zhou, L and Zheng, X and Tong, A},
title = {Genome-scale CRISPR-Cas9 screen reveals novel regulators of B7-H3 in tumor cells.},
journal = {Journal for immunotherapy of cancer},
volume = {10},
number = {6},
pages = {},
pmid = {35768165},
issn = {2051-1426},
mesh = {Animals ; *B7 Antigens/biosynthesis/immunology ; CRISPR-Cas Systems ; Eukaryotic Initiation Factor-4E/immunology/metabolism ; Female ; Humans ; Mice ; *Ovarian Neoplasms/drug therapy/immunology/metabolism ; Transcription Factors/immunology/metabolism ; Tumor Microenvironment ; p38 Mitogen-Activated Protein Kinases/immunology/metabolism ; },
abstract = {BACKGROUND: Despite advances in B7 homolog 3 protein (B7-H3) based immunotherapy, the development of drug resistance remains a major clinical concern. The heterogeneity and emerging loss of B7-H3 expression are the main causes of drug resistance and treatment failure in targeted therapies, which reveals an urgent need to elucidate the mechanism underlying the regulation of B7-H3 expression. In this study, we identified and explored the crucial role of the transcription factor SPT20 homolog (SP20H) in B7-H3 expression and tumor progression.<br><br>METHODS: Here, we performed CRISPR/Cas9-based genome scale loss-of-function screening to identify regulators of B7-H3 in human ovarian cancer cells. Signaling pathways altered by SP20H knockout were revealed by RNA sequencing. The regulatory role and mechanism of SP20H in B7-H3 expression were validated using loss-of-function and gain-of-function assays in vitro. The effects of inhibiting SP20H on tumor growth and efficacy of anti-B7-H3 treatment were evaluated in tumor-bearing mice.<br><br>RESULTS: We identified SUPT20H (SP20H) as negative and eIF4E as positive regulators of B7-H3 expression in various cancer cells. Furthermore, we provided evidence that either SP20H loss or TNF-&#x3b1; stimulation in tumor cells constitutively activates p38 MAPK-eIF4E signaling, thereby upregulating B7-H3 expression. Loss of SP20H upregulated B7-H3 expression both in vitro and in vivo. Additionally, deletion of SP20H significantly suppressed tumor growth and increased immune cells infiltration in tumor microenvironment. More importantly, antibody-drug conjugates targeting B7-H3 exhibited superior antitumor performance against SP20H-deficient tumors relative to control groups.<br><br>CONCLUSIONS: Activation of p38 MAPK-eIF4E signaling serves as a key event in the transcription initiation and B7-H3 protein expression in tumor cells. Genetically targeting SP20H upregulates target antigen expression and sensitizes tumors to anti-B7-H3 treatment. Collectively, our findings provide new insight into the mechanisms underlying B7-H3 expression and introduce a potential synergistic target for existing antibody-based targeted therapy against B7-H3.},
}
@article {pmid35767832,
year = {2022},
author = {Yang, H and Yang, S and Xia, X and Deng, R and Gao, H and Dong, Y},
title = {Sensitive Detection of a Single-Nucleotide Polymorphism in Foodborne Pathogens Using CRISPR/Cas12a-Signaling ARMS-PCR.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {27},
pages = {8451-8457},
doi = {10.1021/acs.jafc.2c03304},
pmid = {35767832},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Polymerase Chain Reaction ; *Salmonella enterica/genetics ; Polymorphism, Single Nucleotide ; },
abstract = {Salmonella infection, particularly that caused by drug-resistant strain, has become a worldwide public health issue. Herein, we presented a CRISPR/Cas12a-signaling ARMS-PCR assay, termed cARMS, capable of sensitively detecting drug-resistant Salmonella enterica (S. enterica) involving single-nucleotide polymorphism (SNP). Owing to the dual-recognition processes, i.e., allele-specific primed polymerization and CRISPR/Cas12 binding, the cARMS assay yielded a high sensitivity for detecting SNP down to ∼0.5%. We used the cARMS assay to investigate the adaptation of SNP-involved drug-resistant S. enterica to salt stress. It was found that the mutants exhibited stronger adaptation to salt stress, indicating the potential risk of using high salt content as a sterilization strategy. The results verified the feasibility of the cARMS assay in controlling SNP-involved bacteria-associated biosafety.},
}
@article {pmid35767740,
year = {2022},
author = {Hutcheson, RL and Hayes, M and Sugden, B},
title = {Optimal LentiCRISPR-Based System for Sequential CRISPR/Cas9 Screens.},
journal = {ACS synthetic biology},
volume = {11},
number = {7},
pages = {2259-2266},
doi = {10.1021/acssynbio.2c00152},
pmid = {35767740},
issn = {2161-5063},
support = {P01 CA022443/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Library ; Genome ; High-Throughput Nucleotide Sequencing ; Humans ; *RNA, Guide/genetics ; },
abstract = {The advent of genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening has advanced the understanding of molecular systems within cells. Here, we demonstrate the utility of sequentially performed CRISPR knockout screens that use an existing library to explore a biological question across the human genome, and then the remaining cells are used to examine each gene candidate against one common gene of interest. We call this approach "Many vs One" CRISPR screening, made possible by a modified 7SK promoter in place of the U6 promoter to drive expression of a single guide RNA. Inserting this novel 7SK promoter into the ubiquitously used lentiCRISPRv2 backbone is crucial, because it overcomes the need for a substantial increase in CRISPR library coverage during screening, sample processing, and next generation sequencing. This new 7SK vector equals the original lentiCRISPRv2 in lentiviral titer, knockout efficiency, and ease of use.},
}
@article {pmid35767601,
year = {2022},
author = {Yuan, G and Lu, H and De, K and Hassan, MM and Liu, Y and Li, Y and Muchero, W and Abraham, PE and Tuskan, GA and Yang, X},
title = {An Intein-Mediated Split-nCas9 System for Base Editing in Plants.},
journal = {ACS synthetic biology},
volume = {11},
number = {7},
pages = {2513-2517},
pmid = {35767601},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; *Inteins/genetics ; Plants/genetics ; },
abstract = {Virus-assisted delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. Among the CRISPR/Cas systems, CRISPR/Cas9 is most widely used; however, to pack the relatively large size of the CRISPR/Cas9 system into viral vectors with confined packaging capacity is challenging. To address this technical challenge, we developed a strategy based on split inteins that splits the required CRISPR/Cas9 components across a dual-vector system. The CRISPR/Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by a successful demonstration of split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-nCas9 can induce efficient base editing in plant cells. We identified several split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.},
}
@article {pmid35766814,
year = {2022},
author = {Liang, Y and Iqbal, Z and Wang, J and Xu, L and Xu, X and Ouyang, K and Zhang, H and Lu, J and Duan, L and Xia, J},
title = {Cell-derived extracellular vesicles for CRISPR/Cas9 delivery: engineering strategies for cargo packaging and loading.},
journal = {Biomaterials science},
volume = {10},
number = {15},
pages = {4095-4106},
doi = {10.1039/d2bm00480a},
pmid = {35766814},
issn = {2047-4849},
mesh = {*CRISPR-Cas Systems/genetics ; *Extracellular Vesicles ; Gene Editing ; Genetic Therapy ; RNA, Guide/genetics ; },
abstract = {Genome editing technology has emerged as a potential therapeutic tool for treating incurable diseases. In particular, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems and the design of single-guide RNAs (sgRNAs) have revolutionized genome editing applications. Unfortunately, compared with the rapid development of gene-editing tools, the progress in the development of delivery technologies is lagging behind and thus limiting the clinical application of genome editing. To overcome these limitations, researchers have investigated various delivery systems, including viral and non-viral vectors for delivering CRISPR/Cas and sgRNA complexes. As natural endogenous nanocarriers, extracellular vesicles (EVs) present advantages of biocompatibility, low immunogenicity, stability, and high permeability, making them one of the most promising drug delivery vehicles. This review provides an overview of the fundamental mechanisms of EVs from the aspects of biogenesis, trafficking, cargo delivery, and function as nanotherapeutic agents. We also summarize the latest trends in EV-based CRISPR/Cas delivery systems and discuss the prospects for future development. In particular, we put our emphasis on the state-of-the-art engineering strategies to realize efficient cargo packaging and loading. Altogether, EVs hold promise in bridging genome editing in the laboratory and clinical applications of gene therapies by providing a safe, effective, and targeted delivery vehicle.},
}
@article {pmid35766512,
year = {2022},
author = {Li, J and Zhang, L and Xu, Q and Zhang, W and Li, Z and Chen, L and Dong, X},
title = {CRISPR-Cas9 Toolkit for Genome Editing in an Autotrophic CO2-Fixing Methanogenic Archaeon.},
journal = {Microbiology spectrum},
volume = {10},
number = {4},
pages = {e0116522},
pmid = {35766512},
issn = {2165-0497},
mesh = {Archaea/genetics ; *CRISPR-Cas Systems ; Carbon Dioxide ; *Gene Editing/methods ; Nucleotides ; },
abstract = {The CRISPR-Cas9 system is a robust genome editing tool that is widely applied in eukaryotes and bacteria. However, use of this technique has only been developed for one species of Archaea, a domain of life ranking in parallel with Eukarya and Bacteria. In this study, we applied the CRISPR-Cas9 genome editing technique to Methanococcus maripaludis, an autotrophic and hydrogenotrophic methanogenic archaeon with a remarkably polyploid genome comprising up to ~55 chromosomal copies per cell. An editing plasmid was designed that encodes small guide RNA (sgRNA), Cas9 protein and an ~1-kb repair template (donor). Highly efficient (75% to 100%) and precise genome editing was achieved following one-step transformation. Significantly, the Cas9-based system efficiently deleted one or two genes and a large DNA fragment (~9 kb) and even synchronously deleted 13 genes located at three loci in all chromosomal copies of M. maripaludis. Moreover, precise in situ genome modifications, such as gene tagging and multiple- and even single-nucleotide mutagenesis, were also introduced with high efficiency. Further, as a proof of concept, precise mutagenesis at the nucleotide level allowed the engineering of both transcriptional and translational activities. Mutations were introduced into an archaeal promoter BRE (transcription factor B [TFB] recognition element), a terminator U-tract region, and a gene coding region. Stop codon introduction into a gene through single-nucleotide substitution shut down its expression, providing an alternative strategy for gene inactivation. In conclusion, the robust CRISPR-Cas9 genetic toolkit developed in this investigation greatly facilitates the application of M. maripaludis as a model system in the study of archaeal biology and biotechnology development, particularly CO2-based biotechnologies. IMPORTANCE Archaea are prokaryotes with intriguing biological characteristics. They possess bacterial cell structures but eukaryotic homologous information processing machinery and eukaryotic featured proteins. Archaea also display excellent adaptability to extreme environments and play pivotal roles in ecological processes, thus exhibiting valuable biotechnological potential. However, the in-depth understanding and practical application of archaea are much lagging, because only a minority of pure cultures are available, and even worse, very few can be genetically manipulated. This work developed CRISPR-Cas9-based genome editing technology in Methanococcus maripaludis, a CO2-fixing methanogenic archaeon. The CRISPR-Cas9 approach developed in this study provides an elegant and efficient genome editing toolkit that can be applied in the knockout of single or multiple genes, in situ gene tagging, multiple- or single-nucleotide mutagenesis, and inactivation of gene expression by introduction of stop codons. The successful development of the CRISPR-Cas9 toolkit will facilitate the application of M. maripaludis in archaeal biology research and biotechnology development, particularly CO2-derived biotechnologies.},
}
@article {pmid35766492,
year = {2022},
author = {Fasching, CL and Servellita, V and McKay, B and Nagesh, V and Broughton, JP and Sotomayor-Gonzalez, A and Wang, B and Brazer, N and Reyes, K and Streithorst, J and Deraney, RN and Stanfield, E and Hendriks, CG and Fung, B and Miller, S and Ching, J and Chen, JS and Chiu, CY},
title = {COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.},
journal = {Journal of clinical microbiology},
volume = {60},
number = {7},
pages = {e0026122},
pmid = {35766492},
issn = {1098-660X},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; Humans ; Mutation ; *SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.},
}
@article {pmid35766425,
year = {2022},
author = {Zheng, L and Lu, H and Zan, B and Li, S and Liu, H and Liu, Z and Huang, J and Liu, Y and Jiang, F and Liu, Q and Feng, Y and Hong, L},
title = {Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity.},
journal = {Nucleic acids research},
volume = {50},
number = {13},
pages = {7529-7544},
pmid = {35766425},
issn = {1362-4962},
mesh = {*Argonaute Proteins/metabolism ; DNA/genetics/metabolism ; *DNA Cleavage ; Humans ; Prokaryotic Cells/metabolism ; RNA/metabolism ; },
abstract = {Prokaryotic Argonaute proteins (pAgos) widely participate in hosts to defend against the invasion of nucleic acids. Compared with the CRISPR-Cas system, which requires a specific motif on the target and can only use RNA as guide, pAgos exhibit precise endonuclease activity on any arbitrary target sequence and can use both RNA and DNA as guide, thus rendering great potential for genome editing applications. Hitherto, most in-depth studies on the structure-function relationship of pAgos were conducted on thermophilic ones, functioning at ∼60 to 100°C, whose structures were, however, determined experimentally at much lower temperatures (20-33°C). It remains unclear whether these low-temperature structures can represent the true conformations of the thermophilic pAgos under their physiological conditions. The present work studied three pAgos, PfAgo, TtAgo and CbAgo, whose physiological temperatures differ significantly (95, 75 and 37°C). By conducting thorough experimental and simulation studies, we found that thermophilic pAgos (PfAgo and TtAgo) adopt a loosely-packed structure with a partially-melted surface at the physiological temperatures, largely different from the compact crystalline structures determined at moderate temperatures. In contrast, the mesophilic pAgo (CbAgo) assumes a compact crystalline structure at its optimal function temperature. Such a partially-disrupted structure endows thermophilic pAgos with great flexibility both globally and locally at the catalytic sites, which is crucial for them to achieve high DNA-cleavage activity. To further prove this, we incubated thermophilic pAgos with urea to purposely disrupt their structures, and the resulting cleavage activity was significantly enhanced below the physiological temperature, even at human body temperature. Further testing of many thermophilic Agos present in various thermophilic prokaryotes demonstrated that their structures are generally disrupted under physiological conditions. Therefore, our findings suggest that the highly dynamical structure with a partially-melted surface, distinct from the low-temperature crystalline structure, could be a general strategy assumed by thermophilic pAgos to achieve the high DNA-cleavage activity.},
}
@article {pmid35766313,
year = {2022},
author = {Hassan, MM and Yuan, G and Liu, Y and Alam, M and Eckert, CA and Tuskan, GA and Golz, JF and Yang, X},
title = {Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies.},
journal = {Biotechnology journal},
volume = {17},
number = {10},
pages = {e2100673},
doi = {10.1002/biot.202100673},
pmid = {35766313},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Gene Targeting ; Genome, Plant/genetics ; Plant Breeding/methods ; Plants/genetics ; RNA, Guide ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.},
}
@article {pmid35764748,
year = {2022},
author = {Tiwari, JK and A, J and Tuteja, N and Khurana, SMP},
title = {Genome editing (CRISPR-Cas)-mediated virus resistance in potato (Solanum tuberosum L.).},
journal = {Molecular biology reports},
volume = {49},
number = {12},
pages = {12109-12119},
pmid = {35764748},
issn = {1573-4978},
mesh = {Gene Editing ; *Solanum tuberosum/genetics ; CRISPR-Cas Systems/genetics ; Plant Breeding ; *Plant Viruses/genetics ; Genome, Plant ; },
abstract = {Plant viruses are the major pathogens that cause heavy yield loss in potato. The important viruses are potato virus X, potato virus Y and potato leaf roll virus around the world. Besides these three viruses, a novel tomato leaf curl New Delhi virus is serious in India. Conventional cum molecular breeding and transgenics approaches have been applied to develop virus resistant potato genotypes. But progress is slow in developing resistant varieties due to lack of host genes and long breeding process, and biosafety concern with transgenics. Hence, CRISPR-Cas mediated genome editing has emerged as a powerful technology to address these issues. CRISPR-Cas technology has been deployed in potato for several important traits. We highlight here CRISPR-Cas approaches of virus resistance through targeting viral genome (DNA or RNA), host factor gene and multiplexing of target genes simultaneously. Further, advancement in CRISPR-Cas research is presented in the area of DNA-free genome editing, virus-induced genome editing, and base editing. CRISPR-Cas delivery, transformation methods, and challenges in tetraploid potato and possible methods are also discussed.},
}
@article {pmid35763567,
year = {2022},
author = {Mahas, A and Marsic, T and Lopez-Portillo Masson, M and Wang, Q and Aman, R and Zheng, C and Ali, Z and Alsanea, M and Al-Qahtani, A and Ghanem, B and Alhamlan, F and Mahfouz, M},
title = {Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {28},
pages = {e2118260119},
pmid = {35763567},
issn = {1091-6490},
mesh = {*Bacterial Proteins/chemistry/classification/genetics ; Biotechnology ; *COVID-19/diagnosis ; *CRISPR-Associated Proteins/chemistry/classification/genetics ; *Clostridiales/enzymology ; *Endodeoxyribonucleases/chemistry/classification/genetics ; Enzyme Stability ; Hot Temperature ; Humans ; Phylogeny ; *Point-of-Care Testing ; *SARS-CoV-2/isolation &amp; purification ; },
abstract = {Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.},
}
@article {pmid35763517,
year = {2022},
author = {Omachi, K and Miner, JH},
title = {Comparative analysis of dCas9-VP64 variants and multiplexed guide RNAs mediating CRISPR activation.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0270008},
pmid = {35763517},
issn = {1932-6203},
support = {R01 DK128660/DK/NIDDK NIH HHS/United States ; R01 DK058366/DK/NIDDK NIH HHS/United States ; R01 DK078314/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; *RNA, Guide/genetics ; Transcription Factors/metabolism ; Transcriptional Activation ; },
abstract = {CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on the VP64 transcriptional activator have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced basal dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.},
}
@article {pmid35763226,
year = {2022},
author = {Kahraman-Ilıkkan, &#xd6;},
title = {Comparison of Propionibacterium genomes: CRISPR-Cas systems, phage/plasmid diversity, and insertion sequences.},
journal = {Archives of microbiology},
volume = {204},
number = {7},
pages = {434},
pmid = {35763226},
issn = {1432-072X},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; *DNA Transposable Elements ; Plasmids/genetics ; Propionibacterium/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems constitute the adaptive immune system in prokaryotes that provide resistance against invasive genetic elements. The genus Propionibacterium comprises gram-positive, facultative anaerobe, non-spore-forming bacteria, and is the source of some B group vitamins such as B12 as well as bacteriocins. Some of the selected species of the genus Propionibacterium spp. were reclassified into the three genera in 2016 (Acidipropionibacterium spp., Pseudopropionibacterium spp., Cutibacterium spp.). Therefore, this study compared CRISPR/Cas systems, Cas 1 and repeat sequences phylogeny, phage/plasmid surveys as well as insertion sequences of new genera members. In this study, a total of 34 genomes of 13 species were observed with a bioinformatic approach. CRISPR-Cas + + and CRISPRDetect were used to detect CRISPR/Cas systems, direct repeats, and spacers. 39 CRISPR-Cas systems were detected. Type I-E, Type I-U, and one incomplete III-B CRISPR-Cas subtypes were identified. Most of the strains had Cas1/Cas4 fusion proteins. Pseudopropionibacterium propionicum strains had two types I-U and one of the CRISPR loci had csx17 cas genes. Common phage invaders were Propionibacterium phage E6, G4, E1, Anatole, and Doucette. The BLSM62 similarity score of all Cas1 sequences was 48.4% while the pairwise identity of repeat sequences was 48.7%. Common insertion sequences were ISL3, IS3, IS30. The diversity analysis of the CRISPR/Cas system in the genus Propionibacterium provided a new perspective for determining the role of the CRISPR-Cas system in the evolution of new genera.},
}
@article {pmid35762828,
year = {2022},
author = {Chen, Y and Xu, X and Wang, J and Zhang, Y and Zeng, W and Liu, Y and Zhang, X},
title = {Photoactivatable CRISPR/Cas12a Strategy for One-Pot DETECTR Molecular Diagnosis.},
journal = {Analytical chemistry},
volume = {94},
number = {27},
pages = {9724-9731},
doi = {10.1021/acs.analchem.2c01193},
pmid = {35762828},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases ; Ultraviolet Rays ; },
abstract = {As a golden partner of recombinase polymerase amplification (RPA), CRISPR/Cas12a has been proven to solve the false-positive problem caused by nonspecific amplification perfectly; meanwhile, its trans-cleave activity has further enhanced the sensitivity. However, the solution transfer operation after tube cap opening greatly increases the risk of aerosol contamination of amplicon, which is inconsistent with point-of-care (POC) diagnostics requirements. This study proposes a photoactivated CRISPR/Cas12a strategy to achieve one-pot high-sensitivity nucleic acid detection. Using photocleavable complementary ssDNA to block crRNA, RPA amplification can smoothly pass through the exponential interval without being affected by activated Cas12a in the critical early stage. After enough amplicons were produced, the Cas12a test was activated by short bursts of ultraviolet radiation at 365 nm. This one-pot method achieved a sensitivity of 2.5 copies within 40 min. This simple and sensitive one-pot method can effectively avoid amplicon contamination and lower the threshold for molecular diagnostics in POC.},
}
@article {pmid35762550,
year = {2022},
author = {Khan, IS and Faiyaz, Z and Khan, AU},
title = {Use of CRISPR in Infection Control.},
journal = {Current protein &amp; peptide science},
volume = {23},
number = {5},
pages = {299-309},
doi = {10.2174/1389203723666220627152112},
pmid = {35762550},
issn = {1875-5550},
mesh = {Bacteria/genetics ; *COVID-19/diagnosis/genetics ; CRISPR-Cas Systems ; *Communicable Diseases/genetics ; Humans ; Infection Control ; Pandemics ; },
abstract = {One of the greatest threats to the global world is infectious diseases. The morbidity and fatality of infectious diseases cause 17 million deaths annually. The recent COVID-19 pandemic describes the uncertain potential of these diseases. Understanding the pathogenesis of infectious agents, including bacteria, viruses, fungi, etc. and the evolution of rapid diagnostic techniques and treatments has become a pressing priority to improve infectious disease outcomes worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) constitute the adaptive immune system of archaea and bacteria along with CRISPR-associated (Cas) proteins that recognize and destroy foreign DNA acting as molecular scissors. Since their discovery, CRISPR systems are classified into 6 types and 22 subtypes. Type II, V, and VI are used for diagnostic purposes. Utilizing the CRISPR-Cas system's capabilities will aid promote the development of novel and improved diagnostics as well as innovative delivery systems and the prevention and treatment of infectious diseases.},
}
@article {pmid35761332,
year = {2022},
author = {Chen, R and Yuan, W and Zheng, Y and Zhu, X and Jin, B and Yang, T and Yan, Y and Xu, W and Chen, H and Gao, J and Li, G and Gokulnath, P and Vulugundam, G and Li, J and Xiao, J},
title = {Delivery of engineered extracellular vesicles with miR-29b editing system for muscle atrophy therapy.},
journal = {Journal of nanobiotechnology},
volume = {20},
number = {1},
pages = {304},
pmid = {35761332},
issn = {1477-3155},
mesh = {Animals ; CRISPR-Cas Systems ; *Extracellular Vesicles ; Mice ; *MicroRNAs/genetics ; *Muscular Atrophy/genetics/therapy ; Tumor Necrosis Factor-alpha ; },
abstract = {Muscle atrophy is a frequently observed complication, characterized by the loss of muscle mass and strength, which diminishes the quality of life and survival. No effective therapy except exercise is currently available. In our previous study, repressing miR-29b has been shown to reduce muscle atrophy. In our current study, we have constructed artificially engineered extracellular vesicles for the delivery of CRISPR/Cas9 to target miR-29b (EVs-Cas9-29b). EVs-Cas9-29b has shown a favorable functional effect with respect to miR-29b repression in a specific and rapid manner by gene editing. In in vitro conditions, EVs-Cas9-29b could protect against muscle atrophy induced by dexamethasone (Dex), angiotensin II (AngII), and tumor necrosis factor-alpha (TNF-α). And EVs-Cas9-29b introduced in vivo preserved muscle function in the well-established immobilization and denervation-induced muscle atrophy mice model. Our work demonstrates an engineered extracellular vesicles delivery of the miR-29b editing system, which could be potentially used for muscle atrophy therapy.},
}
@article {pmid35760907,
year = {2022},
author = {Brunello, L},
title = {Genome-scale single-cell CRISPR screens.},
journal = {Nature reviews. Genetics},
volume = {23},
number = {8},
pages = {459},
pmid = {35760907},
issn = {1471-0064},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome ; RNA, Guide ; },
}
@article {pmid35760782,
year = {2022},
author = {Tao, J and Wang, Q and Mendez-Dorantes, C and Burns, KH and Chiarle, R},
title = {Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3685},
pmid = {35760782},
issn = {2041-1723},
support = {R01 CA222598/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; HEK293 Cells ; Humans ; *Retroelements/genetics ; },
abstract = {CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.},
}
@article {pmid35759403,
year = {2022},
author = {Huyke, DA and Ramachandran, A and Bashkirov, VI and Kotseroglou, EK and Kotseroglou, T and Santiago, JG},
title = {Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics.},
journal = {Analytical chemistry},
volume = {94},
number = {27},
pages = {9826-9834},
doi = {10.1021/acs.analchem.2c01670},
pmid = {35759403},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA, Single-Stranded ; Limit of Detection ; *Nucleic Acids ; RNA, Guide/genetics ; },
abstract = {Interest in CRISPR-Cas12 and CRISPR-Cas13 detection continues to increase as these detection schemes enable the specific recognition of nucleic acids. The fundamental sensitivity limits of these schemes (and their applicability in amplification-free assays) are governed by kinetic rates. However, these kinetic rates remain poorly understood, and their reporting has been inconsistent. We quantify kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a, and LbuCas13a) and their corresponding limits of detection (LoD). Collectively, we present quantification of enzyme kinetics for 14 guide RNAs (gRNAs) and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. We validate the self-consistency of our measurements by comparing trends and limiting behaviors with a Michaelis-Menten trans-cleavage reaction kinetics model. For our assay conditions, activated Cas12 and Cas13 enzymes exhibit trans-cleavage catalytic efficiencies between order 10[5] and 10[6] M[-1] s[-1]. For assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, the kinetic rates at the current assay conditions result in an amplification-free LoD in the picomolar range. The results suggest that successful detection of target requires cleavage (by an activated CRISPR enzyme) of the order of at least 0.1% of the fluorescent reporter molecules. This fraction of reporters cleaved is required to differentiate the signal from the background, and we hypothesize that this required fraction is largely independent of the detection method (e.g., endpoint vs reaction velocity) and detector sensitivity. Our results demonstrate the fundamental nature by which kinetic rates and background signal limit LoDs and thus highlight areas of improvement for the emerging field of CRISPR diagnostics.},
}
@article {pmid35758824,
year = {2022},
author = {Chen, Y and Banie, L and Breyer, BN and Tan, Y and Wang, Z and Zhou, F and Wang, G and Lin, G and Liu, J and Qi, LS and Lue, TF},
title = {Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of a dCas9 Ribonucleoprotein Complex.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {598-608},
pmid = {35758824},
issn = {2573-1602},
support = {R01 DK124609/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Gene Editing ; Glycogen Synthase Kinase 3 beta/genetics ; Humans ; *Muscle Development/genetics/physiology ; *Myostatin/genetics/metabolism ; Rats ; Ribonucleoproteins/genetics ; *Urinary Incontinence, Stress/genetics/metabolism ; },
abstract = {Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.},
}
@article {pmid35758815,
year = {2022},
author = {Elkayam, S and Orenstein, Y},
title = {DeepCRISTL: deep transfer learning to predict CRISPR/Cas9 functional and endogenous on-target editing efficiency.},
journal = {Bioinformatics (Oxford, England)},
volume = {38},
number = {Suppl 1},
pages = {i161-i168},
pmid = {35758815},
issn = {1367-4811},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; Genome ; Machine Learning ; *RNA, Guide/genetics ; },
abstract = {MOTIVATION: CRISPR/Cas9 technology has been revolutionizing the field of gene editing in recent years. Guide RNAs (gRNAs) enable Cas9 proteins to target specific genomic loci for editing. However, editing efficiency varies between gRNAs. Thus, computational methods were developed to predict editing efficiency for any gRNA of interest. High-throughput datasets of Cas9 editing efficiencies were produced to train machine-learning models to predict editing efficiency. However, these high-throughput datasets have low correlation with functional and endogenous editing. Another difficulty arises from the fact that functional and endogenous editing efficiency is more difficult to measure, and as a result, functional and endogenous datasets are too small to train accurate machine-learning models on.<br><br>RESULTS: We developed DeepCRISTL, a deep-learning model to predict the on-target efficiency given a gRNA sequence. DeepCRISTL takes advantage of high-throughput datasets to learn general patterns of gRNA on-target editing efficiency, and then uses transfer learning (TL) to fine-tune the model and fit it to the functional and endogenous prediction task. We pre-trained the DeepCRISTL model on more than 150&#xa0;000 gRNAs, produced through the DeepHF study as a high-throughput dataset of three Cas9 enzymes. We improved the DeepHF model by multi-task and ensemble techniques and achieved state-of-the-art results over each of the three enzymes: up to 0.89 in Spearman correlation between predicted and measured on-target efficiencies. To fine-tune model weights to predict on-target efficiency of functional or endogenous datasets, we tested several TL approaches, with gradual learning being the overall best performer, both when pre-trained on DeepHF and when pre-trained on CRISPROn, another high-throughput dataset. DeepCRISTL outperformed state-of-the-art methods on all functional and endogenous datasets. Using saliency maps, we identified and compared the important features learned by the model in each dataset. We believe DeepCRISTL will improve prediction performance in many other CRISPR/Cas9 editing contexts by leveraging TL to utilize both high-throughput datasets, and smaller and more biologically relevant datasets, such as functional and endogenous datasets.<br><br>DeepCRISTL is available via github.com/OrensteinLab/DeepCRISTL.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid35757746,
year = {2022},
author = {Moretti, A and Ponzo, M and Nicolette, CA and Tcherepanova, IY and Biondi, A and Magnani, CF},
title = {The Past, Present, and Future of Non-Viral CAR T Cells.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {867013},
pmid = {35757746},
issn = {1664-3224},
support = {/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; RNA, Messenger ; *Receptors, Antigen, T-Cell/genetics ; T-Lymphocytes ; },
abstract = {Adoptive transfer of chimeric antigen receptor (CAR) T lymphocytes is a powerful technology that has revolutionized the way we conceive immunotherapy. The impressive clinical results of complete and prolonged response in refractory and relapsed diseases have shifted the landscape of treatment for hematological malignancies, particularly those of lymphoid origin, and opens up new possibilities for the treatment of solid neoplasms. However, the widening use of cell therapy is hampered by the accessibility to viral vectors that are commonly used for T cell transfection. In the era of messenger RNA (mRNA) vaccines and CRISPR/Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) precise genome editing, novel and virus-free methods for T cell engineering are emerging as a more versatile, flexible, and sustainable alternative for next-generation CAR T cell manufacturing. Here, we discuss how the use of non-viral vectors can address some of the limitations of the viral methods of gene transfer and allow us to deliver genetic information in a stable, effective and straightforward manner. In particular, we address the main transposon systems such as Sleeping Beauty (SB) and piggyBac (PB), the utilization of mRNA, and innovative approaches of nanotechnology like Lipid-based and Polymer-based DNA nanocarriers and nanovectors. We also describe the most relevant preclinical data that have recently led to the use of non-viral gene therapy in emerging clinical trials, and the related safety and efficacy aspects. We will also provide practical considerations for future trials to enable successful and safe cell therapy with non-viral methods for CAR T cell generation.},
}
@article {pmid35755158,
year = {2022},
author = {Whitworth, KM and Green, JA and Redel, BK and Geisert, RD and Lee, K and Telugu, BP and Wells, KD and Prather, RS},
title = {Improvements in pig agriculture through gene editing.},
journal = {CABI agriculture and bioscience},
volume = {3},
number = {1},
pages = {41},
pmid = {35755158},
issn = {2662-4044},
abstract = {Genetic modification of animals via selective breeding is the basis for modern agriculture. The current breeding paradigm however has limitations, chief among them is the requirement for the beneficial trait to exist within the population. Desirable alleles in geographically isolated breeds, or breeds selected for a different conformation and commercial application, and more importantly animals from different genera or species cannot be introgressed into the population via selective breeding. Additionally, linkage disequilibrium results in low heritability and necessitates breeding over successive generations to fix a beneficial trait within a population. Given the need to sustainably improve animal production to feed an anticipated 9 billion global population by 2030 against a backdrop of infectious diseases and a looming threat from climate change, there is a pressing need for responsive, precise, and agile breeding strategies. The availability of genome editing tools that allow for the introduction of precise genetic modification at a single nucleotide resolution, while also facilitating large transgene integration in the target population, offers a solution. Concordant with the developments in genomic sequencing approaches, progress among germline editing efforts is expected to reach feverish pace. The current manuscript reviews past and current developments in germline engineering in pigs, and the many advantages they confer for advancing animal agriculture.},
}
@article {pmid35753889,
year = {2022},
author = {Zhang, Y and Fernie, AR},
title = {Dynamically regulating metabolic fluxes with synthetic metabolons.},
journal = {Trends in biotechnology},
volume = {40},
number = {9},
pages = {1019-1020},
doi = {10.1016/j.tibtech.2022.06.005},
pmid = {35753889},
issn = {1879-3096},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Metabolic Engineering ; *RNA/metabolism ; },
abstract = {Enzyme-enzyme assemblies commonly occur naturally, yet the factors that lead to their transient nature are not fully understood. Mitkas et al. have shown how clustered regularly interspaced short palindromic repeats (CRISPR) enzymes and RNA scaffolds allow synthetic enzyme complexes to be formed and disassembled as needed, providing powerful new tools for metabolic engineering.},
}
@article {pmid35752768,
year = {2022},
author = {Guo, G and Wang, Z and Li, Q and Yu, Y and Li, Y and Tan, Z and Zhang, W},
title = {Genomic characterization of Streptococcus parasuis, a close relative of Streptococcus suis and also a potential opportunistic zoonotic pathogen.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {469},
pmid = {35752768},
issn = {1471-2164},
mesh = {Animals ; Cattle ; Genomics ; *Streptococcal Infections/veterinary ; Streptococcus ; *Streptococcus suis/genetics ; Swine ; *Swine Diseases ; Virulence/genetics ; },
abstract = {Streptococcus parasuis (S. parasuis) is a close relative of Streptococcus suis (S. suis), composed of former members of S. suis serotypes 20, 22 and 26. S. parasuis could infect pigs and cows, and recently, human infection cases have been reported, making S. parasuis a potential opportunistic zoonotic pathogen. In this study, we analysed the genomic characteristics of S. parasuis, using pan-genome analysis, and compare some phenotypic determinants such as capsular polysaccharide, integrative conjugative elements, CRISPR-Cas system and pili, and predicted the potential virulence genes by associated analysis of the clinical condition of isolated source animals and genotypes. Furthermore, to discuss the relationship with S. suis, we compared these characteristics of S. parasuis with those of S. suis. We found that the characteristics of S. parasuis are similar to those of S. suis, both of them have "open" pan-genome, their antimicrobial resistance gene profiles are similar and a srtF pilus cluster of S. suis was identified in S. parasuis genome. But S. parasuis still have its unique characteristics, two novel pilus clusters are and three different type CRISPR-Cas system were found. Therefore, this study provides novel insights into the interspecific and intraspecific genetic characteristics of S. parasuis, which can be useful for further study of this opportunistic pathogen, such as serotyping, diagnostics, vaccine development, and study of the pathogenesis mechanism.},
}
@article {pmid35752617,
year = {2022},
author = {de Freitas Almeida, GM and Hoikkala, V and Ravantti, J and Rantanen, N and Sundberg, LR},
title = {Mucin induces CRISPR-Cas defense in an opportunistic pathogen.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3653},
pmid = {35752617},
issn = {2041-1723},
mesh = {Animals ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; Fishes ; Mucins ; },
abstract = {Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies obtain at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the phage-bacterium interactions on mucosal surfaces may select for the diversification of bacterial immune systems.},
}
@article {pmid35752612,
year = {2022},
author = {Qin, S and Xiao, W and Zhou, C and Pu, Q and Deng, X and Lan, L and Liang, H and Song, X and Wu, M},
title = {Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {199},
pmid = {35752612},
issn = {2059-3635},
support = {R01 AI138203-3/NH/NIH HHS/United States ; R01 AI109317/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *COVID-19 ; Drug Resistance, Microbial ; Humans ; Mammals/metabolism ; *Pseudomonas Infections/drug therapy/genetics ; Pseudomonas aeruginosa/genetics ; Technology ; Virulence Factors/genetics/metabolism/pharmacology ; },
abstract = {Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen that infects patients with cystic fibrosis, burn wounds, immunodeficiency, chronic obstructive pulmonary disorder (COPD), cancer, and severe infection requiring ventilation, such as COVID-19. P. aeruginosa is also a widely-used model bacterium for all biological areas. In addition to continued, intense efforts in understanding bacterial pathogenesis of P. aeruginosa including virulence factors (LPS, quorum sensing, two-component systems, 6 type secretion systems, outer membrane vesicles (OMVs), CRISPR-Cas and their regulation), rapid progress has been made in further studying host-pathogen interaction, particularly host immune networks involving autophagy, inflammasome, non-coding RNAs, cGAS, etc. Furthermore, numerous technologic advances, such as bioinformatics, metabolomics, scRNA-seq, nanoparticles, drug screening, and phage therapy, have been used to improve our understanding of P. aeruginosa pathogenesis and host defense. Nevertheless, much remains to be uncovered about interactions between P. aeruginosa and host immune responses, including mechanisms of drug resistance by known or unannotated bacterial virulence factors as well as mammalian cell signaling pathways. The widespread use of antibiotics and the slow development of effective antimicrobials present daunting challenges and necessitate new theoretical and practical platforms to screen and develop mechanism-tested novel drugs to treat intractable infections, especially those caused by multi-drug resistance strains. Benefited from has advancing in research tools and technology, dissecting this pathogen's feature has entered into molecular and mechanistic details as well as dynamic and holistic views. Herein, we comprehensively review the progress and discuss the current status of P. aeruginosa biophysical traits, behaviors, virulence factors, invasive regulators, and host defense patterns against its infection, which point out new directions for future investigation and add to the design of novel and/or alternative therapeutics to combat this clinically significant pathogen.},
}
@article {pmid35752172,
year = {2022},
author = {Hughes, NW and Qu, Y and Zhang, J and Tang, W and Pierce, J and Wang, C and Agrawal, A and Morri, M and Neff, N and Winslow, MM and Wang, M and Cong, L},
title = {Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles.},
journal = {Molecular cell},
volume = {82},
number = {16},
pages = {3103-3118.e8},
doi = {10.1016/j.molcel.2022.06.001},
pmid = {35752172},
issn = {1097-4164},
support = {R35 HG011316/HG/NHGRI NIH HHS/United States ; R01 CA231253/CA/NCI NIH HHS/United States ; S10 OD023452/OD/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Lineage/genetics ; *DNA Barcoding, Taxonomic/methods ; Humans ; Machine Learning ; Phylogeny ; },
abstract = {The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes-at a fraction of the size of Cas9 barcodes-achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.},
}
@article {pmid35751797,
year = {2022},
author = {Sirohi, U and Kumar, M and Sharma, VR and Teotia, S and Singh, D and Chaudhary, V and Priya, and Yadav, MK},
title = {CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops.},
journal = {Molecular biotechnology},
volume = {64},
number = {12},
pages = {1303-1318},
pmid = {35751797},
issn = {1559-0305},
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; Horticulture ; *Oils, Volatile ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {Demand of flowers is increasing with time worldwide. Floriculture has become one of the most important commercial trades in agriculture. Although traditional breeding methods like hybridization and mutation breeding have contributed significantly to the development of important flower varieties, flower production and quality of flowers can be significantly improved by employing modern breeding approaches. Novel traits of significance have interest to consumers and producers, such as fragrance, new floral color, change in floral architecture and morphology, vase life, aroma, and resistance to biotic and abiotic stresses, have been introduced by genetic manipulation. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has recently emerged as a powerful genome-editing tool for accurately changing DNA sequences at specific locations. It provides excellent means of genetically improving floricultural crops. CRISPR/Cas system has been utilized in gene editing in horticultural cops. There are few reports on the utilization of the CRISPR/Cas9 system in flowers. The current review summarizes the research work done by employing the CRISPR/Cas9 system in floricultural crops including improvement in flowering traits such as color modification, prolonging the shelf life of flowers, flower initiation, and development, changes in color of ornamental foliage by genome editing. CRISPR/Cas9 gene editing could be useful in developing novel cultivars with higher fragrance and enhanced essential oil and many other useful traits. The present review also highlights the basic mechanism and key components involved in the CRISPR/Cas9 system.},
}
@article {pmid35751791,
year = {2022},
author = {Datta, A and Sarmah, D and Kaur, H and Chaudhary, A and Vadak, N and Borah, A and Shah, S and Wang, X and Bhattacharya, P},
title = {Advancement in CRISPR/Cas9 Technology to Better Understand and Treat Neurological Disorders.},
journal = {Cellular and molecular neurobiology},
volume = {},
number = {},
pages = {},
pmid = {35751791},
issn = {1573-6830},
abstract = {Neurological disorders have complicated pathophysiology that may involve several genetic mutations. Conventional treatment has limitations as they only treat apparent symptoms. Although, personalized medicine is emerging as a promising neuro-intervention, lack of precision is the major pitfall. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is evolving as a technological platform that may overcome the therapeutic limitations towards precision medicine. In the future, targeting genes in neurological disorders may be the mainstay of modern therapy. The present review on CRISPR/Cas9 and its application in various neurological disorders may provide a platform for its future clinical relevance towards developing precise and personalized medicine.},
}
@article {pmid35751329,
year = {2022},
author = {Khan, SH and Zaidi, SK and Gilani, M},
title = {PCR to CRISPR: Role of Nucleic Acid Tests (NAT) in detection of COVID-19.},
journal = {JPMA. The Journal of the Pakistan Medical Association},
volume = {72},
number = {6},
pages = {1166-1174},
doi = {10.47391/JPMA.2324},
pmid = {35751329},
issn = {0030-9982},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; Clinical Laboratory Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Nucleic Acids ; Polymerase Chain Reaction ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; Uronic Acids ; },
abstract = {COVID-19 infection has emerged as an unparalleled pandemic with morbidity and mortality tolls challenging diagnostic approaches and therapeutic interventions, and raising serious questions for healthcare policy-makers. From the diagnostic perspective, Reverse transcriptase polymerase chain reaction remains the gold standard. However, issues associated with gene primer variation in different countries, low analytical sensitivity, cross-reactivity with certain human coronaviruses have raised serious concerns within the scientific community. Alongside longer turnaround times, requirements of sophisticated equipment and trained technicians are the other challenges for conventional reverse transcriptase polymerase chain reaction testing. The recent biotechnological boom has now allowed newer nucleic acid testing options for diagnosing severe acute respiratory syndrome Coronovairus 2 (SARS-CoV2) with much better diagnostic efficiency, reduced turnaround times and possible benefit for use as a point-of-care test. Isothermal techniques with simple equipment requirements along with uniform temperature for analysis have emerged to be more sensitive and specific with turnaround times as low as 10-15 minutes. Similarly, Cluster Regularly Interspaced Short Palindromic Repeats have also been seen to play a very decisive role in COVID-19 diagnostics with much superior diagnostic efficiency and feasibility as a point-of-care test and its possible use for sequencing. The current narrative review was planned to consolidate data for all possible nucleic acid testing options under research/clinical use, and to provide a comparative assessment from the perspective of both the clinician and the laboratory.},
}
@article {pmid35751058,
year = {2022},
author = {Jiang, J and Zeng, T and Zhang, L and Fan, X and Jin, Q and Ni, H and Ye, Y and Cheng, L and Li, L and Wang, L and Xu, S and Yang, Y and Gu, J and Guo, B and Wang, L and Li, X and Qin, Y and Li, J and Wang, J and Chen, X and Wu, M and Ying, QL and Qin, X and Wang, Y and Wang, Y},
title = {Optimization of Cas9 RNA sequence to reduce its unexpected effects as a microRNA sponge.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {136},
pmid = {35751058},
issn = {1476-4598},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Gene Editing ; HeLa Cells ; Humans ; *MicroRNAs/genetics ; },
abstract = {UNLABELLED: Cas9 RNA functions as a miRNA sponge. Let-7 is the dominant regulated miRNA by Cas9 RNA. RNA sequence optimization of Cas9 by synonymous mutation improves its safety.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-022-01604-x.},
}
@article {pmid35750764,
year = {2022},
author = {Miskel, D and Poirier, M and Beunink, L and Rings, F and Held, E and Tholen, E and Tesfaye, D and Schellander, K and Salilew-Wondim, D and Blaschka, C and Große-Brinkhaus, C and Bertram, B and Hoelker, M},
title = {The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {10793},
pmid = {35750764},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Cattle ; Cell Division ; Electroporation/methods ; Gene Editing/methods ; Mammals/metabolism ; Ribonucleoproteins/metabolism ; *Zygote/metabolism ; },
abstract = {At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote's cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.},
}
@article {pmid35750651,
year = {2022},
author = {Hillary, VE and Ceasar, SA},
title = {Prime editing in plants and mammalian cells: Mechanism, achievements, limitations, and future prospects.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {44},
number = {9},
pages = {e2200032},
doi = {10.1002/bies.202200032},
pmid = {35750651},
issn = {1521-1878},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Endonucleases ; *Gene Editing ; Genome ; Mammals/genetics ; Plants/genetics ; },
abstract = {Clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genetic research in the life sciences. Four classes of CRISPR/Cas-derived genome editing agents, such as nuclease, base editor, recombinase, and prime editor have been introduced for engineering the genomes of diverse organisms. The recently introduced prime editing system offers precise editing without many off-target effects than traditional CRISPR-based systems. Many researchers have successfully applied this gene-editing toolbox in diverse systems for various genome-editing applications. This review presents the mechanism of prime editing and summarizes the details of the prime editing system applied in plants and mammalian cells for precise genome editing. We also discuss the advantages, limitations, and potential future applications of prime editing in these systems. This review enables the researcher to gain knowledge on prime editing tools and their potential applications in plants and mammalian cells.},
}
@article {pmid35749339,
year = {2022},
author = {Lu, ZH and Li, J and Dmitriev, IP and Kashentseva, EA and Curiel, DT},
title = {Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface.},
journal = {ACS nano},
volume = {16},
number = {7},
pages = {10443-10455},
pmid = {35749339},
issn = {1936-086X},
support = {R01 CA211096/CA/NCI NIH HHS/United States ; UG3 TR002851/TR/NCATS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *RNA, Guide/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Adenoviridae/genetics/metabolism ; Capsid/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral "piggyback" approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.},
}
@article {pmid35748558,
year = {2022},
author = {Mohammad-Rafiei, F and Safdarian, E and Adel, B and Vandchali, NR and Navashenaq, JG and Gheibihayat, SM},
title = {CRISPR: A Promising Tool for Cancer Therapy.},
journal = {Current molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.2174/1566524022666220624111311},
pmid = {35748558},
issn = {1875-5666},
abstract = {The clustered regularly interspaced short palindromic repeats system, called CRISPR, as one of the major technological advances, allows geneticists and researchers to perform genome editing. This remarkable technology is quickly eclipsing zinc-finger nucleases (ZFNs) and other editing tools, and its ease of use and accuracy have thus far revolutionized genome editing, from fundamental science projects to medical research and treatment options. This system consists of two key components: a CRISPR-associated (Cas) nuclease, which binds and cuts deoxyribonucleic acid (DNA) and a guide ribonucleic acid (gRNA) sequence, directing the Cas nuclease to its target site. In the research arena, CRISPR has been up to now exploited in various ways alongside gene editing, such as epigenome modifications, genome-wide screening, targeted cancer therapies, and so on. This article reviews the current perceptions of the CRISPR/Cas systems with special attention to studies reflecting on the relationship between the CRISPR/Cas systems and their role in cancer therapy.},
}
@article {pmid35746591,
year = {2022},
author = {Liu, Y and Zhang, X and Chen, D and Yang, D and Zhu, C and Tang, L and Yang, X and Wang, Y and Luo, X and Wang, M and Huang, Y and Hu, Z and Liu, Z},
title = {CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms.},
journal = {Viruses},
volume = {14},
number = {6},
pages = {},
pmid = {35746591},
issn = {1999-4915},
mesh = {Animals ; Animals, Genetically Modified ; *Bombyx ; CRISPR-Cas Systems ; *Nucleopolyhedroviruses/genetics ; },
abstract = {Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.},
}
@article {pmid35744944,
year = {2022},
author = {Zhang, J and Zhou, Q and Zhang, D and Yang, G and Zhang, C and Wu, Y and Xu, Y and Chen, J and Kong, W and Kong, G and Wang, J},
title = {The Agronomic Traits, Alkaloids Analysis, FT-IR and 2DCOS-IR Spectroscopy Identification of the Low-Nicotine-Content Nontransgenic Tobacco Edited by CRISPR-Cas9.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {12},
pages = {},
pmid = {35744944},
issn = {1420-3049},
mesh = {CRISPR-Cas Systems/genetics ; *Nicotine ; Spectrophotometry, Infrared ; Spectroscopy, Fourier Transform Infrared/methods ; *Tobacco/genetics ; },
abstract = {In this study, the agricultural traits, alkaloids content and Fourier transform infrared spectroscopy (FT-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR) analysis of the tobacco after Berberine Bridge Enzyme-Like Proteins (BBLs) knockout were investigated. The knockout of BBLs has limited effect on tobacco agricultural traits. After the BBLs knockout, nicotine and most alkaloids are significantly reduced, but the content of myosmine and its derivatives increases dramatically. In order to identify the gene editing of tobacco, principal component analysis (PCA) was performed on the FT-IR and 2DCOS-IR spectroscopy data. The results showed that FT-IR can distinguish between tobacco roots and leaves but cannot classify the gene mutation tobacco from the wild one. 2DCOS-IR can enhance the characteristics of the samples due to the increased apparent resolution of the spectra. Using the autopeaks in the synchronous map for PCA analysis, we successfully identified the mutants with an accuracy of over 90%.},
}
@article {pmid35743282,
year = {2022},
author = {Chang, YJ and Kang, Z and Bei, J and Chou, SJ and Lu, MJ and Su, YL and Lin, SW and Wang, HH and Lin, S and Chang, CJ},
title = {Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
pmid = {35743282},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Cell Proliferation/genetics ; *Gene Editing ; Gene Expression ; Hemoglobin Subunits/genetics/metabolism ; Humans ; K562 Cells ; *MicroRNAs ; Transcription Factors/metabolism ; Tripartite Motif-Containing Protein 28/metabolism ; },
abstract = {TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.},
}
@article {pmid35743185,
year = {2022},
author = {Voisard, P and Diofano, F and Glazier, AA and Rottbauer, W and Just, S},
title = {CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
pmid = {35743185},
issn = {1422-0067},
mesh = {Adenosine Triphosphatases/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; *Frontotemporal Dementia/genetics/metabolism ; Muscle, Skeletal/metabolism ; *Muscle, Striated/metabolism ; Mutation ; *Myositis, Inclusion Body/genetics/metabolism ; Proteostasis/genetics ; Valosin Containing Protein/genetics/metabolism ; Zebrafish/genetics/metabolism ; },
abstract = {Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo.},
}
@article {pmid35743007,
year = {2022},
author = {Thomson, MJ and Biswas, S and Tsakirpaloglou, N and Septiningsih, EM},
title = {Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
pmid = {35743007},
issn = {1422-0067},
mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant ; Plant Breeding/methods ; },
abstract = {Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.},
}
@article {pmid35742919,
year = {2022},
author = {Merckx, NLL and Van Esch, H},
title = {Human Brain Models of Intellectual Disability: Experimental Advances and Novelties.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
pmid = {35742919},
issn = {1422-0067},
mesh = {Brain ; CRISPR-Cas Systems ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Intellectual Disability/genetics/metabolism/therapy ; },
abstract = {Intellectual disability (ID) is characterized by deficits in conceptual, social and practical domains. ID can be caused by both genetic defects and environmental factors and is extremely heterogeneous, which complicates the diagnosis as well as the deciphering of the underlying pathways. Multiple scientific breakthroughs during the past decades have enabled the development of novel ID models. The advent of induced pluripotent stem cells (iPSCs) enables the study of patient-derived human neurons in 2D or in 3D organoids during development. Gene-editing tools, such as CRISPR/Cas9, provide isogenic controls and opportunities to design personalized gene therapies. In practice this has contributed significantly to the understanding of ID and opened doors to identify novel therapeutic targets. Despite these advances, a number of areas of improvement remain for which novel technologies might entail a solution in the near future. The purpose of this review is to provide an overview of the existing literature on scientific breakthroughs that have been advancing the way ID can be studied in the human brain. The here described human brain models for ID have the potential to accelerate the identification of underlying pathophysiological mechanisms and the development of therapies.},
}
@article {pmid35742831,
year = {2022},
author = {Vuelta, E and Ordoñez, JL and Sanz, DJ and Ballesteros, S and Hernández-Rivas, JM and Méndez-Sánchez, L and Sánchez-Martín, M and García-Tuñón, I},
title = {CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
pmid = {35742831},
issn = {1422-0067},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Chronic Disease ; *Fusion Proteins, bcr-abl/genetics/metabolism ; Humans ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy/therapy ; },
abstract = {Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.},
}
@article {pmid35742811,
year = {2022},
author = {Irshad, F and Li, C and Wu, HY and Yan, Y and Xu, JH},
title = {The Function of DNA Demethylase Gene ROS1a Null Mutant on Seed Development in Rice (Oryza Sativa) Using the CRISPR/CAS9 System.},
journal = {International journal of molecular sciences},
volume = {23},
number = {12},
pages = {},
pmid = {35742811},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; DNA/metabolism ; Endosperm/metabolism ; Gene Expression Regulation, Plant ; Glutens/metabolism ; *Oryza/metabolism ; Plant Proteins/genetics/metabolism ; Seeds/metabolism ; Starch/metabolism ; },
abstract = {The endosperm is the main nutrient source in cereals for humans, as it is a highly specialized storage organ for starch, lipids, and proteins, and plays an essential role in seed growth and development. Active DNA demethylation regulates plant developmental processes and is ensured by cytosine methylation (5-meC) DNA glycosylase enzymes. To find out the role of OsROS1a in seed development, the null mutant of OsROS1a was generated using the CRISPR/Cas9 system. The null mutant of OsROS1a was stable and heritable, which affects the major agronomic traits, particularly in rice seeds. The null mutant of OsROS1a showed longer and narrower grains, and seeds were deformed containing an underdeveloped and less-starch-producing endosperm with slightly irregularly shaped embryos. In contrast to the transparent grains of the wild type, the grains of the null mutant of OsROS1a were slightly opaque and rounded starch granules, with uneven shapes, sizes, and surfaces. A total of 723 differential expression genes (DEGs) were detected in the null mutant of OsROS1a by RNA-Seq, of which 290 were downregulated and 433 were upregulated. The gene ontology (GO) terms with the top 20 enrichment factors were visualized for cellular components, biological processes, and molecular functions. The key genes that are enriched for these GO terms include starch synthesis genes (OsSSIIa and OsSSIIIa) and cellulose synthesis genes (CESA2, CESA3, CESA6, and CESA8). Genes encoding polysaccharides and glutelin were found to be downregulated in the mutant endosperm. The glutelins were further verified by SDS-PAGE, suggesting that glutelin genes could be involved in the null mutant of OsROS1a seed phenotype and OsROS1a could have the key role in the regulation of glutelins. Furthermore, 378 differentially alternative splicing (AS) genes were identified in the null mutant of OsROS1a, suggesting that the OsROS1a gene has an impact on AS events. Our findings indicated that the function on rice endosperm development in the null mutant of OsROS1a could be influenced through regulating gene expression and AS, which could provide the base to properly understand the molecular mechanism related to the OsROS1a gene in the regulation of rice seed development.},
}
@article {pmid35741761,
year = {2022},
author = {Wang, HQ and Wang, T and Gao, F and Ren, WZ},
title = {Application of CRISPR/Cas Technology in Spermatogenesis Research and Male Infertility Treatment.},
journal = {Genes},
volume = {13},
number = {6},
pages = {},
pmid = {35741761},
issn = {2073-4425},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Infertility, Male/genetics/therapy ; Male ; Spermatogenesis/genetics ; Technology ; },
abstract = {As the basis of animal reproductive activity, normal spermatogenesis directly determines the efficiency of livestock production. An in-depth understanding of spermatogenesis will greatly facilitate animal breeding efforts and male infertility treatment. With the continuous development and application of gene editing technologies, they have become valuable tools to study the mechanism of spermatogenesis. Gene editing technologies have provided us with a better understanding of the functions and potential mechanisms of action of factors that regulate spermatogenesis. This review summarizes the applications of gene editing technologies, especially CRISPR/Cas9, in deepening our understanding of the function of spermatogenesis-related genes and disease treatment. The problems of gene editing technologies in the field of spermatogenesis research are also discussed.},
}
@article {pmid35741243,
year = {2022},
author = {Hernandez-Garcia, A and Morales-Moreno, MD and Valdés-Galindo, EG and Jimenez-Nieto, EP and Quezada, A},
title = {Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
pmid = {35741243},
issn = {2075-4418},
abstract = {The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.},
}
@article {pmid35741144,
year = {2022},
author = {Selvam, K and Ahmad Najib, M and Khalid, MF and Ozsoz, M and Aziah, I},
title = {CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {6},
pages = {},
pmid = {35741144},
issn = {2075-4418},
abstract = {Recently, CRISPR-Cas system-based assays for bacterial detection have been developed. The aim of this scoping review is to map existing evidence on the utilization of CRISPR-Cas systems in the development of bacterial detection assays. A literature search was conducted using three databases (PubMed, Scopus, and Cochrane Library) and manual searches through the references of identified full texts based on a PROSPERO-registered protocol (CRD42021289140). Studies on bacterial detection using CRISPR-Cas systems that were published before October 2021 were retrieved. The Critical Appraisal Skills Programme (CASP) qualitative checklist was used to assess the risk of bias for all the included studies. Of the 420 studies identified throughout the search, 46 studies that met the inclusion criteria were included in the final analysis. Bacteria from 17 genera were identified utilising CRISPR-Cas systems. Most of the bacteria came from genera such as Staphylococcus, Escherichia, Salmonella, Listeria, Mycobacterium and Streptococcus. Cas12a (64%) is the most often used Cas enzyme in bacterial detection, followed by Cas13a (13%), and Cas9 (11%). To improve the signal of detection, 83% of the research exploited Cas enzymes' trans-cleavage capabilities to cut tagged reporter probes non-specifically. Most studies used the extraction procedure, whereas only 17% did not. In terms of amplification methods, isothermal reactions were employed in 66% of the studies, followed by PCR (23%). Fluorescence detection (67%) was discovered to be the most commonly used method, while lateral flow biosensors (13%), electrochemical biosensors (11%), and others (9%) were found to be less commonly used. Most of the studies (39) used specific bacterial nucleic acid sequences as a target, while seven used non-nucleic acid targets, including aptamers and antibodies particular to the bacteria under investigation. The turnaround time of the 46 studies was 30 min to 4 h. The limit of detection (LoD) was evaluated in three types of concentration, which include copies per mL, CFU per mL and molarity. Most of the studies used spiked samples (78%) rather than clinical samples (22%) to determine LoD. This review identified the gap in clinical accuracy evaluation of the CRISPR-Cas system in bacterial detection. More research is needed to assess the diagnostic sensitivity and specificity of amplification-free CRISPR-Cas systems in bacterial detection for nucleic acid-based tests.},
}
@article {pmid35739111,
year = {2022},
author = {Lainšček, D and Forstnerič, V and Mikolič, V and Malenšek, &#x160; and Pečan, P and Benčina, M and Sever, M and Podgornik, H and Jerala, R},
title = {Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3604},
pmid = {35739111},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Exonucleases/genetics ; *Gene Editing ; Gene Knockout Techniques ; Humans ; RNA, Guide ; },
abstract = {The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.},
}
@article {pmid35738948,
year = {2022},
author = {Lan, TH and He, L and Huang, Y and Zhou, Y},
title = {Optogenetics for transcriptional programming and genetic engineering.},
journal = {Trends in genetics : TIG},
volume = {38},
number = {12},
pages = {1253-1270},
doi = {10.1016/j.tig.2022.05.014},
pmid = {35738948},
issn = {0168-9525},
support = {R01 CA232017/CA/NCI NIH HHS/United States ; R01 GM112003/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Optogenetics ; *Genetic Engineering ; Genome ; DNA ; CRISPR-Cas Systems ; Mammals/genetics ; },
abstract = {Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200-1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research.},
}
@article {pmid35738528,
year = {2022},
author = {Lee, ABC and Tan, MH and Chai, CLL},
title = {Small-molecule enhancers of CRISPR-induced homology-directed repair in gene therapy: A medicinal chemist's perspective.},
journal = {Drug discovery today},
volume = {27},
number = {9},
pages = {2510-2525},
doi = {10.1016/j.drudis.2022.06.006},
pmid = {35738528},
issn = {1878-5832},
mesh = {*CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing ; Genetic Therapy ; Humans ; *Recombinational DNA Repair ; },
abstract = {CRISPR technologies are increasingly being investigated and utilized for the treatment of human genetic diseases via genome editing. CRISPR-Cas9 first generates a targeted DNA double-stranded break, and a functional gene can then be introduced to replace the defective copy in a precise manner by templated repair via the homology-directed repair (HDR) pathway. However, this is challenging owing to the relatively low efficiency of the HDR pathway compared with a rival random repair pathway known as non-homologous end joining (NHEJ). Small molecules can be employed to increase the efficiency of HDR and decrease that of NHEJ to improve the efficiency of precise knock-in genome editing. This review discusses the potential usage of such small molecules in the context of gene therapy and their drug-likeness, from a medicinal chemist's perspective.},
}
@article {pmid35738280,
year = {2022},
author = {Vijayan, K and Arang, N and Wei, L and Morrison, R and Geiger, R and Parks, KR and Lewis, AJ and Mast, FD and Douglass, AN and Kain, HS and Aitchison, JD and Johnson, JS and Aderem, A and Kaushansky, A},
title = {A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.},
journal = {Cell chemical biology},
volume = {29},
number = {9},
pages = {1419-1433.e5},
pmid = {35738280},
issn = {2451-9448},
support = {R01 AI032972/AI/NIAID NIH HHS/United States ; U19 AI111276/AI/NIAID NIH HHS/United States ; U19 AI100627/AI/NIAID NIH HHS/United States ; R01 GM101183/GM/NIGMS NIH HHS/United States ; R00 AI111785/AI/NIAID NIH HHS/United States ; U19 AI135976/AI/NIAID NIH HHS/United States ; U19 AI106761/AI/NIAID NIH HHS/United States ; P41 GM109824/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Humans ; Liver/metabolism/parasitology ; *Malaria/metabolism ; *Microtubule-Associated Proteins/metabolism ; *Microtubules/metabolism ; Plasmodium/metabolism ; Tubulin/metabolism ; },
abstract = {Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.},
}
@article {pmid35737816,
year = {2022},
author = {Gong, FL and Han, J and Li, S},
title = {MULTI-SCULPT: Multiplex Integration via Selective, CRISPR-Mediated, Ultralong Pathway Transformation in Yeast for Plant Natural Product Synthesis.},
journal = {ACS synthetic biology},
volume = {11},
number = {7},
pages = {2484-2495},
pmid = {35737816},
issn = {2161-5063},
support = {R21 DC019206/DC/NIDCD NIH HHS/United States ; T32 GM138826/GM/NIGMS NIH HHS/United States ; },
mesh = {*Biological Products/metabolism ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Genomics ; *Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {Yeast has been a versatile model host for complex and valuable natural product biosynthesis via the reconstruction of heterologous biosynthetic pathways. Recent advances in natural product pathway elucidation have uncovered many large and complicated plant pathways that contain 10-30 genes for the biosynthesis of structurally complex, valuable natural products. However, the ability to reconstruct ultralong pathways efficiently in yeast does not match the increasing demand for valuable plant natural product biomanufacturing. Here, we developed a one-pot, multigene pathway integration method in yeast, named MULTI-SCULPT for multiplex integration via selective, CRISPR-mediated, ultralong pathway transformation. Leveraging multilocus genomic disruption via CRISPR/Cas9, newly developed native and synthetic genetic parts, and fine-tuned gene integration and characterization methods, we managed to integrate 21 DNA inserts that contain a 12-gene plant isoflavone biosynthetic pathway into yeast with a 90-100% success rate in 12 days. This method enables fast and efficient ultralong biosynthetic pathway integration and can allow for the fast iterative integration of even longer pathways in the future. Ultimately, this method will accelerate combinatorial optimization of elucidated plant natural product pathways and accelerate putative pathway characterization heterologously.},
}
@article {pmid35737584,
year = {2022},
author = {Hao, Y and Li, M and Zhang, Q and Shi, J and Li, J and Li, Q and Fan, C and Wang, F},
title = {DNA Origami-Based Single-Molecule CRISPR Machines for Spatially Resolved Searching.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {34},
pages = {e202205460},
doi = {10.1002/anie.202205460},
pmid = {35737584},
issn = {1521-3773},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *DNA Cleavage ; Endonucleases/metabolism ; Nanotechnology ; RNA, Guide/genetics ; },
abstract = {Repurposing the RNA-guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR-Cas9-based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami-based single-molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially-confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high-throughput nucleic acid detection.},
}
@article {pmid35737245,
year = {2022},
author = {Sayed, S and Sürün, D and Mircetic, J and Sidorova, OA and Buchholz, F},
title = {Using CRISPR-Cas9 to Dissect Cancer Mutations in Cell Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2508},
number = {},
pages = {235-260},
pmid = {35737245},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing ; Humans ; Mutation ; *Neoplasms/genetics ; Precision Medicine ; },
abstract = {The CRISPR-Cas9 technology has revolutionized the scope and pace of biomedical research, enabling the targeting of specific genomic sequences for a wide spectrum of applications. Here we describe assays to functionally interrogate mutations identified in cancer cells utilizing both CRISPR-Cas9 nuclease and base editors. We provide guidelines to interrogate known cancer driver mutations or functionally screen for novel vulnerability mutations with these systems in characterized human cancer cell lines. The proposed platform should be transferable to primary cancer cells, opening up a path for precision oncology on a functional level.},
}
@article {pmid35736067,
year = {2022},
author = {Devanna, BN and Jain, P and Solanke, AU and Das, A and Thakur, S and Singh, PK and Kumari, M and Dubey, H and Jaswal, R and Pawar, D and Kapoor, R and Singh, J and Arora, K and Saklani, BK and AnilKumar, C and Maganti, SM and Sonah, H and Deshmukh, R and Rathour, R and Sharma, TR},
title = {Understanding the Dynamics of Blast Resistance in Rice-Magnaporthe oryzae Interactions.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {8},
number = {6},
pages = {},
pmid = {35736067},
issn = {2309-608X},
abstract = {Rice is a global food grain crop for more than one-third of the human population and a source for food and nutritional security. Rice production is subjected to various stresses; blast disease caused by Magnaporthe oryzae is one of the major biotic stresses that has the potential to destroy total crop under severe conditions. In the present review, we discuss the importance of rice and blast disease in the present and future global context, genomics and molecular biology of blast pathogen and rice, and the molecular interplay between rice-M. oryzae interaction governed by different gene interaction models. We also elaborated in detail on M. oryzae effector and Avr genes, and the role of noncoding RNAs in disease development. Further, rice blast resistance QTLs; resistance (R) genes; and alleles identified, cloned, and characterized are discussed. We also discuss the utilization of QTLs and R genes for blast resistance through conventional breeding and transgenic approaches. Finally, we review the demonstrated examples and potential applications of the latest genome-editing tools in understanding and managing blast disease in rice.},
}
@article {pmid35735623,
year = {2022},
author = {Li, X and Xu, S and Fuhrmann-Aoyagi, MB and Yuan, S and Iwama, T and Kobayashi, M and Miura, K},
title = {CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses.},
journal = {Current issues in molecular biology},
volume = {44},
number = {6},
pages = {2664-2682},
pmid = {35735623},
issn = {1467-3045},
abstract = {Global warming and climate change have severely affected plant growth and food production. Therefore, minimizing these effects is required for sustainable crop yields. Understanding the molecular mechanisms in response to abiotic stresses and improving agricultural traits to make crops tolerant to abiotic stresses have been going on unceasingly. To generate desirable varieties of crops, traditional and molecular breeding techniques have been tried, but both approaches are time-consuming. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALENs) are genome-editing technologies that have recently attracted the attention of plant breeders for genetic modification. These technologies are powerful tools in the basic and applied sciences for understanding gene function, as well as in the field of crop breeding. In this review, we focus on the application of genome-editing systems in plants to understand gene function in response to abiotic stresses and to improve tolerance to abiotic stresses, such as temperature, drought, and salinity stresses.},
}
@article {pmid35732990,
year = {2022},
author = {Kobelt, D and Pahle, J and Walther, W},
title = {A Brief Introduction to Current Cancer Gene Therapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2521},
number = {},
pages = {1-21},
pmid = {35732990},
issn = {1940-6029},
mesh = {Gene Transfer Techniques ; Genes, Neoplasm ; Genetic Therapy ; *Genetic Vectors/genetics ; Humans ; *Neoplasms/genetics/therapy ; Transgenes ; },
abstract = {Gene therapy has started in the late 1980s as novel, clinically applicable therapeutic option. It revolutionized the treatment of genetic diseases with the initial intent to repair or replace defective genes. Gene therapy has been adapted for treatment of malignant diseases to improve the outcome of cancer patients. In fact, cancer gene therapy has rapidly gained great interest and evolved into a research field with highest proportion of research activities in gene therapy. In this context, cancer gene therapy has long entered translation into clinical trials and therefore more than two-thirds of all gene therapy trials worldwide are aiming at the treatment of cancer disease using different therapeutic strategies. During the decades in cancer gene therapy, tremendous knowledge has accumulated. This led to significant improvements in vector design, transgene repertoire, more targeted interventions, use of novel gene therapeutic technologies such as CRISPR/Cas, sleeping beauty vectors, and development of effective cancer immunogene therapies. In this chapter, a brief overview of current key developments in cancer gene therapy is provided to gain insights into the recent directions in research as well as in clinical application of cancer gene therapy.},
}
@article {pmid35732739,
year = {2022},
author = {Chaffin, M and Papangeli, I and Simonson, B and Akkad, AD and Hill, MC and Arduini, A and Fleming, SJ and Melanson, M and Hayat, S and Kost-Alimova, M and Atwa, O and Ye, J and Bedi, KC and Nahrendorf, M and Kaushik, VK and Stegmann, CM and Margulies, KB and Tucker, NR and Ellinor, PT},
title = {Single-nucleus profiling of human dilated and hypertrophic cardiomyopathy.},
journal = {Nature},
volume = {608},
number = {7921},
pages = {174-180},
pmid = {35732739},
issn = {1476-4687},
support = {R01 HL092577/HL/NHLBI NIH HHS/United States ; R01 HL128914/HL/NHLBI NIH HHS/United States ; K24 HL105780/HL/NHLBI NIH HHS/United States ; K01 HL140187/HL/NHLBI NIH HHS/United States ; R01 HL105993/HL/NHLBI NIH HHS/United States ; /AHA/American Heart Association-American Stroke Association/United States ; },
mesh = {CRISPR-Cas Systems ; *Cardiomyopathy, Dilated/genetics/pathology ; *Cardiomyopathy, Hypertrophic/genetics/pathology ; Case-Control Studies ; *Cell Nucleus/genetics ; Cells, Cultured ; *Gene Expression Profiling ; Gene Knockout Techniques ; *Heart Failure/genetics/pathology ; Heart Ventricles/metabolism/pathology ; Humans ; Myocardium/metabolism/pathology ; Myofibroblasts/metabolism/pathology ; RNA-Seq ; *Single-Cell Analysis ; Transcription, Genetic ; Transforming Growth Factor beta1 ; },
abstract = {Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function[1,2] and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts[3,4], but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFβ1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.},
}
@article {pmid35732256,
year = {2022},
author = {Awan, MJA and Pervaiz, K and Rasheed, A and Amin, I and Saeed, NA and Dhugga, KS and Mansoor, S},
title = {Genome edited wheat- current advances for the second green revolution.},
journal = {Biotechnology advances},
volume = {60},
number = {},
pages = {108006},
doi = {10.1016/j.biotechadv.2022.108006},
pmid = {35732256},
issn = {1873-1899},
mesh = {Adenosine ; *CRISPR-Cas Systems/genetics ; Cytidine ; Edible Grain/genetics ; Endonucleases/genetics ; Genome, Plant/genetics ; Plant Breeding ; *Triticum/genetics ; },
abstract = {Common wheat is a major source of nutrition around the globe, but unlike maize and rice hybrids, no breakthrough has been made to enhance wheat yield since Green Revolution. With the availability of reference genome sequence of wheat and advancement of allied genomics technologies, understanding of genes involved in grain yield components and disease resistance/susceptibility has opened new avenues for crop improvement. Wheat has a huge hexaploidy genome of approximately 17 GB with 85% repetition, and it is a daunting task to induce any mutation across three homeologues that can be helpful for the enhancement of agronomic traits. The CRISPR-Cas9 system provides a promising platform for genome editing in a site-specific manner. In wheat, CRISPR-Cas9 is being used in the improvement of yield, grain quality, biofortification, resistance against diseases, and tolerance against abiotic factors. The promising outcomes of the CRISPR-based multiplexing approach circumvent the constraint of targeting merely one gene at a time. Deployment of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 endonuclease (CRISPR-Cas9) and Cas9 variant systems such as cytidine base editing, adenosine base editing, and prime editing in wheat has been used to induce point mutations more precisely. Scientists have acquired major events such as induction of male sterility, fertility restoration, and alteration of seed dormancy through Cas9 in wheat that can facilitate breeding programs for elite variety development. Furthermore, a recent discovery in tissue culturing enables scientists to significantly enhance regeneration efficiency in wheat by transforming the GRF4-GIF1 cassette. Rapid generation advancement by speed breeding technology provides the opportunity for the generation advancement of the desired plants to segregate out unwanted transgenes and allows rapid integration of gene-edited wheat into the breeding pipeline. The combination of these novel technologies addresses some of the most important limiting factors for sustainable and climate-smart wheat that should lead to the second "Green Revolution" for global food security.},
}
@article {pmid35730730,
year = {2022},
author = {Yuan, R and Bai, X and Hu, X and Zhang, H and Hou, C and Long, Q and Luo, Y},
title = {CATCH: high specific transcriptome-focused fusion gene variants discrimination.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {55},
pages = {7618-7621},
doi = {10.1039/d2cc02660k},
pmid = {35730730},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Transcriptome ; },
abstract = {We develop a CRISPR-Cas13a triggered catalytic hairpin assembly (CATCH) approach for accurate and impartial identification of variants by integrating the fusion gene-selected recognition of CRISPR-Cas13a with collateral cleavage-assisted catalytic hairpin assembly amplification. This approach achieved an accuracy of 100% in a pilot experiment involving 34 clinical samples.},
}
@article {pmid35730587,
year = {2022},
author = {Bao, J and de Dios Mateos, E and Scheller, S},
title = {Efficient CRISPR/Cas12a-Based Genome-Editing Toolbox for Metabolic Engineering in Methanococcus maripaludis.},
journal = {ACS synthetic biology},
volume = {11},
number = {7},
pages = {2496-2503},
pmid = {35730587},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Carbon Dioxide/metabolism ; *Gene Editing/methods ; Hydrogen/metabolism ; Metabolic Engineering/methods ; *Methanococcus/genetics/metabolism ; },
abstract = {The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits that affect the primary metabolism and cell growth. Here, we have designed a genome-editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. This toolbox can delete target genes with a success rate of up to 95%, despite the hyperpolyploidy of M. maripaludis. For the purpose of demonstrating a large deletion, the M. maripaludis flagellum operon (∼8.9 kbp) was replaced by the Escherichia coli β-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters was quantified in the presence of two common growth substrates, either formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and quick method for genome editing in a methanogen.},
}
@article {pmid35728759,
year = {2022},
author = {Zhao, Y and Zhu, X and Chen, X and Zhou, JM},
title = {From plant immunity to crop disease resistance.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {49},
number = {8},
pages = {693-703},
doi = {10.1016/j.jgg.2022.06.003},
pmid = {35728759},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural ; *Disease Resistance ; Gene Editing ; Genome, Plant ; },
abstract = {Plant diseases caused by diverse pathogens lead to a serious reduction in crop yield and threaten food security worldwide. Genetic improvement of plant immunity is considered as the most effective and sustainable approach to control crop diseases. In the last decade, our understanding of plant immunity at both molecular and genomic levels has improved greatly. Combined with advances in biotechnologies, particularly clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based genome editing, we can now rapidly identify new resistance genes and engineer disease-resistance crop plants like never before. In this review, we summarize the current knowledge of plant immunity and outline existing and new strategies for disease resistance improvement in crop plants. We also discuss existing challenges in this field and suggest directions for future studies.},
}
@article {pmid35727982,
year = {2022},
author = {Hu, M and Qiu, Z and Bi, Z and Tian, T and Jiang, Y and Zhou, X},
title = {Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {26},
pages = {e2202034119},
pmid = {35727982},
issn = {1091-6490},
mesh = {*Bacterial Proteins ; COVID-19/diagnosis ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/radiation effects ; *Endodeoxyribonucleases ; Humans ; RNA/radiation effects ; *Reagent Kits, Diagnostic ; Recombinases/genetics ; *Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2/isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.},
}
@article {pmid35727866,
year = {2022},
author = {Lee, YY and Park, R and Miller, SM and Li, Y},
title = {Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii.},
journal = {The Plant journal : for cell and molecular biology},
volume = {111},
number = {4},
pages = {1069-1080},
pmid = {35727866},
issn = {1365-313X},
mesh = {Animals ; *Chlamydomonas reinhardtii/genetics/metabolism ; Diacylglycerol O-Acyltransferase/genetics ; *Microalgae/genetics/metabolism ; Plants/metabolism ; Triglycerides/metabolism ; },
abstract = {Genetic compensation has been proposed to explain phenotypic differences between gene knockouts and knockdowns in several metazoan and plant model systems. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in the model alga Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, for example, CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a Δvtc1 mutant, in which PDAT was upregulated under stress, resulting in a 90% increase in TAG content. Knockout of PDAT in the Δvtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in Δvtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis under stress, and provides guidance for future genetic engineering and mutant characterization efforts.},
}
@article {pmid35727555,
year = {2022},
author = {Ametrano, A and Coscia, MR},
title = {Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2498},
number = {},
pages = {337-350},
pmid = {35727555},
issn = {1940-6029},
mesh = {Animals ; Antibodies, Monoclonal/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Fishes/metabolism ; *Gene Editing/methods ; Hybridomas/metabolism ; Mice ; RNA, Guide/genetics ; Technology ; },
abstract = {The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol.},
}
@article {pmid35727554,
year = {2022},
author = {Russo, MT and Santin, A and Rogato, A and Ferrante, MI},
title = {Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2498},
number = {},
pages = {327-336},
pmid = {35727554},
issn = {1940-6029},
mesh = {Biolistics/methods ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Cell Nucleus/genetics ; *Diatoms/genetics ; },
abstract = {The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.},
}
@article {pmid35727450,
year = {2022},
author = {Chen, W and She, W and Li, A and Zhai, C and Ma, L},
title = {Site-Directed Mutagenesis Method Mediated by Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2461},
number = {},
pages = {165-174},
pmid = {35727450},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Mutagenesis ; Mutagenesis, Site-Directed ; Plasmids/genetics ; Polymerase Chain Reaction ; },
abstract = {This study presents an in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants or site-saturation mutagenesis libraries in a PCR-independent manner. The plasmid DNA is double digested with Cas9 bearing specific single guide RNAs to remove the target nucleotides. Next, T5 exonuclease excises both 5'-ends of the linearized plasmid to generate homologous regions of approximately 15 nt. Subsequently, a short dsDNA of approximately 30-50 bp containing the desired mutation cyclizes the plasmid through base pairing and introduces the mutation into the plasmid. The gaps are repaired in Escherichia coli host cells after transformation. This method is highly efficient and accurate. Both single and multiple site-directed mutagenesis can be successfully performed, especially to large sized plasmids. This method demonstrates the great potential for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.},
}
@article {pmid35726663,
year = {2022},
author = {Kapusi, E and Cong, L and Stoger, E},
title = {Editorial: CRISPR and alternative approaches.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2200290},
doi = {10.1002/biot.202200290},
pmid = {35726663},
issn = {1860-7314},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35726625,
year = {2022},
author = {Saito-Tarashima, N and Ueno, M and Murai, A and Matsuo, A and Minakawa, N},
title = {Cas9-mediated DNA cleavage guided by enzymatically prepared 4'-thio-modified RNA.},
journal = {Organic &amp; biomolecular chemistry},
volume = {20},
number = {26},
pages = {5245-5248},
doi = {10.1039/d2ob00742h},
pmid = {35726625},
issn = {1477-0539},
mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; RNA ; RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR-Cas9-mediated DNA editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage by the Cas endonuclease. Because natural gRNA is susceptible to intracellular degradation, it is desirable to chemically protect it for efficient editing. Using 4'-thioribonucleoside 5'-triphosphates and T7 transcription, we have prepared 4'-thio-modified gRNAs that guide Cas9-mediated DNA cleavage. This approach is a simple way to obtain chemically modified RNA suitable for CRISPR-Cas9 DNA editing.},
}
@article {pmid35725215,
year = {2022},
author = {Khademi, Z and Ramezani, M and Alibolandi, M and Zirak, MR and Salmasi, Z and Abnous, K and Taghdisi, SM},
title = {A novel dual-targeting delivery system for specific delivery of CRISPR/Cas9 using hyaluronic acid, chitosan and AS1411.},
journal = {Carbohydrate polymers},
volume = {292},
number = {},
pages = {119691},
doi = {10.1016/j.carbpol.2022.119691},
pmid = {35725215},
issn = {1879-1344},
mesh = {Aptamers, Nucleotide ; *CRISPR-Cas Systems/genetics ; *Chitosan ; Gene Transfer Techniques ; HEK293 Cells ; Humans ; Hyaluronic Acid ; Oligodeoxyribonucleotides ; },
abstract = {A facile method was designed that can specifically deliver CRISPR/Cas9 into target cells nuclei and reduce the off-target effects. A multifunctional delivery vector for FOXM1 knockout was composed by integration of cell targeting polymer (hyaluronic acid) and cell and nuclear targeting group (AS1411 aptamer) on the surface of nanoparticles formed by genome editing plasmid and chitosan (CS) as the core (Apt-HA-CS-CRISPR/Cas9). The data of cytotoxicity experiment and western blot confirmed this issue. The results of flow cytometry analysis and fluorescence imaging demonstrated that Apt-HA-CS-CRISPR/Cas9 was significantly internalized into target cells (MCF-7, SK-MES-1, HeLa) but not into nontarget cells (HEK293). Furthermore, the in vivo studies displayed that the Apt-HA-CS-CRISPR/Cas9 was strongly rendered tumor inhibitory effect and delivered efficiently CRISPR/Cas9 into the tumor with no detectable distribution in other organs compared with naked plasmid. This approach provides an avenue for specific in vivo gene editing therapeutics with the lowest side effect.},
}
@article {pmid35724832,
year = {2022},
author = {Haellman, V and Pirkl, M and Akmammedov, A and Saxena, P and Beerenwinkel, N and Paro, R and Teixeira, AP and Fussenegger, M},
title = {dCas9-mediated dysregulation of gene expression in human induced pluripotent stem cells during primitive streak differentiation.},
journal = {Metabolic engineering},
volume = {73},
number = {},
pages = {70-81},
doi = {10.1016/j.ymben.2022.06.003},
pmid = {35724832},
issn = {1096-7184},
support = {785800/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems ; Gene Expression ; Humans ; *Induced Pluripotent Stem Cells ; Primitive Streak ; },
abstract = {CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.},
}
@article {pmid35724001,
year = {2022},
author = {Montano Gomez, P},
title = {[Not Available].},
journal = {Journal international de bioethique et d'ethique des sciences},
volume = {33},
number = {1},
pages = {85-101},
doi = {10.3917/jibes.331.0085},
pmid = {35724001},
issn = {2608-1008},
mesh = {*Gene Editing ; Humans ; },
abstract = {There are many reasons why we might think that the human species might be in danger. We talk about global warming which produces major unusual natural disasters; increasingly destructive weapons; serious terrorist attacks with chemical weapons, etc. But we cannot ignore one of the threats hanging over the human species, which is transhumanism or posthumanism. It is from the point of view of this last subject, on which much has already been written, that we will try to focus our analysis, paying particular attention to the question relating to gene editing (CRISPR/Cas 9).},
}
@article {pmid35723935,
year = {2022},
author = {Han, L and Haslam, RP and Silvestre, S and Lu, C and Napier, JA},
title = {Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathway.},
journal = {Plant biotechnology journal},
volume = {20},
number = {8},
pages = {1444-1446},
pmid = {35723935},
issn = {1467-7652},
support = {BBS/E/C/000I0420/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Brassicaceae/genetics ; CRISPR-Cas Systems/genetics ; Docosahexaenoic Acids/metabolism ; *Eicosapentaenoic Acid/metabolism ; Plants, Genetically Modified/metabolism ; },
}
@article {pmid35723482,
year = {2022},
author = {Rathbone, T and Ates, I and Stuart, C and Parker, T and Cottle, RN},
title = {Electroporation-mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {184},
pages = {},
doi = {10.3791/63828},
pmid = {35723482},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Electroporation/methods ; Hepatocytes/metabolism ; Mice ; RNA, Messenger/genetics ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 × 10[6] cells per liver and cell viability of >85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of >35% in mouse hepatocytes. To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.},
}
@article {pmid35723254,
year = {2022},
author = {Kanca, O and Zirin, J and Hu, Y and Tepe, B and Dutta, D and Lin, WW and Ma, L and Ge, M and Zuo, Z and Liu, LP and Levis, RW and Perrimon, N and Bellen, HJ},
title = {An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35723254},
issn = {2050-084X},
support = {U54 HD083092/HD/NICHD NIH HHS/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; R01 GM067858/GM/NIGMS NIH HHS/United States ; R01 GM084947/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R24 OD031447/OD/NIH HHS/United States ; U54 NS093793/NS/NINDS NIH HHS/United States ; R24 OD022005/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Drosophila/genetics ; Exons/genetics ; Homologous Recombination ; Plasmids ; },
abstract = {Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.},
}
@article {pmid35722725,
year = {2022},
author = {Zhou, J and Liu, Y and Guo, X and Birchler, JA and Han, F and Su, H},
title = {Centromeres: From chromosome biology to biotechnology applications and synthetic genomes in plants.},
journal = {Plant biotechnology journal},
volume = {20},
number = {11},
pages = {2051-2063},
pmid = {35722725},
issn = {1467-7652},
mesh = {Humans ; *Plant Breeding ; *Centromere/genetics ; Chromosomes, Plant/genetics ; Plants/genetics ; Epigenomics ; Biotechnology ; },
abstract = {Centromeres are the genomic regions that organize and regulate chromosome behaviours during cell cycle, and their variations are associated with genome instability, karyotype evolution and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.},
}
@article {pmid35722296,
year = {2022},
author = {Parra-Flores, J and Holý, O and Acuña, S and Lepuschitz, S and Pietzka, A and Contreras-Fernández, A and Chavarría-Sepulveda, P and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Castillo, A and Ruppitsch, W and Forsythe, S},
title = {Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {884721},
pmid = {35722296},
issn = {1664-302X},
abstract = {This study characterized five Cronobacter spp. and six Salmonella spp. strains that had been isolated from 155 samples of powdered infant formula (PIF) sold in Chile and manufactured in Chile and Mexico in 2018-2020. Two strains of Cronobacter sakazakii sequence type (ST) ST1 and ST31 (serotypes O:1 and O:2) and one strain of Cronobacter malonaticus ST60 (O:1) were identified. All Salmonella strains were identified as Salmonella Typhimurium ST19 (serotype O:4) by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST (cgMLST). The C. sakazakii and C. malonaticus isolates were resistant to cephalothin, whereas the Salmonella isolates were resistant to oxacillin and ampicillin. Nineteen antibiotic resistance genes were detected in the C. sakazakii and C. malonaticus isolates; the most prevalent were mcr-9.1, blaCSA , and blaCMA . In Salmonella, 30 genes encoding for aminoglycoside and cephalosporin resistance were identified, including aac(6')-Iaa, β-lactamases ampH, ampC1, and marA. In the Cronobacter isolates, 32 virulence-associated genes were detected by WGS and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, use of sialic acid, and toxin-antitoxin genes. In the Salmonella strains, 120 virulence associated genes were detected, adherence, magnesium uptake, resistance to antimicrobial peptides, secretion system, stress protein, toxin, resistance to complement killing, and eight pathogenicity islands. The C. sakazakii and C. malonaticus strains harbored I-E and I-F CRISPR-Cas systems and carried Col(pHHAD28) and IncFIB(pCTU1) plasmids, respectively. The Salmonella strains harbored type I-E CRISPR-Cas systems and carried IncFII(S) plasmids. The presence of C. sakazakii and Salmonella in PIF is a health risk for infants aged less than 6 months. For this reason, sanitary practices should be reinforced for its production and retail surveillance.},
}
@article {pmid35722276,
year = {2022},
author = {Panahi, B and Majidi, M and Hejazi, MA},
title = {Genome Mining Approach Reveals the Occurrence and Diversity Pattern of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Systems in Lactobacillus brevis Strains.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {911706},
pmid = {35722276},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) together with their CRISPR-associated (Cas) genes are widely distributed in prokaryotes that provide an adaptive defense mechanism against foreign invasive DNA. There is relatively little knowledge about the CRISPR-Cas diversity and evolution in Lactobacillus brevis strains. Therefore, in this study, a genome-mining approach was employed to investigate the diversity and occurrence of the CRISPR-Cas system in 83 L. brevis strains. Moreover, trans-activating CRISPR RNA (tracrRNA) and protospacer adjacent motif (PAM) as pivotal elements for the successful targeting and inference of phages by the subtype II CRISPR-Cas systems were surveyed. Finally, evolutionary paths of L. brevis strains under selective pressure from foreign invasive DNA such as plasmids and phages of studied strains were surveyed using acquisition and deletion events analysis of spacers. A total of 127 confirmed CRISPRs were identified, which were distributed in 69 strains. Among strains with confirmed CRISPRs, 35 strains only contained one CRISPR locus, 23 strains contained two CRISPR loci, and 12 strains contained three to six CRISPR loci. L. brevis strains frequently harbor more than one CRISPR system. Analysis of confirmed CRISPR arrays showed that 31 out of 127 confirmed CRISPRs included Cas genes which were categorized as one of the II-A, II-C, and I-E subtypes. Analysis of subtype II-A spacers reflected divergent evolution for 18 strains into 16 unique groups. Additional analysis of spacer sequences also confirmed the implication of characterizing CRISPR-Cas systems in targeting of phages and plasmids. The current study highlighted the potential of utilizing CRISPR spacer polymorphism in genotyping lactobacillus strains. Moreover, it provides deep insights into the occurrence, diversity, and functional impacts of the CRISPR-Cas system in L. brevis strains.},
}
@article {pmid35721864,
year = {2022},
author = {Zhang, Y and Ge, H and Marchisio, MA},
title = {A Mutated Nme1Cas9 Is a Functional Alternative RNase to Both LwaCas13a and RfxCas13d in the Yeast S. cerevisiae.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {922949},
pmid = {35721864},
issn = {2296-4185},
abstract = {CRISPR-Cas systems provide powerful biological tools for genetic manipulation and gene expression regulation. Class 2 systems, comprising type II, type V, and type VI, have the significant advantage to require a single effector Cas protein (Cas9, Cas12, and Cas13 respectively) to cleave nucleic acids upon binding the crRNA. Both Cas9 and Cas12 recognize DNA and induce a double-strand break in it. In contrast, Cas13 bind and cleave RNA exclusively. However, some Cas9 homologs have shown RNase activity as well. Here, we harnessed Nme1Cas9, LwaCas13a, and RfxCas13d to carry out gene downregulation in Saccharomyces cerevisiae by triggering mRNA degradation. To avoid potential DNA damage, we mutated Nme1Cas9 into d16ANme1Cas9 that lost the nuclease activity of the RuvC domain but retained the active HNH domain, able to act on the target DNA strand and, therefore, on the corresponding transcript. Our results showed that d16ANme1Cas9 is a functional RNase in vivo, although with moderate activity since it provoked a fluorescence reduction from 21% to 32%. Interestingly, d16ANme1Cas9 works in a PAM-independent way nor demands helper PAMmer molecules. LwaCas13a and RfxCas13d appeared substantially unfunctional in S. cerevisiae, though they were shown to perform well in mammalian cells. To the best of our knowledge, this is the first report about the working in vivo of a variant of Nme1Cas9 as an RNase and the issues connected with the usage of Cas13 proteins in S. cerevisiae.},
}
@article {pmid35719725,
year = {2022},
author = {Chilian, M and Vargas Parra, K and Sandoval, A and Ramirez, J and Yoon, WH},
title = {CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue using sgRNAs that target exon-intron junctions in Drosophila melanogaster.},
journal = {STAR protocols},
volume = {3},
number = {3},
pages = {101465},
pmid = {35719725},
issn = {2666-1667},
support = {P20 GM103636/GM/NIGMS NIH HHS/United States ; R01 NS121298/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA, Complementary/genetics ; Drosophila/genetics ; *Drosophila melanogaster/genetics ; Exons/genetics ; Introns ; },
abstract = {In this protocol, we take CRISPR/Cas9 and Gal4/UAS approaches to achieve tissue-specific knockout in parallel with rescue of the knockout by cDNA expression in Drosophila. We demonstrate that guide RNAs targeting the exon-intron junction of target genes cleave the genomic locus of the genes, but not UAS-cDNA transgenes, in a tissue where Gal4 drives Cas9 expression. The efficiency of this approach enables the determination of pathogenicity of disease-associated variants in human genes in a tissue-specific manner in Drosophila. For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).},
}
@article {pmid35719360,
year = {2022},
author = {Qiu, X and Xu, S and Liu, X and Ren, H and Han, L and Li, Z},
title = {CRISPR/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {884411},
pmid = {35719360},
issn = {2235-2988},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; Nocardia ; *Nocardia Infections ; *Nucleic Acids ; Pandemics ; },
abstract = {Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica (N. farcinica). A highly conserved and species-specific gene pbr1 of N. farcinica, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the N. farcinica CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70°C, 40 min), and CRISPR/Cas12a-mediated detection (37°C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of N. farcinica CRISPR-CLA assay, and no cross-reaction was observed with non-N. farcinica templates. The limit of detection (LoD) of the N. farcinica CRISPR-CLA assay was 100 fg double-strand DNA per reaction. N. farcinica was detected accurately in 41 sputum specimens using the N. farcinica CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the N. farcinica CRISPR-CLA assay is a rapid, economic and accurate method to diagnose N. farcinica infection.},
}
@article {pmid35717841,
year = {2022},
author = {Zhang, H and Yao, S and Sheng, R and Wang, J and Li, H and Fu, Y and Li, J and Zhang, X and Zhao, C},
title = {A cascade amplification strategy for ultrasensitive Salmonella typhimurium detection based on DNA walker coupling with CRISPR-Cas12a.},
journal = {Journal of colloid and interface science},
volume = {625},
number = {},
pages = {257-263},
doi = {10.1016/j.jcis.2022.06.027},
pmid = {35717841},
issn = {1095-7103},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA/genetics ; Limit of Detection ; *Salmonella typhimurium/genetics ; },
abstract = {Salmonella typhimurium (S. typhimurium) infection is one of leading causes of severe foodborne illness, which poses grievous threats to public health. Thus, the detection with ultra-sensitivity is highly demanded for timely prevention and diagnosis of S. typhimurium. In this study, we developed a novel detection machinery based on DNA walker and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas12a technologies. Mechanistically, the S. typhimurium specific sequence triggers Nt.AlwI nicking endonuclease and produces particular signaling nucleotide, which further activates Cas12a for strong fluorescence signal output. This cascade amplification strategy exhibits excellent specificity and successfully decreases the limit of detection (LOD) of DNA walker by 2,000 folds to 5 CFU/mL. Collectively, this combinatorial approach offers great promises to effectively reduce foodborne diseases by ultrasensitive detection of S. typhimurium. As a proof of concept, this innovative design also shows prominent potential in detections of other biomolecules, cells and pathogens.},
}
@article {pmid35717416,
year = {2022},
author = {Velimirovic, M and Zanetti, LC and Shen, MW and Fife, JD and Lin, L and Cha, M and Akinci, E and Barnum, D and Yu, T and Sherwood, RI},
title = {Peptide fusion improves prime editing efficiency.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3512},
pmid = {35717416},
issn = {2041-1723},
support = {R01 HG008754/HG/NHGRI NIH HHS/United States ; R21 HG010391/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Fusion ; Peptides/genetics ; },
abstract = {Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency.},
}
@article {pmid35716656,
year = {2022},
author = {Ali, S and Khan, N and Tang, Y},
title = {Epigenetic marks for mitigating abiotic stresses in plants.},
journal = {Journal of plant physiology},
volume = {275},
number = {},
pages = {153740},
doi = {10.1016/j.jplph.2022.153740},
pmid = {35716656},
issn = {1618-1328},
mesh = {Chromatin ; Epigenesis, Genetic ; *Gene Editing ; *Plants/genetics ; Stress, Physiological/genetics ; },
abstract = {Abiotic stressors are one of the major factors affecting agricultural output. Plants have evolved adaptive systems to respond appropriately to various environmental cues. These responses can be accomplished by modulating or fine-tuning genetic and epigenetic regulatory mechanisms. Understanding the response of plants' molecular features to abiotic stress is a priority in the current period of continued environmental changes. Epigenetic modifications are necessary that control gene expression by changing chromatin status and recruiting various transcription regulators. The present study summarized the current knowledge on epigenetic modifications concerning plant responses to various environmental stressors. The functional relevance of epigenetic marks in regulating stress tolerance has been revealed, and epigenetic changes impact the effector genes. This study looks at the epigenetic mechanisms that govern plant abiotic stress responses, especially DNA methylation, histone methylation/acetylation, chromatin remodeling, and various metabolites. Plant breeders will benefit from a thorough understanding of these processes to create alternative crop improvement approaches. Genome editing with clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins (CRISPR/Cas) provides genetic tools to make agricultural genetic engineering more sustainable and publicly acceptable.},
}
@article {pmid35716290,
year = {2022},
author = {Ferreira, P and Choupina, AB},
title = {CRISPR/Cas9 a simple, inexpensive and effective technique for gene editing.},
journal = {Molecular biology reports},
volume = {49},
number = {7},
pages = {7079-7086},
pmid = {35716290},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; *Gene Editing/methods ; Genetic Therapy ; Genome ; },
abstract = {In recent years, the number of tools and techniques that enable genetic material to be added, removed or altered at specific locations in the genome has increased significantly. The objective is to know the structure of genomes, the function of genes and improve gene therapy.In this work we intend to explain the functioning of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) and the advantages that this technique may have compared to previously developed techniques, such as RNA interference (RNAi), Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in gene and genome editing.We will start with the story of the discovery, then its biological function in the adaptive immune system of bacteria against bacteriophage attack, and ending with a description of the mechanism of action and its use in gene editing. We will also discuss other Cas enzymes with great potential for use in genome editing as an alternative to Cas9.CRISPR/Cas9 is a simple, inexpensive, and effective technique for gene editing with multiple applications from the development of functional genomics and epigenetics. This technique will, in the near future, have great applications in the development of cell models for use in medical and pharmaceutical processes, in targeted therapy, and improvement of agricultural and environmental species.},
}
@article {pmid35715750,
year = {2022},
author = {Vaghari-Tabari, M and Hassanpour, P and Sadeghsoltani, F and Malakoti, F and Alemi, F and Qujeq, D and Asemi, Z and Yousefi, B},
title = {CRISPR/Cas9 gene editing: a new approach for overcoming drug resistance in cancer.},
journal = {Cellular &amp; molecular biology letters},
volume = {27},
number = {1},
pages = {49},
pmid = {35715750},
issn = {1689-1392},
mesh = {CRISPR-Cas Systems/genetics ; Drug Resistance ; *Gene Editing/methods ; Humans ; *Neoplasms/drug therapy/genetics ; RNA ; },
abstract = {The CRISPR/Cas9 system is an RNA-based adaptive immune system in bacteria and archaea. Various studies have shown that it is possible to target a wide range of human genes and treat some human diseases, including cancers, by the CRISPR/Cas9 system. In fact, CRISPR/Cas9 gene editing is one of the most efficient genome manipulation techniques. Studies have shown that CRISPR/Cas9 technology, in addition to having the potential to be used as a new therapeutic approach in the treatment of cancers, can also be used to enhance the effectiveness of existing treatments. Undoubtedly, the issue of drug resistance is one of the main obstacles in the treatment of cancers. Cancer cells resist anticancer drugs by a variety of mechanisms, such as enhancing anticancer drugs efflux, enhancing DNA repair, enhancing stemness, and attenuating apoptosis. Mutations in some proteins of different cellular signaling pathways are associated with these events and drug resistance. Recent studies have shown that the CRISPR/Cas9 technique can be used to target important genes involved in these mechanisms, thereby increasing the effectiveness of anticancer drugs. In this review article, studies related to the applications of this technique in overcoming drug resistance in cancer cells will be reviewed. In addition, we will give a brief overview of the limitations of the CRISP/Cas9 gene-editing technique.},
}
@article {pmid35715648,
year = {2022},
author = {Cao, G and Xiong, Y and Nie, F and Chen, X and Peng, L and Li, Y and Yang, M and Huo, D and Hou, C},
title = {Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {12},
pages = {4695-4704},
pmid = {35715648},
issn = {1432-0614},
mesh = {*African Swine Fever/diagnosis ; *African Swine Fever Virus/genetics ; Animals ; CRISPR-Cas Systems ; *Nucleic Acids ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Swine ; },
abstract = {Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10[-4] ng·μL[-1] (4 copies·μL[-1]). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.},
}
@article {pmid35715136,
year = {2022},
author = {Taghdisi, SM and Ramezani, M and Alibolandi, M and Khademi, Z and Hajihasani, MM and Alinezhad Nameghi, M and Khakshour Abdolabadi, A and Rahimi, H and Abnous, K and Danesh, NM},
title = {A highly sensitive fluorescent aptasensor for detection of prostate specific antigen based on the integration of a DNA structure and CRISPR-Cas12a.},
journal = {Analytica chimica acta},
volume = {1219},
number = {},
pages = {340031},
doi = {10.1016/j.aca.2022.340031},
pmid = {35715136},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/genetics ; DNA, Cruciform ; Humans ; Male ; *Prostate-Specific Antigen ; },
abstract = {Herein, a facile fluorescent CRISPR-Cas12a-based sensing strategy is presented for prostate specific antigen (PSA), as a prostate cancer biomarker, with the assistance of a cruciform DNA nanostructure and PicoGreen (PG) as a fluorochrome. Highly sensitive recognition of PSA is one of the virtues of the proposed method which comes from the use of unique features of both CRISPR-Cas12a and DNA structure in the design of the aptasensor. The presence of PSA creates a cruciform DNA nanostructure in the sample which can be loaded by PG and make sharp fluorescence emission. While, when there is no PSA, the CRISPR-Cas12a digests sequences 1 and 3 as single-stranded DNAs, causing no DNA structure and a negligible fluorescence is detected after addition of PG. This aptasensor presents a sensitive recognition performance with detection limit of 4 pg/mL and a practical use for determination of PSA in serum samples. So, this analytical strategy introduces a convenient and highly sensitive approach for detection of disease biomarkers.},
}
@article {pmid35714703,
year = {2022},
author = {Valashedi, MR and Roushandeh, AM and Tomita, K and Kuwahara, Y and Pourmohammadi-Bejarpasi, Z and Kozani, PS and Sato, T and Roudkenar, MH},
title = {CRISPR/Cas9-mediated knockout of Lcn2 in human breast cancer cell line MDA-MB-231 ameliorates erastin-mediated ferroptosis and increases cisplatin vulnerability.},
journal = {Life sciences},
volume = {304},
number = {},
pages = {120704},
doi = {10.1016/j.lfs.2022.120704},
pmid = {35714703},
issn = {1879-0631},
mesh = {*Breast Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin/pharmacology ; Female ; *Ferroptosis/genetics ; Humans ; Lipocalin-2/genetics ; Piperazines ; Sincalide ; },
abstract = {AIMS: Lipocalin 2 (Lcn2) is an antioxidant-related protein upregulated in various cellular stress conditions, especially cancer. In this study, we abrogated Lcn2 expression in MDA-MB-231 breast cancer cells using the CRISPR/Cas9 technology and evaluated its effect on cellular proliferation, migration, and ferroptotic cell death.<br><br>MAIN METHODS: Validated human Lcn2 CRISPR/Cas9 knockout (KO) and homology-directed repair (HDR) plasmids were co-transfected into MDA-MB-231 breast cancer cells. Lcn2 gene knockout was confirmed at the transcriptional and protein levels using reverse transcription (RT)-PCR and enzyme-linked immunosorbent assay (ELISA). Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cytotoxicity assay was performed in the presence or absence of erastin, cisplatin (CDDP), and ferrostatin-1 using the CCK-8 method. Ferroptosis level was measured using the malondialdehyde assay lipid peroxidation kit. The migration capacity of the cells was also evaluated using the scratch assay.<br><br>KEY FINDINGS: Targeting Lcn2 using CRISPR/Cas9 reduced cellular proliferation and migration capability, and elevated the vulnerability of MDA-MB-231 cells to cisplatin. Furthermore, Lcn2 expression loss effectively promoted erastin-mediated ferroptosis in MDA-MB-231 cells.<br><br>SIGNIFICANCE: Inhibition of Lcn2 is a potentially useful strategy for sensitizing MDA-MB-231 tumor cells to ferroptotic cell death.},
}
@article {pmid35714601,
year = {2022},
author = {Smith, EM and Ferrell, S and Tokars, VL and Mondragón, A},
title = {Structures of an active type III-A CRISPR effector complex.},
journal = {Structure (London, England : 1993)},
volume = {30},
number = {8},
pages = {1109-1128.e6},
pmid = {35714601},
issn = {1878-4186},
support = {P30 CA060553/CA/NCI NIH HHS/United States ; R35 GM118108/GM/NIGMS NIH HHS/United States ; U24 GM129539/GM/NIGMS NIH HHS/United States ; U24 GM129541/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Triphosphate/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; RNA/metabolism ; Staphylococcus epidermidis/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.},
}
@article {pmid35714449,
year = {2022},
author = {Zheng, W and Zhong, Y and Yuan, L and Yu, X and Wang, X and Yang, C and Liu, H and Lv, P and Luo, Y and Qiu, B and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-82) carrying a homozygous c.1538G &gt; A (p.G513D) mutation in the OSMR gene by CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {63},
number = {},
pages = {102842},
doi = {10.1016/j.scr.2022.102842},
pmid = {35714449},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Homologous Recombination ; Homozygote ; *Human Embryonic Stem Cells/metabolism ; Humans ; Kruppel-Like Transcription Factors/genetics ; Mutation/genetics ; Oncostatin M Receptor beta Subunit/genetics/metabolism ; },
abstract = {Mutations in the tumor suppressor M receptor (OSMR) gene are associated with primary localized cutaneous amyloidosis (PLCA). Recently, we confirmed that OSMR loss-of-function mutations enhance epidermal keratinocyte differentiation via inactivation of the STAT5/KLF7 signaling. However, no disease model was available for PLCA. Accordingly, we generated an OSMR c.1538G > A mutant human embryonic stem cell line (SMUDHe010-A-82) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human skin organoid, which may provide a disease model for PLCA.},
}
@article {pmid35713718,
year = {2022},
author = {Rather, GA and Ayzenshtat, D and Teper-Bamnolker, P and Kumar, M and Forotan, Z and Eshel, D and Bocobza, S},
title = {Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.},
journal = {Planta},
volume = {256},
number = {1},
pages = {14},
pmid = {35713718},
issn = {1432-2048},
mesh = {CRISPR-Cas Systems/genetics ; DNA/metabolism ; *Gene Editing/methods ; Genome, Plant ; Kanamycin/metabolism ; Plant Breeding/methods ; Protoplasts/metabolism ; *Solanum tuberosum/genetics/metabolism ; Tetraploidy ; Transcription Factors/genetics ; Transfection ; },
abstract = {An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.},
}
@article {pmid35713402,
year = {2022},
author = {Liu, F and Kambakam, S and Almeida, MP and Ming, Z and Welker, JM and Wierson, WA and Schultz-Rogers, LE and Ekker, SC and Clark, KJ and Essner, JJ and McGrail, M},
title = {Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35713402},
issn = {2050-084X},
support = {R24 OD020166/OD/NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Integrases/genetics/metabolism ; *Zebrafish/genetics/metabolism ; },
abstract = {The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4[off] and rb1[off] integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4[on] and rb1[on] integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.},
}
@article {pmid35713220,
year = {2022},
author = {Yang, H and Wei, Y and Zhang, Q and Yang, Y and Bi, X and Yang, L and Xiao, N and Zang, A and Ren, L and Li, X},
title = {CRISPR/Cas9‑induced saturated mutagenesis identifies Rad51 haplotype as a marker of PARP inhibitor sensitivity in breast cancer.},
journal = {Molecular medicine reports},
volume = {26},
number = {2},
pages = {},
pmid = {35713220},
issn = {1791-3004},
mesh = {*Antineoplastic Agents/pharmacology ; *Breast Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Female ; Haplotypes ; Humans ; Mutagenesis ; Phthalazines/pharmacology/therapeutic use ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; *Rad51 Recombinase/genetics/metabolism ; },
abstract = {Breast cancer treatment with poly(ADP‑ribose)polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination repair (HRR) pathway. The chemical inhibition of many HRR deficiency genes may sensitize cancer cells to PARP inhibitors. In the present study, Rad51, a central player in the HRR pathway, was selected to explore additional low variation and highly representative markers for PARP inhibitor activity. A CRISPR/Cas9‑based saturated mutation approach for the Rad51 WALKER domain was used to evaluate the sensitivity of the PARP inhibitor olaparib. Five amino acid mutation sites were identified in olaparib‑resistant cells. Two Rad51 haplotypes were assembled from the mutations, and may represent useful pharmacogenomic markers of PARP inhibitor sensitivity.},
}
@article {pmid35712599,
year = {2022},
author = {Mattiello, L and Rütgers, M and Sua-Rojas, MF and Tavares, R and Soares, JS and Begcy, K and Menossi, M},
title = {Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {868027},
pmid = {35712599},
issn = {1664-462X},
abstract = {The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The improvement of CRISPR-based technologies efficiency will enable the implementation of genome editing tools in a variety of crop plants, as well as accelerate progress in basic research and molecular breeding.},
}
@article {pmid35712010,
year = {2022},
author = {Novarina, D and Koutsoumpa, A and Milias-Argeitis, A},
title = {A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101358},
pmid = {35712010},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Saccharomyces cerevisiae/genetics ; *Saccharomycetales/genetics ; },
abstract = {CRISPR/Cas9 technology allows accurate, marker-less genome editing. We report a detailed, robust, and streamlined protocol for CRISPR/Cas9 genome editing in Saccharomyces cerevisiae, based on the widely used MoClo-Yeast Toolkit (https://www.addgene.org/kits/moclo-ytk/). This step-by-step protocol guides the reader from sgRNA design to verification of the desired genome editing event and provides preassembled plasmids for cloning the sgRNA(s), making this technology easily accessible to any yeast research group. For complete details on the use and execution of this protocol, please refer to Novarina et al. (2021).},
}
@article {pmid35711363,
year = {2022},
author = {Wang, Q and Park, KH and Geng, B and Chen, P and Yang, C and Jiang, Q and Yi, F and Tan, T and Zhou, X and Bian, Z and Ma, J and Zhu, H},
title = {MG53 Inhibits Necroptosis Through Ubiquitination-Dependent RIPK1 Degradation for Cardiac Protection Following Ischemia/Reperfusion Injury.},
journal = {Frontiers in cardiovascular medicine},
volume = {9},
number = {},
pages = {868632},
pmid = {35711363},
issn = {2297-055X},
abstract = {RATIONALE: While reactive oxygen species (ROS) has been recognized as one of the main causes of cardiac injury following myocardial infarction, the clinical application of antioxidants has shown limited effects on protecting hearts against ischemia-reperfusion (I/R) injury. Thus, the precise role of ROS following cardiac injury remains to be fully elucidated.<br><br>OBJECTIVE: We investigated the role of mitsugumin 53 (MG53) in regulating necroptosis following I/R injury to the hearts and the involvement of ROS in MG53-mediated cardioprotection.<br><br>METHODS AND RESULTS: Antioxidants were used to test the role of ROS in MG53-mediated cardioprotection in the mouse model of I/R injury and induced human pluripotent stem cells (hiPSCs)-derived cardiomyocytes subjected to hypoxia or re-oxygenation (H/R) injury. Western blotting and co-immunoprecipitation were used to identify potential cell death pathways that MG53 was involved in. CRISPR/Cas 9-mediated genome editing and mutagenesis assays were performed to further identify specific interaction amino acids between MG53 and its ubiquitin E3 ligase substrate. We found that MG53 could protect myocardial injury via inhibiting the necroptosis pathway. Upon injury, the generation of ROS in the infarct zone of the hearts promoted interaction between MG53 and receptor-interacting protein kinase 1 (RIPK1). As an E3 ubiquitin ligase, MG53 added multiple ubiquitin chains to RIPK1 at the sites of K316, K604, and K627 for proteasome-mediated RIPK1 degradation and inhibited necroptosis. The application of N-acetyl cysteine (NAC) disrupted the interaction between MG53 and RIPK1 and abolished MG53-mediated cardioprotective effects.<br><br>CONCLUSIONS: Taken together, this study provided a molecular mechanism of a potential beneficial role of ROS following acute myocardial infarction. Thus, fine-tuning ROS levels might be critical for cardioprotection.},
}
@article {pmid35711292,
year = {2022},
author = {Bhattacharjee, R and Nandi, A and Mitra, P and Saha, K and Patel, P and Jha, E and Panda, PK and Singh, SK and Dutt, A and Mishra, YK and Verma, SK and Suar, M},
title = {Theragnostic application of nanoparticle and CRISPR against food-borne multi-drug resistant pathogens.},
journal = {Materials today. Bio},
volume = {15},
number = {},
pages = {100291},
pmid = {35711292},
issn = {2590-0064},
abstract = {Foodborne infection is one of the leading sources of infections spreading across the world. Foodborne pathogens are recognized as multidrug-resistant (MDR) pathogens posing a significant problem in the food industry and healthy consumers resulting in enhanced economic burden, and nosocomial infections. The continued search for enhanced microbial detection tools has piqued the interest of the CRISPR-Cas system and Nanoparticles. CRISPR-Cas system is present in the bacterial genome of some prokaryotes and is repurposed as a theragnostic tool against MDR pathogens. Nanoparticles and composites have also emerged as an efficient tool in theragnostic applications against MDR pathogens. The diagnostic limitations of the CRISPR-Cas system are believed to be overcome by a synergistic combination of the nanoparticles system and CRISPR-Cas using nanoparticles as vehicles. In this review, we have discussed the diagnostic application of CRISPR-Cas technologies along with their potential usage in applications like phage resistance, phage vaccination, strain typing, genome editing, and antimicrobial. we have also elucidated the antimicrobial and detection role of nanoparticles against foodborne MDR pathogens. Moreover, the novel combinatorial approach of CRISPR-Cas and nanoparticles for their synergistic effects in pathogen clearance and drug delivery vehicles has also been discussed.},
}
@article {pmid35711141,
year = {2022},
author = {Tipanee, J and Samara-Kuko, E and Gevaert, T and Chuah, MK and VandenDriessche, T},
title = {Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {10},
pages = {3155-3175},
pmid = {35711141},
issn = {1525-0024},
mesh = {Animals ; Antigens, CD19 ; CRISPR-Cas Systems ; *Graft vs Host Disease/metabolism ; *Hematopoietic Stem Cell Transplantation ; Immunotherapy ; Immunotherapy, Adoptive ; Mice ; *Neoplasms/genetics/therapy ; Receptors, Antigen, T-Cell/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes ; },
abstract = {Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an "off-the-shelf" therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR-Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared with the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR-Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.},
}
@article {pmid35710827,
year = {2022},
author = {Ryu, J and Statz, JP and Chan, W and Burch, FC and Brigande, JV and Kempton, B and Porsov, EV and Renner, L and McGill, T and Burwitz, BJ and Hanna, CB and Neuringer, M and Hennebold, JD},
title = {CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher syndrome type 1B.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {10036},
pmid = {35710827},
issn = {2045-2322},
support = {P51 OD011092/OD/NIH HHS/United States ; R21 DC018126/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Endonucleases/genetics ; Gene Editing ; Humans ; Macaca mulatta/genetics/metabolism ; RNA, Guide/metabolism ; RNA, Messenger ; *Usher Syndromes/genetics ; },
abstract = {Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.},
}
@article {pmid35708998,
year = {2022},
author = {Gemperle, J and Harrison, TS and Flett, C and Adamson, AD and Caswell, PT},
title = {On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35708998},
issn = {2050-084X},
support = {203128/A/16/Z/WT_/Wellcome Trust/United Kingdom ; MR/R009376/1/MRC_/Medical Research Council/United Kingdom ; C147/A25254/CRUK_/Cancer Research UK/United Kingdom ; DCRPGF\100002/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Doxycycline ; Indoleacetic Acids ; Methacrylates ; *rab GTP-Binding Proteins/genetics/metabolism ; },
abstract = {CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.},
}
@article {pmid35708748,
year = {2022},
author = {Xiong, Y and Cao, G and Chen, X and Yang, J and Shi, M and Wang, Y and Nie, F and Huo, D and Hou, C},
title = {One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {12},
pages = {4607-4616},
pmid = {35708748},
issn = {1432-0614},
mesh = {*African Swine Fever Virus/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Nucleotidyltransferases ; Real-Time Polymerase Chain Reaction ; *Recombinases/genetics ; Sensitivity and Specificity ; Swine ; },
abstract = {The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10[-6] ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10[-5] ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. KEY POINTS: • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.},
}
@article {pmid35708734,
year = {2022},
author = {Wilson, J and Loizou, JI},
title = {Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR-based screens.},
journal = {Molecular oncology},
volume = {16},
number = {21},
pages = {3778-3791},
pmid = {35708734},
issn = {1878-0261},
support = {ERC Synergy Grant DDREAMM 855741/ERC_/European Research Council/International ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Mutation ; Genetic Testing ; DNA Damage ; },
abstract = {The concepts of synthetic lethality and viability have emerged as powerful approaches to identify vulnerabilities and resistances within the DNA damage response for the treatment of cancer. Historically, interactions between two genes have had a longstanding presence in genetics and have been identified through forward genetic screens that rely on the molecular basis of the characterized phenotypes, typically caused by mutations in single genes. While such complex genetic interactions between genes have been studied extensively in model organisms, they have only recently been prioritized as therapeutic strategies due to technological advancements in genetic screens. Here, we discuss synthetic lethal and viable interactions within the DNA damage response and present how CRISPR-based genetic screens and chemical compounds have allowed for the systematic identification and targeting of such interactions for the treatment of cancer.},
}
@article {pmid35708612,
year = {2022},
author = {Bean, BDM and Whiteway, M and Martin, VJJ},
title = {The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {8},
pages = {},
pmid = {35708612},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Green Fluorescent Proteins/genetics ; RNA, Guide/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9)-based genome editing tools, we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with 3 components: (1) a set of optimized Streptococcus pyogenes Cas9-guide RNA expression vectors with 5 selectable markers and the option to either preclone or cotransform the gRNAs; (2) vectors for the one-step construction of integration cassettes expressing an untagged or green fluorescent protein/red fluorescent protein/hemagglutinin-tagged gene of interest at one of 3 levels, supporting localization and overexpression studies; and (3) integration cassettes containing moderately expressed green fluorescent protein- or red fluorescent protein-tagged compartmental markers for colocalization experiments. These components allow rapid, high-efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to colabel all target subcellular compartments with green fluorescent protein and red fluorescent protein. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle that allows the stable genomic integration of DNA with the ease of use approaching that of transforming plasmids.},
}
@article {pmid35708478,
year = {2022},
author = {Zeng, R and Xu, J and Lu, L and Lin, Q and Huang, X and Huang, L and Li, M and Tang, D},
title = {Photoelectrochemical bioanalysis of microRNA on yolk-in-shell Au@CdS based on the catalytic hairpin assembly-mediated CRISPR-Cas12a system.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {54},
pages = {7562-7565},
doi = {10.1039/d2cc02821b},
pmid = {35708478},
issn = {1364-548X},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Catalysis ; DNA, Single-Stranded ; Electrochemical Techniques ; *MicroRNAs/analysis ; },
abstract = {This work reports on the proof-of-concept of a photoelectrochemical (PEC) biosensor with a horseradish peroxidase-single stranded DNA-encoded magnetic bead (MB-ssDNA-HRP) signal probe cleaved by the catalytic hairpin assembly (CHA)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for the quantification of microRNA (miR-21) by using yolk-in-shell Au@CdS as a photoactive material.},
}
@article {pmid35708146,
year = {2022},
author = {Segurado, OG and Jiang, R and Pipe, SW},
title = {Challenges and opportunities when transitioning from in vivo gene replacement to in vivo CRISPR/Cas9 therapies - a spotlight on hemophilia.},
journal = {Expert opinion on biological therapy},
volume = {22},
number = {9},
pages = {1091-1098},
doi = {10.1080/14712598.2022.2090241},
pmid = {35708146},
issn = {1744-7682},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; *Hemophilia A/genetics/therapy ; Humans ; },
abstract = {INTRODUCTION: Currently, a few in vivo gene replacement therapies are commercially available, with many in clinical development for the treatment of some inherited monogenic diseases. These disorders arise from mutations in genes encoding essential proteins with a well understood biological function. Wide adoption of gene replacement therapies requires solid safety and efficacy profiles with demonstrable long-term durability and cost-benefit advantages vs standard therapies.<br><br>AREAS COVERED: This expert review outlines the challenges and opportunities in treating hemophilia, including the progression from in vivo gene therapies toward in vivo gene editing, focusing on pre-clinical and emerging clinical data for gene editing and addressing the need for sustained and durable gene expression during hepatocyte proliferation when the liver is unable to maintain steady gene expression and protein production.<br><br>EXPERT OPINION: In vivo gene editing in liver tissues may be able to rescue patients younger than 18&#xa0;years who are not eligible for gene replacement therapies, with hemophilia as a prime example.},
}
@article {pmid35707386,
year = {2022},
author = {Zhang, YY and Li, SQ and Song, Y and Wang, P and Song, XG and Zhu, WF and Wang, DM},
title = {Silencing the ADAM9 Gene through CRISPR/Cas9 Protects Mice from Alcohol-Induced Acute Liver Injury.},
journal = {BioMed research international},
volume = {2022},
number = {},
pages = {5110161},
pmid = {35707386},
issn = {2314-6141},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Gene Editing ; Liver ; Mice ; *RNA, Guide/genetics ; },
abstract = {Alcoholic liver injury is a major global public health concern at present. The ADAM9 gene plays a crucial role in the occurrence and development of various liver diseases, but its role in acute alcoholic liver injury remains ambiguous. In this study, a chimeric single-guide RNA targeting the genomic regions of mouse ADAM9 was designed using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. Next, the role of ADAM9 in acute alcoholic liver injury in vitro in cultured mouse cells and in vivo in a hydrodynamic injection-based alcoholic liver injury mouse model was documented. The findings of this study suggest that ADAM9 induces by regulating cell proliferation, apoptosis, and stress metabolism in mice. Thus, inhibiting the expression of ADAM9 gene using CRISPR/Cas9 can attenuate alcohol-induced acute liver injury in mice.},
}
@article {pmid35705772,
year = {2022},
author = {Wang, Y and Huang, C and Zhao, W},
title = {Recent advances of the biological and biomedical applications of CRISPR/Cas systems.},
journal = {Molecular biology reports},
volume = {49},
number = {7},
pages = {7087-7100},
pmid = {35705772},
issn = {1573-4978},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Escherichia coli/genetics ; Genome ; Mammals ; Mice ; Plants/genetics ; *Zebrafish/genetics ; },
abstract = {The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas) system, referred to as CRISPR/Cas system, has attracted significant interest in scientific community due to its great potential in translating into versatile therapeutic tools in biomedical field. For instance, a myriad of studies has demonstrated that the CRISPR/Cas system is capable of detecting various types of viruses, killing antibiotic-resistant bacteria, treating inherited genetic diseases, and providing new strategies for cancer therapy. Furthermore, CRISPR/Cas systems are also exploited as research tools such as genome engineering tool that allows researchers to interrogate the biological roles of unexplored genes or uncover novel functions of known genes. Additionally, the CRISPR/Cas system has been employed to edit the genome of a wide range of eukaryotic, prokaryotic organisms and experimental models, including but not limited to mammalian cells, mice, zebrafish, plants, yeast, and Escherichia coli. The present review mainly focuses on summarizing recent discoveries regarding the type II CRISPR/Cas9 and type VI CRISPR/Cas13a systems to give researchers a glimpse of their potential applications in the biological and biomedical field.},
}
@article {pmid35705709,
year = {2022},
author = {},
title = {Caribou's first CRISPR CAR-T impresses.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {807},
doi = {10.1038/s41587-022-01371-6},
pmid = {35705709},
issn = {1546-1696},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Immunotherapy, Adoptive ; Receptors, Antigen, T-Cell/genetics ; *Receptors, Chimeric Antigen/metabolism ; *Reindeer/metabolism ; },
}
@article {pmid35705705,
year = {2022},
author = {},
title = {CRISPR technology.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {832},
doi = {10.1038/s41587-022-01359-2},
pmid = {35705705},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Technology ; },
}
@article {pmid35704130,
year = {2022},
author = {Cao, T and Liu, S and Qiu, Y and Gao, M and Wu, J and Wu, G and Liang, P and Huang, J},
title = {Generation of C-to-G transversion in mouse embryos via CG editors.},
journal = {Transgenic research},
volume = {31},
number = {4-5},
pages = {445-455},
pmid = {35704130},
issn = {1573-9368},
mesh = {Animals ; CRISPR-Cas Systems ; Cytosine ; *Gene Editing/methods ; Mice ; *RNA, Guide/genetics ; },
abstract = {Base editors (BEs) are efficient and precise tools for generating single base conversions in living organisms. While most BE systems are limited in mediating C-to-T or A-to-G conversions, recently developed C-to-G base editors (CGBEs) could produce C-to-G transversions. CGBEs convert cytosine within the editing window to abasic intermediates, which would be replaced with any base after base excision repair (BER). By far, though the efficiency and editing scope of CGBEs have been investigated in cultured cells via gRNA library and machine-learning, the viability of CGBEs in generating mouse models has not been adequately tested. In this study, we tested the C-to-G transversion efficiency of the CGBE1 and CGBE-XRCC1 systems in mouse embryos. Our results showed that both of the CGBE systems were able to mediate C-to-G transversion on 2 out of 3 targets tested, with up to 20% frequency within the editing window. Notably, most of the groups showed over 40% of other base conversions, predominantly C-to-T. Lastly, we successfully acquired the F1 mouse carrying a disease-causing mutation. In all, our study suggested that CGBEs systems held great potential in generating mouse models and indicated that XRCC1 based system is applicable in mouse embryos.},
}
@article {pmid35704073,
year = {2022},
author = {Dash, HR and Arora, M},
title = {CRISPR-CasB technology in forensic DNA analysis: challenges and solutions.},
journal = {Applied microbiology and biotechnology},
volume = {106},
number = {12},
pages = {4367-4374},
pmid = {35704073},
issn = {1432-0614},
mesh = {*Biotechnology ; CRISPR-Cas Systems ; DNA, Mitochondrial ; Gene Editing/methods ; Genetic Markers/genetics ; *Genome ; Sequence Analysis, DNA ; },
abstract = {CRISPR-Cas technology has revolutionized the field of biotechnology with its precise therapeutic use from genetic as well as infectious diseases point of view. This technology is rapidly evolving to single tool enabling site-directed cut in the genome and highly specific activation or inhibition of gene expression or the exchange of single bases. Besides clinical applications, CRISPR-Cas technology has also shown promising use in the field of forensic DNA analysis. Enrichment of targeted genetic marker for identification followed by sequencing and non-PCR-dependent technique ensures the use of CRISPR-Cas technology in challenging forensic biological samples. The use of this advanced technology is also deemed helpful in mixed profile attribution, mostly in LCN contributors and the generation of a useful DNA profile in degraded samples. Besides its useful applications in forensic DNA analysis, CRISPR-Cas technology poses a huge threat from the generation of ghost DNA profiles by modification/alteration of target genetic markers. Forensic DNA analysts should carry out analysis of additional markers such as non-CODIS markers, Y-, X-chromosome markers, and mitochondrial DNA sequencing in a suspected ghost DNA profile case. KEY POINTS: • CRISPR-Cas9 technique is useful in analyzing LCN, mixed and degraded samples • Alteration of DNA using this technique can lead to generation of ghost DNA profiles • Alternative genetic markers and methylation pattern may detect a ghost DNA profile.},
}
@article {pmid35703314,
year = {2022},
author = {Inwood, SL and Tian, L and Parratt, K and Maragh, S and Wang, L},
title = {Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing.},
journal = {BioTechniques},
volume = {72},
number = {6},
pages = {279-286},
doi = {10.2144/btn-2022-0015},
pmid = {35703314},
issn = {1940-9818},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Flow Cytometry ; *Gene Editing/methods ; },
abstract = {Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective CRISPR/Cas9-engineered therapies.},
}
@article {pmid35703039,
year = {2022},
author = {Kurushima, J and Tomita, H},
title = {Advances of genetic engineering in streptococci and enterococci.},
journal = {Microbiology and immunology},
volume = {66},
number = {9},
pages = {411-417},
doi = {10.1111/1348-0421.13015},
pmid = {35703039},
issn = {1348-0421},
mesh = {CRISPR-Cas Systems ; *Enterococcus/genetics ; *Gene Editing/methods ; Genetic Engineering/methods ; Streptococcus/genetics ; },
abstract = {In the post-genome era, reverse genetic engineering is an indispensable methodology for experimental molecular biology to provide a deeper understanding of the principal relationship between genomic features and biological phenotypes. Technically, genetic engineering is carried out through allele replacement of a target genomic locus with a designed nucleotide sequence, so called site-directed mutagenesis. To artificially manipulate allele replacement through homologous recombination, researchers have improved various methodologies that are optimized to the bacterial species of interest. Here, we review widely used genetic engineering technologies, particularly for streptococci and enterococci, and recent advances that enable more effective and flexible manipulation. The development of genetic engineering has been promoted by synthetic biology approaches based on basic biological knowledge of horizontal gene transfer systems, such as natural conjugative transfer, natural transformation, and the CRISPR/Cas system. Therefore, this review also describes basic insights into molecular biology that underlie improvements in genetic engineering technology.},
}
@article {pmid35701838,
year = {2022},
author = {Liu, Z and Yan, Q and Jiang, C and Li, J and Jian, H and Fan, L and Zhang, R and Xiao, X and Meng, D and Liu, X and Wang, J and Yin, H},
title = {Growth rate determines prokaryote-provirus network modulated by temperature and host genetic traits.},
journal = {Microbiome},
volume = {10},
number = {1},
pages = {92},
pmid = {35701838},
issn = {2049-2618},
mesh = {Ecosystem ; Prokaryotic Cells ; *Proviruses/genetics ; Temperature ; *Viruses/genetics ; },
abstract = {BACKGROUND: Prokaryote-virus interactions play key roles in driving biogeochemical cycles. However, little is known about the drivers shaping their interaction network structures, especially from the host features. Here, we compiled 7656 species-level genomes in 39 prokaryotic phyla across environments globally and explored how their interaction specialization is constrained by host life history traits, such as growth rate.<br><br>RESULTS: We first reported that host growth rate indicated by the reverse of minimal doubling time was negatively related to interaction specialization for host in host-provirus network across various ecosystems and taxonomy groups. Such a negative linear growth rate-specialization relationship (GrSR) was dependent on host optimal growth temperature (OGT), and stronger toward the two gradient ends of OGT. For instance, prokaryotic species with an OGT &#x2265; 40 &#xb0;C showed a stronger GrSR (Pearson's r = -0.525, P < 0.001). Significant GrSRs were observed with the presences of host genes in promoting the infection cycle at stages of adsorption, establishment, and viral release, but nonsignificant with the presence of immune systems, such as restriction-modification systems and CRISPR-Cas systems. Moreover, GrSR strength was increased with the presence of temperature-dependent lytic switches, which was also confirmed by mathematical modeling.<br><br>CONCLUSIONS: Together, our results advance our understanding of the interactions between prokaryotes and proviruses and highlight the importance of host growth rate in interaction specialization during lysogenization. Video Abstract.},
}
@article {pmid35701478,
year = {2022},
author = {Rosello, M and Serafini, M and Mignani, L and Finazzi, D and Giovannangeli, C and Mione, MC and Concordet, JP and Del Bene, F},
title = {Disease modeling by efficient genome editing using a near PAM-less base editor in vivo.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3435},
pmid = {35701478},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome/genetics ; Zebrafish/genetics/metabolism ; },
abstract = {Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.},
}
@article {pmid35701417,
year = {2022},
author = {Teufel, M and Klein, CA and Mager, M and Sobetzko, P},
title = {A multifunctional system for genome editing and large-scale interspecies gene transfer.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3430},
pmid = {35701417},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Escherichia coli/genetics ; *Gene Editing ; Genetic Therapy ; Genome, Bacterial/genetics ; },
abstract = {CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. We demonstrate the excision, transfer and integration of large chromosomal regions between E. coli, V. natriegens and D. dadantii without size-limiting intermediate DNA extraction. CRISPR SWAPnDROP also provides common genome editing approaches comprising scarless, marker-free, iterative and parallel insertions and deletions. The modular character facilitates DNA library applications, and recycling of standardized parts. Its multi-color scarless co-selection system significantly improves editing efficiency and provides visual quality controls throughout the assembly and editing process.},
}
@article {pmid35701408,
year = {2022},
author = {Kosicki, M and Allen, F and Steward, F and Tomberg, K and Pan, Y and Bradley, A},
title = {Cas9-induced large deletions and small indels are controlled in a convergent fashion.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3422},
pmid = {35701408},
issn = {2041-1723},
support = {098051/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA Repair/genetics ; INDEL Mutation ; Mice ; },
abstract = {Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.},
}
@article {pmid35701400,
year = {2022},
author = {Liang, F and Zhang, Y and Li, L and Yang, Y and Fei, JF and Liu, Y and Qin, W},
title = {SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3421},
pmid = {35701400},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing ; Zebrafish/genetics/metabolism ; },
abstract = {Precise genetic modifications in model organisms are essential for biomedical research. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here we first optimize SpG and SpRY systems in zebrafish by purifying protein combined with synthetically modified gRNA. SpG shows high editing efficiency at NGN PAM sites, whereas SpRY efficiently edit PAM-less sites in the zebrafish genome. Then, we generate the SpRY-mediated cytosine base editor SpRY-CBE4max and SpRY-mediated adenine base editor zSpRY-ABE8e. Both target relaxed PAM with up to 96% editing efficiency and high product purity. With these tools, some previously inaccessible disease-relevant genetic variants are generated in zebrafish, supporting the utility of high-resolution targeting across genome-editing applications. Our study significantly improves CRISPR-Cas targeting in the genomic landscape of zebrafish, promoting the application of this model organism in revealing gene function, physiological mechanisms, and disease pathogenesis.},
}
@article {pmid35700604,
year = {2022},
author = {Kang, W and Liu, L and Yu, P and Zhang, T and Lei, C and Nie, Z},
title = {A switchable Cas12a enabling CRISPR-based direct histone deacetylase activity detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {213},
number = {},
pages = {114468},
doi = {10.1016/j.bios.2022.114468},
pmid = {35700604},
issn = {1873-4235},
mesh = {Acetylation ; *Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Histone Deacetylases/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {The efficient and robust signal reporting ability of CRISPR-Cas system exhibits huge value in biosensing, but its applicability for non-nucleic acid analyte detection relies on the coupling of additional recognition modules. To address this limitation, we described a switchable Cas12a and exploited it for CRISPR-based direct analysis of histone deacetylase (HDAC) activity. Starting from the acetylation-mediated inactivation of Cas12a by anti-CRISPR protein AcrVA5, we demonstrated that the acetyl-inactivated Cas12a could be reversibly activated by HDAC-mediated deacetylation based on computational simulations (e.g., deep learning and protein-protein docking analysis) and experimental verifications. By leveraging this switchable Cas12a for both target sensing and signal amplification, we established a sensitive one-pot assay capable of detecting deacetylase sirtuin-1 with sub-nanomolar sensitivity, which is 50 times lower than the standard two-step peptide-based assay. The versability of this assay was validated by the sensitive assessment of cellular HDAC activities in different cell lines with good accuracy, making it a valuable tool for biochemical studies and clinical diagnostics.},
}
@article {pmid35700146,
year = {2022},
author = {Liu, XH and Li, BR and Ying, ZM and Tang, LJ and Wang, F and Jiang, JH},
title = {Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation.},
journal = {ACS chemical biology},
volume = {17},
number = {7},
pages = {1769-1777},
doi = {10.1021/acschembio.2c00101},
pmid = {35700146},
issn = {1554-8937},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation ; *RNA, Guide/genetics ; S-Adenosylmethionine ; Transcriptional Activation ; },
abstract = {Inducible CRISPR-dCas9 transcription system has become a powerful tool for transcription regulation and sensing. Here, we develop a new concept of small-molecule-mediated split-aptamer assembly for inducible CRISPR-dCas9 transcription activation, allowing quantitative detection and imaging of S-adenosyl methionine (SAM) in live cells. This inducible transcription system is designed by integrating one fragment of a split SAM aptamer to guide RNA (gRNA) and the other to MS2 arrays. SAM-mediated reassembly of the split fragments recruits an MCP-fused transcription activator to the gRNA-dCas9 complex, activating the expression of a near-infrared fluorescent protein for imaging. We demonstrate that this inducible transcription system achieves quantitative detection of SAM with high sensitivity in live cells. Our system shows that methionine adenosyltransferase 1A (MAT1A) and MAT2A can both catalyze SAM production in live cells and the SAM levels in cancer cells can be increased via upregulation of MAT1A mRNA by epigenetic inhibitors. This split-aptamer assembly strategy could afford a new approach for controlling the CRISPR-dCas9 system, enabling conditional transcription regulation in response to endogenous metabolites in live cells.},
}
@article {pmid35699358,
year = {2022},
author = {Takebayashi, K and Wittayarat, M and Lin, Q and Hirata, M and Yoshimura, N and Torigoe, N and Nagahara, M and Do, LTK and Tanihara, F and Otoi, T},
title = {Gene editing in porcine embryos using a combination of electroporation and transfection methods.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {57},
number = {10},
pages = {1136-1142},
doi = {10.1111/rda.14184},
pmid = {35699358},
issn = {1439-0531},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Electroporation/methods/veterinary ; *Gene Editing/methods/veterinary ; Swine ; Transfection/veterinary ; Zygote ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.},
}
@article {pmid35699339,
year = {2022},
author = {Ahator, SD and Sagar, S and Zhu, M and Wang, J and Zhang, LH},
title = {Nutrient Availability and Phage Exposure Alter the Quorum-Sensing and CRISPR-Cas-Controlled Population Dynamics of Pseudomonas aeruginosa.},
journal = {mSystems},
volume = {7},
number = {4},
pages = {e0009222},
pmid = {35699339},
issn = {2379-5077},
abstract = {Quorum sensing (QS) coordinates bacterial communication and cooperation essential for virulence and dominance in polymicrobial settings. QS also regulates the CRISPR-Cas system for targeted defense against parasitic genomes from phages and horizontal gene transfer. Although the QS and CRISPR-Cas systems are vital for bacterial survival, they undergo frequent selection in response to biotic and abiotic factors. Using the opportunistic Pseudomonas aeruginosa with well-established QS and CRISPR-Cas systems, we show how the social interactions between the acyl-homoserine lactone (AHL)-QS signal-blind mutants (ΔlasRrhlR) and the CRISPR-Cas mutants are affected by phage exposure and nutrient availability. We demonstrate that media conditions and phage exposure alter the resistance and relative fitness of ΔlasRrhlR and CRISPR-Cas mutants while tipping the fitness advantage in favor of the QS signal-blind mutants under nutrient-limiting conditions. We also show that the AHL signal-blind mutants are less selected by phages under QS-inducing conditions than the CRISPR-Cas mutants, whereas the mixed population of the CRISPR-Cas and AHL signal-blind mutants reduce phage infectivity, which can improve survival during phage exposure. Our data reveal that phage exposure and nutrient availability reshape the population dynamics between the ΔlasRrhlR QS mutants and CRISPR-Cas mutants, with key indications for cooperation and conflict between the strains. IMPORTANCE The increase in antimicrobial resistance has created the need for alternative interventions such as phage therapy. However, as previously observed with antimicrobial resistance, phage therapy will not be effective if bacteria evolve resistance and persist in the presence of the phages. The QS is commonly known as an arsenal for bacteria communication, virulence, and regulation of the phage defense mechanism, the CRISPR-Cas system. The QS and CRISPR-Cas systems are widespread in bacteria. However, they are known to evolve rapidly under the influence of biotic and abiotic factors in the bacterial environment, resulting in alteration in bacterial genotypes, which enhance phage resistance and fitness. We believe that adequate knowledge of the influence of environmental factors on the bacterial community lifestyle and phage defense mechanisms driven by the QS and CRISPR-Cas system is necessary for developing effective phage therapy.},
}
@article {pmid35698891,
year = {2022},
author = {Uranga, M and Daròs, JA},
title = {Tools and targets: The dual role of plant viruses in CRISPR-Cas genome editing.},
journal = {The plant genome},
volume = {},
number = {},
pages = {e20220},
doi = {10.1002/tpg2.20220},
pmid = {35698891},
issn = {1940-3372},
abstract = {The recent emergence of tools based on the clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have revolutionized targeted genome editing, thus holding great promise to both basic plant science and precision crop breeding. Conventional approaches for the delivery of editing components rely on transformation technologies or transient delivery to protoplasts, both of which are time-consuming, laborious, and can raise legal concerns. Alternatively, plant RNA viruses can be used as transient delivery vectors of CRISPR-Cas reaction components, following the so-called virus-induced genome editing (VIGE). During the last years, researchers have been able to engineer viral vectors for the delivery of CRISPR guide RNAs and Cas nucleases. Considering that each viral vector is limited to its molecular biology properties and a specific host range, here we review recent advances for improving the VIGE toolbox with a special focus on strategies to achieve tissue-culture-free editing in plants. We also explore the utility of CRISPR-Cas technology to enhance biotic resistance with a special focus on plant virus diseases. This can be achieved by either targeting the viral genome or modifying essential host susceptibility genes that mediate in the infection process. Finally, we discuss the challenges and potential that VIGE holds in future breeding technologies.},
}
@article {pmid35697825,
year = {2022},
author = {Presnell, JS and Bubel, M and Knowles, T and Patry, W and Browne, WE},
title = {Multigenerational laboratory culture of pelagic ctenophores and CRISPR-Cas9 genome editing in the lobate Mnemiopsis leidyi.},
journal = {Nature protocols},
volume = {17},
number = {8},
pages = {1868-1900},
pmid = {35697825},
issn = {1750-2799},
mesh = {Animals ; Biological Evolution ; CRISPR-Cas Systems/genetics ; *Ctenophora/genetics ; Gene Editing ; Genome ; },
abstract = {Despite long-standing experimental interest in ctenophores due to their unique biology, ecological influence and evolutionary status, previous work has largely been constrained by the periodic seasonal availability of wild-caught animals and difficulty in reliably closing the life cycle. To address this problem, we have developed straightforward protocols that can be easily implemented to establish long-term multigenerational cultures for biological experimentation in the laboratory. In this protocol, we describe the continuous culture of the Atlantic lobate ctenophore Mnemiopsis leidyi. A rapid 3-week egg-to-egg generation time makes Mnemiopsis suitable for a wide range of experimental genetic, cellular, embryological, physiological, developmental, ecological and evolutionary studies. We provide recommendations for general husbandry to close the life cycle of Mnemiopsis in the laboratory, including feeding requirements, light-induced spawning, collection of embryos and rearing of juveniles to adults. These protocols have been successfully applied to maintain long-term multigenerational cultures of several species of pelagic ctenophores, and can be utilized by laboratories lacking easy access to the ocean. We also provide protocols for targeted genome editing via microinjection with CRISPR-Cas9 that can be completed within ~2 weeks, including single-guide RNA synthesis, early embryo microinjection, phenotype assessment and sequence validation of genome edits. These protocols provide a foundation for using Mnemiopsis as a model organism for functional genomic analyses in ctenophores.},
}
@article {pmid35697344,
year = {2022},
author = {Liu, Y and Chen, Y and Zhang, Y and Zhong, Q and Zhu, X and Wu, Q},
title = {A functionalized magnetic nanoparticle regulated CRISPR-Cas12a sensor for the ultrasensitive detection of alpha-fetoprotein.},
journal = {The Analyst},
volume = {147},
number = {14},
pages = {3186-3192},
doi = {10.1039/d2an00697a},
pmid = {35697344},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; Gold/chemistry ; *Hepatoblastoma/diagnosis/genetics ; Humans ; Limit of Detection ; *Liver Neoplasms/diagnosis ; *Magnetite Nanoparticles/chemistry ; *Nucleic Acids ; alpha-Fetoproteins/analysis ; },
abstract = {Alpha-fetoprotein (AFP) is an important clinical tumor marker of hepatoblastoma, and the concentration of AFP in serum is closely related to the staging of hepatoblastoma. We report a magnetic bead separation platform based on a switching aptamer triggered hybridization chain reaction (SAT-HCR) and the CRISPR-Cas12a sensor for the in vitro detection of AFP. AFP aptamer, as an easily regulated nucleic acid strand, is responsible for binding to AFP into nucleic acid detection, while HCR-CRISPR-Cas12a, regulated by functionalized magnetic nanoparticles, is responsible for highly specific nucleic acid signal amplification. Under the optimal conditions, the fluorescence intensity was proportional to the concentration of AFP in the range of 0.5-10[4] ng mL[-1] and the limit of detection was 0.170 ng mL[-1]. In addition, we have successfully applied this biosensor to detect AFP in clinical samples from patients with hepatoblastoma, with greater sensitivity relative to ELISA. Our proposed method showed great potential application in clinical diagnosis and pharmaceutical-related fields with the properties of high sensitivity, low cost and high selectivity.},
}
@article {pmid35697070,
year = {2022},
author = {Ren, J and Wang, H and Yang, L and Li, F and Wu, Y and Luo, Z and Chen, Z and Zhang, Y and Feng, Y},
title = {Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {7},
pages = {102124},
pmid = {35697070},
issn = {1083-351X},
mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Endonucleases/metabolism ; },
abstract = {Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.},
}
@article {pmid35696906,
year = {2022},
author = {Li, Y and Gao, Z and Lu, J and Wei, X and Qi, M and Yin, Z and Li, T},
title = {SlSnRK2.3 interacts with SlSUI1 to modulate high temperature tolerance via Abscisic acid (ABA) controlling stomatal movement in tomato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {321},
number = {},
pages = {111305},
doi = {10.1016/j.plantsci.2022.111305},
pmid = {35696906},
issn = {1873-2259},
mesh = {*Abscisic Acid/metabolism ; Gene Expression Regulation, Plant ; *Lycopersicon esculentum/metabolism ; Plant Breeding ; Plant Proteins/genetics/metabolism ; Plant Stomata/physiology ; Temperature ; },
abstract = {Tomato is often exposed to high temperature stress during summer cultivation. Stomatal movement plays important roles in photosynthesis and transpiration which restricts the quality and yield of tomato under environmental stress. To elucidate the mechanism of stomatal movement in high temperature tolerance, SlSnRK2s (sucrose non-fermenting 1-related protein kinases) silenced plants were generated in tomato with CRISPR-Cas 9 gene editing techniques. Through the observation of stomatal parameters, SlSnRK2.3 regulated stomatal closure which was responded to ABA (abscisic acid) and activated signaling pathway of ROS (reactive oxygen species) in high temperature stress. Based on the positive functions of SlSnRK2.3, the cDNA library was generated to investigate interaction proteins of SlSnRK2s. The interaction between SlSnRK2.3 and SlSUI1 (protein translation factor SUI1 homolog) was employed by Yeast two hybrid assay (Y2H), Luciferase (LUC), and Bimolecular fluorescence complementation (BiFC). Finally, the specific interactive sites between SlSnRK2.3 and SlSUI1 were verified by site-directed mutagenesis. The consistent mechanism of SlSnRK2.3 and SlSUI1 in stomatal movement, indicating that SlSUI1 interacted with SlSnRK2.3 through ABA-dependent signaling pathway in high temperature stress. Our results provided evidence for improving the photosynthetic capacity of tomato under high temperature stress, and support the breeding and genetic engineering of tomato over summer facility cultivation.},
}
@article {pmid35696867,
year = {2022},
author = {Shen, Y and Jia, F and He, Y and Fu, Y and Fang, W and Wang, J and Li, Y},
title = {A CRISPR-Cas12a-powered magnetic relaxation switching biosensor for the sensitive detection of Salmonella.},
journal = {Biosensors &amp; bioelectronics},
volume = {213},
number = {},
pages = {114437},
doi = {10.1016/j.bios.2022.114437},
pmid = {35696867},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Magnetic Phenomena ; Magnetics ; Salmonella/genetics ; },
abstract = {Magnetic relaxation switching (MRS) biosensors are attractive in the field of food safety owing to their simplicity and high signal-to-noise ratio. But they are less in sensitivity and stability caused by the insufficient crosslinking or non-specific binding of magnetic nanoparticles (MNPs) with targets. To address this problem, the CRISPR-Cas12a system was introduced into an MRS biosensor for the first time, to precisely control the binding of two types of MNPs with sizes of 130 nm (MNP130) and 30 nm (MNP30), for the sensitive detection of Salmonella. Delicately, the biosensor was designed based on the different magnetic properties of the two sizes of MNPs. The target Salmonella activated the collateral cleavage activity of the CRISPR-Cas12a system, which inhibited the binding of the two sizes of MNPs, resulting in an increase of unbound MNP30. After separating MNP130-MNP30 complexes and MNP130 from MNP30, the free MNP30 left in solution acted as transverse relaxation time (T2) signal reporters for Salmonella detection. Under optimized conditions, the CRISPR-MRS biosensor presented a limit of detection of 1.3 × 10[2] CFU mL[-1] for Salmonella, which is lower than most MRS biosensor analogues. It also showed satisfactory specificity and performed well in spiked chicken meat samples. This biosensing strategy not only extends the reach of the CRISPR-Cas12a system in biosensors but also offers an alternative for pathogen detection with satisfactory sensitivity.},
}
@article {pmid35696571,
year = {2022},
author = {Simonetti, B and Daly, JL and Simón-Gracia, L and Klein, K and Weeratunga, S and Antón-Plágaro, C and Tobi, A and Hodgson, L and Lewis, PA and Heesom, KJ and Shoemark, DK and Davidson, AD and Collins, BM and Teesalu, T and Yamauchi, Y and Cullen, PJ},
title = {ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {25},
pages = {e2201980119},
pmid = {35696571},
issn = {1091-6490},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*COVID-19/metabolism/virology ; CRISPR-Cas Systems ; *Endosomes/virology ; Gene Deletion ; *Host-Pathogen Interactions ; Humans ; Nanoparticles ; *Neuropilin-1/genetics/metabolism ; Proteomics ; *SARS-CoV-2/metabolism ; Sorting Nexins/metabolism ; Spike Glycoprotein, Coronavirus/metabolism ; },
abstract = {Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.},
}
@article {pmid35696040,
year = {2022},
author = {Kalds, P and Crispo, M and Li, C and Tesson, L and Anegón, I and Chen, Y and Wang, X and Menchaca, A},
title = {Generation of Double-Muscled Sheep and Goats by CRISPR /Cas9-Mediated Knockout of the Myostatin Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {295-323},
pmid = {35696040},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Goats/genetics/metabolism ; Muscle, Skeletal/metabolism ; *Myostatin/genetics ; Sheep/genetics ; },
abstract = {The myostatin (MSTN) gene has shown to play a critical role in the regulation of skeletal muscle mass, and the translational inhibition of this gene has shown increased muscle mass, generating what is known as "double-muscling phenotype." Disruption of the MSTN gene expression using the CRISPR/Cas9 genome-editing system has shown improved muscle development and growth rates in livestock species, including sheep and goats. Here, we describe procedures for the generation of MSTN knockout sheep and goats using the microinjection approach of the CRISPR/Cas9 system, including the selection of targeting sgRNAs, the construction of CRISPR/Cas9 targeting vector, the in vitro examination of system efficiency, the in vivo targeting to generate MSTN knockout founders, the genomic and phenotypic characterization of the generated offspring, and the assessment of off-target effects in gene-edited founders through targeted validation of predicted off-target sites, as well as genome-wide off-target analysis by whole-genome sequencing. Editing the MSTN gene using the CRISPR/Cas9 system might be a rapid and promising alternative to promote meat production in livestock.},
}
@article {pmid35696038,
year = {2022},
author = {Liu, J and Verma, PJ},
title = {Generating a Heat-Tolerance Mouse Model.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {259-272},
pmid = {35696038},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Female ; Gene Editing ; *Hot Temperature ; Mice ; Recombinational DNA Repair ; Zygote/metabolism ; },
abstract = {Creating mouse models of human genetic disease (Gurumurthy and Lloyd, Dis Models Mech 12(1):dmm029462, 2019) and livestock trait (Schering et al. Arch Physiol Biochem 121(5):194-205, 2015; Habiela et al. J Gen Virol 95 (Pt 11):2329-2345, 2014) have been proven to be a useful tool for understanding the mechanism behind the phenotypes and fundamental and applied research in livestock. A single base pair deletion of prolactin receptor (PRLR) has an impact on hair morphology phenotypes beyond its classical roles in lactation in cattle, the so-called slick cattle (Littlejohn et al. Nat Commun 5:5861, 2014). Here, we generate a knock-in mouse model by targeting the specific locus of PRLR gene using Cas9-mediated genome editing via homology-directed repair (HDR) in mouse zygotes. The mouse model carrying the identical PRLR mutation in slick cattle may provide a useful animal model to study the pathway of thermoregulation and the mechanism of heat-tolerance in the livestock.},
}
@article {pmid35696037,
year = {2022},
author = {Goldsmith, T and Bondareva, A and Webster, D and Voigt, AL and Su, L and Carlson, DF and Dobrinski, I},
title = {Targeted Gene Editing in Porcine Germ Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {245-258},
pmid = {35696037},
issn = {1940-6029},
support = {R01 OD016575/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Male ; Mutation ; Spermatogonia ; Swine ; },
abstract = {As the genetic mutations driving human disease are identified, there is an increasing need for a biomedical model that can accurately represent the disease of interest and provide a platform for potential therapeutic testing. Pigs are a better model for human disease than rodents because of their genetic and physiological similarities to humans. However, current methods to generate porcine models are both technically challenging and expensive. Germline genetic modification through gene edited spermatogonia provides an effective alternative to how these models are developed. Here, we report an improved technique of gene editing in spermatogonia of pigs using CRISPR-Cas9 to generate different edits that reflect the genotypes of human diseases.},
}
@article {pmid35696036,
year = {2022},
author = {Crispo, M and Chenouard, V and Dos Santos-Neto, P and Tesson, L and Souza-Neves, M and Heslan, JM and Cuadro, F and Anegón, I and Menchaca, A},
title = {Generation of a Human Deafness Sheep Model Using the CRISPR/Cas System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {233-244},
pmid = {35696036},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; *Deafness/genetics ; Gene Editing/methods ; Humans ; Mutation ; Recombinational DNA Repair ; Sheep ; },
abstract = {CRISPR/Cas9 system is a promising method for the generation of human disease models by genome editing in non-conventional experimental animals. Medium/large-sized animals like sheep have several advantages to study human diseases and medicine. Here, we present a protocol that describes the generation of an otoferlin edited sheep model via CRISPR-assisted single-stranded oligodinucleotide-mediated Homology-Directed Repair (HDR), through direct cytoplasmic microinjection in in vitro produced zygotes.Otoferlin is a protein expressed in the cochlear inner hair cells, with different mutations at the OTOF gene being the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. By using this protocol, we reported for the first time an OTOF KI model in sheep with 17.8% edited lambs showing indel mutations, and 61.5% of them bearing knock-in mutations by HDR . The reported method establishes the bases to produce a deafness model to test novel therapies in human disorders related to OTOF mutations.},
}
@article {pmid35696035,
year = {2022},
author = {Bunting, MD and Pfitzner, C and Gierus, L and White, M and Piltz, S and Thomas, PQ},
title = {Generation of Gene Drive Mice for Invasive Pest Population Suppression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {203-230},
pmid = {35696035},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Gene Editing/methods ; Introduced Species ; Mice ; Mice, Transgenic ; Transgenes ; },
abstract = {Gene drives are genetic elements that are transmitted to greater than 50% of offspring and have potential for population modification or suppression. While gene drives are known to occur naturally, the recent emergence of CRISPR-Cas9 genome-editing technology has enabled generation of synthetic gene drives in a range of organisms including mosquitos, flies, and yeast. For example, studies in Anopheles mosquitos have demonstrated >95% transmission of CRISPR-engineered gene drive constructs, providing a possible strategy for malaria control. Recently published studies have also indicated that it may be possible to develop gene drive technology in invasive rodents such as mice. Here, we discuss the prospects for gene drive development in mice, including synthetic "homing drive" and X-shredder strategies as well as modifications of the naturally occurring t haplotype. We also provide detailed protocols for generation of gene drive mice through incorporation of plasmid-based transgenes in a targeted and non-targeted manner. Importantly, these protocols can be used for generating transgenic mice for any project that requires insertion of kilobase-scale transgenes such as knock-in of fluorescent reporters, gene swaps, overexpression/ectopic expression studies, and conditional "floxed" alleles.},
}
@article {pmid35696034,
year = {2022},
author = {Paulo, DF and Williamson, ME and Scott, MJ},
title = {CRISPR/Cas9 Genome Editing in the New World Screwworm and Australian Sheep Blowfly.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {173-201},
pmid = {35696034},
issn = {1940-6029},
mesh = {Animals ; Australia ; CRISPR-Cas Systems/genetics ; Calliphoridae ; *Diptera/genetics ; *Gene Editing/methods ; RNA, Guide/genetics ; },
abstract = {Blowflies are of interest for medical applications (maggot therapy), forensic investigations, and for evolutionary developmental studies such as the evolution of parasitism. It is because of the latter that some blowflies such as the New World screwworm and the Australian sheep blowfly are considered major economic pests of livestock. Due to their importance, annotated assembled genomes for several species are now available. Here, we present a detailed guide for using the Streptococcus pyogenes Cas9 RNA-guided nuclease to efficiently generate both knockout and knock-in mutations in screwworm and sheep blowfly. These methods should accelerate genetic investigations in these and other closely related species and lead to a better understanding of the roles of selected genes in blowfly development and behavior.},
}
@article {pmid35696033,
year = {2022},
author = {Choo, A and Fung, E and Nguyen, TNM and Okada, A and Crisp, P},
title = {CRISPR/Cas9 Mutagenesis to Generate Novel Traits in Bactrocera tryoni for Sterile Insect Technique.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {151-171},
pmid = {35696033},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Mutagenesis ; *Tephritidae/genetics ; },
abstract = {Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.},
}
@article {pmid35696032,
year = {2022},
author = {Alberio, V and Savy, V and Salamone, DF},
title = {CRISPR-on for Endogenous Activation of SMARCA4 Expression in Bovine Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {129-148},
pmid = {35696032},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Cell Differentiation ; Cell Line ; Cellular Reprogramming/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR-on system is a programmable, simple, and versatile gene activator that has proven to be efficient in cultured cells from several species and in bovine embryos. This technology allows for the precise and specific activation of single endogenous gene expression and also multiplexed gene expression in a simple fashion. Therefore, CRISPR-on has unique advantages over other activator systems and a wide adaptability for studies in basic and applied science, such as cell reprogramming and cell fate differentiation for regenerative medicine.In this chapter, we describe the materials and methods of the CRISPR-on system for activation of the endogenous SMARCA4 expression in bovine embryos.},
}
@article {pmid35696031,
year = {2022},
author = {Delerue, F and Ittner, LM},
title = {Microinjection of Zygotes for CRISPR/Cas9-Mediated Insertion of Transgenes into the Murine Rosa26 Safe Harbor.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {115-128},
pmid = {35696031},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Microinjections ; RNA, Untranslated/*genetics ; Transgenes ; *Zygote ; },
abstract = {Genetically modified (GM) mice are widely used in biomedical research because they can address complex questions in an in-vivo setting that could not otherwise be addressed in-vitro. Microinjection of zygotes remains the most common technique to generate GM animals to date. Here, we describe the targeted insertion (knock-in) of transgenes by microinjection of 1-cell or 2-cell stage embryos into the murine Rosa26 safe harbor.},
}
@article {pmid35696030,
year = {2022},
author = {Gu, J and Rollo, B and Sumer, H and Cromer, B},
title = {Targeting the AAVS1 Site by CRISPR/Cas9 with an Inducible Transgene Cassette for the Neuronal Differentiation of Human Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {99-114},
pmid = {35696030},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Pluripotent Stem Cells ; Transgenes ; },
abstract = {CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.},
}
@article {pmid35696029,
year = {2022},
author = {Malaver-Ortega, LF and Rosenbluh, J},
title = {Immortalised Cas9-expressing Cell lines for Gene interrogation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {91-97},
pmid = {35696029},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Genome ; },
abstract = {The ability of modifying the genome of multiple species, precisely and without or minimal off-targeted effects, have opened numerous opportunities for the biotechnology industry. In this chapter, we describe an easy to establish, robust, and practical pipeline that can be used to generate immortalized cell lines, from different tissues, to capture cell linage context and validate the tools required for genome editing and genetic modification. This pipeline serves as a reference for similar approaches for gene interrogation in other species.},
}
@article {pmid35696026,
year = {2022},
author = {Shamshirgaran, Y and Liu, J and Sumer, H and Verma, PJ and Taheri-Ghahfarokhi, A},
title = {Tools for Efficient Genome Editing; ZFN, TALEN, and CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {29-46},
pmid = {35696026},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism ; },
abstract = {The last two decades have marked significant advancement in the genome editing field. Three generations of programmable nucleases (ZFNs, TALENs, and CRISPR-Cas system) have been adopted to introduce targeted DNA double-strand breaks (DSBs) in eukaryotic cells. DNA repair machinery of the cells has been exploited to introduce insertion and deletions (indels) at the targeted DSBs to study function of any gene-of-interest. The resulting indels were generally assumed to be "random" events produced by "error-prone" DNA repair pathways. However, recent advances in computational tools developed to study the Cas9-induced mutations have changed the consensus and implied the "non-randomness" nature of these mutations. Furthermore, CRISPR-centric tools are evolving at an unprecedented pace, for example, base- and prime-editors are the newest developments that have been added to the genome editing toolbox. Altogether, genome editing tools have revolutionized our way of conducting research in life sciences. Here, we present a concise overview of genome editing tools and describe the DNA repair pathways underlying the generation of genome editing outcome.},
}
@article {pmid35696025,
year = {2022},
author = {Montoliu, L},
title = {Historical DNA Manipulation Overview.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2495},
number = {},
pages = {3-28},
pmid = {35696025},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; Mammals/genetics ; Mice ; },
abstract = {The history of DNA manipulation for the creation of genetically modified animals began in the 1970s, using viruses as the first DNA molecules microinjected into mouse embryos at different preimplantation stages. Subsequently, simple DNA plasmids were used to microinject into the pronuclei of fertilized mouse oocytes and that method became the reference for many years. The isolation of embryonic stem cells together with advances in genetics allowed the generation of gene-specific knockout mice, later on improved with conditional mutations. Cloning procedures expanded the gene inactivation to livestock and other non-model mammalian species. Lentiviruses, artificial chromosomes, and intracytoplasmic sperm injections expanded the toolbox for DNA manipulation. The last chapter of this short but intense history belongs to programmable nucleases, particularly CRISPR-Cas systems, triggering the development of genomic-editing techniques, the current revolution we are living in.},
}
@article {pmid35695893,
year = {2022},
author = {Coon, BG and Timalsina, S and Astone, M and Zhuang, ZW and Fang, J and Han, J and Themen, J and Chung, M and Yang-Klingler, YJ and Jain, M and Hirschi, KK and Yamamato, A and Trudeau, LE and Santoro, M and Schwartz, MA},
title = {A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells.},
journal = {The Journal of cell biology},
volume = {221},
number = {7},
pages = {},
pmid = {35695893},
issn = {1540-8140},
support = {R01 HL75092/NH/NIH HHS/United States ; ERC-CoG 647057/ERC_/European Research Council/International ; //CIHR/Canada ; },
mesh = {Atherosclerosis/pathology ; CRISPR-Cas Systems ; Calcium Signaling ; *Endothelial Cells/metabolism ; Humans ; Inflammation ; *Kruppel-Like Transcription Factors/genetics/metabolism ; MAP Kinase Kinase 5 ; MAP Kinase Kinase Kinase 2 ; MAP Kinase Kinase Kinase 3 ; *Mitochondria/metabolism ; Mitogen-Activated Protein Kinase 7/genetics/metabolism ; Reactive Oxygen Species ; *Stress, Mechanical ; },
abstract = {Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3-MEK5-ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK-MEK5-ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.},
}
@article {pmid35695482,
year = {2022},
author = {Zhang, RX and Li, BB and Yang, ZG and Huang, JQ and Sun, WH and Bhanbhro, N and Liu, WT and Chen, KM},
title = {Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {24},
pages = {7343-7359},
doi = {10.1021/acs.jafc.2c01754},
pmid = {35695482},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing ; *Genes, Plant ; Genome, Plant ; Plant Breeding ; },
abstract = {The CRISPR-based gene editing technology has become more and more powerful in genome manipulation for agricultural breeding, with numerous improved toolsets springing up. In recent years, many CRISPR toolsets for gene editing, such as base editors (BEs), CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and plant epigenetic editors (PEEs), have been developed to clarify gene function and full-level gene regulation. Here, we comprehensively summarize the application and capacity of the different CRISPR toolsets in the study of plant gene expression regulation, highlighting their potential application in gene regulatory networks' analysis. The general problems in CRISPR application and the optimal solutions in the existing schemes for high-throughput gene function analysis are also discussed. The CRISPR toolsets targeting gene manipulation discussed here provide new solutions for further genetic improvement and molecular breeding of crops.},
}
@article {pmid35694840,
year = {2022},
author = {Mao, X and Zhao, Y and Jiang, J and Du, Q and Tu, B and Li, J and Wang, F},
title = {Sensitive and high-accuracy detection of Salmonella based on CRISPR/Cas12a combined with recombinase polymerase amplification.},
journal = {Letters in applied microbiology},
volume = {75},
number = {4},
pages = {899-907},
doi = {10.1111/lam.13765},
pmid = {35694840},
issn = {1472-765X},
mesh = {*CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction ; *Recombinases/genetics ; Salmonella/genetics ; },
abstract = {Salmonella is a crucial food-borne pathogen causing food poisoning, leading to severe public health events. Here, we developed a technique by integrating recombinase polymerase amplification with CRISPR-LbCas12a and employing two targets with engineered crRNA for detection of Salmonella (RPA-LbCas12a-TTECDS). Our findings revealed that this novel method rapidly detects trace Salmonella in food through fluorescence intensity and provides a template for other food-borne pathogen detection methods. Further, crRNA was optimized to increase detection sensitivity. Double targets were used to enhance the detection accuracy, reaching the level of qPCR, which was superior to fluorescent RPA. The RPA-LbCas12a-TTECDS system specifically detected Salmonella levels as low as 50 CFU per ml at 37°C in 1 h. In summary, a simple, rapid, sensitive and high accuracy detection technique based on CRISPR-Cas12a was created for Salmonella detection without complicated equipment.},
}
@article {pmid35694537,
year = {2022},
author = {Maslennikova, A and Mazurov, D},
title = {Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {880030},
pmid = {35694537},
issn = {2235-2988},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; Genomics ; *HIV Infections/drug therapy ; *HIV-1/genetics ; Humans ; Virus Latency ; },
abstract = {Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.},
}
@article {pmid35694192,
year = {2022},
author = {Bexte, T and Alzubi, J and Reindl, LM and Wendel, P and Schubert, R and Salzmann-Manrique, E and von Metzler, I and Cathomen, T and Ullrich, E},
title = {CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma.},
journal = {Oncoimmunology},
volume = {11},
number = {1},
pages = {2081415},
pmid = {35694192},
issn = {2162-402X},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Killer Cells, Natural/metabolism ; *Multiple Myeloma/genetics/therapy ; *NK Cell Lectin-Like Receptor Subfamily C/genetics/metabolism ; },
abstract = {Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as 'off-the-shelf' product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications.},
}
@article {pmid35693210,
year = {2022},
author = {Liu, Y and Chen, Q and Song, C and Xu, Z and Yang, S and Li, X},
title = {Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101436},
pmid = {35693210},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Embryonic Stem Cells ; Mice ; *Mouse Embryonic Stem Cells ; Puromycin/pharmacology ; Transfection ; },
abstract = {Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.},
}
@article {pmid35693174,
year = {2022},
author = {Silva, FDA and Fontes, EPB},
title = {Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein System for Resistance Against Plant Viruses: Applications and Perspectives.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {904829},
pmid = {35693174},
issn = {1664-462X},
abstract = {Different genome editing approaches have been used to engineer resistance against plant viruses. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas; CRISPR/Cas) systems to create pinpoint genetic mutations have emerged as a powerful tool for molecular engineering of plant immunity and increasing resistance against plant viruses. This review presents (i) recent advances in engineering resistance against plant viruses by CRISPR/Cas and (ii) an overview of the potential host factors as targets for the CRISPR/Cas system-mediated broad-range resistance and immunity. Applications, challenges, and perspectives in enabling the CRISPR/Cas system for crop protection are also outlined.},
}
@article {pmid35693153,
year = {2022},
author = {Chen, Y and Hu, Y and Wang, X and Luo, S and Yang, N and Chen, Y and Li, Z and Zhou, Q and Li, W},
title = {Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing.},
journal = {Innovation (Cambridge (Mass.))},
volume = {3},
number = {4},
pages = {100264},
pmid = {35693153},
issn = {2666-6758},
abstract = {The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed a method termed Improving Editing Activity by Synergistic Engineering (MIDAS). This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions between Cas nuclease with the protospacer adjacent motif (PAM) and the single-stranded DNA (ssDNA) substrate in the catalytic pocket simultaneously. MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i, Cas12b, and CasX in human cells. Notably, a Cas12i variant, Cas12i [Max] , exhibited robust activity with a very broad PAM range (NTNN, NNTN, NAAN, and NCAN) and higher efficiency than the current widely used Cas nucleases. A high-fidelity version of Cas12i [Max] (Cas12i [HiFi]) has been further engineered to minimize off-target effects. Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.},
}
@article {pmid35692726,
year = {2022},
author = {Shim, H},
title = {Investigating the Genomic Background of CRISPR-Cas Genomes for CRISPR-Based Antimicrobials.},
journal = {Evolutionary bioinformatics online},
volume = {18},
number = {},
pages = {11769343221103887},
pmid = {35692726},
issn = {1176-9343},
abstract = {CRISPR-Cas systems are an adaptive immunity that protects prokaryotes against foreign genetic elements. Genetic templates acquired during past infection events enable DNA-interacting enzymes to recognize foreign DNA for destruction. Due to the programmability and specificity of these genetic templates, CRISPR-Cas systems are potential alternative antibiotics that can be engineered to self-target antimicrobial resistance genes on the chromosome or plasmid. However, several fundamental questions remain to repurpose these tools against drug-resistant bacteria. For endogenous CRISPR-Cas self-targeting, antimicrobial resistance genes and functional CRISPR-Cas systems have to co-occur in the target cell. Furthermore, these tools have to outplay DNA repair pathways that respond to the nuclease activities of Cas proteins, even for exogenous CRISPR-Cas delivery. Here, we conduct a comprehensive survey of CRISPR-Cas genomes. First, we address the co-occurrence of CRISPR-Cas systems and antimicrobial resistance genes in the CRISPR-Cas genomes. We show that the average number of these genes varies greatly by the CRISPR-Cas type, and some CRISPR-Cas types (IE and IIIA) have over 20 genes per genome. Next, we investigate the DNA repair pathways of these CRISPR-Cas genomes, revealing that the diversity and frequency of these pathways differ by the CRISPR-Cas type. The interplay between CRISPR-Cas systems and DNA repair pathways is essential for the acquisition of new spacers in CRISPR arrays. We conduct simulation studies to demonstrate that the efficiency of these DNA repair pathways may be inferred from the time-series patterns in the RNA structure of CRISPR repeats. This bioinformatic survey of CRISPR-Cas genomes elucidates the necessity to consider multifaceted interactions between different genes and systems, to design effective CRISPR-based antimicrobials that can specifically target drug-resistant bacteria in natural microbial communities.},
}
@article {pmid35692096,
year = {2022},
author = {Wang, Q and Li, J and Zhu, J and Mao, J and Duan, C and Liang, X and Zhu, L and Zhu, M and Zhang, Z and Lin, F and Guo, R},
title = {Genome-wide CRISPR/Cas9 screening for therapeutic targets in NSCLC carrying wild-type TP53 and receptor tyrosine kinase genes.},
journal = {Clinical and translational medicine},
volume = {12},
number = {6},
pages = {e882},
pmid = {35692096},
issn = {2001-1326},
mesh = {CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology ; Humans ; *Lung Neoplasms/drug therapy/genetics/pathology ; Pemetrexed/therapeutic use ; Receptor Protein-Tyrosine Kinases/genetics/therapeutic use ; Tumor Suppressor Protein p53/genetics/therapeutic use ; },
abstract = {BACKGROUND: Targeted drugs have greatly improved the therapeutic outcome of non-small cell lung cancer (NSCLC) patients compared with conventional chemotherapy, whereas about one-third of patients are so far not suitable for targeted therapy due to lack of known driver oncogenes such as a mutated receptor tyrosine kinase (RTK) genes. In this study, we aimed to identify therapeutic targets for this subgroup of NSCLC patients.<br><br>METHODS: We performed genome-wide CRISPR/Cas9 screens in two NSCLC cell lines carrying wild-type TP53 and receptor tyrosine kinase (wtTP53-RTK) genes using a GeCKO v2.0 lentiviral library (containing 123411 sgRNAs and targeting 19050 genes). MAGeCKFlute was used to analyse and identify candidate genes. Genetic perturbation and pharmacological inhibition were used to validate the result in vitro and in vivo.<br><br>RESULTS: The Genome-wide CRISPR/Cas9 screening identified MDM2 as a potential therapeutic target for wtTP53-RTK NSCLC. Genetic and pharmacological inhibition of MDM2 reduced cell proliferation and impaired tumour growth in the xenograft model, thus confirming the finding of the CRISPR/Cas9 screening. Moreover, treatment by a selective MDM2 inhibitor RG7388 triggered both cell cycle arrest and apoptosis in several NSCLC cell lines. Additionally, RG7388 and pemetrexed synergistically blocked the cell proliferation and growth of wtTP53-RTK tumours but had limited effects for other genotypes.<br><br>CONCLUSIONS: We identified MDM2 as an essential gene and a potential therapeutic target in wtTP53-RTK NSCLC via a genome-wide CRISPR/Cas9 screening. For this subgroup, treatment by RG7388 alone or by its combination with pemetrexed resulted in significant tumour inhibition.},
}
@article {pmid35691789,
year = {2023},
author = {Yuan, G and Xia, X and Zhang, J and Huang, J and Xie, F and Li, X and Chen, D and Peng, C},
title = {A novel "signal on-off-super on" sandwich-type aptamer sensor of CRISPR-Cas12a coupled voltage enrichment assay for VEGF detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {221},
number = {},
pages = {114424},
doi = {10.1016/j.bios.2022.114424},
pmid = {35691789},
issn = {1873-4235},
mesh = {Gold/chemistry ; *Aptamers, Nucleotide/chemistry ; Vascular Endothelial Growth Factor A/genetics ; *Biosensing Techniques ; *Metal Nanoparticles/chemistry ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Reproducibility of Results ; },
abstract = {Vascular endothelial growth factor (VEGF) plays an important role in atherosclerosis, and the detection of VEGF is critical for the prevention, monitoring, and diagnosis of cardiovascular diseases. Here, a novel "signal on-off-super on" sandwich-type aptamer sensor with a triple signal amplification strategy was developed for the first time. Based on the capture aptamer was labeled with methylene blue (MB) on the internal bases, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-coupled voltage enrichment was used to amplify the electrochemical signal. To improve the analytical performance of the aptamer sensor, gold nanoparticles@Ti3C2Tx-Mxene (AuNPs@Ti3C2Tx-Mxene) were synthesized through the electrodeposition of AuNPs on the Ti3C2Tx-Mxene surface, providing active sites for the immobilization of the aptamer and amplifying the electrochemical signals. The excellent trans-cleavage activity of the CRISPR-Cas12a system was harnessed to cleave signal probes. The cleaved signal probes were enriched using an electrochemical signal instead of complicated target amplification steps before detection. Hence, we report a simplified detection process for amplifying electrochemical signals. Under optimal conditions, the aptamer sensor exhibited high sensitivity, acceptable stability, and reproducibility with a wide linear range from 1 pM to 10 μM (R[2] = 0.9917) and an ultralow detection limit of 0.33 pM (S/N = 3). Therefore, we propose a novel strategy of CRISPR-Cas12a-based protein detection that opens a new window for the diagnostic applications of various biomarkers.},
}
@article {pmid35691167,
year = {2022},
author = {Wang, X and Qin, Y and Huang, Y and Hu, K and Zhao, S and Tian, J},
title = {A sensitive and facile microRNA detection based on CRISPR-Cas12a coupled with strand displacement amplification.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {279},
number = {},
pages = {121476},
doi = {10.1016/j.saa.2022.121476},
pmid = {35691167},
issn = {1873-3557},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics ; },
abstract = {MicroRNAs (miRNAs) are important biomarkers that are closely associated with certain diseases. The detection of miRNA is critical because it provides the necessary information for Disease Diagnosis. In this study, we achieved miRNA determination by coupling the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) system with strand displacement amplification (SDA). In the experiment, miRNA was used as the initiator of SDA, and the activator of Cas12a nuclease activity was amplified by SDA. Subsequently, the unique nuclease activity of Cas12a was exploited to carry out trans cleaving on the ssDNA reporting probe modified with carboxyfluorescein(FAM) and BHQ1(dark Quencher: 480-580 nm) to achieve a signal output. In addition to chain design and reaction simplification, this method is lofty sensitive and selective for the determination of miRNA with a good linear range of 250 fmol·L[-1] ∼ 40 pmol·L[-1], the detection limit of 150 fmol·L[-1] (S/N = 3), and the method showed good recovery in spiked human serum. Overall, this method is expected to be applied to diagnosis with miRNA biomarkers because of its rapidity, high sensitivity, and high selectivity.},
}
@article {pmid35690427,
year = {2022},
author = {Zeng, H and Zhang, P and Jiang, X and Duan, C and Yu, Y and Wu, Q and Yang, X},
title = {Rapid RNA detection through intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification.},
journal = {Analytica chimica acta},
volume = {1217},
number = {},
pages = {340009},
doi = {10.1016/j.aca.2022.340009},
pmid = {35690427},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide/metabolism ; RNA, Viral/genetics ; },
abstract = {The clinical methods to detect RNA viruses and disease-related RNAs suffer from time-consuming processes, high false-positive rates, or limited sensitivity. Here, we propose a strategy for rapid RNA detection through intra-enzyme chain replacement-mediated Cas13a cascade cyclic reaction without target amplification. A hairpin RNA mediator (a cleavage substrate for target-activated Cas13a) and a guiding RNA recognized by the cleavage product through intra-enzyme chain replacement were designed and optimized. Upon the recognition and binding of the target RNA to the Cas13a/CrRNA complex, Cas13a is initially activated to cleave the mediator, and the cleavage products recognize the corresponding Cas13a/CrRNA complex by intra-enzyme chain replacement and initiate the circular cascade of Cas13a cleavage and activation. The accumulated active Cas13a cleaves fluorescent reporter probe for achieving target RNA detection. This "mix &amp; read" RNA detection at room temperature was performed in total 30 min. Using miRNA-21 as the target, the changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM, while no significant changes in fluorescence intensity were detected for non-targets. This method applied to the clinical sputum respiratory syncytial virus-positive samples gave results consistent with those from the clinical fluorescence immunoassay. Thus, intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction for detection of RNA viruses in the "mix &amp; read" mode at room temperature is rapid, simple, convenient, and efficient for RNA detection and can be adapted to point-of-care testing for high throughput screening of RNA virus infections.},
}
@article {pmid35690249,
year = {2022},
author = {Santiago-Frangos, A and Nemudryi, A and Nemudraia, A and Wiegand, T and Nichols, JE and Krishna, P and Scherffius, AM and Zahl, TR and Wilkinson, RA and Wiedenheft, B},
title = {CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics.},
journal = {Methods (San Diego, Calif.)},
volume = {205},
number = {},
pages = {1-10},
pmid = {35690249},
issn = {1095-9130},
support = {P20 GM103474/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {*Argonaute Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Polymerase Chain Reaction ; },
abstract = {Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.},
}
@article {pmid35690103,
year = {2022},
author = {Shibata, Y and Suzuki, M and Hirose, N and Takayama, A and Sanbo, C and Inoue, T and Umesono, Y and Agata, K and Ueno, N and Suzuki, KT and Mochii, M},
title = {CRISPR/Cas9-based simple transgenesis in Xenopus laevis.},
journal = {Developmental biology},
volume = {489},
number = {},
pages = {76-83},
doi = {10.1016/j.ydbio.2022.06.001},
pmid = {35690103},
issn = {1095-564X},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Ribonucleoproteins/genetics ; Transgenes ; Xenopus laevis/genetics ; },
abstract = {Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.},
}
@article {pmid35690065,
year = {2022},
author = {Vialetto, E and Yu, Y and Collins, SP and Wandera, KG and Barquist, L and Beisel, CL},
title = {A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {8},
pages = {1151-1162.e6},
pmid = {35690065},
issn = {1934-6069},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {Autoimmunity/genetics ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; Plasmids ; RNA ; },
abstract = {CRISPR-Cas systems must enact robust immunity against foreign genetic material without inducing cytotoxic autoimmunity. For type VI systems that use Cas13 nucleases and recognize RNA targets, immune activation requires extensive CRISPR RNA (crRNA) guide-target complementarity and a target-flanking motif. Here, we report a third requirement shaping the immune response: the expression of the target transcript exceeding a threshold. We found that endogenous non-essential transcripts targeted by crRNAs rarely elicited autoimmunity. Instead, autoimmune induction required over-expressing the targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as a global determinant of cytotoxic autoimmunity and revealed that this threshold shifts with each guide-target pair. This threshold further ensured defense against a lytic bacteriophage yet allowed the tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as an additional criterion for immune defense by RNA-targeting CRISPR-Cas systems, preventing autoimmunity and distinguishing pathogenic and benign invaders.},
}
@article {pmid35689948,
year = {2022},
author = {Fang, B and Jia, Z and Liu, C and Tu, K and Zhang, M and Zhang, L},
title = {A versatile CRISPR Cas12a-based point-of-care biosensor enabling convenient glucometer readout for ultrasensitive detection of pathogen nucleic acids.},
journal = {Talanta},
volume = {249},
number = {},
pages = {123657},
doi = {10.1016/j.talanta.2022.123657},
pmid = {35689948},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; DNA/genetics ; DNA Probes/genetics ; *HIV Infections ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Point-of-Care Systems ; SARS-CoV-2/genetics ; beta-Fructofuranosidase ; },
abstract = {Pathogen nucleic acid detection is of great significance to control the spread of diseases caused by the viruses. Nevertheless, traditional methods for nucleic acid detection such as polymerase chain reaction (PCR) and oligonucleotide microarrays require bulky instruments, which restrain their point-of-care (POC) testing application. Here, we proposed a POC method enabling sensitive detection of pathogen nucleic acids by combining the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a-based assay and personal glucometer readout (PGM). The quantification of target pathogen DNA by PGM was achieved based on pathogen DNA activates Cas12a ssDNase to cleave magnetic bead-DNA-invertase reporter probe, and separated free invertase to catalyze hydrolysis of sucrose to glucose. Without using nucleic acid amplification technology, we demonstrated here dual signal amplifications based on Cas12a and invertase-mediated catalytic reactions, making it possible to sensitively detect HIV-related DNA or SARS-CoV-2 pseudovirus with the limits of detection of 11.0 fM and 50 copies/μL, respectively. This strategy also showed excellent selectivity as well as potential applicability for detection of HIV in human serum samples or of SARS-CoV-2 in saliva samples. Therefore, our CRISPR-PGM-based dual signal amplifications detection platform might offer a great promise in POC diagnosis of pathogen nucleic acids.},
}
@article {pmid35689914,
year = {2022},
author = {Saha, A and Arantes, PR and Palermo, G},
title = {Dynamics and mechanisms of CRISPR-Cas9 through the lens of computational methods.},
journal = {Current opinion in structural biology},
volume = {75},
number = {},
pages = {102400},
pmid = {35689914},
issn = {1879-033X},
support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; DNA Cleavage ; *Gene Editing/methods ; Molecular Dynamics Simulation ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) genome-editing revolution established the beginning of a new era in life sciences. Here, we review the role of state-of-the-art computations in the CRISPR-Cas9 revolution, from the early refinement of cryo-EM data to enhanced simulations of large-scale conformational transitions. Molecular simulations reported a mechanism for RNA binding and the formation of a catalytically competent Cas9 enzyme, in agreement with subsequent structural studies. Inspired by single-molecule experiments, molecular dynamics offered a rationale for the onset of off-target effects, while graph theory unveiled the allosteric regulation. Finally, the use of a mixed quantum-classical approach established the catalytic mechanism of DNA cleavage. Overall, molecular simulations have been instrumental in understanding the dynamics and mechanism of CRISPR-Cas9, contributing to understanding function, catalysis, allostery, and specificity.},
}
@article {pmid35689624,
year = {2022},
author = {Weiss, T and Crisp, PA and Rai, KM and Song, M and Springer, NM and Zhang, F},
title = {Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants.},
journal = {Plant physiology},
volume = {190},
number = {2},
pages = {1153-1164},
pmid = {35689624},
issn = {1532-2548},
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Epigenomics ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.},
}
@article {pmid35689490,
year = {2022},
author = {Bhat, AI and Aman, R and Mahfouz, M},
title = {Onsite detection of plant viruses using isothermal amplification assays.},
journal = {Plant biotechnology journal},
volume = {20},
number = {10},
pages = {1859-1873},
pmid = {35689490},
issn = {1467-7652},
mesh = {*Nucleic Acids ; *Plant Viruses/genetics ; Recombinases ; Sensitivity and Specificity ; Viral Proteins ; },
abstract = {Plant diseases caused by viruses limit crop production and quality, resulting in significant losses. However, options for managing viruses are limited; for example, as systemic obligate parasites, they cannot be killed by chemicals. Sensitive, robust, affordable diagnostic assays are needed to detect the presence of viruses in plant materials such as seeds, vegetative parts, insect vectors, or alternative hosts and then prevent or limit their introduction into the field by destroying infected plant materials or controlling insect hosts. Diagnostics based on biological and physical properties are not very sensitive and are time-consuming, but assays based on viral proteins and nucleic acids are more specific, sensitive, and rapid. However, most such assays require laboratories with sophisticated equipment and technical skills. By contrast, isothermal-based assays such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are simple, easy to perform, reliable, specific, and rapid and do not require specialized equipment or skills. Isothermal amplification assays can be performed using lateral flow devices, making them suitable for onsite detection or testing in the field. To overcome non-specific amplification and cross-contamination issues, isothermal amplification assays can be coupled with CRISPR/Cas technology. Indeed, the collateral activity associated with some CRISPR/Cas systems has been successfully harnessed for visual detection of plant viruses. Here, we briefly describe traditional methods for detecting viruses and then examine the various isothermal assays that are being harnessed to detect viruses.},
}
@article {pmid35688932,
year = {2022},
author = {Harmsen, TJW and Pritchard, CEJ and Riepsaame, J and van de Vrugt, HJ and Huijbers, IJ and Te Riele, H},
title = {HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {9606},
pmid = {35688932},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Endonucleases/genetics ; *Gene Editing/methods ; Mice ; RNA, Guide/genetics ; },
abstract = {Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy.},
}
@article {pmid35688146,
year = {2022},
author = {Replogle, JM and Saunders, RA and Pogson, AN and Hussmann, JA and Lenail, A and Guna, A and Mascibroda, L and Wagner, EJ and Adelman, K and Lithwick-Yanai, G and Iremadze, N and Oberstrass, F and Lipson, D and Bonnar, JL and Jost, M and Norman, TM and Weissman, JS},
title = {Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.},
journal = {Cell},
volume = {185},
number = {14},
pages = {2559-2575.e28},
pmid = {35688146},
issn = {1097-4172},
support = {R44 HG010558/HG/NHGRI NIH HHS/United States ; R00 GM130964/GM/NIGMS NIH HHS/United States ; R44 HG011060/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R01 CA129105/CA/NCI NIH HHS/United States ; F31 NS115380/NS/NINDS NIH HHS/United States ; DP2 GM140925/GM/NIGMS NIH HHS/United States ; R01 GM134539/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromosome Mapping ; *Genomics ; Genotype ; Phenotype ; *Single-Cell Analysis/methods ; },
abstract = {A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.},
}
@article {pmid35687910,
year = {2022},
author = {Zuo, Q and Xu, W and Wan, Y and Feng, D and He, C and Lin, C and Huang, D and Chen, F and Han, L and Sun, Q and Chen, D and Du, H and Huang, L},
title = {Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {152},
number = {},
pages = {113243},
doi = {10.1016/j.biopha.2022.113243},
pmid = {35687910},
issn = {1950-6007},
mesh = {*CRISPR-Cas Systems/genetics ; *Cytochrome P-450 CYP3A/genetics ; Gene Editing/methods ; Gene Knock-In Techniques ; Luciferases/genetics ; },
abstract = {CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.},
}
@article {pmid35687127,
year = {2022},
author = {Tao, R and Wang, Y and Jiao, Y and Hu, Y and Li, L and Jiang, L and Zhou, L and Qu, J and Chen, Q and Yao, S},
title = {Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells.},
journal = {Nucleic acids research},
volume = {50},
number = {11},
pages = {6423-6434},
pmid = {35687127},
issn = {1362-4962},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Deoxyribonuclease I/metabolism ; *Gene Editing ; Mammals/genetics ; *RNA, Guide/genetics ; RNA-Directed DNA Polymerase/metabolism ; },
abstract = {Prime editors consisting of Cas9-nickase and reverse transcriptase enable targeted precise editing of small DNA pieces, including all 12 kinds of base substitutions, insertions and deletions, while without requiring double-strand breaks or donor templates. Current optimized prime editing strategy (PE3) uses two guide RNAs to guide the performance of prime editor. One guide RNA carrying both spacer and templating sequences (pegRNA) guides prime editor to produce ssDNA break and subsequent extension, and the other one produces a nick in the complementary strand. Here, we demonstrated that positioning the nick sgRNA nearby the templating sequences of the pegRNA facilitated targeted large fragment deletion and that engineering both guide RNAs to be pegRNAs to achieve bi-direction prime editing (Bi-PE) further increase the efficiency by up to 16 times and improved the accuracy of editing products by 60 times. In addition, we showed that Bi-PE strategy also increased the efficiency of simultaneous conversion of multiple bases but not single base conversion over PE3. In conclusion, Bi-PE strategy expanded the editing scope and improved the efficiency and the accuracy of prime editing system, which might have a wide range of potential applications.},
}
@article {pmid35686982,
year = {2022},
author = {Sanchez-Baltasar, R and Garcia-Torralba, A and Nieto-Romero, V and Page, A and Molinos-Vicente, A and López-Manzaneda, S and Ojeda-Pérez, I and Ramirez, A and Navarro, M and Segovia, JC and García-Bravo, M},
title = {Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {422-434},
pmid = {35686982},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; Mice, Knockout ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics ; *Zygote/metabolism ; },
abstract = {Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.},
}
@article {pmid35686981,
year = {2022},
author = {Lyons, LA},
title = {It's a Knockout for Cat Allergies?.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {356-357},
doi = {10.1089/crispr.2022.29148.lal},
pmid = {35686981},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; *Hypersensitivity/genetics ; },
}
@article {pmid35686980,
year = {2022},
author = {Shen, X and Lin, Q and Liang, Z and Wang, J and Yang, X and Liang, Y and Liang, H and Pan, H and Yang, J and Zhu, Y and Li, M and Xiang, W and Zhu, H},
title = {Reduction of Pre-Existing Adaptive Immune Responses Against SaCas9 in Humans Using Epitope Mapping and Identification.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {445-456},
doi = {10.1089/crispr.2021.0142},
pmid = {35686980},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Epitope Mapping ; *Gene Editing/methods ; HLA-A Antigens/genetics ; Humans ; Immunity ; Leukocytes, Mononuclear ; *Staphylococcus aureus/genetics ; },
abstract = {The CRISPR-Cas9 system is increasingly being used as a gene editing therapeutic technique in complex diseases but concerns remain regarding the clinical risks of Cas9 immunogenicity. In this study, we detected antibodies against Staphylococcus aureus Cas9 (SaCas9) and anti-SaCas9 T cells in 4.8% and 70% of Chinese donors, respectively. We predicted 135 SaCas9-derived B cell epitopes and 50 SaCas9-derived CD8+ T cell epitopes for HLA-A*24:02, HLA-A*11:01, and HLA-A*02:01. We identified R338 as an immunodominant SaCas9 B cell epitope and SaCas9_200-208 as an immunodominant CD8+ T cell epitope for the three human leukocyte antigen allotypes through immunological assays using sera positive for SaCas9-specific antibodies and peripheral blood mononuclear cells positive for SaCas9-reactive T cells, respectively. We also demonstrated that an SaCas9 variant bearing an R338G substitution reduces B cell immunogenicity and retains its gene-editing function. Our study highlights the immunological risks of the CRISPR-Cas9 system and provides a solution to mitigate pre-existing adaptive immune responses against Cas9 in humans.},
}
@article {pmid35686979,
year = {2022},
author = {Lo Presti, V and Cutilli, A and Dogariu, Y and Müskens, KF and Dünnebach, E and van den Beemt, DAMH and Cornel, AM and Plantinga, M and Nierkens, S},
title = {Gene Editing of Checkpoint Molecules in Cord Blood-Derived Dendritic Cells and CD8[+] T Cells Using CRISPR-Cas9.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {435-444},
doi = {10.1089/crispr.2021.0133},
pmid = {35686979},
issn = {2573-1602},
mesh = {CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Dendritic Cells ; Fetal Blood ; *Gene Editing/methods ; Humans ; *Neoplasms/genetics ; },
abstract = {Immunotherapies targeting checkpoint inhibition and cell therapies are considered breakthroughs for cancer therapy. However, only a part of patients benefit from these treatments and resistance has been observed. Combining both approaches can potentially further enhance their efficacy. With the advent of gene editing techniques, such as clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), the elimination of checkpoint molecules became available as an option in good manufacturing practice conditions to improve persistence and efficacy. However, no data of CRISPR-Cas9 application have been reported in cord blood (CB)-derived immune cells, potentially usable for allogeneic cell therapy purposes. In this article, we describe the optimization of a protocol to deplete checkpoint molecules at the genomic level using CRISPR-Cas9 technology from CB-dendritic cells (DCs) and CB-CD8[+] T cells. The protocol is based on the electroporation of a ribonucleoprotein complex, easily translatable to clinical settings. In both cell types, the knock-out (KO) was successful and did not affect cell viability. CB-DCs showed a decrease in expression of the targeted protein ranging from 50% to 95%, while CB-CD8[+] T cells showed a reduction in the range of 25-45%. The procedure did not affect the stimulatory function of the CB-DCs or the response of CB-CD8[+] T cells (proliferation or TNF-α production). In conclusion, we optimized a protocol to eliminate checkpoint molecules from CB-derived DCs and CD8[+] T cells, with the aim to further implement allogeneic cell therapies for cancer.},
}
@article {pmid35686978,
year = {2022},
author = {Torriano, S and Baulier, E and Garcia Diaz, A and Corneo, B and Farber, DB},
title = {CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {457-471},
pmid = {35686978},
issn = {2573-1602},
mesh = {*Albinism, Ocular/genetics/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Eye Proteins/genetics/metabolism ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Membrane Glycoproteins/genetics/metabolism ; Mutation ; },
abstract = {Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143. This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.},
}
@article {pmid35686977,
year = {2022},
author = {Chen, S and Liu, Z and Xie, W and Yu, H and Lai, L and Li, Z},
title = {Compact Cje3Cas9 for Efficient In Vivo Genome Editing and Adenine Base Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {472-486},
doi = {10.1089/crispr.2021.0143},
pmid = {35686977},
issn = {2573-1602},
mesh = {Adenine ; Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; *Proprotein Convertase 9/genetics ; },
abstract = {Many therapeutic applications of CRISPR-Cas9 gene editing rely on delivery using the highly versatile adeno-associated virus (AAV) vector. The smallest type II Cas9 ortholog-Cje1Cas9, derived from Campylobacter jejuni with <1,000 amino acids-is particularly attractive for AAV delivery. However, the complex protospacer adjacent motif (PAM) of Cje1Cas9 (N3VRYAC) greatly restricts the density of recognition sequences in human genome. In this study, we identify two compact CjeCas9 orthologs designated as Cje2Cas9 and Cje3Cas9, whose PAM-interacting residues are different from those of the well-known Cje1Cas9. They can induce efficient genome editing in human cells, and their simpler trinucleotide PAM (N4CYA) requirements expand the scope of targeting. Moreover, Cje3Cas9 efficiently disrupts the Tyr gene in mice after being micro-injected into zygotes with the corresponding sgRNA. It also successfully disrupts the Pcsk9 gene in 8-week-old mouse liver after delivery with an sgRNA using an all-in-one AAV delivery vehicle. The gene-edited mice showed lower cholesterol level than wild-type mice. Notably, the 8e-nCje3-ABE and an sgRNA targeting Pcsk9 were successfully packaged into a single AAV vector for genome editing in adult mouse liver, with editing efficiency up to 12%. Thus, simple PAMs and a compact size enable Cje2/3Cas9 to expand the target scope of CRISPR-Cas9 toolsets, exhibiting considerable potential for therapeutic applications.},
}
@article {pmid35686976,
year = {2022},
author = {Bradford, J and Chappell, T and Perrin, D},
title = {Rapid Whole-Genome Identification of High Quality CRISPR Guide RNAs with the Crackling Method.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {410-421},
doi = {10.1089/crispr.2021.0102},
pmid = {35686976},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome ; *RNA, Guide/genetics ; Software ; },
abstract = {The design of CRISPR-Cas9 guide RNAs is not trivial and is a computationally demanding task. Design tools need to identify target sequences that will maximize the likelihood of obtaining the desired cut, while minimizing off-target risk. There is a need for a tool that can meet both objectives while remaining practical to use on large genomes. In this study, we present Crackling, a new method that is more suitable for meeting these objectives. We test its performance on 12 genomes and on data from validation studies. Crackling maximizes guide efficiency by combining multiple scoring approaches. On experimental data, the guides it selects are better than those selected by others. It also incorporates Inverted Signature Slice Lists (ISSL) for faster off-target scoring. ISSL provides a gain of an order of magnitude in speed compared with other popular tools, such as Cas-OFFinder, Crisflash, and FlashFry, while preserving the same level of accuracy. Overall, this makes Crackling a faster and better method to design guide RNAs at scale. Crackling is available at https://github.com/bmds-lab/Crackling under the Berkeley Software Distribution (BSD) 3-Clause license.},
}
@article {pmid35686974,
year = {2022},
author = {Barrangou, R},
title = {Celebrating a Decade of CRISPR-Fueled Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {354-355},
doi = {10.1089/crispr.2022.29150.rba},
pmid = {35686974},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35686327,
year = {2022},
author = {Gantz, VM and Bier, E},
title = {Active genetics comes alive: Exploring the broad applications of CRISPR-based selfish genetic elements (or gene-drives): Exploring the broad applications of CRISPR-based selfish genetic elements (or gene-drives).},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {44},
number = {8},
pages = {e2100279},
pmid = {35686327},
issn = {1521-1878},
support = {R01 AI131081/AI/NIAID NIH HHS/United States ; R01 AI162911/AI/NIAID NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Gene Editing ; Inheritance Patterns ; RNA, Guide/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based "active genetic" elements developed in 2015 bypassed the fundamental rules of traditional genetics. Inherited in a super-Mendelian fashion, such selfish genetic entities offered a variety of potential applications including: gene-drives to disseminate gene cassettes carrying desired traits throughout insect populations to control disease vectors or pest species, allelic drives biasing inheritance of preferred allelic variants, neutralizing genetic elements to delete and replace or to halt the spread of gene-drives, split-drives with the core constituent Cas9 endonuclease and guide RNA (gRNA) components inserted at separate genomic locations to accelerate assembly of complex arrays of genetic traits or to gain genetic entry into novel organisms (vertebrates, plants, bacteria), and interhomolog based copying systems in somatic cells to develop tools for treating inherited or infectious diseases. Here, we summarize the substantial advances that have been made on all of these fronts and look forward to the next phase of this rapidly expanding and impactful field.},
}
@article {pmid35686011,
year = {2022},
author = {Parsaeimehr, A and Ebirim, RI and Ozbay, G},
title = {CRISPR-Cas technology a new era in genomic engineering.},
journal = {Biotechnology reports (Amsterdam, Netherlands)},
volume = {34},
number = {},
pages = {e00731},
pmid = {35686011},
issn = {2215-017X},
abstract = {The CRISPR-Cas systems have offered a flexible, easy-to-use platform to precisely modify and control the genomes of organisms in various fields, ranging from agricultural biotechnology to therapeutics. This system is extensively used in the study of infectious, progressive, and life-threatening genetic diseases for the improvement of quality and quantity of major crops and in the development of sustainable methods for the generation of biofuels. As CRISPR-Cas technology continues to evolve, it is becoming more controllable and precise with the addition of molecular regulators, which will provide benefits for everyone and save many lives. Studies on the constant growth of CRISPR technology are important due to its rapid development. In this paper, we present the current applications and progress of CRISPR-Cas genome editing systems in several fields of research, we further highlight the applications of anti-CRISPR molecules to regulate CRISPR-Cas gene editing systems, and we discuss ethical considerations in CRISPR-Cas applications.},
}
@article {pmid35682838,
year = {2022},
author = {Happi Mbakam, C and Rousseau, J and Tremblay, G and Yameogo, P and Tremblay, JP},
title = {Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy.},
journal = {International journal of molecular sciences},
volume = {23},
number = {11},
pages = {},
pmid = {35682838},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; *Dystrophin/genetics/metabolism ; Gene Editing/methods ; HEK293 Cells ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Mutation ; },
abstract = {The Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (DMD) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the DMD gene in HEK293T cells were obtained with the PRIME Editor 2 (PE2) and PE3, respectively. Three repeated treatments increased the percentage of specific mutations with PE2 to 16%. An additional mutation in the protospacer adjacent motif (PAM) sequence improved the PE3 result to 38% after a single treatment. We also carried out the correction of c.428 G>A point mutation in exon 6 of the DMD gene in a patient myoblast. Myoblast electroporation showed up to 8% and 28% modifications, respectively, for one and three repeated treatments using the PE3 system. The myoblast correction led to dystrophin expression in myotubes detected by Western blot. Thus, prime editing can be used for the correction of point mutations in the DMD gene.},
}
@article {pmid35682737,
year = {2022},
author = {Bhardwaj, P and Kant, R and Behera, SP and Dwivedi, GR and Singh, R},
title = {Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection.},
journal = {International journal of molecular sciences},
volume = {23},
number = {11},
pages = {},
pmid = {35682737},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Nucleic Acids/genetics ; RNA ; RNA, Guide/genetics ; },
abstract = {The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.},
}
@article {pmid35682671,
year = {2022},
author = {Smirnikhina, SA and Zaynitdinova, MI and Sergeeva, VA and Lavrov, AV},
title = {Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle.},
journal = {International journal of molecular sciences},
volume = {23},
number = {11},
pages = {},
pmid = {35682671},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Cell Division ; *Gene Editing ; Humans ; Mammals/genetics ; Recombinational DNA Repair ; },
abstract = {Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation.},
}
@article {pmid35681543,
year = {2022},
author = {Liu, X and Wang, S and Ai, D},
title = {Predicting CRISPR/Cas9 Repair Outcomes by Attention-Based Deep Learning Framework.},
journal = {Cells},
volume = {11},
number = {11},
pages = {},
pmid = {35681543},
issn = {2073-4409},
mesh = {*CRISPR-Cas Systems/genetics ; *Deep Learning ; Endonucleases/genetics ; Gene Editing/methods ; Mutation/genetics ; },
abstract = {As a simple and programmable nuclease-based genome editing tool, the CRISPR/Cas9 system has been widely used in target-gene repair and gene-expression regulation. The DNA mutation generated by CRISPR/Cas9-mediated double-strand breaks determines its biological and phenotypic effects. Experiments have demonstrated that CRISPR/Cas9-generated cellular-repair outcomes depend on local sequence features. Therefore, the repair outcomes after DNA break can be predicted by sequences near the cleavage sites. However, existing prediction methods rely on manually constructed features or insufficiently detailed prediction labels. They cannot satisfy clinical-level-prediction accuracy, which limit the performance of these models to existing knowledge about CRISPR/Cas9 editing. We predict 557 repair labels of DNA, covering the vast majority of Cas9-generated mutational outcomes, and build a deep learning model called Apindel, to predict CRISPR/Cas9 editing outcomes. Apindel, automatically, trains the sequence features of DNA with the GloVe model, introduces location information through Positional Encoding (PE), and embeds the trained-word vector matrixes into a deep learning model, containing BiLSTM and the Attention mechanism. Apindel has better performance and more detailed prediction categories than the most advanced DNA-mutation-predicting models. It, also, reveals that nucleotides at different positions relative to the cleavage sites have different influences on CRISPR/Cas9 editing outcomes.},
}
@article {pmid35681058,
year = {2022},
author = {Hartweger, H and Nussenzweig, MC},
title = {CRISPR comes a-knock-in to reprogram antibodies in vivo.},
journal = {Nature biotechnology},
volume = {40},
number = {8},
pages = {1183-1184},
pmid = {35681058},
issn = {1546-1696},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene Knock-In Techniques ; },
}
@article {pmid35679361,
year = {2022},
author = {Liu, N and Olson, EN},
title = {CRISPR Modeling and Correction of Cardiovascular Disease.},
journal = {Circulation research},
volume = {130},
number = {12},
pages = {1827-1850},
pmid = {35679361},
issn = {1524-4571},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 AR071980/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Mice ; },
abstract = {Cardiovascular disease remains the leading cause of morbidity and mortality in the developed world. In recent decades, extraordinary effort has been devoted to defining the molecular and pathophysiological characteristics of the diseased heart and vasculature. Mouse models have been especially powerful in illuminating the complex signaling pathways, genetic and epigenetic regulatory circuits, and multicellular interactions that underlie cardiovascular disease. The advent of CRISPR genome editing has ushered in a new era of cardiovascular research and possibilities for genetic correction of disease. Next-generation sequencing technologies have greatly accelerated the identification of disease-causing mutations, and advances in gene editing have enabled the rapid modeling of these mutations in mice and patient-derived induced pluripotent stem cells. The ability to correct the genetic drivers of cardiovascular disease through delivery of gene editing components in vivo, while still facing challenges, represents an exciting therapeutic frontier. In this review, we provide an overview of cardiovascular disease mechanisms and the potential applications of CRISPR genome editing for disease modeling and correction. We also discuss the extent to which mice can faithfully model cardiovascular disease and the opportunities and challenges that lie ahead.},
}
@article {pmid35679334,
year = {2022},
author = {Fan, S and Zhang, Z and Song, Y and Zhang, J and Wang, P},
title = {CRISPR/Cas9-mediated targeted mutagenesis of GmTCP19L increasing susceptibility to Phytophthora sojae in soybean.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0267502},
pmid = {35679334},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Disease Resistance/genetics ; Mutagenesis ; *Phytophthora/physiology ; Plant Breeding ; Plant Diseases/genetics ; Plant Proteins/metabolism ; Soybeans ; Transcription Factors/genetics/metabolism ; },
abstract = {The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors is one of the superfamilies of plant-specific transcription factors involved in plant growth, development, and biotic and abiotic stress. However, there is no report on the research of the TCP transcription factors in soybean response to Phytophthora sojae. In this study, Agrobacterium-mediated transformation was used to introduce the CRISPR/Cas9 expression vector into soybean cultivar "Williams 82" and generated targeted mutants of GmTCP19L gene, which was previously related to involve in soybean responses to P. sojae. We obtained the tcp19l mutants with 2-bp deletion at GmTCP19L coding region, and the frameshift mutations produced premature translation termination codons and truncated GmTCP19L proteins, increasing susceptibility to P. sojae in the T2-generation. These results suggest that GmTCP19L encodes a TCP transcription factor that affects plant defense in soybean. The new soybean germplasm with homozygous tcp19l mutations but the BAR and Cas9 sequences were undetectable using strip and PCR methods, respectively, suggesting directions for the breeding or genetic engineering of disease-resistant soybean plants.},
}
@article {pmid35678575,
year = {2022},
author = {Sharma, N and Das, A and Raja, P and Marathe, SA},
title = {The CRISPR-Cas System Differentially Regulates Surface-Attached and Pellicle Biofilm in Salmonella enterica Serovar Typhimurium.},
journal = {Microbiology spectrum},
volume = {10},
number = {3},
pages = {e0020222},
pmid = {35678575},
issn = {2165-0497},
mesh = {Bacterial Proteins/genetics/metabolism ; Biofilms ; CRISPR-Cas Systems ; Cellulose/metabolism ; *Gene Expression Regulation, Bacterial ; Lipopolysaccharides/metabolism ; *Salmonella typhimurium/metabolism ; Serogroup ; },
abstract = {The CRISPR-Cas mediated regulation of biofilm by Salmonella enterica serovar Typhimurium was investigated by deleting CRISPR-Cas components ΔcrisprI, ΔcrisprII, ΔΔcrisprI crisprII, and Δcas op. We determined that the system positively regulates surface biofilm while inhibiting pellicle biofilm formation. Results of real-time PCR suggest that the flagellar (fliC, flgK) and curli (csgA) genes were repressed in knockout strains, causing reduced surface biofilm. The mutants displayed altered pellicle biofilm architecture. They exhibited bacterial multilayers and a denser extracellular matrix with enhanced cellulose and less curli, ergo weaker pellicles than those of the wild type. The cellulose secretion was more in the knockout strains due to the upregulation of bcsC, which is necessary for cellulose export. We hypothesized that the secreted cellulose quickly integrates into the pellicle, leading to enhanced pellicular cellulose in the knockout strains. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD and, hence, also of csgA and bcsA. The conflicting upregulation of bcsC, the last gene of the bcsABZC operon, could be caused by independent regulation by the CRISPR-Cas system owing to a partial match between the CRISPR spacers and bcsC gene. The cAMP-regulated protein (CRP)-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of yddx governing the availability of the sigma factor σ[28] that further regulates class 3 flagellar genes (fliC, fljB, and flgK). Additionally, the variations in the lipopolysaccharide (LPS) profile and expression of LPS-related genes (rfaC, rfbG, and rfbI) in knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle biofilm. IMPORTANCE In addition to being implicated in bacterial immunity and genome editing, the CRISPR-Cas system has recently been demonstrated to regulate endogenous gene expression and biofilm formation. While the function of individual cas genes in controlling Salmonella biofilm has been explored, the regulatory role of CRISPR arrays in biofilm is less studied. Moreover, studies have focused on the effects of the CRISPR-Cas system on surface-associated biofilms, and comprehensive studies on the impact of the system on pellicle biofilm remain an unexplored niche. We demonstrate that the CRISPR array and cas genes modulate the expression of various biofilm genes in Salmonella, whereby surface and pellicle biofilm formation is distinctively regulated.},
}
@article {pmid35674887,
year = {2022},
author = {Andreyenkov, OV and Volkova, EI and Andreyenkova, NG and Demakov, SA},
title = {Using the CRISPR/Cas9 System for Dissection of Functional Sites of the Notch Gene in Drosophila melanogaster.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2472},
number = {},
pages = {1-14},
pmid = {35674887},
issn = {1940-6029},
mesh = {Animals ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Drosophila/metabolism ; *Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics/metabolism ; },
abstract = {The Notch gene is a key factor in the signaling cascade that allows communication between neighboring cells in many organisms, from worms and insects to humans. The relative simplicity of the Notch pathway in Drosophila, combined with a powerful set of molecular and cytogenetic methods, makes this model attractive for studying the fundamental principles of Notch regulation and functioning. Here, using the CRISPR/Cas9 system in combination with homologous recombination, for the first time at the level of the whole organism, we obtained a directed deletion of the 5'-regulatory region and the first exon of the Notch gene, which were replaced by the attP integration site of the ΦC31 phage. Based on this approach, we obtained and characterized new Notch mutations. Thus, a new powerful tool is provided for studying the genetic regulation of the Notch gene and the organization of chromatin at this locus.},
}
@article {pmid35674844,
year = {2022},
author = {Bhattacharjee, R and Das Roy, L and Choudhury, A},
title = {Understanding on CRISPR/Cas9 mediated cutting-edge approaches for cancer therapeutics.},
journal = {Discover. Oncology},
volume = {13},
number = {1},
pages = {45},
pmid = {35674844},
issn = {2730-6011},
abstract = {The research focus on CRISPR/Cas9 has gained substantial concentration since the discovery of 'an unusual repeat sequence' reported by Ishino et al. (J Bacteriol 169:5429-5433, 1987) and the journey comprises the recent Nobel Prize award (2020), conferred to Emmanuelle Charpentier and Jennifer Doudna. Cumulatively, the CRISPR has a short, compact, and most discussed success of its application in becoming one of the most versatile and paradigm shifting technologies of Biological Research. Today, the CRISPR/Cas9 genome editing system is almost ubiquitously utilized in many facets of biological research where its tremendous gene manipulation capability has been harnessed to create miracles. From 2012, the CRISPR/Cas 9 system has been showcased in almost 15,000 research articles in the PubMed database, till date. Backed by some strong molecular evidence, the CRISPR system has been utilized in a few clinical trials targeted towards various pathologies. While the area covered by CRISPR is cosmic, this review will focus mostly on the utilization of CRISPR/Cas9 technology in the field of cancer therapy.},
}
@article {pmid35672603,
year = {2022},
author = {Negi, C and Vasistha, NK and Singh, D and Vyas, P and Dhaliwal, HS},
title = {Application of CRISPR-Mediated Gene Editing for Crop Improvement.},
journal = {Molecular biotechnology},
volume = {64},
number = {11},
pages = {1198-1217},
pmid = {35672603},
issn = {1559-0305},
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; Plant Breeding/methods ; RNA ; Transcription Activator-Like Effectors/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {Plant gene editing has become an important molecular tool to revolutionize modern breeding of crops. Over the past years, remarkable advancement has been made in developing robust and efficient editing methods for plants. Despite a variety of available genome editing methods, the discovery of most recent system of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) has been one of the biggest advancement in this path, with being the most efficient approach for genome manipulation. Until recently, genetic manipulations were confined to methods, like Agrobacterium-mediated transformations, zinc-finger nucleases, and TAL effector nucleases. However this technology supersedes all other methods for genetic modification. This RNA-guided CRISPR-Cas system is being rapidly developed with enhanced functionalities for better use and greater possibilities in biological research. In this review, we discuss and sum up the application of this simple yet powerful tool of CRISPR-Cas system for crop improvement with recent advancement in this technology.},
}
@article {pmid35671698,
year = {2022},
author = {Ke, X and Ou, Y and Lin, Y and Hu, T},
title = {Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a.},
journal = {Biosensors &amp; bioelectronics},
volume = {212},
number = {},
pages = {114428},
doi = {10.1016/j.bios.2022.114428},
pmid = {35671698},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Luminescence ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; },
abstract = {CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.},
}
@article {pmid35671288,
year = {2022},
author = {Castle, AR and Wohlgemuth, S and Arce, L and Westaway, D},
title = {Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0269342},
pmid = {35671288},
issn = {1932-6203},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Mammals/genetics ; Mice ; *Prion Diseases/genetics ; Prion Proteins/genetics ; *Prions/genetics ; RNA, Guide ; *Wasting Disease, Chronic/genetics ; },
abstract = {Prion diseases are a group of fatal neurodegenerative disorders that includes chronic wasting disease, which affects cervids and is highly transmissible. Given that chronic wasting disease prevalence exceeds 30% in some endemic areas of North America, and that eventual transmission to other mammalian species, potentially including humans, cannot be ruled out, novel control strategies beyond population management via hunting and/or culling must be investigated. Prion diseases depend upon post-translational conversion of the cellular prion protein, encoded by the Prnp gene, into a disease-associated conformation; ablation of cellular prion protein expression, which is generally well-tolerated, eliminates prion disease susceptibility entirely. Inspired by demonstrations of gene drive in caged mosquito species, we aimed to test whether a CRISPR/Cas9-based gene drive mechanism could, in principle, promote the spread of a null Prnp allele among mammalian populations. First, we showed that transient co-expression of Cas9 and Prnp-directed guide RNAs in RK13 cells generates indels within the Prnp open-reading frame, indicating that repair of Cas9-induced double-strand breaks by non-homologous end-joining had taken place. Second, we integrated a ~1.2 kb donor DNA sequence into the Prnp open-reading frame in N2a cells by homology-directed repair following Cas9-induced cleavages and confirmed that integration occurred precisely in most cases. Third, we demonstrated that electroporation of Cas9/guide RNA ribonucleoprotein complexes into fertilised mouse oocytes resulted in pups with a variety of disruptions to the Prnp open reading frame, with a new coisogenic line of Prnp-null mice obtained as part of this work. However, a technical challenge in obtaining expression of Cas9 in the male germline prevented implementation of a complete gene drive mechanism in mice.},
}
@article {pmid35671066,
year = {2022},
author = {Klinnert, S and Chemnitzer, A and Rusert, P and Metzner, KJ},
title = {Systematic HIV-1 promoter targeting with CRISPR/dCas9-VPR reveals optimal region for activation of the latent provirus.},
journal = {The Journal of general virology},
volume = {103},
number = {6},
pages = {},
doi = {10.1099/jgv.0.001754},
pmid = {35671066},
issn = {1465-2099},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *HIV Infections ; *HIV Seropositivity ; *HIV-1/genetics ; Humans ; Proviruses/genetics ; RNA, Guide/genetics ; Virus Activation/genetics ; Virus Latency/genetics ; },
abstract = {CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the 'shock and kill' cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5'LTR promoter, making them a promising 'shock' strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5'LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3' half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located -165 to -106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the 'shock and kill' cure approach.},
}
@article {pmid35670994,
year = {2022},
author = {Rahimi, H and Zaboli, KA and Thekkiniath, J and Mousavi, SH and Johari, B and Hashemi, MR and Nosrati, H and Goldschneider, D and Bernet, A and Danafar, H and Kaboli, S},
title = {BSA-PEI Nanoparticle Mediated Efficient Delivery of CRISPR/Cas9 into MDA-MB-231 Cells.},
journal = {Molecular biotechnology},
volume = {64},
number = {12},
pages = {1376-1387},
pmid = {35670994},
issn = {1559-0305},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Humans ; Mice ; *Nanoparticles ; Polyethyleneimine ; Ribonucleoproteins/genetics/metabolism ; Serum Albumin, Bovine ; },
abstract = {The discovery of bacterial-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome engineering and gene therapy due to its wide range of applications. One of the major challenging issues in CRISPR/Cas system is the lack of an efficient, safe, and clinically suitable delivery of the system's components into target cells. Here, we describe the development of polyethylenimine coated-bovine serum albumin nanoparticles (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both DNA (px458 plasmid) and ribonucleoprotein (RNP) forms into MDA-MB-231 human breast cancer cell line. Our data showed that synthesized BSA-PEI (BP) NPs delivered plasmid px458 at concentrations of 0.15, 0.25, and 0.35 µg/µl with efficiencies of approximately 29.7, 54.8, and 84.1% into MDA-MB-231 cells, respectively. Our study demonstrated that Cas9/sgRNA RNP complex efficiently (~ 92.6%) delivered by BSA-PEI NPs into the same cells. Analysis of toxicity and biocompatibility of synthesized NPs on human red blood cells, MDA-MB-231 cells, and mice showed that the selected concentration (28 µg/µl) of BSA-PEI NPs for transfection had no remarkable toxicity effects. Thus, obtained results suggest BSA-PEI NPs as one of the most promising carrier for delivering CRISPR/Cas9 to target cells.},
}
@article {pmid35670674,
year = {2022},
author = {Marino, ND and Pinilla-Redondo, R and Bondy-Denomy, J},
title = {CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity.},
journal = {Nucleic acids research},
volume = {50},
number = {11},
pages = {6414-6422},
pmid = {35670674},
issn = {1362-4962},
support = {R01 GM127489/GM/NIGMS NIH HHS/United States ; F32 GM133127/GM/NIGMS NIH HHS/United States ; DP5 OD021344/OD/NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA ; *DNA, Single-Stranded/genetics ; Escherichia coli/genetics/metabolism ; *Immunity/genetics ; Pseudomonas aeruginosa/genetics/metabolism ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting dsDNA phage, ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities do not significantly impact infection outcomes.},
}
@article {pmid35670669,
year = {2022},
author = {Lu, Z and Ni, K and Wang, Y and Zhou, Y and Li, Y and Yan, J and Song, Q and Liu, M and Xu, Y and Yu, Z and Guo, T and Ma, L},
title = {An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations.},
journal = {Nucleic acids research},
volume = {50},
number = {11},
pages = {6575-6586},
pmid = {35670669},
issn = {1362-4962},
mesh = {CD8-Positive T-Lymphocytes/immunology ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Library ; Lymphocyte Activation ; Neoplasms/immunology ; *RNA, Guide/genetics ; },
abstract = {Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors.},
}
@article {pmid35670380,
year = {2022},
author = {Huang, L and Zhou, M and Abbas, G and Li, C and Cui, M and Zhang, XE and Wang, DB},
title = {A Cancer Cell Membrane-Derived Biomimetic Nanocarrier for Synergistic Photothermal/Gene Therapy by Efficient Delivery of CRISPR/Cas9 and Gold Nanorods.},
journal = {Advanced healthcare materials},
volume = {11},
number = {16},
pages = {e2201038},
doi = {10.1002/adhm.202201038},
pmid = {35670380},
issn = {2192-2659},
mesh = {Biomimetics ; CRISPR-Cas Systems/genetics ; Cell Membrane ; Genetic Therapy/methods ; Gold/pharmacology ; Humans ; *Nanotubes ; *Neoplasms/therapy ; Survivin/genetics ; },
abstract = {Bimodal synergistic therapy produces superadditive effect for enhanced therapeutic efficacy. However, how to efficiently and simultaneously deliver several kinds of therapeutic agents is still challenging. A cancer cell membrane-derived nanocarrier (mCas9-sGNRs) is proposed for synergistic photothermal/gene therapy (PTT/GT) by efficient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) and gold nanorods (GNRs). In this approach, Cas9 proteins can be efficiently loaded inside the cell membranes (mCas9) by electrostatic interactions. Similarly, single-guide RNAs, which target survivin, can be loaded onto GNRs (sGNRs) through electrostatic interactions and encapsulated by mCas9. As a result, the nanodelivery systems present advantages in biocompatibility, homologous targeting capacity and loading efficiency of cargoes. In addition, significant antitumor effects is achieved by gene editing of survivin which induces anticancer activity and reduces heat tolerance of cancer cells caused by GNRs mediated PTT due to the downregulation of HSP70. These results indicate the nanotherapeutic platform leads to enhanced PTT/GT efficacy. Therefore, this work not only provides a general strategy to construct a versatile nanoplatform for loading and target delivery of several therapeutic cargos but will also be valuable for PTT/GT and other bimodal synergistic therapy.},
}
@article {pmid35670376,
year = {2022},
author = {Zhang, W and Shi, R and Dong, K and Hu, H and Shu, W and Mu, Y and Yan, B and Li, L and Xiao, X and Wang, H},
title = {The Off-Target Effect of CRISPR-Cas12a System toward Insertions and Deletions between Target DNA and crRNA Sequences.},
journal = {Analytical chemistry},
volume = {94},
number = {24},
pages = {8596-8604},
doi = {10.1021/acs.analchem.1c05499},
pmid = {35670376},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Mutation ; RNA/genetics ; },
abstract = {The CRISPR-Cas12a system is a new type of genome editing tool with high efficiency and targeting. However, other sequences in the genome may also be cleaved nonspecifically, resulting in unavoidable off-target effects. Therefore, it is necessary to learn more about the mechanism of CRISPR-Cas12a to recognize target sequences to avoid its off-target effects. Here, we show that insertion (DNA bubble) or deletion (RNA bubble) of the target dsDNA sequence compared with the crRNA sequence, the CRISPR-Cas12a system can still recognize and cleave the target dsDNA sequence. We conclude that the tolerance of CRISPR-Cas12a to the bubbles is closely related to the location and size of the bubble and the GC base content of crRNA. In addition, we used the unique property of CRISPR-Cas12a to invent a new method to detect mutations and successfully detect the CD41-42(-CTTT) mutation. The detection limit of this method is 0.001%. Overall, our results strongly indicate that in addition to considering off-target effects caused by base mismatches, a comprehensive off-target analysis of the insertion and deletion of the target dsDNA sequence is required, and specific guidelines for effectively reducing potential off-target cleavage are proposed, to improve the safety manual of CRISPR-Cas12a biological application.},
}
@article {pmid35668177,
year = {2022},
author = {Tang, XE and Tan, SX and Hoon, S and Yeo, GW},
title = {Pre-existing adaptive immunity to the RNA-editing enzyme Cas13d in humans.},
journal = {Nature medicine},
volume = {28},
number = {7},
pages = {1372-1376},
pmid = {35668177},
issn = {1546-170X},
mesh = {Adaptive Immunity ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; RNA ; Staphylococcus aureus ; },
abstract = {RNA-guided RNA-targeting nucleases, such as CRISPR-Cas13 proteins, have therapeutic potential for gene editing. Among Cas13d enzymes, Cas13d from the bacteria Ruminococcus flavefaciens (RfxCas13d) is of particular interest owing to its small size and high specificity. However, the existence of pre-existing immunity against RfxCas13d is unclear. In this study, we evaluated antibody and T cell responses to RfxCas13d in healthy donors using ELISA and T cell culture assays. We found RfxCas13d-reactive antibodies and CD4 and CD8 T cell responses in most donors, comparable to responses against Cas9 proteins from Staphylococcus aureus (SaCas9) and Streptococcus pyogenes (SpCas9). RfxCas13d-responding T cells could produce the inflammatory cytokines IFN-γ, TNF-α and IL-17. These findings should be taken into consideration in the development of RfxCas13d for therapy.},
}
@article {pmid35667925,
year = {2022},
author = {Eid, A and Qi, Y},
title = {Prime editor integrase systems boost targeted DNA insertion and beyond.},
journal = {Trends in biotechnology},
volume = {40},
number = {8},
pages = {907-909},
doi = {10.1016/j.tibtech.2022.05.002},
pmid = {35667925},
issn = {1879-3096},
mesh = {Animals ; CRISPR-Cas Systems ; *DNA/genetics ; *Gene Editing/methods ; Genome, Human ; Humans ; *Integrases/genetics/metabolism ; Plasmids ; },
abstract = {Previously developed genome engineering tools cannot efficiently direct site-specific long DNA insertion. Built on the prime editing platform, two recent studies have reported integrase-mediated site-specific long DNA integration in the human genome. These prime editor integrase (PEI) systems will unleash many exciting applications in humans, animals, and plants.},
}
@article {pmid35666940,
year = {2022},
author = {Hou, Y and Wang, D and Lu, S and Guo, D and Li, M and Cui, M and Zhang, XE},
title = {Optogenetic Control of Background Fluorescence Reduction for CRISPR-Based Genome Imaging.},
journal = {Analytical chemistry},
volume = {94},
number = {24},
pages = {8724-8731},
doi = {10.1021/acs.analchem.2c01113},
pmid = {35666940},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Nucleus ; Genome ; Microscopy, Fluorescence ; *Optogenetics ; },
abstract = {The CRISPR/dCas9 system has become an essential tool for live-cell imaging of genomic loci, but it has limited applications in imaging low-/non-repetitive genomic loci due to the strong nuclear background noise emerging from many untargeted fluorescent modules. Here, we propose an optogenetically controlled background fluorescence reduction strategy that combines the CRISPR-SunTag system with a light-inducible nuclear export tag (LEXY). Utilizing the SunTag system, multiple copies of LEXY-tagged sfGFP were recruited to the C-terminal dCas9, recognizing the target genomic loci. As the nuclear export sequence at the C-terminal LEXY could be exposed to pulsed blue light irradiation, the untargeted nuclear labeling modules were light controllably transferred to the cytoplasm. Consequently, genomic loci containing as few as nine copies of repeats were clearly visualized, and a significant increase in the signal-to-noise ratio was achieved. This simple and controllable method is expected to have a wide range of applications in cell biology.},
}
@article {pmid35666449,
year = {2022},
author = {Guerra-Resendez, RS and Hilton, IB},
title = {Harnessing CRISPR-Cas9 for Epigenetic Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {237-251},
pmid = {35666449},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Epigenome ; Epigenomics ; Gene Editing/methods ; *RNA, Guide/genetics/metabolism ; },
abstract = {Epigenome editing has become more precise and effective by coupling epigenetic effectors to the dCas9 protein and targeting regulatory regions such as promoters and enhancers. Here, we describe a basic methodology for performing an epigenome editing experiment, starting from gRNA design and cloning to transiently transfecting the gRNA plasmid and the CRISPR/dCas9-based epigenetic effector and finalizing with chromatin immunoprecipitation (ChIP) to validate changes in epigenetic state at a targeted genomic region.},
}
@article {pmid35666448,
year = {2022},
author = {Jiao, C and Beisel, CL},
title = {Reprogramming TracrRNAs for Multiplexed RNA Detection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {217-235},
pmid = {35666448},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *RNA/genetics ; *RNA, Guide/genetics ; },
abstract = {CRISPR-based detection and recording technologies are gaining increasing attention in disease surveillance and prevention. In this chapter, we describe how our recent discovery of noncanonical crRNAs inspired the engineering of reprogrammed tracrRNAs and led to a powerful platform for multiplexed RNA detection. We provide detailed protocols regarding how to design reprogrammed tracrRNA and carry out assays in vitro and in vivo.},
}
@article {pmid35666447,
year = {2022},
author = {Villegas Kcam, MC and Chappell, J},
title = {Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {203-215},
pmid = {35666447},
issn = {1940-6029},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; *RNA, Guide/genetics/metabolism ; Transcriptional Activation ; },
abstract = {CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.},
}
@article {pmid35666436,
year = {2022},
author = {Simmons, TR and Ellington, AD and Contreras, LM},
title = {RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2518},
number = {},
pages = {1-31},
pmid = {35666436},
issn = {1940-6029},
support = {R01 GM135495/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; Nucleotides ; Ribonucleoproteins/genetics/metabolism ; Synthetic Biology ; },
abstract = {Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.},
}
@article {pmid35666156,
year = {2022},
author = {Kang, M and Zuo, Z and Yin, Z and Gu, J},
title = {Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition.},
journal = {Journal of chemical information and modeling},
volume = {62},
number = {12},
pages = {3057-3066},
doi = {10.1021/acs.jcim.1c01562},
pmid = {35666156},
issn = {1549-960X},
mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; CRISPR-Cas Systems/genetics ; DNA/chemistry ; *RNA, Guide/chemistry/genetics ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {The off-target effects of Streptococcus pyogenes Cas9 (SpCas9) pose a significant challenge to harness it as a therapeutical approach. Two major factors can result in SpCas9 off-targeting: tolerance to target DNA-guide RNA (gRNA) mismatch and less stringent recognition of protospacer adjacent motif (PAM) flanking the target DNA. Despite the abundance of engineered SpCas9-gRNA variants with improved sensitivity to target DNA-gRNA mismatch, studies focusing on enhancing SpCas9 PAM recognition stringency are quite few. A recent pioneering study identified a D1135E variant of SpCas9 that exhibits much-reduced editing activity at the noncanonical NAG/NGA PAM sites while preserving robust on-target activity at the canonical NGG-flanking sites (N is any nucleobase). Herein, we aim to clarify the molecular mechanism by which this single D1135E mutation confers on SpCas9 enhanced specificity for PAM recognition by molecular dynamics simulations. The results suggest that the variant maintains the base-specific recognition for the canonical NGG PAM via four hydrogen bonds, akin to that in the wild type (WT) SpCas9. While the noncanonical NAG PAM is engaged to the two PAM-interacting arginine residues (i.e., R1333 and R1335) in WT SpCas9 via two to three hydrogen bonds, the D1135E variant prefers to establish two hydrogen bonds with the PAM bases, accounting for its minimal editing activity on the off-target sites with an NAG PAM. The impaired NAG recognition by D1135E SpCas9 results from the PAM duplex displacement such that the hydrogen bond of R1333 to the second PAM base is disfavored. We further propose a mechanistic model to delineate how the mutation perturbs the noncanonical PAM recognition. We anticipate that the mechanistic knowledge could be leveraged for continuous optimization of SpCas9 PAM recognition specificity toward high-precision demanding applications.},
}
@article {pmid35665758,
year = {2022},
author = {Bessoltane, N and Charlot, F and Guyon-Debast, A and Charif, D and Mara, K and Collonnier, C and Perroud, PF and Tepfer, M and Nogué, F},
title = {Genome-wide specificity of plant genome editing by both CRISPR-Cas9 and TALEN.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {9330},
pmid = {35665758},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; Plants/genetics ; Plants, Genetically Modified/genetics ; *Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.},
}
@article {pmid35663880,
year = {2022},
author = {Surachat, K and Kantachote, D and Wonglapsuwan, M and Chukamnerd, A and Deachamag, P and Mittraparp-Arthorn, P and Jeenkeawpiam, K},
title = {Complete Genome Sequence of Weissella cibaria NH9449 and Comprehensive Comparative-Genomic Analysis: Genomic Diversity and Versatility Trait Revealed.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {826683},
pmid = {35663880},
issn = {1664-302X},
abstract = {Lactic acid bacteria (LAB) in the genus Weissella spp. contain traits in their genome that confer versatility. In particular, Weissella cibaria encodes several beneficial genes that are useful in biotechnological applications. The complete genome of W. cibaria NH9449 was sequenced and an in silico comparative analysis was performed to gain insight into the genomic diversity among members of the genus Weissella. A total of 219 Weissella genomes were used in a bioinformatics analysis of pan-genomes, phylogenetics, self-defense mechanisms, virulence factors, antimicrobial resistance, and carbohydrate-active enzymes. These investigations showed that the strain NH9449 encodes several restriction-modification-related genes and a CRISPR-Cas region in its genome. The identification of carbohydrate-active enzyme-encoding genes indicated that this strain could be beneficial in biotechnological applications. The comparative genomic analysis reveals the very high genomic diversity in this genus, and some marked differences in genetic variation and genes among Weissella species. The calculated average amino acid identity (AAI) and phylogenetic analysis of core and accessory genes shows the possible existence of three new species in this genus. These new genomic insights into Weissella species and their biological functions could be useful in the food industry and other applications.},
}
@article {pmid35661966,
year = {2022},
author = {Hou, Q and Wang, J and Chen, X},
title = {A novel and powerful approach for designing future crops-target editing promoter overcomes tradeoffs caused by gene pleiotropy.},
journal = {Science China. Life sciences},
volume = {65},
number = {10},
pages = {2128-2130},
pmid = {35661966},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant ; Plants, Genetically Modified/genetics ; },
}
@article {pmid35659862,
year = {2022},
author = {Chen, H and Mayer, A and Balasubramanian, V},
title = {A scaling law in CRISPR repertoire sizes arises from the avoidance of autoimmunity.},
journal = {Current biology : CB},
volume = {32},
number = {13},
pages = {2897-2907.e5},
doi = {10.1016/j.cub.2022.05.021},
pmid = {35659862},
issn = {1879-0445},
mesh = {*Autoimmunity ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; },
abstract = {Some prokaryotes possess CRISPR-Cas systems that use DNA segments called spacers, which are acquired from invading phages, to guide immune defense. Here, we propose that cross-reactive CRISPR targeting can, however, lead to "heterologous autoimmunity," whereby foreign spacers guide self-targeting in a spacer-length-dependent fashion. Balancing antiviral defense against autoimmunity predicts a scaling relation between spacer length and CRISPR repertoire size. We find evidence for this scaling through a comparative analysis of sequenced prokaryotic genomes and show that this association also holds at the level of CRISPR types. By contrast, the scaling is absent in strains with nonfunctional CRISPR loci. Finally, we demonstrate that stochastic spacer loss can explain variations around the scaling relation, even between strains of the same species. Our results suggest that heterologous autoimmunity is a selective factor shaping the evolution of CRISPR-Cas systems, analogous to the trade-offs between immune specificity, breadth, and autoimmunity that constrain the diversity of adaptive immune systems in vertebrates.},
}
@article {pmid35659494,
year = {2022},
author = {Stirm, M and Fonteyne, LM and Shashikadze, B and Stöckl, JB and Kurome, M and Keßler, B and Zakhartchenko, V and Kemter, E and Blum, H and Arnold, GJ and Matiasek, K and Wanke, R and Wurst, W and Nagashima, H and Knieling, F and Walter, MC and Kupatt, C and Fröhlich, T and Klymiuk, N and Blutke, A and Wolf, E},
title = {Pig models for Duchenne muscular dystrophy - from disease mechanisms to validation of new diagnostic and therapeutic concepts.},
journal = {Neuromuscular disorders : NMD},
volume = {32},
number = {7},
pages = {543-556},
doi = {10.1016/j.nmd.2022.04.005},
pmid = {35659494},
issn = {1873-2364},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Dystrophin/genetics ; Exons ; Female ; Gene Editing/methods ; *Muscular Dystrophy, Duchenne/diagnosis/genetics/therapy ; Swine ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.},
}
@article {pmid35659325,
year = {2022},
author = {Huang, CJ and Adler, BA and Doudna, JA},
title = {A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression.},
journal = {Molecular cell},
volume = {82},
number = {11},
pages = {2148-2160.e4},
doi = {10.1016/j.molcel.2022.04.020},
pmid = {35659325},
issn = {1097-4164},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; T32 GM132022/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Antiviral Agents ; Bacteria/genetics ; *Bacteriophages/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/metabolism ; Deoxyribonuclease I/metabolism ; Deoxyribonucleases/genetics ; Gene Expression ; RNA/metabolism ; },
abstract = {Used widely for genome editing, CRISPR-Cas enzymes provide RNA-guided immunity to microbes by targeting foreign nucleic acids for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. These crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Nevertheless, Cas12c represses transcription and can defend bacteria against lytic bacteriophage infection when targeting an essential phage gene. Together, these results show that Cas12c employs targeted DNA binding to provide antiviral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.},
}
@article {pmid35658035,
year = {2022},
author = {Tran, NT and Danner, E and Li, X and Graf, R and Lebedin, M and de la Rosa, K and Kühn, R and Rajewsky, K and Chu, VT},
title = {Precise CRISPR-Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells.},
journal = {Science advances},
volume = {8},
number = {22},
pages = {eabm9106},
pmid = {35658035},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; Dependovirus ; Gene Editing ; Genetic Therapy ; *Hematopoietic Stem Cells ; Humans ; Mutation ; },
abstract = {While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9[D10A] nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations. Using spacer-nick, we developed an approach to repair disease-causing mutations occurring in the HBB, ELANE, IL7R, and PRF1 genes. We achieved gene correction efficiencies of 20 to 50% with minimal NHEJ-mediated on-target mutations. On the basis of in-depth off-target assessment, frequent unintended genetic alterations induced by classical CRISPR-Cas9 were significantly reduced or absent in the HSPCs treated with spacer-nick. Thus, the spacer-nick gene correction approach provides improved safety and suitability for gene therapy.},
}
@article {pmid35657952,
year = {2022},
author = {Spisak, S and O'Brien, B and Ostermeier, M},
title = {A bacterial dual positive and negative selection system for dCas9 activity.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0269270},
pmid = {35657952},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; },
abstract = {The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.},
}
@article {pmid35657788,
year = {2022},
author = {Lee, N and Park, J and Kim, JE and Shin, JY and Min, K and Son, H},
title = {Genome editing using preassembled CRISPR-Cas9 ribonucleoprotein complexes in Fusarium graminearum.},
journal = {PloS one},
volume = {17},
number = {6},
pages = {e0268855},
pmid = {35657788},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; *Fusarium/genetics ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; },
abstract = {Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has greatly facilitated the genetic analysis of fungal pathogens. The head blight fungus, Fusarium graminearum, causes destructive losses of economically important cereal crops. The recent development of the CRISPR-Cas9 system for use with F. graminearum has enabled more efficient genome editing. In this study, we described a CRISPR-Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins (RNPs) into the protoplasts of F. graminearum. The use of RNPs significantly increased both the number of transformants and percentage of transformants in which the target gene was successfully replaced with a selectable marker. We showed that a single double-strand DNA break mediated by the Cas9 ribonucleoprotein was sufficient for gene deletion. In addition, short-homology recombination required only 50 base pair regions flanking the target gene. The high efficiency of Cas9 RNPs enables large-scale functional analysis, the identification of essential genes, and gene deletion that is difficult with conventional methods. We expect that our approach will accelerate genetic studies of F. graminearum.},
}
@article {pmid35657565,
year = {2022},
author = {Zhang, W and Zhao, S and Xie, Z and Chen, S and Huang, Y and Zhao, Z and Yi, G},
title = {The fluorescence amplification strategy based on 3D DNA walker and CRISPR/Cas12a for the rapid detection of BRAF V600E.},
journal = {Analytical sciences : the international journal of the Japan Society for Analytical Chemistry},
volume = {38},
number = {8},
pages = {1057-1066},
pmid = {35657565},
issn = {1348-2246},
mesh = {*CRISPR-Cas Systems ; DNA ; Endonucleases ; Fluorescence ; Humans ; *Proto-Oncogene Proteins B-raf/genetics ; },
abstract = {Circulating tumor DNA (ctDNA) is a fragment of single- or double-stranded DNA originating from tumor or circulating tumor cells and provides accurate information regarding the molecular characteristics of tumors. Therefore, sensitive detection of ctDNA is of great significance to mutation analysis and clinical diagnosis. Among various ctDNAs, the BRAF V600E is related to aggressive behavior, disease recurrence, and disease-specific mortality in papillary thyroid carcinoma. Herein, we selected the BRAF V600E gene sequence as an in vitro biomarker, and established a fluorescence detection strategy combined 3D DNA walker with CRISPR/Cas12a. In the presence of the target ctDNA, 3D DNA walker could identify and bind it, and thus released a large amount of output DNAs through cyclic cleavage with the assistance of specific endonuclease (Nb.BbvCI). The output DNAs were specifically bound to crRNA and activated the non-specific trans-cleavage activity of Cas12a. Finally, the fluorescence signal was significantly enhanced. Notably, this method can detect the BRAF V600E in a range of 1 fM ~ 20 nM with a detection limit of 0.37 fM without DNA polymerase. Due to the powerful amplification capability of 3D DNA walker and high specificity and programmability of CRISPR/Cas12a, the entire process took only a maximum of 70 min. Furthermore, it can be potentially used for the detection of ctDNA in human serum. In summary, this method not only provides a platform for the rapid detection of ctDNA, but also shows good potential for early clinical diagnosis and biomedical research.},
}
@article {pmid35657510,
year = {2022},
author = {Xu, S and Chen, T and Tian, M and Rahantaniaina, MS and Zhang, L and Wang, R and Xuan, W and Han, Y},
title = {Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2526},
number = {},
pages = {25-41},
pmid = {35657510},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant ; Homeostasis ; *Oryza/genetics ; Plants, Genetically Modified/genetics ; Reactive Oxygen Species ; },
abstract = {Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse environmental conditions can either promote ROS production or downregulate antioxidative enzymes, leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover their signaling mechanisms and to characterize related components, genetic modification of ROS homeostasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technology. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-mediated transformation, and mutation characterization are described. Application of this system in editing a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.},
}
@article {pmid35655368,
year = {2022},
author = {Zhao, X and Chen, X and Xue, Y and Wang, X},
title = {Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR-AsCpf1 system.},
journal = {Journal of basic microbiology},
volume = {62},
number = {7},
pages = {824-832},
doi = {10.1002/jobm.202200134},
pmid = {35655368},
issn = {1521-4028},
mesh = {Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; },
abstract = {Bacillus subtilis is a useful chassis in the fields of synthetic biology and metabolic engineering for chemical production. Here, we constructed CRISPR-AsCpf1-based expression plasmids with the temperature-sensitive replicon for iterative genome editing in B. subtilis. This method allowed gene insertion and large genomic deletion with an editing efficiency of up 80%-100% and rapid plasmid curing to facilitate the iterative genome editing in B. subtilis 168. Using the customized CRISPR-AsCpf1 system, we successfully and efficiently implemented the related gene editing in B. subtilis 168 for hyaluronic acid (HA) biosynthesis, HA synthase gene (hasA) insertion, UDP-glucose-dehydrogenase gene (tuaD) insertion, and eps gene cluster (epsA-O) deletion. The heterologous production of HA was realized by the engineered strain with a yield of 1.39 g/L. These results support the finding that the CRISPR-AsCpf1 system is highly efficient in bacteria genome editing and provide valuable guidance and essential references for genome engineering in B. subtilis using the CRISPR-AsCpf1 system.},
}
@article {pmid35655130,
year = {2022},
author = {Rawashdeh, O and Rawashdeh, RY and Kebede, T and Kapp, D and Ralescu, A},
title = {Bio-informatic analysis of CRISPR protospacer adjacent motifs (PAMs) in T4 genome.},
journal = {BMC genomic data},
volume = {23},
number = {1},
pages = {40},
pmid = {35655130},
issn = {2730-6844},
mesh = {Amino Acids ; *Bacteriophages/genetics ; *Computational Biology ; },
abstract = {BACKGROUND: The existence of protospacer adjacent motifs (PAMs) sequences in bacteriophage genome is critical for the recognition and function of the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) machinery system. We further elucidate the significance of PAMs and their function, particularly as a part of transcriptional regulatory regions in T4 bacteriophages.<br><br>METHODS: A scripting language was used to analyze a sequence of T4 phage genome, and a list of few selected PAMs. Mann-Whitney Wilcoxon (MWW) test was used to compare the sequence hits for the PAMs versus the hits of all the possible sequences of equal lengths.<br><br>RESULTS: The results of MWW test show that certain PAMs such as: 'NGG' and 'TATA' are preferably located at the core of phage promoters: around -10 position, whereas the position around -35 appears to have no detectable count variation of any of the tested PAMs. Among all tested PAMs, the following three sequences: 5'-GCTV-3', 5'-TTGAAT-3' and 5'-TTGGGT-3' have higher prevalence in essential genes. By analyzing all the possible ways of reading PAM sequences as codons for the corresponding amino acids, it was found that deduced amino acids of some PAMs have a significant tendency to prefer the surface of proteins.<br><br>CONCLUSION: These results provide novel insights into the location and the subsequent identification of the role of PAMs as transcriptional regulatory elements. Also, CRISPR targeting certain PAM sequences is somehow likely to be connected to the hydrophilicity (water solubility) of amino acids translated from PAM's triplets. Therefore, these amino acids are found at the interacting unit at protein-protein interfaces.},
}
@article {pmid35654791,
year = {2022},
author = {Kobayashi, N and Okae, H and Hiura, H and Kubota, N and Kobayashi, EH and Shibata, S and Oike, A and Hori, T and Kikutake, C and Hamada, H and Kaji, H and Suyama, M and Bortolin-Cavaillé, ML and Cavaillé, J and Arima, T},
title = {The microRNA cluster C19MC confers differentiation potential into trophoblast lineages upon human pluripotent stem cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3071},
pmid = {35654791},
issn = {2041-1723},
mesh = {Animals ; Cell Differentiation/genetics ; Epigenesis, Genetic ; Humans ; Mammals ; *MicroRNAs/genetics ; *Pluripotent Stem Cells ; Trophoblasts ; },
abstract = {The first cell fate commitment during mammalian development is the specification of the inner cell mass and trophectoderm. This irreversible cell fate commitment should be epigenetically regulated, but the precise mechanism is largely unknown in humans. Here, we show that naïve human embryonic stem (hES) cells can transdifferentiate into trophoblast stem (hTS) cells, but primed hES cells cannot. Our transcriptome and methylome analyses reveal that a primate-specific miRNA cluster on chromosome 19 (C19MC) is active in naïve hES cells but epigenetically silenced in primed ones. Moreover, genome and epigenome editing using CRISPR/Cas systems demonstrate that C19MC is essential for hTS cell maintenance and C19MC-reactivated primed hES cells can give rise to hTS cells. Thus, we reveal that C19MC activation confers differentiation potential into trophoblast lineages on hES cells. Our findings are fundamental to understanding the epigenetic regulation of human early development and pluripotency.},
}
@article {pmid35654042,
year = {2022},
author = {Kaczmarska, Z and Czarnocki-Cieciura, M and Górecka-Minakowska, KM and Wingo, RJ and Jackiewicz, J and Zajko, W and Poznański, JT and Rawski, M and Grant, T and Peters, JE and Nowotny, M},
title = {Structural basis of transposon end recognition explains central features of Tn7 transposition systems.},
journal = {Molecular cell},
volume = {82},
number = {14},
pages = {2618-2632.e7},
pmid = {35654042},
issn = {1097-4164},
support = {R01 GM129118/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; Cryoelectron Microscopy ; DNA Transposable Elements/genetics ; DNA, Bacterial/metabolism ; DNA-Binding Proteins/genetics ; Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/genetics ; },
abstract = {Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.},
}
@article {pmid35653473,
year = {2022},
author = {Kaiser, J},
title = {A gentler way to tweak genes: epigenome editing.},
journal = {Science (New York, N.Y.)},
volume = {376},
number = {6597},
pages = {1034-1035},
doi = {10.1126/science.add2703},
pmid = {35653473},
issn = {1095-9203},
mesh = {Animals ; CRISPR-Cas Systems ; *Epigenome ; *Gene Editing ; *Gene Expression Regulation ; *Genetic Therapy ; Mice ; },
abstract = {Flipping genetic on-off switches can treat diseases in mice.},
}
@article {pmid35652435,
year = {2022},
author = {Noureen, A and Zuhaib Khan, M and Amin, I and Zainab, T and Ahmad, N and Haider, S and Mansoor, S},
title = {Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop.},
journal = {GM crops &amp; food},
volume = {13},
number = {1},
pages = {97-111},
pmid = {35652435},
issn = {2164-5701},
mesh = {Plant Diseases/genetics ; Plants, Genetically Modified/genetics ; *Potyvirus/genetics ; RNA, Guide ; *Solanum tuberosum/genetics ; },
abstract = {Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVY[N], PVY[O], and PVY[NTN]. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.},
}
@article {pmid35651625,
year = {2022},
author = {Yim, S and Hwang, W and Han, N and Lee, D},
title = {Computational Discovery of Cancer Immunotherapy Targets by Intercellular CRISPR Screens.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {884561},
pmid = {35651625},
issn = {1664-3224},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Immunotherapy ; T-Lymphocytes, Cytotoxic ; *Triple Negative Breast Neoplasms/genetics ; },
abstract = {Cancer immunotherapy targets the interplay between immune and cancer cells. In particular, interactions between cytotoxic T lymphocytes (CTLs) and cancer cells, such as PD-1 (PDCD1) binding PD-L1 (CD274), are crucial for cancer cell clearance. However, immune checkpoint inhibitors targeting these interactions are effective only in a subset of patients, requiring the identification of novel immunotherapy targets. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening in either cancer or immune cells has been employed to discover regulators of immune cell function. However, CRISPR screens in a single cell type complicate the identification of essential intercellular interactions. Further, pooled screening is associated with high noise levels. Herein, we propose intercellular CRISPR screens, a computational approach for the analysis of genome-wide CRISPR screens in every interacting cell type for the discovery of intercellular interactions as immunotherapeutic targets. We used two publicly available genome-wide CRISPR screening datasets obtained while triple-negative breast cancer (TNBC) cells and CTLs were interacting. We analyzed 4825 interactions between 1391 ligands and receptors on TNBC cells and CTLs to evaluate their effects on CTL function. Intercellular CRISPR screens discovered targets of approved drugs, a few of which were not identifiable in single datasets. To evaluate the method's performance, we used data for cytokines and costimulatory molecules as they constitute the majority of immunotherapeutic targets. Combining both CRISPR datasets improved the recall of discovering these genes relative to using single CRISPR datasets over two-fold. Our results indicate that intercellular CRISPR screens can suggest novel immunotherapy targets that are not obtained through individual CRISPR screens. The pipeline can be extended to other cancer and immune cell types to discover important intercellular interactions as potential immunotherapeutic targets.},
}
@article {pmid35651324,
year = {2022},
author = {Shin, JH and Lee, J and Jung, YK and Kim, KS and Jeong, J and Choi, D},
title = {Therapeutic applications of gene editing in chronic liver diseases: an update.},
journal = {BMB reports},
volume = {55},
number = {6},
pages = {251-258},
pmid = {35651324},
issn = {1976-670X},
mesh = {Animals ; *COVID-19 ; CRISPR-Cas Systems ; Gene Editing/methods ; Humans ; *Liver Diseases/genetics/therapy ; Pandemics ; RNA, Messenger ; },
abstract = {Innovative genome editing techniques developed in recent decades have revolutionized the biomedical research field. Liver is the most favored target organ for genome editing owing to its ability to regenerate. The regenerative capacity of the liver enables ex vivo gene editing in which the mutated gene in hepatocytes isolated from the animal model of genetic disease is repaired. The edited hepatocytes are injected back into the animal to mitigate the disease. Furthermore, the liver is considered as the easiest target organ for gene editing as it absorbs almost all foreign molecules. The mRNA vaccines, which have been developed to manage the COVID-19 pandemic, have provided a novel gene editing strategy using Cas mRNA. A single injection of gene editing components with Cas mRNA is reported to be efficient in the treatment of patients with genetic liver diseases. In this review, we first discuss previously reported gene editing tools and cases managed using them, as well as liver diseases caused by genetic mutations. Next, we summarize the recent successes of ex vivo and in vivo gene editing approaches in ameliorating liver diseases in animals and humans. [BMB Reports 2022; 55(6): 251-258].},
}
@article {pmid35649413,
year = {2022},
author = {Wandera, KG and Alkhnbashi, OS and Bassett, HVI and Mitrofanov, A and Hauns, S and Migur, A and Backofen, R and Beisel, CL},
title = {Anti-CRISPR prediction using deep learning reveals an inhibitor of Cas13b nucleases.},
journal = {Molecular cell},
volume = {82},
number = {14},
pages = {2714-2726.e4},
doi = {10.1016/j.molcel.2022.05.003},
pmid = {35649413},
issn = {1097-4164},
mesh = {Bacteria/genetics/metabolism ; *Bacteriophages/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; *Deep Learning ; Endonucleases/genetics/metabolism ; },
abstract = {As part of the ongoing bacterial-phage arms race, CRISPR-Cas systems in bacteria clear invading phages whereas anti-CRISPR proteins (Acrs) in phages inhibit CRISPR defenses. Known Acrs have proven extremely diverse, complicating their identification. Here, we report a deep learning algorithm for Acr identification that revealed an Acr against type VI-B CRISPR-Cas systems. The algorithm predicted numerous putative Acrs spanning almost all CRISPR-Cas types and subtypes, including over 7,000 putative type IV and VI Acrs not predicted by other algorithms. By performing a cell-free screen for Acr hits against type VI-B systems, we identified a potent inhibitor of Cas13b nucleases we named AcrVIB1. AcrVIB1 blocks Cas13b-mediated defense against a targeted plasmid and lytic phage, and its inhibitory function principally occurs upstream of ribonucleoprotein complex formation. Overall, our work helps expand the known Acr universe, aiding our understanding of the bacteria-phage arms race and the use of Acrs to control CRISPR technologies.},
}
@article {pmid35648481,
year = {2022},
author = {Pothoulakis, G and Nguyen, MTA and Andersen, ES},
title = {Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control.},
journal = {Nucleic acids research},
volume = {50},
number = {12},
pages = {7176-7187},
pmid = {35648481},
issn = {1362-4962},
support = {683305/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/chemistry ; RNA, Guide/genetics ; *Saccharomyces cerevisiae/genetics ; Synthetic Biology ; },
abstract = {Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.},
}
@article {pmid35647578,
year = {2022},
author = {Tripathi, L and Dhugga, KS and Ntui, VO and Runo, S and Syombua, ED and Muiruri, S and Wen, Z and Tripathi, JN},
title = {Genome Editing for Sustainable Agriculture in Africa.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {876697},
pmid = {35647578},
issn = {2673-3439},
abstract = {Sustainable intensification of agriculture in Africa is essential for accomplishing food and nutritional security and addressing the rising concerns of climate change. There is an urgent need to close the yield gap in staple crops and enhance food production to feed the growing population. In order to meet the increasing demand for food, more efficient approaches to produce food are needed. All the tools available in the toolbox, including modern biotechnology and traditional, need to be applied for crop improvement. The full potential of new breeding tools such as genome editing needs to be exploited in addition to conventional technologies. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing has rapidly become the most prevalent genetic engineering approach for developing improved crop varieties because of its simplicity, efficiency, specificity, and easy to use. Genome editing improves crop variety by modifying its endogenous genome free of any foreign gene. Hence, genome-edited crops with no foreign gene integration are not regulated as genetically modified organisms (GMOs) in several countries. Researchers are using CRISPR/Cas-based genome editing for improving African staple crops for biotic and abiotic stress resistance and improved nutritional quality. Many products, such as disease-resistant banana, maize resistant to lethal necrosis, and sorghum resistant to the parasitic plant Striga and enhanced quality, are under development for African farmers. There is a need for creating an enabling environment in Africa with science-based regulatory guidelines for the release and adoption of the products developed using CRISPR/Cas9-mediated genome editing. Some progress has been made in this regard. Nigeria and Kenya have recently published the national biosafety guidelines for the regulation of gene editing. This article summarizes recent advances in developments of tools, potential applications of genome editing for improving staple crops, and regulatory policies in Africa.},
}
@article {pmid35646725,
year = {2022},
author = {Wei, J and Li, Y and Cao, Y and Liu, Q and Yang, K and Song, X and Shao, Y and Qi, K and Tu, J},
title = {Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {879887},
pmid = {35646725},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; *Parvovirus, Porcine/genetics ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Swine ; *Swine Diseases/genetics ; },
abstract = {Porcine parvovirus (PPV) is one of the important causes of pig reproductive diseases. The most prevalent methods for PPV authentication are the polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and quantitative real-time PCR. However, these procedures have downsides, such as the fact that they take a long time and require expensive equipment. As a result, a rapid, visible, and economical clinical diagnostic strategy to detect PPV is necessary. In this study, three pairs of crRNA primers were designed to recognize the VP2 gene, and an ERA-CRISPR/Cas12a system for PPV detection was successfully developed. The approach involved isothermal detection at 37°C, and the method can be used for visual inspection. The detection limit of the ERA-CRISPR/Cas12a system was 3.75 × 10[2] copies/μL, and no cross reactions with other porcine viruses were found. In view of the preceding, a rapid, visible, and low-cost nucleic acid testing approach for PPV has been developed using the ERA-CRISPR/Cas12a system.},
}
@article {pmid35646415,
year = {2022},
author = {Leibrock, NV and Santegoets, J and Mooijman, PJW and Yusuf, F and Zuijdgeest, XCL and Zutt, EA and Jacobs, JGM and Schaart, JG},
title = {The biological feasibility and social context of gene-edited, caffeine-free coffee.},
journal = {Food science and biotechnology},
volume = {31},
number = {6},
pages = {635-655},
pmid = {35646415},
issn = {2092-6456},
abstract = {Coffee, especially the species Coffea arabica and Coffea canephora, is one of the world's most consumed beverages. The consumer demand for caffeine-free coffee is currently being met through chemical decaffeination processes. However, this method leads to loss of beverage quality. In this review, the feasibility of using gene editing to produce caffeine-free coffee plants is reviewed. The genes XMT (7-methylxanthosine methyltransferase) and DXMT (3,7-dimethylxanthine methyltransferase) were identified as candidate target genes for knocking out caffeine production in coffee plants. The possible effect of the knock-out of the candidate genes was assessed. Using Agrobacterium tumefaciens-mediated introduction of the CRISPR-Cas system to Knock out XMT or DXMT would lead to blocking caffeine biosynthesis. The use of CRISPR-Cas to genetically edit consumer products is not yet widely accepted, which may lead to societal hurdles for introducing gene-edited caffeine-free coffee cultivars onto the market. However, increased acceptance of CRISPR-Cas/gene editing on products with a clear benefit for consumers offers better prospects for gene editing efforts for caffeine-free coffee.},
}
@article {pmid35643959,
year = {2022},
author = {Yin, J and Hu, J},
title = {The origin of unwanted editing byproducts in gene editing.},
journal = {Acta biochimica et biophysica Sinica},
volume = {54},
number = {6},
pages = {767-781},
doi = {10.3724/abbs.2022056},
pmid = {35643959},
issn = {1745-7270},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; *Gene Editing ; },
abstract = {The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety.},
}
@article {pmid35643551,
year = {2022},
author = {Baldanta, S and Guevara, G and Navarro-Llorens, JM},
title = {SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {103},
pmid = {35643551},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; *Synechocystis/genetics/metabolism ; },
abstract = {BACKGROUND: Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology.<br><br>RESULTS: In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology.<br><br>CONCLUSIONS: The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.},
}
@article {pmid35643496,
year = {2022},
author = {Xin, Q and Chen, Y and Chen, Q and Wang, B and Pan, L},
title = {Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {99},
pmid = {35643496},
issn = {1475-2859},
mesh = {*Bacillus amyloliquefaciens/genetics ; *CRISPR-Cas Systems ; DNA Restriction-Modification Enzymes/genetics ; Gene Editing/methods ; alpha-Amylases/genetics ; },
abstract = {BACKGROUND: Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus. This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02.<br><br>RESULTS: In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes (aprE, nprE, wprA, and bamHIR) mutant strain, LB1ba02&#x25b3;4. The mutant strain secreted 1.25-fold more &#x3b1;-amylase into the medium than the wild-type strain.<br><br>CONCLUSIONS: The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.},
}
@article {pmid35643083,
year = {2022},
author = {Kato, K and Zhou, W and Okazaki, S and Isayama, Y and Nishizawa, T and Gootenberg, JS and Abudayyeh, OO and Nishimasu, H},
title = {Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.},
journal = {Cell},
volume = {185},
number = {13},
pages = {2324-2337.e16},
doi = {10.1016/j.cell.2022.05.003},
pmid = {35643083},
issn = {1097-4172},
support = {R21 AI149694/AI/NIAID NIH HHS/United States ; R01 EB031957/EB/NIBIB NIH HHS/United States ; R56 HG011857/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/chemistry ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; Humans ; RNA Precursors ; RNA, Bacterial/chemistry ; RNA, Guide/chemistry/genetics ; },
abstract = {The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.},
}
@article {pmid35642647,
year = {2022},
author = {Verma, MK and Roychowdhury, S and Sahu, BD and Mishra, A and Sethi, KK},
title = {CRISPR-based point-of-care diagnostics incorporating Cas9, Cas12, and Cas13 enzymes advanced for SARS-CoV-2 detection.},
journal = {Journal of biochemical and molecular toxicology},
volume = {36},
number = {8},
pages = {e23113},
pmid = {35642647},
issn = {1099-0461},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; CRISPR-Cas Systems ; Humans ; Pandemics ; Point-of-Care Testing ; *SARS-CoV-2/genetics ; },
abstract = {An outbreak of the novel beta coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first came to light in December 2019, which has unfolded rapidly and turned out to be a global pandemic. Early prognosis of viral contamination involves speedy intervention, disorder control, and good-sized management of the spread of disease. Reverse transcription-polymerase chain reaction, considered the gold standard test for detecting nucleic acids and pathogen diagnosis, provides high sensitivity and specificity. However, reliance on high-priced equipped kits, associated reagents, and skilled personnel slow down sickness detection. Lately, the improvement of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated protein)-based diagnostic systems has reshaped molecular diagnosis due to their low cost, simplicity, speed, efficiency, high sensitivity, specificity, and versatility, which is vital for accomplishing point-of-care diagnostics. We reviewed and summarized CRISPR-Cas-based point-of-care diagnostic strategies and research in these paintings while highlighting their characteristics and challenges for identifying SARS-CoV-2.},
}
@article {pmid35642255,
year = {2022},
author = {de Bruijn, R and Wielstra, PCM and Calcines-Cruz, C and van Waveren, T and Hernandez-Garcia, A and van der Schoot, P},
title = {A kinetic model for the impact of packaging signal mimics on genome encapsulation.},
journal = {Biophysical journal},
volume = {121},
number = {13},
pages = {2583-2599},
pmid = {35642255},
issn = {1542-0086},
mesh = {DNA ; *DNA Packaging ; Kinetics ; Proteins/genetics ; *Virus Assembly/genetics ; },
abstract = {Inspired by recent experiments on the spontaneous assembly of virus-like particles from a solution containing a synthetic coat protein and double-stranded DNA, we put forward a kinetic model that has as main ingredients a stochastic nucleation and a deterministic growth process. The efficiency and rate of DNA packaging strongly increase after tiling the DNA with CRISPR-Cas proteins at predesignated locations, mimicking assembly signals in viruses. Our model shows that treating these proteins as nucleation-inducing diffusion barriers is sufficient to explain the experimentally observed increase in encapsulation efficiency, but only if the nucleation rate is sufficiently high. We find an optimum in the encapsulation kinetics for conditions where the number of packaging signal mimics is equal to the number of nucleation events that can occur during the time required to fully encapsulate the DNA template, presuming that the nucleation events can only take place adjacent to a packaging signal. Our theory is in satisfactory agreement with the available experimental data.},
}
@article {pmid35641501,
year = {2022},
author = {Volke, DC and Martino, RA and Kozaeva, E and Smania, AM and Nikel, PI},
title = {Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3026},
pmid = {35641501},
issn = {2041-1723},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; Cytidine/genetics ; *Gene Editing/methods ; Phenotype ; },
abstract = {CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.},
}
@article {pmid35641498,
year = {2022},
author = {Vos, PD and Rossetti, G and Mantegna, JL and Siira, SJ and Gandadireja, AP and Bruce, M and Raven, SA and Khersonsky, O and Fleishman, SJ and Filipovska, A and Rackham, O},
title = {Computationally designed hyperactive Cas9 enzymes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3023},
pmid = {35641498},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Engineering ; Genome ; Mammals ; },
abstract = {The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.},
}
@article {pmid35641106,
year = {2022},
author = {Zhai, H and Cui, L and Xiong, Z and Qi, Q and Hou, J},
title = {CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression in Saccharomyces cerevisiae.},
journal = {Nucleic acids research},
volume = {50},
number = {10},
pages = {5988-6000},
pmid = {35641106},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/metabolism ; Gene Editing/methods ; Lactic Acid/analogs &amp; derivatives/biosynthesis ; *Metabolic Engineering/methods ; *Saccharomyces cerevisiae/genetics ; *Transcriptional Activation ; },
abstract = {Saccharomyces cerevisiae is an important model eukaryotic microorganism and widely applied in fundamental research and the production of various chemicals. Its ability to efficiently and precisely control the expression of multiple genes is valuable for metabolic engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)-mediated regulation enables complex gene expression programming; however, the regulation efficiency is often limited by the efficiency of pertinent regulators. Here, we developed CRISPR-mediated protein-tagging signal amplification system for simultaneous multiplexed gene activation and repression in S. cerevisiae. By introducing protein scaffolds (SPY and SunTag systems) to recruit multiple copies of regulators to different nuclease-deficient CRISPR proteins and design optimization, our system amplified gene regulation efficiency significantly. The gene activation and repression efficiencies reached as high as 34.9-fold and 95%, respectively, being 3.8- and 8.6-fold higher than those observed on the direct fusion of regulators with nuclease-deficient CRISPR proteins, respectively. We then applied the orthogonal bifunctional CRISPR-mediated transcriptional regulation system to regulate the expression of genes associated with 3-hydroxypropanoic acid production to deduce that CRISPR-associated regulator recruiting systems represent a robust method for simultaneously regulating multiple genes and rewiring metabolic pathways.},
}
@article {pmid35640826,
year = {2022},
author = {Molla, KA},
title = {CRISPR-Cas9 helps solve a piece of the puzzle of the biosynthesis of salicinoids and suggests a role in the growth-defense trade-off in poplar.},
journal = {The Plant cell},
volume = {34},
number = {8},
pages = {2819-2820},
pmid = {35640826},
issn = {1532-298X},
mesh = {*CRISPR-Cas Systems/genetics ; *Populus/genetics ; },
}
@article {pmid35640600,
year = {2022},
author = {Shin, CH and Park, SC and Park, IG and Kim, H and An, B and Lee, C and Kim, SH and Lee, J and Lee, JM and Oh, SJ},
title = {Cytosolic microRNA-inducible nuclear translocation of Cas9 protein for disease-specific genome modification.},
journal = {Nucleic acids research},
volume = {50},
number = {10},
pages = {5919-5933},
pmid = {35640600},
issn = {1362-4962},
mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Gene Editing ; *MicroRNAs/genetics ; RNA, Messenger/genetics ; },
abstract = {MicroRNA-dependent mRNA decay plays an important role in gene silencing by facilitating posttranscriptional and translational repression. Inspired by this intrinsic nature of microRNA-mediated mRNA cleavage, here, we describe a microRNA-targeting mRNA as a switch platform called mRNA bridge mimetics to regulate the translocation of proteins. We applied the mRNA bridge mimetics platform to Cas9 protein to confer it the ability to translocate into the nucleus via cleavage of the nuclear export signal. This system performed programmed gene editing in vitro and in vivo. Combinatorial treatment with cisplatin and miR-21-EZH2 axis-targeting CRISPR Self Check-In improved sensitivity to chemotherapeutic drugs in vivo. Using the endogenous microRNA-mediated mRNA decay mechanism, our platform is able to remodel a cell's natural biology to allow the entry of precise drugs into the nucleus, devoid of non-specific translocation. The mRNA bridge mimetics strategy is promising for applications in which the reaction must be controlled via intracellular stimuli and modulates Cas9 proteins to ensure safe genome modification in diseased conditions.},
}
@article {pmid35637351,
year = {2022},
author = {Kempton, HR and Love, KS and Guo, LY and Qi, LS},
title = {Scalable biological signal recording in mammalian cells using Cas12a base editors.},
journal = {Nature chemical biology},
volume = {18},
number = {7},
pages = {742-750},
pmid = {35637351},
issn = {1552-4469},
support = {U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Humans ; Mammals ; *RNA, Guide/genetics ; },
abstract = {Biological signal recording enables the study of molecular inputs experienced throughout cellular history. However, current methods are limited in their ability to scale up beyond a single signal in mammalian contexts. Here, we develop an approach using a hyper-efficient dCas12a base editor for multi-signal parallel recording in human cells. We link signals of interest to expression of guide RNAs to catalyze specific nucleotide conversions as a permanent record, enabled by Cas12's guide-processing abilities. We show this approach is plug-and-play with diverse biologically relevant inputs and extend it for more sophisticated applications, including recording of time-delimited events and history of chimeric antigen receptor T cells' antigen exposure. We also demonstrate efficient recording of up to four signals in parallel on an endogenous safe-harbor locus. This work provides a versatile platform for scalable recording of signals of interest for a variety of biological applications.},
}
@article {pmid35637316,
year = {2022},
author = {Bhargava, N and Thakur, P and Muruganandam, TP and Jaitly, S and Gupta, P and Lohani, N and Goswami, SG and Saravanakumar, V and Bhattacharya, SK and Jain, S and Ramalingam, S},
title = {Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells.},
journal = {Molecular biology reports},
volume = {49},
number = {8},
pages = {7887-7898},
pmid = {35637316},
issn = {1573-4978},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Gene Editing/methods ; Genome, Human ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {BACKGROUND: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications.<br><br>METHODS AND RESULTS: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2&#xa0;weeks.<br><br>CONCLUSION: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.},
}
@article {pmid35637227,
year = {2022},
author = {Corsi, GI and Qu, K and Alkan, F and Pan, X and Luo, Y and Gorodkin, J},
title = {CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {3006},
pmid = {35637227},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Genome ; *RNA, Guide/genetics ; },
abstract = {A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 "sliding" on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design.},
}
@article {pmid35636828,
year = {2022},
author = {Rodino, KG and Smith, KP and Pettengill, MA},
title = {Novel Assays for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2.},
journal = {Clinics in laboratory medicine},
volume = {42},
number = {2},
pages = {299-307},
pmid = {35636828},
issn = {1557-9832},
mesh = {*COVID-19/diagnosis ; Humans ; Pandemics ; *SARS-CoV-2 ; },
abstract = {From the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 pandemic, there has been a major emphasis on molecular laboratory tests for the virus. Shortages in various testing supplies, the desire to increase testing capacity, and a push to make point-of-care or home-based testing available have fostered considerable innovation for SARS-CoV-2 molecular diagnostics, advancements likely to be applicable to other diagnostic uses. The authors attempt to cover some of the most compelling novel types of molecular assays or novel approaches in adapting established molecular methodologies for SARS-CoV-2 detection or characterization.},
}
@article {pmid35635764,
year = {2022},
author = {Nagy, ED and Kuehn, R and Wang, D and Shrawat, A and Duda, DM and Groat, JR and Yang, P and Beach, S and Zhang, Y and Rymarquis, L and Carter, SL and Gaeta, RT and Gilbertson, LA},
title = {Site-directed integration of exogenous DNA into the soybean genome by LbCas12a fused to a plant viral HUH endonuclease.},
journal = {The Plant journal : for cell and molecular biology},
volume = {111},
number = {3},
pages = {905-916},
doi = {10.1111/tpj.15849},
pmid = {35635764},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; DNA ; *Endonucleases/genetics/metabolism ; Gene Editing ; Genome, Plant/genetics ; *Soybeans/genetics/metabolism ; },
abstract = {High efficiency site-directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR-associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR-associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70-nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site-directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four-fold higher than in untethered controls. Integrations via canonical homology-directed repair or non-homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN-associated tethering to improve site-directed DNA integration in plants.},
}
@article {pmid35635176,
year = {2022},
author = {Lin, M and Yue, H and Tian, T and Xiong, E and Zhu, D and Jiang, Y and Zhou, X},
title = {Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay.},
journal = {Analytical chemistry},
volume = {94},
number = {23},
pages = {8277-8284},
doi = {10.1021/acs.analchem.2c00616},
pmid = {35635176},
issn = {1520-6882},
mesh = {*African Swine Fever Virus/genetics ; Animals ; *COVID-19 ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Glycerol ; Nucleic Acid Amplification Techniques/methods ; Recombinases ; SARS-CoV-2 ; Sensitivity and Specificity ; Swine ; },
abstract = {CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.},
}
@article {pmid35635022,
year = {2022},
author = {Hu, T and Ke, X and Ou, Y and Lin, Y},
title = {CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.},
journal = {Analytical chemistry},
volume = {94},
number = {23},
pages = {8506-8513},
doi = {10.1021/acs.analchem.2c01507},
pmid = {35635022},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Hydrogen Peroxide ; Luminescence ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Tyramine ; },
abstract = {CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.},
}
@article {pmid35634923,
year = {2022},
author = {Ansai, S and Kitano, J},
title = {Speciation and adaptation research meets genome editing.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {377},
number = {1855},
pages = {20200516},
pmid = {35634923},
issn = {1471-2970},
mesh = {Acclimatization ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; Genomics ; },
abstract = {Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.},
}
@article {pmid35634921,
year = {2022},
author = {Kitano, J and Ishikawa, A and Ravinet, M and Courtier-Orgogozo, V},
title = {Genetic basis of speciation and adaptation: from loci to causative mutations.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {377},
number = {1855},
pages = {20200503},
pmid = {35634921},
issn = {1471-2970},
mesh = {Acclimatization ; *Adaptation, Physiological/genetics ; *Ecosystem ; Mutation ; },
abstract = {Does evolution proceed in small steps or large leaps? How repeatable is evolution? How constrained is the evolutionary process? Answering these long-standing questions in evolutionary biology is indispensable for both understanding how extant biodiversity has evolved and predicting how organisms and ecosystems will respond to changing environments in the future. Understanding the genetic basis of phenotypic diversification and speciation in natural populations is key to properly answering these questions. The leap forward in genome sequencing technologies has made it increasingly easier to not only investigate the genetic architecture but also identify the variant sites underlying adaptation and speciation in natural populations. Furthermore, recent advances in genome editing technologies are making it possible to investigate the functions of each candidate gene in organisms from natural populations. In this article, we discuss how these recent technological advances enable the analysis of causative genes and mutations and how such analysis can help answer long-standing evolutionary biology questions. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.},
}
@article {pmid35633938,
year = {2022},
author = {Piskunen, P and Latham, R and West, CE and Castronovo, M and Linko, V},
title = {Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond.},
journal = {iScience},
volume = {25},
number = {6},
pages = {104389},
pmid = {35633938},
issn = {2589-0042},
abstract = {Precise genome editing with CRISPR/Cas paves the way for many biochemical, biotechnological, and medical applications, and consequently, it may enable treatment of already known and still-to-be-found genetic diseases. Meanwhile, another rapidly emerging field-structural DNA nanotechnology-provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective.},
}
@article {pmid35633570,
year = {2022},
author = {Huang, L and Xiao, H and Xie, X and Hu, F and Tang, F and Smith, SB and Gan, L},
title = {Generation of Sigmar1 conditional knockout mouse using CRISPR-Cas9 gene targeting.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {60},
number = {6-7},
pages = {e23487},
pmid = {35633570},
issn = {1526-968X},
support = {R01 EY026614/EY/NEI NIH HHS/United States ; R01 EY028103/EY/NEI NIH HHS/United States ; P30 EY031631/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Targeting ; Mice ; Mice, Knockout ; Mitochondria/metabolism ; Retina/metabolism ; },
abstract = {The Sigma 1 receptor (SIGMAR1) is a transmembrane protein located in the mitochondria-associated endoplasmic reticulum membrane, and plays an important role in cell survival as a pluripotent modulator of a variety of signaling pathways related to neurodegeneration. Though SIGMAR1 is a potential target for neurodegenerative diseases, the specific role of SIGMAR1 in different tissue and cell types remains unclear. Here we reported the generation of Sigmar1 conditional knockout (Sigmar1[loxP]) mice using CRISPR-Cas9 method to insert loxP sites into the 5'- and 3'-untranslated regions of Sigmar1. We showed that the insertion of loxP sequences did not affect the expression of Sigmar1 and that Sigmar1[loxP/loxP] mice exhibited no detectable visual defects compared with wild-type mice at the early adult stage. By crossing Sigmar1[loxP] mice with retina-specific Six3-Cre and ubiquitous CMV-Cre mice, we confirmed the deletion of Sigmar1 coding regions of exons 1-4, and the retina-specific and global loss of SIGMAR1 expression, respectively. Thus, Sigmar1[loxP] mice provide a valuable tool for unraveling the tissue and cell-type-specific role of Sigmar1.},
}
@article {pmid35631721,
year = {2022},
author = {Hamdan, MF and Mohd Noor, SN and Abd-Aziz, N and Pua, TL and Tan, BC},
title = {Green Revolution to Gene Revolution: Technological Advances in Agriculture to Feed the World.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {10},
pages = {},
pmid = {35631721},
issn = {2223-7747},
abstract = {Technological applications in agriculture have evolved substantially to increase crop yields and quality to meet global food demand. Conventional techniques, such as seed saving, selective breeding, and mutation breeding (variation breeding), have dramatically increased crop production, especially during the 'Green Revolution' in the 1990s. However, newer issues, such as limited arable lands, climate change, and ever-increasing food demand, pose challenges to agricultural production and threaten food security. In the following 'Gene Revolution' era, rapid innovations in the biotechnology field provide alternative strategies to further improve crop yield, quality, and resilience towards biotic and abiotic stresses. These innovations include the introduction of DNA recombinant technology and applications of genome editing techniques, such as transcription activator-like effector (TALEN), zinc-finger nucleases (ZFN), and clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas) systems. However, the acceptance and future of these modern tools rely on the regulatory frameworks governing their development and production in various countries. Herein, we examine the evolution of technological applications in agriculture, focusing on the motivations for their introduction, technical challenges, possible benefits and concerns, and regulatory frameworks governing genetically engineered product development and production.},
}
@article {pmid35631480,
year = {2022},
author = {He, J and Biswas, R and Bugde, P and Li, J and Liu, DX and Li, Y},
title = {Application of CRISPR-Cas9 System to Study Biological Barriers to Drug Delivery.},
journal = {Pharmaceutics},
volume = {14},
number = {5},
pages = {},
pmid = {35631480},
issn = {1999-4923},
abstract = {In recent years, sequence-specific clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems have been widely used in genome editing of various cell types and organisms. The most developed and broadly used CRISPR-Cas system, CRISPR-Cas9, has benefited from the proof-of-principle studies for a better understanding of the function of genes associated with drug absorption and disposition. Genome-scale CRISPR-Cas9 knockout (KO) screen study also facilitates the identification of novel genes in which loss alters drug permeability across biological membranes and thus modulates the efficacy and safety of drugs. Compared with conventional heterogeneous expression models or other genome editing technologies, CRISPR-Cas9 gene manipulation techniques possess significant advantages, including ease of design, cost-effectiveness, greater on-target DNA cleavage activity and multiplexing capabilities, which makes it possible to study the interactions between membrane proteins and drugs more accurately and efficiently. However, many mechanistic questions and challenges regarding CRISPR-Cas9 gene editing are yet to be addressed, ranging from off-target effects to large-scale genetic alterations. In this review, an overview of the mechanisms of CRISPR-Cas9 in mammalian genome editing will be introduced, as well as the application of CRISPR-Cas9 in studying the barriers to drug delivery.},
}
@article {pmid35630321,
year = {2022},
author = {Layton, AM and Redding, KE},
title = {Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum.},
journal = {Microorganisms},
volume = {10},
number = {5},
pages = {},
pmid = {35630321},
issn = {2076-2607},
abstract = {Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.},
}
@article {pmid35628611,
year = {2022},
author = {Serrano, LJ and Garcia-Arranz, M and De Pablo-Moreno, JA and Segovia, JC and Olivera-Salazar, R and Garcia-Olmo, D and Liras, A},
title = {Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628611},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; *Factor V/genetics ; *Factor V Deficiency/genetics ; Gene Editing ; Humans ; Mutation ; },
abstract = {Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.},
}
@article {pmid35628596,
year = {2022},
author = {Clausing, M and William, D and Preussler, M and Biedermann, J and Grützmann, K and Richter, S and Buchholz, F and Temme, A and Schröck, E and Klink, B},
title = {Different Effects of RNAi-Mediated Downregulation or Chemical Inhibition of NAMPT in an Isogenic IDH Mutant and Wild-Type Glioma Cell Model.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628596},
issn = {1422-0067},
mesh = {*Brain Neoplasms/genetics/metabolism ; *Cytokines/genetics/metabolism ; Down-Regulation ; *Glioblastoma/genetics/metabolism ; *Glioma/genetics/metabolism ; Humans ; *Isocitrate Dehydrogenase/genetics/metabolism ; NAD/metabolism ; NADP/metabolism ; *Nicotinamide Phosphoribosyltransferase/genetics/metabolism ; RNA Interference ; },
abstract = {The IDH1[R132H] mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP[+] were described, the consequences for the NAD[+] synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1[R132H] on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1[wt]) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD[+] in IDH1[R132H] glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD[+] synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1[R132H] glioblastoma cells. Given this dependence of IDH1[R132H] cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1[R132H] and IDH1[wt] cells. Altogether, our results indicate that targeting the NAD[+] synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose.},
}
@article {pmid35628572,
year = {2022},
author = {Pavese, V and Moglia, A and Abbà, S and Milani, AM and Torello Marinoni, D and Corredoira, E and Martínez, MT and Botta, R},
title = {First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628572},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Plant Breeding ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.},
}
@article {pmid35628563,
year = {2022},
author = {Cai, Q and Guo, D and Cao, Y and Li, Y and Ma, R and Liu, W},
title = {Application of CRISPR/CasΦ2 System for Genome Editing in Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628563},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; Plants/genetics ; Tobacco/genetics ; },
abstract = {CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg[2+] irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg[2+] irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg[2+] ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.},
}
@article {pmid35628407,
year = {2022},
author = {Hu, H and Yu, F},
title = {A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628407},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {Transgenic or genetically modified crops have great potential in modern agriculture but still suffer from heavy regulations worldwide due to biosafety concerns. As a promising alternative route, cisgenic crops have received higher public acceptance and better reviews by governing authorities. To serve the purpose of cisgenic plant breeding, we have developed a CRISPR/Cas9-based vector system, which is capable of delivering target gene-of-interest (GOI) into recipient plants while removing undesired genetic traces in the plants. The new system features a controllable auto-excision feature, which is realized by a core design of embedded multi-clonal sequence and the use of inducible promoters controlling the expression of Cas9 nuclease. In the current proof-of-concept study in Arabidopsis thaliana (L.) Heynh., we have successfully incorporated a GOI into the plant and removed the selection marker and CRISPR/Cas9 components from the final product. Following the designed workflow, we have demonstrated that novel cisgenic plant germplasms with desired traits could be developed within one to two generations. Further characterizations of the vector system have shown that heat treatment at 37 °C could significantly improve the editing efficiency (up to 100%), and no off-target mutations were identified in the Arabidopsis background. This novel vector system is the first CRISPR/Cas9-based genome editing tool for cisgenic plant breeding and should prove powerful for other similar applications in the bright future of precision molecular breeding.},
}
@article {pmid35628253,
year = {2022},
author = {Zhang, N and He, J and Muhammad, A and Shao, Y},
title = {CRISPR/Cas9-Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant-Associated Isolates.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628253},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plants/genetics ; Pseudomonas/genetics ; },
abstract = {As one of the most widespread groups of Gram-negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.},
}
@article {pmid35628210,
year = {2022},
author = {Kim, TH and Lee, SW},
title = {Therapeutic Application of Genome Editing Technologies in Viral Diseases.},
journal = {International journal of molecular sciences},
volume = {23},
number = {10},
pages = {},
pmid = {35628210},
issn = {1422-0067},
mesh = {*Gene Editing ; Genome ; Humans ; Technology ; Transcription Activator-Like Effector Nucleases/genetics ; *Virus Diseases/genetics/therapy ; },
abstract = {Viral infections can be fatal and consequently, they are a serious threat to human health. Therefore, the development of vaccines and appropriate antiviral therapeutic agents is essential. Depending on the virus, it can cause an acute or a chronic infection. The characteristics of viruses can act as inhibiting factors for the development of appropriate treatment methods. Genome editing technology, including the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), is a technology that can directly target and modify genomic sequences in almost all eukaryotic cells. The development of this technology has greatly expanded its applicability in life science research and gene therapy development. Research on the use of this technology to develop therapeutics for viral diseases is being conducted for various purposes, such as eliminating latent infections or providing resistance to new infections. In this review, we will look at the current status of the development of viral therapeutic agents using genome editing technology and discuss how this technology can be used as a new treatment approach for viral diseases.},
}
@article {pmid35627307,
year = {2022},
author = {Xiong, Y and Zhuang, R and Zhao, G and Liu, Y and Su, Y and Wang, W and Xi, X and Yang, Y and Han, X and Xie, S and Wang, H and Li, X and Zuo, B and Zhao, S and Feng, Z and Ruan, J},
title = {Identification of the CKM Gene as a Potential Muscle-Specific Safe Harbor Locus in Pig Genome.},
journal = {Genes},
volume = {13},
number = {5},
pages = {},
pmid = {35627307},
issn = {2073-4425},
mesh = {Animals ; *CRISPR-Cas Systems ; *Genome/genetics ; Livestock/genetics ; Muscles ; Promoter Regions, Genetic ; Swine/genetics ; },
abstract = {Genetically modified pigs have shown considerable application potential in the fields of life science research and livestock breeding. Nevertheless, a barrier impedes the production of genetically modified pigs. There are too few safe harbor loci for the insertion of foreign genes into the pig genome. Only a few loci (pRosa26, pH11 and Pifs501) have been successfully identified to achieve the ectopic expression of foreign genes and produce gene-edited pigs. Here, we use CRISPR/Cas9-mediated homologous directed repair (HDR) to accurately knock the exogenous gene-of-interest fragments into an endogenous CKM gene in the porcine satellite cells. After porcine satellite cells are induced to differentiate, the CKM gene promoter simultaneously initiates the expression of the CKM gene and the exogenous gene. We infer preliminarily that the CKM gene can be identified as a potential muscle-specific safe harbor locus in pigs for the integration of exogenous gene-of-interest fragments.},
}
@article {pmid35627242,
year = {2022},
author = {Carrington, B and Bishop, K and Sood, R},
title = {A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases.},
journal = {Genes},
volume = {13},
number = {5},
pages = {},
pmid = {35627242},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Transcription Activator-Like Effector Nucleases ; *Zebrafish/genetics ; },
abstract = {The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.},
}
@article {pmid35627121,
year = {2022},
author = {Jiang, C and Tao, D and Geng, Y and Yang, H and Xu, B and Chen, Y and Hu, C and Chen, H and Xie, S and Guo, A},
title = {Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification.},
journal = {Genes},
volume = {13},
number = {5},
pages = {},
pmid = {35627121},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; DNA, Viral/genetics ; *Lumpy skin disease virus/genetics ; Lysergic Acid Diethylamide ; Nucleotidyltransferases/genetics ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; },
abstract = {Lumpy skin disease (LSD) is a severe and highly infectious pox disease of cattle caused by the lumpy skin disease virus (LSDV). To facilitate early control of LSD, this study aimed to develop a new rapid on-site LSDV detection method using an orf068 gene-based recombinase polymerase amplification assay (RPA) coupled with a CRISPR-Cas12a-based fluorescence assay (RPA-Cas12a-fluorescence assay). The results showed that the sensitivity of our RPA-Cas12a-fluorescence assay for detecting LSDV orf068 gene reached 5 copies/μL with plasmid as a template, and 10[2] TCID50/mL with viral genomic DNA as a template. No cross-reaction with other common bovine viruses was observed. Further, an on-site RPA-Cas12a-fluorescence assay of 40 clinical samples from cattle with or without LSD showed a diagnostic sensitivity of 96.3% (95% CI: 81.0-99.9%) and specificity of 92.31% (95% CI: 62.1-99.6%), which was close to those of the quantitative PCR assay. Therefore, our RPA-Cas12a-fluorescence assay has promising prospects in on-site rapid LSDV detection.},
}
@article {pmid35626649,
year = {2022},
author = {Abuhamad, AY and Mohamad Zamberi, NN and Sheen, L and Naes, SM and Mohd Yusuf, SNH and Ahmad Tajudin, A and Mohtar, MA and Amir Hamzah, AS and Syafruddin, SE},
title = {Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations.},
journal = {Cells},
volume = {11},
number = {10},
pages = {},
pmid = {35626649},
issn = {2073-4409},
mesh = {*Breast Neoplasms/genetics ; *CRISPR-Cas Systems/genetics ; Female ; HEK293 Cells ; Humans ; Mutation/genetics ; Tumor Suppressor Protein p53/genetics ; Workflow ; },
abstract = {Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations' molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.},
}
@article {pmid35625953,
year = {2022},
author = {Coira, IF and Rincón, R and Cuendet, M},
title = {The Multiple Myeloma Landscape: Epigenetics and Non-Coding RNAs.},
journal = {Cancers},
volume = {14},
number = {10},
pages = {},
pmid = {35625953},
issn = {2072-6694},
support = {310030_184790/SNSF_/Swiss National Science Foundation/Switzerland ; },
abstract = {Despite advances in available treatments, multiple myeloma (MM) remains an incurable disease and represents a challenge in oncohematology. New insights into epigenetic factors contributing to MM development and progression have improved the knowledge surrounding its molecular basis. Beyond classical epigenetic factors, including methylation and acetylation, recent genome analyses have unveiled the importance of non-coding RNAs in MM pathogenesis. Non-coding RNAs have become of interest, as their dysregulation opens the door to new therapeutic approaches. The discovery, in the past years, of molecular techniques, such as CRISPR-Cas, has led to innovative therapies with potential benefits to achieve a better outcome for MM patients. This review summarizes the current knowledge on epigenetics and non-coding RNAs in MM pathogenesis.},
}
@article {pmid35623786,
year = {2022},
author = {Zhou, S and Sun, H and Huo, D and Wang, X and Qi, N and Peng, L and Yang, M and Lu, P and Hou, C},
title = {A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation.},
journal = {Analytica chimica acta},
volume = {1212},
number = {},
pages = {339914},
doi = {10.1016/j.aca.2022.339914},
pmid = {35623786},
issn = {1873-4324},
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA/genetics ; *DNA Methylation ; *Deoxyribonuclease I ; Humans ; },
abstract = {Detection of methylation changes associated with oncogenic transformation is essential for early screening and treatment of cancer. Herein, we propose a novel DNA methylation detection assay based on the methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a (GlaI-DC-SDA-CRISPR/Cas12a). The GlaI enables highly specific recognition and digestion of methylated target site (dsDNA) but leaves unmethylated target intact. Therefore, only methylated DNA can be digested to produce two free 3'-OH terminus for triggering the next SDA-CRISPR/Cas12a. Compared with the fluorescence response under single amplification template, DC-SDA with double amplification templates shows higher sensitivity. Benefiting from the high specificity of GlaI and the cascaded amplification effect of DC-SDA combined with CRISPR/Cas12a, the proposed method shows excellent performance for DNA methylation detection with low LOD (1.28 × 10[-13] M), ultra-low background interference and wide detection range (2 × 10[-13] to 4 × 10[-11], 4 × 10[-11] to 1 × 10[-8] M). 0.1% of DNA methylation can be discriminated from the mixture with a mass of unmethylated DNA. Most importantly, the proposed assay can be applied to the actual detection of human serum and genomic DNA, as well as to distinguish normal cells from cancer cells. It can also quantify DNA methylation in genomic DNA (HCT116) with a LOD of 37.95 ng, indicating its great potential in early clinical cancer screening.},
}
@article {pmid35623354,
year = {2022},
author = {Johnson, MC and Hille, LT and Kleinstiver, BP and Meeske, AJ and Bondy-Denomy, J},
title = {Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages.},
journal = {Molecular cell},
volume = {82},
number = {11},
pages = {2161-2166.e3},
pmid = {35623354},
issn = {1097-4164},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; R35 GM142460/GM/NIGMS NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Bacteriophages/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; Leptotrichia/genetics ; Mammals/metabolism ; Prophages/genetics/metabolism ; Ribonucleases/metabolism ; },
abstract = {CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.},
}
@article {pmid35623274,
year = {2022},
author = {Chen, H and Li, ZY and Chen, J and Yu, H and Zhou, W and Shen, F and Chen, Q and Wu, L},
title = {CRISPR/Cas12a-based electrochemical biosensor for highly sensitive detection of cTnI.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {146},
number = {},
pages = {108167},
doi = {10.1016/j.bioelechem.2022.108167},
pmid = {35623274},
issn = {1878-562X},
mesh = {*Aptamers, Nucleotide ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Humans ; Limit of Detection ; Troponin I ; },
abstract = {The successful fabrication of the cTnI detection platform is very meaningful for instant diagnosis of the myocardialinjury and related cardiovascular diseases (CVDs). In this research work, the magnetic nanoparticles and aptamer collaboration with the Cas12a/crRNA are used for the electrochemical detection of cTnI. The aptamer is hybridized with its partially complementary DNA (probe 2, P2) and then is modified on the magnetic nanoparticles. In the presence of cTnI, the cTnI combines with the aptamer and P2 is released. The released P2 is hybridized with the crRNA and the trans-cleavage activity of CRISPR/Cas12a is triggered. Therefore, the methylene blue-modified DNA (probe1, P1) on the surface of the electrode is cleaved, resulting in the decrease of the electrochemical signal. Based on the synergy effect of the high specific target recognition of aptamer, target-specifically triggering trans-cleavage activity of CRISPR/Cas12a, as well as good separation ability of magnetic nanoparticles, the developed electrochemical biosensor enables to detect cTnI with high specificity and sensitivity. The detection limit is low down to 10 pg/mL with a linear range from 100 pg/mL to 50000 pg/mL. The developed sensing platform was successfully applied for the detection of cTnI in human serum. This fabricated CRISPR/Cas12a-based electrochemical biosensor can offer a valuable tool for the diagnosis, prognosis, and treatment of patient with CVDs.},
}
@article {pmid35623245,
year = {2022},
author = {Li, K and Luo, S and Guan, S and Situ, B and Wu, Y and Ou, Z and Tao, M and Zheng, L and Cai, Z},
title = {Tetrahedral framework nucleic acids linked CRISPR/Cas13a signal amplification system for rare tumor cell detection.},
journal = {Talanta},
volume = {247},
number = {},
pages = {123531},
doi = {10.1016/j.talanta.2022.123531},
pmid = {35623245},
issn = {1873-3573},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; RNA ; },
abstract = {The sensitive and accurate detection of rare tumor cells provides precise diagnosis and dynamic assessment information in various tumor spectrums. However, rare tumor cells assay is still a challenge due to the exceedingly rare presence in the blood. In this research, we develop a fluorescent approach for the identification of rare tumor cells based on a combination of immunosorbent capture and a three-step signal amplification strategy. First, rare tumor cells are captured by immunoadsorption on 96-well plates. Second, self-synthesized tetrahedral framework nucleic acids (tFNAs) spontaneously anchor into the lipid bilayer of rare tumor cells, resulting in a "one to more" amplification effect. Then, the double-stranded DNA (dsDNA) binds to the vertices of the tFNAs and generates a large amount of target RNA by T7 polymerase, which is the secondary signal amplification. Finally, the target RNA activates the collateral cleavage ability of CRISPR/Cas13a, and the reporter RNA is cleaved for third signal amplification. The detection limit of the proposed method is down to 1 cell mL[-1]. Furthermore, the tFNAs-Cas13a system is also shown to be capable of detecting rare tumor cells in spiked-in samples and clinical blood samples. This platform enables speedy detection of rare tumor cells with high sensitivity and good specificity, and shows great potential for tumor diagnosis.},
}
@article {pmid35621119,
year = {2022},
author = {Ishizaki, T and Hernandez, S and Paoletta, MS and Sanderson, T and Bushell, ESC},
title = {CRISPR/Cas9 and genetic screens in malaria parasites: small genomes, big impact.},
journal = {Biochemical Society transactions},
volume = {50},
number = {3},
pages = {1069-1079},
pmid = {35621119},
issn = {1470-8752},
support = {210918/Z/18/Z//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Humans ; *Parasites/genetics ; *Plasmodium/genetics ; *Toxoplasma/genetics ; },
abstract = {The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.},
}
@article {pmid35620343,
year = {2022},
author = {Akbari Kordkheyli, V and Rashidi, M and Shokri, Y and Fallahpour, S and Variji, A and Nabipour Ghara, E and Hosseini, SM},
title = {CRISPER/CAS System, a Novel Tool of Targeted Therapy of Drug-Resistant Lung Cancer.},
journal = {Advanced pharmaceutical bulletin},
volume = {12},
number = {2},
pages = {262-273},
pmid = {35620343},
issn = {2228-5881},
abstract = {Lung cancer (LC) is the most common cause of cancer-related death worldwide. Patients with LC are usually diagnosed at advanced phases. Five-year survival rate in LC patients is approximately 16%. Despite decades of research on LC treatments, clinical outcomes are still very poor, necessitating to develop novel technologies to manage the disease. Considering the role of genetic and epigenetic changes in oncogenes and tumor-suppressor genes in cancer progression, gene therapy provides a hot spot in cancer treatment research. Gene therapy offers less side effects compared to conventional methods such as chemotherapy. Unlike the traditional approaches of gene therapy that have temporary effects, using genetic modification tools can offer persistent cure. Over the past a few years, many studies have effectively used the CRISPR-Cas9 approach to modify gene expression in cells. This system is applied to induce site-specific mutagenesis and epigenetic modifications and regulate gene expression. In this review, we discuss recent applications of the CRISPR-Cas9 technology in treating LC.},
}
@article {pmid35619560,
year = {2022},
author = {Li, H and Zhu, Z and Li, S and Li, J and Yan, L and Zhang, C and Ma, Y and Xia, L},
title = {Multiplex precision gene editing by a surrogate prime editor in rice.},
journal = {Molecular plant},
volume = {15},
number = {7},
pages = {1077-1080},
doi = {10.1016/j.molp.2022.05.009},
pmid = {35619560},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Plant ; *Oryza/genetics ; },
}
@article {pmid35619556,
year = {2022},
author = {Majeau, N and Fortin-Archambault, A and Gérard, C and Rousseau, J and Yaméogo, P and Tremblay, JP},
title = {Serum extracellular vesicles for delivery of CRISPR-CAS9 ribonucleoproteins to modify the dystrophin gene.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {7},
pages = {2429-2442},
pmid = {35619556},
issn = {1525-0024},
support = {//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Dystrophin/genetics/metabolism ; *Extracellular Vesicles/metabolism ; Gene Editing/methods ; Genetic Therapy/methods ; Mice ; Mice, Inbred mdx ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Ribonucleoproteins/metabolism ; },
abstract = {Extracellular vesicles (EVs) mediate intercellular biomolecule exchanges in the body, making them promising delivery vehicles for therapeutic cargo. Genetic engineering by the CRISPR system is an interesting therapeutic avenue for genetic diseases such as Duchenne muscular dystrophy (DMD). We developed a simple method for loading EVs with CRISPR ribonucleoproteins (RNPs) consisting of SpCas9 proteins and guide RNAs (gRNAs). EVs were first purified from human or mouse serum using ultrafiltration and size-exclusion chromatography. Using protein transfectant to load RNPs into serum EVs, we showed that EVs are good carriers of RNPs in vitro and restored the expression of the tdTomato fluorescent protein in muscle fibers of Ai9 mice. EVs carrying RNPs targeting introns 22 and 24 of the DMD gene were also injected into muscles of mdx mice having a non-sense mutation in exon 23. Up to 19% of the cDNA extracted from treated mdx mice had the intended deletion of exons 23 and 24, allowing dystrophin expression in muscle fibers. RNPs alone, without EVs, were inefficient in generating detectable deletions in mouse muscles. This method opens new opportunities for rapid and safe delivery of CRISPR components to treat DMD.},
}
@article {pmid35619054,
year = {2022},
author = {Arnan, C and Ullrich, S and Pulido-Quetglas, C and Nurtdinov, R and Esteban, A and Blanco-Fernandez, J and Aparicio-Prat, E and Johnson, R and Pérez-Lluch, S and Guigó, R},
title = {Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {402},
pmid = {35619054},
issn = {1471-2164},
mesh = {CRISPR-Cas Systems ; Cell Transdifferentiation ; Humans ; Macrophages ; *RNA, Guide/genetics ; *RNA, Long Noncoding/genetics ; },
abstract = {CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.},
}
@article {pmid35618895,
year = {2022},
author = {DeLuca, S and Bursac, N},
title = {CRISPR Library Screening in Cultured Cardiomyocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2485},
number = {},
pages = {1-13},
pmid = {35618895},
issn = {1940-6029},
support = {R01 HL132389/HL/NHLBI NIH HHS/United States ; T32 HD040372/HD/NICHD NIH HHS/United States ; U01 EB028901/EB/NIBIB NIH HHS/United States ; U01 HL134764/HL/NHLBI NIH HHS/United States ; U01 HL156348/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Library ; Genome ; High-Throughput Nucleotide Sequencing ; *Myocytes, Cardiac ; },
abstract = {CRISPR-Cas9-based screening technologies enable precise, high-throughput genetic and epigenetic manipulation to study mechanisms of development and disease and identify new therapeutic targets. Here, we describe a general protocol for the generation of custom, pooled CRISPR sgRNA libraries for screening in cardiomyocyte cultures. This methodology can address a variety of lab-specific research questions in cardiomyocytes and other cell types, as the genes to be modified can be curated or whole genomes can be investigated. The use of lentiviral sgRNA delivery followed by high-throughput sequencing allows for rapid comparison and identification of candidate genes and epigenetic modifiers, which can be further validated individually or in sub-pooled libraries following screening.},
}
@article {pmid35618430,
year = {2022},
author = {Woodside, WT and Vantsev, N and Catchpole, RJ and Garrett, SC and Olson, S and Graveley, BR and Terns, MP},
title = {Type III-A CRISPR systems as a versatile gene knockdown technology.},
journal = {RNA (New York, N.Y.)},
volume = {28},
number = {8},
pages = {1074-1088},
pmid = {35618430},
issn = {1469-9001},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Knockdown Techniques ; RNA/genetics ; Staphylococcus epidermidis ; Technology ; },
abstract = {CRISPR-Cas systems are functionally diverse prokaryotic antiviral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis, and S. thermophilus) were each expressed on a single plasmid in E. coli, and the efficiency and specificity of gene knockdown was assessed by northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and noncoding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.},
}
@article {pmid35618221,
year = {2022},
author = {Yu, H and Iqbal, A and Fang, X and Jiang, P and Zhao, Z},
title = {Transcriptome analysis of CRISPR/Cas9-mediated GPAM[-/-] in bovine mammary epithelial cell-line unravelled the effects of GPAM gene on lipid metabolism.},
journal = {Gene},
volume = {834},
number = {},
pages = {146574},
doi = {10.1016/j.gene.2022.146574},
pmid = {35618221},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Epithelial Cells/metabolism ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; Gene Expression Profiling ; Glycerol-3-Phosphate O-Acyltransferase/genetics ; *Lipid Metabolism/genetics ; *Mammary Glands, Animal/metabolism ; Milk/metabolism ; Phosphates/metabolism ; },
abstract = {Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is an enzyme in animal lipid metabolism pathways that catalyzes the initial and most committed step of glycerolipid biosynthesis. The present study mainly focused on exploring the relationship between the GPAM gene and the lipid metabolism of mammary epithelial cells and the effect of GPAM on the related pathways of lipid metabolism. The GPAM gene was knocked out entirely in bovine mammary epithelial cells(BMECs) using CRISPR/Cas9 technology, and the mechanism by which the GPAM gene regulates lipid metabolism in BMECs was confirmed. Furthermore, after the complete loss of GPAM, BMECs' triglycerides (TGs) and cholesterol (CHOL) levels were significantly decreased (p < 0.05). Concurrently, the content of octanoic acid, a medium-chain saturated fatty acid, increased substantially in BMECs. RNA-seq of GPAM[-/-] BMECs revealed that GPAM could affect the expression of genes related to lipid metabolism, downregulated the expression of Acyl-CoA synthetase long-chain family member 5 (ACSL5), Fatty Acid Binding Protein 3 (FABP3), Hormone-sensitive lipase (HSL), Protease, serine-2 (PRSS2), 1-Acylglycerol-3-Phosphate O Acyltransferase 4 (AGPAT4), and regulated the milk synthesis metabolism pathway.The findings revealed that a number of genes were expressed, a number of genes were differentially expressed genes (DEGs), and a number of GO terms were enriched, with a number of GO terms considerably increased. Further, the differentially expressed genes (DEGs) were significantly enriched in Fat digestion and absorption pathway, Fatty acid metabolic pathway, Biosynthesis of unsaturated fatty acids, Biosynthesis of unsaturated fatty acids and steroids, NF-kappa B signalling pathway, MAPK signalling pathway. In conclusion, the current research results show that GPAM is a crucial regulator of BMEC lipid metabolism. GPAM[-/-] BMEC may also become useful genetic materials and tools for future research on gene functions related to lipid and fatty acid metabolism. This study will contribute to the discovery of gene regulation and molecular mechanisms in milk fat synthesis.},
}
@article {pmid35617958,
year = {2022},
author = {Omer-Javed, A and Pedrazzani, G and Albano, L and Ghaus, S and Latroche, C and Manzi, M and Ferrari, S and Fiumara, M and Jacob, A and Vavassori, V and Nonis, A and Canarutto, D and Naldini, L},
title = {Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells.},
journal = {Cell},
volume = {185},
number = {13},
pages = {2248-2264.e21},
pmid = {35617958},
issn = {1097-4172},
mesh = {Animals ; *Gene Editing ; Genetic Therapy/methods ; *Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells ; Humans ; Mice ; },
abstract = {Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.},
}
@article {pmid35617843,
year = {2022},
author = {He, Z and Feng, K and Sun, H and Yu, T and Zhu, D and Yang, Y},
title = {Generation of a human extended pluripotent stem cell line (SKLRMe002-A) carrying a doxycycline-inducible Cas9 expression cassette.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102816},
doi = {10.1016/j.scr.2022.102816},
pmid = {35617843},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; *Doxycycline/pharmacology ; Humans ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Human extended pluripotent stem cell (hEPS) is a novel type of pluripotent stem cell, which possesses bi-potency towards both embryonic and extraembryonic lineages. Here, we generated a hEPS cell line (hEPS1-iCas9-B) from the cell line named hEPS1, carrying a doxycycline-inducible Cas9 expression cassette along with a constitutive reverse tetracycline transactivator (M2rtTA) expression cassette at the AAVS1 locus, thus we could efficiently generate genetically modified hEPS for studies. This cell lined remained self-renewal, differentiation potential and normal karyotype. Meanwhile, it showed robust transcriptional expression of Cas9 with doxycycline induction and could target the site where the sgRNA guided.},
}
@article {pmid35617371,
year = {2022},
author = {Schuler, G and Hu, C and Ke, A},
title = {Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.},
journal = {Science (New York, N.Y.)},
volume = {376},
number = {6600},
pages = {1476-1481},
doi = {10.1126/science.abq7220},
pmid = {35617371},
issn = {1095-9203},
mesh = {Amino Acid Motifs ; *CRISPR-Associated Protein 9/chemistry/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *DNA Cleavage ; Nucleic Acid Conformation ; Protein Domains ; RNA, Bacterial/genetics ; *RNA, Guide/chemistry ; *Ribonucleoproteins/chemistry ; },
abstract = {Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.},
}
@article {pmid35617303,
year = {2022},
author = {Hernandez, VA and Carvajal-Moreno, J and Wang, X and Pietrzak, M and Yalowich, JC and Elton, TS},
title = {Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells.},
journal = {PloS one},
volume = {17},
number = {5},
pages = {e0265794},
pmid = {35617303},
issn = {1932-6203},
support = {R01 CA226906/CA/NCI NIH HHS/United States ; },
mesh = {Antigens, Neoplasm/genetics ; CRISPR-Cas Systems/genetics ; DNA Topoisomerases, Type II/genetics/metabolism ; Etoposide/pharmacology ; Humans ; Introns/genetics ; K562 Cells ; *Leukemia/genetics ; *RNA Splice Sites ; RNA, Messenger ; },
abstract = {DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5' splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5' splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.},
}
@article {pmid35616867,
year = {2022},
author = {Jansing, J and Bortesi, L},
title = {Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2480},
number = {},
pages = {241-284},
pmid = {35616867},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Glycosylation ; Glycosyltransferases/genetics/metabolism ; Humans ; Mammals/genetics ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Polysaccharides/chemistry ; Recombinant Proteins/genetics/metabolism ; *Tobacco/genetics/metabolism ; Xylose/metabolism ; },
abstract = {Plants are excellent production hosts for the in vivo synthesis of complex glycosylated proteins such as antibodies. The plant N-glycosylation machinery is largely similar to that found in humans and other mammalian organisms, which is an advantage in comparison to microbial production systems in particular. However, there are some differences in the identity and chemical linkage of the sugars that plants and mammals use to build their N-glycans. These differences can affect important properties of glycosylated proteins produced recombinantly in plants. Here we describe the complete procedure of multiplex targeted gene knockout with CRISPR/Cas9 in Nicotiana benthamiana in order to eliminate the undesirable sugars α-1,3-fucose and β-1,2-xylose from the plant N-glycans. The workflow includes target gene identification, guide RNA design and testing, plant transformation, and the analysis of the regenerated transgenic plants by Sanger sequencing, immunoblot, and mass-spectrometric analysis of recombinant and endogenous proteins.},
}
@article {pmid35616865,
year = {2022},
author = {González, B and Vazquez-Vilar, M and Sánchez-Vicente, J and Orzáez, D},
title = {Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2480},
number = {},
pages = {193-214},
pmid = {35616865},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Plant Breeding ; *RNA, Guide/genetics ; Tobacco/genetics/metabolism ; },
abstract = {New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results.},
}
@article {pmid35615910,
year = {2022},
author = {Ma, JY and Wang, SY and Du, YC and Wang, DX and Tang, AN and Wang, J and Kong, DM},
title = {"RESET" Effect: Random Extending Sequences Enhance the Trans-Cleavage Activity of CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {94},
number = {22},
pages = {8050-8057},
doi = {10.1021/acs.analchem.2c01401},
pmid = {35615910},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; },
abstract = {The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing applications. However, the lack of exploration on the fundamental properties of CRISPR/Cas12a not only discourages further in-depth studies of the CRISPR/Cas12a system but also limits the design space of CRISPR/Cas12a-based applications. Herein, a "RESET" effect (random extending sequences enhance trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, a single-stranded DNA, which is too short to work as the activator, can efficiently activate CRISPR/Cas12a after being extended a random sequence from its 3'-end, even when the random sequence folds into secondary structures. The finding of the "RESET" effect enriches the CRISPR/Cas12a-based sensing strategies. Based on this effect, two CRISPR/Cas12a-based biosensors are designed for the sensitive and specific detection of two biologically important enzymes.},
}
@article {pmid35614226,
year = {2022},
author = {Nusser, A and Sagar, and Swann, JB and Krauth, B and Diekhoff, D and Calderon, L and Happe, C and Grün, D and Boehm, T},
title = {Developmental dynamics of two bipotent thymic epithelial progenitor types.},
journal = {Nature},
volume = {606},
number = {7912},
pages = {165-171},
pmid = {35614226},
issn = {1476-4687},
support = {/ERC_/European Research Council/International ; },
mesh = {Aging ; Animals ; Autocrine Communication ; CRISPR-Cas Systems ; Cellular Microenvironment ; *Epithelial Cells/cytology/metabolism ; Epithelium ; Fibroblast Growth Factor 7 ; Mice ; RNA-Seq ; Single-Cell Analysis ; *Stem Cells/cytology ; *T-Lymphocytes/cytology/metabolism ; *Thymus Gland/cytology ; },
abstract = {T cell development in the thymus is essential for cellular immunity and depends on the organotypic thymic epithelial microenvironment. In comparison with other organs, the size and cellular composition of the thymus are unusually dynamic, as exemplified by rapid growth and high T cell output during early stages of development, followed by a gradual loss of functional thymic epithelial cells and diminished naive T cell production with age[1-10]. Single-cell RNA sequencing (scRNA-seq) has uncovered an unexpected heterogeneity of cell types in the thymic epithelium of young and aged adult mice[11-18]; however, the identities and developmental dynamics of putative pre- and postnatal epithelial progenitors have remained unresolved[1,12,16,17,19-27]. Here we combine scRNA-seq and a new CRISPR-Cas9-based cellular barcoding system in mice to determine qualitative and quantitative changes in the thymic epithelium over time. This dual approach enabled us to identify two principal progenitor populations: an early bipotent progenitor type biased towards cortical epithelium and a postnatal bipotent progenitor population biased towards medullary epithelium. We further demonstrate that continuous autocrine provision of Fgf7 leads to sustained expansion of thymic microenvironments without exhausting the epithelial progenitor pools, suggesting a strategy to modulate the extent of thymopoietic activity.},
}
@article {pmid35613622,
year = {2022},
author = {Prosser, BL and Helbig, I},
title = {Base editing the synapse: Modeling a complex neurological disorder in non-human primates.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {6},
pages = {2114-2116},
pmid = {35613622},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; *Neuromuscular Diseases ; *Primates/genetics ; RNA Editing ; Synapses ; },
}
@article {pmid35613590,
year = {2022},
author = {López Del Amo, V and Juste, SS and Gantz, VM},
title = {A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila.},
journal = {Cell reports},
volume = {39},
number = {8},
pages = {110843},
pmid = {35613590},
issn = {2211-1247},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 AI162911/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA ; *Deoxyribonuclease I/metabolism ; Drosophila/metabolism ; *Gene Drive Technology ; Gene Editing ; },
abstract = {CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5' overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.},
}
@article {pmid35612314,
year = {2022},
author = {Liu, J and Vogel, AK and Miao, J and Carnahan, JA and Lowes, DJ and Rybak, JM and Peters, BM},
title = {Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction.},
journal = {Microbiology spectrum},
volume = {10},
number = {3},
pages = {e0263021},
pmid = {35612314},
issn = {2165-0497},
support = {R21 AI141829/AI/NIAID NIH HHS/United States ; R01 AI134796/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Candida albicans/genetics ; Homozygote ; Hygromycin B ; Ribonucleoproteins/genetics ; Sequence Deletion ; },
abstract = {As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1-flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene (CaHygB). Cassettes were PCR-amplified from both SAT1-flipper and CaHygB-flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 10[4] homozygous deletion mutants per μg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δals3, Δ/Δcph1 Δ/Δefg1, Δ/Δece1) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1β response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1-flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1- and CaHygB-flipper cassettes were simultaneously integrated at loci with high efficiency (10[4] transformants per μg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.},
}
@article {pmid35611733,
year = {2022},
author = {Shang, L and Song, S and Zhang, T and Yan, K and Cai, H and Yuan, Y and Cheng, Y},
title = {[Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {5},
pages = {1847-1858},
doi = {10.13345/j.cjb.210541},
pmid = {35611733},
issn = {1872-2075},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Muscle, Skeletal/metabolism ; Mutation ; *Myostatin/genetics/metabolism ; Phenotype ; Rabbits ; },
abstract = {Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN[-/-] homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN[-/-] primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN[-/-] homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN[+/+] wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm[2] vs. (1 274.8±327.3) μm[2], P < 0.01), but also significantly higher than that of MSTN[+/-] hemizygous rabbits ((3 512.2±439.2) μm[2] vs. (2 610.4±604.4) μm[2], P < 0.05). In summary, five homozygous mutants rabbits of MSTN[-/-] gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN[-/-] homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.},
}
@article {pmid35610585,
year = {2022},
author = {Sun, L and Wang, J and Yan, F and Wang, G and Li, Y and Huang, J},
title = {CrisprVi: a software for visualizing and analyzing CRISPR sequences of prokaryotes.},
journal = {BMC bioinformatics},
volume = {23},
number = {Suppl 3},
pages = {172},
pmid = {35610585},
issn = {1471-2105},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome ; Prokaryotic Cells ; *Software ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi.<br><br>RESULTS: CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes.<br><br>CONCLUSIONS: CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes.},
}
@article {pmid35610381,
year = {2022},
author = {Zhou, M and Cao, Y and Sui, M and Shu, X and Wan, F and Zhang, B},
title = {Dead Cas(t) light on new life: CRISPRa-mediated reprogramming of somatic cells into neurons.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {6},
pages = {315},
pmid = {35610381},
issn = {1420-9071},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Gene Editing ; Neurons/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Overexpression of exogenous lineage-specific transcription factors could directly induce terminally differentiated somatic cells into target cell types. However, the low conversion efficiency and the concern about introducing exogenous genes limit the clinical application. With the rapid progress in genome editing, the application of CRISPR/dCas9 has been expanding rapidly, including converting somatic cells into other types of cells in vivo and in vitro. Using the CRISPR/dCas9 system, direct neuronal reprogramming could be achieved by activating endogenous genes. Here, we will discuss the latest progress, new insights, and future challenges of the application of the dCas9 system in direct neuronal reprogramming.},
}
@article {pmid35609453,
year = {2022},
author = {Wu, H and Cao, X and Meng, Y and Richards, D and Wu, J and Ye, Z and deMello, AJ},
title = {DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114377},
doi = {10.1016/j.bios.2022.114377},
pmid = {35609453},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids/genetics ; Salmonella typhimurium/genetics ; },
abstract = {Since their discovery, CRISPR/Cas systems have been extensively exploited in nucleic acid biosensing. However, the vast majority of contemporary platforms offer only qualitative detection of nucleic acid, and fail to realize ultrasensitive quantitative detection. Herein, we report a digital droplet-based platform (DropCRISPR), which combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a to realize ultrasensitive and quantitative detection of nucleic acids. This is achieved through a novel two-step microfluidic system which combines droplet LAMP with a picoinjector capable of injecting the required CRISPR/Cas12a reagents into each droplet. This method circumvents the temperature incompatibilities of LAMP and CRISPR/Cas12a and avoids mutual interference between amplification reaction and CRISPR detection. Ultrasensitive detection (at fM level) was achieved for a model plasmid containing the invA gene of Salmonella typhimurium (St), with detection down to 10[2] cfu/mL being achieved in pure bacterial culture. Additionally, we demonstrate that the DropCRISPR platform is capable of detecting St in raw milk samples without additional nucleic acid extraction. The sensitivity and robustness of the DropCRISPR further demonstrates the potential of CRISPR/Cas-based diagnostic platforms, particularly when combined with state-of-the-art microfluidic architectures.},
}
@article {pmid35609450,
year = {2022},
author = {Hang, XM and Liu, PF and Tian, S and Wang, HY and Zhao, KR and Wang, L},
title = {Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114393},
doi = {10.1016/j.bios.2022.114393},
pmid = {35609450},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA ; *Hemorrhagic Fever, Ebola/diagnosis/genetics ; Humans ; RNA ; },
abstract = {A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/μL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.},
}
@article {pmid35608753,
year = {2022},
author = {Qin, Y and Li, S and Li, XJ and Yang, S},
title = {CRISPR-Based Genome-Editing Tools for Huntington's Disease Research and Therapy.},
journal = {Neuroscience bulletin},
volume = {38},
number = {11},
pages = {1397-1408},
pmid = {35608753},
issn = {1995-8218},
mesh = {Humans ; Gene Editing ; *Huntington Disease/genetics/therapy ; CRISPR-Cas Systems/genetics ; *Neurodegenerative Diseases ; },
abstract = {Huntington's disease (HD) is an autosomal dominantly-inherited neurodegenerative disease, which is caused by CAG trinucleotide expansion in exon 1 of the Huntingtin (HTT) gene. Although HD is a rare disease, its monogenic nature makes it an ideal model in which to understand pathogenic mechanisms and to develop therapeutic strategies for neurodegenerative diseases. Clustered regularly-interspaced short palindromic repeats (CRISPR) is the latest technology for genome editing. Being simple to use and highly efficient, CRISPR-based genome-editing tools are rapidly gaining popularity in biomedical research and opening up new avenues for disease treatment. Here, we review the development of CRISPR-based genome-editing tools and their applications in HD research to offer a translational perspective on advancing the genome-editing technology to HD treatment.},
}
@article {pmid35608343,
year = {2022},
author = {Zhang, C and Li, N and Rao, L and Li, J and Liu, Q and Tian, C},
title = {Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.},
journal = {Microbiology spectrum},
volume = {10},
number = {3},
pages = {e0232121},
pmid = {35608343},
issn = {2165-0497},
mesh = {*CRISPR-Cas Systems ; *Cellulase/genetics ; Cytosine ; DNA ; Sordariales ; Transcription Factors/genetics ; },
abstract = {Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi. IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.},
}
@article {pmid35608169,
year = {2022},
author = {Huang, C and Zhang, L and Zhu, Y and Zhang, Z and Liu, Y and Liu, C and Ge, S and Yu, J},
title = {Dual-Engine Powered Paper Photoelectrochemical Platform Based on 3D DNA Nanomachine-Mediated CRISPR/Cas12a for Detection of Multiple miRNAs.},
journal = {Analytical chemistry},
volume = {94},
number = {22},
pages = {8075-8084},
doi = {10.1021/acs.analchem.2c01717},
pmid = {35608169},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Electrochemical Techniques/methods ; *MicroRNAs ; *Quantum Dots/chemistry ; },
abstract = {This work proposed a novel double-engine powered paper photoelectrochemical (PEC) biosensor based on an anode-cathode cooperative amplification strategy and various signal enhancement mechanisms, which realized the monitoring of multiple miRNAs (such as miRNA-141 and miRNA-21). Specifically, C3N4 quantum dots (QDs) sensitized ZnO nanostars and BiOI nanospheres simultaneously to construct a composite photoelectric layer that amplified the original photocurrent of the photoanode and photocathode, respectively. Through the independent design and partition of a flexible paper chip to functionalize injection holes and electrode areas, the bipolar combination completed the secondary upgrade of signals, which also provided biological reaction sites for multitarget detection. With the synergistic participation of a three-dimensional (3D) DNA nanomachine and programmable CRISPR/Cas12a shearing tool, C3N4 QDs lost their attachment away from the electrode surface to quench the signal. Moreover, electrode zoning significantly reduced the spatial cross talk of related substances for multitarget detection, while the universal trans-cleavage capability of CRISPR/Cas12a simplified the operation. The designed PEC biosensor revealed excellent linear ranges for detection of miRNA-141 and miRNA-21, for which the detection limits were 5.5 and 3.4 fM, respectively. With prominent selectivity and sensitivity, the platform established an effective approach for trace multitarget monitoring in clinical applications, and its numerous pioneering attempts owned favorable reference values.},
}
@article {pmid35606745,
year = {2022},
author = {Metzloff, M and Yang, E and Dhole, S and Clark, AG and Messer, PW and Champer, J},
title = {Experimental demonstration of tethered gene drive systems for confined population modification or suppression.},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {119},
pmid = {35606745},
issn = {1741-7007},
support = {R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; F32 AI138476/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila/genetics ; *Gene Drive Technology/methods ; },
abstract = {BACKGROUND: Homing gene drives hold great promise for the genetic control of natural populations. However, current homing systems are capable of spreading uncontrollably between populations connected by even marginal levels of migration. This could represent a substantial sociopolitical barrier to the testing or deployment of such drives and may generally be undesirable when the objective is only local population control, such as suppression of an invasive species outside of its native range. Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive.<br><br>RESULTS: Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a regionally confined CRISPR Toxin-Antidote Recessive Embryo (TARE) drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency).<br><br>CONCLUSIONS: Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.},
}
@article {pmid35605545,
year = {2022},
author = {Jia, HY and Zhao, HL and Wang, T and Chen, PR and Yin, BC and Ye, BC},
title = {A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114382},
doi = {10.1016/j.bios.2022.114382},
pmid = {35605545},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA ; *MicroRNAs/analysis/genetics ; Nucleic Acid Hybridization ; },
abstract = {MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans-cleavage activity of Cas12a for further signal amplification. More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.},
}
@article {pmid35604372,
year = {2022},
author = {Rouatbi, N and McGlynn, T and Al-Jamal, KT},
title = {Pre-clinical non-viral vectors exploited for in vivo CRISPR/Cas9 gene editing: an overview.},
journal = {Biomaterials science},
volume = {10},
number = {13},
pages = {3410-3432},
doi = {10.1039/d1bm01452h},
pmid = {35604372},
issn = {2047-4849},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors/genetics ; },
abstract = {Clustered regulatory interspaced short palindromic repeats or CRISPR/Cas9 has emerged as a potent and versatile tool for efficient genome editing. This technology has been exploited for several applications including disease modelling, cell therapy, diagnosis, and treatment of many diseases including cancer. The in vivo application of CRISPR/Cas9 is hindered by poor stability, pharmacokinetic profile, and the limited ability of the CRISPR payloads to cross biological barriers. Although viral vectors have been implemented as delivery tools for efficient in vivo gene editing, their application is associated with high immunogenicity and toxicity, limiting their clinical translation. Hence, there is a need to explore new delivery methods that can guarantee safe and efficient delivery of the CRISPR/Cas9 components to target cells. In this review, we first provide a brief history and principles of nuclease-mediated gene editing, we then focus on the different CRISPR/Cas9 formats outlining their potentials and limitations. Finally, we discuss the alternative non-viral delivery strategies currently adopted for in vivo CRISPR/Cas9 gene editing.},
}
@article {pmid35603967,
year = {2022},
author = {Li, H and Song, J and He, Y and Liu, Y and Liu, Z and Sun, W and Hu, W and Lei, QY and Hu, X and Chen, Z and He, X},
title = {CRISPR/Cas9 Screens Reveal that Hexokinase 2 Enhances Cancer Stemness and Tumorigenicity by Activating the ACSL4-Fatty Acid β-Oxidation Pathway.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {9},
number = {21},
pages = {e2105126},
pmid = {35603967},
issn = {2198-3844},
mesh = {CRISPR-Cas Systems/genetics ; *Coenzyme A Ligases/genetics/metabolism ; Fatty Acid-Binding Proteins/genetics/metabolism ; Fatty Acids/metabolism ; *Hexokinase/genetics/metabolism ; Humans ; *Liver Neoplasms/genetics/metabolism ; *Neoplastic Stem Cells/metabolism ; },
abstract = {Metabolic reprogramming is often observed in carcinogenesis, but little is known about the aberrant metabolic genes involved in the tumorigenicity and maintenance of stemness in cancer cells. Sixty-seven oncogenic metabolism-related genes in liver cancer by in vivo CRISPR/Cas9 screening are identified. Among them, acetyl-CoA carboxylase 1 (ACC1), aldolase fructose-bisphosphate A (ALDOA), fatty acid binding protein 5 (FABP5), and hexokinase 2 (HK2) are strongly associated with stem cell properties. HK2 further facilitates the maintenance and self-renewal of liver cancer stem cells. Moreover, HK2 enhances the accumulation of acetyl-CoA and epigenetically activates the transcription of acyl-CoA synthetase long-chain family member 4 (ACSL4), leading to an increase in fatty acid β-oxidation activity. Blocking HK2 or ACSL4 effectively inhibits liver cancer growth, and GalNac-siHK2 administration specifically targets the growth of orthotopic tumor xenografts. These results suggest a promising therapeutic strategy for the treatment of liver cancer.},
}
@article {pmid35599436,
year = {2022},
author = {Liu, J and Wang, H and Zhang, L and Lu, Y and Wang, X and Shen, M and Li, N and Feng, L and Jing, J and Cao, B and Zou, X and Cheng, J and Xu, Y},
title = {Sensitive and Rapid Diagnosis of Respiratory Virus Coinfection Using a Microfluidic Chip-Powered CRISPR/Cas12a System.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {18},
number = {26},
pages = {e2200854},
doi = {10.1002/smll.202200854},
pmid = {35599436},
issn = {1613-6829},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; *Coinfection/diagnosis ; Humans ; Microfluidics ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Viruses ; },
abstract = {The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 is profoundly influencing the global healthcare system and people's daily lives. The high resource consumption of coronavirus disease 2019 (COVID-19) is resulting in insufficient surveillance of coinfection or resurgence of other critical respiratory epidemics, which is of public concern. To facilitate evaluation of the current coinfection situation, a microfluidic system (MAPnavi) is developed for the rapid (<40 min) and sensitive diagnosis of multiple respiratory viruses from swab samples in a fully sealed and automated manner, in which a nested-recombinase polymerase amplification and the CRISPR-based amplification system is first proposed to ensure the sensitivity and specificity. This novel system has a remarkably low limit of detection (50-200 copies mL[-1]) and is successfully applied to detect 171 clinical samples (98.5% positive predictive agreement; 100% negative predictive agreement), and the results identify 45.6% coinfection among clinical samples from patients with COVID-19. This approach has the potential to shift diagnostic and surveillance efforts from targeted testing for a high-priority virus to comprehensive testing of multiple virus sets and to greatly benefit the implementation of decentralized testing.},
}
@article {pmid35599136,
year = {2022},
author = {Dima, O and Heyvaert, Y and Inzé, D},
title = {Interactive database of genome editing applications in crops and future policy making in the European Union.},
journal = {Trends in plant science},
volume = {27},
number = {8},
pages = {746-748},
doi = {10.1016/j.tplants.2022.05.002},
pmid = {35599136},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Databases, Genetic ; European Union ; *Gene Editing/methods ; Genome, Plant/genetics ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Policy Making ; },
abstract = {European R&amp;D in plant breeding is lagging behind, bound by strict genetically modified organism (GMO) regulations, applied to all crop varieties obtained with genome editing techniques. We developed an online database of worldwide genome editing applications in crops to support conclusions and to facilitate science-based policy making for this plant breeding innovation.},
}
@article {pmid35598687,
year = {2022},
author = {Gonzalez-Salinas, F and Martinez-Amador, C and Trevino, V},
title = {Characterizing genes associated with cancer using the CRISPR/Cas9 system: A systematic review of genes and methodological approaches.},
journal = {Gene},
volume = {833},
number = {},
pages = {146595},
doi = {10.1016/j.gene.2022.146595},
pmid = {35598687},
issn = {1879-0038},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; Mutation ; *Neoplasms/genetics ; Oncogenes ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system enables a versatile set of genomes editing and genetic-based disease modeling tools due to its high specificity, efficiency, and accessible design and implementation. In cancer, the CRISPR/Cas9 system has been used to characterize genes and explore different mechanisms implicated in tumorigenesis. Different experimental strategies have been proposed in recent years, showing dependency on various intrinsic factors such as cancer type, gene function, mutation type, and technical approaches such as cell line, Cas9 expression, and transfection options. However, the successful methodological approaches, genes, and other experimental factors have not been analyzed. We, therefore, initially considered more than 1,300 research articles related to CRISPR/Cas9 in cancer to finally examine more than 400 full-text research publications. We summarize findings regarding target genes, RNA guide designs, cloning, Cas9 delivery systems, cell enrichment, and experimental validations. This analysis provides valuable information and guidance for future cancer gene validation experiments.},
}
@article {pmid35598011,
year = {2022},
author = {Herrera-Uribe, J and Zaldívar-López, S and Aguilar, C and Entrenas-García, C and Bautista, R and Claros, MG and Garrido, JJ},
title = {Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.},
journal = {Veterinary research},
volume = {53},
number = {1},
pages = {35},
pmid = {35598011},
issn = {1297-9716},
mesh = {Animals ; Ileum ; *MicroRNAs/genetics/metabolism ; *Salmonella Infections, Animal ; Salmonella typhimurium/genetics ; Swine ; Toll-Like Receptor 4/metabolism ; },
abstract = {Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection.},
}
@article {pmid35597342,
year = {2022},
author = {Liu, L and Duan, JJ and Wei, XY and Hu, H and Wang, YB and Jia, PP and Pei, DS},
title = {Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment.},
journal = {The Science of the total environment},
volume = {838},
number = {Pt 2},
pages = {156048},
doi = {10.1016/j.scitotenv.2022.156048},
pmid = {35597342},
issn = {1879-1026},
mesh = {Animals ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; *Recombinases/genetics ; *Staphylococcal Infections ; Staphylococcus aureus/genetics ; },
abstract = {Staphylococcus aureus (S. aureus) is an important opportunistic human and animal pathogen that can cause a wide diversity of infections. Due to its environmental health risks, it is crucial to establish a time-saving, high-throughput, and highly sensitive technique for water quality surveillance. In this study, we developed a novel method to detect S. aureus in the water environment based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a. This method utilizes isothermal amplification of nucleic acids and the trans-cleavage activity of the CRISPR/Cas12a system to generate fluorescence signals with a single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporter and a naked-eye detected lateral flow assay (LFA). Our RPA-CRISPR/Cas12a detection system can reduce the detection time to 35 min and enhance the high-throughput detection threshold to ≥5 copies of pathogen DNA, which is more sensitive than that of reported. Moreover, in the lower reaches of the Jialing River in Chongqing, China, 10 water samples from the mainstream and 7 ones from tributaries were successfully monitored S. aureus for less than 35 min using RPA-CRISPR/Cas12a detection system. Taken together, a novel high-throughput RPA-CRISPR detection was established and firstly applied for sensitively monitoring S. aureus in the natural water environment.},
}
@article {pmid35597144,
year = {2022},
author = {Wei, Y and Tao, Z and Wan, L and Zong, C and Wu, J and Tan, X and Wang, B and Guo, Z and Zhang, L and Yuan, H and Wang, P and Yang, Z and Wan, Y},
title = {Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {211},
number = {},
pages = {114282},
doi = {10.1016/j.bios.2022.114282},
pmid = {35597144},
issn = {1873-4235},
mesh = {*Aptamers, Nucleotide/chemistry ; *Biosensing Techniques ; DNA ; Humans ; *Staphylococcal Infections/diagnosis ; Staphylococcus aureus/chemistry/genetics ; },
abstract = {CRISPR-Cas systems have been employed to detect a large variety of pathogenic microorganisms by simply changing the guide RNA sequence. However, these platforms usually rely on nucleic acid extraction and amplification to achieve good sensitivity. Herein, we developed a new platform for the highly specific and sensitive detection of live staphylococcus aureus (S. aureus) based on an Aptamer-based Cas14a1 Biosensor (ACasB), without the need for nucleic acid extraction or amplification. First, the S. aureus specific aptamer was hybrid with a blocker DNA. After the live S. aureus was added, the blocker can be released upon bacteria-aptamer binding. Finally, the released blocker can activate Cas14a1 protein by binding with the sgRNA to generate a change of fluorescent intensity. The ACasB indicates high specificity and sensitivity: it can directly distinguish 400 CFU/ml live S. aureus cells. Comparable to qPCR, the Cas14a1-aptamer biosensor can detect S. aureus with 100% accuracy in complex samples. Therefore, this ACasB for the on-site detection of live S. aureus can broaden its applications in food safety and environmental monitoring.},
}
@article {pmid35596077,
year = {2022},
author = {Pan, C and Li, G and Malzahn, AA and Cheng, Y and Leyson, B and Sretenovic, S and Gurel, F and Coleman, GD and Qi, Y},
title = {Boosting plant genome editing with a versatile CRISPR-Combo system.},
journal = {Nature plants},
volume = {8},
number = {5},
pages = {513-525},
pmid = {35596077},
issn = {2055-0278},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.},
}
@article {pmid35595842,
year = {2022},
author = {Flegler, A and Lipski, A},
title = {Engineered CRISPR/Cas9 System for Transcriptional Gene Silencing in Arthrobacter Species Indicates Bacterioruberin is Indispensable for Growth at Low Temperatures.},
journal = {Current microbiology},
volume = {79},
number = {7},
pages = {199},
pmid = {35595842},
issn = {1432-0991},
mesh = {*Arthrobacter/genetics/metabolism ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Temperature ; },
abstract = {Pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, which is suspected to be part of the cold adaptation mechanism. In silico analysis of the repertoire of genes encoded by the Arthrobacter agilis and Arthrobacter bussei genome revealed the biosynthetic pathway of bacterioruberin. Although genetic analysis is an essential tool for studying the physiology of Arthrobacter species, genetic manipulation of Arthrobacter is always time and labor intensive due to the lack of genetic engineering tools. Here we report the construction and application of a CRISPR/deadCas9 system (pCasiART) for gene silencing in Arthrobacter species. The engineered system pCasiART is suitable for the Golden Gate assembly of spacers, enabling rapid and accurate construction of adapted systems. In addition, pCasiART has been developed to provide an efficient transcription inhibition system for genome-wide gene silencing. The gene silencing of the phytoene synthase (CrtB), the first enzyme in bacterioruberin biosynthesis, suppressed bacterioruberin biosynthesis in Arthrobacter agilis and Arthrobacter bussei, resulting in a lack of pink pigmentation, reduction of biomass production, and growth rates at low temperatures.},
}
@article {pmid35595757,
year = {2022},
author = {McGaw, C and Garrity, AJ and Munoz, GZ and Haswell, JR and Sengupta, S and Keston-Smith, E and Hunnewell, P and Ornstein, A and Bose, M and Wessells, Q and Jakimo, N and Yan, P and Zhang, H and Alfonse, LE and Ziblat, R and Carte, JM and Lu, WC and Cerchione, D and Hilbert, B and Sothiselvam, S and Yan, WX and Cheng, DR and Scott, DA and DiTommaso, T and Chong, S},
title = {Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2833},
pmid = {35595757},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; HEK293 Cells ; Humans ; RNA/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.},
}
@article {pmid35595728,
year = {2022},
author = {Schwartz, EA and McBride, TM and Bravo, JPK and Wrapp, D and Fineran, PC and Fagerlund, RD and Taylor, DW},
title = {Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2829},
pmid = {35595728},
issn = {2041-1723},
support = {R35 GM138348/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; DNA Cleavage ; *Nucleic Acids ; RNA ; },
abstract = {CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.},
}
@article {pmid35595574,
year = {2022},
author = {Li, L and Shen, G and Wu, M and Jiang, J and Xia, Q and Lin, P},
title = {CRISPR-Cas-mediated diagnostics.},
journal = {Trends in biotechnology},
volume = {40},
number = {11},
pages = {1326-1345},
doi = {10.1016/j.tibtech.2022.04.006},
pmid = {35595574},
issn = {1879-3096},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Molecular Diagnostic Techniques/methods ; *Nucleic Acids ; },
abstract = {An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.},
}
@article {pmid35595297,
year = {2022},
author = {Yaish, O and Asif, M and Orenstein, Y},
title = {A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction.},
journal = {Briefings in bioinformatics},
volume = {23},
number = {5},
pages = {},
doi = {10.1093/bib/bbac157},
pmid = {35595297},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Guide/genetics ; Research Design ; },
abstract = {CRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this editing technique is quite accurate in the target region, there may be many unplanned off-target sites (OTSs). Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of OTSs) produced by experimental techniques to detect OTSs with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect OTSs, was used to produce a dataset of unprecedented scale and quality (>200 000 OTS over 110 guide RNAs). In addition, the same study included in cellula GUIDE-seq experiments for 58 of the guide RNAs. Here, we fill the gap in previous computational methods by utilizing these data to systematically evaluate data processing and formulation of the CRISPR OTSs prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive OTSs to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between guide RNAs and their OTSs as a feature. Finally, we present predictive off-target in cellula models based on both in vitro and in cellula data and compare them to state-of-the-art methods in predicting true OTSs. Our conclusions will be instrumental in any future development of an off-target predictor based on high-throughput datasets.},
}
@article {pmid35594718,
year = {2022},
author = {Nath, A and Bhattacharjee, R and Nandi, A and Sinha, A and Kar, S and Manoharan, N and Mitra, S and Mojumdar, A and Panda, PK and Patro, S and Dutt, A and Ahuja, R and Verma, SK and Suar, M},
title = {Phage delivered CRISPR-Cas system to combat multidrug-resistant pathogens in gut microbiome.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {151},
number = {},
pages = {113122},
doi = {10.1016/j.biopha.2022.113122},
pmid = {35594718},
issn = {1950-6007},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome ; Gene Editing ; },
abstract = {The Host-microbiome interactions that exist inside the gut microbiota operate in a synergistic and abnormal manner. Additionally, the normal homeostasis and functioning of gut microbiota are frequently disrupted by the intervention of Multi-Drug Resistant (MDR) pathogens. CRISPR-Cas (CRISPR-associated protein with clustered regularly interspersed short palindromic repeats) recognized as a prokaryotic immune system has emerged as an effective genome-editing tool to edit and delete specific microbial genes for the expulsion of bacteria through bactericidal action. In this review, we demonstrate many functioning CRISPR-Cas systems against the anti-microbial resistance of multiple pathogens, which infiltrate the gastrointestinal tract. Moreover, we discuss the advancement in the development of a phage-delivered CRISPR-Cas system for killing a gut MDR pathogen. We also discuss a combinatorial approach to use bacteriophage as a delivery system for the CRISPR-Cas gene for targeting a pathogenic community in the gut microbiome to resensitize the drug sensitivity. Finally, we discuss engineered phage as a plausible potential option for the CRISPR-Cas system for pathogenic killing and improvement of the efficacy of the system.},
}
@article {pmid35594500,
year = {2022},
author = {Kazemian, P and Yu, SY and Thomson, SB and Birkenshaw, A and Leavitt, BR and Ross, CJD},
title = {Lipid-Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Components.},
journal = {Molecular pharmaceutics},
volume = {19},
number = {6},
pages = {1669-1686},
pmid = {35594500},
issn = {1543-8392},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; Lipids ; Liposomes ; *Nanoparticles ; },
abstract = {Gene editing mediated by CRISPR/Cas9 systems is due to become a beneficial therapeutic option for treating genetic diseases and some cancers. However, there are challenges in delivering CRISPR components which necessitate sophisticated delivery systems for safe and effective genome editing. Lipid nanoparticles (LNPs) have become an attractive nonviral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility. In this review, we provide a background of CRISPR-mediated gene therapy, as well as LNPs and their applicable characteristics for delivering CRISPR components. We then highlight the challenges of CRISPR delivery, which have driven the significant development of new, safe, and optimized LNP formulations in the past decade. Finally, we discuss considerations for using LNPs to deliver CRISPR and future perspectives on clinical translation of LNP-CRISPR gene editing.},
}
@article {pmid35592579,
year = {2022},
author = {Chen, H and Neubauer, M and Wang, JP},
title = {Enhancing HR Frequency for Precise Genome Editing in Plants.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {883421},
pmid = {35592579},
issn = {1664-462X},
abstract = {Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.},
}
@article {pmid35589728,
year = {2022},
author = {Yuan, Q and Gao, X},
title = {Multiplex base- and prime-editing with drive-and-process CRISPR arrays.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2771},
pmid = {35589728},
issn = {2041-1723},
support = {R01 HL157714/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; RNA, Guide/genetics ; RNA, Transfer/genetics ; },
abstract = {Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. We leverage tRNA as the RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays, of which the individual gRNAs are released by the cellular endogenous tRNA processing machinery. We engineer a 75-nt human cysteine tRNA (hCtRNA) for the DAP array, achieving up to 31-loci MBE and up to 3-loci MPE. By applying MBE or MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, we demonstrate simultaneous editing of multiple disease-relevant genomic loci. Our work streamlines the expression and processing of gRNAs on a single array and establishes efficient MBE and MPE strategies for biomedical research and therapeutic applications.},
}
@article {pmid35589712,
year = {2022},
author = {Philippe, C and Morency, C and Plante, PL and Zufferey, E and Achigar, R and Tremblay, DM and Rousseau, GM and Goulet, A and Moineau, S},
title = {A truncated anti-CRISPR protein prevents spacer acquisition but not interference.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2802},
pmid = {35589712},
issn = {2041-1723},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; Streptococcus thermophilus/genetics ; },
abstract = {CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference activity. Here, we show that S. thermophilus can block ACR-containing phages when the CRISPR immunity specifically targets the acr gene. This in turn selects for phage mutants carrying a deletion within the acr gene. Remarkably, a truncated acrIIA allele, found in a wild-type virulent streptococcal phage, does not block the interference activity of Cas9 but still prevents the acquisition of new immunities, thereby providing an example of an ACR specifically inhibiting spacer acquisition.},
}
@article {pmid35588782,
year = {2022},
author = {Tripathi, R and Sinha, NR and Kempuraj, D and Balne, PK and Landreneau, JR and Juneja, A and Webel, AD and Mohan, RR},
title = {Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro.},
journal = {Experimental eye research},
volume = {220},
number = {},
pages = {109113},
doi = {10.1016/j.exer.2022.109113},
pmid = {35588782},
issn = {1096-0007},
support = {R01 EY017294/EY/NEI NIH HHS/United States ; R01 EY030774/EY/NEI NIH HHS/United States ; },
mesh = {Actins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Co-Repressor Proteins/genetics/metabolism ; Collagen/metabolism ; *Corneal Diseases/pathology ; Fibroblasts/metabolism ; Fibrosis ; *Gene Editing ; Homeodomain Proteins ; Humans ; Myofibroblasts/metabolism ; Repressor Proteins/metabolism ; Transcription Factors/genetics ; Transforming Growth Factor beta1/pharmacology ; },
abstract = {Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5'TG3'-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-β1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.},
}
@article {pmid35588272,
year = {2022},
author = {Meliawati, M and May, T and Eckerlin, J and Heinrich, D and Herold, A and Schmid, J},
title = {Insights in the Complex DegU, DegS, and Spo0A Regulation System of Paenibacillus polymyxa by CRISPR-Cas9-Based Targeted Point Mutations.},
journal = {Applied and environmental microbiology},
volume = {88},
number = {11},
pages = {e0016422},
pmid = {35588272},
issn = {1098-5336},
mesh = {Bacillus subtilis/genetics ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Paenibacillus polymyxa/genetics/metabolism ; Point Mutation ; },
abstract = {Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated, the single mutants DegU Q218*, DegS L99F, and Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild-type and the engineered strains revealed differences in swarming behavior, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365. IMPORTANCE To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate several regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.},
}
@article {pmid35587321,
year = {2022},
author = {Kumar, G and Jagadeeshwari, U and Sreya, P and Shabbir, A and Sasikala, C and Ramana, CV},
title = {A genomic overview including polyphasic taxonomy of Thalassoroseus pseudoceratinae gen. nov., sp. nov. isolated from a marine sponge, Pseudoceratina sp.},
journal = {Antonie van Leeuwenhoek},
volume = {115},
number = {7},
pages = {843-856},
pmid = {35587321},
issn = {1572-9699},
mesh = {Animals ; Bacterial Typing Techniques ; DNA, Bacterial/chemistry/genetics ; Fatty Acids/analysis ; Genomics ; Phospholipids/analysis ; Phylogeny ; *Planctomycetales/genetics ; *Porifera ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; Ubiquinone/analysis ; },
abstract = {A pink-coloured, salt- and alkali-tolerant planctomycetal strain (JC658[T]) with oval to pear-shaped, motile, aerobic, Gram-negative stained cells was isolated from a marine sponge, Pseudoceratina sp. Strain JC658[T] shares the highest 16S rRNA gene sequence identity with Maioricimonas rarisocia Mal4[T] (< 89.2%) in the family Planctomycetaceae. The genomic analysis of the new strain indicates its biotechnological potential for the production of various industrially important enzymes, notably sulfatases and carbohydrate-active enzymes (CAZymes), and also potential antimicrobial compounds. Several genes encoding restriction-modification (RM) and CRISPR-CAS systems are also present. NaCl is obligate for growth, of which strain JC658[T] can tolerate a concentration up to 6% (w/v). Optimum pH and temperature for growth are 8.0 (range 7.0-9.0) and 25 ºC (range 10-40 °C), respectively. The major respiratory quinone of strain JC658[T] is MK6. Major fatty acids are C16:1ω7c/C16:1ω6c, C18:0 and C16:0. Major polar lipids are phosphatidylcholine, phosphatidyl-dimethylethanolamine and phosphatidyl-monomethylethanolamine. The genomic size of strain JC658[T] is 7.36 Mb with a DNA G + C content of 54.6 mol%. Based on phylogenetic, genomic (ANI, AAI, POCP, dDDH), chemotaxonomic, physiological and biochemical characteristics, we conclude that strain JC658[T] belongs to a novel genus and constitutes a novel species within the family Planctomycetaceae, for which we propose the name Thalassoroseus pseudoceratinae gen. nov., sp. nov. The novel species is represented by the type strain JC658[T] (= KCTC 72881[ T] = NBRC 114371[ T]).},
}
@article {pmid35587292,
year = {2022},
author = {Van Vu, T and Das, S and Hensel, G and Kim, JY},
title = {Genome editing and beyond: what does it mean for the future of plant breeding?.},
journal = {Planta},
volume = {255},
number = {6},
pages = {130},
pmid = {35587292},
issn = {1432-2048},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.},
}
@article {pmid35586918,
year = {2022},
author = {He, D and Liu, G and Yang, J and Jiang, X and Wang, H and Fan, Y and Gong, S and Wei, F and Diao, Y and Tang, Y},
title = {Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics.},
journal = {Bioconjugate chemistry},
volume = {33},
number = {6},
pages = {1232-1240},
doi = {10.1021/acs.bioconjchem.2c00200},
pmid = {35586918},
issn = {1520-4812},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Flavivirus/genetics ; RNA ; Recombinases ; },
abstract = {In China, drastic losses in the economy have been caused by the Tembusu virus (TMUV), the causative agent of the egg-drop syndrome, to the duck-raising industry. To succeed in preventing and controlling infections, extant techniques must be upgraded to achieve fast detection of viruses. This work is the first attempt to present the development of a recombinase polymerase amplification (RPA)-based clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas13a approach for the TMUV infection diagnosis, where the CRISPR-Cas13a system is exploited, i.e., the programmability of CRISPR RNA (crRNA) and the promiscuous RNase collateral cleavage of Cas13a upon recognition of target RNAs. A prokaryotic expression system was utilized for the expression of LwCas13a soluble protein, while its purification was accomplished by nickel-nitrilotriacetic acid (Ni-NTA) agarose. In the design of a particular crRNA, the target used was the TMUV NS3 RNA transcribed in vitro. The signals used for the Cas13a activity validation were an RNA-bound fluorescent group (single-stranded) and a quenching fluorophore. In the present work, a specific high-sensitivity enzymatic molecular detection system termed RPA-based CRISPR-Cas13a was established by combining Cas13a with T7 transcription and RPA for sensitive detection of TMUV at room temperature. This system can detect 10[2] copies of the target TMUV DNA standard/μL within 50 min. A comparison revealed that the specificity was superior to that for other avian viruses. Furthermore, the RPA-based CRISPR-Cas13a detection system was successfully applied for clinical samples, and its performance is comparable to the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR). Being satisfyingly reliable, simple, specific, and sensitive, our RPA-based CRISPR-Cas13a detection system could be expanded and universalized for identifying other viruses, enabling quick detection in the field with a portable lateral flow dipstick.},
}
@article {pmid35586709,
year = {2022},
author = {Yin, T and Luo, J and Huang, D and Li, H},
title = {Current Progress of Mitochondrial Genome Editing by CRISPR.},
journal = {Frontiers in physiology},
volume = {13},
number = {},
pages = {883459},
pmid = {35586709},
issn = {1664-042X},
}
@article {pmid35585651,
year = {2022},
author = {Osteikoetxea, X and Silva, A and Lázaro-Ibáñez, E and Salmond, N and Shatnyeva, O and Stein, J and Schick, J and Wren, S and Lindgren, J and Firth, M and Madsen, A and Mayr, LM and Overman, R and Davies, R and Dekker, N},
title = {Engineered Cas9 extracellular vesicles as a novel gene editing tool.},
journal = {Journal of extracellular vesicles},
volume = {11},
number = {5},
pages = {e12225},
pmid = {35585651},
issn = {2001-3078},
mesh = {CRISPR-Cas Systems/genetics ; *Extracellular Vesicles ; *Gene Editing ; HEK293 Cells ; Humans ; Proprotein Convertase 9/genetics ; },
abstract = {Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.},
}
@article {pmid35585381,
year = {2022},
author = {Rasheed, A and Barqawi, AA and Mahmood, A and Nawaz, M and Shah, AN and Bay, DH and Alahdal, MA and Hassan, MU and Qari, SH},
title = {CRISPR/Cas9 is a powerful tool for precise genome editing of legume crops: a review.},
journal = {Molecular biology reports},
volume = {49},
number = {6},
pages = {5595-5609},
pmid = {35585381},
issn = {1573-4978},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Fabaceae/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; Vegetables/genetics ; },
abstract = {Legumes are an imperative source of food and proteins across the globe. They also improve soil fertility through symbiotic nitrogen fixation (SNF). Genome editing (GE) is now a novel way of developing desirable traits in legume crops. Genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR) system permits a defined genome alteration to improve crop performance. This genome editing tool is reliable, cost-effective, and versatile, and it has to deepen in terms of use compared to other tools. Recently, many novel variations have drawn the attention of plant geneticists, and efforts are being made to develop trans-gene-free cultivars for ensuring biosafety measures. This review critically elaborates on the recent development in genome editing of major legumes crops. We hope this updated review will provide essential informations for the researchers working on legumes genome editing. In general, the CRISPR/Cas9 novel GE technique can be integrated with other techniques like omics approaches and next-generation tools to broaden the range of gene editing and develop any desired legumes traits. Regulatory ethics of CRISPR/Cas9 are also discussed.},
}
@article {pmid35584409,
year = {2022},
author = {Lee, HJ and Kim, HJ and Park, YJ and Lee, SJ},
title = {Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.},
journal = {ACS synthetic biology},
volume = {11},
number = {6},
pages = {2134-2143},
pmid = {35584409},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Genome, Microbial ; *Nucleotides/genetics ; },
abstract = {Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3'-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3'-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3'-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3'-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.},
}
@article {pmid35584220,
year = {2022},
author = {Fan, N and Bian, X and Li, M and Chen, J and Wu, H and Peng, Q and Bai, H and Cheng, W and Kong, L and Ding, S and Li, S and Cheng, W},
title = {Hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons for spatial-controlled genome editing and precise cancer therapy.},
journal = {Science advances},
volume = {8},
number = {20},
pages = {eabn7382},
pmid = {35584220},
issn = {2375-2548},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Neoplasms/genetics/therapy ; RNA ; RNA, Messenger/genetics ; },
abstract = {CRISPR-Cas13a holds enormous potential for developing precise RNA editing. However, spatial manipulation of CRISPR-Cas13a activity remains a daunting challenge for elaborately regulating localized RNase function. Here, we designed hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons (RNCOs-D), featuring tumor-specific recognition and spatial-controlled activation of Cas13a, for precise cancer synergistic therapy. RNCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and caging chemotherapeutic drug, and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein (Cas13a RNP) activity. The acidic endo/lysosomal microenvironment stimulates the outer decomposition of NCs with concomitant Cas13a RNP activity revitalization, while the inner disassembly through trans-cleavage of RNSs initiated by cis-recognition and cleavage of EGFR variant III (EGFRvIII) mRNA. RNCOs-D demonstrates the effective EGFRvIII mRNA silencing for synergistic therapy of glioblastoma cancer cells in vitro and in vivo. The engineering of RNSs, together with efficient Cas13a activity regulation, holds immense prospect for multimodal and synergistic cancer therapy.},
}
@article {pmid35583743,
year = {2022},
author = {Vento, JM and Beisel, CL},
title = {Genome Editing with Cas9 in Lactobacilli.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {245-261},
pmid = {35583743},
issn = {1940-6029},
support = {T32 GM008776/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Vectors/genetics ; Genome, Bacterial ; Lactobacillus/genetics ; *Lactobacillus plantarum/genetics ; },
abstract = {The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype-phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli.},
}
@article {pmid35583742,
year = {2022},
author = {Xu, T and Tao, X and Kempher, ML and Zhou, J},
title = {Cas9 Nickase-Based Genome Editing in Clostridium cellulolyticum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {227-243},
pmid = {35583742},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Clostridium cellulolyticum/genetics/metabolism ; Deoxyribonuclease I/metabolism ; *Gene Editing/methods ; RNA, Guide/genetics ; },
abstract = {Clostridium cellulolyticum is a model mesophilic, cellulolytic bacterium, with the potential to produce biofuels from lignocellulose. However, the natural cellulose utilization efficiency is quite low and, therefore, metabolically engineered strains with increased efficiency can decrease both the overall cost and time required for biofuel production. Traditional genetic tools are inefficient, expensive, and time-consuming, but recent developments in the use of CRISPR-Cas genetic editing systems have greatly expanded our ability to reprogram cells. Here we describe an established protocol enabling one-step versatile genome editing in C. cellulolyticum. It integrates Cas9 nickase (Cas9n) which introduces a single nick that triggers repair via homologous recombination (SNHR) to edit genomic loci with high efficiency and accuracy. This one-step editing is achieved by transforming an all-in-one vector to coexpress Cas9n and a single guide RNA (gRNA) and carries a user-defined homologous donor template to promote SNHR at a desired target site. Additionally, this system has high specificity and allows for various types of genomic editing, including markerless insertions, deletions, substitutions, and even multiplex editing.},
}
@article {pmid35583741,
year = {2022},
author = {Tan, LL and Heng, E and Zulkarnain, N and Hsiao, WC and Wong, FT and Zhang, MM},
title = {CRISPR/Cas-Mediated Genome Editing of Streptomyces.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {207-225},
pmid = {35583741},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Engineering ; Plasmids/genetics ; *Streptomyces/genetics ; },
abstract = {Streptomyces are an important source and reservoir of natural products with diverse applications in medicine, agriculture, and food. Engineered Streptomyces strains have also proven to be functional chassis for the discovery and production of bioactive compounds and enzymes. However, genetic engineering of Streptomyces is often laborious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of Streptomyces. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.},
}
@article {pmid35583740,
year = {2022},
author = {Wen, Z and Qian, F and Zhang, J and Jiang, Y and Yang, S},
title = {Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {189-206},
pmid = {35583740},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Corynebacterium glutamicum/genetics ; *Gene Editing/methods ; },
abstract = {Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.},
}
@article {pmid35583739,
year = {2022},
author = {Hong, W and Zhang, J and Cui, G and Zhou, Q and Wang, P and Wang, Y},
title = {Highly Efficient Genome Editing in Clostridium difficile Using the CRISPR-Cpf1 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {175-187},
pmid = {35583739},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Clostridioides difficile/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Here, we describe a streamlined CRISPR-Cpf1-based protocol to achieve precise genome editing in C. difficile with high efficiencies. Our work highlighted the first application of CRISPR-Cpf1 for genome editing in C. difficile, which are both crucial for understanding pathogenic mechanism of C. difficile and developing strategies to fight against C. difficile infection (CDI). In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. Approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.},
}
@article {pmid35583738,
year = {2022},
author = {Wozniak, KJ and Simmons, LA},
title = {Genome Editing Methods for Bacillus subtilis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {159-174},
pmid = {35583738},
issn = {1940-6029},
support = {R35 GM131772/GM/NIGMS NIH HHS/United States ; T32 GM008353/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacillus subtilis/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Engineering ; Humans ; },
abstract = {Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application.},
}
@article {pmid35583737,
year = {2022},
author = {Penewit, K and Salipante, SJ},
title = {Recombineering in Staphylococcus aureus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {135-157},
pmid = {35583737},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; Recombinases/genetics ; *Staphylococcal Infections/genetics ; *Staphylococcus aureus/genetics ; },
abstract = {Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.},
}
@article {pmid35583736,
year = {2022},
author = {Ellington, AJ and Reisch, CR},
title = {Generating Single Nucleotide Point Mutations in E. coli with the No-SCAR System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {119-133},
pmid = {35583736},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Editing/methods ; Nucleotides ; Point Mutation ; },
abstract = {Genetic manipulation of microbial genomes is highly relevant for studying biological systems and the development of biotechnologies. In E. coli, λ-Red recombineering is one of the most widely used gene-editing methods, enabling site-specific insertions, deletions, and point mutations of any genomic locus. The no-SCAR system combines λ-Red recombineering with CRISPR/Cas9 for programmable selection of recombinant cells. Recombineering results in the transient production of heteroduplex DNA, as only one strand of DNA is initially altered, leaving the mismatched bases susceptible to repair by the host methyl-directed mismatch repair (MMR) system and reduces the efficiency of generating single nucleotide point mutations. Here we describe a method, where expression of cas9 and the MMR-inhibiting mutLE32K variant are independently controlled by anhydrotetracycline- and cumate-inducible promoters from the pCas9CyMutL plasmid. Thus, MMR is selectively inhibited until recombinant cells have undergone replication and the desired mutation is permanently incorporated. By transiently inhibiting MMR, the accumulation of off-target mutations typically associated with MMR-deficient cell types is minimized. Methods for designing the editing template and sgRNA, cloning of the sgRNA, induction of λ-Red and MutLE32K, the transformation of editing oligo, and induction of Cas9 for mutant selection are detailed within.},
}
@article {pmid35583735,
year = {2022},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2479},
number = {},
pages = {105-117},
pmid = {35583735},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics ; *Gene Editing/methods ; *Klebsiella pneumoniae/genetics/metabolism ; Technology ; },
abstract = {CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.},
}
@article {pmid35583489,
year = {2022},
author = {Cheng, MHY and Brimacombe, CA and Verbeke, R and Cullis, PR},
title = {Exciting Times for Lipid Nanoparticles: How Canadian Discoveries Are Enabling Gene Therapies.},
journal = {Molecular pharmaceutics},
volume = {19},
number = {6},
pages = {1663-1668},
doi = {10.1021/acs.molpharmaceut.2c00365},
pmid = {35583489},
issn = {1543-8392},
support = {FDN 148469//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; Canada ; *Gene Editing ; Liposomes ; *Nanoparticles ; },
abstract = {In this brief perspective, we describe key events in the history of the lipid-based nanomedicine field, highlight Canadian contributions, and outline areas where lipid nanoparticle technology is poised to have a transformative effect on the future of medicine.},
}
@article {pmid35583202,
year = {2022},
author = {Luo, G and Najafi, J and Correia, PMP and Trinh, MDL and Chapman, EA and Østerberg, JT and Thomsen, HC and Pedas, PR and Larson, S and Gao, C and Poland, J and Knudsen, S and DeHaan, L and Palmgren, M},
title = {Accelerated Domestication of New Crops: Yield is Key.},
journal = {Plant &amp; cell physiology},
volume = {63},
number = {11},
pages = {1624-1640},
pmid = {35583202},
issn = {1471-9053},
mesh = {*Domestication ; *Plant Breeding ; Crops, Agricultural/genetics ; Agriculture ; Biodiversity ; },
abstract = {Sustainable agriculture in the future will depend on crops that are tolerant to biotic and abiotic stresses, require minimal input of water and nutrients and can be cultivated with a minimal carbon footprint. Wild plants that fulfill these requirements abound in nature but are typically low yielding. Thus, replacing current high-yielding crops with less productive but resilient species will require the intractable trade-off of increasing land area under cultivation to produce the same yield. Cultivating more land reduces natural resources, reduces biodiversity and increases our carbon footprint. Sustainable intensification can be achieved by increasing the yield of underutilized or wild plant species that are already resilient, but achieving this goal by conventional breeding programs may be a long-term prospect. De novo domestication of orphan or crop wild relatives using mutagenesis is an alternative and fast approach to achieve resilient crops with high yields. With new precise molecular techniques, it should be possible to reach economically sustainable yields in a much shorter period of time than ever before in the history of agriculture.},
}
@article {pmid35581476,
year = {2022},
author = {Tringe, SG},
title = {A toolkit for microbial community editing.},
journal = {Nature reviews. Microbiology},
volume = {20},
number = {7},
pages = {383},
pmid = {35581476},
issn = {1740-1534},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; *Microbiota ; },
}
@article {pmid35581343,
year = {2022},
author = {Perdigoto, CN},
title = {TALEDs complete the toolkit for editing human mitochondrial DNA.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {5},
pages = {415},
doi = {10.1038/s41594-022-00781-z},
pmid = {35581343},
issn = {1545-9985},
mesh = {CRISPR-Cas Systems/genetics ; *DNA, Mitochondrial/genetics ; *Gene Editing ; Humans ; Mitochondria/genetics ; },
}
@article {pmid35581233,
year = {2022},
author = {Sansbury, BM and Hewes, AM and Tharp, OM and Masciarelli, SB and Kaouser, S and Kmiec, EB},
title = {Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {8132},
pmid = {35581233},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems/genetics ; Catalysis ; Gene Editing/methods ; Mutagenesis ; Mutation ; *Point Mutation ; },
abstract = {Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.},
}
@article {pmid35580855,
year = {2022},
author = {Hu, WX and Rong, Y and Guo, Y and Jiang, F and Tian, W and Chen, H and Dong, SS and Yang, TL},
title = {ExsgRNA: reduce off-target efficiency by on-target mismatched sgRNA.},
journal = {Briefings in bioinformatics},
volume = {23},
number = {5},
pages = {},
doi = {10.1093/bib/bbac183},
pmid = {35580855},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; DNA ; Gene Editing/methods ; Luciferases/genetics/metabolism ; *RNA, Guide/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has been widely used to facilitate efficient genome editing. Current popular sgRNA design tools only consider the sgRNA perfectly matched to the target site and provide the results without any on-target mismatch. We suppose taking on-target gRNA-DNA mismatches into consideration might provide better sgRNA with similar binding activity and reduced off-target sites. Here, we trained a seq2seq-attention model with feedback-loop architecture, to automatically generate sgRNAs with on-target mismatches. Dual-luciferase reporter experiment showed that multiple sgRNAs with three mismatches could achieve the 80% of the relative activity of the perfect matched sgRNA. Meanwhile, it could reduce the number of off-target sites using sgRNAs with on-target mismatches. Finally, we provided a freely accessible web server sgRNA design tool named ExsgRNA. Users could submit their target sequence to this server and get optimal sgRNAs with less off-targets and similar on-target activity compared with the perfect-matched sgRNA.},
}
@article {pmid35580804,
year = {2022},
author = {Petel Légaré, V and Rampal, CJ and Gurberg, TJN and Harji, ZA and Allard-Chamard, X and Rodríguez, EC and Armstrong, GAB},
title = {Development of an endogenously myc-tagged TARDBP (TDP-43) zebrafish model using the CRISPR/Cas9 system and homology directed repair.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {261},
number = {},
pages = {110756},
doi = {10.1016/j.cbpb.2022.110756},
pmid = {35580804},
issn = {1879-1107},
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Epitopes/metabolism ; *Neurodegenerative Diseases/genetics ; *Zebrafish/genetics/metabolism ; },
abstract = {Many of the modern advances in cellular biology have been made by the expression of engineered constructs with epitope tags for subsequent biochemical investigations. While the utility of epitope tags has permitted insights in cellular and animal models, these are often expressed using traditional transgenic approaches. Using the CRISPR/Cas9 system and homology directed repair we recombine a single myc epitope sequence following the start codon of the zebrafish ortholog of TARDBP (TDP-43). TDP-43 is an RNA binding protein that is involved in the neurodegenerative disease amyotrophic lateral sclerosis and frontotemporal dementia. We report that zebrafish expressing the myc-tardbp engendered allele produced a stable protein that was detected by both western blot and immunofluorescence. Furthermore, both heterozygous and homozygous carriers of the myc-tardbp allele developed to sexual maturity. We propose that the methodology used here will be useful for zebrafish researchers and other comparative animal biologists interested in developing animal models expressing endogenously tagged proteins.},
}
@article {pmid35580124,
year = {2022},
author = {Labude, MK and Xafis, V and Lai, PS and Mills, C},
title = {Vulnerability and the Ethics of Human Germline Genome Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {358-363},
pmid = {35580124},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Ethics, Research ; *Gene Editing ; Genome, Human/genetics ; Germ Cells ; Humans ; },
abstract = {The concept of vulnerability has played an important role in theoretical bioethics as well as in numerous authoritative guidelines on research ethics. The concept helps to identify situations in which research participants and other individuals may be at a heightened risk of experiencing harm. However, existing guidance documents on the ethics of human germline gene editing largely fail to make any reference to considerations of vulnerability. In this article, we discuss this oversight and we highlight the role that vulnerability can play in ethical debates about human heritable genome editing. Future guidance documents on germline gene editing should pay attention to considerations of vulnerability and reference these appropriately.},
}
@article {pmid35579838,
year = {2022},
author = {Pavani, G and Klein, JG and French, DL and Gadue, P},
title = {Genome Engineering Human ESCs or iPSCs with Cytosine and Adenine Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2520},
number = {},
pages = {321-333},
pmid = {35579838},
issn = {1940-6029},
support = {U01 HL134696/HL/NHLBI NIH HHS/United States ; R01 DK118155/DK/NIDDK NIH HHS/United States ; R01 DK123162/DK/NIDDK NIH HHS/United States ; UG3 DK122644/DK/NIDDK NIH HHS/United States ; },
mesh = {Adenine ; *CRISPR-Cas Systems/genetics ; Cytosine ; Genome, Human ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {The ability to engineer specific mutations in human embryonic stem cells (ECSs) or induced pluripotent stem cells (iPSCs) is extremely important in the modeling of human diseases and the study of biological processes. While CRISPR/Cas9 can robustly generate gene knockouts (KOs) and gene loci modifications in coding sequences of iPSCs, it remains difficult to produce monoallelic mutations or modify specific nucleotides in noncoding sequences due to technical constraints.Here, we describe how to leverage cytosine (BE4max) and adenine (ABEmax) base editors to introduce precise mutations in iPSCs without inducing DNA double-stranded breaks. This chapter illustrates how to design and clone gRNAs, evaluate editing efficiency, and detect genomic edits at specific sites in iPSCs through the utilization of base editing technology.},
}
@article {pmid35579071,
year = {2022},
author = {Zhang, L and Zhao, X and Hu, X and Zhang, Y and Liu, R and Peng, H and Chen, Y and Zhang, H and Luo, Y},
title = {Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification.},
journal = {The Analyst},
volume = {147},
number = {12},
pages = {2655-2661},
doi = {10.1039/d2an00170e},
pmid = {35579071},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA Methylation ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Aberrant DNA methylation plays a pivotal role in tumor development and metastasis, and is regarded as a valuable non-invasive cancer biomarker. However, the sensitive and accurate quantification of DNA methylation from clinical samples remains a challenge. Herein, we propose an easy-to-operate Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system Assisted Methylation (CAM) approach for the sensitive detection of DNA methylation through the integration of rolling circle amplification and CRISPR-Cas12a-assisted cascade amplification. Briefly, bisulfite was employed to prepare the clinical samples so that the methylated DNA sequences trigger the subsequent triple signal amplifications, whilst the normal counterparts do not. The triple signal amplification procedure consists of methylated DNA sequence-based rolling circle amplification for a preliminary signal enhancement, a nicking enzyme-initiated target cleavage for a secondary amplification, and CRISPR-Cas12a enzyme-mediated trans-cleavage for a tertiary signal enhancement. This proposed approach reveals high sensitivity, which can even distinguish as low as 0.01% methylation levels from mixtures, paving the way towards the acceleration of methylation-based cancer diagnostics and management.},
}
@article {pmid35578991,
year = {2022},
author = {Loo, L and Harris, S and Milton, M and Meena, and Lembke, W and Berisha, F and Bertholet, S and Dessy, F and Dodge, R and Fang, X and Fiscella, M and Garofolo, F and Gorovits, B and Gupta, S and Jawa, V and Ishii-Watabe, A and Long, B and Lu, Y and Mack, T and McGuire, K and Nolan, K and Pan, L and Potthoff, B and Purushothama, S and Smith, D and Solstad, T and Sonderegger, I and Taddeo, F and Tangri, S and Wagner, L and Wu, B and Xu, Y and Kirshner, S and Verthelyi, D and Yan, H and Maxfield, K and Pedras-Vasconcelos, J and Abhari, MR and Gupta, S and Wu, Y and Rajadhyaksha, M and Andisik, M and Baltrukonis, D and Cherry, E and Cludts, I and Gunn, G and Millner, AH and Jordan, G and Kar, S and Kubiak, R and Lotz, GP and Palmer, R and Peng, K and Poetzl, J and Richards, S and Savoie, N and Staack, RF and Stubenrauch, K and Wadhwa, M and Waxenecker, G and Yang, TY and Zhang, L},
title = {2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 &amp; CAR-T Immunogenicity; PCR &amp; Vaccine Assay Performance; ADA Assay Comparability &amp; Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).},
journal = {Bioanalysis},
volume = {14},
number = {11},
pages = {737-793},
doi = {10.4155/bio-2022-0081},
pmid = {35578991},
issn = {1757-6199},
mesh = {Biomarkers/analysis ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Humans ; Immunotherapy, Active ; Polymerase Chain Reaction ; *Receptors, Chimeric Antigen ; *Vaccines ; },
abstract = {The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 &amp; CAR-T Immunogenicity; PCR &amp; Vaccine Assay Performance; ADA Assay Comparability &amp; Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene &amp; Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral &amp; Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.},
}
@article {pmid35578739,
year = {2022},
author = {Mu, K and Ren, X and Yang, H and Zhang, T and Yan, W and Yuan, F and Wu, J and Kang, Z and Han, D and Deng, R and Zeng, Q},
title = {CRISPR-Cas12a-Based Diagnostics of Wheat Fungal Diseases.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {23},
pages = {7240-7247},
doi = {10.1021/acs.jafc.1c08391},
pmid = {35578739},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Fusarium/metabolism ; *Mycoses/genetics ; Plant Diseases/microbiology ; RNA, Guide/metabolism ; Triticum/genetics/microbiology ; },
abstract = {Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum (F. graminearum) infection, reduces crop yield and contaminates grain with mycotoxins. We report a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based nucleic acid assay for an early and rapid diagnosis of wheat FHB. Guide RNA (gRNA) was screened for highly specific recognition of polymerase chain reaction (PCR) amplicon of the internal transcribed spacer (ITS) region and the transcription elongation factor 1α (EF1α) of F. graminearum. The trans-activation of Cas12a protein cleaves the single-stranded DNA probes with the terminal fluorophore and quencher groups, thus allowing us to report the presence of ITS and EF1α of F. graminearum. Owing to the dual recognition process through PCR primers and gRNA hybridization, the approach realized specific discrimination of F. graminearum from other pathogenic fungi. It also allowed us to detect as low as 1 fg/μL total DNA from F. graminearum, which is sufficient to diagnose a 4 day F. graminearum infection. CRISPR-Cas12a-based nucleic acid assay promises the molecular diagnosis of crop diseases and broadens the application of CRISPR tools.},
}
@article {pmid35578138,
year = {2022},
author = {Choi, SH and Ahn, WS and Jie, EY and Cho, HS and Kim, SW},
title = {Development of late-bolting plants by CRISPR/Cas9-mediated genome editing from mesophyll protoplasts of lettuce.},
journal = {Plant cell reports},
volume = {41},
number = {7},
pages = {1627-1630},
pmid = {35578138},
issn = {1432-203X},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing/methods ; Lettuce/genetics ; Protoplasts/metabolism ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR/Cas9-mediated introduction of a single base mutation in SOC1, a transcription factor that regulates flowering time, results in late-bolting phenotypes in lettuce. Lettuce is a widely consumed leafy vegetable crop. One of the molecular approaches that can increase leaf yield of lettuce is to delay the onset of flowering. Flowering time or time-to-bolting is not only a valuable trait for lettuce, but also a sought-after phenotype for other leafy vegetable crops. This is because delayed flowering enables more extensive vegetative growth, which leads to higher leaf numbers, and possibly larger leaves. Here, we deployed the most recent gene-editing technique to reduce the expression of SOC1, which is a gene that encodes one of several transcription factors that regulate the onset of flowering in plants. By inducing a single base mutation in SOC1 through Cas9 protein-gRNA ribonucleoproteins (RNPs) system, we showed that the time to first flower bud formation in lettuce is longer than that of wild type. In addition, expression of the floral regulatory genes including LsLFY, LsFUL, LsAPL1, and LsAPL2, was lower in the SOC1 gene edited plants than that of the wild type. The gene-editing technique established in this study could be directly applied for diverse quality improvement of lettuce by direct RNP transfer from protoplasts. Furthermore, it is expected that direct RNP transfer from protoplasts can be used as a useful mean for developing various gene edited crops.},
}
@article {pmid35577099,
year = {2022},
author = {Panda, G and Ray, A},
title = {Decrypting the mechanistic basis of CRISPR/Cas9 protein.},
journal = {Progress in biophysics and molecular biology},
volume = {172},
number = {},
pages = {60-76},
doi = {10.1016/j.pbiomolbio.2022.05.001},
pmid = {35577099},
issn = {1873-1732},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Gene Editing ; *RNA, Guide/chemistry/genetics/metabolism ; },
abstract = {CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.},
}
@article {pmid35576811,
year = {2022},
author = {Gulimiheranmu, M and Li, S and Zhou, J},
title = {Generation of a MIR5004 knockout cell line from human induced pluripotent stem cells by CRISPR/Cas9 gene editing.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102805},
doi = {10.1016/j.scr.2022.102805},
pmid = {35576811},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {MIR5004 is located in the intronic region of SYNGAP1, a genetic risk factor for Autism Spectrum Disorders (ASD), and co-expressed with SYNGAP1 in brain tissue, which indicates that MIR5004 may play an important role in ASD pathogenesis through the regulation of SYNGAP1. Here, we generated a MIR5004 knockout human induced pluripotent stem cell (iPSC) line SHCDCLi001-B. SHCDCLi001-B shows expression of pluripotent markers, three lineage differentiation capacity, normal morphology and karyotypes, the same DNA origin with wild type iPSC (iPSC-WT) and no off-target effects, making it as a valuable tool for studying the interplay between MIR5004 and SYNGAP1 in ASD pathogenesis.},
}
@article {pmid35575623,
year = {2022},
author = {Yue, SJ and Huang, P and Li, S and Cai, YY and Wang, W and Zhang, XH and Nikel, PI and Hu, HB},
title = {Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.},
journal = {Microbial biotechnology},
volume = {15},
number = {9},
pages = {2324-2336},
pmid = {35575623},
issn = {1751-7915},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/genetics/metabolism ; Deoxyribonuclease I/genetics/metabolism ; Gene Editing/methods ; Genome, Bacterial ; *Pseudomonas chlororaphis/genetics/metabolism ; Rats ; },
abstract = {Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9pp[D10A]) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts.},
}
@article {pmid35575140,
year = {2022},
author = {Liu, Q and Liu, M and Jin, Y and Li, B},
title = {Ratiometric fluorescent probe: a sensitive and reliable reporter for the CRISPR/Cas12a-based biosensing platform.},
journal = {The Analyst},
volume = {147},
number = {11},
pages = {2567-2574},
doi = {10.1039/d2an00613h},
pmid = {35575140},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; *Fluorescent Dyes ; },
abstract = {Due to the excellent activity of trans-cleavage and target recognition, the recently discovered CRISPR/Cas12a systems provide a promising opportunity for designing fluorescence biosensing. In the reported CRISPR/Cas12a-based biosensing platform, TaqMan probe is widely used as the reporter. However, the TaqMan probe provides single-wavelength fluorescence changes, which is easily influenced by various analyte-independent confounding factors to produce false-positive signals. In this study, a ratiometric fluorescent probe was designed to act as the reporter of a CRISPR/Cas12a-based system. As a proof-of-concept, fluorescein (FAM) and tetramethylrhodamine (TAMRA) were chosen as the two fluorescence dyes to label one short ssDNA at 5' and 3' ends, respectively, which was designed as one ratiometric fluorescent probe. When the ratiometric probe excites at 480 nm, duo to FRET effect, the probe emitted the 580 nm-fluorescence of TAMRA. The activated Cas12a can cleave the dual-labeled ssDNA, resulting in a decrease in the TAMRA's fluorescence and an increase in the FAM's fluorescence. This dual-response fluorescent probe can act as the reporter of the CRISPR/Cas12a-based biosensing platform. Compared with the classic TaqMan, CRISPR/Cas12a-based biosensing with the ratiometric probe as the reporter not only exhibited higher sensitivity but also could distinguish and avoid false positive signals.},
}
@article {pmid35573787,
year = {2022},
author = {Shankar, C and Vasudevan, K and Jacob, JJ and Baker, S and Isaac, BJ and Neeravi, AR and Sethuvel, DPM and George, B and Veeraraghavan, B},
title = {Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {875116},
pmid = {35573787},
issn = {2235-2988},
mesh = {Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Humans ; *Klebsiella Infections ; *Klebsiella pneumoniae ; Plasmids/genetics ; Virulence/genetics ; beta-Lactamases/genetics ; },
abstract = {BACKGROUND: Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes.<br><br>METHODS: Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment.<br><br>RESULTS: Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB-IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, blaOXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition.<br><br>CONCLUSION: The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.},
}
@article {pmid35573771,
year = {2022},
author = {Wu, X and Wang, S and Li, C and Shi, J and Peng, Z and Liu, C and Han, H and Ma, Y and Zheng, L and Xu, S and Du, W and Li, J and Zhang, F},
title = {CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {865744},
pmid = {35573771},
issn = {2235-2988},
mesh = {Animals ; Autophagy ; *CRISPR-Cas Systems ; DNA Viruses ; Female ; *Picornaviridae/genetics ; RNA Interference ; Swine ; Virus Replication ; },
abstract = {RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-Dicer[Δ-] and BHK-Ago2[Δ-]. Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.},
}
@article {pmid35573047,
year = {2022},
author = {Kath, J and Du, W and Pruene, A and Braun, T and Thommandru, B and Turk, R and Sturgeon, ML and Kurgan, GL and Amini, L and Stein, M and Zittel, T and Martini, S and Ostendorf, L and Wilhelm, A and Akyüz, L and Rehm, A and Höpken, UE and Pruß, A and Künkele, A and Jacobi, AM and Volk, HD and Schmueck-Henneresse, M and Stripecke, R and Reinke, P and Wagner, DL},
title = {Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {25},
number = {},
pages = {311-330},
pmid = {35573047},
issn = {2329-0501},
abstract = {Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR[+] T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.},
}
@article {pmid35572739,
year = {2022},
author = {Abdullah, M and Okemo, P and Furtado, A and Henry, R},
title = {Potential of Genome Editing to Capture Diversity From Australian Wild Rice Relatives.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {875243},
pmid = {35572739},
issn = {2673-3439},
abstract = {Rice, a staple food worldwide and a model crop, could benefit from the introduction of novel genetics from wild relatives. Wild rice in the AA genome group closely related to domesticated rice is found across the tropical world. Due to their locality outside the range of domesticated rice, Australian wild rice populations are a potential source of unique traits for rice breeding. These rice species provide a diverse gene pool for improvement that could be utilized for desirable traits such as stress resistance, disease tolerance, and nutritional qualities. However, they remain poorly characterized. The CRISPR/Cas system has revolutionized gene editing and has improved our understanding of gene functions. Coupled with the increasing availability of genomic information on the species, genes in Australian wild rice could be modified through genome editing technologies to produce new domesticates. Alternatively, beneficial alleles from these rice species could be incorporated into cultivated rice to improve critical traits. Here, we summarize the beneficial traits in Australian wild rice, the available genomic information and the potential of gene editing to discover and understand the functions of novel alleles. Moreover, we discuss the potential domestication of these wild rice species for health and economic benefits to rice production globally.},
}
@article {pmid35572197,
year = {2022},
author = {Nieland, L and van Solinge, TS and Cheah, PS and Morsett, LM and El Khoury, J and Rissman, JI and Kleinstiver, BP and Broekman, MLD and Breakefield, XO and Abels, ER},
title = {CRISPR-Cas knockout of miR21 reduces glioma growth.},
journal = {Molecular therapy oncolytics},
volume = {25},
number = {},
pages = {121-136},
pmid = {35572197},
issn = {2372-7705},
support = {R35 CA232103/CA/NCI NIH HHS/United States ; R56 NS123271/NS/NINDS NIH HHS/United States ; },
abstract = {Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.},
}
@article {pmid35572153,
year = {2022},
author = {Christian, A},
title = {Addressing Conflicts of Interest and Conflicts of Commitment in Public Advocacy and Policy Making on CRISPR/Cas-Based Human Genome Editing.},
journal = {Frontiers in research metrics and analytics},
volume = {7},
number = {},
pages = {775336},
pmid = {35572153},
issn = {2504-0537},
abstract = {Leading experts on CRISPR/Cas-based genome editing-such as 2020 Nobel laureates Jennifer Doudna and Emmanuelle Charpentier-are not only renowned specialists in their fields, but also public advocates for upcoming regulatory frameworks on CRISPR/Cas. These frameworks will affect large portions of biomedical research on human genome editing. In advocating for particular ways of handling the risks and prospects of this technology, high-profile scientists not only serve as scientific experts, but also as moral advisers. The majority of them currently intend to bring about a "responsible pathway" toward human genome interventions in clinical therapy. Engaging in advocacy for such a pathway, they issue moral judgments on the risks and benefits of this new technology. They declare that there actually is a responsible pathway, they draft resolutions on temporary moratoria, they make judgments on which groups and individuals are credible and should participate in public and semi-public debates, so they also set the standards for deciding who counts as well-informed, as well as the standards of evidence for adopting or rejecting research policies. This degree of influence on public debates and policy making is, at the very least, noteworthy. This contribution sounds a note of caution with regard to the endeavor of a responsible pathway to human genome editing and in particular scrutinizes the legitimacy of expert-driven research policies given commercial conflicts of interest and conflicts of commitment among first-rank scholars.},
}
@article {pmid35569864,
year = {2022},
author = {Islam, MM and Koirala, D},
title = {Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses.},
journal = {Analytica chimica acta},
volume = {1209},
number = {},
pages = {339338},
pmid = {35569864},
issn = {1873-4324},
mesh = {*COVID-19/diagnosis ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA, Viral/analysis ; *SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {As the COVID-19 pandemic continues to affect human health across the globe rapid, simple, point-of-care (POC) diagnosis of infectious viruses such as SARS-CoV-2 remains challenging. Polymerase chain reaction (PCR)-based diagnosis has risen to meet these demands and despite its high-throughput and accuracy, it has failed to gain traction in the rapid, low-cost, point-of-test settings. In contrast, different emerging isothermal amplification-based detection methods show promise in the rapid point-of-test market. In this comprehensive study of the literature, several promising isothermal amplification methods for the detection of SARS-CoV-2 are critically reviewed that can also be applied to other infectious viruses detection. Starting with a brief discussion on the SARS-CoV-2 structure, its genomic features, and the epidemiology of the current pandemic, this review focuses on different emerging isothermal methods and their advancement. The potential of isothermal amplification combined with the revolutionary CRISPR/Cas system for a more powerful detection tool is also critically reviewed. Additionally, the commercial success of several isothermal methods in the pandemic are highlighted. Different variants of SARS-CoV-2 and their implication on isothermal amplifications are also discussed. Furthermore, three most crucial aspects in achieving a simple, fast, and multiplexable platform are addressed.},
}
@article {pmid35569695,
year = {2022},
author = {Zhang, X and Jin, X and Sun, R and Zhang, M and Lu, W and Zhao, M},
title = {Gene knockout in cellular immunotherapy: Application and limitations.},
journal = {Cancer letters},
volume = {540},
number = {},
pages = {215736},
doi = {10.1016/j.canlet.2022.215736},
pmid = {35569695},
issn = {1872-7980},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Humans ; Immunotherapy, Adoptive ; Zinc Finger Nucleases/genetics ; },
abstract = {Cellular immunotherapy has achieved incremental success in recent years. Varieties of cell products are undergoing fundamental research and clinical trials, among which CAR-T cell therapy is approved for marketing. As research progresses, these cells need to be modified to promote their safety and efficacy. Gene-editing technologies have evolved from RNA interference (RNAi), including small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to new generations of zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and clusters of regularly spaced short palindromic repeats (CRISPR/Cas9), and delivery methods are widely used. Here, we summarize the ongoing clinical trials and fundamental research for genome editing therapy. Additionally, we highlight existing in vivo delivery systems and their limitations to find a better method to deliver genes.},
}
@article {pmid35568950,
year = {2022},
author = {Cai, P and Han, M and Zhang, R and Ding, S and Zhang, D and Liu, D and Liu, S and Hu, QN},
title = {SynBioStrainFinder: A microbial strain database of manually curated CRISPR/Cas genetic manipulation system information for biomanufacturing.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {87},
pmid = {35568950},
issn = {1475-2859},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {BACKGROUND: Microbial strain information databases provide valuable data for microbial basic research and applications. However, they rarely contain information on the genetic operating system of microbial strains.<br><br>RESULTS: We established a comprehensive microbial strain database, SynBioStrainFinder, by integrating CRISPR/Cas gene-editing system information with cultivation methods, genome sequence data, and compound-related information. It is presented through three modules, Strain2Gms/PredStrain2Gms, Strain2BasicInfo, and Strain2Compd, which combine to form a rapid strain information query system conveniently curated, integrated, and accessible on a single platform. To date, 1426 CRISPR/Cas gene-editing records of 157 microbial strains have been manually extracted from the literature in the Strain2Gms module. For strains without established CRISPR/Cas systems, the PredStrain2Gms module recommends the system of the most closely related strain as a reference to facilitate the construction of a new CRISPR/Cas gene-editing system. The database contains 139,499 records of strain cultivation and genome sequences, and 773,298 records of strain-related compounds. To facilitate simple and intuitive data application, all microbial strains are also labeled with stars based on the order and availability of strain information. SynBioStrainFinder provides a user-friendly interface for querying, browsing, and visualizing detailed information on microbial strains, and it is publicly available at http://design.rxnfinder.org/biosynstrain/ .<br><br>CONCLUSION: SynBioStrainFinder is the first microbial strain database with manually curated information on the strain CRISPR/Cas system as well as other microbial strain information. It also provides reference information for the construction of new CRISPR/Cas systems. SynBioStrainFinder will serve as a useful resource to extend microbial strain research and application for biomanufacturing.},
}
@article {pmid35568789,
year = {2022},
author = {Chaudhary, M and Mukherjee, TK and Singh, R and Gupta, M and Goyal, S and Singhal, P and Kumar, R and Bhusal, N and Sharma, P},
title = {CRISPR/Cas technology for improving nutritional values in the agricultural sector: an update.},
journal = {Molecular biology reports},
volume = {49},
number = {7},
pages = {7101-7110},
pmid = {35568789},
issn = {1573-4978},
mesh = {Biotechnology ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; Genome, Plant ; Nutritive Value ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system was initially identified in bacteria and archaea as a defense mechanism to confer immunity against phages. Later on, it was developed as a gene editing tool for both prokaryotic and eukaryotic cells including plant cells.<br><br>METHODS AND RESULTS: CRISPR/Cas9 approach has wider applications in reverse genetics as well as in crop improvement. Various characters involved in enhancing economic value and crop sustainability against biotic/abiotic stresses can be targeted through this tool. Currently, CRISPR/Cas9 gene editing mechanism has been applied on around 20 crop species for improvement in several traits including yield enhancement and resistance against biotic and abiotic stresses. In the last five years, maximum genome editing research has been validated in rice, wheat, maize and soybean. Genes targeted in these plants has been involved in causing male sterility, conferring resistance against pathogens or having certain nutritional value.<br><br>CONCLUSIONS: Current review summarizes various applications of CRISPR/Cas system and its future prospects in plant biotechnology targeting crop improvement with higher yield, disease tolerance and enhanced nutritional value.},
}
@article {pmid35567848,
year = {2022},
author = {Zhang, D and Zhou, M and Zhang, Y and Shan, Y and Pan, G},
title = {Generation of an RNF1-deficient human pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102809},
doi = {10.1016/j.scr.2022.102809},
pmid = {35567848},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/physiology ; Cell Line ; *Human Embryonic Stem Cells/metabolism ; Humans ; *Pluripotent Stem Cells ; Technology ; },
abstract = {RNF1 (RING1A) is a catalytic component of the polycomb repressive complex 1 (PRC1) involved in regulation of, among others, embryonic development and disease progression. However, the exact role of RNF1 in self-renewal and differentiation of human embryonic stem cells (ESCs) remains unknown. Here, we derive one RNF1 knockout human ESC line using CRISPR/Cas9 system. The cell line retains the canonical stem cell morphology and normal karyotype. Moreover, the cell line highly expresses pluripotency genes and has three germ-layer differentiation potential. The RNF1 -/- cell line will be useful for studies on the function and role of RNF1 in human embryonic stem cell fate decisions.},
}
@article {pmid35567297,
year = {2022},
author = {Athukoralage, JS and White, MF},
title = {Cyclic Nucleotide Signaling in Phage Defense and Counter-Defense.},
journal = {Annual review of virology},
volume = {9},
number = {1},
pages = {451-468},
doi = {10.1146/annurev-virology-100120-010228},
pmid = {35567297},
issn = {2327-0578},
support = {BB/S000313/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T004789/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Antiviral Agents ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids ; Nucleotides, Cyclic ; Pyrimidines ; },
abstract = {Advances in our understanding of prokaryotic antiphage defense mechanisms in the past few years have revealed a multitude of new cyclic nucleotide signaling molecules that play a crucial role in switching infected cells into an antiviral state. Defense pathways including type III CRISPR (clustered regularly interspaced palindromic repeats), CBASS (cyclic nucleotide-based antiphage signaling system), PYCSAR (pyrimidine cyclase system for antiphage resistance), and Thoeris all use cyclic nucleotides as second messengers to activate a diverse range of effector proteins. These effectors typically degrade or disrupt key cellular components such as nucleic acids, membranes, or metabolites, slowing down viral replication kinetics at great cost to the infected cell. Mechanisms to manipulate the levels of cyclic nucleotides are employed by cells to regulate defense pathways and by viruses to subvert them. Here we review the discovery and mechanism of the key pathways, signaling molecules and effectors, parallels and differences between the systems, open questions, and prospects for future research in this area.},
}
@article {pmid35565854,
year = {2022},
author = {Quéré, M and Alberto, JM and Broly, F and Hergalant, S and Christov, C and Gauchotte, G and Guéant, JL and Namour, F and Battaglia-Hsu, SF},
title = {ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency.},
journal = {Nutrients},
volume = {14},
number = {9},
pages = {},
pmid = {35565854},
issn = {2072-6643},
mesh = {Cell Line, Tumor ; *Glioblastoma/metabolism ; Humans ; Methionine/metabolism ; Neoplastic Stem Cells/metabolism ; Oxidative Stress ; Reactive Oxygen Species/metabolism ; },
abstract = {Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.},
}
@article {pmid35563876,
year = {2022},
author = {Gómez-García, F and Martínez-Pulleiro, R and Carrera, N and Allegue, C and Garcia-Gonzalez, MA},
title = {Genetic Kidney Diseases (GKDs) Modeling Using Genome Editing Technologies.},
journal = {Cells},
volume = {11},
number = {9},
pages = {},
pmid = {35563876},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Female ; *Gene Editing/methods ; Humans ; *Kidney Diseases/genetics/therapy ; Male ; Zinc Finger Nucleases ; },
abstract = {Genetic kidney diseases (GKDs) are a group of rare diseases, affecting approximately about 60 to 80 per 100,000 individuals, for which there is currently no treatment that can cure them (in many cases). GKDs usually leads to early-onset chronic kidney disease, which results in patients having to undergo dialysis or kidney transplant. Here, we briefly describe genetic causes and phenotypic effects of six GKDs representative of different ranges of prevalence and renal involvement (ciliopathy, glomerulopathy, and tubulopathy). One of the shared characteristics of GKDs is that most of them are monogenic. This characteristic makes it possible to use site-specific nuclease systems to edit the genes that cause GKDs and generate in vitro and in vivo models that reflect the genetic abnormalities of GKDs. We describe and compare these site-specific nuclease systems (zinc finger nucleases (ZFNs), transcription activator-like effect nucleases (TALENs) and regularly clustered short palindromic repeat-associated protein (CRISPR-Cas9)) and review how these systems have allowed the generation of cellular and animal GKDs models and how they have contributed to shed light on many still unknown fields in GKDs. We also indicate the main obstacles limiting the application of these systems in a more efficient way. The information provided here will be useful to gain an accurate understanding of the technological advances in the field of genome editing for GKDs, as well as to serve as a guide for the selection of both the genome editing tool and the gene delivery method most suitable for the successful development of GKDs models.},
}
@article {pmid35563479,
year = {2022},
author = {Feser, CJ and Lees, CJ and Lammers, DT and Riddle, MJ and Bingham, JR and Eckert, MJ and Tolar, J and Osborn, MJ},
title = {Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563479},
issn = {1422-0067},
mesh = {*Blood Coagulation Factors/biosynthesis/genetics ; *CRISPR-Cas Systems ; Fibrinogen/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Recombinant Proteins/biosynthesis/genetics ; Transcriptional Activation ; },
abstract = {Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120-4700-fold and 60-680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700-92,000-fold increases and 80-5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.},
}
@article {pmid35563453,
year = {2022},
author = {Shin, NR and Shin, YH and Kim, HS and Park, YD},
title = {Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563453},
issn = {1422-0067},
mesh = {*Brassica/genetics ; *CRISPR-Cas Systems ; China ; Gene Editing ; Mutagenesis ; Plant Breeding ; },
abstract = {Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50-70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.},
}
@article {pmid35563435,
year = {2022},
author = {Fischer, B and Schmidt, V and Ly, TD and Kleine, A and Knabbe, C and Faust-Hinse, I},
title = {First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563435},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; Fibroblasts/cytology/metabolism ; Gene Editing ; Humans ; Infant, Newborn ; *Pentosyltransferases/genetics/metabolism ; *Skin/metabolism ; },
abstract = {BACKGROUND: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue.<br><br>METHODS: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis.<br><br>RESULTS: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization.<br><br>CONCLUSION: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.},
}
@article {pmid35563412,
year = {2022},
author = {Yao, M and Ren, T and Pan, Y and Xue, X and Li, R and Zhang, L and Li, Y and Huang, K},
title = {A New Generation of Lineage Tracing Dynamically Records Cell Fate Choices.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563412},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage/genetics ; Gene Editing ; *Single-Cell Analysis ; *Transcriptome ; },
abstract = {Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing for large-scale lineage tracing has been used to reconstruct the pseudotime trajectory of cells and improve lineage tracing accuracy. This review presents the progress of the latest CbLT (CRISPR-based Lineage Tracing) and discusses the current limitations and potential technical pitfalls in their application and other emerging concepts.},
}
@article {pmid35563297,
year = {2022},
author = {Moniruzzaman, M and Zhong, Y and Huang, Z and Zhong, G},
title = {Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563297},
issn = {1422-0067},
mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Gene Editing ; Mutagenesis ; *RNA, Guide/genetics ; },
abstract = {Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.},
}
@article {pmid35563243,
year = {2022},
author = {Tian, J and Xing, B and Li, M and Xu, C and Huo, YX and Guo, S},
title = {Efficient Large-Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563243},
issn = {1422-0067},
mesh = {*Bacillus subtilis/genetics ; Biofuels ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Bacterial ; Metabolic Engineering ; },
abstract = {Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.},
}
@article {pmid35563030,
year = {2022},
author = {Toinga-Villafuerte, S and Vales, MI and Awika, JM and Rathore, KS},
title = {CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35563030},
issn = {1422-0067},
mesh = {Amylopectin/metabolism ; Amylose/metabolism ; CRISPR-Cas Systems/genetics ; Gold/metabolism ; Mutagenesis ; *Solanum tuberosum/genetics/metabolism ; Starch/metabolism ; *Starch Synthase/genetics ; Yukon Territory ; },
abstract = {Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.},
}
@article {pmid35562887,
year = {2022},
author = {Wang, YY and Hsu, SH and Tsai, HY and Cheng, FY and Cheng, MC},
title = {Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {9},
pages = {},
pmid = {35562887},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Carrier Proteins ; Chromatography, Liquid ; HEK293 Cells ; Humans ; Microfilament Proteins ; Mitochondrial Proteins ; *Proteomics ; Tandem Mass Spectrometry ; *Transcriptome ; },
abstract = {Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.},
}
@article {pmid35562427,
year = {2022},
author = {Wang, JY and Pausch, P and Doudna, JA},
title = {Structural biology of CRISPR-Cas immunity and genome editing enzymes.},
journal = {Nature reviews. Microbiology},
volume = {20},
number = {11},
pages = {641-656},
pmid = {35562427},
issn = {1740-1534},
support = {U01 AI142817/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Antitoxins/genetics ; Bacteria ; Biology ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements ; *Gene Editing ; RNA, Bacterial ; Retroelements ; },
abstract = {CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.},
}
@article {pmid35561628,
year = {2022},
author = {Cai, G and Lin, Z and Shi, S},
title = {Development and expansion of the CRISPR/Cas9 toolboxes for powerful genome engineering in yeast.},
journal = {Enzyme and microbial technology},
volume = {159},
number = {},
pages = {110056},
doi = {10.1016/j.enzmictec.2022.110056},
pmid = {35561628},
issn = {1879-0909},
mesh = {Biotechnology ; *CRISPR-Cas Systems ; Gene Editing/methods ; RNA, Guide/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Yeasts represent a group of the microorganisms most frequently seen in biotechnology. Recently, the class 2 type II CRISPR system (CRISPR/Cas9) has become the principal toolbox for genome editing. By efficiently implementing genetic manipulations such as gene integration/knockout, base editor, and transcription regulation, the development of biotechnological applications in yeasts has been extensively promoted. The genome-level tools based on CRISPR/Cas9, used for screening and identifying functional genes/gene clusters, are also advancing. In general, CRISPR/Cas9-assisted editing tools have gradually become standardized and function as host-orthogonal genetic systems, which results in time-saving for strain engineering and biotechnological application processes. In this review, we summarize the key points of the basic elements in the CRISPR/Cas9 system, including Cas9 variants, guide RNA, donors, and effectors. With a focus on yeast, we have also introduced the development of various CRISPR/Cas9 systems and discussed their future possibilities.},
}
@article {pmid35561459,
year = {2022},
author = {Fu, J and Jiang, L and Yu, B and Liu, Y and Wei, R and Hu, Y and Ho, WI and Yang, B and Chu, M and Tse, HF and Yang, J},
title = {Generation of a human iPSC line CIBi010-A with a reporter for ASGR1 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102800},
doi = {10.1016/j.scr.2022.102800},
pmid = {35561459},
issn = {1876-7753},
mesh = {Asialoglycoprotein Receptor/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Hepatocytes/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Pluripotent Stem Cells/metabolism ; },
abstract = {ASGR1 is a liver-specific surface marker that has been used to purify human pluripotent stem cell (PSC)-derived hepatocytes (iHeps). Furthermore, ASGR1[+] iHeps represents a more mature subpopulation of iHeps. To utilize this marker for optimizing iHep differentiation and purification, we substituted the stop coden of ASGR1 with a fluorescent reporter protein mCherry in a human iPSC line iPSN0052 via CRISPR/Cas9-mediated homologus recombination. The generated CIBi010-A enableds us to monitor ASGR1 expression during hepatic differentiation and thus can be used to optimize our hepatic differentiation procedures.},
}
@article {pmid35561458,
year = {2022},
author = {Chen, CX and You, Z and Abdian, N and Sirois, J and Shlaifer, I and Tabatabaei, M and Boivin, MN and Gaborieau, L and Karamchandani, J and Beitel, LK and Fon, EA and Durcan, TM},
title = {Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102806},
doi = {10.1016/j.scr.2022.102806},
pmid = {35561458},
issn = {1876-7753},
support = {PJT &#x2013; 169095//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mitophagy/genetics ; *Parkinson Disease/genetics ; Protein Kinases/genetics ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson's disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson's disease.},
}
@article {pmid35560779,
year = {2022},
author = {Beyene, G and Chauhan, RD and Villmer, J and Husic, N and Wang, N and Gebre, E and Girma, D and Chanyalew, S and Assefa, K and Tabor, G and Gehan, M and McGrone, M and Yang, M and Lenderts, B and Schwartz, C and Gao, H and Gordon-Kamm, W and Taylor, NJ and MacKenzie, DJ},
title = {CRISPR/Cas9-mediated tetra-allelic mutation of the 'Green Revolution' SEMIDWARF-1 (SD-1) gene confers lodging resistance in tef (Eragrostis tef).},
journal = {Plant biotechnology journal},
volume = {20},
number = {9},
pages = {1716-1729},
pmid = {35560779},
issn = {1467-7652},
mesh = {Alleles ; CRISPR-Cas Systems ; *Eragrostis/genetics ; Gene Editing ; *Genes, Plant ; Mutation ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.},
}
@article {pmid35560156,
year = {2022},
author = {Diorio, C and Murray, R and Naniong, M and Barrera, L and Camblin, A and Chukinas, J and Coholan, L and Edwards, A and Fuller, T and Gonzales, C and Grupp, SA and Ladd, A and Le, M and Messana, A and Musenge, F and Newman, H and Poh, YC and Poulin, H and Ryan, T and Shraim, R and Tasian, SK and Vincent, T and Young, L and Zhang, Y and Ciaramella, G and Gehrke, J and Teachey, DT},
title = {Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL.},
journal = {Blood},
volume = {140},
number = {6},
pages = {619-629},
pmid = {35560156},
issn = {1528-0020},
support = {T32 CA009615/CA/NCI NIH HHS/United States ; UG1 CA233249/CA/NCI NIH HHS/United States ; R03 CA256550/CA/NCI NIH HHS/United States ; R01 CA264837/CA/NCI NIH HHS/United States ; R01 CA193776/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cytosine ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Humans ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics ; },
abstract = {Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.},
}
@article {pmid35560111,
year = {2022},
author = {Worthington, AK and Forsberg, EC},
title = {A CRISPR view of hematopoietic stem cells: Moving innovative bioengineering into the clinic.},
journal = {American journal of hematology},
volume = {97},
number = {9},
pages = {1226-1235},
pmid = {35560111},
issn = {1096-8652},
support = {R01 AG062879/AG/NIA NIH HHS/United States ; T32 GM008646/GM/NIGMS NIH HHS/United States ; F31 HL151199/HL/NHLBI NIH HHS/United States ; F31HL151199/HL/NHLBI NIH HHS/United States ; R01DK100917/DK/NIDDK NIH HHS/United States ; R01 DK100917/DK/NIDDK NIH HHS/United States ; R01AG062879/AG/NIA NIH HHS/United States ; },
mesh = {Bioengineering ; *CRISPR-Cas Systems ; Genomics/methods ; *Hematologic Diseases/genetics/therapy ; Hematopoietic Stem Cells ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome engineering has emerged as a powerful tool to modify precise genomic sequences with unparalleled accuracy and efficiency. Major advances in CRISPR technologies over the last 5 years have fueled the development of novel techniques in hematopoiesis research to interrogate the complexities of hematopoietic stem cell (HSC) biology. In particular, high throughput CRISPR based screens using various "flavors" of Cas coupled with sequencing and/or functional outputs are becoming increasingly efficient and accessible. In this review, we discuss recent achievements in CRISPR-mediated genomic engineering and how these new tools have advanced the understanding of HSC heterogeneity and function throughout life. Additionally, we highlight how these techniques can be used to answer previously inaccessible questions and the challenges to implement them. Finally, we focus on their translational potential to both model and treat hematological diseases in the clinic.},
}
@article {pmid35559673,
year = {2022},
author = {Feng, X and Tang, M and Dede, M and Su, D and Pei, G and Jiang, D and Wang, C and Chen, Z and Li, M and Nie, L and Xiong, Y and Li, S and Park, JM and Zhang, H and Huang, M and Szymonowicz, K and Zhao, Z and Hart, T and Chen, J},
title = {Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors.},
journal = {Science advances},
volume = {8},
number = {19},
pages = {eabm6638},
pmid = {35559673},
issn = {2375-2548},
support = {P01 CA193124/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; R01 CA216911/CA/NCI NIH HHS/United States ; R01 CA216437/CA/NCI NIH HHS/United States ; R01 CA210929/CA/NCI NIH HHS/United States ; },
mesh = {*Antineoplastic Agents ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Tumor Suppressor ; Humans ; *Neoplasms/genetics ; Synthetic Lethal Mutations ; },
abstract = {Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.},
}
@article {pmid35559592,
year = {2022},
author = {Li, H and Yang, J and Wu, G and Weng, Z and Song, Y and Zhang, Y and Vanegas, JA and Avery, L and Gao, Z and Sun, H and Chen, Y and Dieckhaus, KD and Gao, X and Zhang, Y},
title = {Amplification-Free Detection of SARS-CoV-2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field-Effect Transistors.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {32},
pages = {e202203826},
pmid = {35559592},
issn = {1521-3773},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; *Graphite ; Humans ; *Nucleic Acids ; Respiratory Syncytial Viruses ; SARS-CoV-2/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR-based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification-free nucleic acid detection via harnessing the trans-cleavage mechanism of Cas13a and ultrasensitive graphene field-effect transistors (gFETs). CRISPR Cas13a-gFET achieves the detection of SARS-CoV-2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS-CoV-2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a-gFET among the most sensitive amplification-free nucleic acid diagnostic platforms to date.},
}
@article {pmid35559022,
year = {2022},
author = {Mandal, S and Ghorai, M and Anand, U and Roy, D and Kant, N and Mishra, T and Mane, AB and Jha, NK and Lal, MK and Tiwari, RK and Kumar, M and Radha, and Ghosh, A and Bhattacharjee, R and Proćków, J and Dey, A},
title = {Cytokinins: A Genetic Target for Increasing Yield Potential in the CRISPR Era.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {883930},
pmid = {35559022},
issn = {1664-8021},
abstract = {Over the last decade, remarkable progress has been made in our understanding the phytohormones, cytokinin's (CKs) biosynthesis, perception, and signalling pathways. Additionally, it became apparent that interfering with any of these steps has a significant effect on all stages of plant growth and development. As a result of their complex regulatory and cross-talk interactions with other hormones and signalling networks, they influence and control a wide range of biological activities, from cellular to organismal levels. In agriculture, CKs are extensively used for yield improvement and management because of their wide-ranging effects on plant growth, development and physiology. One of the primary targets in this regard is cytokinin oxidase/dehydrogenase (CKO/CKX), which is encoded by CKX gene, which catalyses the irreversible degradation of cytokinin. The previous studies on various agronomically important crops indicated that plant breeders have targeted CKX directly. In recent years, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been increasingly used in editing the CKO/CKX gene and phenomenal results have been achieved. This review provides an updated information on the applications of CRISPR-based gene-editing tools in manipulating cytokinin metabolism at the genetic level for yield improvement. Furthermore, we summarized the current developments of RNP-mediated DNA/transgene-free genomic editing of plants which would broaden the application of this technology. The current review will advance our understanding of cytokinins and their role in sustainably increase crop production through CRISPR/Cas genome editing tool.},
}
@article {pmid35558825,
year = {2022},
author = {Rahman, F and Mishra, A and Gupta, A and Sharma, R},
title = {Spatiotemporal Regulation of CRISPR/Cas9 Enables Efficient, Precise, and Heritable Edits in Plant Genomes.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {870108},
pmid = {35558825},
issn = {2673-3439},
abstract = {CRISPR/Cas-mediated editing has revolutionized crop engineering. Due to the broad scope and potential of this technology, many studies have been carried out in the past decade towards optimizing genome editing constructs. Clearly, the choice of the promoter used to drive gRNA and Cas9 expression is critical to achieving high editing efficiency, precision, and heritability. While some important considerations for choosing a promoter include the number and nature of targets, host organism, mode of transformation and goal of the experiment, spatiotemporal regulation of Cas9 expression using tissue-specific or inducible promoters enables higher heritability and efficiency of targeted mutagenesis with reduced off-target effects. In this review, we discuss specific studies that highlight the prospects and trade-offs associated with the choice of promoters on genome editing and emphasize the need for inductive exploration and discovery to further advance this area of research in crop plants.},
}
@article {pmid35557039,
year = {2022},
author = {Ye, J and Xi, H and Chen, Y and Chen, Q and Lu, X and Lv, J and Chen, Y and Gu, F and Zhao, J},
title = {Can SpRY recognize any PAM in human cells?.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {5},
pages = {382-391},
pmid = {35557039},
issn = {1862-1783},
mesh = {*CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; DNA ; Gene Editing/methods ; Humans ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.},
}
@article {pmid35552388,
year = {2022},
author = {Vicencio, J and Sánchez-Bolaños, C and Moreno-Sánchez, I and Brena, D and Vejnar, CE and Kukhtar, D and Ruiz-López, M and Cots-Ponjoan, M and Rubio, A and Melero, NR and Crespo-Cuadrado, J and Carolis, C and Pérez-Pulido, AJ and Giráldez, AJ and Kleinstiver, BP and Cerón, J and Moreno-Mateos, MA},
title = {Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2601},
pmid = {35552388},
issn = {2041-1723},
support = {R00 CA218870/CA/NCI NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; R35 GM122580/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics/metabolism ; *Gene Editing/methods ; RNA, Guide/genetics ; RNA, Messenger ; Zebrafish/genetics/metabolism ; },
abstract = {The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.},
}
@article {pmid35551911,
year = {2022},
author = {Xu, X and Wu, G},
title = {Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {6},
pages = {102021},
pmid = {35551911},
issn = {1083-351X},
support = {R35 GM136397/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; Gene Knockdown Techniques ; *Golgi Apparatus/metabolism ; Humans ; *Membrane Proteins/genetics/metabolism ; Protein Transport ; RNA, Small Interfering ; *Receptors, Adrenergic, alpha-2/genetics/metabolism ; Receptors, G-Protein-Coupled/metabolism ; },
abstract = {The molecular mechanisms underlying the anterograde surface transport of G protein-coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α2A-adrenergic receptor (α2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER-Golgi-surface transport of α2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β2-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.},
}
@article {pmid35551240,
year = {2022},
author = {Zhang, D and Wang, G and Yu, X and Wei, T and Farbiak, L and Johnson, LT and Taylor, AM and Xu, J and Hong, Y and Zhu, H and Siegwart, DJ},
title = {Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy.},
journal = {Nature nanotechnology},
volume = {17},
number = {7},
pages = {777-787},
pmid = {35551240},
issn = {1748-3395},
support = {R01 CA269787/CA/NCI NIH HHS/United States ; P30 CA142543/CA/NCI NIH HHS/United States ; R01 CA251928/CA/NCI NIH HHS/United States ; R01 DK125396/DK/NIDDK NIH HHS/United States ; R01 DK111588/DK/NIDDK NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; B7-H1 Antigen/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; Liposomes ; Mice ; Nanoparticles ; *Neoplasms/genetics/therapy ; },
abstract = {Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.},
}
@article {pmid35550915,
year = {2022},
author = {Li, Y and Mensah, EO and Fordjour, E and Bai, J and Yang, Y and Bai, Z},
title = {Recent advances in high-throughput metabolic engineering: Generation of oligonucleotide-mediated genetic libraries.},
journal = {Biotechnology advances},
volume = {59},
number = {},
pages = {107970},
doi = {10.1016/j.biotechadv.2022.107970},
pmid = {35550915},
issn = {1873-1899},
mesh = {CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; *Gene Editing ; Genetic Engineering ; *Metabolic Engineering ; Oligonucleotides ; Saccharomyces cerevisiae/genetics ; },
abstract = {The preparation of genetic libraries is an essential step to evolve microorganisms and study genotype-phenotype relationships by high-throughput screening/selection. As the large-scale synthesis of oligonucleotides becomes easy, cheap, and high-throughput, numerous novel strategies have been developed in recent years to construct high-quality oligo-mediated libraries, leveraging state-of-art molecular biology tools for genome editing and gene regulation. This review presents an overview of recent advances in creating and characterizing in vitro and in vivo genetic libraries, based on CRISPR/Cas, regulatory RNAs, and recombineering, primarily for Escherichia coli and Saccharomyces cerevisiae. These libraries' applications in high-throughput metabolic engineering, strain evolution and protein engineering are also discussed.},
}
@article {pmid35550024,
year = {2022},
author = {Mesa, V and Monot, M and Ferraris, L and Popoff, M and Mazuet, C and Barbut, F and Delannoy, J and Dupuy, B and Butel, MJ and Aires, J},
title = {Core-, pan- and accessory genome analyses of Clostridium neonatale: insights into genetic diversity.},
journal = {Microbial genomics},
volume = {8},
number = {5},
pages = {},
pmid = {35550024},
issn = {2057-5858},
mesh = {*Clostridium/genetics ; Genetic Variation ; *Genome, Bacterial ; Humans ; Infant, Newborn ; Phylogeny ; },
abstract = {Clostridium neonatale is a potential opportunistic pathogen recovered from faecal samples in cases of necrotizing enterocolitis (NEC), a gastrointestinal disease affecting preterm neonates. Although the C. neonatale species description and name validation were published in 2018, comparative genomics are lacking. In the present study, we provide the closed genome assembly of the C. neonatale ATCC BAA-265[T] (=250.09) reference strain with a manually curated functional annotation of the coding sequences. Pan-, core- and accessory genome analyses were performed using the complete 250.09 genome (4.7 Mb), three new assemblies (4.6-5.6 Mb), and five publicly available draft genome assemblies (4.6-4.7 Mb). The C. neonatale pan-genome contains 6840 genes, while the core-genome has 3387 genes. Pan-genome analysis revealed an 'open' state and genomic diversity. The strain-specific gene families ranged from five to 742 genes. Multiple mobile genetic elements were predicted, including a total of 201 genomic islands, 13 insertion sequence families, one CRISPR-Cas type I-B system and 15 predicted intact prophage signatures. Primary virulence classes including offensive, defensive, regulation of virulence-associated genes and non-specific virulence factors were identified. The presence of a tet(W/N/W) gene encoding a tetracycline resistance ribosomal protection protein and a 23S rRNA methyltransferase ermQ gene were identified in two different strains. Together, our results revealed a genetic diversity and plasticity of C. neonatale genomes and provide a comprehensive view of this species genomic features, paving the way for the characterization of its biological capabilities.},
}
@article {pmid35549895,
year = {2022},
author = {Bernas, G and Ouellet, M and Barrios, A and Jamann, H and Larochelle, C and Lévy, &#xc9; and Schmouth, JF},
title = {Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.},
journal = {BMC biotechnology},
volume = {22},
number = {1},
pages = {14},
pmid = {35549895},
issn = {1472-6750},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Electroporation/methods ; Embryo, Mammalian ; Exons ; Mammals/genetics ; Mice ; },
abstract = {BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project.<br><br>RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%.<br><br>CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.},
}
@article {pmid35549163,
year = {2022},
author = {Zeng, R and Gong, H and Li, Y and Li, Y and Lin, W and Tang, D and Knopp, D},
title = {CRISPR-Cas12a-Derived Photoelectrochemical Biosensor for Point-Of-Care Diagnosis of Nucleic Acid.},
journal = {Analytical chemistry},
volume = {94},
number = {20},
pages = {7442-7448},
doi = {10.1021/acs.analchem.2c01373},
pmid = {35549163},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; Electrochemical Techniques ; Humans ; Hydrogen Peroxide ; *Nucleic Acids ; Point-of-Care Systems ; },
abstract = {This work presented a point-of-care (POC) photoelectrochemical (PEC) biosensing for the detection of human papillomavirus-16 (HPV-16) on a portable electrochemical detection system by using CRISPR-Cas12a trans-cleaving the G-quadruplex for the biorecognition/amplification and a hollow In2O3-In2S3-modified screen-printed electrode (In2O3-In2S3/SPE) as the photoactive material. G-quadruplexes were capable of biocatalytic precipitation (H2O2-mediated 4-chloro-1-naphthol oxidation) on the In2O3-In2S3/SPE surface, resulting in a weakened photocurrent, but suffered from trans-cleavage when the CRISPR-Cas12a system specifically recognized the analyte. The photocurrent results could be directly observed with the card-sized electrochemical device via a smartphone, which displayed a high-value photocurrent for these positive samples, while a low-value photocurrent for the target-free samples. Such a system exhibited satisfying photocurrent responses toward HPV-16 within a wide working range from 5.0 to 5000 pM and allowed for detection of HPV-16 at a concentration as low as 1.2 pM. The proposed assay provided a smartphone signal readout to enable the rapid screening PEC determination of HPV-16 concentration without sophisticated instruments, thus meeting the requirements of remote areas and resource-limited settings. We envision that combining an efficient biometric PEC sensing platform with a wireless card-sized electrochemical device will enable high-throughput POC diagnostic analysis.},
}
@article {pmid35549133,
year = {2021},
author = {Kanduri, V and LaVigne, D and Larsen, J},
title = {Current Advances Toward the Encapsulation of Cas9.},
journal = {ACS macro letters},
volume = {10},
number = {12},
pages = {1576-1589},
doi = {10.1021/acsmacrolett.1c00538},
pmid = {35549133},
issn = {2161-1653},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Lipids ; Liposomes ; Nanoparticles ; Polymers/metabolism ; Quality of Life ; },
abstract = {Genetic diseases present formidable hurdles in maintaining a good quality of life for those suffering from these ailments. Often, patients look to inadequate treatments to manage symptoms, which can result in harmful effects on the body. Through genetic engineering, scientists utilize the clustered regularly short palindromic repeat (CRISPR)-associated protein, known as Cas9, to treat the root of the problem. The Cas9 protein is often codelivered with guide RNAs or in ribonucleoprotein complexes (RNP) to ensure targeted delivery of the genetic tool as well as to limit off-target effects. This paper provides an overview of the current advances made toward the encapsulation and delivery of Cas9 to desired locations in the body through encapsulating nanoparticles. Several factors must be considered when employing the Cas9 system to allow gene editing to occur. Material selection is crucial to protect the payload of the delivery vector. Current literature indicates that lipid- and polymer-based nanoparticles show the most potential as delivery vessels for Cas9. Lipid nanoparticles greatly outpace polymer-based nanoparticles in the clinic, despite the benefits that polymers may introduce. When developing translatable systems, there are factors that have not yet been considered that are relevant to Cas9 delivery that are highlighted in this Viewpoint. The proper functioning of Cas9 is dependent on maintaining a proper internal environment; however, there are gaps in the literature regarding these optimal conditions. Interactions between charges of the Cas9 protein, codelivered molecules, and delivery vehicles could impact the effectiveness of the gene editing taking place. While the internal charges of nanoparticles and their effects on Cas9 are presently undetermined, nanoparticles currently offer the ideal delivery method for the Cas9 protein due to their adequate size, modifiable external charge, and ability to be modified. Overall, a cationic lipid-/polymer-based nanoparticle system was found to have the most prospects in Cas9 delivery thus far. By understanding the successes of other systems, translatable, polymer-based delivery vehicles may be developed.},
}
@article {pmid35548699,
year = {2022},
author = {Sturme, MHJ and van der Berg, JP and Bouwman, LMS and De Schrijver, A and de Maagd, RA and Kleter, GA and Battaglia-de Wilde, E},
title = {Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants.},
journal = {ACS agricultural science &amp; technology},
volume = {2},
number = {2},
pages = {192-201},
pmid = {35548699},
issn = {2692-1952},
abstract = {CRISPR-Cas-based genome editing allows for precise and targeted genetic modification of plants. Nevertheless, unintended off-target edits can arise that might confer risks when present in gene-edited food crops. Through an extensive literature review we gathered information on CRISPR-Cas off-target edits in plants. Most observed off-target changes were small insertions or deletions (1-22 bp) or nucleotide substitutions, and large deletions (>100 bp) were rare. One study detected the insertion of vector-derived DNA sequences, which is important considering the risk assessment of gene-edited plants. Off-target sites had few mismatches (1-3 nt) with the target sequence and were mainly located in protein-coding regions, often in target gene homologues. Off-targets edits were predominantly detected via biased analysis of predicted off-target sites instead of unbiased genome-wide analysis. CRISPR-Cas-edited plants showed lower off-target mutation frequencies than conventionally bred plants. This Review can aid discussions on the relevance of evaluating off-target modifications for risk assessment of CRISPR-Cas-edited plants.},
}
@article {pmid35547744,
year = {2022},
author = {Chan, YT and Lu, Y and Wu, J and Zhang, C and Tan, HY and Bian, ZX and Wang, N and Feng, Y},
title = {CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives.},
journal = {Theranostics},
volume = {12},
number = {7},
pages = {3329-3344},
pmid = {35547744},
issn = {1838-7640},
mesh = {*Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; *Neoplasms/drug therapy/genetics ; },
abstract = {CRISPR-Cas9 is a Nobel Prize-winning robust gene-editing tool developed in the last decade. This technique enables a stable genetic engineering method with high precision on the genomes of all organisms. The latest advances in the technology include a genome library screening approach, which can detect survival-essential and drug resistance genes via gain or loss of function. The versatile machinery allows genomic screening for gene activation or inhibition, and targets non-coding sequences, such as promoters, miRNAs, and lncRNAs. In this review, we introduce the emerging high-throughput CRISPR-Cas9 library genome screening technology and its working principles to detect survival and drug resistance genes through positive and negative selection. The technology is compared with other existing approaches while focusing on the advantages of its variable applications in anti-cancer drug discovery, including functions and target identification, non-coding RNA information, actions of small molecules, and drug target discoveries. The combination of the CRISPR-Cas9 system with multi-omic platforms represents a dynamic field expected to advance anti-cancer drug discovery and precision medicine in the clinic.},
}
@article {pmid35545708,
year = {2022},
author = {Tang, L},
title = {Spatial CRISPR screens in tumors.},
journal = {Nature methods},
volume = {19},
number = {5},
pages = {517},
pmid = {35545708},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neoplasms/genetics ; RNA, Guide ; },
}
@article {pmid35544771,
year = {2022},
author = {Liu, W and An, C and Shu, X and Meng, X and Yao, Y and Zhang, J and Chen, F and Xiang, H and Yang, S and Gao, X and Gao, SS},
title = {Correction to "A Dual-Plasmid CRISPR/Cas System for Mycotoxin Elimination in Polykaryotic Industrial Fungi".},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1991},
doi = {10.1021/acssynbio.2c00228},
pmid = {35544771},
issn = {2161-5063},
}
@article {pmid35544322,
year = {2022},
author = {Liang, Y and Xie, J and Zhang, Q and Wang, X and Gou, S and Lin, L and Chen, T and Ge, W and Zhuang, Z and Lian, M and Chen, F and Li, N and Ouyang, Z and Lai, C and Liu, X and Li, L and Ye, Y and Wu, H and Wang, K and Lai, L},
title = {AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.},
journal = {Nucleic acids research},
volume = {50},
number = {9},
pages = {5384-5399},
pmid = {35544322},
issn = {1362-4962},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; INDEL Mutation ; Mammals/genetics ; Mutation ; Uracil-DNA Glycosidase/genetics ; },
abstract = {Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.},
}
@article {pmid35543560,
year = {2022},
author = {Li, Y and Zhang, L and Yang, H and Xia, Y and Liu, L and Chen, X and Shen, W},
title = {Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.},
journal = {Microbiology spectrum},
volume = {10},
number = {3},
pages = {e0005922},
pmid = {35543560},
issn = {2165-0497},
mesh = {CRISPR-Cas Systems ; Candida tropicalis/genetics ; *Gene Editing/methods ; *RNA, Guide/genetics ; RNA, Transfer/genetics ; RNA, Transfer, Gly ; beta Carotene ; },
abstract = {Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNA[Gly] sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNA[Gly], and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNA[Gly] and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.},
}
@article {pmid35538629,
year = {2022},
author = {Bellingrath, JS and McClements, ME and Shanks, M and Clouston, P and Fischer, MD and MacLaren, RE},
title = {Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.},
journal = {Ophthalmic genetics},
volume = {43},
number = {5},
pages = {661-670},
doi = {10.1080/13816810.2022.2073599},
pmid = {35538629},
issn = {1744-5094},
support = {/DH_/Department of Health/United Kingdom ; },
mesh = {Adenine ; Adolescent ; Amino Acid Sequence ; CRISPR-Cas Systems ; Codon, Nonsense ; *Eye Proteins/genetics ; Humans ; Male ; Membrane Proteins/genetics ; Mutation ; Nerve Tissue Proteins/genetics ; Nucleotides ; *Retinal Degeneration/genetics ; },
abstract = {BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms.<br><br>PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing.<br><br>METHODS: Case report.<br><br>RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window.<br><br>CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.},
}
@article {pmid35538076,
year = {2022},
author = {Dong, C and Fu, S and Karvas, RM and Chew, B and Fischer, LA and Xing, X and Harrison, JK and Popli, P and Kommagani, R and Wang, T and Zhang, B and Theunissen, TW},
title = {A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2548},
pmid = {35538076},
issn = {2041-1723},
support = {R35 GM142917/GM/NIGMS NIH HHS/United States ; DP2 GM137418/GM/NIGMS NIH HHS/United States ; UL1 TR000448/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; DNA-Binding Proteins/genetics/metabolism ; Female ; Humans ; *Placenta/metabolism ; Pregnancy ; Protein Tyrosine Phosphatases, Non-Receptor/genetics ; Stem Cells/metabolism ; Transcription Factors/genetics/metabolism ; *Trophoblasts/metabolism ; },
abstract = {The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.},
}
@article {pmid35537849,
year = {2022},
author = {Kořínková, N and Fontana, IM and Nguyen, TD and Pouramini, P and Bergougnoux, V and Hensel, G},
title = {Enhancing cereal productivity by genetic modification of root architecture.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100505},
doi = {10.1002/biot.202100505},
pmid = {35537849},
issn = {1860-7314},
mesh = {Agriculture/methods ; CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Food security is one of the main topics of today's agriculture, primarily due to increasingly challenging environmental conditions. As most of humankind has a daily intake of cereal grains, current breeding programs focus on these crop plants. Customized endonucleases have been included in the breeders' toolbox after successfully demonstrating their use. Due to technological restrictions, the main focus of the new technology was on above-ground plant organs. In contrast, the essential below ground components were given only limited attention. In the present review, the knowledge of the root system architecture in cereals and the role of phytohormones during their establishment is summarized, and the underlying molecular mechanisms are outlined. The review summarizes how the use of CRISPR-based genome editing methodology can improve the root system architecture to enhance crop production genetically. Finally, future research directions involving this knowledge and technical advances are suggested.},
}
@article {pmid35537243,
year = {2022},
author = {Zhang, J and Zhou, T and Shan, Y and Pan, G},
title = {Generation of RYBP FLAG-HA knock-in human embryonic stem cell line through CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102803},
doi = {10.1016/j.scr.2022.102803},
pmid = {35537243},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Embryonic Stem Cells/metabolism ; Homologous Recombination ; *Human Embryonic Stem Cells/metabolism ; Humans ; Mice ; Polycomb-Group Proteins ; Repressor Proteins/metabolism ; },
abstract = {RYBP, a critical component of polycomb repressive complex1 (PRC1), is required for the pluripotency and differentiation of mouse embryonic stem cells(mESCs). However, its function and mechanism to regulate human embryonic stem cells(hESCs) remain unknown. Here, to investigate the role of RYBP in hESCs, we generate an hESC line with FLAG-HA tag knock-in to RYBP locus through CRISPR/Cas9-mediated homologous recombination. hESC with RYBP_FLAG-HA knock-in maintains normal morphology and karyotype, while it maintains pluripotency to differentiate into three germ layers.},
}
@article {pmid35536747,
year = {2022},
author = {Gotoh, Y and Atsuta, Y and Taniguchi, T and Nishida, R and Nakamura, K and Ogura, Y and Misawa, N and Hayashi, T},
title = {Helicobacter cinaedi is a human-adapted lineage in the Helicobacter cinaedi/canicola/'magdeburgensis' complex.},
journal = {Microbial genomics},
volume = {8},
number = {5},
pages = {},
pmid = {35536747},
issn = {2057-5858},
mesh = {Animals ; *Bacteremia ; Cricetinae ; Dogs ; *Helicobacter/genetics ; *Helicobacter Infections ; Humans ; Rats ; },
abstract = {Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.},
}
@article {pmid35536186,
year = {2022},
author = {Wagner, A and Seiler, J and Beye, M},
title = {Highly efficient site-specific integration of DNA fragments into the honeybee genome using CRISPR/Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {6},
pages = {},
pmid = {35536186},
issn = {2160-1836},
mesh = {Animals ; Base Sequence ; Bees/genetics ; *CRISPR-Cas Systems ; DNA ; Gene Editing/methods ; *Genome ; Mutation ; },
abstract = {Functional genetic studies in honeybees have been limited to transposon mediated transformation and site directed mutagenesis tools. However, site- and sequence-specific manipulations that insert DNA fragments or replace sequences at specific target sites are lacking. Such tools would enable the tagging of proteins, the expression of reporters and site-specific amino acid changes, which are all gold standard manipulations for physiological, organismal, and genetic studies. However, such manipulations must be very efficient in honeybees since screening and crossing procedures are laborious due to their social organization. Here, we report an accurate and remarkably efficient site-specific integration of DNA-sequences into the honeybee genome using clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9-mediated homology-directed repair. We employed early embryonic injections and selected a highly efficient sgRNA in order to insert 294 and 729 bp long DNA sequences into a specific locus at the dsx gene. These sequences were locus-specifically integrated in 57% and 59% of injected bees. Most importantly, 21% and 25% of the individuals lacked the wildtype sequence demonstrating that we generated homozygous mutants in which all cells are affected (no mosaicism). The highly efficient, locus-specific insertions of nucleotide sequences generating homozygous mutants demonstrate that systematic molecular studies for honeybees are in hand that allow somatic mutation approaches via workers or studies in the next generation using queens with their worker progeny. The employment of early embryonic injections and screenings of highly efficient sgRNAs may offer the prospect of highly successful sequence- and locus-specific mutations also in other organisms.},
}
@article {pmid35534986,
year = {2022},
author = {Tian, Y and Shen, R and Li, Z and Yao, Q and Zhang, X and Zhong, D and Tan, X and Song, M and Han, H and Zhu, JK and Lu, Y},
title = {Efficient C-to-G editing in rice using an optimized base editor.},
journal = {Plant biotechnology journal},
volume = {20},
number = {7},
pages = {1238-1240},
pmid = {35534986},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Oryza/genetics ; },
}
@article {pmid35534557,
year = {2022},
author = {Eisenstein, M},
title = {Base editing marches on the clinic.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {623-625},
doi = {10.1038/s41587-022-01326-x},
pmid = {35534557},
issn = {1546-1696},
mesh = {Ambulatory Care Facilities ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; },
}
@article {pmid35534475,
year = {2022},
author = {Bishop, AL and López Del Amo, V and Okamoto, EM and Bodai, Z and Komor, AC and Gantz, VM},
title = {Double-tap gene drive uses iterative genome targeting to help overcome resistance alleles.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2595},
pmid = {35534475},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 AI162911/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Germ Cells ; Mammals/genetics ; Mice ; RNA, Guide/genetics ; },
abstract = {Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.},
}
@article {pmid35534455,
year = {2022},
author = {Bodai, Z and Bishop, AL and Gantz, VM and Komor, AC},
title = {Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2351},
pmid = {35534455},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 AI162911/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair ; *Gene Editing/methods ; Mammals/genetics ; *RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.},
}
@article {pmid35533514,
year = {2022},
author = {Xu, Q and Zhao, T and Zhang, Y and Fan, W and Yan, Y and Lan, F},
title = {Generation of a human embryonic stem cell line (WAe009-A-78) carrying homozygous TBX18 knockout.},
journal = {Stem cell research},
volume = {62},
number = {},
pages = {102804},
doi = {10.1016/j.scr.2022.102804},
pmid = {35533514},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells/metabolism ; Female ; Homozygote ; *Human Embryonic Stem Cells/metabolism ; Humans ; Pregnancy ; Sinoatrial Node ; T-Box Domain Proteins/genetics/metabolism ; },
abstract = {T-Box Transcription Factor 18 is a member of the T-box family, encoding TBX18 protein. As a transcriptional repressor, it related to developmental processes of a majority of tissues and organs and plays crucial part in the embryonic development of sinoatrial node. Using an episomal vector-based CRISPR/Cas9 system, we have established a homozygous TBX18 knockout (TBX18-KO) human embryonic stem cell (hESC) line. This newly TBX18[-/-] hESC line display normal pluripotency, morphology, karyotype and trilineage differentiating capacity. This cell line may provide a powerful tool to investigate the role of TBX18 gene in sinoatrial node development in future.},
}
@article {pmid35532260,
year = {2022},
author = {Chambers, C and Quan, L and Yi, G and Esquela-Kerscher, A},
title = {CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {182},
pages = {},
doi = {10.3791/63704},
pmid = {35532260},
issn = {1940-087X},
mesh = {CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing/methods ; Humans ; *MicroRNAs/genetics ; RNA, Guide/genetics ; RNA, Untranslated ; },
abstract = {MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30% of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic. The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment. The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.},
}
@article {pmid35532172,
year = {2022},
author = {Gordon, H and Fellenberg, C and Lackus, ND and Archinuk, F and Sproule, A and Nakamura, Y and K Llner, TG and Gershenzon, J and Overy, DP and Constabel, CP},
title = {CRISPR/Cas9 disruption of UGT71L1 in poplar connects salicinoid and salicylic acid metabolism and alters growth and morphology.},
journal = {The Plant cell},
volume = {34},
number = {8},
pages = {2925-2947},
pmid = {35532172},
issn = {1532-298X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cyclopentanes/metabolism ; Herbivory ; *Moths/genetics/metabolism ; Oxylipins/metabolism ; Plant Leaves/metabolism ; Plants, Genetically Modified/metabolism ; *Populus/genetics/metabolism ; Salicylic Acid/metabolism/pharmacology ; },
abstract = {Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore effects found only in poplars and willows. Their biosynthesis is poorly understood, but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here, we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knockouts contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.},
}
@article {pmid35531207,
year = {2022},
author = {Raza, SHA and Hassanin, AA and Pant, SD and Bing, S and Sitohy, MZ and Abdelnour, SA and Alotaibi, MA and Al-Hazani, TM and Abd El-Aziz, AH and Cheng, G and Zan, L},
title = {Potentials, prospects and applications of genome editing technologies in livestock production.},
journal = {Saudi journal of biological sciences},
volume = {29},
number = {4},
pages = {1928-1935},
pmid = {35531207},
issn = {1319-562X},
abstract = {In recent years, significant progress has been achieved in genome editing applications using new programmable DNA nucleases such as zinc finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs) and the clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9). These genome editing tools are capable of nicking DNA precisely by targeting specific sequences, and enable the addition, removal or substitution of nucleotides via double-stranded breakage at specific genomic loci. CRISPR/Cas system, one of the most recent genome editing tools, affords the ability to efficiently generate multiple genomic nicks in single experiment. Moreover, CRISPR/Cas systems are relatively easy and cost effective when compared to other genome editing technologies. This is in part because CRISPR/Cas systems rely on RNA-DNA binding, unlike other genome editing tools that rely on protein-DNA interactions, which affords CRISPR/Cas systems higher flexibility and more fidelity. Genome editing tools have significantly contributed to different aspects of livestock production such as disease resistance, improved performance, alterations of milk composition, animal welfare and biomedicine. However, despite these contributions and future potential, genome editing technologies also have inherent risks, and therefore, ethics and social acceptance are crucial factors associated with implementation of these technologies. This review emphasizes the impact of genome editing technologies in development of livestock breeding and production in numerous species such as cattle, pigs, sheep and goats. This review also discusses the mechanisms behind genome editing technologies, their potential applications, risks and associated ethics that should be considered in the context of livestock.},
}
@article {pmid35527517,
year = {2022},
author = {Cao, G and Dong, J and Chen, X and Lu, P and Xiong, Y and Peng, L and Li, J and Huo, D and Hou, C},
title = {Simultaneous detection of CaMV35S and T-nos utilizing CRISPR/Cas12a and Cas13a with multiplex-PCR (MPT-Cas12a/13a).},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {43},
pages = {6328-6331},
doi = {10.1039/d2cc01300b},
pmid = {35527517},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural ; *Multiplex Polymerase Chain Reaction ; },
abstract = {Here, we established a strategy (MPT-Cas12a/13a) that combined CRISPR/Cas12a and Cas13a for simultaneously detecting CaMV35S and T-nos based on multiplex PCR (M-PCR) and transcription. It realized a simultaneous detection mode with different signals in the same space. The MPT-Cas12a/13a had excellent sensitivity with the limit of detection as low as 11 copies of T-nos and 13 copies of CaMV35S and it had outstanding specificity and anti-interference ability in actual sample analysis. Therefore, it is a potential candidate in the detection of GM crops.},
}
@article {pmid35527391,
year = {2022},
author = {Nardi, F and Pezzella, L and Drago, R and Di Rita, A and Simoncelli, M and Marotta, G and Gozzetti, A and Bocchia, M and Kabanova, A},
title = {Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells.},
journal = {European journal of immunology},
volume = {52},
number = {8},
pages = {1362-1365},
doi = {10.1002/eji.202149784},
pmid = {35527391},
issn = {1521-4141},
mesh = {*CRISPR-Cas Systems ; Electroporation ; *Gene Editing/methods ; Gene Expression ; Humans ; RNA, Messenger/genetics/metabolism ; },
abstract = {Robust methods for manipulation of human B cells, isolated from healthy donors and patients with B cell disorders, has the potential to significantly accelerate B cell research. Our work describes a step-by-step protocol to perform electroporation-based screening of gene function in B cells through the use of Cas9 ribonuclecomplexes and in vitro produced mRNA.},
}
@article {pmid35526203,
year = {2022},
author = {Liu, L and Chen, Z and Tian, X and Chu, J},
title = {Knockout and functional analysis of BSSS-related genes in Acremonium chrysogenum by novel episomal expression vector containing Cas9 and AMA1.},
journal = {Biotechnology letters},
volume = {44},
number = {5-6},
pages = {755-766},
pmid = {35526203},
issn = {1573-6776},
mesh = {*Acremonium/genetics ; CRISPR-Cas Systems/genetics ; Cdc20 Proteins/metabolism ; Cell Cycle Proteins/genetics ; Cephalosporins/metabolism ; GTP-Binding Proteins/genetics/metabolism ; Gene Editing ; Genes, Fungal ; Membrane Glycoproteins/genetics ; Saccharomyces cerevisiae/genetics ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {OBJECTIVE: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum.<br><br>RESULT: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed.<br><br>CONCLUSION: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.},
}
@article {pmid35525736,
year = {2022},
author = {Wang, B and Chang, M and Zhang, R and Wo, J and Wu, B and Zhang, H and Zhou, Z and Li, Z and Zhang, F and Zhong, C and Tang, S and Yang, S and Sun, G},
title = {Spinal cord injury target-immunotherapy with TNF-α autoregulated and feedback-controlled human umbilical cord mesenchymal stem cell derived exosomes remodelled by CRISPR/Cas9 plasmid.},
journal = {Biomaterials advances},
volume = {133},
number = {},
pages = {112624},
doi = {10.1016/j.msec.2021.112624},
pmid = {35525736},
issn = {2772-9508},
mesh = {CRISPR-Cas Systems ; Cytokines/metabolism ; *Exosomes/metabolism ; Feedback ; Humans ; Immunologic Factors/metabolism ; Immunotherapy ; Inflammation/metabolism ; *Mesenchymal Stem Cells/metabolism ; Plasmids ; *Spinal Cord Injuries/therapy ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/metabolism ; },
abstract = {Human umbilical cord mesenchymal stem cell (hucMSC) derived exosomes (EXOs) have been investigated as a new treatment for spinal cord injury (SCI) because of their anti-inflammatory, anti-apoptotic, angiogenesis-promoting, and axonal regeneration properties. The CAQK peptide found in the brains of mice and humans after trauma has recently been found to specifically bind to the injured site after SCI. Thus, we developed a nanocarrier system called EXO-C@P based on hucMSC exosomes remodelled by the CRISPR/Cas9 plasmid to control inflammation and modified by the CAQK peptide. EXO-C@P was shown to effectively accumulate at the injury site and saturate the macrophages to significantly reduce the expression of inflammatory cytokines in a mouse model of SCI. Moreover, EXO-C@P treatment improved the performance of mice in behavioural assessments and upregulated soluble tumour necrosis factor receptor-1 (sTNFR1) in serum and at the trauma site after SCI surgery, but lowered the proportion of iNOS[+] cells and the concentration of proinflammatory factors. In conclusion, EXO-C@P provides an effective alternative to multiple topical administration and drug delivery approaches for the treatment of SCI. STATEMENT OF SIGNIFICANCE: SCI is a serious disease characterised by a high incidence, high disability rate, and high medical costs, and has become a global medical problem. Several studies have shown that the inflammatory response is the critical inducer of secondary injury after SCI. The inflammatory cytokine TNF-α is considered to be one of the most significant therapeutic targets for autoimmune diseases. Antibodies targeting TNF-α and sTNFR1 are capable of neutralising free TNF-α. In this study, exosomes in the CRISPR/Cas9 system were used to establish stem cells with an autoregulated and feedback-controlled TNF-α response, with these cells secreting sTNFR1, which neutralised TNF-α and antagonised the inflammation stimulated by TNF-α. Moreover, the plasmid was combined with CAQK, which targeted the injury site and promoted the recovery of SCI function.},
}
@article {pmid35525561,
year = {2022},
author = {Cassidy, AM and Kuliyev, E and Thomas, DB and Chen, H and Pelletier, S},
title = {Dissecting protein function in vivo: Engineering allelic series in mice using CRISPR-Cas9 technology.},
journal = {Methods in enzymology},
volume = {667},
number = {},
pages = {775-812},
doi = {10.1016/bs.mie.2022.03.053},
pmid = {35525561},
issn = {1557-7988},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Targeting ; Mice ; Mutagenesis ; Technology ; },
abstract = {Allelic series are extremely valuable genetic tools to study gene function and identify essential structural features of gene products. In mice, allelic series have been engineered using conventional gene targeting in embryonic stem cells or chemical mutagenesis. While these approaches have provided valuable information about the function of genes, they remain cumbersome. Modern approaches such as CRISPR-Cas9 technologies now allow for the precise and cost-effective generation of mouse models with specific mutations, facilitating the development of allelic series. Here, we describe procedures for the generation of three types of mutations used to dissect protein function in vivo using CRISPR-Cas9 technology. This step-by-step protocol describes the generation of missense mutations, large in-frame deletions, and insertions of genetic material using SCY1-like 1 (Scyl1) as a model gene.},
}
@article {pmid35525543,
year = {2022},
author = {Jacobsen, AV and Murphy, JM},
title = {CRISPR deletions in cell lines for reconstitution studies of pseudokinase function.},
journal = {Methods in enzymology},
volume = {667},
number = {},
pages = {229-273},
doi = {10.1016/bs.mie.2022.03.054},
pmid = {35525543},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing/methods ; Humans ; *Protein Kinases/genetics ; },
abstract = {The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.},
}
@article {pmid35524464,
year = {2022},
author = {Přibylová, A and Fischer, L and Pyott, DE and Bassett, A and Molnar, A},
title = {DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner.},
journal = {The New phytologist},
volume = {235},
number = {6},
pages = {2285-2299},
pmid = {35524464},
issn = {1469-8137},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Methylation/genetics ; DNA Repair ; Gene Editing ; Mutation/genetics ; },
abstract = {The impact of epigenetic modifications on the efficacy of CRISPR/Cas9-mediated double-stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants. In this study, we investigated the effect of the level of cytosine methylation on the outcome of CRISPR/Cas9-induced mutations at multiple Cas9 target sites in Nicotiana benthamiana leaf cells using next-generation sequencing. We found that high levels of promoter methylation, but not gene-body methylation, decreased the frequency of Cas9-mediated mutations. DNA methylation also influenced the ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target-specific manner. In addition, we detected an over-representation of deletion events governed by a single 5'-terminal nucleotide at Cas9-induced DNA breaks. Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subsequent DNA repair, probably through changes in the local chromatin structure. In addition to the well described Cas9-induced blunt-end double-stranded DNA breaks, we provide evidence for Cas9-mediated staggered DNA cuts in plant cells. Both types of cut may direct microhomology-mediated DNA repair by a novel, as yet undescribed, mechanism.},
}
@article {pmid35524459,
year = {2022},
author = {Wu, Y and Ren, Q and Zhong, Z and Liu, G and Han, Y and Bao, Y and Liu, L and Xiang, S and Liu, S and Tang, X and Zhou, J and Zheng, X and Sretenovic, S and Zhang, T and Qi, Y and Zhang, Y},
title = {Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice.},
journal = {Plant biotechnology journal},
volume = {20},
number = {9},
pages = {1670-1682},
pmid = {35524459},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Gene Editing/methods ; Genome-Wide Association Study ; *Oryza/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; },
abstract = {PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.},
}
@article {pmid35524183,
year = {2022},
author = {Zhou, S and Kalds, P and Luo, Q and Sun, K and Zhao, X and Gao, Y and Cai, B and Huang, S and Kou, Q and Petersen, B and Chen, Y and Ma, B and Wang, X},
title = {Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {348},
pmid = {35524183},
issn = {1471-2164},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; Goats/genetics ; Meat ; *Myostatin/genetics ; RNA, Guide/genetics ; RNA, Messenger ; Sheep/genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner.<br><br>RESULTS: We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency >&#x2009;50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100&#x2009;ng/&#x3bc;L Cas9 mRNA and 200&#x2009;ng/&#x3bc;L sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs.<br><br>CONCLUSIONS: This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.},
}
@article {pmid35524126,
year = {2022},
author = {Després, PC and Dubé, AK and Yachie, N and Landry, CR},
title = {High-Throughput Gene Mutagenesis Screening Using Base Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2477},
number = {},
pages = {331-348},
pmid = {35524126},
issn = {1940-6029},
support = {387697//CIHR/Canada ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutagenesis/genetics ; RNA, Guide/genetics ; },
abstract = {Base editing is a CRISPR-Cas9 genome engineering tool that allows programmable mutagenesis without the creation of double-stranded breaks. Here, we describe the design and execution of large-scale base editing screens using the Target-AID base editor in yeast. Using this approach, thousands of sites can be mutated simultaneously. The effects of these mutations on fitness can be measured using a pooled growth competition assay followed by DNA sequencing of gRNAs as barcodes.},
}
@article {pmid35524121,
year = {2022},
author = {Dubé, AK and Dandage, R and Dibyachintan, S and Dionne, U and Després, PC and Landry, CR},
title = {Deep Mutational Scanning of Protein-Protein Interactions Between Partners Expressed from Their Endogenous Loci In Vivo.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2477},
number = {},
pages = {237-259},
pmid = {35524121},
issn = {1940-6029},
support = {387697//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Point Mutation ; },
abstract = {Deep mutational scanning (DMS) generates mutants of a protein of interest in a comprehensive manner. CRISPR-Cas9 technology enables large-scale genome editing with high efficiency. Using both DMS and CRISPR-Cas9 therefore allows us to investigate the effects of thousands of mutations inserted directly in the genome. Combined with protein-fragment complementation assay (PCA), which enables the quantitative measurement of protein-protein interactions (PPIs) in vivo, these methods allow for the systematic assessment of the effects of mutations on PPIs in living cells. Here, we describe a method leveraging DMS, CRISPR-Cas9, and PCA to study the effect of point mutations on PPIs mediated by protein domains in yeast.},
}
@article {pmid35524052,
year = {2022},
author = {Chhun, A and Alberti, F},
title = {CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2489},
number = {},
pages = {201-222},
pmid = {35524052},
issn = {1940-6029},
mesh = {*Actinobacteria/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Streptomyces/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene.},
}
@article {pmid35524051,
year = {2022},
author = {Massicard, JM and Su, L and Jacob, C and Weissman, KJ},
title = {Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2489},
number = {},
pages = {173-200},
pmid = {35524051},
issn = {1940-6029},
mesh = {Animals ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Polyketides/metabolism ; RNA, Guide/genetics/metabolism ; *Streptomyces/genetics/metabolism ; },
abstract = {The CRISPR/Cas system, which has been widely applied to organisms ranging from microbes to animals, is currently being adapted for use in Streptomyces bacteria. In this case, it is notably applied to rationally modify the biosynthetic pathways giving rise to the polyketide natural products, which are heavily exploited in the medical and agricultural arenas. Our aim here is to provide the potential user with a practical guide to exploit this approach for manipulating polyketide biosynthesis, by treating key experimental aspects including vector choice, design of the basic engineering components, and trouble-shooting.},
}
@article {pmid35524023,
year = {2022},
author = {Nguyen, TTT and Tamai, M and Harama, D and Kagami, K and Kasai, S and Watanabe, A and Akahane, K and Goi, K and Inukai, T},
title = {Introduction of the T315I gatekeeper mutation of BCR/ABL1 into a Philadelphia chromosome-positive lymphoid leukemia cell line using the CRISPR/Cas9 system.},
journal = {International journal of hematology},
volume = {116},
number = {4},
pages = {534-543},
pmid = {35524023},
issn = {1865-3774},
mesh = {*Antineoplastic Agents/therapeutic use ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Dasatinib/therapeutic use ; Drug Resistance, Neoplasm/genetics ; Fusion Proteins, bcr-abl ; Humans ; Imatinib Mesylate/pharmacology/therapeutic use ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy ; Mutation ; Nucleotides/therapeutic use ; Oligodeoxyribonucleotides/therapeutic use ; Philadelphia Chromosome ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/genetics ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; RNA, Guide/therapeutic use ; },
abstract = {Imatinib and second-generation tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, overcoming TKI resistance due to the T315I gatekeeper mutation of BCR/ABL1 is crucial for further improving the prognosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is appropriate for establishing a human model of Ph+ ALL with the T315I mutation, because it can induce specific mutations via homologous recombination (HR) repair in cells with intact endogenous HR pathway. Here we used CRISPR/Cas9 to introduce the T315I mutation into the Ph+ lymphoid leukemia cell line KOPN55bi, which appeared to have an active HR pathway based on its resistance to a poly (ADP-Ribose) polymerase-1 inhibitor. Single-guide RNA targeting at codon 315 and single-strand oligodeoxynucleotide containing ACT to ATT nucleotide transition at codon 315 were electroporated with recombinant Cas9 protein. Dasatinib-resistant sublines were obtained after one-month selection with the therapeutic concentration of dasatinib, leading to T315I mutation acquisition through HR. T315I-acquired sublines were highly resistant to imatinib and second-generation TKIs but moderately sensitive to the therapeutic concentration of ponatinib. This authentic human model is helpful for developing new therapeutic strategies overcoming TKI resistance in Ph+ ALL due to T315I mutation.},
}
@article {pmid35523179,
year = {2022},
author = {Breau, KA and Ok, MT and Gomez-Martinez, I and Burclaff, J and Kohn, NP and Magness, ST},
title = {Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells.},
journal = {Stem cell reports},
volume = {17},
number = {6},
pages = {1493-1506},
pmid = {35523179},
issn = {2213-6711},
support = {R01 DK115806/DK/NIDDK NIH HHS/United States ; P30 DK034987/DK/NIDDK NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; R43 DK125155/DK/NIDDK NIH HHS/United States ; T32 DK007737/DK/NIDDK NIH HHS/United States ; F32 DK124929/DK/NIDDK NIH HHS/United States ; T32 GM133364/GM/NIGMS NIH HHS/United States ; F30 DK126307/DK/NIDDK NIH HHS/United States ; R01 DK109559/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Targeting ; Humans ; Intestine, Small ; Organoids ; Stem Cells ; Transfection ; },
abstract = {Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.},
}
@article {pmid35522691,
year = {2022},
author = {Matozel, EK and Parziale, S and Price, AC},
title = {A programmable DNA roadblock system using dCas9 and multivalent target sites.},
journal = {PloS one},
volume = {17},
number = {5},
pages = {e0268099},
pmid = {35522691},
issn = {1932-6203},
mesh = {Binding Sites ; CRISPR-Cas Systems ; *DNA/metabolism ; DNA-Binding Proteins/genetics ; *Endonucleases/metabolism ; RNA, Guide/genetics ; },
abstract = {A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.},
}
@article {pmid35521548,
year = {2022},
author = {Shor, O and Rabinowitz, R and Offen, D and Benninger, F},
title = {Computational normal mode analysis accurately replicates the activity and specificity profiles of CRISPR-Cas9 and high-fidelity variants.},
journal = {Computational and structural biotechnology journal},
volume = {20},
number = {},
pages = {2013-2019},
pmid = {35521548},
issn = {2001-0370},
abstract = {The CRISPR-Cas system has transformed the field of gene-editing and created opportunities for novel genome engineering therapeutics. The field has significantly progressed, and recently, CRISPR-Cas9 was utilized in clinical trials to target disease-causing mutations. Existing tools aim to predict the on-target efficacy and potential genome-wide off-targets by scoring a particular gRNA according to an array of gRNA design principles or machine learning algorithms based on empirical results of large numbers of gRNAs. However, such tools are unable to predict the editing outcome by variant Cas enzymes and can only assess potential off-targets related to reference genomes. Here, we employ normal mode analysis (NMA) to investigate the structure of the Cas9 protein complexed with its gRNA and target DNA and explore the function of the protein. Our results demonstrate the feasibility and validity of NMA to predict the activity and specificity of SpyCas9 in the presence of mismatches by comparison to empirical data. Furthermore, despite the absence of their exact structures, this method accurately predicts the enzymatic activity of known high-fidelity engineered Cas9 variants.},
}
@article {pmid35513429,
year = {2022},
author = {Li, R and Klingbeil, O and Monducci, D and Young, MJ and Rodriguez, DJ and Bayyat, Z and Dempster, JM and Kesar, D and Yang, X and Zamanighomi, M and Vakoc, CR and Ito, T and Sellers, WR},
title = {Comparative optimization of combinatorial CRISPR screens.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2469},
pmid = {35513429},
issn = {2041-1723},
mesh = {*Acidaminococcus/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide/genetics ; Staphylococcus aureus/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.},
}
@article {pmid35512461,
year = {2022},
author = {Song, G and Li, X and Wang, Z and Dong, C and Xie, X and Yan, X},
title = {Structure of AcrVIA2 and its binding mechanism to CRISPR-Cas13a.},
journal = {Biochemical and biophysical research communications},
volume = {612},
number = {},
pages = {84-90},
doi = {10.1016/j.bbrc.2022.04.091},
pmid = {35512461},
issn = {1090-2104},
mesh = {Bacteria/metabolism ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; *RNA, Guide/genetics ; },
abstract = {Phages and non-phage derived bacteria have evolved many anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes. Thus Acrs can be applied as a regulatory tool for gene edition by CRISPR system. Recently, a non-phage derived AcrVIA2 has been identified as an inhibitor that blocks the editing activity of Cas13a in vitro by binding to Cas13a. Here, we solved the crystal structure of AcrVIA2 at a resolution of 2.59 Å and confirmed that AcrVIA2 can bind to Helical-I domain in LshCas13a. Structural analysis show that the V-shaped acidic groove formed by β3-β3 hairpin of AcrVIA2 dimer is the key region that mediates the interaction between AcrVIA2 and Helical-I domain. In addition, we also reveal that Asp37 of AcrVIA2 plays an essential role in the functioning of the V-shaped acidic groove, and the functional dimer conformation of AcrVIA2 is stabilized by hydrogen bonds formed between Tyr41 of one monomer with Glu35 and Asp37 of the other monomer. These data expand the current understanding of the diverse interaction mechanisms between Acrs and Cas proteins, and also provide new ideas for the development of CRISPR-Cas13a regulatory tool.},
}
@article {pmid35512092,
year = {2022},
author = {Taylor, JH and Walton, JC and McCann, KE and Norvelle, A and Liu, Q and Vander Velden, JW and Borland, JM and Hart, M and Jin, C and Huhman, KL and Cox, DN and Albers, HE},
title = {CRISPR-Cas9 editing of the arginine-vasopressin V1a receptor produces paradoxical changes in social behavior in Syrian hamsters.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {19},
pages = {e2121037119},
pmid = {35512092},
issn = {1091-6490},
support = {R01 MH122622/MH/NIMH NIH HHS/United States ; },
mesh = {Aggression/physiology ; Animals ; Arginine/metabolism ; Arginine Vasopressin/genetics ; *CRISPR-Cas Systems ; Cricetinae ; Mesocricetus ; *Receptors, Vasopressin/genetics/metabolism ; Social Behavior ; },
abstract = {Studies from a variety of species indicate that arginine–vasopressin (AVP) and its V1a receptor (Avpr1a) play a critical role in the regulation of a range of social behaviors by their actions in the social behavior neural network. To further investigate the role of AVPRs in social behavior, we performed CRISPR-Cas9–mediated editing at the Avpr1a gene via pronuclear microinjections in Syrian hamsters (Mesocricetus auratus), a species used extensively in behavioral neuroendocrinology because they produce a rich suite of social behaviors. Using this germ-line gene-editing approach, we generated a stable line of hamsters with a frame-shift mutation in the Avpr1a gene resulting in the null expression of functional Avpr1as. Avpr1a knockout (KO) hamsters exhibited a complete lack of Avpr1a-specific autoradiographic binding throughout the brain, behavioral insensitivity to centrally administered AVP, and no pressor response to a peripherally injected Avpr1a-specific agonist, thus confirming the absence of functional Avpr1as in the brain and periphery. Contradictory to expectations, Avpr1a KO hamsters exhibited substantially higher levels of conspecific social communication (i.e., odor-stimulated flank marking) than their wild-type (WT) littermates. Furthermore, sex differences in aggression were absent, as both male and female KOs exhibited more aggression toward same-sex conspecifics than did their WT littermates. Taken together, these data emphasize the importance of comparative studies employing gene-editing approaches and suggest the startling possibility that Avpr1a-specific modulation of the social behavior neural network may be more inhibitory than permissive.},
}
@article {pmid35510853,
year = {2022},
author = {Liang, X and Boonhok, R and Siddiqui, FA and Xiao, B and Li, X and Qin, J and Min, H and Jiang, L and Cui, L and Miao, J},
title = {A Leak-Free Inducible CRISPRi/a System for Gene Functional Studies in Plasmodium falciparum.},
journal = {Microbiology spectrum},
volume = {10},
number = {3},
pages = {e0278221},
pmid = {35510853},
issn = {2165-0497},
support = {R21 AI149202/AI/NIAID NIH HHS/United States ; },
mesh = {*Antimalarials ; *Artemisinins ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Gene Expression Regulation ; Plasmodium falciparum/genetics ; },
abstract = {By fusing catalytically dead Cas9 (dCas9) to active domains of histone deacetylase (Sir2a) or acetyltransferase (GCN5), this CRISPR interference/activation (CRISPRi/a) system allows gene regulation at the transcriptional level without causing permanent changes in the parasite genome. However, the constitutive expression of dCas9 poses a challenge for studying essential genes, which may lead to adaptive changes in the parasite, masking the true phenotypes. Here, we developed a leak-free inducible CRISPRi/a system by integrating the DiCre/loxP regulon to allow the expression of dCas9-GCN5/-Sir2a upon transient induction with rapamycin, which allows convenient transcriptional regulation of a gene of interest by introducing a guide RNA targeting its transcription start region. Using eight genes that are either silent or expressed from low to high levels during asexual erythrocytic development, we evaluated the robustness and versatility of this system in the asexual parasites. For most genes analyzed, this inducible CRISPRi/a system led to 1.5- to 3-fold up-or downregulation of the target genes at the mRNA level. Alteration in the expression of PfK13 and PfMYST resulted in altered sensitivities to artemisinin. For autophagy-related protein 18, an essential gene related to artemisinin resistance, a >2-fold up- or downregulation was obtained by inducible CRISPRi/a, leading to growth retardation. For the master regulator of gametocytogenesis, PfAP2-G, a >10-fold increase of the PfAP2-G transcripts was obtained by CRISPRa, resulting in >4-fold higher gametocytemia in the induced parasites. Additionally, inducible CRISPRi/a could also regulate gene expression in gametocytes. This inducible epigenetic regulation system offers a fast way of studying gene functions in Plasmodium falciparum. IMPORTANCE Understanding the fundamental biology of malaria parasites through functional genetic/genomic studies is critical for identifying novel targets for antimalarial development. Conditional knockout/knockdown systems are required to study essential genes in the haploid blood stages of the parasite. In this study, we developed an inducible CRISPRi/a system via the integration of DiCre/loxP. We evaluated the robustness and versatility of this system by activating or repressing eight selected genes and achieved up- and downregulation of the targeted genes located in both the euchromatin and heterochromatin regions. This system offers the malaria research community another tool for functional genetic studies.},
}
@article {pmid35510603,
year = {2022},
author = {Li, Y and Zeng, R and Wang, W and Xu, J and Gong, H and Li, L and Li, M and Tang, D},
title = {Size-Controlled Engineering Photoelectrochemical Biosensor for Human Papillomavirus-16 Based on CRISPR-Cas12a-Induced Disassembly of Z-Scheme Heterojunctions.},
journal = {ACS sensors},
volume = {7},
number = {5},
pages = {1593-1601},
doi = {10.1021/acssensors.2c00691},
pmid = {35510603},
issn = {2379-3694},
mesh = {*Alphapapillomavirus ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/chemistry ; DNA, Single-Stranded ; Electrochemical Techniques/methods ; Human papillomavirus 16/genetics ; Humans ; },
abstract = {Photoelectrochemical (PEC) biosensors incorporating biomolecular recognition with photon-to-electron conversion capabilities of the photoactive species have been developed for molecular diagnosis, but most involve difficulty in adjusting band gap positions and are unsuitable for PEC biodetection. In this work, an innovative PEC biosensor combined with quantum size-controlled engineering based on quantum confinement by controlling the quantum size was designed for the detection of human papillomavirus-16 (HPV-16) through CRISPR-Cas12a (Cpf1)-induced disassembly of Z-scheme heterojunction. To the best of our knowledge, quantum size-controlled engineering that precisely tunes the properties of photoactive materials is first utilized in the PEC bioanalysis. Based on the quantum size effect, the light absorption efficiency and charge-transfer rate were tuned to suitable levels to obtain the best PEC performance. After incubation with target HPV-16, the binding of Cas12a-crRNA to the target double-stranded DNA (dsDNA) stimulated the activity of indiscriminate cleavage toward single-stranded DNA (ssDNA), resulting in a decrease in photocurrent due to the blocking of electron transfer through the heterojunction. By optimizing experimental conditions, the Z-scheme sensing system exhibited incredible photocurrent response to HPV-16 in the range from 3.0 pM to 600 nM with a detection limit of 1.0 pM. Impressively, the application of the quantum size effect could stimulate more interest in the precise design of band gap structure to improve PEC performance.},
}
@article {pmid35509467,
year = {2022},
author = {Wang, DX and Wang, YX and Wang, J and Ma, JY and Liu, B and Tang, AN and Kong, DM},
title = {MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a.},
journal = {Chemical science},
volume = {13},
number = {15},
pages = {4364-4371},
pmid = {35509467},
issn = {2041-6520},
abstract = {Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO2 nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO2 nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO2 nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn[2+] as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO2 nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO2 nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.},
}
@article {pmid35509363,
year = {2022},
author = {Ebrahimi, S and Khanbabaei, H and Abbasi, S and Fani, M and Soltani, S and Zandi, M and Najafimemar, Z},
title = {CRISPR-Cas System: A Promising Diagnostic Tool for Covid-19.},
journal = {Avicenna journal of medical biotechnology},
volume = {14},
number = {1},
pages = {3-9},
pmid = {35509363},
issn = {2008-2835},
abstract = {More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people's life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19.},
}
@article {pmid35508982,
year = {2022},
author = {Shojaei Baghini, S and Gardanova, ZR and Abadi, SAH and Zaman, BA and İlhan, A and Shomali, N and Adili, A and Moghaddar, R and Yaseri, AF},
title = {CRISPR/Cas9 application in cancer therapy: a pioneering genome editing tool.},
journal = {Cellular &amp; molecular biology letters},
volume = {27},
number = {1},
pages = {35},
pmid = {35508982},
issn = {1689-1392},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {The progress of genetic engineering in the 1970s brought about a paradigm shift in genome editing technology. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a flexible means to target and modify particular DNA sequences in the genome. Several applications of CRISPR/Cas9 are presently being studied in cancer biology and oncology to provide vigorous site-specific gene editing to enhance its biological and clinical uses. CRISPR's flexibility and ease of use have enabled the prompt achievement of almost any preferred alteration with greater efficiency and lower cost than preceding modalities. Also, CRISPR/Cas9 technology has recently been applied to improve the safety and efficacy of chimeric antigen receptor (CAR)-T cell therapies and defeat tumor cell resistance to conventional treatments such as chemotherapy and radiotherapy. The current review summarizes the application of CRISPR/Cas9 in cancer therapy. We also discuss the present obstacles and contemplate future possibilities in this context.},
}
@article {pmid35508977,
year = {2022},
author = {Trung, NT and Son, LHP and Hien, TX and Quyen, DT and Bang, MH and Song, LH},
title = {CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis.},
journal = {BMC infectious diseases},
volume = {22},
number = {1},
pages = {429},
pmid = {35508977},
issn = {1471-2334},
mesh = {*CRISPR-Cas Systems ; DNA ; Humans ; Molecular Diagnostic Techniques ; *Neisseria meningitidis/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA ; },
abstract = {BACKGROUND: Loop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen's DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.<br><br>METHODS: The MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14&#xa0;N. meningitidis confirmed patients and 37 control subjects).<br><br>RESULTS: In relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2&#xa0;h.<br><br>CONCLUSIONS: In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.},
}
@article {pmid35508460,
year = {2022},
author = {Mac Kain, A and Maarifi, G and Aicher, SM and Arhel, N and Baidaliuk, A and Munier, S and Donati, F and Vallet, T and Tran, QD and Hardy, A and Chazal, M and Porrot, F and OhAinle, M and Carlson-Stevermer, J and Oki, J and Holden, K and Zimmer, G and Simon-Lorière, E and Bruel, T and Schwartz, O and van der Werf, S and Jouvenet, N and Nisole, S and Vignuzzi, M and Roesch, F},
title = {Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2442},
pmid = {35508460},
issn = {2041-1723},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Co-Repressor Proteins/genetics/metabolism ; Humans ; Interferons/metabolism ; Molecular Chaperones/genetics/metabolism ; Proteasome Endopeptidase Complex/metabolism ; *SARS-CoV-2 ; },
abstract = {Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.},
}
@article {pmid35507186,
year = {2022},
author = {Desjardins, J and Cowan, M and Yamanaka, Y},
title = {Designing Genetically Engineered Mouse Models (GEMMs) Using CRISPR Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {515-531},
pmid = {35507186},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome/genetics ; Mammals/genetics ; Mice ; RNA, Guide/genetics ; Zygote ; },
abstract = {Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.},
}
@article {pmid35507174,
year = {2022},
author = {Ford, MJ and Yamanaka, Y},
title = {Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipulation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {367-377},
pmid = {35507174},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Electroporation/methods ; Epithelial Cells ; Fallopian Tubes ; Female ; *Gene Editing/methods ; Humans ; Mice ; },
abstract = {Advances in gene editing tools such as CRISPR/Cas9 have made precise in vivo gene editing possible, opening up avenues of research into somatic cell reprograming to study adult stem cells, homeostasis, and malignant transformation. Here we describe a method for CRISPR/Cas9 mediated in vivo gene editing, in combination with Cre-based lineage tracing via electroporation in the mouse oviduct. This method facilitates the delivery of multiple plasmids into oviduct epithelial cells, sufficient for studying homeostasis and generation of high-grade serous ovarian cancer (HGSOC) models.},
}
@article {pmid35507170,
year = {2022},
author = {Devaraju, N and Rajendiran, V and Ravi, NS and Mohankumar, KM},
title = {Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {307-331},
pmid = {35507170},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Mice ; Transplantation, Autologous ; },
abstract = {Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.},
}
@article {pmid35507169,
year = {2022},
author = {Park, SH and Lee, CM and Bao, G},
title = {Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2429},
number = {},
pages = {281-306},
pmid = {35507169},
issn = {1940-6029},
support = {UG3 HL151545/HL/NHLBI NIH HHS/United States ; R01 HL152314/HL/NHLBI NIH HHS/United States ; OT2 HL154977/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Hematopoietic Stem Cells/metabolism ; High-Throughput Nucleotide Sequencing ; *RNA, Guide/genetics/metabolism ; },
abstract = {Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.},
}
@article {pmid35506993,
year = {2022},
author = {Hong, J and Meng, Z and Zhang, Z and Su, H and Fan, Y and Huang, R and Ding, R and Zhang, N and Li, F and Wang, S},
title = {Comprehensive Analysis of CRISPR-Cas9 Editing Outcomes in Yeast Xanthophyllomyces dendrorhous.},
journal = {The CRISPR journal},
volume = {5},
number = {4},
pages = {558-570},
doi = {10.1089/crispr.2021.0116},
pmid = {35506993},
issn = {2573-1602},
mesh = {Basidiomycota ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HeLa Cells ; Humans ; Nucleotides ; Saccharomyces cerevisiae/genetics ; },
abstract = {DNA repair after Cas9 cutting can result in deletions/insertions, genomic rearrangements, and rare nucleotide substitutions. However, most work has only focused on deletions/insertions resulting from repair after CRISPR-Cas9 action. Here, we comprehensively analyzed the editing outcomes induced by CRISPR-Cas9 treatment in yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing and identified diverse DNA repair patterns, including DNA deletions, interchromosomal translocations, and on-target nucleotide substitutions (point mutations). Some deletions were observed repeatedly, and others, especially large deletions, varied in size. Genome sequencing and structural variation analysis showed that the interchromosomal translocations happened between Cas9 target sites and the endogenous ADH4 promoter. In contrast to previous studies, analysis revealed that the on-target point mutations were not random. Importantly, these point mutations showed strong sequence dependence that is not consistent with previous work in Hela cells, where CRISPR-mediated substitutions were found to lack sequence dependence and conversion preferences. Finally, we found that the non-homologous end joining components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the production of both point mutations and deletions. This work expands our knowledge of CRISPR-Cas9 mediated DNA repair.},
}
@article {pmid35506982,
year = {2022},
author = {Pulman, J and Sahel, JA and Dalkara, D},
title = {New Editing Tools for Gene Therapy in Inherited Retinal Dystrophies.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {377-388},
pmid = {35506982},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Humans ; *Retinal Dystrophies/genetics/therapy ; },
abstract = {Inherited retinal dystrophies (IRDs) are a heterogeneous group of diseases that affect more than 2 million people worldwide. Gene therapy (GT) has emerged as an exciting treatment modality with the potential to provide long-term benefit to patients. Today, gene addition is the most straightforward GT for autosomal recessive IRDs. However, there are three scenarios where this approach falls short. First, in autosomal dominant diseases caused by gain-of-function or dominant-negative mutations, the toxic mutated protein needs to be silenced. Second, a number of IRD genes exceed the limited carrying capacity of adeno-associated virus vectors. Third, there are still about 30% of patients with unknown mutations. In the first two contexts, precise editing tools, such as CRISPR-Cas9, base editors, or prime editors, are emerging as potential GT solutions for the treatment of IRDs. Here, we review gene editing tools based on CRISPR-Cas9 technology that have been used in vivo and the recent first-in-human application of CRISPR-Cas9 in an IRD.},
}
@article {pmid35506667,
year = {2022},
author = {Kever, L and Hardy, A and Luthe, T and Hünnefeld, M and Gätgens, C and Milke, L and Wiechert, J and Wittmann, J and Moraru, C and Marienhagen, J and Frunzke, J},
title = {Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.},
journal = {mBio},
volume = {13},
number = {3},
pages = {e0078322},
pmid = {35506667},
issn = {2150-7511},
mesh = {Aminoglycosides/pharmacology ; Anti-Bacterial Agents/pharmacology ; Antiviral Agents/pharmacology ; *Bacteriophages/genetics ; Escherichia coli ; },
abstract = {In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.},
}
@article {pmid35506451,
year = {2022},
author = {Kalafati, E and Papanikolaou, E and Marinos, E and Anagnou, NP and Pappa, KI},
title = {Mimiviruses: Giant viruses with novel and intriguing features (Review).},
journal = {Molecular medicine reports},
volume = {25},
number = {6},
pages = {},
pmid = {35506451},
issn = {1791-3004},
mesh = {*Amoeba ; CRISPR-Cas Systems ; Capsid ; *Giant Viruses/genetics ; Humans ; *Mimiviridae/genetics ; },
abstract = {The Mimivirus is a giant virus that infects amoebae and was long considered to be a bacterium due to its size. The viral particles are composed of a protein capsid of ~500 nm in diameter, which is enclosed in a polysaccharide layer in which ~120‑140 nm long fibers are embedded, resulting in an overall diameter of 700 nm. The virus has a genome size of 1.2 Mb DNA, and surprisingly, replicates only in the cytoplasm of the infected cells without entering the nucleus, which is a unique characteristic among DNA viruses. Their existence is undeniable; however, as with any novel discovery, there is still uncertainty concerning their pathogenicity mechanisms in humans and the nature of the Mimivirus virophage resistance element system (MIMIVIRE), a term given to describe the immune network of the Mimivirus, which closely resembles the CRISPR‑Cas system. The scope of the present review is to discuss the recent developments derived from structural and functional studies performed on the distinctive characteristics of the Mimivirus, and from studies concerning their putative clinical relevance in humans.},
}
@article {pmid35504953,
year = {2022},
author = {Warmt, C and Yaslanmaz, C and Henkel, J},
title = {Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {7137},
pmid = {35504953},
issn = {2045-2322},
mesh = {DNA Primers ; Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques/methods ; *Nucleotides ; Spectrometry, Fluorescence ; },
abstract = {Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.},
}
@article {pmid35504940,
year = {2022},
author = {Sommerkamp, P and Sommerkamp, AC and Zeisberger, P and Eiben, PL and Narr, A and Korkmaz, A and Przybylla, A and Sohn, M and van der Hoeven, F and Schönig, K and Trumpp, A},
title = {CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {7181},
pmid = {35504940},
issn = {2045-2322},
mesh = {3' Untranslated Regions ; Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Hematopoietic Stem Cells/metabolism ; Mice ; *Poly(A)-Binding Protein I/metabolism ; Polyadenylation ; RNA, Messenger/genetics ; },
abstract = {Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1[flox/flox] mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1[flox/flox] mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.},
}
@article {pmid35503156,
year = {2022},
author = {Zhu, H and Liu, S and He, W and Sun, F and Li, Y and Yang, P and Yu, Q and Zhang, S},
title = {One-Step Genotyping Method in loxP-Based Conditional Knockout Mice Generated by CRISPR-Cas9 Technology.},
journal = {Molecular biotechnology},
volume = {64},
number = {11},
pages = {1227-1233},
pmid = {35503156},
issn = {1559-0305},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genotype ; Mice ; Mice, Knockout ; Technology ; },
abstract = {With the development of CRISPR-Cas9 gene editing and in vitro fertilization (IVF) technology, we can now easily construct genetically modified mouse strains with indels, especially for loxP-based strategy. However, the general genotyping methods are time-consuming and unreliable given the loxP site is only 34 bp long. Here, based on the tetra primer-paired PCR amplification, we describe an efficient genotyping method which can simultaneously generate the internal control band, wild type (wt)-genotype band, and/or loxP-genotype band through one single PCR amplification. It is easy to interpret the mouse genotypes from the pattern of the bands. Further, the results could also help to exclude the possibility of minor cross-contamination, since the ratio between the bands' quantity in wt/wt, wt/loxP, and loxP/loxP mice are relatively constant, which makes the genotyping more reliable when it is performed in a large amount.},
}
@article {pmid35502187,
year = {2022},
author = {Espinoza-Mellado, MDR and Vilchis-Rangel, RE},
title = {Review of CRISPR-Cas Systems in Listeria Species: Current Knowledge and Perspectives.},
journal = {International journal of microbiology},
volume = {2022},
number = {},
pages = {9829770},
pmid = {35502187},
issn = {1687-918X},
abstract = {Listeria spp. are pathogens widely distributed in the environment and Listeria monocytogenes is associated with food-borne illness in humans. Food facilities represent an adverse environment for this bacterium, mainly due to the disinfection and cleaning processes included in good hygiene practices, and its virulence is related to stress responses. One of the recently described stress-response systems is CRISPR-Cas. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes have been found in several bacteria. CRISPR-Cas has revolutionized biotechnology since it acts as an adaptive immune system of bacteria, which also helps in the evasion of the host immune response. There are three CRISPR systems described on Listeria species. Type II is present in many pathogenic bacteria and characterized by the presence of cas9 that becomes the main target of some anti-CRISPR proteins, such as AcrIIA1, encoded on Listeria phages. The presence of Cas9, either alone or in combination with anti-CRISPR proteins, suggests having a main role on the virulence of bacteria. In this review, we describe the most recent information on CRISPR-Cas systems in Listeria spp., particularly in L. monocytogenes, and their relationship with the virulence and pathogenicity of those bacteria. Besides, some applications of CRISPR systems and future challenges in the food processing industry, bacterial vaccination, antimicrobial resistance, pathogens biocontrol by phage therapy, and regulation of gene expression have been explored.},
}
@article {pmid35500431,
year = {2022},
author = {Navarro-Serna, S and Dehesa-Etxebeste, M and Piñeiro-Silva, C and Romar, R and Lopes, JS and López de Munaín, A and Gadea, J},
title = {Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination.},
journal = {Theriogenology},
volume = {186},
number = {},
pages = {175-184},
doi = {10.1016/j.theriogenology.2022.04.012},
pmid = {35500431},
issn = {1879-3231},
mesh = {Animals ; *CRISPR-Cas Systems ; *Calpain/genetics ; Electroporation/methods/veterinary ; Gene Editing/methods/veterinary ; Insemination ; Microinjections/veterinary ; Oocytes ; Swine/genetics ; },
abstract = {Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs.},
}
@article {pmid35500195,
year = {2022},
author = {Zhao, M and Gao, M and Xiong, L and Liu, Y and Tao, X and Gao, B and Liu, M and Wang, FQ and Wei, DZ},
title = {CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1958-1970},
doi = {10.1021/acssynbio.2c00112},
pmid = {35500195},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Engineering ; Mutagenesis ; RNA, Guide/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; beta Carotene/metabolism ; },
abstract = {Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.},
}
@article {pmid35499084,
year = {2022},
author = {Caruso, SM and Quinn, PM and da Costa, BL and Tsang, SH},
title = {CRISPR/Cas therapeutic strategies for autosomal dominant disorders.},
journal = {The Journal of clinical investigation},
volume = {132},
number = {9},
pages = {},
pmid = {35499084},
issn = {1558-8238},
support = {R21 AG050437/AG/NIA NIH HHS/United States ; R01 EY018213/EY/NEI NIH HHS/United States ; U01 EY030580/EY/NEI NIH HHS/United States ; U54 OD020351/OD/NIH HHS/United States ; R01 EY009076/EY/NEI NIH HHS/United States ; R01 EY026682/EY/NEI NIH HHS/United States ; R24 EY027285/EY/NEI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R24 EY028758/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; P30 EY019007/EY/NEI NIH HHS/United States ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; Mutation ; },
abstract = {Autosomal dominant disorders present unique challenges, as therapeutics must often distinguish between healthy and diseased alleles while maintaining high efficiency, specificity, and safety. For this task, CRISPR/Cas remains particularly promising. Various CRISPR/Cas systems, like homology-directed repair, base editors, and prime editors, have been demonstrated to selectively edit mutant alleles either by incorporating these mutations into sgRNA sequences (near the protospacer-adjacent motif ["near the PAM"]) or by targeting a novel PAM generated by the mutation ("in the PAM"). However, these probability-based designs are not always assured, necessitating generalized, mutation-agnostic strategies like ablate-and-replace and single-nucleotide polymorphism editing. Here, we detail recent advancements in CRISPR therapeutics to treat a wide range of autosomal dominant disorders and discuss how they are altering the landscape for future therapies.},
}
@article {pmid35499048,
year = {2022},
author = {Ibrahim, S and Saleem, B and Rehman, N and Zafar, SA and Naeem, MK and Khan, MR},
title = {CRISPR/Cas9 mediated disruption of Inositol Pentakisphosphate 2-Kinase 1 (TaIPK1) reduces phytic acid and improves iron and zinc accumulation in wheat grains.},
journal = {Journal of advanced research},
volume = {37},
number = {},
pages = {33-41},
pmid = {35499048},
issn = {2090-1224},
mesh = {CRISPR-Cas Systems ; Edible Grain ; Humans ; Inositol Phosphates ; Iron ; *Phytic Acid/metabolism ; *Triticum/genetics ; Zinc/metabolism ; },
abstract = {INTRODUCTION: Phytic acid (PA) is an important antinutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, causing malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis.<br><br>OBJECTIVE: A recent genome editing tool CRISPR/Cas9 has been successfully applied to develop biofortified rice by disrupting IPK1 gene, however, it remained a challenge in wheat. The aim of this study was to biofortify wheat using CRISPR/Cas9.<br><br>METHODS: In this study, we isolated 3 TaIPK1 homeologs in wheat designated as TaIPK1.A, TaIPK1.B and TaIPK1.D and found that the expression abundance of TaIPK1.A was stronger in early stages of grain filling. Using CRISPR/Cas9, we have disrupted TaIPK1.A gene in cv. Borlaug-2016 with two guide RNAs targeting the 1st and 2nd exons.<br><br>RESULTS: We got several genome-edited lines in the T0 generation at frequencies of 12.7% and 10.8%. Sequencing analysis revealed deletion of 1-23 nucleotides and even an addition of 1 nucleotide in various lines. Analysis of the genome-edited lines revealed a significant decrease in the PA content and an increase in iron and zinc accumulation in grains compared with control plants.<br><br>CONCLUSION: Our study demonstrates the potential application of CRISPR/Cas9 technique for the rapid generation of biofortified wheat cultivars.},
}
@article {pmid35496799,
year = {2022},
author = {Picchi-Constante, GFA and Hiraiwa, PM and Marek, M and Rogerio, VZ and Guerra-Slompo, EP and Romier, C and Zanchin, NIT},
title = {Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in Trypanosoma cruzi.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101324},
pmid = {35496799},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/genetics ; *Chagas Disease/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Genes, Essential ; Humans ; *Trypanosoma cruzi/genetics ; },
abstract = {This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).},
}
@article {pmid35496795,
year = {2022},
author = {Ling, X and Chang, L and Chen, H and Liu, T},
title = {Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101321},
pmid = {35496795},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; },
abstract = {We recently developed a system to create human chimeric antigen receptor (CAR)-T cells using conjugated Cas12a (cCas12a) in which Cas12a is covalently linked to its CRISPR RNA (crRNA). This protocol describes site-specific modification of Cas12a and the preparation of Cas12a-crRNA complex using bio-orthogonal chemistry, followed by CAR-T cell generation through electroporation and AAV infection. This system shows robust editing efficiency in human cells and can be used for precisely targeted, highly efficient integration of CAR genes into T cell genome. For complete details on the use and execution of this protocol, please refer to Ling et al. (2021).},
}
@article {pmid35495695,
year = {2022},
author = {Zhang, X and An, X},
title = {Adaptation by Type III CRISPR-Cas Systems: Breakthrough Findings and Open Questions.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {876174},
pmid = {35495695},
issn = {1664-302X},
abstract = {CRISPR-Cas systems acquire heritable defense memory against invading nucleic acids through adaptation. Type III CRISPR-Cas systems have unique and intriguing features of defense and are important in method development for Genetics research. We started to understand the common and unique properties of type III CRISPR-Cas adaptation in recent years. This review summarizes our knowledge regarding CRISPR-Cas adaptation with the emphasis on type III systems and discusses open questions for type III adaptation studies.},
}
@article {pmid35495653,
year = {2022},
author = {Wörtz, J and Smith, V and Fallmann, J and König, S and Thuraisingam, T and Walther, P and Urlaub, H and Stadler, PF and Allers, T and Hille, F and Marchfelder, A},
title = {Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {822304},
pmid = {35495653},
issn = {1664-302X},
abstract = {CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.},
}
@article {pmid35491944,
year = {2022},
author = {Fremin, BJ and Kyrpides, NC},
title = {Identifying candidate structured RNAs in CRISPR operons.},
journal = {RNA biology},
volume = {19},
number = {1},
pages = {678-685},
pmid = {35491944},
issn = {1555-8584},
mesh = {*CRISPR-Cas Systems ; Genomics ; Operon ; *RNA/genetics ; Repetitive Sequences, Nucleic Acid ; },
abstract = {Noncoding RNAs with secondary structures play important roles in CRISPR-Cas systems. Many of these structures likely remain undiscovered. We used a large-scale comparative genomics approach to predict 156 novel candidate structured RNAs from 36,111 CRISPR-Cas systems. A number of these were found to overlap with coding genes, including palindromic candidates that overlapped with a variety of Cas genes in type I and III systems. Among these 156 candidates, we identified 46 new models of CRISPR direct repeats and 1 tracrRNA. This tracrRNA model occasionally overlapped with predicted cas9 coding regions, emphasizing the importance of expanding our search windows for novel structure RNAs in coding regions. We also demonstrated that the antirepeat sequence in this tracrRNA model can be used to accurately assign thousands of predicted CRISPR arrays to type II-C systems. This study highlights the importance of unbiased identification of candidate structured RNAs across CRISPR-Cas systems.},
}
@article {pmid35490366,
year = {2022},
author = {Bhunia, RK and Menard, GN and Eastmond, PJ},
title = {A native promoter-gene fusion created by CRISPR/Cas9-mediated genomic deletion offers a transgene-free method to drive oil accumulation in leaves.},
journal = {FEBS letters},
volume = {596},
number = {15},
pages = {1865-1870},
pmid = {35490366},
issn = {1873-3468},
mesh = {*Arabidopsis/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Gene Fusion ; Genomics ; Transgenes ; },
abstract = {Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase-deficient sugar-dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20-fold and the triacylglycerol content increases by around 30-fold. This deletion strategy offers a transgene-free route to engineering traits that rely on transcriptional gain-of-function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.},
}
@article {pmid35489308,
year = {2022},
author = {Lv, X and Hueso-Gil, A and Bi, X and Wu, Y and Liu, Y and Liu, L and Ledesma-Amaro, R},
title = {New synthetic biology tools for metabolic control.},
journal = {Current opinion in biotechnology},
volume = {76},
number = {},
pages = {102724},
doi = {10.1016/j.copbio.2022.102724},
pmid = {35489308},
issn = {1879-0429},
support = {BB/R01602X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T011408/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T013176/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Gene Regulatory Networks ; Metabolic Engineering ; *Synthetic Biology ; },
abstract = {In industrial bioprocesses, microbial metabolism dictates the product yields, and therefore, our capacity to control it has an enormous potential to help us move towards a bio-based economy. The rapid development of multiomics data has accelerated our systematic understanding of complex metabolic regulatory mechanisms, which allow us to develop tools to manipulate them. In the last few years, machine learning-based metabolic modeling, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) derived synthetic biology tools, and synthetic genetic circuits have been widely used to control the metabolism of microorganisms, manipulate gene expression, and build synthetic pathways for bioproduction. This review describes the latest developments for metabolic control, and focuses on the trends and challenges of metabolic engineering strategies.},
}
@article {pmid35487018,
year = {2022},
author = {Klose, SM and Wawegama, N and Sansom, FM and Marenda, MS and Browning, GF},
title = {Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.},
journal = {Veterinary microbiology},
volume = {269},
number = {},
pages = {109436},
doi = {10.1016/j.vetmic.2022.109436},
pmid = {35487018},
issn = {1873-2542},
mesh = {Animals ; *CRISPR-Cas Systems ; *Mycoplasma gallisepticum/genetics ; Plasmids/genetics ; },
abstract = {Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.},
}
@article {pmid35484862,
year = {2022},
author = {Wang, Y and He, X and Qiao, L and Yu, Z and Chen, B and He, Z},
title = {CRISPR/Cas9 mediates efficient site-specific mutagenesis of the odorant receptor co-receptor (Orco) in the malaria vector Anopheles sinensis.},
journal = {Pest management science},
volume = {78},
number = {8},
pages = {3294-3304},
doi = {10.1002/ps.6954},
pmid = {35484862},
issn = {1526-4998},
mesh = {Animals ; *Anopheles/genetics/metabolism ; *CRISPR-Cas Systems ; Malaria/prevention &amp; control ; Mosquito Vectors/genetics ; *Mutagenesis, Site-Directed ; *Receptors, Odorant/genetics/metabolism ; },
abstract = {BACKGROUND: Anopheles sinensis is the most widely distributed mosquito species and is the main transmitter of Plasmodium vivax malaria in China. Most previous research has focused on the mechanistic understanding of biological processes in An. sinensis and novel ways of interrupting malaria transmission. However, the development of functional genomics and genetics-based vector control strategies against An. sinensis remain limited because of insufficient site-specific genome editing tools.<br><br>RESULTS: We report the first successful application of the CRISPR/Cas9 mediated knock-in for highly efficient, site-specific mutagenesis in An. sinensis. The EGFP marker gene driven by the 3&#x2009;&#xd7;&#x2009;P3 promoter was precisely integrated into the odorant receptor co-receptor (Orco) by direct injections of Cas9 protein, double-stranded DNA donor, and Orco-gRNA. We achieved a mutation rate of 3.77%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed by polymerase chain reaction (PCR) amplification and sequencing. The Orco mutation severely impaired mosquito sensitivity to some odors and their ability to locate and discriminate a human host.<br><br>CONCLUSION: Orco was confirmed as a key mediator of multiple olfactory-driven behaviors in the An. sinensis life cycle, highlighting the importance of Orco as a key molecular target for malaria control. The results also demonstrated that CRISPR/Cas9 was a simple and highly efficient genome editing technique for An. sinensis and could be used to develop genetic control tools for this vector. &#xa9; 2022 Society of Chemical Industry.},
}
@article {pmid35483968,
year = {2022},
author = {Ji, H and Xiong, W and Zhang, K and Tian, T and Zhou, X},
title = {Hydrogen Peroxide-triggered Chemical Strategy for Controlling CRISPR systems.},
journal = {Chemistry, an Asian journal},
volume = {17},
number = {16},
pages = {e202200214},
doi = {10.1002/asia.202200214},
pmid = {35483968},
issn = {1861-471X},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Hydrogen Peroxide ; RNA ; *RNA, Guide/genetics ; },
abstract = {The function of the CRISPR system can be conditionally controlled through rationally guided RNA engineering such that the target sequences can be precisely selected and manipulated. In particular, gRNA, as an important component of the CRISPR system, provides a unique tool for multifunctional control of the system based on the structure of the RNA itself. Therefore, we introduced here a protective group on the 2'-OH position of RNA to inhibit RNA-guided nucleic acid cleavage. Next, the modified gRNA can restore its original function under the chemical stimulation of hydrogen peroxide to realize the control of the CRISPR system. The experiment result demonstrated that the operating mechanism of this strategy may be based on chemical modifications that reduce the number of complementary base pairs between RNAs and targets, and the RNA-protein interaction. This further enriches the toolbox of conditional control of CRISPR function and has broad potential for gene editing in living cells and disease treatment using endogenous hydrogen peroxide.},
}
@article {pmid35483740,
year = {2022},
author = {Herman, JA and Arora, S and Carter, L and Zhu, J and Biggins, S and Paddison, PJ},
title = {Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens.},
journal = {Genes &amp; development},
volume = {36},
number = {7-8},
pages = {495-510},
pmid = {35483740},
issn = {1549-5477},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; R01 GM064386/GM/NIGMS NIH HHS/United States ; R01 NS119650/NS/NINDS NIH HHS/United States ; R01 CA190957/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Mutagenesis ; },
abstract = {The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.},
}
@article {pmid35483732,
year = {2022},
author = {Shin, J and Miller, M and Wang, YC},
title = {Recent advances in CRISPR-based systems for the detection of foodborne pathogens.},
journal = {Comprehensive reviews in food science and food safety},
volume = {21},
number = {3},
pages = {3010-3029},
doi = {10.1111/1541-4337.12956},
pmid = {35483732},
issn = {1541-4337},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Food Industry ; },
abstract = {There has long been a need for more advanced forms of pathogen detection in the food industry. Though in its infancy, biosensing based on clustered regularly interspaced short palindromic repeats (CRISPR) has the potential to solve many problems that cannot be addressed using conventional methods. In this review, we briefly introduce and classify the various CRISPR/Cas protein effectors that have thus far been used in biosensors. We then assess the current state of CRISPR technology in food-safety contexts; describe how each Cas effector is utilized in foodborne-pathogen detection; and discuss the limitations of the current technology, as well as how it might usefully be applied in other areas of the food industry. We conclude that, if the limitations of existing CRISPR/Cas-based detection methods are overcome, they can be deployed on a wide scale and produce a range of positive food-safety outcomes.},
}
@article {pmid35483302,
year = {2022},
author = {Li, Y and Yang, F and Yuan, R and Zhong, X and Zhuo, Y},
title = {Electrochemiluminescence covalent organic framework coupling with CRISPR/Cas12a-mediated biosensor for pesticide residue detection.},
journal = {Food chemistry},
volume = {389},
number = {},
pages = {133049},
doi = {10.1016/j.foodchem.2022.133049},
pmid = {35483302},
issn = {1873-7072},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/genetics ; *Metal-Organic Frameworks ; *Pesticide Residues ; },
abstract = {The trace detection of pesticide residue becomes particularly important since increasing attentions have been attached to food safety. Herein, we developed an electrochemiluminescence (ECL) covalent organic framework (COF) based-biosensor for trace pesticide detection coupling with CRISPR/Cas12a-mediated signal accumulation strategy. Firstly, the target conversion was carried out with an aptamer-assembled magnetic spherical nucleic acids, which can convert acetamiprid to activator DNA, triggering the CRISPR/Cas12a to make quenching probes far away from electrode for signal accumulation. The COF with stable and strong ECL was synthesized by a condensation reaction between the perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) and melamine (MA), due to the highly ordered arrangement of the PTCDA luminescence units among COF structure and the pore confinement effect. Moreover, the designed assay method was successfully employed to detect the residual level of acetamiprid in real sample and expected to be widely used in pesticide-related food safety.},
}
@article {pmid35483191,
year = {2022},
author = {Mitra, S and Sarker, J and Mojumder, A and Shibbir, TB and Das, R and Emran, TB and Tallei, TE and Nainu, F and Alshahrani, AM and Chidambaram, K and Simal-Gandara, J},
title = {Genome editing and cancer: How far has research moved forward on CRISPR/Cas9?.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {150},
number = {},
pages = {113011},
doi = {10.1016/j.biopha.2022.113011},
pmid = {35483191},
issn = {1950-6007},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer accounted for almost ten million deaths worldwide in 2020. Metastasis, characterized by cancer cell invasion to other parts of the body, is the main cause of cancer morbidity and mortality. Therefore, understanding the molecular mechanisms of tumor formation and discovery of potential drug targets are of great importance. Gene editing techniques can be used to find novel drug targets and study molecular mechanisms. In this review, we describe how popular gene-editing methods such as CRISPR/Cas9, TALEN and ZFNs work, and, by comparing them, we demonstrate that CRISPR/Cas9 has superior efficiency and precision. We further provide an overview of the recent applications of CRISPR/Cas9 to cancer research, focusing on the most common cancers such as breast cancer, lung cancer, colorectal cancer, and prostate cancer. We describe how these applications will shape future research and treatment of cancer, and propose new ways to overcome current challenges.},
}
@article {pmid35482449,
year = {2022},
author = {Li, YY and Li, HD and Fang, WK and Liu, D and Liu, MH and Zheng, MQ and Zhang, LL and Yu, H and Tang, HW},
title = {Amplification of the Fluorescence Signal with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a Based on Au Nanoparticle-DNAzyme Probe and On-Site Detection of Pb[2+] Via the Photonic Crystal Chip.},
journal = {ACS sensors},
volume = {7},
number = {5},
pages = {1572-1580},
doi = {10.1021/acssensors.2c00516},
pmid = {35482449},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems ; *DNA, Catalytic ; Gold ; Lead ; *Metal Nanoparticles ; },
abstract = {Although great headway has been made in DNAzyme-based detection of Pb[2+], its adaptability, sensitivity, and accessibility in complex media still need to be improved. For this, we introduce new ways to surmount these hurdles. First, a spherical nucleic acid (SNA) fluorescence probe (Au nanoparticles-DNAzyme probe) is utilized to specifically identify Pb[2+] and its suitability for precise detection of Pb[2+] in complex samples due to its excellent nuclease resistance. Second, the sensitivity of Pb[2+] detection is greatly enhanced via the use of a clustered regularly interspaced short palindromic repeats-Cas12a with target recognition accuracy to amplify the fluorescent signal upon the trans cleavage of the SNA (signal probe), and the limit of detection reaches as low as 86 fM. Third, we boost the fluorescence on photonic crystal chips with a bionic periodic arrangement by employing a straightforward detection device (smartphone and portable UV lamp) to achieve on-site detection of Pb[2+] with the limit of detection as low as 24 pM. Based on the abovementioned efforts, the modified Pb[2+] fluorescence sensor has the advantages of higher sensitivity, better specificity, accessibility, less sample consumption, and so forth. Moreover, it can be applied to accurately detect Pb[2+] in complex biological or environmental samples, which is of great promise for widespread applications.},
}
@article {pmid35481381,
year = {2022},
author = {Liang, Y and Zou, L and Lin, H and Li, B and Zhao, J and Wang, H and Sun, J and Chen, J and Mo, Y and Yang, X and Deng, X and Tang, S},
title = {Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1811-1823},
doi = {10.1021/acssynbio.1c00643},
pmid = {35481381},
issn = {2161-5063},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Mutation ; *SARS-CoV-2/genetics ; },
abstract = {Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.},
}
@article {pmid35481139,
year = {2022},
author = {Li, Q and Feng, Q and Snouffer, A and Zhang, B and Rodríguez, GR and van der Knaap, E},
title = {Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {879642},
pmid = {35481139},
issn = {1664-462X},
abstract = {CRISPR/Cas-mediated genome editing is a powerful approach to accelerate yield enhancement to feed growing populations. Most applications focus on "negative regulators" by targeting coding regions and promoters to create nulls or weak loss-of-function alleles. However, many agriculturally important traits are conferred by gain-of-function alleles. Therefore, creating gain-of-function alleles for "positive regulators" by CRISPR will be of great value for crop improvement. CYP78A family members are the positive regulators of organ weight and size in crops. In this study, we engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) that is highly associated with fruit weight. The SNP was located in a conserved putative cis-regulatory element (CRE) as detected by the homology-based prediction and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Twenty-one mutant alleles with various insertion and deletion sizes were generated in the LA1589 background. Five mutant alleles (m2+4bp , m3+1bp , m5-1bp , m13-8bp , and m14-9bp) showed a consistent increase in fruit weight and a significant decrease in the proportion of small fruits in all experimental evaluations. Notably, m2+4bp and m3+1bp homozygote significantly increase fruit weight by 10.7-15.7 and 8.7-16.3%, respectively. Further analysis of fruit weight based on fruit position on the inflorescence indicated that the five beneficial alleles increase the weight of all fruits along inflorescence. We also found that allele types and transcriptional changes of SlKLUH were poor predictors of the changes in fruit weight. This study not only provides a way of identifying conserved CRE but also highlights enormous potential for CRISPR/Cas-mediated cis-engineering of CYP78A members in yield improvement.},
}
@article {pmid35478255,
year = {2022},
author = {Khairkhah, N and Bolhassani, A and Najafipour, R},
title = {Current and future direction in treatment of HPV-related cervical disease.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {100},
number = {6},
pages = {829-845},
pmid = {35478255},
issn = {1432-1440},
mesh = {Female ; Gene Editing ; Humans ; Papillomavirus E7 Proteins/genetics ; *Papillomavirus Infections/complications/genetics/therapy ; *Papillomavirus Vaccines/therapeutic use ; *Uterine Cervical Neoplasms/genetics/prevention &amp; control ; },
abstract = {Human papillomavirus (HPV) is the most common sexually transmitted virus in the world. About 70% of cervical cancers are caused by the most oncogenic HPV genotypes of 16 and 18. Since available prophylactic vaccines do not induce immunity in those with established HPV infections, the development of therapeutic HPV vaccines using E6 and E7 oncogenes, or both as the target antigens remains essential. Also, knocking out the E6 and E7 oncogenes in host genome by genome-editing CRISPR/Cas system can result in tumor growth suppression. These methods have shown promising results in both preclinical and clinical trials and can be used for controlling the progression of HPV-related cervical diseases. This comprehensive review will detail the current treatment of HPV-related cervical precancerous and cancerous diseases. We also reviewed the future direction of treatment including different kinds of therapeutic methods and vaccines, genome-editing CRISPR/Cas system being studied in clinical trials. Although the progress in the development of therapeutic HPV vaccine has been slow, encouraging results from recent trials showed vaccine-induced regression in high-grade CIN lesions. CRISPR/Cas genome-editing system is also a promising strategy for HPV cancer therapy. However, its safety and specificity need to be optimized before it is used in clinical setting.},
}
@article {pmid35478023,
year = {2022},
author = {Mallapaty, S},
title = {China focuses on ethics to deter another 'CRISPR babies' scandal.},
journal = {Nature},
volume = {605},
number = {7908},
pages = {15-16},
doi = {10.1038/d41586-022-01051-0},
pmid = {35478023},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; China ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Ethics, Medical ; *Gene Editing ; Humans ; },
}
@article {pmid35475666,
year = {2022},
author = {Maeda, F and Kato, A and Takeshima, K and Shibazaki, M and Sato, R and Shibata, T and Miyake, K and Kozuka-Hata, H and Oyama, M and Shimizu, E and Imoto, S and Miyano, S and Adachi, S and Natsume, T and Takeuchi, K and Maruzuru, Y and Koyanagi, N and Jun, A and Yasushi, K},
title = {Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.},
journal = {Journal of virology},
volume = {96},
number = {10},
pages = {e0030622},
pmid = {35475666},
issn = {1098-5514},
mesh = {Animals ; CRISPR-Cas Systems ; Chlorocebus aethiops ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; *Herpesvirus 1, Human/genetics/physiology ; Humans ; *Membrane Transport Proteins/metabolism ; Nuclear Envelope/metabolism ; Nuclear Proteins ; Proteomics ; Vero Cells ; Viral Proteins/metabolism ; *Virus Release ; },
abstract = {This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.},
}
@article {pmid35475594,
year = {2022},
author = {Thyssen, C and Johannes, E and Müller, K and Wünn, J},
title = {Non-genetically modified organism in vitro CRISPR/Cas9 gene editing of the lacZα gene: A 4.5 h laboratory course for senior high-school students.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {50},
number = {4},
pages = {393-400},
doi = {10.1002/bmb.21622},
pmid = {35475594},
issn = {1539-3429},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Escherichia coli/genetics ; *Gene Editing/methods ; Humans ; Students ; },
abstract = {The CRISPR/Cas9 system opens new horizons (M. Adli, Nat Commun, 2018) regarding genetic modifications of living organisms but also as an in vitro tool in laboratory protocols. Therefore, it boosts possibilities in research and future medical treatments. As the controversial claim of genomically edited babies by He Jiankui (Cyranoski D., Nature, 2019) demonstrates, the new gene editing potentials entail ethical discussions. A public or social discussion presupposes not only a theoretical knowledge or understanding of the system, but also profits from direct laboratory experiences showing how easy these techniques can be applied. Introducing numerous students and classes into these emerging techniques in a modern biology classroom depends on a suitable course concept, which fits legal and organizational requirements at the same time. Therefore, we implemented an appropriate hands-on laboratory course for senior high-school students, lasting just 4.5 h. Particularly with regard to European regulations concerning the handling of genetically modified organisms, the constructs and protocols avoid the transfer of Cas9 DNA. This normally mandatory transfer was replaced by in vitro gene-editing. This leads to Cas9 induced gene knock-outs due to frame shifts and/or the excision of DNA fragments in common Escherichia coli (E. coli) plasmids, such as pUC19. This gene knock-out concept covers various steps: In vitro plasmid editing with Cas9, ligation and transformation of E. coli cells with the modified plasmid DNA and finally the spread plating of transformed E. coli cells in order to analyze colonies after overnight incubation. The successful excision of DNA fragments by in vitro Cas9 treatment was determined by subsequent gel electrophoresis.},
}
@article {pmid35475170,
year = {2022},
author = {Nemudraia, A and Nemudryi, A and Buyukyoruk, M and Scherffius, A and Zahl, T and Wiegand, T and Pandey, S and Nichols, J and Hall, L and McVey, A and Lee, H and Wilkinson, R and Snyder, L and Jones, J and Koutmou, K and Santiago-Frangos, A and Wiedenheft, B},
title = {Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.},
journal = {Research square},
volume = {},
number = {},
pages = {},
pmid = {35475170},
abstract = {Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.},
}
@article {pmid35474904,
year = {2022},
author = {Cheng, L and Yang, F and Tang, L and Qian, L and Chen, X and Guan, F and Zhang, J and Li, G},
title = {Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage.},
journal = {Research (Washington, D.C.)},
volume = {2022},
number = {},
pages = {9826484},
pmid = {35474904},
issn = {2639-5274},
abstract = {Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.},
}
@article {pmid35473361,
year = {2022},
author = {Chantaravisoot, N and Kaewsapsak, P and Mayuramart, O and Nimsamer, P and Mankhong, S and Chomta, N and Bootsri, R and Alee, I and Wongkongkathep, P and Treeprasertsuk, S and Payungporn, S},
title = {COVID-19 active case findings based on self-collected saliva samples with CRISPR-Cas12a detection.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {247},
number = {14},
pages = {1228-1234},
pmid = {35473361},
issn = {1535-3699},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Pandemics/prevention &amp; control ; SARS-CoV-2/genetics ; Saliva ; Sensitivity and Specificity ; },
abstract = {COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus affecting the world population. Early detection has become one of the most successful strategies to alleviate the epidemic and pandemic of this contagious coronavirus. Surveillance testing programs have been initiated in many countries worldwide to prevent the outbreak of COVID-19. In this study, we demonstrated that our previously established clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay could detect variants of concern during 2021 in Thailand, including Alpha, Beta, and Delta strains as well as Omicron strain in early 2022. In combination with the newly designed saliva collection funnel, we established a safe, simple, economical, and efficient self-collection protocol for the COVID-19 screening process. We successfully utilized the assay in an active case finding with a total number of 578 asymptomatic participants to detect the SARS-CoV-2 in saliva samples. We finally demonstrated that the validation and evaluation in a large-scale setting could provide valuable information and elaborate the practicality of the test in real-world settings. Our optimized protocol yielded effective results with high sensitivity, specificity, and diagnostic accuracy (96.86%). In addition, this study demonstrates COVID-19 active case findings in low-resource settings, which would be feasible and attractive for surveillance and outbreak prevention in the future.},
}
@article {pmid35473305,
year = {2022},
author = {Bindal, G and Amlinger, L and Lundgren, M and Rath, D},
title = {Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae.},
journal = {mSphere},
volume = {7},
number = {3},
pages = {e0003822},
pmid = {35473305},
issn = {2379-5042},
mesh = {Archaea/genetics ; Bacteria/genetics ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Plasmids/genetics ; Saccharomyces cerevisiae/genetics ; *Viruses/genetics ; },
abstract = {Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.},
}
@article {pmid35472302,
year = {2022},
author = {Cho, SI and Lee, S and Mok, YG and Lim, K and Lee, J and Lee, JM and Chung, E and Kim, JS},
title = {Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.},
journal = {Cell},
volume = {185},
number = {10},
pages = {1764-1776.e12},
doi = {10.1016/j.cell.2022.03.039},
pmid = {35472302},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems ; Cytosine/metabolism ; *DNA, Mitochondrial/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; Humans ; Mitochondria/genetics/metabolism ; *Mitochondrial Diseases/genetics ; Purines ; },
abstract = {Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.},
}
@article {pmid35472287,
year = {2022},
author = {Horáčková, V and Voleman, L and Hagen, KD and Petrů, M and Vinopalová, M and Weisz, F and Janowicz, N and Marková, L and Motyčková, A and Najdrová, V and Tůmová, P and Dawson, SC and Doležal, P},
title = {Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.},
journal = {Open biology},
volume = {12},
number = {4},
pages = {210361},
pmid = {35472287},
issn = {2046-2441},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Giardia lamblia/genetics ; Humans ; RNA, Guide ; Tetraploidy ; },
abstract = {CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.},
}
@article {pmid35471824,
year = {2022},
author = {Lei, R and Li, L and Wu, P and Fei, X and Zhang, Y and Wang, J and Zhang, D and Zhang, Q and Yang, N and Wang, X},
title = {RPA/CRISPR/Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1772-1781},
doi = {10.1021/acssynbio.1c00620},
pmid = {35471824},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Toxoplasma/genetics/metabolism ; },
abstract = {Toxoplasma gondii is an opportunistic pathogen widely distributed within the world, poses a huge threat to human health, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection of T. gondii by combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. A glass microfiber filter device used for the first step can efficiently extract T. gondii from low-concentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and both reactions can be performed on a low-cost thermal controller (∼37 °C), avoiding the drawbacks of the step-by-step addition of components. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon of T. gondii over other parasites with a limit of detection of 3.3 copies/μL. The visual signal readout can be easily realized by a fluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas.},
}
@article {pmid35471009,
year = {2022},
author = {Zhu, X and Wu, Y and Lv, X and Liu, Y and Du, G and Li, J and Liu, L},
title = {Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1897-1907},
doi = {10.1021/acssynbio.2c00041},
pmid = {35471009},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Escherichia coli/genetics ; *Gene Editing/methods ; Histidine/genetics ; Metabolic Engineering/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based gene-editing technology has been widely used in various microorganisms due to its advantages of low cost, high efficiency, easy operation, and multiple functions. In this study, an efficient and fast double-plasmid gene-editing system pEcCpf1/pcrEG was constructed in Escherichia coli based on CRISPR/Cpf1. First, gene knockout and integration efficiency were verified in eight different kinds of protospacer adjacent motif (PAM) regions. Then, the transformation method was optimized, and the efficiency of gene knockout or gene integration of this system increased to nearly 100%, and the large-length fragments could be integrated into the genome in E. coli BL21 (DE3). The system was also optimized by replacing the homologous recombination system in plasmid pEcCpf1, resulting in pEcCpf1H, which could perform precise single-point mutation, terminator insertion, short-sequence insertion, or gene knockout with high efficiency using a 90 nt (nucleotide) single-stranded primer. Further, multiple genes could be edited simultaneously. Next, these two systems were demonstrated in other E. coli strains. Finally, as an application, the system was used to engineer the synthesis pathway of l-histidine in the engineered strain. The titer of l-histidine in a shake flask reached 7.16 g/L, a value increased by 84.1% compared to the starting strain. Thus, this study provided an effective tool for metabolic engineering of E. coli.},
}
@article {pmid35470620,
year = {2022},
author = {LE, Y and He, X and Sun, J},
title = {[Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {4},
pages = {1475-1489},
doi = {10.13345/j.cjb.210827},
pmid = {35470620},
issn = {1872-2075},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; },
abstract = {The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.},
}
@article {pmid35470618,
year = {2022},
author = {Chao, S and Hu, X},
title = {[Application of gene editing technology in Escherichia coli].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {4},
pages = {1446-1461},
doi = {10.13345/j.cjb.210680},
pmid = {35470618},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing ; Homologous Recombination ; Technology ; },
abstract = {Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.},
}
@article {pmid35470617,
year = {2022},
author = {Wang, C and Liu, Z and Tang, B and Yang, H and Sun, D},
title = {[Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {4},
pages = {1432-1445},
doi = {10.13345/j.cjb.210348},
pmid = {35470617},
issn = {1872-2075},
mesh = {Anti-Bacterial Agents ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; },
abstract = {Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.},
}
@article {pmid35469710,
year = {2022},
author = {Cury, J and Bernheim, A},
title = {CRISPR-Cas and restriction-modification team up to achieve long-term immunity.},
journal = {Trends in microbiology},
volume = {30},
number = {6},
pages = {513-514},
doi = {10.1016/j.tim.2022.04.001},
pmid = {35469710},
issn = {1878-4380},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; },
abstract = {Bacteria have been shown to harbor a growing arsenal of various defense systems against phages. Maguin et al. have uncovered how two of the most frequent defense systems interact: the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system recycles by-products of the restriction-modification (RM) system to increase bacterial defense in the long run.},
}
@article {pmid35468950,
year = {2022},
author = {Mitkas, AA and Valverde, M and Chen, W},
title = {Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.},
journal = {Nature chemical biology},
volume = {18},
number = {5},
pages = {492-500},
pmid = {35468950},
issn = {1552-4469},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Endonucleases/metabolism ; RNA/metabolism ; RNA Processing, Post-Transcriptional ; },
abstract = {In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR-Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA-RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers and mCherry messenger RNA. Both direct and 'Turn-On' assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.},
}
@article {pmid35468907,
year = {2022},
author = {Thean, DGL and Chu, HY and Fong, JHC and Chan, BKC and Zhou, P and Kwok, CCS and Chan, YM and Mak, SYL and Choi, GCG and Ho, JWK and Zheng, Z and Wong, ASL},
title = {Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2219},
pmid = {35468907},
issn = {2041-1723},
mesh = {*Bacterial Proteins/metabolism ; *CRISPR-Cas Systems/genetics ; DNA/metabolism ; Humans ; Machine Learning ; Mutagenesis ; },
abstract = {The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity.},
}
@article {pmid35468792,
year = {2022},
author = {Huang, H and Huang, G and Tan, Z and Hu, Y and Shan, L and Zhou, J and Zhang, X and Ma, S and Lv, W and Huang, T and Liu, Y and Wang, D and Zhao, X and Lin, Y and Rong, Z},
title = {Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {91},
pmid = {35468792},
issn = {1741-7007},
mesh = {Acidaminococcus/genetics ; *CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing ; Proto-Oncogene Proteins B-raf/genetics ; },
abstract = {BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).<br><br>RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAF[V600E] mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAF[V600E] cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.<br><br>CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.},
}
@article {pmid35467081,
year = {2022},
author = {Sparmann, A and Beisel, CL},
title = {CRISPR memories in single cells.},
journal = {Molecular systems biology},
volume = {18},
number = {4},
pages = {e11011},
pmid = {35467081},
issn = {1744-4292},
mesh = {*Bacteria/genetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas systems allow bacteria to memorize prior infections as a means to combat the same invader if it attempts another attack in the future. While the underlying mechanisms of this bacterial immunity have been intensely studied over the past decade, little attention has been paid to CRISPR defense at the single-cell level. In their recent work, Brouns and colleagues (McKenzie et al, 2022) track memory acquisition and defense in individual cells and find a wide range of temporal dynamics that shape how a cell population experiences and combats an active infection.},
}
@article {pmid35467080,
year = {2022},
author = {McKenzie, RE and Keizer, EM and Vink, JNA and van Lopik, J and Büke, F and Kalkman, V and Fleck, C and Tans, SJ and Brouns, SJJ},
title = {Single cell variability of CRISPR-Cas interference and adaptation.},
journal = {Molecular systems biology},
volume = {18},
number = {4},
pages = {e10680},
doi = {10.15252/msb.202110680},
pmid = {35467080},
issn = {1744-4292},
mesh = {Adaptation, Physiological/genetics ; *Bacteriophages ; *CRISPR-Cas Systems/genetics ; DNA/metabolism ; Escherichia coli/genetics/metabolism ; },
abstract = {While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.},
}
@article {pmid35466160,
year = {2022},
author = {Bajaj, A and Cuchel, M},
title = {Advancements in the Treatment of Homozygous Familial Hypercholesterolemia.},
journal = {Journal of atherosclerosis and thrombosis},
volume = {29},
number = {8},
pages = {1125-1135},
pmid = {35466160},
issn = {1880-3873},
mesh = {Angiopoietin-Like Protein 3 ; Angiopoietin-like Proteins ; *Anticholesteremic Agents/pharmacology ; *Atherosclerosis/drug therapy ; Cholesterol, LDL ; *Homozygous Familial Hypercholesterolemia ; Humans ; *Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use ; *Hyperlipoproteinemia Type II/drug therapy/genetics ; Proprotein Convertase 9 ; },
abstract = {Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder with extreme elevations of low-density lipoprotein cholesterol (LDL-C) leading to premature atherosclerotic cardiovascular disease (ASCVD) as early as in childhood. Management of HoFH centers around aggressive and adequate reduction of LDL-C levels to slow the trajectory of ASCVD development. Historically, lowering LDL-C levels in HoFH has been challenging because of both the markedly elevated LDL-C levels (often ＞400 mg/dL) and reduced response to treatment options, such as statins, for which the mechanism of action requires a functional LDL receptor. However, the treatment landscape for HoFH has rapidly progressed over the last decade. While statins and ezetimibe remain first-line treatment, patients often require addition of multiple therapies to achieve goal LDL-C levels. The PCSK9 inhibitors are an important recent addition to the available treatment options, along with lomitapide, bile acid sequestrants, and, possibly, bempedoic acid. Additionally, ANGPTL3 has emerged as an important therapeutic target, with evinacumab being the first available ANGPTL3 inhibitor on the market for the treatment of patients with HoFH. For patients who cannot achieve adequate LDL-C reduction, lipoprotein apheresis may be necessary, with the added benefit of reducing lipoprotein(a) levels that carries an added risk if also elevated in patients with HoFH. Finally, gene therapy and genome editing using CRISPR/Cas-9 are moving through clinical development and may dramatically alter the future landscape of treatment for HoFH.},
}
@article {pmid35465826,
year = {2022},
author = {Ghosh, A and Myacheva, K and Riester, M and Schmidt, C and Diederichs, S},
title = {Chimeric oligonucleotides combining guide RNA and single-stranded DNA repair template effectively induce precision gene editing.},
journal = {RNA biology},
volume = {19},
number = {1},
pages = {588-593},
pmid = {35465826},
issn = {1555-8584},
mesh = {CRISPR-Cas Systems ; Chimera/metabolism ; DNA, Single-Stranded/genetics ; *Gene Editing/methods ; Oligonucleotides/genetics ; *RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; },
abstract = {The ability to precisely alter the genome holds immense potential for molecular biology, medicine and biotechnology. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) into a genomic editing tool has vastly simplified genome engineering. Here, we explored the use of chemically synthesized chimeric oligonucleotides encoding a target-specific crRNA (CRISPR RNA) fused to a single-stranded DNA repair template for RNP-mediated precision genome editing. By generating three clinically relevant oncogenic driver mutations, two non-stop extension mutations, an FGFRi resistance mutation and a single nucleotide change, we demonstrate the ability of chimeric oligos to form RNPs and direct Cas9 to effectively induce genome editing. Further, we demonstrate that the polarity of the chimeric oligos is crucial: only chimeric oligos with the single-stranded DNA repair template fused to the 3'-end of the crRNA are functional for accurate editing, while templates fused to the 5'-end are ineffective. We also find that chimeras can perform editing with both symmetric and asymmetric single-stranded DNA repair templates. Depending on the target locus, the editing efficiency using chimeric RNPs is similar to or less than the efficiency of editing using the bipartite standard RNPs. Our results indicate that chimeric RNPs comprising RNA-DNA oligos formed from fusing the crRNA and DNA repair templates can successfully induce precise edits. While chimeric RNPs do not display an advantage over standard RNPs, they nonetheless represent a viable approach for one-molecule precision genome editing.},
}
@article {pmid35461863,
year = {2022},
author = {Yadav, M and Atala, A and Lu, B},
title = {Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression.},
journal = {International journal of biological macromolecules},
volume = {209},
number = {Pt A},
pages = {1260-1270},
doi = {10.1016/j.ijbiomac.2022.04.114},
pmid = {35461863},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression ; Gene Products, gag/genetics ; Oligonucleotides ; *RNA, Guide/genetics ; RNA, Messenger/genetics ; },
abstract = {Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.},
}
@article {pmid35461662,
year = {2022},
author = {Le, Y and Sun, J},
title = {CRISPR/Cas genome editing systems in thermophiles: Current status, associated challenges, and future perspectives.},
journal = {Advances in applied microbiology},
volume = {118},
number = {},
pages = {1-30},
doi = {10.1016/bs.aambs.2022.02.001},
pmid = {35461662},
issn = {0065-2164},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Metabolic Engineering ; },
abstract = {Thermophiles, offering an attractive and unique platform for a broad range of applications in biofuels and environment protections, have received a significant attention and growing interest from academy and industry. However, the exploration and exploitation of thermophilic organisms have been hampered by the lack of a powerful genome manipulation tool to improve production efficiency. At current, the clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated (Cas) system has been successfully exploited as a competent, simplistic, and powerful tool for genome engineering both in eukaryotes and prokaryotes. Indeed, with the significant efforts made in recent years, some thermostable Cas9 proteins have been well identified and characterized and further, some thermostable Cas9-based editing tools have been successfully established in some representative obligate thermophiles. In this regard, we reviewed the current status and its progress in CRISPR/Cas-based genome editing system towards a variety of thermophilic organisms. Despite the potentials of these progresses, multiple factors/barriers still have to be overcome and optimized for improving its editing efficiency in thermophiles. Some insights into the roles of thermostable CRISPR/Cas technologies for the metabolic engineering of thermophiles as a thermophilic microbial cell factory were also fully analyzed and discussed.},
}
@article {pmid35461452,
year = {2022},
author = {Bieluszewski, T and Szymanska-Lejman, M and Dziegielewski, W and Zhu, L and Ziolkowski, PA},
title = {Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2484},
number = {},
pages = {161-182},
pmid = {35461452},
issn = {1940-6029},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Seeds/genetics ; },
abstract = {Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined to Col-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.},
}
@article {pmid35461116,
year = {2022},
author = {Li, Y and Deng, F and Goldys, EM},
title = {A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics.},
journal = {Talanta},
volume = {246},
number = {},
pages = {123469},
doi = {10.1016/j.talanta.2022.123469},
pmid = {35461116},
issn = {1873-3573},
mesh = {Antibodies ; *Biosensing Techniques ; *CRISPR-Cas Systems ; DNA, Single-Stranded ; Humans ; Immunoassay ; Oligonucleotides ; },
abstract = {Recent advances in CRISPR/Cas biosensing have led to impressive performance in sensitivity, specificity, and speed for nucleic acid detection. However, the remarkable advantages (such as universality, ultralow, attomolar detection limits) of CRISPR/Cas biosensing systems are limited in testing non-nucleic acid targets. Herein, by synthesizing a functional hybrid conjugate of antibody and single strand DNA oligonucleotide, we had successfully demonstrated the capability to integrate CRISPR/Cas12a-based signal amplification into different types of immunoassay schemes without the need for any additional recognition molecule or molecular synthesis during the detection process, thus providing a simple but generally applicable approach to improve the conventional immunoassays with attomolar sensitivity for small protein detections, referred as the CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). CRUISE is capable of being integrated into various immunoassays either through the primary antibody or the secondary antibody, with sensitivity down to 1 fg mL[-1] (∼50 aM) and 6 logs of linear range for detecting cytokines, such as IFN-γ and EGFR, under 3-4 h. It has a 10[3] times higher sensitivity compared to a commercial IFN-γ ELISA kit, but uses the same experimental scheme. The same 1 fg mL[-1] sensitivity along with 6 logs of linear range was realized for IFN-γ detection in human plasma samples. We are expecting that our CRUISE provides an alternative but simple, user-friendly and effective strategy for those who rely on the use of immunoassays, while struggling with the limits of their sensitivity or detection ranges.},
}
@article {pmid35460444,
year = {2022},
author = {Das, D and Singha, DL and Paswan, RR and Chowdhury, N and Sharma, M and Reddy, PS and Chikkaputtaiah, C},
title = {Recent advancements in CRISPR/Cas technology for accelerated crop improvement.},
journal = {Planta},
volume = {255},
number = {5},
pages = {109},
pmid = {35460444},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Technology ; },
abstract = {Precise genome engineering approaches could be perceived as a second paradigm for targeted trait improvement in crop plants, with the potential to overcome the constraints imposed by conventional CRISPR/Cas technology. The likelihood of reduced agricultural production due to highly turbulent climatic conditions increases as the global population expands. The second paradigm of stress-resilient crops with enhanced tolerance and increased productivity against various stresses is paramount to support global production and consumption equilibrium. Although traditional breeding approaches have substantially increased crop production and yield, effective strategies are anticipated to restore crop productivity even further in meeting the world's increasing food demands. CRISPR/Cas, which originated in prokaryotes, has surfaced as a coveted genome editing tool in recent decades, reshaping plant molecular biology in unprecedented ways and paving the way for engineering stress-tolerant crops. CRISPR/Cas is distinguished by its efficiency, high target specificity, and modularity, enables precise genetic modification of crop plants, allowing for the creation of allelic variations in the germplasm and the development of novel and more productive agricultural practices. Additionally, a slew of advanced biotechnologies premised on the CRISPR/Cas methodologies have augmented fundamental research and plant synthetic biology toolkits. Here, we describe gene editing tools, including CRISPR/Cas and its imitative tools, such as base and prime editing, multiplex genome editing, chromosome engineering followed by their implications in crop genetic improvement. Further, we comprehensively discuss the latest developments of CRISPR/Cas technology including CRISPR-mediated gene drive, tissue-specific genome editing, dCas9 mediated epigenetic modification and programmed self-elimination of transgenes in plants. Finally, we highlight the applicability and scope of advanced CRISPR-based techniques in crop genetic improvement.},
}
@article {pmid35458632,
year = {2022},
author = {Vojnits, K and Nakanishi, M and Porras, D and Kim, Y and Feng, Z and Golubeva, D and Bhatia, M},
title = {Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {8},
pages = {},
pmid = {35458632},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Gene Editing/methods ; Genes, Reporter ; Green Fluorescent Proteins ; Humans ; *Pluripotent Stem Cells ; },
abstract = {Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC-OCT4-EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC-AAVS1-EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC-AAVS1-EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.},
}
@article {pmid35458562,
year = {2022},
author = {Liu, J and Tao, D and Chen, X and Shen, L and Zhu, L and Xu, B and Liu, H and Zhao, S and Li, X and Liu, X and Xie, S and Niu, L},
title = {Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.},
journal = {Viruses},
volume = {14},
number = {4},
pages = {},
pmid = {35458562},
issn = {1999-4915},
mesh = {Alphacoronavirus ; Animals ; CRISPR-Cas Systems ; *Coronavirus/genetics ; *Coronavirus Infections/diagnosis/genetics/veterinary ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; *Porcine epidemic diarrhea virus/genetics ; RNA-Directed DNA Polymerase/genetics ; Sensitivity and Specificity ; Swine ; *Swine Diseases ; },
abstract = {Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.},
}
@article {pmid35458523,
year = {2022},
author = {Calderón, K and Rojas-Neyra, A and Carbajal-Lévano, B and Luján-Valenzuela, L and Ticona, J and Isasi-Rivas, G and Montalvan, A and Criollo-Orozco, M and Huaccachi-Gonzáles, E and Tataje-Lavanda, L and Alvarez, KLF and Fernández-Sánchez, M and Fernández-Díaz, M and Tang, N and Yao, Y and Nair, V},
title = {A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.},
journal = {Viruses},
volume = {14},
number = {4},
pages = {},
pmid = {35458523},
issn = {1999-4915},
support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/OS/NW/000007/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Antibodies, Viral ; CRISPR-Cas Systems ; Chickens ; Genotype ; Herpesvirus 1, Meleagrid/genetics ; *Herpesvirus 2, Gallid ; Integrases ; *Newcastle Disease/prevention &amp; control ; Newcastle disease virus/genetics ; *Poultry Diseases ; Vaccines, Synthetic/genetics ; *Viral Vaccines/genetics ; },
abstract = {In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.},
}
@article {pmid35457940,
year = {2022},
author = {Song, W and Zhang, T and Lin, H and Yang, Y and Zhao, G and Huang, X},
title = {Conventional and Microfluidic Methods for the Detection of Nucleic Acid of SARS-CoV-2.},
journal = {Micromachines},
volume = {13},
number = {4},
pages = {},
pmid = {35457940},
issn = {2072-666X},
abstract = {Nucleic acid testing (NAT) played a crucial role in containing the spread of SARS-CoV-2 during the epidemic. The gold standard technique, the quantitative real-time polymerase chain reaction (qRT-PCR) technique, is currently used by the government and medical boards to detect SARS-CoV-2. Due to the limitations of this technology, it is not capable of meeting the needs of large-scale rapid detection. To solve this problem, many new techniques for detecting nucleic acids of SARS-CoV-2 have been reported. Therefore, a review that systematically and comprehensively introduces and compares various detection technologies is needed. In this paper, we not only review the traditional NAT but also provide an overview of microfluidic-based NAT technologies and summarize and discuss the characteristics and development prospects of these techniques.},
}
@article {pmid35457271,
year = {2022},
author = {Zegeye, WA and Tsegaw, M and Zhang, Y and Cao, L},
title = {CRISPR-Based Genome Editing: Advancements and Opportunities for Rice Improvement.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457271},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant ; *Oryza/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {To increase the potentiality of crop production for future food security, new technologies for plant breeding are required, including genome editing technology-being one of the most promising. Genome editing with the CRISPR/Cas system has attracted researchers in the last decade as a safer and easier tool for genome editing in a variety of living organisms including rice. Genome editing has transformed agriculture by reducing biotic and abiotic stresses and increasing yield. Recently, genome editing technologies have been developed quickly in order to avoid the challenges that genetically modified crops face. Developing transgenic-free edited plants without introducing foreign DNA has received regulatory approval in a number of countries. Several ongoing efforts from various countries are rapidly expanding to adopt the innovations. This review covers the mechanisms of CRISPR/Cas9, comparisons of CRISPR/Cas9 with other gene-editing technologies-including newly emerged Cas variants-and focuses on CRISPR/Cas9-targeted genes for rice crop improvement. We have further highlighted CRISPR/Cas9 vector construction model design and different bioinformatics tools for target site selection.},
}
@article {pmid35457234,
year = {2022},
author = {Rozov, SM and Permyakova, NV and Sidorchuk, YV and Deineko, EV},
title = {Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457234},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Genome, Plant ; Mammals/genetics ; Plants, Genetically Modified/genetics ; Recombinant Proteins/genetics ; Transgenes ; },
abstract = {Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic "safe harbors") using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of "euchromatization" of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.},
}
@article {pmid35457228,
year = {2022},
author = {López-Márquez, A and Morín, M and Fernández-Peñalver, S and Badosa, C and Hernández-Delgado, A and Natera-de Benito, D and Ortez, C and Nascimento, A and Grinberg, D and Balcells, S and Roldán, M and Moreno-Pelayo, M&#xc1; and Jiménez-Mallebrera, C},
title = {CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457228},
issn = {1422-0067},
mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; *Collagen Type VI/genetics/metabolism ; Extracellular Matrix/metabolism ; Fibroblasts/metabolism ; Humans ; Mutation ; },
abstract = {Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.},
}
@article {pmid35457099,
year = {2022},
author = {Vanhoye, X and Janin, A and Caillaud, A and Rimbert, A and Venet, F and Gossez, M and Dijk, W and Marmontel, O and Nony, S and Chatelain, C and Durand, C and Lindenbaum, P and Rieusset, J and Cariou, B and Moulin, P and Di Filippo, M},
title = {APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35457099},
issn = {1422-0067},
mesh = {Apolipoproteins B/metabolism ; CRISPR-Cas Systems ; *Fatty Liver/genetics ; Humans ; *Hypobetalipoproteinemia, Familial, Apolipoprotein B/genetics ; *Hypobetalipoproteinemias/diagnosis/genetics/metabolism ; },
abstract = {Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.},
}
@article {pmid35456898,
year = {2022},
author = {Dunbar, T and Tsakirpaloglou, N and Septiningsih, EM and Thomson, MJ},
title = {Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds.},
journal = {International journal of molecular sciences},
volume = {23},
number = {8},
pages = {},
pmid = {35456898},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; DNA ; Gene Editing/methods ; Genome, Plant ; *Nanotubes, Carbon ; *Oryza/genetics ; Plant Leaves/genetics ; Plants/genetics ; Plants, Genetically Modified/genetics ; Plasmids/genetics ; Seeds/genetics ; },
abstract = {CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.},
}
@article {pmid35453874,
year = {2022},
author = {Li, Y and Shi, Z and Hu, A and Cui, J and Yang, K and Liu, Y and Deng, G and Zhu, C and Zhu, L},
title = {Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {12},
number = {4},
pages = {},
pmid = {35453874},
issn = {2075-4418},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.},
}
@article {pmid35453665,
year = {2022},
author = {Azlan, A and Rajasegaran, Y and Kang Zi, K and Rosli, AA and Yik, MY and Yusoff, NM and Heidenreich, O and Moses, EJ},
title = {Elucidating miRNA Function in Cancer Biology via the Molecular Genetics' Toolbox.},
journal = {Biomedicines},
volume = {10},
number = {4},
pages = {},
pmid = {35453665},
issn = {2227-9059},
abstract = {Micro-RNA (miRNAs) are short non-coding RNAs of about 18-20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this review will highlight the various mechanisms by which microRNAs are dysregulated in cancer. Further highlights include the abundance of molecular genetics tools that are currently available to study miRNA function as well as their advantages and disadvantages with a special focus on various CRISPR/Cas systems This review provides general workflows and some practical considerations when studying miRNA function thus enabling researchers to make informed decisions in regards to the appropriate molecular genetics tool to be utilized for their experiments.},
}
@article {pmid35452778,
year = {2022},
author = {Ceasar, SA and Maharajan, T and Hillary, VE and Ajeesh Krishna, TP},
title = {Insights to improve the plant nutrient transport by CRISPR/Cas system.},
journal = {Biotechnology advances},
volume = {59},
number = {},
pages = {107963},
doi = {10.1016/j.biotechadv.2022.107963},
pmid = {35452778},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Fertilizers ; Genome, Plant ; Nutrients ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {We need to improve food production to feed the ever growing world population especially in a changing climate. Nutrient deficiency in soils is one of the primary bottlenecks affecting the crop production both in developed and developing countries. Farmers are forced to apply synthetic fertilizers to improve the crop production to meet the demand. Understanding the mechanism of nutrient transport is helpful to improve the nutrient-use efficiency of crops and promote the sustainable agriculture. Many transporters involved in the acquisition, export and redistribution of nutrients in plants are characterized. In these studies, heterologous systems like yeast and Xenopus were most frequently used to study the transport function of plant nutrient transporters. CRIPSR/Cas system introduced recently has taken central stage for efficient genome editing in diverse organisms including plants. In this review, we discuss the key nutrient transporters involved in the acquisition and redistribution of nutrients from soil. We draw insights on the possible application CRISPR/Cas system for improving the nutrient transport in plants by engineering key residues of nutrient transporters, transcriptional regulation of nutrient transport signals, engineering motifs in promoters and transcription factors. CRISPR-based engineering of plant nutrient transport not only helps to study the process in native plants with conserved regulatory system but also aid to develop non-transgenic crops with better nutrient use-efficiency. This will reduce the application of synthetic fertilizers and promote the sustainable agriculture strengthening the food and nutrient security.},
}
@article {pmid35452274,
year = {2022},
author = {Anliker, B and Childs, L and Rau, J and Renner, M and Schüle, S and Schuessler-Lenz, M and Sebe, A},
title = {Regulatory Considerations for Clinical Trial Applications with CRISPR-Based Medicinal Products.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {364-376},
doi = {10.1089/crispr.2021.0148},
pmid = {35452274},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Clinical Trials as Topic ; *Gene Editing ; Gene Targeting ; Humans ; },
abstract = {Since first proposed as a new tool for gene targeting and genome editing, CRISPR technology has quickly advanced into the clinical stage. Initial studies highlight the potential for CRISPR-Cas9-mediated therapeutic approaches in human medicine to correct incurable genetic diseases and enhance cell-based therapeutic approaches. While acknowledging the opportunities this technology brings for the treatment of patients with severe diseases, timely development of these innovative medicinal products requires regulatory oversight and adaptation of regulatory requirements to ensure the safety and efficacy of medicinal products based on CRISPR technology. We briefly present the current regulatory framework applicable for CRISPR-Cas-based developments as advanced therapy medicinal products. Moreover, scientific- and regulatory-driven considerations relevant for advancing product development toward clinical trial applications in Germany are highlighted by discussing the key aspects of quality and nonclinical and clinical development requirements.},
}
@article {pmid35452075,
year = {2022},
author = {Oh, SA and Senger, K and Madireddi, S and Akhmetzyanova, I and Ishizuka, IE and Tarighat, S and Lo, JH and Shaw, D and Haley, B and Rutz, S},
title = {High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA.},
journal = {The Journal of experimental medicine},
volume = {219},
number = {5},
pages = {},
pmid = {35452075},
issn = {1540-9538},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Humans ; Plasmids/genetics ; T-Lymphocytes ; },
abstract = {Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.},
}
@article {pmid35451949,
year = {2022},
author = {Han, X and Zhou, X and Pei, Z and Stanton, C and Ross, RP and Zhao, J and Zhang, H and Yang, B and Chen, W},
title = {Characterization of CRISPR-Cas systems in Bifidobacterium breve.},
journal = {Microbial genomics},
volume = {8},
number = {4},
pages = {},
pmid = {35451949},
issn = {2057-5858},
mesh = {*Bifidobacterium breve/genetics ; *CRISPR-Cas Systems/genetics ; DNA ; Sequence Analysis, DNA ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system is an important adaptive immune system for bacteria to resist foreign DNA infection, which has been widely used in genotyping and gene editing. To provide a theoretical basis for the application of the CRISPR-Cas system in Bifidobacterium breve, the occurrence and diversity of CRISPR-Cas systems were analysed in 150 B. breve strains. Specifically, 47 % (71/150) of B. breve genomes possessed the CRISPR-Cas system, and type I-C CRISPR-Cas system was the most widely distributed among those strains. The spacer sequences present in B. breve can be used as a genotyping marker. Additionally, the phage assembly-related proteins were important targets of the type I-C CRISPR-Cas system in B. breve, and the protospacer adjacent motif sequences were further characterized in B. breve type I-C system as 5'-TTC-3'. All these results might provide a molecular basis for the development of endogenous genome editing tools in B. breve.},
}
@article {pmid35451838,
year = {2022},
author = {Luo, T and Li, J and He, Y and Liu, H and Deng, Z and Long, X and Wan, Q and Ding, J and Gong, Z and Yang, Y and Zhong, S},
title = {Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection.},
journal = {Analytical chemistry},
volume = {94},
number = {17},
pages = {6566-6573},
doi = {10.1021/acs.analchem.2c00401},
pmid = {35451838},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA, Single-Stranded/genetics ; *MicroRNAs/genetics ; },
abstract = {Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.},
}
@article {pmid35451132,
year = {2022},
author = {Kershanskaya, OI and Yessenbaeva, GL and Nelidova, DS and Karabekova, AN and Sadullaeva, ZN},
title = {CRISPR/Cas genome editing perspectives for barley breeding.},
journal = {Physiologia plantarum},
volume = {174},
number = {3},
pages = {e13686},
doi = {10.1111/ppl.13686},
pmid = {35451132},
issn = {1399-3054},
mesh = {CRISPR-Cas Systems/genetics ; Eukaryotic Initiation Factor-4E/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; *Hordeum/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {The CRISPR/Cas9 technology shows potential to improve crop breeding efficiency and antiviral defense. The interest in DNA editing in crops has grown due to the possibility of increasing the resistance of different plants to many viruses. Our aim was to create an elite disease-resistant local barley cultivar using CRISPR/Cas9 biotechnology. For this purpose, we used CRISPR/Cas 9-eIF4E with the eukaryotic translation initiation factor 4E (eIF4E) barley gene to edit the genomes of five local Kazakhstan barley cultivars. After identifying the single guide RNA (sgRNA) target sequences, they were synthesized and cloned into the CRISPR-plant vector before being introduced into barley cells via our own patented Agrobacterium germ-line transformation technique. Barley plants eIF4E-modified were successfully obtained and were resistant to virus infection. Based on our research, the CRISPR/Cas9 system for plant genome editing could be a prospect for applying this breakthrough biotechnology in barley breeding.},
}
@article {pmid35448780,
year = {2022},
author = {Haroon, M and Wang, X and Afzal, R and Zafar, MM and Idrees, F and Batool, M and Khan, AS and Imran, M},
title = {Novel Plant Breeding Techniques Shake Hands with Cereals to Increase Production.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {8},
pages = {},
pmid = {35448780},
issn = {2223-7747},
abstract = {Cereals are the main source of human food on our planet. The ever-increasing food demand, continuously changing environment, and diseases of cereal crops have made adequate production a challenging task for feeding the ever-increasing population. Plant breeders are striving their hardest to increase production by manipulating conventional breeding methods based on the biology of plants, either self-pollinating or cross-pollinating. However, traditional approaches take a decade, space, and inputs in order to make crosses and release improved varieties. Recent advancements in genome editing tools (GETs) have increased the possibility of precise and rapid genome editing. New GETs such as CRISPR/Cas9, CRISPR/Cpf1, prime editing, base editing, dCas9 epigenetic modification, and several other transgene-free genome editing approaches are available to fill the lacuna of selection cycles and limited genetic diversity. Over the last few years, these technologies have led to revolutionary developments and researchers have quickly attained remarkable achievements. However, GETs are associated with various bottlenecks that prevent the scaling development of new varieties that can be dealt with by integrating the GETs with the improved conventional breeding methods such as speed breeding, which would take plant breeding to the next level. In this review, we have summarized all these traditional, molecular, and integrated approaches to speed up the breeding procedure of cereals.},
}
@article {pmid35448772,
year = {2022},
author = {Niazian, M and Belzile, F and Torkamaneh, D},
title = {CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {8},
pages = {},
pmid = {35448772},
issn = {2223-7747},
abstract = {Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene's function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is an efficient reverse screen method in plant functional analysis. Precise modification of targeted crop genome sequences is possible through the stable and/or transient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) reagents. Currently, CRISPR/Cas9 is the most powerful reverse genetics approach for fast and precise functional analysis of candidate genes/mutations of interest. Rapid and large-scale analyses of CRISPR/Cas-induced mutagenesis is achievable through Agrobacterium rhizogenes-mediated hairy root transformation. The combination of A. rhizogenes hairy root-CRISPR/Cas provides an extraordinary platform for rapid, precise, easy, and cost-effective "in root" functional analysis of genes of interest in legume plants, including soybean. Both hairy root transformation and CRISPR/Cas9 techniques have their own complexities and considerations. Here, we discuss recent advancements in soybean hairy root transformation and CRISPR/Cas9 techniques. We highlight the critical factors required to enhance mutation induction and hairy root transformation, including the new generation of reporter genes, methods of Agrobacterium infection, accurate gRNA design strategies, Cas9 variants, gene regulatory elements of gRNAs and Cas9 nuclease cassettes and their configuration in the final binary vector to study genes involved in root-related traits in soybean.},
}
@article {pmid35447246,
year = {2022},
author = {Kanafi, MM and Tavallaei, M},
title = {Overview of advances in CRISPR/deadCas9 technology and its applications in human diseases.},
journal = {Gene},
volume = {830},
number = {},
pages = {146518},
doi = {10.1016/j.gene.2022.146518},
pmid = {35447246},
issn = {1879-0038},
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Gene Editing ; Gene Expression ; Humans ; *RNA, Guide ; Technology ; },
abstract = {Prokaryotes possess an adaptive immune system using various CRISPR associated (Cas) genes to make an archive of records from invading phages and eliminate them upon re-exposure when specialized Cas proteins cut foreign DNA into small pieces. On the basis of the different types of Cas proteins, CRISPR systems seen in some prokaryotic genomes, are different to each other. It has been proved that CRISPR has a great potential for genome engineering. Studies have also demonstrated that in comparison to the preceding genome engineering tools CRISPR/Cas systems can be harnessed as a flexible tool with easy multiplexing and scaling ability. Recent studies suggest that CRISPR/Cas systems can also be used for non-genome engineering roles. Isolation and identification of new Cas proteins or modification of existing ones are effectively increasing the number of CRISPR applications and helps its development. D10A and H840A mutations at RuvC and HNH endonuclease domains of wild type Streptococcus pyogenes Cas9 (SpCas9) respectively creates a nuclease, dead Cas9 (dCas9) molecule, that does not cut target DNA but still retains its capability for binding to target DNA based on the gRNA targeting sequence. In this article we review the potentials of this enzyme, dCas9, toward development of the applications of CRISPR/dCas9 technology in fields such as; visualization of genomic loci, disease diagnosis and transcriptional repression and activation.},
}
@article {pmid35446558,
year = {2022},
author = {Chen, Z and Devi, G and Arif, A and Zamore, PD and Sontheimer, EJ and Watts, JK},
title = {Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing.},
journal = {ACS chemical biology},
volume = {17},
number = {5},
pages = {1045-1050},
pmid = {35446558},
issn = {1554-8937},
support = {R35 GM136275/GM/NIGMS NIH HHS/United States ; UG3 TR002668/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *RNA, Guide/chemistry/genetics ; },
abstract = {CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.},
}
@article {pmid35446444,
year = {2022},
author = {Natsuga, K and Furuta, Y and Takashima, S and Nohara, T and Huang, HY and Shinkuma, S and Nakamura, H and Katsuda, Y and Higashi, H and Hsu, CK and Fukushima, S and Ujiie, H},
title = {Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.},
journal = {Human mutation},
volume = {43},
number = {7},
pages = {877-881},
doi = {10.1002/humu.24385},
pmid = {35446444},
issn = {1098-1004},
mesh = {CRISPR-Cas Systems ; *Collagen Type VII/genetics ; *Epidermolysis Bullosa Dystrophica/genetics/pathology/therapy ; Genes, Recessive ; Haplotypes ; Humans ; Mutation ; },
abstract = {An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.},
}
@article {pmid35446403,
year = {2022},
author = {Liu, X and Xiong, W and Qi, Q and Zhang, Y and Ji, H and Cui, S and An, J and Sun, X and Yin, H and Tian, T and Zhou, X},
title = {Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing.},
journal = {Nucleic acids research},
volume = {50},
number = {8},
pages = {4769-4783},
pmid = {35446403},
issn = {1362-4962},
mesh = {Humans ; *RNA, Guide/genetics/metabolism ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Ligands ; Plasmids ; },
abstract = {It is important to control CRISPR/Cas9 when sufficient editing is obtained. In the current study, rational engineering of guide RNAs (gRNAs) is performed to develop small-molecule-responsive CRISPR/Cas9. For our purpose, the sequence of gRNAs are modified to introduce ligand binding sites based on the rational design of ligand-RNA pairs. Using short target sequences, we demonstrate that the engineered RNA provides an excellent scaffold for binding small molecule ligands. Although the 'stem-loop 1' variants of gRNA induced variable cleavage activity for different target sequences, all 'stem-loop 3' variants are well tolerated for CRISPR/Cas9. We further demonstrate that this specific ligand-RNA interaction can be utilized for functional control of CRISPR/Cas9 in vitro and in human cells. Moreover, chemogenetic control of gene editing in human cells transfected with all-in-one plasmids encoding Cas9 and designer gRNAs is demonstrated. The strategy may become a general approach for generating switchable RNA or DNA for controlling other biological processes.},
}
@article {pmid35446391,
year = {2022},
author = {Gleerup, JL and Mogensen, TH},
title = {CRISPR-Cas in Diagnostics and Therapy of Infectious Diseases.},
journal = {The Journal of infectious diseases},
volume = {226},
number = {11},
pages = {1867-1876},
doi = {10.1093/infdis/jiac145},
pmid = {35446391},
issn = {1537-6613},
mesh = {Humans ; CRISPR-Cas Systems ; *Communicable Diseases/diagnosis/therapy ; Bacteria/genetics ; Genome ; *Anti-Infective Agents ; },
abstract = {Infectious diseases are a major threat to the global health. The rise in antimicrobial-resistant organisms, incurable chronic infections, and an increasing demand for rapid accurate diagnostics have prompted researchers to experiment with new approaches. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) is a naturally occurring adaptive immune system in bacteria that has been developed as a tool for performing genomic alterations in any genome of interest, including humans and microbes. Accordingly, several studies have been conducted to investigate how the technology can be utilized in infectious diseases to improve diagnostics, disrupt antimicrobial resistance, and cure chronic infections. This review provides an overview of the CRISPR-Cas system and how it has been applied in studies on infectious diseases. The review also investigates the current challenges of the technology and the improvements that are needed for the platform to be adopted for clinical use in patients.},
}
@article {pmid35445018,
year = {2022},
author = {Hu, Y and Li, W},
title = {Development and Application of CRISPR-Cas Based Tools.},
journal = {Frontiers in cell and developmental biology},
volume = {10},
number = {},
pages = {834646},
pmid = {35445018},
issn = {2296-634X},
abstract = {Abundant CRISPR-Cas systems in nature provide us with unlimited valuable resources to develop a variety of versatile tools, which are powerful weapons in biological discovery and disease treatment. Here, we systematically review the development of CRISPR-Cas based tools from DNA nuclease to RNA nuclease, from nuclease dependent-tools to nucleic acid recognition dependent-tools. Also, considering the limitations and challenges of current CRISPR-Cas based tools, we discuss the potential directions for development of novel CRISPR toolkits in the future.},
}
@article {pmid35443934,
year = {2022},
author = {Zhou, J and Liu, Y and Wei, Y and Zheng, S and Gou, S and Chen, T and Yang, Y and Lan, T and Chen, M and Liao, Y and Zhang, Q and Tang, C and Liu, Y and Wu, Y and Peng, X and Gao, M and Wang, J and Zhang, K and Lai, L and Zou, Q},
title = {Eliminating predictable DNA off-target effects of cytosine base editor by using dual guiders including sgRNA and TALE.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {7},
pages = {2443-2451},
pmid = {35443934},
issn = {1525-0024},
mesh = {CRISPR-Cas Systems ; *Cytosine ; DNA/genetics ; Gene Editing ; *RNA, Guide/genetics ; Transcription Activator-Like Effectors/genetics ; },
abstract = {Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations.},
}
@article {pmid35442747,
year = {2022},
author = {Zou, Y and Sun, X and Yang, Q and Zheng, M and Shimoni, O and Ruan, W and Wang, Y and Zhang, D and Yin, J and Huang, X and Tao, W and Park, JB and Liang, XJ and Leong, KW and Shi, B},
title = {Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.},
journal = {Science advances},
volume = {8},
number = {16},
pages = {eabm8011},
pmid = {35442747},
issn = {2375-2548},
mesh = {Animals ; Blood-Brain Barrier ; CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; *Glioblastoma/genetics/therapy ; Mice ; *Nanocapsules ; RNA, Guide/genetics ; },
abstract = {We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.},
}
@article {pmid35440677,
year = {2022},
author = {de Souza Pacheco, I and Doss, AA and Vindiola, BG and Brown, DJ and Ettinger, CL and Stajich, JE and Redak, RA and Walling, LL and Atkinson, PW},
title = {Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {6428},
pmid = {35440677},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Hemiptera/genetics ; Pigmentation/genetics ; },
abstract = {CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.},
}
@article {pmid35440579,
year = {2022},
author = {de Rooij, MFM and Thus, YJ and Swier, N and Beijersbergen, RL and Pals, ST and Spaargaren, M},
title = {A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {2136},
pmid = {35440579},
issn = {2041-1723},
mesh = {Agammaglobulinaemia Tyrosine Kinase/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Adhesion/genetics ; Humans ; Integrins/metabolism ; *Leukemia/drug therapy ; Protein Kinase Inhibitors/therapeutic use ; Signal Transduction ; Tumor Microenvironment ; },
abstract = {The clinical introduction of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which targets B-cell antigen-receptor (BCR)-controlled integrin-mediated retention of malignant B cells in their growth-supportive lymphoid organ microenvironment, provided a major breakthrough in lymphoma and leukemia treatment. Unfortunately, a significant subset of patients is intrinsically resistant or acquires resistance against ibrutinib. Here, to discover novel therapeutic targets, we present an unbiased loss-of-adhesion CRISPR-Cas9 knockout screening method to identify proteins involved in BCR-controlled integrin-mediated adhesion. Illustrating the validity of our approach, several kinases with an established role in BCR-controlled adhesion, including BTK and PI3K, both targets for clinically applied inhibitors, are among the top hits of our screen. We anticipate that pharmacological inhibitors of the identified targets, e.g. PAK2 and PTK2B/PYK2, may have great clinical potential as therapy for lymphoma and leukemia patients. Furthermore, this screening platform is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.},
}
@article {pmid35440051,
year = {2022},
author = {Tao, R and Wang, Y and Hu, Y and Jiao, Y and Zhou, L and Jiang, L and Li, L and He, X and Li, M and Yu, Y and Chen, Q and Yao, S},
title = {WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {108},
pmid = {35440051},
issn = {2059-3635},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3' terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.},
}
@article {pmid35438517,
year = {2022},
author = {Sherkow, JS},
title = {Immaculate Conception? Priority and Invention in the CRISPR Patent Dispute.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {174-180},
doi = {10.1089/crispr.2022.0033},
pmid = {35438517},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Dissent and Disputes ; *Gene Editing ; Inventions ; RNA, Guide/genetics ; },
abstract = {The U.S. Patent Trial and Appeal Board (PTAB), in an interference proceeding decided in February 2022, concluded that researchers at the Broad Institute (Cambridge, MA) were the first to "conceive" of using single-guide RNA CRISPR-Cas9 genome editing in eukaryotic cells in 2012. The PTAB reached this verdict even though competing researchers at the University of California, Berkeley, among other institutions, could document the idea 7 months earlier. Understanding the basis for the PTAB's decision turns on patent law's particular "conception" requirement. In this study, I explain that requirement, detail the PTAB's interference decision, and discuss the decision's practical effects on CRISPR technology and routine science.},
}
@article {pmid35438515,
year = {2022},
author = {Zuo, Z and Babu, K and Ganguly, C and Zolekar, A and Newsom, S and Rajan, R and Wang, YC and Liu, J},
title = {Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {329-340},
pmid = {35438515},
issn = {2573-1602},
support = {R15 HL147265/HL/NHLBI NIH HHS/United States ; P20 GM103640/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; Endonucleases/genetics ; *Gene Editing ; Humans ; RNA/chemistry ; },
abstract = {The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.},
}
@article {pmid35438514,
year = {2022},
author = {Sun, W and Wang, Y},
title = {SuperFi-Cas9: High Fidelity Meets High Activity.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {171-173},
doi = {10.1089/crispr.2022.29146.ywa},
pmid = {35438514},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide ; },
}
@article {pmid35438513,
year = {2022},
author = {Barrangou, R},
title = {CRISPR à la carte.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {170},
doi = {10.1089/crispr.2022.29147.rba},
pmid = {35438513},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
}
@article {pmid35437598,
year = {2022},
author = {Yagita, Y and Abe, Y and Fujiki, Y},
title = {De novo formation and maintenance of mammalian peroxisomes in cultured PEX16-knockout cells generated by CRISPR/Cas9.},
journal = {Journal of cell science},
volume = {135},
number = {9},
pages = {},
doi = {10.1242/jcs.258377},
pmid = {35437598},
issn = {1477-9137},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; Intracellular Membranes/metabolism ; Mammals/metabolism ; Membrane Proteins/metabolism ; *Peroxisomes/metabolism ; },
abstract = {Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16.},
}
@article {pmid35436106,
year = {2022},
author = {Park, H and Osman, EA and Cromwell, CR and St Laurent, CD and Liu, Y and Kitova, EN and Klassen, JS and Hubbard, BP and Macauley, MS and Gibbs, JM},
title = {CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.},
journal = {Bioconjugate chemistry},
volume = {33},
number = {5},
pages = {858-868},
doi = {10.1021/acs.bioconjchem.2c00106},
pmid = {35436106},
issn = {1520-4812},
mesh = {Alkynes ; Azides/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide/genetics/metabolism ; },
abstract = {Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.},
}
@article {pmid35436041,
year = {2022},
author = {Liu, X and Cui, Z and Su, T and Lu, X and Hou, J and Qi, Q},
title = {Identification of genome integration sites for developing a CRISPR-based gene expression toolkit in Yarrowia lipolytica.},
journal = {Microbial biotechnology},
volume = {15},
number = {8},
pages = {2223-2234},
pmid = {35436041},
issn = {1751-7915},
mesh = {CRISPR-Cas Systems ; Gene Expression ; Lycopene/metabolism ; Metabolic Engineering ; *Yarrowia/genetics/metabolism ; },
abstract = {With the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR-based gene expression toolkit of Y. lipolytica. By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR-based gene expression toolkit developed in this study will facilitate the genetic engineering of Y. lipolytica.},
}
@article {pmid35435904,
year = {2022},
author = {Hu, C and Doerksen, T and Bugbee, T and Wallace, NA and Palinski, R},
title = {Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {181},
pages = {},
doi = {10.3791/62583},
pmid = {35435904},
issn = {1940-087X},
support = {P20 GM130448/GM/NIGMS NIH HHS/United States ; R15 CA242057/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; High-Throughput Nucleotide Sequencing ; Mutation ; },
abstract = {Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.},
}
@article {pmid35435766,
year = {2022},
author = {Vaca, DJ and Thibau, A and Leisegang, MS and Malmström, J and Linke, D and Eble, JA and Ballhorn, W and Schaller, M and Happonen, L and Kempf, VAJ},
title = {Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells.},
journal = {Microbiology spectrum},
volume = {10},
number = {3},
pages = {e0059822},
pmid = {35435766},
issn = {2165-0497},
mesh = {Adhesins, Bacterial/metabolism ; Bacterial Adhesion ; *Bartonella ; *Bartonella henselae/genetics/metabolism ; Cell Adhesion ; Endothelial Cells/metabolism/microbiology ; Fibronectins/metabolism ; Humans ; Type V Secretion Systems/metabolism ; },
abstract = {Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.},
}
@article {pmid35433512,
year = {2022},
author = {Lu, P and Chen, J and Li, Z and Li, Z and Zhang, J and Kan, B and Pang, B},
title = {Visual Identification and Serotyping of Toxigenic Vibrio cholerae Serogroups O1 and O139 With CARID.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {863435},
pmid = {35433512},
issn = {2235-2988},
mesh = {*Cholera/diagnosis/microbiology ; Cholera Toxin ; Humans ; Serogroup ; Serotyping ; *Vibrio cholerae O1/genetics ; },
abstract = {There is a growing demand for rapid, sensitive, field-deployable nucleic acid tests for cholera, which usually occurs in rural areas. In this study, we developed a Cas12a-assisted rapid isothermal detection (CARID) system for the detection of toxigenic V. cholerae serogroups O1 and O139 by combining recombinase-aided amplification and CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). The results can be determined by fluorescence signal and visualized by lateral flow dipstick. We identified 154 V. cholerae strains and 129 strains of other intestinal diarrheagenic bacteria with a 100% coincidence rate. The limit of detection of CARID was 20 copies/reaction of V. cholerae genomic DNA, which is comparable to that of polymerase chain reaction (PCR) and qPCR. Multiple-CARID was also established for efficiency and economic considerations with an acceptable decrease in sensitivity. Simulated sample tests showed that CARID is suitable for complex samples. In conclusion, CARID is a rapid, sensitive, economically efficient, and portable method for the detection of V. cholerae, which makes it suitable for field responses to cholera.},
}
@article {pmid35432328,
year = {2022},
author = {Moraes, L and Trentini, MM and Fousteris, D and Eto, SF and Chudzinski-Tavassi, AM and Leite, LCC and Kanno, AI},
title = {CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {867195},
pmid = {35432328},
issn = {1664-3224},
mesh = {Adjuvants, Immunologic ; Adjuvants, Pharmaceutic ; Animals ; BCG Vaccine/genetics ; CRISPR-Cas Systems ; Escherichia coli ; Mice ; *Tuberculosis ; *Tuberculosis Vaccines/genetics ; },
abstract = {Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.},
}
@article {pmid35430708,
year = {2022},
author = {Bhoria, S and Yadav, J and Yadav, H and Chaudhary, D and Jaiwal, R and Jaiwal, PK},
title = {Current advances and future prospects in production of recombinant insulin and other proteins to treat diabetes mellitus.},
journal = {Biotechnology letters},
volume = {44},
number = {5-6},
pages = {643-669},
pmid = {35430708},
issn = {1573-6776},
mesh = {Animals ; Cell Differentiation ; Cell Line ; *Diabetes Mellitus/drug therapy/metabolism ; Insulin/metabolism ; *Insulin-Secreting Cells/metabolism ; *Islets of Langerhans/metabolism ; Mammals/metabolism ; },
abstract = {Diabetes mellitus is the most prevalent deadly disease caused by the destruction and dysfunction of pancreatic β cells that consequentially increased blood glucose levels. The management of this disease via external administration of insulin/insulin analogs has been difficult and challenging due to their limited production and accessibility at affordable prices. The conventional insulin production platforms (Escherichia coli, Saccharomyces cerevisiae and mammalian cell lines) with limited scalability and high upstream process costs have not been successful in meeting the rapidly increasing insulin demands. However, plants have been used as safe, scalable, environmentally friendly and cost-effective high capacity production platforms for recombinant orally delivered insulin. Recent technological advances in genome engineering and editing technologies for adequate insulin and insulin analogs production, renewable cellular sources of insulin through transplantation of islets or insulin-producing cells and reprogramming or differentiation of non β cells into β-like cells, used either alone or in combination, for diabetes containment are reviewed here along with their future prospects.},
}
@article {pmid35430408,
year = {2022},
author = {Moon, J and Song, J and Jang, H and Kang, H and Huh, YM and Son, HY and Rho, HW and Park, M and Talwar, CS and Park, KH and Woo, E and Lim, J and Lim, EK and Jung, J and Jung, Y and Park, HG and Kang, T},
title = {Ligation-free isothermal nucleic acid amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {209},
number = {},
pages = {114256},
doi = {10.1016/j.bios.2022.114256},
pmid = {35430408},
issn = {1873-4235},
mesh = {Animals ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Mice ; Nucleic Acid Amplification Techniques/methods ; RNA ; RNA, Messenger ; },
abstract = {In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.},
}
@article {pmid35430120,
year = {2022},
author = {Kleinboehl, E and Laoharawee, K and Moriarity, BS},
title = {Primary B cell engineering for therapeutic research.},
journal = {Trends in molecular medicine},
volume = {28},
number = {6},
pages = {528-529},
doi = {10.1016/j.molmed.2022.03.007},
pmid = {35430120},
issn = {1471-499X},
mesh = {B-Lymphocytes ; CRISPR-Cas Systems ; Cell Engineering ; *Gene Editing ; Genetic Engineering ; Humans ; *Therapeutic Human Experimentation ; },
}
@article {pmid35429769,
year = {2022},
author = {Pan, Y and Luan, X and Zeng, F and Xu, Q and Li, Z and Gao, Y and Liu, X and Li, X and Han, X and Shen, J and Song, Y},
title = {Hollow covalent organic framework-sheltering CRISPR/Cas12a as an in-vivo nanosensor for ATP imaging.},
journal = {Biosensors &amp; bioelectronics},
volume = {209},
number = {},
pages = {114239},
doi = {10.1016/j.bios.2022.114239},
pmid = {35429769},
issn = {1873-4235},
mesh = {Adenosine Triphosphate ; Animals ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded ; Gene Editing/methods ; *Metal-Organic Frameworks ; Oligonucleotides ; },
abstract = {In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.},
}
@article {pmid35429445,
year = {2022},
author = {Chen, Q and Wen, Y},
title = {Target recognition triggered CRISPR-Cas12a assisted allosteric scaffold for sensitively analyzing bacterial infection after dental implantation.},
journal = {Analytical biochemistry},
volume = {647},
number = {},
pages = {114666},
doi = {10.1016/j.ab.2022.114666},
pmid = {35429445},
issn = {1096-0309},
mesh = {*Bacterial Infections/diagnosis/genetics ; *CRISPR-Cas Systems ; DNA ; Dental Implantation ; Humans ; Limit of Detection ; },
abstract = {Accurate identification and sensitive quantification of infected bacteria after dental implant both play crucial roles in early-diagnosis of bacterial infection and guiding medicine applications. Herein, we propose a sensitive and accurate bacteria detection method based on CRISPR-Cas12a system assisted allosteric scaffold. In the method, the allosteric scaffold takes the responsibility of specifically identifying target bacteria and inducing CRISPR-Cas12a based signal amplification. Eventually, the method exhibits a wide detection range from 6 × 10[6] cfu/mL to 6 × 10[2] cfu/mL with the limit of detection (LOD) of 47 cfu/mL. Furthermore, the established approach also possesses a high specificity due to high selectivity of aptamer and robust accuracy in recognizing double strand DNA by CRISPR-Cas12a system. We believe that this work can provide new strategies in the field of diagnosing bacterial infections after dental implantation.},
}
@article {pmid35429317,
year = {2022},
author = {Razzaq, MK and Akhter, M and Ahmad, RM and Cheema, KL and Hina, A and Karikari, B and Raza, G and Xing, G and Gai, J and Khurshid, M},
title = {CRISPR-Cas9 based stress tolerance: New hope for abiotic stress tolerance in chickpea (Cicer arietinum).},
journal = {Molecular biology reports},
volume = {49},
number = {9},
pages = {8977-8985},
pmid = {35429317},
issn = {1573-4978},
mesh = {CRISPR-Cas Systems/genetics ; *Cicer/genetics ; Plant Breeding ; Plants ; Stress, Physiological/genetics ; },
abstract = {Plants are subjected to biotic and abiotic stresses regularly, which irreparably harm agricultural production. Eco-friendly and sustainable technology to deal with this challenge is to breed abiotic stress tolerant cultivars. To generate crop plants conferring resistance against stresses, conventional breeding was used in the past, but because of the complex heredity of abiotic stress tolerance traits, such techniques remain insufficient in making greater enhancement. Genome-engineering based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein9) has shown enormous potential in developing climate-resilient cultivars. Likewise, the development of chickpea transgenic lines by knockout of 4CL and REV7 genes exhibits drought tolerance which establishes a foundation for future studies in chickpea. In addition, the CRISPR-Cas9 system can boost yield potential under abiotic stress situations by producing non-transgenic plants having the required characteristics. This review article discusses the validation of gene function based on the CRISPR-Cas9 for the development of abiotic stress-tolerant crop plants, emphasizing the chickpea to open the new ventures of generating abiotic stress-tolerant chickpea varieties.},
}
@article {pmid35429082,
year = {2022},
author = {Rylee, JC and Nin-Velez, A and Mahato, S and Helms, KJ and Wade, MJ and Zentner, GE and Zelhof, AC},
title = {Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum.},
journal = {Insect molecular biology},
volume = {31},
number = {5},
pages = {543-550},
pmid = {35429082},
issn = {1365-2583},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics ; Gene Editing ; *Tribolium/genetics ; },
abstract = {CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3' UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR- mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.},
}
@article {pmid35427904,
year = {2022},
author = {Lee, HM and Kim, AH and Hwang, S and Jung, J and Seol, H and Sung, JJ and Jeong, SM and Choi, YM and Jun, JK and Kim, HS and Jang, J},
title = {Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102779},
doi = {10.1016/j.scr.2022.102779},
pmid = {35427904},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Gene Targeting ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Myosin Heavy Chains/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is a major component of cardiac muscle filaments involved in cardiac muscle contraction. Here, we established an αMHC-enhanced fluorescent protein (EGFP) knock-in human pluripotent stem cell (hPSC) line by linking the EGFP gene to the C-terminal region of αMHC via a 2A non-joining peptide using CRISPR/Cas9 nuclease. The EGFP reporter precisely reflected the endogenous level of αMHC upon the induction of cardiac differentiation. This reporter cell line will be a valuable platform for cardiotoxicity tests, drug screening, and investigating the pathological mechanisms of cardiomyocytes.},
}
@article {pmid35427721,
year = {2022},
author = {Avci-Adali, M and A Santos, H},
title = {Current trends in delivery of non-viral nucleic acid-based therapeutics for improved efficacy.},
journal = {Advanced drug delivery reviews},
volume = {185},
number = {},
pages = {114297},
pmid = {35427721},
issn = {1872-8294},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; Nanomedicine ; *Nucleic Acids ; },
}
@article {pmid35427441,
year = {2022},
author = {Uetz, P and Melnik, S and Grünwald-Gruber, C and Strasser, R and Stoger, E},
title = {CRISPR/Cas9-mediated knockout of a prolyl-4-hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100698},
doi = {10.1002/biot.202100698},
pmid = {35427441},
issn = {1860-7314},
support = {W 1224/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; Plants, Genetically Modified/genetics/metabolism ; Prolyl Hydroxylases/genetics/metabolism ; RNA, Guide/metabolism ; *Tobacco/genetics/metabolism ; },
abstract = {The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the number of peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.},
}
@article {pmid35427438,
year = {2022},
author = {Kelkar, A and Groth, T and Neelamegham, S},
title = {Forward Genetic Screens of Human Glycosylation Pathways Using the GlycoGene CRISPR Library.},
journal = {Current protocols},
volume = {2},
number = {4},
pages = {e402},
pmid = {35427438},
issn = {2691-1299},
support = {R01 HL103411/HL/NHLBI NIH HHS/United States ; R21 GM133195/GM/NIGMS NIH HHS/United States ; HL103411/GF/NIH HHS/United States ; GM133195/GF/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Library ; *Genetic Testing ; Glycosylation ; Humans ; Lectins/genetics ; },
abstract = {CRISPR-Cas9-based forward genetic screens represent a powerful discovery platform to uncover genes regulating specific biological processes. This article describes a method for utilizing a freely available GlycoGene CRISPR library to knock out any gene participating in human glycosylation in arbitrary cell types. The end product is a stable GlycoGene CRISPR knockout cell library, where each cell contains one or more sgRNA and lacks corresponding function. The cell library can be screened using various lectin/antibody reagents. It can also be applied in functional assays to establish glycan structure-glycogene-glycopathway relationships. This is a powerful systems glycobiology strategy for dissecting glycosylation pathways and processes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Scale-up and NGS validation of the GlycoGene CRISPR plasmid library Basic Protocol 2: Preparation of a GlycoGene CRISPR lentivirus pool and an isogenic cell line stably expressing Cas9 nuclease Basic Protocol 3: Preparation of a GlycoGene CRISPR cell library, self-inactivation of Cas9, and library validation by NGS Basic Protocol 4: Enrichment of lectin-binding or non-binding cells and related multiplex NGS data acquisition Basic Protocol 5: Bioinformatics pathway analysis.},
}
@article {pmid35426306,
year = {2022},
author = {Gao, ZF and Zheng, LL and Dong, LM and Li, JZ and Shen, Y and Chen, P and Xia, F},
title = {Label-Free Resonance Rayleigh Scattering Amplification for Lipopolysaccharide Detection and Logical Circuit by CRISPR/Cas12a-Driven Guanine Nanowire Assisted Non-Cross-Linking Hybridization Chain Reaction.},
journal = {Analytical chemistry},
volume = {94},
number = {16},
pages = {6371-6379},
doi = {10.1021/acs.analchem.2c00848},
pmid = {35426306},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; Guanine ; Lipopolysaccharides ; Logic ; *Nanowires ; },
abstract = {Although the CRISPR/Cas system has pioneered a new generation of analytical techniques, there remain many challenges in developing a label-free, accurate, and reliable CRISPR/Cas-based assay for reporting the levels of low abundance biomolecules in complex biological samples. Here, we reported a novel CRISPR-derived resonance Rayleigh scattering (RRS) amplification strategy and logical circuit based on a guanine nanowire (G-wire) assisted non-cross-linking hybridization chain reaction (GWancHCR) for label-free detection of lipopolysaccharide (LPS). In the presence of a target, the protospacer-adjacent motif-inserted aptamer is rationally designed to specifically combine with LPS rather than Cas12a, suppressing the trans-cleavage activity of CRISPR/Cas12a and retaining the reporter probes to trigger non-cross-linking aggregation. Owing to the automatic hybridization chain reaction (HCR), in the presence of Mg[2+], the released G-quadruplex sequence aggregated to assemble the G-wire superstructure through non-cross-linking. As a result, a dramatically amplified RRS intensity is observed, allowing for reporting LPS levels in a low detection limit of 0.17 pg/mL and a wide linear range among 1.0-100.0 ng/mL. Moreover, this reaction event is capable of programming to perform classical Boolean logic tree analysis, including basic logic computing and complex integrated logic circuits. This study comprehensively analyzed with respect to information flow, matter (molecular events), and energy (RRS), revealing the potential promise in designing of molecular-level "Internet of Things", intelligent computing, and sensing systems.},
}
@article {pmid35426186,
year = {2022},
author = {Xu, X and Zhang, X and Peng, X and Liu, C and Li, W and Liu, M},
title = {Comparison of the efficiency and precision of Base editor and CRISPR/Cas9 for inducing defined point mutation (S395F) in ovine embryos.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {57},
number = {8},
pages = {829-838},
doi = {10.1111/rda.14124},
pmid = {35426186},
issn = {1439-0531},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods/veterinary ; Microinjections/veterinary ; Mutation ; *Point Mutation ; RNA, Messenger/genetics ; Sheep/genetics ; },
abstract = {Cytosine base editors (CBEs) and CRISPR/Cas9-mediated HDR method both have the ability to introduce nucleotide substitution into genomes, which exhibit great potential for improving economically important traits in livestock species. The FecG[H] mutation (g. C1184T, p. S395F) of growth differentiation factor 9 (GDF9) gene increases prolificacy in Cambridge sheep and Belclare sheep. In the present study, we aimed to compare the efficiency and precision of BE4-Gam and CRISPR/Cas9 systems on generating FecG[H] mutation in ovine genome. First, the microinjection of BE4-Gam mRNA had no adverse effects on development rate after cleavage, and the efficiencies of total mutants and targeted mutants were 8.9% and 7.1%, respectively. Then, the total mutation and targeted mutation rates were improved from 8.5% to 22.5% (p < .01), and 6.4% to 16.3%, respectively, by adjusting the injection time of BE4-Gam mRNA from 14 to 12 hr post-insemination (hpi). Furthermore, CRISPR/Cas9-mediated HDR method introduced the FecG[H] mutation at the efficiency of 16.1%, which was comparable to BE4-Gam system (16.3%). There was no bystander editing event happened in edited embryos caused by CRISPR/Cas9, but the bystander editing efficiency was as high as 15.0% in BE4-Gam-edited embryos. In summary, our findings demonstrated that CRISPR/Cas9-mediated HDR method was more accurate than BE4-Gam system in introducing FecG[H] into ovine genome, and highlight the potential of the former strategy to modify economically important trait-associated SNPs.},
}
@article {pmid35422794,
year = {2022},
author = {Kamruzzaman, M and Yan, A and Castro-Escarpulli, G},
title = {Editorial: CRISPR-Cas Systems in Bacteria and Archaea.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {887778},
pmid = {35422794},
issn = {1664-302X},
}
@article {pmid35422517,
year = {2022},
author = {Sinan, S and Russell, R},
title = {A tweak and a peek: How Cas9 pries open double-stranded DNA to check its sequence.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {4},
pages = {286-288},
pmid = {35422517},
issn = {1545-9985},
mesh = {*CRISPR-Cas Systems ; *DNA ; },
}
@article {pmid35422516,
year = {2022},
author = {Cofsky, JC and Soczek, KM and Knott, GJ and Nogales, E and Doudna, JA},
title = {CRISPR-Cas9 bends and twists DNA to read its sequence.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {4},
pages = {395-402},
pmid = {35422516},
issn = {1545-9985},
support = {/HHMI/Howard Hughes Medical Institute/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/metabolism ; Endonucleases/metabolism ; Gene Editing ; *RNA, Guide/metabolism ; },
abstract = {In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.},
}
@article {pmid35422459,
year = {2022},
author = {},
title = {Forum: CRISPR screening roundtable with Stegmaier and Doench.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {655},
doi = {10.1038/s41587-022-01303-4},
pmid = {35422459},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *Mass Screening ; Research ; },
}
@article {pmid35422284,
year = {2022},
author = {Li, Q and Lv, X and Tang, C and Yin, C},
title = {Co-delivery of doxorubicin and CRISPR/Cas9 or RNAi-expressing plasmid by chitosan-based nanoparticle for cancer therapy.},
journal = {Carbohydrate polymers},
volume = {287},
number = {},
pages = {119315},
doi = {10.1016/j.carbpol.2022.119315},
pmid = {35422284},
issn = {1879-1344},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Chitosan ; Doxorubicin/pharmacology ; Drug Carriers ; Drug Delivery Systems/methods ; Folic Acid ; *Frontotemporal Dementia ; Humans ; *Nanoparticles ; *Neoplasms/drug therapy/genetics ; Plasmids/genetics ; RNA Interference ; Survivin/genetics/metabolism ; },
abstract = {Folic acid (FA) and 2-(Diisopropylamino) ethyl methacrylate (DPA) double grafted trimethyl chitosan (TMC) nanoparticles (FTD NPs) were synthesized for the co-delivery of doxorubicin (DOX) and Survivin CRISPR/Cas9-expressing plasmid (sgSurvivin pDNA) or Survivin shRNA-expressing plasmid (iSur pDNA). FA modification enhanced the uptake of DOX and pDNA loaded into FTD NPs in tumor cells. A rapid release of DOX was triggered under acidic conditions due to pH-sensitiveness of FTD NPs arising from DPA conjugation. Negligible differences between FTD/sgSurvivin pDNA NPs and FTD/iSur pDNA NPs demonstrated that RNA interference (RNAi) and CRISPR/Cas9 technologies possessed comparable antitumor efficiency. Notably, the in vitro and in vivo antitumor efficacies of FTD/DOX/sgSurvivin pDNA NPs were superior to those of single delivery of DOX or sgSurvivin pDNA, while were comparable to those of FTD/DOX/iSur pDNA NPs. These results suggested that the combination of chemotherapeutics and CRISPR/Cas9 systems would provide a potential modality for cancer therapy.},
}
@article {pmid35421847,
year = {2022},
author = {Zhang, F and Meier, AB and Sinnecker, D and Engelhardt, S and Lipp, P and Laugwitz, KL and Dorn, T and Moretti, A},
title = {Generation of heterozygous (MRli003-A-5) and homozygous (MRli003-A-6) voltage-sensing knock-in human iPSC lines by CRISPR/Cas9 editing of the AAVS1 locus.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102785},
doi = {10.1016/j.scr.2022.102785},
pmid = {35421847},
issn = {1876-7753},
support = {788381/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; },
abstract = {Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.},
}
@article {pmid35421656,
year = {2022},
author = {Fu, R and Wang, Y and Liu, Y and Liu, H and Zhao, Q and Zhang, Y and Wang, C and Li, Z and Jiao, B and He, Y},
title = {CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.},
journal = {Food chemistry},
volume = {387},
number = {},
pages = {132919},
doi = {10.1016/j.foodchem.2022.132919},
pmid = {35421656},
issn = {1873-7072},
mesh = {Acetylcholinesterase/genetics ; *Biosensing Techniques ; CRISPR-Cas Systems ; *DNA, Catalytic ; Manganese Compounds ; Organophosphorus Compounds ; Oxides ; *Pesticides ; },
abstract = {Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn[2+] ions to activate the Mn[2+]-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.},
}
@article {pmid35421582,
year = {2022},
author = {Chen, S and Liu, Z and Yu, H and Lai, L and Li, Z},
title = {Efficient multinucleotide deletions using deaminase-Cas9 fusions in human cells.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {49},
number = {10},
pages = {927-933},
doi = {10.1016/j.jgg.2022.03.007},
pmid = {35421582},
issn = {1673-8527},
mesh = {Humans ; Rats ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Cytidine Deaminase/genetics/metabolism ; INDEL Mutation/genetics ; APOBEC-1 Deaminase/genetics ; },
abstract = {CRISPR/Cas9 system is a robust genome editing platform in biotechnology and medicine. However, it generally produces small insertions/deletions (indels, typically 1-3 bp) but rarely induces larger deletions in specific target sites. Here, we report a cytidine deaminase-Cas9 fusion-induced deletion system (C-DEL) and an adenine deaminase-Cas9 fusion-induced deletion system (A-DEL) by combining Cas9 with rat APOBEC1 (rA1) and TadA 8e, respectively. Both C-DEL and A-DEL improve the efficiency of deletions compared with the conventional Cas9 system in human cells. In addition, the C-DEL system generates a considerable fraction of predictable multinucleotide deletions from 5'-deaminated C bases to the Cas9-cleavage site and increases the proportion of larger deletions at the target loci. Taken together, the C-DEL and A-DEL systems provide a practical strategy for producing efficient multinucleotide deletions, expanding the CRISPR/Cas9 toolsets for gene modifications in human cells.},
}
@article {pmid35421352,
year = {2022},
author = {Bari, SMN and Chou-Zheng, L and Howell, O and Hossain, M and Hill, CM and Boyle, TA and Cater, K and Dandu, VS and Thomas, A and Aslan, B and Hatoum-Aslan, A},
title = {A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {4},
pages = {570-582.e7},
doi = {10.1016/j.chom.2022.03.001},
pmid = {35421352},
issn = {1934-6069},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Multifunctional Enzymes/genetics ; *Nucleic Acids ; Staphylococcus Phages/genetics ; },
abstract = {The perpetual arms race between bacteria and their viruses (phages) has given rise to diverse immune systems, including restriction-modification and CRISPR-Cas, which sense and degrade phage-derived nucleic acids. These complex systems rely upon production and maintenance of multiple components to achieve antiphage defense. However, the prevalence and effectiveness of minimal, single-component systems that cleave DNA remain unknown. Here, we describe a unique mode of nucleic acid immunity mediated by a single enzyme with nuclease and helicase activities, herein referred to as Nhi (nuclease-helicase immunity). This enzyme provides robust protection against diverse staphylococcal phages and prevents phage DNA accumulation in cells stripped of all other known defenses. Our observations support a model in which Nhi targets and degrades phage-specific replication intermediates. Importantly, Nhi homologs are distributed in diverse bacteria and exhibit functional conservation, highlighting the versatility of such compact weapons as major players in antiphage defense.},
}
@article {pmid35421333,
year = {2022},
author = {Huiting, E and Bondy-Denomy, J},
title = {A single bacterial enzyme i(NHI)bits phage DNA replication.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {4},
pages = {417-419},
doi = {10.1016/j.chom.2022.03.025},
pmid = {35421333},
issn = {1934-6069},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; DNA Helicases/genetics ; DNA Replication ; },
abstract = {In this issue of Cell Host &amp; Microbe, Nayeemul Bari et al. discover an anti-phage immune system in bacteria that uses a single enzyme to accomplish the challenging feat of detecting phage DNA and limiting its replication. Unlike CRISPR-Cas and restriction modification (R-M) systems, which use sequence motifs, nuclease-helicase immunity (Nhi) is proposed to target phage-specific replication intermediates.},
}
@article {pmid35420793,
year = {2022},
author = {Wang, J and Skeens, E and Arantes, PR and Maschietto, F and Allen, B and Kyro, GW and Lisi, GP and Palermo, G and Batista, VS},
title = {Structural Basis for Reduced Dynamics of Three Engineered HNH Endonuclease Lys-to-Ala Mutants for the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Associated 9 (CRISPR/Cas9) Enzyme.},
journal = {Biochemistry},
volume = {61},
number = {9},
pages = {785-794},
pmid = {35420793},
issn = {1520-4995},
support = {R01 GM136815/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics ; *Endonucleases/chemistry ; },
abstract = {Many bacteria possess type-II immunity against invading phages or plasmids known as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system to detect and degrade the foreign DNA sequences. The Cas9 protein has two endonucleases responsible for double-strand breaks (the HNH domain for cleaving the target strand of DNA duplexes and RuvC domain for the nontarget strand, respectively) and a single-guide RNA-binding domain where the RNA and target DNA strands are base-paired. Three engineered single Lys-to-Ala HNH mutants (K810A, K848A, and K855A) exhibit an enhanced substrate specificity for cleavage of the target DNA strand. We report in this study that in the wild-type (wt) enzyme, D835, Y836, and D837 within the Y836-containing loop (comprising E827-D837) adjacent to the catalytic site have uncharacterizable broadened [1]H[15]N nuclear magnetic resonance (NMR) features, whereas remaining residues in the loop have different extents of broadened NMR spectra. We find that this loop in the wt enzyme exhibits three distinct conformations over the duration of the molecular dynamics simulations, whereas the three Lys-to-Ala mutants retain only one conformation. The versatility of multiple alternate conformations of this loop in the wt enzyme could help to recruit noncognate DNA substrates into the HNH active site for cleavage, thereby reducing its substrate specificity relative to the three mutants. Our study provides further experimental and computational evidence that Lys-to-Ala substitutions reduce dynamics of proteins and thus increase their stability.},
}
@article {pmid35419381,
year = {2022},
author = {Happi Mbakam, C and Lamothe, G and Tremblay, JP},
title = {Therapeutic Strategies for Dystrophin Replacement in Duchenne Muscular Dystrophy.},
journal = {Frontiers in medicine},
volume = {9},
number = {},
pages = {859930},
pmid = {35419381},
issn = {2296-858X},
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease characterized by progressive muscle wasting due to modifications in the DMD gene (exon deletions, nonsense mutations, intra-exonic insertions or deletions, exon duplications, splice site defects, and deep intronic mutations) that result in a lack of functional dystrophin expression. Many therapeutic approaches have so far been attempted to induce dystrophin expression and improve the patient phenotype. In this manuscript, we describe the relevant updates for some therapeutic strategies for DMD aiming to restore dystrophin expression. We also present and analyze in vitro and in vivo ongoing experimental approaches to treat the disease.},
}
@article {pmid35418245,
year = {2022},
author = {Parashar, A and Bak, K and Murshed, M},
title = {Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {42},
number = {6},
pages = {e155-e167},
doi = {10.1161/ATVBAHA.122.317518},
pmid = {35418245},
issn = {1524-4636},
support = {PJT-168888//CIHR/Canada ; },
mesh = {Alanine ; Animals ; Calcium-Binding Proteins/genetics/metabolism ; Extracellular Matrix Proteins/genetics/metabolism ; *Glutamic Acid ; Mice ; Serine ; *Vascular Calcification/chemically induced/genetics/prevention &amp; control ; X-Ray Microtomography ; },
abstract = {BACKGROUND: Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications-4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)-are thought to be essential for MGP's function.<br><br>METHODS: We pursued a genetic approach to study the roles of MGP's conserved residues. First, a transgenic line (SM22a-GlamutMgp) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp[-/-] mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model (Mgp[S3mut/S3mut]) to mutate MGP's conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging.<br><br>RESULTS: On a regular diet, the arterial walls in the Mgp[-/-]; SM22&#x3b1;-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, Mgp[S3mut/S3mut] mice on a regular diet recapitulated arterial calcification traits of Mgp[-/-] mice, although with lesser severity.<br><br>CONCLUSIONS: For the first time, we show here that MGP's conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.},
}
@article {pmid35418239,
year = {2022},
author = {Orazi, G and Collins, AJ and Whitaker, RJ},
title = {Prediction of Prophages and Their Host Ranges in Pathogenic and Commensal Neisseria Species.},
journal = {mSystems},
volume = {7},
number = {3},
pages = {e0008322},
pmid = {35418239},
issn = {2379-5077},
abstract = {The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one another, primarily via transformation and homologous recombination and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified, and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1,302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from prophages and other MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE Drug-resistant Neisseria gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) are known to infect Neisseria species. In this study, we identified a large number of candidate phages integrated in the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.},
}
@article {pmid35416602,
year = {2022},
author = {Xing, D and Su, B and Li, S and Bangs, M and Creamer, D and Coogan, M and Wang, J and Simora, R and Ma, X and Hettiarachchi, D and Alston, V and Wang, W and Johnson, A and Lu, C and Hasin, T and Qin, Z and Dunham, R},
title = {CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus).},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {24},
number = {3},
pages = {513-523},
pmid = {35416602},
issn = {1436-2236},
mesh = {Actins/genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Docosahexaenoic Acids ; Eicosapentaenoic Acid ; Fatty Acids ; *Fatty Acids, Omega-3 ; Fatty Acids, Unsaturated/metabolism ; Gene Transfer Techniques ; *Ictaluridae/genetics/metabolism ; *Oncorhynchus/genetics ; Salmon/genetics ; },
abstract = {Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish (Ictalurus punctatus) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon (Oncorhynchus masou) driven by a carp β-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P1 transgenic fish with tissue-specific gene expression. Liver and muscle had relative high gene expression (13.4- and 9.2-fold change, respectively). Fatty acid analysis showed DHA content in the muscle from transgenic fish was 1.62-fold higher than in non-transgenic fish (P < 0.05). Additionally, total n-3 PUFAs and omega-6 polyunsaturated fatty acids (n-6 PUFAs) increased to 1.41-fold and 1.50-fold, respectively, suggesting the β-actin-elovl2 transgene improved biosynthesis of PUFAs in channel catfish as a whole. The n-9 fatty acid level decreased in the transgenic fish compared to the control. Morphometric analysis showed that there were significant differences between injected fish with sgRNAs (including positive and negative fish) and sham-injected controls (P < 0.001). Potential off-target effects are likely the major factor responsible for morphological deformities. Optimization of sgRNA design to maximize activity and reduce off-target effects of CRISPR/Cas9 should be examined in future transgenic research, but this research shows a promising first step in the improvement of n-3 PUFAs in channel catfish.},
}
@article {pmid35415942,
year = {2022},
author = {Bao, YY and Zhong, JT and Shen, LF and Dai, LB and Zhou, SH and Fan, J and Yao, HT and Lu, ZJ},
title = {Effect of Glut-1 and HIF-1α double knockout by CRISPR/CAS9 on radiosensitivity in laryngeal carcinoma via the PI3K/Akt/mTOR pathway.},
journal = {Journal of cellular and molecular medicine},
volume = {26},
number = {10},
pages = {2881-2894},
doi = {10.1111/jcmm.17303},
pmid = {35415942},
issn = {1582-4934},
mesh = {Animals ; CRISPR-Cas Systems ; *Carcinoma/genetics/metabolism/radiotherapy ; Cell Line, Tumor ; Glucose ; *Glucose Transporter Type 1/genetics/metabolism ; Humans ; Hypoxia ; *Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; *Laryngeal Neoplasms/genetics/metabolism/radiotherapy ; Phosphatidylinositol 3-Kinases/genetics/metabolism ; Proto-Oncogene Proteins c-akt/genetics/metabolism ; Radiation Tolerance/genetics ; TOR Serine-Threonine Kinases/genetics/metabolism ; Wortmannin ; },
abstract = {Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1α (HIF-1α) and glucose transporter 1 (Glut-1) double knockout reduced tumour biological behaviour in laryngeal carcinoma cells. However, their radioresistance mechanism remains unclear. In this study, cell viability was determined by CCK8 assay. Glucose uptake capability was evaluated by measurement of [18] F-fluorodeoxyglucose radioactivity. A tumour xenograft model was established by subcutaneous injection of Tu212 cells. Tumour histopathology was determined by haematoxylin and eosin staining, immunohistochemical staining, and TUNEL assays. Signalling transduction was evaluated by Western blotting. We found that hypoxia induced radioresistance in Tu212 cells accompanied by increased glucose uptake capability and activation of the PI3K/Akt/mTOR pathway. Inhibition of PI3K/Akt/mTOR activity abolished hypoxia-induced radioresistance and glucose absorption. Mechanistic analysis revealed that hypoxia promoted higher expressions of HIF-1α and Glut-1. Moreover, the PI3K/Akt/mTOR pathway was a positive mediator of HIF-1α and/or Glut-1 in the presence of irradiation. HIF-1α and/or Glut-1 knockout significantly reduced cell viability, glucose uptake and PI3K/Akt/mTOR activity, all of which were induced by hypoxia in the presence of irradiation. In vivo analysis showed that knockout of HIF-1α and/or Glut-1 also inhibited tumour growth by promoting cell apoptosis, more robustly compared with the PI3K inhibitor wortmannin, particularly in tumours with knockout of both HIF-1α and Glut-1. HIF-1α and/or Glut-1 knockout also abrogated PI3K/Akt/mTOR signalling transduction in tumour tissues, in a manner similar to wortmannin. HIF-1α and/or Glut-1 knockout facilitated radiosensitivity in laryngeal carcinoma Tu212 cells by regulation of the PI3K/Akt/mTOR pathway.},
}
@article {pmid35414398,
year = {2022},
author = {López-Valls, M and Escalona-Noguero, C and Rodríguez-Díaz, C and Pardo, D and Castellanos, M and Milán-Rois, P and Martínez-Garay, C and Coloma, R and Abreu, M and Cantón, R and Galán, JC and Miranda, R and Somoza, &#xc1; and Sot, B},
title = {CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles.},
journal = {Analytica chimica acta},
volume = {1205},
number = {},
pages = {339749},
pmid = {35414398},
issn = {1873-4324},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Gold ; Humans ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; },
abstract = {The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.},
}
@article {pmid35414381,
year = {2022},
author = {Sohail, M and Xie, S and Zhang, X and Li, B},
title = {Methodologies in visualizing the activation of CRISPR/Cas: The last mile in developing CRISPR-Based diagnostics and biosensing - A review.},
journal = {Analytica chimica acta},
volume = {1205},
number = {},
pages = {339541},
doi = {10.1016/j.aca.2022.339541},
pmid = {35414381},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Colorimetry ; Molecular Probes ; *Nucleic Acids ; },
abstract = {CRISPR/Cas-based analytical procedures have revolutionized the sensing platform to fulfill the requirements of the current era in terms of sensitivity, selectivity, robustness, user-friendly feature, and cost-effectiveness for the detection of nucleic acid as well as non-nucleic acid analytes. Molecular target monitoring and transduction of the signals is a crucial prerequisite for precise molecular sensing tools. Besides, the reporting systems have become the last milestone for fabricating Cas-based molecular probes to visualize the activation of CRISPR/Cas enzymes. In this review, we have highlighted various CRISPR/Cas reporters, their mechanisms, sensing strategies, merits, and demerits. Moreover, signal transducers, i.e. fluorescent, colorimetric, and electrochemical, have also been discussed in detail along with various sensing strategies to generate recordable signals. It was concluded that there is still a need to overcome issues offered by the reported sensing devices, such as off-target effect, target sequence limitation, multiplexed quantitative detection, the influence of the inhibitor, and reaction kinetic constraint. Additionally, it is required to make them available for commercial use by validating their stability, robustness, safety profile in an off-lab environment as most of the probes have been tested in the controlled atmosphere of the laboratories. We believe that this novel critical interpretation and summary will assist the researchers in designing and validating new CRISPR/Cas reporters and probes for practical applications on a commercial scale.},
}
@article {pmid35414240,
year = {2022},
author = {Liu, Y and Champer, J},
title = {Modelling homing suppression gene drive in haplodiploid organisms.},
journal = {Proceedings. Biological sciences},
volume = {289},
number = {1972},
pages = {20220320},
pmid = {35414240},
issn = {1471-2954},
mesh = {Alleles ; CRISPR-Cas Systems ; Female ; *Gene Drive Technology/methods ; Germ Cells ; Humans ; Male ; RNA, Guide/genetics ; },
abstract = {Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo owing to maternally deposited Cas9 and guide RNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly owing to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.},
}
@article {pmid35414130,
year = {2022},
author = {Tornabene, P and Ferla, R and Llado-Santaeularia, M and Centrulo, M and Dell'Anno, M and Esposito, F and Marrocco, E and Pone, E and Minopoli, R and Iodice, C and Nusco, E and Rossi, S and Lyubenova, H and Manfredi, A and Di Filippo, L and Iuliano, A and Torella, A and Piluso, G and Musacchia, F and Surace, EM and Cacchiarelli, D and Nigro, V and Auricchio, A},
title = {Therapeutic homology-independent targeted integration in retina and liver.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1963},
pmid = {35414130},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; *Dependovirus/genetics ; *Gene Editing/methods ; Genetic Vectors/genetics ; Liver ; Mice ; Retina/metabolism ; Swine ; },
abstract = {Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.},
}
@article {pmid35414049,
year = {2022},
author = {Shi, H and Xu, Y and Tian, N and Yang, M and Liang, FS},
title = {Inducible and reversible RNA N[6]-methyladenosine editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1958},
pmid = {35414049},
issn = {2041-1723},
support = {R01 GM143256/GM/NIGMS NIH HHS/United States ; R21 CA247638/CA/NCI NIH HHS/United States ; },
mesh = {Adenosine/analogs &amp; derivatives ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenesis, Genetic ; Humans ; *RNA/genetics ; },
abstract = {RNA modifications, including N[6]-methyladenosine (m[6]A), have been reported to regulate fundamental RNA processes and properties, and directly linked to various human diseases. Methods enabling temporal and transcript/locus-specific editing of specific RNA modifications are essential, but still limited, to dissect the dynamic and context-dependent functions of these epigenetic modifications. Here, we develop a chemically inducible and reversible RNA m[6]A modification editing platform integrating chemically induced proximity (CIP) and CRISPR methods. We show that m[6]A editing can be temporally controlled at specific sites of individual RNA transcripts by the addition or removal of the CIP inducer, abscisic acid (ABA), in the system. By incorporating a photo-caged ABA, a light-controlled version of m[6]A editing platform can be developed. We expect that this platform and strategy can be generally applied to edit other RNA modifications in addition to m[6]A.},
}
@article {pmid35414015,
year = {2022},
author = {Guo, LY and Bian, J and Davis, AE and Liu, P and Kempton, HR and Zhang, X and Chemparathy, A and Gu, B and Lin, X and Rane, DA and Xu, X and Jamiolkowski, RM and Hu, Y and Wang, S and Qi, LS},
title = {Multiplexed genome regulation in vivo with hyper-efficient Cas12a.},
journal = {Nature cell biology},
volume = {24},
number = {4},
pages = {590-600},
pmid = {35414015},
issn = {1476-4679},
support = {P30 EY026877/EY/NEI NIH HHS/United States ; T32 EY020485/EY/NEI NIH HHS/United States ; R01 NS109990/NS/NINDS NIH HHS/United States ; R01 EY028106/EY/NEI NIH HHS/United States ; R01 EY032518/EY/NEI NIH HHS/United States ; R01 EY023295/EY/NEI NIH HHS/United States ; R01 EY024932/EY/NEI NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Mice ; RNA/metabolism ; },
abstract = {Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.},
}
@article {pmid35413320,
year = {2022},
author = {Chauhan, N and Saxena, K and Jain, U},
title = {Single molecule detection; from microscopy to sensors.},
journal = {International journal of biological macromolecules},
volume = {209},
number = {Pt A},
pages = {1389-1401},
doi = {10.1016/j.ijbiomac.2022.04.038},
pmid = {35413320},
issn = {1879-0003},
mesh = {*Biosensing Techniques/methods ; Microscopy ; *Nanopores ; Nanotechnology/methods ; },
abstract = {Single molecule detection is necessary to find out physical, chemical properties and their mechanism involved in the normal functioning of body cells. In this way, they can provide a new direction to the healthcare system. Various techniques have been developed and employed for their successful detection. Herein, we have emphasized various traditional methods as well as biosensing technology which offer single molecule sensitivity. The various methods including plasmonic resonance, nanopores, whispering gallery mode, Simoa assay and recognition tunneling are discussed in the initial part which has been followed by a discussion about biosensor-based detection. Plasmonic, SERS, CRISPR/Cas, and other types of biosensors are focused in this review and found to be highly sensitive for single molecule detection. This review provides an overview of progression in different techniques employed for single molecule detection.},
}
@article {pmid35412812,
year = {2022},
author = {Scott, H and Sun, D and Beal, J and Kiani, S},
title = {Simulation-Based Engineering of Time-Delayed Safety Switches for Safer Gene Therapies.},
journal = {ACS synthetic biology},
volume = {11},
number = {5},
pages = {1782-1789},
doi = {10.1021/acssynbio.1c00621},
pmid = {35412812},
issn = {2161-5063},
support = {R01 EB024562/EB/NIBIB NIH HHS/United States ; P30 DK120531/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Humans ; },
abstract = {CRISPR-based gene editing is a powerful tool with great potential for applications in the treatment of many inherited and acquired diseases. The longer that CRISPR gene therapy is maintained within a patient, however, the higher the likelihood that it will result in problematic side effects such as off-target editing or immune response. One approach to mitigating these issues is to link the operation of the therapeutic system to a safety switch that autonomously disables its operation and removes the delivered therapeutics after some amount of time. We present here a simulation-based analysis of the potential for regulating the time delay of such a safety switch using one or two transcriptional regulators and/or recombinases. Combinatorial circuit generation identifies 30 potential architectures for such circuits, which we evaluate in simulation with respect to tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation. This modeling predicts one of these circuit architectures to have the desired dynamics and robustness, which can be further tested and applied in the context of CRISPR therapeutics.},
}
@article {pmid35412632,
year = {2022},
author = {Pan, X and Li, H and Zhang, X},
title = {TedSim: temporal dynamics simulation of single-cell RNA sequencing data and cell division history.},
journal = {Nucleic acids research},
volume = {50},
number = {8},
pages = {4272-4288},
pmid = {35412632},
issn = {1362-4962},
support = {R35 GM143070/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Division/genetics ; Cell Lineage/genetics ; Sequence Analysis, RNA/methods ; *Single-Cell Analysis/methods ; },
abstract = {Recently, lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes, which allows for the reconstruction of the cell division tree and makes it possible to reconstruct ancestral cell types and trace the origin of each cell type. Meanwhile, trajectory inference methods are widely used to infer cell trajectories and pseudotime in a dynamic process using gene expression data of present-day cells. Here, we present TedSim (single-cell temporal dynamics simulator), which simulates the cell division events from the root cell to present-day cells, simultaneously generating two data modalities for each single cell: the lineage barcode and gene expression data. TedSim is a framework that connects the two problems: lineage tracing and trajectory inference. Using TedSim, we conducted analysis to show that (i) TedSim generates realistic gene expression and barcode data, as well as realistic relationships between these two data modalities; (ii) trajectory inference methods can recover the underlying cell state transition mechanism with balanced cell type compositions; and (iii) integrating gene expression and barcode data can provide more insights into the temporal dynamics in cell differentiation compared to using only one type of data, but better integration methods need to be developed.},
}
@article {pmid35412236,
year = {2022},
author = {Aksenova, V and Arnaoutov, A and Dasso, M},
title = {Analysis of Nucleoporin Function Using Inducible Degron Techniques.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2502},
number = {},
pages = {129-150},
pmid = {35412236},
issn = {1940-6029},
support = {Z01 HD008754/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Targeting/methods ; *Indoleacetic Acids/pharmacology ; Nuclear Pore ; *Nuclear Pore Complex Proteins/chemistry/genetics/metabolism ; Proteins ; Proteolysis ; },
abstract = {Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.},
}
@article {pmid35412223,
year = {2022},
author = {Li, J and Zhu, D and Hu, S and Nie, Y},
title = {CRISPR-CasRx knock-in mice for RNA degradation.},
journal = {Science China. Life sciences},
volume = {65},
number = {11},
pages = {2248-2256},
pmid = {35412223},
issn = {1869-1889},
mesh = {Mice ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics/metabolism ; Myocytes, Cardiac/metabolism ; RNA Stability ; },
abstract = {The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity. To enable the application of CasRx in vivo, we established a Credependent CasRx knock-in mouse. Using these mice, we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes, which induced cardiac regeneration after myocardial infarction. We also knocked down the lncRNA Mhrt in cardiomyocytes with the CasRx knock-in mice, causing hypertrophic cardiomyopathy. In summary, we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lncRNA expression in specific somatic cells. This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.},
}
@article {pmid35411929,
year = {2022},
author = {Li, X and Wang, X and Sun, W and Huang, S and Zhong, M and Yao, Y and Ji, Q and Huang, X},
title = {Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes.},
journal = {Journal of molecular cell biology},
volume = {14},
number = {4},
pages = {},
pmid = {35411929},
issn = {1759-4685},
mesh = {CRISPR-Cas Systems/genetics ; *G-Quadruplexes ; RNA/genetics ; },
}
@article {pmid35411846,
year = {2022},
author = {Rangarajan, AA and Yilmaz, C and Schnetz, K},
title = {Deletion of FRT-sites by no-SCAR recombineering in Escherichia coli.},
journal = {Microbiology (Reading, England)},
volume = {168},
number = {4},
pages = {},
doi = {10.1099/mic.0.001173},
pmid = {35411846},
issn = {1465-2080},
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded ; *Escherichia coli/genetics ; Genetic Engineering/methods ; },
abstract = {Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in Escherichia coli and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNAFRT, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless E. coli strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNAFRT/Cas9 may be generally applicable to cure FRT scar-sites of E. coli strains constructed by classical λ-Red recombineering.},
}
@article {pmid35411720,
year = {2022},
author = {Xie, L and Hu, Y and Li, L and Jiang, L and Jiao, Y and Wang, Y and Zhou, L and Tao, R and Qu, J and Chen, Q and Yao, S},
title = {Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non-PI domain mutations derived from xCas9.},
journal = {The FEBS journal},
volume = {289},
number = {19},
pages = {5899-5913},
doi = {10.1111/febs.16457},
pmid = {35411720},
issn = {1742-4658},
mesh = {Amino Acid Substitution ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Mutation ; },
abstract = {The recognition of protospacer adjacent motif (PAM) is a key factor for the CRISPR (i.e. clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (Cas9) system to distinguish foreign DNAs from the host genome, and also significantly restricts the targeting scope of the system during genome-editing applications. Structurally, the PAM interacting (PI) domain, which usually is located in the C-terminus of Cas9 proteins, directly binds to PAM and plays a key role in determining the recognition specificity. However, several lines of evidence showed that other regions of Cas9 protein beyond the PI domain might also play roles in PAM interaction. Here, we constructed a mosaic SpCas9 protein (xCas9-NG) by fusing the PI domain of SpCas9 PAM variant, Cas9-NG with the non-PI fragment of xCas9 protein that contains multiple amino acid substitutions. We found that non-PI fragment of xCas9 expanded PAM recognition of the Cas9-NG PI domain. In addition, xCas9-NG showed an improved editing efficiency in the majority of targets harboring xCas9 and Cas9-NG PAMs. Importantly, this finding was also successfully extended to other Cas9 variants, including SpRY and the non-G SpCas9 series. Together, our work expands the target scope of SpCas9 editing system and demonstrates the notion that the non-PI domain fragment plays an important role in PAM restriction.},
}
@article {pmid35411116,
year = {2022},
author = {Kim, Y and Lee, S and Cho, S and Park, J and Chae, D and Park, T and Minna, JD and Kim, HH},
title = {High-throughput functional evaluation of human cancer-associated mutations using base editors.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {874-884},
pmid = {35411116},
issn = {1546-1696},
support = {P50 CA070907/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; Mutation/genetics ; *Neoplasms/genetics ; RNA, Guide/genetics ; },
abstract = {Comprehensive phenotypic characterization of the many mutations found in cancer tissues is one of the biggest challenges in cancer genomics. In this study, we evaluated the functional effects of 29,060 cancer-related transition mutations that result in protein variants on the survival and proliferation of non-tumorigenic lung cells using cytosine and adenine base editors and single guide RNA (sgRNA) libraries. By monitoring base editing efficiencies and outcomes using surrogate target sequences paired with sgRNA-encoding sequences on the lentiviral delivery construct, we identified sgRNAs that induced a single primary protein variant per sgRNA, enabling linking those mutations to the cellular phenotypes caused by base editing. The functions of the vast majority of the protein variants (28,458 variants, 98%) were classified as neutral or likely neutral; only 18 (0.06%) and 157 (0.5%) variants caused outgrowing and likely outgrowing phenotypes, respectively. We expect that our approach can be extended to more variants of unknown significance and other tumor types.},
}
@article {pmid35411005,
year = {2022},
author = {Yang, L and Zhang, L and Yin, P and Ding, H and Xiao, Y and Zeng, J and Wang, W and Zhou, H and Wang, Q and Zhang, Y and Chen, Z and Yang, M and Feng, Y},
title = {Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1931},
pmid = {35411005},
issn = {2041-1723},
mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Viral Proteins/metabolism ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the crRNA-guided surveillance (Csy) complex. The HTH motif of AcrIF24 can bind the Acr promoter region and repress its transcription, suggesting its role as an Aca gene in self-regulation. AcrIF24 forms a homodimer and further induces dimerization of the Csy complex. Apart from blocking the hybridization of target DNA to the crRNA, AcrIF24 also induces the binding of non-sequence-specific dsDNA to the Csy complex, similar to AcrIF9, although this binding seems to play a minor role in AcrIF24 inhibitory capacity. Further structural and biochemical studies of the Csy-AcrIF24-dsDNA complexes and of AcrIF24 mutants reveal that the HTH motif of AcrIF24 and the PAM recognition loop of the Csy complex are structural elements essential for this non-specific dsDNA binding. Moreover, AcrIF24 and AcrIF9 display distinct characteristics in inducing non-specific DNA binding. Together, our findings highlight a multifunctional Acr and suggest potential wide distribution of Acr-induced non-specific DNA binding.},
}
@article {pmid35410430,
year = {2022},
author = {Che, P and Wu, E and Simon, MK and Anand, A and Lowe, K and Gao, H and Sigmund, AL and Yang, M and Albertsen, MC and Gordon-Kamm, W and Jones, TJ},
title = {Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {344},
pmid = {35410430},
issn = {2399-3642},
mesh = {CRISPR-Cas Systems ; Edible Grain/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Regeneration/genetics ; *Sorghum/genetics ; },
abstract = {For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.},
}
@article {pmid35410423,
year = {2022},
author = {Liu, Y and Pinto, F and Wan, X and Yang, Z and Peng, S and Li, M and Cooper, JM and Xie, Z and French, CE and Wang, B},
title = {Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1937},
pmid = {35410423},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/S018875/1/MRC_/Medical Research Council/United Kingdom ; MR/V035401/1/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; },
abstract = {In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.},
}
@article {pmid35410288,
year = {2022},
author = {Oh, Y and Lee, WJ and Hur, JK and Song, WJ and Lee, Y and Kim, H and Gwon, LW and Kim, YH and Park, YH and Kim, CH and Lim, KS and Song, BS and Huh, JW and Kim, SU and Jun, BH and Jung, C and Lee, SH},
title = {Expansion of the prime editing modality with Cas9 from Francisella novicida.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {92},
pmid = {35410288},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; Francisella ; *Gene Editing ; Humans ; RNA-Directed DNA Polymerase ; },
abstract = {Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.},
}
@article {pmid35409000,
year = {2022},
author = {Huang, L and Luo, J and Song, N and Gao, W and Zhu, L and Yao, W},
title = {CRISPR/Cas9-Mediated Knockout of miR-130b Affects Mono- and Polyunsaturated Fatty Acid Content via PPARG-PGC1α Axis in Goat Mammary Epithelial Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {7},
pages = {},
pmid = {35409000},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; *Goats/genetics/metabolism ; Mammary Glands, Animal/metabolism ; *MicroRNAs/genetics/metabolism ; PPAR gamma/metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics/metabolism ; Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; },
abstract = {MicroRNA (miRNA)-130b, as a regulator of lipid metabolism in adipose and mammary gland tissues, is actively involved in lipogenesis, but its endogenous role in fatty acid synthesis remains unclear. Here, we aimed to explore the function and underlying mechanism of miR-130b in fatty acid synthesis using the CRISPR/Cas9 system in primary goat mammary epithelial cells (GMEC). A single clone with deletion of 43 nucleotides showed a significant decrease in miR-130b-5p and miR-130b-3p abundances and an increase of target genes PGC1α and PPARG. In addition, knockout of miR-130b promoted triacylglycerol (TAG) and cholesterol accumulation, and decreased the proportion of monounsaturated fatty acids (MUFA) C16:1, C18:1 and polyunsaturated fatty acids (PUFA) C18:2, C20:3, C20:4, C20:5, C22:6. Similarly, the abundance of fatty acid synthesis genes ACACA and FASN and transcription regulators SREBP1c and SREBP2 was elevated. Subsequently, interference with PPARG instead of PGC1α in knockout cells restored the effect of miR-130b knockout, suggesting that PPARG is responsible for miR-130b regulating fatty acid synthesis. Moreover, disrupting PPARG inhibits PGC1α transcription and translation. These results reveal that miR-130b directly targets the PPARG-PGC1α axis, to inhibit fatty acid synthesis in GMEC. In conclusion, miR-130b could be a potential molecular regulator for improving the beneficial fatty acids content in goat milk.},
}
@article {pmid35408979,
year = {2022},
author = {Tripathi, L and Ntui, VO and Tripathi, JN},
title = {Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing.},
journal = {International journal of molecular sciences},
volume = {23},
number = {7},
pages = {},
pmid = {35408979},
issn = {1422-0067},
mesh = {*Bacterial Infections/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Musa/genetics/microbiology ; Plant Diseases/genetics/microbiology/prevention &amp; control ; *Xanthomonas/genetics ; },
abstract = {Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is a vast yield gap in the production of bananas in regions where bacterial pathogens and several other pathogens and pests are present together in the same field. BXW disease caused by Xanthomonas campestris pv. musacearum is reported to be the most destructive banana disease in East Africa. The disease affects all the banana varieties grown in the region. Only the wild-type diploid banana, Musa balbisiana, is resistant to BXW disease. Developing disease-resistant varieties of bananas is one of the most effective strategies to manage diseases. Recent advances in CRISPR/Cas-based gene editing techniques can accelerate banana improvement. Some progress has been made to create resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes. A synopsis of recent advancements and perspectives on the application of gene editing for the control of bacterial wilt diseases are presented in this article.},
}
@article {pmid35403388,
year = {2022},
author = {Pan, W and Cheng, Z and Han, Z and Yang, H and Zhang, W and Zhang, H},
title = {Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {4},
pages = {339-344},
pmid = {35403388},
issn = {1862-1783},
mesh = {CRISPR-Cas Systems ; *Citrullus/genetics ; *Cucurbitaceae/genetics ; Gene Editing ; Plant Breeding ; Transformation, Genetic ; },
abstract = {Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.},
}
@article {pmid35400359,
year = {2022},
author = {Wang, LY and Jiang, PF and Li, JZ and Hu, JD},
title = {[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD[+]AML].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {2},
pages = {},
doi = {10.19746/j.cnki.issn.1009-2137.2022.02.002},
pmid = {35400359},
issn = {1009-2137},
mesh = {CRISPR-Cas Systems ; Doxorubicin/pharmacology ; Drug Resistance ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics ; *MicroRNAs/genetics ; RNA, Guide/genetics ; fms-Like Tyrosine Kinase 3/genetics ; },
abstract = {OBJECTIVE: Two sgRNAs transfected FLT3-ITD[+]AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.<br><br>METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.<br><br>RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.<br><br>CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.},
}
@article {pmid35398513,
year = {2022},
author = {Shin, S and Kim, SH and Park, JH and Lee, JS and Lee, GM},
title = {Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress.},
journal = {Metabolic engineering},
volume = {72},
number = {},
pages = {247-258},
doi = {10.1016/j.ymben.2022.03.017},
pmid = {35398513},
issn = {1096-7184},
mesh = {Antibodies, Monoclonal ; *CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Kidney/metabolism ; Osmotic Pressure ; *Recombinant Proteins/biosynthesis ; *Recombinases/genetics ; },
abstract = {Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.},
}
@article {pmid35398275,
year = {2022},
author = {Kreuter, J and Stark, G and Mach, RL and Mach-Aigner, AR and Zimmermann, C},
title = {Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.},
journal = {Journal of biotechnology},
volume = {350},
number = {},
pages = {11-16},
doi = {10.1016/j.jbiotec.2022.03.017},
pmid = {35398275},
issn = {1873-4863},
mesh = {*Ascomycota/genetics ; Aureobasidium ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.},
}
@article {pmid35396804,
year = {2022},
author = {Raghav, D and Jyoti, A and Siddiqui, AJ and Saxena, J},
title = {Plant-associated endophytic fungi as potential bio-factories for extracellular enzymes: Progress, Challenges and Strain improvement with precision approaches.},
journal = {Journal of applied microbiology},
volume = {133},
number = {2},
pages = {287-310},
doi = {10.1111/jam.15574},
pmid = {35396804},
issn = {1365-2672},
mesh = {Biotechnology ; *Endophytes/genetics/metabolism ; *Fungi/genetics/metabolism ; },
abstract = {There is an intricate network of relations between endophytic fungi and their hosts that affects the production of various bioactive compounds. Plant-associated endophytic fungi contain industrially important enzymes and have the potential to fulfil their rapid demand in the international market to boost business in technology. Being safe and metabolically active, they have replaced the usage of toxic and harmful chemicals and hold a credible application in biotransformation, bioremediation and industrial processes. Despite these, there are limited reports on fungal endophytes that can directly cater to the demand and supply of industrially stable enzymes. The underlying reasons include low endogenous production and secretion of enzymes from fungal endophytes which have raised concern for widely accepted applications. Hence, it is imperative to augment the biosynthetic and secretory potential of fungal endophytes. Modern state-of-the-art biotechnological technologies aiming at strain improvement using cell factory engineering as well as precise gene editing like Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its Associated proteins (Cas) systems which can provide a boost in fungal endophyte enzyme production. Additionally, it is vital to characterize optimum conditions to grow one strain with multiple enzymes (OSME). The present review encompasses various plants-derived endophytic fungal enzymes and their applications in various sectors. Furthermore, we postulate the feasibility of new precision approaches with an aim for strain improvement and enhanced enzyme production.},
}
@article {pmid35396760,
year = {2022},
author = {Bhatt, A and Fatima, Z and Ruwali, M and Misra, CS and Rangu, SS and Rath, D and Rattan, A and Hameed, S},
title = {CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.},
journal = {Journal of applied microbiology},
volume = {133},
number = {2},
pages = {410-421},
pmid = {35396760},
issn = {1365-2672},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA ; RNA, Viral/genetics ; *SARS-CoV-2/genetics ; Sensitivity and Specificity ; Technology ; },
abstract = {AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved.<br><br>METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100&#x2009;min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively.<br><br>CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis.<br><br>To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.},
}
@article {pmid35396392,
year = {2022},
author = {van Riet, J and Saha, C and Strepis, N and Brouwer, RWW and Martens-Uzunova, ES and van de Geer, WS and Swagemakers, SMA and Stubbs, A and Halimi, Y and Voogd, S and Tanmoy, AM and Komor, MA and Hoogstrate, Y and Janssen, B and Fijneman, RJA and Niknafs, YS and Chinnaiyan, AM and van IJcken, WFJ and van der Spek, PJ and Jenster, G and Louwen, R},
title = {CRISPRs in the human genome are differentially expressed between malignant and normal adjacent to tumor tissue.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {338},
pmid = {35396392},
issn = {2399-3642},
mesh = {Archaea/genetics ; Bacteria/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Human ; Humans ; Male ; *RNA, Small Untranslated ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs.},
}
@article {pmid35395997,
year = {2022},
author = {Yang, F and Jiang, M and Lin, ZH and Xie, ZL and Ma, ZN and Yang, L and Liu, H and Wang, ZY and Zhou, L},
title = {[Effects of the ITGA2B Nonsense Mutation (c.2659C &gt; T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {2},
pages = {559-564},
doi = {10.19746/j.cnki.issn.1009-2137.2022.02.040},
pmid = {35395997},
issn = {1009-2137},
mesh = {Animals ; CRISPR-Cas Systems ; *Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics ; Humans ; *Integrin alpha2/genetics ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics ; RNA, Guide ; *Thrombasthenia/diagnosis/genetics ; Thrombin/genetics ; },
abstract = {OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane.<br><br>METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of &#x3b1;IIb&#x3b2;3 on the surface of platelet.<br><br>RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of &#x3b1;IIb&#x3b2;3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05).<br><br>CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.},
}
@article {pmid35395959,
year = {2022},
author = {Wang, LY and Jiang, PF and Li, JZ and Hu, JD},
title = {[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD[+]AML].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {30},
number = {2},
pages = {334-340},
doi = {10.19746/j.cnki.issn.1009-2137.2022.02.002},
pmid = {35395959},
issn = {1009-2137},
mesh = {CRISPR-Cas Systems ; Doxorubicin/pharmacology ; Drug Resistance ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics ; *MicroRNAs/genetics ; RNA, Guide/genetics ; fms-Like Tyrosine Kinase 3/genetics ; },
abstract = {OBJECTIVE: Two sgRNAs transfected FLT3-ITD[+]AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.<br><br>METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.<br><br>RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.<br><br>CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.},
}
@article {pmid35395152,
year = {2022},
author = {Hobbs, SJ and Wein, T and Lu, A and Morehouse, BR and Schnabel, J and Leavitt, A and Yirmiya, E and Sorek, R and Kranzusch, PJ},
title = {Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity.},
journal = {Nature},
volume = {605},
number = {7910},
pages = {522-526},
pmid = {35395152},
issn = {1476-4687},
support = {F32 GM133063/GM/NIGMS NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; S10 RR029205/RR/NCRR NIH HHS/United States ; S10 OD021527/OD/NIH HHS/United States ; /ERC_/European Research Council/International ; },
mesh = {Bacteria/metabolism ; Bacterial Proteins/metabolism ; Bacteriophage T4/metabolism ; *Bacteriophages/physiology ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Escherichia coli/metabolism ; Nucleotides, Cyclic/metabolism ; Oligonucleotides ; Pyrimidines/metabolism ; },
abstract = {The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation[1,2]. Phages use several strategies to defeat host CRISPR and restriction-modification systems[3-10], but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.},
}
@article {pmid35394860,
year = {2022},
author = {Mamontov, V and Martynov, A and Morozova, N and Bukatin, A and Staroverov, DB and Lukyanov, KA and Ispolatov, Y and Semenova, E and Severinov, K},
title = {Persistence of plasmids targeted by CRISPR interference in bacterial populations.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {15},
pages = {e2114905119},
pmid = {35394860},
issn = {1091-6490},
support = {R01 GM064530/GM/NIGMS NIH HHS/United States ; R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics/physiology ; *Escherichia coli/genetics ; Gene-Environment Interaction ; *Interspersed Repetitive Sequences/genetics ; Models, Genetic ; *Plasmids/genetics ; },
abstract = {CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy and microfluidics analyses we demonstrate that plasmids can persist for multiple generations in some Escherichia coli cell lineages at conditions of continuous targeting by the type I-E CRISPR-Cas system. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to continuous maintenance of mobile genetic elements in some cells, which leads to diversification of phenotypes in the entire community and allows rapid changes in the population structure to meet the demands of a changing environment.},
}
@article {pmid35394503,
year = {2022},
author = {Giordano, A and Santo Domingo, M and Quadrana, L and Pujol, M and Martín-Hernández, AM and Garcia-Mas, J},
title = {CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation.},
journal = {Journal of experimental botany},
volume = {73},
number = {12},
pages = {4022-4033},
doi = {10.1093/jxb/erac148},
pmid = {35394503},
issn = {1460-2431},
mesh = {CRISPR-Cas Systems ; *Cucurbitaceae/genetics ; Epigenesis, Genetic ; Ethylenes ; Fruit/genetics ; Gene Editing ; Gene Expression Regulation, Plant ; *Lycopersicon esculentum/genetics ; Plant Proteins/genetics ; Protein-Tyrosine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; },
abstract = {Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon.},
}
@article {pmid35394026,
year = {2022},
author = {Yang, E and Metzloff, M and Langmüller, AM and Xu, X and Clark, AG and Messer, PW and Champer, J},
title = {A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {6},
pages = {},
pmid = {35394026},
issn = {2160-1836},
support = {R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; W 1225/FWF_/Austrian Science Fund FWF/Austria ; F32 AI138476/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics ; Female ; *Gene Drive Technology/methods ; *RNA, Guide/genetics ; Translocation, Genetic ; },
abstract = {Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The selected gRNA target sites were close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.},
}
@article {pmid35393561,
year = {2022},
author = {Tsuji, S and Stephens, CJ and Bortolussi, G and Zhang, F and Baj, G and Jang, H and de Alencastro, G and Muro, AF and Pekrun, K and Kay, MA},
title = {Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice.},
journal = {Nature biotechnology},
volume = {40},
number = {8},
pages = {1285-1294},
pmid = {35393561},
issn = {1546-1696},
support = {R01 HL064274/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Endonucleases/genetics ; Gene Editing/methods ; *Genetic Vectors ; Homologous Recombination ; Humans ; Mice ; Vidarabine/analogs &amp; derivatives ; },
abstract = {Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.},
}
@article {pmid35390717,
year = {2022},
author = {Ivanov, AV and Safenkova, IV and Zherdev, AV and Dzantiev, BB},
title = {DIRECT[2]: A novel platform for a CRISPR-Cas12-based assay comprising universal DNA-IgG probe and a direct lateral flow test.},
journal = {Biosensors &amp; bioelectronics},
volume = {208},
number = {},
pages = {114227},
doi = {10.1016/j.bios.2022.114227},
pmid = {35390717},
issn = {1873-4235},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA Probes/genetics ; DNA, Single-Stranded ; Gold ; Immunoglobulin G ; *Metal Nanoparticles ; Mice ; },
abstract = {CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT[2]). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR-Cas12 activity detection and its bioanalytical applications.},
}
@article {pmid35389616,
year = {2022},
author = {Abdullah, and Wang, P and Han, T and Liu, W and Ren, W and Wu, Y and Xiao, Y},
title = {Adenine Base Editing System for Pseudomonas and Prediction Workflow for Protein Dysfunction via ABE.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1650-1657},
doi = {10.1021/acssynbio.2c00066},
pmid = {35389616},
issn = {2161-5063},
mesh = {Adenine/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Pseudomonas/genetics/metabolism ; Workflow ; },
abstract = {Pseudomonas is a large genus that inhabits diverse environments due to its distinct metabolic versatility. Its applications range from environmental to industrial biotechnology. Molecular tools that allow precise and efficient genetic manipulation are required to understand and harness its full potential. Here, we report the development of a highly efficient adenine base editing system, i.e., dxABE-PS, for Pseudomonas species. The system allows A:T → G:C transition with up to 100% efficiency along a broad target spectrum because we use xCas9 3.7, which recognizes NG PAM. To enhance the dxABE-PS utility, we develop a prediction workflow for protein dysfunction using ABE, namely, DABE-CSP (dysfunction via ABE through CRISPOR-SIFT prediction). We applied DABE-CSP to inactivate several genes in Pseudomonas putida KT2440 to accumulate a nylon precursor, i.e., muconic acid from catechol with 100% yield. Moreover, we expanded the ABE to non-model Pseudomonas species by developing an nxABE system for P. chengduensisDY56-96, isolated from sediment samples from the seamount area in the West Pacific Ocean. Taken together, the establishment of the ABE systems along with DABE-CSP will fast-track research on Pseudomonas species.},
}
@article {pmid35389493,
year = {2022},
author = {Nagalakshmi, U and Meier, N and Liu, JY and Voytas, DF and Dinesh-Kumar, SP},
title = {High-efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus.},
journal = {Plant physiology},
volume = {189},
number = {3},
pages = {1241-1245},
pmid = {35389493},
issn = {1532-2548},
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Mutation ; *RNA Viruses/genetics/metabolism ; },
}
@article {pmid35389492,
year = {2022},
author = {Carballar-Lejarazú, R and Tushar, T and Pham, TB and James, AA},
title = {Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae.},
journal = {Genetics},
volume = {221},
number = {2},
pages = {},
pmid = {35389492},
issn = {1943-2631},
mesh = {Alleles ; Animals ; *Anopheles/genetics ; CRISPR-Cas Systems ; Female ; *Malaria/prevention &amp; control ; Maternal Inheritance ; Mosquito Vectors/genetics ; },
abstract = {CRISPR/Cas9 technologies are important tools for the development of gene-drive systems to modify mosquito vector populations to control the transmission of pathogens that cause diseases such as malaria. However, one of the challenges for current Cas9-based drive systems is their ability to produce drive-resistant alleles resulting from insertions and deletions (indels) caused principally by nonhomologous end-joining following chromosome cleavage. Rapid increases in the frequency of such alleles may impair gene-drive dynamics. We explored the generation of indels in the germline and somatic cells in female gene-drive lineages using a series of selective crosses between a gene-drive line, AgNosCd-1, and wild-type mosquitoes. We find that potential drive-resistant mutant alleles are generated largely during embryonic development, most likely caused by deposition of the Cas9 endonuclease and guide RNAs in oocytes and resulting embryos by homozygous and hemizygous gene-drive mothers.},
}
@article {pmid35389262,
year = {2022},
author = {Martinez, MG and Combe, E and Inchauspe, A and Mangeot, PE and Delberghe, E and Chapus, F and Neveu, G and Alam, A and Carter, K and Testoni, B and Zoulim, F},
title = {CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants.},
journal = {mBio},
volume = {13},
number = {2},
pages = {e0288821},
pmid = {35389262},
issn = {2150-7511},
mesh = {CRISPR-Cas Systems ; DNA, Circular/genetics ; DNA, Viral/genetics ; *Hepatitis B virus/genetics ; *Hepatitis B, Chronic/drug therapy ; Humans ; RNA, Guide/genetics ; },
abstract = {Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. IMPORTANCE Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.},
}
@article {pmid35389256,
year = {2022},
author = {Govindarajan, S and Borges, A and Karambelkar, S and Bondy-Denomy, J},
title = {Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria.},
journal = {Journal of bacteriology},
volume = {204},
number = {5},
pages = {e0010522},
pmid = {35389256},
issn = {1098-5530},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; DNA Helicases/genetics ; Endonucleases/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remain largely unaddressed. Here, we used fluorescent fusion proteins introduced at the chromosomal CRISPR-Cas locus to study the localization of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. When lacking a target in the cell, the Cascade complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. When targeted to an integrated prophage, however, the CRISPR RNA (crRNA)-guided type I-F Cascade complex and a majority of Cas3 molecules in the cell are recruited to a single focus. Nucleoid association of the Csy proteins that form the Cascade complex is crRNA dependent and specifically inhibited by the expression of anti-CRISPR AcrIF2, which blocks protospacer adjacent motif (PAM) binding. The Cas9 nuclease is also nucleoid localized, only when single guide RNA (sgRNA) bound, which is abolished by the PAM-binding inhibitor AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex. IMPORTANCE CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood. By investigating the cell biology of the type I-F CRISPR-Cas system, we show that the surveillance complex, which "patrols" the cell to find targets, is largely nucleoid bound, while Cas3 nuclease is cytoplasmic. Nucleoid localization is also conserved for class 2 CRISPR-Cas single protein effector Cas9. Our observation of differential localization of the surveillance complex and Cas3 reveals a new layer of posttranslational spatiotemporal regulation to prevent autoimmunity.},
}
@article {pmid35389053,
year = {2022},
author = {Töpfer, R and Trapp, O},
title = {A cool climate perspective on grapevine breeding: climate change and sustainability are driving forces for changing varieties in a traditional market.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {},
number = {},
pages = {},
pmid = {35389053},
issn = {1432-2242},
abstract = {A multitude of diverse breeding goals need to be combined in a new cultivar, which always forces to compromise. The biggest challenge grapevine breeders face is the extraordinarily complex trait of wine quality, which is the all-pervasive and most debated characteristic. Since the 1920s, Germany runs continuous grapevine breeding programmes. This continuity was the key to success and lead to various new cultivars on the market, so called PIWIs. Initially, introduced pests and diseases such as phylloxera, powdery and downy mildew were the driving forces for breeding. However, preconceptions about the wine quality of new resistant selections impeded the market introduction. These preconceptions are still echoing today and may be the reason in large parts of the viticultural community for: (1) ignoring substantial breeding progress, and (2) sticking to successful markets of well-known varietal wines or blends (e.g. Chardonnay, Cabernet Sauvignon, Riesling). New is the need to improve viticulture´s sustainability and to adapt to changing environmental conditions. Climate change with its extreme weather will impose the need for a change in cultivars in many wine growing regions. Therefore, a paradigm shift is knocking on the door: new varieties (PIWIs) versus traditional varieties for climate adapted and sustainable viticulture. However, it will be slow process and viticulture is politically well advised to pave the way to variety innovation. In contrast to the widely available PIWIs, competitive cultivars created by means of new breeding technologies (NBT, e.g. through CRISPR/Cas) are still decades from introduction to the market.},
}
@article {pmid35388425,
year = {2022},
author = {Ezura, H},
title = {Letter to the Editor: The World's First CRISPR Tomato Launched to a Japanese Market: The Social-Economic Impact of its Implementation on Crop Genome Editing.},
journal = {Plant &amp; cell physiology},
volume = {63},
number = {6},
pages = {731-733},
doi = {10.1093/pcp/pcac048},
pmid = {35388425},
issn = {1471-9053},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome, Plant/genetics ; Japan ; *Lycopersicon esculentum/genetics ; },
}
@article {pmid35388178,
year = {2022},
author = {Rönspies, M and Schindele, P and Wetzel, R and Puchta, H},
title = {CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.},
journal = {Nature protocols},
volume = {17},
number = {5},
pages = {1332-1358},
pmid = {35388178},
issn = {1750-2799},
mesh = {*Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Chromosomes ; Gene Editing/methods ; Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.},
}
@article {pmid35388146,
year = {2022},
author = {Fuchs, RT and Curcuru, JL and Mabuchi, M and Noireterre, A and Weigele, PR and Sun, Z and Robb, GB},
title = {Characterization of Cme and Yme thermostable Cas12a orthologs.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {325},
pmid = {35388146},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *DNA Cleavage ; Nucleic Acid Conformation ; RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.},
}
@article {pmid35387989,
year = {2022},
author = {Clow, PA and Du, M and Jillette, N and Taghbalout, A and Zhu, JJ and Cheng, AW},
title = {CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1871},
pmid = {35387989},
issn = {2041-1723},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Chromosomes ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics ; *RNA, Guide/genetics ; },
abstract = {Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.},
}
@article {pmid35387980,
year = {2022},
author = {Zhang, G and Liu, Y and Huang, S and Qu, S and Cheng, D and Yao, Y and Ji, Q and Wang, X and Huang, X and Liu, J},
title = {Enhancement of prime editing via xrRNA motif-joined pegRNA.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1856},
pmid = {35387980},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Genome ; },
abstract = {The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3'-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan-target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications.},
}
@article {pmid35387453,
year = {2022},
author = {Liu, FX and Cui, JQ and Park, H and Chan, KW and Leung, T and Tang, BZ and Yao, S},
title = {Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics.},
journal = {Analytical chemistry},
volume = {94},
number = {15},
pages = {5883-5892},
doi = {10.1021/acs.analchem.2c00067},
pmid = {35387453},
issn = {1520-6882},
mesh = {Biological Assay ; CRISPR-Cas Systems ; *Microfluidics ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Recombinases/metabolism ; },
abstract = {High sensitivity and specificity nucleic acid detection has been achieved by the Cas13a collateral effect in combination with a separate recombinase polymerase amplification (RPA). However, these emerging methods cannot provide accurate quantification of nucleic acids because the two-step assay performance may be compromised if the RPA and Cas13a reactions are simply unified in a single step. In this work, we first addressed the challenges associated with enzymatic incompatibility and the macromolecular crowding effect in the one-pot assay development, making the consolidated RPA-Cas13a assay a facile and robust diagnostic tool. Next, we found that the one-pot reaction cannot precisely quantify the targets at low concentrations. Thus, by leveraging droplet microfluidics, we converted the one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Due to the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-free signals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays are promising for the next generation of nucleic acid quantification methods.},
}
@article {pmid35386853,
year = {2022},
author = {He, X and Zeng, XX},
title = {Immunotherapy and CRISPR Cas Systems: Potential Cure of COVID-19?.},
journal = {Drug design, development and therapy},
volume = {16},
number = {},
pages = {951-972},
pmid = {35386853},
issn = {1177-8881},
mesh = {Antibodies, Neutralizing ; *COVID-19/drug therapy/therapy ; *CRISPR-Cas Systems ; Disease Progression ; Humans ; Immunization, Passive ; Immunologic Factors ; SARS-CoV-2 ; },
abstract = {The COVID-19 has plunged the world into a pandemic that affected millions. The continually emerging new variants of concern raise the question as to whether the existing vaccines will continue to provide sufficient protection for individuals from SARS-CoV-2 during natural infection. This narrative review aims to briefly outline various immunotherapeutic options and discuss the potential of clustered regularly interspaced short palindromic repeat (CRISPR Cas system technology against COVID-19 treatment as specific cure. As the development of vaccine, convalescent plasma, neutralizing antibodies are based on the understanding of human immune responses against SARS-CoV-2, boosting human body immune responses in case of SARS-CoV-2 infection, immunotherapeutics seem feasible as specific cure against COVID-19 if the present challenges are overcome. In cell based therapeutics, apart from the high costs, risks and side effects, there are technical problems such as the production of sufficient potent immune cells and antibodies under limited time to treat the COVID-19 patients in mild conditions prior to progression into a more severe case. The CRISPR Cas technology could be utilized to refine the specificity and safety of CAR-T cells, CAR-NK cells and neutralizing antibodies against SARS-CoV-2 during various stages of the COVID-19 disease progression in infected individuals. Moreover, CRISPR Cas technology are proposed in hypotheses to degrade the viral RNA in order to terminate the infection caused by SARS-CoV-2. Thus personalized cocktails of immunotherapeutics and CRISPR Cas systems against COVID-19 as a strategy might prevent further disease progression and circumvent immunity escape.},
}
@article {pmid35386818,
year = {2022},
author = {Jiang, Y and Hoenisch, RC and Chang, Y and Bao, X and Cameron, CE and Lian, XL},
title = {Robust genome and RNA editing via CRISPR nucleases in PiggyBac systems.},
journal = {Bioactive materials},
volume = {14},
number = {},
pages = {313-320},
pmid = {35386818},
issn = {2452-199X},
abstract = {CRISPR/Cas-mediated genome editing in human pluripotent stem cells (hPSCs) offers unprecedented opportunities for developing in vitro disease modeling, drug screening and cell-based therapies. To efficiently deliver the CRISPR components, here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively. These vectors utilized the PiggyBac transposon system, which allows stable expression of CRISPR components in hPSCs. The Cas9 vector PB-CRISPR exhibited high efficiency (up to 99%) of inducing gene knockout in both protein-coding genes and long non-coding RNAs. The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown (98% knockdown for CD90) with optimized gRNA designs. Taken together, our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.},
}
@article {pmid35386712,
year = {2022},
author = {Scheller, SH and Rashad, Y and Saleh, FM and Willingham, KA and Reilich, A and Lin, D and Izadpanah, R and Alt, EU and Braun, SE},
title = {Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {821190},
pmid = {35386712},
issn = {1664-3224},
mesh = {Alleles ; CRISPR-Cas Systems ; *HIV Infections/genetics ; *HIV Seropositivity/genetics ; *HIV-1/genetics ; Humans ; Receptors, CCR5/genetics ; Virus Replication ; },
abstract = {Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.},
}
@article {pmid35386307,
year = {2022},
author = {Haider, MZ and Shabbir, MAB and Yaqub, T and Sattar, A and Maan, MK and Mahmood, S and Mehmood, T and Aslam, HB},
title = {CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.},
journal = {BioMed research international},
volume = {2022},
number = {},
pages = {9080396},
pmid = {35386307},
issn = {2314-6141},
mesh = {Ampicillin ; Animals ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial ; Microbial Sensitivity Tests ; Poultry ; *Salmonella enterica ; *Salmonella enteritidis/genetics ; },
abstract = {Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.},
}
@article {pmid35385916,
year = {2022},
author = {Mallick, T and Mishra, R and Mohanty, S and Joshi, RK},
title = {Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features.},
journal = {The plant pathology journal},
volume = {38},
number = {2},
pages = {102-114},
pmid = {35385916},
issn = {1598-2254},
abstract = {Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.},
}
@article {pmid35385325,
year = {2022},
author = {Ahmed, ASI and Sheng, MHC and Lau, KW and Wilson, SM and Wongworawat, MD and Tang, X and Ghahramanpouri, M and Nehme, A and Xu, Y and Abdipour, A and Zhang, XB and Wasnik, S and Baylink, DJ},
title = {Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation.},
journal = {American journal of physiology. Cell physiology},
volume = {322},
number = {5},
pages = {C977-C990},
pmid = {35385325},
issn = {1522-1563},
support = {IK6 BX003782/BX/BLRD VA/United States ; },
mesh = {Animals ; *Bone Resorption/metabolism ; Calcium/metabolism ; Calcium Channels/genetics/metabolism ; Cell Differentiation ; Mice ; Osteoclasts/metabolism ; *Osteogenesis ; RANK Ligand/metabolism ; Receptors, Calcium-Sensing/genetics/metabolism ; Vascular Endothelial Growth Factor A/metabolism ; },
abstract = {A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.},
}
@article {pmid35385207,
year = {2022},
author = {Yang, S and Joesaar, A and Bögels, BWA and Mann, S and de Greef, TFA},
title = {Protocellular CRISPR/Cas-Based Diffusive Communication Using Transcriptional RNA Signaling.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {26},
pages = {e202202436},
pmid = {35385207},
issn = {1521-3773},
mesh = {*Artificial Cells ; *CRISPR-Cas Systems/genetics ; Communication ; DNA ; RNA/genetics ; },
abstract = {Protocells containing enzyme-driven biomolecular circuits that can process and exchange information offer a promising approach for mimicking cellular features and developing molecular information platforms. Here, we employ synthetic transcriptional circuits together with CRISPR/Cas-based DNA processing inside semipermeable protein-polymer microcompartments. We first establish a transcriptional protocell that can be activated by external DNA strands and produce functional RNA aptamers. Subsequently, we engineer a transcriptional module to generate RNA strands functioning as diffusive signals that can be sensed by neighboring protocells and trigger the activation of internalized DNA probes or localization of Cas nucleases. Our results highlight the opportunities to combine CRISPR/Cas machinery and DNA nanotechnology for protocellular communication and provide a step towards the development of protocells capable of distributed molecular information processing.},
}
@article {pmid35384405,
year = {2022},
author = {Koch, PJ and Webb, S and Gugger, JA and Salois, MN and Koster, MI},
title = {Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes.},
journal = {Current protocols},
volume = {2},
number = {4},
pages = {e408},
pmid = {35384405},
issn = {2691-1299},
support = {R21EY029081/NH/NIH HHS/United States ; R21 EY029081/EY/NEI NIH HHS/United States ; R01 AR072621/AR/NIAMS NIH HHS/United States ; R01AR072621/NH/NIH HHS/United States ; },
mesh = {Cell Culture Techniques/methods ; Cell Differentiation/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Keratinocytes ; Skin ; },
abstract = {Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create "designer" iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC-derived keratinocytes (iPSC-K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF[™] Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell[™] 400 6-well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells.},
}
@article {pmid35384352,
year = {2022},
author = {Whitley, JA and Kim, S and Lou, L and Ye, C and Alsaidan, OA and Sulejmani, E and Cai, J and Desrochers, EG and Beharry, Z and Rickman, CB and Klingeborn, M and Liu, Y and Xie, ZR and Cai, H},
title = {Encapsulating Cas9 into extracellular vesicles by protein myristoylation.},
journal = {Journal of extracellular vesicles},
volume = {11},
number = {4},
pages = {e12196},
pmid = {35384352},
issn = {2001-3078},
support = {R21 AI157831/AI/NIAID NIH HHS/United States ; U01 CA225784/CA/NCI NIH HHS/United States ; R01 EY031748/EY/NEI NIH HHS/United States ; R21 EY028671/EY/NEI NIH HHS/United States ; P30 EY005722/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Extracellular Vesicles ; Gene Editing ; Genetic Therapy ; },
abstract = {CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.},
}
@article {pmid35382990,
year = {2022},
author = {Horie, M and Yamano-Adachi, N and Kawabe, Y and Kaneoka, H and Fujita, H and Nagamori, E and Iwai, R and Sato, Y and Kanie, K and Ohta, S and Somiya, M and Ino, K},
title = {Recent advances in animal cell technologies for industrial and medical applications.},
journal = {Journal of bioscience and bioengineering},
volume = {133},
number = {6},
pages = {509-514},
doi = {10.1016/j.jbiosc.2022.03.005},
pmid = {35382990},
issn = {1347-4421},
mesh = {Animals ; Bioreactors ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing/methods ; *Genetic Engineering ; Humans ; Mammals/genetics ; Regenerative Medicine ; },
abstract = {The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.},
}
@article {pmid35382978,
year = {2022},
author = {Mou, Z and Zhao, D},
title = {Gene rational design: the dawn of crop breeding.},
journal = {Trends in plant science},
volume = {27},
number = {7},
pages = {633-636},
doi = {10.1016/j.tplants.2022.03.007},
pmid = {35382978},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics ; *Plant Breeding ; },
abstract = {Limited natural genetic diversity restricts the creation of excellent crops. Modeling-guided rational design represents a promising protein engineering technology to optimize existing genes for desired agronomic traits. Rational design coupled with other engineering approaches could also be applied in artificial gene improvement for the creation of economically valuable crops.},
}
@article {pmid35381364,
year = {2022},
author = {Sun, N and Zhao, D and Li, S and Zhang, Z and Bi, C and Zhang, X},
title = {Reconstructed glycosylase base editors GBE2.0 with enhanced C-to-G base editing efficiency and purity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {7},
pages = {2452-2463},
pmid = {35381364},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Mammals ; },
abstract = {Base editing techniques were developed for precise base conversion on cellular genomic DNA, which has great potential for the treatment of human genetic diseases. The glycosylase base editor (GBE) recently developed in our lab was used to perform C-to-G transversions in mammalian cells. To improve the application prospects of GBE, it is necessary to further increase its performance. With this aim, we replaced the human Ung in GBE with Ung1 from Saccharomyces cerevisiae. The resulting editor APOBEC-nCas9-Ung1 was tested at 17 chromosomal loci and was found to have an increased C-to-G editing efficiency ranging from 2.63% to 52.3%, with an average of 23.48%, which was a significant improvement over GBE, with an average efficiency of 15.54%, but with a decreased purity. For further improvement, we constructed APOBEC(R33A)-nCas9-Rad51-Ung1 with two beneficial modifications adapted from previous reports. This base editor was able to achieve even higher editing efficiency ranging from 8.70% to 72.1%, averaging 30.88%, while also exhibiting high C-to-G purity ranging from 35.57% to 92.92%, and was designated GBE2.0. GBE2.0 provides high C-to-G editing efficiency and purity in mammalian cells, making it a powerful genetic tool for scientific research or potential genetic therapies for disease-causing G/C mutations.},
}
@article {pmid35380812,
year = {2022},
author = {Pan, R and Liu, J and Wang, P and Wu, D and Chen, J and Wu, Y and Li, G},
title = {Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {14},
pages = {4484-4491},
doi = {10.1021/acs.jafc.1c08262},
pmid = {35380812},
issn = {1520-5118},
mesh = {Alkalies ; Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; DNA/genetics ; Gold ; *Metal Nanoparticles ; Milk ; *Nucleic Acids ; Spectrum Analysis, Raman ; },
abstract = {An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)6[4-]), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core-shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.},
}
@article {pmid35380342,
year = {2022},
author = {Chen, J and Jiang, S and Shao, H and Li, B and Ji, T and Staiculescu, D and He, J and Zhao, J and Cai, L and Liang, X and Xu, J and Cai, X},
title = {CRISPR-Cas9-based genome-wide screening identified novel targets for treating sorafenib-resistant hepatocellular carcinoma: a cross-talk between FGF21 and the NRF2 pathway.},
journal = {Science China. Life sciences},
volume = {65},
number = {10},
pages = {1998-2016},
pmid = {35380342},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/drug therapy/genetics/metabolism ; Fibroblast Growth Factors ; Humans ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; *Liver Neoplasms/drug therapy/genetics/metabolism ; NF-E2-Related Factor 2/genetics/metabolism/therapeutic use ; Signal Transduction ; Sorafenib/pharmacology/therapeutic use ; },
abstract = {The treatment of hepatocellular carcinoma (HCC) has been dominated by multikinase inhibitors for more than a decade. However, drug resistance can severely restrict the efficacy of these drugs. Using CRISPR/CAS9 genome library screening, we evaluated Kelch-like ECH-associated protein 1 (KEAP1) as a key regulator of sorafenib's susceptibility in HCC. We also investigated whether KEAP1's knockdown can stabilize nuclear factor (erythroid-derived 2)-like 2 (NRF2) protein levels that led to sorafenib's resistance, including an NRF2 inhibitor that can synergize with sorafenib to abolish HCC's growth in vitro and in vivo. Furthermore, we clarified that fibroblast growth factor 21 (FGF21) is an important downstream regulator of NRF2 in HCC. Intriguingly, we observed that FGF21 bound to NRF2 through the C-terminus of FGF21, thereby stabilizing NRF2 by reducing its ubiquitination and generating a positive feedback loop in sorafenib-resistant HCC. These findings, therefore, propose that targeting FGF21 is a promising strategy to combat HCC sorafenib's resistance.},
}
@article {pmid35379962,
year = {2022},
author = {Liu, B and Dong, X and Cheng, H and Zheng, C and Chen, Z and Rodríguez, TC and Liang, SQ and Xue, W and Sontheimer, EJ},
title = {A split prime editor with untethered reverse transcriptase and circular RNA template.},
journal = {Nature biotechnology},
volume = {40},
number = {9},
pages = {1388-1393},
pmid = {35379962},
issn = {1546-1696},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Deoxyribonuclease I/genetics ; Gene Editing ; Mice ; *RNA, Circular/genetics ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; *Tyrosinemias/genetics ; },
abstract = {Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion-deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify β-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein-protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.},
}
@article {pmid35379961,
year = {2022},
author = {Mok, BY and Kotrys, AV and Raguram, A and Huang, TP and Mootha, VK and Liu, DR},
title = {CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA.},
journal = {Nature biotechnology},
volume = {40},
number = {9},
pages = {1378-1387},
pmid = {35379961},
issn = {1546-1696},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 EB027793/EB/NIBIB NIH HHS/United States ; R35 GM122455/GM/NIGMS NIH HHS/United States ; T32 GM095450/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; Cytidine Deaminase/genetics ; DNA, Mitochondrial/genetics ; *Gene Editing ; Humans ; Mitochondria/genetics/metabolism ; },
abstract = {The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15-30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.},
}
@article {pmid35378361,
year = {2022},
author = {Krueger, LA and Morris, AC},
title = {Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing.},
journal = {Biochemical and biophysical research communications},
volume = {608},
number = {},
pages = {8-13},
pmid = {35378361},
issn = {1090-2104},
support = {F30 EY031545/EY/NEI NIH HHS/United States ; R01 EY021769/EY/NEI NIH HHS/United States ; TL1 TR001997/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Knock-In Techniques ; Recombinational DNA Repair ; *Zebrafish/genetics ; },
abstract = {Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and developmental biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.},
}
@article {pmid35378143,
year = {2022},
author = {Kovářová, J and Novotná, M and Faria, J and Rico, E and Wallace, C and Zoltner, M and Field, MC and Horn, D},
title = {CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes.},
journal = {Molecular and biochemical parasitology},
volume = {249},
number = {},
pages = {111476},
doi = {10.1016/j.molbiopara.2022.111476},
pmid = {35378143},
issn = {1872-9428},
support = {217105/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; 203134/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 097945/B/11/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Genes, Essential ; *Trypanosoma/genetics ; *Trypanosoma brucei brucei/genetics ; Untranslated Regions ; },
abstract = {Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; [GFP]HAT2 or [GFP]HDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.},
}
@article {pmid35377968,
year = {2022},
author = {Cheng, Y and Sretenovic, S and Zhang, Y and Pan, C and Huang, J and Qi, Y},
title = {Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100571},
doi = {10.1002/biot.202100571},
pmid = {35377968},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Endonucleases/genetics ; Gene Editing ; Humans ; *Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.},
}
@article {pmid35377421,
year = {2022},
author = {Luo, Z and Dai, W and Wang, C and Ye, Q and Zhou, Q and Wan, QL},
title = {Gene activation in Caenorhabditis elegans using the Campylobacter jejuni CRISPR-Cas9 feeding system.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {6},
pages = {},
pmid = {35377421},
issn = {2160-1836},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Caenorhabditis elegans/genetics ; *Campylobacter jejuni/genetics ; Endonucleases/genetics ; Gene Editing/methods ; Transcriptional Activation ; },
abstract = {Clustered regularly interspaced palindromic repeats-based activation system, a powerful genetic manipulation technology, can modulate endogenous gene transcription in various organisms through fusing nuclease-deficient Cas9 to transcriptional regulatory domains. At present, this clustered regularly interspaced palindromic repeats-based activation system has been applied to activate gene expression by microinjection manner in Caenorhabditis elegans. However, this complicated and time-consuming injection manner is not suitable for efficient and high-throughput gene regulation with clustered regularly interspaced palindromic repeats-Cas9 system. Here, we engineered a Campylobacter jejun clustered regularly interspaced palindromic repeats-Cas9-based gene activation system through bacteria feeding technique to delivering gene-specific sgRNA in C. elegans. It enables to activate various endogenous genes efficiently, as well as induce the corresponding phenotypes with a more efficient and labor-saving manner. Collectively, our results demonstrated that our novel dCjCas9-based activation feeding system holds great promise and potential in C. elegans.},
}
@article {pmid35377365,
year = {2022},
author = {Yan, Q and He, Y and Yue, Y and Jie, L and Wen, T and Zhao, Y and Zhang, M and Zhang, T},
title = {Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {181},
pages = {},
doi = {10.3791/63629},
pmid = {35377365},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Grasshoppers/genetics/metabolism ; Humans ; RNA, Guide/genetics ; Technology ; },
abstract = {The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.},
}
@article {pmid35373735,
year = {2022},
author = {Cornean, A and Gierten, J and Welz, B and Mateo, JL and Thumberger, T and Wittbrodt, J},
title = {Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35373735},
issn = {2050-084X},
mesh = {Adenine ; Animals ; CRISPR-Cas Systems ; Cytosine ; DNA ; *Gene Editing ; Mutation ; *Zebrafish/genetics ; },
abstract = {Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.},
}
@article {pmid35373270,
year = {2022},
author = {},
title = {A Base Editing Platform Shows Function of Cancer-Associated Variants.},
journal = {Cancer discovery},
volume = {12},
number = {4},
pages = {883},
doi = {10.1158/2159-8290.CD-RW2022-034},
pmid = {35373270},
issn = {2159-8290},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {A resource was developed to introduce and interrogate cancer-associated single-nucleotide variants.},
}
@article {pmid35373187,
year = {2022},
author = {Ravendran, S and Hernández, SS and König, S and Bak, RO},
title = {CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {793010},
pmid = {35373187},
issn = {2673-3439},
abstract = {Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.},
}
@article {pmid35371164,
year = {2022},
author = {Ali, Q and Yu, C and Hussain, A and Ali, M and Ahmar, S and Sohail, MA and Riaz, M and Ashraf, MF and Abdalmegeed, D and Wang, X and Imran, M and Manghwar, H and Zhou, L},
title = {Genome Engineering Technology for Durable Disease Resistance: Recent Progress and Future Outlooks for Sustainable Agriculture.},
journal = {Frontiers in plant science},
volume = {13},
number = {},
pages = {860281},
pmid = {35371164},
issn = {1664-462X},
abstract = {Crop production worldwide is under pressure from multiple factors, including reductions in available arable land and sources of water, along with the emergence of new pathogens and development of resistance in pre-existing pathogens. In addition, the ever-growing world population has increased the demand for food, which is predicted to increase by more than 100% by 2050. To meet these needs, different techniques have been deployed to produce new cultivars with novel heritable mutations. Although traditional breeding continues to play a vital role in crop improvement, it typically involves long and laborious artificial planting over multiple generations. Recently, the application of innovative genome engineering techniques, particularly CRISPR-Cas9-based systems, has opened up new avenues that offer the prospects of sustainable farming in the modern agricultural industry. In addition, the emergence of novel editing systems has enabled the development of transgene-free non-genetically modified plants, which represent a suitable option for improving desired traits in a range of crop plants. To date, a number of disease-resistant crops have been produced using gene-editing tools, which can make a significant contribution to overcoming disease-related problems. Not only does this directly minimize yield losses but also reduces the reliance on pesticide application, thereby enhancing crop productivity that can meet the globally increasing demand for food. In this review, we describe recent progress in genome engineering techniques, particularly CRISPR-Cas9 systems, in development of disease-resistant crop plants. In addition, we describe the role of CRISPR-Cas9-mediated genome editing in sustainable agriculture.},
}
@article {pmid35369445,
year = {2022},
author = {Santana de Carvalho, D and Trovatti Uetanabaro, AP and Kato, RB and Aburjaile, FF and Jaiswal, AK and Profeta, R and De Oliveira Carvalho, RD and Tiwar, S and Cybelle Pinto Gomide, A and Almeida Costa, E and Kukharenko, O and Orlovska, I and Podolich, O and Reva, O and Ramos, PIP and De Carvalho Azevedo, VA and Brenig, B and Andrade, BS and de Vera, JP and Kozyrovska, NO and Barh, D and Góes-Neto, A},
title = {The Space-Exposed Kombucha Microbial Community Member Komagataeibacter oboediens Showed Only Minor Changes in Its Genome After Reactivation on Earth.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {782175},
pmid = {35369445},
issn = {1664-302X},
abstract = {Komagataeibacter is the dominant taxon and cellulose-producing bacteria in the Kombucha Microbial Community (KMC). This is the first study to isolate the K. oboediens genome from a reactivated space-exposed KMC sample and comprehensively characterize it. The space-exposed genome was compared with the Earth-based reference genome to understand the genome stability of K. oboediens under extraterrestrial conditions during a long time. Our results suggest that the genomes of K. oboediens IMBG180 (ground sample) and K. oboediens IMBG185 (space-exposed) are remarkably similar in topology, genomic islands, transposases, prion-like proteins, and number of plasmids and CRISPR-Cas cassettes. Nonetheless, there was a difference in the length of plasmids and the location of cas genes. A small difference was observed in the number of protein coding genes. Despite these differences, they do not affect any genetic metabolic profile of the cellulose synthesis, nitrogen-fixation, hopanoid lipids biosynthesis, and stress-related pathways. Minor changes are only observed in central carbohydrate and energy metabolism pathways gene numbers or sequence completeness. Altogether, these findings suggest that K. oboediens maintains its genome stability and functionality in KMC exposed to the space environment most probably due to the protective role of the KMC biofilm. Furthermore, due to its unaffected metabolic pathways, this bacterial species may also retain some promising potential for space applications.},
}
@article {pmid35369433,
year = {2022},
author = {Riedl, A and Fischer, J and Burgert, HG and Ruzsics, Z},
title = {Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {854690},
pmid = {35369433},
issn = {1664-302X},
abstract = {Recombinant adenovirus (rAd) vectors represent one of the most frequently used vehicles for gene transfer applications in vitro and in vivo. rAd genomes are constructed in Escherichia coli where their genomes can be maintained, propagated, and modified in form of circular plasmids or bacterial artificial chromosomes. Although the rescue of rAds from their circular plasmid or bacmid forms is well established, it works with relatively low primary efficiency, preventing this technology for library applications. To overcome this barrier, we tested a novel strategy for the reconstitution of rAds that utilizes the CRISPR/Cas-machinery to cleave the circular rAd genomes in close proximity to their inverted terminal repeats (ITRs) within the producer cells upon transfection. This CRISPR/Cas-mediated in vivo terminal resolution allowed efficient rescue of vectors derived from different human adenovirus (HAdV) species. By this means, it was not only possible to increase the efficiency of virus rescue by about 50-fold, but the presented methodology appeared also remarkably simpler and faster than traditional rAd reconstitution methods.},
}
@article {pmid35368966,
year = {2022},
author = {Cai, J and Wu, D and Jin, Y and Bao, S},
title = {Effect of CMB Carrying PTX and CRISPR/Cas9 on Endometrial Cancer Naked Mouse Model.},
journal = {Journal of healthcare engineering},
volume = {2022},
number = {},
pages = {7119195},
pmid = {35368966},
issn = {2040-2309},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; *Endometrial Neoplasms/genetics ; Female ; Glycogen Synthase Kinase 3 ; Humans ; Mammals ; Mice ; *Paclitaxel/pharmacology/therapeutic use ; },
abstract = {Endometrial cancer, one of the most common gynecological cancers in women. Patients with advanced or recurrent disease have poor long-term outcomes. The current experiment explore the roles of cationic microbubbles (CMBs) carrying paclitaxel (PTX) and CRISPR/Cas9 plasmids on the xenotransplantation model of mice with endometrial cancer. The tumor histology, tumor cell viability, cell cycle, and invasion ability were investigated. Meanwhile, the P27, P21, GSK-3, Bcl-2 associated death promoter (Bad), mammalian target of rapamycin (mTOR), and C-erbB-2 expressions were evaluated by qRT-PCR and western blotting, respectively. CMB-PTX-CRISPR/Cas9 had an inhibitory action on the tumor growth, tumor cell viability, cell cycle, and invasion ability of the mouse xenograft model of endometrial cancer. The CMB-PTX-CRISPR/Cas9 increased the GSK-3, P21, P27, and Bad expression levels, while reduced the C-erbB-2 and mTOR expressions. CMBs loaded with both PTX and CRISPR/Cas9 plasmids may be a new combination treatment with much potential. CMB-PTX-CRISPR/Cas9 may regulate the tumor cell viability, invasion, and metastasis of endometrial cancer naked mouse model by upregulating expressions of GSK-3, P21, P27, and Bad.},
}
@article {pmid35367933,
year = {2022},
author = {Liu, N and Liu, R and Zhang, J},
title = {CRISPR-Cas12a-mediated label-free electrochemical aptamer-based sensor for SARS-CoV-2 antigen detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {146},
number = {},
pages = {108105},
pmid = {35367933},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; DNA ; Humans ; Nucleic Acid Amplification Techniques/methods ; SARS-CoV-2/genetics ; },
abstract = {Serological antigen testing has emerged as an important diagnostic paradigm in COVID-19, but often suffers from potential cross-reactivity. To address this limitation, we herein report a label-free electrochemical aptamer-based sensor for the detection of SARS-CoV-2 antigen by integrating aptamer-based specific recognition with CRISPR-Cas12a-mediated signal amplification. The sensing principle is based on the competitive binding of antigen and the preassembled Cas12a-crRNA complex to the antigen-specific aptamer, resulting in a change in the collateral cleavage activity of Cas12a. To further generate an electrochemical signal, a DNA architecture was fabricated by in situ rolling circle amplification on a gold electrode, which serves as a novel substrate for Cas12a. Upon Cas12a-based collateral DNA cleavage, the DNA architecture was degraded, leading to a significant decrease in impedance that can be measured spectroscopically. Using SARS-CoV-2 nucleocapsid antigen as the model, the proposed CRISPR-Cas12a-based electrochemical sensor (CRISPR-E) showed excellent analytical performance for the quantitative detection of nucleocapsid antigen. Since in vitro selection can obtain aptamers selective for many SARS-CoV-2 antigens, the proposed strategy can expand this powerful CRISPR-E system significantly for quantitative monitoring of a wide range of COVID-19 biomarkers.},
}
@article {pmid35366394,
year = {2022},
author = {Kurihara, N and Nakagawa, R and Hirano, H and Okazaki, S and Tomita, A and Kobayashi, K and Kusakizako, T and Nishizawa, T and Yamashita, K and Scott, DA and Nishimasu, H and Nureki, O},
title = {Structure of the type V-C CRISPR-Cas effector enzyme.},
journal = {Molecular cell},
volume = {82},
number = {10},
pages = {1865-1877.e4},
pmid = {35366394},
issn = {1097-4164},
support = {MC_UP_A025_1012/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/genetics ; *RNA, Guide/metabolism ; Ribonucleases/metabolism ; },
abstract = {RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.},
}
@article {pmid35366350,
year = {2022},
author = {Murugesan, AC and Varughese, HS},
title = {Analysis of CRISPR-Cas system and antimicrobial resistance in Staphylococcus coagulans isolates.},
journal = {Letters in applied microbiology},
volume = {75},
number = {1},
pages = {126-134},
doi = {10.1111/lam.13713},
pmid = {35366350},
issn = {1472-765X},
mesh = {Animals ; *Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; Dogs ; Drug Resistance, Bacterial ; Staphylococcus ; },
abstract = {CRISPR-Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR-Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR-Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR-Cas system. Three isolates from Antarctica seals neither contain CRISPR-Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR-Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR-Cas system, and this Type IIIA CRISPR-Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR-Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR-Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.},
}
@article {pmid35365834,
year = {2022},
author = {Poudel, R and Rodriguez, LT and Reisch, CR and Rivers, AR},
title = {GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes.},
journal = {GigaScience},
volume = {11},
number = {},
pages = {},
pmid = {35365834},
issn = {2047-217X},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genome ; *RNA, Guide/genetics ; Software ; },
abstract = {BACKGROUND: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens.<br><br>FINDINGS: GuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in &#x223c;1-2 minutes.<br><br>CONCLUSIONS: GuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.},
}
@article {pmid35364533,
year = {2022},
author = {Lin, X and Li, C and Meng, X and Yu, W and Duan, N and Wang, Z and Wu, S},
title = {CRISPR-Cas12a-mediated luminescence resonance energy transfer aptasensing platform for deoxynivalenol using gold nanoparticle-decorated Ti3C2Tx MXene as the enhanced quencher.},
journal = {Journal of hazardous materials},
volume = {433},
number = {},
pages = {128750},
doi = {10.1016/j.jhazmat.2022.128750},
pmid = {35364533},
issn = {1873-3336},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer/methods ; Gold ; Luminescence ; *Metal Nanoparticles ; Titanium ; Trichothecenes ; },
abstract = {Deoxynivalenol (DON) is a typical mycotoxin in cereals and poses tremendous threats to the ecological environment and public health. Therefore, exploiting sensitive and robust analytical methods for DON is particularly important. Here, we fabricated a CRISPR-Cas12a-mediated luminescence resonance energy transfer (LRET) aptasensor to detect DON by using single-stranded DNA modified upconversion nanoparticles (ssDNA-UCNPs) as anti-interference luminescence labels and gold nanoparticle-decorated Ti3C2Tx MXene nanosheets (MXene-Au) as enhanced quenchers. The DON aptamer can activate the trans-cleavage activity of Cas12a to indiscriminately cut nearby ssDNA-UCNPs into small fragments, which prevents ssDNA-UCNPs from adsorbing onto MXene-Au, and the upconversion luminescence (UCL) remains. Upon the binding of the aptamer with DON, the trans-cleavage activity of Cas12a was suppressed, and the ssDNA-UCNPs were not cleaved and easily adsorbed onto MXene-Au, which caused UCL quenching. Under optimized conditions, the limit of detection was determined to be 0.64 ng/mL with a linear range of 1 - 500 ng/mL. In addition, the sensor was successfully applied to detect DON in corn flour and Tai Lake water with recoveries of 96.2 - 105% and 95.2 - 104%, respectively. This platform achieves a sensitive and specific analysis of DON and greatly broadens the detection range of CRISPR-Cas sensors for non-nucleic acids hazards in the environment and food.},
}
@article {pmid35363475,
year = {2022},
author = {Malcı, K and Walls, LE and Rios-Solis, L},
title = {Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1555-1567},
pmid = {35363475},
issn = {2161-5063},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA, Viral/analysis/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.},
}
@article {pmid35362932,
year = {2022},
author = {Courtright-Lim, A},
title = {"CRISPR for Disabilities: How to Self-Regulate" or Something?.},
journal = {Journal of bioethical inquiry},
volume = {19},
number = {1},
pages = {151-161},
pmid = {35362932},
issn = {1872-4353},
mesh = {CRISPR-Cas Systems ; *Disabled Persons ; *Gene Editing ; Humans ; Stem Cell Research ; },
abstract = {The development of the CRISPR gene editing technique has been hyped as a technique that could fundamentally change scientific research and its clinical application. Unrecognized is the fact that it joins other technologies that have tried and failed under the same discourse of scientific hype. These technologies, like gene therapy and stem cell research, have moved quickly passed basic research into clinical application with dire consequences. Before hastily moving to clinical applications, it is necessary to consider basic research and determine how CRISPR/Cas systems should be applied. In the case of single gene diseases, that application is expected to have positive impacts, but as we shift to more complex diseases, the impact could be unintentionally negative. In the context of common disabilities, the level of genetic complexity may render this technology useless but potentially toxic, aggravating a social discourse that devalues those with disabilities. This paper intends to define the issues related to disability that are associated with using the CRIPSR/Cas system in basic research. It also aims to provide a decision tree to help determine whether the technology should be utilized or if alternative approaches beyond scientific research could lead to a better use of limited funding resources.},
}
@article {pmid35362263,
year = {2022},
author = {Maniego, J and Pesko, B and Habershon-Butcher, J and Hincks, P and Taylor, P and Tozaki, T and Ohnuma, A and Stewart, G and Proudman, C and Ryder, E},
title = {Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer.},
journal = {Drug testing and analysis},
volume = {14},
number = {8},
pages = {1429-1437},
doi = {10.1002/dta.3267},
pmid = {35362263},
issn = {1942-7611},
mesh = {Animals ; CRISPR-Cas Systems ; *Doping in Sports ; *Gene Editing/methods/veterinary ; Horses/genetics ; Mitochondria/genetics ; Nuclear Transfer Techniques/veterinary ; },
abstract = {Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, have highlighted the need to develop tools to detect potential prohibited use of the technology. One possible method involves the characterisation of the mitochondrial genome (which is not routinely preserved during cloning) and comparing it with the sequence of the registered dam. We present here our approach to whole-mitochondrial sequencing using tiled long-range PCR and next-generation sequencing. To determine whether the background mutation rate in the mitochondrial genome could potentially confound results, we sequenced 10 sets of dam and foal duos. We found variation between duos but none within duos, indicating that this method is feasible for future screening systems. Analysis of WGS data from over 100 Thoroughbred horses revealed wide variation in the mitochondria sequence within the breed, further displaying the utility of this approach.},
}
@article {pmid35358830,
year = {2022},
author = {Liu, Y and Jin, Y and Chen, T and Wu, Y and Peng, X and Li, W and Wei, S and Chen, M and Zou, Q and Guo, S and Xu, J and Tang, C and Zhou, X},
title = {Generation of a homozygous ARHGAP11B knockout hiPSC line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102764},
doi = {10.1016/j.scr.2022.102764},
pmid = {35358830},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; GTPase-Activating Proteins ; Gene Editing ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {The human specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal neocortex and plays a key role in the evolutionary expansion of the neocortex. Here, we generated a homozygous ARHGAP11B knockout human induced pluripotent stem cell (hiPSC) line through CRISPR/Cas9 gene editing system. ARHGAP11B deficient cell line maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The ARHGAP11B knockout cell line can provide a new cell model for studying the evolution of human neocortex.},
}
@article {pmid35358611,
year = {2022},
author = {Bernard, BE and Landmann, E and Jeker, LT and Schumann, K},
title = {CRISPR/Cas-based Human T cell Engineering: Basic Research and Clinical Application.},
journal = {Immunology letters},
volume = {245},
number = {},
pages = {18-28},
doi = {10.1016/j.imlet.2022.03.005},
pmid = {35358611},
issn = {1879-0542},
mesh = {*CRISPR-Cas Systems ; Cell Engineering ; *Gene Editing ; Genetic Engineering ; Humans ; T-Lymphocytes ; },
abstract = {Engineering human T cells for the treatment of cancer, viral infections and autoimmunity has been a long-standing dream of many immunologists and hematologists. Although primary human T cells have been genetically engineered for decades, this process was challenging, time consuming and mostly limited to transgene insertions mediated by viral transduction. The absence of widely accessible tools to efficiently and precisely engineer T cells genetically in a targeted manner limited their applicability as a living drug. This fundamentally changed with the discovery of CRISPR/Cas9 and its adaptation to human T cells. CRISPR/Cas9 has made T cell engineering widely accessible and accelerated the development of engineered adoptive T cell therapies. Only 6 years after the discovery of CRISPR/Cas9 as a biotechnological tool the first CRISPR engineered T cells have been administered to patients with refractory cancers in a phase I clinical trial. Novel Cas proteins - natural and engineered ones - are rapidly emerging. These offer for instance increased flexibility, activity and/or specificity. Moreover, sophisticated protein engineering and fusions of Cas with deaminases or reverse transcriptases enable genomic DNA editing without the need for a double strand cut. Thus, the "CRISPR tool box" for experimental use as well as for novel therapeutic approaches is rapidly expanding. In this review, we will summarize the current state of CRISPR/Cas-based engineering in human T cells for basic research and its clinical applications.},
}
@article {pmid35357193,
year = {2022},
author = {Wu, T and Cao, Y and Liu, Q and Wu, X and Shang, Y and Piao, J and Li, Y and Dong, Y and Liu, D and Wang, H and Liu, J and Ding, B},
title = {Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {14},
pages = {6575-6582},
doi = {10.1021/jacs.2c01760},
pmid = {35357193},
issn = {1520-5126},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/metabolism ; Gene Editing ; *Nanostructures ; Ribonucleoproteins ; },
abstract = {DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.},
}
@article {pmid35357116,
year = {2022},
author = {Beyersdorf, JP and Bawage, S and Iglesias, N and Peck, HE and Hobbs, RA and Wroe, JA and Zurla, C and Gersbach, CA and Santangelo, PJ},
title = {Robust, Durable Gene Activation In Vivo via mRNA-Encoded Activators.},
journal = {ACS nano},
volume = {16},
number = {4},
pages = {5660-5671},
pmid = {35357116},
issn = {1936-086X},
support = {U01 AI146356/AI/NIAID NIH HHS/United States ; },
mesh = {Transcriptional Activation ; RNA, Messenger/genetics ; *CRISPR-Cas Systems ; *Liposomes ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Programmable control of gene expression via nuclease-null Cas9 fusion proteins has enabled the engineering of cellular behaviors. Here, both transcriptional and epigenetic gene activation via synthetic mRNA and lipid nanoparticle delivery was demonstrated in vivo. These highly efficient delivery strategies resulted in high levels of activation in multiple tissues. Finally, we demonstrate durable gene activation in vivo via transient delivery of a single dose of a gene activator that combines VP64, p65, and HSF1 with a SWI/SNF chromatin remodeling complex component SS18, representing an important step toward gene-activation-based therapeutics. This induced sustained gene activation could be inhibited via mRNA-encoded AcrIIA4, further improving the safety profile of this approach.},
}
@article {pmid35355475,
year = {2022},
author = {Zhou, Z and Lü, X and Zhu, L and Zhou, J and Huang, H and Zhang, C and Liu, X},
title = {[Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {3},
pages = {1074-1085},
doi = {10.13345/j.cjb.210635},
pmid = {35355475},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.},
}
@article {pmid35354039,
year = {2022},
author = {Yi, C and Cai, C and Cheng, Z and Zhao, Y and Yang, X and Wu, Y and Wang, X and Jin, Z and Xiang, Y and Jin, M and Han, L and Zhang, A},
title = {Genome-wide CRISPR-Cas9 screening identifies the CYTH2 host gene as a potential therapeutic target of influenza viral infection.},
journal = {Cell reports},
volume = {38},
number = {13},
pages = {110559},
doi = {10.1016/j.celrep.2022.110559},
pmid = {35354039},
issn = {2211-1247},
mesh = {Antiviral Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Endosomes ; Humans ; *Influenza A virus/genetics ; *Influenza, Human/drug therapy/genetics ; },
abstract = {Host genes critical for viral infection are effective antiviral drug targets with tremendous potential due to their universal characteristics against different subtypes of viruses and minimization of drug resistance. Accordingly, we execute a genome-wide CRISPR-Cas9 screen with multiple rounds of survival selection. Enriched in this screen are several genes critical for host sialic acid biosynthesis and transportation, including the cytohesin 2 (CYTH2), tetratricopeptide repeat protein 24 (TTC24), and N-acetylneuraminate synthase (NANS), which we confirm are responsible for efficient influenza viral infection. Moreover, we reveal that CYTH2 is required for the early stage of influenza virus infection by mediating endosomal trafficking. Furthermore, CYTH2 antagonist SecinH3 blunts influenza virus infection in vivo. In summary, these data suggest that CYTH2 is an attractive target for developing host-directed antiviral drugs and therapeutics against influenza virus infection.},
}
@article {pmid35353638,
year = {2022},
author = {Rallapalli, KL and Ranzau, BL and Ganapathy, KR and Paesani, F and Komor, AC},
title = {Combined Theoretical, Bioinformatic, and Biochemical Analyses of RNA Editing by Adenine Base Editors.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {294-310},
pmid = {35353638},
issn = {2573-1602},
support = {R21 GM135736/GM/NIGMS NIH HHS/United States ; R35 GM138317/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine/metabolism ; CRISPR-Cas Systems ; Computational Biology ; *Gene Editing ; RNA/genetics ; *RNA Editing/genetics ; },
abstract = {Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.},
}
@article {pmid35352981,
year = {2022},
author = {Li, Y and Liu, Y and Singh, J and Tangprasertchai, NS and Trivedi, R and Fang, Y and Qin, PZ},
title = {Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {341-352},
pmid = {35352981},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Endonucleases/genetics ; Gene Editing ; *RNA/chemistry/genetics ; },
abstract = {CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. A key determinant in Cas9 target selection is DNA duplex unwinding to form an R-loop, in which the single-stranded RNA guide hybridizes with one of the DNA strands. To advance understanding on DNA unwinding by Cas9, we combined two types of spectroscopic label, 2-aminopurine and nitroxide spin-label, to investigate unwinding at a specific DNA base pair induced by Streptococcus pyogenes Cas9. Data obtained with RNA guide lengths varying from 13 to 20 nucleotide revealed that the DNA segment distal to the protospacer adjacent motif can adopt a "partial unwinding" state, in which a mixture of DNA-paired and DNA-unwound populations exist in equilibrium. Significant unwinding can occur at positions not supported by RNA/DNA pairing, and the degree of unwinding depends on RNA guide length and modulates DNA cleavage activity. The results shed light on Cas9 target selection and may inform developments of genome-engineering strategies.},
}
@article {pmid35351985,
year = {2022},
author = {Sridhara, S and Rai, J and Whyms, C and Goswami, H and He, H and Woodside, W and Terns, MP and Li, H},
title = {Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {279},
pmid = {35351985},
issn = {2399-3642},
support = {S10 RR025080/RR/NCRR NIH HHS/United States ; U24 GM116788/GM/NIGMS NIH HHS/United States ; S10 RR024564/RR/NCRR NIH HHS/United States ; R01 GM099604/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Triphosphate ; Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Lactococcus lactis/genetics/metabolism ; RNA ; },
abstract = {The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.},
}
@article {pmid35351888,
year = {2022},
author = {Jia, K and Cui, YR and Huang, S and Yu, P and Lian, Z and Ma, P and Liu, J},
title = {Phage peptides mediate precision base editing with focused targeting window.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1662},
pmid = {35351888},
issn = {2041-1723},
mesh = {Alleles ; Animals ; *Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; Peptides/genetics ; },
abstract = {Base editors (BEs) are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks (DSBs). A variety of strategies have been developed to improve the targeting scope and window of BEs. In a previous study, we found that a bacteriophage-derived peptide, referred to as G8PPD, could improve the specificity of Cas9 nuclease. Herein, we investigate the applicability of G8PPD as molecular modulators of BEs. We show that G8PPD can improve cytidine base editor (CBEs) and adenine base editor (ABE) to more focused targeting windows. Notably, in a cell-based disease model, G8PPD increases the percentage of perfectly edited gene alleles by BEs from less than 4% to more than 38% of the whole population. In addition, G8PPD can improve the targeting scope of BE in mouse embryos. In summary, our study presents the peptidyl modulators that can improve BEs for precision base editing.},
}
@article {pmid35351879,
year = {2022},
author = {Li, X and Zhou, L and Gao, BQ and Li, G and Wang, X and Wang, Y and Wei, J and Han, W and Wang, Z and Li, J and Gao, R and Zhu, J and Xu, W and Wu, J and Yang, B and Sun, X and Yang, L and Chen, J},
title = {Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1669},
pmid = {35351879},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; INDEL Mutation ; Mutation ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.},
}
@article {pmid35350901,
year = {2022},
author = {Wheatley, MS and Wang, Q and Wei, W and Bottner-Parker, KD and Zhao, Y and Yang, Y},
title = {Cas12a-Based Diagnostics for Potato Purple Top Disease Complex Associated with Infection by 'Candidatus Phytoplasma trifolii'-Related Strains.},
journal = {Plant disease},
volume = {106},
number = {8},
pages = {2039-2045},
doi = {10.1094/PDIS-09-21-2119-RE},
pmid = {35350901},
issn = {0191-2917},
mesh = {CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Phylogeny ; *Phytoplasma/genetics ; Plant Diseases/microbiology ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; *Solanum tuberosum/microbiology ; },
abstract = {'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches' broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive, and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a transcleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA intergenic transcribed spacer sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate readers or lateral flow assays for potentially high-throughput and/or field-deployable disease diagnostics.},
}
@article {pmid35350865,
year = {2022},
author = {Wasala, NB and Million, ED and Watkins, TB and Wasala, LP and Han, J and Yue, Y and Lu, B and Chen, SJ and Hakim, CH and Duan, D},
title = {The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy.},
journal = {Human gene therapy},
volume = {33},
number = {9-10},
pages = {518-528},
pmid = {35350865},
issn = {1557-7422},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics/metabolism ; *Dystrophin/genetics/metabolism ; Gene Editing/methods ; Genetic Therapy/methods ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; RNA, Guide/genetics/metabolism ; },
abstract = {Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.},
}
@article {pmid35349718,
year = {2022},
author = {Konstantakos, V and Nentidis, A and Krithara, A and Paliouras, G},
title = {CRISPR-Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning.},
journal = {Nucleic acids research},
volume = {50},
number = {7},
pages = {3616-3637},
pmid = {35349718},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing/methods ; *RNA, Guide/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR-Cas9 guide design.},
}
@article {pmid35349689,
year = {2022},
author = {Zhao, D and Jiang, G and Li, J and Chen, X and Li, S and Wang, J and Zhou, Z and Pu, S and Dai, Z and Ma, Y and Bi, C and Zhang, X},
title = {Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.},
journal = {Nucleic acids research},
volume = {50},
number = {7},
pages = {4161-4170},
pmid = {35349689},
issn = {1362-4962},
mesh = {Adenine/metabolism ; Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; *RNA, Guide/genetics ; },
abstract = {CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.},
}
@article {pmid35349073,
year = {2022},
author = {Das, S and Bombaywala, S and Srivastava, S and Kapley, A and Dhodapkar, R and Dafale, NA},
title = {Genome plasticity as a paradigm of antibiotic resistance spread in ESKAPE pathogens.},
journal = {Environmental science and pollution research international},
volume = {29},
number = {27},
pages = {40507-40519},
pmid = {35349073},
issn = {1614-7499},
mesh = {*Acinetobacter baumannii ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Drug Resistance, Microbial/genetics ; *Enterococcus faecium/genetics ; Gene Transfer, Horizontal ; Klebsiella pneumoniae ; },
abstract = {The major reason behind the spread of antibiotic resistance genes (ARGs) is persistent selective pressure in the environment encountered by bacteria. Genome plasticity plays a crucial role in dissemination of antibiotic resistance among bacterial pathogens. Mobile genetic elements harboring ARGs are reported to dodge bacterial immune system and mediate horizontal gene transfer (HGT) under selective pressure. Residual antibiotic pollutants develop selective pressures that force the bacteria to lose their defense mechanisms (CRISPR-cas) and acquire resistance. The present study targets the ESKAPE organisms (namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) causing various nosocomial infections and emerging multidrug-resistant species. The role of CRISPR-cas systems in inhibition of HGT in prokaryotes and its loss due to presence of various stressors in the environment is also focused in the study. IncF and IncH plasmids were identified in all strains of E. faecalis and K. pneumoniae, carrying Beta-lactam and fluoroquinolone resistance genes, whereas sal3, phiCTX, and SEN34 prophages harbored aminoglycoside resistance genes (aadA, aac). Various MGEs present in selected environmental niches that aid the bacterial genome plasticity and transfer of ARGs contributing to its spread are also identified.},
}
@article {pmid35348649,
year = {2022},
author = {Dong, C and Wang, X and Ma, C and Zeng, Z and Pu, DK and Liu, S and Wu, CS and Chen, S and Deng, Z and Guo, FB},
title = {Anti-CRISPRdb v2.2: an online repository of anti-CRISPR proteins including information on inhibitory mechanisms, activities and neighbors of curated anti-CRISPR proteins.},
journal = {Database : the journal of biological databases and curation},
volume = {2022},
number = {},
pages = {},
pmid = {35348649},
issn = {1758-0463},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Viral Proteins/genetics ; },
abstract = {UNLABELLED: We previously released the Anti-CRISPRdb database hosting anti-CRISPR proteins (Acrs) and associated information. Since then, the number of known Acr families, types, structures and inhibitory activities has accumulated over time, and Acr neighbors can be used as a candidate pool for screening Acrs in further studies. Therefore, we here updated the database to include the new available information. Our newly updated database shows several improvements: (i) it comprises more entries and families because it includes both Acrs reported in the most recent literatures and Acrs obtained via performing homologous alignment; (ii) the prediction of Acr neighbors is integrated into Anti-CRISPRdb v2.2, and users can identify novel Acrs from these candidates; and (iii) this version includes experimental information on the inhibitory strength and stage for Acr-Cas/Acr-CRISPR pairs, motivating the development of tools for predicting specific inhibitory abilities. Additionally, a parameter, the rank of codon usage bias (CUBRank), was proposed and provided in the new version, which showed a positive relationship with predicted result from AcRanker; hence, it can be used as an indicator for proteins to be Acrs. CUBRank can be used to estimate the possibility of genes occurring within genome island-a hotspot hosting potential genes encoding Acrs. Based on CUBRank and Anti-CRISPRdb, we also gave the first glimpse for the emergence of Acr genes (acrs).<br><br>DATABASE URL: http://guolab.whu.edu.cn/anti-CRISPRdb.},
}
@article {pmid35347694,
year = {2022},
author = {Zhou, Y and Fu, Q and Shi, H and Zhou, G},
title = {CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2549},
number = {},
pages = {233-257},
pmid = {35347694},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Endonucleases/genetics ; Gene Library ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *RNA, Guide/genetics ; },
abstract = {High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection = 0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.},
}
@article {pmid35347685,
year = {2022},
author = {Frey, N and Schwank, G},
title = {CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-β Pathway Regulators.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2488},
number = {},
pages = {99-111},
pmid = {35347685},
issn = {1940-6029},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Intestines ; *Organoids ; *Transforming Growth Factor beta/metabolism ; Wnt Signaling Pathway/genetics ; },
abstract = {The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.},
}
@article {pmid35347684,
year = {2022},
author = {Huang, Z and Loewer, A},
title = {Generating Somatic Knockout Cell Lines with CRISPR-Cas9 Technology to Investigate SMAD Signaling.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2488},
number = {},
pages = {81-97},
pmid = {35347684},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; *Signal Transduction/genetics ; Technology ; },
abstract = {Genome engineering provides a powerful tool to explore TGF-β/SMAD signaling by enabling the deletion and modification of critical components of the pathway. Over the past years, CRISPR-Cas9 technology has matured and can now be used to routinely generate knockout cell lines. Here, we describe a method to design and generate deletions of genes from the SMAD pathway in somatic human cell lines based on homologous recombination.},
}
@article {pmid35344733,
year = {2022},
author = {Rusni, S and Sassa, M and Takagi, T and Kinoshita, M and Takehana, Y and Inoue, K},
title = {Establishment of cytochrome P450 1a gene-knockout Javanese medaka, Oryzias javanicus, which distinguishes toxicity modes of the polycyclic aromatic hydrocarbons, pyrene and phenanthrene.},
journal = {Marine pollution bulletin},
volume = {178},
number = {},
pages = {113578},
doi = {10.1016/j.marpolbul.2022.113578},
pmid = {35344733},
issn = {1879-3363},
mesh = {Animals ; Cytochrome P-450 CYP1A1/genetics/metabolism ; Cytochrome P-450 Enzyme System/metabolism ; Indonesia ; *Oryzias/genetics ; *Phenanthrenes/metabolism/toxicity ; *Polycyclic Aromatic Hydrocarbons/analysis ; Pyrenes/metabolism/toxicity ; *Water Pollutants, Chemical/analysis ; },
abstract = {Cytochrome P450 1a (Cyp1a) is an important enzyme for metabolism of organic pollutants. To understand its reaction to polycyclic aromatic hydrocarbons (PAHs), we knocked out this gene in a marine model fish, Javanese medaka, Oryzias javanicus, using the CRISPR/Cas 9 system. A homozygous mutant (KO) strain with a four-base deletion was established using an environmental DNA (eDNA)-based genotyping technique. Subsequently, KO, heterozygous mutant (HT), and wild-type (WT) fish were exposed to model pollutants, pyrene and phenanthrene, and survivorship and swimming behavior were analyzed. Compared to WT, KO fish were more sensitive to pyrene, suggesting that Cyp1a transforms pyrene into less toxic metabolites. Conversely, WT fish were sensitive to phenanthrene, suggesting that metabolites transformed by Cyp1a are more toxic than the original compound. HT fish showed intermediate results. Thus, comparative use of KO and WT fish can distinguish modes of pollutant toxicity, providing a deeper understanding of fish catabolism of environmental pollutants.},
}
@article {pmid35343817,
year = {2022},
author = {Brackett, NF and Davis, BW and Adli, M and Pomés, A and Chapman, MD},
title = {Evolutionary Biology and Gene Editing of Cat Allergen, Fel d 1.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {213-223},
doi = {10.1089/crispr.2021.0101},
pmid = {35343817},
issn = {2573-1602},
mesh = {*Allergens/chemistry/genetics ; Animals ; Biology ; CRISPR-Cas Systems/genetics ; Cats ; Gene Editing ; Glycoproteins/chemistry/genetics ; *Hypersensitivity/genetics/therapy ; },
abstract = {Allergy to domestic cat affects up to 15% of the population, and sensitization to cat allergen is associated with asthma. Despite the pervasiveness of cat allergic disease, current treatments have limited impact. Here, we present a bioinformatics analysis of the major cat allergen, Fel d 1, and demonstrate proof of principle for CRISPR gene editing of the allergen. Sequence and structural analyses of Fel d 1 from 50 domestic cats identified conserved coding regions in genes CH1 and CH2 suitable for CRISPR editing. Comparative analyses of Fel d 1 and orthologous sequences from eight exotic felid species determined relatively low-sequence identities for CH1 and CH2, and implied that the allergen may be nonessential for cats, given the apparent lack of evolutionary conservation. In vitro knockouts of domestic cat Fel d 1 using CRISPR-Cas9 yielded editing efficiencies of up to 55% and found no evidence of editing at predicted potential off-target sites. Taken together, our data indicate that Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.},
}
@article {pmid35343224,
year = {2022},
author = {Tian, H and Niu, H and Luo, J and Yao, W and Chen, X and Wu, J and Geng, Y and Gao, W and Lei, A and Gao, Z and Tian, X and Zhao, X and Shi, H and Li, C and Hua, J},
title = {Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {13},
pages = {4030-4043},
doi = {10.1021/acs.jafc.2c00642},
pmid = {35343224},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; *Goats/metabolism ; *Milk/chemistry ; *Stearoyl-CoA Desaturase/genetics/metabolism ; },
abstract = {Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism in vivo, SCD1-knockout goats were generated through CRISPR/Cas9 technology for the first time. SCD1 deficiency did not influence goat growth or serum biochemistry. Plasma phosphatidylcholines increased by lipidomics after SCD1 knockout in goats. Whole-blood RNA-seq indicated alterations in biosynthesis of unsaturated fatty acid synthesis, cAMP, ATPase activity, and Wnt signaling pathways. In SCD1-knockout goats, milk fat percentage and unsaturated fatty acid levels were reduced but other milk components were unchanged. Milk lipidomics revealed decreased triacylglycerols and diacylglycerols levels, and the differential abundance of lipids were enriched in glycerolipid, glycerophospholipids, and thermogenesis metabolism pathways. In milk fat globules, the expression levels of genes related to fatty acid and TAG synthesis including SREBP1 were reduced. ATP content and AMPK activity were promoted, and p-p70S6K protein level was suppressed in SCD1-knockout goat mammary epithelial cells, suggesting that SCD1 affected milk lipid metabolism by influencing AMPK-mTORC1/p70S6K-SREBP1 pathway. The integrative analysis of gene expression levels and lipidomics of milk revealed a crucial role of SCD1 in glycerolipids and glycerophospholipids metabolism pathways. Our observations indicated that SCD1 regulated the synthesis of milk fat and unsaturated fatty acid in goat by affecting lipid metabolism gene expression and lipid metabolic pathways. These findings would be essential for improving goat milk nutritional value which is beneficial to human health.},
}
@article {pmid35343100,
year = {2022},
author = {Zheng, F and Chen, Z and Li, J and Wu, R and Zhang, B and Nie, G and Xie, Z and Zhang, H},
title = {A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {9},
number = {14},
pages = {e2105231},
pmid = {35343100},
issn = {2198-3844},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Nucleic Acids ; RNA, Guide/genetics/metabolism ; Surface Plasmon Resonance ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR-associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed with a specific single-guide RNA, enabling the amplification-free detection of target sequences within genomic DNA. The sensor, termed CRISPR-SPR-Chip, is used to successfully analyze recombinant plasmids with only three-base mutations with a limit of detection as low as 1.3 fM. Real-time monitoring CRISPR-SPR-Chip is used to analyze clinical samples of patients with Duchenne muscular dystrophy with two exon deletions, which are detected without any pre-amplification step, yielding significantly positive results within 5 min. The ability of this novel CRISPR-empowered SPR (CRISPR-eSPR) sensing platform to rapidly, precisely, sensitively, and specifically detect a target gene sequence provides a new on-chip optic approach for clinical gene analysis.},
}
@article {pmid35341983,
year = {2022},
author = {Li, C and Chu, W and Gill, RA and Sang, S and Shi, Y and Hu, X and Yang, Y and Zaman, QU and Zhang, B},
title = {Computational tools and resources for CRISPR/Cas genome editing.},
journal = {Genomics, proteomics &amp; bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.gpb.2022.02.006},
pmid = {35341983},
issn = {2210-3244},
abstract = {The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genomic editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduced the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focused on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights on the limitations of available computational tools that surely help researchers of this field for further optimization. Lastly, we suggested a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.},
}
@article {pmid35341958,
year = {2022},
author = {Wang, S and Hu, J and Sui, C and He, G and Qu, Z and Chen, X and Wang, Y and Guo, D and Liu, X},
title = {Accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) to diagnose COVID-19, a meta-analysis.},
journal = {Microbial pathogenesis},
volume = {165},
number = {},
pages = {105498},
pmid = {35341958},
issn = {1096-1208},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; },
abstract = {OBJECTIVE: To estimate the accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) in determining coronavirus disease-19 (COVID-19).<br><br>METHODS: As of January 31, 2022, PubMed, Web of Science, Embase, Science Direct, Wiley and Springer Link were searched. Sensitivity, specificity, likelihood ratio (LR), diagnostic odds ratio (DOR) and area under the summary receiver-operating characteristic (AUC) curve were used to assess the accuracy of CRISPR.<br><br>RESULTS: According to the inclusion criteria, 5857 patients from 54 studies were included in this meta-analysis. The pooled sensitivity, specificity and AUC were 0.98, 1.00 and 1.00, respectively. For CRISPR-associated (Cas) proteins-12, the sensitivity, specificity was 0.96, 1.00, respectively. For Cas-13, the sensitivity and specificity were 0.99 and 0.99.<br><br>CONCLUSION: This meta-analysis showed that the diagnostic performance of CRISPR is close to the gold standard, and it is expected to meet the Point of care requirements in resource poor areas.},
}
@article {pmid35340673,
year = {2022},
author = {Alduhaidhawi, AHM and AlHuchaimi, SN and Al-Mayah, TA and Al-Ouqaili, MTS and Alkafaas, SS and Muthupandian, S and Saki, M},
title = {Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater.},
journal = {Infection and drug resistance},
volume = {15},
number = {},
pages = {1143-1154},
pmid = {35340673},
issn = {1178-6973},
abstract = {PURPOSE: This study aimed to evaluate the presence of CRISPR-Cas system genes and their possible association with antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium species isolated from hospital wastewater (HWW) samples of several hospitals.<br><br>METHODS: HWW samples (200 mL) were collected from wastewater discharged from different hospitals from October 2020 to March 2021. The isolation and identification of enterococci species were performed by standard bacteriology tests and polymerase chain reaction (PCR). Antibiotic resistance was determined using the disc diffusion. The presence of various CRISPR-Cas systems was investigated by PCR. The association of the occurrence of CRISPR-Cas systems with antibiotic resistance was analyzed with appropriate statistical tests.<br><br>RESULTS: In total, 85 different enterococci species were isolated and identified using phenotypic methods. The results of PCR confirmed the prevalence of 50 (58.8%) E. faecalis and 35 (41.2%) E. faecium, respectively. In total, 54 (63.5%) of 85 isolates showed the presence of CRISPR-Cas loci. The incidence of CRISPR-Cas was more common in E. faecalis. CRISPR1, CRISPR2, and CRISPR3 were present in 35 (41.2%), 47 (55.3%), and 30 (35.3%) enterococci isolates, respectively. The CRISPR-Cas positive isolates showed significant lower resistance rates against vancomycin, ampicillin, chloramphenicol, erythromycin, rifampin, teicoplanin, tetracycline, imipenem, tigecycline, and trimethoprim-sulfamethoxazole in comparison with CRISPR-Cas negative isolates. The results showed that the presence of CRISPR-Cas genes was lower in multidrug-resistant (MDR) isolates (53.1%, n = 26/49) compared to the non-MDR enterococci isolates (77.8%, n = 28/36) (P = 0.023).<br><br>CONCLUSION: This study revealed the higher prevalence of E. faecalis than E. faecium in HWWs. Also, the lack of CRISPR-Cas genes was associated with more antibiotic resistance rates and multidrug resistance in E. faecalis and E. faecium isolates with HWW origin.},
}
@article {pmid35339825,
year = {2022},
author = {Geng, B and Wang, X and Park, KH and Lee, KE and Kim, J and Chen, P and Zhou, X and Tan, T and Yang, C and Zou, X and Janssen, PM and Cao, L and Ye, L and Wang, X and Cai, C and Zhu, H},
title = {UCHL1 protects against ischemic heart injury via activating HIF-1α signal pathway.},
journal = {Redox biology},
volume = {52},
number = {},
pages = {102295},
pmid = {35339825},
issn = {2213-2317},
support = {R01 AR067766/AR/NIAMS NIH HHS/United States ; R01 CA166590/CA/NCI NIH HHS/United States ; R01 EY030621/EY/NEI NIH HHS/United States ; R01 HL153876/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Escherichia coli ; *Heart Injuries ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; *Induced Pluripotent Stem Cells ; Mice ; *Myocardial Infarction/genetics/pathology ; Signal Transduction ; Ubiquitin Thiolesterase/genetics ; },
abstract = {Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.},
}
@article {pmid35339823,
year = {2022},
author = {Liu, C and Ren, L and Li, X and Fan, N and Chen, J and Zhang, D and Yang, W and Ding, S and Xu, W and Min, X},
title = {Self-electrochemiluminescence biosensor based on CRISPR/Cas12a and PdCuBP@luminol nanoemitter for highly sensitive detection of cytochrome c oxidase subunit III gene of acute kidney injury.},
journal = {Biosensors &amp; bioelectronics},
volume = {207},
number = {},
pages = {114207},
doi = {10.1016/j.bios.2022.114207},
pmid = {35339823},
issn = {1873-4235},
mesh = {*Acute Kidney Injury ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Electron Transport Complex IV ; Female ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Luminol ; Male ; },
abstract = {The cytochrome c oxidase subunit III (COX III) gene is a powerful biomarker for the early diagnosis of acute kidney injury. However, current methods for COX III gene detection are usually laborious and time-consuming, with limited sensitivity. Herein, we report a novel self-electrochemiluminescence (ECL) biosensor for highly sensitive detection of the COX III gene based on CRISPR/Cas12a and nanoemitters of luminol-loaded multicomponent metal-metalloid PdCuBP alloy mesoporous nanoclusters. The nanoemitter with excellent self-ECL in neutral media exhibited a high specific surface area for binding luminol and outstanding oxidase-like catalytic activity toward dissolved O2. Meanwhile, the CRISPR/Cas12a system, as a target-trigger, was employed to specifically recognize the COX III gene and efficiently cleave the interfacial quencher of dopamine-labeled hairpin DNA. As a result, the ECL biosensor showed superior analytical performance for COX III gene detection without exogenous coreactant. Benefiting from the high-efficiency ECL emission of the nanoemitter and Cas12a-mediated interfacial cleavage of the quencher, the developed ECL biosensor exhibited high sensitivity to COX III with a low detection limit of 0.18 pM. The established ECL biosensing method possessed excellent practical performance in urine samples. Meaningfully, the proposed strategy presents promising prospects for nucleic acid detection in the field of clinical diagnostics.},
}
@article {pmid35339702,
year = {2022},
author = {Clemmensen, SE and Kromphardt, KJK and Frandsen, RJN},
title = {Marker-free CRISPR-Cas9 based genetic engineering of the phytopathogenic fungus, Penicillium expansum.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {160},
number = {},
pages = {103689},
doi = {10.1016/j.fgb.2022.103689},
pmid = {35339702},
issn = {1096-0937},
mesh = {CRISPR-Cas Systems ; Genetic Engineering ; *Malus ; *Penicillium/genetics/metabolism ; },
abstract = {Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000 bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum.},
}
@article {pmid35338974,
year = {2022},
author = {Fapohunda, FO and Qiao, S and Pan, Y and Wang, H and Liu, Y and Chen, Q and Lü, P},
title = {CRISPR Cas system: A strategic approach in detection of nucleic acids.},
journal = {Microbiological research},
volume = {259},
number = {},
pages = {127000},
doi = {10.1016/j.micres.2022.127000},
pmid = {35338974},
issn = {1618-0623},
mesh = {*CRISPR-Cas Systems ; Humans ; *Nucleic Acids ; },
abstract = {Over the decades, rapid nucleic acid detection has been difficult for scientists, especially in microbiology, biotechnology and immunology. Most technologies are finite in sensitivity, specificity or both. Early diagnosis of disease allows swift response, disease monitoring and control of the rapid spread of the disease. However, limited access to test kits, specialized laboratory equipment, and the need for highly skilled personnel has led to a detection downshift. CRISPR-based diagnostic techniques, based on clustered regularly interspaced short palindromic repeats (CRISPR), have recently altered molecular diagnosis. This modern technology is combined or paired with other methods like SHERLOCK, DETECTR, HUDSON, CDetection and so on to detect viruses, bacteria infection and other pathogenic agents, due to its precision and versatility, it can also detect diseases directly from patient samples. Rapid, sensitive, accurate and advanced molecular techniques are much needed since they soothe researchers in diagnosis and detection, and can also be employed in therapeutic treatments. In this review, detailed CRISPR Cas variants were discussed, emphasizing the use of CRISPR system as a tool in diagnostics and nucleic acids detection and the benefits of this robust tool over other amplification methods.},
}
@article {pmid35338712,
year = {2022},
author = {Tanouchi, M and Igawa, T and Suzuki, N and Suzuki, M and Hossain, N and Ochi, H and Ogino, H},
title = {Optimization of CRISPR/Cas9-mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique.},
journal = {Development, growth &amp; differentiation},
volume = {64},
number = {4},
pages = {219-225},
doi = {10.1111/dgd.12778},
pmid = {35338712},
issn = {1440-169X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Phenotype ; RNA, Messenger/genetics ; Xenopus laevis/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 method has become popular for gene disruption experiments in Xenopus laevis. However, the experimental conditions that influence the efficiency of CRISPR/Cas9 remain unclear. To that end, we developed an image analysis technique for the semi-quantitative evaluation of the pigment phenotype resulting from the disruption of tyrosinase genes in X. laevis using a CRISPR/Cas9 approach, and then examined the effects of varying five experimental parameters (timing of the CRISPR reagent injection into developing embryos; amount of Cas9 mRNA in the injection reagent; total injection volume per embryo; number of injection sites per embryo; and the culture temperature of the injected embryos) on the gene disruption efficiency. The results of this systematic analysis suggest that the highest possible efficiency of target gene disruption can be achieved by injecting a total of 20 nL of the CRISPR reagent containing 1500 pg of Cas9 mRNA or 4 ng of Cas9 protein into two separate locations (10 nL each) of one-cell stage embryos cultured at 22°C. This study also highlights the importance of balancing the experimental parameters for increasing gene disruption efficiency and provides valuable insights into the optimal conditions for applying the CRISPR/Cas9 system to new experimental organisms.},
}
@article {pmid35338607,
year = {2022},
author = {Zhou, H and Wang, X and Steer, CJ and Song, G and Niu, J},
title = {Efficient silencing of hepatitis B virus S gene through CRISPR-mediated base editing.},
journal = {Hepatology communications},
volume = {6},
number = {7},
pages = {1652-1663},
pmid = {35338607},
issn = {2471-254X},
mesh = {CRISPR-Cas Systems/genetics ; Codon, Nonsense ; *Hepatitis B/genetics ; Hepatitis B Surface Antigens ; *Hepatitis B virus/genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {Hepatitis B virus (HBV) infection is a major risk factor of liver cirrhosis and hepatocellular carcinoma. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been used to precisely edit the HBV genome and eliminate HBV through non-homologous end-joining repair of double-stranded break (DSB). However, the CRISPR/Cas9-mediated DSB triggers instability of host genome and exhibits low efficiency to edit genome, limiting its application. CRISPR cytidine base editors (CBEs) could silence genes by generating a premature stop codon. Here we developed a CRISPR base editor approach to precisely edit single nucleotide within the HBV genome to impair HBV gene expression. Specifically, a single-guide RNA (sgRNA) was designed to edit the 30th codon of HBV S gene, which encodes HBV surface antigen (HBsAg), from CAG (glutamine) to stop codon TAG. We next used human hepatoma PLC/PRF/5 cells carrying the HBV genome to establish a cell line that expresses a CBE (PLC/PRF/5-CBE). Lentivirus was used to introduce sgRNA into PLC/PRF/5-CBE cells. Phenotypically, 71% of PLC/PRF/5-CBE cells developed a premature stop codon within the S gene. Levels of HBs messenger RNA were significantly decreased. A 92% reduction of HBsAg secretion was observed in PLC/PRF/5-CBE cells. The intracellular HBsAg was also reduced by 84% after treatment of gRNA_S. Furthermore, no off-target effect was detected in predicted off-target loci within the HBV genome. Sequencing confirmed that 95%, 93%, 93%, 9%, and 72% S gene sequences of HBV genotypes B, C, F, G, and H had the binding site of sgRNA. Conclusion: Our findings indicate that CRISPR-mediated base editing is an efficient approach to silence the HBV S gene, suggesting its therapeutic potential to eliminate HBV.},
}
@article {pmid35338523,
year = {2022},
author = {Chen, Y and Jong, TT and Chen, C and Sidransky, E},
title = {CRISPR/Cas9-Based Functional Genomics in Human Induced Pluripotent Stem Cell-Derived Models: Can "the Stars Align" for Neurodegenerative Diseases?.},
journal = {Movement disorders : official journal of the Movement Disorder Society},
volume = {37},
number = {5},
pages = {886-890},
doi = {10.1002/mds.28998},
pmid = {35338523},
issn = {1531-8257},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Genomics ; Humans ; *Induced Pluripotent Stem Cells ; *Neurodegenerative Diseases/genetics ; },
}
@article {pmid35338236,
year = {2022},
author = {Stukenberg, D and Hoff, J and Faber, A and Becker, A},
title = {NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {265},
pmid = {35338236},
issn = {2399-3642},
mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing/methods ; *Vibrio/genetics ; },
abstract = {The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum.},
}
@article {pmid35338140,
year = {2022},
author = {Krysler, AR and Cromwell, CR and Tu, T and Jovel, J and Hubbard, BP},
title = {Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1617},
pmid = {35338140},
issn = {2041-1723},
support = {CIHR-PS-408552//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; },
abstract = {CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.},
}
@article {pmid35337340,
year = {2022},
author = {Das, S and Bano, S and Kapse, P and Kundu, GC},
title = {CRISPR based therapeutics: a new paradigm in cancer precision medicine.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {85},
pmid = {35337340},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; *Precision Medicine ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine.<br><br>MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo.<br><br>CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.},
}
@article {pmid35337108,
year = {2022},
author = {Park, HM and Park, Y and Vankerschaver, J and Van Messem, A and De Neve, W and Shim, H},
title = {Rethinking Protein Drug Design with Highly Accurate Structure Prediction of Anti-CRISPR Proteins.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {35337108},
issn = {1424-8247},
abstract = {Protein therapeutics play an important role in controlling the functions and activities of disease-causing proteins in modern medicine. Despite protein therapeutics having several advantages over traditional small-molecule therapeutics, further development has been hindered by drug complexity and delivery issues. However, recent progress in deep learning-based protein structure prediction approaches, such as AlphaFold2, opens new opportunities to exploit the complexity of these macro-biomolecules for highly specialised design to inhibit, regulate or even manipulate specific disease-causing proteins. Anti-CRISPR proteins are small proteins from bacteriophages that counter-defend against the prokaryotic adaptive immunity of CRISPR-Cas systems. They are unique examples of natural protein therapeutics that have been optimized by the host-parasite evolutionary arms race to inhibit a wide variety of host proteins. Here, we show that these anti-CRISPR proteins display diverse inhibition mechanisms through accurate structural prediction and functional analysis. We find that these phage-derived proteins are extremely distinct in structure, some of which have no homologues in the current protein structure domain. Furthermore, we find a novel family of anti-CRISPR proteins which are structurally similar to the recently discovered mechanism of manipulating host proteins through enzymatic activity, rather than through direct inference. Using highly accurate structure prediction, we present a wide variety of protein-manipulating strategies of anti-CRISPR proteins for future protein drug design.},
}
@article {pmid35336126,
year = {2022},
author = {de Oliveira, IMF and Godoy-Santos, F and Oyama, LB and Moreira, SM and Dias, RG and Huws, SA and Creevey, CJ and Mantovani, HC},
title = {Whole-Genome Sequencing and Comparative Genomic Analysis of Antimicrobial Producing Streptococcus lutetiensis from the Rumen.},
journal = {Microorganisms},
volume = {10},
number = {3},
pages = {},
pmid = {35336126},
issn = {2076-2607},
abstract = {Antimicrobial peptides (AMPs) can efficiently control different microbial pathogens and show the potential to be applied in clinical practice and livestock production. In this work, the aim was to isolate AMP-producing ruminal streptococci and to characterize their genetic features through whole-genome sequencing. We cultured 463 bacterial isolates from the rumen of Nelore bulls, 81 of which were phenotypically classified as being Streptococcaceae. Five isolates with broad-range activity were genome sequenced and confirmed as being Streptococcus lutetiensis. The genetic features linked to their antimicrobial activity or adaptation to the rumen environment were characterized through comparative genomics. The genome of S. lutetiensis UFV80 harbored a putative CRISPR-Cas9 system (Type IIA). Computational tools were used to discover novel biosynthetic clusters linked to the production of bacteriocins. All bacterial genomes harbored genetic clusters related to the biosynthesis of class I and class II bacteriocins. SDS-PAGE confirmed the results obtained in silico and demonstrated that the class II bacteriocins predicted in the genomes of three S. lutetiensis strains had identical molecular mass (5197 Da). These results demonstrate that ruminal bacteria of the Streptococcus bovis/equinus complex represent a promising source of novel antimicrobial peptides.},
}
@article {pmid35335122,
year = {2022},
author = {Jiang, Z and Abdullah, and Zhang, S and Jiang, Y and Liu, R and Xiao, Y},
title = {Development and Optimization of CRISPR Prime Editing System in Photoautotrophic Cells.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {6},
pages = {},
pmid = {35335122},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; INDEL Mutation ; Point Mutation ; RNA, Guide/genetics ; },
abstract = {Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA). It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. This study reports an optimized-prime editor (O-PE) for endogenous gene editing in Arabidopsis thaliana cells, with an average 1.15% editing efficiency, which is 16.4-fold higher than previously reported. Meanwhile, we observed an increase in indels when testing alternative reverse transcriptase and found out that nCas9(H840A) fused to non-functional reverse transcriptase was responsible for the increase. This work develops an efficient prime editor for plant cells and provides a blueprint for applying PE in other photoautotrophic cells, such as microalgae, that have a high industrial value.},
}
@article {pmid35333864,
year = {2022},
author = {Zhang, Q and Zhang, Y and Chai, Y},
title = {Optimization of CRISPR/LbCas12a-mediated gene editing in Arabidopsis.},
journal = {PloS one},
volume = {17},
number = {3},
pages = {e0265114},
pmid = {35333864},
issn = {1932-6203},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Promoter Regions, Genetic ; },
abstract = {CRISPR/LbCas12a system (LbCpf1) has been widely used for genome modification including plant species. However, the efficiency of CRISPR/LbCas12a varied considerably in different plant species and tissues, and the editing efficiency needs to be further improved. In this study, we tried to improve the editing efficiency of CRISPR/LbCas12a in Arabidopsis by optimizing the crRNA expression strategies and Pol II promoters. Notably, the combination of tRNA-crRNA fusion strategy and RPS5A promoter in CRISPR/LbCas12a system has highest editing efficiency, while CRISPR/LbCas12a driven by EC1f-in(crR)p had the highest ratio of homozygous &amp; bi-allelic mutants. In addition, all homozygous &amp; bi-allelic mutants can be stably inherited to the next generation and have no phenotypic separation. In this study, the editing efficiency of the CRISPR/LbCas12a system was improved by selecting the optimal crRNA expression strategies and promoter of LbCas12a in Arabidopsis, which will prove useful for optimization of CRISPR/LbCas12a methods in other plants.},
}
@article {pmid35333620,
year = {2022},
author = {Juríková, K and Sepšiová, R and Ševčovičová, A and Tomáška, &#x13d; and Džugasová, V},
title = {Implementing CRISPR-Cas9 Yeast Practicals into Biology Curricula.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {181-186},
doi = {10.1089/crispr.2021.0125},
pmid = {35333620},
issn = {2573-1602},
mesh = {Biology ; *CRISPR-Cas Systems/genetics ; Curriculum ; Gene Editing/methods ; Humans ; *Saccharomyces cerevisiae/genetics ; },
abstract = {CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast, Saccharomyces cerevisiae. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.},
}
@article {pmid35333175,
year = {2022},
author = {Thumberger, T and Tavhelidse-Suck, T and Gutierrez-Triana, JA and Cornean, A and Medert, R and Welz, B and Freichel, M and Wittbrodt, J},
title = {Boosting targeted genome editing using the hei-tag.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35333175},
issn = {2050-084X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing/methods ; Mammals ; Nuclear Localization Signals ; RNA, Messenger/genetics ; },
abstract = {Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.},
}
@article {pmid35332696,
year = {2022},
author = {Aragonés, V and Aliaga, F and Pasin, F and Daròs, JA},
title = {Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100504},
doi = {10.1002/biot.202100504},
pmid = {35332696},
issn = {1860-7314},
mesh = {Agrobacterium/genetics ; *Gene Editing ; Gene Silencing ; Genetic Vectors/genetics ; Genome, Plant ; *Plant Viruses/genetics ; Tobacco/genetics/metabolism ; },
abstract = {Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we present JoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci.},
}
@article {pmid35332341,
year = {2022},
author = {Zong, Y and Liu, Y and Xue, C and Li, B and Li, X and Wang, Y and Li, J and Liu, G and Huang, X and Cao, X and Gao, C},
title = {An engineered prime editor with enhanced editing efficiency in plants.},
journal = {Nature biotechnology},
volume = {40},
number = {9},
pages = {1394-1402},
pmid = {35332341},
issn = {1546-1696},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; Mice ; *Oryza/genetics ; Plants ; RNA, Guide ; },
abstract = {Prime editing is a versatile genome-editing technology, but it suffers from low editing efficiency. In the present study, we introduce optimized prime editors with substantially improved editing efficiency. We engineered the Moloney-murine leukemia virus reverse transcriptase by removing its ribonuclease H domain and incorporated a viral nucleocapsid protein with nucleic acid chaperone activity. Each modification independently improved prime editing efficiency by ~1.8-3.4-fold in plant cells. When combined in our engineered plant prime editor (ePPE), the two modifications synergistically enhanced the efficiency of base substitutions, deletions and insertions at various endogenous sites by on average 5.8-fold compared with the original PPE in cell culture. No significant increase in byproducts or off-target editing was observed. We used the ePPE to generate rice plants tolerant to sulfonylurea and imidazolinone herbicides, observing an editing frequency of 11.3% compared with 2.1% using PPE. We also combined ePPE with the previously reported dual-prime editing guide (peg) RNAs and engineered pegRNAs to further increase efficiency.},
}
@article {pmid35332138,
year = {2022},
author = {Peterka, M and Akrap, N and Li, S and Wimberger, S and Hsieh, PP and Degtev, D and Bestas, B and Barr, J and van de Plassche, S and Mendoza-Garcia, P and Šviković, S and Sienski, G and Firth, M and Maresca, M},
title = {Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1240},
pmid = {35332138},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA Repair ; Endonucleases/metabolism ; Gene Editing ; Mammals/genetics ; },
abstract = {Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.},
}
@article {pmid35331236,
year = {2022},
author = {Selvakumar, SC and Preethi, KA and Ross, K and Tusubira, D and Khan, MWA and Mani, P and Rao, TN and Sekar, D},
title = {CRISPR/Cas9 and next generation sequencing in the personalized treatment of Cancer.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {83},
pmid = {35331236},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; High-Throughput Nucleotide Sequencing ; Humans ; *Neoplasms/genetics/therapy ; *Precision Medicine ; },
abstract = {BACKGROUND: Cancer is caused by a combination of genetic and epigenetic abnormalities. Current cancer therapies are limited due to the complexity of their mechanism, underlining the need for alternative therapeutic approaches. Interestingly, combining the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system with next-generation sequencing (NGS) has the potential to speed up the identification, validation, and targeting of high-value targets.<br><br>MAIN TEXT: Personalized or precision medicine combines genetic information with phenotypic and environmental characteristics to produce healthcare tailored to the individual and eliminates the constraints of "one-size-fits-all" therapy. Precision medicine is now possible thanks to cancer genome sequencing. Having advantages over limited sample requirements and the recent development of biomarkers have made the use of NGS a major leap in personalized medicine. Tumor and cell-free DNA profiling using NGS, proteome and RNA analyses, and a better understanding of immunological systems, are all helping to improve cancer treatment choices. Finally, direct targeting of tumor genes in cancer cells with CRISPR/Cas9 may be achievable, allowing for eliminating genetic changes that lead to tumor growth and metastatic capability.<br><br>CONCLUSION: With NGS and CRISPR/Cas9, the goal is no longer to match the treatment for the diagnosed tumor but rather to build a treatment method that fits the tumor exactly. Hence, in this review, we have discussed the potential role of CRISPR/Cas9 and NGS in advancing personalized medicine.},
}
@article {pmid35331211,
year = {2022},
author = {Garcia-Perez, E and Diego-Martin, B and Quijano-Rubio, A and Moreno-Giménez, E and Selma, S and Orzaez, D and Vazquez-Vilar, M},
title = {A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.},
journal = {BMC biotechnology},
volume = {22},
number = {1},
pages = {12},
pmid = {35331211},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems/genetics ; Copper ; Gene Expression ; Plants/genetics ; *Tobacco/genetics ; Transcriptional Activation ; },
abstract = {BACKGROUND: CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).<br><br>RESULTS: In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.<br><br>CONCLUSIONS: The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.},
}
@article {pmid35331142,
year = {2022},
author = {Blomme, J and Develtere, W and Köse, A and Arraiza Ribera, J and Brugmans, C and Jaraba-Wallace, J and Decaestecker, W and Rombaut, D and Baekelandt, A and Daniel Fernández Fernández, &#xc1; and Van Breusegem, F and Inzé, D and Jacobs, T},
title = {The heat is on: a simple method to increase genome editing efficiency in plants.},
journal = {BMC plant biology},
volume = {22},
number = {1},
pages = {142},
pmid = {35331142},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Plant/genetics ; Mutagenesis ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress.<br><br>RESULTS: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions.<br><br>CONCLUSIONS: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.},
}
@article {pmid35330963,
year = {2022},
author = {Angelopoulou, A and Papaspyropoulos, A and Papantonis, A and Gorgoulis, VG},
title = {CRISPR-Cas9-mediated induction of large chromosomal inversions in human bronchial epithelial cells.},
journal = {STAR protocols},
volume = {3},
number = {2},
pages = {101257},
pmid = {35330963},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Chromosome Inversion ; Epithelial Cells ; Gene Editing/methods ; Humans ; },
abstract = {The in vitro recapitulation of chromosomal rearrangements is a necessary tool for understanding malignancy at the molecular level. Here, we describe the targeted induction of a large chromosomal inversion (>3.7 Mbp) through CRISPR-Cas9-mediated genome editing. As inversions occur at low frequency following Cas9 cleavage, we provide a detailed screening approach of FACS-sorted, single-cell-derived clonal human bronchial epithelial cell (HBEC) cultures. The protocol provided is tailored to HBECs; however, it can be readily applied to additional adherent cellular models. For complete details on the use and execution of this protocol, please refer to Zampetidis et al. (2021).},
}
@article {pmid35330692,
year = {2022},
author = {Sharma, P and Lew, TTS},
title = {Principles of Nanoparticle Design for Genome Editing in Plants.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {846624},
pmid = {35330692},
issn = {2673-3439},
abstract = {Precise plant genome editing technologies have provided new opportunities to accelerate crop improvement and develop more sustainable agricultural systems. In particular, the prokaryote-derived CRISPR platforms allow precise manipulation of the crop genome, enabling the generation of high-yielding and stress-tolerant crop varieties. Nanotechnology has the potential to catalyze the development of a novel molecular toolbox even further by introducing the possibility of a rapid, universal delivery method to edit the plant genome in a species-independent manner. In this Perspective, we highlight how nanoparticles can help unlock the full potential of CRISPR/Cas technology in targeted manipulation of the plant genome to improve agricultural output. We discuss current challenges hampering progress in nanoparticle-enabled plant gene-editing research and application in the field, and highlight how rational nanoparticle design can overcome them. Finally, we examine the implications of the regulatory frameworks and social acceptance for the future of nano-enabled precision breeding in the developing world.},
}
@article {pmid35330117,
year = {2022},
author = {Zakharevich, NV and Nikitin, MS and Kovtun, AS and Malov, VO and Averina, OV and Danilenko, VN and Artamonova, II},
title = {CRISPR-Cas Systems in Gut Microbiome of Children with Autism Spectrum Disorders.},
journal = {Life (Basel, Switzerland)},
volume = {12},
number = {3},
pages = {},
pmid = {35330117},
issn = {2075-1729},
abstract = {The human gut microbiome is associated with various diseases, including autism spectrum disorders (ASD). Variations of the taxonomical composition in the gut microbiome of children with ASD have been observed repeatedly. However, features and parameters of the microbiome CRISPR-Cas systems in ASD have not been investigated yet. Here, we demonstrate such an analysis in order to describe the overall changes in the microbiome CRISPR-Cas systems during ASD as well as to reveal their potential to be used in diagnostics and therapy. For the systems identification, we used a combination of the publicly available tools suited for completed genomes with subsequent filtrations. In the considered data, the microbiomes of children with ASD contained fewer arrays per Gb of assembly than the control group, but the arrays included more spacers on average. CRISPR arrays from the microbiomes of children with ASD differed from the control group neither in the fractions of spacers with protospacers from known genomes, nor in the sets of known bacteriophages providing protospacers. Almost all bacterial protospacers of the gut microbiome systems for both children with ASD and the healthy ones were located in prophage islands, leaving no room for the systems to participate in the interspecies competition.},
}
@article {pmid35328599,
year = {2022},
author = {Mészár, Z and Kókai, &#xc9; and Varga, R and Ducza, L and Papp, T and Béresová, M and Nagy, M and Szücs, P and Varga, A},
title = {CRISPR/Cas9-Based Mutagenesis of Histone H3.1 in Spinal Dynorphinergic Neurons Attenuates Thermal Sensitivity in Mice.},
journal = {International journal of molecular sciences},
volume = {23},
number = {6},
pages = {},
pmid = {35328599},
issn = {1422-0067},
mesh = {Animals ; *Burns/genetics ; CRISPR-Cas Systems/genetics ; *Histones/genetics/metabolism ; Hyperalgesia/metabolism ; Mice ; Mutagenesis ; Neurons/metabolism ; Spinal Cord/metabolism ; },
abstract = {Burn injury is a trauma resulting in tissue degradation and severe pain, which is processed first by neuronal circuits in the spinal dorsal horn. We have recently shown that in mice, excitatory dynorphinergic (Pdyn) neurons play a pivotal role in the response to burn-injury-associated tissue damage via histone H3.1 phosphorylation-dependent signaling. As Pdyn neurons were mostly associated with mechanical allodynia, their involvement in thermonociception had to be further elucidated. Using a custom-made AAV9_mutH3.1 virus combined with the CRISPR/cas9 system, here we provide evidence that blocking histone H3.1 phosphorylation at position serine 10 (S10) in spinal Pdyn neurons significantly increases the thermal nociceptive threshold in mice. In contrast, neither mechanosensation nor acute chemonociception was affected by the transgenic manipulation of histone H3.1. These results suggest that blocking rapid epigenetic tagging of S10H3 in spinal Pdyn neurons alters acute thermosensation and thus explains the involvement of Pdyn cells in the immediate response to burn-injury-associated tissue damage.},
}
@article {pmid35328559,
year = {2022},
author = {Aulitto, M and Martinez-Alvarez, L and Fiorentino, G and Limauro, D and Peng, X and Contursi, P},
title = {A Comparative Analysis of Weizmannia coagulans Genomes Unravels the Genetic Potential for Biotechnological Applications.},
journal = {International journal of molecular sciences},
volume = {23},
number = {6},
pages = {},
pmid = {35328559},
issn = {1422-0067},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; *Genomics ; },
abstract = {The production of biochemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Weizmannia coagulans species. So far, the genomes of 47 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of Carbohydrate-Active enZymes (CAZymes), secretion systems, and resistance mechanisms to environmental challenges. Moreover, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated (Cas) genes, was also analyzed. Overall, this study expands our understanding of the strain's genomic diversity of W. coagulans to fully exploit its potential in biotechnological applications.},
}
@article {pmid35328421,
year = {2022},
author = {Porika, M and Tippani, R and Saretzki, GC},
title = {CRISPR/Cas: A New Tool in the Research of Telomeres and Telomerase as Well as a Novel Form of Cancer Therapy.},
journal = {International journal of molecular sciences},
volume = {23},
number = {6},
pages = {},
pmid = {35328421},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Neoplasms/genetics/therapy ; *Telomerase/genetics/metabolism ; Telomere/genetics/metabolism ; },
abstract = {Due to their close connection with senescence, aging, and disease, telomeres and telomerase provide a unique and vital research route for boosting longevity and health span. Despite significant advances during the last three decades, earlier studies into these two biological players were impeded by the difficulty of achieving real-time changes inside living cells. As a result of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated system's (Cas) method, targeted genetic studies are now underway to change telomerase, the genes that govern it as well as telomeres. This review will discuss studies that have utilized CRISPR-related technologies to target and modify genes relevant to telomeres and telomerase as well as to develop targeted anti-cancer therapies. These studies greatly improve our knowledge and understanding of cellular and molecular mechanisms that underlie cancer development and aging.},
}
@article {pmid35327601,
year = {2022},
author = {Li, B and Ai, D and Liu, X},
title = {CNN-XG: A Hybrid Framework for sgRNA On-Target Prediction.},
journal = {Biomolecules},
volume = {12},
number = {3},
pages = {},
pmid = {35327601},
issn = {2218-273X},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Neural Networks, Computer ; *RNA, Guide/genetics/metabolism ; },
abstract = {As the third generation gene editing technology, Crispr/Cas9 has a wide range of applications. The success of Crispr depends on the editing of the target gene via a functional complex of sgRNA and Cas9 proteins. Therefore, highly specific and high on-target cleavage efficiency sgRNA can make this process more accurate and efficient. Although there are already many sophisticated machine learning or deep learning models to predict the on-target cleavage efficiency of sgRNA, prediction accuracy remains to be improved. XGBoost is good at classification as the ensemble model could overcome the deficiency of a single classifier to classify, and we would like to improve the prediction efficiency for sgRNA on-target activity by introducing XGBoost into the model. We present a novel machine learning framework which combines a convolutional neural network (CNN) and XGBoost to predict sgRNA on-target knockout efficacy. Our framework, called CNN-XG, is mainly composed of two parts: a feature extractor CNN is used to automatically extract features from sequences and predictor XGBoost is applied to predict features extracted after convolution. Experiments on commonly used datasets show that CNN-XG performed significantly better than other existing frameworks in the predicted classification mode.},
}
@article {pmid35326449,
year = {2022},
author = {Jamehdor, S and Pajouhanfar, S and Saba, S and Uzan, G and Teimoori, A and Naserian, S},
title = {Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions.},
journal = {Cells},
volume = {11},
number = {6},
pages = {},
pmid = {35326449},
issn = {2073-4409},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Viruses ; Host Microbial Interactions ; Humans ; SARS-CoV-2/genetics ; *Virus Diseases/diagnosis/genetics ; *Viruses/genetics ; },
abstract = {Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.},
}
@article {pmid35325722,
year = {2022},
author = {Zhuang, J and Zhao, Z and Lian, K and Yin, L and Wang, J and Man, S and Liu, G and Ma, L},
title = {SERS-based CRISPR/Cas assay on microfluidic paper analytical devices for supersensitive detection of pathogenic bacteria in foods.},
journal = {Biosensors &amp; bioelectronics},
volume = {207},
number = {},
pages = {114167},
doi = {10.1016/j.bios.2022.114167},
pmid = {35325722},
issn = {1873-4235},
mesh = {Bacteria/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Microfluidics ; Recombinases ; Salmonella typhimurium/genetics ; Spectrum Analysis, Raman ; },
abstract = {Rapid and point-of-need (PON) detection of bacteria is crucial to directly provide rapid and reliable diagnostics information during on-site tests, allowing more room for taking proactive measures. By taking the multifaceted advantages of CRISPR/Cas12a and surface-enhanced Raman scattering (SERS), for the first time, we designed a recombinase polymerase amplification (RPA)-integrated microfluidic paper-based analytical device (μPAD), coined RPA-Cas12a-μPAD for supersensitive SERS detection. Single-stranded DNAs were designed to "pull down" SERS nanoprobes. The amplicons of the invA gene triggered the trans-cleavage of Cas12a, resulting in the indiscriminate shredding of linker ssDNA. Thus, the degree of aggregation of SERS nanoprobes was dependent on the concentration of Salmonella typhimurium (S. typhi), which was determined on a μPAD and monitored by a Raman spectrometer. The limit of detection for S. typhi was approximately 3-4 CFU/mL for spiked milk and meat samples with a dynamic detection range from 1 to 10[8] CFU/mL. The RPA-Cas12a-μPAD secured accurate tests for food samples in 45 min. This work expands the reach of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust bacterial PON detection platform.},
}
@article {pmid35325565,
year = {2022},
author = {Huerne, K and Palmour, N and Wu, AR and Beck, S and Berner, A and Siebert, R and Joly, Y},
title = {Auditing the Editor: A Review of Key Translational Issues in Epigenetic Editing.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {203-212},
doi = {10.1089/crispr.2021.0094},
pmid = {35325565},
issn = {2573-1602},
support = {CEE-151618//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems/genetics ; Epigenomics ; *Gene Editing ; Humans ; },
abstract = {Currently, most advances in site-specific epigenetic editing for human use are concentrated in basic research, yet, there is considerable interest to translate this technology beyond the bench. This review highlights recent developments with epigenetic editing technology in comparison with the canonical CRISPR-Cas genome editing, as well as the epistemic and ethical considerations with preemptive translation of epigenetic editing into clinical or commercial use in humans. Key considerations in safety, equity, and access to epigenetic editing are highlighted, with a spotlight on the ethical, legal, and social issues of this technology in the context of global health equity.},
}
@article {pmid35325432,
year = {2022},
author = {Movahedi, A and Hajiahmadi, Z and Wei, H and Yang, L and Ruan, H and Zhuge, Q},
title = {A Method to Reduce off-Targets in CRISPR/Cas9 System in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2408},
number = {},
pages = {317-324},
pmid = {35325432},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Mutagenesis ; Mutation ; Plasmids/genetics ; *Silicon Dioxide ; },
abstract = {One of the strategies to reduce the off-target mutations in CRISPR/Cas9 system is to use the temperature-independent gene transformation method. Mesoporous silica nanoparticles (MSNs)-gene delivery system is temperature-independent; thus, it can transfer the interesting plasmid (pDNA) to the target plant at different temperatures, including 37 °C. Due to the high activity of SpCas9 at 37 °C compared to lower temperatures, on-target mutagenesis increases at 37 °C. Therefore, we describe the synthesis of the functionalized MSNs with the particle size of less than 40 nm, binding pDNA to the MSNs, and transferring of the pDNA-MSNs into the target plants.},
}
@article {pmid35325413,
year = {2022},
author = {Pandey, P and Mysore, KS and Senthil-Kumar, M},
title = {Recent Advances in Plant Gene Silencing Methods.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2408},
number = {},
pages = {1-22},
pmid = {35325413},
issn = {1940-6029},
mesh = {Gene Editing ; *Gene Silencing ; Genes, Plant ; *Plants/genetics ; },
abstract = {With the increasing understanding of fundamentals of gene silencing pathways in plants, various tools and techniques for downregulating the expression of a target gene have been developed across multiple plant species. This chapter provides an insight into the molecular mechanisms of gene silencing and highlights the advancements in various gene silencing approaches. The prominent aspects of different gene silencing methods, their advantages and disadvantages have been discussed. A succinct discussion on the newly emerged microRNA-based technologies like microRNA-induced gene silencing (MIGS) and microRNA-mediated virus-induced gene silencing (MIR-VIGS) are also presented. We have also discussed the gene-editing system like CRISPR-Cas. The prominent bottlenecks in gene silencing methods are the off-target effects and lack of universal applicability. However, the tremendous growth in understanding of this field reflects the potentials for improvements in the currently available approaches and the development of new widely applicable methods for easy, fast, and efficient functional characterization of plant genes.},
}
@article {pmid35325044,
year = {2022},
author = {Gladka, MM and Baker, AH},
title = {Cutting a path to effective delivery of genome engineering machinery.},
journal = {Cardiovascular research},
volume = {118},
number = {6},
pages = {e42-e44},
doi = {10.1093/cvr/cvac034},
pmid = {35325044},
issn = {1755-3245},
support = {/BHF_/British Heart Foundation/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid35323947,
year = {2022},
author = {Liang, M and Liu, L and Xu, F and Zeng, X and Wang, R and Yang, J and Wang, W and Karthik, L and Liu, J and Yang, Z and Zhu, G and Wang, S and Bai, L and Tong, Y and Liu, X and Wu, M and Zhang, LX and Tan, GY},
title = {Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach.},
journal = {Nucleic acids research},
volume = {50},
number = {6},
pages = {3581-3592},
pmid = {35323947},
issn = {1362-4962},
mesh = {*Biological Products ; CRISPR-Cas Systems ; Cloning, Molecular ; Multigene Family ; *Streptomyces/genetics ; },
abstract = {Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.},
}
@article {pmid35323942,
year = {2022},
author = {Yoo, KW and Yadav, MK and Song, Q and Atala, A and Lu, B},
title = {Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9.},
journal = {Nucleic acids research},
volume = {50},
number = {7},
pages = {3944-3957},
pmid = {35323942},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/genetics/metabolism ; DNA-Directed DNA Polymerase/metabolism ; *Gene Editing/methods ; },
abstract = {Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.},
}
@article {pmid35323424,
year = {2022},
author = {Li, C and Chen, X and Wen, R and Ma, P and Gu, K and Li, C and Zhou, C and Lei, C and Tang, Y and Wang, H},
title = {Immunocapture Magnetic Beads Enhanced the LAMP-CRISPR/Cas12a Method for the Sensitive, Specific, and Visual Detection of Campylobacter jejuni.},
journal = {Biosensors},
volume = {12},
number = {3},
pages = {},
pmid = {35323424},
issn = {2079-6374},
mesh = {CRISPR-Cas Systems ; *Campylobacter jejuni ; Magnetic Phenomena ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.},
}
@article {pmid35322386,
year = {2022},
author = {Bykonya, AG and Lavrov, AV and Smirnikhina, SA},
title = {Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {35322386},
issn = {1559-0305},
abstract = {The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.},
}
@article {pmid35322099,
year = {2022},
author = {Heu, CC and Gross, RJ and Le, KP and LeRoy, DM and Fan, B and Hull, JJ and Brent, CS and Fabrick, JA},
title = {CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {4917},
pmid = {35322099},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; *Eye Color/genetics ; Gene Editing ; *Heteroptera/genetics ; Mercury Compounds ; Nymph ; Pigmentation/genetics ; Plants/genetics ; },
abstract = {The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.},
}
@article {pmid35320824,
year = {2022},
author = {Monte Neto, RLD and Moreira, POL and de Sousa, AM and Garcia, MADN and Maran, SR and Moretti, NS},
title = {Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress.},
journal = {Memorias do Instituto Oswaldo Cruz},
volume = {117},
number = {},
pages = {e210403},
pmid = {35320824},
issn = {1678-8060},
mesh = {*Antiprotozoal Agents/chemistry ; Drug Discovery ; Humans ; *Leishmania ; *Leishmaniasis/drug therapy ; Protein Processing, Post-Translational ; },
abstract = {Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.},
}
@article {pmid35320636,
year = {2022},
author = {Alekseeva, AE and Brusnigina, NF and Gordinskaya, NA and Makhova, MA and Kolesnikova, EA},
title = {Molecular genetic characteristics of resistome and virulome of carbapenem-resistant Klebsiella pneumoniae clinical strains.},
journal = {Klinicheskaia laboratornaia diagnostika},
volume = {67},
number = {3},
pages = {186-192},
doi = {10.51620/0869-2084-2022-67-3-186-192},
pmid = {35320636},
issn = {0869-2084},
mesh = {Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; Humans ; *Klebsiella Infections/drug therapy/genetics ; *Klebsiella pneumoniae/genetics ; Microbial Sensitivity Tests ; Molecular Biology ; },
abstract = {The characteristics of resistome and virulome structure of four carbapenem-resistant Klebsiella pneumoniae clinical strains are present in the work. Two strains belonged to the sequence-type ST395, one strain - ST2262, one strain - to the new sequence-type 5816. The genes of fimbriae, enterobactin, beta-lactamase SHV type, resistance to fosfomycin fosA and transport of fluoroquinolones oqxAB in all Klebsiella strains chromosome structure were identified. The determinants of yersineobactin and aerobactin are enriched the virulome of ST395 NNKP315 and NNKP343 strains. The aerobactin genes are located on IncHI1B plasmids (IncHI1B/FIB) which highly homologous to the virulence pLVPK and pK2044 plasmids. IncR, IncL, IncQ plasmids carrying blaOXA-48, blaCTX-M-15, blaOXA-1, blaTEM-1, qnrS1, tetA, sul1, dfrA1, aac(6 ')-Ib-cr, catA1, catB3 etc. were identified in these strains. As a result of in silico analysis, an assumption about the localization of the blaOXA-48 in the structure of the IncHI1B plasmid of NNKP315 strain was made. This plasmid also contains the aminoglycosidases genes inserted into a class 1 integron In822. The mutations were found in the porin proteins OmpK35, OmpK36 and OmpK37 genes, which increases the carbapenem resistance. The virulome of NNKP16 (ST2262) strain additionally includes of the iron utilization system kfuABC chromosomal genes, and the virulome of NNKP15 (ST5816) strain contains of the capsular polysaccharide kvgAS and microcin E492 genes. Additional determinants of resistance were not identified in the resistome structure of K. pneumoniae NNKP16 and only the blaCTX-M-15 gene was found in the NNKP15 strain. The absence of acquired resistance genes seems to be due to the presence of the type I-E CRISPR-Cas system. Multiple drug resistance of the studied strains is associated with mutations identified in the gene structure of porin proteins OmpK36 and OmpK37, as well as the activity of efflux systems. It was showed the stop codon formation in the nucleotide sequence of the regulatory gene ramR to both strains, which can potentially provide overexpression of AcrAB efflux proteins.},
}
@article {pmid35320567,
year = {2022},
author = {Martin, CJ and Calarco, JA},
title = {Approaches for CRISPR/Cas9 Genome Editing in C. elegans.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2468},
number = {},
pages = {215-237},
pmid = {35320567},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *Genome, Helminth ; },
abstract = {The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe our most up-to-date protocols using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. We present several considerations when deciding which strategy best suits the needs of the experiment.},
}
@article {pmid35320539,
year = {2022},
author = {Feng, C and Nita-Lazar, M and González-Montalbán, N and Wang, J and Mancini, J and Wang, S and Ravindran, C and Ahmed, H and Vasta, GR},
title = {Manipulating Galectin Expression in Zebrafish (Danio rerio).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2442},
number = {},
pages = {425-443},
pmid = {35320539},
issn = {1940-6029},
support = {R01 GM070589/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Galectins/metabolism ; Gene Knockdown Techniques ; Mammals/genetics ; Morpholinos/genetics/metabolism ; RNA/metabolism ; *Zebrafish/metabolism ; },
abstract = {Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are β-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.},
}
@article {pmid35320319,
year = {2022},
author = {Snyder, AJ and Abad, AT and Danthi, P},
title = {A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses.},
journal = {PLoS pathogens},
volume = {18},
number = {3},
pages = {e1010398},
pmid = {35320319},
issn = {1553-7374},
support = {R01 AI110637/AI/NIAID NIH HHS/United States ; R03 AI142013/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Endosomes/metabolism ; Mammals ; Mice ; *Reoviridae/genetics ; *Reoviridae Infections/metabolism ; WD40 Repeats ; },
abstract = {Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it reduced viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.},
}
@article {pmid35318087,
year = {2022},
author = {Jiang, J and Sun, Y and Wang, Y and Sabek, A and Shangguan, A and Wang, K and Zhao, S and Li, G and Zhou, A and Zhang, S},
title = {Genome-wide CRISPR/Cas9 screen identifies host factors important for porcine reproductive and respiratory syndrome virus replication.},
journal = {Virus research},
volume = {314},
number = {},
pages = {198738},
doi = {10.1016/j.virusres.2022.198738},
pmid = {35318087},
issn = {1872-7492},
mesh = {Animals ; CRISPR-Cas Systems ; Genome ; *Porcine Reproductive and Respiratory Syndrome/genetics ; *Porcine respiratory and reproductive syndrome virus/genetics ; Receptors, Cell Surface/genetics ; Swine ; Virus Replication ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS), a viral infection caused by PRRS virus (PRRSV) can result in severe reproductive failure, and respiratory disease in the pigs thus causing enormous economic losses to the global swine industry. Although the cellular receptors for PRRSV have been identified, but mechanisms underlying PPRSV replication remain obscure. Here, we have performed a genome-scale CRISPR/Cas9 knockout screen in the pig kidney cells with PRRSV. Several genes were found to be highly enriched post-PRRSV selection, just like KxDL Motif Containing 1(KXD1), Proteasome 26S Subunit, Non-ATPase 3 (PSMD3) and Galectin 2 (LGALS2) and soon on. Importantly, we have identified that loss of KXD1 resulted in the restricted autophagy and inhibited replication of PRRSV. Therefore, our study demonstrates that CRISPR/Cas9 system can be effectively used for the screening of pig factors responsible for PRRSV replication.},
}
@article {pmid35316854,
year = {2022},
author = {Bonillo, M and Pfromm, J and Fischer, MD},
title = {Challenges to Gene Editing Approaches in the Retina.},
journal = {Klinische Monatsblatter fur Augenheilkunde},
volume = {239},
number = {3},
pages = {275-283},
doi = {10.1055/a-1757-9810},
pmid = {35316854},
issn = {1439-3999},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Humans ; Retina ; *Retinal Diseases/genetics ; },
abstract = {Retinal gene therapy has recently been at the cutting edge of clinical development in the diverse field of genetic therapies. The retina is an attractive target for genetic therapies such as gene editing due to the distinctive anatomical and immunological features of the eye, known as immune privilege, so that inherited retinal diseases (IRDs) have been studied in several clinical studies. Thus, rapid strides are being made toward developing targeted treatments for IRDs. Gene editing in the retina faces a group of heterogenous challenges, including editing efficiencies, off-target effects, the anatomy of the target organ, immune responses, inactivation, and identifying optimal application methods. As clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) based technologies are at the forefront of current gene editing advances, their specific editing efficiency challenges and potential off-target effects were assessed. The immune privilege of the eye reduces the likelihood of systemic immune responses following retinal gene therapy, but possible immune responses must not be discounted. Immune responses to gene editing in the retina may be humoral or cell mediated, with immunologically active cells, including microglia, implicated in facilitating possible immune responses to gene editing. Immunogenicity of gene therapeutics may also lead to the inactivation of edited cells, reducing potential therapeutic benefits. This review outlines the broad spectrum of potential challenges currently facing retinal gene editing, with the goal of facilitating further advances in the safety and efficacy of gene editing therapies.},
}
@article {pmid35316773,
year = {2022},
author = {Mir, TUG and Wani, AK and Akhtar, N and Shukla, S},
title = {CRISPR/Cas9: Regulations and challenges for law enforcement to combat its dual-use.},
journal = {Forensic science international},
volume = {334},
number = {},
pages = {111274},
doi = {10.1016/j.forsciint.2022.111274},
pmid = {35316773},
issn = {1872-6283},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; Gene Editing ; Humans ; *Law Enforcement ; Plants ; },
abstract = {For many years, scientists have aspired to edit any DNA segment of living organisms precisely, and with the discovery of CRISPR/Cas9, they have moved closer to achieving their objective. Using this tool, researchers can knock out or knock-in a gene into or from any part of the genome. The CRISPR-Cas9 system is a highly effective and accurate tool and has replaced previous genome editing technologies such as ZFN and TALEN having lesser precision and accuracy. Advances in CRISPR/Cas technology have been remarkable in recent years, with significant applications in various fields of life sciences research. Despite the diverse applications of the CRISPR system, this tool also poses a threat to living organisms and the environment. The intentional misuse of CRISPR could be a disaster to society and the environment. Different countries have imposed laws and guidelines to regulate genome editing through multiple agencies. However, in some countries, these rules and regulations are still under development or don't exist, leading to higher chances of the technology getting misused. Dual-use of this technology could be a threat to biosecurity and biodefense. This article summarizes the potential applications of the CRISPR system in human and plants, and also evaluate the laws and regulations imposed by different countries to keep genome editing technology under check.},
}
@article {pmid35316566,
year = {2022},
author = {Schiermeyer, A and Cerda-Bennasser, P and Schmelter, T and Huang, X and Christou, P and Schillberg, S},
title = {Rapid production of SaCas9 in plant-based cell-free lysate for activity testing.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100564},
doi = {10.1002/biot.202100564},
pmid = {35316566},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; Staphylococcus aureus ; Tobacco/genetics/metabolism ; },
abstract = {Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with 10 different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.},
}
@article {pmid35315725,
year = {2022},
author = {Ye, Y and Shi, Q and Yang, T and Xie, F and Zhang, X and Xu, B and Fang, J and Chen, J and Zhang, Y and Li, J},
title = {In Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System.},
journal = {Technology in cancer research &amp; treatment},
volume = {21},
number = {},
pages = {15330338221085370},
pmid = {35315725},
issn = {1533-0338},
mesh = {CRISPR-Cas Systems/genetics ; Cell Communication ; *Extracellular Vesicles/genetics/metabolism ; Gene Editing ; Humans ; *Melanoma/genetics/metabolism ; },
abstract = {Introduction: Tumor extracellular vesicles (EVs) and their relevance to various processes of tumor growth have been vigorously investigated over the past decade. However, obtaining direct evidence of spontaneous EV transfer in vivo remains challenging. In our previous study, a single-guide RNA (sgRNA): Cas9 ribonucleoprotein complex, which can efficiently delete target genes, was delivered into recipient cells using an engineered EV. Aim: Applying this newly discovered exosomal bio-cargo to track the uptake and distribution of tumor EVs. Methods: Tumor cells of interest were engineered to express and release the sgRNA:Cas9 complex, and a reporter cell/system containing STOP-fluorescent protein (FP) elements was also generated. EV-delivered Cas9 proteins from donor cells were programmed by a pair of sgRNAs to completely delete a blockade sequence and, in turn, recuperated the expression of FP in recipient reporter cells. Thus, fluorescently illuminated cells indicate the uptake of EVs. To improve the efficiency and sensitivity of this tracking system in vivo, we optimized the sgRNA design, which could more efficiently trigger the expression of reporter proteins. Results: We demonstrated the EV-mediated crosstalk between tumor cells, and between tumor cells and normal cells in vitro. In vivo, we showed that intravenously administered EVs can be taken up by the liver. Moreover, we showed that EVs derived from melanoma xenografts in vivo preferentially target the brain and liver. This distribution resembles the manifestation of organotrophic metastasis of melanoma. Conclusion: This study provides an alternative tool to study the distribution and uptake of tumor EVs.},
}
@article {pmid35315229,
year = {2022},
author = {Mukherjee, P and Patino, CA and Pathak, N and Lemaitre, V and Espinosa, HD},
title = {Deep Learning-Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard-to-Transfect Cells.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {18},
number = {20},
pages = {e2107795},
pmid = {35315229},
issn = {1613-6829},
support = {R43 GM128500/GM/NIGMS NIH HHS/United States ; R21 GM132709/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Deep Learning ; Electroporation/methods ; Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.},
}
@article {pmid35314803,
year = {2022},
author = {Lu, S and Tong, X and Han, Y and Zhang, K and Zhang, Y and Chen, Q and Duan, J and Lei, X and Huang, M and Qiu, Y and Zhang, DY and Zhou, X and Zhang, Y and Yin, H},
title = {Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.},
journal = {Nature biomedical engineering},
volume = {6},
number = {3},
pages = {286-297},
pmid = {35314803},
issn = {2157-846X},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; *RNA, Viral/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.},
}
@article {pmid35314780,
year = {2022},
author = {Liao, C and Sharma, S and Svensson, SL and Kibe, A and Weinberg, Z and Alkhnbashi, OS and Bischler, T and Backofen, R and Caliskan, N and Sharma, CM and Beisel, CL},
title = {Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA.},
journal = {Nature microbiology},
volume = {7},
number = {4},
pages = {530-541},
pmid = {35314780},
issn = {2058-5276},
support = {865973/ERC_/European Research Council/International ; },
mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; RNA/genetics ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.},
}
@article {pmid35314679,
year = {2022},
author = {Schindele, A and Gehrke, F and Schmidt, C and Röhrig, S and Dorn, A and Puchta, H},
title = {Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1502},
pmid = {35314679},
issn = {2041-1723},
mesh = {*Arabidopsis/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Mutagenesis ; Tandem Repeat Sequences ; },
abstract = {CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.},
}
@article {pmid35314527,
year = {2022},
author = {Jair Lara-Navarro, I and Rebeca Jaloma-Cruz, A},
title = {Current Therapies in Hemophilia: From Plasma-Derived Factor Modalities to CRISPR/Cas Alternatives.},
journal = {The Tohoku journal of experimental medicine},
volume = {256},
number = {3},
pages = {197-207},
doi = {10.1620/tjem.256.197},
pmid = {35314527},
issn = {1349-3329},
mesh = {CRISPR-Cas Systems/genetics ; Factor IX/genetics/therapeutic use ; *Hemophilia A/genetics/therapy ; *Hemophilia B/drug therapy/therapy ; Humans ; Quality of Life ; },
abstract = {Since the middle of the last century, there have been amazing therapeutic advances for hemophilia such as the development of plasma-derived products and bioengineered recombinant factors VIII and IX (for hemophilia A and B, respectively) with improved stability, higher activity, and extended half-life. The recent use of a monoclonal antibody that mimics factor VIII activity (which is an efficient treatment for all hemophilia A phenotypes with or without inhibitors) has shown the great possibilities of non-factor therapies for improving the quality of life of hemophilia A patients, with a safer application and long-lasting effects. Gene therapy offers the promise of a "true cure" for hemophilia based on the permanent effect that a gene edition may render. Clinical trials developed in the last decade based on adenoviral vectors show modest but consistent results; now, CRISPR/Cas technology (which is considered the most efficient tool for gene edition) is being developed on different hemophilia models. Once the off-target risks are solved and an efficient switch on/off for Cas activity is developed, this strategy might become the most feasible option for gene therapy in hemophilia and other monogenic diseases.},
}
@article {pmid35313109,
year = {2022},
author = {Harrison, PT},
title = {CRISPR gene editing - what are the possibilities for respiratory medicine?.},
journal = {Expert review of respiratory medicine},
volume = {16},
number = {4},
pages = {371-374},
doi = {10.1080/17476348.2022.2056021},
pmid = {35313109},
issn = {1747-6356},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Therapy ; Humans ; *Pulmonary Medicine ; },
}
@article {pmid35312212,
year = {2022},
author = {Nguyen, STT and Vardeh, DP and Nelson, TM and Pearson, LA and Kinsela, AS and Neilan, BA},
title = {Bacterial community structure and metabolic potential in microbialite-forming mats from South Australian saline lakes.},
journal = {Geobiology},
volume = {20},
number = {4},
pages = {546-559},
pmid = {35312212},
issn = {1472-4669},
mesh = {Australia ; *Cyanobacteria/genetics ; Geologic Sediments/chemistry ; Lakes/microbiology ; *Microbiota ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; South Australia ; },
abstract = {Microbialites are sedimentary rocks created in association with benthic microorganisms. While they harbour complex microbial communities, Cyanobacteria perform critical roles in sediment stabilisation and accretion. Microbialites have been described from permanent and ephemeral saline lakes in South Australia; however, the microbial communities that generate and inhabit these biogeological structures have not been studied in detail. To address this knowledge gap, we investigated the composition, diversity and metabolic potential of bacterial communities from different microbialite-forming mats and surrounding sediments in five South Australian saline coastal lakes using 16S rRNA gene sequencing and predictive metagenome analyses. While Proteobacteria and Bacteroidetes were the dominant phyla recovered from the mats and sediments, Cyanobacteria were significantly more abundant in the mat samples. Interestingly, at lower taxonomic levels, the mat communities were vastly different across the five lakes. Comparative analysis of putative mat and sediment metagenomes via PICRUSt2 revealed important metabolic pathways driving the process of carbonate precipitation, including cyanobacterial oxygenic photosynthesis, ureolysis and nitrogen fixation. These pathways were highly conserved across the five examined lakes, although they appeared to be performed by distinct groups of bacterial taxa found in each lake. Stress response, quorum sensing and circadian clock were other important pathways predicted by the in silico metagenome analysis. The enrichment of CRISPR/Cas and phage shock associated genes in these cyanobacteria-rich communities suggests that they may be under selective pressure from viral infection. Together, these results highlight that a very stable ecosystem function is maintained by distinctly different communities in microbialite-forming mats in the five South Australian lakes and reinforce the concept that 'who' is in the community is not as critical as their net metabolic capacity.},
}
@article {pmid35310854,
year = {2022},
author = {Liu, X and Qiu, X and Xu, S and Che, Y and Han, L and Kang, Y and Yue, Y and Chen, S and Li, F and Li, Z},
title = {A CRISPR-Cas12a-Assisted Fluorescence Platform for Rapid and Accurate Detection of Nocardia cyriacigeorgica.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {835213},
pmid = {35310854},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded ; *Nocardia/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Nocardia cyriacigeorgica has gradually become a common pathogen in clinical microbial infections. Identification of Nocardia at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of N. cyriacigeorgica. In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of N. cyriacigeorgica in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for N. cyriacigeorgica was 10[-3] ng and the specificity was 100%. Thus, the N. cyriacigeorgica CRISPR-PCR assay is a rapid and specific method for detecting N. cyriacigeorgica, and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.},
}
@article {pmid35310559,
year = {2022},
author = {Zhang, Y and Li, Z and Milon Essola, J and Ge, K and Dai, X and He, H and Xiao, H and Weng, Y and Huang, Y},
title = {Biosafety materials: Ushering in a new era of infectious disease diagnosis and treatment with the CRISPR/Cas system.},
journal = {Biosafety and health},
volume = {4},
number = {2},
pages = {70-78},
pmid = {35310559},
issn = {2590-0536},
abstract = {Despite multiple virus outbreaks over the past decade, including the devastating coronavirus disease 2019 (COVID-19) pandemic, the lack of accurate and timely diagnosis and treatment technologies has wreaked havoc on global biosecurity. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has the potential to address these critical needs for tackling infectious diseases to detect viral nucleic acids and inhibit viral replication. This review summarizes how the CRISPR/Cas system is being utilized for the treatment and diagnosis of infectious diseases with the help of biosafety materials and highlights the design principle and in vivo and in vitro efficacy of advanced biosafety materials used to deal with virus attacks.},
}
@article {pmid35308851,
year = {2022},
author = {Chen, S and Wang, R and Peng, S and Xie, S and Lei, C and Huang, Y and Nie, Z},
title = {PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing.},
journal = {Chemical science},
volume = {13},
number = {7},
pages = {2011-2020},
pmid = {35308851},
issn = {2041-6520},
abstract = {The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.},
}
@article {pmid35308373,
year = {2022},
author = {Duan, G and Kan, B and Li, D and Song, H},
title = {Editorial: The CRISPR/Cas System in Pathogen Resistance, Virulence, Diagnosis and Typing.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {832152},
pmid = {35308373},
issn = {1664-302X},
}
@article {pmid35308131,
year = {2022},
author = {Lax, C and Navarro-Mendoza, MI and Pérez-Arques, C and Navarro, E and Nicolás, FE and Garre, V},
title = {Transformation and CRISPR-Cas9-mediated homologous recombination in the fungus Rhizopus microsporus.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101237},
pmid = {35308131},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Homologous Recombination ; *Mucormycosis/genetics ; Rhizopus/genetics ; },
abstract = {Here, we describe a reliable approach for targeted DNA integrations in the genome of R. microsporus, one of the main causal agents of mucormycosis. We provide a strategy for stable, targeted integration of DNA templates by homologous recombination (HR) based on the CRISPR-Cas9 technology. This strategy opens a wide range of possibilities for the genetic modification of R. microsporus and will be useful for the study of mucormycosis. For complete details on the use and execution of this protocol, please refer to Lax et al. (2021).},
}
@article {pmid35307599,
year = {2022},
author = {Dong, JF and Feng, CJ and Wang, P and Li, RQ and Zou, QH},
title = {Comparative genomics analysis of Acinetobacter baumannii multi-drug resistant and drug sensitive strains in China.},
journal = {Microbial pathogenesis},
volume = {165},
number = {},
pages = {105492},
doi = {10.1016/j.micpath.2022.105492},
pmid = {35307599},
issn = {1096-1208},
mesh = {*Acinetobacter baumannii ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Multiple, Bacterial/genetics ; Genomics ; Microbial Sensitivity Tests ; Plasmids/genetics ; },
abstract = {The incidence of multidrug-resistant Acinetobacter baumannii has posed a major challenge for clinical treatment. There is still a significant gap in understanding the mechanism causing multi-drug resistance (MDR). In this study, the genomes of 10 drug sensitive and 10 multi-drug resistant A.baumannii strains isolated from a hospital in China were sequenced and compared. The antibiotic resistance genes, virulence factors were determined and CRIPSR-Cas system along with prophages were detected. The results showed that MDR strains are significantly different from the drug sensitive strains in the CARD entries, patterns of sequences matching up to plasmids, VFDB entries and CRISPR-Cas system. MDR strains contain unique CARD items related to antibiotic resistance which are absent in sensitive strains. Furthermore, sequences from genomes of MDR strains can match up with plasmids from more diversified bacteria genera compared to drug sensitive strains. MDR strains also contain a lower level of CRISPR genes and larger amount of prophages, along with higher levels of spacer sequences. These findings provide new experimental evidences for the study of the antibiotic resistance mechanism of A. baumannii.},
}
@article {pmid35306273,
year = {2022},
author = {Chen, P and Wang, L and Qin, P and Yin, BC and Ye, BC},
title = {An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs.},
journal = {Biosensors &amp; bioelectronics},
volume = {207},
number = {},
pages = {114152},
doi = {10.1016/j.bios.2022.114152},
pmid = {35306273},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Endonucleases ; *MicroRNAs/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system.},
}
@article {pmid35306148,
year = {2022},
author = {Sun, X and Wang, DO and Wang, J},
title = {Targeted manipulation of m[6]A RNA modification through CRISPR-Cas-based strategies.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {56-61},
doi = {10.1016/j.ymeth.2022.03.006},
pmid = {35306148},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Methyltransferases/genetics/metabolism ; RNA/genetics ; RNA, Guide/genetics/metabolism ; },
abstract = {N[6]-methyladenosine (m[6]A) is a reversible and prevalent internal modification in RNAs and can be dynamically modulated by methyltransferase and demethylase. Targeted manipulation of m[6]A RNA modification is critical in studying the functions of specific m[6]A sites as well as developing molecular therapies through targeting m[6]A. The CRISPR-Cas systems including CRISPR-Cas9 and CRISPR-Cas13 have been widely used to edit and modify specific nucleotides on DNA and RNA through fusing effective proteins such as enzymes with Cas9/13. Through taking advantage of the m[6]A methyltransferase and demethylase, a series of CRISPR-Cas-based methods have also been developed to manipulate the m[6]A methylation at specific RNA sites. This review summarizes the latest CRISPR-Cas13 and Cas9 toolkits for m[6]A site-specific manipulation, including fundamental components, on-target efficiency, editing window, PAM/PFS requirement, and subcellularly localized targeting as well as potential limitations. We thus aim to provide an overview to assist researchers to choose an optimal tool to manipulate m[6]A for different purposes and also point out possible optimization strategies.},
}
@article {pmid35305903,
year = {2022},
author = {Bekaert, B and Boel, A and Cosemans, G and De Witte, L and Menten, B and Heindryckx, B},
title = {CRISPR/Cas gene editing in the human germline.},
journal = {Seminars in cell &amp; developmental biology},
volume = {131},
number = {},
pages = {93-107},
doi = {10.1016/j.semcdb.2022.03.012},
pmid = {35305903},
issn = {1096-3634},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Female ; *Gene Editing ; Germ Cells ; Humans ; Mosaicism ; Oocytes ; Pregnancy ; },
abstract = {The ease and efficacy of CRISPR/Cas9 germline gene editing in animal models paved the way to human germline gene editing (HGGE), by which permanent changes can be introduced into the embryo. Distinct genes can be knocked out to examine their function during embryonic development. Alternatively, specific sequences can be introduced which can be applied to correct disease-causing mutations. To date, it has been shown that the success of HGGE is dependent on various experimental parameters and that various hurdles (i.e. loss-of-heterozygosity and mosaicism) need to be overcome before clinical applications should be considered. Due to the shortage of human germline material and the ethical constraints concerning HGGE, alternative models such as stem cells have been evaluated as well, in terms of their predictive value on the genetic outcome for HGGE approaches. This review will give an overview of the state of the art of HGGE in oocytes and embryos, and its accompanying challenges.},
}
@article {pmid35304449,
year = {2022},
author = {Wang, Y and Zhang, G and Meng, Q and Huang, S and Guo, P and Leng, Q and Sun, L and Liu, G and Huang, X and Liu, J},
title = {Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1454},
pmid = {35304449},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Regulatory Networks ; Humans ; *Neoplasms/genetics/therapy ; *Transcription Factors/genetics ; },
abstract = {Reinvigoration of antitumor immunity has recently become the central theme for the development of cancer therapies. Nevertheless, the precise delivery of immunotherapeutic activities to the tumors remains challenging. Here, we explore a synthetic gene circuit-based strategy for specific tumor identification, and for subsequently engaging immune activation. By design, these circuits are assembled from two interactive modules, i.e., an oncogenic TF-driven CRISPRa effector, and a corresponding p53-inducible off-switch (NOT gate), which jointly execute an AND-NOT logic for accurate tumor targeting. In particular, two forms of the NOT gate are developed, via the use of an inhibitory sgRNA or an anti-CRISPR protein, with the second form showing a superior performance in gating CRISPRa by p53 loss. Functionally, the optimized AND-NOT logic circuit can empower a highly specific and effective tumor recognition/immune rewiring axis, leading to therapeutic effects in vivo. Taken together, our work presents an adaptable strategy for the development of precisely delivered immunotherapy.},
}
@article {pmid35304271,
year = {2022},
author = {Wen, W and Zhang, XB},
title = {CRISPR-Cas9 gene editing induced complex on-target outcomes in human cells.},
journal = {Experimental hematology},
volume = {110},
number = {},
pages = {13-19},
doi = {10.1016/j.exphem.2022.03.002},
pmid = {35304271},
issn = {1873-2399},
mesh = {*CRISPR-Cas Systems ; DNA Repair ; *Gene Editing/methods ; Genetic Therapy ; Genome ; Humans ; },
abstract = {CRISPR-Cas9 is a powerful tool for editing the genome and holds great promise for gene therapy applications. Initial concerns of gene engineering focus on off-target effects. However, in addition to short indel mutations (often <50 bp), an increasing number of studies have revealed complex on-target results after double-strand break repair by CRISPR-Cas9, such as large deletions, gene rearrangement, and loss of heterozygosity. These unintended mutations are potential safety concerns in clinical gene editing. Here, in this review, we summarize the significant findings of CRISPR-Cas9-induced on-target deleterious outcomes and discuss putative ways to achieve safe gene therapy.},
}
@article {pmid35303871,
year = {2022},
author = {Dimitri, A and Herbst, F and Fraietta, JA},
title = {Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {78},
pmid = {35303871},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; Immunotherapy, Adoptive ; *Neoplasms/drug therapy/therapy ; Receptors, Antigen, T-Cell/genetics ; *Receptors, Chimeric Antigen/metabolism ; T-Lymphocytes ; },
abstract = {Chimeric Antigen Receptor (CAR) T-cells represent a breakthrough in personalized cancer therapy. In this strategy, synthetic receptors comprised of antigen recognition, signaling, and costimulatory domains are used to reprogram T-cells to target tumor cells for destruction. Despite the success of this approach in refractory B-cell malignancies, optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been achieved. Factors such as T-cell exhaustion, lack of CAR T-cell persistence, cytokine-related toxicities, and bottlenecks in the manufacturing of autologous products have hampered the safety, effectiveness, and availability of this approach. With the ease and accessibility of CRISPR-Cas9-based gene editing, it is possible to address many of these limitations. Accordingly, current research efforts focus on precision engineering of CAR T-cells with conventional CRISPR-Cas9 systems or novel editors that can install desired genetic changes with or without introduction of a double-stranded break (DSB) into the genome. These tools and strategies can be directly applied to targeting negative regulators of T-cell function, directing therapeutic transgenes to specific genomic loci, and generating reproducibly safe and potent allogeneic universal CAR T-cell products for on-demand cancer immunotherapy. This review evaluates several of the ongoing and future directions of combining next-generation CRISPR-Cas9 gene editing with synthetic biology to optimize CAR T-cell therapy for future clinical trials toward the establishment of a new cancer treatment paradigm.},
}
@article {pmid35303554,
year = {2022},
author = {Zhang, L and Jiang, H and Zhu, Z and Liu, J and Li, B},
title = {Integrating CRISPR/Cas within isothermal amplification for point-of-Care Assay of nucleic acid.},
journal = {Talanta},
volume = {243},
number = {},
pages = {123388},
doi = {10.1016/j.talanta.2022.123388},
pmid = {35303554},
issn = {1873-3573},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Point-of-Care Systems ; },
abstract = {Nucleic acid detection technology is now widely used in scientific research and clinical testing, such as infectious and genetic diseases screening, molecular diagnosis of tumors and pharmacogenomic research, which is also an important part of in vitro diagnostics (IVD). However, with the increasing requirements of diagnosis and treatment, existing nucleic acid detection technologies are facing challenges in dealing with the current problems (especially since the outbreak of coronavirus disease in 2019 (Covid-19)). Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas)-based diagnostics have become a hot spot of attention. CRISPR/Cas has been developed as a molecular detection tool besides scientific research in biology and medicine fields, and some CRISPR-based products have already been translated. It is known as the "next-generation molecular diagnostic technology" because of its advantages such as easy design and accurate identification. CRISPR/Cas relies on pre-amplification of target sequences and subsequent detection of Cas proteins. Combining the CRISPR/Cas system with various isothermal nucleic acid amplification strategies can generate amplified detection signals, enrich low abundance molecular targets, improve the specificity and sensitivity of analysis, and develop point-of-care (POC) diagnostic techniques. In this review, we analyze the current status of CRISPR/Cas systems and isothermal amplification, report the advantages of combining the two and summarize the recent progress with the integration of both technologies with POC sensors in the nucleic acid field. In addition, the challenges and future prospects of CRISPR technology combined with isothermal amplification strategies in biosensing and clinical applications are discussed.},
}
@article {pmid35303481,
year = {2022},
author = {Roth, MO and Li, H},
title = {"X" marks the spot: Mining the gold in CasX for gene editing.},
journal = {Molecular cell},
volume = {82},
number = {6},
pages = {1083-1085},
pmid = {35303481},
issn = {1097-4164},
support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gold ; Humans ; },
abstract = {In this issue of Molecular Cell, Tsuchida et al. (2022) present a successful structure-guided effort in improving genome-editing efficiencies of CRISPR-CasX from Deltaproteobacteria (DpbCasX) and Planctomycetes (PlmCasX). Engineered variants that stabilize the active conformational state improved the catalytic efficiency by ∼10-20 fold in vitro and mean-editing efficiency by ∼2-3 fold in human cells.},
}
@article {pmid35303047,
year = {2022},
author = {Ceballos-Garzon, A and Roman, E and Pla, J and Pagniez, F and Amado, D and Alméciga-Díaz, CJ and Le Pape, P and Parra-Giraldo, CM},
title = {CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.},
journal = {PloS one},
volume = {17},
number = {3},
pages = {e0265777},
pmid = {35303047},
issn = {1932-6203},
mesh = {Animals ; Antifungal Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Calcineurin/metabolism ; *Candida glabrata/genetics/metabolism ; Drug Resistance, Fungal/genetics ; *Echinocandins/pharmacology/therapeutic use ; Humans ; Mice ; Microbial Sensitivity Tests ; Transcription Factors/genetics/metabolism ; Zinc/metabolism ; Zinc Fingers ; },
abstract = {Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.},
}
@article {pmid35302756,
year = {2022},
author = {Lu, D and Foley, CA and Birla, SV and Hepperla, AJ and Simon, JM and James, LI and Hathaway, NA},
title = {Bioorthogonal Chemical Epigenetic Modifiers Enable Dose-Dependent CRISPR Targeted Gene Activation in Mammalian Cells.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1397-1407},
pmid = {35302756},
issn = {2161-5063},
support = {P30 ES010126/ES/NIEHS NIH HHS/United States ; R01 GM118653/GM/NIGMS NIH HHS/United States ; R61 DA047023/DA/NIDA NIH HHS/United States ; R01 GM132299/GM/NIGMS NIH HHS/United States ; R01 CA242305/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mammals/genetics ; Nuclear Proteins/genetics/metabolism ; Tacrolimus Binding Proteins/genetics/metabolism ; Transcription Factors/genetics ; Transcriptional Activation ; },
abstract = {CRISPR-Cas9 systems have been developed to regulate gene expression by using either fusions to epigenetic regulators or, more recently, through the use of chemically mediated strategies. These approaches have armed researchers with new tools to examine the function of proteins by intricately controlling expression levels of specific genes. Here we present a CRISPR-based chemical approach that uses a new chemical epigenetic modifier (CEM) to hone to a gene targeted with a catalytically inactive Cas9 (dCas9) bridged to an FK506-binding protein (FKBP) in mammalian cells. One arm of the bifunctional CEM recruits BRD4 to the target site, and the other arm is composed of a bumped ligand that binds to a mutant FKBP with a compensatory hole at F36V. This bump-and-hole strategy allows for activation of target genes in a dose-dependent and reversible fashion with increased specificity and high efficacy, providing a new synthetic biology approach to answer important mechanistic questions in the future.},
}
@article {pmid35302142,
year = {2022},
author = {Li, D and Duan, C and Cheng, W and Gong, Y and Yao, Y and Wang, X and Wang, Z and Xiang, Y},
title = {A simple and rapid method to assay SARS-CoV-2 RNA based on a primer exchange reaction.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {28},
pages = {4484-4487},
doi = {10.1039/d2cc00488g},
pmid = {35302142},
issn = {1364-548X},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; *SARS-CoV-2/genetics ; },
abstract = {A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a primer exchange reaction (PER). By ingeniously integrating the PER cascade and CRISPR/cas12a system, this method can achieve convenient detection of the target RNA in 40 min and distinguish a single-base mutation from the target sequence, demonstrating its superior analytical performance.},
}
@article {pmid35302126,
year = {2022},
author = {Zhou, M and Yin, Y and Shi, Y and Huang, Z and Shi, Y and Chen, M and Ke, G and Zhang, XB},
title = {Spherical nucleic acid reporter-based cascade CRISPR/Cas12a amplifier for stable and sensitive biosensing of circulating tumor DNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {28},
pages = {4508-4511},
doi = {10.1039/d2cc00960a},
pmid = {35302126},
issn = {1364-548X},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/genetics ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; },
abstract = {Stable and sensitive ctDNA biosensing in complex biological fluid is highly important but still remains a challenge. Herein, we develop a spherical nucleic acid reporter-based cascade CRISPR/Cas12a amplifier with improved stability and sensitivity (5 orders of magnitude).},
}
@article {pmid35301482,
year = {2022},
author = {Xie, Y and Zhang, L and Gao, Z and Yin, P and Wang, H and Li, H and Chen, Z and Zhang, Y and Yang, M and Feng, Y},
title = {AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition.},
journal = {Nature chemical biology},
volume = {18},
number = {6},
pages = {670-677},
pmid = {35301482},
issn = {1552-4469},
mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; Viral Proteins/chemistry/genetics/metabolism ; },
abstract = {CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring its unique anti-CRISPR mechanism. AcrIF5 shows binding specificity only for the target DNA-bound form of the crRNA-guided surveillance (Csy) complex, but not the apo Csy complex from the type I-F CRISPR-Cas system. We solved the structure of the Csy-dsDNA-AcrIF5 complex, revealing that the conformational changes of the Csy complex caused by dsDNA binding dictate the binding specificity for the Csy-dsDNA complex by AcrIF5. Mechanistically, five AcrIF5 molecules bind one Csy-dsDNA complex, which destabilizes the helical bundle domain of Cas8f, thus preventing subsequent Cas2/3 recruitment. AcrIF5 exists in symbiosis with AcrIF3, which blocks Cas2/3 recruitment. This attack on the recruitment event stands in contrast to the conventional mechanisms of blocking binding of target DNA. Overall, our study reveals an unprecedented mechanism of CRISPR-Cas inhibition by AcrIF5.},
}
@article {pmid35301428,
year = {2022},
author = {Cui, YR and Wang, SJ and Ma, T and Yu, P and Chen, J and Guo, T and Meng, G and Jiang, B and Dong, J and Liu, J},
title = {KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {237},
pmid = {35301428},
issn = {2399-3642},
mesh = {Active Transport, Cell Nucleus ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; RNA, Messenger/genetics/metabolism ; },
abstract = {CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.},
}
@article {pmid35301321,
year = {2022},
author = {Cui, Z and Tian, R and Huang, Z and Jin, Z and Li, L and Liu, J and Huang, Z and Xie, H and Liu, D and Mo, H and Zhou, R and Lang, B and Meng, B and Weng, H and Hu, Z},
title = {FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1425},
pmid = {35301321},
issn = {2041-1723},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome ; RNA, Guide/genetics ; },
abstract = {Genome editing technologies hold tremendous potential in biomedical research and drug development. Therefore, it is imperative to discover gene editing tools with superior cutting efficiency, good fidelity, and fewer genomic restrictions. Here, we report a CRISPR/Cas9 from Faecalibaculum rodentium, which is characterized by a simple PAM (5'-NNTA-3') and a guide RNA length of 21-22 bp. We find that FrCas9 could achieve comparable efficiency and specificity to SpCas9. Interestingly, the PAM of FrCas9 presents a palindromic sequence, which greatly expands its targeting scope. Due to the PAM sequence, FrCas9 possesses double editing-windows for base editor and could directly target the TATA-box in eukaryotic promoters for TATA-box related diseases. Together, our results broaden the understanding of CRISPR/Cas-mediated genome engineering and establish FrCas9 as a safe and efficient platform for wide applications in research, biotechnology and therapeutics.},
}
@article {pmid35300995,
year = {2022},
author = {Hou, Y and Zhang, X and Sun, X and Qin, Q and Chen, D and Jia, M and Chen, Y},
title = {Genetically modified rabbit models for cardiovascular medicine.},
journal = {European journal of pharmacology},
volume = {922},
number = {},
pages = {174890},
doi = {10.1016/j.ejphar.2022.174890},
pmid = {35300995},
issn = {1879-0712},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Cardiovascular Agents ; Gene Editing/methods ; Gene Targeting ; Genetic Engineering/methods ; Models, Animal ; Phenotype ; Rabbits ; },
abstract = {Genetically modified (GM) rabbits are outstanding animal models for studying human genetic and acquired diseases. As such, GM rabbits that express human genes have been extensively used as models of cardiovascular disease. Rabbits are genetically modified via prokaryotic microinjection. Through this process, genes are randomly integrated into the rabbit genome. Moreover, gene targeting in embryonic stem (ES) cells is a powerful tool for understanding gene function. However, rabbits lack stable ES cell lines. Therefore, ES-dependent gene targeting is not possible in rabbits. Nevertheless, the RNA interference technique is rapidly becoming a useful experimental tool that enables researchers to knock down specific gene expression, which leads to the genetic modification of rabbits. Recently, with the emergence of new genetic technology, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated protein 9 (CRISPR/Cas9), major breakthroughs have been made in rabbit gene targeting. Using these novel genetic techniques, researchers have successfully modified knockout (KO) rabbit models. In this paper, we aimed to review the recent advances in GM technology in rabbits and highlight their application as models for cardiovascular medicine.},
}
@article {pmid35300480,
year = {2022},
author = {Lan, XR and Liu, ZL and Niu, DK},
title = {Precipitous Increase of Bacterial CRISPR-Cas Abundance at Around 45°C.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {773114},
pmid = {35300480},
issn = {1664-302X},
abstract = {Although performing adaptive immunity, CRISPR-Cas systems are present in only 40% of bacterial genomes. We observed an abrupt increase of bacterial CRISPR-Cas abundance at around 45°C. Phylogenetic comparative analyses confirmed that the abundance correlates with growth temperature only at the temperature range around 45°C. From the literature, we noticed that the diversities of cellular predators (like protozoa, nematodes, and myxobacteria) have a steep decline at this temperature range. The grazing risk faced by bacteria reduces substantially at around 45°C and almost disappears above 60°C. We propose that viral lysis would become the dominating factor of bacterial mortality, and antivirus immunity has a higher priority at higher temperatures. In temperature ranges where the abundance of cellular predators does not change with temperature, the growth temperatures of bacteria would not significantly affect their CRISPR-Cas contents. The hypothesis predicts that bacteria should also be rich in CRISPR-Cas systems if they live in other extreme conditions inaccessible to grazing predators.},
}
@article {pmid35300341,
year = {2022},
author = {Xu, Y and Chen, C and Guo, Y and Hu, S and Sun, Z},
title = {Effect of CRISPR/Cas9-Edited PD-1/PD-L1 on Tumor Immunity and Immunotherapy.},
journal = {Frontiers in immunology},
volume = {13},
number = {},
pages = {848327},
pmid = {35300341},
issn = {1664-3224},
mesh = {Animals ; *B7-H1 Antigen/genetics ; CRISPR-Cas Systems ; Immunologic Factors ; Immunotherapy ; *Neoplasms/genetics/therapy ; Programmed Cell Death 1 Receptor/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease9 (CRISPR/Cas9) gene editing technology implements precise programming of the human genome through RNA guidance. At present, it has been widely used in the construction of animal tumor models, the study of drug resistance regulation mechanisms, epigenetic control and innovation in cancer treatment. Tumor immunotherapy restores the normal antitumor immune response by restarting and maintaining the tumor-immune cycle. CRISPR/Cas9 technology has occupied a central position in further optimizing anti-programmed cell death 1(PD-1) tumor immunotherapy. In this review, we summarize the recent progress in exploring the regulatory mechanism of tumor immune PD-1 and programmed death ligand 1(PD-L1) based on CRISPR/Cas9 technology and its clinical application in different cancer types. In addition, CRISPR genome-wide screening identifies new drug targets and biomarkers to identify potentially sensitive populations for anti-PD-1/PD-L1 therapy and maximize antitumor effects. Finally, the strong potential and challenges of CRISPR/Cas9 for future clinical applications are discussed.},
}
@article {pmid35299835,
year = {2021},
author = {Parra-Flores, J and Holý, O and Bustamante, F and Lepuschitz, S and Pietzka, A and Contreras-Fernández, A and Castillo, C and Ovalle, C and Alarcón-Lavín, MP and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Troncoso, M and Figueroa, G and Ruppitsch, W},
title = {Virulence and Antibiotic Resistance Genes in Listeria monocytogenes Strains Isolated From Ready-to-Eat Foods in Chile.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {796040},
pmid = {35299835},
issn = {1664-302X},
abstract = {Listeria monocytogenes is causing listeriosis, a rare but severe foodborne infection. Listeriosis affects pregnant women, newborns, older adults, and immunocompromised individuals. Ready-to-eat (RTE) foods are the most common sources of transmission of the pathogen This study explored the virulence factors and antibiotic resistance in L. monocytogenes strains isolated from ready-to-eat (RTE) foods through in vitro and in silico testing by whole-genome sequencing (WGS). The overall positivity of L. monocytogenes in RTE food samples was 3.1% and 14 strains were isolated. L. monocytogenes ST8, ST2763, ST1, ST3, ST5, ST7, ST9, ST14, ST193, and ST451 sequence types were identified by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST. Seven isolates had serotype 1/2a, five 1/2b, one 4b, and one 1/2c. Three strains exhibited in vitro resistance to ampicillin and 100% of the strains carried the fosX, lin, norB, mprF, tetA, and tetC resistance genes. In addition, the arsBC, bcrBC, and clpL genes were detected, which conferred resistance to stress and disinfectants. All strains harbored hlyA, prfA, and inlA genes almost thirty-two the showed the bsh, clpCEP, hly, hpt, iap/cwhA, inlA, inlB, ipeA, lspA, mpl, plcA, pclB, oat, pdgA, and prfA genes. One isolate exhibited a type 11 premature stop codon (PMSC) in the inlA gene and another isolate a new mutation (deletion of A in position 819). The Inc18(rep25), Inc18(rep26), and N1011A plasmids and MGEs were found in nine isolates. Ten isolates showed CAS-Type II-B systems; in addition, Anti-CRISPR AcrIIA1 and AcrIIA3 phage-associated systems were detected in three genomes. These virulence and antibiotic resistance traits in the strains isolated in the RTE foods indicate a potential public health risk for consumers.},
}
@article {pmid35298777,
year = {2022},
author = {Zhang, X and Tian, Y and Xu, L and Fan, Z and Cao, Y and Ma, Y and Li, H and Ren, F},
title = {CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection.},
journal = {Hepatology international},
volume = {16},
number = {2},
pages = {306-315},
pmid = {35298777},
issn = {1936-0541},
mesh = {CRISPR-Cas Systems ; DNA, Circular/genetics ; DNA, Viral/analysis/genetics ; *Hepatitis B/diagnosis ; Hepatitis B virus/genetics ; *Hepatitis B, Chronic ; Humans ; Leukocytes, Mononuclear ; Real-Time Polymerase Chain Reaction/methods ; },
abstract = {BACKGROUND AND AIMS: The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology.<br><br>METHODS: We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs).<br><br>RESULTS: Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/&#x3bc;l HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods.<br><br>CONCLUSIONS: We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.},
}
@article {pmid35298666,
year = {2022},
author = {Garrigues, S and Manzanares, P and Marcos, JF},
title = {Application of recyclable CRISPR/Cas9 tools for targeted genome editing in the postharvest pathogenic fungi Penicillium digitatum and Penicillium expansum.},
journal = {Current genetics},
volume = {68},
number = {3-4},
pages = {515-529},
pmid = {35298666},
issn = {1432-0983},
mesh = {CRISPR-Cas Systems ; Fungal Proteins/genetics ; *Gene Editing ; Humans ; *Penicillium/genetics/metabolism ; },
abstract = {Penicillium digitatum and Penicillium expansum are plant pathogenic fungi that cause the green and blue mold diseases, respectively, leading to serious postharvest economic losses worldwide. Moreover, P. expansum can produce mycotoxins, which are hazardous compounds to human and animal health. The development of tools that allow multiple and precise genetic manipulation of these species is crucial for the functional characterization of their genes. In this sense, CRISPR/Cas9 represents an excellent opportunity for genome editing due to its efficiency, accuracy and versatility. In this study, we developed protoplast generation and transformation protocols and applied them to implement the CRISPR/Cas9 technology in both species for the first time. For this, we used a self-replicative, recyclable AMA1-based plasmid which allows unlimited number of genomic modifications without the limitation of integrative selection markers. As test case, we successfully targeted the wetA gene, which encodes a regulator of conidiophore development. Finally, CRISPR/Cas9-derived ΔwetA strains were analyzed. Mutants showed reduced axenic growth, differential pathogenicity and altered conidiogenesis and germination. Additionally, P. digitatum and P. expansum ΔwetA mutants showed distinct sensitivity to fungal antifungal proteins (AFPs), which are small, cationic, cysteine-rich proteins that have become interesting antifungals to be applied in agriculture, medicine and in the food industry. With this work, we demonstrate the feasibility of the CRISPR/Cas9 system, expanding the repertoire of genetic engineering tools available for these two important postharvest pathogens and open up the possibility to adapt them to other economically relevant phytopathogenic fungi, for which toolkits for genetic modifications are often limited.},
}
@article {pmid35298644,
year = {2022},
author = {Bewg, WP and Harding, SA and Engle, NL and Vaidya, BN and Zhou, R and Reeves, J and Horn, TW and Joshee, N and Jenkins, JW and Shu, S and Barry, KW and Yoshinaga, Y and Grimwood, J and Schmitz, RJ and Schmutz, J and Tschaplinski, TJ and Tsai, CJ},
title = {Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.},
journal = {Plant physiology},
volume = {189},
number = {2},
pages = {516-526},
pmid = {35298644},
issn = {1532-2548},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Populus/genetics ; *RNA, Guide/genetics ; Trichomes ; },
abstract = {As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.},
}
@article {pmid35296857,
year = {2022},
author = {Li, XV and Leonardi, I and Putzel, GG and Semon, A and Fiers, WD and Kusakabe, T and Lin, WY and Gao, IH and Doron, I and Gutierrez-Guerrero, A and DeCelie, MB and Carriche, GM and Mesko, M and Yang, C and Naglik, JR and Hube, B and Scherl, EJ and Iliev, ID},
title = {Immune regulation by fungal strain diversity in inflammatory bowel disease.},
journal = {Nature},
volume = {603},
number = {7902},
pages = {672-678},
pmid = {35296857},
issn = {1476-4687},
support = {R37 DE022550/DE/NIDCR NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; F32 DK120228/DK/NIDDK NIH HHS/United States ; R01 DK113136/DK/NIDDK NIH HHS/United States ; R01 DK121977/DK/NIDDK NIH HHS/United States ; R21 AI146957/AI/NIAID NIH HHS/United States ; R01 AI163007/AI/NIAID NIH HHS/United States ; 214229_Z_18_Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Candida albicans ; *Fungi/genetics/pathogenicity ; *Gastrointestinal Microbiome ; Genetic Variation ; Humans ; Immunity ; Inflammation ; *Inflammatory Bowel Diseases ; Mammals ; *Microbiota ; *Mycobiome ; },
abstract = {The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation[1-6]. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease[3-9], it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1β-dependent mechanisms. Niche-specific inflammatory immunity and interleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.},
}
@article {pmid35294876,
year = {2022},
author = {Garipler, G and Lu, C and Morrissey, A and Lopez-Zepeda, LS and Pei, Y and Vidal, SE and Zen Petisco Fiore, AP and Aydin, B and Stadtfeld, M and Ohler, U and Mahony, S and Sanjana, NE and Mazzoni, EO},
title = {The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.},
journal = {Cell reports},
volume = {38},
number = {11},
pages = {110524},
pmid = {35294876},
issn = {2211-1247},
support = {R01 NS100897/NS/NINDS NIH HHS/United States ; DP2 HG010099/HG/NHGRI NIH HHS/United States ; R00 HG008171/HG/NHGRI NIH HHS/United States ; R01 HD079682/HD/NICHD NIH HHS/United States ; T32 GM102057/GM/NIGMS NIH HHS/United States ; R01 GM125722/GM/NIGMS NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Embryonic Stem Cells/metabolism ; Germ Layers/metabolism ; Humans ; Mice ; *Pluripotent Stem Cells/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.},
}
@article {pmid35294852,
year = {2022},
author = {Gao, R and Fu, ZC and Li, X and Wang, Y and Wei, J and Li, G and Wang, L and Wu, J and Huang, X and Yang, L and Chen, J},
title = {Genomic and Transcriptomic Analyses of Prime Editing Guide RNA-Independent Off-Target Effects by Prime Editors.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {276-293},
doi = {10.1089/crispr.2021.0080},
pmid = {35294852},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Mammals/genetics ; RNA/genetics ; *RNA, Guide/genetics ; Transcriptome ; },
abstract = {Prime editors (PEs) were developed to induce versatile edits at a guide-specified genomic locus. With all RNA-guided genome editors, guide-dependent off-target (OT) mutations can occur at other sites bearing similarity to the intended target. However, whether PEs carry the additional risk of guide-independent mutations elicited by their unique enzymatic moiety (i.e., reverse transcriptase) has not been examined systematically in mammalian cells. Here, we developed a cost-effective sensitive platform to profile guide-independent OT effects in human cells. We did not observe guide-independent OT mutations in the DNA or RNA of prime editor 3 (PE3)-edited cells, or alterations to their telomeres, endogenous retroelements, alternative splicing events, or gene expression. Together, our results showed undetectable prime editing guide RNA-independent OT effects of PE3 in human cells, suggesting the high editing specificity of its reverse-transcriptase moiety.},
}
@article {pmid35294186,
year = {2022},
author = {Shi, Y and Zhang, L and Zhang, M and Chu, J and Xia, Y and Yang, H and Liu, L and Chen, X},
title = {A CRISPR-Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1497-1509},
doi = {10.1021/acssynbio.1c00582},
pmid = {35294186},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Saccharomycetales/genetics ; Yeasts/genetics ; },
abstract = {Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola, a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR-Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola. This CRISPR-Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR-Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR-Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola.},
}
@article {pmid35292641,
year = {2022},
author = {Eslami-Mossallam, B and Klein, M and Smagt, CVD and Sanden, KVD and Jones, SK and Hawkins, JA and Finkelstein, IJ and Depken, M},
title = {A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1367},
pmid = {35292641},
issn = {2041-1723},
support = {F32 AG053051/AG/NIA NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Endonucleases/metabolism ; Gene Editing ; RNA, Guide/genetics ; },
abstract = {The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.},
}
@article {pmid35290826,
year = {2022},
author = {Nguyen, LT and Macaluso, NC and Pizzano, BLM and Cash, MN and Spacek, J and Karasek, J and Miller, MR and Lednicky, JA and Dinglasan, RR and Salemi, M and Jain, PK},
title = {A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.},
journal = {EBioMedicine},
volume = {77},
number = {},
pages = {103926},
pmid = {35290826},
issn = {2352-3964},
support = {R21 AI156321/AI/NIAID NIH HHS/United States ; U01 GH002338/GH/CGH CDC HHS/United States ; },
mesh = {*Brevibacillus/genetics ; *COVID-19/diagnosis ; Humans ; RNA, Guide ; SARS-CoV-2/genetics ; },
abstract = {BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs.<br><br>METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs).<br><br>FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92&#xb7;8% sensitivity, 99&#xb7;4% specificity, and 96&#xb7;7% accuracy within 10-30&#xa0;min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99&#xb7;1% and 95&#xb7;1%, respectively. Interestingly, for samples with high viral load (Ct value &#x2264; 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals.<br><br>INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19.<br><br>FUNDING: This project was funded in part by the United States-India Science &amp; Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., &amp; P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).},
}
@article {pmid35290629,
year = {2022},
author = {Chen, BR and Sleckman, BP},
title = {A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2444},
number = {},
pages = {15-27},
pmid = {35290629},
issn = {1940-6029},
support = {R01 AI074953/AI/NIAID NIH HHS/United States ; R01 AI047829/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Mice ; RNA, Guide/genetics ; },
abstract = {DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of HR or NHEJ is dictated in part by whether the broken DNA ends are resected to generate extended single-stranded DNA (ssDNA) overhangs, which are quickly bound by the trimeric ssDNA binding complex RPA, the first step of HR. Here we describe a series of protocols for generating Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9 that can be used to identify and study novel pathways regulating DNA end processing. These approaches involve gene inactivation by CRISPR/Cas9, whole genome guide RNA (gRNA) library-mediated screen, and flow cytometry-based detection of chromatin-bound RPA after DNA damage.},
}
@article {pmid35290572,
year = {2022},
author = {Wei, W and Gao, C},
title = {Gene editing: from technologies to applications in research and beyond.},
journal = {Science China. Life sciences},
volume = {65},
number = {4},
pages = {657-659},
pmid = {35290572},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Technology ; },
}
@article {pmid35289346,
year = {2022},
author = {Zhou, J and Hu, J and Liu, R and Wang, C and Lv, Y},
title = {Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {26},
pages = {4247-4250},
doi = {10.1039/d2cc00792d},
pmid = {35289346},
issn = {1364-548X},
mesh = {Biological Assay ; CRISPR-Cas Systems/genetics ; Gold ; *HIV Infections ; Humans ; *Metal Nanoparticles ; *Nucleic Acids ; },
abstract = {Nucleic acid amplification strategies have successfully dominated ultrasensitive bioassays, but they sometimes bring high time-consumption, multi-step operation, increased contamination risk, and mismatch-related inaccuracy. We proposed a nucleic acid amplification-free method called the AuNPs-tagging based CRISPR-Cas12a bioassay platform. The signal amplification was realized by integrating the self-amplification effect of CRISPR-Cas12a with the enhancement effect of the large number of detectable atoms inside each gold nanoparticle. The proposed method achieved a low LOD of 1.05 amol in 40 min for HIV-related DNA.},
}
@article {pmid35288985,
year = {2022},
author = {Kabay, G and DeCastro, J and Altay, A and Smith, K and Lu, HW and Capossela, AM and Moarefian, M and Aran, K and Dincer, C},
title = {Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {34},
number = {30},
pages = {e2201085},
doi = {10.1002/adma.202201085},
pmid = {35288985},
issn = {1521-4095},
support = {R01 HL139605/GF/NIH HHS/United States ; },
mesh = {Biomarkers ; *Biosensing Techniques/methods ; *Communicable Diseases/diagnosis ; Humans ; Pandemics ; Point-of-Care Systems ; },
abstract = {Several viral infectious diseases appear limitless since the beginning of the 21[st] century, expanding into pandemic lengths. Thus, there are extensive efforts to provide more efficient means of diagnosis, a better understanding of acquired immunity, and improved monitoring of inflammatory biomarkers, as these are all crucial for controlling the spread of infection while aiding in vaccine development and improving patient outcomes. In this regard, various biosensors have been developed recently to streamline pathogen and immune response detection by addressing the limitations of traditional methods, including isothermal amplification-based systems and lateral flow assays. This review explores state-of-the-art biosensors for detecting viral pathogens, serological assays, and inflammatory biomarkers from the material perspective, by discussing their advantages, limitations, and further potential regarding their analytical performance, clinical utility, and point-of-care adaptability. Additionally, next-generation biosensing technologies that offer better sensitivity and selectivity, and easy handling for end-users are highlighted. An emerging example of these next-generation biosensors are those powered by novel synthetic biology tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated proteins (Cas), in combination with integrated point-of-care devices. Lastly, the current challenges are discussed and a roadmap for furthering these advanced biosensing technologies to manage future pandemics is provided.},
}
@article {pmid35288718,
year = {2022},
author = {Kaltenbacher, T and Löprich, J and Maresch, R and Weber, J and Müller, S and Oellinger, R and Groß, N and Griger, J and de Andrade Krätzig, N and Avramopoulos, P and Ramanujam, D and Brummer, S and Widholz, SA and Bärthel, S and Falcomatà, C and Pfaus, A and Alnatsha, A and Mayerle, J and Schmidt-Supprian, M and Reichert, M and Schneider, G and Ehmer, U and Braun, CJ and Saur, D and Engelhardt, S and Rad, R},
title = {CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver.},
journal = {Nature protocols},
volume = {17},
number = {4},
pages = {1142-1188},
pmid = {35288718},
issn = {1750-2799},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Liver ; Mice ; Mice, Knockout ; *Neoplasms/genetics ; Pancreas ; },
abstract = {Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.},
}
@article {pmid35288688,
year = {2022},
author = {Shaffer, C},
title = {Broad defeats Berkeley CRISPR patent.},
journal = {Nature biotechnology},
volume = {40},
number = {4},
pages = {445},
doi = {10.1038/d41587-022-00004-2},
pmid = {35288688},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35288582,
year = {2022},
author = {Biegler, MT and Fedrigo, O and Collier, P and Mountcastle, J and Haase, B and Tilgner, HU and Jarvis, ED},
title = {Induction of an immortalized songbird cell line allows for gene characterization and knockout by CRISPR-Cas9.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {4369},
pmid = {35288582},
issn = {2045-2322},
support = {R01 GM135247/GM/NIGMS NIH HHS/United States ; RF1 MH121267/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U01 DA053625/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Finches/genetics ; Genome ; Genomics ; },
abstract = {The zebra finch is one of the most commonly studied songbirds in biology, particularly in genomics, neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. We generated a cell line, designated CFS414, from zebra finch embryonic fibroblasts using the SV40 large and small T antigens. This cell line demonstrates an improvement over previous songbird cell lines through continuous and density-independent growth, allowing for indefinite culture and monoclonal line derivation. Cytogenetic, genomic, and transcriptomic profiling established the provenance of this cell line and identified the expression of genes relevant to ongoing songbird research. Using this cell line, we disrupted endogenous gene sequences using S.aureus Cas9 and confirmed a stress-dependent localization response of a song system specialized gene, SAP30L. The utility of CFS414 cells enhances the comprehensive molecular potential of the zebra finch and validates cell immortalization strategies in a songbird species.},
}
@article {pmid35288574,
year = {2022},
author = {Sangree, AK and Griffith, AL and Szegletes, ZM and Roy, P and DeWeirdt, PC and Hegde, M and McGee, AV and Hanna, RE and Doench, JG},
title = {Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1318},
pmid = {35288574},
issn = {2041-1723},
mesh = {Benchmarking ; *CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Proto-Oncogene Proteins c-bcl-2/genetics ; },
abstract = {Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.},
}
@article {pmid35288457,
year = {2022},
author = {Ding, L and Schmitt, LT and Brux, M and Sürün, D and Augsburg, M and Lansing, F and Mircetic, J and Theis, M and Buchholz, F},
title = {DNA methylation-independent long-term epigenetic silencing with dCRISPR/Cas9 fusion proteins.},
journal = {Life science alliance},
volume = {5},
number = {6},
pages = {},
pmid = {35288457},
issn = {2575-1077},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *DNA Methylation/genetics ; Epigenesis, Genetic/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Mice ; RNA, Guide/genetics ; },
abstract = {The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand. Here, we show that expression of promoter-associated short guide (sg)RNAs together with dead Cas9 (dCas9) fused to a Krüppel-associated box domains (KRABd) in combination with the transcription repression domain of methyl CpG-binding protein 2 (MeCP2) can lead to persistent gene silencing in mouse embryonic stem cells and in human embryonic kidney (HEK) 293 cells. Surprisingly, this effect is achievable and even enhanced in DNA (cytosine-5)-methyltransferase 3A and 3B (Dnmt3A[-/-], Dnmt3b[-/-]) depleted cells. Our results suggest that dCas9-KRABd-MeCP2 fusions are useful for long-term epigenetic gene silencing with utility in cell biology and potentially in therapeutical settings.},
}
@article {pmid35288230,
year = {2022},
author = {Zheng, SY and Ma, LL and Wang, XL and Lu, LX and Ma, ST and Xu, B and Ouyang, W},
title = {RPA-Cas12aDS: A visual and fast molecular diagnostics platform based on RPA-CRISPR-Cas12a method for infectious bursal disease virus detection.},
journal = {Journal of virological methods},
volume = {304},
number = {},
pages = {114523},
doi = {10.1016/j.jviromet.2022.114523},
pmid = {35288230},
issn = {1879-0984},
mesh = {Animals ; *Birnaviridae Infections/diagnosis/veterinary ; CRISPR-Cas Systems ; Chickens ; *Infectious bursal disease virus/genetics ; Nucleic Acid Amplification Techniques/methods ; Pathology, Molecular ; *Poultry Diseases/diagnosis ; Recombinases/genetics ; },
abstract = {Infectious bursal disease (IBD), a major disease of birds, is caused by infectious bursal disease virus (IBDV). The disease can lead to immunosuppression, resulting in huge economic losses in the poultry industry. A specific, rapid, and simple detection method is important for the early diagnosis and prevention and control of IBDV. In this study, we established a naked-eye visual IBDV detection method, named "RPA-Cas12aDS", by combining recombinase polymerase amplification (RPA) with CRISPR-Cas12a-based nucleic acid detection. The detection process can be accomplished in 50 min, and uncapping contamination can be avoided. The detection results can be observed under blue or UV light. We used the RPA-Cas12aDS method to detect IBDV in bursa of Fabricius tissue samples of chickens, and the results were consistent with those obtained using commercial RT-PCR kits. This method presents great potential for visual, rapid, and point-of-care molecular diagnostics of IBDV in poultry.},
}
@article {pmid35286975,
year = {2022},
author = {Hommersom, MP and Bijnagte-Schoenmaker, C and Albert, S and van de Warrenburg, BPC and Nadif Kasri, N and van Bokhoven, H},
title = {Generation of induced pluripotent stem cell lines carrying monoallelic (UCSFi001-A-60) or biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {61},
number = {},
pages = {102730},
doi = {10.1016/j.scr.2022.102730},
pmid = {35286975},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Calcium Channels/metabolism ; Cells, Cultured ; Frameshift Mutation ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {CACNA1A encodes a P/Q-type voltage-gated calcium channel. Heterozygous loss-of-function variants in this gene have been associated with episodic ataxia type 2. In this study, we used CRISPR/Cas9 to generate isogenic human induced pluripotent stem cell lines with a gene-dosage dependent deficiency of CACNA1A. We obtained one clone with monoallelic (UCSFi001-A-60) and two clones with biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A. All three lines showed expression of pluripotency markers and a normal karyotype.},
}
@article {pmid35286377,
year = {2022},
author = {Pallaseni, A and Peets, EM and Koeppel, J and Weller, J and Vanderstichele, T and Ho, UL and Crepaldi, L and van Leeuwen, J and Allen, F and Parts, L},
title = {Predicting base editing outcomes using position-specific sequence determinants.},
journal = {Nucleic acids research},
volume = {50},
number = {6},
pages = {3551-3564},
pmid = {35286377},
issn = {1362-4962},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adenine ; *CRISPR-Cas Systems ; Cytosine/metabolism ; *Gene Editing ; Humans ; Nucleotides ; },
abstract = {CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window. The impact of features on editing rate depends on the position, with sequence bias efficacy mainly influencing bases away from the center of the window. We use these observations to train a machine learning model to predict editing activity per position, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of disease mutation correcting guides, and find that most of them suffer from more unwanted editing than pure outcomes. This work unravels the position-specificity of base editing biases and allows more efficient planning of editing campaigns in experimental and therapeutic contexts.},
}
@article {pmid35286371,
year = {2022},
author = {Xiong, X and Li, Z and Liang, J and Liu, K and Li, C and Li, JF},
title = {A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants.},
journal = {Nucleic acids research},
volume = {50},
number = {6},
pages = {3565-3580},
pmid = {35286371},
issn = {1362-4962},
mesh = {Adenine ; Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing ; },
abstract = {CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.},
}
@article {pmid35285719,
year = {2022},
author = {Xu, X and Harvey-Samuel, T and Siddiqui, HA and Ang, JX and Anderson, ME and Reitmayer, CM and Lovett, E and Leftwich, PT and You, M and Alphey, L},
title = {Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {224-236},
doi = {10.1089/crispr.2021.0129},
pmid = {35285719},
issn = {2573-1602},
support = {BB/S506680/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Gene Editing ; Larva/genetics/metabolism ; Mice ; *Moths/genetics ; },
abstract = {Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.},
}
@article {pmid35285707,
year = {2022},
author = {Parcey, M and Gayder, S and Castle, AJ and Svircev, AM},
title = {Function and Application of the CRISPR-Cas System in the Plant Pathogen Erwinia amylovora.},
journal = {Applied and environmental microbiology},
volume = {88},
number = {7},
pages = {e0251321},
pmid = {35285707},
issn = {1098-5336},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; *Erwinia amylovora/genetics ; Plasmids/genetics ; Streptomycin ; },
abstract = {Phage-based biocontrol is an emerging method for managing the plant pathogen Erwinia amylovora. Control of E. amylovora in North America is achieved chiefly through the application of streptomycin and has led to the development of streptomycin resistance. Resistant E. amylovora can be tracked through the analysis of CRISPR spacer sequences. An alternative to antibiotics are bacterial viruses, known as phages, which lyse their hosts during replication to control the bacterial population. Endogenous CRISPR-Cas systems act as phage resistance mechanisms however, preliminary genomic analysis suggests this activity is limited in E. amylovora. This leaves the functionality of the CRISPR-Cas system, any clade-based differences, and the impact which this system may have on phage-based biocontrol in question. In this study, the CRISPR arrays from 127 newly available genomic sequences of E. amylovora were analyzed through a novel bioinformatic pipeline. Through this, the Eastern and Western North American clades were shown to be incompatible with the current PCR-based approaches for tracking E. amylovora given the size and composition of their CRISPR arrays. Two artificial CRISPR arrays were designed to investigate the functionality of the CRISPR-Cas system in E. amylovora. This system was capable of curing a targeted plasmid and providing phage resistance but was not the source of phage resistance observed within the controls. This suggests that while the CRISPR-Cas system is an important defense mechanism for invasive plasmids, an as yet unidentified mechanism is the primary source of phage resistance in E. amylovora. IMPORTANCE Erwinia amylovora is an economically significant agricultural pathogen found throughout the world. In North America, E. amylovora has developed streptomycin resistance and therefore alternative treatments using phages have received increased attention. In this study, we analyzed recently published genomes to determine that two significant groups of E. amylovora are poorly identified using the current, CRISPR-based tracking methods. We also showed that the CRISPR-Cas system and an unidentified mechanism work together to provide a significant degree of resistance against one of the phages proposed for phage-based biocontrol.},
}
@article {pmid35284843,
year = {2022},
author = {Aouida, M and Aljogol, D and Ali, R and Ramotar, D},
title = {A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101216},
pmid = {35284843},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Humans ; Peroxiredoxins/genetics ; *RNA, Guide/genetics ; Transfection ; },
abstract = {Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.},
}
@article {pmid35284835,
year = {2022},
author = {Oh, S and Buisson, R},
title = {A digital PCR-based protocol to detect and quantify RNA editing events at hotspots.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101148},
pmid = {35284835},
issn = {2666-1667},
support = {P30 CA062203/CA/NCI NIH HHS/United States ; R00 CA212154/CA/NCI NIH HHS/United States ; R37 CA252081/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cytidine Deaminase ; *Gene Editing/methods ; Humans ; Polymerase Chain Reaction ; Proteins ; RNA ; *RNA Editing/genetics ; },
abstract = {APOBEC3A, CRISPR programmable RNA base editors, or other enzymes can edit RNA transcripts at specific locations or hotspots. Precise quantification of these RNA-editing events is crucial to determine the activity and efficiency of these enzymes in cells. We have developed a quick method to quantify RNA-editing activity using digital PCR, a sensitive and quantitative technique to detect rare mutations by micro-partitioning bulk PCR reactions. This assay allows rapid absolute quantification of RNA editing events in cell lines or patient samples. For complete details on the use and execution of this protocol, please refer to Jalili et al. (2020) and Oh et al. (2021).},
}
@article {pmid35284406,
year = {2022},
author = {Chen, B and Li, Y and Xu, F and Yang, X},
title = {Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {851712},
pmid = {35284406},
issn = {2296-4185},
abstract = {In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.},
}
@article {pmid35283690,
year = {2022},
author = {Ivanova, E},
title = {How Various Drug Delivery Methods Could Aid in the Translation of Genome Prime Editing Technologies.},
journal = {Genetics research},
volume = {2022},
number = {},
pages = {7301825},
pmid = {35283690},
issn = {1469-5073},
mesh = {*CRISPR-Cas Systems ; Drug Delivery Systems ; *Gene Editing/methods ; Pharmaceutical Preparations ; Technology ; },
abstract = {Drug delivery systems can be engineered to enhance the localization of therapeutics in specific tissues in response to externally applied stimuli and/or local environmental changes. In recent decades, efforts to improve drug delivery techniques at both nano- and macroscale have led to a new era of therapeutic efficacy. Such technological advancements resulted in improved drug delivery systems regularly entering the clinical setting. However, these delivery innovations are unfortunately not always readily applied to newly developed technologies. One of these new and exciting technologies that has been overlooked by drug delivery scientists is prime editing. Prime editing is a novel genome editing technology that exhibits the plug-and-play capability of CRISPR/Cas9 editors while avoiding double-strand DNA breaks throughout the entire process. This article focuses on describing the potential advantages and disadvantages of selecting nanomedicine technologies along with prime editing capabilities for the delivery of cargo.},
}
@article {pmid35281441,
year = {2022},
author = {Garre, V},
title = {Recent Advances and Future Directions in the Understanding of Mucormycosis.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {850581},
pmid = {35281441},
issn = {2235-2988},
mesh = {Antifungal Agents/therapeutic use ; *COVID-19 ; Genomics ; Humans ; *Mucorales/genetics ; *Mucormycosis/diagnosis/microbiology/pathology ; },
abstract = {Mucormycosis is an emerging infection caused by fungi of the order Mucorales that has recently gained public relevance due to the high incidence among COVID-19 patients in some countries. The reduced knowledge about Mucorales pathogenesis is due, in large part, to the historically low interest for these fungi fostered by their reluctance to be genetically manipulated. The recent introduction of more tractable genetic models together with an increasing number of available whole genome sequences and genomic analyses have improved our understanding of Mucorales biology and mucormycosis in the last ten years. This review summarizes the most significant advances in diagnosis, understanding of the innate and acquired resistance to antifungals, identification of new virulence factors and molecular mechanisms involved in the infection. The increased awareness about the disease and the recent successful genetic manipulation of previous intractable fungal models using CRISPR-Cas9 technology are expected to fuel the characterization of Mucorales pathogenesis, facilitating the development of effective treatments to fight this deadly infection.},
}
@article {pmid35279545,
year = {2022},
author = {de Toledo, MAS and Fu, X and Kluge, F and Götz, K and Schmitz, S and Wanek, P and Schüler, HM and Pannen, K and Chatain, N and Koschmieder, S and Brümmendorf, TH and Zenke, M},
title = {CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102732},
doi = {10.1016/j.scr.2022.102732},
pmid = {35279545},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Heterozygote ; Homozygote ; *Human Embryonic Stem Cells/metabolism ; Humans ; },
abstract = {The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.},
}
@article {pmid35278851,
year = {2022},
author = {Xie, Z and Chen, S and Zhang, W and Zhao, S and Zhao, Z and Wang, X and Huang, Y and Yi, G},
title = {A novel fluorescence amplification strategy combining cascade primer exchange reaction with CRISPR/Cas12a system for ultrasensitive detection of RNase H activity.},
journal = {Biosensors &amp; bioelectronics},
volume = {206},
number = {},
pages = {114135},
doi = {10.1016/j.bios.2022.114135},
pmid = {35278851},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; Humans ; Nucleic Acid Amplification Techniques/methods ; RNA ; Ribonuclease H ; },
abstract = {Ribonuclease H (RNase H), which plays a vital role in various cellular processes, is to be closely related to the emergence of many diseases. As an essential therapeutic target, it shows great prospects in the development of associated drugs. Herein, a DNA-RNA chimeric hairpin (DR HP) was designed to introduce a new signal amplification strategy based on cascade primer exchange reaction (cPER) and CRISPR/Cas12a system for sensitive and specific analysis of RNase H activity. In the presence of RNase H, the RNA fragment of DR HP was specifically degraded and the blocked primer DNA was released. The process of enzymatic hydrolysis of substrate hairpin and cyclic signal amplification was completed in a one-step method under isothermal conditions, enriching many activator strands to initiate trans-cleavage of CRISPR/Cas system, thereby restoring the fluorescence signal. Under optimized conditions, the developed strategy exhibited a good linear relationship ranging from 0.005 to 0.1U/mL and offered a detection limit of 0.00061U/mL. Moreover, this method was used for RNase H activity assay in complicated human serum and real cell lysates with good stability and repeatability, and was also demonstrated to apply for RNase H inhibitors screening and inhibitory capability assessment. Therefore, the proposed system is a promising platform not only for determination of RNase H activity, but open up new thoughts for the biological enzyme research and inhibitor screening.},
}
@article {pmid35277676,
year = {2022},
author = {Mullard, A},
title = {Proof-of-principle Intellia trial shows in vivo CRISPR activity.},
journal = {Nature reviews. Drug discovery},
volume = {21},
number = {4},
pages = {249},
doi = {10.1038/d41573-022-00050-0},
pmid = {35277676},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; },
}
@article {pmid35277177,
year = {2022},
author = {Lan, T and Que, H and Luo, M and Zhao, X and Wei, X},
title = {Genome editing via non-viral delivery platforms: current progress in personalized cancer therapy.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {71},
pmid = {35277177},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genes, Neoplasm ; Genetic Therapy ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer is a severe disease that substantially jeopardizes global health. Although considerable efforts have been made to discover effective anti-cancer therapeutics, the cancer incidence and mortality are still growing. The personalized anti-cancer therapies present themselves as a promising solution for the dilemma because they could precisely destroy or fix the cancer targets based on the comprehensive genomic analyses. In addition, genome editing is an ideal way to implement personalized anti-cancer therapy because it allows the direct modification of pro-tumor genes as well as the generation of personalized anti-tumor immune cells. Furthermore, non-viral delivery system could effectively transport genome editing tools (GETs) into the cell nucleus with an appreciable safety profile. In this manuscript, the important attributes and recent progress of GETs will be discussed. Besides, the laboratory and clinical investigations that seek for the possibility of combining non-viral delivery systems with GETs for the treatment of cancer will be assessed in the scope of personalized therapy.},
}
@article {pmid35276091,
year = {2022},
author = {Simkin, D and Papakis, V and Bustos, BI and Ambrosi, CM and Ryan, SJ and Baru, V and Williams, LA and Dempsey, GT and McManus, OB and Landers, JE and Lubbe, SJ and George, AL and Kiskinis, E},
title = {Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.},
journal = {Stem cell reports},
volume = {17},
number = {4},
pages = {993-1008},
pmid = {35276091},
issn = {2213-6711},
support = {P30 AR075049/AR/NIAMS NIH HHS/United States ; R21 NS125503/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Quality Control ; },
abstract = {The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.},
}
@article {pmid35271371,
year = {2022},
author = {Dominguez, AA and Chavez, MG and Urke, A and Gao, Y and Wang, L and Qi, LS},
title = {CRISPR-Mediated Synergistic Epigenetic and Transcriptional Control.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {264-275},
pmid = {35271371},
issn = {2573-1602},
support = {U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenomics ; Gene Editing ; Gene Expression Regulation/genetics ; },
abstract = {Targeted activation of endogenous genes is an important approach for cell engineering. Here, we report that the nuclease-deactivated dCas9 fused to a transcriptional activator (VPR) and an epigenetic effector (the catalytic domain of histone acetyltransferase p300[core]) simultaneously, sequentially, or as a single quadripartite effector can lead to enhanced activation of target genes. The composite activator, VPRP, behaves more efficiently than individual activators across a set of genes in different cell types. We characterize off-target effects for host chromatin acetylation and transcriptome using the effectors. Our work demonstrates that transcriptional and epigenetic effectors can be used together to enhance gene activation and suggests the need for further optimization of epigenetic effectors to reduce off-targets.},
}
@article {pmid35271311,
year = {2022},
author = {Cho, NH and Cheveralls, KC and Brunner, AD and Kim, K and Michaelis, AC and Raghavan, P and Kobayashi, H and Savy, L and Li, JY and Canaj, H and Kim, JYS and Stewart, EM and Gnann, C and McCarthy, F and Cabrera, JP and Brunetti, RM and Chhun, BB and Dingle, G and Hein, MY and Huang, B and Mehta, SB and Weissman, JS and Gómez-Sjöberg, R and Itzhak, DN and Royer, LA and Mann, M and Leonetti, MD},
title = {OpenCell: Endogenous tagging for the cartography of human cellular organization.},
journal = {Science (New York, N.Y.)},
volume = {375},
number = {6585},
pages = {eabi6983},
pmid = {35271311},
issn = {1095-9203},
support = {F31 HL143882/HL/NHLBI NIH HHS/United States ; R01 GM131641/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Cluster Analysis ; Datasets as Topic ; Fluorescent Dyes ; HEK293 Cells ; Humans ; Immunoprecipitation ; Machine Learning ; Mass Spectrometry ; Microscopy, Confocal ; *Protein Interaction Mapping ; Proteins/*metabolism ; Proteome/*metabolism ; Proteomics/*methods ; RNA-Binding Proteins/metabolism ; Spatial Analysis ; },
abstract = {Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.},
}
@article {pmid35271248,
year = {2022},
author = {Bernhards, CB and Liem, AT and Berk, KL and Roth, PA and Gibbons, HS and Lux, MW},
title = {Putative Phenotypically Neutral Genomic Insertion Points in Prokaryotes.},
journal = {ACS synthetic biology},
volume = {11},
number = {4},
pages = {1681-1685},
pmid = {35271248},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Genomics ; Software ; },
abstract = {The barriers to effective genome editing in diverse prokaryotic organisms have been falling at an accelerated rate. As editing becomes easier in more organisms, quickly identifying genomic locations to insert new genetic functions without disrupting organism fitness becomes increasingly useful. When the insertion is noncoding DNA for applications such as information storage or barcoding, a neutral insertion point can be especially important. Here we describe an approach to identify putatively neutral insertion sites in prokaryotes. An algorithm (targetFinder) finds convergently transcribed genes with gap sizes within a specified range, and looks for annotations within the gaps. We report putative editing targets for 10 common synthetic biology chassis organisms, including coverage of available RNA-seq data, and provide software to apply to others. We further experimentally evaluate the neutrality of six identified targets in Escherichia coli through insertion of a DNA barcode. We anticipate this information and the accompanying tool will prove useful for synthetic biologists seeking neutral insertion points for genome editing.},
}
@article {pmid35270040,
year = {2022},
author = {Oh, HJ and Chung, E and Kim, J and Kim, MJ and Kim, GA and Lee, SH and Ra, K and Eom, K and Park, S and Chae, JH and Kim, JS and Lee, BC},
title = {Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study.},
journal = {International journal of molecular sciences},
volume = {23},
number = {5},
pages = {},
pmid = {35270040},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dogs ; *Dystrophin/genetics/metabolism ; Gene Editing ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Nuclear Transfer Techniques ; },
abstract = {Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.},
}
@article {pmid35269691,
year = {2022},
author = {Sato, M and Nakamura, S and Inada, E and Takabayashi, S},
title = {Recent Advances in the Production of Genome-Edited Rats.},
journal = {International journal of molecular sciences},
volume = {23},
number = {5},
pages = {},
pmid = {35269691},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Female ; Gene Editing/methods ; Genome/genetics ; Humans ; *Nucleic Acids ; Rats ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.},
}
@article {pmid35269602,
year = {2022},
author = {Song, H and Ahn, JY and Yan, F and Ran, Y and Koo, O and Lee, GJ},
title = {Genetic Dissection of CRISPR-Cas9 Mediated Inheritance of Independently Targeted Alleles in Tobacco α-1,3-Fucosyltransferase 1 and β-1,2-Xylosyltransferase 1 Loci.},
journal = {International journal of molecular sciences},
volume = {23},
number = {5},
pages = {},
pmid = {35269602},
issn = {1422-0067},
mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; Fucosyltransferases ; Gene Editing/methods ; Genes, Plant ; Humans ; Mutation ; Pentosyltransferases ; Plants, Genetically Modified/genetics ; *Tobacco/genetics ; },
abstract = {We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and β-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T0 were further segregated into chimeric progeny (37-54%) in the next generation, whereas homozygous T0 mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T1 generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T0 generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T2 generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a -4 bp deletion in NbXylT1 was found to produce T2 progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis.},
}
@article {pmid35269584,
year = {2022},
author = {Koo, CZ and Matthews, AL and Harrison, N and Szyroka, J and Nieswandt, B and Gardiner, EE and Poulter, NS and Tomlinson, MG},
title = {The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors.},
journal = {International journal of molecular sciences},
volume = {23},
number = {5},
pages = {},
pmid = {35269584},
issn = {1422-0067},
support = {FS/18/9/33388/BHF_/British Heart Foundation/United Kingdom ; BB/P00783X/1 and MIBTP PhD Studentships/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {ADAM10 Protein/*metabolism ; Amyloid Precursor Protein Secretases/*metabolism ; Blood Platelets/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Membrane Proteins/*metabolism ; Platelet Membrane Glycoproteins/*genetics/metabolism ; Protein Domains ; Proteolysis ; Substrate Specificity ; Tetraspanins/chemistry/*genetics/*metabolism ; },
abstract = {The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a 'molecular scissor' that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.},
}
@article {pmid35269459,
year = {2022},
author = {Freudenberg, RA and Wittemeier, L and Einhaus, A and Baier, T and Kruse, O},
title = {The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing.},
journal = {Cells},
volume = {11},
number = {5},
pages = {},
pmid = {35269459},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/genetics ; *Chlamydomonas reinhardtii/genetics/metabolism ; *Gene Editing/methods ; Spermidine/metabolism ; Spermidine Synthase/genetics/metabolism ; },
abstract = {Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.},
}
@article {pmid35269429,
year = {2022},
author = {Parain, K and Lourdel, S and Donval, A and Chesneau, A and Borday, C and Bronchain, O and Locker, M and Perron, M},
title = {CRISPR/Cas9-Mediated Models of Retinitis Pigmentosa Reveal Differential Proliferative Response of Müller Cells between Xenopus laevis and Xenopus tropicalis.},
journal = {Cells},
volume = {11},
number = {5},
pages = {},
pmid = {35269429},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Ependymoglial Cells/metabolism ; Retinal Rod Photoreceptor Cells/metabolism ; *Retinitis Pigmentosa/metabolism ; *Rhodopsin/genetics/metabolism ; Xenopus laevis/genetics/metabolism ; },
abstract = {Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones. Rhodopsin is the most frequently mutated gene in this disease. We here developed rhodopsin gene editing-based models of retinitis pigmentosa in two Xenopus species, Xenopus laevis and Xenopus tropicalis, by using CRISPR/Cas9 technology. In both of them, loss of rhodopsin function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in Xenopus laevis and Xenopus tropicalis. While a significant proportion of Müller cells re-enter into the cell cycle in Xenopus laevis, their proliferation remains extremely limited in Xenopus tropicalis. This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.},
}
@article {pmid35266687,
year = {2022},
author = {Mahas, A and Wang, Q and Marsic, T and Mahfouz, MM},
title = {Development of Cas12a-Based Cell-Free Small-Molecule Biosensors via Allosteric Regulation of CRISPR Array Expression.},
journal = {Analytical chemistry},
volume = {94},
number = {11},
pages = {4617-4626},
pmid = {35266687},
issn = {1520-6882},
mesh = {Allosteric Regulation ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Single-Stranded ; },
abstract = {Cell-free biosensors can detect various molecules, thus promising to transform the landscape of diagnostics. Here, we developed a simple, rapid, sensitive, and field-deployable small-molecule detection platform based on allosteric transcription factor (aTF)-regulated expression of a clustered regularly interspaced short palindromic repeats (CRISPR) array coupled to Cas12a activity. To this end, we engineered an expression cassette harboring a T7 promoter, an aTF binding sequence, a Cas12a CRISPR array, and protospacer adjacent motif-flanked Cas12a target sequences. In the presence of the ligand, dissociation of the aTF allows transcription of the CRISPR array; this leads to activation of Cas12a collateral activity, which cleaves a single-stranded DNA linker to free a quenched fluorophore, resulting in a rapid, significant increase of fluorescence. As a proof of concept, we used TetR as the aTF to detect different tetracycline antibiotics with high sensitivity and specificity and a simple, hand-held visualizer to develop a fluorescence-based visual readout. We also adapted a mobile phone application to further simplify the interpretation of the results. Finally, we showed that the reagents could be lyophilized to facilitate storage and distribution. This detection platform represents a valuable addition to the toolbox of cell-free, CRISPR-based biosensors, with great potential for in-field deployment to detect non-nucleic acid small molecules.},
}
@article {pmid35266285,
year = {2022},
author = {Nayeri, S and Baghban Kohnehrouz, B and Ahmadikhah, A and Mahna, N},
title = {CRISPR/Cas9-mediated P-CR domain-specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar.},
journal = {Plant biotechnology journal},
volume = {20},
number = {6},
pages = {1197-1212},
pmid = {35266285},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Wall/genetics/metabolism ; Cellulose/metabolism ; Glucosyltransferases/genetics ; *Populus/genetics/metabolism ; },
abstract = {Cellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high-value bioproducts. The large transmembrane-localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para-crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant-conserved region (P-CR) of PalCESA4 subunit, which are involved in the CESA4-CESA8 heterodimerization. The CRISPR/Cas9-knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4-binding affinity resulting in normal roots, 1.5-2-fold higher plant growth and cell wall regeneration rates, 1.7-fold thinner cell wall, high hemicellulose content, 37%-67% decrease in cellulose content, high cellulose DP, 25%-37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2-fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9-mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose-based nanomaterial technologies.},
}
@article {pmid35265984,
year = {2022},
author = {Cremanns, M and Lange, F and Gatermann, SG and Pfennigwerth, N},
title = {Effect of sigma E on carbapenem resistance in OXA-48-producing Klebsiella pneumoniae.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {77},
number = {6},
pages = {1578-1585},
doi = {10.1093/jac/dkac078},
pmid = {35265984},
issn = {1460-2091},
mesh = {Anti-Bacterial Agents/metabolism/pharmacology ; Bacterial Proteins/metabolism ; Carbapenems/metabolism/pharmacology ; Cephalosporins/pharmacology ; Humans ; *Klebsiella Infections/microbiology ; *Klebsiella pneumoniae ; Meropenem/pharmacology ; Microbial Sensitivity Tests ; Porins/genetics/metabolism ; beta-Lactamases/metabolism ; },
abstract = {OBJECTIVES: Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria.<br><br>METHODS: Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels.<br><br>RESULTS: Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; &#x3c3;E) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE.<br><br>CONCLUSIONS: Enhanced synthesis of &#x3c3;E leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated &#x3c3;E stress response also leads to a significant reduction in growth rates.},
}
@article {pmid35265606,
year = {2022},
author = {Shi, Y and Kang, L and Mu, R and Xu, M and Duan, X and Li, Y and Yang, C and Ding, JW and Wang, Q and Li, S},
title = {CRISPR/Cas12a-Enhanced Loop-Mediated Isothermal Amplification for the Visual Detection of Shigella flexneri.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {10},
number = {},
pages = {845688},
pmid = {35265606},
issn = {2296-4185},
abstract = {Shigella flexneri is a serious threat to global public health, and a rapid detection method is urgently needed. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and in vitro diagnosis. Here, we combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a to develop a novel diagnostic test (CRISPR/Cas12a-E-LAMP) for the diagnosis of S. flexneri. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for S. flexneri templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene hypothetical protein (accession: AE014073, region: 4170556-4171,068) of S. flexneri. After the LAMP reaction at 60°C for 20 min, the pre-loaded CRISPR/Cas12a regents were mixed with the LAMP products in one tube at 37°C for 20 min, and the final results can be viewed by naked eyes with a total time of 40 min. The sensitivity of CRISPR/Cas12a-E-LAMP to detect S. flexneri was 4 × 10[0] copies/μl plasmids and without cross-reaction with other six closely related non-S. flexneri. Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of S. flexneri and may be applied in other pathogen infection detection.},
}
@article {pmid35264807,
year = {2022},
author = {Ledford, H},
title = {Major CRISPR patent decision won't end tangled dispute.},
journal = {Nature},
volume = {603},
number = {7901},
pages = {373-374},
doi = {10.1038/d41586-022-00629-y},
pmid = {35264807},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Dissent and Disputes ; Gene Editing ; },
}
@article {pmid35264732,
year = {2022},
author = {},
title = {Fine-tuning epigenome editors.},
journal = {Nature biotechnology},
volume = {40},
number = {3},
pages = {281},
doi = {10.1038/s41587-022-01270-w},
pmid = {35264732},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; *Epigenome ; *Gene Editing ; },
}
@article {pmid35264460,
year = {2022},
author = {Christian, ML and Dapp, MJ and Scharffenberger, SC and Jones, H and Song, C and Frenkel, LM and Krumm, A and Mullins, JI and Rawlings, DJ},
title = {CRISPR/Cas9-Mediated Insertion of HIV Long Terminal Repeat within BACH2 Promotes Expansion of T Regulatory-like Cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {208},
number = {7},
pages = {1700-1710},
pmid = {35264460},
issn = {1550-6606},
support = {R01 DA040532/DA/NIDA NIH HHS/United States ; R61 DA047010/DA/NIDA NIH HHS/United States ; R01 CA206466/CA/NCI NIH HHS/United States ; R33 AI122361/AI/NIAID NIH HHS/United States ; R01 AI134419/AI/NIAID NIH HHS/United States ; R21 AI122361/AI/NIAID NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI125026/AI/NIAID NIH HHS/United States ; },
mesh = {Basic-Leucine Zipper Transcription Factors/genetics ; *CRISPR-Cas Systems ; HIV Long Terminal Repeat/genetics ; *HIV-1/genetics ; Humans ; Virus Integration ; },
abstract = {One key barrier to curative therapies for HIV is the limited understanding of HIV persistence. HIV provirus integration sites (ISs) within BACH2 are common, and almost all sites mapped to date are located upstream of the start codon in the same transcriptional orientation as the gene. These unique features suggest the possibility of insertional mutagenesis at this location. Using CRISPR/Cas9-based homology-directed repair in primary human CD4[+] T cells, we directly modeled the effects of HIV integration within BACH2 Integration of the HIV long terminal repeat (LTR) and major splice donor increased BACH2 mRNA and protein levels, altered gene expression, and promoted selective outgrowth of an activated, proliferative, and T regulatory-like cell population. In contrast, introduction of the HIV-LTR alone or an HIV-LTR-major splice donor construct into STAT5B, a second common HIV IS, had no functional impact. Thus, HIV LTR-driven BACH2 expression modulates T cell programming and leads to cellular outgrowth and unique phenotypic changes, findings that support a direct role for IS-dependent HIV-1 persistence.},
}
@article {pmid35263584,
year = {2022},
author = {Tian, S and Liu, Y and Appleton, E and Wang, H and Church, GM and Dong, M},
title = {Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms.},
journal = {Cell reports},
volume = {38},
number = {10},
pages = {110476},
pmid = {35263584},
issn = {2211-1247},
support = {R21 NS106159/NS/NINDS NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R01 NS117626/NS/NINDS NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; },
mesh = {*Bacterial Toxins/genetics ; *COVID-19 ; CRISPR-Cas Systems ; Gene Editing ; Humans ; RNA, Guide/metabolism ; SARS-CoV-2 ; },
abstract = {Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.},
}
@article {pmid35263578,
year = {2022},
author = {Michl, J and Wang, Y and Monterisi, S and Blaszczak, W and Beveridge, R and Bridges, EM and Koth, J and Bodmer, WF and Swietach, P},
title = {CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH.},
journal = {Cell reports},
volume = {38},
number = {10},
pages = {110493},
pmid = {35263578},
issn = {2211-1247},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Survival/genetics ; Humans ; Hydrogen-Ion Concentration ; *Neoplasms/genetics ; *Oxidative Phosphorylation ; },
abstract = {Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1[-/-] cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.},
}
@article {pmid35263090,
year = {2022},
author = {Kang, Y and Su, G and Yu, Y and Cao, J and Wang, J and Yan, B},
title = {CRISPR-Cas12a-Based Aptasensor for On-Site and Highly Sensitive Detection of Microcystin-LR in Freshwater.},
journal = {Environmental science &amp; technology},
volume = {56},
number = {7},
pages = {4101-4110},
doi = {10.1021/acs.est.1c06733},
pmid = {35263090},
issn = {1520-5851},
mesh = {*Aptamers, Nucleotide/metabolism ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Fresh Water ; Limit of Detection ; Marine Toxins ; Microcystins/metabolism ; },
abstract = {On-site monitoring of trace organic pollutants with facile methods is critical to environmental pollutant prevention and control. Herein, we proposed a CRISPR-Cas12a-based aptasensor platform (named as MC-LR-Casor) for on-site and sensitive detection of microcystin-LR (MC-LR). After hybridization with blocker DNA, the MC-LR aptamers were conjugated to magnetic beads (MBs) to get the MB aptasensor. In the presence of MC-LR, their interactions with aptamers were triggered and the specific binding caused the release of blocker DNA. Using the programmability of the CRISPR-Cas system, the released blocker DNA was designed to activate a Cas12a-crRNA complex. Single strand DNA reporters were rapidly cleaved by the complex. Signal readout could be achieved by fluorometer or lateral flow strips, which were positively correlated to MC-LR concentration. Benefiting from the CRISPR-Cas12a amplification system, the proposed sensing platform exhibited high sensitivity and reached the limit of detection of ∼3 × 10[-6] μg/L (fluorescence method) or 1 × 10[-3] μg/L (lateral flow assay). In addition, the MC-LR-Casor showed excellent selectivity and good recovery rates, demonstrating their good applicability for real water sample analysis. During the whole assay, only two steps of incubation at a constant temperature were required and the results could be visualized when employing flow strips. Therefore, the proposed assay offered a simple and convenient alternative for in situ MC-LR monitoring, which may hold great promise for future environmental surveillance.},
}
@article {pmid35261159,
year = {2022},
author = {Saw, PE and Cui, GH and Xu, X},
title = {Nanoparticles-Mediated CRISPR/Cas Gene Editing Delivery System.},
journal = {ChemMedChem},
volume = {17},
number = {9},
pages = {e202100777},
doi = {10.1002/cmdc.202100777},
pmid = {35261159},
issn = {1860-7187},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; *Nanoparticles ; },
abstract = {CRISPR/Cas system has become one of the most powerful techologies in biomedical research, and has showed great potentials in the gene related diseases. However, efficient delivery systems of CRISPR/Cas to target cells remains challenging. In recent years, nanoparticles have showned great potentials for the delivery of CRISPR/Cas systems. This paper mainly approaches the development and new strategies of CRISPR/Cas delivery systems, as well as their application in the clinical diseases. By summarizing the CRISPR/Cas systems delivery, new strategies are expected for the gene therapy.},
}
@article {pmid35260779,
year = {2022},
author = {Nakagawa, R and Ishiguro, S and Okazaki, S and Mori, H and Tanaka, M and Aburatani, H and Yachie, N and Nishimasu, H and Nureki, O},
title = {Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {211},
pmid = {35260779},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; *Campylobacter jejuni/genetics ; Gene Editing ; Humans ; },
abstract = {The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.},
}
@article {pmid35260651,
year = {2022},
author = {Pini, V and Mariot, V and Dumonceaux, J and Counsell, J and O'Neill, HC and Farmer, S and Conti, F and Muntoni, F},
title = {Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {3756},
pmid = {35260651},
issn = {2045-2322},
support = {/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; Dystrophin/genetics/metabolism ; Endonucleases/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Myoblasts/metabolism ; },
abstract = {Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.},
}
@article {pmid35260581,
year = {2022},
author = {Yin, J and Lu, R and Xin, C and Wang, Y and Ling, X and Li, D and Zhang, W and Liu, M and Xie, W and Kong, L and Si, W and Wei, P and Xiao, B and Lee, HY and Liu, T and Hu, J},
title = {Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1204},
pmid = {35260581},
issn = {2041-1723},
mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing ; Humans ; RNA, Guide/chemistry/genetics ; Translocation, Genetic ; },
abstract = {The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.},
}
@article {pmid35258835,
year = {2022},
author = {Decaestecker, W and Bollier, N and Buono, RA and Nowack, MK and Jacobs, TB},
title = {Protoplast Preparation and Fluorescence-Activated Cell Sorting for the Evaluation of Targeted Mutagenesis in Plant Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2464},
number = {},
pages = {205-221},
pmid = {35258835},
issn = {1940-6029},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; Flow Cytometry/methods ; Mutagenesis ; Plant Cells ; *Protoplasts ; },
abstract = {Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells.},
}
@article {pmid35258833,
year = {2022},
author = {Patil, GB and Stupar, RM and Zhang, F},
title = {Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2464},
number = {},
pages = {173-186},
pmid = {35258833},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plant Breeding ; *Protoplasts/metabolism ; Ribonucleoproteins/metabolism ; Soybeans/genetics/metabolism ; Transfection ; },
abstract = {Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.},
}
@article {pmid35258832,
year = {2022},
author = {Kang, M and Lee, K and Wang, K},
title = {Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense) and Camelina (Camelina sativa).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2464},
number = {},
pages = {153-171},
pmid = {35258832},
issn = {1940-6029},
mesh = {Crops, Agricultural/genetics ; DNA/metabolism ; Protoplasts ; *Thlaspi/genetics/metabolism ; Transfection ; },
abstract = {Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6 × 10[6] cells from pennycress and 3 × 10[6] cells from camelina per gram of fresh leaf tissues. Using a 7.3 kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.},
}
@article {pmid35258825,
year = {2022},
author = {Banakar, R and Rai, KM and Zhang, F},
title = {CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2464},
number = {},
pages = {65-82},
pmid = {35258825},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Genome, Plant ; *Protoplasts ; Ribonucleoproteins/genetics ; Tobacco/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated system) has become the multipurpose tool to manipulate plant genome via their programmable sequence recognition, binding, and cleavage activities. Efficient plant genome modification often requires robust plant transformation. For most plant species, the CRISPR/Cas reagents are delivered into plants as plasmids by Agrobacterium-mediated T-DNA transfer or biolistic approaches. However, these methods are generally inefficient, heavily genotype dependent, and low throughput. Among the alternative plant transformation approaches, the protoplast-based transformation holds the potential to directly deliver DNA, RNA, or protein molecules into plant cells in an efficient and high-throughput manner. Here, we presented a robust and simplified protocol for protoplast-based DNA/ribonucleoprotein (RNP)-mediated genome editing in the model species Nicotiana benthamiana. Using this protocol, we have achieved the gene editing efficiency at 30-60% in protoplasts and 50-80% in regenerated calli and plants. The edited protoplasts can be readily regenerated without selection agents owing to highly efficient DNA or preassembled RNP transformation frequency. Lastly, this protocol utilized an improved culture media regime to overcome the complex media composition used in the previous studies. It offers quick turnaround time and higher throughput to facilitate the development of new genetic engineering technologies and holds the promise to combine with other genetic and genomic tools for fundamental and translational plant research.},
}
@article {pmid35258824,
year = {2022},
author = {Wu, FH and Yuan, YH and Hsu, CT and Cheng, QW and Lin, CS},
title = {Application of Protoplast Regeneration to CRISPR/Cas9 Mutagenesis in Nicotiana tabacum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2464},
number = {},
pages = {49-64},
pmid = {35258824},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Mutagenesis ; *Protoplasts/metabolism ; Tobacco/genetics ; },
abstract = {Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and protein-protein interactions. In order to successfully use protoplast transfection with the newly emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein editing platform, high yield of protoplasts, stable transfection efficiency, and reliable regeneration protocols are necessary. The Nicotiana tabacum transient protoplast transfection and regeneration system can effectively obtain target gene mutations in regenerated plants without transgenes and is thus a very attractive technique for evaluating gene editing reagents using CRISPR/Cas-based systems. Here, we describe in detail sterilized seed germination, culture conditions, isolation of Nicotiana tabacum protoplasts from tissue culture explants, construction of a vector containing the Cas protein and sgRNA cassette, highly efficient polyethylene glycol-calcium transient transfection of plasmids delivered into protoplasts, evaluation of mutagenesis efficiency and genotype analysis from protoplasts and regenerated plants, and the regeneration conditions to obtain CRISPR-edited plants from single protoplasts.},
}
@article {pmid35258601,
year = {2022},
author = {Rybnicky, GA and Fackler, NA and Karim, AS and Köpke, M and Jewett, MC},
title = {Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences.},
journal = {Nucleic acids research},
volume = {50},
number = {6},
pages = {3523-3534},
pmid = {35258601},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems/genetics ; Clostridium/genetics ; Computational Biology/*methods ; Gene Library ; Nucleotide Motifs ; },
abstract = {RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.},
}
@article {pmid35257575,
year = {2022},
author = {Zhang, D and Liu, L and Jin, S and Tota, E and Li, Z and Piao, X and Zhang, X and Fu, XD and Devaraj, NK},
title = {Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.},
journal = {Journal of the American Chemical Society},
volume = {144},
number = {10},
pages = {4487-4495},
pmid = {35257575},
issn = {1520-5126},
support = {R01 GM123285/GM/NIGMS NIH HHS/United States ; R35 GM141939/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mammals/genetics ; *RNA, Guide/genetics ; },
abstract = {Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.},
}
@article {pmid35255977,
year = {2022},
author = {Li, L and Yi, H and Liu, Z and Long, P and Pan, T and Huang, Y and Li, Y and Li, Q and Ma, Y},
title = {Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology.},
journal = {Stem cell research &amp; therapy},
volume = {13},
number = {1},
pages = {102},
pmid = {35255977},
issn = {1757-6512},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Technology ; beta-Globins/genetics/metabolism ; *beta-Thalassemia/genetics/metabolism/therapy ; },
abstract = {BACKGROUND: Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above.<br><br>METHODS: In the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous &#x3b2;41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C&#x2009;>&#x2009;G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays.<br><br>RESULTS: These mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential.<br><br>CONCLUSIONS: We designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent &#x3b1;- and &#x3b2;-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent &#x3b1;- and &#x3b2;-thalassemia.},
}
@article {pmid35255784,
year = {2022},
author = {Wan, HF and Han, WJ and Zhou, L and Wang, S and Sui, C},
title = {New Advances of CRISPR/Cas9 Technique and its Application in Disease Treatment and Medicinal Plants Research.},
journal = {Current pharmaceutical biotechnology},
volume = {23},
number = {14},
pages = {1678-1690},
doi = {10.2174/1389201023666220307104501},
pmid = {35255784},
issn = {1873-4316},
mesh = {Humans ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Plants, Medicinal/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology is widely used in disease treatment and medicinal plant improvements due to its advantages, such as easy operation, low time consumption, and high efficiency. However, potential off-target risks still exist in practical applications and need to be solved urgently.<br><br>OBJECTIVES: This study aimed to review the application progress of CRISPR/Cas9 technology in the field of disease treatment and medicinal agriculture in recent years. Furthermore, the study discusses the ways to reduce the off-target effect of CRISPR/Cas9 technology, providing a reference for the further application of this technology.<br><br>METHODS: CiteSpace software was used to analyze relevant literature data from 2013 to August 2021, and search results were retrieved from Web of Science, PubMed, and CNKI databases.<br><br>RESULTS: In the field of disease treatment, CRISPR/Cas9 technology has great potential to cure challenging human diseases and has been widely used in drug target development, drug design, and screening. In crop breeding, CRISPR/Cas9 accelerates the improvement of crop varieties and shortens the number of years of crop breeding. By adjusting the length and GC content of sgRNA and changing the concentration of Cas9/sgRNA complex to reduce the off-target effect of CRISPR/Cas9 technology, the target genes can be manipulated more accurately.<br><br>CONCLUSION: CRISPR/Cas9 technology is an indispensable and key technology in the field of disease treatment and medicinal plants. With the in-depth study of the off-target effect, CRISPR/Cas9 technology can have broader application prospects in the fields of medicine and medicinal agriculture.},
}
@article {pmid35254919,
year = {2022},
author = {Zheng, Y and VanDusen, NJ and Butler, CE and Ma, Q and King, JS and Pu, WT},
title = {Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes.},
journal = {Circulation},
volume = {145},
number = {10},
pages = {787-789},
pmid = {35254919},
issn = {1524-4539},
support = {K99 HL143194/HL/NHLBI NIH HHS/United States ; R01 HL146634/HL/NHLBI NIH HHS/United States ; UM1 HL098166/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; *Myocytes, Cardiac ; *Recombinational DNA Repair ; },
}
@article {pmid35254721,
year = {2022},
author = {Grotz, S and Schäfer, J and Wunderlich, KA and Ellederova, Z and Auch, H and Bähr, A and Runa-Vochozkova, P and Fadl, J and Arnold, V and Ardan, T and Veith, M and Santamaria, G and Dhom, G and Hitzl, W and Kessler, B and Eckardt, C and Klein, J and Brymova, A and Linnert, J and Kurome, M and Zakharchenko, V and Fischer, A and Blutke, A and Döring, A and Suchankova, S and Popelar, J and Rodríguez-Bocanegra, E and Dlugaiczyk, J and Straka, H and May-Simera, H and Wang, W and Laugwitz, KL and Vandenberghe, LH and Wolf, E and Nagel-Wolfrum, K and Peters, T and Motlik, J and Fischer, MD and Wolfrum, U and Klymiuk, N},
title = {Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.},
journal = {EMBO molecular medicine},
volume = {14},
number = {4},
pages = {e14817},
pmid = {35254721},
issn = {1757-4684},
mesh = {Animals ; Cell Cycle Proteins/genetics ; Cytoskeletal Proteins ; Humans ; Photoreceptor Cells ; Swine ; *Usher Syndromes/genetics/metabolism/therapy ; },
abstract = {Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.},
}
@article {pmid35254529,
year = {2022},
author = {de Souza Moraes, T and van Es, SW and Hernández-Pinzón, I and Kirschner, GK and van der Wal, F and da Silveira, SR and Busscher-Lange, J and Angenent, GC and Moscou, M and Immink, RGH and van Esse, GW},
title = {The TCP transcription factor HvTB2 heterodimerizes with VRS5 and controls spike architecture in barley.},
journal = {Plant reproduction},
volume = {35},
number = {3},
pages = {205-220},
pmid = {35254529},
issn = {2194-7961},
mesh = {Edible Grain/metabolism ; Gene Expression Regulation, Plant ; *Hordeum/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Zea mays/metabolism ; },
abstract = {Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.},
}
@article {pmid35252043,
year = {2022},
author = {Yin, D and Yin, L and Wang, J and Shen, X and Pan, X and Hou, H and Zhao, R and Hu, X and Wang, G and Qi, K and Dai, Y},
title = {Visual Detection of Duck Tembusu Virus With CRISPR/Cas13: A Sensitive and Specific Point-of-Care Detection.},
journal = {Frontiers in cellular and infection microbiology},
volume = {12},
number = {},
pages = {848365},
pmid = {35252043},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; Flavivirus ; *Flavivirus Infections/diagnosis/veterinary ; Point-of-Care Systems ; *Poultry Diseases/diagnosis ; Sensitivity and Specificity ; },
abstract = {Duck tembusu virus (DTMUV), which causes huge economic losses for the poultry industries in Southeast Asia and China, was first identified in 2010. DTMUV disease has become an important disease that endangers the duck industry. A sensitive, accurate, and convenient DTMUV detection method is an important means to reduce the occurrence of the disease. In this study, a CRISPR/Cas13a system was combined with recombinase polymerase amplification to develop a convenient diagnostic method to detect DTMUV. The novel method was based on isothermal detection at 37°C, and the detection was used for visual readout or real-time analysis. The assay was highly sensitive and specific, with a detection limit of 1 copy/μL of the target gene and showed no cross-reactivity with other pathogens. The enhanced Cas13a detection worked well with clinical samples. Overall, a visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a proved to be a powerful tool for detecting DTMUV.},
}
@article {pmid35252020,
year = {2021},
author = {Sharma, R and Avendaño Rangel, F and Reis-Cunha, JL and Marques, LP and Figueira, CP and Borba, PB and Viana, SM and Beneke, T and Bartholomeu, DC and de Oliveira, CI},
title = {Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {790418},
pmid = {35252020},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; *Leishmania ; *Leishmania braziliensis/genetics ; *Leishmaniasis, Cutaneous/parasitology ; Mice ; Mice, Inbred BALB C ; Trimethoprim, Sulfamethoxazole Drug Combination ; },
abstract = {Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen[-/-]) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen[-/-] and wild-type promastigotes were similar, but axenic and intracellular LbCen[-/-] amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen[-/-] were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen[-/-] , differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen[-/-] can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.},
}
@article {pmid35251616,
year = {2022},
author = {Aripova, T and Muratkhodjaev, J},
title = {A novel concept of human antiviral protection: It's all about RNA (Review).},
journal = {Biomedical reports},
volume = {16},
number = {4},
pages = {29},
pmid = {35251616},
issn = {2049-9442},
abstract = {The comparative analysis of the antiviral protective mechanisms, including protozoa and RNA interference in multicellular organisms, has revealed their similarity and provided a basic understanding of adaptive immunity. The present article summarizes the latest studies on RNA-guided gene regulation in human antiviral protection, and its importance. Additionally, the role of both neutralizing antibodies and the interferon system in viral invasion is considered. The interferon system is an additional mechanism for suppressing viral infections in humans, which shifts cells into an 'alarm' mode to attempt to prevent further contagion. The primary task of the human central immune system is to maintain integrity and to protect against foreign organisms. In this review, a novel concept is proposed: Antiviral protection in all organisms can be achieved through an intracellular RNA-guided mechanism. A simple and effective defence against viruses is incorporation of a part of a virus's DNA (spacer) into the hosts chromosomes. Following reinfection, RNA transcripts of this spacer are created to direct nuclease enzymes to destroy the viral genome. This is an example of real-time adaptive immunity potentially possessed by every cell with a full complement of chromosomes, and an indicator that antiviral immunity is not only mediated by the presence of neutralizing antibodies and memory B- and T-cells, but also by the presence of specific spacers in the DNA of individuals who have recovered from a viral infection.},
}
@article {pmid35250902,
year = {2021},
author = {Ambroa, A and Blasco, L and López, M and Pacios, O and Bleriot, I and Fernández-García, L and González de Aledo, M and Ortiz-Cartagena, C and Millard, A and Tomás, M},
title = {Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {784949},
pmid = {35250902},
issn = {1664-302X},
abstract = {To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction-modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.},
}
@article {pmid35250283,
year = {2022},
author = {Xu, H and Tang, H and Li, R and Xia, Z and Yang, W and Zhu, Y and Liu, Z and Lu, G and Ni, S and Shen, J},
title = {A New Method Based on LAMP-CRISPR-Cas12a-Lateral Flow Immunochromatographic Strip for Detection.},
journal = {Infection and drug resistance},
volume = {15},
number = {},
pages = {685-696},
pmid = {35250283},
issn = {1178-6973},
abstract = {INTRODUCTION: Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern. Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options. It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains.<br><br>METHODS: For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-&#x3b2;-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR-Cas12a, and lateral flow immunochromatographic strip (CRISPR-Cas-LAMP-lateral flow strip). The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system.<br><br>RESULTS: The cleavage products are visible to the naked eye on the lateral flow strip. This method is highly sensitive in direct detection of bacteria in samples containing at least 3&#xd7;10[5] CFU/mL without the need for bacterial culture.<br><br>DISCUSSION: It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method. This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.},
}
@article {pmid35248880,
year = {2022},
author = {Kang, B and Cong, Z and Duan, J and Liu, K and Wang, Y},
title = {Generation of a GFI1-flag knock-in human embryonic stem cell line using CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102724},
doi = {10.1016/j.scr.2022.102724},
pmid = {35248880},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; DNA-Binding Proteins/genetics/metabolism ; Embryonic Stem Cells/metabolism ; *Human Embryonic Stem Cells/metabolism ; Humans ; Technology ; Transcription Factors/genetics/metabolism ; },
abstract = {GFI1 is a DNA binding transcriptional repressor, it is shown to be an important gene associated with blood cells development and many blood diseases (Möröy et al., 2015). But the role of GFI1 in human hematopoieticdevelopment has not been known (Thambyrajah et al., 2016). To illustrate the function of GFI1 in human hematopoieticdevelopment, we constructed a GFI1-2 × flag-tag knock-in human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, and it would be the effective tool to study GFI1. The cell line could express GFI1-2 × flag-tag and can be identified with western blot and immunofluorescence. This cell line maintains stem cell morphology, and displays normal karyotype, pluripotent stem cell marker expression and differentiation potential.},
}
@article {pmid35247858,
year = {2022},
author = {Safdar, S and Driesen, S and Leirs, K and De Sutter, D and Eyckerman, S and Lammertyn, J and Spasic, D},
title = {Engineered tracrRNA for enabling versatile CRISPR-dCas9-based biosensing concepts.},
journal = {Biosensors &amp; bioelectronics},
volume = {206},
number = {},
pages = {114140},
doi = {10.1016/j.bios.2022.114140},
pmid = {35247858},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA ; *DNA, Catalytic/genetics ; RNA ; },
abstract = {In recent years, CRISPR-Cas (stands for: clustered regularly interspaced short palindromic repeats - CRISPR associated protein) based technologies have gained increasing attention in the biosensing field. Thanks to excellent sequence specificity, their use is of particular interest for detecting nucleic acid (NA) targets. In this context, signal generation and amplification can be realized by employing the cis-cleavage activity of the Cas9 protein, although other options involving the catalytically inactive dead Cas9 (dCas9) are increasingly explored. The latter are however mostly based on complex protein engineering processes and often lack efficient signal amplification. Here we showed for the first time that flexible signal generation and amplification properties can be integrated into the CRISPR-dCas9 complex based on a straightforward incorporation of a DNA sequence into the trans-activating CRISPR RNA (tracrRNA). The intrinsic nuclease activity of the engineered complex remained conserved, while the incorporated DNA stretch enabled two modes of amplified fluorescent signal generation: (1) as an RNA-cleaving DNA-based enzyme (DNAzyme) or (2) as hybridization site for biotinylated DNA probes, allowing subsequent enzyme labeling. Both signal generation strategies were demonstrated in solution as well as while coupled to a solid surface. Finally, in a proof of concept bioassay, we demonstrated the successful detection of single stranded DNA on magnetic microbeads using the engineered CRISPR-dCas9 complex. Thanks to the flexibility of incorporating different NA-based signal generation and amplification strategies, this novel NA engineering approach holds enormous promise for many new CRISPR-based biosensing applications.},
}
@article {pmid35247841,
year = {2022},
author = {Chiu, W and Li, A and Wang, T and Li, W and Zhang, X},
title = {Generation of a MSX1 knockout human embryonic stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102729},
doi = {10.1016/j.scr.2022.102729},
pmid = {35247841},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Human Embryonic Stem Cells/metabolism ; Humans ; MSX1 Transcription Factor/genetics/metabolism ; Technology ; },
abstract = {The MSX1 gene encodes a transcriptional repressor and plays important roles in limb-pattern formation, craniofacial development, and odontogenesis during vertebrate embryogenesis. Previous studies demonstrated that human MSX1 mutations are associated with tooth agenesis, orofacial clefting, and nail dysplasia. Here, we generated a MSX1 knockout cell line from human embryonic stem cell (hESC) line (H9) by CRISPR/cas9-mediated gene targeting. This cell line may serve as a valuable in vitro cell model for MSX1 mutation-related diseases and help to gain more insight into the biological function of MSX1.},
}
@article {pmid35247837,
year = {2022},
author = {Wang, H and Luo, Y and Li, J and Guan, J and Yang, S and Wang, Q},
title = {Generation of a gene corrected human isogenic iPSC line (CPGHi001-A-1) from a hearing loss patient with the TMC1 p.M418K mutation using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102736},
doi = {10.1016/j.scr.2022.102736},
pmid = {35247837},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Hearing Loss/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Membrane Proteins/genetics ; Mice ; Mutation/genetics ; },
abstract = {TMC1 p.M418K mutation is homologous to that in Beethoven mice, which may induce autosomal dominant non-syndromic progressive hearing loss. Previously, we generated an induced pluripotent stem cells (iPSCs) line (CPGHi001-A) from a hearing loss patient with the TMC1 c.1253 T > A (p.M418K) mutation. Here we genetically corrected the TMC1 c.1253 T > A mutation using CRISPR/Cas9 technology to generate an isogenic control, CPGHi001-A-1. The resulting iPSCs had a normal karyotype, showed pluripotency by immunofluorescence staining, and differentiated into the three germ layers in vitro.},
}
@article {pmid35247836,
year = {2022},
author = {Zhang, Y and Zhu, J and Dai, Y and Wang, L and Liu, R and Guo, X},
title = {Generation of a heterozygous FUS-Q290X knock in human embryonic stem cell line (WAe009-A-83) using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102734},
doi = {10.1016/j.scr.2022.102734},
pmid = {35247836},
issn = {1876-7753},
mesh = {*Amyotrophic Lateral Sclerosis/metabolism ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Humans ; *Liposarcoma/genetics ; Mutation ; RNA, Messenger/metabolism ; RNA-Binding Protein FUS/genetics/metabolism ; },
abstract = {Fused in Sarcoma (FUS) gene encodes FUS RNA binding protein, a multifunctional protein component of the heterogeneous nuclear ribonucleoprotein complex, which is involved in pre-mRNA splicing and the export of fully processed mRNA to the cytoplasm, and it has been implicated in regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. FUS gene mutations result in amyotrophic lateral sclerosis and Liposarcoma. This heterozygous FUS-Q290X knock in hESC line will be a valuable tool to investigate the disease mechanisms of amyotrophic lateral sclerosis and Liposarcoma.},
}
@article {pmid35247835,
year = {2022},
author = {Bray, L and Caillaud, A and Girardeau, A and Patitucci, M and Le May, C and Cariou, B and Rimbert, A},
title = {Generation of a GPR146 knockout human induced pluripotent stem cell line (ITXi001-A-1).},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102721},
doi = {10.1016/j.scr.2022.102721},
pmid = {35247835},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Liver ; Mice ; },
abstract = {Dyslipidemia is a key modifiable causal risk factor involved in the development of atherosclerotic cardiovascular disease. Recently, the G protein-coupled receptor 146 (GPR146), a member of the G-coupled protein receptors' family, has been shown to be a regulator of plasma cholesterol. Inhibition of hepatic GPR146 in mice displays protective effect against both hypercholesterolemia and atherosclerosis. Here, we characterize a genetically engineered human induced pluripotent stem cell (hiPSC) model invalidated for GPR146 (ITXi001-A-1) using CRISPR-Cas9 editing technology. Differentiation of ITXi001-A-1 towards hepatic fate will provide a suitable model for deciphering the molecular mechanisms sustaining the beneficial metabolic effects of GPR146 inhibition.},
}
@article {pmid35247491,
year = {2022},
author = {Chae, SY and Jeong, E and Kang, S and Yim, Y and Kim, JS and Min, DH},
title = {Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {345},
number = {},
pages = {108-119},
doi = {10.1016/j.jconrel.2022.02.035},
pmid = {35247491},
issn = {1873-4995},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Nanoparticles ; Ribonucleoproteins/genetics ; },
abstract = {Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.},
}
@article {pmid35246186,
year = {2022},
author = {Guo, M and Chen, H and Dong, S and Zhang, Z and Luo, H},
title = {CRISPR-Cas gene editing technology and its application prospect in medicinal plants.},
journal = {Chinese medicine},
volume = {17},
number = {1},
pages = {33},
pmid = {35246186},
issn = {1749-8546},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing technology has opened a new era of genome interrogation and genome engineering because of its ease operation and high efficiency. An increasing number of plant species have been subjected to site-directed gene editing through this technology. However, the application of CRISPR-Cas technology to medicinal plants is still in the early stages. Here, we review the research history, structural characteristics, working mechanism and the latest derivatives of CRISPR-Cas technology, and discussed their application in medicinal plants for the first time. Furthermore, we creatively put forward the development direction of CRISPR technology applied to medicinal plant gene editing. The aim is to provide a reference for the application of this technology to genome functional studies, synthetic biology, genetic improvement, and germplasm innovation of medicinal plants. CRISPR-Cas is expected to revolutionize medicinal plant biotechnology in the near future.},
}
@article {pmid35245852,
year = {2022},
author = {Zhang, F and Meier, AB and Lipp, P and Laugwitz, KL and Dorn, T and Moretti, A},
title = {Generation of heterozygous (MRli003-A-3) and homozygous (MRli003-A-4) TRPM4 knockout human iPSC lines.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102731},
doi = {10.1016/j.scr.2022.102731},
pmid = {35245852},
issn = {1876-7753},
support = {788381/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Heterozygote ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; *TRPM Cation Channels/genetics/metabolism ; },
abstract = {TRPM4 is a Ca[2+]-activated channel mediating the transport of monovalent cations across the cell membrane. Mutations in the TRPM4 gene have been associated with cardiac arrhythmias in humans. Using CRISPR/Cas9 gene editing technology, we established two TRPM4 knockout human iPSC lines - one heterozygous (MRli003-A-3) and one homozygous (MRli003-A-4) - by inserting a frameshift mutation in exon 2 of the TRPM4 gene. Both lines maintained pluripotency, a normal karyotype, parental cell morphology, and the ability to differentiate into the three germ layers.},
}
@article {pmid35245313,
year = {2022},
author = {Wen, T and Mao, C and Gao, L},
title = {Analysis of the gut microbiota composition of myostatin mutant cattle prepared using CRISPR/Cas9.},
journal = {PloS one},
volume = {17},
number = {3},
pages = {e0264849},
pmid = {35245313},
issn = {1932-6203},
mesh = {Animals ; Bacteria/genetics ; CRISPR-Cas Systems ; Cattle ; *Gastrointestinal Microbiome/genetics ; Myostatin/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Myostatin (MSTN) negatively regulates muscle development and positively regulates metabolism through various pathways. Although MSTN function in cattle has been widely studied, the changes in the gut microbiota due to MSTN mutation, which contribute to host health by regulating its metabolism, remain unclear. Here, high-throughput sequencing of the 16S rRNA gene was conducted to analyze the gut microbiota of wild-type (WT) and MSTN mutant (MT) cattle. A total of 925 operational taxonomic units (OTUs) were obtained, which were classified into 11 phyla and 168 genera. Alpha diversity results showed no significant differences between MT and WT cattle. Beta diversity analyses suggested that the microbial composition of WT and MT cattle was different. Three dominant phyla and 21 dominant genera were identified. The most abundant bacterial genus had a significant relationship with the host metabolism. Moreover, various bacteria beneficial for health were found in the intestines of MT cattle. Analysis of the correlation between dominant gut bacteria and serum metabolic factors affected by MSTN mutation indicated that MSTN mutation affected the metabolism mainly by three metabolism-related bacteria, Ruminococcaceae_UCG-013, Clostridium_sensu_stricto_1, and Ruminococcaceae_UCG-010. This study provides further insight into MSTN mutation regulating the host metabolism by gut microbes and provides evidence for the safety of gene-edited animals.},
}
@article {pmid35245307,
year = {2022},
author = {Zhang, H and Zhu, S and Xing, Y and Liu, Q and Guo, Z and Cai, Z and Shen, Z and Xia, Q and Sheng, H},
title = {Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out.},
journal = {PloS one},
volume = {17},
number = {3},
pages = {e0263162},
pmid = {35245307},
issn = {1932-6203},
mesh = {*Aquaporins/genetics ; CRISPR-Cas Systems/genetics ; Cryopreservation ; Gene Editing/methods ; *Glioma ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; RNA, Messenger ; Reproducibility of Results ; },
abstract = {Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.},
}
@article {pmid35244719,
year = {2022},
author = {Liu, Y and Ma, G and Gao, Z and Li, J and Wang, J and Zhu, X and Ma, R and Yang, J and Zhou, Y and Hu, K and Zhang, Y and Guo, Y},
title = {Global chromosome rearrangement induced by CRISPR-Cas9 reshapes the genome and transcriptome of human cells.},
journal = {Nucleic acids research},
volume = {50},
number = {6},
pages = {3456-3474},
pmid = {35244719},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Chromosomes/metabolism ; DNA Copy Number Variations ; *Gene Editing/methods ; Genome, Human ; Humans ; RNA, Guide/genetics ; *Transcriptome ; },
abstract = {Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.},
}
@article {pmid35244715,
year = {2022},
author = {Ai, Y and Liang, D and Wilusz, JE},
title = {CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells.},
journal = {Nucleic acids research},
volume = {50},
number = {11},
pages = {e65},
pmid = {35244715},
issn = {1362-4962},
support = {R35 GM119735/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila/genetics ; Eukaryotic Cells ; RNA/genetics ; *RNA, Guide/genetics ; },
abstract = {CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.},
}
@article {pmid35244489,
year = {2022},
author = {Nguyen Tran, MT and Kc, R and Hewitt, AW},
title = {A Taxonomic and Phylogenetic Classification of Diverse Base Editors.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {311-328},
doi = {10.1089/crispr.2021.0095},
pmid = {35244489},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing ; Phylogeny ; },
abstract = {Base editors mediate the targeted conversion of single nucleobases in a therapeutically relevant manner. Herein, we present a hypothetical taxonomic and phylogenetic framework for the classification of more than 200 different DNA base editors, and we categorize them based on their described properties. Following evaluation of their in situ activity windows, which were derived by cataloguing their activity in published literature, organization is done hierarchically, with specific base editor signatures being subcategorized according to their on-target activity or nonspecific, genome- or transcriptome-wide activity. Based on this categorization, we curate a phylogenetic framework, based on protein homology alignment, and describe a taxonomic structure that clusters base editor variants on their target chemistry, endonuclease component, identity of their deaminase component, and their described properties into discrete taxa. Thus, we establish a hypothetical taxonomic structure that can describe and organize current and potentially future base editing variants into clearly defined groups that are defined by their characteristics. Finally, we summarize our findings into a navigable database (ShinyApp in R) that allows users to select through our repository to nominate ideal base editor candidates as a starting point for further testing in their specific application.},
}
@article {pmid35244184,
year = {2022},
author = {Thiruppathi, D},
title = {CRISPR keeps going "wild": a new protocol for DNA-free genome editing of tetraploid wild tomatoes.},
journal = {Plant physiology},
volume = {189},
number = {1},
pages = {10-11},
pmid = {35244184},
issn = {1532-2548},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; *Lycopersicon esculentum/genetics ; *Solanum/genetics ; Tetraploidy ; },
}
@article {pmid35243691,
year = {2022},
author = {Thompson, EL and Pickett-Leonard, M and Riddle, MJ and Chen, W and Albert, FW and Tolar, J},
title = {Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.},
journal = {Experimental dermatology},
volume = {31},
number = {7},
pages = {1065-1075},
pmid = {35243691},
issn = {1600-0625},
support = {T32 AG029796/AG/NIA NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; R21 HG010380/HG/NHGRI NIH HHS/United States ; },
mesh = {Cell Line ; *Collagen Type VII ; *Epidermolysis Bullosa Dystrophica/drug therapy/genetics ; Humans ; Keratinocytes/metabolism ; Mutation ; },
abstract = {Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7 mRNA and protein in DEB patient cells.},
}
@article {pmid35243655,
year = {2022},
author = {Zabaleta, N and Torella, L and Weber, ND and Gonzalez-Aseguinolaza, G},
title = {mRNA and gene editing: Late breaking therapies in liver diseases.},
journal = {Hepatology (Baltimore, Md.)},
volume = {76},
number = {3},
pages = {869-887},
pmid = {35243655},
issn = {1527-3350},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; *Liver Diseases/genetics/therapy ; RNA, Messenger/genetics ; },
abstract = {The efficient delivery of RNA molecules to restore the expression of a missing or inadequately functioning protein in a target cell and the intentional specific modification of the host genome using engineered nucleases represent therapeutic concepts that are revolutionizing modern medicine. The initiation of several clinical trials using these approaches to treat metabolic liver disorders as well as the recently reported remarkable results obtained by patients with transthyretin amyloidosis highlight the advances in this field and show the potential of these therapies to treat these diseases safely and efficaciously. These advances have been possible due, firstly, to significant improvements made in RNA chemistry that increase its stability and prevent activation of the innate immune response and, secondly, to the development of very efficient liver-targeted RNA delivery systems. In parallel, the breakout of CRISPR/CRISPR-associated 9-based technology in the gene editing field has marked a turning point in in vivo modification of the cellular genome with therapeutic purposes, which can be based on gene supplementation, correction, or silencing. In the coming years we are likely to witness the therapeutic potential of these two strategies both separately and in combination. In this review we summarize the preclinical data obtained in animal models treated with mRNA as a therapeutic agent and discuss the different gene editing strategies applied to the treatment of liver diseases, highlighting both their therapeutic efficacy as well as safety concerns.},
}
@article {pmid35241090,
year = {2022},
author = {Rasul, MF and Hussen, BM and Salihi, A and Ismael, BS and Jalal, PJ and Zanichelli, A and Jamali, E and Baniahmad, A and Ghafouri-Fard, S and Basiri, A and Taheri, M},
title = {Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {64},
pmid = {35241090},
issn = {1476-4598},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; Humans ; Mutation ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) shows the opportunity to treat a diverse array of untreated various genetic and complicated disorders. Therapeutic genome editing processes that target disease-causing genes or mutant genes have been greatly accelerated in recent years as a consequence of improvements in sequence-specific nuclease technology. However, the therapeutic promise of genome editing has yet to be explored entirely, many challenges persist that increase the risk of further mutations. Here, we highlighted the main challenges facing CRISPR/Cas9-based treatments and proposed strategies to overcome these limitations, for further enhancing this revolutionary novel therapeutics to improve long-term treatment outcome human health.},
}
@article {pmid35240127,
year = {2022},
author = {Arantes, PR and Patel, AC and Palermo, G},
title = {Emerging Methods and Applications to Decrypt Allostery in Proteins and Nucleic Acids.},
journal = {Journal of molecular biology},
volume = {434},
number = {17},
pages = {167518},
pmid = {35240127},
issn = {1089-8638},
support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {Allosteric Regulation ; CRISPR-Cas Systems ; *DNA/chemistry ; Gene Editing ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; Nucleosomes/chemistry ; Protein Conformation ; *Proteins/chemistry ; Spliceosomes/chemistry ; },
abstract = {Many large protein-nucleic acid complexes exhibit allosteric regulation. In these systems, the propagation of the allosteric signaling is strongly coupled to conformational dynamics and catalytic function, challenging state-of-the-art analytical methods. Here, we review established and innovative approaches used to elucidate allosteric mechanisms in these complexes. Specifically, we report network models derived from graph theory and centrality analyses in combination with molecular dynamics (MD) simulations, introducing novel schemes that implement the synergistic use of graph theory with enhanced simulations methods and ab-initio MD. Accelerated MD simulations are used to construct "enhanced network models", describing the allosteric response over long timescales and capturing the relation between allostery and conformational changes. "Ab-initio network models" combine graph theory with ab-initio MD and quantum mechanics/molecular mechanics (QM/MM) simulations to describe the allosteric regulation of catalysis by following the step-by-step dynamics of biochemical reactions. This approach characterizes how the allosteric regulation changes from reactants to products and how it affects the transition state, revealing a tense-to-relaxed allosteric regulation along the chemical step. Allosteric models and applications are showcased for three paradigmatic examples of allostery in protein-nucleic acid complexes: (i) the nucleosome core particle, (ii) the CRISPR-Cas9 genome editing system and (iii) the spliceosome. These methods and applications create innovative protocols to determine allosteric mechanisms in protein-nucleic acid complexes that show tremendous promise for medicine and bioengineering.},
}
@article {pmid35239244,
year = {2022},
author = {Shimizu, S and Shiraki, A},
title = {CRISPR/Cas9 unveils the dynamics of the endogenous µ-opioid receptors on neuronal cells under continuous opioid stimulation.},
journal = {Pharmacology research &amp; perspectives},
volume = {10},
number = {2},
pages = {e00933},
pmid = {35239244},
issn = {2052-1707},
mesh = {*Analgesics, Opioid/pharmacology ; CRISPR-Cas Systems ; Ligands ; Receptors, Opioid ; *Receptors, Opioid, mu/genetics/metabolism ; },
abstract = {Long-term opioid use develops tolerance and attenuates analgesic effects. Upon activation, µ-opioid receptors (MOPs) are internalized and directed to either recycling or degradation pathway. Ligand stimulation also promotes de novo MOP synthesis. These processes collaboratively regulate MOP expression and play critical roles in tolerance development. However, there is limited understanding of how the endogenous MOP expression changes after prolonged opioid administration because previous analyses have focused on individual processes using overexpression systems, which ignored physiological regulation. Another fundamental problem is the unavailability of commercial antibodies to detect the low expression of endogenous MOP in neuronal systems. Here, we established a neuronal cell line to detect endogenous MOP with sufficient sensitivity using CRISPR/Cas9 technology. We incorporated the hemagglutinin sequence into the MOP gene of the SH-SY5Y cell. The genome-editing did not significantly impair MOP functions such as MOP internalization or the downstream signaling. The clone was differentiated into a state similar to the primary culture undergoing treatment with all-trans retinoic acid, followed by brain-derived neurotrophic factor. Upon continuous stimulation with MOP ligands, endogenous MOP constantly decreased up to 48 h. The expression level was maintained at a certain level following this period, depending on the ligand properties. DAMGO reduced MOP from the cell surface by about 70%, while morphine did so by 40%. Our results indicate that even a few days of opioid administration could significantly reduce the MOP expression level. Our cell line could be a potential tool to investigate the molecular mechanisms underlying the problems caused by long-term opioid use.},
}
@article {pmid35238624,
year = {2022},
author = {Rathbone, T and Ates, I and Fernando, L and Addlestone, E and Lee, CM and Richards, VP and Cottle, RN},
title = {Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {397-409},
pmid = {35238624},
issn = {2573-1602},
support = {P20 GM103499/GM/NIGMS NIH HHS/United States ; P30 GM131959/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Electroporation/methods ; *Gene Editing/methods ; Hepatocytes/metabolism ; Mice ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {Adeno-associated virus vectors are the most used delivery method for liver-directed gene editing. Still, they are associated with significant disadvantages that can compromise the safety and efficacy of therapies. Here, we investigate the effects of electroporating CRISPR-Cas9 as mRNA and ribonucleoproteins (RNPs) into primary hepatocytes regarding on-target activity, specificity, and cell viability. We observed a transfection efficiency of >60% and on-target insertions/deletions (indels) of up to 95% in primary mouse hepatocytes electroporated with Cas9 RNPs targeting Hpd, the gene encoding hydroxyphenylpyruvate dioxygenase. In primary human hepatocytes, we observed on-target indels of 52.4% with Cas9 RNPs and >65% viability after electroporation. These results establish the impact of using electroporation to deliver Cas9 RNPs into primary hepatocytes as a highly efficient and potentially safe approach for therapeutic liver-directed gene editing and the production of liver disease models.},
}
@article {pmid35238621,
year = {2022},
author = {Yu, SY and Birkenshaw, A and Thomson, T and Carlaw, T and Zhang, LH and Ross, CJD},
title = {Increasing the Targeting Scope of CRISPR Base Editing System Beyond NGG.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {187-202},
doi = {10.1089/crispr.2021.0109},
pmid = {35238621},
issn = {2573-1602},
support = {NMIN 2019-T2-05//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; DNA Breaks, Double-Stranded ; *Gene Editing ; Humans ; Nucleotides ; },
abstract = {Genome editing provides a new therapeutic strategy to cure genetic diseases. The recently developed CRISPR-Cas9 base editing technology has shown great potential to repair the majority of pathogenic point mutations in the patient's DNA precisely. Base editor is the fusion of a Cas9 nickase with a base-modifying enzyme that can change a nucleotide on a single strand of DNA without generating double-stranded DNA breaks. However, a major limitation in applying such a system is the prerequisite of a protospacer adjacent motif sequence at the desired position relative to the target site. Progress has been made to increase the targeting scope of base editors by engineering SpCas9 protein variants, establishing systems with broadened editing windows, characterizing new SpCas9 orthologs, and developing prime editing technology. In this review, we discuss recent progress in the development of CRISPR base editing, focusing on its targeting scope, and we provide a workflow for selecting a suitable base editor based on the target nucleotide sequences.},
}
@article {pmid35238619,
year = {2022},
author = {Chen, S and Liu, Z and Lai, L and Li, Z},
title = {Efficient C-to-G Base Editing with Improved Target Compatibility Using Engineered Deaminase-nCas9 Fusions.},
journal = {The CRISPR journal},
volume = {5},
number = {3},
pages = {389-396},
doi = {10.1089/crispr.2021.0124},
pmid = {35238619},
issn = {2573-1602},
mesh = {Adenine ; Animals ; *CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing ; Genome ; Mice ; },
abstract = {CRISPR-guided DNA base editors (BEs) are potent genome editing tools in biotechnology and medicine. However, conventional cytosine and adenine BEs can only induce base transitions (C-to-T and A-to-G) and cannot induce base transversions. Recently, several C-to-G base editors (CGBEs) were generated and applied in human cells. By comparing them, we found that engineered deaminases rather than additional base excision repair proteins significantly improved the C-to-G efficiency. In addition, significant increase in C-to-G transversions in the GC context were determined by using rationally engineered eAID deaminase. The genome-targeting scope of CGBEs were further expanded by using SpRY Cas9 variant, which then successfully induced stop codon (TAC to TAG) to disrupt Tyr gene in mouse embryos. Taken together, these new CGBEs with engineered deaminase-nCas9 fusions broaden the BE toolsets for efficient gene modification and therapeutic applications.},
}
@article {pmid35238530,
year = {2022},
author = {Nouri, R and Dong, M and Politza, AJ and Guan, W},
title = {Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison.},
journal = {ACS sensors},
volume = {7},
number = {3},
pages = {900-911},
pmid = {35238530},
issn = {2379-3694},
support = {R61 AI147419/AI/NIAID NIH HHS/United States ; },
mesh = {Biological Assay ; CRISPR-Cas Systems/genetics ; *Nucleic Acids ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid-sensing systems have grown rapidly in the past few years. Nevertheless, an objective approach to benchmark the performances of different CRISPR sensing systems is lacking due to the heterogeneous experimental setup. Here, we developed a quantitative CRISPR sensing figure of merit (FOM) to compare different CRISPR methods and explore performance improvement strategies. The CRISPR sensing FOM is defined as the product of the limit of detection (LOD) and the associated CRISPR reaction time (T). A smaller FOM means that the method can detect smaller target quantities faster. We found that there is a tradeoff between the LOD of the assay and the required reaction time. With the proposed CRISPR sensing FOM, we evaluated five strategies to improve the CRISPR-based sensing: preamplification, enzymes of higher catalytic efficiency, multiple crRNAs, digitalization, and sensitive readout systems. We benchmarked the FOM performances of 57 existing studies and found that the effectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found that digitalization is the most promising amplification-free method for achieving comparable FOM performances (∼1 fM·min) as those using preamplification. The findings here would have broad implications for further optimization of the CRISPR-based sensing.},
}
@article {pmid35238390,
year = {2022},
author = {Chen, J and Li, S and He, Y and Li, J and Xia, L},
title = {An update on precision genome editing by homology-directed repair in plants.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1780-1794},
pmid = {35238390},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair/genetics ; *Gene Editing ; Recombinational DNA Repair/genetics ; },
abstract = {Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single-nucleotide polymorphisms or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. Clustered regularly interspaced short palindromic repeats-associated endonuclease Cas9 system (CRISPR/Cas9)-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction, and donor delivery, and so on. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement.},
}
@article {pmid35237996,
year = {2022},
author = {De Giorgi, M and Jarrett, KE and de Aguiar Vallim, TQ and Lagor, WR},
title = {In Vivo Gene Editing in Lipid and Atherosclerosis Research.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2419},
number = {},
pages = {673-713},
pmid = {35237996},
issn = {1940-6029},
support = {U42 OD026645/OD/NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; T32 HL069766/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Atherosclerosis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hypercholesterolemia/genetics ; *Hyperlipidemias ; Mice ; },
abstract = {The low-density lipoprotein receptor (Ldlr) and apolipoprotein E (Apoe) germline knockout (KO) models have provided fundamental insights in lipid and atherosclerosis research for decades. However, testing new candidate genes in these models requires extensive breeding, which is highly time and resource consuming. In this chapter, we provide methods for rapidly modeling hypercholesterolemia and atherosclerosis as well as testing new genes in adult mice through somatic gene editing. Adeno-associated viral (AAV) vectors are exploited to deliver the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system (AAV-CRISPR) to the liver. This tool enables rapid and efficient editing of lipid- and atherosclerosis-related genes in the liver.},
}
@article {pmid35237980,
year = {2022},
author = {Xian, X and Wang, Y and Liu, G},
title = {Genetically Engineered Hamster Models of Dyslipidemia and Atherosclerosis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2419},
number = {},
pages = {433-459},
pmid = {35237980},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; *Atherosclerosis/genetics ; CRISPR-Cas Systems ; Cricetinae ; Disease Models, Animal ; *Dyslipidemias/genetics ; Genetic Engineering/methods ; Mice ; Rabbits ; Rats ; Swine ; },
abstract = {Animal models of human diseases play an extremely important role in biomedical research. Among them, mice are widely used animal models for translational research, especially because of ease of generation of genetically engineered mice. However, because of the great differences in biology between mice and humans, translation of findings to humans remains a major issue. Therefore, the exploration of models with biological and metabolic characteristics closer to those of humans has never stopped.Although pig and nonhuman primates are biologically similar to humans, their genetic engineering is technically difficult, the cost of breeding is high, and the experimental time is long. As a result, the application of these species as model animals, especially genetically engineered model animals, in biomedical research is greatly limited.In terms of lipid metabolism and cardiovascular diseases, hamsters have several characteristics different from rats and mice, but similar to those in humans. The hamster is therefore an ideal animal model for studying lipid metabolism and cardiovascular disease because of its small size and short reproduction period. However, the phenomenon of zygote division, which was unexpectedly blocked during the manipulation of hamster embryos for some unknown reasons, had plagued researchers for decades and no genetically engineered hamsters have therefore been generated as animal models of human diseases for a long time. After solving the problem of in vitro development of hamster zygotes, we successfully prepared enhanced green fluorescent protein (eGFP) transgenic hamsters by microinjection of lentiviral vectors into the zona pellucida space of zygotes. On this basis, we started the development of cardiovascular disease models using the hamster embryo culture system combined with the novel genome editing technique of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9). In this chapter, we will introduce some of the genetically engineered hamster models with dyslipidemia and the corresponding characteristics of these models. We hope that the genetically engineered hamster models can be further recognized and complement other genetically engineered animal models such as mice, rats, and rabbits. This will lead to new avenues and pathways for the study of lipid metabolism and its related diseases.},
}
@article {pmid35237818,
year = {2021},
author = {Guiziou, S and Chu, JC and Nemhauser, JL},
title = {Decoding and recoding plant development.},
journal = {Plant physiology},
volume = {187},
number = {2},
pages = {515-526},
pmid = {35237818},
issn = {1532-2548},
support = {R01 GM107084/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; *Cell Lineage ; *Gene Editing ; Genetic Engineering ; Integrases/genetics ; Molecular Biology ; Plant Development/genetics/*physiology ; *Synthetic Biology ; *Systems Biology ; },
abstract = {The development of multicellular organisms has been studied for centuries, yet many critical events and mechanisms of regulation remain challenging to observe directly. Early research focused on detailed observational and comparative studies. Molecular biology has generated insights into regulatory mechanisms, but only for a limited number of species. Now, synthetic biology is bringing these two approaches together, and by adding the possibility of sculpting novel morphologies, opening another path to understanding biology. Here, we review a variety of recently invented techniques that use CRISPR/Cas9 and phage integrases to trace the differentiation of cells over various timescales, as well as to decode the molecular states of cells in high spatiotemporal resolution. Most of these tools have been implemented in animals. The time is ripe for plant biologists to adopt and expand these approaches. Here, we describe how these tools could be used to monitor development in diverse plant species, as well as how they could guide efforts to recode programs of interest.},
}
@article {pmid35237805,
year = {2021},
author = {Ali, Z and Mahfouz, MM},
title = {CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond.},
journal = {Plant physiology},
volume = {186},
number = {4},
pages = {1770-1785},
pmid = {35237805},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/virology ; Disease Resistance/*genetics ; Gene Editing/methods ; Plant Breeding/*methods ; Plant Diseases/*genetics/*virology ; Plant Immunity/*genetics ; Plant Viruses/*pathogenicity ; },
abstract = {Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics.},
}
@article {pmid35236982,
year = {2022},
author = {Bravo, JPK and Liu, MS and Hibshman, GN and Dangerfield, TL and Jung, K and McCool, RS and Johnson, KA and Taylor, DW},
title = {Structural basis for mismatch surveillance by CRISPR-Cas9.},
journal = {Nature},
volume = {603},
number = {7900},
pages = {343-347},
pmid = {35236982},
issn = {1476-4687},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/chemistry/genetics ; *DNA Mismatch Repair ; *Gene Editing ; Nucleic Acid Conformation ; *RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage[1-4], and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood[5-7]. Although Cas9 variants with greater discrimination against mismatches have been designed[8-10], these suffer from substantially reduced rates of on-target DNA cleavage[5,11]. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.},
}
@article {pmid35236841,
year = {2022},
author = {Smargon, AA and Madrigal, AA and Yee, BA and Dong, KD and Mueller, JR and Yeo, GW},
title = {Crosstalk between CRISPR-Cas9 and the human transcriptome.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1125},
pmid = {35236841},
issn = {2041-1723},
support = {R01 EY029166/EY/NEI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; RNA Editing ; *RNA, Guide/metabolism ; Transcriptome ; },
abstract = {CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a cellular and RNA impact likely inherent to CRISPR-Cas systems.},
}
@article {pmid35236570,
year = {2022},
author = {Lee, H and Sashital, DG},
title = {Creating memories: molecular mechanisms of CRISPR adaptation.},
journal = {Trends in biochemical sciences},
volume = {47},
number = {6},
pages = {464-476},
pmid = {35236570},
issn = {0968-0004},
support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; R35 GM140876/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Prokaryotes use clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) proteins as an adaptive immune system. CRISPR-Cas systems preserve molecular memories of infections by integrating short fragments of foreign nucleic acids as spacers into the host CRISPR array in a process termed 'adaptation'. Functional spacers ensure a robust immune response by Cas effectors, which neutralizes subsequent infection through RNA-guided interference pathways. In this review, we summarize recent discoveries that have advanced our understanding of adaptation, with a focus on how functional spacers are generated and incorporated through many widespread, but type-specific, mechanisms. Finally, we highlight future directions and outstanding questions for a more thorough understanding of CRISPR adaptation.},
}
@article {pmid35236300,
year = {2022},
author = {Krohannon, A and Srivastava, M and Rauch, S and Srivastava, R and Dickinson, BC and Janga, SC},
title = {CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {172},
pmid = {35236300},
issn = {1471-2164},
support = {R01 GM123314/GM/NIGMS NIH HHS/United States ; R01 MH122142/MH/NIMH NIH HHS/United States ; R35 GM119840/GM/NIGMS NIH HHS/United States ; R01GM123314/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; HEK293 Cells ; HeLa Cells ; Humans ; *RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {BACKGROUND: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited.<br><br>RESULTS: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner.<br><br>CONCLUSIONS: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.},
}
@article {pmid35235944,
year = {2022},
author = {Economos, NG and Quijano, E and Carufe, KEW and Perera, JDR and Glazer, PM},
title = {Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation.},
journal = {Nucleic acids research},
volume = {50},
number = {10},
pages = {e59},
pmid = {35235944},
issn = {1362-4962},
support = {U01 AI145965/AI/NIAID NIH HHS/United States ; F30 HL149185/HL/NHLBI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; R01 HL139756/HL/NHLBI NIH HHS/United States ; R35 CA197574/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; *Peptide Nucleic Acids/pharmacology ; *RNA, Guide/genetics ; },
abstract = {Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or 'antispacer', PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.},
}
@article {pmid35235462,
year = {2022},
author = {Bakkers, MJG and Moon-Walker, A and Herlo, R and Brusic, V and Stubbs, SH and Hastie, KM and Saphire, EO and Kirchhausen, TL and Whelan, SPJ},
title = {CD164 is a host factor for lymphocytic choriomeningitis virus entry.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {10},
pages = {e2119676119},
pmid = {35235462},
issn = {1091-6490},
support = {F31 AI154700/AI/NIAID NIH HHS/United States ; T32 AI007245/AI/NIAID NIH HHS/United States ; T32 AI125179/AI/NIAID NIH HHS/United States ; U19 AI109740/AI/NIAID NIH HHS/United States ; },
mesh = {A549 Cells ; CRISPR-Cas Systems ; Endolyn/physiology ; Gene Editing ; HEK293 Cells ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Hydrogen-Ion Concentration ; Lymphocytic choriomeningitis virus/pathogenicity/*physiology ; Membrane Fusion ; Virulence Factors ; *Virus Internalization ; },
abstract = {Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne zoonotic arenavirus that causes congenital abnormalities and can be fatal for transplant recipients. Using a genome-wide loss-of-function screen, we identify host factors required for LCMV entry into cells. We identify the lysosomal mucin CD164, glycosylation factors, the heparan sulfate biosynthesis machinery, and the known receptor alpha-dystroglycan (α-DG). Biochemical analysis revealed that the LCMV glycoprotein binds CD164 at acidic pH and requires a sialylated glycan at residue N104. We demonstrate that LCMV entry proceeds by the virus switching binding from heparan sulfate or α-DG at the plasma membrane to CD164 prior to membrane fusion, thus identifying additional potential targets for therapeutic intervention.},
}
@article {pmid35235150,
year = {2022},
author = {Li, G and Li, X and Zhuang, S and Wang, L and Zhu, Y and Chen, Y and Sun, W and Wu, Z and Zhou, Z and Chen, J and Huang, X and Wang, J and Li, D and Li, W and Wang, H and Wei, W},
title = {Gene editing and its applications in biomedicine.},
journal = {Science China. Life sciences},
volume = {65},
number = {4},
pages = {660-700},
pmid = {35235150},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Humans ; },
abstract = {The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.},
}
@article {pmid35234927,
year = {2022},
author = {O'Geen, H and Tomkova, M and Combs, JA and Tilley, EK and Segal, DJ},
title = {Determinants of heritable gene silencing for KRAB-dCas9 + DNMT3 and Ezh2-dCas9 + DNMT3 hit-and-run epigenome editing.},
journal = {Nucleic acids research},
volume = {50},
number = {6},
pages = {3239-3253},
pmid = {35234927},
issn = {1362-4962},
support = {P30 CA093373/CA/NCI NIH HHS/United States ; R21 HG010559/HG/NHGRI NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chromatin ; DNA Methylation/genetics ; Epigenesis, Genetic ; *Epigenome ; *Gene Editing/methods ; Gene Silencing ; },
abstract = {Precision epigenome editing has gained significant attention as a method to modulate gene expression without altering genetic information. However, a major limiting factor has been that the gene expression changes are often transient, unlike the life-long epigenetic changes that occur frequently in nature. Here, we systematically interrogate the ability of CRISPR/dCas9-based epigenome editors (Epi-dCas9) to engineer persistent epigenetic silencing. We elucidated cis regulatory features that contribute to the differential stability of epigenetic reprogramming, such as the active transcription histone marks H3K36me3 and H3K27ac strongly correlating with resistance to short-term repression and resistance to long-term silencing, respectively. H3K27ac inversely correlates with increased DNA methylation. Interestingly, the dependance on H3K27ac was only observed when a combination of KRAB-dCas9 and targetable DNA methyltransferases (DNMT3A-dCas9 + DNMT3L) was used, but not when KRAB was replaced with the targetable H3K27 histone methyltransferase Ezh2. In addition, programmable Ezh2/DNMT3A + L treatment demonstrated enhanced engineering of localized DNA methylation and was not sensitive to a divergent chromatin state. Our results highlight the importance of local chromatin features for heritability of programmable silencing and the differential response to KRAB- and Ezh2-based epigenetic editing platforms. The information gained in this study provides fundamental insights into understanding contextual cues to more predictably engineer persistent silencing.},
}
@article {pmid35234622,
year = {2022},
author = {Zhang, Z and Hou, W and Chen, S},
title = {Updates on CRISPR-based gene editing in HIV-1/AIDS therapy.},
journal = {Virologica Sinica},
volume = {37},
number = {1},
pages = {1-10},
pmid = {35234622},
issn = {1995-820X},
mesh = {*Acquired Immunodeficiency Syndrome/therapy ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Genome, Human ; *HIV-1/genetics ; Humans ; },
abstract = {Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.},
}
@article {pmid35234463,
year = {2022},
author = {Wan, Y and Zong, C and Li, X and Wang, A and Li, Y and Yang, T and Bao, Q and Dubow, M and Yang, M and Rodrigo, LA and Mao, C},
title = {New Insights for Biosensing: Lessons from Microbial Defense Systems.},
journal = {Chemical reviews},
volume = {122},
number = {9},
pages = {8126-8180},
doi = {10.1021/acs.chemrev.1c01063},
pmid = {35234463},
issn = {1520-6890},
support = {BB/R01602X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; *Nanopores ; *Nucleic Acids ; Proteins ; },
abstract = {Microorganisms have gained defense systems during the lengthy process of evolution over millions of years. Such defense systems can protect them from being attacked by invading species (e.g., CRISPR-Cas for establishing adaptive immune systems and nanopore-forming toxins as virulence factors) or enable them to adapt to different conditions (e.g., gas vesicles for achieving buoyancy control). These microorganism defense systems (MDS) have inspired the development of biosensors that have received much attention in a wide range of fields including life science research, food safety, and medical diagnosis. This Review comprehensively analyzes biosensing platforms originating from MDS for sensing and imaging biological analytes. We first describe a basic overview of MDS and MDS-inspired biosensing platforms (e.g., CRISPR-Cas systems, nanopore-forming proteins, and gas vesicles), followed by a critical discussion of their functions and properties. We then discuss several transduction mechanisms (optical, acoustic, magnetic, and electrical) involved in MDS-inspired biosensing. We further detail the applications of the MDS-inspired biosensors to detect a variety of analytes (nucleic acids, peptides, proteins, pathogens, cells, small molecules, and metal ions). In the end, we propose the key challenges and future perspectives in seeking new and improved MDS tools that can potentially lead to breakthrough discoveries in developing a new generation of biosensors with a combination of low cost; high sensitivity, accuracy, and precision; and fast detection. Overall, this Review gives a historical review of MDS, elucidates the principles of emulating MDS to develop biosensors, and analyzes the recent advancements, current challenges, and future trends in this field. It provides a unique critical analysis of emulating MDS to develop robust biosensors and discusses the design of such biosensors using elements found in MDS, showing that emulating MDS is a promising approach to conceptually advancing the design of biosensors.},
}
@article {pmid35234398,
year = {2022},
author = {Lu, H and Zhang, Q and Yu, S and Wang, Y and Kang, M and Han, S and Liu, Y and Wang, M},
title = {[Optimization of CRISPR/Cas9-based multiplex base editing in Corynebacterium glutamicum].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {2},
pages = {780-795},
doi = {10.13345/j.cjb.210109},
pmid = {35234398},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; *Corynebacterium glutamicum/genetics/metabolism ; Gene Editing ; Plasmids ; RNA, Guide/genetics/metabolism ; },
abstract = {As a new CRISPR/Cas-derived genome engineering technology, base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs, requiring exogenous template, or depending on homologous recombination. Recently, researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum, and achieved simultaneous editing of two and three genes. However, the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs, interference of repeated sequence and difficulty of target loci replacement. In this study, multiplex base editing in C. glutamicum was optimized by the following strategies. Firstly, the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized. The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method, which also avoids the interference of repeated sequence. Although the multiple sgRNAs structure is still complicated, the editing efficiency of this strategy is the highest. Then, the multiple gRNA expression cassettes based on Type Ⅱ CRISPR crRNA arrays and tRNA processing were developed. The two strategies only require one single promoter and terminator, and greatly simplify the structure of the expression cassette. Although the editing efficiency has decreased, both methods are still applicable. Taken together, this study provides a powerful addition to the genome editing toolbox of C. glutamicum and facilitates genetic modification of this strain.},
}
@article {pmid35234393,
year = {2022},
author = {Yang, Y and Li, N and Zhou, J and Chen, J},
title = {[A CRISPR/dCpf1-based transcriptional repression system for Gluconobacter oxydans].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {2},
pages = {719-736},
doi = {10.13345/j.cjb.210157},
pmid = {35234393},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene Expression ; *Gluconobacter oxydans/genetics ; Metabolic Engineering ; },
abstract = {Gluconobacter oxydans are widely used in industrial due to its ability of oxidizing carbohydrate rapidly. However, the limited gene manipulation methods and less of efficient gene editing tools impose restrictions on its application in industrial production. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been widely used in genome editing and transcriptional regulation which improves the efficiency of genome editing greatly. Here we constructed a CRISPR/dCpf1-mediated gene transcriptional repression system, the expression of a nuclease inactivation Cpf1 protein (dCpf1) in Gluconobacter oxydans together with a 19 nt direct repeats showed effective repression in gene transcription. This system in single gene repression had strong effect and the relative repression level had been increased to 97.9%. While it could be applied in multiplex gene repression which showed strong repression ability at the same time. Furthermore, this system was used in the metabolic pathway of L-sorbose and the regulatory of respiratory chain. The development of CRISPR transcriptional repression system effectively covered the shortage of current gene regulation methods in G. oxydans and provided an efficient gene manipulation tool for metabolic engineering modification in G. oxydans.},
}
@article {pmid35233982,
year = {2022},
author = {Eun, K and Hwang, SU and Kim, M and Yoon, JD and Kim, E and Choi, H and Kim, G and Jeon, HY and Kim, JK and Kim, JY and Hong, N and Park, MG and Jang, J and Jeong, HJ and Kim, SJ and Ko, BW and Lee, SC and Kim, H and Hyun, SH},
title = {Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100434},
doi = {10.1002/biot.202100434},
pmid = {35233982},
issn = {1860-7314},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; Female ; Fibroblasts/metabolism ; Gene Knockout Techniques ; Humans ; Male ; *Nuclear Transfer Techniques ; Oncogenes ; Swine/genetics ; },
abstract = {Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRAS[G12V] . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.},
}
@article {pmid35233628,
year = {2021},
author = {Bischof, S},
title = {Which factors shape the rice DNA methylome?.},
journal = {The Plant cell},
volume = {33},
number = {9},
pages = {2904-2905},
pmid = {35233628},
issn = {1532-298X},
mesh = {CRISPR-Cas Systems ; DNA ; DNA Methylation/genetics ; *Epigenome ; Methyltransferases/genetics ; *Oryza/genetics ; },
}
@article {pmid35232993,
year = {2022},
author = {Rezazade Bazaz, M and Ghahramani Seno, MM and Dehghani, H},
title = {Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {3390},
pmid = {35232993},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knock-In Techniques ; *Genome, Human ; Humans ; Mammals/metabolism ; Plasmids ; *Transposases/genetics/metabolism ; },
abstract = {Various methods have been used in targeted gene knock-in applications. CRISPR-based knock-in strategies based on homology-independent repair pathways such as CRISPR HITI have been shown to possess the best efficiency for gene knock-in in mammalian cells. However, these methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. In this study, we introduce a new homology-independent knock-in strategy, Transposase-CRISPR mediated Targeted Integration (TransCRISTI), that exploits a fusion of Cas9 nuclease and a double mutant piggyBac transposase. In isogenic mammalian cell lines, we show that the TransCRISTI method demonstrates higher efficiency (72%) for site-specific insertions than the CRISPR HITI (44%) strategy. Application of the TransCRISTI method resulted in site-directed integration in 4.13% and 3.69% of the initially transfected population in the human AAVS1and PML loci, respectively, while the CRISPR HITI strategy resulted in site-directed integration in the PML locus in only 0.6% of cells. We also observed lower off-target and random insertions in the TransCRISTI group than the CRISPR HITI group. The TransCRISTI technology represents a great potential for the accurate and high-efficiency knock-in of the desired transposable elements into the predetermined genomic locations.},
}
@article {pmid35232966,
year = {2022},
author = {Schene, IF and Joore, IP and Baijens, JHL and Stevelink, R and Kok, G and Shehata, S and Ilcken, EF and Nieuwenhuis, ECM and Bolhuis, DP and van Rees, RCM and Spelier, SA and van der Doef, HPJ and Beekman, JM and Houwen, RHJ and Nieuwenhuis, EES and Fuchs, SA},
title = {Mutation-specific reporter for optimization and enrichment of prime editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1028},
pmid = {35232966},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome ; Humans ; Mutation ; RNA, Guide/genetics ; },
abstract = {Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.},
}
@article {pmid35232910,
year = {2021},
author = {Pan, S and Zhang, H},
title = {Discovery in CRISPR-Cas9 system.},
journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences},
volume = {46},
number = {12},
pages = {1392-1402},
doi = {10.11817/j.issn.1672-7347.2021.210169},
pmid = {35232910},
issn = {1672-7347},
mesh = {Animals ; Bacteria/genetics ; *CRISPR-Cas Systems ; DNA ; Gene Editing ; *Neoplasms/genetics ; },
abstract = {The 2020 Nobel Prize in Chemistry was awarded to the American scientist Jennifer A. Doudna and the French scientist Emmanuelle Charpentier, in recognition of their discovery in one of the greatest weapons in genetic technology: CRISPR-Cas9 gene scissors. The CRISPR-Cas system is a bacterial defense immune system against exogenous genetic material. Because the system can specifically recognize and cut DNA, this technology is widely used for precise editing of animal, plant, and microbial DNA. The discovery of CRISPR-Cas9 gene scissors enables the tedious and complicated cell gene editing work to be completed in a few weeks or even less, which has promoted the development of gene editing technology in various fields and brought revolutionary influence to the field of life sciences. At the same time, CRISPR gene editing technology has become one of the new therapies for tumors because of its large number of targets and relatively simple operation, and it also makes gene therapy possible. Although the technology still needs to solve technical problems such as off-target and promoter inefficiency, the CRISPR-Cas system will show its unique advantages in more fields with the continuous development of life science and basic medicine.},
}
@article {pmid35230602,
year = {2022},
author = {Hurley, A and Lagor, WR},
title = {Treating Cardiovascular Disease with Liver Genome Engineering.},
journal = {Current atherosclerosis reports},
volume = {24},
number = {2},
pages = {75-84},
pmid = {35230602},
issn = {1534-6242},
support = {R01 DK124477/DK/NIDDK NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; U42 OD026645/OD/NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing/methods ; Humans ; Liver ; Mice ; },
abstract = {PURPOSE OF REVIEW: This review examines recent progress in somatic genome editing for cardiovascular disease. We briefly highlight new gene editing approaches, delivery systems, and potential targets in the liver.<br><br>RECENT FINDINGS: In recent years, new editing and delivery systems have been applied successfully in model organisms to modify genes within hepatocytes. Disruption of several genes has been shown to dramatically lower plasma cholesterol and triglyceride levels in mice as well as non-human primates. More precise modification of cardiovascular targets has also been achieved through homology-directed repair or base editing. Improved viral vectors and nanoparticle delivery systems are addressing important delivery challenges and helping to mitigate safety concerns. Liver-directed genome editing has the potential to cure both rare and common forms of cardiovascular disease. Exciting progress is already being made, including promising results from preclinical studies and the initiation of human gene therapy trials.},
}
@article {pmid35228726,
year = {2022},
author = {Wang, J and He, Z and Wang, G and Zhang, R and Duan, J and Gao, P and Lei, X and Qiu, H and Zhang, C and Zhang, Y and Yin, H},
title = {Efficient targeted insertion of large DNA fragments without DNA donors.},
journal = {Nature methods},
volume = {19},
number = {3},
pages = {331-340},
pmid = {35228726},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing ; Genome ; Genomics ; *RNA, Guide/genetics ; },
abstract = {Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.},
}
@article {pmid35228123,
year = {2022},
author = {Beppu, K and Tsutsumi, R and Ansai, S and Ochiai, N and Terakawa, M and Mori, M and Kuroda, M and Horikawa, K and Tomoi, T and Sakamoto, J and Kamei, Y and Naruse, K and Sakaue, H},
title = {Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka.},
journal = {Biochemical and biophysical research communications},
volume = {601},
number = {},
pages = {65-72},
doi = {10.1016/j.bbrc.2022.02.082},
pmid = {35228123},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dysgeusia/genetics ; Gene Expression ; Green Fluorescent Proteins/genetics/metabolism ; *Oryzias/genetics ; Quality of Life ; Taste ; },
abstract = {Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.},
}
@article {pmid35228065,
year = {2022},
author = {Najafi, S and Tan, SC and Aghamiri, S and Raee, P and Ebrahimi, Z and Jahromi, ZK and Rahmati, Y and Sadri Nahand, J and Piroozmand, A and Jajarmi, V and Mirzaei, H},
title = {Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {148},
number = {},
pages = {112743},
pmid = {35228065},
issn = {1950-6007},
mesh = {COVID-19/therapy ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genome, Viral ; HIV Infections/therapy ; Hepatitis B/therapy ; Herpesviridae Infections/therapy ; Humans ; Papillomavirus Infections/therapy ; SARS-CoV-2 ; Virus Diseases/*therapy ; },
abstract = {Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.},
}
@article {pmid35227602,
year = {2022},
author = {Lee, E and Shah, D and Porteus, M and Wright, JF and Bacchetta, R},
title = {Design of experiments as a decision tool for cell therapy manufacturing.},
journal = {Cytotherapy},
volume = {24},
number = {6},
pages = {590-596},
doi = {10.1016/j.jcyt.2022.01.009},
pmid = {35227602},
issn = {1477-2566},
mesh = {*CRISPR-Cas Systems/genetics ; Cell- and Tissue-Based Therapy ; Gene Editing ; Humans ; Prospective Studies ; *RNA, Guide/genetics ; },
abstract = {BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost.<br><br>METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4[+] T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.<br><br>RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50&#x2006;000-750&#x2006;000 vg/cell AAV and 0.8-4 &#x3bc;g sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193&#x2006;000 vg/cell AAV and 1.78 &#x3bc;g sgRNA.<br><br>CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.},
}
@article {pmid35227160,
year = {2022},
author = {Chattopadhyay, I and J, RB and Usman, TMM and Varjani, S},
title = {Exploring the role of microbial biofilm for industrial effluents treatment.},
journal = {Bioengineered},
volume = {13},
number = {3},
pages = {6420-6440},
pmid = {35227160},
issn = {2165-5987},
mesh = {*Biodegradation, Environmental ; *Biofilms ; CRISPR-Cas Systems ; Gene Editing ; Genome, Bacterial/genetics ; *Industrial Waste ; *Quorum Sensing ; *Waste Water ; },
abstract = {Biofilm formation on biotic or abiotic surfaces is caused by microbial cells of a single or heterogeneous species. Biofilm protects microbes from stressful environmental conditions, toxic action of chemicals, and antimicrobial substances. Quorum sensing (QS) is the generation of autoinducers (AIs) by bacteria in a biofilm to communicate with one other. QS is responsible for the growth of biofilm, synthesis of exopolysaccharides (EPS), and bioremediation of environmental pollutants. EPS is used for wastewater treatment due to its three-dimensional matrix which is composed of proteins, polysaccharides, humic-like substances, and nucleic acids. Autoinducers mediate significantly the degradation of environmental pollutants. Acyl-homoserine lactone (AHL) producing bacteria as well as quorum quenching enzyme or bacteria can effectively improve the performance of wastewater treatment. Biofilms-based reactors due to their economic and ecofriendly nature are used for the treatment of industrial wastewaters. Electrodes coated with electro-active biofilm (EAB) which are obtained from sewage sludge, activated sludge, or industrial and domestic effluents are getting popularity in bioremediation. Microbial fuel cells are involved in wastewater treatment and production of energy from wastewater. Synthetic biological systems such as genome editing by CRISPR-Cas can be used for the advanced bioremediation process through modification of metabolic pathways in quorum sensing within microbial communities. This narrative review discusses the impacts of QS regulatory approaches on biofilm formation, extracellular polymeric substance synthesis, and role of microbial community in bioremediation of pollutants from industrial effluents.},
}
@article {pmid35225758,
year = {2022},
author = {Jin, CZ and Jin, L and Liu, MJ and Kang, MK and Park, SH and Park, DJ and Kim, CJ},
title = {Salinarimonas soli sp. nov., isolated from soil.},
journal = {International journal of systematic and evolutionary microbiology},
volume = {72},
number = {2},
pages = {},
doi = {10.1099/ijsem.0.005095},
pmid = {35225758},
issn = {1466-5034},
mesh = {Alphaproteobacteria/*classification/isolation &amp; purification ; Bacterial Typing Techniques ; Base Composition ; DNA, Bacterial/genetics ; Fatty Acids/chemistry ; Phospholipids/chemistry ; *Phylogeny ; RNA, Ribosomal, 16S/genetics ; Republic of Korea ; Sequence Analysis, DNA ; *Soil Microbiology ; Ubiquinone/analogs &amp; derivatives/chemistry ; },
abstract = {A light pink coloured bacterium, designated strain BN140002[T], was isolated from a soil sample collected in Goesan-gun, Chungcheongbuk-do, Republic of Korea. Cells of strain BN140002[T] were Gram-stain-negative, aerobic, motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed 94.7, 94.7, 93.9, 93.3, 93.4 and 93.0% similarities to Salinarimonas rosea KCTC 22346[T], Salinarimonas ramus DSM 22962[T], Saliniramus fredricksonii HL-109[T], Microvirga soli R491[T], Chelatococcus caeni EBR-4-1[T] and Chelatococcus composti PC-2[T], respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 1 (C12 : 0 aldehyde and/or unknown 10.98) and the predominant ubiquinone was Q-10. The genomic DNA G+C content of strain BN140002[T] was 70.1 mol%. The genomic orthoANI values between strain BN140002[T] and Salinarimonas rosea KCTC 22346[T] and Salinarimonas ramus DSM 22962[T] were 75.0 and 74.8 %, respectively. Strain BN140002[T] had a class I-C type CRISPR-Cas system (CRISPR-associated helicase Cas3, CRISPR-associated protein Cas8c, CRISPR-associated protein Cas7, CRISPR-associated RecB family exonuclease Cas4, CRISPR-associated protein 1, 2). Based on phenotypic, chemotaxonomic and phylogenetic data, strain BN140002[T] should be assigned as a novel species of the genus Salinarimonas, for which the name Salinarimonas soli sp. nov. is proposed. The type strain is BN140002[T] (=KCTC 42643[T]=CCTCC AB 2017173[T]).},
}
@article {pmid35225591,
year = {2022},
author = {Kotikam, V and Gajula, PK and Coyle, L and Rozners, E},
title = {Amide Internucleoside Linkages Are Well Tolerated in Protospacer Adjacent Motif-Distal Region of CRISPR RNAs.},
journal = {ACS chemical biology},
volume = {17},
number = {3},
pages = {509-512},
pmid = {35225591},
issn = {1554-8937},
support = {R35 GM130207/GM/NIGMS NIH HHS/United States ; },
mesh = {*Amides/chemistry ; CRISPR-Cas Systems/genetics ; DNA Cleavage ; *Gene Editing/methods ; RNA Interference ; RNA, Small Interfering/chemistry ; },
abstract = {The development of CRISPR-Cas9 mediated gene editing technology is revolutionizing molecular biology, biotechnology, and medicine. However, as with other nucleic acid technologies, CRISPR would greatly benefit from chemical modifications that optimize delivery, activity, and specificity of gene editing. Amide modifications at certain positions of short interfering RNAs have been previously shown to improve their RNAi activity and specificity, which motivated the current study on replacement of selected internucleoside phosphates of CRISPR RNAs with amide linkages. Herein, we show that amide modifications did not interfere with CRISPR-Cas9 activity when placed in the protospacer adjacent motif (PAM) distal region of CRISPR RNAs. In contrast, modification of the seed region led to a loss of DNA cleavage activity at most but not all positions. These results are encouraging for future studies on amides as backbone modifications in CRISPR RNAs.},
}
@article {pmid35225354,
year = {2022},
author = {Sun, Y and Zhang, Y and Zhang, D and Wang, G and Song, L and Liu, Z},
title = {In vivo CRISPR-Cas9-mediated DNA chop identifies a cochlear outer hair cell-specific enhancer.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {36},
number = {4},
pages = {e22233},
doi = {10.1096/fj.202100421RR},
pmid = {35225354},
issn = {1530-6860},
mesh = {Animals ; *CRISPR-Cas Systems ; Cochlea/*metabolism ; *Enhancer Elements, Genetic ; Female ; Green Fluorescent Proteins/genetics/metabolism ; Hair Cells, Auditory, Outer/*metabolism ; Humans ; *Introns ; Male ; Mice ; Mice, Transgenic ; Molecular Motor Proteins/genetics/*metabolism ; Sulfate Transporters/antagonists &amp; inhibitors/*genetics/metabolism ; },
abstract = {Cochlear outer hair cells (OHCs) are essential for hearing. A short, OHC-specific enhancer is necessary but not yet available for gene therapeutic applications in OHC damage. Such damage is a major cause of deafness. Prestin is a motor protein exclusively expressed in OHCs. We hypothesized that the cis-regulatory DNA fragment deletion of Slc26a5 would affect its expression. We tested this hypothesis by conducting CRISPR/Cas9-mediated large DNA fragment deletion of mouse Slc26a5 intron regions. First, starting from a ~13 kbp fragment, step-by-step, we narrowed down the sequence to a 1.4 kbp segment. By deleting either a 13 kbp or 1.4 kbp fragment, we observed delayed Prestin expression. Second, we showed that 1.4 kbp was an OHC-specific enhancer because enhanced green fluorescent protein (EGFP) was highly and specifically expressed in OHCs in a transgenic mouse where EGFP was driven by the 1.4 kbp segment. More importantly, specific EGFP was also driven by its homologous 398 bp fragment in human Slc26a5. This suggests that the enhancer is likely to be evolutionarily conserved across different species.},
}
@article {pmid35225345,
year = {2022},
author = {Puchta, H and Jiang, J and Wang, K and Zhao, Y},
title = {Updates on gene editing and its applications.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1725-1730},
pmid = {35225345},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
}
@article {pmid35224232,
year = {2022},
author = {Huang, C and Wang, C and Luo, Y},
title = {Research progress of pathway and genome evolution in microbes.},
journal = {Synthetic and systems biotechnology},
volume = {7},
number = {1},
pages = {648-656},
pmid = {35224232},
issn = {2405-805X},
abstract = {Microbes can produce valuable natural products widely applied in medicine, food and other important fields. Nevertheless, it is usually challenging to achieve ideal industrial yields due to low production rate and poor toxicity tolerance. Evolution is a constant mutation and adaptation process used to improve strain performance. Generally speaking, the synthesis of natural products in microbes is often intricate, involving multiple enzymes or multiple pathways. Individual evolution of a certain enzyme often fails to achieve the desired results, and may lead to new rate-limiting nodes that affect the growth of microbes. Therefore, it is inevitable to evolve the biosynthetic pathways or the whole genome. Here, we reviewed the pathway-level evolution including multi-enzyme evolution, regulatory elements engineering, and computer-aided engineering, as well as the genome-level evolution based on several tools, such as genome shuffling and CRISPR/Cas systems. Finally, we also discussed the major challenges faced by in vivo evolution strategies and proposed some potential solutions.},
}
@article {pmid35222448,
year = {2021},
author = {Nguyen, DV and Hoang, TT and Le, NT and Tran, HT and Nguyen, CX and Moon, YH and Chu, HH and Do, PT},
title = {An Efficient Hairy Root System for Validation of Plant Transformation Vector and CRISPR/Cas Construct Activities in Cucumber (Cucumis sativus L.).},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {770062},
pmid = {35222448},
issn = {1664-462X},
abstract = {Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD650 at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.},
}
@article {pmid35222299,
year = {2021},
author = {Wentz, TG and Tremblay, BJM and Bradshaw, M and Doxey, AC and Sharma, SK and Sauer, JD and Pellett, S},
title = {Endogenous CRISPR-Cas Systems in Group I Clostridium botulinum and Clostridium sporogenes Do Not Directly Target the Botulinum Neurotoxin Gene Cluster.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {787726},
pmid = {35222299},
issn = {1664-302X},
support = {R01 AI139306/AI/NIAID NIH HHS/United States ; R21 AI144060/AI/NIAID NIH HHS/United States ; },
abstract = {Most strains of proteolytic group I Clostridium botulinum (G1 C. botulinum) and some strains of Clostridium sporogenes possess genes encoding botulinum neurotoxin (BoNT), a potent neuroparalytic agent. Within G1 C. botulinum, conserved bont gene clusters of three major toxin serotypes (bont/A/B/F) can be found on conjugative plasmids and/or within chromosomal pathogenicity islands. CRISPR-Cas systems enable site-specific targeting of previously encountered mobile genetic elements (MGE) such as plasmids and bacteriophage through the creation of a spacer library complementary to protospacers within the MGEs. To examine whether endogenous CRISPR-Cas systems restrict the transfer of bont gene clusters across strains we conducted a bioinformatic analysis profiling endogenous CRISPR-Cas systems from 241 G1 C. botulinum and C. sporogenes strains. Approximately 6,200 CRISPR spacers were identified across the strains and Type I-B, III-A/B/D cas genes and CRISPR array features were identified in 83% of the strains. Mapping the predicted spacers against the masked strain and RefSeq plasmid dataset identified 56,000 spacer-protospacer matches. While spacers mapped heavily to targets within bont(+) plasmids, no protospacers were identified within the bont gene clusters. These results indicate the toxin is not a direct target of CRISPR-Cas but the plasmids predominantly responsible for its mobilization are. Finally, while the presence of a CRISPR-Cas system did not reliably indicate the presence or absence of a bont gene cluster, comparative genomics across strains indicates they often occupy the same hypervariable loci common to both species, potentially suggesting similar mechanisms are involved in the acquisition and curation of both genomic features.},
}
@article {pmid35221320,
year = {2022},
author = {Dorgalaleh, A and Kiani, J and Zaker, F and Safa, M},
title = {The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.},
journal = {Blood coagulation &amp; fibrinolysis : an international journal in haemostasis and thrombosis},
volume = {33},
number = {3},
pages = {153-158},
doi = {10.1097/MBC.0000000000001126},
pmid = {35221320},
issn = {1473-5733},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli ; Factor XIII/genetics ; *Factor XIII Deficiency/genetics/therapy ; *Gene Editing ; Humans ; Mutation ; },
abstract = {Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.},
}
@article {pmid35219699,
year = {2022},
author = {Ganguly, K and Cox, JL and Ghersi, D and Grandgenett, PM and Hollingsworth, MA and Jain, M and Kumar, S and Batra, SK},
title = {Mucin 5AC-Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity.},
journal = {Gastroenterology},
volume = {162},
number = {7},
pages = {2032-2046.e12},
pmid = {35219699},
issn = {1528-0012},
support = {R01 CA206444/CA/NCI NIH HHS/United States ; U01 CA200466/CA/NCI NIH HHS/United States ; R01 CA247471/CA/NCI NIH HHS/United States ; R44 CA235991/CA/NCI NIH HHS/United States ; R50 CA211462/CA/NCI NIH HHS/United States ; R01 CA183459/CA/NCI NIH HHS/United States ; R01 CA210637/CA/NCI NIH HHS/United States ; P01 CA217798/CA/NCI NIH HHS/United States ; },
mesh = {Actins/metabolism ; Animals ; Cluster Analysis ; Heterografts ; Humans ; *Hyaluronan Receptors/metabolism ; *Integrin beta1/metabolism ; *Mesenchymal Stem Cells/metabolism/pathology ; Mice ; *Mucin 5AC/metabolism ; *Pancreatic Neoplasms/genetics/metabolism/pathology ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; },
abstract = {BACKGROUND &amp; AIMS: Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes.<br><br>METHODS: Autochthonous murine models of PC (Kras[G12D], Pdx1-Cre [KC] and Kras[G12D], Pdx1-Cre, Muc5ac[-/-] [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay.<br><br>RESULTS: The KCM tumors showed a significant decrease in the expression of &#x3b1;-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-&#x3b2;1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward &#x3b1;-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors.<br><br>CONCLUSION: MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.},
}
@article {pmid35219382,
year = {2022},
author = {Tsuchida, CA and Zhang, S and Doost, MS and Zhao, Y and Wang, J and O'Brien, E and Fang, H and Li, CP and Li, D and Hai, ZY and Chuck, J and Brötzmann, J and Vartoumian, A and Burstein, D and Chen, XW and Nogales, E and Doudna, JA and Liu, JG},
title = {Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.},
journal = {Molecular cell},
volume = {82},
number = {6},
pages = {1199-1209.e6},
pmid = {35219382},
issn = {1097-4164},
support = {F31 HL156468/HL/NHLBI NIH HHS/United States ; P01 GM051487/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Mammals/metabolism ; RNA/genetics ; *RNA, Guide/genetics/metabolism ; },
abstract = {A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.},
}
@article {pmid35219341,
year = {2022},
author = {Stovicek, V and Dato, L and Almqvist, H and Schöpping, M and Chekina, K and Pedersen, LE and Koza, A and Figueira, D and Tjosås, F and Ferreira, BS and Forster, J and Lidén, G and Borodina, I},
title = {Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate.},
journal = {Biotechnology for biofuels and bioproducts},
volume = {15},
number = {1},
pages = {22},
pmid = {35219341},
issn = {2731-3654},
abstract = {BACKGROUND: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars-in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids.<br><br>RESULTS: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (&#xb5;max) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with &#xb5;max between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2&#xa0;mol and 0.12&#xa0;mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL.<br><br>CONCLUSIONS: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1&#xa0;mol and 0.065&#xa0;mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.},
}
@article {pmid35218528,
year = {2022},
author = {Genolet, O and Ravid Lustig, L and Schulz, EG},
title = {Dissecting Molecular Phenotypes Through FACS-Based Pooled CRISPR Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2520},
number = {},
pages = {1-24},
pmid = {35218528},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Flow Cytometry ; Phenotype ; RNA, Guide/genetics/metabolism ; },
abstract = {Pooled CRISPR screens are emerging as a powerful tool to dissect regulatory networks, by assessing how a protein responds to genetic perturbations in a highly multiplexed manner. A large number of genes are perturbed in a cell population through genomic integration of one single-guide RNA (sgRNA) per cell. A subset of cells with the phenotype of interest can then be enriched through fluorescence-activated cell sorting (FACS). SgRNAs with altered abundance after phenotypic enrichment allow identification of genes that either promote or attenuate the investigated phenotype. Here we provide detailed guidelines on how to design and execute a pooled CRISPR screen to investigate molecular phenotypes. We describe how to generate a custom sgRNA library and how to perform a FACS-based screen using readouts such as intracellular antibody staining or Flow-FISH to assess phosphorylation levels or RNA abundance. Through the variety of available perturbation systems and readout options many different molecular and cellular phenotypes can now be tackled with pooled CRISPR screens.},
}
@article {pmid35217837,
year = {2022},
author = {Mallapaty, S},
title = {How to protect the first 'CRISPR babies' prompts ethical debate.},
journal = {Nature},
volume = {603},
number = {7900},
pages = {213-214},
pmid = {35217837},
issn = {1476-4687},
mesh = {*Bioethical Issues ; *CRISPR-Cas Systems/genetics ; *Gene Editing/ethics/legislation &amp; jurisprudence/trends ; Humans ; Infant ; },
}
@article {pmid35217666,
year = {2022},
author = {Toral, MA and Charlesworth, CT and Ng, B and Chemudupati, T and Homma, S and Nakauchi, H and Bassuk, AG and Porteus, MH and Mahajan, VB},
title = {Investigation of Cas9 antibodies in the human eye.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {1053},
pmid = {35217666},
issn = {2041-1723},
support = {P30 EY026877/EY/NEI NIH HHS/United States ; T32 GM139776/GM/NIGMS NIH HHS/United States ; R01 EY030151/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; T32 GM007337/GM/NIGMS NIH HHS/United States ; R01 EY031952/EY/NEI NIH HHS/United States ; R01 EY025225/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Antibodies/metabolism ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Humans ; Mice ; Streptococcus pyogenes/metabolism ; T-Lymphocytes ; },
abstract = {Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.},
}
@article {pmid35217603,
year = {2022},
author = {Petersen, GEL and Buntjer, JB and Hely, FS and Byrne, TJ and Doeschl-Wilson, A},
title = {Modeling suggests gene editing combined with vaccination could eliminate a persistent disease in livestock.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {9},
pages = {},
pmid = {35217603},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; Livestock/*genetics ; Porcine Reproductive and Respiratory Syndrome/*prevention &amp; control ; Porcine respiratory and reproductive syndrome virus/*genetics/immunology ; Proof of Concept Study ; Swine ; *Vaccination ; },
abstract = {Recent breakthroughs in gene-editing technologies that can render individual animals fully resistant to infections may offer unprecedented opportunities for controlling future epidemics in farm animals. Yet, their potential for reducing disease spread is poorly understood as the necessary theoretical framework for estimating epidemiological effects arising from gene-editing applications is currently lacking. Here, we develop semistochastic modeling approaches to investigate how the adoption of gene editing may affect infectious disease prevalence in farmed animal populations and the prospects and time scale for disease elimination. We apply our models to the porcine reproductive and respiratory syndrome (PRRS), one of the most persistent global livestock diseases to date. Whereas extensive control efforts have shown limited success, recent production of gene-edited pigs that are fully resistant to the PRRS virus have raised expectations for eliminating this deadly disease. Our models predict that disease elimination on a national scale would be difficult to achieve if gene editing was used as the only disease control. However, from a purely epidemiological perspective, disease elimination may be achievable within 3 to 6 y, if gene editing were complemented with widespread and sufficiently effective vaccination. Besides strategic distribution of genetically resistant animals, several other key determinants underpinning the epidemiological impact of gene editing were identified.},
}
@article {pmid35217600,
year = {2022},
author = {Kwon, T and Ra, JS and Lee, S and Baek, IJ and Khim, KW and Lee, EA and Song, EK and Otarbayev, D and Jung, W and Park, YH and Wie, M and Bae, J and Cheng, H and Park, JH and Kim, N and Seo, Y and Yun, S and Kim, HE and Moon, HE and Paek, SH and Park, TJ and Park, YU and Rhee, H and Choi, JH and Cho, SW and Myung, K},
title = {Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {9},
pages = {},
pmid = {35217600},
issn = {1091-6490},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Death/genetics ; DNA Breaks, Double-Stranded ; Heterografts ; Humans ; *INDEL Mutation ; Mice ; Neoplasms/*genetics ; },
abstract = {An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.},
}
@article {pmid35217495,
year = {2022},
author = {Zhou, H and Sun, H and Rong, Z and Cui, W},
title = {Generation of Mt3 Homozygote murine ES cell lines via CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102714},
doi = {10.1016/j.scr.2022.102714},
pmid = {35217495},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Homozygote ; Mice ; Phenotype ; *Technology ; },
abstract = {Metallothioneins belong to a superfamily of intracellular metal-binding proteins, have bearing on almost all biochemical processes,include free radical scavenging, apoptosis, regulation of intracellular redox balance, and anti-inflammatory processes. By using a CRISPR/Cas 9 system, we generated Mt3[-/-] mES line. This cell line has contributed to further investigation of the functions of Mtt3 during early development, as well as a cell model for screening for metal-related detoxificationan and antioxidant response phenotype during stem cell differentiation.},
}
@article {pmid35217482,
year = {2022},
author = {Son, JS and Park, CY and Lee, G and Park, JY and Kim, HJ and Kim, G and Chi, KY and Woo, DH and Han, C and Kim, SK and Park, HJ and Kim, DW and Kim, JH},
title = {Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs.},
journal = {Biomaterials},
volume = {283},
number = {},
pages = {121429},
doi = {10.1016/j.biomaterials.2022.121429},
pmid = {35217482},
issn = {1878-5905},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endothelial Cells/metabolism ; *Hemophilia A/genetics/metabolism/therapy ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mice ; Organoids/metabolism ; },
abstract = {The bleeding disorder hemophilia A (HA) is caused by a single-gene (F8) defect and its clinical symptom can be substantially improved by a small increase in the plasma coagulation factor VIII (FVIII) level. In this study, we used F8-defective human induced pluripotent stem cells from an HA patient (F8d-HA hiPSCs) and F8-corrected (F8c) HA hiPSCs produced by CRISPR/Cas9 genome engineering of F8d-HA hiPSCs. We obtained a highly enriched population of CD157[+] cells from CRISPR/Cas9-edited F8c-HA hiPSCs. These cells exhibited multiple cellular and functional phenotypes of endothelial cells (ECs) with significant levels of FVIII activity, which was not observed in F8d-HA hiPSC-ECs. After transplantation, the engineered F8c-HA hiPSC-ECs dramatically changed bleeding episodes in HA animals and restored plasma FVIII activity. Notably, grafting a high dose of ECs substantially reduced the bleeding time during multiple consecutive bleeding challenges in HA mice, demonstrating a robust hemostatic effect (90% survival). Furthermore, the engrafted ECs survived more than 3 months in HA mice and reversed bleeding phenotypes against lethal wounding challenges. We also produced F8c-HA hiPSC-derived 3D liver organoids by assembling three different cell types in microwell devices and confirmed its therapeutic effect in HA animals. Our data demonstrate that the combination of genome-engineering and iPSC technologies represents a novel modality that allows autologous cell-mediated gene therapy for treating HA.},
}
@article {pmid35217297,
year = {2022},
author = {Lu, L and Shen, X and Sun, X and Yan, Y and Wang, J and Yuan, Q},
title = {CRISPR-based metabolic engineering in non-model microorganisms.},
journal = {Current opinion in biotechnology},
volume = {75},
number = {},
pages = {102698},
doi = {10.1016/j.copbio.2022.102698},
pmid = {35217297},
issn = {1879-0429},
mesh = {CRISPR-Cas Systems/genetics ; *Cyanobacteria/genetics ; Gene Editing ; *Metabolic Engineering ; Metabolic Networks and Pathways/genetics ; },
abstract = {Non-model microorganisms possess unique and versatile metabolic characteristics, offering great opportunities as cell factories for biosynthesis of target products. However, lack of efficient genetic tools for pathway engineering represents a big challenge to unlock the full production potential of these microbes. Over the past years, CRISPR systems have been extensively developed and applied to domesticate non-model microorganisms. In this paper, we summarize the current significant advances in designing and constructing CRISPR-mediated genetic modification systems in non-model microorganisms, such as bacteria, fungi and cyanobacteria. We particularly put emphasis on reviewing some successful implementations in metabolic pathway engineering via CRISPR-based genome editing tools. Moreover, the current barriers and future perspectives on improving the editing efficiency of CRISPR systems in non-model microorganisms are also discussed.},
}
@article {pmid35217295,
year = {2022},
author = {Dong, C and Gou, Y and Lian, J},
title = {SgRNA engineering for improved genome editing and expanded functional assays.},
journal = {Current opinion in biotechnology},
volume = {75},
number = {},
pages = {102697},
doi = {10.1016/j.copbio.2022.102697},
pmid = {35217295},
issn = {1879-0429},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas system has been established as the most powerful and practical genome engineering tool for both fundamental researches and biotechnological applications. Great efforts have been devoted to engineering the CRISPR system with better performance and novel functions. As an essential component, single guide RNAs (sgRNAs) have been extensively designed and engineered with desirable functions. This review highlights representative studies that optimize the sgRNA nucleotide sequences for improved genome editing performance (e.g. activity and specificity) as well as add extra aptamers and end extensions for expanded CRISPR-based functional assays (e.g. transcriptional regulation, genome imaging, and prime editor). The perspectives for further sgRNA engineering to establish more powerful and versatile CRISPR/Cas systems are also discussed.},
}
@article {pmid35216669,
year = {2022},
author = {Wimmer, F and Mougiakos, I and Englert, F and Beisel, CL},
title = {Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons.},
journal = {Molecular cell},
volume = {82},
number = {6},
pages = {1210-1224.e6},
doi = {10.1016/j.molcel.2022.01.026},
pmid = {35216669},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; Cell-Free System/metabolism ; DNA/genetics ; *Endonucleases/genetics ; RNA/metabolism ; },
abstract = {CRISPR-Cas biology and technologies have been largely shaped to date by the characterization and use of single-effector nucleases. By contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) systems to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons.},
}
@article {pmid35216463,
year = {2022},
author = {Wu, Q and Liu, Y and Huang, J},
title = {CRISPR-Cas9 Mediated Mutation in OsPUB43 Improves Grain Length and Weight in Rice by Promoting Cell Proliferation in Spikelet Hull.},
journal = {International journal of molecular sciences},
volume = {23},
number = {4},
pages = {},
pmid = {35216463},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; *Cell Proliferation ; Edible Grain/*anatomy &amp; histology/physiology ; Gene Editing ; *Mutation ; Oryza/anatomy &amp; histology/*enzymology/metabolism/physiology ; Plant Proteins/genetics ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Grain weight, a crucial trait that determines the grain yield in rice, is influenced by grain size. Although a series of regulators that control grain size have been identified in rice, the mechanisms underlying grain development are not yet well understood. In this study, we identified OsPUB43, a U-box E3 ubiquitin ligase, as an important negative regulator determining the gain size and grain weight in rice. Phenotypes of large grain are observed in ospub43 mutants, whereas overexpression of OsPUB43 results in short grains. Scanning electron microscopy analysis reveals that OsPUB43 modulates the grain size mainly by inhibiting cell proliferation in the spikelet hull. The OsPUB43 protein is localized in the cytoplasm and nucleus. The ospub43 mutants display high sensitivity to exogenous BR, while OsPUB43-OE lines are hyposensitive to BR. Furthermore, the transient transcriptional activity assay shows that OsBZR1 can activate the expression of OsPUB43. Collectively, our results indicate that OsPUB43 negatively controls the gain size by modulating the expression of BR-responsive genes as well as MADS-box genes that are required for lemma/palea specification, suggesting that OsPUB43 has a potential valuable application in the enlargement of grain size in rice.},
}
@article {pmid35216449,
year = {2022},
author = {Simbulan-Rosenthal, CM and Haribabu, Y and Vakili, S and Kuo, LW and Clark, H and Dougherty, R and Alobaidi, R and Carney, B and Sykora, P and Rosenthal, DS},
title = {Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {4},
pages = {},
pmid = {35216449},
issn = {1422-0067},
support = {5 R42 ES026908 03/ES/NIEHS NIH HHS/United States ; 1R41ES032435-01/ES/NIEHS NIH HHS/United States ; 1R43GM139439-01/GM/NIGMS NIH HHS/United States ; },
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Caspases/metabolism ; Cell Line, Tumor ; Humans ; *Melanoma/drug therapy/genetics/pathology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Proto-Oncogene Proteins c-akt/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA, Small Interfering/pharmacology ; Skin Neoplasms ; Stem Cells/metabolism ; bcl-2-Associated X Protein/metabolism ; },
abstract = {Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRAS[Q61K] and NRAS[Q61R] drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.},
}
@article {pmid35216418,
year = {2022},
author = {Khan, ZA and Kumar, R and Dasgupta, I},
title = {CRISPR/Cas-Mediated Resistance against Viruses in Plants.},
journal = {International journal of molecular sciences},
volume = {23},
number = {4},
pages = {},
pmid = {35216418},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Genome, Viral/*genetics ; Plant Viruses/*genetics ; Plants, Genetically Modified/genetics/virology ; },
abstract = {CRISPR/Cas9 provides a robust and widely adaptable system with enormous potential for genome editing directed towards generating useful products. It has been used extensively to generate resistance against viruses infecting plants with more effective and prolonged efficiency as compared with previous antiviral approaches, thus holding promise to alleviate crop losses. In this review, we have discussed the reports of CRISPR/Cas-based virus resistance strategies against plant viruses. These strategies include approaches targeting single or multiple genes (or non-coding region) in the viral genome and targeting host factors essential for virus propagation. In addition, the utilization of base editing has been discussed to generate transgene-free plants resistant to viruses. This review also compares the efficiencies of these approaches. Finally, we discuss combinatorial approaches, including multiplexing, to increase editing efficiency and bypass the generation of escape mutants.},
}
@article {pmid35216392,
year = {2022},
author = {Tay Fernandez, CG and Nestor, BJ and Danilevicz, MF and Marsh, JI and Petereit, J and Bayer, PE and Batley, J and Edwards, D},
title = {Expanding Gene-Editing Potential in Crop Improvement with Pangenomes.},
journal = {International journal of molecular sciences},
volume = {23},
number = {4},
pages = {},
pmid = {35216392},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Phenotype ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Pangenomes aim to represent the complete repertoire of the genome diversity present within a species or cohort of species, capturing the genomic structural variance between individuals. This genomic information coupled with phenotypic data can be applied to identify genes and alleles involved with abiotic stress tolerance, disease resistance, and other desirable traits. The characterisation of novel structural variants from pangenomes can support genome editing approaches such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein Cas (CRISPR-Cas), providing functional information on gene sequences and new target sites in variant-specific genes with increased efficiency. This review discusses the application of pangenomes in genome editing and crop improvement, focusing on the potential of pangenomes to accurately identify target genes for CRISPR-Cas editing of plant genomes while avoiding adverse off-target effects. We consider the limitations of applying CRISPR-Cas editing with pangenome references and potential solutions to overcome these limitations.},
}
@article {pmid35216252,
year = {2022},
author = {Navarro-Serna, S and Piñeiro-Silva, C and Luongo, C and Parrington, J and Romar, R and Gadea, J},
title = {Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {23},
number = {4},
pages = {},
pmid = {35216252},
issn = {1422-0067},
mesh = {Animals ; Animals, Genetically Modified ; Aphidicolin/*pharmacology ; CRISPR-Cas Systems/*drug effects ; Cell Nucleus/*drug effects ; DNA Replication/*drug effects ; Embryo, Mammalian/*drug effects ; Embryonic Development/drug effects ; Eukaryota/*drug effects ; Gene Editing/methods ; Mosaicism/drug effects ; Swine ; Zygote/drug effects ; },
abstract = {Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.},
}
@article {pmid35216118,
year = {2022},
author = {Rahman, H and Fukushima, C and Kaya, H and Yaeno, T and Kobayashi, K},
title = {Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics.},
journal = {International journal of molecular sciences},
volume = {23},
number = {4},
pages = {},
pmid = {35216118},
issn = {1422-0067},
mesh = {Agrobacterium/drug effects/genetics ; Aminoglycosides/*pharmacology ; Anti-Bacterial Agents/*pharmacology ; Arabidopsis/*genetics/microbiology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Microbial/*genetics ; Gene Editing/methods ; Genome, Plant/genetics ; Mutation/genetics ; Plant Leaves/genetics/microbiology ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Tobacco/*genetics/microbiology ; },
abstract = {To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs-knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.},
}
@article {pmid35216030,
year = {2022},
author = {Prokhorova, D and Zhukova Eschenko, N and Lemza, A and Sergeeva, M and Amirkhanov, R and Stepanov, G},
title = {Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35216030},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chickens ; Gene Editing/*methods ; Humans ; Immunity, Cellular ; Influenza A virus/genetics/*physiology ; Virus Internalization ; Virus Replication ; },
abstract = {Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production.},
}
@article {pmid35216029,
year = {2022},
author = {Li, W and Zhang, Y and Moffat, K and Nair, V and Yao, Y},
title = {V5 and GFP Tagging of Viral Gene pp38 of Marek's Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35216029},
issn = {1999-4915},
support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chickens/virology ; Gene Editing/*methods ; Genome, Viral ; Green Fluorescent Proteins/*genetics ; Mardivirus/*genetics ; Marek Disease/prevention &amp; control ; Marek Disease Vaccines/*genetics ; Phosphoproteins/genetics ; Poultry Diseases/prevention &amp; control ; Viral Envelope Proteins/chemistry/*genetics ; Virus Replication ; },
abstract = {Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.},
}
@article {pmid35216010,
year = {2022},
author = {Yuan, H and Yang, L and Zhang, Y and Xiao, W and Wang, Z and Tang, X and Ouyang, H and Pang, D},
title = {Current Status of Genetically Modified Pigs That Are Resistant to Virus Infection.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35216010},
issn = {1999-4915},
mesh = {African Swine Fever Virus/*genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Classical Swine Fever Virus/*genetics ; Gene Editing/methods ; Herpesvirus 1, Suid/*genetics ; Porcine epidemic diarrhea virus/*genetics ; Porcine respiratory and reproductive syndrome virus/*genetics ; Swine/virology ; Transmissible gastroenteritis virus/*genetics ; Virus Diseases/prevention &amp; control ; },
abstract = {Pigs play an important role in agriculture and biomedicine. The globally developing swine industry must address the challenges presented by swine-origin viruses, including ASFV (African swine fever virus), PRRSV (porcine reproductive and respiratory syndrome virus), PEDV (porcine epidemic diarrhea virus), PRV (pseudorabies virus), CSFV (classical swine fever virus), TGEV (transmissible gastroenteritis virus), et al. Despite sustained efforts by many government authorities, these viruses are still widespread. Currently, gene-editing technology has been successfully used to generate antiviral pigs, which offers the possibility for increasing animal disease tolerance and improving animal economic traits in the future. Here, we summarized the current advance in knowledge regarding the host factors in virus infection and the current status of genetically modified pigs that are resistant to virus infection in the world. There has not been any report on PEDV-resistant pigs, ASFV-resistant pigs, and PRV-resistant pigs owing to the poor understanding of the key host factors in virus infection. Furthermore, we summarized the remaining problems in producing virus-resistant pigs, and proposed several potential methods to solve them. Using genome-wide CRISPR/Cas9 library screening to explore the key host receptors in virus infection may be a feasible method. At the same time, exploring the key amino acids of host factors in virus infection with library screening based on ABEs and CBEs (Bes) may provide creative insight into producing antiviral pigs in the future.},
}
@article {pmid35215977,
year = {2022},
author = {Hussein, M and Andrade Dos Ramos, Z and Berkhout, B and Herrera-Carrillo, E},
title = {In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35215977},
issn = {1999-4915},
mesh = {CRISPR-Cas Systems ; *Computer Simulation ; *Genome, Viral ; Humans ; RNA, Viral/*genetics ; SARS-CoV-2/*genetics ; Spike Glycoprotein, Coronavirus/genetics ; },
abstract = {The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics.},
}
@article {pmid35215971,
year = {2022},
author = {Wu, BW and Yee, MB and Goldstein, RS and Kinchington, PR},
title = {Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35215971},
issn = {1999-4915},
support = {R01 AI151290/AI/NIAID NIH HHS/United States ; T32 AI049820/AI/NIAID NIH HHS/United States ; R01 AI122640/AI/NIAID NIH HHS/United States ; },
mesh = {Antiviral Agents/pharmacology ; *CRISPR-Cas Systems ; Cell Line ; Dependovirus/*genetics ; Drug Discovery ; Herpesvirus 3, Human/drug effects/*physiology ; Human Embryonic Stem Cells ; Humans ; Immediate-Early Proteins ; Neurons/*virology ; Open Reading Frames/*genetics ; Trans-Activators ; Viral Envelope Proteins ; Virus Latency ; Virus Replication ; },
abstract = {Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.},
}
@article {pmid35215968,
year = {2022},
author = {Lu, H and Xie, Q and Zhang, W and Zhang, J and Wang, W and Lian, M and Zhao, Z and Ren, D and Xie, S and Lin, Y and Li, T and Mu, Y and Wan, Z and Shao, H and Qin, A and Ye, J},
title = {A Novel Recombinant FAdV-4 Virus with Fiber of FAdV-8b Provides Efficient Protection against Both FAdV-4 and FAdV-8b.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35215968},
issn = {1999-4915},
mesh = {Adenoviridae Infections/*prevention &amp; control/veterinary/virology ; Animals ; Antibodies, Neutralizing/blood ; Aviadenovirus/*genetics ; CRISPR-Cas Systems ; *Chickens ; Gene Editing ; Poultry Diseases/*prevention &amp; control/virology ; Serogroup ; Vaccines, Synthetic/administration &amp; dosage ; Viral Vaccines/*administration &amp; dosage ; },
abstract = {Since 2015, the outbreaks of hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and serotype 8 fowl adenovirus (FAdV-8), respectively, have caused huge economic losses to the poultry industry. Although several vaccines have been developed to control HHS or IBH, a recombinant genetic engineering vaccine against both FAdV-4 and FAdV-8 has not been reported. In this study, recombinant FAdV-4 expressing the fiber of FAdV-8b, designated as FA4-F8b, expressing fiber of FAdV-8b was generated by the CRISPR-Cas9 and homologous recombinant techniques. Infection studies in vitro and in vivo revealed that the FA4-F8b replicated efficiently in LMH cells and was also highly pathogenic to 2-week-old SPF chickens. Moreover, the inoculation of inactivated the FA4-F8b in chickens could not only induce highly neutralizing antibodies, but also provide efficient protection against both FAdV-4 and FAdV-8b. All these demonstrate that the inactivated recombinant FA4-F8b generated here can act as a vaccine candidate to control HHS and IBH, and FAdV-4 can be an efficient vaccine vector to deliver foreign antigens.},
}
@article {pmid35215773,
year = {2022},
author = {Wei, N and Zheng, B and Niu, J and Chen, T and Ye, J and Si, Y and Cao, S},
title = {Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip.},
journal = {Viruses},
volume = {14},
number = {2},
pages = {},
pmid = {35215773},
issn = {1999-4915},
mesh = {African Swine Fever/*diagnosis/virology ; African Swine Fever Virus/genetics/*isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Genotype ; Reagent Strips ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; Sus scrofa ; Swine ; Time Factors ; },
abstract = {The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 10[1] copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.},
}
@article {pmid35213016,
year = {2022},
author = {López-Márquez, A and Martínez-Pizarro, A and Pérez, B and Richard, E and Desviat, LR},
title = {Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2434},
number = {},
pages = {167-184},
pmid = {35213016},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Exons/genetics ; *Gene Editing/methods ; Hep G2 Cells ; Humans ; RNA Splicing ; },
abstract = {The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defect.},
}
@article {pmid35212386,
year = {2022},
author = {Zhao, Z and Shang, P and Sage, F and Geijsen, N},
title = {Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.},
journal = {Nucleic acids research},
volume = {50},
number = {11},
pages = {e62},
pmid = {35212386},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; *Gene Editing ; Homologous Recombination/genetics ; Recombinational DNA Repair ; },
abstract = {CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy 'Ligation-Assisted Homologous Recombination' (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.},
}
@article {pmid35209078,
year = {2022},
author = {Skripova, V and Vlasenkova, R and Zhou, Y and Astsaturov, I and Kiyamova, R},
title = {Identification of New Regulators of Pancreatic Cancer Cell Sensitivity to Oxaliplatin and Cisplatin.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {4},
pages = {},
pmid = {35209078},
issn = {1420-3049},
mesh = {Antineoplastic Agents/*pharmacology ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Computational Biology/methods ; Drug Resistance, Neoplasm/*drug effects/genetics ; Drug Screening Assays, Antitumor ; Gene Expression Profiling ; Gene Knockdown Techniques ; Gene Ontology ; Gene Regulatory Networks ; Humans ; Oxaliplatin/*pharmacology ; Pancreatic Neoplasms ; Synthetic Lethal Mutations ; },
abstract = {The chemoresistance of tumor cells is one of the most urgent challenges in modern oncology and in pancreatic cancer, in which this problem is the most prominent. Therefore, the identification of new chemosensitizing co-targets may be a path toward increasing chemotherapy efficacy. In this work, we performed high-performance in vitro knockout CRISPR/Cas9 screening to find potential regulators of the sensitivity of pancreatic cancer. For this purpose, MIA PaCa-2 cells transduced with two sgRNA libraries ("cell cycle/nuclear proteins genes" and "genome-wide") were screened by oxaliplatin and cisplatin. In total, 173 candidate genes were identified as potential regulators of pancreatic cancer cell sensitivity to oxaliplatin and/or cisplatin; among these, 25 genes have previously been reported, while 148 genes were identified for the first time as potential platinum drug sensitivity regulators. We found seven candidate genes involved in pancreatic cancer cell sensitivity to both cisplatin and oxaliplatin. Gene ontology enrichment analysis reveals the enrichment of single-stranded DNA binding, damaged DNA binding pathways, and four associated with NADH dehydrogenase activity. Further investigation and validation of the obtained results by in vitro, in vivo, and bioinformatics approaches, as well as literature analysis, will help to identify novel pancreatic cancer platinum sensitivity regulators.},
}
@article {pmid35207459,
year = {2022},
author = {Gutierrez, K and Glanzner, WG and de Macedo, MP and Rissi, VB and Dicks, N and Bohrer, RC and Baldassarre, H and Agellon, LB and Bordignon, V},
title = {Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos.},
journal = {Life (Basel, Switzerland)},
volume = {12},
number = {2},
pages = {},
pmid = {35207459},
issn = {2075-1729},
abstract = {CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14-16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.},
}
@article {pmid35203804,
year = {2022},
author = {Murugaiyan, J and Kumar, PA and Rao, GS and Iskandar, K and Hawser, S and Hays, JP and Mohsen, Y and Adukkadukkam, S and Awuah, WA and Jose, RAM and Sylvia, N and Nansubuga, EP and Tilocca, B and Roncada, P and Roson-Calero, N and Moreno-Morales, J and Amin, R and Kumar, BK and Kumar, A and Toufik, AR and Zaw, TN and Akinwotu, OO and Satyaseela, MP and van Dongen, MBM},
title = {Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {11},
number = {2},
pages = {},
pmid = {35203804},
issn = {2079-6382},
abstract = {Antibiotic resistance, and, in a broader perspective, antimicrobial resistance (AMR), continues to evolve and spread beyond all boundaries. As a result, infectious diseases have become more challenging or even impossible to treat, leading to an increase in morbidity and mortality. Despite the failure of conventional, traditional antimicrobial therapy, in the past two decades, no novel class of antibiotics has been introduced. Consequently, several novel alternative strategies to combat these (multi-) drug-resistant infectious microorganisms have been identified. The purpose of this review is to gather and consider the strategies that are being applied or proposed as potential alternatives to traditional antibiotics. These strategies include combination therapy, techniques that target the enzymes or proteins responsible for antimicrobial resistance, resistant bacteria, drug delivery systems, physicochemical methods, and unconventional techniques, including the CRISPR-Cas system. These alternative strategies may have the potential to change the treatment of multi-drug-resistant pathogens in human clinical settings.},
}
@article {pmid35203404,
year = {2022},
author = {Haase-Kohn, C and Laube, M and Donat, CK and Belter, B and Pietzsch, J},
title = {CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells.},
journal = {Cells},
volume = {11},
number = {4},
pages = {},
pmid = {35203404},
issn = {2073-4409},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Cyclooxygenase 2/genetics ; Gene Knockdown Techniques ; Humans ; *Melanoma/pathology ; Mice ; *Neoplasm Invasiveness ; Tumor Microenvironment ; },
abstract = {The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [[18]F]FDG and [[18]F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.},
}
@article {pmid35202423,
year = {2022},
author = {Addo-Gyan, D and Matsushita, H and Sora, E and Nishi, T and Yuda, M and Shinzawa, N and Iwanaga, S},
title = {Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system.},
journal = {PloS one},
volume = {17},
number = {2},
pages = {e0260176},
pmid = {35202423},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Centromere/genetics ; Chromosomes/*genetics ; Gene Expression Regulation/genetics ; Malaria/*genetics/parasitology ; Plasmodium berghei/*genetics/pathogenicity ; Plasmodium falciparum/genetics/pathogenicity ; Rodentia/parasitology ; Telomere/genetics ; },
abstract = {Spatial arrangement of chromosomes is responsible for gene expression in Plasmodium parasites. However, methods for rearranging chromosomes have not been established, which makes it difficult to investigate its role in detail. Here, we report a method for splitting chromosome in rodent malaria parasite by CRISPR/Cas9 system using fragments in which a telomere and a centromere were incorporated. The resultant split chromosomes segregated accurately into daughter parasites by the centromere. In addition, elongation of de novo telomeres were observed, indicating its proper function. Furthermore, chromosome splitting had no effect on development of parasites. Splitting of the chromosome is expected to alter its spatial arrangement, and our method will thus be useful for investigating its biological role related with gene expression.},
}
@article {pmid35201886,
year = {2022},
author = {Li, SB and Damonte, VM and Chen, C and Wang, GX and Kebschull, JM and Yamaguchi, H and Bian, WJ and Purmann, C and Pattni, R and Urban, AE and Mourrain, P and Kauer, JA and Scherrer, G and de Lecea, L},
title = {Hyperexcitable arousal circuits drive sleep instability during aging.},
journal = {Science (New York, N.Y.)},
volume = {375},
number = {6583},
pages = {eabh3021},
pmid = {35201886},
issn = {1095-9203},
support = {R01 NS106301/NS/NINDS NIH HHS/United States ; P30 EY026877/EY/NEI NIH HHS/United States ; K01 AG061230/AG/NIA NIH HHS/United States ; R01 MH116470/MH/NIMH NIH HHS/United States ; R01 NS104950/NS/NINDS NIH HHS/United States ; P50 HG007735/HG/NHGRI NIH HHS/United States ; R01 DA011289/DA/NIDA NIH HHS/United States ; R01 AG047671/AG/NIA NIH HHS/United States ; R01 MH128140/MH/NIMH NIH HHS/United States ; },
mesh = {*Aging ; Aminopyridines/pharmacology ; Animals ; CRISPR-Cas Systems ; Electroencephalography ; Electromyography ; Female ; Hypothalamic Area, Lateral/physiopathology ; KCNQ2 Potassium Channel/genetics/metabolism ; KCNQ3 Potassium Channel/genetics/metabolism ; Male ; Mice ; Narcolepsy/genetics/physiopathology ; Nerve Tissue Proteins/genetics/metabolism ; Neural Pathways ; Neurons/*physiology ; Optogenetics ; Orexins/*physiology ; Patch-Clamp Techniques ; RNA-Seq ; *Sleep ; Sleep Deprivation/*physiopathology ; Sleep Quality ; *Wakefulness ; },
abstract = {Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.},
}
@article {pmid35200314,
year = {2022},
author = {Díaz-Galicia, E and Grünberg, R and Arold, ST},
title = {How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application.},
journal = {Biosensors},
volume = {12},
number = {2},
pages = {},
pmid = {35200314},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; *RNA ; },
abstract = {CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.},
}
@article {pmid35198566,
year = {2021},
author = {Feng, S and Wang, Z and Li, A and Xie, X and Liu, J and Li, S and Li, Y and Wang, B and Hu, L and Yang, L and Guo, T},
title = {Strategies for High-Efficiency Mutation Using the CRISPR/Cas System.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {803252},
pmid = {35198566},
issn = {2296-634X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.},
}
@article {pmid35197607,
year = {2022},
author = {Gu, X and Hu, X and Wang, D and Xu, Z and Wang, F and Li, D and Li, GL and Yang, H and Li, H and Zuo, E and Shu, Y},
title = {Treatment of autosomal recessive hearing loss via in vivo CRISPR/Cas9-mediated optimized homology-directed repair in mice.},
journal = {Cell research},
volume = {32},
number = {7},
pages = {699-702},
pmid = {35197607},
issn = {1748-7838},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Hearing Loss/genetics/therapy ; Mice ; },
}
@article {pmid35197604,
year = {2022},
author = {Lin, SC and Haga, K and Zeng, XL and Estes, MK},
title = {Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.},
journal = {Nature protocols},
volume = {17},
number = {4},
pages = {1004-1027},
pmid = {35197604},
issn = {1750-2799},
support = {U19 AI144297/AI/NIAID NIH HHS/United States ; U19 AI116497/AI/NIAID NIH HHS/United States ; P30 ES030285/ES/NIEHS NIH HHS/United States ; P01 AI057788/AI/NIAID NIH HHS/United States ; P42 ES027725/ES/NIEHS NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Clone Cells ; Cloning, Molecular ; Gene Editing/methods ; Gene Knockout Techniques ; Humans ; *Lentivirus/genetics ; RNA, Guide/genetics ; },
abstract = {Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.},
}
@article {pmid35197298,
year = {2022},
author = {Xu, B and Kucenas, S and Zong, H},
title = {zMADM (zebrafish mosaic analysis with double markers) for single-cell gene knockout and dual-lineage tracing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {9},
pages = {},
pmid = {35197298},
issn = {1091-6490},
support = {P30 CA044579/CA/NCI NIH HHS/United States ; R21 OD026524/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Lineage ; Gene Knockdown Techniques ; *Genetic Markers ; *Mosaicism ; Single-Cell Analysis/*methods ; Zebrafish/*genetics ; },
abstract = {As a vertebrate model organism, zebrafish has many unique advantages in developmental studies, regenerative biology, and disease modeling. However, tissue-specific gene knockout in zebrafish is challenging due to technical difficulties in making floxed alleles. Even when successful, tissue-level knockout can affect too many cells, making it difficult to distinguish cell autonomous from noncell autonomous gene function. Here, we present a genetic system termed zebrafish mosaic analysis with double markers (zMADM). Through Cre/loxP-mediated interchromosomal mitotic recombination of two reciprocally chimeric fluorescent genes, zMADM generates sporadic (<0.5%), GFP[+] mutant cells along with RFP[+] sibling wild-type cells, enabling phenotypic analysis at single-cell resolution. Using wild-type zMADM, we traced two sibling cells (GFP[+] and RFP[+]) in real time during a dynamic developmental process. Using nf1 mutant zMADM, we demonstrated an overproliferation phenotype of nf1 mutant cells in comparison to wild-type sibling cells in the same zebrafish. The readiness of zMADM to produce sporadic mutant cells without the need to generate floxed alleles should fundamentally improve the throughput of genetic analysis in zebrafish; the lineage-tracing capability combined with phenotypic analysis at the single-cell level should lead to deep insights into developmental and disease mechanisms. Therefore, we are confident that zMADM will enable groundbreaking discoveries once broadly distributed in the field.},
}
@article {pmid35196879,
year = {2022},
author = {Statkute, E and Wang, ECY and Stanton, RJ},
title = {An Optimized CRISPR/Cas9 Adenovirus Vector (AdZ-CRISPR) for High-Throughput Cloning of sgRNA, Using Enhanced sgRNA and Cas9 Variants.},
journal = {Human gene therapy},
volume = {33},
number = {17-18},
pages = {990-1001},
doi = {10.1089/hum.2021.120},
pmid = {35196879},
issn = {1557-7422},
support = {MR/P001602/1/MRC_/Medical Research Council/United Kingdom ; MR/V000489/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adenoviridae/genetics ; *CRISPR-Cas Systems ; Cloning, Molecular ; Gene Editing ; Genetic Vectors/genetics ; *RNA, Guide/genetics ; },
abstract = {Recombinant adenovirus vectors enable highly efficient gene delivery in vitro and in vivo. As a result, they are widely used in gene therapy, vaccination, and anticancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high-throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. In this study, we adapt this vector system to enable high-throughput cloning of sequences for CRISPR/Cas9 editing. Vectors were optimized to ensure efficient cloning, and high editing efficiency using spCas9 and single guide RNA (sgRNA) sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity, but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large-scale CRISPR/Cas9 projects to be undertaken easily and quickly.},
}
@article {pmid35196804,
year = {2022},
author = {Shahi, I and Llaneras, CN and Perelman, SS and Torres, VJ and Ratner, AJ},
title = {Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae β-Hemolysin/Cytolysin-Induced Cell Death.},
journal = {Microbiology spectrum},
volume = {10},
number = {1},
pages = {e0218621},
pmid = {35196804},
issn = {2165-0497},
support = {P30 CA016087/CA/NCI NIH HHS/United States ; R01 AI155476/AI/NIAID NIH HHS/United States ; R01 AI099394/AI/NIAID NIH HHS/United States ; R01 AI105129/AI/NIAID NIH HHS/United States ; R01 AI143290/AI/NIAID NIH HHS/United States ; R01 AI121244/AI/NIAID NIH HHS/United States ; R21 AI147511/AI/NIAID NIH HHS/United States ; },
mesh = {CD59 Antigens/genetics/metabolism ; CRISPR-Cas Systems ; Cell Death ; Cell Line ; Genome, Bacterial ; Hemolysin Proteins/metabolism/*toxicity ; Host-Pathogen Interactions ; Humans ; Perforin/metabolism/*toxicity ; Streptococcal Infections/*genetics/metabolism/microbiology/*physiopathology ; Streptococcus agalactiae/genetics/*metabolism ; },
abstract = {Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT β-hemolysin/cytolysin (βhc). Little is understood about the cellular factors involved in βhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to βhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for βhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of βhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on βhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that βhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to βhc, a toxin required for GBS virulence. These data suggest that βhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.},
}
@article {pmid35196219,
year = {2022},
author = {Simon, DA and Tálas, A and Kulcsár, PI and Biczók, Z and Krausz, SL and Várady, G and Welker, E},
title = {PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35196219},
issn = {2050-084X},
mesh = {Animals ; Bacteria ; *CRISPR-Cas Systems ; Fluorescence ; Gene Editing/*methods ; *Genes, Reporter ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; },
abstract = {Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.},
}
@article {pmid35196217,
year = {2022},
author = {Donà, V and Ramette, A and Perreten, V},
title = {Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.},
journal = {Microbial genomics},
volume = {8},
number = {2},
pages = {},
pmid = {35196217},
issn = {2057-5858},
mesh = {*Actinobacillus pleuropneumoniae/genetics ; Animals ; Genomics/methods ; Lipopolysaccharides ; Multilocus Sequence Typing ; Serogroup ; Swine ; },
abstract = {Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae.},
}
@article {pmid35195982,
year = {2022},
author = {Yan, X and Zhang, J and Jiang, Q and Jiao, D and Cheng, Y},
title = {Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA.},
journal = {Analytical chemistry},
volume = {94},
number = {9},
pages = {4119-4125},
doi = {10.1021/acs.analchem.2c00294},
pmid = {35195982},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Ligase Chain Reaction/methods ; *MicroRNAs/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; *RNA Probes/genetics ; RNA, Guide/genetics ; },
abstract = {The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.},
}
@article {pmid35195954,
year = {2022},
author = {Aralis, Z and Rauch, JN and Audouard, M and Valois, E and Lach, RS and Solley, S and Baxter, NJ and Kosik, KS and Wilson, MZ and Acosta-Alvear, D and Arias, C},
title = {CREST, a Cas13-Based, Rugged, Equitable, Scalable Testing (CREST) for SARS-CoV-2 Detection in Patient Samples.},
journal = {Current protocols},
volume = {2},
number = {2},
pages = {e385},
doi = {10.1002/cpz1.385},
pmid = {35195954},
issn = {2691-1299},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques ; Pandemics ; *SARS-CoV-2 ; },
abstract = {The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets.},
}
@article {pmid35637939,
year = {2021},
author = {Petraitytė, G and Preikšaitienė, E and Mikštienė, V},
title = {Genome Editing in Medicine: Tools and Challenges.},
journal = {Acta medica Lituanica},
volume = {28},
number = {2},
pages = {205-219},
pmid = {35637939},
issn = {1392-0138},
abstract = {Studies which seek fundamental, thorough knowledge of biological processes, and continuous advancement in natural sciences and biotechnology enable the establishment of molecular strategies and tools to treat disorders caused by genetic mutations. Over the years biological therapy evolved from using stem cells and viral vectors to RNA therapy and testing different genome editing tools as promising gene therapy agents. These genome editing technologies (Zinc finger nucleases, TAL effector nucleases), specifically CRISPR-Cas system, revolutionized the field of genetic engineering and is widely applied to create cell and animal models for various hereditary, infectious human diseases and cancer, to analyze and understand the molecular and cellular base of pathogenesis, to find potential drug/treatment targets, to eliminate pathogenic DNA changes in various medical conditions and to create future "precise medication". Although different concerning factors, such as precise system delivery to the target cells, efficacy and accuracy of editing process, different approaches of making the DNA changes as well as worrying bioethical issues remain, the importance of genome editing technologies in medicine is undeniable. The future of innovative genome editing approach and strategies to treat diseases is complicated but interesting and exciting at once for all related parties - researchers, clinicians, and patients.},
}
@article {pmid35548117,
year = {2018},
author = {Zhang, J and Zhou, W and Wang, X and Wang, L},
title = {The CRISPR-Cas9 system: a promising tool for discovering potential approaches to overcome drug resistance in cancer.},
journal = {RSC advances},
volume = {8},
number = {58},
pages = {33464-33472},
pmid = {35548117},
issn = {2046-2069},
abstract = {The CRISPR-Cas system was identified in bacteria as an immune defense mechanism against threats from the external environment. A common form of this system, called CRISPR-Cas9, is now widely used in gene editing, especially in mammalian cells. Through CRISPR-Cas9, gene knock-ins or knock-outs have become more feasible, thus deepening our understanding of the mechanisms of human diseases, including cancers, and suggesting possible treatment strategies. In this review, we discuss how CRISPR-Cas9 can be used as a tool to discover more about drug-resistance in cancers, including both the underlying mechanisms and ways to overcome them.},
}
@article {pmid35194830,
year = {2022},
author = {Eerkens, AL and Vledder, A and van Rooij, N and Foijer, F and Nijman, HW and de Bruyn, M},
title = {Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples.},
journal = {Immunology and cell biology},
volume = {100},
number = {4},
pages = {285-295},
pmid = {35194830},
issn = {1440-1711},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Leukocytes, Mononuclear ; Mice ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9-mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T-cell models for interferon-γ, Cbl proto-oncogene B (CBLB), Fas cell surface death receptor (Fas) and T-cell receptor (TCRαβ) genes. The effect of CRISPR/Cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T-cell viability. Cytokine release and T-cell proliferation were not affected in gene-edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T-cell responses in gene-edited and untouched control cells, making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T-cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen-specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically used immune-monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers.},
}
@article {pmid35194511,
year = {2022},
author = {Dubey, AK and Kumar Gupta, V and Kujawska, M and Orive, G and Kim, NY and Li, CZ and Kumar Mishra, Y and Kaushik, A},
title = {Exploring nano-enabled CRISPR-Cas-powered strategies for efficient diagnostics and treatment of infectious diseases.},
journal = {Journal of nanostructure in chemistry},
volume = {12},
number = {5},
pages = {833-864},
pmid = {35194511},
issn = {2008-9244},
abstract = {UNLABELLED: Biomedical researchers have subsequently been inspired the development of new approaches for precisely changing an organism's genomic DNA in order to investigate customized diagnostics and therapeutics utilizing genetic engineering techniques. Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one such technique that has emerged as a safe, targeted, and effective pharmaceutical treatment against a wide range of disease-causing organisms, including bacteria, fungi, parasites, and viruses, as well as genetic abnormalities. The recent discovery of very flexible engineered nucleic acid binding proteins has changed the scientific area of genome editing in a revolutionary way. Since current genetic engineering technique relies on viral vectors, issues about immunogenicity, insertional oncogenesis, retention, and targeted delivery remain unanswered. The use of nanotechnology has the potential to improve the safety and efficacy of CRISPR/Cas9 component distribution by employing tailored polymeric nanoparticles. The combination of two (CRISPR/Cas9 and nanotechnology) offers the potential to open new therapeutic paths. Considering the benefits, demand, and constraints, the goal of this research is to acquire more about the biology of CRISPR technology, as well as aspects of selective and effective diagnostics and therapies for infectious illnesses and other metabolic disorders. This review advocated combining nanomedicine (nanomedicine) with a CRISPR/Cas enabled sensing system to perform early-stage diagnostics and selective therapy of specific infectious disorders. Such a Nano-CRISPR-powered nanomedicine and sensing system would allow for successful infectious illness control, even on a personal level. This comprehensive study also discusses the current obstacles and potential of the predicted technology.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40097-022-00472-7.},
}
@article {pmid35194225,
year = {2022},
author = {Bouvenot, G},
title = {[Interfering RNA and antisense oligonucleotide treatments currently available in France: An update].},
journal = {Bulletin de l'Academie nationale de medecine},
volume = {206},
number = {4},
pages = {554-558},
pmid = {35194225},
issn = {0001-4079},
abstract = {The arrival of anti-Covid-19 RNA vaccines in 2020 should not obscure the fact that for several years we have already had treatments based on interfering RNA or antisense oligonucleotides in a number of rare diseases with a very poor prognosis such as transthyretin amyloidosis, acute hepatic porphyria, primary hyperoxaluria, spinal muscular atrophy or familial hyperchylomicronemia. If their performance, unlike that of vaccines, is for the moment only qualified as moderate therapeutic progress (moderate clinical added value) in the therapeutic strategies against these diseases, it should be taken into account that their initial evaluation was penalized by a certain number of unfavorable factors: trials of small numbers, therapeutic modalities to be refined, the lack of hindsight on their long-term effects but especially the choice of the moment of the initiation of the treatment in the natural evolution of the sickness. This choice is not trivial because it is hard to imagine that the products used could, beyond a simple stabilization of the disease installed, allow its regression as soon as certain lesions formed are irreversible. This is why their very early implementation, possibly based on genetic screening, is an avenue to be seized in the interest of patients. But, in the competitive context of innovations in the field, interfering RNAs and antisense oligonucleotides will have to reckon with gene therapy and genome editing using the CRISPR-Cas 9 technique.},
}
@article {pmid35194207,
year = {2022},
author = {Anglada-Girotto, M and Handschin, G and Ortmayr, K and Campos, AI and Gillet, L and Manfredi, P and Mulholland, CV and Berney, M and Jenal, U and Picotti, P and Zampieri, M},
title = {Combining CRISPRi and metabolomics for functional annotation of compound libraries.},
journal = {Nature chemical biology},
volume = {18},
number = {5},
pages = {482-491},
pmid = {35194207},
issn = {1552-4469},
support = {866004/ERC_/European Research Council/International ; R01 AI139465/AI/NIAID NIH HHS/United States ; R21 AI133191/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Escherichia coli/genetics/metabolism ; Humans ; Metabolomics/methods ; },
abstract = {Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines.},
}
@article {pmid35194186,
year = {2022},
author = {Dowling, JJ},
title = {CRISPR editing as a therapeutic strategy for Duchenne muscular dystrophy-anti-Cas9 immune response casts its shadow over safety and efficacy.},
journal = {Gene therapy},
volume = {29},
number = {10-11},
pages = {575-577},
pmid = {35194186},
issn = {1476-5462},
mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Gene Editing ; CRISPR-Cas Systems/genetics ; Immunity ; Dystrophin/genetics/metabolism ; },
}
@article {pmid35194172,
year = {2022},
author = {Katti, A and Diaz, BJ and Caragine, CM and Sanjana, NE and Dow, LE},
title = {CRISPR in cancer biology and therapy.},
journal = {Nature reviews. Cancer},
volume = {22},
number = {5},
pages = {259-279},
pmid = {35194172},
issn = {1474-1768},
support = {F31 CA247351/CA/NCI NIH HHS/United States ; F31 CA261061/CA/NCI NIH HHS/United States ; },
mesh = {Biology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genome ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {Over the past decade, CRISPR has become as much a verb as it is an acronym, transforming biomedical research and providing entirely new approaches for dissecting all facets of cell biology. In cancer research, CRISPR and related tools have offered a window into previously intractable problems in our understanding of cancer genetics, the noncoding genome and tumour heterogeneity, and provided new insights into therapeutic vulnerabilities. Here, we review the progress made in the development of CRISPR systems as a tool to study cancer, and the emerging adaptation of these technologies to improve diagnosis and treatment.},
}
@article {pmid35193353,
year = {2022},
author = {Liu, X and Kang, X and Lei, C and Ren, W and Liu, C},
title = {Programming the trans-cleavage Activity of CRISPR-Cas13a by Single-Strand DNA Blocker and Its Biosensing Application.},
journal = {Analytical chemistry},
volume = {94},
number = {9},
pages = {3987-3996},
doi = {10.1021/acs.analchem.1c05124},
pmid = {35193353},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA, Single-Stranded ; },
abstract = {The precise and controllable programming of the trans-cleavage activity of the CRISPR-Cas13a systems is significant but challenging for fabricating high-performance biosensing systems toward various kinds of biomolecule targets. In this work, we have demonstrated that under a critical low Mg[2+] concentration, a simple and short single-stranded DNA (ssDNA) probe free of any modification can efficiently prevent the assembly of crRNA and LwaCas13a only by partially binding with the crRNA repeat region, thereby blocking the trans-cleavage activity of the LwaCas13a system. Furthermore, we have demonstrated that the blocked trans-cleavage activity of the LwaCas13a system can be recovered by various kinds of biologically important substances as long as they could specifically release the blocker DNA from the crRNA in a target-responsive manner, providing a facile route for the quantification of diverse biomarkers such as enzymes, antigens/proteins, and exosomes. To the best of our knowledge, this is reported for the first time that a simple ssDNA can be employed as the switch element to control the crRNA structure and regulate the trans-cleavage activity of Cas13a, which has enriched the CRISPR-Cas13a sensing toolbox and will greatly expand its application scope.},
}
@article {pmid35192680,
year = {2022},
author = {Jiang, C and Qian, M and Gocho, Y and Yang, W and Du, G and Shen, S and Yang, JJ and Zhang, H},
title = {Genome-wide CRISPR/Cas9 screening identifies determinant of panobinostat sensitivity in acute lymphoblastic leukemia.},
journal = {Blood advances},
volume = {6},
number = {8},
pages = {2496-2509},
pmid = {35192680},
issn = {2473-9537},
support = {P50 GM115279/GM/NIGMS NIH HHS/United States ; R01 GM118578/GM/NIGMS NIH HHS/United States ; },
mesh = {Apoptosis ; CRISPR-Cas Systems ; *Histones/metabolism ; Humans ; Panobinostat/pharmacology ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/genetics ; Sirtuin 1/genetics/metabolism/pharmacology ; United States ; },
abstract = {Epigenetic alterations, including histone acetylation, contribute to the malignant transformation of hematopoietic cells and disease progression, as well as the emergence of chemotherapy resistance. Targeting histone acetylation provides new strategies for the treatment of cancers. As a pan-histone deacetylase inhibitor, panobinostat has been approved by the US Food and Drug Administration for the treatment of multiple myeloma and has shown promising antileukemia effects in acute lymphoblastic leukemia (ALL). However, the underlying drug resistance mechanism in ALL remains largely unknown. Using genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9 (CRISPR/Cas9) screening, we identified mitochondrial activity as the driver of panobinostat resistance in ALL. Mechanistically, ectopic SIRT1 expression activated mitochondrial activity and sensitized ALL to panobinostat through activating mitochondria-related apoptosis pathway. Meanwhile, the transcription level of SIRT1 was significantly associated with panobinostat sensitivity across diverse tumor types and thus could be a potential biomarker of panobinostat response in cancers. Our data suggest that patients with higher SIRT1 expression in cancer cells might benefit from panobinostat treatment, supporting the implementation of combinatorial therapy with SIRT1 or mitochondrial activators to overcome panobinostat resistance.},
}
@article {pmid35192362,
year = {2022},
author = {Wei, T and Wen, X and Niu, C and An, S and Wang, D and Xi, Z and Wang, NN},
title = {Design of Acetohydroxyacid Synthase Herbicide-Resistant Germplasm through MB-QSAR and CRISPR/Cas9-Mediated Base-Editing Approaches.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {9},
pages = {2817-2824},
doi = {10.1021/acs.jafc.1c07180},
pmid = {35192362},
issn = {1520-5118},
mesh = {*Acetolactate Synthase/genetics/metabolism ; CRISPR-Cas Systems ; Herbicide Resistance/genetics ; *Herbicides/pharmacology ; Quantitative Structure-Activity Relationship ; },
abstract = {The development of herbicide-resistant germplasm is significant in solving the increasingly severe weed problem in crop fields. In this study, we, for the first time, rationally designed a predictable and effective approach to create herbicide-resistant germplasm by combining mutation-dependent biomacromolecular quantitative structure-activity relationship (MB-QSAR) and CRISPR/Cas9-mediated base-editing strategies. Our results showed that the homozygous P197F-G654D-G655S or P197F-G654N-G655S Arabidopsis plants exhibited high resistance to multiple acetohydroxyacid synthase-inhibiting herbicides, including chlorsulfuron, bispyribac-sodium, and flucarbazone-sodium. Additionally, the plants with the homozygous P197S mutant displayed increased susceptibility to bispyribac-sodium than the wild-type but more resistance to flumetsulam than other mutants. Besides, we found that the herbicide resistance levels of the gene-edited plants have a good correlation with MB-QSAR prediction.},
}
@article {pmid35191754,
year = {2022},
author = {Barrangou, R},
title = {CRISPR Rewrites the Future of Medicine.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {1},
doi = {10.1089/crispr.2022.29144.rba},
pmid = {35191754},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35191752,
year = {2022},
author = {Moreb, EA and Lynch, MD},
title = {A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {146-154},
doi = {10.1089/crispr.2021.0084},
pmid = {35191752},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Library ; *RNA, Guide/genetics ; },
abstract = {CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here, we perform a meta-analysis of 39 published CRISPR-Cas9 data sets to understand better the role of secondary structures in inhibiting gRNA activity. We (1) identify two distinct inhibitory structures that can form, (2) measure the prevalence of these structures in existing gRNA library data sets, and (3) provide free energy cutoffs at which these structures become inhibitory. First, we show that hairpins that form within the targeting portion (spacer) of the gRNA, having a minimum free energy of <-5 kcal/mol, negatively impact gRNA activity. Second, we demonstrate that a longer hairpin can form between the spacer and the nexus portion of the gRNA scaffold. A duplex stability of this longer hairpin of <-15 kcal/mol negatively impacts gRNA activity. These cutoffs help to explain conflicting impacts of free energy values in different data sets, as well as provide a guideline for future gRNA designs.},
}
@article {pmid35191751,
year = {2022},
author = {Cárdenas, P and Esherick, LY and Chambonnier, G and Dey, S and Turlo, CV and Nasamu, AS and Niles, JC},
title = {GeneTargeter: Automated In Silico Design for Genome Editing in the Malaria Parasite, Plasmodium falciparum.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {155-164},
pmid = {35191751},
issn = {2573-1602},
support = {P30 ES002109/ES/NIEHS NIH HHS/United States ; T32 ES007020/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Malaria, Falciparum/genetics/parasitology ; *Parasites/genetics ; Plasmodium falciparum/genetics ; },
abstract = {Functional characterization of the multitude of poorly described proteins in the human malarial pathogen, Plasmodium falciparum, requires tools to enable genome-scale perturbation studies. Here, we present GeneTargeter (genetargeter.mit.edu), a software tool for automating the design of homology-directed repair donor vectors to achieve gene knockouts, conditional knockdowns, and epitope tagging of P. falciparum genes. We demonstrate GeneTargeter-facilitated genome-scale design of six different types of knockout and conditional knockdown constructs for the P. falciparum genome and validate the computational design process experimentally with successful donor vector assembly and transfection. The software's modular nature accommodates arbitrary destination vectors and allows customizable designs that extend the genome manipulation outcomes attainable in Plasmodium and other organisms.},
}
@article {pmid35191750,
year = {2022},
author = {Yoon, HH and Ye, S and Lim, S and Jo, A and Lee, H and Hong, F and Lee, SE and Oh, SJ and Kim, NR and Kim, K and Kim, BJ and Kim, H and Lee, CJ and Nam, MH and Hur, JW and Jeon, SR},
title = {CRISPR-Cas9 Gene Editing Protects from the A53T-SNCA Overexpression-Induced Pathology of Parkinson's Disease In Vivo.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {95-108},
doi = {10.1089/crispr.2021.0025},
pmid = {35191750},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; *Parkinson Disease/genetics/metabolism/pathology/therapy ; Rats ; *alpha-Synuclein/genetics/metabolism ; },
abstract = {Mutations in specific genes, including synuclein alpha (SNCA) that encodes the α-synuclein protein, are known to be risk factors for sporadic Parkinson's disease (PD), as well as critical factors for familial PD. In particular, A53T-mutated SNCA (A53T-SNCA) is a well-studied familial pathologic mutation in PD. However, techniques for deletion of the mutated SNCA gene in vivo have not been developed. Here, we used the CRISPR-Cas9 system to delete A53T-SNCA in vitro as well as in vivo. Adeno-associated virus carrying SaCas9-KKH with a single-guide RNA targeting A53T-SNCA significantly reduced A53T-SNCA expression levels in vitro. Furthermore, we tested its therapeutic potential in vivo in a viral A53T-SNCA-overexpressing rat model of PD. Gene deletion of A53T-SNCA significantly rescued the overexpression of α-synuclein, reactive microgliosis, dopaminergic neurodegeneration, and parkinsonian motor symptoms. Our findings propose CRISPR-Cas9 system as a potential prevention strategy for A53T-SNCA-specific PD.},
}
@article {pmid35191749,
year = {2022},
author = {Miccio, A},
title = {CRISPR's Path to the Clinic.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {2-3},
doi = {10.1089/crispr.2022.29143.am},
pmid = {35191749},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid35191500,
year = {2022},
author = {Kumar, S and Liu, ZB and Sanyour-Doyel, N and Lenderts, B and Worden, A and Anand, A and Cho, HJ and Bolar, J and Harris, C and Huang, L and Xing, A and Richardson, A},
title = {Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.},
journal = {Plant physiology},
volume = {189},
number = {2},
pages = {585-594},
pmid = {35191500},
issn = {1532-2548},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Targeting/methods ; *Ochrobactrum/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; *Soybeans/genetics ; },
abstract = {Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.},
}
@article {pmid35190679,
year = {2022},
author = {Sago, CD and Lokugamage, MP and Loughrey, D and Lindsay, KE and Hincapie, R and Krupczak, BR and Kalathoor, S and Sato, M and Echeverri, ES and Fitzgerald, JP and Gan, Z and Gamboa, L and Paunovska, K and Sanhueza, CA and Hatit, MZC and Finn, MG and Santangelo, PJ and Dahlman, JE},
title = {Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.},
journal = {Nature biomedical engineering},
volume = {6},
number = {2},
pages = {157-167},
pmid = {35190679},
issn = {2157-846X},
mesh = {Animals ; Antigens, CD ; CRISPR-Cas Systems ; Cell Adhesion Molecules/genetics ; Endothelial Cells ; *Gene Editing ; Lipids/chemistry ; Liposomes ; Liver ; Lung ; Mice ; *Nanoparticles/chemistry ; Spleen ; },
abstract = {Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.},
}
@article {pmid35190677,
year = {2022},
author = {Dilliard, SA and Siegwart, DJ},
title = {Disrupting off-target Cas9 activity in the liver.},
journal = {Nature biomedical engineering},
volume = {6},
number = {2},
pages = {106-107},
pmid = {35190677},
issn = {2157-846X},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Liver ; *RNA, Guide ; },
}
@article {pmid35189910,
year = {2022},
author = {Wang, SW and Gao, C and Zheng, YM and Yi, L and Lu, JC and Huang, XY and Cai, JB and Zhang, PF and Cui, YH and Ke, AW},
title = {Current applications and future perspective of CRISPR/Cas9 gene editing in cancer.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {57},
pmid = {35189910},
issn = {1476-4598},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Genomics ; Humans ; *Neoplasms/genetics/therapy ; Oncogenes ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.},
}
@article {pmid35189535,
year = {2022},
author = {Liang, Y and Lin, H and Zou, L and Deng, X and Tang, S},
title = {Rapid detection and tracking of Omicron variant of SARS-CoV-2 using CRISPR-Cas12a-based assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {205},
number = {},
pages = {114098},
pmid = {35189535},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; SARS-CoV-2/genetics ; },
abstract = {BACKGROUND: The newly emerged SARS-CoV-2 variant of concern (VOC) Omicron is spreading quickly worldwide, which manifests an urgent need of simple and rapid assay to detect and diagnose Omicron infection and track its spread.<br><br>METHODS: To design allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of Omicron variant, and to develop a CRISPR-Cas12a-based assay to specifically detect Omicron variant.<br><br>RESULTS: Our system showed a low limit of detection of 2 copies per reaction for the plasmid DNA of Omicron variant, and could readily detect Omicron variant in 5 laboratory-confirmed clinical samples and distinguish them from 57 SARS-CoV-2 positive clinical samples (4 virus isolates and 53 oropharyngeal swab specimens) infected with wild-type (N&#xa0;=&#xa0;8) and the variants of Alpha (N&#xa0;=&#xa0;17), Beta (N&#xa0;=&#xa0;17) and Delta (N&#xa0;=&#xa0;15). The testing results could be measured by fluorescent detector or judged by naked eyes. In addition, no cross-reaction was observed when detecting 16 clinical samples infected with 9 common respiratory pathogens.<br><br>CONCLUSIONS: The rapid assay could be easily set up in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests and implemented routinely in resource-limited settings to monitor and track the spread of Omicron variant.},
}
@article {pmid35188655,
year = {2022},
author = {Kelterborn, S and Boehning, F and Sizova, I and Baidukova, O and Evers, H and Hegemann, P},
title = {Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2379},
number = {},
pages = {45-65},
pmid = {35188655},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Chlamydomonas reinhardtii/genetics ; Electroporation/methods ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; },
abstract = {With the establishment of the CRISPR-Cas9 molecular tool as a DNA editing system in 2012, the handling of gene editing experiments was strongly facilitated pushing reverse genetics approaches forward in many organisms. These new gene editing technologies also drastically increased the possibilities for design-driven synthetic biology. Here, we describe a protocol for gene editing in the green algae Chlamydomonas reinhardtii using preassembled CRISPR-Cas9 ribonucleoproteins.The three sections of the protocol guide through a complete gene editing experiment, starting with the experimental design and the choice of suitable CRISPR target sites and how to perform a Cas9 in vitro test digestion. The second part covers the transformation of algal cells with Cas9 RNPs using electroporation. In the last part, the PCR-based screening for mutants and isolation of clones is explained.},
}
@article {pmid35188654,
year = {2022},
author = {Vazquez-Vilar, M and Juarez, P and Bernabé-Orts, JM and Orzaez, D},
title = {Design of Multiplexing CRISPR/Cas9 Constructs for Plant Genome Engineering Using the GoldenBraid DNA Assembly Standard.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2379},
number = {},
pages = {27-44},
pmid = {35188654},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Genome, Plant/genetics ; RNA, Guide/genetics ; },
abstract = {Due to the huge potential of CRISPR/Cas9 for synthetic biology and genome engineering, many plant researchers are adopting this technology in their laboratories. CRISPR/Cas9 allows multiplexing of guide RNAs (gRNAs), therefore targeting several loci in the genome simultaneously. However, making DNA constructs for this purpose is not always straightforward for first-time users. Here we show how to make multiplex CRISPR/Cas9 constructs using the GoldenBraid (GB) DNA assembly system. As an example, we create a polycistronic gRNA construct that guides a dead version of Cas9 to three different positions of the nopaline synthase promoter, leading to transcriptional repression. After a description of the reagents, the protocol describes step-by-step the considerations for DNA target selection and the molecular cloning process of the final T-DNA construct as well as its testing by transient expression in Nicotiana benthamiana leaves along with a reporter construct for luciferase expression.},
}
@article {pmid35188653,
year = {2022},
author = {Dudley, QM and Raitskin, O and Patron, NJ},
title = {Cas9-Mediated Targeted Mutagenesis in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2379},
number = {},
pages = {1-26},
pmid = {35188653},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Genome, Plant ; Mutagenesis ; Plants, Genetically Modified/genetics ; Protoplasts/metabolism ; },
abstract = {Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target. When the targets are in coding sequences, this often results in loss-of-function mutations. In this chapter, we describe a method to rapidly design and assemble RNA-guided Cas9 constructs for plants and test their ability to induce mutations at their intended targets in rapid assays using both Agrobacterium-mediated transient expression and PEG-mediated DNA delivery to protoplasts, the latter of which can be adapted to a wide range of plant species. We describe a PCR-based method for detecting mutagenesis and outline the steps required to segregate the Cas9 transgene from the targeted mutation to enable the production of transgene-free mutated plants. These techniques are amenable to a range of plant species and should accelerate the application of Cas-9-mediated genome engineering for basic plant science as well as crop development.},
}
@article {pmid35188577,
year = {2022},
author = {Song, G and Zhang, F and Tian, C and Gao, X and Zhu, X and Fan, D and Tian, Y},
title = {Discovery of potent and versatile CRISPR-Cas9 inhibitors engineered for chemically controllable genome editing.},
journal = {Nucleic acids research},
volume = {50},
number = {5},
pages = {2836-2853},
pmid = {35188577},
issn = {1362-4962},
mesh = {Bacteriophages/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Interspersed Repetitive Sequences ; Streptococcus/*genetics/virology ; },
abstract = {Anti-CRISPR (Acr) proteins are encoded by many mobile genetic elements (MGEs) such as phages and plasmids to combat CRISPR-Cas adaptive immune systems employed by prokaryotes, which provide powerful tools for CRISPR-Cas-based applications. Here, we discovered nine distinct type II-A anti-CRISPR (AcrIIA24-32) families from Streptococcus MGEs and found that most Acrs can potently inhibit type II-A Cas9 orthologs from Streptococcus (SpyCas9, St1Cas9 or St3Cas9) in bacterial and human cells. Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 are able to block Cas9 binding to DNA, while AcrIIA24 abrogates DNA cleavage by Cas9. Notably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, exhibiting unique anti-CRISPR characteristics. Importantly, we developed several chemically inducible anti-CRISPR variants based on AcrIIA25.1 and AcrIIA32.1 by comprising hybrids of Acr protein and the 4-hydroxytamoxifen-responsive intein, which enabled post-translational control of CRISPR-Cas9-mediated genome editing in human cells. Taken together, our work expands the diversity of type II-A anti-CRISPR families and the toolbox of Acr proteins for the chemically inducible control of Cas9-based applications.},
}
@article {pmid35187887,
year = {2022},
author = {Li, X and Sun, B and Qian, H and Ma, J and Paolino, M and Zhang, Z},
title = {A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {2},
pages = {141-152},
pmid = {35187887},
issn = {1862-1783},
mesh = {Alleles ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing/methods ; *Recombinational DNA Repair ; },
abstract = {Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.},
}
@article {pmid35186041,
year = {2022},
author = {Tiwari, JK and Buckseth, T and Challam, C and Zinta, R and Bhatia, N and Dalamu, D and Naik, S and Poonia, AK and Singh, RK and Luthra, SK and Kumar, V and Kumar, M},
title = {CRISPR/Cas Genome Editing in Potato: Current Status and Future Perspectives.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {827808},
pmid = {35186041},
issn = {1664-8021},
}
@article {pmid35185818,
year = {2021},
author = {Zhou, Y and Zhou, W and Zhou, J and Yan, J and Xu, D and Zheng, X and Zong, S and Jiang, P and Tian, S and Han, J and Qu, D},
title = {The Clustered Regularly Interspaced Short Palindromic Repeats-Associated System and Its Relationship With Mobile Genetic Elements in Klebsiella.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {790673},
pmid = {35185818},
issn = {1664-302X},
abstract = {Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this study, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS1, IS609, and enzymes of DNA metabolism were closely related to CRISPR-Cas. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS5 were observed near the CRISPR loci in plasmid, which is also confirmed by the results of PCA that they may be important factors affecting the plasmid with CRISPR.},
}
@article {pmid35184547,
year = {2022},
author = {Sun, S and He, Z and Jiang, P and Baral, R and Pandelia, ME},
title = {Metal Dependence and Functional Diversity of Type I Cas3 Nucleases.},
journal = {Biochemistry},
volume = {61},
number = {5},
pages = {327-338},
pmid = {35184547},
issn = {1520-4995},
support = {R00 GM111978/GM/NIGMS NIH HHS/United States ; R01 GM126303/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; DNA Helicases/metabolism ; DNA, Single-Stranded ; Endonucleases/genetics ; Metals/metabolism ; },
abstract = {Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by cleaving foreign DNA. In addition, they impact bacterial physiology by regulating pathogenicity and virulence, making them key players in adaptability and evolution. The signature nuclease Cas3 is a phosphodiesterase belonging to the HD-domain metalloprotein superfamily. By directing specific metal incorporation, we map a promiscuous metal ion cofactor profile for Cas3 from Thermobifida fusca (Tf). Tf Cas3 affords significant ssDNA cleavage with four homo-dimetal centers (Fe[2+], Co[2+], Mn[2+], and Ni[2+]), while the diferrous form is the most active and likely biologically relevant in vivo. Electron paramagnetic resonance (EPR) spectroscopy and Mössbauer spectroscopy show that the diiron cofactor can access three redox forms, while the diferrous form can be readily obtained with mild reductants. We further employ EPR and Mössbauer on Fe-enriched proteins to establish that Cas3″ enzymes harbor a dinuclear cofactor, which was not previously confirmed. We demonstrate that the ancillary His ligand is critical for efficient ssDNA cleavage but not for diiron assembly or small molecule hydrolysis. We further explore the ability of Cas3 to hydrolyze cyclic mononucleotides and show that Tf Cas3 hydrolyzes 2'3'-cAMP with catalytic efficiency comparable to that of the conserved virulence factor A (CvfA), an HD-domain protein hydrolyzing 2'3'-cylic phosphodiester bonds at RNA 3'-termini. Because this CvfA activity is linked to virulence regulation, Cas3 may also utilize 2'3'-cAMP hydrolysis as a possible molecular route to control virulence.},
}
@article {pmid35184131,
year = {2022},
author = {Carpintero-Fernández, P and Borghesan, M and Eleftheriadou, O and Pan-Castillo, B and Fafián-Labora, JA and Mitchell, TP and Yuste, A and Ogrunc, M and Nightingale, TD and Mayan, M and O'Loghlen, A},
title = {Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence.},
journal = {Cell death &amp; disease},
volume = {13},
number = {2},
pages = {163},
pmid = {35184131},
issn = {2041-4889},
support = {MR/K501372/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*Breast Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cellular Senescence/genetics ; Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors/metabolism ; *Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors/metabolism ; *Factor IX/genetics ; Female ; Humans ; MCF-7 Cells ; },
abstract = {During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.},
}
@article {pmid35183175,
year = {2022},
author = {Schultz, JC and Mishra, S and Gaither, E and Mejia, A and Dinh, H and Maranas, C and Zhao, H},
title = {Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {26},
pmid = {35183175},
issn = {1475-2859},
mesh = {Aldehyde Oxidoreductases/genetics/metabolism ; Bioreactors ; CRISPR-Cas Systems ; Culture Media ; Fatty Alcohols/*metabolism ; Fermentation ; Gene Editing ; Lipid Metabolism ; Lipidomics ; *Metabolic Engineering ; Rhodotorula/*genetics/*metabolism ; },
abstract = {BACKGROUND: The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product.<br><br>RESULTS: Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7&#xa0;g/L fatty alcohol titer in the LRO1&#x394; mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption.<br><br>CONCLUSIONS: The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.},
}
@article {pmid35183173,
year = {2022},
author = {Song, I and Kim, S and Kim, J and Oh, H and Jang, J and Jeong, SJ and Baek, K and Shin, WS and Sim, SJ and Jin, E},
title = {Macular pigment-enriched oil production from genome-edited microalgae.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {27},
pmid = {35183173},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics/*metabolism ; Culture Media ; *Gene Editing ; Genome ; Glucose-1-Phosphate Adenylyltransferase/genetics/metabolism ; Lipids/biosynthesis ; Lutein/analysis ; Macular Pigment/*biosynthesis ; Microalgae/*genetics/*metabolism ; Mutation ; Oils/chemistry/*metabolism ; Zeaxanthins/analysis ; },
abstract = {BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain.<br><br>RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93&#x2009;&#xb1;&#x2009;0.22&#xa0;mg&#xa0;g[-1] DCW: dried cell weight), zeaxanthin (3.12&#x2009;&#xb1;&#x2009;0.30&#xa0;mg&#xa0;g[-1] DCW), and lipids (450.09&#x2009;&#xb1;&#x2009;25.48&#xa0;mg&#xa0;g[-1] DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196&#x2009;&#xb1;&#x2009;20.1&#xa0;mg&#xa0;g[-1] DCW of edible microalgal oil containing 8.42&#x2009;&#xb1;&#x2009;0.92&#xa0;mg&#xa0;g[-1] lutein&#xa0;of oil and 7.69&#x2009;&#xb1;&#x2009;1.03&#xa0;mg&#xa0;g[-1] zeaxanthin&#xa0;of oil was produced.<br><br>CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.},
}
@article {pmid35182556,
year = {2022},
author = {Rezazade Bazaz, M and Dehghani, H},
title = {From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods.},
journal = {Life sciences},
volume = {295},
number = {},
pages = {120409},
doi = {10.1016/j.lfs.2022.120409},
pmid = {35182556},
issn = {1879-0631},
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; DNA/chemistry/metabolism ; DNA Breaks/drug effects ; DNA End-Joining Repair/genetics ; DNA Repair/*genetics/*physiology ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Humans ; Recombinational DNA Repair/genetics ; },
abstract = {Various DNA breaks created via programmable CRISPR/Cas9 nuclease activity results in different intracellular DNA break repair pathways. Based on the cellular repair pathways, CRISPR-based gene knock-in methods can be categorized into two major strategies: 1) Homology-independent strategies which are targeted insertion events based on non-homologous end joining, and 2) Homology-dependent strategies which are targeted insertion events based on the homology-directed repair. This review elaborates on various gene knock-in methods in mammalian cells using the CRISPR/Cas9 system and in sync with DNA-break repair pathways. Gene knock-in methods are applied in functional genomics and gene therapy. To compensate or correct genetic defects, different CRISPR-based gene knock-in strategies can be used. Thus, researchers need to make a conscious decision about the most suitable knock-in method. For a successful gene-targeted insertion, some determinant factors should be considered like cell cycle, dominant DNA repair pathway, size of insertions, and donor properties. In this review, different aspects of each gene knock-in strategy are discussed to provide a framework for choosing the most appropriate gene knock-in method in different applications.},
}
@article {pmid35182055,
year = {2022},
author = {Chen, Y and Fang, L and Ying, X and Cheng, M and Wang, L and Sun, P and Zhang, Z and Shi, L and Cao, Y and Song, H},
title = {Development of Whole Genome-Scale Base Editing Toolbox to Promote Efficiency of Extracellular Electron Transfer in Shewanella oneidensis MR-1.},
journal = {Advanced biology},
volume = {6},
number = {3},
pages = {e2101296},
doi = {10.1002/adbi.202101296},
pmid = {35182055},
issn = {2701-0198},
mesh = {*CRISPR-Cas Systems ; Electrons ; Gene Editing ; *Shewanella/genetics ; },
abstract = {Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/dCas9-AID base editor that shows a relatively narrow editing window restricted to the "-20 to -16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "-22 to -9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, the coverage of the editable gene is expanded from 89% to nearly 100% in S. oneidensis MR-1. This whole genome-scale cytidine deaminase-based base editing toolbox (WGcBE) is applied to regulate the cell length and the biofilm morphology, which enhances the EET efficiency by 6.7-fold. WGcBE enables an efficient deactivation of genes with full genome coverage, which would contribute to the in-depth and multi-faceted EET study in Shewanella.},
}
@article {pmid35180691,
year = {2022},
author = {Zhu, Z and Li, R and Zhang, H and Wang, J and Lu, Y and Zhang, D and Yang, L},
title = {PAM-free loop-mediated isothermal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens.},
journal = {Biosensors &amp; bioelectronics},
volume = {204},
number = {},
pages = {114076},
doi = {10.1016/j.bios.2022.114076},
pmid = {35180691},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Pathogenic disease is an important factor affecting rice growth, yield and quality, and the development and application of rapid diagnostic methods will contribute to the prevention and control of rice disease. Herein, we developed a novel protospacer adjacent motif (PAM)-free loop-mediated isothermal amplification (LAMP) assisted CRISPR/Cas12a cleavage (Cas-PfLAMP) assay for detection of three rice pathogens; Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). The Cas-PfLAMP assay showed high specificity due to doubly specific recognition of LAMP primer sets and FnCas12a/sgRNA, and high sensitivity down to 9 or 3 copies due to LAMP amplification and CRISPR/Cas12a trans cleavage activity. Furthermore, a visual on-spot Cas-PfLAMP platform was established for detection of rice pathogens by combining solid-phase nucleic acid extraction and a lateral flow strip (LFS) test. Analysis of rice leaf field samples confirmed the impressive performance of the Cas-PfLAMP platform, demonstrating its suitability for rapid (∼50 min) on-spot detection of rice diseases. The assay could also be extended to detection of other plant diseases, and other nucleic acid field tests.},
}
@article {pmid35180689,
year = {2022},
author = {Liu, S and Wang, C and Wang, Z and Xiang, K and Zhang, Y and Fan, GC and Zhao, L and Han, H and Wang, W},
title = {Binding induced isothermal amplification reaction to activate CRISPR/Cas12a for amplified electrochemiluminescence detection of rabies viral RNA via DNA nanotweezer structure switching.},
journal = {Biosensors &amp; bioelectronics},
volume = {204},
number = {},
pages = {114078},
doi = {10.1016/j.bios.2022.114078},
pmid = {35180689},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Humans ; RNA, Viral/genetics ; *Rabies/genetics ; },
abstract = {Rabies is caused by the infection of Rabies virus, it leads to fatal encephalitis, developing a highly sensitive and specific detection method for Rabies virus remains a challenge. Herein, we report an electrochemiluminescence (ECL) biosensor for Rabies viral RNA based on dual-signal amplification and DNA nanotweezers (DTs). Dual-signal amplification process includes target binding induced isothermal amplification and CRISPR-based amplification. In the presence of target RNA, two assisted probes simultaneously hybridized with it to trigger isothermal amplification with the help of polymerase and nicking enzyme. This process generated a large amount of single-stranded DNA (ssDNA) as products. The products hybridized with CRISPR RNA to activate the trans-cleavage activity of Cas12a to indiscriminately cleave predesigned single-stranded trigger (ST) strands. After mixing the cleavage products with DTs and hemin molecules, DTs cannot be closed by cleaved ST strands to capture hemin to the electrode to quench the ECL signal. Therefore, the higher concentration of the target, the stronger intensity of the ECL signal. The detection limit is as low as 2.8 pM and the detection range is from 5 pM to 5 nM with excellent specificity and stability. The proposed method provides a promising strategy for Rabies detection, and can be easily adapted to other analytes via reasonable design as a valuable and versatile tool in bioanalysis.},
}
@article {pmid35180538,
year = {2022},
author = {Mao, Z and Wang, X and Chen, R and Zhou, Z and Ren, S and Liang, J and Gao, Z},
title = {Upconversion-mediated CRISPR-Cas12a biosensing for sensitive detection of ochratoxin A.},
journal = {Talanta},
volume = {242},
number = {},
pages = {123232},
doi = {10.1016/j.talanta.2022.123232},
pmid = {35180538},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA, Single-Stranded ; *Ochratoxins ; },
abstract = {The CRISPR-Cas system has achieved breakthrough applications in the field of molecular diagnostics. CRISPR/Cas12a can accurately identify subtle changes in a target nucleic acid sequence and has a wide range of applications such as in highly sensitive detection methods. In this study, an upconversion-magnetic probe-DNA-Fe3O4 probe was designed to replace traditional fluorescent probes using nucleic acid aptamers to design a biosensing method powered by CRISPR/Cas12a. The CRISPR/Cas12a technology can be widely used for sensitive, rapid, and stable detection of ochratoxin A. In our sensing strategy, the ochratoxin A capture probe was used to capture ochratoxin A and release the Cas protein activation chain, further triggering the single-stranded DNA degradation activity of CRISPR/Cas12a and removal of the fluorescent probe to generate a fluorescent signal. Compared with traditional commercial kits, our method was more rapid and exhibited comparable detection capability.},
}
@article {pmid35180218,
year = {2022},
author = {Ang, JX and Nevard, K and Ireland, R and Purusothaman, DK and Verkuijl, SAN and Shackleford, L and Gonzalez, E and Anderson, MAE and Alphey, L},
title = {Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source.},
journal = {PLoS genetics},
volume = {18},
number = {2},
pages = {e1010060},
pmid = {35180218},
issn = {1553-7404},
support = {BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Culicidae/genetics ; DNA Repair/genetics ; Genome ; Humans ; Mosquito Vectors/genetics ; },
abstract = {The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59-88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.},
}
@article {pmid35177812,
year = {2022},
author = {Zeng, Z and Zhang, X and Jiang, CQ and Zhang, YG and Wu, X and Li, J and Tang, S and Li, L and Gu, LJ and Xie, XY and Jiang, YA},
title = {Identifying novel therapeutic targets in gastric cancer using genome-wide CRISPR-Cas9 screening.},
journal = {Oncogene},
volume = {41},
number = {14},
pages = {2069-2078},
pmid = {35177812},
issn = {1476-5594},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Transformation, Neoplastic/genetics ; Early Detection of Cancer ; *Genes, Essential ; Humans ; *Stomach Neoplasms/genetics ; },
abstract = {Genome-scale CRISPR-Cas9 screening technology is a powerful tool to systematically identify genes essential for cancer cell survival. Herein, TKOv3, a genome-scale CRISPR-Cas9 knock-out library, was screened in the gastric cancer (GC) cells, and relevant validation experiments were performed. We obtained 854 essential genes for the AGS cell line, and 184 were novel essential genes. After knocking down essential genes: SPC25, DHX37, ABCE1, SNRPB, TOP3A, RUVBL1, CIT, TACC3 and MTBP, cell viability and proliferation were significantly decreased. Then, we analysed the detected essential genes at different time points and proved more characteristic genes might appear with the extension of selection. After progressive selection using a series of open datasets, 41 essential genes were identified as potential drug targets. Among them, methyltransferase 1 (METTL1) was over expressed in GC tissues. High METTL1 expression was associated with poor prognosis among 3 of 6 GC cohorts. Furthermore, GC cells growth was significantly inhibited after the down-regulation of METTL1 in vitro and in vivo. Function analysis revealed that METTL1 might play a role in the cell cycle through AKT/STAT3 pathways. In conclusion, compared with existing genome-scale screenings, we obtained 184 novel essential genes. Among them, METTL1 was validated as a potential therapeutic target of GC.},
}
@article {pmid35177617,
year = {2022},
author = {Baisya, D and Ramesh, A and Schwartz, C and Lonardi, S and Wheeldon, I},
title = {Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {922},
pmid = {35177617},
issn = {2041-1723},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Deep Learning ; Endodeoxyribonucleases/genetics ; Gene Editing/*methods ; Genome, Fungal ; *Models, Genetic ; RNA, Guide/genetics/*metabolism ; Yarrowia/*genetics ; },
abstract = {Genome-wide functional genetic screens have been successful in discovering genotype-phenotype relationships and in engineering new phenotypes. While broadly applied in mammalian cell lines and in E. coli, use in non-conventional microorganisms has been limited, in part, due to the inability to accurately design high activity CRISPR guides in such species. Here, we develop an experimental-computational approach to sgRNA design that is specific to an organism of choice, in this case the oleaginous yeast Yarrowia lipolytica. A negative selection screen in the absence of non-homologous end-joining, the dominant DNA repair mechanism, was used to generate single guide RNA (sgRNA) activity profiles for both SpCas9 and LbCas12a. This genome-wide data served as input to a deep learning algorithm, DeepGuide, that is able to accurately predict guide activity. DeepGuide uses unsupervised learning to obtain a compressed representation of the genome, followed by supervised learning to map sgRNA sequence, genomic context, and epigenetic features with guide activity. Experimental validation, both genome-wide and with a subset of selected genes, confirms DeepGuide's ability to accurately predict high activity sgRNAs. DeepGuide provides an organism specific predictor of CRISPR guide activity that with retraining could be applied to other fungal species, prokaryotes, and other non-conventional organisms.},
}
@article {pmid35176664,
year = {2022},
author = {Kamiya, D and Yamashita, T and Akaboshi, T and Yamaguchi, Y and Toyooka, Y and Ikeya, M},
title = {Generation of human GAPDH knock-in reporter iPSC lines for stable expression of tdTomato in pluripotent and differentiated culture conditions.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102704},
doi = {10.1016/j.scr.2022.102704},
pmid = {35176664},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Luminescent Proteins/metabolism ; },
abstract = {Human induced pluripotent stem cells (iPSCs) can differentiate into multiple cell types and are utilized for research on human development and regenerative medicine. Here, we report the establishment of human GAPDH knock-in reporter iPSC lines (GAPDH-tdT1 and 2), via CRISPR/Cas9-mediated homologous recombination, that stably express tdTomato as a constitutive cell label in both iPSCs and their differentiated derivatives. These cell lines will provide useful tools to trace cell locations and fates in 2D cultures and 3D organoids and will facilitate in vivo experiments.},
}
@article {pmid35176430,
year = {2022},
author = {Fear, VS and Forbes, CA and Anderson, D and Rauschert, S and Syn, G and Shaw, N and Jones, ME and Forrest, AR and Baynam, G and Lassmann, T},
title = {Functional validation of variants of unknown significance using CRISPR gene editing and transcriptomics: A Kleefstra syndrome case study.},
journal = {Gene},
volume = {821},
number = {},
pages = {146287},
doi = {10.1016/j.gene.2022.146287},
pmid = {35176430},
issn = {1879-0038},
mesh = {CRISPR-Cas Systems ; Chromosome Deletion ; Chromosomes, Human, Pair 19/genetics ; Chromosomes, Human, Pair 9/genetics ; Chromosomes, Human, X/genetics ; Craniofacial Abnormalities/*diagnosis/genetics ; Early Diagnosis ; Gene Editing/*methods ; Gene Expression Profiling/*methods ; Gene Expression Regulation ; *Gene Regulatory Networks ; Genetic Variation ; HEK293 Cells ; Heart Defects, Congenital/*diagnosis/genetics ; Histone-Lysine N-Methyltransferase/*genetics ; Humans ; Intellectual Disability/*diagnosis/genetics ; Proof of Concept Study ; Sequence Analysis, RNA ; },
abstract = {There are an estimated > 400 million people living with a rare disease globally, with genetic variants the cause of approximately 80% of cases. Next Generation Sequencing (NGS) rapidly identifies genetic variants however they are often of unknown significance. Low throughput functional validation in specialist laboratories is the current ad hoc approach for functional validation of genetic variants, which creating major bottlenecks in patient diagnosis. This study investigates the application of CRISPR gene editing followed by genome wide transcriptomic profiling to facilitate patient diagnosis. As proof-of-concept, we introduced a variant in the Euchromatin histone methyl transferase (EHMT1) gene into HEK293T cells. We identified changes in the regulation of the cell cycle, neural gene expression and suppression of gene expression changes on chromosome 19 and chromosome X, that are in keeping with Kleefstra syndrome clinical phenotype and/or provide insight into disease mechanism. This study demonstrates the utility of genome editing followed by functional readouts to rapidly and systematically validating the function of variants of unknown significance in patients suffering from rare diseases.},
}
@article {pmid35176052,
year = {2022},
author = {Ashoti, A and Limone, F and van Kranenburg, M and Alemany, A and Baak, M and Vivié, J and Piccioni, F and Dijkers, PF and Creyghton, M and Eggan, K and Geijsen, N},
title = {Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen.},
journal = {PloS one},
volume = {17},
number = {2},
pages = {e0263262},
pmid = {35176052},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; Cell Survival ; *Gene Expression Regulation ; Homeodomain Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism/*pathology ; Muscle Cells/metabolism/*pathology ; Muscular Dystrophy, Facioscapulohumeral/genetics/metabolism/*pathology ; Myoblasts/metabolism/*pathology ; },
abstract = {Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.},
}
@article {pmid35173702,
year = {2022},
author = {Lemaire, C and Le Gallou, B and Lanotte, P and Mereghetti, L and Pastuszka, A},
title = {Distribution, Diversity and Roles of CRISPR-Cas Systems in Human and Animal Pathogenic Streptococci.},
journal = {Frontiers in microbiology},
volume = {13},
number = {},
pages = {828031},
pmid = {35173702},
issn = {1664-302X},
abstract = {Streptococci form a wide group of bacteria and are involved in both human and animal pathologies. Among pathogenic isolates, differences have been highlighted especially concerning their adaptation and virulence profiles. CRISPR-Cas systems have been identified in bacteria and many streptococci harbor one or more systems, particularly subtypes I-C, II-A, and III-A. Since the demonstration that CRISPR-Cas act as an adaptive immune system in Streptococcus thermophilus, a lactic bacteria, the diversity and role of CRISPR-Cas were extended to many germs and functions were enlarged. Among those, the genome editing tool based on the properties of Cas endonucleases is used worldwide, and the recent attribution of the Nobel Prize illustrates the importance of this tool in the scientific world. Another application is CRISPR loci analysis, which allows to easily characterize isolates in order to understand the interactions of bacteria with their environment and visualize species evolution. In this review, we focused on the distribution, diversity and roles of CRISPR-Cas systems in the main pathogenic streptococci.},
}
@article {pmid35173152,
year = {2022},
author = {Liu, J and Liu, M and Shi, T and Sun, G and Gao, N and Zhao, X and Guo, X and Ni, X and Yuan, Q and Feng, J and Liu, Z and Guo, Y and Chen, J and Wang, Y and Zheng, P and Sun, J},
title = {CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {891},
pmid = {35173152},
issn = {2041-1723},
mesh = {Base Sequence ; Bioengineering/*methods ; Bioreactors/*microbiology ; CRISPR-Cas Systems/genetics ; Carrier Proteins/genetics ; Corynebacterium glutamicum/*genetics/*metabolism ; Gene Editing/methods ; Phosphotransferases (Carboxyl Group Acceptor)/genetics ; Proline/*biosynthesis ; Protein Transport/genetics ; },
abstract = {Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards L-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an L-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces L-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.},
}
@article {pmid35173102,
year = {2022},
author = {Tateno, M and Umeyama, T and Inukai, T and Takatsuka, S and Hoshino, Y and Yamagoe, S and Yamagata Murayama, S and Ishino, K and Miyazaki, Y},
title = {Examination of Cyp51A-Mediated Azole Resistance in Aspergillus lentulus Using CRISPR/Cas9 Genome Editing.},
journal = {Medical mycology journal},
volume = {63},
number = {2},
pages = {27-35},
doi = {10.3314/mmj.21-00024},
pmid = {35173102},
issn = {1882-0476},
mesh = {Antifungal Agents/pharmacology ; Aspergillus ; Aspergillus fumigatus/genetics ; *Azoles/pharmacology ; CRISPR-Cas Systems ; *Drug Resistance, Fungal/genetics ; Fungal Proteins/genetics/metabolism ; Gene Editing ; Humans ; Microbial Sensitivity Tests ; },
abstract = {Aspergillus lentulus was first reported in 2005 as a cryptic species of Aspergillus fumigatus, and since then, its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Although it has been reported that P450 14-α sterol demethylase (Cyp51) is involved in azole resistance in A. lentulus, the specific resistance mechanism has not been elucidated. In this study, we successfully introduced the entire A. fumigatus cyp51A gene into the cyp51A locus in A. lentulus using the CRISPR/Cas9 genome-editing system. The A. lentulus strains harboring A. fumigatus cyp51A showed reduced minimum inhibitory concentrations for itraconazole and voriconazole compared with those of the parent strain. This finding suggests that Cyp51A is involved in azole resistance in A. lentulus and may contribute to the elucidation of the mechanism of resistance to azole drugs via Cyp51A and to the development of new antifungal drugs. In addition, our successful application of the CRISPR/Cas9 system to A. lentulus opens the door to examination of other gene functions in this fungus.},
}
@article {pmid35172714,
year = {2022},
author = {Müller Paul, H and Istanto, DD and Heldenbrand, J and Hudson, ME},
title = {CROPSR: an automated platform for complex genome-wide CRISPR gRNA design and validation.},
journal = {BMC bioinformatics},
volume = {23},
number = {1},
pages = {74},
pmid = {35172714},
issn = {1471-2105},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome ; Plant Breeding ; *RNA, Guide/genetics ; Software ; },
abstract = {BACKGROUND: CRISPR/Cas9 technology has become an important tool to generate targeted, highly specific genome mutations. The technology has great potential for crop improvement, as crop genomes are tailored to optimize specific traits over generations of breeding. Many crops have highly complex and polyploid genomes, particularly those used for bioenergy or bioproducts. The majority of tools currently available for designing and evaluating gRNAs for CRISPR experiments were developed based on mammalian genomes that do not share the characteristics or design criteria for crop genomes.<br><br>RESULTS: We have developed an open source tool for genome-wide design and evaluation of gRNA sequences for CRISPR experiments, CROPSR. The genome-wide approach provides a significant decrease in the time required to design a CRISPR experiment, including validation through PCR, at the expense of an overhead compute time required once per genome, at the first run. To better cater to the needs of crop geneticists, restrictions imposed by other packages on design and evaluation of gRNA sequences were lifted. A new machine learning model was developed to provide scores while avoiding situations in which the currently available tools sometimes failed to provide guides for repetitive, A/T-rich genomic regions. We show that our gRNA scoring model provides a significant increase in prediction accuracy over existing tools, even in non-crop genomes.<br><br>CONCLUSIONS: CROPSR provides the scientific community with new methods and a new workflow for performing CRISPR/Cas9 knockout experiments. CROPSR reduces the challenges of working in crops, and helps speed gRNA sequence design, evaluation and validation. We hope that the new software will accelerate discovery and reduce the number of failed experiments.},
}
@article {pmid35172133,
year = {2022},
author = {Sivakumar, S and Qi, S and Cheng, N and Sathe, AA and Kanchwala, M and Kumar, A and Evers, BM and Xing, C and Yu, H},
title = {TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling.},
journal = {Cell reports},
volume = {38},
number = {7},
pages = {110395},
pmid = {35172133},
issn = {2211-1247},
support = {R01 GM124096/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Motifs ; Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; *Cell Lineage ; Cilia/*metabolism ; Cytoskeletal Proteins/genetics/metabolism ; Genome, Human ; Hedgehog Proteins/*metabolism ; Human Embryonic Stem Cells/*cytology/*metabolism ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Neural Stem Cells/metabolism ; Neurogenesis/genetics ; *Organogenesis ; *Signal Transduction ; Teratoma/pathology ; Tumor Suppressor Protein p53/chemistry/*metabolism ; },
abstract = {Aneuploidy, defective differentiation, and inactivation of the tumor suppressor TP53 all occur frequently during tumorigenesis. Here, we probe the potential links among these cancer traits by inactivating TP53 in human embryonic stem cells (hESCs). TP53[-/-] hESCs exhibit increased proliferation rates, mitotic errors, and low-grade structural aneuploidy; produce poorly differentiated immature teratomas in mice; and fail to differentiate into neural progenitor cells (NPCs) in vitro. Genome-wide CRISPR screen reveals requirements of ciliogenesis and sonic hedgehog (Shh) pathways for hESC differentiation into NPCs. TP53 deletion causes abnormal ciliogenesis in neural rosettes. In addition to restraining cell proliferation through CDKN1A, TP53 activates the transcription of BBS9, which encodes a ciliogenesis regulator required for proper Shh signaling and NPC formation. This developmentally regulated transcriptional program of TP53 promotes ciliogenesis, restrains Shh signaling, and commits hESCs to neural lineages.},
}
@article {pmid35171471,
year = {2022},
author = {Guo, X and Kampmann, M},
title = {CRISPR-Based Screening for Stress Response Factors in Mammalian Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2428},
number = {},
pages = {19-40},
pmid = {35171471},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; Genome ; Phenotype ; RNA Interference ; },
abstract = {In the presence of different physiological and environmental stresses, cells rapidly initiate stress responses to re-establish cellular homeostasis. Stress responses usually orchestrate both transcriptional and translational programs via distinct mechanisms. With the advance of transcriptomics and proteomics technologies, transcriptional and translational outputs to a particular stress condition have become easier to measure; however, these technologies lack the ability to reveal the upstream regulatory pathways. Unbiased genetic screens based on a transcriptional or translational reporter are powerful approaches to identify regulatory factors of a specific stress response. CRISPR/Cas-based technologies, together with next-generation sequencing, enable genome-scale pooled screens to systematically elucidate gene function in mammalian cells, with a significant reduction in the rate of off-target effects compared to the previously used RNAi technology. Here, we describe our fluorescence-activated cell sorting (FACS)-based CRISPR interference (CRISPRi) screening platform using a translational reporter to identify novel genetic factors of the mitochondrial stress response in mammalian cells. This protocol provides a general framework for scientists who wish to establish a reporter-based CRISPRi screening platform to address questions in their area of research.},
}
@article {pmid35170139,
year = {2022},
author = {Oikawa, M and Nagae, M and Mizuno, N and Iwatsuki, K and Yoshida, F and Inoue, N and Uenoyama, Y and Tsukamura, H and Nakauchi, H and Hirabayashi, M and Kobayashi, T},
title = {Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting.},
journal = {Molecular reproduction and development},
volume = {89},
number = {3},
pages = {129-132},
doi = {10.1002/mrd.23562},
pmid = {35170139},
issn = {1098-2795},
mesh = {Animals ; *CRISPR-Cas Systems ; *Dependovirus/genetics ; Female ; Gene Editing ; Gene Knock-In Techniques ; Gene Targeting ; Luminescent Proteins ; Mammals/genetics ; Pregnancy ; Rats ; Zygote/metabolism ; },
abstract = {Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.},
}
@article {pmid35169967,
year = {2022},
author = {Widjaya, MA and Ju, JC and Lee, SD},
title = {CRISPR-Edited Stem Cell Transplantation for HIV-Related Gene Modification In Vivo: A Systematic Review.},
journal = {Stem cell reviews and reports},
volume = {18},
number = {5},
pages = {1743-1755},
pmid = {35169967},
issn = {2629-3277},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *HIV Infections/genetics/therapy ; *Hematopoietic Stem Cell Transplantation ; Humans ; Stem Cell Transplantation ; },
abstract = {BACKGROUND: CRISPR is a novel genomic editing technology which can be useful for the treatment of immune diseases such as HIV. However, the application of CRISPR in stem cells for HIV-related research was not effective, and most of the research was done in vivo. This systematic review is to identify a new research idea about increase CRISPR-editing efficiencies in stem cell transplantation for HIV treatment, as well as its future perspective.<br><br>METHOD: Four databases were searched for articles published during 1952 to 2020. PRISMA method was used to select appropriate research papers. CAMARADES was used to identify the paper quality. The outcome was engraftment efficiency, gene disruption percentage, differentiation ability, HIV-resistant efficiency.<br><br>RESULT: Screening method showed 196 papers mentioned the topic. However, only 5 studies were reliable with the research objective. We found that (1) Two research ideas which was double gene knockout and knockout-knockin method to provide HIV-resistant cells, engraftment support and avoid cardiac disease as an HIV disease side effect. (2) Ribonucleoprotein (RNP) delivery was the best way to deliver the CRISPR/Cas9 and Adeno-Associated Virus (AAV) would be effective for knockin purpose. (3) CRISPR/SaCas9 could replace CRISPR/Cas9 role in editing HIV-related gene.<br><br>CONCLUSION: Potential genes to increase HIV resistance and stem cell engraftment should be explored more in the future. Double knockout and knock-in procedures should be applied to set up a better engraftment for improving HIV treatment or resistance of patients. CRISPR/SaCas9 and RNP delivery should be explored more in the future.<br><br>PROSPERO CRD42020203312.},
}
@article {pmid35169221,
year = {2022},
author = {Fatimah, RM and Adhitama, N and Kato, Y and Watanabe, H},
title = {Development of transgenic Daphnia magna for visualizing homology-directed repair of DNA.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {2497},
pmid = {35169221},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems ; DNA/genetics ; DNA End-Joining Repair/genetics ; Daphnia/*genetics ; Gene Editing/methods ; Gene Knock-In Techniques ; Genes, Reporter ; Genotype ; Green Fluorescent Proteins/*genetics/metabolism ; Luminescent Proteins/*genetics/metabolism ; Plasmids ; Promoter Regions, Genetic/genetics ; Recombinational DNA Repair/*genetics ; Signal Transduction/genetics ; },
abstract = {In the crustacean Daphnia magna, studying homology-directed repair (HDR) is important to understand genome maintenance during parthenogenesis, effects of environmental toxicants on the genome, and improvement of HDR-mediated genome editing. Here we developed a transgenic D. magna that expresses green fluorescence protein (GFP) upon HDR occurrence. We utilized the previously established reporter plasmid named DR-GFP that has a mutated eGFP gene (SceGFP) and the tandemly located donor GFP gene fragment (iGFP). Upon double-strand break (DSB) introduction on SceGFP, the iGFP gene fragment acts as the HDR template and restores functional eGFP expression. We customized this reporter plasmid to allow bicistronic expression of the mCherry gene under the control of the D. magna EF1α-1 promoter/enhancer. By CRISPR/Cas-mediated knock-in of this plasmid via non-homologous joining, we generated the transgenic D. magna that expresses mCherry ubiquitously, suggesting that the DR-GFP reporter gene is expressed in most cells. Introducing DSB on the SceGFP resulted in eGFP expression and this HDR event could be detected by fluorescence, genomic PCR, and quantitative reverse-transcription PCR, suggesting this line could be used for evaluating HDR. The established reporter line might expand our understanding of the HDR mechanism and also improve the HDR-based gene-editing system in this species.},
}
@article {pmid35169201,
year = {2022},
author = {Blankestijn, M and Bloks, VW and Struik, D and Huijkman, N and Kloosterhuis, N and Wolters, JC and Wanders, RJA and Vaz, FM and Islinger, M and Kuipers, F and van de Sluis, B and Groen, AK and Verkade, HJ and Jonker, JW},
title = {Mice with a deficiency in Peroxisomal Membrane Protein 4 (PXMP4) display mild changes in hepatic lipid metabolism.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {2512},
pmid = {35169201},
issn = {2045-2322},
mesh = {Animals ; Bile Acids and Salts/metabolism ; CRISPR-Cas Systems ; Diet/methods ; Fatty Acids/*metabolism ; Fatty Acids, Unsaturated/*metabolism ; Female ; Fenofibrate/administration &amp; dosage ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Liver/*metabolism ; Male ; Membrane Proteins/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Oxidation-Reduction/drug effects ; PPAR alpha/metabolism ; Peroxisomes/drug effects/metabolism ; Phytanic Acid/metabolism ; Phytol/administration &amp; dosage ; Signal Transduction/*genetics ; },
abstract = {Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout (Pxmp4[-/-]) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4[-/-] mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4[-/-] mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4[-/-] mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids.},
}
@article {pmid35168613,
year = {2022},
author = {Lu, QSM and Tian, L},
title = {An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes.},
journal = {BMC biotechnology},
volume = {22},
number = {1},
pages = {7},
pmid = {35168613},
issn = {1472-6750},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Plant/genetics ; Mutation ; Oxidoreductases ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; *Soybeans/genetics ; },
abstract = {BACKGROUND: Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies.<br><br>RESULTS: We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used 'half-seed' explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75-100% mutation efficiencies. Indel frequencies varied widely among plants (3-100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27-100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants.<br><br>CONCLUSIONS: Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.},
}
@article {pmid35168603,
year = {2022},
author = {Kim, B and Kim, Y and Shin, S and Lee, ST and Cho, JY and Lee, KA},
title = {Application of CRISPR/Cas9-based mutant enrichment technique to improve the clinical sensitivity of plasma EGFR testing in patients with non-small cell lung cancer.},
journal = {Cancer cell international},
volume = {22},
number = {1},
pages = {82},
pmid = {35168603},
issn = {1475-2867},
abstract = {BACKGROUND: Approximately 50%-60% of secondary resistance to primary EGFR- tyrosine kinase inhibitors (TKI) therapy is caused by acquired p.Thr790Met (T790M) mutation; however, highly fragmented, low-quantity circulating tumor DNA is an obstacle for detecting mutations. Therefore, more sensitive mutation detection techniques are required. Here, we report a new mutant enrichment technology, the CRISPR system combined with post-polymerase chain reaction (PCR) cell-free DNA (cfDNA) (CRISPR-CPPC) to detect the T790M mutation using droplet digital PCR (ddPCR) from cfDNA.<br><br>METHODS: The CRISPR-CPPC process comprises the following three steps: (1) cfDNA PCR, (2) assembly of post-PCR cfDNA and CRISPR/CRISPR associated protein 9 complex, and (3) enrichment of the target DNA template. After CRISPR-CPPC, the target DNA was detected using ddPCR. We optimized and validated CRISPR-CPPC using reference cfDNA standards and cfDNA from patients with non-small cell lung cancer who underwent TKI therapy. We then compared the detection sensitivity of CRISPR-CPPC assay with the results of real-time PCR and those of ddPCR.<br><br>RESULTS: CRISPR-CPPC aided detection of T790M with 93.9% sensitivity and 100% specificity. T790M mutant copies were sensitively detected achieving an approximately 13-fold increase in the detected allele frequency. Furthermore, positive rate of detecting a low T790M copy number (<&#xa0;10 copies/mL)&#xa0;were 93.8% (15/16) and 43.8% (7/16) for CRISPR-CPPC assay and ddPCR, respectively.<br><br>CONCLUSIONS: CRISPR-CPPC is a useful mutant enrichment tool for the sensitive detection of target mutation. When tested in patients with progressive disease, the diagnostic performance of CRISPR-CPPC assay is exceptionally better than that of any other currently available methods.},
}
@article {pmid35168568,
year = {2022},
author = {Wang, Y and Xie, S and Armendariz, D and Hon, GC},
title = {Computational identification of clonal cells in single-cell CRISPR screens.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {135},
pmid = {35168568},
issn = {1471-2164},
support = {DP2GM128203/GM/NIGMS NIH HHS/United States ; UM1 HG011996/HG/NHGRI NIH HHS/United States ; DP2 GM128203/GM/NIGMS NIH HHS/United States ; UM1HG011996/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome ; *RNA, Guide ; Reproducibility of Results ; },
abstract = {BACKGROUND: Single-cell CRISPR screens are powerful tools to understand genome function by linking genetic perturbations to transcriptome-wide phenotypes. However, since few cells can be affordably sequenced in these screens, biased sampling of cells could affect data interpretation. One potential source of biased sampling is clonal cell expansion.<br><br>RESULTS: Here, we identify clonal cells in single cell screens using multiplexed sgRNAs as barcodes. We find that the cells in each clone share transcriptional similarities and bear segmental copy number changes. These analyses suggest that clones are genetically distinct. Finally, we show that the transcriptional similarities of clonally expanded cells contribute to false positives in single-cell CRISPR screens.<br><br>CONCLUSIONS: Experimental conditions that reduce clonal expansion or computational filtering of clonal cells will improve the reliability of single-cell CRISPR screens.},
}
@article {pmid35168043,
year = {2022},
author = {Zhao, G and Wang, J and Yao, C and Xie, P and Li, X and Xu, Z and Xian, Y and Lei, H and Shen, X},
title = {Alkaline lysis-recombinase polymerase amplification combined with CRISPR/Cas12a assay for the ultrafast visual identification of pork in meat products.},
journal = {Food chemistry},
volume = {383},
number = {},
pages = {132318},
doi = {10.1016/j.foodchem.2022.132318},
pmid = {35168043},
issn = {1873-7072},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; DNA ; Humans ; Meat/analysis ; *Meat Products/analysis ; *Pork Meat ; Recombinases/genetics/metabolism ; *Red Meat/analysis ; Swine/genetics ; },
abstract = {The "Horsemeat Scandal" makes people pay more attention to the meat authenticity. However, expensive equipment, complicated operations, and professional personnel of current methods limit their field testing. In this study, CRISPR/Cas12a combined with recombinase polymerase amplification was used to establish a sensitive and rapid detection method for pig-derived component. The detection limit can reach to 10[-3] ng for pig DNA and be completed within 30 min. Beef and pork binary mixture models under different processing conditions of raw meat, boiled, and high-pressure were tested. Combining with two different DNA extraction methods, the detection limits of pork are as low as 0.1% and 0.001% (w/w), respectively. After 125 commercial products are tested, the results are completely consistent with the Chinese national standard real-time PCR method. This method not only has better detectability, but also can quickly and conveniently realize the visual identification of pig-derived ingredients, thus is suitable for on-site detection.},
}
@article {pmid35167623,
year = {2022},
author = {Kim, SS and Kycia, I and Karski, M and Ma, RK and Bordt, EA and Kwan, J and Karki, A and Winter, E and Aktas, RG and Wu, Y and Emili, A and Bauer, DE and Sethupathy, P and Vakili, K},
title = {DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling.},
journal = {PloS one},
volume = {17},
number = {2},
pages = {e0263829},
pmid = {35167623},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*genetics ; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/*genetics/metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; HSP40 Heat-Shock Proteins/*genetics/metabolism ; Humans ; Mitochondria/metabolism ; Models, Biological ; Oncogene Proteins, Fusion/*genetics ; Proteomics ; RNA, Long Noncoding/*genetics ; *Up-Regulation ; },
abstract = {Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.},
}
@article {pmid35167486,
year = {2021},
author = {Lamarthée, B and Burger, C and Leclaire, C and Lebraud, E and Zablocki, A and Morin, L and Lebreton, X and Charreau, B and Snanoudj, R and Charbonnier, S and Blein, T and Hardy, M and Zuber, J and Satchell, S and Gallazzini, M and Terzi, F and Legendre, C and Taupin, JL and Rabant, M and Tinel, C and Anglicheau, D},
title = {CRISPR/Cas9-Engineered HLA-Deleted Glomerular Endothelial Cells as a Tool to Predict Pathogenic Non-HLA Antibodies in Kidney Transplant Recipients.},
journal = {Journal of the American Society of Nephrology : JASN},
volume = {32},
number = {12},
pages = {3231-3251},
pmid = {35167486},
issn = {1533-3450},
mesh = {Adult ; Aged ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Endothelial Cells/immunology ; Female ; Gene Deletion ; Graft Rejection/*etiology ; HLA Antigens/genetics/*immunology ; Humans ; Isoantibodies/*immunology ; Kidney Glomerulus/*immunology ; Kidney Transplantation/*adverse effects ; Male ; Middle Aged ; Nuclear Proteins/genetics ; Reoperation ; Retrospective Studies ; *Tissue Donors ; Trans-Activators/genetics ; beta 2-Microglobulin/genetics ; },
abstract = {BACKGROUND: After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs).<br><br>METHODS: W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II-deficient glomerular endothelial cells (CiGEnC&#x394;HLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens.<br><br>RESULTS: W e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnC&#x394;HLA clone. CiGEnC&#x394;HLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (P<0.001). Stratification of 3-month allograft biopsy specimens (n=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (P=0.002), microvascular inflammation (P=0.003), and ABMRh (P=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (P=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh.<br><br>CONCLUSION: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.},
}
@article {pmid35166844,
year = {2022},
author = {Okada, K and Aoki, K and Tabei, T and Sugio, K and Imai, K and Bonkohara, Y and Kamachi, Y},
title = {Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9.},
journal = {Nucleic acids research},
volume = {50},
number = {5},
pages = {2854-2871},
pmid = {35166844},
issn = {1362-4962},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA/genetics ; *RNA, Guide/genetics ; Ribonucleoproteins/genetics ; Zebrafish/genetics ; },
abstract = {Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.},
}
@article {pmid35166843,
year = {2022},
author = {Hong, SH and Lee, G and Park, C and Koo, J and Kim, EH and Bae, E and Suh, JY},
title = {The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites.},
journal = {Nucleic acids research},
volume = {50},
number = {4},
pages = {2363-2376},
pmid = {35166843},
issn = {1362-4962},
mesh = {*Bacteriophages/genetics ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Viral Proteins/metabolism ; },
abstract = {Bacteria and archaea use the CRISPR-Cas system to fend off invasions of bacteriophages and foreign plasmids. In response, bacteriophages encode anti-CRISPR (Acr) proteins that potently inhibit host Cas proteins to suppress CRISPR-mediated immunity. AcrIE4-F7, which was isolated from Pseudomonas citronellolis, is a fused form of AcrIE4 and AcrIF7 that inhibits both type I-E and type I-F CRISPR-Cas systems. Here, we determined the structure of AcrIE4-F7 and identified its Cas target proteins. The N-terminal AcrIE4 domain adopts a novel α-helical fold that targets the PAM interaction site of the type I-E Cas8e subunit. The C-terminal AcrIF7 domain exhibits an αβ fold like native AcrIF7, which disables target DNA recognition by the PAM interaction site in the type I-F Cas8f subunit. The two Acr domains are connected by a flexible linker that allows prompt docking onto their cognate Cas8 targets. Conserved negative charges in each Acr domain are required for interaction with their Cas8 targets. Our results illustrate a common mechanism by which AcrIE4-F7 inhibits divergent CRISPR-Cas types.},
}
@article {pmid35165856,
year = {2022},
author = {Happi Mbakam, C and Lamothe, G and Tremblay, G and Tremblay, JP},
title = {CRISPR-Cas9 Gene Therapy for Duchenne Muscular Dystrophy.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {19},
number = {3},
pages = {931-941},
pmid = {35165856},
issn = {1878-7479},
support = {//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Editing/methods ; Genetic Therapy/methods ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; },
abstract = {Discovery of the CRISPR-Cas (clustered regularly interspaced short palindromic repeat, CRISPR-associated) system a decade ago has opened new possibilities in the field of precision medicine. CRISPR-Cas was initially identified in bacteria and archaea to play a protective role against foreign genetic elements during viral infections. The application of this technique for the correction of different mutations found in the Duchenne muscular dystrophy (DMD) gene led to the development of several potential therapeutic approaches for DMD patients. The mutations responsible for Duchenne muscular dystrophy mainly include exon deletions (70% of patients) and point mutations (about 30% of patients). The CRISPR-Cas 9 technology is becoming increasingly precise and is acquiring diverse functions through novel innovations such as base editing and prime editing. However, questions remain about its translation to the clinic. Current research addressing off-target editing, efficient muscle-specific delivery, immune response to nucleases, and vector challenges may eventually lead to the clinical use of the CRISPR-Cas9 technology. In this review, we present recent CRISPR-Cas9 strategies to restore dystrophin expression in vitro and in animal models of DMD.},
}
@article {pmid35165826,
year = {2022},
author = {Nakano, Y and Susa, K and Yanagi, T and Hiraoka, Y and Suzuki, T and Mori, T and Ando, F and Mandai, S and Fujiki, T and Rai, T and Uchida, S and Sohara, E},
title = {Generation of NPHP1 knockout human pluripotent stem cells by a practical biallelic gene deletion strategy using CRISPR/Cas9 and ssODN.},
journal = {In vitro cellular &amp; developmental biology. Animal},
volume = {58},
number = {2},
pages = {85-95},
pmid = {35165826},
issn = {1543-706X},
mesh = {Adaptor Proteins, Signal Transducing/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Cytoskeletal Proteins/genetics ; Gene Deletion ; Gene Editing/methods ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {CRISPR/Cas9 genome editing underwent remarkable progress and significantly contributed to the development of life sciences. Induced pluripotent stem cells (iPSCs) have also made a relevant contribution to regenerative medicine, pharmacological research, and genetic disease analysis. However, knockout iPSC generation with CRISPR/Cas9 in general has been difficult to achieve using approaches such as frameshift mutations to reproduce genetic diseases with full-length or nearly full-length gene deletions. Moreover, splicing and illegitimate translation could make complete knockouts difficult. Full-length gene deletion methods in iPSCs might solve these problems, although no such approach has been reported yet. In this study, we present a practical two-step gene-editing strategy leading to the precise, biallelic, and complete deletion of the full-length NPHP1 gene in iPSCs, which is the first report of biallelic (compound heterozygous) full-gene deletion in iPSCs using CRISPR/Cas9 and single-stranded oligodeoxynucleotides mainly via single-strand template repair (SSTR). Our strategy requires no selection or substances to enhance SSTR and can be used for the analysis of genetic disorders that are difficult to reproduce by conventional knockout methods.},
}
@article {pmid35165384,
year = {2022},
author = {Sánchez-Rivera, FJ and Diaz, BJ and Kastenhuber, ER and Schmidt, H and Katti, A and Kennedy, M and Tem, V and Ho, YJ and Leibold, J and Paffenholz, SV and Barriga, FM and Chu, K and Goswami, S and Wuest, AN and Simon, JM and Tsanov, KM and Chakravarty, D and Zhang, H and Leslie, CS and Lowe, SW and Dow, LE},
title = {Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.},
journal = {Nature biotechnology},
volume = {40},
number = {6},
pages = {862-873},
pmid = {35165384},
issn = {1546-1696},
support = {T32 CA160001/CA/NCI NIH HHS/United States ; F31 CA247351/CA/NCI NIH HHS/United States ; R01 CA229773/CA/NCI NIH HHS/United States ; F31 CA192835/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P01 CA129243/CA/NCI NIH HHS/United States ; P01 CA087497/CA/NCI NIH HHS/United States ; R35 CA197588/CA/NCI NIH HHS/United States ; F31 CA261061/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Neoplasms/genetics ; Nucleotides ; RNA, Guide/genetics ; },
abstract = {Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.},
}
@article {pmid35165339,
year = {2022},
author = {Fierro, J and DiPasquale, J and Perez, J and Chin, B and Chokpapone, Y and Tran, AM and Holden, A and Factoriza, C and Sivagnanakumar, N and Aguilar, R and Mazal, S and Lopez, M and Dou, H},
title = {Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {2417},
pmid = {35165339},
issn = {2045-2322},
mesh = {B7-H1 Antigen/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Cell Polarity ; Cell Proliferation ; Exons ; Gene Editing ; Gene Knockdown Techniques ; Glioblastoma/*genetics/metabolism/pathology/*physiopathology ; Humans ; Interleukin-4/genetics/metabolism ; Neoplasm Invasiveness ; RNA, Guide ; Tumor-Associated Macrophages/*cytology/metabolism ; },
abstract = {Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.},
}
@article {pmid35165036,
year = {2022},
author = {Deng, F and Zeng, F and Shen, Q and Abbas, A and Cheng, J and Jiang, W and Chen, G and Shah, AN and Holford, P and Tanveer, M and Zhang, D and Chen, ZH},
title = {Molecular evolution and functional modification of plant miRNAs with CRISPR.},
journal = {Trends in plant science},
volume = {27},
number = {9},
pages = {890-907},
doi = {10.1016/j.tplants.2022.01.009},
pmid = {35165036},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Evolution, Molecular ; Gene Editing ; *MicroRNAs/genetics ; },
abstract = {Gene editing using clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins (CRISPR/Cas) has revolutionized biotechnology and provides genetic tools for medicine and life sciences. However, the application of this technology to miRNAs, with the function as negative gene regulators, has not been extensively reviewed in plants. Here, we summarize the evolution, biogenesis, and structure of miRNAs, as well as their interactions with mRNAs and computational models for predicting target genes. In addition, we review current advances in CRISPR/Cas for functional analysis and for modulating miRNA genes in plants. Extending our knowledge of miRNAs and their manipulation with CRISPR will provide fundamental understanding of the functions of plant miRNAs and facilitate more sustainable and publicly acceptable genetic engineering of crops.},
}
@article {pmid35164736,
year = {2022},
author = {Wang, G and Xu, Z and Wang, F and Huang, Y and Xin, Y and Liang, S and Li, B and Si, H and Sun, L and Wang, Q and Ding, X and Zhu, X and Chen, L and Yu, L and Lindsey, K and Zhang, X and Jin, S},
title = {Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum).},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {45},
pmid = {35164736},
issn = {1741-7007},
mesh = {Adenine/metabolism ; CRISPR-Cas Systems ; Gene Editing ; *Gossypium/genetics/metabolism ; *Oryza/genetics ; RNA ; },
abstract = {BACKGROUND: Base editors (BEs) display diverse applications in a variety of plant species such as Arabidopsis, rice, wheat, maize, soybean, and cotton, where they have been used to mediate precise base pair conversions without the collateral generation of undesirable double-stranded breaks (DSB). Studies of single-nucleotide polymorphisms (SNPs) underpinning plant traits are still challenging, particularly in polyploidy species where such SNPs are present in multiple copies, and simultaneous modification of all alleles would be required for functional analysis. Allotetraploid cotton has a number of homoeologous gene pairs located in the A and D sub-genomes with considerable SNPs, and it is desirable to develop adenine base editors (ABEs) for efficient and precise A-to-G single-base editing without DSB in such complex genome.<br><br>RESULTS: We established various ABE vectors based on different engineered adenosine deaminase (TadA) proteins fused to Cas9 variants (dCas9, nCas9), enabling efficient A to G editing up to 64% efficiency on-target sites of the allotetraploid cotton genome. Comprehensive analysis showed that GhABE7.10n exhibited the highest editing efficiency, with the main editing sites specifically located at the position A5 (counting the PAM as positions 21-23). Furthermore, DNA and RNA off-target analysis of cotton plants edited with GhABE7.10n and GhABE7.10d by whole genome and whole-transcriptome sequencing revealed no DNA off-target mutations, while very low-level RNA off-target mutations were detected. A new base editor, namely GhABE7.10dCpf1 (7.10TadA + dCpf1), that recognizes a T-rich PAM, was developed for the first time. Targeted A-to-G substitutions generated a single amino acid change in the cotton phosphatidyl ethanolamine-binding protein (GhPEBP), leading to a compact cotton plant architecture, an ideotype for mechanized harvesting of modern cotton production.<br><br>CONCLUSIONS: Our data illustrate the robustness of adenine base editing in plant species with complex genomes, which provides efficient and precise toolkit for cotton functional genomics and precise molecular breeding.},
}
@article {pmid35164636,
year = {2022},
author = {Singh, M and Bindal, G and Misra, CS and Rath, D},
title = {The era of Cas12 and Cas13 CRISPR-based disease diagnosis.},
journal = {Critical reviews in microbiology},
volume = {48},
number = {6},
pages = {714-729},
doi = {10.1080/1040841X.2021.2025041},
pmid = {35164636},
issn = {1549-7828},
mesh = {Animals ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; Endonucleases/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) systems, since their discovery, have found growing applications in cell imaging, transcription modulation, therapeutics and diagnostics. Discovery of Cas12 and Cas13 have brought a new dimension to the field of disease diagnosis. These endonucleases have been extensively used for diagnosis of viral diseases in humans and animals and to a lesser extent in plants. The exigency of SARS-CoV-2 pandemic has highlighted the potential of CRISPR-Cas systems and sparked the development of innovative point-of-care diagnostic technologies. Rapid adaptation of CRISPR-chemistry combined with sensitive read-outs for emerging pathogens make them ideal candidates for detection and management of diseases in future. CRISPR-based approaches have been recruited for the challenging task of cancer detection and prognosis. It stands to reason that the field of CRISPR-Cas-based diagnosis is likely to expand with Cas12 and Cas13 playing a pivotal role. Here we focus exclusively on Cas12- and Cas13-based molecular diagnosis in humans, animals and plants including the detection of SARS-coronavirus. The CRISPR-based diagnosis of plant and animal diseases have not found adequate mention in previous reviews. We discuss various advancements, the potential shortfalls and challenges in the widespread adaptation of this technology for disease diagnosis.},
}
@article {pmid35164562,
year = {2022},
author = {Angermeyer, A and Hays, SG and Nguyen, MHT and Johura, FT and Sultana, M and Alam, M and Seed, KD},
title = {Evolutionary Sweeps of Subviral Parasites and Their Phage Host Bring Unique Parasite Variants and Disappearance of a Phage CRISPR-Cas System.},
journal = {mBio},
volume = {13},
number = {1},
pages = {e0308821},
pmid = {35164562},
issn = {2150-7511},
support = {R01 AI127652/AI/NIAID NIH HHS/United States ; R01 AI153303/AI/NIAID NIH HHS/United States ; },
abstract = {Vibrio cholerae is a significant threat to global public health in part due to its propensity for large-scale evolutionary sweeps where lineages emerge and are replaced. These sweeps may originate from the Bay of Bengal, where bacteriophage predation and the evolution of antiphage counterdefenses is a recurring theme. The bacteriophage ICP1 is a key predator of epidemic V. cholerae and is notable for acquiring a CRISPR-Cas system to combat PLE, a defensive subviral parasite encoded by its V. cholerae host. Here, we describe the discovery of four previously unknown PLE variants through a retrospective analysis of >3,000 publicly available sequences as well as one additional variant (PLE10) from recent surveillance of cholera patients in Bangladesh. In recent sampling we also observed a lineage sweep of PLE-negative V. cholerae occurring within the patient population in under a year. This shift coincided with a loss of ICP1's CRISPR-Cas system in favor of a previously prevalent PLE-targeting endonuclease called Odn. Interestingly, PLE10 was resistant to ICP1-encoded Odn, yet it was not found in any recent V. cholerae strains. We also identified isolates from within individual patient samples that revealed both mixed PLE(+)/PLE(-) V. cholerae populations and ICP1 strains possessing CRISPR-Cas or Odn with evidence of in situ recombination. These findings reinforce our understanding of the successive nature of V. cholerae evolution and suggest that ongoing surveillance of V. cholerae, ICP1, and PLE in Bangladesh is important for tracking genetic developments relevant to pandemic cholera that can occur over relatively short timescales. IMPORTANCE With 1 to 4 million estimated cases annually, cholera is a disease of serious global concern in regions where access to safe drinking water is limited by inadequate infrastructure, inequity, or natural disaster. The Global Task Force on Cholera Control (GTFCC.org) considers outbreak surveillance to be a primary pillar in the strategy to reduce mortality from cholera worldwide. Therefore, developing a better understanding of temporal evolutionary changes in the causative agent of cholera, Vibrio cholerae, could help in those efforts. The significance of our research is in tracking the genomic shifts that distinguish V. cholerae outbreaks, with specific attention paid to current and historical trends in the arms race between V. cholerae and a cooccurring viral (bacteriophage) predator. Here, we discover additional diversity of a specific phage defense system in epidemic V. cholerae and document the loss of a phage-encoded CRISPR-Cas system, underscoring the dynamic nature of microbial populations across cholera outbreaks.},
}
@article {pmid35164379,
year = {2022},
author = {Roueinfar, M and Templeton, HN and Sheng, JA and Hong, KL},
title = {An Update of Nucleic Acids Aptamers Theranostic Integration with CRISPR/Cas Technology.},
journal = {Molecules (Basel, Switzerland)},
volume = {27},
number = {3},
pages = {},
pmid = {35164379},
issn = {1420-3049},
mesh = {Aptamers, Nucleotide/*therapeutic use ; Biosensing Techniques/*methods ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Precision Medicine ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is best known for its role in genomic editing. It has also demonstrated great potential in nucleic acid biosensing. However, the specificity limitation in CRISPR/Cas has created a hurdle for its advancement. More recently, nucleic acid aptamers known for their high affinity and specificity properties for their targets have been integrated into CRISPR/Cas systems. This review article gives a brief overview of the aptamer and CRISPR/Cas technology and provides an updated summary and discussion on how the two distinctive nucleic acid technologies are being integrated into modern diagnostic and therapeutic applications.},
}
@article {pmid35163762,
year = {2022},
author = {Wang, J and Zhou, G and Huang, W and Li, W and Feng, D and Liu, L and Xi, P and Jiang, Z and Kong, G},
title = {Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora&amp;nbsp;litchii.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163762},
issn = {1422-0067},
mesh = {Autophagy ; Beclin-1/*genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Litchi/*growth &amp; development/parasitology ; Mycelium/genetics/growth &amp; development/pathogenicity ; Oxidative Stress ; Phytophthora/genetics/*growth &amp; development/pathogenicity ; Plant Leaves/growth &amp; development/parasitology ; Reproduction, Asexual ; Salt Tolerance ; *Up-Regulation ; Virulence Factors/genetics ; },
abstract = {Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.},
}
@article {pmid35163754,
year = {2022},
author = {Erkut, E and Yokota, T},
title = {CRISPR Therapeutics for Duchenne Muscular Dystrophy.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163754},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; Frameshift Mutation ; Gene Editing/*methods ; Humans ; Male ; Muscular Dystrophy, Duchenne/diagnosis/genetics/*therapy ; Reading Frames ; Translational Research, Biomedical ; },
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with a prevalence of approximately 1 in 3500-5000 males. DMD manifests as childhood-onset muscle degeneration, followed by loss of ambulation, cardiomyopathy, and death in early adulthood due to a lack of functional dystrophin protein. Out-of-frame mutations in the dystrophin gene are the most common underlying cause of DMD. Gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising therapeutic for DMD, as it can permanently correct DMD mutations and thus restore the reading frame, allowing for the production of functional dystrophin. The specific mechanism of gene editing can vary based on a variety of factors such as the number of cuts generated by CRISPR, the presence of an exogenous DNA template, or the current cell cycle stage. CRISPR-mediated gene editing for DMD has been tested both in vitro and in vivo, with many of these studies discussed herein. Additionally, novel modifications to the CRISPR system such as base or prime editors allow for more precise gene editing. Despite recent advances, limitations remain including delivery efficiency, off-target mutagenesis, and long-term maintenance of dystrophin. Further studies focusing on safety and accuracy of the CRISPR system are necessary prior to clinical translation.},
}
@article {pmid35163678,
year = {2022},
author = {Puig-Serra, P and Casado-Rosas, MC and Martinez-Lage, M and Olalla-Sastre, B and Alonso-Yanez, A and Torres-Ruiz, R and Rodriguez-Perales, S},
title = {CRISPR Approaches for the Diagnosis of Human Diseases.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163678},
issn = {1422-0067},
mesh = {COVID-19/genetics ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Diagnosis ; Diagnostic Techniques and Procedures/*trends ; Disease/*genetics ; Gene Editing/*methods ; Humans ; Reproducibility of Results ; SARS-CoV-2/genetics/pathogenicity ; },
abstract = {CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.},
}
@article {pmid35163611,
year = {2022},
author = {Rezalotfi, A and Fritz, L and Förster, R and Bošnjak, B},
title = {Challenges of CRISPR-Based Gene Editing in Primary T Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163611},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; *Immunotherapy ; *T-Lymphocytes ; },
abstract = {Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.},
}
@article {pmid35163555,
year = {2022},
author = {Carey-Fung, O and O'Brien, M and Beasley, JT and Johnson, AAT},
title = {A Model to Incorporate the bHLH Transcription Factor OsIRO3 within the Rice Iron Homeostasis Regulatory Network.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163555},
issn = {1422-0067},
mesh = {Basic Helix-Loop-Helix Transcription Factors/*genetics/*metabolism ; CRISPR-Cas Systems ; Ferroptosis ; Gene Editing ; Gene Expression Regulation, Plant ; Homeostasis ; Iron/*metabolism ; Oryza/genetics/*growth &amp; development/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {Iron (Fe) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly understood in monocotyledon species such as rice (Oryza sativa L.). Recent investigation of the rice Fe homeostasis network revealed OsIRO3, a basic Helix-Loop-Helix (bHLH) TF as a putative negative regulator of genes involved in Fe uptake, transport, and storage. We employed CRISPR-Cas9 gene editing to target the OsIRO3 coding sequence and generate two independent T-DNA-free, loss-of-function iro3 mutants in rice cv. Nipponbare. The iro3 mutant plants had similar phenotype under nutrient-sufficient conditions and had stunted growth under Fe-deficient conditions, relative to a T-DNA free, wild-type control (WT). Under Fe deficiency, iro3 mutant shoots had reduced expression of Fe chelator biosynthetic genes (OsNAS1, OsNAS2, and OsNAAT1) and upregulated expression of an Fe transporter gene (OsYSL15), relative to WT shoots. We place our results in the context of the existing literature and generate a model describing the role of OsIRO3 in rice Fe homeostasis and reinforce the essential function of OsIRO3 in the rice Fe deficiency response.},
}
@article {pmid35163510,
year = {2022},
author = {Tan, J and Wang, Y and Chen, S and Lin, Z and Zhao, Y and Xue, Y and Luo, Y and Liu, YG and Zhu, Q},
title = {An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163510},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; DNA Shuffling ; Flowers/genetics/growth &amp; development ; Gene Editing/*methods ; Homologous Recombination ; Oryza/genetics/*growth &amp; development ; Plant Proteins/*genetics ; Plant Shoots/genetics/growth &amp; development ; Plant Stems/genetics/growth &amp; development ; *Promoter Regions, Genetic ; },
abstract = {In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.},
}
@article {pmid35163260,
year = {2022},
author = {Lee, MH and Shin, JI and Yang, JW and Lee, KH and Cha, DH and Hong, JB and Park, Y and Choi, E and Tizaoui, K and Koyanagi, A and Jacob, L and Park, S and Kim, JH and Smith, L},
title = {Genome Editing Using CRISPR-Cas9 and Autoimmune Diseases: A Comprehensive Review.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163260},
issn = {1422-0067},
mesh = {Animals ; Autoimmune Diseases/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Autoimmune diseases are disorders that destruct or disrupt the body's own tissues by its own immune system. Several studies have revealed that polymorphisms of multiple genes are involved in autoimmune diseases. Meanwhile, gene therapy has become a promising approach in autoimmune diseases, and clustered regularly interspaced palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) has become one of the most prominent methods. It has been shown that CRISPR-Cas9 can be applied to knock out proprotein convertase subtilisin/kexin type 9 (PCSK9) or block PCSK9, resulting in lowering low-density lipoprotein cholesterol. In other studies, it can be used to treat rare diseases such as ornithine transcarbamylase (OTC) deficiency and hereditary tyrosinemia. However, few studies on the treatment of autoimmune disease using CRISPR-Cas9 have been reported so far. In this review, we highlight the current and potential use of CRISPR-Cas9 in the management of autoimmune diseases. We summarize the potential target genes for immunomodulation using CRISPR-Cas9 in autoimmune diseases including rheumatoid arthritis (RA), inflammatory bowel diseases (IBD), systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes mellitus (DM), psoriasis, and type 1 coeliac disease. This article will give a new perspective on understanding the use of CRISPR-Cas9 in autoimmune diseases not only through animal models but also in human models. Emerging approaches to investigate the potential target genes for CRISPR-Cas9 treatment may be promising for the tailored immunomodulation of some autoimmune diseases in the near future.},
}
@article {pmid35163069,
year = {2022},
author = {Shelake, RM and Pramanik, D and Kim, JY},
title = {In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163069},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Cloning, Molecular/*methods ; Cytidine Deaminase/genetics ; Escherichia coli/genetics/*growth &amp; development ; Gene Editing/*methods ; Glycoproteins/genetics ; Humans ; Nuclear Proteins/genetics ; Proteins/genetics ; },
abstract = {Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.},
}
@article {pmid35163047,
year = {2022},
author = {Zhdanova, PV and Chernonosov, AA and Prokhorova, DV and Stepanov, GA and Kanazhevskaya, LY and Koval, VV},
title = {Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163047},
issn = {1422-0067},
mesh = {CRISPR-Associated Protein 9/*chemistry/*metabolism ; Hydrogen Deuterium Exchange-Mass Spectrometry ; Models, Molecular ; Molecular Dynamics Simulation ; Protein Binding ; Protein Conformation ; Protein Domains ; RNA, Guide/*metabolism ; Streptococcus pyogenes/chemistry/*enzymology ; },
abstract = {The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR-Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.},
}
@article {pmid35163006,
year = {2022},
author = {Papasavva, PL and Patsali, P and Loucari, CC and Kurita, R and Nakamura, Y and Kleanthous, M and Lederer, CW},
title = {CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation.},
journal = {International journal of molecular sciences},
volume = {23},
number = {3},
pages = {},
pmid = {35163006},
issn = {1422-0067},
mesh = {3' Untranslated Regions ; CRISPR-Cas Systems ; Cell Line ; DNA End-Joining Repair ; Erythroid Cells/*cytology/metabolism ; Gene Editing/*methods ; HEK293 Cells ; Humans ; MicroRNAs/*genetics ; Proof of Concept Study ; Repressor Proteins/*genetics ; },
abstract = {Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor BCL11A in erythroid cells by tagging the 3' untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.},
}
@article {pmid35159251,
year = {2022},
author = {Liu, X and Zhang, S and Jiang, Y and Yan, T and Fang, C and Hou, Q and Wu, S and Xie, K and An, X and Wan, X},
title = {Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize.},
journal = {Cells},
volume = {11},
number = {3},
pages = {},
pmid = {35159251},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/genetics ; Fertility/genetics ; Gene Editing ; *Infertility, Male/genetics ; Plant Infertility/genetics ; Pollen/genetics ; *Zea mays/genetics ; },
abstract = {Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.},
}
@article {pmid35159110,
year = {2022},
author = {Fuziwara, CS and de Mello, DC and Kimura, ET},
title = {Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?.},
journal = {Cancers},
volume = {14},
number = {3},
pages = {},
pmid = {35159110},
issn = {2072-6694},
abstract = {Important advances on the role of genetic alterations in thyroid cancer have been achieved in the last two decades. One key reason is linked to the development of technical approaches that allowed for the mimicking of genetic alterations in vitro and in vivo and, more recently, the gene editing methodology. The CRISPR/Cas methodology has emerged as a tangible tool for editing virtually any DNA sequence in the genome. To induce a double-strand break and programmable gene editing, Cas9 endonuclease is guided by a single-guide RNA (sgRNA) that is complementary to the target sequence in DNA. The gene editing per se occurs as the cells repair the broken DNA and may erroneously change the original DNA sequence. In this review, we explore the principles of the CRISPR/Cas system to facilitate an understanding of the mainstream technique and its applications in gene editing. Furthermore, we explored new applications of CRISPR/Cas for gene modulation without changing the DNA sequence and provided a Dry Lab experience for those who are interested in starting "CRISPRing" any given gene. In the last section, we will discuss the progress in the knowledge of thyroid cancer biology fostered by the CRISPR/Cas gene editing tools.},
}
@article {pmid35157940,
year = {2022},
author = {Ren, XH and Xu, C and Li, LL and Zuo, Y and Han, D and He, XY and Cheng, SX},
title = {A targeting delivery system for effective genome editing in leukemia cells to reverse malignancy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {343},
number = {},
pages = {645-656},
doi = {10.1016/j.jconrel.2022.02.012},
pmid = {35157940},
issn = {1873-4995},
mesh = {Alginates ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Humans ; *Leukemia, Myeloid, Acute/drug therapy/therapy ; Plasmids ; },
abstract = {Therapy resistance associated with relapse is a main cause of death in acute myeloid leukemia (AML). To address this issue, a dual-targeting CRISPR-Cas9 genome editing nanosystem was constructed for CXCR4 knockout to reverse the malignancy of leukemia cells. The surface of the dual-targeting nanosystem is composed of MUC1 specific aptamer incorporated alginate (MUC1 aptamer-alginate) and T22-NLS peptide with T22 sequence targeting CXCR4; the core of the nanosystem consists of protamine complexed with CRISPR-Cas9 plasmid. The in vitro study shows that the nanosystem mediated genome editing induces cell apoptosis, cell cycle arrest, as well as inhibited cell migration and adhesion in edited THP-1 cells after CXCR4 knockout. Further, the unprocessed peripheral blood from acute myeloid leukemia (AML) patients was directly used to carry out ex vivo study. The results show the genome editing nanosystem can effectively knock out CXCR4 in leukemia cells, leading to attenuated CXCR4 protein as studied by antibody labeling and reduced CXCR4 mRNA as probed by a molecular beacon delivery system. In addition to developing a promising delivery vector for gene therapy on AML, this study also provides an effective strategy to evaluate the therapeutic efficiency of particular treatments by peripheral blood-based ex vivo studies.},
}
@article {pmid35157895,
year = {2022},
author = {Qian, S and Chen, Y and Xu, X and Peng, C and Wang, X and Wu, H and Liu, Y and Zhong, X and Xu, J and Wu, J},
title = {Advances in amplification-free detection of nucleic acid: CRISPR/Cas system as a powerful tool.},
journal = {Analytical biochemistry},
volume = {643},
number = {},
pages = {114593},
doi = {10.1016/j.ab.2022.114593},
pmid = {35157895},
issn = {1096-0309},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/*genetics ; RNA/*genetics ; },
abstract = {Amplification technologies such as polymerase chain reaction (PCR) play an important role in nucleic acid detection. However, they require bulky and sophisticated thermal cycling instrument, as well as are prone to get false-positive results due to amplicon contamination. Currently, CRISPR/Cas system has become an increasingly popular diagnostic tool for nucleic acid with the discovery of its trans-cleavage activity which can degrade single-stranded DNA or RNA at a very high turnover rate. This inherent signal amplification capability allows CRISPR/Cas system to detect unamplified nucleic acids. Here, we reviewed the recent advances of CRISPR-based amplification-free methods for nucleic acid detection. With the assistance of various signal enhancement strategies, the detection sensitivity could be comparable to that of amplification-based methods. We then presented the pros and cons of these methods. And the subsistent challenges including sample preparation, off-target effect, sequences limit, quantitative and multiplex detection were further discussed in this review. It is probable for CRISPR-powered detection methods to pave the road for rapid, cheap, highly sensitive and specific on-site detection without amplification.},
}
@article {pmid35157790,
year = {2022},
author = {Chandrasekaran, AP and Karapurkar, JK and Chung, HY and Ramakrishna, S},
title = {The role of the CRISPR-Cas system in cancer drug development: Mechanisms of action and therapy.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100468},
doi = {10.1002/biot.202100468},
pmid = {35157790},
issn = {1860-7314},
mesh = {*Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Drug Discovery ; Gene Editing ; Genomics ; *Neoplasms/drug therapy/genetics ; },
abstract = {BACKGROUND: The recent emergence of gene editing using Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated system (Cas) tools and advances in genomics and proteomics has revolutionized drug discovery and personalized medicine.<br><br>PURPOSE AND SCOPE: The CRISPR-Cas system has enabled gene and cell-based therapies, screening for novel drug targets, a new generation of disease models, elucidation of drug resistance mechanisms, and drug efficacy testing. Here, we summarized recent investigations and strategies involved in cancer-related drug discovery using the CRISPR-Cas system.<br><br>CONCLUSION: CRISPR-Cas-mediated gene editing has shown great potential in the development of next generation drugs for treatment of Mendelian disorders and various cancer types. In this review, we focused on the impact of the CRISPR-Cas system in drug discovery and its application to biomarker identification and validation, high-end target genes, and breakthrough anticancer cell therapies. We also highlighted the role of CRISPR-Cas in precision disease modeling and functional drug screening.},
}
@article {pmid35157372,
year = {2022},
author = {Pan, C and Qi, Y},
title = {CRISPR-Act3.0-Based Highly Efficient Multiplexed Gene Activation in Plants.},
journal = {Current protocols},
volume = {2},
number = {2},
pages = {e365},
doi = {10.1002/cpz1.365},
pmid = {35157372},
issn = {2691-1299},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; *Plant Breeding ; Plants/genetics ; Transcriptional Activation ; },
abstract = {CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double-strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9-mediated multiplexed gene activation system based on the CRISPR-Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC. Basic Protocol 1: sgRNA design and construction of CRISPR-Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR-Act3.0 vectors using rice protoplasts.},
}
@article {pmid35157300,
year = {2022},
author = {Arbabi Zaboli, K and Rahimi, H and Thekkiniath, J and Taromchi, AH and Kaboli, S},
title = {Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line.},
journal = {Folia histochemica et cytobiologica},
volume = {60},
number = {1},
pages = {13-23},
doi = {10.5603/FHC.a2022.0007},
pmid = {35157300},
issn = {1897-5631},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Female ; *Gene Editing/methods ; Humans ; Mutation ; Plasmids ; Tetraspanin 28/genetics ; },
abstract = {INTRODUCTION: Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line.<br><br>MATERIALS AND METHODS: Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively.<br><br>RESULTS: Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique.<br><br>CONCLUSIONS: Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.},
}
@article {pmid35155494,
year = {2022},
author = {Guo, T and Sun, X and Li, M and Wang, Y and Jiao, H and Li, G},
title = {The Involvement of the csy1 Gene in the Antimicrobial Resistance of Acinetobacter baumannii.},
journal = {Frontiers in medicine},
volume = {9},
number = {},
pages = {797104},
pmid = {35155494},
issn = {2296-858X},
abstract = {Acinetobacter baumannii is an important, opportunistic nosocomial pathogen that causes a variety of nosocomial infections, and whose drug resistance rate has increased in recent years. The CRISPR-Cas system exists in several bacteria, providing adaptive immunity to foreign nucleic acid invasion. This study explores whether CRISPR-Cas is related to drug resistance. Antibiotics were used to treat strains ATCC19606 and AB43, and the expression of CRISPR-related genes was found to be changed. The Csy proteins (Csy1-4) were previously detected to promote target recognition; however, the potential function of csy1 gene is still unknown. Thus, the RecAb homologous recombination system was utilized to knock out the csy1 gene from A. baumannii AB43, which carries the Type I-Fb CRISPR-Cas system, and to observe the drug resistance changes in wild-type and csy1-deleted strains. The AB43Δcsy1 mutant strain was found to become resistant to antibiotics, while the wild-type strain was sensitive to antibiotics. Moreover, transcriptome analysis revealed that the csy1 gene regulates genes encoding CRISPR-Cas-related proteins, drug-resistant efflux pumps, membrane proteins, and oxidative phosphorylation-related proteins, inhibiting antimicrobial resistance in A. baumannii. The in vitro resistance development assay revealed that the complete CRISPR-Cas system could inhibit the development of bacterial resistance. Our findings expand our understanding of the role of CRISPR-Cas csy1 gene in A. baumannii and link the CRISPR-Cas system to the biogenesis of bacterial drug-resistant structures.},
}
@article {pmid35154288,
year = {2022},
author = {Lo, N and Xu, X and Soares, F and He, HH},
title = {The Basis and Promise of Programmable RNA Editing and Modification.},
journal = {Frontiers in genetics},
volume = {13},
number = {},
pages = {834413},
pmid = {35154288},
issn = {1664-8021},
abstract = {One key advantage of RNA over genomic editing is its temporary effects. Aside from current use of DNA-targeting CRISPR-Cas9, the more recently discovered CRISPR-Cas13 has been explored as a means of editing due to its RNA-targeting capabilities. Specifically, there has been a recent interest in identifying and functionally characterizing biochemical RNA modifications, which has spurred a new field of research known as "epitranscriptomics". As one of the most frequently occurring transcriptome modifications, N6-methyladenosine (m6A) has generated much interest. The presence of m6A modifications is under the tight control of a series of regulators, and the ability of fusing these proteins or demethylases to catalytically inactive CRISPR proteins have resulted in a new wave of programmable RNA methylation tools. In addition, studies have been conducted to develop different CRISPR/Cas and base editor systems capable of more efficient editing, and some have explored the effects of in vivo editing for certain diseases. As well, the application of CRISPR and base editors for screening shows promise in revealing the phenotypic outcomes from m6A modification, many of which are linked to physiological, and pathological effects. Thus, the therapeutic potential of CRISPR/Cas and base editors for not only m6A related, but other RNA and DNA related disease has also garnered insight. In this review, we summarize/discuss the recent findings on RNA editing with CRISPR, base editors and non-CRISPR related tools and offer a perspective regarding future applications for basic and clinical research.},
}
@article {pmid35154257,
year = {2021},
author = {Piotter, E and McClements, ME and MacLaren, RE},
title = {The Scope of Pathogenic ABCA4 Mutations Targetable by CRISPR DNA Base Editing Systems-A Systematic Review.},
journal = {Frontiers in genetics},
volume = {12},
number = {},
pages = {814131},
pmid = {35154257},
issn = {1664-8021},
support = {MC_PC_19049/MRC_/Medical Research Council/United Kingdom ; },
abstract = {Stargardt macular dystrophy (STGD1) is the most common form of inherited childhood blindness worldwide and for which no current treatments exist. It is an autosomal recessive disease caused by mutations in ABCA4. To date, a variety of gene supplementation approaches have been tested to create a therapy, with some reaching clinical trials. New technologies, such as CRISPR-Cas based editing systems, provide an exciting frontier for addressing genetic disease by allowing targeted DNA or RNA base editing of pathogenic mutations. ABCA4 has ∼1,200 known pathogenic mutations, of which ∼63% are transition mutations amenable to this editing technology. In this report, we screened the known "pathogenic" and "likely pathogenic" mutations in ABCA4 from available data in gnomAD, Leiden Open Variation Database (LOVD), and ClinVar for potential PAM sites of relevant base editors, including Streptococcus pyogenes Cas (SpCas), Staphylococcus aureus Cas (SaCas), and the KKH variant of SaCas (Sa-KKH). Overall, of the mutations screened, 53% (ClinVar), 71% (LOVD), and 71% (gnomAD), were editable, pathogenic transition mutations, of which 35-47% had "ideal" PAM sites. Of these mutations, 16-20% occur within a range of multiple PAM sites, enabling a variety of editing strategies. Further, in relevant patient data looking at three cohorts from Germany, Denmark, and China, we find that 44-76% of patients, depending on the presence of complex alleles, have at least one transition mutation with a nearby SaCas, SpCas, or Sa-KKH PAM site, which would allow for potential DNA base editing as a treatment strategy. Given the complexity of the genetic landscape of Stargardt, these findings provide a clearer understanding of the potential for DNA base editing approaches to be applied as ABCA4 gene therapy strategies.},
}
@article {pmid35154046,
year = {2021},
author = {Liu, X and Li, Y and Wang, X and Song, Y and Wu, L and Yu, B and Ma, X and Ma, P and Liu, M and Huang, X and Wang, X},
title = {Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {820698},
pmid = {35154046},
issn = {1664-302X},
abstract = {Rapid and sensitive nucleic acid detection of SARS-CoV-2 has contributed to the clinical diagnosis and control of COVID-19. Although detection of virus genomic RNA (gRNA) has been commonly used in clinical diagnosis, SARS-CoV-2 gRNA detection could not discriminate between active infectious virus with remnant viral RNA. In contrast to genomic RNA, subgenomic RNAs (sgRNAs) are only produced when the virus is actively replicating and transcription, detection of sgRNA could be an indication to evaluate infectivity. CRISPR/Cas-based nucleic acid detection methods have been considered potential diagnostic tools due to their intrinsic sensitivity, specificity and simplicity. In this study, to specifically detect active virus replication, we developed a CRISPR-based active SARS-CoV-2 (CRISPR-actCoV) detection strategy by detecting sgRNAs of SARS-CoV-2. CRISPR-actCoV with CRISPR Cas12a-assisted fluorescence reporter system enables detection of sgRNAs at 10 copies in 35 min with high specificity and can be read out with naked eyes. Further, we performed CRISPR-actCoV mediated sgRNA detection in 30 SARS-CoV-2 potentially infected clinical samples, and 21 samples were SARS-CoV-2 sgRNA positive. A quantitative RT-PCR assay was also performed to detect gRNA of SARS-CoV-2 in parallel. Among the 30 clinical samples, 27 samples were gRNA positive. Taken together, CRISPR-actCoV provides an alternative for rapid and accurate detection of active SARS-CoV-2 and has great significance in better response of coronavirus causing epidemic disease.},
}
@article {pmid35153078,
year = {2022},
author = {Awan, MJA and Ali, Z and Amin, I and Mansoor, S},
title = {Twin prime editor: seamless repair without damage.},
journal = {Trends in biotechnology},
volume = {40},
number = {4},
pages = {374-376},
doi = {10.1016/j.tibtech.2022.01.013},
pmid = {35153078},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; DNA ; DNA Breaks, Double-Stranded ; DNA Repair ; *Gene Editing ; },
abstract = {CRISPR-Cas9 creates remarkable possibilities to modify targeted regions in genomic DNA. However, CRISPR-Cas-mediated DNA double-stranded breaks (DSBs), that tend to generate random insertions or deletions, limit this technology. Recently, Anzalone et al. developed a 'twin prime editing' tool to replace, integrate, or delete large genomic DNA sequences without generating DNA DSBs.},
}
@article {pmid35152989,
year = {2022},
author = {Ding, R and Chao, CC and Gao, Q},
title = {High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell therapy.},
journal = {Methods in cell biology},
volume = {167},
number = {},
pages = {1-14},
doi = {10.1016/bs.mcb.2021.08.001},
pmid = {35152989},
issn = {0091-679X},
mesh = {CRISPR-Cas Systems/genetics ; Cell- and Tissue-Based Therapy ; *Gene Editing/methods ; Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/metabolism ; },
abstract = {Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR-Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.},
}
@article {pmid35152318,
year = {2022},
author = {Becirovic, E},
title = {Maybe you can turn me on: CRISPRa-based strategies for therapeutic applications.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {2},
pages = {130},
pmid = {35152318},
issn = {1420-9071},
mesh = {CRISPR-Cas Systems/*physiology ; Dependovirus/genetics ; Gene Editing ; *Genetic Therapy ; Humans ; Transcriptional Activation ; },
abstract = {Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological tools that go far beyond its original function as "genetic scissors". One of these further developments of the CRISPR-Cas system allows genes to be activated in a targeted and efficient manner. These gene-activating CRISPR-Cas modules (CRISPRa) are based on a programmable recruitment of transcription factors to specific loci and offer several key advantages that make them particularly attractive for therapeutic applications. These advantages include inter alia low off-target effects, independence of the target gene size as well as the potential to develop gene- and mutation-independent therapeutic strategies. Herein, I will give an overview on the currently available CRISPRa modules and discuss recent developments, future potentials and limitations of this approach with a focus on therapeutic applications and in vivo delivery.},
}
@article {pmid35151447,
year = {2022},
author = {Perroud, PF and Guyon-Debast, A and Veillet, F and Kermarrec, MP and Chauvin, L and Chauvin, JE and Gallois, JL and Nogué, F},
title = {Prime Editing in the model plant Physcomitrium patens and its potential in the tetraploid potato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {316},
number = {},
pages = {111162},
doi = {10.1016/j.plantsci.2021.111162},
pmid = {35151447},
issn = {1873-2259},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; Plant Breeding ; *Solanum tuberosum/genetics ; Tetraploidy ; },
abstract = {Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding. Although the classical CRISPR-Cas9 system is a highly efficient tool for disruptive targeted mutagenesis, this system is mostly inefficient for the introduction of precise and predictable nucleotide substitutions. Recently, Prime Editing technology has been developed, allowing the simultaneous generation of nucleotide transitions and transversions but also short defined indels. In this study, we report on the successful use of Prime Editing in two plants of interest: the plant model Physcomitrium patens and the tetraploid and highly heterozygous potato (Solanum tuberosum). In both cases editing rates were lower than with other CRISPR-Cas9 based techniques, but we were able to successfully introduce nucleotide transversions into targeted genes, a unique feature of Prime Editing. Additionally, the analysis of potential off-target mutation sites in P. patens suggested very high targeting fidelity in this organism. The present work paves the way for the use Prime Editing in Physcomitrium patens and potato, however highlighting the limitations that need to be overcome for more efficient precision plant breeding.},
}
@article {pmid35151407,
year = {2022},
author = {Wu, X and Chan, C and Springs, SL and Lee, YH and Lu, TK and Yu, H},
title = {A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids.},
journal = {Analytica chimica acta},
volume = {1196},
number = {},
pages = {339494},
doi = {10.1016/j.aca.2022.339494},
pmid = {35151407},
issn = {1873-4324},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; RNA, Viral/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA) for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/μl SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital chips were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/μL for DNA and 1.2-18391 copies/μL for RNA (both R[2] values > 0.99). Moreover, WS-RADICA had lower detection limit and higher inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. To prove its performance on nucleic acids derived from live virus, WS-RADICA was also validated to detect and quantify human adenovirus and herpes simplex virus. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification.},
}
@article {pmid35151271,
year = {2022},
author = {Seni-Silva, AC and Maleski, ALA and Souza, MM and Falcao, MAP and Disner, GR and Lopes-Ferreira, M and Lima, C},
title = {Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {123},
pmid = {35151271},
issn = {1471-2164},
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development/genetics ; *Fish Venoms ; Pore Forming Cytotoxic Proteins ; *Zebrafish/genetics ; Zebrafish Proteins/genetics ; },
abstract = {BACKGROUND: The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity.<br><br>RESULTS: Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish.<br><br>CONCLUSIONS: The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.},
}
@article {pmid35150572,
year = {2022},
author = {},
title = {Correction to 'Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs'.},
journal = {Nucleic acids research},
volume = {50},
number = {5},
pages = {2986},
doi = {10.1093/nar/gkac110},
pmid = {35150572},
issn = {1362-4962},
}
@article {pmid35149424,
year = {2022},
author = {Qian, S and Chen, Y and Peng, C and Wang, X and Wu, H and Che, Y and Wang, H and Xu, J and Wu, J},
title = {Dipstick-based rapid nucleic acids purification and CRISPR/Cas12a-mediated isothermal amplification for visual detection of African swine fever virus.},
journal = {Talanta},
volume = {242},
number = {},
pages = {123294},
doi = {10.1016/j.talanta.2022.123294},
pmid = {35149424},
issn = {1873-3573},
mesh = {*African Swine Fever/diagnosis ; *African Swine Fever Virus/genetics ; Animals ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Sensitivity and Specificity ; Swine ; },
abstract = {African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/μL. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.},
}
@article {pmid35149141,
year = {2022},
author = {Bhattacharjee, G and Gohil, N and Khambhati, K and Mani, I and Maurya, R and Karapurkar, JK and Gohil, J and Chu, DT and Vu-Thi, H and Alzahrani, KJ and Show, PL and Rawal, RM and Ramakrishna, S and Singh, V},
title = {Current approaches in CRISPR-Cas9 mediated gene editing for biomedical and therapeutic applications.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {343},
number = {},
pages = {703-723},
doi = {10.1016/j.jconrel.2022.02.005},
pmid = {35149141},
issn = {1873-4995},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Mutation ; Quality of Life ; },
abstract = {A single gene mutation can cause a number of human diseases that affect the quality of life. Until the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems, it was challenging to correct a gene mutation to avoid a disease by reverting phenotypes. The advent of CRISPR technology has changed the field of gene editing, given its simplicity and intrinsic programmability, surpassing the limitations of both zinc-finger nuclease and transcription activator-like effector nuclease and becoming the method of choice for therapeutic gene editing by overcoming the bottlenecks of conventional gene-editing techniques. Currently, there is no commercially available medicinal cure to correct a gene mutation that corrects and reverses the abnormality of a gene's function. Devising reprogramming strategies for faithful recapitulation of normal phenotypes is a crucial aspect for directing the reprogrammed cells toward clinical trials. The CRISPR-Cas9 system has been promising as a tool for correcting gene mutations in maladies including blood disorders and muscular degeneration as well as neurological, cardiovascular, renal, genetic, stem cell, and optical diseases. In this review, we highlight recent developments and utilization of the CRISPR-Cas9 system in correcting or generating gene mutations to create model organisms to develop deeper insights into diseases, rescue normal gene functionality, and curb the progression of a disease. Delivery of CRISPR-components being a pivotal aspect in proving its effectiveness, various proven delivery systems have also been briefly discussed.},
}
@article {pmid35149003,
year = {2022},
author = {Colijn, S and Yin, Y and Stratman, AN},
title = {High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products.},
journal = {Developmental biology},
volume = {484},
number = {},
pages = {22-29},
doi = {10.1016/j.ydbio.2022.02.003},
pmid = {35149003},
issn = {1095-564X},
support = {R00 HL125683/HL/NHLBI NIH HHS/United States ; R35 GM137976/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Polymerase Chain Reaction ; *Zebrafish/genetics ; },
abstract = {Targeted mutagenesis in zebrafish, fruit flies, and C. elegans has been significantly improved over the years through CRISPR technology. CRISPR enables researchers to efficiently examine cellular pathways by inducing small, targeted mutations in vivo. Though these mutations are commonly random insertions or deletions (indels), they often result in functionally disrupted alleles of a target gene if the CRISPR components are appropriately designed. However, current protocols used to identify the presence of CRISPR-generated indels are often labor intensive, time-consuming, or expensive. Here, we describe a straightforward, high-throughput method for identifying the presence of mutations by using a fragment analyzer platform which allows for DNA fragment sizing through high-resolution capillary gel-electrophoresis. Following this protocol, small indels-down to 2 base pairs-can be quickly and reliably identified, thus allowing for large-scale genotyping of newly-generated or stable mutant lines.},
}
@article {pmid35148990,
year = {2022},
author = {Du, S and Wang, G and Zhang, Z and Ma, C and Gao, N and Xiao, J},
title = {Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {3},
pages = {101702},
pmid = {35148990},
issn = {1083-351X},
mesh = {Animals ; Cryoelectron Microscopy ; Drosophila melanogaster ; *Lysosomes/chemistry/genetics/metabolism ; Mammals/metabolism ; *Mucolipidoses/genetics ; Proteins ; Structure-Activity Relationship ; *Transferases (Other Substituted Phosphate Groups)/chemistry/metabolism ; },
abstract = {GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.},
}
@article {pmid35148314,
year = {2022},
author = {Cho, B and Song, S and Wan, JY and Axelrod, JD},
title = {Prickle isoform participation in distinct polarization events in the Drosophila eye.},
journal = {PloS one},
volume = {17},
number = {2},
pages = {e0262328},
pmid = {35148314},
issn = {1932-6203},
support = {R01 GM097081/GM/NIGMS NIH HHS/United States ; R35 GM131914/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Brain/metabolism ; CRISPR-Cas Systems/genetics ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Drosophila/*metabolism ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Eye/*metabolism ; Gene Editing ; Genotype ; LIM Domain Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Membrane Proteins/metabolism ; Phenotype ; Protein Isoforms/antagonists &amp; inhibitors/genetics/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Signal Transduction/genetics ; },
abstract = {Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. Here, by characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, we show that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. We propose that expression dynamics and competitive interactions determine isoform participation in these processes. We propose that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation.},
}
@article {pmid35147495,
year = {2022},
author = {Ravi, NS and Wienert, B and Wyman, SK and Bell, HW and George, A and Mahalingam, G and Vu, JT and Prasad, K and Bandlamudi, BP and Devaraju, N and Rajendiran, V and Syedbasha, N and Pai, AA and Nakamura, Y and Kurita, R and Narayanasamy, M and Balasubramanian, P and Thangavel, S and Marepally, S and Velayudhan, SR and Srivastava, A and DeWitt, MA and Crossley, M and Corn, JE and Mohankumar, KM},
title = {Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35147495},
issn = {2050-084X},
mesh = {Adenine/metabolism ; Anemia, Sickle Cell/*genetics ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine/metabolism ; Fetal Hemoglobin/*genetics ; Gene Editing/*methods ; Hematopoietic Stem Cells/metabolism ; Humans ; Point Mutation ; Promoter Regions, Genetic ; beta-Globins/genetics ; beta-Thalassemia/genetics ; gamma-Globins/genetics ; },
abstract = {Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.},
}
@article {pmid35146856,
year = {2022},
author = {Lu, L and Rao, D and Niu, C and Cheng, L and Ma, D and Xi, Z},
title = {Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {23},
number = {7},
pages = {e202100544},
doi = {10.1002/cbic.202100544},
pmid = {35146856},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Plasmids ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100-200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.},
}
@article {pmid35145304,
year = {2022},
author = {Ortabozkoyun, H and Huang, PY and Cho, H and Narendra, V and LeRoy, G and Gonzalez-Buendia, E and Skok, JA and Tsirigos, A and Mazzoni, EO and Reinberg, D},
title = {CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters.},
journal = {Nature genetics},
volume = {54},
number = {2},
pages = {202-212},
pmid = {35145304},
issn = {1546-1718},
support = {P01 CA229086/CA/NCI NIH HHS/United States ; P30 CA016087/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; F31 HD090892/HD/NICHD NIH HHS/United States ; R01 NS100897/NS/NINDS NIH HHS/United States ; R01 CA229235/CA/NCI NIH HHS/United States ; R35 GM122515/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CCCTC-Binding Factor/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism ; Cell Differentiation ; Cell Line ; Chromatin/metabolism ; DNA-Binding Proteins/chemistry/genetics/*metabolism ; Embryonic Stem Cells/cytology/*metabolism ; Gene Editing ; Gene Expression ; Gene Expression Regulation, Developmental ; *Genes, Homeobox ; Homeodomain Proteins/*genetics ; Mice ; Transcription Factors/chemistry/genetics/*metabolism ; },
abstract = {CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.},
}
@article {pmid35145296,
year = {2022},
author = {Mallapaty, S},
title = {China's approval of gene-edited crops energizes researchers.},
journal = {Nature},
volume = {602},
number = {7898},
pages = {559-560},
pmid = {35145296},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; China ; *Crops, Agricultural/genetics ; *Gene Editing ; },
}
@article {pmid35145236,
year = {2022},
author = {Nishiga, M and Liu, C and Qi, LS and Wu, JC},
title = {The use of new CRISPR tools in cardiovascular research and medicine.},
journal = {Nature reviews. Cardiology},
volume = {19},
number = {8},
pages = {505-521},
pmid = {35145236},
issn = {1759-5010},
support = {R01 HL126527/HL/NHLBI NIH HHS/United States ; R01 HL123968/HL/NHLBI NIH HHS/United States ; R01 HL141371/HL/NHLBI NIH HHS/United States ; R01 HL141851/HL/NHLBI NIH HHS/United States ; R01 HL150693/HL/NHLBI NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.},
}
@article {pmid35145221,
year = {2022},
author = {Wang, C and Qu, Y and Cheng, JKW and Hughes, NW and Zhang, Q and Wang, M and Cong, L},
title = {dCas9-based gene editing for cleavage-free genomic knock-in of long sequences.},
journal = {Nature cell biology},
volume = {24},
number = {2},
pages = {268-278},
pmid = {35145221},
issn = {1476-4679},
support = {R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; S10 OD023452/OD/NIH HHS/United States ; },
mesh = {Actins/genetics/metabolism ; Aptamers, Nucleotide/genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics/metabolism ; Dyneins/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; *Gene Editing ; *Gene Knock-In Techniques ; HEK293 Cells ; HSP90 Heat-Shock Proteins/genetics/metabolism ; HeLa Cells ; Hep G2 Cells ; Humans ; Viral Proteins/genetics/metabolism ; },
abstract = {Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.},
}
@article {pmid35143959,
year = {2022},
author = {Bengtsson, NE and Crudele, JM and Klaiman, JM and Halbert, CL and Hauschka, SD and Chamberlain, JS},
title = {Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {6},
pages = {2176-2185},
pmid = {35143959},
issn = {1525-0024},
support = {P50 AR065139/AR/NIAMS NIH HHS/United States ; P30 DK017047/DK/NIDDK NIH HHS/United States ; },
mesh = {Aging ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Disease Progression ; Dogs ; *Dystrophin/genetics ; Gene Editing/methods ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; },
abstract = {Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.},
}
@article {pmid35143940,
year = {2022},
author = {Ma, SP and Gao, XX and Zhou, GQ and Zhang, HK and Yang, JM and Wang, WJ and Song, XM and Chen, HY and Lu, DR},
title = {Reactivation of γ-globin expression using a minicircle DNA system to treat β-thalassemia.},
journal = {Gene},
volume = {820},
number = {},
pages = {146289},
doi = {10.1016/j.gene.2022.146289},
pmid = {35143940},
issn = {1879-0038},
mesh = {Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; DNA, Circular/metabolism/*therapeutic use ; Fetal Hemoglobin/*metabolism ; Gene Editing ; Genetic Therapy/methods ; Genetic Vectors ; Hematopoietic Stem Cells/metabolism ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; Promoter Regions, Genetic ; RNA, Guide/metabolism/therapeutic use ; Repressor Proteins/*metabolism ; beta-Thalassemia/*genetics/*therapy ; gamma-Globins/*genetics/*metabolism ; },
abstract = {Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for β-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of β-thalassemia. In our study, we found that the transfection efficiencies of CD34[+] hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of β-thalassemia.},
}
@article {pmid35143476,
year = {2022},
author = {Chirichella, M and Bianchi, N and Džafo, E and Foli, E and Gualdrini, F and Kenyon, A and Natoli, G and Monticelli, S},
title = {RFX transcription factors control a miR-150/PDAP1 axis that restrains the proliferation of human T cells.},
journal = {PLoS biology},
volume = {20},
number = {2},
pages = {e3001538},
pmid = {35143476},
issn = {1545-7885},
mesh = {3' Untranslated Regions/genetics ; Blotting, Western ; CD4-Positive T-Lymphocytes/cytology/*metabolism ; Cell Proliferation/*genetics ; Cells, Cultured ; Chromatin Immunoprecipitation Sequencing/methods ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Intercellular Signaling Peptides and Proteins/*genetics/metabolism ; Jurkat Cells ; Lymphocyte Activation/genetics ; MicroRNAs/*genetics ; Regulatory Factor X Transcription Factors/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction/genetics ; },
abstract = {Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.},
}
@article {pmid35143386,
year = {2022},
author = {Huang, Z and Yu, K and Fu, S and Xiao, Y and Wei, Q and Wang, D},
title = {Genomic analysis reveals high intra-species diversity of Shewanella algae.},
journal = {Microbial genomics},
volume = {8},
number = {2},
pages = {},
pmid = {35143386},
issn = {2057-5858},
mesh = {Adaptation, Biological ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems ; China ; Drug Resistance, Bacterial/genetics ; *Genetic Variation ; Genome, Bacterial ; Genomic Islands ; *Genomics ; Humans ; Microbial Sensitivity Tests ; Phylogeny ; Prophages/genetics ; Shewanella/classification/drug effects/*genetics/isolation &amp; purification ; Species Specificity ; Virulence/genetics ; },
abstract = {Shewanella algae is widely distributed in marine and freshwater habitats, and has been proved to be an emerging marine zoonotic and human pathogen. However, the genomic characteristics and pathogenicity of Shewanella algae are unclear. Here, the whole-genome features of 55 S. algae strains isolated from different sources were described. Pan-genome analysis yielded 2863 (19.4 %) genes shared among all strains. Functional annotation of the core genome showed that the main functions are focused on basic lifestyle such as metabolism and energy production. Meanwhile, the phylogenetic tree of the single nucleotide polymorphisms (SNPs) of core genome divided the 55 strains into three clades, with the majority of strains from China falling into the first two clades. As for the accessory genome, 167 genomic islands (GIs) and 65 phage-related elements were detected. The CRISPR-Cas system with a high degree of confidence was predicted in 23 strains. The GIs carried a suite of virulence genes and mobile genetic elements, while prophages contained several transposases and integrases. Horizontal genes transfer based on homology analysis indicated that these GIs and prophages were parts of major drivers for the evolution and the environmental adaptation of S. algae. In addition, a rich putative virulence-associated gene pool was found. Eight classes of antibiotic-associated resistance genes were detected, and the carriage rate of β-lactam resistance genes was 100 %. In conclusion, S. algae exhibits a high intra-species diversity in the aspects of population structure, virulence-associated genes and potential drug resistance, which is helpful for its evolution in pathogenesis and environmental adaptability.},
}
@article {pmid35142491,
year = {2022},
author = {Lee, SW and Tran, KT and Vazquez-Uribe, R and Gotfredsen, CH and Clausen, MH and Mendez, BL and Montoya, G and Bach, A and Sommer, MOA},
title = {Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening.},
journal = {Journal of medicinal chemistry},
volume = {65},
number = {4},
pages = {3266-3305},
doi = {10.1021/acs.jmedchem.1c01834},
pmid = {35142491},
issn = {1520-4804},
mesh = {*CRISPR-Cas Systems ; Drug Design ; Escherichia coli/drug effects ; Gene Editing ; High-Throughput Screening Assays ; Humans ; Ligands ; Small Molecule Libraries ; Structure-Activity Relationship ; },
abstract = {CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.},
}
@article {pmid35142378,
year = {2022},
author = {You, H and Gordon, CA and MacGregor, SR and Cai, P and McManus, DP},
title = {Potential of the CRISPR-Cas system for improved parasite diagnosis: CRISPR-Cas mediated diagnosis in parasitic infections: CRISPR-Cas mediated diagnosis in parasitic infections.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {44},
number = {4},
pages = {e2100286},
doi = {10.1002/bies.202100286},
pmid = {35142378},
issn = {1521-1878},
mesh = {Animals ; *COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems/genetics ; Humans ; *Parasites/genetics ; *Parasitic Diseases ; },
abstract = {CRISPR-Cas technology accelerates development of fast, accurate, and portable diagnostic tools, typified by recent applications in COVID-19 diagnosis. Parasitic helminths cause devastating diseases afflicting 1.5 billion people globally, representing a significant public health and economic burden, especially in developing countries. Currently available diagnostic tests for worm infection are neither sufficiently sensitive nor field-friendly for use in low-endemic or resource-poor settings, leading to underestimation of true prevalence rates. Mass drug administration programs are unsustainable long-term, and diagnostic tools - required to be rapid, specific, sensitive, cost-effective, and user-friendly without specialized equipment and expertise - are urgently needed for rapid mapping of helminthic diseases and monitoring control programs. We describe the key features of the CRISPR-Cas12/13 system and emphasise its potential for the development of effective tools for the diagnosis of parasitic and other neglected tropical diseases (NTDs), a key recommendation of the NTDs 2021-2030 roadmap released by the World Health Organization.},
}
@article {pmid35142306,
year = {2022},
author = {Zhang, S and Liu, M and Cui, H and Ziaee, MA and Sun, R and Chen, L and Chen, D and Garoli, D and Wang, J},
title = {Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system.},
journal = {The Analyst},
volume = {147},
number = {5},
pages = {905-914},
doi = {10.1039/d1an02313f},
pmid = {35142306},
issn = {1364-5528},
mesh = {CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; DNA, Single-Stranded/genetics ; *Nanopores ; },
abstract = {The fabrication of nanopores with a matched pore size, and the existence of multiple interferents make the reproducible detection of small-sized molecules by means of solid-state nanopores still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructures related to the existence of small-sized targets. In particular, a DNA tetrahedron with a well-defined 3D nanostructure is the ideal candidate for use as a signal transducer. Here, we demonstrate the detection of an L1-encoding gene of HPV18 as a test DNA target sequence in a reaction buffer solution, where long single-stranded DNA linking DNA tetrahedra onto the surface of the magnetic beads is cleaved by a target DNA-activated CRISPR-cas12 system. The DNA tetrahedra are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) one signal transducer can be used to detect different targets; (2) a glassy nanopore with a pore size much larger than the target DNA fragment can boost the tolerance of the contaminants and interferents which often degrade the performance of a nanopore sensor.},
}
@article {pmid35142264,
year = {2022},
author = {Bhatt, A and Bumbrah, GS and Ruwali, M and Hameed, S and Fatima, Z},
title = {Diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis.},
journal = {Pathogens and global health},
volume = {116},
number = {7},
pages = {410-420},
pmid = {35142264},
issn = {2047-7732},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; },
abstract = {To address the challenges associated with COVID-19 diagnosis, we need a faster, direct, and more versatile detection method for efficient epidemiological management of the COVID-19 pandemic. RT-qPCR (reverse transcription quantitative real-time Polymerase Chain Reaction) although the most popular diagnostic method suffers from a major drawback of equipment dependency and trained molecular biologists that limits rapid and large-scale screening, particularly in low resource regions. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a feasible alternative for RT-qPCR; however, it also suffers from the drawback of false-positive issues. Recently, RT-LAMP has been integrated with the CRISPR-Cas technique to take care of the problems associated with RT-LAMP for COVID-19 diagnosis. In this study, a meta-analysis was conducted using three scientific databases considering the PRISMA guidelines to assess the diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technology. Out of a total of 1286 studies on COVID-19, we identified 15 articles that met our eligibility criteria of using simultaneous RT-LAMP and CRISPR-Cas technique. Our meta-analysis of the included studies revealed that most of the studies were conducted in the USA with the N gene as the most common target and fluorescence-based detection method. The meta-analysis results of all included studies have further revealed a pooled sensitivity value of higher than 85% and a pooled specificity value of 80% with the confidence interval of 95%, respectively, as revealed from the forest plot and SROC curve. The accuracy rate of included studies was also calculated which varied from 77.4% to 100%. Furthermore, the precision of included studies varied from 75% to 100%. Lastly, a quality assessment of bias and applicability was performed based on QUADAS-2. Taken together, combined RT-LAMP and CRISPR-Cas technique could be a potential alternative to RT-qPCR particularly in low resource regions having a high demand for rapid testing.},
}
@article {pmid35142116,
year = {2022},
author = {DU, Q and Wang, C and Liu, G and Zhang, D and Zhang, S and Qiu, J},
title = {[Plant prime editing technique: a new genome editing tool for plants].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {1},
pages = {26-33},
doi = {10.13345/j.cjb.210291},
pmid = {35142116},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Genome, Plant/genetics ; *Plant Breeding ; Plants/genetics ; },
abstract = {The CRISPR/Cas9 based prime editing (PE) technique enables all 12 types of base substitutions and precise small DNA deletions or insertions without generating DNA double-strand breaks. Prime editing has been successfully applied in plants and plays important roles in plant precision breeding. Although plant prime editing (PPE) can substantially expand the scope and capabilities of precise genome editing in plants, its editing efficiency still needs to be further improved. Here, we review the development of PPE technique, and introduce structural composition, advantages and limitations of PPE. Strategies to improve the PPE editing efficiency, including the Tm-directed PBS length design, the RT template length, the dual-pegRNA strategy, the PlantPegDesigner website, and the strategies for optimizing the target proteins of PPE, were highlighted. Finally, the prospects of future development and application of PPE were discussed.},
}
@article {pmid35142064,
year = {2022},
author = {Zhang, D and Tang, S and Xie, P and Yang, D and Wu, Y and Cheng, S and Du, K and Xin, P and Chu, J and Yu, F and Xie, Q},
title = {Creation of fragrant sorghum by CRISPR/Cas9.},
journal = {Journal of integrative plant biology},
volume = {64},
number = {5},
pages = {961-964},
doi = {10.1111/jipb.13232},
pmid = {35142064},
issn = {1744-7909},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Edible Grain ; Seeds ; *Sorghum/genetics ; },
abstract = {Sorghum, the fifth largest cereal crop, has high value as a staple food and raw material for liquor and vinegar brewing. Due to its high biomass and quality, it is also used as the second most planted silage resource. No fragrant sorghums are currently on the market. Through CRISPR/Cas9-mediated knockout of SbBADH2, we obtained sorghum lines with extraordinary aromatic smell in both seeds and leaves. Animal feeding experiments showed that fragrant sorghum leaves were attractable. We believe this advantage will produce great value in the sorghum market for both grain and whole biomass forage.},
}
@article {pmid35141701,
year = {2022},
author = {Laforest, LC and Nadakuduti, SS},
title = {Advances in Delivery Mechanisms of CRISPR Gene-Editing Reagents in Plants.},
journal = {Frontiers in genome editing},
volume = {4},
number = {},
pages = {830178},
pmid = {35141701},
issn = {2673-3439},
abstract = {Gene-editing by CRISPR/Cas systems has revolutionized plant biology by serving as a functional genomics tool. It has tremendously advanced plant breeding and crop improvement by accelerating the development of improved cultivars, creating genetic variability, and aiding in domestication of wild and orphan crops. Gene-editing is a rapidly evolving field. Several advancements include development of different Cas effectors with increased target range, efficacy, and enhanced capacity for precise DNA modifications with base editing and prime editing. The existing toolbox of various CRISPR reagents facilitate gene knockouts, targeted gene insertions, precise base substitutions, and multiplexing. However, the major challenge in plant genome-editing remains the efficient delivery of these reagents into plant cells. Plants have larger and more complex genome structures compared to other living systems due to the common occurrence of polyploidy and other genome re-arrangements. Further, rigid cell walls surrounding plant cells deter the entry of any foreign biomolecules. Unfortunately, genetic transformation to deliver gene-editing reagents has been established only in a limited number of plant species. Recently, there has been significant progress in CRISPR reagents delivery in plants. This review focuses on exploring these delivery mechanisms categorized into Agrobacterium-mediated delivery and breakthroughs, particle bombardment-based delivery of biomolecules and recent improvements, and protoplasts, a versatile system for gene-editing and regeneration in plants. The ultimate goal in plant gene-editing is to establish highly efficient and genotype-independent reagent delivery mechanisms for editing multiple targets simultaneously and achieve DNA-free gene-edited plants at scale.},
}
@article {pmid35140292,
year = {2022},
author = {Noroozi, Z and Shamsara, M and Valipour, E and Esfandyari, S and Ehghaghi, A and Monfaredan, A and Azizi, Z and Motevaseli, E and Modarressi, MH},
title = {Antiproliferative effects of AAV-delivered CRISPR/Cas9-based degradation of the HPV18-E6 gene in HeLa cells.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {2224},
pmid = {35140292},
issn = {2045-2322},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/*genetics ; Cell Proliferation/genetics ; DNA-Binding Proteins/*genetics/*metabolism ; Dependovirus/*genetics ; Female ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Oncogene Proteins, Viral/*genetics/*metabolism ; Tumor Suppressor Protein p53/metabolism ; Uterine Cervical Neoplasms/genetics/therapy ; },
abstract = {Human papillomavirus infections are associated with most cervical cancers, which are the fourth most common cancer in women. HPV-E6 protein binds to protein p53 and inhibits its function, leading to the switching of normal cells toward cancer cells. Here, we disrupted the HPV-E6 gene and investigated its effects on the proliferation and apoptosis of HeLa cells. The HPV18-E6 gene was targeted with two designed sgRNAs cloned into an AAV-CRISPR-based plasmid. The AAV-E6-CRISPR/Cas9 virions were prepared and titrated in HEK293t cells. The cleavage created in the HPV-E6 gene was detected using the T7E1 assay. Cell cycle profiling, MTT assay, and annexin V/PI staining were performed. Also, the p53 protein level was measured by Western blotting. Our data showed that disruption of the HPV-E6 gene led to increased cell apoptosis and decreased cell proliferation. A significant accumulation of infected cells in sub-G1 phase was observed in the cell profiling assay. Also, HPV-E6 gene disruption resulted in a significant increase in the level of P53 protein. Our findings indicated that AAV-mediated delivery of CRISPR/Cas9 can effectively target the HPV-E6 gene in HeLa cells, and its antiproliferative effects may provide therapeutic benefits of local administration of this gene-editing system for HPV-related cervical cancers.},
}
@article {pmid35140185,
year = {2022},
author = {Ryoden, Y and Segawa, K and Nagata, S},
title = {Requirement of Xk and Vps13a for the P2X7-mediated phospholipid scrambling and cell lysis in mouse T cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {7},
pages = {},
pmid = {35140185},
issn = {1091-6490},
mesh = {Adenosine Triphosphate ; Amino Acid Transport Systems, Neutral/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Death ; Cell Line ; Gene Deletion ; Gene Expression Regulation/drug effects ; Genome-Wide Association Study ; HEK293 Cells ; Humans ; Mice ; Mice, Transgenic ; Mutation ; Phosphatidylserines/pharmacology ; Phospholipids/*metabolism ; Receptors, Purinergic P2X7/genetics/*metabolism ; T-Lymphocytes/*physiology ; Vesicular Transport Proteins/genetics/*metabolism ; },
abstract = {A high extracellular adenosine triphosphate (ATP) concentration rapidly and reversibly exposes phosphatidylserine (PtdSer) in T cells by binding to the P2X7 receptor, which ultimately leads to necrosis. Using mouse T cell transformants expressing P2X7, we herein performed CRISPR/Cas9 screening for the molecules responsible for P2X7-mediated PtdSer exposure. In addition to Eros, which is required for the localization of P2X7 to the plasma membrane, this screening identified Xk and Vps13a as essential components for this process. Xk is present at the plasma membrane, and its paralogue, Xkr8, functions as a phospholipid scramblase. Vps13a is a lipid transporter in the cytoplasm. Blue-native polyacrylamide gel electrophoresis indicated that Xk and Vps13a interacted at the membrane. A null mutation in Xk or Vps13a blocked P2X7-mediated PtdSer exposure, the internalization of phosphatidylcholine, and cytolysis. Xk and Vps13a formed a complex in mouse splenic T cells, and Xk was crucial for ATP-induced PtdSer exposure and cytolysis in CD25[+]CD4[+] T cells. XK and VPS13A are responsible for McLeod syndrome and chorea-acanthocytosis, both characterized by a progressive movement disorder and cognitive and behavior changes. Our results suggest that the phospholipid scrambling activity mediated by XK and VPS13A is essential for maintaining homeostasis in the immune and nerve systems.},
}
@article {pmid35140182,
year = {2022},
author = {Khan, K and Long, B and Baleanu-Gogonea, C and Gogonea, V and Deshpande, GM and Vasu, K and Fox, PL},
title = {Cotranslational interaction of human EBP50 and ezrin overcomes masked binding site during complex assembly.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {7},
pages = {},
pmid = {35140182},
issn = {1091-6490},
support = {R01 AG067146/AG/NIA NIH HHS/United States ; R01 DK123236/DK/NIDDK NIH HHS/United States ; R01 DK124203/DK/NIDDK NIH HHS/United States ; R01 NS124547/NS/NINDS NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems ; Cloning, Molecular ; Cytoskeletal Proteins/genetics/*metabolism ; DNA, Complementary ; Gene Expression Regulation ; Gene Silencing ; HCT116 Cells ; HEK293 Cells ; Humans ; Jurkat Cells ; Models, Molecular ; Phosphoproteins/genetics/*metabolism ; Protein Binding ; Protein Biosynthesis ; Protein Conformation ; Sodium-Hydrogen Exchangers/genetics/*metabolism ; },
abstract = {Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser[339] and Ser[340] abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.},
}
@article {pmid35139891,
year = {2022},
author = {Li, S and Liu, L and Sun, W and Zhou, X and Zhou, H},
title = {A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {51},
pmid = {35139891},
issn = {1474-760X},
mesh = {*Adenine/metabolism ; CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Mutation ; Transcriptome ; },
abstract = {BACKGROUND: The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects.<br><br>RESULTS: In this study, we perform a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, reveal that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicates that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the genome. Furthermore, we detect off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tend to cluster, while off-target A>G DNA mutations rarely clustered.<br><br>CONCLUSION: Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.},
}
@article {pmid35139798,
year = {2022},
author = {Santiago-McRae, E and Oh, SW and Carlo, AM and Bar, O and Guan, E and Zheng, D and Grgicak, C and Fu, J},
title = {Rapid Nucleic Acid Reaction Circuits for Point-of-care Diagnosis of Diseases.},
journal = {Current topics in medicinal chemistry},
volume = {22},
number = {8},
pages = {686-698},
doi = {10.2174/1570163819666220207114148},
pmid = {35139798},
issn = {1873-4294},
mesh = {Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Pandemics ; Point-of-Care Systems ; },
abstract = {An urgent need exists for a rapid, cost-effective, facile, and reliable nucleic acid assay for mass screening to control and prevent the spread of emerging pandemic diseases. This urgent need is not fully met by current diagnostic tools. In this review, we summarize the current state-of-the-art research in novel nucleic acid amplification and detection that could be applied to point-of-care (POC) diagnosis and mass screening of diseases. The critical technological breakthroughs will be discussed for their advantages and disadvantages. Finally, we will discuss the future challenges of developing nucleic acid-based POC diagnosis.},
}
@article {pmid35139706,
year = {2022},
author = {Chu, LL},
title = {CRISPR-Cas system in microbial hosts for terpenoid production.},
journal = {Critical reviews in biotechnology},
volume = {42},
number = {7},
pages = {1116-1133},
doi = {10.1080/07388551.2021.1995318},
pmid = {35139706},
issn = {1549-7801},
mesh = {Biofuels ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; Hormones/metabolism ; Humans ; Pharmaceutical Preparations/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; *Terpenes/metabolism ; Vitamins/metabolism ; },
abstract = {Terpenoids represent the largest group of secondary metabolites with variable structures and functions. Terpenoids are well known for their beneficial application in human life, such as pharmaceutical products, vitamins, hormones, anticancer drugs, cosmetics, flavors and fragrances, foods, agriculture, and biofuels. Recently, engineering microbial cells have been provided with a sustainable approach to produce terpenoids with high yields. Noticeably, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has emerged as one of the most efficient genome-editing technologies to engineer microorganisms for improving terpenoid production. In this review, we summarize the application of the CRISPR-Cas system for the production of terpenoids in microbial hosts such as Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Pseudomonas putida. CRISPR-Cas9 deactivated Cas9 (dCas9)-based CRISPR (CRISPRi), and the dCas9-based activator (CRISPRa) have been used in either individual or combinatorial systems to control the metabolic flux for enhancing the production of terpenoids. Finally, the prospects of using the CRISPR-Cas system in terpenoid production are also discussed.},
}
@article {pmid35139474,
year = {2022},
author = {Takeuchi, S and Yamamoto, M and Matsumoto, S and Kenjo, E and Karashima, M and Ikeda, Y},
title = {Pinpoint modification strategy for stabilization of single guide RNA.},
journal = {Journal of chromatography. B, Analytical technologies in the biomedical and life sciences},
volume = {1192},
number = {},
pages = {123149},
doi = {10.1016/j.jchromb.2022.123149},
pmid = {35139474},
issn = {1873-376X},
mesh = {CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Mass Spectrometry ; RNA Processing, Post-Transcriptional/genetics ; RNA Stability/*genetics ; *RNA, Guide/chemistry/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats-CRISPR associated protein9 (CRISPR-Cas9) system, which includes a single guide RNA (sgRNA) and a Cas9 protein, is an emerging and promising gene editing technology that produces specific changes, including insertions, deletions, or substitutions, in desired targets. This approach can be applied in novel therapeutic areas for multiple cancers and genetic diseases, including Parkinson's disease, sickle cell disease, and muscular dystrophy. However, there are many limitations to its potential application to therapeutics. CRISPR-Cas9 activity without side effects, delivery of CRISPR-Cas9 to the target cell within the desired tissue including liver, lungs, brain and muscle and the expression of Cas9 endonuclease in the target cell are key factors in achieving therapeutic efficacy. Generally, single-stranded RNA is immediately degraded in cells and biological fluids such as serum, as chemically unmodified single-stranded RNA shows extremely poor stability against nuclease degradation. To overcome this limitation, sgRNA is chemically modified to obtain a highly stable sgRNA for efficient gene editing in cells and in vivo. Here, we identified the cleavage site of sgRNA for pinpoint modification in biological tissues using mass spectrometry and improved stability of pinpoint modified sgRNA in these fluids. Although improved efficiency provided by modified sgRNA has already been reported, we identified the cleavage site by mass spectrometry and revealed that the stability increased with the pinpoint modification strategy for the first time in this study. In future studies, the efficiency of pinpoint modification strategy for the potential application of sgRNA by systematic routes, including intravenous and subcutaneous administration will be assessed.},
}
@article {pmid35138842,
year = {2022},
author = {Bai, L and Wang, L and Huang, S and Bai, R and Lv, X and Sun, L and Zhang, F and Xu, X},
title = {Rapid, Visual, and Sequence-Specific Detection of Salmonella in Egg Liquid with vis-NEAA, a CRISPR/Cas12 Empowered New Strategy.},
journal = {Journal of agricultural and food chemistry},
volume = {70},
number = {7},
pages = {2401-2409},
doi = {10.1021/acs.jafc.1c06715},
pmid = {35138842},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; Eggs ; *Foodborne Diseases ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Salmonella/genetics ; },
abstract = {Salmonella is one of the main pathogenic factors that cause foodborne diseases. Rapid and accurate detection of Salmonella in food is of great importance to ensure food safety. Nicking enzyme-assisted amplification (NEAA) is one of the promising isothermal amplification methods finishing the in vitro amplification in ∼10 min; however, it suffers from nonspecific amplification a lot (∼70% products are noises). In this paper, we introduced CRISPR/Cas12a to specifically recognize the NEAA amplicons and transduce the signals into turned-on fluorescent visual readouts (vis-NEAA). Impressively, with this method, the high efficiency of NEAA has been taken great advantage and the nonspecific products were successfully bypassed at the same time. In comparison to NEAA-gel electrophoresis, vis-NEAA showed complete fidelity toward the presence of specific products, while for real-time PCR, it possesses equivalent sensitivity and specificity but saves ∼80% of the time. A level of 80 CFU/mL Salmonella in spiked eggs can be detected on-site in ∼20 min.},
}
@article {pmid35137623,
year = {2022},
author = {Minami, Y and Yuan, Y and Ueda, HR},
title = {High-throughput Genetically Modified Animal Experiments Achieved by Next-generation Mammalian Genetics.},
journal = {Journal of biological rhythms},
volume = {37},
number = {2},
pages = {135-151},
doi = {10.1177/07487304221075002},
pmid = {35137623},
issn = {1552-4531},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Circadian Rhythm ; Gene Editing/methods ; Genome ; Mammals/genetics ; },
abstract = {Animal models are essential tools for modern scientists to conduct biological experiments and investigate their hypotheses in vivo. However, for the past decade, raising the throughput of such animal experiments has been a great challenge. Conventionally, in vivo high-throughput assay was achieved through large-scale mutagen-driven forward genetic screening, which took years to find causal genes. In contrast, reverse genetics accelerated the causal gene identification process, but its throughput was also limited by 2 barriers, that is, the genome modification step and the time-consuming crossing step. Defined as genetics without crossing, next-generation genetics is able to produce gene-modified animals that can be analyzed at the founder generation (F0). This method is or can be accomplished through recent technological advances in gene editing and virus-based efficient gene modifications. Notably, next-generation genetics has accelerated the process of cross-species studies, and it will be a useful technique during animal experiments as it can provide genetic perturbation at an individual level without crossing. In this review, we begin by introducing the history of animal-based high-throughput analysis, with a specific focus on chronobiology. We then describe ways that gene modification efficiency during animal experiments was enhanced and why crossing remained a barrier to reaching higher efficiency. Moreover, we mention the Triple CRISPR as a critical technique for achieving next-generation genetics. Finally, we discuss the potential applications and limitations of next-generation mammalian genetics.},
}
@article {pmid35136148,
year = {2022},
author = {Hennig, SL and Owen, JR and Lin, JC and McNabb, BR and Van Eenennaam, AL and Murray, JD},
title = {A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {2067},
pmid = {35136148},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Dairying/*methods ; Embryo Transfer/methods ; Fetus/*anatomy &amp; histology/embryology ; Genotype ; Horns/*growth &amp; development ; Phenotype ; RNA, Guide/genetics ; Sequence Deletion/*genetics ; },
abstract = {Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.},
}
@article {pmid35135865,
year = {2022},
author = {Clara, JA and Levy, ER and Reger, R and Barisic, S and Chen, L and Cherkasova, E and Chakraborty, M and Allan, DSJ and Childs, R},
title = {High-affinity CD16 integration into a CRISPR/Cas9-edited CD38 locus augments CD38-directed antitumor activity of primary human natural killer cells.},
journal = {Journal for immunotherapy of cancer},
volume = {10},
number = {2},
pages = {},
pmid = {35135865},
issn = {2051-1426},
mesh = {ADP-ribosyl Cyclase 1/*metabolism ; Animals ; CRISPR-Cas Systems/*immunology ; Cell Line, Tumor ; Gene Editing/*methods ; Humans ; Immunotherapy/*methods ; Killer Cells, Natural/*metabolism ; Luciferases, Firefly ; Mice ; Mice, Inbred NOD ; Transfection ; },
abstract = {BACKGROUND: Adoptive transfer of natural killer (NK) cells with augmented antibody-dependent cellular cytotoxicity (ADCC) capabilities and resistance to CD38 targeting has the potential to enhance the clinical anti-myeloma activity of daratumumab (DARA). Therefore, we sought to develop an efficient CRISPR/Cas9-based gene editing platform to disrupt CD38 expression (CD38 knockout (KO)) in ex vivo expanded NK cells and simultaneously arm CD38[KO] NK cells with a high-affinity CD16 (CD16-158V) receptor.<br><br>METHODS: CD38[KO] human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38[KO] NK cells and targeted gene insertion at the CD38 locus to mediate gene knockin (KI). The capacity of these gene-edited NK cells to persist and mediate ADCC in the presence of DARA was tested in vitro and in a MM.1S xenograft mouse model.<br><br>RESULTS: Highly efficient CD38 gene disruption was achieved in ex vivo expanded NK cells without affecting their proliferative or functional capacity. CD38 KO conferred resistance to DARA-induced NK cell fratricide, enabling persistence and augmented ADCC against myeloma cell lines in the presence of DARA in vitro and in a MM.1S xenograft mouse model. CD38[KO] NK cells could be further modified by transfection with mRNA encoding a CD16-158V receptor, resulting in augmented DARA-mediated ADCC. Finally, we observed that a homology-directed repair template targeted to the CD38 locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38[KO]/CD16[KI] NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo.<br><br>CONCLUSIONS: Adoptive immunotherapy using ex vivo expanded CD38[KO]/CD16[KI] NK cells has the potential to boost the clinical efficacy of DARA. By incorporating complementary genetic engineering strategies into a CD38 KO manufacturing platform, we generated NK cells with substantially augmented CD38-directed antitumor activity, establishing a strong rationale for exploring this immunotherapy strategy in the clinic.},
}
@article {pmid35135438,
year = {2021},
author = {Abdallah, NA and Hamwieh, A and Radwan, K and Fouad, N and Prakash, C},
title = {Genome editing techniques in plants: a comprehensive review and future prospects toward zero hunger.},
journal = {GM crops &amp; food},
volume = {12},
number = {2},
pages = {601-615},
pmid = {35135438},
issn = {2164-5701},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; *Hunger ; },
abstract = {Promoting sustainable agriculture and improving nutrition are the main united nation sustainable development goals by 2030. New technologies are required to achieve zero hunger, and genome editing technology is the most promising one. In the last decade, genome editing (GE) using the CRISPR/Cas system has attracted researchers as a safer and easy tool for genome editing in several living organisms. GE has revolutionized the field of agriculture by improving biotic and abiotic stresses and yield improvement. GE technologies were developed fast lately to avoid the obstacles that face GM crops. GE technology, depending on site directed nuclease (SDN), is divided into three categories according to the modification methods. Developing transgenic-free edited plants without introducing foreign DNA meet the acceptance and regulatory ratification of several countries. There are several ongoing efforts from different countries that are rapidly expanding to adopt the current technological innovations. This review summarizes the different GE technologies and their application as a way to help in ending hunger.},
}
@article {pmid35134533,
year = {2022},
author = {Nganso, BT and Pines, G and Soroker, V},
title = {Insights into gene manipulation techniques for Acari functional genomics.},
journal = {Insect biochemistry and molecular biology},
volume = {143},
number = {},
pages = {103705},
doi = {10.1016/j.ibmb.2021.103705},
pmid = {35134533},
issn = {1879-0240},
mesh = {*Acari ; Animals ; *CRISPR-Cas Systems ; Ecosystem ; Gene Editing/methods ; Genomics/methods ; },
abstract = {Functional genomics is an essential tool for elucidating the structure and function of genes in any living organism. Here, we review the use of different gene manipulation techniques in functional genomics of Acari (mites and ticks). Some of these Acari species inflict severe economic losses to managed crops and health problems to humans, wild and domestic animals, but many also provide important ecosystem services worldwide. Currently, RNA interference (RNAi) is the leading gene expression manipulation tool followed by gene editing via the bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 system (CRISPR-Cas9). Whilst RNAi, via siRNA, does not always lead to expected outcomes, the exploitations of the CRISPR systems in Acari are still in their infancy and are limited only to CRISP/Cas9 to date. In this review, we discuss the advantages and disadvantages of RNAi and CRISPR-Cas9 and the technical challenges associated with their exploitations. We also compare the biochemical machinery of RNAi and CRISPR-Cas9 technologies. We highlight some potential solutions for experimental optimization of each mechanism in gene function studies. The potential benefits of adopting various CRISPR-Cas9 systems for expanding on functional genomics experiments in Acari are also discussed.},
}
@article {pmid35134339,
year = {2022},
author = {Maguin, P and Varble, A and Modell, JW and Marraffini, LA},
title = {Cleavage of viral DNA by restriction endonucleases stimulates the type II CRISPR-Cas immune response.},
journal = {Molecular cell},
volume = {82},
number = {5},
pages = {907-919.e7},
pmid = {35134339},
issn = {1097-4164},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages/metabolism ; CRISPR-Cas Systems ; DNA Restriction Enzymes/genetics ; *DNA, Viral/genetics ; Endonucleases/genetics ; Immunity ; },
abstract = {Prokaryotic organisms have developed multiple defense systems against phages; however, little is known about whether and how these interact with each other. Here, we studied the connection between two of the most prominent prokaryotic immune systems: restriction-modification and CRISPR. While both systems employ enzymes that cleave a specific DNA sequence of the invader, CRISPR nucleases are programmed with phage-derived spacer sequences, which are integrated into the CRISPR locus upon infection. We found that restriction endonucleases provide a short-term defense, which is rapidly overcome through methylation of the phage genome. In a small fraction of the cells, however, restriction results in the acquisition of spacer sequences from the cleavage site, which mediates a robust type II-A CRISPR-Cas immune response against the methylated phage. This mechanism is reminiscent of eukaryotic immunity in which the innate response offers a first temporary line of defense and also activates a second and more robust adaptive response.},
}
@article {pmid35134247,
year = {2022},
author = {Gardiner, J and Ghoshal, B and Wang, M and Jacobsen, SE},
title = {CRISPR-Cas-mediated transcriptional control and epi-mutagenesis.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1811-1824},
pmid = {35134247},
issn = {1532-2548},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Mutagenesis ; },
abstract = {Tools for sequence-specific DNA binding have opened the door to new approaches in investigating fundamental questions in biology and crop development. While there are several platforms to choose from, many of the recent advances in sequence-specific targeting tools are focused on developing Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated (CRISPR-Cas)-based systems. Using a catalytically inactive Cas protein (dCas), this system can act as a vector for different modular catalytic domains (effector domains) to control a gene's expression or alter epigenetic marks such as DNA methylation. Recent trends in developing CRISPR-dCas systems include creating versions that can target multiple copies of effector domains to a single site, targeting epigenetic changes that, in some cases, can be inherited to the next generation in the absence of the targeting construct, and combining effector domains and targeting strategies to create synergies that increase the functionality or efficiency of the system. This review summarizes and compares DNA targeting technologies, the effector domains used to target transcriptional control and epi-mutagenesis, and the different CRISPR-dCas systems used in plants.},
}
@article {pmid35134245,
year = {2022},
author = {Huang, Y and Shang, M and Liu, T and Wang, K},
title = {High-throughput methods for genome editing: the more the better.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1731-1745},
pmid = {35134245},
issn = {1532-2548},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plants/genetics ; RNA, Guide ; },
abstract = {During the last decade, targeted genome-editing technologies, especially clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) technologies, have permitted efficient targeting of genomes, thereby modifying these genomes to offer tremendous opportunities for deciphering gene function and engineering beneficial traits in many biological systems. As a powerful genome-editing tool, the CRISPR/Cas systems, combined with the development of next-generation sequencing and many other high-throughput techniques, have thus been quickly developed into a high-throughput engineering strategy in animals and plants. Therefore, here, we review recent advances in using high-throughput genome-editing technologies in animals and plants, such as the high-throughput design of targeted guide RNA (gRNA), construction of large-scale pooled gRNA, and high-throughput genome-editing libraries, high-throughput detection of editing events, and high-throughput supervision of genome-editing products. Moreover, we outline perspectives for future applications, ranging from medication using gene therapy to crop improvement using high-throughput genome-editing technologies.},
}
@article {pmid35133958,
year = {2022},
author = {Chen, S and Jiao, Y and Pan, F and Guan, Z and Cheng, SH and Sun, D},
title = {Knock-In of a Large Reporter Gene via the High-Throughput Microinjection of the CRISPR/Cas9 System.},
journal = {IEEE transactions on bio-medical engineering},
volume = {69},
number = {8},
pages = {2524-2532},
doi = {10.1109/TBME.2022.3149530},
pmid = {35133958},
issn = {1558-2531},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques ; Genes, Reporter/genetics ; Microinjections ; *Zebrafish/genetics ; },
abstract = {The non-viral delivery of the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) nuclease system provides promising solutions for gene therapy. However, traditional chemical and physical delivery approaches for gene knock-in are confronted by significant challenges to overcome the drawbacks of low efficiency and high toxicity. An alternative method for directly delivering CRISPR components into single cells is microinjection. Here, we present the high-throughput robotic microinjection of CRISPR machinery plasmids to produce gene insertions. We demonstrate that the microinjection of CRISPR/Cas9 with an enhanced green fluorescent protein (eGFP) donor template into single HepG2 cells can achieve reporter gene knock-in targeting the adeno-associated virus site 1 locus. Homology-directed repair-mediated knock-in can be observed with an efficiency of 41%. Assessment via T7E1 assay indicates that the eGFP knock-in cells exhibit no detectable changes at potential off-target sites. A case study of injecting the eGFP knock-in cells into zebrafish (Danio rerio) embryos to form an in vivo tumor model is conducted. Results demonstrate the efficiency of combining microinjection with the CRISPR/Cas9 system in achieving gene knock-in.},
}
@article {pmid35133877,
year = {2022},
author = {Tremblay, G and Rousseau, J and Mbakam, CH and Tremblay, JP},
title = {Insertion of the Icelandic Mutation (A673T) by Prime Editing: A Potential Preventive Treatment for Familial and Sporadic Alzheimer's Disease.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {109-122},
pmid = {35133877},
issn = {2573-1602},
support = {U24 AG021886/AG/NIA NIH HHS/United States ; //CIHR/Canada ; },
mesh = {*Alzheimer Disease/genetics/prevention &amp; control ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Iceland ; Mutation ; },
abstract = {Alzheimer's disease (AD) is the result of abnormal processing of the amyloid precursor protein (APP) by β-secretase and γ-secretase, which leads to the formation of toxic β-amyloid peptides. The toxic β-amyloid peptides induce neuron death, memory problems, and AD development. Several APP mutations increase the risk of developing early-onset AD. However, the A673T mutation identified in the Icelandic population prevents AD development by reducing the cleavage of APP by β-secretase. In this study, we inserted the A673T mutation in human cells using the CRISPR prime editing (PE) technique. Repeated PE treatments resulted in the insertion of the A673T mutation in up to 49.2% of APP genes when a second nick was induced in the other DNA strand. When the protospacer adjacent motif used for PE was also mutated, up to 68.9% of the APP genes contained the protective A673T mutation. PE is a promising approach to introduce the A673T mutation precisely without mutating nearby nucleotides.},
}
@article {pmid35133672,
year = {2022},
author = {Park, H and Kim, J},
title = {Activation of melatonin receptor 1 by CRISPR-Cas9 activator ameliorates cognitive deficits in an Alzheimer's disease mouse model.},
journal = {Journal of pineal research},
volume = {72},
number = {3},
pages = {e12787},
doi = {10.1111/jpi.12787},
pmid = {35133672},
issn = {1600-079X},
mesh = {*Alzheimer Disease/drug therapy/genetics/metabolism ; Amyloid beta-Peptides/metabolism ; Animals ; CRISPR-Cas Systems ; Cognition ; Disease Models, Animal ; *Melatonin/metabolism/pharmacology/therapeutic use ; Mice ; Mice, Transgenic ; Receptors, Melatonin/metabolism ; },
abstract = {Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of neurotoxic beta-amyloid (Aβ) in the brain. Melatonin receptors have been reported to associate with aging and AD, and their expression decreased with the progression of AD. As an alternative to AD treatment, overexpression of melatonin receptors may lead to melatonin-like effects to treat alleviate the symptoms of AD. Here, we successfully activated the type 1 melatonin receptor (Mt1) in vivo brain using a Cas9 activator as a novel AD therapeutic strategy. The Cas9 activator efficiently activated the endogenous Mt1 gene in the brain. Activation of Mt1 via Cas9 activators modulated anti-amyloidogenic and anti-inflammatory roles in 5xFAD AD mice brain. Moreover, activation of Mt1 with the CRISPR/Cas9 activator improved cognitive deficits in an AD model. These results demonstrated the therapeutic potential of melatonin receptor activation via CRISPR/Cas9 activator for AD.},
}
@article {pmid35132857,
year = {2022},
author = {Feng, C and Liang, W and Liu, F and Xiong, Y and Chen, M and Feng, P and Guo, M and Wang, Y and Li, Z and Zhang, L},
title = {A Simple and Highly Sensitive Naked-Eye Analysis of EGFR 19del via CRISPR/Cas12a Triggered No-Nonspecific Nucleic Acid Amplification.},
journal = {ACS synthetic biology},
volume = {11},
number = {2},
pages = {867-876},
doi = {10.1021/acssynbio.1c00521},
pmid = {35132857},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics ; *DNA, Catalytic ; ErbB Receptors/genetics ; Humans ; *Lung Neoplasms/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {The mutation status of epidermal growth factor receptor (EGFR) exon 19 is of great importance for predicting sensitivity to tyrosine kinase inhibitors (TKIs) in the treatment of non-small-cell lung cancer (NSCLC). However, the development of simple, sensitive, and no-nonspecific amplification platforms for EGFR 19del detection in NSCLC remains a challenge. Herein, we developed a novel, simple, and highly sensitive naked-eye assay utilizing CRISPR/Cas12a-triggered no-nonspecific nucleic acid amplification (NAA) with rolling circle amplification (RCA) as a model for EGFR 19del detection. Typically, circular padlocks are designed to be the trans-cleavage substrate of Cas12a/crRNA and serve as templates for RCA. Since the target EGFR 19del induces robust trans-cleavage activity of the Cas12a/crRNA duplex, the surrounding circular padlocks are cleaved into random short linear fragments that are unable to initiate RCA, resulting in a colorless solution. However, in the absence of EGFR 19del, the inactivated Cas12a enzymes cannot cleave the circular padlocks, and they remain able to serve as templates to initiate RCA to generate long single-stranded DNA to further fold into G-quadruplex/hemin DNAzymes to catalyze the oxidation of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS[2-]), generating a color response that is obvious to the naked eye. As expected, this strategy with a detection limit as low as 20 fM exhibited robust selectivity and anti-interference ability. Moreover, this method was applicable for detecting EGFR 19del in real serum samples and showed high consistency with real-time quantitative polymerase chain reaction (qPCR) and sequencing results, providing a promising strategy for the early noninvasive diagnosis and guidance of clinical treatment for cancer.},
}
@article {pmid35132255,
year = {2022},
author = {Riedmayr, LM and Hinrichsmeyer, KS and Karguth, N and Böhm, S and Splith, V and Michalakis, S and Becirovic, E},
title = {dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy.},
journal = {Nature protocols},
volume = {17},
number = {3},
pages = {781-818},
pmid = {35132255},
issn = {1750-2799},
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Therapy ; Mice ; Promoter Regions, Genetic ; *RNA, Guide/genetics ; Transcriptional Activation ; },
abstract = {Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the appropriate counterpart(s) to compensate for the missing gene function. This approach offers key advantages over conventional gene therapies because it is mutation- and gene size-independent. Here, we describe a protocol for the design, execution and evaluation of such gene therapies using dCas9-VPR. We offer guidelines on how to identify functionally equivalent genes, design and clone single guide RNAs and evaluate transcriptional activation in vitro. Moreover, focusing on inherited retinal diseases, we provide a detailed protocol on how to apply this strategy in mice using dual recombinant adeno-associated virus vectors and how to evaluate its functionality and off-target effects in the target tissue. This strategy is in principle applicable to all organisms that possess functionally equivalent genes suitable for transcriptional activation and addresses pivotal unmet needs in gene therapy with high translational potential. The protocol can be completed in 15-20 weeks.},
}
@article {pmid35131696,
year = {2022},
author = {Habimana, JD and Huang, R and Muhoza, B and Kalisa, YN and Han, X and Deng, W and Li, Z},
title = {Mechanistic insights of CRISPR/Cas nucleases for programmable targeting and early-stage diagnosis: A review.},
journal = {Biosensors &amp; bioelectronics},
volume = {203},
number = {},
pages = {114033},
doi = {10.1016/j.bios.2022.114033},
pmid = {35131696},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Pandemics ; SARS-CoV-2/genetics ; },
abstract = {Conventional and routine diagnostics such as polymerase chain reaction (PCR) and serological tests are less sensitive, costly, and require sample pretreatment procedures. CRISPR/Cas systems that inherently assist bacteria and archaea in destroying invading phage genetic materials via an RNA-mediated interference strategy have been reconstituted in vitro and harnessed for nucleic and non-nucleic acid diagnostics. CRISPR/Cas-based diagnostics (CRISPR-Dx) are cost-effective, possess excellent sensitivity (attomolar) and specificity (single base distinction), exhibit fast turnaround response, and support nucleic acid extraction-free workflow. However, CRISPR-Dx still needs to address various challenges to translate the laboratory work into end-user tailored solutions. In this perspective, we review the relevant progress of CRISPR/Cas systems-based diagnostics, focusing on the comprehensive customization and applications of leading and trending CRISPR/Cas systems as platform technologies for fluorescence, colorimetric, and electrical signal detection. The impact of the CRISPR game-changing technology on the COVID-19 pandemic is highlighted. We also demonstrate the role of CRISPR/Cas systems for carryover contamination prevention. The advancements in signal amplification strategies using engineered crRNAs, novel reporters, nanoparticles, artificial genetic circuits, microfluidics, and smartphones are also covered. Furthermore, we critically discuss the translation of CRISPR-Dx's basic research into end-user diagnostics for commercialization success in the near future. Finally, we discuss the complex challenges and alternative solutions to harness the CRISPR/Cas potential in detail.},
}
@article {pmid35131266,
year = {2022},
author = {Tsutsumi, R and Chaya, T and Tsujii, T and Furukawa, T},
title = {The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {3},
pages = {101686},
pmid = {35131266},
issn = {1083-351X},
mesh = {Animals ; *Autoantigens/metabolism ; Basal Bodies ; Cilia/metabolism ; *Ciliopathies/genetics/metabolism ; Female ; Homeostasis ; Humans ; *Kidney Diseases, Cystic/metabolism ; Male ; Mammals ; Mice ; Mutation ; *Neoplasm Proteins/metabolism ; Proteins/metabolism ; },
abstract = {In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8[ΔC/ΔC] mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8[ΔC/ΔC] mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8[ΔC/ΔC] mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein-protein interaction networks involved in cilia development.},
}
@article {pmid35129865,
year = {2022},
author = {Hou, Z and Hu, C and Ke, A and Zhang, Y},
title = {Introducing Large Genomic Deletions in Human Pluripotent Stem Cells Using CRISPR-Cas3.},
journal = {Current protocols},
volume = {2},
number = {2},
pages = {e361},
pmid = {35129865},
issn = {2691-1299},
support = {GM137883/NH/NIH HHS/United States ; GM102543/NH/NIH HHS/United States ; R01 GM102543/GM/NIGMS NIH HHS/United States ; GM117268/NH/NIH HHS/United States ; R35 GM118174/GM/NIGMS NIH HHS/United States ; K99 GM117268/GM/NIGMS NIH HHS/United States ; R35 GM137883/GM/NIGMS NIH HHS/United States ; GM118174/NH/NIH HHS/United States ; R00 GM117268/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genomics ; Humans ; *Pluripotent Stem Cells/metabolism ; },
abstract = {CRISPR-Cas systems provide researchers with eukaryotic genome editing tools and therapeutic platforms that make it possible to target disease mutations in somatic organs. Most of these tools employ Type II (e.g., Cas9) or Type V (e.g., Cas12a) CRISPR enzymes to create RNA-guided precise double-strand breaks in the genome. However, such technologies are limited in their capacity to make targeted large deletions. Recently, the Type I CRISPR system, which is prevalent in microbes and displays unique enzymatic features, has been harnessed to effectively create large chromosomal deletions in human cells. Type I CRISPR first uses a multisubunit ribonucleoprotein (RNP) complex called Cascade to find its guide-complementary target site, and then recruits a helicase-nuclease enzyme, Cas3, to travel along and shred the target DNA over a long distance with high processivity. When introduced into human cells as purified RNPs, the CRISPR-Cas3 complex can efficiently induce large genomic deletions of varying lengths (1-100 kb) from the CRISPR-targeted site. Because of this unique editing outcome, CRISPR-Cas3 holds great promise for tasks such as the removal of integrated viral genomes and the interrogation of structural variants affecting gene function and human disease. Here, we provide detailed protocols for introducing large deletions using CRISPR-Cas3. We describe step-by-step procedures for purifying the Type I-E CRISPR proteins Cascade and Cas3 from Thermobifida fusca, electroporating RNPs into human cells, and characterizing DNA deletions using PCR and sequencing. We focus here on human pluripotent stem cells due to their clinical potential, but these protocols will be broadly useful for other cell lines and model organisms for applications including large genomic deletion, full-gene or -chromosome removal, and CRISPR screening for noncoding elements, among others. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of Tfu Cascade RNP Support Protocol 1: Expression and purification of TfuCas3 protein Support Protocol 2: Culture of human pluripotent stem cells Basic Protocol 2: Introduction of Tfu Cascade RNP and Cas3 protein into hPSCs via electroporation Basic Protocol 3: Characterization of genomic DNA lesions using long-range PCR, TOPO cloning, and Sanger sequencing Alternate Protocol: Comprehensive analysis of genomic lesions by Tn5-based next-generation sequencing Support Protocol 3: Single-cell clonal isolation.},
}
@article {pmid35129197,
year = {2022},
author = {Li, R and Meng, Q and Qi, J and Hu, L and Huang, J and Zhang, Y and Yang, J and Sun, J},
title = {Microinjection-based CRISPR/Cas9 mutagenesis in the decapoda crustaceans Neocaridina heteropoda and Eriocheir sinensis.},
journal = {The Journal of experimental biology},
volume = {225},
number = {6},
pages = {},
doi = {10.1242/jeb.243702},
pmid = {35129197},
issn = {1477-9145},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Crustacea ; *Decapoda/genetics ; Humans ; Microinjections ; Mutagenesis ; },
abstract = {CRISPR/Cas9 technology has been applied to many arthropods. However, application of this technology to crustaceans remains limited because of the unique characteristics of embryos. Our group has developed a microinjection system to introduce the CRISPR/Cas9 system into Neocaridina heteropoda embryos (one-cell stage). Using the developed method, we mutated the target gene Nh-scarlet (N. heteropoda scarlet), which functions in eye development and pigmentation. The results showed that both eye color and shape were altered in individuals in which Nh-scarlet was knocked out. Furthermore, this system was also successfully applied to another decapod crustacean, Eriocheir sinensis. DNA sequencing revealed that the zoeae with red eyes had an edited version of Es-scarlet. This study provides a stable microinjection method for freshwater crustaceans, and will contribute to functional genomics studies in various decapods.},
}
@article {pmid35129125,
year = {2022},
author = {Asadian, M and Saadati, M and Bajestani, FB and Beardall, J and Abdolahadi, F and Mahdinezhad, N},
title = {Knockout of Cia5 gene using CRISPR/Cas9 technique in Chlamydomonas reinhardtii and evaluating CO2 sequestration in control and mutant isolates.},
journal = {Journal of genetics},
volume = {101},
number = {},
pages = {},
pmid = {35129125},
issn = {0973-7731},
mesh = {CRISPR-Cas Systems ; Carbon Dioxide/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 technology is one of the common methods of genome editing and targeted gene mutation, which has recently been used for manipulating microalgae such as Chlamydomonas reinhardtii. Besides, this technology can play a role in the fight against greenhouse gases (e.g., carbon dioxide) production by studying genetic pathways to improve algal strains. Among several genes in algae that respond to CO2 and regulators control the expression of each; Cia5 is one of the most critical transcriptional regulators. In this research, we knocked out the Cia5 gene using the CRISPR/Cas9 technique and analysed the ability of C. reinhardtii to perform CO2 sequestration. Our results showed that C. reinhardtii has better performance (i.e., response to CO2 treatment) in both control and mutant species at 0.5% CO2 concentration than other concentrations. However, the difference between the control microalgae species and the mutant species was in the CO2 removal efficiency. Additionally, our findings revealed that the control type isolate in CO2 concentrations of 0.04%, 0.5% and 1% had removal efficiencies of 27%, 37% and 21%, respectively. Nevertheless, for mutant species in the same concentrations, the observed removal efficiencies were 16%, 23% and 9%.},
}
@article {pmid35128523,
year = {2021},
author = {Carlsen, FM and Johansen, IE and Yang, Z and Liu, Y and Westberg, IN and Kieu, NP and Jørgensen, B and Lenman, M and Andreasson, E and Nielsen, KL and Blennow, A and Petersen, BL},
title = {Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {795644},
pmid = {35128523},
issn = {2673-3439},
abstract = {Potato, Solanum tuberosum is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the gene encoding glucan water dikinase (GWD) 1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3-2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. At the same time, high editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids. Furthermore, high editing efficiencies reduce the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNPs) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and the DMR6-1, 6-10 gRNAs were designed to target regions comprising the 5' and the 3' end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs varied significantly, and some generated specific indel patterns. RNP's targeting the 5' end of GWD1 yielded significantly higher editing efficiency as compared to targeting the 3' end. For DMR6-1, such an effect was not seen. Simultaneously targeting each of the two target regions with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3' end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or a slightly negative effect on editing from the single or combined gRNA/RNPs. These initial findings may instigate much larger studies needed for facilitating and optimizing precision breeding in plants.},
}
@article {pmid35127671,
year = {2021},
author = {Aman, R and Marsic, T and Sivakrishna Rao, G and Mahas, A and Ali, Z and Alsanea, M and Al-Qahtani, A and Alhamlan, F and Mahfouz, M},
title = {iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {9},
number = {},
pages = {800104},
pmid = {35127671},
issn = {2296-4185},
abstract = {Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.},
}
@article {pmid35123997,
year = {2022},
author = {Sayed, N and Allawadhi, P and Khurana, A and Singh, V and Navik, U and Pasumarthi, SK and Khurana, I and Banothu, AK and Weiskirchen, R and Bharani, KK},
title = {Gene therapy: Comprehensive overview and therapeutic applications.},
journal = {Life sciences},
volume = {294},
number = {},
pages = {120375},
doi = {10.1016/j.lfs.2022.120375},
pmid = {35123997},
issn = {1879-0631},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Transfer Techniques ; Genetic Diseases, Inborn/genetics/*therapy ; *Genetic Therapy ; Genetic Vectors/*therapeutic use ; Humans ; },
abstract = {Gene therapy is the product of man's quest to eliminate diseases. Gene therapy has three facets namely, gene silencing using siRNA, shRNA and miRNA, gene replacement where the desired gene in the form of plasmids and viral vectors, are directly administered and finally gene editing based therapy where mutations are modified using specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short tandem repeats (CRISPR)/CRISPR-associated protein (Cas)-associated nucleases. Transfer of gene is either through transformation where under specific conditions the gene is directly taken up by the bacterial cells, transduction where a bacteriophage is used to transfer the genetic material and lastly transfection that involves forceful delivery of gene using either viral or non-viral vectors. The non-viral transfection methods are subdivided into physical, chemical and biological. The physical methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound and hydrodynamic gene transfer. The chemical methods utilize calcium- phosphate, DAE-dextran, liposomes and nanoparticles for transfection. The biological methods are increasingly using viruses for gene transfer, these viruses could either integrate within the genome of the host cell conferring a stable gene expression, whereas few other non-integrating viruses are episomal and their expression is diluted proportional to the cell division. So far, gene therapy has been wielded in a plethora of diseases. However, coherent and innocuous delivery of genes is among the major hurdles in the use of this promising therapy. Hence this review aims to highlight the current options available for gene transfer along with the advantages and limitations of every method.},
}
@article {pmid35123478,
year = {2022},
author = {Shukal, S and Lim, XH and Zhang, C and Chen, X},
title = {Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering.},
journal = {Microbial cell factories},
volume = {21},
number = {1},
pages = {19},
pmid = {35123478},
issn = {1475-2859},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics/*metabolism ; Escherichia coli Proteins/genetics/metabolism ; Gene Deletion ; Gene Editing ; Genome, Bacterial ; Lycopene/*metabolism ; *Metabolic Engineering ; Plasmids/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Recombinases/genetics/metabolism ; },
abstract = {BACKGROUND: The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked.<br><br>RESULTS: In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10&#xa0;kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by&#x2009;~&#x2009;threefold.<br><br>CONCLUSIONS: Our work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.},
}
@article {pmid35123001,
year = {2022},
author = {Das, A and Goswami, HN and Whyms, CT and Sridhara, S and Li, H},
title = {Structural principles of CRISPR-Cas enzymes used in nucleic acid detection.},
journal = {Journal of structural biology},
volume = {214},
number = {1},
pages = {107838},
pmid = {35123001},
issn = {1095-8657},
support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing ; RNA/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based technology has revolutionized the field of biomedicine with broad applications in genome editing, therapeutics and diagnostics. While a majority of applications involve the RNA-guided site-specific DNA or RNA cleavage by CRISPR enzymes, recent successes in nucleic acid detection rely on their collateral and non-specific cleavage activated by viral DNA or RNA. Ranging in enzyme composition, the mechanism for distinguishing self- from foreign-nucleic acids, the usage of second messengers, and enzymology, the CRISPR enzymes provide a diverse set of diagnosis tools in further innovations. Structural biology plays an important role in elucidating the mechanisms of these CRISPR enzymes. Here we summarize and compare structures of three types of CRISPR enzymes used in nucleic acid detection captured in their respective functional forms and illustrate the current understanding of their activation mechanism.},
}
@article {pmid35122622,
year = {2022},
author = {Xu, K and Zhang, X and Liu, Z and Ruan, J and Xu, C and Che, J and Fan, Z and Mu, Y and Li, K},
title = {A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection.},
journal = {Science China. Life sciences},
volume = {65},
number = {8},
pages = {1535-1546},
pmid = {35122622},
issn = {1869-1889},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Gene Editing/methods ; Male ; *Nuclear Transfer Techniques ; RNA ; Ribonucleoproteins/genetics ; Swine ; },
abstract = {Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3-4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP's strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.},
}
@article {pmid35122203,
year = {2022},
author = {Quazi, S},
title = {Elucidation of CRISPR-Cas9 application in novel cellular immunotherapy.},
journal = {Molecular biology reports},
volume = {49},
number = {7},
pages = {7069-7077},
pmid = {35122203},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Humans ; Immunotherapy ; Immunotherapy, Adoptive/methods ; *Neoplasms/genetics ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {Novel cellular immunotherapy with engineered T cells has improved cancer treatment and established therapeutic promises to prevent tumor formation in clinical studies. Due to certain restrictions and difficulties, CAR and TCR T-cells therapies were inadequate at points. CRISPR Cas9 genome-editing tool has significant potential for these two cell-based therapies. As a specialized gene-editing technique, CRISPR Cas9 is used to repair genetic alternations with minimal damage. It is used as an adjunct to immunotherapy to stimulate a more robust immune response. CRISPR has long outpaced other target-specific genome editing methods such as ZFNs and TALEN because of its high efficiency, competence in targeting, and stable operating conditions. CRISPR can overcome the two major drawbacks of universal CAR T cells: allorejection and graft-vs-host disease. TCR-based T cell treatment can reduce inappropriate binding between endogenous and transgenic TCR, resulting in a reduction of severe toxicity. The CAR and TCR T based cell therapies uphold an excellent future for tumor malignancies. This article has elucidated the administration of CRISPR Cas9 in novel cellular immunotherapy, CAR, and TCR T cell therapy. However, this article did not fail to observe this technology's ethical concerns, limitations, and challenges. Furthermore, the article compares CRISPR-mediated allogeneic CAR T cell to TCR-T cell therapy.},
}
@article {pmid35121546,
year = {2022},
author = {Abnous, K and Abdolabadi, AK and Ramezani, M and Alibolandi, M and Nameghi, MA and Zavvar, T and Khoshbin, Z and Lavaee, P and Taghdisi, SM and Danesh, NM},
title = {A highly sensitive electrochemical aptasensor for cocaine detection based on CRISPR-Cas12a and terminal deoxynucleotidyl transferase as signal amplifiers.},
journal = {Talanta},
volume = {241},
number = {},
pages = {123276},
doi = {10.1016/j.talanta.2022.123276},
pmid = {35121546},
issn = {1873-3573},
mesh = {*Aptamers, Nucleotide ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; *Cocaine ; DNA Nucleotidylexotransferase ; Electrochemical Techniques/methods ; Limit of Detection ; },
abstract = {Cocaine is one of the mainly used illegal drugs in the world. Using the signal amplification elements of terminal deoxynucleotidyl transferase (TdT) and CRISPR-Cas12a, a highly sensitive and simple electrochemical aptasensor was introduced for cocaine quantification. When, no cocaine existed in the sample, the 3'-end of complementary strand of aptamer (CS) was extended by TdT, leading to the activation of CRISPR-Cas12a and remaining of very short oligonucleotides on the working electrode. So, the current signal was remarkably promoted. With the presence of cocaine, CS left the electrode surface. Thus, nothing changed following the incubation of TdT and CRISPR-Cas12a and the Aptamer/Cocaine complex presented on the electrode. Consequently, the [Fe(CN)6][3-/4-] could not freely reach the electrode surface and the signal response was weak. Under optimal situations, the biosensor revealed a wide linear relation from 40 pM to 150 nM with detection limit of 15 pM for cocaine. The sensitivity of the analytical system was comparable and even better than other reported methods for cocaine detection. The designed method displayed excellent cocaine selectivity. The aptasensor could work well for cocaine assay in serum samples. So, the aptasensor is expected to be an efficient analytical method with broad applications in the determination of diverse analytes.},
}
@article {pmid35121375,
year = {2022},
author = {Asahina, Y and Sakamoto, K and Hisabori, T and Wakabayashi, KI},
title = {The mammalian-type thioredoxin reductase 1 confers a high-light tolerance to the green alga Chlamydomonas reinhardtii.},
journal = {Biochemical and biophysical research communications},
volume = {596},
number = {},
pages = {97-103},
doi = {10.1016/j.bbrc.2022.01.088},
pmid = {35121375},
issn = {1090-2104},
mesh = {Algal Proteins/*genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Chlamydomonas reinhardtii/enzymology/*genetics/radiation effects ; Gene Editing/methods ; Gene Knockout Techniques ; Hydrogen Peroxide/pharmacology ; *Light ; Mammals/genetics/metabolism ; Oxidants/pharmacology ; Photosynthesis/genetics/radiation effects ; Phototaxis/drug effects/radiation effects ; RNA-Seq/methods ; Radiation Tolerance/*genetics ; Reactive Oxygen Species/metabolism ; Thioredoxin Reductase 1/*genetics/metabolism ; },
abstract = {Reactive oxygen species (ROS) can both act as a poison causing cell death and important signaling molecules among various organisms. Photosynthetic organisms inevitably produce ROS, making the appropriate elimination of ROS an essential strategy for survival. Interestingly, the unicellular green alga Chlamydomonas reinhardtii expresses a mammalian form of thioredoxin reductase, TR1, which functions as a ROS scavenger in animal cells. To investigate the properties of TR1 in C. reinhardtii, we generated TR1 knockout strains using CRISPR/Cas9-based genome editing. We found a reduced tolerance to high-light and ROS stresses in the TR1 knockout strains compared to the parental strain. In addition, the regulation of phototactic orientation, known to be regulated by ROS, was affected in the knockout strains. These results suggest that TR1 contributes to a ROS-scavenging pathway in C. reinhardtii.},
}
@article {pmid35121198,
year = {2022},
author = {Gong, X and Zheng, Z and Yang, T and Zheng, H and Xiao, X and Jia, N},
title = {Generation of an isogenic gene-corrected iPSC line (OGHFUi001-A-1) from a type 1 early infantile epileptic encephalopathy (EIEE1) patient with a hemizygous R330L mutation in the ARX gene.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102693},
doi = {10.1016/j.scr.2022.102693},
pmid = {35121198},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Genes, Homeobox ; Homeodomain Proteins/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Male ; Mutation/genetics ; *Spasms, Infantile/genetics ; Transcription Factors/genetics ; },
abstract = {Type 1 early infantile epileptic encephalopathy (EIEE1) is a rare X-link neurodevelopmental disorder caused by mutations in the ARX gene. The mechanism remains unclear due to the lack of cellular models for the disease. We previously have generated an iPSC line (OGHFUi001-A) from a male EIEE1 patient with a hemizygous R330L mutation in the ARX gene. Here we corrected the R330L mutation genetically using CRISPR/Cas9 technology to generate an isogenic control, which was an ideal control to investigate the pathogenesis of the mutation in this disease.},
}
@article {pmid35121196,
year = {2022},
author = {Lahm, H and Heinrich, P and Zierler, E and Dzilic, E and Neb, I and Luzius, T and Doppler, SA and Schneider, S and Lange, R and Krane, M and Dreßen, M},
title = {Generation of a CRISPR/Cas edited human induced pluripotent stem cell line DHMi005-A-1 carrying a patient-specific disease-causing point mutation in the TBX5 gene.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102691},
doi = {10.1016/j.scr.2022.102691},
pmid = {35121196},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Point Mutation ; T-Box Domain Proteins/*genetics/metabolism ; *Upper Extremity Deformities, Congenital/genetics ; },
abstract = {A number of mutations in the human TBX5 gene have been described which cause Holt-Oram syndrome, a severe congenital disease associated with abnormalities in heart and upper limb development. We have used a prime-editing approach to introduce a patient-specific disease-causing TBX5 mutation (c.920_C > A) into an induced pluripotent stem cell (iPSC) line from a healthy donor. The resulting iPSC line provides a powerful tool to identify and analyze the biological and molecular impact of this specific TBX5 mutation in comparison to the isogenic control iPSC line during cardiac development.},
}
@article {pmid35119473,
year = {2022},
author = {Li, Y and Wei, Y and Li, G and Huang, S and Xu, J and Ding, Q and Hong, J},
title = {Targeting NECTIN-1 Based on CRISPR/Cas9 System Attenuated the Herpes Simplex Virus Infection in Human Corneal Epithelial Cells In Vitro.},
journal = {Translational vision science &amp; technology},
volume = {11},
number = {2},
pages = {8},
pmid = {35119473},
issn = {2164-2591},
mesh = {CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism ; *Herpes Simplex/genetics ; *Herpesvirus 1, Human/genetics/metabolism ; Humans ; *Keratitis, Herpetic/genetics/metabolism ; Lentivirus/genetics/metabolism ; Nectins/genetics/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {PURPOSE: Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment.<br><br>METHODS: The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction.<br><br>RESULTS: Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group.<br><br>CONCLUSIONS: We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs.<br><br>TRANSLATIONAL RELEVANCE: This study offered a promising target for the cure of HSK.},
}
@article {pmid35119310,
year = {2022},
author = {Mozumdar, D and Csörgő, B and Bondy-Denomy, J},
title = {Genetic Manipulation of a CAST of Characters in a Microbial Community.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {4-6},
pmid = {35119310},
issn = {2573-1602},
support = {R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Techniques ; *Microbiota/genetics ; },
}
@article {pmid35119294,
year = {2022},
author = {Conant, D and Hsiau, T and Rossi, N and Oki, J and Maures, T and Waite, K and Yang, J and Joshi, S and Kelso, R and Holden, K and Enzmann, BL and Stoner, R},
title = {Inference of CRISPR Edits from Sanger Trace Data.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {123-130},
doi = {10.1089/crispr.2021.0113},
pmid = {35119294},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; RNA, Guide/genetics ; Software ; },
abstract = {Efficient and precise genome editing requires a fast, quantitative, and inexpensive assay to assess genotype following editing. Here, we present ICE (Inference of CRISPR Edits), which enables robust analysis of CRISPR edits using Sanger data. ICE proposes potential outcomes for editing with guide RNAs, and then determines which are supported by the data via regression. The ICE algorithm is robust and reproducible, and it can be used to analyze CRISPR experiments within days after transfection. We also confirm that ICE produces accurate estimates of editing outcomes across a variety of benchmarks, and within the context of other existing Sanger analysis tools. The ICE tool is free to use and open source, and offers several improvements over current analysis tools, such as batch analysis and support for a variety of editing conditions. It is available online at ice.synthego.com, and the source code is available at github.com/synthego-open/ice.},
}
@article {pmid35119139,
year = {2022},
author = {Xie, R and Wang, X and Wang, Y and Ye, M and Zhao, Y and Yandell, BS and Gong, S},
title = {pH-Responsive Polymer Nanoparticles for Efficient Delivery of Cas9 Ribonucleoprotein With or Without Donor DNA.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {34},
number = {23},
pages = {e2110618},
pmid = {35119139},
issn = {1521-4095},
support = {UG3 NS111688/NS/NINDS NIH HHS/United States ; UH3 NS111688/NS/NINDS NIH HHS/United States ; 4-UH3-NS111688/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/metabolism ; Hydrogen-Ion Concentration ; Mice ; *Nanoparticles ; Polymers ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) may offer new therapeutics for genetic diseases through gene disruption via nonhomologous end joining (NHEJ) or gene correction via homology-directed repair (HDR). However, clinical translation of CRISPR technology is limited by the lack of safe and efficient delivery systems. Here, facilely fabricated pH-responsive polymer nanoparticles capable of safely and efficiently delivering Cas9 ribonucleoprotein alone (termed NHEJ-NP, diameter = 29.4 nm), or together with donor DNA (termed HDR-NP, diameter = 33.3 nm) are reported. Moreover, intravenously, intratracheally, and intramuscularly injected NHEJ-NP induces efficient gene editing in mouse liver, lung, and skeletal muscle, respectively. Intramuscularly injected HDR-NP also leads to muscle strength recovery in a Duchenne muscular dystrophy mouse model. NHEJ-NP and HDR-NP possess many desirable properties including high payload loading content, small and uniform sizes, high editing efficiency, good biocompatibility, low immunogenicity, and ease of production, storage, and transport, making them great interest for various genome editing applications with clinical potentials.},
}
@article {pmid35115715,
year = {2022},
author = {Priyadarshini, M and Ni, JZ and Vargas-Velazquez, AM and Gu, SG and Frøkjær-Jensen, C},
title = {Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C. elegans.},
journal = {Nature methods},
volume = {19},
number = {2},
pages = {187-194},
pmid = {35115715},
issn = {1548-7105},
support = {R01 GM111752/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Argonaute Proteins/genetics ; Caenorhabditis elegans/embryology/*genetics ; Caenorhabditis elegans Proteins/genetics ; Cell Cycle Proteins/genetics ; Embryo, Nonmammalian ; Epigenesis, Genetic ; Female ; *Gene Silencing ; Male ; *RNA Interference ; RNA, Small Interfering/*genetics ; },
abstract = {Single-guide RNAs can target exogenous CRISPR-Cas proteins to unique DNA locations, enabling genetic tools that are efficient, specific and scalable. Here we show that short synthetic guide Piwi-interacting RNAs (piRNAs) (21-nucleotide sg-piRNAs) expressed from extrachromosomal transgenes can, analogously, reprogram the endogenous piRNA pathway for gene-specific silencing in the hermaphrodite germline, sperm and embryos of Caenorhabditis elegans. piRNA-mediated interference ('piRNAi') is more efficient than RNAi and can be multiplexed, and auxin-mediated degradation of the piRNA-specific Argonaute PRG-1 allows conditional gene silencing. Target-specific silencing results in decreased messenger RNA levels, amplification of secondary small interfering RNAs and repressive chromatin modifications. Short (300 base pairs) piRNAi transgenes amplified from arrayed oligonucleotide pools also induce silencing, potentially making piRNAi highly scalable. We show that piRNAi can induce transgenerational epigenetic silencing of two endogenous genes (him-5 and him-8). Silencing is inherited for four to six generations after target-specific sg-piRNAs are lost, whereas depleting PRG-1 leads to essentially permanent epigenetic silencing.},
}
@article {pmid35115506,
year = {2022},
author = {Rottinghaus, AG and Ferreiro, A and Fishbein, SRS and Dantas, G and Moon, TS},
title = {Genetically stable CRISPR-based kill switches for engineered microbes.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {672},
pmid = {35115506},
issn = {2041-1723},
support = {R01 AT009741/AT/NCCIH NIH HHS/United States ; },
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/*genetics ; Escherichia coli/*genetics/metabolism/physiology ; Female ; Gene Expression Regulation/drug effects ; Genetic Engineering/*methods ; Mice, Inbred C57BL ; Microbial Viability/drug effects/genetics ; Probiotics/*metabolism/pharmacology ; SOS Response, Genetics/drug effects/genetics ; Streptomycin/pharmacology ; Temperature ; Tetracyclines/pharmacology ; },
abstract = {Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development.},
}
@article {pmid35115009,
year = {2022},
author = {Sentmanat, MF and White, JM and Kouranova, E and Cui, X},
title = {Highly reliable creation of floxed alleles by electroporating single-cell embryos.},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {31},
pmid = {35115009},
issn = {1741-7007},
support = {P01 DK096990/DK/NIDDK NIH HHS/United States ; P30 AR073752/AR/NIAMS NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Mice ; Mice, Knockout ; *RNA, Guide/genetics ; Zygote ; },
abstract = {BACKGROUND: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking.<br><br>RESULTS: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448&#x2009;bp to 160&#x2009;kb reported here).<br><br>CONCLUSIONS: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.},
}
@article {pmid35114359,
year = {2022},
author = {Lee, EG and Kwak, JS and Kim, KH},
title = {CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus).},
journal = {Fish &amp; shellfish immunology},
volume = {122},
number = {},
pages = {98-105},
doi = {10.1016/j.fsi.2022.01.040},
pmid = {35114359},
issn = {1095-9947},
mesh = {Animals ; Bacterial Vaccines ; CRISPR-Cas Systems ; *Edwardsiella/genetics ; *Enterobacteriaceae Infections/prevention &amp; control/veterinary ; *Fish Diseases/prevention &amp; control ; *Flounder ; Immunization ; },
abstract = {Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method. Under the optimal CRISPR engineering condition, we could efficiently produce either alr or asd gene knockout E. piscicida auxotrophic mutants, and this genome editing process was much simpler and faster than the allelic exchange method. The simultaneous knockout of double auxotrophic genes (alr and asd) and the insertion of a foreign gene expression cassette in E. piscicida chromosome were also successfully performed using the established CRISPR/Cas9 system. Furthermore, to enhance the possibility to get permission as a commercial vaccine, we produced an auxotrophic E. piscicida mutant having only one nucleotide-deleted alr gene (E. piscicida △alr-1). Olive flounder (Paralichthys olivaceus) fingerlings immunized with 1 × 10[6] and 1 × 10[5] CFU/fish of E. piscicida △alr-1 showed the superior ability in the induction of serum agglutination activity and in the protection against E. piscicida compared to killed E. piscicida. However, olive flounder immunized with 1 × 10[7] CFU/fish of E. piscicida △alr-1 showed high mortality far before the challenge, and the isolated E. piscicida from moribund and dead fish had the wild type alr gene, suggesting the reversion of one base-deleted alr gene to original form by a second mutation in olive flounder. Therefore, investigation on the minimum number of edited nucleotide for stable maintenance of E. piscicida mutants should be further conducted.},
}
@article {pmid35113911,
year = {2022},
author = {Aslam, S and Khan, SH and Ahmad, A and Walawage, SL and Dandekar, AM},
title = {Founder transformants of cotton (Gossypium hirsutum L.) obtained through the introduction of DS-Red, Rec, Rep and CRISPR/Cas9 expressing constructs for developing base lines of recombinase mediated gene stacking.},
journal = {PloS one},
volume = {17},
number = {2},
pages = {e0263219},
pmid = {35113911},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting/*methods ; *Genome, Plant ; Gossypium/*genetics/growth &amp; development ; Mutagenesis ; Plants, Genetically Modified/*genetics/growth &amp; development ; Recombinases/genetics/*metabolism ; },
abstract = {Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.},
}
@article {pmid35113687,
year = {2022},
author = {Schmidt, R and Steinhart, Z and Layeghi, M and Freimer, JW and Bueno, R and Nguyen, VQ and Blaeschke, F and Ye, CJ and Marson, A},
title = {CRISPR activation and interference screens decode stimulation responses in primary human T cells.},
journal = {Science (New York, N.Y.)},
volume = {375},
number = {6580},
pages = {eabj4008},
pmid = {35113687},
issn = {1095-9203},
support = {R01 HG008140/HG/NHGRI NIH HHS/United States ; S10 OD021822/OD/NIH HHS/United States ; S10 RR028962/RR/NCRR NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Gene Expression Regulation ; *Gene Regulatory Networks ; Genome, Human ; Humans ; Interferon-gamma/*biosynthesis/genetics ; Interleukin-2/*biosynthesis/genetics ; *Lymphocyte Activation ; NF-kappa B/metabolism ; RNA-Seq ; Signal Transduction ; Single-Cell Analysis ; T-Lymphocytes/*immunology/metabolism ; },
abstract = {Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.},
}
@article {pmid35110559,
year = {2022},
author = {Nowakowska, MB and Selby, K and Przykopanski, A and Krüger, M and Krez, N and Dorner, BG and Dorner, MB and Jin, R and Minton, NP and Rummel, A and Lindström, M},
title = {Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {1790},
pmid = {35110559},
issn = {2045-2322},
support = {R01 AI125704/AI/NIAID NIH HHS/United States ; },
mesh = {Botulinum Toxins/*biosynthesis/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clostridium botulinum/genetics/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genotype ; Phenotype ; Point Mutation ; },
abstract = {Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.},
}
@article {pmid35110541,
year = {2022},
author = {Höijer, I and Emmanouilidou, A and Östlund, R and van Schendel, R and Bozorgpana, S and Tijsterman, M and Feuk, L and Gyllensten, U and den Hoed, M and Ameur, A},
title = {CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {627},
pmid = {35110541},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA ; Gene Editing/*methods ; Genetic Therapy ; Germ Cells ; Humans ; Mutation ; RNA, Guide/genetics ; Zebrafish/*genetics ; },
abstract = {CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions ≥50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.},
}
@article {pmid35110534,
year = {2022},
author = {Zhao, B and Rao, Y and Leighow, S and O'Brien, EP and Gilbert, L and Pritchard, JR},
title = {A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {625},
pmid = {35110534},
issn = {2041-1723},
support = {DP2 CA239597/CA/NCI NIH HHS/United States ; R21 EB026617/EB/NIBIB NIH HHS/United States ; R35 GM124818/GM/NIGMS NIH HHS/United States ; U01 CA265709/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; *Genetic Engineering ; Genomics ; Humans ; *Machine Learning ; Synthetic Lethal Mutations ; },
abstract = {A genetic knockout can be lethal to one human cell type while increasing growth rate in another. This context specificity confounds genetic analysis and prevents reproducible genome engineering. Genome-wide CRISPR compendia across most common human cell lines offer the largest opportunity to understand the biology of cell specificity. The prevailing viewpoint, synthetic lethality, occurs when a genetic alteration creates a unique CRISPR dependency. Here, we use machine learning for an unbiased investigation of cell type specificity. Quantifying model accuracy, we find that most cell type specific phenotypes are predicted by the function of related genes of wild-type sequence, not synthetic lethal relationships. These models then identify unexpected sets of 100-300 genes where reduced CRISPR measurements can produce genome-scale loss-of-function predictions across >18,000 genes. Thus, it is possible to reduce in vitro CRISPR libraries by orders of magnitude-with some information loss-when we remove redundant genes and not redundant sgRNAs.},
}
@article {pmid35110402,
year = {2022},
author = {Suhail, Y and Maziarz, JD and Novin, A and Dighe, A and Afzal, J and Wagner, G and Kshitiz, },
title = {Tracing the cis-regulatory changes underlying the endometrial control of placental invasion.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {6},
pages = {},
pmid = {35110402},
issn = {1091-6490},
support = {R37 CA248161/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Endometrium/*metabolism/pathology ; Female ; GATA2 Transcription Factor/genetics/metabolism ; Gene Knockout Techniques ; Humans ; Neoplasm Proteins/genetics/metabolism ; Neoplasms/genetics/metabolism ; Transcription Factor DP1/metabolism ; Trophoblasts/*metabolism/pathology ; },
abstract = {Among eutherian (placental) mammals, placental embedding into the maternal endometrium exhibits great differences, from being deeply invasive (e.g., humans) to noninvasive (e.g., cattle). The degree of invasion of placental trophoblasts is positively correlated with the rate of cancer malignancy. Previously, we have shown that fibroblasts from different species offer different levels of resistance to the invading trophoblasts as well as to cancer cell invasion. Here we present a comparative genomic investigation revealing cis-regulatory elements underlying these interspecies differences in invasibility. We identify transcription factors that regulate proinvasibility and antiinvasibility genes in stromal cells. Using an in vitro invasibility assay combined with CRISPR-Cas9 gene knockout, we found that the transcription factors GATA2 and TFDP1 strongly influence the invasibility of endometrial and skin fibroblasts. This work identifies genomic mechanisms explaining species differences in stromal invasibility, paving the way to therapies targeting stromal characteristics to regulate placental invasion, wound healing, and cancer dissemination.},
}
@article {pmid35107377,
year = {2022},
author = {Zhang, Y and Li, W and Tang, N and Moffat, K and Nair, V and Yao, Y},
title = {Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek's Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1.},
journal = {Journal of virology},
volume = {96},
number = {6},
pages = {e0202721},
pmid = {35107377},
issn = {1098-5514},
support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/OS/NW/000007/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Chick Embryo ; Chickens ; *Herpesvirus 3, Gallid/genetics ; *Lymphoma/veterinary/virology ; *Marek Disease ; *Viral Envelope Proteins/genetics ; },
abstract = {Marek's disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek's disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. IMPORTANCE Marek's disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.},
}
@article {pmid35107356,
year = {2022},
author = {Chua, MJ and Collins, J},
title = {Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a.},
journal = {Microbiology spectrum},
volume = {10},
number = {1},
pages = {e0242721},
pmid = {35107356},
issn = {2165-0497},
support = {P20 GM125504/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Enterococcus faecium/*genetics/metabolism ; Gene Editing/economics/*methods ; Genome, Bacterial ; Mutagenesis, Insertional ; Plasmids/genetics/metabolism ; Sequence Deletion ; },
abstract = {Considered a serious threat by the Centers for Disease Control and Prevention, multidrug-resistant Enterococcus faecium is an increasing cause of hospital-acquired infection. Here, we provide details on a single-plasmid CRISPR-Cas12a system for generating clean deletions and insertions. Single manipulations were carried out in under 2 weeks, with successful deletions/insertions present in >80% of the clones tested. Using this method, we generated three individual clean deletion mutations in the acpH, treA, and lacL genes and inserted codon-optimized unaG, enabling green fluorescent protein (GFP)-like fluorescence under the control of the trehalase operon. The use of in vivo recombination for plasmid construction kept costs to a minimum. IMPORTANCE Enterococcus faecium is increasingly associated with hard-to-treat antibiotic-resistant infections. The ability to generate clean genomic alterations is the first step in generating a complete mechanistic understanding of how E. faecium acquires pathogenic traits and causes disease. Here, we show that CRISPR-Cas12a can be used to quickly (under 2 weeks) and cheaply delete or insert genes into the E. faecium genome. This substantial improvement over current methods should speed up research on this important opportunistic pathogen.},
}
@article {pmid35105899,
year = {2022},
author = {Liu, Z and Chen, S and Lai, L and Li, Z},
title = {Inhibition of base editors with anti-deaminases derived from viruses.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {597},
pmid = {35105899},
issn = {2041-1723},
mesh = {APOBEC Deaminases ; CRISPR-Cas Systems ; Cytidine Deaminase/*genetics/*metabolism ; Cytosine ; DNA ; Deoxyribonuclease I ; Gene Editing ; Genetic Therapy ; HEK293 Cells ; Humans ; Viruses/*genetics/*metabolism ; },
abstract = {Cytosine base editors (CBEs), combining cytidine deaminases with the Cas9 nickase (nCas9), enable targeted C-to-T conversions in genomic DNA and are powerful genome-editing tools used in biotechnology and medicine. However, the overexpression of cytidine deaminases in vivo leads to unexpected potential safety risks, such as Cas9-independent off-target effects. This risk makes the development of deaminase off switches for modulating CBE activity an urgent need. Here, we report the repurpose of four virus-derived anti-deaminases (Ades) that efficiently inhibit APOBEC3 deaminase-CBEs. We demonstrate that they antagonize CBEs by inhibiting the APOBEC3 catalytic domain, relocating the deaminases to the extranuclear region or degrading the whole CBE complex. By rationally engineering the deaminase domain, other frequently used base editors, such as CGBE, A&amp;CBE, A&amp;CGBE, rA1-CBE and ABE8e, can be moderately inhibited by Ades, expanding the scope of their applications. As a proof of concept, the Ades in this study dramatically decrease both Cas9-dependent and Cas9-independent off-target effects of CBEs better than traditional anti-CRISPRs (Acrs). Finally, we report the creation of a cell type-specific CBE-ON switch based on a microRNA-responsive Ade vector, showing its practicality. In summary, these natural deaminase-specific Ades are tools that can be used to regulate the genome-engineering functions of BEs.},
}
@article {pmid35104875,
year = {2022},
author = {Lin-Shiao, E and Pfeifer, WG and Shy, BR and Saffari Doost, M and Chen, E and Vykunta, VS and Hamilton, JR and Stahl, EC and Lopez, DM and Sandoval Espinoza, CR and Deyanov, AE and Lew, RJ and Poirer, MG and Marson, A and Castro, CE and Doudna, JA},
title = {CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.},
journal = {Nucleic acids research},
volume = {50},
number = {3},
pages = {1256-1268},
pmid = {35104875},
issn = {1362-4962},
support = {S10 OD025096/OD/NIH HHS/United States ; F32 GM140637/GM/NIGMS NIH HHS/United States ; P01 AI138962/AI/NIAID NIH HHS/United States ; F32 GM142146/GM/NIGMS NIH HHS/United States ; },
mesh = {Active Transport, Cell Nucleus ; CRISPR-Cas Systems ; DNA/genetics ; Gene Editing/methods ; *Gene Transfer Techniques ; Genome ; Humans ; *Nanostructures ; },
abstract = {DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.},
}
@article {pmid35104106,
year = {2022},
author = {Ma, S and Su, T and Liu, J and Lu, X and Qi, Q},
title = {Reduction of the Bacterial Genome by Transposon-Mediated Random Deletion.},
journal = {ACS synthetic biology},
volume = {11},
number = {2},
pages = {668-677},
doi = {10.1021/acssynbio.1c00353},
pmid = {35104106},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Gene Library ; *Genome, Bacterial/genetics ; Genomics ; Synthetic Biology ; },
abstract = {Genome reduction is an important strategy in synthetic biology for constructing functional chassis cells or minimal genomes. However, the limited knowledge of complex gene functions and interactions makes genome reduction by rational design encounter a bottleneck. Here, we present an iterative and random genome reduction method for Escherichia coli, named "transposon-mediated random deletion (TMRD)". TMRD generates random double-strand breaks (DSBs) in the genome by combining Tn5 transposition with the CRISPR/Cas9 system and allows genomic deletions of various sizes at random positions during DSB repair through the intracellular alternative end-joining mechanism. Using E. coli MG1655 as the original strain, a pool of cells with multiple random genomic deletions were obtained after five reduction cycles. The growth rates of the obtained cells were comparable to that of MG1655, while the electroporation efficiency increased by at least 2 magnitudes. TMRD can generate a small E. coli library carrying multiple and random genomic deletions while enriching the cells with environmental fitness in the population. TMRD has the potential to be widely applied in the construction of minimal genomes or chassis cells for metabolic engineering.},
}
@article {pmid35103962,
year = {2022},
author = {Smith, J and Banerjee, R and Weeks, RJ and Chatterjee, A},
title = {Editing of DNA Methylation Patterns Using CRISPR-Based Tools.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2458},
number = {},
pages = {63-74},
pmid = {35103962},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *DNA Methylation ; Epigenesis, Genetic ; Epigenomics/methods ; *Gene Editing/methods ; Mammals/genetics ; },
abstract = {DNA methylation is an epigenetic modification with an established role in both normal cellular function and mammalian disease. Despite well-characterized associations between aberrant DNA methylation changes and gene expression, evidence for a causal relationship in this context has been difficult to obtain. Early techniques for interrogating the role of DNA methylation in the regulation of gene transcription lack specificity and, where more specific techniques such and ZNFs and TALEs have been developed, they are limited by their extensive cost and labor requirements. However, the recent advent of CRISPR-based technologies has revolutionized our potential for site-specific epigenomic editing. Here, we provide a detailed protocol for the design, construction, and utilization of a transient, CRISPR-based DNA methylation-editing system in mammalian cells.},
}
@article {pmid35103261,
year = {2022},
author = {Lin, Y and Wagner, E and Lächelt, U},
title = {Non-viral delivery of the CRISPR/Cas system: DNA versus RNA versus RNP.},
journal = {Biomaterials science},
volume = {10},
number = {5},
pages = {1166-1192},
doi = {10.1039/d1bm01658j},
pmid = {35103261},
issn = {2047-4849},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Gene Editing/methods ; Humans ; *RNA/genetics ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Since its discovery, the CRISPR/Cas technology has rapidly become an essential tool in modern biomedical research. The opportunities to specifically modify and correct genomic DNA have also raised big hope for therapeutic applications by direct in vivo genome editing. In order to achieve the intended genome modifications, the functional unit of the CRISPR/Cas system finally has to be present in the nucleus of target cells. This can be achieved by delivery of different biomolecular Cas9 and gRNA formats: plasmid DNA (pDNA), RNA or Cas9 ribonucleoproteins (RNPs). While the initial research focussed on pDNA transfections, the currently most promising strategy for systemic non-viral in vivo delivery is based on RNA which has achieved remarkable results in the first clinical trials. RNP delivery receives much attention for ex vivo applications, but the translation to systemic in vivo genome editing in patients has not been reached so far. The article summarises the characteristics and differences of each format, provides an overview of the published delivery strategies and highlights recent examples of delivery systems including the status of clinical applications.},
}
@article {pmid35102294,
year = {2022},
author = {Crunkhorn, S},
title = {Enhancing base editor delivery.},
journal = {Nature reviews. Drug discovery},
volume = {21},
number = {3},
pages = {177},
doi = {10.1038/d41573-022-00021-5},
pmid = {35102294},
issn = {1474-1784},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid35101771,
year = {2022},
author = {Carrasco-Padilla, C and Roda-Navarro, P},
title = {CRISPR/Cas9-mediated genome editing assists protein dynamics studies in live cells.},
journal = {European journal of cell biology},
volume = {101},
number = {2},
pages = {151203},
doi = {10.1016/j.ejcb.2022.151203},
pmid = {35101771},
issn = {1618-1298},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {Spatial and temporal regulation of molecular reactions dictates cell fate. Thus, studying molecular dynamics is essential to understand how cells decide what to do and the fundamental perturbations causing disease. Classically, molecular dynamics has been studied by protocols based in the overexpression of fluorescent fusion proteins. However, overexpression is associated to altered stoichiometry, molecular dynamics and subcellular distribution. We here discuss the necessity to study molecular dynamics of fluorescent fusion proteins expressed under physiological mechanisms in the cell, pointing to CRISPR/Cas9-mediated genome editing as the ideal means to do so. Current genome editing protocols enable us to study molecular dynamics while avoiding drawbacks associated to overexpression.},
}
@article {pmid35101371,
year = {2022},
author = {Katayama, T and Maruyama, JI},
title = {CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae.},
journal = {Journal of bioscience and bioengineering},
volume = {133},
number = {4},
pages = {353-361},
doi = {10.1016/j.jbiosc.2021.12.017},
pmid = {35101371},
issn = {1347-4421},
mesh = {Aspergillus ; *Aspergillus oryzae/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Deletion ; Gene Editing/methods ; Mutagenesis ; },
abstract = {In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A. oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A. sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A. oryzae and A. sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A. oryzae and the AswA gene in A. sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A. oryzae and as a new approach for efficient genetic engineering of A. sojae.},
}
@article {pmid35100423,
year = {2022},
author = {Xiong, W and Liu, X and Qi, Q and Ji, H and Liu, F and Zhong, C and Liu, S and Tian, T and Zhou, X},
title = {Supramolecular CRISPR-OFF switches with host-guest chemistry.},
journal = {Nucleic acids research},
volume = {50},
number = {3},
pages = {1241-1255},
pmid = {35100423},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) technology is a powerful tool in biology and medicine. However, the safety and application of this technology is hampered by excessive activity of CRISPR machinery. It is particularly important to develop methods for switching off CRISPR activity in human cells. The current study demonstrates the concept of supramolecular CRISPR-OFF switches by employing host-guest chemistry. We demonstrate that the CRISPR systems show considerable tolerance to adamantoylation on guide RNAs (gRNAs), whereas supramolecular complexation tremendously affects the function of adamantoyl gRNAs. Host-guest chemistry is demonstrated to be novel and effective tools to reduce unwanted excessive activities of CRISPR complexes in human cells. This work indicates considerable potential of supramolecular strategy for controlling and enhancing CRISPR systems.},
}
@article {pmid35099925,
year = {2022},
author = {Ma, Y and Gao, W and Zhang, Y and Yang, M and Yan, X and Zhang, Y and Li, G and Liu, C and Xu, C and Zhang, M},
title = {Biomimetic MOF Nanoparticles Delivery of C-Dot Nanozyme and CRISPR/Cas9 System for Site-Specific Treatment of Ulcerative Colitis.},
journal = {ACS applied materials &amp; interfaces},
volume = {14},
number = {5},
pages = {6358-6369},
doi = {10.1021/acsami.1c21700},
pmid = {35099925},
issn = {1944-8252},
mesh = {Animals ; Biomimetic Materials/*chemistry ; CRISPR-Cas Systems/*genetics ; Carbon/chemistry ; Colitis, Ulcerative/chemically induced/drug therapy ; Colon/pathology ; Dextran Sulfate/toxicity ; Disease Models, Animal ; Drug Carriers/chemistry ; Female ; Fusion Regulatory Protein-1/genetics/metabolism ; Humans ; Imidazoles/*chemistry ; Metal-Organic Frameworks/*chemistry ; Mice ; Mice, Inbred C57BL ; Nanoparticles/*chemistry ; Plasmids/genetics/metabolism ; Quantum Dots/*chemistry ; Reactive Oxygen Species/metabolism ; Superoxide Dismutase/*chemistry/therapeutic use ; },
abstract = {Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) of unknown etiology affecting the colon and rectum. Previous studies have found that reactive oxygen species (ROS) overproduction and transmembrane glycoprotein CD98 (encoded by SLC3A2) upregulation played important roles in the initiation and progression of UC. On the basis of this, a biomimetic pH-responsive metal organic framework (MOF) carrier was constructed to deliver carbon nanodot-SOD nanozyme and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system for site-specific treatment of UC. In this system, carbon nanodots (C-dots) and CD98 CRISPR/Cas9 plasmid were successfully encapsulated into MOF carrier (ZIF-8 nanoparticles) by a one-pot approach (formed as CCZ), and then camouflaged with macrophage membrane (formed as CCZM). It was worth noting that the C-dot nanozyme showed excellent superoxide dismutase (SOD) enzymatic activity, which could scavenge ROS effectively. As expected, this biomimetic system exhibited pH-responsive, immune escape, and inflammation targeting capability simultaneously. In vitro experiments showed that ROS was significantly eliminated, and CD98 was downregulated by CCZM. In the dextran sulfate sodium salt (DSS)-induced UC model, administration of CCZM significantly ameliorated the inflammation symptoms of mice, including the colon length and pathological parameters such as epithelium integrity and inflammation infiltration. In addition, both in vitro and in vivo results demonstrated that biomimetic nanoparticles effectively reduced the expression of pro-inflammatory cytokines. Overall, this study would provide a promising approach for the precise treatment of UC.},
}
@article {pmid35099818,
year = {2022},
author = {Nazir, R and Mandal, S and Mitra, S and Ghorai, M and Das, N and Jha, NK and Majumder, M and Pandey, DK and Dey, A},
title = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genome-editing toolkit to enhance salt stress tolerance in rice and wheat.},
journal = {Physiologia plantarum},
volume = {174},
number = {2},
pages = {e13642},
doi = {10.1111/ppl.13642},
pmid = {35099818},
issn = {1399-3054},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Edible Grain/genetics ; Genome, Plant/genetics ; *Oryza/genetics ; Salt Tolerance/genetics ; Triticum/genetics ; },
abstract = {The rice and wheat agricultural system is the primary source of food for billions across the world. However, the productivity and long-term sustainability of rice and wheat are threatened by a large number of abiotic stresses, especially salinity stress. Salinity has a significant impact on plant development and productivity and is one of the leading causes of crop yield losses in agricultural soils worldwide. Over the last few decades, several attempts have been undertaken to enhance salinity stress tolerance, most of which have relied on traditional or molecular breeding approaches. These approaches have so far been insufficient in addressing the issues of abiotic stress. However, due to the availability of genome sequences for cereal crops like rice and wheat and the development of genome editing techniques like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9), it is now possible to "edit" genes and influence key traits. Here, we review the application of the CRISPR/Cas9 system in both rice (Oryza sativa L.) and wheat (Triticum aestivum L.) to develop salinity tolerant cultivars. The CRISPR/Cas genome editing toolkit holds great promise of producing cereal crops tolerant to salt stress to increase agriculture resilience with a strong impact on the environment and public health.},
}
@article {pmid35099553,
year = {2022},
author = {Wada, N and Osakabe, K and Osakabe, Y},
title = {Expanding the plant genome editing toolbox with recently developed CRISPR-Cas systems.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1825-1837},
pmid = {35099553},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Plant/genetics ; Humans ; Plants/genetics ; },
abstract = {Since its first appearance, CRISPR-Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR-Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR-Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR-Cas systems have been reported. Among them, several CRISPR-Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR-Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR-Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR-Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR-Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR-Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR-Cas tools are helping expand our plant genome engineering toolbox.},
}
@article {pmid35099480,
year = {2022},
author = {Li, J and Luo, T and He, Y and Liu, H and Deng, Z and Bu, J and Long, X and Zhong, S and Yang, Y},
title = {Discovery of the Rnase activity of CRISPR-Cas12a and its distinguishing cleavage efficiency on various substrates.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {15},
pages = {2540-2543},
doi = {10.1039/d1cc06295f},
pmid = {35099480},
issn = {1364-548X},
mesh = {CRISPR-Cas Systems/genetics ; Ribonucleases/genetics/*metabolism ; },
abstract = {We, herein, indicated for the first time the Rnase activities of LbCas12a on linear ssRNA above 11 bases, and hairpin RNA substrates. Meanwhile, the LbCas12a bound to ssDNA or ssRNA exhibited different cleavage efficiencies on various substrates, including short ssDNA, hairpin DNA, linear ssRNA and hairpin RNA. With hairpin DNA as a reporter, we attained a detection limit of 5 pM and 50 pM for the ssDNA and ssRNA targets, respectively. We believe that these findings will pave a new avenue for expanding the reporter toolbox for Cas12a-based diagnostics in biosensing and biochemistry.},
}
@article {pmid35099280,
year = {2022},
author = {Boutin, J and Cappellen, D and Rosier, J and Amintas, S and Dabernat, S and Bedel, A and Moreau-Gaudry, F},
title = {ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {19-30},
doi = {10.1089/crispr.2021.0120},
pmid = {35099280},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Endonucleases/genetics ; *Gene Editing ; Genomics ; },
abstract = {CRISPR-Cas9 is a highly promising technology for clinical development. However, this powerful tool can induce adverse genomic events. The off-target genotoxicity is well described, predictable, detectable, and resolved by the use of new generations of Cas9 nucleases with high fidelity. In contrast, the ON-target genotoxicity due to a DNA double-strand break at the targeted locus is still underestimated. Here, we review several genomic outcomes induced by CRISPR-Cas9 from the insertion/deletion of a few bases to megabase-scale rearrangements. We hope to highlight this barely detectable complex safety concern to promote further studies to understand the mechanisms better, to detect these unwanted events, and to prevent them for the safety management of future CRISPR-Cas9 clinical trials.},
}
@article {pmid35099270,
year = {2022},
author = {Bachmann, L and Gallego Villarejo, L and Heinen, N and Marks, D and Peters, M and Müller, T},
title = {Gene-Edited Fluorescent and Mixed Cerebral Organoids.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {53-65},
doi = {10.1089/crispr.2021.0070},
pmid = {35099270},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Organoids/metabolism ; },
abstract = {Cerebral organoids are a promising model to study human brain function and disease, although the high inter-organoid variability is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combining gene editing and organoid differentiation offers a unique tool to study gene function in a complex human three-dimensional model. Using a CRISPR-Cas9 gene-editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs, and mixtures of differently edited cells were seeded to induce cerebral organoid differentiation. Consequently, the development of the organoids was detectable by live confocal fluorescence microscopy of whole organoids and immunofluorescence staining in fixed samples. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, thus overcoming the inter-organoid inhomogeneity limitations. Furthermore, the approach enables mosaic analysis of mutant clones in a wild-type three-dimensional cellular environment. It paves the way for the reliable analysis of human genetic disorders using organoids and the gain of fundamental understanding of the molecular mechanisms underlying pathological conditions.},
}
@article {pmid35099107,
year = {2022},
author = {Gomaa, F and Li, ZH and Beaudoin, DJ and Alzan, H and Girguis, PR and Docampo, R and Edgcomb, VP},
title = {CRISPR/Cas9-induced disruption of Bodo saltans paraflagellar rod-2 gene reveals its importance for cell survival.},
journal = {Environmental microbiology},
volume = {24},
number = {7},
pages = {3051-3062},
pmid = {35099107},
issn = {1462-2920},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Survival ; DNA ; *Gene Editing ; Homologous Recombination ; },
abstract = {Developing transfection protocols for marine protists is an emerging field that will allow the functional characterization of protist genes and their roles in organism responses to the environment. We developed a CRISPR/Cas9 editing protocol for Bodo saltans, a free-living kinetoplastid with tolerance to both marine and freshwater conditions and a close non-parasitic relative of trypanosomatids. Our results show that SaCas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex-mediated disruption of the paraflagellar rod 2 gene (BsPFR2) was achieved using electroporation-mediated transfection. The use of CRISPR/Cas9 genome editing can increase the efficiency of targeted homologous recombination when a repair DNA template is provided. Our sequence analysis suggests two mechanisms for repairing double-strand breaks in B. saltans are active; homologous-directed repair (HDR) utilizing an exogenous DNA template that carries an antibiotic resistance gene and likley non-homologous end joining (NHEJ). However, HDR was only achieved when a single (vs. multiple) SaCas9 RNP complex was provided. Furthermore, the biallelic knockout of BsPFR2 was detrimental for the cell, highlighting its essential role for cell survival because it facilitates the movement of food particles into the cytostome. Our Cas9/sgRNA RNP complex protocol provides a new tool for assessing gene functions in B. saltans and perhaps similar protists with polycistronic transcription.},
}
@article {pmid35098362,
year = {2022},
author = {Wang, S and Li, Y and Zhong, L and Wu, K and Zhang, R and Kang, T and Wu, S and Wu, Y},
title = {Efficient gene editing through an intronic selection marker in cells.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {2},
pages = {111},
pmid = {35098362},
issn = {1420-9071},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; DNA/genetics/metabolism ; *DNA Breaks, Double-Stranded ; DNA Repair/*genetics ; Exons/genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Introns/*genetics ; Luminescent Proteins/*genetics/metabolism ; Microscopy, Fluorescence ; Mutation ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factor RelA/genetics ; },
abstract = {BACKGROUND: Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. However, despite the great importance and universality of gene editing, the efficiency of homology-directed DNA repair (HDR) is too low, and base editors (BEs) cannot accomplish desired indel editing tasks.<br><br>RESULTS AND DISCUSSION: Our group has improved HDR gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves a DsRed positive rate as high as 87.5% after two rounds of fluorescence-activated cell sorter (FACS) selection without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and efficiency, and our data suggest that GEIS has the potential to edit approximately 97% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.},
}
@article {pmid35098208,
year = {2021},
author = {Banakar, R and Schubert, M and Kurgan, G and Rai, KM and Beaudoin, SF and Collingwood, MA and Vakulskas, CA and Wang, K and Zhang, F},
title = {Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {760820},
pmid = {35098208},
issn = {2673-3439},
abstract = {Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7-41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0-85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.},
}
@article {pmid35095097,
year = {2022},
author = {Lv, J and Xi, H and Lv, X and Zhou, Y and Wang, J and Chen, H and Yan, T and Jin, J and Zhao, J and Gu, F and Song, Z},
title = {Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?.},
journal = {Gene therapy},
volume = {29},
number = {7-8},
pages = {458-463},
pmid = {35095097},
issn = {1476-5462},
mesh = {CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing/methods ; Genetic Therapy ; Genome, Human ; Humans ; *Staphylococcus aureus/genetics ; },
abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.},
}
@article {pmid35094325,
year = {2022},
author = {Gardner, A and Morgan, D and Al'Khafaji, A and Brock, A},
title = {Functionalized Lineage Tracing for the Study and Manipulation of Heterogeneous Cell Populations.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2394},
number = {},
pages = {109-131},
pmid = {35094325},
issn = {1940-6029},
support = {R21 CA212928/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cell Lineage/genetics ; Genes, Reporter ; *RNA, Guide/genetics ; },
abstract = {The ability to track and isolate unique cell lineages from large heterogeneous populations increases the resolution at which cellular processes can be understood under normal and pathogenic states beyond snapshots obtained from single-cell RNA sequencing (scRNA-seq). Here, we describe the Control of Lineages by Barcode Enabled Recombinant Transcription (COLBERT) method in which unique single guide RNA (sgRNA) barcodes are used as functional tags to identify and recall specific lineages of interest. An sgRNA barcode is stably integrated and actively transcribed, such that all cellular progeny will contain the parental barcode and produce a functional sgRNA. The sgRNA barcode has all the benefits of a DNA barcode and added functionalities. Once a barcode pertaining to a lineage of interest is identified, the lineage of interest can be isolated using an activator variant of Cas9 (such as dCas9-VPR) and a barcode-matched sequence upstream of a fluorescent reporter gene. CRISPR activation of the fluorescent reporter will only occur in cells producing the matched sgRNA barcode, allowing precise identification and isolation of lineages of interest from heterogeneous populations.},
}
@article {pmid35093717,
year = {2022},
author = {Gizon, M and Duboscq-Bidot, L and El Kassar, L and Bobin, P and Ader, F and Giraud-Triboult, K and Charron, P and Villard, E and Fontaine, V and Neyroud, N},
title = {Generation of a heterozygous SCN5A knockout human induced pluripotent stem cell line by CRISPR/Cas9 edition.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102680},
doi = {10.1016/j.scr.2022.102680},
pmid = {35093717},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Myocytes, Cardiac/metabolism ; NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism ; },
abstract = {Mutations leading to haploinsufficiency in SCN5A, the gene encoding the cardiac sodium channel Nav1.5 α-subunit, are involved in life-threatening cardiac disorders. Using CRISPR/Cas9-mediated genome edition, we generated here a human induced-pluripotent stem cell (hiPSC) line carrying a heterozygous mutation in exon 2 of SCN5A, which leads to apparition of a premature stop codon. SCN5A-clone 5 line maintained normal karyotype, morphology and pluripotency and differentiated into three germ layers. Cardiomyocytes derived from these hiPSCs would be a useful model for investigating channelopathies related to SCN5A heterozygous deficiency.},
}
@article {pmid35093620,
year = {2022},
author = {Wei, B and Wang, J and Cadang, L and Goyon, A and Chen, B and Yang, F and Zhang, K},
title = {Development of an ion pairing reversed-phase liquid chromatography-mass spectrometry method for characterization of clustered regularly interspaced short palindromic repeats guide ribonucleic acid.},
journal = {Journal of chromatography. A},
volume = {1665},
number = {},
pages = {462839},
doi = {10.1016/j.chroma.2022.462839},
pmid = {35093620},
issn = {1873-3778},
mesh = {*CRISPR-Cas Systems ; *Chromatography, Reverse-Phase ; Mass Spectrometry ; RNA ; RNA, Guide ; },
abstract = {Guide ribonucleic acid (gRNA) is a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. The single stranded guide RNA (sgRNA) is the most commonly used gRNA in application. Evaluation of the impurity profile of synthetic sgRNA is important for any CRISPR genome editing experiments. However, the large molecular size, complex impurity profile and unique secondary structure pose many challenges in the analysis of sgRNA by ion pairing reversed-phase liquid chromatography (IP-RPLC), the commonly used method. In this work, we developed a generic IP-RPLC method for guide RNA analysis. We found that large pore size of stationary phase was the most critical column parameter to achieve high resolution separation of sgRNA while particle structure, particle size and surface chemistry had less impact. Our results indicated that charge interaction was the most critical mechanism for retention and mass transfer had less impact on the performance of separation. An IP-RPLC/mass spectrometry (MS) method was also developed with a specific practice to reduce adducts and enable intact MS analysis of sgRNAs. The generic IP-RPLC method demonstrates its feasibility to serve as a release, stability, characterization and in-process control testing method for synthetic sgRNA products.},
}
@article {pmid35093594,
year = {2022},
author = {Wang, K and Zhou, H and Qian, Q},
title = {The rice codebook: From reading to editing.},
journal = {Molecular plant},
volume = {15},
number = {4},
pages = {569-572},
doi = {10.1016/j.molp.2022.01.017},
pmid = {35093594},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Oryza/genetics ; Reading ; },
}
@article {pmid35093321,
year = {2022},
author = {Han, Y and Tan, X and Jin, T and Zhao, S and Hu, L and Zhang, W and Kurita, R and Nakamura, Y and Liu, J and Li, D and Zhang, Z and Fang, X and Huang, S},
title = {CRISPR/Cas9-based multiplex genome editing of BCL11A and HBG efficiently induces fetal hemoglobin expression.},
journal = {European journal of pharmacology},
volume = {918},
number = {},
pages = {174788},
doi = {10.1016/j.ejphar.2022.174788},
pmid = {35093321},
issn = {1879-0712},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Gene Expression Regulation ; Humans ; Repressor Proteins/*metabolism ; Whole Genome Sequencing/methods ; beta-Globins/*genetics ; *beta-Thalassemia/genetics/metabolism/therapy ; *gamma-Globins/genetics/metabolism ; },
abstract = {Beta-hemoglobinopathies are caused by mutations in the β-globin gene. One strategy to cure this disease relies on re-activating the γ-globin expression. BCL11A is an important transcription factor that suppresses the γ-globin expression, which makes it one of the most promising therapeutic targets in β-hemoglobinopathies. Here, we performed single-gene editing and multiplex gene editing via CRISPR/Cas9 technology to edit BCL11A erythroid-specific enhancer and BCL11A binding site on γ-globin gene promoter in HUDEP-2 cells and adult human CD34[+] cells. Multiplex gene editing led to higher γ-globin expression than single-gene editing without inhibiting erythroid differentiation. By further optimizing the on-target DNA editing efficiency of multiplex gene editing, the percentage of F-cells exceeded 50% in HUDEP-2 cells. Amplicon deep sequencing and whole genome sequencing were used to detect the editing frequency of on- and potential off-target sites in CD34[+] cells. No off-target mutations were detected, suggesting its accuracy in HSPCs. In summary, our study provides a new approach which can be used for the treatment of β-hemoglobinopathies in the future.},
}
@article {pmid35092938,
year = {2022},
author = {Maurer, W and Hartmann, N and Argyriou, L and Sossalla, S and Streckfuss-Bömeke, K},
title = {Generation of homozygous Nav1.8 knock-out iPSC lines by CRISPR Cas9 genome editing to investigate a potential new antiarrhythmic strategy.},
journal = {Stem cell research},
volume = {60},
number = {},
pages = {102677},
doi = {10.1016/j.scr.2022.102677},
pmid = {35092938},
issn = {1876-7753},
mesh = {Anti-Arrhythmia Agents ; CRISPR-Cas Systems/genetics ; Gene Editing ; Homozygote ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac ; },
abstract = {The sodium channel Nav1.8, encoded by SCN10A, is reported to contribute to arrhythmogenesis by inducing the late INa and thereby enhanced persistent Na[+] current. However, its exact electrophysiological role in cardiomyocytes remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) with a homozygous SCN10A knock-out from a healthy iPSC line by CRISPR Cas9 genome editing. The edited iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro differentiation capacity. The iPSCs are able to differentiate into iPSC-cardiomyocytes, hence making it possible to investigate the role of Nav1.8 in the heart.},
}
@article {pmid35092559,
year = {2022},
author = {Shademan, B and Masjedi, S and Karamad, V and Isazadeh, A and Sogutlu, F and Rad, MHS and Nourazarian, A},
title = {CRISPR Technology in Cancer Diagnosis and Treatment: Opportunities and Challenges.},
journal = {Biochemical genetics},
volume = {60},
number = {5},
pages = {1446-1470},
pmid = {35092559},
issn = {1573-4927},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Therapy ; Humans ; *Neoplasms/diagnosis/genetics/therapy ; Technology ; },
abstract = {A novel gene editing tool, the Cas system, associated with the CRISPR system, is emerging as a potential method for genome modification. This simple method, based on the adaptive immune defense system of prokaryotes, has been developed and used in human cancer research. These technologies have tremendous therapeutic potential, especially in gene therapy, where a patient-specific mutation is genetically corrected to cure diseases that cannot be cured with conventional treatments. However, translating CRISPR/Cas9 into the clinic will be challenging, as we still need to improve the efficiency, specificity, and application of the technology. In this review, we will explain how CRISPR-Cas9 technology can treat cancer at the molecular level, focusing on ordination and the epigenome. We will also focus on the promise and shortcomings of this system to ensure its application in the treatment and prevention of cancer.},
}
@article {pmid35091986,
year = {2022},
author = {Najafabadi, ZY and Fanuel, S and Falak, R and Kaboli, S and Kardar, GA},
title = {The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {1-16},
pmid = {35091986},
issn = {1559-0305},
abstract = {Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR-Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR-Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR-Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR-Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.},
}
@article {pmid35091037,
year = {2022},
author = {Li, Q and Lu, J and Zhang, G and Liu, S and Zhou, J and Du, G and Chen, J},
title = {Recent advances in the development of Aspergillus for protein production.},
journal = {Bioresource technology},
volume = {348},
number = {},
pages = {126768},
doi = {10.1016/j.biortech.2022.126768},
pmid = {35091037},
issn = {1873-2976},
mesh = {Aspergillus/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering ; },
abstract = {Aspergillus had been widely used in the industrial production of recombinant proteins. In addition to the safety and broad substrate utilization spectrum, its efficient post-translational modification and strong protein secretion capacity have significant advantages for developing an excellent protein-producing cell factory in industrial production. However, the difficulties in genetic manipulation of Aspergillus and varying expression levels of different heterologous proteins hampered its further development and application. Recently, the development of CRISPR genome editing and high-throughput screening platforms has facilitated the Aspergillus development of a wide range of modifications and applications. Meanwhile, multi-omics analysis and multiplexed genetic engineering have promoted effective knowledge mining. This paper provides a comprehensive and updated review of these advances, including high-throughput screening, genome editing, protein expression modules, and fermentation optimization. It also highlights and discusses the latest significant progress, aiming to provide a practical guide for implementing Aspergillus as an efficient protein-producing cell factory.},
}
@article {pmid35090653,
year = {2022},
author = {Wang, W and Liu, J and Wu, LA and Ko, CN and Wang, X and Lin, C and Liu, J and Ling, L and Wang, J},
title = {Nicking enzyme-free strand displacement amplification-assisted CRISPR-Cas-based colorimetric detection of prostate-specific antigen in serum samples.},
journal = {Analytica chimica acta},
volume = {1195},
number = {},
pages = {339479},
doi = {10.1016/j.aca.2022.339479},
pmid = {35090653},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems ; Colorimetry ; Gold ; Humans ; Male ; *Metal Nanoparticles ; *Prostate-Specific Antigen ; },
abstract = {Immunosorbent assay is the gold standard diagnostic technique for the detection of protein biomarkers. However, this technique tends to have low sensitivity and requires laborious manipulation. Although advanced CRISPR-Cas-based biosensors offer advantages of simplicity, low cost and high accuracy, the synergy of using CRISPR-Cas-assisted dual signal amplification system for rapid diagnosis of protein biomarkers remains scarce. In this work, we report a synergetic signal amplification system comprising CRISPR-Cas12a and nicking enzyme-free strand displacement amplification (SDA) technique for accurate detection of prostate-specific antigen (PSA). The presence of PSA will initiate the nicking enzyme-free SDA process, generating amplicons that can be recognized by the CRISPR-Cas12a system. The activated CRISPR-Cas system will then mediate trans-ssDNA cleavage of neighboring linker DNA, which unlocks the gold nanoparticles (AuNPs) signal probes and gives a distance-dependent colorimetric readout. This assay could detect PSA in aqueous buffer sensitively and selectively with a limit of detection (LOD) down to 0.030 ng mL[-1]. Importantly, this assay was successfully applied for discriminating four blood samples from prostate cancer patients among thirteen blood samples from normal individuals/cancer patients accurately. This work will open an avenue for the development of SDA-CRISPR-AuNPs hybrid sensing systems, offering great potential for the development of non-invasive point-of-care diagnostic tools for prostate cancer.},
}
@article {pmid35090645,
year = {2022},
author = {Lu, LL and Li, CZ and Guo, HZ and Liu, D and Tang, HW and Zheng, B and Li, CY},
title = {Monitoring of viral myocarditis injury using an energy-confined upconversion nanoparticle and nature-inspired biochip combined CRISPR/Cas12a-powered biosensor.},
journal = {Analytica chimica acta},
volume = {1195},
number = {},
pages = {339455},
doi = {10.1016/j.aca.2022.339455},
pmid = {35090645},
issn = {1873-4324},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer ; Mice ; *Myocarditis/diagnosis ; *Nanospheres ; },
abstract = {The early diagnosis and timely intervention of viral myocarditis urgently require a noninvasive detection approach. Therefore, we present a CRISPR/Cas12a-powered biosensor that integrates an exceptionally efficient upconversion luminescent resonance energy transfer (LRET) with a nature-inspired biochip to determine a golden-standard cardiac biomarker (cardiac troponin I). First, a unique sandwich-structured energy-confined upconversion nanoparticle (acting as the energy donor) is synthesized to dramatically reinforce the LRET's ability. Such a structural improvement endows a relatively high quenching efficiency (as much as 93.8%) toward the surface acceptors and enhances the working adaption in complicated biological media. Moreover, a three-dimensional photonic crystal fabricated using a self-assembly of nanospheres is employed to construct a biochip interface, under which the upconversion luminescence is prominently boosted to approximately 27-fold to achieve signal amplification. Finally, the newly developed luminescence sensing method exhibits remarkable assay performance after introducing these attempts into a dual-aptamer-regulated CRISPR/Cas12a system to transduce the target. More importantly, this biosensor can primarily be a quite useful tracer tool to allow dynamic monitoring of the entire myocardial injury process in a coxsackievirus B3 infected mouse model, paving an attractive venue for medical diagnostic techniques.},
}
@article {pmid35090586,
year = {2022},
author = {Duelen, R and Costamagna, D and Gilbert, G and De Waele, L and Goemans, N and Desloovere, K and Verfaillie, CM and Sipido, KR and Buyse, GM and Sampaolesi, M},
title = {Human iPSC model reveals a central role for NOX4 and oxidative stress in Duchenne cardiomyopathy.},
journal = {Stem cell reports},
volume = {17},
number = {2},
pages = {352-368},
pmid = {35090586},
issn = {2213-6711},
mesh = {Acetylcysteine/pharmacology ; Adenosine Triphosphate/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Survival/drug effects ; Dystrophin/genetics/metabolism ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Mitochondria/drug effects/physiology ; Muscular Dystrophy, Duchenne/genetics/*pathology ; Myocytes, Cardiac/cytology/metabolism ; NADPH Oxidase 4/*metabolism ; Oxadiazoles/pharmacology ; *Oxidative Stress/drug effects ; Reactive Oxygen Species/metabolism ; },
abstract = {Duchenne muscular dystrophy (DMD) is a progressive muscle disorder caused by mutations in the Dystrophin gene. Cardiomyopathy is a major cause of early death. We used DMD-patient-specific human induced pluripotent stem cells (hiPSCs) to model cardiomyopathic features and unravel novel pathologic insights. Cardiomyocytes (CMs) differentiated from DMD hiPSCs showed enhanced premature cell death due to significantly elevated intracellular reactive oxygen species (ROS) resulting from depolarized mitochondria and increased NADPH oxidase 4 (NOX4). CRISPR-Cas9 correction of Dystrophin restored normal ROS levels. ROS reduction by N-acetyl-L-cysteine (NAC), ataluren (PTC124), and idebenone improved hiPSC-CM survival. We show that oxidative stress in DMD hiPSC-CMs was counteracted by stimulating adenosine triphosphate (ATP) production. ATP can bind to NOX4 and partially inhibit the ROS production. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose targeting ROS production and preventing detrimental effects of NOX4 on DMD CMs as promising therapeutic strategy.},
}
@article {pmid35089463,
year = {2022},
author = {Fu, J and Fu, YW and Zhao, JJ and Yang, ZX and Li, SA and Li, GH and Quan, ZJ and Zhang, F and Zhang, JP and Zhang, XB and Sun, CK},
title = {Improved and Flexible HDR Editing by Targeting Introns in iPSCs.},
journal = {Stem cell reviews and reports},
volume = {18},
number = {5},
pages = {1822-1833},
pmid = {35089463},
issn = {2629-3277},
mesh = {*CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; *Induced Pluripotent Stem Cells ; Introns/genetics ; Pyridazines ; Quinazolines ; *Recombinational DNA Repair ; },
abstract = {Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.},
}
@article {pmid35089385,
year = {2022},
author = {Meyer, CM and Goldman, IL and Grzebelus, E and Krysan, PJ},
title = {Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.},
journal = {Plant cell reports},
volume = {41},
number = {4},
pages = {947-960},
pmid = {35089385},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems ; *Daucus carota/genetics/metabolism ; *Gene Editing/methods ; Genome, Plant ; Plants, Genetically Modified/genetics ; Protoplasts ; },
abstract = {We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant's genome. We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.},
}
@article {pmid35088855,
year = {2022},
author = {Lin, CS and Hsu, CT and Yuan, YH and Zheng, PX and Wu, FH and Cheng, QW and Wu, YL and Wu, TL and Lin, S and Yue, JJ and Cheng, YH and Lin, SI and Shih, MC and Sheen, J and Lin, YC},
title = {DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1917-1930},
pmid = {35088855},
issn = {1532-2548},
support = {R01 GM129093/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant/genetics ; *Lycopersicon esculentum/genetics ; Plant Breeding ; Protoplasts ; Regeneration ; *Solanum/genetics ; Tetraploidy ; },
abstract = {Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.},
}
@article {pmid35087864,
year = {2021},
author = {Shademan, B and Nourazarian, A and Hajazimian, S and Isazadeh, A and Biray Avci, C and Oskouee, MA},
title = {CRISPR Technology in Gene-Editing-Based Detection and Treatment of SARS-CoV-2.},
journal = {Frontiers in molecular biosciences},
volume = {8},
number = {},
pages = {772788},
pmid = {35087864},
issn = {2296-889X},
abstract = {Outbreak and rapid spread of coronavirus disease (COVID-19) caused by coronavirus acute respiratory syndrome (SARS-CoV-2) caused severe acute respiratory syndrome (SARS-CoV-2) that started in Wuhan, and has become a global problem because of the high rate of human-to-human transmission and severe respiratory infections. Because of high prevalence of SARS-CoV-2, which threatens many people worldwide, rapid diagnosis and simple treatment are needed. Genome editing is a nucleic acid-based approach to altering the genome by artificially changes in genetic information and induce irreversible changes in the function of target gene. Clustered, regularly interspaced short palindromic repeats (CRISPR/Cas) could be a practical and straightforward approach to this disease. CRISPR/Cas system contains Cas protein, which is controlled by a small RNA molecule to create a double-stranded DNA gap. Evidence suggested that CRISPR/Cas was also usable for diagnosis and treatment of SARS-CoV-2 infection. In this review study, we discoursed on application of CRISPR technology in detection and treatment of SARS-CoV-2 infection. Another aspect of this study was to introduce potential future problems in use of CRISPR/Cas technology.},
}
@article {pmid35087538,
year = {2021},
author = {Radakovics, K and Battin, C and Leitner, J and Geiselhart, S and Paster, W and Stöckl, J and Hoffmann-Sommergruber, K and Steinberger, P},
title = {A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {817604},
pmid = {35087538},
issn = {1664-3224},
mesh = {Bacteria/*metabolism ; Bacterial Infections/*metabolism/*microbiology ; *Bacterial Physiological Phenomena ; Biological Assay/*methods ; Biosensing Techniques ; CRISPR-Cas Systems ; Cell Line ; Gene Expression ; Genes, Reporter ; Humans ; Ligands ; Multigene Family ; Reproducibility of Results ; Sensitivity and Specificity ; Toll-Like Receptors/genetics/*metabolism ; },
abstract = {Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.},
}
@article {pmid35087158,
year = {2022},
author = {Li, Y and Zaheri, S and Nguyen, K and Liu, L and Hassanipour, F and Bleris, L},
title = {Machine learning-based approaches for identifying human blood cells harboring CRISPR-mediated fetal chromatin domain ablations.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {1481},
pmid = {35087158},
issn = {2045-2322},
mesh = {Anemia, Sickle Cell/blood/genetics/*therapy ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Separation/*methods ; Flow Cytometry/methods ; Gene Editing/methods ; Genetic Therapy/methods ; Genotyping Techniques/*methods ; Humans ; Machine Learning ; Mutation ; Protein Domains/genetics ; ROC Curve ; beta-Thalassemia/blood/genetics/*therapy ; gamma-Globins/*genetics ; },
abstract = {Two common hemoglobinopathies, sickle cell disease (SCD) and β-thalassemia, arise from genetic mutations within the β-globin gene. In this work, we identified a 500-bp motif (Fetal Chromatin Domain, FCD) upstream of human ϒ-globin locus and showed that the removal of this motif using CRISPR technology reactivates the expression of ϒ-globin. Next, we present two different cell morphology-based machine learning approaches that can be used identify human blood cells (KU-812) that harbor CRISPR-mediated FCD genetic modifications. Three candidate models from the first approach, which uses multilayer perceptron algorithm (MLP 20-26, MLP26-18, and MLP 30-26) and flow cytometry-derived cellular data, yielded 0.83 precision, 0.80 recall, 0.82 accuracy, and 0.90 area under the ROC (receiver operating characteristic) curve when predicting the edited cells. In comparison, the candidate model from the second approach, which uses deep learning (T2D5) and DIC microscopy-derived imaging data, performed with less accuracy (0.80) and ROC AUC (0.87). We envision that equivalent machine learning-based models can complement currently available genotyping protocols for specific genetic modifications which result in morphological changes in human cells.},
}
@article {pmid35087086,
year = {2022},
author = {Queiroz, LL and Hoffmann, C and Lacorte, GA and de Melo Franco, BDG and Todorov, SD},
title = {Genomic and functional characterization of bacteriocinogenic lactic acid bacteria isolated from Boza, a traditional cereal-based beverage.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {1460},
pmid = {35087086},
issn = {2045-2322},
mesh = {Alcoholic Beverages/*microbiology ; Bacteriocins/*metabolism ; Bulgaria ; Edible Grain ; Fermented Foods/*microbiology ; Food Microbiology ; Lactobacillales/genetics/*isolation &amp; purification/metabolism ; Whole Genome Sequencing ; },
abstract = {Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as Pediococcus acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 to 12,800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37 °C. All bacteriocins remained active after incubation at pH 2.0-10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage β-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining genomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.},
}
@article {pmid35086771,
year = {2022},
author = {Radtke, S and Kiem, HP},
title = {Bringing gene therapy to where it's needed.},
journal = {Trends in molecular medicine},
volume = {28},
number = {3},
pages = {171-172},
doi = {10.1016/j.molmed.2022.01.005},
pmid = {35086771},
issn = {1471-499X},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Humans ; },
abstract = {Gene editing allows the precise modification of cells to correct genetic defects or enhance immunotherapies. A limitation is the delivery of this technology to specific cells or organs. Recently, Banskota et al. reported the use of virus-like particles (VLPs) loaded with gene-editing agents for gene therapy delivery directly inside the body.},
}
@article {pmid35085557,
year = {2022},
author = {Wang, H and Gao, T and Zhou, Y and Ren, J and Guo, J and Zeng, J and Xiao, Y and Zhang, Y and Feng, Y},
title = {Mechanistic insights into the inhibition of the CRISPR-Cas surveillance complex by anti-CRISPR protein AcrIF13.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {3},
pages = {101636},
pmid = {35085557},
issn = {1083-351X},
mesh = {*Bacteriophages/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Viral Proteins/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide prokaryotes with nucleic acid-based adaptive immunity against infections of mobile genetic elements, including phages. To counteract this immune process, phages have evolved various anti-CRISPR (Acr) proteins which deactivate CRISPR-Cas-based immunity. However, the mechanisms of many of these Acr-mediated inhibitions are not clear. Here, we report the crystal structure of AcrIF13 and explore its inhibition mechanism. The structure of AcrIF13 is unique and displays a negatively charged surface. Additionally, biochemical studies identified that AcrIF13 interacts with the type I-F CRISPR-Cas surveillance complex (Csy complex) to block target DNA recognition and that the Cas5f-8f tail and Cas7.6f subunit of the Csy complex are specific binding targets of AcrIF13. Further mutational studies demonstrated that several negatively charged residues of AcrIF13 and positively charged residues of Cas8f and Cas7f of the Csy complex are involved in AcrIF13-Csy binding. Together, our findings provide mechanistic insights into the inhibition mechanism of AcrIF13 and further suggest the prevalence of the function of Acr proteins as DNA mimics.},
}
@article {pmid35085009,
year = {2022},
author = {Yang, F and Zhang, H and Cai, S and Imtiaz, K and Li, M and Wang, M and Liu, Y and Xue, F and Zhang, L and Gu, F},
title = {Green Fluorescent Protein Tagged Polycistronic Reporter System Reveals Functional Editing Characteristics of CRISPR-Cas.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {254-263},
doi = {10.1089/crispr.2021.0056},
pmid = {35085009},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Green Fluorescent Proteins/genetics ; Humans ; },
abstract = {The green fluorescent protein (GFP)-based reporter system has been widely harnessed as a quick quantitative activity assessment method for characterizing CRISPR-Cas via flow cytometry. However, due to the small size (738 nt) of the GFP coding sequence, the targeting sites for certain CRISPR-Cas are greatly restricted. To address this, here we developed a GFP tagged polycistronic reporter system to determine the activity of CRISPR-Cas in human cells. Specifically, the system contains the herpes simplex virus thymidine kinase (TK) gene, bacterial neomycin phosphotransferase (Neo) gene, and green fluorescent protein (GFP), named TNG gene, with a coding sequence of 2,577 nt. To investigate its performance, we generated a human cell line harboring the TNG expression cassette at the AAVS1 locus, and then we tested it with different Cas orthologs (SaCas9, St1Cas9, and AsCas12a). Our results demonstrated that using the TNG reporter system greatly expands the targeting site selection (3- to 13-fold) with CRISPR-Cas genome editing. The study therefore reports an additional method for the characterization of CRISPR-Cas technology.},
}
@article {pmid35083895,
year = {2022},
author = {Lim, JM and Kim, HH},
title = {Basic Principles and Clinical Applications of CRISPR-Based Genome Editing.},
journal = {Yonsei medical journal},
volume = {63},
number = {2},
pages = {105-113},
pmid = {35083895},
issn = {1976-2437},
support = {2017R1A2B3004198//National Research Foundation of Korea/Korea ; 2017M3A9B4062403//National Research Foundation of Korea/Korea ; 2018R1A5A2025079//National Research Foundation of Korea/Korea ; //Yonsei University College of Medicine/Korea ; HI17C0676//Ministry of Health and Welfare/Korea ; 2021-22-0014//Yonsei Signature Research Cluster/Korea ; IBS-R026-D1//Institute for Basic Science/Korea ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Humans ; },
abstract = {Advances in sequencing technologies have facilitated the discovery of previously unknown genetic variants in both inherited and acquired disorders, and tools to correct these pathogenic variants are rapidly evolving. Since the first introduction of CRISPR-Cas9 in 2012, the field of CRISPR-based genome editing has progressed immensely, giving hope to many patients suffering from genetic disorders that lack effective treatment. In this review, we will examine the basic principles of CRISPR-based genome editing, explain the mechanisms of new genome editors, including base editors and prime editors, and evaluate the therapeutic possibilities of CRISPR-based genome editing by focusing on recently published clinical trials and animal studies. Although efficacy and safety issues remain a large concern, we cannot deny that CRISPR-based genome editing will soon be prevalent in clinical practice.},
}
@article {pmid35080853,
year = {2022},
author = {Gao, J and Xu, J and Zuo, Y and Ye, C and Jiang, L and Feng, L and Huang, L and Xu, Z and Lian, J},
title = {Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas9.},
journal = {ACS synthetic biology},
volume = {11},
number = {2},
pages = {623-633},
doi = {10.1021/acssynbio.1c00307},
pmid = {35080853},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Pichia/genetics/metabolism ; Recombinant Proteins/metabolism ; *Saccharomycetales/metabolism ; *Synthetic Biology ; },
abstract = {Pichia pastoris, an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris. First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, β-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.},
}
@article {pmid35080493,
year = {2022},
author = {Li, X and Chen, F and Liu, X and Xiao, J and Andongma, BT and Tang, Q and Cao, X and Chou, SH and Galperin, MY and He, J},
title = {Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response.},
journal = {eLife},
volume = {11},
number = {},
pages = {},
pmid = {35080493},
issn = {2050-084X},
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; DNA-Directed RNA Polymerases/genetics/metabolism ; Endopeptidase Clp/genetics/*metabolism ; Escherichia coli/*enzymology/genetics/metabolism/pathogenicity ; Gene Expression Regulation, Bacterial/*physiology ; RNA, Antisense/genetics/*metabolism ; RNA, Ribosomal/genetics/metabolism ; Transcription Factors/metabolism ; Transcription, Genetic/*physiology ; Virulence ; },
abstract = {Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.},
}
@article {pmid35078991,
year = {2022},
author = {Cheng, W and Liu, F and Ren, Z and Chen, W and Chen, Y and Liu, T and Ma, Y and Cao, N and Wang, J},
title = {Parallel functional assessment of m[6]A sites in human endodermal differentiation with base editor screens.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {478},
pmid = {35078991},
issn = {2041-1723},
mesh = {Adenosine/*analogs &amp; derivatives/genetics/metabolism ; Adrenomedullin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Cell Differentiation ; *Cell Lineage ; Cells, Cultured ; Endoderm/*cytology/metabolism ; *Gene Expression Regulation, Developmental ; Human Embryonic Stem Cells/*metabolism ; Humans ; Proteins/genetics/metabolism ; SOXB1 Transcription Factors/genetics/*metabolism ; },
abstract = {N[6]-methyladenosine (m[6]A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m[6]A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m[6]A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m[6]A sites to screen for m[6]A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m[6]A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m[6]A site on ADM. Our study provides a functional screening of m[6]A sites and paves the way for functional studies of m[6]A at individual m[6]A site level.},
}
@article {pmid35078987,
year = {2022},
author = {Fu, R and He, W and Dou, J and Villarreal, OD and Bedford, E and Wang, H and Hou, C and Zhang, L and Wang, Y and Ma, D and Chen, Y and Gao, X and Depken, M and Xu, H},
title = {Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {474},
pmid = {35078987},
issn = {2041-1723},
support = {R01 HL157714/HL/NHLBI NIH HHS/United States ; R35 GM137927/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence/*genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Humans ; *Mutation ; Neoplasms/genetics/pathology/*therapy ; RNA, Guide/*chemistry/genetics ; },
abstract = {The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an "epistasis-like" combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the "epistasis-like" combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.},
}
@article {pmid35078986,
year = {2022},
author = {Riesenberg, S and Helmbrecht, N and Kanis, P and Maricic, T and Pääbo, S},
title = {Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {489},
pmid = {35078986},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; *Gene Editing ; *Genome ; Humans ; *Nucleic Acid Conformation ; Oligonucleotides/*chemistry ; RNA, Guide/*chemistry/genetics ; },
abstract = {The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The 'Genome-editing Optimized Locked Design' (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.},
}
@article {pmid35078560,
year = {2022},
author = {Zhang, Y and An, MX and Gong, C and Li, YY and Wang, YT and Lin, M and Li, R and Tian, C},
title = {Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 (arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae.},
journal = {Biomedical and environmental sciences : BES},
volume = {35},
number = {1},
pages = {35-44},
doi = {10.3967/bes2022.005},
pmid = {35078560},
issn = {2214-0190},
mesh = {Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/genetics/physiology ; Phenotype ; RNA/isolation &amp; purification ; Real-Time Polymerase Chain Reaction/standards ; Rho Guanine Nucleotide Exchange Factors/*genetics/metabolism ; Sincalide/analysis ; Spectrophotometry/methods ; Zebrafish/genetics/*physiology ; },
abstract = {OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.<br><br>METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 [-/-], arhgef10 [+/-], and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.<br><br>RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 [-/-] zebrafish had more severe symptoms than arhgef10 [+/-] zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.<br><br>CONCLUSION: Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.},
}
@article {pmid35078394,
year = {2022},
author = {Chakravarti, R and Lenka, SK and Gautam, A and Singh, R and Ravichandiran, V and Roy, S and Ghosh, D},
title = {A Review on CRISPR-mediated Epigenome Editing: A Future Directive for Therapeutic Management of Cancer.},
journal = {Current drug targets},
volume = {23},
number = {8},
pages = {836-853},
doi = {10.2174/1389450123666220117105531},
pmid = {35078394},
issn = {1873-5592},
mesh = {CRISPR-Cas Systems ; DNA Methylation ; *Diabetes Mellitus, Type 2/genetics/therapy ; Epigenesis, Genetic ; Epigenome ; Histones/metabolism ; Humans ; *Neoplasms/genetics/therapy ; Obesity ; Transcription Factors/metabolism ; },
abstract = {Recent studies have shed light on the role of epigenetic marks in certain diseases like cancer, type II diabetes mellitus (T2DM), obesity, and cardiovascular dysfunction, to name a few. Epigenetic marks like DNA methylation and histone acetylation are randomly altered in the disease state. It has been seen that methylation of DNA and histones can result in down-regulation of gene expression, whereas histone acetylation, ubiquitination, and phosphorylation are linked to enhanced expression of genes. How can we precisely target such epigenetic aberrations to prevent the advent of diseases? The answer lies in the amalgamation of the efficient genome editing technique, CRISPR, with certain effector molecules that can alter the status of epigenetic marks as well as employ certain transcriptional activators or repressors. In this review, we have discussed the rationale of epigenetic editing as a therapeutic strategy and how CRISPR-Cas9 technology coupled with epigenetic effector tags can efficiently edit epigenetic targets. In the later part, we have discussed how certain epigenetic effectors are tagged with dCas9 to elicit epigenetic changes in cancer. Increased interest in exploring the epigenetic background of cancer and non-communicable diseases like type II diabetes mellitus and obesity accompanied with technological breakthroughs has made it possible to perform large-scale epigenome studies.},
}
@article {pmid35077577,
year = {2022},
author = {Natsuga, K and Furuta, Y and Takashima, S and Nohara, T and Kosumi, H and Mai, Y and Higashi, H and Ujiie, H},
title = {Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing.},
journal = {Human mutation},
volume = {43},
number = {4},
pages = {529-536},
doi = {10.1002/humu.24331},
pmid = {35077577},
issn = {1098-1004},
mesh = {CRISPR-Cas Systems ; Collagen Type VII/genetics ; *Epidermolysis Bullosa Dystrophica/diagnosis/genetics ; Humans ; Mosaicism ; Mutation ; Skin ; },
abstract = {Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM.},
}
@article {pmid35077145,
year = {2022},
author = {Villegas Kcam, MC and Tsong, AJ and Chappell, J},
title = {Uncovering the Distinct Properties of a Bacterial Type I-E CRISPR Activation System.},
journal = {ACS synthetic biology},
volume = {11},
number = {2},
pages = {1000-1003},
doi = {10.1021/acssynbio.1c00496},
pmid = {35077145},
issn = {2161-5063},
mesh = {*Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Protein Domains ; Transcriptional Activation/genetics ; },
abstract = {Synthetic gene regulators based upon CRISPR-Cas systems offer programmable technologies to control gene expression in bacteria. Bacterial CRISPR activators (CRISPRa) have been developed that use engineered type II CRISPR-dCas9 to localize transcription activation domains near promoter elements. However, several reports have demonstrated distance-dependent requirements and periodical activation patterns that overall limit their flexibility. Here, we demonstrate the potential of using an alternative type I-E CRISPR-Cas system to create a CRISPRa with distinct and expanded regulatory properties. Furthermore, we create the first bacterial CRISPRa system based upon a type I-E CRISPR-Cas and characterize the distance-dependent activation patterns to reveal a distinct and more frequent periodicity of activation.},
}
@article {pmid35076284,
year = {2022},
author = {Zhao, B and Chen, SA and Lee, J and Fraser, HB},
title = {Bacterial Retrons Enable Precise Gene Editing in Human Cells.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {31-39},
pmid = {35076284},
issn = {2573-1602},
support = {R01 GM134228/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; *Gene Editing ; HEK293 Cells ; Humans ; *RNA-Directed DNA Polymerase/genetics/metabolism ; },
abstract = {Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.},
}
@article {pmid35076280,
year = {2022},
author = {Sun, W and Liu, H and Yin, W and Qiao, J and Zhao, X and Liu, Y},
title = {Strategies for Enhancing the Homology-Directed Repair Efficiency of CRISPR-Cas Systems.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {7-18},
doi = {10.1089/crispr.2021.0039},
pmid = {35076280},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Endonucleases/genetics ; *Gene Editing ; Recombinational DNA Repair ; },
abstract = {The CRISPR-Cas nuclease has emerged as a powerful genome-editing tool in recent years. The CRISPR-Cas system induces double-strand breaks that can be repaired via the non-homologous end joining or homology-directed repair (HDR) pathway. Compared to non-homologous end joining, HDR can be used for the treatment of incurable monogenetic diseases. Therefore, remarkable efforts have been dedicated to enhancing the efficacy of HDR. In this review, we summarize the currently used strategies for enhancing the HDR efficiency of CRISPR-Cas systems based on three factors: (1) regulation of the key factors in the DNA repair pathways, (2) modulation of the components in the CRISPR machinery, and (3) alteration of the intracellular environment around double-strand breaks. Representative cases and potential solutions for further improving HDR efficiency are also discussed, facilitating the development of new CRISPR technologies to achieve highly precise genetic manipulation in the future.},
}
@article {pmid35076264,
year = {2022},
author = {Du, P and Li, B and Liu, X and Yang, L and Ren, N and Li, Y and Huang, Q},
title = {Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {131-145},
doi = {10.1089/crispr.2021.0105},
pmid = {35076264},
issn = {2573-1602},
mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; Taq Polymerase/metabolism ; },
abstract = {Detection of genome editing with quantitative polymerase chain reaction (PCR) primarily relies on and is limited by its ability to discriminate genome modification from the wild-type sequence. An enhanced DNA polymerase variant with superior specificity is needed for this application. Here, we perform semi-rational molecular evolution on full-length Taq polymerase to screen high-specific variants that meet the requirements of gene variation detection. We substituted each of the 40 polar amino acids in direct contact with the primer/template duplex and conducted extensive random mutagenesis to generate a Taq mutation library. Screening on a quantitative PCR system with insertion and deletion-containing templates identified a series of improved Taq variants. We demonstrate that the Taq388 variant bearing three amino acid substitutions, S577A, W645R, and I707V, has improved sensitivity to insertion and deletion-derived primer/template mismatch by a ΔCt value of 25-26 and is superior for application in evaluating CRISPR-Cas9 editing efficiency and single-cell clone genotyping. In addition, the Taq variant shows substantial potential for single-nucleotide polymorphism detection by means of allele-specific PCR because of its high sensitivity to mismatches.},
}
@article {pmid35074874,
year = {2022},
author = {Fuller, CW and Padayatti, PS and Abderrahim, H and Adamiak, L and Alagar, N and Ananthapadmanabhan, N and Baek, J and Chinni, S and Choi, C and Delaney, KJ and Dubielzig, R and Frkanec, J and Garcia, C and Gardner, C and Gebhardt, D and Geiser, T and Gutierrez, Z and Hall, DA and Hodges, AP and Hou, G and Jain, S and Jones, T and Lobaton, R and Majzik, Z and Marte, A and Mohan, P and Mola, P and Mudondo, P and Mullinix, J and Nguyen, T and Ollinger, F and Orr, S and Ouyang, Y and Pan, P and Park, N and Porras, D and Prabhu, K and Reese, C and Ruel, T and Sauerbrey, T and Sawyer, JR and Sinha, P and Tu, J and Venkatesh, AG and VijayKumar, S and Zheng, L and Jin, S and Tour, JM and Church, GM and Mola, PW and Merriman, B},
title = {Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {5},
pages = {},
pmid = {35074874},
issn = {1091-6490},
mesh = {Biosensing Techniques/*instrumentation ; DNA ; Electronics/*instrumentation ; Enzyme Assays/*instrumentation ; Equipment Design/instrumentation ; Kinetics ; Lab-On-A-Chip Devices ; Miniaturization/instrumentation ; Nanotechnology/instrumentation ; Oligonucleotide Array Sequence Analysis/*instrumentation ; Semiconductors ; },
abstract = {For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.},
}
@article {pmid35074547,
year = {2022},
author = {Chaudhry, N and Muhammad, H and Seidl, C and Downes, D and Young, DA and Hao, Y and Zhu, L and Vincent, TL},
title = {Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.},
journal = {Osteoarthritis and cartilage},
volume = {30},
number = {4},
pages = {596-604},
pmid = {35074547},
issn = {1522-9653},
support = {R476/0516/DMT_/The Dunhill Medical Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chondrocytes/metabolism ; Humans ; Joints/metabolism ; Mice ; *MicroRNAs/metabolism ; *Osteoarthritis/metabolism ; },
abstract = {OBJECTIVE: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes.<br><br>METHOD: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140[-/-] mice.<br><br>RESULTS: High efficiency gene editing of miR-140 (90-98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P&#xa0;<&#xa0;0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium.<br><br>CONCLUSION: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.},
}
@article {pmid35074418,
year = {2022},
author = {Hussain, MS and Kumar, M},
title = {Assembly of Cas7 subunits of Leptospira on the mature crRNA of CRISPR-Cas I-B is modulated by divalent ions.},
journal = {Gene},
volume = {818},
number = {},
pages = {146244},
doi = {10.1016/j.gene.2022.146244},
pmid = {35074418},
issn = {1879-0038},
mesh = {CRISPR-Cas Systems/*genetics ; Cations, Divalent/*pharmacology ; DNA, Bacterial/metabolism ; Endodeoxyribonucleases/chemistry/metabolism ; Leptospira/*genetics ; Magnesium/pharmacology ; Protein Conformation ; Protein Subunits/chemistry/*metabolism ; RNA, Bacterial/*metabolism ; Recombinant Proteins/isolation &amp; purification ; Substrate Specificity/drug effects ; },
abstract = {The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg[2+] ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg[2+] ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg[2+] ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd = 26.81 mM) between rLinCas7 and Mg[2+] ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.},
}
@article {pmid35073397,
year = {2022},
author = {Sun, W and Guo, W and Liu, Z and Qiao, S and Wang, Z and Wang, J and Qu, L and Shan, L and Sun, F and Xu, S and Bai, O and Liang, C},
title = {Direct MYD88[L265P] gene detection for diffuse large B-cell lymphoma (DLBCL) via a miniaturised CRISPR/dCas9-based sensing chip.},
journal = {Lab on a chip},
volume = {22},
number = {4},
pages = {768-776},
doi = {10.1039/d1lc01055g},
pmid = {35073397},
issn = {1473-0189},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; *Lymphoma, Large B-Cell, Diffuse/diagnosis/genetics ; Mutation ; *Myeloid Differentiation Factor 88/genetics ; },
abstract = {Traditional methods for single-nucleotide variants based on amplification and fluorescence signals require expensive reagents and cumbersome instruments, and they are time-consuming for each trial. Here, a porous anodised aluminium (PAA)-based sensing chip modified with deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor is developed, which can be used for the label-free sensing of the diffuse large B-cell lymphoma (DLBCL) MYD88[L265P] gene by integrating with electrochemical ionic current rectification (ICR) measurement. The sgRNA that can specifically identify and capture the MYD88[L265P] gene was screened, which has been proved to be workable to activate dCas9 for the target MYD88[L265P]. In the sensing process, the dCas9 proteins can capture the genome sequence, thus bringing negative charges over the PAA chip and correspondingly resulting in a variation in the ICR value due to the uneven transport of potassium anions through the ion channels of the PAA chip. The whole sensing can be finished within 40 min, and there is no need for gene amplification. The CRISPR/dCas9-based sensor demonstrates ultrasensitive detection performance in the concentration range of 50 to 200 ng μL[-1] and it has been proved to be feasible for the genome sequence of patient tissues. This sensor shows the potential of targeting other mutations by designing the corresponding sgRNAs and expands the applications of CRISPR/dCas9 technology to the on-chip electrical detection of nucleic acids, which will be very valuable for rapid diagnosis of clinically mutated genes. This makes the hybrid CRISPR-PAA chip an ideal candidate for next-generation nucleic acid biosensors.},
}
@article {pmid35073349,
year = {2022},
author = {Carneiro, P and de Freitas, MV and Matte, U},
title = {In silico analysis of potential off-target sites to gene editing for Mucopolysaccharidosis type I using the CRISPR/Cas9 system: Implications for population-specific treatments.},
journal = {PloS one},
volume = {17},
number = {1},
pages = {e0262299},
pmid = {35073349},
issn = {1932-6203},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Computer Simulation ; *Gene Editing/methods ; Gene Targeting/methods ; Humans ; Mucopolysaccharidosis I/genetics/*therapy ; Polymorphism, Genetic ; },
abstract = {Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase deficiency encoded by the IDUA gene. Therapy with CRISPR/Cas9 is being developed for treatment, however a detailed investigation of off-target effects must be performed. This study aims to evaluate possible off-targets for a sgRNA aiming to correct the most common variant found in MPS I patients (p.Trp402*). A total of 272 potential off-target sequences was obtained and 84 polymorphic sites were identified in these sequences with a frequency equal to or greater than 1% in at least one of the populations. In the majority of cases, polymorphic sites decrease the chance of off-target cleavage and a new PAM was created, which indicates the importance of such analysis. This study highlights the importance of screening off-targets in a population-specific context using Mucopolysaccharidosis type I as an example of a problem that concerns all therapeutic treatments. Our results can have broader applications for other targets already clinically in use, as they could affect CRISPR/Cas9 safety and efficiency.},
}
@article {pmid35072325,
year = {2022},
author = {Murakami, Y and Kobayashi, T},
title = {An effective double gene knock-in strategy using small-molecule L755507 in the medaka fish (Oryzias latipes).},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {60},
number = {1-2},
pages = {e23465},
doi = {10.1002/dvg.23465},
pmid = {35072325},
issn = {1526-968X},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knock-In Techniques ; *Oryzias/genetics ; Recombinational DNA Repair ; },
abstract = {Homology-directed repair (HDR)-mediated genome editing has become a powerful method for altering chromosomal sequences in a seamless and accurate manner. However, the low efficiency of HDR in most cells hinders the establishment of desired strains harboring accurately modified genomes. To enhance HDR-mediated knock-in events, we explored two approaches, namely low-temperature incubation and chemical compound administration using medaka embryos after microinjection. We validated the performance of each method by calculating the knock-in efficiencies according to the expression area of fluorescent protein in the embryos. The in vivo assay indicated that the reduction in temperature did not promote HDR events, whereas among the nine compounds screened, the small molecule L755507 could enhance the HDR-mediated targeted integration of reporter cassettes. Additionally, the L755507-based approach allowed for the simultaneous integration of two different DNA fragments into the two targeted loci, that is, double knock-in. Our established knock-in system combining L755507, donor plasmids, and the CRISPR/Cas9 nickase system can reduce the workload for genetically modified strain generation, thus accelerating studies on the molecular mechanisms of biological phenomena.},
}
@article {pmid35070166,
year = {2022},
author = {Hou, Q and Pucci, F and Pan, F and Xue, F and Rooman, M and Feng, Q},
title = {Using metagenomic data to boost protein structure prediction and discovery.},
journal = {Computational and structural biotechnology journal},
volume = {20},
number = {},
pages = {434-442},
pmid = {35070166},
issn = {2001-0370},
abstract = {Over the past decade, metagenomic sequencing approaches have been providing an ever-increasing amount of protein sequence data at an astonishing rate. These constitute an invaluable source of information which has been exploited in various research fields such as the study of the role of the gut microbiota in human diseases and aging. However, only a small fraction of all metagenomic sequences collected have been functionally or structurally characterized, leaving much of them completely unexplored. Here, we review how this information has been used in protein structure prediction and protein discovery. We begin by presenting some widely used metagenomic databases and analyze in detail how metagenomic data has contributed to the impressive improvement in the accuracy of structure prediction methods in recent years. We then examine how metagenomic information can be exploited to annotate protein sequences. More specifically, we focus on the role of metagenomes in the discovery of enzymes and new CRISPR-Cas systems, and in the identification of antibiotic resistance genes. With this review, we provide an overview of how metagenomic data is currently revolutionizing our understanding of protein science.},
}
@article {pmid35069497,
year = {2021},
author = {Liu, P and Wang, X and Liang, J and Dong, Q and Zhang, J and Liu, D and Wang, S and Bi, J and Liu, W and Wang, Z and Chen, L and Liu, L and Huang, X and Zhang, G},
title = {A Recombinase Polymerase Amplification-Coupled Cas12a Mutant-Based Module for Efficient Detection of Streptomycin-Resistant Mutations in Mycobacterium tuberculosis.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {796916},
pmid = {35069497},
issn = {1664-302X},
abstract = {Drug-resistant tuberculosis (TB) is a serious public health problem and threat to global TB prevention and control. Streptomycin (STR) is the earliest and classical anti-TB drug, and it is the earliest drug that generated resistance to anti-TB treatment, which limits its use in treating TB and impedes TB control efforts. The rapid, economical, and highly sensitive detection of STR-resistant TB may help reduce disease transmission and morbimortality. CRISPR/CRISPR-associated protein (Cas) is a new-generation pathogen detection method that can detect single-nucleotide polymorphisms with high sensitivity and good specificity. In this study, a Cas12a RR detection system that can recognize more non-traditional protospacer-adjacent motif-targeting sequences was developed based on Cas12a combined with recombinase polymerase amplification technology. This system detects 0.1% of the target substance, and the entire detection process can be completed within 60 min. Its sensitivity and specificity for detecting clinical STR-resistant Mycobacterium tuberculosis were both 100%. Overall, the Cas12 RR detection system provides a novel alternative for the rapid, simple, sensitive, and specific detection of STR-resistant TB, which may contribute to the prompt treatment and prevention of disease transmission in STR-resistant TB.},
}
@article {pmid35068602,
year = {2022},
author = {Kahraman Ilıkkan, &#xd6;},
title = {Analysis of Probiotic Bacteria Genomes: Comparison of CRISPR/Cas Systems and Spacer Acquisition Diversity.},
journal = {Indian journal of microbiology},
volume = {62},
number = {1},
pages = {40-46},
pmid = {35068602},
issn = {0046-8991},
abstract = {UNLABELLED: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes constitute an adaptive (acquired) defense system of bacteria and archaea. Here 72 probiotic bacteria genomes were investigated in terms of the presence of CRISPR/Cas systems and phage/plasmid invaders through spacer analysis. 49 CRISPR/Cas systems were detected within probiotic strains, namely,17 type II-A, 10 type I-C, 8 type I-E, 5 Type I-U (I-G), 4 type III-A, 2 type I-B, 1 type I-A, 1 type IV-B, and 1 type II-C. The predicted target of spacers was determined in 25 strains and consequently, three different spacer and target patterns were revealed. The diversity of CRISPR spacers provides insight and understanding to determine strain-specific invaders of probiotic bacteria as well as their relationships between strains. CRISPR systems were clarified in many studies for genomic characterization. However, recently, endogenous genome editing with CRISPR has provided an approach for various genome editing projects. Thus, in the future, producing strain-specific phage-resistant starter cultures or probiotics by endogenous genome editing methods according to phage/plasmid survey can be utilized for industrial and pharmaceutical applications. Therefore, this study intended a comprehensive investigation of CRISPR systems of probiotic bacteria.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00971-1.},
}
@article {pmid35067302,
year = {2022},
author = {Nguyen, TM and Lu, CA and Huang, LF},
title = {Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {315},
number = {},
pages = {111132},
doi = {10.1016/j.plantsci.2021.111132},
pmid = {35067302},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/metabolism ; Gene Expression Regulation, Plant ; Genes, Plant ; *Introns ; Mutagenesis, Insertional/*methods ; Oryza/*genetics/*metabolism ; Plant Proteins/*genetics/*metabolism ; Plants, Genetically Modified ; },
abstract = {The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3' splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.},
}
@article {pmid35067222,
year = {2022},
author = {Liu, X and Hussain, M and Dai, J and Li, Y and Zhang, L and Yang, J and Ali, Z and He, N and Tang, Y},
title = {Programmable Biosensors Based on RNA-Guided CRISPR/Cas Endonuclease.},
journal = {Biological procedures online},
volume = {24},
number = {1},
pages = {2},
pmid = {35067222},
issn = {1480-9222},
abstract = {Highly infectious illnesses caused by pathogens constitute severe threats to public health and lead to global economic loss. The use of robust and programmable clustered regularly interspaced short palindromic repeat and CRISPR-associated protein (CRISPR-Cas) systems, repurposed from genome-engineering applications has markedly improved traditional nucleic acid detection for precise identification, independently enabling rapid diagnostics of multiplex biomarker with genetic and mutation related to tumors, and microbial pathogens. In this review, we delineate the utility of the current CRISPR-Cas enzyme as biosensors by which these effector toolkits achieve recognition, signaling amplification, and finally, accurate detection. Additionally, we discuss the details of the dominance and hurdles related to expanding this revolutionary technology into an effective and convenient contraption crucial for improving the rational redesign to CRISPR/Cas biosensing. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems by CRISPR/Cas tools.},
}
@article {pmid35065347,
year = {2022},
author = {Lee, SY and Oh, SW},
title = {Filtration-based LAMP-CRISPR/Cas12a system for the rapid, sensitive and visualized detection of Escherichia coli O157:H7.},
journal = {Talanta},
volume = {241},
number = {},
pages = {123186},
doi = {10.1016/j.talanta.2021.123186},
pmid = {35065347},
issn = {1873-3573},
mesh = {CRISPR-Cas Systems ; *Escherichia coli O157/genetics ; Food Microbiology ; Humans ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Escherichia coli O157:H7 is a major cause of fresh vegetable-associated infections that can threaten human health. A method for rapidly detecting food-borne pathogens should be developed for safe food management. A clustered regularly interspaced short palindromic repeats (CRISPR)-based detection method has the potential to greatly advance biosensing technology through its high sensitivity and specificity. In this study, we developed a rapid, sensitive, and visualized method of detecting E. coli O157:H7 (stx2 gene) based on a loop-mediated isothermal amplification (LAMP)-CRISPR/Cas12a system. The developed method was able to rectify the common false-negative results produced by LAMP, and the detection limit was 1.22 × 10[0] CFU/mL in pure culture. Furthermore, the LAMP-CRISPR/Cas12a system using filtration enrichment successfully detected 4.80 × 10[0] CFU/g of E. coli O157:H7 in romaine lettuce without pre-microbial enrichment culture. Consequently, the LAMP-CRISPR/Cas12a system is a useful technique for rapid and sensitive detection of E. coli O157:H7 in fresh products.},
}
@article {pmid35064613,
year = {2022},
author = {Tang, Y and Qi, L and Liu, Y and Guo, L and Zhao, R and Yang, M and Du, Y and Li, B},
title = {CLIPON: A CRISPR-Enabled Strategy that Turns Commercial Pregnancy Test Strips into General Point-of-Need Test Devices.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {12},
pages = {e202115907},
doi = {10.1002/anie.202115907},
pmid = {35064613},
issn = {1521-3773},
mesh = {*CRISPR-Cas Systems ; DNA/analysis ; Female ; Humans ; Lab-On-A-Chip Devices ; *Point-of-Care Testing ; Pregnancy ; *Pregnancy Tests ; RNA, Viral/analysis ; Reproducibility of Results ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; Viruses/isolation &amp; purification ; },
abstract = {Desirable biosensing assays need to be sensitive, specific, cost-effective, instrument-free, and versatile. Herein we report a new strategy termed CLIPON (CRISPR and Large DNA assembly Induced Pregnancy strips for signal-ON detection) that can deliver these traits. CLIPON integrates a commercial pregnancy test strip (PTS) with four biological elements: the human chorionic gonadotropin (hCG), CRISPR-Cas12a, crRNA and cauliflower-like large-sized DNA assemblies (CLD). CLIPON uses the Cas12a/crRNA complex both to recognize a target of interest and to release CLD-bound hCG so that target presence can translate into a colorimetric signal on the PTS. We demonstrate the versatility of CLIPON through sensitive and specific detection of HPV genomic DNA, SARS-CoV-2 genomic RNA and adenosine. We also engineer a cell phone app and a hand-held microchip to achieve signal quantification. CLIPON represents an attractive option for biosensing and point-of-care diagnostics.},
}
@article {pmid35064336,
year = {2022},
author = {Sarri, N and Wang, K and Tsioumpekou, M and Castillejo-López, C and Lennartsson, J and Heldin, CH and Papadopoulos, N},
title = {Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRβ and affect PDGF-BB-induced STAT3 signalling.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {79},
number = {2},
pages = {85},
pmid = {35064336},
issn = {1420-9071},
mesh = {Becaplermin/*pharmacology ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Endopeptidases/chemistry/genetics/*metabolism ; Humans ; Mutagenesis ; Protein Transport ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Receptor, Platelet-Derived Growth Factor beta/*metabolism ; STAT3 Transcription Factor/*metabolism ; Signal Transduction/*drug effects ; Ubiquitin-Specific Proteases/antagonists &amp; inhibitors/genetics/*metabolism ; Ubiquitination ; },
abstract = {Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.},
}
@article {pmid35064134,
year = {2022},
author = {Liang, SQ and Liu, P and Smith, JL and Mintzer, E and Maitland, S and Dong, X and Yang, Q and Lee, J and Haynes, CM and Zhu, LJ and Watts, JK and Sontheimer, EJ and Wolfe, SA and Xue, W},
title = {Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {437},
pmid = {35064134},
issn = {2041-1723},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; T32 GM107000/GM/NIGMS NIH HHS/United States ; UG3 TR002668/TR/NCATS NIH HHS/United States ; U24 HG010423/HG/NHGRI NIH HHS/United States ; F30 CA239483/CA/NCI NIH HHS/United States ; R01 HL150669/HL/NHLBI NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; T32 CA130807/CA/NCI NIH HHS/United States ; R01 AG040061/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Biotin/metabolism ; Biotinylation ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA/metabolism ; *Gene Editing ; Genes, Reporter ; Genome ; Liver/metabolism ; Lung/metabolism ; Mice ; RNA, Guide/*genetics ; Ribonucleoproteins/metabolism ; },
abstract = {Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.},
}
@article {pmid35064117,
year = {2022},
author = {Tarnowski, MJ and Gorochowski, TE},
title = {Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {434},
pmid = {35064117},
issn = {2041-1723},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Base Pairing ; Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Library ; *Genetic Engineering ; *High-Throughput Nucleotide Sequencing ; Insulator Elements/genetics ; Nanopore Sequencing ; Protein Isoforms/genetics/metabolism ; RNA, Guide/genetics ; RNA, Messenger/*genetics/metabolism ; *Sequence Analysis, RNA ; Terminator Regions, Genetic ; Transcription Termination, Genetic ; },
abstract = {Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as 'transcriptional valves' that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes.},
}
@article {pmid35063774,
year = {2022},
author = {Sui, X and Tang, X and Wu, X and Liu, Y},
title = {Identification of ERCC8 as a novel cisplatin-resistant gene in esophageal cancer based on genome-scale CRISPR/Cas9 screening.},
journal = {Biochemical and biophysical research communications},
volume = {593},
number = {},
pages = {84-92},
doi = {10.1016/j.bbrc.2022.01.033},
pmid = {35063774},
issn = {1090-2104},
mesh = {Antineoplastic Agents/pharmacology ; Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; Cisplatin/*pharmacology ; DNA Repair Enzymes/antagonists &amp; inhibitors/*genetics/metabolism ; *Drug Resistance, Neoplasm ; Esophageal Neoplasms/*drug therapy/genetics/pathology ; *Gene Editing ; *Genome, Human ; Humans ; Transcription Factors/antagonists &amp; inhibitors/*genetics/metabolism ; Tumor Cells, Cultured ; },
abstract = {Esophageal cancer (ESCA) is one of the most common malignant tumors of the digestive system worldwide. As a first-line drug for chemotherapy, cisplatin resistance is the major obstacle in the successful treatment of esophageal cancer. Previous studies largely failed to identify the key genes associated with cisplatin resistance. Hence, the aim of this study was to screen the cisplatin resistance-related genes of esophageal cancer using CRISPR/Cas9 gene-editing technology and Brunello iBar library. Of note, we identified ERCC8 as a novel cisplatin-resistant gene by high-throughput sequencing and cisplatin resistance assays. Based on KEGG and GO analysis, we hypothesized that the mechanism of ERCC8 involvement in cisplatin resistance is through binding to damaged DNA to perform nucleotide excision repair, contributing to the restoration of basic DNA functions and cellular life activities in ESCA. In addition, Cell proliferation and wound healing assay confirmed that ERCC8 had little effect on the proliferation and migration of esophageal cancer cells in vitro. Survival analysis showed that ERCC8 expression was not associated with OS, DSS, or FPI in patients with ESCA. Immuno-infiltration analysis indicated that increased ERCC8 expression is associated with NK cells, macrophages, T helper cells, Th1 cells, and Th2 cells. Collectively, ERCC8 may serve as a new biomarker for predicting cisplatin resistance and have the prospect of becoming an effective target for the clinical treatment of cisplatin resistance in ESCA.},
}
@article {pmid35063130,
year = {2022},
author = {Inoue, H and Nakamura, S and Higo, S and Shiba, M and Kohama, Y and Kondo, T and Kameda, S and Tabata, T and Okuno, S and Ikeda, Y and Li, J and Liu, L and Yamazaki, S and Takeda, M and Ito, E and Takashima, S and Miyagawa, S and Sawa, Y and Hikoso, S and Sakata, Y},
title = {Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes.},
journal = {Stem cell reports},
volume = {17},
number = {2},
pages = {337-351},
pmid = {35063130},
issn = {2213-6711},
mesh = {Arrhythmias, Cardiac/genetics/*pathology ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Desmosomes/*physiology ; Female ; Gene Editing ; Genetic Vectors/genetics/metabolism ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Male ; Models, Biological ; Myocardial Contraction/*physiology ; Myocytes, Cardiac/cytology/metabolism ; Pedigree ; Plakophilins/genetics/*metabolism ; },
abstract = {Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.},
}
@article {pmid35063127,
year = {2022},
author = {Whiteley, JT and Fernandes, S and Sharma, A and Mendes, APD and Racha, V and Benassi, SK and Marchetto, MC},
title = {Reaching into the toolbox: Stem cell models to study neuropsychiatric disorders.},
journal = {Stem cell reports},
volume = {17},
number = {2},
pages = {187-210},
pmid = {35063127},
issn = {2213-6711},
mesh = {CRISPR-Cas Systems/genetics ; Cell Culture Techniques, Three Dimensional ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Mental Disorders/*pathology ; *Models, Biological ; Organoids/metabolism/pathology ; },
abstract = {Recent advances in genetics, molecular biology, and stem cell biology have accelerated our understanding of neuropsychiatric disorders, like autism spectrum disorder (ASD), major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SZ). This progress highlights the incredible complexity of both the human brain and mental illnesses from the biochemical to the cellular level. Contributing to the complexity of neuropsychiatric disorders are their polygenic nature, cellular and brain region interconnectivity, and dysregulation of human-specific neurodevelopmental processes. Here, we discuss available tools, including CRISPR-Cas9, and the applications of these tools to develop cell-based two-dimensional (2D) models and 3D brain organoid models that better represent and unravel the intricacies of neuropsychiatric disorder pathophysiology.},
}
@article {pmid35063100,
year = {2022},
author = {Nambiar, TS and Baudrier, L and Billon, P and Ciccia, A},
title = {CRISPR-based genome editing through the lens of DNA repair.},
journal = {Molecular cell},
volume = {82},
number = {2},
pages = {348-388},
pmid = {35063100},
issn = {1097-4164},
support = {P01 CA174653/CA/NCI NIH HHS/United States ; R01 CA197774/CA/NCI NIH HHS/United States ; R01 CA227450/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; DNA Breaks, Single-Stranded ; *DNA Damage ; *DNA Repair ; *Gene Editing ; *Gene Targeting ; *Genome, Human ; Humans ; },
abstract = {Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases.},
}
@article {pmid35062978,
year = {2022},
author = {Dronina, J and Samukaite-Bubniene, U and Ramanavicius, A},
title = {Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review).},
journal = {Journal of nanobiotechnology},
volume = {20},
number = {1},
pages = {41},
pmid = {35062978},
issn = {1477-3155},
mesh = {Animals ; Biosensing Techniques/methods/trends ; COVID-19/virology ; CRISPR-Cas Systems/*physiology ; DNA, Viral/analysis/*isolation &amp; purification ; Environmental Pollutants/analysis/isolation &amp; purification ; Food Contamination/analysis ; Humans ; Molecular Typing/methods/trends ; *Nucleic Acid Amplification Techniques/methods/trends ; SARS-CoV-2/genetics ; Virology/methods/trends ; Virus Diseases/classification/diagnosis/virology ; },
abstract = {Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.},
}
@article {pmid35062262,
year = {2021},
author = {Liu, Y and Chen, D and Zhang, X and Chen, S and Yang, D and Tang, L and Yang, X and Wang, Y and Luo, X and Wang, M and Hu, Z and Huang, Y},
title = {Construction of Baculovirus-Inducible CRISPR/Cas9 Antiviral System Targeting BmNPV in Bombyx mori.},
journal = {Viruses},
volume = {14},
number = {1},
pages = {},
pmid = {35062262},
issn = {1999-4915},
mesh = {Animals ; Animals, Genetically Modified ; Antiviral Agents/*metabolism/pharmacology ; Baculoviridae/*genetics ; Bombyx/*virology ; *CRISPR-Cas Systems ; DNA Replication ; DNA, Viral/genetics ; Gene Editing ; Gene Expression ; Nucleopolyhedroviruses/*drug effects ; Promoter Regions, Genetic ; RNA, Guide ; Virus Diseases/genetics ; Virus Replication ; },
abstract = {The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.},
}
@article {pmid35062208,
year = {2021},
author = {Yang, YC and Yang, HC},
title = {Recent Progress and Future Prospective in HBV Cure by CRISPR/Cas.},
journal = {Viruses},
volume = {14},
number = {1},
pages = {},
pmid = {35062208},
issn = {1999-4915},
mesh = {Antiviral Agents/pharmacology/therapeutic use ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; DNA, Viral/genetics ; *Gene Editing ; *Genetic Therapy ; *Genome, Viral ; Hepatitis B/*therapy/virology ; Hepatitis B virus/*genetics/physiology ; Hepatitis B, Chronic/*therapy/virology ; Humans ; Protein Engineering ; },
abstract = {Hepatitis B virus (HBV) infection remains an important issue of global public health. Although current antiviral therapy has dramatically reduced the mortality and morbidity of chronic hepatitis B (CHB), it fails to cure it. Rebound viremia often occurs after stopping antiviral therapy. Persistent HBV covalently closed circular DNA (cccDNA) and integrated DNA under antiviral therapy form the major barrier to eradication of HBV infection. CRISPR-mediated genome editing has emerged as a promising therapeutic approach to specifically destroy persistent HBV genomes, both cccDNA and integrated DNA, for HBV cure. However, the cleavage of integrated HBV DNA by CRISPR-Cas9 will cause double-strand break (DSB) of host genome, raising a serious safety concern about genome instability and carcinogenesis. The newly developed CRISPR-derived base editors (BEs), which fuse a catalytically disabled nuclease with a nucleobase deaminase enzyme, can be used to permanently inactivate HBV genome by introducing irreversible point mutations for generation of premature stop codons without DSBs of host genome. Although promising, CRISPR-mediated base editing still faces daunting challenges before its clinical application, including the base-editing efficacy, the off-target effect, the difficulty in finding conserved target HBV sequences, and in vivo delivery efficiency. Several strategies have been adopted to optimize the efficiency and specificity of CRISPR-BEs and to improve in vivo delivery efficacy through novel viral and non-viral delivery approaches. Particularly, the non-viral delivery of Cas9 mRNA and ribonucleoprotein by lipid nanoparticles exhibits attractive potential for liver-targeted delivery in clinical. Along with all progress above, the CRISPR-mediated gene therapy will ultimately achieve HBV cure.},
}
@article {pmid35061906,
year = {2022},
author = {Gao, F and Zheng, K and Li, YB and Jiang, F and Han, CY},
title = {A Cas6-based RNA tracking platform functioning in a fluorescence-activation mode.},
journal = {Nucleic acids research},
volume = {50},
number = {8},
pages = {e46},
pmid = {35061906},
issn = {1362-4962},
mesh = {Binding Sites ; CRISPR-Cas Systems ; *Endoribonucleases/metabolism ; Fluorescence ; Molecular Conformation ; *RNA/chemistry ; },
abstract = {Given the fact that the localization of RNAs is closely associated with their functions, techniques developed for tracking the distribution of RNAs in live cells have greatly advanced the study of RNA biology. Recently, innovative application of fluorescent protein-labelled Cas9 and Cas13 into live-cell RNA tracking further enriches the toolbox. However, the Cas9/Cas13 platform, as well as the widely-used MS2-MCP technique, failed to solve the problem of high background noise. It was recently reported that CRISPR/Cas6 would exhibit allosteric alteration after interacting with the Cas6 binding site (CBS) on RNAs. Here, we exploited this feature and designed a Cas6-based switch platform for detecting target RNAs in vivo. Conjugating split-Venus fragments to both ends of the endoribonuclease-mutated Escherichia coli Cas6(dEcCas6) allowed ligand (CBS)-activated split-Venus complementation. We name this platform as Cas6 based Fluorescence Complementation (Cas6FC). In living cells, Cas6FC could detect target RNAs with nearly free background noise. Moreover, as minimal as one copy of CBS (29nt) tagged in an RNA of interest was able to turn on Cas6FC fluorescence, which greatly reduced the odds of potential alteration of conformation and localization of target RNAs. Thus, we developed a new RNA tracking platform inherently with high sensitivity and specificity.},
}
@article {pmid35061755,
year = {2022},
author = {Montaño, ET and Nideffer, JF and Brumage, L and Erb, M and Busch, J and Fernandez, L and Derman, AI and Davis, JP and Estrada, E and Fu, S and Le, D and Vuppala, A and Tran, C and Luterstein, E and Lakkaraju, S and Panchagnula, S and Ren, C and Doan, J and Tran, S and Soriano, J and Fujita, Y and Gutala, P and Fujii, Q and Lee, M and Bui, A and Villarreal, C and Shing, SR and Kim, S and Freeman, D and Racha, V and Ho, A and Kumar, P and Falah, K and Dawson, T and Enustun, E and Prichard, A and Gomez, A and Khanna, K and Trigg, S and Pogliano, K and Pogliano, J},
title = {Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals.},
journal = {PloS one},
volume = {17},
number = {1},
pages = {e0262354},
pmid = {35061755},
issn = {1932-6203},
support = {K12 GM068524/GM/NIGMS NIH HHS/United States ; T32 GM133351/GM/NIGMS NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Bacteriophages/genetics/*isolation &amp; purification ; Methicillin-Resistant Staphylococcus aureus/drug effects ; Microbial Sensitivity Tests ; Multigene Family/genetics ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Streptomyces/*metabolism/*virology ; },
abstract = {The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.},
}
@article {pmid35061543,
year = {2022},
author = {Han, JP and Kim, M and Choi, BS and Lee, JH and Lee, GS and Jeong, M and Lee, Y and Kim, EA and Oh, HK and Go, N and Lee, H and Lee, KJ and Kim, UG and Lee, JY and Kim, S and Chang, J and Lee, H and Song, DW and Yeom, SC},
title = {In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.},
journal = {Science advances},
volume = {8},
number = {3},
pages = {eabj6901},
pmid = {35061543},
issn = {2375-2548},
mesh = {Animals ; Antithrombins ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hemophilia A/genetics/therapy ; Humans ; Liposomes ; Mice ; *Nanoparticles ; Thrombin/genetics ; },
abstract = {Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.},
}
@article {pmid35061373,
year = {2022},
author = {James, JS and Jones, S and Martella, A and Luo, Y and Fisher, DI and Cai, Y},
title = {Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.},
journal = {ACS synthetic biology},
volume = {11},
number = {2},
pages = {587-595},
doi = {10.1021/acssynbio.1c00330},
pmid = {35061373},
issn = {2161-5063},
support = {BB/P02114X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Automation ; CRISPR-Cas Systems ; *Gene Editing ; Gene Library ; Genetic Vectors/genetics ; Mammals/genetics ; *RNA, Guide/genetics ; },
abstract = {With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.},
}
@article {pmid35060334,
year = {2022},
author = {Barthet, VJA and Mrschtik, M and Kania, E and McEwan, DG and Croft, D and O'Prey, J and Long, JS and Ryan, KM},
title = {DRAM-4 and DRAM-5 are compensatory regulators of autophagy and cell survival in nutrient-deprived conditions.},
journal = {The FEBS journal},
volume = {289},
number = {13},
pages = {3752-3769},
pmid = {35060334},
issn = {1742-4658},
support = {C596/A17196/CRUK_/Cancer Research UK/United Kingdom ; A22903/CRUK_/Cancer Research UK/United Kingdom ; A31287/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Apoptosis/physiology ; Autophagy/physiology ; Cell Survival/genetics ; Humans ; *Membrane Proteins/metabolism ; Nutrients ; *Tumor Suppressor Protein p53/genetics ; },
abstract = {Macroautophagy is a membrane-trafficking process that delivers cytoplasmic material to lysosomes for degradation. The process preserves cellular integrity by removing damaged cellular constituents and can promote cell survival by providing substrates for energy production during hiatuses of nutrient availability. The process is also highly responsive to other forms of cellular stress. For example, DNA damage can induce autophagy and this involves up-regulation of the Damage-Regulated Autophagy Modulator-1 (DRAM-1) by the tumor suppressor p53. DRAM-1 belongs to an evolutionarily conserved protein family, which has five members in humans and we describe here the initial characterization of two members of this family, which we term DRAM-4 and DRAM-5 for DRAM-Related/Associated Member 4/5. We show that the genes encoding these proteins are not regulated by p53, but instead are induced by nutrient deprivation. Similar to other DRAM family proteins, however, DRAM-4 principally localizes to endosomes and DRAM-5 to the plasma membrane and both modulate autophagy flux when over-expressed. Deletion of DRAM-4 using CRISPR/Cas-9 also increased autophagy flux, but we found that DRAM-4 and DRAM-5 undergo compensatory regulation, such that deletion of DRAM-4 does not affect autophagy flux in the absence of DRAM-5. Similarly, deletion of DRAM-4 also promotes cell survival following growth of cells in the absence of amino acids, serum, or glucose, but this effect is also impacted by the absence of DRAM-5. In summary, DRAM-4 and DRAM-5 are nutrient-responsive members of the DRAM family that exhibit interconnected roles in the regulation of autophagy and cell survival under nutrient-deprived conditions.},
}
@article {pmid35060075,
year = {2022},
author = {Song, R and Wang, Y and Zheng, Q and Yao, J and Cao, C and Wang, Y and Zhao, J},
title = {One-step base editing in multiple genes by direct embryo injection for pig trait improvement.},
journal = {Science China. Life sciences},
volume = {65},
number = {4},
pages = {739-752},
pmid = {35060075},
issn = {1869-1889},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Embryo, Mammalian ; *Gene Editing/methods ; Swine ; },
abstract = {The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.},
}
@article {pmid35058954,
year = {2021},
author = {Raza, A and Tabassum, J and Zahid, Z and Charagh, S and Bashir, S and Barmukh, R and Khan, RSA and Barbosa, F and Zhang, C and Chen, H and Zhuang, W and Varshney, RK},
title = {Advances in "Omics" Approaches for Improving Toxic Metals/Metalloids Tolerance in Plants.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {794373},
pmid = {35058954},
issn = {1664-462X},
abstract = {Food safety has emerged as a high-urgency matter for sustainable agricultural production. Toxic metal contamination of soil and water significantly affects agricultural productivity, which is further aggravated by extreme anthropogenic activities and modern agricultural practices, leaving food safety and human health at risk. In addition to reducing crop production, increased metals/metalloids toxicity also disturbs plants' demand and supply equilibrium. Counterbalancing toxic metals/metalloids toxicity demands a better understanding of the complex mechanisms at physiological, biochemical, molecular, cellular, and plant level that may result in increased crop productivity. Consequently, plants have established different internal defense mechanisms to cope with the adverse effects of toxic metals/metalloids. Nevertheless, these internal defense mechanisms are not adequate to overwhelm the metals/metalloids toxicity. Plants produce several secondary messengers to trigger cell signaling, activating the numerous transcriptional responses correlated with plant defense. Therefore, the recent advances in omics approaches such as genomics, transcriptomics, proteomics, metabolomics, ionomics, miRNAomics, and phenomics have enabled the characterization of molecular regulators associated with toxic metal tolerance, which can be deployed for developing toxic metal tolerant plants. This review highlights various response strategies adopted by plants to tolerate toxic metals/metalloids toxicity, including physiological, biochemical, and molecular responses. A seven-(omics)-based design is summarized with scientific clues to reveal the stress-responsive genes, proteins, metabolites, miRNAs, trace elements, stress-inducible phenotypes, and metabolic pathways that could potentially help plants to cope up with metals/metalloids toxicity in the face of fluctuating environmental conditions. Finally, some bottlenecks and future directions have also been highlighted, which could enable sustainable agricultural production.},
}
@article {pmid35058659,
year = {2021},
author = {Zheng, Z and Deng, W and Bai, Y and Miao, R and Mei, S and Zhang, Z and Pan, Y and Wang, Y and Min, R and Deng, F and Wu, Z and Li, W and Chen, P and Ma, T and Lou, X and Lieberman, J and Liu, X},
title = {The Lysosomal Rag-Ragulator Complex Licenses RIPK1 and Caspase-8-mediated Pyroptosis by Yersinia.},
journal = {Science (New York, N.Y.)},
volume = {372},
number = {6549},
pages = {},
pmid = {35058659},
issn = {1095-9203},
support = {R01 CA240955/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caspase 8/*metabolism ; Cells, Cultured ; HEK293 Cells ; Humans ; Inflammasomes/metabolism ; Intracellular Membranes/metabolism ; Lysosomes/*metabolism ; MAP Kinase Kinase Kinases/antagonists &amp; inhibitors/metabolism ; Macrophages/*metabolism/*microbiology ; Mice ; Monomeric GTP-Binding Proteins/metabolism ; Multiprotein Complexes/metabolism ; *Pyroptosis ; Receptor-Interacting Protein Serine-Threonine Kinases/*metabolism ; Signal Transduction ; Yersinia pseudotuberculosis/pathogenicity/*physiology ; },
abstract = {Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor-β-activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen to uncover mediators of caspase-8-dependent pyroptosis identified an unexpected role of the lysosomal FLCN-FNIP2-Rag-Ragulator supercomplex, which regulates metabolic signalling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, FADD, RIPK1 and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag GTPase activity and lysosomal tethering of Rag-Ragulator, but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.},
}
@article {pmid35058543,
year = {2022},
author = {Kick, LM and von Wrisberg, MK and Runtsch, LS and Schneider, S},
title = {Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {71},
pmid = {35058543},
issn = {2399-3642},
mesh = {Bacterial Proteins/*chemistry/genetics/metabolism ; Rhodobacter capsulatus/enzymology/*genetics ; Ribonucleases/*chemistry/genetics/metabolism ; },
abstract = {Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.},
}
@article {pmid35058453,
year = {2022},
author = {Cakir, B and Tanaka, Y and Kiral, FR and Xiang, Y and Dagliyan, O and Wang, J and Lee, M and Greaney, AM and Yang, WS and duBoulay, C and Kural, MH and Patterson, B and Zhong, M and Kim, J and Bai, Y and Min, W and Niklason, LE and Patra, P and Park, IH},
title = {Expression of the transcription factor PU.1 induces the generation of microglia-like cells in human cortical organoids.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {430},
pmid = {35058453},
issn = {2041-1723},
support = {R01 MH118554/MH/NIMH NIH HHS/United States ; R01 AA025080/AA/NIAAA NIH HHS/United States ; R01 CA203011/CA/NCI NIH HHS/United States ; R01 MH118344/MH/NIMH NIH HHS/United States ; },
mesh = {Alzheimer Disease/genetics/pathology ; Amyloid beta-Peptides/toxicity ; Animals ; CRISPR-Cas Systems/genetics ; Cell Lineage/drug effects ; Cells, Cultured ; Cerebral Cortex/*metabolism ; Green Fluorescent Proteins/metabolism ; Human Embryonic Stem Cells/metabolism/ultrastructure ; Humans ; Mice ; Microglia/drug effects/*metabolism/ultrastructure ; Organoids/*cytology/metabolism ; Phagocytosis/drug effects ; Proto-Oncogene Proteins/*metabolism ; Single-Cell Analysis ; Trans-Activators/*metabolism ; },
abstract = {Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-β (Aβ). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aβ. Furthermore, in mhCOs, we observed reduced expression of Aβ-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer's disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aβ using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.},
}
@article {pmid35057957,
year = {2022},
author = {Deng, F and Li, Y and Qiao, L and Goldys, E},
title = {A CRISPR/Cas12a-assisted on-fibre immunosensor for ultrasensitive small protein detection in complex biological samples.},
journal = {Analytica chimica acta},
volume = {1192},
number = {},
pages = {339351},
doi = {10.1016/j.aca.2021.339351},
pmid = {35057957},
issn = {1873-4324},
mesh = {Biological Assay ; *Biosensing Techniques ; CRISPR-Cas Systems ; Humans ; Immunoassay ; },
abstract = {Tracking trace amounts of analytes directly from low volumes of complex biological samples remains an ongoing challenge in precision diagnostics, as the commonly used immunosorbent assays have limited sensitivity. Herein, a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure, in which a single strand DNA aptamer was applied to detect the analyte while triggering the CRISPR/Cas12a fluorescent detection system to amplify the analyte signal. This novel CAFI biosensing system was fabricated on a glass fibre surface with an antifouling PEG polymer brush modified for the detection of a spectrum of small molecules from complex media. In comparison with a conventional ELISA system, CAFI has a 1,000-fold higher sensitivity with the limit of detection for IFN-γ down to 1 fg mL[-1] (58.8 aM). It also has a tuneable linear detection range that can be easily adjusted within the range 1 fg mL[-1] to 100 pg mL[-1] (5 orders of magnitude), meeting the requirements of the demanding diagnostic scenarios. CAFI has successfully been demonstrated by detecting IFN-γ from a diverse complex biological sample type, including human serum, whole blood, perspiration, and saliva. Moreover, CAFI is applicable for the detection of other analytes by simply modifying the capture antibody and detection aptamer, demonstrated here with insulin. All these superior capabilities of CAFI make it a suitable technology to measure proteins in low (100 μL) volume complex biological samples.},
}
@article {pmid35057952,
year = {2022},
author = {Luo, X and Xue, Y and Ju, E and Tao, Y and Li, M and Zhou, L and Yang, C and Zhou, J and Wang, J},
title = {Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA.},
journal = {Analytica chimica acta},
volume = {1192},
number = {},
pages = {339336},
doi = {10.1016/j.aca.2021.339336},
pmid = {35057952},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems ; *Nucleic Acids ; *RNA, Viral/genetics ; Sensitivity and Specificity ; },
abstract = {Early and accurate diagnosis of viruses is critical for control of the pandemic. CRISPR/Cas-based detection of nucleic acid is an emerging technology for molecular diagnostics, and has been applied for virus detection. Though these methods have excellent sensitivity and specificity, most of them were not able to measure the quantity of virus. We here developed a droplet digital reverse transcription loop-mediated isothermal amplification (RT-LAMP) enhanced Cas12b-based RNA detection platform (RECD), for quantitative detection of viral RNA. CRISPR/Cas12b, which is more thermally stable than other family members in CRISPR systems, is combined with digital RT-LAMP. Due to the innate characteristic of digital format detection and CRISPR/Cas system, droplet digital RECD (ddRECD) assay enables absolute quantification of viral RNA, with single-molecule sensitivity. We expect the ddRECD assay will be a powerful tool for molecular diagnostics.},
}
@article {pmid35057922,
year = {2022},
author = {Chen, Y and Wu, H and Qian, S and Yu, X and Chen, H and Wu, J},
title = {Applying CRISPR/Cas system as a signal enhancer for DNAzyme-based lead ion detection.},
journal = {Analytica chimica acta},
volume = {1192},
number = {},
pages = {339356},
doi = {10.1016/j.aca.2021.339356},
pmid = {35057922},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; *DNA, Catalytic/metabolism ; Ions ; Lead/*analysis ; },
abstract = {Heavy metal lead accumulation in the environment pollutes the ecology systems and further threatens the human health. It is necessary to develop a sensitive method to detect it. Here, we propose a highly sensitive lead detection method by combining DNAzyme and CRISPR system. Once the lead ion is recognized, the substrate chain of DNAzyme is cleaved to produce single strand DNA. The produced single strand DNA can be detected by Cas protein/guide RNA complex and further trigger the collateral cleavage effect of CRISPR system, which can indiscriminately cut short single strand DNA reporters. By this way, the detection signals can be greatly amplified. This method can detect lead ions as low as 0.48 nM. The sensitivity is higher than the DNAzyme method. Furthermore, the portable 3D printing device is designed to observe the fluorescent signals so the end-point detection results can be visualized by the naked eyes. The entire detection process can avoid using bulky and expensive instruments, which can promote on-site lead ion detection.},
}
@article {pmid35056644,
year = {2022},
author = {Buttimer, C and Bottacini, F and Shkoporov, AN and Draper, LA and Ross, P and Hill, C},
title = {Selective Isolation of Eggerthella lenta from Human Faeces and Characterisation of the Species Prophage Diversity.},
journal = {Microorganisms},
volume = {10},
number = {1},
pages = {},
pmid = {35056644},
issn = {2076-2607},
abstract = {Eggerthella lenta is an anaerobic, high GC, Gram-positive bacillus commonly found in the human digestive tract that belongs to the class Coriobacteriia of the phylum Actinobacteria. This species has been of increasing interest as an important player in the metabolism of xenobiotics and dietary compounds. However, little is known regarding its susceptibility to bacteriophage predation and how this may influence its fitness. Here, we report the isolation of seven novel E. lenta strains using cefotaxime and ceftriaxone as selective agents. We conducted comparative and pangenome analyses of these strains and those publicly available to investigate the diversity of prophages associated with this species. Prophage gene products represent a minimum of 5.8% of the E. lenta pangenome, comprising at least ten distantly related prophage clades that display limited homology to currently known bacteriophages. All clades possess genes implicated in virion structure, lysis, lysogeny and, to a limited extent, DNA replication. Some prophages utilise tyrosine recombinases and diversity generating retroelements to generate phase variation among targeted genes. The prophages have differing levels of sensitivity to the CRISPR/cas systems of their hosts, with spacers from 44 E. lenta isolates found to target only five out of the ten identified prophage clades. Furthermore, using a PCR-based approach targeting the prophage attP site, we were able to determine that several of these elements can excise from the host chromosome, thus supporting the notion that these are active prophages. The findings of this study provide further insights into the diversity of prophages infecting species of the phylum Actinobacteria.},
}
@article {pmid35056089,
year = {2021},
author = {Perez-SanJose, D and de la Fuente, MA and Serna Pérez, J and Simarro, M and Eiros Bouza, JM and Sanz-Muñoz, I},
title = {CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses?.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {15},
number = {1},
pages = {},
pmid = {35056089},
issn = {1424-8247},
abstract = {Influenza viruses provide a great threat for the human population, causing highly contagious respiratory infections that can lead to serious clinical complications. There are a limited variety of influenza antivirals, and these antivirals are subjected to the constant emergence of resistances. Therefore, the development of new antiviral strategies to combat influenza viruses and other RNA viruses must be promoted. In this work, we design a proof-of-concept of a recently described CRISPR/Cas tool that has been proposed as a possible future RNA virus antiviral, named CRISPR/CasRx. For this, we verified the efficiency of the CasRx endonuclease in the degradation of the eGFP mRNA reporter gene and we established the best conditions for, and the efficient performance of, the CRISPR/CasRx system. The results were measured by fluorescence microscopy, flow cytometry, and qRT-PCR. The analyses demonstrated a reduction in fluorescence, regardless of the amount of eGFP reporter plasmid transfected. The analyses showed an 86-90% reduction in fluorescence by flow cytometry and a 51-80% reduction in mRNA expression by qRT-PCR. Our results demonstrate that the CasRx endonuclease is an efficient tool for eGFP mRNA knockdown. Therefore, subsequent experiments could be useful for the development of a new antiviral tool.},
}
@article {pmid35055871,
year = {2021},
author = {Baci, GM and Cucu, AA and Giurgiu, AI and Muscă, AS and Bagameri, L and Moise, AR and Bobiș, O and Rațiu, AC and Dezmirean, DS},
title = {Advances in Editing Silkworms (Bombyx mori) Genome by Using the CRISPR-Cas System.},
journal = {Insects},
volume = {13},
number = {1},
pages = {},
pmid = {35055871},
issn = {2075-4450},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) represents a powerful genome editing technology that revolutionized in a short period of time numerous natural sciences branches. Therefore, extraordinary progress was made in various fields, such as entomology or biotechnology. Bombyx mori is one of the most important insects, not only for the sericulture industry, but for numerous scientific areas. The silkworms play a key role as a model organism, but also as a bioreactor for the recombinant protein production. Nowadays, the CRISPR-Cas genome editing system is frequently used in order to perform gene analyses, to increase the resistance against certain pathogens or as an imaging tool in B. mori. Here, we provide an overview of various studies that made use of CRISPR-Cas for B. mori genome editing, with a focus on emphasizing the high applicability of this system in entomology and biological sciences.},
}
@article {pmid35055150,
year = {2022},
author = {Cao, HX and Vu, GTH and Gailing, O},
title = {From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees.},
journal = {International journal of molecular sciences},
volume = {23},
number = {2},
pages = {},
pmid = {35055150},
issn = {1422-0067},
mesh = {Acclimatization ; CRISPR-Cas Systems ; Forests ; Gene Editing/*methods ; Genome, Plant ; Plant Breeding ; Trees/genetics/*growth &amp; development ; Whole Genome Sequencing/*methods ; },
abstract = {Due to the economic and ecological importance of forest trees, modern breeding and genetic manipulation of forest trees have become increasingly prevalent. The CRISPR-based technology provides a versatile, powerful, and widely accepted tool for analyzing gene function and precise genetic modification in virtually any species but remains largely unexplored in forest species. Rapidly accumulating genetic and genomic resources for forest trees enabled the identification of numerous genes and biological processes that are associated with important traits such as wood quality, drought, or pest resistance, facilitating the selection of suitable gene editing targets. Here, we introduce and discuss the latest progress, opportunities, and challenges of genome sequencing and editing for improving forest sustainability.},
}
@article {pmid35055037,
year = {2022},
author = {Kaushal, K and Tyagi, A and Karapurkar, JK and Kim, EJ and Tanguturi, P and Kim, KS and Jung, HS and Ramakrishna, S},
title = {Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation.},
journal = {International journal of molecular sciences},
volume = {23},
number = {2},
pages = {},
pmid = {35055037},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Deubiquitinating Enzymes/*genetics/metabolism ; Gene Expression Regulation ; Genome-Wide Association Study/*methods ; Humans ; MSX1 Transcription Factor/genetics/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; Osteogenesis/*genetics ; Proteolysis ; Regenerative Medicine ; Thiolester Hydrolases/*genetics/metabolism ; Transcription Factors/metabolism ; Ubiquitination ; },
abstract = {The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.},
}
@article {pmid35055026,
year = {2022},
author = {Biswas, S and Wahl, NJ and Thomson, MJ and Cason, JM and McCutchen, BF and Septiningsih, EM},
title = {Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.},
journal = {International journal of molecular sciences},
volume = {23},
number = {2},
pages = {},
pmid = {35055026},
issn = {1422-0067},
mesh = {2S Albumins, Plant/*genetics ; Antigens, Plant/*genetics ; Arachis/*genetics/immunology ; CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting ; Genetic Vectors/genetics ; Pilot Projects ; Plant Proteins/genetics/immunology ; Promoter Regions, Genetic ; *Protoplasts ; RNA, Guide ; Seedlings ; Temperature ; Transfection/methods ; },
abstract = {The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.},
}
@article {pmid35054841,
year = {2022},
author = {Vallverdú-Prats, M and Brugada, R and Alcalde, M},
title = {Premature Termination Codon in 5' Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation.},
journal = {International journal of molecular sciences},
volume = {23},
number = {2},
pages = {},
pmid = {35054841},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Codon, Nonsense ; Desmocollins/genetics ; Desmoglein 2/genetics ; Desmoplakins/*genetics/*metabolism ; Frameshift Mutation ; Mice ; Nonsense Mediated mRNA Decay ; Plakophilins/genetics ; Protein Biosynthesis ; gamma Catenin/*genetics/*metabolism ; },
abstract = {Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5'-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5'-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5'-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5' region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5'-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5'-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5'-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.},
}
@article {pmid35054816,
year = {2022},
author = {Ittiprasert, W and Chatupheeraphat, C and Mann, VH and Li, W and Miller, A and Ogunbayo, T and Tran, K and Alrefaei, YN and Mentink-Kane, M and Brindley, PJ},
title = {RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.},
journal = {International journal of molecular sciences},
volume = {23},
number = {2},
pages = {},
pmid = {35054816},
issn = {1422-0067},
support = {Award number 107475/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Catalysis ; Female ; Gene Dosage ; *Gene Knockout Techniques ; *Genes, Protozoan ; *Genetic Loci ; Humans ; Mutation/genetics ; Oligonucleotides/metabolism ; RNA, Guide/*metabolism ; *Recombinational DNA Repair/genetics ; Reference Standards ; Ribonucleases/*genetics ; Schistosoma mansoni/*genetics ; Transcription, Genetic ; Transgenes ; },
abstract = {The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.},
}
@article {pmid35054418,
year = {2021},
author = {Rusanova, A and Fedorchuk, V and Toshchakov, S and Dubiley, S and Sutormin, D},
title = {An Interplay between Viruses and Bacteria Associated with the White Sea Sponges Revealed by Metagenomics.},
journal = {Life (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
pmid = {35054418},
issn = {2075-1729},
abstract = {Sponges are remarkable holobionts harboring extremely diverse microbial and viral communities. However, the interactions between the components within holobionts and between a holobiont and environment are largely unknown, especially for polar organisms. To investigate possible interactions within and between sponge-associated communities, we probed the microbiomes and viromes of cold-water sympatric sponges Isodictya palmata (n = 2), Halichondria panicea (n = 3), and Halichondria sitiens (n = 3) by 16S and shotgun metagenomics. We showed that the bacterial and viral communities associated with these White Sea sponges are species-specific and different from the surrounding water. Extensive mining of bacterial antiphage defense systems in the metagenomes revealed a variety of defense mechanisms. The abundance of defense systems was comparable in the metagenomes of the sponges and the surrounding water, thus distinguishing the White Sea sponges from those inhabiting the tropical seas. We developed a network-based approach for the combined analysis of CRISPR-spacers and protospacers. Using this approach, we showed that the virus-host interactions within the sponge-associated community are typically more abundant (three out of four interactions studied) than the inter-community interactions. Additionally, we detected the occurrence of viral exchanges between the communities. Our work provides the first insight into the metagenomics of the three cold-water sponge species from the White Sea and paves the way for a comprehensive analysis of the interactions between microbial communities and associated viruses.},
}
@article {pmid35053409,
year = {2022},
author = {Clees, AS and Stolp, V and Häupl, B and Fuhrmann, DC and Wempe, F and Seibert, M and Weber, S and Banning, A and Tikkanen, R and Williams, R and Brüne, B and Serve, H and Schnütgen, F and von Metzler, I and Kurrle, N},
title = {Identification of the Cysteine Protease Legumain as a Potential Chronic Hypoxia-Specific Multiple Myeloma Target Gene.},
journal = {Cells},
volume = {11},
number = {2},
pages = {},
pmid = {35053409},
issn = {2073-4409},
mesh = {Basic Helix-Loop-Helix Transcription Factors/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cysteine Endopeptidases/*genetics ; Gene Expression Regulation, Neoplastic ; Hexokinase/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Lactate Dehydrogenase 5/metabolism ; *Molecular Targeted Therapy ; Multiple Myeloma/*enzymology/*genetics ; Proteome/metabolism ; RNA, Messenger/genetics/metabolism ; Signal Transduction/genetics ; Tumor Hypoxia/*genetics ; Up-Regulation/genetics ; },
abstract = {Multiple myeloma (MM) is the second most common hematologic malignancy, which is characterized by clonal proliferation of neoplastic plasma cells in the bone marrow. This microenvironment is characterized by low oxygen levels (1-6% O2), known as hypoxia. For MM cells, hypoxia is a physiologic feature that has been described to promote an aggressive phenotype and to confer drug resistance. However, studies on hypoxia are scarce and show little conformity. Here, we analyzed the mRNA expression of previously determined hypoxia markers to define the temporal adaptation of MM cells to chronic hypoxia. Subsequent analyses of the global proteome in MM cells and the stromal cell line HS-5 revealed hypoxia-dependent regulation of proteins, which directly or indirectly upregulate glycolysis. In addition, chronic hypoxia led to MM-specific regulation of nine distinct proteins. One of these proteins is the cysteine protease legumain (LGMN), the depletion of which led to a significant growth disadvantage of MM cell lines that is enhanced under hypoxia. Thus, herein, we report a methodologic strategy to examine MM cells under physiologic hypoxic conditions in vitro and to decipher and study previously masked hypoxia-specific therapeutic targets such as the cysteine protease LGMN.},
}
@article {pmid35053384,
year = {2022},
author = {Nouri, P and Zimmer, A and Brüggemann, S and Friedrich, R and Kühn, R and Prakash, N},
title = {Generation of a NES-mScarlet Red Fluorescent Reporter Human iPSC Line for Live Cell Imaging and Flow Cytometric Analysis and Sorting Using CRISPR-Cas9-Mediated Gene Editing.},
journal = {Cells},
volume = {11},
number = {2},
pages = {},
pmid = {35053384},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/*genetics ; Calcium/metabolism ; Cell Differentiation/genetics ; Cell Line ; Dopaminergic Neurons/metabolism ; *Flow Cytometry ; *Gene Editing ; Gene Expression Regulation ; *Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Luminescent Proteins/*metabolism ; Mesencephalon/metabolism ; Nestin/*metabolism ; Neural Stem Cells/metabolism ; },
abstract = {Advances in the regenerative stem cell field have propelled the generation of tissue-specific cells in the culture dish for subsequent transplantation, drug screening purposes, or the elucidation of disease mechanisms. One major obstacle is the heterogeneity of these cultures, in which the tissue-specific cells of interest usually represent only a fraction of all generated cells. Direct identification of the cells of interest and the ability to specifically isolate these cells in vitro is, thus, highly desirable for these applications. The type VI intermediate filament protein NESTIN is widely used as a marker for neural stem/progenitor cells (NSCs/NPCs) in the developing and adult central and peripheral nervous systems. Applying CRISPR-Cas9 technology, we have introduced a red fluorescent reporter (mScarlet) into the NESTIN (NES) locus of a human induced pluripotent stem cell (hiPSC) line. We describe the generation and characterization of NES-mScarlet reporter hiPSCs and demonstrate that this line is an accurate reporter of NSCs/NPCs during their directed differentiation into human midbrain dopaminergic (mDA) neurons. Furthermore, NES-mScarlet hiPSCs can be used for direct identification during live cell imaging and for flow cytometric analysis and sorting of red fluorescent NSCs/NPCs in this paradigm.},
}
@article {pmid35053376,
year = {2022},
author = {Schröder, SK and Pinoé-Schmidt, M and Weiskirchen, R},
title = {Lipocalin-2 (LCN2) Deficiency Leads to Cellular Changes in Highly Metastatic Human Prostate Cancer Cell Line PC-3.},
journal = {Cells},
volume = {11},
number = {2},
pages = {},
pmid = {35053376},
issn = {2073-4409},
mesh = {Actins/metabolism ; Biomarkers, Tumor/metabolism ; CRISPR-Cas Systems/genetics ; Carcinogenesis/metabolism/pathology ; Cell Adhesion ; Cell Proliferation ; Cytoskeleton/metabolism ; Endoplasmic Reticulum Stress ; Gene Knockdown Techniques ; Humans ; Interleukin-1beta/metabolism ; Lipocalin-2/*deficiency/metabolism ; Male ; Neoplasm Metastasis ; PC-3 Cells ; Prostatic Neoplasms/*metabolism/*pathology ; RNA, Small Interfering/metabolism ; Stress Fibers/metabolism ; Unfolded Protein Response ; },
abstract = {The transporter protein lipocalin-2 (LCN2) also termed neutrophil-gelatinase-associated lipocalin (NGAL) has pleiotropic effects in tumorigenesis in various cancers. Since the precise role of LCN2 in prostate cancer (PCa) is poorly understood, we aimed to elucidate its functions in PCa in vitro. For this purpose, LCN2 was transiently suppressed or permanently depleted in human PC-3 cells using siRNA or CRISPR/Cas9-mediated knockout. Effects of LCN2 suppression on expression of different tumorigenic markers were investigated by Western blot analysis and RT-qPCR. LCN2 knockout cells were analyzed for cellular changes and their ability to cope endoplasmic stress compared to parenteral PC-3 cells. Reduced LCN2 was accompanied by decreased expression of IL-1β and Cx43. In PC-3 cells, LCN2 deficiency leads to reduced proliferation, diminished expression of pro-inflammatory cytokines, lower adhesion, and disrupted F-actin distribution. In addition, IL-1β expression strongly correlated with LCN2 levels. LCN2 knockout cells showed enhanced and sustained activation of unfolded protein response proteins when treated with tunicamycin or cultured under glucose deprivation. Interestingly, an inverse correlation between phosphorylation of eukaryotic initiation factor 2 α subunit (p-eIF2α) and LCN2 expression was observed suggesting that LCN2 triggers protein synthesis under stress conditions. The finding that LCN2 depletion leads to significant phenotypic and cellular changes in PC-3 cells adds LCN2 as a valuable target for the treatment of PCa.},
}
@article {pmid35053163,
year = {2021},
author = {Christopher, GA and Noort, RJ and Esseltine, JL},
title = {Connexin 43 Gene Ablation Does Not Alter Human Pluripotent Stem Cell Germ Lineage Specification.},
journal = {Biomolecules},
volume = {12},
number = {1},
pages = {},
pmid = {35053163},
issn = {2218-273X},
mesh = {*CRISPR-Cas Systems ; *Cell Lineage ; Connexin 43/*deficiency/metabolism ; Female ; *Gene Deletion ; Germ Layers/*metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; },
abstract = {During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell-cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.},
}
@article {pmid35053161,
year = {2021},
author = {Mandrioli, M},
title = {Genome Editing among Bioethics and Regulatory Practices.},
journal = {Biomolecules},
volume = {12},
number = {1},
pages = {},
pmid = {35053161},
issn = {2218-273X},
mesh = {Bioethics/*history ; *CRISPR-Cas Systems ; Gene Editing/*ethics/history ; History, 20th Century ; History, 21st Century ; Humans ; },
abstract = {In the last decade, genome editing technologies became very effective and several clinical trials have been started in order to use them for treating some genetic diseases. Interestingly, despite more than 50 years of discussion about the frontiers of genetics in human health and evolution, the debate about the bioethics and the regulatory practices of genome editing is still far from satisfactory answers. This delay results from an excessive emphasis on the effectiveness of the genome editing technologies that is relevant for the regulatory practices, but not at a bioethical level. Indeed, other factors (such as accessibility and acceptability) could make these techniques not accepted at the bioethical level, even in the presence of their 100% effectiveness.},
}
@article {pmid35051997,
year = {2022},
author = {Yuen, CTL and Thean, DGL and Chan, BKC and Zhou, P and Kwok, CCS and Chu, HY and Cheung, MSH and Wang, B and Chan, YM and Mak, SYL and Leung, AY and Choi, GCG and Zheng, Z and Wong, ASL},
title = {High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.},
journal = {Nucleic acids research},
volume = {50},
number = {3},
pages = {1650-1660},
pmid = {35051997},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Micrococcal Nuclease/genetics ; RNA, Guide ; *Staphylococcus aureus/genetics ; },
abstract = {The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.},
}
@article {pmid35051627,
year = {2022},
author = {Postma, ED and Hassing, EJ and Mangkusaputra, V and Geelhoed, J and de la Torre, P and van den Broek, M and Mooiman, C and Pabst, M and Daran, JM and Daran-Lapujade, P},
title = {Modular, synthetic chromosomes as new tools for large scale engineering of metabolism.},
journal = {Metabolic engineering},
volume = {72},
number = {},
pages = {1-13},
doi = {10.1016/j.ymben.2021.12.013},
pmid = {35051627},
issn = {1096-7184},
mesh = {*Anthocyanins ; CRISPR-Cas Systems ; Chromosomes/genetics/metabolism ; *Metabolic Engineering ; Metabolic Networks and Pathways ; Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.},
}
@article {pmid35051369,
year = {2022},
author = {Jin, WB and Li, TT and Huo, D and Qu, S and Li, XV and Arifuzzaman, M and Lima, SF and Shi, HQ and Wang, A and Putzel, GG and Longman, RS and Artis, D and Guo, CJ},
title = {Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.},
journal = {Cell},
volume = {185},
number = {3},
pages = {547-562.e22},
pmid = {35051369},
issn = {1097-4172},
support = {R21 AI142213/AI/NIAID NIH HHS/United States ; R01 AI095466/AI/NIAID NIH HHS/United States ; DP2 HD101401/HD/NICHD NIH HHS/United States ; R01 DK126871/DK/NIDDK NIH HHS/United States ; R01 AI151599/AI/NIAID NIH HHS/United States ; R01 DK128257/DK/NIDDK NIH HHS/United States ; U01 AI095608/AI/NIAID NIH HHS/United States ; R01 DK114252/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Bile Acids and Salts/metabolism ; CRISPR-Cas Systems/genetics ; Clostridium/genetics ; Colitis/chemically induced/microbiology/pathology ; Dextran Sulfate ; Drug Resistance, Microbial/genetics ; Female ; Gastrointestinal Microbiome/*genetics ; Gene Expression Regulation, Bacterial ; Gene Transfer Techniques ; *Genes, Bacterial ; Germ-Free Life ; Inflammation/pathology ; Intestines/pathology ; Male ; Metabolome/genetics ; Metagenomics ; Mice, Inbred C57BL ; Mice, Knockout ; Mutagenesis, Insertional/genetics ; Mutation/genetics ; RNA, Ribosomal, 16S/genetics ; Transcription, Genetic ; },
abstract = {Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.},
}
@article {pmid35051352,
year = {2022},
author = {Klompe, SE and Jaber, N and Beh, LY and Mohabir, JT and Bernheim, A and Sternberg, SH},
title = {Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons.},
journal = {Molecular cell},
volume = {82},
number = {3},
pages = {616-628.e5},
pmid = {35051352},
issn = {1097-4164},
support = {DP2 HG011650/HG/NHGRI NIH HHS/United States ; R01 EB031935/EB/NIBIB NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements/*genetics ; DNA, Bacterial/*genetics/metabolism ; Escherichia coli/*genetics/immunology/metabolism ; *Evolution, Molecular ; Gene Editing ; Gene Expression Regulation, Bacterial ; Genetic Variation ; Immunity, Innate ; RNA, Bacterial/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Transposases/*genetics/metabolism ; },
abstract = {Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.},
}
@article {pmid35051351,
year = {2022},
author = {Tan, R and Krueger, RK and Gramelspacher, MJ and Zhou, X and Xiao, Y and Ke, A and Hou, Z and Zhang, Y},
title = {Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems.},
journal = {Molecular cell},
volume = {82},
number = {4},
pages = {852-867.e5},
pmid = {35051351},
issn = {1097-4164},
support = {K99 GM117268/GM/NIGMS NIH HHS/United States ; R00 GM117268/GM/NIGMS NIH HHS/United States ; R35 GM118174/GM/NIGMS NIH HHS/United States ; R35 GM137883/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Deletion ; *Gene Editing ; *Genome, Human ; HEK293 Cells ; HeLa Cells ; Humans ; Neisseria lactamica/enzymology/*genetics ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer ∼95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid- and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.},
}
@article {pmid35051288,
year = {2022},
author = {Tulin, F},
title = {Two stones, one bird: co-targeting facilitates precise gene editing of non-selectable genes in Chlamydomonas.},
journal = {Plant physiology},
volume = {188},
number = {1},
pages = {16-18},
pmid = {35051288},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Genes, Plant ; Transformation, Genetic ; },
}
@article {pmid35051029,
year = {2022},
author = {Gao, Q and Lin, Y and Wang, X and Jing, D and Wang, Z and He, K and Bai, S and Zhang, Y and Zhang, T},
title = {Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis.},
journal = {Toxins},
volume = {14},
number = {1},
pages = {},
pmid = {35051029},
issn = {2072-6651},
mesh = {ATP Binding Cassette Transporter, Subfamily G/*genetics/metabolism ; Animals ; *Bacillus thuringiensis Toxins ; CRISPR-Cas Systems ; *Endotoxins ; Gene Knockout Techniques ; *Hemolysin Proteins ; Insect Control ; Insect Proteins/*genetics/metabolism ; Insecticide Resistance/*genetics ; Larva/drug effects/genetics/growth &amp; development ; Moths/drug effects/*genetics/growth &amp; development ; Pest Control, Biological ; },
abstract = {Ostrinia furnacalis is an important borer on maize. Long-term and large-scale planting of transgenic corn has led O. furnacalis evolving resistance and reducing the control effect. Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily G gene ABCG4 in O. furnacalis. In order to further determine the relationship between ABCG4 gene and the resistance to Cry1 toxins in O. furnacalis, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct ABCG4-KO knockout homozygous strain. Bioassay results indicated that an ABCG4-KO strain had a higher resistance to Cry1 proteins compared with a susceptible strain (ACB-BtS). The result indicates that the ABCG4 gene may act as a receptor of the Bt Cry1 toxin in O. furnacalis. Furthermore, the development time was significantly changed in the early stage ABCG4-KO larvae, and the population parameters were also significantly changed. In summary, our CRISPR/Cas9-mediated genome editing study presents evidence that ABCG4 gene is a functional receptor for Bt Cry1 toxins, laying the foundation for further clarification of the Bt resistance mechanism.},
}
@article {pmid35050652,
year = {2022},
author = {Schraivogel, D and Kuhn, TM and Rauscher, B and Rodríguez-Martínez, M and Paulsen, M and Owsley, K and Middlebrook, A and Tischer, C and Ramasz, B and Ordoñez-Rueda, D and Dees, M and Cuylen-Haering, S and Diebold, E and Steinmetz, LM},
title = {High-speed fluorescence image-enabled cell sorting.},
journal = {Science (New York, N.Y.)},
volume = {375},
number = {6578},
pages = {315-320},
pmid = {35050652},
issn = {1095-9203},
support = {294542/ERC_/European Research Council/International ; 742804/ERC_/European Research Council/International ; },
mesh = {Active Transport, Cell Nucleus ; Animals ; CRISPR-Cas Systems ; Cell Nucleus/metabolism ; Cell Shape ; *Flow Cytometry ; Genetic Techniques ; Genome ; Genome, Human ; Humans ; Microscopy, Fluorescence ; Mitosis ; NF-kappa B/metabolism ; *Optical Imaging ; Organelles/ultrastructure ; Phenotype ; Transcription Factor RelA/metabolism ; },
abstract = {Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.},
}
@article {pmid35050578,
year = {2022},
author = {Zhang, Q and Zhao, S and Tian, X and Qiu, JG and Zhang, CY},
title = {Development of a CRISPR-Cas-Based Biosensor for Rapid and Sensitive Detection of 8-Oxoguanine DNA Glycosylase.},
journal = {Analytical chemistry},
volume = {94},
number = {4},
pages = {2119-2125},
doi = {10.1021/acs.analchem.1c04453},
pmid = {35050578},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *DNA Glycosylases/genetics/metabolism ; DNA Repair ; Guanine/analogs &amp; derivatives ; Humans ; },
abstract = {8-Oxoguanine DNA glycosylase is essential for maintaining genomic integrity and stability, while its abnormal activity may lead to the disturbance in the normal DNA damage repair and the occurrence of carcinogenicity and teratogenicity. Herein, we construct a CRISPR-Cas-based biosensor for rapid and sensitive measurement of 8-oxoguanine DNA glycosylases. This biosensor involves a hairpin probe and integrates quadratic strand displacement amplification (SDA) with a CRISPR/Cas12a effector with the characteristics of rapidity (within 40 min) and isothermal assay. The presence of 8-oxoguanine DNA glycosylase can initiate the quadratic SDA to produce large amounts of activators with the assistance of polynucleotide kinase (PNK). Subsequently, the activators can bind with crRNA to activate Cas12a, cleaving signal probes and recovering Cy5 fluorescence, which can be accurately quantified by single-molecule imaging. Notably, the designed hairpin probes can effectively block the hybridization of the generated activators with free hairpin probes, endowing this biosensor with high sensitivity. In addition, the utilization of PNK instead of apurinic/apyrimidinic endonuclease (APE1) greatly simplifies the experimental procedure to only a one-step reaction. The introduction of a single-molecule detection further reduces the sample consumption and improves the sensitivity. This biosensor displays a detection limit of 4.24 × 10[-9] U μL[-1], and it can accurately quantify cellular human 8-oxoguanine DNA glycosylase at a single-cell level. Furthermore, this biosensor can be applied for the screening of inhibitors, the analysis of kinetic parameters, and the discrimination of cancer cells from normal cells, with potential applications in molecular diagnostic and point-of-care testing.},
}
@article {pmid35050192,
year = {2022},
author = {Katzmann, JL and Cupido, AJ and Laufs, U},
title = {Gene Therapy Targeting PCSK9.},
journal = {Metabolites},
volume = {12},
number = {1},
pages = {},
pmid = {35050192},
issn = {2218-1989},
abstract = {The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable-ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.},
}
@article {pmid35049658,
year = {2022},
author = {Paramasivam, K and Shen, Y and Yuan, J and Waheed, I and Mao, C and Zhou, X},
title = {Advances in the Development of Phage-Based Probes for Detection of Bio-Species.},
journal = {Biosensors},
volume = {12},
number = {1},
pages = {},
pmid = {35049658},
issn = {2079-6374},
mesh = {*Bacteriophages ; *CRISPR-Associated Proteins ; CRISPR-Cas Systems ; },
abstract = {Bacteriophages, abbreviated as "phages", have been developed as emerging nanoprobes for the detection of a wide variety of biological species, such as biomarker molecules and pathogens. Nanosized phages can display a certain length of exogenous peptides of arbitrary sequence or single-chain variable fragments (scFv) of antibodies that specifically bind to the targets of interest, such as animal cells, bacteria, viruses, and protein molecules. Metal nanoparticles generally have unique plasmon resonance effects. Metal nanoparticles such as gold, silver, and magnetism are widely used in the field of visual detection. A phage can be assembled with metal nanoparticles to form an organic-inorganic hybrid probe due to its nanometer-scale size and excellent modifiability. Due to the unique plasmon resonance effect of this composite probe, this technology can be used to visually detect objects of interest under a dark-field microscope. In summary, this review summarizes the recent advances in the development of phage-based probes for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays, and highlighting the commonly used editing technologies of phage genomes such as homologous recombination and clustered regularly interspaced palindromic repeats/CRISPR-associated proteins system (CRISPR-Cas). Finally, we discuss the possible scenarios for clinical application of phage-probe-based detection methods.},
}
@article {pmid35049639,
year = {2021},
author = {Yi, Z and de Dieu Habimana, J and Mukama, O and Li, Z and Odiwuor, N and Jing, H and Nie, C and Hu, M and Lin, Z and Wei, H and Zeng, L},
title = {Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities.},
journal = {Biosensors},
volume = {12},
number = {1},
pages = {},
pmid = {35049639},
issn = {2079-6374},
mesh = {*COVID-19/diagnosis ; *COVID-19 Testing ; *CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; RNA, Viral ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.},
}
@article {pmid35049367,
year = {2022},
author = {Allen, D and Weiss, LE and Saguy, A and Rosenberg, M and Iancu, O and Matalon, O and Lee, C and Beider, K and Nagler, A and Shechtman, Y and Hendel, A},
title = {High-Throughput Imaging of CRISPR- and Recombinant Adeno-Associated Virus-Induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {80-94},
pmid = {35049367},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; DNA Damage/genetics ; *Dependovirus/genetics ; *Gene Editing/methods ; Humans ; Stem Cells ; },
abstract = {CRISPR-Cas technology has revolutionized gene editing, but concerns remain due to its propensity for off-target interactions. This, combined with genotoxicity related to both CRISPR-Cas9-induced double-strand breaks and transgene delivery, poses a significant liability for clinical genome-editing applications. Current best practice is to optimize genome-editing parameters in preclinical studies. However, quantitative tools that measure off-target interactions and genotoxicity are costly and time-consuming, limiting the practicality of screening large numbers of potential genome-editing reagents and conditions. Here, we show that flow-based imaging facilitates DNA damage characterization of hundreds of human hematopoietic stem and progenitor cells per minute after treatment with CRISPR-Cas9 and recombinant adeno-associated virus serotype 6. With our web-based platform that leverages deep learning for image analysis, we find that greater DNA damage response is observed for guide RNAs with higher genome-editing activity, differentiating even single on-target guide RNAs with different levels of off-target interactions. This work simplifies the characterization and screening process of genome-editing parameters toward enabling safer and more effective gene-therapy applications.},
}
@article {pmid35048966,
year = {2022},
author = {Aviram, N and Thornal, AN and Zeevi, D and Marraffini, LA},
title = {Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {50},
number = {3},
pages = {1661-1672},
pmid = {35048966},
issn = {1362-4962},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Plasmids/genetics ; Staphylococcus epidermidis/*genetics/metabolism/virology ; },
abstract = {CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called 'spacers', into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.},
}
@article {pmid35045150,
year = {2022},
author = {Son, H and Park, J and Choi, YH and Jung, Y and Lee, JW and Bae, S and Lee, S},
title = {Exploring the dynamic nature of divalent metal ions involved in DNA cleavage by CRISPR-Cas12a.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {12},
pages = {1978-1981},
doi = {10.1039/d1cc04446j},
pmid = {35045150},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems ; Cations, Divalent ; DNA/*genetics ; Fluorescence Resonance Energy Transfer/methods ; Gene Editing/methods ; Metals/*chemistry ; Nucleic Acid Conformation ; },
abstract = {CRISPR-Cas12a has been widely used in genome editing and nucleic acid detection. In both of these applications, Cas12a cleaves target DNA in a divalent metal ion-dependent manner. However, when and how metal ions contribute to the cleavage reaction is unclear. Here, using a single-molecule FRET assay, we reveal that these metal ions are necessary for stabilising cleavage-competent conformations and that they are easily exchangeable, suggesting that they are dynamically coordinated.},
}
@article {pmid35044815,
year = {2022},
author = {Powell, JE and Lim, CKW and Krishnan, R and McCallister, TX and Saporito-Magriña, C and Zeballos, MA and McPheron, GD and Gaj, T},
title = {Targeted gene silencing in the nervous system with CRISPR-Cas13.},
journal = {Science advances},
volume = {8},
number = {3},
pages = {eabk2485},
pmid = {35044815},
issn = {2375-2548},
support = {R01 GM141296/GM/NIGMS NIH HHS/United States ; T32 EB019944/EB/NIBIB NIH HHS/United States ; },
mesh = {*Amyotrophic Lateral Sclerosis/genetics ; Animals ; *CRISPR-Cas Systems ; Gene Silencing ; Mammals ; Mice ; Spinal Cord ; Superoxide Dismutase ; Superoxide Dismutase-1/genetics ; },
abstract = {Cas13 nucleases are a class of programmable RNA-targeting CRISPR effector proteins that are capable of silencing target gene expression in mammalian cells. Here, we demonstrate that RfxCas13d, a Cas13 ortholog with favorable characteristics to other family members, can be delivered to the mouse spinal cord and brain to silence neurodegeneration-associated genes. Intrathecally delivering an adeno-associated virus vector encoding an RfxCas13d variant programmed to target superoxide dismutase 1 (SOD1), a protein whose mutation can cause amyotrophic lateral sclerosis, reduced SOD1 mRNA and protein in the spinal cord by >50% and improved outcomes in a mouse model of the disorder. We further show that intrastriatally delivering an RfxCas13d variant programmed to target huntingtin (HTT), a protein whose mutation is causative for Huntington’s disease, led to a ~50% reduction in HTT protein in the mouse brain. Our results establish RfxCas13d as a versatile platform for knocking down gene expression in the nervous system.},
}
@article {pmid35044159,
year = {2022},
author = {Fan, W and Han, P and Feng, Q and Sun, Y and Ren, W and Lawson, T and Liu, C},
title = {Nucleic Acid Substrate-Independent DNA Polymerization on the Exosome Membrane: A Mechanism Study and Application in Exosome Analysis.},
journal = {Analytical chemistry},
volume = {94},
number = {4},
pages = {2172-2179},
doi = {10.1021/acs.analchem.1c04636},
pmid = {35044159},
issn = {1520-6882},
mesh = {DNA/metabolism ; DNA Nucleotidylexotransferase/metabolism ; *Exosomes/metabolism ; Humans ; *Nucleic Acids ; Polymerization ; },
abstract = {As generally acknowledged, terminal deoxynucleotidyl transferase (TdT) can only elongate DNA substrates from their 3'-OH ends. Herein, for the first time, we report that TdT-catalyzed DNA polymerization can directly proceed on the exosome membrane without the mediation of any nucleic acids. We prove that both the glycosyl and phenolic hydroxyl groups on the membrane proteins can initiate the DNA polymerization. Accordingly, we have developed powerful strategies for high-sensitive exosome profiling based on a conventional flow cytometer and an emerging CRISPR/Cas system. By using our strategy, the featured membrane protein distributions of different cancer cell-derived exosomes can be figured out, which can clearly distinguish plasma samples of breast cancer patients from those of healthy people. This work paves new ways for exosome profiling and liquid biopsy and expands the understanding of TdT, holding great significance in developing TdT-based sensing systems as well as establishing protein/nucleic acid hybrid biomaterials.},
}
@article {pmid35043239,
year = {2022},
author = {Shi, J and Wei, L},
title = {Rho Kinases in Embryonic Development and Stem Cell Research.},
journal = {Archivum immunologiae et therapiae experimentalis},
volume = {70},
number = {1},
pages = {4},
pmid = {35043239},
issn = {1661-4917},
support = {P01 HL085098/HL/NHLBI NIH HHS/United States ; R01 HL107537/HL/NHLBI NIH HHS/United States ; R01 HL151480/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Embryonic Development ; Mice ; *Neoplasms ; Protein Isoforms ; Signal Transduction ; *Stem Cell Research ; rho-Associated Kinases/metabolism ; },
abstract = {The Rho-associated coiled-coil containing kinases (ROCKs or Rho kinases) belong to the AGC (PKA/PKG/PKC) family of serine/threonine kinases and are major downstream effectors of small GTPase RhoA, a key regulator of actin-cytoskeleton reorganization. The ROCK family contains two members, ROCK1 and ROCK2, which share 65% overall identity and 92% identity in kinase domain. ROCK1 and ROCK2 were assumed to be functionally redundant, based largely on their major common activators, their high degree kinase domain homology, and study results from overexpression with kinase constructs or chemical inhibitors. ROCK signaling research has expanded to all areas of biology and medicine since its discovery in 1996. The rapid advance is befitting ROCK's versatile functions in modulating various cell behavior, such as contraction, adhesion, migration, proliferation, polarity, cytokinesis, and differentiation. The rapid advance is noticeably driven by an extensive linking with clinical medicine, including cardiovascular abnormalities, aberrant immune responsive, and cancer development and metastasis. The rapid advance during the past decade is further powered by novel biotechnologies including CRISPR-Cas and single cell omics. Current consensus, derived mainly from gene targeting and RNA interference approaches, is that the two ROCK isoforms have overlapping and distinct cellular, physiological and pathophysiology roles. In this review, we present an overview of the milestone discoveries in ROCK research. We then focus on the current understanding of ROCK signaling in embryonic development, current research status using knockout and knockin mouse models, and stem cell research.},
}
@article {pmid35042129,
year = {2022},
author = {Hu, F and Liu, Y and Zhao, S and Zhang, Z and Li, X and Peng, N and Jiang, Z},
title = {A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {202},
number = {},
pages = {113994},
pmid = {35042129},
issn = {1873-4235},
mesh = {*African Swine Fever Virus ; Animals ; *Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; RNA, Viral/genetics ; Reproducibility of Results ; SARS-CoV-2 ; Sensitivity and Specificity ; Swine ; },
abstract = {The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.},
}
@article {pmid35042038,
year = {2022},
author = {Tao, Y and Yi, K and Wang, H and Kim, HW and Li, K and Zhu, X and Li, M},
title = {CRISPR-Cas12a-regulated DNA adsorption and metallization on MXenes as enhanced enzyme mimics for sensitive colorimetric detection of hepatitis B virus DNA.},
journal = {Journal of colloid and interface science},
volume = {613},
number = {},
pages = {406-414},
doi = {10.1016/j.jcis.2022.01.038},
pmid = {35042038},
issn = {1095-7103},
mesh = {Adsorption ; *Biosensing Techniques ; CRISPR-Cas Systems ; *Colorimetry ; DNA ; Hepatitis B virus/genetics ; Humans ; },
abstract = {Hepatitis B virus (HBV) infection is closely associated with the high risk of evolving into human hepatitis diseases including chronic hepatitis, liver fibrosis and cirrhosis, as well as hepatoma. Although various methods have been developed for HBV DNA detection, most of them either rely on expensive instruments or laborious procedures involving professional personnel. In this study, we for the first time established the CRISPR-Cas12a based colorimetric biosensor for target HBV detection by utilizing probe DNA regulation of the catalytic behaviors of Mxene-probe DNA-Ag/Pt nanohybrids. In the presence of HBV target, the Cas12a trans-cleavage activity could be efficiently activated to degrade the DNA probes, which led to the inhibition of DNA metallization and enzyme activity enhancer DNA adsorbed on Mxene, resulting in significantly reduced catalytic activity. The Mxene-probe DNA-Ag/Pt nanohybrids exhibited excellent sensitivity and specificity with subpicomolar detection limits, as well as good accuracy and stability for the determination of target HBV DNA in human serum samples. Moreover, this colorimetric sensing strategy could be integrated with the smartphone platform to allow the visible sensitive detection of target DNA. Taken together, the proposed colorimetric method provides a novel approach for HBV DNA diagnosis, especially suitable for the high endemic, developing countries with limited instrumental and medical supports.},
}
@article {pmid35041709,
year = {2022},
author = {Morgens, DW and Nandakumar, D and Didychuk, AL and Yang, KJ and Glaunsinger, BA},
title = {A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains.},
journal = {PLoS pathogens},
volume = {18},
number = {1},
pages = {e1010236},
pmid = {35041709},
issn = {1553-7374},
support = {R01 AI122528/AI/NIAID NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Gene Expression Regulation, Viral/*physiology ; Genes, Viral/*genetics ; HEK293 Cells ; Herpesvirus 8, Human/*genetics ; Humans ; },
abstract = {While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi's sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.},
}
@article {pmid35040978,
year = {2022},
author = {Li, D and Li, P and Peng, M and Zhao, X and Jiang, X and Wang, D and Yuan, Y and Guo, Q and Wang, M and Xu, X and Wang, M},
title = {Transmission barrier of the blaKPC plasmid mediated by type I restriction-modification systems in Escherichia coli.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {77},
number = {4},
pages = {952-956},
doi = {10.1093/jac/dkab489},
pmid = {35040978},
issn = {1460-2091},
mesh = {Bacterial Proteins/genetics ; DNA Restriction-Modification Enzymes ; *Escherichia coli/genetics ; Humans ; *Klebsiella Infections/microbiology ; Klebsiella pneumoniae/genetics ; Plasmids/genetics ; beta-Lactamases/genetics ; },
abstract = {BACKGROUND: Transportation of carbapenem-resistant plasmids contributes to carbapenem resistance in Gram-negative bacteria. KPC enzymes are the most clinically important enzymes among carbapenem-resistant Klebsiella pneumoniae, whereas the rate of blaKPC in Escherichia coli is low. The CRISPR-Cas system and restriction-modification system (R-M system) in bacteria defend against invading genomes. Currently, the role of the immune systems in the low rate of KPC-producing E. coli remains unclear.<br><br>OBJECTIVES: We investigated the relationship between immune systems and the low detection rate of blaKPC in E. coli.<br><br>METHODS: We searched for blaKPC among 1039 E. coli whole genomes available in GenBank using nucleotide BLAST. CRISPR-Cas systems and the R-M system were detected in all strains having the ST as blaKPC-positive strains. Nucleotide BLAST was used to search for protospacers on blaKPC plasmids. A conjugation assay was performed to determine whether the R-M system influences the acquisition of blaKPC plasmids by E. coli.<br><br>RESULTS: ST131 was the dominant ST of KPC-producing E. coli and IncN was the main plasmid type (12/32). CRISPR-Cas systems were frequently present in E. coli carrying blaKPC. Furthermore, CRISPR-Cas systems in E. coli didn't target plasmids with blaKPC. Type I R-M systems were rare in KPC-producing E. coli, but significantly over-represented in KPC-negative strains. E. coli DH5&#x3b1; with hsdR deletion accepted blaKPC-carrying plasmids, whereas those with hsdR complementation impeded blaKPC-carrying plasmid conjugation.<br><br>CONCLUSIONS: Horizontal transmission of blaKPC occurs among E. coli. The type I R-M system is associated with the defence against blaKPC plasmid transport into E. coli.},
}
@article {pmid35040621,
year = {2022},
author = {Hasanzadeh, A and Noori, H and Jahandideh, A and Haeri Moghaddam, N and Kamrani Mousavi, SM and Nourizadeh, H and Saeedi, S and Karimi, M and Hamblin, MR},
title = {Smart Strategies for Precise Delivery of CRISPR/Cas9 in Genome Editing.},
journal = {ACS applied bio materials},
volume = {5},
number = {2},
pages = {413-437},
doi = {10.1021/acsabm.1c01112},
pmid = {35040621},
issn = {2576-6422},
support = {R01 AI050875/AI/NIAID NIH HHS/United States ; R21 AI121700/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing ; Genome ; RNA, Guide/genetics ; },
abstract = {The emergence of CRISPR/Cas technology has enabled scientists to precisely edit genomic DNA sequences. This approach can be used to modulate gene expression for the treatment of genetic disorders and incurable diseases such as cancer. This potent genome-editing tool is based on a single guide RNA (sgRNA) strand that recognizes the targeted DNA, plus a Cas nuclease protein for binding and processing the target. CRISPR/Cas has great potential for editing many genes in different types of cells and organisms both in vitro and in vivo. Despite these remarkable advances, the risk of off-target effects has hindered the translation of CRISPR/Cas technology into clinical applications. To overcome this hurdle, researchers have devised gene regulatory systems that can be controlled in a spatiotemporal manner, by designing special sgRNA, Cas, and CRISPR/Cas delivery vehicles that are responsive to different stimuli, such as temperature, light, magnetic fields, ultrasound (US), pH, redox, and enzymatic activity. These systems can even respond to dual or multiple stimuli simultaneously, thereby providing superior spatial and temporal control over CRISPR/Cas gene editing. Herein, we summarize the latest advances on smart sgRNA, Cas, and CRISPR/Cas nanocarriers, categorized according to their stimulus type (physical, chemical, or biological).},
}
@article {pmid35040444,
year = {2022},
author = {Shin, H and Kim, J},
title = {Nanoparticle-based non-viral CRISPR delivery for enhanced immunotherapy.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {12},
pages = {1860-1870},
doi = {10.1039/d1cc05999h},
pmid = {35040444},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems ; *Genetic Vectors ; Humans ; Immunotherapy/*methods ; Nanoparticles/*administration &amp; dosage ; },
abstract = {The CRISPR Cas9 system has received considerable attention due to its simplicity, efficiency, and high precision for gene editing. The development of various therapeutic applications of the CRISPR system is under active research. In particular, its proven effects and promise in immunotherapy are of note. CRISPR/Cas9 components can be transported in various forms, such as plasmid DNA, mRNA of the Cas9 protein with gRNA, or a ribonucleoprotein complex. Even with its proven gene editing superiority, there are limitations in delivering the CRISPR system to target cells. CRISPR systems can be delivered via physical methods, viral vectors, or non-viral carriers. The development of diverse types of nanoparticles that could be used as non-viral carriers could overcome the disadvantages of physical techniques and viral vectors such as low cell viability, induction of immune response, limited loading capacity, and lack of targeting ability. Herein, we review the recent developments in applications of CRISPR system-mediated non-viral carriers in immunotherapy, depending on the targeting cell types, and discuss future research directions.},
}
@article {pmid35040120,
year = {2022},
author = {Wang, S and Chen, Y and Fang, H and Xu, Y and Ding, M and Ma, C and Lin, Y and Cui, Z and Sun, H and Niu, Q and Sun, S and Zhou, BS and Xiao, N and Li, H},
title = {A γ-glutamyl hydrolase lacking the signal peptide confers susceptibility to folates/antifolates in acute lymphoblastic leukemia cells.},
journal = {FEBS letters},
volume = {596},
number = {4},
pages = {437-448},
doi = {10.1002/1873-3468.14285},
pmid = {35040120},
issn = {1873-3468},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; Drug Resistance, Neoplasm/*genetics ; Folic Acid/*metabolism ; Folic Acid Antagonists/*pharmacology ; Gene Editing/methods ; Glycosylation ; HeLa Cells ; Humans ; Lymphocytes/*drug effects/metabolism/pathology ; Lysosomes/drug effects/metabolism ; Methotrexate/pharmacology ; Polyglutamic Acid/metabolism ; Protein Sorting Signals/*genetics ; Quinazolines/pharmacology ; Thiophenes/pharmacology ; gamma-Glutamyl Hydrolase/deficiency/*genetics ; },
abstract = {A key cofactor of several enzymes implicated in DNA synthesis, repair, and methylation, folate has been shown to be required for normal cell growth and replication and is the basis for cancer chemotherapy using antifolates. γ-Glutamyl hydrolase (GGH) catalyzes the removal of γ-polyglutamate tails of folylpoly-/antifolylpoly-γ-glutamates to facilitate their export out of the cell, thereby maintaining metabolic homeostasis of folates or pharmacological efficacy of antifolates. However, the factors that control or modulate GGH function are not well understood. In this study, we show that intact GGH is not indispensable for the chemosensitivity and growth of acute lymphoblastic leukemia (ALL) cells, whereas GGH lacking N-terminal signal peptide (GGH[-ΔN]) confers the significant drug resistance of ALL cells to the antifolates MTX and RTX. In addition, ALL cells harboring GGH[-ΔN] show high susceptibility to the change in folates, and glycosylation is not responsible for these phenotypes elicited by GGH[-ΔN] . Mechanistically, the loss of signal peptide enhances intracellular retention of GGH and its lysosomal disposition. Our findings clearly define the in vivo role of GGH in ALL cells and indicate a novel modulation of the GGH function, suggesting new avenues for ALL treatment in future.},
}
@article {pmid35039239,
year = {2022},
author = {Maganti, HB and Kirkham, AM and Bailey, AJM and Shorr, R and Kekre, N and Pineault, N and Allan, DS},
title = {Use of CRISPR/Cas9 gene editing to improve chimeric antigen-receptor T cell therapy: A systematic review and meta-analysis of preclinical studies.},
journal = {Cytotherapy},
volume = {24},
number = {4},
pages = {405-412},
doi = {10.1016/j.jcyt.2021.10.010},
pmid = {35039239},
issn = {1477-2566},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Glioma ; *Hematopoietic Stem Cell Transplantation ; Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/metabolism ; },
abstract = {BACKGROUND: Chimeric antigen-receptor T (CAR-T) cells represent great promise in cancer treatment. CRISPR/Cas9 gene editing in preclinical studies has enabled the development of enhanced CAR-T products with improved function and reduced toxicity.<br><br>METHODS: A systematic review of preclinical animal studies was conducted to determine the efficacy and safety of this approach.<br><br>RESULTS: 3753 records were identified (to September 9, 2020), with 11 studies using CRISPR/Cas9 gene editing in combination with CAR-T therapy against human cells in animal models of acute leukemia (four studies), glioma (two studies), melanoma (two studies), and other cancers (three studies). Compared with unedited controls, gene-edited CAR-T cells reduced tumor volume in treated animals and improved survival. No adverse side effects were reported. Use of allogeneic "third-party" CAR-T cells appears feasible. Improved efficacy was achieved through both knock-in and knockout gene editing of various targets implicated in immune function. Targeting multiple genes also appears feasible. Significant heterogeneity in study design and outcome reporting was observed, and potential bias was identified in all studies.<br><br>CONCLUSION: CRISPR/Cas9 gene editing enables manufacturing of CAR-T cells with improved anti-cancer effects. Future studies should reduce unintentional bias and heterogeneity of study designs and strive to augment long-term persistence of edited cells.<br><br>PROTOCOL REGISTRATION: PROSPERO; registration number CRD42020220313 registered November 30, 2020.},
}
@article {pmid35038453,
year = {2022},
author = {Xia, P and Dutta, A and Gupta, K and Batish, M and Parashar, V},
title = {Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {2},
pages = {101591},
pmid = {35038453},
issn = {1083-351X},
support = {R35 GM119504/GM/NIGMS NIH HHS/United States ; S10 OD018483/OD/NIH HHS/United States ; },
mesh = {*Adenine Nucleotides/chemistry/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coenzyme A ; DNA/genetics ; Models, Molecular ; *Oligoribonucleotides/chemistry/metabolism ; Structure-Activity Relationship ; Transcription Factors/chemistry/metabolism ; },
abstract = {RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.},
}
@article {pmid35038442,
year = {2022},
author = {Binti, S and Melinda, RV and Joseph, BB and Edeen, PT and Miller, SD and Fay, DS},
title = {A life cycle alteration can correct molting defects in Caenorhabditis elegans.},
journal = {Developmental biology},
volume = {483},
number = {},
pages = {143-156},
pmid = {35038442},
issn = {1095-564X},
support = {P20 GM103432/GM/NIGMS NIH HHS/United States ; R35 GM136236/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/*metabolism ; Caenorhabditis elegans Proteins/genetics/metabolism ; Endocytosis/genetics ; Epidermis/metabolism ; Forkhead Transcription Factors/genetics/metabolism ; *Gene Expression Regulation, Developmental ; Larva/genetics/metabolism ; Life Cycle Stages/*genetics ; Loss of Function Mutation ; Molting/*genetics ; NIMA-Related Kinases/*genetics/*metabolism ; Signal Transduction/genetics ; Starvation ; Sterols/metabolism ; Up-Regulation/genetics ; },
abstract = {Molting is a widespread feature in the development of many invertebrates, including nematodes and arthropods. In Caenorhabditis elegans, the highly conserved protein kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (NEKLs) promote molting through their involvement in the uptake and intracellular trafficking of epidermal cargos. We found that the relative requirements for NEKL-2 and NEKL-3 differed at different life-cycle stages and under different environmental conditions. Most notably, the transition from the second to the third larval stage (L2→L3 molt) required a higher level of NEKL function than during several other life stages or when animals had experienced starvation at the L1 stage. Specifically, larvae that entered the pre-dauer L2d stage could escape molting defects when transiting to the (non-dauer) L3 stage. Consistent with this, mutations that promote entry into L2d suppressed nekl-associated molting defects, whereas mutations that inhibit L2d entry reduced starvation-mediated suppression. We further showed that loss or reduction of NEKL functions led to defects in the transcription of cyclically expressed molting genes, many of which are under the control of systemic steroid hormone regulation. Moreover, the timing and severity of these transcriptional defects correlated closely with the strength of nekl alleles and with their stage of arrest. Interestingly, transit through L2d rescued nekl-associated expression defects in suppressed worms, providing an example of how life-cycle decisions can impact subsequent developmental events. Given that NEKLs are implicated in the uptake of sterols by the epidermis, we propose that loss of NEKLs leads to a physiological reduction in steroid-hormone signaling and consequent defects in the transcription of genes required for molting.},
}
@article {pmid35036136,
year = {2022},
author = {Ramnarine, SDBJ and Jayaraman, J and Ramsubhag, A},
title = {Comparative genomics of the black rot pathogen Xanthomonas campestris pv. campestris and non-pathogenic co-inhabitant Xanthomonas melonis from Trinidad reveal unique pathogenicity determinants and secretion system profiles.},
journal = {PeerJ},
volume = {9},
number = {},
pages = {e12632},
pmid = {35036136},
issn = {2167-8359},
abstract = {Black-rot disease caused by the phytopathogen Xanthomonas campestris pv. campestris (Xcc) continues to have considerable impacts on the productivity of cruciferous crops in Trinidad and Tobago and the wider Caribbean region. While the widespread occurrence of resistance of Xcc against bactericidal agrochemicals can contribute to the high disease burdens, the role of virulence and pathogenicity features of local strains on disease prevalence and severity has not been investigated yet. In the present study, a comparative genomic analysis was performed on 6 pathogenic Xcc and 4 co-isolated non-pathogenic Xanthomonas melonis (Xmel) strains from diseased crucifer plants grown in fields with heavy chemical use in Trinidad. Native isolates were grouped into two known and four newly assigned ribosomal sequence types (rST). Mobile genetic elements were identified which belonged to the IS3, IS5 family, Tn3 transposon, resolvases, and tra T4SS gene clusters. Additionally, exogenous plasmid derived sequences with origins from other bacterial species were characterised. Although several instances of genomic rearrangements were observed, native Xcc and Xmel isolates shared a significant level of structural homology with reference genomes, Xcc ATCC 33913 and Xmel CFBP4644, respectively. Complete T1SS hlyDB, T2SS, T4SS vir and T5SS xadA, yapH and estA gene clusters were identified in both species. Only Xmel strains contained a complete T6SS but no T3SS. Both species contained a complex repertoire of extracellular cell wall degrading enzymes. Native Xcc strains contained 37 T3SS and effector genes but a variable and unique profile of 8 avr, 4 xop and 1 hpa genes. Interestingly, Xmel strains contained several T3SS effectors with low similarity to references including avrXccA1 (~89%), hrpG (~73%), hrpX (~90%) and xopAZ (~87%). Furthermore, only Xmel genomes contained a CRISPR-Cas I-F array, but no lipopolysaccharide wxc gene cluster. Xmel strains were confirmed to be non-pathogenic by pathogenicity assays. The results of this study will be useful to guide future research into virulence mechanisms, agrochemical resistance, pathogenomics and the potential role of the co-isolated non-pathogenic Xanthomonas strains on Xcc infections.},
}
@article {pmid35034313,
year = {2022},
author = {Li, Y and Zhang, H and Chen, Z and Fan, J and Chen, T and Zeng, B and Zhang, Z},
title = {Construction of single, double, or triple mutants within kojic acid synthesis genes kojA, kojR, and kojT by the CRISPR/Cas9 tool in Aspergillus oryzae.},
journal = {Folia microbiologica},
volume = {67},
number = {3},
pages = {459-468},
pmid = {35034313},
issn = {1874-9356},
mesh = {*Aspergillus oryzae/genetics/metabolism ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Pyrones/metabolism ; },
abstract = {Kojic acid is an industrially important secondary metabolite produced by Aspergillus oryzae. The construction of genetic materials for kojic acid related genes is important for understanding the mechanism of kojic acid synthesis in A. oryzae. However, multigene simultaneous knockout mutants for kojic acid synthesis genes remain limited because A. oryzae is multinuclear and good selectable markers are scarce. Here, we firstly successfully obtained single mutants of kojA, kojR, and kojT by our previously constructed CRISPR/Cas9 system in A. oryzae, which demonstrated the feasibility of the targeting sgRNAs for kojA, kojR, and kojT. Then, the AMA1-based genome-editing system for multiplex gene editing was developed in A. oryzae. In the multiplex gene-editing system, two guide RNA expression cassettes were ligated in tandem and driven by two U6 promoters in the AMA1-based autonomously replicating plasmid with the Cas9-expression cassette. Moreover, the multiplex gene-editing technique was applied to target the kojic acid synthesis genes kojA, kojR, and kojT, and the double and triple mutants within kojA, kojR, and kojT were obtained successfully. Additionally, the selectable marker pyrG was knocked out in the single and triple mutants of kojA, kojR, and kojT to obtain the auxotrophic strains, which can facilitate to introduce a target gene into the single and triple mutants of kojA, kojR, and kojT for investigating their relationship. The multiplex gene-editing system and release of these materials provide a foundation for further kojic acid research and utilization.},
}
@article {pmid35033831,
year = {2022},
author = {Vyas, P and Harish, },
title = {Anti-CRISPR proteins as a therapeutic agent against drug-resistant bacteria.},
journal = {Microbiological research},
volume = {257},
number = {},
pages = {126963},
doi = {10.1016/j.micres.2022.126963},
pmid = {35033831},
issn = {1618-0623},
mesh = {Bacteria/genetics/metabolism ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; *Pharmaceutical Preparations ; Viral Proteins/metabolism ; },
abstract = {The continuous deployment of various antibiotics to treat multiple serious bacterial infections leads to multidrug resistance among the bacterial population. It has failed the standard treatment strategies through different antibacterial agents and serves as a significant threat to public health worldwide at devastating levels. The discovery of anti-CRISPR proteins catches the interest of researchers around the world as a promising therapeutic agent against drug-resistant bacteria. Anti-CRISPR proteins are known to inhibit bacterial CRISPR-Cas defense systems in multiple possible ways. The CRISPR-Cas nucleoprotein assembly provides adaptive immunity in bacteria against diverse categories of phage infections. Parallelly, phages also try to break the CRISPR-Cas barrier by producing anti-CRISPR proteins, leading to growth inhibition and bacterial lysis. This review begins with a brief description of the bacterial CRISPR-Cas system, followed by a detailed portrayal of anti-CRISPR proteins, including their discovery and evolution, mechanism of action, regulation of expression, and potential applications in the healthcare sector as an alternative therapeutic strategy to combat severe bacterial infections.},
}
@article {pmid35033172,
year = {2022},
author = {Zhang, J and Li, Y and Liu, H and Zhang, J and Wang, J and Xia, J and Zhang, Y and Yu, X and Ma, J and Huang, M and Wang, J and Wang, L and Li, Q and Cui, R and Yang, W and Xu, Y and Feng, W},
title = {Genome-wide CRISPR/Cas9 library screen identifies PCMT1 as a critical driver of ovarian cancer metastasis.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {41},
number = {1},
pages = {24},
pmid = {35033172},
issn = {1756-9966},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Female ; Humans ; Mice ; Mice, Nude ; Middle Aged ; Neoplasm Metastasis ; Ovarian Neoplasms/*genetics/pathology ; Protein D-Aspartate-L-Isoaspartate Methyltransferase/*metabolism ; },
abstract = {BACKGROUND: The development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance.<br><br>METHODS: Genome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging.<br><br>RESULTS: We found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors.<br><br>CONCLUSIONS: Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.},
}
@article {pmid35032802,
year = {2022},
author = {Yu, Y and Li, W and Gu, X and Yang, X and Han, Y and Ma, Y and Wang, Z and Zhang, J},
title = {Inhibition of CRISPR-Cas12a trans-cleavage by lead (II)-induced G-quadruplex and its analytical application.},
journal = {Food chemistry},
volume = {378},
number = {},
pages = {131802},
doi = {10.1016/j.foodchem.2021.131802},
pmid = {35032802},
issn = {1873-7072},
mesh = {*CRISPR-Cas Systems ; *G-Quadruplexes ; },
abstract = {In this work, the inhibition of clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR associated protein (Cas) trans-cleavage by Pb[2+]-induced G-quadruplex has been firstly explored to detect Pb[2+] in tea beverage and milk. In absence of Pb[2+], the Na[+]-induced G-quadruplex can be cleaved by CRISPR-Cas12a. In contrast, Pb[2+] can competitively combine with G-quadruplex, resulting in its conformational changes and resistance to trans-cleavage of CRISPR-Cas12a. Therefore, the fluorescence resonance energy transfer can happen. Pb[2+] can be detected in a linear range from 100 nM to 5 µM with a lowest detection limit of 2.6 nM and a relative standard deviation of 4.32%. In summary, this work not only provides a new method for Pb[2+] detection based on its induced G-quadruplex inhibition on CRISPR-Cas12a trans-cleavage, but also broadens the application of CRISPR-Cas system for heavy metal analysis in the field of food safety.},
}
@article {pmid35032250,
year = {2022},
author = {Rehman, F and Gong, H and Bao, Y and Zeng, S and Huang, H and Wang, Y},
title = {CRISPR gene editing of major domestication traits accelerating breeding for Solanaceae crops improvement.},
journal = {Plant molecular biology},
volume = {108},
number = {3},
pages = {157-173},
pmid = {35032250},
issn = {1573-5028},
mesh = {*Breeding ; *CRISPR-Cas Systems ; Gene Expression Regulation, Plant/*physiology ; *Genetic Engineering ; Plant Proteins/genetics/*metabolism ; Solanaceae/*genetics ; },
abstract = {Domestication traits particularly fruit size and plant architecture and flowering are critical in transforming a progenitor's wild stature into a super improved plant. The latest advancements in the CRISPR system, as well as its rapid adoption, are speeding up plant breeding. Solanaceae has a varied range of important crops, with a few model crops, such as tomato and, more recently, groundcherry, serving as a foundation for developing molecular techniques, genome editing tools, and establishing standards for other crops. Domestication traits in agricultural plants are quantified and widely adopted under modern plant breeding to improve small-fruited and bushy crop species like goji berry. The molecular mechanisms of the FW2.2, FW3.2, FW11.3, FAS/CLV3, LC/WUS, SP, SP5G, and CRISPR genome editing technology have been described in detail here. Furthermore, special focus has been placed on CRISPR gene editing achievements for revolutionizing Solanaceae breeding and changing the overall crop landscape. This review seeks to provide a thorough overview of the CRISPR technique's ongoing advancements, particularly in Solanaceae, in terms of domesticated features, future prospects, and regulatory risks. We believe that this vigorous discussion will lead to a broader understanding of CRISPR gene editing as a tool for achieving key breeding goals in other Solanaceae minor crops with significant industrial value.},
}
@article {pmid35032067,
year = {2022},
author = {Vera, LNP and Schuh, RS and Fachel, FNS and Poletto, E and Piovesan, E and Kubaski, F and Couto, E and Brum, B and Rodrigues, G and Souza, H and Giugliani, R and Matte, U and Baldo, G and Teixeira, HF},
title = {Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.},
journal = {The journal of gene medicine},
volume = {24},
number = {4},
pages = {e3410},
doi = {10.1002/jgm.3410},
pmid = {35032067},
issn = {1521-2254},
mesh = {Animals ; Brain/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Iduronidase/genetics/metabolism ; Mice ; *Mucopolysaccharidosis I/genetics/therapy ; Plasmids ; },
abstract = {BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-l-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice.<br><br>METHODS: Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30&#x2009;days in young MPS I mice, which were euthanized at 6&#xa0;months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated.<br><br>RESULTS: Monodisperse mucoadhesive complexes around 110&#x2009;nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage.<br><br>CONCLUSION: Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders.},
}
@article {pmid35031635,
year = {2022},
author = {Diaz, R and Kronenberg, NM and Martinelli, A and Liehm, P and Riches, AC and Gather, MC and Paracchini, S},
title = {KIAA0319 influences cilia length, cell migration and mechanical cell-substrate interaction.},
journal = {Scientific reports},
volume = {12},
number = {1},
pages = {722},
pmid = {35031635},
issn = {2045-2322},
support = {BB/P027148/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 105621/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Actins/metabolism ; CRISPR-Cas Systems ; Cell Communication/*genetics/*physiology ; Cell Line ; Cell Movement/*genetics/*physiology ; Cilia/*genetics/*physiology ; Humans ; Microscopy, Interference ; Models, Genetic ; Nerve Tissue Proteins/*genetics/*physiology ; Podosomes/physiology ; Retinal Pigment Epithelium/*cytology/metabolism ; Vinculin/metabolism ; },
abstract = {Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell-substrate force regulation.},
}
@article {pmid35031563,
year = {2022},
author = {Liao, Y and Chen, CH and Xiao, T and de la Peña Avalos, B and Dray, EV and Cai, C and Gao, S and Shah, N and Zhang, Z and Feit, A and Xue, P and Liu, Z and Yang, M and Lee, JH and Xu, H and Li, W and Mei, S and Pierre, RS and Shu, S and Fei, T and Duarte, M and Zhao, J and Bradner, JE and Polyak, K and Kantoff, PW and Long, H and Balk, SP and Liu, XS and Brown, M and Xu, K},
title = {Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {3},
pages = {},
pmid = {35031563},
issn = {1091-6490},
support = {P50 CA090381/CA/NCI NIH HHS/United States ; K99 CA178199/CA/NCI NIH HHS/United States ; R01 GM137009/GM/NIGMS NIH HHS/United States ; R01 HG008927/HG/NHGRI NIH HHS/United States ; P01 CA163227/CA/NCI NIH HHS/United States ; R00 CA178199/CA/NCI NIH HHS/United States ; K99 CA166507/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Damage/*genetics/*physiology ; DNA Repair/genetics/physiology ; Enhancer of Zeste Homolog 2 Protein/*genetics/*metabolism ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Hepatocyte Nuclear Factor 3-alpha/genetics/metabolism ; Humans ; Male ; Prostatic Neoplasms, Castration-Resistant/genetics/metabolism ; *Transcriptional Activation ; },
abstract = {Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.},
}
@article {pmid35031438,
year = {2022},
author = {Sun, ML and Shi, TQ and Lin, L and Ledesma-Amaro, R and Ji, XJ},
title = {Advancing Yarrowia lipolytica as a superior biomanufacturing platform by tuning gene expression using promoter engineering.},
journal = {Bioresource technology},
volume = {347},
number = {},
pages = {126717},
doi = {10.1016/j.biortech.2022.126717},
pmid = {35031438},
issn = {1873-2976},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Expression ; Metabolic Engineering ; *Yarrowia/genetics ; },
abstract = {Yarrowia lipolytica is recognized as an excellent non-conventional yeast in the field of biomanufacturing, where it is used as a host to produce oleochemicals, terpenes, organic acids, polyols and recombinant proteins. Consequently, metabolic engineering of this yeast is becoming increasingly popular to advance it as a superior biomanufacturing platform, of which promoters are the most basic elements for tuning gene expression. Endogenous promoters of Yarrowia lipolytica were reviewed, which are the basis for promoter engineering. The engineering strategies, such as hybrid promoter engineering, intron enhancement promoter engineering, and transcription factor-based inducible promoter engineering are described. Additionally, the applications of Yarrowia lipolytica promoter engineering to rationally reconstruct biosynthetic gene clusters and improve the genome-editing efficiency of the CRISPR-Cas systems were reviewed. Finally, research needs and future directions for promoter engineering are also discussed in this review.},
}
@article {pmid35031384,
year = {2022},
author = {Abdolhosseini, M and Zandsalimi, F and Moghaddam, FS and Tavoosidana, G},
title = {A review on colorimetric assays for DNA virus detection.},
journal = {Journal of virological methods},
volume = {301},
number = {},
pages = {114461},
doi = {10.1016/j.jviromet.2022.114461},
pmid = {35031384},
issn = {1879-0984},
mesh = {*Colorimetry/methods ; DNA ; DNA Viruses ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Early detection is one of the ways to deal with DNA virus widespread prevalence, and it is necessary to know new diagnostic methods and techniques. Colorimetric assays are one of the most advantageous methods in detecting viruses. These methods are based on color change, which can be seen either with the naked eye or with special devices. The aim of this study is to introduce and evaluate effective colorimetric methods based on amplification, nanoparticle, CRISPR/Cas, and Lateral flow in the diagnosis of DNA viruses and to discuss the effectiveness of each of the updated methods. Compared to the other methods, colorimetric assays are preferred for faster detection, high efficiency, cheaper cost, and high sensitivity and specificity. It is expected that the spread of these viruses can be prevented by identifying and developing new methods.},
}
@article {pmid35031120,
year = {2022},
author = {Miyagawa, S and Watanabe, M and Nagashima, H and Sato, K and Kogata, S and Toyama, C and Masahata, K and Kamiyama, M and Yamamoto, R and Eguchi, H and Maeda, A and Yoshimi, K and Mashimo, T and Okuyama, H},
title = {Study of the CRISPR/Cas3 System for Xenotransplantation.},
journal = {Transplantation proceedings},
volume = {54},
number = {2},
pages = {522-524},
doi = {10.1016/j.transproceed.2021.09.070},
pmid = {35031120},
issn = {1873-2623},
mesh = {Animals ; *CRISPR-Cas Systems ; *Escherichia coli ; Humans ; Swine ; Transfection ; Transplantation, Heterologous ; },
abstract = {The CRISPR/Cas3 system, classified in class I system, was recently focused as a new technology. For application of this system to porcine cells, the plasmids of bpNLS-Cascade, BPNLS-hCas3, and pBS-U6icrRNA were prepared. Initially, 2 crRNAs were established in the exon 9 of pig Gal-T (GGTA1) as #45 and #86. Next, hCas3 + #45 + #86 (group 1, control), Cascade + hCas3 + #45 (group 2), Cascade + hCas3 + #86 (group 3), and Cascade + hCas3 + #45 + #86 (group 4) were set and transfected into pig fibroblasts. Transfected cells were analyzed for bulk expression of α1,3Gal epitope by fluorescence-activated cell sorting (FACS), using a GSI-B4 lectin 2 days after the transfection. As the results, changes of expression are observed in order of G4>G2>G3, indicating the effect of the Cas3 system. Therefore, the nested polymerase chain reaction (PCR) for target region of GGTA1 was performed. Next, the PCR products from each group were checked in blotting, and the products were placed into the cloning sit of TOPO vector and transformed into Escherichia coli. Sixteen colonies of each group were checked by PCR, and clones containing PCR product with slightly varying length were evaluated. The direct sequence of these PCR changes were demonstrated as 294 to 754 bp deletions. In conclusion, we confirmed the effect of the CRISPR/Cas3 system on pig cell, especially in xenotransplantation.},
}
@article {pmid35029964,
year = {2022},
author = {Rybnicky, GA and Dixon, RA and Kuhn, RM and Karim, AS and Jewett, MC},
title = {Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom.},
journal = {ACS synthetic biology},
volume = {11},
number = {2},
pages = {835-842},
doi = {10.1021/acssynbio.1c00503},
pmid = {35029964},
issn = {2161-5063},
mesh = {Biotechnology ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Engineering ; Humans ; Synthetic Biology/education ; *Wolbachia/genetics ; },
abstract = {Training the future synthetic biology workforce requires the opportunity for students to be exposed to biotechnology concepts and activities in secondary education. Detecting Wolbachia bacteria in arthropods using polymerase chain reaction (PCR) has become a common way for secondary students to investigate and apply recombinant DNA technology in the science classroom. Despite this important activity, cutting-edge biotechnologies such as clustered regularly interspaced short palindromic repeat (CRISPR)-based diagnostics have yet to be widely implemented in the classroom. To address this gap, we present a freeze-dried CRISPR-Cas12 sensing reaction to complement traditional recombinant DNA technology education and teach synthetic biology concepts. The reactions accurately detect Wolbachia from arthropod-derived PCR samples in under 2 h and can be stored at room temperature for over a month without appreciable degradation. The reactions are easy-to-use and cost less than $40 to implement for a classroom of 22 students including the cost of reusable equipment. We see these freeze-dried CRISPR-Cas12 reactions as an accessible way to incorporate synthetic biology education into the existing biology curriculum, which will expand biology educational opportunities in science, technology, engineering, and mathematics.},
}
@article {pmid35029639,
year = {2022},
author = {Prescott, JA and Balmanno, K and Mitchell, JP and Okkenhaug, H and Cook, SJ},
title = {IKKα plays a major role in canonical NF-κB signalling in colorectal cells.},
journal = {The Biochemical journal},
volume = {479},
number = {3},
pages = {305-325},
pmid = {35029639},
issn = {1470-8728},
support = {BB/S017062/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013384/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Colorectal Neoplasms/genetics/*metabolism/pathology ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; I-kappa B Kinase/antagonists &amp; inhibitors/genetics/*metabolism ; Interleukin-1alpha/metabolism ; NF-KappaB Inhibitor alpha/metabolism ; NF-kappa B/genetics/*metabolism ; Phosphorylation/genetics ; Protein Kinase Inhibitors/pharmacology ; RNA, Small Interfering/genetics ; Signal Transduction/drug effects/*genetics ; Transfection ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells, IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.},
}
@article {pmid35029089,
year = {2022},
author = {Pan, Q and Luo, J and Jiang, Y and Wang, Z and Lu, K and Chen, T},
title = {Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA.},
journal = {Journal of Zhejiang University. Science. B},
volume = {23},
number = {1},
pages = {74-83},
pmid = {35029089},
issn = {1862-1783},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Oryzias/genetics ; RNA, Guide/genetics ; RNA, Transfer/genetics ; },
abstract = {Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.},
}
@article {pmid35027735,
year = {2022},
author = {Zhang, HL and Hu, BX and Li, ZL and Du, T and Shan, JL and Ye, ZP and Peng, XD and Li, X and Huang, Y and Zhu, XY and Chen, YH and Feng, GK and Yang, D and Deng, R and Zhu, XF},
title = {PKCβII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis.},
journal = {Nature cell biology},
volume = {24},
number = {1},
pages = {88-98},
pmid = {35027735},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Coenzyme A Ligases/*metabolism ; Ferroptosis/*physiology ; Gene Knockout Techniques ; Humans ; Immunotherapy/methods ; Lipid Peroxidation/*physiology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Neoplasms/therapy ; Phosphorylation ; Protein Kinase C beta/genetics/*metabolism ; },
abstract = {The accumulation of lipid peroxides is recognized as a determinant of the occurrence of ferroptosis. However, the sensors and amplifying process of lipid peroxidation linked to ferroptosis remain obscure. Here we identify PKCβII as a critical contributor of ferroptosis through independent genome-wide CRISPR-Cas9 and kinase inhibitor library screening. Our results show that PKCβII senses the initial lipid peroxides and amplifies lipid peroxidation linked to ferroptosis through phosphorylation and activation of ACSL4. Lipidomics analysis shows that activated ACSL4 catalyses polyunsaturated fatty acid-containing lipid biosynthesis and promotes the accumulation of lipid peroxidation products, leading to ferroptosis. Attenuation of the PKCβII-ACSL4 pathway effectively blocks ferroptosis in vitro and impairs ferroptosis-associated cancer immunotherapy in vivo. Our results identify PKCβII as a sensor of lipid peroxidation, and the lipid peroxidation-PKCβII-ACSL4 positive-feedback axis may provide potential targets for ferroptosis-associated disease treatment.},
}
@article {pmid35027730,
year = {2022},
author = {Mirman, Z and Sasi, NK and King, A and Chapman, JR and de Lange, T},
title = {53BP1-shieldin-dependent DSB processing in BRCA1-deficient cells requires CST-Polα-primase fill-in synthesis.},
journal = {Nature cell biology},
volume = {24},
number = {1},
pages = {51-61},
pmid = {35027730},
issn = {1476-4679},
support = {R35 CA210036/CA/NCI NIH HHS/United States ; MC_UU_00016/19/MRC_/Medical Research Council/United Kingdom ; C52690/A19270/CRUK_/Cancer Research UK/United Kingdom ; MR/R017549/1/MRC_/Medical Research Council/United Kingdom ; F99 CA245720/CA/NCI NIH HHS/United States ; K00 CA245720/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; BRCA1 Protein/*genetics ; Binding Sites/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA/genetics ; *DNA Breaks, Double-Stranded ; DNA Polymerase I/*metabolism ; DNA Primase/genetics/metabolism ; DNA Repair/*genetics ; Gene Knockout Techniques ; Humans ; Mice ; Poly(ADP-ribose) Polymerase Inhibitors/metabolism ; Poly(ADP-ribose) Polymerases/metabolism ; Rad51 Recombinase/metabolism ; Shelterin Complex/*metabolism ; Telomere-Binding Proteins/metabolism ; Tumor Suppressor p53-Binding Protein 1/genetics/*metabolism ; },
abstract = {The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Polα-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Polα) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Polα-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Polα-primase is the major effector of shieldin-dependent DSB processing.},
}
@article {pmid35027641,
year = {2022},
author = {Kouranov, A and Armstrong, C and Shrawat, A and Sidorov, V and Huesgen, S and Lemke, B and Boyle, T and Gasper, M and Lawrence, R and Yang, S},
title = {Demonstration of targeted crossovers in hybrid maize using CRISPR technology.},
journal = {Communications biology},
volume = {5},
number = {1},
pages = {53},
pmid = {35027641},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; *Chromosomes, Plant ; *Crossing Over, Genetic ; DNA End-Joining Repair ; Gene Editing/*methods ; *Hybridization, Genetic ; Zea mays/*genetics ; },
abstract = {Naturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding.},
}
@article {pmid35027447,
year = {2022},
author = {Shanmugam, R and Ozturk, MB and Low, JL and Akincilar, SC and Chua, JYH and Thangavelu, MT and Periyasamy, G and DasGupta, R and Tergaonkar, V},
title = {Genome-wide screens identify specific drivers of mutant hTERT promoters.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {3},
pages = {},
pmid = {35027447},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin ; DNA-Binding Proteins ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; Mediator Complex/genetics/metabolism ; *Mutation ; Neoplasms/genetics ; *Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid ; Telomerase/*genetics/metabolism ; Transcription Factors ; Transcription, Genetic ; },
abstract = {Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.},
}
@article {pmid35026545,
year = {2022},
author = {Li, H and Zhao, W and Pu, J and Zhong, S and Wang, S and Yu, R},
title = {Combining functional hairpin probes with disordered cleavage of CRISPR/Cas12a protease to screen for B lymphocytic leukemia.},
journal = {Biosensors &amp; bioelectronics},
volume = {201},
number = {},
pages = {113941},
doi = {10.1016/j.bios.2021.113941},
pmid = {35026545},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Humans ; *Leukemia, B-Cell ; Peptide Hydrolases ; },
abstract = {One of the causes of B lymphocytic leukemia is abnormal expression of the Pax-5a gene. Detection of the Pax-5a gene can provide effective technical means for early screening of B lymphocytic leukemia. In this work, we designed a sensing scheme to detect the Pax-5a gene based on the signal amplification system, which is based on dual-enzyme assisted target gene circulation, and the disordered cleavage of CRISPR/Cas12a protease. The hairpin probe (HP) in this scheme not only contains the binding sites of the target gene and the primer, but also cleverly contains half of the Nt.BbvCI splicing sites. When the target gene is present, through the synergistic effect of KF and Nt.BbvCI, a large number of single strands of a specific sequence can be produced. At the same time, the target gene falls off from the first hairpin and opens the other hairpin to realize the cycle of the target gene. The resulting single-strand can bind to the Cas12a/crRNA binary complex and unlock the anti-cleavage activity of the CRISPR/Cas12a protease. The single strands labeled with the fluorescent group (FAM) and the quenching group (BHQ) around the solution are cleaved, the fluorescence signal of FAM is restored, and a detectable fluorescence signal is generated. The detection limit is as low as 6.77 fM, and the target gene and the mismatch sequence can also be distinguished well. Therefore, the sensing scheme provides a new detection direction for the early diagnosis and screening of B lymphocytic leukemia.},
}
@article {pmid35026470,
year = {2022},
author = {Peleg-Chen, D and Shuvali, G and Brio, L and Ifrach, A and Iancu, O and Barbiro-Michaely, E and Hendel, A and Gerber, D},
title = {Microfluidic tool for rapid functional characterization of CRISPR complexes.},
journal = {New biotechnology},
volume = {68},
number = {},
pages = {1-8},
pmid = {35026470},
issn = {1876-4347},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genomics ; *Microfluidics ; RNA, Guide/genetics/metabolism ; },
abstract = {RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic approach to characterizing Enzymatic Interaction and Function of CRISPR complexes on a microfluidic platform (EnzyMIF) is presented. The method can rapidly detect the kd, koff, km and kcat for various RNA guided nucleases. In this work, two single guide RNAs with significantly different in-cell cleavage efficiency, RAG2 and RAG1, are used as proof-of-concept. The EnzyMIF assay results provide biochemical characterization of these guide RNAs that can explain the difference in cleavage using both wild type (WT) CAS9 and HiFi CAS9. Notably, it is shown that EnzyMIF characterization correlates with cell culture genomic editing efficiency results. It is suggested that EnzyMIF can predict the quality of cleavage rapidly and quantitatively.},
}
@article {pmid35026352,
year = {2022},
author = {Taha, EA and Lee, J and Hotta, A},
title = {Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {342},
number = {},
pages = {345-361},
doi = {10.1016/j.jconrel.2022.01.013},
pmid = {35026352},
issn = {1873-4995},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Liposomes ; *Nanoparticles ; },
abstract = {The discovery of clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technology opened the door to provide a versatile approach for treating multiple diseases. Promising results have been shown in numerous pre-clinical studies and clinical trials. However, a safe and effective method to deliver genome-editing components is still a key challenge for in vivo genome editing therapy. Adeno-associated virus (AAV) is one of the most commonly used vector systems to date, but immunogenicity against capsid, liver toxicity at high dose, and potential genotoxicity caused by off-target mutagenesis and genomic integration remain unsolved. Recently developed transient delivery systems, such as virus-like particle (VLP) and lipid nanoparticle (LNP), may solve some of the issues. This review summarizes existing in vivo delivery systems and possible solutions to overcome their limitations. Also, we highlight the ongoing clinical trials for in vivo genome editing therapy and recently developed genome editing tools for their potential applications.},
}
@article {pmid35026132,
year = {2022},
author = {Barrangou, R and Marraffini, LA},
title = {Turning CRISPR on with antibiotics.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {1},
pages = {12-14},
doi = {10.1016/j.chom.2021.12.013},
pmid = {35026132},
issn = {1934-6069},
mesh = {*Anti-Bacterial Agents/pharmacology ; Bacteria/genetics ; Base Sequence ; CRISPR-Cas Systems ; *Viruses ; },
abstract = {CRISPR-Cas systems have the ability to integrate invasive DNA sequences to build adaptive immunity in bacteria. In this issue Dimitriu et al. show bacteriostatic antibiotics prompt CRISPR acquisition events, illustrating how environmental conditions affect complex dynamics between host and virus and the corresponding biological and genetic arms race.},
}
@article {pmid35025886,
year = {2022},
author = {Simões, ML and Dong, Y and Mlambo, G and Dimopoulos, G},
title = {C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites.},
journal = {PLoS biology},
volume = {20},
number = {1},
pages = {e3001515},
pmid = {35025886},
issn = {1545-7885},
support = {R21 AI131574/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Anopheles/genetics/*immunology/parasitology ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Insect Proteins/genetics/metabolism ; Lectins, C-Type/*genetics/metabolism ; Melanins/genetics/immunology ; Plasmodium berghei/*physiology ; Plasmodium falciparum/*physiology ; },
abstract = {Anopheles gambiae melanization-based refractoriness to the human malaria parasite Plasmodium falciparum has rarely been observed in either laboratory or natural conditions, in contrast to the rodent model malaria parasite Plasmodium berghei that can become completely melanized by a TEP1 complement-like system-dependent mechanism. Multiple studies have shown that the rodent parasite evades this defense by recruiting the C-type lectins CTL4 and CTLMA2, while permissiveness to the human malaria parasite was not affected by partial depletion of these factors by RNAi silencing. Using CRISPR/Cas9-based CTL4 knockout, we show that A. gambiae can mount melanization-based refractoriness to the human malaria parasite, which is independent of the TEP1 complement-like system and the major anti-Plasmodium immune pathway Imd. Our study indicates a hierarchical specificity in the control of Plasmodium melanization and proves CTL4 as an essential host factor for P. falciparum transmission and one of the most potent mosquito-encoded malaria transmission-blocking targets.},
}
@article {pmid35024561,
year = {2021},
author = {Lewis, IC and Yan, Y and Finnigan, GC},
title = {Analysis of a Cas12a-based gene-drive system in budding yeast.},
journal = {Access microbiology},
volume = {3},
number = {12},
pages = {000301},
pmid = {35024561},
issn = {2516-8290},
support = {P20 GM103418/GM/NIGMS NIH HHS/United States ; },
abstract = {The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5' end of the crRNA.},
}
@article {pmid35022402,
year = {2022},
author = {Kaduskar, B and Kushwah, RBS and Auradkar, A and Guichard, A and Li, M and Bennett, JB and Julio, AHF and Marshall, JM and Montell, C and Bier, E},
title = {Reversing insecticide resistance with allelic-drive in Drosophila melanogaster.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {291},
pmid = {35022402},
issn = {2041-1723},
support = {R01 AI165575/AI/NIAID NIH HHS/United States ; R01 DC007864/DC/NIDCD NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {*Alleles ; Animals ; CRISPR-Cas Systems ; Culicidae ; Drosophila melanogaster/*genetics/*physiology ; Female ; Genetic Engineering ; Insecticide Resistance/*genetics ; Insecticides ; Male ; Mutation ; },
abstract = {A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.},
}
@article {pmid35022275,
year = {2022},
author = {Li, W and Duan, X and Zhu, C and Liu, X and Jeyarajan, AJ and Xu, M and Tu, Z and Sheng, Q and Chen, D and Zhu, C and Shao, T and Cheng, Z and Salloum, S and Schaefer, EA and Kruger, AJ and Holmes, JA and Chung, RT and Lin, W},
title = {Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-β1-Induced OCT4/Nanog Pathway.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {208},
number = {3},
pages = {672-684},
pmid = {35022275},
issn = {1550-6606},
support = {U19 AI082630/AI/NIAID NIH HHS/United States ; R01 AI155140/AI/NIAID NIH HHS/United States ; R01 AI069939/AI/NIAID NIH HHS/United States ; R56 DK098079/DK/NIDDK NIH HHS/United States ; R01 DK098079/DK/NIDDK NIH HHS/United States ; R01 DK108370/DK/NIDDK NIH HHS/United States ; },
mesh = {Actins/biosynthesis ; Adult ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/physiology ; Coinfection/pathology ; Collagen Type I, alpha 1 Chain/biosynthesis ; Female ; Gene Knockout Techniques ; Hepacivirus/metabolism ; Hepatic Stellate Cells/pathology/virology ; Hepatitis B/*pathology ; Hepatitis B virus/metabolism ; Hepatitis C/*pathology ; Hepatocytes/pathology/virology ; Humans ; Liver/pathology ; Liver Cirrhosis/*pathology/virology ; Male ; Nanog Homeobox Protein/genetics/*metabolism ; Octamer Transcription Factor-3/genetics/*metabolism ; Organic Anion Transporters, Sodium-Dependent/metabolism ; Symporters/metabolism ; Tissue Inhibitor of Metalloproteinase-1/biosynthesis ; Transforming Growth Factor beta1/*metabolism ; },
abstract = {Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1-induced OCT4/Nanog-dependent pathway.},
}
@article {pmid35021898,
year = {2022},
author = {Mijakovac, A and Miškec, K and Krištić, J and Vičić Bočkor, V and Tadić, V and Bošković, M and Lauc, G and Zoldoš, V and Vojta, A},
title = {A Transient Expression System with Stably Integrated CRISPR-dCas9 Fusions for Regulation of Genes Involved in Immunoglobulin G Glycosylation.},
journal = {The CRISPR journal},
volume = {5},
number = {2},
pages = {237-253},
doi = {10.1089/crispr.2021.0089},
pmid = {35021898},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *Genome-Wide Association Study ; Glycosylation ; Humans ; Immunoglobulin G/genetics/metabolism ; },
abstract = {Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.},
}
@article {pmid35021884,
year = {2022},
author = {Chen, S and Wan, L and Wei, DD and Du, F and Huang, QS and Liu, Y},
title = {High Prevalence and Fitness of IncFrepB Carrying qnrS1 in Hypervirulent Klebsiella pneumoniae Isolates.},
journal = {Microbial drug resistance (Larchmont, N.Y.)},
volume = {28},
number = {3},
pages = {361-369},
doi = {10.1089/mdr.2021.0241},
pmid = {35021884},
issn = {1931-8448},
mesh = {Genes, Bacterial ; Intracellular Signaling Peptides and Proteins ; Klebsiella pneumoniae/*genetics ; *Plasmids ; Prevalence ; Virulence ; Whole Genome Sequencing ; },
abstract = {Objective: This study aimed to reveal the prevalence and fitness of qnrS1-carrying plasmids in hypervirulent Klebsiella pneumoniae (hvKP) isolates. Materials and Methods: Two hundred ninety-nine hvKP strains carrying qnrS1 were collected and screened for resistance genes using PCR and sequencing. The location of qnrS1 and rmpA2 was identified by Southern blotting. The transferability and fitness of qnrS1-carrying plasmids were analyzed by conjugation experiments and plasmid stability assay. Result: In 299 hvKP isolates, the most frequently detected capsular serotype was K64 (81.9%, 245/299), followed by K1 (4.7%, 14/299) and K2 (3.7%, 11/299). All K64-hvKP were sequence type (ST) 11. The qnrS1 and rmpA2 gene mainly was located on the ∼70-210 kb IncFrepB and ∼170-220 kb IncFIB plasmid, respectively. QnrS1-carrying plasmids could be transferred into Escherichia coli J53. However, the plasmid was transferred at a low rate of 13.4% (40/299). The 40 donor isolates belong to 4 STs-ST11, ST700, ST592, and ST86, and none contains the CRISPR-Cas loci. CRISPR-Cas loci were mainly found in ST23 K. pneumoniae. The relative fitness (RF) of qnrS1-carrying plasmids in ST86 and ST11 (cotransfer with blaTEM-1 genes) was more than one and enhanced during cultivation, especially in ST86. However, the RF of qnrS1-carrying plasmids in ST592 and ST700 showed a high fitness cost. Whole-genome sequencing showed that the qnrS1-carrying plasmids in ST86 harbored more maintenance modules (SOS inhibitor protein psiB, parA, and parB partition systems) and insertion sequence (IS) elements (IS91, IS481-like, IS1380), indicating that the qnrS1-carrying plasmid in ST86 is more stable than the other types of qnrS1-carrying plasmids. Conclusion: QnrS1-carrying IncFrepB plasmids were highly prevalent and show polymorphism in hvKP strains. The qnrS1-carrying IncFrepB plasmid in ST86 hvKP should be highlighted due to its remarkable adaptability advantages.},
}
@article {pmid35019836,
year = {2022},
author = {Rodrigues, C and Desai, S and Passet, V and Gajjar, D and Brisse, S},
title = {Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147.},
journal = {Microbial genomics},
volume = {8},
number = {1},
pages = {},
pmid = {35019836},
issn = {2057-5858},
mesh = {*Drug Resistance, Multiple, Bacterial ; Evolution, Molecular ; Genome, Bacterial ; Genomics/*methods ; India ; Interspersed Repetitive Sequences ; Klebsiella pneumoniae/*classification/drug effects/genetics ; Microbial Sensitivity Tests ; Multigene Family ; Phylogeny ; Plasmids/genetics ; Prophages/genetics ; Virulence Factors/genetics ; Whole Genome Sequencing/*methods ; },
abstract = {The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: blaNDM-5 and two copies of blaOXA-181 in the chromosome, and a second copy of blaNDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured blaCTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal blaNDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.},
}
@article {pmid35018532,
year = {2022},
author = {Deb, S and Choudhury, A and Kharbyngar, B and Satyawada, RR},
title = {Applications of CRISPR/Cas9 technology for modification of the plant genome.},
journal = {Genetica},
volume = {150},
number = {1},
pages = {1-12},
pmid = {35018532},
issn = {1573-6857},
mesh = {*CRISPR-Cas Systems ; DNA/metabolism ; *Gene Editing/methods ; Genome, Plant ; Technology ; },
abstract = {The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) system was discovered in bacteria and archea as an acquired immune response to protect the cells from infection. This technology has now evolved to become an efficient genome editing tool, and is replacing older gene editing technologies. This technique uses programmable sgRNAs to guide the Cas9 endonuclease to the target DNA location. sgRNA is a vital component of the CRISPR technology, since without it the Cas nuclease cannot reach to its target location. Over the years, many tools have been developed for designing sgRNAs, the details of which have been extensively reviewed here. It has proven to be a promising tool in the field of genetic engineering and has successfully generated many plant varieties with better and desirable qualities. In the present review, we attempted to collect,collate and summarize information related to the development of CRISPR/Cas9 system as a tool and subsequently into a technique having a wide array of applications in the field of plant genome editing in attaining desirable traits like resistance to various diseases, nutritional enhancement etc. In addition, the probable future prospects and the various bio-safety concerns associated with CRISPR gene editing technology have been discussed in detail.},
}
@article {pmid35018468,
year = {2022},
author = {Habib, O and Habib, G and Hwang, GH and Bae, S},
title = {Comprehensive analysis of prime editing outcomes in human embryonic stem cells.},
journal = {Nucleic acids research},
volume = {50},
number = {2},
pages = {1187-1197},
pmid = {35018468},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; DNA/*metabolism ; Gene Editing/*methods ; Human Embryonic Stem Cells ; Humans ; *alpha 1-Antitrypsin/genetics/metabolism ; alpha 1-Antitrypsin Deficiency/*genetics ; },
abstract = {Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here, we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.},
}
@article {pmid35017728,
year = {2022},
author = {Tang, L},
title = {Seeking more nucleases.},
journal = {Nature methods},
volume = {19},
number = {1},
pages = {27},
doi = {10.1038/s41592-021-01371-5},
pmid = {35017728},
issn = {1548-7105},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Endonucleases/*genetics/metabolism ; Gene Editing/*methods ; Humans ; Microbiota ; Phylogeny ; },
}
@article {pmid35017532,
year = {2022},
author = {Honnell, V and Norrie, JL and Patel, AG and Ramirez, C and Zhang, J and Lai, YH and Wan, S and Dyer, MA},
title = {Identification of a modular super-enhancer in murine retinal development.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {253},
pmid = {35017532},
issn = {2041-1723},
support = {R01 EY018599/EY/NEI NIH HHS/United States ; R01 EY014867/EY/NEI NIH HHS/United States ; R01 CA168875/CA/NCI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; R01 EY030180/EY/NEI NIH HHS/United States ; F31 EY031577/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Proliferation ; Epigenomics ; Female ; *Gene Expression Regulation, Developmental ; Homeodomain Proteins/chemistry/genetics ; Male ; Mice ; Neurogenesis/genetics/*physiology ; Neuroglia/physiology ; Neurons/metabolism ; *Regulatory Sequences, Nucleic Acid/genetics ; Retina/*metabolism ; Stem Cells/physiology ; Transcription Factors/chemistry/physiology ; },
abstract = {Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage- and cell-type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.},
}
@article {pmid35016549,
year = {2022},
author = {Hao, Y and Wang, Q and Li, J and Yang, S and Zheng, Y and Peng, W},
title = {Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering.},
journal = {Open biology},
volume = {12},
number = {1},
pages = {210241},
pmid = {35016549},
issn = {2046-2441},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genome ; Genomics ; *RNA ; },
abstract = {New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.},
}
@article {pmid35016109,
year = {2022},
author = {Heo, W and Lee, K and Park, S and Hyun, KA and Jung, HI},
title = {Electrochemical biosensor for nucleic acid amplification-free and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA via CRISPR/Cas13a trans-cleavage reaction.},
journal = {Biosensors &amp; bioelectronics},
volume = {201},
number = {},
pages = {113960},
pmid = {35016109},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2 ; },
abstract = {The outbreak of the COVID-19 pandemic has led to millions of fatalities worldwide. For preventing epidemic transmission, rapid and accurate virus detection methods to early identify infected people are urgently needed in the current situation. Therefore, an electrochemical biosensor based on the trans-cleavage activity of CRISPR/Cas13a was developed in this study for rapid, sensitive, and nucleic-acid-amplification-free detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a redox probe conjugated with ssRNA is immobilized on the electrode surface modified with a nanocomposite (NC) and gold nanoflower (AuNF) for enhancing the sensing performance. The SARS-CoV-2 RNA is captured by the Cas13a-crRNA complex, which triggers the RNase function of Cas13a. The enzymatically activated Cas13a-crRNA complex is subsequently introduced to the reRNA-conjugated electrochemical sensor, and consequently cleaves the reRNA. A change in current occurs due to the release of the redox molecule labeled on the reRNA, which is trans-cleaved from the Cas13a-crRNA complex. The biosensor can detect as low as 4.4 × 10[-2] fg/mL and 8.1 × 10[-2] fg/mL of ORF and S genes, respectively, over a wide dynamic range (1.0 × 10[-1] to 1.0 × 10[5] fg/mL). Moreover, the biosensor was evaluated by measuring SARS-CoV-2 RNA spiked in artificial saliva. The recovery of the developed sensor was found to be in an agreeable range of 96.54-101.21%. The designed biosensor lays the groundwork for pre-amplification-free detection of ultra-low concentrations of SARS-CoV-2 RNA and on-site and rapid diagnostic testing for COVID-19.},
}
@article {pmid35015844,
year = {2022},
author = {Zhao, L and Zhao, T and Yang, X and Cao, L and Xu, R and Liu, J and Lin, C and Yu, Y and Xuan, D and Zhu, X and Liu, L and Hua, Y and Deng, C and Wan, W and Zou, H and Xue, Y},
title = {IL-37 blocks gouty inflammation by shaping macrophages into a non-inflammatory phagocytic phenotype.},
journal = {Rheumatology (Oxford, England)},
volume = {61},
number = {9},
pages = {3841-3853},
doi = {10.1093/rheumatology/keac009},
pmid = {35015844},
issn = {1462-0332},
mesh = {Animals ; *Arthritis, Gouty/metabolism ; Glycogen Synthase Kinase 3 beta/metabolism ; *Gout/metabolism ; Humans ; Inflammation/metabolism ; Interleukin-1 ; Macrophages/metabolism ; Mice ; Phenotype ; Symptom Flare Up ; Uric Acid/metabolism ; },
abstract = {OBJECTIVE: Interleukin (IL)-37 is a natural suppressor of inflammation. Macrophages play an important role in acute gout flare by dominating the inflammation and spontaneous relief. We have reported that IL-37 could limit runaway inflammation in gout. Here we focus on whether IL-37 inhibits gouty inflammation by altering macrophage functions, and how it does so.<br><br>METHODS: Macrophage functions were evaluated in terms of phagocytosis, pyroptosis, polarization and metabolism. Phagocytosis and polarization of macrophages were detected by side scattering and double-labelling induced nitrogen monoxide synthase (iNOS)/arginase-1 (Arg-1) using flow cytometry, respectively. Transcription of pyroptosis-related molecules was detected by qPCR. Metabolomics was performed by liquid chromatograph mass spectrometer. Human IL-37 knock-in mice and a model with point mutation (S9A) at mouse Gsk3b locus were created by CRISPR/Cas-mediated genome engineering. MSU was injected into the paws and peritoneal cavity to model acute gout. Vernier calliper was used to measure the thickness of the paws. The mice paws and human synovium tissues or tophi were collected for pathological staining. Peritoneal fluid of mice was used to enrich macrophages to detect polarization.<br><br>RESULTS: IL-37 promoted non-inflammatory phagocytic activity of macrophages by enhancing phagocytosis of MSU, reducing transcription of pyroptosis-related proteins and release of inflammatory cytokines, protecting mitochondrial function, and mediating metabolic reprogramming in MSU-treated THP-1 cells. These multifaceted roles of IL-37 were partly depended on the mediation of glycogen synthase kinase-3&#x3b2; (GSK-3&#x3b2;).<br><br>CONCLUSIONS: Our study revealed that IL-37 could shape macrophages into a 'silent' non-inflammatory phagocytic fashion. IL-37 may become a potentially valuable treatment option for patients of chronic gout, especially for those with tophi.},
}
@article {pmid35015821,
year = {2022},
author = {Shi, Q and Fahs, SA and Mattson, JG and Yu, H and Perry, CL and Morateck, PA and Schroeder, JA and Rapten, J and Weiler, H and Montgomery, RR},
title = {A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD.},
journal = {Blood advances},
volume = {6},
number = {9},
pages = {2778-2790},
pmid = {35015821},
issn = {2473-9537},
support = {R01 HL112614/HL/NHLBI NIH HHS/United States ; R01 HL102035/HL/NHLBI NIH HHS/United States ; P01 HL144457/HL/NHLBI NIH HHS/United States ; P01 HL081588/HL/NHLBI NIH HHS/United States ; R01 HL139847/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing ; Hemorrhage/genetics ; *Hemostatics ; Humans ; Mice ; *von Willebrand Disease, Type 2 ; *von Willebrand Diseases/genetics ; von Willebrand Factor/genetics/metabolism ; },
abstract = {Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF-/-) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4 to 6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable but short-lived unprotected in plasma due to dysfunctional 2N VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF-/- mice. Bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N mice, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.},
}
@article {pmid35014191,
year = {2022},
author = {Karunarathne, SD and Han, Y and Zhang, XQ and Li, C},
title = {CRISPR/Cas9 gene editing and natural variation analysis demonstrate the potential for HvARE1 in improvement of nitrogen use efficiency in barley.},
journal = {Journal of integrative plant biology},
volume = {64},
number = {3},
pages = {756-770},
doi = {10.1111/jipb.13214},
pmid = {35014191},
issn = {1744-7909},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome-Wide Association Study ; *Hordeum/genetics ; Nitrogen/metabolism ; },
abstract = {Nitrogen is a major determinant of grain yield and quality. As excessive use of nitrogen fertilizer leads to environmental pollution and high production costs, improving nitrogen use efficiency (NUE) is fundamental for a sustainable agriculture. Here, we dissected the role of the barley abnormal cytokinin response1 repressor 1 (HvARE1) gene, a candidate for involvement in NUE previously identified in a genome-wide association study, through natural variation analysis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. HvARE1 was predominantly expressed in leaves and shoots, with very low expression in roots under low nitrogen conditions. Agrobacterium-mediated genetic transformation of immature embryos (cv. Golden Promise) with single guide RNAs targeting HvARE1 generated 22 T0 plants, from which four T1 lines harbored missense and/or frameshift mutations based on genotyping. Mutant are1 lines exhibited an increase in plant height, tiller number, grain protein content, and yield. Moreover, we observed a 1.5- to 2.8-fold increase in total chlorophyll content in the flag leaf at the grain filling stage. Delayed senescence by 10-14 d was also observed in mutant lines. Barley are1 mutants had high nitrogen content in shoots under low nitrogen conditions. These findings demonstrate the potential of ARE1 in NUE improvement in barley.},
}
@article {pmid35013328,
year = {2022},
author = {Radford, F and Elliott, SD and Schepartz, A and Isaacs, FJ},
title = {Targeted editing and evolution of engineered ribosomes in vivo by filtered editing.},
journal = {Nature communications},
volume = {13},
number = {1},
pages = {180},
pmid = {35013328},
issn = {2041-1723},
support = {R01 GM125951/GM/NIGMS NIH HHS/United States ; T32 GM067543/GM/NIGMS NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems ; Escherichia coli/drug effects/*genetics/metabolism ; Exons ; Gene Editing/*methods ; Genetic Engineering ; *Genome, Bacterial ; Introns ; Mutagenesis, Site-Directed/*methods ; Polymers/chemistry ; Protein Biosynthesis ; *RNA Splicing ; RNA, Ribosomal, 16S/genetics/metabolism ; RNA, Ribosomal, 23S/genetics/metabolism ; Repetitive Sequences, Nucleic Acid ; Ribosomes/*genetics/metabolism ; },
abstract = {Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome's translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.},
}
@article {pmid35012980,
year = {2022},
author = {Ficarrotta, V and Hanly, JJ and Loh, LS and Francescutti, CM and Ren, A and Tunström, K and Wheat, CW and Porter, AH and Counterman, BA and Martin, A},
title = {A genetic switch for male UV iridescence in an incipient species pair of sulphur butterflies.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {3},
pages = {},
pmid = {35012980},
issn = {1091-6490},
mesh = {Animals ; Butterflies/*genetics/*radiation effects ; CRISPR-Cas Systems/genetics ; Chromosomes/genetics ; Genes, Insect ; *Genes, Switch ; Genetic Loci ; Insect Proteins/genetics/metabolism ; Iridescence/*genetics/radiation effects ; Male ; Sexual Behavior, Animal/physiology ; Species Specificity ; Sulfur/*chemistry ; Sympatry/genetics ; *Ultraviolet Rays ; Wings, Animal/metabolism ; },
abstract = {Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.},
}
@article {pmid35012607,
year = {2022},
author = {Seronick, E and Son, J and Michael, C and Fogg, H and Gromley, Z and Gromley, A},
title = {CRISPR/Cas9 genome editing system confirms centriolin's role in cytokinesis.},
journal = {BMC research notes},
volume = {15},
number = {1},
pages = {8},
pmid = {35012607},
issn = {1756-0500},
mesh = {*CRISPR-Cas Systems ; Cytokinesis ; *Gene Editing ; Genome ; RNA, Small Interfering ; },
abstract = {OBJECTIVE: In addition to its function as the microtubule organizing center of the cell, the centrosome has functions in many other cellular processes including primary cilia formation, DNA damage checkpoints, and cell cycle progression. But the role of individual components of the centrosome in these processes remains unclear. Previous studies used siRNA (small interfering RNA) to "knock down" protein levels of the centrosome component centriolin, resulting in failed cytokinesis. Since this approach was transient, only targeting centriolin at the mRNA level, we sought to confirm these findings by permanently disrupting the gene encoding centriolin using the CRISPR/Cas9 system of genome editing.<br><br>RESULTS: This study provides evidence that the CRISPR/Cas9 system is capable of effectively reducing centriolin protein levels in the cell. Furthermore, this disruption leads to a failure of cytokinesis that is reminiscent of the phenotype previously reported for the siRNA-mediated disruption of centriolin. Furthermore, no additional defects in cell division were observed, consistent with results seen with previous siRNA studies. We conclude that the CRISPR/Cas9 system is an effective means of permanently removing the cellular pools of centriolin and that the disruption of centriolin at both the mRNA level and genomic level lead to similar cell division defects.},
}
@article {pmid35011680,
year = {2021},
author = {Cazin, C and Neirijnck, Y and Loeuillet, C and Wehrli, L and Kühne, F and Lordey, I and Mustapha, SFB and Bouker, A and Zouari, R and Thierry-Mieg, N and Nef, S and Arnoult, C and Ray, PF and Kherraf, ZE},
title = {Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B.},
journal = {Cells},
volume = {11},
number = {1},
pages = {},
pmid = {35011680},
issn = {2073-4409},
mesh = {Azoospermia/*etiology/physiopathology ; CRISPR-Cas Systems/*genetics ; Chaperonin Containing TCP-1/*genetics ; Humans ; Male ; Testis/*metabolism ; Whole Exome Sequencing/*methods ; },
abstract = {The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.},
}
@article {pmid35009056,
year = {2021},
author = {Kiryushkin, AS and Ilina, EL and Guseva, ED and Pawlowski, K and Demchenko, KN},
title = {Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.},
journal = {Plants (Basel, Switzerland)},
volume = {11},
number = {1},
pages = {},
pmid = {35009056},
issn = {2223-7747},
abstract = {CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.},
}
@article {pmid35008996,
year = {2022},
author = {Balon, K and Sheriff, A and Jacków, J and Łaczmański, &#x141;},
title = {Targeting Cancer with CRISPR/Cas9-Based Therapy.},
journal = {International journal of molecular sciences},
volume = {23},
number = {1},
pages = {},
pmid = {35008996},
issn = {1422-0067},
mesh = {Animals ; Biomarkers, Tumor ; *CRISPR-Cas Systems ; Combined Modality Therapy ; Disease Susceptibility ; *Gene Editing ; Gene Expression ; Gene Expression Regulation ; Gene Transfer Techniques ; *Genetic Therapy/adverse effects/methods ; Genetic Vectors/classification/genetics ; Humans ; Neoplasms/*genetics/*therapy ; Oncogenes ; Organ Specificity ; Transgenes ; },
abstract = {Cancer is a devastating condition characterised by the uncontrolled division of cells with many forms remaining resistant to current treatment. A hallmark of cancer is the gradual accumulation of somatic mutations which drive tumorigenesis in cancerous cells, creating a mutation landscape distinctive to a cancer type, an individual patient or even a single tumour lesion. Gene editing with CRISPR/Cas9-based tools now enables the precise and permanent targeting of mutations and offers an opportunity to harness this technology to target oncogenic mutations. However, the development of safe and effective gene editing therapies for cancer relies on careful design to spare normal cells and avoid introducing other mutations. This article aims to describe recent advancements in cancer-selective treatments based on the CRISPR/Cas9 system, especially focusing on strategies for targeted delivery of the CRISPR/Cas9 machinery to affected cells, controlling Cas9 expression in tissues of interest and disrupting cancer-specific genes to result in selective death of malignant cells.},
}
@article {pmid35008989,
year = {2022},
author = {Tabebi, M and Kumar Dutta, R and Skoglund, C and Söderkvist, P and Gimm, O},
title = {Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS.},
journal = {International journal of molecular sciences},
volume = {23},
number = {1},
pages = {},
pmid = {35008989},
issn = {1422-0067},
mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line ; *Energy Metabolism/genetics ; Gene Dosage ; Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Glycolysis ; Humans ; Mitochondria/genetics/metabolism ; Mutation ; Neoplasms/genetics/metabolism/pathology ; *Oxidative Phosphorylation ; Phenotype ; Succinate Dehydrogenase/*deficiency ; },
abstract = {BACKGROUND: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis.<br><br>METHODS: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology.<br><br>RESULTS: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs.<br><br>CONCLUSIONS: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.},
}
@article {pmid35008958,
year = {2022},
author = {Rabelo-Fernández, RJ and Santiago-Sánchez, GS and Sharma, RK and Roche-Lima, A and Carrion, KC and Rivera, RAN and Quiñones-Díaz, BI and Rajasekaran, S and Siddiqui, J and Miles, W and Rivera, YS and Valiyeva, F and Vivas-Mejia, PE},
title = {Reduced RBPMS Levels Promote Cell Proliferation and Decrease Cisplatin Sensitivity in Ovarian Cancer Cells.},
journal = {International journal of molecular sciences},
volume = {23},
number = {1},
pages = {},
pmid = {35008958},
issn = {1422-0067},
support = {P20 GM103475/GM/NIGMS NIH HHS/United States ; R25 GM061151/GM/NIGMS NIH HHS/United States ; U24 MD015970/MD/NIMHD NIH HHS/United States ; R01 (WM)/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cellular Senescence/genetics ; Cisplatin/*pharmacology ; Drug Resistance, Neoplasm/*genetics ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Immunohistochemistry ; Neoplasm Grading ; Neoplasm Staging ; Ovarian Neoplasms/*genetics/metabolism/mortality/pathology ; Prognosis ; RNA Splicing ; RNA-Binding Proteins/*genetics/*metabolism ; Tumor Microenvironment/drug effects/genetics ; },
abstract = {Worldwide, the number of cancer-related deaths continues to increase due to the ability of cancer cells to become chemotherapy-resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front-line therapy, cisplatin. Although many mechanisms of cisplatin resistance have been proposed, the key mechanisms of such resistance remain elusive. The RNA binding protein with multiple splicing (RBPMS) binds to nascent RNA transcripts and regulates splicing, transport, localization, and stability. Evidence indicates that RBPMS also binds to protein members of the AP-1 transcription factor complex repressing its activity. Until now, little has been known about the biological function of RBPMS in ovarian cancer. Accordingly, we interrogated available Internet databases and found that ovarian cancer patients with high RBPMS levels live longer compared to patients with low RBPMS levels. Similarly, immunohistochemical (IHC) analysis in a tissue array of ovarian cancer patient samples showed that serous ovarian cancer tissues showed weaker RBPMS staining when compared with normal ovarian tissues. We generated clustered regularly interspaced short palindromic repeats (CRISPR)-mediated RBPMS knockout vectors that were stably transfected in the high-grade serous ovarian cancer cell line, OVCAR3. The knockout of RBPMS in these cells was confirmed via bioinformatics analysis, real-time PCR, and Western blot analysis. We found that the RBPMS knockout clones grew faster and had increased invasiveness than the control CRISPR clones. RBPMS knockout also reduced the sensitivity of the OVCAR3 cells to cisplatin treatment. Moreover, β-galactosidase (β-Gal) measurements showed that RBPMS knockdown induced senescence in ovarian cancer cells. We performed RNAseq in the RBPMS knockout clones and identified several downstream-RBPMS transcripts, including non-coding RNAs (ncRNAs) and protein-coding genes associated with alteration of the tumor microenvironment as well as those with oncogenic or tumor suppressor capabilities. Moreover, proteomic studies confirmed that RBPMS regulates the expression of proteins involved in cell detoxification, RNA processing, and cytoskeleton network and cell integrity. Interrogation of the Kaplan-Meier (KM) plotter database identified multiple downstream-RBPMS effectors that could be used as prognostic and response-to-therapy biomarkers in ovarian cancer. These studies suggest that RBPMS acts as a tumor suppressor gene and that lower levels of RBPMS promote the cisplatin resistance of ovarian cancer cells.},
}
@article {pmid35008709,
year = {2021},
author = {Strohmeier, K and Hofmann, M and Hauser, F and Sivun, D and Puthukodan, S and Karner, A and Sandner, G and Le Renard, PE and Jacak, J and Mairhofer, M},
title = {CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis.},
journal = {International journal of molecular sciences},
volume = {23},
number = {1},
pages = {},
pmid = {35008709},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Extracellular Vesicles/*metabolism/ultrastructure ; Fluorescence ; *Gene Editing ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; *Staining and Labeling ; },
abstract = {Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag[®] with lipid membrane permeable dye, JaneliaFluor[®] 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.},
}
@article {pmid35008665,
year = {2021},
author = {Kim, EJ and Hong, WJ and Kim, YJ and Jung, KH},
title = {Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa).},
journal = {International journal of molecular sciences},
volume = {23},
number = {1},
pages = {},
pmid = {35008665},
issn = {1422-0067},
mesh = {Cell Wall/metabolism ; Down-Regulation/genetics ; *Gene Expression Profiling ; *Gene Expression Regulation, Plant ; Genes, Plant ; Germination/*genetics ; Models, Biological ; Molecular Sequence Annotation ; Mutation/*genetics ; Oryza/*genetics/*growth &amp; development ; Phenotype ; Plant Infertility/genetics ; Plant Proteins/*genetics/metabolism ; Pollen/genetics/*growth &amp; development ; Promoter Regions, Genetic/genetics ; Starch/metabolism ; },
abstract = {The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.},
}
@article {pmid35008103,
year = {2022},
author = {Guo, Y and Tong, C and Su, L and Zhang, W and Jia, H and Liu, Y and Yang, Q and Wu, Z and Wang, Y and Han, W},
title = {CRISPR/Cas9 genome-edited universal CAR T cells in patients with relapsed/refractory lymphoma.},
journal = {Blood advances},
volume = {6},
number = {8},
pages = {2695-2699},
pmid = {35008103},
issn = {2473-9537},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; *Lymphoma/genetics/therapy ; T-Lymphocytes ; },
}
@article {pmid35007156,
year = {2022},
author = {Matthews, LA and Simmons, LA},
title = {The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.},
journal = {Journal of bacteriology},
volume = {204},
number = {3},
pages = {e0053921},
pmid = {35007156},
issn = {1098-5530},
support = {R35 GM131772/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacillus subtilis/genetics/metabolism ; DNA/genetics ; DNA Damage ; DNA Helicases/genetics ; DNA Replication ; Escherichia coli/genetics ; *Escherichia coli Proteins/metabolism ; },
abstract = {DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The important in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.},
}
@article {pmid35006779,
year = {2021},
author = {Hashemzadeh, I and Hasanzadeh, A and Radmanesh, F and Khodadadi Chegeni, B and Hosseini, ES and Kiani, J and Shahbazi, A and Naseri, M and Fatahi, Y and Nourizadeh, H and Kheiri Yeghaneh Azar, B and Aref, AR and Liu, Y and Hamblin, MR and Karimi, M},
title = {Polyethylenimine-Functionalized Carbon Dots for Delivery of CRISPR/Cas9 Complexes.},
journal = {ACS applied bio materials},
volume = {4},
number = {11},
pages = {7979-7992},
doi = {10.1021/acsabm.1c00890},
pmid = {35006779},
issn = {2576-6422},
support = {R01 AI050875/AI/NIAID NIH HHS/United States ; R21 AI121700/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Carbon ; Gene Transfer Techniques ; *Polyethyleneimine ; Transfection ; },
abstract = {Carbon dots (CDs) have become the focus of many studies due to their outstanding optical properties and good biocompatibility. We investigated their potential application to produce a smart and highly efficient yet nontoxic nanovector for gene delivery. This was achieved by conjugating PEI1.8k-functionalized CDs (synthesized by one-step microwave-assisted pyrolysis) with arginine-disulfide linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector could deliver p-CRISPR (9.3 kb) into different types of cell lines with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding transfection efficiency at a high serum concentration and low p-CRISPR dose, compared to PEI25k, which was ineffective under those conditions. Additionally, CD-PEI1.8k-Arg could knock out the GFP gene with great efficiency by delivering the required components of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP cells. Moreover, the nanoparticles showed potential for the local delivery of p-CRISPR into brain tissue. The remarkable properties of CD-PEI1.8k-Arg could enable the development of a safe, highly efficient gene-delivery nanovector for the treatment of various diseases in the near future.},
}
@article {pmid35005648,
year = {2022},
author = {Huang, J and Cook, DE},
title = {CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101072},
pmid = {35005648},
issn = {2666-1667},
mesh = {Ascomycota ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Magnaporthe/genetics ; Ribonucleoproteins/genetics ; },
abstract = {Gene replacements through homologous recombination (HR) have been extensively used for functional genomic studies. However, the general efficiency of HR repair can be low in filamentous fungi and the process laborious. Here, we provide a detailed protocol for efficient gene editing by inserting donor DNA into a region of interest following Cas12a ribonucleoprotein (RNP)-mediated DNA double-strand break. We demonstrate this protocol using Magnaporthe oryzae (synonym of Pyricularia oryzae), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021).},
}
@article {pmid35005644,
year = {2022},
author = {Chen, Y and Ding, Q},
title = {Optimized protocols for efficient gene editing in mouse hepatocytes in vivo using CRISPR-Cas9 technology.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101062},
pmid = {35005644},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Hepatocytes ; Mice ; Plasmids ; Technology ; },
abstract = {We provide a protocol for gene editing in mouse hepatocytes in vivo using the CRISPR-Cas9 technology via AAV delivery. This protocol describes the construction of AAV plasmids, AAV packaging, injection, and the detection of in vivo knockout efficiency. Using this protocol, we can get up to 10[14] AAV and knock out genes in hepatocytes efficiently within 15 days. Moreover, we describe an optimized protocol to simultaneously target two genes via AAV delivery of CRISPR-Cas9 materials in the liver. For complete details on the use and execution of this profile, please refer to Wei et al. (2020).},
}
@article {pmid35005640,
year = {2022},
author = {Hernandez-Huertas, L and Kushawah, G and Diaz-Moscoso, A and Tomas-Gallardo, L and Moreno-Sanchez, I and da Silva Pescador, G and Bazzini, AA and Moreno-Mateos, MA},
title = {Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101058},
pmid = {35005640},
issn = {2666-1667},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; RNA/genetics ; *RNA, Guide/genetics ; RNA, Messenger/genetics ; *Zebrafish/genetics ; },
abstract = {CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates. For complete details on the use and execution of this protocol, please refer to Kushawah et al. (2020).},
}
@article {pmid35004705,
year = {2021},
author = {Wong, RC and Huang, J and Li, D and Amaral, O},
title = {Editorial: Somatic Cell Gene Editing for Treating Diseases.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {828195},
doi = {10.3389/fcell.2021.828195},
pmid = {35004705},
issn = {2296-634X},
}
@article {pmid35002515,
year = {2022},
author = {Song, X and Cui, Y and Wang, Y and Zhang, Y and He, Q and Yu, Z and Xu, C and Ning, H and Han, Y and Cai, Y and Cheng, X and Wang, J and Teng, Y and Yang, X and Wang, J},
title = {Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells.},
journal = {International journal of biological sciences},
volume = {18},
number = {2},
pages = {652-660},
pmid = {35002515},
issn = {1449-2288},
mesh = {Animals ; Blood-Brain Barrier/metabolism ; CRISPR-Cas Systems ; *Dependovirus ; Disease Models, Animal ; Endothelial Cells/*metabolism ; *Gene Editing ; Gene Knockout Techniques ; High-Throughput Nucleotide Sequencing ; Luminescent Proteins/*genetics ; Male ; Mice ; Mice, Transgenic ; NIH 3T3 Cells ; RNA, Guide/genetics ; beta Catenin/*genetics ; },
abstract = {Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2[Cas9]). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2[Cas9] transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.},
}
@article {pmid35002020,
year = {2022},
author = {Li, PY and Li, SQ and Gao, SG and Dong, DY},
title = {A one-step platform for screening high-efficient and minimal off-target CRISPR/Cas13 crRNAs to eradicate SARS-CoV-2 virus for treatment of COVID-19 patients.},
journal = {Medical hypotheses},
volume = {159},
number = {},
pages = {110754},
pmid = {35002020},
issn = {1532-2777},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Genome, Viral ; Humans ; RNA, Viral ; *SARS-CoV-2 ; },
abstract = {Coronavirus disease 2019 (COVID-19) is a new respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and now spreads globally. Currently, therapeutics and effective treatment options remain scarce and there is no proven drug to treat COVID-19. Targeting the positive-sense RNA genome and viral mRNAs of SARS-CoV-2 to simultaneously degrade viral genome templates for replication and viral mRNAs for essential gene expression would be a strategy to completely realize virus elimination. Type VI CRISPR enzymes Cas13 have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allows for RNA cleavage and degradation. The precise viral RNA detection and antiviral application of the CRISPR/Cas13 system depend on high-efficient and minimal off-target crRNAs. Although a computer-based algorithm has been applied for the design of crRNAs targeting SRAS-CoV-2, the experimental screening system to identify optimal crRNA is not available. We develop a one-step experimental screening system to identify high-efficient crRNAs with minimal off-target effects for CRISPR/Cas13-based SARS-CoV-2 elimination. This platform provides the foundation for CRISPR/Cas13-based diagnostics and therapeutics for COVID-19. This platform is versatile and could also be applied for crRNAs screening for other RNA viruses.},
}
@article {pmid35001909,
year = {2021},
author = {Moravec, CE and Voit, GC and Pelegri, F},
title = {Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {178},
pages = {},
pmid = {35001909},
issn = {1940-087X},
support = {R01 GM065303/GM/NIGMS NIH HHS/United States ; R56 GM065303/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Genome ; Germ Cells ; Oogenesis ; *RNA, Guide/genetics ; *Zygote ; },
abstract = {Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary for development until zygotic genome activation. Typically, genetic targeting of these maternal factors requires an additional generation to identify maternal-effect phenotypes, hindering the ability to determine the role of maternally-expressed genes during development. The discovery of the biallelic editing capabilities of CRISPR-Cas9 has allowed screening of embryonic phenotypes in somatic tissues of injected embryos or "crispants," augmenting the understanding of the role zygotically-expressed genes play in developmental programs. This article describes a protocol that is an extension of the crispant method. In this method, the biallelic editing of germ cells allows for the isolation of a maternal-effect phenotype in a single generation, or "maternal crispants." Multiplexing guide RNAs to a single target promotes the efficient production of maternal crispants, while sequence analysis of maternal crispant haploids provides a simple method to corroborate genetic lesions that produce a maternal-effect phenotype. The use of maternal crispants supports the rapid identification of essential maternally-expressed genes, thus facilitating the understanding of early development.},
}
@article {pmid35000262,
year = {2022},
author = {Heitink, L and Whittle, JR and Vaillant, F and Capaldo, BD and Dekkers, JF and Dawson, CA and Milevskiy, MJG and Surgenor, E and Tsai, M and Chen, HR and Christie, M and Chen, Y and Smyth, GK and Herold, MJ and Strasser, A and Lindeman, GJ and Visvader, JE},
title = {In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis.},
journal = {Molecular oncology},
volume = {16},
number = {5},
pages = {1119-1131},
pmid = {35000262},
issn = {1878-0261},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Transformation, Neoplastic/genetics ; *Gene Editing ; Genes, Tumor Suppressor ; Humans ; Mice ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53[+/-] heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.},
}
@article {pmid34999200,
year = {2022},
author = {Han, WK and Yang, YL and Si, YX and Wei, ZQ and Liu, SR and Liu, XL and Yan, Q and Dong, SL},
title = {Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis.},
journal = {Insect biochemistry and molecular biology},
volume = {141},
number = {},
pages = {103719},
doi = {10.1016/j.ibmb.2022.103719},
pmid = {34999200},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; Insect Proteins/*genetics/metabolism ; Larva/genetics/growth &amp; development/metabolism ; Moths/*genetics/growth &amp; development/metabolism ; Receptors, Odorant/*genetics/metabolism ; Sex Attractants/*metabolism ; },
abstract = {General odorant-binding proteins (GOBPs) are long considered responsible for the perception of plant odorants. In this study with the important noctuid pest Spodoptera litura, we functionally characterized that GOBP2 is also involved in the perception of sex pheromone components using in vivo CRISPR/Cas9 technique. First, the GOBP2 sgRNA and Cas9 protein were injected into the newly laid insect eggs, resulting in a 35.6% target mutagenesis in G0 moths. Then, the homozygous GOBP2 knockout strain (GOBP2[-/-]) was obtained after the screening of three generations. The knockout male and female moths displayed a significant reduction in EAG responses to the sex pheromone components, and the knockout females also displayed a significant reduction to plant odorants. In the behavioral assay of food choice, GOBP2[-/-] larvae lost the preference to artificial diet added with the major sex pheromone component Z9, E11-tetradecadienyl acetate (Z9, E11-14:Ac), whereas the WT larvae highly preferred the pheromone diet. Y-tube olfactometer assay and direct pheromone stimulation assay showed that GOBP2[-/-] male adults reduced significantly than WT males in percentages of choice, hair pencil displaying and mating attempt to Z9, E11-14:Ac. In the oviposition test, GOBP2[-/-] females showed significantly reduced preference for the soybean plants compared to the WT females. Our study demonstrated that GOBP2 plays an important role in perceiving sex pheromones in adult and larval stages, providing new insight into sex pheromone perception and a potential target for sex pheromone-based behavioral regulation in the pest.},
}
@article {pmid34999179,
year = {2022},
author = {Ghosh, N and Saha, I and Sharma, N},
title = {Palindromic target site identification in SARS-CoV-2, MERS-CoV and SARS-CoV-1 by adopting CRISPR-Cas technique.},
journal = {Gene},
volume = {818},
number = {},
pages = {146136},
pmid = {34999179},
issn = {1879-0038},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Humans ; Inverted Repeat Sequences/*genetics ; Middle East Respiratory Syndrome Coronavirus/*genetics ; SARS Virus/*genetics ; SARS-CoV-2/*genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas protein (CRISPR-Cas) has turned out to be a very important tool for the rapid detection of viruses. This can be used for the identification of the target site in a virus by identifying a 3-6 nt length Protospacer Adjacent Motif (PAM) adjacent to the potential target site, thus motivating us to adopt CRISPR-Cas technique to identify SARS-CoV-2 as well as other members of Coronaviridae family. In this regard, we have developed a fast and effective method using k-mer technique in order to identify the PAM by scanning the whole genome of the respective virus. Subsequently, palindromic sequences adjacent to the PAM locations are identified as the potential target sites. Palindromes are considered in this work as they are known to identify viruses. Once all the palindrome-PAM combinations are identified, PAMs specific for the RNA-guided DNA Cas9/Cas12 endonuclease are identified to bind and cut the target sites. In this regard, PAMs such as 5'-TGG-3' and 5'-TTTA-3' in NSP3 and Exon for SARS-CoV-2, 5'-GGG-3' and 5'-TGG-3' in Exon and NSP2 for MERS-CoV and 5'-AGG-3' and 5'-TTTG-3' in Helicase and NSP3 respectively for SARS-CoV-1 are identified corresponding to SpCas9 and FnCas12a endonucleases. Finally, to recognise the target sites of Coronaviridae family as cleaved by SpCas9 and FnCas12a, complements of the palindromic target regions are designed as primers or guide RNA (gRNA). Therefore, such complementary gRNAs along with respective Cas proteins can be considered in assays for the identification of SARS-CoV-2, MERS-CoV and SARS-CoV-1.},
}
@article {pmid34998241,
year = {2022},
author = {Fink-Baldauf, IM and Stuart, WD and Brewington, JJ and Guo, M and Maeda, Y},
title = {CRISPRi links COVID-19 GWAS loci to LZTFL1 and RAVER1.},
journal = {EBioMedicine},
volume = {75},
number = {},
pages = {103806},
pmid = {34998241},
issn = {2352-3964},
support = {K08 HL144825/HL/NHLBI NIH HHS/United States ; R01 CA240317/CA/NCI NIH HHS/United States ; U01 HL134745/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; COVID-19/*genetics/metabolism ; *CRISPR-Cas Systems ; Chromosomes, Human, Pair 19/genetics/metabolism ; Chromosomes, Human, Pair 3/genetics/metabolism ; Databases, Nucleic Acid ; *Genetic Loci ; Genome-Wide Association Study ; Haplorhini ; Humans ; *Polymorphism, Single Nucleotide ; RNA-Seq ; Ribonucleoproteins/*genetics/metabolism ; SARS-CoV-2/*genetics/metabolism ; Transcription Factors/*genetics/metabolism ; },
abstract = {BACKGROUND: To identify host genetic variants (SNPs) associated with COVID-19 disease severity, a number of genome-wide association studies (GWAS) have been conducted. Since most of the identified variants are located at non-coding regions, such variants are presumed to affect the expression of neighbouring genes, thereby influencing COVID-19 disease severity. However, it remains largely unknown which genes are influenced by such COVID-19 GWAS loci.<br><br>METHODS: CRISPRi (interference)-mediated gene expression analysis was performed to identify genes functionally regulated by COVID-19 GWAS loci by targeting regions near the loci (SNPs) in lung epithelial cell lines. The expression of CRISPRi-identified genes was investigated using COVID-19-contracted human and monkey lung single-nucleus/cell (sn/sc) RNA-seq datasets.<br><br>FINDINGS: CRISPRi analysis indicated that a region near rs11385942 at chromosome 3p21.31 (locus of highest significance with COVID-19 disease severity at intron 5 of LZTFL1) significantly affected the expression of LZTFL1 (P<0.05), an airway cilia regulator. A region near rs74956615 at chromosome 19p13.2 (locus located at the 3' untranslated exonic region of RAVER1), which is associated with critical illness in COVID-19, affected the expression of RAVER1 (P<0.05), a coactivator of MDA5 (IFIH1), which induces antiviral response genes, including ICAM1. The sn/scRNA-seq datasets indicated that the MDA5/RAVER1-ICAM1 pathway was activated in lung epithelial cells of COVID-19-resistant monkeys but not those of COVID-19-succumbed humans.<br><br>INTERPRETATION: Patients with risk alleles of rs11385942 and rs74956615 may be susceptible to critical illness in COVID-19 in part through weakened airway viral clearance via LZTFL1-mediated ciliogenesis and diminished antiviral immune response via the MDA5/RAVER1 pathway, respectively.<br><br>FUNDING: NIH.},
}
@article {pmid34997407,
year = {2022},
author = {Zhu, XX and Pan, JS and Lin, T and Yang, YC and Huang, QY and Yang, SP and Qu, ZX and Lin, ZS and Wen, JC and Yan, AF and Feng, J and Liu, L and Zhang, XL and Lu, JH and Tang, DS},
title = {Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.},
journal = {Biotechnology letters},
volume = {44},
number = {1},
pages = {59-76},
pmid = {34997407},
issn = {1573-6776},
mesh = {*Adenine/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Exons/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; Swine ; },
abstract = {Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.},
}
@article {pmid34996449,
year = {2022},
author = {Mianné, J and Nasri, A and Van, CN and Bourguignon, C and Fieldès, M and Ahmed, E and Duthoit, C and Martin, N and Parrinello, H and Louis, A and Iché, A and Gayon, R and Samain, F and Lamouroux, L and Bouillé, P and Bourdin, A and Assou, S and De Vos, J},
title = {CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles.},
journal = {BMC biology},
volume = {20},
number = {1},
pages = {8},
pmid = {34996449},
issn = {1741-7007},
mesh = {Alleles ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Gene Conversion ; Gene Editing/methods ; Gene Knockout Techniques ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; RNA/metabolism ; Retroviridae/genetics ; },
abstract = {BACKGROUND: The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications.<br><br>RESULTS: We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion.<br><br>CONCLUSIONS: Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.},
}
@article {pmid34995499,
year = {2022},
author = {Domenig, SA and Bundschuh, N and Lenardič, A and Ghosh, A and Kim, I and Qabrati, X and D'Hulst, G and Bar-Nur, O},
title = {CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.},
journal = {Stem cell reports},
volume = {17},
number = {2},
pages = {321-336},
pmid = {34995499},
issn = {2213-6711},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cellular Reprogramming/*genetics ; Disease Models, Animal ; Dystrophin/genetics/*metabolism ; Fibroblasts/cytology/metabolism ; Gene Editing/*methods ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Muscle Development ; Muscular Dystrophy, Duchenne/genetics/metabolism/*pathology ; Mutation ; MyoD Protein/genetics/metabolism ; Myoblasts/cytology/metabolism ; Stem Cells/cytology/metabolism ; },
abstract = {Genetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.},
}
@article {pmid34995286,
year = {2022},
author = {Kilbas, PO and Can, ND and Kizilboga, T and Ezberci, F and Doganay, HL and Arisan, ED and Dinler Doganay, G},
title = {CRISPR/Cas9-mediated Bag-1 knockout increased mesenchymal characteristics of MCF-7 cells via Akt hyperactivation-mediated actin cytoskeleton remodeling.},
journal = {PloS one},
volume = {17},
number = {1},
pages = {e0261062},
pmid = {34995286},
issn = {1932-6203},
mesh = {Actin Cytoskeleton/genetics/*metabolism ; Actins/metabolism ; Apoptosis/genetics ; Breast Neoplasms/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival ; DNA-Binding Proteins/genetics/*metabolism ; Epithelial-Mesenchymal Transition/physiology ; Female ; Humans ; MCF-7 Cells/metabolism ; Proto-Oncogene Proteins c-akt/genetics/*metabolism ; Signal Transduction/genetics ; Transcription Factors/genetics/*metabolism ; },
abstract = {Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation.},
}
@article {pmid34994551,
year = {2022},
author = {Hunt, VM and Chen, W},
title = {Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {397-405},
doi = {10.1021/acssynbio.1c00479},
pmid = {34994551},
issn = {2161-5063},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Expression ; *Gene Expression Regulation ; HeLa Cells ; Humans ; Mammals/genetics ; Proteolysis ; *RNA, Guide/metabolism ; },
abstract = {A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.},
}
@article {pmid34994540,
year = {2022},
author = {Zhang, Y and Marchisio, MA},
title = {Interaction of Bare dSpCas9, Scaffold gRNA, and Type II Anti-CRISPR Proteins Highly Favors the Control of Gene Expression in the Yeast S. cerevisiae.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {176-190},
doi = {10.1021/acssynbio.1c00352},
pmid = {34994540},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Expression ; *RNA, Guide/genetics/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {Type II CRISPR-(d)SpCas9 and anti-CRISPR proteins (AcrIIs) show evidence of coevolution and competition for survival between bacteria and phages. In biotechnology, CRISPR-(d)SpCas9 is utilized for gene editing and transcriptional regulation. Moreover, its activity is controlled by AcrIIs. However, studies of dSpCas9/AcrII-based transcription regulation in Saccharomyces cerevisiae are rare. In this work, we used dSpCas9 as a template to engineer new transcription activators. We found that the most performant activation system requires the use of bare dSpCas9 in conjunction with scaffold gRNA (scRNA). This means that activation domains shall not be fused to dSpCas9 but rather interact with scRNA. We showed that a low amount of sgRNA is not a limiting factor in dSpCas9-driven transcription regulation. Moreover, a high quantity of sgRNA does not improve, generally, activation (and repression) efficiency. Importantly, we analyzed the performance of AcrIIA2, AcrIIA4, and AcrIIA5 in S. cerevisiae in depth. AcrIIA4 is the strongest of the three AcrIIs and also the only one able to induce high inhibition at low concentrations. However, the activation domains fused to dSpCas9 hindered interactions with the AcrIIs as well and limited their control of gene transcription regulation, confirming that bare dSpCas9 is the best solution for building synthetic genetic networks in yeast.},
}
@article {pmid34992734,
year = {2022},
author = {Mizuki, H and Shimoyama, Y and Ishikawa, T and Sasaki, M},
title = {A genomic sequence of the type II-A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system in Mycoplasma salivarium strain ATCC 29803.},
journal = {Journal of oral microbiology},
volume = {14},
number = {1},
pages = {2008153},
pmid = {34992734},
issn = {2000-2297},
abstract = {INTRODUCTION: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems are RNA-mediated adaptive immune systems that actagainst invading genetic elements such as phages or plasmids. CRISPR/Cas systems exist in nearly half of bacteria. Mycoplasma salivarium is a commensal species of the oropharynx. The American Type Culture Collection maintains five M. salivarium strains: ATCC 14277, 23064, 23557, 29803, and 33130. The genome sequence of ATCC 23064 revealed that it has an incomplete CRISPR/Cas system. However, the genome sequences of the remaining strains have not been analyzed.<br><br>METHODS: We performed polymerase chain reaction-amplicon sequencing and de novo genome sequencing to evaluate the presence of the CRISPR/Cas system in four strains.<br><br>RESULTS: Only ATCC 29803 possessed cas1, cas2, cas9, and csn2 genes, a CRISPR array, and tracrRNA. The sequences of most components were identical between the CRISPR/Cas systems of ATCC 29803 and ATCC 23064, whereas the spacer sequences and a region of the cas9 gene were different. Unlike the CRISPR/Cas system of ATCC 23064, the cas9 gene of ATCC 29803 was not disrupted by the presence of stop codons.<br><br>CONCLUSION: ATCC 29803 possesses genomic components required to express the type II-A CRISPR/Cas system, which potentially functions as an RNA-guided endonuclease.},
}
@article {pmid34992260,
year = {2022},
author = {Mohanraju, P and Saha, C and van Baarlen, P and Louwen, R and Staals, RHJ and van der Oost, J},
title = {Alternative functions of CRISPR-Cas systems in the evolutionary arms race.},
journal = {Nature reviews. Microbiology},
volume = {20},
number = {6},
pages = {351-364},
pmid = {34992260},
issn = {1740-1534},
mesh = {Archaea/physiology ; Bacteria ; Bacterial Physiological Phenomena ; Biological Evolution ; *CRISPR-Cas Systems/genetics ; Humans ; *Viruses/genetics ; },
abstract = {CRISPR-Cas systems of bacteria and archaea comprise chromosomal loci with typical repetitive clusters and associated genes encoding a range of Cas proteins. Adaptation of CRISPR arrays occurs when virus-derived and plasmid-derived sequences are integrated as new CRISPR spacers. Cas proteins use CRISPR-derived RNA guides to specifically recognize and cleave nucleic acids of invading mobile genetic elements. Apart from this role as an adaptive immune system, some CRISPR-associated nucleases are hijacked by mobile genetic elements: viruses use them to attack their prokaryotic hosts, and transposons have adopted CRISPR systems for guided transposition. In addition, some CRISPR-Cas systems control the expression of genes involved in bacterial physiology and virulence. Moreover, pathogenic bacteria may use their Cas nuclease activity indirectly to evade the human immune system or directly to invade the nucleus and damage the chromosomal DNA of infected human cells. Thus, the evolutionary arms race has led to the expansion of exciting variations in CRISPR mechanisms and functionalities. In this Review, we explore the latest insights into the diverse functions of CRISPR-Cas systems beyond adaptive immunity and discuss the implications for the development of CRISPR-based applications.},
}
@article {pmid34991617,
year = {2022},
author = {Wang, D and Chen, L and Li, C and Long, Q and Yang, Q and Huang, A and Tang, H},
title = {CRISPR/Cas9 delivery by NIR-responsive biomimetic nanoparticles for targeted HBV therapy.},
journal = {Journal of nanobiotechnology},
volume = {20},
number = {1},
pages = {27},
pmid = {34991617},
issn = {1477-3155},
mesh = {Animals ; *Biomimetic Materials/chemistry/radiation effects ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Transfer Techniques ; Hepatitis B/*therapy ; Hepatitis B virus ; Infrared Rays ; Mice ; Mice, Transgenic ; *Nanoparticles/chemistry/radiation effects ; },
abstract = {BACKGROUND: Currently, there are no curative drugs for hepatitis B virus (HBV). Complete elimination of HBV covalently closed circular DNA (cccDNA) is key to the complete cure of hepatitis B virus infection. The CRISPR/Cas9 system can directly destroy HBV cccDNA. However, a CRISPR/Cas9 delivery system with low immunogenicity and high efficiency has not yet been established. Moreover, effective implementation of precise remote spatiotemporal operations in CRISPR/Cas9 is a major limitation.<br><br>RESULTS: In this work, we designed NIR-responsive biomimetic nanoparticles (UCNPs-Cas9@CM), which could effectively deliver Cas9 RNP to achieve effective genome editing for HBV therapy. HBsAg, HBeAg, HBV pgRNA and HBV DNA along with cccDNA in HBV-infected cells were found to be inhibited. These findings were confirmed in HBV-Tg mice, which did not exhibit significant cytotoxicity and minimal off-target DNA damage.<br><br>CONCLUSIONS: The UCNPs-based biomimetic nanoplatforms achieved the inhibition of HBV&#xa0;replication via CRISPR therapy and it&#xa0;is a potential system for efficient treatment of human HBV diseases.},
}
@article {pmid34990959,
year = {2022},
author = {Han, C and Li, W and Li, Q and Xing, W and Luo, H and Ji, H and Fang, X and Luo, Z and Zhang, L},
title = {CRISPR/Cas12a-Derived electrochemical aptasensor for ultrasensitive detection of COVID-19 nucleocapsid protein.},
journal = {Biosensors &amp; bioelectronics},
volume = {200},
number = {},
pages = {113922},
doi = {10.1016/j.bios.2021.113922},
pmid = {34990959},
issn = {1873-4235},
mesh = {*Aptamers, Nucleotide ; *Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Electrochemical Techniques ; Electrodes ; Gold ; Humans ; Limit of Detection ; Nucleocapsid Proteins ; SARS-CoV-2 ; },
abstract = {Fast, affordable, portable, and sensitive technology to detect COVID-19 is critical to address the current outbreak. Here, we present a CRISPR/Cas12a-derived electrochemical aptasensor for cost-effective, fast, and ultrasensitive COVID-19 nucleocapsid protein (Np) detection. First, an electrochemical sensing interface was fabricated by immobilizing methylene blue labeled poly adenines DNA sequence (polyA-MB electrochemical reporter) on a gold electrode surface. Second, an arched probe was prepared via hybridization of Np aptamer and an activator strand. In the presence of COVID-19 Np, the activator strand could be released from the arched probe due to the specific interaction between the target and the aptamer, which then activated the trans-cleavage activity of the CRISPR/Cas12a system. Subsequently, the polyA-MB reporters were cleaved from the electrode surface, decreasing the current of differential pulse voltammetry (DPV) at a potential of -0.27 V(vs. Ag/AgCl). The CRISPR/Cas12a-derived electrochemical aptasensor shows a highly efficient performance for COVID-19 Np detection in 50 pg mL[-1] to 100 ng mL[-1] with a limit of detection (LOD) low to 16.5 pg mL[-1]. Notably, the whole process of one test can be completed within 30 min. Simultaneously, the aptasensor displays a high selectivity to other proteins. The further measurements demonstrate that the aptasensor is robust in a natural system for point-of-care testing, such as in tap water, milk, or serum. The aptasensor is universal and expandable and holds great potential in the COVID-19 early diagnosis, environmental surveillance, food security, and other aspects.},
}
@article {pmid34990903,
year = {2022},
author = {Rivas, CM and Schiff, HV and Moutal, A and Khanna, R and Kiela, PR and Dussor, G and Price, TJ and Vagner, J and DeFea, KA and Boitano, S},
title = {Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.},
journal = {Biochemical and biophysical research communications},
volume = {591},
number = {},
pages = {13-19},
pmid = {34990903},
issn = {1090-2104},
support = {R01 NS098826/NS/NINDS NIH HHS/United States ; R56 NS098826/NS/NINDS NIH HHS/United States ; R21 AI140257/AI/NIAID NIH HHS/United States ; R01 NS119263/NS/NINDS NIH HHS/United States ; R41 HL160424/HL/NHLBI NIH HHS/United States ; },
mesh = {Alternaria/*physiology ; Base Sequence ; Bronchi/*pathology ; CRISPR-Cas Systems/genetics ; Cell Line ; Epithelial Cells/metabolism/*microbiology ; Humans ; Inflammation/*pathology ; Receptor, PAR-2/*metabolism ; *Signal Transduction ; },
abstract = {Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR[-][/-]) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR[-][/-] line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2[-/-] grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.},
}
@article {pmid34990594,
year = {2022},
author = {Perveen, S and Sharma, R},
title = {Screening approaches and therapeutic targets: The two driving wheels of tuberculosis drug discovery.},
journal = {Biochemical pharmacology},
volume = {197},
number = {},
pages = {114906},
doi = {10.1016/j.bcp.2021.114906},
pmid = {34990594},
issn = {1873-2968},
mesh = {Animals ; Antitubercular Agents/*administration &amp; dosage/metabolism ; Drug Delivery Systems/*methods ; Drug Discovery/*methods ; Energy Metabolism/drug effects/physiology ; High-Throughput Screening Assays/*methods ; Humans ; Mycobacterium tuberculosis/drug effects/metabolism ; Tuberculosis/*drug therapy/metabolism ; },
abstract = {Tuberculosis (TB) is an infectious disease, infecting a quarter of world's population. Drug resistant TB further exacerbates the grim scenario of the drying TB drug discovery pipeline. The limited arsenal to fight TB presses the need for thorough efforts for identifying promising hits to combat the disease. The review highlights the efforts in the field of tuberculosis drug discovery, with an emphasis on massive drug screening campaigns for identifying novel hits against Mtb in both industry and academia. As an intracellular pathogen, mycobacteria reside in a complicated intracellular environment with multiple factors at play. Here, we outline various strategies employed in an effort to mimic the intracellular milieu for bringing the screening models closer to the actual settings. The review also focuses on the novel targets and pathways that could aid in target-based drug discovery in TB. The recent high throughput screening efforts resulting in the identification of potent hits against Mtb has been summarized in this article. There is a pressing need for effective screening strategies and approaches employing innovative tools and recent technologies; including nanotechnology, gene-editing tools such as CRISPR-cas system, host-directed bacterial killing and high content screening to augment the TB drug discovery pipeline with safer and shorter drug regimens.},
}
@article {pmid34990457,
year = {2022},
author = {Jirawannaporn, S and Limothai, U and Tachaboon, S and Dinhuzen, J and Kiatamornrak, P and Chaisuriyong, W and Bhumitrakul, J and Mayuramart, O and Payungporn, S and Srisawat, N},
title = {Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32.},
journal = {PLoS neglected tropical diseases},
volume = {16},
number = {1},
pages = {e0010112},
pmid = {34990457},
issn = {1935-2735},
mesh = {Bacterial Outer Membrane Proteins/*genetics ; CRISPR-Cas Systems/genetics ; Female ; Humans ; Leptospira/*genetics/isolation &amp; purification ; Leptospirosis/*diagnosis ; Limit of Detection ; Lipoproteins/*genetics ; Male ; Mass Screening/methods ; Middle Aged ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/*methods ; Point-of-Care Testing ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples.<br><br>A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4-6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study.<br><br>CONCLUSIONS/SIGNIFICANCE: The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.},
}
@article {pmid34990206,
year = {2022},
author = {Cheng, P and Wirka, RC and Shoa Clarke, L and Zhao, Q and Kundu, R and Nguyen, T and Nair, S and Sharma, D and Kim, HJ and Shi, H and Assimes, T and Brian Kim, J and Kundaje, A and Quertermous, T},
title = {ZEB2 Shapes the Epigenetic Landscape of Atherosclerosis.},
journal = {Circulation},
volume = {145},
number = {6},
pages = {469-485},
pmid = {34990206},
issn = {1524-4539},
support = {R01 HL109512/HL/NHLBI NIH HHS/United States ; R01 HL151535/HL/NHLBI NIH HHS/United States ; R01 HL139478/HL/NHLBI NIH HHS/United States ; K08 HL133375/HL/NHLBI NIH HHS/United States ; R01 HL134817/HL/NHLBI NIH HHS/United States ; R01 HL145708/HL/NHLBI NIH HHS/United States ; R33 HL120757/HL/NHLBI NIH HHS/United States ; UM1 HG011972/HG/NHGRI NIH HHS/United States ; F32 HL143847/HL/NHLBI NIH HHS/United States ; K08 HL152308/HL/NHLBI NIH HHS/United States ; R01 HL156846/HL/NHLBI NIH HHS/United States ; K08 HL153798/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Atherosclerosis/*genetics/pathology ; Epigenesis, Genetic/*genetics ; Humans ; Mice ; Single-Cell Analysis ; Zinc Finger E-box Binding Homeobox 2/*genetics ; },
abstract = {BACKGROUND: Smooth muscle cells (SMCs) transition into a number of different phenotypes during atherosclerosis, including those that resemble fibroblasts and chondrocytes, and make up the majority of cells in the atherosclerotic plaque. To better understand the epigenetic and transcriptional mechanisms that mediate these cell state changes, and how they relate to risk for coronary artery disease (CAD), we have investigated the causality and function of transcription factors at genome-wide associated loci.<br><br>METHODS: We used CRISPR-Cas 9 genome and epigenome editing to identify the causal gene and cells for a complex CAD genome-wide association study signal at 2q22.3. Single-cell epigenetic and transcriptomic profiling in murine models and human coronary artery smooth muscle cells were used to understand the cellular and molecular mechanism by which this CAD risk gene exerts its function.<br><br>RESULTS: CRISPR-Cas 9 genome and epigenome editing showed that the complex CAD genetic signals within a genomic region at 2q22.3 lie within smooth muscle long-distance enhancers for ZEB2, a transcription factor extensively studied in the context of epithelial mesenchymal transition in development of cancer. Zeb2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and transforming growth factor &#x3b2; signaling, thus altering the epigenetic trajectory of SMC transitions. SMC-specific loss of Zeb2 resulted in an inability of transitioning SMCs to turn off contractile programing and take on a fibroblast-like phenotype, but accelerated the formation of chondromyocytes, mirroring features of high-risk atherosclerotic plaques in human coronary arteries.<br><br>CONCLUSIONS: These studies identify ZEB2 as a new CAD genome-wide association study gene that affects features of plaque vulnerability through direct effects on the epigenome, providing a new therapeutic approach to target vascular disease.},
}
@article {pmid34990118,
year = {2022},
author = {Zheng, L and Tan, Y and Hu, Y and Shen, J and Qu, Z and Chen, X and Ho, CL and Leung, EL and Zhao, W and Dai, L},
title = {CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {464-472},
doi = {10.1021/acssynbio.1c00543},
pmid = {34990118},
issn = {2161-5063},
mesh = {Bacteroides/genetics ; *CRISPR-Cas Systems/genetics ; Escherichia coli ; *Gene Editing ; Genome ; Humans ; },
abstract = {Bacteroides is the most abundant genus in the human gut microbiome and has been increasingly used as model organisms for studying the function and ecology of the gut microbiome. However, genome editing tools for such commensal gut microbes are still lacking. Here we developed a versatile, highly efficient CRISPR/Cas-based genome editing tool that allows markerless gene deletion and insertion in human gut Bacteroides species. We constructed multiple CRISPR/Cas systems in all-in-one Bacteroides-E. coli shuttle plasmids and systematically evaluated the genome editing efficiency in Bacteroides thetaiotaomicron, including the mode of Cas protein expression (constitutive, inducible), different Cas proteins (FnCas12a, SpRY, SpCas9), and sgRNAs. Using the anhydrotetracycline (aTc)-inducible CRISPR/FnCas12a system, we successfully deleted large genomic fragments up to 50 kb to study the function of metabolic gene clusters. Furthermore, we demonstrated that CRISPR/FnCas12a can be broadly applied to engineer multiple human gut Bacteroides species, including Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, and Bacteroides vulgatus. We envision that CRISPR/Cas-based genome editing tools for Bacteroides will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.},
}
@article {pmid34989166,
year = {2022},
author = {Zhang, C and Ren, H and Liu, G and Li, J and Wang, X and Zhang, Y},
title = {Effective Genome Editing Using CRISPR-Cas9 Nanoflowers.},
journal = {Advanced healthcare materials},
volume = {11},
number = {10},
pages = {e2102365},
doi = {10.1002/adhm.202102365},
pmid = {34989166},
issn = {2192-2659},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; Micelles ; *Nanoparticles ; Polymers/metabolism ; },
abstract = {CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, a new mesoporous nanoflower (NF)-like delivery nanoplatform termed Cas9-NF is reported by crosslinking Cas9 and polymeric micelles that enables efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology is flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remains stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibits excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway toward the application of the protein-micelle crosslinked nanoflower for protein delivery, which warrants further investigations for gene regulation and cancer treatment.},
}
@article {pmid34987632,
year = {2022},
author = {Shen, T and Zhang, Y and Mei, L and Zhang, XB and Zhu, G},
title = {Single-stranded circular DNA theranostics.},
journal = {Theranostics},
volume = {12},
number = {1},
pages = {35-47},
pmid = {34987632},
issn = {1838-7640},
support = {KL2 TR002648/TR/NCATS NIH HHS/United States ; R21 NS114455/NS/NINDS NIH HHS/United States ; R35 GM143014/GM/NIGMS NIH HHS/United States ; UL1 TR002649/TR/NCATS NIH HHS/United States ; },
mesh = {DNA, Circular/*therapeutic use ; Gene Editing ; Humans ; Precision Medicine/*methods ; },
abstract = {The past decade has witnessed the blossom of nucleic acid therapeutics and diagnostics (theranostics). Unlike conventional small molecule medicines or protein biologics, nucleic acid theranostics have characteristic features such as the intrinsic ability as "information drugs" to code and execute genetic and theranostic information, ready programmability for nucleic acid engineering, intrinsic stimulatory or regulatory immunomodulation, versatile functionalities, and easy conformational recovery upon thermal or chemical denaturation. Single-stranded circular DNA (circDNA) are a class of single-stranded DNAs (ssDNA) featured with their covalently-closed topology. In addition to the basic advantages of nucleic acids-based materials, such as low cost, biocompatibility, and simplicity of chemical modification, the lack of terminals in circDNA prevents exonuclease degradation, resulting in enhanced biostability relative to the corresponding linear ssDNA. circDNA has been explored for versatile theranostic applications. For instance, circDNA has been extensively studied as templates for bioanalytical signal amplification and the synthesis of nano-/micro-/macro- biomaterials via rolling circle amplification (RCA) and rolling circle transcription (RCT) technologies. circDNA has also been commonly used as the scaffolds for the self-assembly of versatile DNA origami. Finally, circDNA has been implemented as theranostic aptamers, miRNA inhibitors, as well as clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) gene editing donors. In this review article, we will discuss the chemistry, characteristic properties, and the theranostic applications of circDNA (excluding double-stranded circular DNA such as plasmids); we will also envision the challenges and opportunities in this research field.},
}
@article {pmid34986794,
year = {2022},
author = {Uribe-Salazar, JM and Kaya, G and Sekar, A and Weyenberg, K and Ingamells, C and Dennis, MY},
title = {Evaluation of CRISPR gene-editing tools in zebrafish.},
journal = {BMC genomics},
volume = {23},
number = {1},
pages = {12},
pmid = {34986794},
issn = {1471-2164},
support = {DP2 MH119424/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; *Zebrafish/genetics ; },
abstract = {BACKGROUND: Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create 'knockout' models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.<br><br>METHODS: To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.<br><br>RESULTS: We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (<&#x2009;1%). To characterize if commonly used 'mock' CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5&#x2009;days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the 'mock' injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation.<br><br>CONCLUSION: Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.},
}
@article {pmid34986352,
year = {2022},
author = {Zhang, X and Jin, H and Huang, X and Chaurasiya, B and Dong, D and Shanley, TP and Zhao, YY},
title = {Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA.},
journal = {Cell reports},
volume = {38},
number = {1},
pages = {110196},
pmid = {34986352},
issn = {2211-1247},
support = {R01 HL140409/HL/NHLBI NIH HHS/United States ; R01 HL148810/HL/NHLBI NIH HHS/United States ; P01 HL077806/HL/NHLBI NIH HHS/United States ; R01 HL125350/HL/NHLBI NIH HHS/United States ; UL1 TR001422/TR/NCATS NIH HHS/United States ; R01 HL133951/HL/NHLBI NIH HHS/United States ; R01 HL123957/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endothelium, Vascular/*cytology ; Female ; Gene Editing/*methods ; Genetic Therapy/methods ; High-Throughput Nucleotide Sequencing ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nanoparticles/*chemistry ; Plasmids/*genetics ; Polyethyleneimine/chemistry ; RNA, Guide/genetics ; },
abstract = {Vascular endothelium plays a crucial role in vascular homeostasis and tissue fluid balance. To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by CDH5 promoter and guide RNA (U6 promoter) induced highly efficient genome editing in endothelial cells (ECs) of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. Western blotting and immunofluorescent staining demonstrated an ∼80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice. Nanoparticle delivery of plasmid DNA could induce genome editing of two genes or genome editing and transgene expression in ECs simultaneously. Thus, nanoparticle delivery of plasmid DNA is a powerful tool to rapidly and efficiently alter expression of gene(s) in ECs for cardiovascular research and potential gene therapy.},
}
@article {pmid34985759,
year = {2022},
author = {Huang, A and Bryan, B and Kraves, S and Alvarez-Saavedra, E and Stark, JC},
title = {Implementing Hands-On Molecular and Synthetic Biology Education Using Cell-Free Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2433},
number = {},
pages = {413-432},
pmid = {34985759},
issn = {1940-6029},
support = {F32 CA250324/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; Learning ; *Synthetic Biology ; Technology ; },
abstract = {Active, hands-on learning has been shown to improve educational outcomes in STEM subjects. However, implementation of hands-on activities for teaching biology has lagged behind other science disciplines due to challenges associated with the use of living cells. To address this limitation, we developed BioBits[®]: biology education activities enabled by freeze-dried cell-free reactions that can be activated by just adding water. Here, we describe detailed protocols for labs designed to teach the central dogma, biomaterial formation, an important mechanism of antibiotic resistance, and CRISPR-Cas9 gene editing via cell-free synthesis of proteins with visual outputs. The activities described are designed for a range of educational levels and time/resource requirements, so that educators can select the demonstrations that best fit their needs. We anticipate that the availability of BioBits[®] activities will enhance biology instruction by enabling hands-on learning in a variety of educational settings.},
}
@article {pmid34985758,
year = {2022},
author = {Wimmer, F and Englert, F and Beisel, CL},
title = {A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2433},
number = {},
pages = {391-411},
pmid = {34985758},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell-Free System/metabolism ; Gene Library ; Plasmids/genetics ; },
abstract = {Type I CRISPR-Cas systems represent the most common and diverse type of these prokaryotic defense systems and are being harnessed for a growing set of applications. As these systems rely on multi-protein effector complexes, their characterization remains challenging. Here, we report a rapid and straightforward method to characterize these systems in a cell-free transcription-translation (TXTL) system. A ribonucleoprotein complex is produced and binds to its target next to a recognized PAM, thereby preventing the targeted sequence from being cleaved by a restriction enzyme. Selection for uncleaved targeted plasmids leads to an enrichment of recognized sequences within a PAM library. This assay will aid the exploration of CRISPR-Cas diversity and evolution and help contribute new systems for CRISPR technologies and applications.},
}
@article {pmid34985679,
year = {2022},
author = {Herskovitz, J and Hasan, M and Patel, M and Kevadiya, BD and Gendelman, HE},
title = {Pathways Toward a Functional HIV-1 Cure: Balancing Promise and Perils of CRISPR Therapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2407},
number = {},
pages = {429-445},
pmid = {34985679},
issn = {1940-6029},
support = {R01 NS036126/NS/NINDS NIH HHS/United States ; R01 MH115860/MH/NIMH NIH HHS/United States ; P01 DA028555/DA/NIDA NIH HHS/United States ; P30 MH062261/MH/NIMH NIH HHS/United States ; R01 MH121402/MH/NIMH NIH HHS/United States ; R01 AG043540/AG/NIA NIH HHS/United States ; R01 NS033249/NS/NINDS NIH HHS/United States ; R01 AG024353/AG/NIA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *HIV Infections ; *HIV-1/genetics ; RNA, Guide/genetics ; Virus Latency ; },
abstract = {First identified as a viral defense mechanism, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) has been transformed into a gene-editing tool. It now affords promise in the treatment and potential eradication of a range of divergent genetic, cancer, infectious, and degenerative diseases. Adapting CRISPR-Cas into a programmable endonuclease directed guide RNA (gRNA) has attracted international attention. It was recently awarded the 2020 Nobel Prize in Chemistry. The limitations of this technology have also been identified and work has been made in providing potential remedies. For treatment of the human immunodeficiency virus type one (HIV-1), in particular, a CRISPR-Cas9 approach was adapted to target then eliminate latent proviral DNA. To this end, we reviewed the promise and perils of CRISPR-Cas gene-editing strategies for HIV-1 elimination. Obstacles include precise delivery to reservoir tissue and cell sites of latent HIV-1 as well as assay sensitivity and specificity. The detection and consequent excision of common viral strain sequences and the avoidance of off-target activity will serve to facilitate a final goal of HIV-1 DNA elimination and accelerate testing in infected animals ultimately for use in man.},
}
@article {pmid34984879,
year = {2021},
author = {Dong, Z and Qin, Q and Zhang, X and Li, K and Chen, P and Pan, M},
title = {[Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {12},
pages = {4342-4350},
doi = {10.13345/j.cjb.210126},
pmid = {34984879},
issn = {1872-2075},
mesh = {Animals ; *Bombyx/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.},
}
@article {pmid34984801,
year = {2022},
author = {Tan, J and Zeng, D and Zhao, Y and Wang, Y and Liu, T and Li, S and Xue, Y and Luo, Y and Xie, X and Chen, L and Liu, YG and Zhu, Q},
title = {PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.},
journal = {Plant biotechnology journal},
volume = {20},
number = {5},
pages = {934-943},
pmid = {34984801},
issn = {1467-7652},
mesh = {Adenine ; *CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant ; },
abstract = {Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.},
}
@article {pmid34984631,
year = {2022},
author = {Oliveira, FS and da Silva Rodrigues, R and de Carvalho, AF and Nero, LA},
title = {Genomic Analyses of Pediococcus pentosaceus ST65ACC, a Bacteriocinogenic Strain Isolated from Artisanal Raw-Milk Cheese.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {34984631},
issn = {1867-1314},
abstract = {Pediococcus pentosaceus ST65ACC was obtained from a Brazilian artisanal cheese (BAC) and characterized as bacteriocinogenic. This strain presented beneficial properties in previous studies, indicating its potential as a probiotic candidate. In this study, we aimed to carry out a genetic characterization based on whole-genome sequencing (WGS), including taxonomy, biotechnological properties, bacteriocin clusters and safety-related genes. WGS was performed using the Illumina MiSeq platform and the genome was annotated with the Prokaryotic Genome Annotation (Prokka). P. pentosaceus ST65ACC taxonomy was investigated and bacteriocin genes clusters were identified by BAGEL4, metabolic pathways were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and safety-related genes were checked. P. pentosaceus ST65ACC had a total draft genome size of 1,933,194 bp with a GC content of 37.00%, and encoded 1950 protein coding sequences (CDSs), 6 rRNA, 55 tRNA, 1 tmRNA and no plasmids were detected. The analysis revealed absence of a CRISPR/Cas system, bacteriocin gene clusters for pediocin PA-1/AcH and penocin-A were identified. Genes related to beneficial properties, such as stress adaptation genes and adhesion genes, were identified. Furthermore, genes related to biogenic amines and virulence-related genes were not detected. Genes related to antibiotic resistance were identified, but not in prophage regions. Based on the obtained results, the beneficial potential of P. pentosaceus ST65ACC was confirmed, allowing its characterization as a potential probiotic candidate.},
}
@article {pmid34983823,
year = {2022},
author = {Wong, KM and King, DA and Schwartz, EK and Herrera, RE and Morrison, AJ},
title = {Retinoblastoma protein regulates carcinogen susceptibility at heterochromatic cancer driver loci.},
journal = {Life science alliance},
volume = {5},
number = {4},
pages = {},
pmid = {34983823},
issn = {2575-1077},
support = {R21 CA171050/CA/NCI NIH HHS/United States ; R21 CA178529/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Carcinogenesis/drug effects/genetics ; Carcinogens/*pharmacology ; Cell Line ; Gene Knockout Techniques ; Genetic Predisposition to Disease/genetics ; Humans ; Mutation/genetics ; *Neoplasms/genetics/pathology ; Oncogenes/genetics ; Retinoblastoma Binding Proteins/*genetics ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Carcinogenic insult, such as UV light exposure, creates DNA lesions that evolve into mutations if left unrepaired. These resulting mutations can contribute to carcinogenesis and drive malignant phenotypes. Susceptibility to carcinogens (i.e., the propensity to form a carcinogen-induced DNA lesion) is regulated by both genetic and epigenetic factors. Importantly, carcinogen susceptibility is a critical contributor to cancer mutagenesis. It is known that mutations can be prevented by tumor suppressor regulation of DNA damage response pathways; however, their roles carcinogen susceptibility have not yet been reported. In this study, we reveal that the retinoblastoma (RB1) tumor suppressor regulates UV susceptibility across broad regions of the genome. In particular, centromere and telomere-proximal regions exhibit significant increases in UV lesion susceptibility when RB1 is deleted. Several cancer-related genes are located within genomic regions of increased susceptibility, including telomerase reverse transcriptase, TERT, thereby accelerating mutagenic potential in cancers with RB1 pathway alterations. These findings reveal novel genome stability mechanisms of a tumor suppressor and uncover new pathways to accumulate mutations during cancer evolution.},
}
@article {pmid34983546,
year = {2022},
author = {Chen, Y and Li, L and Lan, J and Cui, Y and Rao, X and Zhao, J and Xing, T and Ju, G and Song, G and Lou, J and Liang, J},
title = {CRISPR screens uncover protective effect of PSTK as a regulator of chemotherapy-induced ferroptosis in hepatocellular carcinoma.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {11},
pmid = {34983546},
issn = {1476-4598},
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Antineoplastic Combined Chemotherapy Protocols/adverse effects/therapeutic use ; Biomarkers, Tumor ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/diagnosis/drug therapy/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Drug Resistance, Neoplasm/drug effects ; Drug Synergism ; Ferroptosis/*drug effects/*genetics ; Gene Knockdown Techniques ; *Genetic Testing/methods ; Humans ; Kaplan-Meier Estimate ; Liver Neoplasms/diagnosis/drug therapy/*genetics ; Mice ; Oxidation-Reduction/drug effects ; Phosphotransferases (Alcohol Group Acceptor)/antagonists &amp; inhibitors/chemistry/*genetics ; Prognosis ; Treatment Outcome ; },
abstract = {BACKGROUND: Hepatocellular carcinoma (HCC) is among the most common forms of cancer and is associated with poor patient outcomes. The emergence of therapeutic resistance has hampered the efficacy of targeted treatments employed to treat HCC patients to date. In this study, we conducted a series of CRISPR/Cas9 screens to identify genes associated with synthetic lethality capable of improving HCC patient clinical responses.<br><br>METHODS: CRISPR-based loss-of-function genetic screens were used to target 18,053 protein-coding genes in HCC cells to identify chemotherapy-related synthetic lethal genes in these cells. Synergistic effects were analyzed through in vitro and in vivo analyses, while related mechanisms were explored through RNA-seq and metabolomics analyses. Potential inhibitors of identified genetic targets were selected through high-throughput virtual screening.<br><br>RESULTS: The inhibition of phosphoseryl-tRNA kinase (PSTK) was found to increase HCC cell sensitivity to chemotherapeutic treatment. PSTK was associated with the suppression of chemotherapy-induced ferroptosis in HCC cells, and the depletion of PSTK resulted in the inactivation of glutathione peroxidative 4 (GPX4) and the disruption of glutathione (GSH) metabolism owing to the inhibition of selenocysteine and cysteine synthesis, thus enhancing the induction of ferroptosis upon targeted chemotherapeutic treatment. Punicalin, an agent used to treat hepatitis B virus (HBV), was identified as a possible PSTK inhibitor that exhibited synergistic efficacy when applied together with Sorafenib to treat HCC in vitro and in vivo.<br><br>CONCLUSIONS: These results highlight a key role for PSTK as a mediator of resistance to targeted therapeutic treatment in HCC cells that functions by suppressing ferroptotic induction. PSTK inhibitors may thus represent ideal candidates for overcoming drug resistance in HCC.},
}
@article {pmid34983523,
year = {2022},
author = {Huang, Y and Xu, Z and Gu, S and Nie, M and Wang, Y and Zhao, J and Li, F and Deng, H and Huang, J and Sun, X and Zhu, L},
title = {The recombinant pseudorabies virus expressing porcine deltacoronavirus spike protein is safe and effective for mice.},
journal = {BMC veterinary research},
volume = {18},
number = {1},
pages = {16},
pmid = {34983523},
issn = {1746-6148},
mesh = {Animals ; Antibodies, Viral ; *Coronavirus Infections/prevention &amp; control/veterinary ; Deltacoronavirus ; *Herpesvirus 1, Suid/genetics/immunology ; Mice ; Spike Glycoprotein, Coronavirus/*immunology ; Swine ; *Swine Diseases/prevention &amp; control/virology ; Viral Vaccines/*immunology ; },
abstract = {BACKGROUND: Porcine deltacoronavirus (PDCoV) is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since 2012. PDCoV disease caused acute diarrhea, vomiting, dehydration and death in piglets, resulted in significant economic loss to the pig industry. However, there is no commercially available vaccine for PDCoV. In this study, we constructed recombinant pseudorabies virus (rPRVXJ-delgE/gI/TK-S) expressing PDCoV spike (S) protein and evaluated its safety and immunogenicity in mice.<br><br>RESULTS: The recombinant strain rPRVXJ-delgE/gI/TK-S obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster syrian kidney-21 (BHK-21) cells and is safe to mice. After immunizing mice with rPRVXJ-delgE/gI/TK-S, the expression levels of IFN-&#x3b3; and IL-4 in peripheral blood of mice were up-regulated, the proliferation of spleen-specific T lymphocytes and the percentage of CD4[+] and CD8[+] lymphocytes in mice spleen was increased. rPRVXJ-delgE/gI/TK-S showed good immunogenicity for mice. On the seventh day after booster immunity, PRV gB and PDCoV S specific antibodies were detected in mice, and the antibody level continued to increase, and the neutralizing antibody level reached the maximum at 28&#x2009;days post- immunization (dpi). The recombinant strain can protect mice with 100% from the challenge of virulent strain (PRV XJ) and accelerate the detoxification of PDCoV in mice.<br><br>CONCLUSION: The recombinant rPRVXJ-delgE/gI/TK-S strain is safe and effective with strong immunogenicity and is expected to be a candidate vaccine against PDCoV and PRV.},
}
@article {pmid34982771,
year = {2022},
author = {Smith, JJ and Xiao, Y and Parsan, N and Medwig-Kinney, TN and Martinez, MAQ and Moore, FEQ and Palmisano, NJ and Kohrman, AQ and Chandhok Delos Reyes, M and Adikes, RC and Liu, S and Bracht, SA and Zhang, W and Wen, K and Kratsios, P and Matus, DQ},
title = {The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo.},
journal = {PLoS genetics},
volume = {18},
number = {1},
pages = {e1009981},
pmid = {34982771},
issn = {1553-7404},
support = {F31 HD100091/HD/NICHD NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R01 NS118078/NS/NINDS NIH HHS/United States ; R01 GM121597/GM/NIGMS NIH HHS/United States ; F31 GM128319/GM/NIGMS NIH HHS/United States ; F30 CA257383/CA/NCI NIH HHS/United States ; T32 GM008444/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Basement Membrane/metabolism ; CRISPR-Cas Systems ; Caenorhabditis elegans/metabolism/*physiology ; Caenorhabditis elegans Proteins/genetics/*metabolism ; Cell Cycle ; Cell Movement ; Chromosomal Proteins, Non-Histone/*genetics/metabolism ; Gene Expression Regulation ; Models, Animal ; *Mutation ; Phenotype ; Proto-Oncogene Proteins c-fos/*metabolism ; Single-Cell Analysis ; },
abstract = {Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and chromatin regulating factors that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.},
}
@article {pmid34982531,
year = {2022},
author = {Murawska, GM and Vogel, C and Jan, M and Lu, X and Schild, M and Slabicki, M and Zou, C and Zhanybekova, S and Manojkumar, M and Petzold, G and Kaiser, P and Thomä, N and Ebert, B and Gillingham, D},
title = {Repurposing the Damage Repair Protein Methyl Guanine Methyl Transferase as a Ligand Inducible Fusion Degron.},
journal = {ACS chemical biology},
volume = {17},
number = {1},
pages = {24-31},
doi = {10.1021/acschembio.1c00771},
pmid = {34982531},
issn = {1554-8937},
mesh = {CRISPR-Cas Systems ; Cell Line ; DNA Damage ; DNA Modification Methylases/antagonists &amp; inhibitors/genetics/*metabolism ; *DNA Repair ; DNA Repair Enzymes/antagonists &amp; inhibitors/genetics/*metabolism ; Humans ; Ligands ; Tumor Suppressor Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O[6] position of guanine (O[6]G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRAS[G12C] and a chimeric antigen receptor.},
}
@article {pmid34982495,
year = {2022},
author = {Li, F and Song, N and Dong, Y and Li, S and Li, L and Liu, Y and Li, Z and Yang, D},
title = {A Proton-Activatable DNA-Based Nanosystem Enables Co-Delivery of CRISPR/Cas9 and DNAzyme for Combined Gene Therapy.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {61},
number = {9},
pages = {e202116569},
doi = {10.1002/anie.202116569},
pmid = {34982495},
issn = {1521-3773},
mesh = {Breast Neoplasms/*therapy ; CRISPR-Cas Systems/*genetics ; DNA/*chemistry ; DNA, Catalytic/*genetics/metabolism ; Female ; *Genetic Therapy ; Humans ; Nanotechnology ; *Protons ; RNA/genetics ; },
abstract = {CRISPR/Cas9 is emerging as a platform for gene therapeutics, and the treatment efficiency is expected to be enhanced by combination with other therapeutic agents. Herein, we report a proton-activatable DNA-based nanosystem that enables co-delivery of Cas9/sgRNA and DNAzyme for the combined gene therapy of cancer. Ultra-long ssDNA chains, which contained the recognition sequences of sgRNA in Cas9/sgRNA, DNAzyme sequence and HhaI enzyme cleavage site, were synthesized as the scaffold of the nanosystem. The DNAzyme cofactor Mn[2+] was used to compress DNA chains to form nanoparticles and acid-degradable polymer-coated HhaI enzymes were assembled on the surface of nanoparticles. In response to protons in lysosome, the polymer coating was decomposed and HhaI enzyme was consequently exposed to recognize and cut off the cleavage sites, thus triggering the release of Cas9/sgRNA and DNAzyme to regulate gene expressions to achieve a high therapeutic efficacy of breast cancer.},
}
@article {pmid34981762,
year = {2022},
author = {Lee, SY and Kim, GE and Park, HH},
title = {Molecular basis of transcriptional repression of anti-CRISPR by anti-CRISPR-associated 2.},
journal = {Acta crystallographica. Section D, Structural biology},
volume = {78},
number = {Pt 1},
pages = {59-68},
doi = {10.1107/S2059798321011670},
pmid = {34981762},
issn = {2059-7983},
mesh = {AIDS-Related Complex ; Aeromonadaceae ; CRISPR-Associated Proteins/chemistry/*genetics ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; Epigenetic Repression ; Molecular Structure ; Mutagenesis ; Promoter Regions, Genetic ; Transcription, Genetic ; },
abstract = {CRISPR-Cas systems are well known host defense mechanisms that are conserved in bacteria and archaea. To counteract CRISPR-Cas systems, phages and viruses have evolved to possess multiple anti-CRISPR (Acr) proteins that can inhibit the host CRISPR-Cas system via different strategies. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins that bind to an upstream promoter and regulate the expression of acr genes during transcription. Although the role of Aca as a transcriptional repressor has been demonstrated, the mechanism of action of Aca has not been determined. Here, the molecular mechanism underlying the Aca2-mediated transcriptional control of acr genes was elucidated by determining the crystal structure of Aca2 from Oceanimonas smirnovii at a high resolution of 1.92 Å. Aca2 forms a dimer in solution, and dimerization of Aca2 is critical for specific promoter binding. The promoter-binding strategy of dimeric Aca2 was also revealed by performing mutagenesis studies. The atomic structure of the Aca family shown in this study provides insights into the fine regulation of host defense and immune-escape mechanisms and also demonstrates the conserved working mechanism of the Aca family.},
}
@article {pmid34981101,
year = {2022},
author = {Kim, H and Lee, S and Lee, J and Park, HG},
title = {CRISPR/Cas12a collateral cleavage activity for an ultrasensitive assay of RNase H.},
journal = {Chemical communications (Cambridge, England)},
volume = {58},
number = {16},
pages = {2654-2657},
doi = {10.1039/d1cc06026k},
pmid = {34981101},
issn = {1364-548X},
mesh = {*Biological Assay ; CRISPR-Cas Systems ; Ribonuclease H/*analysis/metabolism ; },
abstract = {We herein describe an ultrasensitive RNase H assay by utilizing CRISPR/Cas12a collateral cleavage activity. Based on this unique design principle, the RNase H activity was successfully determined down to 0.00024 U mL[-1], which is quite superior to those of alternative approaches.},
}
@article {pmid34980910,
year = {2022},
author = {Liu, R and Yang, J and Yao, J and Zhao, Z and He, W and Su, N and Zhang, Z and Zhang, C and Zhang, Z and Cai, H and Zhu, L and Zhao, Y and Quan, S and Chen, X and Yang, Y},
title = {Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {779-786},
pmid = {34980910},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; Genomics ; *Optogenetics ; *RNA/genetics ; RNA-Binding Proteins/genetics/metabolism ; },
abstract = {RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.},
}
@article {pmid34980881,
year = {2022},
author = {Chen, M and Yao, C and Qin, Y and Cui, X and Li, P and Ji, Z and Lin, L and Wu, H and Zhou, Z and Gui, Y and Li, Z and Gao, F},
title = {Mutations of MSH5 in nonobstructive azoospermia (NOA) and rescued via in vivo gene editing.},
journal = {Signal transduction and targeted therapy},
volume = {7},
number = {1},
pages = {1},
pmid = {34980881},
issn = {2059-3635},
mesh = {Animals ; *Azoospermia/genetics/metabolism/therapy ; *CRISPR-Cas Systems ; *Cell Cycle Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; *Gene Editing ; Humans ; Male ; Mice ; Mice, Mutant Strains ; },
}
@article {pmid34980227,
year = {2022},
author = {Sharma, VK and Marla, S and Zheng, W and Mishra, D and Huang, J and Zhang, W and Morris, GP and Cook, DE},
title = {CRISPR guides induce gene silencing in plants in the absence of Cas.},
journal = {Genome biology},
volume = {23},
number = {1},
pages = {6},
pmid = {34980227},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; RNA/genetics ; RNA Interference ; RNA, Guide/genetics ; Sequence Analysis, RNA ; },
abstract = {BACKGROUND: RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity.<br><br>RESULTS: Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design.<br><br>CONCLUSIONS: Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.},
}
@article {pmid34980132,
year = {2022},
author = {Li, Y and Yang, C and Liu, Z and Du, S and Can, S and Zhang, H and Zhang, L and Huang, X and Xiao, Z and Li, X and Fang, J and Qin, W and Sun, C and Wang, C and Chen, J and Chen, H},
title = {Integrative analysis of CRISPR screening data uncovers new opportunities for optimizing cancer immunotherapy.},
journal = {Molecular cancer},
volume = {21},
number = {1},
pages = {2},
pmid = {34980132},
issn = {1476-4598},
mesh = {Antineoplastic Agents, Immunological/administration &amp; dosage/adverse effects/therapeutic use ; *CRISPR-Cas Systems ; Clinical Decision-Making ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; Disease Management ; Gene Expression Profiling ; Gene Regulatory Networks ; Genetic Testing/*methods ; Genomics/*methods ; Humans ; Immunotherapy/methods/standards ; *Medical Oncology/methods/standards ; Prognosis ; Transcriptome ; Treatment Outcome ; },
abstract = {BACKGROUND: In recent years, the application of functional genetic immuno-oncology screens has showcased the striking ability to identify potential regulators engaged in tumor-immune interactions. Although these screens have yielded substantial data, few studies have attempted to systematically aggregate and analyze them.<br><br>METHODS: In this study, a comprehensive data collection of tumor immunity-associated functional screens was performed. Large-scale genomic data sets were exploited to conduct integrative analyses.<br><br>RESULTS: We identified 105 regulator genes that could mediate resistance or sensitivity to immune cell-induced tumor elimination. Further analysis identified MON2 as a novel immune-oncology target with considerable therapeutic potential. In addition, based on the 105 genes, a signature named CTIS (CRISPR screening-based tumor-intrinsic immune score) for predicting response to immune checkpoint blockade (ICB) and several immunomodulatory agents with the potential to augment the efficacy of ICB were also determined.<br><br>CONCLUSION: Overall, our findings provide insights into immune oncology and open up novel opportunities for improving the efficacy of current immunotherapy agents.},
}
@article {pmid34979927,
year = {2022},
author = {Tang, Y and Li, H and Liu, C and He, Y and Wang, H and Zhao, T and Xu, X and Li, J and Yang, H and Jiang, J},
title = {CRISPR-Cas9-mediated mutagenesis of the SlSRM1-like gene leads to abnormal leaf development in tomatoes.},
journal = {BMC plant biology},
volume = {22},
number = {1},
pages = {13},
pmid = {34979927},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems ; Lycopersicon esculentum ; Mutagenesis ; Plant Leaves/genetics/*growth &amp; development ; Plant Proteins/*genetics/metabolism ; },
abstract = {BACKGROUND: Leaves, which are the most important organs of plants, can not only fix carbon sources through photosynthesis, but also absorb nutrients through transpiration. Leaf development directly determines the growth, flowering and fruiting of plants. There are many factors that affect leaf development, such as the growth environment, gene expression, and hormone synthesis. In this study, tomatoes were used to study the role of the transcription factor Solanum lycopersicum salt-related MYB1-like (SlSRM1-like) in the development of tomato leaves.<br><br>RESULTS: Loss-of-function of the SlSRM1-like gene mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) resulted in abnormal tomato leaf morphology, including thinner leaves, wrinkled edges, raised veins, disordered edge veins, and left and right asymmetry. An analysis of the transcription levels of genes related to leaf development revealed that the expression of these genes was significantly altered in the SlSRM1-like mutants (SlSRM1-like-Ms). Moreover, the SlSRM1-like gene was expressed at higher transcription levels in young tissues than in old tissues, and its expression was also induced in response to auxin. In addition, the transcription levels of genes related to the auxin pathway, which regulates tomato growth and development, were severely affected in the SlSRM1-like-Ms. Therefore, it is hypothesized that the SlSRM1-like gene functions in the regulation of tomato leaf development through the auxin-related pathway.<br><br>CONCLUSIONS: In this study, we successfully knocked out the SlSRM1-like gene in the tomato variety Ailsa Craig using CRISPR technology and found that knockout of the SlSRM1-like gene resulted in abnormal development of tomato leaves. Further research indicated that SlSRM1-like regulated tomato leaf development through auxin-related pathways. The results provide an important reference for the functional study of other SRM1-like genes in plants and provide new insights into the regulation of leaf development in tomato and other plants.},
}
@article {pmid34978812,
year = {2022},
author = {Pantoja Angles, A and Ali, Z and Mahfouz, M},
title = {CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {430-440},
doi = {10.1021/acssynbio.1c00516},
pmid = {34978812},
issn = {2161-5063},
mesh = {Biotechnology ; *CRISPR-Cas Systems/genetics ; Chromosomes ; DNA/genetics ; *Synthetic Biology ; },
abstract = {Synthetic biology holds great promise for translating ideas into products to address the grand challenges facing humanity. Molecular biomanufacturing is an emerging technology that facilitates the production of key products of value, including therapeutics and select chemical compounds. Current biomanufacturing technologies require improvements to overcome limiting factors, including efficient production, cost, and safe release; therefore, developing optimum chassis for biomolecular manufacturing is of great interest for enabling diverse synthetic biology applications. Here, we harnessed the power of the CRISPR-Cas12 system to design, build, and test a DNA device for genome shredding, which fragments the native genome to enable the conversion of bacterial cells into nonreplicative, biosynthetically active, and programmable molecular biomanufacturing chassis. As a proof of concept, we demonstrated the efficient production of green fluorescent protein and violacein, an antimicrobial and antitumorigenic compound. Our CRISPR-Cas12-based chromosome-shredder DNA device has built-in biocontainment features providing a roadmap for the conversion of any bacterial cell into a chromosome-shredded chassis amenable to high-efficiency molecular biomanufacturing, thereby enabling exciting and diverse biotechnological applications.},
}
@article {pmid34977860,
year = {2021},
author = {Gong, Z and Cheng, M and Botella, JR},
title = {Non-GM Genome Editing Approaches in Crops.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {817279},
pmid = {34977860},
issn = {2673-3439},
abstract = {CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.},
}
@article {pmid34977677,
year = {2022},
author = {Moosmann, C and Müller, TR and Busch, DH and Schober, K},
title = {Orthotopic T-cell receptor replacement in primary human T cells using CRISPR-Cas9-mediated homology-directed repair.},
journal = {STAR protocols},
volume = {3},
number = {1},
pages = {101031},
pmid = {34977677},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Receptors, Antigen, T-Cell/genetics ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; Recombinational DNA Repair ; T-Lymphocytes ; },
abstract = {Adoptive T cell therapy using T-cell receptor (TCR)-engineered T cells allows to redirect T cell specificity and to target any antigen of interest. Here, we apply advanced genetic engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) for simultaneous editing of TCR α- and β-chains in primary human T cells. Together with non-virally delivered template DNA, this CRISPR-Cas9-system allows for elimination of the endogenous TCR and orthotopic placement of TCR α- and β-chains. For complete details on the use and execution of this protocol, please refer to Schober et al. (2019) and Müller et al. (2021).},
}
@article {pmid34977337,
year = {2021},
author = {Ni, N and Deng, F and He, F and Wang, H and Shi, D and Liao, J and Zou, Y and Wang, H and Zhao, P and Hu, X and Chen, C and Hu, DA and Sabharwal, M and Qin, KH and Wagstaff, W and Qin, D and Hendren-Santiago, B and Haydon, RC and Luu, HH and Reid, RR and Shen, L and He, TC and Fan, J},
title = {A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology.},
journal = {Molecular therapy oncolytics},
volume = {23},
number = {},
pages = {602-611},
pmid = {34977337},
issn = {2372-7705},
abstract = {Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.},
}
@article {pmid34977113,
year = {2021},
author = {Sharma, SK and Gupta, OP and Pathaw, N and Sharma, D and Maibam, A and Sharma, P and Sanasam, J and Karkute, SG and Kumar, S and Bhattacharjee, B},
title = {CRISPR-Cas-Led Revolution in Diagnosis and Management of Emerging Plant Viruses: New Avenues Toward Food and Nutritional Security.},
journal = {Frontiers in nutrition},
volume = {8},
number = {},
pages = {751512},
pmid = {34977113},
issn = {2296-861X},
abstract = {Plant viruses pose a serious threat to agricultural production systems worldwide. The world's population is expected to reach the 10-billion mark by 2057. Under the scenario of declining cultivable land and challenges posed by rapidly emerging and re-emerging plant pathogens, conventional strategies could not accomplish the target of keeping pace with increasing global food demand. Gene-editing techniques have recently come up as promising options to enable precise changes in genomes with greater efficiency to achieve the target of higher crop productivity. Of genome engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins have gained much popularity, owing to their simplicity, reproducibility, and applicability in a wide range of species. Also, the application of different Cas proteins, such as Cas12a, Cas13a, and Cas9 nucleases, has enabled the development of more robust strategies for the engineering of antiviral mechanisms in many plant species. Recent studies have revealed the use of various CRISPR-Cas systems to either directly target a viral gene or modify a host genome to develop viral resistance in plants. This review provides a comprehensive record of the use of the CRISPR-Cas system in the development of antiviral resistance in plants and discusses its applications in the overall enhancement of productivity and nutritional landscape of cultivated plant species. Furthermore, the utility of this technique for the detection of various plant viruses could enable affordable and precise in-field or on-site detection. The futuristic potential of CRISPR-Cas technologies and possible challenges with their use and application are highlighted. Finally, the future of CRISPR-Cas in sustainable management of viral diseases, and its practical utility and regulatory guidelines in different parts of the globe are discussed systematically.},
}
@article {pmid34975951,
year = {2021},
author = {Ahmad, M and Waraich, EA and Skalicky, M and Hussain, S and Zulfiqar, U and Anjum, MZ and Habib Ur Rahman, M and Brestic, M and Ratnasekera, D and Lamilla-Tamayo, L and Al-Ashkar, I and El Sabagh, A},
title = {Adaptation Strategies to Improve the Resistance of Oilseed Crops to Heat Stress Under a Changing Climate: An Overview.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {767150},
pmid = {34975951},
issn = {1664-462X},
abstract = {Temperature is one of the decisive environmental factors that is projected to increase by 1. 5°C over the next two decades due to climate change that may affect various agronomic characteristics, such as biomass production, phenology and physiology, and yield-contributing traits in oilseed crops. Oilseed crops such as soybean, sunflower, canola, peanut, cottonseed, coconut, palm oil, sesame, safflower, olive etc., are widely grown. Specific importance is the vulnerability of oil synthesis in these crops against the rise in climatic temperature, threatening the stability of yield and quality. The natural defense system in these crops cannot withstand the harmful impacts of heat stress, thus causing a considerable loss in seed and oil yield. Therefore, a proper understanding of underlying mechanisms of genotype-environment interactions that could affect oil synthesis pathways is a prime requirement in developing stable cultivars. Heat stress tolerance is a complex quantitative trait controlled by many genes and is challenging to study and characterize. However, heat tolerance studies to date have pointed to several sophisticated mechanisms to deal with the stress of high temperatures, including hormonal signaling pathways for sensing heat stimuli and acquiring tolerance to heat stress, maintaining membrane integrity, production of heat shock proteins (HSPs), removal of reactive oxygen species (ROS), assembly of antioxidants, accumulation of compatible solutes, modified gene expression to enable changes, intelligent agricultural technologies, and several other agronomic techniques for thriving and surviving. Manipulation of multiple genes responsible for thermo-tolerance and exploring their high expressions greatly impacts their potential application using CRISPR/Cas genome editing and OMICS technology. This review highlights the latest outcomes on the response and tolerance to heat stress at the cellular, organelle, and whole plant levels describing numerous approaches applied to enhance thermos-tolerance in oilseed crops. We are attempting to critically analyze the scattered existing approaches to temperature tolerance used in oilseeds as a whole, work toward extending studies into the field, and provide researchers and related parties with useful information to streamline their breeding programs so that they can seek new avenues and develop guidelines that will greatly enhance ongoing efforts to establish heat stress tolerance in oilseeds.},
}
@article {pmid34974332,
year = {2022},
author = {Nakao, J and Yamamoto, T and Yamayoshi, A},
title = {Therapeutic application of sequence-specific binding molecules for novel genome editing tools.},
journal = {Drug metabolism and pharmacokinetics},
volume = {42},
number = {},
pages = {100427},
doi = {10.1016/j.dmpk.2021.100427},
pmid = {34974332},
issn = {1880-0920},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
abstract = {Genome editing has been expected to widely increase the available treatment options for various diseases and permit pharmaceutical interventions in previously untreatable conditions. The availability of genome editing tools was dramatically increased by the development of the CRISPR-Cas9 system. However, a number of issues limit the use of the CRISPR-Cas9 system and other gene-editing tools in the clinical treatment of diseases. This review summarized the history and types of genome editing tools and limitations of their use. In addition, the study addressed several next-generation technologies aiming to overcome the limitations of current gene therapy protocols in an effort to accelerate the clinical development of potential treatment options. This review has provided an extensive foundation of the current state of genome editing technology and its clinical development. This review also indicate that the study additionally highlighted the need for multidisciplinary approaches to overcome current bottlenecks in the development of genome editing.},
}
@article {pmid34974144,
year = {2022},
author = {Chavez-Granados, PA and Manisekaran, R and Acosta-Torres, LS and Garcia-Contreras, R},
title = {CRISPR/Cas gene-editing technology and its advances in dentistry.},
journal = {Biochimie},
volume = {194},
number = {},
pages = {96-107},
doi = {10.1016/j.biochi.2021.12.012},
pmid = {34974144},
issn = {1638-6183},
mesh = {Animals ; *CRISPR-Cas Systems ; Dentistry ; *Gene Editing/methods ; Prospective Studies ; Technology ; },
abstract = {A recent discovery of revolutionary Clustered regularly interspaced palindromic repeats (CRISPR) is a gene-editing tool that provides a type of adaptive immunity in prokaryotic organisms, which is currently used as a revolutionizing tool in biomedical research. It has a mechanism of correcting genome errors, turning on/off genes in cells and organisms. Most importantly playing a crucial function in bacterial defence by identifying and destroying Deoxyribonucleic acid (DNA) segments during bacteriophage invasions since the CRISPR-associated protein 9 (Cas9) enzyme recognizes and cleaves invasive DNA sequences complementary to CRISPR. Therefore, researchers employ this biological device to manipulate the genes to develop new therapies to combat systemic diseases. Currently, the most significant advance at the laboratory level is the generation of cell and animal models, functional genomic screens, live images of the cell genome, and defective DNA repairs to find the cure for genetic disorders. Even though this technology has enormous biomedical applications in various sectors, this review will summarize CRISPR/Cas emphasizing both the therapeutic and diagnostic mechanisms developed in the field of dentistry and the promising attempts to transfer this technology to clinical application. Finally, future developments are also described, which proposes to use CRISPR/Cas systems for prospective clinical dentistry applications.},
}
@article {pmid34973546,
year = {2022},
author = {Li, Z and Li, Y and Xiao, C and Yan, Z and Pan, R and Gao, Y and Li, B and Wei, J and Qiu, Y and Liu, K and Shao, D and Ma, Z},
title = {Genomic and metabolic features of the Lactobacillus sakei JD10 revealed potential probiotic traits.},
journal = {Microbiological research},
volume = {256},
number = {},
pages = {126954},
doi = {10.1016/j.micres.2021.126954},
pmid = {34973546},
issn = {1618-0623},
mesh = {Animals ; *Bacteriocins/genetics ; Genomics ; *Lactobacillus sakei/genetics/metabolism ; *Probiotics ; Swine ; },
abstract = {Lactic acid bacteria that inhabit in the lung play important roles in maintaining the microbiome balance by interacting with the host immune system. Numerous metabolites (e.g., short chain fatty acids, bacteriocins, and hydrogen peroxide) produced by Lactobacillus sakei possess a special inhibitory spectrum against invading pathogens. In this research, the whole genome of L. sakei JD10 strain isolated from the porcine lung was sequenced and investigated. The whole size of the L. sakei JD10 chromosome was 1,989,921 bp, which encoded a total of 1951 predicted genes. Genome analyses revealed that many genes encoded carbohydrate-active enzymes (CAZymes) were predicted, which were responsible for the carbohydrate degradation and short chain fatty acids production. The metabolic profiles of short chain fatty acids in the L. sakei JD10 culture medium were measured by GC/TOFMS, and their regulatory effects on bacterial phagocytosis of RAW264.7 cells were also determined. The bacteriocin-producing genes of the L. sakei JD10 genome were also predicted, and a bacteriocin gene encoding carnocin was characterized and its molecular structure was analyzed. Two CRISPR-Cas system related genes were identified from the L. sakei JD10 genome, revealed that precise and efficient genome editing technologies could be applied for genetic engineering-manipulation. In all, investigation on the genomic features and metabolic features of L. sakei JD10 showed the potential probiotic traits to fight against pathogenic infection and regulate the host immune function.},
}
@article {pmid34973111,
year = {2022},
author = {Chaudhuri, A and Halder, K and Datta, A},
title = {Classification of CRISPR/Cas system and its application in tomato breeding.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {135},
number = {2},
pages = {367-387},
pmid = {34973111},
issn = {1432-2242},
mesh = {Archaea/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Lycopersicon esculentum/genetics ; Plant Breeding ; },
abstract = {Remarkable diversity in the domain of genome loci architecture, structure of effector complex, array of protein composition, mechanisms of adaptation along with difference in pre-crRNA processing and interference have led to a vast scope of detailed classification in bacterial and archaeal CRISPR/Cas systems, their intrinsic weapon of adaptive immunity. Two classes: Class 1 and Class 2, several types and subtypes have been identified so far. While the evolution of the effector complexes of Class 2 is assigned solely to mobile genetic elements, the origin of Class 1 effector molecules is still in a haze. Majority of the types target DNA except type VI, which have been found to target RNA exclusively. Cas9, the single effector protein, has been the primary focus of CRISPR-mediated genome editing revolution and is an integral part of Class 2 (type II) system. The present review focuses on the different CRISPR types in depth and the application of CRISPR/Cas9 for epigenome modification, targeted base editing and improving traits such as abiotic and biotic stress tolerance, yield and nutritional aspects of tomato breeding.},
}
@article {pmid34972984,
year = {2022},
author = {Yu, TT and Zhou, X and Vakifahmetoglu-Norberg, H},
title = {CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2445},
number = {},
pages = {39-50},
pmid = {34972984},
issn = {1940-6029},
mesh = {Autophagy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Lysosome-Associated Membrane Glycoproteins/genetics/metabolism ; Lysosomes/metabolism ; *Neoplasms/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; },
abstract = {Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent protein degradation pathway. A critical molecular component of CMA is the lysosome-associated membrane protein (LAMP) type 2A, which is required for substrate uptake by the lysosome. Defects in the CMA pathway have been associated with various human pathologies, including malignancies, increasing the overall interest in methods to monitor this selective autophagy process. Yet isogenic LAMP-2A knockout cancer cell models are still lacking. This is likely to depend on challenges related to that human LAMP-2 gene undergoes alternative splicing of its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. However, without assessment of the impact of LAMP-2A loss of function specifically in human cells, the involvement of CMA in human pathologies, including carcinogenesis remains speculative. Here, we describe the generation of isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, without affecting the other two isoforms, allowing for experimental evaluation of LAMP-2A, thus CMA in human cancer models.},
}
@article {pmid34970254,
year = {2021},
author = {Roberts, A and Chouhan, RS and Shahdeo, D and Shrikrishna, NS and Kesarwani, V and Horvat, M and Gandhi, S},
title = {A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {732756},
pmid = {34970254},
issn = {1664-3224},
mesh = {COVID-19/*diagnosis/epidemiology/virology ; COVID-19 Testing/*methods ; Clinical Laboratory Techniques/*methods ; Humans ; Immunoassay/methods ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/methods ; Oligonucleotide Probes/genetics ; Pandemics ; RNA, Viral/genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2/*genetics/physiology ; Sensitivity and Specificity ; },
abstract = {Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.},
}
@article {pmid34969984,
year = {2022},
author = {He, Z and Maynard, A and Jain, A and Gerber, T and Petri, R and Lin, HC and Santel, M and Ly, K and Dupré, JS and Sidow, L and Sanchis Calleja, F and Jansen, SMJ and Riesenberg, S and Camp, JG and Treutlein, B},
title = {Lineage recording in human cerebral organoids.},
journal = {Nature methods},
volume = {19},
number = {1},
pages = {90-99},
pmid = {34969984},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; Cell Lineage ; Cerebral Cortex/*cytology ; *Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Microscopy/methods ; Mutation ; Neurons/cytology/physiology ; Organoids/*cytology ; Recombinant Proteins/genetics/metabolism ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; Tuberous Sclerosis Complex 2 Protein/genetics ; },
abstract = {Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.},
}
@article {pmid34968750,
year = {2022},
author = {Jin, X and Zhang, L and Wang, X and An, L and Huang, S and Dai, P and Gao, H and Ma, X},
title = {Novel CRISPR/Cas12a-based genetic diagnostic approach for SLC26A4 mutation-related hereditary hearing loss.},
journal = {European journal of medical genetics},
volume = {65},
number = {2},
pages = {104406},
doi = {10.1016/j.ejmg.2021.104406},
pmid = {34968750},
issn = {1878-0849},
mesh = {*CRISPR-Cas Systems ; Genetic Testing/*methods ; Hearing Loss/diagnosis/*genetics ; Humans ; Mutation ; Nucleic Acid Amplification Techniques/methods ; Sulfate Transporters/*genetics ; },
abstract = {Hereditary hearing loss is a common defect of the auditory nervous system with high-incidence, seriously affecting the quality of life of the patients. The clinical manifestations of SLC26A4 mutation-related hearing loss are congenital sensorineural or mixed deafness. Sensitive and specific SLC26A4 mutation detection in the early clinical stage is key for the early indication of potential hearing loss in the lack of effective treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology, we designed a fast and sensitive detection system for SLC26A4 pathogenic mutations (c.919-2A > G, c.2168A > G and c.1229C > T). This recombinase-aided amplification-based detection system allows rapid target gene amplification and, in combination with the CRISPR-based nucleic acid testing (NAT) system, mutation site detection. Moreover, mismatches were introduced in CRISPR-derived RNA (crRNA) to increase signal differences between the wild-type genes and mutant genes. A total of 64 samples were examined using this approach and all results were verified using Sanger sequencing. The detection results were consistent with the polymerase chain reaction-Sanger sequencing results. Overall, this CRISPR-based NAT technology provides a sensitive and fast new approach for the detection of hereditary deafness and provides a crRNA optimization strategy for single-nucleotide polymorphism detection, which could be helpful for the clinical diagnosis of SLC26A4 mutation-related hereditary hearing loss.},
}
@article {pmid34968443,
year = {2022},
author = {Godden, AM and Antonaci, M and Ward, NJ and van der Lee, M and Abu-Daya, A and Guille, M and Wheeler, GN},
title = {An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.},
journal = {Developmental biology},
volume = {483},
number = {},
pages = {66-75},
pmid = {34968443},
issn = {1095-564X},
support = {BB/K019988/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/J014524/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 101480Z/WT_/Wellcome Trust/United Kingdom ; BB/H003525/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M011216/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques/methods ; Gene Knockout Techniques/*methods ; In Situ Hybridization/methods ; MicroRNAs/*genetics ; Morpholinos/genetics ; Neural Crest/embryology/metabolism ; Neural Plate/embryology/metabolism ; Neurulation/genetics ; Phenotype ; RNA, Guide/genetics ; Transcription Factors/genetics/metabolism ; Transcriptome/genetics ; Xenopus Proteins/genetics/metabolism ; Xenopus laevis/*embryology/*genetics ; },
abstract = {In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.},
}
@article {pmid34968414,
year = {2022},
author = {Liu, G and Lin, Q and Jin, S and Gao, C},
title = {The CRISPR-Cas toolbox and gene editing technologies.},
journal = {Molecular cell},
volume = {82},
number = {2},
pages = {333-347},
doi = {10.1016/j.molcel.2021.12.002},
pmid = {34968414},
issn = {1097-4164},
mesh = {Animals ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Diffusion of Innovation ; *Gene Editing ; Humans ; },
abstract = {The emergence of CRISPR-Cas systems has accelerated the development of gene editing technologies, which are widely used in the life sciences. To improve the performance of these systems, workers have engineered and developed a variety of CRISPR-Cas tools with a broader range of targets, higher efficiency and specificity, and greater precision. Moreover, CRISPR-Cas-related technologies have also been expanded beyond making cuts in DNA by introducing functional elements that permit precise gene modification, control gene expression, make epigenetic changes, and so on. In this review, we introduce and summarize the characteristics and applications of different types of CRISPR-Cas tools. We discuss certain limitations of current approaches and future prospects for optimizing CRISPR-Cas systems.},
}
@article {pmid34968397,
year = {2021},
author = {Chen, A and Liao, P and Li, Q and Zhao, Q and Gao, M and Wang, P and Liu, Z and Meng, G and Dong, Z and Liu, M},
title = {phytanoyl-CoA dioxygenase domain-containing protein 1 plays an important role in egg shell formation of silkworm (Bombyx mori).},
journal = {PloS one},
volume = {16},
number = {12},
pages = {e0261918},
pmid = {34968397},
issn = {1932-6203},
mesh = {Animals ; Bombyx/*physiology ; CRISPR-Cas Systems ; Chorion/chemistry ; Chromosomes ; Coenzyme A/chemistry ; Down-Regulation ; Egg Shell/*physiology ; Female ; Gene Silencing ; Insect Proteins/genetics ; Larva/genetics ; Male ; Models, Genetic ; Mutation ; Oxygenases/*chemistry ; Phenotype ; Phytanic Acid/analogs &amp; derivatives/chemistry ; Polymerase Chain Reaction ; Protein Domains ; RNA-Seq ; Reproduction ; Sex Chromosomes/metabolism ; },
abstract = {Yun7Ge is a giant egg mutant found in the silkworm variety Yun7. In comparison with the giant mutant Ge, the eggs of Yun7Ge are larger. The number of laid eggs and hatching rate of Yun7Ge are reduced, which is not conducive to reproduction. In this work, the target gene controlling giant egg trait is located on the Z chromosome and was determined through genetic analysis. Transcriptome results showed that phytanoyl-CoA dioxygenase domain-containing protein 1 (PHYHD1) on the Z chromosome was silenced, and the 25 chorion genes on chromosome 2 were remarkably downregulated. Sequence analysis showed that the 73.5 kb sequence including the PHYHD1 was replaced by a ~3.0 kb sequence. After knocking out the PHYHD1 by using CRISPR/Cas9, the chorion genes were significantly downregulated. Hence, the silencing of PHYHD1 leads to the downregulation of many chorion protein genes, thus directly causing giant eggs.},
}
@article {pmid34965029,
year = {2022},
author = {Cona, B and Hayashi, T and Yamada, A and Shimizu, N and Yokota, N and Nakato, R and Shirahige, K and Akiyama, T},
title = {The splicing factor DHX38/PRP16 is required for ovarian clear cell carcinoma tumorigenesis, as revealed by a CRISPR-Cas9 screen.},
journal = {FEBS open bio},
volume = {12},
number = {3},
pages = {582-593},
pmid = {34965029},
issn = {2211-5463},
mesh = {*Adenocarcinoma, Clear Cell/genetics/metabolism/pathology ; Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics ; DEAD-box RNA Helicases/genetics/metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; *Ovarian Neoplasms/drug therapy ; *RNA Splicing Factors/genetics/metabolism/therapeutic use ; },
abstract = {Certain cancers, such as ovarian clear cell carcinoma (OCCC), display high levels of genetic variation between patients, making it difficult to develop effective therapies. In order to identify novel genes critical to OCCC growth, we carried out a comprehensive CRISPR-Cas9 knockout screen against cell growth using an OCCC cell line and a normal ovarian surface epithelium cell line. We identified the gene encoding DHX38/PRP16, an ATP-dependent RNA helicase involved in splicing, as critical for the growth and tumorigenesis of OCCC. DHX38/PRP16 knockdown in OCCC cells, but not normal cells, induces apoptosis and impairs OCCC tumorigenesis in a mouse model. Our results suggest that DHX38/PRP16 may play a role in OCCC tumorigenesis and could potentially be a promising therapeutic target.},
}
@article {pmid34964942,
year = {2022},
author = {Moon, HY and Sim, GH and Kim, HJ and Kim, K and Kang, HA},
title = {Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {60},
number = {1},
pages = {18-30},
pmid = {34964942},
issn = {1976-3794},
mesh = {*CRISPR-Cas Systems ; Genetic Markers ; Integrases/genetics/metabolism ; Plasmids/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {We evaluated the Cre-lox and CRISPR-Cas9 systems as marker-recycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4-based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura[-] transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.},
}
@article {pmid34963339,
year = {2022},
author = {Wang, YM and Wang, HZ and Jian, YZ and Luo, ZT and Shao, HW and Zhang, WF},
title = {Strategies for Optimization of the Clustered Regularly Interspaced Short Palindromic Repeat-Based Genome Editing System for Enhanced Editing Specificity.},
journal = {Human gene therapy},
volume = {33},
number = {7-8},
pages = {358-370},
doi = {10.1089/hum.2021.283},
pmid = {34963339},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/genetics ; *Gene Editing ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is inarguably the most valuable gene editing tool ever discovered. Currently, three classes of CRISPR-based genome editing systems have been developed for gene editing, including CRISPR/CRISPR associate system (Cas) nucleases, base editors, and prime editors. Ever-evolving CRISPR technology plays an important role in medicine; however, the biggest obstacle to its use in clinical practice is the induction of off-target effects (OTEs) during targeted editing. Therefore, continuous improvement and optimization of the CRISPR system for reduction of OTEs is a major focus in the field of CRISPR research. This review aims to provide a comprehensive guide for optimization of the CRISPR-based genome editing system.},
}
@article {pmid34963106,
year = {2021},
author = {Oh, Y and Kim, SG},
title = {RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata.},
journal = {Molecules and cells},
volume = {44},
number = {12},
pages = {911-919},
pmid = {34963106},
issn = {0219-1032},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Plant ; RNA, Guide/genetics ; Ribosomal Proteins ; *Tobacco/genetics/metabolism ; },
abstract = {The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M1 progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.},
}
@article {pmid34962995,
year = {2022},
author = {Hua, K and Han, P and Zhu, JK},
title = {Improvement of base editors and prime editors advances precision genome engineering in plants.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1795-1810},
pmid = {34962995},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant/genetics ; *Plant Breeding ; Plants/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-mediated gene disruption has revolutionized biomedical research as well as plant and animal breeding. However, most disease-causing mutations and agronomically important genetic variations are single base polymorphisms (single-nucleotide polymorphisms) that require precision genome editing tools for correction of the sequences. Although homology-directed repair of double-stranded breaks (DSBs) can introduce precise changes, such repairs are inefficient in differentiated animal and plant cells. Base editing and prime editing are two recently developed genome engineering approaches that can efficiently introduce precise edits into target sites without requirement of DSB formation or donor DNA templates. They have been applied in several plant species with promising results. Here, we review the extensive literature on improving the efficiency, target scope, and specificity of base editors and prime editors in plants. We also highlight recent progress on base editing in plant organellar genomes and discuss how these precision genome editing tools are advancing basic plant research and crop breeding.},
}
@article {pmid34962926,
year = {2021},
author = {Kratzel, A and Kelly, JN and V'kovski, P and Portmann, J and Brüggemann, Y and Todt, D and Ebert, N and Shrestha, N and Plattet, P and Staab-Weijnitz, CA and von Brunn, A and Steinmann, E and Dijkman, R and Zimmer, G and Pfaender, S and Thiel, V},
title = {A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets.},
journal = {PLoS biology},
volume = {19},
number = {12},
pages = {e3001490},
pmid = {34962926},
issn = {1545-7885},
mesh = {Antiviral Agents/pharmacology ; Autophagy/*genetics ; *CRISPR-Cas Systems ; Gene Knockdown Techniques ; Host-Pathogen Interactions ; Humans ; Middle East Respiratory Syndrome Coronavirus/drug effects/*genetics/physiology ; SARS-CoV-2/drug effects/*genetics/physiology ; Virus Replication ; },
abstract = {Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.},
}
@article {pmid34962615,
year = {2022},
author = {Li, L and Duan, C and Weng, J and Qi, X and Liu, C and Li, X and Zhu, J and Xie, C},
title = {A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13.},
journal = {Science China. Life sciences},
volume = {65},
number = {7},
pages = {1456-1465},
pmid = {34962615},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems ; DNA ; Endonucleases ; *Nucleic Acids ; *RNA ; },
abstract = {For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.},
}
@article {pmid34962611,
year = {2021},
author = {Singha, DL and Das, D and Sarki, YN and Chowdhury, N and Sharma, M and Maharana, J and Chikkaputtaiah, C},
title = {Harnessing tissue-specific genome editing in plants through CRISPR/Cas system: current state and future prospects.},
journal = {Planta},
volume = {255},
number = {1},
pages = {28},
pmid = {34962611},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Plant/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {In a nutshell, tissue-specific CRISPR/Cas genome editing is the most promising approach for crop improvement which can bypass the hurdle associated with constitutive GE such as off target and pleotropic effects for targeted crop improvement. CRISPR/Cas is a powerful genome-editing tool with a wide range of applications for the genetic improvement of crops. However, the constitutive genome editing of vital genes is often associated with pleiotropic effects on other genes, needless metabolic burden, or interference in the cellular machinery. Tissue-specific genome editing (TSGE), on the other hand, enables researchers to study those genes in specific cells, tissues, or organs without disturbing neighboring groups of cells. Until recently, there was only limited proof of the TSGE concept, where the CRISPR-TSKO tool was successfully used in Arabidopsis, tomato, and cotton, laying a solid foundation for crop improvement. In this review, we have laid out valuable insights into the concept and application of TSGE on relatively unexplored areas such as grain trait improvement under favorable or unfavorable conditions. We also enlisted some of the prominent tissue-specific promoters and described the procedure of their isolation with several TSGE promoter expression systems in detail. Moreover, we highlighted potential negative regulatory genes that could be targeted through TSGE using tissue-specific promoters. In a nutshell, tissue-specific CRISPR/Cas genome editing is the most promising approach for crop improvement which can bypass the hurdle associated with constitutive GE such as off target and pleotropic effects for targeted crop improvement.},
}
@article {pmid34962045,
year = {2022},
author = {Ye, Q and Meng, X and Chen, H and Wu, J and Zheng, L and Shen, C and Guo, D and Zhao, Y and Liu, J and Xue, Q and Dong, J and Wang, T},
title = {Construction of genic male sterility system by CRISPR/Cas9 editing from model legume to alfalfa.},
journal = {Plant biotechnology journal},
volume = {20},
number = {4},
pages = {613-615},
pmid = {34962045},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Infertility, Male/genetics ; Medicago sativa/genetics ; *Medicago truncatula/genetics ; },
}
@article {pmid34960730,
year = {2021},
author = {Liu, Y and Jeeninga, RE and Klaver, B and Berkhout, B and Das, AT},
title = {Transient CRISPR-Cas Treatment Can Prevent Reactivation of HIV-1 Replication in a Latently Infected T-Cell Line.},
journal = {Viruses},
volume = {13},
number = {12},
pages = {},
pmid = {34960730},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; Cell Line ; DNA, Viral/genetics ; Gene Editing ; HIV Infections/*virology ; HIV-1/*genetics/physiology ; Humans ; Lentivirus/genetics/physiology ; Proviruses/genetics/physiology ; T-Lymphocytes/*virology ; Transduction, Genetic ; *Virus Activation ; },
abstract = {Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application.},
}
@article {pmid34960685,
year = {2021},
author = {Nugnes, MV and Targovnik, AM and Mengual-Martí, A and Miranda, MV and Cerrudo, CS and Herrero, S and Belaich, MN},
title = {The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species.},
journal = {Viruses},
volume = {13},
number = {12},
pages = {},
pmid = {34960685},
issn = {1999-4915},
mesh = {Amino Acid Motifs ; Animals ; CRISPR-Cas Systems ; Genetic Complementation Test ; Larva/virology ; Moths/virology ; Nucleopolyhedroviruses/*chemistry/genetics/*physiology ; Phylogeny ; Protein Domains ; Recombinant Fusion Proteins/chemistry/metabolism ; Sf9 Cells ; Spodoptera/*virology ; Viral Envelope Proteins/*chemistry/genetics/metabolism ; },
abstract = {Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.},
}
@article {pmid34958212,
year = {2022},
author = {Camperi, J and Moshref, M and Dai, L and Lee, HY},
title = {Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes.},
journal = {Analytical chemistry},
volume = {94},
number = {2},
pages = {1432-1440},
doi = {10.1021/acs.analchem.1c04795},
pmid = {34958212},
issn = {1520-6882},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; RNA, Guide/genetics ; *Ribonucleoproteins/chemistry/genetics/metabolism ; },
abstract = {Advances in gene-editing technology enable efficient, targeted ex vivo engineering of different cell types, which offer a potential therapeutic platform for most challenging disease areas. CRISPR-Cas9 is a widely used gene-editing tool in therapeutic applications. The quality of gene-editing reagents (i.e., Cas9 nuclease, single guide (sg)RNA) is associated with the final cellular product quality as they can impact the gene-editing accuracy and efficiency. To assess the impact of the quality of Cas9 protein and sgRNA in the formation of a Cas9 ribonucleoprotein (RNP) complex, stability, and functional activities, we developed a size exclusion chromatography method that utilizes multiple detectors and an in vitro DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides new tools to characterize the Cas9 RNP complex formation, stability, and functional activity and provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.},
}
@article {pmid34956138,
year = {2021},
author = {Yang, F and Xu, L and Liang, L and Liang, W and Li, J and Lin, D and Dai, M and Zhou, D and Li, Y and Chen, Y and Zhao, H and Tian, GB and Feng, S},
title = {The Involvement of Mycobacterium Type III-A CRISPR-Cas System in Oxidative Stress.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {774492},
pmid = {34956138},
issn = {1664-302X},
abstract = {Type I and type II CRISPR-Cas systems are employed to evade host immunity by targeting interference of bacteria's own genes. Although Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, possesses integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2∼5, cas10, cas6) were upregulated in Mycobacterium bovis BCG under oxidative stress treatment, indicating the role of type III-A CRISPR-Cas system in oxidative stress. To explore the functional role of type III-A CRISPR-Cas system, TCC (Type III-A CRISPR-Cas system, including cas6, cas10, and csm2-6) mutant was generated. Deletion of TCC results in increased sensitivity in response to hydrogen peroxide and reduced cell envelope integrity. Analysis of RNA-seq dataset revealed that TCC impacted on the oxidation-reduction process and the composition of cell wall which is essential for mycobacterial envelop integrity. Moreover, disrupting TCC led to poor intracellular survival in vivo and in vitro. Finally, we showed for the first time that TCC contributed to the regulation of regulatory T cell population, supporting a role of TCC in modulating host immunity. Our finding reveals the important role of TCC in cell envelop homeostasis. Our work also highlights type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, thus may be a potential target for therapy.},
}
@article {pmid34954876,
year = {2022},
author = {Jiang, D and Zhang, D and Li, S and Liang, Y and Zhang, Q and Qin, X and Gao, J and Qiu, JL},
title = {Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I-C CRISPR-Cas system.},
journal = {Molecular plant pathology},
volume = {23},
number = {4},
pages = {583-594},
pmid = {34954876},
issn = {1364-3703},
mesh = {CRISPR-Cas Systems/genetics ; DNA ; Gene Editing ; *Oryza/microbiology ; Plant Diseases/microbiology ; *Xanthomonas/genetics ; },
abstract = {Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I-C CRISPR-Cas system in the Xoo genome and leveraged this endogenous defence system for high-efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini-CRISPR array, donor DNA, and a phage-derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I-C CRISPR-Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome-editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR-harbouring bacterial plant pathogens.},
}
@article {pmid34954815,
year = {2021},
author = {Liao, B and Chen, X and Zhou, X and Zhou, Y and Shi, Y and Ye, X and Liao, M and Zhou, Z and Cheng, L and Ren, B},
title = {Applications of CRISPR/Cas gene-editing technology in yeast and fungi.},
journal = {Archives of microbiology},
volume = {204},
number = {1},
pages = {79},
pmid = {34954815},
issn = {1432-072X},
mesh = {*CRISPR-Cas Systems ; Fungi/genetics ; *Gene Editing ; Saccharomyces cerevisiae/genetics ; Technology ; },
abstract = {Genome editing technology has progressed rapidly in recent years. Although traditional gene-editing methods, including homologous recombination, zinc finger endonucleases, and transcription activator-like effector nucleases, have substantial implications for research in genetics and molecular biology, but they have remarkable limitations, including their low efficiency, high error rate, and complex design. A new gene-editing technology, the CRISPR/Cas system, was developed based on studies of archaeal and bacterial immune responses to viruses. Owing to its high target efficiency, simple primer design, and wide applications, the CRISPR/Cas system, whose developers were awarded the Nobel Prize in Chemistry in 2020, has become the dominant genomic editing technology in academia and the pharmaceutical industry. Here, we briefly introduce the CRISPR/Cas system and its main applications for genome engineering, metabolic engineering, and transcriptional regulation in yeast, filamentous fungi, and macrofungi. The polygene and polyploid editing, construction of yeast chromosomes, yeast library creation, regulation of metabolic pathways, and CRISPR activation/CRISPR interference systems are mainly summarized and discussed. The potential applications for the treatment of fungal infections and the further transformation and application of the CRISPR/Cas system in fungi are also proposed and discussed.},
}
@article {pmid34954490,
year = {2022},
author = {Wilk, C and Effenberg, L and Abberger, H and Steenpass, L and Hansen, W and Zeschnigk, M and Kirschning, C and Buer, J and Kehrmann, J},
title = {CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.},
journal = {Cellular immunology},
volume = {371},
number = {},
pages = {104471},
doi = {10.1016/j.cellimm.2021.104471},
pmid = {34954490},
issn = {1090-2163},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/immunology ; Cell Line, Tumor ; DNA Methylation/*genetics ; Demethylation ; Forkhead Transcription Factors/genetics/*metabolism ; GATA3 Transcription Factor/genetics ; Gene Editing/*methods ; Humans ; Jurkat Cells ; Mixed Function Oxygenases/*genetics ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics ; Proto-Oncogene Proteins/*genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics ; T-Lymphocytes, Regulatory/*cytology/immunology ; },
abstract = {Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.},
}
@article {pmid34954237,
year = {2022},
author = {Hwang, S and Pan, C and Garcia, B and Davidson, AR and Moraes, TF and Maxwell, KL},
title = {Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa.},
journal = {Journal of molecular biology},
volume = {434},
number = {5},
pages = {167420},
doi = {10.1016/j.jmb.2021.167420},
pmid = {34954237},
issn = {1089-8638},
support = {//CIHR/Canada ; },
mesh = {*Bacteriophages/enzymology ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry ; *DNA Cleavage ; *Haemophilus parainfluenzae/virology ; Prophages/enzymology ; Protein Domains ; *Viral Proteins/chemistry/metabolism ; },
abstract = {Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.},
}
@article {pmid34953139,
year = {2021},
author = {Zhang, E and Zhou, W and Zhou, J and He, Z and Zhou, Y and Han, J and Qu, D},
title = {CRISPR-Cas systems are present predominantly on chromosome and its relationship with MEGs in Vibrio species.},
journal = {Archives of microbiology},
volume = {204},
number = {1},
pages = {76},
pmid = {34953139},
issn = {1432-072X},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Chromosomes ; Phylogeny ; Plasmids/genetics ; *Vibrio/genetics ; },
abstract = {Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. In this study, the structure and function of the CRISPR-Cas system in 120 Vibrio strains were analyzed by bioinformatics methods, as well as the correlation between CRISPR and mobile genetic elements (MEGs). Only 61 Vibrio strains contained one or more CRISPR structures, and finally 102 CRISPRs were identified. The typical repeat size was 28 bp, and the total length of CRISPRs is nearly 60 bp, which was the most stable length of CRISPR in Vibrio strains. The types of CRISPR-Cas present in 61 strains were I-C, I-E, I-F, II-B, III-B, III-D and the rare type IV systems. Through principal component analysis, we found that Cas gene was most closely related to CRISPR. In addition, phages and plasmids were also highly correlated, showing negative correlation with CRISPR-Cas system. CRISPR-Cas predominantly present on chromosome within Vibrio while rarely in plasmids. Comparing the structural characteristics of plasmids containing CRISPR and without CRISPR, we found plasmid pMBL287 with CRISPR contained a bacteriophage f237, with more MGES, suggesting the diversity was greater. In addition, the same mobile genetic elements IS256 and ISL3 were found in the upstream and downstream of CRISPR. This study provides the prevalence, diversity and phylogenetic distribution of CRISPR-Cas in Vibrio, revealing which type of CRISPR-Cas system is predominant, and the factors affecting its function, as well as its relationship with mobile genetic elements.},
}
@article {pmid34952739,
year = {2022},
author = {Modell, AE and Lim, D and Nguyen, TM and Sreekanth, V and Choudhary, A},
title = {CRISPR-based therapeutics: current challenges and future applications.},
journal = {Trends in pharmacological sciences},
volume = {43},
number = {2},
pages = {151-161},
pmid = {34952739},
issn = {1873-3735},
support = {R01 GM132825/GM/NIGMS NIH HHS/United States ; R01 GM137606/GM/NIGMS NIH HHS/United States ; R01GM137606/NH/NIH HHS/United States ; R01GM132825/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; *Genetic Therapy ; Humans ; },
abstract = {The discovery, only a decade ago, of the genome editing power of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases is already reinventing the therapeutic process, from how new drugs are discovered to novel ways to treat diseases. CRISPR-based screens can aid therapeutic development by quickly identifying a drug's mechanism of action and escape mutants. Additionally, CRISPR-Cas has advanced emerging ex vivo therapeutics, such as cell replacement therapies. However, Cas9 is limited as an in vivo therapeutic due to ineffective delivery, unwanted immune responses, off-target effects, unpredictable repair outcomes, and cellular stress. To address these limitations, controls that inhibit or degrade Cas9, biomolecule-Cas9 conjugates, and base editors have been developed. Herein, we discuss CRISPR-Cas systems that advance both conventional and emerging therapeutics.},
}
@article {pmid34952262,
year = {2022},
author = {Park, H and Kim, D and Cho, B and Byun, J and Kim, YS and Ahn, Y and Hur, J and Oh, YK and Kim, J},
title = {In vivo therapeutic genome editing via CRISPR/Cas9 magnetoplexes for myocardial infarction.},
journal = {Biomaterials},
volume = {281},
number = {},
pages = {121327},
doi = {10.1016/j.biomaterials.2021.121327},
pmid = {34952262},
issn = {1878-5905},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Targeting ; Genetic Therapy/methods ; Mice ; *Myocardial Infarction/genetics/therapy ; },
abstract = {CRISPR/Cas9-mediated gene-editing technology has gained attention as a new therapeutic method for intractable diseases. However, the use of CRISPR/Cas9 for cardiac conditions such as myocardial infarction remains challenging due to technical and biological barriers, particularly difficulties in delivering the system and targeting genes in the heart. In the present study, we demonstrated the in vivo efficacy of the CRISPR/Cas9 magnetoplexes system for therapeutic genome editing in myocardial infarction. First, we developed CRISPR/Cas9 magnetoplexes that magnetically guided CRISPR/Cas9 system to the heart for efficient in vivo therapeutic gene targeting during heart failures. We then demonstrated that the in vivo gene targeting of miR34a via these CRISPR/Cas9 magnetoplexes in a mouse model of myocardial infarction significantly improved cardiac repair and regeneration to facilitate improvements in cardiac function. These results indicated that CRISPR/Cas9 magnetoplexes represent an effective in vivo therapeutic gene-targeting platform in the myocardial infarction of heart, and that this strategy may be applicable for the treatment of a broad range of cardiac failures.},
}
@article {pmid34951459,
year = {2022},
author = {Shebanova, R and Nikitchina, N and Shebanov, N and Mekler, V and Kuznedelov, K and Ulashchik, E and Vasilev, R and Sharko, O and Shmanai, V and Tarassov, I and Severinov, K and Entelis, N and Mazunin, I},
title = {Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.},
journal = {Nucleic acids research},
volume = {50},
number = {2},
pages = {1162-1173},
pmid = {34951459},
issn = {1362-4962},
mesh = {*Acidaminococcus/genetics/metabolism ; Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/*metabolism ; DNA Cleavage ; Endodeoxyribonucleases/*metabolism ; Francisella/genetics/metabolism ; Gene Editing ; },
abstract = {CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.},
}
@article {pmid34951457,
year = {2022},
author = {Paul, B and Chaubet, L and Verver, DE and Montoya, G},
title = {Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA.},
journal = {Nucleic acids research},
volume = {50},
number = {9},
pages = {5208-5225},
pmid = {34951457},
issn = {1362-4962},
mesh = {Bacterial Proteins/chemistry/*genetics/metabolism ; Bacteriophage lambda/genetics ; CRISPR-Associated Proteins/chemistry/*genetics/metabolism ; *CRISPR-Cas Systems ; DNA/chemistry ; Endodeoxyribonucleases/chemistry/*genetics/metabolism ; Endonucleases/metabolism ; Gene Editing/*methods ; RNA, Guide/genetics ; },
abstract = {Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage λ-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that λ-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.},
}
@article {pmid34950912,
year = {2021},
author = {Fuertes-Perez, S and Vogel, RF and Hilgarth, M},
title = {Comparative genomics of Photobacterium species from terrestrial and marine habitats.},
journal = {Current research in microbial sciences},
volume = {2},
number = {},
pages = {100087},
pmid = {34950912},
issn = {2666-5174},
abstract = {Photobacterium (P.) is a genus widely studied in regards to its association with and ubiquitous presence in marine environments. However, certain species (P. phosphoreum, P. carnosum, P. iliopiscarium) have been recently described to colonize and spoil raw meats without a marine link. We have studied 27 strains from meat as well as 26 strains from marine environments in order to probe for intraspecies marine/terrestrial subpopulations and identify distinct genomic features acquired by environmental adaptation. We have conducted phylogenetic analysis (MLSA, ANI, fur, codon usage), search of plasmids (plasmidSPADES), phages (PHASTER), CRISPR-cas operons (CRISPR-finder) and secondary metabolites gene clusters (antiSMASH, BAGEL), in addition to a targeted gene search for specific pathways (e.g. TCA cycle, pentose phosphate, respiratory chain) and elements relevant for growth, adaptation and competition (substrate utilization, motility, bioluminescence, sodium and iron transport). P. carnosum appears as a conserved single clade, with one isolate from MAP fish clustering apart that doesn't, however, show distinct features that could indicate different adaptation. The species harbors genes for a wide carbon source utilization (glycogen/starch, maltose, pullulan, fucose) for colonization of diverse niches in its genome. P. phosphoreum is represented by two different clades on the phylogenetic analyses not correlating to their origin or distribution of other features analyzed that can be divided into two novel subspecies based on genome-wide values. A more diverse antimicrobial activity (sactipeptides, microcins), production of secondary metabolites (siderophores and arylpolyenes), stress response and adaptation (bioluminescence, sodium transporters, catalase, high affinity for oxygen cytochrome cbb3 oxidase, DMSO reductase and proton translocating NADH dehydrogenase) is predicted compared to the other species. P. iliopiscarium was divided into two clades based on source of isolation correlating with phylogeny and distribution of several traits. The species shows traits common to the other two species, similar carbon utilization/transport gene conservation as P. carnosum for the meat-isolated strains, and predicted utilization of marine-common DMSO and flagellar cluster for the sea-isolated strains. Results additionally suggest that photobacteria are highly prone to horizontal acquisition/loss of genetic material and genetic transduction, and that it might be a strategy for increasing the frequency of strain- or species-specific features that offers a growth/competition advantage.},
}
@article {pmid34949863,
year = {2022},
author = {Grunwald, HA and Weitzel, AJ and Cooper, KL},
title = {Applications of and considerations for using CRISPR-Cas9-mediated gene conversion systems in rodents.},
journal = {Nature protocols},
volume = {17},
number = {1},
pages = {3-14},
pmid = {34949863},
issn = {1750-2799},
support = {R21 GM129448/GM/NIGMS NIH HHS/United States ; T32 GM007240/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Conversion/*genetics ; Gene Editing/*methods ; Mice ; Mice, Transgenic/genetics ; Rats ; Rats, Transgenic/genetics ; },
abstract = {Genetic elements that are inherited at super-Mendelian frequencies could be used in a 'gene drive' to spread an allele to high prevalence in a population with the goal of eliminating invasive species or disease vectors. We recently demonstrated that the gene conversion mechanism underlying a CRISPR-Cas9-mediated gene drive is feasible in mice. Although substantial technical hurdles remain, overcoming these could lead to strategies that might decrease the spread of rodent-borne Lyme disease or eliminate invasive populations of mice and rats that devastate island ecology. Perhaps more immediately achievable at moderate gene conversion efficiency, applications in a laboratory setting could produce complex genotypes that reduce the time and cost in both dollars and animal lives compared with Mendelian inheritance strategies. Here, we discuss what we have learned from early efforts to achieve CRISPR-Cas9-mediated gene conversion, potential for broader applications in the laboratory, current limitations, and plans for optimizing this potentially powerful technology.},
}
@article {pmid34949839,
year = {2022},
author = {Tang, CP and Clark, O and Ferrarone, JR and Campos, C and Lalani, AS and Chodera, JD and Intlekofer, AM and Elemento, O and Mellinghoff, IK},
title = {GCN2 kinase activation by ATP-competitive kinase inhibitors.},
journal = {Nature chemical biology},
volume = {18},
number = {2},
pages = {207-215},
pmid = {34949839},
issn = {1552-4469},
support = {F31 CA239401/CA/NCI NIH HHS/United States ; R35 NS105109/NS/NINDS NIH HHS/United States ; R01 CA194547/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; UL1 TR002384/TR/NCATS NIH HHS/United States ; R01 GM121505/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Triphosphate/*metabolism ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Delivery Systems ; Gene Deletion ; Gene Expression Regulation, Neoplastic/*drug effects ; Glioblastoma/drug therapy ; Humans ; Protein Kinase Inhibitors/chemistry/*pharmacology ; Quinolines/*pharmacology ; },
abstract = {Small-molecule kinase inhibitors represent a major group of cancer therapeutics, but tumor responses are often incomplete. To identify pathways that modulate kinase inhibitor response, we conducted a genome-wide knockout (KO) screen in glioblastoma cells treated with the pan-ErbB inhibitor neratinib. Loss of general control nonderepressible 2 (GCN2) kinase rendered cells resistant to neratinib, whereas depletion of the GADD34 phosphatase increased neratinib sensitivity. Loss of GCN2 conferred neratinib resistance by preventing binding and activation of GCN2 by neratinib. Several other Food and Drug Administration (FDA)-approved inhibitors, such erlotinib and sunitinib, also bound and activated GCN2. Our results highlight the utility of genome-wide functional screens to uncover novel mechanisms of drug action and document the role of the integrated stress response (ISR) in modulating the response to inhibitors of oncogenic kinases.},
}
@article {pmid34949807,
year = {2022},
author = {Albanese, M and Ruhle, A and Mittermaier, J and Mejías-Pérez, E and Gapp, M and Linder, A and Schmacke, NA and Hofmann, K and Hennrich, AA and Levy, DN and Humpe, A and Conzelmann, KK and Hornung, V and Fackler, OT and Keppler, OT},
title = {Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4[+] T cells allows complex functional analyses.},
journal = {Nature methods},
volume = {19},
number = {1},
pages = {81-89},
pmid = {34949807},
issn = {1548-7105},
support = {R01 AI145753/AI/NIAID NIH HHS/United States ; },
mesh = {CD4-Positive T-Lymphocytes/cytology/*physiology/virology ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Cell Movement/genetics ; Cells, Cultured ; DNA ; Gene Editing/*methods ; Gene Knockout Techniques ; HIV Infections/*genetics/metabolism ; Humans ; Membrane Glycoproteins/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/metabolism ; RNA, Guide ; SAM Domain and HD Domain-Containing Protein 1/genetics ; Transgenes ; mRNA Cleavage and Polyadenylation Factors/genetics/metabolism ; },
abstract = {CD4[+] T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4[+] T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4[+] T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4[+] T cells.},
}
@article {pmid34949803,
year = {2022},
author = {Sretenovic, S and Qi, Y},
title = {Plant prime editing goes prime.},
journal = {Nature plants},
volume = {8},
number = {1},
pages = {20-22},
pmid = {34949803},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Plants ; },
}
@article {pmid34949802,
year = {2022},
author = {Xu, W and Yang, Y and Yang, B and Krueger, CJ and Xiao, Q and Zhao, S and Zhang, L and Kang, G and Wang, F and Yi, H and Ren, W and Li, L and He, X and Zhang, C and Zhang, B and Zhao, J and Yang, J},
title = {A design optimized prime editor with expanded scope and capability in plants.},
journal = {Nature plants},
volume = {8},
number = {1},
pages = {45-52},
pmid = {34949802},
issn = {2055-0278},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; *Oryza/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in plant breeding. Prime editing, a versatile gene-editing approach that directly writes new genetic information into a specified DNA site without requiring double-strand DNA breaks, suffers from low efficiency in plants[1-5]. In this study, N-terminal reverse transcriptase-Cas9 nickase fusion performed better in rice than the commonly applied C-terminal fusion. In addition, introduction of multiple-nucleotide substitutions in the reverse transcriptase template stimulated prime editing with enhanced efficiency. By using these two methods synergistically, prime editing with an average editing frequency as high as 24.3% at 13 endogenous targets in rice transgenic plants, 6.2% at four targets in maize protoplasts and 12.5% in human cells was achieved, which is two- to threefold higher than the original editor, Prime Editor 3. Therefore, our optimized approach has potential to make more formerly non-editable target sites editable, and expands the scope and capabilities of prime editing in the future.},
}
@article {pmid34948465,
year = {2021},
author = {Paolini Sguazzi, G and Muto, V and Tartaglia, M and Bertini, E and Compagnucci, C},
title = {Induced Pluripotent Stem Cells (iPSCs) and Gene Therapy: A New Era for the Treatment of Neurological Diseases.},
journal = {International journal of molecular sciences},
volume = {22},
number = {24},
pages = {},
pmid = {34948465},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Clinical Trials as Topic ; Gene Editing ; Genetic Therapy/*methods ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Nervous System Diseases/genetics/*therapy ; Stem Cell Transplantation/*methods ; },
abstract = {To date, gene therapy has employed viral vectors to deliver therapeutic genes. However, recent progress in molecular and cell biology has revolutionized the field of stem cells and gene therapy. A few years ago, clinical trials started using stem cell replacement therapy, and the induced pluripotent stem cells (iPSCs) technology combined with CRISPR-Cas9 gene editing has launched a new era in gene therapy for the treatment of neurological disorders. Here, we summarize the latest findings in this research field and discuss their clinical applications, emphasizing the relevance of recent studies in the development of innovative stem cell and gene editing therapeutic approaches. Even though tumorigenicity and immunogenicity are existing hurdles, we report how recent progress has tackled them, making engineered stem cell transplantation therapy a realistic option.},
}
@article {pmid34948123,
year = {2021},
author = {Palamarchuk, AI and Alekseeva, NA and Streltsova, MA and Ustiuzhanina, MO and Kobyzeva, PA and Kust, SA and Grechikhina, MV and Boyko, AA and Shustova, OA and Sapozhnikov, AM and Kovalenko, EI},
title = {Increased Susceptibility of the CD57[-] NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response.},
journal = {International journal of molecular sciences},
volume = {22},
number = {24},
pages = {},
pmid = {34948123},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Cell Death ; *Cell Proliferation ; *Gene Expression Regulation ; *Genetic Vectors ; Humans ; K562 Cells ; Killer Cells, Natural ; *Lymphocyte Activation ; NK Cell Lectin-Like Receptor Subfamily C/*biosynthesis/genetics ; Receptors, KIR2DL2/*biosynthesis/genetics ; Receptors, KIR2DL3/*biosynthesis/genetics ; *Retroviridae ; *Transduction, Genetic ; },
abstract = {Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57[-]NKG2C[+] NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57[-] NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3[+]NKG2C[+] NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3[+] cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3[+]NKG2C[+] NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.},
}
@article {pmid34948095,
year = {2021},
author = {Zhu, D and Wang, J and Yang, D and Xi, J and Li, J},
title = {High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity.},
journal = {International journal of molecular sciences},
volume = {22},
number = {24},
pages = {},
pmid = {34948095},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; HEK293 Cells ; HeLa Cells ; Humans ; },
abstract = {CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA-target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position -3 for PAM TATV and at position -4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.},
}
@article {pmid34948046,
year = {2021},
author = {Batur, T and Argundogan, A and Keles, U and Mutlu, Z and Alotaibi, H and Senturk, S and Ozturk, M},
title = {AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest and Polyploidization.},
journal = {International journal of molecular sciences},
volume = {22},
number = {24},
pages = {},
pmid = {34948046},
issn = {1422-0067},
mesh = {Animals ; Benzocycloheptenes ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/metabolism/*pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; G2 Phase Cell Cycle Checkpoints ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Liver Neoplasms/genetics/metabolism/*pathology ; Male ; Mice ; Neoplasm Transplantation ; Phenotype ; Proto-Oncogene Proteins/*genetics/*metabolism ; Receptor Protein-Tyrosine Kinases/*genetics/*metabolism ; Triazoles ; *Up-Regulation ; },
abstract = {AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.},
}
@article {pmid34947996,
year = {2021},
author = {Xue, Y and Li, S and Miao, D and Huang, S and Guo, B and Li, S and An, XM},
title = {Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {22},
number = {24},
pages = {},
pmid = {34947996},
issn = {1422-0067},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Cellulose/*biosynthesis ; Cloning, Molecular/*methods ; Gene Expression Regulation, Plant ; Glucose/metabolism ; Glucosyltransferases/*genetics ; Plant Leaves/genetics/metabolism ; Plant Proteins/genetics ; Plant Stems/genetics/metabolism ; Populus/genetics/*metabolism ; Sucrose/metabolism ; Transformation, Bacterial ; Wood/genetics ; },
abstract = {Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa(P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.},
}
@article {pmid34947888,
year = {2021},
author = {Kwon, S and Shin, HY},
title = {Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19.},
journal = {Life (Basel, Switzerland)},
volume = {11},
number = {12},
pages = {},
pmid = {34947888},
issn = {2075-1729},
abstract = {Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.},
}
@article {pmid34946828,
year = {2021},
author = {Niu, R and Peng, J and Zhang, Z and Shang, X},
title = {R-CRISPR: A Deep Learning Network to Predict Off-Target Activities with Mismatch, Insertion and Deletion in CRISPR-Cas9 System.},
journal = {Genes},
volume = {12},
number = {12},
pages = {},
pmid = {34946828},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/*genetics ; Deep Learning ; Gene Deletion ; Gene Editing/*methods ; Mutagenesis, Insertional/*genetics ; Neural Networks, Computer ; RNA, Guide/genetics ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system is a groundbreaking gene-editing tool, which has been widely adopted in biomedical research. However, the guide RNAs in CRISPR-Cas9 system may induce unwanted off-target activities and further affect the practical application of the technique. Most existing in silico prediction methods that focused on off-target activities possess limited predictive precision and remain to be improved. Hence, it is necessary to propose a new in silico prediction method to address this problem. In this work, a deep learning framework named R-CRISPR is presented, which devises an encoding scheme to encode gRNA-target sequences into binary matrices, a convolutional neural network as feature extractor, and a recurrent neural network to predict off-target activities with mismatch, insertion, or deletion. It is demonstrated that R-CRISPR surpasses six mainstream prediction methods with a significant improvement on mismatch-only datasets verified by GUIDE-seq. Compared with the state-of-art prediction methods, R-CRISPR also achieves competitive performance on datasets with mismatch, insertion, and deletion. Furthermore, experiments show that data concatenate could influence the quality of training data, and investigate the optimal combination of datasets.},
}
@article {pmid34946160,
year = {2021},
author = {Buckley, D and Odamaki, T and Xiao, J and Mahony, J and van Sinderen, D and Bottacini, F},
title = {Diversity of Human-Associated Bifidobacterial Prophage Sequences.},
journal = {Microorganisms},
volume = {9},
number = {12},
pages = {},
pmid = {34946160},
issn = {2076-2607},
support = {SFI/12/RC/2273-P1/SFI_/Science Foundation Ireland/Ireland ; SFI/12/RC/2273-P2/SFI_/Science Foundation Ireland/Ireland ; },
abstract = {Members of Bifidobacterium play an important role in the development of the immature gut and are associated with positive long-term health outcomes for their human host. It has previously been shown that intestinal bacteriophages are detected within hours of birth, and that induced prophages constitute a significant source of such gut phages. The gut phageome can be vertically transmitted from mother to newborn and is believed to exert considerable selective pressure on target prokaryotic hosts affecting abundance levels, microbiota composition, and host characteristics. The objective of the current study was to investigate prophage-like elements and predicted CRISPR-Cas viral immune systems present in publicly available, human-associated Bifidobacterium genomes. Analysis of 585 fully sequenced bifidobacterial genomes identified 480 prophage-like elements with an occurrence of 0.82 prophages per genome. Interestingly, we also detected the presence of very similar bifidobacterial prophages and corresponding CRISPR spacers across different strains and species, thus providing an initial exploration of the human-associated bifidobacterial phageome. Our analyses show that closely related and likely functional prophages are commonly present across four different species of human-associated Bifidobacterium. Further comparative analysis of the CRISPR-Cas spacer arrays against the predicted prophages provided evidence of historical interactions between prophages and different strains at an intra- and inter-species level. Clear evidence of CRISPR-Cas acquired immunity against infection by bifidobacterial prophages across several bifidobacterial strains and species was obtained. Notably, a spacer representing a putative major capsid head protein was found on different genomes representing multiple strains across B. adolescentis, B. breve, and B. bifidum, suggesting that this gene is a preferred target to provide bifidobacterial phage immunity.},
}
@article {pmid34944496,
year = {2021},
author = {Charbonneau, AA and Eckert, DM and Gauvin, CC and Lintner, NG and Lawrence, CM},
title = {Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus.},
journal = {Biomolecules},
volume = {11},
number = {12},
pages = {},
pmid = {34944496},
issn = {2218-273X},
mesh = {Adenosine Monophosphate/metabolism ; Archaeal Proteins/chemistry/metabolism ; Binding Sites ; CRISPR-Cas Systems ; Crystallography, X-Ray ; Models, Molecular ; Protein Conformation ; Protein Domains ; Sulfolobus solfataricus/*immunology ; Transcription Factors/chemistry/*metabolism ; },
abstract = {Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating "ring nuclease" activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.},
}
@article {pmid34944413,
year = {2021},
author = {Toma, L and Barbălată, T and Sanda, GM and Niculescu, LS and Sima, AV and Stancu, CS},
title = {CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction.},
journal = {Biomolecules},
volume = {11},
number = {12},
pages = {},
pmid = {34944413},
issn = {2218-273X},
mesh = {Apolipoprotein A-I/*genetics/metabolism ; Aryldialkylphosphatase/*genetics/metabolism ; CRISPR-Cas Systems ; Caco-2 Cells ; Culture Media, Conditioned/chemistry ; Endothelial Cells/*cytology/metabolism ; Enterocytes/*cytology/metabolism ; Gene Expression Regulation ; Humans ; Lipoproteins, HDL/metabolism ; Oxidative Stress ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were measured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC.},
}
@article {pmid34944345,
year = {2021},
author = {Yum, SY and Jang, G and Koo, O},
title = {Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts.},
journal = {Animals : an open access journal from MDPI},
volume = {11},
number = {12},
pages = {},
pmid = {34944345},
issn = {2076-2615},
abstract = {Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.},
}
@article {pmid34943170,
year = {2021},
author = {Saradadevi, GP and Das, D and Mangrauthia, SK and Mohapatra, S and Chikkaputtaiah, C and Roorkiwal, M and Solanki, M and Sundaram, RM and Chirravuri, NN and Sakhare, AS and Kota, S and Varshney, RK and Mohannath, G},
title = {Genetic, Epigenetic, Genomic and Microbial Approaches to Enhance Salt Tolerance of Plants: A Comprehensive Review.},
journal = {Biology},
volume = {10},
number = {12},
pages = {},
pmid = {34943170},
issn = {2079-7737},
support = {NASF/CRISPR-Cas-7003/2018-19/GATES/Bill &amp; Melinda Gates Foundation/United States ; },
abstract = {Globally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.},
}
@article {pmid34942344,
year = {2022},
author = {Qazi, MA and Wang, Q and Dai, Z},
title = {Sophorolipids bioproduction in the yeast Starmerella bombicola: Current trends and perspectives.},
journal = {Bioresource technology},
volume = {346},
number = {},
pages = {126593},
doi = {10.1016/j.biortech.2021.126593},
pmid = {34942344},
issn = {1873-2976},
mesh = {Glycolipids ; Oleic Acids ; *Saccharomycetales/genetics ; Yeasts ; },
abstract = {Sophorolipids are highly active green surfactants (glycolipid biosurfactants) getting tremendous appreciation worldwide due to their low toxicity, biodegradability, broad spectrum of applications, and significant biotechnological potential. Sophorolipids are mainly produced by an oleaginous budding yeast Starmerella bombicola using low-cost substrates. Therefore, the recent state-of-art literature information about S. bombicola yeast is hereby provided, especially the underlying production pathways, biosynthetic gene cluster, and regulatory enzymes. Moreover, the S. bombicola offers flexibility for regulating the structural diversity of sophorolipids, either genetically or by varying fermentative conditions. The emergence of advanced technologies like 'Omics and CRISPR/Cas have certainly boosted rational engineering research for designing high-performing platform strains. Therefore, currently available genetic engineering tools in S. bombicola were reviewed, thereby opening up exciting new possibilities for improving the overall bioproduction titers, structural variability, and stability of sophorolipids. Finally, some technical perspectives to address the current challenges were discussed.},
}
@article {pmid34942274,
year = {2022},
author = {Bloomer, H and Khirallah, J and Li, Y and Xu, Q},
title = {CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells.},
journal = {Advanced drug delivery reviews},
volume = {181},
number = {},
pages = {114087},
pmid = {34942274},
issn = {1872-8294},
support = {R01 EB027170/EB/NIBIB NIH HHS/United States ; T32 GM008448/GM/NIGMS NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; UH3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Drug Delivery Systems/methods ; Epigenome/genetics ; Gene Editing/*methods ; Genome/*genetics ; Ribonucleoproteins/*metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has revolutionized the ability to edit the mammalian genome, providing a platform for the correction of pathogenic mutations and further investigation into gene function. CRISPR reagents can be delivered into the cell as DNA, RNA, or pre-formed ribonucleoproteins (RNPs). RNPs offer numerous advantages over other delivery approaches due to their ability to rapidly target genomic sites and quickly degrade thereafter. Here, we review the production steps and delivery methods for Cas9 RNPs. Additionally, we discuss how RNPs enhance genome and epigenome editing efficiencies, reduce off-target editing activity, and minimize cellular toxicity in clinically relevant mammalian cell types. We include details on a broad range of editing approaches, including novel base and prime editing techniques. Finally, we summarize key challenges for the use of RNPs, and propose future perspectives on the field.},
}
@article {pmid34942204,
year = {2022},
author = {Hu, J and Yu, M and Chang, Y and Tang, H and Wang, W and Du, L and Wang, K and Yan, Y and Ye, X},
title = {Functional analysis of TaPDI genes on storage protein accumulation by CRISPR/Cas9 edited wheat mutants.},
journal = {International journal of biological macromolecules},
volume = {196},
number = {},
pages = {131-143},
doi = {10.1016/j.ijbiomac.2021.12.048},
pmid = {34942204},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; Edible Grain ; *Gene Editing ; Gene Expression Regulation, Plant ; INDEL Mutation ; *Mutation ; Phenotype ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Protein Disulfide-Isomerases/*genetics/metabolism ; Transformation, Genetic ; Triticum/chemistry/*genetics/metabolism ; },
abstract = {Wheat protein disulfide isomerase (PDI) is involved in the formation of glutenin macropolymers (GMP) and the correct folding and accumulation of storage proteins in endosperm. In present study, seven types of homozygous TaPDI gene edited mutants were obtained by CRISPR/Cas9 technology, which were confirmed by PCR-RE and sequencing. Compared with other mutants and wild type (WT), the grain length and width in mutant PDI-abd-6 which was edited for the three TaPDI homoeologous genes were reduced, and the grain middle parts were slumped. The GMP size in PDI-abd-6 was not significantly different from that in WT, whereas the accumulation of protein bodies (PBs) increased during grain development. The endosperm cells became denser in PDI-abd-6 without sheet-like structure, and the expression level of TaBiP gene was significantly decreased. Particularly, the GMP content in PDI-abd-6 is also decreased significantly. The basic bread and flour rheological parameters in the mutant were negatively changed compared with those in WT. Our results indicated that TaPDI genes affects wheat flour-processing quality by the order of TaPDI-4B, TaPDI-4D, and TaPDI-4A from high to low; the expression of either one TaPDI could be enough to maintain the GMP accumulation and processing properties of wheat dough.},
}
@article {pmid34941944,
year = {2021},
author = {Kruasuwan, W and Puseenam, A and Tanapongpipat, S and Roongsawang, N},
title = {Multiplexed CRISPR-mediated engineering of protein secretory pathway genes in the thermotolerant methylotrophic yeast Ogataea thermomethanolica.},
journal = {PloS one},
volume = {16},
number = {12},
pages = {e0261754},
pmid = {34941944},
issn = {1932-6203},
mesh = {*6-Phytase/genetics/metabolism ; *CRISPR-Cas Systems ; *Endo-1,4-beta Xylanases/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Genome, Fungal ; *Microorganisms, Genetically-Modified/enzymology/genetics ; *Saccharomycetales/enzymology/genetics ; *Secretory Pathway ; },
abstract = {CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.},
}
@article {pmid34941928,
year = {2021},
author = {Yang, J and Barua, N and Rahman, MN and Lo, N and Tsang, TF and Yang, X and Chan, PKS and Zhang, L and Ip, M},
title = {Chimeric crRNA improves CRISPR-Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2.},
journal = {PloS one},
volume = {16},
number = {12},
pages = {e0261778},
pmid = {34941928},
issn = {1932-6203},
mesh = {COVID-19/*diagnosis/genetics ; CRISPR-Cas Systems/genetics ; DNA Primers/genetics ; Diagnostic Tests, Routine/methods ; Humans ; Mutation/genetics ; Nucleic Acid Amplification Techniques/*methods ; RNA, Guide/genetics/metabolism ; SARS-CoV-2/*genetics ; Sensitivity and Specificity ; },
abstract = {Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.},
}
@article {pmid34941868,
year = {2021},
author = {Weitzel, AJ and Grunwald, HA and Weber, C and Levina, R and Gantz, VM and Hedrick, SM and Bier, E and Cooper, KL},
title = {Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline.},
journal = {PLoS biology},
volume = {19},
number = {12},
pages = {e3001478},
pmid = {34941868},
issn = {1545-7885},
support = {R21 GM129448/GM/NIGMS NIH HHS/United States ; T32 GM133351/GM/NIGMS NIH HHS/United States ; R01 AI131081/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; Female ; Gene Conversion/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Gene Expression Regulation, Developmental/genetics ; Genetic Engineering/methods ; Germ Cells/metabolism ; Male ; Meiosis/genetics ; Mice ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; },
abstract = {Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a "gene drive," to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an "insertion/deletion" (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of "super-mendelian" inheritance from both male and female mice.},
}
@article {pmid34940839,
year = {2021},
author = {Banerjee, R and Chakraborty, P and Yu, MC and Gunawardena, S},
title = {A stop or go switch: glycogen synthase kinase 3β phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {24},
pages = {},
pmid = {34940839},
issn = {1477-9129},
support = {R03 NS114731/NS/NINDS NIH HHS/United States ; },
mesh = {Adenosine Triphosphatases/genetics ; Animals ; Axonal Transport/genetics ; Axons/metabolism ; CRISPR-Cas Systems/genetics ; Cell Movement/*genetics/physiology ; Drosophila Proteins/*genetics ; Drosophila melanogaster/genetics ; Dyneins/genetics ; Glycogen Synthase Kinase 3 beta/*genetics ; Kinesins/*genetics ; Larva/genetics ; Microtubules/*genetics ; Neurons/metabolism ; Phosphorylation/genetics ; Protein Domains/genetics ; },
abstract = {It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previously, we found that GSK3β plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3β in vivo. The GSK3β-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3β phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3β non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, we propose that GSK3β phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.},
}
@article {pmid34939826,
year = {2022},
author = {Johansen, KH},
title = {How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research.},
journal = {DNA and cell biology},
volume = {41},
number = {1},
pages = {53-57},
pmid = {34939826},
issn = {1557-7430},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; T-Lymphocytes ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells ex vivo before transferring these back to patients to increase T cell efficacy. This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.},
}
@article {pmid34939798,
year = {2022},
author = {Ali, Z and Sánchez, E and Tehseen, M and Mahas, A and Marsic, T and Aman, R and Sivakrishna Rao, G and Alhamlan, FS and Alsanea, MS and Al-Qahtani, AA and Hamdan, S and Mahfouz, M},
title = {Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {406-419},
pmid = {34939798},
issn = {2161-5063},
mesh = {*COVID-19/diagnosis/genetics ; *COVID-19 Nucleic Acid Testing ; *CRISPR-Cas Systems ; Humans ; *Point-of-Care Systems ; RNA, Viral/*genetics ; SARS-CoV-2/genetics ; },
abstract = {Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/μL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, β, and δ SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.},
}
@article {pmid34937946,
year = {2022},
author = {Thandapani, P and Kloetgen, A and Witkowski, MT and Glytsou, C and Lee, AK and Wang, E and Wang, J and LeBoeuf, SE and Avrampou, K and Papagiannakopoulos, T and Tsirigos, A and Aifantis, I},
title = {Valine tRNA levels and availability regulate complex I assembly in leukaemia.},
journal = {Nature},
volume = {601},
number = {7893},
pages = {428-433},
pmid = {34937946},
issn = {1476-4687},
support = {R01 CA173636/CA/NCI NIH HHS/United States ; R01 CA252239/CA/NCI NIH HHS/United States ; P30 CA016087/CA/NCI NIH HHS/United States ; R01 CA228135/CA/NCI NIH HHS/United States ; R01 CA227649/CA/NCI NIH HHS/United States ; K22 CA201088/CA/NCI NIH HHS/United States ; R01 CA266212/CA/NCI NIH HHS/United States ; P01 CA229086/CA/NCI NIH HHS/United States ; R01 CA216421/CA/NCI NIH HHS/United States ; R01 HL159175/HL/NHLBI NIH HHS/United States ; R01 CA242020/CA/NCI NIH HHS/United States ; R37 CA222504/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Biological Availability ; CRISPR-Cas Systems ; Diet ; Electron Transport Complex I/genetics ; Large Neutral Amino Acid-Transporter 1 ; Mice ; Mitochondria/metabolism ; Oxidative Phosphorylation ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Transfer/genetics ; *Valine/metabolism ; *Valine-tRNA Ligase/metabolism ; },
abstract = {Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers[1,2], the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.},
}
@article {pmid34937346,
year = {2022},
author = {Xie, S and Tao, D and Fu, Y and Xu, B and Tang, Y and Steinaa, L and Hemmink, JD and Pan, W and Huang, X and Nie, X and Zhao, C and Ruan, J and Zhang, Y and Han, J and Fu, L and Ma, Y and Li, X and Liu, X and Zhao, S},
title = {Rapid Visual CRISPR Assay: A Naked-Eye Colorimetric Detection Method for Nucleic Acids Based on CRISPR/Cas12a and a Convolutional Neural Network.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {383-396},
pmid = {34937346},
issn = {2161-5063},
mesh = {*African Swine Fever/diagnosis/genetics ; African Swine Fever Virus/*genetics ; Animals ; *COVID-19/diagnosis/genetics ; *COVID-19 Nucleic Acid Testing ; *CRISPR-Cas Systems ; Colorimetry ; Humans ; SARS-CoV-2/*genetics ; Swine ; },
abstract = {Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.},
}
@article {pmid34936700,
year = {2022},
author = {Lu, A and Hsieh, F and Sharma, BR and Vaughn, SR and Enrich, C and Pfeffer, SR},
title = {CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export.},
journal = {The Journal of cell biology},
volume = {221},
number = {2},
pages = {},
pmid = {34936700},
issn = {1540-8140},
support = {R01 HL134991/HL/NHLBI NIH HHS/United States ; 5R01HL134991-04/HL/NHLBI NIH HHS/United States ; },
mesh = {Biological Transport ; CRISPR-Cas Systems/*genetics ; Cholesterol/*metabolism ; Endoplasmic Reticulum/*metabolism ; Endosomes/metabolism ; *Genetic Testing ; Genome ; Green Fluorescent Proteins/metabolism ; Humans ; K562 Cells ; Lipids/*chemistry ; Lysosomes/*metabolism ; Protein Domains ; Sorting Nexins/chemistry/metabolism ; },
abstract = {We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER-lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.},
}
@article {pmid34936163,
year = {2022},
author = {Qiu, X and Xu, S and Liu, X and Han, L and Zhao, B and Che, Y and Han, L and Hou, X and Li, D and Yue, Y and Chen, S and Kang, Y and Sun, L and Li, Z},
title = {A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica.},
journal = {Journal of applied microbiology},
volume = {132},
number = {5},
pages = {3685-3693},
doi = {10.1111/jam.15424},
pmid = {34936163},
issn = {1365-2672},
mesh = {*CRISPR-Cas Systems ; DNA ; *Nocardia/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {AIMS: To establish a CRISPR-based nucleic acid detection platform and apply it to the detection of Nocardia farcinica.<br><br>METHODS AND RESULTS: A CRISPR-based nucleic acid detection platform, termed CRISPR-CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre-amplification of target sequences and CRISPR-Cas12a-based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR-CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR-CPA. The whole detection process of N. farcinica CRISPR-CPA assay, including sample pre-treatment and DNA extraction (~20&#xa0;min), PCR pre-amplification (60&#xa0;min), CRISPR-based detection (10&#xa0;min), can be completed within 90&#xa0;min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR-CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR-CPA assay is 1&#xa0;pg DNA per reaction in pure cultures and 10[5] &#xa0;CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving.<br><br>CONCLUSIONS: The N. farcinica CRISPR-CPA assay is an economic and specific method to detect N. farcinica and provides a high-efficiency tool for screening of pathogens especially of some hard-to-culture and slow-growth infectious agents.<br><br>In CRISPR-CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5' end of the engineered PCR primer for protecting PAM site, thus the CRISPR-CPA can detect any sequence. Also, we applied CRISPR-CPA to rapidly detect N. farcinica, which is slow-growing bacteria and is firstly detected by a CRISPR-based method.},
}
@article {pmid34935492,
year = {2021},
author = {Liao, F and Tang, YD and Tan, L and Yao, J and Zheng, C and Wang, A},
title = {Using the CRISPR-Cas System to Solve Porcine Viral Infection-related Issues.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {776-788},
doi = {10.1089/crispr.2021.0091},
pmid = {34935492},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Swine ; *Virus Diseases/genetics ; },
abstract = {Viral infection-associated diseases seriously affect the development of the swine industry and pose a potential threat to the health of humans. Fortunately, the emergence of CRISPR-Cas has inspired scientists' efforts to address these viral-related issues in pigs using this technology. Based on progress in the field to date, this review summarizes the applications of the CRISPR-Cas system in dissecting the functions of swine viral genes and host factors related to their infections, improving the antiviral ability of pigs, inactivating porcine endogenous retrovirus prior to xenotransplantation, and detecting swine viruses. We also discuss the challenges of the practice of porcine genetic modification and the CRISPR-Cas system's prospects as an important tool for basic virology research and a promising strategy for controlling swine viral infection-related diseases.},
}
@article {pmid34935491,
year = {2021},
author = {Zhou, A and Zhang, W and Dong, X and Tang, B},
title = {Porcine Genome-Wide CRISPR Screen Identifies the Golgi Apparatus Complex Protein COG8 as a Pivotal Regulator of Influenza Virus Infection.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {872-883},
doi = {10.1089/crispr.2021.0054},
pmid = {34935491},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Golgi Apparatus/genetics/metabolism ; Humans ; *Influenza, Human/genetics/metabolism ; *Orthomyxoviridae/genetics ; Swine ; },
abstract = {Avian and human influenza viruses bind to porcine sialic acid receptors to generate novel viruses that pose a potential pandemic threat to public health. Evidence suggests that the host factors regulating the influenza virus life cycle and viral reassortment are potential broad-spectrum antiviral drug targets, compared to the ineffective seasonal vaccines against highly pathogenic viruses, leading to drug resistance. After performing a genome-wide CRISPR-Cas9 screen targeting 13,735 genes in porcine cell lines, we identified several host factors critical for influenza virus infection-notably, a conserved oligomeric Golgi complex protein, COG8, which regulates viral protein transport and immune factor expression. Viral titers indicated that the loss of COG8 significantly enhanced cellular resistance to influenza (p < 0.005). Moreover, COG8 knockout reduced the colocalization between viral particles and early endosome marker (EEA1), indicating COG8's role in the early endosome trafficking events of the virus. COG8 deletion inhibited the retrograde transport from the endosome to the trans-Golgi network, thereby accumulating the influenza protein M2 in early endosomes. COG8 silencing enhanced the expression of immune-related genes, indicating COG8-mediated host immune responses affect virus replication. Our experiments have revealed COG8 as an essential factor in influenza virus infection.},
}
@article {pmid34935490,
year = {2021},
author = {Barrangou, R},
title = {CRISPR Milestones and Anticipating What Is Next for the Journal.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {771-772},
doi = {10.1089/crispr.2021.29139.rba},
pmid = {34935490},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid34935489,
year = {2021},
author = {Medhi, D and Jasin, M},
title = {Repair-seq: Seeking and Perturbing DNA Repair.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {773-775},
doi = {10.1089/crispr.2021.29140.dme},
pmid = {34935489},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; DNA Damage ; DNA Repair/genetics ; *Gene Editing ; },
}
@article {pmid34935484,
year = {2021},
author = {Gao, L and Yang, L and Zhang, S and Ge, Z and Su, M and Shi, Y and Wang, X and Huang, C},
title = {Engineering NK-92 Cell by Upregulating CXCR2 and IL-2 Via CRISPR-Cas9 Improves Its Antitumor Effects as Cellular Immunotherapy for Human Colon Cancer.},
journal = {Journal of interferon &amp; cytokine research : the official journal of the International Society for Interferon and Cytokine Research},
volume = {41},
number = {12},
pages = {450-460},
doi = {10.1089/jir.2021.0078},
pmid = {34935484},
issn = {1557-7465},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Chemotaxis ; Colonic Neoplasms/etiology/*therapy ; Disease Models, Animal ; *Gene Editing ; *Gene Expression Regulation, Neoplastic ; Humans ; Immunotherapy, Adoptive ; Interleukin-2/*genetics ; Killer Cells, Natural/immunology/*metabolism ; Mice ; Plasmids/genetics ; RNA, Guide ; Receptors, Interleukin-8B/*genetics ; Transduction, Genetic ; Xenograft Model Antitumor Assays ; },
abstract = {Natural killer (NK) cells have shown good application prospects in adoptive cellular immunotherapy against cancer. However, due to its insufficient infiltration and low activity, the therapeutic effect of infused NK cells has been limited in solid tumors, such as colorectal cancer. It has been proved that tumor-produced chemokines regulate the migration of NK cells expressing corresponding chemokine receptors, and cytokines could enhance the antitumor activity of NK cells. In this study, we innovatively upregulated the expression of chemokine receptor CXC chemokine receptor 2 (CXCR2) and cytokine interleukin (IL)-2 on NK-92 cells using CRISPR-Cas9 gene-editing technology. We demonstrated that overexpressing CXCR2 and IL-2 promotes NK-92 cells to increasingly transfer into tumor sites and achieve stronger cell-killing and proliferation activity. Moreover, the inhibitory effects of gene-edited NK-92 cells on the growth of human colon cancer in vivo were also improved. The tumor burden of tumor-bearing mice was reduced, and their survival time was significantly prolonged. Gene-editing modification NK cells are expected to become a novel and promising tumor treatment strategy.},
}
@article {pmid34935462,
year = {2022},
author = {Simone, BW and Lee, HB and Daby, CL and Ata, H and Restrepo-Castillo, S and Martínez-Gálvez, G and Kar, B and Gendron, WAC and Clark, KJ and Ekker, SC},
title = {Chimeric RNA: DNA TracrRNA Improves Homology-Directed Repair In Vitro and In Vivo.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {40-52},
pmid = {34935462},
issn = {2573-1602},
support = {R01 GM134732/GM/NIGMS NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; DNA Breaks, Double-Stranded ; *Gene Editing ; Humans ; RNA/genetics ; },
abstract = {Nearly 90% of human pathogenic mutations are caused by small genetic variations, and methods to correct these errors efficiently are critically important. One way to make small DNA changes is providing a single-stranded oligo deoxynucleotide (ssODN) containing an alteration coupled with a targeted double-strand break (DSB) at the target locus in the genome. Coupling an ssODN donor with a CRISPR-Cas9-mediated DSB is one of the most streamlined approaches to introduce small changes. However, in many systems, this approach is inefficient and introduces imprecise repair at the genetic junctions. We herein report a technology that uses spatiotemporal localization of an ssODN with CRISPR-Cas9 to improve gene alteration. We show that by fusing an ssODN template to the trans-activating RNA (tracrRNA), we recover precise genetic alterations, with increased integration and precision in vitro and in vivo. Finally, we show that this technology can be used to enhance gene conversion with other gene editing tools such as transcription activator like effector nucleases.},
}
@article {pmid34935453,
year = {2021},
author = {Keeler, AM},
title = {Immune Responses to Adeno-Associated Virus-Mediated CRISPR Therapy.},
journal = {Human gene therapy},
volume = {32},
number = {23-24},
pages = {1430-1432},
doi = {10.1089/hum.2021.29193.amk},
pmid = {34935453},
issn = {1557-7422},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Dependovirus/genetics ; Gene Editing ; Genetic Vectors/genetics ; Immunity ; },
}
@article {pmid34934130,
year = {2021},
author = {Nagamoto, S and Agawa, M and Tsuchitani, E and Akimoto, K and Matsushima, SK and Kanegae, Y},
title = {Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {24369},
pmid = {34934130},
issn = {2045-2322},
mesh = {Adenoviridae/*genetics ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Vectors ; HEK293 Cells ; HeLa Cells ; Hep G2 Cells ; Hepatitis B/chemically induced/genetics ; Hepatitis B virus/*genetics ; Hepatocytes/*metabolism/virology ; Humans ; Integrases/genetics/*metabolism ; },
abstract = {Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least 6 months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80-90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.},
}
@article {pmid34934014,
year = {2022},
author = {Muntean, BS and Marwari, S and Li, X and Sloan, DC and Young, BD and Wohlschlegel, JA and Martemyanov, KA},
title = {Members of the KCTD family are major regulators of cAMP signaling.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {119},
number = {1},
pages = {},
pmid = {34934014},
issn = {1091-6490},
support = {R01 DA036596/DA/NIDA NIH HHS/United States ; R01 GM089778/GM/NIGMS NIH HHS/United States ; },
mesh = {Allosteric Regulation ; Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Cation Transport Proteins/metabolism ; Corpus Striatum/cytology/metabolism ; Cyclic AMP/biosynthesis/*metabolism ; Humans ; Mice ; Neurons/metabolism ; Potassium Channels/*metabolism ; Receptors, G-Protein-Coupled/metabolism ; *Signal Transduction ; },
abstract = {Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger with an essential role in neuronal function. cAMP synthesis by adenylyl cyclases (AC) is controlled by G protein-coupled receptor (GPCR) signaling systems. However, the network of molecular players involved in the process is incompletely defined. Here, we used CRISPR/Cas9-based screening to identify that members of the potassium channel tetradimerization domain (KCTD) family are major regulators of cAMP signaling. Focusing on striatal neurons, we show that the dominant isoform KCTD5 exerts its effects through an unusual mechanism that modulates the influx of Zn[2+] via the Zip14 transporter to exert unique allosteric effects on AC. We further show that KCTD5 controls the amplitude and sensitivity of stimulatory GPCR inputs to cAMP production by Gβγ-mediated AC regulation. Finally, we report that KCTD5 haploinsufficiency in mice leads to motor deficits that can be reversed by chelating Zn[2+] Together, our findings uncover KCTD proteins as major regulators of neuronal cAMP signaling via diverse mechanisms.},
}
@article {pmid34933779,
year = {2022},
author = {Zirin, J and Bosch, J and Viswanatha, R and Mohr, SE and Perrimon, N},
title = {State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila.},
journal = {Trends in genetics : TIG},
volume = {38},
number = {5},
pages = {437-453},
pmid = {34933779},
issn = {0168-9525},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila/genetics ; Drosophila melanogaster/genetics ; Gene Editing/methods ; Mutagenesis ; Recombinational DNA Repair ; },
abstract = {For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.},
}
@article {pmid34932986,
year = {2022},
author = {Dimitriu, T and Kurilovich, E and Łapińska, U and Severinov, K and Pagliara, S and Szczelkun, MD and Westra, ER},
title = {Bacteriostatic antibiotics promote CRISPR-Cas adaptive immunity by enabling increased spacer acquisition.},
journal = {Cell host &amp; microbe},
volume = {30},
number = {1},
pages = {31-40.e5},
doi = {10.1016/j.chom.2021.11.014},
pmid = {34932986},
issn = {1934-6069},
mesh = {Adaptive Immunity/*genetics ; Anti-Bacterial Agents/*pharmacology ; Bacteria/*drug effects/*genetics/growth &amp; development/immunology ; Bacteriophages/genetics ; CRISPR-Cas Systems/*immunology ; Genome, Bacterial ; Humans ; Mutation ; Pseudomonas aeruginosa/drug effects/genetics/growth &amp; development/immunology ; },
abstract = {Phages impose strong selection on bacteria to evolve resistance against viral predation. Bacteria can rapidly evolve phage resistance via receptor mutation or using their CRISPR-Cas adaptive immune systems. Acquisition of CRISPR immunity relies on the insertion of a phage-derived sequence into CRISPR arrays in the bacterial genome. Using Pseudomonas aeruginosa and its phage DMS3vir as a model, we demonstrate that conditions that reduce bacterial growth rates, such as exposure to bacteriostatic antibiotics (which inhibit cell growth without killing), promote the evolution of CRISPR immunity. We demonstrate that this is due to slower phage development under these conditions, which provides more time for cells to acquire phage-derived sequences and mount an immune response. Our data reveal that the speed of phage development is a key determinant of the evolution of CRISPR immunity and suggest that use of bacteriostatic antibiotics can trigger elevated levels of CRISPR immunity in human-associated and natural environments.},
}
@article {pmid34930897,
year = {2021},
author = {E, F and Zhang, H and Yin, W and Wang, C and Liu, Y and Li, Y and Wang, L and Wu, Y and Zhang, R and Zou, C and Song, T and Matunda, C and Zou, C and Gao, X},
title = {CPEB3 deficiency in mice affect ovarian follicle development and causes premature ovarian insufficiency.},
journal = {Cell death &amp; disease},
volume = {13},
number = {1},
pages = {21},
pmid = {34930897},
issn = {2041-4889},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Proliferation/genetics ; Disease Models, Animal ; Female ; Fertility/*genetics ; Granulosa Cells/*metabolism ; Growth Differentiation Factor 9/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; *Mutation ; Oocytes/metabolism ; Phenotype ; Pregnancy ; Primary Ovarian Insufficiency/genetics/*metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Signal Transduction/*genetics ; },
abstract = {Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.},
}
@article {pmid34930557,
year = {2022},
author = {Rocafort, M and Arshed, S and Hudson, D and Sidhu, JS and Bowen, JK and Plummer, KM and Bradshaw, RE and Johnson, RD and Johnson, LJ and Mesarich, CH},
title = {CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis.},
journal = {Fungal biology},
volume = {126},
number = {1},
pages = {35-46},
doi = {10.1016/j.funbio.2021.10.001},
pmid = {34930557},
issn = {1878-6146},
mesh = {*Ascomycota/genetics ; CRISPR-Cas Systems ; Fungal Genus Venturia ; Gene Editing ; *Malus/genetics ; Plant Diseases ; },
abstract = {Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.},
}
@article {pmid34930414,
year = {2021},
author = {Barry, T and Wang, X and Morris, JA and Roeder, K and Katsevich, E},
title = {SCEPTRE improves calibration and sensitivity in single-cell CRISPR screen analysis.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {344},
pmid = {34930414},
issn = {1474-760X},
support = {R01 MH123184/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Calibration ; Chromatin Immunoprecipitation Sequencing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Expression ; *Genome, Human ; Humans ; Single-Cell Analysis/*methods ; },
abstract = {Single-cell CRISPR screens are a promising biotechnology for mapping regulatory elements to target genes at genome-wide scale. However, technical factors like sequencing depth impact not only expression measurement but also perturbation detection, creating a confounding effect. We demonstrate on two single-cell CRISPR screens how these challenges cause calibration issues. We propose SCEPTRE: analysis of single-cell perturbation screens via conditional resampling, which infers associations between perturbations and expression by resampling the former according to a working model for perturbation detection probability in each cell. SCEPTRE demonstrates very good calibration and sensitivity on CRISPR screen data, yielding hundreds of new regulatory relationships supported by orthogonal biological evidence.},
}
@article {pmid34930405,
year = {2021},
author = {Dempster, JM and Boyle, I and Vazquez, F and Root, DE and Boehm, JS and Hahn, WC and Tsherniak, A and McFarland, JM},
title = {Chronos: a cell population dynamics model of CRISPR experiments that improves inference of gene fitness effects.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {343},
pmid = {34930405},
issn = {1474-760X},
mesh = {Algorithms ; Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Computational Biology ; Gene Knockout Techniques ; Gene Library ; *Genome ; Humans ; Neoplasms/genetics ; *Population Dynamics ; },
abstract = {CRISPR loss of function screens are powerful tools to interrogate biology but exhibit a number of biases and artifacts that can confound the results. Here, we introduce Chronos, an algorithm for inferring gene knockout fitness effects based on an explicit model of cell proliferation dynamics after CRISPR gene knockout. We test Chronos on two pan-cancer CRISPR datasets and one longitudinal CRISPR screen. Chronos generally outperforms competitors in separation of controls and strength of biomarker associations, particularly when longitudinal data is available. Additionally, Chronos exhibits the lowest copy number and screen quality bias of evaluated methods. Chronos is available at https://github.com/broadinstitute/chronos .},
}
@article {pmid34929443,
year = {2022},
author = {Canac, R and Caillaud, A and Cimarosti, B and Girardeau, A and Hamamy, H and Reversade, B and Bonnard, C and Al Sayed, ZR and David, L and Poschmann, J and Lemarchand, P and Lamirault, G and Gaborit, N},
title = {Generation of three human induced pluripotent stem cell lines with IRX5 knockout and knockin genetic editions using CRISPR-Cas9 system.},
journal = {Stem cell research},
volume = {58},
number = {},
pages = {102627},
doi = {10.1016/j.scr.2021.102627},
pmid = {34929443},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Heterozygote ; Homeodomain Proteins/genetics/metabolism ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Studies on animal models have shown that Irx5 is an important regulator of cardiac development and that it regulates ventricular electrical repolarization gradient in the adult heart. Mutations in IRX5 have also been linked in humans to cardiac conduction defects. In order to fully characterize the role of IRX5 during cardiac development and in cardiomyocyte function, we generated three genetically-modified human induced pluripotent stem cell lines: two knockout lines (heterozygous and homozygous) and a knockin HA-tagged line (homozygous).},
}
@article {pmid34928942,
year = {2021},
author = {Lassègue, B and Kumar, S and Mandavilli, R and Wang, K and Tsai, M and Kang, DW and Demos, C and Hernandes, MS and San Martín, A and Taylor, WR and Jo, H and Griendling, KK},
title = {Characterization of Poldip2 knockout mice: Avoiding incorrect gene targeting.},
journal = {PloS one},
volume = {16},
number = {12},
pages = {e0247261},
pmid = {34928942},
issn = {1932-6203},
support = {P01 HL095070/HL/NHLBI NIH HHS/United States ; R01 HL119798/HL/NHLBI NIH HHS/United States ; R01 HL139757/HL/NHLBI NIH HHS/United States ; R56 HL152167/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Targeting ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Knockout ; Mitochondrial Proteins/*deficiency/metabolism ; Mouse Embryonic Stem Cells/*metabolism ; Nuclear Proteins/*deficiency/metabolism ; *RNA-Seq ; },
abstract = {POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from three limitations: perinatal lethality in homozygotes, constitutive Poldip2 inactivation and inadvertent downregulation of the adjacent Tmem199 gene. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, RNA-seq and RT-qPCR experiments were performed in constitutive knockout arteries. Results show that POLDIP2 inactivation affects multiple cellular processes and provide new opportunities for future in-depth study of its functions.},
}
@article {pmid34928385,
year = {2022},
author = {Malone, LM and Hampton, HG and Morgan, XC and Fineran, PC},
title = {Type I CRISPR-Cas provides robust immunity but incomplete attenuation of phage-induced cellular stress.},
journal = {Nucleic acids research},
volume = {50},
number = {1},
pages = {160-174},
pmid = {34928385},
issn = {1362-4962},
mesh = {Bacterial Proteins/genetics/metabolism ; Bacteriophages/pathogenicity ; *CRISPR-Cas Systems ; Flagella/metabolism ; Serratia/*genetics/metabolism/virology ; *Stress, Physiological ; },
abstract = {During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4 homologue, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.},
}
@article {pmid34928332,
year = {2022},
author = {Rajkumar, AS and Morrissey, JP},
title = {Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.},
journal = {FEMS yeast research},
volume = {22},
number = {1},
pages = {},
pmid = {34928332},
issn = {1567-1364},
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques ; *Kluyveromyces/genetics ; RNA, Guide ; },
abstract = {There is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.},
}
@article {pmid34928030,
year = {2021},
author = {Su, J and Ke, Y and Maboyi, N and Zhi, X and Yan, S and Li, F and Zhao, B and Jia, X and Song, S and Ding, X},
title = {CRISPR/Cas12a Powered DNA Framework-Supported Electrochemical Biosensing Platform for Ultrasensitive Nucleic Acid Analysis.},
journal = {Small methods},
volume = {5},
number = {12},
pages = {e2100935},
doi = {10.1002/smtd.202100935},
pmid = {34928030},
issn = {2366-9608},
mesh = {Bacterial Proteins/*metabolism ; Biosensing Techniques/instrumentation/*methods ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; DNA, Viral/analysis ; Endodeoxyribonucleases/*metabolism ; Human papillomavirus 16/genetics/isolation &amp; purification ; Limit of Detection ; Nucleic Acids/*analysis ; },
abstract = {Nucleic acid analysis using ultrasensitive and simple methods is critically important for the early-stage diagnosis and treatment of diseases. The CRISPR/Cas proteins, guided by a single-stranded RNA have shown incredible capability for sequence-specific targeting and detection. Herein, in order to improve and expand the application of CRISPR/Cas technology to the electrochemical interface-based nucleic acids analysis, the authors develop a CRISPR/Cas12a powered DNA framework-supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a on the heterogeneous carbon interface (the existing publications which commonly adopted trans-cleavage). Their solid-liquid interface is first immobilized by 3D tetrahedral framework nucleic acids (FNAs) with specific DNA recognition probe. Based on the recognition of the complementary target through protospacer adjacent motif (PAM) confirmation and CRISPR-derived RNA (crRNA) matching, the easily formed Cas12a/crRNA duplex can get access to the interface, and the cis and trans cleavage of Cas12a can be easily activated. In combination with the enzyme catalyzed reaction, they achieved an ultralow limit of detection (LOD) of 100 fm in HPV-16 detection without pre-amplification. Furthermore, the platform is compatible with a spike-in human serum sample and has superior stability. Thus, their reported platform offers a practical, versatile, and amplification-free toolbox for ultrasensitive nucleic acid analysis.},
}
@article {pmid34927998,
year = {2021},
author = {Jing, R and Jiao, P and Chen, J and Meng, X and Wu, X and Duan, Y and Shang, K and Qian, L and Huang, Y and Liu, J and Huang, T and Jin, J and Chen, W and Zeng, X and Yin, W and Gao, X and Zhou, C and Sadelain, M and Sun, J},
title = {Cas9-Cleavage Sequences in Size-Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells.},
journal = {Small methods},
volume = {5},
number = {7},
pages = {e2100071},
doi = {10.1002/smtd.202100071},
pmid = {34927998},
issn = {2366-9608},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA ; Gene Editing/methods ; Gene Targeting ; Genetic Vectors ; Homologous Recombination ; Humans ; Immunotherapy/methods ; Immunotherapy, Adoptive/*methods ; Mice ; NIH 3T3 Cells ; *Plasmids ; T-Lymphocytes/*immunology ; },
abstract = {T cell genome editing holds great promise to advance a range of immunotherapies but is encumbered by the dependence on difficult-to-produce and expensive viral vectors. Here, small double-stranded plasmid DNA modified to mediate high-efficiency homologous recombination is designed. The resulting chimeric antigen receptor (CAR)-T cells display a similar phenotype, transcriptional profile, and in vivo potency to CAR-T cells generated using adeno-associated viral vector. This method should simplify and accelerate the use of precision engineering to produce edited T cells for research and clinical purposes.},
}
@article {pmid34927803,
year = {2022},
author = {Monaghan, TI and Baker, JA and Krabben, P and Davies, ET and Jenkinson, ER and Goodhead, IB and Robinson, GK and Shepherd, M},
title = {Deletion of glyceraldehyde-3-phosphate dehydrogenase (gapN) in Clostridium saccharoperbutylacetonicum N1-4(HMT) using CLEAVE™ increases the ATP pool and accelerates solvent production.},
journal = {Microbial biotechnology},
volume = {15},
number = {5},
pages = {1574-1585},
pmid = {34927803},
issn = {1751-7915},
mesh = {Adenosine Triphosphate/metabolism ; *Clostridium/genetics/metabolism ; *Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism ; Solvents/metabolism ; },
abstract = {The development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1-4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde-3-phosphate (GAP) to 3-phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN, demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4-fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.},
}
@article {pmid34927791,
year = {2022},
author = {Manjón, AG and Linder, S and Teunissen, H and Friskes, A and Zwart, W and de Wit, E and Medema, RH},
title = {Unexpected gene activation following CRISPR-Cas9-mediated genome editing.},
journal = {EMBO reports},
volume = {23},
number = {2},
pages = {e53902},
pmid = {34927791},
issn = {1469-3178},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Transcriptional Activation ; },
abstract = {The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSBs) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiently generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are not yet well understood. Here, we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of Taxol-resistant colonies. We show that these colonies upregulate ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 on-target effect that researchers need to be aware of when using lentiviral vectors for genome editing.},
}
@article {pmid34925305,
year = {2021},
author = {Goswami, C and Fox, S and Holden, M and Leanord, A and Evans, TJ},
title = {Genomic Analysis of Global Staphylococcus argenteus Strains Reveals Distinct Lineages With Differing Virulence and Antibiotic Resistance Gene Content.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {795173},
pmid = {34925305},
issn = {1664-302X},
abstract = {Infections due to Staphylococcus argenteus have been increasingly reported worldwide and the microbe cannot be distinguished from Staphylococcus aureus by standard methods. Its complement of virulence determinants and antibiotic resistance genes remain unclear, and how far these are distinct from those produced by S. aureus remains undetermined. In order to address these uncertainties, we have collected 132 publicly available sequences from fourteen different countries, including the United Kingdom, between 2005 and 2018 to study the global genetic structure of the population. We have compared the genomes for antibiotic resistance genes, virulence determinants and mobile genetic elements such as phages, pathogenicity islands and presence of plasmid groups between different clades. 20% (n = 26) isolates were methicillin resistant harboring a mecA gene and 88% were penicillin resistant, harboring the blaZ gene. ST2250 was identified as the most frequent strain, but ST1223, which was the second largest group, contained a marginally larger number of virulence genes compared to the other STs. Novel S. argenteus pathogenicity islands were identified in our isolates harboring tsst-1, seb, sec3, ear, selk, selq toxin genes, as well as chromosomal clusters of enterotoxin and superantigen-like genes. Strain-specific type I modification systems were widespread which would limit interstrain transfer of genetic material. In addition, ST2250 possessed a CRISPR/Cas system, lacking in most other STs. S. argenteus possesses important genetic differences from S. aureus, as well as between different STs, with the potential to produce distinct clinical manifestations.},
}
@article {pmid34922545,
year = {2021},
author = {Zha, Y and Lu, Y and Zhang, T and Yan, K and Zhuang, W and Liang, J and Cheng, Y and Wang, Y},
title = {CRISPR/Cas9-mediated knockout of APOC3 stabilizes plasma lipids and inhibits atherosclerosis in rabbits.},
journal = {Lipids in health and disease},
volume = {20},
number = {1},
pages = {180},
pmid = {34922545},
issn = {1476-511X},
mesh = {Animals ; Apolipoprotein C-III/*metabolism ; Atherosclerosis/*etiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytokines/blood ; Female ; Gene Knockdown Techniques ; Intestine, Small/metabolism ; Lipids/*blood ; Liver/metabolism ; Male ; Plaque, Atherosclerotic/etiology ; Rabbits ; },
abstract = {BACKGROUND: High levels of apolipoprotein C3 (APOC3) can lead to hypertriglyceridemia, which increases the risk of cardiovascular disease. We aim to create APOC3-knockout (KO) rabbits and explore the effects of APOC3 deletion on the occurrence and development of atherosclerosis.<br><br>METHODS: An sgRNA anchored to exon 2 of APOC3 was designed to edit embryo genomes using the CRISPR/Cas9 system. The founder rabbits were sequenced, and their lipid profile, inflammatory cytokines, and atherosclerotic plaques were analyzed.<br><br>RESULTS: When given a normal chow (NC) diet, all APOC3-KO rabbits had 50% lower triglyceride (TG) levels than those of the matched age control group. Additionally, their plasma lipoprotein lipase increased. When fed a high-fat diet, APOC3 deficiency was observed to be more conducive to the maintenance of plasma TG, total cholesterol, and low-density lipoprotein cholesterol levels, and the inhibition of the inflammatory response and the protection against atherosclerosis in rabbits.<br><br>CONCLUSION: APOC3 deficiency can delay the formation of atherosclerosis-induced HFD in rabbits, indicating this is a novel therapeutic target to treat atherosclerosis.},
}
@article {pmid34921769,
year = {2022},
author = {Frederickson, RM and Herzog, RW},
title = {Second installment of Special Issue on expanding the scale and scope and of gene editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {1},
doi = {10.1016/j.ymthe.2021.12.010},
pmid = {34921769},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}
@article {pmid34920978,
year = {2022},
author = {Dong, MB and Tang, K and Zhou, X and Zhou, JJ and Chen, S},
title = {Tumor immunology CRISPR screening: present, past, and future.},
journal = {Trends in cancer},
volume = {8},
number = {3},
pages = {210-225},
pmid = {34920978},
issn = {2405-8025},
support = {RF1 DA048811/DA/NIDA NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; T32 GM007499/GM/NIGMS NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Humans ; Immunotherapy ; *Neoplasms/genetics/therapy ; },
abstract = {Recent advances in immunotherapy have fundamentally changed the landscape of cancer treatment by leveraging the specificity and selectivity of the adaptive immune system to kill cancer cells. These successes have ushered in a new wave of research aimed at understanding immune recognition with the hope of developing newer immunotherapies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technologies and advancement of multiomics modalities have greatly accelerated the discovery process. Here, we review the current literature surrounding CRISPR screens within the context of tumor immunology, provide essential components needed to conduct immune-specific CRISPR screens, and present avenues for future research.},
}
@article {pmid34920928,
year = {2022},
author = {Zhang, F and Huang, Z},
title = {Mechanistic insights into the versatile class II CRISPR toolbox.},
journal = {Trends in biochemical sciences},
volume = {47},
number = {5},
pages = {433-450},
doi = {10.1016/j.tibs.2021.11.007},
pmid = {34920928},
issn = {0968-0004},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
abstract = {The constantly expanding group of class II CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated) effectors and their engineered variants exhibit distinct editing modes and efficiency, fidelity, target range, and molecular size. Their enormous diversity of capabilities provides a formidable toolkit for a large array of technologies. We review the structural and biochemical mechanisms of versatile effector proteins from class II CRISPR-Cas systems to provide mechanistic insights into their target specificity, protospacer adjacent motif (PAM) restriction, and activity regulation, and discuss possible strategies to enhance genome-engineering tools in terms of accuracy, efficiency, applicability, and controllability.},
}
@article {pmid34920098,
year = {2022},
author = {Jamehdor, S and Naserian, S and Teimoori, A},
title = {Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {97},
number = {},
pages = {105188},
pmid = {34920098},
issn = {1567-7257},
mesh = {APOBEC Deaminases/genetics/immunology ; Adenosine Deaminase/genetics/immunology ; Bacterial Proteins/genetics/immunology ; COVID-19/immunology/*prevention &amp; control ; COVID-19 Vaccines/biosynthesis/*genetics ; *CRISPR-Cas Systems ; Endonucleases/genetics/immunology ; Gene Editing/*methods ; Gene Expression ; Genome, Viral ; Humans ; *Mutation Rate ; RNA-Binding Proteins/genetics/immunology ; Recombinant Fusion Proteins/genetics/immunology ; SARS-CoV-2/*genetics/immunology ; Selection, Genetic ; Vaccines, Attenuated ; Viral Proteins/*genetics/immunology ; },
abstract = {The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.},
}
@article {pmid34919973,
year = {2022},
author = {Srivastava, A and Gupta, T and Srivastava, S and Dhir, S and Kumar, P and Singhal, T and Rani, A and Rishi, N},
title = {Development of a new Collateral Cleavage-independent CRISPR/Cas12a based easy detection system for plant viruses.},
journal = {Journal of virological methods},
volume = {300},
number = {},
pages = {114432},
doi = {10.1016/j.jviromet.2021.114432},
pmid = {34919973},
issn = {1879-0984},
mesh = {*Begomovirus/genetics ; CRISPR-Cas Systems ; *Geminiviridae/genetics ; Phylogeny ; Plant Diseases ; *Plant Viruses/genetics ; },
abstract = {Plant virus spread through various means, from mechanically to the insect vectors and act as obligate parasite, therefore, are extremely challenging to eradicate. Geminiviruses are an important class of viruses which have reported extensively in last two decades on several new hosts. They infect wide range of annual crops and perineal shrubs, therefore, essentially required to detect them on field and dispose to check their vector transmission to healthy crops. In this study, we have chosen two important begomovirus viz. Mungbean yellow mosaic India virus which infect wide range of leguminous crops while Ageratum enation virus is reported to infect a wide range of crops from weed to opium poppy. Here, we have utilized the binding and cleaving ability of LbaCas12a protein with target to detect the virus infection on field. We proposed here a new Collateral Cleavage Independent CRISPR/Cas12a based detection system (CCI-CRISPR) for plant viruses.},
}
@article {pmid34919766,
year = {2022},
author = {Wang, L and Xu, D and Scharf, K and Frank, W and Leister, D and Kleine, T},
title = {The RNA-binding protein RBP45D of Arabidopsis promotes transgene silencing and flowering time.},
journal = {The Plant journal : for cell and molecular biology},
volume = {109},
number = {6},
pages = {1397-1415},
doi = {10.1111/tpj.15637},
pmid = {34919766},
issn = {1365-313X},
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; DNA Methylation ; DNA-Directed RNA Polymerases/metabolism ; Flowers/*physiology ; RNA, Plant/genetics ; RNA, Small Interfering/genetics ; *RNA-Binding Proteins/genetics/metabolism ; Transgenes ; },
abstract = {RNA-directed DNA methylation (RdDM) helps to defend plants against invasive nucleic acids. In the canonical form of RdDM, 24-nt small interfering RNAs (siRNAs) are produced by DICER-LIKE 3 (DCL3). The siRNAs are loaded onto ARGONAUTE (AGO) proteins leading ultimately to de novo DNA methylation. Here, we introduce the Arabidopsis thaliana prors1 (LUC) transgenic system, in which 24-nt siRNAs are generated to silence the promoter-LUC construct. A forward genetic screen performed with this system identified, besides known components of RdDM (NRPD2A, RDR2, AGO4 and AGO6), the RNA-binding protein RBP45D. RBP45D is involved in CHH (where H is A, C or T) DNA methylation, and maintains siRNA production originating from the LUC transgene. RBP45D is localized to the nucleus, where it is associated with small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). RNA-Seq analysis showed that in CRISPR/Cas-mediated rbp-ko lines FLOWERING LOCUS C (FLC) mRNA levels are upregulated and several loci differentially spliced, among them FLM. In consequence, loss of RBP45D delays flowering, presumably mediated by the release of FLC levels and/or alternative splicing of FLM. Moreover, because levels and processing of transcripts of known RdDM genes are not altered in rbp-ko lines, RBP45D should have a more direct function in transgene silencing, probably independent of the canonical RdDM pathway. We suggest that RBP45D facilitates siRNA production by stabilizing either the precursor RNA or the slicer protein. Alternatively, RBP45D could be involved in chromatin modifications, participate in retention of Pol IV transcripts and/or in Pol V-dependent lncRNA retention in chromatin to enable their scaffold function.},
}
@article {pmid34919578,
year = {2021},
author = {Hammouda, OT and Wu, MY and Kaul, V and Gierten, J and Thumberger, T and Wittbrodt, J},
title = {In vivo identification and validation of novel potential predictors for human cardiovascular diseases.},
journal = {PloS one},
volume = {16},
number = {12},
pages = {e0261572},
pmid = {34919578},
issn = {1932-6203},
support = {R01 ES029917/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Cardiovascular Diseases/diagnosis/*genetics/pathology ; Gene Editing ; Genome-Wide Association Study ; Heart Rate/*genetics ; Humans ; Myosin Light Chains/*genetics ; Oryzias/*genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.},
}
@article {pmid34919397,
year = {2022},
author = {Li, ZJ and Zhang, ZX and Xu, Y and Shi, TQ and Ye, C and Sun, XM and Huang, H},
title = {CRISPR-Based Construction of a BL21 (DE3)-Derived Variant Strain Library to Rapidly Improve Recombinant Protein Production.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {343-352},
doi = {10.1021/acssynbio.1c00463},
pmid = {34919397},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Escherichia coli/genetics/metabolism ; *Membrane Proteins/genetics ; *Protein Engineering/methods ; Recombinant Proteins/biosynthesis ; },
abstract = {Escherichia coli BL21 (DE3) is the most widely used host for recombinant protein expression. However, not every protein can be highly expressed in BL21 (DE3), so individual optimization strategies are often required for different proteins, which is time-consuming and difficult to apply rapidly for industrial production. Constructing more hosts is a good choice to enrich protein expression selection. The expression level of T7 RNAP is the core control node of the pET expression system, so regulating its expression level is an effective way of improving the production of difficult-to-express proteins. Various BL21 (DE3)-derived variant hosts with different translation levels of T7 RNAP could be obtained by changing the ribosomal binding site (RBS) sequences of T7 RNAP in a genome. Here, a BL21 (DE3)-derived variant strain library with different RBS sequences of T7 RNAP was constructed using a base editor and CRISPR-Cas9. Notably, the CRISPR-Cas9 system combined with degenerate primers enabled the construction of an RBS library with 87.5% of the theoretical coverage in single editing, which is more convenient and efficient than the use of a base editor. The expression level of a target gene in the variant strain library ranged from 28 to 220% of the parental strain. Furthermore, a high-throughput host-screening platform for recombinant protein production was constructed, which enabled us to obtain the best expression host for certain target proteins in only 3 days. As a proof of concept, the production of all eight difficult-to-express proteins was greatly improved, including autolytic protein, membrane proteins, antimicrobial peptides, and hardly soluble proteins. Among them, the expression of glucose dehydrogenase in the best host exhibited a 298-fold increase compared to the parental strain. This strategy is simple and effective, requires no advanced equipment, and can be carried out in any laboratory.},
}
@article {pmid34917979,
year = {2021},
author = {Guarnaccia, AD and Weissmiller, AM and Tansey, WP},
title = {Gene-specific quantification of nascent transcription following targeted degradation of endogenous proteins in cultured cells.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {101000},
pmid = {34917979},
issn = {2666-1667},
support = {R01 CA200709/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Cell Line, Tumor ; Cells, Cultured ; Gene Editing/*methods ; Humans ; *Proteins/analysis/genetics/metabolism ; Proteolysis ; Transcription, Genetic/*genetics ; },
abstract = {Genome-wide nuclear run-ons are a powerful way to determine the impact of a perturbation such as transcription factor degradation on transcriptional patterns. But often investigators are interested in monitoring transcriptional effects at specific sets of genes, rather than the entire genome. Here we describe an approach that couples genome engineering to tag endogenous proteins for degradation with a streamlined nuclear run-on assay to yield gene-specific information on primary transcriptional changes elicited by factor depletion. For complete details on the use and execution of this protocol, please refer to Guarnaccia et al. (2021).},
}
@article {pmid34917099,
year = {2021},
author = {Klein, K and Hölzemer, A and Wang, T and Kim, TE and Dugan, HL and Jost, S and Altfeld, M and Garcia-Beltran, WF},
title = {A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {798235},
pmid = {34917099},
issn = {1664-3224},
support = {R01 AI067031/AI/NIAID NIH HHS/United States ; P01 AI104715/AI/NIAID NIH HHS/United States ; F31 AI116366/AI/NIAID NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Heparan Sulfate Proteoglycans/*agonists ; Humans ; K562 Cells ; Killer Cells, Natural/*immunology ; Ligands ; Receptors, KIR/*agonists ; Receptors, KIR3DS1/*metabolism ; Signal Transduction ; },
abstract = {While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.},
}
@article {pmid34916221,
year = {2022},
author = {Scheidmann, MC and Castro-Giner, F and Strittmatter, K and Krol, I and Paasinen-Sohns, A and Scherrer, R and Donato, C and Gkountela, S and Szczerba, BM and Diamantopoulou, Z and Muenst, S and Vlajnic, T and Kunz, L and Vetter, M and Rochlitz, C and Taylor, V and Giachino, C and Schroeder, T and Platt, RJ and Aceto, N},
title = {An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.},
journal = {Cancer research},
volume = {82},
number = {4},
pages = {681-694},
pmid = {34916221},
issn = {1538-7445},
support = {163938/SNSF_/Swiss National Science Foundation/Switzerland ; #101001652/ERC_/European Research Council/International ; PP00P3_190077/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; Biomarkers, Tumor/*genetics/metabolism ; Breast Neoplasms/blood/*genetics/pathology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Neoplasm Metastasis ; Neoplastic Cells, Circulating/*metabolism/pathology ; Protein Serine-Threonine Kinases/genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide/genetics/metabolism ; RNA-Seq/methods ; Survival Analysis ; Xenograft Model Antitumor Assays/methods ; },
abstract = {UNLABELLED: Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches.<br><br>SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.},
}
@article {pmid34915813,
year = {2021},
author = {Suchý, T and Kaczmarek, I and Maricic, T and Zieschang, C and Schöneberg, T and Thor, D and Liebscher, I},
title = {Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines.},
journal = {Adipocyte},
volume = {10},
number = {1},
pages = {631-645},
pmid = {34915813},
issn = {2162-397X},
mesh = {3T3-L1 Cells ; *Adipocytes ; Adipogenesis/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Feasibility Studies ; Humans ; Mice ; },
abstract = {Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out.},
}
@article {pmid34915706,
year = {2022},
author = {Zhu, X and Yang, H and Wang, M and Wu, M and Khan, MR and Luo, A and Deng, S and Busquets, R and He, G and Deng, R},
title = {Label-Free Detection of Transgenic Crops Using an Isothermal Amplification Reporting CRISPR/Cas12 Assay.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {317-324},
doi = {10.1021/acssynbio.1c00428},
pmid = {34915706},
issn = {2161-5063},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *G-Quadruplexes ; Nucleic Acid Amplification Techniques ; Polymerase Chain Reaction ; },
abstract = {Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars ("Bt11" and "MON89034") from nontransgenic corn cultivars ("yellow", "shenyu", "xianyu", and "jingke"). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.},
}
@article {pmid34914966,
year = {2022},
author = {Lata, S and Mishra, R and Arya, RP and Arora, P and Lahon, A and Banerjea, AC and Sood, V},
title = {Where all the Roads Meet? A Crossover Perspective on Host Factors Regulating SARS-CoV-2 infection.},
journal = {Journal of molecular biology},
volume = {434},
number = {5},
pages = {167403},
pmid = {34914966},
issn = {1089-8638},
mesh = {COVID-19/epidemiology/*virology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Host Microbial Interactions ; Host-Derived Cellular Factors/genetics/*metabolism ; Humans ; *Pandemics ; SARS-CoV-2/genetics/*physiology ; },
abstract = {COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.},
}
@article {pmid34914351,
year = {2022},
author = {Meliawati, M and Teckentrup, C and Schmid, J},
title = {CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {77-84},
doi = {10.1021/acssynbio.1c00565},
pmid = {34914351},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Engineering ; *Paenibacillus polymyxa/genetics ; },
abstract = {The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa. However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12-41 kb by the use of a single targeting sgRNA. Furthermore, we also harnessed the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80% efficiency, while simultaneous integration at two distantly located sites was obtained with 58% efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.},
}
@article {pmid34914197,
year = {2022},
author = {Riepe, C and Zelin, E and Frankino, PA and Meacham, ZA and Fernandez, SG and Ingolia, NT and Corn, JE},
title = {Double stranded DNA breaks and genome editing trigger loss of ribosomal protein RPS27A.},
journal = {The FEBS journal},
volume = {289},
number = {11},
pages = {3101-3114},
pmid = {34914197},
issn = {1742-4658},
support = {DP2 CA195768/CA/NCI NIH HHS/United States ; DP2 HL141006/HL/NHLBI NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; T32 GM007232/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA Damage/genetics ; DNA Repair ; *Gene Editing/methods ; Ribosomal Proteins/genetics ; },
abstract = {DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.},
}
@article {pmid34913119,
year = {2022},
author = {Kumari, P and Sturgeon, M and Bonde, G and Cornell, RA},
title = {Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2403},
number = {},
pages = {91-106},
pmid = {34913119},
issn = {1940-6029},
support = {R01 DE027362/DE/NIDCR NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Cation Transport Proteins ; RNA ; RNA, Guide/genetics ; *Zebrafish/genetics ; Zebrafish Proteins/genetics ; },
abstract = {Danio rerio (zebrafish), traditionally used in forward genetic screens, has in the last decade become a popular model for reverse genetic studies with the introduction of TALENS, zinc finger nucleases, and CRISPR/Cas9. Unexpectedly, homozygous frameshift mutations generated by these tools frequently result in phenotypes that are less penetrant than those seen in embryos injected with antisense morpholino oligonucleotides targeting the same gene. One explanation for the difference is that some frameshift mutations result in nonsense-mediated decay of the gene transcript, a process which can induce expression of homologous genes. This form of genetic compensation, called transcriptional adaptation, does not occur when the mutant allele results in no RNA transcripts being produced from the targeted gene. Such RNA-less mutants can be generated by deleting a gene's promoter using a pair of guide RNAs and Cas9 protein. Here, we present a protocol and use it to generate alleles of arhgap29b and slc41a1 that lack detectable zygotic transcription. In the case of the arhgap29b mutant, an emerging phenotype did not segregate with the promoter deletion mutation, highlighting the potential for off-target mutagenesis with these tools. In summary, this chapter describes a method to generate zebrafish mutants that avoid a form of genetic compensation that occurs in many frameshift mutants.},
}
@article {pmid34913112,
year = {2022},
author = {Gruss, M and Corsi, AK},
title = {Using Caenorhabditis elegans as a Model for Mechanistic Insights of Craniofacial Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2403},
number = {},
pages = {1-18},
pmid = {34913112},
issn = {1940-6029},
support = {R15 DE018519/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors ; *Caenorhabditis elegans/genetics ; Caenorhabditis elegans Proteins/genetics ; Gene Editing ; Humans ; Mutation ; Phenotype ; Skull ; },
abstract = {Caenorhabditis elegans has served as a powerful model for understanding the molecular and cell biology of clinically important human proteins due to the conservation of genes that are associated with human disorders. It is well established that evolution has conserved critical domains of proteins and their cellular functions even though the phenotypic output for analogous mutations can be distinct among organisms. To that end, the genes that are associated with human craniosynostosis such as TWIST1, TCF12, and FGFR2 have homologs in C. elegans hlh-8, hlh-2, and egl-15, respectively. Whereas mutations in these human genes lead to bone defects in the skull, mutations in the C. elegans genes lead to defects primarily in nonstriated muscles that are responsible for laying eggs and controlling defecation. Even though the phenotypes are distinct in nature, the ability to quantify them in C. elegans can give a sense of the severity to provide a genotype-phenotype correlation. With the advent of CRISPR/Cas-9 genome editing in C. elegans, it is possible to model specific patient mutations that affect conserved amino acids in C. elegans proteins. These mutant strains can then be evaluated for their phenotypes in both homozygous and heterozygous animals. The assays that can be used to measure these phenotypes are described in this chapter.},
}
@article {pmid34913067,
year = {2022},
author = {Shimizu, S and Kawahara, R and Simizu, S},
title = {Methionine aminopeptidase‑2 is a pivotal regulator of vasculogenic mimicry.},
journal = {Oncology reports},
volume = {47},
number = {2},
pages = {},
pmid = {34913067},
issn = {1791-2431},
mesh = {Aminopeptidases/*drug effects/genetics ; Angiogenesis Inhibitors/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cyclohexanes/*pharmacology ; Fatty Acids, Unsaturated/*pharmacology ; Fibrosarcoma/drug therapy ; Gene Knockdown Techniques ; Humans ; Metalloendopeptidases/*drug effects/genetics ; Methionyl Aminopeptidases/*drug effects/genetics ; Neovascularization, Pathologic/*drug therapy/genetics ; O-(Chloroacetylcarbamoyl)fumagillol/*pharmacology ; Sesquiterpenes/pharmacology ; },
abstract = {Vasculogenic mimicry (VM) is the formation of a blood supply system that confers aggressive and metastatic properties to tumors and correlates with a poor prognosis in cancer patients. Thus, the inhibition of VM is considered an effective approach for cancer treatment, although such a mechanism remains poorly described. In the present study, we examined methionine aminopeptidase‑2 (MetAP2), a key factor of angiogenesis, and demonstrated that it is pivotal for VM, using pharmacological and genetic approaches. Fumagillin and TNP‑470, angiogenesis inhibitors that target MetAP2, significantly suppressed VM in various human cancer cell lines. We established MetAP2‑knockout (KO) human fibrosarcoma HT1080 cells using the CRISPR/Cas9 system and found that VM was attenuated in these cells. Furthermore, re‑expression of wild‑type MetAP2 restored VM in the MetAP2‑KO HT1080 cells, but the substitution of D251, a conserved amino acid in MetAP2, failed to rescue the VM. Collectively, our results demonstrate that MetAP2 is critical for VM in human cancer cells and suggest fumagillin and TNP‑470 as potent VM‑suppressing agents.},
}
@article {pmid34911820,
year = {2022},
author = {Corkins, ME and DeLay, BD and Miller, RK},
title = {Tissue-Targeted CRISPR-Cas9-Mediated Genome Editing of Multiple Homeologs in F0-Generation Xenopus laevis Embryos.},
journal = {Cold Spring Harbor protocols},
volume = {2022},
number = {3},
pages = {},
doi = {10.1101/pdb.prot107037},
pmid = {34911820},
issn = {1559-6095},
support = {R01 DK115655/DK/NIDDK NIH HHS/United States ; R03 DK118771/DK/NIDDK NIH HHS/United States ; K01 DK092320/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide/genetics ; Xenopus laevis/genetics ; },
abstract = {Xenopus laevis frogs are a powerful developmental model that enables studies combining classical embryology and molecular manipulation. Because of the large embryo size, ease of microinjection, and ability to target tissues through established fate maps, X. laevis has become the predominant amphibian research model. Given that their allotetraploid genome has complicated the generation of gene knockouts, strategies need to be established for efficient mutagenesis of multiple homeologs to evaluate gene function. Here we describe a protocol to use CRISPR-Cas9-mediated genome editing to target either single alleles or multiple alloalleles in F0 X. laevis embryos. A single-guide RNA (sgRNA) is designed to target a specific DNA sequence encoding a critical protein domain. To mutagenize a gene with two alloalleles, the sgRNA is designed against a sequence that is common to both homeologs. This sgRNA, along with the Cas9 protein, is microinjected into the zygote to disrupt the genomic sequences in the whole embryo or into a specific blastomere for tissue-targeted effects. Error-prone repair of CRISPR-Cas9-generated DNA double-strand breaks leads to insertions and deletions creating mosaic gene lesions within the embryos. The genomic DNA isolated from each mosaic F0 embryo is sequenced, and software is applied to assess the nature of the mutations generated and degree of mosaicism. This protocol enables the knockout of genes within the whole embryo or in specific tissues in F0 X. laevis embryos to facilitate the evaluation of resulting phenotypes.},
}
@article {pmid34911780,
year = {2021},
author = {Fong, SH and Munson, BP and Ideker, T},
title = {Uncovering Tumorigenesis Circuitry with Combinatorial CRISPR.},
journal = {Cancer research},
volume = {81},
number = {24},
pages = {6078-6079},
doi = {10.1158/0008-5472.CAN-21-3672},
pmid = {34911780},
issn = {1538-7445},
mesh = {Animals ; *CRISPR-Cas Systems ; *Carcinogenesis/genetics ; Cell Transformation, Neoplastic/genetics ; Gene Expression Profiling ; Gene Regulatory Networks ; Mice ; },
abstract = {Oncogenesis relies on the alteration of multiple driver genes, but precisely which groups of alterations lead to cancer is not well understood. To chart these combinations, Zhao and colleagues use the CRISPR-Cas9 system to knockout all pairwise combinations among 52 tumor suppressor genes, with the goal of identifying groups of alterations that collaborate to promote cell growth. Interaction screens are performed across multiple models of tumorigenesis in cell cultures and mice, revealing clear cooperation among NF2, PTEN, and TP53 in multiple models. These and other strongly synergistic interactions are characterized further by single-cell transcriptomic profiling. This methodology presents a scalable approach to move beyond single-gene drivers to map the complex gene networks that give rise to tumorigenesis.See related article by Zhao et al., p. 6090.},
}
@article {pmid34911411,
year = {2021},
author = {Dey, A and Nandy, S},
title = {CRISPER/Cas in Plant Natural Product Research: Therapeutics as Anticancer and other Drug Candidates and Recent Patents.},
journal = {Recent patents on anti-cancer drug discovery},
volume = {16},
number = {4},
pages = {460-468},
doi = {10.2174/1574892816666210706155602},
pmid = {34911411},
issn = {2212-3970},
mesh = {Antineoplastic Agents, Phytogenic/isolation &amp; purification/pharmacology ; Biological Products/isolation &amp; purification/pharmacology ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Genome, Plant ; Humans ; Patents as Topic ; Plant Preparations/isolation &amp; purification/*pharmacology ; Plants, Genetically Modified/chemistry/genetics ; Plants, Medicinal/*chemistry/genetics ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated9 (Cas9) endonuclease system is a facile, highly efficient and selective site-directed mutagenesis tool for RNA-guided genome-editing. CRISPR/Cas9 genome-editing strategy uses designed guide-RNAs that recognizes a 3 base-pair protospacer adjacent motif (PAM) sequence in the target-DNA. CRISPR/Cas-editing tools have mainly been employed in crop plants in relation to yield and stress tolerance. However, the immense potential of this technology has not yet been fully utilized in medicinal plants in deciphering or modulating secondary metabolic pathways producing therapeutically active phytochemicals against cancer and other diseases.<br><br>OBJECTIVE: The present review elucidates the use of CRISPR-Cas9 as a promising genome-editing tool in plants and plant-derived natural products with anticancer and other therapeutic applications. It also includes recent patents on the therapeutic applications of CRISPR-CAS systems implicated to cancer and other human medical conditions.<br><br>METHODS: Popular search engines, such as PubMed, Scopus, Google Scholar, Google Patents, Medline, ScienceDirect, SpringerLink, EMBASE, Mendeley, etc., were searched in order to retrieve literature using relevant keywords viz. CRISPER/Cas, plant natural product research, anticancer, therapeutics, etc., either singly or in various combinations.<br><br>RESULTS: Retrieved citations and further cross-referencing among the literature have resulted in a total number of 71 publications and 3 patents are being cited in this work. Information presented in this review aims to support further biotechnological and clinical strategies to be carried using CRISPER/ Cas mediated optimization of plant natural products against cancer and an array of other human medical conditions.<br><br>CONCLUSION: Off late, knock-in and knock-out, point mutation, controlled tuning of gene-expression and targeted mutagenesis have enabled the versatile CRISPR/Cas-editing device to engineer medicinal plants' genomes. In addition, by combining CRISPR/Cas-editing tool with next-generation sequencing (NGS) and various tools of system biology, many medicinal plants have been engineered genetically to optimize the production of valuable bioactive compounds of industrial significance.},
}
@article {pmid34911161,
year = {2021},
author = {Cao, C and Liu, T and Zhang, Q and Li, R and Zeng, Z and Cui, Z and Wang, X and Gong, D and Tian, X and Hu, Z},
title = {Somatic mutations and CRISPR/Cas9 library screening integrated analysis identifies cervical cancer drug-resistant pathways.},
journal = {Clinical and translational medicine},
volume = {11},
number = {12},
pages = {e632},
pmid = {34911161},
issn = {2001-1326},
mesh = {CRISPR-Cas Systems/*genetics/physiology ; Early Detection of Cancer/methods/statistics &amp; numerical data ; Female ; Humans ; Mass Screening/methods/statistics &amp; numerical data ; *Medically Unexplained Symptoms ; Odds Ratio ; Signal Transduction/genetics/physiology ; Uterine Cervical Neoplasms/*diagnosis/genetics/physiopathology ; },
}
@article {pmid34910772,
year = {2021},
author = {Mitchell, LJ and Tettamanti, V and Rhodes, JS and Marshall, NJ and Cheney, KL and Cortesi, F},
title = {CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants.},
journal = {PloS one},
volume = {16},
number = {12},
pages = {e0261331},
pmid = {34910772},
issn = {1932-6203},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Fishes/*genetics/physiology ; Gene Editing/*methods ; Gene Frequency/genetics ; Genome/genetics ; Genomics/methods ; Zygote/transplantation ; },
abstract = {Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.},
}
@article {pmid34909513,
year = {2021},
author = {Priya Swetha, PD and Sonia, J and Sapna, K and Prasad, KS},
title = {Towards CRISPR powered electrochemical sensing for smart diagnostics.},
journal = {Current opinion in electrochemistry},
volume = {30},
number = {},
pages = {100829},
pmid = {34909513},
issn = {2451-9103},
abstract = {Even though global health has been steadily improved, the global disease burden associated with communicable and non-communicable diseases extensively increased healthcare expenditure. The present COVID-19 pandemic scenario has again ascertained the importance of clinical diagnostics as a basis to make life-saving decisions. In this context, there is a need for developing next-generation integrated smart real-time responsive biosensors with high selectivity and sensitivity. The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas biosensing systems has shown remarkable potential for developing next-generation biosensors. CRISPR/Cas integrated electrochemical biosensors (E-CRISPR) stands out with excellent properties. In this opinionated review, we illustrate the rapidly evolving applications for E-CRISPR-integrated detection systems towards biosensing and the future scope associated with E-CRISPR based diagnostics.},
}
@article {pmid34908530,
year = {2021},
author = {Nierzwicki, L and East, KW and Morzan, UN and Arantes, PR and Batista, VS and Lisi, GP and Palermo, G},
title = {Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34908530},
issn = {2050-084X},
support = {R01 GM136815/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; R01GM141329/NH/NIH HHS/United States ; R01GM136815/NH/NIH HHS/United States ; },
mesh = {Allosteric Regulation/*genetics ; CRISPR-Cas Systems/*genetics ; *Genetic Variation ; Genotype ; *Molecular Dynamics Simulation ; Molecular Structure ; *Mutation ; Streptococcus pyogenes/*genetics ; },
abstract = {CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.},
}
@article {pmid34908145,
year = {2022},
author = {Kumagai, Y and Liu, Y and Hamada, H and Luo, W and Zhu, J and Kuroki, M and Nagira, Y and Taoka, N and Katoh, E and Imai, R},
title = {Introduction of a second "Green Revolution" mutation into wheat via in planta CRISPR/Cas9 delivery.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1838-1842},
pmid = {34908145},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Mutation/genetics ; RNA, Guide/genetics ; *Triticum/genetics ; },
abstract = {Direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem via particle bombardment enabled introduction of a semidwarf1-orthologous mutation into an elite wheat variety.},
}
@article {pmid34908140,
year = {2022},
author = {Bryson, JW and Auxillos, JY and Rosser, SJ},
title = {Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor.},
journal = {Nucleic acids research},
volume = {50},
number = {1},
pages = {549-560},
pmid = {34908140},
issn = {1362-4962},
support = {BB/M018040/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/*metabolism ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Protein Splicing ; },
abstract = {The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5' of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.},
}
@article {pmid34907381,
year = {2021},
author = {O'Leary, K},
title = {Gene editing advances on all fronts.},
journal = {Nature medicine},
volume = {27},
number = {12},
pages = {2056},
doi = {10.1038/s41591-021-01607-z},
pmid = {34907381},
issn = {1546-170X},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Humans ; },
}
@article {pmid34907351,
year = {2022},
author = {Waltz, E},
title = {GABA-enriched tomato is first CRISPR-edited food to enter market.},
journal = {Nature biotechnology},
volume = {40},
number = {1},
pages = {9-11},
doi = {10.1038/d41587-021-00026-2},
pmid = {34907351},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Lycopersicon esculentum/genetics ; gamma-Aminobutyric Acid/metabolism ; },
}
@article {pmid34907017,
year = {2021},
author = {Yokochi, Y and Fukushi, Y and Wakabayashi, KI and Yoshida, K and Hisabori, T},
title = {Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {51},
pages = {},
pmid = {34907017},
issn = {1091-6490},
mesh = {Arabidopsis/chemistry/genetics/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Chloroplasts/*enzymology/genetics/metabolism ; Gene Expression Regulation, Enzymologic/*physiology ; Gene Expression Regulation, Plant/*physiology ; Light ; Mutation ; Oxidation-Reduction ; Plant Leaves/chemistry/metabolism ; Thioredoxins/genetics/*metabolism ; },
abstract = {Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.},
}
@article {pmid34906838,
year = {2022},
author = {Li, Z and Ding, X and Yin, K and Avery, L and Ballesteros, E and Liu, C},
title = {Instrument-free, CRISPR-based diagnostics of SARS-CoV-2 using self-contained microfluidic system.},
journal = {Biosensors &amp; bioelectronics},
volume = {199},
number = {},
pages = {113865},
pmid = {34906838},
issn = {1873-4235},
support = {R01 EB023607/EB/NIBIB NIH HHS/United States ; R61 AI154642/AI/NIAID NIH HHS/United States ; },
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Humans ; Microfluidics ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {Rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for early diagnostics and timely medical treatment of coronavirus disease 2019 (COVID-19). However, current detection methods typically rely on expensive and bulky instrumentation. Here, we developed a simple, sensitive, instrument-free, CRISPR-based diagnostics of SARS-CoV-2 using a self-contained microfluidic system. The microfluidic chip integrates isothermal amplification, CRISPR cleavage, and lateral flow detection in a single, closed microfluidic platform, enabling contamination-free, visual detection. To simplify the operation and transportation of the device, we lyophilized the CRISPR reagents in the reaction chamber and pre-stored the liquid solutions in blisters. We employed a low-cost, portable hand warmer to incubate the microfluidic chip without the need for electricity. The self-contained microfluidic system can detect down to 100 copies of SARS-CoV-2 RNA. Further, we clinically validated our method by detecting 24 COVID-19 clinical nasopharyngeal swab samples, achieving excellent sensitivity (94.1%), specificity (100%), and accuracy (95.8%). This simple, sensitive, and affordable microfluidic system represents a promising tool for point-of-care diagnostics of COVID-19 and other infectious diseases.},
}
@article {pmid34905767,
year = {2022},
author = {Zhang, T and Li, Y and Yang, Y and Weng, L and Wu, Z and Zhu, J and Qin, J and Liu, Q and Wang, P},
title = {iCRISEE: an integrative analysis of CRISPR screen by reducing false positive hits.},
journal = {Briefings in bioinformatics},
volume = {23},
number = {1},
pages = {},
doi = {10.1093/bib/bbab505},
pmid = {34905767},
issn = {1477-4054},
mesh = {Algorithms ; *CRISPR-Cas Systems ; *Gene Editing ; RNA, Guide/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) technology has become a popular tool for the study of genome function, and the use of this technology can achieve large-scale screening studies of specific phenotypes. Several analysis tools for CRISPR/Cas9 screening data have been developed, while high false positive rate remains a great challenge. To this end, we developed iCRISEE, an integrative analysis of CRISPR ScrEEn by reducing false positive hits. iCRISEE can dramatically reduce false positive hits and it is robust to different single guide RNA (sgRNA) library by introducing precise data filter and normalization, model selection and valid sgRNA number correction in data preprocessing, sgRNA ranking and gene ranking. Furthermore, a powerful web server has been presented to automatically complete the whole CRISPR/Cas9 screening analysis, where we integrated the main hypothesis in multiple algorithms as a full workflow, including quality control, sgRNA extracting, sgRNA alignment, sgRNA ranking, gene ranking and pathway enrichment. In addition, output of iCRISEE, including result mapping, sample clustering, sgRNA ranking and gene ranking, can be easily visualized and downloaded for publication. Taking together, iCRISEE presents to be the state-of-the-art and user-friendly tool for CRISPR screening data analysis. iCRISEE is available at https://www.icrisee.com.},
}
@article {pmid34905700,
year = {2021},
author = {Zhou, Q and Wang, K and Qiu, J and Zhu, D and Tian, T and Zhang, Y and Qin, X},
title = {Comparative transcriptome analysis and CRISPR/Cas9 gene editing reveal that E4BP4 mediates lithium upregulation of Per2 expression.},
journal = {Open biology},
volume = {11},
number = {12},
pages = {210140},
pmid = {34905700},
issn = {2046-2441},
mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/*genetics ; Bipolar Disorder/drug therapy/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cycloheximide/pharmacology ; Gene Editing ; Gene Expression Profiling/*methods ; Gene Expression Regulation/drug effects ; Humans ; Lithium/*administration &amp; dosage/pharmacology ; Mice ; Mutagenesis, Site-Directed ; NIH 3T3 Cells ; Period Circadian Proteins/*genetics ; Promoter Regions, Genetic ; Sequence Analysis, RNA ; Up-Regulation ; },
abstract = {Bipolar disorder (BPD) is a psychiatric disorder characterized by alternate episodes of mania and depression. Disruption of normal circadian clock and abnormal sleep cycles are common symptoms of BPD patients. Lithium salt is currently an effective clinical therapeutic drug for BPD. Animal and cellular studies have found that lithium salt can upregulate the expression of the clock gene Per2, but the mechanism is unknown. We aim to understand the mechanism underlying the Per2 upregulation by lithium treatment. By taking approaches of both comparative transcriptome analysis and comparative qPCR analysis between human and murine cells, Lumicycle assay, luciferase assay and RT-qPCR assay showed that lithium could significantly upregulate the expression of Per2 in both mouse and human cells, and significantly inhibit the expression of E4bp4, which encodes a transcriptional inhibitor of Per2. After knocking out the cis-element upstream on the Per2 promoter that responds to E4BP4, the upregulation effect on Per2 by lithium disappeared. When E4bp4 gene was knocked out, the upregulation effect on Per2 by lithium salt disappeared. This study has found that lithium upregulates Per2 expression by reducing the expression of transcription factor E4BP4, but the mechanism of lithium salt downregulation of E4BP4 remains to be further studied. Our study provides a new therapeutic target and approaches for treating BPD.},
}
@article {pmid34905186,
year = {2022},
author = {Xu, K and Li, Y},
title = {Developing Rice Mutants Using CRISPR/Cas9-Based Genome Editing Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2400},
number = {},
pages = {11-19},
pmid = {34905186},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; *Oryza/genetics ; RNA, Guide/genetics ; Technology ; },
abstract = {Genome editing provides advanced techniques that enable the precise and efficient targeted modification of an organism's genome. It is a powerful tool for understanding gene functions and developing valuable new traits in crops such as rice. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system has recently emerged as an alternative nuclease-based method for efficient and versatile genome editing. In the CRISPR/Cas9 system, only 20 nt within the single guide RNA (sgRNA) needs to be changed. The convenience of operating and few limitations on target loci make the CRISPR/Cas9 system an ideal tool in our research. Here, we describe a detailed protocol of the CRISPR/Cas9 system to generate rice mutants used in our recent gene function studies.},
}
@article {pmid34904653,
year = {2022},
author = {Sharifi, F and Ye, Y},
title = {Identification and classification of reverse transcriptases in bacterial genomes and metagenomes.},
journal = {Nucleic acids research},
volume = {50},
number = {5},
pages = {e29},
pmid = {34904653},
issn = {1362-4962},
support = {R01 AI143254/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophages/genetics ; *Genome, Bacterial ; Humans ; *Metagenome ; *RNA-Directed DNA Polymerase/metabolism ; Retroelements/genetics ; },
abstract = {Reverse transcriptases (RTs) are found in different systems including group II introns, Diversity Generating Retroelements (DGRs), retrons, CRISPR-Cas systems, and Abortive Infection (Abi) systems in prokaryotes. Different classes of RTs can play different roles, such as template switching and mobility in group II introns, spacer acquisition in CRISPR-Cas systems, mutagenic retrohoming in DGRs, programmed cell suicide in Abi systems, and recently discovered phage defense in retrons. While some classes of RTs have been studied extensively, others remain to be characterized. There is a lack of computational tools for identifying and characterizing various classes of RTs. In this study, we built a tool (called myRT) for identification and classification of prokaryotic RTs. In addition, our tool provides information about the genomic neighborhood of each RT, providing potential functional clues. We applied our tool to predict RTs in all complete and draft bacterial genomes, and created a collection that can be used for exploration of putative RTs and their associated protein domains. Application of myRT to metagenomes showed that gut metagenomes encode proportionally more RTs related to DGRs, outnumbering retron-related RTs, as compared to the collection of reference genomes. MyRT is both available as a standalone software (https://github.com/mgtools/myRT) and also through a website (https://omics.informatics.indiana.edu/myRT/).},
}
@article {pmid34904600,
year = {2022},
author = {Kim, HJ and Park, JM and Lee, S and Cho, HB and Park, JI and Kim, JH and Park, JS and Park, KH},
title = {Efficient CRISPR-Cas9-based knockdown of RUNX2 to induce chondrogenic differentiation of stem cells.},
journal = {Biomaterials science},
volume = {10},
number = {2},
pages = {514-523},
doi = {10.1039/d1bm01716k},
pmid = {34904600},
issn = {2047-4849},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Core Binding Factor Alpha 1 Subunit/genetics ; Humans ; *Osteogenesis/genetics ; Stem Cells ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system recognizes and deletes specific nucleotide sequences in cells for gene editing. This study aimed to edit and knockdown the RUNX2 gene, a key transcription factor that is directly involved in all stages of stem cell differentiation into osteoblasts. The RUNX2 gene was depleted using the CRISPR-Cas9 system to inhibit osteoblast differentiation of stem cells. shRNA vectors targeting RUNX2 were used as a control. The surface of nanoparticles (NPs) was coated with the cationic polymer linear polyethyleneimine. Thereafter, negatively charged CRISPR-Cas9 and shRNA vectors were complexed with positively charged NPs via ionic interactions. Several analytical methods were used to determine the size, surface charge, and morphology of NPs and to characterize the complexed genes. NPs complexed with CRISPR-Cas9 and shRNA vectors were delivered into human mesenchymal stem cells (hMSCs) via endocytosis. The mRNA and protein expression patterns of various genes in hMSCs were measured over time following internalization of NPs complexed with CRISPR-Cas9 and shRNA vectors in two- and three-dimensional culture systems. Knockdown of the RUNX2 gene decreased osteogenic differentiation and increased chondrogenic differentiation of hMSCs. As a result of investigating the efficiency of NPs complexed with CRISPR-Cas9 (CASP-NPs), Runx2 effectively knocked down in mesenchymal stem cells to enhance differentiation into chondrocytes, therefore CASP-NPs proved to be an effective gene carrier in hMSCs.},
}
@article {pmid34904260,
year = {2022},
author = {Devi, V and Harjai, K and Chhibber, S},
title = {Self-targeting spacers in CRISPR-array: Accidental occurrence or evolutionarily conserved phenomenon.},
journal = {Journal of basic microbiology},
volume = {62},
number = {1},
pages = {4-12},
doi = {10.1002/jobm.202100514},
pmid = {34904260},
issn = {1521-4028},
mesh = {Accidents ; *Bacteriophages ; *CRISPR-Cas Systems/genetics ; },
abstract = {In recent years, a tremendous amount of inquisitiveness among scientists in the clustered regularly interspaced short palindrome repeats (CRISPR)-CRISPR-associated proteins (Cas) has led to many studies to delineate their exact role in prokaryotes. CRISPR-Cas is an adaptive immune system that protects prokaryotes from phages and mobile genetic elements. It incorporates small DNA fragment of the invader in the CRISPR-array and protects the host from future invasion by them. In a few instances, the CRISPR-array also incorporates self-targeting spacers, most likely by accident or leaky incorporation. A significant number of spacers are found to match with the host genes across the species; however, self-targeting spacers have not been investigated in detail in most of the organisms. The presence of self-targeting spacers in the CRISPR-array led to speculation that the CRISPR-Cas system has a lot more to offer than just being the conventional adaptive immune system. It has been implicated in gene regulation and autoimmunity more or less equally. In this review, an attempt has been made to understand self-targeting spacers in the context of gene regulation, autoimmunity, and its avoidance strategies.},
}
@article {pmid34903813,
year = {2021},
author = {Hirata, M and Wittayarat, M and Namula, Z and Le, QA and Lin, Q and Takebayashi, K and Thongkittidilok, C and Mito, T and Tomonari, S and Tanihara, F and Otoi, T},
title = {Generation of mutant pigs by lipofection-mediated genome editing in embryos.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23806},
pmid = {34903813},
issn = {2045-2322},
mesh = {Animals ; Blastocyst/drug effects/metabolism ; CRISPR-Cas Systems ; Gene Editing/*methods/veterinary ; Lipids/pharmacology ; *Mutation ; Myostatin/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; Swine/*genetics ; Transfection/*methods/veterinary ; },
abstract = {The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.},
}
@article {pmid34903767,
year = {2021},
author = {Brun, NR and Salanga, MC and Mora-Zamorano, FX and Lamb, DC and Goldstone, JV and Stegeman, JJ},
title = {Orphan cytochrome P450 20a1 CRISPR/Cas9 mutants and neurobehavioral phenotypes in zebrafish.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23892},
pmid = {34903767},
issn = {2045-2322},
support = {P01 ES028938/ES/NIEHS NIH HHS/United States ; R01 ES029917/ES/NIEHS NIH HHS/United States ; P01 ES021923/ES/NIEHS NIH HHS/United States ; P42 ES007381/ES/NIEHS NIH HHS/United States ; },
mesh = {Adaptation, Physiological ; Animals ; Anxiety/*genetics ; CRISPR-Cas Systems ; *Cytochrome P-450 Enzyme System/genetics/physiology ; Homozygote ; Loss of Function Mutation ; *Movement ; *Visual Perception ; *Zebrafish/genetics/physiology ; *Zebrafish Proteins/genetics/physiology ; },
abstract = {Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish cyp20a1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.},
}
@article {pmid34903756,
year = {2021},
author = {Fukushima, T and Tanaka, Y and Adachi, K and Masuyama, N and Tsuchiya, A and Asada, S and Ishiguro, S and Mori, H and Seki, M and Yachie, N and Goyama, S and Kitamura, T},
title = {CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23889},
pmid = {34903756},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cytosine Deaminase/genetics/metabolism ; *DNA Repair ; *Gene Editing ; HEK293 Cells ; Humans ; *Mutation ; RNA, Guide/*genetics ; TATA Box/genetics ; Thymidine/genetics ; },
abstract = {Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.},
}
@article {pmid34903170,
year = {2021},
author = {Xiao, LM and Wan, YQ and Jiang, ZR},
title = {AttCRISPR: a spacetime interpretable model for prediction of sgRNA on-target activity.},
journal = {BMC bioinformatics},
volume = {22},
number = {1},
pages = {589},
pmid = {34903170},
issn = {1471-2105},
mesh = {CRISPR-Cas Systems/genetics ; *Machine Learning ; *RNA, Guide ; },
abstract = {BACKGROUND: More and more Cas9 variants with higher specificity are developed to avoid the off-target effect, which brings a significant volume of experimental data. Conventional machine learning performs poorly on these datasets, while the methods based on deep learning often lack interpretability, which makes researchers have to trade-off accuracy and interpretability. It is necessary to develop a method that can not only match deep learning-based methods in performance but also with good interpretability that can be comparable to conventional machine learning methods.<br><br>RESULTS: To overcome these problems, we propose an intrinsically interpretable method called AttCRISPR based on deep learning to predict the on-target activity. The advantage of AttCRISPR lies in using the ensemble learning strategy to stack available encoding-based methods and embedding-based methods with strong interpretability. Comparison with the state-of-the-art methods using WT-SpCas9, eSpCas9(1.1), SpCas9-HF1 datasets, AttCRISPR can achieve an average Spearman value of 0.872, 0.867, 0.867, respectively on several public datasets, which is superior to these methods. Furthermore, benefits from two attention modules-one spatial and one temporal, AttCRISPR has good interpretability. Through these modules, we can understand the decisions made by AttCRISPR at both global and local levels without other post hoc explanations techniques.<br><br>CONCLUSION: With the trained models, we reveal the preference for each position-dependent nucleotide on the sgRNA (short guide RNA) sequence in each dataset at a global level. And at a local level, we prove that the interpretability of AttCRISPR can be used to guide the researchers to design sgRNA with higher activity.},
}
@article {pmid34903162,
year = {2021},
author = {Kolesnik, MV and Fedorova, I and Karneyeva, KA and Artamonova, DN and Severinov, KV},
title = {Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {10},
pages = {1301-1314},
pmid = {34903162},
issn = {1608-3040},
mesh = {*Biological Evolution ; CRISPR-Cas Systems/genetics/*immunology ; Gene Editing/*methods ; Immune System/metabolism ; Prokaryotic Cells/*immunology/metabolism ; Signal Transduction ; },
abstract = {The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit - the so-called CRISPR polymerase - which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.},
}
@article {pmid34902746,
year = {2022},
author = {Wang, L and Zhou, L and Li, M and Zhao, J and Liu, Y and Chen, Y and Qin, X and Wang, S and Chen, H and Piao, Y and Xiang, R and Li, J and Shi, Y},
title = {Genome-wide CRISPR/Cas9 knockout screening uncovers ZNF319 as a novel tumor suppressor critical for breast cancer metastasis.},
journal = {Biochemical and biophysical research communications},
volume = {589},
number = {},
pages = {107-115},
doi = {10.1016/j.bbrc.2021.12.023},
pmid = {34902746},
issn = {1090-2104},
mesh = {Animals ; Breast Neoplasms/*genetics/*pathology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Female ; G2 Phase ; *Gene Knockout Techniques ; *Genes, Tumor Suppressor ; *Genome, Human ; Humans ; Mice ; Mitosis ; Neoplasm Metastasis ; },
abstract = {Breast cancer is prone to relapse and metastasize to many vital organs, contributing to most of the breast cancer-related death and accentuating the importance of systematic identification of key factors regulating the metastasis of breast cancer. In this study, we performed a genome-wide CRISPR/Cas9 knock out screen in an orthotopic murine model of breast cancer for essential genes monitoring the progression and metastasis of breast cancer. We found one member of the zinc finger protein (ZNF) family, i.e., ZNF319, was among the top candidate genes. We further confirmed the lower expression of ZNF319 in the tumor tissue of breast cancer patients by analyzing tissue sections with IHC staining and TCGA database. Consistently, higher expression of ZNF319 correlates with better clinical outcome in almost all subtypes of breast cancer. Moreover, knocking down or overexpressing ZNF319 in breast cancer cells dramatically affects the breast cancer growth and metastasis capacity both in vitro and in vivo, suggesting ZNF319 functions as a strong suppressor of breast cancer progression. Lastly, the transcriptome analysis on ZNF319-silenced breast cancer cells shows that ZNF319 is involved in multiple crucial signaling pathways and biological processes, especially in cell cycle and proliferation. GO and KEGG analyses of our RNA-seq results reveal the up-regulation of E2F and G2/M related genes in ZNF319-silenced cells, suggesting that ZNF319 monitors the cell cycle during the breast cancer progression through the regulation of the E2F target genes and G2/M checkpoint. In summary, our study identifies ZNF319 as a novel metastasis suppressor gene arresting tumor cell cycle in breast cancer and thus presents a novel potential therapeutic target for breast cancer treatment.},
}
@article {pmid34901007,
year = {2021},
author = {Javaid, N and Choi, S},
title = {CRISPR/Cas System and Factors Affecting Its Precision and Efficiency.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {761709},
pmid = {34901007},
issn = {2296-634X},
abstract = {The diverse applications of genetically modified cells and organisms require more precise and efficient genome-editing tool such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas). The CRISPR/Cas system was originally discovered in bacteria as a part of adaptive-immune system with multiple types. Its engineered versions involve multiple host DNA-repair pathways in order to perform genome editing in host cells. However, it is still challenging to get maximum genome-editing efficiency with fewer or no off-targets. Here, we focused on factors affecting the genome-editing efficiency and precision of CRISPR/Cas system along with its defense-mechanism, orthologues, and applications.},
}
@article {pmid34900150,
year = {2021},
author = {Hassani, M and Hesami, S and Maroofi, N and Banan, M},
title = {Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs.},
journal = {Avicenna journal of medical biotechnology},
volume = {13},
number = {4},
pages = {226-229},
pmid = {34900150},
issn = {2008-2835},
abstract = {BACKGROUND: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously.<br><br>METHODS: In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping.<br><br>RESULTS: This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids.<br><br>CONCLUSION: Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.},
}
@article {pmid34900128,
year = {2021},
author = {Li, X and Wang, C and Peng, T and Chai, Z and Ni, D and Liu, Y and Zhang, J and Chen, T and Lu, S},
title = {Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6.},
journal = {Computational and structural biotechnology journal},
volume = {19},
number = {},
pages = {6108-6124},
pmid = {34900128},
issn = {2001-0370},
abstract = {CRISPR-Cas systems are prokaryotic adaptive immunity against invading phages and plasmids. Phages have evolved diverse protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, to neutralize this CRISPR machinery. In response, bacteria have co-evolved Cas variants to escape phage's anti-CRISPR strategies, called anti-anti-CRISPR systems. Here we explore the anti-CRISPR allosteric inhibition and anti-anti-CRISPR rescue mechanisms between Streptococcus thermophilus Cas9 (St1Cas9) and the anti-CRISPR protein AcrIIA6 at the atomic level, by generating mutants of key residues in St1Cas9. Extensive unbiased molecular dynamics simulations show that the functional motions of St1Cas9 in the presence of AcrIIA6 differ substantially from those of St1Cas9 alone. AcrIIA6 binding triggers a shift of St1Cas9 conformational ensemble towards a less catalytically competent state; this state significantly compromises protospacer adjacent motif (PAM) recognition and nuclease activity by altering interdependently conformational dynamics and allosteric signals among nuclease domains, PAM-interacting (PI) regions, and AcrIIA6 binding motifs. Via in vitro DNA cleavage assays, we further elucidate the rescue mechanism of efficiently escaping AcrIIA6 inhibition harboring St1Cas9 triple mutations (G993K/K1008M/K1010E) in the PI domain and identify the evolutionary landscape of such mutational escape within species. Our results provide mechanistic insights into Acr proteins as natural brakes for the CRISPR-Cas systems and a promising potential for the design of allosteric Acr peptidomimetics.},
}
@article {pmid34899631,
year = {2021},
author = {Bayoumi, M and Munir, M},
title = {Potential Use of CRISPR/Cas13 Machinery in Understanding Virus-Host Interaction.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {743580},
pmid = {34899631},
issn = {1664-302X},
abstract = {Prokaryotes have evolutionarily acquired an immune system to fend off invading mobile genetic elements, including viral phages and plasmids. Through recognizing specific sequences of the invading nucleic acid, prokaryotes mediate a subsequent degradation process collectively referred to as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system. The CRISPR-Cas systems are divided into two main classes depending on the structure of the effector Cas proteins. Class I systems have effector modules consisting of multiple proteins, while class II systems have a single multidomain effector. Additionally, the CRISPR-Cas systems can also be categorized into types depending on the spacer acquisition components and their evolutionary features, namely, types I-VI. Among CRISPR/Cas systems, Cas9 is one of the most common multidomain nucleases that identify, degrade, and modulate DNA. Importantly, variants of Cas proteins have recently been found to target RNA, especially the single-effector Cas13 nucleases. The Cas13 has revolutionized our ability to study and perturb RNAs in endogenous microenvironments. The Cas13 effectors offer an excellent candidate for developing novel research tools in virological and biotechnological fields. Herein, in this review, we aim to provide a comprehensive summary of the recent advances of Cas13s for targeting viral RNA for either RNA-mediated degradation or CRISPR-Cas13-based diagnostics. Additionally, we aim to provide an overview of the proposed applications that could revolutionize our understanding of viral-host interactions using Cas13-mediated approaches.},
}
@article {pmid34898428,
year = {2021},
author = {Reint, G and Li, Z and Labun, K and Keskitalo, S and Soppa, I and Mamia, K and Tolo, E and Szymanska, M and Meza-Zepeda, LA and Lorenz, S and Cieslar-Pobuda, A and Hu, X and Bordin, DL and Staerk, J and Valen, E and Schmierer, B and Varjosalo, M and Taipale, J and Haapaniemi, E},
title = {Rapid genome editing by CRISPR-Cas9-POLD3 fusion.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34898428},
issn = {2050-084X},
mesh = {CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured/*physiology ; DNA Polymerase III/*genetics/*metabolism ; DNA Repair/genetics/physiology ; Gene Editing/*methods ; Humans ; },
abstract = {Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.},
}
@article {pmid34896246,
year = {2022},
author = {Eini, O and Schumann, N and Niessen, M and Varrelmann, M},
title = {Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components.},
journal = {New biotechnology},
volume = {67},
number = {},
pages = {1-11},
doi = {10.1016/j.nbt.2021.12.002},
pmid = {34896246},
issn = {1876-4347},
mesh = {*CRISPR-Cas Systems/genetics ; *Geminiviridae/genetics ; Gene Editing ; Mutagenesis ; },
abstract = {Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons.},
}
@article {pmid34895514,
year = {2022},
author = {Yan, Z and Lynch, TJ and Engelhardt, JF},
title = {AAV-mediated gene editing lights up the lung.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {7-9},
pmid = {34895514},
issn = {1525-0024},
support = {K99 HL155843/HL/NHLBI NIH HHS/United States ; P30 DK054759/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Dependovirus/genetics ; *Gene Editing ; Lung ; },
}
@article {pmid34895501,
year = {2022},
author = {Lagor, WR},
title = {Anti-Cas9 immunity: A formidable challenge for muscle genome editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {10-12},
pmid = {34895501},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Muscles ; RNA, Guide ; },
}
@article {pmid34894625,
year = {2022},
author = {He, C and Lin, C and Mo, G and Xi, B and Li, AA and Huang, D and Wan, Y and Chen, F and Liang, Y and Zuo, Q and Xu, W and Feng, D and Zhang, G and Han, L and Ke, C and Du, H and Huang, L},
title = {Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {198},
number = {},
pages = {113857},
pmid = {34894625},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Humans ; Mutation ; SARS-CoV-2 ; },
abstract = {The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10[-17] M (approximately 6 copies/μL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10[-16] M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.},
}
@article {pmid34894597,
year = {2022},
author = {Tang, N and Ning, Q and Wang, Z and Tao, Y and Zhao, X and Tang, S},
title = {Tumor microenvironment based stimuli-responsive CRISPR/Cas delivery systems: A viable platform for interventional approaches.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {210},
number = {},
pages = {112257},
doi = {10.1016/j.colsurfb.2021.112257},
pmid = {34894597},
issn = {1873-4367},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; Tumor Microenvironment ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have emerged as robust tools in cancer gene therapy due to their simplicity and versatility. Nevertheless, the genome editing efficiency in tumor sites and the clinical applications of CRISPR/Cas have been compromised by non-specific delivery and genotoxicity. Recently, intelligent delivery systems incorporating sensitive materials in response to endogenous stimuli of the tumor microenvironment (TME) have represented viable platforms for tumor-specific genome editing and reduced side effects of CRISPR/Cas. Spurred by this promising direction, this review first introduces the CRISPR/Cas systems widely employed in cancer therapeutic explorations. Various types of CRISPR/Cas delivery systems sensitive to the stimuli in TME and typical dual-/multiple-responsive CRISPR/Cas carriers are further discussed, emphasizing the correlations between sensitive components and spatiotemporal delivery mechanisms. The genome editing efficiencies of CRISPR/Cas-loaded stimuli-responsive carriers are also summarized both in vitro and in vivo. Collectively, stimuli-responsive CRISPR/Cas delivery systems hold great promise for potent cancer gene therapy.},
}
@article {pmid34893907,
year = {2022},
author = {Gehrke, F and Schindele, A and Puchta, H},
title = {Nonhomologous end joining as key to CRISPR/Cas-mediated plant chromosome engineering.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1769-1779},
pmid = {34893907},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Chromosomes, Plant/genetics ; DNA End-Joining Repair/genetics ; Gene Editing ; Plant Breeding ; },
abstract = {Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated gene editing has revolutionized biology and plant breeding, large-scale, heritable restructuring of plant chromosomes is still in its infancy. Duplications and inversions within a chromosome, and also translocations between chromosomes, can now be achieved. Subsequently, genetic linkages can be broken or can be newly created. Also, the order of genes on a chromosome can be changed. While natural chromosomal recombination occurs by homologous recombination during meiosis, CRISPR/Cas-mediated chromosomal rearrangements can be obtained best by harnessing nonhomologous end joining (NHEJ) pathways in somatic cells. NHEJ can be subdivided into the classical (cNHEJ) and alternative NHEJ (aNHEJ) pathways, which partially operate antagonistically. The cNHEJ pathway not only protects broken DNA ends from degradation but also suppresses the joining of previously unlinked broken ends. Hence, in the absence of cNHEJ, more inversions or translocations can be obtained which can be ascribed to the unrestricted use of the aNHEJ pathway for double-strand break (DSB) repair. In contrast to inversions or translocations, short tandem duplications can be produced by paired single-strand breaks via a Cas9 nickase. Interestingly, the cNHEJ pathway is essential for these kinds of duplications, whereas aNHEJ is required for patch insertions that can also be formed during DSB repair. As chromosome engineering has not only been accomplished in the model plant Arabidopsis (Arabidopsis thaliana) but also in the crop maize (Zea mays), we expect that this technology will soon transform the breeding process.},
}
@article {pmid34893900,
year = {2022},
author = {Liu, JL and Chen, MM and Chen, WQ and Liu, CM and He, Y and Song, XF},
title = {A CASE toolkit for easy and efficient multiplex transgene-free gene editing.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1843-1847},
pmid = {34893900},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Transgenes ; },
abstract = {An integrated transgene-free multiplex gene-editing toolkit based on the Transgene Killer CRISPR technology greatly saves labor, time, and cost.},
}
@article {pmid34893878,
year = {2022},
author = {Zhang, X and Garrett, S and Graveley, BR and Terns, MP},
title = {Unique properties of spacer acquisition by the type III-A CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {50},
number = {3},
pages = {1562-1582},
pmid = {34893878},
issn = {1362-4962},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/metabolism ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; Plasmids ; Streptococcus thermophilus/*genetics/metabolism/*virology ; },
abstract = {Type III CRISPR-Cas systems have a unique mode of interference, involving crRNA-guided recognition of nascent RNA and leading to DNA and RNA degradation. How type III systems acquire new CRISPR spacers is currently not well understood. Here, we characterize CRISPR spacer uptake by a type III-A system within its native host, Streptococcus thermophilus. Adaptation by the type II-A system in the same host provided a basis for comparison. Cas1 and Cas2 proteins were critical for type III adaptation but deletion of genes responsible for crRNA biogenesis or interference did not detectably change spacer uptake patterns, except those related to host counter-selection. Unlike the type II-A system, type III spacers are acquired in a PAM- and orientation-independent manner. Interestingly, certain regions of plasmids and the host genome were particularly well-sampled during type III-A, but not type II-A, spacer uptake. These regions included the single-stranded origins of rolling-circle replicating plasmids, rRNA and tRNA encoding gene clusters, promoter regions of expressed genes and 5' UTR regions involved in transcription attenuation. These features share the potential to form DNA secondary structures, suggesting a preferred substrate for type III adaptation. Lastly, the type III-A system adapted to and protected host cells from lytic phage infection.},
}
@article {pmid34893860,
year = {2022},
author = {Wang, X and Li, X and Ma, Y and He, J and Liu, X and Yu, G and Yin, H and Zhang, H},
title = {Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18.},
journal = {Nucleic acids research},
volume = {50},
number = {1},
pages = {512-521},
pmid = {34893860},
issn = {1362-4962},
mesh = {Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; RNA, Guide/*metabolism ; Viral Proteins/*metabolism ; },
abstract = {Mobile genetic elements such as phages and plasmids have evolved anti-CRISPR proteins (Acrs) to suppress CRISPR-Cas adaptive immune systems. Recently, several phage and non-phage derived Acrs including AcrIIA17 and AcrIIA18 have been reported to inhibit Cas9 through modulation of sgRNA. Here, we show that AcrIIA17 and AcrIIA18 inactivate Cas9 through distinct mechanisms. AcrIIA17 inhibits Cas9 activity through interference with Cas9-sgRNA binary complex formation. In contrast, AcrIIA18 induces the truncation of sgRNA in a Cas9-dependent manner, generating a shortened sgRNA incapable of triggering Cas9 activity. The crystal structure of AcrIIA18, combined with mutagenesis studies, reveals a crucial role of the N-terminal β-hairpin in AcrIIA18 for sgRNA cleavage. The enzymatic inhibition mechanism of AcrIIA18 is different from those of the other reported type II Acrs. Our results add new insights into the mechanistic understanding of CRISPR-Cas9 inhibition by Acrs, and also provide valuable information in the designs of tools for conditional manipulation of CRISPR-Cas9.},
}
@article {pmid34893621,
year = {2021},
author = {Santana, DJ and O'Meara, TR},
title = {Forward and reverse genetic dissection of morphogenesis identifies filament-competent Candida auris strains.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {7197},
pmid = {34893621},
issn = {2041-1723},
support = {K22 AI137299/AI/NIAID NIH HHS/United States ; T32 AI007528/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antifungal Agents/pharmacology ; CRISPR-Cas Systems ; Candida auris/*cytology/drug effects/*genetics/*physiology ; Candidiasis/microbiology ; Disease Models, Animal ; Drug Resistance, Fungal/drug effects ; Fluconazole ; Fungal Proteins/*genetics ; Gene Expression Regulation, Fungal ; Morphogenesis/drug effects/*genetics ; Moths ; Mutation ; Protein Kinases/genetics ; *Reverse Genetics ; Virulence ; },
abstract = {Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome.},
}
@article {pmid34893609,
year = {2021},
author = {Chen, Y and Liu, Z and Régnière, J and Vasseur, L and Lin, J and Huang, S and Ke, F and Chen, S and Li, J and Huang, J and Gurr, GM and You, M and You, S},
title = {Large-scale genome-wide study reveals climate adaptive variability in a cosmopolitan pest.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {7206},
pmid = {34893609},
issn = {2041-1723},
mesh = {*Adaptation, Physiological ; Animals ; CRISPR-Cas Systems ; *Climate ; Climate Change ; Evolution, Molecular ; Gene Editing ; Genome-Wide Association Study/*methods ; Genomics ; Greenhouse Gases ; Moths/genetics/*physiology ; Survival Rate ; Temperature ; },
abstract = {Understanding the genetic basis of climatic adaptation is essential for predicting species' responses to climate change. However, intraspecific variation of these responses arising from local adaptation remains ambiguous for most species. Here, we analyze genomic data from diamondback moth (Plutella xylostella) collected from 75 sites spanning six continents to reveal that climate-associated adaptive variation exhibits a roughly latitudinal pattern. By developing an eco-genetic index that combines genetic variation and physiological responses, we predict that most P. xylostella populations have high tolerance to projected future climates. Using genome editing, a key gene, PxCad, emerged from our analysis as functionally temperature responsive. Our results demonstrate that P. xylostella is largely capable of tolerating future climates in most of the world and will remain a global pest beyond 2050. This work improves our understanding of adaptive variation along environmental gradients, and advances pest forecasting by highlighting the genetic basis for local climate adaptation.},
}
@article {pmid34893590,
year = {2021},
author = {Gamez, S and Chaverra-Rodriguez, D and Buchman, A and Kandul, NP and Mendez-Sanchez, SC and Bennett, JB and Sánchez C, HM and Yang, T and Antoshechkin, I and Duque, JE and Papathanos, PA and Marshall, JM and Akbari, OS},
title = {Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {7202},
pmid = {34893590},
issn = {2041-1723},
support = {R01 AI151004/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Drosophila melanogaster/genetics ; Endonucleases/genetics ; Female ; Gene Drive Technology/*methods ; Gene Editing/methods ; *Genes, Y-Linked ; Male ; Sex Preselection/*methods ; Sex Ratio ; Synthetic Biology/methods ; Transgenes ; *Y Chromosome ; },
abstract = {CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.},
}
@article {pmid34893527,
year = {2022},
author = {Sun, W and Jia, X and Liesa, M and Tantin, D and Ward, DM},
title = {ABCB10 Loss Reduces CD4[+] T Cell Activation and Memory Formation.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {208},
number = {2},
pages = {328-337},
pmid = {34893527},
issn = {1550-6606},
support = {R01 AA026914/AA/NIAAA NIH HHS/United States ; R01 AI100873/AI/NIAID NIH HHS/United States ; U54 DK110858/DK/NIDDK NIH HHS/United States ; },
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CD4-Positive T-Lymphocytes/cytology/*immunology ; CD8-Positive T-Lymphocytes/cytology/immunology ; CRISPR-Cas Systems/genetics ; Cell Line ; Cytokines/*biosynthesis/immunology ; Glycolysis/physiology ; Humans ; Immunologic Memory/genetics/*immunology ; Jurkat Cells ; Lymphocyte Activation/genetics/immunology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; },
abstract = {T cells must shift their metabolism to respond to infections and tumors and to undergo memory formation. The ATP-binding cassette transporter ABCB10 localizes to the mitochondrial inner membrane, where it is thought to export a substrate important in heme biosynthesis and metabolism, but its role in T cell development and activation is unknown. In this article, we use a combination of methods to study the effect of ABCB10 loss in primary and malignantly transformed T cells. Although Abcb10 is dispensable for development of both CD4[+] and CD8[+] T cells, it is required for expression of specific cytokines in CD4[+], but not CD8[+], T cells activated in vitro. These defects in cytokine expression are magnified on repeated stimulation. In vivo, CD8[+] cells lacking ABCB10 expand more in response to viral infection than their control counterparts, while CD4[+] cells show reductions in both number and percentage. CD4[+] cells lacking ABCB10 show impairment in Ag-specific memory formation and recall responses that become more severe with time. In malignant human CD4[+] Jurkat T cells, we find that CRISPR-mediated ABCB10 disruption recapitulates the same cytokine expression defects upon activation as observed in primary mouse T cells. Mechanistically, ABCB10 deletion in Jurkat T cells disrupts the ability to switch to aerobic glycolysis upon activation. Cumulatively, these results show that ABCB10 is selectively required for specific cytokine responses and memory formation in CD4[+] T cells, suggesting that targeting this molecule could be used to mitigate aberrant T cell activation.},
}
@article {pmid34890975,
year = {2022},
author = {Hu, M and Lei, XY and Larson, JD and McAlonis, M and Ford, K and McDonald, D and Mach, K and Rusert, JM and Wechsler-Reya, RJ and Mali, P},
title = {Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures.},
journal = {Biomaterials},
volume = {280},
number = {},
pages = {121276},
pmid = {34890975},
issn = {1878-5905},
support = {R01 CA159859/CA/NCI NIH HHS/United States ; R01 GM123313/GM/NIGMS NIH HHS/United States ; P30 CA030199/CA/NCI NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; R01 HG009285/HG/NHGRI NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Early Detection of Cancer ; Genome ; Humans ; *Neoplasms/genetics ; *Tissue Engineering ; },
abstract = {Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.},
}
@article {pmid34890109,
year = {2022},
author = {Zhou, Y and Xu, S and Jiang, N and Zhao, X and Bai, Z and Liu, J and Yao, W and Tang, Q and Xiao, G and Lv, C and Wang, K and Hu, X and Tan, J and Yang, Y},
title = {Engineering of rice varieties with enhanced resistances to both blast and bacterial blight diseases via CRISPR/Cas9.},
journal = {Plant biotechnology journal},
volume = {20},
number = {5},
pages = {876-885},
pmid = {34890109},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; Gene Editing ; *Oryza/genetics/microbiology ; Plant Diseases/genetics/microbiology ; },
abstract = {Rice blast and bacterial blight represent two of major diseases having devastating impact on the yield of rice in most rice-growing countries. Developments of resistant cultivars are the most economic and effective strategy to control these diseases. Here, we used CRISPR/Cas9-mediated gene editing to rapidly install mutations in three known broad-spectrum blast-resistant genes, Bsr-d1, Pi21 and ERF922, in an indica thermosensitive genic male sterile (TGMS) rice line Longke638S (LK638S). We obtained transgene-free homozygous single or triple mutants in T1 generations. While all single and triple mutants showed increased resistance to rice blast compared with wild type, the erf922 mutants displayed the strongest blast resistance similar with triple mutants. Surprisingly, we found that Pi21 or ERF922 single mutants conferred enhanced resistance to most of tested bacterial blight. Both resistances in mutants were attribute to the up-regulation of SA- and JA-pathway associated genes. Moreover, phenotypic analysis of these single mutants in paddy fields revealed that there were no trade-offs between resistances and main agricultural traits. Together, our study provides a rapid and effective way to generate rice varieties with resistance to both rice blast and bacterial blight.},
}
@article {pmid34889892,
year = {2021},
author = {Rasheed, A and Gill, RA and Hassan, MU and Mahmood, A and Qari, S and Zaman, QU and Ilyas, M and Aamer, M and Batool, M and Li, H and Wu, Z},
title = {A Critical Review: Recent Advancements in the Use of CRISPR/Cas9 Technology to Enhance Crops and Alleviate Global Food Crises.},
journal = {Current issues in molecular biology},
volume = {43},
number = {3},
pages = {1950-1976},
pmid = {34889892},
issn = {1467-3045},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Disease Resistance/genetics ; Food Quality ; Food Supply ; *Gene Editing ; Genetic Engineering ; Genome, Plant ; Genomics/methods ; *Plant Breeding ; Plants, Genetically Modified ; },
abstract = {Genome editing (GE) has revolutionized the biological sciences by creating a novel approach for manipulating the genomes of living organisms. Many tools have been developed in recent years to enable the editing of complex genomes. Therefore, a reliable and rapid approach for increasing yield and tolerance to various environmental stresses is necessary to sustain agricultural crop production for global food security. This critical review elaborates the GE tools used for crop improvement. These tools include mega-nucleases (MNs), such as zinc-finger nucleases (ZFNs), and transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, this review addresses the latest advancements in the role of CRISPR/Cas9 for genome manipulation for major crop improvement, including yield and quality development of biotic stress- and abiotic stress-tolerant crops. Implementation of this technique will lead to the production of non-transgene crops with preferred characteristics that can result in enhanced yield capacity under various environmental stresses. The CRISPR/Cas9 technique can be combined with current and potential breeding methods (e.g., speed breeding and omics-assisted breeding) to enhance agricultural productivity to ensure food security. We have also discussed the challenges and limitations of CRISPR/Cas9. This information will be useful to plant breeders and researchers in the thorough investigation of the use of CRISPR/Cas9 to boost crops by targeting the gene of interest.},
}
@article {pmid34889094,
year = {2021},
author = {Huang, D and Ni, D and Fang, M and Shi, Z and Xu, Z},
title = {Microfluidic Ruler-Readout and CRISPR Cas12a-Responded Hydrogel-Integrated Paper-Based Analytical Devices (μReaCH-PAD) for Visible Quantitative Point-of-Care Testing of Invasive Fungi.},
journal = {Analytical chemistry},
volume = {93},
number = {50},
pages = {16965-16973},
doi = {10.1021/acs.analchem.1c04649},
pmid = {34889094},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Fungi ; Humans ; Hydrogels ; *Microfluidics ; Point-of-Care Testing ; },
abstract = {Invasive fungi (IF) have become a significant problem affecting human health. However, the culture-based assay of IF, known as the most commonly used clinical diagnostic method, suffers from time consumption, complicated operation, and the requirement of trained operators, which may cause the delay diagnosis of the disease. In this report, a microfluidic ruler-readout and CRISPR Cas12a-responded hydrogel-integrated paper-based analytical device (μReaCH-PAD) was established for visible and quantitative point-of-care testing of IF. Using the genus-conserved fragments of 18s rRNA as the detection target, this platform relied on a CRISPR Cas12a system for target recognition, a DNA hydrogel coupled with a cascade of enzymatic reactions for signal amplification and transduction, and paper-based microfluidic chips for visual quantitative readout by naked eyes. The 18s rRNA fragments of Candida or Aspergillus were employed as a model target and introduced with PAM sites for Cas12a-recognition during reverse transcription recombinase-aided amplification. Using μReaCH-PAD, as low as 10 CFU/mL Candida and Aspergillus were visually identified by unaided eyes. The calculated detection limits were 4.90 and 4.13 CFU/mL (in 1 mL samples), respectively. The quantitative detection results can be obtained in the range from 10 to 10[4] CFU/mL with reasonable specificity and accuracy compared with qRT-PCR. Furthermore, μReaCH-PAD can analyze complex biological samples by Candida, Aspergillus, and Cryptococcus detection systems and identify specific genera of different IF by naked eyes, indicating a good agreement with the culture-based assay and the advantages over G-testing and GM-testing systems. With the benefits of high sensitivity, selectivity, quantitative readout, low cost, and ease of operation, μReaCH-PAD is expected to provide a portable detection tool of IF in resource-limited settings by untrained personnel and technical support for early diagnosis.},
}
@article {pmid34887556,
year = {2022},
author = {Anzalone, AV and Gao, XD and Podracky, CJ and Nelson, AT and Koblan, LW and Raguram, A and Levy, JM and Mercer, JAM and Liu, DR},
title = {Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing.},
journal = {Nature biotechnology},
volume = {40},
number = {5},
pages = {731-740},
pmid = {34887556},
issn = {1546-1696},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Chromosome Inversion ; DNA/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.},
}
@article {pmid34887431,
year = {2021},
author = {Fuselier, KTB and Salbaum, JM and Kappen, C},
title = {Broad spectrum of CRISPR-induced edits in an embryonic lethal gene.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23732},
pmid = {34887431},
issn = {2045-2322},
support = {P30 DK072476/DK/NIDDK NIH HHS/United States ; P30 GM118430/GM/NIGMS NIH HHS/United States ; R01 HD087283/HD/NICHD NIH HHS/United States ; R01 HD037283/HD/NICHD NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Embryonic Development/*genetics ; *Gene Editing ; *Genes, Lethal ; Genetic Engineering ; Genotype ; INDEL Mutation ; Mice ; Mutagenesis ; Phenotype ; RNA, Guide ; },
abstract = {Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype-phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.},
}
@article {pmid34887319,
year = {2022},
author = {Chang, L and Masada, M and Kojima, M and Yamamoto, N},
title = {Involvement of Denervated Midbrain-Derived Factors in the Formation of Ectopic Cortico-Mesencephalic Projection after Hemispherectomy.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {42},
number = {5},
pages = {749-761},
pmid = {34887319},
issn = {1529-2401},
mesh = {Animals ; Brain Injuries/genetics/*metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cerebral Cortex/chemistry/cytology/*metabolism ; Denervation/trends ; Gene Knockout Techniques/methods ; Hemispherectomy/*trends ; Mesencephalon/chemistry/cytology/*metabolism ; Mice ; Mice, Inbred ICR ; Nerve Regeneration/physiology ; Neural Pathways/cytology/metabolism ; Neuronal Plasticity/*physiology ; Organ Culture Techniques ; Receptor, trkB/analysis/genetics/metabolism ; },
abstract = {Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit β three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors.SIGNIFICANCE STATEMENT After brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury.},
}
@article {pmid34886891,
year = {2021},
author = {Rezaei-Lotfi, S and Vujovic, F and Simonian, M and Hunter, N and Farahani, RM},
title = {Programmed genomic instability regulates neural transdifferentiation of human brain microvascular pericytes.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {334},
pmid = {34886891},
issn = {1474-760X},
mesh = {Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; *Brain ; CRISPR-Cas Systems ; Cell Transdifferentiation/*genetics ; Chromatin/metabolism ; *Genomic Instability ; Humans ; Neurogenesis ; Neurons/metabolism ; Pericytes/*physiology ; Transcriptome ; },
abstract = {BACKGROUND: Transdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming.<br><br>RESULTS: We show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome.<br><br>CONCLUSIONS: These findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state.},
}
@article {pmid34886764,
year = {2021},
author = {Jiao, J and Zheng, N and Wei, W and Fleming, J and Wang, X and Li, Z and Zhang, L and Liu, Y and Zhang, Z and Shen, A and Chuanyou, L and Bi, L and Zhang, H},
title = {M. tuberculosis CRISPR/Cas proteins are secreted virulence factors that trigger cellular immune responses.},
journal = {Virulence},
volume = {12},
number = {1},
pages = {3032-3044},
pmid = {34886764},
issn = {2150-5608},
mesh = {Animals ; CRISPR-Cas Systems ; Humans ; Immunity, Cellular ; Mice ; *Mycobacterium tuberculosis/genetics/metabolism ; *Tuberculosis ; Virulence Factors/genetics/metabolism ; },
abstract = {The role of prokaryotic CRISPR/Cas system proteins as a defensive shield against invasive nucleic acids has been studied extensively. Non-canonical roles in pathogenesis involving intracellular targeting of certain virulence-associated endogenous mRNA have also been reported for some Type I and Type II CRISPR/Cas proteins, but no such roles have yet been established for Type III system proteins. Here, we demonstrate that M. tuberculosis (Type III-A system) CRISPR/Cas proteins Csm1, Csm3, Csm5, Csm6, and Cas6 are secreted and induce host immune responses. Using cell and animal experiments, we show that Cas6, in particular, provokes IFN-γ release from PBMCs from active tuberculosis (TB) patients, and its deletion markedly attenuates virulence in a murine M. tuberculosis challenge model. Recombinant MTBCas6 induces apoptosis of macrophages and lung fibroblasts, and interacts with the surface of cells in a caspase and TLR-2 independent manner. Transcriptomic and signal pathway studies using THP-1 macrophages stimulated with MTBCas6 indicated that MTBCas6 upregulates expression of genes associated with the NF-κB pathway leading to higher levels of IL-6, IL-1β, and TNF-α release, cytokines known to activate immune system cells in response to M. tuberculosis infection. Our findings suggest that, in addition to their intracellular shielding role, M. tuberculosis CRISPR/Cas proteins have non-canonical extracellular roles, functioning like a virulent sword, and activating host immune responses.},
}
@article {pmid34884880,
year = {2021},
author = {Guzmán-López, MH and Marín-Sanz, M and Sánchez-León, S and Barro, F},
title = {A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {22},
number = {23},
pages = {},
pmid = {34884880},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Computational Biology ; *Gene Editing ; *Genes, Plant ; Genome, Plant ; *Genomics ; Gliadin/*genetics ; INDEL Mutation ; Sequence Analysis, DNA ; Triticum/genetics/*metabolism ; },
abstract = {The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.},
}
@article {pmid34884871,
year = {2021},
author = {Muñoz-Sanz, JV and Tovar-Méndez, A and Lu, L and Dai, R and McClure, B},
title = {A Cysteine-Rich Protein, SpDIR1L, Implicated in S-RNase-Independent Pollen Rejection in the Tomato (Solanum Section Lycopersicon) Clade.},
journal = {International journal of molecular sciences},
volume = {22},
number = {23},
pages = {},
pmid = {34884871},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Cysteine ; Flowers/genetics/physiology ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/*genetics/physiology ; Mutation ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Pollen/*genetics ; Reproduction/genetics ; Ribonucleases/genetics ; Solanum/*genetics/physiology ; },
abstract = {Tomato clade species (Solanum sect. Lycopersicon) display multiple interspecific reproductive barriers (IRBs). Some IRBs conform to the SI x SC rule, which describes unilateral incompatibility (UI) where pollen from SC species is rejected on SI species' pistils, but reciprocal pollinations are successful. However, SC x SC UI also exists, offering opportunities to identify factors that contribute to S-RNase-independent IRBs. For instance, SC Solanum pennellii LA0716 pistils only permit SC Solanum lycopersicum pollen tubes to penetrate to the top third of the pistil, while S. pennellii pollen penetrates to S. lycopersicum ovaries. We identified candidate S. pennellii LA0716 pistil barrier genes based on expression profiles and published results. CRISPR/Cas9 mutants were created in eight candidate genes, and mutants were assessed for changes in S. lycopersicum pollen tube growth. Mutants in a gene designated Defective in Induced Resistance 1-like (SpDIR1L), which encodes a small cysteine-rich protein, permitted S. lycopersicum pollen tubes to grow to the bottom third of the style. We show that SpDIR1L protein accumulation correlates with IRB strength and that species with weak or no IRBs toward S. lycopersicum pollen share a 150 bp deletion in the upstream region of SpDIR1L. These results suggest that SpDIR1L contributes to an S-RNase-independent IRB.},
}
@article {pmid34884583,
year = {2021},
author = {Tsujino, T and Komura, K and Inamoto, T and Azuma, H},
title = {CRISPR Screen Contributes to Novel Target Discovery in Prostate Cancer.},
journal = {International journal of molecular sciences},
volume = {22},
number = {23},
pages = {},
pmid = {34884583},
issn = {1422-0067},
mesh = {Antineoplastic Agents/*pharmacology ; Biomarkers, Tumor/*antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Drug Discovery ; *Gene Editing ; Humans ; Male ; Precision Medicine ; Prostatic Neoplasms/*drug therapy/genetics/pathology ; },
abstract = {Prostate cancer (PCa) is one of the common malignancies in male adults. Recent advances in omics technology, especially in next-generation sequencing, have increased the opportunity to identify genes that correlate with cancer diseases, including PCa. In addition, a genetic screen based on CRISPR/Cas9 technology has elucidated the mechanisms of cancer progression and drug resistance, which in turn has enabled the discovery of new targets as potential genes for new therapeutic targets. In the era of precision medicine, such knowledge is crucial for clinicians in their decision-making regarding patient treatment. In this review, we focus on how CRISPR screen for PCa performed to date has contributed to the identification of biologically critical and clinically relevant target genes.},
}
@article {pmid34883448,
year = {2022},
author = {Ader, F and Duboscq-Bidot, L and Marteau, S and Hamlin, M and Richard, P and Fontaine, V and Villard, E},
title = {Generation of CRISPR-Cas9 edited human induced pluripotent stem cell line carrying FLNC exon skipping variant.},
journal = {Stem cell research},
volume = {58},
number = {},
pages = {102616},
doi = {10.1016/j.scr.2021.102616},
pmid = {34883448},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Cardiomyopathy, Dilated/genetics ; Exons/genetics ; Filamins/genetics/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; },
abstract = {Loss-of-function (LoF) mutations in FLNC are strongly associated with dilated cardiomyopathy (DCM). Using CRISPR/Cas9 mediated edition in an healthy donor derived iPSC (ICAN-403.3) we subcloned 1 iPSC line harboring LoF mutation in FLNC. All lines are fully pluripotent and isogenic except at edited site where it presents a homozygous (ICAN-FLNC42.1) deletion of splice site leading to skipping of exon 42 traduced into a short filamin form with reduced expression in derived cardiomyocytes. This line would serve for FLNC mutation DCM modeling after differentiation into cardiocytes or beating organoids.},
}
@article {pmid34882991,
year = {2022},
author = {Biswas, P and Anand, U and Ghorai, M and Pandey, DK and Jha, NK and Behl, T and Kumar, M and Chauhan, R and Shekhawat, MS and Dey, A},
title = {Unraveling the promise and limitations of CRISPR/Cas system in natural product research: Approaches and challenges.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100507},
doi = {10.1002/biot.202100507},
pmid = {34882991},
issn = {1860-7314},
mesh = {*Biological Products ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; Plants/genetics ; },
abstract = {An incredible array of natural products is produced by plants that serve several ecological functions, including protecting them from herbivores and microbes, attracting pollinators, and dispersing seeds. In addition to their obvious medical applications, natural products serve as flavoring agents, fragrances, and many other uses by humans. With the increasing demand for natural products and the development of various gene engineering systems, researchers are trying to modify the plant genome to increase the biosynthetic pathway of the compound of interest or blocking the pathway of unwanted compound synthesis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has had widespread success in genome editing due to the system's high efficiency, ease of use, and accuracy which revolutionized the genome editing system in living organisms. This study highlights the method of the CRISPR/Cas system, its application in different organisms including microbes, algae, fungi, and also higher plants in natural product research, and its shortcomings and future prospects.},
}
@article {pmid34882804,
year = {2022},
author = {Hao, X and Chen, W and Amato, A and Jouhet, J and Maréchal, E and Moog, D and Hu, H and Jin, H and You, L and Huang, F and Moosburner, M and Allen, AE and Gong, Y},
title = {Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids.},
journal = {The New phytologist},
volume = {233},
number = {4},
pages = {1797-1812},
doi = {10.1111/nph.17911},
pmid = {34882804},
issn = {1469-8137},
mesh = {CRISPR-Cas Systems/genetics ; Coenzyme A/genetics/metabolism ; Coenzyme A Ligases/genetics/metabolism ; *Diatoms/genetics/metabolism ; Fatty Acids/metabolism ; Mitochondria/metabolism ; },
abstract = {Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown. In P. tricornutum, a family of five genes (ptACSL1-ptACSL5) encodes LACS activities. We generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.},
}
@article {pmid34882409,
year = {2022},
author = {Dalvie, NC and Lorgeree, T and Biedermann, AM and Love, KR and Love, JC},
title = {Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination.},
journal = {ACS synthetic biology},
volume = {11},
number = {1},
pages = {497-501},
pmid = {34882409},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Gene Editing ; Gene Knockout Techniques ; Genetic Engineering ; *Homologous Recombination/genetics ; },
abstract = {Genetic engineering of industrial cell lines often requires knockout of multiple endogenous genes. Tools like CRISPR-Cas9 have enabled serial or parallelized gene disruption in a wide range of industrial organisms, but common practices for the screening and validation of genome edits are lacking. For gene disruption, DNA repair by homologous recombination offers several advantages over nonhomologous end joining, including more efficient screening for knockout clones and improved genomic stability. Here we designed and characterized a knockout fragment intended to repair Cas9-induced gene disruptions by homologous recombination. We identified knockout clones of Komagataella phaffii with high fidelity by PCR, removing the need for Sanger sequencing. Short overlap sequences for homologous recombination (30 bp) enabled the generation of gene-specific knockout fragments by PCR, removing the need for subcloning. Finally, we demonstrated that the genotype conferred by the knockout fragment is stable under common cultivation conditions.},
}
@article {pmid34882002,
year = {2022},
author = {Antony, JS and Daniel-Moreno, A and Lamsfus-Calle, A and Raju, J and Kaftancioglu, M and Ureña-Bailén, G and Rottenberger, J and Hou, Y and Santhanakumaran, V and Lee, JH and Heumos, L and Böhringer, J and Krägeloh-Mann, I and Handgretinger, R and Mezger, M},
title = {A Mutation-Agnostic Hematopoietic Stem Cell Gene Therapy for Metachromatic Leukodystrophy.},
journal = {The CRISPR journal},
volume = {5},
number = {1},
pages = {66-79},
doi = {10.1089/crispr.2021.0075},
pmid = {34882002},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Hematopoietic Stem Cells/metabolism ; Humans ; *Leukodystrophy, Metachromatic/genetics/therapy ; Mutation ; Prospective Studies ; },
abstract = {Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in the Arylsulfatase-A (ARSA) gene. The enzyme plays a key role in sulfatide metabolism in brain cells, and its deficiency leads to neurodegeneration. The clinical manifestations of MLD include stagnation and decline of motor and cognitive function, leading to premature death with limited standard treatment options. Here, we describe a mutation-agnostic hematopoietic stem and progenitor cell (HSPC) gene therapy using CRISPR-Cas9 and AAV6 repair template as a prospective treatment option for MLD. Our strategy achieved efficient insertions and deletions (>87%) and a high level of gene integration (>47%) at the ARSA locus in human bone marrow-derived HSPCs, with no detectable off-target editing. As a proof of concept, we tested our mutation-agnostic therapy in HSPCs derived from two MLD patients with distinct mutations and demonstrated restoration of ARSA enzyme activity (>30-fold improvement) equivalent to healthy adults. In summary, our investigation enabled a mutation-agnostic therapy for MLD patients with proven efficacy and strong potential for clinical translation.},
}
@article {pmid34881774,
year = {2021},
author = {Colic, M and Hart, T},
title = {Common computational tools for analyzing CRISPR screens.},
journal = {Emerging topics in life sciences},
volume = {5},
number = {6},
pages = {779-788},
pmid = {34881774},
issn = {2397-8554},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome ; Genomics/methods ; },
abstract = {CRISPR-Cas technology offers a versatile toolbox for genome editing, with applications in various cancer-related fields such as functional genomics, immunotherapy, synthetic lethality and drug resistance, metastasis, genome regulation, chromatic accessibility and RNA-targeting. The variety of screening platforms and questions in which they are used have caused the development of a wide array of analytical methods for CRISPR analysis. In this review, we focus on the algorithms and frameworks used in the computational analysis of pooled CRISPR knockout (KO) screens and highlight some of the most significant target discoveries made using these methods. Lastly, we offer perspectives on the design and analysis of state-of-art multiplex screening for genetic interactions.},
}
@article {pmid34881529,
year = {2021},
author = {Selvaraj, D and Dawar, R and Sivakumar, PK and Devi, A},
title = {Clustered regularly interspaced short palindromic repeats, a glimpse - impacts in molecular biology, trends and highlights.},
journal = {Hormone molecular biology and clinical investigation},
volume = {43},
number = {1},
pages = {105-112},
doi = {10.1515/hmbci-2021-0062},
pmid = {34881529},
issn = {1868-1891},
mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Therapy/methods ; Humans ; Molecular Biology ; SARS-CoV-2/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a novel molecular tool. In recent days, it has been highlighted a lot, as the Nobel prize was awarded for this sector in 2020, and also for its recent use in Covid-19 related diagnostics. Otherwise, it is an eminent gene-editing technique applied in diverse medical zones of therapeutics in genetic diseases, hematological diseases, infectious diseases, etc., research related to molecular biology, cancer, hereditary diseases, immune and inflammatory diseases, etc., diagnostics related to infectious diseases like viral hemorrhagic fevers, Covid-19, etc. In this review, its discovery, working mechanisms, challenges while handling the technique, recent advancements, applications, alternatives have been discussed. It is a cheaper, faster technique revolutionizing the medicinal field right now. However, their off-target effects and difficulties in delivery into the desired cells make CRISPR, not easily utilizable. We conclude that further robust research in this field may promise many interesting, useful results.},
}
@article {pmid34880423,
year = {2022},
author = {Koch, L},
title = {CRISPR editing within microbial communities.},
journal = {Nature reviews. Genetics},
volume = {23},
number = {2},
pages = {72},
pmid = {34880423},
issn = {1471-0064},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *Microbiota/genetics ; },
}
@article {pmid34880381,
year = {2021},
author = {Manoj, F and Tai, LW and Wang, KSM and Kuhlman, TE},
title = {Targeted insertion of large genetic payloads using cas directed LINE-1 reverse transcriptase.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23625},
pmid = {34880381},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/genetics ; Gene Editing/*methods ; *Long Interspersed Nucleotide Elements ; RNA-Directed DNA Polymerase/*genetics ; Reverse Transcription ; },
abstract = {A difficult genome editing goal is the site-specific insertion of large genetic constructs. Here we describe the GENEWRITE system, where site-specific targetable activity of Cas endonucleases is coupled with the reverse transcriptase activity of the ORF2p protein of the human retrotransposon LINE-1. This is accomplished by providing two RNAs: a guide RNA targeting Cas endonuclease activity and an appropriately designed payload RNA encoding the desired insertion. Using E. coli as a simple platform for development and deployment, we show that with proper payload design and co-expression of helper proteins, GENEWRITE can enable insertion of large genetic payloads to precise locations, although with off-target effects, using the described approach. Based upon these results, we describe a potential strategy for implementation of GENEWRITE in more complex systems.},
}
@article {pmid34880355,
year = {2021},
author = {Kuzin, A and Redler, B and Onuska, J and Slesarev, A},
title = {RGEN-seq for highly sensitive amplification-free screen of off-target sites of gene editors.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23600},
pmid = {34880355},
issn = {2045-2322},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems ; Cricetulus ; *Gene Editing ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Limit of Detection ; Polymerase Chain Reaction/methods ; },
abstract = {Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.},
}
@article {pmid34880227,
year = {2021},
author = {Liu, Y and Wu, Z and Zhou, J and Ramadurai, DKA and Mortenson, KL and Aguilera-Jimenez, E and Yan, Y and Yang, X and Taylor, AM and Varley, KE and Gertz, J and Choi, PS and Cherniack, AD and Chen, X and Bass, AJ and Bailey, SD and Zhang, X},
title = {A predominant enhancer co-amplified with the SOX2 oncogene is necessary and sufficient for its expression in squamous cancer.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {7139},
pmid = {34880227},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Carcinoma, Squamous Cell/*genetics ; Cell Cycle Proteins/genetics/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Chromatin ; Enhancer Elements, Genetic ; Epigenomics ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Heterografts ; Humans ; Neoplasms, Squamous Cell/*genetics ; Oncogenes/genetics ; SOXB1 Transcription Factors/*genetics/*metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3' noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.},
}
@article {pmid34880218,
year = {2021},
author = {Kenjo, E and Hozumi, H and Makita, Y and Iwabuchi, KA and Fujimoto, N and Matsumoto, S and Kimura, M and Amano, Y and Ifuku, M and Naoe, Y and Inukai, N and Hotta, A},
title = {Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {7101},
pmid = {34880218},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Disease Models, Animal ; Dystrophin/genetics/metabolism ; Exons ; Gene Editing/*methods ; Genetic Therapy ; Humans ; Liposomes ; Mice ; Muscle, Skeletal/*metabolism ; Muscular Dystrophy, Duchenne/genetics/therapy ; Nanoparticles ; Neuromuscular Diseases/genetics/therapy ; *RNA, Messenger ; },
abstract = {Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.},
}
@article {pmid34880157,
year = {2022},
author = {Nakano, K and Shimizu, Y and Arai, T and Kaneko, T and Okamura, T},
title = {The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator.},
journal = {Experimental animals},
volume = {71},
number = {2},
pages = {214-223},
pmid = {34880157},
issn = {1881-7122},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Mice, Knockout ; },
abstract = {Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genome-editing efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.},
}
@article {pmid34880108,
year = {2022},
author = {Dong, H and Zhang, Y and Wang, J and Xiang, H and Lv, T and Wei, L and Yang, S and Liu, X and Ren, B and Zhang, X and Liu, L and Cao, J and Wang, M and Shi, J and Yang, N},
title = {Cas9-Based Local Enrichment and Genomics Sequence Revision of Megabase-Sized Shark IgNAR Loci.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {208},
number = {1},
pages = {181-189},
doi = {10.4049/jimmunol.2100844},
pmid = {34880108},
issn = {1550-6606},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Fish Proteins/*genetics ; Genetic Loci/*genetics ; Genome ; Genomics ; High-Throughput Nucleotide Sequencing ; Immunoglobulins/*genetics ; Receptors, Antigen/*genetics ; Sequence Analysis, DNA ; Sharks/*immunology ; },
abstract = {The 0.8-Mb Ig new Ag receptor (IgNAR) region of the whitespotted bamboo shark (Chiloscyllium plagiosum) is incompletely assembled in Chr_44 of the reference genome. Here we used Cas9-assisted targeting of chromosome segments (CATCH) to enrich the 2 Mb region of the Chr_44 IgNAR loci and sequenced it by PacBio and next-generation sequencing. A fragment >3.13 Mb was isolated intact from the RBCs of sharks. The target was enriched 245.531-fold, and sequences had up to 94% coverage with a 255× mean depth. Compared with the previously published sequences, 20 holes were filled, with a total length of 3508 bp. In addition, we report five potential germline V alleles of IgNAR1 from six sharks that may belong to two clusters of the IgNAR. Our results provide a new method to research the germline of large Ig gene segments, as well as provide the enhanced bamboo shark IgNAR gene loci with fewer gaps.},
}
@article {pmid34879274,
year = {2021},
author = {Zhao, H and Liu, Y and Wang, L and Jin, G and Zhao, X and Xu, J and Zhang, G and Ma, Y and Yin, N and Peng, M},
title = {Genome-wide fitness gene identification reveals Roquin as a potent suppressor of CD8 T cell expansion and anti-tumor immunity.},
journal = {Cell reports},
volume = {37},
number = {10},
pages = {110083},
doi = {10.1016/j.celrep.2021.110083},
pmid = {34879274},
issn = {2211-1247},
mesh = {Animals ; CD8-Positive T-Lymphocytes/immunology/metabolism/*transplantation ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Cell Proliferation ; *Cytotoxicity, Immunologic ; Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study ; *Immunotherapy, Adoptive ; Interferon Regulatory Factors/genetics/metabolism ; *Lymphocyte Activation ; Mice, Inbred C57BL ; Mice, Transgenic ; Neoplasms/genetics/immunology/metabolism/*therapy ; Phenotype ; Signal Transduction ; Tumor Escape ; Ubiquitin-Protein Ligases/*genetics/metabolism ; },
abstract = {Robust expansion of adoptively transferred T cells is a prerequisite for effective cancer immunotherapy, but how many genes in the genome modulate T cell expansion remains unknown. Here, we perform in vivo and in vitro CRISPR screens to systematically identify genes influencing CD8 T cell expansion. In the mouse genome, ∼2,600 and ∼1,500 genes are required for optimal CD8 T cell expansion in vivo and in vitro, respectively. In vivo-specific CD8 T cell essential genes are enriched in metabolic pathways, including mitochondrial metabolism. The strongest repressor of CD8 T cell expansion is Roquin, the ablation of which drastically boosts T cell proliferation by enhancing cell-cycle progression and upregulation of IRF4. Roquin deficiency or IRF4 overexpression potently enhances anti-tumor immunity. These data provide a functional catalog of CD8 T cell fitness genes and suggest that targeting the Roquin-IRF4 axis is an effective strategy to enhance efficacy of adoptive transfer therapy for cancer.},
}
@article {pmid34878784,
year = {2021},
author = {Yuan, G and Hassan, MM and Yao, T and Lu, H and Vergara, MM and Labbé, JL and Muchero, W and Pan, C and Chen, JG and Tuskan, GA and Qi, Y and Abraham, PE and Yang, X},
title = {Plant-Based Biosensors for Detecting CRISPR-Mediated Genome Engineering.},
journal = {ACS synthetic biology},
volume = {10},
number = {12},
pages = {3600-3603},
doi = {10.1021/acssynbio.1c00455},
pmid = {34878784},
issn = {2161-5063},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant/genetics ; Plants/genetics ; },
abstract = {CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with the CRISPR/Cas technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRISPR/Cas tools in biological systems is therefore very necessary. Here, we developed four real-time detection systems that can spontaneously indicate the presence of active CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.},
}
@article {pmid34876522,
year = {2021},
author = {Muto, M and Chakraborty, D and Varberg, KM and Moreno-Irusta, A and Iqbal, K and Scott, RL and McNally, RP and Choudhury, RH and Aplin, JD and Okae, H and Arima, T and Matsumoto, S and Ema, M and Mast, AE and Grundberg, E and Soares, MJ},
title = {Intersection of regulatory pathways controlling hemostasis and hemochorial placentation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {50},
pages = {},
pmid = {34876522},
issn = {1091-6490},
support = {R01 HD020676/HD/NICHD NIH HHS/United States ; F32 HD096809/HD/NICHD NIH HHS/United States ; F31 HD104495/HD/NICHD NIH HHS/United States ; R01 HD099638/HD/NICHD NIH HHS/United States ; S10 OD021743/OD/NIH HHS/United States ; P01 HD079363/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Endothelial Cells/physiology ; Female ; Gene Editing ; Humans ; Lipoproteins/genetics/*metabolism ; Mutation ; Placenta/metabolism ; Placentation/*physiology ; Pregnancy ; RNA Interference ; Rats ; Rats, Sprague-Dawley ; Stem Cells/*physiology ; Trophoblasts/*physiology ; },
abstract = {Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.},
}
@article {pmid34876467,
year = {2022},
author = {Larbalestier, H and Keatinge, M and Watson, L and White, E and Gowda, S and Wei, W and Koler, K and Semenova, SA and Elkin, AM and Rimmer, N and Sweeney, ST and Mazzolini, J and Sieger, D and Hide, W and McDearmid, J and Panula, P and MacDonald, RB and Bandmann, O},
title = {GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {42},
number = {4},
pages = {702-716},
pmid = {34876467},
issn = {1529-2401},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/R011354/1/MRC_/Medical Research Council/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Animals, Genetically Modified ; Brain/*enzymology/immunology ; Dopaminergic Neurons/enzymology/immunology ; GTP Cyclohydrolase/*deficiency/genetics ; Genetic Predisposition to Disease/genetics ; Homeostasis/*physiology ; Immunity, Innate/*physiology ; Parkinson Disease/enzymology/genetics/immunology ; Sequence Analysis, RNA/methods ; Tyrosine 3-Monooxygenase/antagonists &amp; inhibitors/genetics/*metabolism ; Zebrafish ; },
abstract = {The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1[-/-]), using CRISPR/Cas technology. gch1[-/-] zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1[-/-] larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1[-/-] larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1[-/-] The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.},
}
@article {pmid34874915,
year = {2022},
author = {Lane, BM and Chryst-Stangl, M and Wu, G and Shalaby, M and El Desoky, S and Middleton, CC and Huggins, K and Sood, A and Ochoa, A and Malone, AF and Vancini, R and Miller, SE and Hall, G and Kim, SY and Howell, DN and Kari, JA and Gbadegesin, R},
title = {Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis.},
journal = {JCI insight},
volume = {7},
number = {2},
pages = {},
pmid = {34874915},
issn = {2379-3708},
support = {K08 DK111940/DK/NIDDK NIH HHS/United States ; R21 HD104176/HD/NICHD NIH HHS/United States ; },
mesh = {Adrenal Cortex Hormones ; Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems/genetics ; Carrier Proteins/*genetics ; Cells, Cultured ; *Endocytosis/drug effects/genetics ; Gene Knockout Techniques ; Genetic Association Studies ; High-Throughput Nucleotide Sequencing/methods ; Humans ; *Nephrotic Syndrome/drug therapy/genetics/metabolism/pathology ; *Oxidative Stress/drug effects/genetics ; *Podocytes/drug effects/metabolism ; Reactive Oxygen Species/antagonists &amp; inhibitors ; Zebrafish ; Zebrafish Proteins ; },
abstract = {We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.},
}
@article {pmid34874743,
year = {2022},
author = {Magdy, T and Jouni, M and Kuo, HH and Weddle, CJ and Lyra-Leite, D and Fonoudi, H and Romero-Tejeda, M and Gharib, M and Javed, H and Fajardo, G and Ross, CJD and Carleton, BC and Bernstein, D and Burridge, PW},
title = {Identification of Drug Transporter Genomic Variants and Inhibitors That Protect Against Doxorubicin-Induced Cardiotoxicity.},
journal = {Circulation},
volume = {145},
number = {4},
pages = {279-294},
pmid = {34874743},
issn = {1524-4539},
support = {R01 CA220002/CA/NCI NIH HHS/United States ; T32 CA009560/CA/NCI NIH HHS/United States ; R01 CA261898/CA/NCI NIH HHS/United States ; F31 CA247395/CA/NCI NIH HHS/United States ; K99 HL121177/HL/NHLBI NIH HHS/United States ; R33 HL123655/HL/NHLBI NIH HHS/United States ; R00 HL121177/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Cardiotoxicity/*physiopathology ; Disease Models, Animal ; Doxorubicin/*adverse effects ; Genetic Variation/*genetics ; Genomics ; Humans ; Male ; Mice ; },
abstract = {BACKGROUND: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated.<br><br>METHODS: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes using a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9). Fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of the potential causal variant was done using cytosine base editor. SLC28A3-AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-sequencing after ribosomal RNA depletion. Drug screening was done using the Prestwick Chemical Library (n = 1200), followed by in vivo validation in mice. The effect of desipramine on doxorubicin cytotoxicity was also investigated in 8 cancer cell lines.<br><br>RESULTS: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore-based fine-mapping and base editing, we identify a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus; its effect is exerted via regulation of an antisense long noncoding RNA (SLC28A3-AS1) that overlaps with SLC28A3. Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC.<br><br>CONCLUSIONS: This work demonstrates the power of the human induced pluripotent stem cell model to take a single nucleotide polymorphism from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.},
}
@article {pmid34873327,
year = {2021},
author = {Lim, JM and Kim, HH},
title = {Improving CRISPR tools by elucidating DNA repair.},
journal = {Nature biotechnology},
volume = {39},
number = {12},
pages = {1512-1514},
pmid = {34873327},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Repair/genetics ; *Gene Editing ; },
}
@article {pmid34873292,
year = {2022},
author = {Rubin, BE and Diamond, S and Cress, BF and Crits-Christoph, A and Lou, YC and Borges, AL and Shivram, H and He, C and Xu, M and Zhou, Z and Smith, SJ and Rovinsky, R and Smock, DCJ and Tang, K and Owens, TK and Krishnappa, N and Sachdeva, R and Barrangou, R and Deutschbauer, AM and Banfield, JF and Doudna, JA},
title = {Species- and site-specific genome editing in complex bacterial communities.},
journal = {Nature microbiology},
volume = {7},
number = {1},
pages = {34-47},
pmid = {34873292},
issn = {2058-5276},
support = {F32 GM131654/GM/NIGMS NIH HHS/United States ; F32 GM134694/GM/NIGMS NIH HHS/United States ; R01 AI092531/AI/NIAID NIH HHS/United States ; },
mesh = {Archaea/genetics ; Bacteria/classification ; CRISPR-Cas Systems ; Gastrointestinal Microbiome/*genetics ; Gene Editing/*methods ; *Genome, Bacterial ; Humans ; Infant ; Microbial Consortia/*genetics ; RNA, Guide ; *Soil Microbiology ; },
abstract = {Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control.},
}
@article {pmid34872747,
year = {2022},
author = {Li, X and Huang, X},
title = {Towards precise large genomic fragment deletion.},
journal = {Trends in genetics : TIG},
volume = {38},
number = {3},
pages = {214-215},
doi = {10.1016/j.tig.2021.11.004},
pmid = {34872747},
issn = {0168-9525},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing ; Genome/genetics ; Genomics ; },
abstract = {The prime editing (PE) system can install small insertions and deletions in addition to various base substitutions. Two recent studies published in Nature Biotechnology by Choi et al. and Jiang et al. report that the system can also be tweaked for efficient and precise deletions of large DNA fragments.},
}
@article {pmid34872358,
year = {2021},
author = {Gallo, G and Mougiakos, I and Bianco, M and Carbonaro, M and Carpentieri, A and Illiano, A and Pucci, P and Bartolucci, S and van der Oost, J and Fiorentino, G},
title = {A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.},
journal = {mBio},
volume = {12},
number = {6},
pages = {e0281321},
pmid = {34872358},
issn = {2150-7511},
mesh = {Amino Acid Sequence ; Arsenic/chemistry/*metabolism ; Bacterial Proteins/*chemistry/*genetics/metabolism ; Binding Sites ; CRISPR-Cas Systems ; Enzyme Stability ; Gene Editing ; Methyltransferases/*chemistry/*genetics/metabolism ; Sequence Alignment ; Thermus thermophilus/chemistry/*enzymology/genetics ; },
abstract = {Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.},
}
@article {pmid34871741,
year = {2022},
author = {Belato, HB and D'Ordine, AM and Nierzwicki, L and Arantes, PR and Jogl, G and Palermo, G and Lisi, GP},
title = {Structural and dynamic insights into the HNH nuclease of divergent Cas9 species.},
journal = {Journal of structural biology},
volume = {214},
number = {1},
pages = {107814},
pmid = {34871741},
issn = {1095-8657},
support = {P20 GM109035/GM/NIGMS NIH HHS/United States ; R01 GM136815/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; *Molecular Dynamics Simulation ; RNA, Guide/chemistry/genetics/metabolism ; Streptococcus pyogenes/metabolism ; },
abstract = {CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, which drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.},
}
@article {pmid34871408,
year = {2021},
author = {Grüschow, S and Adamson, CS and White, MF},
title = {Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector.},
journal = {Nucleic acids research},
volume = {49},
number = {22},
pages = {13122-13134},
pmid = {34871408},
issn = {1362-4962},
support = {BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; COVID-19 ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chlorocebus aethiops ; Endodeoxyribonucleases/*metabolism ; Endoribonucleases/*metabolism ; Humans ; Prophages/genetics ; RNA, Viral/*genetics ; SARS-CoV-2/*genetics ; Vero Cells ; Vibrio/virology ; },
abstract = {Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.},
}
@article {pmid34871331,
year = {2021},
author = {Song, Y and Huang, H and Hu, Y and Zhang, J and Li, F and Yin, X and Shi, J and Li, Y and Li, C and Zhao, D and Chen, H},
title = {A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis.},
journal = {PLoS pathogens},
volume = {17},
number = {12},
pages = {e1010141},
pmid = {34871331},
issn = {1553-7374},
mesh = {A549 Cells ; Animals ; CRISPR-Cas Systems ; DCC Receptor/*metabolism ; Endocytosis/*physiology ; Gene Knockout Techniques ; Humans ; Influenza A Virus, H5N1 Subtype/*pathogenicity ; Mice ; Mice, Knockout ; Orthomyxoviridae Infections/*virology ; *Virus Internalization ; },
abstract = {Influenza virus infection is dependent on host cellular factors, and identification of these factors and their underlying mechanisms can provide important information for the development of strategies to inhibit viral infection. Here, we used a highly pathogenic H5N1 influenza virus to perform a genome-wide CRISPR/Cas9 gene knockout screen in human lung epithelial cells (A549 cells), and found that knockout of transmembrane protein immunoglobulin superfamily DCC subclass member 4 (IGDCC4) significantly reduced the replication of the virus in A549 cells. Further studies showed that IGDCC4 interacted with the viral hemagglutinin protein and facilitated virus internalization into host cells. Animal infection studies showed that replication of H5N1 virus in the nasal turbinates, lungs, and kidneys of IGDCC4-knockout mice was significantly lower than that in the corresponding organs of wild-type mice. Half of the IGDCC4-knockout mice survived a lethal H5N1 virus challenge, whereas all of the wild-type mice died within 11 days of infection. Our study identifies a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.},
}
@article {pmid34871328,
year = {2021},
author = {Sun, L and Zhao, C and Fu, Z and Fu, Y and Su, Z and Li, Y and Zhou, Y and Tan, Y and Li, J and Xiang, Y and Nie, X and Zhang, J and Liu, F and Zhao, S and Xie, S and Peng, G},
title = {Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication.},
journal = {PLoS pathogens},
volume = {17},
number = {12},
pages = {e1010113},
pmid = {34871328},
issn = {1553-7374},
mesh = {Animals ; *CRISPR-Cas Systems ; Gastroenteritis, Transmissible, of Swine/genetics/transmission/*virology ; *Host-Pathogen Interactions ; Membrane Proteins/antagonists &amp; inhibitors/*physiology ; Mice ; Mice, Inbred C57BL ; Organelles/*virology ; Swine ; Transmissible gastroenteritis virus/*physiology ; *Virus Replication ; },
abstract = {Emerging coronaviruses (CoVs) pose a severe threat to human and animal health worldwide. To identify host factors required for CoV infection, we used α-CoV transmissible gastroenteritis virus (TGEV) as a model for genome-scale CRISPR knockout (KO) screening. Transmembrane protein 41B (TMEM41B) was found to be a bona fide host factor involved in infection by CoV and three additional virus families. We found that TMEM41B is critical for the internalization and early-stage replication of TGEV. Notably, our results also showed that cells lacking TMEM41B are unable to form the double-membrane vesicles necessary for TGEV replication, indicating that TMEM41B contributes to the formation of CoV replication organelles. Lastly, our data from a mouse infection model showed that the KO of this factor can strongly inhibit viral infection and delay the progression of a CoV disease. Our study revealed that targeting TMEM41B is a highly promising approach for the development of broad-spectrum anti-viral therapeutics.},
}
@article {pmid34870593,
year = {2021},
author = {Seleit, A and Aulehla, A and Paix, A},
title = {Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34870593},
issn = {2050-084X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Developmental Biology ; Gene Editing/*methods ; Gene Knock-In Techniques ; Oryzias/*genetics/*physiology ; Protein Transport/*genetics/*physiology ; },
abstract = {The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology-directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient, and precise strategy for CRISPR/Cas9-mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR-amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.},
}
@article {pmid34870354,
year = {2022},
author = {Raza, A and Tabassum, J and Mubarik, MS and Anwar, S and Zahra, N and Sharif, Y and Hafeez, MB and Zhang, C and Corpas, FJ and Chen, H},
title = {Hydrogen sulfide: an emerging component against abiotic stress in plants.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {24},
number = {4},
pages = {540-558},
doi = {10.1111/plb.13368},
pmid = {34870354},
issn = {1438-8677},
mesh = {*Hydrogen Sulfide ; Nitric Oxide ; Plant Physiological Phenomena ; Plants/genetics ; Stress, Physiological/physiology ; },
abstract = {As a result of climate change, abiotic stresses are the most common cause of crop losses worldwide. Abiotic stresses significantly impair plants' physiological, biochemical, molecular and cellular mechanisms, limiting crop productivity under adverse climate conditions. However, plants can implement essential mechanisms against abiotic stressors to maintain their growth and persistence under such stressful environments. In nature, plants have developed several adaptations and defence mechanisms to mitigate abiotic stress. Moreover, recent research has revealed that signalling molecules like hydrogen sulfide (H2 S) play a crucial role in mitigating the adverse effects of environmental stresses in plants by implementing several physiological and biochemical mechanisms. Mainly, H2 S helps to implement antioxidant defence systems, and interacts with other molecules like nitric oxide (NO), reactive oxygen species (ROS), phytohormones, etc. These molecules are well-known as the key players that moderate the adverse effects of abiotic stresses. Currently, little progress has been made in understanding the molecular basis of the protective role of H2 S; however, it is imperative to understand the molecular basis using the state-of-the-art CRISPR-Cas gene-editing tool. Subsequently, genetic engineering could provide a promising approach to unravelling the molecular basis of stress tolerance mediated by exogenous/endogenous H2 S. Here, we review recent advances in understanding the beneficial roles of H2 S in conferring multiple abiotic stress tolerance in plants. Further, we also discuss the interaction and crosstalk between H2 S and other signal molecules; as well as highlighting some genetic engineering-based current and future directions.},
}
@article {pmid34868276,
year = {2021},
author = {Joo, KA and Muszynski, MG and Kantar, MB and Wang, ML and He, X and Del Valle Echevarria, AR},
title = {Utilizing CRISPR-Cas in Tropical Crop Improvement: A Decision Process for Fitting Genome Engineering to Your Species.},
journal = {Frontiers in genetics},
volume = {12},
number = {},
pages = {786140},
pmid = {34868276},
issn = {1664-8021},
abstract = {Adopting modern gene-editing technologies for trait improvement in agriculture requires important workflow developments, yet these developments are not often discussed. Using tropical crop systems as a case study, we describe a workflow broken down into discrete processes with specific steps and decision points that allow for the practical application of the CRISPR-Cas gene editing platform in a crop of interest. While we present the steps of developing genome-edited plants as sequential, in practice parts can be done in parallel, which are discussed in this perspective. The main processes include 1) understanding the genetic basis of the trait along with having the crop's genome sequence, 2) testing and optimization of the editing reagents, development of efficient 3) tissue culture and 4) transformation methods, and 5) screening methods to identify edited events with commercial potential. Our goal in this perspective is to help any lab that wishes to implement this powerful, easy-to-use tool in their pipeline, thus aiming to democratize the technology.},
}
@article {pmid34867924,
year = {2021},
author = {Abdel-Glil, MY and Fischer, U and Steinhagen, D and McCarthy, U and Neubauer, H and Sprague, LD},
title = {Phylogenetic Relatedness and Genome Structure of Yersinia ruckeri Revealed by Whole Genome Sequencing and a Comparative Analysis.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {782415},
pmid = {34867924},
issn = {1664-302X},
abstract = {Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a serious infection that affects global aquaculture with high economic impact. The present study used whole genome sequences to perform a comparative analysis on 10 Y. ruckeri strains and to explore their genetic relatedness to other members of the genus. Y. ruckeri, Yersinia entomophaga, and Yersinia nurmii formed a species complex that constitutes the most basal lineage of the genus. The results showed that the taxonomy of Y. ruckeri strains is better defined by using a core genome alignment and phylogenetic analysis. The distribution of accessory genes in all Yersinia species revealed the presence of 303 distinctive genes in Y. ruckeri. Of these, 169 genes were distributed in 17 genomic islands potentially involved in the pathogenesis of ERM via (1) encoding virulence factors such as Afp18, Yrp1, phage proteins and (2) improving the metabolic capabilities by enhancing utilization and metabolism of iron, amino acids (specifically, arginine and histidine), and carbohydrates. The genome of Y. ruckeri is highly conserved regarding gene structure, gene layout and functional categorization of genes. It contains various components of mobile genetic elements but lacks the CRISPR-Cas system and possesses a stable set of virulence genes possibly playing a critical role in pathogenicity. Distinct virulence plasmids were exclusively restricted to a specific clonal group of Y. ruckeri (CG4), possibly indicating a selective advantage. Phylogenetic analysis of Y. ruckeri genomes revealed the co-presence of multiple genetically distant lineages of Y. ruckeri strains circulating in Germany. Our results also suggest a possible dissemination of a specific group of strains in the United States, Peru, Germany, and Denmark. In conclusion, this study provides new insights into the taxonomy and evolution of Y. ruckeri and contributes to a better understanding of the pathogenicity of ERM in aquaculture. The genomic analysis presented here offers a framework for the development of more efficient control strategies for this pathogen.},
}
@article {pmid34865958,
year = {2022},
author = {Yamada, S and Suzuki, Y and Kouzuma, A and Watanabe, K},
title = {Development of a CRISPR interference system for selective gene knockdown in Acidithiobacillus ferrooxidans.},
journal = {Journal of bioscience and bioengineering},
volume = {133},
number = {2},
pages = {105-109},
doi = {10.1016/j.jbiosc.2021.10.012},
pmid = {34865958},
issn = {1347-4421},
mesh = {*Acidithiobacillus/genetics ; *CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques ; Plasmids ; },
abstract = {Acidithiobacillus ferrooxidans is an iron-oxidizing chemolithotroph used for bioleaching of precious metals and is also regarded as a potential host for bioelectrochemical production of value-added chemicals. Despite its industrial utility, however, it is difficult to genetically engineer A. ferrooxidans due to low transformation and recombination efficiencies. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system that can selectively repress the expression of a target gene in A. ferrooxidans. The mutated gene encoding a nuclease-deactivated Cas9 protein was cloned into the broad-host-range plasmid pBBR1-MCS2, and the applicability of the CRISPRi system was examined using the nitrogenase nifH gene as a knockdown target. Introduction of the CRISPRi plasmid into A. ferrooxidans resulted in decreased nifH transcription and retarded cell growth in the absence of nitrogen sources, demonstrating that the CRISPRi system altered the phenotype of this bacterium via selective gene knockdown. We suggest that the CRISPRi system developed in this study provides an efficient technique for constructing A. ferrooxidans knockdown mutants that are useful for the genetic dissection of this bacterium.},
}
@article {pmid34865411,
year = {2021},
author = {Fan, F and Wu, JH and Luo, Y},
title = {[Current researches and prospects of human induced pluripotent stem cells and gene editing technology of CRISPR/Cas9 in inherited ocular diseases].},
journal = {[Zhonghua yan ke za zhi] Chinese journal of ophthalmology},
volume = {57},
number = {9},
pages = {712-716},
doi = {10.3760/cma.j.cn112142-20210407-00163},
pmid = {34865411},
issn = {0412-4081},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genome, Human ; Humans ; *Induced Pluripotent Stem Cells ; Technology ; },
abstract = {Human induced pluripotent stem cells have the ability to differentiate into specific cell types or tissues of the eye in humans and have application prospects in retinal cell transplantation, corneal transplantation, and lens regeneration. Gene editing technology of clustered regularly interspaced short palindromic repeats/crispr-associated protein-9 nuclease (CRISPR/Cas9) can introduce causative mutations of inherited ocular diseases into human induced pluripotent stem cells effectively. Then they can be further differentiated into specific somatic cells maintaining a genetic disease background, which can mimic the occurrence of inherited ocular diseases in vitro. The cell model may help scientists study the mechanism of human disease development, establish an in vitro screening platform to find therapeutic drugs, and correct genetic mutations in the human genome for cell therapy. The combination of stem cells and gene editing technology is revolutionizing the regenerative medicine in ophthalmology and gene therapy of inherited ocular diseases. This review summarizes the current application of human induced pluripotent stem cells and its combination with gene editing technology in the study of inherited ocular diseases. (Chin J Ophthalmol, 2021, 57: 712-716).},
}
@article {pmid34864205,
year = {2022},
author = {Kuang, C and Xiao, Y and Hondmann, D},
title = {Cleavage-free human genome editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {268-282},
pmid = {34864205},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *Gene Editing ; Genome, Human ; Humans ; },
abstract = {Most gene editing technologies introduce breaks or nicks into DNA, leading to the generation of mutagenic insertions and deletions by non-homologous end-joining repair. Here, we report a new, cleavage-free gene editing approach based on replication interrupted template-driven DNA modification (RITDM). The RITDM system makes use of sequence-specific DLR fusion molecules that are specifically designed to enable localized, temporary blockage of DNA replication fork progression, thereby exposing single-stranded DNA that can be bound by DNA sequence modification templates for precise editing. We evaluate the use of zinc-finger arrays for sequence recognition. We demonstrate that RITDM can be used for gene editing at endogenous genomic loci in human cells and highlight its safety profile of low indel frequencies and undetectable off-target side effects in RITDM-edited clones and pools of cells.},
}
@article {pmid34863708,
year = {2022},
author = {Perillo, M and Swartz, SZ and Wessel, GM},
title = {A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification.},
journal = {Developmental biology},
volume = {482},
number = {},
pages = {28-33},
pmid = {34863708},
issn = {1095-564X},
support = {K99 HD099315/HD/NICHD NIH HHS/United States ; R35 GM140897/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DEAD-box RNA Helicases/*metabolism ; Embryo Culture Techniques ; Embryo, Nonmammalian/embryology ; Germ Cells/*cytology ; Protein Processing, Post-Translational ; Sea Urchins/*embryology ; Starfish/*embryology ; Ubiquitin-Protein Ligases/*metabolism ; },
abstract = {Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.},
}
@article {pmid34863214,
year = {2021},
author = {Wu, Y and Battalapalli, D and Hakeem, MJ and Selamneni, V and Zhang, P and Draz, MS and Ruan, Z},
title = {Engineered CRISPR-Cas systems for the detection and control of antibiotic-resistant infections.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {401},
pmid = {34863214},
issn = {1477-3155},
mesh = {Animals ; *Bacteria/genetics/pathogenicity ; *Bacterial Infections/diagnosis/microbiology/prevention &amp; control ; CRISPR-Cas Systems/*genetics ; Drug Delivery Systems ; *Drug Resistance, Bacterial ; Humans ; Mice ; },
abstract = {Antibiotic resistance is spreading rapidly around the world and seriously impeding efforts to control microbial infections. Although nucleic acid testing is widely deployed for the detection of antibiotic resistant bacteria, the current techniques-mainly based on polymerase chain reaction (PCR)-are time-consuming and laborious. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance (AMR). The CRISPR-Cas system is an adaptive immune system found in many prokaryotes that presents attractive opportunities to target and edit nucleic acids with high precision and reliability. Engineered CRISPR-Cas systems are reported to effectively kill bacteria or even revert bacterial resistance to antibiotics (resensitizing bacterial cells to antibiotics). Strategies for combating antimicrobial resistance using CRISPR (i.e., Cas9, Cas12, Cas13, and Cas14) can be of great significance in detecting bacteria and their resistance to antibiotics. This review discusses the structures, mechanisms, and detection methods of CRISPR-Cas systems and how these systems can be engineered for the rapid and reliable detection of bacteria using various approaches, with a particular focus on nanoparticles. In addition, we summarize the most recent advances in applying the CRISPR-Cas system for virulence modulation of bacterial infections and combating antimicrobial resistance.},
}
@article {pmid34862751,
year = {2021},
author = {Feng, Q and Xiao, L and He, Y and Liu, M and Wang, J and Tian, S and Zhang, X and Yuan, L},
title = {Highly efficient, genotype-independent transformation and gene editing in watermelon (Citrullus lanatus) using a chimeric ClGRF4-GIF1 gene.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {12},
pages = {2038-2042},
doi = {10.1111/jipb.13199},
pmid = {34862751},
issn = {1744-7909},
mesh = {CRISPR-Cas Systems/genetics ; *Citrullus/genetics ; *Gene Editing ; Genotype ; Plant Breeding ; Triticum/genetics ; },
abstract = {Efficient genetic transformation has the potential to advance research and breeding in watermelon (Citrullus lanatus), but regeneration from tissue culture remains challenging. Previous work showed that expressing a fusion of two interacting transcription factors, GROWTH-REGULATING FACTOR4 (GRF4) and GRF-INTERACTING FACTOR1 (GIF1), improved regeneration in wheat (Triticum aestivum). By overexpressing a chimeric fusion of ClGRF4 and ClGIF1, we achieved highly efficient transformation in watermelon. Mutating the mi396 microRNA target site in ClGRF further boosted the transformation efficiency up to 67.27% in a genotype-independent manner. ClGRF4-GIF1 can also be combined with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing tools to achieve highly efficient gene editing in watermelon, which we used to successfully create diploid seedless watermelon. This research thus puts forward a powerful transformation tool for future watermelon research and breeding.},
}
@article {pmid34862378,
year = {2021},
author = {Pallarès-Masmitjà, M and Ivančić, D and Mir-Pedrol, J and Jaraba-Wallace, J and Tagliani, T and Oliva, B and Rahmeh, A and Sánchez-Mejías, A and Güell, M},
title = {Find and cut-and-transfer (FiCAT) mammalian genome engineering.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {7071},
pmid = {34862378},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA Transposable Elements/*genetics ; Electroporation ; Female ; Gene Editing/*methods ; Humans ; Liver ; Male ; Mice ; Protein Engineering ; Transposases/*genetics/metabolism ; },
abstract = {While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes.},
}
@article {pmid34862376,
year = {2021},
author = {Douglas, C and Maciulyte, V and Zohren, J and Snell, DM and Mahadevaiah, SK and Ojarikre, OA and Ellis, PJI and Turner, JMA},
title = {CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6926},
pmid = {34862376},
issn = {2041-1723},
support = {FC001193/WT_/Wellcome Trust/United Kingdom ; BB/N000463/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; FC001193/ARC_/Arthritis Research UK/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; /CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Animal Husbandry ; Animals ; CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*methods ; Litter Size/genetics ; Male ; Mice ; Mice, Transgenic ; Models, Animal ; Pregnancy ; Selective Breeding ; Sex Determination Processes/*genetics ; Synthetic Lethal Mutations ; },
abstract = {Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.},
}
@article {pmid34860215,
year = {2021},
author = {Citartan, M},
title = {The dynamicity of light-up aptamers in one-pot in vitro diagnostic assays.},
journal = {The Analyst},
volume = {147},
number = {1},
pages = {10-21},
doi = {10.1039/d1an01690c},
pmid = {34860215},
issn = {1364-5528},
mesh = {*Oligonucleotides ; },
abstract = {Light-up aptamers are aptamers that ignite the fluorescence emission of certain dyes upon binding. Widely harnessed in in vivo imaging, the binding capacity of the light-up aptamers can also be deployed in in vitro diagnostic assays, engendering a mix-and-read format. Intrigued by this, I intend to provide an overview of the various formats of diagnostic assays developed using light-up aptamers from the direct modulation of the light-up aptamers, split aptamer-based configuration, strand displacement, in vitro transcription-based one-pot diagnostic assay, CRISPR-Cas system to the measurement of the ion reliance. The incorporation of the light-up aptamers into each configuration is expounded and further supported by describing the exemplary assays developed thus far. It is anticipated that the present study can be enlightening to any researchers who aspire to embark on the development of one-pot in vitro diagnostic assays based on light-up aptamers.},
}
@article {pmid34860046,
year = {2021},
author = {Lu, Q and Bhat, D and Stepanenko, D and Pigolotti, S},
title = {Search and Localization Dynamics of the CRISPR-Cas9 System.},
journal = {Physical review letters},
volume = {127},
number = {20},
pages = {208102},
doi = {10.1103/PhysRevLett.127.208102},
pmid = {34860046},
issn = {1079-7114},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; DNA/*metabolism ; },
abstract = {The CRISPR-Cas9 system acts as the prokaryotic immune system and has important applications in gene editing. The protein Cas9 is one of its crucial components. The role of Cas9 is to search for specific target sequences on the DNA and cleave them. In this Letter, we introduce a model of facilitated diffusion for Cas9 and fit its parameters to single-molecule experiments. Our model confirms that Cas9 search for targets by sliding, but shows that its sliding length is rather short. We then investigate how Cas9 explores a long stretch of DNA containing randomly placed targets. We solve this problem by mapping it into the theory of Anderson localization in condensed matter physics. Our theoretical approach rationalizes experimental evidence on the distribution of Cas9 molecules along the DNA.},
}
@article {pmid34858961,
year = {2021},
author = {Dong, H and Cui, Y and Zhang, D},
title = {CRISPR/Cas Technologies and Their Applications in Escherichia coli.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {9},
number = {},
pages = {762676},
pmid = {34858961},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have revolutionized genome editing and greatly promoted the development of biotechnology. However, these systems unfortunately have not been developed and applied in bacteria as extensively as in eukaryotic organism. Here, the research progress on the most widely used CRISPR/Cas tools and their applications in Escherichia coli is summarized. Genome editing based on homologous recombination, non-homologous DNA end-joining, transposons, and base editors are discussed. Finally, the state of the art of transcriptional regulation using CRISPRi is briefly reviewed. This review provides a useful reference for the application of CRISPR/Cas systems in other bacterial species.},
}
@article {pmid34858958,
year = {2021},
author = {Lin, X and Wang, G and Ma, L and Liu, G},
title = {Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {9},
number = {},
pages = {752514},
pmid = {34858958},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas is now playing a significant role in biosensing applications, especially when the trans-cleavage activity of several Cas effectors is discovered. Taking advantages of both CRISPR/Cas and the enzyme-linked immunosorbent assay (ELISA) in analytical and clinical investigations, CRISPR/Cas-powered ELISA has been successfully designed to detect a spectrum of analytes beyond nucleic acid. Herein, we developed a CRISPR/Cas12a-assisted new immunoassay (CANi) for detection of salivary insulin as an example. Specifically, factors (antibody selection, temperature, and assay time) affecting the CRISPR/Cas-based ELISA system's performance were investigated. It was observed that the concentration of blocking solution, selection of the capture antibody pairs, and the sequences of triggering ssDNA and guiding RNA affected this immunoassay sensitivity. In contrast, the preincubation of CRISPR/Cas12a working solution and pre-mixture of detection antibody with anti-IgG-ssDNA did not show influence on the performance of CANi for the detection of insulin. Under optimized conditions, the sensitivity for detection of salivary insulin was 10 fg/ml with a linear range from 10 fg/ml to 1 ng/ml.},
}
@article {pmid34858879,
year = {2021},
author = {Espada, CR and Quilles, JC and Albuquerque-Wendt, A and Cruz, MC and Beneke, T and Lorenzon, LB and Gluenz, E and Cruz, AK and Uliana, SRB},
title = {Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {772311},
pmid = {34858879},
issn = {2235-2988},
support = {15/16_MSD_836338/MRC_/Medical Research Council/United Kingdom ; 104111/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; DNA-Directed RNA Polymerases ; Gene Editing ; *Leishmania braziliensis/genetics ; *Leishmania major ; Viral Proteins ; },
abstract = {Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.},
}
@article {pmid34857736,
year = {2021},
author = {Zhong, L and Huang, Y and He, J and Yang, N and Xu, B and Ma, Y and Liu, J and Tang, C and Luo, C and Wu, P and Lai, Z and Huo, Y and Lu, T and Huang, D and Gong, W and Gan, L and Luo, Y and Zhang, Z and Liu, X and Zhao, Y},
title = {Generation of in situ CRISPR-mediated primary and metastatic cancer from monkey liver.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {411},
pmid = {34857736},
issn = {2059-3635},
mesh = {Animals ; *CRISPR-Cas Systems ; *INDEL Mutation ; *Liver Neoplasms/genetics/metabolism/pathology ; Macaca fascicularis ; Male ; Neoplasm Metastasis ; *PTEN Phosphohydrolase/genetics/metabolism ; *Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {Non-human primates (NHPs) represent the most valuable animals for drug discovery. However, the current main challenge remains that the NHP has not yet been used to develop an efficient translational medicine platform simulating human diseases, such as cancer. This study generated an in situ gene-editing approach to induce efficient loss-of-function mutations of Pten and p53 genes for rapid modeling primary and metastatic liver tumors using the CRISPR/Cas9 in the adult cynomolgus monkey. Under ultrasound guidance, the CRISPR/Cas9 was injected into the cynomolgus monkey liver through the intrahepatic portal vein. The results showed that the ultrasound-guided CRISPR/Cas9 resulted in indels of the Pten and p53 genes in seven out of eight monkeys. The best mutation efficiencies for Pten and p53 were up to 74.71% and 74.68%, respectively. Furthermore, the morbidity of primary and extensively metastatic (lung, spleen, lymph nodes) hepatoma in CRISPR-treated monkeys was 87.5%. The ultrasound-guided CRISPR system could have great potential to successfully pursue the desired target genes, thereby reducing possible side effects associated with hitting non-specific off-target genes, and significantly increasing more efficiency as well as higher specificity of in situ gene editing in vivo, which holds promise as a powerful, yet feasible tool, to edit disease genes to build corresponding human disease models in adult NHPs and to greatly accelerate the discovery of new drugs and save economic costs.},
}
@article {pmid34856977,
year = {2021},
author = {Liu, Y and Chen, Y and Dang, L and Liu, Y and Huang, S and Wu, S and Ma, P and Jiang, H and Li, Y and Pan, Y and Wei, Y and Ma, X and Liu, M and Ji, Q and Chi, T and Huang, X and Wang, X and Zhou, F},
title = {EasyCatch, a convenient, sensitive and specific CRISPR detection system for cancer gene mutations.},
journal = {Molecular cancer},
volume = {20},
number = {1},
pages = {157},
pmid = {34856977},
issn = {1476-4598},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genes, Neoplasm ; Genetic Testing/*methods/standards ; Humans ; Mutation ; Neoplasms/*diagnosis/*genetics ; Reproducibility of Results ; Sensitivity and Specificity ; },
}
@article {pmid34856461,
year = {2022},
author = {Byeon, E and Kim, MS and Kim, DH and Lee, Y and Jeong, H and Lee, JS and Hong, SA and Park, JC and Kang, HM and Sayed, AEH and Kato, Y and Bae, S and Watanabe, H and Lee, YH and Lee, JS},
title = {The freshwater water flea Daphnia magna NIES strain genome as a resource for CRISPR/Cas9 gene targeting: The glutathione S-transferase omega 2 gene.},
journal = {Aquatic toxicology (Amsterdam, Netherlands)},
volume = {242},
number = {},
pages = {106021},
doi = {10.1016/j.aquatox.2021.106021},
pmid = {34856461},
issn = {1879-1514},
mesh = {Animals ; *CRISPR-Cas Systems ; *Daphnia/genetics ; *Gene Targeting ; Glutathione Transferase/genetics ; },
abstract = {The water flea Daphnia magna is a small freshwater planktonic animal in the Cladocera. In this study, we assembled the genome of the D. magna NIES strain, which is widely used for gene targeting but has no reported genome. We used the long-read sequenced data of the Oxford nanopore sequencing tool for assembly. Using 3,231 genetic markers, the draft genome of the D. magna NIES strain was built into ten linkage groups (LGs) with 483 unanchored contigs, comprising a genome size of 173.47 Mb. The N50 value of the genome was 12.54 Mb and the benchmarking universal single-copy ortholog value was 98.8%. Repeat elements in the D. magna NIES genome were 40.8%, which was larger than other Daphnia spp. In the D. magna NIES genome, 15,684 genes were functionally annotated. To assess the genome of the D. magna NIES strain for CRISPR/Cas9 gene targeting, we selected glutathione S-transferase omega 2 (GST-O2), which is an important gene for the biotransformation of arsenic in aquatic organisms, and targeted it with an efficient make-up (25.0%) of mutant lines. In addition, we measured reactive oxygen species and antioxidant enzymatic activity between wild type and a mutant of the GST-O2 targeted D. magna NIES strain in response to arsenic. In this study, we present the genome of the D. magna NIES strain using GST-O2 as an example of gene targeting, which will contribute to the construction of deletion mutants by CRISPR/Cas9 technology.},
}
@article {pmid34854968,
year = {2022},
author = {Massel, K and Lam, Y and Hintzsche, J and Lester, N and Botella, JR and Godwin, ID},
title = {Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals.},
journal = {Plant cell reports},
volume = {41},
number = {2},
pages = {489-492},
pmid = {34854968},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems ; Edible Grain/genetics ; Gene Editing/*methods ; Plants, Genetically Modified ; *Promoter Regions, Genetic ; Sorghum/*genetics ; },
abstract = {Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.},
}
@article {pmid34854166,
year = {2022},
author = {Jiao, G and Wang, C and Chen, Y and Dai, M and Zhang, Y and Li, W},
title = {Enhancing targeted transgene knock-in by donor recruitment.},
journal = {Cell proliferation},
volume = {55},
number = {1},
pages = {e13163},
pmid = {34854166},
issn = {1365-2184},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Genetic Therapy ; HEK293 Cells ; Humans ; Patient Selection ; Recombinational DNA Repair/genetics ; Transgenes/*genetics ; },
}
@article {pmid34853819,
year = {2022},
author = {Li, C and Yang, T and Weng, Y and Zhang, M and Zhao, D and Guo, S and Hu, B and Shao, W and Wang, X and Hussain, A and Liang, XJ and Huang, Y},
title = {Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy.},
journal = {Bioactive materials},
volume = {9},
number = {},
pages = {590-601},
pmid = {34853819},
issn = {2452-199X},
abstract = {CRISPR/Cas9-based gene editing has emerged as a powerful biotechnological tool, that relies on Cas9 protein and single guided RNA (sgRNA) to edit target DNA. However, the lack of safe and efficient delivery carrier is one of the crucial factors restricting its clinical transformation. Here, we report an ionizable lipid nanoparticle (iLP181, pKa = 6.43) based on iLY1809 lipid enabling robust gene editing in vitro and in vivo. The iLP181 effectively encapsulate psgPLK1, the best-performing plasmid expressing for both Cas9 protein and sgRNA targeting Polo-like kinase 1 (PLK1). The iLP181/psgPLK1 nanoformulation showed uniformity in size, regular nanostructure and nearly neutral zeta potential at pH 7.4. The nanoformulation effectively triggered editing of PLK1 gene with more than 30% efficiency in HepG2-Luc cells. iLP181/psgPLK1 significantly accumulated in the tumor for more than 5 days after a single intravenous injection. In addition, it also achieved excellent tumor growth suppression compared to other nucleic acid modalities such as siRNA, without inducing adverse effects to the main organs including the liver and kidneys. This study not only provides a clinically-applicable lipid nanocarrier for delivering CRISPR/Cas system (even other bioactive molecules), but also constitutes a potential cancer treatment regimen base on DNA editing of oncogenes.},
}
@article {pmid34853396,
year = {2021},
author = {Bermúdez-Guzmán, L},
title = {Pan-cancer analysis of non-oncogene addiction to DNA repair.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {23264},
pmid = {34853396},
issn = {2045-2322},
mesh = {Biomarkers, Tumor/metabolism ; CRISPR-Cas Systems ; Cluster Analysis ; DNA Damage ; DNA Repair/*genetics ; Gene Expression Regulation, Neoplastic ; Genome, Human ; Humans ; Neoplasms/*genetics/*metabolism ; Oncogene Addiction/genetics ; Oncogenes ; RNA, Small Interfering/metabolism ; Transcriptome ; },
abstract = {Cancer cells usually depend on the aberrant function of one or few driver genes to initiate and promote their malignancy, an attribute known as oncogene addiction. However, cancer cells might become dependent on the normal cellular functions of certain genes that are not oncogenes but ensure cell survival (non-oncogene addiction). The downregulation or silencing of DNA repair genes and the consequent genetic and epigenetic instability is key to promote malignancy, but the activation of the DNA-damage response (DDR) has been shown to become a type of non-oncogene addiction that critically supports tumour survival. In the present study, a systematic evaluation of DNA repair addiction at the pan-cancer level was performed using data derived from The Cancer Dependency Map and The Cancer Genome Atlas (TCGA). From 241 DDR genes, 59 were identified as commonly essential in cancer cell lines. However, large differences were observed in terms of dependency scores in 423 cell lines and transcriptomic alterations across 18 cancer types. Among these 59 commonly essential genes, 14 genes were exclusively associated with better overall patient survival and 19 with worse overall survival. Notably, a specific molecular signature among the latter, characterized by DDR genes like UBE2T, RFC4, POLQ, BRIP1, and H2AFX showing the weakest dependency scores, but significant upregulation was strongly associated with worse survival. The present study supports the existence and importance of non-oncogenic addiction to DNA repair in cancer and may facilitate the identification of prognostic biomarkers and therapeutic opportunities.},
}
@article {pmid34853172,
year = {2021},
author = {Son, H and Park, J and Hwang, I and Jung, Y and Bae, S and Lee, S},
title = {Mg[2+]-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {49},
pages = {},
pmid = {34853172},
issn = {1091-6490},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/physiology ; DNA/chemistry/*metabolism ; DNA Cleavage/drug effects ; Deoxyribonuclease I/metabolism ; Endodeoxyribonucleases/*metabolism ; Gene Editing ; Magnesium/metabolism ; Models, Molecular ; Nucleic Acid Conformation/drug effects ; R-Loop Structures/*genetics ; RNA, Guide/metabolism ; Spectroscopy, Fourier Transform Infrared/methods ; },
abstract = {CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg[2+] ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg[2+]-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.},
}
@article {pmid34853037,
year = {2021},
author = {Vazquez, F and Sellers, WR},
title = {Are CRISPR Screens Providing the Next Generation of Therapeutic Targets?.},
journal = {Cancer research},
volume = {81},
number = {23},
pages = {5806-5809},
doi = {10.1158/0008-5472.CAN-21-1784},
pmid = {34853037},
issn = {1538-7445},
mesh = {*CRISPR-Cas Systems ; Humans ; *Molecular Targeted Therapy ; Neoplasm Proteins/*antagonists &amp; inhibitors/genetics ; Neoplasms/genetics/pathology/*therapy ; },
abstract = {CRISPR screens combined with molecular and genetic profiling of large panels of cell lines are helping to systematically identify cancer vulnerabilities. These large-scale screens, together with focused in vivo and isogenic cell line screens, have identified a growing number of promising targets and led directly to numerous target-specific drug discovery programs, several of which have reached clinical testing. However, systematic loss-of-function studies are still in their early stages. Genetic redundancy, the limitation of cell line models for many cancer types, and the difficulty of conducting complex in vitro and in vivo screens remain opportunities for discovery. We expect that over the next few years, efforts like the Cancer Dependency Map along with more focused screens will play a significant role in the creation of a roadmap of oncology therapeutic targets.},
}
@article {pmid34851176,
year = {2021},
author = {Kao, CY and Lu, JJ and Lin, LC and Lin, HC and Chang, SC},
title = {Phylogenetic Distribution of CRISPR-Cas Systems in Staphylococcus lugdunensis.},
journal = {Microbiology spectrum},
volume = {9},
number = {3},
pages = {e0124721},
pmid = {34851176},
issn = {2165-0497},
mesh = {Anti-Bacterial Agents/*pharmacology ; CRISPR-Cas Systems/*genetics ; DNA, Intergenic/genetics ; Drug Resistance, Bacterial/*genetics ; Humans ; Microbial Sensitivity Tests ; Multilocus Sequence Typing/methods ; Oxacillin/*pharmacology ; Phylogeny ; Staphylococcal Infections/drug therapy ; Staphylococcus lugdunensis/*drug effects/*genetics/isolation &amp; purification ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) genes (CRISPR-Cas) are present in many bacterial genomes with functions beyond adaptive immunity. We aimed to characterize the CRISPR-Cas system in the pathogenic Gram-positive bacterium Staphylococcus lugdunensis and determine its association with sequence types (STs) determined by multilocus sequence typing (MLST) and oxacillin susceptibility. Primers were designed to detect and sequence types IIIA and IIC CRISPR-Cas in 199 S. lugdunensis isolates. MLST and oxacillin susceptibility tests were also performed on the isolates. We found that 84 S. lugdunensis isolates had type IIIA CRISPR-Cas, while 46 had type IIC. The results showed a strong association between STs and CRISPR-Cas types. The ST1, ST6, ST12, and ST15 isolates had type IIIA CRISPR-Cas systems, and the ST4, ST27, and ST29 isolates had type IIC CRISPR-Cas. Interestingly, of 83 isolates containing type IIIA CRISPR-Cas, 17 (20.5%) were oxacillin-resistant S. lugdunensis (ORSL), and all of these ORSL isolates belonged to ST6 cluster 1. Moreover, spacers 23 and 21 were found in 16 and 17 ORSL isolates, respectively. In contrast, all 46 isolates with type IIC CRISPR-Cas were susceptible to oxacillin. Our results showed that 41.3% of CRISPR-Cas IIIA spacers were homologous to plasmids and 20.2% were homologous to phages. However, in type IIC CRISPR-Cas, 11.8% and 39.9% of spacers showed sequence homology with plasmids and phages, respectively. In conclusion, we found that the distribution and composition of the CRISPR-Cas system in S. lugdunensis was associated with STs and oxacillin susceptibility. IMPORTANCE CRISPR-Cas systems have been characterized as playing several biological roles in many bacterial genomes. Moreover, CRISPR-Cas systems are useful for epidemiological, diagnostic, and evolutionary studies of pathogenic bacteria. However, the characteristics of CRISPR-Cas systems in Staphylococcus lugdunensis have been rarely reported. In this study, we revealed that type IIIA CRISPR-Cas was dominant in S. lugdunensis isolates, followed by type IIC CRISPR-Cas. Moreover, the composition of CRISPR-Cas spacers was strongly associated with multilocus sequence typing and oxacillin susceptibility of S. lugdunensis. These results advance our understanding of the evolution of CRISPR-Cas systems; however, the biological functions of CRISPR-Cas systems in S. lugdunensis remain to be further characterized.},
}
@article {pmid34850950,
year = {2022},
author = {Wang, L and Wang, Y and Makhmoudova, A and Nitschke, F and Tetlow, IJ and Emes, MJ},
title = {CRISPR-Cas9-mediated editing of starch branching enzymes results in altered starch structure in Brassica napus.},
journal = {Plant physiology},
volume = {188},
number = {4},
pages = {1866-1886},
pmid = {34850950},
issn = {1532-2548},
mesh = {*1,4-alpha-Glucan Branching Enzyme/genetics ; *Brassica napus/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Starch ; },
abstract = {Starch branching enzymes (SBEs) are one of the major classes of enzymes that catalyze starch biosynthesis in plants. Here, we utilized the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene editing system to investigate the effects of SBE mutation on starch structure and turnover in the oilseed crop Brassica napus. Multiple single-guide RNA (sgRNA) expression cassettes were assembled into a binary vector and two rounds of transformation were employed to edit all six BnaSBE genes. All mutations were heterozygous monoallelic or biallelic, and no chimeric mutations were detected from a total of 216 editing events. Previously unannotated gene duplication events associated with two BnaSBE genes were characterized through analysis of DNA sequencing chromatograms, reflecting the complexity of genetic information in B. napus. Five Cas9-free homozygous mutant lines carrying two to six mutations of BnaSBE were obtained, allowing us to compare the effect of editing different BnaSBE isoforms. We also found that in the sextuple sbe mutant, although indels were introduced at the genomic DNA level, an alternate transcript of one BnaSBE2.1 gene bypassed the indel-induced frame shift and was translated to a modified full-length protein. Subsequent analyses showed that the sextuple mutant possesses much lower SBE enzyme activity and starch branching frequency, higher starch-bound phosphate content, and altered pattern of amylopectin chain length distribution relative to wild-type (WT) plants. In the sextuple mutant, irregular starch granules and a slower rate of starch degradation during darkness were observed in rosette leaves. At the pod-filling stage, the sextuple mutant was distinguishable from WT plants by its thick main stem. This work demonstrates the applicability of the CRISPR-Cas9 system for the study of multi-gene families and for investigation of gene-dosage effects in the oil crop B. napus. It also highlights the need for rigorous analysis of CRISPR-Cas9-mutated plants, particularly with higher levels of ploidy, to ensure detection of gene duplications.},
}
@article {pmid34850861,
year = {2021},
author = {Nitahara-Kasahara, Y and Mizumoto, S and Inoue, YU and Saka, S and Posadas-Herrera, G and Nakamura-Takahashi, A and Takahashi, Y and Hashimoto, A and Konishi, K and Miyata, S and Masuda, C and Matsumoto, E and Maruoka, Y and Yoshizawa, T and Tanase, T and Inoue, T and Yamada, S and Nomura, Y and Takeda, S and Watanabe, A and Kosho, T and Okada, T},
title = {A new mouse model of Ehlers-Danlos syndrome generated using CRISPR/Cas9-mediated genomic editing.},
journal = {Disease models &amp; mechanisms},
volume = {14},
number = {12},
pages = {},
pmid = {34850861},
issn = {1754-8411},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Ehlers-Danlos Syndrome/genetics/pathology ; Female ; Genomics ; Mice ; Mice, Knockout ; Pregnancy ; Sulfotransferases/genetics/metabolism ; },
abstract = {Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies.},
}
@article {pmid34850143,
year = {2021},
author = {Molina, R and Jensen, ALG and Marchena-Hurtado, J and López-Méndez, B and Stella, S and Montoya, G},
title = {Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.},
journal = {Nucleic acids research},
volume = {49},
number = {21},
pages = {12577-12590},
pmid = {34850143},
issn = {1362-4962},
mesh = {Adenine Nucleotides/chemistry/*metabolism ; Binding Sites/genetics ; Biocatalysis ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; Chromatography, Liquid ; Crystallography, X-Ray ; Endonucleases/chemistry/genetics/*metabolism ; Kinetics ; Mass Spectrometry/methods ; Models, Molecular ; Mutation ; Nucleotides, Cyclic/chemistry/*metabolism ; Oligoribonucleotides/chemistry/*metabolism ; Protein Domains ; Sulfolobus solfataricus/*enzymology/genetics ; },
abstract = {Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.},
}
@article {pmid34850137,
year = {2022},
author = {Corsi, GI and Gadekar, VP and Gorodkin, J and Seemann, SE},
title = {CRISPRroots: on- and off-target assessment of RNA-seq data in CRISPR-Cas9 edited cells.},
journal = {Nucleic acids research},
volume = {50},
number = {4},
pages = {e20},
pmid = {34850137},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; RNA, Guide/genetics ; RNA-Seq ; },
abstract = {The CRISPR-Cas9 genome editing tool is used to study genomic variants and gene knockouts, and can be combined with transcriptomic analyses to measure the effects of such alterations on gene expression. But how can one be sure that differential gene expression is due to a successful intended edit and not to an off-target event, without performing an often resource-demanding genome-wide sequencing of the edited cell or strain? To address this question we developed CRISPRroots: CRISPR-Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites. Our method combines Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells to discover potential off-targets. Applied on seven public datasets, CRISPRroots identified critical off-target candidates that were overlooked in all of the corresponding previous studies. CRISPRroots is available via https://rth.dk/resources/crispr.},
}
@article {pmid34849868,
year = {2021},
author = {Huang, R and Shi, M and Luo, L and Yang, C and Ou, M and Zhang, W and Liao, L and Li, Y and Xia, XQ and Zhu, Z and Wang, Y},
title = {De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.},
journal = {GigaScience},
volume = {10},
number = {11},
pages = {},
pmid = {34849868},
issn = {2047-217X},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosomes ; *Cyprinidae/genetics ; Gene Editing/methods ; Mutation ; },
abstract = {BACKGROUND: Mutants are important for the discovery of functional genes and creation of germplasm resources. Mutant acquisition depends on the efficiency of mutation technology and screening methods. CRISPR-Cas9 technology is an efficient gene editing technology mainly used for editing a few genes or target sites, which has not been applied for the construction of random mutant libraries and for the de novo discovery of functional genes.<br><br>RESULTS: In this study, we first sequenced and assembled the chromosome-level genome of wild-type rare minnow (Gobiocypris rarus) as a susceptible model of hemorrhagic disease, obtained a 956.05&#xa0;Mb genome sequence, assembled the sequence into 25 chromosomes, and annotated 26,861 protein-coding genes. Thereafter, CRISPR-Cas9 technology was applied to randomly mutate the whole genome of rare minnow with the conserved bases (TATAWAW and ATG) of the promoter and coding regions as the target sites. The survival rate of hemorrhagic disease in the rare minnow gradually increased from 0% (the entire wild-type population died after infection) to 38.24% (F3 generation). Finally, 7 susceptible genes were identified via genome comparative analysis and cell-level verification based on the rare minnow genome.<br><br>CONCLUSIONS: The results provided the genomic resources for wild-type rare minnow, and confirmed that the random mutation system designed using CRISPR-Cas9 technology in this study is simple and efficient and is suitable for the de novo discovery of functional genes and creation of a germplasm resource related to qualitative traits.},
}
@article {pmid34849815,
year = {2021},
author = {Iwata, S and Sasaki, T and Nagahara, M and Iwamoto, T},
title = {An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {8},
pages = {},
pmid = {34849815},
issn = {2160-1836},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; Gonads ; Mice ; Mice, Inbred C3H ; *Nucleic Acids ; },
abstract = {As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.},
}
@article {pmid34849607,
year = {2021},
author = {Donnelly, LL and Hogan, TC and Lenahan, SM and Nandagopal, G and Eaton, JG and Lebeau, MA and McCann, CL and Sarausky, HM and Hampel, KJ and Armstrong, JD and Cameron, MP and Sidiropoulos, N and Deming, P and Seward, DJ},
title = {Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers.},
journal = {Carcinogenesis},
volume = {42},
number = {12},
pages = {1428-1438},
pmid = {34849607},
issn = {1460-2180},
mesh = {AMP-Activated Protein Kinase Kinases/*genetics/metabolism ; Alternative Splicing ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/diagnosis/*genetics/mortality ; DNA Mutational Analysis ; Disease Susceptibility ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease ; Humans ; Lung Neoplasms/diagnosis/*genetics/mortality ; Mutagenesis, Site-Directed ; *Mutation ; Mutation, Missense ; Phosphorylation ; Prognosis ; RNA Splice Sites ; },
abstract = {Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.},
}
@article {pmid34849487,
year = {2021},
author = {Adhikari, B and Narain, A and Wolf, E},
title = {Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100949},
pmid = {34849487},
issn = {2666-1667},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cloning, Molecular/*methods ; Gene Knock-In Techniques/*methods ; Humans ; Plant Proteins/*genetics ; *Proteolysis ; RNA, Guide/genetics ; Transfection ; },
abstract = {Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021).},
}
@article {pmid34848728,
year = {2021},
author = {Wang, L and Liu, H and Zhang, X and Song, E and Wang, Y and Xu, T and Li, Z},
title = {WFS1 functions in ER export of vesicular cargo proteins in pancreatic β-cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6996},
pmid = {34848728},
issn = {2041-1723},
mesh = {Animals ; Biological Transport ; COP-Coated Vesicles ; CRISPR-Cas Systems ; Diabetes Mellitus ; Endoplasmic Reticulum/*metabolism ; Golgi Apparatus ; HEK293 Cells ; Humans ; Insulin Secretion ; Insulin-Secreting Cells/*metabolism ; Membrane Proteins/*genetics/*metabolism ; Mice ; Mice, Knockout ; Proinsulin ; },
abstract = {The sorting of soluble secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex is mediated by coat protein complex II (COPII) vesicles and thought to required specific ER membrane cargo-receptor proteins. However, these receptors remain largely unknown. Herein, we show that ER to Golgi transfer of vesicular cargo proteins requires WFS1, an ER-associated membrane protein whose loss of function leads to Wolfram syndrome. Mechanistically, WFS1 directly binds to vesicular cargo proteins including proinsulin via its ER luminal C-terminal segment, whereas pathogenic mutations within this region disrupt the interaction. The specific ER export signal encoded in the cytosolic N-terminal segment of WFS1 is recognized by the COPII subunit SEC24, generating mature COPII vesicles that traffic to the Golgi complex. WFS1 deficiency leads to abnormal accumulation of proinsulin in the ER, impeding the proinsulin processing as well as insulin secretion. This work identifies a vesicular cargo receptor for ER export and suggests that impaired peptide hormone transport underlies diabetes resulting from pathogenic WFS1 mutations.},
}
@article {pmid34848542,
year = {2021},
author = {Grubisha, MJ and Sun, T and Eisenman, L and Erickson, SL and Chou, S and Helmer, CD and Trudgen, MT and Ding, Y and Homanics, GE and Penzes, P and Wills, ZP and Sweet, RA},
title = {A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {49},
pages = {},
pmid = {34848542},
issn = {1091-6490},
support = {R01 MH097216/MH/NIMH NIH HHS/United States ; K08 MH118513/MH/NIMH NIH HHS/United States ; R56 MH071316/MH/NIMH NIH HHS/United States ; U01 AA020889/AA/NIAAA NIH HHS/United States ; R01 MH071316/MH/NIMH NIH HHS/United States ; R56 AG058593/AG/NIA NIH HHS/United States ; R01 MH071533/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cerebral Cortex/*cytology ; Dendrites/*physiology ; GPI-Linked Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental/*physiology ; Genotype ; Guanine Nucleotide Exchange Factors/genetics/*metabolism ; Humans ; Mice ; Mice, Transgenic ; Mutation, Missense ; Myelin Proteins/genetics/metabolism ; Neurons/*physiology ; Nogo Receptor 1/genetics/metabolism ; Sexual Maturation ; Signal Transduction/*physiology ; },
abstract = {Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.},
}
@article {pmid34847747,
year = {2021},
author = {Sabharwal, A and Kar, B and Restrepo-Castillo, S and Holmberg, SR and Mathew, ND and Kendall, BL and Cotter, RP and WareJoncas, Z and Seiler, C and Nakamaru-Ogiso, E and Clark, KJ and Ekker, SC},
title = {The FusX TALE Base Editor (FusXTBE) for Rapid Mitochondrial DNA Programming of Human Cells In Vitro and Zebrafish Disease Models In Vivo.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {799-821},
pmid = {34847747},
issn = {2573-1602},
support = {R01 GM063904/GM/NIGMS NIH HHS/United States ; U01 AI142773/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *DNA, Mitochondrial/genetics ; Gene Editing ; Humans ; Mitochondria/genetics ; *Zebrafish/genetics ; },
abstract = {Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo. Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.},
}
@article {pmid34847745,
year = {2021},
author = {Perez-Leal, O and Nixon-Abell, J and Barrero, CA and Gordon, JC and Oesterling, J and Rico, MC},
title = {Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, Mitochondrial Dynamics, and Cell Growth.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {854-871},
pmid = {34847745},
issn = {2573-1602},
support = {R03 DK105267/DK/NIDDK NIH HHS/United States ; },
mesh = {*Autophagy ; *COVID-19/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Targeting ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Humans ; Microscopy ; *Mitochondrial Dynamics ; *SARS-CoV-2/genetics/metabolism ; *Viroporin Proteins/genetics/metabolism ; },
abstract = {The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.},
}
@article {pmid34847743,
year = {2021},
author = {Wimmer, T and Bonthu, D and Moeschl, V and Kleekamp, P and Thiel, C and Lytvynchuk, L and Ellinwood, M and Stieger, K},
title = {A Bioluminescence Resonance Energy Transfer-Based Reporter System: Characterization and Applications.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {884-895},
doi = {10.1089/crispr.2021.0023},
pmid = {34847743},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Energy Transfer ; *Gene Editing/methods ; RNA, Guide/genetics ; },
abstract = {Genome editing strategies and DNA repair research need powerful analytical tools. We generated a bioluminescence resonance energy transfer (BRET)-based reporter for the quantification of indel frequencies induced by DNA repair. The BRET reporter, expressed as a single molecule, consists of a mutated Renilla reniformis luciferase domain and a GFP2 domain separated by a shuttle-cloning box for the integration of any given endonuclease target sequence. The luciferase activity acts both as energy donor and as the internal standard, while the loss of GFP2 fluorescence acts as a reporter for the out-of-frame sequence alterations that result from the DNA repair via the non-homologous end joining/microhomology-mediated end joining DNA repair pathways of the endonuclease-mediated DNA double-strand break. This results in a decrease of the fluorescence/luminescence ratio. Employing this reporter in different experimental scenarios, using different cell lines and diseases targeted, we quantified the influence of both protein knockdown of DNA repair pathways as well as guide RNA mismatches on CRISPR-mediated nuclease activity and subsequent repair based on mutagenic repair on the reporter. In conclusion, we demonstrated this BRET-based reporter to be a robust and sensitive analytical tool for assessment of variety of different genome editing-based approaches.},
}
@article {pmid34847741,
year = {2021},
author = {Gao, M and Zhu, X and Yang, G and Bao, J and Bu, H},
title = {CRISPR/Cas9-Mediated Gene Editing in Porcine Models for Medical Research.},
journal = {DNA and cell biology},
volume = {40},
number = {12},
pages = {1462-1475},
doi = {10.1089/dna.2020.6474},
pmid = {34847741},
issn = {1557-7430},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Humans ; Models, Animal ; Swine/*genetics ; },
abstract = {Pigs have been extensively used as the research models for human disease pathogenesis and gene therapy. They are also the optimal source of cells, tissues, and organs for xenotransplantation due to anatomical and physiological similarities to humans. Several breakthroughs in gene-editing technologies, including the advent of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9), have greatly improved the efficiency of genetic manipulation and significantly broadened the application of gene-edited large animal models. In this review, we have not only outlined the important applications of the CRISPR/Cas9 system in pigs as a means to study human diseases but also discussed the potential challenges of the use of CRISPR/Cas9 in large animals.},
}
@article {pmid34847728,
year = {2021},
author = {Yang, J and Yang, J and Zhang, Y and Yang, S and Zhang, J and Jiang, Y and Yang, S},
title = {CRISPR-Associated Transposase System Can Insert Multiple Copies of Donor DNA into the Same Target Locus.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {789-798},
doi = {10.1089/crispr.2021.0019},
pmid = {34847728},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; Gene Editing ; *Transposases/genetics/metabolism ; },
abstract = {The CRISPR-associated transposase system enables site-specific DNA integration on the genome independent of homologous recombination. Previous studies have demonstrated that the type V-K CRISPR-associated Tn7-like transposase system from Scytonema hofmanni and the type I-F system from Vibrio cholerae have strong target immunity like Tn7, and therefore two or more copies of the donor DNA would not be inserted into the same target location in theory. In this paper, we report that the type I-F system can insert multiple donor copies into one site, which was identified and confirmed by single-strain identification and high-throughput sequencing. This result is beneficial for our application of multicopy chromosomal integration by CRISPR-associated transposases, allowing more donor insertions into the chromosome. This unexpected result shows that the target immunity mechanism of this system has not been fully understood. Attention should be paid to the possibility of multiple insertions and their effects in related research.},
}
@article {pmid34846104,
year = {2022},
author = {Vu, TV and Das, S and Nguyen, CC and Kim, J and Kim, JY},
title = {Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100413},
doi = {10.1002/biot.202100413},
pmid = {34846104},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated.<br><br>PURPOSE AND SCOPE: Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing.<br><br>SUMMARY AND CONCLUSION: The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.},
}
@article {pmid34845024,
year = {2021},
author = {Rybarski, JR and Hu, K and Hill, AM and Wilke, CO and Finkelstein, IJ},
title = {Metagenomic discovery of CRISPR-associated transposons.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {49},
pages = {},
pmid = {34845024},
issn = {1091-6490},
support = {R01 GM088344/GM/NIGMS NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/physiology ; DNA Transposable Elements/*genetics/physiology ; Endonucleases/genetics ; Gene Editing ; Metagenome ; Metagenomics/methods ; RNA, Guide/genetics ; Transposases/genetics ; },
abstract = {CRISPR-associated Tn7 transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover architectures for all known CASTs, including arrangements of the Cascade effectors, target homing modalities, and minimal V-K systems. We also describe families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These systems shed light on how CRISPR systems have coevolved with transposases and expand the programmable gene-editing toolkit.},
}
@article {pmid34845012,
year = {2021},
author = {Oberhofer, G and Ivy, T and Hay, BA},
title = {Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {49},
pages = {},
pmid = {34845012},
issn = {1091-6490},
support = {P40 OD018537/OD/NIH HHS/United States ; T32 GM007616/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; Gene Drive Technology/*methods ; Gene Expression Regulation/genetics ; Genes, Essential/*genetics/physiology ; Models, Genetic ; Nuclear Proteins/genetics/metabolism ; Population Control/*methods ; RNA, Guide/genetics/metabolism ; Temperature ; Transgenes ; },
abstract = {One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. As a consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition dependent, so too will be the survival of that population. To test this idea, we created a ClvR in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvR[dbe]). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvR[db][e] are shifted to 29 °C, death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in homing-based drive and to bring about suppression and/or killing of specific individuals in response to other stimuli.},
}
@article {pmid34844120,
year = {2022},
author = {Qiu, Z and Park, A and Wang, L and Wilsey, R and Lee, M},
title = {The RGD (Arg-Gly-Asp) is a potential cell-binding motif of UNC-52/PERLECAN.},
journal = {Biochemical and biophysical research communications},
volume = {586},
number = {},
pages = {143-149},
doi = {10.1016/j.bbrc.2021.11.083},
pmid = {34844120},
issn = {1090-2104},
mesh = {Amino Acid Motifs ; Amino Acid Substitution ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/metabolism ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Conserved Sequence ; Embryo, Nonmammalian ; Gene Expression Regulation ; Integrin beta Chains/*genetics/metabolism ; Membrane Proteins/*genetics/metabolism ; Mutation ; Oligopeptides/*metabolism ; Phenotype ; Protein Binding ; Proteoglycans/*genetics/metabolism ; Signal Transduction ; Talin/*genetics/metabolism ; },
abstract = {UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian β integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the [2021]RGD[2023] motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE[2023] caused defective actin filaments and aberrant localization of PAT-3 β integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD[2023] resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 β integrin null allele. These results indicate that RGD[2023] is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.},
}
@article {pmid34843938,
year = {2022},
author = {Li, H and Huang, X and Yang, Y and Chen, X and Yang, Y and Wang, J and Jiang, H},
title = {The short neuropeptide F receptor regulates olfaction-mediated foraging behavior in the oriental fruit fly Bactrocera dorsalis (Hendel).},
journal = {Insect biochemistry and molecular biology},
volume = {140},
number = {},
pages = {103697},
doi = {10.1016/j.ibmb.2021.103697},
pmid = {34843938},
issn = {1879-0240},
mesh = {Animals ; Arthropod Antennae/metabolism ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/metabolism ; Feeding Behavior/*physiology ; Immunohistochemistry ; Insect Proteins/genetics/metabolism ; Neurons/metabolism ; Neuropeptides/metabolism ; Receptors, G-Protein-Coupled/genetics/metabolism ; *Receptors, Neuropeptide/genetics/metabolism ; Receptors, Odorant/genetics/metabolism ; Smell/*physiology ; *Tephritidae/genetics/metabolism/physiology ; },
abstract = {The short neuropeptide F (sNPF) signaling system, consisting of sNPF and its receptor (sNPFR), influences many physiological processes in insects, including feeding, growth and olfactory memory. We previously showed that sNPF regulates olfactory sensitivity in the oriental fruit fly Bactrocera dorsalis (Hendel) during starvation. However, the functional analysis of sNPFR is constrained by the failure of RNA interference in this species. Here, we generated a null sNPFR mutant using the CRISPR/Cas9 system to investigate the physiological roles of this receptor in more detail. G0 adults were produced at a frequency of 60.8%, and sNPFR[-/-] mutants were obtained after several generations of backcrossing followed by self-crossing among heterozygous flies. We found that the mutants were significantly less successful at foraging for certain foods and showed increased foraging latency. Electroantennogram (EAG) assays indicated that the mutants had significantly lower electrophysiological responses to three tested odorants. Furthermore, qPCR data revealed the inhibition of several olfactory receptor genes, including Orco. Immunohistochemistry showed that BdsNPFR was localized in cells under the sensillum on the antennae. Based on their shape and size, the BdsNPFR[+] cells differ from odorant receptor neurons (ORNs), which were labeled using a Drosophila melanogaster Orco antibody. Our data suggest that sNPFR regulates olfaction-mediated foraging behavior by mediating interactions between BdsNPFR[+] cells and selected ORNs.},
}
@article {pmid34843826,
year = {2022},
author = {Fathi Karkan, S and Maleki Baladi, R and Shahgolzari, M and Gholizadeh, M and Shayegh, F and Arashkia, A},
title = {The evolving direct and indirect platforms for the detection of SARS-CoV-2.},
journal = {Journal of virological methods},
volume = {300},
number = {},
pages = {114381},
pmid = {34843826},
issn = {1879-0984},
mesh = {Antibodies, Viral ; *COVID-19 ; Clinical Laboratory Techniques ; Humans ; Point-of-Care Testing ; *SARS-CoV-2 ; },
abstract = {Diagnosis of SARS-CoV-2 by standard screening measures can reduce the chance of COVID-19 spread before the symptoms become severe. Detecting viral RNA and antigens, anti-viral antibodies, and CT-scan are the most routine diagnostic methods. Accordingly, several diagnostic platforms including thermal and isothermal amplifications, CRISPR/Cas‑based approaches, digital PCR, ELISA, NGS, and point-of-care testing methods with variable sensitivities, have been developed that may facilitate managing and preventing the further spread of the infection. Here, we summarized the currently available direct and indirect testing platforms in research and clinical settings, including recent progress in the methods to detect viral RNA, antigens, and specific antibodies. This summary may help in selecting the effective method for a special application sucha as routine laboratory diagnosis, point-of-care tests or tracing the the virus spread and mutations.},
}
@article {pmid34842768,
year = {2021},
author = {Lister, NC and Milton, AM and Hanrahan, BJ and Waters, PD},
title = {Between the Devil and the Deep Blue Sea: Non-Coding RNAs Associated with Transmissible Cancers in Tasmanian Devil, Domestic Dog and Bivalves.},
journal = {Non-coding RNA},
volume = {7},
number = {4},
pages = {},
pmid = {34842768},
issn = {2311-553X},
abstract = {Currently there are nine known examples of transmissible cancers in nature. They have been observed in domestic dog, Tasmanian devil, and six bivalve species. These tumours can overcome host immune defences and spread to other members of the same species. Non-coding RNAs (ncRNAs) are known to play roles in tumorigenesis and immune system evasion. Despite their potential importance in transmissible cancers, there have been no studies on ncRNA function in this context to date. Here, we present possible applications of the CRISPR/Cas system to study the RNA biology of transmissible cancers. Specifically, we explore how ncRNAs may play a role in the immortality and immune evasion ability of these tumours.},
}
@article {pmid34842509,
year = {2022},
author = {Wang, D and Chen, G and Lyu, Y and Feng, E and Zhu, L and Pan, C and Zhang, W and Liu, X and Wang, H},
title = {A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis.},
journal = {Emerging microbes &amp; infections},
volume = {11},
number = {1},
pages = {428-437},
pmid = {34842509},
issn = {2222-1751},
mesh = {Anthrax/*diagnosis ; Antisense Elements (Genetics)/genetics ; Bacillus anthracis/*genetics/isolation &amp; purification ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; DNA, Bacterial/genetics ; DNA, Catalytic/*genetics ; Endodeoxyribonucleases/*genetics/metabolism ; G-Quadruplexes ; Plasmids/genetics ; },
abstract = {As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.},
}
@article {pmid34842230,
year = {2021},
author = {Zhang, L and Huang, R and Lu, L and Fu, R and Guo, G and Gu, Y and Liu, Z and He, L and Malissen, M and Liang, Y},
title = {Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {177},
pages = {},
doi = {10.3791/62328},
pmid = {34842230},
issn = {1940-087X},
mesh = {*COVID-19 ; *CRISPR-Cas Systems ; Cell Line ; Gene Knock-In Techniques ; Humans ; Macrophages ; Pandemics ; SARS-CoV-2 ; T-Lymphocytes ; },
abstract = {Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.},
}
@article {pmid34841793,
year = {2021},
author = {Liu, T and Tian, Y and Liu, C and Fang, S and Wu, Y and Wu, M and Li, B and Yang, H and Liu, Q},
title = {[Application of CRISPR/Cas-based biosensors for detecting nucleic acid of pathogens].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {11},
pages = {3890-3904},
doi = {10.13345/j.cjb.200679},
pmid = {34841793},
issn = {1872-2075},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Nucleic Acids/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats -associated protein (CRISPR/Cas) has been developed as a precise, efficient, affordable and sensitive nucleic acid detection tool due to its efficient targeted binding ability and programmability. At present, biosensors based on CRISPR-Cas system have shown excellent performance in the detection of nucleic acid of pathogens, which has attracted widespread attention, and is expected to replace the conventional detection methods. This review summarizes the latest research progress of biosensors based on CRISPR/Cas system for detecting nucleic acid of pathogens.},
}
@article {pmid34841792,
year = {2021},
author = {Hu, X and Wang, S and Yu, L and Zhang, X and Chen, W},
title = {[Advances of Cas9/sgRNA delivery system for gene editing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {11},
pages = {3880-3889},
doi = {10.13345/j.cjb.210040},
pmid = {34841792},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; },
abstract = {In the application of CRISPR genome editing, direct cellular delivery of non-replicable Cas9/sgRNA may reduce unwanted gene targeting and integrational mutagenesis, thus offering greater specificity and safety. Cas9/sgRNA delivery system holds great potential for treating genetic diseases. This review summarizes the advances of Cas9/sgRNA delivery systems and its therapeutic applications, providing new understandings and inspirations for vector design and future clinical applications.},
}
@article {pmid34841437,
year = {2022},
author = {Tanaka, K and Kandori, S and Sakka, S and Nitta, S and Tanuma, K and Shiga, M and Nagumo, Y and Negoro, H and Kojima, T and Mathis, BJ and Shimazui, T and Watanabe, M and Sato, TA and Miyamoto, T and Matsuzaka, T and Shimano, H and Nishiyama, H},
title = {ELOVL2 promotes cancer progression by inhibiting cell apoptosis in renal cell carcinoma.},
journal = {Oncology reports},
volume = {47},
number = {2},
pages = {},
pmid = {34841437},
issn = {1791-2431},
mesh = {Apoptosis/*genetics ; CRISPR-Cas Systems ; Carcinoma, Renal Cell/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Disease Progression ; Fatty Acid Elongases/*genetics ; Gene Knockdown Techniques ; Humans ; Kidney Neoplasms/*genetics/pathology ; Lipid Metabolism/*genetics ; },
abstract = {Renal cell carcinoma (RCC) is an aggressive genitourinary malignancy which has been associated with a poor prognosis, particularly in patients with metastasis, its major subtypes being clear cell RCC (ccRCC), papillary PCC (pRCC) and chromophobe RCC (chRCC). The presence of intracellular lipid droplets (LDs) is considered to be a hallmark of ccRCC. The importance of an altered lipid metabolism in ccRCC has been widely recognized. The elongation of very‑long‑chain fatty acid (ELOVL) catalyzes the elongation of fatty acids (FAs), modulating lipid composition, and is required for normal bodily functions. However, the involvement of elongases in RCC remains unclear. In the present study, the expression of ELOVL2 in ccRCC was examined; in particular, high levels of seven ELOVL isozymes were observed in primary tumors. Of note, elevated ELOVL2 expression levels were observed in ccRCC, as well as in pRCC and chRCC. Furthermore, a higher level of ELOVL2 was significantly associated with the increased incidence of a poor prognosis of patients with ccRCC and pRCC. The CRISPR/Cas9‑mediated knockdown of ELOVL2 resulted in the suppression of the elongation of long‑chain polyunsaturated FAs and increased LD production in renal cancer cells. Moreover, ELOVL2 ablation resulted in the suppression of cellular proliferation via the induction of apoptosis in vitro and the attenuation of tumor growth in vivo. On the whole, the present study provides new insight into the tumor proliferation mechanisms involving lipid metabolism, and suggests that ELOVL2 may be an attractive novel target for RCC therapy.},
}
@article {pmid34841349,
year = {2021},
author = {Dixit, B and Prakash, A and Kumar, P and Gogoi, P and Kumar, M},
title = {The core Cas1 protein of CRISPR-Cas I-B in Leptospira shows metal-tunable nuclease activity.},
journal = {Current research in microbial sciences},
volume = {2},
number = {},
pages = {100059},
pmid = {34841349},
issn = {2666-5174},
abstract = {Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 is the causative agent of leptospirosis in animals and humans. This organism carries a functional cas1 gene classified under CRISPR-Cas I-B. In this study, using various nuclease assays and bioinformatics analysis, we report that the recombinant Cas1 (LinCas1) possesses metal-ion dependent DNase activity, which is inhibited upon substitution or chelation of metal-ion and/or interaction with recombinant Cas2 (LinCas2) of L. interrogans. Model of LinCas1 structure shows a shorter N-terminal domain unlike other Cas1 orthologs reported to date. The C-terminal domain of LinCas1 contains conserved divalent-metal binding residues (Glu108, His176, and Glu191) and the mutation of these residues leads to abolition in DNase activity. Immunoassay using anti-LinCas2 demonstrates that LinCas1 interacts with LinCas2 and attains a saturation point. Moreover, the nuclease activity of the LinCas1-Cas2 mixture on ds-DNA displayed a reduction in activity compared to the pure core LinCas proteins under in vitro condition. The DNase activity for LinCas1 is consistent with a role for this protein in the recognition/cleavage of foreign DNA and integration of foreign DNA as spacer into the CRISPR array.},
}
@article {pmid34841331,
year = {2021},
author = {Dixit, B and Anand, V and Hussain, MS and Kumar, M},
title = {The CRISPR-associated Cas4 protein from Leptospira interrogans demonstrate versatile nuclease activity.},
journal = {Current research in microbial sciences},
volume = {2},
number = {},
pages = {100040},
pmid = {34841331},
issn = {2666-5174},
abstract = {The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the adaptation module in many variants of the CRISPR-Cas system in prokaryotes against the invading genetic elements. Cas4 is recognized as a DNA exonuclease that contains a RecB nuclease domain and a Fe-S cluster-binding module. In Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130, the cas4 gene is functionally transcribed as an active component of the CRISPR-Cas I-B system. Investigation of nuclease activity of Cas4 (LinCas4) of the L. interrogans illustrated divalent-metal cofactor (Mn[2+] or Mg[2+]) dependent endonuclease activity on the DNA substrate. In agreement, mutation of the selective metal interacting residues (Asp[74] and Glu[87]) curtails the DNA cleavage activity in LinCas4. Computational modeling shows metal-ion interacting residues (Asp[74] and Glu[87]) in the LinCas4 to be a part of the RecB motifs II and III, the same as other Cas4 orthologs. The mutation of a potential DNA interacting residue in the LinCas4 (LinCas4[Y132A]) or one of the four cysteine residues (LinCas4[C18A]) involved in coordinating the 4Fe-4S cluster did not perturb its DNase activity. Iron chelation assay of the purified LinCas4 demonstrated it in the apostate conformation. Reconstitution of the Fe-S cluster in the LinCas4 under in vitro condition displayed its coordination with four iron atoms per LinCas4 monomer and was confirmed by the UV and CD spectroscopy studies.},
}
@article {pmid34840925,
year = {2021},
author = {Singh, S and Ramakrishna, W},
title = {Application of CRISPR-Cas9 in plant-plant growth-promoting rhizobacteria interactions for next Green Revolution.},
journal = {3 Biotech},
volume = {11},
number = {12},
pages = {492},
pmid = {34840925},
issn = {2190-572X},
abstract = {Agriculture's beginnings resulted in the domestication of numerous plant species as well as the use of natural resources. Food grain production took about 10,000 years to reach a billion tonnes in 1960, however, it took only 40 years to achieve 2 billion tonnes in year 2000. The creation of genetically modified crops, together with the use of enhanced agronomic practices, resulted in this remarkable increase, dubbed the "Green Revolution". Plants and bacteria that interact with each other in nature are co-evolving, according to Red Queen dynamics. Plant microorganisms, also known as plant microbiota, are an essential component of plant life. Plant-microbe (PM) interactions can be beneficial or harmful to hosts, depending on the health impact. The significance of microbiota in plant growth promotion (PGP) and stress resistance is well known. Our understanding of the community composition of the plant microbiome and important driving forces has advanced significantly. As a result, utilising the plant microbiota is a viable strategy for the next Green Revolution for meeting food demand. The utilisation of newer methods to understand essential genetic and molecular components of the multiple PM interactions is required for their application. The use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing (GE) techniques to investigate PM interactions is of tremendous interest. The implementation of GE techniques to boost the ability of microorganisms or plants for agronomic trait development will be enabled by a comprehensive understanding of PM interactions. This review focuses on using GE approaches to investigate the principles of PM interactions, disease resistance, PGP activity, and future implications in agriculture in plants or associated microbiota.},
}
@article {pmid34839714,
year = {2022},
author = {Pursey, E and Dimitriu, T and Paganelli, FL and Westra, ER and van Houte, S},
title = {CRISPR-Cas is associated with fewer antibiotic resistance genes in bacterial pathogens.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {377},
number = {1842},
pages = {20200464},
pmid = {34839714},
issn = {1471-2970},
support = {BB/S017674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R010781/10/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Anti-Bacterial Agents/pharmacology ; Bacteria/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Microbial ; Gene Transfer, Horizontal ; Genome, Bacterial ; Humans ; },
abstract = {The acquisition of antibiotic resistance (ABR) genes via horizontal gene transfer (HGT) is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of ABR. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of ABR. We analysed approximately 40 000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens, including Enterococcus, Staphylococcus, Acinetobacter and Pseudomonas. We modelled the association between CRISPR-Cas and indicators of HGT, and found that pathogens with a CRISPR-Cas system were less likely to carry ABR genes than those lacking this defence system. Analysis of the mobile genetic elements (MGEs) targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of ABR. These results suggest a potential 'immunocompromised' state for multidrug-resistant strains that may be exploited in tailored interventions that rely on MGEs, such as phages or phagemids, to treat infections caused by bacterial pathogens. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.},
}
@article {pmid34839262,
year = {2021},
author = {Bunnell, BA},
title = {Excision of latent HIV-1: CRISPR technology overcomes viral strain diversity.},
journal = {EBioMedicine},
volume = {74},
number = {},
pages = {103720},
pmid = {34839262},
issn = {2352-3964},
mesh = {*CRISPR-Cas Systems ; Genetic Variation ; Genome, Viral ; HIV Infections/diagnosis/*virology ; HIV-1/*classification/*physiology ; Humans ; Molecular Typing/*methods ; *Virus Latency/genetics ; },
}
@article {pmid34839010,
year = {2022},
author = {Schneider, N and Sundaresan, Y and Gopalakrishnan, P and Beryozkin, A and Hanany, M and Levanon, EY and Banin, E and Ben-Aroya, S and Sharon, D},
title = {Inherited retinal diseases: Linking genes, disease-causing variants, and relevant therapeutic modalities.},
journal = {Progress in retinal and eye research},
volume = {89},
number = {},
pages = {101029},
doi = {10.1016/j.preteyeres.2021.101029},
pmid = {34839010},
issn = {1873-1635},
mesh = {Genetic Association Studies ; Humans ; Mutation ; Pedigree ; Retina ; *Retinal Diseases/genetics/therapy ; *Retinal Dystrophies/genetics ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically complex and heterogenous group of visual impairment phenotypes caused by pathogenic variants in at least 277 nuclear and mitochondrial genes, affecting different retinal regions, and depleting the vision of affected individuals. Genes that cause IRDs when mutated are unique by possessing differing genotype-phenotype correlations, varying inheritance patterns, hypomorphic alleles, and modifier genes thus complicating genetic interpretation. Next-generation sequencing has greatly advanced the identification of novel IRD-related genes and pathogenic variants in the last decade. For this review, we performed an in-depth literature search which allowed for compilation of the Global Retinal Inherited Disease (GRID) dataset containing 4,798 discrete variants and 17,299 alleles published in 31 papers, showing a wide range of frequencies and complexities among the 194 genes reported in GRID, with 65% of pathogenic variants being unique to a single individual. A better understanding of IRD-related gene distribution, gene complexity, and variant types allow for improved genetic testing and therapies. Current genetic therapeutic methods are also quite diverse and rely on variant identification, and range from whole gene replacement to single nucleotide editing at the DNA or RNA levels. IRDs and their suitable therapies thus require a range of effective disease modelling in human cells, granting insight into disease mechanisms and testing of possible treatments. This review summarizes genetic and therapeutic modalities of IRDs, provides new analyses of IRD-related genes (GRID and complexity scores), and provides information to match genetic-based therapies such as gene-specific and variant-specific therapies to the appropriate individuals.},
}
@article {pmid34838817,
year = {2022},
author = {Mabashi-Asazuma, H and Jarvis, DL},
title = {A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans.},
journal = {The Journal of biological chemistry},
volume = {298},
number = {1},
pages = {101454},
pmid = {34838817},
issn = {1083-351X},
support = {R43 GM102982/GM/NIGMS NIH HHS/United States ; R43 GM136071/GM/NIGMS NIH HHS/United States ; R44 GM102982/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Baculoviridae/genetics/metabolism ; CRISPR-Cas Systems ; *Cell Line ; Glycoproteins/biosynthesis/genetics ; Humans ; *Insecta/cytology/genetics/metabolism ; Polysaccharides/metabolism ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)-deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR-Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN[Lec1]. We found that Sf-RVN and Sf-RVN[Lec1] cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN[Lec1] cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN[Lec1] cells were Endo H-cleavable Man5GlcNAc2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.},
}
@article {pmid34838536,
year = {2022},
author = {Akram, F and Haq, IU and Aqeel, A and Ahmed, Z and Shah, FI and Nawaz, A and Zafar, J and Sattar, R},
title = {Insights into the evolutionary and prophylactic analysis of SARS-CoV-2: A review.},
journal = {Journal of virological methods},
volume = {300},
number = {},
pages = {114375},
pmid = {34838536},
issn = {1879-0984},
mesh = {Antiviral Agents ; *COVID-19 ; Humans ; Pandemics ; Phylogeny ; *SARS-CoV-2 ; },
abstract = {In late 2019, following the emergence of a β-originated SARS-CoV-2, phylogenetic and evolutionary approaches have been demonstrated to strengthen the diagnostic and prophylactic stratagem of COVID-19 at an unprecedented level. Despite its clinical prominence, the SARS-CoV-2 gene set remains largely irrefutable by impeding the dissection of COVID-19 biology. However, many pieces of molecular and serological evidence have predicted that SARS-CoV-2 related viruses carry their roots from bats and pangolins of South East Asia. Analysis of viral genome predicts that point mutations at a rate of 10[-4] nucleotides per base in the receptor-binding domain allow the emergence of new SARS-CoV-2 genomic variants at regular intervals. Research in the evolution of molecular pathways involved in emergence of pandemic is critical for the development of therapeutics and vaccines as well as the prevention of future zoonosis. By determining the phyletic lineages of the SARS-CoV-2 genomic variants and those of the conserved regions in the accessory and spike proteins of all the SARS-related coronaviruses, a universal vaccine against all human coronaviruses could be formulated which would revolutionize the field of medicine. This review highlighted the current development and future prospects of antiviral drugs, inhibitors, mesenchymal stem cells, passive immunization, targeted immune therapy and CRISPR-Cas-based prophylactic and therapeutic strategies against SARS-CoV-2. However, further investigations on Covid-19 pathogenesis is required for the successful fabrication of successful antivirals.},
}
@article {pmid34838059,
year = {2021},
author = {Alishah, K and Birtel, M and Masoumi, E and Jafarzadeh, L and Mirzaee, HR and Hadjati, J and Voss, RH and Diken, M and Asad, S},
title = {CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.},
journal = {Journal of translational medicine},
volume = {19},
number = {1},
pages = {482},
pmid = {34838059},
issn = {1479-5876},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; Immunotherapy, Adoptive ; Mesothelin ; *Neoplasms/genetics ; *Receptors, Chimeric Antigen/genetics/metabolism ; },
abstract = {BACKGROUND: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells.<br><br>METHODS: In this study, we hypothesized that knocking out the TGF&#x3b2; receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGF&#x3b2;RII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGF&#x3b2;RII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells.<br><br>RESULTS: Our experiments demonstrated that TGF&#x3b2;RII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGF&#x3b2; in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGF&#x3b2;RII KO CAR T-cells has been recorded. TGF&#x3b2;RII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGF&#x3b2;RII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart.<br><br>CONCLUSION: The TGF&#x3b2;RII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.},
}
@article {pmid34837040,
year = {2022},
author = {Andergassen, D and Rinn, JL},
title = {From genotype to phenotype: genetics of mammalian long non-coding RNAs in vivo.},
journal = {Nature reviews. Genetics},
volume = {23},
number = {4},
pages = {229-243},
pmid = {34837040},
issn = {1471-0064},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Gene Editing ; Genome, Human ; Genotype ; Humans ; Mammals/genetics ; Mice ; Phenotype ; *RNA, Long Noncoding/genetics ; },
abstract = {Genome-wide sequencing has led to the discovery of thousands of long non-coding RNA (lncRNA) loci in the human genome, but evidence of functional significance has remained controversial for many lncRNAs. Genetically engineered model organisms are considered the gold standard for linking genotype to phenotype. Recent advances in CRISPR-Cas genome editing have led to a rapid increase in the use of mouse models to more readily survey lncRNAs for functional significance. Here, we review strategies to investigate the physiological relevance of lncRNA loci by highlighting studies that have used genetic mouse models to reveal key in vivo roles for lncRNAs, from fertility to brain development. We illustrate how an investigative approach, starting with whole-gene deletion followed by transcription termination and/or transgene rescue strategies, can provide definitive evidence for the in vivo function of mammalian lncRNAs.},
}
@article {pmid34836963,
year = {2021},
author = {Tsagkaraki, E and Nicoloro, SM and DeSouza, T and Solivan-Rivera, J and Desai, A and Lifshitz, LM and Shen, Y and Kelly, M and Guilherme, A and Henriques, F and Amrani, N and Ibraheim, R and Rodriguez, TC and Luk, K and Maitland, S and Friedline, RH and Tauer, L and Hu, X and Kim, JK and Wolfe, SA and Sontheimer, EJ and Corvera, S and Czech, MP},
title = {CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6931},
pmid = {34836963},
issn = {2041-1723},
support = {UH3 TR002668/TR/NCATS NIH HHS/United States ; R01 DK089101/DK/NIDDK NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; R01 DK123028/DK/NIDDK NIH HHS/United States ; F31 HL147482/HL/NHLBI NIH HHS/United States ; R37 DK030898/DK/NIDDK NIH HHS/United States ; R01 DK030898/DK/NIDDK NIH HHS/United States ; UG3 TR002668/TR/NCATS NIH HHS/United States ; U2C DK093000/DK/NIDDK NIH HHS/United States ; },
mesh = {Adipocytes, Brown/metabolism/*transplantation ; Adipocytes, White/metabolism ; Adult Stem Cells/physiology ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; Cell Differentiation ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Fatty Liver/etiology/metabolism/prevention &amp; control ; Gene Editing/methods ; Glucose Intolerance/etiology/metabolism/*therapy ; Humans ; Lipid Metabolism/genetics ; Male ; Mice ; Nuclear Receptor Interacting Protein 1/genetics/metabolism ; Obesity/complications/metabolism/*therapy ; RNA, Guide/genetics ; Subcutaneous Fat/cytology ; Thermogenesis/*genetics ; },
abstract = {Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.},
}
@article {pmid34836797,
year = {2021},
author = {Esmail, GA and Al-Dhabi, NA and AlDawood, B and Somily, AM},
title = {Shotgun whole genome sequencing of drug-resistance Streptococcus anginosus strain 47S1 isolated from a patient with pharyngitis in Saudi Arabia.},
journal = {Journal of infection and public health},
volume = {14},
number = {12},
pages = {1740-1749},
doi = {10.1016/j.jiph.2021.11.010},
pmid = {34836797},
issn = {1876-035X},
mesh = {Humans ; *Pharmaceutical Preparations ; *Pharyngitis ; RNA, Ribosomal, 16S/genetics ; Saudi Arabia ; Streptococcus anginosus/genetics ; Whole Genome Sequencing ; },
abstract = {BACKGROUND: Streptococcus anginosus is an emergence opportunistic pathogen that colonize the human upper respiratory tract (URT), S. anginosus alongside with S. intermedius and S. constellatus, members of S. anginosus group, are implicated in several human infections. However, our understanding this bacterium to the genotype level with determining the genes associated with pathogenicity and antimicrobial resistance (AMR) is scarce. S. anginosus 47S1 strain was isolated from sore throat infection, the whole genome was characterized and the virulence &amp; AMR genes contributing in pathogenicity were investigated.<br><br>METHODOLOGY: The whole genome of 47S1 was sequenced by Illumina sequencing technology. Strain 47S1 genome was de novo assembled with different strategies and annotated via PGAP, PROKKA and RAST pipelines. Identifying the CRISPR-Cass system and prophages sequences was performed using CRISPRloci and PhiSpy tools respectively. Prediction the virulence genes were performed with the VFDB database. AMR genes were detected in silico using NCBI AMRFinderPlus pipeline and CARD database and compared with in vitro AST findings.<br><br>RESULTS: &#x3b2;-hemolytic strain 47S1 was identified with conventional microbiology techniques and confirmed by the sequences of 16S rRNA gene. Genome of 47S1 comprised of 1981512 bp. Type I-C CRISPR-Cas system and 4 prophages were detected among the genome of 47S1. Several virulence genes were predicted, most of these genes are found in other pathogenic streptococci, mainly lmb, pavA, htrA/degP, eno, sagA, psaA and cpsI which play a significant role in colonizing, invading host tissues and evade form immune system. In silico AMR findings showed that 47S1 gnome harbors (tetA, tetB &amp;tet32), (aac(6')-I, aadK &amp;aph(3')-IVa), fusC, and PmrA genes that mediated-resistance to tetracyclines, aminoglycosides, fusidic acid, and fluoroquinolone respectively which corresponds with in vitro AST obtained results. In conclusion, WGS is a key approach to predict the virulence and AMR genes, results obtained in this study may contribute for a better understanding of the opportunistic S. anginosus pathogenicity.},
}
@article {pmid34835366,
year = {2021},
author = {Cui, L and Wang, X and Zhao, Y and Peng, Z and Gao, P and Cao, Z and Feng, J and Zhang, F and Guo, K and Wu, M and Chen, H and Dai, M},
title = {Virulence Comparison of Salmonella enterica Subsp. enterica Isolates from Chicken and Whole Genome Analysis of the High Virulent Strain S. Enteritidis 211.},
journal = {Microorganisms},
volume = {9},
number = {11},
pages = {},
pmid = {34835366},
issn = {2076-2607},
abstract = {BACKGROUND: Salmonellaenterica is one of the common pathogens in both humans and animals that causes salmonellosis and threatens public health all over the world.<br><br>METHODS AND RESULTS: Here we determined the virulence phenotypes of nine Salmonellaenterica subsp. enterica (S. enterica) isolates in vitro and in vivo, including pathogenicity to chicken, cell infection, biofilm formation and virulence gene expressions. S. Enteritidis 211 (SE211) was highly pathogenic with notable virulence features among the nine isolates. The combination of multiple virulence genes contributed to the conferring of the high virulence in SE211. Importantly, many mobile genetic elements (MGEs) were found in the genome sequence of SE211, including a virulence plasmid, genomic islands, and prophage regions. The MGEs and CRISPR-Cas system might function synergistically for gene transfer and immune defense. In addition, the neighbor joining tree and the minimum spanning tree were constructed in this study.<br><br>CONCLUSIONS: This study provided both the virulence phenotypes and genomic features, which might contribute to the understanding of bacterial virulence mechanisms in Salmonella enterica subsp. enterica. The first completed genomic sequence for the high virulent S. Enteritidis isolate SE211 and the comparative genomics and phylogenetic analyses provided a preliminary understanding of S. enterica genetics and laid the foundation for further study.},
}
@article {pmid34835095,
year = {2021},
author = {Braspenning, SE and Lebbink, RJ and Depledge, DP and Schapendonk, CME and Anderson, LA and Verjans, GMGM and Sadaoka, T and Ouwendijk, WJD},
title = {Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection.},
journal = {Viruses},
volume = {13},
number = {11},
pages = {},
pmid = {34835095},
issn = {1999-4915},
support = {R01 AI151290/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Ganglia/pathology/virology ; *Gene Expression Regulation, Viral ; Herpesvirus 3, Human/*genetics ; Humans ; Mutagenesis ; Neurons/pathology/virology ; Open Reading Frames/genetics ; Transcription, Genetic/drug effects ; Viral Proteins/*genetics/metabolism ; Virus Latency/*genetics ; Virus Physiological Phenomena ; },
abstract = {Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.},
}
@article {pmid34835064,
year = {2021},
author = {Chulanov, V and Kostyusheva, A and Brezgin, S and Ponomareva, N and Gegechkori, V and Volchkova, E and Pimenov, N and Kostyushev, D},
title = {CRISPR Screening: Molecular Tools for Studying Virus-Host Interactions.},
journal = {Viruses},
volume = {13},
number = {11},
pages = {},
pmid = {34835064},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; Drug Discovery ; *Genetic Techniques ; Genome-Wide Association Study/*methods ; High-Throughput Screening Assays/*methods ; Host Microbial Interactions ; Humans ; Virus Diseases/*genetics/*virology ; Viruses/*genetics ; },
abstract = {CRISPR/Cas is a powerful tool for studying the role of genes in viral infections. The invention of CRISPR screening technologies has made it possible to untangle complex interactions between the host and viral agents. Moreover, whole-genome and pathway-specific CRISPR screens have facilitated identification of novel drug candidates for treating viral infections. In this review, we highlight recent developments in the fields of CRISPR/Cas with a focus on the use of CRISPR screens for studying viral infections and identifying new candidate genes to aid development of antivirals.},
}
@article {pmid34834963,
year = {2021},
author = {Kirby, EN and Shue, B and Thomas, PQ and Beard, MR},
title = {CRISPR Tackles Emerging Viral Pathogens.},
journal = {Viruses},
volume = {13},
number = {11},
pages = {},
pmid = {34834963},
issn = {1999-4915},
mesh = {COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Communicable Diseases, Emerging/diagnosis/*virology ; Coronavirus/*genetics/physiology ; Coronavirus Infections/diagnosis/*virology ; *Gene Editing ; Host-Pathogen Interactions ; Humans ; SARS-CoV-2/genetics ; Zika Virus/*genetics/physiology ; Zika Virus Infection/diagnosis/*virology ; },
abstract = {Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.},
}
@article {pmid34834954,
year = {2021},
author = {Workman, AM and Heaton, MP and Webster, DA and Harhay, GP and Kalbfleisch, TS and Smith, TPL and Falkenberg, SM and Carlson, DF and Sonstegard, TS},
title = {Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance.},
journal = {Viruses},
volume = {13},
number = {11},
pages = {},
pmid = {34834954},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Line ; Diarrhea/virology ; Diarrhea Viruses, Bovine Viral/*physiology ; Dogs ; GTPase-Activating Proteins/genetics ; *Gene Deletion ; Gene Knockout Techniques ; Nerve Tissue Proteins/genetics ; Protein Tyrosine Phosphatase, Non-Receptor Type 12/genetics ; Receptors, Glutamate/genetics ; Virus Internalization ; Virus Replication ; Whole Genome Sequencing ; },
abstract = {Bovine viral diarrhea virus's (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.},
}
@article {pmid34834920,
year = {2021},
author = {Luu, AP and Yao, Z and Ramachandran, S and Azzopardi, SA and Miles, LA and Schneider, WM and Hoffmann, HH and Bozzacco, L and Garcia, G and Gong, D and Damoiseaux, R and Tang, H and Morizono, K and Rudin, CM and Sun, R and Arumugaswami, V and Poirier, JT and MacDonald, MR and Rice, CM and Li, MMH},
title = {A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.},
journal = {Viruses},
volume = {13},
number = {11},
pages = {},
pmid = {34834920},
issn = {1999-4915},
support = {R01 AI145044/AI/NIAID NIH HHS/United States ; R01 AI091707/AI/NIAID NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R01 AI158704/AI/NIAID NIH HHS/United States ; P30 AI028697/AI/NIAID NIH HHS/United States ; },
mesh = {A549 Cells ; CRISPR-Cas Systems ; GTP-Binding Proteins/genetics/*metabolism ; Humans ; Neoplasm Proteins/genetics/*metabolism ; Transcriptional Coactivator with PDZ-Binding Motif Proteins/genetics/metabolism ; Virus Internalization ; Virus Replication ; Zika Virus/genetics/*physiology ; Zika Virus Infection/*enzymology/genetics/virology ; p21-Activated Kinases/genetics/metabolism ; rhoB GTP-Binding Protein/genetics/*metabolism ; },
abstract = {Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.},
}
@article {pmid34834620,
year = {2021},
author = {Kawall, K},
title = {The Generic Risks and the Potential of SDN-1 Applications in Crop Plants.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {11},
pages = {},
pmid = {34834620},
issn = {2223-7747},
abstract = {The use of site-directed nucleases (SDNs) in crop plants to alter market-oriented traits is expanding rapidly. At the same time, there is an on-going debate around the safety and regulation of crops altered with the site-directed nuclease 1 (SDN-1) technology. SDN-1 applications can be used to induce a variety of genetic alterations ranging from fairly 'simple' genetic alterations to complex changes in plant genomes using, for example, multiplexing approaches. The resulting plants can contain modified alleles and associated traits, which are either known or unknown in conventionally bred plants. The European Commission recently published a study on new genomic techniques suggesting an adaption of the current GMO legislation by emphasizing that targeted mutagenesis techniques can produce genomic alterations that can also be obtained by natural mutations or conventional breeding techniques. This review highlights the need for a case-specific risk assessment of crop plants derived from SDN-1 applications considering both the characteristics of the product and the process to ensure a high level of protection of human and animal health and the environment. The published literature on so-called market-oriented traits in crop plants altered with SDN-1 applications is analyzed here to determine the types of SDN-1 application in plants, and to reflect upon the complexity and the naturalness of such products. Furthermore, it demonstrates the potential of SDN-1 applications to induce complex alterations in plant genomes that are relevant to generic SDN-associated risks. In summary, it was found that nearly half of plants with so-called market-oriented traits contain complex genomic alterations induced by SDN-1 applications, which may also pose new types of risks. It further underscores the need for data on both the process and the end-product for a case-by-case risk assessment of plants derived from SDN-1 applications.},
}
@article {pmid34833086,
year = {2021},
author = {Chan, KG and Ang, GY and Yu, CY and Yean, CY},
title = {Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics.},
journal = {Life (Basel, Switzerland)},
volume = {11},
number = {11},
pages = {},
pmid = {34833086},
issn = {2075-1729},
abstract = {The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR-Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR-Cas-based nucleic acid detection strategies and the application of CRISPR-Cas as a potential antiviral agent in the context of COVID-19.},
}
@article {pmid34832953,
year = {2021},
author = {Serajian, S and Ahmadpour, E and Oliveira, SMR and Pereira, ML and Heidarzadeh, S},
title = {CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {14},
number = {11},
pages = {},
pmid = {34832953},
issn = {1424-8247},
abstract = {Through the years, many promising tools for gene editing have been developed including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and homing endonucleases (HEs). These novel technologies are now leading new scientific advancements and practical applications at an inimitable speed. While most work has been performed in eukaryotes, CRISPR systems also enable tools to understand and engineer bacteria. The increase in the number of multi-drug resistant strains highlights a necessity for more innovative approaches to the diagnosis and treatment of infections. CRISPR has given scientists a glimmer of hope in this area that can provide a novel tool to fight against antimicrobial resistance. This system can provide useful information about the functions of genes and aid us to find potential targets for antimicrobials. This paper discusses the emerging use of CRISPR-Cas systems in the fields of clinical microbiology and infectious diseases with a particular emphasis on future prospects.},
}
@article {pmid34831382,
year = {2021},
author = {Synowiec, A and Jedrysik, M and Branicki, W and Klajmon, A and Lei, J and Owczarek, K and Suo, C and Szczepanski, A and Wang, J and Zhang, P and Labaj, PP and Pyrc, K},
title = {Identification of Cellular Factors Required for SARS-CoV-2 Replication.},
journal = {Cells},
volume = {10},
number = {11},
pages = {},
pmid = {34831382},
issn = {2073-4409},
mesh = {Angiotensin-Converting Enzyme 2/genetics/metabolism ; CRISPR-Cas Systems ; Computational Biology ; Genome, Human/genetics ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; SARS-CoV-2/pathogenicity/*physiology ; *Virus Replication ; },
abstract = {Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the recently emerged virus responsible for the COVID-19 pandemic. Clinical presentation can range from asymptomatic disease and mild respiratory tract infection to severe disease with lung injury, multiorgan failure, and death. SARS-CoV-2 is the third animal coronavirus to emerge in humans in the 21st century, and coronaviruses appear to possess a unique ability to cross borders between species and infect a wide range of organisms. This is somewhat surprising as, except for the requirement of host cell receptors, cell-pathogen interactions are usually species-specific. Insights into these host-virus interactions will provide a deeper understanding of the process of SARS-CoV-2 infection and provide a means for the design and development of antiviral agents. In this study, we describe a complex analysis of SARS-CoV-2 infection using a genome-wide CRISPR-Cas9 knock-out system in HeLa cells overexpressing entry receptor angiotensin-converting enzyme 2 (ACE2). This platform allows for the identification of factors required for viral replication. This study was designed to include a high number of replicates (48 replicates; 16 biological repeats with 3 technical replicates each) to prevent data instability, remove sources of bias, and allow multifactorial bioinformatic analyses in order to study the resulting interaction network. The results obtained provide an interesting insight into the replication mechanisms of SARS-CoV-2.},
}
@article {pmid34831379,
year = {2021},
author = {Bahuguna, S and Redhai, S and Zhou, J and Wang, T and Port, F and Boutros, M},
title = {Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors.},
journal = {Cells},
volume = {10},
number = {11},
pages = {},
pmid = {34831379},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Digestive System/metabolism/pathology ; Drosophila melanogaster/*genetics ; *Gene Editing ; Genes, Tumor Suppressor ; Intestinal Neoplasms/*genetics/pathology ; Mitosis ; Mutation/genetics ; Stem Cells/metabolism ; Time Factors ; },
abstract = {CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We generated conditional tissue-specific CRISPR knockouts using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, we observed regional differences in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. Our findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.},
}
@article {pmid34831239,
year = {2021},
author = {Nath, A and Rayabaram, J and Ijee, S and Bagchi, A and Chaudhury, AD and Roy, D and Chambayil, K and Singh, J and Nakamura, Y and Velayudhan, SR},
title = {Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis.},
journal = {Cells},
volume = {10},
number = {11},
pages = {},
pmid = {34831239},
issn = {2073-4409},
mesh = {Adult ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Chromatin/metabolism ; Erythroid Cells/cytology/metabolism ; Erythropoiesis/*genetics ; Gene Editing ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; MicroRNAs/*genetics/metabolism ; Signal Transduction ; Transcription Factors/metabolism ; },
abstract = {MicroRNAs (miRNAs) are small non-coding RNAs, which play an important role in various cellular and developmental processes. The study of miRNAs in erythropoiesis is crucial to uncover the cellular pathways that are modulated during the different stages of erythroid differentiation. Using erythroid cells derived from human CD34+ hematopoietic stem and progenitor cells (HSPCs)and small RNA sequencing, our study unravels the various miRNAs involved in critical cellular pathways in erythroid maturation. We analyzed the occupancy of erythroid transcription factors and chromatin accessibility in the promoter and enhancer regions of the differentially expressed miRNAs to integrate miRNAs in the transcriptional circuitry of erythropoiesis. Analysis of the targets of the differentially expressed miRNAs revealed novel pathways in erythroid differentiation. Finally, we described the application of Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) based editing of miRNAs to study their function in human erythropoiesis.},
}
@article {pmid34830492,
year = {2021},
author = {Schröpfer, S and Flachowsky, H},
title = {Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830492},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Electrophoresis, Capillary ; *Gene Editing ; Genotype ; Genotyping Techniques ; Malus/*genetics ; Mutation/genetics ; },
abstract = {The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.},
}
@article {pmid34830457,
year = {2021},
author = {Yao, K and Peng, D and Jiang, C and Zhao, W and Li, G and Huang, W and Kong, L and Gao, H and Zheng, J and Peng, H},
title = {Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830457},
issn = {1422-0067},
mesh = {Animals ; Bacterial Proteins/*genetics ; Beta vulgaris/genetics/*parasitology ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Endodeoxyribonucleases/*genetics ; Nucleic Acid Amplification Techniques ; Recombinases/chemistry/genetics ; Tylenchoidea/genetics/*isolation &amp; purification/pathogenicity ; },
abstract = {Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10[-4] single cysts and single females, 4[-3] single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.},
}
@article {pmid34830426,
year = {2021},
author = {Binyameen, B and Khan, Z and Khan, SH and Ahmad, A and Munawar, N and Mubarik, MS and Riaz, H and Ali, Z and Khan, AA and Qusmani, AT and Abd-Elsalam, KA and Qari, SH},
title = {Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830426},
issn = {1422-0067},
mesh = {Agrobacterium/genetics ; Begomovirus/*genetics/pathogenicity ; CRISPR-Cas Systems/*genetics ; Disease Resistance/*genetics ; Gossypium/*genetics/growth &amp; development/virology ; Plant Diseases/genetics/virology ; Plants, Genetically Modified/genetics/growth &amp; development/virology ; Solanaceae/genetics/growth &amp; development/virology ; },
abstract = {In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3-5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20-40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60-70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.},
}
@article {pmid34830347,
year = {2021},
author = {Feoktistova, M and Makarov, R and Yazdi, AS and Panayotova-Dimitrova, D},
title = {RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830347},
issn = {1422-0067},
mesh = {Apoptosis/drug effects/*genetics ; Baculoviral IAP Repeat-Containing 3 Protein/genetics/metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/genetics/metabolism ; CRISPR-Cas Systems ; Cycloheximide/pharmacology ; Gene Deletion ; Gene Expression Regulation ; HeLa Cells ; Humans ; I-kappa B Kinase/genetics/metabolism ; Inhibitor of Apoptosis Proteins/genetics/metabolism ; NF-kappa B/genetics/metabolism ; Necroptosis/drug effects/*genetics ; Proteasome Endopeptidase Complex/metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/deficiency/*genetics ; Signal Transduction ; TNF Receptor-Associated Death Domain Protein/deficiency/*genetics ; TNF Receptor-Associated Factor 2/genetics/metabolism ; Tumor Necrosis Factor-alpha/*metabolism/pharmacology ; Ubiquitination ; },
abstract = {TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.},
}
@article {pmid34830309,
year = {2021},
author = {Gracz-Bernaciak, J and Mazur, O and Nawrot, R},
title = {Functional Studies of Plant Latex as a Rich Source of Bioactive Compounds: Focus on Proteins and Alkaloids.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830309},
issn = {1422-0067},
mesh = {Anti-Infective Agents/*chemistry ; Antineoplastic Agents/*chemistry ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chelidonium/genetics/*metabolism ; Drug Discovery/methods ; Gene Editing/methods ; Herbivory/drug effects ; Humans ; Immunologic Factors/*chemistry ; Latex/*chemistry ; Opiate Alkaloids/*chemistry ; Papaver/genetics/*metabolism ; Phytochemicals/*chemistry ; Plant Proteins/*chemistry ; Plants, Genetically Modified ; },
abstract = {Latex, a sticky emulsion produced by specialized cells called laticifers, is a crucial part of a plant's defense system against herbivory and pathogens. It consists of a broad spectrum of active compounds, which are beneficial not only for plants, but for human health as well, enough to mention the use of morphine or codeine from poppy latex. Here, we reviewed latex's general role in plant physiology and the significance of particular compounds (alkaloids and proteins) to its defense system with the example of Chelidonium majus L. from the poppy family. We further attempt to present latex chemicals used so far in medicine and then focus on functional studies of proteins and other compounds with potential pharmacological activities using modern techniques such as CRISPR/Cas9 gene editing. Despite the centuries-old tradition of using latex-bearing plants in therapies, there are still a lot of promising molecules waiting to be explored.},
}
@article {pmid34830212,
year = {2021},
author = {Crescenzi, E and Leonardi, A and Pacifico, F},
title = {NGAL as a Potential Target in Tumor Microenvironment.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830212},
issn = {1422-0067},
mesh = {Animals ; Antibodies, Monoclonal/therapeutic use ; CRISPR-Cas Systems ; Gene Editing/methods ; Humans ; Lipocalin-2/antagonists &amp; inhibitors/genetics/immunology/*metabolism ; Neoplasms/*metabolism/therapy ; RNA, Small Interfering/genetics ; RNAi Therapeutics/methods ; Secretome/metabolism ; *Senescence-Associated Secretory Phenotype ; *Signal Transduction/drug effects/genetics ; Stromal Cells/metabolism ; *Tumor Microenvironment ; },
abstract = {The signaling network between cancer and stromal cells plays a crucial role in tumor microenvironment. The fate of tumor progression mainly depends on the huge amount of information that these cell populations exchange from the onset of neoplastic transformation. Interfering with such signaling has been producing exciting results in cancer therapy: just think of anti-PD-1/anti-PD-L1/anti-CTLA-4 antibodies that, acting as immune checkpoint inhibitors, interrupt the inhibitory signaling exerted by cancer cells on immune cells or the CAR-T technology that fosters the reactivation of anti-tumoral immunity in a restricted group of leukemias and lymphomas. Nevertheless, many types of cancers, in particular solid tumors, are still refractory to these treatments, so the identification of novel molecular targets in tumor secretome would benefit from implementation of current anti-cancer therapeutical strategies. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a secreted protein abundantly expressed in the secretome of various human tumors. It represents a promising target for the multiple roles that are played inside cancer and stromal cells, and also overall in their cross-talk. The review focuses on the different roles of NGAL in tumor microenvironment and in cancer senescence-associated secretory phenotype (SASP), highlighting the most crucial functions that could be eventually targetable in cancer therapy.},
}
@article {pmid34830205,
year = {2021},
author = {Onishi, I and Yamamoto, K and Kinowaki, Y and Kitagawa, M and Kurata, M},
title = {To Discover the Efficient and Novel Drug Targets in Human Cancers Using CRISPR/Cas Screening and Databases.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830205},
issn = {1422-0067},
mesh = {Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems ; *Databases, Genetic ; Drug Discovery/*methods ; Early Detection of Cancer/*methods ; Gene Editing/methods ; Gene Library ; Genes, Essential ; Genome, Human ; Humans ; Molecular Targeted Therapy/*methods ; Neoplasms/*drug therapy/*genetics ; RNA, Guide/genetics ; Synthetic Lethal Mutations ; },
abstract = {CRISPR/Cas has emerged as an excelle nt gene-editing technology and is used worldwide for research. The CRISPR library is an ideal tool for identifying essential genes and synthetic lethality targeted for cancer therapies in human cancers. Synthetic lethality is defined as multiple genetic abnormalities that, when present individually, do not affect function or survival, but when present together, are lethal. Recently, many CRISPR libraries are available, and the latest libraries are more accurate and can be applied to few cells. However, it is easier to efficiently search for cancer targets with their own screenings by effectively using databases of CRISPR screenings, such as Depmap portal, PICKLES (Pooled In-Vitro CRISPR Knockout Library Essentiality Screens), iCSDB, Project Score database, and CRISP-view. This review will suggest recent optimal CRISPR libraries and effective databases for Novel Approaches in the Discovery and Design of Targeted Therapies.},
}
@article {pmid34830019,
year = {2021},
author = {Li, P and Li, Z and Xie, G and Zhang, J},
title = {Trihelix Transcription Factor ZmThx20 Is Required for Kernel Development in Maize.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34830019},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Edible Grain/genetics/growth &amp; development ; Endosperm/genetics/growth &amp; development ; Gene Expression Regulation, Plant/genetics ; Plant Proteins/*genetics ; Seeds/genetics/growth &amp; development ; Transcription Factors/*genetics ; Transcriptome/genetics ; Zea mays/*genetics/growth &amp; development ; Zein/*genetics ; },
abstract = {Maize kernels are the harvested portion of the plant and are related to the yield and quality of maize. The endosperm of maize is a large storage organ that constitutes 80-90% of the dry weight of mature kernels. Maize kernels have long been the study of cereal grain development to increase yield. In this study, a natural mutation that causes abnormal kernel development, and displays a shrunken kernel phenotype, was identified and named "shrunken 2008 (sh2008)". The starch grains in sh2008 are loose and have a less proteinaceous matrix surrounding them. The total storage protein and the major storage protein zeins are ~70% of that in the wild-type control (WT); in particular, the 19 kDa and 22 kDa α-zeins. Map-based cloning revealed that sh2008 encodes a GT-2 trihelix transcription factor, ZmThx20. Using CRISPR/Cas9, two other alleles with mutated ZmThx20 were found to have the same abnormal kernel. Shrunken kernels can be rescued by overexpressing normal ZmThx20. Comparative transcriptome analysis of the kernels from sh2008 and WT showed that the GO terms of translation, ribosome, and nutrient reservoir activity were enriched in the down-regulated genes (sh2008/WT). In short, these changes can lead to defects in endosperm development and storage reserve filling in seeds.},
}
@article {pmid34829981,
year = {2021},
author = {Filler-Hayut, S and Kniazev, K and Melamed-Bessudo, C and Levy, AA},
title = {Targeted Inter-Homologs Recombination in Arabidopsis Euchromatin and Heterochromatin.},
journal = {International journal of molecular sciences},
volume = {22},
number = {22},
pages = {},
pmid = {34829981},
issn = {1422-0067},
mesh = {Arabidopsis/*genetics/growth &amp; development ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Euchromatin/*genetics ; Genome, Plant/genetics ; Heterochromatin/*genetics ; Homologous Recombination/*genetics ; Recombinational DNA Repair/genetics ; },
abstract = {Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both "hot and cold" regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.},
}
@article {pmid34829328,
year = {2021},
author = {Guaman-Bautista, LP and Moreta-Urbano, E and Oña-Arias, CG and Torres-Arias, M and Kyriakidis, NC and Malcı, K and Jonguitud-Borrego, N and Rios-Solis, L and Ramos-Martinez, E and López-Cortés, A and Barba-Ostria, C},
title = {Tracking SARS-CoV-2: Novel Trends and Diagnostic Strategies.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {11},
number = {11},
pages = {},
pmid = {34829328},
issn = {2075-4418},
abstract = {The COVID-19 pandemic has had an enormous impact on economies and health systems globally, therefore a top priority is the development of increasingly better diagnostic and surveillance alternatives to slow down the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In order to establish massive testing and contact tracing policies, it is crucial to have a clear view of the diagnostic options available and their principal advantages and drawbacks. Although classical molecular methods such as RT-qPCR are broadly used, diagnostic alternatives based on technologies such as LAMP, antigen, serological testing, or the application of novel technologies such as CRISPR-Cas for diagnostics, are also discussed. The present review also discusses the most important automation strategies employed to increase testing capability. Several serological-based diagnostic kits are presented, as well as novel nanotechnology-based diagnostic methods. In summary, this review provides a clear diagnostic landscape of the most relevant tools to track COVID-19.},
}
@article {pmid34828396,
year = {2021},
author = {Dolgalev, G and Poverennaya, E},
title = {Applications of CRISPR-Cas Technologies to Proteomics.},
journal = {Genes},
volume = {12},
number = {11},
pages = {},
pmid = {34828396},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Protein Interaction Mapping/methods ; Proteomics/*methods ; },
abstract = {CRISPR-Cas-based genome editing is a revolutionary approach that has provided an unprecedented investigational power for the life sciences. Rapid and efficient, CRISPR-Cas technologies facilitate the generation of complex biological models and at the same time provide the necessary methods required to study these models in depth. The field of proteomics has already significantly benefited from leveraging the power of CRISPR-Cas technologies, however, many potential applications of these technologies in the context of proteomics remain unexplored. In this review, we intend to provide an introduction to the CRISPR-Cas technologies and demonstrate how they can be applied to solving proteome-centric questions. To achieve this goal, we begin with the description of the modern suite of CRISPR-Cas-based tools, focusing on the more mature CRISPR-Cas9 system. In the second part of this review, we highlight both established and potential applications of the CRISPR-Cas technologies to proteomics.},
}
@article {pmid34828367,
year = {2021},
author = {Cheng, L and Zhou, X and Zheng, Y and Tang, C and Liu, Y and Zheng, S and Liu, Y and Zhou, J and Li, C and Chen, M and Lai, L and Zou, Q},
title = {Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats.},
journal = {Genes},
volume = {12},
number = {11},
pages = {},
pmid = {34828367},
issn = {2073-4425},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Codon ; DNA/*genetics ; Epigenesis, Genetic ; Gene Editing ; Gene Expression Regulation ; Genetic Engineering/*methods ; Transcription Activator-Like Effectors/*genetics ; },
abstract = {Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.},
}
@article {pmid34828273,
year = {2021},
author = {Weuring, W and Geerligs, J and Koeleman, BPC},
title = {Gene Therapies for Monogenic Autism Spectrum Disorders.},
journal = {Genes},
volume = {12},
number = {11},
pages = {},
pmid = {34828273},
issn = {2073-4425},
mesh = {Animals ; Autism Spectrum Disorder/genetics/*therapy ; CRISPR-Cas Systems ; Gene Editing/methods ; *Genetic Therapy/methods/trends ; Humans ; Mutation ; },
abstract = {Novel genome editing and transient gene therapies have been developed the past ten years, resulting in the first in-human clinical trials for monogenic disorders. Syndromic autism spectrum disorders can be caused by mutations in a single gene. Given the monogenic aspect and severity of syndromic ASD, it is an ideal candidate for gene therapies. Here, we selected 11 monogenic ASD syndromes, validated by animal models, and reviewed current gene therapies for each syndrome. Given the wide variety and novelty of some forms of gene therapy, the best possible option must be decided based on the gene and mutation.},
}
@article {pmid34827712,
year = {2021},
author = {Rabiee, N and Rabiee, M and Sojdeh, S and Fatahi, Y and Dinarvand, R and Safarkhani, M and Ahmadi, S and Daneshgar, H and Radmanesh, F and Maghsoudi, S and Bagherzadeh, M and Varma, RS and Mostafavi, E},
title = {Porphyrin Molecules Decorated on Metal-Organic Frameworks for Multi-Functional Biomedical Applications.},
journal = {Biomolecules},
volume = {11},
number = {11},
pages = {},
pmid = {34827712},
issn = {2218-273X},
mesh = {Animals ; COVID-19/*diagnosis ; COVID-19 Testing ; CRISPR-Cas Systems ; DNA, Single-Stranded ; HEK293 Cells ; HeLa Cells ; Hep G2 Cells ; Humans ; Hydrogen Bonding ; Limit of Detection ; Metal-Organic Frameworks/*chemistry ; Nanocomposites ; Nanostructures ; Nitrogen/chemistry ; PC12 Cells ; Porosity ; Porphyrins/*chemistry ; RNA, Guide ; RNA, Viral/metabolism ; Rats ; SARS-CoV-2 ; Sensitivity and Specificity ; Surface Properties ; },
abstract = {Metal-organic frameworks (MOFs) have been widely used as porous nanomaterials for different applications ranging from industrial to biomedicals. An unpredictable one-pot method is introduced to synthesize NH2-MIL-53 assisted by high-gravity in a greener media for the first time. Then, porphyrins were deployed to adorn the surface of MOF to increase the sensitivity of the prepared nanocomposite to the genetic materials and in-situ cellular protein structures. The hydrogen bond formation between genetic domains and the porphyrin' nitrogen as well as the surface hydroxyl groups is equally probable and could be considered a milestone in chemical physics and physical chemistry for biomedical applications. In this context, the role of incorporating different forms of porphyrins, their relationship with the final surface morphology, and their drug/gene loading efficiency were investigated to provide a predictable pattern in regard to the previous works. The conceptual phenomenon was optimized to increase the interactions between the biomolecules and the substrate by reaching the limit of detection to 10 pM for the Anti-cas9 protein, 20 pM for the single-stranded DNA (ssDNA), below 10 pM for the single guide RNA (sgRNA) and also around 10 nM for recombinant SARS-CoV-2 spike antigen. Also, the MTT assay showed acceptable relative cell viability of more than 85% in most cases, even by increasing the dose of the prepared nanostructures.},
}
@article {pmid34827239,
year = {2021},
author = {Tyumentseva, M and Mikhaylova, Y and Prelovskaya, A and Karbyshev, K and Tyumentsev, A and Petrova, L and Mironova, A and Zamyatin, M and Shelenkov, A and Akimkin, V},
title = {CRISPR Element Patterns vs. Pathoadaptability of Clinical Pseudomonas aeruginosa Isolates from a Medical Center in Moscow, Russia.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {11},
pages = {},
pmid = {34827239},
issn = {2079-6382},
abstract = {Pseudomonas aeruginosa is a member of the ESKAPE opportunistic pathogen group, which includes six species of the most dangerous microbes. This pathogen is characterized by the rapid acquisition of antimicrobial resistance, thus causing major healthcare concerns. This study presents a comprehensive analysis of clinical P. aeruginosa isolates based on whole-genome sequencing data. The isolate collection studied was characterized by a variety of clonal lineages with a domination of high-risk epidemic clones and different CRISPR/Cas element patterns. This is the first report on the coexistence of two and even three different types of CRISPR/Cas systems simultaneously in Russian clinical strains of P. aeruginosa. The data include molecular typing and genotypic antibiotic resistance determination, as well as the phylogenetic analysis of the full-length cas gene and anti-CRISPR genes sequences, predicted prophage sequences, and conducted a detailed CRISPR array analysis. The differences between the isolates carrying different types and quantities of CRISPR/Cas systems were investigated. The pattern of virulence factors in P. aeruginosa isolates lacking putative CRISPR/Cas systems significantly differed from that of samples with single or multiple putative CRISPR/Cas systems. We found significant correlations between the numbers of prophage sequences, antibiotic resistance genes, and virulence genes in P. aeruginosa isolates with different patterns of CRISPR/Cas-elements. We believe that the data presented will contribute to further investigations in the field of bacterial pathoadaptability, including antimicrobial resistance and the role of CRISPR/Cas systems in the plasticity of the P. aeruginosa genome.},
}
@article {pmid34826338,
year = {2022},
author = {Xu, H and Look, T and Prithiviraj, S and Lennartz, D and Cáceres, MD and Götz, K and Wanek, P and Häcker, H and Kramann, R and Seré, K and Zenke, M},
title = {CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells.},
journal = {European journal of immunology},
volume = {52},
number = {11},
pages = {1859-1862},
doi = {10.1002/eji.202149482},
pmid = {34826338},
issn = {1521-4141},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Cell Line ; Gene Expression Regulation ; Dendritic Cells ; Homeodomain Proteins/genetics ; },
abstract = {HoxB8 multipotent progenitors (MPP) are obtained by expression of the estrogen receptor hormone binding domain (ERHBD) HoxB8 fusion gene in mouse BM cells. HoxB8 MPP generate (i) the full complement of DC subsets (cDC1, cDC2, and pDC) in vitro and in vivo and (ii) allow CRISPR/Cas9-mediated gene editing, for example, generating homozygous deletions in cis-acting DNA elements at high precision, and (iii) efficient gene repression by dCas9-KRAB for studying gene regulation in DC differentiation.},
}
@article {pmid34826009,
year = {2022},
author = {Wang, Z and Yan, X and Zhang, H and Meng, Y and Pan, Y and Cui, H},
title = {NtCycB2 negatively regulates tobacco glandular trichome formation, exudate accumulation, and aphid resistance.},
journal = {Plant molecular biology},
volume = {108},
number = {1-2},
pages = {65-76},
pmid = {34826009},
issn = {1573-5028},
mesh = {Animals ; *Aphids ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Down-Regulation ; Gene Editing ; *Plant Defense Against Herbivory ; Plant Proteins/genetics/*physiology ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; Tobacco/genetics/growth &amp; development/immunology/*metabolism ; Transcription Factors/genetics/*physiology ; Trichomes/*growth &amp; development/metabolism ; },
abstract = {NtCycB2 negatively regulates the initiation of tobacco long stalk glandular trichomes and influences the expression of diterpenoid biosynthesis- and environmental stress resistance-related genes. Many asterid plants possess multicellular trichomes on their surface, both glandular and non-glandular. The CycB2 gene plays a key role in multicellular trichome initiation, but has distinct effects on different types of trichomes; its mechanisms remain unknown. In tomato (Solanum lycopersicum), SlCycB2 negatively regulates non-glandular trichome formation, but its effects on glandular trichomes are ambiguous. In this study, we cloned the SlCycB2 homolog of Nicotiana tabacum, NtCycB2, and analyzed its effect on three types of trichomes, long stalk glandular trichomes (LGT), short stalk glandular trichomes (SGT), and non-glandular trichomes (NGT). Knocking out NtCycB2 (NtCycB2-KO) promoted LGT formation, while overexpression of NtCycB2 (NtCycB2-OE) decreased LGT density. SGT and NGT were not significantly influenced in either NtCycB2-KO or NtCycB2-OE plants, indicating that NtCycB2 regulated only LGT formation in tobacco. In addition, compared with NtCycB2-OE and control plants, NtCycB2-KO plants produced more trichome exudates, including diterpenoids and sugar esters, and exhibited stronger aphid resistance. To further elucidate the function of NtCycB2, RNA-Seq analysis of the NtCycB2-KO, NtCycB2-OE, and control plants was conducted. 2,552 and 1,933 differentially expressed genes (DEGs) were found in NtCycB2-KO and NtCycB2-OE plants, respectively. Gene Ontology analysis of the common DEGs revealed that ion transport, carbohydrate and amino acid metabolism, photosynthesis, and transcription regulation processes were significantly enriched. Among these DEGs, diterpenoid biosynthesis genes were upregulated in NtCycB2-KO plants and downregulated in NtCycB2-OE plants. Two MYB transcription factors and several stress resistance-related genes were also identified, suggesting they may participate in regulating LGT formation and aphid resistance.},
}
@article {pmid34825222,
year = {2021},
author = {Xu, H and Kita, Y and Bang, U and Gee, P and Hotta, A},
title = {Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100965},
pmid = {34825222},
issn = {2666-1667},
mesh = {Adult ; CRISPR-Cas Systems/*genetics ; DNA, Single-Stranded/genetics ; Electroporation/*methods ; Female ; Gene Editing/*methods ; Homologous Recombination/genetics ; Humans ; Male ; *Pluripotent Stem Cells/cytology/metabolism ; RNA, Guide/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).},
}
@article {pmid34824292,
year = {2021},
author = {Wang, J and Teng, Y and Zhang, R and Wu, Y and Lou, L and Zou, Y and Li, M and Xie, ZR and Yan, Y},
title = {Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6916},
pmid = {34824292},
issn = {2041-1723},
support = {R35 GM128620/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/*genetics ; *CRISPR-Cas Systems ; Codon, Initiator ; DNA Cleavage ; Escherichia coli/genetics ; *Gene Expression ; Genetic Engineering/*methods ; Molecular Dynamics Simulation ; Mutation ; Prokaryotic Cells ; RNA, Guide ; Saccharomyces cerevisiae/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {The RNA-guided CRISPR-associated Cas9 proteins have been widely applied in programmable genome recombination, base editing or gene regulation in both prokaryotes and eukaryotes. SpCas9 from Streptococcus pyogenes is the most extensively engineered Cas9 with robust and manifold functionalities. However, one inherent limitation of SpCas9 is its stringent 5'-NGG-3' PAM requirement that significantly restricts its DNA target range. Here, to repurpose SpCas9 as a universal gene repressor, we generate and screen variants of the deactivated SpCas9 (SpdCas9) with relaxed 5'-CAT-3' PAM compatibility that can bind to the start codon ATG of almost any gene. Stepwise structure-guided mutations of the PAM-interacting residues and auxiliary PAM-proximal residues of the SpdNG (5'-NG-3' PAM) create a PAM-flexible variant SpdNG-LWQT that preferentially accommodates 5'-NRN-3' PAMs. SpdNG-LWQT is demonstrated to be effective in gene repression with the advantage of customizable sgRNA design in both Escherichia coli and Saccharomyces cerevisiae. This work validates the feasibility of purposeful PAM expansion of Cas9 towards signature PAMs and establishes a universal SpdCas9-based gene repressor.},
}
@article {pmid34824279,
year = {2021},
author = {Rice, S and Jackson, T and Crump, NT and Fordham, N and Elliott, N and O'Byrne, S and Fanego, MDML and Addy, D and Crabb, T and Dryden, C and Inglott, S and Ladon, D and Wright, G and Bartram, J and Ancliff, P and Mead, AJ and Halsey, C and Roberts, I and Milne, TA and Roy, A},
title = {A human fetal liver-derived infant MLL-AF4 acute lymphoblastic leukemia model reveals a distinct fetal gene expression program.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6905},
pmid = {34824279},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 099175/Z/ 12/Z/WT_/Wellcome Trust/United Kingdom ; MC_UU_12009/6/MRC_/Medical Research Council/United Kingdom ; MC_UU_00016/6/MRC_/Medical Research Council/United Kingdom ; MC_UU_12009/14/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins ; Female ; *Fetus ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Histone-Lysine N-Methyltransferase ; Humans ; Liver ; Mice ; Myeloid-Lymphoid Leukemia Protein/*genetics/metabolism ; Oncogene Proteins, Fusion/*genetics/metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics/*metabolism ; Transcriptional Elongation Factors ; },
abstract = {Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured, the prognosis for infant-ALL remains dismal. Infant-ALL is usually caused by a single genetic hit that arises in utero: an MLL/KMT2A gene rearrangement (MLL-r). This is sufficient to induce a uniquely aggressive and treatment-refractory leukemia compared to older children. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Using the most common MLL-r in infant-ALL, MLL-AF4, as a disease model, we show that fetal-specific gene expression programs are maintained in MLL-AF4 infant-ALL but not in MLL-AF4 childhood-ALL. We use CRISPR-Cas9 gene editing of primary human fetal liver hematopoietic cells to produce a t(4;11)/MLL-AF4 translocation, which replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data support the hypothesis that fetal-specific gene expression programs cooperate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.},
}
@article {pmid34824277,
year = {2021},
author = {Laudenbach, BT and Krey, K and Emslander, Q and Andersen, LL and Reim, A and Scaturro, P and Mundigl, S and Dächert, C and Manske, K and Moser, M and Ludwig, J and Wohlleber, D and Kröger, A and Binder, M and Pichlmair, A},
title = {NUDT2 initiates viral RNA degradation by removal of 5'-phosphates.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6918},
pmid = {34824277},
issn = {2041-1723},
mesh = {Adaptation, Physiological ; Animals ; Antiviral Agents ; Bone Marrow Cells ; CRISPR-Cas Systems ; Exonucleases ; Exoribonucleases ; Female ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Immunity, Innate ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins ; Phosphoric Monoester Hydrolases/*genetics/*metabolism ; Polyphosphates ; *RNA Stability ; RNA, Bacterial ; RNA, Messenger ; RNA, Viral/*metabolism ; Virus Replication ; },
abstract = {While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5'-triphosphate (PPP-) group that impairs degradation by the canonical 5'-3' degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5'-3' exonuclease XRN1. NUDT2 removes 5'-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5'-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.},
}
@article {pmid34824225,
year = {2021},
author = {Thong, WL and Zhang, Y and Zhuo, Y and Robins, KJ and Fyans, JK and Herbert, AJ and Law, BJC and Micklefield, J},
title = {Gene editing enables rapid engineering of complex antibiotic assembly lines.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6872},
pmid = {34824225},
issn = {2041-1723},
support = {BB/L013754/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N023536/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Anti-Bacterial Agents/*biosynthesis/chemistry ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing/*methods ; Lipopeptides/biosynthesis/chemistry ; Multienzyme Complexes/chemistry/*genetics/metabolism ; Mutation ; Peptide Synthases/chemistry/genetics/metabolism ; Peptides, Cyclic/biosynthesis/chemistry ; Protein Domains ; Streptomyces/genetics/metabolism ; Synthetic Biology ; },
abstract = {Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.},
}
@article {pmid34823604,
year = {2021},
author = {Zhong, KX and Cho, A and Deeg, CM and Chan, AM and Suttle, CA},
title = {Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS).},
journal = {Microbiome},
volume = {9},
number = {1},
pages = {230},
pmid = {34823604},
issn = {2049-2618},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Eukaryota/genetics ; High-Throughput Nucleotide Sequencing/methods ; *Microbiota/genetics ; *Ostreidae/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {BACKGROUND: The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences.<br><br>RESULTS: To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected.<br><br>CONCLUSION: CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Video Abstract.},
}
@article {pmid34823004,
year = {2022},
author = {Chen, S and Chen, D and Liu, B and Haisma, HJ},
title = {Modulating CRISPR/Cas9 genome-editing activity by small molecules.},
journal = {Drug discovery today},
volume = {27},
number = {4},
pages = {951-966},
doi = {10.1016/j.drudis.2021.11.018},
pmid = {34823004},
issn = {1878-5832},
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genome engineering has become a standard procedure for creating genetic and epigenetic changes of DNA molecules in basic biology, biotechnology, and medicine. However, its versatile applications have been hampered by its overall low precise gene modification efficiency and uncontrollable prolonged Cas9 activity. Therefore, overcoming these problems could broaden the therapeutic use of CRISPR/Cas9-based technologies. Here, we review small molecules with the clinical potential to precisely modulate CRISPR/Cas9-mediated genome-editing activity and discuss their mechanisms of action. Based on these data, we suggest that direct-acting small molecules for Cas9 are more suitable for precisely regulating Cas9 activity. These findings provide useful information for the identification of novel small-molecule enhancers and inhibitors of Cas9 and Cas9-associated endonucleases.},
}
@article {pmid34822926,
year = {2022},
author = {Raguraman, R and Shanmugarama, S and Mehta, M and Elle Peterson, J and Zhao, YD and Munshi, A and Ramesh, R},
title = {Drug delivery approaches for HuR-targeted therapy for lung cancer.},
journal = {Advanced drug delivery reviews},
volume = {180},
number = {},
pages = {114068},
pmid = {34822926},
issn = {1872-8294},
support = {R01 CA167516/CA/NCI NIH HHS/United States ; R01 CA254192/CA/NCI NIH HHS/United States ; P30 CA225520/CA/NCI NIH HHS/United States ; I01 BX003420/BX/BLRD VA/United States ; P20 GM103639/GM/NIGMS NIH HHS/United States ; R01 CA233201/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Drug Delivery Systems ; ELAV-Like Protein 1/*antagonists &amp; inhibitors/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/*drug therapy/pathology ; Molecular Targeted Therapy ; Nanomedicine ; RNA, Small Interfering/administration &amp; dosage ; },
abstract = {Lung cancer (LC) is often diagnosed at an advanced stage and conventional treatments for disease management have limitations associated with them. Novel therapeutic targets are thus avidly sought for the effective management of LC. RNA binding proteins (RBPs) have been convincingly established as key players in tumorigenesis, and their dysregulation is linked to multiple cancers, including LC. In this context, we review the role of Human antigen R (HuR), an RBP that is overexpressed in LC, and further associated with various aspects of LC tumor growth and response to therapy. Herein, we describe the role of HuR in LC progression and outline the evidences supporting various pharmacologic and biologic approaches for inhibiting HuR expression and function. These approaches, including use of small molecule inhibitors, siRNAs and shRNAs, have demonstrated favorable results in reducing tumor cell growth, invasion and migration, angiogenesis and metastasis. Hence, HuR has significant potential as a key therapeutic target in LC. Use of siRNA-based approaches, however, have certain limitations that prevent their maximal exploitation as cancer therapies. To address this, in the conclusion of this review, we provide a list of nanomedicine-based HuR targeting approaches currently being employed for siRNA and shRNA delivery, and provide a rationale for the immense potential therapeutic benefits offered by nanocarrier-based HuR targeting and its promise for treating patients with LC.},
}
@article {pmid34822716,
year = {2021},
author = {Schifferl, D and Scholze-Wittler, M and Wittler, L and Veenvliet, JV and Koch, F and Herrmann, BG},
title = {A 37 kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {23},
pages = {},
pmid = {34822716},
issn = {1477-9129},
mesh = {Amino Acid Sequence/genetics ; Animals ; CRISPR-Cas Systems/genetics ; Embryonic Development/*genetics ; Enhancer Elements, Genetic/*genetics ; Fetal Proteins/*genetics ; Gene Editing/methods ; Gene Expression Regulation, Developmental/genetics ; Mesoderm/growth &amp; development/metabolism ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Notochord/growth &amp; development/metabolism ; Promoter Regions, Genetic/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; T-Box Domain Proteins/*genetics ; Tail/*growth &amp; development/metabolism ; },
abstract = {The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.},
}
@article {pmid34820908,
year = {2021},
author = {Ma, X and Takahashi, Y and Wu, W and Chen, J and Dehdarani, M and Liang, W and Shin, YH and Benyajati, S and Ma, JX},
title = {Soluble very low-density lipoprotein receptor (sVLDLR) inhibits fibrosis in neovascular age-related macular degeneration.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {12},
pages = {e22058},
pmid = {34820908},
issn = {1530-6860},
support = {R01 EY032931/EY/NEI NIH HHS/United States ; R01 EY032930/EY/NEI NIH HHS/United States ; P30 GM122744/GM/NIGMS NIH HHS/United States ; P30 EY021725/EY/NEI NIH HHS/United States ; R01 EY012231/EY/NEI NIH HHS/United States ; R01 EY028949/EY/NEI NIH HHS/United States ; R01 EY019309/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Choroidal Neovascularization/*complications ; Connective Tissue Growth Factor/genetics/metabolism ; *Disease Models, Animal ; Fibrosis/etiology/metabolism/pathology/*prevention &amp; control ; Low Density Lipoprotein Receptor-Related Protein-6/antagonists &amp; inhibitors/genetics/*metabolism ; Macular Degeneration/*complications ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Receptors, LDL/*physiology ; Retinal Pigment Epithelium/metabolism/*pathology ; Smad Proteins/genetics/metabolism ; Transforming Growth Factor beta/genetics/metabolism ; Wnt Signaling Pathway ; },
abstract = {Subretinal fibrosis is a key pathological feature in neovascular age-related macular degeneration (nAMD). Previously, we identified soluble very low-density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti-fibrogenic role in nAMD models, including Vldlr[-/-] mice and laser-induced choroidal neovascularization (CNV). We found that fibrosis factors including P-Smad2/3, α-SMA, and CTGF were upregulated in the subretinal area of Vldlr[-/-] mice and the laser-induced CNV model. The antibody blocking Wnt co-receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser-induced CNV model. A transgenic strain (sVLDLR-Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser-induced CNV model. In addition, Wnt and TGF-β signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9-mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF-β-induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF-β pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.},
}
@article {pmid34819726,
year = {2021},
author = {Xiong, J and Tan, S and Yu, L and Shen, H and Qu, S and Zhang, C and Ren, C and Zhu, D and Wang, H},
title = {E7-Targeted Nanotherapeutics for Key HPV Afflicted Cervical Lesions by Employing CRISPR/Cas9 and Poly (Beta-Amino Ester).},
journal = {International journal of nanomedicine},
volume = {16},
number = {},
pages = {7609-7622},
pmid = {34819726},
issn = {1178-2013},
mesh = {Animals ; CRISPR-Cas Systems ; Early Detection of Cancer ; Esters ; Female ; Humans ; Mice ; Mice, Nude ; Papillomavirus E7 Proteins/genetics ; *Papillomavirus Infections ; *Uterine Cervical Neoplasms/genetics ; },
abstract = {INTRODUCTION: Persistent HR-HPV (high-risk human papillomavirus) infection is the main cause of cervical cancer. The HPV oncogene E7 plays a key role in HPV tumorigenesis. At present, HPV preventive vaccines are not effective for patients who already have a cervical disease, and implementation of the recommended regular cervical screening is difficult in countries and regions lacking medical resources. Therefore, patients need medications to treat existing HPV infections and thus block the progression of cervical disease.<br><br>METHODS: In this study, we developed nanoparticles (NPs) composed of the non-viral vector PBAE546 and a CRISPR/Cas9 recombinant plasmid targeting HPV16 E7 as a vaginal treatment for HPV infection and related cervical malignancies.<br><br>RESULTS: Our NPs showed low toxicity and high biological safety both in vitro (cell line viability) and in vivo (various important organs of mice). Our NPs significantly inhibited the growth of xenograft tumors derived from cervical cancer cell lines in nude mice and significantly reversed the cervical epithelial malignant phenotype of HPV16 transgenic mice.<br><br>CONCLUSION: Our NPs have great potential to be developed as a drug for the treatment of HPV-related cervical cancer and precancerous lesions.},
}
@article {pmid34819671,
year = {2021},
author = {Rogier, M and Moritz, J and Robert, I and Lescale, C and Heyer, V and Abello, A and Martin, O and Capitani, K and Thomas, M and Thomas-Claudepierre, AS and Laffleur, B and Jouan, F and Pinaud, E and Tarte, K and Cogné, M and Conticello, SG and Soutoglou, E and Deriano, L and Reina-San-Martin, B},
title = {Fam72a enforces error-prone DNA repair during antibody diversification.},
journal = {Nature},
volume = {600},
number = {7888},
pages = {329-333},
pmid = {34819671},
issn = {1476-4687},
mesh = {Animals ; B-Lymphocytes/*metabolism ; CRISPR-Cas Systems/genetics ; *DNA Mismatch Repair ; Female ; Genome/genetics ; Immunoglobulin Class Switching/*genetics ; Immunoglobulin Switch Region/*genetics ; Male ; Mice ; *Mutation ; Somatic Hypermutation, Immunoglobulin/*genetics ; Up-Regulation ; Uracil/metabolism ; },
abstract = {Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)[1] and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins[1-3]. Although uracils arising in DNA are accurately repaired[1-4], how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown[1-3]. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)[5] and is overexpressed in several cancers[5]. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a[-/-] B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a[-/-] B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.},
}
@article {pmid34819670,
year = {2021},
author = {Feng, Y and Li, C and Stewart, JA and Barbulescu, P and Seija Desivo, N and Álvarez-Quilón, A and Pezo, RC and Perera, MLW and Chan, K and Tong, AHY and Mohamad-Ramshan, R and Berru, M and Nakib, D and Li, G and Kardar, GA and Carlyle, JR and Moffat, J and Durocher, D and Di Noia, JM and Bhagwat, AS and Martin, A},
title = {FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.},
journal = {Nature},
volume = {600},
number = {7888},
pages = {324-328},
pmid = {34819670},
issn = {1476-4687},
support = {R21 AI144708/AI/NIAID NIH HHS/United States ; R21 CA252858/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/*metabolism ; CRISPR-Cas Systems ; DNA Glycosylases/*antagonists &amp; inhibitors/metabolism ; *DNA Mismatch Repair ; Epistasis, Genetic ; Female ; HEK293 Cells ; Humans ; Immunoglobulin Class Switching/*genetics ; Immunoglobulin Switch Region/genetics ; Membrane Proteins/deficiency/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; MutS Homolog 2 Protein/genetics/metabolism ; *Mutation ; Neoplasm Proteins/deficiency/genetics/*metabolism ; Somatic Hypermutation, Immunoglobulin/*genetics ; },
abstract = {Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination[1,2]. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells[3-6]. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a[-/-] mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a[-/-] mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)[7], which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers[8] could promote mutagenesis.},
}
@article {pmid34819659,
year = {2021},
author = {Marx, V},
title = {The CRISPR children.},
journal = {Nature biotechnology},
volume = {39},
number = {12},
pages = {1486-1490},
doi = {10.1038/s41587-021-01138-5},
pmid = {34819659},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; Child ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; },
}
@article {pmid34819641,
year = {2021},
author = {Bondy-Denomy, J},
title = {CRISPR inactivation by integration.},
journal = {Nature microbiology},
volume = {6},
number = {12},
pages = {1475-1476},
pmid = {34819641},
issn = {2058-5276},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
}
@article {pmid34819640,
year = {2021},
author = {Varble, A and Campisi, E and Euler, CW and Maguin, P and Kozlova, A and Fyodorova, J and Rostøl, JT and Fischetti, VA and Marraffini, LA},
title = {Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes.},
journal = {Nature microbiology},
volume = {6},
number = {12},
pages = {1516-1525},
pmid = {34819640},
issn = {2058-5276},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Lysogeny ; Plasmids/genetics/metabolism ; Prophages/genetics/*physiology ; Streptococcus Phages/genetics/*physiology ; Streptococcus pyogenes/genetics/*immunology/*virology ; Virus Integration ; },
abstract = {CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR-cas locus is a recurrent mechanism to counteract immunity.},
}
@article {pmid34819517,
year = {2021},
author = {Viswanatha, R and Mameli, E and Rodiger, J and Merckaert, P and Feitosa-Suntheimer, F and Colpitts, TM and Mohr, SE and Hu, Y and Perrimon, N},
title = {Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6825},
pmid = {34819517},
issn = {2041-1723},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Anopheles/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Computational Biology/methods ; Gene Knockout Techniques ; Gene Library ; Genes, Essential ; Humans ; Mosquito Control/*methods ; Mosquito Vectors/*genetics ; Pest Control, Biological/*methods ; RNA, Guide/genetics ; Vector Borne Diseases/*prevention &amp; control/transmission ; },
abstract = {Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.},
}
@article {pmid34819506,
year = {2021},
author = {Hakim, CH and Kumar, SRP and Pérez-López, DO and Wasala, NB and Zhang, D and Yue, Y and Teixeira, J and Pan, X and Zhang, K and Million, ED and Nelson, CE and Metzger, S and Han, J and Louderman, JA and Schmidt, F and Feng, F and Grimm, D and Smith, BF and Yao, G and Yang, NN and Gersbach, CA and Chen, SJ and Herzog, RW and Duan, D},
title = {Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6769},
pmid = {34819506},
issn = {2041-1723},
support = {R01 NS090634/NS/NINDS NIH HHS/United States ; R35 GM134919/GM/NIGMS NIH HHS/United States ; U01 EB028901/EB/NIBIB NIH HHS/United States ; R01 HL131093/HL/NHLBI NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; U01 HL156348/HL/NHLBI NIH HHS/United States ; R01 AR070517/AR/NIAMS NIH HHS/United States ; T32 GM008396/GM/NIGMS NIH HHS/United States ; R01 AI051390/AI/NIAID NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; R01 GM117059/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/*immunology ; Dependovirus/genetics ; Disease Models, Animal ; Dogs ; Dystrophin/genetics/immunology ; Gene Editing/methods ; Genes, Reporter/genetics/immunology ; Genetic Therapy/*adverse effects/methods ; Genetic Vectors/administration &amp; dosage/*immunology ; Humans ; Muscle, Skeletal/*immunology/pathology ; Muscular Dystrophy, Duchenne/genetics/immunology/pathology/*therapy ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics/immunology ; },
abstract = {Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.},
}
@article {pmid34819011,
year = {2022},
author = {Kues, WA and Kumar, D and Selokar, NL and Talluri, TR},
title = {Applications of Genome Editing Tools in Stem Cells Towards Regenerative Medicine: An Update.},
journal = {Current stem cell research &amp; therapy},
volume = {17},
number = {3},
pages = {267-279},
doi = {10.2174/1574888X16666211124095527},
pmid = {34819011},
issn = {2212-3946},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Regenerative Medicine ; Stem Cells ; Zinc Finger Nucleases/genetics ; },
abstract = {Precise and site-specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/ Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulation of genes in various types of cells with target-specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, making alterations, and curing diseases permanently, which are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also been shown to have a significant improvement in clinical trials. However, their widespread use in the research revealed potential safety issues, which need to be addressed before implementing such techniques for clinical purposes. Significant and valiant attempts need to be made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.},
}
@article {pmid34818890,
year = {2021},
author = {Zhang, T and Zhao, W and Zhao, W and Si, Y and Chen, N and Chen, X and Zhang, X and Fan, L and Sui, G},
title = {Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y.},
journal = {Analytical chemistry},
volume = {93},
number = {48},
pages = {16184-16193},
pmid = {34818890},
issn = {1520-6882},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *SARS-CoV-2 ; },
abstract = {Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/μL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.},
}
@article {pmid34815789,
year = {2021},
author = {Liu, C and Wang, N and Luo, R and Li, L and Yang, W and Wang, X and Shen, M and Wu, Q and Gong, C},
title = {A programmable hierarchical-responsive nanoCRISPR elicits robust activation of endogenous target to treat cancer.},
journal = {Theranostics},
volume = {11},
number = {20},
pages = {9833-9846},
pmid = {34815789},
issn = {1838-7640},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nanoparticle Drug Delivery System/chemistry/*pharmacology ; Neoplasms/genetics/therapy ; RNA, Guide/genetics ; Transcriptional Activation/genetics ; Tumor Microenvironment/drug effects ; },
abstract = {Despite promising progress of cancer gene therapy made, these therapeutics were still limited by the diversity of gene sizes and types. CRISPR/dCas9 mediated activation of tumor endogenous gene has shown great potential to surmount hinders of genetic varieties during the process of cancer gene therapy. However, the blood interference along with complicated tumor extra/intracellular microenvironment substantially compromise the performance of CRISPR/dCas9-based therapeutics in vivo. Methods: In this study, we constructed a programmable hierarchical-responsive nanoCRISPR (PICASSO) that can achieve sequential responses to the multiple physiological barriers in vivo. The core-shell structure endows PICASSO with long blood circulation capacity and tumor target accumulation as well as efficient cellular uptake and lysosomal escape, leading to high-performance of CRISPR/dCas9-mediated gene activation, which favors the antitumor efficacy. Results: Owing to these properties, PICASSO facilitated CRISPR/dCas9 mediated efficient transcriptional activation of various types of endogenous gene, and long non-protein-coding genes (LncRNA) containing targets ranging in size from ~1 kb to ~2000 kb in tumor cells. Intravenous administration of PICASSO to the tumor-bearing mice can achieve effective transcriptional activation of therapeutic endogenous gene, resulting in remarkable CRISPR/dCas9-mediate tumor inhibition with minimal adverse effect. Conclusions: Taken together, these characteristics allow PICASSO to unleash the potential of CRISPR/dCas9-based therapeutics in oncological treatment. The study provides a simple and versatile strategy to break through the restriction of sizes and types against cancer by utilization of tumor endogenous gene.},
}
@article {pmid34813932,
year = {2022},
author = {Hu, M and Zhu, D and Zhou, X},
title = {M-CDC: Magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {259-267},
doi = {10.1016/j.ymeth.2021.11.009},
pmid = {34813932},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; Centers for Disease Control and Prevention, U.S. ; Colorimetry ; DNA ; Magnetic Phenomena ; *Nucleic Acid Amplification Techniques/methods ; United States ; },
abstract = {The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M-CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M-CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments. In this paper, step-by-step instructions for M-CDC assay, including recombinase polymerase amplification (RPA)/reverse transcription-polymerase chain reaction (RT-RPA) of DNA or RNA, Cas12a-mediated target recognition and cleavage, and subsequent magnetic beads-mediated colorimetric readouts are provided. In addition, the protocol for the expression and purification of Lachnospiraceae bacterium-Cas12a (LbCas12a) protein, the design and synthesis of high-efficient crRNA, and the preparation of AuNP bioprobe are also offered.},
}
@article {pmid34813661,
year = {2021},
author = {Hiraga, H and Ishita, Y and Chihara, T and Okumura, M},
title = {Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus.},
journal = {Development, growth &amp; differentiation},
volume = {63},
number = {9},
pages = {488-500},
doi = {10.1111/dgd.12761},
pmid = {34813661},
issn = {1440-169X},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Nematoda/genetics ; },
abstract = {CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.},
}
@article {pmid34813372,
year = {2021},
author = {Marr, E and Potter, CJ},
title = {Base Editing of Somatic Cells Using CRISPR-Cas9 in Drosophila.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {836-845},
pmid = {34813372},
issn = {2573-1602},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Drosophila/genetics ; *Gene Editing/methods ; Genome ; Mammals/genetics ; RNA, Guide/genetics ; },
abstract = {Cas9 and a guide RNA (gRNA) function to target specific genomic loci for generation of a double-stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double-stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase (CD) to induce C > T (or G>A) mutations at targeted sites. In this study, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (based on the mammalian BE2) that consists of a fusion protein of CD, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila base editor (Actin5C-BE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C > T were found in positions 3-9 of the gRNA-targeted site, with conversion reaching ∼100% of targeted DNA in somatic tissues. Surprisingly, the simultaneous use of two gRNAs targeting a genomic region spaced ∼50 bp apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.},
}
@article {pmid34813072,
year = {2022},
author = {Liu, S and Narancic, T and Davis, C and O'Connor, KE},
title = {CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2397},
number = {},
pages = {341-358},
pmid = {34813072},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Polyesters ; Polyhydroxyalkanoates/metabolism ; *Pseudomonas putida/genetics/metabolism ; },
abstract = {Genome editing technologies allow us to study the metabolic pathways of cells and the contribution of each associated enzyme to various processes, including polyhydroxyalkanoate (PHA) synthesis. These biodegradable polyesters accumulated by a range of bacteria are thermoplastic, elastomeric, and biodegradable, thus have great applicative potential. However, several challenges are associated with PHA production, mainly the cost and shortcomings in their physical properties. The advances in synthetic biology and metabolic engineering provide us with a tool to improve the production process and allow the synthesis of tailor-made PHAs. CRISPR/Cas9 technology represents a new generation of genome editing tools capable of application in nearly all organisms. However, off-target activity is a crucial issue for CRISPR/Cas9 technology, as it can cause genomic instability and disruption of functions of otherwise normal genes. Here, we provide a detailed protocol for scarless deletion of the genes implicated in PHA metabolism of Pseudomonas putida KT2440 using modified CRISPR/Cas9 systems and methodology.},
}
@article {pmid34813019,
year = {2022},
author = {Guan, L and Han, Y and Yang, C and Lu, S and Du, J and Li, H and Lin, J},
title = {CRISPR-Cas9-Mediated Gene Therapy in Neurological Disorders.},
journal = {Molecular neurobiology},
volume = {59},
number = {2},
pages = {968-982},
pmid = {34813019},
issn = {1559-1182},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Humans ; *Muscular Dystrophy, Duchenne ; },
abstract = {Neurological disorders are primarily diseases with sophisticated etiology that are always refractory and recrudescent. The major obstruction to effective therapies for neurological disorders is the poor understanding of their pathogenic mechanisms. CRISPR-Cas9 technology, which allows precise and effective gene editing in almost any cell type and organism, is accelerating the pace of basic biological research. An increasing number of groups are focusing on uncovering the molecular mechanisms of neurological disorders and developing novel therapies using the CRISPR-Cas9 system. This review highlights the application of CRISPR-Cas9 technology in the treatment of neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis and/or frontotemporal dementia, Duchenne muscular dystrophy, Dravet syndrome, epilepsy, Huntington's disease, and Parkinson's disease. Hopefully, it will improve our understanding of neurological disorders and give insights into future treatments for neurological disorders.},
}
@article {pmid34811797,
year = {2022},
author = {Lin, G and Zhang, K and Han, Y and Peng, R and Li, J},
title = {Preparation of multiplexed control materials for cancer mutation analysis by genome editing in GM12878 cells.},
journal = {Journal of clinical laboratory analysis},
volume = {36},
number = {1},
pages = {e24139},
pmid = {34811797},
issn = {1098-2825},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *DNA Mutational Analysis/methods/standards ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/methods/standards ; Humans ; Mutation/genetics ; Neoplasms/*genetics ; Quality Control ; Reference Standards ; },
abstract = {BACKGROUND: Quality control materials are necessary for assay development, test validation, and proficiency testing in cancer mutation analysis. Most of the existing controls for somatic mutations only harbor a single variant and are derived from unstable cell lines. This study aimed to establish a method to create stable multianalyte controls in a defined background by genome editing in GM12878 cells, which also can be applied for the reference of next-generation sequencing.<br><br>METHODS: GM12878 cells were electroporated with a donor plasmid containing a mutant DNA sequence and a Cas9/sgRNA expressing vector. The genome-edited GM12878 cell was validated with Sanger sequencing, amplification refractory mutation system (ARMS), and next-generation sequencing (NGS).<br><br>RESULTS: We have successfully generated a mutant GM12878 cell line harboring the defined variants including single-nucleotide variants (SNVs), small insertions and deletions (indels), and structural variants (SVs). The introduction of intended mutations in GM12878 cell line was confirmed by both ARMS and sequencing methods.<br><br>CONCLUSIONS: We developed a method for the preparation of the multiplexed controls for reference mutations in cancer gene by genome editing in GM12878 cells. This methodology can be used to generate other stable cancer reference materials with an unlimited supply.},
}
@article {pmid34810375,
year = {2022},
author = {Houston, BJ},
title = {Commentary on "CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice".},
journal = {Asian journal of andrology},
volume = {24},
number = {3},
pages = {273},
pmid = {34810375},
issn = {1745-7262},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Fertility/genetics ; *Gene Editing ; Male ; Mice ; Spermatogenesis/genetics ; *Testis ; },
}
@article {pmid34810254,
year = {2021},
author = {Bumpus, TW and Huang, S and Tei, R and Baskin, JM},
title = {Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {48},
pages = {},
pmid = {34810254},
issn = {1091-6490},
support = {T32 GM138826/GM/NIGMS NIH HHS/United States ; },
mesh = {Biological Phenomena ; CRISPR-Cas Systems/genetics ; Click Chemistry/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Glycogen Synthase Kinase 3/*metabolism/physiology ; HEK293 Cells ; Humans ; K562 Cells ; Phosphatidic Acids/metabolism ; Phospholipase D/*metabolism/physiology ; Protein Kinase C-alpha/*metabolism/physiology ; Second Messenger Systems ; Signal Transduction ; },
abstract = {Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.},
}
@article {pmid34810201,
year = {2022},
author = {Hong, D and Knelson, EH and Li, Y and Durmaz, YT and Gao, W and Walton, E and Vajdi, A and Thai, T and Sticco-Ivins, M and Sabet, AH and Jones, KL and Schinzel, AC and Bronson, RT and Nguyen, QD and Tolstorukov, MY and Vivero, M and Signoretti, S and Barbie, DA and Oser, MG},
title = {Plasticity in the Absence of NOTCH Uncovers a RUNX2-Dependent Pathway in Small Cell Lung Cancer.},
journal = {Cancer research},
volume = {82},
number = {2},
pages = {248-263},
pmid = {34810201},
issn = {1538-7445},
support = {K08 CA222657/CA/NCI NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Cell Line, Tumor ; Cell Plasticity/*genetics ; Core Binding Factor Alpha 1 Subunit/*metabolism ; Disease Models, Animal ; Humans ; Loss of Function Mutation ; Lung Neoplasms/genetics/*metabolism/pathology ; Mice ; Receptor, Notch1/genetics/*metabolism ; Receptor, Notch2/genetics/*metabolism ; Signal Transduction/*genetics ; Small Cell Lung Carcinoma/genetics/*metabolism/pathology ; Transfection ; },
abstract = {Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations. SIGNIFICANCE: A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST-dependent pathway and innate immune signaling.},
}
@article {pmid34807726,
year = {2022},
author = {Bishop, CE and Shadid, TM and Lavey, NP and Kempher, ML and Ballard, JD and Duerfeldt, AS},
title = {Identification of ClpP Dual Isoform Disruption as an Antisporulation Strategy for Clostridioides difficile.},
journal = {Journal of bacteriology},
volume = {204},
number = {2},
pages = {e0041121},
pmid = {34807726},
issn = {1098-5530},
support = {P20 GM103640/GM/NIGMS NIH HHS/United States ; R01 AI119048/AI/NIAID NIH HHS/United States ; R01AI119048/AI/NIAID NIH HHS/United States ; P20GM103640/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; Bortezomib/pharmacology ; Clostridioides difficile/chemistry/drug effects/*genetics/*metabolism ; Clostridium Infections/microbiology ; Gene Editing/methods ; *Gene Expression Regulation, Bacterial ; Humans ; Mutation ; Phenotype ; Protein Isoforms/genetics ; Spores, Bacterial/*genetics/metabolism ; Virulence ; },
abstract = {The Gram-positive bacterium Clostridioides difficile is a primary cause of hospital-acquired diarrhea, threatening both immunocompromised and healthy individuals. An important aspect of defining mechanisms that drive C. difficile persistence and virulence relies on developing a more complete understanding of sporulation. C. difficile sporulation is the single determinant of transmission and complicates treatment and prevention due to the chemical and physical resilience of spores. By extension, the identification of druggable targets that significantly attenuate sporulation would have a significant impact on thwarting C. difficile infection. By use of a new CRISPR-Cas9 nickase genome editing methodology, stop codons were inserted early in the coding sequence for clpP1 and clpP2 to generate C. difficile mutants that no longer produced the corresponding isoforms of caseinolytic protease P (ClpP). The data show that genetic ablation of ClpP isoforms leads to altered sporulation phenotypes with the clpP1/clpP2 double mutant exhibiting asporogenic behavior. A small screen of known ClpP inhibitors in a fluorescence-based biochemical assay identified bortezomib as an inhibitor of C. difficile ClpP that produces dose-dependent inhibition of purified ClpP. Incubation of C. difficile cultures in the presence of bortezomib reveals antisporulation effects approaching that observed in the clpP1/clpP2 double mutant. This work identifies ClpP as a key contributor to C. difficile sporulation and provides compelling support for the pursuit of small-molecule ClpP inhibitors as C. difficile antisporulating agents. IMPORTANCE Due to diverse roles of ClpP and the reliance of pathogens upon this system for infection, it has emerged as a target for antimicrobial development. Biology regulated by ClpP is organism dependent and has not been defined in Clostridioides difficile. This work identifies ClpP as a key contributor to C. difficile sporulation and provides compelling support for the pursuit of small-molecule ClpP inhibitors as antisporulating agents. The identification of new approaches and/or drug targets that reduce C. difficile sporulation would be transformative and are expected to find high utility in prophylaxis, transmission attenuation, and relapse prevention. Discovery of the ClpP system as a major driver to sporulation also provides a new avenue of inquiry for advancing the understanding of sporulation.},
}
@article {pmid34807447,
year = {2022},
author = {Hay, AN and Farrell, K and Leeth, CM and Lee, K},
title = {Use of Genome Editing Techniques to Produce Transgenic Farm Animals.},
journal = {Advances in experimental medicine and biology},
volume = {1354},
number = {},
pages = {279-297},
pmid = {34807447},
issn = {0065-2598},
support = {R21 OD019934/OD/NIH HHS/United States ; R21 OD027062/OD/NIH HHS/United States ; },
mesh = {Animals ; *Animals, Domestic ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Engineering ; Humans ; Livestock ; },
abstract = {Recombinant proteins are essential for the treatment and diagnosis of clinical human ailments. The availability and biological activity of recombinant proteins is heavily influenced by production platforms. Conventional production platforms such as yeast, bacteria, and mammalian cells have biological and economical challenges. Transgenic livestock species have been explored as an alternative production platform for recombinant proteins, predominantly through milk secretion; the strategy has been demonstrated to produce large quantities of biologically active proteins. The major limitation of utilizing livestock species as bioreactors has been efforts required to alter the genome of livestock. Advancements in the genome editing field have drastically improved the ability to genetically engineer livestock species. Specifically, genome editing tools such as the CRISPR/Cas9 system have lowered efforts required to generate genetically engineered livestock, thus minimizing restrictions on the type of genetic modification in livestock. In this review, we discuss characteristics of transgenic animal bioreactors and how the use of genome editing systems enhances design and availability of the animal models.},
}
@article {pmid34807378,
year = {2022},
author = {Shah, P and Magar, ND and Barbadikar, KM},
title = {Current technological interventions and applications of CRISPR/Cas for crop improvement.},
journal = {Molecular biology reports},
volume = {49},
number = {6},
pages = {5751-5770},
pmid = {34807378},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant/genetics ; *Plant Breeding ; Plants/genetics ; Technology ; },
abstract = {Efficient and innovative breeding strategies are immensely required to meet the global food demand, nutritional security and sustainable agriculture. Genome editing tools have emerged as an effective technology for site-directed genome modification causing the change in gene expression and protein function for the improvement of various important traits in particular the CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein). As the technology evolved with time, advances have been observed like prime editing, base editing, PAMless editing, Drosha based editing with multiple targets having the potential to fulfill the regulatory processes around the world. These recent interventions are highly proficient, cost-efficient, user-friendly, and holds promise for a major revolution in basic and applied plant biology research in the ever-evolving climatic conditions. In the review, we have discussed the most recent technologies and advances for CRISPR/Cas editing in plants.},
}
@article {pmid34803983,
year = {2021},
author = {Cobian, N and Garlet, A and Hidalgo-Cantabrana, C and Barrangou, R},
title = {Comparative Genomic Analyses and CRISPR-Cas Characterization of Cutibacterium acnes Provide Insights Into Genetic Diversity and Typing Applications.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {758749},
pmid = {34803983},
issn = {1664-302X},
abstract = {Cutibacterium acnes is an important member of the human skin microbiome and plays a critical role in skin health and disease. C. acnes encompasses different phylotypes that have been found to be associated with different skin phenotypes, suggesting a genetic basis for their impact on skin health. Here, we present a comprehensive comparative analysis of 255 C. acnes genomes to provide insights into the species genetic diversity and identify unique features that define various phylotypes. Results revealed a relatively small and open pan genome (6,240 genes) with a large core genome (1,194 genes), and three distinct phylogenetic clades, with multiple robust sub-clades. Furthermore, we identified several unique gene families driving differences between distinct C. acnes clades. Carbohydrate transporters, stress response mechanisms and potential virulence factors, potentially involved in competitive growth and host colonization, were detected in type I strains, which are presumably responsible for acne. Diverse type I-E CRISPR-Cas systems and prophage sequences were detected in select clades, providing insights into strain divergence and adaptive differentiation. Collectively, these results enable to elucidate the fundamental differences among C. acnes phylotypes, characterize genetic elements that potentially contribute to type I-associated dominance and disease, and other key factors that drive the differentiation among clades and sub-clades. These results enable the use of comparative genomics analyses as a robust method to differentiate among the C. acnes genotypes present in the skin microbiome, opening new avenues for the development of biotherapeutics to manipulate the skin microbiota.},
}
@article {pmid34803491,
year = {2021},
author = {Zhao, M and Quan, Y and Zeng, J and Lyu, X and Wang, H and Lei, JH and Feng, Y and Xu, J and Chen, Q and Sun, H and Xu, X and Lu, L and Deng, CX},
title = {Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.},
journal = {International journal of biological sciences},
volume = {17},
number = {15},
pages = {4176-4191},
pmid = {34803491},
issn = {1449-2288},
mesh = {Animals ; Antibodies/administration &amp; dosage/*therapeutic use ; Antineoplastic Agents/administration &amp; dosage/therapeutic use ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cholangiocarcinoma/*pathology ; Cullin Proteins/genetics/*metabolism ; Gene Expression Regulation ; Gene Knockdown Techniques ; Liver/metabolism ; Liver Neoplasms/*pathology ; Mice ; Mutation ; PTEN Phosphohydrolase/genetics/metabolism ; Programmed Cell Death 1 Receptor/genetics/metabolism ; Smad4 Protein/genetics/metabolism ; Sorafenib/administration &amp; dosage/*therapeutic use ; *Tumor Microenvironment ; },
abstract = {Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4[co/co];Pten[co/co];Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1[high] CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.},
}
@article {pmid34803454,
year = {2021},
author = {Demir, E},
title = {The potential use of Drosophila as an in vivo model organism for COVID-19-related research: a review.},
journal = {Turkish journal of biology = Turk biyoloji dergisi},
volume = {45},
number = {4},
pages = {559-569},
pmid = {34803454},
issn = {1303-6092},
abstract = {The world urgently needs effective antiviral approaches against emerging viruses, as shown by the coronavirus disease 2019 (COVID-19) pandemic, which has become an exponentially growing health crisis. Scientists from diverse backgrounds have directed their efforts towards identifying key features of SARS-CoV-2 and clinical manifestations of COVID-19 infection. Reports of more transmissible variants of SARS-CoV-2 also raise concerns over the possibility of an explosive trajectory of the pandemic, so scientific attention should focus on developing new weapons to help win the fight against coronaviruses that may undergo further mutations in the future. Drosophila melanogaster offers a powerful and potential in vivo model that can significantly increase the efficiency of drug screening for viral and bacterial infections. Thanks to its genes with functional human homologs, Drosophila could play a significant role in such gene-editing studies geared towards designing vaccines and antiviral drugs for COVID-19. It can also help rectify current drawbacks of CRISPR-based therapeutics like off-target effects and delivery issues, representing another momentous step forward in healthcare. Here I present an overview of recent literature and the current state of knowledge, explaining how it can open up new avenues for Drosophila in our battle against infectious diseases.},
}
@article {pmid34800362,
year = {2022},
author = {Tickman, BI and Burbano, DA and Chavali, VP and Kiattisewee, C and Fontana, J and Khakimzhan, A and Noireaux, V and Zalatan, JG and Carothers, JM},
title = {Multi-layer CRISPRa/i circuits for dynamic genetic programs in cell-free and bacterial systems.},
journal = {Cell systems},
volume = {13},
number = {3},
pages = {215-229.e8},
doi = {10.1016/j.cels.2021.10.008},
pmid = {34800362},
issn = {2405-4720},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; Gene Regulatory Networks/genetics ; RNA, Guide/metabolism ; Transcriptional Activation ; },
abstract = {CRISPR-Cas transcriptional circuits hold great promise as platforms for engineering metabolic networks and information processing circuits. Historically, prokaryotic CRISPR control systems have been limited to CRISPRi. Creating approaches to integrate CRISPRa for transcriptional activation with existing CRISPRi-based systems would greatly expand CRISPR circuit design space. Here, we develop design principles for engineering prokaryotic CRISPRa/i genetic circuits with network topologies specified by guide RNAs. We demonstrate that multi-layer CRISPRa/i cascades and feedforward loops can operate through the regulated expression of guide RNAs in cell-free expression systems and E. coli. We show that CRISPRa/i circuits can program complex functions by designing type 1 incoherent feedforward loops acting as fold-change detectors and tunable pulse-generators. By investigating how component characteristics relate to network properties such as depth, width, and speed, this work establishes a framework for building scalable CRISPRa/i circuits as regulatory programs in cell-free expression systems and bacterial hosts. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid34799728,
year = {2021},
author = {Hochstrasser, ML and Nuñez, JK},
title = {CRISPR meets caspase.},
journal = {Nature microbiology},
volume = {6},
number = {12},
pages = {1481-1482},
pmid = {34799728},
issn = {2058-5276},
mesh = {CRISPR-Cas Systems ; *Caspases/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
}
@article {pmid34799652,
year = {2021},
author = {Rahman, ML and Hyodo, T and Karnan, S and Ota, A and Hasan, MN and Mihara, Y and Wahiduzzaman, M and Tsuzuki, S and Hosokawa, Y and Konishi, H},
title = {Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {22627},
pmid = {34799652},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; DNA/*analysis ; DNA Breaks, Double-Stranded ; Gene Editing ; Gene Knock-In Techniques ; Gene Targeting/methods ; Genes, Reporter ; Genetic Engineering ; HCT116 Cells ; Homologous Recombination ; Humans ; Plasmids/metabolism ; RNA, Guide/*genetics ; Recombination, Genetic ; },
abstract = {Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700-2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.},
}
@article {pmid34799578,
year = {2021},
author = {Ferenczi, A and Chew, YP and Kroll, E and von Koppenfels, C and Hudson, A and Molnar, A},
title = {Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6751},
pmid = {34799578},
issn = {2041-1723},
support = {PHYCIBSC-03/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/V509188/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R506163/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Chlamydomonas reinhardtii/*genetics ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA, Plant/genetics/*metabolism ; DNA, Single-Stranded/genetics/*metabolism ; DNA-Directed DNA Polymerase/metabolism ; Endodeoxyribonucleases/metabolism ; Gene Editing/methods ; Genomic Instability ; Oligodeoxyribonucleotides/genetics/metabolism ; RNA, Guide/metabolism ; },
abstract = {Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.},
}
@article {pmid34798542,
year = {2022},
author = {Wellhausen, N and Agarwal, S and Rommel, PC and Gill, SI and June, CH},
title = {Better living through chemistry: CRISPR/Cas engineered T cells for cancer immunotherapy.},
journal = {Current opinion in immunology},
volume = {74},
number = {},
pages = {76-84},
pmid = {34798542},
issn = {1879-0372},
support = {P01 CA214278/CA/NCI NIH HHS/United States ; T32 CA009140/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Humans ; Immunotherapy ; Immunotherapy, Adoptive/methods ; *Neoplasms/genetics/therapy ; T-Lymphocytes ; },
abstract = {T cells engineered to express transgenes such as chimeric antigen receptors (CAR) or modified T cell receptors (TCR) represent a new pillar of cancer therapy. Use of CRISPR/Cas gene-editing tools now allows even stronger and more precise control over the fate and function of engineered T cell therapies, including multiplex genome editing to facilitate use of off-the-shelf allogeneic T cells and novel approaches which have the potential to overcome some of the limitations of canonical Cas9-mediated DNA cleavage. This review summarizes the CRISPR/Cas techniques that have been used in preclinical research and outlines those that currently being tested in clinical trials.},
}
@article {pmid34797942,
year = {2021},
author = {Long, J and Liu, N and Tang, W and Xie, L and Qin, F and Zhou, L and Tao, R and Wang, Y and Hu, Y and Jiao, Y and Li, L and Jiang, L and Qu, J and Chen, Q and Yao, S},
title = {A split cytosine deaminase architecture enables robust inducible base editing.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {12},
pages = {e22045},
doi = {10.1096/fj.202100123R},
pmid = {34797942},
issn = {1530-6860},
mesh = {*CRISPR-Cas Systems ; Cytidine Deaminase/*chemistry/*genetics ; DNA/*chemistry/genetics ; *Gene Editing ; Humans ; Proteins/*chemistry/*genetics ; },
abstract = {Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.},
}
@article {pmid34797876,
year = {2021},
author = {Petit, MJ and Kenaston, MW and Pham, OH and Nagainis, AA and Fishburn, AT and Shah, PS},
title = {Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes.},
journal = {PLoS pathogens},
volume = {17},
number = {11},
pages = {e1010100},
pmid = {34797876},
issn = {1553-7374},
support = {S10 OD010786/OD/NIH HHS/United States ; S10 RR019266/RR/NCRR NIH HHS/United States ; T32 AI060555/AI/NIAID NIH HHS/United States ; T32 GM007377/GM/NIGMS NIH HHS/United States ; },
mesh = {A549 Cells ; CRISPR-Cas Systems ; Dengue/genetics/metabolism/*virology ; Dengue Virus/physiology ; Humans ; *Mutation ; *Protein Interaction Domains and Motifs ; RNA-Seq ; STAT2 Transcription Factor/antagonists &amp; inhibitors/genetics/*metabolism ; Subcellular Fractions/*metabolism ; Transcription Factors/antagonists &amp; inhibitors/genetics/*metabolism ; Viral Nonstructural Proteins/genetics/*metabolism ; },
abstract = {Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.},
}
@article {pmid34797701,
year = {2022},
author = {Kostyushev, D and Kostyusheva, A and Ponomareva, N and Brezgin, S and Chulanov, V},
title = {CRISPR/Cas and Hepatitis B Therapy: Technological Advances and Practical Barriers.},
journal = {Nucleic acid therapeutics},
volume = {32},
number = {1},
pages = {14-28},
doi = {10.1089/nat.2021.0075},
pmid = {34797701},
issn = {2159-3345},
mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; DNA, Circular ; DNA, Viral/genetics ; *Hepatitis B/drug therapy/genetics ; Humans ; },
abstract = {After almost a decade of using CRISPR/Cas9 systems to edit target genes, CRISPR/Cas9 and related technologies are rapidly moving to clinical trials. Hepatitis B virus (HBV), which causes severe liver disease, cannot be cleared by modern antivirals, but represents an ideal target for CRISPR/Cas9 systems. Early studies demonstrated very high antiviral potency of CRISPR/Cas9 and supported its use for developing a cure against chronic HBV infection. This review discusses the key issues that must be solved to make CRISPR/Cas9 an anti-HBV therapy.},
}
@article {pmid34795452,
year = {2021},
author = {Long, L and Wei, J and Lim, SA and Raynor, JL and Shi, H and Connelly, JP and Wang, H and Guy, C and Xie, B and Chapman, NM and Fu, G and Wang, Y and Huang, H and Su, W and Saravia, J and Risch, I and Wang, YD and Li, Y and Niu, M and Dhungana, Y and Kc, A and Zhou, P and Vogel, P and Yu, J and Pruett-Miller, SM and Peng, J and Chi, H},
title = {CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.},
journal = {Nature},
volume = {600},
number = {7888},
pages = {308-313},
pmid = {34795452},
issn = {1476-4687},
support = {R01 AI131703/AI/NIAID NIH HHS/United States ; R01 CA250533/CA/NCI NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; R01 AG053987/AG/NIA NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; R35 CA253188/CA/NCI NIH HHS/United States ; R01 GM134382/GM/NIGMS NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Carrier Proteins/metabolism ; Female ; Forkhead Transcription Factors/metabolism ; *Gene Editing ; Genome/genetics ; Homeostasis ; Immune Tolerance ; Inflammation/pathology ; Male ; Mechanistic Target of Rapamycin Complex 1/metabolism ; Mice ; Neoplasms/immunology ; Nuclear Proteins/metabolism ; Nutrients/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; *Protein Interaction Maps ; Proteolysis ; S-Phase Kinase-Associated Proteins/metabolism ; T-Lymphocytes, Regulatory/*immunology/*metabolism ; Trans-Activators/metabolism ; },
abstract = {Nutrients are emerging regulators of adaptive immunity[1]. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism[2-4], but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.},
}
@article {pmid34795250,
year = {2021},
author = {Bar, S and Vershkov, D and Keshet, G and Lezmi, E and Meller, N and Yilmaz, A and Yanuka, O and Nissim-Rafinia, M and Meshorer, E and Eldar-Geva, T and Benvenisty, N},
title = {Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6718},
pmid = {34795250},
issn = {2041-1723},
mesh = {Animals ; Apoptosis Regulatory Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Chromatin Immunoprecipitation Sequencing/methods ; DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; DNA Methylation ; DNA-Binding Proteins/genetics/metabolism ; *Gene Expression Regulation ; *Genomic Imprinting ; *Haploidy ; HeLa Cells ; Human Embryonic Stem Cells/cytology/*metabolism ; Humans ; MAP Kinase Signaling System/genetics ; Male ; Parthenogenesis/genetics ; RNA-Binding Proteins/genetics/metabolism ; Repressor Proteins/genetics/metabolism ; Spermatogenesis/genetics ; },
abstract = {In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.},
}
@article {pmid34794576,
year = {2021},
author = {Cao, G and Deng, Y and Chen, X and Huo, D and Li, J and Yang, M and Hou, C},
title = {The fluorescent biosensor for detecting N[6] methyladenine FzD5 mRNA and MazF activity.},
journal = {Analytica chimica acta},
volume = {1188},
number = {},
pages = {339185},
doi = {10.1016/j.aca.2021.339185},
pmid = {34794576},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics ; },
abstract = {N[6] methyladenine (m[6]A) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (C-SDA) and CRISPR/Cas12a to detect m[6]A FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to m[6]A RNA. Take advantage of it, the biosensor achieved antibody-independent and gene-specific detection for m[6]A RNA. Moreover, compared with traditional amplification methods, the more efficient C-SDA and the CRISPR/Cas12a system with trans-cleavage activity gave the fluorescent biosensor an excellent sensitivity with the detection limit of 0.64 fM. In addition, MazF, as a new antibacterial target, was detected by the biosensor based on C-SDA and CRISPR/Cas12a with the detection limit of 1.127 × 10[-4] U mL[-1]. More importantly, the biosensor has good performance in complex samples. Therefore, the biosensor is a potential tool in detecting m[6]A FzD5 mRNA and MazF activity.},
}
@article {pmid34794559,
year = {2021},
author = {Xu, ZH and Zhao, ZY and Wang, H and Wang, SM and Chen, HY and Xu, JJ},
title = {CRISPR-Cas12a-based efficient electrochemiluminescence biosensor for ATP detection.},
journal = {Analytica chimica acta},
volume = {1188},
number = {},
pages = {339180},
doi = {10.1016/j.aca.2021.339180},
pmid = {34794559},
issn = {1873-4324},
mesh = {Adenosine Triphosphate ; *Biosensing Techniques ; *CRISPR-Cas Systems ; DNA ; Electrodes ; Luminescent Measurements ; },
abstract = {CRISPR-Cas12a system exhibits tremendous potential in accurate recognition and quantitation of nucleic acids and non-nucleic-acid targets thanks to the discovery of its cleavage capability toward single-stranded DNA (ssDNA). In this study, we developed an efficient electrochemiluminescence (ECL) sensing platform based on CRISPR-Cas12a for the analysis of adenosine triphosphate (ATP). In the presence of the target, the successful release of the DNA activator is specially recognized by Cas12a-crRNA duplex and activates the cleavage of ferrocene (Fc) labeled-ssDNA (Fc-ssDNA) modified on the cathode of bipolar electrode (BPE), resulting in a decrease of ECL intensity of [Ru(bpy)3][2+]/TPrA in the anodic cell of BPE. By means of the unique combination of Cas12a with ECL technique based on BPE, it can convert the recognition of target ATP into a detectable ECL signal. The detection limit of ATP was determined to be 0.48 nM under the optimal conditions. This work will expand the application of CRISPR-Cas detection system and propose a potential method for the analysis of non-nucleic-acid targets.},
}
@article {pmid34793837,
year = {2021},
author = {Doran, CG and Sugisawa, R and Carty, M and Roche, F and Fergus, C and Hokamp, K and Kelly, VP and Bowie, AG},
title = {CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {6},
pages = {101417},
pmid = {34793837},
issn = {1083-351X},
mesh = {Animals ; *Armadillo Domain Proteins/biosynthesis/genetics ; *CRISPR-Cas Systems ; *Cytoskeletal Proteins/biosynthesis/genetics ; *Epitopes/genetics/metabolism ; *Gene Expression Regulation ; Macrophages/*metabolism ; Mice ; Mice, Knockout ; Neurons/metabolism ; *Transcription, Genetic ; Vincristine/metabolism ; },
abstract = {SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1[-/-] mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1[-/-] mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.},
}
@article {pmid34793711,
year = {2021},
author = {Zampetidis, CP and Galanos, P and Angelopoulou, A and Zhu, Y and Polyzou, A and Karamitros, T and Kotsinas, A and Lagopati, N and Mourkioti, I and Mirzazadeh, R and Polyzos, A and Garnerone, S and Mizi, A and Gusmao, EG and Sofiadis, K and Gál, Z and Larsen, DH and Pefani, DE and Demaria, M and Tsirigos, A and Crosetto, N and Maya-Mendoza, A and Papaspyropoulos, A and Evangelou, K and Bartek, J and Papantonis, A and Gorgoulis, VG},
title = {A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.},
journal = {Molecular cell},
volume = {81},
number = {23},
pages = {4907-4923.e8},
doi = {10.1016/j.molcel.2021.10.017},
pmid = {34793711},
issn = {1097-4164},
mesh = {Animals ; Bronchi/metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Transformation, Neoplastic ; *Cellular Senescence ; *Chromosome Inversion ; Chromosomes/*ultrastructure ; Circadian Rhythm ; Computational Biology ; Epithelial Cells/metabolism ; *Epithelial-Mesenchymal Transition ; Flow Cytometry ; Genomics ; Humans ; Karyotyping ; Mice ; Mice, SCID ; Neoplasms/*genetics/metabolism ; *Oncogenes ; Phenotype ; Protein Binding ; Protein Domains ; *Recombination, Genetic ; Senescence-Associated Secretory Phenotype ; },
abstract = {Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.},
}
@article {pmid34793143,
year = {2021},
author = {Naduthodi, MIS and Südfeld, C and Avitzigiannis, EK and Trevisan, N and van Lith, E and Alcaide Sancho, J and D'Adamo, S and Barbosa, M and van der Oost, J},
title = {Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems.},
journal = {ACS synthetic biology},
volume = {10},
number = {12},
pages = {3369-3378},
pmid = {34793143},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; *Microalgae/genetics ; *Stramenopiles/genetics ; },
abstract = {Microalgae can produce industrially relevant metabolites using atmospheric CO2 and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoproteins (RNPs) and homology-directed repair (HDR) to generate scarless and markerless mutants of the microalga Nannochloropsis oceanica. We also developed an episomal plasmid-based Cas12a system for efficiently introducing indels at the target site. Additionally, we exploited the ability of Cas12a to process an associated CRISPR array to perform multiplexed genome engineering. We efficiently targeted three sites in the host genome in a single transformation, thereby making a major step toward high-throughput genome engineering in microalgae. Furthermore, a CRISPR interference (CRISPRi) tool based on Cas9 and Cas12a was developed for effective downregulation of target genes. We observed up to 85% reduction in the transcript levels upon performing CRISPRi with dCas9 in N. oceanica. Overall, these developments substantially accelerate genome engineering efforts in N. oceanica and potentially provide a general toolbox for improving other microalgal strains.},
}
@article {pmid34791430,
year = {2021},
author = {Song, Y and Hou, G and Diep, J and Ooi, YS and Akopyants, NS and Beverley, SM and Carette, JE and Greenberg, HB and Ding, S},
title = {Inhibitor of growth protein 3 epigenetically silences endogenous retroviral elements and prevents innate immune activation.},
journal = {Nucleic acids research},
volume = {49},
number = {22},
pages = {12706-12715},
pmid = {34791430},
issn = {1362-4962},
support = {R01 AI029646/GF/NIH HHS/United States ; R00 AI135031/AI/NIAID NIH HHS/United States ; R01 AI125249/AI/NIAID NIH HHS/United States ; R01 AI130222/AI/NIAID NIH HHS/United States ; R01 AI141970/AI/NIAID NIH HHS/United States ; R01 AI150796/AI/NIAID NIH HHS/United States ; P30 DK052574/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Endogenous Retroviruses ; *Gene Silencing ; HT29 Cells ; HeLa Cells ; Histone Code ; Homeodomain Proteins/metabolism/*physiology ; Humans ; Immunity, Innate/*genetics ; Tumor Suppressor Proteins/metabolism/*physiology ; },
abstract = {Endogenous retroviruses (ERVs) are subject to transcriptional repression in adult tissues, in part to prevent autoimmune responses. However, little is known about the epigenetic silencing of ERV expression. Here, we describe a new role for inhibitor of growth family member 3 (ING3), to add to an emerging group of ERV transcriptional regulators. Our results show that ING3 binds to several ERV promoters (for instance MER21C) and establishes an EZH2-mediated H3K27 trimethylation modification. Loss of ING3 leads to decreases of H3K27 trimethylation enrichment at ERVs, induction of MDA5-MAVS-interferon signaling, and functional inhibition of several virus infections. These data demonstrate an important new function of ING3 in ERV silencing and contributing to innate immune regulation in somatic cells.},
}
@article {pmid34791389,
year = {2022},
author = {Kirillov, B and Savitskaya, E and Panov, M and Ogurtsov, AY and Shabalina, SA and Koonin, EV and Severinov, KV},
title = {Uncertainty-aware and interpretable evaluation of Cas9-gRNA and Cas12a-gRNA specificity for fully matched and partially mismatched targets with Deep Kernel Learning.},
journal = {Nucleic acids research},
volume = {50},
number = {2},
pages = {e11},
pmid = {34791389},
issn = {1362-4962},
mesh = {Algorithms ; *CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing/methods ; *Gene Targeting/methods ; Genomics/methods ; Humans ; Neural Networks, Computer ; RNA, Guide/*genetics ; Reproducibility of Results ; },
abstract = {The choice of guide RNA (gRNA) for CRISPR-based gene targeting is an essential step in gene editing applications, but the prediction of gRNA specificity remains challenging. Lack of transparency and focus on point estimates of efficiency disregarding the information on possible error sources in the model limit the power of existing Deep Learning-based methods. To overcome these problems, we present a new approach, a hybrid of Capsule Networks and Gaussian Processes. Our method predicts the cleavage efficiency of a gRNA with a corresponding confidence interval, which allows the user to incorporate information regarding possible model errors into the experimental design. We provide the first utilization of uncertainty estimation in computational gRNA design, which is a critical step toward accurate decision-making for future CRISPR applications. The proposed solution demonstrates acceptable confidence intervals for most test sets and shows regression quality similar to existing models. We introduce a set of criteria for gRNA selection based on off-target cleavage efficiency and its variance and present a collection of pre-computed gRNAs for human chromosome 22. Using Neural Network Interpretation methods, we show that our model rediscovers an established biological factor underlying cleavage efficiency, the importance of the seed region in gRNA.},
}
@article {pmid34791161,
year = {2022},
author = {Terradas, G and Hermann, A and James, AA and McGinnis, W and Bier, E},
title = {High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes.},
journal = {G3 (Bethesda, Md.)},
volume = {12},
number = {1},
pages = {},
pmid = {34791161},
issn = {2160-1836},
support = {P30 NS047101/NS/NINDS NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Anopheles/genetics ; CRISPR-Cas Systems ; Female ; *Gene Drive Technology ; Germ Cells ; Male ; Mosquito Vectors/genetics ; },
abstract = {Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNosCd-1 drive (nos-Cas9) in Anopheles gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly coexpressed with endogenous genes in stem and premeiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct colocalization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.},
}
@article {pmid34789569,
year = {2021},
author = {Yu, H and Xue, L and Barrangou, R and Chen, S and Huang, Y},
title = {Opinion: Toward inclusive global governance of human genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {47},
pages = {},
pmid = {34789569},
issn = {1091-6490},
mesh = {Bioethics ; Biomedical Research/ethics ; CRISPR-Cas Systems ; Clinical Governance/*ethics/standards ; Gene Editing/*ethics ; Genome, Human ; Government ; Humans ; Publications ; },
}
@article {pmid34789568,
year = {2021},
author = {Naert, T and Tulkens, D and Van Nieuwenhuysen, T and Przybyl, J and Demuynck, S and van de Rijn, M and Al-Jazrawe, M and Alman, BA and Coucke, PJ and De Leeneer, K and Vanhove, C and Savvides, SN and Creytens, D and Vleminckx, K},
title = {CRISPR-SID: Identifying EZH2 as a druggable target for desmoid tumors via in vivo dependency mapping.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {47},
pages = {},
pmid = {34789568},
issn = {1091-6490},
support = {P30 CA014236/CA/NCI NIH HHS/United States ; },
mesh = {Abdominal Neoplasms/genetics ; Adenomatous Polyposis Coli/genetics ; Animals ; *CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cyclic AMP Response Element-Binding Protein ; Enhancer of Zeste Homolog 2 Protein/*genetics/*isolation &amp; purification/*metabolism ; Fibromatosis, Aggressive/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Proteins/genetics/metabolism ; Nerve Tissue Proteins ; Oncogenes ; Polycomb Repressive Complex 2/metabolism ; Transcription Factors/genetics/metabolism ; Wnt Signaling Pathway ; Xenopus ; beta Catenin ; },
abstract = {Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/β-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification.},
}
@article {pmid34789333,
year = {2021},
author = {Wilson, FM and Harrison, RJ},
title = {CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12.},
journal = {Fungal biology and biotechnology},
volume = {8},
number = {1},
pages = {15},
pmid = {34789333},
issn = {2054-3085},
support = {BB/P020364/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {BACKGROUND: Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).<br><br>RESULTS: We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.<br><br>CONCLUSIONS: Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.},
}
@article {pmid34789150,
year = {2021},
author = {Vinceti, A and Karakoc, E and Pacini, C and Perron, U and De Lucia, RR and Garnett, MJ and Iorio, F},
title = {CoRe: a robustly benchmarked R package for identifying core-fitness genes in genome-wide pooled CRISPR-Cas9 screens.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {828},
pmid = {34789150},
issn = {1471-2164},
mesh = {Benchmarking ; *CRISPR-Cas Systems ; Genes, Essential ; Humans ; *Neoplasms/genetics ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome-wide screens are being increasingly performed, allowing systematic explorations of cancer dependencies at unprecedented accuracy and scale. One of the major computational challenges when analysing data derived from such screens is to identify genes that are essential for cell survival invariantly across tissues, conditions, and genomic-contexts (core-fitness genes), and to distinguish them from context-specific essential genes. This is of paramount importance to assess the safety profile of candidate therapeutic targets and for elucidating mechanisms involved in tissue-specific genetic diseases.<br><br>RESULTS: We have developed CoRe: an R package implementing existing and novel methods for the identification of core-fitness genes (at two different level of stringency) from joint analyses of multiple CRISPR-Cas9 screens. We demonstrate, through a fully reproducible benchmarking pipeline, that CoRe outperforms state-of-the-art tools, yielding more reliable and biologically relevant sets of core-fitness genes.<br><br>CONCLUSIONS: CoRe offers a flexible pipeline, compatible with many pre-processing methods for the analysis of CRISPR data, which can be tailored onto different use-cases. The CoRe package can be used for the identification of high-confidence novel core-fitness genes, as well as a means to filter out potentially cytotoxic hits while analysing cancer dependency datasets for identifying and prioritising novel selective therapeutic targets.},
}
@article {pmid34788687,
year = {2021},
author = {Gago, JF and Viver, T and Urdiain, M and Pastor, S and Kämpfer, P and Robledo, PA and Ferreira, E and Rosselló-Móra, R},
title = {Comparative genome analysis of the genus Hydrotalea and proposal of the novel species Hydrotalea lipotrueae sp. nov., isolated from a groundwater aquifer in the south of Mallorca Island, Spain.},
journal = {Systematic and applied microbiology},
volume = {44},
number = {6},
pages = {126277},
doi = {10.1016/j.syapm.2021.126277},
pmid = {34788687},
issn = {1618-0984},
mesh = {Bacterial Typing Techniques ; DNA, Bacterial/genetics ; Fatty Acids/analysis ; *Genomics ; *Groundwater ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; Spain ; },
abstract = {From a collection of > 140 strains isolated from groundwater with thermal anomalies for the purpose of obtaining good candidates with applications in the cosmetic industry, two strains were selected because of their taxonomic novelty. Among the isolates, strains TMF_100[T] and TFM_099 stood out for their potential biotechnological relevance, and a comparative analysis of 16S rRNA gene sequences indicated that these strains represented a new species of the genus Hydrotalea. In addition, from the public genomic databases, metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) could be retrieved that affiliated with this genus. These MAGs and SAGs had been obtained from different environmental samples, such as acid mine drainage or marine sediments. In addition to the description of the new species, the ecological relevance of the members of this genus was demonstrated by means of denitrification, CRISPR-Cas system diversity and heavy metal resistance, as well as their wide geographical distribution and environmental versatility. Supported by the taxonomic study, together with physiological and morphological differences and ecological features, we concluded that strain TMF_100[T] represented a novel species within the genus Hydrotalea, for which we propose the name Hydrotalea lipotrueae sp. nov.},
}
@article {pmid34788305,
year = {2021},
author = {Ahmed, S and Roy, MC and Al Baki, MA and Jung, JK and Lee, D and Kim, Y},
title = {CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.},
journal = {PloS one},
volume = {16},
number = {11},
pages = {e0259322},
pmid = {34788305},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems ; Insect Control ; *Sex Attractants ; *Spodoptera ; },
abstract = {Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.},
}
@article {pmid34788038,
year = {2021},
author = {Lin, J and Wang, WJ and Wang, Y and Liu, Y and Xu, L},
title = {Building Endogenous Gene Connections through RNA Self-Assembly Controlled CRISPR/Cas9 Function.},
journal = {Journal of the American Chemical Society},
volume = {143},
number = {47},
pages = {19834-19843},
doi = {10.1021/jacs.1c09041},
pmid = {34788038},
issn = {1520-5126},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics/metabolism ; Gene Expression Regulation/physiology ; *Gene Regulatory Networks ; HEK293 Cells ; Humans ; Phenotype ; RNA, Guide/*genetics ; RNA, Messenger/genetics ; },
abstract = {Construction of synthetic circuits that can artificially establish endogenous gene connections is essential to introduce new phenotypes for cellular behaviors. Given the diversity of endogenous genes, it lacks a general and easy-to-design toolbox to manipulate the genetic network. Here we present a type of self-assembly-induced RNA circuit that can directly build regulatory connections between endogenous genes. Inspired from the natural assembling process of guide RNA in the CRISPR/Cas9 complex, this design employs an independent trigger RNA strand to induce the formation of a ternary guide RNA assembly for functional control of CRISPR/Cas9. With this general principle, expressional regulations of endogenous genes can be controlled by totally independent endogenous small RNAs and mRNAs in E. coli via activatable CRISPR/Cas9 function. Moreover, the cellular phenotype of E. coli is successfully programmed with introduction of new gene connections. In addition, the functionality of this design is also verified in the mammalian system. This self-assembly-based RNA circuit exhibits a great flexibility and simplicity of design and provides a unique approach to build endogenous gene connections, which paves a broad way toward manipulation of cellular genetic networks.},
}
@article {pmid34787487,
year = {2021},
author = {Liang, Y and Lin, H and Zou, L and Zhao, J and Li, B and Wang, H and Lu, J and Sun, J and Yang, X and Deng, X and Tang, S},
title = {CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern.},
journal = {Microbiology spectrum},
volume = {9},
number = {3},
pages = {e0101721},
pmid = {34787487},
issn = {2165-0497},
mesh = {Alleles ; COVID-19/diagnosis/*virology ; COVID-19 Testing/*methods ; *CRISPR-Cas Systems ; Databases, Nucleic Acid ; Humans ; Mass Screening ; Mutation ; Polymerase Chain Reaction ; Public Health ; SARS-CoV-2/*classification/*genetics/*isolation &amp; purification ; Spike Glycoprotein, Coronavirus/genetics ; },
abstract = {A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.},
}
@article {pmid34787450,
year = {2021},
author = {Jalal, D and Elzayat, MG and Diab, AA and El-Shqanqery, HE and Samir, O and Bakry, U and Hassan, R and Elanany, M and Shalaby, L and Sayed, AA},
title = {Deciphering Multidrug-Resistant Acinetobacter baumannii from a Pediatric Cancer Hospital in Egypt.},
journal = {mSphere},
volume = {6},
number = {6},
pages = {e0072521},
pmid = {34787450},
issn = {2379-5042},
mesh = {Acinetobacter Infections/epidemiology/*microbiology/transmission ; Acinetobacter baumannii/drug effects/*genetics/*isolation &amp; purification/pathogenicity ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Cancer Care Facilities ; Carbapenems/pharmacology ; Colistin/pharmacology ; Cross Infection ; Drug Resistance, Multiple, Bacterial/*genetics ; Egypt ; Hospitals, Pediatric ; Humans ; Microbial Sensitivity Tests ; *Multilocus Sequence Typing ; Whole Genome Sequencing ; beta-Lactamases/genetics ; },
abstract = {Infection by multidrug-resistant (MDR) Acinetobacter baumannii is one of the major causes of hospital-acquired infections worldwide. The ability of A. baumannii to survive in adverse conditions as well as its extensive antimicrobial resistance make it one of the most difficult to treat pathogens associated with high mortality rates. The aim of this study was to investigate MDR A. baumannii that has spread among pediatric cancer patients in the Children's Cancer Hospital Egypt 57357. Whole-genome sequencing was used to characterize 31 MDR A. baumannii clinical isolates. Phenotypically, the isolates were MDR, with four isolates showing resistance to the last-resort antibiotic colistin. Multilocus sequence typing showed the presence of eight clonal groups, two of which were previously reported to cause outbreaks in Egypt, and one novel sequence type (ST), Oxf-ST2246. Identification of the circulating plasmids showed the presence of two plasmid lineages in the isolates, strongly governed by sequence type. A large number of antimicrobial genes with a range of resistance mechanisms were detected in the isolates, including β-lactamases and antibiotic efflux pumps. Analysis of insertion sequences (ISs) revealed the presence of ISAba1 and ISAba125 in all the samples, which amplify β-lactamase expression, causing extensive carbapenem resistance. Mutation analysis was used to decipher underlying mutations responsible for colistin resistance and revealed novel mutations in several outer membrane proteins, in addition to previously reported mutations in pmrB. Altogether, understanding the transmissibility of A. baumannii as well as its resistance and virulence mechanisms will help develop novel treatment options for better management of hospital-acquired infections. IMPORTANCE Acinetobacter baumannii represents a major health threat, in particular among immunocompromised cancer patients. The rise in carbapenem-resistant A. baumannii, and the development of resistance to the last-resort antimicrobial agent colistin, complicates the management of A. baumannii outbreaks and increases mortality rates. Here, we investigate 31 multidrug resistant A. baumannii isolates from pediatric cancer patients in Children's Cancer Hospital Egypt (CCHE) 57357 via whole-genome sequencing. Multilocus sequence typing (MLST) showed the presence of eight clonal groups including a novel sequence type. In silico detection of antimicrobial-resistant genes and virulence factors revealed a strong correlation between certain virulence genes and mortality as well as several point mutations in outer membrane proteins contributing to colistin resistance. Detection of CRISPR/Cas sequences in the majority of the samples was strongly correlated with the presence of prophage sequences and associated with failure of bacteriophage therapy. Altogether, understanding the genetic makeup of circulating A. baumannii is essential for better management of outbreaks.},
}
@article {pmid34785123,
year = {2022},
author = {Awan, MJA and Amin, I and Mansoor, S},
title = {Mini CRISPR-Cas12f1: a new genome editing tool.},
journal = {Trends in plant science},
volume = {27},
number = {2},
pages = {110-112},
doi = {10.1016/j.tplants.2021.11.002},
pmid = {34785123},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; *Nanoparticles ; },
abstract = {The clustered regularly interspaced short palindromic repeats and associated protein (CRISPR-Cas) toolbox enables targeted mutations to be introduced into a genome. However, the delivery of appropriately sized Cas effectors to develop transgene-free edited plants is a limiting factor. A novel mini CRISPR-Cas12f1 system recently reported by Wu et al. overcomes this challenge by deploying viral-based vectors and nanoparticles (NPs) as carriers.},
}
@article {pmid34784388,
year = {2021},
author = {Inbar, E and Eappen, AG and Alford, RT and Reid, W and Harrell, RA and Hosseini, M and Chakravarty, S and Li, T and Sim, BKL and Billingsley, PF and Hoffman, SL},
title = {Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence.},
journal = {PLoS pathogens},
volume = {17},
number = {11},
pages = {e1009770},
pmid = {34784388},
issn = {1553-7374},
support = {R43 AI077262/AI/NIAID NIH HHS/United States ; R44 AI077262/AI/NIAID NIH HHS/United States ; R44 AI131806/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Anopheles/*physiology ; Bacteria/growth &amp; development ; *CRISPR-Cas Systems ; Digestive System/microbiology ; Female ; Insect Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Malaria/*parasitology ; Male ; Mosquito Vectors/genetics/*growth &amp; development/parasitology ; Plasmodium/*growth &amp; development ; *Reproduction ; },
abstract = {PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1's regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.},
}
@article {pmid34784073,
year = {2022},
author = {Assou, J and Zhang, D and Roth, KDR and Steinke, S and Hust, M and Reinard, T and Winkelmann, T and Boch, J},
title = {Removing the major allergen Bra j I from brown mustard (Brassica juncea) by CRISPR/Cas9.},
journal = {The Plant journal : for cell and molecular biology},
volume = {109},
number = {3},
pages = {649-663},
doi = {10.1111/tpj.15584},
pmid = {34784073},
issn = {1365-313X},
mesh = {Agrobacterium tumefaciens ; Allergens/*genetics ; CRISPR-Cas Systems ; Crops, Agricultural/chemistry/genetics ; Food Hypersensitivity/*prevention &amp; control ; Gene Editing/*methods ; Genes, Plant ; Genetic Variation ; Genotype ; Mustard Plant/*genetics ; Plant Breeding/*methods ; Plants, Genetically Modified ; Seed Storage Proteins/*chemistry/*genetics ; Transformation, Genetic ; },
abstract = {Food allergies are a major health issue worldwide. Modern breeding techniques such as genome editing via CRISPR/Cas9 have the potential to mitigate this by targeting allergens in plants. This study addressed the major allergen Bra j I, a seed storage protein of the 2S albumin class, in the allotetraploid brown mustard (Brassica juncea). Cotyledon explants of an Indian gene bank accession (CR2664) and the German variety Terratop were transformed using Agrobacterium tumefaciens harboring binary vectors with multiple single guide RNAs to induce either large deletions or frameshift mutations in both Bra j I homoeologs. A total of 49 T0 lines were obtained with up to 3.8% transformation efficiency. Four lines had large deletions of 566 up to 790 bp in the Bra j IB allele. Among 18 Terratop T0 lines, nine carried indels in the targeted regions. From 16 analyzed CR2664 T0 lines, 14 held indels and three had all four Bra j I alleles mutated. The majority of the CRISPR/Cas9-induced mutations were heritable to T1 progenies. In some edited lines, seed formation and viability were reduced and seeds showed a precocious development of the embryo leading to a rupture of the testa already in the siliques. Immunoblotting using newly developed Bra j I-specific antibodies revealed the amount of Bra j I protein to be reduced or absent in seed extracts of selected lines. Removing an allergenic determinant from mustard is an important first step towards the development of safer food crops.},
}
@article {pmid34783883,
year = {2022},
author = {Matsuo, K},
title = {CRISPR/Cas9-mediated knockout of the DCL2 and DCL4 genes in Nicotiana benthamiana and its productivity of recombinant proteins.},
journal = {Plant cell reports},
volume = {41},
number = {2},
pages = {307-317},
pmid = {34783883},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems ; Fibroblast Growth Factor 1/genetics/metabolism ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified/genetics ; Recombinant Proteins/genetics/*metabolism ; Tobacco/*genetics/metabolism ; },
abstract = {DCL2 and DCL4 genes in Nicotiana benthamiana plants were successfully edited using the CRISPR/Cas9 system. Recently, plants have been utilized for recombinant protein production similar to other expression systems, i.e., bacteria, yeast, insect, and mammal cells. However, insufficient amounts of recombinant proteins are often produced in plant cells. The repression of RNA silencing within plant cells could improve production levels of recombinant protein because RNA silencing frequently decomposes mRNAs from transgenes. In this study, the genes dicer-like protein 2 and 4 (NbDCL2 and NbDCL4) were successfully edited to produce double-knockout transgenic Nicotiana benthamiana plants (dcl2dcl4 plants) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. A transient green fluorescent protein (GFP) gene expression assay revealed that the dcl2dcl4 plants accumulated higher amounts of GFP and GFP mRNA than wild type (WT) and RNA-dependent RNA polymerase 6-knockout N. benthamiana plants (ΔRDR6 plants). Small RNA sequencing also showed that dcl2dcl4 plants accumulated lower amounts of small interfering RNAs (siRNAs) against the GFP gene than WT plants. The dcl2dcl4 plants might also produce higher amounts of human fibroblast growth factor 1 (FGF1) than WT and ΔRDR6 plants. These observations appear to reflect differences between DCLs and RDR6 in plant cell biological mechanisms. These results reveal that dcl2dcl4 plants would be suitable as platform plants for recombinant protein production.},
}
@article {pmid34783536,
year = {2021},
author = {Marqués, MC and Ruiz, R and Montagud-Martínez, R and Márquez-Costa, R and Albert, S and Domingo-Calap, P and Rodrigo, G},
title = {CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation.},
journal = {ACS synthetic biology},
volume = {10},
number = {12},
pages = {3595-3599},
pmid = {34783536},
issn = {2161-5063},
mesh = {Biosensing Techniques ; COVID-19/*diagnosis/virology ; COVID-19 Testing/*methods ; *CRISPR-Cas Systems ; DNA/analysis ; DNA, Viral/*genetics ; Genetic Techniques ; HEK293 Cells ; Humans ; Immunoglobulin G/chemistry ; *Mutation ; Peptide Library ; Polymers/chemistry ; SARS-CoV-2/*genetics ; Spain/epidemiology ; Surface Plasmon Resonance ; },
abstract = {The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.},
}
@article {pmid34782773,
year = {2021},
author = {Lu, Y and Wang, J and Chen, B and Mo, S and Lian, L and Luo, Y and Ding, D and Ding, Y and Cao, Q and Li, Y and Li, Y and Liu, G and Hou, Q and Cheng, T and Wei, J and Zhang, Y and Chen, G and Song, C and Hu, Q and Sun, S and Fan, G and Wang, Y and Liu, Z and Song, B and Zhu, JK and Li, H and Jiang, L},
title = {A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice.},
journal = {Nature plants},
volume = {7},
number = {11},
pages = {1445-1452},
pmid = {34782773},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; DNA ; *Gene Editing ; Genes, Plant ; *Oryza/genetics ; Plant Breeding ; Promoter Regions, Genetic ; Ubiquitin/genetics ; },
abstract = {Structural variations (SVs), such as inversion and duplication, contribute to important agronomic traits in crops[1]. Pan-genome studies revealed that SVs were a crucial and ubiquitous force driving genetic diversification[2-4]. Although genome editing can effectively create SVs in plants and animals[5-8], the potential of designed SVs in breeding has been overlooked. Here, we show that new genes and traits can be created in rice by designed large-scale genomic inversion or duplication using CRISPR/Cas9. A 911 kb inversion on chromosome 1 resulted in a designed promoter swap between CP12 and PPO1, and a 338 kb duplication between HPPD and Ubiquitin2 on chromosome 2 created a novel gene cassette at the joint, promoterUbiquitin2::HPPD. Since the original CP12 and Ubiquitin2 genes were highly expressed in leaves, the expression of PPO1 and HPPD in edited plants with homozygous SV alleles was increased by tens of folds and conferred sufficient herbicide resistance in field trials without adverse effects on other important agronomic traits. CRISPR/Cas-based genome editing for gene knock-ups has been generally considered very difficult without inserting donor DNA as regulatory elements. Our study challenges this notion by providing a donor-DNA-free strategy, thus greatly expanding the utility of CRISPR/Cas in plant and animal improvements.},
}
@article {pmid34782715,
year = {2022},
author = {Christen, F and Hablesreiter, R and Hoyer, K and Hennch, C and Maluck-Böttcher, A and Segler, A and Madadi, A and Frick, M and Bullinger, L and Briest, F and Damm, F},
title = {Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9.},
journal = {Leukemia},
volume = {36},
number = {4},
pages = {1102-1110},
pmid = {34782715},
issn = {1476-5551},
mesh = {*CRISPR-Cas Systems ; Clonal Hematopoiesis ; *Fetal Blood ; Hematopoiesis/genetics ; Hematopoietic Stem Cells ; Humans ; },
abstract = {To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34[+] progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2[mut]) cells, whereas ASXL1[mut] as well as DNMT3A[mut] cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.},
}
@article {pmid34782635,
year = {2021},
author = {Ageely, EA and Chilamkurthy, R and Jana, S and Abdullahu, L and O'Reilly, D and Jensik, PJ and Damha, MJ and Gagnon, KT},
title = {Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6591},
pmid = {34782635},
issn = {2041-1723},
support = {R01 GM135646/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/*genetics/*metabolism ; CRISPR-Associated Proteins/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/chemistry/*genetics/*metabolism ; *Gene Editing ; HEK293 Cells ; Humans ; Nucleic Acids ; Pathology, Molecular/methods ; RNA ; RNA, Guide/genetics ; Ribose/chemistry/*metabolism ; },
abstract = {CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5' handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2'-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2'-hydroxyl sensitivity. Modified 5' pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5' pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5' pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.},
}
@article {pmid34782600,
year = {2021},
author = {Bengel, P and Dybkova, N and Tirilomis, P and Ahmad, S and Hartmann, N and A Mohamed, B and Krekeler, MC and Maurer, W and Pabel, S and Trum, M and Mustroph, J and Gummert, J and Milting, H and Wagner, S and Ljubojevic-Holzer, S and Toischer, K and Maier, LS and Hasenfuss, G and Streckfuss-Bömeke, K and Sossalla, S},
title = {Detrimental proarrhythmogenic interaction of Ca[2+]/calmodulin-dependent protein kinase II and NaV1.8 in heart failure.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6586},
pmid = {34782600},
issn = {2041-1723},
mesh = {Animals ; Arrhythmias, Cardiac/metabolism ; CRISPR-Cas Systems ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/*metabolism ; Heart Failure/*metabolism/pathology ; Homeostasis/*genetics ; Humans ; Mice ; Mice, Knockout ; Mice, Transgenic ; Molecular Medicine ; Myocytes, Cardiac ; NAV1.8 Voltage-Gated Sodium Channel/*genetics/*metabolism ; },
abstract = {An interplay between Ca[2+]/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na[+] current (INaL) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform NaV1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of NaV1.8, we demonstrate that NaV1.8 contributes to INaL formation. In addition, we reveal a direct interaction between NaV1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of NaV1.8 and CaMKIIδc, we show that NaV1.8-driven INaL is CaMKIIδc-dependent and that NaV1.8-inhibtion reduces diastolic SR-Ca[2+] leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a NaV1.8 knock-out is introduced. Cellular and in vivo experiments reveal reduced ventricular arrhythmias without changes in HF progression. Our work therefore identifies a proarrhythmic CaMKIIδc downstream target which may constitute a prognostic and antiarrhythmic strategy.},
}
@article {pmid34781949,
year = {2021},
author = {Bender, G and Fahrioglu Yamaci, R and Taneri, B},
title = {CRISPR and KRAS: a match yet to be made.},
journal = {Journal of biomedical science},
volume = {28},
number = {1},
pages = {77},
pmid = {34781949},
issn = {1423-0127},
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Mice ; *Mutation ; Proto-Oncogene Proteins p21(ras)/*therapeutic use ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) systems are one of the most fascinating tools of the current era in molecular biotechnology. With the ease that they provide in genome editing, CRISPR systems generate broad opportunities for targeting mutations. Specifically in recent years, disease-causing mutations targeted by the CRISPR systems have been of main research interest; particularly for those diseases where there is no current cure, including cancer. KRAS mutations remain untargetable in cancer. Mutations in this oncogene are main drivers in common cancers, including lung, colorectal and pancreatic cancers, which are severe causes of public health burden and mortality worldwide, with no cure at hand. CRISPR systems provide an opportunity for targeting cancer causing mutations. In this review, we highlight the work published on CRISPR applications targeting KRAS mutations directly, as well as CRISPR applications targeting mutations in KRAS-related molecules. In specific, we focus on lung, colorectal and pancreatic cancers. To date, the limited literature on CRISPR applications targeting KRAS, reflect promising results. Namely, direct targeting of mutant KRAS variants using various CRISPR systems resulted in significant decrease in cell viability and proliferation in vitro, as well as tumor growth inhibition in vivo. In addition, the effect of mutant KRAS knockdown, via CRISPR, has been observed to exert regulatory effects on the downstream molecules including PI3K, ERK, Akt, Stat3, and c-myc. Molecules in the KRAS pathway have been subjected to CRISPR applications more often than KRAS itself. The aim of using CRISPR systems in these studies was mainly to analyze the therapeutic potential of possible downstream and upstream effectors of KRAS, as well as to discover further potential molecules. Although there have been molecules identified to have such potential in treatment of KRAS-driven cancers, a substantial amount of effort is still needed to establish treatment strategies based on these discoveries. We conclude that, at this point in time, despite being such a powerful directed genome editing tool, CRISPR remains to be underutilized for targeting KRAS mutations in cancer. Efforts channelled in this direction, might pave the way in solving the long-standing challenge of targeting the KRAS mutations in cancers.},
}
@article {pmid34781740,
year = {2021},
author = {Dhasmana, N and Ram, G and McAllister, KN and Chupalova, Y and Lopez, P and Ross, HF and Novick, RP},
title = {Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.},
journal = {mBio},
volume = {12},
number = {6},
pages = {e0208321},
pmid = {34781740},
issn = {2150-7511},
support = {R01 AI139613/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Genomic Islands ; Staphylococcal Infections/microbiology ; Staphylococcus Phages/genetics/*physiology ; Staphylococcus aureus/drug effects/*genetics/metabolism/*virology ; Tetracycline/pharmacology ; },
abstract = {The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tc[r]) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tc[r]. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.},
}
@article {pmid34779571,
year = {2022},
author = {Mennuni, M and Filograna, R and Felser, A and Bonekamp, NA and Giavalisco, P and Lytovchenko, O and Larsson, NG},
title = {Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen.},
journal = {EMBO reports},
volume = {23},
number = {1},
pages = {e53054},
pmid = {34779571},
issn = {1469-3178},
mesh = {*CRISPR-Cas Systems ; *DNA, Mitochondrial/genetics ; Down-Regulation ; Gene Editing ; Mitochondria/genetics/metabolism ; Transcription, Genetic ; },
abstract = {Cancer cells depend on mitochondria to sustain their increased metabolic need and mitochondria therefore constitute possible targets for cancer treatment. We recently developed small-molecule inhibitors of mitochondrial transcription (IMTs) that selectively impair mitochondrial gene expression. IMTs have potent antitumor properties in vitro and in vivo, without affecting normal tissues. Because therapy-induced resistance is a major constraint to successful cancer therapy, we investigated mechanisms conferring resistance to IMTs. We employed a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats)-(CRISP-associated protein 9) whole-genome screen to determine pathways conferring resistance to acute IMT1 treatment. Loss of genes belonging to von Hippel-Lindau (VHL) and mammalian target of rapamycin complex 1 (mTORC1) pathways caused resistance to acute IMT1 treatment and the relevance of these pathways was confirmed by chemical modulation. We also generated cells resistant to chronic IMT treatment to understand responses to persistent mitochondrial gene expression impairment. We report that IMT1-acquired resistance occurs through a compensatory increase of mitochondrial DNA (mtDNA) expression and cellular metabolites. We found that mitochondrial transcription factor A (TFAM) downregulation and inhibition of mitochondrial translation impaired survival of resistant cells. The identified susceptibility and resistance mechanisms to IMTs may be relevant for different types of mitochondria-targeted therapies.},
}
@article {pmid34779353,
year = {2021},
author = {Wikandari, R and Manikharda, and Baldermann, S and Ningrum, A and Taherzadeh, MJ},
title = {Application of cell culture technology and genetic engineering for production of future foods and crop improvement to strengthen food security.},
journal = {Bioengineered},
volume = {12},
number = {2},
pages = {11305-11330},
pmid = {34779353},
issn = {2165-5987},
mesh = {Animals ; Biotechnology ; Cell Culture Techniques/*methods ; Crops, Agricultural/*growth &amp; development ; *Food Security ; *Genetic Engineering ; Plants, Genetically Modified ; },
abstract = {The growing population and the climate changes put a pressure on food production globally, therefore a fundamental transformation of food production is required. One approach to accelerate food production is application of modern biotechnology such as cell culture, marker assisted selection, and genetic engineering. Cell culture technology reduces the usage of arable land, while marker-assisted selection increases the genetic gain of crop breeding and genetic engineering enable to introduce a desired traits to crop. The cell culture technology has resulted in development of cultured meat, fungal biomass food (mycoprotein), and bioactive compounds from plant cell culture. Except cultured meat which recently begin to penetrate the market, the other products have been in the market for years. The marker-assisted selection and genetic engineering have contributed significantly to increase the resiliency against emerging pests and abiotic stresses. This review addresses diverse techniques of cell culture technology as well as advanced genetic engineering technology CRISPR Cas-9 and its application for crop improvement. The pros and cons of different techniques as well as the challenges and future perspective of application of modern biotechnology for strengthening food security are also discussed.},
}
@article {pmid34779326,
year = {2022},
author = {Jia, YL and Geng, SS and Du, F and Xu, YS and Wang, LR and Sun, XM and Wang, QZ and Li, Q},
title = {Progress of metabolic engineering for the production of eicosapentaenoic acid.},
journal = {Critical reviews in biotechnology},
volume = {42},
number = {6},
pages = {838-855},
doi = {10.1080/07388551.2021.1971621},
pmid = {34779326},
issn = {1549-7801},
mesh = {Biosynthetic Pathways ; Eicosapentaenoic Acid/metabolism ; *Fatty Acids, Omega-3/metabolism ; Metabolic Engineering ; *Microalgae/metabolism ; },
abstract = {Eicosapentaenoic Acid (EPA) is an essential ω-3 polyunsaturated fatty acid for human health. Currently, high-quality EPA production is largely dependent on the extraction of fish oil, but this unsustainable approach cannot meet its rising market demand. Biotechnological approaches for EPA production from microorganisms have received increasing attention due to their suitability for large-scale production and independence of the seasonal or climate restrictions. This review summarizes recent research on different microorganisms capable of producing EPA, such as microalgae, bacteria, and fungi, and introduces the different EPA biosynthesis pathways. Notably, some novel engineering strategies have been applied to endow and improve the abilities of microorganisms to synthesize EPA, including the construction and optimization of the EPA biosynthesis pathway, an increase in the acetyl-CoA pool supply, the increase of NADPH and the inhibition of competing pathways. This review aims to provide an updated summary of EPA production.},
}
@article {pmid34778871,
year = {2021},
author = {Botto, C and Dalkara, D and El-Amraoui, A},
title = {Progress in Gene Editing Tools and Their Potential for Correcting Mutations Underlying Hearing and Vision Loss.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {737632},
pmid = {34778871},
issn = {2673-3439},
abstract = {Blindness and deafness are the most frequent sensory disorders in humans. Whatever their cause - genetic, environmental, or due to toxic agents, or aging - the deterioration of these senses is often linked to irreversible damage to the light-sensing photoreceptor cells (blindness) and/or the mechanosensitive hair cells (deafness). Efforts are increasingly focused on preventing disease progression by correcting or replacing the blindness and deafness-causal pathogenic alleles. In recent years, gene replacement therapies for rare monogenic disorders of the retina have given positive results, leading to the marketing of the first gene therapy product for a form of childhood hereditary blindness. Promising results, with a partial restoration of auditory function, have also been reported in preclinical models of human deafness. Silencing approaches, including antisense oligonucleotides, adeno-associated virus (AAV)-mediated microRNA delivery, and genome-editing approaches have also been applied to various genetic forms of blindness and deafness The discovery of new DNA- and RNA-based CRISPR/Cas nucleases, and the new generations of base, prime, and RNA editors offers new possibilities for directly repairing point mutations and therapeutically restoring gene function. Thanks to easy access and immune-privilege status of self-contained compartments, the eye and the ear continue to be at the forefront of developing therapies for genetic diseases. Here, we review the ongoing applications and achievements of this new class of emerging therapeutics in the sensory organs of vision and hearing, highlighting the challenges ahead and the solutions to be overcome for their successful therapeutic application in vivo.},
}
@article {pmid34777759,
year = {2021},
author = {Hu, J and Liu, R and Zhou, J and Lv, Y},
title = {Element coding based accurate evaluation of CRISPR/Cas9 initial cleavage.},
journal = {Chemical science},
volume = {12},
number = {40},
pages = {13404-13412},
pmid = {34777759},
issn = {2041-6520},
abstract = {As a powerful gene editing tool, the kinetic mechanism of CRISPR/Cas9 has been the focus for its further application. Initial cleavage events as the first domino followed by nuclease end trimming significantly affect the final on-target rate. Here we propose EC-CRISPR, element coding CRISPR, an accurate evaluation platform for initial cleavage that directly characterizes the cleavage efficiency and breaking sites. We benchmarked the influence of 19 single mismatch and 3 multiple mismatch positions of DNA-sgRNA on initial cleavage, as well as various reaction conditions. Results from EC-CRISPR demonstrate that the PAM-distal single mismatch is relatively acceptable compared to the proximal one. And multiple mismatches will not only affect the cleavage efficiency, but also generate more non-site #3 cleavage. Through in-depth research of kinetic behavior, we uncovered an abnormally higher non-#3 proportion at the initial stage of cleavage by using EC-CRISPR. Together, our results provided insights into cleavage efficiency and breaking sites, demonstrating that EC-CRISPR as a novel quantitative platform for initial cleavage enables accurate comparison of efficiencies and specificities among multiple CRISPR/Cas enzymes.},
}
@article {pmid34777318,
year = {2021},
author = {Cheng, YH and Chou, SH and Huang, PH and Yang, TC and Juan, YF and Kreiswirth, BN and Lin, YT and Chen, L},
title = {Characterization of a mcr-1 and CRISPR-Cas System Co-harboring Plasmid in a Carbapenemase-Producing High-Risk ST11 Klebsiella pneumoniae Strain.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {762947},
pmid = {34777318},
issn = {1664-302X},
abstract = {We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring bla KPC- 2 and bla OXA- 48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.},
}
@article {pmid34776215,
year = {2021},
author = {Benamozig, O and Baudrier, L and Billon, P},
title = {A detection method for the capture of genomic signatures: From disease diagnosis to genome editing.},
journal = {Methods in enzymology},
volume = {661},
number = {},
pages = {251-282},
doi = {10.1016/bs.mie.2021.08.012},
pmid = {34776215},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome ; Genomics ; Mutation ; },
abstract = {Variations in the genetic information originate from errors during DNA replication, error-prone repair of DNA damages, or genome editing. The most common approach to detect changes in DNA sequences employs sequencing technologies. However, they remain expensive and time-consuming, limiting their utility for routine laboratory experiments. We recently developed DinucleoTidE Signature CapTure (DTECT). DTECT is a marker-free and versatile detection method that captures targeted dinucleotide signatures resulting from the digestion of genomic amplicons by the type IIS restriction enzyme AcuI. Here, we describe the DTECT protocol to identify mutations introduced by CRISPR-based precision genome editing technologies or resulting from genetic variation. DTECT enables accurate detection of mutations using basic laboratory equipment and off-the-shelf reagents with qualitative or quantitative capture of signatures.},
}
@article {pmid34776214,
year = {2021},
author = {Zou, RS and Ha, T},
title = {Light activation and deactivation of Cas9 for DNA repair studies.},
journal = {Methods in enzymology},
volume = {661},
number = {},
pages = {219-249},
doi = {10.1016/bs.mie.2021.08.007},
pmid = {34776214},
issn = {1557-7988},
support = {R35 GM122569/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U01 DK127432/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA Damage ; *DNA Repair ; Gene Editing/methods ; },
abstract = {DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response: DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.},
}
@article {pmid34775359,
year = {2022},
author = {Hussain, Y and Khan, H and Ahmad, I and Efferth, T and Alam, W},
title = {Nanoscale delivery of phytochemicals targeting CRISPR/Cas9 for cancer therapy.},
journal = {Phytomedicine : international journal of phytotherapy and phytopharmacology},
volume = {94},
number = {},
pages = {153830},
doi = {10.1016/j.phymed.2021.153830},
pmid = {34775359},
issn = {1618-095X},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Neoplasms/drug therapy/genetics ; Phytochemicals ; },
abstract = {BACKGROUND: With growing global prevalence, cancer is a major cause of disease-related deaths. The understanding of the fundamental tumor pathology has contributed to the development of agents targeting oncogenic signaling pathways. Although these agents have increased survival for defined cancers, the therapeutic choices are still limited due to the development of drug resistance. CRISPR/Cas9 is a powerful new technology in cancer therapy by facilitating the identification of novel treatment targets and development of cell-based treatment strategies.<br><br>PURPOSE: We focused on applications of the CRISPR/Cas9 system in cancer therapy and discuss nanoscale delivery of cytotoxic phytochemical targeting the CRISPR/Cas9 system.<br><br>RESULTS: Genome engineering has been significantly accelerated by the advancement of the CRISPR/Cas9 technique. Phytochemicals play a key role in treating cancer by targeting various mechanisms and pathways.<br><br>CONCLUSIONS: The use of CRISPR/Cas9 for nanoscale delivery of phytochemicals opens new avenues in cancer therapy. One of the main obstacles in the clinical application of CRISPR/Cas9 is safe and efficient delivery. As viral delivery methods have certain drawbacks, there is an urgent need to develop non-viral delivery systems for therapeutic applications.},
}
@article {pmid34774990,
year = {2022},
author = {Shi, M and Wang, C and Ji, J and Cai, Q and Zhao, Q and Xi, W and Zhang, J},
title = {CRISPR/Cas9-mediated knockout of SGLT1 inhibits proliferation and alters metabolism of gastric cancer cells.},
journal = {Cellular signalling},
volume = {90},
number = {},
pages = {110192},
doi = {10.1016/j.cellsig.2021.110192},
pmid = {34774990},
issn = {1873-3913},
mesh = {CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Glucose/metabolism ; Humans ; *Stomach Neoplasms/genetics ; },
abstract = {BACKGROUND: The roles played by sodium/glucose cotransporters 1 (SGLT1) that transport glucose in cells independent of extracellular glucose concentration in gastric cancer are unknown.<br><br>METHODS: The expression of SGLT1 in 75 primary gastric cancer and paired adjacent normal tissue specimens was determined. Also, the underlying mechanism of the altered SGLT1 expression and its impact on the proliferation of the gastric cancer cells and their metabolism were investigated.<br><br>RESULTS: SGLT1 expression was found to be positively associated with pT, pN, TNM staging, histological differentiation, and a worse overall survival. CRISPR/Cas9 mediated knockout of SGLT1 could inhibit proliferation of gastric cancer cells, promote their apoptosis, and could also alter the metabolism of gastric cancer cells. Mechanistically, the transcription activity of SGLT1 could be negatively regulated by p53.<br><br>CONCLUSIONS: Besides identifying the important role of SGLT1 in gastric cancer, the underlying regulation mechanism in play was also elucidated. These make SGLT1 a promising new molecular target for the design of novel therapeutic modalities to control gastric cancer.},
}
@article {pmid34774862,
year = {2021},
author = {Sharma, A and Balda, S and Apreja, M and Kataria, K and Capalash, N and Sharma, P},
title = {COVID-19 Diagnosis: Current and Future Techniques.},
journal = {International journal of biological macromolecules},
volume = {193},
number = {Pt B},
pages = {1835-1844},
pmid = {34774862},
issn = {1879-0003},
mesh = {*COVID-19/epidemiology/genetics ; *COVID-19 Nucleic Acid Testing ; Humans ; *Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques ; *Pandemics ; *Point-of-Care Systems ; RNA, Viral/*genetics ; SARS-CoV-2/*genetics ; },
abstract = {COVID-19 pandemic continues to be a global threat, affecting more than 200 countries/territories at both human and economic level. This necessitates the rapid development of highly reliable diagnostic methods in order to effectively and accurately diagnose the pathology to prevent the spread of COVID-19. Currently, RT-PCR is the most widely used method worldwide for SARS-CoV-2 detection. Serological assays are being used for sero-surveys of SARS-CoV-2 antibody prevalence in the community. Radiology imaging has been useful in the clinical diagnosis of COVID-19. These methods have their own limitations and there are continued efforts to develop easier, economic, highly sensitive and specific, point-of-care methods. Reverse transcription-loop mediated isothermal amplification (RT-LAMP), nucleic acid sequence-based amplification (NASBA), CRISPR-Cas-based detection, and digital PCR are such techniques being employed in research laboratories, with many awaiting diagnostic approval from competent authorities. This review highlights the rapidly expanding array of existing and in-development diagnostic tests/strategies that may be used to diagnose SARS-CoV-2 infection in both clinical and research settings.},
}
@article {pmid34774798,
year = {2021},
author = {Romanelli, SM and Lewis, KT and Nishii, A and Rupp, AC and Li, Z and Mori, H and Schill, RL and Learman, BS and Rhodes, CJ and MacDougald, OA},
title = {BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {6},
pages = {101402},
pmid = {34774798},
issn = {1083-351X},
support = {T32 DK071212/DK/NIDDK NIH HHS/United States ; R01 DK125513/DK/NIDDK NIH HHS/United States ; F32 DK123887/DK/NIDDK NIH HHS/United States ; F32 DK122654/DK/NIDDK NIH HHS/United States ; T32 DK101357/DK/NIDDK NIH HHS/United States ; P30 DK089503/DK/NIDDK NIH HHS/United States ; P30 DK034933/DK/NIDDK NIH HHS/United States ; F31 DK122723/DK/NIDDK NIH HHS/United States ; P30 DK020572/DK/NIDDK NIH HHS/United States ; R01 DK121759/DK/NIDDK NIH HHS/United States ; U2C DK110768/DK/NIDDK NIH HHS/United States ; },
mesh = {Adipose Tissue, Brown/*metabolism ; Animals ; *CRISPR-Cas Systems ; Fibroblast Growth Factors/genetics/metabolism ; Gene Knockout Techniques ; Mice ; Mice, Knockout ; Uncoupling Protein 1/genetics/metabolism ; },
abstract = {CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.},
}
@article {pmid34774454,
year = {2021},
author = {Herskovitz, J and Hasan, M and Patel, M and Blomberg, WR and Cohen, JD and Machhi, J and Shahjin, F and Mosley, RL and McMillan, J and Kevadiya, BD and Gendelman, HE},
title = {CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination.},
journal = {EBioMedicine},
volume = {73},
number = {},
pages = {103678},
pmid = {34774454},
issn = {2352-3964},
support = {R01 NS036126/NS/NINDS NIH HHS/United States ; P20 GM103427/GM/NIGMS NIH HHS/United States ; P30 MH062261/MH/NIMH NIH HHS/United States ; R01 AI158160/AI/NIAID NIH HHS/United States ; R01 MH121402/MH/NIMH NIH HHS/United States ; P30 CA036727/CA/NCI NIH HHS/United States ; P01 MH064570/MH/NIMH NIH HHS/United States ; R01 NS126089/NS/NINDS NIH HHS/United States ; R01 NS034239/NS/NINDS NIH HHS/United States ; P01 DA028555/DA/NIDA NIH HHS/United States ; R01 DA054535/DA/NIDA NIH HHS/United States ; T32 NS105594/NS/NINDS NIH HHS/United States ; P30 GM106397/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Conserved Sequence ; *Exons ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Targeting ; Genes, Reporter ; Genetic Therapy ; Genetic Vectors/administration &amp; dosage/genetics ; Genome, Viral ; HIV Infections/*therapy/*virology ; HIV-1/*genetics ; Humans ; Liposomes ; Macrophages/metabolism/virology ; Nanoparticles ; Proviruses/genetics ; RNA Interference ; RNA, Guide ; RNA, Messenger/administration &amp; dosage/genetics ; rev Gene Products, Human Immunodeficiency Virus/genetics ; tat Gene Products, Human Immunodeficiency Virus/genetics ; },
abstract = {BACKGROUND: A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery.<br><br>METHODS: A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels.<br><br>FINDINGS: Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing.<br><br>INTERPRETATION: These results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing.<br><br>FUNDING: The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.},
}
@article {pmid34773154,
year = {2021},
author = {Liao, X and Li, L and Jameel, A and Xing, XH and Zhang, C},
title = {A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris.},
journal = {Applied microbiology and biotechnology},
volume = {105},
number = {24},
pages = {9211-9218},
pmid = {34773154},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Metabolic Engineering ; *Pichia/genetics ; Saccharomycetales ; },
abstract = {Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III-driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology. KEY POINTS: • An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes. • Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes. • CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.},
}
@article {pmid34773119,
year = {2021},
author = {Malinin, NL and Lee, G and Lazzarotto, CR and Li, Y and Zheng, Z and Nguyen, NT and Liebers, M and Topkar, VV and Iafrate, AJ and Le, LP and Aryee, MJ and Joung, JK and Tsai, SQ},
title = {Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.},
journal = {Nature protocols},
volume = {16},
number = {12},
pages = {5592-5615},
pmid = {34773119},
issn = {1750-2799},
support = {DP1 GM105378/GM/NIGMS NIH HHS/United States ; 2020154/DDCF/Doris Duke Charitable Foundation/United States ; U01 AI157189/AI/NIAID NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; 2017093/DDCF/Doris Duke Charitable Foundation/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Primers/chemical synthesis/metabolism ; Deoxyribonucleases, Type II Site-Specific/chemistry ; Electroporation/methods ; Gene Editing/*methods ; *Genome, Human ; Humans ; Osteoblasts/cytology/metabolism ; Plasmids/chemistry/metabolism ; Polymerase Chain Reaction/*methods ; Primary Cell Culture ; RNA, Guide/*genetics/metabolism ; T-Lymphocytes/cytology/metabolism ; },
abstract = {Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.},
}
@article {pmid34773059,
year = {2021},
author = {Eggenschwiler, R and Gschwendtberger, T and Felski, C and Jahn, C and Langer, F and Sterneckert, J and Hermann, A and Lühmann, J and Steinemann, D and Haase, A and Martin, U and Petri, S and Cantz, T},
title = {A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {22154},
pmid = {34773059},
issn = {2045-2322},
mesh = {Amyotrophic Lateral Sclerosis/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mutation ; Superoxide Dismutase-1/genetics ; Transfection/*methods ; },
abstract = {CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.},
}
@article {pmid34773056,
year = {2021},
author = {Palma, MB and Tronik-Le Roux, D and Amín, G and Castañeda, S and Möbbs, AM and Scarafia, MA and La Greca, A and Daouya, M and Poras, I and Inda, AM and Moro, LN and Carosella, ED and García, MN and Miriuka, SG},
title = {HLA-G gene editing in tumor cell lines as a novel alternative in cancer immunotherapy.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {22158},
pmid = {34773056},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; HLA-G Antigens/*genetics/immunology/*metabolism ; Humans ; Immunotherapy/methods ; RNA, Guide ; Transfection ; },
abstract = {Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.},
}
@article {pmid34769484,
year = {2021},
author = {Klementieva, N and Goliusova, D and Krupinova, J and Yanvarev, V and Panova, A and Mokrysheva, N and Kiselev, SL},
title = {A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34769484},
issn = {1422-0067},
mesh = {Adult ; CRISPR-Cas Systems ; Cells, Cultured ; Female ; Fibroblasts/*metabolism ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Multiple Endocrine Neoplasia Type 1/genetics/metabolism/*pathology ; *Mutation ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors/genetics ; },
abstract = {Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype-genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.},
}
@article {pmid34769267,
year = {2021},
author = {Lim, SH and Baek, JI and Jeon, BM and Seo, JW and Kim, MS and Byun, JY and Park, SH and Kim, SJ and Lee, JY and Lee, JH and Kim, SC},
title = {CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34769267},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering ; *Metabolic Networks and Pathways ; *Saccharomyces cerevisiae/genetics/metabolism ; Sapogenins/*metabolism ; },
abstract = {Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4-6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.},
}
@article {pmid34769204,
year = {2021},
author = {Ahmad, A and Munawar, N and Khan, Z and Qusmani, AT and Khan, SH and Jamil, A and Ashraf, S and Ghouri, MZ and Aslam, S and Mubarik, MS and Munir, A and Sultan, Q and Abd-Elsalam, KA and Qari, SH},
title = {An Outlook on Global Regulatory Landscape for Genome-Edited Crops.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34769204},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; *Genome, Plant ; *Plant Breeding ; Plants, Genetically Modified/*genetics ; Transgenes ; },
abstract = {The revolutionary technology of CRISPR/Cas systems and their extraordinary potential to address fundamental questions in every field of biological sciences has led to their developers being awarded the 2020 Nobel Prize for Chemistry. In agriculture, CRISPR/Cas systems have accelerated the development of new crop varieties with improved traits-without the need for transgenes. However, the future of this technology depends on a clear and truly global regulatory framework being developed for these crops. Some CRISPR-edited crops are already on the market, and yet countries and regions are still divided over their legal status. CRISPR editing does not require transgenes, making CRISPR crops more socially acceptable than genetically modified crops, but there is vigorous debate over how to regulate these crops and what precautionary measures are required before they appear on the market. This article reviews intended outcomes and risks arising from the site-directed nuclease CRISPR systems used to improve agricultural crop plant genomes. It examines how various CRISPR system components, and potential concerns associated with CRISPR/Cas, may trigger regulatory oversight of CRISPR-edited crops. The article highlights differences and similarities between GMOs and CRISPR-edited crops, and discusses social and ethical concerns. It outlines the regulatory framework for GMO crops, which many countries also apply to CRISPR-edited crops, and the global regulatory landscape for CRISPR-edited crops. The article concludes with future prospects for CRISPR-edited crops and their products.},
}
@article {pmid34769169,
year = {2021},
author = {Teodoro, JS and Machado, IF and Castela, AC and Amorim, JA and Jarak, I and Carvalho, RA and Palmeira, CM and Rolo, AP},
title = {Chenodeoxycholic Acid Has Non-Thermogenic, Mitodynamic Anti-Obesity Effects in an In Vitro CRISPR/Cas9 Model of Bile Acid Receptor TGR5 Knockdown.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34769169},
issn = {1422-0067},
mesh = {3T3-L1 Cells ; Animals ; Anti-Obesity Agents/*pharmacology ; *CRISPR-Cas Systems ; Chenodeoxycholic Acid/*pharmacology ; Gene Knockdown Techniques ; Mice ; Mitochondria/genetics/*metabolism ; Obesity/*drug therapy/genetics/metabolism ; Receptors, G-Protein-Coupled/*deficiency/metabolism ; Thermogenesis/drug effects/genetics ; },
abstract = {Bile acids (BA) have shown promising effects in animal models of obesity. However, the said effects are thought to rely on a thermogenic effect, which is questionably present in humans. A previous work has shown that the BA chenodeoxycholic acid (CDCA) can revert obesity and accelerate metabolism in animal and cell culture models. Thus, the aim of this study was to understand if this obesity reduction is indeed thermogenically-dependent. A CRISPR/Cas9 model of TGR5 (BA receptor) knockdown in 3T3-L1 adipocytes was developed to diminish thermogenic effects. Various parameters were assessed, including mitochondrial bioenergetics by Seahorse flux analysis, oxidative stress and membrane potential by fluorometry, intermediary metabolism by NMR, protein content assessment by Western Blot, gene expression by qPCR, and confocal microscopy evaluation of mitophagy. CDCA was still capable, for the most part, of reversing the harmful effects of cellular obesity, elevating mitophagy and leading to the reduction of harmed mitochondria within the cells, boosting mitochondrial activity, and thus energy consumption. In summary, CDCA has a non-thermogenic, obesity reducing capacity that hinges on a healthy mitochondrial population, explaining at least some of these effects and opening avenues of human treatment for metabolic diseases.},
}
@article {pmid34769160,
year = {2021},
author = {Chou, CH and Chiang, CF and Yang, CC and Liu, YC and Chang, SR and Chang, KW and Lin, SC},
title = {miR-31-NUMB Cascade Modulates Monocarboxylate Transporters to Increase Oncogenicity and Lactate Production of Oral Carcinoma Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34769160},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Head and Neck Neoplasms/*genetics/metabolism ; Humans ; Lactic Acid/metabolism ; Membrane Proteins/*genetics/metabolism ; MicroRNAs/*genetics/metabolism ; Monocarboxylic Acid Transporters/*genetics/metabolism ; Muscle Proteins/*genetics/metabolism ; Nerve Tissue Proteins/*genetics/metabolism ; Squamous Cell Carcinoma of Head and Neck/*genetics/metabolism ; Symporters/*genetics/metabolism ; },
abstract = {Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated death worldwide. miR-31 is an oncogenic miRNA in OSCC. NUMB is an adaptor protein capable of suppressing malignant transformation. Disruption of the miR-31-NUMB regulatory axis has been demonstrated in malignancies. Mitochondrial dysfunction and adaptation to glycolytic respiration are frequent events in malignancies. Monocarboxylate transporters (MCTs) function to facilitate lactate flux in highly glycolytic cells. Upregulation of MCT1 and MCT4 has been shown to be a prognostic factor of OSCC. Here, we reported that miR-31-NUMB can modulate glycolysis in OSCC. Using the CRISPR/Cas9 gene editing strategy, we identified increases in oncogenic phenotypes, MCT1 and MCT4 expression, lactate production, and glycolytic respiration in NUMB-deleted OSCC subclones. Transfection of the Numb1 or Numb4 isoform reversed the oncogenic induction elicited by NUMB deletion. This study also showed, for the first time, that NUMB4 binds MCT1 and MCT4 and that this binding increases their ubiquitination, which may decrease their abundance in cell lysates. The disruptions in oncogenicity and metabolism associated with miR-31 deletion and NUMB deletion were partially rescued by MCT1/MCT4 expression or knockdown. This study demonstrated that NUMB is a novel binding partner of MCT1 and MCT4 and that the miR-31-NUMB-MCT1/MCT4 regulatory cascade is present in oral carcinoma.},
}
@article {pmid34768853,
year = {2021},
author = {Liu, C and Zhang, Y and Tan, Y and Zhao, T and Xu, X and Yang, H and Li, J},
title = {CRISPR/Cas9-Mediated SlMYBS2 Mutagenesis Reduces Tomato Resistance to Phytophthora infestans.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34768853},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Disease Resistance/*genetics ; Gene Expression Regulation, Plant/genetics ; Lycopersicon esculentum/genetics/*parasitology ; Phytophthora infestans/*pathogenicity ; Plant Diseases/*parasitology ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; Reactive Oxygen Species/metabolism ; Transcription Factors/*genetics ; },
abstract = {Phytophthora infestans (P. infestans) recently caused epidemics of tomato late blight. Our study aimed to identify the function of the SlMYBS2 gene in response to tomato late blight. To further investigate the function of SlMYBS2 in tomato resistance to P. infestans, we studied the effects of SlMYBS2 gene knock out. The SlMYBS2 gene was knocked out by CRISPR-Cas9, and the resulting plants (SlMYBS2 gene knockout, slmybs2-c) showed reduced resistance to P. infestans, accompanied by increases in the number of necrotic cells, lesion sizes, and disease index. Furthermore, after P. infestans infection, the expression levels of pathogenesis-related (PR) genes in slmybs2-c plants were significantly lower than those in wild-type (AC) plants, while the number of necrotic cells and the accumulation of reactive oxygen species (ROS) were higher than those in wild-type plants. Taken together, these results indicate that SlMYBS2 acts as a positive regulator of tomato resistance to P. infestans infection by regulating the ROS level and the expression level of PR genes.},
}
@article {pmid34768819,
year = {2021},
author = {Kim, ST and Choi, M and Bae, SJ and Kim, JS},
title = {The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34768819},
issn = {1422-0067},
mesh = {Arabidopsis/*genetics/physiology ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; *Multigene Family ; Plants, Genetically Modified ; Salt Tolerance/*genetics ; },
abstract = {Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.},
}
@article {pmid34768751,
year = {2021},
author = {Dehshahri, A and Biagioni, A and Bayat, H and Lee, EHC and Hashemabadi, M and Fekri, HS and Zarrabi, A and Mohammadinejad, R and Kumar, AP},
title = {Editing SOX Genes by CRISPR-Cas: Current Insights and Future Perspectives.},
journal = {International journal of molecular sciences},
volume = {22},
number = {21},
pages = {},
pmid = {34768751},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods/trends ; Genetic Engineering/methods ; Genome ; Humans ; Neoplasms/genetics/therapy ; SOX Transcription Factors/*genetics/*metabolism ; Stem Cells/metabolism ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins (Cas) is an adaptive immune system in archaea and most bacteria. By repurposing these systems for use in eukaryote cells, a substantial revolution has arisen in the genome engineering field. In recent years, CRISPR-Cas technology was rapidly developed and different types of DNA or RNA sequence editors, gene activator or repressor, and epigenome modulators established. The versatility and feasibility of CRISPR-Cas technology has introduced this system as the most suitable tool for discovering and studying the mechanism of specific genes and also for generating appropriate cell and animal models. SOX genes play crucial roles in development processes and stemness. To elucidate the exact roles of SOX factors and their partners in tissue hemostasis and cell regeneration, generating appropriate in vitro and in vivo models is crucial. In line with these premises, CRISPR-Cas technology is a promising tool for studying different family members of SOX transcription factors. In this review, we aim to highlight the importance of CRISPR-Cas and summarize the applications of this novel, promising technology in studying and decoding the function of different members of the SOX gene family.},
}
@article {pmid34767923,
year = {2022},
author = {Okafor, IC and Choi, J and Ha, T},
title = {Single molecule methods for studying CRISPR Cas9-induced DNA unwinding.},
journal = {Methods (San Diego, Calif.)},
volume = {204},
number = {},
pages = {319-326},
doi = {10.1016/j.ymeth.2021.11.003},
pmid = {34767923},
issn = {1095-9130},
support = {T32 GM080189/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; DNA/chemistry ; *DNA Cleavage ; RNA, Guide/genetics/metabolism ; },
abstract = {Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9's HNH domain before and after cleavage.},
}
@article {pmid34767917,
year = {2022},
author = {Yeh, TK and Jean, SS and Lee, YL and Lu, MC and Ko, WC and Lin, HJ and Liu, PY and Hsueh, PR},
title = {Bacteriophages and phage-delivered CRISPR-Cas system as antibacterial therapy.},
journal = {International journal of antimicrobial agents},
volume = {59},
number = {1},
pages = {106475},
doi = {10.1016/j.ijantimicag.2021.106475},
pmid = {34767917},
issn = {1872-7913},
mesh = {Anti-Bacterial Agents/*therapeutic use ; Bacterial Infections/*drug therapy ; *Bacteriophages ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial ; Humans ; Phage Therapy/*methods ; },
abstract = {Multidrug-resistant (MDR) bacterial infections in humans are increasing worldwide. The global spread of antimicrobial resistance poses a considerable threat to human health. Phage therapy is a promising approach to combat MDR bacteria. An increasing number of reports have been published on phage therapy and the successful application of antibacterials derived using this method. Additionally, the CRISPR-Cas system has been used to develop antimicrobials with bactericidal effects in vivo. The CRISPR-Cas system can be delivered into target bacteria in various ways, with phage-based vectors being reported as an effective method. In this review, we briefly summarise the results of randomised control trials on bacteriophage therapy. Moreover, we integrated mechanisms of the CRISPR-Cas system antimicrobials in a schematic diagram and consolidated the research on phage-delivered CRISPR-Cas system antimicrobials.},
}
@article {pmid34767864,
year = {2022},
author = {Yan, J and Kang, DD and Turnbull, G and Dong, Y},
title = {Delivery of CRISPR-Cas9 system for screening and editing RNA binding proteins in cancer.},
journal = {Advanced drug delivery reviews},
volume = {180},
number = {},
pages = {114042},
pmid = {34767864},
issn = {1872-8294},
support = {R01 HL136652/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Early Detection of Cancer ; Genetic Therapy ; Humans ; Neoplasms/*genetics ; RNA-Binding Proteins/*genetics ; },
abstract = {RNA-binding proteins (RBPs) play an important role in RNA metabolism, regulating the stability, localization, and functional dynamics of RNAs. Alternation in the RBP-RNA network has profound implications in cellular physiology, and is related to the development and spread of cancer in certain cases. To regulate the expression of specific genes and their biological activities, various strategies have been applied to target RBPs for cancer treatments, including small-molecule inhibitors, small-interfering RNA, peptides, and aptamers. Recently, the deployment of the CRISPR-Cas9 technology has provided a new platform for RBP screening and regulation. This review summarizes the delivery systems of the CRISPR-Cas9 system and their role in RBP-based cancer therapeutics, including identification of novel RBPs and regulation of cancer-associated RBPs. The efficient delivery of the CRISPR-Cas9 system is important to the profound understanding and clinical transition of RBPs as cancer therapeutic targets.},
}
@article {pmid34767749,
year = {2021},
author = {Ho, BX and Yu, H and Pang, JKS and Hor, JH and Liew, LC and Szyniarowski, P and Lim, CYY and An, O and Yang, HH and Stewart, CL and Chan, WK and Ng, SY and Soh, BS},
title = {Upregulation of the JAK-STAT pathway promotes maturation of human embryonic stem cell-derived cardiomyocytes.},
journal = {Stem cell reports},
volume = {16},
number = {12},
pages = {2928-2941},
pmid = {34767749},
issn = {2213-6711},
mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation/drug effects ; Cell Line ; Electrophysiological Phenomena/drug effects ; Genes, Reporter ; Green Fluorescent Proteins/metabolism ; Human Embryonic Stem Cells/*cytology/drug effects/*metabolism ; Humans ; Interferon-gamma/metabolism/pharmacology ; Janus Kinases/*metabolism ; Myocytes, Cardiac/*cytology ; STAT Transcription Factors/*metabolism ; *Signal Transduction/drug effects ; *Up-Regulation/drug effects ; },
abstract = {The immature characteristics and metabolic phenotypes of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) restrict their applications for disease modeling, drug discovery, and cell-based therapy. Leveraging on the metabolic shifts from glycolysis to fatty acid oxidation as CMs mature, a human hexokinase1-GFP metabolic reporter cell line (H7 HK1-GFP) was generated to facilitate the isolation of fetal or more matured hPSC-CMs. RNA sequencing of fetal versus more matured CMs uncovered a potential role of interferon-signaling pathway in regulating CM maturation. Indeed, IFN-γ-treated CMs resulted in an upregulation of the JAK-STAT pathway, which was found to be associated with increased expression of CM maturation genes, shift from MYH6 to MYH7 expression, and improved sarcomeric structure. Functionally, IFN-γ-treated CMs exhibited a more matured electrophysiological profile, such as increased calcium dynamics and action potential upstroke velocity, demonstrated through calcium imaging and MEA. Expectedly, the functional improvements were nullified with a JAK-STAT inhibitor, ruxolitinib.},
}
@article {pmid34767550,
year = {2021},
author = {Behrmann, MS and Perera, HM and Hoang, JM and Venkat, TA and Visser, BJ and Bates, D and Trakselis, MA},
title = {Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness.},
journal = {PLoS genetics},
volume = {17},
number = {11},
pages = {e1009886},
pmid = {34767550},
issn = {1553-7404},
support = {R01 GM135368/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Chromosomes, Bacterial ; DNA, Bacterial/chemistry/genetics ; DnaB Helicases/chemistry/genetics/*metabolism ; Escherichia coli/enzymology/*genetics ; *Genomic Instability ; Mutation ; },
abstract = {Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork.},
}
@article {pmid34767372,
year = {2021},
author = {Wan, T and Pan, Q and Liu, C and Guo, J and Li, B and Yan, X and Cheng, Y and Ping, Y},
title = {A Duplex CRISPR-Cas9 Ribonucleoprotein Nanomedicine for Colorectal Cancer Gene Therapy.},
journal = {Nano letters},
volume = {21},
number = {22},
pages = {9761-9771},
doi = {10.1021/acs.nanolett.1c03708},
pmid = {34767372},
issn = {1530-6992},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Colorectal Neoplasms/genetics/therapy ; Genetic Therapy ; Mice ; Nanomedicine ; Ribonucleoproteins/genetics/metabolism ; Wnt Signaling Pathway ; },
abstract = {Based on the high frequency of concurrent adenomatous polyposis coli (APC) and KRAS mutations and their strong cooperative interaction in human colorectal cancer (CRC) promotion, we herein develop a CRISPR-Cas9-based genome-editing nanomedicine to target both APC and KRAS mutations for the treatment of CRC. To this end, a hyaluronic acid (HA)-decorated phenylboronic dendrimer (HAPD) was designed for the targeted delivery of Cas9 ribonucleoprotein (RNP), by which both APC and KRAS genetic mutations harboring in CRC cells can be synergistically disrupted. Systemic administration of Cas9 RNP targeting APC and KRAS enabled by HAPD significantly inhibits tumor growth on xenografted and orthotopic CRC mouse models and also greatly prevents CRC-induced liver metastasis and lung metastasis. Thus, this duplex genome-editing system provides a promising gene therapy strategy for the treatment of human CRC and can be extended to other types of cancers with activated Wnt/β-catenin and RAS/extracellular signal-regulated kinase (ERK) pathways.},
}
@article {pmid34766904,
year = {2021},
author = {van Vliet, AHM and Charity, OJ and Reuter, M},
title = {A Campylobacter integrative and conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare?.},
journal = {Microbial genomics},
volume = {7},
number = {11},
pages = {},
pmid = {34766904},
issn = {2057-5858},
support = {/WT_/Wellcome Trust/United Kingdom ; BB/J004529/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M011216/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Campylobacter ; Conjugation, Genetic ; Plasmids/genetics ; },
abstract = {Microbial genomes are highly adaptable, with mobile genetic elements (MGEs) such as integrative conjugative elements (ICEs) mediating the dissemination of new genetic information throughout bacterial populations. This is countered by defence mechanisms such as CRISPR-Cas systems, which limit invading MGEs by sequence-specific targeting. Here we report the distribution of the pVir, pTet and PCC42 plasmids and a new 70–129 kb ICE (CampyICE1) in the foodborne bacterial pathogens Campylobacter jejuni and Campylobacter coli . CampyICE1 contains a degenerated Type II-C CRISPR system consisting of a sole Cas9 protein, which is distinct from the previously described Cas9 proteins from C. jejuni and C. coli . CampyICE1 is conserved in structure and gene order, containing blocks of genes predicted to be involved in recombination, regulation and conjugation. CampyICE1 was detected in 134/5829 (2.3 %) C . jejuni genomes and 92/1347 (6.8 %) C . coli genomes. Similar ICEs were detected in a number of non-jejuni/coli Campylobacter species, although these lacked a CRISPR-Cas system. CampyICE1 carries three separate short CRISPR spacer arrays containing a combination of 108 unique spacers and 16 spacer-variant families. A total of 69 spacers and 10 spacer-variant families (63.7 %) were predicted to target Campylobacter plasmids. The presence of a functional CampyICE1 Cas9 protein and matching anti-plasmid spacers was associated with the absence of the pVir, pTet and pCC42 plasmids (188/214 genomes, 87.9 %), suggesting that the CampyICE1-encoded CRISPR-Cas has contributed to the exclusion of competing plasmids. In conclusion, the characteristics of the CRISPR-Cas9 system on CampyICE1 suggests a history of plasmid warfare in Campylobacter .},
}
@article {pmid34766316,
year = {2022},
author = {Falato, L and Vunk, B and Langel, &#xdc;},
title = {CRISPR/Cas9 Plasmid Delivery Through the CPP: PepFect14.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2383},
number = {},
pages = {587-593},
pmid = {34766316},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell-Penetrating Peptides ; Gene Editing ; Plasmids/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Gene editing is increasing its popularity day by day especially as an essential tool for the research. It is based on two recombination mechanisms in mammalian cells: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). The first one can be used to silence a specific gene or a portion of it and the second one to insert new DNA, in presence of a donor template, in a targeted position in the genome. In order to exploit one of these two mechanisms, three major targeted nucleases have been developed: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein). The last one seems to be the most promising tool among the others for gene editing. By using the properties and versatility of the Cell Penetrating Peptide (CPP) PepFect14, we developed a protocol to deliver a plasmid encoding for CRISPR-Cas9 and Green Fluorescent Protein (GFP) in BHM cell line expressing luciferase (Bomirsky Hamster Melanoma pLuc). Aiming to knocking down the luciferase gene in the cell line and to expressing GFP. Having two fast and easy read-outs of the plasmid's activity at the same time. Furthermore, by labeling the CRISPR plasmid with Cy5 it is possible to have a visual confirmation of the cellular uptake of the pDNA/CPP complex, via fluorescent microscopy, as described.},
}
@article {pmid34764969,
year = {2021},
author = {Tussipkan, D and Manabayeva, SA},
title = {Employing CRISPR/Cas Technology for the Improvement of Potato and Other Tuber Crops.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {747476},
pmid = {34764969},
issn = {1664-462X},
abstract = {New breeding technologies have not only revolutionized biological science, but have also been employed to generate transgene-free products. Genome editing is a powerful technology that has been used to modify genomes of several important crops. This review describes the basic mechanisms, advantages and disadvantages of genome editing systems, such as ZFNs, TALENs, and CRISPR/Cas. Secondly, we summarize in detail all studies of the CRISPR/Cas system applied to potato and other tuber crops, such as sweet potato, cassava, yam, and carrot. Genes associated with self-incompatibility, abiotic-biotic resistance, nutrient-antinutrient content, and post-harvest factors targeted utilizing the CRISPR/Cas system are analyzed in this review. We hope that this review provides fundamental information that will be useful for future breeding of tuber crops to develop novel cultivars.},
}
@article {pmid34762653,
year = {2021},
author = {Xie, B and Shi, X and Li, Y and Xia, B and Zhou, J and Du, M and Xing, X and Bai, L and Liu, E and Alvarez, F and Jin, L and Deng, S and Mitchell, GA and Pan, D and Li, M and Wu, J},
title = {Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans.},
journal = {PLoS genetics},
volume = {17},
number = {11},
pages = {e1009891},
pmid = {34762653},
issn = {1553-7404},
mesh = {Animals ; Asialoglycoprotein Receptor/*genetics ; CRISPR-Cas Systems ; Cardiovascular Diseases/*prevention &amp; control ; Cholesterol/biosynthesis ; Disease Models, Animal ; Humans ; Risk Factors ; Swine ; },
abstract = {Genetic variants in the asialoglycoprotein receptor 1 (ASGR1) are associated with a reduced risk of cardiovascular disease (CVD) in humans. However, the underlying molecular mechanism remains elusive. Given the cardiovascular similarities between pigs and humans, we generated ASGR1-deficient pigs using the CRISPR/Cas9 system. These pigs show age-dependent low levels of non-HDL-C under standard diet. When received an atherogenic diet for 6 months, ASGR1-deficient pigs show lower levels of non-HDL-C and less atherosclerotic lesions than that of controls. Furthermore, by analysis of hepatic transcriptome and in vivo cholesterol metabolism, we show that ASGR1 deficiency reduces hepatic de novo cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and increases cholesterol clearance by upregulating the hepatic low-density lipoprotein receptor (LDLR), which together contribute to the low levels of non-HDL-C. Despite the cardioprotective effect, we unexpectedly observed mild to moderate hepatic injury in ASGR1-deficient pigs, which has not been documented in humans with ASGR1 variants. Thus, targeting ASGR1 might be an effective strategy to reduce hypercholesterolemia and atherosclerosis, whereas further clinical evidence is required to assess its hepatic impact.},
}
@article {pmid34761361,
year = {2022},
author = {Panula, P and Chen, YC and Baronio, D and Lewis, S and Sundvik, M},
title = {The Histamine System in Zebrafish Brain: Organization, Receptors, and Behavioral Roles.},
journal = {Current topics in behavioral neurosciences},
volume = {59},
number = {},
pages = {291-302},
pmid = {34761361},
issn = {1866-3370},
mesh = {Animals ; Brain/metabolism ; *Histamine/metabolism/pharmacology ; Histidine Decarboxylase/genetics/metabolism ; Orexins/metabolism ; Presenilin-1/metabolism ; Receptors, Histamine/genetics/metabolism ; *Zebrafish/metabolism ; },
abstract = {Three of the four histamine receptors have been identified in zebrafish. Whereas only one histamine receptor 1 gene (hrh1) is known, two copies of histamine receptor 2 (hrh2a and hrh2b) have been identified. Although initially only one gene encoding for histamine receptor 3 (hrh3) was recognized in zebrafish, the genome database contains information for two more hrh3-like genes, whereas no genes corresponding for histamine receptor 4 with expression mainly in the immune system have been identified. Hrh1 and hrh3 show prominent uneven expression in the zebrafish brain, with the strongest expression in the dorsal telencephalon. Quantitatively significant expression of hrh1, hrh2, and hrh3 can also be found in several peripheral organs. Whereas antagonists of hrh1, hrh2, and hrh3 all affect the locomotor activity of zebrafish larvae, interpretation of the data is hampered by a lack of information on receptor binding and signaling characteristics. Zebrafish mutants lacking any of the three histamine receptors have shown modest behavioral phenotypes, possibly due to genetic compensation. None of the receptor mutant fish have shown significant sleep phenotypes. Adult zebrafish lacking hrh3 display decreased locomotor activity. The zebrafish histamine system shows significant life-long plasticity: presenilin 1 mutant zebrafish develop an abnormally large number of histamine neurons and increased thigmotaxis and anxiety-related phenotype. Overexpression of histidine decarboxylase (hdc) in larval zebrafish is associated with an increased number of hypocretin neurons, whereas translation inhibition of hdc or exposure to α-fluoromethylhistidine leads to decreased numbers of hypocretin neurons. Current pharmacological evidence suggests that this may be mediated by hrh1. Further studies using acute, e.g., pharmacogenetic or optogenetic manipulation of selected components of brain circuits, are required to understand the full range of physiological functions of zebrafish histamine receptors.},
}
@article {pmid34759957,
year = {2021},
author = {Mushtaq, M and Dar, AA and Basu, U and Bhat, BA and Mir, RA and Vats, S and Dar, MS and Tyagi, A and Ali, S and Bansal, M and Rai, GK and Wani, SH},
title = {Integrating CRISPR-Cas and Next Generation Sequencing in Plant Virology.},
journal = {Frontiers in genetics},
volume = {12},
number = {},
pages = {735489},
pmid = {34759957},
issn = {1664-8021},
abstract = {Plant pathology has been revolutionized by the emergence and intervention of next-generation sequencing technologies (NGS) which provide a fast, cost-effective, and reliable diagnostic for any class of pathogens. NGS has made tremendous advancements in the area of research and diagnostics of plant infecting viromes and has bridged plant virology with other advanced research fields like genome editing technologies. NGS in a broader perspective holds the potential for plant health improvement by diagnosing and mitigating the new or unusual symptoms caused by novel/unidentified viruses. CRISPR-based genome editing technologies can enable rapid engineering of efficient viral/viroid resistance by directly targeting specific nucleotide sites of plant viruses and viroids. Critical genes such as eIf (iso) 4E or eIF4E have been targeted via the CRISPR platform to produce plants resistant to single-stranded RNA (ssRNA) viruses. CRISPR/Cas-based multi-target DNA or RNA tests can be used for rapid and accurate diagnostic assays for plant viruses and viroids. Integrating NGS with CRISPR-based genome editing technologies may lead to a paradigm shift in combating deadly disease-causing plant viruses/viroids at the genomic level. Furthermore, the newly discovered CRISPR/Cas13 system has unprecedented potential in plant viroid diagnostics and interference. In this review, we have highlighted the application and importance of sequencing technologies on covering the viral genomes for precise modulations. This review also provides a snapshot vision of emerging developments in NGS technologies for the characterization of plant viruses and their potential utilities, advantages, and limitations in plant viral diagnostics. Furthermore, some of the notable advances like novel virus-inducible CRISPR/Cas9 system that confers virus resistance with no off-target effects have been discussed.},
}
@article {pmid34759910,
year = {2021},
author = {Mortensen, K and Lam, TJ and Ye, Y},
title = {Comparison of CRISPR-Cas Immune Systems in Healthcare-Related Pathogens.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {758782},
pmid = {34759910},
issn = {1664-302X},
support = {R01 AI143254/AI/NIAID NIH HHS/United States ; },
abstract = {The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and Clostridium difficile have been identified as the leading global cause of multidrug-resistant bacterial infections in hospitals. CRISPR-Cas systems are bacterial immune systems, empowering the bacteria with defense against invasive mobile genetic elements that may carry the antimicrobial resistance (AMR) genes, among others. On the other hand, the CRISPR-Cas systems are themselves mobile. In this study, we annotated and compared the CRISPR-Cas systems in these pathogens, utilizing their publicly available large numbers of sequenced genomes (e.g., there are more than 12 thousands of S. aureus genomes). The presence of CRISPR-Cas systems showed a very broad spectrum in these pathogens: S. aureus has the least tendency of obtaining the CRISPR-Cas systems with only 0.55% of its isolates containing CRISPR-Cas systems, whereas isolates of C. difficile we analyzed have CRISPR-Cas systems each having multiple CRISPRs. Statistical tests show that CRISPR-Cas containing isolates tend to have more AMRs for four of the pathogens (A. baumannii, E. faecium, P. aeruginosa, and S. aureus). We made available all the annotated CRISPR-Cas systems in these pathogens with visualization at a website (https://omics.informatics.indiana.edu/CRISPRone/pathogen), which we believe will be an important resource for studying the pathogens and their arms-race with invaders mediated through the CRISPR-Cas systems, and for developing potential clinical applications of the CRISPR-Cas systems for battles against the antibiotic resistant pathogens.},
}
@article {pmid34759907,
year = {2021},
author = {Gaur, RK and Ali, A and Cheng, X and Mäkinen, K and Agindotan, B and Wang, X},
title = {Editorial: Plant Viruses, Volume II: Molecular Plant Virus Epidemiology and Its Management.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {756807},
doi = {10.3389/fmicb.2021.756807},
pmid = {34759907},
issn = {1664-302X},
}
@article {pmid34759315,
year = {2021},
author = {Querques, I and Schmitz, M and Oberli, S and Chanez, C and Jinek, M},
title = {Target site selection and remodelling by type V CRISPR-transposon systems.},
journal = {Nature},
volume = {599},
number = {7885},
pages = {497-502},
pmid = {34759315},
issn = {1476-4687},
support = {182567/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Adenosine Triphosphatases/metabolism/ultrastructure ; Bacterial Proteins/metabolism/ultrastructure ; Biopolymers ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *Cyanobacteria/enzymology/genetics ; DNA Transposable Elements/*genetics ; DNA, Bacterial/genetics/metabolism/ultrastructure ; Gene Editing/*methods ; Models, Molecular ; Mutagenesis, Insertional ; Polymerization ; RNA/genetics/metabolism ; Substrate Specificity ; Transposases/metabolism/ultrastructure ; Zinc Fingers ; },
abstract = {Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements[1]. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion[2-7]. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ[7], but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.},
}
@article {pmid34759314,
year = {2021},
author = {Gaidt, MM and Morrow, A and Fairgrieve, MR and Karr, JP and Yosef, N and Vance, RE},
title = {Self-guarding of MORC3 enables virulence factor-triggered immunity.},
journal = {Nature},
volume = {600},
number = {7887},
pages = {138-142},
pmid = {34759314},
issn = {1476-4687},
support = {P01 AI063302/AI/NIAID NIH HHS/United States ; R01 AI075039/AI/NIAID NIH HHS/United States ; R37 AI075039/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenosine Triphosphatases/deficiency/*immunology/metabolism ; CRISPR-Cas Systems ; Cell Line ; DNA-Binding Proteins/deficiency/*immunology/metabolism ; Gene Editing ; Herpesvirus 1, Human/immunology/pathogenicity ; Humans ; Immediate-Early Proteins/immunology ; Immunity, Innate ; Interferon Regulatory Factor-3/metabolism ; Interferon Regulatory Factor-7/metabolism ; Interferon Type I/antagonists &amp; inhibitors/genetics/immunology ; *Models, Immunological ; Monocytes/immunology ; Receptor, Interferon alpha-beta ; Repressor Proteins/deficiency/immunology/metabolism ; Response Elements/genetics ; Ubiquitin-Protein Ligases/immunology ; Virulence Factors/*immunology ; },
abstract = {Pathogens use virulence factors to inhibit the immune system[1]. The guard hypothesis[2,3] postulates that hosts monitor (or 'guard') critical innate immune pathways such that their disruption by virulence factors provokes a secondary immune response[1]. Here we describe a 'self-guarded' immune pathway in human monocytes, in which guarding and guarded functions are combined in one protein. We find that this pathway is triggered by ICP0, a key virulence factor of herpes simplex virus type 1, resulting in robust induction of anti-viral type I interferon (IFN). Notably, induction of IFN by ICP0 is independent of canonical immune pathways and the IRF3 and IRF7 transcription factors. A CRISPR screen identified the ICP0 target MORC3[4] as an essential negative regulator of IFN. Loss of MORC3 recapitulates the IRF3- and IRF7-independent IFN response induced by ICP0. Mechanistically, ICP0 degrades MORC3, which leads to de-repression of a MORC3-regulated DNA element (MRE) adjacent to the IFNB1 locus. The MRE is required in cis for IFNB1 induction by the MORC3 pathway, but is not required for canonical IFN-inducing pathways. As well as repressing the MRE to regulate IFNB1, MORC3 is also a direct restriction factor of HSV-1[5]. Our results thus suggest a model in which the primary anti-viral function of MORC3 is self-guarded by its secondary IFN-repressing function-thus, a virus that degrades MORC3 to avoid its primary anti-viral function will unleash the secondary anti-viral IFN response.},
}
@article {pmid34758284,
year = {2022},
author = {Meaden, S and Biswas, A and Arkhipova, K and Morales, SE and Dutilh, BE and Westra, ER and Fineran, PC},
title = {High viral abundance and low diversity are associated with increased CRISPR-Cas prevalence across microbial ecosystems.},
journal = {Current biology : CB},
volume = {32},
number = {1},
pages = {220-227.e5},
pmid = {34758284},
issn = {1879-0445},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; Ecosystem ; Metagenomics ; Prevalence ; },
abstract = {CRISPR-Cas are adaptive immune systems that protect their hosts against viruses and other parasitic mobile genetic elements.[1] Although widely distributed among prokaryotic taxa, CRISPR-Cas systems are not ubiquitous.[2-4] Like most defense-system genes, CRISPR-Cas are frequently lost and gained, suggesting advantages are specific to particular environmental conditions.[5] Selection from viruses is assumed to drive the acquisition and maintenance of these immune systems in nature, and both theory[6-8] and experiments have identified phage density and diversity as key fitness determinants.[9][,][10] However, these approaches lack the biological complexity inherent in nature. Here, we exploit metagenomic data from 324 samples across diverse ecosystems to analyze CRISPR abundance in natural environments. For each metagenome, we quantified viral abundance and diversity to test whether these contribute to CRISPR-Cas abundance across ecosystems. We find a strong positive association between CRISPR-Cas abundance and viral abundance. In addition, when controlling for differences in viral abundance, CRISPR-Cas systems are more abundant when viral diversity is low, suggesting that such adaptive immune systems may offer limited protection when required to target a diverse viral community. CRISPR-Cas abundance also differed among environments, with environmental classification explaining roughly a quarter of the variation in CRISPR-Cas relative abundance. The relationships between CRISPR-Cas abundance, viral abundance, and viral diversity are broadly consistent across environments, providing robust evidence from natural ecosystems that supports predictions of when CRISPR is beneficial. These results indicate that viral abundance and diversity are major ecological factors that drive the selection and maintenance of CRISPR-Cas in microbial ecosystems.},
}
@article {pmid34757726,
year = {2021},
author = {Babu, K and Kathiresan, V and Kumari, P and Newsom, S and Parameshwaran, HP and Chen, X and Liu, J and Qin, PZ and Rajan, R},
title = {Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.},
journal = {Biochemistry},
volume = {60},
number = {49},
pages = {3783-3800},
pmid = {34757726},
issn = {1520-4995},
support = {P20 GM103640/GM/NIGMS NIH HHS/United States ; R15 HL147265/HL/NHLBI NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Associated Protein 9/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; DNA/*chemistry/genetics/metabolism ; DNA Cleavage ; Gene Expression ; Kinetics ; Molecular Dynamics Simulation ; Mutation ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Domains ; Protein Interaction Domains and Motifs ; RNA/*chemistry/genetics/metabolism ; RNA, Guide/*chemistry/genetics/metabolism ; Streptococcus pyogenes/*chemistry/enzymology/genetics ; Substrate Specificity ; Thermodynamics ; },
abstract = {CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas9[2Pro]) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.},
}
@article {pmid34757047,
year = {2021},
author = {Jeon, S and Kim, MM},
title = {The down-regulation of melanogenesis via MITF and FOXO1 signaling pathways in SIRT1 knockout cells using CRISPR/Cas9 system.},
journal = {Journal of biotechnology},
volume = {342},
number = {},
pages = {114-127},
doi = {10.1016/j.jbiotec.2021.10.005},
pmid = {34757047},
issn = {1873-4863},
mesh = {CRISPR-Cas Systems ; Down-Regulation ; Kelch-Like ECH-Associated Protein 1 ; Melanins/metabolism ; *Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Monophenol Monooxygenase/metabolism ; NF-E2-Related Factor 2/metabolism ; *Sirtuin 1/genetics ; },
abstract = {Hair graying is processed by the inactivation of tyrosinase caused by the accumulation of oxidative stress and a decrease in the number of melanocytes. Therefore, the purpose of this study was to investigate the effect of SIRT1 gene knockout using the CRISPR/Cas9 system on the protein and gene expressions related to melanogenesis. In this study, the mutation in the SIRT1 knockout(KO) gene was verified by T7EI assay and Sanger DNA sequencing. Furthermore, the expression levels of SIRT1 protein and gene in KO cells were remarkably decreased compared with normal cells. Therefore, the SIRT1 gene KO cell line was successfully established for further study. The KO cells also increased SA-β-galactosidase and decreased melanin production and the scavenging activity of hydrogen peroxide. In particular, the down-regulation of p38 and c-kit as well as the up-regulation of ERK resulted in the inactivation of MITF in the KO cells. Thus, KO cells reduced the expressions of Tyrosinase, Tyrosine hydroxylase, TRP-1 and TRP-2 through the negative modulation of MITF. Furthermore, SIRT1 gene KO cells negatively modulated antioxidant proteins such as Catalase, MnSOD, MsrA and MsrB3 through FOXO1 and Keap1. Therefore, it is suggested that SIRT1 could play a positive role in melanogenesis via MITF and FOXO1.},
}
@article {pmid34756887,
year = {2021},
author = {Liu, Y and Zhang, L and Guo, M and Chen, L and Wu, B and Huang, H},
title = {Structural basis for anti-CRISPR repression mediated by bacterial operon proteins Aca1 and Aca2.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {6},
pages = {101357},
pmid = {34756887},
issn = {1083-351X},
mesh = {Bacteriophages/chemistry/genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Models, Molecular ; *Operon ; Pectobacterium carotovorum/genetics/metabolism/*virology ; Protein Conformation ; Protein Multimerization ; Pseudomonas Phages/chemistry/genetics/metabolism ; Pseudomonas aeruginosa/genetics/metabolism/*virology ; Viral Proteins/chemistry/genetics/*metabolism ; },
abstract = {It has been shown that phages have evolved anti-CRISPR (Acr) proteins to inhibit host CRISPR-Cas systems. Most acr genes are located upstream of anti-CRISPR-associated (aca) genes, which is instrumental for identifying these acr genes. Thus far, eight Aca families (Aca1-Aca8) have been identified, all proteins of which share low sequence homology and bind to different target DNA sequences. Recently, Aca1 and Aca2 proteins were discovered to function as repressors by binding to acr-aca promoters, thus implying a potential anti-anti-CRISPR mechanism. However, the structural basis for the repression roles of Aca proteins is still unknown. Here, we elucidated apo-structures of Aca1 and Aca2 proteins and their complex structures with their cognate operator DNA in two model systems, the Pseudomonas phage JBD30 and the Pectobacterium carotovorum template phage ZF40. In combination with biochemical and cellular assays, our study unveils dimerization and DNA-recognition mechanisms of Aca1 and Aca2 family proteins, thus revealing the molecular basis for Aca1-and Aca2-mediated anti-CRISPR repression. Our results also shed light on understanding the repression roles of other Aca family proteins and autoregulation roles of acr-aca operons.},
}
@article {pmid34753955,
year = {2021},
author = {Achary, VMM and Reddy, MK},
title = {CRISPR-Cas9 mediated mutation in GRAIN WIDTH and WEIGHT2 (GW2) locus improves aleurone layer and grain nutritional quality in rice.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {21941},
pmid = {34753955},
issn = {2045-2322},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Edible Grain/*genetics ; Gene Expression Regulation, Plant ; Gene Knockdown Techniques ; Genes, Plant ; *Mutation ; },
abstract = {Enhancing crop productivity and their nutritional quality are the key components and primary focus of crop improvement strategy for fulfilling future food demand and improving human health. Grain filling and endosperm development are the key determinants of grain yield and nutritional quality. GRAIN WIDTH and WEIGHT2 (GW2) gene encodes a RING-type E3 ubiquitin ligase and determines the grain weight in cereal crops. Here we report GW2 knockout (KO) mutants in Indica (var. MTU1010) through CRISPR/Cas9 genome editing. The endosperm of GW2-KO mutant seed displays a thick aleurone layer with enhanced grain protein content. Further the loss of function of OsGW2 results in improved accumulation of essential dietary minerals (Fe, Zn, K, P, Ca) in the endosperm of rice grain. Additionally, the mutants displayed an early growth vigour phenotype with an improved root and shoot architecture. The hull morphology of GW2-KO lines also showed improved, grain filling thereby promoting larger grain architecture. Together, our findings indicate that GW2 may serve as a key regulator of improved grain architecture, grain nutritional quality and an important modulator of plant morphology. The study offers a strategy for the development of improved rice cultivars with enriched nutritional quality and its possible implementation in other cereals as well.},
}
@article {pmid34753924,
year = {2021},
author = {Perez, AR and Sala, L and Perez, RK and Vidigal, JA},
title = {CSC software corrects off-target mediated gRNA depletion in CRISPR-Cas9 essentiality screens.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6461},
pmid = {34753924},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Computational Biology/methods ; Gene Editing ; Humans ; RNA, Guide/genetics/*metabolism ; Software ; },
abstract = {Off-target effects are well established confounders of CRISPR negative selection screens that impair the identification of essential genomic loci. In particular, non-coding regulatory elements and repetitive regions are often difficult to target with specific gRNAs, effectively precluding the unbiased screening of a large portion of the genome. To address this, we developed CRISPR Specificity Correction (CSC), a computational method that corrects for the effect of off-targeting on gRNA depletion. We benchmark CSC with data from the Cancer Dependency Map and show that it significantly improves the overall sensitivity and specificity of viability screens while preserving known essentialities, particularly for genes targeted by highly promiscuous gRNAs. We believe this tool will further enable the functional annotation of the genome as it represents a robust alternative to the traditional filtering strategy of discarding unspecific guides from the analysis. CSC is an open-source software that can be seamlessly integrated into current CRISPR analysis pipelines.},
}
@article {pmid34753073,
year = {2021},
author = {Khan, WA and Barney, RE and Tsongalis, GJ},
title = {CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting.},
journal = {Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology},
volume = {145},
number = {},
pages = {105019},
pmid = {34753073},
issn = {1873-5967},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral ; *SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: The well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-PCR assays has led to the use of alternate testing options. Here we evaluate the accuracy and performance of a novel fluorescence-based assay that received EUA authorization for detecting SARS-CoV-2 in clinical samples.<br><br>METHODS: The Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-PCR assays. Positive samples were selected based on a broad range of cycle thresholds.<br><br>RESULTS: A total of 60 COVID-19 patient samples were correctly diagnosed with 100% detection accuracy (relative fluorescence ratios: N gene 95% CI 29.9-43.8, ORF1ab gene 95% CI 30.1-46.3). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence.<br><br>CONCLUSIONS: We have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads, including low titers, using SHERLOCK CRISPR collateral detection in a clinical setting. These findings demonstrate encouraging results, at a time when COVID-19 clinical diagnosis and screening protocols remain in demand; especially as new variants emerge and vaccine mandates evolve. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.},
}
@article {pmid34752910,
year = {2021},
author = {Ebrahimi, S and Kalantari, M and Alipour, H and Azizi, K and Asgari, Q and Bahreini, MS},
title = {In vitro evaluation of CRISPR PX-LmGP63 vector effect on pathogenicity of Leishmania major as a primary step to control leishmaniasis.},
journal = {Microbial pathogenesis},
volume = {161},
number = {Pt A},
pages = {105281},
doi = {10.1016/j.micpath.2021.105281},
pmid = {34752910},
issn = {1096-1208},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Editing ; *Leishmania major/genetics ; *Leishmaniasis, Cutaneous ; Virulence ; },
abstract = {Cutaneous leishmaniasis (CL) is caused by intracellular obligate parasites (Leishmania spp.) carried by the blood-sucking of female sandflies and transmitted between mammalian hosts. Despite the high incidence and prevalence of Leishmania cases in many countries, it has been a neglected tropical disease. The current treatment approaches are limited by the complications such as loss of fertility and drug resistance. It is, therefore, essential to find new medicines to treat leishmaniasis. CRISPR/Cas9 as a powerful genome-editing tool provides the opportunity to create precise genetic manipulation to investigate the molecular basis of different leishmaniasis cases. Therefore, our main goal was to evaluate the CRISPR PX-LmGP63 vector effect on pathogenicity of Leishmania majorin vitroto challenge for using CRISPR/Cas9 as a therapeutic CL through the reduction of L. major pathogenicity by manipulating the GP63 gene. In this study, L. major parasites were transfected with CRISPR/Cas9 vectors constructed by electroporation and then added to macrophage cells on RPMI. The effect of CRISPR/Cas9 constructs on GP63 mutation, viability, and status of L. major was investigated by counting phagocytic parasites into macrophages and DNA sequence analysis. Our data validate that the use of CRISPR/Cas9 in L. major creates a new stop codon and disrupts the frame sheet of the gene by creating a new insertion (thymine), which prevents its expression. In addition, the parasite count was significantly different in the case and control of infected macrophages (P < 0.05). This study shows the successfully targeted manipulation of the L. major GP63 gene via the adaptation of the CRISPR/Cas9 editing tool. The manipulation of GP63 revealed a reduction in the infection load compared to wild-type parasite infection. Therefore, more studies are necessary for this field to help achieve a new method for the prevention and treatment of CL disease.},
}
@article {pmid34752487,
year = {2021},
author = {Tyumentseva, MA and Tyumentsev, AI and Akimkin, VG},
title = {Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells.},
journal = {PloS one},
volume = {16},
number = {11},
pages = {e0259812},
pmid = {34752487},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Cell Culture Techniques ; Cell Line ; Cell- and Tissue-Based Therapy ; Cloning, Molecular/*methods ; Cost-Benefit Analysis ; Electroporation ; Gene Editing/*methods ; Gene Transfer Techniques/instrumentation/trends ; Humans ; RNA, Guide/genetics ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {BACKGROUND: Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based methods.<br><br>METHODS: In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques.<br><br>DISCUSSION: This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered 'research-use only', others will be recommended for scaling and application in the development of cell-based therapies.},
}
@article {pmid34752296,
year = {2021},
author = {McGaw, C and Chong, S},
title = {Cell-free protein synthesis of CRISPR ribonucleoproteins (RNP).},
journal = {Methods in enzymology},
volume = {659},
number = {},
pages = {371-389},
doi = {10.1016/bs.mie.2021.05.010},
pmid = {34752296},
issn = {1557-7988},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; RNA, Guide/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; Streptococcus pyogenes/genetics ; },
abstract = {Cell-free protein synthesis is a powerful tool to produce recombinant proteins, and as an open system, can often integrate all or part of downstream assays. Here we describe in vitro synthesis of the Streptococcus pyogenes type II-A CRISPR-Cas9 ribonucleoproteins (SpCas9 RNPs), consisting of the effector protein and the single guide RNAs (sgRNAs). In spite of its large molecular weight (160kDa), the SpCas9 effector is expressed relatively well from linear DNA templates under T7 promoter in commercial reconstituted cell-free protein synthesis systems. sgRNAs can be added before the effector synthesis reaction, or transcribed directly from linear DNA templates during the synthesis reaction. The newly synthesized SpCas9 effector forms an active RNP complex with sgRNAs. When a reconstituted cell-free system is used, the target DNA templates can also be added in the reactions, thereby combining CRISPR-Cas synthesis and activity assay.},
}
@article {pmid34752293,
year = {2021},
author = {Feng, M and She, Q},
title = {Purification and characterization of ribonucleoprotein effector complexes of Sulfolobus islandicus CRISPR-Cas systems.},
journal = {Methods in enzymology},
volume = {659},
number = {},
pages = {327-347},
doi = {10.1016/bs.mie.2021.05.007},
pmid = {34752293},
issn = {1557-7988},
mesh = {Archaea ; CRISPR-Cas Systems ; Ribonucleoproteins/genetics ; *Sulfolobus/genetics ; },
abstract = {Archaea are preferred hosts for CRISPR-Cas systems. This adaptive immune system is not only widespread in archaeal organisms, but different types of CRISPR-Cas also co-exist in the same organism. Sulfolobus islandicus provides a good model for CRISPR research as genetic assays have been developed for revealing CRISPR immunity for the crenarchaeal model, and native ribonucleoprotein effector complexes have been expressed in this crenarchaeon and purified for characterization. Here we report a detailed protocol of purification and characterization of the Sulfolobus islandicus Cmr-β, the largest CRISPR effector known to date. The method can readily be applied to the purification of effectors encoded by other CRISPR-Cas systems in this organism, with the possibility to extend the application to other Sulfolobales.},
}
@article {pmid34751850,
year = {2021},
author = {Singh, M and Nara, U and Kumar, A and Choudhary, A and Singh, H and Thapa, S},
title = {Salinity tolerance mechanisms and their breeding implications.},
journal = {Journal, genetic engineering &amp; biotechnology},
volume = {19},
number = {1},
pages = {173},
pmid = {34751850},
issn = {2090-5920},
abstract = {BACKGROUND: The era of first green revolution brought about by the application of chemical fertilizers surely led to the explosion of food grains, but left behind the notable problem of salinity. Continuous application of these fertilizers coupled with fertilizer-responsive crops make the country self-reliant, but continuous deposition of these led to altered the water potential and thus negatively affecting the proper plant functioning from germination to seed setting.<br><br>MAIN BODY: Increased concentration of anion and cations and their accumulation and distribution cause cellular toxicity and ionic imbalance. Plants respond to salinity stress by any one of two mechanisms, viz., escape or tolerate, by either limiting their entry via root system or controlling their distribution and storage. However, the understanding of tolerance mechanism at the physiological, biochemical, and molecular levels will provide an insight for the identification of related genes and their introgression to make the crop more resilient against salinity stress.<br><br>SHORT CONCLUSION: Novel emerging approaches of plant breeding and biotechnologies such as genome-wide association studies, mutational breeding, marker-assisted breeding, double haploid production, hyperspectral imaging, and CRISPR/Cas serve as engineering tools for dissecting the in-depth physiological mechanisms. These techniques have well-established implications to understand plants' adaptions to develop more tolerant varieties and lower the energy expenditure in response to stress and, constitutively fulfill the void that would have led to growth resistance and yield penalty.},
}
@article {pmid34751285,
year = {2021},
author = {Li, P and Zhang, J and Lin, Q and Kong, J and Fang, X},
title = {Rapid differential diagnosis of the B.1.617.2 (delta) variant of SARS-CoV-2 using an automated Cas12a-microfluidic system.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {92},
pages = {12270-12272},
doi = {10.1039/d1cc04874k},
pmid = {34751285},
issn = {1364-548X},
mesh = {Automation ; COVID-19/*diagnosis/virology ; CRISPR-Cas Systems/*genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Microfluidics/*methods ; Mutation ; Polymerase Chain Reaction ; RNA, Guide/genetics/metabolism ; RNA, Viral/metabolism ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {An automated Cas12a-microfluidic system was constructed to distinguish the B.1.617.2 (delta) variant of SARS-CoV-2 from the wild-type virus rapidly and was validated using 30 clinical samples, showing 100% consistency with next-generation sequencing. It will be a potential tool for the rapid differential diagnosis of the delta variant of SARS-CoV-2.},
}
@article {pmid34751223,
year = {2021},
author = {Wang, Y and Mao, T and Li, Y and Xiao, W and Liang, X and Duan, G and Yang, H},
title = {Characterization of 67 Confirmed Clustered Regularly Interspaced Short Palindromic Repeats Loci in 52 Strains of Staphylococci.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {736565},
pmid = {34751223},
issn = {1664-302X},
abstract = {Staphylococcus aureus (S. aureus), which is one of the most important species of Staphylococci, poses a great threat to public health. Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune platform to combat foreign mobile genetic elements (MGEs) such as plasmids and phages. The aim of this study is to describe the distribution and structure of CRISPR-Cas system in S. aureus, and to explore the relationship between CRISPR and horizontal gene transfer (HGT). Here, we analyzed 67 confirmed CRISPR loci and 15 companion Cas proteins in 52 strains of Staphylococci with bioinformatics methods. Comparing with the orphan CRISPR loci in Staphylococci, the strains harboring complete CRISPR-Cas systems contained multiple CRISPR loci, direct repeat sequences (DR) forming stable RNA secondary structures with lower minimum free energy (MFE), and variable spacers with detectable protospacers. In S. aureus, unlike the orphan CRISPRs away from Staphylococcal cassette chromosome mec (SCCmec), the complete CRISPR-Cas systems were in J1 region of SCCmec. In addition, we found a conserved motif 5'-TTCTCGT-3' that may protect their downstream sequences from DNA interference. In general, orphan CRISPR locus in S. aureus differed greatly from the structural characteristics of the CRISPR-Cas system. Collectively, our results provided new insight into the diversity and characterization of the CRISPR-Cas system in S. aureus.},
}
@article {pmid34750615,
year = {2021},
author = {Rahman, ML and Hyodo, T and Hasan, MN and Mihara, Y and Karnan, S and Ota, A and Tsuzuki, S and Hosokawa, Y and Konishi, H},
title = {Flow cytometry-based quantification of targeted knock-in events in human cell lines using a GPI-anchor biosynthesis gene PIGP.},
journal = {Bioscience reports},
volume = {41},
number = {12},
pages = {},
pmid = {34750615},
issn = {1573-4935},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Membrane/*genetics/metabolism ; *Flow Cytometry ; GPI-Linked Proteins/biosynthesis/*genetics ; Gene Expression Regulation ; *Gene Knock-In Techniques ; *Genes, Reporter ; HCT116 Cells ; Hexosyltransferases/*genetics/metabolism ; Humans ; Membrane Proteins/*genetics/metabolism ; Mutation ; },
abstract = {Targeted knock-in supported by the CRISPR/Cas systems enables the insertion, deletion, and substitution of genome sequences exactly as designed. Although this technology is considered to have wide range of applications in life sciences, one of its prerequisites for practical use is to improve the efficiency, precision, and specificity achieved. To improve the efficiency of targeted knock-in, there first needs to be a reporter system that permits simple and accurate monitoring of targeted knock-in events. In the present study, we created such a system using the PIGP gene, an autosomal gene essential for GPI-anchor biosynthesis, as a reporter gene. We first deleted a PIGP allele using Cas9 nucleases and then incorporated a truncating mutation into the other PIGP allele in two near-diploid human cell lines. The resulting cell clones were used to monitor the correction of the PIGP mutations by detecting GPI anchors distributed over the cell membrane via flow cytometry. We confirmed the utility of these reporter clones by performing targeted knock-in in these clones via a Cas9 nickase-based strategy known as tandem paired nicking, as well as a common process using Cas9 nucleases, and evaluating the efficiencies of the achieved targeted knock-in. We also leveraged these reporter clones to test a modified procedure for tandem paired nicking and demonstrated a slight increase in the efficiency of targeted knock-in by the new procedure. These data provide evidence for the utility of our PIGP-based assay system to quantify the efficiency of targeted knock-in and thereby help improve the technology of targeted knock-in.},
}
@article {pmid34750099,
year = {2022},
author = {Jiang, L and Ingelshed, K and Shen, Y and Boddul, SV and Iyer, VS and Kasza, Z and Sedimbi, S and Lane, DP and Wermeling, F},
title = {CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies.},
journal = {Cancer research},
volume = {82},
number = {1},
pages = {36-45},
doi = {10.1158/0008-5472.CAN-21-1692},
pmid = {34750099},
issn = {1538-7445},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA Damage/*genetics ; Humans ; Mice ; Mutation ; Transfection ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.},
}
@article {pmid34749799,
year = {2021},
author = {Billaud, A and Chevalier, LM and Augereau, P and Frenel, JS and Passot, C and Campone, M and Morel, A},
title = {Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes.},
journal = {Genome medicine},
volume = {13},
number = {1},
pages = {174},
pmid = {34749799},
issn = {1756-994X},
mesh = {BRCA1 Protein/genetics ; BRCA2 Protein/genetics ; Breast Neoplasms/*genetics ; CRISPR-Cas Systems ; Female ; *Gene Editing ; *Genes, Tumor Suppressor ; Genetic Predisposition to Disease ; Genetic Testing/*methods ; *Genetic Variation ; Humans ; Middle Aged ; Ovarian Neoplasms/genetics ; Poly(ADP-ribose) Polymerase Inhibitors ; Retrospective Studies ; },
abstract = {BACKGROUND: Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients' access to targeted therapies.<br><br>METHODS: We first evaluated the functional impact of 26 BRCA1 and 7 BRCA2 variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of POLE VUS.<br><br>RESULTS: For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients' responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three POLE VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four POLE variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal.<br><br>CONCLUSIONS: Our new rapid functional assay can be used to characterize the functional implication of BRCA1 and BRCA2 variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients' responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact.},
}
@article {pmid34749073,
year = {2021},
author = {Yan, T and Yang, K and Chen, C and Zhou, Z and Shen, P and Jia, Y and Xue, Y and Zhang, Z and Shen, X and Han, X},
title = {Synergistic photothermal cancer immunotherapy by Cas9 ribonucleoprotein-based copper sulfide nanotherapeutic platform targeting PTPN2.},
journal = {Biomaterials},
volume = {279},
number = {},
pages = {121233},
doi = {10.1016/j.biomaterials.2021.121233},
pmid = {34749073},
issn = {1878-5905},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Copper ; *Hyperthermia, Induced ; Immunotherapy ; Mice ; *Nanoparticles ; *Neoplasms/therapy ; Phototherapy ; Protein Tyrosine Phosphatase, Non-Receptor Type 2 ; Ribonucleoproteins ; Sulfides ; },
abstract = {Photothermal therapy (PTT) is a promising strategy for the treatment of advanced malignant neoplasm. However, the anti-tumor efficacy by PTT alone is insufficient to control tumor growth and metastasis. Here, we report a multifunctional nanotherapeutic system exerting a combined PTT and immunotherapy to synergistically enhance the therapeutic effect on melanoma. In particular, we selected the semiconductor nanomaterial copper sulfide (CuS), which served not only as a near-infrared (NIR) light-triggered photothermal converter for tumor hyperthermia but as a basic carrier to modify Cas9 ribonucleoprotein targeting PTPN2 on its surface. Efficient PTPN2 depletion was observed after the treatment of CuS-RNP@PEI nanoparticles, which caused the accumulation of intratumoral infiltrating CD8 T lymphocytes in tumor-bearing mice and upregulated the expression levels of IFN-ᵧ and TNF-α in tumor tissue, thus sensitizing tumors to immunotherapy. In addition, the effect worked synergistically with tumor ablation and immunogenic cell death (ICD) induced by PTT to amplify anti-tumor efficacy. Taken together, this exogenously controlled method provides a simple and effective treatment option for advanced malignant neoplasm.},
}
@article {pmid34748964,
year = {2022},
author = {Francis, C and Wroblewska, L and Pegman, P and Amiji, M},
title = {Systemic biodistribution and hepatocyte-specific gene editing with CRISPR/Cas9 using hyaluronic acid-based nanoparticles.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {40},
number = {},
pages = {102488},
doi = {10.1016/j.nano.2021.102488},
pmid = {34748964},
issn = {1549-9642},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Hepatocytes ; Hyaluronic Acid ; Mice ; *Nanoparticles ; Tissue Distribution ; },
abstract = {The goal of this study was to evaluate hepatocyte-specific gene editing, via systemic administration of hyaluronic acid (HA)-based nanoparticles in naïve CD-1 mice. Using HA-poly(ethylene imine) (HA-PEI) and HA-PEI-mannose nanoparticles with differential mannose density (1X and 2X), we have evaluated systemic biodistribution and hepatocyte-specific delivery using IVIS imaging and flow cytometry. Additionally, we have investigated hepatocyte-specific delivery and transfection of CRISPR/Cas9 gene editing plasmid and eGFP gene payload to integrate at the Rosa26 locus. IVIS imaging showed uptake of HA-PEI nanoparticles primarily by the liver, and with addition of mannose at different concentrations, the nanoparticles showed increased uptake in both the liver and spleen. HA-PEI-mannose nanoparticles showed 55-65% uptake by hepatocytes, along with uptake by resident macrophage regardless of the mannose concentration. One of two gRNA targets showed 15% genome editing and obtained similar results for all three nanoparticle formulations. Cells positive for our gene payload were greatest with HA-PEI-mannose-1X nanoparticles where 16.2% of cells were GFP positive. The results were encouraging as proof of concept for the development of a non-viral biodegradable and biocompatible polymeric delivery system for gene editing specifically targeting hepatocytes upon systemic administration.},
}
@article {pmid34748534,
year = {2021},
author = {Schwartz, ML and Davis, MW and Rich, MS and Jorgensen, EM},
title = {High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome.},
journal = {PLoS genetics},
volume = {17},
number = {11},
pages = {e1009755},
pmid = {34748534},
issn = {1553-7404},
support = {F32 GM133139/GM/NIGMS NIH HHS/United States ; R01 GM095817/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Gene Editing/*methods ; *Genome, Helminth ; },
abstract = {Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50-100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.},
}
@article {pmid34748284,
year = {2021},
author = {Atri, DS and Lee-Kim, VS and Vellarikkal, SK and Sias-Garcia, O and Yanamandala, M and Schniztler, GR and Gupta, RM},
title = {CRISPR-Cas9 Genome Editing of Primary Human Vascular Cells In Vitro.},
journal = {Current protocols},
volume = {1},
number = {11},
pages = {e291},
pmid = {34748284},
issn = {2691-1299},
support = {K08 HL128810/HL/NHLBI NIH HHS/United States ; DP2 HL152423/HL/NHLBI NIH HHS/United States ; R03 HL148483/HL/NHLBI NIH HHS/United States ; T32 HL007604/HL/NHLBI NIH HHS/United States ; T32 HL007917/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Expression ; Humans ; Plasmids ; Transfection ; },
abstract = {Genome editing of primary human cells with CRISPR-Cas9 is a powerful tool to study gene function. For many cell types, there are efficient protocols for editing with optimized plasmids for Cas9 and sgRNA expression. Vascular cells, however, remain refractory to plasmid-based delivery of CRISPR machinery for in vitro genome editing due to low transfection efficiency, poor expression of the Cas9 machinery, and toxic effects of the selection antibiotics. Here, we describe a method for high-efficiency editing of primary human vascular cells in vitro using nucleofection for direct delivery of sgRNA:Cas9-NLS ribonucleoprotein complexes. This method is more rapid and its high editing efficiency eliminates the need for additional selection steps. The edited cells can be employed in diverse applications, such as gene expression measurement or functional assays to assess various genetic perturbation effects in vitro. This method proves effective in vascular cells that are refractory to standard genome manipulation techniques using viral plasmid delivery. We anticipate that this technique will be applied to other non-vascular cell types that face similar barriers to efficient genome editing. © 2021 Wiley Periodicals LLC. Basic Protocol: CRISPR-Cas9 genome editing of primary human vascular cells in vitro.},
}
@article {pmid34748280,
year = {2021},
author = {Wardyn, JD and Chan, ASY and Jeyasekharan, AD},
title = {A Robust Protocol for CRISPR-Cas9 Gene Editing in Human Suspension Cell Lines.},
journal = {Current protocols},
volume = {1},
number = {11},
pages = {e286},
doi = {10.1002/cpz1.286},
pmid = {34748280},
issn = {2691-1299},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Endonucleases/genetics ; *Gene Editing ; Humans ; RNA, Guide/genetics ; },
abstract = {The implementation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems in mammalian cells has sparked an exciting new era in targeted gene editing. CRISPR-Cas9 technologies allow gene function to be interrogated by gene deletions, mutations, and truncations, and by epitope tagging and promoter activity modulation. Many robust protocols have been published to date on CRISPR-Cas9 techniques, however, most of these focus on adherent cell lines. Suspension cell lines, typically of hematolymphoid origin, such as Jurkat, Daudi, and TOLEDO, pose unique challenges to the setup of CRISPR experiments. Here, using B cell lymphoma cells as a primary model, we describe a comprehensive protocol for targeted gene manipulations using the CRISPR-Cas9 system in suspension cells. We also highlight necessary optimization steps to make this approach universal to other suspension cell lines. We first describe a detailed protocol for transient expression of the Cas9 nuclease and guide RNAs. We then suggest workflows for obtaining single-cell clones and for screening for successful homozygous knockout (KO) clones in suspension lines. This protocol aims to serve as a comprehensive resource to facilitate gene editing experiments for users starting CRISPR-Cas9 gene editing protocols on suspension cell lines or those looking to optimize their current workflows. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Transient CRISPR Cas9-gRNA delivery for gene knockout by Neon[TM] electroporation Support Protocol 1: Designing and preparing gene-specific gRNA Support Protocol 2: Preparation of conditioned medium and culture vessels for single-cell FACS Alternate Protocol 1: Transient CRISPR Cas9-gRNA delivery for gene knockout by Nepagene electroporator Basic Protocol 2: FACS and single-cell clone generation Alternate Protocol 2: Manual cell dilution to obtain single-cell clones Basic Protocol 3: Confirming indels status in single-cell clones by PCR on genomic DNA and Sanger sequencing.},
}
@article {pmid34747695,
year = {2021},
author = {Kiritsy, MC and Ankley, LM and Trombley, J and Huizinga, GP and Lord, AE and Orning, P and Elling, R and Fitzgerald, KA and Olive, AJ},
title = {A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34747695},
issn = {2050-084X},
support = {P01 AI132130/AI/NIAID NIH HHS/United States ; R21 AI146504/AI/NIAID NIH HHS/United States ; T32 AI095213/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Histocompatibility Antigens Class II/genetics/*metabolism ; Interferon-gamma/*metabolism ; Lymphocyte Activation/*genetics ; Macrophages/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Nuclear Proteins/metabolism ; T-Lymphocytes ; Trans-Activators/metabolism ; },
abstract = {Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNγ) is a key cytokine in protective immunity that induces major histocompatibility complex class II molecules (MHCII) to amplify CD4[+] T cell activation and effector function. Despite its central role, the dynamic regulation of IFNγ-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages, we identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase three beta (GSK3β) or the mediator complex subunit 16 (MED16). Both pathways control distinct aspects of the IFNγ response and are necessary for IFNγ-mediated induction of the MHCII transactivator Ciita, MHCII expression, and CD4[+] T cell activation. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4[+] T cell responses.},
}
@article {pmid34747603,
year = {2021},
author = {Wang, P and Yi, Y and Lü, X},
title = {CRISPR/Cas9-Based Genome Editing Platform for Companilactobacillus crustorum to Reveal the Molecular Mechanism of Its Probiotic Properties.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {50},
pages = {15279-15289},
doi = {10.1021/acs.jafc.1c05389},
pmid = {34747603},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Genetic Engineering ; *Probiotics ; },
abstract = {Companilactobacillus crustorum usually serves as a starter culture for the food industry. Recent studies revealed that this species also possesses probiotic properties. Genome engineering, including point mutation or gene deletion, is desired to understand the mechanisms of its probiotic and fermentation properties. To tackle the hurdle in genetic manipulation in C. crustorum, here, we established a fast and easy CRISPR/Cas9-based platform for precise genome editing in this species. The platform includes two CRISPR/Cas9 systems and a CRISPR/Cas9-based editing system. Using the developed methods, we were able to knockout 12 genes in C. crustorum by deleting a fragment located in the open reading frames. The editing efficiency ranged from 14.3 to 100%. Moreover, we developed a CRISPR-assisted cytidine base-editing system, enabling programmed C to T conversion in the chromosome for gene inactivation or point mutation. To further exploit this platform, we investigated the role of nine putative bacteriocin-encoding genes and found that bacteriocins BM173 and BM1157 mostly contributed to the antimicrobial activity of C. crustorum MN047 against Staphylococcus aureus and Escherichia coli. In addition, the regulation of bacteriocin expression was also revealed to be linked with the quorum-sensing modulator luxS. This work will dramatically accelerate the genetic engineering of C. crustorum and close-related species.},
}
@article {pmid34747560,
year = {2021},
author = {Anderson, DA and Voigt, CA},
title = {Competitive dCas9 binding as a mechanism for transcriptional control.},
journal = {Molecular systems biology},
volume = {17},
number = {11},
pages = {e10512},
pmid = {34747560},
issn = {1744-4292},
mesh = {Binding, Competitive ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; Promoter Regions, Genetic ; *RNA, Guide/genetics ; },
abstract = {Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27-fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence-independent control by adopting it for elongation control, achieving 8-fold repression and 4-fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis-regulatory logic on CRISPRi-targeted native genes.},
}
@article {pmid34747197,
year = {2022},
author = {Long, KR and Rbaibi, Y and Bondi, CD and Ford, BR and Poholek, AC and Boyd-Shiwarski, CR and Tan, RJ and Locker, JD and Weisz, OA},
title = {Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells.},
journal = {American journal of physiology. Renal physiology},
volume = {322},
number = {1},
pages = {F14-F26},
pmid = {34747197},
issn = {1522-1466},
support = {R01 DK118726/DK/NIDDK NIH HHS/United States ; T32 CA082084/CA/NCI NIH HHS/United States ; K08 DK118211/DK/NIDDK NIH HHS/United States ; P30 DK079307/DK/NIDDK NIH HHS/United States ; R21 AI135027/AI/NIAID NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Agenesis of Corpus Callosum/genetics/metabolism/pathology ; Animals ; Apoptosis Regulatory Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cells, Cultured ; Databases, Genetic ; *Gene Knockout Techniques ; Gene Regulatory Networks ; Hearing Loss, Sensorineural/genetics/metabolism/pathology ; Hernias, Diaphragmatic, Congenital/genetics/metabolism/pathology ; Humans ; Kidney Tubules, Proximal/*metabolism/pathology ; Low Density Lipoprotein Receptor-Related Protein-2/genetics/*metabolism ; Male ; Mice, Knockout ; Monodelphis ; Myopia/genetics/metabolism/pathology ; Proteinuria/genetics/metabolism/pathology ; Receptors, Cell Surface/genetics/*metabolism ; Renal Tubular Transport, Inborn Errors/genetics/metabolism/pathology ; *Transcription, Genetic ; },
abstract = {The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW &amp; NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.},
}
@article {pmid34746867,
year = {2021},
author = {Zou, RS and Liu, Y and Ha, T},
title = {CRISPR deactivation in mammalian cells using photocleavable guide RNAs.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100909},
pmid = {34746867},
issn = {2666-1667},
support = {T32 GM136577/GM/NIGMS NIH HHS/United States ; F30 CA254160/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism/radiation effects ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; INDEL Mutation/genetics ; Photolysis ; *RNA, Guide/genetics/metabolism/radiation effects ; },
abstract = {The ability to deactivate CRISPR-Cas systems on demand would improve the safety and applicability of genome editing. Here, we detail a protocol using photocleavable guide RNAs (pcRNAs) to deactivate CRISPR-Cas9 inside cells. We verify that deactivation is both rapid and complete by checking for insertion-deletion (indel) mutations using Sanger sequencing. This protocol will be useful for researchers interested in using pcRNAs to improve genome editing specificity, characterize the timescales of genome editing, and study cellular DNA damage responses. For complete details on the use and execution of this protocol, please refer to Zou et al. (2021).},
}
@article {pmid34746857,
year = {2021},
author = {Kumar, A and Lee, SJ and Liu, Q and Chan, AKN and Pokharel, SP and Yu, J and Chen, CW and Swaminathan, S},
title = {Generation and validation of CRISPR-engineered human natural killer cell lines for research and therapeutic applications.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100874},
pmid = {34746857},
issn = {2666-1667},
support = {R01 AI129582/AI/NIAID NIH HHS/United States ; R01 NS106170/NS/NINDS NIH HHS/United States ; R21 CA223400/CA/NCI NIH HHS/United States ; R01 CA266457/CA/NCI NIH HHS/United States ; R37 CA233691/CA/NCI NIH HHS/United States ; P01 CA163205/CA/NCI NIH HHS/United States ; R01 CA247550/CA/NCI NIH HHS/United States ; R01 CA265095/CA/NCI NIH HHS/United States ; P50 CA107399/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line ; Gene Editing/*methods ; Humans ; K562 Cells ; Killer Cells, Natural/*cytology ; },
abstract = {Cytotoxic natural killer cells kill tumors and infected cells. We carried out CRISPR-based gene editing and transcriptional regulation in hard-to-manipulate NK-92 cells. NK-92-based therapies were found to be safe and efficacious in preclinical studies of cancers. Here, we have pioneered the generation and validation of NK-92 cells constitutively expressing Cas9 or dCas9 for knockout (CRISPRko), transcriptional activation (CRISPRa), or transcriptional repression (CRISPRi) of genes. Our CRISPR-engineered NK-92 cell platforms can be modified for research and off-the-shelf therapeutic applications.},
}
@article {pmid34746853,
year = {2021},
author = {Farooq, U and Notani, D},
title = {Optimized protocol to create deletion in adherent cell lines using CRISPR/Cas9 system.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100857},
pmid = {34746853},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Deletion ; Gene Editing/*methods ; HeLa Cells ; Humans ; MCF-7 Cells ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for genome editing and is adaptable for a wide range of applications. Here, we have put together a step-by-step protocol for generating knockout cell lines (coding or non-coding region) using CRISPR/Cas9 tool. The protocol below has been tested on adherent cell lines such as HeLa and MCF7. However, it may easily be adapted to other adherent cell lines with minor variations. For complete details on the use and execution of this protocol, please refer to Farooq et al. (2021).},
}
@article {pmid34745851,
year = {2021},
author = {Lu, J and Liu, J and Guo, Y and Zhang, Y and Xu, Y and Wang, X},
title = {CRISPR-Cas9: A method for establishing rat models of drug metabolism and pharmacokinetics.},
journal = {Acta pharmaceutica Sinica. B},
volume = {11},
number = {10},
pages = {2973-2982},
pmid = {34745851},
issn = {2211-3835},
abstract = {The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9, a super-selective and precise gene editing tool. CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell, and presents great potential in disease treatment and animal model construction. In recent years, CRISPR-Cas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics (DMPK), such as Cyp, Abcb1, Oatp1b2 gene knockout rats. These new rat models are not only widely used in the study of drug metabolism, chemical toxicity, and carcinogenicity, but also promote the study of DMPK related mechanism, and further strengthen the relationship between drug metabolism and pharmacology/toxicology. This review systematically introduces the advantages and disadvantages of CRISPR-Cas9, summarizes the methods of establishing DMPK rat models, discusses the main challenges in this field, and proposes strategies to overcome these problems.},
}
@article {pmid34743972,
year = {2021},
author = {Li, JJ and Shi, Y and Wu, JN and Li, H and Smagghe, G and Liu, TX},
title = {CRISPR/Cas9 in lepidopteran insects: Progress, application and prospects.},
journal = {Journal of insect physiology},
volume = {135},
number = {},
pages = {104325},
doi = {10.1016/j.jinsphys.2021.104325},
pmid = {34743972},
issn = {1879-1611},
mesh = {Animals ; Bombyx/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Lepidoptera/*genetics ; },
abstract = {Clustered regularly spaced short palindrome repeats (CRISPR) structure family forms the acquired immune system in bacteria and archaea. Recent advances in CRISPR/Cas genome editing as derived from prokaryotes, confirmed the characteristics of robustness, high target specificity and programmability, and also revolutionized the insect sciences field. The successful application of CRISPR in a wide variety of lepidopteran insects, with a high genetic diversity, provided opportunities to explore gene functions, insect modification and pest control. In this review, we present a detailed overview on the recent progress of CRISPR in lepidopteran insects, and described the basic principles of the system and its application. Major interest is on wing development, pigmentation, mating, reproduction, sex determination, metamorphosis, resistance and silkworm breeding innovation. Finally, we outlined the limitations of CRISPR/Cas system and discussed its application prospects in lepidopteran insects.},
}
@article {pmid34742871,
year = {2022},
author = {Haq, S and Sarodaya, N and Karapurkar, JK and Suresh, B and Jo, JK and Singh, V and Bae, YS and Kim, KS and Ramakrishna, S},
title = {CYLD destabilizes NoxO1 protein by promoting ubiquitination and regulates prostate cancer progression.},
journal = {Cancer letters},
volume = {525},
number = {},
pages = {146-157},
doi = {10.1016/j.canlet.2021.10.032},
pmid = {34742871},
issn = {1872-7980},
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Deubiquitinating Enzyme CYLD/*genetics ; Deubiquitinating Enzymes/genetics ; Disease Progression ; Gene Expression Regulation, Neoplastic/genetics ; Genome, Human/genetics ; Heterografts ; Humans ; Male ; Prostatic Neoplasms/*genetics/pathology ; Reactive Oxygen Species/metabolism ; Signal Transduction/genetics ; Ubiquitination/*genetics ; },
abstract = {The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.},
}
@article {pmid34741959,
year = {2022},
author = {Phan, QA and Truong, LB and Medina-Cruz, D and Dincer, C and Mostafavi, E},
title = {CRISPR/Cas-powered nanobiosensors for diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {197},
number = {},
pages = {113732},
doi = {10.1016/j.bios.2021.113732},
pmid = {34741959},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA, Single-Stranded ; *Metal Nanoparticles ; },
abstract = {CRISPR diagnostics (CRISPR-Dx) offer a wide range of enhancements compared to traditional nanobiosensors by taking advantage of the excellent trans-cleavage activity of the CRISPR/Cas systems. However, the single-stranded DNA/RNA reporters of the current CRISPR-Dx suffer from poor stability and limited sensitivity, which make their application in complex biological environments difficult. In comparison, nanomaterials, especially metal nanoparticles, exhibits robust stability and desirable optical and electrocatalytical properties, which make them ideal as reporter molecules. Therefore, biosensing research is moving towards the use of the trans-cleavage activity of CRISPR/Cas effectors on metal nanoparticles and apply the new phenomenon to develop novel nanobiosensors to target various targets such as viral infections, genetic mutations and tumor biomarkers, by using different sensing methods, including, but not limited to fluorescence, luminescence resonance, colorimetric and electrochemical signal readout. In this review, we explore some of the most recent advances in the field of CRISPR-powered nanotechnological biosensors. Demonstrating high accuracy, sensitivity, selectivity and versatility, nanobiosensors along with CRISPR/Cas technology offer tremendous potential for next-generation diagnostics of multiple targets, especially at the point of care and without any target amplification.},
}
@article {pmid34741801,
year = {2021},
author = {Kumar, S and Javed, R and Mudd, M and Pallikkuth, S and Lidke, KA and Jain, A and Tangavelou, K and Gudmundsson, SR and Ye, C and Rusten, TE and Anonsen, JH and Lystad, AH and Claude-Taupin, A and Simonsen, A and Salemi, M and Phinney, B and Li, J and Guo, LW and Bradfute, SB and Timmins, GS and Eskelinen, EL and Deretic, V},
title = {Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.},
journal = {Cell},
volume = {184},
number = {24},
pages = {5950-5969.e22},
pmid = {34741801},
issn = {1097-4172},
support = {P20 GM121176/GM/NIGMS NIH HHS/United States ; R37 AI042999/AI/NIAID NIH HHS/United States ; R01 AI111935/AI/NIAID NIH HHS/United States ; S10 OD026918/OD/NIH HHS/United States ; R01 EY029809/EY/NEI NIH HHS/United States ; S10 OD021801/OD/NIH HHS/United States ; },
mesh = {Autophagosomes/*virology ; Autophagy ; COVID-19/metabolism/*virology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Endoplasmic Reticulum/metabolism ; Endosomes/physiology/virology ; Golgi Apparatus/physiology ; HEK293 Cells ; HeLa Cells ; Humans ; Membrane Fusion ; Microscopy, Confocal ; Phagosomes/metabolism/virology ; Qa-SNARE Proteins/biosynthesis ; Receptors, sigma/biosynthesis ; SARS-CoV-2 ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/biosynthesis ; Synaptotagmins/biosynthesis ; },
abstract = {The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.},
}
@article {pmid34741732,
year = {2022},
author = {Bharathkumar, N and Sunil, A and Meera, P and Aksah, S and Kannan, M and Saravanan, KM and Anand, T},
title = {CRISPR/Cas-Based Modifications for Therapeutic Applications: A Review.},
journal = {Molecular biotechnology},
volume = {64},
number = {4},
pages = {355-372},
pmid = {34741732},
issn = {1559-0305},
mesh = {*CRISPR-Cas Systems ; DNA/metabolism ; *Gene Editing ; RNA ; RNA, Guide/genetics ; },
abstract = {The CRISPR-Cas genome editing system is an intrinsic property of a bacteria-based immune system. This employs a guide RNA to detect and cleave the PAM-associated target DNA or RNA in subsequent infections, by the invasion of a similar bacteriophage. The discovery of Cas systems has paved the way to overcome the limitations of existing genome editing tools. In this review, we focus on Cas proteins that are available for gene modifications among which Cas9, Cas12a, and Cas13 have been widely used in the areas of medicine, research, and diagnostics. Since CRISPR has been already proven for its potential research applications, the next milestone for CRISPR will be proving its efficacy and safety. In this connection, we systematically review recent advances in exploring multiple variants of Cas proteins and their modifications for therapeutic applications.},
}
@article {pmid34740777,
year = {2021},
author = {Mohsin, M and Li, Y and Zhang, X and Wang, Y and Huang, Z and Yin, G and Zhang, Z},
title = {Development of CRISPR-CAS9 based RNA drugs against Eimeria tenella infection.},
journal = {Genomics},
volume = {113},
number = {6},
pages = {4126-4135},
doi = {10.1016/j.ygeno.2021.10.019},
pmid = {34740777},
issn = {1089-8646},
mesh = {Animals ; CRISPR-Cas Systems ; Chickens/genetics ; *Eimeria tenella/genetics ; RNA ; RNA, Messenger/genetics ; },
abstract = {Parasitic diseases are major trouble in many parts of the world. We consider that if a chemical can break a DNA barcode sequence, it might be used to develop a species-specific anti-parasitic agent. To examine this hypothesis, we constructed sgRNAs that target both the control (5.8S rDNA) and a DNA barcode (ITS) sequence in Eimeria tenella. In vitro experiment showed that Cas9 mRNA combined with sgRNAs could reduce the sporulation percentage of oocysts and the survival rate of sporulated oocysts and sporozoites. Quantitative real-time PCR showed that the DNAs of parasites exposed to Cas9 mRNA and sgRNAs were significantly affected, regardless of whether they were exposed to a combination of two sgRNAs or just a single sgRNA. The DNA sequencing also indicated that the experimental group exposed to two sgRNAs mixed with Cas9-induced deletion of large parts and a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments. In vivo trial, the effect of sgRNA and Cas9 RNA on the pathogenicity of E. tenella in chicken showed less lesion score and oocysts score (P < 0.05) in experimental groups than control groups. The results and concepts presented in this research can lead to discovering novel nucleic acid therapeutic drugs for Eimeriasis and other parasitic infections, which provide insights into the development of species-specific anti-parasitic agents.},
}
@article {pmid34740249,
year = {2021},
author = {Chen, J and Luo, J and Gurav, AS and Chen, Z and Wang, Y and Montell, C},
title = {A DREaMR system to simplify combining mutations with rescue transgenes in Aedes aegypti.},
journal = {Genetics},
volume = {219},
number = {3},
pages = {},
pmid = {34740249},
issn = {1943-2631},
support = {R01 AI165575/AI/NIAID NIH HHS/United States ; R01 DC007864/DC/NIDCD NIH HHS/United States ; R01 EY008117/EY/NEI NIH HHS/United States ; R01 EY010852/EY/NEI NIH HHS/United States ; },
mesh = {Aedes/*genetics ; Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems/genetics ; Drosophila melanogaster/genetics ; Gene Editing/*methods ; Genes, Insect/genetics ; Mosquito Control/*methods ; Mosquito Vectors/*genetics ; Mutation ; },
abstract = {In most experimental animals, it is challenging to combine mutations and rescue transgenes and to use bipartite systems to assess gene expression. To circumvent the difficulties in combining multiple genetic elements, we developed the DREaMR (Drug-on, REporter, Mutant, Rescue) system. Using Drosophila white as the initial model, we demonstrated that introduction of a single insertion by CRISPR/Cas9 created a null mutation, a tagged rescue construct, which could be induced with doxycycline, and which allowed assessment of protein expression. To create a DREaMR in an organism in which combining multiple genetic elements is more problematic than in Drosophila, we tested the mosquito, Aedes aegypti-the insect vector for dengue, yellow fever, Zika, and other viral diseases. We generated a DREaMR allele in the kh gene, which permitted us to induce expression of the rescue construct, and detect expression of Kh. Thus, this system avoids the need to perform genetic crosses to introduce an inducible rescue transgene in a mutant background, or to combine driver and reporter lines to examine expression of the targeted protein. We propose that DREaMR provides a system that can be applied to additional mosquito vectors as well as other organisms in which CRISPR/Cas9 is effective.},
}
@article {pmid34740248,
year = {2021},
author = {Cohen, JD and Cadena Del Castillo, CE and Serra, ND and Kaech, A and Spang, A and Sundaram, MV},
title = {The Caenorhabditis elegans Patched domain protein PTR-4 is required for proper organization of the precuticular apical extracellular matrix.},
journal = {Genetics},
volume = {219},
number = {3},
pages = {},
pmid = {34740248},
issn = {1943-2631},
support = {P40 OD010440/OD/NIH HHS/United States ; R35 GM136315/GM/NIGMS NIH HHS/United States ; R01 GM125959/GM/NIGMS NIH HHS/United States ; T32 AR007465/AR/NIAMS NIH HHS/United States ; R01 GM058540/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics/metabolism ; Caenorhabditis elegans Proteins/metabolism ; Extracellular Matrix/*metabolism/ultrastructure ; Membrane Proteins/genetics/*metabolism ; Microscopy, Electron, Transmission ; Molting/genetics ; Mucins/metabolism ; Mutation ; Protein Domains/genetics ; },
abstract = {The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.},
}
@article {pmid34739981,
year = {2021},
author = {Park, H and Hwang, Y and Kim, J},
title = {Transcriptional activation with Cas9 activator nanocomplexes rescues Alzheimer's disease pathology.},
journal = {Biomaterials},
volume = {279},
number = {},
pages = {121229},
doi = {10.1016/j.biomaterials.2021.121229},
pmid = {34739981},
issn = {1878-5905},
mesh = {*Alzheimer Disease/drug therapy/genetics ; Animals ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Genetic Therapy ; Mice ; Transcriptional Activation ; },
abstract = {CRISPR/Cas9-mediated gene activation is a potential therapeutic strategy that does not induce double-strand break (DSB) DNA damage. However, in vivo gene activation via a Cas9 activator remains a challenge, currently limiting its therapeutic applications. We developed a Cas9 activator nanocomplex that efficiently activates an endogenous gene in the brain in vivo, suggesting its possible application in novel therapeutics. We demonstrated a potential treatment application of the Cas9 activator nanocomplex by activating Adam10 in the mouse brain without introducing insertions and deletions (inDels). Remarkably, in vivo activation of Adam10 with the Cas9 activator nanocomplex improved cognitive deficits in an Alzheimer's disease (AD) mouse model. These results demonstrate the therapeutic potential of Cas9 activator nanocomplexes for a wide range of neurological diseases.},
}
@article {pmid34739827,
year = {2021},
author = {Catchpole, RJ and Terns, MP},
title = {New Type III CRISPR variant and programmable RNA targeting tool: Oh, thank heaven for Cas7-11.},
journal = {Molecular cell},
volume = {81},
number = {21},
pages = {4354-4356},
pmid = {34739827},
issn = {1097-4164},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/genetics ; RNA/genetics ; RNA Cleavage ; },
abstract = {Özcan et al. (2021) and van Beljouw et al. (2021) characterize a novel Type III-E CRISPR-Cas subtype, composed of a single polypeptide with crRNA processing and sequence-specific RNA cleavage activities, that provides a new RNA knockdown tool for mammalian cells with fewer off-target effects than current technologies.},
}
@article {pmid34739076,
year = {2021},
author = {Okada, S and Doi, G and Nakagawa, S and Kusumoto, E and Ito, T},
title = {Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {12},
pages = {},
pmid = {34739076},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome ; Saccharomyces cerevisiae/genetics ; },
abstract = {Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Because each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here, we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.},
}
@article {pmid34736322,
year = {2021},
author = {Gong, S and Zhang, S and Wang, X and Li, J and Pan, W and Li, N and Tang, B},
title = {Strand Displacement Amplification Assisted CRISPR-Cas12a Strategy for Colorimetric Analysis of Viral Nucleic Acid.},
journal = {Analytical chemistry},
volume = {93},
number = {45},
pages = {15216-15223},
doi = {10.1021/acs.analchem.1c04133},
pmid = {34736322},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Colorimetry ; DNA ; DNA, Single-Stranded/genetics ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; },
abstract = {The development of a sensitive, facile, and cost-effective colorimetric method is of great significance for the point-of-care testing of viral nucleic acid. Herein, we reported a strand displacement amplification assisted CRISPR-Cas12a (SDACC) method for the colorimetric analysis of viral nucleic acid. The hepatitis B virus (HBV) DNA was chosen as the target to trigger strand displacement amplification (SDA) and generate abundant single-strand DNA (ssDNA) products. The ssDNA amplicon hybridized with template DNA to activate the trans-cleavage activity of CRISPR-Cas12a, leading to the nonspecific cleavage of ssDNA on GOx-ssDNA-modified magnetic beads and the release of GOx. The released GOx was capable of catalyzing the substrate solution to generate a color change, which could be directly observed by naked eyes. The SDACC strategy could identify a single-base mismatch located in the DNA sequence and achieve a sensitive detection for HBV DNA with the limit of detection as low as 41.8 fM. Notably, the sophisticated primer design for target amplification and complicated detection process could be circumvented. The current approach realizes a simple, low-cost, and sensitive colorimetric detection for viral nucleic acid and holds great promise for the practical application of virus infection diagnosis.},
}
@article {pmid34735288,
year = {2021},
author = {Kandul, NP and Liu, J and Akbari, OS},
title = {Temperature-Inducible Precision-Guided Sterile Insect Technique.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {822-835},
pmid = {34735288},
issn = {2573-1602},
support = {R01 AI151004/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; Female ; *Gene Editing ; Male ; RNA, Guide/genetics ; Temperature ; },
abstract = {Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale.},
}
@article {pmid34735227,
year = {2021},
author = {Avagyan, S and Henninger, JE and Mannherz, WP and Mistry, M and Yoon, J and Yang, S and Weber, MC and Moore, JL and Zon, LI},
title = {Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis.},
journal = {Science (New York, N.Y.)},
volume = {374},
number = {6568},
pages = {768-772},
doi = {10.1126/science.aba9304},
pmid = {34735227},
issn = {1095-9203},
support = {P01 HL131477/HL/NHLBI NIH HHS/United States ; F31 HL126338/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Clonal Hematopoiesis ; Cytokines/genetics/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Frameshift Mutation ; Genes, p53 ; Hematopoietic Stem Cells/*physiology ; *Inflammation/genetics ; Mutation ; Myeloid Cells/*physiology ; Nuclear Receptor Subfamily 4, Group A, Member 1/genetics/metabolism ; RNA-Seq ; Repressor Proteins/genetics ; Selection, Genetic ; Single-Cell Analysis ; Zebrafish/embryology/genetics ; Zebrafish Proteins/genetics ; },
abstract = {Clonal hematopoiesis results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. We developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in clonal hematopoiesis–associated genes such as asxl1 promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1, abrogated the ability of asxl1-mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny.},
}
@article {pmid34735006,
year = {2022},
author = {Takasugi, PR and Wang, S and Truong, KT and Drage, EP and Kanishka, SN and Higbee, MA and Bamidele, N and Ojelabi, O and Sontheimer, EJ and Gagnon, JA},
title = {Orthogonal CRISPR-Cas tools for genome editing, inhibition, and CRISPR recording in zebrafish embryos.},
journal = {Genetics},
volume = {220},
number = {1},
pages = {},
pmid = {34735006},
issn = {1943-2631},
support = {R35 GM142950/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is the most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from S. pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1) are orthogonal systems capable of operating simultaneously in zebrafish. CRISPR systems from Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) were also active in embryos. We implemented multichannel CRISPR recording using three CRISPR systems and show that LbaCas12a may provide superior information density compared with previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. Our results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.},
}
@article {pmid34734710,
year = {2021},
author = {Jiang, YH and Liu, YF and Wang, K and Zhou, JY and Guo, F and Zhao, QW and Mao, XM},
title = {Fine-Tuning Cas9 Activity with a Cognate Inhibitor AcrIIA4 to Improve Genome Editing in Streptomyces.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {2833-2841},
doi = {10.1021/acssynbio.1c00141},
pmid = {34734710},
issn = {2161-5063},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Engineering/methods ; RNA, Guide/genetics ; Streptomyces/*genetics ; },
abstract = {Efficient enabling technology is required for synthetic biology in Streptomyces due to its natural product reservoir. Though the CRISPR-Cas9 system is powerful for genome editing in this genus, the proposed Cas9 toxicity has limited its application. Here on the basis of previous inducible Cas9 expression at the transcriptional and translational levels coupled with atpD overexpression, a Cas9 cognate inhibitor AcrIIA4 was further introduced to fine-tune the Cas9 activity. In both laboratory and industrial Streptomyces species, we showed that, compared to the constitutively expressed Cas9, incorporating AcrIIA4 increased the conjugation efficiency from 700- to 7000-fold before induction, while a comparable 65%-90% editing efficiency was obtained even on multiple loci for simultaneous deletion after Cas9 expression was induced, along with no significant off-targets. Thus, AcrIIA4 could be a modulator to control Cas9 activity to significantly improve genome editing, and this new toolkit would be widely adaptable and fasten genetic engineering in Streptomyces.},
}
@article {pmid34732750,
year = {2021},
author = {Wessel, GM and Wada, Y and Yajima, M and Kiyomoto, M},
title = {Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {21583},
pmid = {34732750},
issn = {2045-2322},
support = {S10 OD023461/OD/NIH HHS/United States ; 1R01GM126043/NH/NIH HHS/United States ; 1R35GM140897/NH/NIH HHS/United States ; },
mesh = {Acrosome/metabolism ; Acrosome Reaction ; Animals ; CRISPR-Cas Systems ; Developmental Biology ; Female ; Fertilization ; Glycoproteins/genetics ; Infertility, Male/*genetics/metabolism ; Male ; Mutation ; Ovum/physiology ; Phenotype ; Receptors, Cell Surface/*metabolism ; Sea Urchins/physiology ; Species Specificity ; Sperm Motility/physiology ; Sperm-Ovum Interactions ; Spermatozoa/metabolism/*physiology/*ultrastructure ; },
abstract = {Cell-cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete-specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species-specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species-specific sperm-egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.},
}
@article {pmid34732717,
year = {2021},
author = {Tálas, A and Simon, DA and Kulcsár, PI and Varga, &#xc9; and Krausz, SL and Welker, E},
title = {BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6353},
pmid = {34732717},
issn = {2041-1723},
mesh = {Adenine/*metabolism ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Cytosine/*metabolism ; DNA ; Deoxyribonuclease I/genetics ; Gene Editing/methods ; *Genetic Engineering ; Genome ; HEK293 Cells ; Humans ; Mutation ; RNA ; },
abstract = {Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.},
}
@article {pmid34731631,
year = {2021},
author = {Lam, KN and Spanogiannopoulos, P and Soto-Perez, P and Alexander, M and Nalley, MJ and Bisanz, JE and Nayak, RR and Weakley, AM and Yu, FB and Turnbaugh, PJ},
title = {Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome.},
journal = {Cell reports},
volume = {37},
number = {5},
pages = {109930},
pmid = {34731631},
issn = {2211-1247},
support = {T32 GM007810/GM/NIGMS NIH HHS/United States ; T32 AI060537/AI/NIAID NIH HHS/United States ; R01 AT011117/AT/NCCIH NIH HHS/United States ; K08 AR073930/AR/NIAMS NIH HHS/United States ; //CIHR/Canada ; P30 DK098722/DK/NIDDK NIH HHS/United States ; R01 HL122593/HL/NHLBI NIH HHS/United States ; F32 AI147456/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bacteriophage M13/*genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Chromosome Deletion ; *Chromosomes, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/*genetics/growth &amp; development ; Feces/microbiology ; Female ; *Gastrointestinal Microbiome ; *Gene Editing ; Gene Expression Regulation, Bacterial ; Mice, Inbred BALB C ; Mice, Transgenic ; Proof of Concept Study ; },
abstract = {Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.},
}
@article {pmid34731163,
year = {2021},
author = {Haswell, JR and Mattioli, K and Gerhardinger, C and Maass, PG and Foster, DJ and Peinado, P and Wang, X and Medina, PP and Rinn, JL and Slack, FJ},
title = {Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency.},
journal = {PloS one},
volume = {16},
number = {11},
pages = {e0252848},
pmid = {34731163},
issn = {1932-6203},
support = {P01 GM099117/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Forkhead Transcription Factors/genetics ; Humans ; RNA Interference ; RNA, Long Noncoding ; },
abstract = {Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.},
}
@article {pmid34730409,
year = {2021},
author = {Ennis, CL and Hernday, AD and Nobile, CJ},
title = {A Markerless CRISPR-Mediated System for Genome Editing in Candida auris Reveals a Conserved Role for Cas5 in the Caspofungin Response.},
journal = {Microbiology spectrum},
volume = {9},
number = {3},
pages = {e0182021},
pmid = {34730409},
issn = {2165-0497},
support = {F31 DE028488/DE/NIDCR NIH HHS/United States ; R15 AI137975/AI/NIAID NIH HHS/United States ; R35 GM124594/GM/NIGMS NIH HHS/United States ; },
mesh = {Antifungal Agents/*pharmacology ; CRISPR-Cas Systems/genetics ; Candida auris/drug effects/*genetics ; Candidiasis/drug therapy ; Caspofungin/*pharmacology ; Drug Resistance, Multiple, Fungal/*genetics ; Gene Deletion ; Gene Editing/*methods ; Genome, Fungal/genetics ; Humans ; Microbial Sensitivity Tests ; Transcription Factors/*genetics ; },
abstract = {Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide. It can cause life-threatening disseminated infections in humans, with mortality rates upwards of 50%. The molecular mechanisms underlying its multidrug resistance and pathogenic properties are largely unknown. Few methods exist for genome editing in C. auris, all of which rely on selectable markers that limit the number of modifications that can be made. Here, we present a markerless CRISPR/Cas9-mediated genome editing system in C. auris. Using this system, we successfully deleted genes of interest and subsequently reconstituted them at their native loci in isolates across all five C. auris clades. This system also enabled us to introduce precision genome edits to create translational fusions and single point mutations. Using Cas5 as a test case for this system, we discovered a conserved role for Cas5 in the caspofungin response between Candida albicans and C. auris. Overall, the development of a system for precise and facile genome editing in C. auris that can allow edits to be made in a high-throughput manner is a major step forward in improving our understanding of this important human fungal pathogen. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris. Here, we present a markerless genome editing system for C. auris that relies on CRISPR/Cas9 technology and works to modify the genomes of all known C. auris clades. Using this system, we discovered a conserved role for Cas5 in the caspofungin response between C. albicans and C. auris. Overall, the development of a system for facile genome editing in C. auris is a major step forward in improving our understanding of this important human fungal pathogen.},
}
@article {pmid34728724,
year = {2021},
author = {Kawaguchi, K and Takei-Hoshi, R and Yoshikawa, I and Nishida, K and Kobayashi, M and Kusano, M and Lu, Y and Ariizumi, T and Ezura, H and Otagaki, S and Matsumoto, S and Shiratake, K},
title = {Functional disruption of cell wall invertase inhibitor by genome editing increases sugar content of tomato fruit without decrease fruit weight.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {21534},
pmid = {34728724},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell Wall/enzymology ; Fruit/genetics/growth &amp; development/*metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/genetics/growth &amp; development/*metabolism ; Plant Proteins/genetics/*metabolism ; Plants, Genetically Modified/genetics/growth &amp; development/metabolism ; Sugars/*metabolism ; beta-Fructofuranosidase/*antagonists &amp; inhibitors/genetics ; },
abstract = {Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wall INVINH in tomato (SlINVINH1) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar 'Suzukoma', while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193-3, 199-2, and 247-2), whose SSC was significantly higher than 'Suzukoma' and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193-3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out of SlINVINH1 using genome editing technology. This study showed that functional disruption of SlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.},
}
@article {pmid34727527,
year = {2021},
author = {Moradi, P and Hasanzadeh, A and Radmanesh, F and Rajai Daryasarei, S and Hosseini, ES and Kiani, J and Shahbazi, A and Nourizadeh, H and Eslami, M and Dorgalaleh, A and Sahlolbei, M and Hamblin, MR and Karimi, M},
title = {Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.},
journal = {Nanotechnology},
volume = {33},
number = {7},
pages = {},
doi = {10.1088/1361-6528/ac357a},
pmid = {34727527},
issn = {1361-6528},
mesh = {Animals ; Arginine/*chemistry ; Brain/metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Editing ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Nanoparticle Drug Delivery System/*chemistry ; Nanoparticles/*chemistry ; PC12 Cells ; Plasmids/chemistry/pharmacokinetics ; Polyelectrolytes/*chemistry ; Rats ; Transfection/*methods ; },
abstract = {An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.},
}
@article {pmid34727007,
year = {2021},
author = {Dong, Y and Ma, K and Cao, Q and Huang, H and Nie, M and Liu, G and Jiang, M and Lu, C and Liu, Y},
title = {CRISPR-dependent endogenous gene regulation is required for virulence in piscine Streptococcus agalactiae.},
journal = {Emerging microbes &amp; infections},
volume = {10},
number = {1},
pages = {2113-2124},
pmid = {34727007},
issn = {2222-1751},
mesh = {Animals ; Bacterial Proteins/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Fish Diseases/*microbiology ; Fishes ; *Gene Expression Regulation, Bacterial ; Mice ; Mice, Inbred C57BL ; Perforin/genetics/metabolism ; Streptococcal Infections/microbiology/*veterinary ; Streptococcus agalactiae/genetics/*pathogenicity/physiology ; Virulence ; },
abstract = {The clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) system is a prokaryotic defence against invading mobile genetic elements, such as bacteriophages or exogenous plasmids. Beyond this, this system has been shown to play an important role in controlling the virulence of some bacterial pathogens. Streptococcus agalactiae strain GD201008-001, a causative agent of septicemia and meningitis in tilapia, contains a single type II CRISPR-Cas system with Cas9 as a signature protein. In this study, we found that the deletion of CRISPR significantly reduced adhesion, invasion, cytotoxicity and haemolysis, and caused severely attenuated virulence in the piscine S. agalactiae strain. RNA-Seq identified 236 endogenous genes regulated by CRISPR, with 159 genes upregulated and 77 genes downregulated. The resulting change in gene transcription by CRISPR was much more pronounced than that by cas9 in this bacterium, indicating CRISPR-mediated endogenous gene regulation was mostly independently of cas9. Subsequent studies showed that CovR/S two-component system was transcriptionally upregulated due to CRISPR deletion, which repressed the expression of the cylE gene coding for a cytolytic toxin, and thus decreased the activity of β-haemolysin/cytolysin. However, upregulation of CovR/S was not the contributor to the attenuation phenotype of ΔCRISPR. Further, we demonstrated that CRISPR is capable of repressing the expression of Toll-like receptor 2 (TLR2)-activating lipoprotein Sag0671 and thus dampens the innate immune response. This study revealed that the CRISPR system of S. agalactiae exhibited extraordinary potential capability in the regulation of endogenous transcripts, which contributes to bacterial innate immune evasion and virulence.},
}
@article {pmid34726841,
year = {2022},
author = {Yang, N and Yan, L and Zheng, Z and Zhang, Y and Zhan, H and Tian, Y and Zhang, T and Li, R and Gong, X and Xu, M and Zhu, M and Wu, J and He, Y and Zhao, Y},
title = {Editing gene families by CRISPR/Cas9: accelerating the isolation of multiple transgene-free null mutant combinations with much reduced labor-intensive analysis.},
journal = {Plant biotechnology journal},
volume = {20},
number = {2},
pages = {241-243},
pmid = {34726841},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Transgenes ; },
}
@article {pmid34726388,
year = {2021},
author = {Mózsik, L and Pohl, C and Meyer, V and Bovenberg, RAL and Nygård, Y and Driessen, AJM},
title = {Modular Synthetic Biology Toolkit for Filamentous Fungi.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {2850-2861},
pmid = {34726388},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular/methods ; Fungi/*genetics ; Genetic Engineering/methods ; Genetic Vectors/genetics ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; Synthetic Biology/*methods ; },
abstract = {Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.},
}
@article {pmid34724929,
year = {2021},
author = {Aoshima, T and Kobayashi, Y and Takagi, H and Iijima, K and Sato, M and Takabayashi, S},
title = {Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {63},
pmid = {34724929},
issn = {1472-6750},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Electroporation ; Female ; *Gene Editing ; Gonads ; Humans ; Mice ; *Nucleic Acids ; Rats ; },
abstract = {BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency.<br><br>RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p&#x2009;<&#x2009;0.05) or crRNA3 (0%; p&#x2009;<&#x2009;0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15&#xa0;&#x3bc;M) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different.<br><br>CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.},
}
@article {pmid34723968,
year = {2021},
author = {Zhang, B and Ding, Z and Li, L and Xie, LK and Fan, YJ and Xu, YZ},
title = {Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila.},
journal = {PLoS genetics},
volume = {17},
number = {11},
pages = {e1009861},
pmid = {34723968},
issn = {1553-7404},
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila/*genetics/immunology ; Drosophila Proteins/*genetics ; *Introns ; *Mutation ; },
abstract = {SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and-H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.},
}
@article {pmid34723963,
year = {2021},
author = {Azpurua, J and El-Karim, EG and Tranquille, M and Dubnau, J},
title = {A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia.},
journal = {PLoS genetics},
volume = {17},
number = {11},
pages = {e1009882},
pmid = {34723963},
issn = {1553-7404},
support = {R01 NS091748/NS/NINDS NIH HHS/United States ; RF1 AG057338/AG/NIA NIH HHS/United States ; },
mesh = {Aging/*metabolism ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; DNA-Binding Proteins/*metabolism ; Humans ; Motor Neurons/*metabolism ; Neuroglia/*metabolism ; Serine-Arginine Splicing Factors/*metabolism ; },
abstract = {Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer's disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export- pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator.},
}
@article {pmid34723510,
year = {2021},
author = {Cliff, ER and Kirkpatrick, RL and Cunningham-Bryant, D and Fernandez, B and Harman, JL and Zalatan, JG},
title = {CRISPR-Cas-Mediated Tethering Recruits the Yeast HMR Mating-Type Locus to the Nuclear Periphery but Fails to Silence Gene Expression.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {2870-2877},
pmid = {34723510},
issn = {2161-5063},
support = {R35 GM124773/GM/NIGMS NIH HHS/United States ; T32 HG000035/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Nucleus/*genetics ; DNA-Binding Proteins/genetics ; Gene Expression/*genetics ; Gene Silencing/*physiology ; Genes, Reporter/genetics ; Genetic Techniques ; Genome, Bacterial/genetics ; Saccharomyces cerevisiae/*genetics ; },
abstract = {To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both of which are well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not detectably silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing effect has an unexpected dependence on the properties of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.},
}
@article {pmid34723245,
year = {2021},
author = {Baddeley, HJE and Isalan, M},
title = {The Application of CRISPR/Cas Systems for Antiviral Therapy.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {745559},
pmid = {34723245},
issn = {2673-3439},
abstract = {As CRISPR/Cas systems have been refined over time, there has been an effort to apply them to real world problems, such as developing sequence-targeted antiviral therapies. Viruses pose a major threat to humans and new tools are urgently needed to combat these rapidly mutating pathogens. Importantly, a variety of CRISPR systems have the potential to directly cleave DNA and RNA viral genomes, in a targeted and easily-adaptable manner, thus preventing or treating infections. This perspective article highlights recent studies using different Cas effectors against various RNA viruses causing acute infections in humans; a latent virus (HIV-1); a chronic virus (hepatitis B); and viruses infecting livestock and animal species of industrial importance. The outlook and remaining challenges are discussed, particularly in the context of tacking newly emerging viruses, such as SARS-CoV-2.},
}
@article {pmid34721373,
year = {2021},
author = {Somma, D and Kok, FO and Kerrigan, D and Wells, CA and Carmody, RJ},
title = {Defining the Role of Nuclear Factor (NF)-κB p105 Subunit in Human Macrophage by Transcriptomic Analysis of NFKB1 Knockout THP1 Cells.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {669906},
pmid = {34721373},
issn = {1664-3224},
support = {MR/M010694/1/MRC_/Medical Research Council/United Kingdom ; BB/M003671/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T007427/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity ; CRISPR-Cas Systems ; Cytokines/genetics/metabolism ; *Gene Expression Profiling ; *Gene Knockout Techniques ; Humans ; Immunity, Cellular ; Inflammation/*genetics/immunology/metabolism ; Macrophage Activation ; Macrophages/immunology/*metabolism ; NF-kappa B p50 Subunit/deficiency/*genetics ; Phenotype ; RNA-Seq ; THP-1 Cells ; Toll-Like Receptors/genetics/metabolism ; *Transcriptome ; },
abstract = {Since its discovery over 30 years ago the NF-ĸB family of transcription factors has gained the status of master regulator of the immune response. Much of what we understand of the role of NF-ĸB in immune development, homeostasis and inflammation comes from studies of mice null for specific NF-ĸB subunit encoding genes. The role of inflammation in diseases that affect a majority of individuals with health problems globally further establishes NF-ĸB as an important pathogenic factor. More recently, genomic sequencing has revealed loss of function mutations in the NFKB1 gene as the most common monogenic cause of common variable immunodeficiencies in Europeans. NFKB1 encodes the p105 subunit of NF-ĸB which is processed to generate the NF-ĸB p50 subunit. NFKB1 is the most highly expressed transcription factor in macrophages, key cellular drivers of inflammation and immunity. Although a key role for NFKB1 in the control of the immune system is apparent from Nfkb1[-/-] mouse studies, we know relatively little of the role of NFKB1 in regulating human macrophage responses. In this study we use the THP1 monocyte cell line and CRISPR/Cas9 gene editing to generate a model of NFKB1[-/-] human macrophages. Transcriptomic analysis reveals that activated NFKB1[-/-] macrophages are more pro-inflammatory than wild type controls and express elevated levels of TNF, IL6, and IL1B, but also have reduced expression of co-stimulatory factors important for the activation of T cells and adaptive immune responses such as CD70, CD83 and CD209. NFKB1[-/-] THP1 macrophages recapitulate key observations in individuals with NFKB1 haploinsufficiency including decreased IL10 expression. These data supporting their utility as an in vitro model for understanding the role of NFKB1 in human monocytes and macrophages and indicate that of loss of function NFKB1 mutations in these cells is an important component in the associated pathology.},
}
@article {pmid34719552,
year = {2021},
author = {Chatterjee, N and Zhang, X},
title = {CRISPR approach in environmental chemical screening focusing on population variability.},
journal = {The Journal of toxicological sciences},
volume = {46},
number = {11},
pages = {499-507},
doi = {10.2131/jts.46.499},
pmid = {34719552},
issn = {1880-3989},
mesh = {*CRISPR-Cas Systems ; Environmental Exposure/statistics &amp; numerical data ; Gene Editing ; *Genome-Wide Association Study ; Phenotype ; },
abstract = {A significant barrier to include population variability in risk assessment is our incomplete understanding of inter-individual variability and the differential susceptibility to environmental exposures induced adverse outcomes. By combining genome editing tools with the population diversity model, this article intended to highlight a potential strategy to identify and characterize the inter-individual variability factors, the determinant gene anchoring to a particular phenotype. The goal could be achieved by integrating the perturbed CRISPR-based unbiased functional genomics screening, genome-wide or a focused subset of genes, in a population-based in vitro model system (such as the lymphoblastoid cell lines, LCL, available from HapMap and 1000 Genomes project). Then data can be translated to genetic variability and individual (or subpopulation) susceptibility by incorporating ethnicity and corresponding genome-wide association studies (GWAS) with functional genomics screening results. This approach can provide complementary data for next-generation risk assessment, in particular, for environmental stressors. The current paper outlined the previous work conducted with a population-based in vitro model system, perturbed CRISPR-based functional toxicogenomic screening of environmental chemicals, and finally, the potential strategies to combine these two platforms with their opportunities and challenges to achieve a mechanistic understanding of population variability.},
}
@article {pmid34719304,
year = {2022},
author = {Zocca, VFB and Corrêa, GG and Lins, MRDCR and de Jesus, VN and Tavares, LF and Amorim, LADS and Kundlatsch, GE and Pedrolli, DB},
title = {The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis.},
journal = {Critical reviews in biotechnology},
volume = {42},
number = {6},
pages = {813-826},
doi = {10.1080/07388551.2021.1983516},
pmid = {34719304},
issn = {1549-7801},
mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {CRISPR has revolutionized the way we engineer genomes. Its simplicity and modularity have enabled the development of a great number of tools to edit genomes and to control gene expression. This powerful technology was first adapted to Bacillus subtilis in 2016 and has been intensely upgraded since then. Many tools have been successfully developed to build a CRISPR toolbox for this Gram-positive model and important industrial chassis. The toolbox includes tools, such as double-strand and single-strand cutting CRISPR for point mutation, gene insertion, and gene deletion up to 38 kb. Moreover, catalytic dead Cas proteins have been used for base editing, as well as for the control of gene expression (CRISPRi and CRISPRa). Many of these tools have been used for multiplex CRISPR with the most successful one targeting up to six loci simultaneously for point mutation. However, tools for efficient multiplex CRISPR for other functionalities are still missing in the toolbox. CRISPR engineering has already resulted in efficient protein and metabolite-producing strains, demonstrating its great potential. In this review, we cover all the important additions made to the B. subtilis CRISPR toolbox since 2016, and strain developments fomented by the technology.},
}
@article {pmid34719216,
year = {2021},
author = {Dubey, A and Kumar, A and Malla, MA and Chowdhary, K and Singh, G and Ravikanth, G and Harish, and Sharma, S and Saati-Santamaria, Z and Menéndez, E and Dames, JF},
title = {Approaches for the amelioration of adverse effects of drought stress on crop plants.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {26},
number = {10},
pages = {928-947},
doi = {10.52586/4998},
pmid = {34719216},
issn = {2768-6698},
mesh = {*CRISPR-Cas Systems ; *Droughts ; Gene Editing ; Plants ; Stress, Physiological ; },
abstract = {Climate change, water scarcity, population growth, and food shortage are some of the threatening challenges being faced in today's world. Among different types of stresses, drought stress presents a persistent challenge for global food production, however, its harshness and intensity are supposed to expand in the imminent future. The most striking effects of drought stress on plants are stunted growth, severe damage to photosynthetic apparatus, reduction in photosynthesis, reduction in seed germination, and nutrient uptake. To deal with the destructive effect of drought stress on plants, it is necessary to consider its effects, mechanisms of action, the agronomic and genetic basis for sustainable management. Therefore, there is an urgent need for sustainable solutions to cope up with the negative impact of drought stress. This review focuses on the detrimental effects of drought stress on plants' morphological, physiological, and biochemical characteristics and recommends suitable drought management techniques to reduce the severity of drought stress. We summarize the effect of drought stress on physiological and biochemical parameters (such as germination, photosynthesis, biomass, water status, and nutrient uptake) and yield. Overall, in this article, we have reviewed the role of different phytohormones, osmolytes, exogenous compounds, proteins, plant growth-promoting microbes (PGPM), omics approaches, and genome editing technologies like clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) in alleviating drought effects in plants. We also proposed that developing drought-tolerant plant varieties requires the combined use of biotechnological and agronomic approaches and cutting-edge genome editing (GE) tools.},
}
@article {pmid34719215,
year = {2021},
author = {Huang, S and Yan, Y and Su, F and Huang, X and Xia, D and Jiang, X and Dong, Y and Lv, P and Chen, F and Lv, Y},
title = {Research progress in gene editing technology.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {26},
number = {10},
pages = {916-927},
doi = {10.52586/4997},
pmid = {34719215},
issn = {2768-6698},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Technology ; },
abstract = {As a tool for modifying the genome, gene editing technology has developed rapidly in recent years, especially in the past two years. With the emergence of new gene editing technologies, such as transposon editing tools, numerous advancements have been made including precise editing of the genome, double base editing, and pilot editing. This report focuses on the development of gene editing tools in recent years, elaborates the progress made in classic editing tools, base editor and other new editing tools, and provides insights into challenges and opportunities.},
}
@article {pmid34718995,
year = {2022},
author = {Dhanjal, JK and Vora, D and Radhakrishnan, N and Sundar, D},
title = {Computational Approaches for Designing Highly Specific and Efficient sgRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2349},
number = {},
pages = {147-166},
pmid = {34718995},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Computational Biology ; Endonucleases ; RNA, Guide ; },
abstract = {The easily programmable CRISPR/Cas9 system has found applications in biomedical research as well as microbial and crop applications, due to its ability to create site-specific edits. This powerful and flexible system has also been modified to enable inducible gene regulation, epigenome modifications and high-throughput screens. Designing efficient and specific guides for the nuclease is a key step and also a major challenge in effective application. This chapter describes rules for sgRNA design and important features to consider while touching upon bioinformatics advances in predicting efficient guides. Computational tools that suggest improved guides, depending on application, or predict off-targets have also been mentioned and compared.},
}
@article {pmid34718481,
year = {2021},
author = {Manning, BJ and Khan, WA and Peña, JM and Fiore, ES and Boisvert, H and Tudino, MC and Barney, RE and Wilson, MK and Singh, S and Mowatt, JA and Thompson, HJ and Tsongalis, GJ and Blake, WJ},
title = {High-Throughput CRISPR-Cas13 SARS-CoV-2 Test.},
journal = {Clinical chemistry},
volume = {68},
number = {1},
pages = {172-180},
doi = {10.1093/clinchem/hvab238},
pmid = {34718481},
issn = {1530-8561},
mesh = {*COVID-19/diagnosis ; COVID-19 Testing/*methods ; *CRISPR-Cas Systems ; High-Throughput Screening Assays ; Humans ; RNA, Viral/isolation &amp; purification ; SARS-CoV-2/isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: The ability to control the spread of COVID-19 continues to be hampered by a lack of rapid, scalable, and easily deployable diagnostic solutions.<br><br>METHODS: We developed a diagnostic method based on CRISPR (clustered regularly interspaced short palindromic repeats) that can deliver sensitive, specific, and high-throughput detection of Sudden Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). The assay utilizes SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the qualitative detection of SARS-CoV-2 RNA and may be performed directly on a swab or saliva sample without nucleic acid extraction. The assay uses a 384-well format and provides results in <1&#x2009;hour.<br><br>RESULTS: Assay performance was evaluated with 105 (55 negative, 50 positive) remnant SARS-CoV-2 specimens previously tested using Food and Drug Administration emergency use authorized assays and retested with a modified version of the Centers for Disease Control and Prevention (CDC) quantitative PCR with reverse transcription (RT-qPCR) assay. When combined with magnetic bead-based extraction, the high-throughput SHERLOCK SARS-CoV-2 assay was 100% concordant (n&#x2009;=&#x2009;60) with the CDC RT-qPCR. When used with direct sample addition the high-throughput assay was also 100% concordant with the CDC RT-qPCR direct method (n&#x2009;=&#x2009;45). With direct saliva sample addition, the negative and positive percentage agreements were 100% (15/15, 95% CI: 81.8-100%) and 88% (15/17, 95% CI: 63.6-98.5%), respectively, compared with results from a collaborating clinical laboratory.<br><br>CONCLUSIONS: This high-throughput assay identifies SARS-CoV-2 from patient samples with or without nucleic acid extraction with high concordance to RT-qPCR methods. This test enables high complexity laboratories to rapidly increase their testing capacities with simple equipment.},
}
@article {pmid34718347,
year = {2022},
author = {Yu, C and Luo, D and Yu, J and Zhang, M and Zheng, X and Xu, G and Wang, J and Wang, H and Xu, Y and Jiang, K and Xu, J and Ma, X and Jing, J and Shi, H},
title = {Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer.},
journal = {Oncogene},
volume = {41},
number = {2},
pages = {191-203},
pmid = {34718347},
issn = {1476-5594},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms/*genetics/pathology ; Early Detection of Cancer ; Genome-Wide Association Study/*methods ; Humans ; Mice ; Signal Transduction ; },
abstract = {Targeting the KRAS pathway is a promising but challenging approach for colorectal cancer therapy. Despite showing potent efficacy in BRAF-mutated melanoma, MEK inhibitors appeared to be tolerated by colorectal cancer cells due to their intrinsic compensatory signaling. Here, we performed genome-wide CRISPR/Cas9 screening in the presence of MEK inhibitor to identify genes that are synthetically lethal with MEK inhibition in CRC models harboring KRAS mutations. Several genes were identified as potential functional drivers, which were significantly enriched in the GRB7-mediated RTK pathway. Loss-of-function and gain-of-function assays validated that GRB7 potently rendered CRC cells primary resistance to MEK inhibitors through the RTK pathway. Mass spectrum analysis of GRB7 immunoprecipitates revealed that PLK1 was the predominant interacting kinase of GRB7. Inhibition of PLK1 suppressed downstream signaling of RTK, including FAK, STAT3, AKT, and 4EBP1. The combination of PLK1 and MEK inhibitors synergistically inhibited CRC cell proliferation and induced apoptosis in vitro and in vivo. In conclusion, we identified GRB7-PLK1 as a pivotal axis mediating RTKs, resulting in MEK inhibitor tolerance. PLK1 is therefore a promising target for synergizing MEK inhibitors in the clinical treatment of CRC patients harboring KRAS mutations.},
}
@article {pmid34717656,
year = {2021},
author = {Dronina, J and Samukaite-Bubniene, U and Ramanavicius, A},
title = {Advances and insights in the diagnosis of viral infections.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {348},
pmid = {34717656},
issn = {1477-3155},
mesh = {Biosensing Techniques ; COVID-19/diagnosis/epidemiology ; *Clinical Laboratory Techniques ; Humans ; Nucleic Acid Amplification Techniques ; Pandemics ; SARS-CoV-2/genetics/immunology/isolation &amp; purification ; Viral Proteins/genetics/immunology ; Virus Diseases/*diagnosis/epidemiology ; Viruses/classification/genetics/immunology/*isolation &amp; purification ; },
abstract = {Viral infections are the most common among diseases that globally require around 60 percent of medical care. However, in the heat of the pandemic, there was a lack of medical equipment and inpatient facilities to provide all patients with viral infections. The detection of viral infections is possible in three general ways such as (i) direct virus detection, which is performed immediately 1-3 days after the infection, (ii) determination of antibodies against some virus proteins mainly observed during/after virus incubation period, (iii) detection of virus-induced disease when specific tissue changes in the organism. This review surveys some global pandemics from 1889 to 2020, virus types, which induced these pandemics, and symptoms of some viral diseases. Non-analytical methods such as radiology and microscopy also are overviewed. This review overlooks molecular analysis methods such as nucleic acid amplification, antibody-antigen complex determination, CRISPR-Cas system-based viral genome determination methods. Methods widely used in the certificated diagnostic laboratory for SARS-CoV-2, Influenza A, B, C, HIV, and other viruses during a viral pandemic are outlined. A comprehensive overview of molecular analytical methods has shown that the assay's sensitivity, accuracy, and suitability for virus detection depends on the choice of the number of regions in the viral open reading frame (ORF) genome sequence and the validity of the selected analytical method.},
}
@article {pmid34715203,
year = {2021},
author = {Wang, Z and Zhong, C},
title = {Cas12c-DETECTOR: A specific and sensitive Cas12c-based DNA detection platform.},
journal = {International journal of biological macromolecules},
volume = {193},
number = {Pt A},
pages = {441-449},
doi = {10.1016/j.ijbiomac.2021.10.167},
pmid = {34715203},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/*genetics ; DNA, Bacterial/*isolation &amp; purification ; DNA, Viral/*isolation &amp; purification ; Humans ; Plants/*microbiology ; },
abstract = {The robust and precise nucleic acid detection platform enormously influences the clinical diagnosis of human and plant pathogens, drastically affecting disease pandemic control. CRISPR-based nucleic acid detection tools have been successfully applied for rapid and sensitive nucleic acid detection. However, the T-rich protospacer adjacent motif (PAM), specificity, and sensitivity of the CRISPR-based nucleic acid detection tools limited its wide application. We first developed a new Cas12c-based nucleic acid detection platform (Cas12c-DETECTOR), recognizing a 5'-TG PAM and showing high sensitivity and specificity on examined targets. Our results indicate that Cas12c-DETECTOR coupling with the optimized single-guide RNA (sgRNA) can be applied to specifically identity single nucleotide polymorphism (SNP). Moreover, combined with pre-amplification and lateral flow strips or the visual fluorescence detection method, Cas12c-DETECTOR can be used to diagnose human and plant pathogens in practice. Therefore, our findings illustrated that Cas12c-DETECTOR is a robust, sensitive, precise, and practiced nucleic acid detection platform.},
}
@article {pmid34714068,
year = {2021},
author = {Yuan, M and Ding, R and Chen, S and Duan, G},
title = {Advances in Field Detection Based on CRISPR/Cas System.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {2824-2832},
doi = {10.1021/acssynbio.1c00401},
pmid = {34714068},
issn = {2161-5063},
mesh = {*COVID-19/diagnosis/genetics ; *COVID-19 Nucleic Acid Testing ; *CRISPR-Cas Systems ; Humans ; *Point-of-Care Systems ; SARS-CoV-2/*genetics ; },
abstract = {Rapid and accurate diagnostic methods are essential to interrupt outbreaks of infectious diseases such as COVID-19. However, the most commonly used nucleic acid detection method, qPCR or RT-qPCR, takes several hours to complete and requires highly sophisticated equipment. Recently, an emerging nucleic acid detection method based on the CRISPR/Cas system has reduced the reliance on qPCR. It has several important features that make it suitable for on-site POCT (point-of-care testing), including short detection cycles, low cost, high sensitivity, and the ability to be combined with different readout methods. This review briefly introduces the steps of CRISPR/Cas detection and then summarizes the current advances of CRISPR/Cas-based POCT from four steps: nucleic acid extraction, target amplification, CRISPR/Cas-based signal generation, and signal output. Finally, we discuss the advantages and challenges of CRISPR-based POCT and describe the future research perspectives for CRISPR.},
}
@article {pmid34713271,
year = {2021},
author = {Harwood, W and Chen, QJ and Zhang, F and Yang, B},
title = {Editorial: New Genome Editing Tools and Resources: Enabling Gene Discovery and Functional Genomics.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {771622},
pmid = {34713271},
issn = {2673-3439},
support = {BB/L014130/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
}
@article {pmid34713270,
year = {2021},
author = {Lederer, CW and Genovese, P and Miccio, A and Philipsen, S},
title = {Editorial: Mutation-Specific Gene Editing for Blood Disorders.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {761771},
pmid = {34713270},
issn = {2673-3439},
}
@article {pmid34713263,
year = {2021},
author = {Yue, JJ and Yuan, JL and Wu, FH and Yuan, YH and Cheng, QW and Hsu, CT and Lin, CS},
title = {Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {717017},
pmid = {34713263},
issn = {2673-3439},
abstract = {In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.},
}
@article {pmid34713262,
year = {2021},
author = {Liu, S and Striebel, J and Pasquini, G and Ng, AHM and Khoshakhlagh, P and Church, GM and Busskamp, V},
title = {Neuronal Cell-type Engineering by Transcriptional Activation.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {715697},
pmid = {34713262},
issn = {2673-3439},
abstract = {Gene activation with the CRISPR-Cas system has great implications in studying gene function, controlling cellular behavior, and modulating disease progression. In this review, we survey recent studies on targeted gene activation and multiplexed screening for inducing neuronal differentiation using CRISPR-Cas transcriptional activation (CRISPRa) and open reading frame (ORF) expression. Critical technical parameters of CRISPRa and ORF-based strategies for neuronal programming are presented and discussed. In addition, recent progress on in vivo applications of CRISPRa to the nervous system are highlighted. Overall, CRISPRa represents a valuable addition to the experimental toolbox for neuronal cell-type programming.},
}
@article {pmid34713259,
year = {2021},
author = {Dong, S and Qin, YL and Vakulskas, CA and Collingwood, MA and Marand, M and Rigoulot, S and Zhu, L and Jiang, Y and Gu, W and Fan, C and Mangum, A and Chen, Z and Yarnall, M and Zhong, H and Elumalai, S and Shi, L and Que, Q},
title = {Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {670529},
pmid = {34713259},
issn = {2673-3439},
abstract = {Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.},
}
@article {pmid34713250,
year = {2021},
author = {Ferrari, S and Vavassori, V and Canarutto, D and Jacob, A and Castiello, MC and Javed, AO and Genovese, P},
title = {Gene Editing of Hematopoietic Stem Cells: Hopes and Hurdles Toward Clinical Translation.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {618378},
pmid = {34713250},
issn = {2673-3439},
abstract = {In the field of hematology, gene therapies based on integrating vectors have reached outstanding results for a number of human diseases. With the advent of novel programmable nucleases, such as CRISPR/Cas9, it has been possible to expand the applications of gene therapy beyond semi-random gene addition to site-specific modification of the genome, holding the promise for safer genetic manipulation. Here we review the state of the art of ex vivo gene editing with programmable nucleases in human hematopoietic stem and progenitor cells (HSPCs). We highlight the potential advantages and the current challenges toward safe and effective clinical translation of gene editing for the treatment of hematological diseases.},
}
@article {pmid34713247,
year = {2021},
author = {Wang, L and Kaya, HB and Zhang, N and Rai, R and Willmann, MR and Carpenter, SCD and Read, AC and Martin, F and Fei, Z and Leach, JE and Martin, GB and Bogdanove, AJ},
title = {Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants.},
journal = {Frontiers in genome editing},
volume = {3},
number = {},
pages = {617553},
pmid = {34713247},
issn = {2673-3439},
abstract = {Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.},
}
@article {pmid34713236,
year = {2020},
author = {Blattner, G and Cavazza, A and Thrasher, AJ and Turchiano, G},
title = {Gene Editing and Genotoxicity: Targeting the Off-Targets.},
journal = {Frontiers in genome editing},
volume = {2},
number = {},
pages = {613252},
pmid = {34713236},
issn = {2673-3439},
abstract = {Gene editing technologies show great promise for application to human disease as a result of rapid developments in targeting tools notably based on ZFN, TALEN, and CRISPR-Cas systems. Precise modification of a DNA sequence is now possible in mature human somatic cells including stem and progenitor cells with increasing degrees of efficiency. At the same time new technologies are required to evaluate their safety and genotoxicity before widespread clinical application can be confidently implemented. A number of methodologies have now been developed in an attempt to predict expected and unexpected modifications occurring during gene editing. This review surveys the techniques currently available as state of the art, highlighting benefits and limitations, and discusses approaches that may achieve sufficient accuracy and predictability for application in clinical settings.},
}
@article {pmid34713235,
year = {2020},
author = {Vu, TV and Das, S and Tran, MT and Hong, JC and Kim, JY},
title = {Precision Genome Engineering for the Breeding of Tomatoes: Recent Progress and Future Perspectives.},
journal = {Frontiers in genome editing},
volume = {2},
number = {},
pages = {612137},
pmid = {34713235},
issn = {2673-3439},
abstract = {Currently, poor biodiversity has raised challenges in the breeding and cultivation of tomatoes, which originated from the Andean region of Central America, under global climate change. Meanwhile, the wild relatives of cultivated tomatoes possess a rich source of genetic diversity but have not been extensively used for the genetic improvement of cultivated tomatoes due to the possible linkage drag of unwanted traits from their genetic backgrounds. With the advent of new plant breeding techniques (NPBTs), especially CRISPR/Cas-based genome engineering tools, the high-precision molecular breeding of tomato has become possible. Further, accelerated introgression or de novo domestication of novel and elite traits from/to the wild tomato relatives to/from the cultivated tomatoes, respectively, has emerged and has been enhanced with high-precision tools. In this review, we summarize recent progress in tomato precision genome editing and its applications for breeding, with a special focus on CRISPR/Cas-based approaches. Future insights and precision tomato breeding scenarios in the CRISPR/Cas era are also discussed.},
}
@article {pmid34713221,
year = {2020},
author = {Félix, AJ and Solé, A and Noé, V and Ciudad, CJ},
title = {Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology.},
journal = {Frontiers in genome editing},
volume = {2},
number = {},
pages = {583577},
pmid = {34713221},
issn = {2673-3439},
abstract = {Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.},
}
@article {pmid34711982,
year = {2022},
author = {Ploessl, D and Zhao, Y and Cao, M and Ghosh, S and Lopez, C and Sayadi, M and Chudalayandi, S and Severin, A and Huang, L and Gustafson, M and Shao, Z},
title = {A repackaged CRISPR platform increases homology-directed repair for yeast engineering.},
journal = {Nature chemical biology},
volume = {18},
number = {1},
pages = {38-46},
pmid = {34711982},
issn = {1552-4469},
mesh = {*CRISPR-Cas Systems ; DNA End-Joining Repair ; Fermentation ; Genes, Fungal ; *Genetic Engineering ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.},
}
@article {pmid34711981,
year = {2022},
author = {Zhuang, Y and Liu, J and Wu, H and Zhu, Q and Yan, Y and Meng, H and Chen, PR and Yi, C},
title = {Increasing the efficiency and precision of prime editing with guide RNA pairs.},
journal = {Nature chemical biology},
volume = {18},
number = {1},
pages = {29-37},
pmid = {34711981},
issn = {1552-4469},
mesh = {CRISPR-Cas Systems ; HEK293 Cells ; Humans ; *RNA Editing ; RNA, Guide/*genetics ; },
abstract = {The recently reported prime editor (PE) can produce all types of base substitution, insertion and deletion, greatly expanding the scope of genome editing. However, improving the editing efficiency and precision of PE represents a major challenge. Here, we report an approach termed the homologous 3' extension mediated prime editor (HOPE). HOPE uses paired prime editing guide RNAs (pegRNAs) encoding the same edits in both sense and antisense DNA strands to achieve high editing efficiency in human embryonic kidney 293T cells as well as mismatch repair-deficient human colorectal carcinoma 116 cells. In addition, we found that HOPE shows greatly improved product purity compared to the original PE3 system. We envision that this enhanced tool could broaden both fundamental research and therapeutic applications of prime editing.},
}
@article {pmid34711951,
year = {2021},
author = {de Almeida, M and Hinterndorfer, M and Brunner, H and Grishkovskaya, I and Singh, K and Schleiffer, A and Jude, J and Deswal, S and Kalis, R and Vunjak, M and Lendl, T and Imre, R and Roitinger, E and Neumann, T and Kandolf, S and Schutzbier, M and Mechtler, K and Versteeg, GA and Haselbach, D and Zuber, J},
title = {AKIRIN2 controls the nuclear import of proteasomes in vertebrates.},
journal = {Nature},
volume = {599},
number = {7885},
pages = {491-496},
pmid = {34711951},
issn = {1476-4687},
support = {/ERC_/European Research Council/International ; },
mesh = {Active Transport, Cell Nucleus ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; DNA-Binding Proteins/*metabolism ; Female ; Genes, myc ; Humans ; Male ; Mitosis ; Nuclear Proteins/*metabolism ; Proteasome Endopeptidase Complex/chemistry/*metabolism ; Protein Binding ; Proteolysis ; Transcription Factors/*metabolism ; },
abstract = {Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth[1-3]. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.},
}
@article {pmid34711342,
year = {2021},
author = {Brandts, J and Ray, KK},
title = {Familial Hypercholesterolemia: JACC Focus Seminar 4/4.},
journal = {Journal of the American College of Cardiology},
volume = {78},
number = {18},
pages = {1831-1843},
doi = {10.1016/j.jacc.2021.09.004},
pmid = {34711342},
issn = {1558-3597},
mesh = {*Cardiovascular Diseases/etiology/prevention &amp; control ; Cholesterol, LDL/blood ; Drug Development ; Early Diagnosis ; Humans ; *Hyperlipoproteinemia Type II/genetics/metabolism/therapy ; *Lipid Regulating Agents/classification/pharmacology ; Therapies, Investigational ; },
abstract = {Detecting familial hypercholesterolemia (FH) early and "normalizing" low-density lipoprotein (LDL) cholesterol values are the 2 pillars for effective cardiovascular disease prevention in FH. Combining lipid-lowering therapies targeting synergistic/complementary metabolic pathways makes this feasible, even among severe phenotypes. For LDL receptor-dependent treatments, PCSK9 remains the main target for adjunctive therapy to statins and ezetimibe through a variety of approaches. These include protein inhibition (adnectins), inhibition of translation at mRNA level (antisense oligonucleotides or small interfering RNA), and creation of loss-of-function mutations through base-pair editing. For patients with little LDL receptor function, LDL receptor-independent treatment targeting ANGPTL3 through monoclonal therapies are now available, or in the future, antisense/small interfering RNA-based approaches offer alternative approaches. Finally, first-in-human studies are ongoing, testing adenovirus-mediated gene therapy transducing healthy LDLR DNA in patients with HoFH. Further development of the CRISPR cas technology, which has shown promising results in vivo on introducing PCSK9 loss-of-function mutations, will move a single-dose, curative treatment for FH closer.},
}
@article {pmid34711331,
year = {2021},
author = {Xie, S and Ji, Z and Suo, T and Li, B and Zhang, X},
title = {Advancing sensing technology with CRISPR: From the detection of nucleic acids to a broad range of analytes - A review.},
journal = {Analytica chimica acta},
volume = {1185},
number = {},
pages = {338848},
doi = {10.1016/j.aca.2021.338848},
pmid = {34711331},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Nucleic Acids ; Technology ; },
abstract = {The CRISPR/Cas technology, derived from an adaptive immune system in bacteria, has been awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Increasing reports reveal that CRISPR/Cas technology has a wide scope of applications and it could be incorporated into biosensors for detecting critical analytes. CRISPR-powered biosensors have attracted significant research interest due to their advantages including high accuracy, good specificity, rapid response, and superior integrity. Now the CRISPR technology is not only admirable in nucleic acid monitoring, but also promising for other kinds of biomarkers' detection, including metal ions, small molecules, peptides, and proteins. Therefore, it is of great worth to explore the prospect, and summarize the strategies in applying CRISPR technology for the recognition of a broad range of targets. In this review, we summarized the strategies of CRISPR biosensing for non-nucleic-acid analytes, the latest development of nucleic acid detection, and proposed the challenges and outlook of CRISPR-powered biosensors.},
}
@article {pmid34711321,
year = {2021},
author = {Wang, SY and Du, YC and Wang, DX and Ma, JY and Tang, AN and Kong, DM},
title = {Signal amplification and output of CRISPR/Cas-based biosensing systems: A review.},
journal = {Analytica chimica acta},
volume = {1185},
number = {},
pages = {338882},
doi = {10.1016/j.aca.2021.338882},
pmid = {34711321},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Nucleic Acids ; Sensitivity and Specificity ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) proteins are powerful gene-editing tools because of their ability to accurately recognize and manipulate nucleic acids. Besides gene-editing function, they also show great promise in biosensing applications due to the superiority of easy design and precise targeting. To improve the performance of CRISPR/Cas-based biosensing systems, various nucleic acid-based signal amplification techniques are elaborately incorporated. The incorporation of these amplification techniques not only greatly increases the detection sensitivity and specificity, but also extends the detectable target range, as well as makes the use of various signal output modes possible. Therefore, summarizing the use of signal amplification techniques in sensing systems and elucidating their roles in improving sensing performance are very necessary for the development of more superior CRISPR/Cas-based biosensors for various applications. In this review, CRISPR/Cas-based biosensors are summarized from two aspects: the incorporation of signal amplification techniques in three kinds of CRISPR/Cas-based biosensing systems (Cas9, Cas12 and Cas13-based ones) and the signal output modes used by these biosensors. The challenges and prospects for the future development of CRISPR/Cas-based biosensors are also discussed.},
}
@article {pmid34710352,
year = {2021},
author = {Urnov, FD},
title = {Imagine CRISPR cures.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3103-3106},
pmid = {34710352},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; },
}
@article {pmid34710162,
year = {2021},
author = {Wigger, M and Tröder, SE and Zevnik, B},
title = {A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm.},
journal = {PloS one},
volume = {16},
number = {10},
pages = {e0259202},
pmid = {34710162},
issn = {1932-6203},
mesh = {Animals ; Costs and Cost Analysis ; Cryopreservation/economics/*methods/veterinary ; Female ; Fertilization in Vitro/methods/veterinary ; Male ; Mice ; Mice, Inbred C57BL ; Semen Preservation/economics/*methods/veterinary ; },
abstract = {The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.},
}
@article {pmid34709615,
year = {2022},
author = {Zhang, JY and Niu, TC and Lin, GM and Zhang, CC},
title = {A CRISPR-Based Method for Constructing Conditional Mutations of Essential Genes in Cyanobacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {143-157},
pmid = {34709615},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; *Cyanobacteria/genetics ; *Genes, Essential ; Mutation ; Nitrogen Fixation ; Photosynthesis ; },
abstract = {Cyanobacteria, a group of diverse bacteria capable of oxygenic photosynthesis, are excellent models for investigating many important cellular processes, such as photosynthesis, nitrogen fixation, and prokaryotic cell differentiation. They also have great potential to become the next-generation cell factories for sustainable biosynthesis of valuable products. However, genetic manipulation in cyanobacteria is not as convenient as in other model bacteria. Particularly, handling essential genes in cyanobacteria has been difficult due to the lack of appropriate tools, limiting our understanding of many important cellular functions encoded by them. We recently develop a CRISPR-based method for constructing the conditional mutants of cyanobacterial essential genes by engineering the ribosome binding site to a theophylline-responsive riboswitch. Here, we provide the details of this method. The principle of this method could be used to construct conditional mutants in a wide range of bacterial species.},
}
@article {pmid34709614,
year = {2022},
author = {Liao, X and Xing, XH and Zhang, C},
title = {New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {123-141},
pmid = {34709614},
issn = {1940-6029},
mesh = {Bacteria ; *CRISPR-Cas Systems/genetics ; Genome ; Genome, Bacterial ; Genomics ; RNA, Guide/genetics ; },
abstract = {High-throughput genetic screens based on CRISPR/Cas9 technology are powerful tools to genome-wide identify gene function and genotype-phenotype association. Here, we describe a detailed protocol for conducting and evaluating pooled CRISPR screens interfering with gene expression in Escherichia coli. We provide step-by-step instructions for guide RNA library design and construction, genome-scale screening and next-generation sequencing data processing. This tool outperforms transposon sequencing (Tn-seq) with similar library sizes and short gene length. The workflow can be used in follow-up studies implemented in other bacteria systems.},
}
@article {pmid34709613,
year = {2022},
author = {Nishimura, K and Fukagawa, T},
title = {A Simple Method that Combines CRISPR and AID to Quickly Generate Conditional Knockouts for Essential Genes in Various Vertebrate Cell Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {109-122},
pmid = {34709613},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Gene Knockout Techniques ; *Genes, Essential ; Indoleacetic Acids ; Vertebrates ; },
abstract = {Cells with a loss-of-function mutation in a gene (knockout cells) are powerful tools for characterizing the function of such gene product. However, for essential genes, conditional knockout cell lines must be generated. The auxin-inducible degron (AID) technique enables us to conditionally and rapidly deplete a target protein from various eukaryotic cell lines. A combination of CRISPR-/Cas9-based gene editing and AID technique allows us to generate AID-based conditional knockout cell lines. Using these two techniques, we recently proposed a simple and quick way to generate conditional knockout cells for essential genes. In this chapter, we introduce the reader to the experimental procedures to generate these AID-based conditional knockout cell lines.},
}
@article {pmid34709612,
year = {2022},
author = {DeHart, L and Yockey, OP and Bakke, J},
title = {Identification of Essential Genes Using Sequential CRISPR and siRNA Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {89-107},
pmid = {34709612},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genes, Essential ; RNA, Small Interfering/genetics ; },
abstract = {Genome-wide CRISPR and siRNA screening methodologies are powerful tools that are aptly suited to the discovery of essential genes. In this chapter, we outline our methods to conduct sequential CRISPR and siRNA screens to quickly and efficiently identify essential genes within a collection of cell lines. The utilization of both screening methodologies provides a pipeline that minimizes costs and time while enabling the robust detection of candidate genes.},
}
@article {pmid34709611,
year = {2022},
author = {Wensing, L and Shapiro, RS},
title = {Design and Generation of a CRISPR Interference System for Genetic Repression and Essential Gene Analysis in the Fungal Pathogen Candida albicans.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {69-88},
pmid = {34709611},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Candida albicans/genetics ; Gene Expression ; Genes, Essential ; RNA, Guide/genetics ; },
abstract = {Studying life-threatening fungal pathogens such as Candida albicans is of critical importance, yet progress can be hindered by challenges associated with manipulating these pathogens genetically. CRISPR-based technologies have significantly improved our ability to manipulate the genomes of countless organisms, including fungal pathogens such as C. albicans. CRISPR interference (CRISPRi) is a modified variation of CRISPR technology that enables the targeted genetic repression of specific genes of interest and can be used as a technique for studying essential genes. We recently developed tools to enable CRISPRi in C. albicans and the repression of essential genes in this fungus. Here, we describe a protocol for CRISPRi in C. albicans, including the design of the single-guide RNAs (sgRNAs) for targeting essential genes, the high-efficiency cloning of sgRNAs into C. albicans-optimized CRISPRi plasmids, transformation into fungal strains, and testing to monitor the repression capabilities of these constructs. Together, this protocol will illuminate efficient strategies for targeted genetic repression of essential genes in C. albicans using a novel CRISPRi platform.},
}
@article {pmid34709610,
year = {2022},
author = {Kwanten, B and Neggers, JE and Daelemans, D},
title = {Target Identification of Small Molecules Using Large-Scale CRISPR-Cas Mutagenesis Scanning of Essential Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {43-67},
pmid = {34709610},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Genes, Essential ; Mutagenesis ; RNA, Guide/genetics ; },
abstract = {Target deconvolution of new bioactive agents identified from phenotypic screens remains a challenging task. The discovery of mutations that confer resistance to such agents is regarded as the gold standard proof of target identification. Here, we describe a method that exploits the error-prone repair of CRISPR-induced DNA double-strand breaks to enhance mutagenesis and increase the incidence of drug resistance mutations in essential genes. As each DNA double-strand break is introduced at a targeted genomic site predefined by the presence of a protospacer adjacent motif (PAM) and a particular CRISPR single guide RNA (sgRNA), the genetic location of drug resistance mutations can be easily uncovered through targeted sequencing of CRISPR sgRNAs. Moreover, the method allows for the identification of not only the drug target gene, but also the drug-binding domain within the target gene.},
}
@article {pmid34709609,
year = {2022},
author = {Huggler, KS and Rossiter, NJ and Flickinger, KM and Cantor, JR},
title = {CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {29-42},
pmid = {34709609},
issn = {1940-6029},
support = {K22 CA225864/CA/NCI NIH HHS/United States ; T32 HG002760/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; DNA ; *Genes, Essential ; High-Throughput Nucleotide Sequencing ; Humans ; RNA, Guide/genetics ; },
abstract = {Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells. However, most such screens have been performed in vitro with little consideration into how medium composition might affect gene essentiality. This protocol describes a method to use CRISPR/Cas9-based loss-of-function screens to ask how gene essentiality in human cell lines varies with medium composition. First, a single-guide RNA (sgRNA) library is packaged into lentivirus, and an optimal infection titer is determined for the target cells. Following selection, genomic DNA (gDNA) is extracted from an aliquot of the transduced cells. The remaining transduced cells are then screened in at least two distinct cell culture media. At the conclusion of the screening period, gDNA is collected from each cell population. Next, high-throughput sequencing is used to determine sgRNA barcode abundances from the initial and each of the final populations. Finally, an analytical pipeline is used to identify medium-essential candidate genes from these screen results.},
}
@article {pmid34709608,
year = {2022},
author = {Mair, B and Aregger, M and Tong, AHY and Chan, KSK and Moffat, J},
title = {A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2377},
number = {},
pages = {1-27},
pmid = {34709608},
issn = {1940-6029},
support = {R01 CA260170/CA/NCI NIH HHS/United States ; MOP-142375//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Feeder Cells ; Genes, Essential ; Humans ; *Pluripotent Stem Cells ; },
abstract = {Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.},
}
@article {pmid34708030,
year = {2021},
author = {Shan, L and Dai, Z and Wang, Q},
title = {Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {9},
number = {},
pages = {765396},
pmid = {34708030},
issn = {2296-4185},
abstract = {Non-conventional yeasts have attracted a growing interest on account of their excellent characteristics. In recent years, the emerging of CRISPR/Cas technology has improved the efficiency and accuracy of genome editing. Utilizing the advantages of CRISPR/Cas in bioengineering of non-conventional yeasts, quite a few advancements have been made. Due to the diversity in their genetic background, the ways for building a functional CRISPR/Cas system of various species non-conventional yeasts were also species-specific. Herein, we have summarized the different strategies for optimizing CRISPR/Cas systems in different non-conventional yeasts and their biotechnological applications in the construction of cell factories. In addition, we have proposed some potential directions for broadening and improving the application of CRISPR/Cas technology in non-conventional yeasts.},
}
@article {pmid34706599,
year = {2022},
author = {Fu, J and Li, J and Chen, J and Li, Y and Liu, J and Su, X and Shi, S},
title = {Ultra-specific nucleic acid testing by target-activated nucleases.},
journal = {Critical reviews in biotechnology},
volume = {42},
number = {7},
pages = {1061-1078},
doi = {10.1080/07388551.2021.1983757},
pmid = {34706599},
issn = {1549-7801},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing/methods ; },
abstract = {Specific and sensitive detection of nucleic acids is essential to clinical diagnostics and biotechnological applications. Currently, amplification steps are necessary for most detection methods due to the low concentration of nucleic acid targets in real samples. Although amplification renders high sensitivity, poor specificity is prevalent because of the lack of highly accurate precise strategies, resulting in significant false positives and false negatives. Nucleases exhibit high catalytic activity for nucleic acid cleavage which is regulated in a programmable manner. This review focuses on the latest progress in nucleic acid testing methods based on the target-activated nucleases. It summarizes the property of enzymes such as CRISPR/Cas, Argonautes, and some gene-editing irrelevant nucleases, which have been leveraged to create highly specific and sensitive nucleic acid testing tools. We elaborate on recent advances in the field of nuclease-mediated DNA recognition techniques for nucleic acid detection, and discuss its future applications and challenges in molecular diagnostics.},
}
@article {pmid34706226,
year = {2021},
author = {Ulferts, R and Marcassa, E and Timimi, L and Lee, LC and Daley, A and Montaner, B and Turner, SD and Florey, O and Baillie, JK and Beale, R},
title = {Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation.},
journal = {Cell reports},
volume = {37},
number = {4},
pages = {109899},
pmid = {34706226},
issn = {2211-1247},
support = {FC001827/MRC_/Medical Research Council/United Kingdom ; FC001827/CRUK_/Cancer Research UK/United Kingdom ; //Wellcome Trust/United Kingdom ; MR/M00869X/1/MRC_/Medical Research Council/United Kingdom ; FC001827//Arthritis Research UK/United Kingdom ; FC001827/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Autophagosomes/genetics/metabolism ; *Autophagy-Related Proteins/genetics/metabolism ; CRISPR-Cas Systems ; HCT116 Cells ; HEK293 Cells ; Humans ; Influenza A virus/genetics/metabolism ; *Lipoylation ; *Microtubule-Associated Proteins/genetics/metabolism ; Salmonella/genetics/metabolism ; Viral Matrix Proteins/genetics/metabolism ; Viroporin Proteins/genetics/metabolism ; },
abstract = {Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.},
}
@article {pmid34706181,
year = {2021},
author = {Gillmore, JD and Maitland, ML and Lebwohl, D},
title = {CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. Reply.},
journal = {The New England journal of medicine},
volume = {385},
number = {18},
pages = {1722-1723},
doi = {10.1056/NEJMc2114592},
pmid = {34706181},
issn = {1533-4406},
mesh = {*Amyloid Neuropathies, Familial/genetics/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; RNA, Guide ; },
}
@article {pmid34706180,
year = {2021},
author = {Rim, JH and Gopalappa, R and Gee, HY},
title = {CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.},
journal = {The New England journal of medicine},
volume = {385},
number = {18},
pages = {1722},
doi = {10.1056/NEJMc2114592},
pmid = {34706180},
issn = {1533-4406},
mesh = {*Amyloid Neuropathies, Familial/genetics/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; RNA, Guide ; },
}
@article {pmid34706179,
year = {2021},
author = {Solomon, A},
title = {CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.},
journal = {The New England journal of medicine},
volume = {385},
number = {18},
pages = {1721-1722},
doi = {10.1056/NEJMc2114592},
pmid = {34706179},
issn = {1533-4406},
mesh = {*Amyloid Neuropathies, Familial/genetics/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; RNA, Guide ; },
}
@article {pmid34705337,
year = {2022},
author = {Yu, C and Zhong, H and Yang, X and Li, G and Wu, Z and Yang, H},
title = {Establishment of a pig CRISPR/Cas9 knockout library for functional gene screening in pig cells.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100408},
doi = {10.1002/biot.202100408},
pmid = {34705337},
issn = {1860-7314},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Gene Editing ; Gene Knockout Techniques ; Gene Library ; *Lentivirus/genetics ; RNA, Guide/genetics ; Swine ; },
abstract = {BACKGROUND: As an important farm animal, pig functional genomic study can help understand the molecular mechanism related to the key economic traits of pig, such as growth, reproduction, or disease. The genome-scale library based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system facilitates discovery of key genes involved in a specific function or phenotype, allowing for an effective "phenotype-to-genotype" strategy for functional genomic study.<br><br>METHODS AND RESULTS: We designed and constructed a pig genome-scale CRISPR/Cas9 knockout library targeting 16,888 genes with 970,001 unique sgRNAs. The library is a single-vector system including both Cas9 and sgRNA, and packaged into lentivirus for an easy cell delivery for screening. To establish a screening method in pig cells, we used diphtheria toxin (DT)-induced cell death as a model to screen the host genes critical for DT toxicity in pig PK-15 cells. After lentiviral transduction and two sequential screening with DT treatment, the highest-ranking candidates we identified were previously validated genes, HBEGF, DPH1, DPH2, DPH3, DPH5, DNAJC24, and ZBTB17, which are DT receptor and the key factors involved in biosynthesis of diphthamide, the target of DT action. The function and gene essentiality of candidates were further confirmed by gene knockout and DT toxicity assay in PK-15 cells.<br><br>CONCLUSIONS: Our CRISPR knockout library targeting pig whole genome establishes a promising platform for pig functional genomic analysis.},
}
@article {pmid34704742,
year = {2021},
author = {Zhang, J and Liu, C},
title = {CRISPR-Powered DNA Computing and Digital Display.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {3148-3157},
doi = {10.1021/acssynbio.1c00431},
pmid = {34704742},
issn = {2161-5063},
support = {R01 EB023607/EB/NIBIB NIH HHS/United States ; },
mesh = {Biosensing Techniques/methods ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics ; Genetic Techniques ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology has unique specificity for recognizing and cleaving target DNA complementary to the CRISPR guide sequence. Here, we report on a CRISPR-powered DNA computing and digital display system in which programmed DNA targets serve as the input and an ON/OFF fluorescence signal indicates a TRUE/FALSE output. This system allows the establishment of a one-to-one relationship between input and output, enabling multilevel DNA logic computing. Applying pre-CRISPR reactions that selectively maintain or inhibit CRISPR reactivity can further improve the computing capability by expanding input size. In particular, we present a paper-based microfluidic chip with freeze-dried CRISPR reaction mixtures that are programmed to digitally display the results of functional operations, including square, cube, and square-root operations. This strategy allows information decoding and displaying as well, which brings potential in next-generation DNA steganography and cryptography. We envision that the intrinsic orthogonality of CRISPR provides a new paradigm for DNA computing and molecular programming.},
}
@article {pmid34704559,
year = {2021},
author = {Shao, M and Qi, Y and Sui, D and Xu, FJ},
title = {Phenylboronic acid-functionalized polyaminoglycoside as an effective CRISPR/Cas9 delivery system.},
journal = {Biomaterials science},
volume = {9},
number = {21},
pages = {7104-7114},
doi = {10.1039/d1bm00185j},
pmid = {34704559},
issn = {2047-4849},
mesh = {Boronic Acids ; *CRISPR-Cas Systems ; *Gene Editing ; Plasmids ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a promising approach for cancer therapy, and its application practice urgently requires a safe and effective gene carrier. In this work, we focus on the design of a phenylboronic acid (PBA)-functionalized, disulfide bonded branched polyaminoglycoside (SS-HPT-P) as a robust delivery vector of the CRISPR-Cas9 system. SS-HPT-P showed great tumor-targeting performance, reduction-responsive degradability, and gene transfection ability. The typical pCas9-surv (one CRISPR-Cas9 plasmid that targets and knocks out the survivin gene) delivery mediated by SS-HPT-P exhibited gene editing performance in the A549 cell line, confirming the feasibility of SS-HPT-P to effectively deliver the CRISPR-Cas9 system. SS-HPT-P/pCas9-surv could effectively inhibit the proliferation of tumor cells both in vitro and in vivo, suggesting the potential of PBA-functionalized nanocarriers for cancer gene therapy. The present work provides a promising approach for the treatment of malignant tumors.},
}
@article {pmid34703560,
year = {2021},
author = {Suea-Ngam, A and Howes, PD and deMello, AJ},
title = {An amplification-free ultra-sensitive electrochemical CRISPR/Cas biosensor for drug-resistant bacteria detection.},
journal = {Chemical science},
volume = {12},
number = {38},
pages = {12733-12743},
pmid = {34703560},
issn = {2041-6520},
abstract = {Continued development of high-performance and cost-effective in vitro diagnostic tools is vital for improving infectious disease treatment and transmission control. For nucleic acid diagnostics, moving beyond enzyme-mediated amplification assays will be critical in reducing the time and complexity of diagnostic technologies. Further, an emerging area of threat, in which in vitro diagnostics will play an increasingly important role, is antimicrobial resistance (AMR) in bacterial infections. Herein, we present an amplification-free electrochemical CRISPR/Cas biosensor utilizing silver metallization (termed E-Si-CRISPR) to detect methicillin-resistant Staphylococcus aureus (MRSA). Using a custom-designed guide RNA (gRNA) targeting the mecA gene of MRSA, the Cas12a enzyme allows highly sensitive and specific detection when employed with silver metallization and square wave voltammetry (SWV). Our biosensor exhibits excellent analytical performance, with detection and quantitation limits of 3.5 and 10 fM, respectively, and linearity over five orders of magnitude (from 10 fM to 0.1 nM). Importantly, we observe no degradation in performance when moving from buffer to human serum samples, and achieve excellent selectivity for MRSA in human serum in the presence of other common bacteria. The E-Si-CRISPR method shows significant promise as an ultrasensitive field-deployable device for nucleic acid-based diagnostics, without requiring nucleic acid amplification. Finally, adjustment to a different disease target can be achieved by simple modification of the gRNA protospacer.},
}
@article {pmid34702830,
year = {2021},
author = {Bigelyte, G and Young, JK and Karvelis, T and Budre, K and Zedaveinyte, R and Djukanovic, V and Van Ginkel, E and Paulraj, S and Gasior, S and Jones, S and Feigenbutz, L and Clair, GS and Barone, P and Bohn, J and Acharya, A and Zastrow-Hayes, G and Henkel-Heinecke, S and Silanskas, A and Seidel, R and Siksnys, V},
title = {Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6191},
pmid = {34702830},
issn = {2041-1723},
mesh = {Bacillales/enzymology ; CRISPR-Associated Proteins/chemistry/*metabolism ; CRISPR-Cas Systems ; Clostridiales/enzymology ; DNA/*metabolism ; Endodeoxyribonucleases/chemistry/*metabolism ; *Gene Editing ; HEK293 Cells ; Humans ; Plant Cells ; Protein Multimerization ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/chemistry/metabolism ; Zea mays ; },
abstract = {Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.},
}
@article {pmid34702578,
year = {2022},
author = {Hofvander, P and Andreasson, E and Andersson, M},
title = {Potato trait development going fast-forward with genome editing.},
journal = {Trends in genetics : TIG},
volume = {38},
number = {3},
pages = {218-221},
doi = {10.1016/j.tig.2021.10.004},
pmid = {34702578},
issn = {0168-9525},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; *Solanum tuberosum/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Implementations and improvements of genome editing techniques used in plant science have increased exponentially. For some crops, such as potato, the use of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) has moved to the next step of trait development and field trials, and should soon be applied to commercial cultivation.},
}
@article {pmid34700022,
year = {2021},
author = {Yang, X and Zhang, X and Liu, Y and Yang, D and Liu, Z and Chen, K and Tang, L and Wang, M and Hu, Z and Zhang, S and Huang, Y},
title = {Transgenic genome editing-derived antiviral therapy to nucleopolyhedrovirus infection in the industrial strain of the silkworm.},
journal = {Insect biochemistry and molecular biology},
volume = {139},
number = {},
pages = {103672},
doi = {10.1016/j.ibmb.2021.103672},
pmid = {34700022},
issn = {1879-0240},
mesh = {Animals ; Animals, Genetically Modified/genetics ; Antiviral Agents/*pharmacology ; Bombyx/*genetics ; CRISPR-Cas Systems ; Female ; *Gene Editing ; Genome ; Nucleopolyhedroviruses/*drug effects ; },
abstract = {The silkworm (Bombyx mori) is a domesticated and economically important insect. During the whole growth period, silkworm suffers various pathogen infection. Nearly 80% of silk cocoon crop losses are attributed to viral diseases. The circular double-stranded DNA virus Bombyx mori nuclepolyhedrovirus (BmNPV) is the major viral pathogen responsible for massive silkworm death and huge economic losses in the sericulture industry. Up to now, almost all the new strategies for developing immunity against BmNPV are in laboratory strains because of the lack of transgenic technology in industrial silkworm strains. We previously demonstrated that modification of BmNPV genome DNA with the antivirus transgenic CRISPR/Cas9 system effectively improved the resistance of laboratory silkworm strains to viral pathogens. The industrial strains are monovoltine or bivoltine. It is very difficult to break the diapause before microinjection for genetic transformation. Here, we show that the anti-BmNPV transgenic CRISPR/Cas9 system also works in the industrial silkworm strain Jingsong. In this study, we successfully broke diapause and obtained generation-skipping embryos and constructed two TG Jingsong lines. Both lines exhibited significantly enhanced immunity to BmNPV without significant changes in most of the commercially important traits. These results demonstrate that the construction of TG silkworm lines carrying a heritable immune defense system against BmNPV could be an effective strategy to enhance the resistance of industrial silkworm strains to the most devastating DNA virus. The research opened a window for genetic modification of the important strains from laboratory strains to industrial strains.},
}
@article {pmid34699779,
year = {2021},
author = {Cannon, PM and Kiem, HP},
title = {The genome-editing decade.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3093-3094},
pmid = {34699779},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; *Gene Editing/methods/trends ; Genetic Therapy ; Humans ; Research ; Translational Research, Biomedical ; },
}
@article {pmid34699745,
year = {2021},
author = {Pourtoy-Brasselet, S and Sciauvaud, A and Boza-Moran, MG and Cailleret, M and Jarrige, M and Polvèche, H and Polentes, J and Chevet, E and Martinat, C and Peschanski, M and Aubry, L},
title = {Human iPSC-derived neurons reveal early developmental alteration of neurite outgrowth in the late-occurring neurodegenerative Wolfram syndrome.},
journal = {American journal of human genetics},
volume = {108},
number = {11},
pages = {2171-2185},
pmid = {34699745},
issn = {1537-6605},
mesh = {*Age of Onset ; CRISPR-Cas Systems ; Case-Control Studies ; Endoplasmic Reticulum Stress ; Gene Expression Regulation ; Humans ; Induced Pluripotent Stem Cells/*cytology ; *Neurites/drug effects ; Neurons/*cytology ; Valproic Acid/pharmacology ; Wolfram Syndrome/genetics/*pathology ; },
abstract = {Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response-which is activated in late Wolfram degenerative processes-was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS.},
}
@article {pmid34699644,
year = {2022},
author = {Yan, Y and Zhu, X and Yu, Y and Li, C and Zhang, Z and Wang, F},
title = {Nanotechnology Strategies for Plant Genetic Engineering.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {34},
number = {7},
pages = {e2106945},
doi = {10.1002/adma.202106945},
pmid = {34699644},
issn = {1521-4095},
mesh = {*CRISPR-Cas Systems ; Genetic Engineering ; *Genome, Plant ; Nanotechnology ; Plants, Genetically Modified/genetics ; },
abstract = {Plant genetic engineering is essential for improving crop yield, quality, and resistance to abiotic/biotic stresses for sustainable agriculture. Agrobacterium-, biolistic bombardment-, electroporation-, and poly(ethylene glycol) (PEG)-mediated genetic-transformation systems are extensively used in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, nanotechnology-based gene-delivery methods have been developed for plant genetic transformation. This nanostrategy shows excellent transformation efficiency, good biocompatibility, adequate protection of exogenous nucleic acids, and the potential for plant regeneration. However, the nanomaterial-mediated gene-delivery system in plants is still in its infancy, and there are many challenges for its broad applications. Herein, the conventional genetic transformation techniques used in plants are briefly discussed. After that, the progress in the development of nanomaterial-based gene-delivery systems is considered. CRISPR-Cas-mediated genome editing and its combined applications with plant nanotechnology are also discussed. The conceptual innovations, methods, and practical applications of nanomaterial-mediated genetic transformation summarized herein will be beneficial for promoting plant genetic engineering in modern agriculture.},
}
@article {pmid34699173,
year = {2021},
author = {Goyon, A and Scott, B and Kurita, K and Crittenden, CM and Shaw, D and Lin, A and Yehl, P and Zhang, K},
title = {Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography-High-Resolution Mass Spectrometry Analysis.},
journal = {Analytical chemistry},
volume = {93},
number = {44},
pages = {14792-14801},
doi = {10.1021/acs.analchem.1c03533},
pmid = {34699173},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Chromatography, Liquid ; Digestion ; Humans ; Hydrophobic and Hydrophilic Interactions ; Mass Spectrometry ; *RNA, Guide ; Ribonucleases ; },
abstract = {CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach.},
}
@article {pmid34698513,
year = {2021},
author = {Li, J and Wang, K and Zhang, Y and Qi, T and Yuan, J and Zhang, L and Qiu, H and Wang, J and Yang, HT and Dai, Y and Song, Y and Chang, X},
title = {Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo.},
journal = {Circulation},
volume = {144},
number = {22},
pages = {1760-1776},
doi = {10.1161/CIRCULATIONAHA.121.054628},
pmid = {34698513},
issn = {1524-4539},
mesh = {*APOBEC Deaminases/biosynthesis/genetics ; Animals ; *CRISPR-Cas Systems ; *Cardiomyopathies/genetics/metabolism ; Dependovirus ; *Dystrophin/biosynthesis/genetics ; *Exons ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred mdx ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; },
abstract = {BACKGROUND: Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adulthood. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, because of the lack of a suitable preclinical model.<br><br>METHODS: We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as Dmd[E4*] mice). Echocardiography, microcomputed tomography, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo. AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal Dmd[E4*] mice, which were analyzed 2 or 12 months after treatment to evaluate the extent of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles.<br><br>RESULTS: Dmd[E4*] mice recapitulated many aspects of human DMD, including shortened life span (by &#x2248;50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted >50% targeted exon skipping in the Dmd transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased life span of the Dmd[E4*] mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least 1 year.<br><br>CONCLUSIONS: Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).},
}
@article {pmid34698496,
year = {2021},
author = {Ge, H and Wang, X and Xu, J and Lin, H and Zhou, H and Hao, T and Wu, Y and Guo, Z},
title = {A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean.},
journal = {Analytical chemistry},
volume = {93},
number = {44},
pages = {14885-14891},
doi = {10.1021/acs.analchem.1c04022},
pmid = {34698496},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Crops, Agricultural ; Gold ; *Metal Nanoparticles ; Plants, Genetically Modified/genetics ; Polymerase Chain Reaction ; Reproducibility of Results ; Soybeans/genetics ; },
abstract = {A clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-mediated dual-mode electrochemical biosensor without polymerase chain reaction (PCR) amplification was designed for sensitive and reliable detection of genetically modified soybean SHZD32-1. A functionalized composite bionanomaterial Fe3O4@AuNPs/DNA-Fc&amp;Ru was synthesized as the signal unit, while a characteristic gene fragment of SHZD32-1 was chosen as the target DNA (tDNA). When Cas12a, crRNA, and tDNA were present simultaneously, a ternary complex Cas12a-crRNA-tDNA was formed, and the nonspecific cleavage ability of the CRISPR/Cas12a system toward single-stranded DNA was activated. Thus, the single-stranded DNA-Fc in the signal unit was cleaved, resulting in the decrease in the fast scan voltammetric (FSV) signal from ferrocene (Fc) and the increase in the electrochemiluminescence (ECL) signal from ruthenium complex (Ru) inhibited by Fc. The linear range was 1-10[7] fmol/L for ECL and 10-10[8] fmol/L for FSV, and the limit of detection (LOD) was 0.3 fmol/L for ECL and 3 fmol/L for FSV. Accuracy, precision, stability, selectivity, and reliability were all satisfied. In addition, PCR-free detection could be completed in an hour at room temperature without requiring complicated operation and sample processing, showing great potential in the field detection of genetically modified crops.},
}
@article {pmid34697825,
year = {2022},
author = {Cao, Y and Yan, X and Ran, S and Ralph, J and Smith, RA and Chen, X and Qu, C and Li, J and Liu, L},
title = {Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.},
journal = {Plant, cell &amp; environment},
volume = {45},
number = {1},
pages = {248-261},
pmid = {34697825},
issn = {1365-3040},
mesh = {Ascomycota/*pathogenicity ; Brassica napus/*genetics/metabolism/*microbiology ; CRISPR-Cas Systems ; Cell Wall/chemistry/genetics ; Disease Resistance/genetics ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Genome, Plant ; Lignin/metabolism ; Multigene Family ; Mutation ; Plant Diseases/genetics/*microbiology ; Plant Proteins/*genetics/metabolism ; Plant Stems/cytology/genetics ; Plants, Genetically Modified ; },
abstract = {Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.},
}
@article {pmid34697499,
year = {2022},
author = {Hofer, U},
title = {Cyclic pyrimidines defend against phages.},
journal = {Nature reviews. Microbiology},
volume = {20},
number = {1},
pages = {1},
pmid = {34697499},
issn = {1740-1534},
mesh = {*Bacteriophages ; CRISPR-Cas Systems ; Pyrimidines ; },
}
@article {pmid34696664,
year = {2022},
author = {Zhang, W and Wang, Q and Du, H and Jiang, S},
title = {CRISPR/Cas9-mediated overexpression of long non-coding RNA SRY-box transcription factor 21 antisense divergent transcript 1 regulates the proliferation of osteosarcoma by increasing the expression of mechanistic target of rapamycin kinase and Kruppel-like factor 4.},
journal = {Bioengineered},
volume = {13},
number = {3},
pages = {6678-6687},
pmid = {34696664},
issn = {2165-5987},
mesh = {*Bone Neoplasms/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Humans ; Kruppel-Like Factor 4 ; *MicroRNAs/metabolism ; *Osteosarcoma/metabolism ; *RNA, Long Noncoding/metabolism ; Sirolimus ; TOR Serine-Threonine Kinases/genetics/metabolism ; },
abstract = {Osteosarcoma, derived from primitive mesenchymal cells, is the most common primary solid malignant tumor of bone. The cause of osteosarcoma remains unclear. In recent years, the role of non-coding sequences in regulating protein expression in tumors has been paid more and more attention, especially long non-coding RNA (lncRNA). We speculate that SRY-box transcription factor 21 antisense divergent transcript 1 (SOX21-AS1) can regulate the expression of the mechanistic target of rapamycin kinase (mTOR) and Kruppel-like factor 4 (KLF4) through sponging hsa-mir-7-5p and hsa-mir-145-5p. We knocked lncRNA SOX21-AS1 into the genome of 143B cells through CRISPR/Cas9, then screened out a monoclonal cell line. Detect the transcription level and protein expression level of the above-mentioned related genes, and cell proliferation. Then, ginsenoside Rg3 was added to culture the cell line knocked into lncRNA SOX21-AS1, and the expression levels of lncRNA SOX21-AS1, hsa-mir-7-5p, hsa-mir-145-5p, mTOR, and KLF4 were detected by RT-qPCR and Western blot. Cell proliferation method detects cell viability, explores the molecular mechanism of lncRNA SOX21-AS1 in osteosarcoma, and checks whether it can be used as a potential drug target for the treatment of osteosarcoma. Our results demonstrate that the overexpression of lncRNA SOX21-AS1 up-regulates mTOR and KLF4 by sponging hsa-mir-7-5p and hsa-mir-145-5p, and ultimately regulates the proliferation of osteosarcoma. It is proved that ginsenoside Rg3 can inhibit the cell proliferation of osteosarcoma by reducing the expression level of lncRNA SOX21-AS1. It provides an alternative for the treatment of osteosarcoma in the future.},
}
@article {pmid34696426,
year = {2021},
author = {Söllner, JH and Mettenleiter, TC and Petersen, B},
title = {Genome Editing Strategies to Protect Livestock from Viral Infections.},
journal = {Viruses},
volume = {13},
number = {10},
pages = {},
pmid = {34696426},
issn = {1999-4915},
mesh = {Animal Husbandry/methods ; Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genetic Engineering ; Host Microbial Interactions/genetics ; Livestock/*virology ; Virus Diseases/prevention &amp; control ; Viruses/*genetics/pathogenicity ; },
abstract = {The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been repurposed as genome editors to induce targeted double-strand breaks at almost any location in the genome. Thus, CRISPR/Cas genome editors can also be utilized to generate disease-resistant or resilient livestock, develop vaccines, and further understand virus-host interactions. Genes of interest in animals and viruses can be targeted to understand their functions during infection. Furthermore, transgenic animals expressing CRISPR/Cas can be generated to target the viral genome upon infection. Genetically modified livestock can thereby reduce disease outbreaks and decrease zoonotic threats.},
}
@article {pmid34696364,
year = {2021},
author = {Weidenbach, K and Wolf, S and Kupczok, A and Kern, T and Fischer, MA and Reetz, J and Urbańska, N and Künzel, S and Schmitz, RA and Rother, M},
title = {Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.},
journal = {Viruses},
volume = {13},
number = {10},
pages = {},
pmid = {34696364},
issn = {1999-4915},
mesh = {Archaea/virology ; Archaeal Viruses/classification/*genetics/*metabolism ; Base Sequence/genetics ; Genome, Viral/genetics ; Host Specificity/genetics ; Methanomicrobiaceae/genetics/metabolism/*virology ; Methanomicrobiales/genetics/virology ; Phylogeny ; Sequence Analysis, DNA/methods ; Viruses/genetics ; },
abstract = {Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.},
}
@article {pmid34696355,
year = {2021},
author = {Bruder, MR and Walji, SD and Aucoin, MG},
title = {Comparison of CRISPR-Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS.},
journal = {Viruses},
volume = {13},
number = {10},
pages = {},
pmid = {34696355},
issn = {1999-4915},
mesh = {Animals ; Baculoviridae/*genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; *Gene Expression Regulation, Viral ; Humans ; Sf9 Cells ; },
abstract = {The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR-Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR-Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR-Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.},
}
@article {pmid34695545,
year = {2022},
author = {Liang, SQ and Walkey, CJ and Martinez, AE and Su, Q and Dickinson, ME and Wang, D and Lagor, WR and Heaney, JD and Gao, G and Xue, W},
title = {AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {238-243},
pmid = {34695545},
issn = {1525-0024},
support = {UH3 HL147367/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; U42 OD026645/OD/NIH HHS/United States ; P50 HD103555/HD/NICHD NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Lung ; Mice ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed ∼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types.},
}
@article {pmid34695439,
year = {2021},
author = {Lauwen, S and Baerenfaenger, M and Ruigrok, S and de Jong, EK and Wessels, HJCT and den Hollander, AI and Lefeber, DJ},
title = {Loss of the AMD-associated B3GLCT gene affects glycosylation of TSP1 without impairing secretion in retinal pigment epithelial cells.},
journal = {Experimental eye research},
volume = {213},
number = {},
pages = {108798},
doi = {10.1016/j.exer.2021.108798},
pmid = {34695439},
issn = {1096-0007},
mesh = {Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/*metabolism ; Blotting, Western ; CRISPR-Cas Systems ; Cell Line ; Galactosyltransferases/*genetics ; Gene Expression/physiology ; Gene Knockout Techniques ; Glucosyltransferases/*genetics ; Glycosylation ; Humans ; Macular Degeneration/*genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Retinal Pigment Epithelium/*metabolism ; Tandem Mass Spectrometry ; },
abstract = {Age-related macular degeneration (AMD) has been associated with protective genetic variants in the β1-3 glucosyltransferase (B3GLCT) locus through genome-wide association studies. B3GLCT mediates modification of proteins with thrombospondin type I repeats (TSR) that contain O-linked glucose β1-3 fucose and C-linked mannose glycosylation motifs. B3GLCT-mediated modification is required for proper secretion of TSR-containing proteins. We aimed to start understanding the role of B3GLCT in AMD by evaluating its effect on glycosylation and secretion of proteins from retinal pigment epithelium (RPE) cells. We generated B3GLCT knockout (KO) RPE cells and analyzed glycosylation and secretion of thrombospondin 1 (TSP1), a protein involved in cellular processes highly relevant to AMD. Glycopeptide analysis confirmed the presence of the glucose-β1,3-fucose product of B3GLCT on TSP1 in wildtype (WT) cells and its absence in KO cells. C-mannosylation was variably present on WT TSP1 and increased on TSR domains 1 and 3 in KO cells. Secretion of TSP1 was not affected by the absence of B3GLCT, even not when TSP1 was upregulated by TNFα treatment or when TSP1 was overexpressed in HEK293T cells. Future research is needed to elucidate the effect of the observed glycosylation defects in the context of AMD, which might involve functional loss of TSP1 or effects on other TSR proteins.},
}
@article {pmid34695132,
year = {2021},
author = {Schlott, AC and Knuepfer, E and Green, JL and Hobson, P and Borg, AJ and Morales-Sanfrutos, J and Perrin, AJ and Maclachlan, C and Collinson, LM and Snijders, AP and Tate, EW and Holder, AA},
title = {Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.},
journal = {PLoS biology},
volume = {19},
number = {10},
pages = {e3001408},
pmid = {34695132},
issn = {1545-7885},
support = {C29637/A20183/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Acyltransferases/antagonists &amp; inhibitors/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Cell Survival/drug effects ; Enzyme Inhibitors/pharmacology ; Erythrocytes/drug effects/*parasitology ; Lipoylation/drug effects ; Merozoites/drug effects/metabolism ; Myristic Acid/*metabolism ; Parasites/drug effects/growth &amp; development ; Plasmodium falciparum/drug effects/enzymology/*metabolism/ultrastructure ; Protozoan Proteins/*metabolism ; Solubility ; Substrate Specificity/drug effects ; },
abstract = {We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.},
}
@article {pmid34694813,
year = {2021},
author = {Kumar, A and Satpati, P},
title = {Divalent-Metal-Ion Selectivity of the CRISPR-Cas System-Associated Cas1 Protein: Insights from Classical Molecular Dynamics Simulations and Electronic Structure Calculations.},
journal = {The journal of physical chemistry. B},
volume = {125},
number = {43},
pages = {11943-11954},
doi = {10.1021/acs.jpcb.1c07744},
pmid = {34694813},
issn = {1520-5207},
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Electronics ; Ions ; Molecular Dynamics Simulation ; },
abstract = {CRISPR-associated protein 1 (Cas1) is a universally conserved essential metalloenzyme of the clustered regularly interspaced short palindromic repeat (CRISPR) immune system of prokaryotes (bacteria, archaea) that can cut and integrate a part of viral DNA to its host genome with the help of other proteins. The integrated DNA acts as a memory of viral infection, which can be transcribed to RNA and stop future infection by recognition (based on the RNA/DNA complementarity principle) followed by protein-mediated degradation of the viral DNA. It has been proposed that the presence of a single manganese (Mn[2+]) ion in a conserved divalent-metal-ion binding pocket (key residues: E190, H254, D265, D268) of Cas1 is crucial for its function. Cas1-mediated DNA degradation was proposed to be hindered by metal substitution, metal chelation, or mutation of the binding pocket residues. Cas1 is active toward dsDNA degradation with both Mn[2+] and Mg[2+]. X-ray structures of Cas1 revealed an intricate atomic interaction network of the divalent-metal-ion binding pocket and opened up the possibility of modeling related metal ions (viz., Mg[2+], Ca[2+]) in the binding pocket of wild-type (WT) and mutated Cas1 proteins for computational analysis, which includes (1) quantitative estimation of the energetics of the divalent-metal-ion preference and (2) exploring the structural and dynamical aspects of the protein in response to divalent-metal-ion substitution or amino acid mutation. Using the X-ray structure of the Cas1 protein from Pseudomonas aeruginosa as a template (PDB 3GOD), we performed (∼2.23 μs) classical molecular dynamics (MD) simulations to compare structural and dynamical differences between Mg[2+]- and Ca[2+]-bound binding pockets of wild-type (WT) and mutant (E190A, H254A, D265A, D268A) Cas1. Furthermore, reduced binding pocket models were generated from X-ray and molecular dynamics (MD) trajectories, and the resulting structures were subjected to quantum chemical calculations. Results suggest that Cas1 prefers Mg[2+] binding relative to Ca[2+] and the preference is the strongest for WT and the weakest for the D268A mutant. Quantum chemical calculations indicate that Mn[2+] is the most preferred relative to both Mg[2+] and Ca[2+] in the wild-type and mutant Cas1. Substitution of Mg[2+] by Ca[2+] does not alter the interaction network between Cas1 and the divalent metal ion but increases the wetness of the binding pocket by introducing a single water molecule in the first coordination shell of the latter. The strength of metal-ion preference (Mg[2+] versus Ca[2+]) seems to be dependent on the solvent accessibility of the divalent-metal-ion binding pocket, strongest for wild-type Cas1 (in which the metal-ion binding pocket is dry, which includes two water molecules) and the weakest for the D268A mutant (in which the metal-ion binding pocket is wet, which includes four water molecules).},
}
@article {pmid34694607,
year = {2022},
author = {Wandera, KG and Beisel, CL},
title = {Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2404},
number = {},
pages = {135-153},
pmid = {34694607},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell-Free System/metabolism ; Endonucleases/genetics/metabolism ; RNA ; },
abstract = {Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.},
}
@article {pmid34694606,
year = {2022},
author = {Sharma, S and Sharma, CM},
title = {Identification of RNA Binding Partners of CRISPR-Cas Proteins in Prokaryotes Using RIP-Seq.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2404},
number = {},
pages = {111-133},
pmid = {34694606},
issn = {1940-6029},
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; DNA ; Endonucleases ; *Gene Editing ; RNA/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome. Here, we describe RIP-seq as a method to globally identify the direct RNA binding partners of CRISPR-Cas RNPs using the Cas9 nuclease as an example. RIP-seq combines co-immunoprecipitation (coIP) of an epitope-tagged Cas9 followed by isolation and deep sequencing analysis of its co-purified bound RNAs. This method can not only be used to study interactions of Cas9 with its known interaction partners, crRNAs and tracrRNA in native systems, but also to reveal potential additional RNA substrates of Cas9. For example, in RIP-seq analysis of Cas9 from the foodborne pathogen Campylobacter jejuni (CjeCas9), we recently identified several endogenous RNAs bound to CjeCas9 RNP in a crRNA-dependent manner, leading to the discovery of PAM-independent RNA cleavage activity of CjeCas9 as well as non-canonical crRNAs. RIP-seq can be easily adapted to any other effector RNP of choice from other CRISPR-Cas systems, allowing for the identification of target RNAs. Deciphering novel RNA-protein interactions for CRISPR-Cas proteins within host bacterial genomes will lead to a better understanding of the molecular mechanisms and functions of these systems and enable us to use the in vivo identified interaction rules as design principles for nucleic acid-targeting applications, fitted to each nuclease of interest.},
}
@article {pmid34694123,
year = {2021},
author = {Zhang, T and Li, HT and Xia, X and Liu, J and Lu, Y and Khan, MR and Deng, S and Busquets, R and He, G and He, Q and Zhang, J and Deng, R},
title = {Direct Detection of Foodborne Pathogens via a Proximal DNA Probe-Based CRISPR-Cas12 Assay.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {43},
pages = {12828-12836},
doi = {10.1021/acs.jafc.1c04663},
pmid = {34694123},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; DNA Probes ; Food Microbiology ; *Foodborne Diseases ; Humans ; Nucleic Acid Amplification Techniques ; *Salmonella enterica/genetics ; },
abstract = {Foodborne pathogens can cause illnesses. Existing tools for detecting foodborne pathogens are typically time-consuming or require complex protocols. Here, we report an assay to directly analyze pathogenic genes based on CRISPR-Cas12. This new test, termed proximal DNA probe-based CRISPR-Cas12 (PPCas12), facilitates the detection of foodborne pathogens without amplification steps. The elimination of the nucleic acid amplification process dramatically reduced the processing time, complexity, and costs in the analysis of foodborne pathogens. The substitution of the frequently used dually labeled DNA reporter with a proximal DNA probe in the PPCas12 assay led to a 4-fold sensitivity enhancement. PPCas12 offered a limit of detection of 619 colony-forming units in the detection of Salmonella enterica (S. enterica) without the nucleic acid amplification process. The specific recognition of genes via PPCas12 allowed distinguishing S. enterica from other foodborne pathogens. The PPCas12 assay was applied in the screening of S. enterica contamination on fresh eggs with high precision. Hence, the new PPCas12 assay will be a valuable tool for on-site monitoring of foodborne pathogens.},
}
@article {pmid34693471,
year = {2022},
author = {Liu, X and Bu, S and Feng, J and Wei, H and Wang, Z and Li, X and Zhou, H and He, X and Wan, J},
title = {Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a.},
journal = {Analytical and bioanalytical chemistry},
volume = {414},
number = {2},
pages = {1073-1080},
pmid = {34693471},
issn = {1618-2650},
mesh = {Biosensing Techniques/*methods ; *CRISPR-Cas Systems ; Electrochemical Techniques/*methods ; Electrophoresis, Polyacrylamide Gel ; Salmonella typhimurium/*isolation &amp; purification/pathogenicity ; },
abstract = {In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium. Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR.},
}
@article {pmid34692743,
year = {2021},
author = {Shahzad, R and Jamil, S and Ahmad, S and Nisar, A and Khan, S and Amina, Z and Kanwal, S and Aslam, HMU and Gill, RA and Zhou, W},
title = {Biofortification of Cereals and Pulses Using New Breeding Techniques: Current and Future Perspectives.},
journal = {Frontiers in nutrition},
volume = {8},
number = {},
pages = {721728},
pmid = {34692743},
issn = {2296-861X},
abstract = {Cereals and pulses are consumed as a staple food in low-income countries for the fulfillment of daily dietary requirements and as a source of micronutrients. However, they are failing to offer balanced nutrition due to deficiencies of some essential compounds, macronutrients, and micronutrients, i.e., cereals are deficient in iron, zinc, some essential amino acids, and quality proteins. Meanwhile, the pulses are rich in anti-nutrient compounds that restrict the bioavailability of micronutrients. As a result, the population is suffering from malnutrition and resultantly different diseases, i.e., anemia, beriberi, pellagra, night blindness, rickets, and scurvy are common in the society. These facts highlight the need for the biofortification of cereals and pulses for the provision of balanced diets to masses and reduction of malnutrition. Biofortification of crops may be achieved through conventional approaches or new breeding techniques (NBTs). Conventional approaches for biofortification cover mineral fertilization through foliar or soil application, microbe-mediated enhanced uptake of nutrients, and conventional crossing of plants to obtain the desired combination of genes for balanced nutrient uptake and bioavailability. Whereas, NBTs rely on gene silencing, gene editing, overexpression, and gene transfer from other species for the acquisition of balanced nutritional profiles in mutant plants. Thus, we have highlighted the significance of conventional and NBTs for the biofortification of cereals and pulses. Current and future perspectives and opportunities are also discussed. Further, the regulatory aspects of newly developed biofortified transgenic and/or non-transgenic crop varieties via NBTs are also presented.},
}
@article {pmid34691102,
year = {2021},
author = {Abdelrahman, M and Wei, Z and Rohila, JS and Zhao, K},
title = {Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {721203},
pmid = {34691102},
issn = {1664-462X},
abstract = {Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.},
}
@article {pmid34690674,
year = {2021},
author = {Kim, HJ and Bayarsaikhan, D and Lee, J and Bayarsaikhan, G and Lee, B},
title = {Brain-Derived Neurotrophic Factor Secreting Human Mesenchymal Stem Cells Improve Outcomes in Rett Syndrome Mouse Models.},
journal = {Frontiers in neuroscience},
volume = {15},
number = {},
pages = {725398},
pmid = {34690674},
issn = {1662-4548},
abstract = {Rett syndrome (RTT) is a severe X-linked dominant neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene; MeCP2 regulates the expression of brain-derived neurotrophic factor (BDNF) and increasing BDNF levels ameliorates RTT symptoms. However, the clinical application of BDNF is limited, because of its short half-life and low penetrance across the blood-brain barrier. In this study, we generated BDNF-secreting mesenchymal stem cells (MSCs) from the human umbilical cord cells, using CRISPR-Cas9. We studied the effects of BDNF-MSCs in MECP2 knockout and MECP2-deficient mice. BDNF-MSCs upregulated the expression of BDNF, pAKT, and pERK1/2 and downregulated that of pp38, both in vitro and in vivo. In our in vivo experiments, BDNF-MSCs increased the body and brain weights in mice. BDNF-MSCs increased the neuronal cell numbers in the hippocampus, cortex, and striatum; in addition, they increased the number of synapses. BDNF-MSCs upregulated BDNF and the activity of BDNF downstream effectors, such as pAKT and pERK 1/2; this upregulation was persistent. In conclusion, BDNF-MSCs generated using CRISPR-Cas9 could be a therapeutic strategy for treating RTT.},
}
@article {pmid34689795,
year = {2021},
author = {Sui, X and Wang, X and Liu, T and Ye, Q and Wu, B and Hu, G and Yang, S and He, M and Peng, N},
title = {Endogenous CRISPR-assisted microhomology-mediated end joining enables rapid genome editing in Zymomonas mobilis.},
journal = {Biotechnology for biofuels},
volume = {14},
number = {1},
pages = {208},
pmid = {34689795},
issn = {1754-6834},
abstract = {BACKGROUND: Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required.<br><br>RESULTS: We developed a novel gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat-Spacer-Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500&#xa0;bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a &#x3c3;[28] deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem.<br><br>CONCLUSIONS: Here, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.},
}
@article {pmid34687846,
year = {2022},
author = {Zhou, J and Chen, P and Wang, H and Liu, H and Li, Y and Zhang, Y and Wu, Y and Paek, C and Sun, Z and Lei, J and Yin, L},
title = {Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {244-255},
pmid = {34687846},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Gene Editing/methods ; Mammals ; RNA/genetics ; },
abstract = {Cas12a is an RNA-guided endonuclease that has been widely used for convenient multiplex gene editing with low off-target effects. To minimize off-targeting in gene editing, we engineered a variant of LbCas12a (termed Lb-K538R) with more stringent PAM recognition, lower off-targeting capability, and similar editing efficiency in vivo compared with LbCas12a. We also demonstrated that Lb2Cas12a from Lachnospiraceae bacterium MA2020 has extensive gene-editing activities in mammalian cells. Similar to Lb-K538R, the designed Lb2Cas12a variant (termed Lb2-K518R) not only had a more stringent PAM sequence change from YYN to TYN (Y is T or C, N is A, T, C, or G), but also displayed lower off-target effects, thereby enabling more potential target site selections with low off-targeting than the common TTTV (V is A, G, or C) PAM. To determine whether this type of mutation at the homologous position had similar effects in other Cas12a, As-K548R was evaluated. Based on the results of the genome-wide off-target test, As-K548R displayed lower off-target effects. Collectively, our findings indicate that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects, which suggests a promising and practical approach for minimizing off-targets effects in genome editing.},
}
@article {pmid34687393,
year = {2022},
author = {Dilip Kumar, S and Aashabharathi, M and KarthigaDevi, G and Subbaiya, R and Saravanan, M},
title = {Insights of CRISPR-Cas systems in stem cells: progress in regenerative medicine.},
journal = {Molecular biology reports},
volume = {49},
number = {1},
pages = {657-673},
pmid = {34687393},
issn = {1573-4978},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Humans ; Regenerative Medicine/*methods ; Signal Transduction/*genetics ; Stem Cells/*metabolism ; },
abstract = {Regenerative medicine, a therapeutic approach using stem cells, aims to rejuvenate and restore the normalized function of the cells, tissues, and organs that are injured, malfunctioning, and afflicted. This influential technology reaches its zenith when it is integrated with the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) technology of genome editing. This tool acts as a programmable restriction enzyme system, which targets DNA as well as RNA and gets redeployed for the customization of DNA/RNA sequences. The dynamic behaviour of nuclear manipulation and transcriptional regulation by CRISPR-Cas technology renders it with numerous employment in the field of biologics and research. Here, the possible impact of the commonly practiced CRISPR-Cas systems in regenerative medicines is being reviewed. Primarily, the discussion of the working mechanism of this system and the fate of stem cells will be scrutinized. A detailed description of the CRISPR based regenerative therapeutic approaches for a horde of diseases like genetic disorders, neural diseases, and blood-related diseases is elucidated.},
}
@article {pmid34687379,
year = {2021},
author = {Gunes, S and Soykan, MN and Sariboyaci, AE and Uysal, O and Sevimli, TS},
title = {Enhancement of Apo2L/TRAIL signaling pathway receptors by the activation of Klotho gene with CRISPR/Cas9 in Caco-2 colon cancer cells.},
journal = {Medical oncology (Northwood, London, England)},
volume = {38},
number = {12},
pages = {146},
pmid = {34687379},
issn = {1559-131X},
mesh = {Apoptosis ; CRISPR-Cas Systems/*physiology ; Caco-2 Cells ; Colonic Neoplasms/genetics/*pathology ; *Gene Expression Regulation, Neoplastic ; Humans ; Klotho Proteins/*genetics ; Receptors, TNF-Related Apoptosis-Inducing Ligand/*physiology ; Signal Transduction/physiology ; },
abstract = {Human Klotho gene has many known functions such as anti-aging and anti-tumor, and decreased expression of this gene causes malignant formations in most types of cancer, including colon cancer. Interacting with TRAIL death receptors (DR4 and DR5) induces an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. The present study aimed to investigate downstream effect of overexpression of Klotho gene, which is known to have an antitumor effect on resistant human colon cancer cells, by examining its action on TRAIL death and decoy (DcR1 and DcR2) receptors for the first time. For this purpose, upregulation of human Klotho gene was achieved with CRISPR/Cas9-mediated system in resistant human colon cancer Caco-2 cells. To determine the effect of upregulation of Klotho gene on cancer cells evaluations with flow cytometry, WST-8, qRT-PCR, ELISA, and immunohistochemical analysis were performed. Then, Klotho gene was knocked out and its apoptotic effect was tested to find out whether it is due to overexpression of Klotho gene or not. Our results indicate that overexpression of Klotho gene in Caco-2 cells via CRISPR/Cas9-sensitized TRAIL death receptor DR4 suppresses the proliferation of cells by leading to apoptosis. Thus, this study conducted on apoptosis-resistant colon cancer cells may bring new insights about the role of Klotho gene in colon cancer.},
}
@article {pmid34686979,
year = {2022},
author = {Wang, Q and Coleman, JJ},
title = {CRISPR/Cas9 RNP-Mediated Gene Fusion to Assess Protein Quantification and Subcellular Localization in Fusarium oxysporum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2391},
number = {},
pages = {89-98},
pmid = {34686979},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Fusarium ; Gene Fusion ; Homologous Recombination ; Humans ; },
abstract = {F. oxysporum is a notorious filamentous pathogenic fungus that causes serious problems in agriculture and animal/human health. Knowing how the fungus interacts throughout the course of an infection is necessary to propose an effective control strategy, and consequently the manipulation of the F. oxysporum genome is essential to investigate the molecular interplay between the host and fungus. To facilitate assessing protein quantification and subcellular localization, we developed a simple, economical CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). Reporter genes, including GFP and LacZ, can be inserted at the N- or C-terminus of an endogenous gene of interest at the original chromosomal locus, allowing partial characterization of the gene function.},
}
@article {pmid34686978,
year = {2022},
author = {Pokhrel, A and Seo, S and Wang, Q and Coleman, JJ},
title = {Targeted Gene Disruption Via CRISPR/Cas9 Ribonucleoprotein Complexes in Fusarium oxysporum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2391},
number = {},
pages = {75-87},
pmid = {34686978},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Fungal Proteins ; *Fusarium/genetics/metabolism ; Homologous Recombination ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Characterization of a gene of interest frequently relies on generation of a mutant as a critical component. Transformation to disrupt a gene has been previously accomplished by several methods in Fusarium oxysporum. Here we provide a detailed method to generate a gene mutation mediated by a CRISPR/Cas9 ribonucleoprotein (RNP) complex. The Cas9 RNP cleaves the DNA at the target site, and during DNA repair integration of a dominant selectable marker is incorporated via homologous recombination generating the desired gene disruption.},
}
@article {pmid34686895,
year = {2021},
author = {Yang, K and Liang, Y and Li, Y and Liu, Q and Zhang, W and Yin, D and Song, X and Shao, Y and Tu, J and Qi, K},
title = {Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains.},
journal = {Analytical and bioanalytical chemistry},
volume = {413},
number = {30},
pages = {7521-7529},
pmid = {34686895},
issn = {1618-2650},
mesh = {Animals ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Coronavirus Infections/diagnosis/*veterinary/virology ; Endodeoxyribonucleases/genetics ; Porcine epidemic diarrhea virus/*classification/*genetics/isolation &amp; purification ; Recombinases/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/*veterinary ; Swine ; Swine Diseases/*diagnosis/virology ; Vaccines, Attenuated/*genetics ; Viral Proteins/genetics ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription-enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation.},
}
@article {pmid34686672,
year = {2021},
author = {Wong, NM and Frias, E and Sigoillot, FD and Letendre, JH and Hild, M and Wong, WW},
title = {Engineering digitizer circuits for chemical and genetic screens in human cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6150},
pmid = {34686672},
issn = {2041-1723},
support = {DP2 CA186574/CA/NCI NIH HHS/United States ; F31 HL149334/HL/NHLBI NIH HHS/United States ; R01 EB029483/EB/NIBIB NIH HHS/United States ; R01 GM129011/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Genes, Reporter ; Genomics/*methods ; High-Throughput Screening Assays/*methods ; Humans ; Promoter Regions, Genetic ; Small Molecule Libraries/pharmacology ; Transcription Factors/chemistry/genetics/metabolism ; },
abstract = {Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.},
}
@article {pmid34686666,
year = {2021},
author = {Buddhachat, K and Paenkaew, S and Sripairoj, N and Gupta, YM and Pradit, W and Chomdej, S},
title = {Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. &amp; Thonn.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20888},
pmid = {34686666},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA Barcoding, Taxonomic/*methods ; Phyllanthus/*genetics ; RNA, Guide/genetics ; },
abstract = {Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.},
}
@article {pmid34686207,
year = {2021},
author = {Mei, H and Zha, Z and Wang, W and Xie, Y and Huang, Y and Li, W and Wei, D and Zhang, X and Qu, J and Liu, J},
title = {Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {297},
pmid = {34686207},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Genome, Human ; Glycoproteins/*metabolism/physiology ; HeLa Cells ; Humans ; Immunity, Innate ; Interferon Type I/*antagonists &amp; inhibitors ; Rhinovirus/*physiology ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein/metabolism ; rab5 GTP-Binding Proteins/physiology ; },
abstract = {BACKGROUND: Rhinoviruses (RVs) cause more than half of common colds and, in some cases, more severe diseases. Functional genomics analyses of RVs using siRNA or genome-wide CRISPR screen uncovered a limited set of host factors, few of which have proven clinical relevance.<br><br>RESULTS: Herein, we systematically compare genome-wide CRISPR screen and surface protein-focused CRISPR screen, referred to as surfaceome CRISPR screen, for their efficiencies in identifying RV host factors. We find that surfaceome screen outperforms the genome-wide screen in the success rate of hit identification. Importantly, using the surfaceome screen, we identify olfactomedin-like 3 (OLFML3) as a novel host factor of RV serotypes A and B, including a clinical isolate. We find that OLFML3 is a RV-inducible suppressor of the innate immune response and that OLFML3 antagonizes type I interferon (IFN) signaling in a SOCS3-dependent manner.<br><br>CONCLUSION: Our study suggests that RV-induced OLFML3 expression is an important mechanism for RV to hijack the immune system and underscores surfaceome CRISPR screen in identifying viral host factors.},
}
@article {pmid34685392,
year = {2021},
author = {Alok, A and Chauhan, H and Upadhyay, SK and Pandey, A and Kumar, J and Singh, K},
title = {Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages.},
journal = {Life (Basel, Switzerland)},
volume = {11},
number = {10},
pages = {},
pmid = {34685392},
issn = {2075-1729},
abstract = {CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.},
}
@article {pmid34684206,
year = {2021},
author = {Magro, G and Calistri, A and Parolin, C},
title = {Targeting and Understanding HIV Latency: The CRISPR System against the Provirus.},
journal = {Pathogens (Basel, Switzerland)},
volume = {10},
number = {10},
pages = {},
pmid = {34684206},
issn = {2076-0817},
abstract = {The presence of latently infected cells and reservoirs in HIV-1 infected patients constitutes a significant obstacle to achieve a definitive cure. Despite the efforts dedicated to solve these issues, the mechanisms underlying viral latency are still under study. Thus, on the one hand, new strategies are needed to elucidate which factors are involved in latency establishment and maintenance. On the other hand, innovative therapeutic approaches aimed at eradicating HIV infection are explored. In this context, advances of the versatile CRISPR-Cas gene editing technology are extremely promising, by providing, among other advantages, the possibility to target the HIV-1 genome once integrated into cellular DNA (provirus) and/or host-specific genes involved in virus infection/latency. This system, up to now, has been employed with success in numerous in vitro and in vivo studies, highlighting its increasing significance in the field. In this review, we focus on the progresses made in the use of different CRISPR-Cas strategies to target the HIV-1 provirus, and we then discuss recent advancements in the use of CRISPR screens to elucidate the role of host-specific factors in viral latency.},
}
@article {pmid34681959,
year = {2021},
author = {Naeem, M and Hoque, MZ and Ovais, M and Basheer, C and Ahmad, I},
title = {Stimulus-Responsive Smart Nanoparticles-Based CRISPR-Cas Delivery for Therapeutic Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
pmid = {34681959},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; Nanoparticles/*administration &amp; dosage/*chemistry ; },
abstract = {The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of "genome surgery". Despite these advances, the barriers of CRISPR-Cas9 techniques in clinical applications include efficient delivery of CRISPR/Cas9 and risk of off-target effects. Various types of viral and non-viral vectors are designed to deliver the CRISPR/Cas9 machinery into the desired cell. These methods still suffer difficulties such as immune response, lack of specificity, and efficiency. The extracellular and intracellular environments of cells and tissues differ in pH, redox species, enzyme activity, and light sensitivity. Recently, smart nanoparticles have been synthesized for CRISPR/Cas9 delivery to cells based on endogenous (pH, enzyme, redox specie, ATP) and exogenous (magnetic, ultrasound, temperature, light) stimulus signals. These methodologies can leverage genome editing through biological signals found within disease cells with less off-target effects. Here, we review the recent advances in stimulus-based smart nanoparticles to deliver the CRISPR/Cas9 machinery into the desired cell. This review article will provide extensive information to cautiously utilize smart nanoparticles for basic biomedical applications and therapeutic genome editing.},
}
@article {pmid34681705,
year = {2021},
author = {Crouzier, L and Denus, M and Richard, EM and Tavernier, A and Diez, C and Cubedo, N and Maurice, T and Delprat, B},
title = {Sigma-1 Receptor Is Critical for Mitochondrial Activity and Unfolded Protein Response in Larval Zebrafish.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
pmid = {34681705},
issn = {1422-0067},
mesh = {Animals ; Animals, Genetically Modified/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Larva/physiology ; Locomotion ; Membrane Proteins/metabolism ; Mitochondria/*metabolism ; Phenotype ; Receptors, sigma/chemistry/genetics/*metabolism ; *Unfolded Protein Response ; Zebrafish/growth &amp; development/*metabolism ; Zebrafish Proteins/chemistry/genetics/*metabolism ; },
abstract = {The sigma-1 receptor (S1R) is a highly conserved transmembrane protein highly enriched in mitochondria-associated endoplasmic reticulum (ER) membranes, where it interacts with several partners involved in ER-mitochondria Ca[2+] transfer, activation of the ER stress pathways, and mitochondria function. We characterized a new S1R deficient zebrafish line and analyzed the impact of S1R deficiency on visual, auditory and locomotor functions. The s1r[+25/+25] mutant line showed impairments in visual and locomotor functions compared to s1r[WT]. The locomotion of the s1r[+25/+25] larvae, at 5 days post fertilization, was increased in the light and dark phases of the visual motor response. No deficit was observed in acoustic startle response. A critical role of S1R was shown in ER stress pathways and mitochondrial activity. Using qPCR to analyze the unfolded protein response genes, we observed that loss of S1R led to decreased levels of IRE1 and PERK-related effectors and increased over-expression of most of the effectors after a tunicamycin challenge. Finally, S1R deficiency led to alterations in mitochondria bioenergetics with decreased in basal, ATP-linked and non-mitochondrial respiration and following tunicamycin challenge. In conclusion, this new zebrafish model confirmed the importance of S1R activity on ER-mitochondria communication. It will be a useful tool to further analyze the physiopathological roles of S1R.},
}
@article {pmid34681646,
year = {2021},
author = {Schreurs, J and Sacchetto, C and Colpaert, RMW and Vitiello, L and Rampazzo, A and Calore, M},
title = {Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
pmid = {34681646},
issn = {1422-0067},
mesh = {AMP-Activated Protein Kinases/genetics/metabolism ; Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Heart Diseases/*genetics/pathology ; Humans ; Models, Biological ; Myocytes, Cardiac/cytology/metabolism ; RNA, Guide/metabolism ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {In the past two decades, genome editing has proven its value as a powerful tool for modeling or even treating numerous diseases. After the development of protein-guided systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which for the first time made DNA editing an actual possibility, the advent of RNA-guided techniques has brought about an epochal change. Based on a bacterial anti-phage system, the CRISPR/Cas9 approach has provided a flexible and adaptable DNA-editing system that has been able to overcome several limitations associated with earlier methods, rapidly becoming the most common tool for both disease modeling and therapeutic studies. More recently, two novel CRISPR/Cas9-derived tools, namely base editing and prime editing, have further widened the range and accuracy of achievable genomic modifications. This review aims to provide an overview of the most recent developments in the genome-editing field and their applications in biomedical research, with a particular focus on models for the study and treatment of cardiac diseases.},
}
@article {pmid34681578,
year = {2021},
author = {Semikolenova, O and Sakovina, L and Akhmetova, E and Kim, D and Vokhtantsev, I and Golyshev, V and Vorobyeva, M and Novopashin, S and Novopashina, D},
title = {Photoactivatable nanoCRISPR/Cas9 System Based on crRNA Reversibly Immobilized on Carbon Nanoparticles.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
pmid = {34681578},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Carbon/*chemistry ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Magnetic Iron Oxide Nanoparticles/chemistry ; Ultraviolet Rays ; },
abstract = {Here, we proposed a new approach to engineering a photoactivatable CRISPR/Cas9 gene-editing system. The novel nanoCRISPR/Cas9 system is based on the use of auxiliary photocleavable oligodeoxyribonucleotides (PC-DNAs) complementary to crRNA. PC-DNAs contained up to three UV-sensitive linkers made of 1-(2-nitrophenyl)-1,2-ethanediol inside the oligonucleotide chain. Immobilizing PC-DNAs on the surface of carbon nanoparticles through 3'-terminal pyrene residue provided sufficient blocking of crRNA (and corresponding Cas9 activity) before UV irradiation and allows for crRNA release after UV irradiation at 365 nm, which restores Cas9 activity. We optimized the length of blocking photocleavable oligonucleotide, number of linkers, time of irradiation, and the type of carbon nanoparticles. Based on the results, we consider the nanoCRISPR/Cas9 system involving carbon-encapsulated iron nanoparticles the most promising. It provides the greatest difference of functional activity before/after irradiation and can be used in prospective for magnetic field-controlled delivery of CRISPR system into the target cells or tissues and spatiotemporal gene editing induced by UV irradiation.},
}
@article {pmid34681568,
year = {2021},
author = {Molina-Risco, M and Ibarra, O and Faion-Molina, M and Kim, B and Septiningsih, EM and Thomson, MJ},
title = {Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.},
journal = {International journal of molecular sciences},
volume = {22},
number = {20},
pages = {},
pmid = {34681568},
issn = {1422-0067},
mesh = {Agrobacterium/*physiology ; Biolistics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques ; Oryza/*genetics/growth &amp; development/microbiology ; Oxidoreductases/*genetics ; Phenotype ; Plant Proteins/genetics ; Plants, Genetically Modified/growth &amp; development/microbiology ; Sequence Analysis, DNA ; Transformation, Genetic ; },
abstract = {Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.},
}
@article {pmid34680998,
year = {2021},
author = {Tang, JX and Pyle, A and Taylor, RW and Oláhová, M},
title = {Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing.},
journal = {Genes},
volume = {12},
number = {10},
pages = {},
pmid = {34680998},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Humans ; Mitochondria/*genetics/pathology ; Mitochondrial Diseases/*genetics/pathology/therapy ; },
abstract = {Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.},
}
@article {pmid34680899,
year = {2021},
author = {Lu, W and Pei, Z and Zang, M and Lee, YK and Zhao, J and Chen, W and Wang, H and Zhang, H},
title = {Comparative Genomic Analysis of Bifidobacterium bifidum Strains Isolated from Different Niches.},
journal = {Genes},
volume = {12},
number = {10},
pages = {},
pmid = {34680899},
issn = {2073-4425},
mesh = {Adult ; Bifidobacterium bifidum/*genetics/isolation &amp; purification ; Feces/microbiology ; Genes, Bacterial ; Humans ; Infant ; },
abstract = {The potential probiotic benefits of Bifidobacterium bifidum have received increasing attention recently. We used comparative genomic analysis to explore the differences in the genome and the physiological characteristics of B. bifidum isolated from the fecal samples of Chinese adults and infants. The relationships between genotypes and phenotypes were analyzed to assess the effects of isolation sources on the genetic variation of B. bifidum. The phylogenetic tree results indicated that the phylogeny of B. bifidum may be related to the geographical features of its isolation source. B. bifidum was found to have an open pan-genome and a conserved core genome. The genetic diversity of B. bifidum is mainly reflected in carbohydrate metabolism- and immune/competition-related factors, such as the glycoside hydrolase gene family, bacteriocin operons, antibiotic resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Additionally, the type III A CRISPR-Cas system was discovered in B. bifidum for the first time. B. bifidum strains exhibited niche-specific characteristics, and the results of this study provide an improved understanding of the genetics of this species.},
}
@article {pmid34680159,
year = {2021},
author = {Kowalczyk, JE and Saha, S and Mäkelä, MR},
title = {Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.},
journal = {Biomolecules},
volume = {11},
number = {10},
pages = {},
pmid = {34680159},
issn = {2218-273X},
mesh = {Basidiomycota/*genetics/growth &amp; development ; CRISPR-Cas Systems/*genetics ; Fungal Proteins/*genetics ; Gene Editing/methods ; Lignin/genetics ; Polyporaceae/*genetics ; RNA, Guide/genetics ; Wood/genetics/microbiology ; },
abstract = {Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.},
}
@article {pmid34680045,
year = {2021},
author = {Basak, I and Hansen, RA and Ward, ME and Hughes, SM},
title = {Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement.},
journal = {Biomolecules},
volume = {11},
number = {10},
pages = {},
pmid = {34680045},
issn = {2218-273X},
mesh = {Adolescent ; Adult ; CRISPR-Cas Systems/genetics ; Cathepsin B/pharmacology ; Cell Line ; Cerebellar Cortex/growth &amp; development/metabolism ; Child ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Lysosome-Associated Membrane Glycoproteins/antagonists &amp; inhibitors/*genetics ; Lysosomes/genetics ; Mutation/genetics ; Neurodegenerative Diseases/complications/*genetics/physiopathology ; Neuronal Ceroid-Lipofuscinoses/complications/*genetics/physiopathology ; Neurons/drug effects/*metabolism/pathology ; Phenotype ; Young Adult ; },
abstract = {Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement-a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.},
}
@article {pmid34677325,
year = {2021},
author = {Rezaei, M and Razavi Bazaz, S and Morshedi Rad, D and Shimoni, O and Jin, D and Rawlinson, W and Ebrahimi Warkiani, M},
title = {A Portable RT-LAMP/CRISPR Machine for Rapid COVID-19 Screening.},
journal = {Biosensors},
volume = {11},
number = {10},
pages = {},
pmid = {34677325},
issn = {2079-6374},
mesh = {COVID-19/*diagnosis/virology ; COVID-19 Testing/instrumentation/methods ; CRISPR-Cas Systems/*genetics ; Humans ; Limit of Detection ; Molecular Diagnostic Techniques/instrumentation/*methods ; Nucleic Acid Amplification Techniques/instrumentation/*methods ; Point-of-Care Systems ; RNA, Viral/analysis/metabolism ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf[®] PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.},
}
@article {pmid34676610,
year = {2021},
author = {Kim, GE and Lee, SY and Park, HH},
title = {Crystal structure of the anti-CRISPR, AcrIIC4.},
journal = {Protein science : a publication of the Protein Society},
volume = {30},
number = {12},
pages = {2474-2481},
pmid = {34676610},
issn = {1469-896X},
mesh = {Antibiosis/*genetics ; Bacterial Proteins/*chemistry/genetics/metabolism ; Binding Sites ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; Crystallography, X-Ray ; DNA/chemistry/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Gene Editing ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; Haemophilus parainfluenzae/*genetics/metabolism ; Models, Molecular ; Neisseria meningitidis/genetics/metabolism ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Interaction Domains and Motifs ; RNA, Guide/chemistry/genetics/metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti-CRISPRs (Acrs) that are capable of inhibiting the host CRISPR-Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti-CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.},
}
@article {pmid34675410,
year = {2021},
author = {Xiong, R and Hua, D and Van Hoeck, J and Berdecka, D and Léger, L and De Munter, S and Fraire, JC and Raes, L and Harizaj, A and Sauvage, F and Goetgeluk, G and Pille, M and Aalders, J and Belza, J and Van Acker, T and Bolea-Fernandez, E and Si, T and Vanhaecke, F and De Vos, WH and Vandekerckhove, B and van Hengel, J and Raemdonck, K and Huang, C and De Smedt, SC and Braeckmans, K},
title = {Photothermal nanofibres enable safe engineering of therapeutic cells.},
journal = {Nature nanotechnology},
volume = {16},
number = {11},
pages = {1281-1291},
pmid = {34675410},
issn = {1748-3395},
support = {648124/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Survival/drug effects ; Cell- and Tissue-Based Therapy ; Humans ; *Immunotherapy, Adoptive ; MCF-7 Cells ; Melanoma, Experimental/genetics/immunology/therapy ; Mice ; Nanofibers/chemistry ; Nanoparticles/*chemistry/therapeutic use ; Neoplasms/genetics/immunology/pathology/*therapy ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/immunology ; RNA, Small Interfering/chemistry/genetics/immunology/*pharmacology ; Transfection ; },
abstract = {Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR-Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.},
}
@article {pmid34675221,
year = {2021},
author = {Baek, HS and Kwon, TU and Shin, S and Kwon, YJ and Chun, YJ},
title = {Steroid sulfatase deficiency causes cellular senescence and abnormal differentiation by inducing Yippee-like 3 expression in human keratinocytes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20867},
pmid = {34675221},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Cellular Senescence ; Humans ; Ichthyosis, X-Linked/*genetics/pathology ; Keratinocytes/metabolism/*pathology ; Steryl-Sulfatase/*genetics ; Tumor Suppressor Proteins/*genetics ; Up-Regulation ; },
abstract = {Human steroid sulfatase (STS) is an enzyme that catalyzes the hydrolysis of dehydroepiandrosterone sulfate (DHEAS), estrone sulfate (E1S), and cholesterol sulfate. Abnormal expression of STS causes several diseases including colorectal, breast, and prostate cancer and refractory skin disease. In particular, accumulation of intracellular cholesterol sulfate by STS deficiency leads to a skin disorder with abnormal keratinization called X-linked ichthyosis (XLI). To determine the detailed mechanisms of XLI, we performed RNA sequencing (RNA-seq) analysis using human keratinocyte HaCaT cells treated with cholesterol and cholesterol sulfate. Of the genes with expression changes greater than 1.5-fold, Yippee-like 3 (YPEL3), a factor expected to affect cell differentiation, was found. Induction of YPEL3 causes permanent growth arrest, cellular senescence, and inhibition of metastasis in normal and tumor cells. In this study, we demonstrate that YPEL3 expression was induced by STS deficiency and, using the CRISPR/Cas9 system, a partial knock-out (STS[+/-]) cell line was constructed to establish a disease model for XLI studies. Furthermore, we show that increased expression of YPEL3 in STS-deficient cell lines promoted cellular senescence and expression of keratinization-related proteins such as involucrin and loricrin. Our results suggest that upregulation of YPEL3 expression by STS deficiency may play a crucial role in inducing cellular senescence and abnormal differentiation in human keratinocytes.},
}
@article {pmid34674587,
year = {2022},
author = {Kühn, R},
title = {Genome engineering in rodents - status quo and perspectives.},
journal = {Laboratory animals},
volume = {56},
number = {1},
pages = {83-87},
doi = {10.1177/00236772211051842},
pmid = {34674587},
issn = {1758-1117},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Mice ; Rats ; *Rodentia/genetics ; },
abstract = {The introduction of the CRISPR-Cas9 system in 2013 has revolutionized experimental genetics in mice and rats. This commentary gives an overview on the use of CRISPR either for gene editing in the germline or for editing and beyond editing in somatic cells. Future perspectives are opened by emerging CRISPR technologies that could enable genome engineering at larger scale.},
}
@article {pmid34674375,
year = {2022},
author = {Namula, Z and Hirata, M and Le, QA and Lin, Q and Takebayashi, K and Yoshimura, N and Tanihara, F and Thongkittidilok, C and Otoi, T},
title = {Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes.},
journal = {Veterinary medicine and science},
volume = {8},
number = {1},
pages = {164-169},
pmid = {34674375},
issn = {2053-1095},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Female ; *Gene Editing/methods/veterinary ; Pregnancy ; Swine ; Zona Pellucida/metabolism ; *Zygote/metabolism ; },
abstract = {BACKGROUND: Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated.<br><br>METHODS: The ZP of zygotes was weakened or removed by exposure to 0.5% actinase E, followed by electroporation of the Cas9 protein with guide RNA targeting GGTA1.<br><br>RESULTS: The blastocyst formation rate of ZP-free zygotes after electroporation was significantly lower (p&#xa0;<&#xa0;0.05) than that of ZP-intact zygotes. The mutation rate in blastocysts from ZP-weakened zygotes was similar to that in ZP-intact zygotes, whereas ZP removal increased the mutation rate. The mutation efficiency in blastocysts from electroporated zygotes did not differ among ZP treatment groups.<br><br>CONCLUSIONS: Our results indicate that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, whereas ZP removal has a detrimental effect on embryonic development but may increase the mutation rate.},
}
@article {pmid34673785,
year = {2021},
author = {Noel, EA and Weeks, DP and Van Etten, JL},
title = {Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing.},
journal = {PloS one},
volume = {16},
number = {10},
pages = {e0252696},
pmid = {34673785},
issn = {1932-6203},
mesh = {Agrobacterium/virology ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Chlorella/virology ; DNA Viruses/genetics ; Electroporation/methods ; Gene Editing/methods ; Phycodnaviridae/*genetics ; Ribonucleoproteins/genetics ; Transformation, Genetic/*genetics ; Viral Proteins/genetics ; },
abstract = {Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.},
}
@article {pmid34673274,
year = {2021},
author = {Murty, T and Mackall, CL},
title = {Gene editing to enhance the efficacy of cancer cell therapies.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3153-3162},
pmid = {34673274},
issn = {1525-0024},
support = {T32 GM007365/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cell- and Tissue-Based Therapy ; *Gene Editing ; Gene Transfer Techniques ; Genetic Engineering ; *Genetic Therapy ; Genetic Vectors ; Humans ; Neoplasms/*therapy ; T-Lymphocytes/immunology/metabolism ; Transgenes ; },
abstract = {Adoptive T cell therapies have shown impressive signals of activity, but their clinical impact could be enhanced by technologies to increase T cell potency and diminish the cost and labor involved in manufacturing these products. Gene editing platforms are under study in this arena to (1) enhance immune cell potency by knocking out molecules that inhibit immune responses; (2) deliver genetic payloads into precise genomic locations and thereby enhance safety and/or improve the gene expression profile by leveraging physiologic promoters, enhancers, and repressors; and (3) enable off-the-shelf therapies by preventing alloreactivity and immune rejection. This review discusses gene editing approaches that have been the best studied in the context of human T cells and adoptive T cell therapies, summarizing their current status and near-term potential for translation.},
}
@article {pmid34673207,
year = {2022},
author = {Komissarov, A and Sergeeva, M and Zhuravlev, E and Medvedev, S and Malakhova, A and Andreeva, E and Shurygina, AP and Gorshkov, A and Timofeeva, M and Balakhonova, E and Grudinin, M and Zakian, S and Richter, V and Stepanov, G},
title = {CRISPR-Cas9 mediated knockout of AnxA6 gene enhances influenza A virus replication in low-permissive HEK293FT cell line.},
journal = {Gene},
volume = {809},
number = {},
pages = {146024},
doi = {10.1016/j.gene.2021.146024},
pmid = {34673207},
issn = {1879-0038},
mesh = {Annexin A6/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; HEK293 Cells ; Host-Pathogen Interactions/*genetics ; Humans ; Influenza A virus/pathogenicity/*physiology ; Virion/physiology ; Virus Replication/*physiology ; },
abstract = {Using cell cultures of human origin for the propagation of influenza virus is an attractive way to preserve its glycosylation profile and antigenic properties, which is essential in influenza surveillance and vaccine production. However, only few cell lines are highly permissive to influenza virus, and none of them are of human origin. The barrier might be associated with host restriction factors inhibiting influenza growth, such as AnxA6 protein counteracting the process of influenza virion packaging. In the presented work we explore the CRISPR-Cas9 mediated knockout of ANXA6 gene as a way to overcome the host restriction barrier and increase the susceptibility of human cell line to influenza infection. By CRISPR-Cas9 genome editing we modified HEK293FT cells and obtained several clones defective in the ANXA6 gene. The replication of the influenza A virus in original HEK293FT cells and the HEK293FT-ANXA6[-/-] mutant cells was compared in growth curve experiments. By combination of methods including TCID assay and flow cytometry we showed that accumulation of influenza A virus in the mutant HEK293FT-ANXA6[-/-] cells significantly exceeded the virus titer in the original HEK293FT cells.},
}
@article {pmid34671329,
year = {2021},
author = {Hadidi, A and Czosnek, H and Randles, JW},
title = {Editorial: Next-Generation Sequencing and CRISPR-Cas Editing in Plant Virology.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {723278},
pmid = {34671329},
issn = {1664-302X},
}
@article {pmid34671252,
year = {2021},
author = {Zhang, L and Wang, J and Zhao, YT and Dubielecka, P and Qin, G and Zhuang, S and Chin, EY and Liu, PY and Zhao, TC},
title = {Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.},
journal = {Frontiers in pharmacology},
volume = {12},
number = {},
pages = {698714},
pmid = {34671252},
issn = {1663-9812},
support = {R01 GM141339/GM/NIGMS NIH HHS/United States ; R01 HL089405/HL/NHLBI NIH HHS/United States ; R01 HL115265/HL/NHLBI NIH HHS/United States ; },
abstract = {Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK[-/-] C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK[-/-] cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.},
}
@article {pmid34669232,
year = {2022},
author = {Wu, Y and He, Y and Sretenovic, S and Liu, S and Cheng, Y and Han, Y and Liu, G and Bao, Y and Fang, Q and Zheng, X and Zhou, J and Qi, Y and Zhang, Y and Zhang, T},
title = {CRISPR-BETS: a base-editing design tool for generating stop codons.},
journal = {Plant biotechnology journal},
volume = {20},
number = {3},
pages = {499-510},
pmid = {34669232},
issn = {1467-7652},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Codon, Terminator/genetics ; Cytosine ; *Gene Editing/methods ; Plants/genetics ; RNA, Guide/genetics ; },
abstract = {Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user-friendly design tool named CRISPR-BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein-coding genes of interest. We demonstrated in rice and tomato that CRISPR-BETS is easy-to-use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR-BETS can also serve non-plant species.},
}
@article {pmid34669078,
year = {2021},
author = {Yang, FY and Wei, N and Zhang, ZH and Wang, M and Liu, YC and Zhang, LF and Gu, F},
title = {Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD.},
journal = {Biotechnology letters},
volume = {43},
number = {12},
pages = {2273-2281},
pmid = {34669078},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems/*genetics ; Corynebacterium glutamicum/*genetics/metabolism ; DNA End-Joining Repair/*genetics ; Gene Editing ; Gene Knockout Techniques ; Genome, Bacterial/genetics ; Glutamic Acid/biosynthesis ; Ku Autoantigen/genetics ; Lysine/biosynthesis ; *Metabolic Engineering ; Mycobacterium tuberculosis/genetics ; },
abstract = {OBJECTIVES: Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application.<br><br>RESULTS: In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand&#xa0;breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00&#x2009;&#xb1;&#x2009;5.56%, or something like that.<br><br>CONCLUSION: The present study reported a novel genome manipulation method for C. glutamicum.},
}
@article {pmid34668968,
year = {2022},
author = {Dehdilani, N and Yousefi Taemeh, S and Goshayeshi, L and Dehghani, H},
title = {Genetically engineered birds; pre-CRISPR and CRISPR era†.},
journal = {Biology of reproduction},
volume = {106},
number = {1},
pages = {24-46},
doi = {10.1093/biolre/ioab196},
pmid = {34668968},
issn = {1529-7268},
mesh = {Animals ; Animals, Genetically Modified ; Biological Products ; Birds/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Gene Knock-In Techniques/methods ; Gene Transfer Techniques/trends/veterinary ; Genetic Engineering/methods/*veterinary ; Genetic Vectors ; Male ; Spermatozoa ; },
abstract = {Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR era in the field of avian genome engineering.},
}
@article {pmid34668889,
year = {2021},
author = {Shear, MA and Massa, A},
title = {In Utero Fetal Therapy: Stem Cells, Cell Transplantation, Gene Therapy, and CRISPR-Cas9.},
journal = {Clinical obstetrics and gynecology},
volume = {64},
number = {4},
pages = {861-875},
pmid = {34668889},
issn = {1532-5520},
mesh = {CRISPR-Cas Systems ; Female ; *Fetal Diseases/genetics/therapy ; *Fetal Therapies ; Genetic Therapy ; *Hematopoietic Stem Cell Transplantation ; Humans ; Pregnancy ; },
abstract = {In utero fetal therapy offers the opportunity to prevent and treat diseases with a cellular or genetic basis. Components of successful fetal treatment include isolation of a replacement cell population, in utero stem cell transplantation, cell engraftment with fetal immune tolerance, and ongoing cell function. Fetal gene therapy with CRISPR-Cas9 represents an exciting potential therapy for genetic diseases not amenable to gene supplementation via adenoviral vector transduction. These fetal therapies have unique ethical and safety considerations. Clinical trials for in utero cell therapy are underway, as additional discoveries in stem cell biology and gene therapy move closer to clinical translation.},
}
@article {pmid34668031,
year = {2021},
author = {Zhu, T and Zhao, Y},
title = {Correlation between type IIIA CRISPR-Cas system and SCCmec in Staphylococcus epidermidis.},
journal = {Archives of microbiology},
volume = {203},
number = {10},
pages = {6275-6286},
pmid = {34668031},
issn = {1432-072X},
mesh = {CRISPR-Cas Systems ; Humans ; Multilocus Sequence Typing ; Phylogeny ; *Staphylococcal Infections ; *Staphylococcus epidermidis/genetics ; },
abstract = {A subculture of S.epidermidis strain ATCC35984 that is amenable to genetically manipulate was occasionally found in our laboratory. This mutant exhibited susceptibility to methicillin in contrast to its parent strain. To unveil the underlying mechanism, whole-genome sequencing of the mutant was performed. A comparative analysis revealed that a large DNA fragment encompassing the CRISPR-Cas system, type I R-M system and the SCCmec element was deleted from the mutant. The large chromosomal deletion associated with CRISPR-Cas system was also observed to occur spontaneously in S. epidermidis in another independent laboratory, or artificially induced by introducing engineering crRNAs in other bacterial species. These findings imply the CRISPR-Cas systems can affect bacterial genome remodeling through deletion of the integrated MGEs (mobile genetic elements). Further bioinformatics analysis identified a higher carriage rate of SCCmec element in the S. epidermidis strains harboring the CRISPR-Cas system. MLST typing and phylogenetic analysis of those CRIPSR-Cas-positive S. epidermidis strains revealed multiple origins. In addition, distinct types of SCCmec carried in those strains suggested that acquisition of this MGE originated from multiple independent recombination events. Intriguingly, CRISPR-Cas systems are found to be always located in the vicinity of orfX gene among staphylococci. Allelic analysis of CRISPR loci flanking cas genes disclosed that the loci distal to the orfX gene are considerably stable and conserved, which probably serve as recombination hotspot between CRISPR-Cas system and phage or plasmid. Therefore, the findings generally support the notion that incomplete immune protection of CRISPR-Cas system can promote dissemination of its neighboring SCCmec element.},
}
@article {pmid34667116,
year = {2021},
author = {Genzor, P and Konstantinidou, P and Stoyko, D and Manzourolajdad, A and Marlin Andrews, C and Elchert, AR and Stathopoulos, C and Haase, AD},
title = {Cellular abundance shapes function in piRNA-guided genome defense.},
journal = {Genome research},
volume = {31},
number = {11},
pages = {2058-2068},
pmid = {34667116},
issn = {1549-5469},
support = {ZIA DK075111/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; Mice ; *RNA, Guide/genetics ; RNA, Small Interfering/genetics/metabolism ; },
abstract = {Defense against genome invaders universally relies on RNA-guided immunity. Prokaryotic CRISPR-Cas and eukaryotic RNA interference pathways recognize targets by complementary base-pairing, which places the sequences of their guide RNAs at the center of self/nonself discrimination. Here, we explore the sequence space of PIWI-interacting RNAs (piRNAs), the genome defense of animals, and establish functional priority among individual sequences. Our results reveal that only the topmost abundant piRNAs are commonly present in every cell, whereas rare sequences generate cell-to-cell diversity in flies and mice. We identify a skewed distribution of sequence abundance as a hallmark of piRNA populations and show that quantitative differences of more than a 1000-fold are established by conserved mechanisms of biogenesis. Finally, our genomics analyses and direct reporter assays reveal that abundance determines function in piRNA-guided genome defense. Taken together, we identify an effective sequence space and untangle two classes of piRNAs that differ in complexity and function. The first class represents the topmost abundant sequences and drives silencing of genomic parasites. The second class sparsely covers an enormous sequence space. These rare piRNAs cannot function in every cell, every individual, or every generation but create diversity with potential for adaptation in the ongoing arms race with genome invaders.},
}
@article {pmid34666996,
year = {2021},
author = {Xu, F and Tong, M and Tong, CSW and Chan, BKC and Chu, HY and Wong, TL and Fong, JHC and Cheung, MSH and Mak, KH and Pardeshi, L and Huang, Y and Wong, KH and Choi, GCG and Ma, S and Wong, ASL},
title = {A Combinatorial CRISPR-Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment.},
journal = {Cancer research},
volume = {81},
number = {24},
pages = {6219-6232},
doi = {10.1158/0008-5472.CAN-21-1017},
pmid = {34666996},
issn = {1538-7445},
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology ; Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*drug therapy/metabolism/pathology ; Cell Proliferation ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; Liver Neoplasms/*drug therapy/metabolism/pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Nude ; Mice, SCID ; Piperidines/administration &amp; dosage ; Sorafenib/administration &amp; dosage ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.},
}
@article {pmid34666523,
year = {2021},
author = {Weissman, JL and Alseth, EO and Meaden, S and Westra, ER and Fuhrman, JA},
title = {Immune lag is a major cost of prokaryotic adaptive immunity during viral outbreaks.},
journal = {Proceedings. Biological sciences},
volume = {288},
number = {1961},
pages = {20211555},
pmid = {34666523},
issn = {1471-2954},
mesh = {Archaea ; Bacteria/genetics ; *Bacteriophages ; CRISPR-Cas Systems ; Disease Outbreaks ; *Viruses ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptive immune systems enable bacteria and archaea to efficiently respond to viral pathogens by creating a genomic record of previous encounters. These systems are broadly distributed across prokaryotic taxa, yet are surprisingly absent in a majority of organisms, suggesting that the benefits of adaptive immunity frequently do not outweigh the costs. Here, combining experiments and models, we show that a delayed immune response which allows viruses to transiently redirect cellular resources to reproduction, which we call 'immune lag', is extremely costly during viral outbreaks, even to completely immune hosts. Critically, the costs of lag are only revealed by examining the early, transient dynamics of a host-virus system occurring immediately after viral challenge. Lag is a basic parameter of microbial defence, relevant to all intracellular, post-infection antiviral defence systems, that has to-date been largely ignored by theoretical and experimental treatments of host-phage systems.},
}
@article {pmid34665940,
year = {2021},
author = {Neil, K and Allard, N and Roy, P and Grenier, F and Menendez, A and Burrus, V and Rodrigue, S},
title = {High-efficiency delivery of CRISPR-Cas9 by engineered probiotics enables precise microbiome editing.},
journal = {Molecular systems biology},
volume = {17},
number = {10},
pages = {e10335},
pmid = {34665940},
issn = {1744-4292},
support = {159817//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Conjugation, Genetic ; Gene Editing ; Mice ; *Microbiota ; *Probiotics ; },
abstract = {Antibiotic resistance threatens our ability to treat infectious diseases, spurring interest in alternative antimicrobial technologies. The use of bacterial conjugation to deliver CRISPR-cas systems programmed to precisely eliminate antibiotic-resistant bacteria represents a promising approach but requires high in situ DNA transfer rates. We have optimized the transfer efficiency of conjugative plasmid TP114 using accelerated laboratory evolution. We hence generated a potent conjugative delivery vehicle for CRISPR-cas9 that can eliminate > 99.9% of targeted antibiotic-resistant Escherichia coli in the mouse gut microbiota using a single dose. We then applied this system to a Citrobacter rodentium infection model, achieving full clearance within four consecutive days of treatment.},
}
@article {pmid34665590,
year = {2021},
author = {Wu, H and Qian, S and Peng, C and Wang, X and Wang, T and Zhong, X and Chen, Y and Yang, Q and Xu, J and Wu, J},
title = {Rotary Valve-Assisted Fluidic System Coupling with CRISPR/Cas12a for Fully Integrated Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {6},
number = {11},
pages = {4048-4056},
doi = {10.1021/acssensors.1c01468},
pmid = {34665590},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems ; DNA ; *Vibrio parahaemolyticus/genetics ; },
abstract = {Of late, many nucleic acid analysis platforms have been established, but there is still room for constructing integrated nucleic acid detection systems with high nucleic acid extraction efficiency, low detection cost, and convenient operation. In this work, a simple rotary valve-assisted fluidic chip coupling with CRISPR/Cas12a was established to achieve fully integrated nucleic acid detection. All of the detection reagents were prestored on the fluidic chip. With the aid of the rotary valve and syringe, the liquid flow and stirring can be precisely controlled. The nucleic acid extraction, loop-mediated isothermal amplification (LAMP) reaction, and CRISPR detection could be completed in 80 min. A clean reservoir and an air reservoir on the fluidic chip were designed to effectively remove the remaining ethanol. With Vibrio parahaemolyticus as the targets, the detection sensitivity of the fluidic chip could reach 3.1 × 10[1] copies of target DNA per reaction. A positive sample could be sensitively detected by CRISPR/Cas12a to produce a green fluorescent signal, while a negative sample generated no fluorescent signal. Further, the fluidic chip was successfully applied for detection of spiked shrimp samples, which showed the same detection sensitivity. A great feasibility for real-sample detection was showed by the fluidic chip. The proposed detection platform did not need expensive centrifugal instruments or pumps, which displayed its potential to become a powerful tool for food safety analysis and clinical diagnostics, especially in the resource-limited areas.},
}
@article {pmid34665528,
year = {2021},
author = {Kong, H and Ju, E and Yi, K and Xu, W and Lao, YH and Cheng, D and Zhang, Q and Tao, Y and Li, M and Ding, J},
title = {Advanced Nanotheranostics of CRISPR/Cas for Viral Hepatitis and Hepatocellular Carcinoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {8},
number = {24},
pages = {e2102051},
pmid = {34665528},
issn = {2198-3844},
mesh = {CRISPR-Cas Systems/*genetics ; Carcinoma, Hepatocellular/genetics/*therapy ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hepatitis, Viral, Human/genetics/*therapy ; Humans ; Liver Neoplasms/genetics/*therapy ; Theranostic Nanomedicine/*methods ; },
abstract = {Liver disease, particularly viral hepatitis and hepatocellular carcinoma (HCC), is a global healthcare burden and leads to more than 2 million deaths per year worldwide. Despite some success in diagnosis and vaccine development, there are still unmet needs to improve diagnostics and therapeutics for viral hepatitis and HCC. The emerging clustered regularly interspaced short palindromic repeat/associated proteins (CRISPR/Cas) technology may open up a unique avenue to tackle these two diseases at the genetic level in a precise manner. Especially, liver is a more accessible organ over others from the delivery point of view, and many advanced strategies applied for nanotheranostics can be adapted in CRISPR-mediated diagnostics or liver gene editing. In this review, the focus is on these two aspects of viral hepatitis and HCC applications. An overview on CRISPR editor development and current progress in clinical trials is first given, followed by highlighting the recent advances integrating the merits of gene editing and nanotheranostics. The promising systems that are used in other applications but may hold potentials in liver gene editing are also discussed. This review concludes with the perspectives on rationally designing the next-generation CRISPR approaches and improving the editing performance.},
}
@article {pmid34664392,
year = {2021},
author = {Coppenrath, K and Tavares, ALP and Shaidani, NI and Wlizla, M and Moody, SA and Horb, M},
title = {Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {59},
number = {12},
pages = {e23453},
pmid = {34664392},
issn = {1526-968X},
support = {P40 OD010997/OD/NIH HHS/United States ; R01 DE026434/DE/NIDCR NIH HHS/United States ; R01 HD084409/HD/NICHD NIH HHS/United States ; R24 OD030008/OD/NIH HHS/United States ; },
mesh = {Animals ; Branchial Region/growth &amp; development/pathology ; Branchio-Oto-Renal Syndrome/*genetics/physiopathology ; CRISPR-Cas Systems/genetics ; Congenital Abnormalities/*genetics/pathology ; Embryonic Development/genetics ; Ganglia, Parasympathetic/growth &amp; development/pathology ; Gene Expression ; Gene Expression Regulation, Developmental/genetics ; Hearing Loss/*genetics/physiopathology ; Homeodomain Proteins/*genetics ; Humans ; Neural Tube/growth &amp; development/pathology ; Skull/growth &amp; development/pathology ; Transcription Factors/genetics ; Xenopus/genetics/growth &amp; development ; Xenopus Proteins/*genetics ; },
abstract = {The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors plays critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of Branchio-oto-renal (BOR) syndrome, which is characterized by moderate-to-severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch, and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckel's, ceratohyal, and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.},
}
@article {pmid34664124,
year = {2021},
author = {Yadav, I and Rautela, A and Kumar, S},
title = {Approaches in the photosynthetic production of sustainable fuels by cyanobacteria using tools of synthetic biology.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {37},
number = {12},
pages = {201},
pmid = {34664124},
issn = {1573-0972},
mesh = {Alkanes/metabolism ; *Biofuels ; Butanols ; CRISPR-Cas Systems ; Carrier Proteins ; Cyanobacteria/genetics/*metabolism ; Fatty Acids ; Limonene/metabolism ; Metabolic Engineering ; Photosynthesis/genetics/*physiology ; Sesquiterpenes ; Squalene ; Synechococcus/metabolism ; Synechocystis ; Synthetic Biology/*methods ; },
abstract = {Cyanobacteria, photosynthetic prokaryotic microorganisms having a simple genetic composition are the prospective photoautotrophic cell factories for the production of a wide range of biofuel molecules. The simple genetic composition of cyanobacteria allows effortless genetic manipulation which leads to increased research endeavors from the synthetic biology approach. Various unicellular model cyanobacterial strains like Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been successfully engineered for biofuels generation. Improved development of synthetic biology tools, genetic modification methods and advancement in transformation techniques to construct a strain that can contain multiple foreign genes in a single operon have vastly expanded the functions that can be used for engineering photosynthetic cyanobacteria for the generation of various biofuel molecules. In this review, recent advancements and approaches in synthetic biology tools used for cyanobacterial genome editing have been discussed. Apart from this, cyanobacterial productions of various fuel molecules like isoprene, limonene, α-farnesene, squalene, alkanes, butanol, and fatty acids, which can be a substitute for petroleum and fossil fuels in the future, have been elaborated.},
}
@article {pmid34663942,
year = {2021},
author = {Berríos, KN and Evitt, NH and DeWeerd, RA and Ren, D and Luo, M and Barka, A and Wang, T and Bartman, CR and Lan, Y and Green, AM and Shi, J and Kohli, RM},
title = {Controllable genome editing with split-engineered base editors.},
journal = {Nature chemical biology},
volume = {17},
number = {12},
pages = {1262-1270},
pmid = {34663942},
issn = {1552-4469},
support = {T32 GM007170/GM/NIGMS NIH HHS/United States ; R01 HG010646/HG/NHGRI NIH HHS/United States ; R01 GM138908/GM/NIGMS NIH HHS/United States ; K08 CA212299/CA/NCI NIH HHS/United States ; F30 HG011578/HG/NHGRI NIH HHS/United States ; },
mesh = {Biotechnology ; CRISPR-Cas Systems ; Cytosine/chemistry ; DNA/chemistry ; DNA Breaks, Double-Stranded ; Escherichia coli ; Gene Editing ; Nucleic Acid Conformation ; Nucleoside Deaminases/*chemistry/genetics ; Sirolimus/chemistry ; },
abstract = {DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR-Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.},
}
@article {pmid34663886,
year = {2021},
author = {Lucas-Elío, P and Molina-Quintero, LR and Xu, H and Sánchez-Amat, A},
title = {A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20564},
pmid = {34663886},
issn = {2045-2322},
support = {R01 GM37949 and R35 GM136216/NH/NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Gene Expression ; Histidine Kinase/genetics/*metabolism ; Marinomonas/genetics/*metabolism ; RNA-Binding Proteins/metabolism ; Repressor Proteins/genetics ; Transcription Factors/metabolism ; },
abstract = {CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I-F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.},
}
@article {pmid34663594,
year = {2021},
author = {Garton, J and Shankar, M and Chapman, B and Rose, K and Gaffney, PM and Webb, CF},
title = {Deficiencies in the DNA Binding Protein ARID3a Alter Chromatin Structures Important for Early Human Erythropoiesis.},
journal = {ImmunoHorizons},
volume = {5},
number = {10},
pages = {802-817},
pmid = {34663594},
issn = {2573-7732},
support = {R01 AI118836/AI/NIAID NIH HHS/United States ; R03 AI123951/AI/NIAID NIH HHS/United States ; R56 AI118836/AI/NIAID NIH HHS/United States ; T32 AI007633/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/genetics/*metabolism ; Chromatin Immunoprecipitation Sequencing ; DNA-Binding Proteins/*deficiency/genetics ; Erythropoiesis/*genetics ; Fetal Globulins/biosynthesis ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Humans ; K562 Cells ; RNA-Seq ; Transcription Factors/*deficiency/genetics ; },
abstract = {ARID3a is a DNA-binding protein important for normal hematopoiesis in mice and for in vitro lymphocyte development in human cultures. ARID3a knockout mice die in utero with defects in both early hematopoietic stem cell populations and erythropoiesis. Recent transcriptome analyses in human erythropoietic systems revealed increases in ARID3a transcripts implicating potential roles for ARID3a in human erythrocyte development. However, ARID3a transcript levels do not faithfully reflect protein levels in many cells, and the functions and requirements for ARID3a protein in those systems have not been explored. We used the erythroleukemic cell line K562 as a model to elucidate functions of ARID3a protein in early human erythropoiesis. ARID3a knockdown of hemin-stimulated K562 cells resulted in lack of fetal globin production and modifications in gene expression. Temporal RNA sequencing data link ARID3a expression with the important erythroid regulators Gata1, Gata2, and Klf1 Ablation of ARID3a using CRISPR-Cas9 further demonstrated it is required to maintain chromatin structures associated with erythropoietic differentiation potential. These data demonstrate that the ARID3a protein is required for early erythropoietic events and provide evidence for the requirement of ARID3a functions for proper maintenance of appropriate chromatin structures.},
}
@article {pmid34662386,
year = {2022},
author = {Tsuji-Hosokawa, A and Ogawa, Y and Tsuchiya, I and Terao, M and Takada, S},
title = {Human SRY Expression at the Sex-determining Period is Insufficient to Drive Testis Development in Mice.},
journal = {Endocrinology},
volume = {163},
number = {1},
pages = {},
doi = {10.1210/endocr/bqab217},
pmid = {34662386},
issn = {1945-7170},
mesh = {Animals ; CRISPR-Cas Systems ; Exons ; Female ; Gene Expression Regulation, Developmental ; Genotype ; Gonads/metabolism ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Nuclear Proteins/metabolism ; Ovary/metabolism ; Phenotype ; Protein Domains ; Sex Determination Processes ; Sex Differentiation ; Sex-Determining Region Y Protein/*biosynthesis/genetics ; Signal Transduction ; Testis/*growth &amp; development/*metabolism/physiology ; Transgenes ; },
abstract = {The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1-Sry(SRY) mice-and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry(SRY) mice was detected. The external and internal genitalia of adult Sry(SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry(SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry(SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry(SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.},
}
@article {pmid34713215,
year = {2020},
author = {Schmitz, DJ and Ali, Z and Wang, C and Aljedaani, F and Hooykaas, PJJ and Mahfouz, M and de Pater, S},
title = {CRISPR/Cas9 Mutagenesis by Translocation of Cas9 Protein Into Plant Cells via the Agrobacterium Type IV Secretion System.},
journal = {Frontiers in genome editing},
volume = {2},
number = {},
pages = {6},
pmid = {34713215},
issn = {2673-3439},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNA-guided Cas9 endonuclease is usually delivered into plant cells as a DNA construct encoding Cas9 and the single guide RNA (sgRNA). However, constitutive expression of nucleases may cause off target mutations. In addition, DNA constructs can integrate into the host genome, causing mutations and complicating regulatory approval. Instead of DNA, here we deliver Cas9 through the Agrobacterium T4SS, accomplished by fusion of the VirF T4SS translocation peptide to Cas9 (NCas9F). Co-cultivation of Agrobacteria expressing NCas9F with yeast (Saccharomyces cerevisiae) harboring a sgRNA targeting CAN1 showed that NCas9F was translocated via T4SS and induced targeted mutations in the yeast genome. Infiltration of Nicotiana benthamiana leaves with Agrobacteria expressing NCas9F and sgRNA-PHYTOENE DESATURASE (PDS) resulted in targeted modifications at the PDS locus, albeit at a very low rate. In order to increase the mutation frequency NCas9F protein was co-transported with a T-DNA encoding sgRNA-PDS1. Next generation sequencing confirmed that this resulted in targeted mutations at the PDS locus with a similar distribution but at a 5-fold lower frequency as the mutations obtained with a T-DNA encoding both Cas9 and sgRNA-PDS1. Similarly, infection with Tobacco rattle virus (TRV) encoding sgRNA-PDS2 combined with NCas9F protein translocation resulted in an equally high frequency of PDS mutations in N. benthamiana compared to T-DNA encoded sgRNA-PDS1 combined with NCas9F protein translocation. Our results revealed that translocation of NCas9F protein via the Agrobacterium T4SS can be used for targeted mutagenesis in host cells instead of the permanent and constitutive expression of Cas9 from a T-DNA.},
}
@article {pmid34713211,
year = {2020},
author = {Yang, B},
title = {Grand Challenges in Genome Editing in Plants.},
journal = {Frontiers in genome editing},
volume = {2},
number = {},
pages = {2},
pmid = {34713211},
issn = {2673-3439},
}
@article {pmid35117677,
year = {2020},
author = {Bhatkar, D and Sarode, SC and Sarode, GS and Patil, S and Sharma, NK},
title = {CRISPR-Cas genome editing tool: a narrow lane of cancer therapeutics with potential blockades.},
journal = {Translational cancer research},
volume = {9},
number = {4},
pages = {3135-3141},
pmid = {35117677},
issn = {2219-6803},
abstract = {In recent, clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas) system is emerging as a versatile genome editing tool with applications in basic science, preclinical and translational biology. This CRISPR-Cas genome editing tool is known as a precise and effective option to correct a part of the genome that may have implications in many human diseases including cancer associated genes such as oncogenes and onco-suppressors. Besides robust potential to edit target genes, CRISPR-Cas editing technology displays cellular alterations in the form of activation of DNA double strand break repair system and bringing genomic instability. As a consequence of repair of DNA double strand breaks, highly mitotically active cells may face hyper-DNA repair systems and there may be sometimes a situation leading to error prone mutations and unwanted genomic integrity. Additionally, the use of CRISPR-Cas editing technology in cancer therapy is limited in the backdrop of genotype and epigenomic heterogeneity in tumors. Therefore, a precaution should be considered to employ CRISPR-Cas technology in cancer therapy in view of tumor heterogeneity and environmental pressure.},
}
@article {pmid34691894,
year = {2019},
author = {Zhao, W and Zhao, KT},
title = {An exciting time for genome editing: an interview with David R. Liu.},
journal = {National science review},
volume = {6},
number = {3},
pages = {452-454},
doi = {10.1093/nsr/nwy146},
pmid = {34691894},
issn = {2053-714X},
abstract = {In 1987, several Osaka University researchers discovered a special kind of clustered DNA repeats in bacteria. Within a few years, two other groups independently discovered the same phenomenon but no one knew its function at the time. Only a small handful of scientists studied this property from its discovery in 1987 to 2005. It was then that the function of these DNA repeats, which were named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), was finally elucidated. Researchers found that CRISPR, when combined with its CRISPR-associated partner (Cas), is crucial for the functioning of the bacterial adaptive immune system against viral phage infection. CRISPR sequences can be transcribed into targeting RNA molecules, and Cas enzymes are guided by these RNAs to cut specific viral DNA loci, rendering resistance against the viral infection. Scientists realized that this natural bacterial immune response system could be engineered to become a powerful genome editing tool. Prior to CRISPR, existing genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) relied solely upon protein-DNA interactions to target an enzyme to specific DNA sequences. The design, engineering and evolution of proteins for various DNA sequences is difficult and time-consuming. In contrast, the CRISPR-Cas system uses Watson-Crick base pairing between a guide RNA and the target DNA to localize the complex to specific DNA sequences. This feature enables users to simply change an RNA sequence to match a DNA target to reposition the whole complex. Since then, numerous talented scientists have headed into this field. Within a single decade, they have developed the CRISPR-Cas system into a powerful genome editing tool and applied it to the editing of microorganisms, plants, animals and even human embryos. David R. Liu, Professor of Harvard University and the Broad Institute, and an investigator of the Howard Hughes Medical Institute, is one of them. One of his major contributions to the field is the development of 'base editing'. His group engineered the CRISPR system to transform it from being DNA scissors that cut DNA into specific DNA base pair rewriters that directly convert one base pair to a different base pair. This development opens the door to precision genome editing, raising the possibility of treating thousands of genetic diseases that are caused by single point mutations in the human genome. Here, David talks about this exciting time for genome editing.},
}
@article {pmid34691893,
year = {2019},
author = {Zhu, Y and Huang, Z},
title = {Recent advances in structural studies of the CRISPR-Cas-mediated genome editing tools.},
journal = {National science review},
volume = {6},
number = {3},
pages = {438-451},
pmid = {34691893},
issn = {2053-714X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated (Cas) proteins provide RNA-guided adaptive immunity for prokaryotes to defend themselves against viruses. The CRISPR-Cas systems have attracted much attention in recent years for their power in aiding the development of genome editing tools. Based on the composition of the CRISPR RNA-effector complex, the CRISPR-Cas systems can be divided into two classes and six types. In this review, we summarize recent advances in the structural biology of the CRISPR-Cas-mediated genome editing tools, which helps us to understand the mechanism of how the guide RNAs assemble with diverse Cas proteins to cleave target nucleic acids.},
}
@article {pmid34824864,
year = {2015},
author = {Baltes, NJ and Hummel, AW and Konecna, E and Cegan, R and Bruns, AN and Bisaro, DM and Voytas, DF},
title = {Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system.},
journal = {Nature plants},
volume = {1},
number = {10},
pages = {},
pmid = {34824864},
issn = {2055-0278},
support = {T32 GM008347/GM/NIGMS NIH HHS/United States ; },
abstract = {To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR-Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.},
}
@article {pmid34661431,
year = {2021},
author = {Barrangou, R},
title = {As CRISPR Technologies Ripen, What Should We Prioritize?.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {627},
doi = {10.1089/crispr.2021.29138.rba},
pmid = {34661431},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods/trends ; Humans ; },
}
@article {pmid34661430,
year = {2021},
author = {Knott, GJ and Lapinaite, A},
title = {CRISPR Gets Its Origin Story.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {631-633},
doi = {10.1089/crispr.2021.29137.knl},
pmid = {34661430},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
}
@article {pmid34661429,
year = {2021},
author = {Kandul, NP and Belikoff, EJ and Liu, J and Buchman, A and Li, F and Yamamoto, A and Yang, T and Shriner, I and Scott, MJ and Akbari, OS},
title = {Genetically Encoded CRISPR Components Yield Efficient Gene Editing in the Invasive Pest Drosophila suzukii.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {739-751},
doi = {10.1089/crispr.2021.0032},
pmid = {34661429},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Drosophila/*genetics/pathogenicity ; Drosophila Proteins/genetics ; Fruit/parasitology ; Gene Editing/*methods ; Introduced Species ; Pest Control, Biological/*methods ; },
abstract = {Originally from Asia, Drosophila suzukii Matsumura is a global pest of economically important soft-skinned fruits. Also commonly known as spotted wing drosophila, it is largely controlled through repeated applications of broad-spectrum insecticides by which resistance has been observed in the field. There is a pressing need for a better understanding of D. suzukii biology and for developing alternative environmentally friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here, we describe genetically modified strains that encode three different terminators and four different promoters to express Cas9 robustly in both the soma and/or germline of D. suzukii. The Cas9 strains were rigorously evaluated through genetic crossing to transgenic strains that encode single-guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA strains display remarkably high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in D. suzukii and may even provide tools useful for the development of new genetic strategies for control of this invasive species.},
}
@article {pmid34661428,
year = {2021},
author = {Rykachevsky, A and Stepakov, A and Muzyukina, P and Medvedeva, S and Dobrovolski, M and Burnaev, E and Severinov, K and Savitskaya, E},
title = {SCRAMBLER: A Tool for De Novo CRISPR Array Reconstruction and Its Application for Analysis of the Structure of Prokaryotic Populations.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {673-685},
doi = {10.1089/crispr.2021.0012},
pmid = {34661428},
issn = {2573-1602},
mesh = {Archaea/*genetics ; Bacteria/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Metagenomics/methods ; *Microbiota ; Sequence Analysis, DNA/*methods ; *Software ; },
abstract = {CRISPR arrays are prokaryotic genomic loci consisting of repeat sequences alternating with unique spacers acquired from foreign nucleic acids. As one of the fastest-evolving parts of the genome, CRISPR arrays can be used to differentiate closely related prokaryotic lineages and track individual strains in prokaryotic communities. However, the assembly of full-length CRISPR arrays sequences remains a problem. Here, we developed SCRAMBLER, a tool that includes several pipelines for assembling CRISPR arrays from high-throughput short-read sequencing data. We assessed its performance with model data sets (Escherichia coli strains containing different CRISPR arrays and imitating prokaryotic communities of different complexities) and intestinal microbiomes of extant and extinct pachyderms. Evaluation of SCRAMBLER's performance using model data sets demonstrated its ability to assemble CRISPR arrays correctly from reads containing pairs of spacers, yielding a precision rate of >80% and a recall rate of 60-85% when checked against ground-truth data. Likewise, SCRAMBLER successfully assembled CRISPR arrays from the environmental samples, as attested by their matching with database entries. SCRAMBLER, an open-source software (github.com/biolab-tools/SCRAMBLER), can facilitate analysis of the composition and dynamics of CRISPR arrays in complex communities.},
}
@article {pmid34661427,
year = {2021},
author = {Huang, Y and Zhang, Y and Wu, M and Porter, A and Barrangou, R},
title = {Determination of Factors Driving the Genome Editing Field in the CRISPR Era Using Bibliometrics.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {728-738},
doi = {10.1089/crispr.2021.0001},
pmid = {34661427},
issn = {2573-1602},
mesh = {Animals ; *Bibliometrics ; *CRISPR-Cas Systems ; Gene Editing/*trends ; Humans ; },
abstract = {Over the past two decades, the discovery of CRISPR-Cas immune systems and the repurposing of their effector nucleases as biotechnological tools have revolutionized genome editing. The corresponding work has been captured by 90,000 authors representing 7,600 affiliations in 126 countries, who have published more than 19,000 papers spanning medicine, agriculture, and biotechnology. Here, we use tech mining and an integrated bibliometric and networks framework to investigate the CRISPR literature over three time periods. The analysis identified seminal papers, leading authors, influential journals, and rising applications and topics interconnected through collaborative networks. A core set of foundational topics gave rise to diverging avenues of research and applications, reflecting a bona fide disruptive emerging technology. This analysis illustrates how bibliometrics can identify key factors, decipher rising trends, and untangle emerging applications and technologies that dynamically shape a morphing field, and provides insights into the trajectory of genome editing.},
}
@article {pmid34661426,
year = {2021},
author = {Liu, X and Zhou, X and Li, G and Huang, S and Sun, W and Sun, Q and Li, L and Huang, X and Liu, J and Wang, L},
title = {Editing Properties of Base Editors with SpCas9-NG in Discarded Human Tripronuclear Zygotes.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {710-727},
doi = {10.1089/crispr.2021.0036},
pmid = {34661426},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing/*methods ; HEK293 Cells ; Hep G2 Cells ; Humans ; Point Mutation ; Zygote/*metabolism ; },
abstract = {DNA base editors, comprising nucleotide deaminases and catalytically impaired Cas9 nickase, have been widely used in various organisms for the efficient creation of point mutations, providing researchers with powerful tools in precise genome editing. However, they have been limited by the scope of the editing. The discovery and engineering of various CRISPR-Cas systems, especially SpCas9 variants xCas9, Cas9-NG, and Cas9-SpRY, have diversified the range of targetable DNA sequences and expanded the targeting scope of genomic base editing. To understand the editing properties comprehensively, we conducted an analysis of the editing properties of adenine base editors and cytosine base editors with xCas9, Cas9-NG, and Cas9-SpRY at endogenous sites with NGN protospacer adjacent motifs (PAM). Then, human genetic disease-associated DNA point mutations were installed at a single site or at dual sites with NGH PAM using base editors with SpCas9-NG (ABEmax-NG and Anc-BE4max-NG [BEs-NG]) in cultured human cell lines. Finally, the editing properties of BEs-NG in discarded human tripronuclear embryos were characterized. This study investigated the editing properties of DNA base editors with a relaxed PAM requirement and demonstrated the potential of BEs-NG in human genetic disease-related research and treatment.},
}
@article {pmid34659160,
year = {2021},
author = {Thomas, P and Abdel-Glil, MY and Eichhorn, I and Semmler, T and Werckenthin, C and Baumbach, C and Murmann, W and Bodenthin-Drauschke, A and Zimmermann, P and Schotte, U and Galante, D and Slavic, D and Wagner, M and Wieler, LH and Neubauer, H and Seyboldt, C},
title = {Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {732106},
pmid = {34659160},
issn = {1664-302X},
abstract = {Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.},
}
@article {pmid34658640,
year = {2022},
author = {Ullah, MF and Ali, Y and Khan, MR and Khan, IU and Yan, B and Ijaz Khan, M and Malik, MY},
title = {A review of COVID-19: Treatment strategies and CRISPR/Cas9 gene editing technology approaches to the coronavirus disease.},
journal = {Saudi journal of biological sciences},
volume = {29},
number = {2},
pages = {860-871},
pmid = {34658640},
issn = {1319-562X},
abstract = {The new coronavirus SARS-CoV-2 pandemic has put the world on lockdown for the first time in decades. This has wreaked havoc on the global economy, put additional burden on local and global public health resources, and, most importantly, jeopardised human health. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and the CRISPR associated (Cas) protein (CRISPR/Cas) was identified to have structures in E. coli. The most modern of these systems is CRISPR/Cas. Editing the genomes of plants and animals took several years and cost hundreds of thousands of dollars until the CRISPR approach was discovered in 2012. As a result, CRISPR/Cas has piqued the scientific community's attention, particularly for disease diagnosis and treatment, because it is faster, less expensive, and more precise than previous genome editing technologies. Data from gene mutations in specific patients gathered using CRISPR/Cas can aid in the identification of the best treatment strategy for each patient, as well as other research domains such as coronavirus replication in cell culture, such as SARS-CoV2. The implications of the most prevalent driver mutations, on the other hand, are often unknown, making treatment interpretation difficult. For detecting a wide range of target genes, the CRISPR/Cas categories provide highly sensitive and selective tools. Genome-wide association studies are a relatively new strategy to discovering genes involved in human disease when it comes to the next steps in genomic research. Furthermore, CRISPR/Cas provides a method for modifying non-coding portions of the genome, which will help advance whole genome libraries by speeding up the analysis of these poorly defined parts of the genome.},
}
@article {pmid34655691,
year = {2021},
author = {Quan, FB and Gaillard, AL and Alejevski, F and Pézeron, G and Tostivint, H},
title = {Urotensin II-related peptide (Urp) is expressed in motoneurons in zebrafish, but is dispensable for locomotion in larva.},
journal = {Peptides},
volume = {146},
number = {},
pages = {170675},
doi = {10.1016/j.peptides.2021.170675},
pmid = {34655691},
issn = {1873-5169},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Editing/methods ; In Situ Hybridization ; Larva/*metabolism ; *Locomotion ; Motor Neurons/*metabolism ; Peptides/*metabolism ; Urotensins/*metabolism ; Zebrafish/growth &amp; development/*metabolism ; },
abstract = {The urotensin 2 (uts2) gene family consists of four paralogs called uts2, uts2-related peptide (urp), urp1 and urp2. uts2 is known to exert a large array of biological effects, including osmoregulation, control of cardiovascular functions and regulation of endocrine activities. Lately, urp1 and urp2 have been shown to regulate axial straightening during embryogenesis. In contrast, much less is known about the roles of urp. The aim of the present study was to investigate the expression and the functions of urp by using the zebrafish as a model. For this purpose, we determined the expression pattern of the urp gene. We found that urp is expressed in motoneurons of the brainstem and the spinal cord, as in tetrapods. This was confirmed with a new Tg(urp:gfp) fluorescent reporter line. We also generated a urp knockout mutant by using CRISPR/Cas9-mediated genome editing and analysed its locomotor activity in larvae. urp mutant did not exhibit any apparent defect of spontaneous swimming when compared to wild-type. We also tested the idea that urp may represent an intermediary of urp1 and urp2 in their role on axial straightening. We found that the upward bending of the tail induced by the overexpression of urp2 in 24-hpf embryos was not altered in urp mutants. Our results indicate that urp does probably not act as a relay downstream of urp2. In conclusion, the present study showed that zebrafish urp gene is primarily expressed in motoneurons but is apparently dispensable for locomotor activity in the early larval stages.},
}
@article {pmid34655593,
year = {2021},
author = {Hariprabu, KNG and Sathya, M and Vimalraj, S},
title = {CRISPR/Cas9 in cancer therapy: A review with a special focus on tumor angiogenesis.},
journal = {International journal of biological macromolecules},
volume = {192},
number = {},
pages = {913-930},
doi = {10.1016/j.ijbiomac.2021.10.029},
pmid = {34655593},
issn = {1879-0003},
mesh = {Animals ; Biomarkers, Tumor ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Disease Management ; Disease Models, Animal ; *Gene Editing ; Gene Expression ; Gene Expression Regulation ; Genetic Therapy/*methods ; Humans ; Neoplasms/*genetics/pathology/*therapy ; Neovascularization, Pathologic/genetics/therapy ; Treatment Outcome ; },
abstract = {Tumor angiogenesis is a critical target for cancer treatment and its inhibition has become a common anticancer approach following chemotherapy. However, due to the simultaneous activation of different compensatory molecular mechanisms that enhance tumor angiogenesis, clinically authorized anti-angiogenic medicines are ineffective. Additionally, medications used to treat cancer have an effect on normal body cells; nonetheless, more research is needed to create new cancer therapeutic techniques. With advances in molecular biology, it is now possible to use gene-editing technology to alter the genome and study the functional changes resulting from genetic manipulation. With the development of CRISPR/Cas9 technology, it has become a very powerful tool for altering the genomes of many organisms. It was determined that CRISPR/Cas9, which first appeared in bacteria as a part of an adaptive immune system, could be used, in modified forms, to alter genomes and function. In conclusion, CRISPR/Cas9 could be a major step forward to cancer management by providing patients with an effective method for dealing with cancers by dissecting the carcinogenesis pathways, identifying new biologic targets, and perhaps arming cancer cells with drugs. Hence, this review will discuss the current applications of CRISPR/Cas9 technology in tumor angiogenesis research for the purpose of cancer treatment.},
}
@article {pmid34655396,
year = {2022},
author = {Entesari, M and Zamani, M and Heidarizadeh, M and Moradi, R and Khakdan, F and Rafiei, F},
title = {An Insight Into Detection Pathways/Biosensors of Highly Infectious Coronaviruses.},
journal = {Molecular biotechnology},
volume = {64},
number = {4},
pages = {339-354},
pmid = {34655396},
issn = {1559-0305},
mesh = {Biosensing Techniques/methods ; COVID-19 Testing/instrumentation/*methods ; CRISPR-Cas Systems ; Electrochemical Techniques ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique/methods ; Humans ; Molecular Diagnostic Techniques/instrumentation/methods ; Neutralization Tests ; Nucleic Acid Amplification Techniques/instrumentation/methods ; Polymerase Chain Reaction/instrumentation/methods ; SARS-CoV-2/pathogenicity ; Surface Plasmon Resonance ; },
abstract = {The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.},
}
@article {pmid34653951,
year = {2022},
author = {Pfeiffer, ML and Winkler, J and Van Damme, D and Jacobs, TB and Nowack, MK},
title = {Conditional and tissue-specific approaches to dissect essential mechanisms in plant development.},
journal = {Current opinion in plant biology},
volume = {65},
number = {},
pages = {102119},
pmid = {34653951},
issn = {1879-0356},
support = {639234/ERC_/European Research Council/International ; 864952/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; *Gene Editing ; Plant Development/genetics ; RNA ; },
abstract = {Reverse genetics approaches are routinely used to investigate gene function. However, mutations, especially in critical genes, can lead to pleiotropic effects as severe as lethality, thus limiting functional studies in specific contexts. Approaches that allow for modifications of genes or gene products in a specific spatial or temporal setting can overcome these limitations. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technologies has not only revolutionized targeted genome modification in plants but also enabled new possibilities for inducible and tissue-specific manipulation of gene functions at the DNA and RNA levels. In addition, novel approaches for the direct manipulation of target proteins have been introduced in plant systems. Here, we review the current development in tissue-specific and conditional manipulation approaches at the DNA, RNA, and protein levels.},
}
@article {pmid34653714,
year = {2022},
author = {Tian, T and Qiu, Z and Jiang, Y and Zhu, D and Zhou, X},
title = {Exploiting the orthogonal CRISPR-Cas12a/Cas13a trans-cleavage for dual-gene virus detection using a handheld device.},
journal = {Biosensors &amp; bioelectronics},
volume = {196},
number = {},
pages = {113701},
doi = {10.1016/j.bios.2021.113701},
pmid = {34653714},
issn = {1873-4235},
mesh = {*African Swine Fever Virus ; Animals ; *Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; SARS-CoV-2 ; Swine ; },
abstract = {Although CRISPR-Cas12a and CRISPR-Cas13a systems work individually effective on gene detection, their multiplex detection capability is limited due to the lack of specific probe cleavage mechanism. Herein we present a high-efficient dual-gene diagnostic technique based on the orthogonal DNA/RNA collateral cleavage mechanism of Cas12a/Cas13a system. In this design, dual-gene amplified products from the multiplex recombinase polymerase amplification (RPA) were simultaneously detected by Cas12a and Cas13a assay in a single tube. The resulting orthogonal DNA/RNA collateral cleavage can specifically illuminate two spectral differentiated DNA and RNA probes, respectively. By integrating with the smartphone-based fluorescence readout, a portable detection platform is achieved. As a proof-of-concept, reliable dual-gene detection of SARS-CoV-2 and African Swine fever virus (ASFV) were demonstrated, exhibiting 100% sensitivity and specificity for clinical samples analysis (32 swab specimens for SARS-CoV-2 and 35 ASFV suspected swine blood samples). This developed portable dual-gene detection platform can provide accurate point-of-care screening of infectious diseases in resources-limited settings.},
}
@article {pmid34653700,
year = {2022},
author = {Liu, K and Gao, Y and Li, ZH and Liu, M and Wang, FQ and Wei, DZ},
title = {CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum.},
journal = {New biotechnology},
volume = {66},
number = {},
pages = {61-69},
doi = {10.1016/j.nbt.2021.10.003},
pmid = {34653700},
issn = {1876-4347},
mesh = {*CRISPR-Cas Systems/genetics ; DNA End-Joining Repair ; *Gene Editing ; Mycobacteriaceae/*genetics ; Plasmids ; },
abstract = {Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.},
}
@article {pmid34653674,
year = {2021},
author = {Katayama, S and Andou, M},
title = {Editing of DNA methylation using CRISPR/Cas9 and a ssDNA template in human cells.},
journal = {Biochemical and biophysical research communications},
volume = {581},
number = {},
pages = {20-24},
doi = {10.1016/j.bbrc.2021.10.018},
pmid = {34653674},
issn = {1090-2104},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Clone Cells ; Cytosine/metabolism ; *DNA Methylation ; DNA, Single-Stranded/*genetics/metabolism ; *Epigenesis, Genetic ; Gene Editing/*methods ; Gene Knock-In Techniques ; Genome ; HEK293 Cells ; Humans ; Neoplasm Proteins/*genetics/metabolism ; Plasmids/chemistry/metabolism ; Promoter Regions, Genetic ; RNA, Guide/genetics/metabolism ; Sp3 Transcription Factor/*genetics/metabolism ; Transcription, Genetic ; },
abstract = {Programmable DNA methylation is required for understanding of transcriptional regulation and elucidating gene functions. We previously reported that MMEJ-based promoter replacement enabled targeted DNA methylation in human cells. ssDNA-mediated knock-in has recently been reported to completely reduce random integrations. We speculated that by changing MMEJ-to ssDNA-based knock-in, targeted DNA methylation may be achieved through a hemimethylation-symmetric methylation pathway. We herein successfully developed a new system that enables the replacement of an unmethylated promoter with a methylated ssDNA promoter through ssDNA-based knock-in. A DNA methylation ratio of approximately 100% was achieved at the cancer-associated gene SP3 in HEK293 cells. The present results provide a promising framework for artificial epigenetic modifications.},
}
@article {pmid34653673,
year = {2021},
author = {Guo, Y and Wang, B and Chen, Y and Liang, M and Wang, H and Wang, C and Liang, H and Zhou, Y and Xi, J and Ci, L and Sun, R and Fei, J and Shen, R},
title = {A bioluminescence reporter mouse strain for in vivo imaging of CD8[+] T cell localization and function.},
journal = {Biochemical and biophysical research communications},
volume = {581},
number = {},
pages = {12-19},
doi = {10.1016/j.bbrc.2021.10.022},
pmid = {34653673},
issn = {1090-2104},
mesh = {Animals ; CD8 Antigens/genetics/metabolism ; CD8-Positive T-Lymphocytes/immunology/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Colonic Neoplasms/*diagnostic imaging/genetics/immunology ; Diagnostic Imaging/methods ; *Founder Effect ; Gene Editing/*methods ; Gene Knock-In Techniques ; Genes, Reporter ; *Genome ; Green Fluorescent Proteins/genetics/metabolism ; Heterografts ; Luciferases/genetics/metabolism ; Luminescent Measurements/methods ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Transgenic/*genetics/immunology ; RNA, Guide/genetics/metabolism ; Zygote/immunology/metabolism ; },
abstract = {CD8[+] T cells play a critical role during adaptive immune response, which often change locations and expand or contract in numbers under different states. In the past, many attempts to develop CD8[+]T cells that express luciferase in vivo have involved the use of viral transduction, which has drawbacks of hardly tracked via detection of luciferase signal in untouched natural states. Here, we generate a transgenic mouse model via CRISPR-mediated genome editing, C57BL/6-CD8a[em(IRES-AkaLuci-2A-EGFP)] knock-in mice(CD8a-Aka mice), as a novel tool for non-invasive imaging of CD8[+] T cells, which expressed a highly sensitive luciferase-Akaluciferase. Our study offers a convenient and robust tool for understanding fundamental CD8[+] T cell biology in experimental applications and preclinical translational studies.},
}
@article {pmid34653350,
year = {2021},
author = {Chen, PJ and Hussmann, JA and Yan, J and Knipping, F and Ravisankar, P and Chen, PF and Chen, C and Nelson, JW and Newby, GA and Sahin, M and Osborn, MJ and Weissman, JS and Adamson, B and Liu, DR},
title = {Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.},
journal = {Cell},
volume = {184},
number = {22},
pages = {5635-5652.e29},
pmid = {34653350},
issn = {1097-4172},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; R35 GM138167/GM/NIGMS NIH HHS/United States ; P30 CA072720/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; U54 HD090255/HD/NICHD NIH HHS/United States ; P50 HD105351/HD/NICHD NIH HHS/United States ; P01 CA065493/CA/NCI NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; DNA/metabolism ; DNA Mismatch Repair/genetics ; Female ; *Gene Editing ; Genes, Dominant ; Genome, Human ; Humans ; Male ; Models, Biological ; MutL Protein Homolog 1/genetics ; Mutation/genetics ; RNA/metabolism ; Reproducibility of Results ; },
abstract = {While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.},
}
@article {pmid34653285,
year = {2021},
author = {Piedrafita, A and Balayssac, S and Casemayou, A and Saulnier-Blache, JS and Lucas, A and Iacovoni, JS and Breuil, B and Chauveau, D and Decramer, S and Malet-Martino, M and Schanstra, JP and Faguer, S},
title = {Hepatocyte nuclear factor-1β shapes the energetic homeostasis of kidney tubule cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {11},
pages = {e21931},
doi = {10.1096/fj.202100782RR},
pmid = {34653285},
issn = {1530-6860},
mesh = {Acute Kidney Injury/metabolism ; Animals ; CRISPR-Cas Systems ; Cell Hypoxia/genetics ; Cell Line ; Cell Proliferation/genetics ; Cell Survival/genetics ; Epithelial Cells/*metabolism ; *Gene Deletion ; Gene Expression Regulation ; Gene Knockout Techniques/methods ; Glycolysis/*genetics ; Hepatocyte Nuclear Factor 1-beta/genetics/*metabolism ; Homeostasis/*genetics ; Humans ; Kidney Tubules, Proximal/*cytology ; Metabolome ; Mice ; Signal Transduction/*genetics ; Transcriptome ; },
abstract = {Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1β (HNF-1β) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1β deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined [1] H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b[-/-]), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1β in metabolism. Hnf1b[-/-] cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b[-/-] cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b[-/-] cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1β. HNF-1β shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI.},
}
@article {pmid34653201,
year = {2021},
author = {Mall, EM and Lecanda, A and Drexler, HCA and Raz, E and Schöler, HR and Schlatt, S},
title = {Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs.},
journal = {PloS one},
volume = {16},
number = {10},
pages = {e0258427},
pmid = {34653201},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage ; Cell Proliferation ; Gene Editing ; Gene Expression ; Germ Cells/cytology/*metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Neoplasm Proteins/genetics/*metabolism ; Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism ; Principal Component Analysis ; RNA/chemistry/genetics/metabolism ; RNA, Guide/metabolism ; RNA-Binding Proteins/genetics/metabolism ; Single-Cell Analysis ; },
abstract = {The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.},
}
@article {pmid34652874,
year = {2021},
author = {Morishita, M and Arimoto-Matsuzaki, K and Kitamura, M and Niimura, K and Iwasa, H and Maruyama, J and Hiraoka, Y and Yamamoto, K and Kitagawa, M and Miyamura, N and Nishina, H and Hata, Y},
title = {Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice shows the implication of Rassf6 in the regulation of NF-κB signaling.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {26},
number = {12},
pages = {999-1013},
doi = {10.1111/gtc.12901},
pmid = {34652874},
issn = {1365-2443},
mesh = {Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; Fibroblasts/metabolism ; Mice ; Mice, Knockout ; *Monomeric GTP-Binding Proteins/metabolism ; *NF-kappa B/genetics/metabolism ; Tumor Suppressor Protein p53/genetics ; },
abstract = {RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised, and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis showed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.},
}
@article {pmid34650270,
year = {2022},
author = {Jiang, T and Zhang, XO and Weng, Z and Xue, W},
title = {Deletion and replacement of long genomic sequences using prime editing.},
journal = {Nature biotechnology},
volume = {40},
number = {2},
pages = {227-234},
pmid = {34650270},
issn = {1546-1696},
support = {UH3 HL147367/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; K99 HL153940/HL/NHLBI NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Genome ; Mice ; RNA, Guide/genetics ; },
abstract = {Genomic insertions, duplications and insertion/deletions (indels), which account for ~14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100 base pairs (bp)). Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from ~1 kilobases (kb) to ~10 kb with a desired sequence (up to 60 bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method. PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.},
}
@article {pmid34650269,
year = {2022},
author = {Choi, J and Chen, W and Suiter, CC and Lee, C and Chardon, FM and Yang, W and Leith, A and Daza, RM and Martin, B and Shendure, J},
title = {Precise genomic deletions using paired prime editing.},
journal = {Nature biotechnology},
volume = {40},
number = {2},
pages = {218-226},
pmid = {34650269},
issn = {1546-1696},
support = {UM1 HG009408/HG/NHGRI NIH HHS/United States ; T32 HG000035/HG/NHGRI NIH HHS/United States ; T32 GM007270/GM/NIGMS NIH HHS/United States ; T32 GM136534/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Gene Editing/methods ; Genome ; Genomics ; *RNA, Guide/genetics/metabolism ; },
abstract = {Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements. In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1-30% editing efficiency. PRIME-Del can also be used to couple genomic deletions with short insertions, enabling deletions with junctions that do not fall at protospacer-adjacent motif sites. Finally, extended expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful for precise, flexible programming of genomic deletions, epitope tagging and, potentially, programming genomic rearrangements.},
}
@article {pmid34650077,
year = {2021},
author = {Lozito, TP and Londono, R and Sun, AX and Hudnall, ML},
title = {Introducing dorsoventral patterning in adult regenerating lizard tails with gene-edited embryonic neural stem cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6010},
pmid = {34650077},
issn = {2041-1723},
support = {R01 GM115444/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Body Patterning/genetics/physiology ; CRISPR-Cas Systems ; Cartilage ; Embryonic Stem Cells/*physiology ; Ependyma ; *Gene Editing ; Lizards/embryology/*genetics/*physiology ; Neural Stem Cells/*physiology ; Regeneration/*physiology ; Signal Transduction/genetics ; Smoothened Receptor/genetics ; Spinal Cord/physiology ; Tail/*physiology ; },
abstract = {Lizards regenerate amputated tails but fail to recapitulate the dorsoventral patterning achieved during embryonic development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. However, adult ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tubes. Both NTs and ETs contain neural stem cells (NSCs), but only embryonic NSC populations differentiate into roof plate identities when protected from endogenous Hedgehog signaling. NSCs were isolated from parthenogenetic lizard embryos, rendered unresponsive to Hedgehog signaling via CRISPR/Cas9 gene knockout of smoothened (Smo), and implanted back into clonally-identical adults to regulate tail regeneration. Here we report that Smo knockout embryonic NSCs oppose cartilage formation when engrafted to adult ETs, representing an important milestone in the creation of regenerated lizard tails with dorsoventrally patterned skeletal tissues.},
}
@article {pmid34650052,
year = {2021},
author = {Junqueira Alves, C and Dariolli, R and Haydak, J and Kang, S and Hannah, T and Wiener, RJ and DeFronzo, S and Tejero, R and Gusella, GL and Ramakrishnan, A and Alves Dias, R and Wojcinski, A and Kesari, S and Shen, L and Sobie, EA and Rodrigues Furtado de Mendonça, JP and Azeloglu, EU and Zou, H and Friedel, RH},
title = {Plexin-B2 orchestrates collective stem cell dynamics via actomyosin contractility, cytoskeletal tension and adhesion.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {6019},
pmid = {34650052},
issn = {2041-1723},
support = {R01 DK118222/DK/NIDDK NIH HHS/United States ; T32 HD075735/HD/NICHD NIH HHS/United States ; R01 NS107462/NS/NINDS NIH HHS/United States ; R56 NS073596/NS/NINDS NIH HHS/United States ; R01 NS073596/NS/NINDS NIH HHS/United States ; R01 NS092735/NS/NINDS NIH HHS/United States ; R01 DK106035/DK/NIDDK NIH HHS/United States ; },
mesh = {Actins ; Actomyosin/*metabolism ; CRISPR-Cas Systems ; Cell Adhesion/*physiology ; Cell Differentiation ; Cell-Matrix Junctions/metabolism ; Cytoskeleton/*metabolism ; Embryonic Stem Cells ; Gene Editing ; Gene Expression ; Humans ; Mechanotransduction, Cellular ; Morphogenesis ; Nerve Tissue Proteins/*genetics/*metabolism ; Neural Stem Cells ; Semaphorins ; Signal Transduction ; Stem Cells/*metabolism ; },
abstract = {During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thus establish a significant function of Plexin-B2 in orchestrating cytoskeletal tension and cell-cell/cell-matrix adhesion, therefore solidifying the importance of collective cell mechanics in governing stem cell physiology and tissue morphogenesis.},
}
@article {pmid34648747,
year = {2021},
author = {Ling, X and Chang, L and Chen, H and Gao, X and Yin, J and Zuo, Y and Huang, Y and Zhang, B and Hu, J and Liu, T},
title = {Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates.},
journal = {Molecular cell},
volume = {81},
number = {22},
pages = {4747-4756.e7},
doi = {10.1016/j.molcel.2021.09.021},
pmid = {34648747},
issn = {1097-4164},
mesh = {Acidaminococcus ; Animals ; Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism ; Endodeoxyribonucleases/*genetics ; Endonucleases/metabolism ; Escherichia coli/metabolism ; *Gene Editing ; Gene Knock-In Techniques ; Genetic Techniques ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; In Vitro Techniques ; K562 Cells ; Mice ; Mutagenesis ; RNA/metabolism ; Receptors, Chimeric Antigen/*metabolism ; Tandem Mass Spectrometry ; },
abstract = {The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.},
}
@article {pmid34647883,
year = {2021},
author = {Jordan, B},
title = {[In vivo gene editing for gene therapy].},
journal = {Medecine sciences : M/S},
volume = {37},
number = {10},
pages = {933-935},
doi = {10.1051/medsci/2021140},
pmid = {34647883},
issn = {1958-5381},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; Humans ; },
abstract = {In vivo gene editing has been achieved in a phase I clinical trial and results in a strong reduction of the level of a pathogenic protein. While preliminary, these results open the way for many applications in gene therapy.},
}
@article {pmid34646010,
year = {2021},
author = {Krause, WC and Rodriguez, R and Gegenhuber, B and Matharu, N and Rodriguez, AN and Padilla-Roger, AM and Toma, K and Herber, CB and Correa, SM and Duan, X and Ahituv, N and Tollkuhn, J and Ingraham, HA},
title = {Oestrogen engages brain MC4R signalling to drive physical activity in female mice.},
journal = {Nature},
volume = {599},
number = {7883},
pages = {131-135},
pmid = {34646010},
issn = {1476-4687},
support = {R01 DK121657/DK/NIDDK NIH HHS/United States ; R01 MH109907/MH/NIMH NIH HHS/United States ; 2T32 GM065094/NH/NIH HHS/United States ; K12 GM081266/GM/NIGMS NIH HHS/United States ; T32 GM065094/GM/NIGMS NIH HHS/United States ; R01 EY030138/EY/NEI NIH HHS/United States ; P30 DK098722/DK/NIDDK NIH HHS/United States ; F31 MH124365/MH/NIMH NIH HHS/United States ; /AHA/American Heart Association-American Stroke Association/United States ; R01 AG062331/AG/NIA NIH HHS/United States ; R01 MH113628/MH/NIMH NIH HHS/United States ; K01 DK098320/DK/NIDDK NIH HHS/United States ; R01 CA197139/CA/NCI NIH HHS/United States ; UL1 TR001881/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Brain/*physiology ; CRISPR-Cas Systems ; Energy Metabolism ; Estrogen Receptor alpha/metabolism ; Estrogens/deficiency/*metabolism ; Female ; Gene Editing ; Hippocampus/metabolism ; Male ; Melanocortins/metabolism ; Mice ; Neurons/metabolism ; Obesity/metabolism ; Physical Exertion/*physiology ; Receptor, Melanocortin, Type 4/*metabolism ; Rhombencephalon/metabolism ; Sedentary Behavior ; Sex Characteristics ; *Signal Transduction ; Ventromedial Hypothalamic Nucleus/cytology/physiology ; },
abstract = {Oestrogen depletion in rodents and humans leads to inactivity, fat accumulation and diabetes[1,2], underscoring the conserved metabolic benefits of oestrogen that inevitably decrease with age. In rodents, the preovulatory surge in 17β-oestradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we report that a subset of oestrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)[3-7] projects to arousal centres in the hippocampus and hindbrain, and enables oestrogen to rebalance energy allocation in female mice. Surges in E2 increase melanocortin-4 receptor (MC4R) signalling in these VMHvl neurons by directly recruiting oestrogen receptor-α (ERα) to the Mc4r gene. Sedentary behaviour and obesity in oestrogen-depleted female mice were reversed after chemogenetic stimulation of VMHvl neurons expressing both MC4R and ERα. Similarly, a long-term increase in physical activity is observed after CRISPR-mediated activation of this node. These data extend the effect of MC4R signalling - the most common cause of monogenic human obesity[8] - beyond the regulation of food intake and rationalize reported sex differences in melanocortin signalling, including greater disease severity of MC4R insufficiency in women[9]. This hormone-dependent node illuminates the power of oestrogen during the reproductive cycle in motivating behaviour and maintaining an active lifestyle in women.},
}
@article {pmid34645977,
year = {2021},
author = {Boddu, PC and Gupta, AK and Kim, JS and Neugebauer, KM and Waldman, T and Pillai, MM},
title = {Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {1184},
pmid = {34645977},
issn = {2399-3642},
support = {R01 HL133406/HL/NHLBI NIH HHS/United States ; T32 CA233414/CA/NCI NIH HHS/United States ; U54 DK106857/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*physiology ; DNA Breaks, Double-Stranded ; Dependovirus ; Homologous Recombination ; Humans ; Integrases/*physiology ; Introns/*physiology ; Neoplasms/enzymology/genetics/*physiopathology ; },
abstract = {Scalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.},
}
@article {pmid34645928,
year = {2021},
author = {Noguchi, H and Miyagi-Shiohira, C and Kinjo, T and Saitoh, I and Watanabe, M},
title = {In vivo evaluation of GG2-GG1/A2 element activity in the insulin promoter region using the CRISPR-Cas9 system.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20290},
pmid = {34645928},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Glucose Tolerance Test ; Homozygote ; Insulin/*genetics ; Insulin-Secreting Cells/*metabolism ; Islets of Langerhans/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mutation ; Pancreas/metabolism ; *Promoter Regions, Genetic ; Transcription Factors/metabolism ; Transcription, Genetic ; },
abstract = {The insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2-GG1/A2-C1 (bases - 149 to - 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2-GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2-GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR-Cas9 technology. The mice with homozygous mutations in the GG2-GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2-GG1/A2 element in mice is important for Ins transcription in vivo.},
}
@article {pmid34645668,
year = {2022},
author = {Graniel, JV and Bisht, K and Friedman, A and White, J and Perkey, E and Vanderbeck, A and Moroz, A and Carrington, LJ and Brandstadter, JD and Allen, F and Shami, AN and Thomas, P and Crayton, A and Manzor, M and Mychalowych, A and Chase, J and Hammoud, SS and Keegan, CE and Maillard, I and Nandakumar, J},
title = {Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline.},
journal = {Life science alliance},
volume = {5},
number = {1},
pages = {},
pmid = {34645668},
issn = {2575-1077},
support = {T32 AG000114/AG/NIA NIH HHS/United States ; T32 HL007439/HL/NHLBI NIH HHS/United States ; F30 HD097961/HD/NICHD NIH HHS/United States ; P30 CA046592/CA/NCI NIH HHS/United States ; DP2 HD091949/HD/NICHD NIH HHS/United States ; T32 CA009140/CA/NCI NIH HHS/United States ; T32 HD079342/HD/NICHD NIH HHS/United States ; T32 GM007863/GM/NIGMS NIH HHS/United States ; R25 GM086262/GM/NIGMS NIH HHS/United States ; R01 HD092084/HD/NICHD NIH HHS/United States ; R01 AG050509/AG/NIA NIH HHS/United States ; R01 GM120094/GM/NIGMS NIH HHS/United States ; F30 AI136315/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Dyskeratosis Congenita/*diagnosis/*genetics ; Fertility/genetics ; Gene Editing ; Germ Cells/*metabolism ; Hematopoiesis/*genetics ; Homozygote ; Humans ; Lymphopoiesis/genetics ; Male ; Mice ; Mice, Knockout ; Models, Molecular ; *Mutation ; Organ Specificity/genetics/immunology ; Sperm Count ; Structure-Activity Relationship ; Telomere-Binding Proteins/*genetics ; },
abstract = {Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.},
}
@article {pmid34645259,
year = {2021},
author = {Chen, X and Wang, L and He, F and Chen, G and Bai, L and He, K and Zhang, F and Xu, X},
title = {Label-Free Colorimetric Method for Detection of Vibrio parahaemolyticus by Trimming the G-Quadruplex DNAzyme with CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {93},
number = {42},
pages = {14300-14306},
doi = {10.1021/acs.analchem.1c03468},
pmid = {34645259},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems ; Colorimetry ; *DNA, Catalytic ; Humans ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; *Vibrio parahaemolyticus/genetics ; },
abstract = {Vibrio parahaemolyticus (V. parahaemolyticus), which may cause gastrointestinal disorders in humans, is a pathogen commonly found in seafood. There are many methods for detecting V. parahaemolyticus, yet they have some shortcomings, such as high cost, labor-intensiveness, and complicated operation, which are impractical for resource-limited settings. Herein, we present a sequence-specific, label-free, and colorimetric method for visual detection of V. parahaemolyticus. This method utilizes CRISPR/Cas12a to specifically recognize the loop-mediated isothermal amplification (LAMP) products for further trans-cleaving the G-quadruplex DNAzyme and depriving its peroxidase-mimicking activity. In this way, the results can be directly observed with the naked eyes via the color development of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS[2-]), which displays colorless for positive samples while green for target-free samples. We term such Cas12a-crRNA preventing ABTS[2-] from developing color by trimming the G-quadruplex DNAzyme as Cascade. The proposed method can detect 9.8 CFU (per reaction) of pure cultured V. parahaemolyticus, and the sensitivity is comparable to real-time LAMP. It has been applied for practical use and showed the capability to detect 6.1 × 10[2] CFU/mL V. parahaemolyticus in shrimp samples. Based on this, the newly established Cascade method can be employed as a universal biosensing strategy for pathogenic bacterial testing in the field.},
}
@article {pmid34644300,
year = {2021},
author = {Forsberg, KJ and Schmidtke, DT and Werther, R and Uribe, RV and Hausman, D and Sommer, MOA and Stoddard, BL and Kaiser, BK and Malik, HS},
title = {The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity.},
journal = {PLoS biology},
volume = {19},
number = {10},
pages = {e3001428},
pmid = {34644300},
issn = {1545-7885},
support = {R01 GM105691/GM/NIGMS NIH HHS/United States ; T32 AI007640/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; Contig Mapping ; DNA/*metabolism ; DNA, Superhelical/metabolism ; Genome, Bacterial ; Metagenomics ; Plasmids ; Prophages/genetics ; Protein Multimerization ; },
abstract = {To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid-binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs.},
}
@article {pmid34644057,
year = {2021},
author = {Zhang, X and Gu, S and Zheng, X and Peng, S and Li, Y and Lin, Y and Liang, S},
title = {A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a/Cpf1 for Pichia pastoris.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {2927-2937},
doi = {10.1021/acssynbio.1c00172},
pmid = {34644057},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/methods ; Genome, Bacterial/*genetics ; Metabolic Engineering/methods ; Pichia/*genetics ; RNA, Guide/genetics ; Synthetic Biology/methods ; },
abstract = {Pichia pastoris has been widely exploited for the heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value chemicals and pharmaceuticals. Effective synthetic biology tools for genetic engineering are essential for industrial and biotechnological research in this yeast. Here, we describe a novel and efficient genome editing method mediated by the CRISPR-Cpf1 system, which could facilitate the deletion of large DNA fragments and integration of multiplexed gene fragments. The CRISPR-Cpf1 system exhibited a precise and high editing efficiency for single-gene disruption (99 ± 0.8%), duplex genome editing (65 ± 2.5% to 80 ± 3%), and triplex genome editing (30 ± 2.5%). In addition, the deletion of large DNA fragments of 20kb and one-step integration of multiple genes were first achieved using the developed CRISPR-Cpf1 system. Taken together, this study provides an efficient and simple gene editing tool for P. pastoris. The novel multiloci gene integration method mediated by CRISPR-Cpf1 may accelerate the ability to engineer this methylotrophic yeast for metabolic engineering and genome evolution in both biotechnological and biomedical applications.},
}
@article {pmid34644046,
year = {2021},
author = {Hu, J and Song, H and Zhou, J and Liu, R and Lv, Y},
title = {Metal-Tagged CRISPR/Cas12a Bioassay Enables Ultrasensitive and Highly Selective Evaluation of Kanamycin Bioaccumulation in Fish Samples.},
journal = {Analytical chemistry},
volume = {93},
number = {42},
pages = {14214-14222},
doi = {10.1021/acs.analchem.1c03094},
pmid = {34644046},
issn = {1520-6882},
mesh = {Bioaccumulation ; Biological Assay ; *Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Humans ; Kanamycin ; },
abstract = {The abuse of antibiotics in modern life and aquaculture has become a worldwide problem. Trace amounts of antibiotics discharged into natural water are increased in organisms through bioaccumulation and ultimately harm human health. Herein, we report a metal-tagged CRISPR/Cas12a bioassay and apply it to an ultrasensitive and highly selective evaluation of antibiotics bioaccumulation in wild fish samples. We integrated an element-tagging report probe and collateral cleavage activity of CRISPR/Cas12a. With the recognition and capture of target kanamycin by a "locked-activated" system, the activator strand was subsequently released to activate the collateral cleavage activity of Cas12a, followed by the cleavage of free Tm-Rep. After SA-MB capture, the biotin terminal was modified, and the uncleaved probe of Tm-Rep was removed. The acidized supernate containing the element tag fragment could be directly detected with [169]Tm isotope monitoring by inductively coupled plasma mass spectrometry (ICPMS). With CRISPR/Cas12a biosensing and metal isotope detection by ICPMS, ultrasensitive and fast antibiotics analysis was realized with multiplex detection potential. Taking kanamycin as a modal analyte, a limit of detection as low as 4.06 pM was provided in a 30 min detection workflow. Besides, the bioaccumulation effect of kanamycin in a wild fish sample was also evaluated using the proposed strategy. We investigated the geographical distribution with Pseudorasbora parva samples collected in four different locations along a 600 km stretch of the Yangtze River. In addition, the bioaccumulation kinetics of antibiotics was evaluated in serum, muscle, and liver tissues of Pseudorasbora parva with 7 days of continuous feeding in a kanamycin-enriched environment.},
}
@article {pmid34643870,
year = {2022},
author = {Bhagwat, AC and Patil, AM and Saroj, SD},
title = {CRISPR/Cas 9-Based Editing in the Production of Bioactive Molecules.},
journal = {Molecular biotechnology},
volume = {64},
number = {3},
pages = {245-251},
pmid = {34643870},
issn = {1559-0305},
mesh = {Biological Factors/*metabolism ; CRISPR-Cas Systems ; Fungi/*genetics/metabolism ; Gene Editing/*methods ; Plant Breeding ; Plants/*genetics/metabolism ; Secondary Metabolism ; },
abstract = {Plants, fungi, and bacteria synthesize a wide range of secondary metabolites that exhibit diverse biological activities. These bioactives, due to their potential benefits in research and therapeutics, have gained immense industrial importance. There is a need to synthesize these bioactives at significantly higher concentrations using cost-effective measures to be economically viable. However, the broader study of industrially important secondary metabolites has been hindered, thus, far due to a shortage of reliable, comparatively easy, and highly effective gene manipulation techniques. With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas), there is a revolution in the field of genetic engineering. CRISPR/Cas system, due to its simplicity and ease of use. This has widened its application in plant breeding, strain improvement, and engineering the metabolic pathways involved in the biochemical synthesis of industrially valuable bioactive. This review briefly introduces the CRISPR/Cas9 system and summarizes the applications of CRISPR/Cas9-mediated editing tools for the production of plant and fungal-derived bioactives.},
}
@article {pmid34643819,
year = {2021},
author = {Gao, Y and Gao, K and Yang, H},
title = {Correction to: CRISPR/Cas: a potential gene-editing tool in the nervous system.},
journal = {Cell regeneration (London, England)},
volume = {10},
number = {1},
pages = {35},
pmid = {34643819},
issn = {2045-9769},
}
@article {pmid34643787,
year = {2021},
author = {Krogerus, K and Fletcher, E and Rettberg, N and Gibson, B and Preiss, R},
title = {Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching.},
journal = {Applied microbiology and biotechnology},
volume = {105},
number = {21-22},
pages = {8359-8376},
pmid = {34643787},
issn = {1432-0614},
mesh = {Beer ; *CRISPR-Cas Systems ; Fermentation ; Hybridization, Genetic ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase β-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high β-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for β-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high β-lyase activity, efficient wort fermentation, and lack of POF, all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. KEY POINTS: • CRISPR/Cas9-based mating-type switching was applied to industrial yeast strains. • Transformed strains could be readily mated to form intraspecific hybrids. • Hybrids exhibited heterosis for a number of brewing-relevant traits.},
}
@article {pmid34643425,
year = {2021},
author = {Markus, BM and Boydston, EA and Lourido, S},
title = {CRISPR-Mediated Transcriptional Repression in Toxoplasma gondii.},
journal = {mSphere},
volume = {6},
number = {5},
pages = {e0047421},
pmid = {34643425},
issn = {2379-5042},
support = {R01 AI144369/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression ; Gene Knockout Techniques ; RNA, Guide/*genetics ; Toxoplasma/*genetics ; },
abstract = {Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.},
}
@article {pmid34643250,
year = {2021},
author = {Wang, MY and Zhou, Y and Lai, GS and Huang, Q and Cai, WQ and Han, ZW and Wang, Y and Ma, Z and Wang, XW and Xiang, Y and Fang, SX and Peng, XC and Xin, HW},
title = {DNA barcode to trace the development and differentiation of cord blood stem cells (Review).},
journal = {Molecular medicine reports},
volume = {24},
number = {6},
pages = {},
pmid = {34643250},
issn = {1791-3004},
mesh = {Adult Stem Cells ; Animals ; Antigens, CD34 ; CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Barcoding, Taxonomic ; *Fetal Blood ; Humans ; *Stem Cells ; T-Lymphocytes ; Tissue Engineering ; },
abstract = {Umbilical cord blood transplantation was first reported in 1980. Since then, additional research has indicated that umbilical cord blood stem cells (UCBSCs) have various advantages, such as multi‑lineage differentiation potential and potent renewal activity, which may be induced to promote their differentiation into a variety of seed cells for tissue engineering and the treatment of clinical and metabolic diseases. Recent studies suggested that UCBSCs are able to differentiate into nerve cells, chondrocytes, hepatocyte‑like cells, fat cells and osteoblasts. The culture of UCBSCs has developed from feeder‑layer to feeder‑free culture systems. The classical techniques of cell labeling and tracing by gene transfection and fluorescent dye and nucleic acid analogs have evolved to DNA barcode technology mediated by transposon/retrovirus, cyclization recombination‑recombinase and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 strategies. DNA barcoding for cell development tracing has advanced to include single cells and single nucleic acid mutations. In the present study, the latest research findings on the development and differentiation, culture techniques and labeling and tracing of UCBSCs are reviewed. The present study may increase the current understanding of UCBSC biology and its clinical applications.},
}
@article {pmid34642480,
year = {2021},
author = {Mullard, A},
title = {CRISPR technologies are going to need a bigger toolbox.},
journal = {Nature reviews. Drug discovery},
volume = {20},
number = {11},
pages = {808-809},
doi = {10.1038/d41573-021-00177-6},
pmid = {34642480},
issn = {1474-1784},
mesh = {Biomedical Research/trends ; Biomedical Technology/instrumentation/*trends ; *CRISPR-Cas Systems ; Forecasting ; Humans ; },
}
@article {pmid34642413,
year = {2021},
author = {Abe, Y and Nakao, H and Goto, M and Tamano, M and Koebis, M and Nakao, K and Aiba, A},
title = {Efficient marmoset genome engineering by autologous embryo transfer and CRISPR/Cas9 technology.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {20234},
pmid = {34642413},
issn = {2045-2322},
mesh = {Animals ; Autografts ; CRISPR-Cas Systems ; Callithrix/*genetics ; Embryo Culture Techniques ; Embryo Transfer/*methods ; Female ; Fragile X Mental Retardation Protein/*genetics ; Genetic Engineering/*methods ; Models, Animal ; Mutation ; },
abstract = {Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for "Reduction" in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.},
}
@article {pmid34639221,
year = {2021},
author = {Kaur, M and Manchanda, P and Kalia, A and Ahmed, FK and Nepovimova, E and Kuca, K and Abd-Elsalam, KA},
title = {Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34639221},
issn = {1422-0067},
mesh = {Agrobacterium/*genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/*genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; *Genome, Plant ; Mutagenesis ; Plant Proteins/*genetics ; Plants/*genetics/metabolism ; Transformation, Genetic ; },
abstract = {Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.},
}
@article {pmid34639145,
year = {2021},
author = {Tan, KS and Wang, D and Lu, Z and Zhang, Y and Li, S and Lin, Y and Tan, W},
title = {CNPase, a 2',3'-Cyclic-nucleotide 3'-phosphodiesterase, as a Therapeutic Target to Attenuate Cardiac Hypertrophy by Enhancing Mitochondrial Energy Production.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34639145},
issn = {1422-0067},
mesh = {2',3'-Cyclic-Nucleotide Phosphodiesterases/*antagonists &amp; inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; Cardiomegaly/*prevention &amp; control ; *Disease Models, Animal ; *Energy Metabolism ; Male ; Mitochondria/*physiology ; Nucleotides, Cyclic/*metabolism ; Rats ; Rats, Sprague-Dawley ; Zebrafish ; },
abstract = {Heart failure is the end-stage of all cardiovascular diseases with a ~25% 5-year survival rate, and insufficient mitochondrial energy production to meet myocardial demand is the hallmark of heart failure. Mitochondrial components involved in the regulation of ATP production remain to be fully elucidated. Recently, roles of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) in the pathophysiological processes of heart diseases have emerged, implicated by evidence that mitochondrial CNPase proteins are associated with mitochondrial integrity under metabolic stress. In this study, a zebrafish heart failure model was established, by employing antisense morpholino oligonucleotides and the CRISPR-Cas9 gene-editing system, which recapitulates heart failure phenotypes including heart dysfunction, pericardial edema, ventricular enlargement, bradycardia, and premature death. The translational implications of CNPase in the pathophysiological process of heart failure were tested in a pressure overload-induced heart hypertrophy model, which was carried out in rats through transverse abdominal aorta constriction (TAAC). AAV9-mediated myocardial delivery of CNPase mitigated the hypertrophic response through the specific hydrolysis of 2'-3'-cyclic nucleotides, supported by the decrease of cardiac hypertrophy and fibrosis, the integrity of mitochondrial ultrastructure, and indicators of heart contractility in the AAV9-TAAC group. Finally, the biometrics of a mitochondrial respiration assay carried out on a Seahorse cellular energy analyzer demonstrated that CNPase protects mitochondrial respiration and ATP production from AngII-induced metabolic stress. In summary, this study provides mechanistic insights into CNPase-2',3'-cyclic nucleotide metabolism that protects the heart from energy starvation and suggests novel therapeutic approaches to treat heart failure by targeting CNPase activity.},
}
@article {pmid34639081,
year = {2021},
author = {Klimek-Chodacka, M and Gieniec, M and Baranski, R},
title = {Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34639081},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Daucus carota/*genetics/growth &amp; development/metabolism ; *Gene Editing ; Genetic Engineering/*methods ; Genome, Plant ; Polyethylene Glycols/*chemistry ; Protoplasts ; *RNA, Guide ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33-152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.},
}
@article {pmid34638976,
year = {2021},
author = {Lakatos, Z and Benkő, P and Juhász, G and Lőrincz, P},
title = {Drosophila Rab39 Attenuates Lysosomal Degradation.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34638976},
issn = {1422-0067},
mesh = {Animals ; Animals, Genetically Modified ; Autophagy/*genetics ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/*enzymology/*genetics ; Endocytosis/*genetics ; Larva/enzymology/genetics ; Lysosomes/*metabolism ; Phenotype ; rab GTP-Binding Proteins/genetics/*metabolism ; },
abstract = {Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, we found no failure in the early steps of endocytosis and autophagy. On the contrary, we found that Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. Our data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.},
}
@article {pmid34638922,
year = {2021},
author = {Ahmar, S and Ballesta, P and Ali, M and Mora-Poblete, F},
title = {Achievements and Challenges of Genomics-Assisted Breeding in Forest Trees: From Marker-Assisted Selection to Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34638922},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; *Forests ; Gene Editing/*methods ; Genome, Plant/*genetics ; Genome-Wide Association Study/methods ; Genomics/*methods ; Genotype ; Plant Breeding/*methods ; *Selection, Genetic ; Trees/*genetics ; },
abstract = {Forest tree breeding efforts have focused mainly on improving traits of economic importance, selecting trees suited to new environments or generating trees that are more resilient to biotic and abiotic stressors. This review describes various methods of forest tree selection assisted by genomics and the main technological challenges and achievements in research at the genomic level. Due to the long rotation time of a forest plantation and the resulting long generation times necessary to complete a breeding cycle, the use of advanced techniques with traditional breeding have been necessary, allowing the use of more precise methods for determining the genetic architecture of traits of interest, such as genome-wide association studies (GWASs) and genomic selection (GS). In this sense, main factors that determine the accuracy of genomic prediction models are also addressed. In turn, the introduction of genome editing opens the door to new possibilities in forest trees and especially clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). It is a highly efficient and effective genome editing technique that has been used to effectively implement targetable changes at specific places in the genome of a forest tree. In this sense, forest trees still lack a transformation method and an inefficient number of genotypes for CRISPR/Cas9. This challenge could be addressed with the use of the newly developing technique GRF-GIF with speed breeding.},
}
@article {pmid34638909,
year = {2021},
author = {Kato, Y and Tabata, H and Sato, K and Nakamura, M and Saito, I and Nakanishi, T},
title = {Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34638909},
issn = {1422-0067},
mesh = {Adenoviridae/*genetics/physiology ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/therapy/virology ; Cell Line, Tumor ; Gene Editing/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Hep G2 Cells ; Hepatitis B virus/*genetics/metabolism ; Hepatitis B, Chronic/genetics/therapy/virology ; Humans ; Liver Neoplasms/genetics/therapy/virology ; RNA, Guide/*genetics/metabolism ; Trans-Activators/*genetics/metabolism ; Viral Regulatory and Accessory Proteins/*genetics/metabolism ; Virus Replication/genetics ; },
abstract = {Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.},
}
@article {pmid34638696,
year = {2021},
author = {González Castro, N and Bjelic, J and Malhotra, G and Huang, C and Alsaffar, SH},
title = {Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34638696},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Feasibility Studies ; *Gene Editing ; Humans ; *Safety ; },
abstract = {Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.},
}
@article {pmid34638562,
year = {2021},
author = {Averina, OA and Permyakov, OA and Grigorieva, OO and Starshin, AS and Mazur, AM and Prokhortchouk, EB and Dontsova, OA and Sergiev, PV},
title = {Comparative Analysis of Genome Editors Efficiency on a Model of Mice Zygotes Microinjection.},
journal = {International journal of molecular sciences},
volume = {22},
number = {19},
pages = {},
pmid = {34638562},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Single-Stranded ; Gene Editing/*methods ; Homologous Recombination ; Mice ; Microinjections/methods ; Models, Animal ; RNA, Guide ; Zygote/*metabolism ; },
abstract = {Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.},
}
@article {pmid34635250,
year = {2022},
author = {Zhang, K and Fan, Z and Huang, Y and Ding, Y and Xie, M},
title = {A strategy combining 3D-DNA Walker and CRISPR-Cas12a trans-cleavage activity applied to MXene based electrochemiluminescent sensor for SARS-CoV-2 RdRp gene detection.},
journal = {Talanta},
volume = {236},
number = {},
pages = {122868},
pmid = {34635250},
issn = {1873-3573},
mesh = {COVID-19 ; *CRISPR-Cas Systems ; DNA ; Gold ; Humans ; Metal Nanoparticles ; RNA, Viral ; RNA-Dependent RNA Polymerase/*isolation &amp; purification ; *SARS-CoV-2 ; },
abstract = {Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.},
}
@article {pmid34634943,
year = {2021},
author = {Dong, S and Ye, Z and Tikhe, CV and Tu, ZJ and Zwiebel, LJ and Dimopoulos, G},
title = {Pleiotropic Odorant-Binding Proteins Promote Aedes aegypti Reproduction and Flavivirus Transmission.},
journal = {mBio},
volume = {12},
number = {5},
pages = {e0253121},
pmid = {34634943},
issn = {2150-7511},
support = {R01 AI121253/AI/NIAID NIH HHS/United States ; R01 AI141532/AI/NIAID NIH HHS/United States ; R56 AI127693/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/genetics/physiology/*virology ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Female ; Flavivirus/*physiology ; Flavivirus Infections/*transmission/virology ; Insect Proteins/*genetics/metabolism ; Male ; Mosquito Vectors/physiology/*virology ; Receptors, Odorant/classification/*genetics/metabolism ; Reproduction/genetics ; },
abstract = {Insect odorant-binding proteins (OBPs) are small soluble proteins that have been assigned roles in olfaction, but their other potential functions have not been extensively explored. Using CRISPR/Cas9-mediated disruption of Aedes aegypti Obp10 and Obp22, we demonstrate the pleiotropic contribution of these proteins to multiple processes that are essential for vectorial capacity. Mutant mosquitoes have impaired host-seeking and oviposition behavior, reproduction, and arbovirus transmission. Here, we show that Obp22 is linked to the male-determining sex locus (M) on chromosome 1 and is involved in male reproduction, likely by mediating the development of spermatozoa. Although OBP10 and OBP22 are not involved in flavivirus replication, abolition of these proteins significantly reduces transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. These results extend our current understanding of the role of insect OBPs in insect reproduction and transmission of human pathogens, making them essential determinants of vectorial capacity. IMPORTANCE Aedes aegypti is the major vector for many arthropod-borne viral diseases, such as dengue, Zika, and chikungunya viruses. Previous studies suggested that odorant-binding proteins (OBPs) may have diverse physiological functions beyond the olfactory system in mosquitoes; however, these hypothesized functions have not yet been demonstrated. Here, we have used CRISPR/Cas9-based genome editing to functionally delete (knock out) Obp10 and Obp22 in Aedes aegypti. We showed that disruption of Obp10 or Obp22 significantly impairs female and male reproductive capacity by adversely affecting blood feeding, oviposition, fecundity and fertility, and the development of spermatozoa. We also showed that disruption of Obp10 or Obp22 significantly reduces the transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. Thus, our study is not only significant in understanding the functions of OBPs in mosquito biology, but also shows that OBPs may represent potent flavivirus transmission-blocking targets. Our study is in this regard particularly timely and important from a translational and public health perspective.},
}
@article {pmid34634934,
year = {2021},
author = {Silvis, MR and Rajendram, M and Shi, H and Osadnik, H and Gray, AN and Cesar, S and Peters, JM and Hearne, CC and Kumar, P and Todor, H and Huang, KC and Gross, CA},
title = {Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli.},
journal = {mBio},
volume = {12},
number = {5},
pages = {e0256121},
pmid = {34634934},
issn = {2150-7511},
support = {F32 GM108222/GM/NIGMS NIH HHS/United States ; T32 GM007810/GM/NIGMS NIH HHS/United States ; F32 AI133917/AI/NIAID NIH HHS/United States ; R35 GM118061/GM/NIGMS NIH HHS/United States ; K22 AI137122/AI/NIAID NIH HHS/United States ; RM1 GM135102/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/*genetics ; Gene Knockdown Techniques/*methods ; *Gene Library ; Genes, Essential/*genetics ; High-Throughput Screening Assays ; *Transcription, Genetic ; },
abstract = {CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.},
}
@article {pmid34634758,
year = {2021},
author = {Qin, Z and Sun, L and Sun, X and Su, H and Gao, X},
title = {A CRISPR/Cas9 strategy for the generation of a FLNC knockout hESC line (WAe009-A-70) to model dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102562},
doi = {10.1016/j.scr.2021.102562},
pmid = {34634758},
issn = {1876-7753},
mesh = {*Arrhythmogenic Right Ventricular Dysplasia ; CRISPR-Cas Systems/genetics ; *Cardiomyopathy, Dilated/genetics ; Filamins/genetics ; *Human Embryonic Stem Cells ; Humans ; Mutation ; },
abstract = {The FLNC gene encodes the sarcomeric protein filamin C which plays a central role in cardiomyocytes. Truncating FLNC mutations (stop or frameshift etc.) also cause dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To further understand the exact role of FLNC in DCM, we have generated a human FLNC knockout cell line from the original embryonic stem cell line H9 by CRISPR/Cas9 gene editing technology in this study. The establishment cell line WAe009-A-70 have a compound heterozygous 4 bp deletion/13 bp deletion in the exon 1 of FLNC which resulted in a frameshift in the translation of FLNC. No filamin C protein was detected in cardiomyocytes differentiated from this cell line. Moreover, WAe009-A-70 also expressed pluripotency markers, maintained the ability to differentiate into the three germ layers in vitro and had a normal karyotype.},
}
@article {pmid34633749,
year = {2022},
author = {Yu, Y and Pan, Z and Wang, X and Bian, X and Wang, W and Liang, Q and Kou, M and Ji, H and Li, Y and Ma, D and Li, Z and Sun, J},
title = {Targeting of SPCSV-RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease.},
journal = {Molecular plant pathology},
volume = {23},
number = {1},
pages = {104-117},
pmid = {34633749},
issn = {1364-3703},
mesh = {CRISPR-Cas Systems/genetics ; *Ipomoea batatas/genetics ; Plant Diseases/genetics ; RNA Interference ; *Virus Diseases ; },
abstract = {Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis-related factors (i.e., SPCSV-RNase3) by using the CRISPR-Cas13 technique. First, the RNA targeting activity of four CRISPR-Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV-RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3-targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV-GFP and cucumber mosaic virus CMV-RNase3 co-infection. Moreover, transgenic sweet potato plants carrying an RNase3-targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD-prevalent regions.},
}
@article {pmid34633046,
year = {2021},
author = {Tanaka, N and Mogi, Y and Fujiwara, T and Yabe, K and Toyama, Y and Higashiyama, T and Yoshida, Y},
title = {CZON-cutter - a CRISPR-Cas9 system for multiplexed organelle imaging in a simple unicellular alga.},
journal = {Journal of cell science},
volume = {134},
number = {21},
pages = {},
doi = {10.1242/jcs.258948},
pmid = {34633046},
issn = {1477-9137},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Nucleus/genetics ; Gene Editing ; Humans ; RNA, Guide ; *Rhodophyta ; },
abstract = {The unicellular alga Cyanidioschyzon merolae has a simple cellular structure; each cell has one nucleus, one mitochondrion, one chloroplast and one peroxisome. This simplicity offers unique advantages for investigating organellar proliferation and the cell cycle. Here, we describe CZON-cutter, an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease for targeted editing of any locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants using fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows us to validate phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid34632687,
year = {2022},
author = {Calvache, C and Vazquez-Vilar, M and Selma, S and Uranga, M and Fernández-Del-Carmen, A and Daròs, JA and Orzáez, D},
title = {Strong and tunable anti-CRISPR/Cas activities in plants.},
journal = {Plant biotechnology journal},
volume = {20},
number = {2},
pages = {399-408},
pmid = {34632687},
issn = {1467-7652},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Indoleacetic Acids ; Plants/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.},
}
@article {pmid34632125,
year = {2021},
author = {Chen, Z and Sun, J and Guan, Y and Li, M and Lou, C and Wu, B},
title = {Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E. coli.},
journal = {Synthetic and systems biotechnology},
volume = {6},
number = {4},
pages = {326-334},
pmid = {34632125},
issn = {2405-805X},
abstract = {The development of base editing (BE) technology has opened a new avenue for research studies in bacteriology, particularly for bacterial species in which the DNA double-strand breaks (DSBs) introduced by CRISPR/Cas system would lead to cell death. However, a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions, such as GC content. Herein, we developed a broad-spectrum DNase-inactive Cpf1 (dCpf1) variant from Francisella novicida (bsdFnCpf1) through directed evolution. The resulting optimized mutant showed a substantially expanded targeting range, including previously non-canonical protospacer-adjacent motifs (PAMs), especially the GC-rich PAMs. Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor (UGI) were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E. coli genome without compromising cell growth. We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1.},
}
@article {pmid34631795,
year = {2021},
author = {Feng, R and Patil, S and Zhao, X and Miao, Z and Qian, A},
title = {RNA Therapeutics - Research and Clinical Advancements.},
journal = {Frontiers in molecular biosciences},
volume = {8},
number = {},
pages = {710738},
pmid = {34631795},
issn = {2296-889X},
abstract = {RNA therapeutics involve the use of coding RNA such as mRNA as well as non-coding RNAs such as small interfering RNAs (siRNA), antisense oligonucleotides (ASO) to target mRNA, aptamers, ribozymes, and clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR/Cas) endonuclease to target proteins and DNA. Due to their diverse targeting ability and research in RNA modification and delivery systems, RNA-based formulations have emerged as suitable treatment options for many diseases. Therefore, in this article, we have summarized different RNA therapeutics, their targeting strategies, and clinical progress for various diseases as well as limitations; so that it might help researchers formulate new and advanced RNA therapeutics for various diseases. Additionally, U.S. Food and Drug Administration (USFDA)-approved RNA-based therapeutics have also been discussed.},
}
@article {pmid34631280,
year = {2021},
author = {Villiger, L and Schmidheini, L and Mathis, N and Rothgangl, T and Marquart, K and Schwank, G},
title = {Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope.},
journal = {Molecular therapy. Nucleic acids},
volume = {26},
number = {},
pages = {502-510},
pmid = {34631280},
issn = {2162-2531},
abstract = {Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.},
}
@article {pmid34630494,
year = {2021},
author = {Venezia, M and Creasey Krainer, KM},
title = {Current Advancements and Limitations of Gene Editing in Orphan Crops.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {742932},
pmid = {34630494},
issn = {1664-462X},
abstract = {Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world's most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops.},
}
@article {pmid34628852,
year = {2021},
author = {Qian, H and Wei, W and Chen, XA and Mo, XT and Ge, M and Zhao, QW and Li, YQ},
title = {Strategy for Producing the High-Quality Glycopeptide Antibiotic A82846B in Amycolatopsis orientalis Based on the CRISPR-Cas12a System.},
journal = {ACS synthetic biology},
volume = {10},
number = {11},
pages = {3009-3016},
doi = {10.1021/acssynbio.1c00317},
pmid = {34628852},
issn = {2161-5063},
mesh = {Actinomycetales/genetics/metabolism ; Amycolatopsis/genetics/metabolism ; Anti-Bacterial Agents/*metabolism ; CRISPR-Cas Systems/*genetics ; Drug Resistance, Microbial/genetics ; Glycopeptides/*genetics/*metabolism ; Lipoglycopeptides/genetics/metabolism ; Multigene Family/genetics ; Vancomycin/metabolism ; },
abstract = {Oritavancin is a new-generation semisynthetic lipoglycopeptide antibiotic used to prevent the spread of vancomycin-resistant Gram-positive bacteria. The glycopeptide A82846B is the direct precursor of oritavancin. Considering the structural similarity between A82846B and vancomycin, the vancomycin producer Amycolatopsis orientalis was used as a chassis for the construction of a strain producing high-quality A82846B. To construct the A82846B synthetic pathway, we established a highly efficient CRISPR-Cas12a system by optimizing the conditions of conjugation and by screening the regulatory elements in the A. orientalis, which is difficult to be genetically manipulated. The efficiency of gene knockout was almost 100%. The glycosyltransferases module (gtfDE) and glycosyl synthesis module (vcaAEBD) in the vancomycin gene cluster were replaced with the corresponding glycosyltransferases module (gtfABC) and glycosyl synthesis module (evaAEBD) in the A82846B cluster, respectively. A82846B was successfully produced by the artificially constructed synthetic pathway. Moreover, the titer of A82846B was increased 80% by expressing the pathway-specific regulatory strR. This strategy has excellent potential for remodification of natural products to solve antibiotic resistance.},
}
@article {pmid34628247,
year = {2021},
author = {Thongsin, N and Wattanapanitch, M},
title = {Generation of B2M bi-allelic knockout human induced pluripotent stem cells (MUSIi001-A-1) using a CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102551},
doi = {10.1016/j.scr.2021.102551},
pmid = {34628247},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; HLA Antigens ; Humans ; *Induced Pluripotent Stem Cells ; Regenerative Medicine ; Transplantation, Homologous ; *beta 2-Microglobulin/genetics ; },
abstract = {Allogeneic cell-based therapy is emerging as a promising approach in regenerative medicine. However, rejection of allograft due to mismatch of human leukocyte antigens (HLAs) remains a major concern after transplantation. Here, we generated a homozygous B2M knockout induced pluripotent stem cell (iPSC) line, lacking the expression of HLA class I (HLA-I) molecules, using a CRISPR/Cas9 system. The established iPSC line, MUSIi001-A-1, can serve as an in vitro model for studying immunological responses against allogeneic grafts and provides a prototype for "off-the-shelf" allogeneic cell products for future cell-based therapy.},
}
@article {pmid34628053,
year = {2021},
author = {Rosanwo, TO and Bauer, DE},
title = {Editing outside the body: Ex vivo gene-modification for β-hemoglobinopathy cellular therapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3163-3178},
pmid = {34628053},
issn = {1525-0024},
support = {OT2 HL154984/HL/NHLBI NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/genetics/therapy ; Animals ; Biomarkers ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/adverse effects/*methods ; Disease Management ; *Gene Editing/methods ; Genetic Predisposition to Disease ; Genetic Therapy/adverse effects/*methods ; Genetic Vectors ; Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells/metabolism ; Hemoglobinopathies/*genetics/*therapy ; Humans ; beta-Globins/*genetics ; beta-Thalassemia/genetics/therapy ; },
abstract = {Genome editing produces genetic modifications in somatic cells, offering novel curative possibilities for sickle cell disease and β-thalassemia. These opportunities leverage clinical knowledge of hematopoietic stem cell transplant and gene transfer. Advantages to this mode of ex vivo therapy include locus-specific alteration of patient hematopoietic stem cell genomes, lack of allogeneic immune response, and avoidance of insertional mutagenesis. Despite exciting progress, many aspects of this approach remain to be optimized for ideal clinical implementation, including the efficiency and specificity of gene modification, delivery to hematopoietic stem cells, and robust and nontoxic engraftment of gene-modified cells. This review highlights genome editing as compared to other genetic therapies, the differences between editing strategies, and the clinical prospects and challenges of implementing genome editing as a novel treatment. As the world's most common monogenic disorders, the β-hemoglobinopathies are at the forefront of bringing genome editing to the clinic and hold promise for molecular medicine to address human disease at its root.},
}
@article {pmid34627381,
year = {2021},
author = {Lin, Q and Le, QA and Takebayashi, K and Thongkittidilok, C and Wittayarat, M and Hirata, M and Tanihara, F and Otoi, T},
title = {Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events.},
journal = {BMC research notes},
volume = {14},
number = {1},
pages = {389},
pmid = {34627381},
issn = {1756-0500},
mesh = {Animals ; Blastocyst ; CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide ; Swine ; *Zygote ; },
abstract = {OBJECTIVE: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection.<br><br>RESULTS: Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15&#xa0;h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5&#xa0;h. Lipofection of zygotes collected at 10 and 15&#xa0;h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10&#xa0;h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20&#xa0;h. The blastocyst formation rate of zygotes treated for 20&#xa0;h was significantly lower (p&#x2009;<&#x2009;0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.},
}
@article {pmid34625344,
year = {2021},
author = {Siddiqui, HA and Harvey-Samuel, T and Mansoor, S},
title = {Gene drive: a faster route to plant improvement.},
journal = {Trends in plant science},
volume = {26},
number = {12},
pages = {1204-1206},
doi = {10.1016/j.tplants.2021.09.005},
pmid = {34625344},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems ; *Gene Drive Technology ; Gene Editing ; Plant Breeding ; Plants/genetics ; },
abstract = {Gene drives for control of vector-borne diseases have been demonstrated in insects but remain challenging in plants. Theoretically, they could be transformative in speeding breeding programs and contributing to food security through providing novel weed control methods. Zhang et al. now report the possibility of implementing gene drive in plants for the first time.},
}
@article {pmid34624801,
year = {2022},
author = {Ma, L and Yin, L and Li, X and Chen, S and Peng, L and Liu, G and Ye, S and Zhang, W and Man, S},
title = {A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {195},
number = {},
pages = {113646},
pmid = {34624801},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Gold ; Humans ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; SARS-CoV-2 ; Smartphone ; },
abstract = {The pandemic of coronavirus disease 2019 (COVID-19) resulted from novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide concern. It is imperative to develop rapid, sensitive, and specific biosensing methods. Herein, we developed a CRISPR-Cas12a powered visual biosensor with a smartphone readout for ultrasensitive and selective detection of SARS-CoV-2. Simply, the SARS-CoV-2 derived nucleic acids triggered CRISPR-Cas12a based indiscriminate degradation of a single-stranded DNA that was supposed to link two gold nanoparticles, inducing the dis-aggregation of gold nanoparticles and thus generating observable color changes. This change can be readily distinguished by naked eyes as well as a smartphone with a Color Picker App. The proposed biosensor was successfully applied to detect SARS-CoV-2 gene in synthetic vectors, transcribed RNA and SARS-CoV-2 pseudoviruses. It rendered "single copy resolution" as evidenced by the 1 copy/μL limit of detection of pseudoviruses with no cross-reactivity. When the developed biosensor was challenged with SARS-CoV-2 clinical bio-samples, it provided 100% agreement (both positive and negative) with qPCR results. The sample-to-result time was roughly 90 min. Our work provides a novel and robust technology for ultrasensitive detection of SARS-CoV-2 that could be used clinically.},
}
@article {pmid34624800,
year = {2022},
author = {Fu, X and Sun, J and Ye, Y and Zhang, Y and Sun, X},
title = {A rapid and ultrasensitive dual detection platform based on Cas12a for simultaneous detection of virulence and resistance genes of drug-resistant Salmonella.},
journal = {Biosensors &amp; bioelectronics},
volume = {195},
number = {},
pages = {113682},
doi = {10.1016/j.bios.2021.113682},
pmid = {34624800},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; *Pharmaceutical Preparations ; Salmonella/genetics ; Virulence ; },
abstract = {Accurate, sensitive, and rapid detection of Salmonella and determination of whether it carries drug resistance genes plays an important role in guiding the clinical medication of salmonellosis and laying a foundation for studying the mechanism of drug resistance transmission of Salmonella. Here, a novel nontransferable, ultrasensitive dual detection platform (Cas12a-Ddp) was developed. The round cap allowed for temporary storage of more Cas12a detection solution than flat cap, enabling one-pot assays and reducing aerosol contamination. The results were read out in dual mode by the microplate reader and UV visualization to achieve sensitive dual-target detection of the virulence genes and drug resistance genes of Salmonella simultaneously, with the possibility of onsite detection. Cas12a-Ddp was combined with multiple polymerase chain reactions and recombinase polymerase amplifications successively. An ultrasensitive dual detection limit of 1 CFU/mL was obtained without any cross-reaction within 40 min. This was an improvement of 1-2 orders of magnitude over the existing methods. Cas12a-Ddp overcame the influence of proteins and fat in liquid matrix foods. It was used for the detection of drug-resistant Salmonella in milk and skim milk powder, also with the dual detection limit of 1 CFU/mL and spiked recovery of 68.58%-158.49%. It was also used for the analysis of Salmonella resistance rate analysis. The Cas12a-Ddp provided a reliable, fast, sensitive, and practical multi-CRISPR detection platform.},
}
@article {pmid34624524,
year = {2021},
author = {Urban, MW and Lo, C and Bodinayake, KK and Brunswick, CA and Murakami, S and Heimann, AC and Kwapis, JL},
title = {The circadian clock gene Per1 modulates context fear memory formation within the retrosplenial cortex in a sex-specific manner.},
journal = {Neurobiology of learning and memory},
volume = {185},
number = {},
pages = {107535},
pmid = {34624524},
issn = {1095-9564},
support = {R00 AG056596/AG/NIA NIH HHS/United States ; R21 AG068444/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Circadian Clocks/genetics/physiology ; Conditioning, Classical/physiology ; Fear/*physiology ; Female ; Gene Editing ; Gyrus Cinguli/metabolism/*physiology ; Male ; *Memory Consolidation/physiology ; Mice ; Mice, Inbred C57BL ; Period Circadian Proteins/*physiology ; Sex Factors ; },
abstract = {Context memory formation is a complex process that requires transcription in many subregions of the brain including the dorsal hippocampus and retrosplenial cortex. One critical gene necessary for memory formation is the circadian gene Period1 (Per1), which has been shown to function in the dorsal hippocampus to modulate spatial memory in addition to its well-documented role in regulating the diurnal clock within the suprachiasmatic nucleus (SCN). We recently found that alterations in Per1 expression in the dorsal hippocampus can modulate spatial memory formation, with reduced hippocampal Per1 impairing memory and overexpression of Per1 ameliorating age-related impairments in spatial memory. Whether Per1 similarly functions within other memory-relevant brain regions is currently unknown. Here, to test whether Per1 is a general mechanism that modulates memory across the brain, we tested the role of Per1 in the retrosplenial cortex (RSC), a brain region necessary for context memory formation. First, we demonstrate that context fear conditioning drives a transient increase in Per1 mRNA expression within the anterior RSC that peaks 60 m after training. Next, using HSV-CRISPRi-mediated knockdown of Per1, we show that reducing Per1 within the anterior RSC before context fear acquisition impairs memory in both male and female mice. In contrast, overexpressing Per1 with either HSV-CRISPRa or HSV-Per1 before context fear acquisition drives a sex-specific memory impairment; males show impaired context fear memory whereas females are not affected by Per1 overexpression. Finally, as Per1 levels are known to rhythmically oscillate across the day/night cycle, we tested the possibility that Per1 overexpression might have different effects on memory depending on the time of day. In contrast to the impairment in memory we observed during the daytime, Per1 overexpression has no effect on context fear memory during the night in either male or female mice. Together, our results indicate that Per1 modulates memory in the anterior retrosplenial cortex in addition to its documented role in regulating memory within the dorsal hippocampus, although this role may differ between males and females.},
}
@article {pmid34622618,
year = {2021},
author = {Yu, C and Mo, J and Zhao, X and Li, G and Zhang, X},
title = {[CRISPR/Cas-mediated DNA base editing technology and its application in biomedicine and agriculture].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {9},
pages = {3071-3087},
doi = {10.13345/j.cjb.200693},
pmid = {34622618},
issn = {1872-2075},
mesh = {Agriculture ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing ; Technology ; },
abstract = {In recent years, the genome editing technologies based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) have developed rapidly. The system can use homologous directed recombination (HDR) to achieve precise editing that it medicated, but the efficiency is extremely low, which limits its application in agriculture and biomedical fields. As an emerging genome editing technology, the CRISPR/Cas-mediated DNA base editing technologies can achieve targeted mutations of bases without generating double-strand breaks, and has higher editing efficiency and specificity compared with CRISPR/Cas-mediated HDR editing. At present, cytidine base editors (CBEs) that can mutate C to T, adenine base editors (ABEs) that can mutate A to G, and prime editors (PEs) that enable arbitrary base conversion and precise insertion and deletion of small fragments, have been developed. In addition, glycosylase base editors (GBEs) capable of transitioning from C to G and double base editors capable of editing both A and C simultaneously, have been developed. This review summarizes the development, advances, advantages and limitations of several DNA base editors. The successful applications of DNA base editing technology in biomedicine and agriculture, together with the prospects for further optimization and selection of DNA base editors, are discussed.},
}
@article {pmid34622617,
year = {2021},
author = {Lin, S and Zhong, X and Ma, L and Qiao, J and Liu, Y},
title = {[Advances of CRISPR/dCas9 system in live cell imaging].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {9},
pages = {3061-3070},
doi = {10.13345/j.cjb.200694},
pmid = {34622617},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; Chromatin ; Endonucleases ; *Gene Editing ; },
abstract = {The study of distinct genes, chromosomes and the spatio-temporal relationships between them is of great significance in genetics, developmental biology and biomedicine. CRISPR/Cas9 has become the most widely used gene editing tool due to its excellent targeting ability. Recently, researchers have developed a series of advanced live cell imaging techniques based on the nuclease-inactivated mutant of Cas9 (dCas9), providing rapid and convenient tools for high-resolution imaging of specific sites in the chromatin and genome. This review summarizes the advances of CRISPR/dCas9 system in live cell imaging from three aspects, including the strategies of cell delivery, optimization of the fluorescence signals, as well as orthogonal and multicolor imaging. Furthermore, we shed light on the development trends and prospects of this field.},
}
@article {pmid34621019,
year = {2021},
author = {Gao, S and Soares, F and Wang, S and Wong, CC and Chen, H and Yang, Z and Liu, W and Go, MYY and Ahmed, M and Zeng, Y and O'Brien, CA and Sung, JJY and He, HH and Yu, J},
title = {CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer.},
journal = {Oncogene},
volume = {40},
number = {48},
pages = {6601-6613},
pmid = {34621019},
issn = {1476-5594},
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology ; Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; Cholesterol/*biosynthesis/chemistry ; Colonic Neoplasms/*drug therapy/genetics/metabolism/pathology ; Dimethylallyltranstransferase/*antagonists &amp; inhibitors ; Drug Resistance, Neoplasm/*drug effects ; Farnesyltranstransferase/*antagonists &amp; inhibitors ; Fluorouracil/administration &amp; dosage ; Geranyltranstransferase/*antagonists &amp; inhibitors ; Humans ; Lovastatin/administration &amp; dosage ; Male ; Mice ; Mice, Nude ; Neoplastic Stem Cells/*drug effects/metabolism/pathology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; Zoledronic Acid/administration &amp; dosage ; },
abstract = {Cancer stem cells (CSCs) are responsible for tumor progression, recurrence, and drug resistance. To identify genetic vulnerabilities of colon cancer, we performed targeted CRISPR dropout screens comprising 657 Drugbank targets and 317 epigenetic regulators on two patient-derived colon CSC-enriched spheroids. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells yielded therapeutic candidates. We unraveled 44 essential genes for colon CSC-enriched spheroids propagation, including key cholesterol biosynthetic genes (HMGCR, FDPS, and GGPS1). Cholesterol biosynthesis was induced in colon cancer tissues, especially CSC-enriched spheroids. The genetic and pharmacological inhibition of HMGCR/FDPS impaired self-renewal capacity and tumorigenic potential of the spheroid models in vitro and in vivo. Mechanistically, HMGCR or FDPS depletion impaired cancer stemness characteristics by activating TGF-β signaling, which in turn downregulated expression of inhibitors of differentiation (ID) proteins, key regulators of cancer stemness. Cholesterol and geranylgeranyl diphosphate (GGPP) rescued the growth inhibitory and signaling effect of HMGCR/FDPS blockade, implying a direct role of these metabolites in modulating stemness. Finally, cholesterol biosynthesis inhibitors and 5-FU demonstrated antitumor synergy in colon CSC-enriched spheroids, tumor organoids, and xenografts. Taken together, our study unravels novel genetic vulnerabilities of colon CSC-enriched spheroids and suggests cholesterol biosynthesis as a potential target in conjunction with traditional chemotherapy for colon cancer treatment.},
}
@article {pmid34620839,
year = {2021},
author = {Zhu, XR and Peng, SQ and Wang, L and Chen, XY and Feng, CX and Liu, YY and Chen, MB},
title = {Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer.},
journal = {Cell death &amp; disease},
volume = {12},
number = {10},
pages = {918},
pmid = {34620839},
issn = {2041-4889},
mesh = {Adult ; Aged ; Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Cohort Studies ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Male ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; *Molecular Targeted Therapy ; Pancreatic Neoplasms/*drug therapy/*enzymology/genetics/pathology ; Phosphoenolpyruvate Carboxykinase (ATP)/genetics/*metabolism ; RNA, Messenger/genetics/metabolism ; Signal Transduction ; Xenograft Model Antitumor Assays ; },
abstract = {Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.},
}
@article {pmid34620062,
year = {2022},
author = {Stevanovic, M and Piotter, E and McClements, ME and MacLaren, RE},
title = {CRISPR Systems Suitable for Single AAV Vector Delivery.},
journal = {Current gene therapy},
volume = {22},
number = {1},
pages = {1-14},
doi = {10.2174/1566523221666211006120355},
pmid = {34620062},
issn = {1875-5631},
support = {MC_PC_18059/MRC_/Medical Research Council/United Kingdom ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; *Gene Editing ; Genetic Vectors/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/- Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.},
}
@article {pmid34619744,
year = {2021},
author = {Karvelis, T and Druteika, G and Bigelyte, G and Budre, K and Zedaveinyte, R and Silanskas, A and Kazlauskas, D and Venclovas, &#x10c; and Siksnys, V},
title = {Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease.},
journal = {Nature},
volume = {599},
number = {7886},
pages = {692-696},
pmid = {34619744},
issn = {1476-4687},
mesh = {Base Sequence ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA Transposable Elements/*genetics ; Deinococcus/*enzymology/*genetics ; Deoxyribonuclease I/*genetics/*metabolism ; Escherichia coli/genetics ; Gene Editing ; HEK293 Cells ; Humans ; Nucleotide Motifs ; RNA/*genetics ; },
abstract = {Transposition has a key role in reshaping genomes of all living organisms[1]. Insertion sequences of IS200/IS605 and IS607 families[2] are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established[3-5]. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases[6-8]. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5'-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.},
}
@article {pmid34619643,
year = {2021},
author = {Pavlinov, I and Farkhondeh, A and Yang, S and Xu, M and Cheng, YS and Beers, J and Zou, J and Liu, C and Might, M and Rodems, S and Baumgärtel, K and Zheng, W},
title = {Generation of two gene corrected human isogenic iPSC lines (NCATS-CL6104 and NCATS-CL6105) from a patient line (NCATS-CL6103) carrying a homozygous p.R401X mutation in the NGLY1 gene using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102554},
pmid = {34619643},
issn = {1876-7753},
support = {Z99 TR999999/ImNIH/Intramural NIH HHS/United States ; ZIA TR000018/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Congenital Disorders of Glycosylation/genetics ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; Mutation/genetics ; National Center for Advancing Translational Sciences (U.S.) ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/*deficiency/genetics ; United States ; },
abstract = {NGLY1 deficiency is a rare recessive genetic disease caused by mutations in the NGLY1 gene which codes for N-glycanase 1 (NGLY1). Here, we report the generation of two gene corrected iPSC lines using a patient-derived iPSC line (NCATS-CL6103) that carried a homozygous p.R401X mutation in the NGLY1 gene. These lines contain either one (NCATS-CL6104) or two (NCATS-CL6105) CRISPR/Cas9 corrected alleles of NGLY1. This pair of NGLY1 mutation corrected iPSC lines can be used as a control for the NCATS-CL6103 which serves as a cell-based NGLY1 disease model for the study of the disease pathophysiology and evaluation of therapeutics under development.},
}
@article {pmid34619370,
year = {2021},
author = {Rees, HA and Minella, AC and Burnett, CA and Komor, AC and Gaudelli, NM},
title = {CRISPR-derived genome editing therapies: Progress from bench to bedside.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3125-3139},
pmid = {34619370},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering ; *Genetic Therapy/methods/trends ; Humans ; Models, Animal ; RNA, Guide ; Recombinational DNA Repair ; Translational Research, Biomedical/methods/trends ; },
abstract = {The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.},
}
@article {pmid34619328,
year = {2022},
author = {Chen, K and Ke, R and Du, M and Yi, Y and Chen, Y and Wang, X and Yao, L and Liu, H and Hou, X and Xiong, L and Yang, Y and Xie, K},
title = {A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases.},
journal = {Molecular plant},
volume = {15},
number = {2},
pages = {243-257},
doi = {10.1016/j.molp.2021.09.015},
pmid = {34619328},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Oryza/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs (gRNAs) (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. The identities of gRNAs in Agrobacterium mixtures and transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of the receptor-like kinase (RLK) family in rice. One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs, and 74.3% (710/955) of the target genes had three or more independent T0 lines. Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly (92.1%) associated with frameshift mutations in the target genes. In addition, the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. Furthermore, we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes. This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.},
}
@article {pmid34619091,
year = {2021},
author = {Xu, J and Richard, S},
title = {Cellular pathways influenced by protein arginine methylation: Implications for cancer.},
journal = {Molecular cell},
volume = {81},
number = {21},
pages = {4357-4368},
pmid = {34619091},
issn = {1097-4164},
support = {R01 DE026468/DE/NIDCR NIH HHS/United States ; R01 DE030928/DE/NIDCR NIH HHS/United States ; R21 DE028617/DE/NIDCR NIH HHS/United States ; FDN-154303//CIHR/Canada ; },
mesh = {Alternative Splicing ; Animals ; Arginine/*chemistry ; B7-H1 Antigen/metabolism ; CRISPR-Cas Systems ; Cell Communication ; Cell Line, Tumor ; DNA Damage ; DNA Repair ; Enzyme Inhibitors/pharmacology ; Epigenesis, Genetic ; Histones ; Humans ; Immune System ; Immunotherapy/methods ; *Methylation ; Mice ; Mice, Knockout ; Neoplasms/*metabolism ; *Protein Processing, Post-Translational ; Protein-Arginine N-Methyltransferases/*antagonists &amp; inhibitors/chemistry ; RNA Splicing ; RNA, Messenger/metabolism ; Signal Transduction ; },
abstract = {Arginine methylation is an influential post-translational modification occurring on histones, RNA binding proteins, and many other cellular proteins, affecting their function by altering their protein-protein and protein-nucleic acid interactions. Recently, a wealth of information has been gathered, implicating protein arginine methyltransferases (PRMTs), enzymes that deposit arginine methylation, in transcription, pre-mRNA splicing, DNA damage signaling, and immune signaling with major implications for cancer therapy, especially immunotherapy. This review summarizes this recent progress and the current state of PRMT inhibitors, some in clinical trials, as promising drug targets for cancer.},
}
@article {pmid34618092,
year = {2021},
author = {Akella, S and Ma, X and Bacova, R and Harmer, ZP and Kolackova, M and Wen, X and Wright, DA and Spalding, MH and Weeks, DP and Cerutti, H},
title = {Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas.},
journal = {Plant physiology},
volume = {187},
number = {4},
pages = {2637-2655},
pmid = {34618092},
issn = {1532-2548},
mesh = {Algal Proteins/*genetics ; *CRISPR-Cas Systems ; Chlamydomonas/*genetics ; Gene Editing/*methods ; Ribonucleoproteins/*genetics ; },
abstract = {Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)-in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.},
}
@article {pmid34618074,
year = {2021},
author = {Gupta, SK and Vishwakarma, A and Kenea, HD and Galsurker, O and Cohen, H and Aharoni, A and Arazi, T},
title = {CRISPR/Cas9 mutants of tomato MICRORNA164 genes uncover their functional specialization in development.},
journal = {Plant physiology},
volume = {187},
number = {3},
pages = {1636-1652},
pmid = {34618074},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; Fruit/genetics/*growth &amp; development ; *Genes, Plant ; Lycopersicon esculentum/*genetics/growth &amp; development/metabolism ; RNA, Plant/*genetics/metabolism ; },
abstract = {Plant MICRORNA164 (miR164) plays diverse regulatory functions by post-transcriptional repression of certain NAM/ATAF/CUC-domain transcription factors. However, the involvement of miR164 in fleshy fruit development and ripening remains poorly understood. Here, de novo prediction of tomato (Solanum lycopersicum) MIR164 genes identified four genes (SlMIR164a-d), of which SlMIR164d has an atypically long pre-miRNA. The roles of the fruit expressed SlMIR164a, b, and d were studied by analysis of their Clustered Regularly Interspaced Short Palindromic Repeats mutants. The slmir164bCR mutant plants exhibited shoot and flower abnormalities characteristic of ectopic boundary specification, whereas the shoot and flower development of slmir164aCR and slmir164dCR mutants were indistinguishable from wild-type. Strikingly, the knockout of SlMIR164a practically eliminated sly-miR164 from the developing and ripening fruit pericarp. The sly-miR164-deficient slmir164aCR fruits were smaller than the wild-type, due to reduced pericarp cell division and expansion, and displayed intense red color and matte, instead of glossy appearance, upon ripening. We found that the fruit skin phenotypes were associated with morphologically abnormal outer epidermis and thicker cuticle. Quantitation of sly-miR164 target transcripts in slmir164aCR ripening fruits demonstrated the upregulation of SlNAM3 and SlNAM2. Specific expression of their miR164-resistant versions in the pericarp resulted in the formation of extremely small fruits with abnormal epidermis, highlighting the importance of their negative regulation by sly-miR164a. Taken together, our results demonstrate that SlMIR164a and SlMIR164b play specialized roles in development: SlMIR164b is required for shoot and flower boundary specification, and SlMIR164a is required for fruit growth including the expansion of its outer epidermis, which determines the properties of the fruit skin.},
}
@article {pmid34616406,
year = {2021},
author = {Yang, X and Huang, T and Wang, T and Gao, H and Zhang, H and Peng, W and Zhao, J and Hu, S and Lu, P and Hong, Z and Li, B and Deng, K},
title = {MAT2A-Mediated S-Adenosylmethionine Level in CD4[+] T Cells Regulates HIV-1 Latent Infection.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {745784},
pmid = {34616406},
issn = {1664-3224},
mesh = {Adult ; Anti-HIV Agents/therapeutic use ; CD4-Positive T-Lymphocytes/*enzymology/immunology ; CRISPR-Cas Systems ; Carbon/metabolism ; DNA, Viral/blood ; Gene Knockout Techniques ; Gene Library ; HEK293 Cells ; HIV Infections/blood/drug therapy/*immunology ; HIV Long Terminal Repeat ; HIV-1/*physiology ; Histone Code ; Humans ; Jurkat Cells ; Latent Infection/blood/*immunology ; Methionine Adenosyltransferase/*physiology ; RNA Interference ; RNA, Small Interfering/genetics ; S-Adenosylmethionine/*blood ; Virus Activation ; },
abstract = {Antiretroviral drugs effectively halt HIV-1 replication and disease progression, however, due to the presence of a stable viral latent reservoir, the infection cannot be cured by antiretroviral drugs alone. Elucidating the molecular mechanisms underlying HIV-1 latent infection remains a critical hurdle that precludes the development of novel therapeutic strategies aiming for a potential functional cure. Cellular metabolism has been reported to affect HIV-1 replication in CD4[+] T cells, but it remains largely unclear whether it is involved in the regulation of HIV-1 latency. Here, we performed a sub-pooled CRISPR library knockout screen targeting 1773 metabolic-related genes in a cell model of HIV-1 latent infection and found that Methionine Adenosyltransferase 2A (MAT2A) contributes to HIV-1 latency. MAT2A knockout enhanced the reactivation of latent HIV-1 while MAT2A overexpression did the opposite. Mechanistically, MAT2A modulates HIV-1 latency through S-Adenosylmethionine (SAM)-mediated one-carbon flux. MAT2A knockout resulted in a significant downregulation of DNA and histone methylation at the HIV-1 5'-LTR. Importantly, we found that the plasma level of SAM is positively correlated with HIV-1 DNA in PBMCs from ART-treated infected individuals, suggesting SAM could serve as a potential biomarker for the latent viral reservoir. Overall, this study reveals an important role of MAT2A-mediated one-carbon metabolism in regulating HIV-1 latency and provides a promising target for the development of new strategies for a functional cure of HIV-1.},
}
@article {pmid34615950,
year = {2021},
author = {Lim, J and Sakai, E and Sakurai, F and Mizuguchi, H},
title = {miR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {19820},
pmid = {34615950},
issn = {2045-2322},
mesh = {Bone Morphogenetic Proteins/*genetics/*metabolism ; CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Endoderm/metabolism ; *Gene Expression Regulation, Developmental ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Mesoderm/metabolism ; MicroRNAs/genetics ; RNA Interference ; *Signal Transduction ; },
abstract = {Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.},
}
@article {pmid34615907,
year = {2021},
author = {Suzuki, DG and Wada, H and Higashijima, SI},
title = {Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {19836},
pmid = {34615907},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Knock-In Techniques ; Genes, Reporter ; *Genetic Engineering ; HSP70 Heat-Shock Proteins/genetics ; Lampreys/*genetics ; Promoter Regions, Genetic ; Recombinant Fusion Proteins ; },
abstract = {The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other "non-model" organisms.},
}
@article {pmid34615869,
year = {2021},
author = {Papathanasiou, S and Markoulaki, S and Blaine, LJ and Leibowitz, ML and Zhang, CZ and Jaenisch, R and Pellman, D},
title = {Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5855},
pmid = {34615869},
issn = {2041-1723},
support = {K22 CA216319/CA/NCI NIH HHS/United States ; R01 MH104610/MH/NIMH NIH HHS/United States ; U19 AI131135/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 CA213404/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosome Segregation ; *Chromosome Structures ; Embryo, Mammalian ; Embryonic Development/genetics ; Gene Editing/*methods ; *Genomic Instability ; Karyotype ; Mice ; Whole Genome Sequencing ; },
abstract = {Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.},
}
@article {pmid34615858,
year = {2021},
author = {Sun, Y and Xie, J and Cai, S and Wang, Q and Feng, Z and Li, Y and Lu, JJ and Chen, W and Ye, Z},
title = {Elevated expression of nuclear receptor-binding SET domain 3 promotes pancreatic cancer cell growth.},
journal = {Cell death &amp; disease},
volume = {12},
number = {10},
pages = {913},
pmid = {34615858},
issn = {2041-4889},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Survival/genetics ; Cohort Studies ; *Gene Expression Regulation, Neoplastic ; Histone-Lysine N-Methyltransferase/*genetics/metabolism ; Histones/metabolism ; Humans ; Methylation ; Mice, Nude ; Mutation/genetics ; Neoplasm Invasiveness ; Nuclear Proteins/*genetics/metabolism ; Pancreatic Neoplasms/*genetics/*pathology ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/metabolism ; TOR Serine-Threonine Kinases/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {The nuclear receptor-binding SET domain 3 (NSD3) catalyzes methylation of histone H3 at lysine 36 (H3K36), and promotes malignant transformation and progression of human cancer. Its expression, potential functions and underlying mechanisms in pancreatic cancer are studied. Bioinformatics studies and results from local human tissues show that NSD3 is upregulated in human pancreatic cancer tissues, which is correlated with poor overall survival. In primary and established pancreatic cancer cells, NSD3 silencing (by shRNAs) or CRISPR/Cas9-induced NSD3 knockout potently inhibited cell proliferation, migration and invasion, while provoking cell cycle arrest and apoptosis. Conversely, ectopic expression of NSD3-T1232A mutation significantly accelerated proliferation, migration, and invasion of pancreatic cancer cells. H3K36 dimethylation, expression of NSD3-dependent genes (Prkaa2, Myc, Irgm1, Adam12, and Notch3), and mTOR activation (S6K1 phosphorylation) were largely inhibited by NSD3 silencing or knockout. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD3 shRNA potently inhibited pancreatic cancer xenograft growth in nude mice. These results suggest that elevated NSD3 could be an important driver for the malignant progression of pancreatic cancer.},
}
@article {pmid34614191,
year = {2021},
author = {Patinios, C and Creutzburg, SCA and Arifah, AQ and Adiego-Pérez, B and Gyimah, EA and Ingham, CJ and Kengen, SWM and van der Oost, J and Staals, RHJ},
title = {Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas.},
journal = {Nucleic acids research},
volume = {49},
number = {19},
pages = {11392-11404},
pmid = {34614191},
issn = {1362-4962},
mesh = {Base Pairing ; Base Sequence ; CRISPR-Cas Systems ; Escherichia coli/*genetics/metabolism ; Flavobacterium/*genetics/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; *Genome, Bacterial ; Homologous Recombination ; Introns ; Nucleic Acid Conformation ; Pseudomonas putida/*genetics/metabolism ; RNA Splicing ; RNA, Bacterial/*genetics/metabolism ; Riboswitch ; },
abstract = {CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.},
}
@article {pmid34613377,
year = {2021},
author = {He, YZ and Kuang, X and Long, TF and Li, G and Ren, H and He, B and Yan, JR and Liao, XP and Liu, YH and Chen, L and Sun, J},
title = {Re-engineering a mobile-CRISPR/Cas9 system for antimicrobial resistance gene curing and immunization in Escherichia coli.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {77},
number = {1},
pages = {74-82},
doi = {10.1093/jac/dkab368},
pmid = {34613377},
issn = {1460-2091},
mesh = {Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics ; Plasmids/genetics ; },
abstract = {OBJECTIVES: In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes.<br><br>METHODS: A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts.<br><br>RESULTS: This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids.<br><br>CONCLUSIONS: The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.},
}
@article {pmid34612665,
year = {2021},
author = {Janssens, J and Blokken, J and Lampi, Y and De Wit, F and Zurnic Bonisch, I and Nombela, I and Van de Velde, P and Van Remoortel, B and Gijsbers, R and Christ, F and Debyser, Z},
title = {CRISPR/Cas9-Induced Mutagenesis Corroborates the Role of Transportin-SR2 in HIV-1 Nuclear Import.},
journal = {Microbiology spectrum},
volume = {9},
number = {2},
pages = {e0133621},
pmid = {34612665},
issn = {2165-0497},
mesh = {Active Transport, Cell Nucleus ; CRISPR-Cas Systems ; Cell Nucleus/metabolism/*virology ; HIV Infections/genetics/virology ; HIV Integrase/genetics/metabolism ; HIV-1/genetics/*metabolism ; Humans ; Protein Binding ; Serine-Arginine Splicing Factors/genetics/metabolism ; beta Karyopherins/genetics/*metabolism ; mRNA Cleavage and Polyadenylation Factors/genetics/metabolism ; },
abstract = {To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3. Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import.},
}
@article {pmid34612202,
year = {2021},
author = {Sun, D and Evans, L and Perrone, F and Sokleva, V and Lim, K and Rezakhani, S and Lutolf, M and Zilbauer, M and Rawlins, EL},
title = {A functional genetic toolbox for human tissue-derived organoids.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34612202},
issn = {2050-084X},
support = {102175/B/13/Z/WT_/Wellcome Trust/United Kingdom ; C6946/A24843/CRUK_/Cancer Research UK/United Kingdom ; 109146/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; PhD studentship 102175/B/13/Z/WT_/Wellcome Trust/United Kingdom ; MR/P009581/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; MR/T001917/1/MRC_/Medical Research Council/United Kingdom ; 203144/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Gene Knockdown Techniques/methods ; Gene Targeting/methods ; Humans ; Lung/cytology ; *Organoids ; },
abstract = {Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example, we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag', our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly targeted cells. Our toolbox also incorporates conditional gene knockdown or overexpression using tightly inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many ongoing descriptive studies, such as the Human Cell Atlas Project.},
}
@article {pmid34611682,
year = {2021},
author = {Tang, Y and Gao, L and Feng, W and Guo, C and Yang, Q and Li, F and Le, XC},
title = {The CRISPR-Cas toolbox for analytical and diagnostic assay development.},
journal = {Chemical Society reviews},
volume = {50},
number = {21},
pages = {11844-11869},
doi = {10.1039/d1cs00098e},
pmid = {34611682},
issn = {1460-4744},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acids ; Pandemics ; SARS-CoV-2 ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems have revolutionized biological and biomedical sciences in many ways. The last few years have also seen tremendous interest in deploying the CRISPR-Cas toolbox for analytical and diagnostic assay development because CRISPR-Cas is one of the most powerful classes of molecular machineries for the recognition and manipulation of nucleic acids. In the short period of development, many CRISPR-enabled assays have already established critical roles in clinical diagnostics, biosensing, and bioimaging. We describe in this review the recent advances and design principles of CRISPR mediated analytical tools with an emphasis on the functional roles of CRISPR-Cas machineries as highly efficient binders and molecular scissors. We highlight the diverse engineering approaches for molecularly modifying CRISPR-Cas machineries and for devising better readout platforms. We discuss the potential roles of these new approaches and platforms in enhancing assay sensitivity, specificity, multiplexity, and clinical outcomes. By illustrating the biochemical and analytical processes, we hope this review will help guide the best use of the CRISPR-Cas toolbox in detecting, quantifying and imaging biologically and clinically important molecules and inspire new ideas, technological advances and engineering strategies for addressing real-world challenges such as the on-going COVID-19 pandemic.},
}
@article {pmid34611198,
year = {2021},
author = {Miyata, Y and Fuse, H and Tokumoto, S and Hiki, Y and Deviatiiarov, R and Yoshida, Y and Yamada, TG and Cornette, R and Gusev, O and Shagimardanova, E and Funahashi, A and Kikawada, T},
title = {Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {19698},
pmid = {34611198},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Calcium/metabolism ; *Calcium Signaling ; Cell Line ; Computational Biology/methods ; *Dehydration ; *Gene Editing ; Gene Expression Profiling ; Gene Knock-In Techniques ; Gene Ontology ; Insecta ; Larva ; RNA, Guide ; Stress, Physiological ; Trehalose/metabolism/pharmacology ; },
abstract = {Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca[2+] mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca[2+] concentration, and further experiments revealed that the calmodulin-calcineurin-NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin-calmodulin kinase-CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca[2+] surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.},
}
@article {pmid34610011,
year = {2021},
author = {Fuchs, S and Garrood, WT and Beber, A and Hammond, A and Galizi, R and Gribble, M and Morselli, G and Hui, TJ and Willis, K and Kranjc, N and Burt, A and Crisanti, A and Nolan, T},
title = {Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.},
journal = {PLoS genetics},
volume = {17},
number = {10},
pages = {e1009740},
pmid = {34610011},
issn = {1553-7404},
mesh = {Animals ; Anopheles/genetics ; Biological Evolution ; CRISPR-Cas Systems/*genetics ; Conserved Sequence/*genetics ; Gene Drive Technology/methods ; Genes, Essential/genetics ; Genotype ; Malaria/parasitology ; Mosquito Control/methods ; Mosquito Vectors/genetics ; },
abstract = {CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ('homing') therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.},
}
@article {pmid34609846,
year = {2021},
author = {Gao, C and Guo, L and Hu, G and Liu, J and Chen, X and Xia, X and Liu, L},
title = {Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2661-2671},
doi = {10.1021/acssynbio.1c00294},
pmid = {34609846},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*metabolism ; Metabolic Engineering/*methods ; Promoter Regions, Genetic ; Quorum Sensing ; Shikimic Acid/metabolism ; },
abstract = {Building autonomous switches is an effective approach for rewiring metabolic flux during microbial synthesis of chemicals. However, current autonomous switches largely rely on metabolite-responsive biosensors or quorum-sensing circuits. In this study, a stationary phase promoter (SPP) and a protein degradation tag (PDT) were combined with the CRISPR interference (CRISPRi) system to construct an autonomous repression system that could shut down multiple-gene expression depending on the cellular physiological state. With this autonomous CRISPRi system to regulate one target gene, a fermenter-scale titer of shikimic acid reached 21 g/L, which was the highest titer ever reported by Escherichia coli in a minimal medium without any chemical inducers. With three target genes repressed, 26 g/L glutaric acid could be achieved with decreased byproduct accumulation. These results highlight the applicability of the autonomous CRISPRi system for microbial production of value-added chemicals.},
}
@article {pmid34609046,
year = {2021},
author = {Zhang, T and Li, J and Wang, T and Zhao, F and Sui, T},
title = {No apparent p53 activation in CRISPR-engineered gene-edited rabbits.},
journal = {Journal of cellular and molecular medicine},
volume = {25},
number = {21},
pages = {10313-10317},
pmid = {34609046},
issn = {1582-4934},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression ; Genotype ; Rabbits ; Tumor Suppressor Protein p53/*genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) and base editors (BEs) are revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR-Cas9-mediated genome editing can induce a p53-mediated stress response and cell cycle arrest in human cells, while not illustrated in gene-editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene-edited rabbits using CRISPR-Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE-edited, suggesting that p53 may not need to consider for application in gene-edited animals.},
}
@article {pmid34608972,
year = {2021},
author = {Kong, X and Pan, W and Sun, N and Zhang, T and Liu, L and Zhang, H},
title = {GLABRA2-based selection efficiently enriches Cas9-generated nonchimeric mutants in the T1 generation.},
journal = {Plant physiology},
volume = {187},
number = {2},
pages = {758-768},
pmid = {34608972},
issn = {1532-2548},
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Homeodomain Proteins/*genetics ; *Mutation ; *Selection, Genetic ; },
abstract = {The CRISPR/Cas9 system is a widely used tool for genome editing in plants. In Arabidopsis (Arabidopsis thaliana), egg cell-specific promoters driving Cas9 expression have been applied to reduce the proportion of T1 transformants that are chimeras; however, this approach generally leads to relatively low mutagenesis rates. In this study, a GLABRA2 mutation-based visible selection (GBVS) system was established to enrich nonchimeric mutants among T1 plants generated by an egg cell-specific CRISPR/Cas9 system. GBVS generally enhanced mutation screening, increasing the frequency by 2.58- to 7.50-fold, and 25%-48.15% of T1 plants selected through the GBVS system were homozygous or biallelic mutants, which was 1.71- to 7.86-fold higher than the percentage selected using the original system. The mutant phenotypes of T2 plants were not obviously affected by the glabrous background for all four target genes used in this study. Additionally, the nonchimeric pyrabactin resistance 1 (PYR1)/PYR1-like 1 (PYL1) and PYL2 triple mutant pyr1/pyl1/pyl2 could be obtained in the T1 generation with a ratio of 26.67% when GBVS was applied. Collectively, our results show that compared with the known CRISPR/Cas9 systems, the GBVS system described here saves more time and labor when used for the obtainment of homozygous or biallelic monogenic mutants and nonchimeric polygenic mutants in Arabidopsis.},
}
@article {pmid34608327,
year = {2022},
author = {Nelson, JW and Randolph, PB and Shen, SP and Everette, KA and Chen, PJ and Anzalone, AV and An, M and Newby, GA and Chen, JC and Hsu, A and Liu, DR},
title = {Engineered pegRNAs improve prime editing efficiency.},
journal = {Nature biotechnology},
volume = {40},
number = {3},
pages = {402-410},
pmid = {34608327},
issn = {1546-1696},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; },
abstract = {Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3' terminus of pegRNAs that enhance their stability and prevent degradation of the 3' extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3-4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3' structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3' motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.},
}
@article {pmid34608130,
year = {2021},
author = {Wang, Y and Kui, L and Wang, G},
title = {Combination therapy for HCC: from CRISPR screening to the design of clinical therapies.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {359},
pmid = {34608130},
issn = {2059-3635},
mesh = {CRISPR-Cas Systems/genetics ; *Carcinoma, Hepatocellular/genetics/therapy ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Humans ; *Liver Neoplasms/genetics/therapy ; },
}
@article {pmid34607509,
year = {2021},
author = {Bui, LM and Thi Thu Phung, H and Ho Thi, TT and Singh, V and Maurya, R and Khambhati, K and Wu, CC and Uddin, MJ and Trung, DM and Chu, DT},
title = {Recent findings and applications of biomedical engineering for COVID-19 diagnosis: a critical review.},
journal = {Bioengineered},
volume = {12},
number = {1},
pages = {8594-8613},
pmid = {34607509},
issn = {2165-5987},
mesh = {Artificial Intelligence ; Biomedical Engineering/*methods ; Biosensing Techniques ; COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*methods ; COVID-19 Serological Testing/*methods ; CRISPR-Cas Systems ; Humans ; Immunoassay ; Microfluidics ; Public Health ; SARS-CoV-2 ; },
abstract = {COVID-19 is one of the most severe global health crises that humanity has ever faced. Researchers have restlessly focused on developing solutions for monitoring and tracing the viral culprit, SARS-CoV-2, as vital steps to break the chain of infection. Even though biomedical engineering (BME) is considered a rising field of medical sciences, it has demonstrated its pivotal role in nurturing the maturation of COVID-19 diagnostic technologies. Within a very short period of time, BME research applied to COVID-19 diagnosis has advanced with ever-increasing knowledge and inventions, especially in adapting available virus detection technologies into clinical practice and exploiting the power of interdisciplinary research to design novel diagnostic tools or improve the detection efficiency. To assist the development of BME in COVID-19 diagnosis, this review highlights the most recent diagnostic approaches and evaluates the potential of each research direction in the context of the pandemic.},
}
@article {pmid34607105,
year = {2021},
author = {Al-Mansoori, L and Elsinga, P and Goda, SK},
title = {Bio-vehicles of cytotoxic drugs for delivery to tumor specific targets for cancer precision therapy.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {144},
number = {},
pages = {112260},
doi = {10.1016/j.biopha.2021.112260},
pmid = {34607105},
issn = {1950-6007},
mesh = {Animals ; Antineoplastic Agents/*administration &amp; dosage/metabolism ; CRISPR-Cas Systems ; Cancer Vaccines/adverse effects/*therapeutic use ; *Drug Carriers ; Drug Compounding ; *Drug Resistance, Neoplasm/genetics ; *Genetic Therapy ; Humans ; Molecular Targeted Therapy ; Nanoparticles ; Nanotechnology ; Neoplasms/diagnosis/genetics/immunology/*therapy ; *Precision Medicine ; },
abstract = {Abnormal structural and molecular changes in malignant tissues were thoroughly investigated and utilized to target tumor cells, hence rescuing normal healthy tissues and lowering the unwanted side effects as non-specific cytotoxicity. Various ligands for cancer cell specific markers have been uncovered and inspected for directional delivery of the anti-cancer drug to the tumor site, in addition to diagnostic applications. Over the past few decades research related to the ligand targeted therapy (LTT) increased tremendously aiming to treat various pathologies, mainly cancers with well exclusive markers. Malignant tumors are known to induce elevated levels of a variety of proteins and peptides known as cancer "markers" as certain antigens (e.g., Prostate specific membrane antigen "PSMA", carcinoembryonic antigen "CEA"), receptors (folate receptor, somatostatin receptor), integrins (Integrin αvβ3) and cluster of differentiation molecules (CD13). The choice of an appropriate marker to be targeted and the design of effective ligand-drug conjugate all has to be carefully selected to generate the required therapeutic effect. Moreover, since some tumors express aberrantly high levels of more than one marker, some approaches investigated targeting cancer cells with more than one ligand (dual or multi targeting). We aim in this review to report an update on the cancer-specific receptors and the vehicles to deliver cytotoxic drugs, including recent advancements on nano delivery systems and their implementation in targeted cancer therapy. We will discuss the advantages and limitations facing this approach and possible solutions to mitigate these obstacles. To achieve the said aim a literature search in electronic data bases (PubMed and others) using keywords "Cancer specific receptors, cancer specific antibody, tumor specific peptide carriers, cancer overexpressed proteins, gold nanotechnology and gold nanoparticles in cancer treatment" was carried out.},
}
@article {pmid34606606,
year = {2021},
author = {Payne, LJ and Todeschini, TC and Wu, Y and Perry, BJ and Ronson, CW and Fineran, PC and Nobrega, FL and Jackson, SA},
title = {Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.},
journal = {Nucleic acids research},
volume = {49},
number = {19},
pages = {10868-10878},
pmid = {34606606},
issn = {1362-4962},
mesh = {Adenosine Triphosphatases/genetics/metabolism ; Antibiosis/*genetics ; Archaea/classification/*genetics/metabolism/virology ; Archaeal Proteins/*genetics/metabolism ; Bacteria/classification/*genetics/metabolism/virology ; Bacterial Proteins/*genetics/metabolism ; Bacteriophages/*genetics/growth &amp; development ; CRISPR-Cas Systems ; DNA Helicases/genetics/metabolism ; DNA Modification Methylases/genetics/metabolism ; Markov Chains ; Phylogeny ; *Software ; Terminology as Topic ; },
abstract = {To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).},
}
@article {pmid34606604,
year = {2022},
author = {Pinilla-Redondo, R and Russel, J and Mayo-Muñoz, D and Shah, SA and Garrett, RA and Nesme, J and Madsen, JS and Fineran, PC and Sørensen, SJ},
title = {CRISPR-Cas systems are widespread accessory elements across bacterial and archaeal plasmids.},
journal = {Nucleic acids research},
volume = {50},
number = {8},
pages = {4315-4328},
pmid = {34606604},
issn = {1362-4962},
mesh = {*Archaea/genetics ; Bacteria/genetics ; *CRISPR-Cas Systems ; Plasmids/genetics ; Prokaryotic Cells ; },
abstract = {Many prokaryotes encode CRISPR-Cas systems as immune protection against mobile genetic elements (MGEs), yet a number of MGEs also harbor CRISPR-Cas components. With a few exceptions, CRISPR-Cas loci encoded on MGEs are uncharted and a comprehensive analysis of their distribution, prevalence, diversity, and function is lacking. Here, we systematically investigated CRISPR-Cas loci across the largest curated collection of natural bacterial and archaeal plasmids. CRISPR-Cas loci are widely but heterogeneously distributed across plasmids and, in comparison to host chromosomes, their mean prevalence per Mbp is higher and their distribution is distinct. Furthermore, the spacer content of plasmid CRISPRs exhibits a strong targeting bias towards other plasmids, while chromosomal arrays are enriched with virus-targeting spacers. These contrasting targeting preferences highlight the genetic independence of plasmids and suggest a major role for mediating plasmid-plasmid conflicts. Altogether, CRISPR-Cas are frequent accessory components of many plasmids, which is an overlooked phenomenon that possibly facilitates their dissemination across microbiomes.},
}
@article {pmid34606423,
year = {2021},
author = {Yang, L and Zhang, Y and Yin, P and Feng, Y},
title = {Structural insights into the inactivation of the type I-F CRISPR-Cas system by anti-CRISPR proteins.},
journal = {RNA biology},
volume = {18},
number = {sup2},
pages = {562-573},
pmid = {34606423},
issn = {1555-8584},
mesh = {Animals ; Bacteriophages/*physiology ; Binding Sites ; CRISPR-Associated Proteins/chemistry/metabolism ; *CRISPR-Cas Systems ; DNA, Bacterial/chemistry/metabolism ; DNA-Binding Proteins/chemistry/metabolism ; Enzyme Activation ; Gene Editing ; *Host-Pathogen Interactions ; Humans ; Microbial Interactions ; Models, Molecular ; Protein Conformation ; Protein Interaction Domains and Motifs ; Structure-Activity Relationship ; Viral Proteins/*chemistry/*metabolism ; },
abstract = {Phage infection is one of the major threats to prokaryotic survival, and prokaryotes in turn have evolved multiple protection approaches to fight against this challenge. Various delicate mechanisms have been discovered from this eternal arms race, among which the CRISPR-Cas systems are the prokaryotic adaptive immune systems and phages evolve diverse anti-CRISPR (Acr) proteins to evade this immunity. Until now, about 90 families of Acr proteins have been identified, out of which 24 families were verified to fight against subtype I-F CRISPR-Cas systems. Here, we review the structural and biochemical mechanisms of the characterized type I-F Acr proteins, classify their inhibition mechanisms into two major groups and provide insights for future studies of other Acr proteins. Understanding Acr proteins in this context will lead to a variety of practical applications in genome editing and also provide exciting insights into the molecular arms race between prokaryotes and phages.},
}
@article {pmid34605326,
year = {2021},
author = {Lv, W and Li, T and Wang, S and Wang, H and Li, X and Zhang, S and Wang, L and Xu, Y and Wei, W},
title = {The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer.},
journal = {Technology in cancer research &amp; treatment},
volume = {20},
number = {},
pages = {15330338211045206},
pmid = {34605326},
issn = {1533-0338},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing ; *Genetic Therapy ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/*complications ; Humans ; Liver Neoplasms/*genetics/*therapy/virology ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was originally discovered in prokaryotes and functions as part of the adaptive immune system. The experimental research of many scholars, as well as scientific and technological advancements, has allowed prokaryote-derived CRISPR/Cas genome-editing systems to transform our ability to manipulate, detect, image, and annotate specific DNA and RNA sequences in the living cells of diverse species. Through modern genetic engineering editing technology and high-throughput gene sequencing, we can edit and splice covalently closed circular DNA to silence it, and correct the mutation and deletion of liver cancer genes to achieve precise in situ repair of defective genes and prohibit viral infection or replication. Such manipulations do not destroy the structure of the entire genome and facilitate the cure of diseases. In this review, we discussed the possibility that CRISPR/Cas could be used as a treatment for patients with liver cancer caused by hepatitis B virus infection, and reviewed the challenges incurred by this effective gene-editing technology.},
}
@article {pmid34604812,
year = {2021},
author = {Alcántara, R and Peñaranda, K and Mendoza-Rojas, G and Nakamoto, JA and Dueñas, E and Alvarez, D and Adaui, V and Milón, P},
title = {UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100878},
pmid = {34604812},
issn = {2666-1667},
mesh = {COVID-19/*diagnosis/genetics/virology ; *CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/*methods ; RNA, Viral/*analysis/*genetics ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021.},
}
@article {pmid34604217,
year = {2021},
author = {Vicente, MM and Chaves-Ferreira, M and Jorge, JMP and Proença, JT and Barreto, VM},
title = {The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {718466},
pmid = {34604217},
issn = {2296-634X},
abstract = {The repurposing of the CRISPR/Cas bacterial defense system against bacteriophages as simple and flexible molecular tools has revolutionized the field of gene editing. These tools are now widely used in basic research and clinical trials involving human somatic cells. However, a global moratorium on all clinical uses of human germline editing has been proposed because the technology still lacks the required efficacy and safety. Here we focus on the approaches developed since 2013 to decrease the frequency of unwanted mutations (the off-targets) during CRISPR-based gene editing.},
}
@article {pmid34603259,
year = {2021},
author = {Li, C and Wang, Y and Gao, Y and Li, C and Ma, B and Wang, H},
title = {Antimicrobial Resistance and CRISPR Typing Among Salmonella Isolates From Poultry Farms in China.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {730046},
pmid = {34603259},
issn = {1664-302X},
abstract = {Although knowledge of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been applied in many research areas, comprehensive studies of this system in Salmonella, particularly in analysis of antibiotic resistance, have not been reported. In this work, 75 Salmonella isolates obtained from broilers or broilers products were characterized to determine their antimicrobial susceptibilities, antibiotic resistance gene profiles, and CRISPR array diversities, and genotyping was explored. In total, 80.00% (60/75) of the strains were multidrug resistant, and the main pattern observed in the isolates was CN-AZM-AMP-AMC-CAZ-CIP-ATM-TE-SXT-FOS-C. The resistance genes of streptomycin (aadA), phenicol (floR-like and catB3-like), β-lactams (bla TEM, bla OXA, and bla CTX), tetracycline [tet(A)-like], and sulfonamides (sul1 and sul2) appeared at higher frequencies among the corresponding resistant isolates. Subsequently, we analyzed the CRISPR arrays and found 517 unique spacer sequences and 31 unique direct repeat sequences. Based on the CRISPR spacer sequences, we developed a novel typing method, CRISPR locus three spacer sequences typing (CLTSST), to help identify sources of Salmonella outbreaks especially correlated with epidemiological data. Compared with multi-locus sequence typing (MLST), conventional CRISPR typing (CCT), and CRISPR locus spacer pair typing (CLSPT), discrimination using CLTSST was weaker than that using CCT but stronger than that using MLST and CLSPT. In addition, we also found that there were no close correlations between CRISPR loci and antibiotics but had close correlations between CRISPR loci and antibiotic resistance genes in Salmonella isolates.},
}
@article {pmid34602188,
year = {2021},
author = {Cao, G and Chen, X and Deng, Y and Nie, F and Liu, Y and Wang, G and Huo, D and Hou, C},
title = {Single-nucleotide variant of PIK3CA [H1047R] gene assay by CRISPR/Cas12a combined with rolling circle amplification.},
journal = {Analytica chimica acta},
volume = {1182},
number = {},
pages = {338943},
doi = {10.1016/j.aca.2021.338943},
pmid = {34602188},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Class I Phosphatidylinositol 3-Kinases/genetics ; Humans ; Mutation ; Nucleotides ; *Phosphatidylinositol 3-Kinases/genetics ; },
abstract = {PIK3CA [H1047R] gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA [H1047R] mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA [H1047R] gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA [H1047R] gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways.},
}
@article {pmid34602042,
year = {2021},
author = {Rajeev, M and Ratan, C and Krishnan, K and Vijayan, M},
title = {Hutchinson-Gilford Progeria Syndrome (Hgps) and Application of Gene Therapy Based Crispr/Cas Technology as A Promising Innovative Treatment Approach.},
journal = {Recent patents on biotechnology},
volume = {15},
number = {4},
pages = {266-285},
doi = {10.2174/1872208315666210928114720},
pmid = {34602042},
issn = {2212-4012},
mesh = {CRISPR-Cas Systems ; Genetic Therapy ; Humans ; Patents as Topic ; *Progeria/genetics ; Technology ; },
abstract = {BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS), also known as progeria of childhood or progeria is a rare, rapid, autosomal dominant genetic disorder characterized by premature aging which occurs shortly after birth. HGPS occurs as a result of de novo point mutation in the gene recognized as LMNA gene that encodes two proteins, Lamin A protein and Lamin C protein which are the structural components of the nuclear envelope. Mutations in the gene trigger abnormal splicing and induce internal deletion of 50 amino acids leading to the development of a truncated form of Lamin A protein known as Progerin. Progerin generation can be considered the crucial step in HGPS since the protein is highly toxic to human cells, permanently farnesylated, and exhibits variation in several biochemical and structural properties within the individual. HGPS also produces complications such as skin alterations, growth failure, atherosclerosis, hair and fat loss, and bone and joint diseases. We have also revised all relevant patents relating to Hutchinson-Gilford progeria syndrome and its therapy in the current article.<br><br>METHODS: The goal of the present review article is to provide information about Hutchinson- Gilford progeria syndrome (HGPS) and the use of CRISPR/Cas technology as a promising treatment approach in the treatment of the disease. The review also discusses about different pharmacological and non-pharmacological methods of treatment currently used for HGPS.<br><br>RESULTS: The main limitation associated with progeria is the lack of a definitive cure. The existing treatment modality provides only symptomatic relief. Therefore, it is high time to develop a therapeutic method that hastens premature aging in such patients.<br><br>CONCLUSION: CRISPR/Cas technology is a novel gene-editing tool that allows genome editing at specific loci and is found to be a promising therapeutic approach for the treatment of genetic disorders such as HGPS where dominant-negative mutations take place.},
}
@article {pmid34601868,
year = {2021},
author = {Velázquez, E and Al-Ramahi, Y and Tellechea-Luzardo, J and Krasnogor, N and de Lorenzo, V},
title = {Targetron-Assisted Delivery of Exogenous DNA Sequences into Pseudomonas putida through CRISPR-Aided Counterselection.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2552-2565},
pmid = {34601868},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; DNA/*administration &amp; dosage/genetics ; DNA Barcoding, Taxonomic ; Gene Editing/methods ; Genes, Bacterial ; Introns ; Plasmids ; Pseudomonas putida/*genetics ; },
abstract = {Genome editing methods based on group II introns (known as targetron technology) have long been used as a gene knockout strategy in a wide range of organisms, in a fashion independent of homologous recombination. Yet, their utility as delivery systems has typically been suboptimal due to the reduced efficiency of insertion when carrying exogenous sequences. We show that this limitation can be tackled and targetrons can be adapted as a general tool in Gram-negative bacteria. To this end, a set of broad-host-range standardized vectors were designed for the conditional expression of the Ll.LtrB intron. After establishing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths, to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. With these novel tools, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its ΔrecA derivative. Finally, we applied this technology to successfully tag P. putida with an orthogonal short sequence barcode that acts as a unique identifier for tracking this microorganism in biotechnological settings. These results show the value of the targetron approach for the unrestricted delivery of small DNA fragments to precise locations in the genomes of Gram-negative bacteria, which will be useful for a suite of genome editing endeavors.},
}
@article {pmid34601577,
year = {2021},
author = {Dutta, D and Ravichandiran, V and Sukla, S},
title = {Virophages: association with human diseases and their predicted role as virus killers.},
journal = {Pathogens and disease},
volume = {79},
number = {8},
pages = {},
doi = {10.1093/femspd/ftab049},
pmid = {34601577},
issn = {2049-632X},
mesh = {Amoeba/virology ; *Antibiosis ; Biological Evolution ; *Disease Susceptibility ; Genome, Viral ; Genomics/methods ; Giant Viruses/physiology ; *Host-Pathogen Interactions ; Humans ; Microbial Interactions ; Phage Therapy/methods ; Virophages/classification/*physiology/ultrastructure ; },
abstract = {The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.},
}
@article {pmid34601182,
year = {2021},
author = {Qian, W and Huang, J and Wang, X and Wang, T and Li, Y},
title = {CRISPR-Cas12a combined with reverse transcription recombinase polymerase amplification for sensitive and specific detection of human norovirus genotype GII.4.},
journal = {Virology},
volume = {564},
number = {},
pages = {26-32},
doi = {10.1016/j.virol.2021.09.008},
pmid = {34601182},
issn = {1096-0341},
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Caliciviridae Infections/*diagnosis ; Endodeoxyribonucleases/metabolism ; Gastroenteritis/*diagnosis ; Genotype ; Humans ; Molecular Diagnostic Techniques/*methods ; Norovirus/genetics/*isolation &amp; purification ; Nucleic Acid Amplification Techniques/*methods ; Point-of-Care Testing ; RNA, Viral/genetics ; Recombinases/metabolism ; Reverse Transcription ; Sensitivity and Specificity ; Viral Replicase Complex Proteins/genetics ; },
abstract = {Human norovirus (NOV) is a common and serious virus that accounts for sporadic cases and outbreaks of gastroenteritis. This study aimed to develop rapid, reliable and portable detection systems by coupling reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for NOV genotype GII.4. Here, three primers for RNA-dependent RNA polymerase gene of NOV were designed and screened. Then, RT-RPA products were detected using CRISPR-Cas12a system by combing with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 40 min, with the detection limit of up to 9.65 × 10[2]copies/mL and no cross-reactivity with metapneumovirus, bocavirus, seoul virus, and respiratory syncytial virus. Furthermore, the detection coincidence rates of RT-RPA-Cas12a-based fluorescence and LF with qRT-PCR were 98.3%. Therefore, the present study suggests that both RT-RPA-Cas12a-based fluorescence and LF are promising sensitive, specific and alternative method for diagnosis of NOV genotype GII.4 without ancillary equipment.},
}
@article {pmid34601132,
year = {2021},
author = {Maynard, LH and Humbert, O and Peterson, CW and Kiem, HP},
title = {Genome editing in large animal models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3140-3152},
pmid = {34601132},
issn = {1525-0024},
support = {R01 HL136135/HL/NHLBI NIH HHS/United States ; R01 AI135953/AI/NIAID NIH HHS/United States ; U19 AI149680/AI/NIAID NIH HHS/United States ; R01 HL151765/HL/NHLBI NIH HHS/United States ; UM1 AI126623/AI/NIAID NIH HHS/United States ; U19 HL156247/HL/NHLBI NIH HHS/United States ; U01 AI138329/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clinical Studies as Topic ; *Disease Models, Animal ; *Gene Editing ; Gene Transfer Techniques ; Genetic Diseases, Inborn/genetics/therapy ; *Genetic Therapy/methods/trends ; Genetic Vectors/genetics ; Humans ; },
abstract = {Although genome editing technologies have the potential to revolutionize the way we treat human diseases, barriers to successful clinical implementation remain. Increasingly, preclinical large animal models are being used to overcome these barriers. In particular, the immunogenicity and long-term safety of novel gene editing therapeutics must be evaluated rigorously. However, short-lived small animal models, such as mice and rats, cannot address secondary pathologies that may arise years after a gene editing treatment. Likewise, immunodeficient mouse models by definition lack the ability to quantify the host immune response to a novel transgene or gene-edited locus. Large animal models, including dogs, pigs, and non-human primates (NHPs), bear greater resemblance to human anatomy, immunology, and lifespan and can be studied over longer timescales with clinical dosing regimens that are more relevant to humans. These models allow for larger scale and repeated blood and tissue sampling, enabling greater depth of study and focus on rare cellular subsets. Here, we review current progress in the development and evaluation of novel genome editing therapies in large animal models, focusing on applications in human immunodeficiency virus 1 (HIV-1) infection, cancer, and genetic diseases including hemoglobinopathies, Duchenne muscular dystrophy (DMD), hypercholesterolemia, and inherited retinal diseases.},
}
@article {pmid34601018,
year = {2021},
author = {Chin, YW and Shin, SC and Han, S and Jang, HW and Kim, HJ},
title = {CRISPR/Cas9-mediated Inactivation of arginase in a yeast strain isolated from Nuruk and its impact on the whole genome.},
journal = {Journal of biotechnology},
volume = {341},
number = {},
pages = {163-167},
doi = {10.1016/j.jbiotec.2021.09.019},
pmid = {34601018},
issn = {1873-4863},
mesh = {Arginase/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics ; Urethane ; },
abstract = {Despite the advantages of CRISPR/Cas9 technology in the food industry, controversy over its off-target effects exists. We engineered an industrial Saccharomyces cerevisiae strain isolated from a Korean rice wine starter, Nuruk, using CRISPR/Cas9 to decrease ethyl carbamate (EC) formation. We disrupted the CAR1 gene encoding arginase, which plays a key role in EC formation. Subsequently, we compared the whole genome of the engineered strain to that of the wild type by analyzing heterozygous and homozygous mutations through variant calling. Homozygous mutations in the genome of the engineered strains were identified as the target mutations in CAR1 induced by CRISPR/Cas9, and no other off-target effects were observed. Our findings have critical implications for the use of CRISRP/Cas9 technology in yeasts in the food industry.},
}
@article {pmid34599606,
year = {2022},
author = {Nicolas, HA and Hua, K and Quigley, H and Ivare, J and Tesson, F and Akimenko, MA},
title = {A CRISPR/Cas9 zebrafish lamin A/C mutant model of muscular laminopathy.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {251},
number = {4},
pages = {645-661},
doi = {10.1002/dvdy.427},
pmid = {34599606},
issn = {1097-0177},
support = {DEV312217//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; *Lamin Type A/genetics/metabolism ; *Laminopathies ; Muscle, Skeletal ; Mutation ; Zebrafish/genetics/metabolism ; },
abstract = {BACKGROUND: Lamin A/C gene (LMNA) mutations frequently cause cardiac and/or skeletal muscle diseases called striated muscle laminopathies. We created a zebrafish muscular laminopathy model using CRISPR/Cas9 technology to target the zebrafish lmna gene.<br><br>RESULTS: Heterozygous and homozygous lmna mutants present skeletal muscle damage at 1&#xa0;day post-fertilization (dpf), and mobility impairment at 4 to 7 dpf. Cardiac structure and function analyses between 1 and 7 dpf show mild and transient defects in the lmna mutants compared to wild type (WT). Quantitative RT-PCR analysis of genes implicated in striated muscle laminopathies show a decrease in jun and nf&#x3ba;b2 expression in 7 dpf homozygous lmna mutants compared to WT. Homozygous lmna mutants have a 1.26-fold protein increase in activated Erk 1/2, kinases associated with striated muscle laminopathies, compared to WT at 7 dpf. Activated Protein Kinase C alpha (Pkc &#x3b1;), a kinase that interacts with lamin A/C and Erk 1/2, is also upregulated in 7 dpf homozygous lmna mutants compared to WT.<br><br>CONCLUSIONS: This study presents an animal model of skeletal muscle laminopathy where heterozygous and homozygous lmna mutants exhibit prominent skeletal muscle abnormalities during the first week of development. Furthermore, this is the first animal model that potentially implicates Pkc &#x3b1; in muscular laminopathies.},
}
@article {pmid34598686,
year = {2021},
author = {Zhang, H and Qin, C and An, C and Zheng, X and Wen, S and Chen, W and Liu, X and Lv, Z and Yang, P and Xu, W and Gao, W and Wu, Y},
title = {Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer.},
journal = {Molecular cancer},
volume = {20},
number = {1},
pages = {126},
pmid = {34598686},
issn = {1476-4598},
mesh = {Animals ; Biomarkers, Tumor ; *CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Clinical Decision-Making ; Disease Management ; Disease Susceptibility ; *Gene Editing/methods ; Humans ; Molecular Diagnostic Techniques ; Molecular Targeted Therapy ; Neoplasms/*diagnosis/*etiology/*therapy ; Precision Medicine/methods ; Research ; },
abstract = {The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for the development of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease9 (CRISPR/Cas9) gene editing technology that provided new tools for precise gene editing. It is possible to target any genomic locus virtually using only a complex nuclease protein with short RNA as a site-specific endonuclease. Since cancer is caused by genomic changes in tumor cells, CRISPR/Cas9 can be used in the field of cancer research to edit genomes for exploration of the mechanisms of tumorigenesis and development. In recent years, the CRISPR/Cas9 system has been increasingly used in cancer research and treatment and remarkable results have been achieved. In this review, we introduced the mechanism and development of the CRISPR/Cas9-based gene editing system. Furthermore, we summarized current applications of this technique for basic research, diagnosis and therapy of cancer. Moreover, the potential applications of CRISPR/Cas9 in new emerging hotspots of oncology research were discussed, and the challenges and future directions were highlighted.},
}
@article {pmid34597596,
year = {2021},
author = {Adeyemi, RO and Willis, NA and Elia, AEH and Clairmont, C and Li, S and Wu, X and D'Andrea, AD and Scully, R and Elledge, SJ},
title = {The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair.},
journal = {Molecular cell},
volume = {81},
number = {21},
pages = {4440-4456.e7},
pmid = {34597596},
issn = {1097-4164},
support = {R01 CA234600/CA/NCI NIH HHS/United States ; R01 CA187052/CA/NCI NIH HHS/United States ; R01 CA095175/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 CA217991/CA/NCI NIH HHS/United States ; R01 GM134425/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; BRCA2 Protein/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Helicases/*metabolism ; DNA Repair ; DNA Repair Enzymes/*metabolism ; DNA, Single-Stranded/chemistry/metabolism ; DNA-Binding Proteins/*chemistry ; DNA-Directed DNA Polymerase/*chemistry ; Exodeoxyribonucleases/*metabolism ; HeLa Cells ; Humans ; Mevalonic Acid ; Mice ; Multiprotein Complexes ; Mutation ; Protein Binding ; Protein Conformation ; RNA, Guide/metabolism ; RNA, Small Interfering/metabolism ; Recombination, Genetic ; Transcription Factors/*chemistry ; },
abstract = {Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.},
}
@article {pmid34597584,
year = {2021},
author = {Gao, M and He, Y and Yin, X and Zhong, X and Yan, B and Wu, Y and Chen, J and Li, X and Zhai, K and Huang, Y and Gong, X and Chang, H and Xie, S and Liu, J and Yue, J and Xu, J and Zhang, G and Deng, Y and Wang, E and Tharreau, D and Wang, GL and Yang, W and He, Z},
title = {Ca[2+] sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector.},
journal = {Cell},
volume = {184},
number = {21},
pages = {5391-5404.e17},
doi = {10.1016/j.cell.2021.09.009},
pmid = {34597584},
issn = {1097-4172},
mesh = {CRISPR-Cas Systems/genetics ; Calcium/*metabolism ; Cell Membrane/metabolism ; Disease Resistance/genetics ; Free Radical Scavengers/*metabolism ; Fungal Proteins/*metabolism ; Models, Biological ; Oryza/genetics/*immunology ; Plant Diseases/immunology ; *Plant Immunity ; Plant Proteins/genetics/*metabolism ; Protein Binding ; Protein Stability ; Reactive Oxygen Species/*metabolism ; Reproduction ; Species Specificity ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination ; Zea mays/immunology ; },
abstract = {Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca[2+]-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca[2+] sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.},
}
@article {pmid34596864,
year = {2021},
author = {Ramakrishna, G and Babu, PE and Singh, R and Trehanpati, N},
title = {Application of CRISPR-Cas9 based gene editing to study the pathogenesis of colon and liver cancer using organoids.},
journal = {Hepatology international},
volume = {15},
number = {6},
pages = {1309-1317},
pmid = {34596864},
issn = {1936-0541},
mesh = {Animals ; CRISPR-Cas Systems ; Colon ; *Gene Editing ; Humans ; *Liver Neoplasms/genetics ; Organoids ; },
abstract = {Two breakthrough techniques that have totally revolutionized biology in last 1 decade are the discovery of genome editing tools and growing the stem cells/primary tissue explants in defined 3D culture. In this regard the discovery of CRISPR-Cas9 as a specific gene editing tool and organoid culture from adult stem cell has provided easy handy tools to uncover the process of organ development and also modeling cancer. Genetically modified organoids have been developed by sequential knockout and knockin of driver mutations by genome editing followed by niche-based selection. The modified organoids when xenotransplanted in animal models faithfully recapitulate the neoplastic events of human tumors. The present review focuses on the merging of these two powerful technologies in understanding the complexities of colon and liver cancer.},
}
@article {pmid34596650,
year = {2021},
author = {Liu, C and Xu, X and Koivisto, O and Zhou, W and Jacquemet, G and Rosenholm, JM and Zhang, H},
title = {Improving the knock-in efficiency of the MOF-encapsulated CRISPR/Cas9 system through controllable embedding structures.},
journal = {Nanoscale},
volume = {13},
number = {39},
pages = {16525-16532},
doi = {10.1039/d1nr02872c},
pmid = {34596650},
issn = {2040-3372},
mesh = {CRISPR-Cas Systems/genetics ; *Metal-Organic Frameworks ; *Nanostructures ; Plasmids/genetics ; Transfection ; },
abstract = {Appropriate tuning of robust artificial coatings can not only enhance intracellular delivery but also preserve the biological functions of genetic molecules in gene based therapies. Here, we report a strategy to synthesize controllable nanostructures in situ by encapsulating CRISPR/Cas9 plasmids into metal-organic frameworks (MOFs) via biomimetic mineralization. The structure-functionality relationship studies indicate that MOF-coated nanostructures dramatically impact the biological features of the contained plasmids through different embedding structures. The plasmids are homogeneously distributed within the heterogeneous nanoarchitecture and protected from enzymatic degradation. In addition, the plasmid-MOF structure exhibits excellent loading capability, pH-responsive release, and affinity for plasmid binding. Through in vitro assays it was found that the superior MOF vector can greatly enhance cellular endocytosis and endo/lysosomal escape of sheltered plasmids, resulting in successful knock-in of GFP-tagged paxillin genomic sequences in cancer cell lines with high transfection potency compared to our previous studies. Thus, the development of new cost-effective approaches for MOF-based intracellular delivery systems offers an attractive option for overcoming the physiological barriers to CRISPR/Cas9 delivery, which shows great potential for investigating paxillin-associated focal adhesions and signal regulation.},
}
@article {pmid34596508,
year = {2021},
author = {Alkam, D and Wongsurawat, T and Nookaew, I and Richardson, AR and Ussery, D and Smeltzer, MS and Jenjaroenpun, P},
title = {Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.},
journal = {Microbial genomics},
volume = {7},
number = {10},
pages = {},
pmid = {34596508},
issn = {2057-5858},
support = {P20 GM125503/GM/NIGMS NIH HHS/United States ; P30 GM103450/GM/NIGMS NIH HHS/United States ; R01 AI119380/AI/NIAID NIH HHS/United States ; UL1 TR000039/TR/NCATS NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA Transposable Elements ; Gene Library ; Genes, Essential ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; Humans ; Mutagenesis, Insertional ; Polymerase Chain Reaction/*methods ; Staphylococcus aureus/*genetics ; },
abstract = {As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall's correlation=0.896-0.897) although the latter remain superior owing to their accessibility and high sequencing depth.},
}
@article {pmid34594334,
year = {2021},
author = {Bruijnesteijn, J and van der Wiel, M and de Groot, NG and Bontrop, RE},
title = {Rapid Characterization of Complex Killer Cell Immunoglobulin-Like Receptor (KIR) Regions Using Cas9 Enrichment and Nanopore Sequencing.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {722181},
pmid = {34594334},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Methylation ; Epigenesis, Genetic ; Genome ; Genotype ; Haplotypes ; Humans ; Leukocytes, Mononuclear ; Macaca ; Multigene Family ; Nanopore Sequencing/*methods ; Receptors, KIR/*genetics ; Sequence Analysis, DNA ; },
abstract = {Long-read sequencing approaches have considerably improved the quality and contiguity of genome assemblies. Such platforms bear the potential to resolve even extremely complex regions, such as multigenic immune families and repetitive stretches of DNA. Deep sequencing coverage, however, is required to overcome low nucleotide accuracy, especially in regions with high homopolymer density, copy number variation, and sequence similarity, such as the MHC and KIR gene clusters of the immune system. Therefore, we have adapted a targeted enrichment protocol in combination with long-read sequencing to efficiently annotate complex KIR gene regions. Using Cas9 endonuclease activity, segments of the KIR gene cluster were enriched and sequenced on an Oxford Nanopore Technologies platform. This provided sufficient coverage to accurately resolve and phase highly complex KIR haplotypes. Our strategy eliminates PCR-induced amplification errors, facilitates rapid characterization of large and complex multigenic regions, including its epigenetic footprint, and is applicable in multiple species, even in the absence of a reference genome.},
}
@article {pmid34593991,
year = {2022},
author = {Yaméogo, P and Duchêne, BL and Majeau, N and Tremblay, JP},
title = {CRISPR-SCReT (CRISPR-Stop Codon Read Through) method to control Cas9 expression for gene editing.},
journal = {Gene therapy},
volume = {29},
number = {3-4},
pages = {171-177},
pmid = {34593991},
issn = {1476-5462},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Codon, Terminator/genetics ; *Gene Editing ; HEK293 Cells ; Humans ; },
abstract = {CRISPR/Cas9 has paved the way for the development of therapies that correct genetic mutations. However, constitutive expression of the Cas9 gene can increase off-target mutations and induce an immune response against the Cas9 protein. To limit the time during which the Cas9 nuclease is expressed, we proposed a simple drug inducible system. The approach consists of introducing a premature termination codon (PTC) in the Cas9 gene and subsequently treating with an aminoglycoside drug, which allows readthrough of the complete protein. To validate that system, HEK293T cells were co-transfected with a PX458 plasmid, which was mutated to introduce a PTC in the SpCas9 gene and two sgRNAs targeting the DMD gene (exons 50 and 54). Cells were treated with different doses of geneticin (G418) for 48 h. Western blot confirmed that the Cas9 protein expression, which was shut down by the PTC mutation, can be induced by the drug. The hybrid exon 50-54 formed by the deletion of part of the DMD gene was detected by PCR only in the cells treated with G418. The approach was also used successfully with CjCas9 to edit the FXN gene. Our results show that it is possible to control SpCas9 and CjCas9 expression by CRISPR-SCReT (CRISPR-Stop Codon Read Through) method.},
}
@article {pmid34593942,
year = {2021},
author = {Schubert, MS and Thommandru, B and Woodley, J and Turk, R and Yan, S and Kurgan, G and McNeill, MS and Rettig, GR},
title = {Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {19482},
pmid = {34593942},
issn = {2045-2322},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Cell Line ; Endodeoxyribonucleases/*metabolism ; *Gene Editing ; Humans ; Mutation ; RNA, Guide/genetics ; *Recombinational DNA Repair ; },
abstract = {CRISPR-Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR.},
}
@article {pmid34593874,
year = {2021},
author = {Naboulsi, R and Larsson, M and Andersson, L and Younis, S},
title = {ZBED6 regulates Igf2 expression partially through its regulation of miR483 expression.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {19484},
pmid = {34593874},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Computational Biology/methods ; Flow Cytometry ; Gene Editing ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Targeting ; Insulin-Like Growth Factor II/*genetics ; Mice, Knockout ; Mice, Transgenic ; MicroRNAs/*genetics ; Models, Biological ; *RNA Interference ; Repressor Proteins/*genetics ; Transcriptome ; },
abstract = {The expression of Igf2 in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. We previously identified a novel regulatory mechanism based on the interaction between the transcriptional factor ZBED6 and Igf2 intron. Disruption of the ZBED6-Igf2 interaction leads to a dramatic up-regulation of IGF2 expression postnatally. In the current study we characterize an additional layer of regulation involving miR483 encoded by another Igf2 intron. We found a highly significant up-regulation of miR483 expression when the ZBED6-Igf2 axis is disrupted in transgenic mice. Furthermore, CRISPR/Cas9 mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf2 axis (Igf2[dGGCT]), resulted in down-regulation of Igf2 expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. RNA-seq analysis revealed a significant enrichment of genes involved in the PI3K-Akt signaling pathway among genes down-regulated in miR483[-/-] cells, including Igf2 down-regulation. The opposite pattern was observed in Igf2[dGGCT] cells, where Igf2 is up-regulated. Our data suggest a positive feedback between miR483 and Igf2 promoter activity, strongly affecting how ZBED6 controls Igf2 expression in various cell types.},
}
@article {pmid34593768,
year = {2021},
author = {Chang, J and Guo, Y and Yan, J and Zhang, Z and Yuan, L and Wei, C and Zhang, Y and Ma, J and Yang, J and Zhang, X and Li, H},
title = {The role of watermelon caffeic acid O-methyltransferase (ClCOMT1) in melatonin biosynthesis and abiotic stress tolerance.},
journal = {Horticulture research},
volume = {8},
number = {1},
pages = {210},
pmid = {34593768},
issn = {2662-6810},
abstract = {Melatonin is a pleiotropic signaling molecule that regulates plant growth and responses to various abiotic stresses. The last step of melatonin synthesis in plants can be catalyzed by caffeic acid O-methyltransferase (COMT), a multifunctional enzyme reported to have N-acetylserotonin O-methyltransferase (ASMT) activity; however, the ASMT activity of COMT has not yet been characterized in nonmodel plants such as watermelon (Citrullus lanatus). Here, a total of 16 putative O-methyltransferase (ClOMT) genes were identified in watermelon. Among them, ClOMT03 (Cla97C07G144540) was considered a potential COMT gene (renamed ClCOMT1) based on its high identities (60.00-74.93%) to known COMT genes involved in melatonin biosynthesis, expression in almost all tissues, and upregulation under abiotic stresses. The ClCOMT1 protein was localized in the cytoplasm. Overexpression of ClCOMT1 significantly increased melatonin contents, while ClCOMT1 knockout using the CRISPR/Cas-9 system decreased melatonin contents in watermelon calli. These results suggest that ClCOMT1 plays an essential role in melatonin biosynthesis in watermelon. In addition, ClCOMT1 expression in watermelon was upregulated by cold, drought, and salt stress, accompanied by increases in melatonin contents. Overexpression of ClCOMT1 enhanced transgenic Arabidopsis tolerance against such abiotic stresses, indicating that ClCOMT1 is a positive regulator of plant tolerance to abiotic stresses.},
}
@article {pmid34593753,
year = {2021},
author = {Zhang, Z and Zeng, D and Zhang, W and Chen, A and Lei, J and Liu, F and Deng, B and Zhuo, J and He, B and Yan, M and Lei, X and Wang, S and Lam, EW and Liu, Q and Wang, Z},
title = {Modulation of oxidative phosphorylation augments antineoplastic activity of mitotic aurora kinase inhibition.},
journal = {Cell death &amp; disease},
volume = {12},
number = {10},
pages = {893},
pmid = {34593753},
issn = {2041-4889},
mesh = {Adenosine Triphosphate/metabolism ; Animals ; Antineoplastic Agents/*pharmacology ; Aurora Kinase A/*antagonists &amp; inhibitors/metabolism ; Azepines/pharmacology ; Breast Neoplasms/pathology ; CRISPR-Cas Systems/genetics ; Cell Death/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Respiration/drug effects ; Cytosol/metabolism ; Drug Synergism ; Energy Metabolism/drug effects ; Female ; Homeostasis/drug effects ; Humans ; Metformin/pharmacology ; Mice, Inbred BALB C ; Mitochondria/drug effects/metabolism ; *Mitosis/drug effects ; *Oxidative Phosphorylation/drug effects ; Oxygen Consumption/drug effects ; Pyrimidines/pharmacology ; },
abstract = {Uncontrolled mitosis is one of the most important features of cancer, and mitotic kinases are thought to be ideal targets for anticancer therapeutics. However, despite numerous clinical attempts spanning decades, clinical trials for mitotic kinase-targeting agents have generally stalled in the late stages due to limited therapeutic effectiveness. Alisertib (MLN8237) is a promising oral mitotic aurora kinase A (AURKA, Aurora-A) selective inhibitor, which is currently under several clinical evaluations but has failed in its first Phase III trial due to inadequate efficacy. In this study, we performed genome-wide CRISPR/Cas9-based screening to identify vulnerable biological processes associated with alisertib in breast cancer MDA-MB-231 cells. The result indicated that alisertib treated cancer cells are more sensitive to the genetic perturbation of oxidative phosphorylation (OXPHOS). Mechanistic investigation indicated that alisertib treatment, as well as other mitotic kinase inhibitors, rapidly reduces the intracellular ATP level to generate a status that is highly addictive to OXPHOS. Furthermore, the combinational inhibition of mitotic kinase and OXPHOS by alisertib, and metformin respectively, generates severe energy exhaustion in mitotic cells that consequently triggers cell death. The combination regimen also enhanced tumor regression significantly in vivo. This suggests that targeting OXPHOS by metformin is a potential strategy for promoting the therapeutic effects of mitotic kinase inhibitors through the joint targeting of mitosis and cellular energy homeostasis.},
}
@article {pmid34593043,
year = {2021},
author = {Senevirathne, A and Hewawaduge, C and Lee, JH},
title = {Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek's disease virus in chickens.},
journal = {Veterinary research},
volume = {52},
number = {1},
pages = {125},
pmid = {34593043},
issn = {1297-9716},
mesh = {Animals ; *CRISPR-Cas Systems ; *Chickens ; Female ; Herpesvirus 2, Gallid/*physiology ; Leukocytes, Mononuclear/virology ; Marek Disease/pathology/*prevention &amp; control/virology ; Plasmids/*therapeutic use ; Poultry Diseases/pathology/*prevention &amp; control/virology ; Salmonella/*physiology/virology ; },
abstract = {Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6[th] day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.},
}
@article {pmid34593010,
year = {2021},
author = {Vink, JNA and Baijens, JHL and Brouns, SJJ},
title = {PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {281},
pmid = {34593010},
issn = {1474-760X},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Conserved Sequence ; Position-Specific Scoring Matrices ; Species Specificity ; },
abstract = {BACKGROUND: The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database.<br><br>RESULTS: By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders.<br><br>CONCLUSIONS: This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.},
}
@article {pmid34592602,
year = {2021},
author = {Li, T and Ma, B and Yang, H and Zhu, G and Shu, C and Luo, M and Zhou, Z},
title = {Generation of a CRISPR/Cas9-corrected-hiPSC (NCCDFWi001-A-1) from a Marfan syndrome patient hiPSC with a heterozygous c.2613A&gt;C variant in the fibrillin 1 (FBN1) gene.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102543},
doi = {10.1016/j.scr.2021.102543},
pmid = {34592602},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Fibrillin-1/genetics ; Humans ; *Induced Pluripotent Stem Cells ; *Marfan Syndrome/genetics ; Mutation ; },
abstract = {Patient-specific hiPSCs (NCCDFWi001-A) were generated from a patient with Marfan syndrome carrying a compound heterozygous variant (c.684_736 + 4del, p.Pro228fs and c.2613A>C, p.Leu871Phe). Here, we used CRISPR/ Cas9 to correct the FBN1 c.2613A>C variant, which generated an hiPSC line (NCCDFWi001-A-1) that maintained normal karyotype, pluripotency markers and demonstrated potential for trilineage differentiation.},
}
@article {pmid34592310,
year = {2021},
author = {Lemak, S and Serbanescu, MA and Khusnutdinova, AN and Ruszkowski, M and Beloglazova, N and Xu, X and Brown, G and Cui, H and Tan, K and Joachimiak, A and Cvitkovitch, DG and Savchenko, A and Yakunin, AF},
title = {Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {5},
pages = {101251},
pmid = {34592310},
issn = {1083-351X},
mesh = {Bacterial Proteins/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Models, Molecular ; *RNA Processing, Post-Transcriptional ; RNA, Bacterial/*chemistry/metabolism ; Ribonucleases/*chemistry/genetics/metabolism ; Streptococcus mutans/*chemistry/genetics/metabolism ; },
abstract = {The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted β-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.},
}
@article {pmid34592134,
year = {2021},
author = {Kong, L and Feng, B and Yan, Y and Zhang, C and Kim, JH and Xu, L and Rack, JGM and Wang, Y and Jang, JC and Ahel, I and Shan, L and He, P},
title = {Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.},
journal = {Molecular cell},
volume = {81},
number = {22},
pages = {4591-4604.e8},
pmid = {34592134},
issn = {1097-4164},
support = {210634/WT_/Wellcome Trust/United Kingdom ; R01 GM092893/GM/NIGMS NIH HHS/United States ; BB/R007195/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; R01 GM097247/GM/NIGMS NIH HHS/United States ; 101794/WT_/Wellcome Trust/United Kingdom ; C35050/A22284/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {ADP Ribose Transferases/metabolism ; *ADP-Ribosylation ; Adenosine Diphosphate/chemistry ; Arabidopsis/*immunology/metabolism ; Arabidopsis Proteins/*metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/*metabolism ; Genes, Plant ; Glycoside Hydrolases/metabolism ; Homeostasis ; Humans ; Hydrolysis ; Intracellular Signaling Peptides and Proteins/*metabolism ; Mutation ; *Plant Immunity ; Plants, Genetically Modified ; Poly Adenosine Diphosphate Ribose/metabolism ; Poly(ADP-ribose) Polymerases/metabolism ; Proteostasis ; Seedlings/metabolism ; Substrate Specificity ; Tristetraprolin/chemistry ; Two-Hybrid System Techniques ; Ubiquitin/chemistry ; *Ubiquitination ; *Zinc Fingers ; },
abstract = {Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD[+] onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.},
}
@article {pmid34591643,
year = {2021},
author = {Altae-Tran, H and Kannan, S and Demircioglu, FE and Oshiro, R and Nety, SP and McKay, LJ and Dlakić, M and Inskeep, WP and Makarova, KS and Macrae, RK and Koonin, EV and Zhang, F},
title = {The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.},
journal = {Science (New York, N.Y.)},
volume = {374},
number = {6563},
pages = {57-65},
pmid = {34591643},
issn = {1095-9203},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Conserved Sequence ; DNA Transposable Elements/*genetics ; Endodeoxyribonucleases/*genetics ; *Evolution, Molecular ; Genetic Code ; Genetic Variation ; *RNA, Guide ; RNA, Untranslated/genetics ; },
abstract = {IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.},
}
@article {pmid34590692,
year = {2021},
author = {Chen, J and Qiu, T and Mauk, MG and Fan, Y and Jiang, Y and Ying, J and Zhou, Q and Qiao, Y and Bau, HH and Song, J},
title = {CRISPR Cas9-Mediated Selective Isothermal Amplification for Sensitive Detection of Rare Mutant Alleles.},
journal = {Clinical chemistry},
volume = {67},
number = {11},
pages = {1569-1571},
pmid = {34590692},
issn = {1530-8561},
support = {K01 TW011190/TW/FIC NIH HHS/United States ; R21 CA228614-01A1/GF/NIH HHS/United States ; R21 TW011496/TW/FIC NIH HHS/United States ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; *Nucleic Acid Amplification Techniques ; },
}
@article {pmid34590280,
year = {2021},
author = {Kerek, EM and Cromwell, CR and Hubbard, BP},
title = {Identification of Drug Resistance Genes Using a Pooled Lentiviral CRISPR/Cas9 Screening Approach.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2381},
number = {},
pages = {227-242},
pmid = {34590280},
issn = {1940-6029},
support = {PS-408552//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems/genetics ; Drug Resistance ; Gene Editing ; Genome ; Lentivirus ; Pharmaceutical Preparations ; },
abstract = {In addition to advancing the development of gene-editing therapeutics, CRISPR/Cas9 is transforming how functional genetic studies are carried out in the lab. By increasing the ease with which genetic information can be inserted, deleted, or edited in cell and organism models, it facilitates genotype-phenotype analysis. Moreover, CRISPR/Cas9 has revolutionized the speed at which new genes underlying a particular phenotype can be identified through its application in genomic screens. Arrayed high-throughput and pooled lentiviral-based CRISPR/Cas9 screens have now been used in a wide variety of contexts, including the identification of essential genes, genes involved in cancer metastasis and tumor growth, and even genes involved in viral response. This technology has also been successfully used to identify drug targets and drug resistance mechanisms. Here, we provide a detailed protocol for performing a genome-wide pooled lentiviral CRISPR/Cas9 knockout screen to identify genetic modulators of a small-molecule drug. While we exemplify how to identify genes involved in resistance to a cytotoxic histone deacetylase inhibitor, Trichostatin A (TSA), the workflow we present can easily be adapted to different types of selections and other types of exogenous ligands or drugs.},
}
@article {pmid34590276,
year = {2021},
author = {Zhang, DY and Gui, X and Yang, X},
title = {Mapping Genetic Interactions in Human Cancer Cells Using a One-Step tRNA-CRISPR System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2381},
number = {},
pages = {175-187},
pmid = {34590276},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epistasis, Genetic ; Humans ; *Neoplasms/genetics ; RNA, Transfer/genetics ; },
abstract = {While well studied in yeast, mapping genetic interactions in mammalian cells has been limited due to many technical obstacles. We have recently developed a new one-step tRNA-CRISPR method called TCGI (tRNA-CRISPR for genetic interactions) which generates high-efficiency, barcode-free, and scalable pairwise CRISPR libraries to identify genetic interactions in mammalian cells. Here we describe this method in detail regarding the construction of the pairwise CRISPR libraries and performing high throughput genetic interacting screening and data analysis. This novel TCGI dramatically improves upon the current methods for mapping genetic interactions and screening drug targets for combinational therapies.},
}
@article {pmid34590274,
year = {2021},
author = {MacAuley, MJ and Abuhussein, O and Vizeacoumar, FS},
title = {Identification of Synthetic Lethal Interactions Using High-Throughput, Arrayed CRISPR/Cas9-Based Platforms.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2381},
number = {},
pages = {135-149},
pmid = {34590274},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; Synthetic Lethal Mutations ; },
abstract = {Over the past two decades, the concept of synthetic lethality (SL) that queries genetic relationships between gene pairs has gradually emerged as one of the best strategies to selectively eliminate cancer cells. Some of the most successful approaches to identify synthetic lethal interactions (SLIs) were largely dependent on pooled screening formats that require heavy validation in order to mitigate false positives. Here, we describe a high-throughput method to identify SLIs using CRISPR-based strategy that covers, high-throughput production of plasmid DNA preparations, lentiviral production, and subsequent cellular transduction using single guide RNAs (sgRNAs). This method could be adopted to query hundreds of SLIs. As an example, we describe the methods associated with building an interaction map for DNA damage and repair (DDR) genes. The use of multiwell plates and image-based quantification allows a comparative measurement of SLIs at a high-resolution on a one-by-one basis. Furthermore, this scalable, arrayed CRISPR screening method can be applied to multiple cancer cell types, and genes of interest, resulting in new functional discoveries that can be exploited therapeutically.},
}
@article {pmid34590222,
year = {2021},
author = {Weng, S and Zhao, Y and Yu, C and Wang, X and Xiao, X and Han, L and Zhang, K and Wang, J and Yang, G},
title = {Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass.},
journal = {Biotechnology letters},
volume = {43},
number = {11},
pages = {2111-2129},
pmid = {34590222},
issn = {1573-6776},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dependovirus/*genetics ; Gene Editing/*methods ; Genetic Vectors/*genetics ; Green Fluorescent Proteins/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Skeletal/*physiology ; Myostatin/genetics ; },
abstract = {An ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic risks. This is important for gene editing research at specific site in vivo. We report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and the editing efficiency was similar to that of the classical Cas9 system in vitro and in vivo. And we inserted the sequence of the green fluorescent protein eGFP into rAAV. The number of cells infected with the rAAV and the region in which the rAAV spreads were known by the fluorescent expression of eGFP in cells. In addition, we demonstrated that myostatin gene in the thigh muscles of C57BL/10 mice was knocked out by the rAAV9-SaCas9 system to make muscle mass increased obviously. The protein eGFP into rAAV has significant implications for our indirect analysis of the editing efficiency of SaCas9 in the genome of the target tissue and reduces the harm caused by off-target editing and prevents other tissue mutations. The rAAV system has substantial potential in improving muscle mass and preventing muscle atrophy.},
}
@article {pmid34590151,
year = {2021},
author = {Peng, S and Cai, J and Bao, S},
title = {CMBs carrying PTX and CRISPR/Cas9 targeting C‑erbB‑2 plasmids interfere with endometrial cancer cells.},
journal = {Molecular medicine reports},
volume = {24},
number = {6},
pages = {},
pmid = {34590151},
issn = {1791-3004},
mesh = {Antineoplastic Agents, Phytogenic/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Disease Progression ; Drug Therapy/methods ; Endometrial Neoplasms/*drug therapy ; Female ; Gene Editing/methods ; Gene Expression Regulation, Neoplastic/*drug effects ; Gene Knockout Techniques/methods ; Humans ; Microbubbles/*therapeutic use ; Paclitaxel/*pharmacology ; Plasmids ; Proliferating Cell Nuclear Antigen/metabolism ; Receptor, ErbB-2/*genetics/*metabolism ; },
abstract = {Development of combination therapy to decrease side effects of chemotherapeutic drugs and increase their utilization rate in combination with gene editing is a key research topic in tumor treatment. The present study aimed to investigate the effect of cationic microbubbles (CMBs) carrying paclitaxel (PTX) and C‑erbB‑2 knockout plasmid on the endometrial cancer cell line HEC‑1A and to determine how C‑erbB‑2 regulates the function of endometrial cancer cells. Cells were treated with CMB, PTX, PTX‑CMBs, cationic plasmid‑carrying or cationic PTX‑carrying plasmid groups. After verifying the most effective combination of PTX‑CMBs and plasmids, HEC‑1A cells were transfected. Reverse transcription‑quantitative (RT‑q)PCR and western blotting were used to measure C‑erbB‑2 and protein expression. After verifying C‑erbB‑2 knockout, invasion, healing, clone formation and proliferation of HEC‑1A cells were assessed. Simultaneously, expression levels of the genes for P21, P27, mammalian target of rapamycin (mTOR), and Bcl‑2 associated death promoter (Bad) were measured by RT‑qPCR. Compared with the PTX group, CMBs significantly enhanced the absorption efficiency of PTX by HEC‑1A cells. C‑erbB‑2 knockout had an inhibitory effect on the proliferation, migration and invasion of HEC‑1A cells; cell proliferation and invasion of the group carrying PTX and plasmids simultaneously were significantly weakened. The C‑erbB‑2‑knockout group exhibited increased expression of P21 and P27. Simultaneously loading PTX and plasmid may be novel combination therapy with great potential. C‑erbB‑2 may regulate the proliferation of HEC‑1A cells by downregulating expression of P21 and P27.},
}
@article {pmid34589070,
year = {2021},
author = {Le Gratiet, T and Le Marechal, C and Devaere, M and Chemaly, M and Woudstra, C},
title = {Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {711413},
pmid = {34589070},
issn = {1664-302X},
abstract = {Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I-IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.},
}
@article {pmid34588691,
year = {2021},
author = {Hu, C and Almendros, C and Nam, KH and Costa, AR and Vink, JNA and Haagsma, AC and Bagde, SR and Brouns, SJJ and Ke, A},
title = {Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.},
journal = {Nature},
volume = {598},
number = {7881},
pages = {515-520},
pmid = {34588691},
issn = {1476-4687},
support = {R35 GM118174/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Databases, Genetic ; Endonucleases/*metabolism ; Geobacter/*enzymology ; Models, Molecular ; Molecular Conformation ; Nucleotide Motifs ; },
abstract = {Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array[1]. Spacer insertion is carried out by the Cas1-Cas2 integrase complex[2-4]. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM)[5,6] and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.},
}
@article {pmid34588447,
year = {2021},
author = {Sapozhnikov, DM and Szyf, M},
title = {Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5711},
pmid = {34588447},
issn = {2041-1723},
support = {PJT-159583//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; CpG Islands/genetics ; *DNA Demethylation ; DNA Methylation ; DNA Modification Methylases/genetics/*metabolism ; *Epigenesis, Genetic ; Fragile X Mental Retardation Protein/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Mice ; Mixed Function Oxygenases/genetics/*metabolism ; NIH 3T3 Cells ; Promoter Regions, Genetic/genetics ; Proto-Oncogene Proteins/genetics/*metabolism ; RNA, Guide/metabolism ; Serpins/genetics ; },
abstract = {Despite four decades of research to support the association between DNA methylation and gene expression, the causality of this relationship remains unresolved. Here, we reaffirm that experimental confounds preclude resolution of this question with existing strategies, including recently developed CRISPR/dCas9 and TET-based epigenetic editors. Instead, we demonstrate a highly effective method using only nuclease-dead Cas9 and guide RNA to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzyme, thereby enabling the examination of the role of DNA demethylation per se in living cells, with no evidence of off-target activity. Using this method, we probe a small number of inducible promoters and find the effect of DNA demethylation to be small, while demethylation of CpG-rich FMR1 produces larger changes in gene expression. This method could be used to reveal the extent and nature of the contribution of DNA methylation to gene regulation.},
}
@article {pmid34588320,
year = {2021},
author = {Chaudhary, N and Im, JK and Nho, SH and Kim, H},
title = {Visualizing Live Chromatin Dynamics through CRISPR-Based Imaging Techniques.},
journal = {Molecules and cells},
volume = {44},
number = {9},
pages = {627-636},
pmid = {34588320},
issn = {0219-1032},
mesh = {CRISPR-Cas Systems/*genetics ; Chromatin/*metabolism ; Genomics/*methods ; Humans ; },
abstract = {The three-dimensional organization of chromatin and its time-dependent changes greatly affect virtually every cellular function, especially DNA replication, genome maintenance, transcription regulation, and cell differentiation. Sequencing-based techniques such as ChIP-seq, ATAC-seq, and Hi-C provide abundant information on how genomic elements are coupled with regulatory proteins and functionally organized into hierarchical domains through their interactions. However, visualizing the time-dependent changes of such organization in individual cells remains challenging. Recent developments of CRISPR systems for site-specific fluorescent labeling of genomic loci have provided promising strategies for visualizing chromatin dynamics in live cells. However, there are several limiting factors, including background signals, off-target binding of CRISPR, and rapid photobleaching of the fluorophores, requiring a large number of target-bound CRISPR complexes to reliably distinguish the target-specific foci from the background. Various modifications have been engineered into the CRISPR system to enhance the signal-to-background ratio and signal longevity to detect target foci more reliably and efficiently, and to reduce the required target size. In this review, we comprehensively compare the performances of recently developed CRISPR designs for improved visualization of genomic loci in terms of the reliability of target detection, the ability to detect small repeat loci, and the allowed time of live tracking. Longer observation of genomic loci allows the detailed identification of the dynamic characteristics of chromatin. The diffusion properties of chromatin found in recent studies are reviewed, which provide suggestions for the underlying biological processes.},
}
@article {pmid34587248,
year = {2022},
author = {Sasaki, K and Yamauchi, T and Semba, Y and Nogami, J and Imanaga, H and Terasaki, T and Nakao, F and Akahane, K and Inukai, T and Verhoeyen, E and Akashi, K and Maeda, T},
title = {Genome-wide CRISPR-Cas9 screen identifies rationally designed combination therapies for CRLF2-rearranged Ph-like ALL.},
journal = {Blood},
volume = {139},
number = {5},
pages = {748-760},
pmid = {34587248},
issn = {1528-0020},
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Rearrangement/drug effects ; Humans ; Mice ; Nitriles/pharmacology ; Philadelphia Chromosome ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/*genetics ; Protein Kinase Inhibitors/pharmacology ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Receptors, Cytokine/*genetics ; Signal Transduction/drug effects ; },
abstract = {Acute lymphoblastic leukemia (ALL) harboring the IgH-CRLF2 rearrangement (IgH-CRLF2-r) exhibits poor clinical outcomes and is the most common subtype of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). While multiple chemotherapeutic regimens, including ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. To identify molecules/pathways relevant for IgH-CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of ruxolitinib using 2 IgH-CRLF2-r ALL lines that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled, for the first time, highly efficient transduction of human B cells. While single-guide RNAs (sgRNAs) targeting CRLF2, IL7RA, or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B, or STAT3 did not, suggesting that STAT signaling is largely dispensable for IgH-CRLF2-r ALL cell survival. We show that regulators of RAS signaling are critical for cell fitness and ruxolitinib sensitivity and that CRKL depletion enhances ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that blocks CRKL phosphorylation, effectively killed RAS WT IgH-CRLF2-r ALL cells in vitro and in vivo, either alone or combined with ruxolitinib. We further show that combining gilteritinib with trametinib, a MEK1/2 inhibitor, is an effective means to target IgH-CRLF2-r ALL cells regardless of RAS mutational status. Our study delineates molecules/pathways relevant for CRLF2-r ALL pathogenesis and could suggest rationally designed combination therapies appropriate for disease subtypes.},
}
@article {pmid34586867,
year = {2021},
author = {Shue, B and Chiramel, AI and Cerikan, B and To, TH and Frölich, S and Pederson, SM and Kirby, EN and Eyre, NS and Bartenschlager, RFW and Best, SM and Beard, MR},
title = {Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.},
journal = {Journal of virology},
volume = {95},
number = {24},
pages = {e0059621},
pmid = {34586867},
issn = {1098-5514},
mesh = {A549 Cells ; Aedes ; Animals ; COVID-19 ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; Culicidae ; Dengue Virus/genetics ; Flavivirus/*genetics ; Genome-Wide Association Study ; HEK293 Cells ; Host-Pathogen Interactions/genetics ; Humans ; Neoplasm Proteins/*genetics ; RNA, Small Interfering/metabolism ; RNA, Viral/metabolism ; Receptors for Activated C Kinase/*genetics ; SARS-CoV-2 ; Vero Cells ; *Virus Replication ; West Nile virus/genetics ; Zika Virus/genetics ; Zika Virus Infection/virology ; },
abstract = {Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.},
}
@article {pmid34586743,
year = {2021},
author = {Cao, C and Yao, L and Li, A and Zhang, Q and Zhang, Z and Wang, X and Gan, Y and Liu, Y and Zhang, Q},
title = {A CRISPR/dCasX-mediated transcriptional programming system for inhibiting the progression of bladder cancer cells by repressing c-MYC or activating TP53.},
journal = {Clinical and translational medicine},
volume = {11},
number = {9},
pages = {e537},
pmid = {34586743},
issn = {2001-1326},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Gene Editing/*methods ; Humans ; Mice ; Proto-Oncogene Proteins c-myc/*genetics ; Tumor Suppressor Protein p53/*genetics ; *Urinary Bladder Neoplasms/genetics/therapy ; },
}
@article {pmid34586111,
year = {2021},
author = {Zhang, C and Yao, H and Ma, Q and Yu, B},
title = {Ultrasensitive glucose detection from tears and saliva through integrating a glucose oxidase-coupled DNAzyme and CRISPR-Cas12a.},
journal = {The Analyst},
volume = {146},
number = {21},
pages = {6576-6581},
doi = {10.1039/d1an01385h},
pmid = {34586111},
issn = {1364-5528},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; *DNA, Catalytic ; Glucose ; Glucose Oxidase ; Hydrogen Peroxide ; Saliva ; },
abstract = {The accurate and sensitive detection of glucose from secretory clinical samples, such as tears and saliva, remains a great challenge. In this research, a novel ultrasensitive glucose detection method consisting of a glucose oxidase (GOx), pistol-like DNAzyme (PLDz), and CRISPR-Cas12a system is proposed. First, the oxidation of glucose catalyzed by GOx leads to the production of H2O2; the self-cleavage activity of PLDz is activated after recognition of the produced H2O2. The two procedures triggered by COx and PLDz play an important role in accurately identifying glucose and converting glucose signals to nucleic acids. The obtained PLDz fragments can be recognized by the Cas12 enzyme and thus activate the trans-cleavage activity of the Cas12a enzyme. Finally, the surrounding reporter probes are cut by the Cas12a enzyme to produce fluorescence signals. In summary, an ultra-sensitive and specific fluorescence method has been developed for glucose detection from secretory clinical samples, which could potentially contribute to the noninvasive diagnosis of diabetes mellitus.},
}
@article {pmid34585988,
year = {2021},
author = {Turra, GL and Liedgens, L and Sommer, F and Schneider, L and Zimmer, D and Vilurbina Perez, J and Koncarevic, S and Schroda, M and Mühlhaus, T and Deponte, M},
title = {In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv.},
journal = {Microbiology spectrum},
volume = {9},
number = {2},
pages = {e0080921},
pmid = {34585988},
issn = {2165-0497},
mesh = {CRISPR-Cas Systems/genetics ; Leishmania/classification/genetics/*metabolism ; Mitochondria/*metabolism ; Mitochondrial Precursor Protein Import Complex Proteins/*metabolism ; Oxidation-Reduction ; Oxidoreductases Acting on Sulfur Group Donors/*metabolism ; Protein Domains/genetics ; Protein Folding ; Protein Transport/genetics ; Structure-Activity Relationship ; },
abstract = {Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys[17] or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErv[ΔKISS] using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.},
}
@article {pmid34585814,
year = {2021},
author = {Yu, L and Ma, S and Zhang, X and Tian, D and Yang, S and Jia, X and Traw, MB},
title = {Ancient rapid functional differentiation and fixation of the duplicated members in rice Dof genes after whole genome duplication.},
journal = {The Plant journal : for cell and molecular biology},
volume = {108},
number = {5},
pages = {1365-1381},
doi = {10.1111/tpj.15516},
pmid = {34585814},
issn = {1365-313X},
mesh = {Biological Evolution ; Edible Grain/genetics ; Evolution, Molecular ; Gene Duplication ; Genome, Plant/*genetics ; Oryza/*genetics ; Plant Proteins/*genetics ; Sorghum/*genetics ; Synteny ; Transcription Factors/genetics ; },
abstract = {Whole genome duplication (WGD) in plants is typically followed by genomic downsizing, where large portions of the new genome are lost. Whether this downsizing is accompanied by increased or decreased evolutionary rates of the remaining genes is poorly known, not least because homeolog pairings are often obscured by chromosomal rearrangement. Here, we use the newly published genome from a sedge, namely Kobresia littledalei, and CRISPR/Cas-9 editing to investigate how the Rho WGD event 70 million years ago (MYA) affected transcription factor evolutionary rates, fates, and function in rice (Oryza sativa) and sorghum (Sorghum bicolor). We focus on the 30-member DNA-binding with one zinc finger (Dof) transcription factor family in both crops due to their agronomic importance. Using the known speciation dates of rice from Kobresia (97 MYA) and sorghum (50 MYA), we find that rates of amino acid substitution in the critical Dof domain region were over twofold higher during the 20-million-year period following the WGD than before or afterward. Through comparison of synteny blocks, we report that at least 11% of Dof genes were purged from 70 to 50 MYA, while only 6% have been lost in the most recent 50-million-year interval. CRISPR/Cas9 editing revealed widespread fitness-related defects in flowering and lack of redundancy of paired members, as well as significant differences in expression between gene pairs. Together these findings demonstrate the strength of Dof genes as a model for deep evolutionary study and offer one of the most detailed portraits yet of the Rho WGD impact on a gene lineage.},
}
@article {pmid34585154,
year = {2021},
author = {Alfastsen, L and Peng, X and Bhoobalan-Chitty, Y},
title = {Genome editing in archaeal viruses and endogenous viral protein purification.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100791},
pmid = {34585154},
issn = {2666-1667},
mesh = {Archaeal Viruses/*genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Viral/*genetics ; Host Microbial Interactions/genetics ; Sulfolobus/virology ; Viral Proteins/genetics/*isolation &amp; purification/metabolism ; },
abstract = {Archaea-infecting viruses are morphologically and genomically among the most diverse entities. Unfortunately, they are also fairly understudied due to a lack of efficient genetic tools. Here, we present a detailed protocol for the CRISPR/Cas-based genome editing of the virus SIRV2 infecting the genus Sulfolobus, which could easily be adapted to other archaeal viruses. This protocol also includes the procedure for endogenous viral protein purification and identification, allowing for assessing the molecular mechanisms behind virus life cycle and virus-host interactions. For complete details on the use and execution of this protocol, please refer to Mayo-Muñoz et al. (2018) and Bhoobalan-Chitty et al. (2019).},
}
@article {pmid34585153,
year = {2021},
author = {Vasu, K and Fox, PL},
title = {Screening of CRISPR-Cas9-generated point mutant mice using MiSeq and locked nucleic acid probe PCR.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100785},
pmid = {34585153},
issn = {2666-1667},
support = {P01 HL076491/HL/NHLBI NIH HHS/United States ; R01 DK123236/DK/NIDDK NIH HHS/United States ; R01 AG067146/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Mutational Analysis ; Female ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing ; Male ; Mice ; Mutagenesis, Site-Directed/*methods ; Mutation/genetics ; Oligonucleotides/genetics ; },
abstract = {CRISPR-Cas9-mediated, site-directed mutagenesis in mice generates mosaic founder mice with varied efficiency of desired point mutation and other non-homologous end-joined variants. Here, we present a protocol for design, sample preparation, and analysis for identification of mice with the desired mutation. Deep sequencing provides the proportion of reads of a particular allele for each mouse line. Locked nucleic acid probe-based qPCR provides rapid identification of the mutant allele and can be used for genotyping offspring during subsequent breeding for colony establishment. For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021).},
}
@article {pmid34585151,
year = {2021},
author = {Okamoto, T and Natsume, Y and Yamanaka, H and Fukuda, M and Yao, R},
title = {A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids.},
journal = {STAR protocols},
volume = {2},
number = {4},
pages = {100780},
pmid = {34585151},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; *Colorectal Neoplasms/genetics/pathology ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Humans ; Neoplastic Stem Cells/cytology ; Organoids/*pathology ; Tumor Cells, Cultured ; },
abstract = {Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of the potential cancer stem cell marker gene, which allows us to investigate their potential for self-replication and pluripotency. We describe the procedure for generating mutant PDOs and their application for stem cell research. For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021).},
}
@article {pmid34584990,
year = {2021},
author = {Li, X and Fargue, S and Challa, AK and Poore, W and Knight, J and Wood, KD},
title = {Generation of a GLO-2 deficient mouse reveals its effects on liver carbonyl and glutathione levels.},
journal = {Biochemistry and biophysics reports},
volume = {28},
number = {},
pages = {101138},
pmid = {34584990},
issn = {2405-5808},
support = {K01 DK114332/DK/NIDDK NIH HHS/United States ; },
abstract = {OBJECTIVE: Hydroxyacylglutathione hydrolase (aka as GLO-2) is a component of the glyoxalase pathway involved in the detoxification of the reactive oxoaldehydes, glyoxal and methylglyoxal. These reactive metabolites have been linked to a variety of pathological conditions, including diabetes, cancer and heart disease and may be involved in the aging process. The objective of this study was to generate a mouse model deficient in GLO-2 to provide insight into the function of GLO-2 and to determine if it is potentially linked to endogenous oxalate synthesis which could influence urinary oxalate excretion.<br><br>METHODS: A GLO-2 knock out mouse was generated using CRISPR/Cas 9 techniques. Tissue and 24-h urine samples were collected under baseline conditions from adult male and female animals for biochemical analyses, including chromatographic measurement of glycolate, oxalate, glyoxal, methylglyoxal, D-lactate, ascorbic acid and glutathione levels.<br><br>RESULTS: The GLO-2 KO animals developed normally and there were no changes in 24-h urinary oxalate excretion, liver levels of methylglyoxal, glyoxal, ascorbic acid and glutathione, or plasma d-lactate levels. GLO-2 deficient males had lower plasma glycolate levels than wild type males while this relationship was not observed in females.<br><br>CONCLUSIONS: The lack of a unique phenotype in a GLO-2 KO mouse model under baseline conditions is consistent with recent evidence, suggesting a functional glyoxalase pathway is not required for optimal health. A lower plasma glycolate in male GLO-2 KO animals suggests glyoxal production may be a significant contributor to circulating glycolate levels, but not to endogenous oxalate synthesis.},
}
@article {pmid34584219,
year = {2021},
author = {Roukens, MG and Frederiks, CL and Seinstra, D and Braccioli, L and Khalil, AA and Pals, C and De Neck, S and Bornes, L and Beerling, E and Mokry, M and de Bruin, A and Westendorp, B and van Rheenen, J and Coffer, PJ},
title = {Regulation of a progenitor gene program by SOX4 is essential for mammary tumor proliferation.},
journal = {Oncogene},
volume = {40},
number = {45},
pages = {6343-6353},
pmid = {34584219},
issn = {1476-5594},
mesh = {Animals ; Breast Neoplasms/genetics/*pathology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Epithelial-Mesenchymal Transition ; Female ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Lung Neoplasms/genetics/*pathology/*secondary ; Mice ; Neoplasm Transplantation ; Organoids/pathology/*transplantation ; SOXC Transcription Factors/*genetics ; },
abstract = {In breast cancer the transcription factor SOX4 has been shown to be associated with poor survival, increased tumor size and metastasis formation. This has mostly been attributed to the ability of SOX4 to regulate Epithelial-to-Mesenchymal-Transition (EMT). However, SOX4 regulates target gene transcription in a context-dependent manner that is determined by the cellular and epigenetic state. In this study we have investigated the loss of SOX4 in mammary tumor development utilizing organoids derived from a PyMT genetic mouse model of breast cancer. Using CRISPR/Cas9 to abrogate SOX4 expression, we found that SOX4 is required for inhibiting differentiation by regulating a subset of genes that are highly activated in fetal mammary stem cells (fMaSC). In this way, SOX4 re-activates an oncogenic transcriptional program that is regulated in many progenitor cell-types during embryonic development. SOX4-knockout organoids are characterized by the presence of more differentiated cells that exhibit luminal or basal gene expression patterns, but lower expression of cell cycle genes. In agreement, primary tumor growth and metastatic outgrowth in the lungs are impaired in SOX4[KO] tumors. Finally, SOX4[KO] tumors show a severe loss in competitive capacity to grow out compared to SOX4-proficient cells in primary tumors. Our study identifies a novel role for SOX4 in maintaining mammary tumors in an undifferentiated and proliferative state. Therapeutic manipulation of SOX4 function could provide a novel strategy for cancer differentiation therapy, which would promote differentiation and inhibit cycling of tumor cells.},
}
@article {pmid34583280,
year = {2021},
author = {Barndt, RJ and Ma, N and Tang, Y and Haugh, MP and Alamri, LS and Chan, SY and Wu, H},
title = {Modeling of dilated cardiomyopathy by establishment of isogenic human iPSC lines carrying phospholamban C25T (R9C) mutation (UPITTi002-A-1) using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102544},
pmid = {34583280},
issn = {1876-7753},
support = {R00 HL133473/HL/NHLBI NIH HHS/United States ; UH3 TR002073/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Calcium-Binding Proteins ; *Cardiomyopathy, Dilated/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Mutation/genetics ; },
abstract = {As the most common cause of heart failure, dilated cardiomyopathy (DCM) is characterized by dilated ventricles and weakened contractile force. Mutations in the calcium handling protein phospholamban (PLN) are known to cause inherited DCM. Here, we introduced a PLN-R9C mutation in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9. The genome-edited iPSC line showed typical pluripotent cell morphology, robust expression of pluripotency markers, normal karyotype, and the capacity to differentiate into all three germ layers in vitro. The PLN-R9C iPSC line provides a valuable resource to dissect the molecular mechanisms underlying PLN mutation-related DCM.},
}
@article {pmid34583196,
year = {2021},
author = {Yamasaki, F and Umezawa, F and Sensui, T and Anzo, M and Abo, H and Kuo, CW and Khoo, KH and Kato, K and Yagi, H and Kawashima, H},
title = {Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans.},
journal = {Biochemical and biophysical research communications},
volume = {579},
number = {},
pages = {8-14},
doi = {10.1016/j.bbrc.2021.09.043},
pmid = {34583196},
issn = {1090-2104},
mesh = {Animals ; Antibodies, Monoclonal/*chemistry ; Bacillus subtilis ; CRISPR-Cas Systems ; Chromatography, Liquid ; DNA, Complementary/metabolism ; Dystroglycans/*chemistry ; Female ; Glucuronic Acid/chemistry ; Glycopeptides/chemistry ; HCT116 Cells ; Humans ; Laminin/*chemistry ; Mass Spectrometry ; Mice ; Mice, Inbred BALB C ; Phosphates ; Polysaccharides ; Protein Binding ; Protein Conformation ; Protein Isoforms/*chemistry ; Recombinant Proteins/chemistry ; Ribitol/chemistry ; Xylose ; },
abstract = {α-Dystroglycan (α-DG) is a glycoprotein specifically modified with O-mannosyl glycans bearing long polysaccharides, termed matriglycans, which comprise repeating units of glucuronic acid and xylose. The matriglycan is linked to the O-mannosyl glycan core through two ribitol phosphate units that can be replaced with glycerol phosphate (GroP) units synthesized by fukutin and fukutin-related protein that transfer GroP from CDP-Gro. Here, we found that forced expression of the bacterial CDP-Gro synthase, TagD, from Bacillus subtilis could result in the overproduction of CDP-Gro in human colon carcinoma HCT116 cells. Western blot and liquid chromatography-tandem mass spectrometry analyses indicated that α-DG prepared from the TagD-expressing HCT116 cells contained abundant GroP and lacked matriglycans. Using the GroP-containing recombinant α-DG-Fc, we developed a novel monoclonal antibody, termed DG2, that reacts with several truncated glycoforms of α-DG, including GroP-terminated glycoforms lacking matriglycans; we verified the reactivity of DG2 against various types of knockout cells deficient in the biosynthesis of matriglycans. Accordingly, forced expression of TagD in HCT116 cells resulted in the reduction of matriglycans and an increase in DG2 reactivity. Collectively, our results indicate that DG2 could serve as a useful tool to determine tissue distribution and function of α-DG lacking matriglycans under physiological and pathophysiological conditions.},
}
@article {pmid34582991,
year = {2021},
author = {Chen, X and Lei, Y and Li, H and Xu, L and Yang, H and Wang, J and Jiang, H},
title = {CRISPR/Cas9 mutagenesis abolishes odorant-binding protein BdorOBP56f-2 and impairs the perception of methyl eugenol in Bactrocera dorsalis (Hendel).},
journal = {Insect biochemistry and molecular biology},
volume = {139},
number = {},
pages = {103656},
doi = {10.1016/j.ibmb.2021.103656},
pmid = {34582991},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; Eugenol/*analogs &amp; derivatives/metabolism ; Insect Proteins/*genetics/metabolism ; Male ; Mutagenesis ; Olfactory Perception/*genetics ; Receptors, Odorant/*genetics/metabolism ; Tephritidae/genetics/*physiology ; },
abstract = {Olfaction underpins many insect behaviors, such as foraging, host location, mating, and predator avoidance. In the first step of insect olfaction, odorant-binding proteins (OBPs) bind hydrophobic odorants and transport them to odorant receptors. Methyl eugenol (ME) is a powerful attractant for mature males of the oriental fruit fly Bactrocera dorsalis (Hendel), one of the most destructive fruit pests. The underlying molecular mechanism is unclear, but there is in vitro evidence that BdorOBP56f-2 is involved in ME perception. We used microscale thermophoresis to confirm that BdorOBP56f-2 directly binds ME with strong affinity in vitro. We then used CRISPR/Cas9 to knock out the BdorOBP56f-2 gene, allowing us to establish a homozygous mutant B. dorsalis line. The electroantennogram response and behavioral attraction to ME were significantly reduced in the mutant, providing in vivo evidence that BdorOBP56f-2 is necessary for efficient ME perception. Our results offer insight into the molecular mechanism of ME perception in B. dorsalis and provide a theoretical basis for the functional analysis of other OBPs.},
}
@article {pmid34582782,
year = {2021},
author = {Hossain, AA and McGinn, J and Meeske, AJ and Modell, JW and Marraffini, LA},
title = {Viral recombination systems limit CRISPR-Cas targeting through the generation of escape mutations.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {10},
pages = {1482-1495.e12},
pmid = {34582782},
issn = {1934-6069},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophage lambda/*genetics/immunology/physiology ; *CRISPR-Cas Systems ; Escherichia coli/*genetics/immunology/virology ; Escherichia coli Proteins/genetics/immunology ; Exodeoxyribonuclease V/genetics/immunology ; Host-Pathogen Interactions ; *Mutation ; *Recombination, Genetic ; Viral Proteins/genetics/immunology ; },
abstract = {CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.},
}
@article {pmid34582696,
year = {2021},
author = {Moya-Beltrán, A and Makarova, KS and Acuña, LG and Wolf, YI and Covarrubias, PC and Shmakov, SA and Silva, C and Tolstoy, I and Johnson, DB and Koonin, EV and Quatrini, R},
title = {Evolution of Type IV CRISPR-Cas Systems: Insights from CRISPR Loci in Integrative Conjugative Elements of Acidithiobacillia.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {656-672},
pmid = {34582696},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*genetics ; *Evolution, Molecular ; Genes, Bacterial ; Phylogeny ; Polymorphism, Genetic ; Proteobacteria/classification/*genetics ; },
abstract = {Type IV CRISPR-Cas are a distinct variety of highly derived CRISPR-Cas systems that appear to have evolved from type III systems through the loss of the target-cleaving nuclease and partial deterioration of the large subunit of the effector complex. All known type IV CRISPR-Cas systems are encoded on plasmids, integrative and conjugative elements (ICEs), or prophages, and are thought to contribute to competition between these elements, although the mechanistic details of their function remain unknown. There is a clear parallel between the compositions and likely origin of type IV and type I systems recruited by Tn7-like transposons and mediating RNA-guided transposition. We investigated the diversity and evolutionary relationships of type IV systems, with a focus on those in Acidithiobacillia, where this variety of CRISPR is particularly abundant and always found on ICEs. Our analysis revealed remarkable evolutionary plasticity of type IV CRISPR-Cas systems, with adaptation and ancillary genes originating from different ancestral CRISPR-Cas varieties, and extensive gene shuffling within the type IV loci. The adaptation module and the CRISPR array apparently were lost in the type IV ancestor but were subsequently recaptured by type IV systems on several independent occasions. We demonstrate a high level of heterogeneity among the repeats with type IV CRISPR arrays, which far exceed the heterogeneity of any other known CRISPR repeats and suggest a unique adaptation mechanism. The spacers in the type IV arrays, for which protospacers could be identified, match plasmid genes, in particular those encoding the conjugation apparatus components. Both the biochemical mechanism of type IV CRISPR-Cas function and their role in the competition among mobile genetic elements remain to be investigated.},
}
@article {pmid34582693,
year = {2021},
author = {van Essen, M and Riepsaame, J and Jacob, J},
title = {CRISPR-Cas Gene Perturbation and Editing in Human Induced Pluripotent Stem Cells.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {634-655},
doi = {10.1089/crispr.2021.0063},
pmid = {34582693},
issn = {2573-1602},
support = {/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; DNA Repair ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; },
abstract = {Directing the fates of human pluripotent stem cells (hPSC) to generate a multitude of differentiated cell types allows the study of the genetic regulation of human development and disease. The translational potential of hPSC is maximized by exploiting CRISPR to silence or activate genes with spatial and temporal precision permanently or reversibly. Here, we summarize the increasingly refined and diverse CRISPR toolkit for the latter forms of gene perturbation in hPSC and their downstream applications. We discuss newer methods to install edits efficiently with single nucleotide resolution and describe pooled CRISPR screens as a powerful means of unbiased discovery of genes associated with a phenotype of interest. Last, we discuss the potential of these combined technologies in the treatment of hitherto intractable human diseases and the challenges to their implementation in the clinic.},
}
@article {pmid34582499,
year = {2021},
author = {Kruasuwan, W and Puseenam, A and Phithakrotchanakoon, C and Tanapongpipat, S and Roongsawang, N},
title = {Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.},
journal = {PloS one},
volume = {16},
number = {9},
pages = {e0258005},
pmid = {34582499},
issn = {1932-6203},
mesh = {Autophagy ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Electrophoresis, Polyacrylamide Gel ; Endoplasmic Reticulum Stress ; Fungal Proteins/*metabolism ; Gene Editing ; Genes, Fungal/genetics ; Oxidative Stress ; Protein Translocation Systems/genetics ; Protein Transport/genetics ; Real-Time Polymerase Chain Reaction ; Saccharomycetales/*genetics/metabolism ; Thermotolerance ; },
abstract = {The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.},
}
@article {pmid34582479,
year = {2021},
author = {Leoni, L and Tonelli, F and Besio, R and Gioia, R and Moccia, F and Rossi, A and Forlino, A},
title = {Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV.},
journal = {PloS one},
volume = {16},
number = {9},
pages = {e0257254},
pmid = {34582479},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; Calcium/metabolism ; Cell Line ; Cell Proliferation ; Collagen/chemistry ; Electrophysiology ; Extracellular Matrix/metabolism ; Gene Knockout Techniques ; Humans ; In Vitro Techniques ; Ion Channels/*genetics ; Mice ; Mutation ; Osteoblasts/*metabolism ; Osteogenesis Imperfecta/*genetics/*metabolism ; },
abstract = {Osteogenesis imperfecta (OI) type XIV is a rare recessive bone disorder characterized by variable degree of severity associated to osteopenia. It is caused by mutations in TMEM38B encoding for the trimeric intracellular cation channel TRIC-B, specific for potassium and ubiquitously present in the endoplasmic reticulum (ER) membrane. OI type XIV molecular basis is largely unknown and, due to the rarity of the disease, the availability of patients' osteoblasts is challenging. Thus, CRISPR/Cas9 was used to knock out (KO) TMEM38B in the human Foetal Osteoblast hFOB 1.19 to obtain an OI type XIV model. CRISPR/Cas9 is a powerful technology to generate in vitro and in vivo models for heritable disorders. Its limited cost and ease of use make this technique widely applicable in most laboratories. Nevertheless, to fully take advantage of this approach, it is important to be aware of its strengths and limitations. Three gRNAs were used and several KO clones lacking the expression of TRIC-B were obtained. Few clones were validated as good models for the disease since they reproduce the altered ER calcium flux, collagen I structure and impaired secretion and osteoblastic markers expression detected in patients' cells. Impaired proliferation and mineralization in KO clones unveiled the relevance of TRIC-B in osteoblasts functionality.},
}
@article {pmid34582079,
year = {2022},
author = {Xu, R and Qin, R and Xie, H and Li, J and Liu, X and Zhu, M and Sun, Y and Yu, Y and Lu, P and Wei, P},
title = {Genome editing with type II-C CRISPR-Cas9 systems from Neisseria meningitidis in rice.},
journal = {Plant biotechnology journal},
volume = {20},
number = {2},
pages = {350-359},
pmid = {34582079},
issn = {1467-7652},
mesh = {APOBEC-1 Deaminase/genetics ; Adenine ; Animals ; CRISPR-Cas Systems/genetics ; Cytidine Deaminase ; Gene Editing/methods ; Mammals/genetics ; *Neisseria meningitidis/genetics ; *Oryza/genetics ; Proteins ; Rats ; },
abstract = {Two type II-C Cas9 orthologs (Nm1Cas9 and Nm2Cas9) were recently identified from Neisseria meningitidis and have been extensively used in mammalian cells, but whether these NmCas9 orthologs or other type II-C Cas9 proteins can mediate genome editing in plants remains unclear. In this study, we developed and optimized targeted mutagenesis systems from NmCas9s for plants. Efficient genome editing at the target with N4 GATT and N4 CC protospacer adjacent motifs (PAMs) was achieved with Nm1Cas9 and Nm2Cas9 respectively. These results indicated that a highly active editing system could be developed from type II-C Cas9s with distinct PAM preferences, thus providing a reliable strategy to extend the scope of genome editing in plants. Base editors (BEs) were further developed from the NmCas9s. The editing efficiency of adenine BEs (ABEs) of TadA*-7.10 and cytosine BEs (CBEs) of rat APOBEC1 (rAPO1) or human APOBEC3a (hA3A) were extremely limited, whereas ABEs of TadA-8e and CBEs of Petromyzon marinus cytidine deaminase 1 (PmCDA1) exhibited markedly improved performance on the same targets. In addition, we found that fusion of a single-stranded DNA-binding domain from the human Rad51 protein enhanced the base editing capability of rAPO1-CBEs of NmCas9s. Together, our results suggest that the engineering of NmCas9s or other type II-C Cas9s can provide useful alternatives for crop genome editing.},
}
@article {pmid34580310,
year = {2021},
author = {Shams, A and Higgins, SA and Fellmann, C and Laughlin, TG and Oakes, BL and Lew, R and Kim, S and Lukarska, M and Arnold, M and Staahl, BT and Doudna, JA and Savage, DF},
title = {Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5664},
pmid = {34580310},
issn = {2041-1723},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; S10 RR029668/RR/NCRR NIH HHS/United States ; S10 RR027303/RR/NCRR NIH HHS/United States ; T32 GM066698/GM/NIGMS NIH HHS/United States ; R01 GM127463/GM/NIGMS NIH HHS/United States ; K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism/ultrastructure ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cryoelectron Microscopy ; DNA/metabolism ; Gene Editing/methods ; Humans ; Protein Interaction Domains and Motifs/*genetics ; RNA, Guide/*metabolism ; Single Molecule Imaging ; },
abstract = {Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.},
}
@article {pmid34580296,
year = {2021},
author = {Sridhara, S and Goswami, HN and Whyms, C and Dennis, JH and Li, H},
title = {Virus detection via programmable Type III-A CRISPR-Cas systems.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5653},
pmid = {34580296},
issn = {2041-1723},
support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; },
mesh = {COVID-19/blood/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Cas Systems/*genetics ; Humans ; Limit of Detection ; RNA, Viral/genetics/isolation &amp; purification ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.},
}
@article {pmid34580180,
year = {2021},
author = {Asthana, J and Cade, NI and Normanno, D and Lim, WM and Surrey, T},
title = {Gradual compaction of the central spindle decreases its dynamicity in PRC1 and EB1 gene-edited cells.},
journal = {Life science alliance},
volume = {4},
number = {12},
pages = {},
pmid = {34580180},
issn = {2575-1077},
support = {//Wellcome Trust/United Kingdom ; 323042/ERC_/European Research Council/International ; FC001163//Arthritis Research UK/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cell Line, Transformed ; Chromosome Segregation/genetics ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Microtubule-Associated Proteins/genetics/*metabolism ; Microtubules/metabolism ; Mitosis/*genetics ; Protein Binding/genetics ; Retinal Pigment Epithelium/*metabolism ; Signal Transduction/*genetics ; Spindle Apparatus/*metabolism ; Transfection/methods ; },
abstract = {During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent "bundling, sliding, and compaction" model of antiparallel midzone bundle formation in the central spindle during late mitosis.},
}
@article {pmid34579446,
year = {2021},
author = {Rajput, M and Choudhary, K and Kumar, M and Vivekanand, V and Chawade, A and Ortiz, R and Pareek, N},
title = {RNA Interference and CRISPR/Cas Gene Editing for Crop Improvement: Paradigm Shift towards Sustainable Agriculture.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {9},
pages = {},
pmid = {34579446},
issn = {2223-7747},
abstract = {With the rapid population growth, there is an urgent need for innovative crop improvement approaches to meet the increasing demand for food. Classical crop improvement approaches involve, however, a backbreaking process that cannot equipoise with increasing crop demand. RNA-based approaches i.e., RNAi-mediated gene regulation and the site-specific nuclease-based CRISPR/Cas9 system for gene editing has made advances in the efficient targeted modification in many crops for the higher yield and resistance to diseases and different stresses. In functional genomics, RNA interference (RNAi) is a propitious gene regulatory approach that plays a significant role in crop improvement by permitting the downregulation of gene expression by small molecules of interfering RNA without affecting the expression of other genes. Gene editing technologies viz. the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) have appeared prominently as a powerful tool for precise targeted modification of nearly all crops' genome sequences to generate variation and accelerate breeding efforts. In this regard, the review highlights the diverse roles and applications of RNAi and CRISPR/Cas9 system as powerful technologies to improve agronomically important plants to enhance crop yields and increase tolerance to environmental stress (biotic or abiotic). Ultimately, these technologies can prove to be important in view of global food security and sustainable agriculture.},
}
@article {pmid34579360,
year = {2021},
author = {Citiulo, F and Crosatti, C and Cattivelli, L and Biselli, C},
title = {Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {9},
pages = {},
pmid = {34579360},
issn = {2223-7747},
abstract = {The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles-VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.},
}
@article {pmid34578431,
year = {2021},
author = {Nguyen, H and Wilson, H and Jayakumar, S and Kulkarni, V and Kulkarni, S},
title = {Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System.},
journal = {Viruses},
volume = {13},
number = {9},
pages = {},
pmid = {34578431},
issn = {1999-4915},
support = {R21 AI140956/AI/NIAID NIH HHS/United States ; R56 AI150371/AI/NIAID NIH HHS/United States ; },
mesh = {CD4-Positive T-Lymphocytes/virology ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Endodeoxyribonucleases/*metabolism ; HEK293 Cells ; HIV-1/*genetics/*physiology ; Humans ; Proviruses/physiology ; RNA, Guide/genetics/metabolism ; RNA, Viral/metabolism ; Virus Replication ; },
abstract = {Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13-mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct, non-overlapping sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4[+] T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.},
}
@article {pmid34578378,
year = {2021},
author = {Naimo, E and Zischke, J and Schulz, TF},
title = {Recent Advances in Developing Treatments of Kaposi's Sarcoma Herpesvirus-Related Diseases.},
journal = {Viruses},
volume = {13},
number = {9},
pages = {},
pmid = {34578378},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems ; Castleman Disease/drug therapy ; Clinical Trials as Topic ; DNA-Directed DNA Polymerase ; Exodeoxyribonucleases/antagonists &amp; inhibitors ; Gene Expression Regulation, Viral ; Herpesviridae Infections/classification/*drug therapy ; Herpesvirus 8, Human/*drug effects/genetics ; Humans ; Lymphoma, Primary Effusion/drug therapy ; Mice ; Sarcoma, Kaposi/*drug therapy/virology ; Viral Proteins/antagonists &amp; inhibitors ; Virus Latency/genetics ; Virus Replication/drug effects/*genetics ; },
abstract = {Kaposi-sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is the causative agent of several malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Active KSHV replication has also been associated with a pathological condition called KSHV inflammatory cytokine syndrome (KICS), and KSHV may play a role in rare cases of post-transplant polyclonal lymphoproliferative disorders. Several commonly used herpesviral DNA polymerase inhibitors are active against KSHV in tissue culture. Unfortunately, they are not always efficacious against KSHV-induced diseases. To improve the outcome for the patients, new therapeutics need to be developed, including treatment strategies that target either viral proteins or cellular pathways involved in tumor growth and/or supporting the viral life cycle. In this review, we summarize the most commonly established treatments against KSHV-related diseases and review recent developments and promising new compounds that are currently under investigation or on the way to clinical use.},
}
@article {pmid34578279,
year = {2021},
author = {Venkataraman, S and Hefferon, K},
title = {Application of Plant Viruses in Biotechnology, Medicine, and Human Health.},
journal = {Viruses},
volume = {13},
number = {9},
pages = {},
pmid = {34578279},
issn = {1999-4915},
mesh = {Animals ; Biotechnology/*methods ; CRISPR-Cas Systems ; Comovirus/physiology ; *Global Health ; Humans ; Mice ; Nanoparticles/chemistry ; Nanotechnology/*methods ; Pharmaceutical Preparations ; Plant Viruses/classification/*genetics/*physiology ; Potexvirus/physiology ; Tobacco Mosaic Virus/physiology ; },
abstract = {Plant-based nanotechnology programs using virus-like particles (VLPs) and virus nanoparticles (VNPs) are emerging platforms that are increasingly used for a variety of applications in biotechnology and medicine. Tobacco mosaic virus (TMV) and potato virus X (PVX), by virtue of having high aspect ratios, make ideal platforms for drug delivery. TMV and PVX both possess rod-shaped structures and single-stranded RNA genomes encapsidated by their respective capsid proteins and have shown great promise as drug delivery systems. Cowpea mosaic virus (CPMV) has an icosahedral structure, and thus brings unique benefits as a nanoparticle. The uses of these three plant viruses as either nanostructures or expression vectors for high value pharmaceutical proteins such as vaccines and antibodies are discussed extensively in the following review. In addition, the potential uses of geminiviruses in medical biotechnology are explored. The uses of these expression vectors in plant biotechnology applications are also discussed. Finally, in this review, we project future prospects for plant viruses in the fields of medicine, human health, prophylaxis, and therapy of human diseases.},
}
@article {pmid34576887,
year = {2021},
author = {Huang, Z and Zhou, X and Stanton, C and Ross, RP and Zhao, J and Zhang, H and Yang, B and Chen, W},
title = {Comparative Genomics and Specific Functional Characteristics Analysis of Lactobacillus acidophilus.},
journal = {Microorganisms},
volume = {9},
number = {9},
pages = {},
pmid = {34576887},
issn = {2076-2607},
abstract = {Lactobacillus acidophilus is a common kind of lactic acid bacteria usually found in the human gastrointestinal tract, oral cavity, vagina, and various fermented foods. At present, many studies have focused on the probiotic function and industrial application of L. acidophilus. Additionally, dozens of L. acidophilus strains have been genome sequenced, but there has been no research to compare them at the genomic level. In this study, 46 strains of L. acidophilus were performed comparative analyses to explore their genetic diversity. The results showed that all the L. acidophilus strains were divided into two clusters based on ANI values, phylogenetic analysis and whole genome comparison, due to the difference of their predicted gene composition of bacteriocin operon, CRISPR-Cas systems and prophages mainly. Additionally, L. acidophilus was a pan-genome open species with a difference in carbohydrates utilization, antibiotic resistance, EPS operon, surface layer protein operon and other functional gene composition. This work provides a better understanding of L. acidophilus from a genetic perspective, and offers a frame for the biotechnological potentiality of this species.},
}
@article {pmid34576226,
year = {2021},
author = {Balla, B and Tripon, F and Banescu, C},
title = {From Descriptive to Functional Genomics of Leukemias Focusing on Genome Engineering Techniques.},
journal = {International journal of molecular sciences},
volume = {22},
number = {18},
pages = {},
pmid = {34576226},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clinical Trials as Topic ; DNA Damage ; Gene Editing/*methods ; *Gene Expression Regulation, Leukemic ; Gene Knockout Techniques ; Genetic Engineering/methods ; Genome, Human ; Genomics/methods ; Humans ; Immune System ; Leukemia/*genetics ; Mutation ; Patient Safety ; RNA/metabolism ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Genome engineering makes the precise manipulation of DNA sequences possible in a cell. Therefore, it is essential for understanding gene function. Meganucleases were the start of genome engineering, and it continued with the discovery of Zinc finger nucleases (ZFNs), followed by Transcription activator-like effector nucleases (TALENs). They can generate double-strand breaks at a desired target site in the genome, and therefore can be used to knock in mutations or knock out genes in the same way. Years later, genome engineering was transformed by the discovery of clustered regularly interspaced short palindromic repeats (CRISPR). Implementation of CRISPR systems involves recognition guided by RNA and the precise cleaving of DNA molecules. This property proves its utility in epigenetics and genome engineering. CRISPR has been and is being continuously successfully used to model mutations in leukemic cell lines and control gene expression. Furthermore, it is used to identify targets and discover drugs for immune therapies. The descriptive and functional genomics of leukemias is discussed in this study, with an emphasis on genome engineering methods. The CRISPR/Cas9 system's challenges, viewpoints, limits, and solutions are also explored.},
}
@article {pmid34576035,
year = {2021},
author = {Chuang, CK and Lin, WM},
title = {Points of View on the Tools for Genome/Gene Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {18},
pages = {},
pmid = {34576035},
issn = {1422-0067},
mesh = {Amino Acid Sequence/genetics ; Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/instrumentation/*trends ; Genome/*genetics ; Humans ; Mutation/genetics ; RNA, Guide/genetics ; },
abstract = {Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.},
}
@article {pmid34573392,
year = {2021},
author = {Bhowmik, P and Bilichak, A},
title = {Advances in Gene Editing of Haploid Tissues in Crops.},
journal = {Genes},
volume = {12},
number = {9},
pages = {},
pmid = {34573392},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/*genetics ; Gene Editing/methods/*trends ; *Haploidy ; Phenotype ; Plant Breeding/*methods ; Plants, Genetically Modified ; Zea mays/genetics ; },
abstract = {Emerging threats of climate change require the rapid development of improved varieties with a higher tolerance to abiotic and biotic factors. Despite the success of traditional agricultural practices, novel techniques for precise manipulation of the crop's genome are needed. Doubled haploid (DH) methods have been used for decades in major crops to fix desired alleles in elite backgrounds in a short time. DH plants are also widely used for mapping of the quantitative trait loci (QTLs), marker-assisted selection (MAS), genomic selection (GS), and hybrid production. Recent discoveries of genes responsible for haploid induction (HI) allowed engineering this trait through gene editing (GE) in non-inducer varieties of different crops. Direct editing of gametes or haploid embryos increases GE efficiency by generating null homozygous plants following chromosome doubling. Increased understanding of the underlying genetic mechanisms responsible for spontaneous chromosome doubling in haploid plants may allow transferring this trait to different elite varieties. Overall, further improvement in the efficiency of the DH technology combined with the optimized GE could accelerate breeding efforts of the major crops.},
}
@article {pmid34572866,
year = {2021},
author = {Durán-Vinet, B and Araya-Castro, K and Calderón, J and Vergara, L and Weber, H and Retamales, J and Araya-Castro, P and Leal-Rojas, P},
title = {CRISPR/Cas13-Based Platforms for a Potential Next-Generation Diagnosis of Colorectal Cancer through Exosomes Micro-RNA Detection: A Review.},
journal = {Cancers},
volume = {13},
number = {18},
pages = {},
pmid = {34572866},
issn = {2072-6694},
abstract = {Colorectal cancer (CRC) is the third most prevalent cancer with the second highest mortality rate worldwide. CRC is a heterogenous disease with multiple risk factors associated, including obesity, smoking, and use of alcohol. Of total CRC cases, 60% are diagnosed in late stages, where survival can drop to about 10%. CRC screening programs are based primarily on colonoscopy, yet this approach is invasive and has low patient adherence. Therefore, there is a strong incentive for developing molecular-based methods that are minimally invasive and have higher patient adherence. Recent reports have highlighted the importance of extracellular vesicles (EVs), specifically exosomes, as intercellular communication vehicles with a broad cargo, including micro-RNAs (miRNAs). These have been syndicated as robust candidates for diagnosis, primarily for their known activities in cancer cells, including immunoevasion, tumor progression, and angiogenesis, whereas miRNAs are dysregulated by cancer cells and delivered by cancer-derived exosomes (CEx). Quantitative polymerase chain reaction (qPCR) has shown good results detecting specific cancer-derived exosome micro-RNAs (CEx-miRNAs) associated with CRC, but qPCR also has several challenges, including portability and sensitivity/specificity issues regarding experiment design and sample quality. CRISPR/Cas-based platforms have been presented as cost-effective, ultrasensitive, specific, and robust clinical detection tools in the presence of potential inhibitors and capable of delivering quantitative and qualitative real-time data for enhanced decision-making to healthcare teams. Thereby, CRISPR/Cas13-based technologies have become a potential strategy for early CRC diagnosis detecting CEx-miRNAs. Moreover, CRISPR/Cas13-based platforms' ease of use, scalability, and portability also showcase them as a potential point-of-care (POC) technology for CRC early diagnosis. This study presents two potential CRISPR/Cas13-based methodologies with a proposed panel consisting of four CEx-miRNAs, including miR-126, miR-1290, miR-23a, and miR-940, to streamline novel applications which may deliver a potential early diagnosis and prognosis of CRC.},
}
@article {pmid34571991,
year = {2021},
author = {Al-Sammarraie, N and Ray, SK},
title = {Applications of CRISPR-Cas9 Technology to Genome Editing in Glioblastoma Multiforme.},
journal = {Cells},
volume = {10},
number = {9},
pages = {},
pmid = {34571991},
issn = {2073-4409},
support = {R01 CA091460/CA/NCI NIH HHS/United States ; R01 NS057811/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Gene Expression Regulation/genetics ; Gene Transfer Techniques/trends ; Genetic Therapy/methods ; Glioblastoma/*genetics/metabolism/*therapy ; Humans ; Mutation/genetics ; Oncogenes/genetics ; },
abstract = {Glioblastoma multiforme (GBM) is an aggressive malignancy of the brain and spinal cord with a poor life expectancy. The low survivability of GBM patients can be attributed, in part, to its heterogeneity and the presence of multiple genetic alterations causing rapid tumor growth and resistance to conventional therapy. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) nuclease 9 (CRISPR-Cas9) system is a cost-effective and reliable gene editing technology, which is widely used in cancer research. It leads to novel discoveries of various oncogenes that regulate autophagy, angiogenesis, and invasion and play important role in pathogenesis of various malignancies, including GBM. In this review article, we first describe the principle and methods of delivery of CRISPR-Cas9 genome editing. Second, we summarize the current knowledge and major applications of CRISPR-Cas9 to identifying and modifying the genetic regulators of the hallmark of GBM. Lastly, we elucidate the major limitations of current CRISPR-Cas9 technology in the GBM field and the future perspectives. CRISPR-Cas9 genome editing aids in identifying novel coding and non-coding transcriptional regulators of the hallmarks of GBM particularly in vitro, while work using in vivo systems requires further investigation.},
}
@article {pmid34571882,
year = {2021},
author = {Springer, C and Zakhartchenko, V and Wolf, E and Simmet, K},
title = {Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG.},
journal = {Cells},
volume = {10},
number = {9},
pages = {},
pmid = {34571882},
issn = {2073-4409},
mesh = {Animals ; Apoptosis/drug effects ; Blastocyst/cytology/metabolism ; CRISPR-Cas Systems ; Cattle ; Cell Differentiation ; Cell Lineage ; Embryo, Mammalian/cytology/*metabolism ; Gene Editing ; Germ Layers/cytology/drug effects/*metabolism ; Mice ; Mice, Knockout ; Mitogen-Activated Protein Kinase Kinases/antagonists &amp; inhibitors/metabolism ; Nanog Homeobox Protein/deficiency/*genetics/metabolism ; Protein Kinase Inhibitors/pharmacology ; SOXF Transcription Factors/metabolism ; Signal Transduction ; },
abstract = {The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse Nanog-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another-so far unknown-pathway regulates HB differentiation.},
}
@article {pmid34571881,
year = {2021},
author = {Spek, CA and Aberson, HL and Butler, JM and de Vos, AF and Duitman, J},
title = {CEBPD Potentiates the Macrophage Inflammatory Response but CEBPD Knock-Out Macrophages Fail to Identify CEBPD-Dependent Pro-Inflammatory Transcriptional Programs.},
journal = {Cells},
volume = {10},
number = {9},
pages = {},
pmid = {34571881},
issn = {2073-4409},
mesh = {Animals ; CCAAT-Enhancer-Binding Protein-delta/deficiency/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; Gene Expression Regulation/drug effects ; Interleukin-6/metabolism ; Lipopolysaccharides/pharmacology ; Macrophage Colony-Stimulating Factor/pharmacology ; Macrophages/cytology/drug effects/*metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutagenesis ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.},
}
@article {pmid34571006,
year = {2021},
author = {Zhang, Y and Nishiyama, T and Olson, EN and Bassel-Duby, R},
title = {CRISPR/Cas correction of muscular dystrophies.},
journal = {Experimental cell research},
volume = {408},
number = {1},
pages = {112844},
pmid = {34571006},
issn = {1090-2422},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Dystrophin/metabolism ; *Gene Editing/methods ; *Genetic Therapy ; Humans ; Muscular Dystrophy, Duchenne/*genetics/therapy ; Mutation/genetics ; },
abstract = {Muscular dystrophies are a heterogeneous group of monogenic neuromuscular disorders which lead to progressive muscle loss and degeneration of the musculoskeletal system. The genetic causes of muscular dystrophies are well characterized, but no effective treatments have been developed so far. The discovery and application of the CRISPR/Cas system for genome editing offers a new path for disease treatment with the potential to permanently correct genetic mutations. The post-mitotic and multinucleated features of skeletal muscle provide an ideal target for CRISPR/Cas therapeutic genome editing because correction of a subpopulation of nuclei can provide benefit to the whole myofiber. In this review, we provide an overview of the CRISPR/Cas system and its derivatives in genome editing, proposing potential CRISPR/Cas-based therapies to correct diverse muscular dystrophies, and we discuss challenges for translating CRISPR/Cas genome editing to a viable therapy for permanent correction of muscular dystrophies.},
}
@article {pmid34570096,
year = {2021},
author = {Kojin, BB and Tsujimoto, H and Jakes, E and O'Leary, S and Adelman, ZN},
title = {Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {175},
pages = {},
pmid = {34570096},
issn = {1940-087X},
support = {R01 AI137112/AI/NIAID NIH HHS/United States ; R21 AI115138/AI/NIAID NIH HHS/United States ; },
mesh = {*Aedes/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; Humans ; Mutagenesis ; },
abstract = {Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.},
}
@article {pmid34569891,
year = {2021},
author = {Khosravi, HM and Jantsch, MF},
title = {Site-directed RNA editing: recent advances and open challenges.},
journal = {RNA biology},
volume = {18},
number = {sup1},
pages = {41-50},
pmid = {34569891},
issn = {1555-8584},
support = {P 30505/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Engineering/*methods ; Humans ; *Mutation ; *RNA Editing ; },
abstract = {RNA editing by cytosine and adenosine deaminases changes the identity of the edited bases. While cytosines are converted to uracils, adenines are converted to inosines. If coding regions of mRNAs are affected, the coding potential of the RNA can be changed, depending on the codon affected. The recoding potential of nucleotide deaminases has recently gained attention for their ability to correct genetic mutations by either reverting the mutation itself or by manipulating processing steps such as RNA splicing. In contrast to CRISPR-based DNA-editing approaches, RNA editing events are transient in nature, therefore reducing the risk of long-lasting inadvertent side-effects. Moreover, some RNA-based therapeutics are already FDA approved and their use in targeting multiple cells or organs to restore genetic function has already been shown. In this review, we provide an overview on the current status and technical differences of site-directed RNA-editing approaches. We also discuss advantages and challenges of individual approaches.},
}
@article {pmid34567840,
year = {2021},
author = {Wongpayak, P and Meesungnoen, O and Saejang, S and Subsoontorn, P},
title = {A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection.},
journal = {PeerJ},
volume = {9},
number = {},
pages = {e11996},
pmid = {34567840},
issn = {2167-8359},
abstract = {The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.},
}
@article {pmid34563675,
year = {2021},
author = {Rogers, GL and Cannon, PM},
title = {Genome edited B cells: a new frontier in immune cell therapies.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3192-3204},
pmid = {34563675},
issn = {1525-0024},
support = {U19 HL129902/HL/NHLBI NIH HHS/United States ; U19 HL156247/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Antibodies/genetics/immunology ; B-Lymphocytes/immunology/*metabolism ; *CRISPR-Cas Systems ; Cell Engineering ; Cell- and Tissue-Based Therapy/*methods ; Cellular Reprogramming/genetics/immunology ; *Gene Editing ; Gene Expression Regulation ; Genetic Engineering ; Genetic Therapy/*methods ; Humans ; Immunotherapy/*methods ; T-Lymphocyte Subsets/immunology/metabolism ; },
abstract = {Cell therapies based on reprogrammed adaptive immune cells have great potential as "living drugs." As first demonstrated clinically for engineered chimeric antigen receptor (CAR) T cells, the ability of such cells to undergo clonal expansion in response to an antigen promotes both self-renewal and self-regulation in vivo. B cells also have the potential to be developed as immune cell therapies, but engineering their specificity and functionality is more challenging than for T cells. In part, this is due to the complexity of the immunoglobulin (Ig) locus, as well as the requirement for regulated expression of both cell surface B cell receptor and secreted antibody isoforms, in order to fully recapitulate the features of natural antibody production. Recent advances in genome editing are now allowing reprogramming of B cells by site-specific engineering of the Ig locus with preformed antibodies. In this review, we discuss the potential of engineered B cells as a cell therapy, the challenges involved in editing the Ig locus and the advances that are making this possible, and envision future directions for this emerging field of immune cell engineering.},
}
@article {pmid34563399,
year = {2021},
author = {Christie, KA and Kleinstiver, BP},
title = {Making the cut with PAMless CRISPR-Cas enzymes.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {12},
pages = {1053-1055},
pmid = {34563399},
issn = {0168-9525},
support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
abstract = {Genome editing technologies simplify our ability to rewrite genetic blueprints of life. However, CRISPR-Cas enzymes found in nature can only manipulate a fraction of the genome. To overcome this limitation, new Cas variants have been developed that unlock nearly the entire genome for editing.},
}
@article {pmid34562891,
year = {2021},
author = {Talwar, CS and Park, KH and Ahn, WC and Kim, YS and Kwon, OS and Yong, D and Kang, T and Woo, E},
title = {Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay.},
journal = {Biosensors},
volume = {11},
number = {9},
pages = {},
pmid = {34562891},
issn = {2079-6374},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; Endodeoxyribonucleases/*metabolism ; Fluorescent Dyes/chemistry ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/*isolation &amp; purification ; Nose/virology ; Point-of-Care Testing ; RNA, Guide/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus/genetics/*isolation &amp; purification ; SARS-CoV-2/genetics/*isolation &amp; purification ; Sensitivity and Specificity ; Sputum/virology ; Virus Diseases/*diagnosis ; },
abstract = {The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.},
}
@article {pmid34561855,
year = {2022},
author = {Lam, AJ and Uday, P and Gillies, JK and Levings, MK},
title = {Helios is a marker, not a driver, of human Treg stability.},
journal = {European journal of immunology},
volume = {52},
number = {1},
pages = {75-84},
doi = {10.1002/eji.202149318},
pmid = {34561855},
issn = {1521-4141},
support = {FDN-154304//CIHR/Canada ; },
mesh = {Animals ; Antigens, Differentiation/genetics/*immunology ; CRISPR-Cas Systems ; Cell Line ; Gene Expression Regulation/*immunology ; Gene Knockout Techniques ; Humans ; Ikaros Transcription Factor/genetics/*immunology ; Mice ; T-Lymphocytes, Regulatory/*immunology ; },
abstract = {Treg therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T-cells and, thus, exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Treg lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part, due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Helios[mid])-expressing population correlated with Treg destabilization. We used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-KO and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T-cells.},
}
@article {pmid34561273,
year = {2021},
author = {Zhao, X and Li, J and Liu, Z and Powers, S},
title = {Combinatorial CRISPR/Cas9 Screening Reveals Epistatic Networks of Interacting Tumor Suppressor Genes and Therapeutic Targets in Human Breast Cancer.},
journal = {Cancer research},
volume = {81},
number = {24},
pages = {6090-6105},
doi = {10.1158/0008-5472.CAN-21-2555},
pmid = {34561273},
issn = {1538-7445},
support = {R21 HG009255/HG/NHGRI NIH HHS/United States ; R01 CA217206/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Biomarkers, Tumor/*genetics/metabolism ; Breast Neoplasms/genetics/metabolism/*pathology ; *CRISPR-Cas Systems ; *Epistasis, Genetic ; Female ; *Gene Regulatory Networks ; *Genes, Tumor Suppressor ; Humans ; Mice, Inbred C57BL ; Mice, Nude ; Protein Interaction Maps ; Single-Cell Analysis ; Transcriptome ; },
abstract = {The majority of cancers are driven by multiple genetic alterations, but how these changes collaborate during tumorigenesis remains largely unknown. To gain mechanistic insights into tumor-promoting genetic interactions among tumor suppressor genes (TSG), we conducted combinatorial CRISPR screening coupled with single-cell transcriptomic profiling in human mammary epithelial cells. As expected, different driver gene alterations in mammary epithelial cells influenced the repertoire of tumor suppressor alterations capable of inducing tumor formation. More surprisingly, TSG interaction networks were comprised of numerous cliques-sets of three or four genes such that each TSG within the clique showed oncogenic cooperation with all other genes in the clique. Genetic interaction profiling indicated that the predominant cooperating TSGs shared overlapping functions rather than distinct or complementary functions. Single-cell transcriptomic profiling of CRISPR double knockouts revealed that cooperating TSGs that synergized in promoting tumorigenesis and growth factor independence showed transcriptional epistasis, whereas noncooperating TSGs did not. These epistatic transcriptional changes, both buffering and synergistic, affected expression of oncogenic mediators and therapeutic targets, including CDK4, SRPK1, and DNMT1. Importantly, the epistatic expression alterations caused by dual inactivation of TSGs in this system, such as PTEN and TP53, were also observed in patient tumors, establishing the relevance of these findings to human breast cancer. An estimated 50% of differentially expressed genes in breast cancer are controlled by epistatic interactions. Overall, our study indicates that transcriptional epistasis is a central aspect of multigenic breast cancer progression and outlines methodologies to uncover driver gene epistatic networks in other human cancers. SIGNIFICANCE: This study provides a roadmap for moving beyond discovery and development of therapeutic strategies based on single driver gene analysis to discovery based on interactions between multiple driver genes.See related commentary by Fong et al., p. 6078.},
}
@article {pmid34560185,
year = {2021},
author = {Kwak, JS and Kim, MS and Kim, KH},
title = {Harnessing snakehead rhabdovirus (SHRV) for gene editing by installment of CRISPR/Cas9 in viral genome.},
journal = {Virus research},
volume = {305},
number = {},
pages = {198578},
doi = {10.1016/j.virusres.2021.198578},
pmid = {34560185},
issn = {1872-7492},
mesh = {Animals ; CRISPR-Cas Systems ; Cricetinae ; *Gene Editing ; Genome, Viral ; Mammals ; *Novirhabdovirus/genetics ; },
abstract = {As there is no risk of viral genome integration into host chromosome, cytoplasmic RNA viruses can be a safer vehicle to deliver CRISPR/Cas system. Snakehead rhabdovirus (SHRV) is a piscine RNA virus belonging to the family Rhabdoviridae, and, in the present study, we evaluated the availability of SHRV as a tool for CRISPR/Cas9 delivery in mammalian cells. SHRV was grown well in baby hamster kidney (BHK-21) cells at 28 °C, and the replication ability was greatly reduced by temperature up-shift to 37 °C. We rescued a recombinant SHRV that harboring not only the interferon regulatory factor 9 (IRF9) gene-targeting single-guide RNA (sgRNA) but also Cas9 gene in the genome using the reverse genetic technology. The IRF9 gene of BHK-21 cells was knocked-out by the infection with the IRF9 gene-targeting rSHRV. Moreover, the rSHRVs were sharply disappeared in the cells by elevating temperature to 37 °C, suggesting the possible regulation of knockout efficiency before virus infection-caused cell damage. Although further optimization researches are needed to enhance the editing efficiency using the recombinant SHRV, to our knowledge, this is the first report on the possible applicability of piscine RNA virus for the gene editing in mammalian cells.},
}
@article {pmid34559985,
year = {2021},
author = {Ganaie, SS and Schwarz, MM and McMillen, CM and Price, DA and Feng, AX and Albe, JR and Wang, W and Miersch, S and Orvedahl, A and Cole, AR and Sentmanat, MF and Mishra, N and Boyles, DA and Koenig, ZT and Kujawa, MR and Demers, MA and Hoehl, RM and Moyle, AB and Wagner, ND and Stubbs, SH and Cardarelli, L and Teyra, J and McElroy, A and Gross, ML and Whelan, SPJ and Doench, J and Cui, X and Brett, TJ and Sidhu, SS and Virgin, HW and Egawa, T and Leung, DW and Amarasinghe, GK and Hartman, AL},
title = {Lrp1 is a host entry factor for Rift Valley fever virus.},
journal = {Cell},
volume = {184},
number = {20},
pages = {5163-5178.e24},
pmid = {34559985},
issn = {1097-4172},
support = {T32 AI060525/AI/NIAID NIH HHS/United States ; T32 AI106688/AI/NIAID NIH HHS/United States ; P41 GM103422/GM/NIGMS NIH HHS/United States ; R01 NS101100/NS/NINDS NIH HHS/United States ; R37 AI059371/AI/NIAID NIH HHS/United States ; K08 AI144033/AI/NIAID NIH HHS/United States ; R01 AI123926/AI/NIAID NIH HHS/United States ; P01 AI120943/AI/NIAID NIH HHS/United States ; R24 GM136766/GM/NIGMS NIH HHS/United States ; R01 AI130152/AI/NIAID NIH HHS/United States ; R01 AI161765/AI/NIAID NIH HHS/United States ; R01 AI107056/AI/NIAID NIH HHS/United States ; R01 AI150792/AI/NIAID NIH HHS/United States ; R21 AI163603/AI/NIAID NIH HHS/United States ; U19 AI142784/AI/NIAID NIH HHS/United States ; R01 AI143292/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antibody Specificity/immunology ; Base Sequence ; Brain/pathology/virology ; CRISPR-Cas Systems/genetics ; Cell Membrane/metabolism ; Cells, Cultured ; Glycoproteins/metabolism ; Glycosaminoglycans/metabolism ; Glycosylation ; *Host-Pathogen Interactions ; Humans ; LDL-Receptor Related Protein-Associated Protein/metabolism ; Ligands ; Low Density Lipoprotein Receptor-Related Protein-1/deficiency/*metabolism ; Membrane Glycoproteins/metabolism ; Mice ; Protein Binding ; Protein Denaturation ; Rift Valley Fever/pathology/prevention &amp; control/virology ; Rift Valley fever virus/immunology/*physiology ; *Virus Internalization ; },
abstract = {Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.},
}
@article {pmid34559312,
year = {2021},
author = {Niazian, M and Molaahmad Nalousi, A and Azadi, P and Ma'mani, L and Chandler, SF},
title = {Perspectives on new opportunities for nano-enabled strategies for gene delivery to plants using nanoporous materials.},
journal = {Planta},
volume = {254},
number = {4},
pages = {83},
pmid = {34559312},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Gene Editing ; Genetic Engineering ; Genome, Plant ; *Nanopores ; },
abstract = {Engineered nanocarriers have great potential to deliver different genetic cargos to plant cells and increase the efficiency of plant genetic engineering. Genetic engineering has improved the quality and quantity of crops by introducing desired DNA sequences into the plant genome. Traditional transformation strategies face constraints such as low transformation efficiency, damage to plant tissues, and genotype dependency. Smart nanovehicle-based delivery is a newly emerged method for direct DNA delivery to plant genomes. The basis of this new approach of plant genetic transformation, nanomaterial-mediated gene delivery, is the appropriate protection of transferred DNA from the nucleases present in the cell cytoplasm through the nanocarriers. The conjugation of desired nucleic acids with engineered nanocarriers can solve the problem of genetic manipulation in some valuable recalcitrant plant genotypes. Combining nano-enabled genetic transformation with the new and powerful technique of targeted genome editing, CRISPR (clustered regularly interspaced short palindromic repeats), can create new protocols for efficient improvement of desired plants. Silica-based nanoporous materials, especially mesoporous silica nanoparticles (MSNs), are currently regarded as exciting nanoscale platforms for genetic engineering as they possess several useful properties including ordered and porous structure, biocompatibility, biodegradability, and surface chemistry. These specific features have made MSNs promising candidates for the design of smart, controlled, and targeted delivery systems in agricultural sciences. In the present review, we discuss the usability, challenges, and opportunities for possible application of nano-enabled biomolecule transformation as part of innovative approaches for target delivery of genes of interest into plants.},
}
@article {pmid34558988,
year = {2021},
author = {Shaw, BC and Estus, S},
title = {Pseudogene-Mediated Gene Conversion After CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {699-709},
pmid = {34558988},
issn = {2573-1602},
support = {R21 AG068370/AG/NIA NIH HHS/United States ; F99 NS120365/NS/NINDS NIH HHS/United States ; P30 AG028383/AG/NIA NIH HHS/United States ; P30 CA177558/CA/NCI NIH HHS/United States ; RF1 AG059717/AG/NIA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Conversion ; Gene Editing/*methods ; HEK293 Cells ; Humans ; *Pseudogenes ; Recombinational DNA Repair ; Sialic Acid Binding Ig-like Lectin 3/*genetics ; },
abstract = {Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines, which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.},
}
@article {pmid34558981,
year = {2021},
author = {Li, Y and Mikkelsen, K and Lluch I Grané, O and Wang, Z and Tang, Y and Jiao, X and Ingmer, H and Høyland-Kroghsbo, NM and Li, Q},
title = {Functional Characterization of Type III-A CRISPR-Cas in a Clinical Human Methicillin-R Staphylococcus aureus Strain.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {686-698},
doi = {10.1089/crispr.2021.0046},
pmid = {34558981},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Methicillin-Resistant Staphylococcus aureus/*genetics/isolation &amp; purification/virology ; },
abstract = {CRISPR with its cas genes is an adaptive immune system that protects prokaryotes against foreign genetic elements. The type III-A CRISPR-Cas system is rarely found in Staphylococcus aureus, and little is known about its function in S. aureus. Here, we describe the genome characteristics of the clinical methicillin-resistant S. aureus (MRSA) strain TZ0912, carrying a type III-A CRISPR-Cas system. Phylogenetic analysis of 35 reported CRISPR-Cas-positive S. aureus strains revealed that the CRISPR-Cas system is prevalent in CC8 clones (10/35) and is located in the staphylococcal cassette chromosome mec (SCCmec) V, which confers methicillin resistance. Plasmid transformation and phage infection assays reveal that the type III-A CRISPR-Cas system protects TZ0912 against foreign DNA with sequence homology to the spacers located in the CRISPR array. We observed that the CRISPR-Cas immune system could effectively protect MRSA against phage attacks in both liquid culture and solid medium. In accordance with previous reports, using RNA-seq analysis and plasmid transformation assays, we find that the crRNAs close to the leading sequence of the CRISPR array are more highly expressed and are more effective at directing plasmid elimination compared to the distant spacers. This study established a model for evaluating the efficiency of naive CRISPR-Cas system in MRSA against phage, which could contribute to future research on the function of CRISPR-Cas in clinical MRSA isolates and improve phage therapy against MRSA infections.},
}
@article {pmid34558964,
year = {2021},
author = {Uranga, M and Vazquez-Vilar, M and Orzáez, D and Daròs, JA},
title = {CRISPR-Cas12a Genome Editing at the Whole-Plant Level Using Two Compatible RNA Virus Vectors.},
journal = {The CRISPR journal},
volume = {4},
number = {5},
pages = {761-769},
doi = {10.1089/crispr.2021.0049},
pmid = {34558964},
issn = {2573-1602},
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Genetic Vectors/genetics ; Potexvirus/genetics ; Potyvirus/genetics ; Tobacco/*genetics ; },
abstract = {The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.},
}
@article {pmid34558600,
year = {2022},
author = {Egido, JE and Costa, AR and Aparicio-Maldonado, C and Haas, PJ and Brouns, SJJ},
title = {Mechanisms and clinical importance of bacteriophage resistance.},
journal = {FEMS microbiology reviews},
volume = {46},
number = {1},
pages = {},
pmid = {34558600},
issn = {1574-6976},
mesh = {*Bacterial Infections ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Humans ; *Phage Therapy ; },
abstract = {We are in the midst of a golden age of uncovering defense systems against bacteriophages. Apart from the fundamental interest in these defense systems, and revolutionary applications that have been derived from them (e.g. CRISPR-Cas9 and restriction endonucleases), it is unknown how defense systems contribute to resistance formation against bacteriophages in clinical settings. Bacteriophages are now being reconsidered as therapeutic agents against bacterial infections due the emergence of multidrug resistance. However, bacteriophage resistance through defense systems and other means could hinder the development of successful phage-based therapies. Here, we review the current state of the field of bacteriophage defense, highlight the relevance of bacteriophage defense for potential clinical use of bacteriophages as therapeutic agents and suggest new directions of research.},
}
@article {pmid34556814,
year = {2021},
author = {Bolado-Carrancio, A and Lee, M and Ewing, A and Muir, M and Macleod, KG and Gallagher, WM and Nguyen, LK and Carragher, NO and Semple, CA and Brunton, VG and Caswell, PT and von Kriegsheim, A},
title = {ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling.},
journal = {Oncogene},
volume = {40},
number = {44},
pages = {6235-6247},
pmid = {34556814},
issn = {1476-5594},
support = {MR/R026017/1/MRC_/Medical Research Council/United Kingdom ; 14-1226/AICR_/Worldwide Cancer Research/United Kingdom ; MC_UU_00007/16/MRC_/Medical Research Council/United Kingdom ; MR/R009376/1/MRC_/Medical Research Council/United Kingdom ; 208402/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 203128/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Breast Neoplasms/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytokines/*genetics/*metabolism ; Endocytosis ; ErbB Receptors/metabolism ; Female ; Gene Knockout Techniques ; Guanine Nucleotide Dissociation Inhibitors/*metabolism ; Humans ; Phosphorylation ; Prognosis ; Proteomics ; Proto-Oncogene Proteins c-akt/metabolism ; Signal Transduction ; Survival Analysis ; Ubiquitins/*genetics/*metabolism ; },
abstract = {ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.},
}
@article {pmid34556671,
year = {2021},
author = {Song, M and Lim, JM and Min, S and Oh, JS and Kim, DY and Woo, JS and Nishimasu, H and Cho, SR and Yoon, S and Kim, HH},
title = {Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5617},
pmid = {34556671},
issn = {2041-1723},
mesh = {*Algorithms ; Amino Acid Sequence ; Binding Sites/genetics ; *CRISPR-Cas Systems ; DNA/genetics/*metabolism ; Gene Editing/*methods ; HCT116 Cells ; HEK293 Cells ; Humans ; *Models, Genetic ; Rad51 Recombinase/genetics/*metabolism ; Reproducibility of Results ; },
abstract = {Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency < 1%), hyPE2 enables prime editing with efficiencies ranging from 1.1% to 2.9% at up to 34% of target sequences, potentially facilitating prime editing applications.},
}
@article {pmid34556174,
year = {2021},
author = {Hutton, ER and Vakoc, CR and Siepel, A},
title = {ACE: a probabilistic model for characterizing gene-level essentiality in CRISPR screens.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {278},
pmid = {34556174},
issn = {1474-760X},
support = {P01 CA013106/CA/NCI NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; R01 CA174793/CA/NCI NIH HHS/United States ; R35 GM127070/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Genes, Essential ; Genes, p53 ; Genotype ; *Models, Statistical ; Mutation ; *Software ; },
abstract = {High-throughput CRISPR-Cas9 knockout screens are widely used to evaluate gene essentiality in cancer research. Here we introduce a probabilistic modeling framework, Analysis of CRISPR-based Essentiality (ACE), that accounts for multiple sources of variation in CRISPR-Cas9 screens and enables new statistical tests for essentiality. We show using simulations that ACE is effective at predicting both absolute and differential essentiality. When applied to publicly available data, ACE identifies known and novel candidates for genotype-specific essentiality, including RNA m[6]-A methyltransferases that exhibit enhanced essentiality in the presence of inactivating TP53 mutations. ACE provides a robust framework for identifying genes responsive to subtype-specific therapeutic targeting.},
}
@article {pmid34556164,
year = {2021},
author = {Nishizono, H and Hayano, Y and Nakahata, Y and Ishigaki, Y and Yasuda, R},
title = {Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research.},
journal = {Molecular brain},
volume = {14},
number = {1},
pages = {148},
pmid = {34556164},
issn = {1756-6606},
support = {R01 MH080047/MH/NIMH NIH HHS/United States ; R35 NS116804/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Calcium-Calmodulin-Dependent Protein Kinase Type 1/genetics ; Electroporation/*methods ; Embryo Transfer ; Exons/genetics ; Gene Editing/economics/*methods ; Gene Targeting/economics/*methods ; Integrases ; Mice ; Mice, Inbred C57BL ; *Mice, Knockout ; Neurosciences/economics/*methods ; Transgenes ; },
abstract = {The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.},
}
@article {pmid34555442,
year = {2021},
author = {Qian, W and Huang, J and Wang, T and He, X and Xu, G and Li, Y},
title = {Visual detection of human metapneumovirus using CRISPR-Cas12a diagnostics.},
journal = {Virus research},
volume = {305},
number = {},
pages = {198568},
doi = {10.1016/j.virusres.2021.198568},
pmid = {34555442},
issn = {1872-7492},
mesh = {CRISPR-Cas Systems ; Humans ; *Metapneumovirus/genetics ; Nucleic Acid Amplification Techniques/methods ; Reverse Transcription ; Sensitivity and Specificity ; },
abstract = {Human metapneumovirus (HmPV) is a common and serious virus that causes respiratory tract infection. This study aimed to develop a detection technique by combining reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for clinical diagnosis of HmPV. Herein, four primer pairs targeting partial nucleoprotein (N) gene of HmPV were designed and evaluated. Then, the products amplified by RT-RPA were detected using CRISPR-Cas12a combined with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 35 min or 45 min, and the detection limit was up to 6.97 × 10[2] copies/mL. And there was no cross reaction with human bocavirus, respiratory syncytial virus, adenovirus and parainfluenza virus. By combining with LF, the detection results were evaluated by naked eyes. Furthermore, 28 clinical samples were applied to examine the performance of RT-RPA-Cas12a system. The detection coincidence rates of RT-RPA-Cas12a-fluorescence and RT-RPA-Cas12a-LF with quantitative RT-PCR were 96.4% and 92.9%, respectively. Together, the new method for detecting HmPV with high sensitivity and specificity based on RT-RPA-Cas12a-fluorescence or LF shows promising potential for clinical diagnosis of HmPV without professional skills or ancillary equipment.},
}
@article {pmid34555356,
year = {2021},
author = {Sengupta, D and Zeng, L and Li, Y and Hausmann, S and Ghosh, D and Yuan, G and Nguyen, TN and Lyu, R and Caporicci, M and Morales Benitez, A and Coles, GL and Kharchenko, V and Czaban, I and Azhibek, D and Fischle, W and Jaremko, M and Wistuba, II and Sage, J and Jaremko, &#x141; and Li, W and Mazur, PK and Gozani, O},
title = {NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis.},
journal = {Molecular cell},
volume = {81},
number = {21},
pages = {4481-4492.e9},
pmid = {34555356},
issn = {1097-4164},
support = {R01 CA193466/CA/NCI NIH HHS/United States ; R01 CA228140/CA/NCI NIH HHS/United States ; R35 GM139569/GM/NIGMS NIH HHS/United States ; K99 CA255936/CA/NCI NIH HHS/United States ; P50 CA070907/CA/NCI NIH HHS/United States ; R01 CA236118/CA/NCI NIH HHS/United States ; R01 HG007538/HG/NHGRI NIH HHS/United States ; R01 CA236949/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma of Lung/*metabolism/mortality ; Animals ; Biopsy ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; *DNA Methylation ; Disease Progression ; Epigenesis, Genetic ; Epigenomics ; Female ; Histone-Lysine N-Methyltransferase/*chemistry ; Histones/*chemistry ; Humans ; Lung Neoplasms/*metabolism/mortality ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Transplantation ; Oncogenes ; Prognosis ; Repressor Proteins/*chemistry ; Signal Transduction ; Treatment Outcome ; },
abstract = {The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.},
}
@article {pmid34555355,
year = {2021},
author = {Paes Dias, M and Tripathi, V and van der Heijden, I and Cong, K and Manolika, EM and Bhin, J and Gogola, E and Galanos, P and Annunziato, S and Lieftink, C and Andújar-Sánchez, M and Chakrabarty, S and Smith, GCM and van de Ven, M and Beijersbergen, RL and Bartkova, J and Rottenberg, S and Cantor, S and Bartek, J and Ray Chaudhuri, A and Jonkers, J},
title = {Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.},
journal = {Molecular cell},
volume = {81},
number = {22},
pages = {4692-4708.e9},
pmid = {34555355},
issn = {1097-4164},
support = {R01 CA254037/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; BRCA1 Protein/*genetics ; Biopsy ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/metabolism ; Cell Proliferation ; Chromosome Aberrations ; DNA Damage ; DNA Ligase ATP/*genetics/metabolism ; *DNA, Single-Stranded ; Female ; Humans ; Lentivirus/genetics ; MRE11 Homologue Protein/*genetics ; Mammary Neoplasms, Animal ; Mice ; Mutation ; Ovarian Neoplasms/*metabolism ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Poly-ADP-Ribose Binding Proteins/*genetics/metabolism ; RNA, Small Interfering/metabolism ; Transgenes ; Triple Negative Breast Neoplasms/*metabolism ; Tumor Suppressor p53-Binding Protein 1/*genetics ; },
abstract = {Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.},
}
@article {pmid34555252,
year = {2022},
author = {Zhou, J and Zhang, R and Jia, X and Tang, X and Guo, Y and Yang, H and Zheng, X and Qian, Q and Qi, Y and Zhang, Y},
title = {CRISPR-Cas9 mediated OsMIR168a knockout reveals its pleiotropy in rice.},
journal = {Plant biotechnology journal},
volume = {20},
number = {2},
pages = {310-322},
pmid = {34555252},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant/genetics ; *MicroRNAs/genetics/metabolism ; *Oryza/genetics/metabolism ; Plant Breeding ; },
abstract = {MicroRNA168 (MIR168) is a key miRNA that targets the main RNA-induced silencing complex component Argonaute 1 (AGO1) to regulate plant growth and environmental stress responses. However, the regulatory functions of MIR168 need to be further elucidated in rice. In this paper, we generated clean OsMIR168a deletion mutants by CRISPR-Cas9 strategy. We then phenotypically and molecularly characterized these mutants. The rice OsMIR168a mutants grew rapidly at the seedling stage, produced more tillers and matured early. Compared to the wild-type plants, the mutants were shorter at maturity and produced smaller spikelets and seeds. Analysis of gene expression showed that the transcription levels of OsMIR168a's target genes such as OsAGO1a, OsAGO1b and OsAGO1d were elevated significantly in the OsMIR168a mutants. Intriguingly, OsAGO18, a member of a new AGO clade that is conserved in monocots, was confirmed to be a target of OsMIR168a not only by informatic prediction but also by expression analysis and a cell-based cleavage assay in the OsMIR168a mutants. Many protein-coding genes and miRNAs showed differential expression in the OsMIR168a mutants, suggesting OsMIR168a exerts a major transcriptional regulatory role, likely through its potential target genes such as OsAGO1s and OsAGO18. KEGG enrichment analysis of these differentially expressed genes pointed to OsMIR168a's involvement in important processes such as plant hormone signalling transduction and plant-pathogen interaction. These data collectively support that the complex regulation module of OsMIR168a-OsAGO1/OsAGO18-miRNAs-target genes contributes to agronomically important traits, which sheds light on miRNA-mediated crop breeding.},
}
@article {pmid34555248,
year = {2021},
author = {Mao, Y and Wang, M and Zhao, Y and Huang, B and Wu, Q and Zheng, Q and Botella, JR and Zhu, JK},
title = {Expanding the target range of base editing in plants without loss of efficiency by blocking RNA-silencing.},
journal = {Plant biotechnology journal},
volume = {19},
number = {12},
pages = {2389-2391},
pmid = {34555248},
issn = {1467-7652},
mesh = {*CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Plants/genetics/metabolism ; RNA ; },
}
@article {pmid34555130,
year = {2021},
author = {Zhou, X and Zhang, JL and Chang, MH and Fan, GT and Liu, XZ and Wu, SJ and Shi, X},
title = {Sensitive osteosarcoma diagnosis through five-base telomerase product-triggered CRISPR-Cas12a enhanced rolling circle amplification.},
journal = {Analytical methods : advancing methods and applications},
volume = {13},
number = {36},
pages = {4063-4068},
doi = {10.1039/d1ay00952d},
pmid = {34555130},
issn = {1759-9679},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Osteosarcoma/diagnosis ; *Telomerase/genetics ; },
abstract = {Osteosarcoma is the most frequent primary malignant bone tumor, composed of mesenchymal cells producing osteoid and immature bone. The sensitive detection of telomerase plays a pivotal role in the early diagnosis and therapeutic treatment of osteosarcoma. We report here an in vitro strategy for sensitive telomerase activity detection through the integration of rolling circle amplification (RCA) and a clustered regularly spaced short palindrome repeats (CRISPR)-Cas12a system. In the proposed strategy, telomerase substrate (TS) primers are easily controlled to extend five bases (GGGTT) to give short telomerase extension products (TEP) with definite lengths without adding dATP. The resulting short TEPs can then cyclize the padlock through hybridizing with its two terminals and thus initiate the following RCA. To obtain an improved sensitivity, the CRISPR-Cas12a system is attached to collaterally cut surrounding DNA reporter probes after recognizing the target single strand DNA sequence in the RCA products. The highlights of this strategy are as follows: (i) the short TEP triggered strategy is excellent at detecting low telomerase activity and thus contributes to the early diagnosis of malignant tumors; (ii) highly sensitive telomerase activity detection which is easy to operate from RCA initiated CRISPR-Cas12a; (iii) opening up of a new avenue for telomerase activity detection with a CRISPR-Cas12a system. Finally, the proposed strategy exhibited sensitive telomerase activity detection under optimized experimental parameters and has great application potential for the clinical diagnosis of malignant tumors and the development of anti-cancer drugs.},
}
@article {pmid34554293,
year = {2021},
author = {Tang, X and Chen, S and Yu, H and Zheng, X and Zhang, F and Deng, X and Xu, Q},
title = {Development of a gRNA-tRNA array of CRISPR/Cas9 in combination with grafting technique to improve gene-editing efficiency of sweet orange.},
journal = {Plant cell reports},
volume = {40},
number = {12},
pages = {2453-2456},
pmid = {34554293},
issn = {1432-203X},
mesh = {Agrobacterium/genetics ; *CRISPR-Cas Systems ; Citrus sinensis/*genetics ; Gene Editing/*methods ; Heat-Shock Response/genetics ; Mutation ; Plant Proteins/genetics ; Plants, Genetically Modified ; RNA, Guide/*genetics ; Seedlings/genetics ; Transformation, Bacterial ; },
abstract = {Here, we developed a reliable protocol for the fast and efficient gene-edited Anliu sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90%. In addition, the mutation efficiency of the grafted geneedited sweet orange was significantly improved by short-term heat stress treatments. Thus, the combination strategy of grafting and heat stress treatments provided a reference for the fast and efficient multiplex gene editing of sweet orange.},
}
@article {pmid34554233,
year = {2021},
author = {Yates, JD and Russell, RC and Barton, NJ and Yost, HJ and Hill, JT},
title = {A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries.},
journal = {Nucleic acids research},
volume = {49},
number = {22},
pages = {e131},
pmid = {34554233},
issn = {1362-4962},
support = {U01 HL098160/HL/NHLBI NIH HHS/United States ; U01 HL131003/HL/NHLBI NIH HHS/United States ; UM1 HL098160/HL/NHLBI NIH HHS/United States ; R15 HD098969/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; DNA Restriction Enzymes ; DNA-Directed DNA Polymerase ; Escherichia coli/genetics ; Fluorescent Dyes ; Genetic Techniques ; Genome ; Green Fluorescent Proteins ; Humans ; Myocardium/metabolism ; Oligonucleotides ; RNA/biosynthesis ; Transcriptome ; Zebrafish ; },
abstract = {CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries.},
}
@article {pmid34553925,
year = {2021},
author = {Wang, Q and Liu, Y and Yan, J and Liu, Y and Gao, C and Ge, S and Yu, J},
title = {3D DNA Walker-Assisted CRISPR/Cas12a Trans-Cleavage for Ultrasensitive Electrochemiluminescence Detection of miRNA-141.},
journal = {Analytical chemistry},
volume = {93},
number = {39},
pages = {13373-13381},
doi = {10.1021/acs.analchem.1c03183},
pmid = {34553925},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *MicroRNAs ; },
abstract = {In this study, a CRISPR/Cas12a (LbCpf1)-mediated electrochemiluminescence (ECL) paper-based platform on the basis of a three-dimensional (3D) DNA walker was proposed for the ultrasensitive detection of miRNA-141. Initially, 3D-rGO with a tremendous loading space was modified on the paper working electrode (PWE) to construct an excellent conductive substrate and facilitate the growth of AuPd nanoparticles (NPs). Afterward, the AuPd NPs were introduced as the coreaction emitter medium of the 3D-rGO/PWE to provide convenience for the transformation between S2O8[2-] and SO4[2-], amplifying the ECL emission of g-C3N4 nanosheets (NSs). Meanwhile, with the help of Nt.BsmAI nicking endonuclease, a 3D DNA walker signal amplifier was designed to convert and magnify the target miRNA-141 into a particular trigger sequence, which could act as activator DNA to motivate the trans-acting deoxyribonuclease activity of CRISPR/Cas12a to further achieve efficient annihilation of the ECL signal. Furthermore, the proposed multimechanism-driven biosensor exhibited excellent sensitivity and specificity, with a relatively low detection limit at 0.331 fM (S/N = 3) in the concentration range between 1 fM and 10 nM. Consequently, the designed strategy not only extended the application scope of CRISPR/Cas12a but also devoted a new approach for the clinical diagnosis of modern medicine.},
}
@article {pmid34553495,
year = {2022},
author = {Kordyś, M and Sen, R and Warkocki, Z},
title = {Applications of the versatile CRISPR-Cas13 RNA targeting system.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {13},
number = {3},
pages = {e1694},
doi = {10.1002/wrna.1694},
pmid = {34553495},
issn = {1757-7012},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; RNA/genetics ; RNA Editing ; RNA Processing, Post-Transcriptional ; },
abstract = {CRISPR-Cas are adaptable natural prokaryotic defense systems that act against invading viruses and plasmids. Among the six currently known major CRISPR-Cas types, the type VI CRISPR-Cas13 is the only one known to exclusively bind and cleave foreign RNA. Within the last couple of years, this system has been adapted to serve numerous, and sometimes not obvious, applications, including some that might be developed as effective molecular therapies. Indeed, Cas13 has been adapted to kill antibiotic-resistant bacteria. In a cell-free environment, Cas13 has been used in the development of highly specific, sensitive, multiplexing-capable, and field-adaptable detection tools. Importantly, Cas13 can be reprogrammed and applied to eukaryotes to either combat pathogenic RNA viruses or in the regulation of gene expression, facilitating the knockdown of mRNA, circular RNA, and noncoding RNA. Furthermore, Cas13 has been harnessed for in vivo RNA modifications including programmable regulation of alternative splicing, A-to-I and C to U editing, and m6A modifications. Finally, approaches allowing for the detection and characterization of RNA-interacting proteins have also been demonstrated. Here, we provide a comprehensive overview of the applications utilizing CRISPR-Cas13 that illustrate its versatility. We also discuss the most important limitations of the CRISPR-Cas13-based technologies, and controversies regarding them. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.},
}
@article {pmid34553468,
year = {2021},
author = {Wei, Y and Yang, Z and Zong, C and Wang, B and Ge, X and Tan, X and Liu, X and Tao, Z and Wang, P and Ma, C and Wan, Y and Li, J},
title = {trans Single-Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {45},
pages = {24241-24247},
doi = {10.1002/anie.202110384},
pmid = {34553468},
issn = {1521-3773},
mesh = {CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; DNA Cleavage ; DNA, Single-Stranded/chemistry/*metabolism ; RNA/analysis/*metabolism ; },
abstract = {As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNA cleavage, ATCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high-efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.},
}
@article {pmid34551826,
year = {2021},
author = {Goullée, H and Taylor, RL and Forrest, ARR and Laing, NG and Ravenscroft, G and Clayton, JS},
title = {Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel.},
journal = {Skeletal muscle},
volume = {11},
number = {1},
pages = {23},
pmid = {34551826},
issn = {2044-5040},
mesh = {*CRISPR-Cas Systems ; Collagen ; Drug Combinations ; *Gene Editing ; Humans ; Laminin ; Myoblasts ; Proteoglycans ; Transfection ; },
abstract = {BACKGROUND: CRISPR/Cas9 is an invaluable tool for studying cell biology and the development of molecular therapies. However, delivery of CRISPR/Cas9 components into some cell types remains a major hurdle. Primary human myoblasts are a valuable cell model for muscle studies, but are notoriously difficult to transfect. There are currently no commercial lipofection protocols tailored for primary myoblasts, and most generic guidelines simply recommend transfecting healthy cells at high confluency. This study aimed to maximize CRISPR/Cas9 transfection and editing in primary human myoblasts.<br><br>METHODS: Since increased cell proliferation is associated with increased transfection efficiency, we investigated two factors known to influence myoblast proliferation: cell confluency, and a basement membrane matrix, Matrigel. CRISPR/Cas9 editing was performed by delivering Cas9 ribonucleoprotein complexes via lipofection into primary human myoblasts, cultured in wells with or without a Matrigel coating, at low (~ 40%) or high (~ 80%) confluency.<br><br>RESULTS: Cells transfected at low confluency on Matrigel-coated wells had the highest levels of transfection, and were most effectively edited across three different target loci, achieving a maximum editing efficiency of 93.8%. On average, editing under these conditions was >4-fold higher compared to commercial recommendations (high confluency, uncoated wells).<br><br>CONCLUSION: This study presents a simple, effective and economical method of maximizing CRISPR/Cas9-mediated gene editing in primary human myoblasts. This protocol could be a valuable tool for improving the genetic manipulation of cultured human skeletal muscle cells, and potentially be adapted for use in other cell types.},
}
@article {pmid34551428,
year = {2021},
author = {Cheng, F and Wang, R and Yu, H and Liu, C and Yang, J and Xiang, H and Li, M},
title = {Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.},
journal = {Nucleic acids research},
volume = {49},
number = {18},
pages = {10677-10688},
pmid = {34551428},
issn = {1362-4962},
mesh = {Bacterial Toxins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Engineering ; Genes, Archaeal ; Genes, Bacterial ; Haloarcula/*genetics ; Halobacterium/*genetics ; Protein Biosynthesis ; RNA, Small Untranslated/chemistry/genetics/*metabolism ; RNA, Transfer, Arg/metabolism ; RNA, Transfer, Ile/*metabolism ; },
abstract = {Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.},
}
@article {pmid34551298,
year = {2021},
author = {Chen, Q and Zhang, M and Pan, X and Yuan, X and Zhou, L and Yan, L and Zeng, LH and Xu, J and Yang, B and Zhang, L and Huang, J and Lu, W and Fukagawa, T and Wang, F and Yan, H},
title = {Bub1 and CENP-U redundantly recruit Plk1 to stabilize kinetochore-microtubule attachments and ensure accurate chromosome segregation.},
journal = {Cell reports},
volume = {36},
number = {12},
pages = {109740},
doi = {10.1016/j.celrep.2021.109740},
pmid = {34551298},
issn = {2211-1247},
mesh = {Aurora Kinase B/metabolism ; Benzimidazoles/pharmacology ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Centromere/metabolism ; Chromosome Segregation/drug effects/*physiology ; HeLa Cells ; Histones/antagonists &amp; inhibitors/genetics/*metabolism ; Humans ; Kinetochores/*metabolism ; Microscopy, Fluorescence ; Microtubules/*metabolism ; Poly-ADP-Ribose Binding Proteins/metabolism ; Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics/*metabolism ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/*metabolism ; RNA Interference ; RNA, Guide/metabolism ; RNA, Small Interfering/metabolism ; Thiophenes/pharmacology ; Time-Lapse Imaging ; },
abstract = {Bub1 is required for the kinetochore/centromere localization of two essential mitotic kinases Plk1 and Aurora B. Surprisingly, stable depletion of Bub1 by ∼95% in human cells marginally affects whole chromosome segregation fidelity. We show that CENP-U, which is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex, is required to prevent chromosome mis-segregation in Bub1-depleted cells. Mechanistically, Bub1 and CENP-U redundantly recruit Plk1 to kinetochores to stabilize kinetochore-microtubule attachments, thereby ensuring accurate chromosome segregation. Furthermore, unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B. Consistently, depletion of Bub1 or CENP-U sensitizes cells to the inhibition of Plk1 but not Aurora B kinase activity. Taken together, our findings provide mechanistic insight into the regulation of kinetochore function, which may have implications for targeted treatment of cancer cells with mutations perturbing kinetochore recruitment of Plk1 by Bub1 or the CENP-O complex.},
}
@article {pmid34550780,
year = {2021},
author = {Philippe-Auguste, J and Berdecio, I and Terry, SF},
title = {Learning from the Past: Discussing Lessons from Reproductive Justice in the Gene-Editing Sphere.},
journal = {Genetic testing and molecular biomarkers},
volume = {25},
number = {9},
pages = {571-572},
doi = {10.1089/gtmb.2021.0217},
pmid = {34550780},
issn = {1945-0257},
mesh = {Abortion, Induced/ethics ; CRISPR-Cas Systems/genetics ; Eugenics/history/*methods ; Female ; Gene Editing/*ethics ; History, 20th Century ; History, 21st Century ; Humans ; Patient Participation ; Reproduction/*genetics ; Social Justice/*ethics ; Sterilization, Involuntary/ethics/history ; Trust ; United States ; *Vulnerable Populations ; },
}
@article {pmid34550573,
year = {2022},
author = {Horie, K and Nishimori, K},
title = {CRISPR/Cas9-Mediated Genetic Engineering to Generate a Disease Model Prairie Vole, Based on Species-Optimized Assisted Reproductive Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2384},
number = {},
pages = {139-152},
pmid = {34550573},
issn = {1940-6029},
mesh = {Animals ; Arvicolinae/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Grassland ; Mice ; Oxytocin/genetics ; Rats ; Receptors, Oxytocin/genetics ; Reproductive Techniques, Assisted ; Social Behavior ; },
abstract = {Social and prosocial behaviors, including communication, social bonding, and affiliation, parental behaviors, and empathy are key features of a highly social mammalian species. However, the neuronal mechanism in the brain underlying these behaviors remains unclear because of limited information on the social and prosocial behavioral levels in rodent models generally used in behavioral neuroscience studies.The rodent species, prairie vole (Microtus ochrogaster), is one of the nontraditional animal models with several advantages in experimental science over other rodent models, such as mice or rats. Additionally, it demonstrates characteristics advantageous in the study of social and prosocial behaviors, such as monogamous pair bonding behavior, biparental care, and consoling behavior toward partners stressed by aversive foot shock stimulus. Recent studies of prairie voles have highlighted the importance of oxytocin (OXT) and oxytocin receptor (OXTR)-mediated mechanisms in the regulation of these behaviors.Recently, we established assisted reproductive technologies for prairie voles, and successfully and efficiently generated an OXTR gene knockout (KO) prairie vole using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a powerful genome editing tool with artificially developed single-strand guide RNAs (sgRNA) and Cas9 endonucleases.Herein, we describe the method for CRISPR /Cas9-mediated generation of OXTR KO prairie vole. This OXTR KO prairie vole can be a valuable tool to understand their unique social and prosocial behaviors and elucidate how the oxytocin system influences or modulates these behaviors in the brain.},
}
@article {pmid34549986,
year = {2021},
author = {Lai, M and Maori, E and Quaranta, P and Matteoli, G and Maggi, F and Sgarbanti, M and Crucitta, S and Pacini, S and Turriziani, O and Antonelli, G and Heeney, JL and Freer, G and Pistello, M},
title = {CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.},
journal = {Journal of virology},
volume = {95},
number = {23},
pages = {e0135821},
pmid = {34549986},
issn = {1098-5514},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *DNA, Circular ; Gene Editing ; Gene Expression Regulation, Viral ; Genetic Therapy ; HEK293 Cells ; HIV Infections/virology ; HIV-1/*genetics ; Humans ; Proviruses/*genetics ; RNA, Guide/genetics ; *Terminal Repeat Sequences ; },
abstract = {Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.},
}
@article {pmid34549979,
year = {2021},
author = {Smith, LM and Ladner, JT and Hodara, VL and Parodi, LM and Harris, RA and Callery, JE and Lai, Z and Zou, Y and Raveedran, M and Rogers, J and Giavedoni, LD},
title = {Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA.},
journal = {Journal of virology},
volume = {95},
number = {23},
pages = {e0088221},
pmid = {34549979},
issn = {1098-5514},
support = {P51 OD011133/OD/NIH HHS/United States ; UL1 TR001120/TR/NCATS NIH HHS/United States ; P51 OD011133/CD/ODCDC CDC HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *DNA ; Deoxyribonuclease I/*genetics/*metabolism ; Endonucleases/genetics ; Gene Editing ; HEK293 Cells ; Humans ; Macaca mulatta/metabolism ; Mutagenesis ; Plasmids ; Proviruses/*genetics ; RNA, Guide/*genetics ; Simian Immunodeficiency Virus/*genetics ; },
abstract = {Human and simian immunodeficiency virus (HIV and SIV) infections establish lifelong reservoirs of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactivate integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells cotransfected with SIV provirus and plasmids coding for RGNus identified SIV long terminal repeat (LTR), trans-activation response (TAR) element, and ribosome slip site (RSS) regions as the most effective at virus suppression; RGNi targeting these regions inhibited virus production significantly. Multiplex plasmids that coexpressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole-genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations, making these tools a desirable prospect for moving into animal studies. IMPORTANCE Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole-genome sequencing demonstrated effective SIV sequence mutation and inactivation and the absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.},
}
@article {pmid34549224,
year = {2021},
author = {Hu, J and Zhou, J and Liu, R and Lv, Y},
title = {Element probe based CRISPR/Cas14 bioassay for non-nucleic-acid targets.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {80},
pages = {10423-10426},
doi = {10.1039/d1cc03992j},
pmid = {34549224},
issn = {1364-548X},
mesh = {Adenosine Monophosphate/analysis/chemistry ; Ampicillin/*analysis/chemistry ; Anti-Bacterial Agents/*analysis/chemistry ; Aptamers, Nucleotide/chemistry ; Biosensing Techniques/*methods ; CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/chemistry ; Limit of Detection ; Rivers/chemistry ; Water Pollutants, Chemical/analysis/chemistry ; },
abstract = {Herein, we propose an element probe based CRISPR/Cas14 detection platform and apply it to the detection of non-nucleic-acid targets. Combining metal isotope detection and CRISPR/Cas14 biosensing, the sensitive detection of non-nucleic-acid targets could be realized. We designed and optimized the element probe, which proved that Cas14 has a preference for longer lengths in element probe cleavage. Using this method, the quantitative detection of trace aqueous ampicillin can be achieved within 45 minutes at room temperature (25 °C). A detection limit as low as 2.06 nM is obtained with excellent performance in anti-interference tests and complex matrix detection.},
}
@article {pmid34547891,
year = {2021},
author = {Asin-Garcia, E and Martin-Pascual, M and Garcia-Morales, L and van Kranenburg, R and Martins Dos Santos, VAP},
title = {ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2672-2688},
pmid = {34547891},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; *Gene Editing ; *Genes, Bacterial ; Pseudomonas putida/*genetics ; *Recombination, Genetic ; },
abstract = {Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5'-NNG-3' of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.},
}
@article {pmid34547166,
year = {2021},
author = {Pandey, P and Zhang, N and Curtis, BR and Newman, PJ and Denomme, GA},
title = {Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.},
journal = {Journal of cellular and molecular medicine},
volume = {25},
number = {19},
pages = {9340-9349},
pmid = {34547166},
issn = {1582-4934},
support = {R35 HL139937/HL/NHLBI NIH HHS/United States ; HL139937/NH/NIH HHS/United States ; },
mesh = {Biomarkers ; Blood Group Antigens/genetics/metabolism ; *CRISPR-Cas Systems ; *Cell Differentiation ; Cell Line ; *Cell Lineage ; Erythroblasts/cytology/*metabolism ; *Gene Editing ; Gene Knockdown Techniques ; Hematopoiesis ; Histocytochemistry ; Humans ; Immunophenotyping ; Induced Pluripotent Stem Cells/cytology/*metabolism ; RNA, Guide/genetics ; },
abstract = {Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34[+] CD41[+] CD235ab[+] hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers-CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.},
}
@article {pmid34547052,
year = {2021},
author = {Aparicio-Prat, E and Yan, D and Mariotti, M and Bassik, M and Hess, G and Fortin, JP and Weston, A and Xi, HS and Stanton, R},
title = {Roadmap for the use of base editors to decipher drug mechanism of action.},
journal = {PloS one},
volume = {16},
number = {9},
pages = {e0257537},
pmid = {34547052},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genes, Reporter ; HEK293 Cells ; Humans ; Isoproterenol/chemistry/*metabolism ; Luminescent Proteins/genetics/metabolism ; Mutagenesis, Site-Directed ; RNA Interference ; RNA, Guide/metabolism ; RNA, Small Interfering/metabolism ; Receptors, Adrenergic, beta-2/chemistry/genetics/*metabolism ; },
abstract = {CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The β2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.},
}
@article {pmid34546709,
year = {2021},
author = {Mahas, A and Wang, Q and Marsic, T and Mahfouz, MM},
title = {A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2541-2551},
pmid = {34546709},
issn = {2161-5063},
mesh = {COVID-19/*diagnosis/genetics ; COVID-19 Nucleic Acid Testing ; *CRISPR-Cas Systems ; Humans ; *Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques ; RNA, Viral/analysis/*genetics ; SARS-CoV-2/chemistry/*genetics ; },
abstract = {Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes' RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.},
}
@article {pmid34546072,
year = {2021},
author = {Flores Ramos, S and Brugger, SD and Escapa, IF and Skeete, CA and Cotton, SL and Eslami, SM and Gao, W and Bomar, L and Tran, TH and Jones, DS and Minot, S and Roberts, RJ and Johnston, CD and Lemon, KP},
title = {Genomic Stability and Genetic Defense Systems in Dolosigranulum pigrum, a Candidate Beneficial Bacterium from the Human Microbiome.},
journal = {mSystems},
volume = {6},
number = {5},
pages = {e0042521},
pmid = {34546072},
issn = {2379-5077},
support = {R01 DE027850/DE/NIDCR NIH HHS/United States ; R01 GM117174/GM/NIGMS NIH HHS/United States ; R01 DE027850/DE/NIDCR NIH HHS/United States ; R01 GM117174/GM/NIGMS NIH HHS/United States ; },
abstract = {Dolosigranulum pigrum is positively associated with indicators of health in multiple epidemiological studies of human nasal microbiota. Knowledge of the basic biology of D. pigrum is a prerequisite for evaluating its potential for future therapeutic use; however, such data are very limited. To gain insight into D. pigrum's chromosomal structure, pangenome, and genomic stability, we compared the genomes of 28 D. pigrum strains that were collected across 20 years. Phylogenomic analysis showed closely related strains circulating over this period and closure of 19 genomes revealed highly conserved chromosomal synteny. Gene clusters involved in the mobilome and in defense against mobile genetic elements (MGEs) were enriched in the accessory genome versus the core genome. A systematic analysis for MGEs identified the first candidate D. pigrum prophage and insertion sequence. A systematic analysis for genetic elements that limit the spread of MGEs, including restriction modification (RM), CRISPR-Cas, and deity-named defense systems, revealed strain-level diversity in host defense systems that localized to specific genomic sites, including one RM system hot spot. Analysis of CRISPR spacers pointed to a wealth of MGEs against which D. pigrum defends itself. These results reveal a role for horizontal gene transfer and mobile genetic elements in strain diversification while highlighting that in D. pigrum this occurs within the context of a highly stable chromosomal organization protected by a variety of defense mechanisms. IMPORTANCE Dolosigranulum pigrum is a candidate beneficial bacterium with potential for future therapeutic use. This is based on its positive associations with characteristics of health in multiple studies of human nasal microbiota across the span of human life. For example, high levels of D. pigrum nasal colonization in adults predicts the absence of Staphylococcus aureus nasal colonization. Also, D. pigrum nasal colonization in young children is associated with healthy control groups in studies of middle ear infections. Our analysis of 28 genomes revealed a remarkable stability of D. pigrum strains colonizing people in the United States across a 20-year span. We subsequently identified factors that can influence this stability, including genomic stability, phage predators, the role of MGEs in strain-level variation, and defenses against MGEs. Finally, these D. pigrum strains also lacked predicted virulence factors. Overall, these findings add additional support to the potential for D. pigrum as a therapeutic bacterium.},
}
@article {pmid34546037,
year = {2021},
author = {Dong, J and Chen, C and Liu, Y and Zhu, J and Li, M and Rao, VB and Tao, P},
title = {Engineering T4 Bacteriophage for In Vivo Display by Type V CRISPR-Cas Genome Editing.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2639-2648},
doi = {10.1021/acssynbio.1c00251},
pmid = {34546037},
issn = {2161-5063},
support = {R01 AI111538/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophage T4/*genetics ; *CRISPR-Cas Systems ; *Cell Surface Display Techniques ; Escherichia coli/genetics ; Gene Editing/*methods ; },
abstract = {Bacteriophage T4 has enormous potential for biomedical applications due to its large size, capsid architecture, and high payload capability for protein and DNA delivery. However, it is not very easy to genetically engineer its genome heavily modified by cytosine hydroxymethylation and glucosylation. The glucosyl hydroxymethyl cytosine (ghmC) genome of phage is completely resistant to most restriction endonucleases and exhibits various degrees of resistance to CRISPR-Cas systems. Here, we found that the type V CRISPR-Cas12a system, which shows efficient cleavage of ghmC-modified genome when compared to the type II CRISPR-Cas9 system, can be synergistically employed to generate recombinant T4 phages. Focused on surface display, we analyzed the ability of phage T4 outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) to tether, in vivo, foreign peptides and proteins to T4 capsid. Our data show that while these could be successfully expressed and displayed during the phage infection, shorter peptides are present at a much higher copy number than full-length proteins. However, the copy number of the latter could be elevated by driving the expression of the transgene using the strong T7 RNA polymerase expression system. This CRISPR-inspired approach has the potential to expand the application of phages to various basic and translational research projects.},
}
@article {pmid34545248,
year = {2022},
author = {Przybyla, L and Gilbert, LA},
title = {A new era in functional genomics screens.},
journal = {Nature reviews. Genetics},
volume = {23},
number = {2},
pages = {89-103},
pmid = {34545248},
issn = {1471-0064},
support = {DP2 CA239597/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression Profiling/methods ; Gene Regulatory Networks/genetics ; Genetic Association Studies/methods ; Genome, Human/*genetics ; Genome-Wide Association Study/*methods ; Genomics/*methods/trends ; Humans ; *Polymorphism, Single Nucleotide ; },
abstract = {The past 25 years of genomics research first revealed which genes are encoded by the human genome and then a detailed catalogue of human genome variation associated with many diseases. Despite this, the function of many genes and gene regulatory elements remains poorly characterized, which limits our ability to apply these insights to human disease. The advent of new CRISPR functional genomics tools allows for scalable and multiplexable characterization of genes and gene regulatory elements encoded by the human genome. These approaches promise to reveal mechanisms of gene function and regulation, and to enable exploration of how genes work together to modulate complex phenotypes.},
}
@article {pmid34544832,
year = {2021},
author = {Peck, LJ and Patel, R and Diaz, P and Wintle, YM and Dickenson, AH and Todd, AJ and Calvo, M and Bennett, DLH},
title = {Studying Independent Kcna6 Knock-out Mice Reveals Toxicity of Exogenous LacZ to Central Nociceptor Terminals and Differential Effects of Kv1.6 on Acute and Neuropathic Pain Sensation.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {41},
number = {44},
pages = {9141-9162},
pmid = {34544832},
issn = {1529-2401},
support = {202747/WT_/Wellcome Trust/United Kingdom ; 109117/WT_/Wellcome Trust/United Kingdom ; 102645/WT_/Wellcome Trust/United Kingdom ; 219433/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Knockout Techniques/*adverse effects/methods ; *Genes, Reporter ; Integrases/metabolism ; Kv1.6 Potassium Channel/*genetics/metabolism ; *Lac Operon ; Male ; Mice ; Mice, Inbred C57BL ; Neuralgia/*metabolism ; Nociceptors/*metabolism/pathology ; Synapses/metabolism/pathology ; },
abstract = {The potassium channel Kv1.6 has recently been implicated as a major modulatory channel subunit expressed in primary nociceptors. Furthermore, its expression at juxtaparanodes of myelinated primary afferents is induced following traumatic nerve injury as part of an endogenous mechanism to reduce hyperexcitability and pain-related hypersensitivity. In this study, we compared two mouse models of constitutive Kv1.6 knock-out (KO) achieved by different methods: traditional gene trap via homologous recombination and CRISPR-mediated excision. Both Kv1.6 KO mouse lines exhibited an unexpected reduction in sensitivity to noxious heat stimuli, to differing extents: the Kv1.6 mice produced via gene trap had a far more significant hyposensitivity. These mice (Kcna6[lacZ]) expressed the bacterial reporter enzyme LacZ in place of Kv1.6 as a result of the gene trap mechanism, and we found that their central primary afferent presynaptic terminals developed a striking neurodegenerative phenotype involving accumulation of lipid species, development of "meganeurites," and impaired transmission to dorsal horn wide dynamic range neurons. The anatomic defects were absent in CRISPR-mediated Kv1.6 KO mice (Kcna6[-/-]) but were present in a third mouse model expressing exogenous LacZ in nociceptors under the control of a Nav1.8-promoted Cre recombinase. LacZ reporter enzymes are thus intrinsically neurotoxic to sensory neurons and may induce pathologic defects in transgenic mice, which has confounding implications for the interpretation of gene KOs using lacZ Nonetheless, in Kcna6[-/-] mice not affected by LacZ, we demonstrated a significant role for Kv1.6 regulating acute noxious thermal sensitivity, and both mechanical and thermal pain-related hypersensitivity after nerve injury.SIGNIFICANCE STATEMENT In recent decades, the expansion of technologies to experimentally manipulate the rodent genome has contributed significantly to the field of neuroscience. While introduction of enzymatic or fluorescent reporter proteins to label neuronal populations is now commonplace, often potential toxicity effects are not fully considered. We show a role of Kv1.6 in acute and neuropathic pain states through analysis of two mouse models lacking Kv1.6 potassium channels: one with additional expression of LacZ and one without. We show that LacZ reporter enzymes induce unintended defects in sensory neurons, with an impact on behavioral data outcomes. To summarize we highlight the importance of Kv1.6 in recovery of normal sensory function following nerve injury, and careful interpretation of data from LacZ reporter models.},
}
@article {pmid34544395,
year = {2021},
author = {Zou, YL and Ye, AJ and Liu, S and Wu, WT and Xu, LF and Dai, FY and Tong, XL},
title = {Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {54},
pmid = {34544395},
issn = {1472-6750},
mesh = {Animals ; *Bombyx/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *RNA, Guide/genetics ; Ribonucleoproteins/genetics ; },
abstract = {BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome.<br><br>RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation.<br><br>CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.},
}
@article {pmid34544122,
year = {2021},
author = {Seher, TD and Nguyen, N and Ramos, D and Bapat, P and Nobile, CJ and Sindi, SS and Hernday, AD},
title = {AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {9},
pages = {},
pmid = {34544122},
issn = {2160-1836},
support = {R35 GM124594/GM/NIGMS NIH HHS/United States ; R15 AI137975/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering ; Genomics ; Software ; },
abstract = {CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however, targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an "AddTag" sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene-editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.},
}
@article {pmid34543885,
year = {2021},
author = {Lilianty, J and Bateman, JF and Lamandé, SR},
title = {Generation of a heterozygous COL2A1 (p.G1113C) hypochondrogenesis mutation iPSC line, MCRIi019-A-7, using CRISPR/Cas9 gene editing.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102515},
doi = {10.1016/j.scr.2021.102515},
pmid = {34543885},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Collagen Type II/genetics ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Mutation ; Osteochondrodysplasias ; Polyenes ; },
abstract = {The human inherited cartilage disease, Hypochondrogenesis, is caused by mutations in the collagen type II gene, COL2A1. To produce an in vitro disease model, we generated a heterozygous patient mutation, COL2A1 p.G1113C, in an established control human induced pluripotent stem cell (iPSC) line, MCRIi019-A, using CRISPR-Cas9 gene editing. The gene-edited mutant line, MCRIi019-A-7, exhibited normal iPSC characteristics, including normal cell morphology, expression of pluripotency markers, the ability to differentiate into three embryonic germ layers, and normal karyotype. Together with its parental isogenic control, this cell line will be useful for Hypochondrogenesis disease modelling and drug testing.},
}
@article {pmid34543570,
year = {2021},
author = {Liu, S and Tao, D and Liao, Y and Yang, Y and Sun, S and Zhao, Y and Yang, P and Tang, Y and Chen, B and Liu, Y and Xie, S and Tang, Z},
title = {Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus.},
journal = {ACS synthetic biology},
volume = {10},
number = {10},
pages = {2499-2507},
doi = {10.1021/acssynbio.1c00103},
pmid = {34543570},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; Fluorescence ; Genes, Reporter ; Limit of Detection ; Porcine respiratory and reproductive syndrome virus/genetics/*isolation &amp; purification ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of swine that is caused by PRRS virus (PRRSV). In this study, we established a fluorescence assay for highly sensitive detection of PRRSV through integration of the reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled Cas12a system with an optical property of single stranded DNA-fluorescently quenched (ssDNA-FQ) reporter. This technique can achieve isothermal and visual detection of PRRSV in 25 min. In particular, the assay reaction can be completed in a single tube. The limit of sensitivity for PRRSV detection was single copy without cross-reactivity of other porcine viruses. Correlation between 11 PRRSV clinical samples measured by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) and CRISPR/Cas12a assay was determined; the result showed that our results were highly accurate. To sum up, this study developed a visual, sensitive, and specific method of nucleic acid detection based on a CRISPR-Cas12a technique for the on-site detection of PRRSV.},
}
@article {pmid34543054,
year = {2022},
author = {Chen, X and Tan, Q and Lyu, Q and Yu, C and Jiang, N and Li, J and Luo, L},
title = {Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {35},
number = {1},
pages = {4-14},
doi = {10.1094/MPMI-07-21-0179-TA},
pmid = {34543054},
issn = {0894-0282},
mesh = {*CRISPR-Cas Systems/genetics ; Clavibacter ; Flucytosine ; *Gene Editing ; },
abstract = {Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 μg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.},
}
@article {pmid34542728,
year = {2021},
author = {Wang, Y and Li, J and Li, S and Zhu, X and Wang, X and Huang, J and Yang, X and Tai, J},
title = {LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test.},
journal = {Mikrochimica acta},
volume = {188},
number = {10},
pages = {347},
pmid = {34542728},
issn = {1436-5073},
mesh = {Bacterial Proteins/*genetics ; Biosensing Techniques ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; DNA, Bacterial ; Endodeoxyribonucleases/*genetics ; Fluorescence ; Humans ; Molecular Diagnostic Techniques ; Mycobacterium/genetics/*isolation &amp; purification ; Nucleic Acid Amplification Techniques ; Sputum/*microbiology ; },
abstract = {A CRISPR-based nucleic acid detection platform, termed LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic) has been developed. In the LACD system, the core primer used in conventional LAMP (forward inner primer or backward inner primer) was engineered to contain a PAM (protospacer adjacent motif) site (TTTT) at the linker region. As a result, the LAMP amplicons contained a specific PAM site for CRISPR-Cas12a recognition. At the CRISPR-mediated detection stage, the resulting LAMP products can activate the corresponding CRISPR-Cas12a effector upon the formation of the CRISPR-Cas12a/gRNA/target DNA complex. The single-strand DNA (ssDNA) reporter molecules are then rapidly cleaved due to the CRISPR-Cas12a's trans-enzyme activity. The ssDNA degradation can then be visualized on a lateral flow biosensor or measured by a real-time fluorescence instrument. Our LACD assay allows any target sequence to be detected (even targets which do not contain any PAM sites) as long as they met the design requirement for LAMP. The feasibility of the LACD methodology for nucleic acid detection was validated on the Mycobacterium tuberculosis complex (MTC). This proof-of-concept assay can be reconfigured to detect a variety of target sequences by redesigning the engineered LAMP primers.},
}
@article {pmid34542686,
year = {2021},
author = {Gupta, R and Kazi, TA and Dey, D and Ghosh, A and Ravichandiran, V and Swarnakar, S and Roy, S and Biswas, SR and Ghosh, D},
title = {CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout.},
journal = {Applied microbiology and biotechnology},
volume = {105},
number = {20},
pages = {7593-7605},
pmid = {34542686},
issn = {1432-0614},
mesh = {Antiviral Agents ; *COVID-19 ; CRISPR-Cas Systems ; Humans ; RNA, Viral ; Real-Time Polymerase Chain Reaction ; *SARS-CoV-2 ; },
abstract = {The emergence of SARS-CoV-2 has brought the world to a standstill, and till date, effective treatments and diagnostics against this idiosyncratic pathogen are lacking. As compared to the standard WHO/CDC qPCR detection method, which consumes several hours for detection, CRISPR-based SHERLOCK, DETECTR, and FELUDA have emerged as rapid diagnostic tools for the detection of the RNA genome of SARS-CoV-2 within an hour with 100% accuracy, specificity, and sensitivity. These attributes of CRISPR-based detection technologies have taken themselves one step ahead of available detection systems and are emerging as an inevitable tool for quick detection of the virus. Further, the discovery of Cas13s nucleases and their orthologs has opened a new corridor for exploitation of Cas13s as an antiviral therapy against SARS-CoV-2 and other viral diseases. One such approach is Prophylactic Antiviral CRISPR in huMAN cells (PACMAN), which needs a long haul to bring into therapy. The approval of SHERLOCK as the first CRISPR-based SARS-CoV-2 test kit by the FDA, for emergency diagnosis of COVID-19 patients, has given positive hope to scientists that sooner human trials of CRISPR-based therapy will be ratified. In this review, we have extensively reviewed the present CRISPR-based approaches, challenges, and future prospects in the light of diagnostics and therapeutics against SARS-CoV-2. KEY POINTS: • The discovery of Cas12 and Cas13 siblings allowed scientists to detect the viral genes. • Cas13d's identification aided scientists in precisely cleaving the SARS-CoV-2 ssRNA. • CRISPR-Cas system acts as "molecular detector and antiviral proctor."},
}
@article {pmid34542605,
year = {2021},
author = {Wang, L and Astone, M and Alam, SK and Zhu, Z and Pei, W and Frank, DA and Burgess, SM and Hoeppner, LH},
title = {Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability.},
journal = {Disease models &amp; mechanisms},
volume = {14},
number = {11},
pages = {},
pmid = {34542605},
issn = {1754-8411},
mesh = {Animals ; CRISPR-Cas Systems ; *Capillary Permeability ; Endothelium, Vascular/*metabolism ; Humans ; Intercellular Adhesion Molecule-1/metabolism ; Janus Kinase 2/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Phosphorylation ; STAT3 Transcription Factor/*genetics/metabolism ; Signal Transduction ; Vascular Endothelial Growth Factor A/*metabolism ; Zebrafish ; },
abstract = {Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, including cancer, stroke, cardiovascular disease, retinal conditions and COVID-19-associated pulmonary edema, sepsis and acute lung injury. Understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics to inhibit vascular permeability, while preserving tissue-restorative angiogenesis. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. We show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9-generated Stat3 knockout zebrafish. Intercellular adhesion molecule 1 (ICAM-1) expression is transcriptionally regulated by STAT3, and VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse and human endothelium. Collectively, our findings suggest that VEGF/VEGFR-2/JAK2/STAT3 signaling regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability. This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid34542533,
year = {2021},
author = {Šafarič Tepeš, P and Sordella, R},
title = {A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {175},
pages = {},
doi = {10.3791/60685},
pmid = {34542533},
issn = {1940-087X},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Endonucleases ; Gene Editing ; Genome ; Mutagenesis, Site-Directed ; },
abstract = {The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.},
}
@article {pmid34542296,
year = {2021},
author = {Nouri, R and Jiang, Y and Tang, Z and Lian, XL and Guan, W},
title = {Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores.},
journal = {Nano letters},
volume = {21},
number = {19},
pages = {8393-8400},
pmid = {34542296},
issn = {1530-6992},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; *Nanopores ; Nucleic Acid Amplification Techniques ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/μL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.},
}
@article {pmid34541453,
year = {2021},
author = {Liu, W and Li, L and Jiang, J and Wu, M and Lin, P},
title = {Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics.},
journal = {Precision clinical medicine},
volume = {4},
number = {3},
pages = {179-191},
pmid = {34541453},
issn = {2516-1571},
support = {R01 AI138203/AI/NIAID NIH HHS/United States ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) are efficient tools for targeting specific genes for laboratory research, agricultural engineering, biotechnology, and human disease treatment. Cas9, by far the most extensively used gene-editing nuclease, has shown great promise for the treatment of hereditary diseases, viral infection, cancers, and so on. Recent reports have revealed that some other types of CRISPR-Cas systems may also have surprising potential to join the fray as gene-editing tools for various applications. Despite the rapid progress in basic research and clinical tests, some underlying problems present continuous, significant challenges, such as editing efficiency, relative difficulty in delivery, off-target effects, immunogenicity, etc. This article summarizes the applications of CRISPR-Cas from bench to bedside and highlights the current obstacles that may limit the usage of CRISPR-Cas systems as gene-editing toolkits in precision medicine and offer some viewpoints that may help to tackle these challenges and facilitate technical development. CRISPR-Cas systems, as a powerful gene-editing approach, will offer great hopes in clinical treatments for many individuals with currently incurable diseases.},
}
@article {pmid34538022,
year = {2022},
author = {Carver, J and Kern, M and Ko, P and Greenwood-Goodwin, M and Yu, XC and Duan, D and Tang, D and Misaghi, S and Auslaender, S and Haley, B and Yuk, IH and Shen, A},
title = {A ribonucleoprotein-based decaplex CRISPR/Cas9 knockout strategy for CHO host engineering.},
journal = {Biotechnology progress},
volume = {38},
number = {1},
pages = {e3212},
doi = {10.1002/btpr.3212},
pmid = {34538022},
issn = {1520-6033},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; *RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Chinese hamster ovary (CHO) cell engineering based on CRISPR/Cas9 knockout (KO) technology requires the delivery of guide RNA (gRNA) and Cas9 enzyme for efficient gene targeting. With an ever-increasing list of promising gene targets, developing, and optimizing a multiplex gene KO protocol is crucial for rapid CHO cell engineering. Here, we describe a method that can support efficient targeting and KO of up to 10 genes through sequential transfections. This method utilizes Cas9 protein to first screen multiple synthetic gRNAs per gene, followed by Sanger sequencing indel analysis, to identify effective gRNA sequences. Using sequential transfections of these potent gRNAs led to the isolation of single cell clones with the targeted deletion of all 10 genes (as confirmed by Sanger sequencing at the DNA level and mass spectrometry at the protein level). Screening 704 single cell clones yielded 6 clones in which all 10 genes were deleted through sequential transfections, demonstrating the success of this decaplex gene editing strategy. This pragmatic approach substantially reduces the time and effort required to generate multiple gene knockouts in CHO cells.},
}
@article {pmid34537403,
year = {2022},
author = {Ricroch, AE and Martin-Laffon, J and Rault, B and Pallares, VC and Kuntz, M},
title = {Next biotechnological plants for addressing global challenges: The contribution of transgenesis and new breeding techniques.},
journal = {New biotechnology},
volume = {66},
number = {},
pages = {25-35},
doi = {10.1016/j.nbt.2021.09.001},
pmid = {34537403},
issn = {1876-4347},
mesh = {Biotechnology ; CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; Genome, Plant ; *Plant Breeding ; *Plants, Genetically Modified/genetics ; Recombinant Proteins/biosynthesis ; Vaccines/biosynthesis ; },
abstract = {The aim of this survey is to identify and characterize new products in plant biotechnology since 2015, especially in relation to the advent of New Breeding Techniques (NBTs) such as gene editing based on the CRISPR-Cas system. Transgenic (gene transfer or gene silencing) and gene edited traits which are approved or marketed in at least one country, or which have a non-regulated status in the USA, are collected, as well as related patents worldwide. In addition, to shed light on potential innovation for Africa, field trials on the continent are examined. The compiled data are classified in application categories, including agronomic improvements, industrial use and medical use, namely production of recombinant therapeutic molecules or vaccines (including against Covid-19). The data indicate that gene editing appears to be an effective complement to 'classical' transgenesis, the use of which is not declining, rather than a replacement, a trend also observed in the patenting landscape. Nevertheless, increased use of gene editing is apparent. Compared to transgenesis, gene editing has increased the proportion of some crop species and decreased others amongst approved, non-regulated or marketed products. A similar differential trend is observed for breeding traits. Gene editing has also favored the emergence of new private companies. China, and prevalently its public sector, overwhelmingly dominates the patenting landscape, but not the approved/marketed one, which is dominated by the USA. The data point in the direction that regulatory environments will favor or discourage innovation.},
}
@article {pmid34537287,
year = {2021},
author = {Deligianni, E and Kiamos, IS},
title = {Gene editing in Plasmodium berghei made easy: Development of a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation.},
journal = {Molecular and biochemical parasitology},
volume = {246},
number = {},
pages = {111415},
doi = {10.1016/j.molbiopara.2021.111415},
pmid = {34537287},
issn = {1872-9428},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Plasmids ; Plasmodium berghei/genetics ; *RNA, Catalytic/genetics ; },
abstract = {Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600-700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.},
}
@article {pmid34536041,
year = {2021},
author = {Pu, Y and Yin, H and Dong, C and Xiang, H and Wu, W and Zhou, B and Du, D and Chen, Y and Xu, H},
title = {Sono-Controllable and ROS-Sensitive CRISPR-Cas9 Genome Editing for Augmented/Synergistic Ultrasound Tumor Nanotherapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {33},
number = {45},
pages = {e2104641},
doi = {10.1002/adma.202104641},
pmid = {34536041},
issn = {1521-4095},
mesh = {Animals ; Antineoplastic Agents/*chemistry/pharmacology/therapeutic use ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; DNA Repair Enzymes/genetics/metabolism ; Gene Editing/*methods ; Humans ; Metal-Organic Frameworks/chemistry ; Mice ; Mice, Nude ; Nanoparticles/chemistry ; Neoplasms/pathology/therapy ; Phosphoric Monoester Hydrolases/genetics/metabolism ; Polymers/chemistry ; Porphyrins/chemistry ; RNA, Guide/chemistry/metabolism ; Reactive Oxygen Species/*metabolism ; Transplantation, Heterologous ; Ultrasonic Therapy/*methods ; },
abstract = {The potential of the cluster regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9)-based therapeutic genome editing is severely hampered by the difficulties in precise regulation of the in vivo activity of the CRISPR-Cas9 system. Herein, sono-controllable and reactive oxygen species (ROS)-sensitive sonosensitizer-integrated metal-organic frameworks (MOFs), denoted as P/M@CasMTH1, are developed for augmented sonodynamic therapy (SDT) efficacy using the genome-editing technology. P/M@CasMTH1 nanoparticles comprise singlet oxygen ([1] O2)-generating MOF structures anchored with CRISPR-Cas9 systems via [1] O2 -cleavable linkers, which serve not only as a delivery vector of CRISPR-Cas9 targeting MTH1, but also as a sonoregulator to spatiotemporally activate the genome editing. P/M@CasMTH1 escapes from the lysosomes, harvests the ultrasound (US) energy and converts it into abundant [1] O2 to induce SDT. The generated ROS subsequently trigger cleavage of ROS-responsive thioether bonds, thus inducing controllable release of the CRISPR-Cas9 system and initiation of genome editing. The genomic disruption of MTH1 conspicuously augments the therapeutic efficacy of SDT by destroying the self-defense system in tumor cells, thereby causing cellular apoptosis and tumor suppression. This therapeutic strategy for synergistic MTH1 disruption and abundant [1] O2 generation provides a paradigm for augmenting SDT efficacy based on the emerging nanomedicine-enabled genome-editing technology.},
}
@article {pmid34535663,
year = {2021},
author = {Gu, P and Jia, S and Takasugi, T and Tesmer, VM and Nandakumar, J and Chen, Y and Chang, S},
title = {Distinct functions of POT1 proteins contribute to the regulation of telomerase recruitment to telomeres.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5514},
pmid = {34535663},
issn = {2041-1723},
support = {R01 CA202816/CA/NCI NIH HHS/United States ; R01 AG050509/AG/NIA NIH HHS/United States ; R01 GM120094/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Mutational Analysis ; DNA-Binding Proteins/*metabolism ; Mice ; Protein Binding ; Rad51 Recombinase/metabolism ; Sarcoma/pathology ; Telomerase/*metabolism ; Telomere/*metabolism ; Telomere-Binding Proteins/*metabolism ; },
abstract = {Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.},
}
@article {pmid34535662,
year = {2021},
author = {Beumer, J and Geurts, MH and Lamers, MM and Puschhof, J and Zhang, J and van der Vaart, J and Mykytyn, AZ and Breugem, TI and Riesebosch, S and Schipper, D and van den Doel, PB and de Lau, W and Pleguezuelos-Manzano, C and Busslinger, G and Haagmans, BL and Clevers, H},
title = {A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5498},
pmid = {34535662},
issn = {2041-1723},
mesh = {Angiotensin-Converting Enzyme 2/*genetics ; *Biological Specimen Banks ; COVID-19 ; *CRISPR-Cas Systems ; Cell Line ; *Coronavirus ; Dipeptidyl Peptidase 4/*genetics ; Humans ; Middle East Respiratory Syndrome Coronavirus ; Organoids/*metabolism ; SARS-CoV-2 ; Serine Endopeptidases/*genetics ; Transcriptome ; Virus Replication ; },
abstract = {Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.},
}
@article {pmid34535658,
year = {2021},
author = {Blombach, F and Fouqueau, T and Matelska, D and Smollett, K and Werner, F},
title = {Promoter-proximal elongation regulates transcription in archaea.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5524},
pmid = {34535658},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; WT 207446/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA/metabolism ; DNA-Directed RNA Polymerases/metabolism ; Oxidative Stress/genetics ; *Promoter Regions, Genetic ; Regression Analysis ; Sulfolobus solfataricus/*genetics/growth &amp; development ; *Transcription Elongation, Genetic ; },
abstract = {Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.},
}
@article {pmid34535635,
year = {2021},
author = {He, L and Wang, S and Peng, L and Zhao, H and Li, S and Han, X and Habimana, JD and Chen, Z and Wang, C and Peng, Y and Peng, H and Xie, Y and Lei, L and Deng, Q and Wan, L and Wan, N and Yuan, H and Gong, Y and Zou, G and Li, Z and Tang, B and Jiang, H},
title = {CRISPR/Cas9 mediated gene correction ameliorates abnormal phenotypes in spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cells.},
journal = {Translational psychiatry},
volume = {11},
number = {1},
pages = {479},
pmid = {34535635},
issn = {2158-3188},
mesh = {CRISPR-Cas Systems ; Humans ; *Induced Pluripotent Stem Cells ; *Machado-Joseph Disease/genetics ; *Neurodegenerative Diseases ; Phenotype ; },
abstract = {Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3. The accumulation of the mutant ataxin-3 proteins carrying expanded polyglutamine (polyQ) leads to selective degeneration of neurons. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies have been identified, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3. Induced pluripotent stem cells (iPSCs) can be used as the ideal cell model for the molecular pathogenesis of polyQ diseases. Abnormal CAG expansions mediated by CRISPR/Cas9 genome engineering technologies have shown promising potential for the treatment of polyQ diseases, including SCA3. In this study, SCA3-iPSCs can be corrected by the replacement of the abnormal CAG expansions (74 CAG) with normal repeats (17 CAG) using CRISPR/Cas9-mediated homologous recombination (HR) strategy. Besides, corrected SCA3-iPSCs retained pluripotent and normal karyotype, which can be differentiated into a neural stem cell (NSCs) and neuronal cells, and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the neuronal differentiation from normal control iPSCs (Ctrl-iPSCs), SCA3-iPSCs, and isogenic control SCA3-iPSCs. Furthermore, this study proved that the phenotypic abnormalities in SCA3 neurons, including aggregated IC2-polyQ protein, decreased mitochondrial membrane potential (MMP) and glutathione expressions, increased reactive oxygen species (ROS), intracellular Ca[2+] concentrations, and lipid peroxidase malondialdehyde (MDA) levels, all were rescued in the corrected SCA3-NCs. For the first time, this study demonstrated the feasibility of CRISPR/Cas9-mediated HR strategy to precisely repair SCA3-iPSCs, and reverse the corresponding abnormal disease phenotypes. In addition, the importance of genetic control using CRISPR/Cas9-mediated iPSCs for disease modeling. Our work may contribute to providing a potential ideal model for molecular mechanism research and autologous stem cell therapy of SCA3 or other polyQ diseases, and offer a good gene therapy strategy for future treatment.},
}
@article {pmid34534948,
year = {2021},
author = {van Dongen, JE and Berendsen, JTW and Eijkel, JCT and Segerink, LI},
title = {A CRISPR/Cas12a-assisted in vitro diagnostic tool for identification and quantification of single CpG methylation sites.},
journal = {Biosensors &amp; bioelectronics},
volume = {194},
number = {},
pages = {113624},
doi = {10.1016/j.bios.2021.113624},
pmid = {34534948},
issn = {1873-4235},
mesh = {Bacterial Proteins ; *Biosensing Techniques ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems/genetics ; CpG Islands ; DNA Cleavage ; *DNA Methylation ; Endodeoxyribonucleases ; Endonucleases/metabolism ; },
abstract = {The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a-MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.},
}
@article {pmid34534448,
year = {2021},
author = {He, L and Ding, Y and Zhao, Y and So, KK and Peng, XL and Li, Y and Yuan, J and He, Z and Chen, X and Sun, H and Wang, H},
title = {CRISPR/Cas9/AAV9-mediated in vivo editing identifies MYC regulation of 3D genome in skeletal muscle stem cell.},
journal = {Stem cell reports},
volume = {16},
number = {10},
pages = {2442-2458},
pmid = {34534448},
issn = {2213-6711},
mesh = {Animals ; CRISPR-Cas Systems ; Chromatin/*metabolism ; Gene Editing/methods ; Gene Expression Regulation ; *Genes, myc ; *Genome ; Mice ; MyoD Protein/genetics/*metabolism ; Nucleic Acid Conformation ; Proto-Oncogene Proteins c-bcl-6/genetics/*metabolism ; RNA, Guide/genetics/*metabolism ; Satellite Cells, Skeletal Muscle/*physiology ; Transcription Factors/genetics/metabolism ; },
abstract = {Skeletal muscle satellite cells (SCs) are stem cells responsible for muscle development and regeneration. Although CRISPR/Cas9 has been widely used, its application in endogenous SCs remains elusive. Here, we generate mice expressing Cas9 in SCs and achieve robust editing in juvenile SCs at the postnatal stage through AAV9-mediated short guide RNA (sgRNA) delivery. Additionally, we reveal that quiescent SCs are resistant to CRISPR/Cas9-mediated editing. As a proof of concept, we demonstrate efficient editing of master transcription factor (TF) Myod1 locus using the CRISPR/Cas9/AAV9-sgRNA system in juvenile SCs. Application on two key TFs, MYC and BCL6, unveils distinct functions in SC activation and muscle regeneration. Particularly, we reveal that MYC orchestrates SC activation through regulating 3D genome architecture. Its depletion results in strengthening of the topologically associating domain boundaries thus may affect gene expression. Altogether, our study establishes a platform for editing endogenous SCs that can be harnessed to elucidate the functionality of key regulators governing SC activities.},
}
@article {pmid34533940,
year = {2021},
author = {Lv, Z and Wang, Q and Yang, M},
title = {Multivalent Duplexed-Aptamer Networks Regulated a CRISPR-Cas12a System for Circulating Tumor Cell Detection.},
journal = {Analytical chemistry},
volume = {93},
number = {38},
pages = {12921-12929},
doi = {10.1021/acs.analchem.1c02228},
pmid = {34533940},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA ; Humans ; Immunomagnetic Separation ; *Neoplastic Cells, Circulating ; Oligonucleotides ; },
abstract = {Although circulating tumor cells (CTCs) have great potential to act as the mini-invasive liquid biopsy cancer biomarker, a rapid and sensitive CTC detection method remains lacking. CRISPR-Cas12a has recently emerged as a promising tool in biosensing applications with the characteristic of fast detection, easy operation, and high sensitivity. Herein, we reported a CRISPR-Cas12a-based CTC detection sensor that is regulated by the multivalent duplexed-aptamer networks (MDANs). MDANs were synthesized on a magnetic bead surface by rolling circle amplification (RCA), which contain multiple duplexed-aptamer units that allow structure switching induced by cell-binding events. The presence of target cells can trigger the release of free "activator DNA" from the MDANs structure to activate the downstream CRISPR-Cas12a for signal amplification. Furthermore, the 3D DNA network formed by RCA products also provided significantly higher sensitivity than the monovalent aptamer. As a proof-of-concept study, we chose the most widely used sgc8 aptamer that specifically recognizes CCRF-CEM cells to validate the proposed approach. The MDANs-Cas12a system could afford a simple and fast CTC detection workflow with a detection limit of 26 cells mL[-1]. We also demonstrated that the MDANs-Cas12a could directly detect the CTCs in human blood samples, indicating a great potential of the MDANs-Cas12a in clinical CTC-based liquid biopsy.},
}
@article {pmid34533605,
year = {2022},
author = {Pulido-Quetglas, C and Johnson, R},
title = {Designing libraries for pooled CRISPR functional screens of long noncoding RNAs.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {33},
number = {2},
pages = {312-327},
pmid = {34533605},
issn = {1432-1777},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Library ; Genome ; Humans ; RNA, Guide/genetics ; *RNA, Long Noncoding/genetics ; },
abstract = {Human and other genomes encode tens of thousands of long noncoding RNAs (lncRNAs), the vast majority of which remain uncharacterised. High-throughput functional screening methods, notably those based on pooled CRISPR-Cas perturbations, promise to unlock the biological significance and biomedical potential of lncRNAs. Such screens are based on libraries of single guide RNAs (sgRNAs) whose design is critical for success. Few off-the-shelf libraries are presently available, and lncRNAs tend to have cell-type-specific expression profiles, meaning that library design remains in the hands of researchers. Here we introduce the topic of pooled CRISPR screens for lncRNAs and guide readers through the three key steps of library design: accurate annotation of transcript structures, curation of optimal candidate sets, and design of sgRNAs. This review is a starting point and reference for researchers seeking to design custom CRISPR screening libraries for lncRNAs.},
}
@article {pmid34532823,
year = {2021},
author = {Xiao, G and Fu, X and Zhang, J and Liu, S and Wang, Z and Ye, T and Zhang, G},
title = {Rapid and cost-effective screening of CRISPR/Cas9-induced mutants by DNA-guided Argonaute nuclease.},
journal = {Biotechnology letters},
volume = {43},
number = {11},
pages = {2105-2110},
pmid = {34532823},
issn = {1573-6776},
mesh = {Animals ; Archaeal Proteins/genetics/metabolism ; *Argonaute Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; *Gene Editing/economics/methods ; INDEL Mutation/*genetics ; Pyrococcus furiosus/enzymology/genetics ; },
abstract = {OBJECTIVE: With the widespread application of CRISPR/Cas9 gene editing technology, new methods are needed to screen mutants quickly and effectively. Here, we aimed to develop a simple and cost-effective method to screen CRISPR/Cas9-induced mutants.<br><br>RESULT: We report a novel method to identify CRISPR/Cas9-induced mutants through a DNA-guided Argonaute nuclease derived from the archaeon Pyrococcus furiosus. We demonstrated that the Pyrococcus furiosus Argonaute (PfAgo)-based method could distinguish among biallelic mutants, monoallelic mutants and wild type (WT). Furthermore, this method was able to identify 1&#xa0;bp indel mutations.<br><br>CONCLUSION: The PfAgo-based method is simple to implement and can be applied to screen biallelic mutants and mosaic mutants generated by CRISPR-Cas9 or other kinds of gene editing tools.},
}
@article {pmid34531512,
year = {2021},
author = {Liu, T and Huang, Y and Jiang, L and Dong, C and Gou, Y and Lian, J},
title = {Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {1089},
pmid = {34531512},
issn = {2399-3642},
mesh = {Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Indole Alkaloids/*metabolism ; *Metabolic Engineering ; Quinolines/*metabolism ; Saccharomyces cerevisiae/genetics/*metabolism ; Vinblastine/*analogs &amp; derivatives/biosynthesis ; },
abstract = {Vindoline is a plant derived monoterpene indole alkaloid (MIA) with potential therapeutic applications and more importantly serves as the precursor to vinblastine and vincristine. To obtain a yeast strain for high yield production of vindoline from tabersonine, multiple metabolic engineering strategies were employed via the CRISPR/Cas9 mediated multiplex genome integration technology in the present study. Through increasing and tuning the copy numbers of the pathway genes, pairing cytochrome P450 enzymes (CYPs) with appropriate cytochrome P450 reductases (CPRs), engineering the microenvironment for functional expression of CYPs, enhancing cofactor supply, and optimizing fermentation conditions, the production of vindoline was increased to a final titer as high as ∼16.5 mg/L, which is more than 3,800,000-fold higher than the parent strain and the highest tabersonine to vindoline conversion yield ever reported. This work represents a key step of the engineering efforts to establish de novo biosynthetic pathways for vindoline, vinblastine, and vincristine.},
}
@article {pmid34531479,
year = {2021},
author = {Nishi, T and Shinzawa, N and Yuda, M and Iwanaga, S},
title = {Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {18501},
pmid = {34531479},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Malaria, Falciparum/genetics ; Mutation ; Plasmodium falciparum/*genetics ; },
abstract = {The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.},
}
@article {pmid34531330,
year = {2022},
author = {Willsey, HR and Guille, M and Grainger, RM},
title = {Modeling Human Genetic Disorders with CRISPR Technologies in Xenopus.},
journal = {Cold Spring Harbor protocols},
volume = {2022},
number = {3},
pages = {},
doi = {10.1101/pdb.prot106997},
pmid = {34531330},
issn = {1559-6095},
support = {BB/R014841/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 212942/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; U01 MH115747/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Xenopus laevis/genetics ; },
abstract = {Combining the power of Xenopus developmental biology with CRISPR-based technologies promises great discoveries in understanding and treating human genetic disorders. Here we provide a practical pipeline for how to go from known disease gene(s) or risk gene(s) of interest to methods for gaining functional insight into the contribution of these genes to disorder etiology in humans.},
}
@article {pmid34531254,
year = {2022},
author = {Lin, S and Larrue, C and Scheidegger, NK and Seong, BKA and Dharia, NV and Kuljanin, M and Wechsler, CS and Kugener, G and Robichaud, AL and Conway, AS and Mashaka, T and Mouche, S and Adane, B and Ryan, JA and Mancias, JD and Younger, ST and Piccioni, F and Lee, LH and Wunderlich, M and Letai, A and Tamburini, J and Stegmaier, K},
title = {An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML.},
journal = {Cancer discovery},
volume = {12},
number = {2},
pages = {432-449},
pmid = {34531254},
issn = {2159-8290},
support = {K99 CA263161/CA/NCI NIH HHS/United States ; P50 CA206963/CA/NCI NIH HHS/United States ; R35 CA210030/CA/NCI NIH HHS/United States ; R50 CA211404/CA/NCI NIH HHS/United States ; S10 OD023410/OD/NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*therapeutic use ; *CRISPR-Cas Systems ; Humans ; Leukemia, Myeloid, Acute/*drug therapy/genetics ; *Precision Medicine ; *Xenograft Model Antitumor Assays ; },
abstract = {CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275.},
}
@article {pmid34530641,
year = {2021},
author = {Zhang, H and Li, T and Sun, Y and Yang, H},
title = {Perfecting Targeting in CRISPR.},
journal = {Annual review of genetics},
volume = {55},
number = {},
pages = {453-477},
doi = {10.1146/annurev-genet-071719-030438},
pmid = {34530641},
issn = {1545-2948},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing ; Genome/genetics ; Transcriptome ; },
abstract = {CRISPR-based genome editing holds promise for genome engineering and other applications in diverse organisms. Defining and improving the genome-wide and transcriptome-wide specificities of these editing tools are essential for realizing their full potential in basic research and biomedical therapeutics. This review provides an overview of CRISPR-based DNA- and RNA-editing technologies, methods to quantify their specificities, and key solutions to reduce off-target effects for research and improve therapeutic applications.},
}
@article {pmid34529907,
year = {2022},
author = {Zeng, XX and Zeng, J and Zhu, B},
title = {Future generation of combined multimodal approach to treat brain glioblastoma multiforme and potential impact on micturition control.},
journal = {Reviews in the neurosciences},
volume = {33},
number = {3},
pages = {313-326},
doi = {10.1515/revneuro-2021-0068},
pmid = {34529907},
issn = {2191-0200},
mesh = {Brain/metabolism ; *Glioblastoma/genetics/therapy ; Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; Social Responsibility ; T-Lymphocytes ; Urination ; },
abstract = {Glioblastoma remains lethal even when treated with standard therapy. This review aims to outline the recent development of various advanced therapeutics for glioblastoma and briefly discuss the potential impact of glioblastoma and some of its therapeutic approaches on the neurological function micturition control. Although immunotherapy led to success in treating hematological malignancies, but no similar success occurred in treatment for brain glioblastoma. Neither regenerative medicine nor stem cell therapy led to astounding success in glioblastoma. However, CRISPR Cas system holds potential in multiple applications due to its capacity to knock-in and knock-out genes, modify immune cells and cell receptors, which will enable it to address clinical challenges in immunotherapy such as CAR-T and regenerative therapy for brain glioblastoma, improving the precision and safety of these approaches. The studies mentioned in this review could indicate that glioblastoma is a malignant disease with multiple sophisticated barriers to be overcome and more challenges might arise in the attempt of researchers to yield a successful cure. A multimodal approach of future generation of refined and safe therapeutics derived from CRISPR Cas therapeutics, immunotherapy, and regenerative therapeutics mentioned in this review might prolong survival or even contribute towards a potential cure for glioblastoma.},
}
@article {pmid34529719,
year = {2021},
author = {Kim, DV and Kulishova, LM and Torgasheva, NA and Melentyev, VS and Dianov, GL and Medvedev, SP and Zakian, SM and Zharkov, DO},
title = {Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line.},
journal = {PloS one},
volume = {16},
number = {9},
pages = {e0257473},
pmid = {34529719},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints ; *DNA Repair/drug effects ; DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry/*genetics/metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Hydrogen Peroxide/chemistry ; Methyl Methanesulfonate/pharmacology ; Phenotype ; RNA, Guide/metabolism ; },
abstract = {The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein's catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5-2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.},
}
@article {pmid34529489,
year = {2021},
author = {Martin, TD and Patel, RS and Cook, DR and Choi, MY and Patil, A and Liang, AC and Li, MZ and Haigis, KM and Elledge, SJ},
title = {The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6561},
pages = {1327-1335},
doi = {10.1126/science.abg5784},
pmid = {34529489},
issn = {1095-9203},
support = {U01 CA199252/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Carcinogenesis ; Cell Line, Tumor ; Chemokine CCL2/metabolism ; Female ; GTP-Binding Protein alpha Subunits, G12-G13/genetics/metabolism ; *Gene Silencing ; *Genes, Tumor Suppressor ; Humans ; *Immune Evasion/genetics ; Mammary Neoplasms, Experimental/genetics/immunology/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental/*genetics/*immunology/pathology ; Selection, Genetic ; Tumor Microenvironment ; },
abstract = {During tumorigenesis, tumors must evolve to evade the immune system and do so by disrupting the genes involved in antigen processing and presentation or up-regulating inhibitory immune checkpoint genes. We performed in vivo CRISPR screens in syngeneic mouse tumor models to examine requirements for tumorigenesis both with and without adaptive immune selective pressure. In each tumor type tested, we found a marked enrichment for the loss of tumor suppressor genes (TSGs) in the presence of an adaptive immune system relative to immunocompromised mice. Nearly one-third of TSGs showed preferential enrichment, often in a cancer- and tissue-specific manner. These results suggest that clonal selection of recurrent mutations found in cancer is driven largely by the tumor’s requirement to avoid the adaptive immune system.},
}
@article {pmid34529358,
year = {2021},
author = {Huang, RS and Lai, MC and Lin, S},
title = {Ex Vivo Expansion and CRISPR-Cas9 Genome Editing of Primary Human Natural Killer Cells.},
journal = {Current protocols},
volume = {1},
number = {9},
pages = {e246},
doi = {10.1002/cpz1.246},
pmid = {34529358},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Knockout Techniques ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; },
abstract = {Natural killer (NK) cells are potent innate immune cells that provide the surveillance and elimination of infected, stressed, and malignant cells. The unique immune recognition mechanisms and functions of NK cells make them an attractive cell type for immunology research and adoptive immunotherapy. However, primary NK cells are challenging to culture ex vivo and lack efficient genetic tools, hindering the research of NK cells and the development of NK cell therapeutics. Here we describe methods for the freeze-thaw process, feeder-free ex vivo expansion, CRISPR-Cas9 genome editing, and functional characterizations of primary human NK cells. Our protocol enables ∼30-fold and ∼2000-fold average expansion rates from 1 × 10[7] cryopreserved NK cells in 14 and 28 days, respectively. We also detail methods for CRISPR gene knockout and knockin by nucleofection of Cas9 ribonucleoproteins (RNP) and DNA repair templates. Gene knockout by Cas9 RNP nucleofection can be multiplexed to simultaneously target three genes. The CRISPR-edited cells can be cryopreserved and rethawed with high viability for future studies. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Thawing of natural killer cells Basic Protocol 2: Ex vivo expansion of natural killer cells Basic Protocol 3: Cryopreservation of expanded natural killer cells Basic Protocol 4: Characterization of natural killer cells: Flow cytometry and surface marker analysis Basic Protocol 5: Cytotoxicity and degranulation assays Basic Protocol 6: Preparation of homology-directed repair templates Basic Protocol 7: Nucleofection of CRISPR-Cas9 ribonucleoproteins Basic Protocol 8: Genotyping of gene-edited natural killer cells Basic Protocol 9: Phenotyping of gene-edited natural killer cells.},
}
@article {pmid34528946,
year = {2021},
author = {Balachandran, YL and Li, X and Jiang, X},
title = {Biodegradable freestanding rare-earth nanosheets promote multimodal imaging and delivers CRISPR-Cas9 plasmid against tumor.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {74},
pages = {9386-9389},
doi = {10.1039/d1cc03228c},
pmid = {34528946},
issn = {1364-548X},
mesh = {Biocompatible Materials/chemistry/metabolism/*pharmacology ; CRISPR-Cas Systems/drug effects ; *Drug Delivery Systems ; Humans ; Metals, Rare Earth/chemistry/metabolism/*pharmacology ; *Multimodal Imaging ; Nanostructures/*chemistry ; Neoplasms/diagnostic imaging/*drug therapy ; },
abstract = {Designing nanomaterials for bio-imaging and drug delivery for advanced cancer therapy with biodegradability and biocompatibility is a promising but challenging frontier. Herein, we assembled biodegradable and biocompatible ultrathin rare-earth erbium/dysprosium nanosheets that improve contrast in multimodal bio-imaging settings (MRI and X-ray CT) and deliver CRISPR-Cas9 plasmid to treat tumors.},
}
@article {pmid34528710,
year = {2022},
author = {Rogava, M and Braun, AD and van der Sluis, TC and Shridhar, N and Tüting, T and Gaffal, E},
title = {Tumor cell intrinsic Toll-like receptor 4 signaling promotes melanoma progression and metastatic dissemination.},
journal = {International journal of cancer},
volume = {150},
number = {1},
pages = {142-151},
doi = {10.1002/ijc.33804},
pmid = {34528710},
issn = {1097-0215},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; *Cell Movement ; Humans ; Lung Neoplasms/genetics/metabolism/*secondary ; Melanoma/genetics/metabolism/*pathology ; Mice ; Mice, Inbred C57BL ; Toll-Like Receptor 4/antagonists &amp; inhibitors/genetics/*metabolism ; Tumor Cells, Cultured ; },
abstract = {Most melanoma-associated deaths result from the early development of metastasis. Toll-like receptor 4 (TLR4) expression on nontumor cells is well known to contribute to tumor development and metastatic progression. The role of TLR4 expression on tumor cells however is less well understood. Here we describe TLR4 as a driver of tumor progression and metastatic spread of melanoma cells by employing a transplantable mouse melanoma model. HCmel12 melanoma cells lacking functional TLR4 showed increased sensitivity to tumor necrosis factor α induced cell killing in vitro compared to cells with intact TLR4. Interestingly, TLR4 knockout melanoma cells also showed impaired migratory capacity in vitro and a significantly reduced ability to metastasize to the lungs after subcutaneous transplantation in vivo. Finally, we demonstrate that activation of TLR4 also promotes migration in a subset of human melanoma cell lines. Our work describes TLR4 as an important mediator of melanoma migration and metastasis and provides a rationale for therapeutic inhibition of TLR4 in melanoma.},
}
@article {pmid34528284,
year = {2021},
author = {Targa, A and Larrimore, KE and Wong, CK and Chong, YL and Fung, R and Lee, J and Choi, H and Rancati, G},
title = {Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene.},
journal = {The EMBO journal},
volume = {40},
number = {21},
pages = {e107839},
pmid = {34528284},
issn = {1460-2075},
mesh = {Adaptation, Physiological/*genetics ; *Alleles ; CRISPR-Cas Systems ; Cell Line, Tumor ; G-Protein-Coupled Receptor Kinase 1/*genetics/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Regulatory Networks ; Genes, Reporter ; *Genetic Fitness ; Green Fluorescent Proteins/genetics/metabolism ; HCT116 Cells ; HEK293 Cells ; Haploidy ; Humans ; Karyopherins/genetics/metabolism ; Luminescent Proteins/genetics/metabolism ; Mutation ; Myeloid Cells/metabolism/pathology ; N-Glycosyl Hydrolases/*genetics/metabolism ; Nuclear Pore Complex Proteins/*genetics/metabolism ; Signal Transduction ; Transcriptome ; },
abstract = {Adaptive evolution to cellular stress is a process implicated in a wide range of biological and clinical phenomena. Two major routes of adaptation have been identified: non-genetic changes, which allow expression of different phenotypes in novel environments, and genetic variation achieved by selection of fitter phenotypes. While these processes are broadly accepted, their temporal and epistatic features in the context of cellular evolution and emerging drug resistance are contentious. In this manuscript, we generated hypomorphic alleles of the essential nuclear pore complex (NPC) gene NUP58. By dissecting early and long-term mechanisms of adaptation in independent clones, we observed that early physiological adaptation correlated with transcriptome rewiring and upregulation of genes known to interact with the NPC; long-term adaptation and fitness recovery instead occurred via focal amplification of NUP58 and restoration of mutant protein expression. These data support the concept that early phenotypic plasticity allows later acquisition of genetic adaptations to a specific impairment. We propose this approach as a genetic model to mimic targeted drug therapy in human cells and to dissect mechanisms of adaptation.},
}
@article {pmid34526543,
year = {2021},
author = {da Silva Santos, R and Pinheiro, DP and Teixeira, LPR and Sales, SLA and Dos Santos Luciano, MC and de Lima Melo, MM and Pinheiro, RF and Tavares, KCS and Furtado, GP and Pessoa, C and Furtado, CLM},
title = {CRISPR/Cas9 small promoter deletion in H19 lncRNA is associated with altered cell morphology and proliferation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {18380},
pmid = {34526543},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; Biomarkers, Tumor ; *CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Cycle/genetics ; Cell Proliferation/genetics ; Cytogenetic Analysis ; Gene Editing ; Gene Knockdown Techniques ; Humans ; Mice ; Neoplasms/*genetics/*pathology ; *Promoter Regions, Genetic ; RNA, Long Noncoding/chemistry/*genetics ; *Sequence Deletion ; },
abstract = {The imprinted H19 long non-coding RNA, a knowing oncofetal gene, presents a controversial role during the carcinogenesis process since its tumor suppressor or oncogenic activity is not completely elucidated. Since H19 lncRNA is involved in many biological pathways related to tumorigenesis, we sought to develop a non-cancer lineage with CRISPR-Cas9-mediated H19 knockdown (H19-) and observe the changes in a cellular context. To edit the promoter region of H19, two RNA guides were designed, and the murine C2C12 myoblast cells were transfected. H19 deletion was determined by DNA sequencing and gene expression by qPCR. We observed a small deletion (~ 60 bp) in the promoter region that presented four predicted transcription binding sites. The deletion reduced H19 expression (30%) and resulted in increased proliferative activity, altered morphological patterns including cell size and intracellular granularity, without changes in viability. The increased proliferation rate in the H19- cell seems to facilitate chromosomal abnormalities. The H19- myoblast presented characteristics similar to cancer cells, therefore the H19 lncRNA may be an important gene during the initiation of the tumorigenic process. Due to CRISPR/Cas9 permanent edition, the C2C12 H19- knockdown cells allows functional studies of H19 roles in tumorigenesis, prognosis, metastases, as well as drug resistance and targeted therapy.},
}
@article {pmid34524900,
year = {2021},
author = {Tu, JL and Bai, XY and Xu, YL and Li, N and Xu, JW},
title = {Targeted Gene Insertion and Replacement in the Basidiomycete Ganoderma lucidum by Inactivation of Nonhomologous End Joining Using CRISPR/Cas9.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {23},
pages = {e0151021},
pmid = {34524900},
issn = {1098-5336},
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Genomics ; *Mutagenesis, Insertional ; *Reishi/genetics ; },
abstract = {Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.},
}
@article {pmid34524446,
year = {2022},
author = {Hacker, L and Dorn, A and Enderle, J and Puchta, H},
title = {The repair of topoisomerase 2 cleavage complexes in Arabidopsis.},
journal = {The Plant cell},
volume = {34},
number = {1},
pages = {287-301},
pmid = {34524446},
issn = {1532-298X},
mesh = {Arabidopsis/enzymology/*genetics/metabolism ; Arabidopsis Proteins/*genetics/metabolism ; DNA Topoisomerases/*genetics/metabolism ; },
abstract = {DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.},
}
@article {pmid34524200,
year = {2021},
author = {León, Y and Faherty, CS},
title = {Bacteriophages against enteropathogens: rediscovery and refinement of novel antimicrobial therapeutics.},
journal = {Current opinion in infectious diseases},
volume = {34},
number = {5},
pages = {491-499},
pmid = {34524200},
issn = {1473-6527},
support = {R21 AI146405/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/therapeutic use ; Bacteria ; *Bacteriophages ; CRISPR-Cas Systems ; Dysbiosis/drug therapy ; Humans ; },
abstract = {PURPOSE OF REVIEW: Alarming rates of antibiotic resistance in bacteria and gastrointestinal dysbiosis associated with traditional antimicrobial therapy have led to renewed interests in developing bacteriophages as novel therapeutics. In this review, we highlight some of the recent advances in bacteriophage therapeutic development targeting important enteropathogens of the gastrointestinal tract.<br><br>RECENT FINDINGS: Bacteriophages are viruses that infect bacteria, either to utilize the bacterial machinery to produce new progeny or stably integrate into the bacterial chromosome to ensure maintenance of the viral genome. With recent advances in synthetic biology and the discovery of CRISPR-Cas systems used by bacteria to protect against bacteriophages, novel molecular applications are taking us beyond the discovery of bacteriophages and toward innovative applications, including the targeting of bacterial virulence factors, the use of temperate bacteriophages, and the production of bacteriophage proteins as antimicrobial agents. These technologies offer promise to target enteropathogens without disrupting the healthy microbiota of the gastrointestinal tract. Moreover, the use of nanoparticle technology and other modifications are helping researchers circumvent the harsh gastrointestinal conditions that could limit the efficacy of bacteriophages against enteric pathogens.<br><br>SUMMARY: This era of discovery and development offers significant potential to modify bacteriophages and overcome the global impact of enteropathogens.},
}
@article {pmid34523974,
year = {2021},
author = {Sachla, AJ and Alfonso, AJ and Helmann, JD},
title = {A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.},
journal = {Microbiology spectrum},
volume = {9},
number = {2},
pages = {e0075421},
pmid = {34523974},
issn = {2165-0497},
support = {R35 GM122461/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacillus subtilis/*genetics ; Bacterial Proteins/*genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Plasmids ; RNA, Guide ; Streptococcus pyogenes/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.},
}
@article {pmid34523847,
year = {2021},
author = {Wolthuis, RMF and van de Vrugt, HJ and Cornel, MC},
title = {[CRISPR gene therapy enters the clinic: the future starts now].},
journal = {Nederlands tijdschrift voor geneeskunde},
volume = {165},
number = {},
pages = {},
pmid = {34523847},
issn = {1876-8784},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Female ; *Gene Editing ; Genetic Therapy ; Humans ; },
abstract = {In 2020, the Nobel Prize in Chemistry was awarded to American molecular biologist Jennifer Doudna and her French colleague Emmanuelle Charpentier for their fundamental research on CRISPR, an ingenious bacterial immune system. Studies into the working mechanism of CRISPR led to many Eureka moments. Through smart biotechnological engineering, CRISPR became suitable for applications in 'DNA surgery': the targeted editing of the genetic code. Here, we discuss emerging medical CRISPR applications for the treatment of human genetic disorders, including in vivo therapy. This Nobel Prize-winning discovery is powerful, adaptable and accurate, and clinical trials are being launched at an amazing pace. However, extensive research is needed on safe clinical use and possible side effects of CRISPR. In addition, the regulations on market authorization and reimbursement are not yet tailored to this very personal and potentially expensive therapy. Whereas challenges remain, CRISPR gene therapy will continue to rapidly mature as a clinical reality.},
}
@article {pmid34522785,
year = {2021},
author = {Rao, GS and Jiang, W and Mahfouz, M},
title = {Synthetic directed evolution in plants: unlocking trait engineering and improvement.},
journal = {Synthetic biology (Oxford, England)},
volume = {6},
number = {1},
pages = {ysab025},
pmid = {34522785},
issn = {2397-7000},
abstract = {Genetic variation accelerates adaptation and resilience and enables the survival of species in their changing environment. Increasing the genetic diversity of crop species is essential to improve their yield and enhance food security. Synthetic directed evolution (SDE) employs localized sequence diversification (LSD) of gene sequence and selection pressure to evolve gene variants with better fitness, improved properties and desired phenotypes. Recently, CRISPR-Cas-dependent and -independent technologies have been applied for LSD to mediate synthetic evolution in diverse species, including plants. SDE holds excellent promise to discover, accelerate and expand the range of traits of the value in crop species. Here, we highlight the efficient SDE approaches for the LSD of plant genes, selection strategies and critical traits for targeted improvement. We discuss the potential of emerging technologies, including CRISPR-Cas base editing, retron editing, EvolvR and prime editing, to establish efficient SDE in plants. Moreover, we cover CRISPR-Cas-independent technologies, including T7 polymerase editor for continuous evolution. We highlight the key challenges and potential solutions of applying SDE technologies to improve the plant traits of the value.},
}
@article {pmid34522033,
year = {2021},
author = {Koch, L},
title = {CRISPR systems go mini.},
journal = {Nature reviews. Genetics},
volume = {22},
number = {11},
pages = {690},
pmid = {34522033},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
}
@article {pmid34521928,
year = {2021},
author = {Soblechero-Martín, P and Albiasu-Arteta, E and Anton-Martinez, A and de la Puente-Ovejero, L and Garcia-Jimenez, I and González-Iglesias, G and Larrañaga-Aiestaran, I and López-Martínez, A and Poyatos-García, J and Ruiz-Del-Yerro, E and Gonzalez, F and Arechavala-Gomeza, V},
title = {Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {18188},
pmid = {34521928},
issn = {2045-2322},
mesh = {3' Untranslated Regions/genetics ; CRISPR-Cas Systems ; Cells, Cultured ; Cytoskeletal Proteins/metabolism ; Drug Discovery/*methods ; Dystroglycans/metabolism ; Dystrophin/genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Muscular Dystrophy, Duchenne/*genetics/metabolism ; Myoblasts/*drug effects/metabolism ; Myogenic Regulatory Factor 5/metabolism ; Primary Cell Culture/*methods ; Sarcoglycans/metabolism ; Utrophin/*genetics/metabolism ; },
abstract = {Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient's immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.},
}
@article {pmid34521192,
year = {2021},
author = {Wang, C and Han, C and Du, X and Guo, W},
title = {Versatile CRISPR-Cas12a-Based Biosensing Platform Modulated with Programmable Entropy-Driven Dynamic DNA Networks.},
journal = {Analytical chemistry},
volume = {93},
number = {38},
pages = {12881-12888},
doi = {10.1021/acs.analchem.1c01597},
pmid = {34521192},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Entropy ; Humans ; *Nucleic Acids ; },
abstract = {In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.},
}
@article {pmid34520771,
year = {2022},
author = {Mirzaei, S and Paskeh, MDA and Hashemi, F and Zabolian, A and Hashemi, M and Entezari, M and Tabari, T and Ashrafizadeh, M and Raee, P and Aghamiri, S and Aref, AR and Leong, HC and Kumar, AP and Samarghandian, S and Zarrabi, A and Hushmandi, K},
title = {Long non-coding RNAs as new players in bladder cancer: Lessons from pre-clinical and clinical studies.},
journal = {Life sciences},
volume = {288},
number = {},
pages = {119948},
doi = {10.1016/j.lfs.2021.119948},
pmid = {34520771},
issn = {1879-0631},
mesh = {Animals ; Clinical Trials as Topic/*statistics &amp; numerical data ; *Disease Models, Animal ; Humans ; RNA, Long Noncoding/*genetics ; Urinary Bladder Neoplasms/genetics/*pathology/therapy ; },
abstract = {The clinical management of bladder cancer (BC) has become an increasing challenge due to high incidence rate of BC, malignant behavior of cancer cells and drug resistance. The non-coding RNAs are considered as key factors involved in BC progression. The long non-coding RNAs (lncRNAs) are RNA molecules and do not encode proteins. They have more than 200 nucleotides in length and affect gene expression at epigenetic, transcriptional and post-transcriptional phases. The lncRNAs demonstrate abnormal expression in BC cells and tissues. The present aims to identifying lncRNAs with tumor-suppressor and tumor-promoting roles, and evaluating their roles as regulatory of growth and migration. Apoptosis, glycolysis and EMT are tightly regulated by lncRNAs in BC. Response of BC cells to cisplatin, doxorubicin and gemcitabine chemotherapy is modulated by lncRNAs. LncRNAs regulate immune cell infiltration in tumor microenvironment and affect response of BC cells to immunotherapy. Besides, lncRNAs are able to regulate microRNAs, STAT3, Wnt, PTEN and PI3K/Akt pathways in affecting both proliferation and migration of BC cells. Noteworthy, anti-tumor compounds and genetic tools such as siRNA, shRNA and CRISPR/Cas systems can regulate lncRNA expression in BC. Finally, lncRNAs and exosomal lncRNAs can be considered as potential diagnostic and prognostic tools in BC.},
}
@article {pmid34519839,
year = {2022},
author = {Kruglov, O and Johnson, LDS and Minic, A and Jordan, K and Uger, RA and Wong, M and Sievers, EL and Shou, Y and Akilov, OE},
title = {The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {71},
number = {4},
pages = {919-932},
pmid = {34519839},
issn = {1432-0851},
mesh = {Animals ; CD47 Antigen ; Humans ; Interferon-gamma ; Killer Cells, Natural ; Mice ; *Mycosis Fungoides/drug therapy/pathology ; *Skin Neoplasms/drug therapy/pathology ; },
abstract = {CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.},
}
@article {pmid34518669,
year = {2021},
author = {Molla, KA and Sretenovic, S and Bansal, KC and Qi, Y},
title = {Precise plant genome editing using base editors and prime editors.},
journal = {Nature plants},
volume = {7},
number = {9},
pages = {1166-1187},
pmid = {34518669},
issn = {2055-0278},
mesh = {CRISPR-Associated Protein 9/*genetics ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; *Genome, Plant ; Plant Breeding/*methods ; },
abstract = {The development of CRISPR-Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications.},
}
@article {pmid34518609,
year = {2021},
author = {Arai, D and Nakao, Y},
title = {Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {18132},
pmid = {34518609},
issn = {2045-2322},
mesh = {Alleles ; Animals ; Base Sequence ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA-Activated Protein Kinase/*genetics/metabolism ; DNA-Directed DNA Polymerase/*genetics/metabolism ; Gene Editing ; Gene Expression ; *Gene Knock-In Techniques ; Gene Order ; Genes, Reporter ; Genetic Markers ; Humans ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; Phenotype ; Plasmids/genetics ; },
abstract = {CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.},
}
@article {pmid34518536,
year = {2021},
author = {Franke, M and De la Calle-Mustienes, E and Neto, A and Almuedo-Castillo, M and Irastorza-Azcarate, I and Acemel, RD and Tena, JJ and Santos-Pereira, JM and Gómez-Skarmeta, JL},
title = {CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5415},
pmid = {34518536},
issn = {2041-1723},
mesh = {Animals ; Body Patterning/genetics ; CCCTC-Binding Factor/deficiency/*genetics ; CRISPR-Cas Systems ; Chromatin/genetics/metabolism ; Embryo, Nonmammalian/embryology/*metabolism ; Enhancer Elements, Genetic/genetics ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques/*methods ; Organogenesis/genetics ; Promoter Regions, Genetic/genetics ; RNA-Seq/methods ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/deficiency/*genetics ; },
abstract = {Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes.},
}
@article {pmid34517958,
year = {2021},
author = {Pickerill, ES and Bernstein, DA},
title = {CRISPR mediated genome editing, a tool to dissect RNA modification processes.},
journal = {Methods in enzymology},
volume = {658},
number = {},
pages = {435-452},
doi = {10.1016/bs.mie.2021.06.013},
pmid = {34517958},
issn = {1557-7988},
support = {R15 AI130950/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; RNA/genetics ; RNA, Guide/genetics ; },
abstract = {Though over 100 distinct RNA modifications have been identified, the roles for many of these modifications in vivo remain unknown. Genome editing is one tool investigators are using to better understand the roles these modifications play and the consequences of their absence. In this chapter, we describe how CRISPR mediated genome editing can be used to interrogate the process of RNA modification in C. albicans. Furthermore, we discuss how the protocols described can be altered to meet experimental demands. The underlying theory on which these protocols are based are applicable to a variety of model systems. The protocols described utilize the widely used S. pyogenes Cas9, but the field of genome editing is quickly evolving. We discuss the recent developments of more flexible CRISPR systems that can target a greater number of sites in the genome. These and other advancements make CRISPR mediated genome editing a practical methodology to investigate RNA modification.},
}
@article {pmid34517842,
year = {2021},
author = {Luo, Y and Na, R and Nowak, JS and Qiu, Y and Lu, QS and Yang, C and Marsolais, F and Tian, L},
title = {Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.},
journal = {BMC plant biology},
volume = {21},
number = {1},
pages = {419},
pmid = {34517842},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/genetics/virology ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Gene Expression Regulation, Viral ; Genome, Plant ; Host-Pathogen Interactions/*genetics ; Mutagenesis ; Plant Viruses/*genetics ; RNA, Guide ; RNA, Plant ; RNA, Viral ; Soybeans/*genetics/*virology ; Virus Diseases/*genetics ; },
abstract = {BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing.<br><br>RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected.<br><br>CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.},
}
@article {pmid34517795,
year = {2022},
author = {Zaib, S and Saleem, MA and Khan, I},
title = {CRISPR-Cas9 Genome Engineering: Trends in Medicine and Health.},
journal = {Mini reviews in medicinal chemistry},
volume = {22},
number = {3},
pages = {410-421},
doi = {10.2174/1389557521666210913112030},
pmid = {34517795},
issn = {1875-5607},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; *RNA, Guide/genetics ; },
abstract = {The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine, and biotechnology. Over the past few decades, the development of CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the whole genetic engineering process utilizing the principles of Watson-Crick base pairing. CRISPRCas9 technology offers the simplest, fastest, most versatile, reliable, and precise method of genetic manipulation, thus enabling geneticists and medical researchers to edit parts of the genome by removing, adding, or altering sections of the DNA sequence. The current review focuses on the applications of CRISPR-Cas9 in the field of medical research. Compared with other gene-editing technologies, CRISPR/Cas9 demonstrates numerous advantages for the treatment of various medical conditions, including cancer, hepatitis B, cardiovascular diseases, or even high cholesterol. Given its promising performance, CRISPR/Cas9 gene-editing technology will surely help in the therapy of several disorders while addressing the issues pertaining to the minimization of the off-target effects of gene editing and incomplete matches between sgRNA and genomic DNA by Cas9.},
}
@article {pmid34517762,
year = {2021},
author = {Wang, L and Sola, I and Enjuanes, L and Zuñiga, S},
title = {MOV10 Helicase Interacts with Coronavirus Nucleocapsid Protein and Has Antiviral Activity.},
journal = {mBio},
volume = {12},
number = {5},
pages = {e0131621},
pmid = {34517762},
issn = {2150-7511},
support = {P01 AI060699/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antiviral Agents/*pharmacology ; Blotting, Western ; Cell Line, Tumor ; Chlorocebus aethiops ; Coronavirus Nucleocapsid Proteins/*metabolism ; Fluorescent Antibody Technique ; Humans ; Immunoprecipitation ; RNA Helicases/*metabolism/*pharmacology ; RNA, Viral/metabolism ; Vero Cells ; Virus Replication/drug effects ; },
abstract = {Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.},
}
@article {pmid34517092,
year = {2021},
author = {Savadi, S and Mangalassery, S and Sandesh, MS},
title = {Advances in genomics and genome editing for breeding next generation of fruit and nut crops.},
journal = {Genomics},
volume = {113},
number = {6},
pages = {3718-3734},
doi = {10.1016/j.ygeno.2021.09.001},
pmid = {34517092},
issn = {1089-8646},
mesh = {*Fruit/genetics ; *Gene Editing ; Genome, Plant ; Genomics ; Nuts ; Plant Breeding ; },
abstract = {Fruit tree crops are an essential part of the food production systems and are key to achieve food and nutrition security. Genetic improvement of fruit trees by conventional breeding has been slow due to the long juvenile phase. Advancements in genomics and molecular biology have paved the way for devising novel genetic improvement tools like genome editing, which can accelerate the breeding of these perennial crops to a great extent. In this article, advancements in genomics of fruit trees covering genome sequencing, transcriptome sequencing, genome editing technologies (GET), CRISPR-Cas system based genome editing, potential applications of CRISPR-Cas9 in fruit tree crops improvement, the factors influencing the CRISPR-Cas editing efficiency and the challenges for CRISPR-Cas9 applications in fruit tree crops improvement are reviewed. Besides, base editing, a recently emerging more precise editing system, and the future perspectives of genome editing in the improvement of fruit and nut crops are covered.},
}
@article {pmid34516550,
year = {2021},
author = {Suzzi, S and Ahrendt, R and Hans, S and Semenova, SA and Chekuru, A and Wirsching, P and Kroehne, V and Bilican, S and Sayed, S and Winkler, S and Spieß, S and Machate, A and Kaslin, J and Panula, P and Brand, M},
title = {Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.},
journal = {PLoS genetics},
volume = {17},
number = {9},
pages = {e1009794},
pmid = {34516550},
issn = {1553-7404},
mesh = {Animals ; Anxiety/genetics ; Biogenic Monoamines/*metabolism ; Brain/embryology/enzymology/*metabolism ; CRISPR-Cas Systems ; *Gene Deletion ; Larva/metabolism ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/*genetics ; Monoamine Oxidase/metabolism ; Smell/genetics ; Swimming ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.},
}
@article {pmid34516103,
year = {2021},
author = {Xia, X and Ma, B and Zhang, T and Lu, Y and Khan, MR and Hu, Y and Lei, C and Deng, S and He, Q and He, G and Zhang, K and Deng, R},
title = {G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization In Vivo.},
journal = {ACS sensors},
volume = {6},
number = {9},
pages = {3295-3302},
doi = {10.1021/acssensors.1c01061},
pmid = {34516103},
issn = {2379-3694},
mesh = {Animals ; *CRISPR-Cas Systems ; *Chickens ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; },
abstract = {Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for Salmonella enterica (S. enterica) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of S. enterica and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore S. enterica colonization in the intestinal tract and organs of chickens and showed the risk of S. enterica infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.},
}
@article {pmid34515826,
year = {2022},
author = {Slesarenko, YS and Lavrov, AV and Smirnikhina, SA},
title = {Off-target effects of base editors: what we know and how we can reduce it.},
journal = {Current genetics},
volume = {68},
number = {1},
pages = {39-48},
pmid = {34515826},
issn = {1432-0983},
mesh = {*CRISPR-Cas Systems ; *Cytosine/metabolism ; DNA Breaks, Double-Stranded ; Gene Editing/methods ; Humans ; Mutation ; },
abstract = {The recently discovered CRISPR-Cas9 modification, base editors (BEs), is considered as one of the most promising tools for correcting disease-causing mutations in humans, since it allows point substitutions to be edited without generating double-stranded DNA breaks, and, therefore, with a significant decrease in non-specific activity. Until recently, this method was considered the safest, but at the same time, it is quite effective. However, recent studies of non-specific activity of BEs revealed that some of them lead to the formation of a huge number of off-targets in both DNA and RNA, occurring due to the nature of the Cas9-fused proteins used. In this review article, we have considered and combined data from numerous studies about the most commonly used and more described in detail APOBEC-based BEs and Target-AID version of CBE, as well as ABE7 and ABE8 with their basic modifications into TadA to improve BEs' specificity. In our opinion, modern advances in molecular genetics make it possible to dramatically reduce the off-target activity of base editors due to introducing mutations into the domains of deaminases or inhibition of Cas9 by anti-CRISPR proteins, which returns BEs to the leading position in genome editing technologies.},
}
@article {pmid34514290,
year = {2021},
author = {Jordan, WT and Currie, S and Schmitz, RJ},
title = {Multiplex genome editing in Arabidopsis thaliana using Mb3Cas12a.},
journal = {Plant direct},
volume = {5},
number = {9},
pages = {e344},
pmid = {34514290},
issn = {2475-4455},
abstract = {The use of CRISPR-Cas proteins for the creation of multiplex genome engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR-Cas9 system for multiplex genome engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome editing in Arabidopsis thaliana. We identified optimized cis-regulatory sequences for driving expression of single-transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex genome engineering in plant-based systems.},
}
@article {pmid34514084,
year = {2021},
author = {Silva, FSR and Erdogmus, E and Shokr, A and Kandula, H and Thirumalaraju, P and Kanakasabapathy, MK and Hardie, JM and Pacheco, LGC and Li, JZ and Kuritzkes, DR and Shafiee, H},
title = {SARS-CoV-2 RNA Detection by a Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) Assay.},
journal = {Advanced materials technologies},
volume = {6},
number = {12},
pages = {2100602},
pmid = {34514084},
issn = {2365-709X},
support = {R01 AI118502/AI/NIAID NIH HHS/United States ; R01 AI138800/AI/NIAID NIH HHS/United States ; R61 AI140489/AI/NIAID NIH HHS/United States ; U54 HL119145/HL/NHLBI NIH HHS/United States ; },
abstract = {CRISPR (Clustered regularly interspaced short palindromic repeats)-based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS-CoV-2 molecular detection at the point of need. However, efficient translation of CRISPR-diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence-based results readout. Herein, an amplification-free CRISPR/Cas12a-based diagnostic technology for SARS-CoV-2 RNA detection is presented using a smartphone camera for results readout. This method, termed Cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase-generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS-CoV-2 reverse-transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL[-1]) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 - 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR-positive COVID-19 patients.},
}
@article {pmid34513721,
year = {2021},
author = {Lin, H and Li, G and Peng, X and Deng, A and Ye, L and Shi, L and Wang, T and He, J},
title = {The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {590989},
pmid = {34513721},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; *Communicable Diseases ; Hepatitis B virus/genetics ; Humans ; *Virus Diseases/genetics ; *Viruses/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a set of versatile gene-editing toolkit that perform diverse revolutionary functions in various fields of application such as agricultural practices, food industry, biotechnology, biomedicine, and clinical research. Specially, as a novel antiviral method of choice, CRISPR/Cas9 system has been extensively and effectively exploited to fight against human infectious viruses. Infectious diseases including human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papillomavirus (HPV), and other viruses are still global threats with persistent potential to probably cause pandemics. To facilitate virus removals, the CRISPR/Cas9 system has already been customized to confer new antiviral capabilities into host animals either by modifying host genome or by directly targeting viral inherent factors in the form of DNA. Although several limitations and difficulties still need to be conquered, this technology holds great promises in the treatment of human viral infectious diseases. In this review, we will first present a brief biological feature of CRISPR/Cas9 systems, which includes a description of CRISPR/Cas9 structure and composition; thereafter, we will focus on the investigations and applications that employ CRISPR/Cas9 system to combat several human infectious viruses and discuss challenges and future perspectives of using this new platform in the preclinical and clinical settings as an antiviral strategy.},
}
@article {pmid34511901,
year = {2021},
author = {Piperno, A and Sciortino, MT and Giusto, E and Montesi, M and Panseri, S and Scala, A},
title = {Recent Advances and Challenges in Gene Delivery Mediated by Polyester-Based Nanoparticles.},
journal = {International journal of nanomedicine},
volume = {16},
number = {},
pages = {5981-6002},
pmid = {34511901},
issn = {1178-2013},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Gene Transfer Techniques ; Genetic Therapy ; Humans ; *Nanoparticles ; Polyesters ; SARS-CoV-2 ; },
abstract = {Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly-interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.},
}
@article {pmid34509921,
year = {2021},
author = {Jelinkova, S and Martyniak, A and Dulak, J and Stępniewski, J},
title = {Derivation of human pluripotent stem cell line via CRISPR/Cas9 mediated deletion of exon 3 LAMA2 gene (DMBi001-A-1).},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102529},
doi = {10.1016/j.scr.2021.102529},
pmid = {34509921},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Exons/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Laminin/genetics/metabolism ; Muscle Fibers, Skeletal/metabolism ; *Muscular Dystrophies/genetics ; },
abstract = {LAMA2-related muscular dystrophy (LAMA2-MD) results from mutations in LAMA2 gene, encoding laminin α-2. It is a congenital disease characterized by muscle wasting, with the most severe version being diagnosed within first few months after birth. To generate LAMA2-DM in vitro model, we excised exon 3 from the LAMA2 gene in our previously derived healthy human induced pluripotent stem cells (hiPSCs). Obtained hiPSCs show expression of pluripotency markers, differentiation capacity into all three germ layers, normal karyotype and lack of LAMA2 expression on mRNA and protein level after differentiation into skeletal myocytes. Accordingly, it may provide novel insight into the molecular basis of LAMA2-MD.},
}
@article {pmid34509918,
year = {2021},
author = {Zu, B and Zhang, X and You, G and Fu, Q},
title = {Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for PTCH1 gene.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102517},
doi = {10.1016/j.scr.2021.102517},
pmid = {34509918},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Hedgehog Proteins ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; *Patched-1 Receptor ; },
abstract = {PTCH1 is the receptor protein of Hedgehog signaling pathway, and Hedgehog pathway plays a vital role in mammalian embryonic development. However, the specific biological role of PTCH1 is incompletely understood for embryonic development. Here, we used a CRISPR/Cas9 genome editing approach to generate a homozygous PTCH1 knock-out iPSC line (SCMCi001-A-1) from a healthy donor, which will be a valuable in vitro model to study the pathogenic mechanism of PTCH1 dysfunction in congenital disease.},
}
@article {pmid34509917,
year = {2021},
author = {Zhou, M and Xing, Q and Zhang, D and Zhang, C and Zhang, Y and Zhang, J and Shan, Y},
title = {Generation of an Akaluc knock-in human embryonic stem cell reporter line using CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102532},
doi = {10.1016/j.scr.2021.102532},
pmid = {34509917},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; Luciferases ; Technology ; },
abstract = {Akaluc, an enzyme engineered from luciferase, provides a potential powerful tool for tracing transplanted cells in vivo because of its near-infrared emission light. To enable evaluation of its potency, we inserted the Akaluc gene at AAVS1 locus using CRISPR/Cas9 technology and generated a clonal human embryonic stem stable cell line (Named H1-AAVS1-EF1α-Akaluc-KI or AkalucHES). AkalucHES could efficiently express Akaluc and were traced easily in vivo. We verified that AkalucHES expressed the pluripotency markers and showed normal stem cell morphology. Furthermore, AkalucHES maitains normal karyotype and is able to differentiate toward three germ-layer in vivo. So the Akaluc is effective for tracing transplanted cells in vivo.},
}
@article {pmid34509669,
year = {2021},
author = {Newby, GA and Liu, DR},
title = {In vivo somatic cell base editing and prime editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3107-3124},
pmid = {34509669},
issn = {1525-0024},
support = {R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *Gene Editing/methods ; Gene Rearrangement ; Gene Transfer Techniques ; Genetic Diseases, Inborn/genetics/therapy ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Humans ; INDEL Mutation ; RNA, Guide ; },
abstract = {Recent advances in genome editing technologies have magnified the prospect of single-dose cures for many genetic diseases. For most genetic disorders, precise DNA correction is anticipated to best treat patients. To install desired DNA changes with high precision, our laboratory developed base editors (BEs), which can correct the four most common single-base substitutions, and prime editors, which can install any substitution, insertion, and/or deletion over a stretch of dozens of base pairs. Compared to nuclease-dependent editing approaches that involve double-strand DNA breaks (DSBs) and often result in a large percentage of uncontrolled editing outcomes, such as mixtures of insertions and deletions (indels), larger deletions, and chromosomal rearrangements, base editors and prime editors often offer greater efficiency with fewer byproducts in slowly dividing or non-dividing cells, such as those that make up most of the cells in adult animals. Both viral and non-viral in vivo delivery methods have now been used to deliver base editors and prime editors in animal models, establishing that base editors and prime editors can serve as effective agents for in vivo therapeutic genome editing in animals. This review summarizes examples of in vivo somatic cell (post-natal) base editing and prime editing and prospects for future development.},
}
@article {pmid34509668,
year = {2021},
author = {Pickar-Oliver, A and Gough, V and Bohning, JD and Liu, S and Robinson-Hamm, JN and Daniels, H and Majoros, WH and Devlin, G and Asokan, A and Gersbach, CA},
title = {Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3243-3257},
pmid = {34509668},
issn = {1525-0024},
support = {T32 GM008555/GM/NIGMS NIH HHS/United States ; U01 EB028901/EB/NIBIB NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; UL1 TR002553/TR/NCATS NIH HHS/United States ; U01 HL156348/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/*genetics ; Disease Models, Animal ; Dystrophin/*genetics ; *Exons ; *Gene Editing ; Gene Expression ; Gene Order ; Gene Transfer Techniques ; Genetic Engineering ; Genetic Therapy/methods ; Genetic Vectors/*genetics ; Humans ; Mice ; Mice, Transgenic ; Muscle, Skeletal/metabolism ; Muscular Dystrophy, Duchenne/*genetics/*therapy ; Mutation ; Myocardium/metabolism ; Virus Integration ; },
abstract = {Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITI-mediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease.},
}
@article {pmid34509667,
year = {2021},
author = {Lee, BC and Lozano, RJ and Dunbar, CE},
title = {Understanding and overcoming adverse consequences of genome editing on hematopoietic stem and progenitor cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3205-3218},
pmid = {34509667},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/adverse effects/methods ; *Gene Editing/methods ; Gene Expression Regulation ; Genetic Engineering ; Genetic Therapy/*adverse effects/*methods ; Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Hematopoietic Stem Cells/*metabolism ; Humans ; Transgenes ; Transplantation Conditioning ; Treatment Outcome ; },
abstract = {Hematopoietic stem and progenitor cell (HSPC) gene therapies have recently moved beyond gene-addition approaches to encompass targeted genome modification or correction, based on the development of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas technologies. Advances in ex vivo HSPC manipulation techniques have greatly improved HSPC susceptibility to genetic modification. Targeted gene-editing techniques enable precise modifications at desired genomic sites. Numerous preclinical studies have already demonstrated the therapeutic potential of gene therapies based on targeted editing. However, several significant hurdles related to adverse consequences of gene editing on HSPC function and genomic integrity remain before broad clinical potential can be realized. This review summarizes the status of HSPC gene editing, focusing on efficiency, genomic integrity, and long-term engraftment ability related to available genetic editing platforms and HSPC delivery methods. The response of long-term engrafting HSPCs to nuclease-mediated DNA breaks, with activation of p53, is a significant challenge, as are activation of innate and adaptive immune responses to editing components. Lastly, we propose alternative strategies that can overcome current hurdles to HSPC editing at various stages from cell collection to transplantation to facilitate successful clinical applications.},
}
@article {pmid34509634,
year = {2021},
author = {Zhang, W and Xu, L and Liu, Q and Cao, Y and Yang, K and Song, X and Shao, Y and Tu, J and Qi, K},
title = {Enzymatic recombinase amplification coupled with CRISPR-Cas12a for ultrasensitive, rapid, and specific Porcine circovirus 3 detection.},
journal = {Molecular and cellular probes},
volume = {59},
number = {},
pages = {101763},
doi = {10.1016/j.mcp.2021.101763},
pmid = {34509634},
issn = {1096-1194},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Circovirus/genetics ; Nucleic Acid Amplification Techniques ; Recombinases/genetics ; Swine ; *Swine Diseases/diagnosis ; },
abstract = {Porcine circovirus type 3 (PCV3) is a disease associated with porcine dermatitis and nephrotic syndrome (PDNS) that has caused significant economic losses to swine herds since its discovery in 2016. To develop a simple, on-site, rapid, and sensitive assay to combat the spread of PCV3, we optimized the CRISPR/Cas12a (also known as Cpf1) system combined with enzymatic recombinase amplification (ERA) nucleic acid amplification to diagnose PCV3. The results showed that the ERA-CRISPR/Cas12a reaction could detect PCV3 within 1 h in genomic DNA harboring a minimum of seven copies. Additionally, we confirmed no cross-reactivity with PCV2, PCV4, or other porcine viruses, revealing the good specificity of this technique. These results demonstrated the ability of ERA-CRISPR/Cas12a to detect DNA at the single-molecule level and provide a rapid, simple, ultrasensitive, one-pot point-of-care test for PCV3 and suggest its potential for a variety of nucleic acid detection applications.},
}
@article {pmid34509299,
year = {2021},
author = {Guo, H and Liu, L and Nishiga, M and Cong, L and Wu, JC},
title = {Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {12},
pages = {1109-1123},
pmid = {34509299},
issn = {0168-9525},
support = {R01 HL145676/HL/NHLBI NIH HHS/United States ; R01 HL146690/HL/NHLBI NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; R01 HL141371/HL/NHLBI NIH HHS/United States ; K99 HL150319/HL/NHLBI NIH HHS/United States ; R01 HL126527/HL/NHLBI NIH HHS/United States ; R01 HL145708/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genome-Wide Association Study ; Humans ; *Induced Pluripotent Stem Cells ; Virulence ; },
abstract = {Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.},
}
@article {pmid34508657,
year = {2022},
author = {Ke, J and Robinson, D and Wu, ZY and Kuftin, A and Louie, K and Kosina, S and Northen, T and Cheng, JF and Yoshikuni, Y},
title = {CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria.},
journal = {Cell chemical biology},
volume = {29},
number = {4},
pages = {696-710.e4},
doi = {10.1016/j.chembiol.2021.08.009},
pmid = {34508657},
issn = {2451-9448},
mesh = {Bacteria ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genome, Bacterial ; Multigene Family ; *Recombinases ; },
abstract = {With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.},
}
@article {pmid34508276,
year = {2022},
author = {Ding, J and Frantzeskos, A and Orozco, G},
title = {Functional interrogation of autoimmune disease genetics using CRISPR/Cas9 technologies and massively parallel reporter assays.},
journal = {Seminars in immunopathology},
volume = {44},
number = {1},
pages = {137-147},
pmid = {34508276},
issn = {1863-2300},
support = {/WT_/Wellcome Trust/United Kingdom ; 207491/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 21754/VAC_/Versus Arthritis/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {*Autoimmune Diseases/diagnosis/genetics/therapy ; CRISPR-Cas Systems ; *Genome-Wide Association Study ; Genomics ; Humans ; },
abstract = {Genetic studies, including genome-wide association studies, have identified many common variants that are associated with autoimmune diseases. Strikingly, in addition to being frequently observed in healthy individuals, a number of these variants are shared across diseases with diverse clinical presentations. This highlights the potential for improved autoimmune disease understanding which could be achieved by characterising the mechanism by which variants lead to increased risk of disease. Of particular interest is the potential for identifying novel drug targets or of repositioning drugs currently used in other diseases. The majority of autoimmune disease variants do not alter coding regions and it is often difficult to generate a plausible hypothetical mechanism by which variants affect disease-relevant genes and pathways. Given the interest in this area, considerable effort has been invested in developing and applying appropriate methodologies. Two of the most important technologies in this space include both low- and high-throughput genomic perturbation using the CRISPR/Cas9 system and massively parallel reporter assays. In this review, we introduce the field of autoimmune disease functional genomics and use numerous examples to demonstrate the recent and potential future impact of these technologies.},
}
@article {pmid34508260,
year = {2021},
author = {Aregger, M and Xing, K and Gonatopoulos-Pournatzis, T},
title = {Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening.},
journal = {Nature protocols},
volume = {16},
number = {10},
pages = {4722-4765},
pmid = {34508260},
issn = {1750-2799},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Deletion ; *Gene Editing ; Humans ; },
abstract = {CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15-20 weeks.},
}
@article {pmid34508259,
year = {2021},
author = {Ward, HN and Aregger, M and Gonatopoulos-Pournatzis, T and Billmann, M and Ohsumi, TK and Brown, KR and Blencowe, BJ and Moffat, J and Myers, CL},
title = {Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.},
journal = {Nature protocols},
volume = {16},
number = {10},
pages = {4766-4798},
pmid = {34508259},
issn = {1750-2799},
support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; MOP-142375//CIHR/Canada ; FDN-148434//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; *RNA, Guide ; },
abstract = {The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.},
}
@article {pmid34508128,
year = {2021},
author = {Abe, T and Suzuki, Y and Ikeya, T and Hirota, K},
title = {Targeting chromosome trisomy for chromosome editing.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {18054},
pmid = {34508128},
issn = {2045-2322},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Line ; Chickens ; *Chromosome Aberrations ; DEAD-box RNA Helicases/genetics ; *Gene Editing ; Gene Targeting ; *Genetic Engineering ; Genetic Markers ; High-Throughput Nucleotide Sequencing ; Trisomy/*genetics ; },
abstract = {A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.},
}
@article {pmid34507144,
year = {2021},
author = {Lu, JZ and Qiao, ZB and Zhang, L and Cao, HX and Bai, ZH and Qi, YY and Zhu, HY and Chen, YQ and Zhang, SM and Yan, XH and Bao, Y and Jia, WW and Liu, ZM},
title = {Generation of a laminopathies-specific iPSC line EHTJUi005-A-3 with homozygous knockout of the LMNA gene by CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102530},
doi = {10.1016/j.scr.2021.102530},
pmid = {34507144},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Lamin Type A/genetics/metabolism ; *Laminopathies ; *Muscular Dystrophy, Emery-Dreifuss/genetics ; Mutation ; Technology ; },
abstract = {LAMIN A/C, encoded by the LMNA gene, supports the normal structure of the cell nucleus and regulates the connection between the nucleus and the cytoskeleton as a component of the nucleus envelope. The loss of expression and function of the LMNA gene would lead to the occurrence of congenital muscular dystrophy and Emery-Dreifuss muscular dystrophy which are collectively named as laminopathies. Here, we report a human induced pluripotent stem cell (iPSC) line (EHTJUi005-A-3) generated from a wild iPSC (EHTJUi005-A) with homozygous knockout of the gene LMNA through CRISPR/Cas9. This iPSC line provides a useful research model for studying laminopathies disease.},
}
@article {pmid34506262,
year = {2021},
author = {Sami, A and Xue, Z and Tazein, S and Arshad, A and He Zhu, Z and Ping Chen, Y and Hong, Y and Tian Zhu, X and Jin Zhou, K},
title = {CRISPR-Cas9-based genetic engineering for crop improvement under drought stress.},
journal = {Bioengineered},
volume = {12},
number = {1},
pages = {5814-5829},
pmid = {34506262},
issn = {2165-5987},
mesh = {Abscisic Acid/metabolism ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; *Droughts ; Ethylenes/metabolism ; Gene Editing/*methods ; Genome, Plant/genetics ; Osmotic Pressure/physiology ; Plants, Genetically Modified/genetics ; Stress, Physiological/*genetics ; },
abstract = {In several parts of the world, the prevalence and severity of drought are predicted to increase, creating considerable pressure on global agricultural yield. Among all abiotic stresses, drought is anticipated to produce the most substantial impact on soil biota and plants, along with complex environmental impacts on other ecological systems. Being sessile, plants tend to be the least resilient to drought-induced osmotic stress, which reduces nutrient accessibility due to soil heterogeneity and limits nutrient access to the root system. Drought tolerance is a complex quantitative trait regulated by multiple genes, and it is one of the most challenging characteristics to study and classify. Fortunately, the clustered regularly interspaced short palindromic repeat (CRISPR) technology has paved the way as a new frontier in crop improvement, thereby revolutionizing plant breeding. The application of CRISPER systems has proven groundbreaking across numerous biological fields, particularly in biomedicine and agriculture. The present review highlights the principle and optimization of CRISPR systems and their implementation for crop improvement, particularly in terms of drought tolerance, yield, and domestication. Furthermore, we address the ways in which innovative genome editing tools can help recognize and modify novel genes coffering drought tolerance. We anticipate the establishment of effective strategies of crop yield improvement in water-limited regions through collaborative efforts in the near future.},
}
@article {pmid34506127,
year = {2021},
author = {Feng, W and Peng, H and Xu, J and Liu, Y and Pabbaraju, K and Tipples, G and Joyce, MA and Saffran, HA and Tyrrell, DL and Babiuk, S and Zhang, H and Le, XC},
title = {Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.},
journal = {Analytical chemistry},
volume = {93},
number = {37},
pages = {12808-12816},
doi = {10.1021/acs.analchem.1c03456},
pmid = {34506127},
issn = {1520-6882},
mesh = {*COVID-19 ; COVID-19 Testing ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; Recombinases/genetics ; *Reverse Transcription ; SARS-CoV-2 ; Sensitivity and Specificity ; Technology ; },
abstract = {CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.},
}
@article {pmid34505742,
year = {2022},
author = {Verosloff, MS and Shapiro, SJ and Hawkins, EM and Alpay, E and Verma, D and Stanfield, EG and Kreindler, L and Jain, S and McKay, B and Hubbell, SA and Hendriks, CG and Blizard, BA and Broughton, JP and Chen, JS},
title = {CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100304},
doi = {10.1002/biot.202100304},
pmid = {34505742},
issn = {1860-7314},
mesh = {*COVID-19/diagnosis ; *CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Gene Editing/methods ; Humans ; Pandemics ; },
abstract = {The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications.},
}
@article {pmid34505269,
year = {2022},
author = {Stellon, D and Tran, MTN and Talbot, J and Chear, S and Khalid, MKNM and Pébay, A and Vickers, JC and King, AE and Hewitt, AW and Cook, AL},
title = {CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2549},
number = {},
pages = {379-398},
pmid = {34505269},
issn = {1940-6029},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Genetic Engineering ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models.},
}
@article {pmid34505086,
year = {2021},
author = {Ghanta, KS and Ishidate, T and Mello, CC},
title = {Microinjection for precision genome editing in Caenorhabditis elegans.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100748},
pmid = {34505086},
issn = {2666-1667},
support = {P40 OD010440/OD/NIH HHS/United States ; R37 GM058800/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Caenorhabditis elegans Proteins/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Microinjections/*methods ; },
abstract = {In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).},
}
@article {pmid34503976,
year = {2021},
author = {Liu, J and Shang, X and Huang, S and Xu, Y and Lu, J and Zhang, Y and Liu, Z and Wang, X},
title = {Construction and Characterization of CRISPR/Cas9 Knockout Rat Model of Carboxylesterase 2a Gene.},
journal = {Molecular pharmacology},
volume = {100},
number = {5},
pages = {480-490},
doi = {10.1124/molpharm.121.000357},
pmid = {34503976},
issn = {1521-0111},
mesh = {Animals ; Antihypertensive Agents/pharmacology ; Base Sequence ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Carboxylesterase/*deficiency/*genetics ; Diltiazem/pharmacology ; Dose-Response Relationship, Drug ; Female ; Gene Knockdown Techniques/*methods ; Insulin Resistance/physiology ; Male ; Microsomes, Liver/drug effects/enzymology ; Rats ; Rats, Sprague-Dawley ; },
abstract = {Carboxylesterase (CES) 2, an important metabolic enzyme, plays a critical role in drug biotransformation and lipid metabolism. Although CES2 is very important, few animal models have been generated to study its properties and functions. Rat Ces2 is similar to human CES2A-CES3A-CES4A gene cluster, with highly similar gene structure, function, and substrate. In this report, CRISPR-associated protein-9 (CRISPR/Cas9) technology was first used to knock out rat Ces2a, which is a main subtype of Ces2 mostly distributed in the liver and intestine. This model showed the absence of CES2A protein expression in the liver. Further pharmacokinetic studies of diltiazem, a typical substrate of CES2A, confirmed the loss of function of CES2A both in vivo and in vitro. At the same time, the expression of CES2C and CES2J protein in the liver decreased significantly. The body and liver weight of Ces2a knockout rats also increased, but the food intake did not change. Moreover, the deficiency of Ces2a led to obesity, insulin resistance, and liver fat accumulation, which are consistent with the symptoms of nonalcoholic fatty liver disease (NAFLD). Therefore, this rat model is not only a powerful tool to study drug metabolism mediated by CES2 but also a good disease model to study NAFLD. SIGNIFICANCE STATEMENT: Human carboxylesterase (CES) 2 plays a key role in the first-pass hydrolysis metabolism of most oral prodrugs as well as lipid metabolism. In this study, CRISPR/Cas9 technology was used to knock out Ces2a gene in rats for the first time. This model can be used not only in the study of drug metabolism and pharmacokinetics but also as a disease model of nonalcoholic fatty liver disease (NAFLD) and other metabolic disorders.},
}
@article {pmid34503839,
year = {2021},
author = {Toledo-Hernández, M and Lander, TA and Bao, C and Xie, K and Atta-Boateng, A and Wanger, TC},
title = {Genome-edited tree crops: mind the socioeconomic implementation gap.},
journal = {Trends in ecology &amp; evolution},
volume = {36},
number = {11},
pages = {972-975},
doi = {10.1016/j.tree.2021.08.007},
pmid = {34503839},
issn = {1872-8383},
mesh = {*CRISPR-Cas Systems ; Genome, Plant ; Plants, Genetically Modified/genetics ; Socioeconomic Factors ; *Trees ; },
abstract = {The discussion about CRISPR/Cas genome editing is focused mostly on technical aspects to improve productivity and climate resilience in major tree crops such as cocoa, coffee, and citrus. We suggest a solution to the largely ignored socioeconomic impacts for farmers, when new genome-edited varieties are introduced from the laboratory to the field.},
}
@article {pmid34503562,
year = {2021},
author = {Azhagiri, MKK and Babu, P and Venkatesan, V and Thangavel, S},
title = {Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy.},
journal = {Stem cell research &amp; therapy},
volume = {12},
number = {1},
pages = {500},
pmid = {34503562},
issn = {1757-6512},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; },
abstract = {The advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.},
}
@article {pmid34502462,
year = {2021},
author = {Zheng, S and Ye, C and Lu, J and Liufu, J and Lin, L and Dong, Z and Li, J and Zhuang, C},
title = {Improving the Rice Photosynthetic Efficiency and Yield by Editing OsHXK1 via CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502462},
issn = {1422-0067},
mesh = {Biomass ; CRISPR-Cas Systems ; Edible Grain/*growth &amp; development ; Hexokinase/*genetics/metabolism ; Oryza/*physiology ; Photosynthesis/*genetics ; Plants, Genetically Modified ; },
abstract = {Rice (Oryza sativa L.) is an important food crop species in China. Cultivating high-yielding rice varieties that have a high photosynthetic efficiency is an important goal of rice breeding in China. In recent years, due to the continual innovation of molecular breeding methods, many excellent genes have been applied in rice breeding, which is highly important for increasing rice yields. In this paper, the hexokinase gene OsHXK1 was knocked out via the CRISPR/Cas9 gene-editing method in the indica rice varieties Huanghuazhan, Meixiangzhan, and Wushansimiao, and OsHXK1-CRISPR/Cas9 lines were obtained. According to the results of a phenotypic analysis and agronomic trait statistics, the OsHXK1-CRISPR/Cas9 plants presented increased light saturation points, stomatal conductance, light tolerance, photosynthetic products, and rice yields. Moreover, transcriptome analysis showed that the expression of photosynthesis-related genes significantly increased. Taken together, our results revealed that knocking out OsHXK1 via the CRISPR/Cas9 gene-editing method could effectively lead to the cultivation of high-photosynthetic efficiency and high-yielding rice varieties. They also revealed the important roles of OsHXK1 in the regulation of rice yield and photosynthesis.},
}
@article {pmid34502441,
year = {2021},
author = {Lyu, R and Ahmed, S and Fan, W and Yang, J and Wu, X and Zhou, W and Zhang, P and Yuan, L and Wang, H},
title = {Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502441},
issn = {1422-0067},
mesh = {*Gene Editing ; Ipomoea batatas/*genetics ; Starch/*biosynthesis/*genetics ; },
abstract = {Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes.},
}
@article {pmid34502338,
year = {2021},
author = {de Vrieze, E and de Bruijn, SE and Reurink, J and Broekman, S and van de Riet, V and Aben, M and Kremer, H and van Wijk, E},
title = {Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502338},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; *Gene Editing ; Gene Knock-In Techniques/*methods ; Genetic Diseases, Inborn/*genetics ; Genetic Engineering/*methods ; Mutagenesis ; Ribonucleoproteins/genetics/metabolism ; Zebrafish ; Zebrafish Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; },
abstract = {CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3-8% of alleles, and 30-45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.},
}
@article {pmid34502319,
year = {2021},
author = {Hassan, JJ and Lieske, A and Dörpmund, N and Klatt, D and Hoffmann, D and Kleppa, MJ and Kustikova, OS and Stahlhut, M and Schwarzer, A and Schambach, A and Maetzig, T},
title = {A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502319},
issn = {1422-0067},
mesh = {Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Proliferation ; *Gene Expression Regulation, Neoplastic ; Group IV Phospholipases A2/*antagonists &amp; inhibitors/genetics ; High-Throughput Screening Assays ; Homeodomain Proteins/genetics/*metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics/*metabolism ; Tumor Cells, Cultured ; },
abstract = {HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.},
}
@article {pmid34502191,
year = {2021},
author = {Lee, HH and Kim, D and Jung, J and Kang, H and Cho, H},
title = {NLRP3 Deficiency in Hepatocellular Carcinoma Enhances Surveillance of NK-92 through a Modulation of MICA/B.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502191},
issn = {1422-0067},
mesh = {Animals ; Apoptosis ; Biomarkers, Tumor/genetics/metabolism ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*immunology/metabolism/pathology ; Cell Proliferation ; Cytotoxicity, Immunologic/*immunology ; Disease Models, Animal ; Female ; Gene Expression Regulation, Neoplastic ; Histocompatibility Antigens Class I/genetics/*metabolism ; Humans ; Killer Cells, Natural/*immunology ; Liver Neoplasms/immunology/metabolism/pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Monitoring, Immunologic/*methods ; NLR Family, Pyrin Domain-Containing 3 Protein/*deficiency/genetics ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Human hepatocellular carcinoma (HCC) is the most common and even worse at prognosis. The patients with HCC which accompanied by other diseases, such as cirrhosis, can be limited in various treatments, such as chemotherapy, not HCC patients without other diseases. NLRP3 inflammasome plays an important role in the innate immune response, but emerging evidence has indicated that the NLRP3 inflammasome is implicated in all stages of cancer development. Various cells express NLRP3 protein through the autocrine or paracrine signaling in their environment, but NK cells do not. The expanding evidence shows that patients who suffer from liver cancers have a low frequency of natural killer (NK) cells, and the function of these cells is also impaired. Thus, we examined how the expression of NLRP3 in HCC cells affects cancer surveillance by NK cells in a state of a co-culture of both cells. When the expression of NLRP3 in HCC cells was ablated, MICA/B on the surface of HCC cells was upregulated through the lowered expression of matrix metalloproteinase. The expression of MICA on the surface of HCC cells interacted with the NKG2D receptor on NK-92 cells, which led to NK cytotoxicity. Furthermore, in a xenograft mice model, NLRP3 KO HCC cells delayed tumor development and metastasis as well as increased the sensitivity to NK cell cytotoxicity. Taken together, NLRP3 KO in HCC could enhance NK immunosurveillance through an interaction of NKG2D-MICA.},
}
@article {pmid34502143,
year = {2021},
author = {Arango, D and Bittar, A and Esmeral, NP and Ocasión, C and Muñoz-Camargo, C and Cruz, JC and Reyes, LH and Bloch, NI},
title = {Understanding the Potential of Genome Editing in Parkinson's Disease.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502143},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Parkinson Disease/*genetics/therapy ; },
abstract = {CRISPR is a simple and cost-efficient gene-editing technique that has become increasingly popular over the last decades. Various CRISPR/Cas-based applications have been developed to introduce changes in the genome and alter gene expression in diverse systems and tissues. These novel gene-editing techniques are particularly promising for investigating and treating neurodegenerative diseases, including Parkinson's disease, for which we currently lack efficient disease-modifying treatment options. Gene therapy could thus provide treatment alternatives, revolutionizing our ability to treat this disease. Here, we review our current knowledge on the genetic basis of Parkinson's disease to highlight the main biological pathways that become disrupted in Parkinson's disease and their potential as gene therapy targets. Next, we perform a comprehensive review of novel delivery vehicles available for gene-editing applications, critical for their successful application in both innovative research and potential therapies. Finally, we review the latest developments in CRISPR-based applications and gene therapies to understand and treat Parkinson's disease. We carefully examine their advantages and shortcomings for diverse gene-editing applications in the brain, highlighting promising avenues for future research.},
}
@article {pmid34502064,
year = {2021},
author = {Schellens, R and de Vrieze, E and Graave, P and Broekman, S and Nagel-Wolfrum, K and Peters, T and Kremer, H and Collin, RWJ and van Wijk, E},
title = {Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
pmid = {34502064},
issn = {1422-0067},
support = {PPA-0517-0717-RAD/FFB/Foundation Fighting Blindness/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Exons ; Eye Proteins/chemistry/*genetics/metabolism ; Genetic Therapy/methods ; Phenotype ; Protein Domains ; Retinitis Pigmentosa/*genetics/pathology/therapy ; Zebrafish ; Zebrafish Proteins/chemistry/*genetics/metabolism ; },
abstract = {Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactly one Laminin G and two EGF domains. Although the eys[Δexon40-44] transcript was found at levels comparable to wild-type eys, and no unwanted off-target modifications were identified within the eys coding sequence after single-molecule sequencing, Eys[Δexon40-44] protein expression could not be detected. Visual motor response experiments revealed that eys[Δexon40-44] larvae were visually impaired and histological analysis revealed a progressive degeneration of the retinal outer nuclear layer in these zebrafish. Altogether, the data obtained in our zebrafish model currently provide no indications for the skipping of EYS exons 37-41 as an effective future treatment strategy for EYS-associated RP.},
}
@article {pmid34500849,
year = {2021},
author = {Klabenkova, K and Fokina, A and Stetsenko, D},
title = {Chemistry of Peptide-Oligonucleotide Conjugates: A Review.},
journal = {Molecules (Basel, Switzerland)},
volume = {26},
number = {17},
pages = {},
pmid = {34500849},
issn = {1420-3049},
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems ; Cell Membrane Permeability ; Cell-Penetrating Peptides/*chemistry/metabolism ; Click Chemistry ; Delayed-Action Preparations/*chemistry ; Drug Liberation ; Humans ; Nucleic Acids ; Oligonucleotides/*chemistry/metabolism ; RNA, Small Interfering/chemistry ; Solid-Phase Synthesis Techniques ; },
abstract = {Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.},
}
@article {pmid34500148,
year = {2021},
author = {Zhi, L and Su, X and Yin, M and Zhang, Z and Lu, H and Niu, Z and Guo, C and Zhu, W and Zhang, X},
title = {Genetical engineering for NK and T cell immunotherapy with CRISPR/Cas9 technology: Implications and challenges.},
journal = {Cellular immunology},
volume = {369},
number = {},
pages = {104436},
doi = {10.1016/j.cellimm.2021.104436},
pmid = {34500148},
issn = {1090-2163},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; Killer Cells, Natural/*transplantation ; Receptors, Chimeric Antigen/*therapeutic use ; T-Lymphocytes/*transplantation ; },
abstract = {Immunotherapy has become one of the most promising strategies in cancer therapies. Among the therapeutic alternatives, genetically engineered NK/T cell therapies have emerged as powerful and innovative therapeutic modalities for cancer patients with precise targeting and impressive efficacy. Nonetheless, this approach still faces multiple challenges, such as immunosuppressive tumor microenvironment, exhaustion of immune effector cells in tumors, off-target effects manufacturing complexity, and poor infiltration of effector cells, all of which need to be overcome for further utilization to cancers. Recently, CRISPR/Cas9 genome editing technology, with the goal of enhancing the efficacy and increasing the availability of engineered effector cell therapies, has shown considerable potential in the novel strategies and options to overcome these limitations. Here we review the current progress of the applications of CRISPR in cancer immunotherapy. Furthermore, we discuss issues related to the NK/T cell applications, gene delivery methods, efficiency, challenges, and implications of CRISPR/Cas9.},
}
@article {pmid34499925,
year = {2021},
author = {Yoast, RE and Emrich, SM and Zhang, X and Xin, P and Arige, V and Pathak, T and Benson, JC and Johnson, MT and Abdelnaby, AE and Lakomski, N and Hempel, N and Han, JM and Dupont, G and Yule, DI and Sneyd, J and Trebak, M},
title = {The Mitochondrial Ca[2+] uniporter is a central regulator of interorganellar Ca[2+] transfer and NFAT activation.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {4},
pages = {101174},
pmid = {34499925},
issn = {1083-351X},
support = {R01 CA242021/CA/NCI NIH HHS/United States ; R01 DE019245/DE/NIDCR NIH HHS/United States ; R01 HL137852/HL/NHLBI NIH HHS/United States ; R35 HL150778/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Channels/genetics/*metabolism ; *Calcium Signaling ; Cytosol/*metabolism ; Endoplasmic Reticulum ; Gene Knockout Techniques ; HCT116 Cells ; HEK293 Cells ; Humans ; Jurkat Cells ; Lymphocyte Activation ; Mitochondria/*metabolism ; NFATC Transcription Factors/genetics/*metabolism ; T-Lymphocytes/metabolism ; },
abstract = {Mitochondrial Ca[2+] uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca[2+] uptake by the mitochondrial Ca[2+] uniporter (MCU) shapes receptor-evoked interorganellar Ca[2+] signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca[2+] imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca[2+] signaling across mammalian cell types. MCU activity sustains cytosolic Ca[2+] signaling by preventing Ca[2+]-dependent inactivation of store-operated Ca[2+] release-activated Ca[2+] channels and by inhibiting Ca[2+] extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca[2+] responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca[2+] oscillations, endoplasmic reticulum Ca[2+] refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca[2+] clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca[2+] transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca[2+] buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca[2+] signals that regulate cellular transcription and function.},
}
@article {pmid34499670,
year = {2021},
author = {Duy, PN and Lan, DT and Pham Thu, H and Thi Thu, HP and Nguyen Thanh, H and Pham, NP and Auguy, F and Bui Thi Thu, H and Manh, TB and Cunnac, S and Pham, XH},
title = {Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter.},
journal = {PloS one},
volume = {16},
number = {9},
pages = {e0255470},
pmid = {34499670},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Disease Resistance/genetics/*immunology ; Disease Susceptibility ; *Gene Expression Regulation, Plant ; Monosaccharide Transport Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Mutation ; Oryza/growth &amp; development/*immunology/microbiology ; Plant Diseases/*immunology/microbiology ; Plant Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; *Promoter Regions, Genetic ; Xanthomonas/*physiology ; },
abstract = {TBR225 is one of the most popular commercial rice varieties in Northern Vietnam. However, this variety is highly susceptible to bacterial leaf blight (BLB), a disease caused by Xanthomonas oryzae pv. oryzae (Xoo) which can lead to important yield losses. OsSWEET14 belongs to the SWEET gene family that encodes sugar transporters. Together with other Clade III members, it behaves as a susceptibility (S) gene whose induction by Asian Xoo Transcription-Activator-Like Effectors (TALEs) is absolutely necessary for disease. In this study, we sought to introduce BLB resistance in the TBR225 elite variety. First, two Vietnamese Xoo strains were shown to up-regulate OsSWEET14 upon TBR225 infection. To investigate if this induction is connected with disease susceptibility, nine TBR225 mutant lines with mutations in the AvrXa7, PthXo3 or TalF TALEs DNA target sequences of the OsSWEET14 promoter were obtained using the CRISPR/Cas9 editing system. Genotyping analysis of T0 and T1 individuals showed that mutations were stably inherited. None of the examined agronomic traits of three transgene-free T2 edited lines were significantly different from those of wild-type TBR225. Importantly, one of these T2 lines, harboring the largest homozygous 6-bp deletion, displayed decreased OsSWEET14 expression as well as a significantly reduced susceptibility to a Vietnamese Xoo strains and complete resistance to another one. Our findings indicate that CRISPR/Cas9 editing conferred an improved BLB resistance to a Vietnamese commercial elite rice variety.},
}
@article {pmid34499198,
year = {2021},
author = {Liu, J and Li, TT and Liang, QL and Elsheikha, HM and Zhao, DY and Zhang, ZW and Xu, XP and Zhu, XQ and Wang, M},
title = {Characterization of functions in parasite growth and virulence of four Toxoplasma gondii genes involved in lipid synthesis by CRISPR-Cas9 system.},
journal = {Parasitology research},
volume = {120},
number = {11},
pages = {3749-3759},
pmid = {34499198},
issn = {1432-1955},
mesh = {Animals ; CRISPR-Cas Systems ; Fatty Acids ; *Parasites/metabolism ; Protozoan Proteins/genetics/metabolism ; *Toxoplasma/genetics/metabolism ; Virulence ; },
abstract = {Fatty acid uptake is extremely important for the survival and growth of the intracellular parasite Toxoplasma gondii. In this study, CRISPR-Cas9 gene editing technology was used to investigate the role of four lipid synthesis enzymes, namely, glycerol-3-phosphate dehydrogenase (G3PDH), malonyl CoA-acyl carrier protein transacylase (FabD), acyl-ACP thiolesterase (TE), and diacylglycerol acyltransferase (DGAT), in the virulence and infectivity of Type I RH and Type II Prugniaud (Pru) strains of T. gondii. Immunofluorescence analysis of the tachyzoite stage showed that FabD protein was located in the apicoplast; however, the expression level of the other three proteins was undetectable. Compared with wild-type (WT) strains, the growth of RHΔG3PDH, RHΔTE, and RHΔDGAT in vitro and their virulence in vivo were not significantly different. However, RHΔFabD exhibited a significantly reduced growth rate, compared with the WT strain. The deletion of FabD attenuated the virulence of Type II Pru strain and reduced the formation of cysts in vivo. These data improved our understanding of the role of lipid synthesis enzymes in the pathogenesis of T. gondii.},
}
@article {pmid34499171,
year = {2021},
author = {Tromp, A and Robinson, K and Hall, TE and Mowry, B and Giacomotto, J},
title = {Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {12},
pages = {},
pmid = {34499171},
issn = {2160-1836},
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; Genomics ; RNA, Guide/genetics ; *Zebrafish/genetics ; },
abstract = {Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four single guide RNAs (sgRNAs) targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase, we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons, or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150 bp deletion in average) to their entire genomic locus (300 kb deletion for nrxn1a for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that in our study, their transmission frequency does not seem to decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions. Note that we are also describing here a custom method to produce the sgRNAs, which proved to be faster and more robust than the ones traditionally used in the community to date.},
}
@article {pmid34498373,
year = {2021},
author = {Fan, S and Zhang, L and Tang, M and Cai, Y and Liu, J and Liu, H and Liu, J and Terzaghi, W and Wang, H and Hua, W and Zheng, M},
title = {CRISPR/Cas9-targeted mutagenesis of the BnaA03.BP gene confers semi-dwarf and compact architecture to rapeseed (Brassica napus L.).},
journal = {Plant biotechnology journal},
volume = {19},
number = {12},
pages = {2383-2385},
pmid = {34498373},
issn = {1467-7652},
mesh = {*Brassica napus/genetics ; *Brassica rapa ; CRISPR-Cas Systems/genetics ; Mutagenesis ; },
}
@article {pmid34498163,
year = {2021},
author = {Rao, MJ and Wang, L},
title = {CRISPR/Cas9 technology for improving agronomic traits and future prospective in agriculture.},
journal = {Planta},
volume = {254},
number = {4},
pages = {68},
pmid = {34498163},
issn = {1432-2048},
mesh = {Agriculture ; *CRISPR-Cas Systems/genetics ; Genome, Plant ; *Plant Breeding ; Plants, Genetically Modified/genetics ; Technology ; },
abstract = {In this review, we have focused on the CRISPR/Cas9 technology for improving the agronomic traits in plants through point mutations, knockout, and single base editing, and we highlighted the recent progress in plant metabolic engineering. CRISPR/Cas9 technology has immense power to reproduce plants with desired characters and revolutionizing the field of genome engineering by erasing the barriers in targeted genome editing. Agriculture fields are using this advance genome editing tool to get the desired traits in the crops plants such as increase yield, improve product quality attributes, and enhance resistance against biotic and abiotic stresses by identifying and editing genes of interest. This review focuses on CRISPR/Cas-based gene knockout for trait improvement and single base editing to boost yield, quality, stress tolerance, and disease resistance traits in crops. Use of CRISPR/Cas9 system to facilitate crop domestication and hybrid breeding are also touched. We summarize recent developments and up-gradation of delivery mechanism (nanotechnology and virus particle-based delivery system) and progress in multiplex gene editing. We also shed lights in advances and challenges of engineering the important metabolic pathways that contain a variety of dietary metabolites and phytochemicals. In addition, we endorsed substantial technical hurdles and possible ways to overcome the unpredictability of CRISPR/Cas technology for broader application across various crop species. We speculated that by making a strong interconnection among all genomic fields will give a gigantic bunt of knowledge to develop crop expressing desired traits.},
}
@article {pmid34497417,
year = {2021},
author = {Kamber, RA and Nishiga, Y and Morton, B and Banuelos, AM and Barkal, AA and Vences-Catalán, F and Gu, M and Fernandez, D and Seoane, JA and Yao, D and Liu, K and Lin, S and Spees, K and Curtis, C and Jerby-Arnon, L and Weissman, IL and Sage, J and Bassik, MC},
title = {Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.},
journal = {Nature},
volume = {597},
number = {7877},
pages = {549-554},
pmid = {34497417},
issn = {1476-4687},
support = {DP2 HD084069/HD/NICHD NIH HHS/United States ; R01 AI143889/AI/NIAID NIH HHS/United States ; R35 CA231997/CA/NCI NIH HHS/United States ; R35 CA220434/CA/NCI NIH HHS/United States ; U01 CA213273/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antibodies, Monoclonal/immunology ; Antibody-Dependent Cell Cytotoxicity/*genetics/immunology ; Antigens, Neoplasm/immunology ; CD47 Antigen/antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Cytophagocytosis/*genetics ; Female ; Gene Editing ; Gene Knockout Techniques ; Humans ; Lymphoma, T-Cell/immunology/pathology ; Macrophages/cytology/*immunology/metabolism ; Male ; Membrane Glycoproteins/deficiency/genetics ; Mice ; Neoplasms/*immunology/*pathology ; Receptors, G-Protein-Coupled/metabolism ; },
abstract = {Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells[1-7]. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.},
}
@article {pmid34497366,
year = {2022},
author = {Zhou, Y and Zhang, L and Xie, YH and Wu, J},
title = {Advancements in detection of SARS-CoV-2 infection for confronting COVID-19 pandemics.},
journal = {Laboratory investigation; a journal of technical methods and pathology},
volume = {102},
number = {1},
pages = {4-13},
pmid = {34497366},
issn = {1530-0307},
mesh = {Antibodies, Viral/analysis ; Antigens, Viral/analysis ; COVID-19/*diagnosis/epidemiology/virology ; COVID-19 Nucleic Acid Testing/methods/trends ; COVID-19 Serological Testing/methods/trends ; COVID-19 Testing/*methods/trends ; Genome, Viral ; Humans ; Molecular Diagnostic Techniques/methods/trends ; Mutation ; Nucleic Acid Amplification Techniques/methods/trends ; Open Reading Frames ; *Pandemics ; RNA, Viral/analysis/genetics ; Reverse Transcriptase Polymerase Chain Reaction/methods/trends ; *SARS-CoV-2/classification/genetics/immunology/isolation &amp; purification ; Sequence Analysis, RNA/methods/trends ; },
abstract = {As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.},
}
@article {pmid34496881,
year = {2021},
author = {Liang, J and Teng, P and Xiao, W and He, G and Song, Q and Zhang, Y and Peng, B and Li, G and Hu, L and Cao, D and Tang, Y},
title = {Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of SARS-CoV-2 from clinical samples.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {273},
pmid = {34496881},
issn = {1477-3155},
mesh = {COVID-19/diagnosis/virology ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation, Fungal ; Genes, Viral ; Humans ; *Nucleic Acid Amplification Techniques ; RNA, Viral/analysis ; SARS-CoV-2/*genetics/*isolation &amp; purification ; Sensitivity and Specificity ; *Spectrum Analysis, Raman ; },
abstract = {The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).},
}
@article {pmid34495758,
year = {2021},
author = {Kesavan, G and Machate, A and Brand, M},
title = {CRISPR/Cas9-Based Split Fluorescent Protein Tagging.},
journal = {Zebrafish},
volume = {18},
number = {6},
pages = {369-373},
doi = {10.1089/zeb.2021.0031},
pmid = {34495758},
issn = {1557-8542},
mesh = {Animals ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Green Fluorescent Proteins/genetics/metabolism ; Zebrafish/genetics/metabolism ; },
abstract = {Genetically encoded fluorescent tags such as green fluorescent protein fused to protein have revolutionized cell biology as they permit high-resolution protein imaging in live systems. Split fluorescent proteins, with a small fragment of 16 amino acids, can be inserted in the coding sequence to label proteins. We demonstrate successful integration of two bright and fast maturing split fluorescent proteins, mNeon green and sfCherry2, in zebrafish, and show that they are suitable for live imaging, including time-lapse series, and that they have a high signal-to-noise ratio. Furthermore, we show that CRISPR/Cas9 can be used to generate fluorescently tagged proteins in vivo.},
}
@article {pmid34495650,
year = {2021},
author = {Wei, G and Peng, Z and Liu, J and Yang, K and Zhao, C and Xie, W and Huang, T and Liu, J and Li, J and An, G},
title = {Accurate Identification and Early Diagnosis of Osteosarcoma through CRISPR-Cas12a-Based Average Telomerase Activity Detection.},
journal = {ACS synthetic biology},
volume = {10},
number = {9},
pages = {2409-2416},
doi = {10.1021/acssynbio.1c00389},
pmid = {34495650},
issn = {2161-5063},
mesh = {Bone Neoplasms/*diagnosis/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA Primers/metabolism ; Early Detection of Cancer ; Fluorescent Dyes/chemistry ; Humans ; Nucleic Acid Amplification Techniques ; Osteosarcoma/*diagnosis/metabolism ; RNA, Guide/metabolism ; Telomerase/*metabolism ; },
abstract = {Sensitive and reliable analysis of telomerase activity is important for clinical diagnosis, therapy, and prognosis of osteosarcoma. Telomerase activity is a complicated concept including both the amount of active telomerases and the length of the telomerases extension product. Still, few of the strategies formerly proposed distinguish the two aspects of telomerase activity. Herein, we propose a novel CRISPR-Cas12a-based fluorescent sensing platform that can output signals of both the amounts of telomerase and length of telomerase extension products with the assistance of an elegantly designed stem-loop probe and CRISPR-Cas12a system. On this basis, we induced a novel index, average telomerase activity, for accurate cancer reporting. Through systematic laboratory and clinical experiments, we have demonstrated that average telomerase activity can accurately distinguish cancer cells and has the potential for osteosarcoma staging.},
}
@article {pmid34495525,
year = {2022},
author = {Yan, R and Lin, X},
title = {CRISPR/Cas9-Mediated Genome Editing System in Insect Genomics and Pest Management.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2360},
number = {},
pages = {347-366},
pmid = {34495525},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genomics ; Pest Control ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.},
}
@article {pmid34494640,
year = {2021},
author = {Losito, M and Smith, QM and Newton, MD and Cuomo, ME and Rueda, DS},
title = {Cas12a target search and cleavage on force-stretched DNA.},
journal = {Physical chemistry chemical physics : PCCP},
volume = {23},
number = {47},
pages = {26640-26644},
pmid = {34494640},
issn = {1463-9084},
support = {/WT_/Wellcome Trust/United Kingdom ; 206292/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Bacterial Proteins/*chemistry ; CRISPR-Associated Proteins/*chemistry ; CRISPR-Cas Systems ; DNA/*chemistry ; Endodeoxyribonucleases/*chemistry ; Models, Molecular ; Optical Tweezers ; },
abstract = {Using optical tweezers, we investigate target search and cleavage by CRISPR-Cas12a on force-stretched λ-DNA. Cas12a uses fast, one-dimensional hopping to locate its target. Binding and cleavage occur rapidly and specifically at low forces (≤5 pN), with a 1.8 nm rate-limiting conformational change. Mechanical distortion slows diffusion, increases off-target binding but hinders cleavage.},
}
@article {pmid34494480,
year = {2021},
author = {Amendola, M and Bedel, A and Buj-Bello, A and Carrara, M and Concordet, JP and Frati, G and Gilot, D and Giovannangeli, C and Gutierrez-Guerrero, A and Laurent, M and Miccio, A and Moreau-Gaudry, F and Sourd, C and Valton, J and Verhoeyen, E},
title = {Recent Progress in Genome Editing for Gene Therapy Applications: The French Perspective.},
journal = {Human gene therapy},
volume = {32},
number = {19-20},
pages = {1059-1075},
doi = {10.1089/hum.2021.191},
pmid = {34494480},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing ; Gene Transfer Techniques ; Genetic Therapy ; },
abstract = {Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.},
}
@article {pmid34494285,
year = {2022},
author = {Oemer, G and Koch, J and Wohlfarter, Y and Lackner, K and Gebert, REM and Geley, S and Zschocke, J and Keller, MA},
title = {The lipid environment modulates cardiolipin and phospholipid constitution in wild type and tafazzin-deficient cells.},
journal = {Journal of inherited metabolic disease},
volume = {45},
number = {1},
pages = {38-50},
doi = {10.1002/jimd.12433},
pmid = {34494285},
issn = {1573-2665},
mesh = {Acyltransferases/*genetics ; Barth Syndrome/genetics/*metabolism/pathology ; CRISPR-Cas Systems ; Cardiolipins/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Mitochondria/metabolism ; Mutation ; Phospholipids/*metabolism ; },
abstract = {Deficiency of the transacylase tafazzin due to loss of function variants in the X-chromosomal TAFAZZIN gene causes Barth syndrome (BTHS) with severe neonatal or infantile cardiomyopathy, neutropenia, myopathy, and short stature. The condition is characterized by drastic changes in the composition of cardiolipins, a mitochondria-specific class of phospholipids. Studies examining the impact of tafazzin deficiency on the metabolism of other phospholipids have so far generated inhomogeneous and partly conflicting results. Recent studies showed that the cardiolipin composition in cells and different murine tissues is highly dependent on the surrounding lipid environment. In order to study the relevance of different lipid states and tafazzin function for cardiolipin and phospholipid homeostasis we conducted systematic modulation experiments in a CRISPR/Cas9 knock-out model for BTHS. We found that-irrespective of tafazzin function-the composition of cardiolipins strongly depends on the nutritionally available lipid pool. Tafazzin deficiency causes a consistent shift towards cardiolipin species with more saturated and shorter acyl chains. Interestingly, the typical biochemical BTHS phenotype in phospholipid profiles of HEK 293T TAZ knock-out cells strongly depends on the cellular lipid context. In response to altered nutritional lipid compositions, we measured more pronounced changes on phospholipids that were largely masked under standard cell culturing conditions, therewith giving a possible explanation for the conflicting results reported so far on BTHS lipid phenotypes.},
}
@article {pmid34493846,
year = {2021},
author = {},
title = {License CRISPR patents for free to share gene editing globally.},
journal = {Nature},
volume = {597},
number = {7875},
pages = {152},
doi = {10.1038/d41586-021-02420-x},
pmid = {34493846},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}
@article {pmid34493749,
year = {2021},
author = {Gupta, S and Kumar, P and Rathi, B and Verma, V and Dhanda, RS and Devi, P and Yadav, M},
title = {Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {17801},
pmid = {34493749},
issn = {2045-2322},
mesh = {Adhesins, Escherichia coli/*genetics ; Animals ; Bacterial Adhesion/drug effects ; Biofilms/drug effects ; CRISPR-Associated Protein 9/administration &amp; dosage/genetics ; *CRISPR-Cas Systems ; Caenorhabditis elegans/microbiology ; Carbon ; Drug Delivery Systems ; Escherichia coli Infections/*microbiology ; Escherichia coli K12/drug effects/genetics ; Fimbriae Proteins/*genetics ; Gene Editing/*methods ; HeLa Cells ; Hemagglutination/drug effects ; Humans ; Mannose/pharmacology ; Pharmaceutical Vehicles ; Quantum Dots/*administration &amp; dosage/toxicity ; RNA, Guide/administration &amp; dosage/genetics ; THP-1 Cells ; Urinary Tract Infections/*microbiology ; Uropathogenic Escherichia coli/drug effects/*genetics/isolation &amp; purification/pathogenicity ; Virulence/genetics ; },
abstract = {Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.},
}
@article {pmid34490965,
year = {2021},
author = {Chen, HY and Hu, Y and Xu, XB and Zhou, YA and Li, NS and He, C and Xie, C and Lu, NH and Zhu, Y},
title = {Upregulation of oncogene Activin A receptor type I by Helicobacter pylori infection promotes gastric intestinal metaplasia via regulating CDX2.},
journal = {Helicobacter},
volume = {26},
number = {6},
pages = {e12849},
doi = {10.1111/hel.12849},
pmid = {34490965},
issn = {1523-5378},
mesh = {*Activin Receptors, Type I ; Activins ; Animals ; *CDX2 Transcription Factor/genetics/metabolism ; Gastric Mucosa/metabolism ; *Helicobacter Infections ; *Helicobacter pylori/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Humans ; Metaplasia ; Mice ; Mice, Inbred C57BL ; Oncogenes ; *Stomach Neoplasms/genetics ; Up-Regulation ; },
abstract = {BACKGROUND: Activin A receptor type I (ACVR1) is involved in tumorigenesis. However, the underlying molecular mechanisms of ACVR1 in gastric cancer (GC) and its association with Helicobacter pylori remained unclear.<br><br>MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) database were utilized to explore the ACVR1 expression in GC and normal control and its association with survival. The ACVR1 was knocked out using CRISPR/Cas-9; RNA sequencing analysis was performed in AGS cells with ACVR1&#xa0;knockout and normal control. Functional experiments (CCK-8, colony-forming, and transwell assays) were conducted to demonstrate the role of ACVR1 in cell proliferation, invasion, and metastasis. H.&#xa0;pylori-infected C57/BL6&#xa0;models were established. ACVR1, p-Smad1/5, and CDX2 were detected in AGS cells cocultured with H.&#xa0;pylori strains. The CDX2 and key elements of BMP signaling pathway were detected in AGS cells with ACVR1&#xa0;knockout and normal control. In addition, Immunohistochemistry was performed to detect the ACVR1 and CDX2 expression in gastric samples.<br><br>RESULTS: ACVR1 expression was higher in GC than normal control from TCGA, GEPIA, and samples collected from our hospital (p&#xa0;<&#xa0;0.05). ACVR1 promoted cell proliferation, migration, and invasion in vitro. Both cagA[+] and cagA[-] H.&#xa0;pylori could upregulate the expression ACVR1 (p&#xa0;<&#xa0;0.05). Downregulation of ACVR1 inhibited the H. pylori-induced cell proliferation, migration, and invasion (p&#xa0;<&#xa0;0.05). H. pylori increased the expression of p-Smad 1/5 and CDX2. The CDX2 and key elements of BMP signaling pathway were downregulated in AGS cells with ACVR1&#xa0;knockout. ACVR1 and CDX2 were upregulated in the stage of intestinal metaplasia (IM). Moreover, ACVR1 and CDX2 expressions were higher in H. pylori-positive group than H.&#xa0;pylori-negative group (p&#xa0;<&#xa0;0.05).<br><br>CONCLUSION: Our data indicate that H.&#xa0;pylori infection increases ACVR1 expression, promoting gastric IM via regulating CDX2, which is an essential step in H.&#xa0;pylori carcinogenesis.},
}
@article {pmid34490773,
year = {2021},
author = {Li, CY and Zheng, B and Lu, LL and Fang, WK and Zheng, MQ and Gao, JL and Yuheng, L and Pang, DW and Tang, HW},
title = {Biomimetic Chip Enhanced Time-Gated Luminescent CRISPR-Cas12a Biosensors under Functional DNA Regulation.},
journal = {Analytical chemistry},
volume = {93},
number = {37},
pages = {12514-12523},
doi = {10.1021/acs.analchem.1c01403},
pmid = {34490773},
issn = {1520-6882},
mesh = {Biomimetics ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; *DNA, Catalytic ; Humans ; Luminescence ; },
abstract = {Despite that the currently discovered CRISPR-Cas12a system is beneficial for improving the detection accuracy and design flexibility of luminescent biosensors, there are still challenges to extend target species and strengthen adaptability in complicated biological media. To conquer these obstacles, we present here some useful strategies. For the former, the limitation to nucleic acids assay is broken through by introducing a simple functional DNA regulation pathway to activate the unique trans-cleavage effect of this CRISPR system, under which the expected biosensors are capable of effectively transducing a protein (employing dual aptamers) and a metal ion (employing DNAzyme). For the latter, a time-gated luminescence resonance energy transfer imaging manner using a long-persistent nanophosphor as the energy donor is performed to completely eliminate the background interference and a nature-inspired biomimetic periodic chip constructed by photonic crystals is further combined to enhance the persistent luminescence. In line with the above efforts, the improved CRISPR-Cas12a luminescent biosensor not only exhibits a sound analysis performance toward the model targets (carcinoembryonic antigen and Na[+]) but also owns a strong anti-interference feature to actualize accurate sensing in human plasma samples, offering a new and applicative analytical tool for laboratory medicine.},
}
@article {pmid34489905,
year = {2021},
author = {Duan, C and Cao, H and Zhang, LH and Xu, Z},
title = {Harnessing the CRISPR-Cas Systems to Combat Antimicrobial Resistance.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {716064},
pmid = {34489905},
issn = {1664-302X},
abstract = {The emergence of antimicrobial-resistant (AMR) bacteria has become one of the most serious threats to global health, necessitating the development of novel antimicrobial strategies. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system, known as a bacterial adaptive immune system, can be repurposed to selectively target and destruct bacterial genomes other than invasive genetic elements. Thus, the CRISPR-Cas system offers an attractive option for the development of the next-generation antimicrobials to combat infectious diseases especially those caused by AMR pathogens. However, the application of CRISPR-Cas antimicrobials remains at a very preliminary stage and numerous obstacles await to be solved. In this mini-review, we summarize the development of using type I, type II, and type VI CRISPR-Cas antimicrobials to eradicate AMR pathogens and plasmids in the past a few years. We also discuss the most common challenges in applying CRISPR-Cas antimicrobials and potential solutions to overcome them.},
}
@article {pmid34489903,
year = {2021},
author = {Hoffert, M and Anderson, RE and Reveillaud, J and Murphy, LG and Stepanauskas, R and Huber, JA},
title = {Genomic Variation Influences Methanothermococcus Fitness in Marine Hydrothermal Systems.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {714920},
pmid = {34489903},
issn = {1664-302X},
abstract = {Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus lineages and their transcripts were more abundant than others in individual vent sites, indicating differential fitness among lineages. The relative abundances of lineages represented by SAGs in each of the samples matched phylogenetic relationships based on single-copy universal genes, and genes related to nitrogen fixation and the CRISPR/Cas immune system were among those differentiating the clades. Lineages possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.},
}
@article {pmid34489594,
year = {2021},
author = {Özcan, A and Krajeski, R and Ioannidi, E and Lee, B and Gardner, A and Makarova, KS and Koonin, EV and Abudayyeh, OO and Gootenberg, JS},
title = {Programmable RNA targeting with the single-protein CRISPR effector Cas7-11.},
journal = {Nature},
volume = {597},
number = {7878},
pages = {720-725},
pmid = {34489594},
issn = {1476-4687},
support = {R21 AI149694/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Computational Biology ; Deltaproteobacteria/genetics ; Escherichia coli ; *Gene Editing ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; RNA/*genetics ; RNA Interference ; },
abstract = {CRISPR-Cas interference is mediated by Cas effector nucleases that are either components of multisubunit complexes-in class 1 CRISPR-Cas systems-or domains of a single protein-in class 2 systems[1-3]. Here we show that the subtype III-E effector Cas7-11 is a single-protein effector in the class 1 CRISPR-Cas systems originating from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D. Cas7-11 from Desulfonema ishimotonii (DiCas7-11), when expressed in Escherichia coli, has substantial RNA interference effectivity against mRNAs and bacteriophages. Similar to many class 2 effectors-and unique among class 1 systems-DiCas7-11 processes pre-CRISPR RNA into mature CRISPR RNA (crRNA) and cleaves RNA at positions defined by the target:spacer duplex, without detectable non-specific activity. We engineered Cas7-11 for RNA knockdown and editing in mammalian cells. We show that Cas7-11 has no effects on cell viability, whereas other RNA-targeting tools (such as short hairpin RNAs and Cas13) show substantial cell toxicity[4,5]. This study illustrates the evolution of a single-protein effector from multisubunit class 1 effector complexes, expanding our understanding of the diversity of CRISPR systems. Cas7-11 provides the basis for new programmable RNA-targeting tools that are free of collateral activity and cell toxicity.},
}
@article {pmid34489579,
year = {2021},
author = {van der Oost, J and Fresco, LO},
title = {Waive CRISPR patents to meet food needs in low-income countries.},
journal = {Nature},
volume = {597},
number = {7875},
pages = {178},
doi = {10.1038/d41586-021-02397-7},
pmid = {34489579},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; Developing Countries/*economics ; *Food Supply/methods/standards ; Gene Editing/economics/*legislation &amp; jurisprudence ; Humans ; Licensure/economics/*legislation &amp; jurisprudence ; Patents as Topic/*legislation &amp; jurisprudence ; Plant Breeding/economics/legislation &amp; jurisprudence/methods ; Poverty ; },
}
@article {pmid34487614,
year = {2022},
author = {Galli, M and Martiny, E and Imani, J and Kumar, N and Koch, A and Steinbrenner, J and Kogel, KH},
title = {CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity, transcriptional gene silencing and plant growth.},
journal = {Plant biotechnology journal},
volume = {20},
number = {1},
pages = {89-102},
pmid = {34487614},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant/genetics ; Gene Silencing ; *Hordeum/metabolism ; Plant Diseases/microbiology ; Plant Immunity ; Plant Proteins/genetics/metabolism ; },
abstract = {The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.},
}
@article {pmid34487299,
year = {2021},
author = {Guzmán, NM and Esquerra-Ruvira, B and Mojica, FJM},
title = {Digging into the lesser-known aspects of CRISPR biology.},
journal = {International microbiology : the official journal of the Spanish Society for Microbiology},
volume = {24},
number = {4},
pages = {473-498},
pmid = {34487299},
issn = {1618-1905},
mesh = {*Archaea/genetics ; Bacteria/genetics ; Biology ; *CRISPR-Cas Systems ; },
abstract = {A long time has passed since regularly interspaced DNA repeats were discovered in prokaryotes. Today, those enigmatic repetitive elements termed clustered regularly interspaced short palindromic repeats (CRISPR) are acknowledged as an emblematic part of multicomponent CRISPR-Cas (CRISPR associated) systems. These systems are involved in a variety of roles in bacteria and archaea, notably, that of conferring protection against transmissible genetic elements through an adaptive immune-like response. This review summarises the present knowledge on the diversity, molecular mechanisms and biology of CRISPR-Cas. We pay special attention to the most recent findings related to the determinants and consequences of CRISPR-Cas activity. Research on the basic features of these systems illustrates how instrumental the study of prokaryotes is for understanding biology in general, ultimately providing valuable tools for diverse fields and fuelling research beyond the mainstream.},
}
@article {pmid34486619,
year = {2021},
author = {Gong, H and Wu, Y and Zeng, R and Zeng, Y and Liu, X and Tang, D},
title = {CRISPR/Cas12a-mediated liposome-amplified strategy for the photoelectrochemical detection of nucleic acid.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {71},
pages = {8977-8980},
doi = {10.1039/d1cc03743a},
pmid = {34486619},
issn = {1364-548X},
mesh = {Bacterial Proteins/*chemistry ; Biosensing Techniques/*methods ; CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry ; DNA, Viral/*analysis ; Dopamine/chemistry ; Electrochemical Techniques/methods ; Endodeoxyribonucleases/*chemistry ; Fluoresceins/chemistry ; Fluorescent Dyes/chemistry ; Human papillomavirus 16/chemistry ; Liposomes/chemistry ; Magnetic Phenomena ; Photochemical Processes ; },
abstract = {This study reports a photoelectrochemical biosensor for dopamine-loaded liposome-encoded magnetic beads cleaved by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 12a system for the quantification of human papilloma virus (HPV)-related DNA using neodymium-doped BiOBr nanosheets (Nd-BiOBr) as a photoactive matrix. Magnetic beads and dopamine-loaded liposomes are covalently attached to the both ends of ssDNA to construct dumbbell-shaped dopamine-loaded liposome-encoded magnetic bead (DLL-MB) probes. When the guide RNA binds to the target HPV-16, the ssDNA will be cleaved by Cas12a, thereby degrading the double dumbbell probes. After magnetic separation, the dissolved DLLs are treated with Triton X-100 to release the dopamine (as an electron donor), which was then detected by an amplified photocurrent using the Nd-BiOBr-based photoelectrode.},
}
@article {pmid34486151,
year = {2021},
author = {Baliga, P and Shekar, M and Tg, P and Sk, G},
title = {Investigation into the prevalent CRISPR-Cas systems among the Aeromonas genus.},
journal = {Journal of basic microbiology},
volume = {61},
number = {10},
pages = {874-882},
doi = {10.1002/jobm.202100234},
pmid = {34486151},
issn = {1521-4028},
mesh = {Aeromonas/*genetics ; Bacteriophages/genetics ; *CRISPR-Cas Systems ; Computational Biology/methods ; Genome, Bacterial ; Phylogeny ; Plasmids ; Repetitive Sequences, Nucleic Acid ; Sequence Analysis, DNA ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune system that checks invasion by mobile genetic elements through nuclease targeting. In this study, we investigated the occurrence, diversity, and features of the CRISPR system in the genus Aeromonas using bioinformatics tools. Only 13 out of 122 complete genomes (10.66%) of the genus Aeromonas from the NCBI GenBank database harbored the CRISPR system. The Type I-F system was the most prevalent CRISPR system among the Aeromonads, followed by the Type I-E system. Only one strain harbored a Type I-C CRISPR system. Among the Aeromonads, Aeromonas caviae (22.7%) and Aeromonas veronii (20%) had a higher prevalence rate of the complete CRISPR system. The analysis of direct repeat (DR) sequences showed that all could form stable RNA secondary structures. A phylogenetic tree generated for the Cas1 protein classified CRISPR subtypes into three distinct clusters. Among the 748 spacers investigated, 41.98% and 17.25% showed perfect homology to phage and plasmid sequences, respectively. Some arrays had duplicated spacers. The CRISPR loci are closely linked to antibiotic resistance genes in most strains. Collectively, our results would contribute to research on antibiotic resistance in the Aeromonas group, and provide new insights into the diversity and evolution of the CRISPR-Cas system.},
}
@article {pmid34485933,
year = {2021},
author = {Marinova, IN and Wandall, HH and Dabelsteen, S},
title = {Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100668},
pmid = {34485933},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems ; Fibroblasts ; Gene Knockout Techniques/*methods ; Glycosylation ; HEK293 Cells ; Humans ; Keratinocytes/cytology ; Lentivirus/genetics ; Organoids/*cytology/physiology ; Skin/*cytology ; },
abstract = {Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids. For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020).},
}
@article {pmid34485609,
year = {2021},
author = {Głów, D and Meyer, S and García Roldán, I and Akingunsade, LM and Riecken, K and Fehse, B},
title = {LATE-a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {22},
number = {},
pages = {249-262},
pmid = {34485609},
issn = {2329-0501},
abstract = {Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.},
}
@article {pmid34485599,
year = {2021},
author = {Zhang, Y and Nishiyama, T and Li, H and Huang, J and Atmanli, A and Sanchez-Ortiz, E and Wang, Z and Mireault, AA and Mammen, PPA and Bassel-Duby, R and Olson, EN},
title = {A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {22},
number = {},
pages = {122-132},
pmid = {34485599},
issn = {2329-0501},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
abstract = {Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD.},
}
@article {pmid34485380,
year = {2021},
author = {Chen, CC and Guan, G and Qi, X and Abulaiti, A and Zhang, T and Liu, J and Lu, F and Chen, X},
title = {Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System.},
journal = {Frontiers in molecular biosciences},
volume = {8},
number = {},
pages = {676957},
pmid = {34485380},
issn = {2296-889X},
abstract = {The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.},
}
@article {pmid34484175,
year = {2021},
author = {Dawes, JC and Uren, AG},
title = {Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {670280},
pmid = {34484175},
issn = {1664-3224},
support = {MC_UP_A652_1001/MRC_/Medical Research Council/United Kingdom ; MC_A652_5PZ20/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Humans ; Leukemia, B-Cell/*genetics ; Lymphoma, B-Cell/*genetics ; Mice ; Mutagenesis, Insertional/methods ; },
abstract = {Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.},
}
@article {pmid34482543,
year = {2021},
author = {Xu, H and Wali, R and Cheruiyot, C and Bodenschatz, J and Hasenfuss, G and Janshoff, A and Habeck, M and Ebert, A},
title = {Non-negative blind deconvolution for signal processing in a CRISPR-edited iPSC-cardiomyocyte model of dilated cardiomyopathy.},
journal = {FEBS letters},
volume = {595},
number = {20},
pages = {2544-2557},
doi = {10.1002/1873-3468.14189},
pmid = {34482543},
issn = {1873-3468},
mesh = {*CRISPR-Cas Systems ; Cardiomyopathy, Dilated/genetics/*pathology ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*pathology ; *Models, Biological ; Mutation ; Myocytes, Cardiac/*pathology ; Troponin T/genetics ; },
abstract = {We developed an integrated platform for analysis of parameterized data from human disease models. We report a non-negative blind deconvolution (NNBD) approach to quantify calcium (Ca[2+]) handling, beating force and contractility in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at the single-cell level. We employed CRISPR/Cas gene editing to introduce a dilated cardiomyopathy (DCM)-causing mutation in troponin T (TnT), TnT-R141W, into wild-type control iPSCs (MUT). The NNDB-based method enabled data parametrization, fitting and analysis in wild-type controls versus isogenic MUT iPSC-CMs. Of note, Cas9-edited TnT-R141W iPSC-CMs revealed significantly reduced beating force and prolonged contractile event duration. The NNBD-based platform provides an alternative framework for improved quantitation of molecular disease phenotypes and may contribute to the development of novel diagnostic tools.},
}
@article {pmid34481190,
year = {2021},
author = {Schoger, E and Zimmermann, WH and Cyganek, L and Zelarayán, LC},
title = {Establishment of a second generation homozygous CRISPRa human induced pluripotent stem cell (hiPSC) line for enhanced levels of endogenous gene activation.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102518},
doi = {10.1016/j.scr.2021.102518},
pmid = {34481190},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases ; Humans ; *Induced Pluripotent Stem Cells ; Transcriptional Activation ; Transgenes ; },
abstract = {CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.},
}
@article {pmid34481189,
year = {2021},
author = {Jo, S and Kim, JW and Noh, H and Kim, H and Kim, JH and Park, HJ},
title = {Generation of an ACTA2-EGFP reporter human induced pluripotent stem cell line, KITi001-C-41, using CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102524},
doi = {10.1016/j.scr.2021.102524},
pmid = {34481189},
issn = {1876-7753},
mesh = {Actins/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Genes, Reporter ; Homologous Recombination ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Alpha-smooth muscle actin (α-SMA) is encoded by ACTA2 and is a key protein in the cellular contractile system of various mesodermal cell types, including hepatic stellate cells (HSCs), smooth muscle cells, and cardiomyocytes. α-SMA, which is a key protein in the development of hepatic fibrosis, is widely used as a reliable marker of HSC activation. Here, we generated an ACTA2-EGFP reporter human induced pluripotent stem cell line, KITi001-C-41, using a CRISPR/Cas9-based knock-in system. These reporter hiPSC lines can be used for lineage tracing of mesodermal cells and for screening of HSC activation factors.},
}
@article {pmid34481123,
year = {2021},
author = {Halbgebauer, D and Roos, J and Funcke, JB and Neubauer, H and Hamilton, BS and Simon, E and Amri, EZ and Debatin, KM and Wabitsch, M and Fischer-Posovszky, P and Tews, D},
title = {Latent TGFβ-binding proteins regulate UCP1 expression and function via TGFβ2.},
journal = {Molecular metabolism},
volume = {53},
number = {},
pages = {101336},
pmid = {34481123},
issn = {2212-8778},
mesh = {Adipose Tissue, White/metabolism ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Humans ; Latent TGF-beta Binding Proteins/deficiency/*metabolism ; Transforming Growth Factor beta2/*metabolism ; Uncoupling Protein 1/genetics/*metabolism ; },
abstract = {OBJECTIVE: Activation of brown adipose tissue (BAT) in humans has been proposed as a new treatment approach for combating obesity and its associated diseases, as BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood.<br><br>METHODS: Profiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting that these proteins are capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes.<br><br>RESULTS: We demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation, but diminishes UCP1 expression and function in the obtained mature adipocytes. We further show that these effects are dependent on TGF&#x3b2;2 but not TGF&#x3b2;1 signaling: TGF&#x3b2;2 deficiency decreases adipocyte UCP1 expression, whereas TGF&#x3b2;2 treatment increases it. The activity of the LTBP3-TGF&#x3b2;2 axis that we delineate herein also significantly correlates with UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well.<br><br>CONCLUSIONS: These results provide evidence that LTBP3, via TGF&#x3b2;2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.},
}
@article {pmid34480847,
year = {2021},
author = {Xu, X and Chemparathy, A and Zeng, L and Kempton, HR and Shang, S and Nakamura, M and Qi, LS},
title = {Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing.},
journal = {Molecular cell},
volume = {81},
number = {20},
pages = {4333-4345.e4},
doi = {10.1016/j.molcel.2021.08.008},
pmid = {34480847},
issn = {1097-4164},
mesh = {CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Mutation ; Promoter Regions, Genetic ; *Protein Engineering ; RNA, Guide/genetics/metabolism ; *Transcriptional Activation ; },
abstract = {Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.},
}
@article {pmid34480604,
year = {2021},
author = {Snijders, KE and Fehér, A and Táncos, Z and Bock, I and Téglási, A and van den Berk, L and Niemeijer, M and Bouwman, P and Le Dévédec, SE and Moné, MJ and Van Rossom, R and Kumar, M and Wilmes, A and Jennings, P and Verfaillie, CM and Kobolák, J and Ter Braak, B and Dinnyés, A and van de Water, B},
title = {Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.},
journal = {Archives of toxicology},
volume = {95},
number = {10},
pages = {3285-3302},
pmid = {34480604},
issn = {1432-0738},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cells, Cultured ; Dose-Response Relationship, Drug ; Green Fluorescent Proteins/genetics ; Heme Oxygenase-1/*genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Male ; Maleates/administration &amp; dosage/toxicity ; Middle Aged ; Oleanolic Acid/administration &amp; dosage/analogs &amp; derivatives/toxicity ; Oxidative Stress/*drug effects ; RNA, Messenger/genetics ; Time Factors ; },
abstract = {Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.},
}
@article {pmid34480322,
year = {2022},
author = {Orf, GS and Gisriel, CJ and Granstrom, J and Baker, PL and Redding, KE},
title = {The PshX subunit of the photochemical reaction center from Heliobacterium modesticaldum acts as a low-energy antenna.},
journal = {Photosynthesis research},
volume = {151},
number = {1},
pages = {11-30},
pmid = {34480322},
issn = {1573-5079},
mesh = {*Bacteriochlorophylls ; *Clostridiales ; },
abstract = {The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. To assess the function of PshX, we produced a ∆pshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ∆pshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer toward the P800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.},
}
@article {pmid34479645,
year = {2021},
author = {Zhou, K and Xu, Y and Zhang, R and Qian, PY},
title = {Arms race in a cell: genomic, transcriptomic, and proteomic insights into intracellular phage-bacteria interplay in deep-sea snail holobionts.},
journal = {Microbiome},
volume = {9},
number = {1},
pages = {182},
pmid = {34479645},
issn = {2049-2618},
mesh = {Animals ; Bacteria/genetics ; *Bacteriophages/genetics ; Genomics ; Proteomics ; Snails ; Transcriptome/genetics ; },
abstract = {BACKGROUND: Deep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown.<br><br>RESULTS: We conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR-Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction-modification systems and dormancy-inducing toxin-antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication.<br><br>CONCLUSIONS: This study provides new insights into phage-bacteria interplay in intracellular environments of a deep-sea vent snail. Video Abstract.},
}
@article {pmid34479069,
year = {2021},
author = {Krumm, L and Pozner, T and Kaindl, J and Regensburger, M and Günther, C and Turan, S and Asadollahi, R and Rauch, A and Winner, B},
title = {Generation and characterization of an endogenously tagged SPG11-human iPSC line by CRISPR/Cas9 mediated knock-in.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102520},
doi = {10.1016/j.scr.2021.102520},
pmid = {34479069},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Proteins/genetics ; *Spastic Paraplegia, Hereditary/genetics ; },
abstract = {Pathogenic bi-allelic variants in the SPG11 gene result in rare motor neuron disorders such as Hereditary Spastic Paraplegia type 11, Charcot-Marie Tooth, and Juvenile Amyotrophic Lateral Sclerosis-5. The main challenge in SPG11-linked disease research is the lack of antibodies against SPG11 encoded spatacsin. Here, we describe the CRISPR/Cas9 mediated generation and validation of an endogenously tagged SPG11- human iPSC line that contains an HA tag at the C-terminus of SPG11. The line exhibits multi-lineage differentiation potential and holds promise for studying the role of spatacsin and for the elucidation of SPG11-associated pathogenesis. Resource Table.},
}
@article {pmid34478654,
year = {2021},
author = {Donohoue, PD and Pacesa, M and Lau, E and Vidal, B and Irby, MJ and Nyer, DB and Rotstein, T and Banh, L and Toh, MS and Gibson, J and Kohrs, B and Baek, K and Owen, ALG and Slorach, EM and van Overbeek, M and Fuller, CK and May, AP and Jinek, M and Cameron, P},
title = {Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.},
journal = {Molecular cell},
volume = {81},
number = {17},
pages = {3637-3649.e5},
doi = {10.1016/j.molcel.2021.07.035},
pmid = {34478654},
issn = {1097-4164},
mesh = {CRISPR-Associated Protein 9/*metabolism/physiology ; CRISPR-Associated Proteins/metabolism/physiology ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Endonucleases/genetics ; Gene Editing/methods ; Genetic Techniques ; Genome/genetics ; Genomics/methods ; Humans ; Leukocytes, Mononuclear/metabolism ; Molecular Conformation ; RNA, Guide/genetics ; Structure-Activity Relationship ; T-Lymphocytes/*metabolism/physiology ; },
abstract = {The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.},
}
@article {pmid34478558,
year = {2021},
author = {Lee, KZ and Mechikoff, MA and Kikla, A and Liu, A and Pandolfi, P and Fitzgerald, K and Gimble, FS and Solomon, KV},
title = {NgAgo possesses guided DNA nicking activity.},
journal = {Nucleic acids research},
volume = {49},
number = {17},
pages = {9926-9937},
pmid = {34478558},
issn = {1362-4962},
mesh = {Argonaute Proteins/*metabolism ; *DNA Cleavage ; DNA Helicases/genetics ; DNA, Bacterial/genetics/*metabolism ; Escherichia coli/genetics ; Gene Editing/*methods ; Homologous Recombination/genetics ; Natronobacterium/*enzymology/genetics/metabolism ; Trans-Activators/genetics ; },
abstract = {Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.},
}
@article {pmid34478496,
year = {2021},
author = {Yang, S and Zhang, Y and Xu, J and Zhang, J and Zhang, J and Yang, J and Jiang, Y and Yang, S},
title = {Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration.},
journal = {Nucleic acids research},
volume = {49},
number = {17},
pages = {10192-10202},
pmid = {34478496},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Engineering/*methods ; Escherichia coli/genetics ; Pseudoalteromonas/*genetics ; RNA, Bacterial/genetics ; Synthetic Biology/methods ; Transposases/genetics/*metabolism ; Vibrio cholerae/*genetics ; },
abstract = {Cell engineering is commonly limited to the serial manipulation of a single gene or locus. The recently discovered CRISPR-associated transposases (CASTs) could manipulate multiple sets of genes to achieve predetermined cell diversity, with orthogonal CASTs being able to manipulate them in parallel. Here, a novel CAST from Pseudoalteromonas translucida KMM520 (PtrCAST) was characterized without a protospacer adjacent motif (PAM) preference which can achieve a high insertion efficiency for larger cargo and multiplexed transposition and tolerate mismatches out of 4-nucleotide seed sequence. More importantly, PtrCAST operates orthogonally with CAST from Vibrio cholerae Tn6677 (VchCAST), though both belonging to type I-F3. The two CASTs were exclusively active on their respective mini-Tn substrate with their respective crRNAs that target the corresponding 5 and 2 loci in one Escherichia coli cell. The multiplexed orthogonal MUCICAT (MUlticopy Chromosomal Integration using CRISPR-Associated Transposases) is a powerful tool for cell programming and appears promising with applications in synthetic biology.},
}
@article {pmid34477520,
year = {2021},
author = {Singh, DD and Verma, R and Tripathi, SK and Sahu, R and Trivedi, P and Yadav, DK},
title = {Breast Cancer Transcriptional Regulatory Network Reprogramming by using the CRISPR/Cas9 System: An Oncogenetics Perspective.},
journal = {Current topics in medicinal chemistry},
volume = {21},
number = {31},
pages = {2800-2813},
doi = {10.2174/1568026621666210902120754},
pmid = {34477520},
issn = {1873-4294},
mesh = {Animals ; Breast Neoplasms/*genetics/*therapy ; CRISPR-Cas Systems/*genetics ; Carcinogenesis/genetics ; *Gene Editing ; *Gene Expression Regulation, Neoplastic ; *Gene Regulatory Networks ; Humans ; Oncogenes/*genetics ; },
abstract = {Breast cancer (BC) is the second most commonly diagnosed cancer in the world. BC develops due to dysregulation of transcriptional profiles, substantial interpatient variations, genetic mutations, and dysregulation of signaling pathways in breast cells. These events are regulated by many genes such as BRCA1/2, PTEN, TP53, mTOR, TERT, AKT, PI3K and others genes. Treatment options for BC remain a hurdle, which warrants a comprehensive understanding that establishes an interlinking connection between these genes in BC tumorigenesis. Consequently, there is an increasing demand for alternative treatment approaches and the design of more effective treatments. In this regard, it is crucial to build the corresponding transcriptional regulatory networks governing BC by using advanced genetic tools and techniques. In the past, several molecular editing technologies have been used to edit genes with several limitations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (CRISPR/Cas9) recently received wise attention due to its potential in biomedical and therapeutic applications. Here, we review the role of various molecular signalling pathways dysregulated in BC development such as PTEN/PI3K/AKT/mTOR as well as BRCA1/BRCA2/TP53/TERT and their interplay between the related gene networks in BC initiation, progression and development of resistance against available targeted therapeutic agents. Use of CRISPR/Cas9 gene-editing technology to generate BC gene-specific transgenic cell lines and animal models to decipher their role and interactions with other gene products has been employed successfully. Moreover, the significance of using CRISPR/Cas9 technology to develop early BC diagnostic tools and treatments is discussed here.},
}
@article {pmid34477109,
year = {2021},
author = {Sun, S and Li, S and Luo, Z and Ren, M and He, S and Wang, G and Liu, Z},
title = {Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34477109},
issn = {2050-084X},
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cochlea/*cytology ; Computational Biology ; Estrogen Antagonists/pharmacology ; Gene Expression Regulation/drug effects/physiology ; Gene Knock-In Techniques ; Hair Cells, Auditory/*physiology ; Ikaros Transcription Factor/genetics/*metabolism ; Mice ; Microscopy, Electron, Scanning ; Molecular Motor Proteins/genetics/metabolism ; RNA/genetics/metabolism ; RNA, Untranslated/genetics/*metabolism ; Sequence Analysis, RNA ; Single-Cell Analysis ; Sulfate Transporters/genetics/*metabolism ; Tamoxifen/pharmacology ; },
abstract = {Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.},
}
@article {pmid34475857,
year = {2021},
author = {Liu, Q and Wang, S and Long, J and Chen, Z and Yang, B and Lin, F},
title = {Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {686715},
pmid = {34475857},
issn = {1664-302X},
abstract = {The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5'-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99[A] strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.},
}
@article {pmid34475598,
year = {2021},
author = {},
title = {Kazuto Kato: the ethics of editing humanity.},
journal = {Bulletin of the World Health Organization},
volume = {99},
number = {9},
pages = {616-617},
doi = {10.2471/BLT.21.030921},
pmid = {34475598},
issn = {1564-0604},
mesh = {*Bioethical Issues/history ; Biotechnology/*ethics ; CRISPR-Cas Systems ; Ethical Theory ; *Ethics, Medical/history ; Gene Editing/*ethics/history ; History, 21st Century ; Human Characteristics ; Humans ; },
abstract = {Gary Humphreys talks to Kazuto Kato about the ethical and societal challenges posed by biotechnologies that allow for the editing of the human genome.},
}
@article {pmid34475565,
year = {2021},
author = {Wu, Z and Zhang, Y and Yu, H and Pan, D and Wang, Y and Wang, Y and Li, F and Liu, C and Nan, H and Chen, W and Ji, Q},
title = {Programmed genome editing by a miniature CRISPR-Cas12f nuclease.},
journal = {Nature chemical biology},
volume = {17},
number = {11},
pages = {1132-1138},
pmid = {34475565},
issn = {1552-4469},
mesh = {Bacillales/*chemistry ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; Gene Editing ; },
abstract = {The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.},
}
@article {pmid34475560,
year = {2022},
author = {Kim, DY and Lee, JM and Moon, SB and Chin, HJ and Park, S and Lim, Y and Kim, D and Koo, T and Ko, JH and Kim, YS},
title = {Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus.},
journal = {Nature biotechnology},
volume = {40},
number = {1},
pages = {94-102},
pmid = {34475560},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; Endonucleases/genetics ; Gene Editing ; Humans ; RNA ; *RNA, Guide/genetics ; },
abstract = {Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5' terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA-crRNA complementary region, a penta(uridinylate) sequence, the 3' terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.},
}
@article {pmid34475519,
year = {2022},
author = {Bartlau, N and Wichels, A and Krohne, G and Adriaenssens, EM and Heins, A and Fuchs, BM and Amann, R and Moraru, C},
title = {Highly diverse flavobacterial phages isolated from North Sea spring blooms.},
journal = {The ISME journal},
volume = {16},
number = {2},
pages = {555-568},
pmid = {34475519},
issn = {1751-7370},
support = {BBS/E/F/000PR10353/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/F/000PR10356/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Bacteriophages/genetics ; Eutrophication ; *Flavobacteriaceae/genetics ; Humans ; Metagenome ; North Sea ; },
abstract = {It is generally recognized that phages are a mortality factor for their bacterial hosts. This could be particularly true in spring phytoplankton blooms, which are known to be closely followed by a highly specialized bacterial community. We hypothesized that phages modulate these dense heterotrophic bacteria successions following phytoplankton blooms. In this study, we focused on Flavobacteriia, because they are main responders during these blooms and have an important role in the degradation of polysaccharides. A cultivation-based approach was used, obtaining 44 lytic flavobacterial phages (flavophages), representing twelve new species from two viral realms. Taxonomic analysis allowed us to delineate ten new phage genera and ten new families, from which nine and four, respectively, had no previously cultivated representatives. Genomic analysis predicted various life styles and genomic replication strategies. A likely eukaryote-associated host habitat was reflected in the gene content of some of the flavophages. Detection in cellular metagenomes and by direct-plating showed that part of these phages were actively replicating in the environment during the 2018 spring bloom. Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years suggested that, at least part of the new flavophages are stable components of the microbial community in the North Sea. Together, our results indicate that these diverse flavophages have the potential to modulate their respective host populations.},
}
@article {pmid34474931,
year = {2021},
author = {Jacquet, K and Vidal-Cruchez, O and Rezzonico, R and Nicolini, VJ and Mograbi, B and Hofman, P and Vassaux, G and Mari, B and Brest, P},
title = {New technologies for improved relevance in miRNA research.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {12},
pages = {1060-1063},
doi = {10.1016/j.tig.2021.08.006},
pmid = {34474931},
issn = {0168-9525},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *MicroRNAs/genetics ; Reproducibility of Results ; },
abstract = {After a number of years of research in the field of miRNA, the robustness and biological relevance of many published articles is increasingly being questioned. We propose the use of new RNA-seq approaches, genome editing technologies, and updated public databases to improve the quality, reliability, and relevance of published data.},
}
@article {pmid34474054,
year = {2021},
author = {Chandrasekaran, M and Boopathi, T and Paramasivan, M},
title = {A status-quo review on CRISPR-Cas9 gene editing applications in tomato.},
journal = {International journal of biological macromolecules},
volume = {190},
number = {},
pages = {120-129},
doi = {10.1016/j.ijbiomac.2021.08.169},
pmid = {34474054},
issn = {1879-0003},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Engineering ; Lycopersicon esculentum/*genetics/growth &amp; development ; Stress, Physiological/genetics ; },
abstract = {Epigenetic changes are emancipated in horticultural crops including tomato due to a variety of environmental factors. These modifications rely on plant phenotypes mediated by genetic architecture consequently resulting in hereditary epigenetic memory. Genome editing strategies like CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/CRISPR-associated protein 9 (Cas9) technologies have revolutionized plants biology foreseeing stable inheritance of epigenetic modifications. CRISPR/Cas9 strategy poses as explicit advancement in providing precise genome editing with minimal off-target mutations, ease of experimental design, higher efficiency, versatility, and cost-effectiveness. Dicot crops, especially tomato remain an ideal candidate for CRISPR/Cas9 based gene modulations thereby augmenting productivity and yields. In the present review, key questions on CRISPR/Cas9 applications aid in enhanced growth based on optimal gene discovery, de novo modification, trait improvement, and biotic/abiotic stress management are discussed. In addition, comparative scenario in tomato and similar horticultural crops are adequately summarized for the pros and cons. Further, limitations hampering potential benefits and success phenomena of the lab to field transition of gene editing alterations are discussed collaterally in addressing futuristic optimization for CRISPR/Cas9 research in tomato.},
}
@article {pmid34473995,
year = {2021},
author = {Enríquez, P and Krajewski, K and Strahl, BD and Rothbart, SB and Dowen, RH and Rose, RB},
title = {Binding specificity and function of the SWI/SNF subunit SMARCA4 bromodomain interaction with acetylated histone H3K14.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {4},
pages = {101145},
pmid = {34473995},
issn = {1083-351X},
support = {S10 RR025528/RR/NCRR NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; S10 RR028976/RR/NCRR NIH HHS/United States ; R35 GM126900/GM/NIGMS NIH HHS/United States ; R35 GM124736/GM/NIGMS NIH HHS/United States ; },
mesh = {Acetylation ; Animals ; Caenorhabditis elegans/genetics/*metabolism ; Caenorhabditis elegans Proteins/genetics/*metabolism ; Histones/genetics/*metabolism ; Humans ; Protein Binding ; Transcription Factors/genetics/*metabolism ; },
abstract = {Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD-H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases.},
}
@article {pmid34472610,
year = {2021},
author = {Fujikawa, T and Ogura, Y and Ishigami, K and Kawano, Y and Nagamine, M and Hayashi, T and Inoue, K},
title = {Unexpected genomic features of high current density-producing Geobacter sulfurreducens strain YM18.},
journal = {FEMS microbiology letters},
volume = {368},
number = {17},
pages = {},
doi = {10.1093/femsle/fnab119},
pmid = {34472610},
issn = {1574-6968},
mesh = {Electrodes ; Electron Transport ; *Genetic Variation ; *Genome, Bacterial/genetics ; Genomics ; *Geobacter/genetics ; },
abstract = {Geobacter sulfurreducens produces high current densities and it has been used as a model organism for extracellular electron transfer studies. Nine G. sulfurreducens strains were isolated from biofilms formed on an anode poised at -0.2 V (vs SHE) in a bioelectrochemical system in which river sediment was used as an inoculum. The maximum current density of an isolate, strain YM18 (9.29 A/m2), was higher than that of the strain PCA (5.72 A/m2), the type strain of G. sulfurreducens, and comparable to strain KN400 (8.38 A/m2), which is another high current-producing strain of G. sulfurreducens. Genomic comparison of strains PCA, KN400 and YM18 revealed that omcB, xapD, spc and ompJ, which are known to be important genes for iron reduction and current production in PCA, were not present in YM18. In the PCA and KN400 genomes, two and one region(s) encoding CRISPR/Cas systems were identified, respectively, but they were missing in the YM18 genome. These results indicate that there is genetic variation in the key components involved in extracellular electron transfer among G. sulfurreducens strains.},
}
@article {pmid34472225,
year = {2021},
author = {Cui, Y and Liu, ZL and Li, CC and Wei, XM and Lin, YJ and You, L and Zhu, ZD and Deng, HM and Feng, QL and Huang, YP and Xiang, H},
title = {Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication.},
journal = {Zoological research},
volume = {42},
number = {5},
pages = {637-649},
pmid = {34472225},
issn = {2095-8137},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; Bombyx/growth &amp; development/*metabolism ; Brain/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation, Developmental ; Genotype ; Insect Proteins/genetics/*metabolism ; Integumentary System/physiology ; Juvenile Hormones/*metabolism ; Larva/growth &amp; development/metabolism ; Phylogeny ; Protein Conformation ; },
abstract = {The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 (Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.},
}
@article {pmid34471706,
year = {2021},
author = {O'Leary, JK and Sleator, RD and Lucey, B},
title = {Cryptosporidium spp. diagnosis and research in the 21[st] century.},
journal = {Food and waterborne parasitology},
volume = {24},
number = {},
pages = {e00131},
pmid = {34471706},
issn = {2405-6766},
abstract = {The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21[st] century. Herein, we provide a review of these advances.},
}
@article {pmid34471126,
year = {2021},
author = {Tong, Y and Jørgensen, TS and Whitford, CM and Weber, T and Lee, SY},
title = {A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5206},
pmid = {34471126},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial ; Escherichia coli/*genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial ; Plasmids ; RNA, Guide/genetics ; },
abstract = {CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.},
}
@article {pmid34469777,
year = {2021},
author = {You, J and Xi, H and Ma, S and Yang, XJ and Chen, L},
title = {Generation of a homozygous LRPAP1 knockout human embryonic stem cell line (FDCHDPe009-B) by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102516},
doi = {10.1016/j.scr.2021.102516},
pmid = {34469777},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; Low Density Lipoprotein Receptor-Related Protein-1 ; },
abstract = {The homozygous autosomal recessive truncating mutations of LDL receptor related protein associated protein 1 (LRPAP1) is a possible reason for Nonsyndromic Extreme Myopia, patients with which show typical chorioretinal degeneration. We generated an LRPAP1 knockout FDCHDPe009-B embryonic stem cell line to study mechanisms of retinal degeneration underlying LRPAP1 deficiency with the help of the CRISPR/Cas9 system. Two distinct biallelic deletions in the cell line have been confirmed, which causing a frameshift and premature stop codons thus influence the translation of LRPAP1. FDCHDPe009-B has maintained normal stem cell morphology, pluripotent gene expression, parental karyotype, and ability to differentiate into three germ layers.},
}
@article {pmid34469736,
year = {2021},
author = {Parrish, PCR and Thomas, JD and Gabel, AM and Kamlapurkar, S and Bradley, RK and Berger, AH},
title = {Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome.},
journal = {Cell reports},
volume = {36},
number = {9},
pages = {109597},
pmid = {34469736},
issn = {2211-1247},
support = {R37 CA252050/CA/NCI NIH HHS/United States ; K99 CA197762/CA/NCI NIH HHS/United States ; P30 CA015704/CA/NCI NIH HHS/United States ; S10 OD028685/OD/NIH HHS/United States ; R01 CA251138/CA/NCI NIH HHS/United States ; R01 DK103854/DK/NIDDK NIH HHS/United States ; R01 HL128239/HL/NHLBI NIH HHS/United States ; T32 HG000035/HG/NHGRI NIH HHS/United States ; R00 CA197762/CA/NCI NIH HHS/United States ; R56 DK103854/DK/NIDDK NIH HHS/United States ; R01 HL151651/HL/NHLBI NIH HHS/United States ; },
mesh = {Adult ; Antineoplastic Agents/pharmacology ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Female ; *Gene Duplication ; Gene Expression Regulation, Neoplastic ; *Genes, Lethal ; *Genes, Synthetic ; *Genes, Tumor Suppressor ; *Genome, Human ; HEK293 Cells ; HeLa Cells ; Humans ; Male ; Middle Aged ; Molecular Targeted Therapy ; Neoplasms/drug therapy/*genetics/pathology ; RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR screens have accelerated the discovery of important cancer vulnerabilities. However, single-gene knockout phenotypes can be masked by redundancy among related genes. Paralogs constitute two-thirds of the human protein-coding genome, so existing methods are likely inadequate for assaying a large portion of gene function. Here, we develop paired guide RNAs for paralog genetic interaction mapping (pgPEN), a pooled CRISPR-Cas9 single- and double-knockout approach targeting more than 2,000 human paralogs. We apply pgPEN to two cell types and discover that 12% of human paralogs exhibit synthetic lethality in at least one context. We recover known synthetic lethal paralogs MEK1/MEK2, important drug targets CDK4/CDK6, and other synthetic lethal pairs including CCNL1/CCNL2. Additionally, we identify ten tumor suppressor paralog pairs whose compound loss promotes cell proliferation. These findings nominate drug targets and suggest that paralog genetic interactions could shape the landscape of positive and negative selection in cancer.},
}
@article {pmid34466691,
year = {2021},
author = {Chen, J and Zhuang, X and Zheng, J and Yang, R and Wu, F and Zhang, A and Fang, B},
title = {Aptamer-based cell-free detection system to detect target protein.},
journal = {Synthetic and systems biotechnology},
volume = {6},
number = {3},
pages = {209-215},
pmid = {34466691},
issn = {2405-805X},
abstract = {Biomarkers of disease, especially protein, show great potential for diagnosis and prognosis. For detecting a certain protein, a binding assay implementing antibodies is commonly performed. However, antibodies are not thermally stable and may cause false-positive when the sample composition is complicated. In recent years, a functional nucleic acid named aptamer has been used in many biochemical analysis cases, which is commonly selected from random sequence libraries by using the systematic evolution of ligands by exponential enrichment (SELEX) techniques. Compared to antibodies, the aptamer is more thermal stable, easier to be modified, conjugated, and amplified. Herein, an Aptamer-Based Cell-free Detection (ABCD) system was proposed to detect target protein, using epithelial cell adhesion molecule (EpCAM) as an example. We combined the robustness of aptamer in binding specificity with the signal amplification ability of CRISPR-Cas12a's trans-cleavage activity in the ABCD system. We also demonstrated that the ABCD system could work well to detect target protein in a relatively low limit of detection (50-100 nM), which lay a foundation for the development of portable detection devices. This work highlights the superiority of the ABCD system in detecting target protein with low abundance and offers new enlightenment for future design and development.},
}
@article {pmid34465535,
year = {2021},
author = {Jung, C and Till, B},
title = {Mutagenesis and genome editing in crop improvement: perspectives for the global regulatory landscape.},
journal = {Trends in plant science},
volume = {26},
number = {12},
pages = {1258-1269},
doi = {10.1016/j.tplants.2021.08.002},
pmid = {34465535},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant/genetics ; Mutagenesis/genetics ; Plant Breeding ; Plants, Genetically Modified ; },
abstract = {Plant breeding depends on broad genetic variation. New allelic variation can be produced by targeted or random mutagenesis. Seemingly, random mutagenesis is outdated because clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology is much more precise and potentially faster. Unfortunately, genome editing is not accessible to breeders in many countries due to legal constraints. Therefore, random mutagenesis remains a vital method to create new allelic variation. Mutant offspring, however, suffer from a heavy mutation load, and application in polyploid crops is limited because multiple mutations are typically required. Exploiting random mutations became more efficient due to recent technological advancements, such as sequence-based mutant screening and genomic background selection. In this review, random and targeted mutagenesis will be compared, highlighting the legal situation.},
}
@article {pmid34465216,
year = {2021},
author = {Ongenae, V and Briegel, A and Claessen, D},
title = {Cell wall deficiency as an escape mechanism from phage infection.},
journal = {Open biology},
volume = {11},
number = {9},
pages = {210199},
pmid = {34465216},
issn = {2046-2441},
mesh = {Bacteria/*virology ; *Bacterial Physiological Phenomena ; Bacterial Proteins/*antagonists &amp; inhibitors/genetics ; Bacteriophages/*physiology ; CRISPR-Cas Systems ; Cell Membrane/*physiology ; Cell Wall/*physiology ; },
abstract = {The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.},
}
@article {pmid34465000,
year = {2021},
author = {Kohm, K and Basu, S and Nawaz, MM and Hertel, R},
title = {Chances and limitations when uncovering essential and non-essential genes of Bacillus subtilis phages with CRISPR-Cas9.},
journal = {Environmental microbiology reports},
volume = {13},
number = {6},
pages = {934-944},
doi = {10.1111/1758-2229.13005},
pmid = {34465000},
issn = {1758-2229},
mesh = {*Bacillus Phages/genetics ; Bacillus subtilis/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; },
abstract = {Virulent bacterial viruses, also known as phages or bacteriophages, are considered as a potential option to fight antibiotic-resistant bacteria. However, their biology is still poorly understood, and only a fraction of phage genes is assigned with a function. To enable the first classification, we explored new options to test phage genes for their requirement on viral replication. As a model, we used the smallest known Bacillus subtilis phage Goe1, and the Cas9-based mutagenesis vector pRH030 as a genetic tool. All phage genes were specifically disrupted, and individual survival rates and mutant genotypes were investigated. Surviving phages relied on the genome integrity through host intrinsic non-homologues end joining system or a natural alteration of the Cas9 target sequence. Quantification of phage survivors and verifying the underlying genetic situation enables the classification of genes in essential or non-essential sets for viral replication. We also observed structural genes to hold more natural mutations than genes of the genome replication machinery.},
}
@article {pmid34464854,
year = {2021},
author = {Qiao, ZB and Lu, JZ and Zhang, L and Cao, HX and Bai, ZH and Qi, YY and Zhu, HY and Zhang, SM and Bao, Y and Jia, WW and Liu, ZM},
title = {CRISPR/Cas9 mediated generation of a iPSC line EHTJUi005-A-1 with homozygous knockout of the SUV39H1 gene.},
journal = {Stem cell research},
volume = {56},
number = {},
pages = {102519},
doi = {10.1016/j.scr.2021.102519},
pmid = {34464854},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; Histone Methyltransferases ; Histones/genetics/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Methyltransferases/genetics/metabolism ; Repressor Proteins/genetics ; },
abstract = {SUV39H1 is a histone methyltransferase involve numerous biological processes, including of aging, embryo development, tumor growth and mitosis via catalysis of dimethylation and trimethylation of lysine 9 of histone H3. Here we report a human induced pluripotent stem cell line (EHTJUi005-A-1) which is generated from a wildtype human iPSC previously established in our laboratory, and this iPSC has a homozygous knockout of 8 bp in Exon 2 of SUV39H1. This iPSC model provides a valuable resource to study epigenetic regulation in extensive biological processes as mentioned above.},
}
@article {pmid34463742,
year = {2021},
author = {Moravec, CE and Voit, GC and Otterlee, J and Pelegri, F},
title = {Identification of maternal-effect genes in zebrafish using maternal crispants.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {19},
pages = {},
pmid = {34463742},
issn = {1477-9129},
support = {R01 GM065303/GM/NIGMS NIH HHS/United States ; R56 GM065303/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; *Maternal Inheritance ; Ovum/cytology/metabolism ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {In animals, early development is dependent on a pool of maternal factors, both RNA and proteins, which are required for basic cellular processes and cell differentiation until zygotic genome activation. The role of the majority of these maternally expressed factors is not fully understood. By exploiting the biallelic editing ability of CRISPR-Cas9, we identify and characterize maternal-effect genes in a single generation, using a maternal crispant technique. We validated the ability to generate biallelic mutations in the germ line by creating maternal crispants that phenocopied previously characterized maternal-effect genes: birc5b, tmi and mid1ip1. Additionally, by targeting maternally expressed genes of unknown function in zebrafish, we identified two maternal-effect zebrafish genes, kpna7 and fhdc3. The genetic identity of these maternal crispants was confirmed by sequencing haploid progeny from F0 females, which allowed the analysis of newly induced lesions in the maternal germ line. Our studies show that maternal crispants allow for the effective identification and primary characterization of maternal-effect genes in a single generation, facilitating the reverse genetics analysis of maternal factors that drive embryonic development.},
}
@article {pmid34463385,
year = {2021},
author = {Kira, S and Noda, T},
title = {A CRISPR/Cas9-based method for seamless N-terminal protein tagging in Saccharomyces cerevisiae.},
journal = {Yeast (Chichester, England)},
volume = {38},
number = {11},
pages = {592-600},
doi = {10.1002/yea.3666},
pmid = {34463385},
issn = {1097-0061},
mesh = {CRISPR-Cas Systems ; Plasmids ; RNA, Guide ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {Protein tagging is an effective method for characterizing a gene of interest. Tagging can be accomplished in vivo in Saccharomyces cerevisiae by chromosomal integration of a PCR-amplified cassette. However, common tagging cassettes are not suitable for in situ N-terminal tagging when we aim to preserve the gene's endogenous promoter. Existing methods require either two rounds of homologous recombination or a relatively complex cloning process to construct strains with N-terminal protein tags. Here, we describe a simple CRISPR/Cas9-based method for seamless N-terminal tagging of yeast genes that preserves their endogenous promoter. This method enables the generation of N-terminally tagged strains by introducing an expression vector containing the cas9 gene and a specific gRNA for cleaving the 5' end of the target gene's protein-coding sequence, along with donor DNA containing the tag sequence and homology arms. gRNA cloning was executed by inverse PCR instead of the conventional method. After verifying the tag, the Cas9 and gRNA expression plasmids were eliminated without using antibiotic-containing medium. By this method, we generated strains that express N-terminally tagged subunits of the TORC1 protein kinase complex and found that these strains are comparable to strains made by conventional methods. Thus, our method provides a cost-effective alternative for seamless N-terminal tagging in baker's yeast.},
}
@article {pmid34463266,
year = {2021},
author = {Dutta, RK and Chinnapaiyan, S and Santiago, MJ and Rahman, I and Unwalla, HJ},
title = {Gene-specific MicroRNA antagonism protects against HIV Tat and TGF-β-mediated suppression of CFTR mRNA and function.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {142},
number = {},
pages = {112090},
pmid = {34463266},
issn = {1950-6007},
support = {R01 HL147715/HL/NHLBI NIH HHS/United States ; R21 HL128141/HL/NHLBI NIH HHS/United States ; T32 GM132054/GM/NIGMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions/genetics ; Bronchi/cytology ; CRISPR-Cas Systems/genetics ; Cell Line ; Cells, Cultured ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Epithelial Cells/metabolism ; Gene Editing ; Humans ; MicroRNAs/*genetics ; RNA, Messenger/*genetics ; Signal Transduction/genetics ; Transforming Growth Factor beta/metabolism ; Up-Regulation ; tat Gene Products, Human Immunodeficiency Virus/*metabolism ; },
abstract = {BACKGROUND: MicroRNAs play an important role in health and disease. TGF-β signaling, upregulated by HIV Tat, and in chronic airway diseases and smokers upregulates miR-145-5p to suppress cystic fibrosis transmembrane conductance regulator (CFTR). CFTR suppression in chronic airway diseases like Cystic Fibrosis, COPD and smokers has been associated with suppressed MCC and recurrent lung infections and inflammation. This can explain the emergence of recurrent lung infections and inflammation in people living with HIV.<br><br>METHODS: Tat-induced aberrant microRNAome was identified by miRNA expression analysis. microRNA mimics and antagomirs were used to validate the identified miRNAs involved in Tat mediated CFTR mRNA suppression. CRISPR-based editing of the miRNA target sites in CFTR 3'UTR was used to determine rescue of CFTR mRNA and function in airway epithelial cell lines and in primary human bronchial epithelial cells exposed to TGF-&#x3b2; and Tat.<br><br>FINDINGS: HIV Tat upregulates miR-145-5p and miR-509-3p. The two miRNAs demonstrate co-operative effects in suppressing CFTR. CRISPR-based editing of the miRNA target site preserves CFTR mRNA and function in airway epithelial cells INTERPRETATION: Given the important roles of TGF-&#x3b2; signaling and the multitude of genes regulated by miRNAs, we demonstrate that CRISPR-based gene-specific microRNA antagonism approach can preserve CFTR mRNA and function in the context of HIV Tat and TGF-&#x3b2; signaling without suppressing expression of other genes regulated by miR-145-5p.},
}
@article {pmid34462600,
year = {2021},
author = {Büning, H and Schambach, A},
title = {A first step toward in vivo gene editing in patients.},
journal = {Nature medicine},
volume = {27},
number = {9},
pages = {1515-1517},
pmid = {34462600},
issn = {1546-170X},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; *Genetic Therapy ; Humans ; },
}
@article {pmid34462587,
year = {2022},
author = {Kannan, S and Altae-Tran, H and Jin, X and Madigan, VJ and Oshiro, R and Makarova, KS and Koonin, EV and Zhang, F},
title = {Compact RNA editors with small Cas13 proteins.},
journal = {Nature biotechnology},
volume = {40},
number = {2},
pages = {194-197},
pmid = {34462587},
issn = {1546-1696},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenosine/genetics ; Adenosine Deaminase/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Mammals/genetics ; *RNA/genetics ; RNA Editing/genetics ; },
abstract = {CRISPR-Cas13 systems have been developed for precise RNA editing, and can potentially be used therapeutically when temporary changes are desirable or when DNA editing is challenging. We have identified and characterized an ultrasmall family of Cas13b proteins-Cas13bt-that can mediate mammalian transcript knockdown. We have engineered compact variants of REPAIR and RESCUE RNA editors by functionalizing Cas13bt with adenosine and cytosine deaminase domains, and demonstrated packaging of the editors within a single adeno-associated virus.},
}
@article {pmid34461727,
year = {2021},
author = {Shen, H and Qileng, A and Yang, H and Liang, H and Zhu, H and Liu, Y and Lei, H and Liu, W},
title = {"Dual-Signal-On" Integrated-Type Biosensor for Portable Detection of miRNA: Cas12a-Induced Photoelectrochemistry and Fluorescence Strategy.},
journal = {Analytical chemistry},
volume = {93},
number = {34},
pages = {11816-11825},
doi = {10.1021/acs.analchem.1c02395},
pmid = {34461727},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA ; Horseradish Peroxidase ; *MicroRNAs/genetics ; },
abstract = {The abnormal expression of microRNA (miRNA) can affect the RNA transcription and protein translation, leading to tumor progression and metastasis. Currently, the accurate detection of aberrant expression of miRNA, particularly using a portable detection system, remains a great challenge. Herein, a novel dual-mode biosensor with high sensitivity and robustness for miR-21 detection was developed based on the cis-cleavage and trans-cleavage activities of Cas12a. miRNA can be combined with hairpin DNA-horseradish peroxidase anchored on a CdS/g-C3N4/B-TiO2 photoelectrode, thus the nonenzymatic amplification was triggered to form numerous HRP-modified double-stranded DNA (HRP-dsDNA). Then, HRP-dsDNA can be specifically recognized and efficiently cis-cleaved by Cas12a nucleases to detach HRP from the substrate, while the remaining HRP on HRP-dsDNA can catalyze 4-chloro-1-naphthol (4-CN) to form biocatalytic precipitation (BCP) on the surface of the photoelectrode, and thus the photocurrent can be changed. Meanwhile, the trans-cleavage ability of Cas12a was activated, and nonspecifically degrade the FQ-reporter and a significant fluorescence signal can be generated. Such two different kinds of signals with independent transmission paths can mutually support to improve the performance of the detection platform. Besides, a portable device was constructed for the point-of-care (POC) detection of miR-21. Moreover, the dual-mode detection platform can be easily expanded for the specific detection of other types of biomarkers by changing the sequence of hairpin DNA, thereby promoting the establishment of POC detection for early cancer diagnosis.},
}
@article {pmid34461306,
year = {2022},
author = {Del Rio, ML and Nguyen, TH and Tesson, L and Heslan, JM and Gutierrez-Adan, A and Fernandez-Gonzalez, R and Gutierrez-Arroyo, J and Buhler, L and Pérez-Simón, JA and Anegon, I and Rodriguez-Barbosa, JI},
title = {The impact of CD160 deficiency on alloreactive CD8 T cell responses and allograft rejection.},
journal = {Translational research : the journal of laboratory and clinical medicine},
volume = {239},
number = {},
pages = {103-123},
doi = {10.1016/j.trsl.2021.08.006},
pmid = {34461306},
issn = {1878-1810},
mesh = {4-1BB Ligand/metabolism ; Allografts ; Animals ; Antigens, CD/genetics/*immunology/metabolism ; Antigens, Differentiation, T-Lymphocyte/metabolism ; CD8-Positive T-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Cell Differentiation ; Female ; GPI-Linked Proteins/genetics/immunology/metabolism ; Gene Expression Regulation ; Genes, MHC Class I ; Graft Rejection/*etiology/immunology ; Killer Cells, Natural/immunology ; Lectins, C-Type/metabolism ; Mice, Inbred Strains ; Mice, Knockout ; Receptors, Immunologic/genetics/*immunology/metabolism ; Receptors, Tumor Necrosis Factor, Member 14/metabolism ; Skin Transplantation ; Thymocytes/immunology ; },
abstract = {CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.},
}
@article {pmid34461102,
year = {2021},
author = {Pollecker, K and Sylvester, M and Voos, W},
title = {Proteomic analysis demonstrates the role of the quality control protease LONP1 in mitochondrial protein aggregation.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {4},
pages = {101134},
pmid = {34461102},
issn = {1083-351X},
mesh = {ATP-Dependent Proteases/genetics/*metabolism ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Mitochondria/genetics/*metabolism ; Mitochondrial Proteins/genetics/*metabolism ; *Protein Aggregates ; Proteome/genetics/*metabolism ; *Proteomics ; },
abstract = {The mitochondrial matrix protease LONP1 is an essential part of the organellar protein quality control system. LONP1 has been shown to be involved in respiration control and apoptosis. Furthermore, a reduction in LONP1 level correlates with aging. Up to now, the effects of a LONP1 defect were mostly studied by utilizing transient, siRNA-mediated knockdown approaches. We generated a new cellular model system for studying the impact of LONP1 on mitochondrial protein homeostasis by a CRISPR/Cas-mediated genetic knockdown (gKD). These cells showed a stable reduction of LONP1 along with a mild phenotype characterized by absent morphological differences and only small negative effects on mitochondrial functions under normal culture conditions. To assess the consequences of a permanent LONP1 depletion on the mitochondrial proteome, we analyzed the alterations of protein levels by quantitative mass spectrometry, demonstrating small adaptive changes, in particular with respect to mitochondrial protein biogenesis. In an additional proteomic analysis, we determined the temperature-dependent aggregation behavior of mitochondrial proteins and its dependence on a reduction of LONP1 activity, demonstrating the important role of the protease for mitochondrial protein homeostasis in mammalian cells. We identified a significant number of mitochondrial proteins that are affected by a reduced LONP1 activity especially with respect to their stress-induced solubility. Taken together, our results suggest a very good applicability of the LONP1 gKD cell line as a model system for human aging processes.},
}
@article {pmid34456921,
year = {2021},
author = {Alves, E and McLeish, E and Blancafort, P and Coudert, JD and Gaudieri, S},
title = {Manipulating the NKG2D Receptor-Ligand Axis Using CRISPR: Novel Technologies for Improved Host Immunity.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {712722},
pmid = {34456921},
issn = {1664-3224},
support = {R01 CA170370/CA/NCI NIH HHS/United States ; R01 DA036906/DA/NIDA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Disease Resistance ; Disease Susceptibility ; Epigenesis, Genetic ; *Gene Editing/methods ; Genetic Therapy ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Immunity/genetics ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K/*genetics/*metabolism ; Neoplasms/etiology ; Protein Binding ; Virus Diseases/etiology ; },
abstract = {The activating immune receptor natural killer group member D (NKG2D) and its cognate ligands represent a fundamental surveillance system of cellular distress, damage or transformation. Signaling through the NKG2D receptor-ligand axis is critical for early detection of viral infection or oncogenic transformation and the presence of functional NKG2D ligands (NKG2D-L) is associated with tumor rejection and viral clearance. Many viruses and tumors have developed mechanisms to evade NKG2D recognition via transcriptional, post-transcriptional or post-translational interference with NKG2D-L, supporting the concept that circumventing immune evasion of the NKG2D receptor-ligand axis may be an attractive therapeutic avenue for antiviral therapy or cancer immunotherapy. To date, the complexity of the NKG2D receptor-ligand axis and the lack of specificity of current NKG2D-targeting therapies has not allowed for the precise manipulation required to optimally harness NKG2D-mediated immunity. However, with the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins, novel opportunities have arisen in the realm of locus-specific gene editing and regulation. Here, we give a brief overview of the NKG2D receptor-ligand axis in humans and discuss the levels at which NKG2D-L are regulated and dysregulated during viral infection and oncogenesis. Moreover, we explore the potential for CRISPR-based technologies to provide novel therapeutic avenues to improve and maximize NKG2D-mediated immunity.},
}
@article {pmid34456559,
year = {2021},
author = {Asmamaw, M and Zawdie, B},
title = {Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing.},
journal = {Biologics : targets &amp; therapy},
volume = {15},
number = {},
pages = {353-361},
pmid = {34456559},
issn = {1177-5475},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas-9 nuclease makes double-stranded breaks at a site 3 base pair upstream to protospacer adjacent motif, then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value. In medicine, it is being investigated for cancers, HIV, and gene therapy such as sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. The technology is also being utilized in the regulation of specific genes through the advanced modification of Cas-9 protein. However, immunogenicity, effective delivery systems, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. Although CRISPR/Cas-9 becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications, there are still challenges to rub in the practical applications and various improvements are needed to overcome obstacles.},
}
@article {pmid34456394,
year = {2021},
author = {Novikov, RV and Gribkova, AK and Kacher, JG and Zaytsev, PA and Armeev, GA and Gluhov, GS and Shaytan, AK},
title = {Design of Nucleic Acid Biosensors Based on CRISPR/Cas Systems and Reporter Split Proteins.},
journal = {Moscow University biological sciences bulletin},
volume = {76},
number = {2},
pages = {52-58},
pmid = {34456394},
issn = {0096-3925},
abstract = {Highly sensitive, specific, rapid, and easy-to-use diagnostic methods for the detection of nucleic acids of pathogens are required for the diagnosis of many human, animal, and plant diseases and environmental monitoring. The approaches based on the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with a high specificity under isothermal conditions are an alternative to the polymerase chain reaction method, which requires expensive laboratory equipment. The development of the methods for signal registration with the formation of a DNA/RNA/Cas9 protein complex is a separate bioengineering task. In this work, a design was developed and the applicability of a biosensor system based on the binding of two dCas9 proteins with target DNA sequences (without their cutting) and detection of their colocalization using reporter systems based on split enzymes was studied. Using the methods of molecular modeling, possible mutual positions of two dCas9 proteins at a detectable locus of genomic DNA, allowing the split enzyme domains attached to them to interact in an optimal way, were determined. The optimal distances on DNA between binding sites of dCas9 proteins in different orientations were determined, and the dependence of the complex structure on the distance between the binding sites of dCas9 proteins was modeled. Using the methods of bioinformatics, the genomes of a number of viruses (including SARS-CoV-2) were analyzed, and the presence of genomic loci unique to the species, allowing the possibility of landing pairs of dCas9 proteins in optimal positions, was demonstrated. The possibility of a combined use of dCas9 proteins from different bacteria to expand the spectrum of detected loci was analyzed. The results of the work indicate a fundamental possibility of the creation of highly specific nucleic acid biosensors based on a combination of CRISPR/Cas9 technologies and split enzymes.},
}
@article {pmid34455394,
year = {2021},
author = {Lam, WY and Tang, CS and So, MT and Yue, H and Hsu, JS and Chung, PH and Nicholls, JM and Yeung, F and Lee, CD and Ngo, DN and Nguyen, PAH and Mitchison, HM and Jenkins, D and O'Callaghan, C and Garcia-Barceló, MM and Lee, SL and Sham, PC and Lui, VC and Tam, PK},
title = {Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism.},
journal = {EBioMedicine},
volume = {71},
number = {},
pages = {103530},
pmid = {34455394},
issn = {2352-3964},
mesh = {Animals ; Biliary Atresia/diagnosis/*etiology ; CRISPR-Cas Systems ; Cell Line ; Cilia/*genetics ; Computational Biology/methods ; Gene Editing ; Gene Knockdown Techniques ; Gene Ontology ; *Genetic Association Studies/methods ; Genetic Heterogeneity ; Genetic Loci ; *Genetic Predisposition to Disease ; Humans ; Liver/metabolism/pathology ; *Mutation ; *Phenotype ; Sequence Analysis, DNA ; Whole Exome Sequencing ; Zebrafish ; },
abstract = {BACKGROUND: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined.<br><br>METHODS: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients' liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy.<br><br>FINDINGS: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]=&#xa0;2.58 [1.15-6.07], adjusted p&#xa0;=&#xa0;0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects.<br><br>INTERPRETATION: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis.<br><br>FUNDING: The study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.},
}
@article {pmid34455224,
year = {2021},
author = {Kim, H and Lee, S and Yoon, J and Song, J and Park, HG},
title = {CRISPR/Cas12a collateral cleavage activity for simple and rapid detection of protein/small molecule interaction.},
journal = {Biosensors &amp; bioelectronics},
volume = {194},
number = {},
pages = {113587},
doi = {10.1016/j.bios.2021.113587},
pmid = {34455224},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; DNA Cleavage ; DNA, Single-Stranded ; Gene Editing ; Humans ; },
abstract = {To realize the full potential of the CRISPR/Cas system and expand its applicability up to the detection of molecular interactions, we herein describe a novel method to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a collateral cleavage activity. This technique employs a single-stranded activator DNA modified with a specific small molecule, which would switch on the CRISPR/Cas12a collateral cleavage activity upon binding to crRNA within the CRISPR/Cas12a system. When the target protein binds to the small molecule on the activator DNA, the bound protein sterically hinders the access of the activator DNA to crRNA, thereby promoting less collateral cleavage activity of CRISPR/Cas12a. As a consequence, fewer reporter probes nearby are cleaved to produce accordingly reduced fluorescence signals in response to target protein. Based on this unique design principle, the two model protein/small molecule interactions, streptavidin/biotin and anti-digoxigenin/digoxigenin, were successfully determined down to 0.03 nM and 0.09 nM, respectively, with a fast and simple detection workflow (11 min). The practical applicability of this method was also verified by reliably detecting target streptavidin spiked in heterogeneous human serum. This work would provide great insight to construct novel strategies to identify protein/small molecule interaction by making the most of the CRISPR/Cas12a system beyond its superior capabilities in genome editing and molecular diagnostics.},
}
@article {pmid34454980,
year = {2021},
author = {Racharaks, R and Arnold, W and Peccia, J},
title = {Development of CRISPR-Cas9 knock-in tools for free fatty acid production using the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973.},
journal = {Journal of microbiological methods},
volume = {189},
number = {},
pages = {106315},
doi = {10.1016/j.mimet.2021.106315},
pmid = {34454980},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems ; Fatty Acids, Nonesterified/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Metabolic Engineering/methods ; Plasmids/genetics ; Synechococcus/*genetics/growth &amp; development/*metabolism ; },
abstract = {Synechococcus elongatus UTEX 2973 has one of the fastest measured doubling time of cyanobacteria making it an important candidate for metabolic engineering. Traditional genetic engineering methods, which rely on homologous recombination, however, are inefficient, labor-intensive, and time-consuming due to the oligoploidy or polyploidy nature of cyanobacteria and the reliance on unique antibiotic resistance markers. CRISPR-Cas9 has emerged as an effective and versatile editing platform in a wide variety of organisms, but its application for cyanobacterial engineering is limited by the inherent toxicity of Cas9 resulting in poor transformation efficiencies. Here, we demonstrated that a single-plasmid CRISPR-Cas9 system, pCRISPOmyces-2, can effectively knock-in a truncated thioesterase gene from Escherichia coli to generate free fatty acid (FFA) producing mutants of Syn2973. To do so, three parameters were evaluated on the effect of generating recipient colonies after conjugation with pCRISPOmyces-2-based plasmids: 1) a modified conjugation protocol termed streaked conjugation, 2) the deletion of the gene encoding RecJ exonuclease, and 3) single guide RNA (sgRNA) sequence. With the use of the streaked conjugation protocol and a ΔrecJ mutant strain of Syn2973, the conjugation efficiency for the pCRISPomyces-2 plasmid could be improved by 750-fold over the wildtype (WT) for a conjugation efficiency of 2.0 × 10[-6] transconjugants/recipient cell. While deletion of the RecJ exonuclease alone increased the conjugation efficiency by 150-fold over the WT, FFA generation was impaired in FFA-producing mutants with the ΔrecJ background, and the large number of poor FFA-producing isolates indicated the potential increase in spontaneous mutation rates. The sgRNA sequence was found to be critical in achieving the desired CRISPR-Cas9-mediated knock-in mutation as the sgRNA impacts conjugation efficiency, likelihood of homogenous recombinants, and free fatty acid production in engineered strains.},
}
@article {pmid34454311,
year = {2021},
author = {Huang, M and Liu, M and Huang, L and Wang, M and Jia, R and Zhu, D and Chen, S and Zhao, X and Zhang, S and Gao, Q and Zhang, L and Cheng, A},
title = {The activation and limitation of the bacterial natural transformation system: The function in genome evolution and stability.},
journal = {Microbiological research},
volume = {252},
number = {},
pages = {126856},
doi = {10.1016/j.micres.2021.126856},
pmid = {34454311},
issn = {1618-0623},
mesh = {Bacteria/genetics ; *Evolution, Molecular ; *Genome, Bacterial/genetics ; *Transformation, Bacterial/genetics ; },
abstract = {Bacteria can take up exogenous naked DNA and integrate it into their genomes, which has been regarded as a main contributor to bacterial evolution. The competent status of bacteria is influenced by environmental cues and by the immune systems of bacteria. Here, we review recent advances in understanding the working mechanisms underlying activation of the natural transformation system and limitations thereof. Environmental stresses including the presence of antimicrobials can activate the natural transformation system. However, bacterial enzymes (nucleases), non-coding RNAs, specific DNA sequences, the restriction-modification (R-M) systems, CRISPR-Cas systems and prokaryotic Argonaute proteins (Agos) are have been found to be involved in the limitation of the natural transformation system. Together, this review represents an opportunity to gain insight into bacterial genome stability and evolution.},
}
@article {pmid34454111,
year = {2021},
author = {Ahmed, MZ and Badani, P and Reddy, R and Mishra, G},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas Advancement in Molecular Diagnostics and Signal Readout Approaches.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {23},
number = {11},
pages = {1433-1442},
doi = {10.1016/j.jmoldx.2021.07.025},
pmid = {34454111},
issn = {1943-7811},
mesh = {Biosensing Techniques/methods ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Humans ; Molecular Diagnostic Techniques/*methods ; Point-of-Care Testing ; Precision Medicine/methods ; },
abstract = {Discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has greatly improved the gene editing technology. Their applications in the area of diagnostic innovation are gaining much attention. The key characteristics of CRISPR/Cas system that allowed its extensive exploitation in the detection platform are their programmable and highly selective target recognition scheme. Herein, this review presents the major three Cas effectors (Cas9, Cas12, and Cas13) and their significance in various detection assays. The CRISPR/Cas detection strategies, based on their target hybridization, cleavage activity, sensor capabilities, and signal readout methods, are discussed. Some of the recent progressions, challenges, and improvement strategies of CRISPR/Cas technology are highlighted and their biosensing detection platforms toward the development of simple, sensitive, and portable point-of-care diagnostic devices are presented.},
}
@article {pmid34454034,
year = {2022},
author = {Zhou, H and Xu, L and Li, F and Li, Y},
title = {Transcriptional regulation by CRISPR/dCas9 in common wheat.},
journal = {Gene},
volume = {807},
number = {},
pages = {145919},
doi = {10.1016/j.gene.2021.145919},
pmid = {34454034},
issn = {1879-0038},
mesh = {CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Catalytic Domain/genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Gene Expression Regulation, Plant/genetics ; Genes, Essential/genetics ; Multigene Family/genetics ; RNA Processing, Post-Transcriptional ; RNA, Guide/genetics ; Transcription Factors/genetics ; Transcriptional Activation/*genetics ; Triticum/*genetics/metabolism ; },
abstract = {The application of CRISPR/Cas9 system for gene editing, as a technical coup for biotechnology, is worldwide and encompasses multiple of species. The inactivation of catalytical site in Cas9 (dCas9) has been reprogrammed as an effective approach to regulate the transcriptional level of target genes, especially for the functionally essential genes and redundant genes. Here, we exploited the CRISPR/dCas9 system to manipulate the transcriptional level of target genes in common wheat. To improve target gene's expression, we generated transcriptional activator by fusing 6×TAL-VP128 activation domain to the C-terminus of dCas9 in frame. For target gene's repressing expression transcriptionally, 3×SRDX repression domain was conjugated to the C-terminus of dCas9 in frame. Our results showed that dCas9 fused activation or repression domain could increase or decrease the transcriptional level of target gene effectively in stable transgenic lines of wheat. The study on the tRNA-processing system in CRISPR/dCas9 based transcriptional regulation system demonstrated that this robust multiplex targeted tool can be incorporated to the CRISPR/dCas9 system to facilitate the target regulation of several genes' transcriptional level. Our data broaden the application of CRISPR/dCas9 based transcriptional regulation and provide great opportunities to investigate the function of essential genes in common wheat.},
}
@article {pmid34453947,
year = {2021},
author = {Kim, JH and Kim, MM},
title = {The relationship between melanin production and lipofuscin formation in Tyrosinase gene knockout melanocytes using CRISPR/Cas9 system.},
journal = {Life sciences},
volume = {284},
number = {},
pages = {119915},
doi = {10.1016/j.lfs.2021.119915},
pmid = {34453947},
issn = {1879-0631},
mesh = {Animals ; Antioxidants/metabolism ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Survival/radiation effects ; Gene Expression Regulation, Neoplastic ; *Gene Knockout Techniques ; Lipofuscin/*metabolism ; Melanins/*biosynthesis ; Melanocytes/*enzymology ; Melanoma, Experimental/genetics/pathology ; Mice ; Monophenol Monooxygenase/*genetics ; Ultraviolet Rays ; },
abstract = {Age spots are a significant phenotypic marker of aging formed by lipofuscin. Melanin is another skin pigment molecule responsible for skin aging. The present study aims to investigate the relationship between melanin production and lipofuscin synthesis in normal mouse melanoma cell line B16F1 cells and Tyrosinase (TYR) gene knockout cells. TYR gene KO cells were successfully developed using CRISPR/Cas9 system and confirmed by Sanger DNA sequencing analysis. Furthermore, the melanin production and lipofuscin formation were validated through RT-PCR and Western blot analysis. The expression levels of gene microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosine-related protein-1 (TRP-1), tyrosine-related protein-2 (TRP-2), and antioxidant proteins such as methionine sulfoxide reductase A (MSRA), Catalase and Glutathione reductase (GR) related to melanogenesis was found to be decreased in TYR gene KO cells compared with normal cells. Moreover, lipofuscin formation was increased in TYR gene KO cells compared to normal cells. Therefore, the above findings suggest that melanin production and lipofuscin formation could be linked by the TYR gene in melanocytes.},
}
@article {pmid34453943,
year = {2021},
author = {Luthra, R and Kaur, S and Bhandari, K},
title = {Applications of CRISPR as a potential therapeutic.},
journal = {Life sciences},
volume = {284},
number = {},
pages = {119908},
doi = {10.1016/j.lfs.2021.119908},
pmid = {34453943},
issn = {1879-0631},
mesh = {Animals ; COVID-19/therapy/virology ; CRISPR-Cas Systems/*genetics ; Clinical Trials as Topic ; Disease/genetics ; Gene Editing ; Humans ; SARS-CoV-2/physiology ; },
abstract = {Genetic disorders and congenital abnormalities are present in 2-5% of births all over the world and can cause up to 50% of all early childhood deaths. The establishment of sophisticated and controlled techniques for customizing DNA manipulation is significant for the therapeutic role in such disorders and further research on them. One such technique is CRISPR that is significant towards optimizing genome editing and therapies, metabolic fluxes as well as artificial genetic systems. CRISPR-Cas9 is a molecular appliance that is applied in the areas of genetic and protein engineering. The CRISPR-CAS system is an integral element of prokaryotic adaptive immunity that allows prokaryotic cells to identify and kill any foreign DNA. The Gene editing property of CRISPR finds various applications like diagnostics and therapeutics in cancer, neurodegenerative disorders, genetic diseases, blindness, etc. This review discusses applications of CRISPR as a therapeutic in various disorders including several genetic diseases (including sickle cell anemia, blindness, thalassemia, cystic fibrosis, hereditary tyrosinemia type I, duchenne muscular dystrophy, mitochondrial disorders), Cancer, Huntington's disease and viral infections (like HIV, COVID, etc.) along with the prospects concerning them. CRISPR-based therapy is also being researched and defined for COVID-19. The related mechanism of CRISPR has been discussed alongside highlighting challenges involved in therapeutic applications of CRISPR.},
}
@article {pmid34452911,
year = {2021},
author = {Jang, HK and Jo, DH and Lee, SN and Cho, CS and Jeong, YK and Jung, Y and Yu, J and Kim, JH and Woo, JS and Bae, S},
title = {High-purity production and precise editing of DNA base editing ribonucleoproteins.},
journal = {Science advances},
volume = {7},
number = {35},
pages = {},
pmid = {34452911},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems ; Cytosine/metabolism ; DNA/genetics ; *Gene Editing ; Mice ; RNA ; *Ribonucleoproteins/genetics ; },
abstract = {Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM-targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.},
}
@article {pmid34452500,
year = {2021},
author = {Helfer-Hungerbuehler, AK and Shah, J and Meili, T and Boenzli, E and Li, P and Hofmann-Lehmann, R},
title = {Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro.},
journal = {Viruses},
volume = {13},
number = {8},
pages = {},
pmid = {34452500},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems ; Cats ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics/metabolism ; Gene Editing ; *Genetic Therapy ; Genetic Vectors/*genetics/metabolism ; Leukemia Virus, Feline/genetics/*physiology ; Leukemia, Feline/genetics/*therapy/*virology ; Viral Load ; *Virus Replication ; },
abstract = {Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat's immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.},
}
@article {pmid34452438,
year = {2021},
author = {Hölper, JE and Grey, F and Baillie, JK and Regan, T and Parkinson, NJ and Höper, D and Thamamongood, T and Schwemmle, M and Pannhorst, K and Wendt, L and Mettenleiter, TC and Klupp, BG},
title = {A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD[-]Pass.},
journal = {Viruses},
volume = {13},
number = {8},
pages = {},
pmid = {34452438},
issn = {1999-4915},
support = {BB/N021738/1, BBS/E/D/20002172/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 211496/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing ; *Genome, Viral ; Herpesvirus 1, Suid/*genetics ; *Host Microbial Interactions ; Kidney/cytology/virology ; *Mutation ; Swine ; Transferases (Other Substituted Phosphate Groups)/*genetics/metabolism ; Virus Replication ; },
abstract = {Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD[-]Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD[-]Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD[-]Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD[-]Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.},
}
@article {pmid34450246,
year = {2021},
author = {van Bergen, MGJM and van Oorschot, R and Bergevoet, SM and de Graaf, AO and Tönnissen, ELRTM and Stevens-Linders, E and Neveling, K and Jansen, PWTC and Baltissen, MPA and Vermeulen, M and Mandoli, A and Martens, JHA and Preijers, F and Jansen, JH and van der Reijden, BA},
title = {Characterization of a genomic region 8 kb downstream of GFI1B associated with myeloproliferative neoplasms.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1867},
number = {12},
pages = {166259},
doi = {10.1016/j.bbadis.2021.166259},
pmid = {34450246},
issn = {1879-260X},
mesh = {Adult ; Aged ; Alleles ; CRISPR-Cas Systems/genetics ; Female ; Gene Expression Regulation/genetics ; *Genetic Predisposition to Disease ; Genome, Human/genetics ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; K562 Cells ; Male ; Middle Aged ; Myeloid Cells/metabolism/pathology ; Myeloproliferative Disorders/*genetics/pathology ; Neoplasms/*genetics/pathology ; Phagocytosis/genetics ; Polymorphism, Single Nucleotide/genetics ; Proto-Oncogene Proteins/*genetics ; Repressor Proteins/*genetics ; Young Adult ; },
abstract = {A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.},
}
@article {pmid34450195,
year = {2021},
author = {Wen, X and Zhang, Y and Cheng, H and An, J and Guo, Y and Wang, L and Wang, M},
title = {A CRISPR/dCas9-assisted system to clone toxic genes in Escherichia coli.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1865},
number = {11},
pages = {129994},
doi = {10.1016/j.bbagen.2021.129994},
pmid = {34450195},
issn = {1872-8006},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Corynebacterium glutamicum/*genetics ; Escherichia coli/*genetics ; },
abstract = {BACKGROUND: The cloning of toxic genes in E. coli requires strict regulation of the target genes' leaky expression. Many methods facilitating successful gene cloning of toxic genes are commonly exploited, but the applicability is severely limited.<br><br>METHODS: A CRISPR/dCas9-assisted system was used to clone toxic genes in E. coli. The plasmid-based and genome-integrated systems were designed in this study. And the green fluorescent protein characterization system was used to test the repression efficiency of the two systems.<br><br>RESULTS: We optimized the plasmid-based CRISPR/dCas9-assisted repression system via testing different sgRNAs targeting the Ptrc promoter and achieved inhibition efficiency up to 64.8%. The genome-integrated system represented 35.9% decreased GFP expression and was successfully employed to cloned four toxic genes from Corynebacterium glutamicum in E. coli.<br><br>CONCLUSIONS: Using this method, we successfully cloned four C. glutamicum-derived toxic genes that had been failed to clone in conventional ways. The CRISPR/dCas9-assisted gene cloning method was a promising tool to facilitate precise gene cloning of different origins in E. coli.<br><br>GENERAL SIGNIFICANCE: This system will be useful for cloning toxic genes from different origins in E. coli, and can accelerate the related research of gene characterization and heterologous expression in the metagenomic era.},
}
@article {pmid34450043,
year = {2021},
author = {Xiao, R and Wang, S and Han, R and Li, Z and Gabel, C and Mukherjee, IA and Chang, L},
title = {Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.},
journal = {Molecular cell},
volume = {81},
number = {21},
pages = {4457-4466.e5},
pmid = {34450043},
issn = {1097-4164},
support = {R01 GM138675/GM/NIGMS NIH HHS/United States ; T32 GM132024/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Motifs ; *CRISPR-Cas Systems ; Cryoelectron Microscopy/*methods ; Cyanobacteria ; DNA/*chemistry/metabolism ; Gene Editing ; Genetic Techniques ; Mutagenesis ; Mutagenesis, Site-Directed ; Mutation ; Protein Conformation ; Protein Domains ; RNA/*chemistry ; *RNA, Guide ; Recombination, Genetic ; },
abstract = {The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.},
}
@article {pmid34450036,
year = {2021},
author = {El-Khairi, R and Olszanowski, E and Muraro, D and Madrigal, P and Tilgner, K and Chhatriwala, M and Vyas, S and Chia, CY and Vallier, L and Rodríguez-Seguí, SA},
title = {Modeling HNF1B-associated monogenic diabetes using human iPSCs reveals an early stage impairment of the pancreatic developmental program.},
journal = {Stem cell reports},
volume = {16},
number = {9},
pages = {2289-2304},
pmid = {34450036},
issn = {2213-6711},
support = {/WT_/Wellcome Trust/United Kingdom ; MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; BRC-1215-20014/DH_/Department of Health/United Kingdom ; },
mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Cell Lineage/genetics ; Diabetes Mellitus/etiology ; Disease Susceptibility ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Haploinsufficiency ; Hepatocyte Nuclear Factor 1-beta/*genetics/metabolism ; Humans ; Immunophenotyping ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Insulin-Secreting Cells/metabolism ; *Models, Biological ; Organogenesis/*genetics ; Pancreas/*embryology/*metabolism ; Signal Transduction ; },
abstract = {Heterozygous mutations in HNF1B in humans result in a multisystem disorder, including pancreatic hypoplasia and diabetes mellitus. Here we used a well-controlled human induced pluripotent stem cell pancreatic differentiation model to elucidate the molecular mechanisms underlying HNF1B-associated diabetes. Our results show that lack of HNF1B blocks specification of pancreatic fate from the foregut progenitor (FP) stage, but HNF1B haploinsufficiency allows differentiation of multipotent pancreatic progenitor cells (MPCs) and insulin-secreting β-like cells. We show that HNF1B haploinsufficiency impairs cell proliferation in FPs and MPCs. This could be attributed to impaired induction of key pancreatic developmental genes, including SOX11, ROBO2, and additional TEAD1 target genes whose function is associated with MPC self-renewal. In this work we uncover an exhaustive list of potential HNF1B gene targets during human pancreas organogenesis whose downregulation might underlie HNF1B-associated diabetes onset in humans, thus providing an important resource to understand the pathogenesis of this disease.},
}
@article {pmid34448690,
year = {2021},
author = {Perez, M and Angers, B and Young, CR and Juniper, SK},
title = {Shining light on a deep-sea bacterial symbiont population structure with CRISPR.},
journal = {Microbial genomics},
volume = {7},
number = {8},
pages = {},
pmid = {34448690},
issn = {2057-5858},
mesh = {Bacteria/*classification/*genetics ; Bacterial Typing Techniques/methods ; *CRISPR-Cas Systems ; Ecosystem ; Genes, Bacterial/genetics ; Genome, Bacterial ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Seawater/*microbiology ; Sequence Analysis, DNA ; Symbiosis ; },
abstract = {Many foundation species in chemosynthesis-based ecosystems rely on environmentally acquired symbiotic bacteria for their survival. Hence, understanding the biogeographic distributions of these symbionts at regional scales is key to understanding patterns of connectivity and predicting resilience of their host populations (and thus whole communities). However, such assessments are challenging because they necessitate measuring bacterial genetic diversity at fine resolutions. For this purpose, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR) constitutes a promising new genetic marker. These DNA sequences harboured by about half of bacteria hold their viral immune memory, and as such, might allow discrimination of different lineages or strains of otherwise indistinguishable bacteria. In this study, we assessed the potential of CRISPR as a hypervariable phylogenetic marker in the context of a population genetic study of an uncultured bacterial species. We used high-throughput CRISPR-based typing along with multi-locus sequence analysis (MLSA) to characterize the regional population structure of the obligate but environmentally acquired symbiont species Candidatus Endoriftia persephone on the Juan de Fuca Ridge. Mixed symbiont populations of Ca. Endoriftia persephone were sampled across individual Ridgeia piscesae hosts from contrasting habitats in order to determine if environmental conditions rather than barriers to connectivity are more important drivers of symbiont diversity. We showed that CRISPR revealed a much higher symbiont genetic diversity than the other housekeeping genes. Several lines of evidence imply this diversity is indicative of environmental strains. Finally, we found with both CRISPR and gene markers that local symbiont populations are strongly differentiated across sites known to be isolated by deep-sea circulation patterns. This research showed the high power of CRISPR to resolve the genetic structure of uncultured bacterial populations and represents a step towards making keystone microbial species an integral part of conservation policies for upcoming mining operations on the seafloor.},
}
@article {pmid34448168,
year = {2021},
author = {Chauvin, L and Sevestre, F and Lukan, T and Nogué, F and Gallois, JL and Chauvin, JE and Veillet, F},
title = {Gene Editing in Potato Using CRISPR-Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2354},
number = {},
pages = {331-351},
pmid = {34448168},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Plants ; RNA, Guide/genetics ; *Solanum tuberosum/genetics ; Technology ; },
abstract = {Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis.},
}
@article {pmid34446939,
year = {2021},
author = {Mihaila, RG and Topircean, D},
title = {The high-performance technology CRISPR/Cas9 improves knowledge and management of acute myeloid leukemia.},
journal = {Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia},
volume = {165},
number = {3},
pages = {249-257},
doi = {10.5507/bp.2021.048},
pmid = {34446939},
issn = {1804-7521},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Proliferation ; Humans ; *Leukemia, Myeloid, Acute/genetics/therapy ; Mutation ; Protein Kinase Inhibitors ; Technology ; fms-Like Tyrosine Kinase 3 ; },
abstract = {Knowledge on acute myeloid leukemia pathogenesis and treatment has progressed recently, but not enough to provide ideal management. Improving the prognosis of acute myeloid leukemia patients depends on advances in molecular biology for the detection of new therapeutic targets and the production of effective drugs. The CRISPR/Cas9 technology allows gene insertions and deletions and it is the first step in investigating the function of their encoded proteins. Thus, new experimental models have been developed and progress has been made in understanding protein metabolism, antitumor activity, leukemic cell maintenance, differentiation, growth, apoptosis, and self-renewal, the combined pathogenetic mechanisms involved in leukemogenesis. The CRISPR/Cas9 system is used to understand drug resistance and find solutions to overcome it. The therapeutic progress achieved using the CRISPR/Cas9 system is remarkable. FST gene removal inhibited acute myeloid leukemia cell growth. Lysine acetyltransferase gene deletion contributed to decreased proliferation rate, increased apoptosis, and favored differentiation of acute myelid leukemia cells carrying MLL-X gene fusions. The removal of CD38 gene from NK cells decreased NK fratricidal cells contributing to increased efficacy of new CD38 CAR-NK cells to target leukemic blasts. BCL2 knockout has synergistic effects with FLT3 inhibitors. Exportin 1 knockout is synergistic with midostaurin treatment in acute myeloid leukemia with FLT3-ITD mutation. Using the results of CRISPR/Cas9 libraries and technology application will allow us to get closer to achieving the goal of curing acute myeloid leukemia in the coming decades.},
}
@article {pmid34446818,
year = {2021},
author = {Uribe, RV and Rathmer, C and Jahn, LJ and Ellabaan, MMH and Li, SS and Sommer, MOA},
title = {Bacterial resistance to CRISPR-Cas antimicrobials.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {17267},
pmid = {34446818},
issn = {2045-2322},
mesh = {Anti-Infective Agents/*pharmacology ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/*genetics ; Escherichia coli/*drug effects/genetics ; Gene Editing/*methods ; Genome, Bacterial/genetics ; Microbial Viability/drug effects/genetics ; Mutation ; Plasmids/genetics ; Streptococcus pyogenes/*drug effects/genetics ; Whole Genome Sequencing/methods ; },
abstract = {In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.},
}
@article {pmid34446442,
year = {2021},
author = {van Beljouw, SPB and Haagsma, AC and Rodríguez-Molina, A and van den Berg, DF and Vink, JNA and Brouns, SJJ},
title = {The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6561},
pages = {1349-1353},
doi = {10.1126/science.abk2718},
pmid = {34446442},
issn = {1095-9203},
mesh = {Bacteria/*enzymology/*genetics ; Bacterial Proteins/chemistry/*metabolism ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; Caspases/chemistry/metabolism ; Endoribonucleases/chemistry/genetics/*metabolism ; Interspersed Repetitive Sequences ; Peptide Hydrolases/chemistry/*metabolism ; Protein Domains ; RNA, Bacterial/metabolism ; RNA, Viral/metabolism ; Substrate Specificity ; },
abstract = {Type III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” (Sb-gRAMP), which is natively encoded by a single gene with several type III domains fused together. This effector uses CRISPR RNA to guide target RNA recognition and cleaves single-stranded RNA at two defined positions six nucleotides apart. Sb-gRAMP physically combines with the caspase-like TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) complex, suggesting a potential mechanism of target RNA–induced protease activity to gain viral immunity.},
}
@article {pmid34446204,
year = {2021},
author = {Han, W and Tang, F and Zhong, Y and Zhang, J and Liu, Z},
title = {Identification of yellow gene family and functional analysis of Spodoptera frugiperda yellow-y by CRISPR/Cas9.},
journal = {Pesticide biochemistry and physiology},
volume = {178},
number = {},
pages = {104937},
doi = {10.1016/j.pestbp.2021.104937},
pmid = {34446204},
issn = {1095-9939},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Insecticide Resistance ; Larva/genetics ; *Moths ; Spodoptera/genetics ; },
abstract = {For a devastating agricultural pest, functional genomics promotes the finding of novel technology to control Spodoptera frugiperda, such as the genetics-based strategies. In the present study, 11 yellow genes were identified in Spodoptera frugiperda. The transcriptome analysis showed the tissue-specific expression of part yellow genes, which suggested the importance of yellow genes in some biological processes in S. frugiperda, such as pigmentation. Among these yellow genes, the expression profiles of yellow-y gene showed that it was expressed in all life stages. In order to realize the further study of yellow-y, we employed CRISPR/Cas9 system to knock out this gene. Following knock out, diverse phenotypes were observed, such as color changes in both larvae and adults. Different from the wild-type larvae and adults, G0 mutants were yellowed since hatching. However, no color difference was observed with the pupal cuticle between the wild-type and mutant pupae before the 8th day. On the basis of the single-pair strategy of G0 generation, the yellow-y gene was proved to be a recessive gene. The G1 yellowish larvae with biallelic mutations displayed a relatively longer development period than wild-type, and often generated abnormal pupae and moths. The deletion of yellow-y also resulted in a decline in the fecundity. The results revealed that yellow-y gene was important for S. frugiperda pigmentation, as well as in its development and reproduction. Besides, the present study set up a standard procedure to knock out genes in S. frugiperda, which could be helpful for our understanding some key molecular processes, such as functional roles of detoxification genes as insecticide resistance mechanisms or modes of action of insecticides to facilitate the management of this insect pest.},
}
@article {pmid34446084,
year = {2021},
author = {Ghaffari, S and Khalili, N and Rezaei, N},
title = {CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {40},
number = {1},
pages = {269},
pmid = {34446084},
issn = {1756-9966},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; Humans ; Immunotherapy, Adoptive/*methods ; Neoplasms/immunology/*therapy ; Receptors, Antigen, T-Cell/immunology ; T-Lymphocytes/*immunology ; },
abstract = {Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers.},
}
@article {pmid34445589,
year = {2021},
author = {Shaw, AM and Qasem, A and Naser, SA},
title = {Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445589},
issn = {1422-0067},
mesh = {Arthritis, Rheumatoid/genetics ; *CRISPR-Cas Systems ; Crohn Disease/genetics ; Gene Expression Regulation, Leukemic/*drug effects ; Genetic Predisposition to Disease ; Humans ; Jurkat Cells ; Leukemia, T-Cell/drug therapy/genetics/*pathology ; Lymphocyte Activation ; *Polymorphism, Single Nucleotide ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/antagonists &amp; inhibitors/*genetics/metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22/antagonists &amp; inhibitors/*genetics/metabolism ; Spermidine/*pharmacology ; },
abstract = {Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 μM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.},
}
@article {pmid34445569,
year = {2021},
author = {Sarkar, H and Toms, M and Moosajee, M},
title = {Involvement of Oxidative and Endoplasmic Reticulum Stress in RDH12-Related Retinopathies.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445569},
issn = {1422-0067},
support = {205174/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alcohol Oxidoreductases/antagonists &amp; inhibitors/genetics/*metabolism ; Animals ; *Apoptosis ; Autophagy ; CRISPR-Cas Systems ; *Endoplasmic Reticulum Stress ; HEK293 Cells ; Humans ; *Mutation ; *Oxidative Stress ; Retinal Diseases/etiology/metabolism/*pathology ; Zebrafish ; },
abstract = {Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol (atROL), as part of the visual cycle. Mutations in RDH12 are primarily associated with autosomal recessive Leber congenital amaurosis. To further our understanding of the disease mechanisms, HEK-293 cell lines expressing wildtype (WT) and mutant RDH12 were created. The WT cells afforded protection from atRAL-induced toxicity and oxidative stress. Mutant RDH12 cells displayed reduced protein expression and activity, with an inability to protect cells from atRAL toxicity, inducing oxidative and endoplasmic reticulum (ER) stress, with upregulation of sXBP1, CHOP, and ATF4. Pregabalin, a retinal scavenger, attenuated atRAL-induced ER stress in the mutant RDH12 cell lines. A zebrafish rdh12 mutant model (rdh12[u533] c.17_23del; p.(Val6AlafsTer5)) was generated through CRISPR-Cas9 gene editing. Mutant fish showed disrupted phagocytosis through transmission electron microscopy, with increased phagosome size at 12 months post-fertilisation. Rhodopsin mislocalisation and reduced expression of atg12 and sod2 indicated early signs of a rod-predominant degeneration. A lack of functional RDH12 results in ER and oxidative stress representing key pathways to be targeted for potential therapeutics.},
}
@article {pmid34445458,
year = {2021},
author = {Khusnutdinov, E and Sukhareva, A and Panfilova, M and Mikhaylova, E},
title = {Anthocyanin Biosynthesis Genes as Model Genes for Genome Editing in Plants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445458},
issn = {1422-0067},
mesh = {*Anthocyanins/biosynthesis/genetics ; *Gene Editing ; *Genes, Plant ; *Models, Genetic ; *Plants/genetics/metabolism ; },
abstract = {CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3'H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists.},
}
@article {pmid34445407,
year = {2021},
author = {Prieto-Bermejo, R and Romo-González, M and Pérez-Fernández, A and García-Macías, MC and Sánchez-Bernal, C and García-Tuñón, I and Sánchez-Yagüe, J and Sánchez-Martín, M and Hernández-Hernández, &#xc1;},
title = {Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445407},
issn = {1422-0067},
mesh = {Animals ; B-Lymphocytes/*metabolism ; CRISPR-Cas Systems ; Cell Lineage ; Cytochrome b Group/*genetics ; Disease Models, Animal ; Female ; Gene Knockdown Techniques ; Granulomatous Disease, Chronic/genetics/immunology/*pathology ; Humans ; Hyperplasia ; Male ; Mice ; Myeloid Cells/immunology/*pathology ; NADPH Oxidases/*genetics ; },
abstract = {Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba[-/-] mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb[-/-] and Ncf1[-/-] models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba[-/-] mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.},
}
@article {pmid34445274,
year = {2021},
author = {Denes, CE and Cole, AJ and Aksoy, YA and Li, G and Neely, GG and Hesselson, D},
title = {Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445274},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Gene Editing ; Humans ; *Models, Genetic ; *Recombinational DNA Repair ; },
abstract = {Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.},
}
@article {pmid34445221,
year = {2021},
author = {Abashkin, DA and Kurishev, AO and Karpov, DS and Golimbet, VE},
title = {Cellular Models in Schizophrenia Research.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445221},
issn = {1422-0067},
mesh = {Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation ; Genome-Wide Association Study ; Humans ; *Models, Neurological ; *Schizophrenia/genetics/metabolism ; },
abstract = {Schizophrenia (SZ) is a prevalent functional psychosis characterized by clinical behavioural symptoms and underlying abnormalities in brain function. Genome-wide association studies (GWAS) of schizophrenia have revealed many loci that do not directly identify processes disturbed in the disease. For this reason, the development of cellular models containing SZ-associated variations has become a focus in the post-GWAS research era. The application of revolutionary clustered regularly interspaced palindromic repeats CRISPR/Cas9 gene-editing tools, along with recently developed technologies for cultivating brain organoids in vitro, have opened new perspectives for the construction of these models. In general, cellular models are intended to unravel particular biological phenomena. They can provide the missing link between schizophrenia-related phenotypic features (such as transcriptional dysregulation, oxidative stress and synaptic dysregulation) and data from pathomorphological, electrophysiological and behavioural studies. The objectives of this review are the systematization and classification of cellular models of schizophrenia, based on their complexity and validity for understanding schizophrenia-related phenotypes.},
}
@article {pmid34445123,
year = {2021},
author = {Siew, WS and Tang, YQ and Kong, CK and Goh, BH and Zacchigna, S and Dua, K and Chellappan, DK and Duangjai, A and Saokaew, S and Phisalprapa, P and Yap, WH},
title = {Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445123},
issn = {1422-0067},
mesh = {Animals ; Atherosclerosis/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Epigenesis, Genetic/genetics ; Gene Editing/methods ; Gene Expression/genetics ; Genome/genetics ; Humans ; RNA/genetics ; RNA, Guide/genetics ; },
abstract = {Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.},
}
@article {pmid34442792,
year = {2021},
author = {Li, M and Zhou, X and Stanton, C and Ross, RP and Zhao, J and Zhang, H and Yang, B and Chen, W},
title = {Comparative Genomics Analyses Reveal the Differences between B. longum subsp. infantis and B. longum subsp. longum in Carbohydrate Utilisation, CRISPR-Cas Systems and Bacteriocin Operons.},
journal = {Microorganisms},
volume = {9},
number = {8},
pages = {},
pmid = {34442792},
issn = {2076-2607},
abstract = {Bifidobacterium longum is one of the most widely distributed and abundant Bifidobacterium in the human intestine, and has been proven to have a variety of physiological functions. In this study, 80 strains of B. longum isolated from human subjects were classified into subspecies by ANI and phylogenetic analyses, and the functional genes were compared. The results showed that there were significant differences in carbohydrate metabolism between the two subspecies, which determined their preference for human milk oligosaccharides or plant-derived carbohydrates. The predicted exopolysaccharide (EPS) gene clusters had large variability within species but without difference at the subspecies level. Four subtype CRISPR-Cas systems presented in B. longum, while the subtypes I-U and II-C only existed in B. longum subsp. longum. The bacteriocin operons in B. longum subsp. infantis were more widely distributed compared with B. longum subsp. longum. In conclusion, this study revealed the similarities and differences between B. longum subsp. infantis and B. longum subsp. longum, which could provide a theoretical basis for further exploring the probiotic characteristics of B. longum.},
}
@article {pmid34441495,
year = {2021},
author = {Chen, Y and Li, N and Zhao, S and Zhang, C and Qiao, N and Duan, H and Xiao, Y and Yan, B and Zhao, J and Tian, F and Zhai, Q and Yu, L and Chen, W},
title = {Integrated Phenotypic-Genotypic Analysis of Latilactobacillus sakei from Different Niches.},
journal = {Foods (Basel, Switzerland)},
volume = {10},
number = {8},
pages = {},
pmid = {34441495},
issn = {2304-8158},
abstract = {Increasing attention has been paid to the potential probiotic effects of Latilactobacillus sakei. To explore the genetic diversity of L. sakei, 14 strains isolated from different niches (feces, fermented kimchi, and meat products) and 54 published strains were compared and analyzed. The results showed that the average genome size and GC content of L.&amp;nbsp;sakei were 1.98 Mb and 41.22%, respectively. Its core genome mainly encodes translation and transcription, amino acid synthesis, glucose metabolism, and defense functions. L.&amp;nbsp;sakei has open pan-genomic characteristics, and its pan-gene curve shows an upward trend. The genetic diversity of L.&amp;nbsp;sakei is mainly reflected in carbohydrate utilization, antibiotic tolerance, and immune/competition-related factors, such as clustering regular interval short palindromic repeat sequence (CRISPR)-Cas. The CRISPR system is mainly IIA type, and a few are IIC types. This work provides a basis for the study of this species.},
}
@article {pmid34440868,
year = {2021},
author = {Liu, Y and Liang, F and Dong, Z and Li, S and Ye, J and Qin, W},
title = {Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM.},
journal = {Cells},
volume = {10},
number = {8},
pages = {},
pmid = {34440868},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing ; Genome/genetics ; Mutation ; Nucleotide Motifs ; RNA, Guide/genetics/metabolism ; Streptococcus/*enzymology ; Zebrafish/*genetics ; Zebrafish Proteins/genetics ; },
abstract = {The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5'-NNG-3' PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.},
}
@article {pmid34440461,
year = {2021},
author = {Sun, S and Wang, Y and Goh, PT and Lopes-Marques, M and Castro, LFC and Monroig, &#xd3; and Kuah, MK and Cao, X and Shu-Chien, AC and Gao, J},
title = {Evolution and Functional Characteristics of the Novel elovl8 That Play Pivotal Roles in Fatty Acid Biosynthesis.},
journal = {Genes},
volume = {12},
number = {8},
pages = {},
pmid = {34440461},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cloning, Molecular ; *Evolution, Molecular ; Fatty Acid Elongases/*genetics ; Fatty Acids/biosynthesis/*genetics ; Fatty Acids, Unsaturated/genetics/metabolism ; Fish Proteins/*genetics ; Gene Expression Regulation, Enzymologic/genetics ; Lipogenesis/genetics ; Zebrafish/genetics ; },
abstract = {Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a[-][/-]) and elovl8b (elovl8b[-][/-]) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.},
}
@article {pmid34440379,
year = {2021},
author = {Gendron, WAC and Rubin, JD and Hansen, MJ and Nace, RA and Simone, BW and Ekker, SC and Barry, MA},
title = {Unlocking loxP to Track Genome Editing In Vivo.},
journal = {Genes},
volume = {12},
number = {8},
pages = {},
pmid = {34440379},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; Green Fluorescent Proteins/genetics ; Integrases/*genetics ; Luciferases/genetics ; Mice ; },
abstract = {The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR "fingerprinting" approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.},
}
@article {pmid34440321,
year = {2021},
author = {Tanmoy, AM and Saha, C and Sajib, MSI and Saha, S and Komurian-Pradel, F and van Belkum, A and Louwen, R and Saha, SK and Endtz, HP},
title = {Reply to Fabre et al. Comment on "Tanmoy et al. CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries. Genes 2020, 11, 1365".},
journal = {Genes},
volume = {12},
number = {8},
pages = {},
pmid = {34440321},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; *Salmonella typhi/genetics ; },
abstract = {We respectfully thank Fabre et al. [...].},
}
@article {pmid34440320,
year = {2021},
author = {Fabre, L and Njamkepo, E and Weill, FX},
title = {Comment on Tanmoy et al. CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries. Genes 2020, 11, 1365.},
journal = {Genes},
volume = {12},
number = {8},
pages = {},
pmid = {34440320},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; *Salmonella typhi/genetics ; },
abstract = {Tanmoy et al. [...].},
}
@article {pmid34439828,
year = {2021},
author = {Kim, S and Ji, S and Koh, HR},
title = {CRISPR as a Diagnostic Tool.},
journal = {Biomolecules},
volume = {11},
number = {8},
pages = {},
pmid = {34439828},
issn = {2218-273X},
mesh = {Aptamers, Nucleotide/genetics/metabolism ; Autoimmune Diseases/diagnosis/genetics/metabolism/*therapy ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Methylation ; DNA, Single-Stranded/genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/methods ; Humans ; MicroRNAs/genetics/metabolism ; Molecular Targeted Therapy/*methods ; Neoplasms/diagnosis/genetics/metabolism/*therapy ; Polymorphism, Single Nucleotide ; RNA, Guide/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.},
}
@article {pmid34439766,
year = {2021},
author = {Ikram, AU and Ding, Y and Su, Y},
title = {OsARP6 Is Involved in Internode Elongation by Regulating Cell-Cycle-Related Genes.},
journal = {Biomolecules},
volume = {11},
number = {8},
pages = {},
pmid = {34439766},
issn = {2218-273X},
mesh = {Adenosine Triphosphatases/metabolism ; Arabidopsis/genetics ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Chromatin/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Exons ; Flowers/genetics ; Gene Expression Regulation, Plant ; Green Fluorescent Proteins/metabolism ; Histones/metabolism ; Homozygote ; Microfilament Proteins/genetics/*metabolism ; Mutation ; Open Reading Frames ; Oryza/*metabolism ; Phenotype ; Phylogeny ; Seeds/metabolism ; Transcription Factors/metabolism ; Two-Hybrid System Techniques ; },
abstract = {The SWR1 complex (SWR1-C) is important for the deposition of histone variant H2A.Z into chromatin to regulate gene expression. Characterization of SWR1-C subunits in Arabidopsis thaliana has revealed their role in variety of developmental processes. Oryza sativa actin related protein 6 (OsARP6) is a subunit of rice SWR1-C. Its role in rice plant development is unknown. Here, we examined the subcellular localization, expression patterns, and loss of function phenotypes for this protein and found that OsARP6 is a nuclear localized protein, and is broadly expressed. OsARP6 interacted with OsPIE1, a central ATPase subunit of rice SWR1-C. The osarp6 knockout mutants displayed pleiotropic phenotypic alterations in vegetative and reproductive traits, including semi-dwarf phenotype, lower tillers number, short leaf length, changes in spikelet morphology, and seed abortion. Microscopic thin sectioning of the top internode revealed that the dwarf phenotype of osarp6 was due to reduced number of cells rather than reduced cell length. The altered transcript level of genes involved in cell division suggested that OsARP6 affects cell cycle regulation. In addition, H2A.Z levels were reduced at the promoters and transcription start sites (TSS) of the regulated genes in osarp6 plants. Together, these results suggest that OsARP6 is involved in rice plant development, and H2A.Z deposition.},
}
@article {pmid34439060,
year = {2021},
author = {Shelenkov, A and Petrova, L and Zamyatin, M and Mikhaylova, Y and Akimkin, V},
title = {Diversity of International High-Risk Clones of Acinetobacter baumannii Revealed in a Russian Multidisciplinary Medical Center during 2017-2019.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {8},
pages = {},
pmid = {34439060},
issn = {2079-6382},
abstract = {Acinetobacter baumannii is a dangerous bacterial pathogen possessing the ability to persist on various surfaces, especially in clinical settings, and to rapidly acquire the resistance to a broad spectrum of antibiotics. Thus, the epidemiological surveillance of A. baumannii within a particular hospital, region, and across the world is an important healthcare task that currently usually includes performing whole-genome sequencing (WGS) of representative isolates. During the past years, the dissemination of A. baumannii across the world was mainly driven by the strains belonging to two major groups called the global clones or international clones (ICs) of high risk (IC1 and IC2). However, currently nine ICs are already considered. Although some clones were previously thought to spread in particular regions of the world, in recent years this is usually not the case. In this study, we determined five ICs, as well as three isolates not belonging to the major ICs, in one multidisciplinary medical center within the period 2017-2019. We performed WGS using both short- and long-read sequencing technologies of nine representative clinical A. baumannii isolates, which allowed us to determine the antibiotic resistance and virulence genomic determinants, reveal the CRISPR/Cas systems, and obtain the plasmid structures. The phenotypic and genotypic antibiotic resistance profiles are compared, and the possible ways of isolate and resistance spreading are discussed. We believe that the data obtained will provide a better understanding of the spreading and resistance acquisition of the ICs of A. baumannii and further stress the necessity for continuous genomic epidemiology surveillance of this problem-causing bacterial species.},
}
@article {pmid34438319,
year = {2021},
author = {Tang, Y and Ren, J and Li, CC},
title = {Establishment of a GFP::LMNB1 knockin cell line (CSUi002-A-1) from a dystonia patient-specific iPSC by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102505},
doi = {10.1016/j.scr.2021.102505},
pmid = {34438319},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; *Dystonia ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {LMNB1, as one of the major components of nuclear lamina, anchors heterochromatin and associates with transcription regulation. LMNB1 was previously demonstrated to be upregulated and nuclear-to-cytoplasmic mislocalized in DYT1 dystonia specific neurons. Here, we established a knockin cell line with GFP::LMNB1 fusion expression from a DYT1 patient derived iPSC line, by CRISPR/Cas9 editing. The generated iPSCs displayed GFP and LMNB1 co-localization, reminiscent of successful genomic editing. They remained pluripotent and normal karyotype, and possessed the potential to differentiate into three germ layers. This GFP::LMNB1 knockin iPSC will be used for studying the lamina-pathophysiology of DYT1 dystonia, and other nucleus-centered questions.},
}
@article {pmid34437126,
year = {2021},
author = {Gaudet, RG and Zhu, S and Halder, A and Kim, BH and Bradfield, CJ and Huang, S and Xu, D and Mamiñska, A and Nguyen, TN and Lazarou, M and Karatekin, E and Gupta, K and MacMicking, JD},
title = {A human apolipoprotein L with detergent-like activity kills intracellular pathogens.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6552},
pages = {},
pmid = {34437126},
issn = {1095-9203},
support = {R01 AI068041/AI/NIAID NIH HHS/United States ; R01 AI108834/AI/NIAID NIH HHS/United States ; R01 NS113236/NS/NINDS NIH HHS/United States ; },
mesh = {Apolipoproteins L/chemistry/genetics/*metabolism ; Bacterial Outer Membrane/metabolism ; Bacteriolysis ; CRISPR-Cas Systems ; Cell Membrane/chemistry/*metabolism/ultrastructure ; Cell Membrane Permeability ; Cells, Cultured ; Cytosol/*microbiology ; Detergents/metabolism ; GTP-Binding Proteins/metabolism ; Gene Editing ; Gram-Negative Bacteria/immunology/pathogenicity/*physiology/ultrastructure ; Humans ; Immunity, Innate ; Interferon-gamma/*immunology ; Lipoproteins/chemistry ; Microbial Viability ; O Antigens/metabolism ; Protein Domains ; Salmonella typhimurium/immunology/pathogenicity/physiology/ultrastructure ; Solubility ; },
abstract = {Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.},
}
@article {pmid34436881,
year = {2021},
author = {Wu, Y and Dong, Y and Shi, Y and Yang, H and Zhang, J and Khan, MR and Deng, S and He, G and He, Q and Lv, Y and Deng, R},
title = {CRISPR-Cas12-Based Rapid Authentication of Halal Food.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {35},
pages = {10321-10328},
doi = {10.1021/acs.jafc.1c03078},
pmid = {34436881},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Food Contamination/analysis ; Meat/analysis ; *Meat Products/analysis ; *Red Meat ; Reproducibility of Results ; },
abstract = {The halal food market is globally growing along with the increased risk of adulteration. We proposed an amplification-free and mix-to-read CRISPR-Cas12-based nucleic acid analytical strategy allowing rapid identification and analysis of pork components, thus enriching the toolbox for ensuring halal food authenticity. We designed and optimized guide RNA (gRNA) targeting the pork cytochrome b (Cyt b) gene. gRNA allowed specific identification of the target Cyt b gene from pork components followed by activation of Cas12 protein to abundantly cleave single-stranded DNA probes with terminally labeled fluorophore and quencher groups, thus turning on fluorescence. The presence of the pork Cyt b gene thus can be mix-and-read- and only-one-step-detected, which may indicate the risk of halal food adulteration. The method allowed specific discrimination of pork meat from beef, mutton, and chicken and yielded a detection limit of 2.7 ng/μL of total DNA from pork meat. The reliability of the method was tested using the following processed meat products: halal foods beef luncheon meat and spiced beef and non-halal foods sausage and dried pork slices. The CRISPR-Cas12-based nucleic acid test strategy is promising for rapid food authentication.},
}
@article {pmid34436049,
year = {2021},
author = {Chen, M and Zhang, C and Hu, Z and Li, Z and Li, M and Wu, L and Zhou, M and Liang, D},
title = {CRISPR/Cas12a-Based Ultrasensitive and Rapid Detection of JAK2 V617F Somatic Mutation in Myeloproliferative Neoplasms.},
journal = {Biosensors},
volume = {11},
number = {8},
pages = {},
pmid = {34436049},
issn = {2079-6374},
mesh = {CRISPR-Cas Systems ; Humans ; Janus Kinase 2/genetics ; Mutation ; *Myeloproliferative Disorders/diagnosis/genetics ; *Neoplasms ; },
abstract = {The JAK2 V617F mutation is a major diagnostic, therapeutic, and monitoring molecular target of Philadelphia-negative myeloproliferative neoplasms (MPNs). To date, numerous methods of detecting the JAK2 V617F mutation have been reported, but there is no gold-standard diagnostic method for clinical applications. Here, we developed and validated an efficient Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 12a (Cas12a)-based assay to detect the JAK2 V617F mutation. Our results showed that the sensitivity of the JAK2 V617F/Cas12a fluorescence detection system was as high as 0.01%, and the JAK2 V617F/Cas12a lateral flow strip assay could unambiguously detect as low as 0.5% of the JAK2 V617F mutation, which was much higher than the sensitivity required for clinical application. The minimum detectable concentration of genomic DNA achieved was 0.01 ng/μL (~5 aM, ~3 copies/μL). In addition, the whole process only took about 1.5 h, and the cost of an individual test was much lower than that of the current assays. Thus, our methods can be applied to detect the JAK2 V617F mutation, and they are highly sensitive, rapid, cost-effective, and convenient.},
}
@article {pmid34435379,
year = {2021},
author = {Tang, Q and Liu, M and Liu, Y and Hwang, RD and Zhang, T and Wang, J},
title = {NDST3 deacetylates α-tubulin and suppresses V-ATPase assembly and lysosomal acidification.},
journal = {The EMBO journal},
volume = {40},
number = {19},
pages = {e107204},
pmid = {34435379},
issn = {1460-2075},
support = {I01 BX002466/BX/BLRD VA/United States ; R01 NS074324/NS/NINDS NIH HHS/United States ; R01 NS110098/NS/NINDS NIH HHS/United States ; R01 NS089616/NS/NINDS NIH HHS/United States ; },
mesh = {Acetylation ; Amyotrophic Lateral Sclerosis/genetics/metabolism ; Animals ; C9orf72 Protein/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Fluorescent Antibody Technique ; Gene Expression ; Gene Knockdown Techniques ; Gene Library ; Genes, Reporter ; Humans ; Hydrogen-Ion Concentration ; Lysosomes/*metabolism ; Macrolides/pharmacology/toxicity ; Mice ; Microtubules/metabolism ; Models, Biological ; Neurons/cytology/drug effects/metabolism ; Pharmacogenomic Testing/methods ; Pharmacogenomic Variants ; Protein Binding ; Sulfotransferases/genetics/*metabolism ; Tubulin/*metabolism ; Vacuolar Proton-Translocating ATPases/genetics/*metabolism ; },
abstract = {Lysosomes are key organelles maintaining cellular homeostasis in health and disease. Here, we report the identification of N-deacetylase and N-sulfotransferase 3 (NDST3) as a potent regulator of lysosomal functions through an unbiased genetic screen. NDST3 constitutes a new member of the histone deacetylase (HDAC) family and catalyzes the deacetylation of α-tubulin. Loss of NDST3 promotes assembly of the V-ATPase holoenzyme on the lysosomal membrane and thereby increases the acidification of the organelle. NDST3 is downregulated in tissues and cells from patients carrying the C9orf72 hexanucleotide repeat expansion linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Deficiency in C9orf72 decreases the level of NDST3, and downregulation of NDST3 exacerbates the proteotoxicity of poly-dipeptides generated from the C9orf72 hexanucleotide repeats. These results demonstrate a previously unknown regulatory mechanism through which microtubule acetylation regulates lysosomal activities and suggest that NDST3 could be targeted to modulate microtubule and lysosomal functions in relevant diseases.},
}
@article {pmid34435370,
year = {2021},
author = {Cable, J and Ronald, PC and Voytas, D and Zhang, F and Levy, AA and Takatsuka, A and Arimura, SI and Jacobsen, SE and Toki, S and Toda, E and Gao, C and Zhu, JK and Boch, J and Van Eck, J and Mahfouz, M and Andersson, M and Fridman, E and Weiss, T and Wang, K and Qi, Y and Jores, T and Adams, T and Bagchi, R},
title = {Plant genome engineering from lab to field-a Keystone Symposia report.},
journal = {Annals of the New York Academy of Sciences},
volume = {1506},
number = {1},
pages = {35-54},
doi = {10.1111/nyas.14675},
pmid = {34435370},
issn = {1749-6632},
mesh = {CRISPR-Cas Systems/genetics ; *Congresses as Topic/trends ; Crops, Agricultural/*genetics ; Gene Editing/methods/trends ; Gene Targeting/methods/trends ; Genetic Engineering/*methods/trends ; Genome, Plant/*genetics ; Plant Breeding/*methods ; *Research Report ; },
abstract = {Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium "Plant Genome Engineering: From Lab to Field" to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.},
}
@article {pmid34433872,
year = {2021},
author = {Lin, L and Chen, J and Mitra, R and Gao, Q and Cheng, F and Xu, T and Zuo, Z and Xiang, H and Han, J},
title = {Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {1007},
pmid = {34433872},
issn = {2399-3642},
mesh = {Biopolymers/biosynthesis ; *Carbon Cycle ; Clustered Regularly Interspaced Short Palindromic Repeats ; Haloferax mediterranei/*metabolism ; Polyesters/*metabolism ; },
abstract = {The haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.},
}
@article {pmid34433825,
year = {2021},
author = {Bermudez-Cabrera, HC and Culbertson, S and Barkal, S and Holmes, B and Shen, MW and Zhang, S and Gifford, DK and Sherwood, RI},
title = {Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5111},
pmid = {34433825},
issn = {2041-1723},
support = {R01 HG008754/HG/NHGRI NIH HHS/United States ; R21 HG010391/HG/NHGRI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Ataxia Telangiectasia Mutated Proteins/*antagonists &amp; inhibitors/genetics/*metabolism ; CRISPR-Cas Systems/*drug effects ; Cell Line ; Gene Editing ; Humans ; Morpholines/*pharmacology ; Mutagenesis, Insertional/*drug effects ; Protein Kinase Inhibitors/*pharmacology ; Sequence Deletion/drug effects ; Thioxanthenes/*pharmacology ; },
abstract = {Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.},
}
@article {pmid34433819,
year = {2021},
author = {Marquart, KF and Allam, A and Janjuha, S and Sintsova, A and Villiger, L and Frey, N and Krauthammer, M and Schwank, G},
title = {Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5114},
pmid = {34433819},
issn = {2041-1723},
mesh = {Algorithms ; Base Pairing ; *Deep Learning ; Gene Editing ; *Gene Library ; Genome ; High-Throughput Nucleotide Sequencing ; Humans ; },
abstract = {Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable transition of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have great potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.},
}
@article {pmid34433012,
year = {2021},
author = {Kumon, T and Ma, J and Akins, RB and Stefanik, D and Nordgren, CE and Kim, J and Levine, MT and Lampson, MA},
title = {Parallel pathways for recruiting effector proteins determine centromere drive and suppression.},
journal = {Cell},
volume = {184},
number = {19},
pages = {4904-4918.e11},
pmid = {34433012},
issn = {1097-4172},
support = {R35 GM122475/GM/NIGMS NIH HHS/United States ; R35 GM124684/GM/NIGMS NIH HHS/United States ; RM1 HG010023/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Amino Acid Sequence ; Animals ; Biological Evolution ; CRISPR-Cas Systems/genetics ; Centromere/*metabolism ; Centromere Protein A/metabolism ; Centromere Protein B/*metabolism ; Chromosomal Proteins, Non-Histone/chemistry/*metabolism ; Chromosomes, Mammalian/metabolism ; Female ; Heterochromatin/metabolism ; Kinetochores/metabolism ; Male ; Mice, Inbred C57BL ; Models, Biological ; Oocytes/metabolism ; Protein Domains ; },
abstract = {Selfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this "centromere drive." In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting the kinetochore pathway with a divergent allele of CENP-C reduces functional differences between centromeres, whereas disrupting heterochromatin by CENP-B deletion amplifies the differences. Molecular evolution analyses using Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.},
}
@article {pmid34433009,
year = {2021},
author = {Socha, M and Sowińska-Seidler, A and Melo, US and Kragesteen, BK and Franke, M and Heinrich, V and Schöpflin, R and Nagel, I and Gruchy, N and Mundlos, S and Sreenivasan, VKA and López, C and Vingron, M and Bukowska-Olech, E and Spielmann, M and Jamsheer, A},
title = {Position effects at the FGF8 locus are associated with femoral hypoplasia.},
journal = {American journal of human genetics},
volume = {108},
number = {9},
pages = {1725-1734},
pmid = {34433009},
issn = {1537-6605},
mesh = {Adolescent ; Alleles ; Animals ; CRISPR-Cas Systems ; Child, Preschool ; Chromatin/chemistry/metabolism ; *Chromosome Duplication ; Chromosomes, Human, Pair 10/*chemistry/metabolism ; *DNA Copy Number Variations ; Enhancer Elements, Genetic ; Family ; Female ; Femur/abnormalities/diagnostic imaging/metabolism ; Fibroblast Growth Factor 8/*genetics/metabolism ; Gene Editing ; Heterozygote ; Humans ; Infant ; Lower Extremity Deformities, Congenital/diagnostic imaging/*genetics/metabolism/pathology ; Male ; Mice ; Mice, Transgenic ; Pedigree ; Phenotype ; },
abstract = {Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.},
}
@article {pmid34432885,
year = {2021},
author = {Namba, M and Kobayashi, T and Koyano, T and Kohno, M and Ohtsuka, M and Matsuyama, M},
title = {GONAD: A new method for germline genome editing in mice and rats.},
journal = {Development, growth &amp; differentiation},
volume = {63},
number = {8},
pages = {439-447},
doi = {10.1111/dgd.12746},
pmid = {34432885},
issn = {1440-169X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Electroporation ; Female ; *Gene Editing ; Gonads ; Humans ; Mice ; *Nucleic Acids ; Oviducts ; Rats ; Zygote ; },
abstract = {Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene-editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome-engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome-editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low-cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as "improved GONAD (i-GONAD)" for mice, and "rat improved GONAD (rGONAD)" for rats.},
}
@article {pmid34432851,
year = {2021},
author = {Hanson, MA and Cohen, LB and Marra, A and Iatsenko, I and Wasserman, SA and Lemaitre, B},
title = {The Drosophila Baramicin polypeptide gene protects against fungal infection.},
journal = {PLoS pathogens},
volume = {17},
number = {8},
pages = {e1009846},
pmid = {34432851},
issn = {1553-7374},
support = {R01 GM050545/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Antifungal Agents/*pharmacology ; Beauveria/*drug effects/growth &amp; development/immunology ; Drosophila Proteins/*pharmacology ; Drosophila melanogaster/*drug effects/genetics/immunology/microbiology ; Female ; Male ; Mycoses/*drug therapy/immunology/microbiology ; Peptides/*pharmacology ; },
abstract = {The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.},
}
@article {pmid34432381,
year = {2021},
author = {Bower, OJ and McCarthy, A and Lea, RA and Alanis-Lobato, G and Zohren, J and Gerri, C and Turner, JMA and Niakan, KK},
title = {Generating CRISPR-Cas9-Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells.},
journal = {Current protocols},
volume = {1},
number = {8},
pages = {e232},
doi = {10.1002/cpz1.232},
pmid = {34432381},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Loss of Function Mutation ; *Pluripotent Stem Cells ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.},
}
@article {pmid34432044,
year = {2021},
author = {Liu, X and Zhang, L and Xiu, Y and Gao, T and Huang, L and Xie, Y and Yang, L and Wang, W and Wang, P and Zhang, Y and Yang, M and Feng, Y},
title = {Insights into the dual functions of AcrIF14 during the inhibition of type I-F CRISPR-Cas surveillance complex.},
journal = {Nucleic acids research},
volume = {49},
number = {17},
pages = {10178-10191},
pmid = {34432044},
issn = {1362-4962},
mesh = {Bacteriophages/genetics/growth &amp; development ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; DNA/genetics/*metabolism ; DNA-Binding Proteins/antagonists &amp; inhibitors/*metabolism ; Endodeoxyribonucleases/*metabolism ; Protein Conformation ; Pseudomonas aeruginosa/*genetics/virology ; Viral Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of both AcrIF14 and its complex with the crRNA-guided surveillance (Csy) complex. Our study demonstrates that apart from interacting with the Csy complex to block the hybridization of target DNA to the crRNA, AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA as AcrIF9 does. Further structural studies of the Csy-AcrIF14-dsDNA complex and biochemical studies uncover that the PAM recognition loop of the Cas8f subunit of the Csy complex and electropositive patches within the N-terminal domain of AcrIF14 are essential for the non-sequence-specific dsDNA binding to the Csy-AcrIF14 complex, which is different from the mechanism of AcrIF9. Our findings highlight the prevalence of Acr-induced non-specific DNA binding and shed light on future studies into the mechanisms of such Acr proteins.},
}
@article {pmid34431041,
year = {2022},
author = {Wang, X and Petersen, B},
title = {More abundant and healthier meat: will the MSTN editing epitome empower the commercialization of gene editing in livestock?.},
journal = {Science China. Life sciences},
volume = {65},
number = {2},
pages = {448-450},
pmid = {34431041},
issn = {1869-1889},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Livestock ; Meat ; },
}
@article {pmid34430917,
year = {2021},
author = {Morrow, CS and Porter, TJ and Moore, DL},
title = {Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100744},
pmid = {34430917},
issn = {2666-1667},
support = {DP2 NS111818/NS/NINDS NIH HHS/United States ; T32 AG000213/AG/NIA NIH HHS/United States ; T32 GM008688/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Fluorescent Antibody Technique/*methods ; Gene Editing/methods ; Mice ; Neural Stem Cells/*metabolism ; Protein Engineering/*methods ; },
abstract = {Although exogenous overexpression of a protein fused to a fluorescent tag can provide insight for the protein's function, it also can produce artifacts attributed to its upregulation and may not fully report the endogenous regulation of the protein of interest. To circumvent these issues, we adapted a protocol to label endogenous proteins with fluorescent tags in primary adult mouse neural stem cells in vitro. Here, we describe reagent construction, reagent delivery, and a screening strategy to isolate edited cells. For complete details on the use and execution of this protocol, please refer to Morrow et al. (2020).},
}
@article {pmid34430908,
year = {2021},
author = {Zhao, L and Liu, P and Xie, W and Zhang, S and Thieme, S and Zitvogel, L and Kroemer, G and Kepp, O},
title = {A genotype-phenotype screening system using conditionally immortalized immature dendritic cells.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100732},
pmid = {34430908},
issn = {2666-1667},
mesh = {Animals ; Antigen Presentation/physiology ; CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Cell Differentiation/physiology ; Cell- and Tissue-Based Therapy ; Cells, Cultured ; Cytokines/metabolism ; Dendritic Cells/*classification/*cytology/immunology/physiology ; Female ; Gene Knockout Techniques/*methods ; Genotype ; Humans ; Mice ; Mice, Inbred C57BL ; Phagocytosis/physiology ; Phenotype ; },
abstract = {Here, we describe a protocol for CRISPR/Cas9-mediated gene knockout in conditionally immortalized immature dendritic cells (DCs), which can be limitlessly expanded before differentiation. This facilitates the genetic screening of DC functions in vitro including assessment of phagocytosis, cytokine production, expression of co-stimulatory or co-inhibitory molecules, and antigen presentation, as well as evaluation of the capacity to elicit anticancer immune responses in vivo. Altogether, these approaches described in this protocol allow investigators to link the genotype of DCs to their phenotype. For complete details on the use and execution of this protocol, please refer to Le Naour et al. (2020).},
}
@article {pmid34429454,
year = {2021},
author = {Akasu, M and Shimada, S and Kabashima, A and Akiyama, Y and Shimokawa, M and Akahoshi, K and Kudo, A and Yamaoka, S and Tanabe, M and Tanaka, S},
title = {Intrinsic activation of β-catenin signaling by CRISPR/Cas9-mediated exon skipping contributes to immune evasion in hepatocellular carcinoma.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {16732},
pmid = {34429454},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Carcinoma, Hepatocellular/immunology/*metabolism/pathology ; *Exons ; Humans ; *Immune Evasion ; Liver Neoplasms/immunology/*metabolism/pathology ; Mutation ; Wnt Signaling Pathway/*genetics ; beta Catenin/genetics/*metabolism ; },
abstract = {Comprehensive analysis of clinical samples has recently identified molecular and immunological classification of hepatocellular carcinoma (HCC), and the CTNNB1 (β-catenin)-mutated subtype exhibits distinctive characteristics of immunosuppressive tumor microenvironment. For clarifying the molecular mechanisms, we first established human and mouse HCC cells with exon 3 skipping of β-catenin, which promoted nuclear translocation and activated the Wnt/β-catenin signaling pathway, by using newly developed multiplex CRISPR/Cas9-based genome engineering system. Gene set enrichment analysis indicated downregulation of immune-associated gene sets in the HCC cells with activated β-catenin signaling. Comparative analysis of gene expression profiles between HCC cells harboring wild-type and exon 3 skipping β-catenin elucidated that the expression levels of four cytokines were commonly decreased in human and mouse β-catenin-mutated HCC cells. Public exome and transcriptome data of 373 human HCC samples showed significant downregulation of two candidate cytokine genes, CCL20 and CXCL2, in HCC tumors with β-catenin hotspot mutations. T cell killing assays and immunohistochemical analysis of grafted tumor tissues demonstrated that the mouse Ctnnb1[Δex3] HCC cells evaded immunosurveillance. Taken together, this study discovered that cytokine controlled by β-catenin signaling activation could contribute to immune evasion, and provided novel insights into cancer immunotherapy for the β-catenin-mutated HCC subtype.},
}
@article {pmid34429421,
year = {2021},
author = {Simhadri, VL and Hopkins, L and McGill, JR and Duke, BR and Mukherjee, S and Zhang, K and Sauna, ZE},
title = {Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4[+] T-cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5090},
pmid = {34429421},
issn = {2041-1723},
mesh = {Amino Acid Sequence ; CD4-Positive T-Lymphocytes/*immunology ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Proliferation/*genetics ; Cytokines ; Gene Editing ; Histocompatibility Antigens Class II/*metabolism ; Humans ; Peptides/*genetics/*metabolism ; Staphylococcus aureus/genetics ; T-Lymphocytes/immunology ; },
abstract = {CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4[+] T-cells.},
}
@article {pmid34429033,
year = {2021},
author = {Hung, C and Livesey, FJ},
title = {Endolysosome and autophagy dysfunction in Alzheimer disease.},
journal = {Autophagy},
volume = {17},
number = {11},
pages = {3882-3883},
pmid = {34429033},
issn = {1554-8635},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; WT101052MA/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alzheimer Disease/etiology/metabolism/*pathology ; *Autophagy ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Humans ; LDL-Receptor Related Proteins/metabolism ; Lysosomes/metabolism/*pathology ; Membrane Transport Proteins/metabolism ; Neurons/metabolism/pathology ; },
abstract = {Abnormalities of the neuronal endolysosome and macroautophagy/autophagy system are an early and prominent feature of Alzheimer disease (AD). SORL1 is notable as a gene in which mutations are causal for a rare, autosomal dominant form of AD, and also variants that increase the risk of developing the common form of late-onset AD. In our recent study, we used patient-derived stem cells and CRISPR engineering to study the effects of SORL1 mutations on the endolysosome and autophagy system in human forebrain neurons. SORL1 mutations causal for monogenic AD are typically truncating mutations, and we found, using stem cells generated from an individual with dementia due to a heterozygous SORL1 truncation mutation, that this class of mutation results in SORL1 haploinsufficiency. Reducing SORL1 protein by half results in disrupted endosomal trafficking in patient-derived neurons, which we confirmed by studying the endolysosomal system in isogenic CRISPR-engineered SORL1 heterozygous null neurons. We also found that SORL1 homozygous null neurons develop more severe phenotypes, with endosome abnormalities, lysosome dysfunction and defects in the degradative phase of autophagy. Endolysosome and autophagy defects in SORL1 mutant neurons are dependent on APP, a key AD gene, as they are rescued by extracellular antisense oligonucleotides that reduce APP protein.},
}
@article {pmid34428454,
year = {2021},
author = {Gao, Y and Han, M and Shang, S and Wang, H and Qi, LS},
title = {Interrogation of the dynamic properties of higher-order heterochromatin using CRISPR-dCas9.},
journal = {Molecular cell},
volume = {81},
number = {20},
pages = {4287-4299.e5},
pmid = {34428454},
issn = {1097-4164},
support = {U01 DK127405/DK/NIDDK NIH HHS/United States ; U01 EB021240/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Chromatin Assembly and Disassembly ; Chromobox Protein Homolog 5/genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Heterochromatin/genetics/*metabolism ; Humans ; Nucleic Acid Conformation ; Protein Conformation ; RNA, Guide/genetics/metabolism ; Structure-Activity Relationship ; Time Factors ; },
abstract = {Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells because of a lack of appropriate tools. Here, we develop CRISPR-engineered chromatin organization (EChO), combining live-cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1α tiled across kilobase-scale genomic DNA form novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kb. These findings support a polymer model of HP1α-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.},
}
@article {pmid34428286,
year = {2021},
author = {Wang, X and Yuan, Q and Zhang, W and Ji, S and Lv, Y and Ren, K and Lu, M and Xiao, Y},
title = {Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1.},
journal = {Nucleic acids research},
volume = {49},
number = {17},
pages = {9938-9952},
pmid = {34428286},
issn = {1362-4962},
mesh = {Archaea/genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Helix-Turn-Helix Motifs/genetics ; High-Throughput Nucleotide Sequencing ; Integrases/*genetics/*metabolism ; Oligonucleotides/genetics ; Terminal Repeat Sequences/*genetics ; },
abstract = {Casposase, a homolog of Cas1 integrase, is encoded by a superfamily of mobile genetic elements known as casposons. While family 2 casposase has been well documented in both function and structure, little is known about the other three casposase families. Here, we studied the family 1 casposase lacking the helix-turn-helix (HTH) domain from Candidatus Nitrosopumilus koreensis AR1 (Ca. N. koreensis). The determinants for integration by Ca. N. koreensis casposase were extensively investigated, and it was found that a 13-bp target site duplication (TSD) sequence, a minimal 3-bp leader and three different nucleotides of the TSD sequences are indispensable for target specific integration. Significantly, the casposase can site-specifically integrate a broad range of terminal inverted repeat (TIR)-derived oligonucleotides ranging from 7-nt to ∼4000-bp, and various oligonucleotides lacking the 5'-TTCTA-3' motif at the 3' end of TIR sequence can be integrated efficiently. Furthermore, similar to some Cas1 homologs, the casposase utilizes a 5'-ATAA-3' motif in the TSD as a molecular ruler to dictate nucleophilic attack at 9-bp downstream of the end of the ruler during the spacer-side integration. By characterizing the family 1 Ca. N. koreensis casposase, we have extended our understanding on mechanistic similarities and evolutionary connections between casposons and the adaptation elements of CRISPR-Cas immunity.},
}
@article {pmid34427973,
year = {2021},
author = {Ma, G and Kuang, Y and Lu, Z and Li, X and Xu, Z and Ren, B and Zhou, X and Zhou, H},
title = {CRISPR/Sc[++] -mediated genome editing in rice.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {9},
pages = {1606-1610},
doi = {10.1111/jipb.13166},
pmid = {34427973},
issn = {1744-7909},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant ; Oryza/*genetics ; Streptococcus/genetics ; },
abstract = {Streptococcus canis Cas9 (ScCas9) is an RNA-guided endonuclease with NNG protospacer adjacent motif (PAM) specificity whose genome-editing activity in rice is locus-dependent. Here we investigated the performance of a ScCas9 variant named Sc[++] at different NNG PAM sites in the rice genome; Sc[++] harbors a T1227K mutation and the substitution of a positively charged loop (residues 367-376). Sc[++] nuclease achieved broader genome editing compared to the original ScCas9, and its nickase improved targeted base editing in transgenic rice plants. Using the high-efficiency adenine base editor rBE73b, we generated many new OsGS1 alleles suitable for screening of rice germplasm for potential herbicide resistance in the future. The CRISPR/Sc[++] system expands the genome-editing toolkit for rice.},
}
@article {pmid34427889,
year = {2022},
author = {Kashtwari, M and Mansoor, S and Wani, AA and Najar, MA and Deshmukh, RK and Baloch, FS and Abidi, I and Zargar, SM},
title = {Random mutagenesis in vegetatively propagated crops: opportunities, challenges and genome editing prospects.},
journal = {Molecular biology reports},
volume = {49},
number = {6},
pages = {5729-5749},
pmid = {34427889},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; Mutagenesis/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {In order to meet the growing human food and nutrition demand a perpetual process of crop improvement is idealized. It has seen changing trends and varying concepts throughout human history; from simple selection to complex gene-editing. Among these techniques, random mutagenesis has been shown to be a promising technology to achieve desirable genetic gain with less time and minimal efforts. Over the decade, several hundred varieties have been released through random mutagenesis, but the production is falling behind the demand. Several food crops like banana, potato, cassava, sweet potato, apple, citrus, and others are vegetatively propagated. Since such crops are not propagated through seed, genetic improvement through classical breeding is impractical for them. Besides, in the case of polyploids, accomplishment of allelic homozygosity requires a considerable land area, extensive fieldwork with huge manpower, and hefty funding for an extended period of time. Apart from induction, mapping of induced genes to facilitate the knowledge of biological processes has been performed only in a few selected facultative vegetative crops like banana and cassava which can form a segregating population. During the last few decades, there has been a shift in the techniques used for crop improvement. With the introduction of the robust technologies like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) more and more crops are being subjected to gene editing. However, more work needs to be done in case of vegetatively propagated crops.},
}
@article {pmid34427091,
year = {2021},
author = {Gong, S and Zhang, S and Lu, F and Pan, W and Li, N and Tang, B},
title = {CRISPR/Cas-Based In Vitro Diagnostic Platforms for Cancer Biomarker Detection.},
journal = {Analytical chemistry},
volume = {93},
number = {35},
pages = {11899-11909},
doi = {10.1021/acs.analchem.1c02533},
pmid = {34427091},
issn = {1520-6882},
mesh = {Biomarkers, Tumor/genetics ; CRISPR-Cas Systems/genetics ; Humans ; *Neoplasms/diagnosis/genetics ; *Nucleic Acids ; },
abstract = {Timely diagnosis is of great benefit to improve the survival rate of cancer patients. Body fluid cancer biomarker detection is a critical kind of noninvasive method for cancer diagnosis. Nevertheless, traditional methods for cancer biomarker detection always rely on a large-scale instrument and involve sophisticated operation. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, holding great promise as the next-generation molecular diagnostic technology. In this Feature, we introduce the working mechanisms of different kinds of CRISPR/Cas systems for biosensing and CRISPR/Cas-mediated detection strategies for different kinds of cancer biomarkers including nucleic acids, proteins, and extracellular vesicles. In addition, the perspective and challenges of CRISPR/Cas-based strategies for cancer biomarkers are discussed.},
}
@article {pmid34426588,
year = {2021},
author = {Carlessi, M and Mariotti, L and Giaume, F and Fornara, F and Perata, P and Gonzali, S},
title = {Targeted knockout of the gene OsHOL1 removes methyl iodide emissions from rice plants.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {17010},
pmid = {34426588},
issn = {2045-2322},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; *Gene Knockout Techniques ; *Genes, Plant ; Hydrocarbons, Iodinated/*isolation &amp; purification ; Luciferases/metabolism ; Methyltransferases/genetics/metabolism ; Mutagenesis/genetics ; Oryza/*genetics ; Plant Leaves/genetics ; Protein Multimerization ; Subcellular Fractions/metabolism ; },
abstract = {Iodine deficiency represents a public health problem worldwide. To increase the amount of iodine in the diet, biofortification strategies of plants have been tried. They rely on the exogenous administration of iodine to increase its absorption and accumulation. However, iodine is not stable in plants and can be volatilized as methyl iodide through the action of specific methyltransferases encoded by the HARMLESS TO OZONE LAYER (HOL) genes. The release of methyl iodide in the atmosphere represents a threat for the environment due to its ozone depletion potential. Rice paddies are among the strongest producers of methyl iodide. Thus, the agronomic approach of iodine biofortification is not appropriate for this crop, leading to further increases of iodine emissions. In this work, we used the genome editing CRISPR/Cas9 technology to knockout the rice HOL genes and investigate their function. OsHOL1 resulted a major player in methyl iodide production, since its knockout abolished the process. Moreover, its overexpression reinforced it. Conversely, knockout of OsHOL2 did not produce effects. Our experiments helped elucidating the function of the rice HOL genes, providing tools to develop new rice varieties with reduced iodine emissions and thus more suitable for biofortification programs without further impacting on the environment.},
}
@article {pmid34426509,
year = {2021},
author = {Becker, A and Wardas, B and Salah, H and Amini, M and Fecher-Trost, C and Sen, Q and Martus, D and Beck, A and Philipp, SE and Flockerzi, V and Belkacemi, A},
title = {Cavβ3 Regulates Ca[2+] Signaling and Insulin Expression in Pancreatic β-Cells in a Cell-Autonomous Manner.},
journal = {Diabetes},
volume = {70},
number = {11},
pages = {2532-2544},
pmid = {34426509},
issn = {1939-327X},
mesh = {Animals ; CREB-Binding Protein ; CRISPR-Cas Systems ; Calcium Channels/genetics/*metabolism ; Calcium Channels, L-Type/genetics/*metabolism ; Calcium Signaling/genetics/*physiology ; Cell Line, Tumor ; Cytosol/metabolism ; Gene Expression Regulation ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Insulin/*metabolism ; Insulin Secretion/*physiology ; Insulin-Secreting Cells/*metabolism ; Insulinoma/metabolism ; Rats ; },
abstract = {Voltage-gated Ca[2+] (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavβ3 could affect cytosolic calcium concentration ([Ca[2] [+]]) in pancreatic β-cells. In this study, we deleted the Cavβ3-encoding gene Cacnb3 in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca[2+] signaling, glucose-induced insulin secretion (GIIS), Cav channel activity, and gene expression in wild-type cells in which Cavβ3 and the IP3 receptor were coimmunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, proprotein convertase subtilisin/kexin type-1, and nitric oxide synthase-1 in Cavβ3-knockout cells. In the absence of Cavβ3, Cav currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca[2+] release and the frequency of Ca[2+] oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3 receptor in isolated β-cells, controls IP3-dependent Ca[2+]-signaling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.},
}
@article {pmid34425703,
year = {2021},
author = {Maikova, A and Boudry, P and Shiriaeva, A and Vasileva, A and Boutserin, A and Medvedeva, S and Semenova, E and Severinov, K and Soutourina, O},
title = {Protospacer-Adjacent Motif Specificity during Clostridioides difficile Type I-B CRISPR-Cas Interference and Adaptation.},
journal = {mBio},
volume = {12},
number = {4},
pages = {e0213621},
pmid = {34425703},
issn = {2150-7511},
mesh = {Adaptation, Physiological/*genetics ; CRISPR-Associated Proteins/classification/*genetics ; *CRISPR-Cas Systems ; Clostridioides difficile/*genetics/metabolism/pathogenicity ; DNA, Bacterial/genetics ; Gene Editing/*methods ; *Genome, Bacterial ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.},
}
@article {pmid34425437,
year = {2021},
author = {Li, Y and Deng, F and Hall, T and Vesey, G and Goldys, EM},
title = {CRISPR/Cas12a-powered immunosensor suitable for ultra-sensitive whole Cryptosporidium oocyst detection from water samples using a plate reader.},
journal = {Water research},
volume = {203},
number = {},
pages = {117553},
doi = {10.1016/j.watres.2021.117553},
pmid = {34425437},
issn = {1879-2448},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; *Cryptosporidiosis ; *Cryptosporidium/genetics ; Immunoassay ; Oocysts ; },
abstract = {Waterborne pathogens, such as Cryptosporidium parvum, pose a major threat to public health globally, and this requires screening of drinking and environmental water for low number of contaminating microbes. However, current detection approaches generally require trained experts with sophisticated instruments, and are not suitable for large-scale screening and rapid outbreak response. Recent advances in ultrasensitive CRISPR/Cas-based biosensing continue to expand the range of detectable molecular targets, however single microbes could not be directly detected so far, especially in environmental samples. Here, we report an ultrasensitive CRISPR/Cas12a-powered immunosensing method suitable for microbial detection which links antibody-based recognition with CRISPR/Cas12a-based fluorescent signal amplification through an antibody-DNA conjugate. This approach is shown here to detect whole 4 µm size Cryptosporidium parvum oocysts with a linear range from 6.25 - 1600 oocysts/mL, at a maximum sensitivity of single oocyst per sample. Its potential to apply to various complex sample matrices has also been demonstrated. After sample dilution by factor of 10, we were able to detect 10 oocysts from a back-wash mud samples from water treatment plate. This method uses the same experimental setup (plate reader) as a conventional ELISA assay thus reducing the need for microscopy-based identification of Cryptosporidium, which represents the gold-standard but requires high level expertise and time-consuming manual counting. This work highlights the potential of CRISPR/Cas-based biosensing for water quality assessment and ultrasensitive whole pathogen detection.},
}
@article {pmid34425195,
year = {2021},
author = {Li, L and Wu, Y and Zhao, C and Miao, Y and Cai, J and Song, L and Wei, J and Chakraborty, T and Wu, L and Wang, D and Zhou, L},
title = {The role of StAR2 gene in testicular differentiation and spermatogenesis in Nile tilapia (Oreochromis niloticus).},
journal = {The Journal of steroid biochemistry and molecular biology},
volume = {214},
number = {},
pages = {105974},
doi = {10.1016/j.jsbmb.2021.105974},
pmid = {34425195},
issn = {1879-1220},
mesh = {Androgens/metabolism ; Animals ; Apoptosis ; CRISPR-Cas Systems ; *Cell Differentiation ; Cichlids/*metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gonads/metabolism ; Leydig Cells/cytology ; Male ; Mutation ; Phosphoproteins/*physiology ; RNA-Binding Proteins/*physiology ; *Spermatogenesis ; Spermatozoa/physiology ; Testis/*embryology/metabolism ; Testosterone/metabolism ; },
abstract = {Sex steroids play critical roles in sex differentiation and gonadal development in teleosts. Steroidogenic acute regulatory protein (StAR), transporting cholesterol (the substrate for steroidogenesis) from the outer mitochondrial membrane to the inner membrane, is the first rate-limiting factor of steroidogenesis. Interestingly, two StAR genes (named as StAR1 and StAR2) have been isolated from non-mammalian vertebrates. To characterize the functions of the novel StAR2 gene in the gonadal differentiation and fertility, we generated a StAR2 homozygous mutant line in Nile tilapia (Oreochromis niloticus). StAR2 gene knockout in male tilapia impeded meiotic initiation, associate with the down-regulation of meiosis related gene expressions of vasa, sycp3 and dazl at 90 days after hatching (dah). Meanwhile, cyp11b2 expression and serum 11-KT production significantly declined in StAR2[-/-] XY fish at 90 dah. From 120-300 dah, spermatogenesis gradually recovered, and so did the expressions of vasa, sycp3 and dazl in StAR2[-/-] XY fish testes. However, seminiferous lobules arranged disorderly in StAR2[-/-] XY fish testes at 300 dah. The number of Leydig cells and expressions of downstream steroidogenesis enzymes including cyp11a1, 3β-HSD-I, 3β-HSD-II, cyp17a1 and cyp17a2 decreased in StAR2[-/-] XY fish testes at 300 dah. Serum testosterone and 11-KT levels were significantly lower in StAR2[-/-] XY fish than that of their control counterparts. Furthermore, significantly elevated ar, fsh and lh expressions in StAR2-deficient XY fish testes and pituitaries were found when compared with the control XY fish. Testes degeneration and spermatogenic cell apoptosis were observed, while no sperm were squeezed out in StAR2[-/-] XY fish testes at 540 dah. Taken together, our results suggest that StAR2 has a role in testicular development, spermatogenesis and spermiation by regulating androgen production in tilapia, but may not be essential and could be compensated.},
}
@article {pmid34424982,
year = {2021},
author = {Fleming, TJ and Schrankel, CS and Vyas, H and Rosenblatt, HD and Hamdoun, A},
title = {CRISPR/Cas9 mutagenesis reveals a role for ABCB1 in gut immune responses to Vibrio diazotrophicus in sea urchin larvae.},
journal = {The Journal of experimental biology},
volume = {224},
number = {7},
pages = {},
doi = {10.1242/jeb.232272},
pmid = {34424982},
issn = {1477-9145},
support = {ES 027921/NH/NIH HHS/United States ; F32 ES029843/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Immunity ; Larva/genetics ; Mutagenesis ; *Sea Urchins ; Vibrio ; },
abstract = {The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host-microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment.},
}
@article {pmid34424032,
year = {2021},
author = {Liu, S and Tang, L and Zhao, X and Nguyen, B and Heallen, TR and Li, M and Wang, J and Wang, J and Martin, JF},
title = {Yap Promotes Noncanonical Wnt Signals From Cardiomyocytes for Heart Regeneration.},
journal = {Circulation research},
volume = {129},
number = {8},
pages = {782-797},
pmid = {34424032},
issn = {1524-4571},
support = {R01 HL142704/HL/NHLBI NIH HHS/United States ; R01 HL118761/HL/NHLBI NIH HHS/United States ; R56 HL142704/HL/NHLBI NIH HHS/United States ; R01 HL127717/HL/NHLBI NIH HHS/United States ; K01 DE026561/DE/NIDCR NIH HHS/United States ; R03 DE025873/DE/NIDCR NIH HHS/United States ; R01 HL130804/HL/NHLBI NIH HHS/United States ; R01 DE029014/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Cells, Cultured ; Humans ; Mice ; Mice, Inbred C57BL ; Myocardial Infarction/*metabolism/pathology ; Myocytes, Cardiac/*metabolism/physiology ; Myofibroblasts/metabolism ; Receptors, G-Protein-Coupled/genetics/*metabolism ; *Regeneration ; Wnt Proteins/genetics/metabolism ; Wnt Signaling Pathway ; YAP-Signaling Proteins/genetics/*metabolism ; },
abstract = {[Figure: see text].},
}
@article {pmid34421833,
year = {2021},
author = {Asante, J and Hetsa, BA and Amoako, DG and Abia, ALK and Bester, LA and Essack, SY},
title = {Genomic Analysis of Antibiotic-Resistant Staphylococcus epidermidis Isolates From Clinical Sources in the Kwazulu-Natal Province, South Africa.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {656306},
pmid = {34421833},
issn = {1664-302X},
abstract = {Staphylococcus epidermidis has become an important nosocomial pathogen. Multidrug resistance makes S. epidermidis infections difficult to treat. The study aims to describe the genomic characteristics of methicillin-resistant S. epidermidis (MRSE) isolated from clinical sources, to comprehend the genetic basis of antibiotic resistance, virulence, and potential pathogenicity. Sixteen MRSE underwent whole-genome sequencing, and bioinformatics analyses were carried out to ascertain their resistome, virulome, mobilome, clonality, and phylogenomic relationships. In all, 75% of isolates displayed multidrug resistance and were associated with the carriage of multiple resistance genes including mecA, blaZ, tet(K), erm(A), erm(B), erm(C), dfrG, aac(6')-aph(2''), and cat(pC221) conferring resistance to β-lactams, tetracyclines, macrolide-lincosamide-streptogramin B, aminoglycosides, and phenicols, which were located on both plasmids and chromosomes. Their virulence profiles were evidenced by the presence of genes involved in adherence/biofilm formation (icaA, icaB, icaC, atl, ebh, and ebp), immune evasion (adsA, capC, and manA), and antiphagocytosis (rmlC, cdsA, and A). The community-acquired SCCmec type IV was the most common SCCmec type. The CoNS belonged to seven multilocus sequence types (MLSTs) and carried a diversity of mobile genetic elements such as phages, insertion sequences, and plasmids. The bacterial anti-phage defense systems clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) immunity phage system and restriction-modification system (R-M system) and the arginine catabolic mobile element (ACME) involved in immune evasion and transport of virulence genes were also found. The insertion sequence, IS256, linked with virulence, was found in 56.3% of isolates. Generally, the isolates clustered according to STs, with some similarity but also considerable variability within isolates. Whole-genome sequencing and bioinformatics analysis provide insights into the likely pathogenicity and antibiotic resistance of S. epidermidis, necessitating surveillance of this emerging pathogen.},
}
@article {pmid34421355,
year = {2021},
author = {Li, L and Yu, S and Hu, Q and Hai, Y and Li, Y},
title = {Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy.},
journal = {International journal of biological sciences},
volume = {17},
number = {12},
pages = {3133-3144},
pmid = {34421355},
issn = {1449-2288},
mesh = {Animals ; Antineoplastic Agents/*therapeutic use ; Autophagy/*drug effects ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/drug therapy/*genetics/pathology ; Cell Proliferation ; Cells, Cultured ; Drug Resistance, Neoplasm/*drug effects ; Flow Cytometry ; Gain of Function Mutation ; Gene Expression Regulation, Neoplastic/physiology ; Humans ; Liver Neoplasms/drug therapy/*genetics/pathology ; Mice ; Mice, Nude ; Microscopy, Electron ; Mitochondrial Membrane Transport Proteins/*genetics ; Real-Time Polymerase Chain Reaction ; Sorafenib/*therapeutic use ; Xenograft Model Antitumor Assays ; },
abstract = {Sorafenib is the standard first-line drug for the treatment of advanced hepatocellular carcinoma (HCC), however, its therapeutic efficacy is not satisfactory due to primary or secondary resistance of HCC cells. In the present study, we identified Metaxin 1 (MTX1) as a new regulator of sorafenib resistance in HCC through genome-scale CRISPR activation (CRISPRa) screening. We found that MTX1 was frequently upregulated in HCC tissues and overexpression of MTX1 promoted HCC cell proliferation in vitro and in vivo. As well, MTX1 overexpression increased cell growth rate and decreased cell apoptosis upon sorafenib treatment. Consistently, the resistance induced by MTX1 was also observed in subcutaneous xenograft tumor model. Clinically, high expression of MTX1 was closely related with poor outcomes in HCC patients who received sorafenib treatment. Mechanistically, overexpression of MTX1 could promote HCC cell autophagy via interacting with and inhibiting CDGSH iron sulfur domain 1 (CISD1), an autophagy negative regulator. Taken together, our findings suggest that MTX1 is upregulated in HCC and contributes to sorafenib resistance via a possible mechanism involving CISD1 mediated autophagy.},
}
@article {pmid34420406,
year = {2021},
author = {O'Halloran, DM},
title = {CRISPR-PN2: a flexible and genome-aware platform for diverse CRISPR experiments in parasitic nematodes.},
journal = {BioTechniques},
volume = {71},
number = {3},
pages = {495-498},
doi = {10.2144/btn-2021-0056},
pmid = {34420406},
issn = {1940-9818},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Nematoda/genetics ; RNA, Guide ; },
abstract = {Parasitic nematodes represent a significant threat to human health, causing diseases of major socioeconomic importance worldwide. Central to controlling infections of parasitic nematodes is a more detailed molecular picture of host specificity, parasite activation and immune suppression. CRISPR technology holds huge potential for researchers in the field of parasitic nematology, as it provides a powerful genetic tool to dissect questions in parasite biology. To expedite the development of CRISPR technology in parasitic nematodes, software is required to facilitate the design of effective and specific sgRNA sequences. Here, the author introduces CRISPR-PN2, a comprehensive web-based platform that provides flexible use control over the automated design of specific gRNA sequences for CRISPR experiments in parasitic nematodes.},
}
@article {pmid34420149,
year = {2021},
author = {Gupta, D and Sharma, G and Saraswat, P and Ranjan, R},
title = {Synthetic Biology in Plants, a Boon for Coming Decades.},
journal = {Molecular biotechnology},
volume = {63},
number = {12},
pages = {1138-1154},
pmid = {34420149},
issn = {1559-0305},
mesh = {CRISPR-Cas Systems ; Metabolic Engineering ; Plants, Genetically Modified/*growth &amp; development/metabolism ; Synthetic Biology/*methods ; },
abstract = {Recently an enormous expansion of knowledge is seen in various disciplines of science. This surge of information has given rise to concept of interdisciplinary fields, which has resulted in emergence of newer research domains, one of them is 'Synthetic Biology' (SynBio). It captures basics from core biology and integrates it with concepts from the other areas of study such as chemical, electrical, and computational sciences. The essence of synthetic biology is to rewire, re-program, and re-create natural biological pathways, which are carried through genetic circuits. A genetic circuit is a functional assembly of basic biological entities (DNA, RNA, proteins), created using typical design, built, and test cycles. These circuits allow scientists to engineer nearly all biological systems for various useful purposes. The development of sophisticated molecular tools, techniques, genomic programs, and ease of nucleic acid synthesis have further fueled several innovative application of synthetic biology in areas like molecular medicines, pharmaceuticals, biofuels, drug discovery, metabolomics, developing plant biosensors, utilization of prokaryotic systems for metabolite production, and CRISPR/Cas9 in the crop improvement. These applications have largely been dominated by utilization of prokaryotic systems. However, newer researches have indicated positive growth of SynBio for the eukaryotic systems as well. This paper explores advances of synthetic biology in the plant field by elaborating on its core components and potential applications. Here, we have given a comprehensive idea of designing, development, and utilization of synthetic biology in the improvement of the present research state of plant system.},
}
@article {pmid34419749,
year = {2021},
author = {Tian, L and Wu, F and Bai, R and Ma, S and Zheng, H and Liu, WJ and Wang, Y},
title = {Generation of a COL4A5 heterozygous mutation human embryonic stem cell line (WAe009-A-58) using an episomal vector-based CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102481},
doi = {10.1016/j.scr.2021.102481},
pmid = {34419749},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Collagen Type IV/genetics ; *Human Embryonic Stem Cells ; Humans ; Mutation ; *Nephritis, Hereditary/genetics ; },
abstract = {X-linked Alport syndrome (XLAS) is the second most common inherited kidney disease which pathogenic variants related to a mutation in the COL4A5 gene encoding the type IV collagen α5 chain. Here, we have generated a COL4A5 heterozygous mutant human embryonic stem cell (hESC) line (H9-COL4A5[+/-)] by an episomal vector-based CRISPR/Cas9 system. The generated H9-COL4A5[+/-] maintained a normal stem cell morphology, stably expressed pluripotent markers, and could differentiate into all three germ layers in vivo. This cell line offers an in vitro efficient platform to explore pathogenic mechanisms in XLAS and provides a cell-based disease model for drug testing.},
}
@article {pmid34419551,
year = {2021},
author = {Li, B and Li, J and Chai, Y and Huang, Y and Li, L and Wang, D and Wang, Z},
title = {Targeted mutagenesis of CYP76AK2 and CYP76AK3 in Salvia miltiorrhiza reveals their roles in tanshinones biosynthetic pathway.},
journal = {International journal of biological macromolecules},
volume = {189},
number = {},
pages = {455-463},
doi = {10.1016/j.ijbiomac.2021.08.112},
pmid = {34419551},
issn = {1879-0003},
mesh = {Abietanes/*biosynthesis ; Amino Acid Motifs ; Amino Acid Sequence ; Base Sequence ; Biosynthetic Pathways/*genetics ; CRISPR-Cas Systems/genetics ; Chromosomes, Plant/genetics ; Conserved Sequence ; Cytochrome P-450 Enzyme System/chemistry/*genetics ; Gene Expression Regulation, Plant/drug effects ; Genes, Plant ; Genetic Vectors/metabolism ; Mutagenesis/*genetics ; Mutation/genetics ; Phylogeny ; Plant Growth Regulators/pharmacology ; Plant Proteins/chemistry/*genetics ; Salvia miltiorrhiza/*enzymology/*genetics ; },
abstract = {Salvia miltiorrhiza Bunge, belonging to Lamiaceae family, is one of the most important Chinese medicinal herbs. The dried roots, also called Danshen in Chinese, are usually used in the formula of Chinese traditional medicine due to the bioactive constituents known as phenolic acids and tanshinones, which are a group of abietane nor-diterpenoid quinone natural products. Cytochrome P450 enzymes (CYPs) usually play crucial roles in terpenoids synthesis, especially in hydroxylation processes. Up to now, several important P450 enzymes, such as CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, and CYP71D375, have been functionally characterized in the tanshinones biosynthetic pathway. Nevertheless, the tanshinones biosynthesis is a so complex network that more P450 enzymes should be identified and characterized. Here, we report two novel P450 enzymes CYP76AK2 and CYP76AK3 that are involved in tanshinones biosynthetic pathway. These two P450 enzymes were highly homologous to previously reported CYP76AK1 and showed the same expression profile as CYP76AK1. Also, CYP76AK2 and CYP76AK3 could be stimulated by MeJA and SA, resulting in increased expression. We used a triple-target CRISPR/Cas9 system to generate targeted mutagenesis of CYP76AK2 and CYP76AK3 in S. miltiorrhiza. The content of five major tanshinones was significantly reduced in both cyp76ak2 and cyp76ak3 mutants, indicating that the two enzymes might be involved in the biosynthesis of tanshinones. This study would provide a foundation for the catalytic function identification of CYP76AK2 and CYP76AK3, and further enrich the understanding of the network of tanshinones secondary metabolism synthesis as well.},
}
@article {pmid34418634,
year = {2021},
author = {Yin, L and Man, S and Ye, S and Liu, G and Ma, L},
title = {CRISPR-Cas based virus detection: Recent advances and perspectives.},
journal = {Biosensors &amp; bioelectronics},
volume = {193},
number = {},
pages = {113541},
pmid = {34418634},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acids ; SARS-CoV-2 ; },
abstract = {Viral infections are one of the most intimidating threats to human beings. One convincing example is the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. Rapid, sensitive, specific and field-deployable identification of causal viruses is critical for disease surveillance, control and treatment. The shortcomings of current methods create an impending need for developing novel biosensing platforms. CRISPR-Cas systems, especially CRISPR-Cas12a and CRISPR-Cas13a, characterized by their sensitivity, specificity, high base resolution and programmability upon nucleic acid recognition, have been repurposed for molecular diagnostics, surging a new path forward in biosensing. They, as the core of some robust diagnostic tools, are revolutionizing the way that virus can be detected. This review focuses on recent advances in virus detection with CRISPR-Cas systems especially CRISPR-Cas12a/Cas13a. We started with a short introduction to CRISPR-Cas systems and the properties of Cas12a and Cas13a effectors, and continued with reviewing the current advances of virus detection utilizing CRISPR-Cas systems. The significance and advantages of such methods were then discussed. Finally, the challenges and perspectives were proposed. We tried to provide readers with a concise profile of emerging and fast-expanding CRISPR-Cas based biosensing technology, and highlighted its potential applications in a range of scenarios with regard to virus detection.},
}
@article {pmid34418266,
year = {2021},
author = {Duan, L and Ouyang, K and Wang, J and Xu, L and Xu, X and Wen, C and Xie, Y and Liang, Y and Xia, J},
title = {Exosomes as Targeted Delivery Platform of CRISPR/Cas9 for Therapeutic Genome Editing.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {22},
number = {24},
pages = {3360-3368},
doi = {10.1002/cbic.202100359},
pmid = {34418266},
issn = {1439-7633},
mesh = {CRISPR-Cas Systems/*genetics ; Exosomes/*genetics ; Gene Editing ; *Gene Transfer Techniques ; *Genetic Therapy ; Humans ; },
abstract = {Therapeutic genome editing harnesses the power of genome editing tools to correct erroneous genes associated with disease pathology. To bring the CRISPR/Cas9 tool from the bench to the bedside, a critical hurdle is the safe and efficient delivery of this nucleic acid tool to the desired type of cells in patients. This review discusses the use of natural carriers, extracellular vesicles (EVs), in particular exosomes, to fill the gap. Exosomes are lipid-containing nanovesicle released by various types of cells to mediate cell-cell communications. Their inherent long-distance transportation capability, biocompatibility, and engineerability have made EVs potential vehicles for delivering therapeutic drugs. We summarize the recent progress of harnessing exosomes as delivery vehicles for the CRISPR/Cas system to achieve therapeutic gene editing for disease treatment, with a focus on various strategies to achieve selective delivery to a particular type of cell and efficient packaging of the genome editing tools in the vesicles. Critical issues and possible solutions in the design and engineering of the targeting vehicles are highlighted. Taken together, we demonstrate EV/exosome-mediated packaging of the nucleic acid/protein tools and the cell/tissue-targeted delivery to be a viable way towards the clinical translation of the CRISPR/Cas9 technology.},
}
@article {pmid34418226,
year = {2021},
author = {Takeda, I and Araki, M and Ishiguro, KI and Ohga, T and Takada, K and Yamaguchi, Y and Hashimoto, K and Kai, T and Nakagata, N and Imasaka, M and Yoshinobu, K and Araki, K},
title = {Gene trapping reveals a new transcriptionally active genome element: The chromosome-specific clustered trap region.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {26},
number = {11},
pages = {874-890},
doi = {10.1111/gtc.12890},
pmid = {34418226},
issn = {1365-2443},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Chromosomes/genetics ; Genes, Reporter ; *Genome ; Mice ; *Repetitive Sequences, Nucleic Acid ; Software ; },
abstract = {Nearly half of the human genome consists of repetitive sequences such as long interspersed nuclear elements. The relationship between these repeating sequences and diseases has remained unclear. Gene trapping is a useful technique for disrupting a gene and expressing a reporter gene by using the promoter activity of the gene. The analysis of trapped genes revealed a new genome element-the chromosome-specific clustered trap (CSCT) region. For any examined sequence within this region, an equivalent was found using the BLAT of the University of California, Santa Cruz (UCSC) Genome Browser. CSCT13 mapped to chromosome 13 and contained only three genes. To elucidate its in vivo function, the whole CSCT13 region (1.6 Mbp) was deleted using the CRISPR/Cas9 system in mouse embryonic stem cells, and subsequently, a CSCT13 knockout mouse line was established. The rate of homozygotes was significantly lower than expected according to Mendel's laws. In addition, the number of offspring obtained by mating homozygotes was significantly smaller than that obtained by crossing controls. Furthermore, CSCT13 might have an effect on meiotic homologous recombination. This study identifies a transcriptionally active CSCT with an important role in mouse development.},
}
@article {pmid34417751,
year = {2021},
author = {Larter, K and Yi, B and Xi, Y},
title = {Genetic Editing of Long Noncoding RNA Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2372},
number = {},
pages = {169-177},
pmid = {34417751},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; RNA Interference ; RNA, Long Noncoding/genetics ; Technology ; },
abstract = {Long noncoding RNAs (lncRNAs) are a class of RNA transcripts greater than 200 nucleotides in length and makeup a considerable part of the human genome. LncRNAs are well established as crucial players in a myriad of physiological and pathological processes; however, despite their abundance and versatility, the functional characteristics of lncRNAs remain largely unknown predominantly due to the lack of suitable genetic editing strategies. The complexity of their genetic structure and regulation combined with their unique functionality poses several limitations in the application of classic genetic manipulation methods in lncRNA functional studies. Several reports have demonstrated the successful implementation of CRISPR/Cas9 technology in screening and identifying the function of specific lncRNAs. Here, we describe a detailed protocol utilizing CRISPR/Cas9 genetic editing technology for knocking down lncRNAs in vitro.},
}
@article {pmid34417549,
year = {2021},
author = {Sajib, AM and Agarwal, P and Patton, DJ and Nance, RL and Stahr, NA and Kretzschmar, WP and Sandey, M and Smith, BF},
title = {In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9.},
journal = {Laboratory investigation; a journal of technical methods and pathology},
volume = {101},
number = {12},
pages = {1627-1636},
pmid = {34417549},
issn = {1530-0307},
mesh = {Adenoviruses, Canine/*genetics ; Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Dogs ; *Gene Editing ; Genome, Viral ; Oncolytic Virotherapy ; Recombinational DNA Repair ; },
abstract = {Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.},
}
@article {pmid34417284,
year = {2021},
author = {VanRyzin, JW and Arambula, SE and Ashton, SE and Blanchard, AC and Burzinski, MD and Davis, KT and Edwards, S and Graham, EL and Holley, A and Kight, KE and Marquardt, AE and Perez-Pouchoulen, M and Pickett, LA and Reinl, EL and McCarthy, MM},
title = {Generation of an Iba1-EGFP Transgenic Rat for the Study of Microglia in an Outbred Rodent Strain.},
journal = {eNeuro},
volume = {8},
number = {5},
pages = {},
pmid = {34417284},
issn = {2373-2822},
support = {F32 HD097816/HD/NICHD NIH HHS/United States ; P01 HD085928/HD/NICHD NIH HHS/United States ; R01 DA039062/DA/NIDA NIH HHS/United States ; R01 MH091424/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; *Microglia ; Rats ; Rats, Sprague-Dawley ; Rats, Transgenic ; *Rodentia ; },
abstract = {Neuroscience has been transformed by the ability to genetically modify inbred mice, including the ability to express fluorescent markers specific to cell types or activation states. This approach has been put to particularly good effect in the study of the innate immune cells of the brain, microglia. These specialized macrophages are exceedingly small and complex, but also highly motile and mobile. To date, there have been no tools similar to those in mice available for studying these fundamental cells in the rat brain, and we seek to fill that gap with the generation of the genetically modified Sprague Dawley rat line: SD-Tg(Iba1-EGFP)Mmmc Using CRISPR-Cas/9 technology, we knocked in EGFP to the promoter of the gene Iba1 With four male and three female founders confirmed by quantitative PCR analysis to have appropriate and specific insertion, we established a breeding colony with at least three generations of backcrosses to obtain stable and reliable Iba1-EGFP expression. The specificity of EGFP expression to microglia was established by flow cytometry for CD45[low]/CD11b[+] cells and by immunohistochemistry. Microglial EGFP expression was detected in neonates and persisted into adulthood. Blood macrophages and monocytes were found to express low levels of EGFP, as expected. Last, we show that EGFP expression is suitable for live imaging of microglia processes in acute brain slices and via intravital two-photon microscopy.},
}
@article {pmid34416913,
year = {2021},
author = {Wen, W and Quan, ZJ and Li, SA and Yang, ZX and Fu, YW and Zhang, F and Li, GH and Zhao, M and Yin, MD and Xu, J and Zhang, JP and Cheng, T and Zhang, XB},
title = {Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {236},
pmid = {34416913},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems ; DNA/genetics ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *DNA Repair ; Gene Editing ; Gene Knock-In Techniques ; Genome ; HEK293 Cells ; Hematopoietic Stem Cells ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Nanopore Sequencing ; Recombinational DNA Repair ; },
abstract = {BACKGROUND: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences.<br><br>RESULTS: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%.<br><br>CONCLUSIONS: Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.},
}
@article {pmid34416907,
year = {2021},
author = {Luo, J and Lu, C and Feng, M and Dai, L and Wang, M and Qiu, Y and Zheng, H and Liu, Y and Li, L and Tang, B and Xu, C and Wang, Y and Yang, X},
title = {Cooperation between liver-specific mutations of pten and tp53 genetically induces hepatocarcinogenesis in zebrafish.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {40},
number = {1},
pages = {262},
pmid = {34416907},
issn = {1756-9966},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*genetics/metabolism/pathology ; Cell Transformation, Neoplastic/*genetics/metabolism ; Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Gene Targeting ; *Genes, p53 ; Genetic Vectors ; Humans ; Immunohistochemistry ; Liver Neoplasms/*genetics/metabolism/pathology ; *Mutation ; Neoplasm Grading ; Organ Specificity/genetics ; PTEN Phosphohydrolase/*genetics ; RNA, Guide ; Signal Transduction ; Zebrafish ; },
abstract = {BACKGROUND: Liver cancer, mainly hepatocellular carcinoma, is one of the deadliest cancers worldwide and has a poor prognosis due to insufficient understanding of hepatocarcinogenesis. Previous studies have revealed that the mutations in PTEN and TP53 are the two most common genetic events in hepatocarcinogenesis. Here, we illustrated the crosstalk between aberrant Pten and Tp53 pathways during hepatocarcinogenesis in zebrafish.<br><br>METHODS: We used the CRISPR/Cas9 system to establish several transgenic zebrafish lines with single or double tissue-specific mutations of pten and tp53 to genetically induce liver tumorigenesis. Next, the morphological and histological determination were performed to investigate the roles of Pten and Tp53 signalling pathways in hepatocarcinogenesis in zebrafish.<br><br>RESULTS: We demonstrated that Pten loss alone induces hepatocarcinogenesis with only low efficiency, whereas single mutation of tp53 failed to induce tumour formation in liver tissue in zebrafish. Moreover, zebrafish with double mutations of pten and tp53 exhibits a much higher tumour incidence, higher-grade histology, and a shorter survival time than single-mutant zebrafish, indicating that these two signalling pathways play important roles in dynamic biological events critical for the initiation and progression of hepatocarcinogenesis in zebrafish. Further histological and pathological analyses showed significant similarity between the tumours generated from liver tissues of zebrafish and humans. Furthermore, the treatment with MK-2206, a specific Akt inhibitor, effectively suppressed hepatocarcinogenesis in zebrafish.<br><br>CONCLUSION: Our findings will offer a preclinical animal model for genetically investigating hepatocarcinogenesis and provide a useful platform for high-throughput anticancer drug screening.},
}
@article {pmid34416117,
year = {2021},
author = {Liao, C and Beisel, CL},
title = {The tracrRNA in CRISPR Biology and Technologies.},
journal = {Annual review of genetics},
volume = {55},
number = {},
pages = {161-181},
doi = {10.1146/annurev-genet-071719-022559},
pmid = {34416117},
issn = {1545-2948},
mesh = {Archaea/genetics ; Biology ; *CRISPR-Cas Systems/genetics ; RNA/genetics ; *RNA, Guide/genetics ; },
abstract = {CRISPR-Cas adaptive immune systems in bacteria and archaea utilize short CRISPR RNAs (crRNAs) to guide sequence-specific recognition and clearance of foreign genetic material. Multiple crRNAs are stored together in a compact format called a CRISPR array that is transcribed and processed into the individual crRNAs. While the exact processing mechanisms vary widely, some CRISPR-Cas systems, including those encoding the Cas9 nuclease, rely on a trans-activating crRNA (tracrRNA). The tracrRNA was discovered in 2011 and was quickly co-opted to create single-guide RNAs as core components of CRISPR-Cas9 technologies. Since then, further studies have uncovered processes extending beyond the traditional role of tracrRNA in crRNA biogenesis, revealed Cas nucleases besides Cas9 that are dependent on tracrRNAs, and established new applications based on tracrRNA engineering. In this review, we describe the biology of the tracrRNA and how its ongoing characterization has garnered new insights into prokaryotic immune defense and enabled key technological advances.},
}
@article {pmid34415905,
year = {2021},
author = {Casey, T and Suarez-Trujillo, A and Cummings, S and Huff, K and Crodian, J and Bhide, K and Aduwari, C and Teeple, K and Shamay, A and Mabjeesh, SJ and San Miguel, P and Thimmapuram, J and Plaut, K},
title = {Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.},
journal = {PloS one},
volume = {16},
number = {8},
pages = {e0248199},
pmid = {34415905},
issn = {1932-6203},
mesh = {ARNTL Transcription Factors/metabolism/*physiology ; Anemia, Sickle Cell ; Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Epithelial Cells/*metabolism ; Female ; Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques ; Mammary Glands, Animal/*metabolism/physiology ; Mice ; Milk Proteins/*metabolism ; },
abstract = {The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.},
}
@article {pmid34415182,
year = {2021},
author = {Philippidis, A},
title = {First Positive Clinical Data for In Vivo Genome Editing in Humans Opens "New Era of Medicine".},
journal = {Human gene therapy},
volume = {32},
number = {15-16},
pages = {783-786},
doi = {10.1089/hum.2021.29170.bfs},
pmid = {34415182},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; },
}
@article {pmid34414615,
year = {2021},
author = {Xu, X and Harvey-Samuel, T and Yang, J and You, M and Alphey, L},
title = {CRISPR/Cas9-based functional characterization of the pigmentation gene ebony in Plutella xylostella.},
journal = {Insect molecular biology},
volume = {30},
number = {6},
pages = {615-623},
doi = {10.1111/imb.12730},
pmid = {34414615},
issn = {1365-2583},
mesh = {Animals ; *CRISPR-Cas Systems ; Dopamine/metabolism ; Insect Proteins/genetics/metabolism ; Larva/genetics/metabolism ; *Moths/genetics/metabolism ; *Pigmentation/genetics ; },
abstract = {Body pigmentation is an important character of insects in adapting to biotic and abiotic environmental challenges. Additionally, based on the relative ease of screening, several genes involved in insect melanization have been used in classic genetic studies or as visual markers in constructing transgenic insects. Here, a homologue of the Bombyx mori melanization-inhibiting gene ebony, associated with the conversion of dopamine to N-β-alanyl dopamine, was identified in a global pest, Plutella xylostella. The CRISPR/Cas9 system was applied to generate multiple Pxebony knockout alleles which were crossed to produce a Pxebony knockout strain, showing darker pigmentation in larvae, pupae and adults, compared with wildtype. Interestingly, we observed that Pxebony heterozygotes displayed an intermediate darkened phenotype, indicating partial dominance between the knockout and wildtype alleles. The fitness costs of Pxebony deficiency were also assessed in the mutant strain, indicating that embryo hatchability and larval survival were significantly reduced, while the eclosion rate was not obviously affected. Our work provides a potential target for exploring CRISPR-based genetics-control systems in this economically important pest lepidopteran.},
}
@article {pmid34413448,
year = {2021},
author = {Sun, D},
title = {Design of time-delayed safety switches for CRISPR gene therapy.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {16908},
pmid = {34413448},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Division ; DNA Repair ; *Genetic Therapy ; Time Factors ; },
abstract = {CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. Inspired by previously reported Cas9 self-elimination systems, time-delayed safety switches are designed in this work. Firstly, ultrasensitive relationship is constructed between Cas9-sgRNA (enzyme) and Cas9 plasmids (substrate), which generates the artificial time delay. Then intrinsic time delay in biomolecular activities is revealed by data fitting and utilized in constructing safety switches. The time-delayed safety switches function by separating the gene editing process and self-elimination process, and the tunable delay time may ensure a good balance between gene editing efficiency and side effect minimization. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.},
}
@article {pmid34413309,
year = {2021},
author = {Moreb, EA and Lynch, MD},
title = {Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5034},
pmid = {34413309},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Gene Editing/*methods ; Humans ; *RNA, Guide ; Retrospective Studies ; Species Specificity ; },
abstract = {CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.},
}
@article {pmid34413302,
year = {2021},
author = {Steens, JA and Zhu, Y and Taylor, DW and Bravo, JPK and Prinsen, SHP and Schoen, CD and Keijser, BJF and Ossendrijver, M and Hofstra, LM and Brouns, SJJ and Shinkai, A and van der Oost, J and Staals, RHJ},
title = {SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5033},
pmid = {34413302},
issn = {2041-1723},
support = {R35 GM138348/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine Nucleotides/*chemistry ; COVID-19/*diagnosis/genetics/metabolism/virology ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Diagnostic Tests, Routine/methods ; Humans ; Oligoribonucleotides/*chemistry ; RNA, Bacterial/*genetics ; Ribonucleases/*metabolism ; SARS-CoV-2/*genetics/isolation &amp; purification/pathogenicity ; },
abstract = {Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.},
}
@article {pmid34413171,
year = {2021},
author = {Parvez, S and Herdman, C and Beerens, M and Chakraborti, K and Harmer, ZP and Yeh, JJ and MacRae, CA and Yost, HJ and Peterson, RT},
title = {MIC-Drop: A platform for large-scale in vivo CRISPR screens.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6559},
pages = {1146-1151},
pmid = {34413171},
issn = {1095-9203},
support = {R01 GM134069/GM/NIGMS NIH HHS/United States ; UM1 HL098160/HL/NHLBI NIH HHS/United States ; U01 HL098160/HL/NHLBI NIH HHS/United States ; U54 NS112107/NS/NINDS NIH HHS/United States ; R24 OD017870/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cardiovascular System/growth &amp; development ; Cell Culture Techniques ; *Genetic Testing ; High-Throughput Nucleotide Sequencing ; *Microfluidic Analytical Techniques ; Zebrafish/*genetics/growth &amp; development ; },
abstract = {CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms.},
}
@article {pmid34412673,
year = {2021},
author = {Li, Y and Chen, J and Tsai, SQ and Cheng, Y},
title = {Easy-Prime: a machine learning-based prime editor design tool.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {235},
pmid = {34412673},
issn = {1474-760X},
support = {R35 GM133614/GM/NIGMS NIH HHS/United States ; U01 EB029373/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA ; *Gene Editing ; HEK293 Cells ; Humans ; *Machine Learning ; RNA, Guide/genetics ; },
abstract = {Prime editing is a revolutionary genome-editing technology that can make a wide range of precise edits in DNA. However, designing highly efficient prime editors (PEs) remains challenging. We develop Easy-Prime, a machine learning-based program trained with multiple published data sources. Easy-Prime captures both known and novel features, such as RNA folding structure, and optimizes feature combinations to improve editing efficiency. We provide optimized PE design for installation of 89.5% of 152,351 GWAS variants. Easy-Prime is available both as a command line tool and an interactive PE design server at: http://easy-prime.cc/ .},
}
@article {pmid34412573,
year = {2021},
author = {Guzmán-Zapata, D and Vargas-Morales, BV and Loyola-Vargas, VM},
title = {From genome scissors to molecular scalpel: evolution of CRISPR systems.},
journal = {Biotechnology &amp; genetic engineering reviews},
volume = {37},
number = {1},
pages = {82-104},
doi = {10.1080/02648725.2021.1962071},
pmid = {34412573},
issn = {2046-5556},
mesh = {Biotechnology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genome ; },
abstract = {From bizarre palindromic repeats to a bacterial defense mechanism, to genome editing tool, and more, Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR has significantly impacted the way we study genome modification in less than a decade. In this review, we would like to highlight some key players over 30 years of research and explain this biotechnological tool's basic mechanisms. We also refer to the evolution of the CRISPR variants and some of the applications derived from them. The understanding and upgrading of this system will be a valuable tool in the years to come to solve some of the challenges in diverse fields from pharmaceuticals to therapeutics, from basic plant genetics to crop improvement, from metabolic engineering to waste management and industrial processing.},
}
@article {pmid34411650,
year = {2021},
author = {Malik, D and Mahendiratta, S and Kaur, H and Medhi, B},
title = {Futuristic approach to cancer treatment.},
journal = {Gene},
volume = {805},
number = {},
pages = {145906},
doi = {10.1016/j.gene.2021.145906},
pmid = {34411650},
issn = {1879-0038},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods/trends ; Genetic Therapy/methods ; Humans ; Neoplasms/*genetics/*therapy ; Precision Medicine/methods/*trends ; },
abstract = {Cancer is becoming one of the deadliest disease in both developed as well as developing countries and continuous effort is being made to find innovative therapies for myriad types of cancers that afflict the human body. Therapeutic options for cancer have grown exponentially over the time but we are quite a way off from finding a magic bullet that can help cure cancer and based on the current evidence we may never find a catch all cure ever and it becomes crucial that we keep on innovating and find multiple ways to attack the menace of this dreaded disease. Many patients suffer recurrence of disease and require second-line or in some cases more than two lines of treatment. In this review article we have discussed the available therapies along with the newer advancements that have been made in cancer therapy. Latest developments in treatment of various cancers that have been discussed include gene editing using CRISPR/Cas9, theranostics, viral mediated therapy, artificial intelligence, tumor infiltrating lymphocyte therapy, etc.},
}
@article {pmid34411422,
year = {2021},
author = {Liu, T and Zeng, D and Zheng, Z and Lin, Z and Xue, Y and Li, T and Xie, X and Ma, G and Liu, YG and Zhu, Q},
title = {The ScCas9[++] variant expands the CRISPR toolbox for genome editing in plants.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {9},
pages = {1611-1619},
doi = {10.1111/jipb.13164},
pmid = {34411422},
issn = {1744-7909},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Oryza/*genetics ; Streptococcus/genetics ; },
abstract = {The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9[+] and ScCas9[++] variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9[+] and ScCas9[++] and a nickase variant ScCas9n[++] to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9[++] has higher editing efficiency than ScCas9 and ScCas9[+] in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n[++] to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n[++] . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C- 1 -C17) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9[++] represents a crucial new tool for improving plant editing.},
}
@article {pmid34410625,
year = {2022},
author = {Espinosa-Camacho, LF and Delgado, G and Cravioto, A and Morales-Espinosa, R},
title = {Diversity in the composition of the accessory genome of Mexican Pseudomonas aeruginosa strains.},
journal = {Genes &amp; genomics},
volume = {44},
number = {1},
pages = {53-77},
pmid = {34410625},
issn = {2092-9293},
mesh = {Adult ; Bacteremia/microbiology ; Child ; Computational Biology/methods ; DNA Transposable Elements/genetics ; *Genetic Variation ; Genome Size/*genetics ; Genome, Bacterial/*genetics ; Genomic Islands/*genetics ; Genomics/*methods ; Humans ; Mexico ; Phylogeny ; Pneumonia, Bacterial/microbiology ; Pseudomonas Infections/microbiology ; Pseudomonas aeruginosa/classification/*genetics/pathogenicity ; Sequence Analysis, DNA/methods ; Virulence/genetics ; },
abstract = {BACKGROUND: Pseudomonas aeruginosa is an important opportunistic pathogen especially in nosocomial infections due to its easy adaptation to different environments; this characteristic is due to the great genetic diversity that presents its genome. In addition, it is considered a pathogen of critical priority due to the high antimicrobial resistance.<br><br>OBJECTIVES: The aim of this study was to characterize the mobile genetic elements present in the chromosome of six Mexican P. aeruginosa strains isolated from adults with pneumonia and children with bacteremia.<br><br>METHODS: The genomic DNA of six P. aeruginosa strains were isolated and sequenced using PacBio RS-II platform. They were annotated using Prokaryotic Genome Annotation Pipeline and manually curated and analyzed for the presence of mobile genetic elements, antibiotic resistances genes, efflux pumps and virulence factors using several bioinformatics programs and databases.<br><br>RESULTS: The global analysis of the strains chromosomes showed a novel chromosomal rearrangement in two strains, possibly mediated by subsequent recombination and inversion events. They have a high content of mobile genetic elements: 21 genomic islands, four new islets, four different integrative conjugative elements, 28 different prophages, one CRISPR-Cas arrangements, and one class 1 integron. The acquisition of antimicrobials resistance genes into these elements are in concordance with their phenotype of multi-drug resistance.<br><br>CONCLUSION: The accessory genome increased the ability of the strains to adapt or survive to the hospital environment, promote genomic plasticity and chromosomal rearrangements, which may affect the expression or functionality of the gene and might influence the clinical outcome, having an impact on the treatment.},
}
@article {pmid34410383,
year = {2021},
author = {Jiao, X and Lyu, L and Zhang, Y and Huang, Q and Zhou, R and Wang, S and Wang, S and Zhang, S and Zhao, ZK},
title = {Reduction of lipid-accumulation of oleaginous yeast Rhodosporidium toruloides through CRISPR/Cas9-mediated inactivation of lipid droplet structural proteins.},
journal = {FEMS microbiology letters},
volume = {368},
number = {16},
pages = {},
doi = {10.1093/femsle/fnab111},
pmid = {34410383},
issn = {1574-6968},
mesh = {CRISPR-Cas Systems ; *Lipid Droplet Associated Proteins/metabolism ; *Lipids ; *Rhodotorula/metabolism ; },
abstract = {The basidiomycetous yeast Rhodosporidium toruloides is an important chassis organism for producing microbial lipids and terpenoids. However, excess carbon flux flows towards lipid synthesis than terpenoid synthesis. Thus, it is essential to limit lipid accumulation so that R. toruloides can be explored as an advanced cell factory for producing non-lipid derivatives. In this study, we knocked out two lipid droplet (LD) structural proteins (Ldp1 and Cals) of R. toruloides NP11 through the CRISPR/Cas9 system to reduce lipid production. The results showed that lipid content of LD protein-disrupted strains dropped by over 40%. LDP1-disrupted mutants harbored small-sized LDs. This study provided valuable information to study about microbial lipid metabolism and platform strains for constructing advanced cell factories.},
}
@article {pmid34409907,
year = {2021},
author = {Hillary, VE and Ignacimuthu, S and Ceasar, SA},
title = {Potential of CRISPR/Cas system in the diagnosis of COVID-19 infection.},
journal = {Expert review of molecular diagnostics},
volume = {21},
number = {11},
pages = {1179-1189},
pmid = {34409907},
issn = {1744-8352},
mesh = {COVID-19/*diagnosis/*virology ; CRISPR-Cas Systems/*genetics ; Communicable Diseases/diagnosis/virology ; Humans ; Pandemics/prevention &amp; control ; RNA, Viral/genetics ; SARS-CoV-2/*genetics ; },
abstract = {INTRODUCTION: Emerging novel infectious diseases and persistent pandemics with potential to destabilize normal life remain a public health concern for the whole world. The recent outbreak of pneumonia caused by Coronavirus infectious disease-2019 (COVID-19) resulted in high mortality due to a lack of effective drugs or vaccines. With a constantly increasing number of infections with mutated strains and deaths across the globe, rapid, affordable and specific detections with more accurate diagnosis and improved health treatments are needed to combat the spread of this novel pathogen COVID-19.<br><br>AREAS COVERED: Researchers have started to utilize the recently invented clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas)-based tools for the rapid detection of novel COVID-19. In this review, we summarize the potential of CRISPR/Cas system for the diagnosis and enablement of efficient control of COVID-19.<br><br>EXPERT OPINION: Multiple groups have demonstrated the potential of utilizing CRISPR-based diagnosis tools for the detection of SARS-CoV-2. In coming months, we expect more novel and rapid CRISPR-based kits for mass detection of COVID-19-infected persons within a fraction of a second. Therefore, we believe science will conquer COVID-19 in the near future.},
}
@article {pmid34408743,
year = {2021},
author = {Van Zeebroeck, L and Arroyo Hornero, R and Côrte-Real, BF and Hamad, I and Meissner, TB and Kleinewietfeld, M},
title = {Fast and Efficient Genome Editing of Human FOXP3[+] Regulatory T Cells.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {655122},
pmid = {34408743},
issn = {1664-3224},
mesh = {Blood Buffy Coat/cytology ; CRISPR-Cas Systems/genetics ; Forkhead Transcription Factors/metabolism ; Gene Editing/*methods ; Gene Knockdown Techniques ; HEK293 Cells ; Healthy Volunteers ; Humans ; Immunotherapy, Adoptive/methods ; Interleukin-2 Receptor alpha Subunit/*genetics ; Primary Cell Culture ; RNA, Guide/genetics ; Receptors, Interleukin-6/*genetics ; T-Lymphocytes, Regulatory/*metabolism ; Time Factors ; },
abstract = {FOXP3[+] regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.},
}
@article {pmid34407984,
year = {2021},
author = {Jensen, TI and Mikkelsen, NS and Gao, Z and Foßelteder, J and Pabst, G and Axelgaard, E and Laustsen, A and König, S and Reinisch, A and Bak, RO},
title = {Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi.},
journal = {Genome research},
volume = {31},
number = {11},
pages = {2120-2130},
pmid = {34407984},
issn = {1549-5469},
mesh = {*CRISPR-Cas Systems ; *Endonucleases/genetics ; Gene Expression Regulation ; Genome ; RNA, Guide/genetics ; Transcriptional Activation ; },
abstract = {Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34[+] hematopoietic stem and progenitor cells (HSPCs) and human CD3[+] T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.},
}
@article {pmid34407706,
year = {2022},
author = {Singh, V and Jain, M},
title = {Recent advancements in CRISPR-Cas toolbox for imaging applications.},
journal = {Critical reviews in biotechnology},
volume = {42},
number = {4},
pages = {508-531},
doi = {10.1080/07388551.2021.1950608},
pmid = {34407706},
issn = {1549-7801},
mesh = {*CRISPR-Cas Systems/genetics ; Chromatin ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; RNA ; },
abstract = {The imaging of chromatin, genomic loci, RNAs, and proteins is very important to study their localization, interaction, and coordinated regulation. Recently, several clustered regularly interspaced short palindromic repeats (CRISPR) based imaging methods have been established. The refurbished tool kits utilizing deactivated Cas9 (dCas9) and dCas13 have been established to develop applications of CRISPR-Cas technology beyond genome editing. Here, we review recent advancements in CRISPR-based methods that enable efficient imaging and visualization of chromatin, genomic loci, RNAs, and proteins. RNA aptamers, Pumilio, SuperNova tagging system, molecular beacons, halotag, bimolecular fluorescence complementation, RNA-guided endonuclease in situ labeling, and oligonucleotide-based imaging methods utilizing fluorescent proteins, organic dyes, or quantum dots have been developed to achieve improved fluorescence and signal-to-noise ratio for the imaging of chromatin or genomic loci. RNA-guided RNA targeting CRISPR systems (CRISPR/dCas13) and gene knock-in strategies based on CRISPR/Cas9 mediated site-specific cleavage and DNA repair mechanisms have been employed for efficient RNA and protein imaging, respectively. A few CRISPR-Cas-based methods to investigate the coordinated regulation of DNA-protein, DNA-RNA, or RNA-protein interactions for understanding chromatin dynamics, transcription, and protein function are also available. Overall, the CRISPR-based methods offer a significant improvement in elucidating chromatin organization and dynamics, RNA visualization, and protein imaging. The current and future advancements in CRISPR-based imaging techniques can revolutionize genome biology research for various applications.},
}
@article {pmid34407138,
year = {2021},
author = {Lin, HW and Lee, JY and Chou, NL and Shih, TW and Chang, MS},
title = {Phosphorylation of PUF-A/PUM3 on Y259 modulates PUF-A stability and cell proliferation.},
journal = {PloS one},
volume = {16},
number = {8},
pages = {e0256282},
pmid = {34407138},
issn = {1932-6203},
mesh = {Atlases as Topic ; CRISPR-Cas Systems ; Carcinogenesis/genetics/*metabolism/pathology ; Cell Movement ; Cell Nucleolus/genetics/metabolism ; Cell Proliferation ; Databases, Genetic ; Female ; Gene Deletion ; HeLa Cells ; Humans ; Minor Histocompatibility Antigens/genetics/*metabolism ; Mutation ; Neoplasms/genetics/*metabolism/mortality/pathology ; Phosphoproteins/genetics/*metabolism ; Phosphorylation ; *Protein Processing, Post-Translational ; Protein Stability ; Survival Analysis ; Tyrosine/*metabolism ; },
abstract = {Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A's phosphorylation on Y259 in tumorigenesis. PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation.},
}
@article {pmid34406572,
year = {2022},
author = {Qiu, F and Xing, S and Xue, C and Liu, J and Chen, K and Chai, T and Gao, C},
title = {Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing.},
journal = {Science China. Life sciences},
volume = {65},
number = {4},
pages = {731-738},
pmid = {34406572},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Expression Regulation, Plant ; Genome, Plant/genetics ; *MicroRNAs/metabolism ; Plants, Genetically Modified/genetics ; Triticum/genetics/metabolism ; },
abstract = {Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1 (TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex (mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of mTaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, mTaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.},
}
@article {pmid34406570,
year = {2022},
author = {Liu, ZH and Tang, S and Hu, W and Lv, R and Mei, H and Yang, R and Song, X and Cao, X and Wang, D},
title = {Precise editing of methylated cytosine in Arabidopsis thaliana using a human APOBEC3Bctd-Cas9 fusion.},
journal = {Science China. Life sciences},
volume = {65},
number = {1},
pages = {219-222},
pmid = {34406570},
issn = {1869-1889},
mesh = {APOBEC Deaminases/*physiology ; Arabidopsis/*genetics ; CRISPR-Cas Systems ; Cytosine/*chemistry ; DNA Methylation ; *Gene Editing ; In Vitro Techniques ; *Plants, Genetically Modified ; },
}
@article {pmid34406049,
year = {2021},
author = {Segev-Hadar, A and Slosman, T and Rozen, A and Sherman, A and Cnaani, A and Biran, J},
title = {Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus).},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {583-594},
doi = {10.1089/crispr.2020.0115},
pmid = {34406049},
issn = {2573-1602},
mesh = {Alleles ; Animals ; *Animals, Genetically Modified ; Base Sequence ; *CRISPR-Cas Systems ; Cloning, Molecular ; *Gene Editing ; *Genes, Reporter ; Genome ; Germ Cells/*metabolism ; Humans ; Membrane Transport Proteins/genetics/metabolism ; Microinjections ; Mutation ; Phenotype ; Phylogeny ; RNA, Guide ; Sequence Analysis, DNA ; Tilapia/*genetics ; Zygote ; },
abstract = {In recent years, there has been increasing demand for red tilapia, which are commercial strains of hybrids of different tilapiine species or red variants of highly inbred Nile tilapia. However, red tilapia phenotypes are genetically unstable and affected by environmental factors, resulting in nonuniform coloration with black or dark-red color blotches that reduce their market value. Solute carrier family 45 member 2 (SLC45A2) is a membrane transporter that mediates melanin biosynthesis and is evolutionarily conserved from fish to humans. In the present study, we describe the generation of a stable and heritable red tilapia phenotype by inducing loss-of-function mutations in the slc45a2 gene. For this purpose, we identified the slc45a2 gene in Nile tilapia and designed highly specific guide RNAs (gRNA) for its genomic sequence. Multiplex microinjection of slc45a2-specific ribonucleoproteins to Nile tilapia zygotes induced up to 97-99% albinism, including loss of melanin in the eye. Next-generation sequencing of the injected zygotes demonstrated that all injected fish carried mutant alleles with variable mutagenesis efficiencies. Sanger sequencing of the genomic target region in the slc45a2 gene from fin clips, sperm, and F1 offspring of a highly mutant male identified various genomic indels and germline transmission of the sperm-identified indels. Overall, this work demonstrates the generation of somatic and germline slc45a2 mutant alleles, which leads to complete albinism in Nile tilapia.},
}
@article {pmid34406048,
year = {2021},
author = {Naeem, MA},
title = {Establishing CRISPR-Cas Technology in Pakistan.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {467-468},
doi = {10.1089/crispr.2021.29132.nae},
pmid = {34406048},
issn = {2573-1602},
mesh = {*Biotechnology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; History, 21st Century ; Humans ; Pakistan ; },
}
@article {pmid34406047,
year = {2021},
author = {Nethery, MA and Korvink, M and Makarova, KS and Wolf, YI and Koonin, EV and Barrangou, R},
title = {CRISPRclassify: Repeat-Based Classification of CRISPR Loci.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {558-574},
pmid = {34406047},
issn = {2573-1602},
mesh = {Algorithms ; Area Under Curve ; Base Sequence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; Databases, Genetic ; *Gene Editing ; *Genetic Loci ; Genome, Bacterial ; Genomics/methods ; Reproducibility of Results ; },
abstract = {Detection and classification of CRISPR-Cas systems in metagenomic data have become increasingly prevalent in recent years due to their potential for diverse applications in genome editing. Traditionally, CRISPR-Cas systems are classified through reference-based identification of proximate cas genes. Here, we present a machine learning approach for the detection and classification of CRISPR loci using repeat sequences in a cas-independent context, enabling identification of unclassified loci missed by traditional cas-based approaches. Using biological attributes of the CRISPR repeat, the core element in CRISPR arrays, and leveraging methods from natural language processing, we developed a machine learning model capable of accurate classification of CRISPR loci in an extensive set of metagenomes, resulting in an F1 measure of 0.82 across all predictions and an F1 measure of 0.97 when limiting to classifications with probabilities >0.85. Furthermore, assessing performance on novel repeats yielded an F1 measure of 0.96. Although the performance of cas-based identification will exceed that of a repeat-based approach in many cases, CRISPRclassify provides an efficient approach to classification of CRISPR loci for cases in which cas gene information is unavailable, such as metagenomes and fragmented genome assemblies.},
}
@article {pmid34406045,
year = {2021},
author = {Barrangou, R},
title = {Clinical Milestone for CRISPR Medicines.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {459},
doi = {10.1089/crispr.2021.29135.rba},
pmid = {34406045},
issn = {2573-1602},
mesh = {Amyloid Neuropathies, Familial/genetics/therapy ; *CRISPR-Cas Systems ; Clinical Trials, Phase I as Topic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Genetic Therapy/methods ; Humans ; Treatment Outcome ; },
}
@article {pmid34406044,
year = {2021},
author = {Nishida, K and Kondo, A},
title = {Transversion Expansion of Base Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {462-463},
doi = {10.1089/crispr.2021.29134.kni},
pmid = {34406044},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Cytosine ; Gene Editing/*methods ; Guanidine ; Humans ; *Mutagenesis ; *Point Mutation ; },
}
@article {pmid34406043,
year = {2021},
author = {Kieper, SN and Almendros, C and Haagsma, AC and Barendregt, A and Heck, AJR and Brouns, SJJ},
title = {Cas4-Cas1 Is a Protospacer Adjacent Motif-Processing Factor Mediating Half-Site Spacer Integration During CRISPR Adaptation.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {536-548},
doi = {10.1089/crispr.2021.0011},
pmid = {34406043},
issn = {2573-1602},
mesh = {*Binding Sites ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA, Bacterial ; *Gene Editing/methods ; Gene Order ; Multiprotein Complexes ; *Nucleotide Motifs ; Plasmids/chemistry/genetics ; Protein Binding ; Protein Multimerization ; },
abstract = {The immunization of bacteria and archaea against invading viruses via CRISPR adaptation is critically reliant on the efficient capture, accurate processing, and integration of CRISPR spacers into the host genome. The adaptation proteins Cas1 and Cas2 are sufficient for successful spacer acquisition in some CRISPR-Cas systems. However, many CRISPR-Cas systems additionally require the Cas4 protein for efficient adaptation. Cas4 has been implied in the selection and processing of spacer precursors, but the detailed mechanistic understanding of how Cas4 contributes to CRISPR adaptation is lacking. Here, we biochemically reconstitute the CRISPR-Cas type I-D adaptation system and show two functionally distinct adaptation complexes: Cas4-Cas1 and Cas1-Cas2. The Cas4-Cas1 complex recognizes and cleaves protospacer adjacent motif (PAM) sequences in 3' overhangs in a sequence-specific manner, while the Cas1-Cas2 complex defines the cleavage of non-PAM sites via host-factor nucleases. Both sub-complexes are capable of mediating half-site integration, facilitating the integration of processed spacers in the correct interference-proficient orientation. We provide a model in which an asymmetric adaptation complex differentially acts on PAM- and non-PAM-containing overhangs, providing cues for the correct orientation of spacer integration.},
}
@article {pmid34406042,
year = {2021},
author = {Shahryari, A and Moya, N and Siehler, J and Wang, X and Burtscher, I and Lickert, H},
title = {Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {491-501},
doi = {10.1089/crispr.2021.0014},
pmid = {34406042},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Cell Differentiation ; Cell Survival ; DNA End-Joining Repair ; Flow Cytometry ; *Gene Editing/methods ; *Gene Expression ; Gene Expression Regulation ; Genetic Engineering/methods ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Plasmids/genetics ; Pluripotent Stem Cells/cytology/*metabolism ; RNA, Guide ; *RNA, Small Untranslated ; Recombinational DNA Repair ; Transfection ; Transgenes ; },
abstract = {Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector. Either we inserted short hairpin RNA expression cassettes to downregulate DNAPK and XRCC4, two main players of the NHEJ pathway, or we increased cell survival by inserting an anti-apoptotic expression cassette of miRNA-21 into the Cas9 vector. For an easy readout, the pluripotency gene SOX2 was targeted with a T2A-tdTomato reporter construct. In vitro downregulating DNAPK and XRCC4 increased the targeting efficiency of SOX2 KI by around twofold. Furthermore, co-expression of miRNA-21 and Cas9 improved the efficiency of SOX2 KI by around threefold. Altogether, our strategies provide a simple and valuable approach for efficient CRISPR-Cas9 gene editing in iPSCs.},
}
@article {pmid34406041,
year = {2021},
author = {Musunuru, K},
title = {CRISPR Hits Home in a First-in-Human Study.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {460-461},
doi = {10.1089/crispr.2021.29131.mus},
pmid = {34406041},
issn = {2573-1602},
mesh = {Amyloid Neuropathies, Familial/genetics/therapy ; Animals ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Genetic Therapy/methods ; Humans ; Treatment Outcome ; },
}
@article {pmid34406040,
year = {2021},
author = {Weuring, WJ and Dilevska, I and Hoekman, J and van de Vondervoort, J and Koetsier, M and van 't Slot, RH and Braun, KPJ and Koeleman, BPC},
title = {CRISPRa-Mediated Upregulation of scn1laa During Early Development Causes Epileptiform Activity and dCas9-Associated Toxicity.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {575-582},
doi = {10.1089/crispr.2021.0013},
pmid = {34406040},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; Disease Susceptibility ; Embryonic Development/*genetics ; Epilepsies, Myoclonic/diagnosis/etiology ; Epilepsy/diagnosis/*etiology ; Gene Editing ; *Gene Expression Regulation ; Genetic Association Studies ; NAV1.1 Voltage-Gated Sodium Channel/*genetics ; *Phenotype ; RNA, Guide ; RNA, Messenger ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene SCN1A. DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous SCN1A can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene scn1Laa in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9-VPR and dCas9-VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of scn1Laa mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published scn1Laa and scn1Lab loss-of-function models, we conclude that gain of scn1Laa function can have an equally severe phenotype. Upregulation of scn1Laa in the current zebrafish model for DS, scn1Lab-KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.},
}
@article {pmid34406037,
year = {2021},
author = {Cornuault, JK and Moineau, S},
title = {Induction and Elimination of Prophages Using CRISPR Interference.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {549-557},
doi = {10.1089/crispr.2021.0026},
pmid = {34406037},
issn = {2573-1602},
mesh = {Bacteriophage lambda/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics/virology ; *Gene Editing ; Gene Order ; Gene Targeting ; Genetic Engineering ; Genome, Viral ; Plasmids/genetics ; Prophages/*genetics ; *Virus Activation ; },
abstract = {Prophages are widely spread among bacterial genomes, and they can have positive or negative effects on their hosts. A key aspect in the study of prophages is the discovery of their induction signals. Prophage induction can occur by inactivating a phage transcriptional repressor, which is responsible for maintaining the lysogenic state. This repressor can be inactivated through the bacterial SOS response. However, the induction signals for numerous prophages do not involve the SOS system, and therefore significant efforts are needed to identify these conditions. Similarly, curing bacterial strains of inducible prophages is a tedious process, requiring the screening of several colonies. Here, we investigated whether transcriptional silencing of a prophage repressor using CRISPR interference (CRISPRi) would lead to prophage induction. Using Escherichia coli phages λ and P2 as models, we demonstrated the efficiency of CRISPRi for prophage induction and for curing lysogenic strains of their prophages.},
}
@article {pmid34406036,
year = {2021},
author = {Nami, F and Ramezankhani, R and Vandenabeele, M and Vervliet, T and Vogels, K and Urano, F and Verfaillie, C},
title = {Fast and Efficient Generation of Isogenic Induced Pluripotent Stem Cell Lines Using Adenine Base Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {502-518},
doi = {10.1089/crispr.2021.0006},
pmid = {34406036},
issn = {2573-1602},
mesh = {*Adenine ; *CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Line ; Flow Cytometry ; *Gene Editing/methods ; Gene Targeting ; Genetic Vectors ; High-Throughput Nucleotide Sequencing ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Membrane Proteins/genetics ; Mutation ; Plasmids ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {Isogenic induced pluripotent stem cell (iPSC) lines are currently mostly created by homology directed repair evoked by a double-strand break (DSB) generated by CRISPR-Cas9. However, this process is in general lengthy and inefficient. This problem can be overcome, specifically for correction or insertion of transition mutations, by using base editing (BE). BE does not require DSB formation, hence avoiding creation of genomic off-target breaks and insertions and deletions, and as it is highly efficient, it also does not require integration of selection cassettes in the genome to enrich for edited cells. BE has been successfully used in many cell types as well as in some in vivo settings to correct or insert mutations, but very few studies have reported generation of isogenic iPSC lines using BE. Here, we describe a simple and fast workflow to generate isogenic iPSCs efficiently with a compound heterozygous or a homozygous Wolfram syndrome 1 (WFS1) mutation using adenine BE, without the need to include a genomic selection cassette and without off-target modifications. We demonstrated that correctly base-edited clones can be generated by screening only five cell clones in less than a month, provided that the mutation is positioned in a correct place with regards to the protospacer adjacent motif sequence and no putative bystander bases exist.},
}
@article {pmid34406035,
year = {2021},
author = {Tang, N and Zhang, Y and Shen, Z and Yao, Y and Nair, V},
title = {Application of CRISPR-Cas9 Editing for Virus Engineering and the Development of Recombinant Viral Vaccines.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {477-490},
doi = {10.1089/crispr.2021.0017},
pmid = {34406035},
issn = {2573-1602},
support = {BB/P016472/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014262/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R012865/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Antigens, Viral/genetics/immunology ; Biotechnology ; *CRISPR-Cas Systems ; Cell Culture Techniques ; *Gene Editing ; Gene Expression ; Gene Transfer Techniques ; Genetic Engineering/*methods ; Genetic Vectors/*genetics ; Humans ; Organ Specificity ; Vaccines, Synthetic/*genetics/immunology ; Vaccinology/methods ; Viral Vaccines/*genetics/immunology ; Viruses/classification/*genetics ; },
abstract = {CRISPR-Cas technology, discovered originally as a bacterial defense system, has been extensively repurposed as a powerful tool for genome editing for multiple applications in biology. In the field of virology, CRISPR-Cas9 technology has been widely applied on genetic recombination and engineering of genomes of various viruses to ask some fundamental questions about virus-host interactions. Its high efficiency, specificity, versatility, and low cost have also provided great inspiration and hope in the field of vaccinology to solve a series of bottleneck problems in the development of recombinant viral vaccines. This review highlights the applications of CRISPR editing in the technological advances compared to the traditional approaches used for the construction of recombinant viral vaccines and vectors, the main factors affecting their application, and the challenges that need to be overcome for further streamlining their effective usage in the prevention and control of diseases. Factors affecting efficiency, target specificity, and fidelity of CRISPR-Cas editing in the context of viral genome editing and development of recombinant vaccines are also discussed.},
}
@article {pmid34406033,
year = {2021},
author = {Vuelta, E and Ordoñez, JL and Alonso-Pérez, V and Méndez, L and Hernández-Carabias, P and Saldaña, R and Sevilla, J and Sebastián, E and Muntión, S and Sánchez-Guijo, F and Hernández-Rivas, JM and García-Tuñón, I and Sánchez-Martín, M},
title = {CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {519-535},
doi = {10.1089/crispr.2021.0009},
pmid = {34406033},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Models, Animal ; Fusion Proteins, bcr-abl/genetics ; *Gene Editing ; Gene Expression ; Gene Targeting/methods ; Gene Transfer Techniques ; *Genetic Therapy/methods ; Hematopoiesis/genetics ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology/metabolism ; Heterografts ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/therapy ; Mice ; Neoplastic Stem Cells/metabolism ; *Oncogenes ; Proof of Concept Study ; },
abstract = {Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.},
}
@article {pmid34404952,
year = {2021},
author = {Sankaranarayanan, G and Berriman, M and Rinaldi, G},
title = {An uneven race: genome editing for parasitic worms.},
journal = {Nature reviews. Microbiology},
volume = {19},
number = {10},
pages = {621},
pmid = {34404952},
issn = {1740-1534},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Helminth/*genetics ; Helminths/*genetics ; },
}
@article {pmid34404810,
year = {2021},
author = {Shen, Y and Verboon, JM and Zhang, Y and Liu, N and Kim, YJ and Marglous, S and Nandakumar, SK and Voit, RA and Fiorini, C and Ejaz, A and Basak, A and Orkin, SH and Xu, J and Sankaran, VG},
title = {A unified model of human hemoglobin switching through single-cell genome editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4991},
pmid = {34404810},
issn = {2041-1723},
support = {R56 DK125234/DK/NIDDK NIH HHS/United States ; R01 HL032259/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 CA230631/CA/NCI NIH HHS/United States ; R01 HL146500/HL/NHLBI NIH HHS/United States ; R01 DK103794/DK/NIDDK NIH HHS/United States ; R01 DK111430/DK/NIDDK NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chromatin ; Chromosomes ; DNA-Binding Proteins/metabolism ; Fetal Hemoglobin/genetics/metabolism ; *Gene Editing ; Gene Expression ; Globins ; Hemoglobins/*genetics/*metabolism ; Humans ; Mutation ; Repressor Proteins ; Transcription Factors/metabolism ; beta-Globins/genetics ; },
abstract = {Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the β-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.},
}
@article {pmid34404802,
year = {2021},
author = {Arandjelovic, S and Perry, JSA and Zhou, M and Ceroi, A and Smirnov, I and Walk, SF and Shankman, LS and Cambré, I and Onengut-Gumuscu, S and Elewaut, D and Conrads, TP and Ravichandran, KS},
title = {ELMO1 signaling is a promoter of osteoclast function and bone loss.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4974},
pmid = {34404802},
issn = {2041-1723},
support = {K99 CA237728/CA/NCI NIH HHS/United States ; R35 GM122542/GM/NIGMS NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/deficiency/*genetics/*metabolism ; Animals ; Arthritis/pathology ; Bone Diseases, Metabolic/*metabolism ; Bone Resorption/metabolism ; CRISPR-Cas Systems ; Female ; Mice ; Mice, Knockout ; Osteoclasts/*metabolism ; Osteoporosis/*metabolism ; Osteoprotegerin/deficiency ; Ovariectomy ; *Signal Transduction ; Transcriptome ; X-Ray Microtomography ; },
abstract = {Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important 'signaling node' in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the 'ELMO1 interactome' in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis.},
}
@article {pmid34404790,
year = {2021},
author = {Fang, L and Fan, J and Luo, S and Chen, Y and Wang, C and Cao, Y and Song, H},
title = {Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4976},
pmid = {34404790},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/*genetics/*isolation &amp; purification/*metabolism ; Escherichia coli Proteins/genetics/metabolism ; Fatty Acids/metabolism ; Fatty Acids, Nonesterified/*biosynthesis ; Fermentation ; Gene Expression Regulation, Bacterial ; Metabolic Engineering ; Metabolic Networks and Pathways/genetics ; Transcriptome ; },
abstract = {To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfA[L-]-aidB[+]-ryfA[M-]-gadA[H-], producing 30.0 g L[-1] FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.},
}
@article {pmid34403853,
year = {2021},
author = {Chen, D and Song, B and Cheng, Y and Zhu, L and Lu, D and Liu, N and Yang, Y and Sun, X},
title = {Generation of a homozygous ZBTB7A knockout human induced pluripotent stem line by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102494},
doi = {10.1016/j.scr.2021.102494},
pmid = {34403853},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA-Binding Proteins ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Transcription Factors/genetics ; },
abstract = {ZBTB7A plays important roles in several biological processes, including silencing of the fetal γ-globin genes, hematopoiesis, primed-to-naive transition, etc. Meanwhile, it is also associated with Oncogenic transformation and tumor progression. However, the mechanism of ZBTB7A function is not fully understood yet. Here, we generated a homozygous ZBTB7A knockout human induced pluripotent stem cell (iPSC) line, GZHMCi007-A by the CRISPR/Cas9-mediated homology-dependent DNA repair method. The iPSCs of ZBTB7A-/- established by us is a powerful tool for related research.},
}
@article {pmid34403806,
year = {2021},
author = {Li, PY and Li, SQ and Gao, SG and Dong, DY},
title = {CRISPR/Cas9-mediated gene editing on Sox2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development in mice.},
journal = {Biochemical and biophysical research communications},
volume = {573},
number = {},
pages = {107-111},
doi = {10.1016/j.bbrc.2021.08.029},
pmid = {34403806},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryonic Development/genetics ; Gene Editing ; Mice ; Neural Tube/*metabolism ; Promoter Regions, Genetic/*genetics ; RNA, Long Noncoding/*genetics ; },
abstract = {Sox2 overlapping transcript (Sox2ot) is a long non-coding RNA (lncRNA), which harbors one of the major regulators of pluripotency, the Sox2 gene, in its intronic region. Sox2ot is primarily expressed in the developing neuroepithelium. However, its role in neural tube closure and embryonic development remains unclear. To investigate if Sox2ot is required for neural tube closure and embryonic development, Sox2ot promoter was deleted by CRISPR-Cas9 genome editing technology to prevent Sox2ot gene expression in mice. We designed 9 guide RNAs to specifically target the Sox2ot promoter and 3 gRNAs induced gene editing on the promoter of the Sox2ot gene in cells transfected with Cas9 mRNA and gRNAs. Then, these gRNAs and Cas9 mRNA were injected into mouse zygotes and implanted into pseudopregnant mice. A Sox2ot promoter-deleted mouse line was identified with complete deletion of promoter as well as deletion of exon 1 and exon 2. Sox2ot transcript was truncated with a lack of exon 1 and exon 2 in Sox2ot promoter-deleted mice. Furthermore, neural tube closure and embryonic development were checked at E9.5, E10.5, E14.5, E17.5 and after-birth (P2) and we did not find any failure of neural tube closure and aberrant embryonic development in Sox2ot promoter-deleted mice. Thus, our study demonstrated that CRISPR-Cas9 gene editing in Sox2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development.},
}
@article {pmid34403547,
year = {2021},
author = {De Meester, B and Vanholme, R and de Vries, L and Wouters, M and Van Doorsselaere, J and Boerjan, W},
title = {Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar.},
journal = {The Plant journal : for cell and molecular biology},
volume = {108},
number = {3},
pages = {752-765},
doi = {10.1111/tpj.15468},
pmid = {34403547},
issn = {1365-313X},
mesh = {Aldehyde Oxidoreductases/genetics ; CRISPR-Cas Systems ; Cell Wall/genetics/ultrastructure ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Lignin/biosynthesis/*metabolism ; Plant Stems/cytology/genetics ; Plants, Genetically Modified ; Populus/*genetics/growth &amp; development/*metabolism ; Sugars/*metabolism ; },
abstract = {Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as 'good neighbors' in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of 'good neighbors' in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.},
}
@article {pmid34403463,
year = {2021},
author = {Campa, AR and Smith, LM and Hampton, HG and Sharma, S and Jackson, SA and Bischler, T and Sharma, CM and Fineran, PC},
title = {The Rsm (Csr) post-transcriptional regulatory pathway coordinately controls multiple CRISPR-Cas immune systems.},
journal = {Nucleic acids research},
volume = {49},
number = {16},
pages = {9508-9525},
pmid = {34403463},
issn = {1362-4962},
mesh = {Adaptive Immunity/genetics ; Bacterial Proteins/*genetics ; Bacteriophages/genetics/pathogenicity ; CRISPR-Cas Systems/*genetics ; Flagella/genetics ; Gene Expression Regulation, Bacterial/genetics ; Plasmids/genetics ; RNA Processing, Post-Transcriptional/genetics ; RNA, Messenger/genetics ; RNA-Binding Proteins ; Repressor Proteins ; Serratia/*genetics ; Signal Transduction/*genetics ; Virulence/genetics ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR-Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR-Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR-Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR-Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR-Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.},
}
@article {pmid34403080,
year = {2022},
author = {de Jesus, LCL and de Jesus Sousa, T and Coelho-Rocha, ND and Profeta, R and Barroso, FAL and Drumond, MM and Mancha-Agresti, P and Ferreira, &#xca; and Brenig, B and Aburjaile, FF and Azevedo, V},
title = {Safety Evaluation of Lactobacillus delbrueckii subsp. lactis CIDCA 133: a Health-Promoting Bacteria.},
journal = {Probiotics and antimicrobial proteins},
volume = {14},
number = {5},
pages = {816-829},
pmid = {34403080},
issn = {1867-1314},
mesh = {Animals ; Anti-Bacterial Agents/metabolism/pharmacology ; Lactobacillus/genetics ; *Lactobacillus delbrueckii/genetics ; Mice ; *Probiotics/pharmacology ; },
abstract = {Lactobacillus delbrueckii subsp. lactis CIDCA is a new potential probiotic strain whose molecular basis attributed to the host's benefit has been reported. This study investigated the safety aspects of Lactobacillus delbrueckii subsp. lactis CIDCA 133 based on whole-genome sequence and phenotypic analysis to avoid future questions about the harmful effects of this strain consumption. Genomic analysis showed that L. delbrueckii subsp. lactis CIDCA 133 harbors virulence, harmful metabolites, and antimicrobial resistance-associated genes. However, none of these genetic elements is flanked or located within prophage regions and plasmid sequence. At a phenotypic level, it was observed L. delbrueckii subsp. lactis CIDCA 133 antimicrobial resistance to aminoglycosides streptomycin and gentamicin antibiotics, but no hemolytic and mucin degradation activity was exhibited by strain. Furthermore, no adverse effects were observed regarding mice clinical and histopathological analysis after the strain consumption (5 × 10[7] CFU/mL). Overall, these findings reveal the safety of Lactobacillus delbrueckii subsp. lactis CIDCA 133 for consumption and future probiotic applications.},
}
@article {pmid34400685,
year = {2021},
author = {Shamshirgaran, Y and Jonebring, A and Svensson, A and Leefa, I and Bohlooly-Y, M and Firth, M and Woollard, KJ and Hofherr, A and Rogers, IM and Hicks, R},
title = {Rapid target validation in a Cas9-inducible hiPSC derived kidney model.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {16532},
pmid = {34400685},
issn = {2045-2322},
mesh = {A549 Cells ; Animals ; *CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Doxycycline/pharmacology ; Drug Discovery/*methods ; Gene Editing/*methods ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*drug effects ; Kidney/cytology ; *Molecular Targeted Therapy ; Organoids/drug effects ; Polycystic Kidney, Autosomal Dominant/drug therapy/*genetics ; RNA, Guide/genetics ; Swine ; TRPP Cation Channels/genetics ; },
abstract = {Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.},
}
@article {pmid34400073,
year = {2022},
author = {Koseoglou, E and van der Wolf, JM and Visser, RGF and Bai, Y},
title = {Susceptibility reversed: modified plant susceptibility genes for resistance to bacteria.},
journal = {Trends in plant science},
volume = {27},
number = {1},
pages = {69-79},
doi = {10.1016/j.tplants.2021.07.018},
pmid = {34400073},
issn = {1878-4372},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; *Disease Resistance/genetics ; Genome, Plant ; Plant Breeding ; Plant Diseases/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Plants have evolved complex defence mechanisms to avoid invasion of potential pathogens. Despite this, adapted pathogens deploy effector proteins to manipulate host susceptibility (S) genes, rendering plant defences ineffective. The identification and mutation of plant S genes exploited by bacterial pathogens are important for the generation of crops with durable and broad-spectrum resistance. Application of mutant S genes in the breeding of resistant crops is limited because of potential pleiotropy. New genome editing techniques open up new possibilities for the modification of S genes. In this review, we focus on S genes manipulated by bacteria and propose ways for their identification and precise modification. Finally, we propose that genes coding for transporter proteins represent a new group of S genes.},
}
@article {pmid34399899,
year = {2021},
author = {Tian, G and Zhang, D and Wang, Y and Hu, T and Lin, Y and Wang, Y and Cheng, W and Xia, Q},
title = {A universal CRISPR/Cas12a nucleic acid sensing platform based on proximity extension and transcription-unleashed self-supply crRNA.},
journal = {Analytica chimica acta},
volume = {1176},
number = {},
pages = {338755},
doi = {10.1016/j.aca.2021.338755},
pmid = {34399899},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; DNA, Single-Stranded ; *Nucleic Acids ; },
abstract = {The extraordinary genome-editing tool CRISPR/Cas12a has also been utilized as a powerful sensing technology owing to its highly-specificity and isothermal signal amplification. Nevertheless, the widespread application of Cas12a-based sensing methods in nucleic acid detection is limited by the targeting range and high undesired background. Herein, we established a universal Cas12a-based nucleic acid sensing strategy by using proximity extension and transcription-unleashed self-suppling of crRNA. The target was recognized and bound to a pair of adjacent probes, and then triggered the proximity-induced primer extension and transcription amplification to produce numerous crRNAs. The amplified abundant crRNAs assembled with Cas12a and dsDNA activators containing PAM to form a ternary complex, which trans-cleaved ssDNA-FQ reporters continuously to generate a strong fluorescent signal. Thus, the cascade enzymatic amplification was performed and subsequently applied for detecting target DNA down to 41.7 amol with a low nonspecific background. The application of this strategy in RNA detection has also been demonstrated, and it is expected to provide a universal and sensitive sensing platform for molecular diagnosis applications.},
}
@article {pmid34396711,
year = {2021},
author = {Liu, R and Jia, Y and Guo, P and Jiang, W and Bai, R and Liu, C},
title = {In Vivo Clonal Analysis Reveals Development Heterogeneity of Oligodendrocyte Precursor Cells Derived from Distinct Germinal Zones.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {8},
number = {20},
pages = {e2102274},
pmid = {34396711},
issn = {2198-3844},
mesh = {Animals ; Animals, Newborn ; Brain/growth &amp; development/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Count ; Cell Differentiation/genetics ; Cell Lineage/*genetics ; Cells, Cultured ; Clonal Evolution/*genetics ; DNA Transposable Elements/genetics ; Electroporation ; *Genetic Heterogeneity ; Humans ; Mice ; Oligodendrocyte Precursor Cells/*metabolism ; Oligodendroglia/metabolism ; Stem Cells/metabolism ; },
abstract = {Mounting evidence supports that oligodendrocyte precursor cells (OPCs) play important roles in maintaining the integrity of normal brains, and that their dysfunction is the etiology of numerous severe neurological diseases. OPCs exhibit diverse heterogeneity in the adult brain, and distinct germinal zones of the embryonic brain contribute to OPC genesis. However, it remains obscure whether developmental origins shape OPC heterogeneity in the adult brain. Here, an in vivo clonal analysis approach is developed to address this. By combining OPC-specific transgenes, in utero electroporation, and the PiggyBac transposon system, the lineages of individual neonatal OPCs derived from either dorsal or ventral embryonic germinal zones are traced, and the landscape of their trajectories is comprehensively described throughout development. Surprisingly, despite behaving indistinguishably in the brain before weaning, dorsally derived OPCs continuously expand throughout life, but ventrally derived OPCs eventually diminish. Importantly, clonal analysis supports the existence of an intrinsic cellular "clock" to control OPC expansion. Moreover, knockout of NF1 could circumvent the distinction of ventrally derived OPCs in the adult brain. Together, this work shows the importance of in vivo clonal analysis in studying stem/progenitor cell heterogeneity, and reveals that developmental origins play a role in determining OPC fate.},
}
@article {pmid34395736,
year = {2021},
author = {Welker, JM and Wierson, WA and Almeida, MP and Mann, CM and Torrie, ME and Ming, Z and Ekker, SC and Clark, KJ and Dobbs, DL and Essner, JJ and McGrail, M},
title = {GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish.},
journal = {Bio-protocol},
volume = {11},
number = {14},
pages = {e4100},
pmid = {34395736},
issn = {2331-8325},
support = {R01 GM055877/GM/NIGMS NIH HHS/United States ; R01 GM088424/GM/NIGMS NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; },
abstract = {Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish. Graphic abstract: GeneWeld method for CRISPR/Cas9 targeted integration.},
}
@article {pmid34395730,
year = {2021},
author = {Nishimura, K and Fukagawa, T},
title = {A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines.},
journal = {Bio-protocol},
volume = {11},
number = {14},
pages = {e4092},
pmid = {34395730},
issn = {2331-8325},
abstract = {Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to essential genes, which require a conditional knockout system. The auxin-inducible degron (AID) system enables rapid depletion of the target protein in an auxin-dependent manner and has been used to generate conditional mutants in various eukaryotic cell lines. One problem with the AID system is the use of high auxin concentrations for protein degradation, which can cause cytotoxicity. Recently, we established a super-sensitive AID (ssAID) system that allowed a reduction in the amount of auxin required by more than 1,000-fold. We also utilized a single-step method to generate AID-based conditional knockout cells with a ssAID system in various cell lines. In this protocol, we introduce our improved method, which provides a powerful tool for the investigation of the roles of essential genes.},
}
@article {pmid34395722,
year = {2021},
author = {Ogawa, H and Sano, S and Walsh, K},
title = {Employing the CRISPR-Cas System for Clonal Hematopoiesis Research.},
journal = {International journal of physical medicine &amp; rehabilitation},
volume = {9},
number = {1},
pages = {},
pmid = {34395722},
issn = {2329-9096},
support = {R01 HL138014/HL/NHLBI NIH HHS/United States ; R21 AG072095/AG/NIA NIH HHS/United States ; R01 HL141256/HL/NHLBI NIH HHS/United States ; R01 HL139819/HL/NHLBI NIH HHS/United States ; R01 HL152174/HL/NHLBI NIH HHS/United States ; },
abstract = {Clonal hematopoiesis is a state in which substantial fraction of hematopoietic stem cells acquire mutations in specific driver genes and expand in the absence of an overt hematological malignancy. Recent clinical studies have shown that clonal hematopoiesis increases likelihood of hematological malignancy and cardiovascular disease. While clinical studies have identified countless candidate driver genes associated with clonal hematopoiesis, experimental studies are required to evaluate causal and mechanistic relationships with disease processes. This task is technically difficult and expensive to achieve with traditional genetically engineered mice. The versatility and programmability of CRISPR-Cas system enables investigators to evaluate the pathogenesis of each mutation in experimental systems. Technical refinements have enabled gene editing in a cell type specific manner and at a single base pair resolution. Here, we summarize strategies to apply CRISPR-Cas system to experimental studies of clonal hematopoiesis and concerns that should be addressed.},
}
@article {pmid34395100,
year = {2021},
author = {Yang, S and Huang, J and He, B},
title = {CASPredict: a web service for identifying Cas proteins.},
journal = {PeerJ},
volume = {9},
number = {},
pages = {e11887},
pmid = {34395100},
issn = {2167-8359},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins constitute the CRISPR-Cas systems, which play a key role in prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas systems have also been designed to facilitate target gene editing in eukaryotic genomes. As one of the important components of the CRISPR-Cas system, Cas protein plays an irreplaceable role. The effector module composed of Cas proteins is used to distinguish the type of CRISPR-Cas systems. Effective prediction and identification of Cas proteins can help biologists further infer the type of CRISPR-Cas systems. Moreover, the class 2 CRISPR-Cas systems are gradually applied in the field of genome editing. The discovery of Cas protein will help provide more candidates for genome editing. In this paper, we described a web service named CASPredict (http://i.uestc.edu.cn/caspredict/cgi-bin/CASPredict.pl) for identifying Cas proteins. CASPredict first predicts Cas proteins based on support vector machine (SVM) by using the optimal dipeptide composition and then annotates the function of Cas proteins based on the hmmscan search algorithm. The ten-fold cross-validation results showed that the 84.84% of Cas proteins were correctly classified. CASPredict will be a useful tool for the identification of Cas proteins, or at least can play a complementary role to the existing methods in this area.},
}
@article {pmid34392967,
year = {2021},
author = {Ferreira da Silva, J and Meyenberg, M and Loizou, JI},
title = {Tissue specificity of DNA repair: the CRISPR compass.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {11},
pages = {958-962},
doi = {10.1016/j.tig.2021.07.010},
pmid = {34392967},
issn = {0168-9525},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Repair/genetics ; *Gene Editing ; Humans ; Organ Specificity/genetics ; },
abstract = {CRISPR-Cas9-mediated genome editing holds great promise for the correction of pathogenic variants in humans. However, its therapeutic implementation is hampered due to unwanted editing outcomes. A better understanding of cell type- and tissue-specific DNA repair processes will ultimately enable precise control of editing outcomes for safer and effective therapies.},
}
@article {pmid34391646,
year = {2021},
author = {Aquino-Jarquin, G},
title = {Current advances in overcoming obstacles of CRISPR/Cas9 off-target genome editing.},
journal = {Molecular genetics and metabolism},
volume = {134},
number = {1-2},
pages = {77-86},
doi = {10.1016/j.ymgme.2021.08.002},
pmid = {34391646},
issn = {1096-7206},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods/standards ; Humans ; },
abstract = {CRISPR/Cas9-based technology has revolutionized biomedical research by providing a high-fidelity gene-editing method, foreshadowing a significant impact on the therapeutics of many human genetic disorders previously considered untreatable. However, off-target events represent a critical hurdle before genome editing can be fully established in clinical practice. This mini-review recapitulates some recent advances for detecting and overcoming off-target effects mediated by the CRISPR/Cas9 system that could increase the likelihood of clinical success of the CRISPR-based approaches.},
}
@article {pmid34391098,
year = {2021},
author = {Boehnke, J and Atakhanov, S and Toledo, MAS and Schüler, HM and Sontag, S and Chatain, N and Koschmieder, S and Brümmendorf, TH and Kramann, R and Zenke, M},
title = {CRISPR/Cas9 mediated CXCL4 knockout in human iPS cells of polycythemia vera patient with JAK2 V617F mutation.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102490},
doi = {10.1016/j.scr.2021.102490},
pmid = {34391098},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Janus Kinase 2/genetics/metabolism ; Mutation ; *Polycythemia Vera/genetics ; },
abstract = {The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4[KO] iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4[KO] iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4[KO] iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases.},
}
@article {pmid34390675,
year = {2021},
author = {Barber, KW and Shrock, E and Elledge, SJ},
title = {CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays.},
journal = {Molecular cell},
volume = {81},
number = {17},
pages = {3650-3658.e5},
doi = {10.1016/j.molcel.2021.07.027},
pmid = {34390675},
issn = {1097-4164},
support = {R24 GM137763/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics/immunology ; Epitopes/*genetics/immunology ; Gene Editing/*methods ; Humans ; Mutagenesis/genetics ; *Peptide Library ; Protein Binding/genetics/immunology ; RNA, Guide/*genetics/immunology ; },
abstract = {CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.},
}
@article {pmid34390349,
year = {2021},
author = {Hao, W and Cui, W and Cheng, Z and Han, L and Suo, F and Liu, Z and Zhou, L and Zhou, Z},
title = {Development of a base editor for protein evolution via in situ mutation in vivo.},
journal = {Nucleic acids research},
volume = {49},
number = {16},
pages = {9594-9605},
pmid = {34390349},
issn = {1362-4962},
mesh = {Bacillus subtilis/*genetics ; CRISPR-Cas Systems/genetics ; Cytidine/genetics ; Cytidine Deaminase/*genetics ; *Evolution, Molecular ; Gene Editing ; Genome, Bacterial/genetics ; Genomic Instability/genetics ; Mutation/genetics ; Proteins/*genetics ; Thymidine/genetics ; },
abstract = {Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.},
}
@article {pmid34389779,
year = {2021},
author = {Ohtani, F and Miyazaki, D and Shimizu, Y and Haruki, T and Yamagami, S and Inoue, Y},
title = {Role of interferon regulatory factor 7 in corneal endothelial cells after HSV-1 infection.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {16487},
pmid = {34389779},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cornea/metabolism/*virology ; Endothelial Cells/metabolism/*virology ; Gene Editing ; *Herpesvirus 1, Human ; Histocompatibility Antigens Class I/metabolism ; Humans ; Interferon Regulatory Factor-7/genetics/*metabolism ; Keratitis, Herpetic/*metabolism ; },
abstract = {Viral infections of the cornea including herpes simplex virus 1 (HSV-1) cause visual morbidity, and the corneal endothelial cell damage leads to significant visual impairment. Interferon regulatory factor 7 (IRF7) has been identified as a significant regulator in corneal endothelial cells after an HSV-1 infection. To examine the role played by IRF7, the DNA binding domain (DBD) of IRF7 of human corneal endothelial cells (HCEn) was disrupted. An RNAi inhibition of IRF7 and IRF7 DBD disruption (IRF7 ∆DBD) led to an impairment of IFN-β production. Impaired IFN-β production by IRF7 ∆DBD was regained by IRF7 DNA transfection. Transcriptional network analysis indicated that IRF7 plays a role in antigen presentation function of corneal endothelial cells. When the antigen presentation activity of HCEn cells were examined for priming of memory CD8 T cells, IRF7 disruption abolished the anti-viral cytotoxic T lymphocyte (CTL) response which was dependent on the major histocompatibility complex (MHC) class I. To further examine the roles played by IRF7 in CTL induction as acquired immunity, the contribution of IRF7 to MHC class I-mediated antigen presentation was assessed. Analysis of IRF7 ∆DBD cells indicated that IRF7 played an unrecognized role in MHC class I induction, and the viral infection induced-MHC class I induction was abolished by IRF7 disruption. Collectively, the IRF7 in corneal endothelial cells not only contributed to type I IFN response, but also to the mediation of viral infection-induced MHC class I upregulation and priming of CD8 arm of acquired immunity.},
}
@article {pmid34389729,
year = {2021},
author = {Boutin, J and Rosier, J and Cappellen, D and Prat, F and Toutain, J and Pennamen, P and Bouron, J and Rooryck, C and Merlio, JP and Lamrissi-Garcia, I and Cullot, G and Amintas, S and Guyonnet-Duperat, V and Ged, C and Blouin, JM and Richard, E and Dabernat, S and Moreau-Gaudry, F and Bedel, A},
title = {CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4922},
pmid = {34389729},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Chromosome Deletion ; Chromosomes, Human, Pair 11/genetics ; DNA Methylation ; Gene Editing/*methods ; Gene Expression ; Globins/*genetics ; HEK293 Cells ; Hematopoietic Stem Cells/cytology/*metabolism ; Humans ; Insulin-Like Growth Factor II/genetics ; Loss of Heterozygosity/*genetics ; Polymorphism, Single Nucleotide ; RNA, Long Noncoding/genetics ; *Sequence Deletion ; },
abstract = {CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.},
}
@article {pmid34389557,
year = {2021},
author = {Dennison, L and Ruggieri, A and Mohan, A and Leatherman, J and Cruz, K and Woolman, S and Azad, N and Lesinski, GB and Jaffee, EM and Yarchoan, M},
title = {Context-Dependent Immunomodulatory Effects of MEK Inhibition Are Enhanced with T-cell Agonist Therapy.},
journal = {Cancer immunology research},
volume = {9},
number = {10},
pages = {1187-1201},
pmid = {34389557},
issn = {2326-6074},
support = {P01 CA247886/CA/NCI NIH HHS/United States ; P30 CA138292/CA/NCI NIH HHS/United States ; R01 CA228414/CA/NCI NIH HHS/United States ; P50 CA062924/CA/NCI NIH HHS/United States ; P30 CA006973/CA/NCI NIH HHS/United States ; UM1 CA186691/CA/NCI NIH HHS/United States ; R01 CA197296/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; B7-H1 Antigen/biosynthesis/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; Colorectal Neoplasms/*drug therapy/*immunology/pathology ; Female ; Humans ; Immunomodulation/*drug effects ; Lymphocytes, Tumor-Infiltrating/*immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mitogen-Activated Protein Kinase Kinases/antagonists &amp; inhibitors ; Protein Kinase Inhibitors/administration &amp; dosage/*pharmacology ; Signal Transduction/drug effects ; Tumor Microenvironment/drug effects ; Xenograft Model Antitumor Assays ; },
abstract = {MEK inhibition (MEKi) is proposed to enhance antitumor immunity but has demonstrated mixed results as an immunomodulatory strategy in human clinical trials. MEKi exerts direct immunomodulatory effects on tumor cells and tumor-infiltrating lymphocytes (TIL), but these effects have not been independently investigated. Here we modeled tumor-specific MEKi through CRISPR/Cas-mediated genome editing of tumor cells [MEK1 knockout (KO)] and pharmacologic MEKi with cobimetinib in a RAS-driven model of colorectal cancer. This approach allowed us to distinguish tumor-mediated and tumor-independent mechanisms of MEKi immunomodulation. MEK1 KO tumors demonstrated upregulation of JAK/STAT signaling, enhanced MHCI expression, CD8[+] T-cell infiltration and T-cell activation, and impaired tumor growth that is immune dependent. Pharmacologic MEKi recapitulated tumor-intrinsic effects but simultaneously impaired T-cell activation in the tumor microenvironment. We confirmed a reduction in human peripheral-lymphocyte activation from a clinical trial of anti-PD-L1 (atezolizumab) with or without cobimetinib in biliary tract cancers. Impaired activation of TILs treated with pharmacologic MEKi was reversible and was rescued with the addition of a 4-1BB agonist. Collectively, these data underscore the ability of MEKi to induce context-dependent immunomodulatory effects and suggest that T cell-agonist therapy maximizes the beneficial effects of MEKi on the antitumor immune response.},
}
@article {pmid34389459,
year = {2021},
author = {Lei, H and Liu, GS},
title = {The current CRISPR methods.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {1-2},
doi = {10.1016/j.ymeth.2021.08.002},
pmid = {34389459},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
}
@article {pmid34389365,
year = {2021},
author = {Pan, X and Pei, X and Huang, H and Su, N and Wu, Z and Wu, Z and Qi, X},
title = {One-in-one individual package and delivery of CRISPR/Cas9 ribonucleoprotein using apoferritin.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {337},
number = {},
pages = {686-697},
doi = {10.1016/j.jconrel.2021.08.015},
pmid = {34389365},
issn = {1873-4995},
mesh = {Apoferritins/genetics ; *CRISPR-Cas Systems ; Gene Editing ; HeLa Cells ; Humans ; *Ribonucleoproteins/genetics ; },
abstract = {So far, most reported delivery of CRISPR/Cas9 is achieved by internalized or encapsulated multiple ribonucleoprotein units into only one carrier unit, with relatively large size. Here, we report a novel, small-sized, individual package of CRISPR/Cas9, via using tetralysine modified H-chian apoferritin (TL-HFn) as packaging material. In this paper, each CRISPR/Cas9 complex is proved to be successfully installed into one TL-HFn (~26 nm), and delivered into the targeting cell via TfR1-mediated endocytosis. We found that after 6 h of treatment, the CRISPR/Cas9 complex can be tracked within the nuclear of Hela cells for the purpose of gene editing of enhanced green fluorescent protein (EGFP). Moreover, TL-HFn individually packed CRISPR/Cas9 displayed higher genome editing activity compared with that of free CRISPR/Cas9 treated group both in vitro (up to 28.96%) and in vivo. Such satisfied genome editing efficiency could be attributed to the endosomal escape and pH-induced disassembly abilities given by TL-HFn after uptake into cytoplasm, which had been verified in our previous research. In all, those results prompted that TL-HFn possessed more potential for intracellular delivery of CRISPR/Cas9, with potential biocompatibility, stability and delivery efficiency.},
}
@article {pmid34387498,
year = {2021},
author = {Uddin, N and Warriner, LW and Pack, DW and DeRouchey, JE},
title = {Enhanced Gene Delivery and CRISPR/Cas9 Homology-Directed Repair in Serum by Minimally Succinylated Polyethylenimine.},
journal = {Molecular pharmaceutics},
volume = {18},
number = {9},
pages = {3452-3463},
doi = {10.1021/acs.molpharmaceut.1c00368},
pmid = {34387498},
issn = {1543-8392},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Knock-In Techniques ; *Gene Transfer Techniques ; Genetic Therapy/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Polyethyleneimine/analogs &amp; derivatives/*chemistry ; Recombinational DNA Repair ; },
abstract = {Gene therapy aims to treat patients by altering or controlling gene expression. The field of gene therapy has had increasing success in recent years primarily using viral-based approaches; however, there is still significant interest toward the use of polymeric materials due to their potential as flexible, low-cost scaffolds for gene delivery that do not suffer the mutagenesis and immunogenicity concerns of viral vectors. To address the challenges of efficiency and biocompatibility, a series of zwitterion-like polyethylenimine derivatives (zPEIs) were produced via the succinylation of 2-11.5% of polyethylenimine (PEI) amines. With increasing modification, zPEI polyplexes exhibited decreased serum-protein aggregation and dissociated more easily in the presence of a competitor polyanion when compared to unmodified PEI. Surprisingly, the gene delivery mediated in the presence of serum showed that succinylation of as few as 2% of PEI amines resulted in transgene expression 260- to 480-fold higher than that of unmodified PEI and 50- to 65-fold higher than that of commercial PEI-PEG2k in HEK293 and HeLa cells, respectively. Remarkably, the same zPEIs also produced 16-fold greater efficiency of CRISPR/Cas9 gene knock-in compared to unmodified PEI in the presence of serum. In addition, we show that 2% succinylation does not significantly decrease polymer/DNA binding ability or serum protein interaction to a significant extent, yet this small modification is still sufficient to provide a remarkable increase in transgene expression and gene knock-in in the presence of serum.},
}
@article {pmid34385461,
year = {2021},
author = {Yuan, T and Yan, N and Fei, T and Zheng, J and Meng, J and Li, N and Liu, J and Zhang, H and Xie, L and Ying, W and Li, D and Shi, L and Sun, Y and Li, Y and Li, Y and Sun, Y and Zuo, E},
title = {Optimization of C-to-G base editors with sequence context preference predictable by machine learning methods.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4902},
pmid = {34385461},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Base Sequence ; Binding Sites/genetics ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Codon/genetics ; Cytidine Deaminase/genetics/*metabolism ; Escherichia coli/genetics ; Female ; Gene Editing/*methods ; Gene Library ; HEK293 Cells ; Humans ; *Machine Learning ; Mice ; Reproducibility of Results ; Uracil-DNA Glycosidase/genetics/*metabolism ; },
abstract = {Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.},
}
@article {pmid34385391,
year = {2021},
author = {Park, JU and Tsai, AW and Mehrotra, E and Petassi, MT and Hsieh, SC and Ke, A and Peters, JE and Kellogg, EH},
title = {Structural basis for target site selection in RNA-guided DNA transposition systems.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6556},
pages = {768-774},
pmid = {34385391},
issn = {1095-9203},
support = {R01 GM144566/GM/NIGMS NIH HHS/United States ; R01 GM129118/GM/NIGMS NIH HHS/United States ; R00 GM124463/GM/NIGMS NIH HHS/United States ; R21 AI148941/AI/NIAID NIH HHS/United States ; R35 GM118174/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Diphosphate/metabolism ; Adenosine Triphosphate/metabolism ; Bacterial Proteins/*chemistry/metabolism ; CRISPR-Associated Proteins/*chemistry/metabolism ; Cryoelectron Microscopy ; Cyanobacteria/*chemistry/genetics/metabolism ; *DNA Transposable Elements ; DNA, Bacterial/metabolism ; Models, Molecular ; Protein Conformation ; Protein Folding ; RNA, Bacterial/metabolism ; Transposases/chemistry/metabolism ; },
abstract = {CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.},
}
@article {pmid34382198,
year = {2021},
author = {Cebola, I},
title = {Deletion of Regulatory Elements with All-in-One CRISPR-Cas9 Vectors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2351},
number = {},
pages = {321-334},
pmid = {34382198},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; *Gene Editing ; Gene Order ; *Gene Targeting/methods ; Genetic Vectors/*genetics ; Humans ; Insulin-Secreting Cells/metabolism ; RNA, Guide/chemistry/genetics ; *Regulatory Sequences, Nucleic Acid ; *Sequence Deletion ; },
abstract = {Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning using Golden Gate, by adapting the primer design.},
}
@article {pmid34382013,
year = {2021},
author = {Hundley, FV and Toczyski, DP},
title = {Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100685},
pmid = {34382013},
issn = {2666-1667},
support = {T32 GM007810/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing/*methods ; Gene Library ; Genetic Testing/*methods ; High-Throughput Screening Assays/*methods ; Humans ; RNA, Guide/genetics ; },
abstract = {The development of CRISPR-Cas9 screening techniques coupled with chemical inhibition of specific biological processes enables high-throughput investigation into many areas of molecular biology. We present a protocol to conduct ubiquitin proteasome system-specific chemical-genetic CRISPR-Cas9 screens in the human HAP1 cell line. This protocol can be adapted for use in other cell lines, with other compounds and types of treatments, and with any other sgRNA library. For complete details on the use and execution of this protocol, please refer to Hundley et al. (2021).},
}
@article {pmid34381246,
year = {2021},
author = {Pausch, P and Soczek, KM and Herbst, DA and Tsuchida, CA and Al-Shayeb, B and Banfield, JF and Nogales, E and Doudna, JA},
title = {DNA interference states of the hypercompact CRISPR-CasΦ effector.},
journal = {Nature structural &amp; molecular biology},
volume = {28},
number = {8},
pages = {652-661},
pmid = {34381246},
issn = {1545-9985},
support = {F31 HL156468/HL/NHLBI NIH HHS/United States ; P01 GM051487/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophages/genetics ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/metabolism ; *DNA Cleavage ; DNA-Binding Proteins/metabolism ; Gene Editing ; Genetic Techniques ; *Molecular Conformation ; RNA, Guide/metabolism ; },
abstract = {CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.},
}
@article {pmid34380739,
year = {2021},
author = {Hines, KM and Chaudhari, V and Edgeworth, KN and Owens, TG and Hanson, MR},
title = {Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {33},
pages = {},
pmid = {34380739},
issn = {1091-6490},
support = {S10 RR025502/RR/NCRR NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carbonic Anhydrases/*metabolism ; Chloroplasts/*enzymology/metabolism ; Gene Deletion ; Gene Expression Regulation, Plant/physiology ; Mutation ; Plant Leaves/growth &amp; development/metabolism ; Plants, Genetically Modified ; Tobacco/*enzymology/genetics ; },
abstract = {The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δβ-ca1 and Δβ-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δβ-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δβ-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δβ-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δβ-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δβ-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the Δβ-ca1ca5 mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δβ-ca1 and Δβ-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.},
}
@article {pmid34380502,
year = {2021},
author = {Li, G and Wang, Y and Li, X and Wang, Y and Huang, X and Gao, J and Hu, X},
title = {Developing PspCas13b-based enhanced RESCUE system, eRESCUE, with efficient RNA base editing.},
journal = {Cell communication and signaling : CCS},
volume = {19},
number = {1},
pages = {84},
pmid = {34380502},
issn = {1478-811X},
mesh = {Adenosine/genetics ; Adenosine Deaminase/*genetics ; CRISPR-Cas Systems/genetics ; Cytidine/genetics ; Genetic Diseases, Inborn/*genetics/therapy ; HEK293 Cells ; Humans ; I-kappa B Kinase/genetics ; Inosine/genetics ; RNA/*genetics ; RNA Editing/*genetics ; RNA-Binding Proteins/*genetics ; Uridine/genetics ; },
abstract = {RNA base editing is potential for cellular function research and genetic diseases treating. There are two main RNA base editors, REPAIR and RESCUE, for in vitro use. REPAIR was developed by fusing inactivated Cas13 (dCas13) with the adenine deaminase domain of ADAR2, which efficiently performs adenosine-to-inosine (A-to-I) RNA editing. RESCUE, which performs both cytidine-to-uridine (C-to-U) and A-to-I RNA editing, was developed by fusing inactivated Cas13 (dCas13) with the evolved ADAR2. However, the relatively low editing efficiency of the RESCUE system limits its broad application. Here, we constructed an enhanced RESCUE (eRESCUE) system; this dPspCas13b-RESCUE-NES system was generated by fusing inactivated PspCas13b with the evolved ADAR2. We determined the endogenous mRNA A-to-I and C-to-U editing efficiency mediated by the dPspCas13b-RESCUE-NES system in HEK-293T cells. This new RNA base editor was then used to induce 177Ser/Gly conversion of inhibitor kappa B kinase β (IKKβ) by changing the genetic code from AGU to GGU. The results showed that the eRESCUE editor mediates more efficient A-to-I and C-to-U RNA editing than the RESCUE RNA editor, as was previously reported. The 177Ser/Gly conversion of IKKβ, accomplished by converting the genetic code from AGU to GGU, resulted in a decrease in the phosphorylation of IKKβ and downregulation of downstream IKKβ-related genes. In summary, we developed a more efficient RNA base editor, eRESCUE, which may provide a useful tool for biomedical research and genetic disease treatment. Video Abstract.},
}
@article {pmid34379992,
year = {2022},
author = {Terceiro, LEL and Blanchard, AAA and Edechi, CA and Freznosa, A and Triggs-Raine, B and Leygue, E and Myal, Y},
title = {Generation of prolactin-inducible protein (Pip) knockout mice by CRISPR/Cas9-mediated gene engineering.},
journal = {Canadian journal of physiology and pharmacology},
volume = {100},
number = {1},
pages = {86-91},
doi = {10.1139/cjpp-2021-0306},
pmid = {34379992},
issn = {1205-7541},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques/*methods ; Genetic Engineering/*methods ; Mice, Inbred C57BL ; *Mice, Knockout ; Models, Animal ; Proteins/*genetics ; },
abstract = {Prolactin-inducible protein (PIP) is a multifunctional glycoprotein that is highly expressed and found in the secretions of apocrine glands such as salivary, lacrimal, and sweat glands including the mammary glands. PIP has been implicated in various diseases, including breast cancer, gross cystic disease of the breast, keratoconus of the eye, and the autoimmune Sjögren's syndrome. Here we have generated a Pip knockout (KO) mouse using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRSPR-associated (Cas)9 system. The Cas9 protein and two single guide RNAs targeting specific regions for both exons 1 and 2 of the Pip gene were microinjected into mouse embryos. The deletions and insertions promoted by CRISPR/Cas9 system on the Pip gene successfully disrupted Pip protein coding, as confirmed by PCR genotyping, sequencing, and ultimately Western blot analysis. This mouse model was generated in the inbred C57Bl/6J mouse, which exhibits lower genetic variation. This novel CRISPR Pip KO mouse model will not only be useful for future studies to interrogate the multifunctional role of PIP in physiological processes but will facilitate a broader understanding of the function of PIP in vivo while providing unprecedented insight into its role in a spectrum of diseases attributed to the deregulation of the PIP gene.},
}
@article {pmid34378567,
year = {2021},
author = {Zheng, Q and Li, W and Mao, L and Wang, M},
title = {Nanoscale metal-organic frameworks for the intracellular delivery of CRISPR/Cas9 genome editing machinery.},
journal = {Biomaterials science},
volume = {9},
number = {21},
pages = {7024-7033},
doi = {10.1039/d1bm00790d},
pmid = {34378567},
issn = {2047-4849},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Editing ; *Metal-Organic Frameworks ; RNA, Guide/genetics ; },
abstract = {The discovery of CRISPR/Cas9 genome-editing technology enables the precise manipulation of mammalian DNA sequences for treating genetic disorders. Despite its high efficiency for genome editing, the introduction of CRISPR/Cas9 machinery, which is composed of Cas9 nuclease protein and guide RNA, into cells challenges its clinical translation potential. Therefore, the intracellular delivery of genome-editing machinery determines the efficacy of gene manipulation via the CRISPR/Cas9 technology. Recently, metallosupramolecules including metal-organic frameworks (MOFs) and metal-organic cages (MOCs) have been designed to selfassemble with Cas9 nuclease and guide RNA for CRISPR/Cas9 delivery and genome editing. Herein, we review the most recent advances and strategies of constructing metallosupramolecules for CRISPR/Cas9 delivery. In particular, we discuss nanoscale MOFs and MOCs that could be assembled and regulated by the intracellular environment for the spatiotemporal delivery of genome editing machinery. We also provide a perspective view of the future development of metallosupramolecules for genome editing and gene therapy in vivo.},
}
@article {pmid34376731,
year = {2021},
author = {Nanasato, Y and Mikami, M and Futamura, N and Endo, M and Nishiguchi, M and Ohmiya, Y and Konagaya, KI and Taniguchi, T},
title = {CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don).},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {16186},
pmid = {34376731},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cryptomeria/*genetics/growth &amp; development ; *Gene Editing ; Genetic Vectors ; Genome, Plant ; Japan ; Lyases/*antagonists &amp; inhibitors/genetics ; *Mutagenesis ; *Mutation ; Plants, Genetically Modified/*genetics/growth &amp; development ; },
abstract = {Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.},
}
@article {pmid34376675,
year = {2021},
author = {Boltryk, SD and Passecker, A and Alder, A and Carrington, E and van de Vegte-Bolmer, M and van Gemert, GJ and van der Starre, A and Beck, HP and Sauerwein, RW and Kooij, TWA and Brancucci, NMB and Proellochs, NI and Gilberger, TW and Voss, TS},
title = {CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4806},
pmid = {34376675},
issn = {2041-1723},
mesh = {Animals ; Anopheles/parasitology ; *CRISPR-Cas Systems ; Cells, Cultured ; Erythrocytes/parasitology ; Hepatocytes/cytology/parasitology ; Host-Parasite Interactions ; Humans ; Malaria, Falciparum/*blood/parasitology/transmission ; Microscopy, Fluorescence ; Mosquito Vectors/parasitology ; Plasmodium falciparum/*genetics/physiology ; Spores, Protozoan/*genetics/physiology ; Sporozoites/genetics/physiology ; },
abstract = {The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.},
}
@article {pmid34376346,
year = {2021},
author = {Roy, SW},
title = {CRISPR-Cas Toxin-Antitoxin Systems: Selfishness as a Constructive Evolutionary Force.},
journal = {Trends in microbiology},
volume = {29},
number = {10},
pages = {869-870},
doi = {10.1016/j.tim.2021.07.005},
pmid = {34376346},
issn = {1878-4380},
mesh = {*Antitoxins/genetics ; CRISPR-Cas Systems/genetics ; Genome ; Genomics ; *Toxin-Antitoxin Systems/genetics ; },
abstract = {A recent paper (Li et al.) reports a novel RNA-based Cas-dependent toxin-antitoxin system with the effect of 'addicting' cells to the cassette. Broadly-defined addiction systems could stabilize diverse genomic features, raising the question of the role of selfish elements and intragenomic conflict in the evolution of biological complexity.},
}
@article {pmid34376154,
year = {2021},
author = {Poovaiah, C and Phillips, L and Geddes, B and Reeves, C and Sorieul, M and Thorlby, G},
title = {Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don).},
journal = {BMC plant biology},
volume = {21},
number = {1},
pages = {363},
pmid = {34376154},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; DNA, Plant ; Gene Editing/*methods ; *Genome, Plant ; INDEL Mutation ; Pinus/*genetics ; RNA, Guide ; Ribonucleoproteins/genetics ; },
abstract = {BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies.<br><br>RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15&#x2009;% using a single gRNA. 12&#x2009;% of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3&#xa0;kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33&#x2009;%, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic.<br><br>CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.},
}
@article {pmid34374341,
year = {2021},
author = {Weißenbruch, K and Grewe, J and Hippler, M and Fladung, M and Tremmel, M and Stricker, K and Schwarz, US and Bastmeyer, M},
title = {Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34374341},
issn = {2050-084X},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/physiology ; *Elasticity ; Homeostasis ; Humans ; Models, Theoretical ; Myosin Type II/classification/genetics/*metabolism ; Nonmuscle Myosin Type IIA/genetics/*metabolism ; Nonmuscle Myosin Type IIB/genetics/*metabolism ; Protein Isoforms ; },
abstract = {Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.},
}
@article {pmid34373891,
year = {2021},
author = {Presnell, JS and Browne, WE},
title = {Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {17},
pages = {},
doi = {10.1242/dev.199771},
pmid = {34373891},
issn = {1477-9129},
mesh = {Animals ; Body Patterning ; CRISPR-Cas Systems ; Ctenophora/cytology/*embryology/genetics/metabolism ; Embryonic Development ; Endoderm/cytology/embryology ; Gene Editing ; Gene Expression ; Kruppel-Like Transcription Factors/genetics/*metabolism ; },
abstract = {The Krüppel-like factor (Klf) gene family encodes transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation and development in bilaterians. Although Klf genes have been shown to specify functionally various cell types in non-bilaterian animals, their role in early-diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here, we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis resulted in abnormal development of several organs, including the pharynx, tentacle bulbs and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation.},
}
@article {pmid34373448,
year = {2021},
author = {Zhu, Q and Ma, Y and Liang, J and Wei, Z and Li, M and Zhang, Y and Liu, M and He, H and Qu, C and Cai, J and Wang, X and Zeng, Y and Jiao, Y},
title = {AHR mediates the aflatoxin B1 toxicity associated with hepatocellular carcinoma.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {299},
pmid = {34373448},
issn = {2059-3635},
mesh = {Active Transport, Cell Nucleus/drug effects ; Aflatoxin B1/pharmacology ; Animals ; B7-H1 Antigen/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/drug effects ; Carcinoma, Hepatocellular/*genetics/pathology/therapy/virology ; Cell Line, Tumor ; Genome, Human/drug effects ; Hepatitis Viruses/pathogenicity ; Hepatocytes/drug effects ; Humans ; Liver Neoplasms/*genetics/pathology/therapy/virology ; Mice ; Receptors, Aryl Hydrocarbon/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR-deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.},
}
@article {pmid34373320,
year = {2021},
author = {Geurts, MH and de Poel, E and Pleguezuelos-Manzano, C and Oka, R and Carrillo, L and Andersson-Rolf, A and Boretto, M and Brunsveld, JE and van Boxtel, R and Beekman, JM and Clevers, H},
title = {Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids.},
journal = {Life science alliance},
volume = {4},
number = {10},
pages = {},
pmid = {34373320},
issn = {2575-1077},
support = {C6307/A29058/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Alleles ; Amino Acid Substitution ; Animals ; *CRISPR-Cas Systems ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Epithelial Cells/metabolism ; *Gene Editing ; Genetic Therapy/methods ; Hepatocytes/metabolism ; Humans ; *Mutation ; Oncogenes/*genetics ; *Organoids ; Sequence Analysis, DNA ; },
abstract = {Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell-derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing-repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.},
}
@article {pmid34372664,
year = {2021},
author = {Atmanli, A and Chai, AC and Cui, M and Wang, Z and Nishiyama, T and Bassel-Duby, R and Olson, EN},
title = {Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy.},
journal = {Circulation research},
volume = {129},
number = {6},
pages = {602-616},
pmid = {34372664},
issn = {1524-4571},
support = {K99 HL153683/HL/NHLBI NIH HHS/United States ; R01 HL138426/HL/NHLBI NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cardiomyopathies/etiology/*therapy ; Cell Line ; Cells, Cultured ; Dependovirus/genetics ; Dystrophin/genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Muscular Dystrophy, Duchenne/complications/*therapy ; Myocytes, Cardiac/cytology/metabolism ; Transcriptome ; },
abstract = {[Figure: see text].},
}
@article {pmid34372578,
year = {2021},
author = {Brezgin, S and Kostyusheva, A and Bayurova, E and Volchkova, E and Gegechkori, V and Gordeychuk, I and Glebe, D and Kostyushev, D and Chulanov, V},
title = {Immunity and Viral Infections: Modulating Antiviral Response via CRISPR-Cas Systems.},
journal = {Viruses},
volume = {13},
number = {7},
pages = {},
pmid = {34372578},
issn = {1999-4915},
mesh = {Animals ; Antiviral Agents/*immunology/*pharmacology ; *CRISPR-Cas Systems ; Exoribonucleases/metabolism ; Host Microbial Interactions/immunology ; Humans ; Immune Evasion ; Immunity, Innate ; Interferons/genetics/immunology ; RNA Editing ; Transcriptome ; Virus Diseases/*immunology/virology ; Virus Internalization ; Virus Replication/drug effects ; },
abstract = {Viral infections cause a variety of acute and chronic human diseases, sometimes resulting in small local outbreaks, or in some cases spreading across the globe and leading to global pandemics. Understanding and exploiting virus-host interactions is instrumental for identifying host factors involved in viral replication, developing effective antiviral agents, and mitigating the severity of virus-borne infectious diseases. The diversity of CRISPR systems and CRISPR-based tools enables the specific modulation of innate immune responses and has contributed impressively to the fields of virology and immunology in a very short time. In this review, we describe the most recent advances in the use of CRISPR systems for basic and translational studies of virus-host interactions.},
}
@article {pmid34372494,
year = {2021},
author = {Dong, W and Kantor, B},
title = {Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives.},
journal = {Viruses},
volume = {13},
number = {7},
pages = {},
pmid = {34372494},
issn = {1999-4915},
support = {K01 DA031747/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; *Genetic Vectors ; Humans ; Integrases/metabolism ; Lentivirus/*genetics ; Mice ; Transgenes ; },
abstract = {CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector's expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.},
}
@article {pmid34371684,
year = {2021},
author = {Hahn, F and Sanjurjo Loures, L and Sparks, CA and Kanyuka, K and Nekrasov, V},
title = {Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {7},
pages = {},
pmid = {34371684},
issn = {2223-7747},
support = {BB/P016855/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {CRISPR/Cas technology has recently become the molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. Primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. While BAK1 is an important regulator of plant immunity and development, Ta-eIF4E and Ta-eIF(iso)4E act as susceptibility (S) factors required for plant viruses from the Potyviridae family to complete their life cycle. We anticipate the resultant homozygous tabak1-2 mutant lines will facilitate studies on the involvement of BAK1 in immune responses in wheat, while ta-eif4e and ta-eif(iso)4e mutant lines have the potential to become a source of resistance to wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV), both of which are important pathogens of wheat. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.},
}
@article {pmid34371550,
year = {2021},
author = {Sattar, MN and Iqbal, Z and Al-Khayri, JM and Jain, SM},
title = {Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {7},
pages = {},
pmid = {34371550},
issn = {2223-7747},
abstract = {Fruit trees provide essential nutrients to humans by contributing to major agricultural outputs and economic growth globally. However, major constraints to sustainable agricultural productivity are the uncontrolled proliferation of the population, and biotic and abiotic stresses. Tree mutation breeding has been substantially improved using different physical and chemical mutagens. Nonetheless, tree plant breeding has certain crucial bottlenecks including a long life cycle, ploidy level, occurrence of sequence polymorphisms, nature of parthenocarpic fruit development and linkage. Genetic engineering of trees has focused on boosting quality traits such as productivity, wood quality, and resistance to biotic and abiotic stresses. Recent technological advances in genome editing provide a unique opportunity for the genetic improvement of woody plants. This review examines application of the CRISPR-Cas system to reduce disease susceptibility, alter plant architecture, enhance fruit quality, and improve yields. Examples are discussed of the contemporary CRISPR-Cas system to engineer easily scorable PDS genes, modify lignin, and to alter the flowering onset, fertility, tree architecture and certain biotic stresses.},
}
@article {pmid34368464,
year = {2020},
author = {Sachse, G and Haythorne, E and Proks, P and Stewart, M and Cater, H and Ellard, S and Davies, B and Ashcroft, FM},
title = {Phenotype of a transient neonatal diabetes point mutation (SUR1-R1183W) in mice.},
journal = {Wellcome open research},
volume = {5},
number = {},
pages = {15},
pmid = {34368464},
issn = {2398-502X},
support = {/WT_/Wellcome Trust/United Kingdom ; MC_U142661184/MRC_/Medical Research Council/United Kingdom ; MC_UP_1502/1/MRC_/Medical Research Council/United Kingdom ; },
abstract = {Background: The K ATP channel plays a key role in glucose homeostasis by coupling metabolically generated changes in ATP to insulin secretion from pancreatic beta-cells. Gain-of-function mutations in either the pore-forming (Kir6.2) or regulatory (SUR1) subunit of this channel are a common cause of transient neonatal diabetes mellitus (TNDM), in which diabetes presents shortly after birth but remits within the first few years of life, only to return in later life. The reasons behind this time dependence are unclear. Methods: In an attempt to understand the mechanism behind diabetes remission and relapse, we generated mice expressing the common TNDM mutation SUR1-R1183W. We employed Cre/LoxP technology for both inducible and constitutive expression of SUR1-R1183W specifically in mouse beta-cells, followed by investigation of their phenotype using glucose tolerance tests and insulin secretion from isolated islets. Results: We found that the R1183W mutation impaired inhibition of K ATP channels by ATP when heterologously expressed in human embryonic kidney cells. However, neither induced nor constitutive expression of SUR1-R1183W in mice resulted in changes in blood glucose homeostasis, compared to littermate controls. When challenged with a high fat diet, female mice expressing SUR1-R1183W showed increased weight gain, elevated blood glucose and impaired glycaemic control, but glucose-stimulated insulin secretion from pancreatic islets appeared unchanged. Conclusions: The mouse model of TNDM did not recapitulate the human phenotype. We discuss multiple potential reasons why this might be the case. Based on our findings, we recommend future TNDM mouse models employing a gain-of-function SUR1 mutation should be created using the minimally invasive CRISPR/Cas technology, which avoids many potential pitfalls associated with the Cre/LoxP system.},
}
@article {pmid34371182,
year = {2022},
author = {Dastidar, S and Majumdar, D and Tipanee, J and Singh, K and Klein, AF and Furling, D and Chuah, MK and VandenDriessche, T},
title = {Comprehensive transcriptome-wide analysis of spliceopathy correction of myotonic dystrophy using CRISPR-Cas9 in iPSCs-derived cardiomyocytes.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {75-91},
pmid = {34371182},
issn = {1525-0024},
mesh = {Alternative Splicing ; CRISPR-Cas Systems ; Calmodulin-Binding Proteins/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; *Myotonic Dystrophy/genetics/therapy ; Myotonin-Protein Kinase/genetics ; RNA-Binding Proteins/genetics/metabolism ; Transcriptome ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.},
}
@article {pmid34371049,
year = {2021},
author = {Ye, X and Tang, X and Zhao, S and Ruan, J and Wu, M and Wang, X and Li, H and Zhong, B},
title = {Mechanism of the growth and development of the posterior silk gland and silk secretion revealed by mutation of the fibroin light chain in silkworm.},
journal = {International journal of biological macromolecules},
volume = {188},
number = {},
pages = {375-384},
doi = {10.1016/j.ijbiomac.2021.08.009},
pmid = {34371049},
issn = {1879-0003},
mesh = {Animals ; Bombyx/*genetics/growth &amp; development ; CRISPR-Cas Systems/genetics ; Fibroins/chemistry/*genetics ; Insect Proteins/genetics ; Larva/genetics/growth &amp; development ; Mutation/genetics ; *Proteomics ; Silk/*biosynthesis/genetics ; Transcriptome/genetics ; },
abstract = {Silkworm, as a model organism, has very high economic value due to its silk secretion ability. Although a large number of studies have attempted to elucidate the mechanism of silk secretion, it remains unclear. In this study, the fibroin light chain (Fib-L) gene of silkworm was subjected to CRISPR/Cas9 editing, which yielded premature termination of translation at 135 aa. Compared with those of the wild type, the posterior silk glands (PSGs) of the homozygous mutants on the third day of the fifth instar showed obvious premature degeneration. Comparative transcriptome and proteomic analyses of the PSGs of wild-type individuals, heterozygous mutants and homozygous mutants were performed on the fourth day of the fifth instar. A GO enrichment analysis showed that the differentially expressed genes (DEGs) between homozygous mutants and wild-type individuals were enriched in cytoskeleton-related terms, and a KEGG enrichment analysis showed that the upregulated DEGs between homozygous mutants and wild-type individuals were enriched in the phagosome and apoptosis pathways. These results indicated that apoptosis was activated prematurely in the PSGs of homozygous mutants. Furthermore, autophagy and heat shock response were activated in the PSGs of homozygous mutants, as demonstrated by an analysis of the DEGs related to autophagy and heat shock. A comparative proteomic analysis further confirmed that autophagy, apoptosis and the heat shock response were activated in the PSGs of homozygous mutants, which led to premature degradation of the PSGs. These results provide insights for obtaining a more in-depth understanding of the mechanism of silk secretion in silkworms.},
}
@article {pmid34370561,
year = {2021},
author = {Johnson, CN and Sheriff, EK and Duerkop, BA and Chatterjee, A},
title = {Let Me Upgrade You: Impact of Mobile Genetic Elements on Enterococcal Adaptation and Evolution.},
journal = {Journal of bacteriology},
volume = {203},
number = {21},
pages = {e0017721},
pmid = {34370561},
issn = {1098-5530},
support = {F31 AI157050/AI/NIAID NIH HHS/United States ; R01 AI141479/AI/NIAID NIH HHS/United States ; T32 AI052066/AI/NIAID NIH HHS/United States ; R01AI141479/AI/NIAID NIH HHS/United States ; },
mesh = {Biological Evolution ; CRISPR-Cas Systems ; Enterococcus faecalis/*genetics ; Enterococcus faecium/*genetics ; Interspersed Repetitive Sequences/*genetics ; },
abstract = {Enterococci are Gram-positive bacteria that have evolved to thrive as both commensals and pathogens, largely due to their accumulation of mobile genetic elements via horizontal gene transfer (HGT). Common agents of HGT include plasmids, transposable elements, and temperate bacteriophages. These vehicles of HGT have facilitated the evolution of the enterococci, specifically Enterococcus faecalis and Enterococcus faecium, into multidrug-resistant hospital-acquired pathogens. On the other hand, commensal strains of Enterococcus harbor CRISPR-Cas systems that prevent the acquisition of foreign DNA, restricting the accumulation of mobile genetic elements. In this review, we discuss enterococcal mobile genetic elements by highlighting their contributions to bacterial fitness, examine the impact of CRISPR-Cas on their acquisition, and identify key areas of research that can improve our understanding of enterococcal evolution and ecology.},
}
@article {pmid34370406,
year = {2021},
author = {Kouprina, N and Kim, JH and Larionov, V},
title = {Highly Selective, CRISPR/Cas9-Mediated Isolation of Genes and Genomic Loci from Complex Genomes by TAR Cloning in Yeast.},
journal = {Current protocols},
volume = {1},
number = {8},
pages = {e207},
pmid = {34370406},
issn = {2691-1299},
support = {Z99 CA999999/ImNIH/Intramural NIH HHS/United States ; ZIA BC010413/ImNIH/Intramural NIH HHS/United States ; /CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Chromosomes, Artificial, Yeast ; Cloning, Molecular ; Escherichia coli/genetics ; Genetic Vectors ; Genomics ; Humans ; Recombination, Genetic ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Here we describe an updated TAR cloning protocol for the selective and efficient isolation of any genomic fragment or gene of interest up to 280 kb in size from genomic DNA. The method exploits the special recombination machinery of the yeast Saccharomyces cerevisiae. TAR cloning is based on the high level of in vivo recombination that occurs between a specific genomic DNA fragment of interest and targeting sequences (hooks) in a TAR vector that are homologous to the 5' and 3' ends of the targeted region. Upon co-transformation into yeast, this results in the isolation of the chromosomal region of interest as a circular YAC molecule, which then propagates and segregates in yeast cells and can be selected for. In the updated TAR cloning protocol described here, the fraction of region-positive clones typically obtained is increased from 1% up to 35% by pre-treatment of the genomic DNA with specifically designed CRISPR/Cas9 endonucleases that create double-strand breaks (DSBs) bracketing the target genomic DNA sequence, thereby making the ends of the chromosomal region of interest highly recombinogenic. In addition, a new TAR vector was constructed that contains YAC and BAC cassettes, permitting direct transfer of a TAR-cloned DNA from yeast to bacterial cells. Once the TAR vector with the hooks is constructed and genomic DNA is prepared, the entire procedure takes 3 weeks to complete. The updated TAR protocol does not require significant yeast experience or extensively time-consuming yeast work because screening only about a dozen yeast transformants is typically enough to find a clone with the region of interest. TAR cloning of chromosomal fragments, individual genes, or gene families can be used for functional, structural, and population studies, for comparative genomics, and for long-range haplotyping, and has potential for gene therapy. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of CRISPR/Cas9-treated genomic DNA for TAR cloning Basic Protocol 2: Isolation of a gene or genomic locus by TAR cloning Basic Protocol 3: Transfer of TAR/YAC/BAC isolates from yeast to E. coli.},
}
@article {pmid34370390,
year = {2022},
author = {Yang, B and Shi, Z and Ma, Y and Wang, L and Cao, L and Luo, J and Wan, Y and Song, R and Yan, Y and Yuan, K and Tian, H and Zheng, H},
title = {LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus.},
journal = {Transboundary and emerging diseases},
volume = {69},
number = {4},
pages = {e216-e223},
doi = {10.1111/tbed.14285},
pmid = {34370390},
issn = {1865-1682},
mesh = {*African Swine Fever/diagnosis ; *African Swine Fever Virus/genetics ; Animals ; CRISPR-Cas Systems ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques/methods/veterinary ; Sensitivity and Specificity ; Swine ; *Swine Diseases/genetics ; },
abstract = {African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a loop-mediated isothermal amplification (LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was established in one tube for the detection of the African swine fever virus (ASFV) p72 gene. The single-stranded DNA-fluorophore quencher reporter and CRISPR-derived RNA were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples; a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.},
}
@article {pmid34369760,
year = {2021},
author = {Choi, JH and Shin, M and Yang, L and Conley, B and Yoon, J and Lee, SN and Lee, KB and Choi, JW},
title = {Clustered Regularly Interspaced Short Palindromic Repeats-Mediated Amplification-Free Detection of Viral DNAs Using Surface-Enhanced Raman Spectroscopy-Active Nanoarray.},
journal = {ACS nano},
volume = {15},
number = {8},
pages = {13475-13485},
doi = {10.1021/acsnano.1c03975},
pmid = {34369760},
issn = {1936-086X},
mesh = {Humans ; Spectrum Analysis, Raman/methods ; DNA, Viral/genetics ; Gold/chemistry ; *COVID-19 ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; *Nucleic Acids ; },
abstract = {Nucleic acid biomarkers have been widely used to detect various viral-associated diseases, including the recent pandemic COVID-19. The CRISPR-Cas-based trans-activating phenomenon has shown excellent potential for developing sensitive and selective detection of nucleic acids. However, the nucleic acid amplification steps are typically required when sensitive and selective monitoring of the target nucleic acid is needed. To overcome the aforementioned challenges, we developed a CRISPR-Cas12a-based nucleic acid amplification-free biosensor by a surface-enhanced Raman spectroscopy (SERS)-assisted ultrasensitive detection system. We integrated the activated CRISPR-Cas12a by viral DNA with a Raman-sensitive system composed of ssDNA-immobilized Raman probe-functionalized Au nanoparticles (RAuNPs) on the graphene oxide (GO)/triangle Au nanoflower array. Using this CRISPR-based Raman-sensitive system improved the detection sensitivity of the multiviral DNAs such as hepatitis B virus (HBV), human papillomavirus 16 (HPV-16), and HPV-18 with an extremely low detection limit and vast detection range from 1 aM to 100 pM without the amplification steps. We suggest that this ultrasensitive amplification-free detection system for nucleic acids can be widely applied to the precise and early diagnosis of viral infections, cancers, and several genetic diseases.},
}
@article {pmid34369095,
year = {2021},
author = {Shaikh, KM and Odaneth, AA},
title = {Metabolic engineering of Yarrowia lipolytica for the production of isoprene.},
journal = {Biotechnology progress},
volume = {37},
number = {6},
pages = {e3201},
doi = {10.1002/btpr.3201},
pmid = {34369095},
issn = {1520-6033},
mesh = {Butadienes/*metabolism ; CRISPR-Cas Systems ; Gene Editing ; Hemiterpenes/*metabolism ; Metabolic Engineering/*methods ; Mevalonic Acid/metabolism ; *Yarrowia/genetics/metabolism ; },
abstract = {Yarrowia lipolytica has recently emerged as a prominent microbial host for production of terpenoids. Its robust metabolism and growth in wide range of substrates offer several advantages at industrial scale. In the present study, we investigate the metabolic potential of Y. lipolytica to produce isoprene. Sustainable production of isoprene has been attempted through engineering several microbial hosts; however, the engineering studies performed so far are challenged with low titers. Engineering of Y. lipolytica, which have inherent high acetyl-CoA flux could fuel precursors into the biosynthesis of isoprene and thus is an approach that would offer sustainable production opportunities. The present work, therefore, explores this opportunity wherein a codon-optimized IspS gene (single copy) of Pueraria montana was integrated into the Y. lipolytica genome. With no detectable isoprene level during the growth or stationary phase of modified strain, attempts were made to overexpress enzymes from MVA pathway. GC-FID analyses of gas collected during stationary phase revealed that engineered strains were able to produce detectable isoprene only after overexpressing HMGR (or tHMGR). The significant role of HMGR (tHMGR) in diverting the pathway flux toward DMAPP is thus highlighted in our study. Nevertheless, the final recombinant strains overexpressing HMGR (tHMGR) along with Erg13 and IDI showed isoprene titers of ~500 μg/L and yields of ~80 μg/g. Further characterization of the recombinant strains revealed high lipid and squalene content compared to the unmodified strain. Overall, the preliminary results of our laboratory-scale studies represent Y. lipolytica as a promising host for fermentative production of isoprene.},
}
@article {pmid34367194,
year = {2021},
author = {Razzaq, A and Kaur, P and Akhter, N and Wani, SH and Saleem, F},
title = {Next-Generation Breeding Strategies for Climate-Ready Crops.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {620420},
pmid = {34367194},
issn = {1664-462X},
abstract = {Climate change is a threat to global food security due to the reduction of crop productivity around the globe. Food security is a matter of concern for stakeholders and policymakers as the global population is predicted to bypass 10 billion in the coming years. Crop improvement via modern breeding techniques along with efficient agronomic practices innovations in microbiome applications, and exploiting the natural variations in underutilized crops is an excellent way forward to fulfill future food requirements. In this review, we describe the next-generation breeding tools that can be used to increase crop production by developing climate-resilient superior genotypes to cope with the future challenges of global food security. Recent innovations in genomic-assisted breeding (GAB) strategies allow the construction of highly annotated crop pan-genomes to give a snapshot of the full landscape of genetic diversity (GD) and recapture the lost gene repertoire of a species. Pan-genomes provide new platforms to exploit these unique genes or genetic variation for optimizing breeding programs. The advent of next-generation clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) systems, such as prime editing, base editing, and de nova domestication, has institutionalized the idea that genome editing is revamped for crop improvement. Also, the availability of versatile Cas orthologs, including Cas9, Cas12, Cas13, and Cas14, improved the editing efficiency. Now, the CRISPR/Cas systems have numerous applications in crop research and successfully edit the major crop to develop resistance against abiotic and biotic stress. By adopting high-throughput phenotyping approaches and big data analytics tools like artificial intelligence (AI) and machine learning (ML), agriculture is heading toward automation or digitalization. The integration of speed breeding with genomic and phenomic tools can allow rapid gene identifications and ultimately accelerate crop improvement programs. In addition, the integration of next-generation multidisciplinary breeding platforms can open exciting avenues to develop climate-ready crops toward global food security.},
}
@article {pmid34367143,
year = {2021},
author = {Majumder, S and Jugovic, I and Saul, D and Bell, L and Hundhausen, N and Seal, R and Beilhack, A and Rosenwald, A and Mougiakakos, D and Berberich-Siebelt, F},
title = {Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9[+] T Cells.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {683631},
pmid = {34367143},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Cas Systems/*genetics/immunology ; Electroporation ; *Gene Editing ; Graft vs Host Disease/genetics/*immunology/metabolism ; Hematopoietic Stem Cell Transplantation/adverse effects ; Interferon Regulatory Factors/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NFATC Transcription Factors/genetics/metabolism ; T-Lymphocytes/*immunology ; },
abstract = {Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3[+] T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9[+]CD3[+] T cells, CD4[+] and CD8[+] conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9[+]CD3[+] T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.},
}
@article {pmid34365511,
year = {2021},
author = {Liu, M and Zhang, W and Xin, C and Yin, J and Shang, Y and Ai, C and Li, J and Meng, FL and Hu, J},
title = {Global detection of DNA repair outcomes induced by CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {49},
number = {15},
pages = {8732-8742},
pmid = {34365511},
issn = {1362-4962},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cells, Cultured ; Computer Simulation ; *DNA Repair ; *Gene Editing ; Humans ; Mice ; Plasmids/genetics ; Sequence Deletion ; Translocation, Genetic ; },
abstract = {CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.},
}
@article {pmid34365302,
year = {2021},
author = {Ryu, AJ and Jeong, BR and Kang, NK and Jeon, S and Sohn, MG and Yun, HJ and Lim, JM and Jeong, SW and Park, YI and Jeong, WJ and Park, S and Chang, YK and Jeong, KJ},
title = {Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.},
journal = {Bioresource technology},
volume = {340},
number = {},
pages = {125676},
doi = {10.1016/j.biortech.2021.125676},
pmid = {34365302},
issn = {1873-2976},
mesh = {*CRISPR-Cas Systems/genetics ; Fatty Acid Desaturases/genetics/metabolism ; Genetic Engineering ; *Stramenopiles/genetics/metabolism ; Transgenes ; },
abstract = {Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.},
}
@article {pmid34364918,
year = {2021},
author = {Sun, B and Chen, H and Gao, X},
title = {Versatile modification of the CRISPR/Cas9 ribonucleoprotein system to facilitate in vivo application.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {337},
number = {},
pages = {698-717},
doi = {10.1016/j.jconrel.2021.08.007},
pmid = {34364918},
issn = {1873-4995},
mesh = {*CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; RNA, Guide/genetics ; *Ribonucleoproteins/genetics ; },
abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.},
}
@article {pmid34363850,
year = {2021},
author = {Huang, X and Zhang, F and Zhu, K and Lin, W and Ma, W},
title = {dsmCRISPR: Dual synthetic mismatches CRISPR/Cas12a-based detection of SARS-CoV-2 D614G mutation.},
journal = {Virus research},
volume = {304},
number = {},
pages = {198530},
pmid = {34363850},
issn = {1872-7492},
mesh = {*COVID-19/diagnosis/virology ; CRISPR-Cas Systems ; Humans ; Mutation ; *Pandemics ; *SARS-CoV-2/genetics/isolation &amp; purification ; Spike Glycoprotein, Coronavirus/*genetics ; },
abstract = {Fast evolving of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has caused the spreading of COVID-19 disease rapidly around the globe. The mutation, especially in the gene encoding spike protein has helped the virus adapt and evade human immune system, as well as affect the efficacy of the immunizations and treatments. SARS-CoV-2 variant carrying D614G amino acid change at the spike protein is the most dominant strain in the pandemic. Therefore, efficient detection of the SARS-CoV-2 variants including D614G mutation is critical to control the COVID-19 pandemic. Herein, we report a dual synthetic mismatches CRISPR/Cas12a (dsmCRISPR) method to detect the SARS-CoV-2 D614G mutation with high sensitivity and specificity. By targeting SARS-CoV-2 D614G mutation, synthetic mismatch crRNAs were designed from -3 to +3 position around the mutation site. To improve the sensitivity and specificity, a synthetic mismatch primer with a 3'-terminal base complementary to the D614G point mutation and a mismatch next to 3'-terminal base was used to specifically amplify the D614G mutation site with higher annealing temperature. Using synthetic mismatch crRNA-(-1), a higher ratio (13.45) of the fluorescence between G614 and D614 was observed. When combined with mismatch primer to amplify D614G mutation, the fluorescence ratio of G614/D164 template detected was increased by 73.53% to 23.12. This method can detect the SARS-CoV-2 D614G mutation nucleic acid with high sensitivity, which was validated with synthetic SARS-CoV-2 D614G RNA. Therefore, the dsmCRIPSR method has significant potential to serve as a sensitive and specific assay for SARS-CoV-2 D614G detection and could be further extended for the detection of other SARS-CoV-2 variants of interest.},
}
@article {pmid34363067,
year = {2022},
author = {Bier, E},
title = {Gene drives gaining speed.},
journal = {Nature reviews. Genetics},
volume = {23},
number = {1},
pages = {5-22},
pmid = {34363067},
issn = {1471-0064},
support = {R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Gene Drive Technology/*methods/trends ; Gene Editing/*methods/trends ; Genetics, Population/*methods ; Humans ; Models, Genetic ; Mutation ; RNA, Guide/*genetics/metabolism ; },
abstract = {Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR-Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.},
}
@article {pmid34363036,
year = {2022},
author = {O'Keeffe Ahern, J and Lara-Sáez, I and Zhou, D and Murillas, R and Bonafont, J and Mencía, &#xc1; and García, M and Manzanares, D and Lynch, J and Foley, R and Xu, Q and Sigen, A and Larcher, F and Wang, W},
title = {Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.},
journal = {Gene therapy},
volume = {29},
number = {3-4},
pages = {157-170},
pmid = {34363036},
issn = {1476-5462},
mesh = {*CRISPR-Cas Systems ; Collagen Type VII/genetics ; *Epidermolysis Bullosa Dystrophica/genetics/metabolism/therapy ; Gene Editing ; HEK293 Cells ; Humans ; Polymers/metabolism ; },
abstract = {Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.},
}
@article {pmid34362733,
year = {2021},
author = {Zhang, C and Lu, T and Zhang, Y and Li, J and Tarique, I and Wen, F and Chen, A and Wang, J and Zhang, Z and Zhang, Y and Shi, DL and Shao, M},
title = {Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish.},
journal = {Science advances},
volume = {7},
number = {32},
pages = {},
pmid = {34362733},
issn = {2375-2548},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Gene Editing ; Oocytes ; RNA, Guide/genetics ; *Zebrafish/genetics ; },
abstract = {Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site-spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.},
}
@article {pmid34360895,
year = {2021},
author = {Matboli, M and Kamel, MM and Essawy, N and Bekhit, MM and Abdulrahman, B and Mohamed, GF and Eissa, S},
title = {Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {22},
number = {15},
pages = {},
pmid = {34360895},
issn = {1422-0067},
mesh = {Adult ; Aged ; Aged, 80 and over ; *CRISPR-Cas Systems ; Case-Control Studies ; Cell Line ; Computational Biology/*methods ; Diabetes Mellitus, Type 2/blood/*genetics ; Female ; Gene Editing/*methods ; *Gene Expression ; Gene Regulatory Networks ; Hospitals, University ; Humans ; Insulin Resistance/*genetics ; Lymphocytes/metabolism ; Male ; MicroRNAs/*genetics ; Middle Aged ; RNA, Long Noncoding/*genetics ; RNA, Messenger/*genetics ; },
abstract = {BACKGROUND: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients', matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence.<br><br>RESULTS: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing.<br><br>CONCLUSIONS: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.},
}
@article {pmid34360856,
year = {2021},
author = {Kumar, A and Anju, T and Kumar, S and Chhapekar, SS and Sreedharan, S and Singh, S and Choi, SR and Ramchiary, N and Lim, YP},
title = {Integrating Omics and Gene Editing Tools for Rapid Improvement of Traditional Food Plants for Diversified and Sustainable Food Security.},
journal = {International journal of molecular sciences},
volume = {22},
number = {15},
pages = {},
pmid = {34360856},
issn = {1422-0067},
mesh = {Food Security/*methods ; Gene Editing ; Genomics/methods ; Humans ; Metabolomics ; Plants, Edible/chemistry/*genetics/*metabolism ; Proteomics ; },
abstract = {Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. Traditional food plants are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in situations such as COVID-19 and in times of other pandemics. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realizing the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of TFPs for their improvements, cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in several TFPs. This suggests that there is ample scope for improvement of TFPs for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.},
}
@article {pmid34360757,
year = {2021},
author = {Santa-Inez, DC and Fuziwara, CS and Saito, KC and Kimura, ET},
title = {Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer.},
journal = {International journal of molecular sciences},
volume = {22},
number = {15},
pages = {},
pmid = {34360757},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cell Movement/genetics ; Cell Survival/genetics ; *Gene Editing ; *Gene Targeting ; Heterografts ; Humans ; Mice ; *MicroRNAs/biosynthesis/genetics ; Neoplasm Transplantation ; *RNA, Neoplasm/biosynthesis/genetics ; *Thyroid Carcinoma, Anaplastic/genetics/metabolism/pathology ; *Thyroid Neoplasms/genetics/metabolism/pathology ; },
abstract = {Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.},
}
@article {pmid34360584,
year = {2021},
author = {Choi, SH and Lee, MH and Jin, DM and Ju, SJ and Ahn, WS and Jie, EY and Lee, JM and Lee, J and Kim, CY and Kim, SW},
title = {TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.},
journal = {International journal of molecular sciences},
volume = {22},
number = {15},
pages = {},
pmid = {34360584},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Cell Division ; Gene Editing/*methods ; Genome, Plant ; Hydroxamic Acids/*pharmacology ; Lettuce/drug effects/genetics/growth &amp; development/*metabolism ; Plant Cells ; Plant Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Protein Synthesis Inhibitors/pharmacology ; Protoplasts/drug effects/*metabolism ; Tobacco/drug effects/genetics/growth &amp; development/*metabolism ; },
abstract = {Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.},
}
@article {pmid34359875,
year = {2021},
author = {Arnst, N and Belio-Mairal, P and García-González, L and Arnaud, L and Greetham, L and Nivet, E and Rivera, S and Dityatev, A},
title = {Deficiency in MT5-MMP Supports Branching of Human iPSCs-Derived Neurons and Reduces Expression of GLAST/S100 in iPSCs-Derived Astrocytes.},
journal = {Cells},
volume = {10},
number = {7},
pages = {},
pmid = {34359875},
issn = {2073-4409},
mesh = {Action Potentials/physiology ; Amyloid beta-Protein Precursor/genetics/metabolism ; Astrocytes/cytology/*metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Excitatory Amino Acid Transporter 1/*genetics/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Matrix Metalloproteinases, Membrane-Associated/deficiency/*genetics ; Neural Stem Cells/cytology/*metabolism ; Neurons/cytology/*metabolism ; Patch-Clamp Techniques ; S100 Calcium Binding Protein beta Subunit/*genetics/metabolism ; Signal Transduction ; },
abstract = {For some time, it has been accepted that the β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the β-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer's disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100β expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.},
}
@article {pmid34359846,
year = {2021},
author = {Abaandou, L and Quan, D and Shiloach, J},
title = {Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes.},
journal = {Cells},
volume = {10},
number = {7},
pages = {},
pmid = {34359846},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems ; Cell Adhesion ; Cell Engineering/*methods ; Cell Proliferation/*genetics ; Clone Cells ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Vectors/chemistry/metabolism ; HEK293 Cells ; Humans ; Recombinant Proteins/biosynthesis/genetics ; Transfection ; *Transgenes ; },
abstract = {The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line's most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.},
}
@article {pmid34359825,
year = {2021},
author = {Granados-Riveron, JT and Aquino-Jarquin, G},
title = {CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs.},
journal = {Cells},
volume = {10},
number = {7},
pages = {},
pmid = {34359825},
issn = {2073-4409},
mesh = {Animals ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Colorimetry ; Electrochemistry ; Genetic Engineering ; Humans ; MicroRNAs/*analysis/genetics ; },
abstract = {MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.},
}
@article {pmid34359076,
year = {2022},
author = {Messling, JE and Agger, K and Andersen, KL and Kromer, K and Kuepper, HM and Lund, AH and Helin, K},
title = {Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.},
journal = {Blood},
volume = {139},
number = {2},
pages = {245-255},
pmid = {34359076},
issn = {1528-0020},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Adenosine Triphosphatases/antagonists &amp; inhibitors/genetics/metabolism ; Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Gene Expression Regulation, Leukemic/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Leukemia, Myeloid, Acute/*drug therapy/genetics/*metabolism ; Mice ; Molecular Targeted Therapy ; Protein Biosynthesis/drug effects ; Protein Kinase Inhibitors/*pharmacology ; },
abstract = {Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed, because current treatments do not cure most patients with AML. We report a domain-focused, kinome-wide CRISPR-Cas9 screening that identified protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. Loss of RIOK2 led to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, the ATPase function of RIOK2 was necessary for cell survival. When a small-molecule inhibitor was used, pharmacological inhibition of RIOK2 similarly led to loss of protein synthesis and apoptosis and affected leukemic cell growth in vivo. Our results provide proof of concept for targeting RIOK2 as a potential treatment of patients with AML.},
}
@article {pmid34359002,
year = {2021},
author = {Schwarz, L and Casadei, N and Fitzgerald, JC},
title = {Generation of R272Q, S156A and K572R RHOT1/Miro1 point mutations in iPSCs from a healthy individual using FACS-assisted CRISPR/Cas9 genome editing.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102469},
doi = {10.1016/j.scr.2021.102469},
pmid = {34359002},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Mitochondrial Proteins/genetics ; *Point Mutation ; Protein Kinases/genetics ; Ubiquitin-Protein Ligases/genetics ; rho GTP-Binding Proteins/metabolism ; },
abstract = {The GTPase Miro1 is tail anchored into the mitochondrial outer membrane and tethers mitochondria to molecular motors which is crucial for mitochondrial transport. Miro1 contains two EF hand, ion binding domains important for calcium sequestration. Miro1 is associated with Parkinson's disease (PD) due to its suggested interaction with PINK1 and Parkin. Rare variants in RHOT1 (encoding Miro1) were found in PD patients but Miro1's function in the brain is understudied. We gene edited three point mutations in healthy iPSCS EF hand R272Q was identified in a PD patient, S156A abolishes the proposed PINK1 phosphorylation site, K572R abolishes the main lysine targeted by pSer65-parkin.},
}
@article {pmid34358458,
year = {2021},
author = {Chatterjee, P},
title = {BARBEKO'ing in the lab: Versatile CRISPR screens with barcoded base editors.},
journal = {Molecular cell},
volume = {81},
number = {15},
pages = {3046-3047},
doi = {10.1016/j.molcel.2021.07.007},
pmid = {34358458},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine ; *Gene Editing ; Reproducibility of Results ; },
abstract = {Xu et al. (2021) describe a novel two-pronged CRISPR screen, termed BARBEKO, by coupling cytosine base editors and internally barcoded sgRNAs to eliminate double-stranded break-induced toxicity, enable high multiplicities of infection, and ensure experimental reproducibility.},
}
@article {pmid34357544,
year = {2022},
author = {Haghighi, N and Doosti, A and Kiani, J},
title = {Evaluation of CRISPR/Cas9 System Effects on Knocking Out NEAT1 Gene in AGS Gastric Cancer Cell Line with Therapeutic Perspective.},
journal = {Journal of gastrointestinal cancer},
volume = {53},
number = {3},
pages = {623-631},
pmid = {34357544},
issn = {1941-6636},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; Humans ; *MicroRNAs/genetics ; RNA, Long Noncoding ; *Stomach Neoplasms/genetics/metabolism/therapy ; },
abstract = {AIM: Gastric cancer (GC) is one of the most common malignant tumors globally, with an increasing incidence rate. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNAs) responsible for regulating cell cycle progression, apoptosis, cell growth, proliferation, and migration in various cells. The present survey was performed to assess the effects of NEAT1 gene knocking out by CRISPR/Cas9 system in human gastric cancer cells.<br><br>METHODS: The CRISPR/Cas9 genome editing technique was used to knockout NEAT1 in AGS cells as a gastric cancer model. After the design and construction of the vector, transfection was performed. The expression levels of mRNA, the survival of cells, apoptosis, and cell migration were evaluated by real-time quantitative polymerase chain reaction, flow cytometry, and scratch wound.<br><br>RESULTS: Degradation of NEAT1 by CRISPR/cas9 significantly suppressed the gene's expression rate, arrested cell cycle in the G0/G1 phase, and a significant reduction in cell number in the S phase (P&#x2009;<&#x2009;0.05). Degradation of NEAT1 by CRISPR/cas9 also restrained the ability to migrate in transfected cells compared to the control group (P&#x2009;<&#x2009;0.01). Knockout of NEAT1 via impact on miR-34a gene expression induced apoptosis of AGS cells (P&#x2009;<&#x2009;0.05) with increasing in the FAS level and total apoptosis (P&#x2009;<&#x2009;0.001).<br><br>CONCLUSIONS: Findings suggest that NEAT1 plays a vital role in cellular mechanisms of GC's occurrence and can serve as a new treatment target in GC.},
}
@article {pmid34356991,
year = {2021},
author = {Maradiaga, ODH and Mok, PL and Sivapragasam, G and Samrot, AV and Ali Khan, MS and Farhana, A and Alzahrani, B and Tong, J and Karuppiah, T and Joseph, NMS and Subbiah, SK},
title = {Lipofection of Single Guide RNA Targeting MMP8 Decreases Proliferation and Migration in Lung Adenocarcinoma Cells.},
journal = {Medicina (Kaunas, Lithuania)},
volume = {57},
number = {7},
pages = {},
pmid = {34356991},
issn = {1648-9144},
mesh = {*Adenocarcinoma of Lung/genetics ; *Carcinoma, Non-Small-Cell Lung ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Humans ; *Lung Neoplasms/genetics ; Matrix Metalloproteinase 8 ; Neoplasm Invasiveness ; RNA, Guide ; },
abstract = {Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.},
}
@article {pmid34356775,
year = {2021},
author = {Wegener, A and Broens, EM and van der Graaf-van Bloois, L and Zomer, AL and Visser, CE and van Zeijl, J and van der Meer, C and Kusters, JG and Friedrich, AW and Kampinga, GA and Sips, GJ and Smeets, L and van Kerckhoven, MEJ and Timmerman, AJ and Wagenaar, JA and Duim, B},
title = {Absence of Host-Specific Genes in Canine and Human Staphylococcus pseudintermedius as Inferred from Comparative Genomics.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {7},
pages = {},
pmid = {34356775},
issn = {2079-6382},
abstract = {Staphylococcus pseudintermedius is an important pathogen in dogs that occasionally causes infections in humans as an opportunistic pathogen of elderly and immunocompromised people. This study compared the genomic relatedness and antimicrobial resistance genes using genome-wide association study (GWAS) to examine host association of canine and human S. pseudintermedius isolates. Canine (n = 25) and human (n = 32) methicillin-susceptible S. pseudintermedius (MSSP) isolates showed a high level of genetic diversity with an overrepresentation of clonal complex CC241 in human isolates. This clonal complex was associated with carriage of a plasmid containing a bacteriocin with cytotoxic properties, a CRISPR-cas domain and a pRE25-like mobile element containing five antimicrobial resistance genes. Multi-drug resistance (MDR) was predicted in 13 (41%) of human isolates and 14 (56%) of canine isolates. CC241 represented 54% of predicted MDR isolates from humans and 21% of predicted MDR canine isolates. While it had previously been suggested that certain host-specific genes were present the current GWAS analysis did not identify any genes that were significantly associated with human or canine isolates. In conclusion, this is the first genomic study showing that MSSP is genetically diverse in both hosts and that multidrug resistance is important in dog and human-associated S. pseudintermedius isolates.},
}
@article {pmid34356045,
year = {2021},
author = {Kerstens, M and Hesen, V and Yalamanchili, K and Bimbo, A and Grigg, S and Opdenacker, D and Beeckman, T and Heidstra, R and Willemsen, V},
title = {Nature and Nurture: Genotype-Dependent Differential Responses of Root Architecture to Agar and Soil Environments.},
journal = {Genes},
volume = {12},
number = {7},
pages = {},
pmid = {34356045},
issn = {2073-4425},
mesh = {*Agar ; Arabidopsis/genetics ; Arabidopsis Proteins/genetics ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; *Genotype ; Phenotype ; Plant Roots/*genetics/*growth &amp; development ; *Soil ; Transcription Factors/genetics ; },
abstract = {Root development is crucial for plant growth and therefore a key factor in plant performance and food production. Arabidopsis thaliana is the most commonly used system to study root system architecture (RSA). Growing plants on agar-based media has always been routine practice, but this approach poorly reflects the natural situation, which fact in recent years has led to a dramatic shift toward studying RSA in soil. Here, we directly compare RSA responses to agar-based medium (plates) and potting soil (rhizotrons) for a set of redundant loss-of-function plethora (plt) CRISPR mutants with variable degrees of secondary root defects. We demonstrate that plt3plt7 and plt3plt5plt7 plants, which produce only a handful of emerged secondary roots, can be distinguished from other genotypes based on both RSA shape and individual traits on plates and rhizotrons. However, in rhizotrons the secondary root density and the total contribution of the side root system to the RSA is increased in these two mutants, effectively rendering their phenotypes less distinct compared to WT. On the other hand, plt3, plt3plt5, and plt5plt7 mutants showed an opposite effect by having reduced secondary root density in rhizotrons. This leads us to believe that plate versus rhizotron responses are genotype dependent, and these differential responses were also observed in unrelated mutants short-root and scarecrow. Our study demonstrates that the type of growth system affects the RSA differently across genotypes, hence the optimal choice of growth conditions to analyze RSA phenotype is not predetermined.},
}
@article {pmid34356043,
year = {2021},
author = {You, W and Li, M and Qi, Y and Wang, Y and Chen, Y and Liu, Y and Li, L and Ouyang, H and Pang, D},
title = {CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus.},
journal = {Genes},
volume = {12},
number = {7},
pages = {},
pmid = {34356043},
issn = {2073-4425},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics ; Fatty Acids/analysis ; Gene Editing/methods ; Gene Knock-In Techniques ; Gene Targeting/methods ; Genotype ; Insulin-Like Growth Factor I/*genetics ; Phenotype ; Swine ; },
abstract = {Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.},
}
@article {pmid34355716,
year = {2021},
author = {Cai, Q and Wang, R and Qiao, Z and Yang, W},
title = {Single-digit Salmonella detection with the naked eye using bio-barcode immunoassay coupled with recombinase polymerase amplification and a CRISPR-Cas12a system.},
journal = {The Analyst},
volume = {146},
number = {17},
pages = {5271-5279},
doi = {10.1039/d1an00717c},
pmid = {34355716},
issn = {1364-5528},
mesh = {CRISPR-Cas Systems ; Gold ; Immunoassay ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques ; *Recombinases/genetics ; Salmonella typhimurium/genetics ; },
abstract = {The ability to visually detect low numbers of Salmonella in food samples is highly valuable but remains a challenge. Here we present a novel platform for ultrasensitive and visual detection of Salmonella Typhimurium by integrating the bio-barcode immunoassay (BCA), recombinase polymerase amplification (RPA), and CRISPR-Cas12a cleavage in a single reaction system (termed as BCA-RPA-Cas12a). In the system, the target bacteria were separated by immunomagnetic nanoparticles and labeled with numerous barcode AuNPs, which carry abundant bio-barcode DNA molecules to amplify the signal. Afterwards, the bio-barcode DNA molecules were amplified by RPA and subsequently triggered the cleavage activity of Cas12a to generate the fluorescence signal. Due to this triplex signal amplification, the BCA-RPA-Cas12a system can selectively detect Salmonella Typhimurium at the single-digit level with the naked eye under blue light within 60 min. Meanwhile, this novel platform was successfully applied to detect Salmonella Typhimurium in spiked milk samples with a similar sensitivity and satisfactory recovery, indicating its potential application in real samples. Furthermore, in virtue of the versatility of the antibody in the stage of BCA, the BCA-RPA-Cas12a system can be extended to further application in other bacteria detection and food safety monitoring.},
}
@article {pmid34354262,
year = {2021},
author = {Liu, TY and Knott, GJ and Smock, DCJ and Desmarais, JJ and Son, S and Bhuiya, A and Jakhanwal, S and Prywes, N and Agrawal, S and Díaz de León Derby, M and Switz, NA and Armstrong, M and Harris, AR and Charles, EJ and Thornton, BW and Fozouni, P and Shu, J and Stephens, SI and Kumar, GR and Zhao, C and Mok, A and Iavarone, AT and Escajeda, AM and McIntosh, R and Kim, S and Dugan, EJ and , and Pollard, KS and Tan, MX and Ott, M and Fletcher, DA and Lareau, LF and Hsu, PD and Savage, DF and Doudna, JA},
title = {Accelerated RNA detection using tandem CRISPR nucleases.},
journal = {Nature chemical biology},
volume = {17},
number = {9},
pages = {982-988},
pmid = {34354262},
issn = {1552-4469},
mesh = {COVID-19/*genetics ; CRISPR-Cas Systems/*genetics ; Humans ; RNA, Viral/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/*genetics ; },
abstract = {Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.},
}
@article {pmid34353334,
year = {2021},
author = {Li, X and Guo, M and Hou, B and Zheng, B and Wang, Z and Huang, M and Xu, Y and Chang, J and Wang, T},
title = {CRISPR/Cas9 nanoeditor of double knockout large fragments of E6 and E7 oncogenes for reversing drugs resistance in cervical cancer.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {231},
pmid = {34353334},
issn = {1477-3155},
mesh = {Animals ; Apoptosis/drug effects ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Drug Resistance, Neoplasm ; Female ; *Gene Knockout Techniques ; Gene Targeting ; Genetic Therapy ; HeLa Cells ; Humans ; Mice ; Mice, Nude ; Nanomedicine ; Nanoparticles ; Oncogenes/*genetics ; Uterine Cervical Neoplasms/genetics/*therapy ; },
abstract = {Drug resistance of tumor cells is always a headache problem in clinical treatment. In order to combat chemotherapy-resistance in cervical cancer and improve treatment effect, we design a CRISPR/Cas9 nanoeditor to knock out two key oncogenes E6 and E7 that lead to drug tolerance. Meanwhile, the deletion of these two oncogenes can effectively reactivate p53 and pRB signaling pathways that inhibit the growth of tumor cells. Our results demonstrated the nanoeditor could simultaneously delete two oncogenes, and the size of DNA fragments knocked out reaches an unprecedented 563 bp. After the preparation of cationic liposomes combined with chemotherapy drug docetaxel (DOC), this nanosystem can significantly inhibit the drug tolerance of cancer cells and improve the therapeutic effect of cervical cancer. Therefore, this study provides a promising strategy for the treatment of cervical cancer by combining chemotherapy and double-target gene therapy. This strategy can also be applied in other disease models to customize personalized anti-tumor strategies by simply changing chemotherapy drugs and targeted genes.},
}
@article {pmid34353330,
year = {2021},
author = {Jiang, Q and Zheng, N and Bu, L and Zhang, X and Zhang, X and Wu, Y and Su, Y and Wang, L and Zhang, X and Ren, S and Dai, X and Wu, D and Xie, W and Wei, W and Zhu, Y and Guo, J},
title = {SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions.},
journal = {Molecular cancer},
volume = {20},
number = {1},
pages = {100},
pmid = {34353330},
issn = {1476-4598},
mesh = {3-Phosphoinositide-Dependent Protein Kinases/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Transformation, Neoplastic/*metabolism ; Disease Models, Animal ; Glycogen Synthase Kinase 3/metabolism ; Heterografts ; Humans ; Mice ; Models, Biological ; Mutation ; Nuclear Proteins/genetics/*metabolism ; Phosphorylation ; Protein Binding ; Proteolysis ; Proto-Oncogene Proteins c-akt/*metabolism ; Repressor Proteins/genetics/*metabolism ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination ; },
abstract = {BACKGROUND: 3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet.<br><br>METHOD: A clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models.<br><br>RESULTS: We identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3&#x3b2;-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3&#x3b2;-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase.<br><br>CONCLUSIONS: Our results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.},
}
@article {pmid34352617,
year = {2021},
author = {Ma, S and Xu, Q and Bai, R and Dong, T and Peng, Z and Liu, X},
title = {Generation of a TPM1 homozygous knockout embryonic stem cell line by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102470},
doi = {10.1016/j.scr.2021.102470},
pmid = {34352617},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; Mice ; Tropomyosin/genetics ; },
abstract = {Alpha-Tropomyosin (TPM1) plays a crucial role in actin regulation and stability and contributes fundamental functions to heart development: without TPM1 expressing, mice embryos will die early in embryogenesis. To further identify the role of TPM1 in human cardiac development, here we generated a homozygous TPM1 knockout (TPM1[-/-]) human embryonic stem cell (hESC) line using CRISPR/Cas9-based genome editing system. The generated TPM1[-/-] hESC line maintained normal karyotype, highly expressed pluripotency markers and was able to differentiate into all three germ layers in vivo. This cell line provides a powerful tool to investigate the role of TPM1 in heart development in future.},
}
@article {pmid34351541,
year = {2021},
author = {Li, P and Li, X and Jiang, M},
title = {CRISPR/Cas9-mediated mutagenesis of WRKY3 and WRKY4 function decreases salt and Me-JA stress tolerance in Arabidopsis thaliana.},
journal = {Molecular biology reports},
volume = {48},
number = {8},
pages = {5821-5832},
pmid = {34351541},
issn = {1573-4978},
mesh = {Arabidopsis/genetics/metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Droughts ; Gene Expression/genetics ; Gene Expression Regulation, Plant/genetics ; Genetic Engineering/methods ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; Polymorphism, Single Nucleotide/genetics ; Salt Stress/*genetics/physiology ; Salt Tolerance/genetics ; Sodium Chloride/metabolism ; Stress, Physiological/genetics ; Transcription Factors/genetics/*metabolism ; },
abstract = {BACKGROUND: WRKY transcription factor is involved in regulation of plant growth and development, response to biotic and abiotic stresses, including homologous WRKY3 and WRKY4 genes which play a vital role in regulating plants defense response to pathogen and drought stress.<br><br>METHODS AND RESULTS: To investigate the function of AtWRKY3 and AtWRKY4 genes in regulating salt and Me-JA stresses, the loss-of-function mutations were generated by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system in Arabidopsis thaliana. Several independent transgenic lines with single or double mutations were obtained via Agrobacterium-mediated transformation. The knockout lines of AtWRKY3 and AtWRKY4 genes were successfully achieved and confirmed by qRT-PCR technology. Expression analysis showed that AtWRKY3 and AtWRKY4 genes had significantly up-regulated under salt and Me-JA stresses. The growth of double mutant plants under salt or Me-JA stresses were significantly inhibited compared with corresponding wild type (WT) plants, especially their root lengths. Moreover, the double mutant plants displayed salt and Me-JA sensitivity phenotypic characteristics, such as the increased relative electrolyte leakage (REL) and a substantial reduce in the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities.<br><br>CONCLUSION: Taken together, these data suggested that the simultaneous modification of homologous gene copies of WRKY are established using CRISPR/Cas9 system in A. thaliana and the loss of AtWRKY3 and AtWRKY4 has an effect on ROS scavenging pathways to reduce stress tolerance.},
}
@article {pmid34350049,
year = {2021},
author = {Sioson, VA and Kim, M and Joo, J},
title = {Challenges in delivery systems for CRISPR-based genome editing and opportunities of nanomedicine.},
journal = {Biomedical engineering letters},
volume = {11},
number = {3},
pages = {217-233},
pmid = {34350049},
issn = {2093-985X},
abstract = {The CRISPR-based genome editing technology has opened extremely useful strategies in biological research and clinical therapeutics, thus attracting great attention with tremendous progress in the past decade. Despite its robust potential in personalized and precision medicine, the CRISPR-based gene editing has been limited by inefficient in vivo delivery to the target cells and by safety concerns of viral vectors for clinical setting. In this review, recent advances in tailored nanoparticles as a means of non-viral delivery vector for CRISPR/Cas systems are thoroughly discussed. Unique characteristics of the nanoparticles including controllable size, surface tunability, and low immune response lead considerable potential of CRISPR-based gene editing as a translational medicine. We will present an overall view on essential elements in CRISPR/Cas systems and the nanoparticle-based delivery carriers including advantages and challenges. Perspectives to advance the current limitations are also discussed toward bench-to-bedside translation in engineering aspects.},
}
@article {pmid34349737,
year = {2021},
author = {Beauruelle, C and Treluyer, L and Pastuszka, A and Cochard, T and Lier, C and Mereghetti, L and Glaser, P and Poyart, C and Lanotte, P},
title = {CRISPR Typing Increases the Discriminatory Power of Streptococcus agalactiae Typing Methods.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {675597},
pmid = {34349737},
issn = {1664-302X},
abstract = {We explored the relevance of a Clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping tool for Streptococcus agalactiae typing and we compared this method to current molecular methods [multi locus sequence typing (MLST) and capsular typing]. To this effect, we developed two CRISPR marker schemes (using 94 or 25 markers, respectively). Among the 255 S. agalactiae isolates tested, 229 CRISPR profiles were obtained. The 94 and 25 markers made it possible to efficiently separate isolates with a high diversity index (0.9947 and 0.9267, respectively), highlighting a high discriminatory power, superior to that of both capsular typing and MLST (diversity index of 0.9017 for MLST). This method has the advantage of being correlated with MLST [through analysis of the terminal direct repeat (TDR) and ancestral spacers] and to possess a high discriminatory power (through analysis of the leader-end spacers recently acquired, which are the witnesses of genetic mobile elements encountered by the bacteria). Furthermore, this "one-shot" approach presents the benefit of much-reduced time and cost in comparison with MLST. On the basis of these data, we propose that this method could become a reference method for group B Streptococcus (GBS) typing.},
}
@article {pmid34349239,
year = {2022},
author = {Gao, C and Wu, P and Yu, L and Liu, L and Liu, H and Tan, X and Wang, L and Huang, X and Wang, H},
title = {The application of CRISPR/Cas9 system in cervical carcinogenesis.},
journal = {Cancer gene therapy},
volume = {29},
number = {5},
pages = {466-474},
pmid = {34349239},
issn = {1476-5500},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Female ; Human papillomavirus 16/genetics/metabolism ; Humans ; Mice ; Mice, Transgenic ; *Oncogene Proteins, Viral/genetics ; Papillomavirus E7 Proteins/genetics ; *Papillomavirus Infections/complications/genetics/therapy ; *Precancerous Conditions/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; *Uterine Cervical Neoplasms/genetics/pathology/therapy ; },
abstract = {Integration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.},
}
@article {pmid34348163,
year = {2021},
author = {Lam, AJ and Lin, DTS and Gillies, JK and Uday, P and Pesenacker, AM and Kobor, MS and Levings, MK},
title = {Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity.},
journal = {Cell reports},
volume = {36},
number = {5},
pages = {109494},
doi = {10.1016/j.celrep.2021.109494},
pmid = {34348163},
issn = {2211-1247},
support = {21738/VAC_/Versus Arthritis/United Kingdom ; FDN-154304//CIHR/Canada ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; DNA Methylation/genetics ; DNA Repair ; Dependovirus/metabolism ; Forkhead Transcription Factors/*metabolism ; *Gene Knock-In Techniques ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; Immunosuppression Therapy ; Interleukin-2/metabolism ; Lymphocyte Subsets/immunology ; Phenotype ; Plasmids/metabolism ; T-Lymphocytes, Regulatory/*immunology ; Time Factors ; Transcription, Genetic ; Transgenes ; },
abstract = {Regulatory T cell (Treg) therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knockin in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.},
}
@article {pmid34347875,
year = {2021},
author = {Kurokawa, S and Rahman, H and Yamanaka, N and Ishizaki, C and Islam, S and Aiso, T and Hirata, S and Yamamoto, M and Kobayashi, K and Kaya, H},
title = {A Simple Heat Treatment Increases SpCas9-Mediated Mutation Efficiency in Arabidopsis.},
journal = {Plant &amp; cell physiology},
volume = {62},
number = {11},
pages = {1676-1686},
doi = {10.1093/pcp/pcab123},
pmid = {34347875},
issn = {1471-9053},
mesh = {Arabidopsis/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Plant ; *Hot Temperature ; Meristem/metabolism ; Mutation ; Plants, Genetically Modified/genetics/metabolism ; Promoter Regions, Genetic ; },
abstract = {The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37°C for 24 h increased the mutation efficiency at all four target sites from 3 to 42%, 43 to 62%, 54 to 75% and 89 to 91%, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37°C for 24 h as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.},
}
@article {pmid34347520,
year = {2021},
author = {Liu, L and Yang, D and Zhang, Z and Liu, T and Hu, G and He, M and Zhao, S and Peng, N},
title = {High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {20},
pages = {e0094821},
pmid = {34347520},
issn = {1098-5336},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Fermentation ; *Gene Editing ; L-Lactate Dehydrogenase/genetics ; Lactic Acid/*metabolism ; Metabolic Engineering ; *Pediococcus acidilactici/genetics/growth &amp; development/metabolism ; Point Mutation ; },
abstract = {Pediococcus acidilactici is commonly used for pediocin production and lactic acid fermentation. However, a high-efficiency genome editing tool is unavailable for this species. In this study, we constructed endogenous subtype II-A CRISPR-Cas system-based genome interference plasmids which carried a "repeat-spacer-repeat" cassette in the pMG36e shuttle vector. These plasmids exhibited self-interference activities in P. acidilactici LA412. Then, the genome-editing plasmids were constructed by cloning the upstream/downstream donor DNA into the corresponding interference plasmids to exert high-efficiency markerless gene deletion, gene integration, and point mutation in P. acidilactici LA412. We found that endogenous CRISPR-mediated depletion of the native plasmids enhanced the cell growth and that integration of an l-lactate dehydrogenase gene into the chromosome enhanced both cell growth and lactic acid production. IMPORTANCE A rapid and precise genome editing tool will promote the practical application of Pediococcus acidilactici, one type of lactic acid bacterium with excellent stress tolerance and probiotic characteristics. This study established a high-efficiency endogenous CRISPR-Cas system-based genome editing tool for P. acidilactici and achieved different genetic manipulations, including gene deletion, gene insertion, mononucleotide mutation, and endogenous plasmid depletion. The engineered strain edited by this tool showed significant advantages in cell growth and lactic acid fermentation. Therefore, our tool can satisfy the requirements for genetic manipulations of P. acidilactici, thus making it a sophisticated chassis species for synthetic biology and bioindustry.},
}
@article {pmid34344987,
year = {2022},
author = {Yamauchi, T and Miyawaki, K and Semba, Y and Takahashi, M and Izumi, Y and Nogami, J and Nakao, F and Sugio, T and Sasaki, K and Pinello, L and Bauer, DE and Bamba, T and Akashi, K and Maeda, T},
title = {Targeting leukemia-specific dependence on the de novo purine synthesis pathway.},
journal = {Leukemia},
volume = {36},
number = {2},
pages = {383-393},
pmid = {34344987},
issn = {1476-5551},
mesh = {Animals ; Apoptosis ; *CRISPR-Cas Systems ; Carboxy-Lyases/*antagonists &amp; inhibitors ; Cell Proliferation ; Enzyme Inhibitors/*pharmacology ; Gene Expression Regulation, Enzymologic/*drug effects ; Gene Expression Regulation, Leukemic/*drug effects ; Genome-Wide Association Study ; Humans ; Leukemia, Myeloid, Acute/*drug therapy/genetics/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, SCID ; Purines/*metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival.},
}
@article {pmid34343828,
year = {2021},
author = {Schoger, E and Zimmermann, WH and Cyganek, L and Zelarayán, LC},
title = {Establishment of two homozygous CRISPR interference (CRISPRi) knock-in human induced pluripotent stem cell (hiPSC) lines for titratable endogenous gene repression.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102473},
doi = {10.1016/j.scr.2021.102473},
pmid = {34343828},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases ; Gene Expression ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Using nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types.},
}
@article {pmid34343827,
year = {2021},
author = {Liu, X and Zhang, S and Chang, Y and Wu, F and Bai, R},
title = {Establishment of a KCNQ1 homozygous knockout human embryonic stem cell line by episomal vector-based CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102467},
doi = {10.1016/j.scr.2021.102467},
pmid = {34343827},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; KCNQ1 Potassium Channel/genetics ; *Long QT Syndrome ; },
abstract = {As a member of the voltage-gated potassium ion channels, KCNQ1 plays an important role in heart physiological functions. Numerous mutations in KCNQ1 were identified as primary causes to hereditary long-QT syndrome. To further study the role of KCNQ1 in human cardiac functions, here we generated a homozygous KCNQ1 knockout human embryonic stem cell line (KCNQ1-KO) using episomal vector-based CRISPR/Cas9 system. This generated cell line presented typical stem cells colony morphology, maintained highly pluripotency and normal karyotype, also was able to differentiate into all three germ layers in vivo.},
}
@article {pmid34343484,
year = {2022},
author = {Méndez-Mancilla, A and Wessels, HH and Legut, M and Kadina, A and Mabuchi, M and Walker, J and Robb, GB and Holden, K and Sanjana, NE},
title = {Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells.},
journal = {Cell chemical biology},
volume = {29},
number = {2},
pages = {321-327.e4},
pmid = {34343484},
issn = {2451-9448},
support = {DP2 HG010099/HG/NHGRI NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; R01 GM138635/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Editing ; Humans ; RNA, Guide/chemical synthesis/chemistry/*genetics ; },
abstract = {RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation.},
}
@article {pmid34343415,
year = {2021},
author = {Brant, EJ and Baloglu, MC and Parikh, A and Altpeter, F},
title = {CRISPR/Cas9 mediated targeted mutagenesis of LIGULELESS-1 in sorghum provides a rapidly scorable phenotype by altering leaf inclination angle.},
journal = {Biotechnology journal},
volume = {16},
number = {11},
pages = {e2100237},
doi = {10.1002/biot.202100237},
pmid = {34343415},
issn = {1860-7314},
mesh = {CRISPR-Cas Systems/genetics ; Edible Grain ; Gene Editing ; Mutagenesis ; Phenotype ; Plant Leaves/genetics ; Plants, Genetically Modified/genetics ; *Sorghum/genetics ; },
abstract = {Sorghum (Sorghum bicolor L. Moench) is one of the world's most cultivated cereal crops. Biotechnology approaches have great potential to complement traditional crop improvement. Earlier studies in rice and maize revealed that LIGULELESS-1 (LG1) is responsible for formation of the ligule and auricle, which determine the leaf inclination angle. However, generation and analysis of lg1 mutants in sorghum has so far not been described. Here, we describe CRISPR/Cas9 mediated targeted mutagenesis of LG1 in sorghum and phenotypic changes in mono- and bi-allelic lg1 mutants. Genome editing reagents were co-delivered to sorghum (var. Tx430) with the nptII selectable marker via particle bombardment of immature embryos followed by regeneration of transgenic plants. Sanger sequencing confirmed a single nucleotide insertion in the sgRNA LG1 target site. Monoallelic edited plantlets displayed more upright leaves in tissue culture and after transfer to soil when compared to wild type. T1 progeny plants with biallelic lg1 mutation lacked ligules entirely and displayed a more severe reduction in leaf inclination angle than monoallelic mutants. Transgene-free lg1 mutants devoid of the genome editing vector were also recovered in the segregating T1 generation. Targeted mutagenesis of LG1 provides a rapidly scorable phenotype in tissue culture and will facilitate optimization of genome editing protocols. Altering leaf inclination angle also has the potential to elevate yield in high-density plantings.},
}
@article {pmid34342578,
year = {2021},
author = {Soveg, FW and Schwerk, J and Gokhale, NS and Cerosaletti, K and Smith, JR and Pairo-Castineira, E and Kell, AM and Forero, A and Zaver, SA and Esser-Nobis, K and Roby, JA and Hsiang, TY and Ozarkar, S and Clingan, JM and McAnarney, ET and Stone, AE and Malhotra, U and Speake, C and Perez, J and Balu, C and Allenspach, EJ and Hyde, JL and Menachery, VD and Sarkar, SN and Woodward, JJ and Stetson, DB and Baillie, JK and Buckner, JH and Gale, M and Savan, R},
title = {Endomembrane targeting of human OAS1 p46 augments antiviral activity.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34342578},
issn = {2050-084X},
support = {R01 AI108765/AI/NIAID NIH HHS/United States ; R01 AI150214/AI/NIAID NIH HHS/United States ; AI127463/NH/NIH HHS/United States ; T32 AI106677/AI/NIAID NIH HHS/United States ; T32 HL007312/HL/NHLBI NIH HHS/United States ; AI145974/NH/NIH HHS/United States ; R21 AI135437/AI/NIAID NIH HHS/United States ; AI104002/NH/NIH HHS/United States ; GM007270/NH/NIH HHS/United States ; T32 GM007270/GM/NIGMS NIH HHS/United States ; AI118916/NH/NIH HHS/United States ; AI106677/NH/NIH HHS/United States ; F31 AI140530/AI/NIAID NIH HHS/United States ; R01 AI145296/AI/NIAID NIH HHS/United States ; AI145974,AI108765,AI135437/NH/NIH HHS/United States ; R01 AI084914/AI/NIAID NIH HHS/United States ; P51 OD010425/OD/NIH HHS/United States ; AI145296/NH/NIH HHS/United States ; R01 AI118916/AI/NIAID NIH HHS/United States ; AI108765/NH/NIH HHS/United States ; AI106677,GM007270,AI140530/NH/NIH HHS/United States ; T32 HL007312/NH/NIH HHS/United States ; R56 AI145974/AI/NIAID NIH HHS/United States ; AI135437/NH/NIH HHS/United States ; AI100625/NH/NIH HHS/United States ; S10 OD026741/OD/NIH HHS/United States ; R01 AI104002/AI/NIAID NIH HHS/United States ; AI104002,AI118916,AI145296,AI127463,AI100625/NH/NIH HHS/United States ; R01 AI127463/AI/NIAID NIH HHS/United States ; AI140530/NH/NIH HHS/United States ; U19 AI100625/AI/NIAID NIH HHS/United States ; },
mesh = {2',5'-Oligoadenylate Synthetase/*metabolism ; Animals ; COVID-19/immunology/*virology ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Humans ; Polymorphism, Single Nucleotide ; SARS-CoV-2/isolation &amp; purification/*metabolism ; },
abstract = {Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.},
}
@article {pmid34341582,
year = {2021},
author = {Gemberling, MP and Siklenka, K and Rodriguez, E and Tonn-Eisinger, KR and Barrera, A and Liu, F and Kantor, A and Li, L and Cigliola, V and Hazlett, MF and Williams, CA and Bartelt, LC and Madigan, VJ and Bodle, JC and Daniels, H and Rouse, DC and Hilton, IB and Asokan, A and Ciofani, M and Poss, KD and Reddy, TE and West, AE and Gersbach, CA},
title = {Transgenic mice for in vivo epigenome editing with CRISPR-based systems.},
journal = {Nature methods},
volume = {18},
number = {8},
pages = {965-974},
pmid = {34341582},
issn = {1548-7105},
support = {R01 DA036865/DA/NIDA NIH HHS/United States ; R33 DA041878/DA/NIDA NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; F31 DA049442/DA/NIDA NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; R01 NS099371/NS/NINDS NIH HHS/United States ; R01 GM115474/GM/NIGMS NIH HHS/United States ; R01 DA047115/DA/NIDA NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Brain/metabolism ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Epigenome ; Female ; Fibroblasts/metabolism ; Gene Editing/*methods ; *Gene Expression Regulation ; Humans ; Liver/metabolism ; Male ; Mice ; Mice, Transgenic ; T-Lymphocytes/metabolism ; },
abstract = {CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.},
}
@article {pmid34341563,
year = {2021},
author = {Doerfler, PA and Feng, R and Li, Y and Palmer, LE and Porter, SN and Bell, HW and Crossley, M and Pruett-Miller, SM and Cheng, Y and Weiss, MJ},
title = {Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.},
journal = {Nature genetics},
volume = {53},
number = {8},
pages = {1177-1186},
pmid = {34341563},
issn = {1546-1718},
support = {F32 DK118822/DK/NIDDK NIH HHS/United States ; R01 HL156647/HL/NHLBI NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; R35 GM133614/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CCAAT-Binding Factor/*genetics ; COS Cells ; CRISPR-Cas Systems ; Cell Line ; Chlorocebus aethiops ; Erythroid Cells ; Fetal Hemoglobin/*genetics ; GATA1 Transcription Factor/*genetics ; Gene Editing/methods ; Gene Expression Regulation, Developmental ; Humans ; Promoter Regions, Genetic ; Repressor Proteins/genetics/metabolism ; gamma-Globins/*genetics ; },
abstract = {Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.},
}
@article {pmid34341479,
year = {2022},
author = {Pikman, Y and Ocasio-Martinez, N and Alexe, G and Dimitrov, B and Kitara, S and Diehl, FF and Robichaud, AL and Conway, AS and Ross, L and Su, A and Ling, F and Qi, J and Roti, G and Lewis, CA and Puissant, A and Vander Heiden, MG and Stegmaier, K},
title = {Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy.},
journal = {Leukemia},
volume = {36},
number = {2},
pages = {348-360},
pmid = {34341479},
issn = {1476-5551},
support = {P30 CA014051/CA/NCI NIH HHS/United States ; 758848/ERC_/European Research Council/International ; K08 CA222684/CA/NCI NIH HHS/United States ; R35 CA210030/CA/NCI NIH HHS/United States ; F31 CA236036/CA/NCI NIH HHS/United States ; R35 CA242379/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antimetabolites, Antineoplastic/pharmacology ; Apoptosis ; *CRISPR-Cas Systems ; Cell Cycle ; Cell Proliferation ; Drug Resistance, Neoplasm/*drug effects ; Enzyme Inhibitors/*pharmacology ; Female ; Folic Acid/*metabolism ; Glycine Hydroxymethyltransferase/*antagonists &amp; inhibitors ; Humans ; Methotrexate/*pharmacology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/*drug therapy/enzymology/pathology ; Prognosis ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.},
}
@article {pmid34341073,
year = {2021},
author = {Zhao, Z and Szczepanski, AP and Tsuboyama, N and Abdala-Valencia, H and Goo, YA and Singer, BD and Bartom, ET and Yue, F and Wang, L},
title = {PAX9 Determines Epigenetic State Transition and Cell Fate in Cancer.},
journal = {Cancer research},
volume = {81},
number = {18},
pages = {4696-4708},
pmid = {34341073},
issn = {1538-7445},
support = {R01 HL153122/HL/NHLBI NIH HHS/United States ; P41 GM108569/GM/NIGMS NIH HHS/United States ; P30 CA060553/CA/NCI NIH HHS/United States ; P01 HL154998/HL/NHLBI NIH HHS/United States ; S10 OD025194/OD/NIH HHS/United States ; R01 HL149883/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Enhancer Elements, Genetic ; *Epigenesis, Genetic ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study/methods ; Histone Deacetylase Inhibitors/pharmacology ; Humans ; Mice ; Models, Biological ; Neoplasms/*genetics/*metabolism/pathology ; PAX9 Transcription Factor/genetics/*metabolism ; Repressor Proteins/genetics/metabolism ; Small Cell Lung Carcinoma/genetics ; Transcription Factors/genetics ; },
abstract = {Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and cofunctioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacologic HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that reactivation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9. SIGNIFICANCE: A genome-wide screen in small cell lung cancer reveals PAX9/NuRD-mediated epigenetic enhancer silencing and tumor progression, supporting the development of novel personalized therapeutic approaches targeting the PAX9-regulated network.},
}
@article {pmid34341072,
year = {2021},
author = {Sole, A and Grossetête, S and Heintzé, M and Babin, L and Zaïdi, S and Revy, P and Renouf, B and De Cian, A and Giovannangeli, C and Pierre-Eugène, C and Janoueix-Lerosey, I and Couronné, L and Kaltenbach, S and Tomishima, M and Jasin, M and Grünewald, TGP and Delattre, O and Surdez, D and Brunet, E},
title = {Unraveling Ewing Sarcoma Tumorigenesis Originating from Patient-Derived Mesenchymal Stem Cells.},
journal = {Cancer research},
volume = {81},
number = {19},
pages = {4994-5006},
pmid = {34341072},
issn = {1538-7445},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R35 CA253174/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Biomarkers ; CRISPR-Cas Systems ; *Cell Transformation, Neoplastic ; Cells, Cultured ; Computational Biology/methods ; Disease Models, Animal ; *Disease Susceptibility ; Gene Editing ; Gene Expression Profiling ; Gene Rearrangement ; Gene Targeting ; Heterografts ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Mesenchymal Stem Cells/*metabolism/pathology ; Mice ; Mutation ; Sarcoma, Ewing/*etiology/*metabolism/pathology ; Translocation, Genetic ; },
abstract = {Ewing sarcoma is characterized by pathognomonic translocations, most frequently fusing EWSR1 with FLI1. An estimated 30% of Ewing sarcoma tumors also display genetic alterations in STAG2, TP53, or CDKN2A (SPC). Numerous attempts to develop relevant Ewing sarcoma models from primary human cells have been unsuccessful in faithfully recapitulating the phenotypic, transcriptomic, and epigenetic features of Ewing sarcoma. In this study, by engineering the t(11;22)(q24;q12) translocation together with a combination of SPC mutations, we generated a wide collection of immortalized cells (EWIma cells) tolerating EWSR1-FLI1 expression from primary mesenchymal stem cells (MSC) derived from a patient with Ewing sarcoma. Within this model, SPC alterations strongly favored Ewing sarcoma oncogenicity. Xenograft experiments with independent EWIma cells induced tumors and metastases in mice, which displayed bona fide features of Ewing sarcoma. EWIma cells presented balanced but also more complex translocation profiles mimicking chromoplexy, which is frequently observed in Ewing sarcoma and other cancers. Collectively, these results demonstrate that bone marrow-derived MSCs are a source of origin for Ewing sarcoma and also provide original experimental models to investigate Ewing sarcomagenesis. SIGNIFICANCE: These findings demonstrate that Ewing sarcoma can originate from human bone-marrow-derived mesenchymal stem cells and that recurrent mutations support EWSR1-FLI1 translocation-mediated transformation.},
}
@article {pmid34341066,
year = {2021},
author = {Yang, Y and McCloskey, JE and Yang, H and Puc, J and Alcaina, Y and Vedvyas, Y and Gomez Gallegos, AA and Ortiz-Sánchez, E and de Stanchina, E and Min, IM and von Hofe, E and Jin, MM},
title = {Bispecific CAR T Cells against EpCAM and Inducible ICAM-1 Overcome Antigen Heterogeneity and Generate Superior Antitumor Responses.},
journal = {Cancer immunology research},
volume = {9},
number = {10},
pages = {1158-1174},
pmid = {34341066},
issn = {2326-6074},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA217059/CA/NCI NIH HHS/United States ; R01 CA254035/CA/NCI NIH HHS/United States ; U54 OD020355/OD/NIH HHS/United States ; },
mesh = {Animals ; Antigenic Drift and Shift ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Epithelial Cell Adhesion Molecule/genetics/*metabolism ; Humans ; Immunotherapy, Adoptive/adverse effects/*methods ; Intercellular Adhesion Molecule-1/genetics/*metabolism ; Male ; Mice ; Neoplasms/immunology/*metabolism/therapy ; Receptors, Antigen, T-Cell/genetics/*metabolism ; Receptors, Chimeric Antigen/genetics/*metabolism ; T-Lymphocytes/immunology/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated unparalleled responses in hematologic cancers, yet antigen escape and tumor relapse occur frequently. CAR T-cell therapy for patients with solid tumors faces even greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity. Here, we developed a bispecific CAR to simultaneously target epithelial cell adhesion molecule (EpCAM) and intercellular adhesion molecule 1 (ICAM-1) to overcome antigen escape and to improve the durability of tumor responses. ICAM-1 is an adhesion molecule inducible by inflammatory cytokines and elevated in many types of tumors. Our study demonstrates superior efficacy of bispecific CAR T cells compared with CAR T cells targeting a single primary antigen. Bispecific CAR T achieved more durable antitumor responses in tumor models with either homogenous or heterogenous expression of EpCAM. We also showed that the activation of CAR T cells against EpCAM in tumors led to upregulation of ICAM-1, which rendered tumors more susceptible to ICAM-1 targeting by bispecific CAR T cells. Our strategy of additional targeting of ICAM-1 may have broad applications in augmenting the activity of CAR T cells against primary tumor antigens that are prone to antigen loss or downregulation.},
}
@article {pmid34340931,
year = {2021},
author = {Hassan, MM and Zhang, Y and Yuan, G and De, K and Chen, JG and Muchero, W and Tuskan, GA and Qi, Y and Yang, X},
title = {Construct design for CRISPR/Cas-based genome editing in plants.},
journal = {Trends in plant science},
volume = {26},
number = {11},
pages = {1133-1152},
doi = {10.1016/j.tplants.2021.06.015},
pmid = {34340931},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Plant/genetics ; Plants/genetics ; },
abstract = {CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.},
}
@article {pmid34339270,
year = {2021},
author = {He, YZ and Yan, JR and He, B and Ren, H and Kuang, X and Long, TF and Chen, CP and Liao, XP and Liu, YH and Sun, J},
title = {A Transposon-Associated CRISPR/Cas9 System Specifically Eliminates both Chromosomal and Plasmid-Borne mcr-1 in Escherichia coli.},
journal = {Antimicrobial agents and chemotherapy},
volume = {65},
number = {10},
pages = {e0105421},
pmid = {34339270},
issn = {1098-6596},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Chromosomes ; Colistin/pharmacology ; Drug Resistance, Bacterial/genetics ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics ; Humans ; Plasmids/genetics ; },
abstract = {The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous mcr-1 containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.},
}
@article {pmid34338757,
year = {2021},
author = {Findlay, GM},
title = {Linking genome variants to disease: scalable approaches to test the functional impact of human mutations.},
journal = {Human molecular genetics},
volume = {30},
number = {R2},
pages = {R187-R197},
pmid = {34338757},
issn = {1460-2083},
support = {/WT_/Wellcome Trust/United Kingdom ; FC011142/ARC_/Arthritis Research UK/United Kingdom ; /CRUK_/Cancer Research UK/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Databases, Genetic ; Gene Editing ; *Genetic Association Studies/methods ; *Genetic Predisposition to Disease ; *Genetic Variation ; *Genome, Human ; Genomics ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Phenotype ; Quantitative Trait Loci ; },
abstract = {The application of genomics to medicine has accelerated the discovery of mutations underlying disease and has enhanced our knowledge of the molecular underpinnings of diverse pathologies. As the amount of human genetic material queried via sequencing has grown exponentially in recent years, so too has the number of rare variants observed. Despite progress, our ability to distinguish which rare variants have clinical significance remains limited. Over the last decade, however, powerful experimental approaches have emerged to characterize variant effects orders of magnitude faster than before. Fueled by improved DNA synthesis and sequencing and, more recently, by CRISPR/Cas9 genome editing, multiplex functional assays provide a means of generating variant effect data in wide-ranging experimental systems. Here, I review recent applications of multiplex assays that link human variants to disease phenotypes and I describe emerging strategies that will enhance their clinical utility in coming years.},
}
@article {pmid34338666,
year = {2021},
author = {He, X and Ma, Q and Jian, B and Liu, Y and Wu, Q and Chen, M and Feng, Q and Zhao, P and Liu, L},
title = {Microsurgical Obstruction of Testes Fusion in Spodoptera litura.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {173},
pages = {},
doi = {10.3791/62524},
pmid = {34338666},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; Male ; Spermatozoa ; Spodoptera/genetics ; *Testis/surgery ; },
abstract = {Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.},
}
@article {pmid34337911,
year = {2021},
author = {Ren, Y and Zhao, Y and Sun, W and Chen, Y and Yang, J and Li, Z and Wu, X and Zhao, L and Sun, W and Lv, C and Huang, N and Li, X},
title = {Effect of CRISPR/Cas9 system-mediated NF-κB knockdown on CNE-2 immune function in nasopharyngeal carcinoma.},
journal = {Journal of biological regulators and homeostatic agents},
volume = {35},
number = {4},
pages = {},
doi = {10.23812/21-171-L},
pmid = {34337911},
issn = {0393-974X},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Humans ; Immunity ; *NF-kappa B/genetics/metabolism ; Nasopharyngeal Carcinoma/genetics ; *Nasopharyngeal Neoplasms/genetics ; },
}
@article {pmid34336867,
year = {2021},
author = {Zhang, Y and Li, M},
title = {Genome Editing Technologies as Cellular Defense Against Viral Pathogens.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {716344},
pmid = {34336867},
issn = {2296-634X},
abstract = {Viral infectious diseases are significant threats to the welfare of world populations. Besides the widespread acute viral infections (e.g., dengue fever) and chronic infections [e.g., those by the human immunodeficiency virus (HIV) and hepatitis B virus (HBV)], emerging viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose great challenges to the world. Genome editing technologies, including clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), have played essential roles in the study of new treatment for viral infectious diseases in cell lines, animal models, and clinical trials. Genome editing tools have been used to eliminate latent infections and provide resistance to new infections. Increasing evidence has shown that genome editing-based antiviral strategy is simple to design and can be quickly adapted to combat infections by a wide spectrum of viral pathogens, including the emerging coronaviruses. Here we review the development and applications of genome editing technologies for preventing or eliminating infections caused by HIV, HBV, HPV, HSV, and SARS-CoV-2, and discuss how the latest advances could enlighten further development of genome editing into a novel therapy for viral infectious diseases.},
}
@article {pmid34335974,
year = {2021},
author = {Zhang, Q and Wadgaonkar, P and Xu, L and Thakur, C and Fu, Y and Bi, Z and Qiu, Y and Almutairy, B and Zhang, W and Stemmer, P and Chen, F},
title = {Environmentally-induced mdig contributes to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan metabolism.},
journal = {Theranostics},
volume = {11},
number = {16},
pages = {7970-7983},
pmid = {34335974},
issn = {1838-7640},
support = {R01 ES028263/ES/NIEHS NIH HHS/United States ; R01 ES028335/ES/NIEHS NIH HHS/United States ; R01 ES031822/ES/NIEHS NIH HHS/United States ; },
mesh = {Alveolar Epithelial Cells/metabolism ; Animals ; COVID-19/epidemiology/*metabolism/*virology ; Cathepsins/metabolism ; Cell Line ; Cells, Cultured ; Dioxygenases/biosynthesis/genetics/*metabolism ; Environmental Exposure ; Histone Demethylases/biosynthesis/genetics/*metabolism ; Histones/metabolism ; Humans ; Neuropilin-1/*metabolism ; Nuclear Proteins/biosynthesis/genetics/*metabolism ; Pandemics ; Polysaccharides/*metabolism ; Rats ; SARS-CoV-2/*metabolism/pathogenicity ; Spike Glycoprotein, Coronavirus/metabolism ; },
abstract = {The novel β-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 177 million people and resulted in 3.84 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provided evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is an important regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. Meanwhile, we also validated that environmental factor arsenic is able to induce mdig, NRP1 and NRP2, and genetic disruption of mdig lowered expression of NRP1 and NRP2. Furthermore, mdig may coordinate with the Neanderthal variants linked to an elevated mortality of COVID-19. These data, thus, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be the one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.},
}
@article {pmid34335512,
year = {2021},
author = {Alves-Barroco, C and Caço, J and Roma-Rodrigues, C and Fernandes, AR and Bexiga, R and Oliveira, M and Chambel, L and Tenreiro, R and Mato, R and Santos-Sanches, I},
title = {New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {686413},
pmid = {34335512},
issn = {1664-302X},
abstract = {Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002-2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011-2013 and compare them with SDSD isolates collected in 2002-2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies "dysgalactiae" and "equisimilis" should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.},
}
@article {pmid34332602,
year = {2021},
author = {Wang, Y and Li, G and Li, X and Wang, Y and Huang, X and Hu, X and Gao, J},
title = {ecRESCUE: a novel ecDHFR-regulated RESCUE system with reduced RNA off-targeting activity.},
journal = {Cell communication and signaling : CCS},
volume = {19},
number = {1},
pages = {81},
pmid = {34332602},
issn = {1478-811X},
mesh = {CRISPR-Cas Systems/genetics ; Genetic Diseases, Inborn/*genetics/pathology/therapy ; HEK293 Cells ; Humans ; Mutation/genetics ; RNA/*genetics ; RNA Editing/*genetics ; },
abstract = {The currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity. Video Abstract.},
}
@article {pmid34332328,
year = {2021},
author = {Wang, W and Bai, R and Song, X},
title = {Generating dHAND homozygous knockout human embryonic stem cell line (WAe009-A-59) by episomal vector-based CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102471},
doi = {10.1016/j.scr.2021.102471},
pmid = {34332328},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; Homozygote ; *Human Embryonic Stem Cells ; Humans ; Mice ; },
abstract = {In order to determine the function of dHAND in a specific subset of cardiomyocyte progenitor cells responsible for the heart and its function in the cellular mechanism of Hand2-/- mouse ventricular hypoplasia, we established an allelic knockout model of dHAND in human embryonic stem cells (hESCs-H9) by an episomal vector-based CRISPR/Cas9 system. This dHAND KO hESC line maintained normal karyotype and typical primed pluripotent human stem cell morphology, and maintained pluripotency, could differentiate into all three germ layers in vivo.},
}
@article {pmid34331750,
year = {2021},
author = {Duan, K and Cheng, Y and Ji, J and Wang, C and Wei, Y and Wang, Y},
title = {Large chromosomal segment deletions by CRISPR/LbCpf1-mediated multiplex gene editing in soybean.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {9},
pages = {1620-1631},
doi = {10.1111/jipb.13158},
pmid = {34331750},
issn = {1744-7909},
mesh = {*Base Sequence ; *CRISPR-Cas Systems ; Gene Editing/*methods ; *Sequence Deletion ; Soybeans/*genetics ; },
abstract = {The creation of new soybean varieties has been limited by genomic duplication and redundancy. Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are promising strategies for overcoming these obstacles. CRISPR/Cpf1 is a robust tool for multiplex gene editing. However, large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species. Here, we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting. The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA (crRNA) array. The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter. The optimized system exhibited editing efficiencies of up to 91.7%. Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed, with an efficiency of up to 17.1%. We successfully employed CRISPR/LbCpf1 to produce small fragments (<1 Kb) and large chromosomal segment deletions (10 Kb-1 Mb) involving four different gene clusters in soybean. Together, these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean, supporting the use of this technology in both basic research and agricultural applications.},
}
@article {pmid34331200,
year = {2021},
author = {Pesonen, L and Svartsjö, S and Bäck, V and de Thonel, A and Mezger, V and Sabéran-Djoneidi, D and Roos-Mattjus, P},
title = {Gambogic acid and gambogenic acid induce a thiol-dependent heat shock response and disrupt the interaction between HSP90 and HSF1 or HSF2.},
journal = {Cell stress &amp; chaperones},
volume = {26},
number = {5},
pages = {819-833},
pmid = {34331200},
issn = {1466-1268},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism ; Heat Shock Transcription Factors/*metabolism ; Heat-Shock Proteins/*metabolism ; *Heat-Shock Response/drug effects ; Humans ; Protein Binding/drug effects ; Sulfhydryl Compounds/*metabolism ; Transcription Factors/*metabolism ; Xanthenes/chemistry/*pharmacology ; Xanthones/chemistry/*pharmacology ; },
abstract = {Cancer cells rely on heat shock proteins (HSPs) for growth and survival. Especially HSP90 has multiple client proteins and plays a critical role in malignant transformation, and therefore different types of HSP90 inhibitors are being developed. The bioactive natural compound gambogic acid (GB) is a prenylated xanthone with antitumor activity, and it has been proposed to function as an HSP90 inhibitor. However, there are contradicting reports whether GB induces a heat shock response (HSR), which is cytoprotective for cancer cells and therefore a potentially problematic feature for an anticancer drug. In this study, we show that GB and a structurally related compound, called gambogenic acid (GBA), induce a robust HSR, in a thiol-dependent manner. Using heat shock factor 1 (HSF1) or HSF2 knockout cells, we show that the GB or GBA-induced HSR is HSF1-dependent. Intriguingly, using closed form ATP-bound HSP90 mutants that can be co-precipitated with HSF1, a known facilitator of cancer, we show that also endogenous HSF2 co-precipitates with HSP90. GB and GBA treatment disrupt the interaction between HSP90 and HSF1 and HSP90 and HSF2. Our study implies that these compounds should be used cautiously if developed for cancer therapies, since GB and its derivative GBA are strong inducers of the HSR, in multiple cell types, by involving the dissociation of a HSP90-HSF1/HSF2 complex.},
}
@article {pmid34329468,
year = {2021},
author = {Zhao, T and Li, Q and Zhou, C and Lv, X and Liu, H and Tu, T and Tang, N and Cheng, Y and Liu, X and Liu, C and Zhao, J and Song, Z and Wang, H and Li, J and Gu, F},
title = {Small-molecule compounds boost genome-editing efficiency of cytosine base editor.},
journal = {Nucleic acids research},
volume = {49},
number = {15},
pages = {8974-8986},
pmid = {34329468},
issn = {1362-4962},
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Cytosine/*metabolism ; Gene Editing/trends ; HEK293 Cells ; Histone Deacetylase 6/*antagonists &amp; inhibitors/genetics ; Humans ; Hydroxamic Acids/pharmacology ; Mice ; Mutation/drug effects ; Phenylurea Compounds/pharmacology ; Pyrimidines/pharmacology ; Small Molecule Libraries/pharmacology ; Stargardt Disease/drug therapy/*genetics/pathology ; T-Lymphocytes/drug effects ; Zygote/drug effects ; },
abstract = {Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.},
}
@article {pmid34328110,
year = {2021},
author = {Kozovska, Z and Rajcaniova, S and Munteanu, P and Dzacovska, S and Demkova, L},
title = {CRISPR: History and perspectives to the future.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {141},
number = {},
pages = {111917},
doi = {10.1016/j.biopha.2021.111917},
pmid = {34328110},
issn = {1950-6007},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*physiology ; Forecasting ; Gene Editing/*methods/trends ; Genetic Engineering/methods/trends ; Genetic Therapy/*methods/trends ; Humans ; },
abstract = {This review summarizes the information about the history and future of the CRISPR/Cas9 method. Genome editing can be perceived as a group of technologies that allow scientists to change the DNA of an organism. These technologies involve the deletion, insertion, or modification of the genome at a specific site in a DNA sequence. Gene therapy in humans has a perspective to be used to eliminate the gene responsible for a particular genetic disorder. The review focuses on the key elements of this promising method and the possibility of its application in the treatment of cancer and genetic diseases.},
}
@article {pmid34327906,
year = {2021},
author = {Bai, Z and Wang, W and Ji, X and Xiao, Y and Zhang, S and Wang, Z and Li, H and Dong, Q},
title = {[Application of CRISPR in evolution analysis, detecting and typing, virulence and antibiotic resistance regulation in food-borne pathogens].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {7},
pages = {2414-2424},
doi = {10.13345/j.cjb.200539},
pmid = {34327906},
issn = {1872-2075},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drug Resistance, Microbial/genetics ; Virulence/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein gene system can limit the horizontal gene transfer, thereby effectively preventing the invasion of foreign gene elements such as bacteriophages. CRISPR arrays of different bacteria are diverse. Based on the differences in the CRISPR system, this review summarizes the application of CRISPR in food-borne pathogen evolution analysis, detection and typing, virulence and antibiotic resistance in recent years. We also address bacterial detection typing method developed based on the characteristics of CRISPR arrays and the association of CRISPR with virulence and drug resistance of food-borne pathogens. The shortcomings of CRISPR in evolution, detection and typing, virulence and resistance applications are analyzed. In addition, we suggest standardizing CRISPR typing methods, improving and expanding the CRISPR database of pathogenic bacteria, and further exploring the co-evolution relationship between phages and bacteria, to provide references for further exploration of CRISPR functions.},
}
@article {pmid34327897,
year = {2021},
author = {Xu, X and Liu, M},
title = {[Recent advances and applications of base editing systems].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {7},
pages = {2307-2321},
doi = {10.13345/j.cjb.200480},
pmid = {34327897},
issn = {1872-2075},
mesh = {Adenine ; Animals ; *CRISPR-Cas Systems/genetics ; Cytosine ; DNA Breaks, Double-Stranded ; *Gene Editing ; },
abstract = {The CRISPR system is able to accomplish precise base editing in genomic DNA, but relies on the cellular homology-directed recombination repair pathway and is therefore extremely inefficient. Base editing is a new genome editing technique developed based on the CRISPR/Cas9 system. Two base editors (cytosine base editor and adenine base editor) were developed by fusing catalytically disabled nucleases with different necleobase deaminases. These two base editors are able to perform C>T (G>A) or A>G (T>C) transition without generating DNA double-stranded breaks. The base editing technique has been widely used in gene therapy, animal models construction, precision animal breeding and gene function analysis, providing a powerful tool for basic and applied research. This review summarized the development process, technical advantages, current applications, challenges and perspectives for base editing technique, aiming to help the readers better understand and use the base editing technique.},
}
@article {pmid34327721,
year = {2021},
author = {Li, Y and Zhou, LQ},
title = {dCas9 techniques for transcriptional repression in mammalian cells: Progress, applications and challenges.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {43},
number = {9},
pages = {e2100086},
doi = {10.1002/bies.202100086},
pmid = {34327721},
issn = {1521-1878},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Expression ; Gene Knockdown Techniques ; *Transcription Factors ; },
abstract = {Innovative loss-of-function techniques developed in recent years have made it much easier to target specific genomic loci at transcriptional levels. CRISPR interference (CRISPRi) has been proven to be the most effective and specific tool to knock down any gene of interest in mammalian cells. The catalytically deactivated Cas9 (dCas9) can be fused with transcription repressors to downregulate gene expression specified by sgRNA complementary to target genomic sequence. Although CRISPRi has huge potential for gene knockdown, there is still a lack of systematic guidelines for efficient and widespread use. Here we describe the working mechanism and development of CRISPRi, designing principles of sgRNA, delivery methods and applications in mammalian cells in detail. Finally, we propose possible solutions and future directions with regard to current challenges.},
}
@article {pmid34327324,
year = {2021},
author = {Li, S and Zhang, C and Li, J and Yan, L and Wang, N and Xia, L},
title = {Present and future prospects for wheat improvement through genome editing and advanced technologies.},
journal = {Plant communications},
volume = {2},
number = {4},
pages = {100211},
pmid = {34327324},
issn = {2590-3462},
mesh = {*Gene Editing ; *Genome, Plant ; *Genomics ; *Plant Breeding ; Triticum/*genetics ; },
abstract = {Wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) is one of the most important staple food crops in the world. Despite the fact that wheat production has significantly increased over the past decades, future wheat production will face unprecedented challenges from global climate change, increasing world population, and water shortages in arid and semi-arid lands. Furthermore, excessive applications of diverse fertilizers and pesticides are exacerbating environmental pollution and ecological deterioration. To ensure global food and ecosystem security, it is essential to enhance the resilience of wheat production while minimizing environmental pollution through the use of cutting-edge technologies. However, the hexaploid genome and gene redundancy complicate advances in genetic research and precision gene modifications for wheat improvement, thus impeding the breeding of elite wheat cultivars. In this review, we first introduce state-of-the-art genome-editing technologies in crop plants, especially wheat, for both functional genomics and genetic improvement. We then outline applications of other technologies, such as GWAS, high-throughput genotyping and phenotyping, speed breeding, and synthetic biology, in wheat. Finally, we discuss existing challenges in wheat genome editing and future prospects for precision gene modifications using advanced genome-editing technologies. We conclude that the combination of genome editing and other molecular breeding strategies will greatly facilitate genetic improvement of wheat for sustainable global production.},
}
@article {pmid34326689,
year = {2021},
author = {Azhar, M and Altaf, S and Uddin, I and Cheng, J and Wu, L and Tong, X and Qin, W and Bao, J},
title = {Towards Post-Meiotic Sperm Production: Genetic Insight into Human Infertility from Mouse Models.},
journal = {International journal of biological sciences},
volume = {17},
number = {10},
pages = {2487-2503},
pmid = {34326689},
issn = {1449-2288},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Genetic Variation ; High-Throughput Nucleotide Sequencing ; Humans ; Infertility, Male/*etiology ; Male ; Meiosis/*genetics ; Mice ; Mutation ; Spermatogenesis/genetics ; Spermatozoa/*pathology ; },
abstract = {Declined quality and quantity of sperm is currently the major cause of patients suffering from infertility. Male germ cell development is spatiotemporally regulated throughout the whole developmental process. While it has been known that exogenous factors, such as environmental exposure, diet and lifestyle, et al, play causative roles in male infertility, recent progress has revealed abundant genetic mutations tightly associated with defective male germline development. In mammals, male germ cells undergo dramatic morphological change (i.e., nuclear condensation) and chromatin remodeling during post-meiotic haploid germline development, a process termed spermiogenesis; However, the molecular machinery players and functional mechanisms have yet to be identified. To date, accumulated evidence suggests that disruption in any step of haploid germline development is likely manifested as fertility issues with low sperm count, poor sperm motility, aberrant sperm morphology or combined. With the continually declined cost of next-generation sequencing and recent progress of CRISPR/Cas9 technology, growing studies have revealed a vast number of disease-causing genetic variants associated with spermiogenic defects in both mice and humans, along with mechanistic insights partially attained and validated through genetically engineered mouse models (GEMMs). In this review, we mainly summarize genes that are functional at post-meiotic stage. Identification and characterization of deleterious genetic variants should aid in our understanding of germline development, and thereby further improve the diagnosis and treatment of male infertility.},
}
@article {pmid34326503,
year = {2022},
author = {Hines, PA and Agricola, E and Llinares Garcia, J and O'Dwyer, L and Herold, R},
title = {Therapeutic genome editing: regulatory horizons.},
journal = {Nature reviews. Drug discovery},
volume = {21},
number = {1},
pages = {1-2},
pmid = {34326503},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Humans ; },
}
@article {pmid34326397,
year = {2021},
author = {Akter, MS and Hada, M and Shikata, D and Watanabe, G and Ogura, A and Matoba, S},
title = {CRISPR/Cas9-based genetic screen of SCNT-reprogramming resistant genes identifies critical genes for male germ cell development in mice.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {15438},
pmid = {34326397},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Electron Transport Complex IV/genetics/metabolism ; Female ; Fertilization in Vitro/methods ; Genes, Essential ; Genetic Testing/*methods ; Male ; Meiosis/genetics ; Membrane Proteins/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Knockout ; *Nuclear Transfer Techniques ; Spermatogenesis/*genetics ; Spermatozoa/*growth &amp; development ; Testis/metabolism ; },
abstract = {Male germ cells undergo complex developmental processes eventually producing spermatozoa through spermatogenesis, although the molecular mechanisms remain largely elusive. We have previously identified somatic cell nuclear transfer-reprogramming resistant genes (SRRGs) that are highly enriched for genes essential for spermatogenesis, although many of them remain uncharacterized in knockout (KO) mice. Here, we performed a CRISPR-based genetic screen using C57BL/6N mice for five uncharacterized SRRGs (Cox8c, Cox7b2, Tuba3a/3b, Faiml, and Gm773), together with meiosis essential gene Majin as a control. RT-qPCR analysis of mouse adult tissues revealed that the five selected SRRGs were exclusively expressed in testis. Analysis of single-cell RNA-seq datasets of adult testis revealed stage-specific expression (pre-, mid-, or post-meiotic expression) in testicular germ cells. Examination of testis morphology, histology, and sperm functions in CRISPR-injected KO adult males revealed that Cox7b2, Gm773, and Tuba3a/3b are required for the production of normal spermatozoa. Specifically, Cox7b2 KO mice produced poorly motile infertile spermatozoa, Gm773 KO mice produced motile spermatozoa with limited zona penetration abilities, and Tuba3a/3b KO mice completely lost germ cells at the early postnatal stages. Our genetic screen focusing on SRRGs efficiently identified critical genes for male germ cell development in mice, which also provides insights into human reproductive medicine.},
}
@article {pmid34326359,
year = {2021},
author = {Rodriguez-Polo, I and Mißbach, S and Petkov, S and Mattern, F and Maierhofer, A and Grządzielewska, I and Tereshchenko, Y and Urrutia-Cabrera, D and Haaf, T and Dressel, R and Bartels, I and Behr, R},
title = {A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {15439},
pmid = {34326359},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Cell Line ; Cellular Reprogramming/*genetics ; DNA Transposable Elements/*genetics ; Female ; Fibroblasts/cytology ; Gene Editing/*methods ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; Induced Pluripotent Stem Cells/*cytology ; Macaca mulatta/*genetics ; Male ; Mice ; Phylogeny ; Skin/cytology ; Transfection ; Transgenes ; Transposases/genetics ; },
abstract = {Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.},
}
@article {pmid34326322,
year = {2021},
author = {Das, R and Sjöström, M and Shrestha, R and Yogodzinski, C and Egusa, EA and Chesner, LN and Chen, WS and Chou, J and Dang, DK and Swinderman, JT and Ge, A and Hua, JT and Kabir, S and Quigley, DA and Small, EJ and Ashworth, A and Feng, FY and Gilbert, LA},
title = {An integrated functional and clinical genomics approach reveals genes driving aggressive metastatic prostate cancer.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4601},
pmid = {34326322},
issn = {2041-1723},
support = {R01 CA227025/CA/NCI NIH HHS/United States ; R00 CA204602/CA/NCI NIH HHS/United States ; K99 CA204602/CA/NCI NIH HHS/United States ; R01 CA230516/CA/NCI NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle/physiology ; Cell Cycle Proteins/*genetics/metabolism ; Cell Movement/physiology ; Cells, Cultured ; Databases, Genetic ; Gene Expression Regulation, Neoplastic ; Heterografts ; Humans ; Kinesins/*genetics/metabolism ; Male ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Metastasis ; Neoplasm Staging ; Nerve Tissue Proteins/*genetics/metabolism ; Prostatic Neoplasms/*genetics/metabolism/*pathology ; Survival Rate ; },
abstract = {Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.},
}
@article {pmid34324887,
year = {2021},
author = {Xu, X and Liu, C and Wang, Y and Koivisto, O and Zhou, J and Shu, Y and Zhang, H},
title = {Nanotechnology-based delivery of CRISPR/Cas9 for cancer treatment.},
journal = {Advanced drug delivery reviews},
volume = {176},
number = {},
pages = {113891},
doi = {10.1016/j.addr.2021.113891},
pmid = {34324887},
issn = {1872-8294},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Humans ; Nanotechnology/*methods ; Neoplasms/genetics/*therapy ; },
abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9) is a potent technology for gene-editing. Owing to its high specificity and efficiency, CRISPR/Cas9 is extensity used for human diseases treatment, especially for cancer, which involves multiple genetic alterations. Different concepts of cancer treatment by CRISPR/Cas9 are established. However, significant challenges remain for its clinical applications. The greatest challenge for CRISPR/Cas9 therapy is how to safely and efficiently deliver it to target sites in vivo. Nanotechnology has greatly contributed to cancer drug delivery. Here, we present the action mechanisms of CRISPR/Cas9, its application in cancer therapy and especially focus on the nanotechnology-based delivery of CRISPR/Cas9 for cancer gene editing and immunotherapy to pave the way for its clinical translation. We detail the difficult barriers for CRISIR/Cas9 delivery in vivo and discuss the relative solutions for encapsulation, target delivery, controlled release, cellular internalization, and endosomal escape.},
}
@article {pmid34324266,
year = {2021},
author = {Yin, X and Hao, J and Yao, Y},
title = {CRISPR/Cas9 in zebrafish: An attractive model for FBN1 genetic defects in humans.},
journal = {Molecular genetics &amp; genomic medicine},
volume = {9},
number = {10},
pages = {e1775},
pmid = {34324266},
issn = {2324-9269},
mesh = {Alleles ; Animals ; *Animals, Genetically Modified ; Base Sequence ; *CRISPR-Cas Systems ; Conserved Sequence ; Fibrillin-1/genetics ; *Gene Editing ; Genes, Reporter ; Genetic Association Studies ; Genetic Predisposition to Disease ; Genotype ; Humans ; Loss of Function Mutation ; Mutation ; Zebrafish/*genetics ; },
abstract = {BACKGROUND: Mutations in the fibrillin-1 gene (FBN1) are associated with various heritable connective tissue disorders (HCTD). The most studied HCTD is Marfan syndrome. Ninety percent of Marfan syndrome is caused by mutations in the FBN1 gene. The zebrafish share high genetic similarity to humans, representing an ideal model for genetic research of human diseases. This study aimed to generate and characterize fbn1[+/-] mutant zebrafish using the CRISPR/Cas9 gene-editing technology.<br><br>METHODS: CRISPR/Cas9 was applied to generate an fbn1 frameshift mutation (fbn1[+/-]) in zebrafish. F1 fbn1[+/-] heterozygotes were crossed with transgenic fluorescent zebrafish to obtain F2 fbn1[+/-] zebrafish. Morphological abnormalities were assessed in F2 fbn1[+/-] zebrafish by comparing with the Tuebingen (TU) wild-type controls at different development stages.<br><br>RESULTS: We successfully generated a transgenic line of fbn1[+/-] zebrafish. Compared with TU wild-type zebrafish, F2 fbn1[+/-] zebrafish exhibited noticeably decreased pigmentation, increased lengths, slender body shape, and abnormal cardiac blood flow from atrium to ventricle.<br><br>CONCLUSION: We generated the first fbn1[+/-] zebrafish model using CRISPR/Cas9 gene-editing approach to mimic FBN1 genetic defects in humans, providing an attractive model of Marfan syndrome and a method to determine the pathogenicity of gene mutation sites.},
}
@article {pmid34324191,
year = {2021},
author = {Canals, I and Ahlenius, H},
title = {CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem Cells for Modeling of Neurological Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2352},
number = {},
pages = {237-251},
pmid = {34324191},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Cell Differentiation/genetics ; Cloning, Molecular ; *Gene Editing ; Humans ; Models, Biological ; Nervous System Diseases/*etiology/metabolism ; Pluripotent Stem Cells/*cytology/*metabolism ; RNA, Guide ; Transfection ; },
abstract = {Recent advances in genome editing have brought new hopes for personalized and precision medicine but have also dramatically facilitated disease modeling studies. Combined with reprogramming approaches, stem cells and differentiation toward neural lineages, genome engineering holds great potential for regenerative approaches and to model neurological disorders. The use of patient-specific induced pluripotent stem cells combined with neural differentiation allows studying the effect of specific mutations in different brain cells. New genome editing tools such as CRISPR/Cas9 represent a step further by facilitating the introduction or correction of specific mutations within the same cell line, thus eliminating variability due to differences in the genetic background. Here, we present a step-by-step protocol from design to generation of human pluripotent stem cell lines with specific mutations introduced or corrected with CRISPR/Cas9 gene editing that can be used in combination with transcription factor-based protocols to dissect underlying mechanisms of neurological disorders.},
}
@article {pmid34323910,
year = {2021},
author = {Wang, CS and Chang, CH and Tzeng, TY and Lin, AM and Lo, YL},
title = {Gene-editing by CRISPR-Cas9 in combination with anthracycline therapy via tumor microenvironment-switchable, EGFR-targeted, and nucleus-directed nanoparticles for head and neck cancer suppression.},
journal = {Nanoscale horizons},
volume = {6},
number = {9},
pages = {729-743},
doi = {10.1039/d1nh00254f},
pmid = {34323910},
issn = {2055-6764},
mesh = {Animals ; Anthracyclines ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; ErbB Receptors/genetics/metabolism ; Mice ; *Nanoparticles ; *Tongue Neoplasms ; Tumor Microenvironment ; },
abstract = {Head and neck cancer (HNC) has a high incidence and a poor prognosis. Epirubicin, a topoisomerase inhibitor, is a potential anthracycline chemotherapeutic for HNC treatment. HuR (ELAVL1), an RNA-binding protein, plays a critical role in promoting tumor survival, invasion, and resistance. HuR knockout via CRISPR/Cas9 (HuR CRISPR) is a possible strategy for the simultaneous modulation of the various pathways of tumor progression. Multifunctional nanoparticles modified with pH-sensitive epidermal growth factor receptor (EGFR)-targeting and nucleus-directed peptides were designed for the efficient delivery of HuR CRISPR and epirubicin to human tongue squamous carcinoma SAS cells and SAS tumor-bearing mice. The pH-sensitive nanoparticles responded to the acidic pH value as a switch to expose the targeting peptides. The cellular uptake and transfection efficiency of these nanoparticles in SAS cells increased via EGFR targeting, ligand-mediated endocytosis, and endosomal escape. These nanoparticles showed low cytotoxicity towards normal oral keratinocyte NOK cells. CRISPR/Cas9 was transported into the nucleus via the nuclear directing peptide and successfully knocked out HuR to suppress proliferation, metastasis, and resistance in SAS cells. The multiple inhibition of EGFR/β-catenin/epithelial-mesenchymal transition pathways was mediated through modulating the EGFR/PI3K/mTOR/AKT axis. The co-treatment of epirubicin and HuR CRISPR in SAS cells further facilitated apoptosis/necroptosis/autophagy and caused cancer cell death. In combination with HuR CRISPR nanoparticles, the efficacy and safety of epirubicin nanoparticles against cancer in SAS tumor-bearing mice improved significantly. Collectively, these nanoparticles showed a tumor pH response, active EGFR targeting, and nuclear localization and thus offered a combinatorial spatiotemporal platform for chemotherapy and the CRISPR/Cas gene-editing system.},
}
@article {pmid34321476,
year = {2021},
author = {Hammond, A and Pollegioni, P and Persampieri, T and North, A and Minuz, R and Trusso, A and Bucci, A and Kyrou, K and Morianou, I and Simoni, A and Nolan, T and Müller, R and Crisanti, A},
title = {Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4589},
pmid = {34321476},
issn = {2041-1723},
mesh = {Animals ; Animals, Genetically Modified ; Anopheles/*genetics ; Bayes Theorem ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Drive Technology ; Housing, Animal ; Malaria/transmission ; Mosquito Control ; Mosquito Vectors/*genetics ; Retrospective Studies ; },
abstract = {CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.},
}
@article {pmid34321286,
year = {2021},
author = {Jeske, AM and Boucher, P and Curiel, DT and Voss, JE},
title = {Vector Strategies to Actualize B Cell-Based Gene Therapies.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {207},
number = {3},
pages = {755-764},
pmid = {34321286},
issn = {1550-6606},
support = {R01 AI165143/AI/NIAID NIH HHS/United States ; R01 AI167003/AI/NIAID NIH HHS/United States ; R01 CA211096/CA/NCI NIH HHS/United States ; UG3 TR002851/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Antibodies/genetics/metabolism ; B-Lymphocytes/*physiology ; CRISPR-Cas Systems ; Epitopes ; Genetic Engineering ; Genetic Therapy/*trends ; Humans ; Immunotherapy ; Receptors, Antigen, B-Cell/*genetics ; },
abstract = {Recent developments in genome editing and delivery systems have opened new possibilities for B cell gene therapy. CRISPR-Cas9 nucleases have been used to introduce transgenes into B cell genomes for subsequent secretion of exogenous therapeutic proteins from plasma cells and to program novel B cell Ag receptor specificities, allowing for the generation of desirable Ab responses that cannot normally be elicited in animal models. Genome modification of B cells or their progenitor, hematopoietic stem cells, could potentially substitute Ab or protein replacement therapies that require multiple injections over the long term. To date, B cell editing using CRISPR-Cas9 has been solely employed in preclinical studies, in which cells are edited ex vivo. In this review, we discuss current B cell engineering efforts and strategies for the eventual safe and economical adoption of modified B cells into the clinic, including in vivo viral delivery of editing reagents to B cells.},
}
@article {pmid34321217,
year = {2021},
author = {Carullo, NVN and Hinds, JE and Revanna, JS and Tuscher, JJ and Bauman, AJ and Day, JJ},
title = {A Cre-Dependent CRISPR/dCas9 System for Gene Expression Regulation in Neurons.},
journal = {eNeuro},
volume = {8},
number = {4},
pages = {},
pmid = {34321217},
issn = {2373-2822},
support = {DP1 DA039650/DA/NIDA NIH HHS/United States ; K99 DA034681/DA/NIDA NIH HHS/United States ; R00 DA034681/DA/NIDA NIH HHS/United States ; R01 MH114990/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Integrases ; Neurons ; Rats ; },
abstract = {Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customization for different applications. The simplicity and modularity of clustered regularly interspaced short palindromic repeats (CRISPR)-based systems have transformed genome editing and expanded the gene regulatory toolbox. However, there are few available tools for cell-selective CRISPR regulation in neurons. We designed, validated, and optimized CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems for Cre recombinase-dependent gene regulation. Unexpectedly, CRISPRa systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction even without Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate numerous rat or human genes (GRM2, Tent5b, Fos, Sstr2, and Gadd45b) in a Cre-specific manner. To illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from a luciferase reporter in HEK293T cells only in the presence of Cre. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.},
}
@article {pmid34321099,
year = {2021},
author = {Razeghian, E and Nasution, MKM and Rahman, HS and Gardanova, ZR and Abdelbasset, WK and Aravindhan, S and Bokov, DO and Suksatan, W and Nakhaei, P and Shariatzadeh, S and Marofi, F and Yazdanifar, M and Shamlou, S and Motavalli, R and Khiavi, FM},
title = {A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies.},
journal = {Stem cell research &amp; therapy},
volume = {12},
number = {1},
pages = {428},
pmid = {34321099},
issn = {1757-6512},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Immunotherapy ; *Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; },
abstract = {To date, two chimeric antigen receptors (CAR)-T cell products from autologous T cells have been approved by The United States Food and Drug Administration (FDA). The case-by-case autologous T cell generation setting is largely considered as a pivotal restraining cause for its large-scale clinical use because of the costly and prolonged manufacturing procedure. Further, activated CAR-T cells mainly express immune checkpoint molecules, including CTLA4, PD1, LAG3, abrogating CAR-T anti-tumor activity. In addition, CAR-T cell therapy potently results in some toxicity, such as cytokine releases syndrome (CRS). Therefore, the development of the universal allogeneic T cells with higher anti-tumor effects is of paramount importance. Thus, genome-editing technologies, in particular, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 are currently being used to establish "off-the-shelf" CAR-T cells with robust resistance to immune cell-suppressive molecules. In fact, that simultaneous ablation of PD-1, T cell receptor alpha constant (TRAC or TCR), and also β-2 microglobulin (B2M) by CRISPR-Cas9 technique can support the manufacture of universal CAR-T cells with robust resistance to PD-L1. . Indeed, the ablation of β2M or TARC can severely hinder swift elimination of allogeneic T cells those express foreign HLA-I molecules, and thereby enables the generation of CAR-T cells from allogeneic healthy donors T cells with higher persistence in vivo. Herein, we will deliver a brief overview of the CAR-T cell application in the context of tumor immunotherapy. More importantly, we will discuss recent finding concerning the application of genome editing technologies for preparing universal CAR-T cells or cells that can effectively counter tumor escape, with a special focus on CRISPR-Cas9 technology.},
}
@article {pmid34321002,
year = {2021},
author = {Gholizadeh, P and Aghazadeh, M and Ghotaslou, R and Rezaee, MA and Pirzadeh, T and Cui, L and Watanabe, S and Feizi, H and Kadkhoda, H and Kafil, HS},
title = {Role of CRISPR-Cas system on antibiotic resistance patterns of Enterococcus faecalis.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {20},
number = {1},
pages = {49},
pmid = {34321002},
issn = {1476-0711},
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Multiple, Bacterial/*genetics ; Enterococcus faecalis/*drug effects/*genetics/isolation &amp; purification ; Genotype ; Gentamicins ; Humans ; Urinary Tract Infections ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in Enterococcus faecalis. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among E. faecalis isolates. A total of 151 isolates of E. faecalis were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant E. faecalis strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive strains. These data indicate a negative correlation between CRISPR-cas loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.},
}
@article {pmid34320241,
year = {2021},
author = {Kanack, AJ and Aoki, K and Tiemeyer, M and Dahms, NM},
title = {Platelet and myeloid cell phenotypes in a rat model of Fabry disease.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {8},
pages = {e21818},
pmid = {34320241},
issn = {1530-6860},
support = {R01 DK042667/DK/NIDDK NIH HHS/United States ; K12 HL141954/HL/NHLBI NIH HHS/United States ; R24 HL114474/HL/NHLBI NIH HHS/United States ; P41 GM103490/GM/NIGMS NIH HHS/United States ; R21 AI129873/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Blood Platelets/*cytology ; Bone Marrow/enzymology ; CRISPR-Cas Systems ; Fabry Disease/*metabolism ; Female ; Leukocytes/physiology ; Male ; Megakaryocytes/physiology ; Myeloid Cells/*classification/*physiology ; Platelet Activation ; Platelet Aggregation ; Rats ; alpha-Galactosidase/genetics/*metabolism ; },
abstract = {Fabry disease results from a deficiency of the lysosomal enzyme ⍺-Galactosidase-A (⍺-Gal A) and is estimated to occur in approximately 1:4100 live births. Characteristic of the disease is the accumulation of α-Gal-A substrates, primarily the glycosphingolipids (GSLs) globotriaosylceramide and globotriaosylsphingosine. Thrombotic events are a significant concern for Fabry patients, with strokes contributing to a significant decrease in overall lifespan. Currently, the mechanisms underlying the increased risk of thrombotic events experienced by Fabry patients are incompletely defined. Using a rat model of Fabry disease, we provide an improved understanding of the mechanisms linking GSL accumulation to thrombotic risk. We found that ⍺-Gal A-deficient rats accumulate myeloid-derived leukocytes at sites of GSL accumulation, including in the bone marrow and circulation, and that myeloid-derived leukocyte and megakaryocyte populations were prominent among cell types that accumulated GSLs. In the circulation, ⍺-Gal A-deficient rats had increases in cytokine-producing cell types and a corresponding elevation of pro-inflammatory cytokines. Lastly, circulating platelets from ⍺-Gal A-deficient rats accumulated a similar set of ⍺-Galactosidase-A substrates as was observed in megakaryocytes in the bone marrow, and exhibited increased platelet binding to fibrinogen in microfluidic and flow cytometric assays.},
}
@article {pmid34320216,
year = {2021},
author = {Mushimiyimana, I and Niskanen, H and Beter, M and Laakkonen, JP and Kaikkonen, MU and Ylä-Herttuala, S and Laham-Karam, N},
title = {Characterization of a functional endothelial super-enhancer that regulates ADAMTS18 and angiogenesis.},
journal = {Nucleic acids research},
volume = {49},
number = {14},
pages = {8078-8096},
pmid = {34320216},
issn = {1362-4962},
mesh = {ADAMTS Proteins/*genetics ; CRISPR-Cas Systems/genetics ; Cell Division/genetics ; Cell Lineage/genetics ; Chromatin/*genetics ; Endothelial Cells/metabolism ; Enhancer Elements, Genetic/*genetics ; Humans ; Neovascularization, Physiologic/*genetics ; RNA, Small Interfering/genetics ; Signal Transduction/genetics ; },
abstract = {Super-enhancers are clusters of enhancers associated with cell lineage. They can be powerful gene-regulators and may be useful in cell-type specific viral-vector development. Here, we have screened for endothelial super-enhancers and identified an enhancer from within a cluster that conferred 5-70-fold increase in transgene expression. Importantly, CRISPR/Cas9 deletion of enhancers demonstrated regulation of ADAMTS18, corresponding to evidence of chromatin contacts between these genomic regions. Cell division-related pathways were primarily affected by the enhancer deletions, which correlated with significant reduction in cell proliferation. Furthermore, we observed changes in angiogenesis-related genes consistent with the endothelial specificity of this SE. Indeed, deletion of the enhancers affected tube formation, resulting in reduced or shortened sprouts. The super-enhancer angiogenic role is at least partly due to its regulation of ADAMTS18, as siRNA knockdown of ADAMTS18 resulted in significantly shortened endothelial sprouts. Hence, functional characterization of a novel endothelial super-enhancer has revealed substantial downstream effects from single enhancer deletions and led to the discovery of the cis-target gene ADAMTS18 and its role in endothelial function.},
}
@article {pmid34320214,
year = {2021},
author = {Wang, C and Tang, M and Chen, Z and Nie, L and Li, S and Xiong, Y and Szymonowicz, KA and Park, JM and Zhang, H and Feng, X and Huang, M and Su, D and Hart, T and Chen, J},
title = {Genetic vulnerabilities upon inhibition of DNA damage response.},
journal = {Nucleic acids research},
volume = {49},
number = {14},
pages = {8214-8231},
pmid = {34320214},
issn = {1362-4962},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA216437/CA/NCI NIH HHS/United States ; },
mesh = {14-3-3 Proteins/*genetics ; Apoptosis/drug effects ; Ataxia Telangiectasia Mutated Proteins/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/genetics ; Checkpoint Kinase 1/antagonists &amp; inhibitors/*genetics ; DNA Damage/*genetics ; DNA-Activated Protein Kinase/*genetics ; Genomic Instability/genetics ; Humans ; Microfilament Proteins/genetics ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; },
abstract = {Because of essential roles of DNA damage response (DDR) in the maintenance of genomic integrity, cellular homeostasis, and tumor suppression, targeting DDR has become a promising therapeutic strategy for cancer treatment. However, the benefits of cancer therapy targeting DDR are limited mainly due to the lack of predictive biomarkers. To address this challenge, we performed CRISPR screens to search for genetic vulnerabilities that affect cells' response to DDR inhibition. By undertaking CRISPR screens with inhibitors targeting key DDR mediators, i.e. ATR, ATM, DNAPK and CHK1, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Specifically, we identified YWHAE loss as a key determinant of sensitivity to CHK1 inhibition. We showed that KLHL15 loss protects cells from DNA damage induced by ATM inhibition. Moreover, we validated that APEX1 loss sensitizes cells to DNAPK inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results enhance the understanding of DDR pathways and will facilitate the use of DDR-targeting agents in cancer therapy.},
}
@article {pmid34315855,
year = {2021},
author = {Lei, H and Wang, Z and Jiang, D and Liu, F and Liu, M and Lei, X and Yang, Y and He, B and Yan, M and Huang, H and Liu, Q and Pang, J},
title = {CRISPR screening identifies CDK12 as a conservative vulnerability of prostate cancer.},
journal = {Cell death &amp; disease},
volume = {12},
number = {8},
pages = {740},
pmid = {34315855},
issn = {2041-4889},
mesh = {Androgen Antagonists/pharmacology ; Anilides/pharmacology ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Lineage/drug effects/genetics ; Cell Survival/drug effects/genetics ; Cyclin-Dependent Kinases/antagonists &amp; inhibitors/*metabolism ; Drug Synergism ; Epigenesis, Genetic/drug effects ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Male ; Prostatic Neoplasms/*enzymology/genetics/pathology ; Pyrimidines/pharmacology ; Receptors, Androgen/metabolism ; Signal Transduction/drug effects ; },
abstract = {Androgen receptor (AR) signaling inhibitors provide limited survival benefits to patients with prostate cancer (PCa), and worse, few feasible genomic lesions restrict targeted treatment to PCa. Thus, a better understanding of the critical dependencies of PCa may enable more feasible therapeutic approaches to the dilemma. We performed a kinome-scale CRISPR/Cas9 screen and identified cyclin-dependent kinase 12 (CDK12) as being conservatively required for PCa cell survival. Suppression of CDK12 by the covalent inhibitor THZ531 led to an obvious anti-PCa effect. Mechanistically, THZ531 downregulated AR signaling and preferentially repressed a distinct class of CDK12 inhibition-sensitive transcripts (CDK12-ISTs), including prostate lineage-specific genes, and contributed to cellular survival processes. Integration of the super-enhancer (SE) landscape and CDK12-ISTs indicated a group of potential PCa oncogenes, further conferring the sensitivity of PCa cells to CDK12 inhibition. Importantly, THZ531 strikingly synergized with multiple AR antagonists. The synergistic effect may be driven by attenuated H3K27ac signaling on AR targets and an intensive SE-associated apoptosis pathway. In conclusion, we highlight the validity of CDK12 as a druggable target in PCa. The synergy of THZ531 and AR antagonists suggests a potential combination therapy for PCa.},
}
@article {pmid34315458,
year = {2021},
author = {Edmondson, C and Zhou, Q and Liu, X},
title = {Analysis of conventional and alternative CRISPR/Cas9 genome editing to enhance a single-base pair knock-in mutation.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {45},
pmid = {34315458},
issn = {1472-6750},
support = {R01 CA075180/CA/NCI NIH HHS/United States ; },
mesh = {Base Pairing ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Humans ; *Mutation ; Phosphorylation ; Ribonucleoproteins/genetics/metabolism ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {BACKGROUND: The use of CRISPR/Cas9 technologies in generating single-base pair knock-in mutations has recently exploded in the number of methods available. However, with the growing expansion of new technologies, it can be difficult to determine the best method for genome editing.<br><br>RESULTS: In this study, we evaluated a number of CRISPR/Cas9 approaches for deriving cell lines with knock-in base pair edits to create a phosphorylation mutation and provide a breakdown of editing efficiencies and suggestions for improvement. Overall, our studies suggest that using pre-formed ribonucleoprotein (RNP) complexes is a reliable editing method to generate homozygous single-base pair mutations. We also show that antibiotic selection coupled homologous recombination is an efficient tool for generating highly specific heterozygous mutations.<br><br>CONCLUSION: The methods and/or combination of methods outlined in this study can be used to help other researchers with similar goals in single-base pair genome editing.},
}
@article {pmid34315245,
year = {2021},
author = {Yura, Y and Miura-Yura, E and Katanasaka, Y and Min, KD and Chavkin, N and Polizio, AH and Ogawa, H and Horitani, K and Doviak, H and Evans, MA and Sano, M and Wang, Y and Boroviak, K and Philippos, G and Domingues, AF and Vassiliou, G and Sano, S and Walsh, K},
title = {The Cancer Therapy-Related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-Ischemic Heart Failure in Mice.},
journal = {Circulation research},
volume = {129},
number = {6},
pages = {684-698},
pmid = {34315245},
issn = {1524-4571},
support = {MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; R01 HL138014/HL/NHLBI NIH HHS/United States ; R01 HL141256/HL/NHLBI NIH HHS/United States ; T32 HL007284/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiotensin II/toxicity ; Animals ; Clonal Hematopoiesis/*genetics ; DNA Damage ; *Gain of Function Mutation ; Heart Failure/etiology/*genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; Inflammasomes/*metabolism ; Interleukin-18/metabolism ; Interleukin-1beta/metabolism ; Macrophages/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Protein Phosphatase 2C/*genetics/metabolism ; },
abstract = {[Figure: see text].},
}
@article {pmid34314597,
year = {2021},
author = {Jakutis, G and Stainier, DYR},
title = {Genotype-Phenotype Relationships in the Context of Transcriptional Adaptation and Genetic Robustness.},
journal = {Annual review of genetics},
volume = {55},
number = {},
pages = {71-91},
doi = {10.1146/annurev-genet-071719-020342},
pmid = {34314597},
issn = {1545-2948},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Genotype ; Phenotype ; },
abstract = {Genetic manipulations with a robust and predictable outcome are critical to investigate gene function, as well as for therapeutic genome engineering. For many years, knockdown approaches and reagents including RNA interference and antisense oligonucleotides dominated functional studies; however, with the advent of precise genome editing technologies, CRISPR-based knockout systems have become the state-of-the-art tools for such studies. These technologies have helped decipher the role of thousands of genes in development and disease. Their use has also revealed how limited our understanding of genotype-phenotype relationships is. The recent discovery that certain mutations can trigger the transcriptional modulation of other genes, a phenomenon called transcriptional adaptation, has provided an additional explanation for the contradicting phenotypes observed in knockdown versus knockout models and increased awareness about the use of each of these approaches. In this review, we first cover the strengths and limitations of different gene perturbation strategies. Then we highlight the diverse ways in which the genotype-phenotype relationship can be discordant between these different strategies. Finally, we review the genetic robustness mechanisms that can lead to such discrepancies, paying special attention to the recently discovered phenomenon of transcriptional adaptation.},
}
@article {pmid34314595,
year = {2021},
author = {Boyd, CM and Angermeyer, A and Hays, SG and Barth, ZK and Patel, KM and Seed, KD},
title = {Bacteriophage ICP1: A Persistent Predator of Vibrio cholerae.},
journal = {Annual review of virology},
volume = {8},
number = {1},
pages = {285-304},
pmid = {34314595},
issn = {2327-0578},
support = {R01 AI127652/AI/NIAID NIH HHS/United States ; R01 AI153303/AI/NIAID NIH HHS/United States ; T32 GM132022/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages ; CRISPR-Cas Systems ; *Cholera/genetics ; Humans ; *Vibrio cholerae/genetics ; },
abstract = {Bacteriophages or phages-viruses of bacteria-are abundant and considered to be highly diverse. Interestingly, a particular group of lytic Vibrio cholerae-specific phages (vibriophages) of the International Centre for Diarrheal Disease Research, Bangladesh cholera phage 1 (ICP1) lineage show high levels of genome conservation over large spans of time and geography, despite a constant coevolutionary arms race with their host. From a collection of 67 sequenced ICP1 isolates, mostly from clinical samples, we find these phages have mosaic genomes consisting of large, conserved modules disrupted by variable sequences that likely evolve mostly through mobile endonuclease-mediated recombination during coinfection. Several variable regions have been associated with adaptations against antiphage elements in V. cholerae; notably, this includes ICP1's CRISPR-Cas system. The ongoing association of ICP1 and V. cholerae in cholera-endemic regions makes this system a rich source for discovery of novel defense and counterdefense strategies in bacteria-phage conflicts in nature.},
}
@article {pmid34313953,
year = {2021},
author = {Christou, P and Dhingra, A and Slamet-Loedin, IH and Oliveira, M and Chakraborty, S and Buyel, J and Stoger, E and Schillberg, S and Orzaez, D and Quemada, H},
title = {Preface: Genome editing in plants.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {317-320},
pmid = {34313953},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Plant ; *Plant Physiological Phenomena ; Plants/*genetics ; },
}
@article {pmid34312508,
year = {2022},
author = {Duan, Y and Ye, T and Qu, Z and Chen, Y and Miranda, A and Zhou, X and Lok, KC and Chen, Y and Fu, AKY and Gradinaru, V and Ip, NY},
title = {Brain-wide Cas9-mediated cleavage of a gene causing familial Alzheimer's disease alleviates amyloid-related pathologies in mice.},
journal = {Nature biomedical engineering},
volume = {6},
number = {2},
pages = {168-180},
pmid = {34312508},
issn = {2157-846X},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Amyloid beta-Protein Precursor/genetics/metabolism ; Animals ; Brain/metabolism ; CRISPR-Cas Systems ; Mice ; Mice, Transgenic ; },
abstract = {The pathology of familial Alzheimer's disease, which is caused by dominant mutations in the gene that encodes amyloid-beta precursor protein (APP) and in those that encode presenilin 1 and presenilin 2, is characterized by extracellular amyloid plaques and intracellular neurofibrillary tangles in multiple brain regions. Here we show that the brain-wide selective disruption of a mutated APP allele in transgenic mouse models carrying the human APP Swedish mutation alleviates amyloid-beta-associated pathologies for at least six months after a single intrahippocampal administration of an adeno-associated virus that encodes both Cas9 and a single-guide RNA that targets the mutation. We also show that the deposition of amyloid-beta, as well as microgliosis, neurite dystrophy and the impairment of cognitive performance, can all be ameliorated when the CRISPR-Cas9 construct is delivered intravenously via a modified adeno-associated virus that can cross the blood-brain barrier. Brain-wide disease-modifying genome editing could represent a viable strategy for the treatment of familial Alzheimer's disease and other monogenic diseases that affect multiple brain regions.},
}
@article {pmid34311897,
year = {2021},
author = {Briggiler Marcó, M and Mercanti, DJ},
title = {Bacteriophages in dairy plants.},
journal = {Advances in food and nutrition research},
volume = {97},
number = {},
pages = {1-54},
doi = {10.1016/bs.afnr.2021.02.015},
pmid = {34311897},
issn = {1043-4526},
mesh = {Animals ; *Bacteriophages ; Dairying ; Milk ; },
abstract = {Bacteriophages represent the main microbiological threat for the manufacture of fermented foods. The dairy industry is the most affected by this problem, as phages are naturally present in raw milk, surfaces, vats, tanks, floors, and distributed by air displacements. Cheese whey may also contain high phage concentrations. Prophages harbored by lysogenic strains could be induced, generating new lytic phages. In this context, where phages cannot be eradicated from dairies, methods of phage monitoring are mandatory. These are mainly based in microbiological features, like classical methods, that are the most used, economic and simple to carry out. Phage DNA detection and quantification by PCR and qPCR, more complex and expensive, are faster, although not able to discern between viable and non-viable virions. Electron microscopy allows direct visualization and characterization of phage morphology, but the apparatus is expensive. Alternative methods based in other phage traits also exist, though less studied and not applicable on a daily basis. Recognition of contamination sources and correct phage monitoring in dairy factories allow a correct application of control measures. These include general measures such as proper factory design, efficient programs of sanitization, good treatment of raw materials, especially milk, and careful handling of by-products. Additionally, the use of starts cultures should be adequate, with application of rotation schemes when possible. Finally, the selection of bacteriophage insensitive mutants (BIM) is essential, and can be achieved simply and empirically, though the study of CRISPR-Cas and other newly discovered mechanisms provide a more rational basis to obtain BIMs with optimized features.},
}
@article {pmid34310946,
year = {2021},
author = {Matsuzawa, T and Morita, M and Shimane, A and Otsuka, R and Mei, Y and Irie, F and Yamaguchi, Y and Yanai, K and Yoshikawa, T},
title = {Heparan sulfate promotes differentiation of white adipocytes to maintain insulin sensitivity and glucose homeostasis.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {3},
pages = {101006},
pmid = {34310946},
issn = {1083-351X},
support = {R01 AR055670/AR/NIAMS NIH HHS/United States ; },
mesh = {3T3-L1 Cells ; Adipocytes, White/*cytology/metabolism ; Animals ; Bone Morphogenetic Protein 4/metabolism ; CRISPR-Cas Systems ; Cell Differentiation/*physiology ; Fibroblast Growth Factor 1/metabolism ; Glucose/*metabolism ; Heparitin Sulfate/*physiology ; *Homeostasis ; *Insulin Resistance ; Mice ; Signal Transduction ; },
abstract = {Heparan sulfate (HS), a highly sulfated linear polysaccharide, is involved in diverse biological functions in various tissues. Although previous studies have suggested a possible contribution of HS to the differentiation of white adipocytes, there has been no direct evidence supporting this. Here, we inhibited the synthesis of HS chains in 3T3-L1 cells using CRISPR-Cas9 technology, resulting in impaired differentiation of adipocytes with attenuated bone morphogenetic protein 4 (BMP4)-fibroblast growth factor 1 (FGF1) signaling pathways. HS reduction resulted in reduced glucose uptake and decreased insulin-dependent intracellular signaling. We then made heterozygous mutant mice for the Ext1 gene, which encodes an enzyme essential for the HS biosynthesis, specifically in the visceral white adipose tissue (Fabp4-Cre[+]::Ext1[flox/WT] mice, hereafter called Ext1[Δ/WT]) to confirm the importance of HS in vivo. The expression levels of transcription factors that control adipocyte differentiation, such as peroxisome proliferator-activated receptor gamma, were reduced in Ext1[Δ/WT] adipocytes, which contained smaller, unilocular lipid droplets, reduced levels of enzymes involved in lipid synthesis, and altered expression of BMP4-FGF1 signaling molecules. Furthermore, we examined the impact of HS reduction in visceral white adipose tissue on systemic glucose homeostasis. We observed that Ext1[Δ/WT] mice showed glucose intolerance because of insulin resistance. Our results demonstrate that HS plays a crucial role in the differentiation of white adipocytes through BMP4-FGF1 signaling pathways, thereby contributing to insulin sensitivity and glucose homeostasis.},
}
@article {pmid34310625,
year = {2021},
author = {Zemmour, A and Dali-Yahia, R and Maatallah, M and Saidi-Ouahrani, N and Rahmani, B and Benhamouche, N and Al-Farsi, HM and Giske, CG},
title = {High-risk clones of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011-2012).},
journal = {PloS one},
volume = {16},
number = {7},
pages = {e0254805},
pmid = {34310625},
issn = {1932-6203},
mesh = {Algeria ; Anti-Bacterial Agents/adverse effects/pharmacology ; Bacterial Proteins/*genetics/metabolism ; Clone Cells/cytology/drug effects ; Drug Resistance, Multiple, Bacterial/drug effects/genetics ; Genome, Bacterial/genetics ; Hospitals, University ; Humans ; Klebsiella Infections/*drug therapy/enzymology/microbiology/pathology ; Klebsiella pneumoniae/*enzymology/pathogenicity ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Phylogeny ; Plasmids/drug effects ; Whole Genome Sequencing ; beta-Lactamases/*genetics/metabolism ; },
abstract = {The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum β-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.},
}
@article {pmid34310056,
year = {2021},
author = {He, C and Liu, H and Chen, D and Xie, WZ and Wang, M and Li, Y and Gong, X and Yan, W and Chen, LL},
title = {CRISPR-Cereal: a guide RNA design tool integrating regulome and genomic variation for wheat, maize and rice.},
journal = {Plant biotechnology journal},
volume = {19},
number = {11},
pages = {2141-2143},
pmid = {34310056},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Edible Grain/genetics ; Gene Editing ; Genomics ; *Oryza/genetics ; *RNA, Guide ; Triticum/genetics ; Zea mays/genetics ; },
}
@article {pmid34305865,
year = {2021},
author = {You, Y and Zhang, P and Wu, G and Tan, Y and Zhao, Y and Cao, S and Song, Y and Yang, R and Du, Z},
title = {Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {700016},
pmid = {34305865},
issn = {1664-302X},
abstract = {The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT-LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 10[2] colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.},
}
@article {pmid34305843,
year = {2021},
author = {Ramachandran, V and Weiland, JJ and Bolton, MD},
title = {CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {679994},
pmid = {34305843},
issn = {1664-302X},
abstract = {Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.},
}
@article {pmid34304411,
year = {2021},
author = {Tan, JJ and Peng, YZ and Huang, GT},
title = {[Research advances on the development and application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein system].},
journal = {Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns},
volume = {37},
number = {7},
pages = {681-687},
doi = {10.3760/cma.j.cn501120-20200329-00201},
pmid = {34304411},
issn = {1009-2587},
mesh = {*CRISPR-Associated Proteins ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Nucleic Acids ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein system, as an emerging gene editing system, can be divided into class 1 and class 2 systems according to the number of Cas protein. The CRISPR/Cas9 in class 2 system can cleave target nucleic acid only with the help of Cas9 protein and single-stranded guide RNA, which is currently the most widely used CRISPR/Cas system. In addition to gene editing in the treatment of genetic diseases, a variety of CRISPR/Cas system derived technologies have vast application prospect in the fields of disease-related gene screening, gene expression regulation, and rapid detection, prevention, and control of pathogens. This article summarizes the discovery process of CRISPR/Cas system and applications of several major CRISPR/Cas derived technologies, aiming to provide a reference for researchers in the field of life science.},
}
@article {pmid34304349,
year = {2022},
author = {Okoli, AS and Blix, T and Myhr, AI and Xu, W and Xu, X},
title = {Sustainable use of CRISPR/Cas in fish aquaculture: the biosafety perspective.},
journal = {Transgenic research},
volume = {31},
number = {1},
pages = {1-21},
pmid = {34304349},
issn = {1573-9368},
mesh = {Animals ; Aquaculture ; *CRISPR-Cas Systems ; *Containment of Biohazards ; Fishes/genetics ; Gene Editing ; },
abstract = {Aquaculture is becoming the primary source of seafood for human diets, and farmed fish aquaculture is one of its fastest growing sectors. The industry currently faces several challenges including infectious and parasitic diseases, reduced viability, fertility reduction, slow growth, escapee fish and environmental pollution. The commercialization of the growth-enhanced AquAdvantage salmon and the CRISPR/Cas9-developed tilapia (Oreochromis niloticus) proffers genetic engineering and genome editing tools, e.g. CRISPR/Cas, as potential solutions to these challenges. Future traits being developed in different fish species include disease resistance, sterility, and enhanced growth. Despite these notable advances, off-target effect and non-clarification of trait-related genes among other technical challenges hinder full realization of CRISPR/Cas potentials in fish breeding. In addition, current regulatory and risk assessment frameworks are not fit-for purpose regarding the challenges of CRISPR/Cas notwithstanding that public and regulatory acceptance are key to commercialization of products of the new technology. In this study, we discuss how CRISPR/Cas can be used to overcome some of these limitations focusing on diseases and environmental release in farmed fish aquaculture. We further present technical limitations, regulatory and risk assessment challenges of the use of CRISPR/Cas, and proffer research strategies that will provide much-needed data for regulatory decisions, risk assessments, increased public awareness and sustainable applications of CRISPR/Cas in fish aquaculture with emphasis on Atlantic salmon (Salmo salar) breeding.},
}
@article {pmid34303808,
year = {2021},
author = {Siddiqui, MF and Kim, MM},
title = {SIRT7 gene knockout using CRISPR/Cas9 system enhances melanin production in the melanoma cells.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1867},
number = {11},
pages = {166219},
doi = {10.1016/j.bbadis.2021.166219},
pmid = {34303808},
issn = {1879-260X},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation ; Gene Knockout Techniques ; Humans ; Melanins/*biosynthesis ; Mice ; Pigmentation Disorders/drug therapy/genetics ; Sirtuins/antagonists &amp; inhibitors/genetics/*metabolism ; Skin/metabolism ; Skin Pigmentation/*genetics ; },
abstract = {Melanin is a prominent pigment of skin and hair, and its deficiency can cause various disorders such as hair graying and albinism. The improvement of melanin production at a genetic level could offer an effective and permanent solution. Recently, SIRT7 has evoked an interest in the study of hair follicle stem cells, but its role in melanin synthesis remains unclear. In the present study, we have first successfully developed SIRT7 gene KO melanoma cells using the CRISPR/Cas9 system. It was found that the SIRT7 gene KO enhanced melanin production in melanoma cells. To validate the role of SIRT7 in melanin production, RT-PCR, western blot, and immunofluorescence staining assays were performed. The expression levels of melanin-producing genes and proteins (MITF, TRP1, TRP-2, TYR, TH) were significantly increased in SIRT7 gene KO cells compared to normal cells. In addition, melanin production was increased in KO cells higher than in normal cells through the image analysis. All these results suggest that SIRT7 could play an essential role in regulating melanin production, providing an alternative drug target to treat pigmentary disorders.},
}
@article {pmid34303705,
year = {2021},
author = {Safren, N and Tank, EM and Malik, AM and Chua, JP and Santoro, N and Barmada, SJ},
title = {Development of a specific live-cell assay for native autophagic flux.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {3},
pages = {101003},
pmid = {34303705},
issn = {1083-351X},
support = {P30 AG053760/AG/NIA NIH HHS/United States ; R01 NS097542/NS/NINDS NIH HHS/United States ; T32 GM007863/GM/NIGMS NIH HHS/United States ; P30 AG072931/AG/NIA NIH HHS/United States ; R01 NS113943/NS/NINDS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics ; Amyotrophic Lateral Sclerosis/genetics/therapy ; *Autophagy/drug effects ; Autophagy-Related Proteins/genetics ; C9orf72 Protein/genetics ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Drug Screening Assays, Antitumor ; Frontotemporal Dementia/genetics/therapy ; HEK293 Cells ; High-Throughput Screening Assays ; Humans ; Luminescent Proteins/genetics ; Microtubule-Associated Proteins/genetics ; Models, Biological ; Motor Neurons/metabolism ; Mutation ; },
abstract = {Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.},
}
@article {pmid34303184,
year = {2022},
author = {Shi, S and Qi, N and Nielsen, J},
title = {Microbial production of chemicals driven by CRISPR-Cas systems.},
journal = {Current opinion in biotechnology},
volume = {73},
number = {},
pages = {34-42},
doi = {10.1016/j.copbio.2021.07.002},
pmid = {34303184},
issn = {1879-0429},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; *Nucleic Acids ; },
abstract = {Microorganisms have provided an attractive route for biosynthesis of various chemicals from renewable resources. CRISPR-Cas systems have served as powerful mechanisms for generating cell factories with desirable properties by manipulating nucleic acids quickly and efficiently. The CRISPR-Cas system provides a toolbox with excellent opportunities for identifying better biocatalysts, multiplexed fine-tuning of metabolic flux, efficient utilization of low-cost substrates, and improvement of metabolic robustness. The overall goal of this review highlights recent advances in the development of microbial cell factories for chemical production using various CRISPR-Cas systems. The perspectives for further development or applications of CRISPR-Cas systems for strain improvement are also discussed.},
}
@article {pmid34302664,
year = {2021},
author = {Nishiga, M and Qi, LS and Wu, JC},
title = {CRISPRi/a Screening with Human iPSCs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2320},
number = {},
pages = {261-281},
pmid = {34302664},
issn = {1940-6029},
support = {R01 HL113006/HL/NHLBI NIH HHS/United States ; R01 HL123968/HL/NHLBI NIH HHS/United States ; R01 HL141851/HL/NHLBI NIH HHS/United States ; UH3 TR002588/TR/NCATS NIH HHS/United States ; R01 HL126527/HL/NHLBI NIH HHS/United States ; U01 EB021240/EB/NIBIB NIH HHS/United States ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Causality ; Cells, Cultured ; Chromatography, Liquid/methods ; DNA/isolation &amp; purification ; Doxycycline/pharmacology ; Flow Cytometry ; Gene Editing/*methods ; Genetic Association Studies ; Genetic Vectors/genetics ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Lentivirus/genetics ; Myocytes, Cardiac/cytology/metabolism ; RNA, Guide/genetics ; Transfection ; },
abstract = {Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.},
}
@article {pmid34302663,
year = {2021},
author = {Anzai, T and Hara, H and Chanthra, N and Sadahiro, T and Ieda, M and Hanazono, Y and Uosaki, H},
title = {Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2320},
number = {},
pages = {247-259},
pmid = {34302663},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Clone Cells ; Culture Media ; DNA Primers ; Drug Resistance/genetics ; Electroporation ; Embryonic Stem Cells/cytology ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Mice ; Mice, Inbred C57BL ; Puromycin/pharmacology ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; },
abstract = {A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.},
}
@article {pmid34302662,
year = {2021},
author = {Higo, S and Hikoso, S and Miyagawa, S and Sakata, Y},
title = {Genome Editing in Human Induced Pluripotent Stem Cells (hiPSCs).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2320},
number = {},
pages = {235-245},
pmid = {34302662},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cell Differentiation ; Clone Cells/cytology/metabolism ; Electroporation/methods ; Equipment Design ; Gene Editing/*methods ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Myocytes, Cardiac/*metabolism ; Puromycin/pharmacology ; Recombinational DNA Repair ; },
abstract = {Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) are powerful tools for elucidating the pathology behind inherited cardiomyopathies. Genome editing technologies enable targeted genome replacement and the generation of isogenic hiPSCs, allowing investigators to precisely determine the roles of identified mutations. Here, we describe a protocol to obtain isogenic hiPSCs with the corrected allele via homology-directed repair (HDR) using CRISPR/Cas9 genome editing under feeder-free conditions. Seeding hiPSCs in a 24-well plate and conducting the initial evaluation using direct genomic sequencing after 1 week is cost- and time-effective. Following optimization of the protocol, sequence confirmation of the corrected HDR clone is completed within 21 days.},
}
@article {pmid34302285,
year = {2021},
author = {Qu, J and Xie, Y and Guo, Z and Liu, X and Jiang, J and Chen, T and Li, K and Hu, Z and Luo, D},
title = {Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets.},
journal = {Molecular biotechnology},
volume = {63},
number = {12},
pages = {1183-1191},
pmid = {34302285},
issn = {1559-0305},
mesh = {Archaeal Proteins/genetics ; Argonaute Proteins/*genetics ; CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Natronobacterium/genetics/*metabolism ; RNA Splicing ; RNA, Guide/genetics ; },
abstract = {Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5' phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3' of the target RNA which is recognized by 5' sequence of the gDNA. These results confirmed that NgAgo-gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3' of the target RNA, which is a new finding that has not been reported in the past.},
}
@article {pmid34302019,
year = {2021},
author = {Li, CY and Steighner, JR and Sweatt, G and Thiele, TR and Juntti, SA},
title = {Manipulation of the Tyrosinase gene permits improved CRISPR/Cas editing and neural imaging in cichlid fish.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {15138},
pmid = {34302019},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cichlids/*genetics ; Evolution, Molecular ; Gene Editing/methods ; Lakes ; Monophenol Monooxygenase/*genetics ; Phenotype ; Tanzania ; },
abstract = {Direct tests of gene function have historically been performed in a limited number of model organisms. The CRISPR/Cas system is species-agnostic, offering the ability to manipulate genes in a range of models, enabling insights into evolution, development, and physiology. Astatotilapia burtoni, a cichlid fish from the rivers and shoreline around Lake Tanganyika, has been extensively studied in the laboratory to understand evolution and the neural control of behavior. Here we develop protocols for the creation of CRISPR-edited cichlids and create a broadly useful mutant line. By manipulating the Tyrosinase gene, which is necessary for eumelanin pigment production, we describe a fast and reliable approach to quantify and optimize gene editing efficiency. Tyrosinase mutants also remove a major obstruction to imaging, enabling visualization of subdermal structures and fluorophores in situ. These protocols will facilitate broad application of CRISPR/Cas9 to studies of cichlids as well as other non-traditional model aquatic species.},
}
@article {pmid34301761,
year = {2021},
author = {Teng, K and Ford, MJ and Harwalkar, K and Li, Y and Pacis, AS and Farnell, D and Yamanaka, N and Wang, YC and Badescu, D and Ton Nu, TN and Ragoussis, J and Huntsman, DG and Arseneau, J and Yamanaka, Y},
title = {Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing.},
journal = {Cancer research},
volume = {81},
number = {20},
pages = {5147-5160},
doi = {10.1158/0008-5472.CAN-20-1518},
pmid = {34301761},
issn = {1538-7445},
mesh = {AMP-Activated Protein Kinases/*physiology ; Animals ; BRCA1 Protein/physiology ; *CRISPR-Cas Systems ; Cystadenocarcinoma, Serous/genetics/*pathology ; DNA Copy Number Variations ; *Disease Models, Animal ; Electroporation ; Fallopian Tubes/metabolism/*pathology ; Female ; *Gene Editing ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Ovarian Neoplasms/genetics/*pathology ; PTEN Phosphohydrolase/physiology ; Tumor Suppressor Protein p53/physiology ; },
abstract = {Ovarian cancer is the most lethal gynecologic cancer to date. High-grade serous ovarian carcinoma (HGSOC) accounts for most ovarian cancer cases, and it is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR-Cas9-mediated genome editing. Mutation of tumor suppressor genes associated with HGSOC in two different combinations (Brca1, Tp53, Pten with and without Lkb1) resulted in successfully generation of HGSOC, albeit with different latencies and pathophysiology. Implementing Cre lineage tracing in this system enabled visualization of peritoneal micrometastases in an immune-competent environment. In addition, these models displayed copy number alterations and phenotypes similar to human HGSOC. Because this strategy is flexible in selecting mutation combinations and targeting areas, it could prove highly useful for generating mouse models to advance the understanding and treatment of ovarian cancer. SIGNIFICANCE: This study unveils a new strategy to generate genetic mouse models of ovarian cancer with high flexibility in selecting mutation combinations and targeting areas.},
}
@article {pmid34299081,
year = {2021},
author = {Alghuthaymi, MA and Ahmad, A and Khan, Z and Khan, SH and Ahmed, FK and Faiz, S and Nepovimova, E and Kuča, K and Abd-Elsalam, KA},
title = {Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {14},
pages = {},
pmid = {34299081},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Exosomes/*chemistry ; *Gene Editing ; *Genetic Therapy ; Genome, Plant ; Liposomes/*chemistry ; Nanoparticles/*chemistry ; Plants/*genetics ; },
abstract = {Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.},
}
@article {pmid34298471,
year = {2021},
author = {Bonini, A and Poma, N and Vivaldi, F and Biagini, D and Bottai, D and Tavanti, A and Di Francesco, F},
title = {A label-free impedance biosensing assay based on CRISPR/Cas12a collateral activity for bacterial DNA detection.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {204},
number = {},
pages = {114268},
doi = {10.1016/j.jpba.2021.114268},
pmid = {34298471},
issn = {1873-264X},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Electric Impedance ; Escherichia coli/genetics ; Humans ; Staphylococcus aureus/genetics ; },
abstract = {The rapid and selective identification in the clinical setting of pathogenic bacteria causing healthcare associated infections (HAIs) and in particular blood stream infections (BSIs) is a major challenge, as the number of people affected worldwide and the associated mortality are on the rise. In fact, traditional laboratory techniques such culture and polymerase chain reaction (PCR)-based methodologies are often associated to long turnaround times, which justify the pressing need for the development of rapid, specific and portable point of care devices. The recently discovered clustered regularly interspaced short palindromic repeat loci (CRISPR) and the new class of programmable endonuclease enzymes called CRISPR associated proteins (Cas) have revolutionised molecular diagnostics. The use of Cas proteins in optical and electrochemical biosensing devices has significantly improved the detection of nucleic acids in clinical samples. In this study, a CRISPR/Cas12a system was coupled with electrochemical impedance spectroscopy (EIS) measurements to develop a label-free biosensing assay for the detection of Escherichia coli and Staphylococcus aureus, two bacterial species commonly associated to BSI infections. The programmable Cas12a endonuclease activity, induced by a specific guide RNA (gRNA), and the triggered collateral activity were assessed in in vitro restriction analyses, and evaluated thanks to impedance measurements using a modified gold electrode. The Cas12a/gRNA system was able to specifically recognize amplicons from different clinical isolates of E. coli and S. aureus with a limit of detection of 3 nM and a short turnaround time approximately of 1.5 h. To the best of our knowledge, this is the first biosensing device based on CRISPR/Cas12a label free impedance assay.},
}
@article {pmid34298016,
year = {2021},
author = {Fleck, N and Grundner, C},
title = {A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria.},
journal = {The Journal of biological chemistry},
volume = {297},
number = {2},
pages = {100990},
pmid = {34298016},
issn = {1083-351X},
support = {R01 AI117023/AI/NIAID NIH HHS/United States ; R03 AI131223/AI/NIAID NIH HHS/United States ; R21 AI137571/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Endodeoxyribonucleases/genetics/*metabolism ; Gene Editing/*methods ; Gene Expression ; *Gene Expression Regulation, Bacterial ; Gene Knockdown Techniques/*methods ; Humans ; Mycobacterium tuberculosis/*metabolism ; },
abstract = {Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.},
}
@article {pmid34297925,
year = {2021},
author = {Bosch, B and DeJesus, MA and Poulton, NC and Zhang, W and Engelhart, CA and Zaveri, A and Lavalette, S and Ruecker, N and Trujillo, C and Wallach, JB and Li, S and Ehrt, S and Chait, BT and Schnappinger, D and Rock, JM},
title = {Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis.},
journal = {Cell},
volume = {184},
number = {17},
pages = {4579-4592.e24},
pmid = {34297925},
issn = {1097-4172},
support = {DP2 AI144850/AI/NIAID NIH HHS/United States ; P41 GM103314/GM/NIGMS NIH HHS/United States ; P41 GM109824/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acyl-tRNA Synthetases/metabolism ; Antitubercular Agents/pharmacology ; Bayes Theorem ; Biological Evolution ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Expression Regulation, Bacterial/drug effects ; Gene Silencing/drug effects ; *Genome, Bacterial ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis/drug effects/*genetics ; RNA, Guide/genetics ; },
abstract = {Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.},
}
@article {pmid34297462,
year = {2021},
author = {Li, X and Pan, Y and Chen, C and Gao, Y and Liu, X and Yang, K and Luan, X and Zhou, D and Zeng, F and Han, X and Song, Y},
title = {Hypoxia-Responsive Gene Editing to Reduce Tumor Thermal Tolerance for Mild-Photothermal Therapy.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {39},
pages = {21200-21204},
doi = {10.1002/anie.202107036},
pmid = {34297462},
issn = {1521-3773},
mesh = {A549 Cells ; Antineoplastic Agents/chemistry/*pharmacology ; Azo Compounds/chemistry/*pharmacology ; CRISPR-Cas Systems/*drug effects/genetics ; Cell Hypoxia/*drug effects ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Dicarboxylic Acids/chemistry/*pharmacology ; Drug Screening Assays, Antitumor ; Gene Editing ; Gold/chemistry/*pharmacology ; Humans ; Infrared Rays ; Metal Nanoparticles/chemistry ; Particle Size ; *Photothermal Therapy ; },
abstract = {Near-infrared (NIR)-light-triggered photothermal therapy (PTT) is usually associated with undesirable damage to healthy organs nearby due to the high temperatures (>50 °C) available for tumor ablation. Low-temperature PTT would therefore have tremendous value for clinical application. Here, we construct a hypoxia-responsive gold nanorods (AuNRs)-based nanocomposite of CRISPR-Cas9 for mild-photothermal therapy via tumor-targeted gene editing. AuNRs are modified with azobenzene-4,4'-dicarboxylic acid (p-AZO) to achieve on-demand release of CRISPR-Cas9 using hypoxia-responsive azo bonds. In the hypoxic tumor microenvironment, the azo groups of the hypoxia-activated CRISPR-Cas9 nanosystem based on gold nanorods (APACPs) are selectively reduced by the overexpression of reductases, leading to the release of Cas9 and subsequent gene editing. Owing to the knockout of HSP90α for reducing the thermal resistance of cancer cells, highly effective tumor ablation both in vitro and in vivo was achieved with APACPs under mild PTT.},
}
@article {pmid34297039,
year = {2021},
author = {Herzner, AM and Khan, Z and Van Nostrand, EL and Chan, S and Cuellar, T and Chen, R and Pechuan-Jorge, X and Komuves, L and Solon, M and Modrusan, Z and Haley, B and Yeo, GW and Behrens, TW and Albert, ML},
title = {ADAR and hnRNPC deficiency synergize in activating endogenous dsRNA-induced type I IFN responses.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {9},
pages = {},
pmid = {34297039},
issn = {1540-9538},
support = {U54HG007005/HG/NHGRI NIH HHS/United States ; HG009530/HG/NHGRI NIH HHS/United States ; R00 HG009530/HG/NHGRI NIH HHS/United States ; U54 HG007005/HG/NHGRI NIH HHS/United States ; U41 HG009889/HG/NHGRI NIH HHS/United States ; K99 HG009530/HG/NHGRI NIH HHS/United States ; },
mesh = {Adenosine Deaminase/*genetics/metabolism ; Alu Elements ; CRISPR-Cas Systems ; Cytosol/physiology ; Heterogeneous-Nuclear Ribonucleoprotein Group C/*genetics/metabolism ; Humans ; Interferon Type I/*genetics/metabolism ; Interferon-Induced Helicase, IFIH1/genetics/metabolism ; Introns ; MCF-7 Cells ; Membrane Proteins/genetics/metabolism ; RNA Editing ; RNA, Double-Stranded/*metabolism ; RNA-Binding Proteins/*genetics/metabolism ; THP-1 Cells ; },
abstract = {Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element-induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.},
}
@article {pmid34296745,
year = {2021},
author = {Isiaku, AI and Zhang, Z and Pazhakh, V and Manley, HR and Thompson, ER and Fox, LC and Yerneni, S and Blombery, P and Lieschke, GJ},
title = {Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions.},
journal = {Disease models &amp; mechanisms},
volume = {14},
number = {7},
pages = {},
pmid = {34296745},
issn = {1754-8411},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Macrophages/metabolism ; Neutrophils/metabolism ; Transcription Factors/metabolism ; *Zebrafish/genetics/metabolism ; },
abstract = {Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid34296348,
year = {2021},
author = {Palihati, M and Tsubouchi, H and Argunhan, B and Kajitani, R and Bakenova, O and Han, YW and Murayama, Y and Itoh, T and Iwasaki, H},
title = {Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens.},
journal = {Current genetics},
volume = {67},
number = {6},
pages = {919-936},
pmid = {34296348},
issn = {1432-0983},
mesh = {Basidiomycota/*genetics ; CRISPR-Cas Systems ; Fungal Proteins/*metabolism ; Gene Editing ; *Gene Targeting ; Genetic Complementation Test ; Genetic Engineering ; Genetic Loci ; Homologous Recombination ; Intramolecular Oxidoreductases/genetics/metabolism ; Ku Autoantigen/genetics ; Rad51 Recombinase/*metabolism ; Rad52 DNA Repair and Recombination Protein/*metabolism ; Transformation, Genetic ; },
abstract = {Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.},
}
@article {pmid34295340,
year = {2021},
author = {Xia, Z and Xu, J and Lu, E and He, W and Deng, S and Gong, AY and Strass-Soukup, J and Martins, GA and Lu, G and Chen, XM},
title = {m[6]A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {705232},
pmid = {34295340},
issn = {1664-3224},
support = {R01 AI116323/AI/NIAID NIH HHS/United States ; R01 AI136877/AI/NIAID NIH HHS/United States ; R21 AI141325/AI/NIAID NIH HHS/United States ; R21 AI156370/AI/NIAID NIH HHS/United States ; G20 RR024001/RR/NCRR NIH HHS/United States ; },
mesh = {Adenosine/*analogs &amp; derivatives/physiology ; AlkB Homolog 5, RNA Demethylase/antagonists &amp; inhibitors/biosynthesis/genetics ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/biosynthesis/genetics ; Animals ; CRISPR-Cas Systems ; Cryptosporidiosis/*immunology ; Cryptosporidium parvum/*immunology ; Epithelium/*immunology ; GTP Phosphohydrolases/biosynthesis/genetics ; GTP-Binding Proteins/biosynthesis/genetics ; Gene Expression Regulation/immunology ; Humans ; *Immunity, Innate ; Intestinal Mucosa/cytology/*immunology ; Methylation ; Mice ; Mice, Inbred C57BL ; NF-kappa B/metabolism ; RNA Interference ; *RNA Processing, Post-Transcriptional ; RNA, Messenger/*immunology ; RNA, Small Interfering/genetics ; },
abstract = {Increasing evidence supports that N6-methyladenosine (m[6]A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m[6]A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m[6]A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m[6]A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m[6]A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m[6]A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.},
}
@article {pmid34294769,
year = {2021},
author = {Purusothaman, DK and Shackleford, L and Anderson, MAE and Harvey-Samuel, T and Alphey, L},
title = {CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14964},
pmid = {34294769},
issn = {2045-2322},
support = {BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Culex/genetics/*growth &amp; development/virology ; DNA Repair ; Disease Vectors ; Female ; Gene Knock-In Techniques/*veterinary ; Genes, Recessive ; Germ Cells/growth &amp; development/metabolism ; Insect Proteins/genetics ; Kynurenine 3-Monooxygenase/*genetics ; Male ; Pest Control, Biological ; Promoter Regions, Genetic ; RNA Polymerase III/*genetics ; West Nile virus/pathogenicity ; },
abstract = {Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.},
}
@article {pmid34294706,
year = {2021},
author = {Carabias, A and Fuglsang, A and Temperini, P and Pape, T and Sofos, N and Stella, S and Erlendsson, S and Montoya, G},
title = {Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4476},
pmid = {34294706},
issn = {2041-1723},
mesh = {Bacteriophages/enzymology/genetics ; CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Catalytic Domain ; Cryoelectron Microscopy ; DNA Cleavage ; Endodeoxyribonucleases/*chemistry/genetics/metabolism ; Escherichia coli Proteins/chemistry/genetics/metabolism ; *Gene Editing ; Models, Molecular ; Mutagenesis, Site-Directed ; Protein Conformation ; RNA, Guide/genetics/metabolism ; RNA, Viral/genetics/metabolism ; },
abstract = {CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.},
}
@article {pmid34294050,
year = {2021},
author = {Straume, AH and Kjærner-Semb, E and Skaftnesmo, KO and Güralp, H and Lillico, S and Wargelius, A and Edvardsen, RB},
title = {Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {563},
pmid = {34294050},
issn = {1471-2164},
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Nucleotides ; Oligonucleotides ; *Salmo salar/genetics ; },
abstract = {BACKGROUND: New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon.<br><br>RESULTS: Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively).<br><br>CONCLUSIONS: In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.},
}
@article {pmid34293821,
year = {2022},
author = {Benamar, M and Charbonnier, LM},
title = {Large scale regulatory T cells screening.},
journal = {Allergy},
volume = {77},
number = {2},
pages = {705-707},
doi = {10.1111/all.15019},
pmid = {34293821},
issn = {1398-9995},
mesh = {*CRISPR-Cas Systems ; Forkhead Transcription Factors/metabolism ; Humans ; *T-Lymphocytes, Regulatory/metabolism ; },
}
@article {pmid34293450,
year = {2021},
author = {Yu, EPY and Perin, S and Saxena, V and Ekker, M},
title = {Novel cross-regulation interactions between dlx genes in larval zebrafish.},
journal = {Gene},
volume = {801},
number = {},
pages = {145848},
doi = {10.1016/j.gene.2021.145848},
pmid = {34293450},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems ; DNA, Intergenic ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/*genetics ; Larva ; Mutation ; Transcription Factors/*genetics ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {The homeodomain-containing transcription factors dlx1a, dlx2a, dlx5a and dlx6a are expressed in the zebrafish brain in overlapping patterns and are important in vertebrate development. Previous work in mice have suggested the overlapping expression pattern is in part due to cross-regulatory interactions among the aforementioned dlx genes. However, the extent of these interactions and whether they are conserved among vertebrates remains to be determined. Through whole-mount in situ hybridization in zebrafish dlx mutants produced by CRISPR-Cas9 mutagenesis, cross-regulatory interactions between dlx1a, dlx2a, dlx5a and dlx6a were examined from 24 to 72 h post fertilization (hpf). Notably, and different from previous work done in mouse, zebrafish dlx2a[-/-] mutants continue to express dlx5a until 72hpf, whereas deletion of both enhancers within the dlx5a/dlx6a locus resulted in delayed dlx5a/dlx6a expression and relative increased dlx2a expression. These results suggest alternative regulatory elements and pathways exist to mediate dlx expression in zebrafish and may highlight evolutionary differences in gene interactions between vertebrates.},
}
@article {pmid34293331,
year = {2021},
author = {Kiyonari, H and Kaneko, M and Abe, T and Shiraishi, A and Yoshimi, R and Inoue, KI and Furuta, Y},
title = {Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing.},
journal = {Current biology : CB},
volume = {31},
number = {17},
pages = {3956-3963.e4},
doi = {10.1016/j.cub.2021.06.056},
pmid = {34293331},
issn = {1879-0445},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Editing ; Genome ; Mice ; *Monodelphis/genetics ; Rats ; },
abstract = {Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.[1][,][2] However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.},
}
@article {pmid34291668,
year = {2021},
author = {Hasanzadeh, A and Radmanesh, F and Hosseini, ES and Hashemzadeh, I and Kiani, J and Naseri, M and Nourizadeh, H and Fatahi, Y and Azar, BKY and Marani, BG and Beyzavi, A and Mahabadi, VP and Karimi, M},
title = {Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery.},
journal = {Nanomedicine (London, England)},
volume = {16},
number = {19},
pages = {1673-1690},
doi = {10.2217/nnm-2021-0038},
pmid = {34291668},
issn = {1748-6963},
mesh = {*CRISPR-Cas Systems ; Carbon ; Cholecalciferol ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Transfer Techniques ; Humans ; },
abstract = {Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials &amp; methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.},
}
@article {pmid34291612,
year = {2021},
author = {Mehmood, A and Ali, W and Din, ZU and Song, S and Sohail, M and Shah, W and Guo, J and Guo, RY and Ilahi, I and Shah, S and Al-Shaebi, F and Zeb, L and Asiamah, EA and Al-Dhamin, Z and Bilal, H and Li, B},
title = {Clustered regularly interspaced short palindromic repeats as an advanced treatment for Parkinson's disease.},
journal = {Brain and behavior},
volume = {11},
number = {8},
pages = {e2280},
pmid = {34291612},
issn = {2162-3279},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Neurodegenerative Diseases ; *Parkinson Disease/genetics/therapy ; alpha-Synuclein ; },
abstract = {Recently, genome-editing technology like clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has improved the translational gap in the treatments mediated through gene therapy. The advantages of the CRISPR system, such as, work in the living cells and tissues, candidate this technique for the employing in experiments and the therapy of central nervous system diseases. Parkinson's disease (PD) is a widespread, disabling, neurodegenerative disease induced by dopaminergic neuron loss and linked to progressive motor impairment. Pathophysiological basis knowledge of PD has modified the PD classification model and expresses in the sporadic and familial types. Analyses of the earliest genetic linkage have shown in PD the inclusion of synuclein alpha (SNCA) genomic duplication and SNCA mutations in the familial types of PD pathogenesis. This review analyzes the structure, development, and function in genome editing regulated through the CRISPR/Cas9. Also, it explains the genes associated with PD pathogenesis and the appropriate modifications to favor PD. This study follows the direction by understanding the PD linking analyses in which the CRISPR technique is applied. Finally, this study explains the limitations and future trends of CRISPR service in relation to the genome-editing process in PD patients' induced pluripotent stem cells.},
}
@article {pmid34290274,
year = {2021},
author = {Innamorati, G and Wilkie, TM and Malpeli, G and Paiella, S and Grasso, S and Rusev, B and Leone, BE and Valenti, MT and Carbonare, LD and Cheri, S and Giacomazzi, A and Zanotto, M and Guardini, V and Deiana, M and Zipeto, D and Serena, M and Parenti, M and Guzzi, F and Lawlor, RT and Malerba, G and Mori, A and Malleo, G and Giacomello, L and Salvia, R and Bassi, C},
title = {Gα15 in early onset of pancreatic ductal adenocarcinoma.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14922},
pmid = {34290274},
issn = {2045-2322},
support = {CA192381/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/*genetics/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; GTP-Binding Protein alpha Subunits, Gq-G11/*genetics ; GTP-Binding Proteins/metabolism ; Gene Expression/genetics ; Gene Expression Regulation, Neoplastic/*genetics ; Humans ; Methylation ; Neoplasm Invasiveness/genetics ; Pancreatic Neoplasms/*genetics/pathology ; Prognosis ; Promoter Regions, Genetic/genetics ; RNA, Messenger ; RNA, Small Interfering ; Signal Transduction ; },
abstract = {The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5' GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.},
}
@article {pmid34290256,
year = {2021},
author = {VanDusen, NJ and Lee, JY and Gu, W and Butler, CE and Sethi, I and Zheng, Y and King, JS and Zhou, P and Suo, S and Guo, Y and Ma, Q and Yuan, GC and Pu, WT},
title = {Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4442},
pmid = {34290256},
issn = {2041-1723},
support = {R01 HL146634/HL/NHLBI NIH HHS/United States ; R01 HG009663/HG/NHGRI NIH HHS/United States ; K99 HL143194/HL/NHLBI NIH HHS/United States ; T32 HL007572/HL/NHLBI NIH HHS/United States ; S10 OD026880/OD/NIH HHS/United States ; UM1 HL098166/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; Gene Expression Regulation, Developmental ; Histones/metabolism ; Mice ; Mutagenesis ; Myocytes, Cardiac/metabolism/*physiology ; Phenotype ; Reproducibility of Results ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Ubiquitination ; },
abstract = {The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.},
}
@article {pmid34290231,
year = {2021},
author = {Zhu, GD and Yu, J and Sun, ZY and Chen, Y and Zheng, HM and Lin, ML and Ou-Yang, S and Liu, GL and Zhang, JW and Shao, FM},
title = {Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma.},
journal = {Cell death &amp; disease},
volume = {12},
number = {8},
pages = {724},
pmid = {34290231},
issn = {2041-4889},
mesh = {Brain Neoplasms/*genetics/pathology/radiotherapy ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA-Binding Proteins/genetics/*metabolism ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; *Genome, Human ; Glioblastoma/*genetics/pathology/radiotherapy ; Humans ; Phosphoproteins/genetics/*metabolism ; RNA, Messenger/genetics/metabolism ; Radiation Tolerance/*genetics ; Transcription Factors/genetics/*metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Up-Regulation/genetics ; },
abstract = {Glioblastomas (GBM) is the most common primary malignant brain tumor, and radiotherapy plays a critical role in its therapeutic management. Unfortunately, the development of radioresistance is universal. Here, we identified calcium-regulated heat-stable protein 1 (CARHSP1) as a critical driver for radioresistance utilizing genome-wide CRISPR activation screening. This is a protein with a cold-shock domain (CSD)-containing that is highly similar to cold-shock proteins. CARHSP1 mRNA level was upregulated in irradiation-resistant GBM cells and knockdown of CARHSP1 sensitized GBM cells to radiotherapy. The high expression of CARHSP1 upon radiation might mediate radioresistance by activating the inflammatory signaling pathway. More importantly, patients with high levels of CARHSP1 had poorer survival when treated with radiotherapy. Collectively, our findings suggested that targeting the CARHSP1/TNF-α inflammatory signaling activation induced by radiotherapy might directly affect radioresistance and present an attractive therapeutic target for GBM, particularly for patients with high levels of CARHSP1.},
}
@article {pmid34290169,
year = {2022},
author = {Oyama, Y and Miyata, H and Shimada, K and Fujihara, Y and Tokuhiro, K and Garcia, TX and Matzuk, MM and Ikawa, M},
title = {CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice.},
journal = {Asian journal of andrology},
volume = {24},
number = {3},
pages = {266-272},
pmid = {34290169},
issn = {1745-7262},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; R01 HD095341/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Fertility/genetics ; *Gene Editing ; Humans ; Male ; Mice ; Mice, Knockout ; *Testis/metabolism ; },
abstract = {Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.},
}
@article {pmid34289313,
year = {2022},
author = {Cameranesi, MM and Kurth, D and Repizo, GD},
title = {Acinetobacter defence mechanisms against biological aggressors and their use as alternative therapeutic applications.},
journal = {Critical reviews in microbiology},
volume = {48},
number = {1},
pages = {21-41},
doi = {10.1080/1040841X.2021.1939266},
pmid = {34289313},
issn = {1549-7828},
mesh = {*Acinetobacter Infections/drug therapy ; *Acinetobacter baumannii/genetics ; Bacteria ; Humans ; },
abstract = {Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii being the species of greatest worldwide concern due to its multi-drug resistance and the recent appearance of hyper-virulent strains in the clinical setting. Colonisation of this environment is associated with a multitude of bacterial factors, and the molecular features that promote environmental persistence in abiotic surfaces, including intrinsic desiccation resistance, biofilm formation and motility, have been previously addressed. On the contrary, mechanisms enabling Acinetobacter spp. survival when faced against other biological competitors are starting to be characterised. Among them, secretion systems (SS) of different types, such as the T5bSS (Contact-dependent inhibition systems) and the T6SS, confer adaptive advantages against bacterial aggressors. Regarding mechanisms of defence against bacteriophages, such as toxin-antitoxin, restriction-modification, Crispr-Cas and CBASS, among others, have been identified but remain poorly characterised. In view of this, we aimed to summarise the present knowledge on defence mechanisms that enable niche establishment in members of the Acinetobacter genus. Different proposals are also described for the use of some components of these systems as molecular tools to treat Acinetobacter infections.},
}
@article {pmid34289223,
year = {2021},
author = {Cai, Z and Xian, P and Cheng, Y and Ma, Q and Lian, T and Nian, H and Ge, L},
title = {CRISPR/Cas9-mediated gene editing of GmJAGGED1 increased yield in the low-latitude soybean variety Huachun 6.},
journal = {Plant biotechnology journal},
volume = {19},
number = {10},
pages = {1898-1900},
pmid = {34289223},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Fabaceae ; *Gene Editing ; Plants, Genetically Modified/genetics ; Soybeans/genetics ; },
}
@article {pmid34288688,
year = {2021},
author = {Ahmad, S and Tang, L and Shahzad, R and Mawia, AM and Rao, GS and Jamil, S and Wei, C and Sheng, Z and Shao, G and Wei, X and Hu, P and Mahfouz, MM and Hu, S and Tang, S},
title = {CRISPR-Based Crop Improvements: A Way Forward to Achieve Zero Hunger.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {30},
pages = {8307-8323},
doi = {10.1021/acs.jafc.1c02653},
pmid = {34288688},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Genome, Plant ; Hunger ; Plant Breeding ; },
abstract = {Zero hunger is one of the sustainable development goals set by the United Nations in 2015 to achieve global food security by 2030. The current harvest of crops is insufficient; feeding the world's population and meeting the goal of zero hunger by 2030 will require larger and more consistent crop production. Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) technology is widely used for the plant genome editing. In this review, we consider this technology as a potential tool for achieving zero hunger. We provide a comprehensive overview of CRISPR-Cas technology and its most important applications for food crops' improvement. We also conferred current and potential technological breakthroughs that will help in breeding future crops to end global hunger. The regulatory aspects of deploying this technology in commercial sectors, bioethics, and the production of transgene-free plants are also discussed. We hope that the CRISPR-Cas system will accelerate the breeding of improved crop cultivars compared with conventional breeding and pave the way toward the zero hunger goal.},
}
@article {pmid34288010,
year = {2021},
author = {Tilson, SG and Morell, CM and Lenaerts, AS and Park, SB and Hu, Z and Jenkins, B and Koulman, A and Liang, TJ and Vallier, L},
title = {Modeling PNPLA3-Associated NAFLD Using Human-Induced Pluripotent Stem Cells.},
journal = {Hepatology (Baltimore, Md.)},
volume = {74},
number = {6},
pages = {2998-3017},
doi = {10.1002/hep.32063},
pmid = {34288010},
issn = {1527-3350},
support = {MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; NC/T2T0419/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; NC/R001987/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; NC/N001540/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Ethanol/toxicity ; Gene Knockout Techniques ; Genetic Predisposition to Disease ; Hepatocytes/drug effects/*pathology ; Humans ; Induced Pluripotent Stem Cells ; Lipase/*genetics ; Lipid Metabolism/genetics ; Loss of Function Mutation ; Membrane Proteins/*genetics ; Methotrexate/toxicity ; Non-alcoholic Fatty Liver Disease/*genetics/pathology ; Polymorphism, Single Nucleotide ; Toxicity Tests, Acute ; },
abstract = {BACKGROUND AND AIMS: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human-induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology.<br><br>APPROACH AND RESULTS: We used isogenic hiPSC lines with either a knockout (PNPLA3[KO]) of the PNPLA3 gene or with the I148M variant (PNPLA3[I148M]) to model PNPLA3-associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3[KO] hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid-induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3[KO] cells to be more susceptible to ethanol-induced and methotrexate-induced toxicity. The PNPLA3[I148M] cells exhibited an intermediate phenotype between the wild-type and PNPLA3[KO] cells.<br><br>CONCLUSIONS: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.},
}
@article {pmid34287045,
year = {2021},
author = {Wang, Q and Liu, Y and Han, C and Yang, M and Huang, F and Duan, X and Wang, S and Yu, Y and Liu, J and Yang, H and Lu, D and Zhao, H and Zhang, Y and Qin, Q},
title = {Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.},
journal = {Journal of virology},
volume = {95},
number = {19},
pages = {e0046121},
pmid = {34287045},
issn = {1098-5514},
mesh = {Animals ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Fish Diseases/*virology ; Nodaviridae/*genetics/physiology ; Perciformes/*virology ; *RNA Interference ; RNA Virus Infections/*veterinary/virology ; RNA, Viral/genetics ; },
abstract = {The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.},
}
@article {pmid34286737,
year = {2021},
author = {Fan, Z and Ding, Y and Yao, B and Wang, J and Zhang, K},
title = {Electrochemiluminescence platform for transcription factor diagnosis by using CRISPR-Cas12a trans-cleavage activity.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {65},
pages = {8015-8018},
doi = {10.1039/d1cc03071j},
pmid = {34286737},
issn = {1364-548X},
mesh = {Bacterial Proteins/chemistry ; Biosensing Techniques/*methods ; CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; DNA/chemistry ; Electrochemical Techniques/methods ; Endodeoxyribonucleases/chemistry ; Gold/chemistry ; Graphite/chemistry ; Immobilized Nucleic Acids/chemistry ; Limit of Detection ; Luminescence ; Luminescent Agents/*chemistry ; Luminescent Measurements/methods ; Metal Nanoparticles/chemistry ; NF-kappa B p50 Subunit/*analysis ; Nucleic Acid Conformation ; Organometallic Compounds/chemistry ; },
abstract = {Herein, we exploited the double-stranded DNA (dsDNA) binding property of transcription factor (TF), combined with the trans cleavage characteristic of CRISPR-Cas12a, for the detection of NF-κB p50.},
}
@article {pmid34286726,
year = {2021},
author = {Xie, R and Wang, Y and Gong, S},
title = {External stimuli-responsive nanoparticles for spatially and temporally controlled delivery of CRISPR-Cas genome editors.},
journal = {Biomaterials science},
volume = {9},
number = {18},
pages = {6012-6022},
pmid = {34286726},
issn = {2047-4849},
support = {R01 HL129785/HL/NHLBI NIH HHS/United States ; R01 HL143469/HL/NHLBI NIH HHS/United States ; R24 EY032434/EY/NEI NIH HHS/United States ; UG3 NS111688/NS/NINDS NIH HHS/United States ; },
mesh = {*Biological Phenomena ; CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Transfer Techniques ; *Nanoparticles ; },
abstract = {The CRISPR-Cas9 system is a powerful tool for genome editing, which can potentially lead to new therapies for genetic diseases. To date, various viral and non-viral delivery systems have been developed for the delivery of CRISPR-Cas9 in vivo. However, spatially and temporally controlled genome editing is needed to enhance the specificity in organs/tissues and minimize the off-target effects of editing. In this review, we summarize the state-of-the-art non-viral vectors that exploit external stimuli (i.e., light, magnetic field, and ultrasound) for spatially and temporally controlled genome editing and their in vitro and in vivo applications.},
}
@article {pmid34285780,
year = {2021},
author = {Tay, AP and Hosking, B and Hosking, C and Bauer, DC and Wilson, LOW},
title = {INSIDER: alignment-free detection of foreign DNA sequences.},
journal = {Computational and structural biotechnology journal},
volume = {19},
number = {},
pages = {3810-3816},
pmid = {34285780},
issn = {2001-0370},
abstract = {External DNA sequences can be inserted into an organism's genome either through natural processes such as gene transfer, or through targeted genome engineering strategies. Being able to robustly identify such foreign DNA is a crucial capability for health and biosecurity applications, such as anti-microbial resistance (AMR) detection or monitoring gene drives. This capability does not exist for poorly characterised host genomes or with limited information about the integrated sequence. To address this, we developed the INserted Sequence Information DEtectoR (INSIDER). INSIDER analyses whole genome sequencing data and identifies segments of potentially foreign origin by their significant shift in k-mer signatures. We demonstrate the power of INSIDER to separate integrated DNA sequences from normal genomic sequences on a synthetic dataset simulating the insertion of a CRISPR-Cas gene drive into wild-type yeast. As a proof-of-concept, we use INSIDER to detect the exact AMR plasmid in whole genome sequencing data from a Citrobacter freundii patient isolate. INSIDER streamlines the process of identifying integrated DNA in poorly characterised wild species or when the insert is of unknown origin, thus enhancing the monitoring of emerging biosecurity threats.},
}
@article {pmid34285418,
year = {2021},
author = {Paulusma, CC and Bosma, PJ},
title = {Therapeutic base editing in the adult liver.},
journal = {Nature reviews. Gastroenterology &amp; hepatology},
volume = {18},
number = {9},
pages = {597-598},
pmid = {34285418},
issn = {1759-5053},
mesh = {Adult ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Liver ; },
}
@article {pmid34285320,
year = {2021},
author = {Jung, KM and Kim, YM and Kim, JL and Han, JY},
title = {Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14746},
pmid = {34285320},
issn = {2045-2322},
mesh = {Adenoviridae/*genetics ; Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Female ; Finches/*genetics ; Gene Editing ; *Gene Transfer Techniques ; Genetic Vectors/genetics/*metabolism ; Germ Cells/cytology/metabolism ; Male ; Mutagenesis ; Transcription Factors/genetics/metabolism ; },
abstract = {Zebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.},
}
@article {pmid34284989,
year = {2021},
author = {Wang, HT and Li, TT and Huang, X and Ma, RL and Liu, QY},
title = {Application of genetic modification technologies in molecular design breeding of sheep.},
journal = {Yi chuan = Hereditas},
volume = {43},
number = {6},
pages = {580-600},
doi = {10.16288/j.yczz.21-087},
pmid = {34284989},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plant Breeding ; Sheep/genetics ; Technology ; },
abstract = {Genetic modification technologies can be used for modifying animal genome to express exogenous genes or affect the function of endogenous genes. In animal breeding, genetic modification technologies allow the rapid generation of germplasms with beneficial traits. It includes traditional genetic modification, virus or sperm carrier-mediated genetic modification and nuclease-mediated genome editing, especially the CRISPR/Cas9, one of the artificial nuclease genome editing technologies, have been applied in genome editing in many domestic animals including sheep (Ovis aries). Compared with conventional strategies used for animal breeding, there is great value for sheep breeding improvement by using genome editing technology, which is more effective and timesaving. In this review, we summarize the approaches of genetic modification in sheep and discuss the possibility of molecular design and breeding of sheep by genome editing technologies. We also identify the potential bottlenecks and challenges of these technologies in sheep breeding.},
}
@article {pmid34284985,
year = {2021},
author = {Jiang, J and Zhao, AQ and Xie, T and Chen, SW and Li, JS},
title = {Construction of genome-wide protein tagging cell and mouse libraries.},
journal = {Yi chuan = Hereditas},
volume = {43},
number = {7},
pages = {704-714},
doi = {10.16288/j.yczz.21-163},
pmid = {34284985},
issn = {0253-9772},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome ; Male ; Mice ; },
abstract = {Mice are the most widely used model organism for the study of gene functions and disease mechanisms through the generation of gene-modified mice. Since the 1980s, different genetic manipulation technologies have been developed to reveal gene functions in vivo, including homologous recombination strategies mediated by embryonic stem cells, transgenic strategies mediated by gametes, and the latest genetic modification strategies based on CRISPR/Cas9 technology. Semi-cloning technology mediated by "artificial spermatids" (androgenetic haploid embryonic stem cells, also termed sperm-like stem cells) is developed by Chinese scientists in 2012. In combination with CRISPR/Cas9, semi-cloning technology enables one-step generation of gene-modified mice through injection of "artificial spermatids" with specific gene modifications into oocytes. It has the characteristics of short construction cycle, high efficiency, low cost, and high application compatibility. In 2017, the Center for Excellence in Molecular Cell Science (CEMCS) of CAS has launched the genome tagging project (GTP) based on "artificial spermatid"-mediated semi-cloning technology. The ambitious goal of GTP is to tag every protein in mice and construct a unique mouse library that maintains the genome-wide protein-tagging mouse models. Subsequently, the GTP center was established at CEMCS to pursue the project. GTP center developed strategies to generate protein-tagging cells and mice. Briefly, a tag sequence is precisely inserted in a specific protein- coding gene endogenously in cultured "artificial spermatids"in vitro to build a cell library, in which, each cell line carrying a specific protein tag. The tagged cells could be further used as a sperm replacement to produce tagged mice in one step upon injection into oocytes. The tagged mouse library enables global analysis of protein expression, localization, and complexes using standard tag-based assays in vivo. By April 2021, the GTP center has generated 1532 tagged cell lines, 277 of which have been successfully used to produce tagged mice through oocyte injection. A total of 242 tagged mouse strains have been distributed to 66 research teams in 32 research institutions of 15 districts in 3 countries. The database of tagging product resources has been established and released regularly on the GTP website for scientists to inquire and order. Later, more information about GTP products, such as mouse breeding, protein tissue expression map, published literature, etc., will also be successively published on the GTP website. The GTP center will provide a standardized platform for protein function research, which may dramatically promote the development of life science and clinical transformation.},
}
@article {pmid34284709,
year = {2021},
author = {Sherstyuk, VV and Zakian, SM},
title = {Generation of Transgenic Rat Embryonic Stem Cells Using the CRISPR/Cpf1 System for Inducible Gene Knockout.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {7},
pages = {843-851},
doi = {10.1134/S0006297921070051},
pmid = {34284709},
issn = {1608-3040},
mesh = {Animals ; CRISPR-Cas Systems ; *Embryonic Stem Cells ; *Gene Editing ; *Gene Knockout Techniques ; Integrases ; Rats ; *Rats, Transgenic ; Tamoxifen/analogs &amp; derivatives ; },
abstract = {Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.},
}
@article {pmid34284273,
year = {2021},
author = {Castaño, J and Romero-Moya, D and Richaud-Patin, Y and Giorgetti, A},
title = {Generation of two heterozygous GATA2 CRISPR/Cas9-edited iPSC lines, R398W and R396Q, for modeling GATA2 deficiency.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102445},
doi = {10.1016/j.scr.2021.102445},
pmid = {34284273},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *GATA2 Deficiency ; GATA2 Transcription Factor/genetics ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells ; *Myelodysplastic Syndromes/genetics ; },
abstract = {Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.},
}
@article {pmid34284170,
year = {2021},
author = {Lee, H and Hong, C and Hwang, J and Seo, PJ},
title = {Go green with plant organelle genome editing.},
journal = {Molecular plant},
volume = {14},
number = {9},
pages = {1415-1417},
doi = {10.1016/j.molp.2021.07.012},
pmid = {34284170},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant/*genetics ; Organelles/*genetics ; Plant Breeding/*methods ; RNA, Plant/genetics ; },
}
@article {pmid34283641,
year = {2021},
author = {Flotte, TR},
title = {Base Editing to the Rescue.},
journal = {Human gene therapy},
volume = {32},
number = {13-14},
pages = {647-648},
doi = {10.1089/hum.2021.29168.trf},
pmid = {34283641},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; },
}
@article {pmid34283640,
year = {2021},
author = {Philippidis, A},
title = {CRISPR-Cas9 Gene-Edited Therapy Shows Sustained Treatment Response.},
journal = {Human gene therapy},
volume = {32},
number = {13-14},
pages = {642-644},
doi = {10.1089/hum.2021.29166.bfs},
pmid = {34283640},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; },
}
@article {pmid34283216,
year = {2021},
author = {Lin, Y and Zhu, Y and Cui, Y and Chen, R and Chen, Z and Li, G and Fan, M and Chen, J and Li, Y and Guo, X and Zheng, X and Chen, L and Wang, F},
title = {Derepression of specific miRNA-target genes in rice using CRISPR/Cas9.},
journal = {Journal of experimental botany},
volume = {72},
number = {20},
pages = {7067-7077},
pmid = {34283216},
issn = {1460-2431},
mesh = {CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; *MicroRNAs/genetics ; *Oryza/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {MicroRNAs (miRNAs) target specific mRNA molecules based on sequence complementarity for their degradation or repression of translation, thereby regulating various developmental and physiological processes in eukaryotic organisms. Expressing the target mimicry (MIM) and short tandem target mimicry (STTM) can block endogenous activity of mature miRNAs and eliminate the inhibition of their target genes, resulting in phenotypic changes due to higher expression of the target genes. Here, we report a strategy to achieve derepression of interested miRNA-target genes through CRISPR/Cas9-based generation of in-frame mutants within the miRNA-complementary sequence of the target gene. We show that two rice genes, OsGRF4 (GROWTH REGULATING FACTOR 4) and OsGRF8 carrying in-frame mutants with disruption of the miR396 recognition sites, escape from miR396-mediated post-transcriptional silencing, resulting in enlarged grain size and increase in brown planthopper (BPH) resistance, in their respective transgenic rice lines. These results demonstrate that CRISPR/Cas9-mediated disruption of miRNA target sites can be effectively employed to precisely derepress particular target genes of functional importance for trait improvement in plants.},
}
@article {pmid34282149,
year = {2021},
author = {Wang, GH and Gamez, S and Raban, RR and Marshall, JM and Alphey, L and Li, M and Rasgon, JL and Akbari, OS},
title = {Combating mosquito-borne diseases using genetic control technologies.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4388},
pmid = {34282149},
issn = {2041-1723},
support = {R01 AI151004/AI/NIAID NIH HHS/United States ; R01 AI116636/AI/NIAID NIH HHS/United States ; R21 AI149161/AI/NIAID NIH HHS/United States ; DP2 AI152071/AI/NIAID NIH HHS/United States ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Female ; Humans ; *Insecticides ; Malaria/prevention &amp; control/transmission ; Male ; Mosquito Control/*methods ; Mosquito Vectors ; Nucleic Acid Amplification Techniques/*methods ; Pest Control, Biological ; Vector Borne Diseases/*genetics ; Wolbachia/genetics ; },
abstract = {Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.},
}
@article {pmid34282051,
year = {2021},
author = {McAndrews, KM and Xiao, F and Chronopoulos, A and LeBleu, VS and Kugeratski, FG and Kalluri, R},
title = {Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic Kras[G12D] in pancreatic cancer.},
journal = {Life science alliance},
volume = {4},
number = {9},
pages = {},
pmid = {34282051},
issn = {2575-1077},
mesh = {Alleles ; Allografts ; Animals ; Biological Transport ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Disease Models, Animal ; Exosomes/*metabolism ; *Gene Editing/methods ; Gene Expression Regulation, Neoplastic ; *Gene Targeting/methods ; Gene Transfer Techniques ; Genes, Reporter ; MAP Kinase Signaling System ; Mice ; Oncogenes ; Pancreatic Neoplasms/*genetics ; Plasmids/administration &amp; dosage/genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; },
abstract = {CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant Kras [G12D] oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.},
}
@article {pmid34281340,
year = {2021},
author = {Ma, L and Peng, L and Yin, L and Liu, G and Man, S},
title = {CRISPR-Cas12a-Powered Dual-Mode Biosensor for Ultrasensitive and Cross-validating Detection of Pathogenic Bacteria.},
journal = {ACS sensors},
volume = {6},
number = {8},
pages = {2920-2927},
doi = {10.1021/acssensors.1c00686},
pmid = {34281340},
issn = {2379-3694},
mesh = {Bacteria ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Gold ; Humans ; *Metal Nanoparticles ; },
abstract = {Pathogenic bacteria infection severely threatens human health and causes substantial medical and financial concern. Rapid, sensitive, specific, and reliable detection of pathogenic bacteria is crucial. In the current study, a CRISPR-Cas12a-powered dual-mode biosensor was developed for ultrasensitive and cross-validating detection of pathogenic bacteria. Simply, the amplicons of Salmonella (used as a model)-specific invA sequence triggered CRISPR-Cas12a-based indiscriminate degradation of single-stranded DNAs that were supposed to link two gold nanoparticle (AuNP) probe pairs, inducing an aggregation-to-dispersion change. This generated observable color changes that became even more apparent after centrifugation. The color changes can be discerned by naked eyes and recorded using a portable colorimeter. Meanwhile, the photothermal effect of CRISPR-Cas12-powered AuNPs was explored for the first time through 808 nm near-infrared irradiation such that quantitative measurement can be carried out by recording temperatures using a thermal camera. For both modes, a limit of detection of 1 CFU/mL and a dynamic range of detection from 10[0] to 10[8] CFU/mL were obtained, which were comparable with or better than previously reported methods. Our proposed biosensor demonstrated satisfactory selectivity for Salmonella against other interfering cells. Furthermore, this biosensor proved to be capable of Salmonella detection in food samples. Regarding the real applications, the result from each mode can be used for cross-validation. Only the case having doubly confirmed positive or negative results can be assured, which rendered a more dependable biosensing conclusion. This technology not only expands the reach of the CRISPR-Cas system in biosensing but also provides a general method for bacteria sensing with desirable sensitivity, specificity, and cross-validating capacity.},
}
@article {pmid34281288,
year = {2021},
author = {Koster, C and van den Hurk, KT and Lewallen, CF and Talib, M and Ten Brink, JB and Boon, CJF and Bergen, AA},
title = {The Lrat[-/-] Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34281288},
issn = {1422-0067},
mesh = {Acyltransferases/*deficiency/*genetics ; Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Disease Models, Animal ; Electroretinography ; Female ; Gene Knockout Techniques ; Humans ; Male ; Mice ; Ophthalmoscopy ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Transgenic ; Retinitis Pigmentosa/diagnostic imaging/*genetics/physiopathology ; Sequence Deletion ; Tomography, Optical Coherence ; Vision, Ocular ; },
abstract = {PURPOSE: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle.<br><br>METHODS: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat[-/-] and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays.<br><br>RESULTS: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat[-/-] animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat[-/-] animals. Lrat[-/-] animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat[-/-] animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals.<br><br>CONCLUSIONS: Our Lrat[-/-] rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.},
}
@article {pmid34280810,
year = {2021},
author = {Inturi, R and Jemth, P},
title = {CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 or E7 induces senescence in cervical cancer cells.},
journal = {Virology},
volume = {562},
number = {},
pages = {92-102},
doi = {10.1016/j.virol.2021.07.005},
pmid = {34280810},
issn = {1096-0341},
mesh = {CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Cellular Senescence/*genetics ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; DNA-Binding Proteins/*genetics/metabolism ; Female ; Gene Editing ; HeLa Cells ; Humans ; Lamin Type B/metabolism ; Oncogene Proteins, Viral/*genetics/metabolism ; Retinoblastoma Protein/metabolism ; Signal Transduction ; Tumor Suppressor Protein p53/metabolism ; Uterine Cervical Neoplasms/*genetics/metabolism/pathology ; beta-Galactosidase/metabolism ; },
abstract = {Human papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, anogenital and oropharyngeal sites. Continuous expression of the HPV oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 or E7 genes can potentially treat HPV-related cancers. Here we report that CRISPR/Cas9-based knockout of E6 or E7 can trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, E6 or E7-inactivated HeLa cells exhibited characteristic senescence markers like enlarged cell surface area, increased β-galactosidase expression and loss of lamin B1. Since E6 and E7 are bicistronic transcripts, inactivation of HPV18 E6 resulted in knockout of both E6 and E7 and increasing levels of p53/p21 and pRb/p21, respectively. Knockout of HPV18 E7 resulted in decreased E6 expression with activation of pRb/p21 pathway. Taken together, our study demonstrates cellular senescence as an alternative outcome of HPV oncogene inactivation by CRISPR/Cas9.},
}
@article {pmid34280652,
year = {2021},
author = {Li, Z and Ding, X and Yin, K and Xu, Z and Cooper, K and Liu, C},
title = {Electric field-enhanced electrochemical CRISPR biosensor for DNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {192},
number = {},
pages = {113498},
pmid = {34280652},
issn = {1873-4235},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R61 AI154642/AI/NIAID NIH HHS/United States ; },
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Electrochemical Techniques ; Humans ; *Nucleic Acids ; },
abstract = {DNA detection plays an important role in the rapid screening of cancers and early diagnosis of infectious diseases. Here, we developed a simple, versatile, electric field-enhanced (EFE), electrochemical CRISPR biosensor to detect DNA targets in a homogeneous solution phase. To improve the detection sensitivity, we applied a pulsed electric field to enrich nucleic acids on the electrode surface. The EFE electrochemical CRISPR biosensor takes advantage of the diffusivity difference between electrochemical oligonucleotide probes and CRISPR-cleaved probes toward a negatively charged working electrode, enabling simple and sensitive electrochemical detection of DNA without the need for complicated immobilization processing of electrochemical probes. Our developed CRISPR biosensor directly detects unamplified human papillomavirus-16 (HPV-16) DNA with a sensitivity of 1 pM. Further, the EFE electrochemical CRISPR biosensor coupled with recombinase polymerase amplification (RPA) successfully detects HPV-16 DNA in clinical samples. Thus, the EFE electrochemical CRISPR biosensor provides a simple, robust, and sensitive detection method for nucleic acid-based molecular diagnostics.},
}
@article {pmid34280549,
year = {2021},
author = {Wang, Y and Lecourieux, F and Zhang, R and Dai, Z and Lecourieux, D and Li, S and Liang, Z},
title = {Data Comparison and Software Design for Easy Selection and Application of CRISPR-based Genome Editing Systems in Plants.},
journal = {Genomics, proteomics &amp; bioinformatics},
volume = {19},
number = {6},
pages = {937-948},
pmid = {34280549},
issn = {2210-3244},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Software Design ; },
abstract = {CRISPR-based genome editing systems have been successfully and effectively used in many organisms. However, only a few studies have reported the comparison between CRISPR/Cas9 and CRISPR/Cpf1 systems in the whole-genome applications. Although many web-based toolkits are available, there is still a shortage of comprehensive, user-friendly, and plant-specific CRISPR databases and desktop software. In this study, we identified and analyzed the similarities and differences between CRISPR/Cas9 and CRISPR/Cpf1 systems by considering the abundance of proto-spacer adjacent motif (PAM) sites, the effects of GC content, optimal proto-spacer length, potential universality within the plant kingdom, PAM-rich region (PARR) inhibiting ratio, and the effects of G-quadruplex (G-Q) structures. Using this information, we built a comprehensive CRISPR database (including 138 plant genome data sources, www.grapeworld.cn/pc/index.html), which provides search tools for the identification of CRISPR editing sites in both CRISPR/Cas9 and CRISPR/Cpf1 systems. We also developed a desktop software on the basis of the Perl/Tk tool, which facilitates and improves the detection and analysis of CRISPR editing sites at the whole-genome level on Linux and/or Windows platform. Therefore, this study provides helpful data and software for easy selection and application of CRISPR-based genome editing systems in plants.},
}
@article {pmid34280234,
year = {2021},
author = {Fu, J and Zhang, Y and Cai, G and Meng, G and Shi, S},
title = {Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus.},
journal = {PloS one},
volume = {16},
number = {7},
pages = {e0254815},
pmid = {34280234},
issn = {1932-6203},
mesh = {African Swine Fever/*diagnosis/genetics/virology ; African Swine Fever Virus/genetics/*isolation &amp; purification ; Animals ; Bacterial Proteins/chemistry/*genetics ; CRISPR-Associated Proteins/chemistry/*genetics ; CRISPR-Cas Systems ; DNA-Directed DNA Polymerase/chemistry ; Endodeoxyribonucleases/chemistry/*genetics ; Molecular Diagnostic Techniques ; Point-of-Care Systems ; Recombinases/chemistry ; Swine ; Swine Diseases/*diagnosis/genetics/pathology/virology ; },
abstract = {African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.},
}
@article {pmid34280034,
year = {2021},
author = {Feng, X and Kambic, L and Nishimoto, JHK and Reed, FA and Denton, JA and Sutton, JT and Gantz, VM},
title = {Evaluation of Gene Knockouts by CRISPR as Potential Targets for the Genetic Engineering of the Mosquito Culex quinquefasciatus.},
journal = {The CRISPR journal},
volume = {4},
number = {4},
pages = {595-608},
pmid = {34280034},
issn = {2573-1602},
support = {DP5 OD023098/OD/NIH HHS/United States ; P20 GM125508/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Culex/*genetics ; Culicidae/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques ; Gene Targeting ; *Genetic Engineering/methods ; Genetic Loci ; Microinjections ; Mutation ; Phenotype ; RNA, Guide ; },
abstract = {Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia, a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Cx. quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.},
}
@article {pmid34279819,
year = {2022},
author = {Guo, S and Chen, Y and Liu, J and Zhang, X and Liu, Z and Zhou, Z and Wei, W},
title = {Low-density lipoprotein receptor-related protein 1 is a CROPs-associated receptor for Clostridioides infection toxin B.},
journal = {Science China. Life sciences},
volume = {65},
number = {1},
pages = {107-118},
pmid = {34279819},
issn = {1869-1889},
mesh = {Bacterial Proteins/*metabolism ; Bacterial Toxins/*metabolism ; CRISPR-Cas Systems ; Chondroitin Sulfate Proteoglycans/deficiency/metabolism ; Clostridioides difficile/*metabolism/pathogenicity ; Clostridium Infections ; Frizzled Receptors ; HeLa Cells ; Humans ; Low Density Lipoprotein Receptor-Related Protein-1/*metabolism ; Membrane Proteins/deficiency/metabolism ; Oligopeptides/metabolism ; },
abstract = {As the leading cause of worldwide hospital-acquired infection, Clostridioides difficile (C. difficile) infection has caused heavy economic and hospitalized burden, while its pathogenesis is not fully understood. Toxin B (TcdB) is one of the major virulent factors of C. difficile. Recently, CSPG4 and FZD2 were reported to be the receptors that mediate TcdB cellular entry. However, genetic ablation of genes encoding these receptors failed to completely block TcdB entry, implicating the existence of alternative receptor(s) for this toxin. Here, by employing the CRISPR-Cas9 screen in CSPG4-deficient HeLa cells, we identified LDL receptor-related protein-1 (LRP1) as a novel receptor for TcdB. Knockout of LRP1 in both CSPG4-deficient HeLa cells and colonic epithelium Caco2 cells conferred cells with increased TcdB resistance, while LRP1 overexpression sensitized cells to TcdB at a low concentration. Co-immunoprecipitation assay showed that LRP1 interacts with full-length TcdB. Moreover, CROPs domain, which is dispensable for TcdB's interaction with CSPG4 and FZD2, is sufficient for binding to LRP1. As such, our study provided evidence for a novel mechanism of TcdB entry and suggested potential therapeutic targets for treating C. difficile infection.},
}
@article {pmid34279489,
year = {2021},
author = {Ai, D and Wang, B and Fan, Z and Fu, Y and Yu, C and Wang, G},
title = {Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner).},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {173},
pages = {},
doi = {10.3791/62068},
pmid = {34279489},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Microinjections ; *Moths/genetics ; RNA, Guide/genetics ; },
abstract = {The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.},
}
@article {pmid34279073,
year = {2021},
author = {Gong, S and Li, J and Pan, W and Li, N and Tang, B},
title = {Duplex-Specific Nuclease-Assisted CRISPR-Cas12a Strategy for MicroRNA Detection Using a Personal Glucose Meter.},
journal = {Analytical chemistry},
volume = {93},
number = {30},
pages = {10719-10726},
doi = {10.1021/acs.analchem.1c02478},
pmid = {34279073},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Glucose ; *MicroRNAs/genetics ; },
abstract = {A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. Nevertheless, the current CRISPR-Cas12a-based detection strategies always need bulky instruments or auxiliary devices to obtain a quantitative signal output, which restrains its point-of-care testing application. Herein, we proposed a duplex-specific nuclease-assisted CRISPR-Cas12a strategy to detect microRNA (miRNA) with a personal glucose meter. The target miRNA was first converted into an amplified initiator DNA via duplex-specific nuclease. Afterward, the initiator DNA activated the collateral cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA-modified magnetic beads, which led to the release of sucrase. The released sucrase was collected and then utilized to catalyze sucrose to glucose, which could be quantitatively detected by a personal glucose meter. The change in the glucose signal directly reflected the concentration of miRNA, which avoided expensive equipment for signal quantification. Two different miRNAs (miRNA21 and miRNA205) could be detected by simply changing the sequence of the template strand (H strand). The developed strategy showed high sensitivity with a limit of detection (LOD) of 2.4 and 1.1 pM for miRNA21 and miRNA205, respectively. In addition, good selectivity and anti-interference ability were achieved using this method, which enabled it promising for miRNA detection at the point-of-care.},
}
@article {pmid34278781,
year = {2021},
author = {Liu, J and Chen, J and Wu, D and Huang, M and Chen, J and Pan, R and Wu, Y and Li, G},
title = {CRISPR-/Cas12a-Mediated Liposome-Amplified Strategy for the Surface-Enhanced Raman Scattering and Naked-Eye Detection of Nucleic Acid and Application to Food Authenticity Screening.},
journal = {Analytical chemistry},
volume = {93},
number = {29},
pages = {10167-10174},
doi = {10.1021/acs.analchem.1c01163},
pmid = {34278781},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gold ; Liposomes ; *Metal Nanoparticles ; *Nucleic Acids ; Spectrum Analysis, Raman ; },
abstract = {Surface-enhanced Raman scattering (SERS) has been recognized as a powerful tool for biosensors due to the ultrahigh sensitivity and unique fingerprint information. However, there are some limitations in trace target nucleic acid detection for the restricted signal-transducing and amplification strategies. Inspired by CRISPR/Cas12a with specific target DNA-activated collateral single-strand DNA (ssDNA) cleavage activity and liposome with signal molecule-loading properties, we first proposed a sensitive SERS-based on-site nucleic acid detection strategy mediated by CRISPR/Cas12a with trans-cleavage activity on ssDNA linkers utilized to capture liposomes. Liposomes loading two kinds of signal molecules, 4-nitrothiophenol (4-NTP) and cysteine, could achieve the dual-mode detection of target DNA with SERS and naked eye, respectively. The promptly amplified signals were initiated by the triggered breakdown of signal molecule-loaded liposomes. Emancipated 4-NTP, a biological-silent Raman reporter, would achieve highly selective and sensitive SERS measurement. Released cysteine induced the aggregation of plasmonic gold nanoparticles, leading to an obvious red to blue colorimetric shift to realize portable naked-eye detection. With this strategy, target nucleic acid concentration was dexterously converted into SERS and visualization signals and could be detected as low as 100 aM and 10 pM, respectively. The approach was also successfully applied to determine meat adulteration, achieving the detection of a low adulteration ratio in the complicated food matrix. We anticipate that this strategy will not only be regarded as a universal platform for the on-site detection of food authenticity but also broaden SERS application for the accurate determination of diverse biomarkers.},
}
@article {pmid34278778,
year = {2021},
author = {Hasanzadeh, A and Radmanesh, F and Hosseini, ES and Hashemzadeh, I and Kiani, J and Nourizadeh, H and Naseri, M and Fatahi, Y and Chegini, F and Madjd, Z and Beyzavi, A and Kowalski, PS and Karimi, M},
title = {Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.},
journal = {Bioconjugate chemistry},
volume = {32},
number = {8},
pages = {1875-1887},
doi = {10.1021/acs.bioconjchem.1c00309},
pmid = {34278778},
issn = {1520-4812},
mesh = {*CRISPR-Cas Systems ; Carbon/*chemistry ; Cell Survival/drug effects ; Drug Delivery Systems ; Fluorescent Dyes ; Gene Editing ; Genetic Therapy/methods ; HEK293 Cells ; Humans ; Nitrogen/*chemistry ; Plasmids/chemistry ; *Quantum Dots ; *RNA, Messenger ; Serum Albumin, Bovine ; Zinc/*chemistry ; },
abstract = {Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.},
}
@article {pmid34276739,
year = {2021},
author = {Vazquez-Vilar, M and Garcia-Carpintero, V and Selma, S and Bernabé-Orts, JM and Sanchez-Vicente, J and Salazar-Sarasua, B and Ressa, A and de Paola, C and Ajenjo, M and Quintela, JC and Fernández-Del-Carmen, A and Granell, A and Orzáez, D},
title = {The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {689937},
pmid = {34276739},
issn = {1664-462X},
abstract = {CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering.},
}
@article {pmid34276629,
year = {2021},
author = {Parra-Flores, J and Holý, O and Riffo, F and Lepuschitz, S and Maury-Sintjago, E and Rodríguez-Fernández, A and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Troncoso, M and Figueroa, G and Ruppitsch, W and Forsythe, S},
title = {Profiling the Virulence and Antibiotic Resistance Genes of Cronobacter sakazakii Strains Isolated From Powdered and Dairy Formulas by Whole-Genome Sequencing.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {694922},
pmid = {34276629},
issn = {1664-302X},
abstract = {Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enterocolitis in preterm infants and newborns with a mortality rate of 15 to 80%. Powdered and dairy formulas (P-DF) have been implicated as major transmission vehicles and subsequently the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to use whole genome sequencing (WGS) and laboratory studies to characterize Cronobacter strains from the contaminated products. Seven strains were identified as C. sakazakii, and the remaining strain was Franconibacter helveticus. All C. sakazakii strains adhered to a neuroblastoma cell line, and 31 virulence genes were predicted by WGS. The antibiograms varied between strains. and included mcr-9.1 and bla CSA genes, conferring resistance to colistin and cephalothin, respectively. The C. sakazakii strains encoded I-E and I-F CRISPR-Cas systems, and carried IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. In summary, WGS enabled the identification of C. sakazakii strains and revealed multiple antibiotic resistance and virulence genes. These findings support the decision to recall the contaminated powdered and dairy formulas from the Chilean market in 2017.},
}
@article {pmid34275631,
year = {2021},
author = {Song, X and Liu, L and Liu, XX and Xiong, ZQ and Xie, CL and Wang, SJ and Ai, LZ},
title = {Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis.},
journal = {Journal of dairy science},
volume = {104},
number = {10},
pages = {10576-10585},
doi = {10.3168/jds.2020-19901},
pmid = {34275631},
issn = {1525-3198},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/veterinary ; *Lactococcus lactis/genetics ; Plasmids/genetics ; RNA, Guide ; },
abstract = {Lactococcus lactis is a food-grade lactic acid bacterial species that is widely used in food and medical industries. Due to its relatively small genome and simple metabolism, L. lactis is commonly engineered to produce large quantities of recombinant proteins. The most common single-gene knockout strategy in L. lactis involves RecA-dependent homologous double-crossover recombination, which is relatively time-consuming and laborious. In this study, a precise and efficient genome-editing plasmid for L. lactis NZ9000 genome engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects of different single guide RNA (sgRNA) promoters, the efficiency of gene deletion was optimized. For LLNZ_02045 (ldh), gene deletion efficiency of up to 50% was achieved. Effective sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1) was also demonstrated using this tool. Additionally, the gene that encodes for uracil phosphoribosyltransferase was identified using this system. Similar robust gene deletion efficiencies of sgRNA that targeted different regions of a single gene suggested that gene deletion was not affected by the location of sgRNA binding. Thus, our study established a new gene-editing tool that may allow further investigation and understanding of the L. lactis NZ9000 genome.},
}
@article {pmid34275489,
year = {2021},
author = {Widjaya, MA and Lee, SD and Ho, YS},
title = {Impactful factors and research design in CRISPR-edited stem cell research from top 10 highly cited articles.},
journal = {Stem cell research &amp; therapy},
volume = {12},
number = {1},
pages = {411},
pmid = {34275489},
issn = {1757-6512},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Research Design ; *Stem Cell Research ; },
abstract = {Our objective in this review was to determine (1) impactful research articles about CRISPR-edited stem cells, (2) factors that affected CRISPR method performance in stem cell, and (3) research design related to CRISPR-edited stem cells. Screening research papers of related topic was carried out by using the Science Citation Index Expanded (SCIE) database of the Clarivate Analytics Web of Science Core Collection updated. We screened impactful CRISPR/Cas9-edited stem cells based on total citation until 2020. The result showed the title "RNA-guided human genome engineering via Cas9" was the highest citation in stem cell research using the CRISPR method with total citation 4789 from Web of Science Core Collection until 2020. It became the most influenced paper because this was the first research using CRISPR method for modifying human cells. On the other hand, cell type, CRISPR/Cas9 delivery, and gene target affected CRISPR/Cas9 performance in stem cells. The more complex the cell structure, the more difficult for CRISPR/Cas9 to mutate the host cells. This problem could be solved by modifying the CRISPR/Cas9 delivery by liposome and SaCas9 modification. Another way was using ribonucleoprotein (RNP) as a delivery method. Then, double gene target was more difficult to execute than single gene target. Although it is difficult, CRISPR/Cas9 had the capability to target any genome region from promoter until intron. Research design used a combination of dry lab and wet lab. The dry lab is usually used for sequence analysis and gRNA design. The wet lab which consisted of in vitro and in vivo was used for gene characterization. In particular, colony selection, DNA analysis, and sequencing were important parts for in vitro research design, while DNA analysis and sequencing were crucial parts for in vivo research design. We hoped these findings could give researchers, investor, and students a guideline to conduct CRISPR-edited stem cells in the future.},
}
@article {pmid34275185,
year = {2021},
author = {Ntui, VO and Uyoh, EA and Ita, EE and Markson, AA and Tripathi, JN and Okon, NI and Akpan, MO and Phillip, JO and Brisibe, EA and Ene-Obong, EE and Tripathi, L},
title = {Strategies to combat the problem of yam anthracnose disease: Status and prospects.},
journal = {Molecular plant pathology},
volume = {22},
number = {10},
pages = {1302-1314},
pmid = {34275185},
issn = {1364-3703},
mesh = {*Dioscorea ; *Fungicides, Industrial ; Genomics ; Plant Leaves ; Plant Tubers ; },
abstract = {Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devastating fungal disease of yam in West Africa, leading to 50%-90% of tuber yield losses in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. alatae affects all parts of the yam plant at all stages of development, including leaves, stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region and other yam-producing countries in the world. Traditional methods adopted by farmers to control the disease have not been very successful. Fungicides have also failed to provide long-lasting control. Although conventional breeding and genomics-assisted breeding have been used to develop some level of resistance to anthracnose in Dioscorea alata, the appearance of new and more virulent strains makes the development of improved varieties with broad-spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted researchers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus-resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose-resistant yam varieties for improved food security in West Africa.},
}
@article {pmid34274537,
year = {2021},
author = {Wu, Y and Jin, W and Wang, Q and Zhou, J and Wang, Y and Tan, Y and Cui, X and Tong, F and Yang, E and Wang, J and Kang, C},
title = {Precise editing of FGFR3-TACC3 fusion genes with CRISPR-Cas13a in glioblastoma.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3305-3318},
pmid = {34274537},
issn = {1525-0024},
mesh = {Animals ; Biomarkers, Tumor ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Models, Animal ; Disease Progression ; *Gene Editing ; Gene Expression ; Gene Expression Profiling ; Glioblastoma/*genetics/pathology ; Heterografts ; Humans ; Hydrogen Bonding ; Mice ; Microtubule-Associated Proteins/chemistry/*genetics ; Models, Molecular ; Nucleic Acid Conformation ; Oncogene Proteins, Fusion/chemistry/*genetics ; Protein Binding ; Protein Conformation ; RNA, Messenger/chemistry/genetics ; Receptor, Fibroblast Growth Factor, Type 3/chemistry/*genetics ; },
abstract = {FGFR3-TACC3 (F3-T3) gene fusions are regarded as a "low-hanging fruit" paradigm for precision therapy in human glioblastoma (GBM). Small molecules designed to target the kinase in FGFR currently serve as one form of potential treatment but cause off-target effects and toxicity. Here, CRISPR-Cas13a, which is known to directly suppress gene expression at the transcriptional level and induce a collateral effect in eukaryotes, was leveraged as a possible precision therapy in cancer cells harboring F3-T3 fusion genes. A library consisting of crRNAs targeting the junction site of F3-T3 was designed, and an in silico simulation scheme was created to select the optimal crRNA candidates. An optimal crRNA, crRNA1, showed efficiency and specificity in inducing the collateral effect in only U87 cells expressing F3-T3 (U87-F3-T3). Expression profiles obtained with microarray analysis were consistent with induction of the collateral effect by the CRISPR-Cas13a system. Tumor cell proliferation and colony formation were decreased in U87-F3-T3 cells expressing the Cas13a-based tool, and tumor growth was suppressed in an orthotopic tumor model in mice. These findings demonstrate that the CRISPR-Cas13a system induces the collateral damage effect in cancer cells and provides a viable strategy for precision tumor therapy based on the customized design of a CRISPR-Cas13a-based tool against F3-T3 fusion genes.},
}
@article {pmid34274311,
year = {2021},
author = {Schonfeld, E and Schonfeld, E and Schonfeld, D},
title = {Autocatalytic-protection for an unknown locus CRISPR-Cas countermeasure for undesired mutagenic chain reactions.},
journal = {Journal of theoretical biology},
volume = {528},
number = {},
pages = {110831},
doi = {10.1016/j.jtbi.2021.110831},
pmid = {34274311},
issn = {1095-8541},
mesh = {*CRISPR-Cas Systems/genetics ; Computer Simulation ; Genome ; Mutagenesis ; *Mutagens ; },
abstract = {The mutagenic chain reaction (MCR) is a genetic tool to use a CRISPR-Cas construct to introduce a homing endonuclease, allowing gene drive to influence whole populations in a minimal number of generations (Esvelt et al., 2014; Gantz and Bier, 2015; Gantz and Bier, 2016). The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele (Gantz, et al., 2015; Webber et al., 2015)? Thus far, effective protection methods require knowledge of the terror locus (Grunwald et al., 2019). Here we introduce a novel approach, an autocatalytic-Protection for an Unknown Locus (a-PUL), whose aim is to spread through a population and arrest and decrease an active terror event's spread without any prior knowledge of the terror-modified locus, thus allowing later natural selection and ERACR drives to restore the normal locus (Hammond et al., 2017). a-PUL, using a mutagenic chain reaction, includes (i) a segment encoding a non-Cas9 endonuclease capable of homology-directed repair suggested as Type II endonuclease Cpf1 (Cas12a), (ii) a ubiquitously-expressed gene encoding a gRNA (gRNA1) with a U4AU4 3'-overhang specific to Cpf1 and with crRNA specific to some desired genomic sequence of non-coding DNA, (iii) a ubiquitously-expressed gene encoding two gRNAs (gRNA2/gRNA3) both with tracrRNA specific to Cas9 and crRNA specific to two distinct sites of the Cas9 locus, and (iv) homology arms flanking the Cpf1/gRNA1/gRNA2/gRNA3 cassette that are identical to the region surrounding the target cut directed by gRNA1 (Khan, 2016; Zetsche et al., 2015). We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.},
}
@article {pmid34272525,
year = {2021},
author = {Kaminski, MM and Abudayyeh, OO and Gootenberg, JS and Zhang, F and Collins, JJ},
title = {CRISPR-based diagnostics.},
journal = {Nature biomedical engineering},
volume = {5},
number = {7},
pages = {643-656},
pmid = {34272525},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems/*genetics ; Communicable Diseases/*diagnosis/microbiology/virology ; Genetic Diseases, Inborn/diagnosis ; Humans ; Nucleic Acid Amplification Techniques/economics/*methods ; Nucleic Acids/*analysis/metabolism ; Point-of-Care Systems ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; },
abstract = {The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.},
}
@article {pmid34272378,
year = {2021},
author = {Montavon, T and Shukeir, N and Erikson, G and Engist, B and Onishi-Seebacher, M and Ryan, D and Musa, Y and Mittler, G and Meyer, AG and Genoud, C and Jenuwein, T},
title = {Complete loss of H3K9 methylation dissolves mouse heterochromatin organization.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4359},
pmid = {34272378},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Chromatin Immunoprecipitation Sequencing ; Chromatography, Liquid ; Demethylation ; Epigenesis, Genetic ; Fibroblasts/enzymology/*metabolism ; Gene Deletion ; Heterochromatin/enzymology/genetics/*metabolism/ultrastructure ; Histone-Lysine N-Methyltransferase/genetics/*metabolism ; Histones/*metabolism ; In Situ Hybridization, Fluorescence ; Lysine/*metabolism ; Mass Spectrometry ; Methylation ; Mice ; Microscopy, Electron, Transmission ; Mutation ; Protein Processing, Post-Translational/genetics ; RNA-Seq ; Repetitive Sequences, Nucleic Acid/genetics ; Retroelements/genetics ; Signal Transduction/genetics ; },
abstract = {Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.},
}
@article {pmid34271398,
year = {2021},
author = {Li, Z and Zhao, W and Ma, S and Li, Z and Yao, Y and Fei, T},
title = {A chemical-enhanced system for CRISPR-Based nucleic acid detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {192},
number = {},
pages = {113493},
pmid = {34271398},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; Pandemics ; RNA, Viral ; SARS-CoV-2 ; },
abstract = {The CRISPR-based nucleic acid detection systems have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance, and reveal a crucial role of bovine serum albumin in enhancing trans-cleavage activity of Cas12a/Cas13a effectors. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness, and helps to develop enhanced reagent formula or test kit towards practical application of CRISPR-based diagnostics.},
}
@article {pmid34271056,
year = {2021},
author = {Kholosy, WM and Visscher, M and Ogink, K and Buttstedt, H and Griffin, K and Beier, A and Gerlach, JP and Molenaar, JJ and Geijsen, N and de Boer, M and Chatsisvili, A},
title = {Simple, fast and efficient iTOP-mediated delivery of CRISPR/Cas9 RNP in difficult-to-transduce human cells including primary T cells.},
journal = {Journal of biotechnology},
volume = {338},
number = {},
pages = {71-80},
doi = {10.1016/j.jbiotec.2021.07.006},
pmid = {34271056},
issn = {1873-4863},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Human ; HEK293 Cells ; Humans ; *T-Lymphocytes ; },
abstract = {The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40-95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70-95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.},
}
@article {pmid34271038,
year = {2021},
author = {Zhao, J and Ao, C and Wan, Z and Dzakah, EE and Liang, Y and Lin, H and Wang, H and Tang, S},
title = {A point-of-care rapid HIV-1 test using an isothermal recombinase-aided amplification and CRISPR Cas12a-mediated detection.},
journal = {Virus research},
volume = {303},
number = {},
pages = {198505},
doi = {10.1016/j.virusres.2021.198505},
pmid = {34271038},
issn = {1872-7492},
mesh = {CRISPR-Cas Systems ; *HIV Infections/diagnosis/genetics ; *HIV-1/genetics/metabolism ; Humans ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Point-of-Care Testing ; Recombinases/metabolism ; Sensitivity and Specificity ; },
abstract = {Human immunodeficiency virus type one (HIV-1) infection is one of the major public health problems worldwide. Effective control of HIV-1 epidemic relies on early diagnosis of HIV-1 infection by using simple, rapid point-of-care test (POCT). An integrated assay was developed and evaluated in this study to combine a real-time isothermal reverse-transcription recombinase-aided amplification (rRT-RAA) and CRISPR Cas12a-mediated detection for HIV-1. The testing results could be directly observed with naked eye using a blue light imager, making it a suitable on-site testing assay. Our preliminary data indicated that the assay was capable of detecting 20 copies of purified HIV-1 DNA or RNA per reaction or as low as 123 copies/ml of HIV-1 viral load in clinical samples. When screening 155 clinical samples with or without HIV-1 infection, the sensitivity and specificity of the rRT-RAA assay were 98.95% (94/95) and 100% (60/60), respectively. The coefficient value was 0.986 when compared with the Chinese FDA approved HIV-1 RT-qPCR assay. Furthermore, the newly developed HIV-1 rRT-RAA assay could detect the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, CRF08_BC and subtype B in China. Our preliminary results indicated that the rRT-RAA assay or its combination with CRISPR Cas12a-mediated detection could serve as a rapid, convenient, and robust assay for HIV-1 detection.},
}
@article {pmid34270859,
year = {2021},
author = {Cankar, K and Bundock, P and Sevenier, R and Häkkinen, ST and Hakkert, JC and Beekwilder, J and van der Meer, IM and de Both, M and Bosch, D},
title = {Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.},
journal = {Plant biotechnology journal},
volume = {19},
number = {12},
pages = {2442-2453},
pmid = {34270859},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Chicory/genetics/metabolism ; Lactones/metabolism/pharmacology ; Plant Breeding ; *Sesquiterpenes/metabolism ; Sesquiterpenes, Germacrane ; },
abstract = {Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.},
}
@article {pmid34270616,
year = {2021},
author = {Kenney, J and Ndoye, A and Lamar, JM and DiPersio, CM},
title = {Comparative use of CRISPR and RNAi to modulate integrin α3β1 in triple negative breast cancer cells reveals that some pro-invasive/pro-metastatic α3β1 functions are independent of global regulation of the transcriptome.},
journal = {PloS one},
volume = {16},
number = {7},
pages = {e0254714},
pmid = {34270616},
issn = {1932-6203},
support = {R01 CA129637/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/*genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Datasets as Topic ; Female ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Humans ; Integrin alpha3beta1/*genetics ; Lung Neoplasms/*genetics/secondary ; Mice ; Neoplasm Invasiveness/genetics ; RNA Interference ; RNA-Seq ; Transcriptome/genetics ; Triple Negative Breast Neoplasms/*genetics/pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Integrin receptors for the extracellular matrix play critical roles at all stages of carcinogenesis, including tumor growth, tumor progression and metastasis. The laminin-binding integrin α3β1 is expressed in all epithelial tissues where it has important roles in cell survival, migration, proliferation, and gene expression programs during normal and pathological tissue remodeling. α3β1 signaling and adhesion functions promote tumor growth and metastasis in a number of different types of cancer cells. Previously, we used RNA interference (RNAi) technology to suppress the expression of the ITGA3 gene (encoding the α3 subunit) in the triple-negative breast cancer cell line, MDA-MB-231, thereby generating variants of this line with reduced expression of integrin α3β1. This approach revealed that α3β1 promotes pro-tumorigenic functions such as cell invasion, lung metastasis, and gene regulation. In the current study, we used CRISPR technology to knock out the ITGA3 gene in MDA-MB-231 cells, thereby ablating expression of integrin α3β1 entirely. RNA-seq analysis revealed that while the global transcriptome was altered substantially by RNAi-mediated suppression of α3β1, it was largely unaffected following CRISPR-mediated ablation of α3β1. Moreover, restoring α3β1 to the latter cells through inducible expression of α3 cDNA failed to alter gene expression substantially, suggesting that use of CRISPR to abolish α3β1 led to a decoupling of the integrin from its ability to regulate the transcriptome. Interestingly, both cell invasion in vitro and metastatic colonization in vivo were reduced when α3β1 was abolished using CRISPR, as we observed previously using RNAi to suppress α3β1. Taken together, our results show that pro-invasive/pro-metastatic roles for α3β1 are not dependent on its ability to regulate the transcriptome. Moreover, our finding that use of RNAi versus CRISPR to target α3β1 produced distinct effects on gene expression underlines the importance of using multiple approaches to obtain a complete picture of an integrin's functions in cancer cells.},
}
@article {pmid34270168,
year = {2021},
author = {Wang, W and Tian, B and Pan, Q and Chen, Y and He, F and Bai, G and Akhunova, A and Trick, HN and Akhunov, E},
title = {Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases.},
journal = {Plant biotechnology journal},
volume = {19},
number = {12},
pages = {2428-2441},
pmid = {34270168},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; Gene Editing ; Genome, Plant/genetics ; *Triticum/genetics/metabolism ; },
abstract = {The development of CRISPR-based editors recognizing distinct protospacer-adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a-induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guides flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides and showed substantial increase after subjecting transgenic plants to high-temperature treatment. We successfully applied LbCas12a-MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease.},
}
@article {pmid34270164,
year = {2021},
author = {Zhang, J and Zhang, H and Li, S and Li, J and Yan, L and Xia, L},
title = {Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {9},
pages = {1649-1663},
doi = {10.1111/jipb.13151},
pmid = {34270164},
issn = {1744-7909},
mesh = {*CRISPR-Cas Systems ; Edible Grain/*growth &amp; development ; *Gene Editing ; Nitrogen/*metabolism ; Triticum/*genetics/growth &amp; development/metabolism ; },
abstract = {Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles. All transgene-free mutant lines showed enhanced tolerance to N starvation, and showed delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.},
}
@article {pmid34270141,
year = {2021},
author = {Ma, S and Lv, J and Feng, Z and Rong, Z and Lin, Y},
title = {Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs.},
journal = {The journal of gene medicine},
volume = {23},
number = {11},
pages = {e3377},
doi = {10.1002/jgm.3377},
pmid = {34270141},
issn = {1521-2254},
mesh = {Animals ; *CRISPR-Cas Systems ; Endonucleases/genetics ; Gene Editing/*methods/*standards ; Genetic Engineering/*methods/*standards ; Humans ; *RNA, Guide ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.},
}
@article {pmid34270031,
year = {2021},
author = {Hoffie, RE and Otto, I and Hisano, H and Kumlehn, J},
title = {Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2287},
number = {},
pages = {199-214},
pmid = {34270031},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Culture Media ; Endonucleases/genetics/*metabolism ; Gene Editing ; Genetic Engineering ; Genome, Plant ; *Haploidy ; Homozygote ; Hordeum/embryology/*genetics/*growth &amp; development ; Mutagenesis, Site-Directed/*methods ; Plant Breeding/*methods ; Plants, Genetically Modified ; Pollen/genetics/growth &amp; development ; RNA, Guide/*genetics ; },
abstract = {In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley (Hordeum vulgare) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.},
}
@article {pmid34269521,
year = {2021},
author = {Chen, C and Ma, Y and Du, S and Wu, Y and Shen, P and Yan, T and Li, X and Song, Y and Zha, Z and Han, X},
title = {Controlled CRISPR-Cas9 Ribonucleoprotein Delivery for Sensitized Photothermal Therapy.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {17},
number = {33},
pages = {e2101155},
doi = {10.1002/smll.202101155},
pmid = {34269521},
issn = {1613-6829},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Copper ; Doxorubicin ; *Nanoparticles ; *Phototherapy ; Photothermal Therapy ; Ribonucleoproteins ; },
abstract = {Manipulation of CRISPR delivery for stimuli-responsive gene editing is crucial for cancer therapeutics through maximizing efficacy and minimizing side-effects. However, realizing controlled gene editing for synergistic combination therapy remains a key challenge. Here, a near-infrared (NIR) light-triggered thermo-responsive copper sulfide (CuS) multifunctional nanotherapeutic platform is constructed to achieve controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and doxorubicin for tumor synergistic combination therapy involving in gene therapy, mild-photothermal therapy (PTT), and chemotherapy. The semiconductor CuS serves as a "photothermal converter" and can stably convert NIR light (808 nm) into local thermal effect to provide photothermal stimulation. The double-strand formed between CuS nanoparticle-linked DNA fragments and single-guide RNA is employed as a controlled element in response to photothermal stimulation for controlled gene editing and drug release. Hsp90α, one subunit of heat shock protein 90 (Hsp90), is targeted by Cas9 RNP to reduce tumor heat tolerance for enhanced mild-PTT effects (≈43 °C). Significant synergistic therapy efficacy can be observed by twice NIR light irradiation both in vitro and in vivo, compared to PTT alone. Overall, this exogenously controlled method provides a versatile strategy for controlled gene editing and drug release with potentially synergistic combination therapy.},
}
@article {pmid34268641,
year = {2021},
author = {Chhichholiya, Y and Suman, P and Singh, S and Munshi, A},
title = {The genomic architecture of metastasis in breast cancer: focus on mechanistic aspects, signalling pathways and therapeutic strategies.},
journal = {Medical oncology (Northwood, London, England)},
volume = {38},
number = {8},
pages = {95},
pmid = {34268641},
issn = {1559-131X},
mesh = {Antineoplastic Agents/administration &amp; dosage ; Biomarkers, Tumor/*genetics/metabolism ; Breast Neoplasms/*genetics/metabolism/*therapy ; CRISPR-Cas Systems/physiology ; Cell Proliferation/physiology ; Female ; Genetic Therapy/methods ; *Genomics ; Humans ; Hyperbaric Oxygenation/methods ; Signal Transduction/*genetics ; },
abstract = {Breast cancer is a multifactorial, heterogeneous disease and the second most frequent cancer amongst women worldwide. Metastasis is one of the most leading causes of death in these patients. Early-stage or locally advanced breast cancer is limited to the breast or nearby lymph nodes. When breast cancer spreads to farther tissues/organs from its original site, it is referred to as metastatic or stage IV breast cancer. Normal breast development is regulated by specific genes and signalling pathways controlling cell proliferation, cell death, cell differentiation and cell motility. Dysregulation of genes involved in various signalling pathways not only leads to the formation of primary tumour but also to the metastasis as well. The metastatic cascade is represented by a multi-step process including invasion of the local tumour cell followed by its entry into the vasculature, exit of malignant cells from the circulation and ultimately their colonization at the distant sites. These stages are referred to as formation of primary tumour, angiogenesis, invasion, intravasation and extravasation, respectively. The major sites of metastasis of breast cancer are the lymph nodes, bone, brain and lung. Only about 28% five-year survival rate has been reported for stage IV breast cancer. Metastasis is a serious concern for breast cancer and therefore, various therapeutic strategies such as tyrosine kinase inhibitors have been developed to target specific dysregulated genes and various signalling pathways involved in different steps of metastasis. In addition, other therapies like hyperbaric oxygen therapy, RNA interference and CRISPR/Cas9 are also being explored as novel strategies to cure the stage IV/metastatic breast cancer. Therefore, the current review has been compiled with an aim to evaluate the genetic basis of stage IV breast cancer with a focus on the molecular mechanisms. In addition, the therapeutic strategies targeting these dysregulated genes involved in various signalling pathways have also been discussed. Genome editing technologies that can target specific genes in the affected areas by making knock-in and knock-out alternations and thereby bring significant treatment outcomes in breast cancer have also been summarized.},
}
@article {pmid34268626,
year = {2021},
author = {Kwak, JS and Kim, KH},
title = {Generation of Self-Inhibitory Recombinant Viral Hemorrhagic Septicemia Virus (VHSV) by Insertion of Viral P Gene-Targeting Artificial MicroRNA into Viral Genome and Effect of Dicer Gene Knockout on the Recombinant VHSV Replication.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {23},
number = {4},
pages = {546-559},
pmid = {34268626},
issn = {1436-2236},
mesh = {Animals ; CRISPR-Cas Systems ; Carps ; Cell Line ; Cell Line, Tumor ; Cricetinae ; DEAD-box RNA Helicases/genetics ; Fish Diseases/virology ; Gene Knockout Techniques ; Genome, Viral/*genetics ; Hemorrhagic Septicemia, Viral ; MicroRNAs ; Novirhabdovirus/*genetics ; Phosphoproteins/genetics/metabolism ; Ribonuclease III/genetics/metabolism ; Virus Replication/*genetics ; },
abstract = {To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P. The replication of rVHSV-A-amiR-C in EPC-ΔDicer cells was not different from that in control EPC cells, while the copy number of rVHSV-A-amiR-P was increasingly risen up in EPC-ΔDicer cells compared to that in control EPC cells, and the final viral titer of rVHSV-A-amiR-P was enhanced by culture in EPC-ΔDicer cells. These results indicate that VHSV can be attenuated by the equipment of self-mRNA-targeting microRNA sequence in the genome, and the titer of artificial miRNA-expressing attenuated recombinant VHSVs can be enhanced by the knockout of Dicer gene in EPC cells.},
}
@article {pmid34267391,
year = {2021},
author = {Sheridan, C},
title = {CRISPR therapies march into clinic, but genotoxicity concerns linger.},
journal = {Nature biotechnology},
volume = {39},
number = {8},
pages = {897-899},
doi = {10.1038/d41587-021-00017-3},
pmid = {34267391},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; *Chromothripsis ; Clinical Trials as Topic ; *DNA Breaks, Double-Stranded ; *Gene Editing ; Genetic Therapy/*adverse effects/methods ; Humans ; },
}
@article {pmid34267243,
year = {2021},
author = {Heymans, C and Delcorte, O and Spourquet, C and Villacorte-Tabelin, M and Dupasquier, S and Achouri, Y and Mahibullah, S and Lemoine, P and Balda, MS and Matter, K and Pierreux, CE},
title = {Spatio-temporal expression pattern and role of the tight junction protein MarvelD3 in pancreas development and function.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14519},
pmid = {34267243},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Female ; Gene Expression Regulation, Developmental ; MAP Kinase Signaling System/genetics ; MARVEL Domain-Containing Proteins/*genetics/metabolism ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Pancreas/cytology/*embryology/*physiology ; Salivary Glands/physiology ; Spatio-Temporal Analysis ; Tight Junction Proteins/*genetics/metabolism ; },
abstract = {Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.},
}
@article {pmid34265443,
year = {2021},
author = {Li, R and Char, SN and Liu, B and Liu, H and Li, X and Yang, B},
title = {High-efficiency plastome base editing in rice with TAL cytosine deaminase.},
journal = {Molecular plant},
volume = {14},
number = {9},
pages = {1412-1414},
doi = {10.1016/j.molp.2021.07.007},
pmid = {34265443},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems ; *Cytosine ; Cytosine Deaminase/*genetics ; *Gene Editing ; Oryza/*genetics ; },
}
@article {pmid34265107,
year = {2021},
author = {Augustine, SM and Cherian, AV and Seiling, K and Di Fiore, S and Raven, N and Commandeur, U and Schillberg, S},
title = {Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.},
journal = {Physiologia plantarum},
volume = {173},
number = {3},
pages = {993-1007},
doi = {10.1111/ppl.13499},
pmid = {34265107},
issn = {1399-3054},
mesh = {*CRISPR-Cas Systems ; Destrin ; Mutagenesis ; Osmotic Pressure ; Ribonucleoproteins/genetics ; *Tobacco/genetics/metabolism ; },
abstract = {DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.},
}
@article {pmid34264302,
year = {2021},
author = {Zhao, H and Li, Y and Dong, N and Zhang, L and Chen, X and Mao, H and Al-Ameri, SAA and Wang, X and Wang, Q and Du, L and Wang, C and Mao, H},
title = {LncRNA LINC01088 inhibits the function of trophoblast cells, activates the MAPK-signaling pathway and associates with recurrent pregnancy loss.},
journal = {Molecular human reproduction},
volume = {27},
number = {8},
pages = {},
doi = {10.1093/molehr/gaab047},
pmid = {34264302},
issn = {1460-2407},
mesh = {Abortion, Habitual/*genetics/physiopathology ; Adult ; Apoptosis ; Arginase/metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Division ; Cell Line ; Cell Movement ; Chorionic Villi/metabolism/pathology ; Female ; HEK293 Cells ; Humans ; MAP Kinase Signaling System/*physiology ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/metabolism ; Pregnancy ; Pregnancy Trimester, First ; RNA, Guide/genetics ; RNA, Long Noncoding/biosynthesis/*genetics ; Trophoblasts/*metabolism/pathology ; Up-Regulation ; Young Adult ; },
abstract = {Long noncoding RNAs (lncRNAs) have been reported to be involved in various cellular processes and to participate in a variety of human diseases. Recently, increasing studies have reported that lncRNAs are related to many reproductive diseases, such as pathogenesis of recurrent pregnancy loss (RPL), preeclampsia (PE) and gestational diabetes mellitus (GDM). In this study, we aimed to investigate the effect of LINC01088 in trophoblast cells and its potential role in pathogenesis of RPL. LINC01088 was found to be upregulated in first-trimester chorionic villi tissues from RPL patients. Increased LINC01088 repressed proliferation, migration and invasion of trophoblast cells, and promoted apoptosis of trophoblast cells. Further exploration indicated that LINC01088 decreased the production of nitric oxide (NO) by binding and increasing Arginase-1 and decreasing eNOS protein levels. Importantly, JNK and p38 MAPK-signaling pathways were active after overexpression of LINC01088. In conclusion, our studies demonstrated that LINC01088 plays an important role in the pathogenesis of RPL, and is a potential therapeutic target for the treatment of RPL.},
}
@article {pmid34263445,
year = {2021},
author = {Matres, JM and Hilscher, J and Datta, A and Armario-Nájera, V and Baysal, C and He, W and Huang, X and Zhu, C and Valizadeh-Kamran, R and Trijatmiko, KR and Capell, T and Christou, P and Stoger, E and Slamet-Loedin, IH},
title = {Genome editing in cereal crops: an overview.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {461-498},
pmid = {34263445},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Edible Grain/*genetics ; *Gene Editing ; Gene Targeting ; *Genome, Plant ; Plant Breeding/*methods ; Plants, Genetically Modified/*genetics ; },
abstract = {Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations.},
}
@article {pmid34261850,
year = {2021},
author = {Lee, HJ and Lee, SJ},
title = {Advances in Accurate Microbial Genome-Editing CRISPR Technologies.},
journal = {Journal of microbiology and biotechnology},
volume = {31},
number = {7},
pages = {903-911},
doi = {10.4014/jmb.2106.06056},
pmid = {34261850},
issn = {1738-8872},
mesh = {Base Pair Mismatch ; CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome, Microbial/*genetics ; Nucleotide Motifs ; RNA, Guide/chemistry/metabolism ; },
abstract = {Previous studies have modified microbial genomes by introducing gene cassettes containing selectable markers and homologous DNA fragments. However, this requires several steps including homologous recombination and excision of unnecessary DNA regions, such as selectable markers from the modified genome. Further, genomic manipulation often leaves scars and traces that interfere with downstream iterative genome engineering. A decade ago, the CRISPR/Cas system (also known as the bacterial adaptive immune system) revolutionized genome editing technology. Among the various CRISPR nucleases of numerous bacteria and archaea, the Cas9 and Cas12a (Cpf1) systems have been largely adopted for genome editing in all living organisms due to their simplicity, as they consist of a single polypeptide nuclease with a target-recognizing RNA. However, accurate and fine-tuned genome editing remains challenging due to mismatch tolerance and protospacer adjacent motif (PAM)-dependent target recognition. Therefore, this review describes how to overcome the aforementioned hurdles, which especially affect genome editing in higher organisms. Additionally, the biological significance of CRISPR-mediated microbial genome editing is discussed, and future research and development directions are also proposed.},
}
@article {pmid34261503,
year = {2021},
author = {Karcher, N and Nigro, E and Punčochář, M and Blanco-Míguez, A and Ciciani, M and Manghi, P and Zolfo, M and Cumbo, F and Manara, S and Golzato, D and Cereseto, A and Arumugam, M and Bui, TPN and Tytgat, HLP and Valles-Colomer, M and de Vos, WM and Segata, N},
title = {Genomic diversity and ecology of human-associated Akkermansia species in the gut microbiome revealed by extensive metagenomic assembly.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {209},
pmid = {34261503},
issn = {1474-760X},
support = {U01 CA230551/CA/NCI NIH HHS/United States ; },
mesh = {Akkermansia/classification/genetics/metabolism/virology ; Animals ; Bacteriophages/growth &amp; development ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gastrointestinal Microbiome/*genetics ; Genetic Variation ; *Genome, Bacterial ; Humans ; *Metagenome ; Mice ; Operon ; *Phylogeny ; RNA, Ribosomal, 16S/genetics ; },
abstract = {BACKGROUND: Akkermansia muciniphila is a human gut microbe with a key role in the physiology of the intestinal mucus layer and reported associations with decreased body mass and increased gut barrier function and health. Despite its biomedical relevance, the genomic diversity of A. muciniphila remains understudied and that of closely related species, except for A. glycaniphila, unexplored.<br><br>RESULTS: We present a large-scale population genomics analysis of the Akkermansia genus using 188 isolate genomes and 2226 genomes assembled from 18,600 metagenomes from humans and other animals. While we do not detect A. glycaniphila, the Akkermansia strains in the human gut can be grouped into five distinct candidate species, including A. muciniphila, that show remarkable whole-genome divergence despite surprisingly similar 16S rRNA gene sequences. These candidate species are likely human-specific, as they are detected in mice and non-human primates almost exclusively when kept in captivity. In humans, Akkermansia candidate species display ecological co-exclusion, diversified functional capabilities, and distinct patterns of associations with host body mass. Analysis of CRISPR-Cas loci reveals new variants and spacers targeting newly discovered putative bacteriophages. Remarkably, we observe an increased relative abundance of Akkermansia when cognate predicted bacteriophages are present, suggesting ecological interactions. A. muciniphila further exhibits subspecies-level genetic stratification with associated functional differences such as a putative exo/lipopolysaccharide operon.<br><br>CONCLUSIONS: We uncover a large phylogenetic and functional diversity of the Akkermansia genus in humans. This variability should be considered in the ongoing experimental and metagenomic efforts to characterize the health-associated properties of A. muciniphila and related bacteria.},
}
@article {pmid34260911,
year = {2021},
author = {Kumari, R and Roy, U and Desai, S and Nilavar, NM and Van Nieuwenhuijze, A and Paranjape, A and Radha, G and Bawa, P and Srivastava, M and Nambiar, M and Balaji, KN and Liston, A and Choudhary, B and Raghavan, SC},
title = {MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development.},
journal = {Cell reports},
volume = {36},
number = {2},
pages = {109390},
doi = {10.1016/j.celrep.2021.109390},
pmid = {34260911},
issn = {2211-1247},
mesh = {3' Untranslated Regions/genetics ; Animals ; B-Lymphocytes/cytology/*metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Gene Expression Regulation ; Homeodomain Proteins/*genetics/metabolism ; Humans ; Luciferases/metabolism ; Lymphocytes/metabolism ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; MicroRNAs/genetics/*metabolism ; RNA Processing, Post-Transcriptional/genetics ; V(D)J Recombination/*genetics ; },
abstract = {Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes. We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer.},
}
@article {pmid34259977,
year = {2021},
author = {Bae, EK and Choi, H and Choi, JW and Lee, H and Kim, SG and Ko, JH and Choi, YI},
title = {Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.},
journal = {Transgenic research},
volume = {30},
number = {6},
pages = {837-849},
pmid = {34259977},
issn = {1573-9368},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Oxidoreductases ; Plants, Genetically Modified/genetics ; *Populus/genetics ; *RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.},
}
@article {pmid34257417,
year = {2022},
author = {Gumerson, JD and Alsufyani, A and Yu, W and Lei, J and Sun, X and Dong, L and Wu, Z and Li, T},
title = {Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing.},
journal = {Gene therapy},
volume = {29},
number = {1-2},
pages = {81-93},
pmid = {34257417},
issn = {1476-5462},
support = {ZIA EY000490/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Eye Proteins/genetics/metabolism ; Gene Editing ; Mice ; Mutation ; *Retinal Degeneration/genetics/therapy ; *Retinitis Pigmentosa/genetics/metabolism/therapy ; },
abstract = {Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGR[ORF15], through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGR[ORF15] protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGR[ORF15] in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGR[ORF15] mutations in vivo.},
}
@article {pmid34257367,
year = {2021},
author = {Mansilla, M and Wang, Y and Lim, R and Palmer, K and Nie, G},
title = {HtrA4 is up-regulated during trophoblast syncytialization and BeWo cells fail to syncytialize without HtrA4.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {14363},
pmid = {34257367},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cadherins/biosynthesis ; Cell Line ; Cell Line, Tumor ; Colforsin/chemistry/pharmacology ; Down-Regulation ; Female ; *Gene Expression Regulation ; Gene Products, env/biosynthesis ; Humans ; Placenta/metabolism ; Pregnancy ; Pregnancy Proteins/biosynthesis ; RNA, Messenger/metabolism ; Serine Proteases/*biosynthesis ; Time Factors ; Trophoblasts/*cytology/*metabolism ; *Up-Regulation ; },
abstract = {The outer layer of the human placenta comprises syncytiotrophoblast, which forms through fusion of cytotrophoblasts (syncytialization), and plays a critical role in maternal-fetal communication including nutrient/oxygen transportation and hormone secretion. Impairment in syncytialization inevitably affects pregnancy outcomes. High temperature requirement factor A 4 (HtrA4) is a placental-specific protease, expressed by various trophoblasts including syncytiotrophoblast, and significantly elevated in preeclampsia at disease presentation. However, it is unknown whether HtrA4 is important for syncytialization. Here we first examined HtrA4 expression in primary human cytotrophoblasts during syncytialization which occurs spontaneously in culture, and in BeWo cells which syncytialize upon forskolin stimulation. The success of syncytialization in each model was confirmed by significant up-regulation/secretion of β-hCG, and the concurrent down-regulation of E-cadherin. In both models, HtrA4 mRNA and protein increased concomitantly with syncytialization. Furthermore, the secreted levels of β-hCG and HtrA4 correlated significantly and positively in both models. We next knocked out HtrA4 in BeWo by CRISPR/Cas9. Upon forskolin treatment, control BeWo profoundly up-regulated β-hCG and syncytin-1, down-regulated E-cadherin, and at the same time increased the formation of multinucleated cells, whereas BeWo cells without HtrA4 did not alter any of these parameters. Our data thus suggest that HtrA4 plays an essential role in syncytialization.},
}
@article {pmid34257311,
year = {2021},
author = {Fareh, M and Zhao, W and Hu, W and Casan, JML and Kumar, A and Symons, J and Zerbato, JM and Fong, D and Voskoboinik, I and Ekert, PG and Rudraraju, R and Purcell, DFJ and Lewin, SR and Trapani, JA},
title = {Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4270},
pmid = {34257311},
issn = {2041-1723},
mesh = {Animals ; Antiviral Agents/pharmacology ; COVID-19/drug therapy/virology ; CRISPR-Cas Systems ; Chlorocebus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Development ; Genome, Viral ; HEK293 Cells ; Humans ; *Mutation ; SARS-CoV-2/genetics/*physiology ; Spike Glycoprotein, Coronavirus/genetics/metabolism ; Vero Cells ; Virus Replication/genetics/*physiology ; },
abstract = {The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.},
}
@article {pmid34254038,
year = {2021},
author = {Perčulija, V and Lin, J and Zhang, B and Ouyang, S},
title = {Functional Features and Current Applications of the RNA-Targeting Type VI CRISPR-Cas Systems.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {8},
number = {13},
pages = {2004685},
pmid = {34254038},
issn = {2198-3844},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas systems are a form of prokaryotic adaptive immunity that employs RNA-guided endonucleases (Cas effectors) to cleave foreign genetic elements. Due to their simplicity, targeting programmability, and efficiency, single-effector CRISPR-Cas systems have great potential for application in research, biotechnology, and therapeutics. While DNA-targeting Cas effectors such as Cas9 and Cas12a have become indispensable tools for genome editing in the past decade, the more recent discovery of RNA-targeting CRISPR-Cas systems has opened the door for implementation of CRISPR-Cas technology in RNA manipulation. With an increasing number of studies reporting their application in transcriptome engineering, viral interference, nucleic acid detection, and RNA imaging, type VI CRISPR-Cas systems and the associated Cas13 effectors particularly hold promise as RNA-targeting or RNA-binding tools. However, even though previous structural and biochemical characterization provided a firm basis for leveraging type VI CRISPR-Cas systems into such tools, the lack of comprehension of certain mechanisms underlying their functions hinders more sophisticated and conventional use. This review will summarize current knowledge on structural and mechanistic properties of type VI CRISPR-Cas systems, give an overview on the reported applications, and discuss functional features that need further investigation in order to improve performance of Cas13-based tools.},
}
@article {pmid34254007,
year = {2021},
author = {Yan, K and Feng, J and Liu, X and Wang, H and Li, Q and Li, J and Xu, T and Sajid, M and Ullah, H and Zhou, L and Zhou, L and Chen, Y},
title = {Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {665184},
pmid = {34254007},
issn = {1664-302X},
abstract = {Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR/Staphylococcus aureus Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both in vitro and in vivo. Three functional guide RNAs (gRNAs), T2, T3, and T6, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.},
}
@article {pmid34253365,
year = {2021},
author = {Shandilya, UK and Sharma, A and Mallikarjunappa, S and Guo, J and Mao, Y and Meade, KG and Karrow, NA},
title = {CRISPR-Cas9-mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate-induced inflammation in bovine mammary epithelial cells.},
journal = {Journal of dairy science},
volume = {104},
number = {10},
pages = {11135-11146},
doi = {10.3168/jds.2021-20305},
pmid = {34253365},
issn = {1525-3198},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; *Cattle Diseases/genetics ; Epithelial Cells ; Female ; Inflammation/veterinary ; *Mycobacterium avium subsp. paratuberculosis ; *Paratuberculosis/genetics ; Toll-Like Receptor 4 ; },
abstract = {Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.},
}
@article {pmid34252931,
year = {2021},
author = {Li, VR and Zhang, Z and Troyanskaya, OG},
title = {CROTON: an automated and variant-aware deep learning framework for predicting CRISPR/Cas9 editing outcomes.},
journal = {Bioinformatics (Oxford, England)},
volume = {37},
number = {Suppl_1},
pages = {i342-i348},
pmid = {34252931},
issn = {1367-4811},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Deep Learning ; Gene Editing ; Neural Networks, Computer ; RNA, Guide ; },
abstract = {MOTIVATION: CRISPR/Cas9 is a revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. CRISPR/Cas9 editing outcomes depend on local DNA sequences at the target site and are thus predictable. However, existing prediction methods are dependent on both feature and model engineering, which restricts their performance to existing knowledge about CRISPR/Cas9 editing.<br><br>RESULTS: Herein, deep multi-task convolutional neural networks (CNNs) and neural architecture search (NAS) were used to automate both feature and model engineering and create an end-to-end deep-learning framework, CROTON (CRISPR Outcomes Through cONvolutional neural networks). The CROTON model architecture was tuned automatically with NAS on a synthetic large-scale construct-based dataset and then tested on an independent primary T cell genomic editing dataset. CROTON outperformed existing expert-designed models and non-NAS CNNs in predicting 1 base pair insertion and deletion probability as well as deletion and frameshift frequency. Interpretation of CROTON revealed local sequence determinants for diverse editing outcomes. Finally, CROTON was utilized to assess how single nucleotide variants (SNVs) affect the genome editing outcomes of four clinically relevant target genes: the viral receptors ACE2 and CCR5 and the immune checkpoint inhibitors CTLA4 and PDCD1. Large SNV-induced differences in CROTON predictions in these target genes suggest that SNVs should be taken into consideration when designing widely applicable gRNAs.<br><br>https://github.com/vli31/CROTON.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid34251367,
year = {2021},
author = {Carballar-Lejarazú, R and Pham, TB and Kelsey, A and Tushar, T and James, AA},
title = {Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {172},
pages = {},
doi = {10.3791/62607},
pmid = {34251367},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Culicidae/genetics ; *Gene Editing ; INDEL Mutation ; Polymerase Chain Reaction ; RNA, Guide ; },
abstract = {Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.},
}
@article {pmid34250657,
year = {2021},
author = {Lee, M and Choi, KH and Oh, JN and Kim, SH and Lee, DK and Choe, GC and Jeong, J and Lee, CK},
title = {SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development.},
journal = {Cell proliferation},
volume = {54},
number = {8},
pages = {e13097},
pmid = {34250657},
issn = {1365-2184},
mesh = {Animals ; Blastocyst/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Cell Proliferation ; Embryo, Mammalian/cytology/metabolism ; *Embryonic Development ; Female ; RNA, Guide/metabolism ; SOXB1 Transcription Factors/genetics/*metabolism ; Swine ; Transcription Factors/genetics/metabolism ; Transcriptome ; },
abstract = {OBJECTIVES: Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species.<br><br>MATERIALS AND METHODS: To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR.<br><br>RESULTS: Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.<br><br>CONCLUSIONS: Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis.},
}
@article {pmid34248284,
year = {2021},
author = {Zhang, Y and Chen, M and Liu, C and Chen, J and Luo, X and Xue, Y and Liang, Q and Zhou, L and Tao, Y and Li, M and Wang, D and Zhou, J and Wang, J},
title = {Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP.},
journal = {Sensors and actuators. B, Chemical},
volume = {345},
number = {},
pages = {130411},
pmid = {34248284},
issn = {0925-4005},
abstract = {The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called Cas12a-assisted RT-LAMP/AuNP (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/μL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.},
}
@article {pmid34247734,
year = {2021},
author = {Wang, P and Liu, Y and Yu, Y and Zhang, Y and Peng, J and Niu, L and Zhang, J},
title = {Hydrazone ligation assisted DNAzyme walking nanomachine coupled with CRISPR-Cas12a for lipopolysaccharide analysis.},
journal = {Analytica chimica acta},
volume = {1174},
number = {},
pages = {338747},
doi = {10.1016/j.aca.2021.338747},
pmid = {34247734},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA, Catalytic ; Hydrazones ; Lipopolysaccharides ; Walking ; },
abstract = {In this work, hydrazone ligation assisted DNAzyme walking nanomachine is explored to couple with CRISPR-Cas12a trans-cleavage. Hydrazone ligation with high efficiency can mediate signal input which can be induced by target binding, thereby regulating the performance of DNAzyme walking nanomachine. The product strand from DNAzyme walking nanomachine can further activate the trans-cleavage of Cas12a. So, cascade signal amplification can be achieved to enhance the sensitivity for target detection. Subsequently, hydrazone ligation assisted DNAzyme walking nanomachine coupled with CRISPR-Cas12a has been further developed as a biosensor to analyze lipopolysaccharides. The developed biosensor exhibits a linear range from 0.05 ng/mL to 10[6] ng/mL and a lowest limit of detection of 7.31 fg/mL. This research provides a new mode for the signal output of DNAzyme walking nanomachine, so as to sensitively analyze different biomolecules.},
}
@article {pmid34247443,
year = {2022},
author = {Gim, GM and Kwon, DH and Eom, KH and Moon, J and Park, JH and Lee, WW and Jung, DJ and Kim, DH and Yi, JK and Ha, JJ and Lim, KY and Kim, JS and Jang, G},
title = {Production of MSTN-mutated cattle without exogenous gene integration using CRISPR-Cas9.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100198},
doi = {10.1002/biot.202100198},
pmid = {34247443},
issn = {1860-7314},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; Cattle/genetics ; Gene Editing/methods ; *Myostatin/genetics/metabolism ; Phenotype ; },
abstract = {Many genome-edited animals have been produced using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique to cattle. The goal of this study was to produce trait-improved cattle using the genome-editing technology CRISPR-Cas9. Myostatin (MSTN) was selected as a target locus, and synthetic mRNA of sgRNA and Cas9 were microinjected into fertilized bovine embryos in vitro. As a result, 17 healthy calves were born, and three of them showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had a biallelic mutation (-12 bps) and a double-muscling phenotype. In conclusion, we demonstrate that the genome-editing technology CRISPR-Cas9 can produce genetically modified calves with improved traits.},
}
@article {pmid34246805,
year = {2021},
author = {Bond, ST and Zhuang, A and Yang, C and Gould, EAM and Sikora, T and Liu, Y and Fu, Y and Watt, KI and Tan, Y and Kiriazis, H and Lancaster, GI and Gregorevic, P and Henstridge, DC and McMullen, JR and Meikle, PJ and Calkin, AC and Drew, BG},
title = {Tissue-specific expression of Cas9 has no impact on whole-body metabolism in four transgenic mouse lines.},
journal = {Molecular metabolism},
volume = {53},
number = {},
pages = {101292},
pmid = {34246805},
issn = {2212-8778},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Phenotype ; },
abstract = {OBJECTIVE: CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly.<br><br>METHODS: In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs.<br><br>RESULTS: We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass.<br><br>CONCLUSIONS: Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.},
}
@article {pmid34245257,
year = {2021},
author = {Harris, A},
title = {Human Molecular Genetics and the long road to treating cystic fibrosis.},
journal = {Human molecular genetics},
volume = {30},
number = {R2},
pages = {R264-R273},
pmid = {34245257},
issn = {1460-2083},
support = {R01 HL094585/HL/NHLBI NIH HHS/United States ; R01 HD068901/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cystic Fibrosis/*diagnosis/*genetics/*therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Disease Management ; Gene Editing ; Gene Expression Regulation ; *Genetic Association Studies/methods ; *Genetic Predisposition to Disease ; Genetic Therapy ; Humans ; Molecular Diagnostic Techniques ; Mutation ; Single-Cell Analysis ; },
abstract = {The causative gene in cystic fibrosis (CF) was identified in 1989, 3 years before the publication of the first issue of Human Molecular Genetics. The cystic fibrosis transmembrane conductance regulator (CFTR) gene was among the first underlying a common inherited disorder to be cloned, and hence, its subsequent utilization toward a cure for CF provides a roadmap for other monogenic diseases. Over the past 30 years, the advances that built upon knowledge of the gene and the CFTR protein to develop effective therapeutics have been remarkable, and yet, the setbacks have also been challenging. Technological progress in other fields has often circumvented the barriers. This review focuses on key aspects of CF diagnostics and current approaches to develop new therapies for all CFTR mutations. It also highlights the major research advances that underpinned progress toward treatments and considers the remaining obstacles.},
}
@article {pmid34244519,
year = {2021},
author = {Zhu, C and Wu, J and Sun, H and Briganti, F and Meder, B and Wei, W and Steinmetz, LM},
title = {Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4203},
pmid = {34244519},
issn = {2041-1723},
mesh = {*Alternative Splicing ; CRISPR-Cas Systems/genetics ; Cardiomyopathy, Dilated/*genetics/pathology ; Cell Differentiation/genetics ; Cell Line ; Feasibility Studies ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells ; Mitochondrial Proteins/genetics ; Molecular Sequence Annotation ; Muscle Proteins/genetics ; Mutation ; Myocytes, Cardiac/*pathology ; RNA Isoforms/genetics ; RNA, Guide/genetics ; RNA-Binding Proteins/*genetics ; RNA-Seq/*methods ; },
abstract = {Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.},
}
@article {pmid34244505,
year = {2021},
author = {Schmidt, MJ and Gupta, A and Bednarski, C and Gehrig-Giannini, S and Richter, F and Pitzler, C and Gamalinda, M and Galonska, C and Takeuchi, R and Wang, K and Reiss, C and Dehne, K and Lukason, MJ and Noma, A and Park-Windhol, C and Allocca, M and Kantardzhieva, A and Sane, S and Kosakowska, K and Cafferty, B and Tebbe, J and Spencer, SJ and Munzer, S and Cheng, CJ and Scaria, A and Scharenberg, AM and Cohnen, A and Coco, WM},
title = {Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4219},
pmid = {34244505},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/isolation &amp; purification ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Dependovirus ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors/administration &amp; dosage/genetics ; HEK293 Cells ; Humans ; Macaca fascicularis ; Male ; Mice ; Parvovirinae/genetics ; Protein Engineering ; RNA, Guide/*genetics ; Ribonucleases ; Staphylococcus/*enzymology/genetics ; Substrate Specificity ; Usher Syndromes/genetics/therapy ; },
abstract = {Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.},
}
@article {pmid34244352,
year = {2022},
author = {Blitz, IL and Nakayama, T},
title = {CRISPR-Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in the F0 Generation and Beyond.},
journal = {Cold Spring Harbor protocols},
volume = {2022},
number = {3},
pages = {},
doi = {10.1101/pdb.prot106971},
pmid = {34244352},
issn = {1559-6095},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Chromosomes, Human, Y ; Gene Editing/methods ; Humans ; Male ; Mosaicism ; Mutagenesis ; Phenotype ; Xenopus/genetics ; },
abstract = {CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Xenopus tropicalis Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR-Cas9 can be used to create either mosaic loss-of-function Xenopus embryos that display F0 generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR-Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.},
}
@article {pmid34244351,
year = {2022},
author = {Cha, SW},
title = {Generating Nonmosaic Mutants in Xenopus Using CRISPR-Cas in Oocytes.},
journal = {Cold Spring Harbor protocols},
volume = {2022},
number = {6},
pages = {Pdb.prot106989},
doi = {10.1101/pdb.prot106989},
pmid = {34244351},
issn = {1559-6095},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA ; *Gene Editing/methods ; Oocytes ; Xenopus laevis/genetics ; },
abstract = {In CRISPR-Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR-Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR-Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR-Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR-Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73-104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.},
}
@article {pmid34241908,
year = {2021},
author = {Sadeqi Nezhad, M and Yazdanifar, M and Abdollahpour-Alitappeh, M and Sattari, A and Seifalian, A and Bagheri, N},
title = {Strengthening the CAR-T cell therapeutic application using CRISPR/Cas9 technology.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {10},
pages = {3691-3705},
doi = {10.1002/bit.27882},
pmid = {34241908},
issn = {1097-0290},
mesh = {*Adoptive Transfer ; *CRISPR-Cas Systems ; Hematologic Neoplasms/genetics/*therapy ; Humans ; *Receptors, Chimeric Antigen/genetics/therapeutic use ; },
abstract = {Adoptive cell immunotherapy with chimeric antigen receptor T (CAR-T) cell has brought a revolutionary means of treatment for aggressive diseases such as hematologic malignancies and solid tumors. Over the last decade, the United States Food and Drug Administration (FDA) approved five types of CAR-T cell therapies for hematologic malignancies, including Idecabtagene vicleucel (Abecma), Lisocabtagene maraleucel (Breyanzi), Brexucabtagene autoleucel (Tecartus), Tisagenlecleucel (Kymriah), and Axicabtagene ciloleucel (Yescarta). Despite outstanding results gained from different clinical trials, CAR-T cell therapy is not free from side effects and toxicities, and needs careful investigations and improvements. Gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, has emerged as a promising tool to address some of the CAR-T therapy hurdles. Using CRISPR/Cas9 technology, CAR expression as well as other cellular pathways can be modified in various ways to enhance CAR-T cells antitumor function and persistence in immunosuppressive tumor microenvironment. CRISPR/Cas9 technology can also be used to decrease CAR-T cell toxicities and side effects. Hereby, we discussed the practical challenges and hurdles related to the accuracy, efficiency, efficacy, safety, and delivery of CRISPR/Cas9 technology to the genetically engineered-T cells. Combining of these two state-of-the-art technologies, CRISPR/Cas9 and CAR-T cells, the field of oncology has an extraordinary opportunity to enter a new era of immunotherapy, which offers novel therapeutic options for different types of tumors.},
}
@article {pmid34240255,
year = {2021},
author = {Li, Y and Zhang, H and Fan, J and Chen, Z and Chen, T and Zeng, B and Zhang, Z},
title = {A highly efficient identification of mutants generated by CRISPR/Cas9 using the non‑functional DsRed assisted selection in Aspergillus oryzae.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {37},
number = {8},
pages = {132},
pmid = {34240255},
issn = {1573-0972},
mesh = {Aspergillus oryzae/enzymology/*genetics/metabolism ; *CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; *Genetic Techniques ; Luminescent Proteins/*genetics/metabolism ; Mutation ; Plasmids/genetics/metabolism ; Pyrones/metabolism ; },
abstract = {The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae. Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon "ATG" of DsRed, yielding the non‑functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing. Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.},
}
@article {pmid34238812,
year = {2021},
author = {Adams, AB and Lovasik, BP and Faber, DA and Burlak, C and Breeden, C and Estrada, JL and Reyes, LM and Vianna, RM and Tector, MF and Tector, AJ},
title = {Anti-C5 Antibody Tesidolumab Reduces Early Antibody-mediated Rejection and Prolongs Survival in Renal Xenotransplantation.},
journal = {Annals of surgery},
volume = {274},
number = {3},
pages = {473-480},
pmid = {34238812},
issn = {1528-1140},
support = {P51 OD011132/OD/NIH HHS/United States ; R01 AI026322/AI/NIAID NIH HHS/United States ; R01 AI126322/AI/NIAID NIH HHS/United States ; U01 AI126322/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Antibiotic Prophylaxis ; Antibodies, Monoclonal/*pharmacology ; Antibodies, Monoclonal, Humanized/*pharmacology ; Graft Rejection/*immunology/*prevention &amp; control ; Immune Tolerance ; Immunosuppressive Agents/*pharmacology ; *Kidney Transplantation ; Macaca mulatta ; Models, Animal ; Rituximab/pharmacology ; Swine ; Tacrolimus/pharmacology ; *Transplantation, Heterologous ; },
abstract = {OBJECTIVE: Pig-to-primate renal xenotransplantation is plagued by early antibody-mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody-mediated rejection in rhesus monkeys receiving pig kidneys receiving costimulatory blockade-based immunosuppression.<br><br>METHODS: Double (Gal and Sda) and triple xenoantigen (Gal, Sda, and SLA I) pigs were created using CRISPR/Cas. Kidneys from DKO and TKO pigs were transplanted into rhesus monkeys that had the least reactive crossmatches. Recipients received anti-C5 antibody weekly for 70&#x200a;days, and T cell depletion, anti-CD154, mycophenolic acid, and steroids as baseline immunosuppression (n = 7). Control recipients did not receive anti-C5 therapy (n = 10).<br><br>RESULTS: Temporary anti-C5 therapy reduced early graft loss secondary to antibody-mediated rejection and improved graft survival (P < 0.01). Deleting class I MHC (SLA I) in donor pigs did not ameliorate early antibody-mediated rejection (table). Anti-C5 therapy did not allow for the use of tacrolimus instead of anti-CD154 (table), prolonging survival to a maximum of 62&#x200a;days.<br><br>CONCLUSION: Inhibition of the C5 complement subunit prolongs renal xenotransplant survival in a pig to non-human primate model.},
}
@article {pmid34237591,
year = {2021},
author = {Bogomiakova, ME and Sekretova, EK and Eremeev, AV and Shuvalova, LD and Bobrovsky, PA and Zerkalenkova, EA and Lebedeva, OS and Lagarkova, MA},
title = {Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing.},
journal = {Stem cell research},
volume = {55},
number = {},
pages = {102451},
doi = {10.1016/j.scr.2021.102451},
pmid = {34237591},
issn = {1876-7753},
mesh = {CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {The mismatch of HLA haplotypes between donor and recipient adversely affects the outcome of tissue transplantation. TheB2Mgene knockout (B2M-KO) disrupts the HLA I heterodimer formation; therefore,B2M-KO cells have reduced immunogenicity to allogeneic CD8[+] T cells. Thus, theB2M-KO IPSCs and their derivatives can potentially solve a problem of the immunological compatibility in allogeneic transplantations. Using CRISPR/Cas9-mediated genome editing, we generated a human B2M-KO iPSC line (RCPCMi007-A-1). The RCPCMi007-A-1 iPSCs express pluripotency markers, have typical stem cell morphology, maintain normal karyotype, and the ability to differentiate into three germ layers.},
}
@article {pmid34237097,
year = {2021},
author = {Palacios Araya, D and Palmer, KL and Duerkop, BA},
title = {CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria.},
journal = {PLoS pathogens},
volume = {17},
number = {7},
pages = {e1009672},
pmid = {34237097},
issn = {1553-7374},
support = {R01 AI116610/AI/NIAID NIH HHS/United States ; R01 AI141479/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Infective Agents/*pharmacology ; *CRISPR-Cas Systems ; *Drug Resistance, Microbial ; Humans ; },
}
@article {pmid34237032,
year = {2021},
author = {González-Martínez, J and Cwetsch, AW and Martínez-Alonso, D and López-Sainz, LR and Almagro, J and Melati, A and Gómez, J and Pérez-Martínez, M and Megías, D and Boskovic, J and Gilabert-Juan, J and Graña-Castro, O and Pierani, A and Behrens, A and Ortega, S and Malumbres, M},
title = {Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias.},
journal = {JCI insight},
volume = {6},
number = {16},
pages = {},
pmid = {34237032},
issn = {2379-3708},
mesh = {Animals ; Brain/cytology/pathology ; CRISPR-Cas Systems/genetics ; Calmodulin-Binding Proteins/genetics/metabolism ; Cell Cycle Proteins/genetics/metabolism ; Centrioles/*genetics/pathology ; *Chromosomal Instability ; Disease Models, Animal ; Embryo, Mammalian ; Female ; Humans ; Male ; Mice ; Mice, Knockout ; Microcephaly/*genetics/pathology ; Microscopy, Electron, Transmission ; Molecular Imaging ; Mutation ; Nerve Tissue Proteins/genetics/metabolism ; Neural Stem Cells/cytology/*pathology/ultrastructure ; Primary Cell Culture ; Time-Lapse Imaging ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.},
}
@article {pmid34236684,
year = {2021},
author = {Arazoe, T},
title = {Genome Editing Using CRISPR/Cas9 System in the Rice Blast Fungus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2356},
number = {},
pages = {149-160},
pmid = {34236684},
issn = {1940-6029},
mesh = {Ascomycota ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Magnaporthe/genetics ; RNA, Guide/genetics ; },
abstract = {Fast and flexible genome manipulation is a powerful strategy for an in-depth understanding of molecular mechanisms in biological research. In recent years, CRISPR/Cas9-mediated genome editing has been used as a reliable genome manipulation method in a broad range of biological research including studies of filamentous fungi. The CRISPR/Cas9 system comprises a single-guide RNA (sgRNA) and a Cas9 protein, and the Cas9/sgRNA complex catalyzes a DNA double-strand break at the desired genomic locus. This protocol describes a fundamental CRISPR/Cas9 methodology that includes the design of the target sequence, construction of the CRISPR/Cas9 expression vector, and transformation for genome editing in Pyricularia (Magnaporthe) oryzae. This allows efficient targeted gene disruption, base editing, and reporter gene knock-in without any additional modifications of the host components. This protocol would be suitable for applying other CRISPR/Cas technologies and various functional genomics in P. oryzae.},
}
@article {pmid34236584,
year = {2021},
author = {Fang, X and Sun, X and Yang, X and Li, Q and Lin, C and Xu, J and Gong, W and Wang, Y and Liu, L and Zhao, L and Liu, B and Qin, J and Zhang, M and Zhang, C and Kong, F and Li, M},
title = {MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean.},
journal = {Science China. Life sciences},
volume = {64},
number = {9},
pages = {1533-1545},
pmid = {34236584},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems ; *Genes, Plant ; Hybridization, Genetic ; Phenotype ; Plant Breeding ; Plant Infertility/*genetics ; Plant Proteins/*genetics ; Seeds/*genetics ; Soybeans/*genetics ; },
abstract = {Male sterility is an essential trait in hybrid seed production, especially for monoclinous and autogamous food crops. Soybean male-sterile ms1 mutant has been known for more than 50 years and could be instrumental in making hybrid seeds. However, the gene responsible for the male-sterile phenotype has remained unknown. Here, we report the map-based cloning and characterization of the MS1 gene in soybean. MS1 encodes a kinesin protein and localizes to the nucleus, where it is required for the male meiotic cytokinesis after telophase II. We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining. Both ms1 and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal, making them perfect tools for producing hybrid seeds. The identification of MS1 has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.},
}
@article {pmid34235662,
year = {2021},
author = {Wong, AI and Rock, JM},
title = {CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2314},
number = {},
pages = {343-364},
pmid = {34235662},
issn = {1940-6029},
mesh = {Bacterial Proteins/*antagonists &amp; inhibitors/*genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; *Gene Silencing ; Genetic Engineering/*methods ; Genetic Techniques ; Mycobacterium tuberculosis/*genetics/growth &amp; development ; },
abstract = {The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close this gap, we recently developed a robust CRISPR interference (CRISPRi) platform for programmable gene silencing in mycobacteria. In this chapter, we: (1) discuss some of the advantages and disadvantages of CRISPRi relative to more traditional genetic approaches; and (2) provide a protocol for the application of CRISPRi to reduce transcription of target genes in mycobacteria.},
}
@article {pmid34235638,
year = {2021},
author = {Carpenter, JC and Lignani, G},
title = {Gene Editing and Modulation: the Holy Grail for the Genetic Epilepsies?.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {18},
number = {3},
pages = {1515-1523},
pmid = {34235638},
issn = {1878-7479},
support = {MR/S011005/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Epilepsy/*genetics/*therapy ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Precision Medicine/*methods ; },
abstract = {Epilepsy is a complex neurological disorder for which there are a large number of monogenic subtypes. Monogenic epilepsies are often severe and disabling, featuring drug-resistant seizures and significant developmental comorbidities. These disorders are potentially amenable to a precision medicine approach, of which genome editing using CRISPR/Cas represents the holy grail. Here we consider mutations in some of the most 'common' rare epilepsy genes and discuss the different CRISPR/Cas approaches that could be taken to cure these disorders. We consider scenarios where CRISPR-mediated gene modulation could serve as an effective therapeutic strategy and discuss whether a single gene corrective approach could hold therapeutic potential in the context of homeostatic compensation in the developing, highly dynamic brain. Despite an incomplete understanding of the mechanisms of the genetic epilepsies and current limitations of gene editing tools, CRISPR-mediated approaches have game-changing potential in the treatment of genetic epilepsy over the next decade.},
}
@article {pmid34235515,
year = {2021},
author = {Liu, J and Wan, Q and Zeng, R and Tang, D},
title = {An ultrasensitive homogeneous electrochemical biosensor based on CRISPR-Cas12a.},
journal = {Analytical methods : advancing methods and applications},
volume = {13},
number = {29},
pages = {3227-3232},
doi = {10.1039/d1ay00725d},
pmid = {34235515},
issn = {1759-9679},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Electrodes ; Humans ; },
abstract = {Taking advantage of the high-efficiency indiscriminate ssDNA cleavage activity of Cas12a in combination with the diffusivity difference of methylene blue (MB)-labeled probes (short oligonucleotides/mononucleotides) toward the negatively-charged indium tin oxide (ITO) electrode, a simple, immobilization-free, highly sensitive, and homogeneous electrochemical biosensor for the detection of human papillomavirus (HPV-16) has been fabricated. At the core of the detection process, Cas12a employed ssDNA trans-cleavage capability to achieve short-strand nucleotide cleavage, while MB-labeled probes served as high-efficiency homogeneous electrochemical emitters to achieve differential pulse voltammetric (DPV) signal. Specifically, due to strong electrostatic repulsion, MB-labeled short oligonucleotides (reporter) cannot diffuse freely to the surface of the negatively charged ITO electrode, and only weak electrochemical signals can be detected. The presence of the target HPV-16 can activate the Cas12a complex to perform indiscriminate ssDNA cleavage of the reporter to produce MB-labeled mononucleotides. The MB-labeled mononucleotides with a smaller size have almost no negative charge, so they very easily diffuse to the surface of the ITO electrode and result in an enhanced electrochemical signal response. Different electrochemical responses (DPV peak intensity) of the CRISPR-Cas12a-assisted amplification strategy can be obtained through the diffusion rate of different MB-labeled DNA on the electrode, which is also positively correlated with the input HPV-16 concentration. Given the unique combination of the CRISPR-Cas12a system with the homogeneous electrochemical solution phase, the detection limit is determined to be 3.22 pM (wide dynamic working range from 0.01 nM to 100 nM) and the two-step detection workflow could be completed within 50 min at ambient temperature, which is superior to that of the HPV-based biosensors previously reported.},
}
@article {pmid34234346,
year = {2021},
author = {Fu, G and Guy, CS and Chapman, NM and Palacios, G and Wei, J and Zhou, P and Long, L and Wang, YD and Qian, C and Dhungana, Y and Huang, H and Kc, A and Shi, H and Rankin, S and Brown, SA and Johnson, A and Wakefield, R and Robinson, CG and Liu, X and Sheyn, A and Yu, J and Jackowski, S and Chi, H},
title = {Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine.},
journal = {Nature},
volume = {595},
number = {7869},
pages = {724-729},
pmid = {34234346},
issn = {1476-4687},
support = {R01 AI131703/AI/NIAID NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; R01 CA221290/CA/NCI NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; R01 AI105887/AI/NIAID NIH HHS/United States ; R01 GM134382/GM/NIGMS NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/immunology ; CRISPR-Cas Systems ; Cell Differentiation ; Cytidine Diphosphate ; Female ; Gene Expression Regulation ; Humans ; *Immunity, Humoral ; Leukocytes, Mononuclear/immunology ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Phosphatidylethanolamines/*metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; RNA Nucleotidyltransferases ; Receptors, CXCR5/*immunology ; Signal Transduction ; T-Lymphocytes, Helper-Inducer/*immunology ; },
abstract = {T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity[1]. Although transcription factors such as BCL6 drive the differentiation of TFH cells[2,3], it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.},
}
@article {pmid34234315,
year = {2021},
author = {Kwisda, K and Cantz, T and Hoppe, N},
title = {Regulatory and intellectual property conundrums surrounding xenotransplantation.},
journal = {Nature biotechnology},
volume = {39},
number = {7},
pages = {796-798},
pmid = {34234315},
issn = {1546-1696},
mesh = {Animals ; Biotechnology/*legislation &amp; jurisprudence ; CRISPR-Cas Systems ; European Union ; Genetic Engineering ; Humans ; *Intellectual Property ; Organisms, Genetically Modified ; Swine/genetics ; Transplantation, Heterologous/*legislation &amp; jurisprudence ; World Health Organization ; },
}
@article {pmid34233005,
year = {2021},
author = {Poggi, L and Emmenegger, L and Descorps-Declère, S and Dumas, B and Richard, GF},
title = {Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations.},
journal = {Nucleic acids research},
volume = {49},
number = {14},
pages = {8120-8134},
pmid = {34233005},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; Endonucleases/*genetics ; Gene Editing ; Genetic Diseases, Inborn/*genetics ; Humans ; Microsatellite Repeats/*genetics ; Mutation/genetics ; Streptococcus pyogenes/enzymology/genetics ; Trinucleotide Repeats/genetics ; },
abstract = {Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.},
}
@article {pmid34232987,
year = {2021},
author = {Wang, Z and Guo, R and Trudeau, SJ and Wolinsky, E and Ast, T and Liang, JH and Jiang, C and Ma, Y and Teng, M and Mootha, VK and Gewurz, BE},
title = {CYB561A3 is the key lysosomal iron reductase required for Burkitt B-cell growth and survival.},
journal = {Blood},
volume = {138},
number = {22},
pages = {2216-2230},
pmid = {34232987},
issn = {1528-0020},
support = {R01 AI137337/AI/NIAID NIH HHS/United States ; R01 CA228700/CA/NCI NIH HHS/United States ; },
mesh = {B-Lymphocytes/metabolism/pathology ; Burkitt Lymphoma/genetics/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cytochromes b/*genetics ; Epstein-Barr Virus Infections/complications ; FMN Reductase/genetics ; *Gene Expression Regulation, Neoplastic ; HEK293 Cells ; Herpesvirus 4, Human/isolation &amp; purification ; Humans ; Lysosomes/genetics/*pathology ; Mitochondria/genetics/pathology ; },
abstract = {Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.},
}
@article {pmid34232860,
year = {2021},
author = {Barth, ZK and Nguyen, MH and Seed, KD},
title = {A chimeric nuclease substitutes a phage CRISPR-Cas system to provide sequence-specific immunity against subviral parasites.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34232860},
issn = {2050-084X},
support = {R01 AI127652/AI/NIAID NIH HHS/United States ; R01 AI153303/AI/NIAID NIH HHS/United States ; S10 OD028685/OD/NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; Bacteriophages/*physiology ; CRISPR-Cas Systems ; Endonucleases/*genetics/metabolism ; Vibrio cholerae/*genetics/virology ; },
abstract = {Mobile genetic elements, elements that can move horizontally between genomes, have profound effects on their host's fitness. The phage-inducible chromosomal island-like element (PLE) is a mobile element that integrates into the chromosome of Vibrio cholerae and parasitizes the bacteriophage ICP1 to move between cells. This parasitism by PLE is such that it abolishes the production of ICP1 progeny and provides a defensive boon to the host cell population. In response to the severe parasitism imposed by PLE, ICP1 has acquired an adaptive CRISPR-Cas system that targets the PLE genome during infection. However, ICP1 isolates that naturally lack CRISPR-Cas are still able to overcome certain PLE variants, and the mechanism of this immunity against PLE has thus far remained unknown. Here, we show that ICP1 isolates that lack CRISPR-Cas encode an endonuclease in the same locus, and that the endonuclease provides ICP1 with immunity to a subset of PLEs. Further analysis shows that this endonuclease is of chimeric origin, incorporating a DNA-binding domain that is highly similar to some PLE replication origin-binding proteins. This similarity allows the endonuclease to bind and cleave PLE origins of replication. The endonuclease appears to exert considerable selective pressure on PLEs and may drive PLE replication module swapping and origin restructuring as mechanisms of escape. This work demonstrates that new genome defense systems can arise through domain shuffling and provides a greater understanding of the evolutionary forces driving genome modularity and temporal succession in mobile elements.},
}
@article {pmid34232726,
year = {2021},
author = {Havlik, LP and Das, A and Mietzsch, M and Oh, DK and Ark, J and McKenna, R and Agbandje-McKenna, M and Asokan, A},
title = {Receptor Switching in Newly Evolved Adeno-associated Viruses.},
journal = {Journal of virology},
volume = {95},
number = {19},
pages = {e0058721},
pmid = {34232726},
issn = {1098-5514},
support = {R01 GM127708/GM/NIGMS NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; R01 NS099371/NS/NINDS NIH HHS/United States ; U24 GM116788/GM/NIGMS NIH HHS/United States ; R01 HL089221/HL/NHLBI NIH HHS/United States ; U24 GM116792/GM/NIGMS NIH HHS/United States ; R01 GM082946/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Motifs ; CRISPR-Cas Systems ; Capsid/chemistry/ultrastructure ; Capsid Proteins/chemistry/metabolism ; Cell Line ; Cell Membrane/metabolism ; Cryoelectron Microscopy ; Dependovirus/chemistry/*genetics/*metabolism/ultrastructure ; *Directed Molecular Evolution ; Genetic Variation ; Glycosaminoglycans/metabolism ; Humans ; Integrin beta1/chemistry/metabolism ; Polysaccharides/metabolism ; Receptors, Cell Surface/metabolism ; Receptors, Virus/chemistry/*metabolism ; Virus Internalization ; },
abstract = {Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduction are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake, and transduction, but through distinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycosaminoglycans, while another displays receptor switching from AAVR to integrin β1 (ITGB1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITGB1 receptor over AAVR. Visualization of the AAVhum.8 capsid by cryoelectron microscopy at 2.49-Å resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. IMPORTANCE Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines, and gene transfer vectors. Here, we demonstrate that distinct epitopes on the surface of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the three-dimensional structure of one such newly evolved AAV capsid, which provides a roadmap for designing viruses with improved attributes for gene therapy applications.},
}
@article {pmid34232706,
year = {2021},
author = {Bouchard, D and Wang, W and Yang, WC and He, S and Garcia, A and Matunis, MJ},
title = {SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.},
journal = {Molecular biology of the cell},
volume = {32},
number = {19},
pages = {1849-1866},
pmid = {34232706},
issn = {1939-4586},
support = {R01 GM060980/GM/NIGMS NIH HHS/United States ; T32 CA009110/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Gene Expression Profiling/*methods ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Gene Ontology ; Humans ; Immunoblotting/methods ; Microscopy, Fluorescence/methods ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SUMO-1 Protein/*genetics/metabolism ; Small Ubiquitin-Related Modifier Proteins/*genetics/metabolism ; Ubiquitins/*genetics/metabolism ; },
abstract = {The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.},
}
@article {pmid34232061,
year = {2021},
author = {Chen, V and Griffin, ME and Maguin, P and Varble, A and Hang, HC},
title = {RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {18},
pages = {e0084421},
pmid = {34232061},
issn = {1098-5336},
support = {T32 AI070084/AI/NIAID NIH HHS/United States ; R01 GM087544/GM/NIGMS NIH HHS/United States ; R21 AI156674/AI/NIAID NIH HHS/United States ; R01 GM103593/GM/NIGMS NIH HHS/United States ; R01 CA245292/CA/NCI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Enterococcus faecium/*genetics ; *Gene Editing ; Recombinases/*genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.},
}
@article {pmid34230973,
year = {2021},
author = {Eleveld, TF and Bakali, C and Eijk, PP and Stathi, P and Vriend, LE and Poddighe, PJ and Ylstra, B},
title = {Engineering large-scale chromosomal deletions by CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {49},
number = {21},
pages = {12007-12016},
pmid = {34230973},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosome Aberrations ; Chromosome Deletion ; DNA/*metabolism ; Humans ; Neuroblastoma/*genetics ; },
abstract = {Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.},
}
@article {pmid34230963,
year = {2021},
author = {Kim, D and An, H and Fan, C and Park, Y},
title = {Identifying oligodendrocyte enhancers governing Plp1 expression.},
journal = {Human molecular genetics},
volume = {30},
number = {23},
pages = {2225-2239},
pmid = {34230963},
issn = {1460-2083},
support = {R01 NS094181/NS/NINDS NIH HHS/United States ; R21 NS102558/NS/NINDS NIH HHS/United States ; R21 NS112608/NS/NINDS NIH HHS/United States ; R21 NS114476/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Binding Sites ; CRISPR-Cas Systems ; Chromatin Immunoprecipitation Sequencing ; Chromosome Mapping ; DNA-Binding Proteins/metabolism ; *Enhancer Elements, Genetic ; *Gene Expression Regulation ; Humans ; Membrane Proteins/metabolism ; Mice ; Myelin Proteolipid Protein/*genetics/metabolism ; Nucleotide Motifs ; Oligodendroglia/*metabolism ; Promoter Regions, Genetic ; Transcription Factors/metabolism ; },
abstract = {Oligodendrocytes (OLs) produce myelin in the central nervous system (CNS), which accelerates the propagation of action potentials and supports axonal integrity. As a major component of CNS myelin, proteolipid protein 1 (Plp1) is indispensable for the axon-supportive function of myelin. Notably, this function requires the continuous high-level expression of Plp1 in OLs. Equally important is the controlled expression of Plp1, as illustrated by Pelizaeus-Merzbacher disease for which the most common cause is PLP1 overexpression. Despite a decade-long search, promoter-distal OL enhancers that govern Plp1 remain elusive. We have recently developed an innovative method that maps promoter-distal enhancers to genes in a principled manner. Here, we applied it to Plp1, uncovering two OL enhancers for it (termed Plp1-E1 and Plp1-E2). Remarkably, clustered regularly interspaced short palindromic repeats (CRISPR) interference epigenome editing showed that Plp1-E1 and Plp1-E2 do not regulate two genes in their vicinity, highlighting their exquisite specificity to Plp1. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) data show that Plp1-E1 and Plp1-E2 are OL-specific enhancers that are conserved among human, mouse and rat. Hi-C data reveal that the physical interactions between Plp1-E1/2 and PLP1 are among the strongest in OLs and specific to OLs. We also show that Myrf, a master regulator of OL development, acts on Plp1-E1 and Plp1-E2 to promote Plp1 expression.},
}
@article {pmid34230949,
year = {2021},
author = {Yan, P and Liang, DY and Xu, WH and Xue, L and Yu, MF and Shen, JH and Liu, QH and Peng, YB},
title = {[Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {73},
number = {3},
pages = {482-490},
pmid = {34230949},
issn = {0371-0874},
mesh = {Animals ; Bronchoalveolar Lavage Fluid ; CRISPR-Cas Systems/genetics ; Calgranulin B ; Disease Models, Animal ; Gene Knockout Techniques ; *Gene Targeting ; Lung ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Ovalbumin ; Phenotype ; },
abstract = {S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F0 mice were obtained and identified by PCR identification and gene sequencing. F0 mice were further mated with wild-type C57BL/6 mice to obtain F1 heterozygous mice, and then homozygous offspring were obtained through F1 mice self-crossing. Real-time PCR, Western blot and immunohistochemistry (IHC) were used to verify the expression and distribution of S100A9. In order to observe the pathological changes of mouse lung tissue using HE staining, an allergic asthma model was induced by ovalbumin from chicken egg white (OVA). The results showed that the 2 492 bp of exons 2, 3 of the S100A9 gene was successfully knocked out, and S100A9[-/-] mice with stable inheritance were obtained. Furthermore, it was found that S100A9 gene was highly expressed in the lung and spleen of wild-type mice. The expression of S100A9 mRNA and protein was not detected in the lung and spleen of S100A9[-/-] mice. However, compared with wild-type mice, the lungs of S100A9[-/-] mice showed a significantly worse inflammatory phenotype, and the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly increased in response to the treatment of OVA. These results suggest we have successfully constructed a new strain of S100A9[-/-] mice, and preliminarily confirmed that the lack of S100A9 function can aggravate airway inflammation in asthmatic mice, providing a new mouse model for further study of S100A9 gene function.},
}
@article {pmid34230602,
year = {2021},
author = {Wang, S and Zhang, F and Mei, M and Wang, T and Yun, Y and Yang, S and Zhang, G and Yi, L},
title = {A split protease-E. coli ClpXP system quantifies protein-protein interactions in Escherichia coli cells.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {841},
pmid = {34230602},
issn = {2399-3642},
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Endopeptidase Clp/genetics/*metabolism ; Enterovirus/enzymology/genetics ; Escherichia coli/genetics/*metabolism ; Escherichia coli Proteins/genetics/*metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Histidine Kinase/genetics/metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Conformation ; *Protein Interaction Maps ; Proteolysis ; Serine Endopeptidases/chemistry/genetics/metabolism ; Viral Proteins/genetics/metabolism ; },
abstract = {Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.},
}
@article {pmid34230586,
year = {2021},
author = {Sapp, V and Aguirre, A and Mainkar, G and Ding, J and Adler, E and Liao, R and Sharma, S and Jain, M},
title = {Genome-wide CRISPR/Cas9 screening in human iPS derived cardiomyocytes uncovers novel mediators of doxorubicin cardiotoxicity.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {13866},
pmid = {34230586},
issn = {2045-2322},
support = {T32GM007752/NH/NIH HHS/United States ; K01HL135464/NH/NIH HHS/United States ; K23HL107755/NH/NIH HHS/United States ; R01HL128135/NH/NIH HHS/United States ; R01CA199376/NH/NIH HHS/United States ; R03HL133720/NH/NIH HHS/United States ; R01 ES027595/ES/NIEHS NIH HHS/United States ; S10 OD020025/OD/NIH HHS/United States ; R01 HL132511/HL/NHLBI NIH HHS/United States ; P30 NS047101/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cardiotoxicity/*pathology ; Cell Line, Tumor ; Doxorubicin/*adverse effects ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Lentivirus/metabolism ; Myocytes, Cardiac/*metabolism ; Phenotype ; RNA, Guide/genetics ; },
abstract = {Human induced pluripotent stem (iPS) cell technologies coupled with genetic engineering now facilitate the study of the molecular underpinnings of disease in relevant human cell types. Application of CRISPR/Cas9-based approaches for genome-scale functional screening in iPS-derived cells, however, has been limited by technical constraints, including inefficient transduction in pooled format, loss of library representation, and poor cellular differentiation. Herein, we present optimized approaches for whole-genome CRISPR/Cas9 based screening in human iPS derived cardiomyocytes with near genome-wide representation at both the iPS and differentiated cell stages. As proof-of-concept, we perform a screen to investigate mechanisms underlying doxorubicin mediated cell death in iPS derived cardiomyocytes. We identified two poorly characterized, human-specific transporters (SLCO1A2, SLCO1B3) whose loss of function protects against doxorubicin-cardiotoxicity, but does not affect cell death in cancer cells. This study provides a technical framework for genome-wide functional screening in iPS derived cells and identifies new targets to mitigate doxorubicin-cardiotoxicity in humans.},
}
@article {pmid34230583,
year = {2021},
author = {Serifi, I and Besta, S and Karetsou, Z and Giardoglou, P and Beis, D and Niewiadomski, P and Papamarcaki, T},
title = {Targeting of SET/I2PP2A oncoprotein inhibits Gli1 transcription revealing a new modulator of Hedgehog signaling.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {13940},
pmid = {34230583},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryo, Nonmammalian/metabolism ; HEK293 Cells ; Hedgehog Proteins/*metabolism ; Humans ; Mice ; Morpholinos/pharmacology ; NIH 3T3 Cells ; Receptors, Cell Surface/genetics/*metabolism ; *Signal Transduction ; *Transcription, Genetic ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/genetics/*metabolism ; Zinc Finger Protein GLI1/*genetics/metabolism ; },
abstract = {The Hedgehog (Hh)/Gli signaling pathway controls cell proliferation and differentiation, is critical for the development of nearly every tissue and organ in vertebrates and is also involved in tumorigenesis. In this study, we characterize the oncoprotein SET/I2PP2A as a novel regulator of Hh signaling. Our previous work has shown that the zebrafish homologs of SET are expressed during early development and localized in the ciliated organs. In the present work, we show that CRISPR/Cas9-mediated knockdown of setb gene in zebrafish embryos resulted in cyclopia, a characteristic patterning defect previously reported in Hh mutants. Consistent with these findings, targeting setb gene using CRISPR/Cas9 or a setb morpholino, reduced Gli1-dependent mCherry expression in the Hedgehog reporter zebrafish line Tg(12xGliBS:mCherry-NLS). Likewise, SET loss of function by means of pharmacological inhibition and gene knockdown prevented the increase of Gli1 expression in mammalian cells in vitro. Conversely, overexpression of SET resulted in an increase of the expression of a Gli-dependent luciferase reporter, an effect likely attributable to the relief of the Sufu-mediated inhibition of Gli1. Collectively, our data support the involvement of SET in Gli1-mediated transcription and suggest the oncoprotein SET/I2PP2A as a new modulator of Hedgehog signaling.},
}
@article {pmid34230471,
year = {2021},
author = {Eggers, EJ and van der Burgt, A and van Heusden, SAW and de Vries, ME and Visser, RGF and Bachem, CWB and Lindhout, P},
title = {Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4141},
pmid = {34230471},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Plant ; Diploidy ; Genes, Plant/*genetics ; Genotype ; Heterozygote ; Magnoliopsida ; *Plant Breeding ; Plant Proteins/*genetics ; Pollen/genetics ; Seeds/metabolism ; Self-Incompatibility in Flowering Plants/genetics ; Solanum tuberosum/*genetics ; },
abstract = {Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.},
}
@article {pmid34229745,
year = {2021},
author = {Liu, Y and Lin, Y and Guo, Y and Wu, F and Zhang, Y and Qi, X and Wang, Z and Wang, Q},
title = {Stress tolerance enhancement via SPT15 base editing in Saccharomyces cerevisiae.},
journal = {Biotechnology for biofuels},
volume = {14},
number = {1},
pages = {155},
pmid = {34229745},
issn = {1754-6834},
abstract = {BACKGROUND: Saccharomyces cerevisiae is widely used in traditional brewing and modern fermentation industries to produce biofuels, chemicals and other bioproducts, but challenged by various harsh industrial conditions, such as hyperosmotic, thermal and ethanol stresses. Thus, its stress tolerance enhancement has been attracting broad interests. Recently, CRISPR/Cas-based genome editing technology offers unprecedented tools to explore genetic modifications and performance improvement of S. cerevisiae.<br><br>RESULTS: Here, we presented that the Target-AID (activation-induced cytidine deaminase) base editor of enabling C-to-T substitutions could be harnessed to generate in situ nucleotide changes on the S. cerevisiae genome, thereby introducing protein point mutations in cells. The general transcription factor gene SPT15 was targeted, and total 36 mutants with diversified stress tolerances were obtained. Among them, the 18 tolerant mutants against hyperosmotic, thermal and ethanol stresses showed more than 1.5-fold increases of fermentation capacities. These mutations were mainly enriched at the N-terminal region and the convex surface of the saddle-shaped structure of Spt15. Comparative transcriptome analysis of three most stress-tolerant (A140G, P169A and R238K) and two most stress-sensitive (S118L and L214V) mutants revealed common and distinctive impacted global transcription reprogramming and transcriptional regulatory hubs in response to stresses, and these five amino acid changes had different effects on the interactions of Spt15 with DNA and other proteins in the RNA Polymerase II transcription machinery according to protein structure alignment analysis.<br><br>CONCLUSIONS: Taken together, our results demonstrated that the Target-AID base editor provided a powerful tool for targeted in situ mutagenesis in S. cerevisiae and more potential targets of Spt15 residues for enhancing yeast stress tolerance.},
}
@article {pmid34228718,
year = {2021},
author = {Grigoraki, L and Cowlishaw, R and Nolan, T and Donnelly, M and Lycett, G and Ranson, H},
title = {CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes.},
journal = {PLoS genetics},
volume = {17},
number = {7},
pages = {e1009556},
pmid = {34228718},
issn = {1553-7404},
support = {/WT_/Wellcome Trust/United Kingdom ; BB/F021933/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/V001264/1/MRC_/Medical Research Council/United Kingdom ; 215894/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Animals, Genetically Modified ; Anopheles/*drug effects/*genetics ; *CRISPR-Cas Systems ; DDT/pharmacology ; Female ; Fertility/genetics ; Genome, Insect ; Glutathione Transferase/genetics ; Insect Proteins/genetics ; Insecticide Resistance/*genetics ; Male ; *Mutation ; Nitriles/pharmacology ; Permethrin/pharmacology ; Piperonyl Butoxide/pharmacology ; Pyrethrins/pharmacology ; },
abstract = {Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.},
}
@article {pmid34228299,
year = {2021},
author = {Tsunekawa, Y and Terhune, RK and Matsuzaki, F},
title = {Protocol for De Novo Gene Targeting Via In Utero Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2312},
number = {},
pages = {309-320},
pmid = {34228299},
issn = {1940-6029},
mesh = {Animals ; Brain/embryology/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Electroporation ; Female ; *Gene Expression Regulation, Developmental ; Gene Knock-In Techniques ; *Gene Targeting ; *Gene Transfer Techniques ; Genes, Reporter ; Gestational Age ; Green Fluorescent Proteins/genetics/metabolism ; Mice ; Mice, Inbred ICR ; Pregnancy ; RNA, Guide/genetics/metabolism ; },
abstract = {Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.},
}
@article {pmid34228293,
year = {2021},
author = {Otabe, T and Nihongaki, Y and Sato, M},
title = {Optical Control of Genome Editing by Photoactivatable Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2312},
number = {},
pages = {225-233},
pmid = {34228293},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism/*radiation effects ; CRISPR-Cas Systems/*radiation effects ; Cell Culture Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; *Gene Editing ; Gene Expression Regulation/*radiation effects ; HEK293 Cells ; Humans ; INDEL Mutation ; *Light ; *Optogenetics ; Point Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.},
}
@article {pmid34228292,
year = {2021},
author = {Ishiguro, S and Yachie, N},
title = {Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2312},
number = {},
pages = {193-223},
pmid = {34228292},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Gene Expression Regulation ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; *Multiplex Polymerase Chain Reaction ; Transfection ; },
abstract = {CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.},
}
@article {pmid34228291,
year = {2021},
author = {Weisbach, NR and Meijs, A and Platt, RJ},
title = {Multiplexed Genome Engineering with Cas12a.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2312},
number = {},
pages = {171-192},
pmid = {34228291},
issn = {1940-6029},
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Culture Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/*genetics/metabolism ; *Gene Editing ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Polymerase Chain Reaction ; *Transcription, Genetic ; Transfection ; },
abstract = {Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.},
}
@article {pmid34227298,
year = {2021},
author = {Liu, J and Zhang, Y and He, W},
title = {[Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {6},
pages = {2116-2126},
doi = {10.13345/j.cjb.200763},
pmid = {34227298},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; Genetic Engineering ; Ribosomes ; *Spiramycin ; *Streptomyces/genetics ; },
abstract = {Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.},
}
@article {pmid34225628,
year = {2021},
author = {Liu, N and Zhou, L and Qu, J and Yao, S},
title = {Recent Advances in CRISPR/Cas9 Directed Base Editing.},
journal = {Current gene therapy},
volume = {21},
number = {4},
pages = {327-337},
doi = {10.2174/1566523221666210322090638},
pmid = {34225628},
issn = {1875-5631},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Genomics ; Humans ; },
abstract = {Recently, CRISPR-based techniques have significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled the irreversible conversion of a specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, the high efficiency of these editing tools also provided great promise in clinical applications. In this review, we discuss the recent progress and challenges of base editing tools.},
}
@article {pmid34225486,
year = {2021},
author = {Bell, SL and Lopez, KL and Cox, JS and Patrick, KL and Watson, RO},
title = {Galectin-8 Senses Phagosomal Damage and Recruits Selective Autophagy Adapter TAX1BP1 To Control Mycobacterium tuberculosis Infection in Macrophages.},
journal = {mBio},
volume = {12},
number = {4},
pages = {e0187120},
pmid = {34225486},
issn = {2150-7511},
support = {R01 AI145287/AI/NIAID NIH HHS/United States ; R21 AI140004/AI/NIAID NIH HHS/United States ; R01 AI120694/AI/NIAID NIH HHS/United States ; R01 AI155621/AI/NIAID NIH HHS/United States ; R01 AI125512/AI/NIAID NIH HHS/United States ; P01 AI063302/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Autophagy ; CRISPR-Cas Systems ; Cell Line ; Galectins/*genetics/immunology ; HEK293 Cells ; Host-Pathogen Interactions ; Humans ; *Immunity, Innate ; Intracellular Signaling Peptides and Proteins/*genetics/immunology ; Macrophages/immunology/*microbiology ; Mice ; Neoplasm Proteins/*genetics/immunology ; Phagocytosis ; Phagosomes/*microbiology ; RAW 264.7 Cells ; U937 Cells ; },
abstract = {Mycobacterium tuberculosis (Mtb) causes one of the deadliest infectious diseases worldwide. Upon infection, Mtb is phagocytosed by macrophages and uses its virulence-associated ESX-1 secretion system to modulate the host cell. We showed previously that the ESX-1 secretion system perturbs the Mtb-containing phagosome, and a population (∼30%) of intracellular Mtb is tagged with ubiquitin and targeted to selective autophagy. However, our understanding of how macrophages sense and respond to damaged Mtb-containing phagosomes remains incomplete. Here, we demonstrate that several cytosolic glycan-binding proteins called galectins recognize Mtb-containing phagosomes; in macrophage cell lines and in primary macrophages, galectin-3, -8, and -9 are all recruited to the same Mtb population that colocalizes with selective autophagy markers (ubiquitin, p62, and LC3). To test whether galectins are required for controlling Mtb replication in macrophages, we generated CRISPR/Cas9 knockouts and found that galectin-8[-/-] and galectin-3/8/9[-/-] macrophages were similarly defective in targeting Mtb to selective autophagy and controlling replication. This suggests galectin-8 plays a unique role in anti-Mtb autophagy. In investigating galectin-8's role, we identified a novel and specific interaction between galectin-8 and the selective autophagy adapter TAX1BP1 and found that this galectin-8/TAX1BP1 interaction was necessary for macrophages to efficiently target Mtb to selective autophagy. Remarkably, overexpressing galectin-8 increased targeting of Mtb to autophagy and limited Mtb replication. Taken together, these data demonstrate that while several galectins are capable of recognizing damaged Mtb-containing phagosomes, galectin-8 plays a privileged role in recruiting downstream autophagy machinery and may represent a promising target for host-directed tuberculosis therapies. IMPORTANCE Mycobacterium tuberculosis (Mtb) infects one-quarter of the global population and causes one of the deadliest infectious diseases worldwide. Macrophages are the first line of defense against Mtb infection and are typically incredibly efficient at destroying intracellular pathogens, but Mtb has evolved to survive and replicate in this harsh environment. Previous work has found that a portion of intracellular Mtb bacilli damage their phagosomes, leaving them vulnerable to detection by the host and delivery to an antibacterial pathway called selective autophagy. Here, we show that in macrophages, galectin-8 recognizes damaged Mtb-containing phagosomes and targets Mtb to selective autophagy; we found that galectin-8, unlike other highly similar and closely related galectins, is required for targeting and controlling Mtb in macrophages. The specific role for galectin-8 appears to stem from its interaction with TAX1BP1, a selective autophagy adapter protein. Interestingly, overexpressing galectin-8 helps macrophages target and control Mtb, highlighting the importance of galectin-8 in the innate immune response to Mtb.},
}
@article {pmid34222000,
year = {2021},
author = {Du, X and Wang, JM and Zhang, DL and Wu, T and Zeng, XY and Jiang, JY and Du, ZX},
title = {AUF1 Promotes Proliferation and Invasion of Thyroid Cancer via Downregulation of ZBTB2 and Subsequent TRIM58.},
journal = {Frontiers in oncology},
volume = {11},
number = {},
pages = {681736},
pmid = {34222000},
issn = {2234-943X},
abstract = {The pathogenesis of papillary thyroid cancer (PTC), the most common type of thyroid cancer, is not yet fully understood. This limits the therapeutic options for approximately 7% of invasive PTC patients. The critical role of AUF1 in the progression of thyroid cancer was first reported in 2009, however, its molecular mechanism remained unclear. Our study used CRISPR/Cas 9 system to knockdown AUF1 in IHH4 and TPC1 cells. We noticed that the expression of TRIM58 and ZBTB2 were increased in the AUF1 knockdown IHH4 and TPC1 cells. When TRIM58 and ZBTB2 were inhibited by small hairpin RNAs (shRNAs) against TRIM58 (shTRIM58) and ZBTB2 (shZBTB2), respectively, the proliferation, migration, and invasion ability of the AUF1-knockdown IHH4 and TPC1 cells were increased. In addition, two ZBTB2 binding sites (-719~-709 and -677~-668) on TRIM58 promoter and two AUF1 binding sites (1250-1256 and 1258-1265) on ZBTB2 3'-UTR were identified. These results suggested that AUF1 affecting thyroid cancer cells via regulating the expression of ZBTB2 and TRIM58.},
}
@article {pmid34218480,
year = {2021},
author = {Cecchin, M and Jeong, J and Son, W and Kim, M and Park, S and Zuliani, L and Cazzaniga, S and Pompa, A and Young Kang, C and Bae, S and Ballottari, M and Jin, E},
title = {LPA2 protein is involved in photosystem II assembly in Chlamydomonas reinhardtii.},
journal = {The Plant journal : for cell and molecular biology},
volume = {107},
number = {6},
pages = {1648-1662},
pmid = {34218480},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; Chlamydomonas reinhardtii/genetics/growth &amp; development/*metabolism ; Chlorophyll/metabolism ; Electron Transport/genetics ; Mutation ; Photosynthesis/genetics ; Photosystem I Protein Complex/metabolism ; Photosystem II Protein Complex/genetics/*metabolism ; Proteins/genetics/*metabolism ; Thylakoids/metabolism ; },
abstract = {Photosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR-Cas9 target-specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse-chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function.},
}
@article {pmid34218353,
year = {2021},
author = {Hu, KJ and Yin, ETS and Hu, YX and Huang, H},
title = {Combination of CRISPR/Cas9 System and CAR-T Cell Therapy: A New Era for Refractory and Relapsed Hematological Malignancies.},
journal = {Current medical science},
volume = {41},
number = {3},
pages = {420-430},
pmid = {34218353},
issn = {2523-899X},
mesh = {CRISPR-Cas Systems/*genetics ; Cell- and Tissue-Based Therapy/trends ; Gene Editing ; Hematologic Neoplasms/genetics/pathology/*therapy ; Humans ; Immunotherapy/trends ; Immunotherapy, Adoptive/*trends ; Receptors, Chimeric Antigen/*genetics ; },
abstract = {Chimeric antigen receptor T (CAR-T) cell therapy is the novel treatment strategy for hematological malignancies such as acute lymphoblastic leukemia (ALL), lymphoma and multiple myeloma. However, treatment-related toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) have become significant hurdles to CAR-T treatment. Multiple strategies were established to alter the CAR structure on the genomic level to improve efficacy and reduce toxicities. Recently, the innovative gene-editing technology-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease9 (Cas9) system, which particularly exhibits preponderance in knock-in and knockout at specific sites, is widely utilized to manufacture CAR-T products. The application of CRISPR/Cas9 to CAR-T cell therapy has shown promising clinical results with minimal toxicity. In this review, we summarized the past achievements of CRISPR/Cas9 in CAR-T therapy and focused on the potential CAR-T targets.},
}
@article {pmid34217945,
year = {2021},
author = {Jbara, A and Siegfried, Z and Karni, R},
title = {Splice-switching as cancer therapy.},
journal = {Current opinion in pharmacology},
volume = {59},
number = {},
pages = {140-148},
doi = {10.1016/j.coph.2021.05.008},
pmid = {34217945},
issn = {1471-4973},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Humans ; *Neoplasms/drug therapy/genetics ; RNA Splicing ; },
abstract = {In light of recent advances in RNA splicing modulation as therapy for specific genetic diseases, there is great optimism that this approach can be applied to treatment of cancer as well. Dysregulation of alternative RNA splicing is a common aberration detected in many cancers and thus, provides an attractive target for therapeutics. Here, we present and compare two promising approaches that are currently being investigated to manipulate alternative splicing and their potential use in therapy. The first strategy makes use of splice-switching oligonucleotides, whereas the second strategy uses CRISPR (clustered regularly interspaced short palindromic repeat Cas (CRISPR-associated) technology. We will discuss both the challenges and limitations of these technologies and progress being made to implement splice-switching as a potential cancer therapy.},
}
@article {pmid34217893,
year = {2021},
author = {Werder, RB and Kaserman, JE and Packer, MS and Lindstrom-Vautrin, J and Villacorta-Martin, C and Young, LE and Aratyn-Schaus, Y and Gregoire, F and Wilson, AA},
title = {Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3219-3229},
pmid = {34217893},
issn = {1525-0024},
support = {R01 DK101501/DK/NIDDK NIH HHS/United States ; R35 GM131726/GM/NIGMS NIH HHS/United States ; S10 OD021587/OD/NIH HHS/United States ; U01 TR001810/TR/NCATS NIH HHS/United States ; },
mesh = {*Adenine ; Biomarkers ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; Endoplasmic Reticulum Stress ; *Gene Editing ; Gene Expression ; Hepatocytes/cytology/*metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Mutation ; alpha 1-Antitrypsin/chemistry/*genetics ; alpha 1-Antitrypsin Deficiency/*genetics/*therapy ; },
abstract = {Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.},
}
@article {pmid34217781,
year = {2021},
author = {Muto, Y and Humphreys, BD},
title = {Recent advances in lineage tracing for the kidney.},
journal = {Kidney international},
volume = {100},
number = {6},
pages = {1179-1184},
pmid = {34217781},
issn = {1523-1755},
support = {P30 DK020579/DK/NIDDK NIH HHS/United States ; R01 DK103740/DK/NIDDK NIH HHS/United States ; UC2 DK126024/DK/NIDDK NIH HHS/United States ; UH3 DK107374/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cell Lineage ; Gene Expression ; Genes, Reporter ; *Genomics ; Humans ; *Kidney ; },
abstract = {Lineage tracing was originally developed by developmental biologists to identify all progeny of a single cell during morphogenesis. More recently this approach has been applied to other fields, including organ homeostasis and recovery from injury. Modern lineage tracing techniques typically rely on reporter gene expression induced by cell-specific DNA recombination. There have been important scientific advances in the last 10 years that have impacted lineage tracing approaches, including intersectional genetics, optical clearing techniques, and the use of sequencing-based genomic lineage tracing. The latter combines CRISPR-Cas9-based genetic scarring with single-cell RNA-sequencing that, in theory, could allow comprehensive reconstruction of a lineage tree for an entire organism. This review summarizes recent advances in lineage tracing technologies and outlines potential applications for kidney research.},
}
@article {pmid34216590,
year = {2021},
author = {Gresakova, V and Novosadova, V and Prochazkova, M and Prochazka, J and Sedlacek, R},
title = {Dual role of Fam208a during zygotic cleavage and early embryonic development.},
journal = {Experimental cell research},
volume = {406},
number = {2},
pages = {112723},
doi = {10.1016/j.yexcr.2021.112723},
pmid = {34216590},
issn = {1090-2422},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Methylation ; *Embryonic Development ; *Epigenesis, Genetic ; Female ; *Genomic Instability ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; *Mitosis ; Nuclear Proteins/antagonists &amp; inhibitors/*physiology ; Phosphoproteins/genetics/*metabolism ; Pregnancy ; Zygote/*physiology ; },
abstract = {Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining genome stability during cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 led to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and the occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by RT-qPCR profiling and in situ hybridization [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is a multi-interacting protein affecting genome stability on the cell division level at the earliest stages of development and by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an important role in the zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Svil, and Itgb3bp.},
}
@article {pmid34215850,
year = {2021},
author = {Xia, Y and Ji, X and Jang, IS and Surka, C and Hsu, C and Wang, K and Rolfe, M and Bence, N and Lu, G},
title = {Genetic and pharmacological interrogation of cancer vulnerability using a multiplexed cell line screening platform.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {834},
pmid = {34215850},
issn = {2399-3642},
mesh = {Antineoplastic Agents/*pharmacology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Cell Survival/drug effects/genetics ; Drug Screening Assays, Antitumor/methods ; Early Detection of Cancer/*methods ; Gene Editing/*methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Neoplasms/diagnosis/*genetics ; },
abstract = {The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.},
}
@article {pmid34215845,
year = {2021},
author = {Huang, H and Hu, Y and Huang, G and Ma, S and Feng, J and Wang, D and Lin, Y and Zhou, J and Rong, Z},
title = {Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {830},
pmid = {34215845},
issn = {2399-3642},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/genetics ; *DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Genome, Human/genetics ; Genome-Wide Association Study/methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Oligodeoxyribonucleotides/genetics ; Reproducibility of Results ; },
abstract = {Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.},
}
@article {pmid34215619,
year = {2021},
author = {Maifrede, S and Le, BV and Nieborowska-Skorska, M and Golovine, K and Sullivan-Reed, K and Dunuwille, WMB and Nacson, J and Hulse, M and Keith, K and Madzo, J and Caruso, LB and Gazze, Z and Lian, Z and Padella, A and Chitrala, KN and Bartholdy, BA and Matlawska-Wasowska, K and Di Marcantonio, D and Simonetti, G and Greiner, G and Sykes, SM and Valent, P and Paietta, EM and Tallman, MS and Fernandez, HF and Litzow, MR and Minden, MD and Huang, J and Martinelli, G and Vassiliou, GS and Tempera, I and Piwocka, K and Johnson, N and Challen, GA and Skorski, T},
title = {TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.},
journal = {Cancer research},
volume = {81},
number = {19},
pages = {5089-5101},
pmid = {34215619},
issn = {1538-7445},
support = {UG1 CA233290/CA/NCI NIH HHS/United States ; R01 DK102428/DK/NIDDK NIH HHS/United States ; UG1 CA232760/CA/NCI NIH HHS/United States ; R01 GM124449/GM/NIGMS NIH HHS/United States ; R01 HL147978/HL/NHLBI NIH HHS/United States ; R01 CA227830/CA/NCI NIH HHS/United States ; R01 CA244044/CA/NCI NIH HHS/United States ; R01 DK124883/DK/NIDDK NIH HHS/United States ; UG1 CA189859/CA/NCI NIH HHS/United States ; P30 CA010815/CA/NCI NIH HHS/United States ; U24 CA196172/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA214799/CA/NCI NIH HHS/United States ; R01 GM135293/GM/NIGMS NIH HHS/United States ; R01 CA247707/CA/NCI NIH HHS/United States ; R01 CA186238/CA/NCI NIH HHS/United States ; U10 CA180820/CA/NCI NIH HHS/United States ; R01 CA237286/CA/NCI NIH HHS/United States ; T32 CA009035/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Methyltransferase 3A/*genetics ; *DNA Repair ; DNA-Binding Proteins/*genetics ; Dioxygenases/*genetics ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/*genetics ; Gene Knockdown Techniques ; Genotype ; Humans ; Leukemia ; Mice ; Mice, Transgenic ; Models, Biological ; *Mutation ; Neoplastic Stem Cells ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Xenograft Model Antitumor Assays ; },
abstract = {Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3[ITD], BCR-ABL1, JAK2[V617F] , and MPL[W515L] , or with mutations affecting related signaling pathways such as NRAS[G12D] and CALR[del52] . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.},
}
@article {pmid34215080,
year = {2021},
author = {Li, S and Huang, J and Ren, L and Jiang, W and Wang, M and Zhuang, L and Zheng, Q and Yang, R and Zeng, Y and Luu, LDW and Wang, Y and Tai, J},
title = {A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.},
journal = {Talanta},
volume = {233},
number = {},
pages = {122591},
pmid = {34215080},
issn = {1873-3573},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.},
}
@article {pmid34215057,
year = {2021},
author = {Wang, X and Chen, X and Chu, C and Deng, Y and Yang, M and Huo, D and Xu, F and Hou, C and Lv, J},
title = {Naked-eye detection of site-specific ssRNA and ssDNA using PAMmer-assisted CRISPR/Cas9 coupling with exponential amplification reaction.},
journal = {Talanta},
volume = {233},
number = {},
pages = {122554},
doi = {10.1016/j.talanta.2021.122554},
pmid = {34215057},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/genetics ; Humans ; *Hydrogen Peroxide ; RNA ; },
abstract = {Accurate and effective detection of single-stranded nucleic acids is vital in both disease diagnosis and pathological studies. Hence, we develop a PAMmer-assisted CRISPR/Cas9 system mediated G4-EXPAR (Cas-G4EX) strategy for site-specific detection of ssRNA and ssDNA. PAMmer-assisted CRISPR/Cas9 executes the site-specific cleavage of target ssRNA or ssDNA and released product fragment with the desired sequence at the 3'-terminal. This fragment serves as a primer to activate subsequent sequence-dependent exponential amplification reaction (EXPAR). The G-rich EXPAR products assembles with hemin to form a G-Quadruplex (G4/hemin). G4/hemin catalyzes ABTS-H2O2 system with the appearance of vivid green color, realizing naked-eye analysis. Cas-G4EX integrates the superiority of CRISPR/Cas9 and EXPAR, presenting outstanding site-specific recognition and high-performance amplification efficiency. Meanwhile, the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection paradigm for any ssRNA or ssDNA. Cas-G4EX assay shows the linear relationship from 250 aM to 2.5 nM for ssRNA detection with the actual LOD of 250 aM, and that ranges from 100 aM to 1 nM for ssDNA detection with the actual LOD of 100 aM. Additionally, the acceptable recoveries of 101.48%-109.61% for ssRNA and 93.25%-111.98% for ssDNA in real detection of human serum are obtained for detection of single-strand nucleic acid in real samples. Cas-G4EX also exhibits the excellent discrimination for single-base mutation of single-stranded nucleic acids. Therefore, Cas-G4EX assay provides a promising platform in the applications of molecular diagnosis and pathological analysis.},
}
@article {pmid34215024,
year = {2021},
author = {Gillmore, JD and Gane, E and Taubel, J and Kao, J and Fontana, M and Maitland, ML and Seitzer, J and O'Connell, D and Walsh, KR and Wood, K and Phillips, J and Xu, Y and Amaral, A and Boyd, AP and Cehelsky, JE and McKee, MD and Schiermeier, A and Harari, O and Murphy, A and Kyratsous, CA and Zambrowicz, B and Soltys, R and Gutstein, DE and Leonard, J and Sepp-Lorenzino, L and Lebwohl, D},
title = {CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.},
journal = {The New England journal of medicine},
volume = {385},
number = {6},
pages = {493-502},
doi = {10.1056/NEJMoa2107454},
pmid = {34215024},
issn = {1533-4406},
mesh = {Amyloid Neuropathies, Familial/*genetics/*therapy ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; Gene Transfer Techniques ; Humans ; Infusions, Intravenous ; Liposomes/*therapeutic use ; Male ; Middle Aged ; Nanoparticles/*therapeutic use ; Prealbumin/analysis/*genetics ; RNA, Guide/*therapeutic use ; RNA, Messenger ; },
abstract = {BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR.<br><br>METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study.<br><br>RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram.<br><br>CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).},
}
@article {pmid34213958,
year = {2022},
author = {Jia, H and Omar, AA and Orbović, V and Wang, N},
title = {Biallelic Editing of the LOB1 Promoter via CRISPR/Cas9 Creates Canker-Resistant 'Duncan' Grapefruit.},
journal = {Phytopathology},
volume = {112},
number = {2},
pages = {308-314},
doi = {10.1094/PHYTO-04-21-0144-R},
pmid = {34213958},
issn = {0031-949X},
mesh = {CRISPR-Cas Systems ; *Citrus/genetics ; *Citrus paradisi/genetics ; Plant Diseases/genetics ; Promoter Regions, Genetic ; *Xanthomonas/genetics ; },
abstract = {Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker.},
}
@article {pmid34212856,
year = {2021},
author = {Dave, N and Cetiner, U and Arroyo, D and Fonbuena, J and Tiwari, M and Barrera, P and Lander, N and Anishkin, A and Sukharev, S and Jimenez, V},
title = {A novel mechanosensitive channel controls osmoregulation, differentiation, and infectivity in Trypanosoma cruzi.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34212856},
issn = {2050-084X},
support = {T34 GM008612/GM/NIGMS NIH HHS/United States ; R15 AI122153/AI/NIAID NIH HHS/United States ; },
mesh = {Antibodies, Protozoan ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Differentiation/*physiology ; Cloning, Molecular ; Computational Biology ; Electrophysiological Phenomena ; Gene Expression Regulation/physiology ; HEK293 Cells ; Humans ; Ion Channels ; Mechanotransduction, Cellular/*physiology ; Mutation ; Osmoregulation/*physiology ; Osmotic Pressure ; Protein Conformation ; Protozoan Proteins/chemistry/*metabolism ; Trypanosoma cruzi/genetics/*physiology ; },
abstract = {The causative agent of Chagas disease undergoes drastic morphological and biochemical modifications as it passes between hosts and transitions from extracellular to intracellular stages. The osmotic and mechanical aspects of these cellular transformations are not understood. Here we identify and characterize a novel mechanosensitive channel in Trypanosoma cruzi (TcMscS) belonging to the superfamily of small-conductance mechanosensitive channels (MscS). TcMscS is activated by membrane tension and forms a large pore permeable to anions, cations, and small osmolytes. The channel changes its location from the contractile vacuole complex in epimastigotes to the plasma membrane as the parasites develop into intracellular amastigotes. TcMscS knockout parasites show significant fitness defects, including increased cell volume, calcium dysregulation, impaired differentiation, and a dramatic decrease in infectivity. Our work provides mechanistic insights into components supporting pathogen adaptation inside the host, thus opening the exploration of mechanosensation as a prerequisite for protozoan infectivity.},
}
@article {pmid34211162,
year = {2021},
author = {Jeong, YK and Lee, S and Hwang, GH and Hong, SA and Park, SE and Kim, JS and Woo, JS and Bae, S},
title = {Adenine base editor engineering reduces editing of bystander cytosines.},
journal = {Nature biotechnology},
volume = {39},
number = {11},
pages = {1426-1433},
pmid = {34211162},
issn = {1546-1696},
mesh = {Adenine ; CRISPR-Cas Systems/genetics ; *Cytosine ; *Gene Editing ; RNA Editing/genetics ; },
abstract = {Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.},
}
@article {pmid34211036,
year = {2021},
author = {Palit, SAL and van Dorp, J and Vis, D and Lieftink, C and Linder, S and Beijersbergen, R and Bergman, AM and Zwart, W and van der Heijden, MS},
title = {A kinome-centered CRISPR-Cas9 screen identifies activated BRAF to modulate enzalutamide resistance with potential therapeutic implications in BRAF-mutated prostate cancer.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {13683},
pmid = {34211036},
issn = {2045-2322},
mesh = {Antineoplastic Agents/*pharmacology ; Benzamides/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Drug Resistance, Neoplasm ; Enzyme Activation ; Humans ; Male ; Mutation ; Nitriles/*pharmacology ; Phenylthiohydantoin/*pharmacology ; Prostatic Neoplasms/drug therapy/*genetics/metabolism ; Proto-Oncogene Proteins B-raf/*genetics/metabolism ; },
abstract = {Resistance to drugs targeting the androgen receptor (AR) signaling axis remains an important challenge in the treatment of prostate cancer patients. Activation of alternative growth pathways is one mechanism used by cancer cells to proliferate despite treatment, conferring drug resistance. Through a kinome-centered CRISPR-Cas9 screen in CWR-R1 prostate cancer cells, we identified activated BRAF signaling as a determinant for enzalutamide resistance. Combined pharmaceutical targeting of AR and MAPK signaling resulted in strong synergistic inhibition of cell proliferation. The association between BRAF activation and enzalutamide resistance was confirmed in two metastatic prostate cancer patients harboring activating mutations in the BRAF gene, as both patients were unresponsive to enzalutamide. Our findings suggest that co-targeting of the MAPK and AR pathways may be effective in patients with an activated MAPK pathway, particularly in patients harboring oncogenic BRAF mutations. These results warrant further investigation of the response to AR inhibitors in BRAF-mutated prostate tumors in clinical settings.},
}
@article {pmid34210975,
year = {2021},
author = {Yang, L and Chan, AKN and Miyashita, K and Delaney, CD and Wang, X and Li, H and Pokharel, SP and Li, S and Li, M and Xu, X and Lu, W and Liu, Q and Mattson, N and Chen, KY and Wang, J and Yuan, YC and Horne, D and Rosen, ST and Soto-Feliciano, Y and Feng, Z and Hoshii, T and Xiao, G and Müschen, M and Chen, J and Armstrong, SA and Chen, CW},
title = {High-resolution characterization of gene function using single-cell CRISPR tiling screen.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {4063},
pmid = {34210975},
issn = {2041-1723},
support = {P50 CA206963/CA/NCI NIH HHS/United States ; K99 CA197498/CA/NCI NIH HHS/United States ; R01 CA233922/CA/NCI NIH HHS/United States ; R01 CA176745/CA/NCI NIH HHS/United States ; R37 CA233691/CA/NCI NIH HHS/United States ; P30 CA033572/CA/NCI NIH HHS/United States ; R01 CA236626/CA/NCI NIH HHS/United States ; R00 CA197498/CA/NCI NIH HHS/United States ; P01 CA066996/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drug Screening Assays, Antitumor ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genetic Testing ; Genome, Human ; Histone-Lysine N-Methyltransferase/chemistry/genetics ; Histones ; Humans ; Models, Molecular ; Mutagenesis ; Neoplasms/*genetics ; *Phenotype ; Transcriptome ; },
abstract = {Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present "sc-Tiling," which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response.},
}
@article {pmid34210861,
year = {2021},
author = {Kaiser, J},
title = {Gene editor injected into the body treats disease.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6550},
pages = {16},
doi = {10.1126/science.373.6550.16},
pmid = {34210861},
issn = {1095-9203},
mesh = {Aged ; Amyloid Neuropathies, Familial/genetics/*therapy ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Female ; Gene Editing/*methods ; Humans ; Injections ; Liver ; Male ; Middle Aged ; Mutation ; Prealbumin/chemistry/genetics/*metabolism ; Protein Folding ; RNA, Messenger/*administration &amp; dosage/genetics ; },
}
@article {pmid34210100,
year = {2021},
author = {Michalski, K and Hertig, C and Mańkowski, DR and Kumlehn, J and Zimny, J and Linkiewicz, AM},
title = {Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8'OH1 loci.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34210100},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/genetics/*metabolism ; Gene Editing/*methods ; Genes, Reporter ; INDEL Mutation ; Mutagenesis, Site-Directed ; Plant Proteins/genetics/*metabolism ; Transfection ; Triticale/genetics/*metabolism ; },
abstract = {Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.},
}
@article {pmid34209672,
year = {2021},
author = {Liang, Y and Biswas, S and Kim, B and Bailey-Serres, J and Septiningsih, EM},
title = {Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34209672},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics ; Gene Editing ; *Genes, Plant ; Oryza/*physiology ; Phenotype ; Plant Development/genetics ; Plants, Genetically Modified ; *Regeneration ; Reproducibility of Results ; Seeds/genetics/growth &amp; development ; *Transformation, Genetic ; },
abstract = {Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.},
}
@article {pmid34208669,
year = {2021},
author = {Lee, HJ and Kim, HJ and Lee, SJ},
title = {Mismatch Intolerance of 5'-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {12},
pages = {},
pmid = {34208669},
issn = {1422-0067},
mesh = {*5' Flanking Region ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; DNA Mismatch Repair ; Escherichia coli/genetics/metabolism ; *Gene Editing/methods ; Genome, Microbial ; Genomics/methods ; RNA, Guide/*genetics ; },
abstract = {The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5'-truncated sgRNAs inhibited target recognition. These results suggest that a 5'-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5'-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5' end of truncated sgRNAs. Further, 5'-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5'-NG), which may enable whole-genome single-base editing.},
}
@article {pmid34207502,
year = {2021},
author = {Peyravian, N and Malekzadeh Kebria, M and Kiani, J and Brouki Milan, P and Mozafari, M},
title = {CRISPR-Associated (CAS) Effectors Delivery via Microfluidic Cell-Deformation Chip.},
journal = {Materials (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
pmid = {34207502},
issn = {1996-1944},
abstract = {Identifying new and even more precise technologies for modifying and manipulating selectively specific genes has provided a powerful tool for characterizing gene functions in basic research and potential therapeutics for genome regulation. The rapid development of nuclease-based techniques such as CRISPR/Cas systems has revolutionized new genome engineering and medicine possibilities. Additionally, the appropriate delivery procedures regarding CRISPR/Cas systems are critical, and a large number of previous reviews have focused on the CRISPR/Cas9-12 and 13 delivery methods. Still, despite all efforts, the in vivo delivery of the CAS gene systems remains challenging. The transfection of CRISPR components can often be inefficient when applying conventional delivery tools including viral elements and chemical vectors because of the restricted packaging size and incompetency of some cell types. Therefore, physical methods such as microfluidic systems are more applicable for in vitro delivery. This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations.},
}
@article {pmid34207150,
year = {2021},
author = {Dunning, K and Martz, A and Peralta, FA and Cevoli, F and Boué-Grabot, E and Compan, V and Gautherat, F and Wolf, P and Chataigneau, T and Grutter, T},
title = {P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation.},
journal = {International journal of molecular sciences},
volume = {22},
number = {12},
pages = {},
pmid = {34207150},
issn = {1422-0067},
mesh = {Adenosine Triphosphate/metabolism ; Algorithms ; Animals ; Anoctamins/chemistry/*metabolism ; CRISPR-Cas Systems ; Cell Membrane/metabolism ; Cell Membrane Permeability ; Cholesterol/metabolism ; HEK293 Cells ; Humans ; Immunohistochemistry ; Models, Biological ; Oocytes ; Receptors, Purinergic P2X7/chemistry/*metabolism ; },
abstract = {P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca[2+]-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.},
}
@article {pmid34206474,
year = {2021},
author = {González de Aledo, M and González-Bardanca, M and Blasco, L and Pacios, O and Bleriot, I and Fernández-García, L and Fernández-Quejo, M and López, M and Bou, G and Tomás, M},
title = {CRISPR-Cas, a Revolution in the Treatment and Study of ESKAPE Infections: Pre-Clinical Studies.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {7},
pages = {},
pmid = {34206474},
issn = {2079-6382},
abstract = {One of the biggest threats we face globally is the emergence of antimicrobial-resistant (AMR) bacteria, which runs in parallel with the lack in the development of new antimicrobials. Among these AMR bacteria pathogens belonging to the ESKAPE group can be highlighted (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) due to their profile of drug resistance and virulence. Therefore, innovative lines of treatment must be developed for these bacteria. In this review, we summarize the different strategies for the treatment and study of molecular mechanisms of AMR in the ESKAPE pathogens based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins' technologies: loss of plasmid or cellular viability, random mutation or gene deletion as well directed mutations that lead to a gene's loss of function.},
}
@article {pmid34204760,
year = {2021},
author = {Dong, H and Huang, Y and Wang, K},
title = {The Development of Herbicide Resistance Crop Plants Using CRISPR/Cas9-Mediated Gene Editing.},
journal = {Genes},
volume = {12},
number = {6},
pages = {},
pmid = {34204760},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; Herbicide Resistance/*genetics ; Plant Breeding/*methods ; },
abstract = {The rapid increase in herbicide-resistant weeds creates a huge challenge to global food security because it can reduce crop production, causing considerable losses. Combined with a lack of novel herbicides, cultivating herbicide-resistant crops becomes an effective strategy to control weeds because of reduced crop phytotoxicity, and it expands the herbicidal spectrum. Recently developed clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing techniques enable efficiently targeted modification and hold great potential in creating desired plants with herbicide resistance. In the present review, we briefly summarize the mechanism responsible for herbicide resistance in plants and then discuss the applications of traditional mutagenesis and transgenic breeding in cultivating herbicide-resistant crops. We mainly emphasize the development and use of CRISPR/Cas technology in herbicide-resistant crop improvement. Finally, we discuss the future applications of the CRISPR/Cas system for developing herbicide-resistant crops.},
}
@article {pmid34204633,
year = {2021},
author = {Kharel, A and Islam, MT and Rookes, J and Cahill, D},
title = {How to Unravel the Key Functions of Cryptic Oomycete Elicitin Proteins and Their Role in Plant Disease.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {6},
pages = {},
pmid = {34204633},
issn = {2223-7747},
abstract = {Pathogens and plants are in a constant battle with one another, the result of which is either the restriction of pathogen growth via constitutive or induced plant defense responses or the pathogen colonization of plant cells and tissues that cause disease. Elicitins are a group of highly conserved proteins produced by certain oomycete species, and their sterol binding ability is recognized as an important feature in sterol-auxotrophic oomycetes. Elicitins also orchestrate other aspects of the interactions of oomycetes with their plant hosts. The function of elicitins as avirulence or virulence factors is controversial and is dependent on the host species, and despite several decades of research, the function of these proteins remains elusive. We summarize here our current understanding of elicitins as either defense-promoting or defense-suppressing agents and propose that more recent approaches such as the use of 'omics' and gene editing can be used to unravel the role of elicitins in host-pathogen interactions. A better understanding of the role of elicitins is required and deciphering their role in host-pathogen interactions will expand the strategies that can be adopted to improve disease resistance and reduce crop losses.},
}
@article {pmid34204559,
year = {2021},
author = {Oleszkiewicz, T and Klimek-Chodacka, M and Kruczek, M and Godel-Jędrychowska, K and Sala, K and Milewska-Hendel, A and Zubko, M and Kurczyńska, E and Qi, Y and Baranski, R},
title = {Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.},
journal = {International journal of molecular sciences},
volume = {22},
number = {12},
pages = {},
pmid = {34204559},
issn = {1422-0067},
mesh = {Base Sequence ; Biosynthetic Pathways/*genetics ; *CRISPR-Cas Systems ; Carotenoids/*metabolism ; Cell Wall/*metabolism/ultrastructure ; Daucus carota/*physiology/ultrastructure ; *Gene Editing ; Gene Targeting ; Genes, Plant ; Genetic Vectors/genetics ; Mutation ; Phenotype ; Plastids/genetics/ultrastructure ; },
abstract = {Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.},
}
@article {pmid34204013,
year = {2021},
author = {Son, GH and Moon, J and Shelake, RM and Vuong, UT and Ingle, RA and Gassmann, W and Kim, JY and Kim, SH},
title = {Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1.},
journal = {International journal of molecular sciences},
volume = {22},
number = {12},
pages = {},
pmid = {34204013},
issn = {1422-0067},
mesh = {Alleles ; Arabidopsis/genetics/*immunology/*microbiology ; Arabidopsis Proteins/genetics/*metabolism ; Base Sequence ; Botrytis/*physiology ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics/*immunology ; Fusarium/*physiology ; Gene Editing ; Gene Expression Regulation, Plant ; Genes, Plant ; Genetic Vectors/metabolism ; Lycopersicon esculentum/genetics ; Mutation/genetics ; *Plant Immunity/genetics ; Plasmids/genetics ; },
abstract = {Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.},
}
@article {pmid34202963,
year = {2021},
author = {Avramucz, &#xc1; and Møller-Olsen, C and Grigonyte, AM and Paramalingam, Y and Millard, A and Sagona, AP and Fehér, T},
title = {Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7.},
journal = {Biology},
volume = {10},
number = {6},
pages = {},
pmid = {34202963},
issn = {2079-7737},
abstract = {The recognition and binding of host bacteria by bacteriophages is most often enabled by a highly specific receptor-ligand type of interaction, with the receptor-binding proteins (RBPs) of phages being the primary determinants of host specificity. Specifically modifying the RBPs could alter or extend the host range of phages otherwise exhibiting desired phenotypic properties. This study employed two different strategies to reprogram T7 phages ordinarily infecting commensal K12 Escherichia coli strains to infect pathogen-associated K1-capsule-expressing strains. The strategies were based on either plasmid-based homologous recombination or bacteriophage recombineering using electroporated DNA (BRED). Our work pursued the construction of two genetic designs: one replacing the gp17 gene of T7, the other replacing gp11, gp12, and gp17 of T7 with their K1F counterparts. Both strategies displayed successful integration of the K1F sequences into the T7 genome, detected by PCR screening. Multiple methods were utilised to select or enrich for chimeric phages incorporating the K1F gp17 alone, including trxA, host-specificity, and CRISPR-Cas-based selection. Irrespective of the selection method, the above strategy yielded poorly reproducible phage propagation on the new host, indicating that the chimeric phage was less fit than the wild type and could not promote continual autonomous reproduction. Chimeric phages obtained from BRED incorporating gp11-12 and gp17, however, all displayed infection in a 2-stage pattern, indicating the presence of both K1F and T7 phenotypes. This study shows that BRED can be used as a tool to quickly access the potential of new RBP constructs without the need to engineer sustainably replicating phages. Additionally, we show that solely repurposing the primary RBP is, in some cases, insufficient to produce a viable chimeric phage.},
}
@article {pmid34201896,
year = {2021},
author = {Shafaroudi, AM and Sharifi-Zarchi, A and Rahmani, S and Nafissi, N and Mowla, SJ and Lauria, A and Oliviero, S and Matin, MM},
title = {Expression and Function of C1orf132 Long-Noncoding RNA in Breast Cancer Cell Lines and Tissues.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34201896},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Nucleus/genetics/metabolism ; Down-Regulation ; Epithelial-Mesenchymal Transition/genetics ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; MCF-7 Cells ; Mice ; MicroRNAs/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Long Noncoding/*genetics/metabolism ; Triple Negative Breast Neoplasms/*genetics/metabolism ; },
abstract = {miR-29b2 and miR-29c play a suppressive role in breast cancer progression. C1orf132 (also named MIR29B2CHG) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher C1orf132 expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted C1orf132 distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that C1orf132 long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of C1orf132 whose functionality was demonstrated by transfecting MCF7 cells with a C1orf132 promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes CD46 and CD34, while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.},
}
@article {pmid34201772,
year = {2021},
author = {DeLeo, KR and Baral, SS and Houser, A and James, A and Sewell, P and Pandey, S and DiMario, PJ},
title = {Drosophila to Explore Nucleolar Stress.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34201772},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Nucleolus/*genetics ; Coiled Bodies/genetics/*pathology ; Drosophila Proteins/antagonists &amp; inhibitors/*genetics ; Drosophila melanogaster/*genetics/physiology ; *Gene Expression Regulation, Developmental ; Larva/genetics/growth &amp; development ; Phosphoproteins ; RNA-Binding Proteins/antagonists &amp; inhibitors/*genetics ; Ribosomes/genetics/metabolism ; *Stress, Physiological ; },
abstract = {Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.},
}
@article {pmid34201604,
year = {2021},
author = {Yan, Y and Kobayashi, Y and Huang, C and Liu, B and Qian, W and Wan, F and Schetelig, MF},
title = {Highly Efficient Temperature Inducible CRISPR-Cas9 Gene Targeting in Drosophila suzukii.},
journal = {International journal of molecular sciences},
volume = {22},
number = {13},
pages = {},
pmid = {34201604},
issn = {1422-0067},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Drosophila/embryology/*genetics ; Drosophila Proteins/genetics ; Embryo, Nonmammalian ; Female ; Gene Targeting/*methods ; Green Fluorescent Proteins/genetics ; Heat-Shock Response/genetics ; Male ; Mutagenesis ; Pigmentation/genetics ; Temperature ; Transgenes ; },
abstract = {The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.},
}
@article {pmid34201194,
year = {2021},
author = {Challagulla, A and Schat, KA and Doran, TJ},
title = {In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells.},
journal = {Methods and protocols},
volume = {4},
number = {2},
pages = {},
pmid = {34201194},
issn = {2409-9279},
abstract = {Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.},
}
@article {pmid34201141,
year = {2021},
author = {Vangheluwe, N and Beeckman, T},
title = {Lateral Root Initiation and the Analysis of Gene Function Using Genome Editing with CRISPR in Arabidopsis.},
journal = {Genes},
volume = {12},
number = {6},
pages = {},
pmid = {34201141},
issn = {2073-4425},
mesh = {Arabidopsis/*genetics/growth &amp; development ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/*methods ; Plant Root Cap/*genetics/growth &amp; development ; },
abstract = {Lateral root initiation is a post-embryonic process that requires the specification of a subset of pericycle cells adjacent to the xylem pole in the primary root into lateral root founder cells. The first visible event of lateral root initiation in Arabidopsis is the simultaneous migration of nuclei in neighbouring founder cells. Coinciding cell cycle activation is essential for founder cells in the pericycle to undergo formative divisions, resulting in the development of a lateral root primordium (LRP). The plant signalling molecule, auxin, is a major regulator of lateral root development; the understanding of the molecular mechanisms controlling lateral root initiation has progressed tremendously by the use of the Arabidopsis model and a continual improvement of molecular methodologies. Here, we provide an overview of the visible events, cell cycle regulators, and auxin signalling cascades related to the initiation of a new LRP. Furthermore, we highlight the potential of genome editing technology to analyse gene function in lateral root initiation, which provides an excellent model to answer fundamental developmental questions such as coordinated cell division, growth axis establishment as well as the specification of cell fate and cell polarity.},
}
@article {pmid34200331,
year = {2021},
author = {Yoo, HM and Kim, IH and Kim, S},
title = {Nucleic Acid Testing of SARS-CoV-2.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34200331},
issn = {1422-0067},
mesh = {Animals ; COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; Molecular Diagnostic Techniques ; Nanotechnology ; Nucleic Acid Amplification Techniques/*methods ; Polymerase Chain Reaction ; RNA, Viral ; Recombinases ; Reverse Transcription ; SARS-CoV-2/*isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.},
}
@article {pmid34200006,
year = {2021},
author = {Zhao, Y and Zou, J and Gao, Q and Xie, S and Cao, J and Zhou, H},
title = {CMAS and ST3GAL4 Play an Important Role in the Adsorption of Influenza Virus by Affecting the Synthesis of Sialic Acid Receptors.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34200006},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Influenza A virus/*physiology ; N-Acetylneuraminic Acid/metabolism ; N-Acylneuraminate Cytidylyltransferase/*antagonists &amp; inhibitors/genetics/metabolism ; Orthomyxoviridae Infections/metabolism/pathology/*virology ; Receptors, Cell Surface/*metabolism ; Sialyltransferases/*antagonists &amp; inhibitors ; Swine ; *Virus Replication ; },
abstract = {Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.},
}
@article {pmid34199901,
year = {2021},
author = {Horodecka, K and Düchler, M},
title = {CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34199901},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Extracellular Vesicles/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; Genetic Therapy/*methods ; Humans ; },
abstract = {The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.},
}
@article {pmid34198749,
year = {2021},
author = {Amend, B and Harland, N and Knoll, J and Stenzl, A and Aicher, WK},
title = {Large Animal Models for Investigating Cell Therapies of Stress Urinary Incontinence.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34198749},
issn = {1422-0067},
mesh = {Animals ; *Cell- and Tissue-Based Therapy ; *Disease Models, Animal ; Dogs ; Humans ; Rabbits ; Swine ; Urethra/*physiopathology ; Urinary Incontinence, Stress/*genetics/physiopathology ; },
abstract = {Stress urinary incontinence (SUI) is a significant health concern for patients affected, impacting their quality of life severely. To investigate mechanisms contributing to SUI different animal models were developed. Incontinence was induced under defined conditions to explore the pathomechanisms involved, spontaneous recovery, or efficacy of therapies over time. The animal models were coined to mimic known SUI risk factors such as childbirth or surgical injury. However, animal models neither reflect the human situation completely nor the multiple mechanisms that ultimately contribute to the pathogenesis of SUI. In the past, most SUI animal studies took advantage of rodents or rabbits. Recent models present for instance transgenic rats developing severe obesity, to investigate metabolic interrelations between the disorder and incontinence. Using recombinant gene technologies, such as transgenic, gene knock-out or CRISPR-Cas animals may narrow the gap between the model and the clinical situation of patients. However, to investigate surgical regimens or cell therapies to improve or even cure SUI, large animal models such as pig, goat, dog and others provide several advantages. Among them, standard surgical instruments can be employed for minimally invasive transurethral diagnoses and therapies. We, therefore, focus in this review on large animal models of SUI.},
}
@article {pmid34198536,
year = {2021},
author = {Koniali, L and Lederer, CW and Kleanthous, M},
title = {Therapy Development by Genome Editing of Hematopoietic Stem Cells.},
journal = {Cells},
volume = {10},
number = {6},
pages = {},
pmid = {34198536},
issn = {2073-4409},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Diseases, Inborn/*genetics/*therapy ; *Genetic Therapy ; *Hematopoietic Stem Cells ; Humans ; },
abstract = {Accessibility of hematopoietic stem cells (HSCs) for the manipulation and repopulation of the blood and immune systems has placed them at the forefront of cell and gene therapy development. Recent advances in genome-editing tools, in particular for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and CRISPR/Cas-derived editing systems, have transformed the gene therapy landscape. Their versatility and the ability to edit genomic sequences and facilitate gene disruption, correction or insertion, have broadened the spectrum of potential gene therapy targets and accelerated the development of potential curative therapies for many rare diseases treatable by transplantation or modification of HSCs. Ongoing developments seek to address efficiency and precision of HSC modification, tolerability of treatment and the distribution and affordability of corresponding therapies. Here, we give an overview of recent progress in the field of HSC genome editing as treatment for inherited disorders and summarize the most significant findings from corresponding preclinical and clinical studies. With emphasis on HSC-based therapies, we also discuss technical hurdles that need to be overcome en route to clinical translation of genome editing and indicate advances that may facilitate routine application beyond the most common disorders.},
}
@article {pmid34197999,
year = {2021},
author = {Zhao, Q and Pan, Y and Luan, X and Gao, Y and Zhao, X and Liu, Y and Wang, Y and Song, Y},
title = {Nano-immunosorbent assay based on Cas12a/crRNA for ultra-sensitive protein detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {190},
number = {},
pages = {113450},
doi = {10.1016/j.bios.2021.113450},
pmid = {34197999},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Gold ; Immunosorbents ; *Metal Nanoparticles ; },
abstract = {Apart from the great potential in genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has recently been widely used in biosensing. However, due to the complex and inefficient signal conversion strategies, most of the works focused on nucleic acid analysis rather than protein biomarkers. Herein, by employing DNA-AuNPs (gold nanoparticles) nanotechnology to activate trans-cleavage activity of CRISPR/Cas12a, a universal signal transduction strategy was established between trans-cleavage of CRISPR/Cas12a and protein analytes. As a result, a sensitive platform was developed for sensing carcinoembryonic antigen (CEA) and prostate specific-antigen (PSA) biomarkers, which was designated as Nano-CLISA (Nano-immunosorbent assay based on Cas12a/crRNA). Nano-CLISA was directly employed to test PSA in clinical samples, indicating its great potential in practical detection. This platform has been used to quantitatively analyze protein at attomolar levels, which was 1000-fold more sensitive than traditional ELISA, and the detection range is 15 times wider than that of traditional ELISA.},
}
@article {pmid34197800,
year = {2021},
author = {Long, C and Dai, L and E, C and Da, LT and Yu, J},
title = {Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2.},
journal = {Biophysical journal},
volume = {120},
number = {15},
pages = {3126-3137},
pmid = {34197800},
issn = {1542-0086},
mesh = {Allosteric Regulation ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Escherichia coli/metabolism ; },
abstract = {Cas1 and Cas2 are highly conserved proteins across clustered-regularly-interspaced-short-palindromic-repeat-Cas systems and play a significant role in protospacer acquisition. Based on crystal structure of twofold symmetric Cas1-Cas2 in complex with dual-forked protospacer DNA (psDNA), we conducted all-atom molecular dynamics simulations to study the psDNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence, or PAMc, of Cas1-Cas2. In the simulation, we noticed that two active sites of Cas1 and Cas1' bind asymmetrically to two identical PAMc on the psDNA captured from the crystal structure. For the modified psDNA containing only one PAMc, as that to be recognized by Cas1-Cas2 in general, our simulations show that the non-PAMc association site of Cas1-Cas2 remains destabilized until after the stably bound PAMc being cleaved at the corresponding association site. Thus, long-range correlation appears to exist upon the PAMc cleavage between the two active sites (∼10 nm apart) on Cas1-Cas2, which can be allosterically mediated by psDNA and Cas2 and Cas2' in bridging. To substantiate such findings, we conducted repeated runs and further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high frequency in acquisition, respectively. Notably, such intersite correlation becomes even more pronounced for the Cas1-Cas2 in complex with psH but remains low for the Cas1-Cas2 in complex with psL. Hence, our studies demonstrate that PAMc recognition and cleavage at one active site of Cas1-Cas2 may allosterically regulate non-PAMc association or even cleavage at the other site, and such regulation can be mediated by noncatalytic Cas2 and DNA protospacer to possibly support the ensued psDNA acquisition.},
}
@article {pmid34197615,
year = {2021},
author = {Cai, P and Duan, X and Wu, X and Gao, L and Ye, M and Zhou, YJ},
title = {Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris.},
journal = {Nucleic acids research},
volume = {49},
number = {13},
pages = {7791-7805},
pmid = {34197615},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Fatty Alcohols/metabolism ; Gene Editing ; Gene Expression ; *Homologous Recombination ; *Metabolic Engineering ; Metabolic Networks and Pathways/genetics ; Promoter Regions, Genetic ; Saccharomycetales/*genetics/metabolism ; },
abstract = {The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68-90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6-380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.},
}
@article {pmid34197614,
year = {2021},
author = {Tang, M and Pei, G and Su, D and Wang, C and Feng, X and Srivastava, M and Chen, Z and Zhao, Z and Chen, J},
title = {Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2.},
journal = {Nucleic acids research},
volume = {49},
number = {13},
pages = {7476-7491},
pmid = {34197614},
issn = {1362-4962},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA216437/CA/NCI NIH HHS/United States ; R01 CA210929/CA/NCI NIH HHS/United States ; R01 CA216911/CA/NCI NIH HHS/United States ; },
mesh = {BRCA2 Protein/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Survival ; Cyclin C/*genetics ; DNA Damage ; DNA Replication ; Gene Expression Regulation ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Mediator Complex/genetics/physiology ; Recombinational DNA Repair ; Stress, Physiological/genetics ; },
abstract = {Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.},
}
@article {pmid34197613,
year = {2021},
author = {Ciurkot, K and Gorochowski, TE and Roubos, JA and Verwaal, R},
title = {Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.},
journal = {Nucleic acids research},
volume = {49},
number = {13},
pages = {7775-7790},
pmid = {34197613},
issn = {1362-4962},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Proteins/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Down-Regulation ; Endodeoxyribonucleases/*chemistry/genetics/metabolism ; *Gene Expression Regulation ; Green Fluorescent Proteins/genetics ; Nuclear Localization Signals ; Promoter Regions, Genetic ; Protein Domains ; RNA/metabolism ; RNA Polymerase II/metabolism ; Saccharomyces cerevisiae/*genetics ; beta Carotene/biosynthesis ; },
abstract = {CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.},
}
@article {pmid34197611,
year = {2021},
author = {Lin, J and Shen, Y and Ni, J and She, Q},
title = {A type III-A CRISPR-Cas system mediates co-transcriptional DNA cleavage at the transcriptional bubbles in close proximity to active effectors.},
journal = {Nucleic acids research},
volume = {49},
number = {13},
pages = {7628-7643},
pmid = {34197611},
issn = {1362-4962},
mesh = {Adenosine Triphosphate/metabolism ; CRISPR-Associated Proteins/chemistry/metabolism ; *CRISPR-Cas Systems ; DNA Cleavage ; DNA, Single-Stranded/metabolism ; Deoxyribonucleases/*metabolism ; Ligands ; Plasmids/metabolism ; RNA/analysis ; *Transcription, Genetic ; },
abstract = {Many type III CRISPR-Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.},
}
@article {pmid34197093,
year = {2021},
author = {Lau, MSH and Sheng, L and Zhang, Y and Minton, NP},
title = {Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains.},
journal = {ACS synthetic biology},
volume = {10},
number = {7},
pages = {1739-1749},
doi = {10.1021/acssynbio.1c00138},
pmid = {34197093},
issn = {2161-5063},
support = {BB/L016478/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N022718/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacillaceae/*genetics ; CRISPR-Cas Systems ; *Gene Editing ; Genes, Bacterial ; Genes, Reporter ; *Genetic Engineering ; Green Fluorescent Proteins/genetics ; Homologous Recombination ; *Plasmids ; Promoter Regions, Genetic ; },
abstract = {The relentless rise in the levels of atmospheric greenhouse gases caused by the exploitation of fossil fuel necessitates the development of more environmentally friendly routes to the manufacture of chemicals and fuels. The exploitation of a fermentative process that uses a thermophilic chassis represents an attractive option. Its use, however, is hindered by a dearth of genetic tools. Here we expand on those available for the engineering of the industrial chassis Parageobacillus thermoglucosidasius through the assembly and testing of a range of promoters, ribosome binding sites, reporter genes, and the implementation of CRISPR/Cas9 genome editing based on two different thermostable Cas9 nucleases. The latter were used to demonstrate that the deletion of the two native plasmids carried by P. thermoglucosidasius, pNCI001 and pNCI002, either singly or in combination, had no discernible effects on the overall phenotypic characteristics of the organism. Through the CRISPR/Cas9-mediated insertion of the gene encoding a novel fluorescent reporter, eCGP123, we showed that pNCI001 exhibited a high degree of segregational stability. As the relatively higher copy number of pNCI001 led to higher levels of eCGP123 expression than when the same gene was integrated into the chromosome, we propose that pNCI001 represents the preferred option for the integration of metabolic operons when stable commercial strains are required.},
}
@article {pmid34195678,
year = {2021},
author = {Uchimura, T and Asano, T and Nakata, T and Hotta, A and Sakurai, H},
title = {A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs.},
journal = {Cell reports. Medicine},
volume = {2},
number = {6},
pages = {100298},
pmid = {34195678},
issn = {2666-3791},
mesh = {Boron Compounds/pharmacology ; CRISPR-Cas Systems ; Cell Differentiation ; Collagen/chemistry ; Creatine/pharmacology ; Dantrolene/pharmacology ; Dystrophin/deficiency/*genetics ; Electric Stimulation/*methods ; Gels ; Gene Expression ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Models, Biological ; Muscle Contraction/drug effects ; Muscle Fibers, Skeletal/cytology/drug effects/*metabolism ; Muscular Dystrophy, Duchenne/*genetics/metabolism/pathology ; Optogenetics ; Primary Cell Culture ; Tumor Necrosis Factor-alpha/genetics/metabolism ; },
abstract = {Duchenne muscular dystrophy (DMD) is a muscle degenerating disease caused by dystrophin deficiency, for which therapeutic options are limited. To facilitate drug development, it is desirable to develop in vitro disease models that enable the evaluation of DMD declines in contractile performance. Here, we show MYOD1-induced differentiation of hiPSCs into functional skeletal myotubes in vitro with collagen gel and electrical field stimulation (EFS). Long-term EFS training (0.5 Hz, 20 V, 2 ms, continuous for 2 weeks) mimicking muscle overuse recapitulates declines in contractile performance in dystrophic myotubes. A screening of clinically relevant drugs using this model detects three compounds that ameliorate this decline. Furthermore, we validate the feasibility of adapting the model to a 96-well culture system using optogenetic technology for large-scale screening. Our results support a disease model using patient-derived iPSCs that allows for the recapitulation of the contractile pathogenesis of DMD and a screening strategy for drug development.},
}
@article {pmid34194490,
year = {2021},
author = {Zhao, X and Henderson, HJ and Wang, T and Liu, B and Li, Y},
title = {Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.},
journal = {Neural plasticity},
volume = {2021},
number = {},
pages = {9979157},
pmid = {34194490},
issn = {1687-5443},
mesh = {Animals ; Auditory Threshold ; Base Sequence ; CRISPR-Cas Systems ; Cellular Senescence ; Clusterin/biosynthesis/*deficiency/genetics/physiology ; Drug Synergism ; Evoked Potentials, Auditory, Brain Stem ; Furosemide/administration &amp; dosage/toxicity ; Hair Cells, Auditory/drug effects/*physiology ; Hearing Loss, Sensorineural/chemically induced/*prevention &amp; control ; Kanamycin/administration &amp; dosage/toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Organ Culture Techniques ; Organ of Corti/pathology ; Otoacoustic Emissions, Spontaneous ; Presbycusis/*prevention &amp; control ; RNA, Messenger/biosynthesis/genetics ; },
abstract = {Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.},
}
@article {pmid34193462,
year = {2021},
author = {Ruf, B and Catania, VV and Wabitsch, S and Ma, C and Diggs, LP and Zhang, Q and Heinrich, B and Subramanyam, V and Cui, LL and Pouzolles, M and Evans, CN and Chari, R and Sakai, S and Oh, S and Barry, CE and Barber, DL and Greten, TF},
title = {Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response.},
journal = {Cancer immunology research},
volume = {9},
number = {9},
pages = {1024-1034},
pmid = {34193462},
issn = {2326-6074},
support = {ZIA BC011345/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD ; Antigens, Differentiation, T-Lymphocyte ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Histocompatibility Antigens Class I/genetics/*metabolism ; Humans ; Lectins, C-Type ; Lymphocyte Activation/*immunology ; Male ; Mice ; Minor Histocompatibility Antigens/genetics/*metabolism ; Mucosal-Associated Invariant T Cells/*drug effects/metabolism ; Neoplasms/*drug therapy/metabolism ; Ribitol/administration &amp; dosage/analogs &amp; derivatives ; Riboflavin/biosynthesis/chemistry/pharmacology ; Uracil/administration &amp; dosage/analogs &amp; derivatives ; },
abstract = {Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro-activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway-derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1 [-/-] mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy.See related Spotlight by Lantz, p. 996.},
}
@article {pmid34193153,
year = {2021},
author = {Lin, YW and Nhieu, J and Wei, CW and Lin, YL and Kagechika, H and Wei, LN},
title = {Regulation of exosome secretion by cellular retinoic acid binding protein 1 contributes to systemic anti-inflammation.},
journal = {Cell communication and signaling : CCS},
volume = {19},
number = {1},
pages = {69},
pmid = {34193153},
issn = {1478-811X},
support = {DK60521/NH/NIH HHS/United States ; R01 DK060521/DK/NIDDK NIH HHS/United States ; F31 DK123999/DK/NIDDK NIH HHS/United States ; R01 DK054733/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Communication/genetics ; Disease Models, Animal ; Exosomes/*genetics ; Extracellular Vesicles/genetics ; Homeostasis/genetics ; Humans ; Inflammation/*genetics/pathology ; Mice ; Mice, Knockout ; Neurons/*metabolism/pathology ; Nuclear Receptor Interacting Protein 1/*genetics ; RAW 264.7 Cells ; Receptors, Retinoic Acid/*genetics ; Signal Transduction/genetics ; Tretinoin/metabolism ; },
abstract = {BACKGROUND: Intercellular communications are important for maintaining normal physiological processes. An important intercellular communication is mediated by the exchange of membrane-enclosed extracellular vesicles. Among various vesicles, exosomes can be detected in a wide variety of biological systems, but the regulation and biological implication of exosome secretion/uptake remains largely unclear.<br><br>METHODS: Cellular retinoic acid (RA) binding protein 1 (Crabp1) knockout (CKO) mice were used for in vivo studies. Extracellular exosomes were monitored in CKO mice and relevant cell cultures including embryonic stem cell (CJ7), macrophage (Raw 264.7) and hippocampal cell (HT22) using Western blot and flow cytometry. Receptor Interacting Protein 140 (RIP140) was depleted by Crispr/Cas9-mediated gene editing. Anti-inflammatory maker was analyzed using qRT-PCR. Clinical relevance was accessed by mining multiple clinical datasets.<br><br>RESULTS: This study uncovers Crabp1 as a negative regulator of exosome secretion from neurons. Specifically, RIP140, a pro-inflammatory regulator, can be transferred from neurons, via Crabp1-regulated exosome secretion, into macrophages to promote their inflammatory polarization. Consistently, CKO mice, defected in the negative control of exosome secretion, have significantly elevated RIP140-containing exosomes in their blood and cerebrospinal fluid, and exhibit an increased vulnerability to systemic inflammation. Clinical relevance of this pathway is supported by patients' data of multiple inflammatory diseases. Further, the action of Crabp1 in regulating exosome secretion involves its ligand and is mediated by its downstream target, the MAPK signaling pathway.<br><br>CONCLUSIONS: This study presents the first evidence for the regulation of exosome secretion, which mediates intercellular communication, by RA-Crabp1 signaling. This novel mechanism can contribute to the control of systemic inflammation by transferring an inflammatory regulator, RIP140, between cells. This represents a new mechanism of vitamin A action that can modulate the homeostasis of system-wide innate immunity without involving gene regulation. Video Abstract.},
}
@article {pmid34193127,
year = {2021},
author = {Madhavan, A and Arun, KB and Sindhu, R and Krishnamoorthy, J and Reshmy, R and Sirohi, R and Pugazhendi, A and Awasthi, MK and Szakacs, G and Binod, P},
title = {Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up.},
journal = {Microbial cell factories},
volume = {20},
number = {1},
pages = {124},
pmid = {34193127},
issn = {1475-2859},
mesh = {Biological Products/*metabolism ; CRISPR-Cas Systems ; Fermentation ; Glycosylation ; *Metabolic Engineering ; Promoter Regions, Genetic ; Recombinant Proteins/*biosynthesis/metabolism ; Yeasts/*genetics/growth &amp; development/metabolism ; },
abstract = {The manufacture of recombinant therapeutics is a fastest-developing section of therapeutic pharmaceuticals and presently plays a significant role in disease management. Yeasts are established eukaryotic host for heterologous protein production and offer distinctive benefits in synthesising pharmaceutical recombinants. Yeasts are proficient of vigorous growth on inexpensive media, easy for gene manipulations, and are capable of adding post translational changes of eukaryotes. Saccharomyces cerevisiae is model yeast that has been applied as a main host for the manufacture of pharmaceuticals and is the major tool box for genetic studies; nevertheless, numerous other yeasts comprising Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Yarrowia lipolytica have attained huge attention as non-conventional partners intended for the industrial manufacture of heterologous proteins. Here we review the advances in yeast gene manipulation tools and techniques for heterologous pharmaceutical protein synthesis. Application of secretory pathway engineering, glycosylation engineering strategies and fermentation scale-up strategies in customizing yeast cells for the synthesis of therapeutic proteins has been meticulously described.},
}
@article {pmid34192988,
year = {2021},
author = {Arlt, B and Mastrobuoni, G and Wuenschel, J and Astrahantseff, K and Eggert, A and Kempa, S and Deubzer, HE},
title = {Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.},
journal = {Journal of enzyme inhibition and medicinal chemistry},
volume = {36},
number = {1},
pages = {1282-1289},
pmid = {34192988},
issn = {1475-6374},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Citric Acid Cycle/drug effects ; Enzyme Inhibitors/*pharmacology ; Gas Chromatography-Mass Spectrometry/methods ; Glucose/metabolism ; Humans ; Metabolomics ; Phosphoglycerate Dehydrogenase/*antagonists &amp; inhibitors/genetics ; Piperazines/*pharmacology ; Pyridines/*pharmacology ; Thioamides/*pharmacology ; },
abstract = {The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.},
}
@article {pmid34192695,
year = {2022},
author = {Butiuc-Keul, A and Farkas, A and Carpa, R and Iordache, D},
title = {CRISPR-Cas System: The Powerful Modulator of Accessory Genomes in Prokaryotes.},
journal = {Microbial physiology},
volume = {32},
number = {1-2},
pages = {2-17},
doi = {10.1159/000516643},
pmid = {34192695},
issn = {2673-1673},
mesh = {Archaea/genetics ; Bacteria/genetics ; *COVID-19/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; SARS-CoV-2/genetics ; },
abstract = {Being frequently exposed to foreign nucleic acids, bacteria and archaea have developed an ingenious adaptive defense system, called CRISPR-Cas. The system is composed of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array, together with CRISPR (cas)-associated genes. This system consists of a complex machinery that integrates fragments of foreign nucleic acids from viruses and mobile genetic elements (MGEs), into CRISPR arrays. The inserted segments (spacers) are transcribed and then used by cas proteins as guide RNAs for recognition and inactivation of the targets. Different types and families of CRISPR-Cas systems consist of distinct adaptation and effector modules with evolutionary trajectories, partially independent. The origin of the effector modules and the mechanism of spacer integration/deletion is far less clear. A review of the most recent data regarding the structure, ecology, and evolution of CRISPR-Cas systems and their role in the modulation of accessory genomes in prokaryotes is proposed in this article. The CRISPR-Cas system's impact on the physiology and ecology of prokaryotes, modulation of horizontal gene transfer events, is also discussed here. This system gained popularity after it was proposed as a tool for plant and animal embryo editing, in cancer therapy, as antimicrobial against pathogenic bacteria, and even for combating the novel coronavirus - SARS-CoV-2; thus, the newest and promising applications are reviewed as well.},
}
@article {pmid34192540,
year = {2021},
author = {Kuzuoglu-Ozturk, D and Hu, Z and Rama, M and Devericks, E and Weiss, J and Chiang, GG and Worland, ST and Brenner, SE and Goodarzi, H and Gilbert, LA and Ruggero, D},
title = {Revealing molecular pathways for cancer cell fitness through a genetic screen of the cancer translatome.},
journal = {Cell reports},
volume = {35},
number = {13},
pages = {109321},
pmid = {34192540},
issn = {2211-1247},
support = {DP2 CA239597/CA/NCI NIH HHS/United States ; R35 CA242986/CA/NCI NIH HHS/United States ; },
mesh = {5' Untranslated Regions/genetics ; Animals ; Apoptosis/genetics ; Autophagy ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Eukaryotic Initiation Factor-4E/genetics/metabolism ; Exons/genetics ; *Genetic Testing ; Genome, Human ; Humans ; Male ; Metalloendopeptidases/metabolism ; Mice ; Mitochondria/metabolism ; Mitochondrial Proteins/metabolism ; Neoplasms/*genetics/pathology ; Peptide Hydrolases/metabolism ; *Protein Biosynthesis/genetics ; *Signal Transduction/genetics ; Stress, Physiological ; bcl-X Protein/metabolism ; },
abstract = {The major cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), an ancient protein required for translation of all eukaryotic genomes, is a surprising yet potent oncogenic driver. The genetic interactions that maintain the oncogenic activity of this key translation factor remain unknown. In this study, we carry out a genome-wide CRISPRi screen wherein we identify more than 600 genetic interactions that sustain eIF4E oncogenic activity. Our data show that eIF4E controls the translation of Tfeb, a key executer of the autophagy response. This autophagy survival response is triggered by mitochondrial proteotoxic stress, which allows cancer cell survival. Our screen also reveals a functional interaction between eIF4E and a single anti-apoptotic factor, Bcl-xL, in tumor growth. Furthermore, we show that eIF4E and the exon-junction complex (EJC), which is involved in many steps of RNA metabolism, interact to control the migratory properties of cancer cells. Overall, we uncover several cancer-specific vulnerabilities that provide further resolution of the cancer translatome.},
}
@article {pmid34191829,
year = {2021},
author = {Stahl-Rommel, S and Li, D and Sung, M and Li, R and Vijayakumar, A and Atabay, KD and Bushkin, GG and Castro, CL and Foley, KD and Copeland, DS and Castro-Wallace, SL and Alvarez Saavedra, E and Gleason, EJ and Kraves, S},
title = {A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station.},
journal = {PloS one},
volume = {16},
number = {6},
pages = {e0253403},
pmid = {34191829},
issn = {1932-6203},
mesh = {Astronauts ; CRISPR-Cas Systems/*genetics ; Cosmic Radiation/*adverse effects ; DNA Breaks, Double-Stranded/*radiation effects ; DNA Repair/*radiation effects ; DNA, Fungal/genetics/radiation effects ; Gene Editing ; Humans ; Mutagenesis ; Mutation ; Occupational Exposure/*adverse effects ; Saccharomyces cerevisiae/genetics/radiation effects ; Saccharomyces cerevisiae Proteins/genetics ; Spacecraft ; },
abstract = {As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.},
}
@article {pmid34189746,
year = {2021},
author = {Hurt, SC and Dickson, PI and Curiel, DT},
title = {Mucopolysaccharidoses type I gene therapy.},
journal = {Journal of inherited metabolic disease},
volume = {44},
number = {5},
pages = {1088-1098},
pmid = {34189746},
issn = {1573-2665},
support = {R01 EB026468/EB/NIBIB NIH HHS/United States ; R01 NS088766/NS/NINDS NIH HHS/United States ; R61 NS111079/NS/NINDS NIH HHS/United States ; UG3 TR002851/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Gene Expression ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Glycosaminoglycans/urine ; Humans ; Iduronidase/analysis/*genetics/metabolism ; Mucopolysaccharidosis I/pathology/*therapy ; },
abstract = {Mucopolysaccharidoses type I (MPS I) is an inherited metabolic disease characterized by a malfunction of the α-l-iduronidase (IDUA) enzyme leading to the storage of glycosaminoglycans in the lysosomes. This disease has longtime been studied as a therapeutic target for those studying gene therapy and many studies have been done using various vectors to deliver the IDUA gene for corrective treatment. Many vectors have difficulties with efficacy and insertional mutagenesis concerns including adeno-associated viral (AAV) vectors. Studies of AAV vectors treating MPS I have seemed promising, but recent deaths in gene therapy clinical trials for other inherited diseases using AAV vectors have left questions about their safety. Additionally, the recent modifications to adenoviral vectors leading them to target the vascular endothelium minimizing the risk of hepatotoxicity could lead to them being a viable option for MPS I gene therapy when coupled with gene editing technologies like CRISPR/Cas9.},
}
@article {pmid34189671,
year = {2021},
author = {Pan, JS and Lin, ZS and Wen, JC and Guo, JF and Wu, XH and Liu, YY and Lai, WJ and Liang, QY and Xie, YS and Chen, YR and Chen, YH and Yan, AF and Feng, J and Liu, L and Gong, DY and Zhu, XX and Lu, JH and Tang, DS},
title = {Application of the modified cytosine base-editing in the cultured cells of bama minipig.},
journal = {Biotechnology letters},
volume = {43},
number = {9},
pages = {1699-1714},
pmid = {34189671},
issn = {1573-6776},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Codon, Terminator ; Cytosine/*metabolism ; Fibroblasts/*cytology/metabolism ; Gene Editing/*methods ; Myostatin/*genetics ; Plasmids/genetics ; Swine ; Swine, Miniature ; Transfection ; },
abstract = {Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.},
}
@article {pmid34189476,
year = {2021},
author = {Wang, X and Xu, BL and Chen, XW},
title = {Acute gene inactivation in the adult mouse liver using the CRISPR-Cas9 technology.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100611},
pmid = {34189476},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knockout Techniques ; Liver/*metabolism ; Mice ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {Genetic manipulation in mice allows the discovery of gene function and biological mechanisms in vivo. The widely used Cre/LoxP system usually takes months to years especially when starting with the production of floxed alleles of a new gene of interest (GOI). Here, we describe a protocol using the CRISPR-Cas9 system to acutely inactivate the GOI in adult mice. This protocol enables hepatocyte-specific gene editing within 4 weeks in adult mice and avoids compensatory effects of traditional gene inactivation initiated during various developmental stages. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).},
}
@article {pmid34188205,
year = {2021},
author = {Baglaenko, Y and Macfarlane, D and Marson, A and Nigrovic, PA and Raychaudhuri, S},
title = {Genome editing to define the function of risk loci and variants in rheumatic disease.},
journal = {Nature reviews. Rheumatology},
volume = {17},
number = {8},
pages = {462-474},
pmid = {34188205},
issn = {1759-4804},
support = {UH2 AR067677/AR/NIAMS NIH HHS/United States ; U01 HG009379/HG/NHGRI NIH HHS/United States ; R01 AR063759/AR/NIAMS NIH HHS/United States ; R01 AR065538/AR/NIAMS NIH HHS/United States ; R01 AR075906/AR/NIAMS NIH HHS/United States ; R01 AR073201/AR/NIAMS NIH HHS/United States ; P30 AR070253/AR/NIAMS NIH HHS/United States ; R21 AR076630/AR/NIAMS NIH HHS/United States ; R21 HL150575/HL/NHLBI NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; R01 DK119979/DK/NIDDK NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *Gene Editing/methods ; Genetic Loci/genetics ; Genetic Predisposition to Disease/*genetics ; Genetic Variation/genetics ; Humans ; Rheumatic Diseases/etiology/*genetics ; Risk Factors ; },
abstract = {Discoveries in human genetic studies have revolutionized our understanding of complex rheumatic and autoimmune diseases, including the identification of hundreds of genetic loci and single nucleotide polymorphisms that potentially predispose individuals to disease. However, in most cases, the exact disease-causing variants and their mechanisms of action remain unresolved. Functional follow-up of these findings is most challenging for genomic variants that are in non-coding genomic regions, where the large majority of common disease-associated variants are located, and/or that probably affect disease progression via cell type-specific gene regulation. To deliver on the therapeutic promise of human genetic studies, defining the mechanisms of action of these alleles is essential. Genome editing technology, such as CRISPR-Cas, has created a vast toolbox for targeted genetic and epigenetic modifications that presents unprecedented opportunities to decipher disease-causing loci, genes and variants in autoimmunity. In this Review, we discuss the past 5-10 years of progress in resolving the mechanisms underlying rheumatic disease-associated alleles, with an emphasis on how genomic editing techniques can enable targeted dissection and mechanistic studies of causal autoimmune risk variants.},
}
@article {pmid34187974,
year = {2021},
author = {Kumar, R and Sharma, AK},
title = {Transcription factor stoichiometry in cell fate determination.},
journal = {Journal of genetics},
volume = {100},
number = {},
pages = {},
pmid = {34187974},
issn = {0973-7731},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Cell Lineage/genetics ; Cell Transdifferentiation/genetics ; Cellular Reprogramming/*genetics ; Gene Expression Regulation, Developmental/genetics ; Humans ; Transcription Factors/*genetics ; *Transcription, Genetic ; },
abstract = {Transcription factors play very important role in cell fate determination. There are many cell specific transcription factors which when expressed ectopically may lead to cell fate conversion or transdifferentiation. Many of these transcription factors function differently based on their levels and stoichiometry. Many different types of differentiated cells have been generated from other differentiated cell types by expressing different levels and stoichiometry of reprogramming factors. Many methodologies have been developed for efficient cell fate conversion by regulating the levels and stoichiometry of transcription factors in a particular cocktail that have therapeutic values. An approach called phenotypic activation which involves overexpression of putative transcription factors has been developed as a tool to discover new transcription factors and their targets. Transcription factor overexpression may also have toxic effects where nonspecific electrostatic interactions and 'squelching' may lead to inhibition of many genes. Altered levels of transcription factors may have disastrous consequences like cancer. Recent developments like designing of artificial transcription factors, nanotechnology-based transcriptional tools and CRISPR-based transcription modules with capabilities of precise regulation of gene expression patterns hold huge potentials in the field of transcriptional therapeutics.},
}
@article {pmid34184636,
year = {2021},
author = {Kulkarni, S and Marquez, J and Date, P and Ventrella, R and Mitchell, BJ and Khokha, MK},
title = {Mechanical stretch scales centriole number to apical area via Piezo1 in multiciliated cells.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34184636},
issn = {2050-084X},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; K99 HL133606/HL/NHLBI NIH HHS/United States ; UL1 TR001863/TR/NCATS NIH HHS/United States ; R00 HL133606/HL/NHLBI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; T32 AR060710/AR/NIAMS NIH HHS/United States ; R01 HD102186/HD/NICHD NIH HHS/United States ; R01 GM089970/GM/NIGMS NIH HHS/United States ; P30 DK034989/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Biomechanical Phenomena ; CRISPR-Cas Systems ; Cell Cycle Proteins ; Centrioles/*physiology ; Gene Silencing ; Ion Channels ; Morpholinos ; RNA, Messenger ; Xenopus/embryology ; },
abstract = {How cells count and regulate organelle number is a fundamental question in cell biology. For example, most cells restrict centrioles to two in number and assemble one cilium; however, multiciliated cells (MCCs) synthesize hundreds of centrioles to assemble multiple cilia. Aberration in centriole/cilia number impairs MCC function and can lead to pathological outcomes. Yet how MCCs control centriole number remains unknown. Using Xenopus, we demonstrate that centriole number scales with apical area over a remarkable 40-fold change in size. We find that tensile forces that shape the apical area also trigger centriole amplification based on both cell stretching experiments and disruption of embryonic elongation. Unexpectedly, Piezo1, a mechanosensitive ion channel, localizes near each centriole suggesting a potential role in centriole amplification. Indeed, depletion of Piezo1 affects centriole amplification and disrupts its correlation with the apical area in a tension-dependent manner. Thus, mechanical forces calibrate cilia/centriole number to the MCC apical area via Piezo1. Our results provide new perspectives to study organelle number control essential for optimal cell function.},
}
@article {pmid34184004,
year = {2021},
author = {Sha, L and Han, Y and Wang, M and Wu, S and Yang, J and Li, G},
title = {A robust CRISPR-Cas12a biosensor coated with metal-organic framework.},
journal = {Journal of materials chemistry. B},
volume = {9},
number = {27},
pages = {5451-5455},
doi = {10.1039/d1tb01126j},
pmid = {34184004},
issn = {2050-7518},
mesh = {Bacterial Proteins/*metabolism ; *Biosensing Techniques ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*drug effects/genetics ; Coated Materials, Biocompatible/chemistry/*pharmacology ; Endodeoxyribonucleases/*metabolism ; Hydrogen-Ion Concentration ; Metal-Organic Frameworks/chemistry/*pharmacology ; Protective Agents/chemistry/*pharmacology ; RNA/genetics/metabolism ; },
abstract = {Metal-organic frameworks (MOFs) are proposed to protect a CRISPR-associated enzyme/RNA complex from harsh environments, while the complex can be quickly released from MOFs with high efficiency. Therefore, the application of CRISPR-powered biosensing can be more feasible.},
}
@article {pmid34183861,
year = {2021},
author = {Koblan, LW and Arbab, M and Shen, MW and Hussmann, JA and Anzalone, AV and Doman, JL and Newby, GA and Yang, D and Mok, B and Replogle, JM and Xu, A and Sisley, TA and Weissman, JS and Adamson, B and Liu, DR},
title = {Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning.},
journal = {Nature biotechnology},
volume = {39},
number = {11},
pages = {1414-1425},
pmid = {34183861},
issn = {1546-1696},
support = {UG3 AI150551/AI/NIAID NIH HHS/United States ; R35 GM138167/GM/NIGMS NIH HHS/United States ; P30 CA072720/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; F31 NS115380/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Machine Learning ; Mammals/genetics ; RNA, Guide/genetics ; },
abstract = {Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE-single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.},
}
@article {pmid34183837,
year = {2021},
author = {Scott, BM and Gutiérrez-Vázquez, C and Sanmarco, LM and da Silva Pereira, JA and Li, Z and Plasencia, A and Hewson, P and Cox, LM and O'Brien, M and Chen, SK and Moraes-Vieira, PM and Chang, BSW and Peisajovich, SG and Quintana, FJ},
title = {Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.},
journal = {Nature medicine},
volume = {27},
number = {7},
pages = {1212-1222},
pmid = {34183837},
issn = {1546-170X},
mesh = {Adenosine Triphosphate/*metabolism ; Animals ; Apyrase/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Dysbiosis/prevention &amp; control ; Female ; Fibrosis/prevention &amp; control ; Gastrointestinal Microbiome/physiology ; Gastrointestinal Tract/microbiology/pathology ; Humans ; Inflammatory Bowel Diseases/pathology/*therapy ; Male ; Mice ; Mice, Inbred C57BL ; Probiotics/*therapeutic use ; Receptors, Purinergic P2Y2/genetics/*metabolism ; Saccharomyces cerevisiae/genetics/*metabolism ; },
abstract = {Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.},
}
@article {pmid34183132,
year = {2021},
author = {Mitschka, S and Fansler, MM and Mayr, C},
title = {Generation of 3'UTR knockout cell lines by CRISPR/Cas9-mediated genome editing.},
journal = {Methods in enzymology},
volume = {655},
number = {},
pages = {427-457},
doi = {10.1016/bs.mie.2021.03.014},
pmid = {34183132},
issn = {1557-7988},
support = {DP1 GM123454/GM/NIGMS NIH HHS/United States ; T32 GM083937/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Humans ; Polyadenylation ; },
abstract = {In addition to the protein code, messenger RNAs (mRNAs) also contain untranslated regions (UTRs). 3'UTRs span the region between the translational stop codon and the poly(A) tail. Sequence elements located in 3'UTRs are essential for pre-mRNA processing. 3'UTRs also contain elements that can regulate protein abundance, localization, and function. At least half of all human genes use alternative cleavage and polyadenylation (APA) to further diversify the regulatory potential of protein functions. Traditional gene editing approaches are designed to disrupt the production of functional proteins. Here, we describe a method that allows investigators to manipulate 3'UTR sequences of endogenous genes for both single- 3'UTR and multi-3'UTR genes. As 3'UTRs can regulate individual functions of proteins, techniques to manipulate 3'UTRs at endogenous gene loci will help to disentangle multi-functionality of proteins. Furthermore, the ability to directly examine the impact of gene regulatory elements in 3'UTRs will provide further insights into their functional significance.},
}
@article {pmid34182254,
year = {2021},
author = {Ma, L and Schmidt, M and Morrow, EM},
title = {Human iPSC lines from a Christianson syndrome patient with NHE6 W523X mutation, a biologically-related control, and CRISPR/Cas9 gene-corrected isogenic controls.},
journal = {Stem cell research},
volume = {54},
number = {},
pages = {102435},
pmid = {34182254},
issn = {1876-7753},
support = {R01 MH105442/MH/NIMH NIH HHS/United States ; R01 NS113141/NS/NINDS NIH HHS/United States ; R21 MH115392/MH/NIMH NIH HHS/United States ; },
mesh = {Ataxia ; CRISPR-Cas Systems/genetics ; Epilepsy ; Genetic Diseases, X-Linked ; Humans ; *Induced Pluripotent Stem Cells ; Intellectual Disability ; Leukocytes, Mononuclear ; Microcephaly ; Mutation ; Ocular Motility Disorders ; },
abstract = {Loss-of-function mutations in Na+/H + exchanger 6 (NHE6) (also termed SLC9A6) cause the X-linked neurogenetic disorder Christianson syndrome (CS). Using peripheral blood mononuclear cells, we developed induced pluripotent stem cell (iPSC) lines from a patient with the NHE6 nonsense mutation c.1569G > A (p.(W523X)) and diagnosed with CS and from a biologically-related control. Using CRISPR/Cas9 gene editing, we generated two isogenic control lines in which the c.1569G > A mutation was corrected. All lines were verified by DNA sequencing and for NHE6 protein expression, pluripotency, and differentiation potential. These lines will serve as a valuable resource for both basic and translational studies in CS.},
}
@article {pmid34181731,
year = {2021},
author = {deYMartín Garrido, N and Orekhova, M and Lai Wan Loong, YTE and Litvinova, A and Ramlaul, K and Artamonova, T and Melnikov, AS and Serdobintsev, P and Aylett, CHS and Yakunina, M},
title = {Structure of the bacteriophage PhiKZ non-virion RNA polymerase.},
journal = {Nucleic acids research},
volume = {49},
number = {13},
pages = {7732-7739},
pmid = {34181731},
issn = {1362-4962},
support = {/WT_/Wellcome Trust/United Kingdom ; 206212/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 206175/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Cryoelectron Microscopy ; DNA-Directed RNA Polymerases/*chemistry/metabolism ; Models, Molecular ; Promoter Regions, Genetic ; Protein Subunits/chemistry/metabolism ; Pseudomonas Phages/*enzymology ; Viral Proteins/*chemistry/metabolism ; },
abstract = {Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a 'nucleus', erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial β/β' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.},
}
@article {pmid34181658,
year = {2021},
author = {Xue, WH and Xu, N and Chen, SJ and Liu, XY and Zhang, JL and Xu, HJ},
title = {Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens.},
journal = {PLoS genetics},
volume = {17},
number = {6},
pages = {e1009653},
pmid = {34181658},
issn = {1553-7404},
mesh = {Adaptation, Physiological/genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Energy Metabolism/genetics ; *Evolution, Molecular ; Gene Dosage ; Gene Editing/methods ; *Gene Expression Regulation, Developmental ; Hemiptera/anatomy &amp; histology/*genetics/growth &amp; development/metabolism ; Insect Proteins/*genetics/metabolism ; Longevity/genetics ; Nymph/genetics/growth &amp; development/metabolism ; Phenotype ; Receptor, Insulin/*genetics/metabolism ; Signal Transduction ; Starvation/genetics/metabolism ; Wings, Animal/anatomy &amp; histology/growth &amp; development/*metabolism ; },
abstract = {A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged-destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates.},
}
@article {pmid34180815,
year = {2021},
author = {Baron, M and Bonnefond, A},
title = {[Metabolic syndrome: Towards cell therapy by using CRISPR/Cas9-engineered human adipocytes].},
journal = {Medecine sciences : M/S},
volume = {37},
number = {6-7},
pages = {585-587},
doi = {10.1051/medsci/2021071},
pmid = {34180815},
issn = {1958-5381},
mesh = {Adipocytes ; *CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Gene Editing ; Humans ; *Metabolic Syndrome/genetics/therapy ; },
}
@article {pmid34180073,
year = {2022},
author = {Linder, B and Klein, C and Hoffmann, ME and Bonn, F and Dikic, I and Kögel, D},
title = {BAG3 is a negative regulator of ciliogenesis in glioblastoma and triple-negative breast cancer cells.},
journal = {Journal of cellular biochemistry},
volume = {123},
number = {1},
pages = {77-90},
doi = {10.1002/jcb.30073},
pmid = {34180073},
issn = {1097-4644},
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Apoptosis Regulatory Proteins/genetics/*metabolism ; Aurora Kinase A/metabolism ; Brain Neoplasms/*metabolism/pathology ; CDC2 Protein Kinase/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/genetics ; Cilia/*enzymology ; Drug Resistance, Neoplasm/genetics ; Epithelial-Mesenchymal Transition/genetics ; Gene Knockout Techniques/methods ; Glioblastoma/*metabolism/pathology ; Humans ; Proteomics/methods ; Signal Transduction/*genetics ; Snail Family Transcription Factors/metabolism ; Triple Negative Breast Neoplasms/*metabolism/pathology ; Zinc Finger E-box-Binding Homeobox 1/metabolism ; },
abstract = {By regulating several hallmarks of cancer, BAG3 exerts oncogenic functions in a wide variety of malignant diseases including glioblastoma (GBM) and triple-negative breast cancer (TNBC). Here we performed global proteomic/phosphoproteomic analyses of CRISPR/Cas9-mediated isogenic BAG3 knockouts of the two GBM lines U343 and U251 in comparison to parental controls. Depletion of BAG3 evoked major effects on proteins involved in ciliogenesis/ciliary function and the activity of the related kinases aurora-kinase A and CDK1. Cilia formation was significantly enhanced in BAG3 KO cells, a finding that could be confirmed in BAG3-deficient versus -proficient BT-549 TNBC cells, thus identifying a completely novel function of BAG3 as a negative regulator of ciliogenesis. Furthermore, we demonstrate that enhanced ciliogenesis and reduced expression of SNAI1 and ZEB1, two key transcription factors regulating epithelial to mesenchymal transition (EMT) are correlated to decreased cell migration, both in the GBM and TNBC BAG3 knockout cells. Our data obtained in two different tumor entities identify suppression of EMT and ciliogenesis as putative synergizing mechanisms of BAG3-driven tumor aggressiveness in therapy-resistant cancers.},
}
@article {pmid34179901,
year = {2021},
author = {Zhao, J and Tan, Z and Wang, L and Lei, C and Nie, Z},
title = {A ligation-driven CRISPR-Cas biosensing platform for non-nucleic acid target detections.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {57},
pages = {7051-7054},
doi = {10.1039/d1cc02578c},
pmid = {34179901},
issn = {1364-548X},
mesh = {Adenosine Triphosphate/*analysis/metabolism ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; DNA/chemistry/metabolism ; DNA Ligases/chemistry/metabolism ; NAD/*analysis/metabolism ; Polynucleotide 5'-Hydroxyl-Kinase/metabolism ; Spectrometry, Fluorescence ; },
abstract = {Herein, we describe a CRISPR-Cas12a sensing platform activated by a DNA ligation reaction for the sensitive detection of non-nucleic acid targets, including NAD[+], ATP and polynucleotide kinase (PNK). In this design, the DNA ligation reaction triggered by these biomolecules generates DNA duplexes, which can activate the nuclease activity of Cas12a to produce amplified fluorescence signals. As a result, this work provides an alternative strategy to expand the applicability of the CRISPR-Cas system into the detection of non-nucleic acid biomolecules.},
}
@article {pmid34179152,
year = {2021},
author = {Yao, R and Xu, Y and Wang, L and Wang, D and Ren, L and Ren, C and Li, C and Li, X and Ni, W and He, Y and Hu, R and Guo, T and Li, Y and Li, L and Wang, X and Hu, S},
title = {CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.},
journal = {Frontiers in veterinary science},
volume = {8},
number = {},
pages = {603919},
pmid = {34179152},
issn = {2297-1769},
abstract = {Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 10[3] pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.},
}
@article {pmid34178991,
year = {2021},
author = {Chuang, YF and Wang, PY and Kumar, S and Lama, S and Lin, FL and Liu, GS},
title = {Methods for in vitro CRISPR/CasRx-Mediated RNA Editing.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {667879},
pmid = {34178991},
issn = {2296-634X},
abstract = {Specific changes in the genome have been accomplished by the revolutionary gene-editing tool known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The advent of programmable RNA editing CRISPR/Cas nucleases has made this gene-editing tool safer and more precise. Specifically, CasRx, a family member of the Cas13d family, has shown great therapeutic potential. Here, we describe the in vitro methods of utilizing this powerful RNA editing platform and determine the RNA editing efficiencies for CasRx with different forms of guide RNAs (also known as gRNA or sgRNA).},
}
@article {pmid34178729,
year = {2020},
author = {Farhud, DD and Zarif-Yeganeh, M},
title = {CRISPR Pioneers Win 2020 Nobel Prize for Chemistry.},
journal = {Iranian journal of public health},
volume = {49},
number = {12},
pages = {2235-2239},
pmid = {34178729},
issn = {2251-6093},
abstract = {Over the last few years, the development of genome editing has revolutionized research on the human genome. Recent advances in developing programmable nucleases, such as meganucleases, ZFNs, TALENs and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas, has greatly expedited the progress of gene editing from concept to clinical practice. The CRISPR has advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Eight years after CRISPR application for human genome edition by Emmanuelle Charpentier and Jennifer A. Doudna, the 2020 Nobel Prize in Chemistry has been jointly given to them for development of CRISPR-Cas9 gene editing, allows scientists to precisely cut and edit of DNA.},
}
@article {pmid34178006,
year = {2021},
author = {Kim, YC and Kang, Y and Yang, EY and Cho, MC and Schafleitner, R and Lee, JH and Jang, S},
title = {Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {688980},
pmid = {34178006},
issn = {1664-462X},
abstract = {The emergence of genome-editing technology has allowed manipulation of DNA sequences in genomes to precisely remove or replace specific sequences in organisms resulting in targeted mutations. In plants, genome editing is an attractive method to alter gene functions to generate improved crop varieties. Genome editing is thought to be simple to use and has a lower risk of off-target effects compared to classical mutation breeding. Furthermore, genome-editing technology tools can also be applied directly to crops that contain complex genomes and/or are not easily bred using traditional methods. Currently, highly versatile genome-editing tools for precise and predictable editing of almost any locus in the plant genome make it possible to extend the range of application, including functional genomics research and molecular crop breeding. Vegetables are essential nutrient sources for humans and provide vitamins, minerals, and fiber to diets, thereby contributing to human health. In this review, we provide an overview of the brief history of genome-editing technologies and the components of genome-editing tool boxes, and illustrate basic modes of operation in representative systems. We describe the current and potential practical application of genome editing for the development of improved nutritious vegetables and present several case studies demonstrating the potential of the technology. Finally, we highlight future directions and challenges in applying genome-editing systems to vegetable crops for research and product development.},
}
@article {pmid34177818,
year = {2021},
author = {Wang, X and Lyu, Y and Wang, S and Zheng, Q and Feng, E and Zhu, L and Pan, C and Wang, S and Wang, D and Liu, X and Wang, H},
title = {Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {536357},
pmid = {34177818},
issn = {1664-302X},
abstract = {The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some "scissors plasmids" via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96-100%), rather than the non-replication initiation region (88-92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.},
}
@article {pmid34177802,
year = {2021},
author = {Alhaidan, Y and Christesen, HT and Lundberg, E and Balwi, MAA and Brusgaard, K},
title = {CRISPR/Cas9 ADCY7 Knockout Stimulates the Insulin Secretion Pathway Leading to Excessive Insulin Secretion.},
journal = {Frontiers in endocrinology},
volume = {12},
number = {},
pages = {657873},
pmid = {34177802},
issn = {1664-2392},
mesh = {Adenylyl Cyclases/deficiency/*genetics ; Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Cell Line ; Child, Preschool ; Congenital Hyperinsulinism/*enzymology/genetics/*metabolism ; Gene Knockout Techniques ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Humans ; Insulin/genetics/*metabolism ; Insulin Secretion ; Insulin-Secreting Cells/*metabolism ; Male ; Rats ; Sequence Alignment ; Trans-Activators/genetics/metabolism ; },
abstract = {AIM: Despite the enormous efforts to understand Congenital hyperinsulinism (CHI), up to 50% of the patients are genetically unexplained. We aimed to functionally characterize a novel candidate gene in CHI.<br><br>PATIENT: A 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative.<br><br>METHODS: A trio-based whole-exome sequencing (WES) was performed. Gene knockout in the RIN-m cell line was established by CRISPR/Cas9. Gene expression was performed using real-time PCR.<br><br>RESULTS: Hyperinsulinemic hypoglycemia with diffuse beta-cell involvement was demonstrated in the patient, who was diazoxide-responsive. By WES, compound heterozygous variants were identified in the adenylyl cyclase 7, ADCY7 gene p.(Asp439Glu) and p.(Gly1045Arg). ADCY7 is calcium-sensitive, expressed in beta-cells and converts ATP to cAMP. The variants located in the cytoplasmic domains C1 and C2 in a highly conserved and functional amino acid region. RIN-m[(-/-][Adcy7][)] cells showed a significant increase in insulin secretion reaching 54% at low, and 49% at high glucose concentrations, compared to wild-type. In genetic expression analysis Adcy7 loss of function led to a 34.1-fold to 362.8-fold increase in mRNA levels of the insulin regulator genes Ins1 and Ins2 (p &#x2264; 0.0002), as well as increased glucose uptake and sensing indicated by higher mRNA levels of Scl2a2 and Gck via upregulation of Pdx1, and Foxa2 leading to the activation of the glucose stimulated-insulin secretion (GSIS) pathway.<br><br>CONCLUSION: This study identified a novel candidate gene, ADCY7, to cause CHI via activation of the GSIS pathway.},
}
@article {pmid34176774,
year = {2021},
author = {Bose, SK and Menon, P and Peranteau, WH},
title = {InUtero Gene Therapy: Progress and Challenges.},
journal = {Trends in molecular medicine},
volume = {27},
number = {8},
pages = {728-730},
pmid = {34176774},
issn = {1471-499X},
support = {DP2 HL152427/HL/NHLBI NIH HHS/United States ; R01 DK123049/DK/NIDDK NIH HHS/United States ; R01 HL151352/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Embryonic Development/*genetics ; Female ; Fetus/*metabolism ; Gene Editing ; *Gene Transfer Techniques ; Genetic Therapy/*methods ; Humans ; Nanotechnology ; Pregnancy ; Theranostic Nanomedicine ; Translational Research, Biomedical ; },
abstract = {In utero gene therapy has the potential to treat lethal and morbid perinatal diseases before birth. Small fetal size, a tolerogenic immune system, and dosing efficiency make the fetus a compelling patient. Numerous clinical, social, and institutional factors must be considered to achieve the promise of genetic treatment before birth.},
}
@article {pmid34175380,
year = {2021},
author = {Zhang, H and Zhang, Y and Williams, RO and Smyth, HDC},
title = {Development of PEGylated chitosan/CRISPR-Cas9 dry powders for pulmonary delivery via thin-film freeze-drying.},
journal = {International journal of pharmaceutics},
volume = {605},
number = {},
pages = {120831},
doi = {10.1016/j.ijpharm.2021.120831},
pmid = {34175380},
issn = {1873-3476},
mesh = {Administration, Inhalation ; Aerosols ; CRISPR-Cas Systems ; *Chitosan ; Dry Powder Inhalers ; Particle Size ; Polyethylene Glycols ; Powders ; },
abstract = {Gene therapy and more recently, gene editing is attractive via pulmonary delivery for enhanced regional targeting. However, processing of sensitive therapeutics into dry powders for inhalation can be problematic due to relatively stressful spraying or milling steps. Thin-film freeze-drying (TFFD) has attracted attention with its promising application in the production of DPI formulations possessing respirable particle size range (1-5 µm) particularly for thermally or shear sensitive therapeutics. In this study, gene editing dry powder formulations containing PEGylated chitosan/CRISPR-Cas9 nanocomplexes were prepared by TFFD. To evaluate stability during processing, nanocomplex size, zeta potential and transfection efficiency of reconstituted formulations were evaluated, and six potential DPI formulations were identified and characterized in terms of geometric particle size, powder surface morphology, and crystallinity. It was found that two formulations containing 3% mannitol with or without leucine were identified as suitable for inhalation with a desired aerodynamic performance. The flow rate dependency and inhaler dependency of these two formulations were also evaluated at different flow rates (60 L/min and 45 L/min) and different inhaler devices (RS01 DPI and HandiHaler) using NGI testing. This study demonstrated that TFFD processing of CRISPR-Cas9 polymer nanocomplexes resulted in a suitable dry powder for inhalation.},
}
@article {pmid34175052,
year = {2021},
author = {Kocher, T and Koller, U},
title = {Advances in gene editing strategies for epidermolysis bullosa.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {81-109},
doi = {10.1016/bs.pmbts.2020.12.007},
pmid = {34175052},
issn = {1878-0814},
mesh = {CRISPR-Cas Systems ; *Epidermolysis Bullosa/genetics/therapy ; *Gene Editing ; Genetic Therapy ; Humans ; Skin ; },
abstract = {Epidermolysis bullosa represents a monogenetic disease comprising a variety of heterogeneous mutations in at least 16 genes encoding structural proteins crucial for skin integrity. Due to well-defined mutations but still lacking causal treatment options for the disease, epidermolysis bullosa represents an ideal candidate for gene therapeutic interventions. Recent developments and improvements in the genome editing field have paved the way for the translation of various gene repair strategies into the clinic. With the ability to accurately predict and monitor targeting events within the human genome, the translation might soon be possible. Here, we describe current advancements in the genome editing field for epidermolysis bullosa, along with a discussion of aspects and strategies for precise and personalized gene editing-based medicine, in order to develop efficient and safe ex vivo as well as in vivo genome editing therapies for epidermolysis bullosa patients in the future.},
}
@article {pmid34175051,
year = {2021},
author = {Grazioli, S and Petris, G},
title = {Synthetic genomics for curing genetic diseases.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {477-520},
doi = {10.1016/bs.pmbts.2021.02.002},
pmid = {34175051},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome ; Genomics ; Humans ; Mutation ; },
abstract = {From the beginning of the genome sequencing era, it has become increasingly evident that genetics plays a role in all diseases, of which only a minority are single-gene disorders, the most common target of current gene therapies. However, the majority of people have some kind of health problems resulting from congenital genetic mutations (over 6000 diseases have been associated to genes, https://www.omim.org/statistics/geneMap) and most genetic disorders are rare and only incompletely understood. The vision and techniques applied to the synthesis of genomes may help to address unmet medical needs from a chromosome and genome-scale perspective. In this chapter, we address the potential therapy of genetic diseases from a different outlook, in which we no longer focus on small gene corrections but on higher-order tools for genome manipulation. These will play a crucial role in the next years, as they prelude to a much deeper understanding of the architecture of the human genome and a more accurate modeling of human diseases, offering new therapeutic opportunities.},
}
@article {pmid34175050,
year = {2021},
author = {Ambrosini, C and Garilli, F and Quattrone, A},
title = {Reprogramming translation for gene therapy.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {439-476},
doi = {10.1016/bs.pmbts.2021.01.028},
pmid = {34175050},
issn = {1878-0814},
mesh = {5' Untranslated Regions ; *Gene Editing ; *Genetic Therapy ; Open Reading Frames ; Protein Biosynthesis/genetics ; },
abstract = {Translational control plays a fundamental role in the regulation of gene expression in eukaryotes. Modulating translational efficiency allows the cell to fine-tune the expression of genes, spatially control protein localization, and trigger fast responses to environmental stresses. Translational regulation involves mechanisms acting on multiple steps of the protein synthesis pathway: initiation, elongation, and termination. Many cis-acting elements present in the 5' UTR of transcripts can influence translation at the initiation step. Among them, the Kozak sequence impacts translational efficiency by regulating the recognition of the start codon; upstream open reading frames (uORFs) are associated with inhibition of translation of the downstream protein; internal ribosomal entry sites (IRESs) can promote cap-independent translation. CRISPR-Cas technology is a revolutionary gene-editing tool that has also been applied to the regulation of gene expression. In this chapter, we focus on the genome editing approaches developed to modulate the translational efficiency with the aim to find novel therapeutic approaches, in particular acting on the cis-elements, that regulate the initiation of protein synthesis.},
}
@article {pmid34175048,
year = {2021},
author = {Molinari, E and Sayer, JA},
title = {Gene and epigenetic editing in the treatment of primary ciliopathies.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {353-401},
doi = {10.1016/bs.pmbts.2021.01.027},
pmid = {34175048},
issn = {1878-0814},
support = {213473/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Ciliopathies ; *Gene Editing ; Genetic Therapy ; Humans ; Retina ; },
abstract = {Primary ciliopathies are inherited human disorders that arise from mutations in ciliary genes. They represent a spectrum of severe, incurable phenotypes, differentially involving several organs, including the kidney and the eye. The development of gene-based therapies is opening up new avenues for the treatment of ciliopathies. Particularly attractive is the possibility of correcting in situ the causative genetic mutation, or pathological epigenetic changes, through the use of gene editing tools. Due to their versatility and efficacy, CRISPR/Cas-based systems represent the most promising gene editing toolkit for clinical applications. However, delivery and specificity issues have so far held back the translatability of CRISPR/Cas-based therapies into clinical practice, especially where systemic administration is required. The eye, with its characteristics of high accessibility and compartmentalization, represents an ideal target for in situ gene correction. Indeed, studies for the evaluation of a CRISPR/Cas-based therapy for in vivo gene correction to treat a retinal ciliopathy have reached the clinical stage. Further technological advances may be required for the development of in vivo CRISPR-based treatments for the kidney. We discuss here the possibilities and the challenges associated to the implementation of CRISPR/Cas-based therapies for the treatment of primary ciliopathies with renal and retinal phenotypes.},
}
@article {pmid34175046,
year = {2021},
author = {Kantor, A and McClements, ME and Peddle, CF and Fry, LE and Salman, A and Cehajic-Kapetanovic, J and Xue, K and MacLaren, RE},
title = {CRISPR genome engineering for retinal diseases.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {29-79},
doi = {10.1016/bs.pmbts.2021.01.024},
pmid = {34175046},
issn = {1878-0814},
support = {MC_PC_18059/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Therapy ; Humans ; *Retinal Diseases/genetics/therapy ; },
abstract = {Novel gene therapy treatments for inherited retinal diseases have been at the forefront of translational medicine over the past couple of decades. Since the discovery of CRISPR mechanisms and their potential application for the treatment of inherited human conditions, it seemed inevitable that advances would soon be made using retinal models of disease. The development of CRISPR technology for gene therapy and its increasing potential to selectively target disease-causing nucleotide changes has been rapid. In this chapter, we discuss the currently available CRISPR toolkit and how it has been and can be applied in the future for the treatment of inherited retinal diseases. These blinding conditions have until now had limited opportunity for successful therapeutic intervention, but the discovery of CRISPR has created new hope of achieving such, as we discuss within this chapter.},
}
@article {pmid34175045,
year = {2021},
author = {Pimentel-Vera, LN and Poletto, E and Gonzalez, EA and de Oliveira Poswar, F and Giugliani, R and Baldo, G},
title = {Genome editing in lysosomal disorders.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {289-325},
doi = {10.1016/bs.pmbts.2021.02.001},
pmid = {34175045},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Genome ; Lysosomes ; },
abstract = {Lysosomal disorders are a group of heterogenous diseases caused by mutations in genes that encode for lysosomal proteins. With exception of some cases, these disorders still lack both knowledge of disease pathogenesis and specific therapies. In this sense, genome editing arises as a technique that allows both the creation of specific cell lines, animal models and gene therapy protocols for these disorders. Here we explain the main applications of genome editing for lysosomal diseases, with examples based on the literature. The ability to rewrite the genome will be of extreme importance to study and potentially treat these rare disorders.},
}
@article {pmid34175044,
year = {2021},
author = {Ginn, SL and Christina, S and Alexander, IE},
title = {Genome editing in the human liver: Progress and translational considerations.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {257-288},
doi = {10.1016/bs.pmbts.2021.01.030},
pmid = {34175044},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Genome ; Humans ; Liver ; },
abstract = {Liver-targeted genome editing offers the prospect of life-long therapeutic benefit following a single treatment and is set to rapidly supplant conventional gene addition approaches. Combining progress in liver-targeted gene delivery with genome editing technology, makes this not only feasible but realistically achievable in the near term. However, important challenges remain to be addressed. These include achieving therapeutic levels of editing, particularly in vivo, avoidance of off-target effects on the genome and the potential impact of pre-existing immunity to bacteria-derived nucleases, when used to improve editing rates. In this chapter, we outline the unique features of the liver that make it an attractive target for genome editing, the impact of liver biology on therapeutic efficacy, and disease specific challenges, including whether the approach targets a cell autonomous or non-cell autonomous disease. We also discuss strategies that have been used successfully to achieve genome editing outcomes in the liver and address translational considerations as genome editing technology moves into the clinic.},
}
@article {pmid34175039,
year = {2021},
author = {Greenfield, A},
title = {Making sense of heritable human genome editing: Scientific and ethical considerations.},
journal = {Progress in molecular biology and translational science},
volume = {182},
number = {},
pages = {1-28},
doi = {10.1016/bs.pmbts.2020.12.008},
pmid = {34175039},
issn = {1878-0814},
support = {MC_U142684167/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Human/genetics ; Germ Cells ; Humans ; Reproductive Techniques, Assisted ; },
abstract = {Genome editing, particularly the use of CRISPR-Cas9-based methodologies, is revolutionizing biology through its impacts on research and the translation of these into applications in biomedicine. Somatic genome editing aimed at treating individuals with disease raises some significant ethical issues, but proposed heritable interventions, through the use of genome editing in gametes or embryos, raise a number of distinct social, ethical and political issues. This review will consider some proposed uses of heritable human genome editing (HHGE) and several of the objections to these that have been raised. Making sense of such proposed uses requires viewing HHGE as an assisted reproductive technology (ART) that, like preimplantation genetic testing (PGT) and mitochondrial replacement techniques (MRT), aims to prevent disease transmission during sexual reproduction, rather than acting as a therapy for an existing individual. Applications beyond the paradigm of disease prevention raise even more difficult scientific and ethical questions. Here, I will discuss various themes that are prominent in discussions of the science and ethics of HHGE, including impacts on human dignity and society, the language of HHGE used for public dialogue and the governance of HHGE.},
}
@article {pmid34174954,
year = {2021},
author = {Fu, PF and Cheng, X and Su, BQ and Duan, LF and Wang, CR and Niu, XR and Wang, J and Yang, GY and Chu, BB},
title = {CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo.},
journal = {Veterinary research},
volume = {52},
number = {1},
pages = {95},
pmid = {34174954},
issn = {1297-9716},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Herpesvirus 1, Suid/drug effects/*pathogenicity/*physiology ; Mice ; Mice, Inbred BALB C ; Pseudorabies/virology ; Virulence ; },
abstract = {Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.},
}
@article {pmid34174445,
year = {2022},
author = {Liu, Z and Chen, S and Xie, W and Song, Y and Li, J and Lai, L and Li, Z},
title = {Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {256-267},
pmid = {34174445},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Editing ; Mice ; *Proprotein Convertase 9/genetics ; RNA, Guide/genetics ; Streptococcus ; },
abstract = {Compact CRISPR-Cas9 systems that can be packaged into an adeno-associated virus (AAV) show promise for gene therapy. However, the requirement of protospacer adjacent motifs (PAMs) restricts the target scope. To expand this repertoire, we revisited and optimized a small Cas9 ortholog derived from Streptococcus pasteurianus (SpaCas9) for efficient genome editing in vivo. We found that SpaCas9 enables potent targeting of 5'-NNGYRA-3' PAMs, which are distinct from those recognized by currently used small Cas9s; the Spa-cytosine base editor (CBE) and Spa-adenine base editor (ABE) systems efficiently generated robust C-to-T and A-to-G conversions both in vitro and in vivo. In addition, by exploiting natural variation in the PAM-interacting domain, we engineered three SpaCas9 variants to further expand the targeting scope of compact Cas9 systems. Moreover, mutant mice with efficient disruption of the Tyr gene were successfully generated by microinjection of SpaCas9 mRNA and the corresponding single guide RNA (sgRNA) into zygotes. Notably, all-in-one AAV delivery of SpaCas9 targeting the Pcsk9 gene in adult mouse liver produced efficient genome-editing events and reduced its serum cholesterol. Thus, with distinct PAMs and a small size, SpaCas9 will broaden the CRISPR-Cas9 toolsets for efficient gene modifications and therapeutic applications.},
}
@article {pmid34174442,
year = {2022},
author = {Khajanchi, N and Saha, K},
title = {Controlling CRISPR with small molecule regulation for somatic cell genome editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {17-31},
pmid = {34174442},
issn = {1525-0024},
support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; T32 HG002760/HG/NHGRI NIH HHS/United States ; U01 EY032333/EY/NEI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Endonucleases/metabolism ; *Gene Editing ; Genetic Vectors ; Humans ; },
abstract = {Biomedical research has been revolutionized by the introduction of many CRISPR-Cas systems that induce programmable edits to nearly any gene in the human genome. Nuclease-based CRISPR-Cas editors can produce on-target genomic changes but can also generate unwanted genotoxicity and adverse events, in part by cleaving non-targeted sites in the genome. Additional translational challenges for in vivo somatic cell editing include limited packaging capacity of viral vectors and host immune responses. Altogether, these challenges motivate recent efforts to control the expression and activity of different Cas systems in vivo. Current strategies utilize small molecules, light, magnetism, and temperature to conditionally control Cas systems through various activation, inhibition, or degradation mechanisms. This review focuses on small molecules that can be incorporated as regulatory switches to control Cas genome editors. Additional development of CRISPR-Cas-based therapeutic approaches with small molecule regulation have high potential to increase editing efficiency with less adverse effects for somatic cell genome editing strategies in vivo.},
}
@article {pmid34174439,
year = {2022},
author = {AlJanahi, AA and Lazzarotto, CR and Chen, S and Shin, TH and Cordes, S and Fan, X and Jabara, I and Zhou, Y and Young, DJ and Lee, BC and Yu, KR and Li, Y and Toms, B and Tunc, I and Hong, SG and Truitt, LL and Klermund, J and Andrieux, G and Kim, MY and Cathomen, T and Gill, S and Tsai, SQ and Dunbar, CE},
title = {Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {209-222},
pmid = {34174439},
issn = {1525-0024},
support = {U01 AI157189/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Macaca mulatta/genetics ; RNA, Guide/genetics ; },
abstract = {The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.},
}
@article {pmid34174352,
year = {2021},
author = {Wang, Y and Shahi, PK and Wang, X and Xie, R and Zhao, Y and Wu, M and Roge, S and Pattnaik, BR and Gong, S},
title = {In vivo targeted delivery of nucleic acids and CRISPR genome editors enabled by GSH-responsive silica nanoparticles.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {336},
number = {},
pages = {296-309},
pmid = {34174352},
issn = {1873-4995},
support = {R01 HL129785/HL/NHLBI NIH HHS/United States ; UG3 NS111688/NS/NINDS NIH HHS/United States ; R24 EY032434/EY/NEI NIH HHS/United States ; R01 EY024995/EY/NEI NIH HHS/United States ; R01 HL143469/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Glutathione ; Mice ; *Nanoparticles ; RNA, Messenger ; *Silicon Dioxide ; },
abstract = {The rapid development of gene therapy and genome editing techniques brings up an urgent need to develop safe and efficient nanoplatforms for nucleic acids and CRISPR genome editors. Herein we report a stimulus-responsive silica nanoparticle (SNP) capable of encapsulating biomacromolecules in their active forms with a high loading content and loading efficiency as well as a well-controlled nanoparticle size (~50 nm). A disulfide crosslinker was integrated into the silica network, endowing SNP with glutathione (GSH)-responsive cargo release capability when internalized by target cells. An imidazole-containing component was incorporated into the SNP to enhance the endosomal escape capability. The SNP can deliver various cargos, including nucleic acids (e.g., DNA and mRNA) and CRISPR genome editors (e.g., Cas9/sgRNA ribonucleoprotein (RNP), and RNP with donor DNA) with excellent efficiency and biocompatibility. The SNP surface can be PEGylated and functionalized with different targeting ligands. In vivo studies showed that subretinally injected SNP conjugated with all-trans-retinoic acid (ATRA) and intravenously injected SNP conjugated with GalNAc can effectively deliver mRNA and RNP to murine retinal pigment epithelium (RPE) cells and liver cells, respectively, leading to efficient genome editing. Overall, the SNP is a promising nanoplatform for various applications including gene therapy and genome editing.},
}
@article {pmid34174210,
year = {2021},
author = {Santiago-Frangos, A and Buyukyoruk, M and Wiegand, T and Krishna, P and Wiedenheft, B},
title = {Distribution and phasing of sequence motifs that facilitate CRISPR adaptation.},
journal = {Current biology : CB},
volume = {31},
number = {16},
pages = {3515-3524.e6},
pmid = {34174210},
issn = {1879-0445},
support = {P20 GM103474/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Endonucleases/genetics/metabolism ; },
abstract = {CRISPR-associated proteins (Cas1 and Cas2) integrate foreign DNA at the "leader" end of CRISPR loci. Several CRISPR leader sequences are reported to contain a binding site for a DNA-bending protein called integration host factor (IHF). IHF-induced DNA bending kinks the leader of type I-E CRISPRs, recruiting an upstream sequence motif that helps dock Cas1-2 onto the first repeat of the CRISPR locus. To determine the prevalence of IHF-directed CRISPR adaptation, we analyzed 15,274 bacterial and archaeal CRISPR leaders. These experiments reveal multiple IHF binding sites and diverse upstream sequence motifs in a subset of the I-C, I-E, I-F, and II-C CRISPR leaders. We identify subtype-specific motifs and show that the phase of these motifs is critical for CRISPR adaptation. Collectively, this work clarifies the prevalence and mechanism(s) of IHF-dependent CRISPR adaptation and suggests that leader sequences and adaptation proteins may coevolve under the selective pressures of foreign genetic elements like plasmids or phages.},
}
@article {pmid34172748,
year = {2021},
author = {Taxiarchi, C and Beaghton, A and Don, NI and Kyrou, K and Gribble, M and Shittu, D and Collins, SP and Beisel, CL and Galizi, R and Crisanti, A},
title = {A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3977},
pmid = {34172748},
issn = {2041-1723},
mesh = {Animals ; Animals, Genetically Modified ; Anopheles/embryology/*genetics ; Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors ; CRISPR-Cas Systems/*genetics ; Female ; Fertility/genetics ; Gene Drive Technology/*methods ; Genetic Fitness ; Genetics, Population ; Listeria monocytogenes ; Male ; },
abstract = {CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.},
}
@article {pmid34171821,
year = {2021},
author = {Chen, FE and Lee, PW and Trick, AY and Park, JS and Chen, L and Shah, K and Mostafa, H and Carroll, KC and Hsieh, K and Wang, TH},
title = {Point-of-care CRISPR-Cas-assisted SARS-CoV-2 detection in an automated and portable droplet magnetofluidic device.},
journal = {Biosensors &amp; bioelectronics},
volume = {190},
number = {},
pages = {113390},
pmid = {34171821},
issn = {1873-4235},
support = {R01 AI138978/AI/NIAID NIH HHS/United States ; R61 AI154628/AI/NIAID NIH HHS/United States ; },
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Point-of-Care Systems ; RNA, Viral ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but their lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR - a single-step CRISPR-Cas-assisted assay that incoporates sample preparation with minimal manual operation via facile magnetic-based nucleic acid concentration and transport. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/μL SARS-CoV-2 RNA from a sample volume of 100 μL in < 30 min. When evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (Cq values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (Cq < 25) in 20 min.},
}
@article {pmid34171554,
year = {2021},
author = {Schibeci Natoli Scialli, N and Colitti, B and Bertolotti, L and Pezzoni, G and Martignani, E and Melega, M and Brocchi, E and Rosati, S},
title = {Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system.},
journal = {Veterinary immunology and immunopathology},
volume = {238},
number = {},
pages = {110286},
doi = {10.1016/j.vetimm.2021.110286},
pmid = {34171554},
issn = {1873-2534},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Cell Line ; Cloning, Molecular ; DNA/genetics ; *Gene Editing ; Gene Expression Regulation ; Green Fluorescent Proteins/genetics/*metabolism ; Hybridomas ; Immunoglobulin Light Chains/*chemistry/genetics ; Mice ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; },
abstract = {The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.},
}
@article {pmid34171270,
year = {2021},
author = {The Lancet Haematology, },
title = {The evolving promise and potential of gene therapy.},
journal = {The Lancet. Haematology},
volume = {8},
number = {7},
pages = {e467},
doi = {10.1016/S2352-3026(21)00172-1},
pmid = {34171270},
issn = {2352-3026},
mesh = {CRISPR-Cas Systems ; *Genetic Therapy ; Genetic Vectors ; Hematologic Diseases/genetics/*therapy ; Humans ; },
}
@article {pmid34169944,
year = {2021},
author = {Cao, Y and Wu, J and Pang, B and Zhang, H and Le, XC},
title = {CRISPR/Cas12a-mediated gold nanoparticle aggregation for colorimetric detection of SARS-CoV-2.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {56},
pages = {6871-6874},
doi = {10.1039/d1cc02546e},
pmid = {34169944},
issn = {1364-548X},
mesh = {COVID-19/*diagnosis/virology ; *CRISPR-Cas Systems ; Colorimetry ; Cross-Linking Reagents ; Genes, Viral/*genetics ; Gold/*chemistry ; Metal Nanoparticles/*chemistry ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/*methods ; RNA/chemistry ; SARS-CoV-2/*genetics ; },
abstract = {The trans-cleavage activity of the target-activated CRISPR/Cas12a liberated an RNA crosslinker from a molecular transducer, which facilitated the assembly of gold nanoparticles. Integration of the molecular transducer with isothermal amplification and CRISPR/Cas12a resulted in visual detection of the N gene and E gene of SARS-CoV-2 in 45 min.},
}
@article {pmid34168346,
year = {2021},
author = {van Kampen, SJ and van Rooij, E},
title = {CRISPR base editing lowers cholesterol in monkeys.},
journal = {Nature biotechnology},
volume = {39},
number = {8},
pages = {920-921},
pmid = {34168346},
issn = {1546-1696},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cardiovascular Diseases/genetics ; *Cholesterol/blood/genetics ; Gene Editing/*methods ; Macaca fascicularis ; Proprotein Convertase 9/genetics ; RNA, Guide/administration &amp; dosage/genetics ; },
}
@article {pmid34168320,
year = {2021},
author = {Pan, C and Wu, X and Markel, K and Malzahn, AA and Kundagrami, N and Sretenovic, S and Zhang, Y and Cheng, Y and Shih, PM and Qi, Y},
title = {CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.},
journal = {Nature plants},
volume = {7},
number = {7},
pages = {942-953},
pmid = {34168320},
issn = {2055-0278},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Expression Regulation, Plant ; Genetic Engineering/*methods ; Genetic Variation ; Genotype ; Lycopersicon esculentum/*genetics ; Oryza/*genetics ; Plant Breeding/*methods ; Transcriptional Activation/*genetics ; },
abstract = {RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR-Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9). The CRISPR-Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA-gR2.0 (single guide RNA 2.0) expression system enabling CRISPR-Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR-Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR-Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR-dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR-Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9-NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.},
}
@article {pmid34165913,
year = {2021},
author = {Calderón, M and Hedtrich, S},
title = {The Delivery Challenge of Genome Editing in Human Epithelia.},
journal = {Advanced healthcare materials},
volume = {10},
number = {19},
pages = {e2100847},
doi = {10.1002/adhm.202100847},
pmid = {34165913},
issn = {2192-2659},
support = {CIHR PJT-166035//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; Humans ; },
abstract = {Despite exciting advances in gene editing, their clinical translation is still hampered by the lack of delivery systems that can encapsulate and deliver gene editing tools like CRISPR-Cas9 or prime editors to the target side. This is particularly challenging in human epithelia, such as the skin and the lung; the latter of which being a mucosal surface that is covered by a mucus layer. In this perspective, the design and biological assessment of delivery systems for gene editing tools like CRISPR in skin and mucosal surfaces are discussed. The current state-of-the-art, current knowledge, and translational gaps, and guide toward improved translation are highlighted.},
}
@article {pmid34165772,
year = {2021},
author = {Li, H and Wu, S and Ma, X and Li, X and Cheng, T and Chen, Z and Wu, J and Lv, L and Li, L and Xu, L and Wang, W and Hu, Y and Jiang, H and Yin, Y and Qiu, Z and Hu, X},
title = {Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype.},
journal = {Neuroscience bulletin},
volume = {37},
number = {9},
pages = {1271-1288},
pmid = {34165772},
issn = {1995-8218},
mesh = {Animals ; Brain ; *CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; Haplorhini ; Phenotype ; Protein Kinases/genetics ; },
abstract = {Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.},
}
@article {pmid34165721,
year = {2021},
author = {Vitiello, M and Poliseno, L},
title = {CRISPR/Cas Technologies Applied to Pseudogenes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2324},
number = {},
pages = {265-284},
pmid = {34165721},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; DNA/metabolism ; Forecasting ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Targeting/methods ; Humans ; Molecular Diagnostic Techniques ; Mutation ; Protein Domains ; *Pseudogenes ; RNA/genetics ; RNA, Messenger/antagonists &amp; inhibitors/isolation &amp; purification ; Substrate Specificity ; Transcription, Genetic ; },
abstract = {Pseudogenes have been considered as nonfunctional copies of their parental genes for a long time. Indeed, they have been often defined "junk DNA" or "transcriptional noise." However, with the identification of their involvement in several biological processes, the necessity of their study is inevitably growing up. The manipulation of pseudogene expression is complicated by their high homology with parental genes and by the fact that most of them work at the transcriptional level as noncoding RNAs. With the advent of CRISPR/Cas technology, these problems can be overcome. Particularly, as we describe in this chapter, it is possible: To perform genome editing, obtaining the complete elimination of the pseudogene genomic sequence (knock-out), preventing pseudogene transcription, introducing specific mutations in the pseudogene sequence, or introducing a specific sequence (knock-in). To positively or negatively manipulate pseudogene transcription. To target pseudogene RNA and negatively regulate its expression. To edit pseudogene DNA and RNA and alter a specific sequence. Moreover, CRISPR/Cas technology can be used as an RNA Binding Protein system for molecular biology techniques (such as RNA immunoprecipitation and pull-down), as well as for transcript tracking and live imaging.},
}
@article {pmid34165717,
year = {2021},
author = {Schoeftner, S and Scarola, M and Benetti, R},
title = {Chromatin Regulation at Parental Gene Promoters by Pseudogene Sense lncRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2324},
number = {},
pages = {203-217},
pmid = {34165717},
issn = {1940-6029},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Capsid Proteins/metabolism ; Chromatin/genetics/*metabolism/ultrastructure ; Chromatin Immunoprecipitation ; Embryonic Stem Cells ; Epigenesis, Genetic/*genetics ; Gene Expression Regulation ; Humans ; Levivirus/genetics ; Methyltransferases/metabolism ; Mice ; Octamer Transcription Factor-3/genetics ; Promoter Regions, Genetic/*genetics ; Pseudogenes/*genetics ; RNA, Long Noncoding/*genetics ; RNA, Messenger/metabolism ; RNA, Viral/metabolism ; RNA-Binding Protein FUS/metabolism ; Repressor Proteins/metabolism ; },
abstract = {There is accumulating evidence that pseudogenes can produce functionally relevant lncRNAs in a tightly controlled manner. This class of transcripts has been demonstrated to play an important role in development and disease, by controlling parental gene expression. Classically, pseudogene derived lncRNAs compete with parental transcripts for miRNAs or factors that control parental mRNA metabolisms. Recently, pseudogene lncRNAs were demonstrated to take over the control of classic chromatin modifying enzymes and alter parental gene promoter activity or genome wide gene expression. Here, we discuss a new mechanism of parental gene expression controlled by the mOct4P4 lncRNA, a sense transcript derived from the murine Oct4 pseudogene 4. mOct4P4 lncRNA specifically interacts with the RNA binding protein FUS and the Histone Methyltransferase SUV39H1 to target heterochromatin formation at the parental Oct4 promoter in trans. In addition, we will address key issues for the functional dissection of epigenetic control of parental gene promoters by pseudogene lncRNAs.},
}
@article {pmid34164771,
year = {2021},
author = {Song, M and Koo, T},
title = {Recent advances in CRISPR technologies for genome editing.},
journal = {Archives of pharmacal research},
volume = {44},
number = {6},
pages = {537-552},
pmid = {34164771},
issn = {1976-3786},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/*genetics ; Gene Editing/*methods/trends ; Humans ; Protein Engineering/*methods/trends ; },
abstract = {The discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, and its development into a set of powerful tools for manipulating the genome, has revolutionized genome editing. Precise, targeted CRISPR/Cas-based genome editing has become the most widely used platform in organisms ranging from plants to animals. The CRISPR/Cas system has been extensively modified to increase its efficiency and fidelity. In addition, the fusion of various protein motifs to Cas effector proteins has facilitated diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation. The CRISPR/Cas system is undergoing continuous development to overcome current limitations, including off-target effects, narrow targeting scope, and issues associated with the delivery of CRISPR components for genome engineering and therapeutic approaches. Here, we review recent progress in a diverse array of CRISPR/Cas-based tools. We also describe limitations and concerns related to the use of CRISPR/Cas technologies.},
}
@article {pmid34163728,
year = {2021},
author = {Feng, W and Newbigging, AM and Tao, J and Cao, Y and Peng, H and Le, C and Wu, J and Pang, B and Li, J and Tyrrell, DL and Zhang, H and Le, XC},
title = {CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules.},
journal = {Chemical science},
volume = {12},
number = {13},
pages = {4683-4698},
pmid = {34163728},
issn = {2041-6520},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.},
}
@article {pmid34163450,
year = {2021},
author = {Tenea, GN and Hurtado, P},
title = {Next-Generation Sequencing for Whole-Genome Characterization of Weissella cibaria UTNGt21O Strain Originated From Wild Solanum quitoense Lam. Fruits: An Atlas of Metabolites With Biotechnological Significance.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {675002},
pmid = {34163450},
issn = {1664-302X},
abstract = {The whole genome of Weissella cibaria strain UTNGt21O isolated from wild fruits of Solanum quitoense (naranjilla) shrub was sequenced and annotated. The similarity proportions based on the genus level, as a result of the best hits for the entire contig, were 54.84% with Weissella, 6.45% with Leuconostoc, 3.23% with Lactococcus, and 35.48% no match. The closest genome was W. cibaria SP7 (GCF_004521965.1) with 86.21% average nucleotide identity (ANI) and 3.2% alignment coverage. The genome contains 1,867 protein-coding genes, among which 1,620 were assigned with the EggNOG database. On the basis of the results, 438 proteins were classified with unknown function from which 247 new hypothetical proteins have no match in the nucleotide Basic Local Alignment Search Tool (BLASTN) database. It also contains 78 tRNAs, six copies of 5S rRNA, one copy of 16S rRNA, one copy of 23S rRNA, and one copy of tmRNA. The W. cibaria UTNGt21O strain harbors several genes responsible for carbohydrate metabolism, cellular process, general stress responses, cofactors, and vitamins, conferring probiotic features. A pangenome analysis indicated the presence of various strain-specific genes encoded for proteins responsible for the defense mechanisms as well as gene encoded for enzymes with biotechnological value, such as penicillin acylase and folates; thus, W. cibaria exhibited high genetic diversity. The genome characterization indicated the presence of a putative CRISPR-Cas array and five prophage regions and the absence of acquired antibiotic resistance genes, virulence, and pathogenic factors; thus, UTNGt21O might be considered a safe strain. Besides, the interaction between the peptide extracts from UTNGt21O and Staphylococcus aureus results in cell death caused by the target cell integrity loss and the release of aromatic molecules from the cytoplasm. The results indicated that W. cibaria UTNGt21O can be considered a beneficial strain to be further exploited for developing novel antimicrobials and probiotic products with improved technological characteristics.},
}
@article {pmid34163064,
year = {2021},
author = {Chien, JC and Badr, CE and Lai, CP},
title = {Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo.},
journal = {Nature protocols},
volume = {16},
number = {8},
pages = {3933-3953},
pmid = {34163064},
issn = {1750-2799},
support = {K22 CA197053/CA/NCI NIH HHS/United States ; R01 NS113822/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *DNA Breaks, Double-Stranded ; DNA Repair/*physiology ; Humans ; Luminescent Measurements/*methods ; Mice ; Mice, Transgenic ; Neoplasms, Experimental ; },
abstract = {The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing. We also describe complementation with orthogonal DSB repair assays and omics analyses to validate the reporter readouts. The bioluminescent repair reporter system provides longitudinal and rapid readout (~seconds per sample) to accurately and efficiently measure the efficacy of genome-editing tools and small-molecule modulators on DSB repair. This protocol takes ~2-4 weeks to establish, and as little as 2 h to complete the assay. The entire bioluminescent repair reporter procedure can be performed by one person with standard molecular biology expertise and equipment. However, orthogonal DNA repair assays would require a specialized facility that performs Sanger sequencing or next-generation sequencing.},
}
@article {pmid34163045,
year = {2021},
author = {Wang, C and Cao, Y and Yang, C and Bernards, R and Qin, W},
title = {Exploring liver cancer biology through functional genetic screens.},
journal = {Nature reviews. Gastroenterology &amp; hepatology},
volume = {18},
number = {10},
pages = {690-704},
pmid = {34163045},
issn = {1759-5053},
mesh = {CRISPR-Cas Systems ; Carcinoma, Hepatocellular/drug therapy/*genetics ; Gain of Function Mutation ; Genetic Testing/*methods ; High-Throughput Screening Assays ; Humans ; Immune Checkpoint Inhibitors/therapeutic use ; Liver Neoplasms/drug therapy/*genetics ; Loss of Function Mutation ; Molecular Targeted Therapy ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-met/antagonists &amp; inhibitors ; Receptor, Fibroblast Growth Factor, Type 4/antagonists &amp; inhibitors ; },
abstract = {As the fourth leading cause of cancer-related death in the world, liver cancer poses a major threat to human health. Although a growing number of therapies have been approved for the treatment of hepatocellular carcinoma in the past few years, most of them only provide a limited survival benefit. Therefore, an urgent need exists to identify novel targetable vulnerabilities and powerful drug combinations for the treatment of liver cancer. The advent of functional genetic screening has contributed to the advancement of liver cancer biology, uncovering many novel genes involved in tumorigenesis and cancer progression in a high-throughput manner. In addition, this unbiased screening platform also provides an efficient tool for the exploration of the mechanisms involved in therapy resistance as well as identifying potential targets for therapy. In this Review, we describe how functional screens can help to deepen our understanding of liver cancer and guide the development of new therapeutic strategies.},
}
@article {pmid34162956,
year = {2021},
author = {Bertin, B and Renaud, Y and Jagla, T and Lavergne, G and Dondi, C and Da Ponte, JP and Junion, G and Jagla, K},
title = {Gelsolin and dCryAB act downstream of muscle identity genes and contribute to preventing muscle splitting and branching in Drosophila.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {13197},
pmid = {34162956},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cell Fusion ; Cell Shape ; Disease Models, Animal ; Drosophila Proteins/genetics/*physiology ; Drosophila melanogaster/embryology/*genetics/metabolism ; Embryo, Nonmammalian ; Gelsolin/genetics/*physiology ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Expression Regulation, Developmental ; *Genes, Insect ; Larva ; Loss of Function Mutation ; Multigene Family ; Muscle Cells/metabolism ; Muscles/metabolism/*ultrastructure ; Muscular Dystrophy, Animal/*genetics/pathology ; Myoblasts/metabolism/ultrastructure ; RNA, Messenger/metabolism ; Transcription Factors/physiology ; Transcription, Genetic ; alpha-Crystallin B Chain/genetics/*physiology ; },
abstract = {A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.},
}
@article {pmid34162938,
year = {2021},
author = {Li, P and Walsh, JR and Lopez, K and Isidan, A and Zhang, W and Chen, AM and Goggins, WC and Higgins, NG and Liu, J and Brutkiewicz, RR and Smith, LJ and Hara, H and Cooper, DKC and Ekser, B},
title = {Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {13131},
pmid = {34162938},
issn = {2045-2322},
support = {R21 AI164002/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*immunology ; Antibodies, Heterophile/immunology ; Antigen-Antibody Reactions ; Antigens, Heterophile/genetics/*immunology ; CRISPR-Cas Systems ; Cell Degranulation ; Cell Line, Transformed ; Cytokines/pharmacology ; Endothelial Cells/drug effects/*immunology ; Galactosyltransferases/genetics/immunology ; Gene Knockout Techniques ; Graft Rejection/immunology/prevention &amp; control ; Histocompatibility Antigens Class I/genetics/immunology ; Humans ; Killer Cells, Natural/immunology ; Liver/cytology ; Lymphocyte Activation ; Mixed Function Oxygenases/genetics/immunology ; N-Acetylgalactosaminyltransferases/genetics/immunology ; Swine/*immunology ; Transplantation, Heterologous/*methods ; beta 2-Microglobulin/genetics/immunology ; },
abstract = {Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.},
}
@article {pmid34162911,
year = {2021},
author = {Jaiswal, AK and Truong, H and Tran, TM and Lin, TL and Casero, D and Alberti, MO and Rao, DS},
title = {Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {13158},
pmid = {34162911},
issn = {2045-2322},
support = {R03 CA251845/CA/NCI NIH HHS/United States ; T32 CA009056/BC/NCI NIH HHS/United States ; R21 AI132869/AI/NIAID NIH HHS/United States ; T32CA009120/BC/NCI NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R01CA166450/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Leukemic ; Genes, Reporter ; Genetic Predisposition to Disease ; Genetic Testing ; Golgi Matrix Proteins/genetics/*physiology ; Homeostasis ; Humans ; Mice ; Mice, Inbred C57BL ; Myeloid-Lymphoid Leukemia Protein/genetics/*physiology ; Neoplasm Proteins/genetics/*physiology ; Oncogene Proteins, Fusion/genetics/*physiology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*genetics ; RNA Processing, Post-Transcriptional ; RNA, Neoplasm/genetics/metabolism ; Transgenes ; Translocation, Genetic ; Tumor Stem Cell Assay ; Vesicular Transport Proteins/genetics/*physiology ; },
abstract = {Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.},
}
@article {pmid34162850,
year = {2021},
author = {Zhang, L and Zuris, JA and Viswanathan, R and Edelstein, JN and Turk, R and Thommandru, B and Rube, HT and Glenn, SE and Collingwood, MA and Bode, NM and Beaudoin, SF and Lele, S and Scott, SN and Wasko, KM and Sexton, S and Borges, CM and Schubert, MS and Kurgan, GL and McNeill, MS and Fernandez, CA and Myer, VE and Morgan, RA and Behlke, MA and Vakulskas, CA},
title = {AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3908},
pmid = {34162850},
issn = {2041-1723},
mesh = {Acidaminococcus/*enzymology/genetics ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Endonucleases/genetics/*metabolism ; Gene Editing/*methods ; HEK293 Cells ; Hematopoietic Stem Cells/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Jurkat Cells ; Killer Cells, Natural/metabolism ; Reproducibility of Results ; T-Lymphocytes/metabolism ; },
abstract = {Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.},
}
@article {pmid34162685,
year = {2021},
author = {Yi, P and Morrow, N},
title = {Applying CRISPR Screen in Diabetes Research.},
journal = {Diabetes},
volume = {70},
number = {9},
pages = {1962-1969},
pmid = {34162685},
issn = {1939-327X},
support = {P30 DK036836/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Diabetes Mellitus/*genetics/metabolism ; Gene Editing ; Humans ; *Research ; },
abstract = {The CRISPR/Cas9 genome editing system has been one of the greatest scientific discoveries in the last decade. The highly efficient and precise editing ability of this technology is of great therapeutic value and benefits the basic sciences as an advantageous research tool. In recent years, forward genetic screens using CRISPR technology have been widely adopted, with genome-wide or pathway-focused screens leading to important and novel discoveries. CRISPR screens have been used primarily in cancer biology, virology, and basic cell biology, but they have rarely been applied to diabetes research. A potential reason for this is that diabetes-related research can be more complicated, often involving cross talk between multiple organs or cell types. Nevertheless, many questions can still be reduced to the study of a single cell type if assays are carefully designed. Here we review the application of CRISPR screen technology and provide perspective on how it can be used in diabetes research.},
}
@article {pmid34160888,
year = {2021},
author = {de Vries, L and Brouckaert, M and Chanoca, A and Kim, H and Regner, MR and Timokhin, VI and Sun, Y and De Meester, B and Van Doorsselaere, J and Goeminne, G and Chiang, VL and Wang, JP and Ralph, J and Morreel, K and Vanholme, R and Boerjan, W},
title = {CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba.},
journal = {Plant biotechnology journal},
volume = {19},
number = {11},
pages = {2221-2234},
pmid = {34160888},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Carboxylesterase ; Gene Expression Regulation, Plant ; Lignin/metabolism ; Plants, Genetically Modified/genetics/metabolism ; *Populus/genetics/metabolism ; },
abstract = {Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery.},
}
@article {pmid34160810,
year = {2021},
author = {Dangelmaier, EA and Lal, A},
title = {Experimental Validation of the Noncoding Potential for lncRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2348},
number = {},
pages = {221-230},
pmid = {34160810},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Computational Biology/methods ; *Gene Expression Regulation ; Humans ; Molecular Sequence Annotation ; Open Reading Frames ; Polyribosomes/metabolism ; *Protein Biosynthesis ; RNA, Long Noncoding/*genetics ; },
abstract = {In recent years, long noncoding RNAs (lncRNAs) have been increasingly recognized as critical regulators of a broad spectrum of cellular processes. Recent advancements in proteomic technologies have uncovered that an abundance of noncoding genes, including lncRNAs, have been misannotated and in reality encode proteins. This revelation underscores the need to accurately determine the coding potential of lncRNAs prior to assessment of their functional mechanisms. Here, we detail numerous experimental techniques useful in the determination of lncRNA coding potential. Several of these methods are doubly useful in that they may also be employed in studying the function of a lncRNA, be it via an RNA, protein, or both.},
}
@article {pmid34160809,
year = {2021},
author = {Vitiello, M and Poliseno, L and Pandolfi, PP},
title = {In Vivo Silencing/Overexpression of lncRNAs by CRISPR/Cas System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2348},
number = {},
pages = {205-220},
pmid = {34160809},
issn = {1940-6029},
mesh = {Adult Stem Cells/cytology/metabolism ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression ; *Gene Silencing ; Gene Targeting ; Mice ; Mice, Transgenic ; Microinjections ; RNA, Long Noncoding/*genetics ; },
abstract = {Long noncoding RNAs (lncRNAs) are implicated in several biological processes and it has been observed that their expression is altered in several diseases. The generation of animal models where selective silencing or overexpression of lncRNAs can be attained is crucial for their biological characterization, since it offers the opportunity to analyze their function at the tissue specific or organismal level. CRISPR/Cas technology is a newly developed tool that allows to easily manipulate the mouse genome, in turn allowing to discover lncRNAs functions in an in vivo context. Here, we provide an overview of how CRISPR/Cas technology can be used to generate transgenic mouse models in which lncRNAs can be studied.},
}
@article {pmid34160808,
year = {2021},
author = {Morelli, E and Gulla', A and Amodio, N and Taiana, E and Neri, A and Fulciniti, M and Munshi, NC},
title = {CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa) to Explore the Oncogenic lncRNA Network.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2348},
number = {},
pages = {189-204},
pmid = {34160808},
issn = {1940-6029},
support = {P01 CA155258/CA/NCI NIH HHS/United States ; P50 CA100707/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Genetic Vectors/genetics ; Humans ; Multiple Myeloma/genetics ; Mutation ; Oncogenes/*genetics ; RNA, Guide ; RNA, Long Noncoding/*genetics ; Transduction, Genetic ; },
abstract = {The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-coding genes. These molecules can play a pivotal role in the development and progression of human disease, including cancer, and are susceptible to therapeutic intervention. Evidence of biologic function, however, is still missing for the vast majority of them. Both loss-of-function (LOF) and gain-of-function (GOF) studies are therefore necessary to advance our understanding of lncRNA networks and programs driving tumorigenesis. Here, we describe a protocol to perform lncRNA's LOF or GOF studies in multiple myeloma (MM) cells, using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) technologies, respectively. These approaches have many advantages, including applicability to large-scale genetic screens in mammalian cells and possible reversibility of modulating effects; moreover, CRISPRa offers the unique opportunity to enhance lncRNA expression at the site of transcription, with relevant biologic implications.},
}
@article {pmid34160807,
year = {2021},
author = {Rosenlund, IA and Calin, GA and Dragomir, MP and Knutsen, E},
title = {CRISPR/Cas9 to Silence Long Non-Coding RNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2348},
number = {},
pages = {175-187},
pmid = {34160807},
issn = {1940-6029},
mesh = {Binding Sites ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Electroporation ; *Gene Editing ; Gene Knockdown Techniques ; *Gene Silencing ; Gene Targeting ; Polymerase Chain Reaction ; RNA Interference ; RNA, Guide/genetics ; RNA, Long Noncoding/*genetics ; },
abstract = {Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.},
}
@article {pmid34160096,
year = {2021},
author = {Zhang, M and Wei, H and Liu, J and Bian, Y and Ma, Q and Mao, G and Wang, H and Wu, A and Zhang, J and Chen, P and Ma, L and Fu, X and Yu, S},
title = {Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.).},
journal = {The Plant journal : for cell and molecular biology},
volume = {107},
number = {4},
pages = {1198-1212},
doi = {10.1111/tpj.15378},
pmid = {34160096},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; Chromosomes, Plant ; Flowers/genetics ; Gene Expression Regulation, Plant ; Gossypium/genetics/*physiology ; Loss of Function Mutation ; Mucoproteins/genetics/metabolism ; Plant Infertility/*genetics/physiology ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Pollen/genetics/physiology ; Tetraploidy ; },
abstract = {Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.},
}
@article {pmid34160014,
year = {2021},
author = {Wall, JM and Basu, A and Zunica, ERM and Dubuisson, OS and Pergola, K and Broussard, JP and Kirwan, JP and Axelrod, CL and Johnson, AE},
title = {CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases.},
journal = {Disease models &amp; mechanisms},
volume = {14},
number = {7},
pages = {},
pmid = {34160014},
issn = {1754-8411},
support = {K99 NS100988/NS/NINDS NIH HHS/United States ; R00 NS100988/NS/NINDS NIH HHS/United States ; R35 GM138116/GM/NIGMS NIH HHS/United States ; U54 GM104940/GM/NIGMS NIH HHS/United States ; },
mesh = {*Amyotrophic Lateral Sclerosis/pathology ; Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics/metabolism ; Disease Models, Animal ; *Drosophila ; Female ; Humans ; Male ; Mutation/genetics ; Valosin Containing Protein/genetics/metabolism ; },
abstract = {Mutations in Valosin Containing Protein (VCP) are associated with several degenerative diseases, including multisystem proteinopathy (MSP-1) and amyotrophic lateral sclerosis. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. A deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis. Using CRISPR/Cas9, we have generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. Our models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. We also made some novel and unexpected findings, including nuclear morphology defects and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models generated in this study will be useful for studying the etiology of individual VCP patient mutations and testing potential genetic and/or pharmacological therapies.},
}
@article {pmid34158624,
year = {2021},
author = {Wu, J and Wu, C and Xing, F and Cao, L and Zeng, W and Guo, L and Li, P and Zhong, Y and Jiang, H and Luo, M and Shi, G and Bu, L and Ji, Y and Hou, P and Peng, H and Huang, J and Li, C and Guo, D},
title = {Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells.},
journal = {Cell research},
volume = {31},
number = {9},
pages = {998-1010},
pmid = {34158624},
issn = {1748-7838},
mesh = {Animals ; *Antiviral Agents ; Embryonic Stem Cells ; Mice ; RNA, Viral ; *RNA-Directed DNA Polymerase ; Ribonuclease H ; },
abstract = {Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.},
}
@article {pmid34158505,
year = {2021},
author = {Zhang, T and Mudgett, M and Rambabu, R and Abramson, B and Dai, X and Michael, TP and Zhao, Y},
title = {Selective inheritance of target genes from only one parent of sexually reproduced F1 progeny in Arabidopsis.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3854},
pmid = {34158505},
issn = {2041-1723},
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/*genetics ; *CRISPR-Cas Systems ; Crosses, Genetic ; Cryptochromes/*genetics ; DNA, Plant/genetics ; Gene Drive Technology/*methods ; Gene Editing/*methods ; Genome, Plant/genetics ; Genotype ; Plants, Genetically Modified ; Recombinational DNA Repair/genetics ; Reproduction/genetics ; Selection, Genetic ; Whole Genome Sequencing/methods ; },
abstract = {Sexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1[drive] lines. After crossing the cry1[drive]/cry1[drive] lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1[drive]/cry1[drive] parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.},
}
@article {pmid34158393,
year = {2021},
author = {Schepers, A and Jochems, F and Lieftink, C and Wang, L and Pogacar, Z and Leite de Oliveira, R and De Conti, G and Beijersbergen, RL and Bernards, R},
title = {Identification of Autophagy-Related Genes as Targets for Senescence Induction Using a Customizable CRISPR-Based Suicide Switch Screen.},
journal = {Molecular cancer research : MCR},
volume = {19},
number = {10},
pages = {1613-1621},
pmid = {34158393},
issn = {1557-3125},
support = {787925/ERC_/European Research Council/International ; },
mesh = {A549 Cells ; Apoptosis/drug effects/genetics ; Autophagy/drug effects/*genetics ; Autophagy-Related Protein-1 Homolog/genetics ; Autophagy-Related Proteins/*genetics ; CRISPR-Cas Systems/drug effects/*genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Cellular Senescence/drug effects/*genetics ; HEK293 Cells ; Humans ; Neoplasms/drug therapy/genetics ; Small Molecule Libraries/pharmacology ; },
abstract = {Pro-senescence therapies are increasingly being considered for the treatment of cancer. Identifying additional targets to induce senescence in cancer cells could further enable such therapies. However, screening for targets whose suppression induces senescence on a genome-wide scale is challenging, as senescent cells become growth arrested, and senescence-associated features can take 1 to 2 weeks to develop. For a screen with a whole-genome CRISPR library, this would result in billions of undesirable proliferating cells by the time the senescent features emerge in the growth arrested cells. Here, we present a suicide switch system that allows genome-wide CRISPR screening in growth-arrested subpopulations by eliminating the proliferating cells during the screen through activation of a suicide switch in proliferating cells. Using this system, we identify in a genome-scale CRISPR screen several autophagy-related proteins as targets for senescence induction. We show that inhibiting macroautophagy with a small molecule ULK1 inhibitor can induce senescence in cancer cell lines of different origin. Finally, we show that combining ULK1 inhibition with the senolytic drug ABT-263 leads to apoptosis in a panel of cancer cell lines. IMPLICATIONS: Our suicide switch approach allows for genome-scale identification of pro-senescence targets, and can be adapted to simplify other screens depending on the nature of the promoter used to drive the switch.},
}
@article {pmid34158378,
year = {2021},
author = {Besse, L and Besse, A and Stolze, SC and Sobh, A and Zaal, EA and van der Ham, AJ and Ruiz, M and Phuyal, S and Büchler, L and Sathianathan, M and Florea, BI and Borén, J and Ståhlman, M and Huber, J and Bolomsky, A and Ludwig, H and Hannich, JT and Loguinov, A and Everts, B and Berkers, CR and Pilon, M and Farhan, H and Vulpe, CD and Overkleeft, HS and Driessen, C},
title = {Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma.},
journal = {Cancer research},
volume = {81},
number = {17},
pages = {4581-4593},
pmid = {34158378},
issn = {1538-7445},
support = {177130/SNSF_/Swiss National Science Foundation/Switzerland ; 182492/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {ATP Binding Cassette Transporter, Subfamily B/metabolism ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Endoplasmic Reticulum/metabolism ; Genome ; Glucose/metabolism ; Golgi Apparatus/metabolism ; HEK293 Cells ; HIV Protease Inhibitors/*pharmacology ; Humans ; Lipidomics ; Lipids/chemistry ; *Membrane Lipids ; Multiple Myeloma/*drug therapy/*metabolism ; Nelfinavir/*pharmacology ; Phospholipids/chemistry ; Phosphorylation ; Receptors, Adiponectin/metabolism ; Signal Transduction ; },
abstract = {The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.},
}
@article {pmid34157973,
year = {2021},
author = {Martínez-Ruiz, EB and Cooper, M and Barrero-Canosa, J and Haryono, MAS and Bessarab, I and Williams, RBH and Szewzyk, U},
title = {Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {464},
pmid = {34157973},
issn = {1471-2164},
mesh = {*Alkaloids ; Burkholderiales/*enzymology/genetics ; Cyanobacteria Toxins ; Genome, Bacterial ; Leptothrix ; *Manganese ; Oxidation-Reduction ; *Oxidoreductases/metabolism ; Pseudomonas/*enzymology/genetics ; },
abstract = {BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn[2+]. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn[2+]. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment.<br><br>RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn[2+] oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn[2+] oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity.<br><br>CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.},
}
@article {pmid34157351,
year = {2021},
author = {Pourtabatabaei, S and Ghanbari, S and Damavandi, N and Bayat, E and Raigani, M and Zeinali, S and Davami, F},
title = {Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9.},
journal = {Journal of biotechnology},
volume = {337},
number = {},
pages = {1-7},
doi = {10.1016/j.jbiotec.2021.06.018},
pmid = {34157351},
issn = {1873-4863},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; Recombinant Proteins/genetics ; Transgenes ; },
abstract = {Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.},
}
@article {pmid34157315,
year = {2021},
author = {Zamaraev, AV and Volik, PI and Sukhikh, GT and Kopeina, GS and Zhivotovsky, B},
title = {Long non-coding RNAs: A view to kill ovarian cancer.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1876},
number = {1},
pages = {188584},
doi = {10.1016/j.bbcan.2021.188584},
pmid = {34157315},
issn = {1879-2561},
mesh = {Animals ; Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm ; Female ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Humans ; Oligonucleotides, Antisense/therapeutic use ; Ovarian Neoplasms/genetics/*metabolism/pathology/therapy ; RNA, Long Noncoding/genetics/*metabolism ; Signal Transduction ; },
abstract = {An emerging role of long non-coding RNAs (lncRNAs) in tumor progression has been revealed in the last decade. Through interactions with nucleic acids and proteins, lncRNAs could act as enhancers, scaffolds or decoys for a number of oncoproteins and tumor suppressors. The aberrant lncRNA expression or mutations are often associated with changes in a variety of cellular processes, including proliferation, stress response and cell death. Here, we will focus on the tumor-associated lncRNAs in ovarian cancer according to their contribution to cancer hallmarks, such as intense proliferation, cell death resistance, altered energy metabolism, invasion and metastasis, and immune evasion. Moreover, the potential clinical implications of lncRNAs and their significance for the diagnosis, prognosis and therapy of ovarian cancer will be discussed.},
}
@article {pmid34157103,
year = {2021},
author = {Xu, Z and Li, Y and Cao, H and Si, M and Zhang, G and Woo, PCY and Yan, A},
title = {A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation.},
journal = {Nucleic acids research},
volume = {49},
number = {16},
pages = {e94},
pmid = {34157103},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Pseudomonas/*genetics ; Recombination, Genetic ; Transcription, Genetic ; },
abstract = {The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λred system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an 'all-in-one' I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in 'non-model' bacterial species.},
}
@article {pmid34156847,
year = {2021},
author = {Lyu, M and Kong, L and Yang, Z and Wu, Y and McGhee, CE and Lu, Y},
title = {PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity.},
journal = {Journal of the American Chemical Society},
volume = {143},
number = {26},
pages = {9724-9728},
pmid = {34156847},
issn = {1520-5126},
support = {R35 GM141931/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*chemistry ; DNA Breaks, Double-Stranded ; DNA, Catalytic/*chemistry ; DNA, Single-Stranded/chemistry ; Genetic Engineering ; RNA/chemistry ; },
abstract = {DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.},
}
@article {pmid34156066,
year = {2021},
author = {Boontawon, T and Nakazawa, T and Xu, H and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {Gene targeting using pre-assembled Cas9 ribonucleoprotein and split-marker recombination in Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {368},
number = {13},
pages = {},
doi = {10.1093/femsle/fnab080},
pmid = {34156066},
issn = {1574-6968},
mesh = {*CRISPR-Cas Systems ; Fungal Proteins/genetics/*metabolism ; Gene Editing ; Gene Targeting/*methods ; Genome, Fungal ; Pleurotus/*genetics/metabolism ; *Recombination, Genetic ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes.},
}
@article {pmid34155407,
year = {2021},
author = {Xu, P and Liu, Z and Liu, Y and Ma, H and Xu, Y and Bao, Y and Zhu, S and Cao, Z and Wu, Z and Zhou, Z and Wei, W},
title = {Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.},
journal = {Nature biotechnology},
volume = {39},
number = {11},
pages = {1403-1413},
pmid = {34155407},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems/genetics ; Cytosine ; DNA Breaks, Double-Stranded ; *Gene Editing/methods ; Genome ; },
abstract = {Canonical CRISPR-knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR-KO screens in various settings.},
}
@article {pmid34155263,
year = {2021},
author = {Lindner, B and Martin, E and Steininger, M and Bundalo, A and Lenter, M and Zuber, J and Schuler, M},
title = {A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {12973},
pmid = {34155263},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Gene Knockout Techniques ; Gene Ontology ; *Genome-Wide Association Study/methods ; Humans ; Monocytes/*immunology/*metabolism ; Phagocytosis/*genetics ; RNA, Guide ; Reproducibility of Results ; THP-1 Cells ; Workflow ; },
abstract = {Phagocytosis of microbial pathogens, dying or dead cells, and cell debris is essential to maintain tissue homeostasis. Impairment of these processes is associated with autoimmunity, developmental defects and toxic protein accumulation. However, the underlying molecular mechanisms of phagocytosis remain incompletely understood. Here, we performed a genome-wide CRISPR knockout screen to systematically identify regulators involved in phagocytosis of Staphylococcus (S.) aureus by human monocytic THP-1 cells. The screen identified 75 hits including known regulators of phagocytosis, e.g. members of the actin cytoskeleton regulation Arp2/3 and WAVE complexes, as well as genes previously not associated with phagocytosis. These novel genes are involved in translational control (EIF5A and DHPS) and the UDP glycosylation pathway (SLC35A2, SLC35A3, UGCG and UXS1) and were further validated by single gene knockout experiments. Whereas the knockout of EIF5A and DHPS impaired phagocytosis, knocking out SLC35A2, SLC35A3, UGCG and UXS1 resulted in increased phagocytosis. In addition to S. aureus phagocytosis, the above described genes also modulate phagocytosis of Escherichia coli and yeast-derived zymosan A. In summary, we identified both known and unknown genetic regulators of phagocytosis, the latter providing a valuable resource for future studies dissecting the underlying molecular and cellular mechanisms and their role in human disease.},
}
@article {pmid34155128,
year = {2021},
author = {Woo, HH and Chambers, SK},
title = {Regulation of closely juxtaposed proto-oncogene c-fms and HMGXB3 gene expression by mRNA 3' end polymorphism in breast cancer cells.},
journal = {RNA (New York, N.Y.)},
volume = {27},
number = {9},
pages = {1068-1081},
pmid = {34155128},
issn = {1469-9001},
mesh = {3' Untranslated Regions ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosomes, Human, Pair 5 ; DNA, Intergenic/genetics/metabolism ; ELAV-Like Protein 1/*genetics/metabolism ; Female ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; *Genes, fms ; High Mobility Group Proteins/*genetics/metabolism ; Humans ; Mammary Glands, Human/metabolism/pathology ; MicroRNAs/*genetics/metabolism ; Polymorphism, Genetic ; Proto-Oncogene Mas ; RNA, Antisense/genetics/metabolism ; Signal Transduction ; Transcription, Genetic ; },
abstract = {Sense-antisense mRNA pairs generated by convergent transcription is a way of gene regulation. c-fms gene is closely juxtaposed to the HMGXB3 gene in the opposite orientation, in chromosome 5. The intergenic region (IR) between c-fms and HMGXB3 genes is 162 bp. We found that a small portion (∼4.18%) of HMGXB3 mRNA is transcribed further downstream, including the end of the c-fms gene generating antisense mRNA against c-fms mRNA. Similarly, a small portion (∼1.1%) of c-fms mRNA is transcribed further downstream, including the end of the HMGXB3 gene generating antisense mRNA against the HMGXB3 mRNA. Insertion of the strong poly(A) signal sequence in the IR results in decreased c-fms and HMGXB3 antisense mRNAs, resulting in up-regulation of both c-fms and HMGXB3 mRNA expression. miR-324-5p targets HMGXB3 mRNA 3' UTR, and as a result, regulates c-fms mRNA expression. HuR stabilizes c-fms mRNA, and as a result, down-regulates HMGXB3 mRNA expression. UALCAN analysis indicates that the expression pattern between c-fms and HMGXB3 proteins are opposite in vivo in breast cancer tissues. Together, our results indicate that the mRNA encoded by the HMGXB3 gene can influence the expression of adjacent c-fms mRNA, or vice versa.},
}
@article {pmid34154416,
year = {2021},
author = {Wu, X and Zhu, J and Tao, P and Rao, VB},
title = {Bacteriophage T4 Escapes CRISPR Attack by Minihomology Recombination and Repair.},
journal = {mBio},
volume = {12},
number = {3},
pages = {e0136121},
pmid = {34154416},
issn = {2150-7511},
support = {R01 AI081726/AI/NIAID NIH HHS/United States ; R56 AI081726/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophage T4/*genetics ; CRISPR-Cas Systems/*genetics ; *DNA Repair ; Genome, Viral/*genetics ; *Recombination, Genetic ; },
abstract = {Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications.},
}
@article {pmid34153439,
year = {2021},
author = {Boontawon, T and Nakazawa, T and Horii, M and Tsuzuki, M and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {154},
number = {},
pages = {103599},
doi = {10.1016/j.fgb.2021.103599},
pmid = {34153439},
issn = {1096-0937},
mesh = {CRISPR-Cas Systems ; Fungal Proteins/*genetics ; Gene Expression Regulation, Fungal ; Genes, Mating Type, Fungal ; Pleurotus/*genetics ; },
abstract = {Understanding the molecular mechanisms controlling dikaryon formation in Agaricomycetes, which is basically controlled by A and B mating-type loci, contributes to improving mushroom cultivation and breeding. In Coprinopsis cinerea, various mutations in the SRY-type high mobility group protein-encoding gene, pcc1, were shown to activate the A-regulated pathway to induce pseudoclamp (clamp cells without clamp connection) and fruiting body formation in monokaryons. The formation of clamp cells was blocked in AmutBmut strain 326 with clp1-1 mutation in C. cinerea. However, considering the diverse mechanisms of sexual development among Agaricomycetes, it remains unclear whether similar phenotypes are also observed in clp1 or pcc1 mutants in cultivated mushrooms. Therefore, phenotypic analyses of Pleurotus ostreatus pcc1 or clp1 (Popcc1 or Poclp1) mutants generated using CRISPR/Cas9 were performed in this study. Plasmids with Cas9 expression cassette and different single guide RNAs targeting Popcc1 or Poclp1 were individually introduced into a monokaryotic P. ostreatus strain PC9 to obtain the mutants. Unlike in C. cinerea, the pseudoclamp cell was not observed in monokaryotic Popcc1 mutants, but it was observed after crossing two compatible strains with Popcc1 mutations. In Poclp1 mutants, dikaryosis was impaired as clamp cells were not observed after crossing, suggesting that Poclp1 functions may be essential for clamp cell formation, like in C. cinerea. These results provided a clue with respect to conserved and diverse mechanisms underlying sexual development in Agaricomycetes (at least between C. cinerea and P. ostreatus).},
}
@article {pmid34152311,
year = {2021},
author = {Graham, S and Gao, L and Wang, R},
title = {Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {172},
pages = {},
doi = {10.3791/61618},
pmid = {34152311},
issn = {1940-087X},
mesh = {Animals ; Antibodies ; CRISPR-Cas Systems ; *Colitis/chemically induced/genetics ; Endonucleases ; *Gene Editing ; Mice ; },
abstract = {The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.},
}
@article {pmid34152223,
year = {2021},
author = {Gao, C and Chen, J},
title = {CRISPR Adventures in China.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {304-306},
doi = {10.1089/crispr.2021.29129.gao},
pmid = {34152223},
issn = {2573-1602},
mesh = {Animals ; COVID-19 ; CRISPR-Cas Systems ; China ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; Humans ; Models, Animal ; Plants/genetics ; },
}
@article {pmid34152222,
year = {2021},
author = {Zaman, QU and Wen, C and Yuqin, S and Mengyu, H and Desheng, M and Jacqueline, B and Baohong, Z and Chao, L and Qiong, H},
title = {Characterization of SHATTERPROOF Homoeologs and CRISPR-Cas9-Mediated Genome Editing Enhances Pod-Shattering Resistance in Brassica napus L.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {360-370},
doi = {10.1089/crispr.2020.0129},
pmid = {34152222},
issn = {2573-1602},
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins ; Brassica napus/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genes, Plant ; Lignin ; MADS Domain Proteins/*genetics ; Mutation ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Brassica napus is the most important oil crop plant for edible oil and renewable energy source worldwide. Yield loss caused by pod shattering is a main problem during B. napus harvest. In this study, six BnSHP1 and two BnSHP2 homoeologs were targeted by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome editing system and multiple SHP1 and SHP2 mutated lines were identified for evaluating the contribution for pod-shattering resistance. Our data suggest that BnSHP1A09 is probably a promising homoeolog for controlling lignin contents at dehiscence zone. Simultaneous mutation of BnSHP1A09/C04-B/A04 and BnSHP2A05/C04-A exhibited reduced lignified layer and separation layer adjacent to valves and replum. The pod-shattering resistance index (SRI) subsequently increased to 0.31 in five homoeolog mutation lines compared with the wild type (SRI = 0.036), which provide the theoretical basis for breeding of commercial pod-shattering resistance variety.},
}
@article {pmid34152221,
year = {2021},
author = {Balderston, S and Clouse, G and Ripoll, JJ and Pratt, GK and Gasiunas, G and Bock, JO and Bennett, EP and Aran, K},
title = {Diversification of the CRISPR Toolbox: Applications of CRISPR-Cas Systems Beyond Genome Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {400-415},
pmid = {34152221},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression ; Genetic Techniques ; Genome ; Pathology, Molecular/methods ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {The discovery of CRISPR has revolutionized the field of genome engineering, but the potential of this technology is far from reaching its limits. In this review, we explore the broad range of applications of CRISPR technology to highlight the rapid expansion of the field beyond gene editing alone. It has been demonstrated that CRISPR technology can control gene expression, spatiotemporally image the genome in vivo, and detect specific nucleic acid sequences for diagnostics. In addition, new technologies are under development to improve CRISPR quality controls for gene editing, thereby improving the reliability of these technologies for therapeutics and beyond. These are just some of the many CRISPR tools that have been developed in recent years, and the toolbox continues to diversify.},
}
@article {pmid34152220,
year = {2021},
author = {Zhou, W and Yuan, Y and Zhang, Y and Chen, D},
title = {A Decade of CRISPR Gene Editing in China and Beyond: A Scientometric Landscape.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {313-320},
doi = {10.1089/crispr.2020.0148},
pmid = {34152220},
issn = {2573-1602},
mesh = {Biotechnology ; *CRISPR-Cas Systems ; China ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural ; Gene Editing/*history/methods/*trends ; Genome, Plant ; History, 21st Century ; Nobel Prize ; },
abstract = {Since its Nobel Prize-winning breakthrough in 2012, CRISPR-Cas-based gene-editing system has emerged as one of the most promising biotechnologies in decades. In this article, we present an objective and comprehensive evaluation of CRISPR-based gene-editing technologies, including base editing and prime editing, based on the bibliometric analysis of 22,902 published records. We also assessed the status of CRISPR gene-editing technologies in academia from 2010 to 2020 globally, with respect to countries, institutions, and researchers, and used text clustering methods to assess technical trends and research hotspots. Our results indicate, not surprisingly, that this is a thriving and prominent area of research. By comparing the relevance and growth of CRISPR gene-editing technologies in China with other countries by several metrics, we show that the Chinese scientific community attaches considerable importance to the field of plant genome engineering, with more scholars from agricultural sectors than other sectors.},
}
@article {pmid34152219,
year = {2021},
author = {Li, H and Dong, X and Wang, Y and Yang, L and Cai, K and Zhang, X and Kou, Z and He, L and Sun, S and Li, T and Nie, Y and Li, X and Sun, Y},
title = {Sensitive and Easy-Read CRISPR Strip for COVID-19 Rapid Point-of-Care Testing.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {392-399},
doi = {10.1089/crispr.2020.0138},
pmid = {34152219},
issn = {2573-1602},
mesh = {COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/*instrumentation ; CRISPR-Cas Systems/*genetics ; Humans ; Limit of Detection ; *Point-of-Care Testing ; Predictive Value of Tests ; RNA, Viral/genetics/isolation &amp; purification ; Reagent Kits, Diagnostic ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {Rapid and clinically sensitive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) play an important role in the contact tracing and containment of the COVID-19 pandemic. A recently developed field-deployable clustered regularly interspaced short palindromic repeats (CRISPR) detection assay with lateral flow strips shows promise for point-of-care detection of SARS-CoV-2. However, the limit of detection of paper strip-based assays (10-100 copies/μL) is much lower than that of fluorescence-based detection methods. In this study, we developed an easy-readout and sensitive enhanced (ERASE) strip to visualize the results of CRISPR detection and improve the sensitivity to 1 copy/μL with an unambiguous easy-read result. Using 649 clinical samples from blind specimens collected from patients in China, we validated our ERASE assay for SARS-CoV-2 RNA detection with 90.67% positive predictive agreement and 99.21% negative predictive agreement. In conclusion, our study provided a customized CRISPR strip for use in a simple, rapid, ultrasensitive, and highly specific assay for SARS-CoV-2 detection. (Clinical Trial Registration number: 2020-008-01; [2020]IEC(ZD01); PJ-NBEY-2020-009-01; 2020#34).},
}
@article {pmid34152218,
year = {2021},
author = {Mack, S and Russell, IA},
title = {CRISPR and Chromothripsis: Proceed with Caution.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {309-312},
doi = {10.1089/crispr.2021.29128.sma},
pmid = {34152218},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; *Chromothripsis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Damage ; Gene Editing ; Humans ; },
}
@article {pmid34152217,
year = {2021},
author = {Bire, S and Buhan, CL and Palazzoli, F},
title = {The CRISPR Patent Landscape: Focus on Chinese Researchers.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {339-349},
doi = {10.1089/crispr.2021.0020},
pmid = {34152217},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; China ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Humans ; *Research Personnel ; },
abstract = {Despite the strong presence of Chinese scientists in genome-editing research, little attention has been paid to the legal, economic, and scientific development of patented CRISPR technologies in China. In this study, we focus on CRISPR patent documents from academic and industrial Chinese players to assess their positioning on this breakthrough technology. We review the fields of application and the CRISPR components claimed in the relevant patent documents. Our results show different profiles observed for academic or industrial assignees. Most of the patent families in our data set cover applications in genome editing and nucleic-acid detection for human therapeutic and diagnostic purposes. Trends in the patent data since 2014 confirm that China' R&amp;D has rapidly developed a significant CRISPR patent landscape of its own, covering a diverse range of systems and applications. These recent developments deserve closer scrutiny from the international CRISPR community.},
}
@article {pmid34152216,
year = {2021},
author = {Napier, JA},
title = {A Field Day for Gene-Edited Brassicas and Crop Improvement.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {307-312},
doi = {10.1089/crispr.2021.29130.kad},
pmid = {34152216},
issn = {2573-1602},
mesh = {Brassica/*genetics ; CRISPR-Cas Systems ; *Crops, Agricultural ; *Gene Editing ; Plants, Genetically Modified/genetics ; United Kingdom ; },
}
@article {pmid34152215,
year = {2021},
author = {Levrier, G},
title = {Tracking and Mapping the Use of CRISPR-Cas Systems for Research in China.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {321-338},
doi = {10.1089/crispr.2021.0015},
pmid = {34152215},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; China ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genome ; Humans ; *Research ; },
abstract = {Scientists have floated the idea of a "Sputnik 2.0" technological race between the People's Republic of China (PRC) and the United States of America in CRISPR-based genome editing research. This quantitative analysis of articles published between 2010 and 2020 shows that research centers based in the PRC have succeeded in making CRISPR-based genome editing a standard tool. A corpus of 18,863 academic documents containing the acronym CRISPR in their abstract shows that although PRC-based research institutions were slower to start publishing on CRISPR, they have now outpaced the publication rate of institutions located in the European Union (EU). While U.S.-based institutions have kept their leading position in basic research, PRC-based research has become momentous in agriculture-related fields. This corpus hence illustrates how deeply the international landscape of life sciences research has shifted since the Human Genome Project, mostly to the PRC's advantage.},
}
@article {pmid34152214,
year = {2021},
author = {Neequaye, M and Stavnstrup, S and Harwood, W and Lawrenson, T and Hundleby, P and Irwin, J and Troncoso-Rey, P and Saha, S and Traka, MH and Mithen, R and Østergaard, L},
title = {CRISPR-Cas9-Mediated Gene Editing of MYB28 Genes Impair Glucoraphanin Accumulation of Brassica oleracea in the Field.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {416-426},
doi = {10.1089/crispr.2021.0007},
pmid = {34152214},
issn = {2573-1602},
support = {BB/P013511/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R012512/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M011216/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N019466/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Arabidopsis Proteins ; Brassica/*genetics/*metabolism ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics/metabolism ; Gene Editing/*methods ; Gene Expression ; Glucosinolates/*biosynthesis/*genetics ; Histone Acetyltransferases/*genetics/*metabolism ; Oximes ; Plants, Genetically Modified ; Sulfoxides/metabolism ; United Kingdom ; },
abstract = {Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of MYB28 in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated sulfur metabolism in field-grown Brassica oleracea with the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene-editing technology. We describe the first myb28 knockout mutant in B. oleracea, and the first CRISPR field trial in the United Kingdom approved and regulated by the UK Department for Environment, Food &amp; Rural Affairs after the reclassification of gene-edited crops as genetically modified organisms by the European Court of Justice on July 25, 2018. We report that knocking out myb28 results in downregulation of A-GSL biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants, whereas accumulation of sulfate, S-methyl cysteine sulfoxide, and indole glucosinolate in leaf and floret tissues remained unchanged. These results demonstrate the potential of gene-editing approaches to translate discoveries in fundamental biological processes for improved crop performance.},
}
@article {pmid34152213,
year = {2021},
author = {Zhang, Y and Yang, J and Yang, S and Zhang, J and Chen, J and Tao, R and Jiang, Y and Yang, J and Yang, S},
title = {Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {350-359},
doi = {10.1089/crispr.2021.0018},
pmid = {34152213},
issn = {2573-1602},
mesh = {Acetylglucosamine/metabolism ; CRISPR-Cas Systems ; China ; *Chromosomes ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Fermentation ; Gene Editing/methods ; Plasmids ; *Transposases/genetics ; },
abstract = {Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.},
}
@article {pmid34152211,
year = {2021},
author = {Dooley, SK and Baken, EK and Moss, WN and Howe, A and Young, JK},
title = {Identification and Evolution of Cas9 tracrRNAs.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {438-447},
pmid = {34152211},
issn = {2573-1602},
support = {R01 GM133810/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaea/genetics ; Bacteria/genetics ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Evolution, Molecular ; Phylogeny ; RNA/*chemistry ; RNA, Guide/genetics ; Sequence Homology ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas)9 transactivating CRISPR RNAs (tracrRNAs) form distinct structures essential for target recognition and cleavage and dictate exchangeability between orthologous proteins. As noncoding RNAs that are often apart from the CRISPR array, their identification can be arduous. In this article, a new bioinformatic method for the detection of Cas9 tracrRNAs is presented. The approach utilizes a covariance model based on both sequence homology and predicted secondary structure to locate tracrRNAs. This method predicts a tracrRNA for 98% of CRISPR-Cas9 systems identified by us. To ensure accuracy, we also benchmark our approach against biochemically vetted tracrRNAs finding false-positive and false-negative rates of 5.5% and 7.1%, respectively. Finally, the association between Cas9 amino acid sequence-based phylogeny and tracrRNA secondary structure is evaluated, revealing strong evidence that secondary structure is evolutionarily conserved among Cas9 lineages. Altogether, our findings provide insight into Cas9 tracrRNA evolution and efforts to characterize the tracrRNA of Cas9 systems.},
}
@article {pmid34151300,
year = {2021},
author = {Li, A and Cartwright, S and Yu, A and Ho, SM and Schrode, N and Deans, PJM and Matos, MR and Garcia, MF and Townsley, KG and Zhang, B and Brennand, KJ},
title = {Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100580},
pmid = {34151300},
issn = {2666-1667},
support = {R56 MH101454/MH/NIMH NIH HHS/United States ; R01 MH106056/MH/NIMH NIH HHS/United States ; R01 MH109897/MH/NIMH NIH HHS/United States ; U01 AG046170/AG/NIA NIH HHS/United States ; RF1 AG054014/AG/NIA NIH HHS/United States ; RF1 AG057440/AG/NIA NIH HHS/United States ; R01 AG057907/AG/NIA NIH HHS/United States ; U01 AG052411/AG/NIA NIH HHS/United States ; R01 AG062355/AG/NIA NIH HHS/United States ; R01 AG068030/AG/NIA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Expression Regulation ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Nerve Tissue Proteins/*genetics ; Neurons/cytology/*metabolism ; Transcriptome ; },
abstract = {We describe a CRISPR inhibition (CRISPRi) protocol to repress endogenous gene expression (e.g., ATP6V1A) in human induced pluripotent stem cell-derived NGN2-induced glutamatergic neurons. CRISPRi enables efficient and precise gene repression of one or multiple target genes via delivering gRNA(s) to direct a dCas9-KRAB fusion protein to the gene(s) of interest. This protocol can also be adapted for gene activation and high-throughput gene manipulation, allowing assessment of the transcriptomic and phenotypic impact of candidate gene(s) associated with neurodevelopment or brain disease. For complete details on the use and execution of this protocol, please refer to Ho et al. (2017) and Wang et al. (2021).},
}
@article {pmid34151296,
year = {2021},
author = {Martens, YA and Xu, S and Tait, R and Li, G and Zhao, XC and Lu, W and Liu, CC and Kanekiyo, T and Bu, G and Zhao, J},
title = {Generation and validation of APOE knockout human iPSC-derived cerebral organoids.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100571},
pmid = {34151296},
issn = {2666-1667},
support = {P01 NS074969/NS/NINDS NIH HHS/United States ; P30 AG062677/AG/NIA NIH HHS/United States ; R01 AG027924/AG/NIA NIH HHS/United States ; RF1 AG046205/AG/NIA NIH HHS/United States ; U19 AG069701/AG/NIA NIH HHS/United States ; R01 AG066395/AG/NIA NIH HHS/United States ; RF1 AG057181/AG/NIA NIH HHS/United States ; },
mesh = {Brain/*metabolism ; CRISPR-Cas Systems ; *Gene Knockdown Techniques ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Organoids/*metabolism ; },
abstract = {Apolipoprotein E (apoE) is a major lipid carrier in the brain and closely associated with the pathogenesis of Alzheimer's disease (AD). Here, we describe a protocol for efficient knockout of APOE in human induced pluripotent stem cells (iPSCs) using the CRISPR-Cas9 system. We obtain homozygous APOE knockout (APOE[-/-]) iPSCs and further validate the deficiency of apoE in iPSC-derived cerebral organoids. APOE[-/-] cerebral organoids can serve as a useful tool to study apoE functions and apoE-related pathogenic mechanisms in AD. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).},
}
@article {pmid34149780,
year = {2021},
author = {Iswanto, ABB and Shelake, RM and Vu, MH and Kim, JY and Kim, SH},
title = {Genome Editing for Plasmodesmal Biology.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {679140},
pmid = {34149780},
issn = {1664-462X},
abstract = {Plasmodesmata (PD) are cytoplasmic canals that facilitate intercellular communication and molecular exchange between adjacent plant cells. PD-associated proteins are considered as one of the foremost factors in regulating PD function that is critical for plant development and stress responses. Although its potential to be used for crop engineering is enormous, our understanding of PD biology was relatively limited to model plants, demanding further studies in crop systems. Recently developed genome editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associate protein (CRISPR/Cas) might confer powerful approaches to dissect the molecular function of PD components and to engineer elite crops. Here, we assess several aspects of PD functioning to underline and highlight the potential applications of CRISPR/Cas that provide new insight into PD biology and crop improvement.},
}
@article {pmid34149645,
year = {2021},
author = {Cruz-López, EA and Rivera, G and Cruz-Hernández, MA and Martínez-Vázquez, AV and Castro-Escarpulli, G and Flores-Magallón, R and Vázquez, K and Cruz-Pulido, WL and Bocanegra-García, V},
title = {Identification and Characterization of the CRISPR/Cas System in Staphylococcus aureus Strains From Diverse Sources.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {656996},
pmid = {34149645},
issn = {1664-302X},
abstract = {The CRISPR-Cas [clustered regularly interspaced short palindromic repeats and the CRISPR-associated genes (Cas)] system provides defense mechanisms in bacteria and archaea vs. mobile genetic elements (MGEs), such as plasmids and bacteriophages, which can either be harmful or add sequences that can provide virulence or antibiotic resistance. Staphylococcus aureus is a Gram-positive bacterium that could be the etiological agent of important soft tissue infections that can lead to bacteremia and sepsis. The role of the CRISPR-Cas system in S. aureus is not completely understood since there is a lack of knowledge about it. We analyzed 716 genomes and 1 genomic island from GENOMES-NCBI and ENA-EMBL searching for the CRISPR-Cas systems and their spacer sequences (SSs). Our bioinformatic analysis shows that only 0.83% (6/716) of the analyzed genomes harbored the CRISPR-Cas system, all of them were subtype III-A, which is characterized by the presence of the cas10/csm1 gene. Analysis of SSs showed that 91% (40/44) had no match to annotated MGEs and 9% of SSs corresponded to plasmids and bacteriophages, indicating that those phages had infected those S. aureus strains. Some of those phages have been proposed as an alternative therapy in biofilm-forming or infection with S. aureus strains, but these findings indicate that such antibiotic phage strategy would be ineffective. More research about the CRISPR/Cas system is necessary for a bigger number of S. aureus strains from different sources, so additional features can be studied.},
}
@article {pmid34148586,
year = {2021},
author = {de Melo-Martín, I and Rosenwaks, Z},
title = {Human embryo genetic editing: hope or pipe dream?.},
journal = {Fertility and sterility},
volume = {116},
number = {1},
pages = {25-26},
doi = {10.1016/j.fertnstert.2021.04.017},
pmid = {34148586},
issn = {1556-5653},
mesh = {*CRISPR-Cas Systems ; Female ; *Fertilization in Vitro ; *Gene Editing ; Gene Expression Regulation, Developmental ; Genetic Diseases, Inborn/diagnosis/genetics/*therapy ; *Genetic Therapy ; Humans ; Infertility/diagnosis/physiopathology/*therapy ; Male ; Pregnancy ; },
}
@article {pmid34148538,
year = {2022},
author = {Huang, X and Li, X and Yang, L and Wang, P and Yan, J and Nie, Z and Gao, Y and Li, Z and Wen, J and Cao, X},
title = {Construction and Optimization of Herpes Simplex Virus Vectors for Central Nervous System Gene Delivery based on CRISPR/Cas9-mediated Genome Editing.},
journal = {Current gene therapy},
volume = {22},
number = {1},
pages = {66-77},
doi = {10.2174/1566523219666210618154326},
pmid = {34148538},
issn = {1875-5631},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Central Nervous System ; Gene Editing ; *Herpes Simplex/genetics ; *Herpesvirus 1, Human/genetics ; Mice ; },
abstract = {AIMS: We aim to define parameters that affect the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS.<br><br>BACKGROUND: Engineered, attenuated Herpes Simplex Virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and longterm transgene expression of attenuated HSV-1 vectors have not been fully understood.<br><br>OBJECTIVE: The study aimed at using CRISPR-Cas9 system to construct attenuated HSV-1 vectors and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety.<br><br>METHODS: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and construct two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in mouse hippocampus gene transduction model.<br><br>RESULTS: The in vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacking Poly (A) induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation.<br><br>CONCLUSION: Our results indicated that the integrity of LAT transcripts was not necessary for establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, which suggests an important role of LAT in maintaining viral reactivation potential.},
}
@article {pmid34148061,
year = {2021},
author = {Zhuo, C and Zhang, J and Lee, JH and Jiao, J and Cheng, D and Liu, L and Kim, HW and Tao, Y and Li, M},
title = {Spatiotemporal control of CRISPR/Cas9 gene editing.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {238},
pmid = {34148061},
issn = {2059-3635},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Transfer Techniques ; *Genetic Therapy ; Humans ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.},
}
@article {pmid34147719,
year = {2021},
author = {Nguyen, NTK and Chang, YH and Truong, VA and Hsu, MN and Pham, NN and Chang, CW and Wu, YH and Chang, YH and Li, H and Hu, YC},
title = {CRISPR activation of long non-coding RNA DANCR promotes bone regeneration.},
journal = {Biomaterials},
volume = {275},
number = {},
pages = {120965},
doi = {10.1016/j.biomaterials.2021.120965},
pmid = {34147719},
issn = {1878-5905},
mesh = {Animals ; *Bone Regeneration ; CRISPR-Cas Systems ; Cell Differentiation ; Chondrogenesis ; RNA, Guide ; *RNA, Long Noncoding ; Rats ; },
abstract = {Healing of large calvarial bone defects in adults adopts intramembranous pathway and is difficult. Implantation of adipose-derived stem cells (ASC) that differentiate towards chondrogenic lineage can switch the bone repair pathway and improve calvarial bone healing. Long non-coding RNA DANCR was recently uncovered to promote chondrogenesis, but its roles in rat ASC (rASC) chondrogenesis and bone healing stimulation have yet to be explored. Here we first verified that DANCR expression promoted rASC chondrogenesis, thus we harnessed CRISPR activation (CRISPRa) technology to upregulate endogenous DANCR, stimulate rASC chondrogenesis and improve calvarial bone healing in rats. We generated 4 different dCas9-VPR orthologues by fusing a tripartite transcription activator domain VPR to catalytically dead Cas9 (dCas9) derived from 4 different bacteria, and compared the degree of activation using the 4 different dCas9-VPR. We unveiled surprisingly that the most commonly used dCas9-VPR derived from Streptococcus pyogenes barely activated DANCR. Nonetheless dCas9-VPR from Staphylococcus aureus (SadCas9-VPR) triggered efficient activation of DANCR in rASC. Delivery of SadCas9-VPR and the associated guide RNA into rASC substantially enhanced chondrogenic differentiation of rASC and augmented cartilage formation in vitro. Implantation of the engineered rASC remarkably potentiated the calvarial bone healing in rats. Furthermore, we identified that DANCR improved the rASC chondrogenesis through inhibition of miR-203a and miR-214. These results collectively proved that DANCR activation by SadCas9-VPR-based CRISPRa provides a novel therapeutic approach to improving calvarial bone healing.},
}
@article {pmid34147688,
year = {2021},
author = {Aquino-Jarquin, G},
title = {Recent progress on rapid SARS-CoV-2/COVID-19 detection by CRISPR-Cas13-based platforms.},
journal = {Drug discovery today},
volume = {26},
number = {8},
pages = {2025-2035},
pmid = {34147688},
issn = {1878-5832},
mesh = {*COVID-19/diagnosis/virology ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Associated Proteins ; CRISPR-Cas Systems/*physiology ; Humans ; Point-of-Care Testing/trends ; RNA Cleavage ; RNA, Viral/*isolation &amp; purification ; SARS-CoV-2/*isolation &amp; purification ; },
abstract = {The limitations of conventional diagnostic procedures, such as real-time PCR-based methods and serological tests, have led the scientific community to innovate alternative nucleic acid detection approaches for SARS-CoV-2 RNA, thereby addressing the dire need for increased testing. Such approaches aim to provide rapid, accurate, cost-effective, sensitive, and high-throughput detection of SARS-CoV-2 RNA, on multiple specimen types, and without specialized equipment and expertise. The CRISPR-Cas13 system functions as a sequence-specific RNA-sensing tool that has recently been harnessed to develop simplified and flexible testing formats. This review recapitulates technical advances in the most recent CRISPR-Cas13-based methods for SARS-CoV-2/COVID-19 diagnosis. The challenges and opportunities for implementing mass testing using these novel CRISPR-Cas13 platforms are critically analyzed.},
}
@article {pmid34146516,
year = {2021},
author = {Baxter, JS and Johnson, N and Tomczyk, K and Gillespie, A and Maguire, S and Brough, R and Fachal, L and Michailidou, K and Bolla, MK and Wang, Q and Dennis, J and Ahearn, TU and Andrulis, IL and Anton-Culver, H and Antonenkova, NN and Arndt, V and Aronson, KJ and Augustinsson, A and Becher, H and Beckmann, MW and Behrens, S and Benitez, J and Bermisheva, M and Bogdanova, NV and Bojesen, SE and Brenner, H and Brucker, SY and Cai, Q and Campa, D and Canzian, F and Castelao, JE and Chan, TL and Chang-Claude, J and Chanock, SJ and Chenevix-Trench, G and Choi, JY and Clarke, CL and , and Colonna, S and Conroy, DM and Couch, FJ and Cox, A and Cross, SS and Czene, K and Daly, MB and Devilee, P and Dörk, T and Dossus, L and Dwek, M and Eccles, DM and Ekici, AB and Eliassen, AH and Engel, C and Fasching, PA and Figueroa, J and Flyger, H and Gago-Dominguez, M and Gao, C and García-Closas, M and García-Sáenz, JA and Ghoussaini, M and Giles, GG and Goldberg, MS and González-Neira, A and Guénel, P and Gündert, M and Haeberle, L and Hahnen, E and Haiman, CA and Hall, P and Hamann, U and Hartman, M and Hatse, S and Hauke, J and Hollestelle, A and Hoppe, R and Hopper, JL and Hou, MF and , and , and Ito, H and Iwasaki, M and Jager, A and Jakubowska, A and Janni, W and John, EM and Joseph, V and Jung, A and Kaaks, R and Kang, D and Keeman, R and Khusnutdinova, E and Kim, SW and Kosma, VM and Kraft, P and Kristensen, VN and Kubelka-Sabit, K and Kurian, AW and Kwong, A and Lacey, JV and Lambrechts, D and Larson, NL and Larsson, SC and Le Marchand, L and Lejbkowicz, F and Li, J and Long, J and Lophatananon, A and Lubiński, J and Mannermaa, A and Manoochehri, M and Manoukian, S and Margolin, S and Matsuo, K and Mavroudis, D and Mayes, R and Menon, U and Milne, RL and Mohd Taib, NA and Muir, K and Muranen, TA and Murphy, RA and Nevanlinna, H and O'Brien, KM and Offit, K and Olson, JE and Olsson, H and Park, SK and Park-Simon, TW and Patel, AV and Peterlongo, P and Peto, J and Plaseska-Karanfilska, D and Presneau, N and Pylkäs, K and Rack, B and Rennert, G and Romero, A and Ruebner, M and Rüdiger, T and Saloustros, E and Sandler, DP and Sawyer, EJ and Schmidt, MK and Schmutzler, RK and Schneeweiss, A and Schoemaker, MJ and Shah, M and Shen, CY and Shu, XO and Simard, J and Southey, MC and Stone, J and Surowy, H and Swerdlow, AJ and Tamimi, RM and Tapper, WJ and Taylor, JA and Teo, SH and Teras, LR and Terry, MB and Toland, AE and Tomlinson, I and Truong, T and Tseng, CC and Untch, M and Vachon, CM and van den Ouweland, AMW and Wang, SS and Weinberg, CR and Wendt, C and Winham, SJ and Winqvist, R and Wolk, A and Wu, AH and Yamaji, T and Zheng, W and Ziogas, A and Pharoah, PDP and Dunning, AM and Easton, DF and Pettitt, SJ and Lord, CJ and Haider, S and Orr, N and Fletcher, O},
title = {Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element.},
journal = {American journal of human genetics},
volume = {108},
number = {7},
pages = {1190-1203},
pmid = {34146516},
issn = {1537-6605},
support = {MC_UU_00004/01/MRC_/Medical Research Council/United Kingdom ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Breast Neoplasms/genetics ; CRISPR-Cas Systems ; Cell Line ; Chromosome Mapping ; Chromosomes, Human, Pair 2 ; Female ; Genetic Association Studies ; Genetic Variation ; Humans ; Insulin-Like Growth Factor Binding Protein 5/*genetics ; *Molecular Sequence Annotation ; *Promoter Regions, Genetic ; Risk Factors ; Sequence Deletion ; },
abstract = {A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 × 10[-31]).},
}
@article {pmid34146158,
year = {2021},
author = {Nagahara, S and Higashiyama, T and Mizuta, Y},
title = {Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube.},
journal = {Plant reproduction},
volume = {34},
number = {3},
pages = {191-205},
pmid = {34146158},
issn = {2194-7961},
mesh = {*Biolistics ; *CRISPR-Cas Systems ; Gene Editing ; Plant Breeding ; Plants, Genetically Modified/genetics ; Pollen Tube/genetics ; },
abstract = {Biolistic delivery into pollen. In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.},
}
@article {pmid34145278,
year = {2021},
author = {Gabriel, CH and Del Olmo, M and Zehtabian, A and Jäger, M and Reischl, S and van Dijk, H and Ulbricht, C and Rakhymzhan, A and Korte, T and Koller, B and Grudziecki, A and Maier, B and Herrmann, A and Niesner, R and Zemojtel, T and Ewers, H and Granada, AE and Herzel, H and Kramer, A},
title = {Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3796},
pmid = {34145278},
issn = {2041-1723},
mesh = {CLOCK Proteins/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Circadian Clocks/*physiology ; Circadian Rhythm/physiology ; Cryptochromes/genetics/*metabolism ; Gene Knock-In Techniques/methods ; Genes, Reporter/genetics ; HCT116 Cells ; Humans ; Period Circadian Proteins/genetics/*metabolism ; Single-Cell Analysis/*methods ; },
abstract = {The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.},
}
@article {pmid34144928,
year = {2021},
author = {Zhang, J and Khazalwa, EM and Abkallo, HM and Zhou, Y and Nie, X and Ruan, J and Zhao, C and Wang, J and Xu, J and Li, X and Zhao, S and Zuo, E and Steinaa, L and Xie, S},
title = {The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {5},
pages = {347-360},
doi = {10.1016/j.jgg.2021.03.015},
pmid = {34144928},
issn = {1673-8527},
mesh = {Animals ; Animals, Genetically Modified ; Breeding ; *CRISPR-Cas Systems ; Disease Resistance ; Gene Editing/*methods/*trends ; Genetic Association Studies/methods/trends ; Genetic Engineering/methods/trends ; Humans ; Models, Animal ; Quantitative Trait Loci ; Quantitative Trait, Heritable ; Research/*trends ; Swine ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.},
}
@article {pmid34143608,
year = {2021},
author = {Bao, M and Chen, Q and Xu, Z and Jensen, EC and Liu, C and Waitkus, JT and Yuan, X and He, Q and Qin, P and Du, K},
title = {Challenges and Opportunities for Clustered Regularly Interspaced Short Palindromic Repeats Based Molecular Biosensing.},
journal = {ACS sensors},
volume = {6},
number = {7},
pages = {2497-2522},
doi = {10.1021/acssensors.1c00530},
pmid = {34143608},
issn = {2379-3694},
mesh = {Biological Assay ; *Biosensing Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats, CRISPR, has recently emerged as a powerful molecular biosensing tool for nucleic acids and other biomarkers due to its unique properties such as collateral cleavage nature, room temperature reaction conditions, and high target-recognition specificity. Numerous platforms have been developed to leverage the CRISPR assay for ultrasensitive biosensing applications. However, to be considered as a new gold standard, several key challenges for CRISPR molecular biosensing must be addressed. In this paper, we briefly review the history of biosensors, followed by the current status of nucleic acid-based detection methods. We then discuss the current challenges pertaining to CRISPR-based nucleic acid detection, followed by the recent breakthroughs addressing these challenges. We focus upon future advancements required to enable rapid, simple, sensitive, specific, multiplexed, amplification-free, and shelf-stable CRISPR-based molecular biosensors.},
}
@article {pmid34143557,
year = {2021},
author = {Zhang, S and Zhang, R and Gao, J and Song, G and Li, J and Li, W and Qi, Y and Li, Y and Li, G},
title = {CRISPR/Cas9-mediated genome editing for wheat grain quality improvement.},
journal = {Plant biotechnology journal},
volume = {19},
number = {9},
pages = {1684-1686},
pmid = {34143557},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Quality Improvement ; RNA, Guide ; *Triticum/genetics ; },
}
@article {pmid34143395,
year = {2021},
author = {Prasad, K and George, A and Ravi, NS and Mohankumar, KM},
title = {CRISPR/Cas based gene editing: marking a new era in medical science.},
journal = {Molecular biology reports},
volume = {48},
number = {5},
pages = {4879-4895},
pmid = {34143395},
issn = {1573-4978},
mesh = {Biomedical Research/*trends ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering ; *Genetic Therapy ; Genome ; Humans ; },
abstract = {CRISPR/Cas9 system, a bacterial adaptive immune system developed into a genome editing technology, has emerged as a powerful tool revolutionising genome engineering in all branches of biological science including agriculture, research and medicine. Rapid evolution of CRISPR/Cas9 system from the generation of double strand breaks to more advanced applications on gene regulation has made the wide-spread use of this technology possible. Medical science has benefited greatly from CRISPR/Cas9; being both a versatile and economical tool, it has brought gene therapy closer to reality. In this review, the development of CRISPR/Cas9 system, variants thereof and its application in different walks of medical science- research, diagnostics and therapy, will be discussed.},
}
@article {pmid34143358,
year = {2021},
author = {Rato, C and Carvalho, MF and Azevedo, C and Oblessuc, PR},
title = {Genome editing for resistance against plant pests and pathogens.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {427-459},
pmid = {34143358},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/genetics/*immunology ; *Gene Editing ; *Genome, Plant ; Plant Diseases/genetics/*immunology/microbiology/parasitology ; Plants/genetics/*immunology ; Plants, Genetically Modified/*genetics ; },
abstract = {The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.},
}
@article {pmid34142793,
year = {2021},
author = {Smith, CW and Kachwala, MJ and Nandu, N and Yigit, MV},
title = {Recognition of DNA Target Formulations by CRISPR-Cas12a Using a dsDNA Reporter.},
journal = {ACS synthetic biology},
volume = {10},
number = {7},
pages = {1785-1791},
pmid = {34142793},
issn = {2161-5063},
support = {R15 GM128115/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/*genetics ; DNA, Single-Stranded/genetics ; *Genes, Reporter ; },
abstract = {CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched via contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.},
}
@article {pmid34142658,
year = {2021},
author = {Zhou, X and Li, JW and Chen, Z and Ni, W and Li, X and Yang, R and Shen, H and Liu, J and DeMayo, FJ and Lu, J and Kaye, FJ and Wu, L},
title = {Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34142658},
issn = {2050-084X},
support = {R01 CA234351/CA/NCI NIH HHS/United States ; R01 DE023641/DE/NIDCR NIH HHS/United States ; },
mesh = {A549 Cells ; AMP-Activated Protein Kinase Kinases ; AMP-Activated Protein Kinases ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cyclic AMP Response Element-Binding Protein/*genetics/metabolism ; Gene Editing ; Heterografts ; Humans ; *Lung Neoplasms/genetics/metabolism/pathology ; Mice ; Protein Serine-Threonine Kinases/*genetics/metabolism ; Transcription Factors/*genetics/metabolism ; Transcriptome/genetics ; },
abstract = {Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here, we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of human and murine LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.},
}
@article {pmid34140632,
year = {2021},
author = {Gong, J and Zhang, G and Wang, W and Liang, L and Li, Q and Liu, M and Xue, L and Tang, G},
title = {A simple and rapid diagnostic method for 13 types of high-risk human papillomavirus (HR-HPV) detection using CRISPR-Cas12a technology.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {12800},
pmid = {34140632},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Primers/metabolism ; Female ; Humans ; Limit of Detection ; Papillomaviridae/*isolation &amp; purification ; Papillomavirus Infections/*diagnosis/*virology ; Plasmids/genetics ; Risk Factors ; },
abstract = {Cervical carcinoma is the second most common cancer in women worldwide with greater than 99% of the cases caused by human papillomaviruses (HPVs). Early detection of HPVs especially the high risk types (HR-HPVs) are essential to prevent the disease progression. The existing methods for HPV detection, such as qPCR are of high sensitivity and specificity, but the need for expensive machinery and well-trained personnel slow down the disease detection. The emerging Cas12a-based method presents a new technique for nucleic acid detection. However, it is time-consuming and labor-intensive when used for HPV detection, as several reactions are required in order to identify multiple HPV infections. We herein present a non-genotyping method for 13 types of HR-HPV detection in a single reaction by combining the isothermal recombinase polymerase amplification (RPA) method with CRISPR-Cas12a technology. The result could be achieved in 35 min with high sensitivity (500 copies per reaction). This assay represents great advances for the application of RPA-Cas12a system and holds a great potential to address the key challenges facing the HPV diagnostics.},
}
@article {pmid34140489,
year = {2021},
author = {Xu, L and Zhang, C and Li, H and Wang, P and Gao, Y and Mokadam, NA and Ma, J and Arnold, WD and Han, R},
title = {Efficient precise in vivo base editing in adult dystrophic mice.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3719},
pmid = {34140489},
issn = {2041-1723},
support = {R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Dependovirus ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred mdx ; Muscle Fibers, Skeletal/*metabolism/pathology ; Muscle, Skeletal/*metabolism/pathology ; Muscular Dystrophy, Duchenne/*genetics/metabolism/pathology/therapy ; Mutation ; RNA, Guide/genetics ; RNA-Seq ; },
abstract = {Recent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx[4cv] mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases.},
}
@article {pmid34140468,
year = {2021},
author = {Zabrady, K and Zabrady, M and Kolesar, P and Li, AWH and Doherty, AJ},
title = {CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3690},
pmid = {34140468},
issn = {2041-1723},
support = {BB/F013795/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/J018643/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M004236/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacteria/enzymology/*genetics ; Bacterial Proteins/genetics/*metabolism ; Bacteroidetes/enzymology/*genetics ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Computational Biology ; DNA Primase/genetics/*metabolism ; DNA Primers/biosynthesis ; DNA-Directed DNA Polymerase/genetics/*metabolism ; Dimerization ; Escherichia coli/metabolism ; Gene Expression ; Mutation ; Phylogeny ; Prokaryotic Cells/metabolism ; Recombinant Proteins ; Ribonucleotides/metabolism ; },
abstract = {CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.},
}
@article {pmid34139989,
year = {2021},
author = {Jivanji, S and Harland, C and Cole, S and Brophy, B and Garrick, D and Snell, R and Littlejohn, M and Laible, G},
title = {The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {457},
pmid = {34139989},
issn = {1471-2164},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome ; Mutagenesis ; Mutation ; },
abstract = {BACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour.<br><br>RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C&#x2009;>&#x2009;A mutations, and in cloned calves they were predominantly T&#x2009;>&#x2009;G mutations, deviating from the expected excess of C&#x2009;>&#x2009;T mutations.<br><br>CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.},
}
@article {pmid34139668,
year = {2021},
author = {Benler, S and Koonin, EV},
title = {Fishing for phages in metagenomes: what do we catch, what do we miss?.},
journal = {Current opinion in virology},
volume = {49},
number = {},
pages = {142-150},
doi = {10.1016/j.coviro.2021.05.008},
pmid = {34139668},
issn = {1879-6265},
mesh = {Animals ; Bacteria/*virology ; Bacteriophages/classification/*genetics/physiology ; CRISPR-Cas Systems ; *Genome, Viral ; Host Specificity ; Humans ; *Metagenome ; *Metagenomics ; Transcriptome ; },
abstract = {Metagenomics and metatranscriptomics have become the principal approaches for discovery of novel bacteriophages and preliminary characterization of their ecology and biology. Metagenomic sequencing dramatically expanded the known diversity of tailed and non-tailed phages with double-stranded DNA genomes and those with single-stranded DNA genomes, whereas metatranscriptomics led to the discovery of thousands of new single-stranded RNA phages. Apart from expanding phage diversity, metagenomics studies discover major novel groups of phages with unique features of genome organization, expression strategy and virus-host interaction, such as the putative order 'crAssvirales', which includes the most abundant human-associated viruses. The continued success of metagenomics hinges on the combination of the most powerful computational methods for phage genome assembly and analysis including harnessing CRISPR spacers for the discovery of novel phages and host assignment. Together, these approaches could make a comprehensive characterization of the earth phageome a realistic goal.},
}
@article {pmid34467103,
year = {2020},
author = {Crowgey, EL and Lee, MM and Sansbury, B and Kmiec, EB},
title = {Perspectives on Molecular Diagnostic Testing for the COVID-19 Pandemic in Delaware.},
journal = {Delaware journal of public health},
volume = {6},
number = {2},
pages = {20-24},
pmid = {34467103},
issn = {2639-6378},
abstract = {The United States has quickly transitioned into one of the epicenters for the coronavirus pandemic. Limitations for rapid testing for the virus responsible for the pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the single most important barrier for early detection and prevention of future outbreaks. Combining innovative molecular biology techniques, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease systems and next generation sequencing (NGS) may prove to be an effective solution to establish a high-throughput diagnostic and genomic surveillance workflow for COVID-19 in the State of Delaware. Integrating key expertise across the medical institutions in Delaware, including ChristianaCare and Nemours/Alfred I. duPont Hospital for Children, is one potential solution for overcoming current barriers and driving a successful implementation of these techniques.},
}
@article {pmid34395778,
year = {2018},
author = {Borca, MV and Berggren, KA and Ramirez-Medina, E and Vuono, EA and Gladue, DP},
title = {CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus.},
journal = {Bio-protocol},
volume = {8},
number = {16},
pages = {e2978},
pmid = {34395778},
issn = {2331-8325},
abstract = {Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are then able to purify our recombinant virus from the parental by limiting dilution and plaque purification. We previously reported comparing the traditional homologous recombination methodology with CRISPR/Cas9, which resulted in over a 4 log increase in recombination.},
}
@article {pmid34178139,
year = {2018},
author = {Polkoff, K and Piedrahita, JA},
title = {The transformational impact of site-specific DNA modifiers on biomedicine and agriculture.},
journal = {Animal reproduction},
volume = {15},
number = {3},
pages = {171-179},
pmid = {34178139},
issn = {1984-3143},
abstract = {The development of genetically modified livestock has been dependent on incremental technological advances such as embryo transfer, homologous recombination, and somatic cell nuclear transfer (SCNT). This development rate has increased exponentially with the advent of targeted gene modifiers such as zinc finger nucleases, TAL-effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas). CRISPR-Cas based systems in particular have broad applicability, and have low technical and economic barriers for their implementation. As a result, they are having, and will continue to have, a transformational impact in the field of gene editing in domestic animals. With these advances also comes the responsibility to properly apply this technology so it has a beneficial effect throughout all levels of society.},
}
@article {pmid34286046,
year = {2018},
author = {Mohanraju, P and Van Der Oost, J and Jinek, M and Swarts, DC},
title = {Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1.},
journal = {Bio-protocol},
volume = {8},
number = {9},
pages = {e2842},
pmid = {34286046},
issn = {2331-8325},
abstract = {This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments. Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli.},
}
@article {pmid34595323,
year = {2017},
author = {Murakami, Y and Ansai, S and Yonemura, A and Kinoshita, M},
title = {Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins.},
journal = {Bio-protocol},
volume = {7},
number = {24},
pages = {e2665},
pmid = {34595323},
issn = {2331-8325},
abstract = {This protocol describes a simple genotyping using two different colors of fluorescent protein genes inserted at the target locus. This method makes it possible to determine the genotype of each individual simply by observing the fluorescence later than F1 generation.},
}
@article {pmid34595293,
year = {2017},
author = {Hopes, A and Nekrasov, V and Belshaw, N and Grouneva, I and Kamoun, S and Mock, T},
title = {Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions.},
journal = {Bio-protocol},
volume = {7},
number = {23},
pages = {e2625},
pmid = {34595293},
issn = {2331-8325},
abstract = {Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.},
}
@article {pmid34541154,
year = {2017},
author = {Chen, F and Ding, X and Feng, Y and Seebeck, T and Jiang, Y and Davis, GD},
title = {Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting.},
journal = {Bio-protocol},
volume = {7},
number = {15},
pages = {e2432},
pmid = {34541154},
issn = {2331-8325},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012 ; Cong et al., 2013 ; Mali et al., 2013 ; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of the chromatin, were also found to impede target DNA access and cleavage by SpCas9 in vitro (Knight et al., 2015 ; Hinz et al., 2015 ; Horlbeck et al., 2016 ; Isaac et al., 2016). Moreover, many CRISPR-Cas systems characterized to date often exhibit inactivity in mammalian cells and are thus precluded from gene editing applications even though they are active in bacteria or on purified DNA substrates. Thus, there is a need to devise a means to alleviate chromatin inhibition to increase gene editing efficiency, especially on difficult-to-access genomic sites, and to enable use of otherwise inactive CRISPR-Cas nucleases for gene editing need. Here we describe a proxy-CRISPR protocol for restoring nuclease activity of various class 2 CRISPR-Cas nucleases on otherwise inaccessible genomic sites in human cells via proximal targeting of a catalytically dead Cas9 (Chen et al., 2017). This protocol is exemplified here by using Campylobacter jejuni Cas9 (CjCas9) as nuclease and catalytically dead SpCas9 (SpdCas9) as proximal DNA binding protein to enable CjCas9 to cleave the target for gene editing using single stranded DNA oligo templates.},
}
@article {pmid34541105,
year = {2017},
author = {Nomura, T and Sakakibara, H},
title = {Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses.},
journal = {Bio-protocol},
volume = {7},
number = {12},
pages = {e2359},
pmid = {34541105},
issn = {2331-8325},
abstract = {RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.},
}
@article {pmid34150504,
year = {2015},
author = {Stovicek, V and Borodina, I and Forster, J},
title = {CRISPR-Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains.},
journal = {Metabolic engineering communications},
volume = {2},
number = {},
pages = {13-22},
pmid = {34150504},
issn = {2214-0301},
abstract = {There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR-Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories.},
}
@article {pmid34138730,
year = {2021},
author = {Wan, C and Mahara, S and Sun, C and Doan, A and Chua, HK and Xu, D and Bian, J and Li, Y and Zhu, D and Sooraj, D and Cierpicki, T and Grembecka, J and Firestein, R},
title = {Genome-scale CRISPR-Cas9 screen of Wnt/β-catenin signaling identifies therapeutic targets for colorectal cancer.},
journal = {Science advances},
volume = {7},
number = {21},
pages = {},
pmid = {34138730},
issn = {2375-2548},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; *Colorectal Neoplasms/pathology ; Gene Expression Regulation, Neoplastic ; Humans ; TCF Transcription Factors/metabolism ; Wnt Signaling Pathway/genetics ; *beta Catenin/genetics/metabolism ; },
abstract = {Aberrant activation of Wnt/β-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/β-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and β-catenin-mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing β-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of β-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of β-catenin to consensus DNA motifs and the transcription of β-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for β-catenin-driven malignancies.},
}
@article {pmid34138728,
year = {2021},
author = {Ho, TC and Kim, HS and Chen, Y and Li, Y and LaMere, MW and Chen, C and Wang, H and Gong, J and Palumbo, CD and Ashton, JM and Kim, HW and Xu, Q and Becker, MW and Leong, KW},
title = {Scaffold-mediated CRISPR-Cas9 delivery system for acute myeloid leukemia therapy.},
journal = {Science advances},
volume = {7},
number = {21},
pages = {},
pmid = {34138728},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism/therapy ; RNA, Guide/genetics ; Ribonucleoproteins/genetics ; Tumor Microenvironment ; },
abstract = {Leukemia stem cells (LSCs) sustain the disease and contribute to relapse in acute myeloid leukemia (AML). Therapies that ablate LSCs may increase the chance of eliminating this cancer in patients. To this end, we used a bioreducible lipidoid-encapsulated Cas9/single guide RNA (sgRNA) ribonucleoprotein [lipidoid nanoparticle (LNP)-Cas9 RNP] to target the critical gene interleukin-1 receptor accessory protein (IL1RAP) in human LSCs. To enhance LSC targeting, we loaded LNP-Cas9 RNP and the chemokine CXCL12α onto mesenchymal stem cell membrane-coated nanofibril (MSCM-NF) scaffolds mimicking the bone marrow microenvironment. In vitro, CXCL12α release induced migration of LSCs to the scaffolds, and LNP-Cas9 RNP induced efficient gene editing. IL1RAP knockout reduced LSC colony-forming capacity and leukemic burden. Scaffold-based delivery increased the retention time of LNP-Cas9 in the bone marrow cavity. Overall, sustained local delivery of Cas9/IL1RAP sgRNA via CXCL12α-loaded LNP/MSCM-NF scaffolds provides an effective strategy for attenuating LSC growth to improve AML therapy.},
}
@article {pmid34137224,
year = {2021},
author = {Yan, K and Zhang, T and Zha, Y and Liang, J and Cheng, Y},
title = {Construction of point mutation rabbits using CRISPR/Cas9.},
journal = {Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences},
volume = {50},
number = {2},
pages = {229-238},
pmid = {34137224},
issn = {1008-9292},
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; Point Mutation ; *Proprotein Convertase 9/genetics ; Rabbits ; },
abstract = {To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.},
}
@article {pmid34137093,
year = {2021},
author = {Farbiak, L and Cheng, Q and Wei, T and Álvarez-Benedicto, E and Johnson, LT and Lee, S and Siegwart, DJ},
title = {All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {33},
number = {30},
pages = {e2006619},
doi = {10.1002/adma.202006619},
pmid = {34137093},
issn = {1521-4095},
support = {R01 EB025192-01A1/EB/NIBIB NIH HHS/United States ; P30 CA142543/CA/NCI NIH HHS/United States ; P30CA142543/CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; DNA/metabolism ; Dendrimers/*chemistry ; Gene Editing ; HEK293 Cells ; Humans ; In Vitro Techniques ; Liposomes/*chemistry ; Mice, Nude ; Mutation ; Nanoparticles/*chemistry ; RNA, Guide/metabolism ; Recombinational DNA Repair ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91% of all cells with 56% HDR efficiency in vitro and >20% HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.},
}
@article {pmid34135367,
year = {2021},
author = {Liu, WH and Völse, K and Senft, D and Jeremias, I},
title = {A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {12649},
pmid = {34135367},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genes, Reporter ; Heterografts ; Humans ; Tumor Cells, Cultured ; },
abstract = {CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.},
}
@article {pmid34135108,
year = {2021},
author = {Lattanzi, A and Camarena, J and Lahiri, P and Segal, H and Srifa, W and Vakulskas, CA and Frock, RL and Kenrick, J and Lee, C and Talbott, N and Skowronski, J and Cromer, MK and Charlesworth, CT and Bak, RO and Mantri, S and Bao, G and DiGiusto, D and Tisdale, J and Wright, JF and Bhatia, N and Roncarolo, MG and Dever, DP and Porteus, MH},
title = {Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.},
journal = {Science translational medicine},
volume = {13},
number = {598},
pages = {},
pmid = {34135108},
issn = {1946-6242},
support = {R01 HL135607/HL/NHLBI NIH HHS/United States ; R01 HL152314/HL/NHLBI NIH HHS/United States ; },
mesh = {*Anemia, Sickle Cell/genetics/therapy ; Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells ; *Heterocyclic Compounds ; Humans ; Mice ; Reproducibility of Results ; beta-Globins/genetics ; },
abstract = {Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (HBB). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34[+] cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.},
}
@article {pmid34135000,
year = {2021},
author = {Sharif, GM and Campbell, MJ and Nasir, A and Sengupta, S and Graham, GT and Kushner, MH and Kietzman, WB and Schmidt, MO and Pearson, GW and Loudig, O and Fineberg, S and Wellstein, A and Riegel, AT},
title = {An AIB1 Isoform Alters Enhancer Access and Enables Progression of Early-Stage Triple-Negative Breast Cancer.},
journal = {Cancer research},
volume = {81},
number = {16},
pages = {4230-4241},
pmid = {34135000},
issn = {1538-7445},
support = {R21 CA226542/CA/NCI NIH HHS/United States ; P30 CA051008/CA/NCI NIH HHS/United States ; R01 CA205632/CA/NCI NIH HHS/United States ; T32 CA009686/CA/NCI NIH HHS/United States ; F31 CA232664/CA/NCI NIH HHS/United States ; R01 CA218670/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques, Three Dimensional ; Cell Line, Tumor ; Dexamethasone/chemistry ; Disease Progression ; Electric Impedance ; Enhancer Elements, Genetic ; Female ; Humans ; Lung Neoplasms/pathology ; Mice ; Mice, SCID ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Transplantation ; Nuclear Receptor Coactivator 3/chemistry/*genetics ; Phenotype ; Protein Isoforms ; RNA Splicing ; Receptors, Glucocorticoid/metabolism ; Signal Transduction ; Thiazolidinediones/pharmacology ; Triple Negative Breast Neoplasms/*genetics/*metabolism ; Zebrafish ; },
abstract = {AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an "enabler" phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. SIGNIFICANCE: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.},
}
@article {pmid34134774,
year = {2021},
author = {Robbins, GM and Wang, M and Pomeroy, EJ and Moriarity, BS},
title = {Nonviral genome engineering of natural killer cells.},
journal = {Stem cell research &amp; therapy},
volume = {12},
number = {1},
pages = {350},
pmid = {34134774},
issn = {1757-6512},
mesh = {*Genetic Engineering ; Humans ; Immunotherapy ; Immunotherapy, Adoptive ; Killer Cells, Natural ; *Neoplasms/genetics/therapy ; Receptors, Antigen, T-Cell/genetics ; Tumor Microenvironment ; },
abstract = {Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.},
}
@article {pmid34134705,
year = {2021},
author = {D'Souza, R and Nguyen, LP and Pinto, NA and Lee, H and Vu, TN and Kim, H and Cho, HS and Yong, D},
title = {Role of AmpG in the resistance to β-lactam agents, including cephalosporins and carbapenems: candidate for a novel antimicrobial target.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {20},
number = {1},
pages = {45},
pmid = {34134705},
issn = {1476-0711},
mesh = {Amino Acid Sequence ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; Carbapenems/*pharmacology ; Cephalosporins/*pharmacology ; DNA Transposable Elements ; Democratic People's Republic of Korea ; Gene Expression Regulation, Bacterial/drug effects ; Gene Knockout Techniques ; Humans ; Klebsiella pneumoniae/genetics ; Membrane Transport Proteins/chemistry/*genetics/metabolism ; Microbial Sensitivity Tests ; Mutagenesis ; Sequence Alignment ; beta-Lactam Resistance/drug effects/*genetics ; beta-Lactams/*pharmacology ; },
abstract = {BACKGROUND: A complex cascade of genes, enzymes, and transcription factors regulates AmpC β-lactamase overexpression. We investigated the network of AmpC β-lactamase overexpression in Klebsiella aerogenes and identified the role of AmpG in resistance to β-lactam agents, including cephalosporins and carbapenems.<br><br>METHODS: A transposon mutant library was created for carbapenem-resistant K. aerogenes YMC2008-M09-943034 (KE-Y1) to screen for candidates with increased susceptibility to carbapenems, which identified the susceptible mutant derivatives KE-Y3 and KE-Y6. All the strains were subjected to highly contiguous de novo assemblies using PacBio sequencing to investigate the loss of resistance due to transposon insertion. Complementation and knock-out experiments using lambda Red-mediated homologous recombinase and CRISPR-Cas9 were performed to confirm the role of gene of interest.<br><br>RESULTS: In-depth analysis of KE-Y3 and KE-Y6 revealed the insertion of a transposon at six positions in each strain, at which truncation of the AmpG permease gene was common in both. The disruption of the AmpG permease leads to carbapenem susceptibility, which was further confirmed by complementation. We generated an AmpG permease gene knockout using lambda Red-mediated recombineering in K. aerogenes KE-Y1 and a CRISPR-Cas9-mediated gene knockout in multidrug-resistant Klebsiella pneumoniae-YMC/2013/D to confer carbapenem susceptibility.<br><br>CONCLUSIONS: These findings suggest that inhibition of the AmpG is a potential strategy to increase the efficacy of &#x3b2;-lactam agents against Klebsiella aerogenes.},
}
@article {pmid34133740,
year = {2021},
author = {Zhang, W and Yin, J and Zhang-Ding, Z and Xin, C and Liu, M and Wang, Y and Ai, C and Hu, J},
title = {In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.},
journal = {Nucleic acids research},
volume = {49},
number = {15},
pages = {8785-8795},
pmid = {34133740},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Plant/genetics ; Mutation/genetics ; Oryza/*genetics ; Streptococcus pyogenes/*enzymology ; },
abstract = {A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.},
}
@article {pmid34133710,
year = {2021},
author = {Alkhnbashi, OS and Mitrofanov, A and Bonidia, R and Raden, M and Tran, VD and Eggenhofer, F and Shah, SA and Öztürk, E and Padilha, VA and Sanches, DS and de Carvalho, ACPLF and Backofen, R},
title = {CRISPRloci: comprehensive and accurate annotation of CRISPR-Cas systems.},
journal = {Nucleic acids research},
volume = {49},
number = {W1},
pages = {W125-W130},
pmid = {34133710},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/classification/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Machine Learning ; *Molecular Sequence Annotation ; *Software ; },
abstract = {CRISPR-Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR-Cas system characterization that offers annotation quality previously available only after manual inspection.},
}
@article {pmid34132917,
year = {2021},
author = {Mishra, R and Mohanty, JN and Mahanty, B and Joshi, RK},
title = {A single transcript CRISPR/Cas9 mediated mutagenesis of CaERF28 confers anthracnose resistance in chilli pepper (Capsicum annuum L.).},
journal = {Planta},
volume = {254},
number = {1},
pages = {5},
pmid = {34132917},
issn = {1432-2048},
mesh = {CRISPR-Cas Systems ; *Capsicum/genetics ; Colletotrichum ; Mutagenesis ; Plant Diseases/genetics ; },
abstract = {T-DNA-free homozygous mutant lines developed through a single transcript CRISPR/Cas9 system harboring the desired modification in the CaERF28 locus exhibited significantly enhanced resistance to the anthracnose pathogen Colletotrichum truncatum coupled with the improved expression of defense responsive genes. Anthracnose, caused by Colletotrichum species, is a major disease of chilli (Capsicum annuum) accounting for significant pre- and post-harvest yield losses across the tropical and subtropical regions of the world. Management of chilli anthracnose using traditional methods have not met with noticeable success. In the present study, we have demonstrated an enhanced anthracnose resistance through a single transcript unit CRISPR/Cas9 mediated alteration of the susceptibility gene CaERF28 in C. annuum. A construct with a single Pol II promoter-driven expression of Cas9, sgRNA and a hammerhead ribozyme (RZ) was designed to modify the CaERF28 gene in the susceptible chilli genotype Arka Lohit. Fourty-five C-ERF28-induced mutant lines (72.5%) were identified from 62 T0 transgenic plants. Further, simultaneously targeted multiple sites within CaERF28 showed increased mutation (85.7%) efficiency. DNA sequence analysis showed that these plants harboured multiple InDels at the target site. The allelic mutants of C-ERF28 were transferred to the following generations by simple Mendelian inheritance. Segregation in the T1 and T2 generations resulted in the identification of T-DNA free and marker-free C-ERF28 mutant lines. Five homozygous mutants demonstrated enhanced resistance to anthracnose compared to wild type as demonstrated by reduced spore count and fungal growth as well as induced expression of defense-related genes. Our results demonstrated that the STU-CRISPR/Cas9 mediated editing of the CaERF28 gene is a rapid, safe and versatile approach for enhancing anthracnose resistance in chilli pepper and pave way for its utilization in the improvement of other solanaceous crops.},
}
@article {pmid34132590,
year = {2021},
author = {Deecker, SR and Urbanus, ML and Nicholson, B and Ensminger, AW},
title = {Legionella pneumophila CRISPR-Cas Suggests Recurrent Encounters with One or More Phages in the Family Microviridae.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {17},
pages = {e0046721},
pmid = {34132590},
issn = {1098-5336},
mesh = {Bacteriophages/classification/genetics/*isolation &amp; purification ; CRISPR-Cas Systems ; DNA Transposable Elements ; Humans ; Legionella pneumophila/genetics/*virology ; Legionnaires' Disease/microbiology ; Microviridae/classification/genetics/*isolation &amp; purification ; Phylogeny ; },
abstract = {Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires' disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella mobile element 1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila's adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants-all targeted by previously and newly identified CRISPR-Cas spacers-but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae. These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires' disease is an often-fatal pneumonia caused by Legionella pneumophila, which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the "missing" L. pneumophila phages belong to the microvirus subfamily Gokushovirinae. Gokushoviruses are known to infect another intracellular pathogen, Chlamydia. How do gokushoviruses access L. pneumophila (and Chlamydia) inside their "cozy niches"? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.},
}
@article {pmid34131407,
year = {2021},
author = {Zhan, Y and Li, XP and Yin, JY},
title = {COVID-19 one year later: a retrospect of CRISPR-Cas system in combating COVID-19.},
journal = {International journal of biological sciences},
volume = {17},
number = {8},
pages = {2080-2088},
pmid = {34131407},
issn = {1449-2288},
mesh = {Animals ; COVID-19/*diagnosis/*therapy ; COVID-19 Nucleic Acid Testing/*methods ; *CRISPR-Cas Systems ; Fluorometry/methods ; Forecasting ; Gene Editing/*methods ; Gene Expression Regulation, Viral/*genetics ; Gene Knockout Techniques ; High-Throughput Nucleotide Sequencing ; Host-Pathogen Interactions ; Humans ; Mice ; Mice, Transgenic ; Models, Animal ; Molecular Targeted Therapy ; Nasopharynx/virology ; Oropharynx/virology ; RNA, Viral/*analysis/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; SARS-CoV-2/*genetics/isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {Coronavirus disease 2019 (COVID-19), an infectious disease caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a persistent global threat. The transmission of SARS-CoV-2 is wide and swift. Rapid detection of the viral RNA and effective therapy are imperative to prevent the worldwide spread of the new infectious disease. Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein (Cas) system is an RNA-directed adaptive immune system, and it has been transformed into a gene editing tool. Applications of CRISPR-Cas system involves in many fields, such as human gene therapy, drug discovery and disease diagnosis. Under the background of COVID-19 pandemic, CRISPR-Cas system shows hidden capacity to fight the emergency in many aspects. This review will focus on the role of gene editing in COVID-19 diagnosis and treatment. We will describe the potential use of CRISPR-Cas-based system in combating COVID-19, from diagnosis to treatment. Furthermore, the limitation and perspectives of this novel technology are also evaluated.},
}
@article {pmid34131248,
year = {2021},
author = {Das, S and Chadwick, BP},
title = {CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {12598},
pmid = {34131248},
issn = {2045-2322},
support = {R21 NS080779/NS/NINDS NIH HHS/United States ; },
mesh = {Adult ; CRISPR-Cas Systems/genetics ; Epigenomics ; Female ; Gene Targeting ; HCT116 Cells ; Heterochromatin/genetics ; Homeodomain Proteins/*genetics ; Humans ; Male ; Muscle Cells/metabolism ; Muscular Dystrophy, Facioscapulohumeral/*genetics/pathology ; Primary Cell Culture ; RNA, Messenger ; Regulatory Sequences, Nucleic Acid/*genetics ; *Transcription, Genetic ; },
abstract = {Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.},
}
@article {pmid34130596,
year = {2022},
author = {Pacheco, MB and Camilo, V and Henrique, R and Jerónimo, C},
title = {Epigenetic Editing in Prostate Cancer: Challenges and Opportunities.},
journal = {Epigenetics},
volume = {17},
number = {5},
pages = {564-588},
pmid = {34130596},
issn = {1559-2308},
mesh = {CRISPR-Cas Systems ; Chromatin ; DNA Methylation ; Epigenesis, Genetic ; *Gene Editing ; Humans ; Male ; *Prostatic Neoplasms/genetics ; },
abstract = {Epigenome editing consists of fusing a predesigned DNA recognition unit to the catalytic domain of a chromatin modifying enzyme leading to the introduction or removal of an epigenetic mark at a specific locus. These platforms enabled the study of the mechanisms and roles of epigenetic changes in several research domains such as those addressing pathogenesis and progression of cancer. Despite the continued efforts required to overcome some limitations, which include specificity, off-target effects, efficacy, and longevity, these tools have been rapidly progressing and improving.Since prostate cancer is characterized by multiple genetic and epigenetic alterations that affect different signalling pathways, epigenetic editing constitutes a promising strategy to hamper cancer progression. Therefore, by modulating chromatin structure through epigenome editing, its conformation might be better understood and events that drive prostate carcinogenesis might be further unveiled.This review describes the different epigenome engineering tools, their mechanisms concerning gene's expression and regulation, highlighting the challenges and opportunities concerning prostate cancer research.},
}
@article {pmid34128206,
year = {2022},
author = {Getachew, H and Chinchilla, B and Fernandez-Godino, R},
title = {Genome Editing of Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes to Model Genetic Ocular Diseases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2549},
number = {},
pages = {321-334},
pmid = {34128206},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Clinical Protocols ; *Eye Diseases, Hereditary/genetics ; *Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells ; Ribonucleoproteins/genetics ; },
abstract = {Genome editing with the use of CRISPR/Cas9 ribonucleoprotein complexes of induced pluripotent stem cells can be used to model many diseases. The combination of stem cells and gene editing technologies is a valuable tool to study ocular disorders, as many have been identified to be caused by specific genetic mutations. This protocol provides experimentally derived guidelines for genome editing of human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 ribonucleoprotein complexes to generate iPSCs harboring single nucleotide variants from ocular disorders. Edited iPSC can be further differentiated into retinal cells in order to study disease mechanisms as well as screen potential therapies.},
}
@article {pmid34127790,
year = {2021},
author = {Sanchez, CG and Acker, CM and Gray, A and Varadarajan, M and Song, C and Cochran, NR and Paula, S and Lindeman, A and An, S and McAllister, G and Alford, J and Reece-Hoyes, J and Russ, C and Craig, L and Capre, K and Doherty, C and Hoffman, GR and Luchansky, SJ and Polydoro, M and Dolmetsch, R and Elwood, F},
title = {Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {736},
pmid = {34127790},
issn = {2399-3642},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Genes/genetics/physiology ; Genetic Testing/methods ; Genome-Wide Association Study ; Humans ; Metabolic Networks and Pathways/*genetics ; Mice ; Neuroblastoma/metabolism ; Neurons/*metabolism ; Rats ; TOR Serine-Threonine Kinases/metabolism ; tau Proteins/*metabolism ; },
abstract = {Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.},
}
@article {pmid34127203,
year = {2021},
author = {Uslu, M and Siyah, P and Harvey, AJ and Kocabaş, F},
title = {Modulating Cas9 activity for precision gene editing.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {89-127},
doi = {10.1016/bs.pmbts.2021.01.015},
pmid = {34127203},
issn = {1878-0814},
mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genomics ; Humans ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 is a RNA-guided nuclease complex that can be specifically programmed to target a user-specified DNA sequence. It has been a powerful and effective tool of genome editing. However, off-target activity of the Cas9 nuclease limits its potential use in the correction of inherited diseases and bona fide gene editing. Various protein engineering and guide RNA selection strategies have been utilized to improve Cas9-based genome-editing specificity and efficiency. We, however, have not yet achieved a degree of safety such that Cas9 gene editing approaches could be applicable in clinical settings. Here, we discuss the recently developed and precise gene editing technologies based on spCas9. Furthermore, we describe Cas9 modulating tools to increase the fidelity of the CRISPR/Cas9 system. These studies suggest that there is still a need for pharmaceutical modulation of Cas9 activity during gene editing procedures. Pharmaceutical modulation of Cas9 nuclease activity at on-target or off-target genomic loci could 1 day allow researchers to develop robust and precise therapeutical strategies in gene editing.},
}
@article {pmid34127202,
year = {2021},
author = {Poletto, E and Baldo, G},
title = {Creating cell lines for mimicking diseases.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {59-87},
doi = {10.1016/bs.pmbts.2021.01.014},
pmid = {34127202},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Phenotype ; Transfection ; },
abstract = {Cell lines can be good models for the disease they are derived from but can also be used to study general physiological and pathological processes. They can also be used to generate cell models of diseases when primary cultures are not available. Recent genome editing tools have been very promising tools toward creating cell models to mimic diseases in vitro. In this chapter, we highlight techniques used to obtain genome-edited cell lines, including cell line selection, transfection and gene editing tools available, together with methods of phenotype characterization and, lastly, a few examples of how in vitro disease models were created using CRISPR-Cas9.},
}
@article {pmid34127201,
year = {2021},
author = {Bhattacharjee, G and Gohil, N and Lam, NL and Singh, V},
title = {CRISPR-based diagnostics for detection of pathogens.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {45-57},
doi = {10.1016/bs.pmbts.2021.01.013},
pmid = {34127201},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {The improved sensitivity and superior specificity associated with the use of molecular assays has improved the fate of disease diagnosis by bestowing the clinicians with outcomes that are both rapid and precise. In recent years, CRISPR has made considerable progress in in vitro diagnostic platform which has paved its way for developing rapid and sensitive CRISPR-based diagnostic tools. Improved perception and better understanding of diverse CRISPR-Cas systems has broadened the reach of CRISPR applications for not just early detection of pathogens but also for early onset of diseases such as cancer. The inherent allele specificity of CRISPR is the predominant reason for its application in designing a diagnostic-tool that is field-deployable, portable, sensitive, specific and rapid. In this chapter, we highlight various CRISPR-based diagnostic platforms, its applications, challenges and future prospects of the CRISPR-Cas system.},
}
@article {pmid34127200,
year = {2021},
author = {Nxumalo, Z and Takundwa, MM and Thimiri Govinda Raj, DB},
title = {Patents, ethics, biosafety and regulation using CRISPR technology.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {345-365},
doi = {10.1016/bs.pmbts.2021.01.023},
pmid = {34127200},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; China ; *Containment of Biohazards ; Humans ; Technology ; },
abstract = {In this review chapter, we provide full comprehensive analysis on the patent, ethics and biosafety regulation with respect to the application of CRISPR technology in mammalian systems. We focused on recent development in CRISPR technology and its patent landscape between countries such as US, European Union, China and Australia. Further, we emphasized on the current scenarios on the ethics regulations with respect to CRISPR research, its applicability in patent and technology transfer. Finally, we elaborated on the biosafety regulation on CRISPR/Cas9 technology application in both mammalian and non-mammalian host system.},
}
@article {pmid34127199,
year = {2021},
author = {Rodríguez, TC and Dadafarin, S and Pratt, HE and Liu, P and Amrani, N and Zhu, LJ},
title = {Genome-wide detection and analysis of CRISPR-Cas off-targets.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {31-43},
doi = {10.1016/bs.pmbts.2021.01.012},
pmid = {34127199},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; *RNA, Guide ; },
abstract = {The clustered, regularly interspersed, short palindromic repeats (CRISPR) technology is revolutionizing biological studies and holds tremendous promise for treating human diseases. However, a significant limitation of this technology is that modifications can occur on off-target sites lacking perfect complementarity to the single guide RNA (sgRNA) or canonical protospacer-adjacent motif (PAM) sequence. Several in vivo and in vitro genome-wide off-target profiling approaches have been developed to inform on the fidelity of gene editing. Of these, GUIDE-seq has become one of the most widely adopted and reproducible methods. To allow users to easily analyze GUIDE-seq data generated on any sequencing platform, we developed an open-source pipeline, GS-Preprocess, that takes standard base-call output in bcl format and generate all required input data for off-target identification using bioconductor package GUIDEseq for off-target identification. Furthermore, we created a Docker image with GS-Proprocess, GUIDE-seq, and all its R and system dependencies already installed. The bundled pipeline will empower end users to streamline the analysis of GUIDE-seq data and motivate their use of higher throughput sequencing with increased multiplexing for GUIDE-seq experiments.},
}
@article {pmid34127198,
year = {2021},
author = {Sar, P and Dalai, S},
title = {CRISPR/Cas9 in epigenetics studies of health and disease.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {309-343},
doi = {10.1016/bs.pmbts.2021.01.022},
pmid = {34127198},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Methylation ; Epigenesis, Genetic ; Epigenomics ; *Gene Editing ; },
abstract = {Epigenetics is the heritable phenotypic changes without altering the genotype. Epigenetic processes are such as histone methylation, acetylation, ubiquitination, sumoylation, phosphorylation, ADP ribosylation, DNA methylation and non-coding RNAs interactions associated with structural changes in chromatin. The change of structure is either open chromatin for "active" state or closed chromatin for "inactive" state, that regulates important biological phenomenon like chromatin condensation, gene expression, DNA repair, cellular development, differentiation and homeostasis, etc. However, dysregulation of epigenetic patterns causes diseases like cancer, diabetes, neurological disorder, infectious diseases, autoimmunity etc. Besides, the most important clinical uses of Epigenetics studies are i. identification of disease biomarkers and ii. development of their therapeutics. Epigenetic therapies include epi-drugs, combinatorial therapy, nanocarriers, plant-derived products that are being used for changing the epigenetic pattern to reverse gene expression. However, the developed epi- drugs cause off-target gene and transposable elements activation; promote mutagenesis and carcinogenesis in normal cells, are the major hurdles regarding their clinical use. Therefore, advanced epigenetic therapeutics are required to develop target-specific epigenetic modifications to reverse gene expression pattern. CRISPR-Cas9 (Clustered Regularly Interspaced Palindrome Repeats-associated protein 9) system-mediated gene activation mechanism paves new methods of target-specific epigenetic therapeutics to cure diseases. In this chapter, we discuss how CRISPR/Cas9 and dCas9 have recently been engineered for epigenome editing. Different strategies have been discussed used for epigenome editing based on their efficacy and complexity. Last but not least we have discussed the limitations, different uses of CRISPR/Cas9 and dCas9 in the area of genetic engineering.},
}
@article {pmid34127197,
year = {2021},
author = {Mani, I},
title = {Genome editing in cardiovascular diseases.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {289-308},
doi = {10.1016/bs.pmbts.2021.01.021},
pmid = {34127197},
issn = {1878-0814},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Cardiovascular Diseases/genetics/therapy ; *Gene Editing ; Humans ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases ; },
abstract = {Genetic modification at the molecular level in somatic cells, germline, and animal models requires for different purposes, such as introducing desired mutation, deletion of alleles, and insertion of novel genes in the genome. Various genome-editing tools are available to accomplish these alterations, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) system. CRISPR-Cas system is an emerging technology, which is being used in biological and medical sciences, including in the cardiovascular field. It assists to identify the mechanism of various cardiovascular disease occurrence, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic cardiomyopathy (ACM). Furthermore, it has been advantages to edit various genes simultaneously and can also be used to treat and prevent several human diseases. This chapter explores the use of the scientific and therapeutic potential of a CRISPR-Cas system to edit the various cardiovascular disease-associated genes to understand the pathways involved in disease progression and treatment.},
}
@article {pmid34127196,
year = {2021},
author = {Choi, DK and Kim, YK and HoonYu, J and Min, SH and Park, SW},
title = {Genome editing of hPSCs: Recent progress in hPSC-based disease modeling for understanding disease mechanisms.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {271-287},
doi = {10.1016/bs.pmbts.2021.01.020},
pmid = {34127196},
issn = {1878-0814},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Generation of proper models for studying human genetic diseases has been hindered until recently by the scarcity of primary cell samples from genetic disease patients and inefficient genetic modification tools. However, recent advances in clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and human induced pluripotent stem cells (hiPSCs) have provided an opportunity to explore the function of pathogenic variants and obtain gene-corrected cells for autologous cell therapy. In this chapter, we address recent applications of CRISPR/Cas9 to hiPSCs in genetic diseases, including neurodegenerative, cardiovascular, and rare diseases.},
}
@article {pmid34127195,
year = {2021},
author = {Bayarsaikhan, D and Bayarsaikhan, G and Lee, B},
title = {Recent advances in stem cells and gene editing: Drug discovery and therapeutics.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {231-269},
doi = {10.1016/bs.pmbts.2021.01.019},
pmid = {34127195},
issn = {1878-0814},
mesh = {CRISPR-Cas Systems/genetics ; *Drug Discovery ; *Gene Editing ; Humans ; Stem Cells/metabolism ; Transcription Activator-Like Effector Nucleases/metabolism ; },
abstract = {The recently introduced genome editing technology has had a remarkable impact on genetic medicine. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas nucleases are the three major platforms used for priming of stem cells or correction of mutated genes. Among these nucleases, CRISPR/Cas is the most easily applicable. Various CRISPR/Cas variants such as base editors, prime editors, mad7 nucleases, RESCUE, REPAIR, digenome sequencing, and SHERLOCK are being developed and considered as a promising tool for gene therapy and drug discovery. These advances in the CRISPR/Cas platform have enabled the correction of gene mutations from DNA to RNA level and validation of the safety of genome editing performance at a very precise level by allowing the detection of one base-pair mismatch. These promising alternatives of the CRISPR/Cas system can benefit millions of patients with intractable diseases. Although the therapeutic effects of stem cells have been confirmed in a wide range of disease models, their safety still remains an issue. Hence, scientists are concentrating on generating functionally improved stem cells by using programmable nucleases such as CRISPR. Therefore, in this chapter, we have summarized the applicable options of the CRISPR/Cas platforms by weighing their advantages and limitations in drug discovery and gene therapy.},
}
@article {pmid34127194,
year = {2021},
author = {Karapurkar, JK and Antao, AM and Kim, KS and Ramakrishna, S},
title = {CRISPR-Cas9 based genome editing for defective gene correction in humans and other mammals.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {185-229},
doi = {10.1016/bs.pmbts.2021.01.018},
pmid = {34127194},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing ; Gene Targeting ; Humans ; Mammals/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR/Cas9), derived from bacterial and archean immune systems, has received much attention from the scientific community as a powerful, targeted gene editing tool. The CRISPR/Cas9 system enables a simple, relatively effortless and highly specific gene targeting strategy through temporary or permanent genome regulation or editing. This endonuclease has enabled gene correction by taking advantage of the endogenous homology directed repair (HDR) pathway to successfully target and correct disease-causing gene mutations. Numerous studies using CRISPR support the promise of efficient and simple genome manipulation, and the technique has been validated in in vivo and in vitro experiments, indicating its potential for efficient gene correction at any genomic loci. In this chapter, we detailed various strategies related to gene editing using the CRISPR/Cas9 system. We also outlined strategies to improve the efficiency of gene correction via the HDR pathway and to improve viral and non-viral mediated gene delivery methods, with an emphasis on their therapeutic potential for correcting genetic disorder in humans and other mammals.},
}
@article {pmid34127193,
year = {2021},
author = {Mani, I},
title = {CRISPR-Cas9 for treating hereditary diseases.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {165-183},
doi = {10.1016/bs.pmbts.2021.01.017},
pmid = {34127193},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Genetic Therapy ; Humans ; Mutation ; },
abstract = {This chapter analyzes to use of the genome editing tool to the treatment of various genetic diseases. The genome editing method could be used to change the DNA in cells or organisms to understand their physiological response. Therefore, a key objective is to present general information about the use of the genome editing tool in a pertinent way. An emerging genome editing technology like a clustered regularly short palindromic repeats (CRISPR) is an extensively expended in biological sciences. CRISPR and CRISPR-associated protein 9 (CRISPR-Cas9) technique is being utilized to edit any DNA mutations associated with hereditary diseases to study in cells (in vitro) and animals (in vivo). Interestingly, CRISPR-Cas9 could be used to the investigation of treatments of various human hereditary diseases such as hemophila, β-thalassemia, cystic fibrosis, Alzheimer's, Huntington's, Parkinson's, tyrosinemia, Duchnene muscular dystrophy, Tay-Sachs, and fragile X syndrome disorders. Furthermore, CRISPR-Cas9 could also be used in other diseases to the improvement of human health. Finally, this chapter discuss current progress to treatment for hereditary diseases using CRISPR-Cas9 technology and highlights associated challenges and future prospects.},
}
@article {pmid34127192,
year = {2021},
author = {Mani, I and Arazoe, T and Singh, V},
title = {CRISPR-Cas systems for genome editing of mammalian cells.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {15-30},
doi = {10.1016/bs.pmbts.2021.01.011},
pmid = {34127192},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Humans ; Phenotype ; Transcription Activator-Like Effector Nucleases ; },
abstract = {In the past decade, ZFNs and TALENs have been used for targeted genome engineering and have gained scientific attention. It has demonstrated huge potential for gene knockout, knock-in, and indels in desired locations of genomes to understand molecular mechanism of diseases and also discover therapy. However, both the genome engineering techniques are still suffering from design, screening and validation in cell and higher organisms. CRISPR-Cas9 is a rapid, simple, specific, and versatile technology and it has been applied in many organisms including mammalian cells. CRISPR-Cas9 has been used for animal models to modify animal cells for understanding human disease for novel drug discovery and therapy. Additionally, base editing has also been discussed herewith for conversion of C/G-to-T/A or A/T-to-G/C without DNA cleavage or donor DNA templates for correcting mutations or altering gene functions. In this chapter, we highlight CRISPR-Cas9 and base editing for desired genome editing in mammalian cells for a better understanding of molecular mechanisms, and biotechnological and therapeutic applications.},
}
@article {pmid34127191,
year = {2021},
author = {Randhawa, S},
title = {CRISPR-Cas9 in cancer therapeutics.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {129-163},
doi = {10.1016/bs.pmbts.2021.01.016},
pmid = {34127191},
issn = {1878-0814},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Humans ; Mutation ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer is a disease mainly caused by an accumulation of mutations in cells. Consequently, correcting those genetic aberrations could be a potential treatment strategy. The traditional route for cancer drug development is tedious, laborious, and time-consuming. Due to target identification, drug formulation, pre-clinical testing, clinical testing, and regulatory hurdles, on average, it takes 10-15 years for a cancer drug to go from target discovery to a marketable oncology drug. The advent of CRISPR-Cas9 technology has greatly expedited this procedure. CRISPR-Cas9 has single-handedly accelerated target identification and pre-clinical testing. Furthermore, CRISPR-Cas9 has also been used in ex vivo editing of T-cells to specifically target tumor cells. In this chapter, we will discuss the various ways in which CRISPR-Cas9 has been used for the betterment of the cancer drug development process. Additionally, we will discuss various ways in which it is currently being used as therapy and the drawbacks which restrict the use of this groundbreaking technology as direct therapy.},
}
@article {pmid34127190,
year = {2021},
author = {Singh, V},
title = {An introduction to CRISPR-Cas systems for reprogramming the genome of mammalian cells.},
journal = {Progress in molecular biology and translational science},
volume = {181},
number = {},
pages = {1-13},
doi = {10.1016/bs.pmbts.2021.01.010},
pmid = {34127190},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; Phenotype ; },
abstract = {In the past few decades, it has been possible to introduce unprecedented mutations in genes of the mammalian cells owing to the development of advanced technologies/methods/assays. Sometimes, these mutations occurring at the cellular level may even cost the life of organisms. A number of diseases in mammals have shown to leave some serious impact on their lives. There are no drugs or medicines available in market for the correction or repair of these mutated genes in order to reverse gene function. A pressing need therefore arises to develop a next generation technology that cannot just corrects gene mutations but also restores gene function. Recent advances in CRISPR-Cas9 technology play a key role for correction of defective genes in wide range of mammalian cells. This chapter highlights CRISPR-Cas systems for basic, biomedical, biotechnological and therapeutic applications.},
}
@article {pmid34125584,
year = {2021},
author = {Backes, N and Phillips, GJ},
title = {Repurposing CRISPR-Cas Systems as Genetic Tools for the Enterobacteriales.},
journal = {EcoSal Plus},
volume = {9},
number = {2},
pages = {eESP00062020},
doi = {10.1128/ecosalplus.ESP-0006-2020},
pmid = {34125584},
issn = {2324-6200},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome ; Plasmids/genetics ; },
abstract = {Over the last decade, the study of CRISPR-Cas systems has progressed from a newly discovered bacterial defense mechanism to a diverse suite of genetic tools that have been applied across all domains of life. While the initial applications of CRISPR-Cas technology fulfilled a need to more precisely edit eukaryotic genomes, creative "repurposing" of this adaptive immune system has led to new approaches for genetic analysis of microorganisms, including improved gene editing, conditional gene regulation, plasmid curing and manipulation, and other novel uses. The main objective of this review is to describe the development and current state-of-the-art use of CRISPR-Cas techniques specifically as it is applied to members of the Enterobacteriales. While many of the applications covered have been initially developed in Escherichia coli, we also highlight the potential, along with the limitations, of this technology for expanding the availability of genetic tools in less-well-characterized non-model species, including bacterial pathogens.},
}
@article {pmid34125434,
year = {2021},
author = {Dovgan, T and Golghalyani, V and Zurlo, F and Hatton, D and Lindo, V and Turner, R and Harris, C and Cui, T},
title = {Targeted CHO cell engineering approaches can reduce HCP-related enzymatic degradation and improve mAb product quality.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {10},
pages = {3821-3831},
doi = {10.1002/bit.27857},
pmid = {34125434},
issn = {1097-0290},
mesh = {Animals ; *Antibodies, Monoclonal/biosynthesis/genetics ; CHO Cells ; *CRISPR-Cas Systems ; Cricetulus ; *Gene Expression ; Humans ; *Metabolic Engineering ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable short hairpin RNA interference or monoallelic deletion of the target gene using CRISPR-Cas9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification.},
}
@article {pmid34125173,
year = {2021},
author = {Katerndahl, CDS and Rogers, ORS and Day, RB and Cai, MA and Rooney, TP and Helton, NM and Hoock, M and Ramakrishnan, SM and Nonavinkere Srivatsan, S and Wartman, LD and Miller, CA and Ley, TJ},
title = {Tumor suppressor function of Gata2 in acute promyelocytic leukemia.},
journal = {Blood},
volume = {138},
number = {13},
pages = {1148-1161},
pmid = {34125173},
issn = {1528-0020},
support = {R50 CA211782/CA/NCI NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; P01 CA101937/CA/NCI NIH HHS/United States ; R35 CA197561/CA/NCI NIH HHS/United States ; T32 HL007088/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Progression ; GATA2 Transcription Factor/*genetics/metabolism ; Gene Expression Regulation, Leukemic ; Genes, Tumor Suppressor ; Humans ; Leukemia, Promyelocytic, Acute/*genetics/metabolism/pathology ; Mice ; Mutation ; },
abstract = {Most patients with acute promyelocytic leukemia (APL) can be cured with combined all-trans retinoic acid (ATRA) and arsenic trioxide therapy, which induces the destruction of PML-RARA, the initiating fusion protein for this disease. However, the underlying mechanisms by which PML-RARA initiates and maintains APL cells are still not clear. Therefore, we identified genes that are dysregulated by PML-RARA in mouse and human APL cells and prioritized GATA2 for functional studies because it is highly expressed in preleukemic cells expressing PML-RARA, its high expression persists in transformed APL cells, and spontaneous somatic mutations of GATA2 occur during APL progression in mice and humans. These and other findings suggested that GATA2 may be upregulated to thwart the proliferative signal generated by PML-RARA and that its inactivation by mutation (and/or epigenetic silencing) may accelerate disease progression in APL and other forms of acute myeloid leukemia (AML). Indeed, biallelic knockout of Gata2 with CRISPR/Cas9-mediated gene editing increased the serial replating efficiency of PML-RARA-expressing myeloid progenitors (as well as progenitors expressing RUNX1-RUNX1T1, or deficient for Cebpa), increased mouse APL penetrance, and decreased latency. Restoration of Gata2 expression suppressed PML-RARA-driven aberrant self-renewal and leukemogenesis. Conversely, addback of a mutant GATA2R362G protein associated with APL and AML minimally suppressed PML-RARA-induced aberrant self-renewal, suggesting that it is a loss-of-function mutation. These studies reveal a potential role for Gata2 as a tumor suppressor in AML and suggest that restoration of its function (when inactivated) may provide benefit for AML patients.},
}
@article {pmid34124996,
year = {2021},
author = {Mubarik, MS and Wang, X and Khan, SH and Ahmad, A and Khan, Z and Amjid, MW and Razzaq, MK and Ali, Z and Azhar, MT},
title = {Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants.},
journal = {GM crops &amp; food},
volume = {12},
number = {2},
pages = {647-658},
pmid = {34124996},
issn = {2164-5701},
mesh = {*Begomovirus/genetics ; *CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Gene Editing/methods ; Plant Breeding ; Plant Diseases/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Advances in genome editing technologies have tremendous potential to address the limitations of classical resistance breeding. CRISPR-Cas9 based gene editing has been applied successfully in plants to tolerate virus infections. In this study, we successfully tested CRISPR-Cas9 system to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs). We also analyzed the ability of CLCuV to escape the Cas9 endonuclease activity. Targeting overlapping genes of most prevalent CLCuVs with three gRNAs resulted in virus interference, as validated by low virus titer. Furthermore, multiplex CRISPR-Cas9 construct simultaneously targeting six genes of CLCuV, was found more effective to interfere with virus proliferation compared to targeting single region individually. Additionally, transgenic N. benthamiana plants expressing multiple gRNAs simultaneously showed enhanced tolerance against CLCuV infection when compared to wild-type plants. T7 Endonuclease-I (T7EI) assay, showing indels in the CLCuV genome, confirmed the occurrence of double strand breaks (DSBs) in DNA at target sequence induced by Cas9 endonuclease. We observed that targeting CLCuV genome at multiple sites simultaneously resulted in better interference, also with inefficient recovery of altered virus molecules. Next, we tested multiplex construct in cotton to interfere CLCuV infection. We found significant decrease in virus accumulation in cotton leaves co-infiltrated with multiplex cassette and virus compared to cotton leaves infiltrated with virus only. The results demonstrate future use of CRISPR-Cas9 system for engineering virus resistance in crops. Moreover, our results also advocate that resistance to mixed virus infections can be engineered using multiplex genome editing.},
}
@article {pmid34122485,
year = {2021},
author = {Ahmar, S and Mahmood, T and Fiaz, S and Mora-Poblete, F and Shafique, MS and Chattha, MS and Jung, KH},
title = {Advantage of Nanotechnology-Based Genome Editing System and Its Application in Crop Improvement.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {663849},
pmid = {34122485},
issn = {1664-462X},
abstract = {Agriculture is an important source of human food. However, current agricultural practices need modernizing and strengthening to fulfill the increasing food requirements of the growing worldwide population. Genome editing (GE) technology has been used to produce plants with improved yields and nutritional value as well as with higher resilience to herbicides, insects, and diseases. Several GE tools have been developed recently, including clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases, a customizable and successful method. The main steps of the GE process involve introducing transgenes or CRISPR into plants via specific gene delivery systems. However, GE tools have certain limitations, including time-consuming and complicated protocols, potential tissue damage, DNA incorporation in the host genome, and low transformation efficiency. To overcome these issues, nanotechnology has emerged as a groundbreaking and modern technique. Nanoparticle-mediated gene delivery is superior to conventional biomolecular approaches because it enhances the transformation efficiency for both temporal (transient) and permanent (stable) genetic modifications in various plant species. However, with the discoveries of various advanced technologies, certain challenges in developing a short-term breeding strategy in plants remain. Thus, in this review, nanobased delivery systems and plant genetic engineering challenges are discussed in detail. Moreover, we have suggested an effective method to hasten crop improvement programs by combining current technologies, such as speed breeding and CRISPR/Cas, with nanotechnology. The overall aim of this review is to provide a detailed overview of nanotechnology-based CRISPR techniques for plant transformation and suggest applications for possible crop enhancement.},
}
@article {pmid34122418,
year = {2021},
author = {Dudek, AM and Porteus, MH},
title = {Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {660302},
pmid = {34122418},
issn = {1664-3224},
support = {F32 HL154667/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Capsid/metabolism ; Capsid Proteins/genetics ; *DNA Damage ; Dependovirus/*genetics/immunology ; *Gene Editing ; Genetic Therapy ; *Genetic Vectors ; Genome, Viral ; Humans ; *Immunity, Innate ; Mice ; },
abstract = {Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of ex-vivo gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.},
}
@article {pmid34122373,
year = {2021},
author = {Gonzalez-Avila, LU and Vega-López, JM and Pelcastre-Rodríguez, LI and Cabrero-Martínez, OA and Hernández-Cortez, C and Castro-Escarpulli, G},
title = {The Challenge of CRISPR-Cas Toward Bioethics.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {657981},
pmid = {34122373},
issn = {1664-302X},
abstract = {Since determining the structure of the DNA double helix, the study of genes and genomes has revolutionized contemporary science; with the decoding of the human genome, new findings have been achieved, including the ability that humans have developed to modify genetic sequences in vitro. The discovery of gene modification mechanisms, such as the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR associated). Derived from the latest discoveries in genetics, the idea that science has no limits has exploded. However, improvements in genetic engineering allowed access to new possibilities to save lives or generate new treatment options for diseases that are not treatable by using genes and their modification in the genome. With this greater knowledge, the immediate question is who governs the limits of genetic science? The first answer would be the intervention of a legislative branch, with adequate scientific advice, from which the logical answer, bioethics, should result. This term was introduced for the first time by Van Rensselaer Potter, who in 1970 combined the Greek words bios and ethos, Bio-Ethik, which determined the study of the morality of human behavior in science. The approach to this term was introduced to avoid the natural tension that results from the scientific technical development and the ethics of limits. Therefore, associating the use of biotechnology through the CRISPR-Cas system and the regulation through bioethics, aims to monitor the use of techniques and technology, with benefits for humanity, without altering fundamental rights, acting with moral and ethical principles.},
}
@article {pmid34122005,
year = {2021},
author = {Duarte, F and Déglon, N},
title = {Corrigendum: Genome Editing for CNS Disorders.},
journal = {Frontiers in neuroscience},
volume = {15},
number = {},
pages = {698879},
pmid = {34122005},
issn = {1662-4548},
abstract = {[This corrects the article DOI: 10.3389/fnins.2020.579062.].},
}
@article {pmid34121564,
year = {2021},
author = {Wu, H and Zhang, L and Zhu, Z and Ding, C and Chen, S and Liu, R and Fan, H and Chen, Y and Li, H},
title = {Novel CD123 polyaptamer hydrogel edited by Cas9/sgRNA for AML-targeted therapy.},
journal = {Drug delivery},
volume = {28},
number = {1},
pages = {1166-1178},
pmid = {34121564},
issn = {1521-0464},
mesh = {Animals ; Apoptosis ; Aptamers, Nucleotide/*administration &amp; dosage/*pharmacology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Chemistry, Pharmaceutical ; Cytokines/drug effects ; Drug Carriers ; Humans ; Hydrogels/*chemistry ; Interleukin-3 Receptor alpha Subunit/*administration &amp; dosage/*metabolism ; Janus Kinase 2/biosynthesis ; Leukemia, Myeloid, Acute/*drug therapy ; Mice ; Signal Transduction/drug effects ; Xenograft Model Antitumor Assays ; },
abstract = {CD123 targeting molecules have been widely applied in acute myelocytic leukemia (AML) therapeutics. Although antibodies have been more widely used as targeting molecules, aptamer have unique advantages for CD123 targeting therapy. In this study, we constructed an aptamer hydrogel termed as SSFH which could be precisely cut by Cas9/sgRNA for programmed SS30 release. To construct hydrogel, rolling-circle amplification (RCA) was used to generate hydrogel containing CD123 aptamer SS30 and sgRNA-targeting sequence. After incubation with Cas9/sgRNA, SSFH could lose its gel property and liberated the SS30 aptamer sequence, and released SS30 has been confirmed by gel electrophoresis. In addition, SS30 released from SSFH could inhibit cell proliferation and induce cell apoptosis in vitro. Moreover, SSFH could prolong survival rate and inhibit tumor growth via JAK2/STAT5 signaling pathway in vivo. Additionally, molecular imaging revealed SSFH co-injected with Cas9/sgRNA remained at the injection site longer than free aptamer. Furthermore, once the levels of cytokines were increasing, the complementary sequences of aptamers injection could neutralize SS30 and relieve side effect immediately. This study suggested that CD123 aptamer hydrogel SSFH and Cas9/sgRNA system has strong potential for CD123-positive AML anticancer therapy.},
}
@article {pmid34120856,
year = {2021},
author = {Lin, Q and Zhu, Z and Liu, G and Sun, C and Lin, D and Xue, C and Li, S and Zhang, D and Gao, C and Wang, Y and Qiu, JL},
title = {Genome editing in plants with MAD7 nuclease.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {6},
pages = {444-451},
doi = {10.1016/j.jgg.2021.04.003},
pmid = {34120856},
issn = {1673-8527},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/*metabolism ; Eubacterium/enzymology ; Gene Editing/*methods ; *Genome, Plant ; INDEL Mutation ; Oryza/genetics ; Plants, Genetically Modified ; Protoplasts/metabolism ; RNA, Guide ; Triticum/genetics ; },
abstract = {MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-LbCas12a system. We develop two variants, MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5'-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.},
}
@article {pmid34120537,
year = {2022},
author = {Jia, YL and Wang, LR and Zhang, ZX and Gu, Y and Sun, XM},
title = {Recent advances in biotechnological production of polyunsaturated fatty acids by Yarrowia lipolytica.},
journal = {Critical reviews in food science and nutrition},
volume = {62},
number = {32},
pages = {8920-8934},
doi = {10.1080/10408398.2021.1937041},
pmid = {34120537},
issn = {1549-7852},
mesh = {Humans ; *Yarrowia/genetics/metabolism ; Metabolic Engineering ; Biotechnology ; Fatty Acids, Unsaturated/metabolism ; Gene Editing ; Fatty Acids/metabolism ; },
abstract = {Owing to the important physiological functions, polyunsaturated fatty acids (PUFAs) play a vital role in protecting human health, such as preventing cancer, cardiovascular disease, and diabetes. Specifically, Yarrowia lipolytica has been identified as the most popular non-conventional oleaginous yeast, which can accumulate the abundant intracellular lipids, indicating that has great potential as an industrial host for production of PUFAs. Notably, some novel engineering strategies have been applied to endow and improve the abilities of Y. lipolytica to synthesize PUFAs, including construction and optimization of PUFAs biosynthetic pathways, improvement of preucrsors acetyl-coA and NADPH supply, inhibition of competing pathways, knockout of β-oxidation pathways, regulation of oxidative stress defense pathways, and regulation of genes involved in upstream lipid metabolism. Besides, some bypass approaches, such as strain mating, evolutionary engineering, and computational model based on omics, also have been proposed to improve the performance of engineering strains. Generally, in this review, we summarized the recent advances in engineering strategies and bypass approaches for improving PUFAs production by Y. lipolytica. In addition, we further summarized the latest efforts of CRISPR/Cas genome editing technology in Y. lipolytica, which is aimed to provide its potential applications in PUFAs production.},
}
@article {pmid34119838,
year = {2021},
author = {Yang, Y and Liu, J and Zhou, X},
title = {A CRISPR-based and post-amplification coupled SARS-CoV-2 detection with a portable evanescent wave biosensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {190},
number = {},
pages = {113418},
pmid = {34119838},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The continuing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has spread globally and its reliable diagnosis is one of the foremost priorities for protecting public health. Herein a rapid (<1 h), easy-to-implement, and accurate CRISPR-based evanescent wave fluorescence biosensing platform for detection of SARS-CoV-2 is reported. The collateral effect of Cas13a is combined with a universal autonomous enzyme-free hybridization chain reaction (HCR) by designing a cleavage hairpin reporter, which is cleaved upon target recognition, and hence releasing the initiator sequence to trigger the downstream HCR circuits. Detection of HCR assemblies is accomplished by first adsorbing to the desthiobiotin-modified optical fiber, followed by fluorescence emission induced by an evanescent field. Three Cas13a crRNAs targeting the genes of S, N and Orf1ab of SARS-CoV-2 are programmed to specifically target SARS-CoV-2 or broadly detect related coronavirus strains, such as MERS-CoV and SARS-CoV. The HCR amplification coupled Cas13a-based biosensing platform is capable of rapid detection of SARS-CoV-2 with attomolar sensitivity. This method is further validated by adding target RNA of SARS-CoV-2 in negative oropharyngeal swabs. The good discrimination capability of this technique demonstrates its promising potential for point-of-care diagnosis of COVID-19.},
}
@article {pmid34119663,
year = {2021},
author = {Wang, C and Rollins, JA},
title = {Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {154},
number = {},
pages = {103598},
doi = {10.1016/j.fgb.2021.103598},
pmid = {34119663},
issn = {1096-0937},
mesh = {Ascomycota/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; RNA, Small Nuclear/*genetics ; },
abstract = {We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of genemutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.},
}
@article {pmid34118887,
year = {2021},
author = {Bandara, AB and Drake, JC and Brown, DA},
title = {Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog.},
journal = {BMC molecular and cell biology},
volume = {22},
number = {1},
pages = {35},
pmid = {34118887},
issn = {2661-8850},
mesh = {CRISPR-Cas Systems ; Cell Proliferation/drug effects/*genetics ; Electron Transport Complex II ; HEK293 Cells ; Humans ; Mitochondria/*metabolism ; Mutation ; Succinate Dehydrogenase/*genetics ; Ubiquinone/*analogs &amp; derivatives/pharmacology ; },
abstract = {BACKGROUND: Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist.<br><br>RESULTS: Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293&#x394;SDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells.<br><br>CONCLUSIONS: Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.},
}
@article {pmid34118444,
year = {2021},
author = {Yamazaki, M and Kawai, M and Kinoshita, S and Tachikawa, K and Nakanishi, T and Ozono, K and Michigami, T},
title = {Clonal osteoblastic cell lines with CRISPR/Cas9-mediated ablation of Pit1 or Pit2 show enhanced mineralization despite reduced osteogenic gene expression.},
journal = {Bone},
volume = {151},
number = {},
pages = {116036},
doi = {10.1016/j.bone.2021.116036},
pmid = {34118444},
issn = {1873-2763},
mesh = {Biological Transport ; *CRISPR-Cas Systems/genetics ; Cell Line ; Gene Expression ; *Sodium-Phosphate Cotransporter Proteins, Type III/genetics/metabolism ; },
abstract = {Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na[+]/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.},
}
@article {pmid34117872,
year = {2021},
author = {Hu, D and Yu, Y and Wang, C and Long, Y and Liu, Y and Feng, L and Lu, D and Liu, B and Jia, J and Xia, R and Du, J and Zhong, X and Gong, L and Wang, K and Zhai, J},
title = {Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions.},
journal = {The Plant cell},
volume = {33},
number = {9},
pages = {2950-2964},
pmid = {34117872},
issn = {1532-298X},
mesh = {CRISPR-Cas Systems ; *DNA Methylation ; Gene Editing ; Methyltransferases/*genetics/metabolism ; Oryza/*genetics/metabolism ; Plant Proteins/*genetics/metabolism ; },
abstract = {DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.},
}
@article {pmid34117481,
year = {2021},
author = {Weber, CM and Hafner, A and Kirkland, JG and Braun, SMG and Stanton, BZ and Boettiger, AN and Crabtree, GR},
title = {mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.},
journal = {Nature structural &amp; molecular biology},
volume = {28},
number = {6},
pages = {501-511},
pmid = {34117481},
issn = {1545-9985},
support = {DP2 GM132935/GM/NIGMS NIH HHS/United States ; R01 CA163915/CA/NCI NIH HHS/United States ; R37 NS046789/NS/NINDS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Chromatin Assembly and Disassembly/genetics/*physiology ; DNA Helicases/genetics/metabolism ; DNA-Binding Proteins/physiology ; Embryonic Stem Cells/metabolism ; Epigenesis, Genetic ; Epigenetic Repression/genetics/*physiology ; Gene Editing ; Gene Expression Regulation/genetics/*physiology ; Genes, Homeobox ; Genome ; HEK293 Cells ; Homeodomain Proteins/genetics/metabolism ; Humans ; Loss of Function Mutation ; Mice ; Multiprotein Complexes/*physiology ; Nuclear Proteins/genetics/metabolism ; Polycomb-Group Proteins/*physiology ; Proteolysis ; Recombinant Fusion Proteins/metabolism ; Transcription Factors/genetics/metabolism/physiology ; },
abstract = {The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.},
}
@article {pmid34117247,
year = {2021},
author = {McDonald, TL and Zhou, W and Castro, CP and Mumm, C and Switzenberg, JA and Mills, RE and Boyle, AP},
title = {Cas9 targeted enrichment of mobile elements using nanopore sequencing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3586},
pmid = {34117247},
issn = {2041-1723},
support = {R21 HG011493/HG/NHGRI NIH HHS/United States ; T32 GM007544/GM/NIGMS NIH HHS/United States ; T32 GM070449/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; DNA-Binding Proteins ; Genome, Human ; *Genomics ; Humans ; *Interspersed Repetitive Sequences ; Nanopore Sequencing/*methods ; Repetitive Sequences, Nucleic Acid ; Ribonucleoproteins/metabolism ; Sequence Analysis, DNA ; },
abstract = {Mobile element insertions (MEIs) are repetitive genomic sequences that contribute to genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9-targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on-targeted signals and exhibiting a 13.4-54x enrichment over whole-genome approaches. We show an individual flow cell can recover most MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify seventeen non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.},
}
@article {pmid34117224,
year = {2021},
author = {Dahlet, T and Truss, M and Frede, U and Al Adhami, H and Bardet, AF and Dumas, M and Vallet, J and Chicher, J and Hammann, P and Kottnik, S and Hansen, P and Luz, U and Alvarez, G and Auclair, G and Hecht, J and Robinson, PN and Hagemeier, C and Weber, M},
title = {E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3582},
pmid = {34117224},
issn = {2041-1723},
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Cell Differentiation ; CpG Islands/*genetics ; DNA Methylation ; E2F6 Transcription Factor/*genetics/*metabolism ; Embryonic Development/*genetics ; *Epigenesis, Genetic ; Gene Silencing ; Germ Cells/*metabolism ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells ; Polycomb Repressive Complex 1/metabolism ; RNA, Small Interfering ; },
abstract = {In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development.},
}
@article {pmid34116143,
year = {2021},
author = {Usher, B and Birkholz, N and Beck, IN and Fagerlund, RD and Jackson, SA and Fineran, PC and Blower, TR},
title = {Crystal structure of the anti-CRISPR repressor Aca2.},
journal = {Journal of structural biology},
volume = {213},
number = {3},
pages = {107752},
pmid = {34116143},
issn = {1095-8657},
mesh = {Bacteria ; *Bacteriophages/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Protein Binding ; Transcription Factors/genetics ; },
abstract = {Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.},
}
@article {pmid34115987,
year = {2021},
author = {Simeonov, KP and Byrns, CN and Clark, ML and Norgard, RJ and Martin, B and Stanger, BZ and Shendure, J and McKenna, A and Lengner, CJ},
title = {Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.},
journal = {Cancer cell},
volume = {39},
number = {8},
pages = {1150-1162.e9},
pmid = {34115987},
issn = {1878-3686},
support = {R00 HG010152/HG/NHGRI NIH HHS/United States ; T32 AI070077/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F30 DK120135/DK/NIDDK NIH HHS/United States ; T32 HD083185/HD/NICHD NIH HHS/United States ; P30 CA023108/CA/NCI NIH HHS/United States ; R01 CA168654/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Lineage ; Cell Proliferation/genetics ; *Epithelial-Mesenchymal Transition/genetics ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice, Inbred NOD ; Pancreatic Neoplasms/genetics/*pathology ; S100 Proteins/genetics ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; Stem Cells/pathology ; Xenograft Model Antitumor Assays ; },
abstract = {The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.},
}
@article {pmid34115079,
year = {2021},
author = {Yan, J and Kang, DD and Dong, Y},
title = {Harnessing lipid nanoparticles for efficient CRISPR delivery.},
journal = {Biomaterials science},
volume = {9},
number = {18},
pages = {6001-6011},
pmid = {34115079},
issn = {2047-4849},
support = {R01 HL136652/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Humans ; Lipids ; *Nanoparticles ; },
abstract = {The CRISPR-Cas system has revolutionized the biomedical research field with its simple and flexible genome editing method. In October 2020, Emmanuelle Charpentier and Jennifer A. Doudna were awarded the 2020 Nobel Prize in chemistry in recognition of their outstanding contributions to the discovery of CRISPR-Cas9 genetic scissors, which allow scientists to alter DNA sequences with high precision. Recently, the first phase I clinical trials in cancer patients affirmed the safety and feasibility of ex vivo CRISPR-edited T cells. However, specific and effective CRISPR delivery in vivo remains challenging due to the multiple extracellular and intracellular barriers. Here, we discuss the recent advances in novel lipid nanomaterials for CRISPR delivery and describe relevant examples of potential therapeutics in cancers, genetic disorders, and infectious diseases.},
}
@article {pmid34114949,
year = {2021},
author = {Li, J and Qin, X and Shi, J and Wang, X and Li, T and Xu, M and Chen, X and Zhao, Y and Han, J and Piao, Y and Zhang, W and Qu, P and Wang, L and Xiang, R and Shi, Y},
title = {A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34114949},
issn = {2050-084X},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Epithelium/immunology ; Female ; Gene Knockdown Techniques ; Humans ; Interleukins/*genetics/metabolism ; Macrophages/immunology ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis/*genetics ; Ovarian Neoplasms/*genetics/immunology/metabolism ; Peritoneal Cavity/pathology ; Receptors, Interleukin/*genetics/metabolism ; Signal Transduction ; Tumor Microenvironment ; },
abstract = {Transcoelomic spread of cancer cells across the peritoneal cavity occurs in most initially diagnosed ovarian cancer (OC) patients and accounts for most cancer-related death. However, how OC cells interact with peritoneal stromal cells to evade the immune surveillance remains largely unexplored. Here, through an in vivo genome-wide CRISPR/Cas9 screen, we identified IL20RA, which decreased dramatically in OC patients during peritoneal metastasis, as a key factor preventing the transcoelomic metastasis of OC. Reconstitution of IL20RA in highly metastatic OC cells greatly suppresses the transcoelomic metastasis. OC cells, when disseminate into the peritoneal cavity, greatly induce peritoneum mesothelial cells to express IL-20 and IL-24, which in turn activate the IL20RA downstream signaling in OC cells to produce mature IL-18, eventually resulting in the polarization of macrophages into the M1-like subtype to clear the cancer cells. Thus, we show an IL-20/IL20RA-mediated crosstalk between OC and mesothelial cells that supports a metastasis-repressing immune microenvironment.},
}
@article {pmid34114251,
year = {2021},
author = {Li, C and Pei, J and Yan, X and Cui, X and Tsuruta, M and Liu, Y and Lian, C},
title = {A poplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light.},
journal = {Plant, cell &amp; environment},
volume = {44},
number = {9},
pages = {3015-3033},
doi = {10.1111/pce.14127},
pmid = {34114251},
issn = {1365-3040},
mesh = {Anthocyanins/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Plant ; Gene Knockdown Techniques ; Light ; Phylogeny ; Plant Proteins/metabolism/*physiology ; Populus/genetics/metabolism/*radiation effects ; Proanthocyanidins/*metabolism ; Real-Time Polymerase Chain Reaction ; Transcription Factors/metabolism/*physiology ; Transcriptome ; },
abstract = {Flavonoids, which modulate plant resistance to various stresses, can be induced by high light. B-box (BBX) transcription factors (TFs) play crucial roles in the transcriptional regulation of flavonoids biosynthesis, but limited information is available on the association of BBX proteins with high light. We present a detailed overview of 45 Populus trichocarpa BBX TFs. Phylogenetic relationships, gene structure, tissue-specific expression patterns and expression profiles were determined under 10 stress or phytohormone treatments to screen candidate BBX proteins associated with the flavonoid pathway. Sixteen candidate genes were identified, of which five were expressed predominantly in young leaves and roots, and BBX23 showed the most distinct response to high light. Overexpression of BBX23 in poplar activated expression of MYB TFs and structural genes in the flavonoid pathway, thereby promoting the accumulation of proanthocyanidins and anthocyanins. CRISPR/Cas9-generated knockout of BBX23 resulted in the opposite trend. Furthermore, the phenotype induced by BBX23 overexpression was enhanced under exposure to high light. BBX23 was capable of binding directly to the promoters of proanthocyanidin- and anthocyanin-specific genes, and its interaction with HY5 enhanced activation activity. We identified novel regulators of flavonoid biosynthesis in poplar, thereby enhancing our general understanding of the transcriptional regulatory mechanisms involved.},
}
@article {pmid34114124,
year = {2021},
author = {Gupta, S and Kumar, A and Patel, R and Kumar, V},
title = {Genetically modified crop regulations: scope and opportunity using the CRISPR-Cas9 genome editing approach.},
journal = {Molecular biology reports},
volume = {48},
number = {5},
pages = {4851-4863},
pmid = {34114124},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Crops, Agricultural/economics/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; Genome, Plant ; *Government Regulation ; Plant Breeding/methods ; *Plants, Genetically Modified/classification/genetics ; Transgenes ; },
abstract = {Global demand for food is increasing day by day due to an increase in population and shrinkage of the arable land area. To meet this increasing demand, there is a need to develop high-yielding varieties that are nutritionally enriched and tolerant against environmental stresses. Various techniques are developed for improving crop quality such as mutagenesis, intergeneric crosses, and translocation breeding. Later, with the development of genetic engineering, genetically modified crops came up with the transgene insertion approach which helps to withstand adverse conditions. The process or product-focused approaches are used for regulating genetically modified crops with their risk analysis on the environment and public health. However, recent advances in gene-editing technologies have led to a new era of plant breeding by developing techniques including site-directed nucleases, zinc finger nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) that involve precise gene editing without the transfer of foreign genes. But these techniques always remain in debate for their regulation status and public acceptance. The European countries and New Zealand, consider the gene-edited plants under the category of genetically modified organism (GMO) regulation while the USA frees the gene-edited plants from such type of regulations. Considering them under the category of GMO makes a long and complicated approval process to use them, which would decrease their immediate commercial value. There is a need to develop strong regulatory approaches for emerging technologies that expedite crop research and attract people to adopt these new varieties without hesitation.},
}
@article {pmid34112918,
year = {2022},
author = {Fan, W and Yu, M and Wang, X and Xie, W and Tian, R and Cui, Z and Jin, Z and Huang, Z and Das, BC and Severinov, K and Hitzeroth, II and Debata, PR and Tian, X and Xie, H and Lang, B and Tan, J and Xu, H and Hu, Z},
title = {Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells.},
journal = {Cancer gene therapy},
volume = {29},
number = {6},
pages = {758-769},
pmid = {34112918},
issn = {1476-5500},
mesh = {CRISPR-Cas Systems/genetics ; Female ; Humans ; Mutagens ; Oligodeoxyribonucleotides ; Oncogenes ; *Papillomavirus Infections/genetics ; *Uterine Cervical Neoplasms/genetics/therapy ; },
abstract = {Genome editing tools targeting high-risk human papillomavirus (HPV) oncogene could be a promising therapeutic strategy for the treatment of HPV-related cervical cancer. We aimed to improve the editing efficiency and detect off-target effects concurrently for the clinical translation strategy by using CRISPR-Cas9 system co-transfected with 34nt non-homologous double-stranded oligodeoxynucleotide (dsODN). We firstly tested this strategy on targeting the Green Fluorescent Protein (GFP) gene, of which the expression is easily observed. Our results showed that the GFP+ cells were significantly decreased when using GFP-sgRNAs with dsODN, compared to using GFP-sgRNAs without donors. By PCR and Sanger sequencing, we verified the dsODN integration into the break sites of the GFP gene. And by amplicon sequencing, we observed that the indels% of the targeted site on the GFP gene was increased by using GFP-sgRNAs with dsODN. Next, we went on to target the HPV18 E7 oncogene by using single E7-sgRNA and multiplexed E7-sgRNAs respectively. Whenever using single sgRNA or multiplexed sgRNAs, the mRNA expression of HPV18 E7 oncogene was significantly decreased when adding E7-sgRNAs with dsODN, compared to E7-sgRNAs without donor. And the indels% of the targeted sites on the HPV18 E7 gene was markedly increased by adding dsODN with E7-sgRNAs. Finally, we performed GUIDE-Seq to verify that the integrated dsODN could serve as the marker to detect off-target effects in using single or multiplexed two sgRNAs. And we detected fewer on-target reads and off-target sites in multiplexes compared to the single sgRNAs when targeting the GFP and the HPV18 E7 genes. Together, CRISPR-Cas9 system co-transfected with 34nt dsODN concurrently improved the editing efficiency and monitored off-target effects, which might provide new insights in the treatment of HPV infections and related cervical cancer.},
}
@article {pmid34111225,
year = {2021},
author = {Hasley, JAR and Navet, N and Tian, M},
title = {CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.},
journal = {PloS one},
volume = {16},
number = {6},
pages = {e0253245},
pmid = {34111225},
issn = {1932-6203},
mesh = {Arabidopsis/genetics ; CRISPR-Cas Systems/*genetics ; Disease Resistance/*genetics ; Mutagenesis/genetics ; Ocimum basilicum/*genetics/microbiology ; Plant Diseases/*genetics/microbiology ; Plant Leaves/genetics/growth &amp; development/microbiology ; Seedlings/genetics/microbiology ; },
abstract = {Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61-68% compared to the wild-type plants and 69-93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.},
}
@article {pmid34108663,
year = {2021},
author = {van der Weegen, Y and de Lint, K and van den Heuvel, D and Nakazawa, Y and Mevissen, TET and van Schie, JJM and San Martin Alonso, M and Boer, DEC and González-Prieto, R and Narayanan, IV and Klaassen, NHM and Wondergem, AP and Roohollahi, K and Dorsman, JC and Hara, Y and Vertegaal, ACO and de Lange, J and Walter, JC and Noordermeer, SM and Ljungman, M and Ogi, T and Wolthuis, RMF and Luijsterburg, MS},
title = {ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation.},
journal = {Nature cell biology},
volume = {23},
number = {6},
pages = {595-607},
pmid = {34108663},
issn = {1476-4679},
support = {R01 CA213214/CA/NCI NIH HHS/United States ; R01 HL098316/HL/NHLBI NIH HHS/United States ; UM1 HG009382/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Damage ; DNA Helicases/genetics/metabolism ; *DNA Repair ; DNA Repair Enzymes/genetics/metabolism ; Humans ; Peptide Elongation Factor 1/genetics/*metabolism ; Poly-ADP-Ribose Binding Proteins/genetics/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; RNA Polymerase II/genetics/*metabolism ; Transcription Elongation, Genetic ; Transcription Factors/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/*metabolism ; *Ubiquitination ; },
abstract = {Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4[CSA] ubiquitin ligase. How CRL4[CSA] is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4[CSA] for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.},
}
@article {pmid34108662,
year = {2021},
author = {Geijer, ME and Zhou, D and Selvam, K and Steurer, B and Mukherjee, C and Evers, B and Cugusi, S and van Toorn, M and van der Woude, M and Janssens, RC and Kok, YP and Gong, W and Raams, A and Lo, CSY and Lebbink, JHG and Geverts, B and Plummer, DA and Bezstarosti, K and Theil, AF and Mitter, R and Houtsmuller, AB and Vermeulen, W and Demmers, JAA and Li, S and van Vugt, MATM and Lans, H and Bernards, R and Svejstrup, JQ and Ray Chaudhuri, A and Wyrick, JJ and Marteijn, JA},
title = {Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability.},
journal = {Nature cell biology},
volume = {23},
number = {6},
pages = {608-619},
pmid = {34108662},
issn = {1476-4679},
support = {NWO_VI.C.182.025/NWO_/Dutch Research Council/Netherlands ; R21 ES029655/ES/NIEHS NIH HHS/United States ; FC001166/CRUK_/Cancer Research UK/United Kingdom ; R01 ES028698/ES/NIEHS NIH HHS/United States ; FC001166/WT_/Wellcome Trust/United Kingdom ; FC001166/ARC_/Arthritis Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; FC001166/MRC_/Medical Research Council/United Kingdom ; R21 ES029302/ES/NIEHS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carrier Proteins/genetics/metabolism ; *DNA Damage ; *DNA Repair ; Evolution, Molecular ; *Genomic Instability ; HCT116 Cells ; Humans ; Peptide Elongation Factor 1/genetics/*metabolism ; RNA Polymerase II/metabolism ; *Transcription Elongation, Genetic ; Transcription Factor TFIIH/genetics/metabolism ; Ubiquitination ; },
abstract = {Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.},
}
@article {pmid34108610,
year = {2021},
author = {Geng, Y and Pertsinidis, A},
title = {Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {12220},
pmid = {34108610},
issn = {2045-2322},
support = {DP2 GM105443/GM/NIGMS NIH HHS/United States ; R01 GM135545/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R21 GM134342/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chromatin/*genetics ; Embryo, Mammalian/*cytology/metabolism ; *Genetic Loci ; Genomics ; HeLa Cells ; Humans ; In Situ Hybridization, Fluorescence/*methods ; Mice ; RNA, Guide/*genetics ; },
abstract = {Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.},
}
@article {pmid34108490,
year = {2021},
author = {Zhang, B and Luo, D and Li, Y and Perčulija, V and Chen, J and Lin, J and Ye, Y and Ouyang, S},
title = {Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3476},
pmid = {34108490},
issn = {2041-1723},
mesh = {Base Pairing ; CRISPR-Associated Proteins/*chemistry/genetics/metabolism ; Catalytic Domain ; DNA/chemistry/metabolism ; *DNA Cleavage ; Endonucleases/*chemistry/genetics/metabolism ; Enzyme Activation ; Magnesium/chemistry ; Models, Biological ; Models, Molecular ; Protein Conformation ; Protein Structure, Tertiary ; *R-Loop Structures ; RNA, Guide/chemistry/metabolism ; Temperature ; },
abstract = {Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.},
}
@article {pmid34107351,
year = {2021},
author = {Yu, M and Liu, X and Cheng, H and Kuang, L and Zhang, S and Yan, X},
title = {Latest progress in the study of nanoparticle-based delivery of the CRISPR/Cas9 system.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {48-55},
doi = {10.1016/j.ymeth.2021.06.004},
pmid = {34107351},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; *Nanoparticles ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system has been harnessed to cleave a targeted DNA fragment via its Cas nuclease activity under the direction of guide RNA for rendering gene insertions, deletions, and point mutations in basic research and clinical applications. There are a number of vehicles, including lipofectamine, viruses, and nanoparticles, that can deliver the CRISPR/Cas9 system, but all these methods face numerous challenges during their application in life science contexts. Here, we focus on the delivery of CRISPR/Cas9 via nanoparticles because this method has shown great advantages in terms of safety, simplicity and flexibility.},
}
@article {pmid34107197,
year = {2021},
author = {Frangoul, H and Ho, TW and Corbacioglu, S},
title = {CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. Reply.},
journal = {The New England journal of medicine},
volume = {384},
number = {23},
pages = {e91},
doi = {10.1056/NEJMc2103481},
pmid = {34107197},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *beta-Thalassemia/genetics/therapy ; },
}
@article {pmid34107196,
year = {2021},
author = {Mehta, J},
title = {CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.},
journal = {The New England journal of medicine},
volume = {384},
number = {23},
pages = {e91},
doi = {10.1056/NEJMc2103481},
pmid = {34107196},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *beta-Thalassemia/genetics/therapy ; },
}
@article {pmid34107195,
year = {2021},
author = {Meisel, R},
title = {CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.},
journal = {The New England journal of medicine},
volume = {384},
number = {23},
pages = {e91},
doi = {10.1056/NEJMc2103481},
pmid = {34107195},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *beta-Thalassemia/genetics/therapy ; },
}
@article {pmid34107040,
year = {2021},
author = {Liu, H and Zhu, Y and Lu, Z and Huang, Z},
title = {Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14.},
journal = {Nucleic acids research},
volume = {49},
number = {11},
pages = {6587-6595},
pmid = {34107040},
issn = {1362-4962},
mesh = {Allosteric Regulation ; Bacterial Proteins/*chemistry ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/*chemistry ; Crystallography, X-Ray ; DNA/chemistry ; Models, Molecular ; Protein Domains ; RNA/chemistry ; Staphylococcus aureus/*enzymology ; },
abstract = {Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9-sgRNA-dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.},
}
@article {pmid34107026,
year = {2021},
author = {Jensen, ED and Laloux, M and Lehka, BJ and Pedersen, LE and Jakočiūnas, T and Jensen, MK and Keasling, JD},
title = {A synthetic RNA-mediated evolution system in yeast.},
journal = {Nucleic acids research},
volume = {49},
number = {15},
pages = {e88},
pmid = {34107026},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; *Directed Molecular Evolution ; Genome, Fungal/genetics ; Humans ; Mutagenesis/genetics ; Mutation/genetics ; RNA, Guide/*genetics ; RNA-Dependent RNA Polymerase/*genetics ; Saccharomyces cerevisiae/*genetics ; Selection, Genetic/genetics ; },
abstract = {Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker's yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.},
}
@article {pmid34106206,
year = {2021},
author = {Rogers, BM and Smith, L and Dezso, Z and Shi, X and DiGiammarino, E and Nguyen, D and Sethuraman, S and Zheng, P and Choi, D and Zhang, D and Nguyen, A and McGuire, K and Liu, W and Chung, N and Chao, DT and Ye, S and Starbeck-Miller, GR},
title = {VISTA is an activating receptor in human monocytes.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {8},
pages = {},
pmid = {34106206},
issn = {1540-9538},
mesh = {Antibodies, Monoclonal/metabolism ; Antibody Specificity/immunology ; B7 Antigens/*metabolism ; CRISPR-Cas Systems/genetics ; Heparitin Sulfate/metabolism ; Humans ; Monocytes/*metabolism ; Protein Binding ; Receptors, Fc/metabolism ; Receptors, Immunologic/*metabolism ; Syndecan-2/metabolism ; Transcription, Genetic ; Transcriptome/genetics ; },
abstract = {As indicated by its name, V-domain Ig suppressor of T cell activation (VISTA) is thought to serve primarily as an inhibitory protein that limits immune responses. VISTA antibodies can dampen the effects of several concomitantly elicited activation signals, including TCR and TLR activation, but it is currently unclear if VISTA agonism could singly affect immune cell biology. In this study, we discovered two novel VISTA antibodies and characterized their effects on human peripheral blood mononuclear cells by scRNA/CITE-seq. Both antibodies appeared to agonize VISTA in an Fc-functional manner to elicit transcriptional and functional changes in monocytes consistent with activation. We also used pentameric VISTA to identify Syndecan-2 and several heparan sulfate proteoglycan synthesis genes as novel regulators of VISTA interactions with monocytic cells, adding further evidence of bidirectional signaling. Together, our study highlights several novel aspects of VISTA biology that have yet to be uncovered in myeloid cells and serves as a foundation for future research.},
}
@article {pmid34106048,
year = {2021},
author = {Kumar, M and Gulati, S and Ansari, AH and Phutela, R and Acharya, S and Azhar, M and Murthy, J and Kathpalia, P and Kanakan, A and Maurya, R and Vasudevan, JS and S, A and Pandey, R and Maiti, S and Chakraborty, D},
title = {FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34106048},
issn = {2050-084X},
mesh = {COVID-19/*genetics ; *COVID-19 Nucleic Acid Testing ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; SARS-CoV-2/*genetics ; },
abstract = {The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.},
}
@article {pmid34105245,
year = {2021},
author = {Ban, Q and Yang, P and Chou, SJ and Qiao, L and Xia, H and Xue, J and Wang, F and Xu, X and Sun, N and Zhang, RY and Zhang, C and Lee, A and Liu, W and Lin, TY and Ko, YL and Antovski, P and Zhang, X and Chiou, SH and Lee, CF and Hui, W and Liu, D and Jonas, SJ and Weiss, PS and Tseng, HR},
title = {Supramolecular Nanosubstrate-Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {17},
number = {28},
pages = {e2100546},
pmid = {34105245},
issn = {1613-6829},
support = {DP5 OD028181/OD/NIH HHS/United States ; R21 EB016270/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Vectors ; Humans ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) is an efficient and precise gene-editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Dose- and time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.},
}
@article {pmid34103715,
year = {2021},
author = {Urnov, FD},
title = {CRISPR-Cas9 can cause chromothripsis.},
journal = {Nature genetics},
volume = {53},
number = {6},
pages = {768-769},
pmid = {34103715},
issn = {1546-1718},
mesh = {*CRISPR-Cas Systems/genetics ; *Chromothripsis ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Humans ; },
}
@article {pmid34103663,
year = {2021},
author = {Liu, Y and Yang, G and Huang, S and Li, X and Wang, X and Li, G and Chi, T and Chen, Y and Huang, X and Wang, X},
title = {Enhancing prime editing by Csy4-mediated processing of pegRNA.},
journal = {Cell research},
volume = {31},
number = {10},
pages = {1134-1136},
pmid = {34103663},
issn = {1748-7838},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; *RNA, Guide ; },
}
@article {pmid34103633,
year = {2021},
author = {Jaladanki, SK and Elmas, A and Malave, GS and Huang, KL},
title = {Genetic dependency of Alzheimer's disease-associated genes across cells and tissue types.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {12107},
pmid = {34103633},
issn = {2045-2322},
support = {R35 GM138113/GM/NIGMS NIH HHS/United States ; },
mesh = {ATP-Binding Cassette Transporters/genetics ; Actins/genetics ; Adaptor Proteins, Signal Transducing/genetics ; Alzheimer Disease/*genetics/*physiopathology ; *CRISPR-Cas Systems ; Cell Lineage ; Gene Expression Profiling ; Gene Regulatory Networks ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; MEF2 Transcription Factors/genetics ; Microglia/metabolism ; Nervous System Diseases/genetics ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins/genetics ; RNA Interference ; Risk ; Trans-Activators/genetics ; },
abstract = {Effective treatments targeting disease etiology are urgently needed for Alzheimer's disease (AD). Although candidate AD genes have been identified and altering their levels may serve as therapeutic strategies, the consequence of such alterations remain largely unknown. Herein, we analyzed CRISPR knockout/RNAi knockdown screen data for over 700 cell lines and evaluated cellular dependencies of 104 AD-associated genes previously identified by genome-wide association studies (GWAS) and gene expression network studies. Multiple genes showed widespread cell dependencies across tissue lineages, suggesting their inhibition may yield off-target effects. Meanwhile, several genes including SPI1, MEF2C, GAB2, ABCC11, ATCG1 were identified as genes of interest since their genetic knockouts specifically affected high-expressing cells whose tissue lineages are relevant to cell types found in AD. Overall, analyses of genetic screen data identified AD-associated genes whose knockout or knockdown selectively affected cell lines of relevant tissue lineages, prioritizing targets for potential AD treatments.},
}
@article {pmid34103250,
year = {2022},
author = {Santos, L and Mention, K and Cavusoglu-Doran, K and Sanz, DJ and Bacalhau, M and Lopes-Pacheco, M and Harrison, PT and Farinha, CM},
title = {Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation.},
journal = {Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society},
volume = {21},
number = {1},
pages = {181-187},
doi = {10.1016/j.jcf.2021.05.014},
pmid = {34103250},
issn = {1873-5010},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cystic Fibrosis/*genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Endodeoxyribonucleases/*genetics ; Gene Editing/*methods ; Humans ; Mutation ; },
abstract = {BACKGROUND: W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations.<br><br>METHODS: We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors.<br><br>RESULTS: Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR.<br><br>CONCLUSION: Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.},
}
@article {pmid34103093,
year = {2021},
author = {Vo, PLH and Acree, C and Smith, ML and Sternberg, SH},
title = {Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing.},
journal = {Mobile DNA},
volume = {12},
number = {1},
pages = {13},
pmid = {34103093},
issn = {1759-8753},
support = {DP2 HG011650/HG/NHGRI NIH HHS/United States ; },
abstract = {Bacterial transposons propagate through either non-replicative (cut-and-paste) or replicative (copy-and-paste) pathways, depending on how the mobile element is excised from its donor source. In the well-characterized E. coli transposon Tn7, a heteromeric TnsA-TnsB transposase directs cut-and-paste transposition by cleaving both strands at each transposon end during the excision step. Whether a similar pathway is involved for RNA-guided transposons, in which CRISPR-Cas systems confer DNA target specificity, has not been determined. Here, we apply long-read, population-based whole-genome sequencing (WGS) to unambiguously resolve transposition products for two evolutionarily distinct transposon types that employ either Cascade or Cas12k for RNA-guided DNA integration. Our results show that RNA-guided transposon systems lacking functional TnsA primarily undergo copy-and-paste transposition, generating cointegrate products that comprise duplicated transposon copies and genomic insertion of the vector backbone. Finally, we report natural and engineered transposon variants encoding a TnsAB fusion protein, revealing a novel strategy for achieving RNA-guided transposition with fewer molecular components.},
}
@article {pmid34102625,
year = {2021},
author = {Khakimzhan, A and Garenne, D and Tickman, B and Fontana, J and Carothers, J and Noireaux, V},
title = {Complex dependence of CRISPR-Cas9 binding strength on guide RNA spacer lengths.},
journal = {Physical biology},
volume = {18},
number = {5},
pages = {},
doi = {10.1088/1478-3975/ac091e},
pmid = {34102625},
issn = {1478-3975},
mesh = {*CRISPR-Cas Systems ; Models, Biological ; RNA, Guide/*chemistry ; Thermodynamics ; },
abstract = {It is established that for CRISPR-Cas9 applications guide RNAs with 17-20 bp long spacer sequences are optimal for accurate target binding and cleavage. In this work we perform cell-free CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition) experiments to demonstrate the existence of a complex dependence of CRISPR-Cas9 binding as a function of the spacer length and complementarity. Our results show that significantly truncated or mismatched spacer sequences can form stronger guide-target bonds than the conventional 17-20 bp long spacers. To explain this phenomenon, we take into consideration previous structural and single-molecule CRISPR-Cas9 experiments and develop a novel thermodynamic model of CRISPR-Cas9 target recognition.},
}
@article {pmid34102014,
year = {2022},
author = {Oh, Y and Kim, H and Lee, HJ and Kim, SG},
title = {Ribozyme-processed guide RNA enhances virus-mediated plant genome editing.},
journal = {Biotechnology journal},
volume = {17},
number = {7},
pages = {e2100189},
doi = {10.1002/biot.202100189},
pmid = {34102014},
issn = {1860-7314},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Genome, Plant/genetics ; Plants/genetics ; *RNA, Catalytic/genetics ; *RNA, Guide/genetics ; },
abstract = {In virus-induced gene-editing system, subgenomic promoters have been used to express guide RNAs (gRNAs). However, the transcription initiation site of the subgenomic promoters remains elusive. Here, we examined the sequence of gRNAs expressed by subgenomic promoters and found the variable length of overhangs at 5'-end of gRNAs. The overhangs at 5'-end of gRNA decrease the cleavage activity of SpCas9. To overcome this problem, we inserted hammerhead ribozyme between the subgenomic promoter and gRNA and confirmed that gRNAs with a precise 5'-end increase the editing efficacy in wild tobacco. This system will be widely used for editing target genes in plants with high efficiency.},
}
@article {pmid34101863,
year = {2021},
author = {Ge, C and You, W and Li, R and Li, W and Shao, Y},
title = {Construction of the PG-deficient mutant of Fusarium equiseti by CRISPR/Cas9 and its pathogenicity of pitaya.},
journal = {Journal of basic microbiology},
volume = {61},
number = {8},
pages = {686-696},
doi = {10.1002/jobm.202100120},
pmid = {34101863},
issn = {1521-4028},
mesh = {Antioxidants ; *CRISPR-Cas Systems ; Cactaceae/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fruit/microbiology ; Fusarium/*genetics ; Gene Editing/methods ; Plant Diseases/microbiology ; Virulence/*genetics ; },
abstract = {Fusarium is an important plant pathogen and many cell wall-degrading enzymes (CWDEs) are produced in Fusarium-infected plant tissues. To investigate the role of CWDEs in the pathogenicity of pitaya pathogen, we isolated a Fusarium equiseti strain from the diseased pitaya fruit and the activities of CWDEs were determined. The higher polygalacturonase (PG) activity was confirmed both in vitro and vivo. Aiming at the PG gene, the CRISPR/Cas9 system of F. equiseti was constructed and optimized for the first time. Through the process of microhomology-mediated end joining, the flanking region containing 30 bp was used to mediate the homologous recombination of Cas9 double-strand breaks, and the PG gene knockout mutants were obtained by protoplast transformation. Through the phenotypic and pathogenicity experiments of the wild-type strain and mutant strain, the results showed that the colony growth rate and spore production of the strain without the PG gene decreased to some extent, and the lesion diameter and the degree of pericarp cell damage decreased, which showed that the CRISPR/Cas9 system could be used in F. equiseti and PG enzyme and can play a significant role in the interaction between F. equiseti and pitaya fruit.},
}
@article {pmid34101430,
year = {2021},
author = {Zhang, Y and Song, X and Lai, Y and Mo, Q and Yuan, J},
title = {High-Yielding Terpene-Based Biofuel Production in Rhodobacter capsulatus.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1545-1552},
doi = {10.1021/acssynbio.1c00146},
pmid = {34101430},
issn = {2161-5063},
mesh = {Batch Cell Culture Techniques/methods ; *Biofuels ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Fermentation ; Gene Editing/methods ; Glucose/metabolism ; Metabolic Engineering/*methods ; Mevalonic Acid/metabolism ; NADP/genetics/metabolism ; Photosynthesis ; Promoter Regions, Genetic/genetics ; Rhodobacter capsulatus/genetics/*metabolism ; Rhodospirillaceae/genetics/*metabolism ; Terpenes/*metabolism ; },
abstract = {Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus. The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.},
}
@article {pmid34100716,
year = {2021},
author = {Uehara, H and Zhang, X and Pereira, F and Narendran, S and Choi, S and Bhuvanagiri, S and Liu, J and Ravi Kumar, S and Bohner, A and Carroll, L and Archer, B and Zhang, Y and Liu, W and Gao, G and Ambati, J and Jun, AS and Ambati, BK},
title = {Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34100716},
issn = {2050-084X},
support = {R01 EY017950/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Codon, Initiator/*genetics ; Collagen Type VIII/genetics ; Disease Models, Animal ; *Fuchs' Endothelial Dystrophy/genetics/prevention &amp; control ; Gene Editing/*methods ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Guide/genetics ; },
abstract = {A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.},
}
@article {pmid34100715,
year = {2021},
author = {Zhong, H and Ceballos, CC and Massengill, CI and Muniak, MA and Ma, L and Qin, M and Petrie, SK and Mao, T},
title = {High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34100715},
issn = {2050-084X},
support = {R01 NS081071/NS/NINDS NIH HHS/United States ; R01 NS104944/NS/NINDS NIH HHS/United States ; RF1 MH120119/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Exons/genetics ; Gene Editing/*methods ; Humans ; Mice ; Mutagenesis, Insertional/*genetics ; Neurons/cytology ; Proteins/*analysis/chemistry/*genetics/metabolism ; },
abstract = {Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.},
}
@article {pmid34100058,
year = {2021},
author = {Yin, K and Ding, X and Li, Z and Sfeir, MM and Ballesteros, E and Liu, C},
title = {Autonomous lab-on-paper for multiplexed, CRISPR-based diagnostics of SARS-CoV-2.},
journal = {Lab on a chip},
volume = {21},
number = {14},
pages = {2730-2737},
pmid = {34100058},
issn = {1473-0189},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R61 AI154642/AI/NIAID NIH HHS/United States ; },
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; *Pandemics ; RNA, Viral/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.},
}
@article {pmid34099937,
year = {2021},
author = {Lei, Z and Meng, H and Lv, Z and Liu, M and Zhao, H and Wu, H and Zhang, X and Liu, L and Zhuang, Y and Yin, K and Yan, Y and Yi, C},
title = {Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.},
journal = {Nature methods},
volume = {18},
number = {6},
pages = {643-651},
pmid = {34099937},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; Cytosine/*metabolism ; Gene Editing/*methods ; Humans ; MCF-7 Cells ; Mutation ; RNA/genetics ; Whole Genome Sequencing ; },
abstract = {Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.},
}
@article {pmid34099936,
year = {2021},
author = {Tang, L},
title = {Prime editing progress.},
journal = {Nature methods},
volume = {18},
number = {6},
pages = {592},
doi = {10.1038/s41592-021-01188-2},
pmid = {34099936},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid34099821,
year = {2021},
author = {Takahashi, K and Niki, T and Ogawa, E and Fumika, K and Nishioka, Y and Sawa, M and Arai, H and Mukai, K and Taguchi, T},
title = {A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {11996},
pmid = {34099821},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cholesterol/*metabolism ; Complex Mixtures/*metabolism ; Cytosol/metabolism/ultrastructure ; DNA/*metabolism ; Endoplasmic Reticulum/metabolism ; Gene Knockdown Techniques ; Golgi Apparatus/metabolism ; Humans ; Interferon Regulatory Factor-3/metabolism ; Interferon Type I/metabolism ; Lipoylation ; Phosphorylation/*drug effects ; Protein Serine-Threonine Kinases/metabolism ; Recombinant Proteins/metabolism ; Signal Transduction ; Sphingomyelin Phosphodiesterase/metabolism ; Sphingomyelins/*metabolism ; beta-Cyclodextrins/metabolism ; },
abstract = {Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.},
}
@article {pmid34099548,
year = {2021},
author = {Gonzalez-Pena, V and Natarajan, S and Xia, Y and Klein, D and Carter, R and Pang, Y and Shaner, B and Annu, K and Putnam, D and Chen, W and Connelly, J and Pruett-Miller, S and Chen, X and Easton, J and Gawad, C},
title = {Accurate genomic variant detection in single cells with primary template-directed amplification.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {24},
pages = {},
pmid = {34099548},
issn = {1091-6490},
support = {DP2 CA239145/CA/NCI NIH HHS/United States ; L40 CA162153/CA/NCI NIH HHS/United States ; },
mesh = {Base Pairing/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Genetic Variation ; *Genome, Human ; Humans ; Mutagens/metabolism ; *Nucleic Acid Amplification Techniques ; Polymorphism, Single Nucleotide/genetics ; *Single-Cell Analysis ; *Templates, Genetic ; },
abstract = {Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.},
}
@article {pmid34098567,
year = {2021},
author = {Xu, J and Xu, K and Jung, S and Conte, A and Lieberman, J and Muecksch, F and Lorenzi, JCC and Park, S and Schmidt, F and Wang, Z and Huang, Y and Luo, Y and Nair, MS and Wang, P and Schulz, JE and Tessarollo, L and Bylund, T and Chuang, GY and Olia, AS and Stephens, T and Teng, IT and Tsybovsky, Y and Zhou, T and Munster, V and Ho, DD and Hatziioannou, T and Bieniasz, PD and Nussenzweig, MC and Kwong, PD and Casellas, R},
title = {Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.},
journal = {Nature},
volume = {595},
number = {7866},
pages = {278-282},
pmid = {34098567},
issn = {1476-4687},
support = {HHSN261200800001C/RC/CCR NIH HHS/United States ; HHSN261200800001E/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antibodies, Neutralizing/chemistry/genetics/*immunology/isolation &amp; purification ; CRISPR-Cas Systems ; Camelids, New World/genetics/*immunology ; Female ; Gene Editing ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Models, Molecular ; Mutation ; Neutralization Tests ; SARS-CoV-2/chemistry/genetics/*immunology ; Single-Domain Antibodies/*chemistry/genetics/*immunology/isolation &amp; purification ; Somatic Hypermutation, Immunoglobulin/genetics ; Spike Glycoprotein, Coronavirus/*chemistry/*immunology ; },
abstract = {Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization[1-3]. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies[4]. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.},
}
@article {pmid34097395,
year = {2021},
author = {Maximiano, MR and Távora, FTPK and Prado, GS and Dias, SC and Mehta, A and Franco, OL},
title = {CRISPR Genome Editing Technology: A Powerful Tool Applied to Developing Agribusiness.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {23},
pages = {6379-6395},
doi = {10.1021/acs.jafc.1c01062},
pmid = {34097395},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; Humans ; Plants, Genetically Modified/genetics ; },
abstract = {The natural increase of the world's population implies boosting agricultural demand. In the current non-optimistic global scenario, where adverse climate changes come associated with substantial population growth, the main challenge in agribusiness is food security. Recently, the CRISPR/Cas system has emerged as a friendly gene editing biotechnological tool, enabling a precise manipulation of genomes and enhancement of desirable traits in several organisms. This review highlights the CRISPR/Cas system as a paramount tool for the improvement of agribusiness products and brings up-to-date findings showing its potential applications in improving agricultural-related traits in major plant crops and farm animals, all representing economic-relevant commodities responsible for feeding the world. Several applied pieces of research have successfully demonstrated the CRISPR/Cas ability in boosting interesting traits in agribusiness products, including animal productivity and welfare, crop yield growth, and seed quality, reflecting positive impacts in both socioeconomics and human health aspects. Hence, the CRISPR/Cas system has revolutionized bioscience and biotechnology, and its concrete application in agribusiness goods is on the horizon.},
}
@article {pmid34097133,
year = {2021},
author = {Hu, L and Feng, S and Liang, G and Du, J and Li, A and Niu, C},
title = {CRISPR/Cas9-induced β-carotene hydroxylase mutation in Dunaliella salina CCAP19/18.},
journal = {AMB Express},
volume = {11},
number = {1},
pages = {83},
pmid = {34097133},
issn = {2191-0855},
abstract = {Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.},
}
@article {pmid34096883,
year = {2021},
author = {Bi, D and Yao, J and Wang, Y and Qin, G and Zhang, Y and Wang, Y and Zhao, J},
title = {CRISPR/Cas13d-mediated efficient KDM5B mRNA knockdown in porcine somatic cells and parthenogenetic embryos.},
journal = {Reproduction (Cambridge, England)},
volume = {162},
number = {2},
pages = {149-160},
pmid = {34096883},
issn = {1741-7899},
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo, Mammalian/cytology/*metabolism ; *Embryonic Development ; *Gene Expression Regulation, Developmental ; Histones/genetics/metabolism ; Jumonji Domain-Containing Histone Demethylases/*antagonists &amp; inhibitors/genetics/metabolism ; *Parthenogenesis ; RNA, Messenger/*antagonists &amp; inhibitors/genetics ; Swine ; },
abstract = {An efficient mRNA knockdown strategy is needed to explore gene function in cells and embryos, especially to understand the process of maternal mRNA decay during early embryo development. Cas13, a novel RNA-targeting CRISPR effector protein, could bind and cleave complementary single-strand RNA, which has been employed for mRNA knockdown in mouse and human cells and RNA-virus interference in plants. Cas13 has not yet been reported to be used in pigs. In the current study, we explored the feasibility of CRISPR/Cas13d-mediated endogenous RNA knockdown in pigs. KDM5B, a histone demethylase of H3K4me3, was downregulated at the transcriptional level by 50% with CRISPR/Cas13d in porcine fibroblast cells. Knockdown of KDM5B-induced H3K4me3 expression and decreased the abundance of H3K27me3, H3K9me3, H3K4ac, H4K8ac, and H4K12ac. These changes affected cell proliferation and cell cycle. Furthermore, stable integration of the CRISPR/Cas13d system into the porcine genome resulted in the continuous expression of Cas13d and persistent knockdown of KDM5B. Finally, the RNA-targeting potential of Cas13d was further validated in porcine parthenogenetic embryos. By microinjection of Cas13d mRNA and gRNA targeting KDM5B into porcine oocytes, the expression of KDM5B was downregulated, the abundance of H3K4me3 increased as expected, and the expression of embryonic development-related genes was changed accordingly. These results indicate that CRISPR/Cas13d provides an easily programmable platform for spatiotemporal transcriptional manipulation in pigs.},
}
@article {pmid34096786,
year = {2021},
author = {Turner, DJ and Turner, M},
title = {RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {427-437},
pmid = {34096786},
issn = {2573-1602},
support = {200823/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Helicases/genetics ; Eukaryotic Initiation Factor-4E ; Gene Knockout Techniques ; Genomic Library ; Humans ; Oxidative Stress/*physiology ; Poly-ADP-Ribose Binding Proteins/genetics ; RNA Helicases/genetics ; RNA Recognition Motif Proteins/genetics ; RNA, Guide/*genetics ; RNA-Binding Proteins/genetics/*metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). We demonstrated the utility of our reagent in a genetic screen for RBPs that conveyed cellular resistance or sensitivity to oxidative stress induced by paraquat. This identified that CSDE1 and STRAP, proteins that interact with each other, convey sensitivity to oxidative stress and that Pumilio homologues (PUM1 and PUM2) convey resistance. Targeting eIF4-E1 and -A1 protected cells from high-dose paraquat, whereas eIF4E2 targeted cells did less well. We also found that G3BP1 promoted sensitivity to a low dose of paraquat but protected cells at a higher dose. Our study highlights the use of genetic screens to identify roles of RBPs and identifies novel genes regulating sensitivity to oxidative stress.},
}
@article {pmid34096572,
year = {2021},
author = {Yamada, K and Maeno, A and Araki, S and Kikuchi, M and Suzuki, M and Ishizaka, M and Satoh, K and Akama, K and Kawabe, Y and Suzuki, K and Kobayashi, D and Hamano, N and Kawamura, A},
title = {An atlas of seven zebrafish hox cluster mutants provides insights into sub/neofunctionalization of vertebrate Hox clusters.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {11},
pages = {},
doi = {10.1242/dev.198325},
pmid = {34096572},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development/genetics ; Evolution, Molecular ; Female ; Gene Duplication ; Gene Expression Regulation, Developmental ; Genes, Homeobox/*genetics ; Male ; *Multigene Family ; Mutation ; Skeleton/anatomy &amp; histology/growth &amp; development ; Species Specificity ; X-Ray Microtomography ; Zebrafish/embryology/*genetics/*physiology ; },
abstract = {Vertebrate Hox clusters are comprised of multiple Hox genes that control morphology and developmental timing along multiple body axes. Although results of genetic analyses using Hox-knockout mice have been accumulating, genetic studies in other vertebrates have not been sufficient for functional comparisons of vertebrate Hox genes. In this study, we isolated all of the seven hox cluster loss-of-function alleles in zebrafish using the CRISPR-Cas9 system. Comprehensive analysis of the embryonic phenotype and X-ray micro-computed tomography scan analysis of adult fish revealed several species-specific functional contributions of homologous Hox clusters along the appendicular axis, whereas important shared general principles were also confirmed, as exemplified by serial anterior vertebral transformations along the main body axis, observed in fish for the first time. Our results provide insights into discrete sub/neofunctionalization of vertebrate Hox clusters after quadruplication of the ancient Hox cluster. This set of seven complete hox cluster loss-of-function alleles provide a formidable resource for future developmental genetic analysis of the Hox patterning system in zebrafish.},
}
@article {pmid34095868,
year = {2021},
author = {Yu, JJS and Vincent, JP and McGough, IJ},
title = {One-step CRISPR-Cas9 protocol for the generation of plug &amp; play conditional knockouts in Drosophila melanogaster.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100560},
pmid = {34095868},
issn = {2666-1667},
support = {206341/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; FC001204/CRUK_/Cancer Research UK/United Kingdom ; FC001204/MRC_/Medical Research Council/United Kingdom ; FC001204/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Drosophila melanogaster/*genetics ; Gene Editing/methods ; *Gene Knockdown Techniques ; },
abstract = {This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required. For complete details on the use and execution of this protocol, please refer to the DWnt4[cKO] example in Yu et al. (2020).},
}
@article {pmid34095862,
year = {2021},
author = {Zhou, Z and Ma, L and Zhang, X},
title = {Protocol for genome-scale CRISPR screening in engineered lineage reporter hPSCs to study cell fate determination.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100548},
pmid = {34095862},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Culture Techniques ; Cell Differentiation/*genetics ; Cell Lineage/*genetics ; Cells, Cultured ; Genome/genetics ; Genomics/*methods ; Humans ; Pluripotent Stem Cells/*cytology ; },
abstract = {PAX6 is a key determinant of human neuroectoderm cell fate. Here, we describe a protocol for genome-scale CRISPR screening for use in genetically engineered human pluripotent stem cells (hPSCs). Using the germ layer reporter PAX6 and an inducible CRISPR/Cas9 knockout system, we describe how to identify lineage-specific preventing genes. This protocol can be applied for use with other reporter genes to study cell fate determination in hPSCs. For complete details on the use and execution of this protocol, please refer to Xu et al. (2021).},
}
@article {pmid34095617,
year = {2021},
author = {Gong, J and Kan, L and Zhang, X and He, Y and Pan, J and Zhao, L and Li, Q and Liu, M and Tian, J and Lin, S and Lu, Z and Xue, L and Wang, C and Tang, G},
title = {An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip.},
journal = {Bioactive materials},
volume = {6},
number = {12},
pages = {4580-4590},
pmid = {34095617},
issn = {2452-199X},
abstract = {CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.},
}
@article {pmid34093491,
year = {2021},
author = {Taylor, HN and Laderman, E and Armbrust, M and Hallmark, T and Keiser, D and Bondy-Denomy, J and Jackson, RN},
title = {Positioning Diverse Type IV Structures and Functions Within Class 1 CRISPR-Cas Systems.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {671522},
pmid = {34093491},
issn = {1664-302X},
support = {R35 GM138080/GM/NIGMS NIH HHS/United States ; },
abstract = {Type IV CRISPR systems encode CRISPR associated (Cas)-like proteins that combine with small RNAs to form multi-subunit ribonucleoprotein complexes. However, the lack of Cas nucleases, integrases, and other genetic features commonly observed in most CRISPR systems has made it difficult to predict type IV mechanisms of action and biological function. Here we summarize recent bioinformatic and experimental advancements that collectively provide the first glimpses into the function of specific type IV subtypes. We also provide a bioinformatic and structural analysis of type IV-specific proteins within the context of multi-subunit (class 1) CRISPR systems, informing future studies aimed at elucidating the function of these cryptic systems.},
}
@article {pmid34093484,
year = {2021},
author = {Tambong, JT and Xu, R and Gerdis, S and Daniels, GC and Chabot, D and Hubbard, K and Harding, MW},
title = {Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {666689},
pmid = {34093484},
issn = {1664-302X},
abstract = {Xanthomonas translucens is the etiological agent of the wheat bacterial leaf streak (BLS) disease. The isolation of this pathogen is usually based on the Wilbrink's-boric acid-cephalexin semi-selective medium which eliminates 90% of other bacteria, some of which might be novel species. In our study, a general purpose nutrient agar was used to isolate 49 bacterial strains including X. translucens from necrotic wheat leaf tissues. Maximum likelihood cluster analysis of 16S rRNA sequences grouped the strains into 10 distinct genera. Pseudomonas (32.7%) and Pantoea (28.6%) were the dominant genera while Xanthomonas, Clavibacter and Curtobacterium had 8.2%, each. Erwinia and Sphingomonas had two strains, each. BLAST and phylogenetic analyses of multilocus sequence analysis (MLSA) of specific housekeeping genes taxonomically assigned all the strains to validly described bacterial species, except three strains (10L4B, 12L4D and 32L3A) of Pseudomonas and two (23L3C and 15L3B) of Sphingomonas. Strains 10L4B and12L4D had Pseudomonas caspiana as their closest known type strain while strain 32L3A was closest to Pseudomonas asturiensis. Sphingomonas sp. strains 23L3C and 15L3B were closest to S. faeni based on MLSA analysis. Our data on MLSA, whole genome-based cluster analysis, DNA-DNA hybridization and average nucleotide identity, matrix-assisted laser desorption/ionization-time-of-flight, chemotaxonomy and phenotype affirmed that these 5 strains constitute three novel lineages and are taxonomically described in this study. We propose the names, Sphingomonas albertensis sp. nov. (type strain 23L3C[T] = DOAB 1063[T] = CECT 30248[T] = LMG 32139[T]), Pseudomonas triticumensis sp. nov. (type strain 32L3A[T] = DOAB 1067[T] = CECT 30249[T] = LMG 32140[T]) and Pseudomonas foliumensis sp. nov. (type strain 10L4B[T] = DOAB 1069[T] = CECT 30250[T] = LMG 32142[T]). Comparative genomics of these novel species, relative to their closest type strains, revealed unique repertoires of core secretion systems and secondary metabolites/antibiotics. Also, the detection of CRISPR-Cas systems in the genomes of these novel species suggests an acquired mechanism for resistance against foreign mobile genetic elements. The results presented here revealed a cohabitation, within the BLS lesions, of diverse bacterial species, including novel lineages.},
}
@article {pmid34093475,
year = {2021},
author = {Valente, S and Piombo, E and Schroeckh, V and Meloni, GR and Heinekamp, T and Brakhage, AA and Spadaro, D},
title = {CRISPR-Cas9-Based Discovery of the Verrucosidin Biosynthesis Gene Cluster in Penicillium polonicum.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {660871},
pmid = {34093475},
issn = {1664-302X},
abstract = {Penicillium polonicum, commonly found on food matrices, is a mycotoxigenic species able to produce a neurotoxin called verrucosidin. This methylated α-pyrone polyketide inhibits oxidative phosphorylation in mitochondria and thereby causes neurological diseases. Despite the importance of verrucosidin as a toxin, its biosynthetic genes have not been characterized yet. By similarity analysis with the polyketide synthase (PKS) genes for the α-pyrones aurovertin (AurA) and citreoviridin (CtvA), 16 PKS genes for putative α-pyrones were identified in the P. polonicum genome. A single PKS gene, verA, was found to be transcribed under verrucosidin-producing growth conditions. The annotated functions of the genes neighboring verA correspond to those required for verrucosidin biosynthesis. To prove the involvement of verA in verrucosidin biosynthesis, the clustered regularly interspaced short palindrome repeats (CRISPR) technology was applied to P. polonicum. In vitro reconstituted CRISPR-Cas9 was used to induce targeted gene deletions in P. polonicum. This approach allowed identifying and characterizing the verrucosidin biosynthetic gene cluster. VerA deletion mutants were no longer able to produce verrucosidin, whereas they were displaying morphological characteristics comparable with the wild-type strain. The available CRISPR-Cas9 technology allows characterizing the biosynthetic potential of P. polonicum as a valuable source of novel compounds.},
}
@article {pmid34092790,
year = {2021},
author = {Wang, X and Xu, J and Zhang, B and Hou, Y and Song, F and Lyu, H and Yue, F},
title = {Genome-wide detection of enhancer-hijacking events from chromatin interaction data in rearranged genomes.},
journal = {Nature methods},
volume = {18},
number = {6},
pages = {661-668},
pmid = {34092790},
issn = {1548-7105},
support = {R01 HG009906/HG/NHGRI NIH HHS/United States ; R35 GM124820/GM/NIGMS NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems ; Chromatin/*metabolism ; Developmental Disabilities/genetics ; *Enhancer Elements, Genetic ; *Genome, Human ; *Genomic Structural Variation ; Humans ; K562 Cells ; Protein Binding ; Real-Time Polymerase Chain Reaction ; },
abstract = {Recent efforts have shown that structural variations (SVs) can disrupt three-dimensional genome organization and induce enhancer hijacking, yet no computational tools exist to identify such events from chromatin interaction data. Here, we develop NeoLoopFinder, a computational framework to identify the chromatin interactions induced by SVs, including interchromosomal translocations, large deletions and inversions. Our framework can automatically resolve complex SVs, reconstruct local Hi-C maps surrounding the breakpoints, normalize copy number variation and allele effects and predict chromatin loops induced by SVs. We applied NeoLoopFinder in Hi-C data from 50 cancer cell lines and primary tumors and identified tens of recurrent genes associated with enhancer hijacking. To experimentally validate NeoLoopFinder, we deleted the hijacked enhancers in prostate adenocarcinoma cells using CRISPR-Cas9, which significantly reduced expression of the target oncogene. In summary, NeoLoopFinder enables identification of critical oncogenic regulatory elements that can potentially reveal therapeutic targets.},
}
@article {pmid34091588,
year = {2021},
author = {Sachinidis, A},
title = {High-throughput base editing: a promising technology for precision medicine and drug discovery.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {221},
pmid = {34091588},
issn = {2059-3635},
mesh = {CRISPR-Cas Systems ; Drug Discovery ; *Gene Editing ; *Precision Medicine ; Technology ; },
}
@article {pmid34091053,
year = {2022},
author = {van Hees, M and Slott, S and Hansen, AH and Kim, HS and Ji, HP and Astakhova, K},
title = {New approaches to moderate CRISPR-Cas9 activity: Addressing issues of cellular uptake and endosomal escape.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {32-46},
pmid = {34091053},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Endosomes/metabolism ; Gene Editing/methods ; Gold/metabolism ; Liposomes ; *Metal Nanoparticles ; Nanoparticles ; },
abstract = {CRISPR-Cas9 is rapidly entering molecular biology and biomedicine as a promising gene-editing tool. A unique feature of CRISPR-Cas9 is a single-guide RNA directing a Cas9 nuclease toward its genomic target. Herein, we highlight new approaches for improving cellular uptake and endosomal escape of CRISPR-Cas9. As opposed to other recently published works, this review is focused on non-viral carriers as a means to facilitate the cellular uptake of CRISPR-Cas9 through endocytosis. The majority of non-viral carriers, such as gold nanoparticles, polymer nanoparticles, lipid nanoparticles, and nanoscale zeolitic imidazole frameworks, is developed with a focus toward optimizing the endosomal escape of CRISPR-Cas9 by taking advantage of the acidic environment in the late endosomes. Among the most broadly used methods for in vitro and ex vivo ribonucleotide protein transfection are electroporation and microinjection. Thus, other delivery formats are warranted for in vivo delivery of CRISPR-Cas9. Herein, we specifically revise the use of peptide and nanoparticle-based systems as platforms for CRISPR-Cas9 delivery in vivo. Finally, we highlight future perspectives of the CRISPR-Cas9 gene-editing tool and the prospects of using non-viral vectors to improve its bioavailability and therapeutic potential.},
}
@article {pmid34089034,
year = {2021},
author = {Subbaraman, N},
title = {First science adviser in US president's cabinet talks COVID, spying and more.},
journal = {Nature},
volume = {594},
number = {7863},
pages = {311},
doi = {10.1038/d41586-021-01501-1},
pmid = {34089034},
issn = {1476-4687},
mesh = {*COVID-19/epidemiology ; CRISPR-Cas Systems ; China ; Disaster Planning/organization &amp; administration ; Humans ; International Cooperation ; Pandemics/prevention &amp; control/statistics &amp; numerical data ; Policy Making ; *Politics ; Prejudice/prevention &amp; control ; Research Support as Topic/organization &amp; administration ; Science/ethics/*legislation &amp; jurisprudence/*organization &amp; administration/standards ; *Security Measures ; United States/epidemiology ; },
}
@article {pmid34089013,
year = {2021},
author = {Jia, N and Patel, DJ},
title = {Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins.},
journal = {Nature reviews. Molecular cell biology},
volume = {22},
number = {8},
pages = {563-579},
pmid = {34089013},
issn = {1471-0080},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Bacteriophages/metabolism ; *Biotechnology ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/chemistry/metabolism ; CRISPR-Cas Systems/*immunology ; Gene Editing ; Protein Binding ; Viral Proteins/*chemistry/*metabolism ; },
abstract = {CRISPR loci and Cas proteins provide adaptive immunity in prokaryotes against invading bacteriophages and plasmids. In response, bacteriophages have evolved a broad spectrum of anti-CRISPR proteins (anti-CRISPRs) to counteract and overcome this immunity pathway. Numerous anti-CRISPRs have been identified to date, which suppress single-subunit Cas effectors (in CRISPR class 2, type II, V and VI systems) and multisubunit Cascade effectors (in CRISPR class 1, type I and III systems). Crystallography and cryo-electron microscopy structural studies of anti-CRISPRs bound to effector complexes, complemented by functional experiments in vitro and in vivo, have identified four major CRISPR-Cas suppression mechanisms: inhibition of CRISPR-Cas complex assembly, blocking of target binding, prevention of target cleavage, and degradation of cyclic oligonucleotide signalling molecules. In this Review, we discuss novel mechanistic insights into anti-CRISPR function that have emerged from X-ray crystallography and cryo-electron microscopy studies, and how these structures in combination with function studies provide valuable tools for the ever-growing CRISPR-Cas biotechnology toolbox, to be used for precise and robust genome editing and other applications.},
}
@article {pmid34087992,
year = {2021},
author = {Tsukamoto, S and Nakade, K and Wakabayashi, T and Nakashima, K and Takami, M and Hemmi, Y and Kuramochi, Y and Shimizu, T and Arai, Y and Matsuo-Takasaki, M and Noguchi, M and Nakamura, Y and Miwa, Y and Hayashi, Y},
title = {Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102363},
doi = {10.1016/j.scr.2021.102363},
pmid = {34087992},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Luminescent Proteins ; },
abstract = {ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.},
}
@article {pmid34087991,
year = {2021},
author = {Wei, C and Luo, Q and Wang, B and Long, Y and Zhang, M and Shan, W and Yu, X and Xu, Y and Qian, P and Huang, H},
title = {Generation of a FTO gene knockout human embryonic stem cell line using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102362},
doi = {10.1016/j.scr.2021.102362},
pmid = {34087991},
issn = {1876-7753},
mesh = {Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics ; *CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing ; Gene Knockout Techniques ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.},
}
@article {pmid34087989,
year = {2021},
author = {Kato, H and Maezawa, Y and Ouchi, Y and Takayama, N and Sone, M and Sone, K and Takada-Watanabe, A and Tsujimura, K and Koshizaka, M and Nagasawa, S and Saitoh, H and Ohtaka, M and Nakanishi, M and Tahara, H and Shimamoto, A and Iwama, A and Eto, K and Yokote, K},
title = {Generation of disease-specific and CRISPR/Cas9-mediated gene-corrected iPS cells from a patient with adult progeria Werner syndrome.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102360},
doi = {10.1016/j.scr.2021.102360},
pmid = {34087989},
issn = {1876-7753},
mesh = {Adult ; CRISPR-Cas Systems/genetics ; Exodeoxyribonucleases/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Werner Syndrome/genetics ; Werner Syndrome Helicase/genetics/metabolism ; },
abstract = {Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.},
}
@article {pmid34087984,
year = {2021},
author = {Luo, Q and Wei, C and Long, Y and Zhang, M and Shan, W and Li, H and Cai, S and Xu, Y and Qian, P and Huang, H},
title = {Generation of an ELTD1 knockout human embryonic stem cell line by the iCRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102350},
doi = {10.1016/j.scr.2021.102350},
pmid = {34087984},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Epidermal Growth Factor ; *Human Embryonic Stem Cells ; Humans ; *Neoplasms ; Neovascularization, Pathologic ; },
abstract = {Human ELTD1 (Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has been reported as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. However, little is known about the function of ELTD1, especially its undiscovered ligands. In this experiment, an ELTD1 homozygous knockout human embryonic stem cell line, FAHZUe001-A, was generated by the iCRISPR/Cas9 system to achieve a deeper understanding of ELTD1. The FAHZUe001-A was confirmed with normal karyotype, typical undifferentiated morphology, pluripotency and trilineage differentiation potential in vitro.},
}
@article {pmid34087983,
year = {2021},
author = {Feng, H and Li, Q and Margolis, RL and Li, PP},
title = {Generation of a human induced pluripotent stem cell line JHUi003-A with homozygous mutation for spinocerebellar ataxia type 12 using genome editing.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102346},
doi = {10.1016/j.scr.2021.102346},
pmid = {34087983},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; Mutation/genetics ; *Spinocerebellar Ataxias/genetics ; },
abstract = {Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding a brain-specific regulatory unit of protein phosphatase 2A (PP2A); while normal alleles carry 4 to 31 triplets, the disease alleles carry 43 to 78 triplets. Here, by CRISPR/Cas9 genome editing, we have generated a human homozygous SCA12 iPSC line with 69 and 72 triplets for each allele. The homozygous SCA12 iPSCs have normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.},
}
@article {pmid34087725,
year = {2021},
author = {Lee Yu, H and Cao, Y and Lu, X and Hsing, IM},
title = {Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity.},
journal = {Biosensors &amp; bioelectronics},
volume = {189},
number = {},
pages = {113382},
doi = {10.1016/j.bios.2021.113382},
pmid = {34087725},
issn = {1873-4235},
mesh = {Alleles ; Bacterial Proteins/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {The sensitive and accurate detection of rare mutations has profound clinical implications; however, current methods require expensive instrumentation and are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations. Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection but its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. Here, we report an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans-cleavage activity. We show that our strategy can allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.},
}
@article {pmid34086963,
year = {2021},
author = {Pedroza-Garcia, JA and Eekhout, T and Achon, I and Nisa, MU and Coussens, G and Vercauteren, I and Van den Daele, H and Pauwels, L and Van Lijsebettens, M and Raynaud, C and De Veylder, L},
title = {Maize ATR safeguards genome stability during kernel development to prevent early endosperm endocycle onset and cell death.},
journal = {The Plant cell},
volume = {33},
number = {8},
pages = {2662-2684},
pmid = {34086963},
issn = {1532-298X},
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/genetics ; Ataxia Telangiectasia Mutated Proteins/genetics ; CRISPR-Cas Systems ; Cell Death/genetics ; DNA Breaks, Double-Stranded ; DNA Repair/*genetics ; DNA Replication/genetics ; Endosperm/cytology/*genetics ; Genomic Instability ; Mutation ; Plant Cells ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Seeds/cytology/genetics/growth &amp; development ; Zea mays/cytology/*genetics/growth &amp; development ; },
abstract = {The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.},
}
@article {pmid34086942,
year = {2021},
author = {Jung, SB and Lee, CY and Lee, KH and Heo, K and Choi, SH},
title = {A cleavage-based surrogate reporter for the evaluation of CRISPR-Cas9 cleavage efficiency.},
journal = {Nucleic acids research},
volume = {49},
number = {15},
pages = {e85},
pmid = {34086942},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; Endonucleases/*genetics ; *Gene Editing ; Genes, Reporter/genetics ; Green Fluorescent Proteins/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Lac Repressors/*genetics ; Peptide Elongation Factor 1/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR-Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.},
}
@article {pmid34086870,
year = {2021},
author = {Brault, J and Liu, T and Bello, E and Liu, S and Sweeney, CL and Meis, RJ and Koontz, S and Corsino, C and Choi, U and Vayssiere, G and Bosticardo, M and Dowdell, K and Lazzarotto, CR and Clark, AB and Notarangelo, LD and Ravell, JC and Lenardo, MJ and Kleinstiver, BP and Tsai, SQ and Wu, X and Dahl, GA and Malech, HL and De Ravin, SS},
title = {CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.},
journal = {Blood},
volume = {138},
number = {26},
pages = {2768-2780},
pmid = {34086870},
issn = {1528-0020},
mesh = {Animals ; CRISPR-Cas Systems ; Cation Transport Proteins/deficiency/*genetics ; Cells, Cultured ; Female ; *Gene Editing/methods ; Genetic Therapy ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism/pathology ; Humans ; Lymphocytes/*metabolism/pathology ; Male ; Mice, Inbred NOD ; X-Linked Combined Immunodeficiency Diseases/*genetics/pathology/therapy ; },
abstract = {XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.},
}
@article {pmid34086867,
year = {2021},
author = {Psatha, N and Georgakopoulou, A and Li, C and Nandakumar, V and Georgolopoulos, G and Acosta, R and Paschoudi, K and Nelson, J and Chee, D and Athanasiadou, A and Kouvatsi, A and Funnell, APW and Lieber, A and Yannaki, E and Papayannopoulou, T},
title = {Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo.},
journal = {Blood},
volume = {138},
number = {17},
pages = {1540-1553},
pmid = {34086867},
issn = {1528-0020},
support = {R01 DK101328/DK/NIDDK NIH HHS/United States ; RM1 HG007743/HG/NHGRI NIH HHS/United States ; },
mesh = {Adult ; Animals ; Antigens, CD34/*genetics ; CRISPR-Cas Systems ; Cells, Cultured ; Fetal Hemoglobin/*genetics ; Gene Editing ; Genetic Therapy ; Humans ; Mice ; *Mutagenesis ; beta-Thalassemia/*genetics/therapy ; gamma-Globins/genetics ; },
abstract = {Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe β-globin phenotype.},
}
@article {pmid34086743,
year = {2021},
author = {Foreman, HC and Kirillov, V and Paniccia, G and Catalano, D and Andrunik, T and Gupta, S and Krug, LT and Zhang, Y},
title = {RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.},
journal = {PloS one},
volume = {16},
number = {6},
pages = {e0252313},
pmid = {34086743},
issn = {1932-6203},
support = {R43 AI131958/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/virology ; CRISPR-Cas Systems/genetics ; Cell Line ; Gammaherpesvirinae/*genetics ; Gene Editing/methods ; Gene Expression Regulation, Viral/genetics ; Genome, Viral/*genetics ; HEK293 Cells ; Herpesviridae Infections/virology ; Herpesvirus 8, Human/genetics ; Humans ; Mice ; Models, Animal ; NIH 3T3 Cells ; RNA, Guide/*genetics ; Virus Activation/genetics ; Virus Latency/genetics ; Virus Replication/genetics ; },
abstract = {Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.},
}
@article {pmid34086167,
year = {2021},
author = {Čermák, T},
title = {Sequence modification on demand: search and replace tools for precise gene editing in plants.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {353-379},
pmid = {34086167},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; *DNA Repair ; Endonucleases/genetics/metabolism ; *Gene Editing ; Gene Targeting ; *Genome, Plant ; Plant Breeding/*methods ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {Until recently, our ability to generate allelic diversity in plants was limited to introduction of variants from domesticated and wild species by breeding via uncontrolled recombination or the use of chemical and physical mutagens-processes that are lengthy and costly or lack specificity, respectively. Gene editing provides a faster and more precise way to create new variation, although its application in plants has been dominated by the creation of short insertion and deletion mutations leading to loss of gene function, mostly due to the dependence of editing outcomes on DNA repair pathway choices intrinsic to higher eukaryotes. Other types of edits such as point mutations and precise and pre-designed targeted sequence insertions have rarely been implemented, despite providing means to modulate the expression of target genes or to engineer the function and stability of their protein products. Several advancements have been developed in recent years to facilitate custom editing by regulation of repair pathway choices or by taking advantage of alternative types of DNA repair. We have seen the advent of novel gene editing tools that are independent of DNA double-strand break repair, and methods completely independent of host DNA repair processes are being increasingly explored. With the aim to provide a comprehensive review of the state-of-the-art methodology for allele replacement in plants, I discuss the adoption of these improvements for plant genome engineering.},
}
@article {pmid34086068,
year = {2021},
author = {Li, X and Zuo, X and Li, M and Yang, X and Zhi, J and Sun, H and Xie, C and Zhang, Z and Wang, F},
title = {Efficient CRISPR/Cas9-mediated genome editing in Rehmannia glutinosa.},
journal = {Plant cell reports},
volume = {40},
number = {9},
pages = {1695-1707},
pmid = {34086068},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems ; Carotenoids/metabolism ; Chlorophyll/genetics/metabolism ; Cloning, Molecular ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genome, Plant ; Mutation ; Oxidoreductases/*genetics ; Plant Proteins/genetics ; Plants, Genetically Modified ; Rehmannia/*genetics/metabolism ; },
abstract = {Here, we cloned a phytoene desaturase (PDS) gene from Rehmannia glutinosa, and realized RgPDS1 knock out in R. glutinosa resulted in the generation of albino plants. Rehmannia glutinosa is a highly important traditional Chinese medicine (TCM) with specific pharmacology and economic value. R. glutinosa is a tetraploid plant, to date, no report has been published on gene editing of R. glutinosa. In this study, we combined the transcriptome database of R. glutinosa and the reported phytoene desaturase (PDS) gene sequences to obtain the PDS gene of R. glutinosa. Then, the PDS gene was used as a marker gene to verify the applicability and gene editing efficiency of the CRISPR/Cas9 system in R. glutinosa. The constructed CRISPR/Cas9 system was mediated by Agrobacterium to genetically transform into R. glutinosa, and successfully regenerated fully albino and chimeric albino plants. The next-generation sequencing (NGS) confirmed that the albino phenotype was indeed caused by RgPDS gene target site editing, and it was found that base deletion was more common than insertion or replacement. Our results revealed that zCas9 has a high editing efficiency on the R. glutinosa genome. This research lays a foundation for further use of gene editing technology to study the molecular functions of genes, create excellent germplasm, accelerate domestication, and improve the yield and quality of R. glutinosa.},
}
@article {pmid34086055,
year = {2021},
author = {Zong, X and Hao, X and Xu, B and Crawford, JC and Wright, S and Li, J and Zhang, Y and Bai, L and He, M and Jiang, M and Fan, Y and Connelly, JP and Pruett-Miller, SM and Berns, H and Janke, L and Li, C and Feng, Y},
title = {Foxp3 enhancers synergize to maximize regulatory T cell suppressive capacity.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {8},
pages = {},
pmid = {34086055},
issn = {1540-9538},
support = {P30 CA021765/CA/NCI NIH HHS/United States ; R01 AI153138/AI/NIAID NIH HHS/United States ; R21 AI146614/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Enhancer Elements, Genetic/*genetics ; Epigenesis, Genetic ; Epistasis, Genetic ; Female ; Forkhead Transcription Factors/genetics/*metabolism ; Lymphocyte Activation/immunology ; Mice, Inbred C57BL ; Mice, Transgenic ; Receptors, Antigen, T-Cell/metabolism ; T-Lymphocytes, Regulatory/*immunology ; Thymus Gland/immunology ; },
abstract = {T reg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of T reg lineage-specifying factor Foxp3. Foxp3 enhancers are known as distinct readers of environmental cues controlling T reg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby T reg cell suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize T reg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers leads to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable T reg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cell-intrinsic factors and cell-extrinsic cues in determining T reg cell suppressive capacity.},
}
@article {pmid34085701,
year = {2021},
author = {Smith, BRC and Nyström, A and Nowell, CJ and Hausser, I and Gretzmeier, C and Robertson, SJ and Varigos, GA and Has, C and Kern, JS and Pang, KC},
title = {Mouse models for dominant dystrophic epidermolysis bullosa carrying common human point mutations recapitulate the human disease.},
journal = {Disease models &amp; mechanisms},
volume = {14},
number = {6},
pages = {},
pmid = {34085701},
issn = {1754-8411},
mesh = {Animals ; CRISPR-Cas Systems ; Collagen Type VII/genetics ; Disease Models, Animal ; Epidermolysis Bullosa Dystrophica/*genetics ; Humans ; Mice ; *Point Mutation ; },
abstract = {Heterozygous missense mutations in the human COL7A1 gene - coding for collagen VII - lead to the rare, dominantly inherited skin disorder dominant dystrophic epidermolysis bullosa (DDEB), which is characterised by skin fragility, blistering, scarring and nail dystrophy. To better understand the pathophysiology of DDEB and develop more effective treatments, suitable mouse models for DDEB are required but to date none have existed. We identified the two most common COL7A1 mutations in DDEB patients (p.G2034R and p.G2043R) and used CRISPR-Cas9 to introduce the corresponding mutations into mouse Col7a1 (p.G2028R and p.G2037R). Dominant inheritance of either of these two alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of epidermolysis bullosa, which frequently show early lethality and severe disease, these mouse models, which to our knowledge are the first for DDEB, show no reduction in growth and survival, and - together with a relatively mild phenotype - represent a practically and ethically tractable tool for better understanding and treating epidermolysis bullosa. This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid34085257,
year = {2021},
author = {Liu, XM and Qian, SB},
title = {Targeted RNA m[6]A Editing Using Engineered CRISPR-Cas9 Conjugates.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2298},
number = {},
pages = {399-414},
pmid = {34085257},
issn = {1940-6029},
support = {R21 CA227917/CA/NCI NIH HHS/United States ; 55108556/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenosine/*analogs &amp; derivatives/genetics ; Base Sequence/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; HeLa Cells ; Humans ; Methylation ; RNA/*genetics ; RNA, Messenger/genetics ; },
abstract = {N[6]-methyladenosine (m[6]A) is a major epitranscriptomic mark exerting crucial diverse roles in RNA metabolisms, including RNA stability, mRNA translation, and RNA structural rearrangement. m[6]A modifications at different RNA regions may have distinct molecular effects. Here, we describe a CRISPR-Cas9-based approach that enables targeted m[6]A addition or removal on endogenous RNA molecules without altering the nucleotide sequence. By fusing a catalytically inactive Cas9 with engineered m[6]A modification enzymes, the programmable m[6]A editors are capable of achieving RNA methylation and demethylation at desired sites, facilitating dissection of regional effects of m[6]A and diversifying the toolkits for RNA manipulation.},
}
@article {pmid34085233,
year = {2021},
author = {Ma, HT},
title = {The Conditional Knockout Analogous System: CRISPR-Mediated Knockout Together with Inducible Degron and Transcription-Controlled Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2329},
number = {},
pages = {323-335},
pmid = {34085233},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cyclin A2/*genetics/*metabolism ; Frameshift Mutation ; Gene Expression Regulation/drug effects ; Gene Knockout Techniques/*methods ; HeLa Cells ; Humans ; Indoleacetic Acids/*pharmacology ; Proteolysis ; Retroviridae/genetics ; Tetracycline/*pharmacology ; Transcription, Genetic/drug effects ; },
abstract = {The revolutionary CRISPR technology opens a new era of cell biology in mammalian cells. The InDel mutation is induced by CRISPR and results in the frameshift mutation of the gene. Owing to the nature of CRISPR induced knockout, the conditional knockout using CRISPR technology is not common. With the recent development of the small molecule-inducible degron system, an analogous system to the classical genetic conditional knockout has become feasible. By integrating CRISPR-knockout, the tetracycline-controlled transcriptional and auxin-induced degradation post-translational control of protein expression, a method imitating the conditional knockout is developed. We herein describe the detailed protocol for the generation of a conditional protein inactivation in human cancer cells. The system is especially useful to study essential gene function in aneuploidy cancer cells where gain in copy number is common.},
}
@article {pmid34085224,
year = {2021},
author = {Geisinger, JM and Stearns, T},
title = {Assaying Cell Cycle Progression via Flow Cytometry in CRISPR/Cas9-Treated Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2329},
number = {},
pages = {195-204},
pmid = {34085224},
issn = {1940-6029},
support = {R35 GM130286/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cell Cycle ; Cell Line ; Click Chemistry ; Flow Cytometry/*methods ; Fluorescent Antibody Technique ; Gene Editing/*methods ; Humans ; Transfection ; },
abstract = {CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.},
}
@article {pmid34083785,
year = {2021},
author = {Truong, DJ and Phlairaharn, T and Eßwein, B and Gruber, C and Tümen, D and Baligács, E and Armbrust, N and Vaccaro, FL and Lederer, EM and Beck, EM and Geilenkeuser, J and Göppert, S and Krumwiede, L and Grätz, C and Raffl, G and Schwarz, D and Zirngibl, M and Živanić, M and Beyer, M and Körner, JD and Santl, T and Evsyukov, V and Strauß, T and Schwarz, SC and Höglinger, GU and Heutink, P and Doll, S and Conrad, M and Giesert, F and Wurst, W and Westmeyer, GG},
title = {Non-invasive and high-throughput interrogation of exon-specific isoform expression.},
journal = {Nature cell biology},
volume = {23},
number = {6},
pages = {652-663},
pmid = {34083785},
issn = {1476-4679},
mesh = {*Alternative Splicing ; CRISPR-Cas Systems ; Exons ; Forkhead Transcription Factors/genetics/*metabolism ; HEK293 Cells ; *High-Throughput Screening Assays ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Protein Isoforms ; Proteome ; *Proteomics ; *RNA Stability ; RNA, Messenger/genetics/*metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Repressor Proteins/genetics/*metabolism ; Single-Cell Analysis ; tau Proteins/genetics/*metabolism ; },
abstract = {Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.},
}
@article {pmid34083685,
year = {2021},
author = {Jarrell, JA and Sytsma, BJ and Wilson, LH and Pan, FL and Lau, KHWJ and Kirby, GTS and Lievano, AA and Pawell, RS},
title = {Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {11818},
pmid = {34083685},
issn = {2045-2322},
support = {75N91020C00030/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Cell Survival ; *Gene Editing/methods ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Hydrodynamics ; Microfluidics/*methods ; Models, Theoretical ; T-Lymphocytes/*metabolism ; Transfection/methods ; Transgenes ; },
abstract = {Microfluidic vortex shedding (µVS) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The mechanistic underpinning of µVS intracellular delivery remains undefined and µVS-Cas9 genome editing requires further studies. Herein, we evaluated a series of µVS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a µVS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field (eµVS). µVS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eµVS with negligible impact on cell viability. Herein, we demonstrate efficient processing of 5 × 10[6] cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 10[6] to 10[8] in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of µVS and eµVS for genome editing human primary T cells with Cas9 RNPs.},
}
@article {pmid34083438,
year = {2021},
author = {Serra, SA and Stojakovic, P and Amat, R and Rubio-Moscardo, F and Latorre, P and Seisenbacher, G and Canadell, D and Böttcher, R and Aregger, M and Moffat, J and de Nadal, E and Valverde, MA and Posas, F},
title = {LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {23},
pages = {},
pmid = {34083438},
issn = {1091-6490},
mesh = {Biological Transport ; CRISPR-Cas Systems ; Cell Death ; *Cell Size ; Cell Survival ; Chloride Channels/*metabolism ; HeLa Cells ; Humans ; Membrane Proteins/*genetics/*metabolism ; Osmotic Pressure ; Phosphorylation ; Potassium/metabolism ; Ribosomal Protein S6 Kinases, 90-kDa/metabolism ; Sodium/metabolism ; },
abstract = {Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl[-] efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na[+]/K[+]/2Cl[-] cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.},
}
@article {pmid34083435,
year = {2021},
author = {Tisza, MJ and Buck, CB},
title = {A catalog of tens of thousands of viruses from human metagenomes reveals hidden associations with chronic diseases.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {23},
pages = {},
pmid = {34083435},
issn = {1091-6490},
mesh = {Adolescent ; Adult ; CRISPR-Cas Systems ; Case-Control Studies ; *Chronic Disease ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Viruses/genetics ; Data Management ; Genome, Viral ; Genomics ; Humans ; *Metagenome ; Metagenomics ; Microbiota ; Parkinson Disease ; Virome ; Viruses/*genetics ; Young Adult ; },
abstract = {Despite remarkable strides in microbiome research, the viral component of the microbiome has generally presented a more challenging target than the bacteriome. This gap persists, even though many thousands of shotgun sequencing runs from human metagenomic samples exist in public databases, and all of them encompass large amounts of viral sequence data. The lack of a comprehensive database for human-associated viruses has historically stymied efforts to interrogate the impact of the virome on human health. This study probes thousands of datasets to uncover sequences from over 45,000 unique virus taxa, with historically high per-genome completeness. Large publicly available case-control studies are reanalyzed, and over 2,200 strong virus-disease associations are found.},
}
@article {pmid34082797,
year = {2021},
author = {Ray, U and Roy, D and Jin, L and Thirusangu, P and Staub, J and Xiao, Y and Kalogera, E and Wahner Hendrickson, AE and Cullen, GD and Goergen, K and Oberg, AL and Shridhar, V},
title = {Group III phospholipase A2 downregulation attenuated survival and metastasis in ovarian cancer and promotes chemo-sensitization.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {40},
number = {1},
pages = {182},
pmid = {34082797},
issn = {1756-9966},
support = {P50 CA136393/CA/NCI NIH HHS/United States ; P50CA136393/NH/NIH HHS/United States ; },
mesh = {Animals ; Autophagy/drug effects ; CRISPR-Cas Systems/genetics ; Cell Proliferation/*drug effects ; Cell Survival/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Group III Phospholipases A2/*genetics ; Heterografts ; Humans ; Lipid Droplets/drug effects ; Lipogenesis/*drug effects ; Mice ; Microscopy, Electron, Transmission ; Neoplasm Metastasis ; Ovarian Neoplasms/*drug therapy/genetics/pathology ; Platinum/pharmacology ; Pyridines/pharmacology ; Quinolines/pharmacology ; },
abstract = {BACKGROUND: Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined.<br><br>METHODS: CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis.<br><br>RESULTS: Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited cancer progression. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy.<br><br>CONCLUSIONS: Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.},
}
@article {pmid34081960,
year = {2021},
author = {Nie, HY and Liang, LQ and Li, QF and Li, ZH and Zhu, YN and Guo, YK and Zheng, QL and Lin, Y and Yang, DL and Li, ZG and Su, SK},
title = {CRISPR/Cas9 mediated knockout of Amyellow-y gene results in melanization defect of the cuticle in adult Apis mellifera.},
journal = {Journal of insect physiology},
volume = {132},
number = {},
pages = {104264},
doi = {10.1016/j.jinsphys.2021.104264},
pmid = {34081960},
issn = {1879-1611},
mesh = {Animals ; Bees/*genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genes, Insect ; *Genetic Markers ; Insect Proteins/genetics/metabolism ; Melanins ; Pigmentation/genetics ; Transcription Factors/genetics ; },
abstract = {Visible genetic markers are critical to gene function studies using genome editing technology in insects. However, there is no report about visible phenotypic markers in Apis mellifera, which extremely influences the application of genomic editing in honey bees. Here, we cloned and characterized the Amyellow-y gene in A. mellifera. Stage expression profiles showed that Amyellow-y gene was highly expressed in 2-, 4-day-old pupae, and newly emerged bees, and a high expression level was detected in the leg, thorax, wing and sting. To understand its functional role in pigmentation, Amyellow-y edited honeybees were created using CRISPR/Cas9, and it was found that the black pigment was decreased in the cuticle of mosaic workers and mutant drones. In particular, mutant drones manifested an overall appearance of yellowish cuticle in the body and appendages, including antennae, wings and legs, indicating that mutagenesis induced by disruption of Amyellow-y with CRISPR/Cas9 are heritable. Furthermore, the expression levels of genes associated with melanin pigmentation was investigated in mutant and wild-type drones using quantitative reverse transcription PCR. Transcription levels of Amyellow-y and aaNAT decreased markedly in mutant drones than that in wild-type ones, whereas laccase 2 was significantly up-regulated. Our results provide the first evidence, to our knowledge, that CRISPR/Cas9 edited G1 mutant drones of A. mellifera have a dramatic body pigmentation defect that can be visualized in adults, suggesting that Amyellow-y may serve as a promising visible phenotypic marker for genome editing in honey bees.},
}
@article {pmid34081824,
year = {2021},
author = {An, HW and Kim, SY and Kwon, JW and Seok, SH and Woo, SH and Kim, DY and Park, JW},
title = {In vivo CRISPR-Cas9 knockout screening using quantitative PCR identifies thymosin beta-4 X-linked that promotes diffuse-type gastric cancer metastasis.},
journal = {Molecular carcinogenesis},
volume = {60},
number = {9},
pages = {597-606},
doi = {10.1002/mc.23326},
pmid = {34081824},
issn = {1098-2744},
mesh = {Animals ; *Biomarkers, Tumor ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Expression ; *Gene Knockout Techniques ; Humans ; Mice ; Neoplasm Metastasis ; *Real-Time Polymerase Chain Reaction ; Signal Transduction ; Stomach Neoplasms/*genetics/*pathology ; Thymosin/*genetics ; },
abstract = {Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.},
}
@article {pmid34081459,
year = {2021},
author = {Liu, Y and Yuan, H and Ding, D and Dong, H and Wang, Q and Zhang, D},
title = {Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1373-1383},
doi = {10.1021/acssynbio.0c00647},
pmid = {34081459},
issn = {2161-5063},
mesh = {Biosensing Techniques/*methods ; CRISPR-Cas Systems ; Escherichia coli/genetics/*metabolism ; Fermentation/genetics ; Fluorescence ; Gene Editing/methods ; High-Throughput Screening Assays/*methods ; Metabolic Engineering/*methods ; Microorganisms, Genetically-Modified ; Mutagenesis ; Plasmids/genetics ; Polymorphism, Single Nucleotide ; Transcriptome/genetics ; Tryptophan/*biosynthesis ; },
abstract = {With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli.},
}
@article {pmid34080208,
year = {2021},
author = {Matsumoto, H and Kawashima, N and Yamamoto, T and Nakama, M and Otsuka, H and Ago, Y and Sasai, H and Kubota, K and Ozeki, M and Kawamoto, N and Esaka, Y and Ohnishi, H},
title = {In vitro functional analysis of four variants of human asparagine synthetase.},
journal = {Journal of inherited metabolic disease},
volume = {44},
number = {5},
pages = {1226-1234},
doi = {10.1002/jimd.12408},
pmid = {34080208},
issn = {1573-2665},
mesh = {Asparagine/metabolism ; CRISPR-Cas Systems ; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/deficiency/*genetics ; Genetic Variation ; HEK293 Cells ; Humans ; },
abstract = {The loss-of-function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild-type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild-type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild-type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell-based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants.},
}
@article {pmid34080099,
year = {2021},
author = {Ramirez-Phillips, AC and Liu, D},
title = {Therapeutic Genome Editing and In Vivo Delivery.},
journal = {The AAPS journal},
volume = {23},
number = {4},
pages = {80},
pmid = {34080099},
issn = {1550-7416},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods/trends ; Gene Transfer Techniques/*trends ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy/*methods/trends ; Humans ; Mutation ; },
abstract = {Improvements in the understanding of human genetics and its roles in disease development and prevention have led to an increased interest in therapeutic genome editing via the use of engineered nucleases. Various approaches have been explored in the past focusing on the development of an effective and safe system for sequence-specific editing. Compared to earlier nucleases such as zinc finger nuclease and transcription activator-like effector nuclease, the relatively low cost and ease of producing clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) systems have made therapeutic genome editing significantly more feasible. CRISPR/Cas9 genome editing has shown great potential to correct genetic mutations implicated in monogenic diseases and to eradicate latent or chronic viral infections in preclinical studies. Several CRISPR/Cas9-based therapeutics have reached the clinical stage, including treatments for inherited red blood cell disorders and Leber Congenital Amaurosis 10, as well as CRISPR/Cas9-edited T cells designed to target and destroy cancer cells. Further advances in therapeutic genome editing will rely on a safe and more efficient method of in vivo CRISPR/Cas9 delivery and improved efficiency of homology-directed repair for site-specific gene insertion or replacement. While other reviews have focused on one or two aspects of CRISPR/Cas9 genome editing, this review aims to provide a summary of the mechanisms of genome editing, the reasons for the emerging interest in CRISPR/Cas9 compared to other engineered nucleases, the current progress in developing CRISPR/Cas9 delivery systems, and the current preclinical and clinical applications of CRISPR/Cas9 genome editing.},
}
@article {pmid34079523,
year = {2021},
author = {Heller, S and Melzer, MK and Azoitei, N and Julier, C and Kleger, A},
title = {Human Pluripotent Stem Cells Go Diabetic: A Glimpse on Monogenic Variants.},
journal = {Frontiers in endocrinology},
volume = {12},
number = {},
pages = {648284},
pmid = {34079523},
issn = {1664-2392},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage ; Diabetes Mellitus, Type 1/*genetics/*metabolism ; Diabetes Mellitus, Type 2/*genetics/*metabolism ; Embryonic Stem Cells/cytology ; Epigenesis, Genetic ; Gene Editing ; Genetic Variation ; Heterozygote ; Humans ; Insulin/metabolism ; Insulin-Secreting Cells/metabolism ; Mice ; Mice, Knockout ; *Mutation ; Pancreas/embryology/pathology ; Phenotype ; Pluripotent Stem Cells/*cytology ; Regeneration ; },
abstract = {Diabetes, as one of the major diseases in industrial countries, affects over 350 million people worldwide. Type 1 (T1D) and type 2 diabetes (T2D) are the most common forms with both types having invariable genetic influence. It is accepted that a subset of all diabetes patients, generally estimated to account for 1-2% of all diabetic cases, is attributed to mutations in single genes. As only a subset of these genes has been identified and fully characterized, there is a dramatic need to understand the pathophysiological impact of genetic determinants on β-cell function and pancreatic development but also on cell replacement therapies. Pluripotent stem cells differentiated along the pancreatic lineage provide a valuable research platform to study such genes. This review summarizes current perspectives in applying this platform to study monogenic diabetes variants.},
}
@article {pmid34077774,
year = {2021},
author = {Anantayanon, J and Jeennor, S and Panchanawaporn, S and Chutrakul, C and Laoteng, K},
title = {Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.},
journal = {Gene},
volume = {793},
number = {},
pages = {145745},
doi = {10.1016/j.gene.2021.145745},
pmid = {34077774},
issn = {1879-0038},
mesh = {Aspergillus oryzae/classification/*enzymology/genetics ; CRISPR-Cas Systems ; Fatty Acids/*biosynthesis/genetics ; Fungal Proteins/*genetics/metabolism ; Gene Deletion ; Gene Expression Regulation, Fungal ; Humans ; Industrial Microbiology ; Isoenzymes/genetics/metabolism ; Kinetics ; Lipase/*genetics/metabolism ; Lipid Metabolism/genetics ; Mycelium/enzymology/genetics ; Phylogeny ; Plasmids/chemistry/metabolism ; Saccharomyces cerevisiae/classification/enzymology/genetics ; Spores, Fungal/*enzymology/genetics ; Triglycerides/*biosynthesis/genetics ; },
abstract = {Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.},
}
@article {pmid34077734,
year = {2021},
author = {Hamilton, JR and Tsuchida, CA and Nguyen, DN and Shy, BR and McGarrigle, ER and Sandoval Espinoza, CR and Carr, D and Blaeschke, F and Marson, A and Doudna, JA},
title = {Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering.},
journal = {Cell reports},
volume = {35},
number = {9},
pages = {109207},
pmid = {34077734},
issn = {2211-1247},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {A549 Cells ; CD4-Positive T-Lymphocytes/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; *Cell Engineering ; Gene Editing ; *Gene Transfer Techniques ; HIV-1/physiology ; Humans ; Jurkat Cells ; Lentivirus/genetics ; Receptors, Chimeric Antigen/metabolism ; Ribonucleoproteins/metabolism ; *Transgenes ; Virion/metabolism ; env Gene Products, Human Immunodeficiency Virus ; },
abstract = {As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4[+] cells while sparing co-cultured CD8[+] cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.},
}
@article {pmid34077687,
year = {2021},
author = {Davis, AE and Castranova, D and Weinstein, BM},
title = {Rapid Generation of Pigment Free, Immobile Zebrafish Embryos and Larvae in Any Genetic Background Using CRISPR-Cas9 dgRNPs.},
journal = {Zebrafish},
volume = {18},
number = {4},
pages = {235-242},
pmid = {34077687},
issn = {1557-8542},
support = {ZIA HD001011/ImNIH/Intramural NIH HHS/United States ; ZIA HD008915/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo, Nonmammalian ; Genetic Background ; Larva ; *Pigmentation/genetics ; Ribonucleoproteins/genetics ; *Zebrafish/genetics ; },
abstract = {The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, increasing amounts of pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges for live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals, but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. In this study, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.},
}
@article {pmid34077671,
year = {2021},
author = {Bi, Y and Gu, L and Wang, J and Chang, Y and Jin, M and Mao, Y and Wang, H and Ji, G},
title = {A Novel System for Simple Rapid Adenoviral Vector Construction to Facilitate CRISPR/Cas9-Mediated Genome Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {381-391},
doi = {10.1089/crispr.2020.0110},
pmid = {34077671},
issn = {2573-1602},
mesh = {Adenoviridae/*genetics ; *CRISPR-Cas Systems ; Cell Line ; Endonucleases/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Gene Transfer Techniques ; Genetic Therapy ; *Genetic Vectors ; Humans ; RNA, Guide ; },
abstract = {Recombinant adenoviruses have broad applications for gene delivery and expression. Furthermore, the adenovirus packaging system facilitates the expression of RNA-guided CRISPR/Cas9 nuclease complexes. In this study, we developed a novel system, named AdBlue, for the construction of recombinant adenoviruses using an enzymatic assembly strategy. This system could significantly reduce the time and labor required to generate adenoviral vectors. When applied to CRISPR/Cas9 design, it simplifies the preparation of recombinant adenoviruses carrying nuclease complexes and can induce high levels of site-specific mutagenesis. Our system has outstanding advantages for adenovirus preparation and could be a useful molecular engineering tool for gene delivery and editing.},
}
@article {pmid34076889,
year = {2021},
author = {Badis, Y and Scornet, D and Harada, M and Caillard, C and Godfroy, O and Raphalen, M and Gachon, CMM and Coelho, SM and Motomura, T and Nagasato, C and Cock, JM},
title = {Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus.},
journal = {The New phytologist},
volume = {231},
number = {5},
pages = {2077-2091},
doi = {10.1111/nph.17525},
pmid = {34076889},
issn = {1469-8137},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Eukaryota ; Gene Knockout Techniques ; *Phaeophyta/genetics ; },
abstract = {Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.},
}
@article {pmid34076866,
year = {2021},
author = {Liu, S and Huang, M and Xu, Y and Kang, J and Ye, S and Liu, S and Wang, Z and Liu, H and Yu, J and Hu, K and Wang, T},
title = {CRISPR/Cas12a Technology Combined with RT-ERA for Rapid and Portable SARS-CoV-2 Detection.},
journal = {Virologica Sinica},
volume = {36},
number = {5},
pages = {1083-1087},
pmid = {34076866},
issn = {1995-820X},
mesh = {*COVID-19/diagnosis ; *CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques ; *SARS-CoV-2/isolation &amp; purification ; },
}
@article {pmid34076729,
year = {2021},
author = {Xia, X and Cheng, X and Li, R and Yao, J and Li, Z and Cheng, Y},
title = {Advances in application of genome editing in tomato and recent development of genome editing technology.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {134},
number = {9},
pages = {2727-2747},
pmid = {34076729},
issn = {1432-2242},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics/growth &amp; development ; *Gene Editing ; *Genes, Plant ; *Genome, Plant ; Lycopersicon esculentum/*genetics/growth &amp; development ; *Quantitative Trait Loci ; },
abstract = {Genome editing, a revolutionary technology in molecular biology and represented by the CRISPR/Cas9 system, has become widely used in plants for characterizing gene function and crop improvement. Tomato, serving as an excellent model plant for fruit biology research and making a substantial nutritional contribution to the human diet, is one of the most important applied plants for genome editing. Using CRISPR/Cas9-mediated targeted mutagenesis, the re-evaluation of tomato genes essential for fruit ripening highlights that several aspects of fruit ripening should be reconsidered. Genome editing has also been applied in tomato breeding for improving fruit yield and quality, increasing stress resistance, accelerating the domestication of wild tomato, and recently customizing tomato cultivars for urban agriculture. In addition, genome editing is continuously innovating, and several new genome editing systems such as the recent prime editing, a breakthrough in precise genome editing, have recently been applied in plants. In this review, these advances in application of genome editing in tomato and recent development of genome editing technology are summarized, and their leaving important enlightenment to plant research and precision plant breeding is also discussed.},
}
@article {pmid34076237,
year = {2021},
author = {Osakabe, K and Wada, N and Murakami, E and Miyashita, N and Osakabe, Y},
title = {Genome editing in mammalian cells using the CRISPR type I-D nuclease.},
journal = {Nucleic acids research},
volume = {49},
number = {11},
pages = {6347-6363},
pmid = {34076237},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/chemistry/genetics/*metabolism ; *Gene Editing ; HEK293 Cells ; Humans ; Mutagenesis ; Mutation ; },
abstract = {Adoption of CRISPR-Cas systems, such as CRISPR-Cas9 and CRISPR-Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteria-remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense ('Cascade': Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR-Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR-Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR-Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.},
}
@article {pmid34075364,
year = {2021},
author = {Santiago-Frangos, A and Hall, LN and Nemudraia, A and Nemudryi, A and Krishna, P and Wiegand, T and Wilkinson, RA and Snyder, DT and Hedges, JF and Cicha, C and Lee, HH and Graham, A and Jutila, MA and Taylor, MP and Wiedenheft, B},
title = {Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.},
journal = {Cell reports. Medicine},
volume = {2},
number = {6},
pages = {100319},
pmid = {34075364},
issn = {2666-3791},
mesh = {COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Cas Systems/*genetics ; Colorimetry ; Humans ; Molecular Diagnostic Techniques ; Nasopharynx/virology ; Nucleic Acid Amplification Techniques ; RNA, Guide/metabolism ; RNA, Viral/chemistry/*metabolism ; SARS-CoV-2/isolation &amp; purification/metabolism ; },
abstract = {There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 10[7] copies/μL for a single guide RNA to 10[6] copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min.},
}
@article {pmid34074795,
year = {2021},
author = {Ghoshal, B and Picard, CL and Vong, B and Feng, S and Jacobsen, SE},
title = {CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG-specific DNA methyltransferase.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {23},
pages = {},
pmid = {34074795},
issn = {1091-6490},
support = {F32 GM136115/GM/NIGMS NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R35 GM130272/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Arabidopsis/enzymology/genetics ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *DNA Methylation ; DNA, Plant/genetics/*metabolism ; *DNA-Cytosine Methylases/genetics/metabolism ; *Plants, Genetically Modified/genetics/metabolism ; Tenericutes/enzymology/*genetics ; },
abstract = {CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.},
}
@article {pmid34074765,
year = {2021},
author = {Kajioka, D and Suzuki, K and Matsushita, S and Hino, S and Sato, T and Takada, S and Isono, K and Takeo, T and Kajimoto, M and Nakagata, N and Nakao, M and Suyama, M and DeFalco, T and Miyagawa, S and Yamada, G},
title = {Sexual fate of murine external genitalia development: Conserved transcriptional competency for male-biased genes in both sexes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {23},
pages = {},
pmid = {34074765},
issn = {1091-6490},
mesh = {Acetylation ; Androgens ; Animals ; CRISPR-Cas Systems ; Female ; Gene Expression Regulation ; Genitalia, Male/*metabolism ; Histones/metabolism ; MafB Transcription Factor ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Knockout ; Receptors, Androgen ; *Sex Differentiation ; Transcription Factors/metabolism ; },
abstract = {Testicular androgen is a master endocrine factor in the establishment of external genital sex differences. The degree of androgenic exposure during development is well known to determine the fate of external genitalia on a spectrum of female- to male-specific phenotypes. However, the mechanisms of androgenic regulation underlying sex differentiation are poorly defined. Here, we show that the genomic environment for the expression of male-biased genes is conserved to acquire androgen responsiveness in both sexes. Histone H3 at lysine 27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1) are enriched at the enhancer of male-biased genes in an androgen-independent manner. Specificity protein 1 (Sp1), acting as a collaborative transcription factor of androgen receptor, regulates H3K27ac enrichment to establish conserved transcriptional competency for male-biased genes in both sexes. Genetic manipulation of MafB, a key regulator of male-specific differentiation, and Sp1 regulatory MafB enhancer elements disrupts male-type urethral differentiation. Altogether, these findings demonstrate conservation of androgen responsiveness in both sexes, providing insights into the regulatory mechanisms underlying sexual fate during external genitalia development.},
}
@article {pmid34074728,
year = {2021},
author = {Sun, D and Lu, J and Zhang, Y and Liu, J and Liu, Z and Yao, B and Guo, Y and Wang, X},
title = {Characterization of a Novel CYP1A2 Knockout Rat Model Constructed by CRISPR/Cas9.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {49},
number = {8},
pages = {638-647},
doi = {10.1124/dmd.121.000403},
pmid = {34074728},
issn = {1521-009X},
mesh = {Animals ; *Animals, Genetically Modified/genetics/metabolism ; Biotransformation ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Cytochrome P-450 CYP1A2/*genetics ; Cytochrome P-450 Enzyme System/genetics ; Gene Knockout Techniques/*methods ; Isoenzymes/genetics ; Models, Animal ; Pharmacokinetics ; Rats ; },
abstract = {CYP1A2, as one of the most important cytochrome P450 isoforms, is involved in the biotransformation of many important endogenous and exogenous substances. CYP1A2 also plays an important role in the development of many diseases because it is involved in the biotransformation of precancerous substances and poisons. Although the generation of Cyp1a2 knockout (KO) mouse model has been reported, there are still no relevant rat models for the study of CYP1A2-mediated pharmacokinetics and diseases. In this report, CYP1A2 KO rat model was established successfully by CRISPR/Cas9 without any detectable off-target effect. Compared with wild-type rats, this model showed a loss of CYP1A2 protein expression in the liver. The results of pharmacokinetics in vivo and incubation in vitro of specific substrates of CYP1A2 confirmed the lack of function of CYP1A2 in KO rats. In further studies of potential compensatory effects, we found that CYP1A1 was significantly upregulated, and CYP2E1, CYP3A2, and liver X receptor β were downregulated in KO rats. In addition, CYP1A2 KO rats exhibited a significant increase in serum cholesterol and free testosterone accompanied by mild liver damage and lipid deposition, suggesting that CYP1A2 deficiency affects lipid metabolism and liver function to a certain extent. In summary, we successfully constructed the CYP1A2 KO rat model, which provides a useful tool for studying the metabolic function and physiologic function of CYP1A2. SIGNIFICANCE STATEMENT: Human CYP1A2 not only metabolizes clinical drugs and pollutants but also mediates the biotransformation of endogenous substances and plays an important role in the development of many diseases. However, there are no relevant CYP1A2 rat models for the research of pharmacokinetics and diseases. This study successfully established CYP1A2 knockout rat model by using CRISPR/Cas9. This rat model provides a powerful tool to study the function of CYP1A2 in drug metabolism and diseases.},
}
@article {pmid34074727,
year = {2021},
author = {Olson, EN},
title = {Toward the correction of muscular dystrophy by gene editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34074727},
issn = {1091-6490},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Dystrophin/genetics/metabolism ; *Gene Editing ; *Genetic Therapy ; Humans ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Myocardium/metabolism ; },
abstract = {Recent advances in gene editing technologies are enabling the potential correction of devastating monogenic disorders through elimination of underlying genetic mutations. Duchenne muscular dystrophy (DMD) is an especially severe genetic disorder caused by mutations in the gene encoding dystrophin, a membrane-associated protein required for maintenance of muscle structure and function. Patients with DMD succumb to loss of mobility early in life, culminating in premature death from cardiac and respiratory failure. The disease has thus far defied all curative strategies. CRISPR gene editing has provided new opportunities to ameliorate the disease by eliminating DMD mutations and thereby restore dystrophin expression throughout skeletal and cardiac muscle. Proof-of-concept studies in rodents, large mammals, and human cells have validated the potential of this approach, but numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene editing components throughout the musculature, and mitigation of possible immune responses. This paper provides an overview of recent work from our laboratory and others toward the genetic correction of DMD and considers the opportunities and challenges in the path to clinical translation. Lessons learned from these studies will undoubtedly enable further applications of gene editing to numerous other diseases of muscle and other tissues.},
}
@article {pmid34074403,
year = {2021},
author = {Deng, Y and Cao, G and Chen, X and Yang, M and Huo, D and Hou, C},
title = {Ultrasensitive detection of gene-PIK3CA[H1047R] mutation based on cascaded strand displacement amplification and trans-cleavage ability of CRISPR/Cas12a.},
journal = {Talanta},
volume = {232},
number = {},
pages = {122415},
doi = {10.1016/j.talanta.2021.122415},
pmid = {34074403},
issn = {1873-3573},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Mutation ; },
abstract = {Low abundance gene-PIK3CA[H1047R] mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CA[H1047R] mutation. The mutated gene-PIK3CA[H1047R] can combine with complementary sequence to form an intact recognition site for endonuclease FspI. Mediated by FspI, it breaks at the mutation site to produce DNA fragment to trigger SDA or C-SDA. Then, the fluorescent biosensors based on SDA-CRISPR/Cas12a or C-SDA-CRISPR/Cas12a were constructed. Compared with biosensor based on SDA-CRISPR/Cas12a (5 pM), the minimum detection of the biosensor based on C-SDA-CRISPR/Cas12a is reduced two orders of magnitude (50 fM). In range of 0.001%-50%, we achieved the ultrasensitive detection of gene-PIK3CA[H1047R] mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CA[H1047R] mutation detection.},
}
@article {pmid34073848,
year = {2021},
author = {Mushtaq, M and Ahmad Dar, A and Skalicky, M and Tyagi, A and Bhagat, N and Basu, U and Bhat, BA and Zaid, A and Ali, S and Dar, TU and Rai, GK and Wani, SH and Habib-Ur-Rahman, M and Hejnak, V and Vachova, P and Brestic, M and Çığ, A and Çığ, F and Erman, M and El Sabagh, A},
title = {CRISPR-Based Genome Editing Tools: Insights into Technological Breakthroughs and Future Challenges.},
journal = {Genes},
volume = {12},
number = {6},
pages = {},
pmid = {34073848},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant ; Plant Breeding/*methods ; },
abstract = {Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the notable advances highlighted here include the development of transgene (DNA)-free genome plants, the availability of compatible nucleases, and the development of safe and effective CRISPR delivery vehicles for plant genome editing, multi-gene targeting and complex genome editing, base editing and prime editing to achieve more complex genetic engineering. Additionally, new avenues that facilitate fine-tuning plant gene regulation have also been addressed. In spite of the tremendous potential of CRISPR and other gene editing tools, major challenges remain. Some of the challenges are related to the practical advances required for the efficient delivery of CRISPR reagents and for precision genome editing, while others come from government policies and public acceptance. This review will therefore be helpful to gain insights into technological advances, its applications, and future challenges for crop improvement.},
}
@article {pmid34073522,
year = {2021},
author = {Hesami, M and Baiton, A and Alizadeh, M and Pepe, M and Torkamaneh, D and Jones, AMP},
title = {Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34073522},
issn = {1422-0067},
mesh = {Agrobacterium ; *CRISPR-Cas Systems ; *Cannabis/genetics/metabolism ; *Gene Editing ; *Plants, Genetically Modified/genetics/metabolism ; },
abstract = {For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.},
}
@article {pmid34073486,
year = {2021},
author = {Wang, Z and Liu, X and Xie, X and Deng, L and Zheng, H and Pan, H and Li, D and Li, L and Zhong, C},
title = {ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34073486},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Plants, Genetically Modified/*genetics ; RNA, Guide/*genetics ; *Tetraploidy ; Tobacco/*genetics ; },
abstract = {Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.},
}
@article {pmid34073189,
year = {2021},
author = {Trompet, E and Temblador, A and Gillemot, S and Topalis, D and Snoeck, R and Andrei, G},
title = {An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness.},
journal = {Viruses},
volume = {13},
number = {6},
pages = {},
pmid = {34073189},
issn = {1999-4915},
mesh = {Amino Acid Substitution ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Codon ; DNA-Directed DNA Polymerase/chemistry/*genetics ; *Gene Editing ; *Genes, Viral ; Genetic Fitness ; Genotype ; Humans ; Mice ; Models, Molecular ; *Mutation ; Phenotype ; Protein Conformation ; Rhadinovirus/drug effects/*physiology ; Structure-Activity Relationship ; },
abstract = {Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383S[GCV], Q827R[HPMP-5azaC], G302W[PFA], K442T[PFA], G302W+K442T[PFA], C297W[HPMPO-DAPy] and C981Y[CDV]. Without antiviral pressure, viral clones Q827R[HPMP-5azaC], G302W[PFA], K442T[PFA] and G302W+K442T[PFA] grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383S[GCV] mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.},
}
@article {pmid34072515,
year = {2021},
author = {Tomkowiak, A and Bocianowski, J and Spychała, J and Grynia, J and Sobiech, A and Kowalczewski, P&#x141;},
title = {DArTseq-Based High-Throughput SilicoDArT and SNP Markers Applied for Association Mapping of Genes Related to Maize Morphology.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34072515},
issn = {1422-0067},
mesh = {*Chromosome Mapping ; *Genetic Markers ; Genome, Plant ; Genome-Wide Association Study ; *High-Throughput Nucleotide Sequencing ; Inbreeding ; Phylogeny ; Plant Breeding ; *Polymorphism, Single Nucleotide ; Quantitative Trait Loci ; *Quantitative Trait, Heritable ; Zea mays/*anatomy &amp; histology/*genetics ; },
abstract = {Today, agricultural productivity is essential to meet the needs of a growing population, and is also a key tool in coping with climate change. Innovative plant breeding technologies such as molecular markers, phenotyping, genotyping, the CRISPR/Cas method and next-generation sequencing can help agriculture meet the challenges of the 21st century more effectively. Therefore, the aim of the research was to identify single-nucleotide polymorphisms (SNPs) and SilicoDArT markers related to select morphological features determining the yield in maize. The plant material consisted of ninety-four inbred lines of maize of various origins. These lines were phenotyped under field conditions. A total of 14 morphological features was analyzed. The DArTseq method was chosen for genotyping because this technique reduces the complexity of the genome by restriction enzyme digestion. Subsequently, short fragment sequencing was used. The choice of a combination of restrictases allowed the isolation of highly informative low copy fragments of the genome. Thanks to this method, 90% of the obtained DArTseq markers are complementary to the unique sequences of the genome. All the observed features were normally distributed. Analysis of variance indicated that the main effect of lines was statistically significant (p < 0.001) for all 14 traits of study. Thanks to the DArTseq analysis with the use of next-generation sequencing (NGS) in the studied plant material, it was possible to identify 49,911 polymorphisms, of which 33,452 are SilicoDArT markers and the remaining 16,459 are SNP markers. Among those mentioned, two markers associated with four analyzed traits deserved special attention: SNP (4578734) and SilicoDArT (4778900). SNP marker 4578734 was associated with the following features: anthocyanin coloration of cob glumes, number of days from sowing to anthesis, number of days from sowing to silk emergence and anthocyanin coloration of internodes. SilicoDArT marker 4778900 was associated with the following features: number of days from sowing to anthesis, number of days from sowing to silk emergence, tassel: angle between the axis and lateral branches and plant height. Sequences with a length of 71 bp were used for physical mapping. The BLAST and EnsemblPlants databases were searched against the maize genome to identify the positions of both markers. Marker 4578734 was localized on chromosome 7, the closest gene was Zm00001d022467, approximately 55 Kb apart, encoding anthocyanidin 3-O-glucosyltransferase. Marker 4778900 was located on chromosome 7, at a distance of 45 Kb from the gene Zm00001d045261 encoding starch synthase I. The latter observation indicated that these flanking SilicoDArT and SNP markers were not in a state of linkage disequilibrium.},
}
@article {pmid34071237,
year = {2021},
author = {Chandrasekaran, AP and Woo, SH and Sarodaya, N and Rhie, BH and Tyagi, A and Das, S and Suresh, B and Ko, NR and Oh, SJ and Kim, KS and Ramakrishna, S},
title = {Ubiquitin-Specific Protease 29 Regulates Cdc25A-Mediated Tumorigenesis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34071237},
issn = {1422-0067},
support = {HI18C2383//Korea Health Industry Development Institute/Republic of Korea ; },
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Carcinogenesis/genetics/*metabolism ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cell Survival ; Cell Transformation, Neoplastic ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; HEK293 Cells ; HeLa Cells ; Heterografts ; Humans ; Male ; Mice ; Mice, Nude ; Mice, SCID ; Oncogenes ; Ubiquitin/metabolism ; Ubiquitin-Specific Proteases/genetics/*metabolism ; Ubiquitination ; cdc25 Phosphatases/genetics/*metabolism ; },
abstract = {Cell division cycle 25A (Cdc25A) is a dual-specificity phosphatase that is overexpressed in several cancer cells and promotes tumorigenesis. In normal cells, Cdc25A expression is regulated tightly, but the changes in expression patterns in cancer cells that lead to tumorigenesis are unknown. In this study, we showed that ubiquitin-specific protease 29 (USP29) stabilized Cdc25A protein expression in cancer cell lines by protecting it from ubiquitin-mediated proteasomal degradation. The presence of USP29 effectively blocked polyubiquitination of Cdc25A and extended its half-life. CRISPR-Cas9-mediated knockdown of USP29 in HeLa cells resulted in cell cycle arrest at the G0/G1 phase. We also showed that USP29 knockdown hampered Cdc25A-mediated cell proliferation, migration, and invasion of cancer cells in vitro. Moreover, NSG nude mice transplanted with USP29-depleted cells significantly reduced the size of the tumors, whereas the reconstitution of Cdc25A in USP29-depleted cells significantly increased the tumor size. Altogether, our results implied that USP29 promoted cell cycle progression and oncogenic transformation by regulating protein turnover of Cdc25A.},
}
@article {pmid34071140,
year = {2021},
author = {Ryu, HG and Jung, Y and Lee, N and Seo, JY and Kim, SW and Lee, KH and Kim, DY and Kim, KT},
title = {HNRNP A1 Promotes Lung Cancer Cell Proliferation by Modulating VRK1 Translation.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34071140},
issn = {1422-0067},
mesh = {3' Untranslated Regions ; Base Sequence ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line ; Cyclin D1/biosynthesis/genetics ; Eukaryotic Initiation Factor-3/metabolism ; Gene Expression Regulation, Neoplastic ; Genes, Reporter ; Heterogeneous Nuclear Ribonucleoprotein A1/chemistry/*physiology ; Humans ; Intracellular Signaling Peptides and Proteins/biosynthesis/*genetics ; Lung Neoplasms/genetics/*pathology ; Neoplasm Proteins/biosynthesis/chemistry/genetics/*physiology ; Protein Binding ; *Protein Biosynthesis ; Protein Domains ; Protein Interaction Mapping ; Protein Serine-Threonine Kinases/biosynthesis/*genetics ; RNA Interference ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/metabolism ; Sequence Deletion ; Up-Regulation ; },
abstract = {THeterogeneous nuclear ribonucleoprotein (HNRNP) A1 is the most abundant and ubiquitously expressed member of the HNRNP protein family. In recent years, it has become more evident that HNRNP A1 contributes to the development of neurodegenerative diseases. However, little is known about the underlying role of HNRNP A1 in cancer development. Here, we report that HNRNP A1 expression is significantly increased in lung cancer tissues and is negatively correlated with the overall survival of patients with lung cancer. Additionally, HNRNP A1 positively regulates vaccinia-related kinase 1 (VRK1) translation via binding directly to the 3' untranslated region (UTR) of VRK1 mRNA, thus increasing cyclin D1 (CCND1) expression by VRK1-mediated phosphorylation of the cAMP response element-binding protein (CREB). Furthermore, HNRNP A1 binding to the cis-acting region of the 3'UTR of VRK1 mRNA contributes to increased lung cancer cell proliferation. Thus, our study unveils a novel role of HNRNP A1 in lung carcinogenesis via post-transcriptional regulation of VRK1 expression and suggests its potential as a therapeutic target for patients with lung cancer.},
}
@article {pmid34070997,
year = {2021},
author = {Zapolnik, P and Pyrkosz, A},
title = {Gene Therapy for Mucopolysaccharidosis Type II-A Review of the Current Possibilities.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34070997},
issn = {1422-0067},
mesh = {Adolescent ; Animals ; Blood-Brain Barrier ; CRISPR-Cas Systems ; Child ; Child, Preschool ; Clinical Trials as Topic ; Dependovirus/genetics ; Disease Models, Animal ; Drug Carriers ; Electroporation ; Enzyme Replacement Therapy/methods ; Gene Editing ; Genetic Therapy ; Genetic Vectors/adverse effects/therapeutic use ; Glycoproteins/*genetics/pharmacokinetics/therapeutic use ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunoconjugates/administration &amp; dosage/therapeutic use ; Infant ; Injections, Intraventricular ; Injections, Spinal ; Lentivirus/genetics ; Mice ; Mucopolysaccharidosis II/genetics/*therapy ; Multicenter Studies as Topic ; Recombinant Proteins/administration &amp; dosage/therapeutic use ; Retroviridae/genetics ; Transcription Activator-Like Effector Nucleases ; },
abstract = {Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder based on a mutation in the IDS gene that encodes iduronate 2-sulphatase. As a result, there is an accumulation of glycosaminoglycans-heparan sulphate and dermatan sulphate-in almost all body tissues, which leads to their dysfunction. Currently, the primary treatment is enzyme replacement therapy, which improves the course of the disease by reducing somatic symptoms, including hepatomegaly and splenomegaly. The enzyme, however, does not cross the blood-brain barrier, and no improvement in the function of the central nervous system has been observed in patients with the severe form of the disease. An alternative method of treatment that solves typical problems of enzyme replacement therapy is gene therapy, i.e., delivery of the correct gene to target cells through an appropriate vector. Much progress has been made in applying gene therapy for MPS II, from cellular models to human clinical trials. In this article, we briefly present the history and basics of gene therapy and discuss the current state of knowledge about the methods of this therapy in mucopolysaccharidosis type II.},
}
@article {pmid34070855,
year = {2021},
author = {Liedtke, V and Schröder, C and Roggenbuck, D and Weiss, R and Stohwasser, R and Schierack, P and Rödiger, S and Schenk, L},
title = {LEDGF/p75 Is Required for an Efficient DNA Damage Response.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34070855},
issn = {1422-0067},
mesh = {Adaptor Proteins, Signal Transducing/deficiency/*genetics ; Antineoplastic Agents, Phytogenic/pharmacology ; Autoantigens/genetics/metabolism ; BRCA1 Protein/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; DNA/*genetics/metabolism ; DNA Damage ; Epithelial Cells/cytology/drug effects/metabolism ; Etoposide/pharmacology ; *Gene Expression Regulation ; Gene Knockout Techniques ; Histones/genetics/metabolism ; Humans ; Osteoblasts/cytology/drug effects/metabolism ; Proteasome Endopeptidase Complex/genetics/metabolism ; *Recombinational DNA Repair ; Replication Protein A/genetics/metabolism ; Signal Transduction ; Transcription Factors/deficiency/*genetics ; Ubiquitin-Conjugating Enzymes/genetics/metabolism ; },
abstract = {Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.},
}
@article {pmid34070435,
year = {2021},
author = {Liu, X and Lillywhite, J and Zhu, W and Huang, Z and Clark, AM and Gosstola, N and Maguire, CT and Dykxhoorn, D and Chen, ZY and Yang, J},
title = {Generation and Genetic Correction of USH2A c.2299delG Mutation in Patient-Derived Induced Pluripotent Stem Cells.},
journal = {Genes},
volume = {12},
number = {6},
pages = {},
pmid = {34070435},
issn = {2073-4425},
support = {R01 DC012115/DC/NIDCD NIH HHS/United States ; R21 EY030198/EY/NEI NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; UL1 TR002538/TR/NCATS NIH HHS/United States ; R01 DC016875/DC/NIDCD NIH HHS/United States ; R01 DC005575/DC/NIDCD NIH HHS/United States ; P30 EY014800/EY/NEI NIH HHS/United States ; R01 EY026521/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Extracellular Matrix Proteins/*genetics/metabolism ; Female ; Gene Deletion ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Primary Cell Culture/*methods ; Usher Syndromes/*genetics/metabolism/pathology ; },
abstract = {Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient's healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient's iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.},
}
@article {pmid34070430,
year = {2021},
author = {Fiaz, S and Ahmar, S and Saeed, S and Riaz, A and Mora-Poblete, F and Jung, KH},
title = {Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34070430},
issn = {1422-0067},
mesh = {Agriculture/*methods ; *CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Edible Grain/genetics ; Gene Editing/*methods ; *Genome, Plant ; Plant Breeding/*methods ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; },
abstract = {A world with zero hunger is possible only through a sustainable increase in food production and distribution and the elimination of poverty. Scientific, logistical, and humanitarian approaches must be employed simultaneously to ensure food security, starting with farmers and breeders and extending to policy makers and governments. The current agricultural production system is facing the challenge of sustainably increasing grain quality and yield and enhancing resistance to biotic and abiotic stress under the intensifying pressure of climate change. Under present circumstances, conventional breeding techniques are not sufficient. Innovation in plant breeding is critical in managing agricultural challenges and achieving sustainable crop production. Novel plant breeding techniques, involving a series of developments from genome editing techniques to speed breeding and the integration of omics technology, offer relevant, versatile, cost-effective, and less time-consuming ways of achieving precision in plant breeding. Opportunities to edit agriculturally significant genes now exist as a result of new genome editing techniques. These range from random (physical and chemical mutagens) to non-random meganucleases (MegaN), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein system 9 (CRISPR/Cas9), the CRISPR system from Prevotella and Francisella1 (Cpf1), base editing (BE), and prime editing (PE). Genome editing techniques that promote crop improvement through hybrid seed production, induced apomixis, and resistance to biotic and abiotic stress are prioritized when selecting for genetic gain in a restricted timeframe. The novel CRISPR-associated protein system 9 variants, namely BE and PE, can generate transgene-free plants with more frequency and are therefore being used for knocking out of genes of interest. We provide a comprehensive review of the evolution of genome editing technologies, especially the application of the third-generation genome editing technologies to achieve various plant breeding objectives within the regulatory regimes adopted by various countries. Future development and the optimization of forward and reverse genetics to achieve food security are evaluated.},
}
@article {pmid34070420,
year = {2021},
author = {Rhie, BH and Antao, AM and Karapurkar, JK and Kim, MS and Jo, WJ and Ramakrishna, S and Kim, KS},
title = {Ubiquitin-Specific Protease 3 Deubiquitinates and Stabilizes Oct4 Protein in Human Embryonic Stem Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {11},
pages = {},
pmid = {34070420},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Carcinoma, Embryonal/*genetics ; Cell Differentiation/genetics/physiology ; Cell Line, Tumor ; Deubiquitinating Enzymes/genetics/*metabolism ; Embryonic Stem Cells/cytology/enzymology/*metabolism ; Gene Knockout Techniques ; Humans ; Octamer Transcription Factor-3/genetics/*metabolism ; Protein Binding ; Protein Processing, Post-Translational ; Protein Stability ; Single-Cell Analysis ; Ubiquitin-Specific Proteases/genetics/*metabolism ; Ubiquitination/genetics ; },
abstract = {Oct4 is an important mammalian POU family transcription factor expressed by early human embryonic stem cells (hESCs). The precise level of Oct4 governs the pluripotency and fate determination of hESCs. Several post-translational modifications (PTMs) of Oct4 including phosphorylation, ubiquitination, and SUMOylation have been reported to regulate its critical functions in hESCs. Ubiquitination and deubiquitination of Oct4 should be well balanced to maintain the pluripotency of hESCs. The protein turnover of Oct4 is regulated by several E3 ligases through ubiquitin-mediated degradation. However, reversal of ubiquitination by deubiquitinating enzymes (DUBs) has not been reported for Oct4. In this study, we generated a ubiquitin-specific protease 3 (USP3) gene knockout using the CRISPR/Cas9 system and demonstrated that USP3 acts as a protein stabilizer of Oct4 by deubiquitinating Oct4. USP3 interacts with endogenous Oct4 and co-localizes in the nucleus of hESCs. The depletion of USP3 leads to a decrease in Oct4 protein level and loss of pluripotent morphology in hESCs. Thus, our results show that USP3 plays an important role in controlling optimum protein level of Oct4 to retain pluripotency of hESCs.},
}
@article {pmid34070077,
year = {2021},
author = {Ferreira, FJ and Carvalho, L and Logarinho, E and Bessa, J},
title = {foxm1 Modulates Cell Non-Autonomous Response in Zebrafish Skeletal Muscle Homeostasis.},
journal = {Cells},
volume = {10},
number = {5},
pages = {},
pmid = {34070077},
issn = {2073-4409},
support = {ERC-2015-StG-680156-ZPR/ERC_/European Research Council/International ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Death ; Cell Differentiation ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Forkhead Box Protein M1/genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Developmental ; *Muscle Development ; Muscle Fibers, Fast-Twitch/metabolism/pathology ; Muscle, Skeletal/*metabolism/pathology ; Satellite Cells, Skeletal Muscle/metabolism/pathology ; Zebrafish/embryology/genetics/*metabolism ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {foxm1 is a master regulator of the cell cycle, contributing to cell proliferation. Recent data have shown that this transcription factor also modulates gene networks associated with other cellular mechanisms, suggesting non-proliferative functions that remain largely unexplored. In this study, we used CRISPR/Cas9 to disrupt foxm1 in the zebrafish terminally differentiated fast-twitching muscle cells. foxm1 genomic disruption increased myofiber death and clearance. Interestingly, this contributed to non-autonomous satellite cell activation and proliferation. Moreover, we observed that Cas9 expression alone was strongly deleterious to muscle cells. Our report shows that foxm1 modulates a muscle non-autonomous response to myofiber death and highlights underreported toxicity to high expression of Cas9 in vivo.},
}
@article {pmid34069872,
year = {2021},
author = {Martens, MC and Edelkamp, J and Seebode, C and Schäfer, M and Stählke, S and Krohn, S and Jung, O and Murua Escobar, H and Emmert, S and Boeckmann, L},
title = {Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.},
journal = {International journal of molecular sciences},
volume = {22},
number = {10},
pages = {},
pmid = {34069872},
issn = {1422-0067},
mesh = {Animals ; Autophagy/genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Fibroblasts/*metabolism/physiology ; Gene Knockout Techniques/*methods ; Humans ; Keratoderma, Palmoplantar/genetics ; Membrane Fusion/genetics ; Mutation/genetics ; Neurocutaneous Syndromes/genetics ; Qb-SNARE Proteins/*genetics/metabolism ; Qc-SNARE Proteins/*genetics/metabolism ; SNARE Proteins/genetics/metabolism ; },
abstract = {Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.},
}
@article {pmid34069056,
year = {2021},
author = {Gallo, G and Puopolo, R and Carbonaro, M and Maresca, E and Fiorentino, G},
title = {Extremophiles, a Nifty Tool to Face Environmental Pollution: From Exploitation of Metabolism to Genome Engineering.},
journal = {International journal of environmental research and public health},
volume = {18},
number = {10},
pages = {},
pmid = {34069056},
issn = {1660-4601},
mesh = {Biodegradation, Environmental ; Ecosystem ; *Extremophiles/genetics ; Metabolic Networks and Pathways ; *Metals, Heavy ; },
abstract = {Extremophiles are microorganisms that populate habitats considered inhospitable from an anthropocentric point of view and are able to tolerate harsh conditions such as high temperatures, extreme pHs, high concentrations of salts, toxic organic substances, and/or heavy metals. These microorganisms have been broadly studied in the last 30 years and represent precious sources of biomolecules and bioprocesses for many biotechnological applications; in this context, scientific efforts have been focused on the employment of extremophilic microbes and their metabolic pathways to develop biomonitoring and bioremediation strategies to face environmental pollution, as well as to improve biorefineries for the conversion of biomasses into various chemical compounds. This review gives an overview on the peculiar metabolic features of certain extremophilic microorganisms, with a main focus on thermophiles, which make them attractive for biotechnological applications in the field of environmental remediation; moreover, it sheds light on updated genetic systems (also those based on the CRISPR-Cas tool), which expand the potentialities of these microorganisms to be genetically manipulated for various biotechnological purposes.},
}
@article {pmid34068886,
year = {2021},
author = {Numan, M and Serba, DD and Ligaba-Osena, A},
title = {Alternative Strategies for Multi-Stress Tolerance and Yield Improvement in Millets.},
journal = {Genes},
volume = {12},
number = {5},
pages = {},
pmid = {34068886},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics/growth &amp; development ; Edible Grain/genetics/growth &amp; development ; Gene Editing/methods ; Genome, Plant/genetics ; Millets/*genetics/*growth &amp; development ; Oryza/genetics/growth &amp; development ; Plant Development/*genetics ; Stress, Physiological/*genetics ; Triticum/genetics/growth &amp; development ; Zea mays/genetics/growth &amp; development ; },
abstract = {Millets are important cereal crops cultivated in arid and semiarid regions of the world, particularly Africa and southeast Asia. Climate change has triggered multiple abiotic stresses in plants that are the main causes of crop loss worldwide, reducing average yield for most crops by more than 50%. Although millets are tolerant to most abiotic stresses including drought and high temperatures, further improvement is needed to make them more resilient to unprecedented effects of climate change and associated environmental stresses. Incorporation of stress tolerance traits in millets will improve their productivity in marginal environments and will help in overcoming future food shortage due to climate change. Recently, approaches such as application of plant growth-promoting rhizobacteria (PGPRs) have been used to improve growth and development, as well as stress tolerance of crops. Moreover, with the advance of next-generation sequencing technology, genome editing, using the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system are increasingly used to develop stress tolerant varieties in different crops. In this paper, the innate ability of millets to tolerate abiotic stresses and alternative approaches to boost stress resistance were thoroughly reviewed. Moreover, several stress-resistant genes were identified in related monocots such as rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays), and other related species for which orthologs in millets could be manipulated by CRISPR/Cas9 and related genome-editing techniques to improve stress resilience and productivity. These cutting-edge alternative strategies are expected to bring this group of orphan crops at the forefront of scientific research for their potential contribution to global food security.},
}
@article {pmid34068874,
year = {2021},
author = {Zhang, C and Li, Z and Chen, M and Hu, Z and Wu, L and Zhou, M and Liang, D},
title = {Cas12a and Lateral Flow Strip-Based Test for Rapid and Ultrasensitive Detection of Spinal Muscular Atrophy.},
journal = {Biosensors},
volume = {11},
number = {5},
pages = {},
pmid = {34068874},
issn = {2079-6374},
mesh = {Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; Muscular Atrophy, Spinal/*diagnosis ; },
abstract = {Spinal muscular atrophy (SMA) is characterized by severe lethality and irreversible progression. Early diagnosis of SMA is of more practical significance with the emergence of effective therapy. However, existing techniques to identify SMA patients rely on cumbersome instruments, hindering their accessibility and application. An SMA-Cas12a-strip assay was developed with the integration of Cas12a-based nucleic acid detection, isothermal amplification, and lateral flow strip. The analytical performance of the assay was assessed with clinical samples. To explore its extensible utility, various specimens were tested. Validated with 168 clinical samples, the sensitivity and specificity of the SMA-Cas12a-strip assay were both 100%. The minimum detectable concentration of genomic DNA containing the target gene achieved 526 aM. The assay was compatible with specimens from several sources, and the turnaround time could be within 1.5 h. We developed a simple, cost-effective, and highly sensitive and specific assay to detect SMA patients. With little and field-portable equipment, the assay holds great promise in the detection of SMA patients, particularly in low-resource regions.},
}
@article {pmid34067383,
year = {2021},
author = {Lee, HH and Park, J and Jung, H and Seo, YS},
title = {Pan-Genome Analysis Reveals Host-Specific Functional Divergences in Burkholderia gladioli.},
journal = {Microorganisms},
volume = {9},
number = {6},
pages = {},
pmid = {34067383},
issn = {2076-2607},
abstract = {Burkholderia gladioli has high versatility and adaptability to various ecological niches. Here, we constructed a pan-genome using 14 genome sequences of B. gladioli, which originate from different niches, including gladiolus, rice, humans, and nature. Functional roles of core and niche-associated genomes were investigated by pathway enrichment analyses. Consequently, we inferred the uniquely important role of niche-associated genomes in (1) selenium availability during competition with gladiolus host; (2) aromatic compound degradation in seed-borne and crude oil-accumulated environments, and (3) stress-induced DNA repair system/recombination in the cystic fibrosis-niche. We also identified the conservation of the rhizomide biosynthetic gene cluster in all the B. gladioli strains and the concentrated distribution of this cluster in human isolates. It was confirmed the absence of complete CRISPR/Cas system in both plant and human pathogenic B. gladioli and the presence of the system in B. gladioli living in nature, possibly reflecting the inverse relationship between CRISPR/Cas system and virulence.},
}
@article {pmid34067003,
year = {2021},
author = {Lundy, SR and Abney, K and Ellerson, D and Igietseme, JU and Carroll, D and Eko, FO and Omosun, YO},
title = {MiR-378b Modulates Chlamydia-Induced Upper Genital Tract Pathology.},
journal = {Pathogens (Basel, Switzerland)},
volume = {10},
number = {5},
pages = {},
pmid = {34067003},
issn = {2076-0817},
abstract = {Genital Chlamydia trachomatis infection causes severe reproductive pathologies such as salpingitis and pelvic inflammatory disease that can lead to tubal factor infertility. MicroRNAs (miRNAs) are evolutionarily conserved regulators of mammalian gene expression in development, immunity and pathophysiologic processes during inflammation and infection, including Chlamydia infection. Among the miRNAs involved in regulating host responses and pathologic outcome of Chlamydia infection, we have shown that miR-378b was significantly differentially expressed during primary infection and reinfection. In this study, we tested the hypothesis that miR-378b is involved in the pathological outcome of Chlamydia infection. We developed miR-378b knockout mice (miR-378b[-/-]) using Crispr/Cas and infected them along with their wild-type (WT) control with Chlamydia to compare the infectivity and reproductive pathologies. The results showed that miR-378b[-/-] mice were unable to clear the infection compared to WT mice; also, miR-378b[-/-] mice exhibited a relatively higher Chlamydia burden throughout the duration of infection. However, gross pathology results showed that miR-378b[-/-] mice had significantly reduced uterine dilatations and pathologic lesions after two infections compared to WT mice. In addition, the pregnancy and fertility rates for infected miR-378b[-/-] mice showed protection from Chlamydia-induced infertility with fertility rate that was comparable to uninfected WT mice. These results are intriguing as they suggest that miR-378b is important in regulating host immune responses that control Chlamydial replication and drive the inflammation that causes complications such as infertility. The finding has important implications for biomarkers of Chlamydial complications and targets for prevention of disease.},
}
@article {pmid34066520,
year = {2021},
author = {Sakuma, C and Sekizuka, T and Kuroda, M and Hanada, K and Yamaji, T},
title = {Identification of SYS1 as a Host Factor Required for Shiga Toxin-Mediated Cytotoxicity in Vero Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {9},
pages = {},
pmid = {34066520},
issn = {1422-0067},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Cell Death/drug effects ; Chlorocebus aethiops ; Drug Resistance, Bacterial/drug effects/genetics ; Humans ; Membrane Proteins/chemistry/*metabolism ; Polysaccharides/biosynthesis ; RNA-Binding Proteins/metabolism ; Shiga Toxin/*toxicity ; Vero Cells ; trans-Golgi Network/metabolism ; },
abstract = {Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome-TGN retrograde transports.},
}
@article {pmid34066014,
year = {2021},
author = {Ahmed, M and Daoud, GH and Mohamed, A and Harati, R},
title = {New Insights into the Therapeutic Applications of CRISPR/Cas9 Genome Editing in Breast Cancer.},
journal = {Genes},
volume = {12},
number = {5},
pages = {},
pmid = {34066014},
issn = {2073-4425},
mesh = {Breast Neoplasms/genetics/*therapy ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Female ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; },
abstract = {Breast cancer is one of the most prevalent forms of cancer globally and is among the leading causes of death in women. Its heterogenic nature is a result of the involvement of numerous aberrant genes that contribute to the multi-step pathway of tumorigenesis. Despite the fact that several disease-causing mutations have been identified, therapy is often aimed at alleviating symptoms rather than rectifying the mutation in the DNA sequence. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a groundbreaking tool that is being utilized for the identification and validation of genomic targets bearing tumorigenic potential. CRISPR/Cas9 supersedes its gene-editing predecessors through its unparalleled simplicity, efficiency and affordability. In this review, we provide an overview of the CRISPR/Cas9 mechanism and discuss genes that were edited using this system for the treatment of breast cancer. In addition, we shed light on the delivery methods-both viral and non-viral-that may be used to deliver the system and the barriers associated with each. Overall, the present review provides new insights into the potential therapeutic applications of CRISPR/Cas9 for the advancement of breast cancer treatment.},
}
@article {pmid34064241,
year = {2021},
author = {Mlaga, KD and Garcia, V and Colson, P and Ruimy, R and Rolain, JM and Diene, SM},
title = {Extensive Comparative Genomic Analysis of Enterococcus faecalis and Enterococcus faecium Reveals a Direct Association between the Absence of CRISPR-Cas Systems, the Presence of Anti-Endonuclease (ardA) and the Acquisition of Vancomycin Resistance in E. faecium.},
journal = {Microorganisms},
volume = {9},
number = {6},
pages = {},
pmid = {34064241},
issn = {2076-2607},
abstract = {Here, we performed a comparative genomic analysis of all available genomes of E. faecalis (n = 1591) and E. faecium (n = 1981) and investigated the association between the presence or absence of CRISPR-Cas systems, endonuclease/anti-endonuclease systems and the acquisition of antimicrobial resistance, especially vancomycin resistance genes. Most of the analysed Enterococci were isolated from humans and less than 14% of them were from foods and animals. We analysed and detected CRISPR-Cas systems in 75.36% of E. faecalis genomes and only 4.89% of E. faecium genomes with a significant difference (p-value < 10[-5]). We found a negative correlation between the number of CRISPR-Cas systems and genome size (r = -0.397, p-value < 10[-5]) and a positive correlation between the genome %GC content and the number of CRISPR-Cas systems (r = 0.215, p-value < 10[-5]). Our findings showed that the presence of the anti-endonuclease ardA gene may explain the decrease in the number of CRISPR-Cas systems in E. faecium, known to deactivate the endonucleases' protective activities and enable the E. faecium genome to be versatile in acquiring mobile genetic elements, including carriers of antimicrobial resistance genes, especially vanB. Most importantly, we observed that there was a direct association between the absence of CRISPR-Cas, the presence of the anti-CRISPR ardA gene and the acquisition of vancomycin resistance genes.},
}
@article {pmid34063629,
year = {2021},
author = {Ding, R and Long, J and Yuan, M and Zheng, X and Shen, Y and Jin, Y and Yang, H and Li, H and Chen, S and Duan, G},
title = {CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV.},
journal = {International journal of molecular sciences},
volume = {22},
number = {9},
pages = {},
pmid = {34063629},
issn = {1422-0067},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Fluorescence ; Hepatitis B/*diagnosis/genetics/virology ; Hepatitis B virus/*isolation &amp; purification/pathogenicity ; Humans ; Point-of-Care Systems ; *Point-of-Care Testing ; },
abstract = {Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.},
}
@article {pmid34063152,
year = {2021},
author = {Alonso, CA and de Toro, M and de la Cruz, F and Torres, C},
title = {Genomic Insights into Drug Resistance and Virulence Platforms, CRISPR-Cas Systems and Phylogeny of Commensal E. coli from Wildlife.},
journal = {Microorganisms},
volume = {9},
number = {5},
pages = {},
pmid = {34063152},
issn = {2076-2607},
abstract = {Commensal bacteria act as important reservoirs of virulence and resistance genes. However, existing data are generally only focused on the analysis of human or human-related bacterial populations. There is a lack of genomic studies regarding commensal bacteria from hosts less exposed to antibiotics and other selective forces due to human activities, such as wildlife. In the present study, the genomes of thirty-eight E. coli strains from the gut of various wild animals were sequenced. The analysis of their accessory genome yielded a better understanding of the role of the mobilome on inter-bacterial dissemination of mosaic virulence and resistance plasmids. The study of the presence and composition of the CRISPR/Cas systems in E. coli from wild animals showed some viral and plasmid sequences among the spacers, as well as the relationship between CRISPR/Cas and E. coli phylogeny. Further, we constructed a single nucleotide polymorphisms-based core tree with E. coli strains from different sources (humans, livestock, food and extraintestinal environments). Bacteria from humans or highly human-influenced settings exhibit similar genetic patterns in CRISPR-Cas systems, plasmids or virulence/resistance genes-carrying modules. These observations, together with the absence of significant genetic changes in their core genome, suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems.},
}
@article {pmid34062874,
year = {2021},
author = {Gupta, N and Augustine, S and Narayan, T and O'Riordan, A and Das, A and Kumar, D and Luong, JHT and Malhotra, BD},
title = {Point-of-Care PCR Assays for COVID-19 Detection.},
journal = {Biosensors},
volume = {11},
number = {5},
pages = {},
pmid = {34062874},
issn = {2079-6374},
mesh = {Animals ; COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*instrumentation/methods ; CRISPR-Cas Systems ; Equipment Design ; Humans ; *Point-of-Care Testing ; Polymerase Chain Reaction/*instrumentation/methods ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.},
}
@article {pmid34062417,
year = {2021},
author = {Rahman, MR and Hossain, MA and Mozibullah, M and Mujib, FA and Afrose, A and Shahed-Al-Mahmud, M and Apu, MAI},
title = {CRISPR is a useful biological tool for detecting nucleic acid of SARS-CoV-2 in human clinical samples.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {140},
number = {},
pages = {111772},
pmid = {34062417},
issn = {1950-6007},
mesh = {COVID-19/*virology ; COVID-19 Testing/methods ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; Pandemics/prevention &amp; control ; SARS-CoV-2/*genetics ; },
abstract = {The recent pandemic of novel coronavirus disease (COVID-19) has spread globally and infected millions of people. The quick and specific detection of the nucleic acid of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) remains a challenge within healthcare providers. Currently, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is the widely used method to detect the SARS-CoV-2 from the human clinical samples. RT-qPCR is expensive equipment and needs skilled personnel as well as lengthy detection time. RT-qPCR limitation needed an alternative healthcare technique to overcome with a fast and cheaper detection method. By applying the principles of CRISPR technology, several promising detection methods giving hope to the healthcare community. CRISPR-based detection methods include SHERLOCK-Covid, STOP-Covid, AIOD-CRISPR, and DETECTR platform. These methods have comparative advantages and drawbacks. Among these methods, AIOD-CRISPR and DETECTR are reasonably better diagnostic methods than the others if we compare the time taken for the test, the cost associated with each test, and their capability of detecting SARS-CoV-2 in the clinical samples. It may expect that the promising CRISPR-based methods would facilitate point-of-care (POC) applications in the CRISPR-built next-generation novel coronavirus diagnostics.},
}
@article {pmid34061172,
year = {2021},
author = {Stewart, S and Le Bleu, HK and Yette, GA and Henner, AL and Robbins, AE and Braunstein, JA and Stankunas, K},
title = {longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {11},
pages = {},
pmid = {34061172},
issn = {1477-9129},
support = {R01 GM127761/GM/NIGMS NIH HHS/United States ; F31 GM139343/GM/NIGMS NIH HHS/United States ; T32 GM007413/GM/NIGMS NIH HHS/United States ; R03 AR067522/AR/NIAMS NIH HHS/United States ; T32 HD007348/HD/NICHD NIH HHS/United States ; F31 AR071283/AR/NIAMS NIH HHS/United States ; },
mesh = {Animal Fins/anatomy &amp; histology/*physiology ; Animals ; CRISPR-Cas Systems ; Calcineurin/metabolism ; Cell Proliferation ; Ectopic Gene Expression/genetics/*physiology ; Ether ; Ether-A-Go-Go Potassium Channels/genetics/*metabolism ; Gene Expression Regulation, Developmental ; Mesoderm/metabolism ; Organ Size ; Regeneration/physiology ; Signal Transduction/genetics ; Zebrafish/genetics ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.},
}
@article {pmid34061021,
year = {2021},
author = {Zhao, Q and Kong, Y and Kittredge, A and Li, Y and Shen, Y and Zhang, Y and Tsang, SH and Yang, T},
title = {Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34061021},
issn = {2050-084X},
support = {R21 AG050437/AG/NIA NIH HHS/United States ; R01 EY018213/EY/NEI NIH HHS/United States ; U01 EY030580/EY/NEI NIH HHS/United States ; U54 OD020351/OD/NIH HHS/United States ; R01 GM127652/GM/NIGMS NIH HHS/United States ; R01 EY026682/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; P30 EY019007/EY/NEI NIH HHS/United States ; R24 EY027285/EY/NEI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R24 EY028758/EY/NEI NIH HHS/United States ; },
mesh = {Bestrophins/*genetics/metabolism ; CRISPR-Cas Systems ; Chlorides/metabolism ; *Gain of Function Mutation ; Genetic Predisposition to Disease ; Genetic Therapy ; HEK293 Cells ; Humans ; *Loss of Function Mutation ; Membrane Potentials ; Phenotype ; Retinal Degeneration/diagnosis/*genetics/metabolism/therapy ; Retinal Pigment Epithelium/metabolism ; },
abstract = {Genetic mutation of the human BEST1 gene, which encodes a Ca[2+]-activated Cl[-] channel (BEST1) predominantly expressed in retinal pigment epithelium (RPE), causes a spectrum of retinal degenerative disorders commonly known as bestrophinopathies. Previously, we showed that BEST1 plays an indispensable role in generating Ca[2+]-dependent Cl[-] currents in human RPE cells, and the deficiency of BEST1 function in patient-derived RPE is rescuable by gene augmentation (Li et al., 2017). Here, we report that BEST1 patient-derived loss-of-function and gain-of-function mutations require different mutant to wild-type (WT) molecule ratios for phenotypic manifestation, underlying their distinct epigenetic requirements in bestrophinopathy development, and suggesting that some of the previously classified autosomal dominant mutations actually behave in a dominant-negative manner. Importantly, the strong dominant effect of BEST1 gain-of-function mutations prohibits the restoration of BEST1-dependent Cl[-] currents in RPE cells by gene augmentation, in contrast to the efficient rescue of loss-of-function mutations via the same approach. Moreover, we demonstrate that gain-of-function mutations are rescuable by a combination of gene augmentation with CRISPR/Cas9-mediated knockdown of endogenous BEST1 expression, providing a universal treatment strategy for all bestrophinopathy patients regardless of their mutation types.},
}
@article {pmid34060909,
year = {2021},
author = {Pei, Z and Sadiq, FA and Han, X and Zhao, J and Zhang, H and Ross, RP and Lu, W and Chen, W},
title = {Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems.},
journal = {mSystems},
volume = {6},
number = {3},
pages = {e0121120},
pmid = {34060909},
issn = {2379-5077},
abstract = {Prophage integration, release, and dissemination exert various effects on host bacteria. In the genus Lactobacillus, they may cause bacteriophage contamination during fermentation and even regulate bacterial populations in the gut. However, little is known about their distribution, genetic architecture, and relationships with their hosts. Here, we conducted prophage prediction analysis on 1,472 genomes from 16 different Lactobacillus species and found prophage fragments in almost all lactobacilli (99.8%), with 1,459 predicted intact prophages identified in 64.1% of the strains. We present an uneven prophage distribution among Lactobacillus species; multihabitat species retained more prophages in their genomes than restricted-habitat species. Characterization of the genome features, average nucleotide identity, and landscape visualization presented a high genome diversity of Lactobacillus prophages. We detected antibiotic resistance genes in more than 10% of Lactobacillus prophages and validated that the occurrence of resistance genes conferred by prophage integration was possibly associated with phenotypic resistance in Lactobacillus plantarum. Furthermore, our broad and comprehensive examination of the distribution of CRISPR-Cas systems across the genomes predicted type I and type III systems as potential antagonistic elements of Lactobacillus prophage. IMPORTANCE Lactobacilli are inherent microorganisms in the human gut and are widely used in the food processing industries due to their probiotic properties. Prophages were reportedly hidden in numerous Lactobacillus genomes and can potentially contaminate entire batches of fermentation or modulate the intestinal microecology once they are released. Therefore, a comprehensive scanning of prophages in Lactobacillus is essential for the safety evaluation and application development of probiotic candidates. We show that prophages are widely distributed among lactobacilli; however, intact prophages are more common in multihabitat species and display wide variations in genome feature, integration site, and genomic organization. Our data of the prophage-mediated antibiotic resistance genes (ARGs) and the resistance phenotype of lactobacilli provide evidence for deciphering the putative role of prophages as vectors of the ARGs. Furthermore, understanding the association between prophages and CRISPR-Cas systems is crucial to appreciate the coevolution of phages and Lactobacillus.},
}
@article {pmid34060719,
year = {2021},
author = {Kondrashov, A},
title = {The precise magic of CRISPR.},
journal = {FEBS open bio},
volume = {11},
number = {6},
pages = {1520-1523},
pmid = {34060719},
issn = {2211-5463},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Plasmids ; },
abstract = {In this issue of FEBS Open Bio, Shen Li et al., in the laboratory of Hector L. Franco (University of North Carolina), provide a proof-of-principle solution for correcting all copies of a gene in the widely used MCF7 breast cancer cell line. The gene for the FOXA1 pioneer transcription factor is localised on chromosome 14, which is present at least 4-5 times in MCF7 cells. To achieve their goal, the authors used a 'classical' version of the CRISPR/Cas9 system. Both sgRNA and Cas9 components were expressed from a single vector, which also has a puromycin resistance cassette; this is an essential module for the chosen strategy, because it ensures expression of both sgRNA and Cas9 in selected cells. A targeting template in the form of nonlinearised plasmid was shown to have the best efficiency and was used to introduce a substitution at position 295 in the gene encoding FOXA1 to change a codon encoding lysine into a codon encoding glutamine (K295Q). The strategy suggested by Li and co-authors is an important development towards genome editing of multiple copy genes in a polyploid environment like cancer cells. One important application of the technique could be in creating models to study the role of single nucleotide polymorphisms in cancer progression and metastasis. Isogenic cancer lines carrying polymorphic variants of key drug targets could be used to optimise anticancer treatment protocols, laying a foundation for personalised therapy.},
}
@article {pmid34059824,
year = {2021},
author = {Mohassel, P and Donkervoort, S and Lone, MA and Nalls, M and Gable, K and Gupta, SD and Foley, AR and Hu, Y and Saute, JAM and Moreira, AL and Kok, F and Introna, A and Logroscino, G and Grunseich, C and Nickolls, AR and Pourshafie, N and Neuhaus, SB and Saade, D and Gangfuß, A and Kölbel, H and Piccus, Z and Le Pichon, CE and Fiorillo, C and Ly, CV and Töpf, A and Brady, L and Specht, S and Zidell, A and Pedro, H and Mittelmann, E and Thomas, FP and Chao, KR and Konersman, CG and Cho, MT and Brandt, T and Straub, V and Connolly, AM and Schara, U and Roos, A and Tarnopolsky, M and Höke, A and Brown, RH and Lee, CH and Hornemann, T and Dunn, TM and Bönnemann, CG},
title = {Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.},
journal = {Nature medicine},
volume = {27},
number = {7},
pages = {1197-1204},
pmid = {34059824},
issn = {1546-170X},
support = {UM1 HG008900/HG/NHGRI NIH HHS/United States ; R01 NS072446/NS/NINDS NIH HHS/United States ; R01 HG009141/HG/NHGRI NIH HHS/United States ; K08 NS107621/NS/NINDS NIH HHS/United States ; R01 GM143282/GM/NIGMS NIH HHS/United States ; F32 NS010762/NS/NINDS NIH HHS/United States ; },
mesh = {Adolescent ; Adult ; Alleles ; Amino Acid Sequence ; Amyotrophic Lateral Sclerosis/enzymology/genetics/*metabolism ; CRISPR-Cas Systems ; Child ; Female ; Genes, Dominant ; HEK293 Cells ; Humans ; Male ; Middle Aged ; Mutation ; Serine C-Palmitoyltransferase/genetics/metabolism ; Sphingolipids/*biosynthesis ; Young Adult ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.},
}
@article {pmid34059674,
year = {2021},
author = {Sincennes, MC and Brun, CE and Lin, AYT and Rosembert, T and Datzkiw, D and Saber, J and Ming, H and Kawabe, YI and Rudnicki, MA},
title = {Acetylation of PAX7 controls muscle stem cell self-renewal and differentiation potential in mice.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3253},
pmid = {34059674},
issn = {2041-1723},
support = {R01 AR044031/AR/NIAMS NIH HHS/United States ; FDN-148387//CIHR/Canada ; },
mesh = {Acetylation ; Animals ; COS Cells ; CRISPR-Cas Systems ; Cardiotoxins/administration &amp; dosage/toxicity ; Cell Differentiation/genetics ; Cell Self Renewal/*genetics ; Chlorocebus aethiops ; Disease Models, Animal ; Gene Knockdown Techniques ; Histone Acetyltransferases/genetics/metabolism ; Humans ; Mice ; Mice, Transgenic ; Muscle, Skeletal/cytology/drug effects/*injuries ; Mutagenesis ; PAX7 Transcription Factor/*metabolism ; Primary Cell Culture ; Promoter Regions, Genetic ; Regeneration/*genetics ; Satellite Cells, Skeletal Muscle/*physiology ; Sf9 Cells ; Sirtuin 2/genetics/metabolism ; Spodoptera ; Transcriptional Activation ; },
abstract = {Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.},
}
@article {pmid34058685,
year = {2021},
author = {Getachew, A and Yang, Z and Huang, X and Wu, F and Liu, YY and Yan, C and Yang, F and Li, Y},
title = {Generation of a TLR2 homozygous knockout human embryonic stem cell line WAe001-A-64 using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {54},
number = {},
pages = {102401},
doi = {10.1016/j.scr.2021.102401},
pmid = {34058685},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; *Toll-Like Receptor 2/genetics ; },
abstract = {Toll-like receptor 2 (TLR2) is a pattern recognition receptor which plays an important role in innate immune system. In humans it's encoded by the TLR2 gene and also been designated as CD282. Using CRISPR/Cas9 gene editing technology, we have established a TLR2 mutant WAe001-A-64 cell line from the original embryonic stem cell line H1. It has adopted two biallelic deletions in exon 3 of TLR2 which resulted in a frame shift and early termination in the translation of TLR2. Moreover, WAe001-A-64 has maintained the normal karyotype, pluripotent phenotype, parental cell morphology and the ability to differentiate into three germ layers.},
}
@article {pmid34057656,
year = {2021},
author = {Rasoulinejad, SA and Maroufi, F},
title = {CRISPR-Based Genome Editing as a New Therapeutic Tool in Retinal Diseases.},
journal = {Molecular biotechnology},
volume = {63},
number = {9},
pages = {768-779},
pmid = {34057656},
issn = {1559-0305},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Dependovirus/genetics/metabolism ; Diabetic Retinopathy/genetics/metabolism/pathology/*therapy ; Eye Proteins/*genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors/chemistry/metabolism ; Humans ; Leber Congenital Amaurosis/genetics/metabolism/pathology/*therapy ; Macular Degeneration/genetics/metabolism/pathology/*therapy ; Mutation ; RNA, Guide/genetics/metabolism ; Retinitis Pigmentosa/genetics/metabolism/pathology/*therapy ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Vitreoretinopathy, Proliferative/genetics/metabolism/pathology/*therapy ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {Retinal diseases are the primary reasons for severe visual defects and irreversible blindness. Retinal diseases are also inherited and acquired. Both of them are caused by mutations in genes or disruptions in specific gene expression, which can be treated by gene-editing therapy. Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) system is a frontier of gene-editing tools with great potential for therapeutic applications in the ophthalmology field to modify abnormal genes and treat the genome or epigenome-related retinal diseases. The CRISPR system is able to edit and trim the gene include deletion, insertion, inhibition, activation, replacing, remodeling, epigenetic alteration, and modify the gene expression. CRISPR-based genome editing techniques have indicated the enormous potential to treat retinal diseases that previous treatment was not available for them. Also, recent CRISPR genome surgery experiments have shown the improvement of patient's vision who suffered from severe visual loss. In this article, we review the applications of the CRISPR-Cas9 system in human or animal models for treating retinal diseases such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), then we survey limitations of CRISPR system for clinical therapy.},
}
@article {pmid34055875,
year = {2021},
author = {Märkle, P and Maier, LK and Maaß, S and Hirschfeld, C and Bartel, J and Becher, D and Voß, B and Marchfelder, A},
title = {A Small RNA Is Linking CRISPR-Cas and Zinc Transport.},
journal = {Frontiers in molecular biosciences},
volume = {8},
number = {},
pages = {640440},
pmid = {34055875},
issn = {2296-889X},
abstract = {The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon Haloferax volcanii, a plethora of sRNAs have been identified; however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from Haloferax volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, and the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild-type Haloferax cells and the deletion mutant revealed upregulation of six genes in the deletion strain, showing that this sRNA has a clearly defined function. Three of the six upregulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, and ZnuC1) suggesting the involvement of s479 in the regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znuC1 mRNA. Proteome comparison of wild-type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-cas genes, but the mature s479 contains a crRNA-like 5' handle, and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together, this might suggest that the CRISPR-Cas system is involved in s479 function.},
}
@article {pmid34054265,
year = {2021},
author = {Zhang, Y and Restall, J and Crisp, P and Godwin, I and Liu, G},
title = {Current status and prospects of plant genome editing in Australia.},
journal = {In vitro cellular &amp; developmental biology. Plant : journal of the Tissue Culture Association},
volume = {57},
number = {4},
pages = {574-583},
pmid = {34054265},
issn = {1475-2689},
abstract = {Plant genome editing, particularly CRISPR-Cas biotechnologies, has rapidly evolved and drawn enormous attention all around the world in the last decade. The cutting-edge technologies have had substantial impact on precise genome editing for manipulating gene expression, stacking gene mutations, and improving crop agronomic traits. Following the global trends, investigations on CRISPR-Cas have been thriving in Australia, especially in agriculture sciences. Importantly, CRISPR-edited plants, classified as SDN-1 organisms (SDN: site-directed nuclease), have been given a green light in Australia, with regulatory bodies indicating they will not be classified as a genetically modified organism (GMO) if no foreign DNA is present in an edited plant. As a result, genome-edited products would not attract the onerous regulation required for the introduction of a GMO, which could mean more rapid deployment of new varieties and products that could be traded freely in Australia, and potentially to export markets. In the present review, we discuss the current status and prospects of plant genome editing in Australia by highlighting several species of interest. Using these species as case studies, we discuss the priorities and potential of plant genome editing, as well as the remaining challenges.},
}
@article {pmid34051576,
year = {2021},
author = {Ronayne, CT and Jonnalagadda, SK and Jonnalagadda, S and Nelson, GL and Solano, LN and Palle, H and Mani, C and Rumbley, J and Holy, J and Mereddy, VR},
title = {Synthesis and biological evaluation of a novel anticancer agent CBISC that induces DNA damage response and diminishes levels of mutant-p53.},
journal = {Biochemical and biophysical research communications},
volume = {562},
number = {},
pages = {127-132},
doi = {10.1016/j.bbrc.2021.05.062},
pmid = {34051576},
issn = {1090-2104},
mesh = {Animals ; Antineoplastic Agents/*chemical synthesis/*pharmacology ; Apoptosis/drug effects ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chlorambucil/chemistry/pharmacology ; Chloramphenicol/chemistry/pharmacology ; *DNA Damage ; Female ; Mice, Nude ; Mutant Proteins/*metabolism ; Poly(ADP-ribose) Polymerases/metabolism ; Tumor Suppressor Protein p53/*genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.},
}
@article {pmid34050941,
year = {2021},
author = {Grandhi, TSP and To, J and Romero, A and Luna, F and Barnes, W and Walker, J and Moran, R and Newlin, R and Miraglia, L and Orth, AP and Horman, SR},
title = {High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {8},
pages = {3187-3199},
doi = {10.1002/bit.27844},
pmid = {34050941},
issn = {1097-0290},
mesh = {Antineoplastic Agents/*pharmacology ; *Breast Neoplasms/drug therapy/genetics/metabolism ; *CRISPR-Cas Systems ; *Cell Culture Techniques ; Cell Line, Tumor ; *Drug Resistance, Neoplasm ; Drug Screening Assays, Antitumor ; Female ; Humans ; Spheroids, Cellular/*metabolism ; *Tumor Microenvironment ; },
abstract = {Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.},
}
@article {pmid34050182,
year = {2021},
author = {Xiang, X and Corsi, GI and Anthon, C and Qu, K and Pan, X and Liang, X and Han, P and Dong, Z and Liu, L and Zhong, J and Ma, T and Wang, J and Zhang, X and Jiang, H and Xu, F and Liu, X and Xu, X and Wang, J and Yang, H and Bolund, L and Church, GM and Lin, L and Gorodkin, J and Luo, Y},
title = {Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3238},
pmid = {34050182},
issn = {2041-1723},
support = {RM1 HG008525/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Computational Biology/*methods ; *Deep Learning ; *Gene Editing ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Plasmids/genetics ; RNA, Guide/genetics ; Software ; },
abstract = {The design of CRISPR gRNAs requires accurate on-target efficiency predictions, which demand high-quality gRNA activity data and efficient modeling. To advance, we here report on the generation of on-target gRNA activity data for 10,592 SpCas9 gRNAs. Integrating these with complementary published data, we train a deep learning model, CRISPRon, on 23,902 gRNAs. Compared to existing tools, CRISPRon exhibits significantly higher prediction performances on four test datasets not overlapping with training data used for the development of these tools. Furthermore, we present an interactive gRNA design webserver based on the CRISPRon standalone software, both available via https://rth.dk/resources/crispr/ . CRISPRon advances CRISPR applications by providing more accurate gRNA efficiency predictions than the existing tools.},
}
@article {pmid34050151,
year = {2021},
author = {Giuffrida, L and Sek, K and Henderson, MA and Lai, J and Chen, AXY and Meyran, D and Todd, KL and Petley, EV and Mardiana, S and Mølck, C and Stewart, GD and Solomon, BJ and Parish, IA and Neeson, PJ and Harrison, SJ and Kats, LM and House, IG and Darcy, PK and Beavis, PA},
title = {CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3236},
pmid = {34050151},
issn = {2041-1723},
mesh = {Adenosine/metabolism ; Adenosine A2 Receptor Antagonists/pharmacology ; Animals ; CRISPR-Cas Systems/genetics ; Cell Engineering/methods ; Cell Line, Tumor/transplantation ; Disease Models, Animal ; Female ; Gene Editing ; Gene Expression Regulation, Neoplastic/immunology ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Immunotherapy, Adoptive/*methods ; Lymphocytes, Tumor-Infiltrating/immunology ; Mice ; Mice, Transgenic ; Neoplasms/genetics/immunology/*therapy ; RNA, Small Interfering/metabolism ; RNA-Seq ; Receptor, Adenosine A2A/*genetics/metabolism ; Receptor, ErbB-2/genetics ; Receptors, Chimeric Antigen/immunology/metabolism ; Signal Transduction/drug effects/genetics/immunology ; T-Lymphocytes/immunology/metabolism/*transplantation ; Tumor Escape/drug effects/genetics ; },
abstract = {Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.},
}
@article {pmid34050031,
year = {2021},
author = {Carroll, D and Meyer, BJ},
title = {Life 2.0-A CRISPR path to a sustainable planet.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34050031},
issn = {1091-6490},
support = {R35 GM131845/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Organisms, Genetically Modified ; Sustainable Development ; },
}
@article {pmid34050019,
year = {2021},
author = {Nasti, RA and Voytas, DF},
title = {Attaining the promise of plant gene editing at scale.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34050019},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; *Crops, Agricultural ; *Gene Editing ; *Genome, Plant ; *Plant Breeding ; Plants, Genetically Modified/*genetics ; },
abstract = {Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.},
}
@article {pmid34050017,
year = {2021},
author = {Garrood, WT and Kranjc, N and Petri, K and Kim, DY and Guo, JA and Hammond, AM and Morianou, I and Pattanayak, V and Joung, JK and Crisanti, A and Simoni, A},
title = {Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34050017},
issn = {1091-6490},
mesh = {Animals ; Anopheles/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome, Insect ; Humans ; *Malaria ; Mosquito Vectors/*genetics ; },
abstract = {CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations' exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.},
}
@article {pmid34050012,
year = {2021},
author = {Power, ME},
title = {Synthetic threads through the web of life.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34050012},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; *Ecosystem ; *Gene Editing ; *Models, Biological ; *Software ; *Synthetic Biology ; },
abstract = {CRISPR-Cas gene editing tools have brought us to an era of synthetic biology that will change the world. Excitement over the breakthroughs these tools have enabled in biology and medicine is balanced, justifiably, by concern over how their applications might go wrong in open environments. We do not know how genomic processes (including regulatory and epigenetic processes), evolutionary change, ecosystem interactions, and other higher order processes will affect traits, fitness, and impacts of edited organisms in nature. However, anticipating the spread, change, and impacts of edited traits or organisms in heterogeneous, changing environments is particularly important with "gene drives on the horizon." To anticipate how "synthetic threads" will affect the web of life on Earth, scientists must confront complex system interactions across many levels of biological organization. Currently, we lack plans, infrastructure, and funding for field science and scientists to track new synthetic organisms, with or without gene drives, as they move through open environments.},
}
@article {pmid34050011,
year = {2021},
author = {Alanis-Lobato, G and Zohren, J and McCarthy, A and Fogarty, NME and Kubikova, N and Hardman, E and Greco, M and Wells, D and Turner, JMA and Niakan, KK},
title = {Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34050011},
issn = {1091-6490},
support = {FC001120/CRUK_/Cancer Research UK/United Kingdom ; FC001120/ARC_/Arthritis Research UK/United Kingdom ; FC001193/ARC_/Arthritis Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; FC001193/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Blastocyst/*metabolism ; *CRISPR-Cas Systems ; Cell Line ; Chromosomes, Human, Pair 6/genetics/metabolism ; *Gene Editing ; Human Embryonic Stem Cells/*metabolism ; Humans ; *Loss of Heterozygosity ; *Octamer Transcription Factor-3 ; },
abstract = {CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.},
}
@article {pmid34050010,
year = {2021},
author = {Epstein, LR and Lee, SS and Miller, MF and Lombardi, HA},
title = {CRISPR, animals, and FDA oversight: Building a path to success.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34050010},
issn = {1091-6490},
mesh = {Animals ; *Animals, Genetically Modified ; *Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; United States ; United States Food and Drug Administration ; },
abstract = {Technological advances, such as genome editing and specifically CRISPR, offer exciting promise for the creation of products that address public health concerns, such as disease transmission and a sustainable food supply and enable production of human therapeutics, such as organs and tissues for xenotransplantation or recombinant human proteins to treat disease. The Food and Drug Administration recognizes the need for such innovative solutions and plays a key role in bringing safe and effective animal biotechnology products to the marketplace. In this article, we (the Food and Drug Administration/Center for Veterinary Medicine) describe the current state of the science, including advances in technology as well as scientific limitations and considerations for how researchers and commercial developers working to create intentional genomic alterations in animals can work within these limitations. We also describe our risk-based approach and how it strikes a balance between our regulatory responsibilities and the need to get innovative products to market efficiently. We continue to seek input from our stakeholders and hope to use this feedback to improve the transparency, predictability, and efficiency of our process. We think that working together, using appropriate science- and risk-based oversight, is the foundation to a successful path forward.},
}
@article {pmid34049825,
year = {2022},
author = {Kabadi, AM and Machlin, L and Dalal, N and Lee, RE and McDowell, I and Shah, NN and Drowley, L and Randell, SH and Reddy, TE},
title = {Epigenome editing of the CFTR-locus for treatment of cystic fibrosis.},
journal = {Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society},
volume = {21},
number = {1},
pages = {164-171},
pmid = {34049825},
issn = {1873-5010},
support = {F31 HL158197/HL/NHLBI NIH HHS/United States ; P30 DK065988/DK/NIDDK NIH HHS/United States ; T32 GM133364/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cystic Fibrosis/drug therapy/*genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/therapeutic use ; Epigenome ; Gene Editing/*methods ; Gene Expression Regulation ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; },
abstract = {BACKGROUND: Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues.<br><br>METHODS: Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9[p300] and dCas9[KRAB] for its ability to enhance or repress CFTR expression, respectively.<br><br>RESULTS: Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9[p300] mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9[KRAB]-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9[p300] was used to induce elevated levels of CFTR from the endogenous locus in &#x394;F508/&#x394;F508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of &#x394;F508 CFTR mRNA along with VX809 treatment.<br><br>CONCLUSIONS: CRISPR/dCas9-based epigenome-editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.},
}
@article {pmid34049503,
year = {2021},
author = {Ramaker, RC and Hardigan, AA and Gordon, ER and Wright, CA and Myers, RM and Cooper, SJ},
title = {Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition.},
journal = {BMC cancer},
volume = {21},
number = {1},
pages = {632},
pmid = {34049503},
issn = {1471-2407},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Pancreatic Ductal/*drug therapy/genetics/mortality/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Chromatin Immunoprecipitation Sequencing ; Co-Repressor Proteins/genetics/metabolism ; Drug Resistance, Multiple/*genetics ; Drug Resistance, Neoplasm/*genetics ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Histone Deacetylase 1/genetics/metabolism ; Humans ; Pancreatic Neoplasms/*drug therapy/genetics/mortality/pathology ; RNA-Seq ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) patients suffer poor outcomes, including a five-year survival of below 10%. Poor outcomes result in part from therapeutic resistance that limits the impact of cytotoxic first-line therapy. Novel therapeutic approaches are needed, but currently no targeted therapies exist to treat PDAC.<br><br>METHODS: To assess cellular resistance mechanisms common to four cytotoxic chemotherapies (gemcitabine, 5-fluorouracil, irinotecan, and oxaliplatin) used to treat PDAC patients, we performed four genome-wide CRISPR activation (CRISPRact) and CRISPR knock-out (CRISPRko) screens in two common PDAC cell lines (Panc-1 and BxPC3). We used pathway analysis to identify gene sets enriched among our hits and conducted RNA-sequencing and chromatin immunoprecipitation-sequencing (ChIP-seq) to characterize top hits from our screen. We used scratch assays to assess changes in cellular migration with HDAC1 overexpression.<br><br>RESULTS: Our data revealed activation of ABCG2, a well-described efflux pump, as the most consistent mediator of resistance in each of our screens. CRISPR-mediated activation of genes involved in transcriptional co-repressor complexes also conferred resistance to multiple drugs. Expression of many of these genes, including HDAC1, is associated with reduced survival in PDAC patients. Up-regulation of HDAC1 in vitro increased promoter occupancy and expression of several genes involved in the epithelial-to-mesenchymal transition (EMT). These cells also displayed phenotypic changes in cellular migration consistent with activation of the EMT pathway. The expression changes resulting from HDAC1 activation were also observed with activation of several other co-repressor complex members. Finally, we developed a publicly available analysis tool, PancDS, which integrates gene expression profiles with our screen results to predict drug sensitivity in resected PDAC tumors and cell lines.<br><br>CONCLUSION: Our results provide a comprehensive resource for identifying cellular mechanisms of drug resistance in PDAC, mechanistically implicate HDAC1, and co-repressor complex members broadly, in multi-drug resistance, and provide an analytical tool for predicting treatment response in PDAC tumors and cell lines.},
}
@article {pmid34049081,
year = {2021},
author = {Ge, X and Meng, T and Tan, X and Wei, Y and Tao, Z and Yang, Z and Song, F and Wang, P and Wan, Y},
title = {Cas14a1-mediated nucleic acid detectifon platform for pathogens.},
journal = {Biosensors &amp; bioelectronics},
volume = {189},
number = {},
pages = {113350},
doi = {10.1016/j.bios.2021.113350},
pmid = {34049081},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases ; *Nucleic Acids ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) based biosensing system provides a novel genomic diagnostic tool for pathogenic detection. However, most of the discovered Cas effectors have poor single strand DNA (ssDNA) target recognition capability with the constraint of protospacer adjacent motif (PAM) sites, which are not suitable for universal pathogenic diagnosis. Herein, we developed a highly sensitive and specific fluorescence tool for bacterial detection by utilizing the unique collateral cleavage activity of a Cas14a1-mediated nucleic acid detection platform (CMP). We combine CMP with molecular amplification to build a CRISPR-Cas based bioanalysis technique, offering fast nucleic acid detection with high sensitivity and specificity. This technique can identify different species of pathogens in milk samples with excellent accuracy. The CMP technique is a promising platform for pathogenic genomic diagnostic in biomedicine and food safety field.},
}
@article {pmid34047246,
year = {2021},
author = {Kmiec, EB and Bloh, K},
title = {A toolmaker's perspective on CRISPR-directed gene editing as a therapeutic strategy for leukemia and beyond.},
journal = {Expert review of hematology},
volume = {14},
number = {7},
pages = {587-592},
doi = {10.1080/17474086.2021.1935853},
pmid = {34047246},
issn = {1747-4094},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Therapy ; Humans ; *Leukemia/genetics/therapy ; },
}
@article {pmid34045481,
year = {2021},
author = {Asano, Y and Yamashita, K and Hasegawa, A and Ogasawara, T and Iriki, H and Muramoto, T},
title = {Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {11163},
pmid = {34045481},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Dictyostelium/genetics/*metabolism ; Gene Editing ; Nucleotides/*metabolism ; },
abstract = {The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.},
}
@article {pmid34045344,
year = {2021},
author = {Abudayyeh, OO and Gootenberg, JS},
title = {CRISPR diagnostics.},
journal = {Science (New York, N.Y.)},
volume = {372},
number = {6545},
pages = {914-915},
doi = {10.1126/science.abi9335},
pmid = {34045344},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Diagnostic Tests, Routine ; Gene Editing ; RNA ; },
}
@article {pmid34043946,
year = {2021},
author = {Nemudryi, A and Nemudraia, A and Wiegand, T and Nichols, J and Snyder, DT and Hedges, JF and Cicha, C and Lee, H and Vanderwood, KK and Bimczok, D and Jutila, MA and Wiedenheft, B},
title = {SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression.},
journal = {Cell reports},
volume = {35},
number = {9},
pages = {109197},
pmid = {34043946},
issn = {2211-1247},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; COVID-19/*immunology/*virology ; Chlorocebus aethiops ; Genome, Viral ; HEK293 Cells ; Humans ; *Immunity ; Interferon Type I/immunology ; Mutation ; Phylogeny ; SARS-CoV-2/*genetics/pathogenicity ; Vero Cells ; Viral Proteins/*genetics/*immunology ; Viral Regulatory and Accessory Proteins/genetics ; },
abstract = {Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7a[Δ115]) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response.},
}
@article {pmid34043288,
year = {2021},
author = {Sobh, A and Russo, M and Vulpe, CD},
title = {CRISPR Screens in Toxicology Research: An Overview.},
journal = {Current protocols},
volume = {1},
number = {5},
pages = {e136},
doi = {10.1002/cpz1.136},
pmid = {34043288},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene-Environment Interaction ; Humans ; Sequence Analysis ; },
abstract = {The use of genome editing tools is expanding our understanding of various human diseases by providing insight into gene-disease interactions. Despite the recognized role of toxicants in the development of human health issues and conditions, there is currently limited characterization of their mechanisms of action, and the application of CRISPR-based genome editing to the study of toxicants could help in the identification of novel gene-environment interactions. CRISPR-based functional screens enable identification of cellular mechanisms fundamental for response and susceptibility to a given toxicant. The aim of this review is to inform future directions in the application of CRISPR technologies in toxicological studies. We review and compare different types of CRISPR-based methods including pooled, anchored, combinatorial, and perturb-sequencing screens in vitro, in addition to pooled screenings in model organisms. © 2021 Wiley Periodicals LLC.},
}
@article {pmid34042501,
year = {2021},
author = {Ma, S and Wang, A and Chen, X and Zhang, T and Xing, W and Xia, Q},
title = {Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {371-380},
doi = {10.1089/crispr.2021.0003},
pmid = {34042501},
issn = {2573-1602},
mesh = {Animals ; Bombyx/*genetics/metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; DNA ; Female ; Gene Editing/methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Mutation ; RNA, Guide/genetics ; },
abstract = {Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or single-base deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.},
}
@article {pmid34042500,
year = {2021},
author = {Kim, Y and Lee, SJ and Park, C and Koo, J and Bae, E and Lee, BJ and Suh, JY},
title = {Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2.},
journal = {The CRISPR journal},
volume = {4},
number = {3},
pages = {448-458},
doi = {10.1089/crispr.2020.0119},
pmid = {34042500},
issn = {2573-1602},
mesh = {Bacteriophages/metabolism ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Bacterial ; Mutagenesis ; Neisseria/virology ; Neisseria meningitidis/genetics/metabolism ; RNA, Guide/genetics ; Viral Proteins/*metabolism ; },
abstract = {Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant KD ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.},
}
@article {pmid34042262,
year = {2021},
author = {Ren, Q and Sretenovic, S and Liu, G and Zhong, Z and Wang, J and Huang, L and Tang, X and Guo, Y and Liu, L and Wu, Y and Zhou, J and Zhao, Y and Yang, H and He, Y and Liu, S and Yin, D and Mayorga, R and Zheng, X and Zhang, T and Qi, Y and Zhang, Y},
title = {Improved plant cytosine base editors with high editing activity, purity, and specificity.},
journal = {Plant biotechnology journal},
volume = {19},
number = {10},
pages = {2052-2068},
pmid = {34042262},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; *Cytosine ; *Gene Editing ; Mutation ; RNA, Guide ; Whole Genome Sequencing ; },
abstract = {Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-to-T base editing in plants, we first compared seven cytidine deaminases in the BE3-like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-to-T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-to-T base editing at A-rich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as pre-mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-to-T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.},
}
@article {pmid34041717,
year = {2021},
author = {Jabbar, A and Zulfiqar, F and Mahnoor, M and Mushtaq, N and Zaman, MH and Din, ASU and Khan, MA and Ahmad, HI},
title = {Advances and Perspectives in the Application of CRISPR-Cas9 in Livestock.},
journal = {Molecular biotechnology},
volume = {63},
number = {9},
pages = {757-767},
pmid = {34041717},
issn = {1559-0305},
mesh = {Animals ; *Animals, Genetically Modified ; *CRISPR-Cas Systems ; Cattle ; Female ; Gene Editing/*methods ; Genome ; Goats/genetics/metabolism ; Livestock/*genetics/metabolism ; Mastitis/genetics/prevention &amp; control ; Meat/analysis/*supply &amp; distribution ; Milk/chemistry/supply &amp; distribution ; Organ Transplantation/methods ; Polymerase Chain Reaction/methods ; Sheep, Domestic/genetics/metabolism ; Swine/genetics/metabolism ; Transcription Activator-Like Effector Nucleases/*genetics/metabolism ; Transplantation, Heterologous ; },
abstract = {The sophistication and revolution in genome editing and manipulation have revolutionized livestock by harvesting essential biotechnological products such as drugs, proteins, and serum. It laid down areas for the large production of transgenic food, resistance against certain diseases such as mastitis, and large production of milk and leaner meat. Nowadays, the increasing demand for animal food and protein is fulfilled using genome-editing technologies. The recent genome-editing techniques have overcome the earlier methods of animal reproduction, such as cloning and artificial embryo transfer. The genome of animals now is modified using the recent alteration techniques such as ZFNs, TALENS technique, and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR-Cas9) system. The literature was illustrated for identifying the researchers to address the advances and perspectives in the application of Cas9 in Livestock. Cas9 is considered better than the previously identified techniques in livestock because of the production of resilience against diseases, improvement of reproductive traits, and animal production to act as a model biomedical research.},
}
@article {pmid34041045,
year = {2021},
author = {Nuss, A and Sharma, A and Gulia-Nuss, M},
title = {Genetic Manipulation of Ticks: A Paradigm Shift in Tick and Tick-Borne Diseases Research.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {678037},
pmid = {34041045},
issn = {2235-2988},
support = {R21 AI128393/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Arthropod Vectors ; Humans ; *Ixodes ; Mosquito Vectors ; *Tick-Borne Diseases ; },
abstract = {Ticks are obligate hematophagous arthropods that are distributed worldwide and are one of the most important vectors of pathogens affecting humans and animals. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind other arthropod vectors, such as mosquitoes. This is largely because of challenges in applying functional genomics and genetic tools to the idiosyncrasies unique to tick biology, particularly techniques for stable genetic transformations. CRISPR-Cas9 is transforming non-model organism research; however, successful germline editing has yet to be accomplished in ticks. Here, we review the ancillary methods needed for transgenic tick development and the use of CRISPR/Cas9, the most promising gene-editing approach, for tick genetic transformation.},
}
@article {pmid34040899,
year = {2021},
author = {Han, RC and Fry, LE and Kantor, A and McClements, ME and Xue, K and MacLaren, RE},
title = {Is subretinal AAV gene replacement still the only viable treatment option for choroideremia?.},
journal = {Expert opinion on orphan drugs},
volume = {9},
number = {1},
pages = {13-24},
pmid = {34040899},
issn = {2167-8707},
support = {/WT_/Wellcome Trust/United Kingdom ; 216593/WT_/Wellcome Trust/United Kingdom ; },
abstract = {INTRODUCTION: Choroideremia is an X-linked inherited retinal degeneration resulting from mutations in the CHM gene, encoding Rab escort protein-1 (REP1), a protein regulating intracellular vesicular transport. Loss-of-function mutations in CHM lead to progressive loss of retinal pigment epithelium (RPE) with photoreceptor and choriocapillaris degeneration, leading to progressive visual field constriction and loss of visual acuity. Three hundred and fifty-four unique mutations have been reported in CHM. While gene augmentation remains an ideal therapeutic option for choroideremia, other potential future clinical strategies may exist.<br><br>AREAS COVERED: The authors examine the pathophysiology and genetic basis of choroideremia. They summarize the status of ongoing gene therapy trials and discuss CHM mutations amenable to other therapeutic approaches including CRISPR/Cas-based DNA and RNA editing, nonsense suppression of premature termination codons, and antisense oligonucleotides for splice modification. The authors undertook a literature search in PubMed and NIH Clinical Trials in October 2020.<br><br>EXPERT OPINION: The authors conclude that AAV-mediated gene augmentation remains the most effective approach for choroideremia. Given the heterogeneity of CHM mutations and potential risks and benefits, genome-editing approaches currently do not offer significant advantages. Nonsense suppression strategies and antisense oligonucleotides are exciting novel therapeutic options; however, their clinical viability remains to be determined.},
}
@article {pmid34040254,
year = {2021},
author = {Oh, S and Shao, J and Mitra, J and Xiong, F and D'Antonio, M and Wang, R and Garcia-Bassets, I and Ma, Q and Zhu, X and Lee, JH and Nair, SJ and Yang, F and Ohgi, K and Frazer, KA and Zhang, ZD and Li, W and Rosenfeld, MG},
title = {Enhancer release and retargeting activates disease-susceptibility genes.},
journal = {Nature},
volume = {595},
number = {7869},
pages = {735-740},
pmid = {34040254},
issn = {1476-4687},
support = {R21 GM132778/GM/NIGMS NIH HHS/United States ; R37 DK039949/DK/NIDDK NIH HHS/United States ; F32 HD008118/HD/NICHD NIH HHS/United States ; R01 HL150521/HL/NHLBI NIH HHS/United States ; R01 CA184770/CA/NCI NIH HHS/United States ; R01 NS093066/NS/NINDS NIH HHS/United States ; U01 HL156059/HL/NHLBI NIH HHS/United States ; K22 CA204468/CA/NCI NIH HHS/United States ; R01 HG008153/HG/NHGRI NIH HHS/United States ; R01 GM136922/GM/NIGMS NIH HHS/United States ; R01 DK018477/DK/NIDDK NIH HHS/United States ; },
mesh = {CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Cells, Cultured ; Chromatin ; Chromosomal Proteins, Non-Histone/genetics ; *Enhancer Elements, Genetic ; Gene Deletion ; Gene Expression Regulation, Neoplastic ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; MCF-7 Cells ; Neoplasms/genetics ; Neural Stem Cells ; Oncogenes ; Parkinson Disease/genetics ; *Promoter Regions, Genetic ; },
abstract = {The functional engagement between an enhancer and its target promoter ensures precise gene transcription[1]. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).},
}
@article {pmid34039988,
year = {2021},
author = {Wang, Q and Hernández-Ochoa, EO and Viswanathan, MC and Blum, ID and Do, DC and Granger, JM and Murphy, KR and Wei, AC and Aja, S and Liu, N and Antonescu, CM and Florea, LD and Talbot, CC and Mohr, D and Wagner, KR and Regot, S and Lovering, RM and Gao, P and Bianchet, MA and Wu, MN and Cammarato, A and Schneider, MF and Bever, GS and Anderson, ME},
title = {CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3175},
pmid = {34039988},
issn = {2041-1723},
support = {R01 NS079584/NS/NINDS NIH HHS/United States ; R01 GM129085/GM/NIGMS NIH HHS/United States ; R21 NS108842/NS/NINDS NIH HHS/United States ; R01 AR059179/AR/NIAMS NIH HHS/United States ; R01 AR075726/AR/NIAMS NIH HHS/United States ; R21 AR067872/AR/NIAMS NIH HHS/United States ; R37 AR055099/AR/NIAMS NIH HHS/United States ; R35 HL140034/HL/NHLBI NIH HHS/United States ; R01 HL124091/HL/NHLBI NIH HHS/United States ; },
mesh = {Aging/*physiology ; Animals ; Animals, Genetically Modified ; *Biological Evolution ; CRISPR-Cas Systems/genetics ; Calcium Signaling/physiology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics/*metabolism ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster ; Female ; Gene Editing ; Gene Knock-In Techniques ; Male ; Mice ; Models, Animal ; Oxidation-Reduction ; Phylogeny ; Physical Fitness/physiology ; Point Mutation ; Reactive Oxygen Species/*metabolism ; Vertebrates/*physiology ; },
abstract = {Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca[2+] and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.},
}
@article {pmid34039408,
year = {2021},
author = {Liu, X and Li, C and Li, X and Ehsan, M and Lu, M and Li, K and Xu, L and Yan, R and Song, X and Li, X},
title = {Proteomics analysis reveals that the proto-oncogene eIF-5A indirectly influences the growth, invasion and replication of Toxoplasma gondii tachyzoite.},
journal = {Parasites &amp; vectors},
volume = {14},
number = {1},
pages = {283},
pmid = {34039408},
issn = {1756-3305},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Replication ; Female ; Gene Deletion ; Mice, Inbred BALB C ; Peptide Initiation Factors/*genetics/*metabolism ; Protein Interaction Maps ; Proteomics/*methods ; Proto-Oncogenes/genetics ; Protozoan Proteins/genetics/metabolism ; RNA-Binding Proteins/*genetics/*metabolism ; Rats ; Rats, Sprague-Dawley ; Toxoplasma/*genetics/*physiology ; Toxoplasmosis/parasitology ; Virulence ; },
abstract = {BACKGROUND: The proliferative stage (tachyzoite) of Toxoplasma gondii (T. gondii) is critical for its transmission and pathogenesis, and a proto-oncogene eukaryotic translation initiation factor (eIF-5A) plays an important role in various cellular processes such as cell multiplication.<br><br>METHODS: We performed a proteomic study to evaluate the specific roles of eIF-5A involved in invasion and replication of T. gondii, and both in vivo and in vitro trials using eIF-5A-interfered and wild tachyzoites were performed to verify the proteomic results.<br><br>RESULTS: The results of our study showed that T. gondii eIF-5A affected tachyzoite growth and also participated in the synthesis of proteins through regulation of both ribosomal and splicing pathways. Inhibition of eIF-5A in T. gondii resulted in the downregulated expression of soluble adhesions, such as microneme protein 1 (MIC1) and MIC4, which in turn decreased the parasite population that adhered to the surface of host cells. The reduced attachment, combined with lower expression of some rhoptry proteins (ROPs) and dense granule antigens (GRAs) involved in different stages of&#xa0;T. gondii invasion such as ROP4 and GRA3, ultimately reduce the invasion efficiency. These processes regulated by eIF-5A eventually affect the replication of tachyzoites.<br><br>CONCLUSIONS: Our findings showed that eIF-5A influenced tachyzoite survival and was also involved in the process of parasite invasion and replication. These results will provide new clues for further development of targeted drugs to control T. gondii infection.},
}
@article {pmid34039112,
year = {2021},
author = {An, SB and Yang, K and Kim, CW and Choi, SH and Kim, E and Kim, SD and Koh, JS},
title = {Longitudinal Imaging of Liver Cancer Using MicroCT and Nanoparticle Contrast Agents in CRISPR/Cas9-Induced Liver Cancer Mouse Model.},
journal = {Technology in cancer research &amp; treatment},
volume = {20},
number = {},
pages = {15330338211016466},
pmid = {34039112},
issn = {1533-0338},
mesh = {Animals ; *CRISPR-Cas Systems ; Carcinogenesis/*pathology ; Contrast Media/*metabolism ; Liver Neoplasms, Experimental/diagnostic imaging/*pathology ; Longitudinal Studies ; Mice ; Mice, Inbred C57BL ; Nanoparticles/*chemistry ; Plasmids/*genetics ; X-Ray Microtomography/*methods ; },
abstract = {INTRODUCTION: Micro-computed tomography with nanoparticle contrast agents may be a suitable tool for monitoring the time course of the development and progression of tumors. Here, we suggest a practical and convenient experimental method for generating and longitudinally imaging murine liver cancer models.<br><br>METHODS: Liver cancer was induced in 6 experimental mice by injecting clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 plasmids causing mutations in genes expressed by hepatocytes. Nanoparticle agents are captured by Kupffer cells and detected by micro-computed tomography, thereby enabling longitudinal imaging. A total of 9 mice were used for the experiment. Six mice were injected with both plasmids and contrast, 2 injected with contrast alone, and one not injected with either agent. Micro-computed tomography images were acquired every 2- up to 14-weeks after cancer induction.<br><br>RESULTS: Liver cancer was first detected by micro-computed tomography at 8 weeks. The mean value of hepatic parenchymal attenuation remained almost unchanged over time, although the standard deviation of attenuation, reflecting heterogeneous contrast enhancement of the hepatic parenchyma, increased slowly over time in all mice. Histopathologically, heterogeneous distribution and aggregation of Kupffer cells was more prominent in the experimental group than in the control group. Heterogeneous enhancement of hepatic parenchyma, which could cause image quality deterioration and image misinterpretation, was observed and could be due to variation in Kupffer cells distribution.<br><br>CONCLUSION: Micro-computed tomography with nanoparticle contrast is useful in evaluating the induction and characteristics of liver cancer, determining appropriate size of liver cancer for testing, and confirming therapeutic response.},
}
@article {pmid34038837,
year = {2021},
author = {Wu, H and Chen, Y and Yang, Q and Peng, C and Wang, X and Zhang, M and Qian, S and Xu, J and Wu, J},
title = {A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemolyticus.},
journal = {Biosensors &amp; bioelectronics},
volume = {188},
number = {},
pages = {113352},
doi = {10.1016/j.bios.2021.113352},
pmid = {34038837},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; *Vibrio parahaemolyticus/genetics ; },
abstract = {Vibrio parahaemolyticus (V. parahaemolyticus) is regarded as a major cause of seafood-associated illnesses, which has aroused widespread public concern. Here, a rapid and convenient detection method for V. parahaemolyticus detection was established by a reversible valve-assisted chip coupling with CRISPR/Cas12a. With optimized lysis buffer, loop mediated isothermal amplification (LAMP) reagents and CRISPR reagents, the whole detection process from sampling to results could be finished within 50 min. The structure of chip was simple and the cost was low. By relying on three reversible rotary valves and the rotation direction-dependent Coriolis pseudo force, the flow order of liquid and the direction of liquid flow could be precisely controlled. The LAMP amplicons were specifically and sensitively identified by CRISPR/Cas12a. Positive amplification would produce green fluorescent signal while negative amplification generated no fluorescent signal, which could be clearly distinguished by the naked eye. With 600 μL of samples processed, the limit of detection (LOD) for both pure cultured V. parahaemolyticus or spiked shrimp samples could achieve 30 copies/reaction. These illustrated the established method displayed great feasibility for real samples detection. In the future, the chip could also combine with other amplification reactions, like PCR or recombinase polymerase amplification reaction (RPA), to conduct detection by changing the corresponding lyophilized amplification reagents. Overall, the proposed detection platform displays great potential for food safety analysis and clinical diagnostics, especially in resource-limited areas.},
}
@article {pmid34038740,
year = {2021},
author = {Wong, HH and Seet, SH and Maier, M and Gurel, A and Traspas, RM and Lee, C and Zhang, S and Talim, B and Loh, AYT and Chia, CY and Teoh, TS and Sng, D and Rensvold, J and Unal, S and Shishkova, E and Cepni, E and Nathan, FM and Sirota, FL and Liang, C and Yarali, N and Simsek-Kiper, PO and Mitani, T and Ceylaner, S and Arman-Bilir, O and Mbarek, H and Gumruk, F and Efthymiou, S and Uğurlu Çi Men, D and Georgiadou, D and Sotiropoulou, K and Houlden, H and Paul, F and Pehlivan, D and Lainé, C and Chai, G and Ali, NA and Choo, SC and Keng, SS and Boisson, B and Yılmaz, E and Xue, S and Coon, JJ and Ly, TTN and Gilani, N and Hasbini, D and Kayserili, H and Zaki, MS and Isfort, RJ and Ordonez, N and Tripolszki, K and Bauer, P and Rezaei, N and Seyedpour, S and Khotaei, GT and Bascom, CC and Maroofian, R and Chaabouni, M and Alsubhi, A and Eyaid, W and Işıkay, S and Gleeson, JG and Lupski, JR and Casanova, JL and Pagliarini, DJ and Akarsu, NA and Maurer-Stroh, S and Cetinkaya, A and Bertoli-Avella, A and Mathuru, AS and Ho, L and Bard, FA and Reversade, B},
title = {Loss of C2orf69 defines a fatal autoinflammatory syndrome in humans and zebrafish that evokes a glycogen-storage-associated mitochondriopathy.},
journal = {American journal of human genetics},
volume = {108},
number = {7},
pages = {1301-1317},
pmid = {34038740},
issn = {1537-6605},
support = {R35 GM131795/GM/NIGMS NIH HHS/United States ; R35 NS105078/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Biological Evolution ; CRISPR-Cas Systems ; Cell Line ; Encephalitis/*genetics/mortality ; Female ; Genes, Recessive ; Glycogen/metabolism ; Humans ; Inflammation/genetics ; Male ; Membrane Proteins/genetics ; Mitochondrial Diseases/*genetics/mortality ; Pedigree ; Seizures/genetics/mortality ; Zebrafish/genetics ; },
abstract = {Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.},
}
@article {pmid34038533,
year = {2021},
author = {Ozkan, J},
title = {Jennifer A. Doudna and Emmanuelle Charpentier.},
journal = {European heart journal},
volume = {42},
number = {22},
pages = {2143-2145},
doi = {10.1093/eurheartj/ehaa1054},
pmid = {34038533},
issn = {1522-9645},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid34038136,
year = {2021},
author = {Yue, H and Shu, B and Tian, T and Xiong, E and Huang, M and Zhu, D and Sun, J and Liu, Q and Wang, S and Li, Y and Zhou, X},
title = {Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.},
journal = {Nano letters},
volume = {21},
number = {11},
pages = {4643-4653},
doi = {10.1021/acs.nanolett.1c00715},
pmid = {34038136},
issn = {1530-6992},
mesh = {*African Swine Fever Virus/genetics ; Animals ; CRISPR-Cas Systems ; DNA/genetics ; *Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; Swine ; },
abstract = {DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.},
}
@article {pmid34038095,
year = {2021},
author = {Zhang, M and Wang, H and Wang, H and Wang, F and Li, Z},
title = {CRISPR/Cas12a-Assisted Ligation-Initiated Loop-Mediated Isothermal Amplification (CAL-LAMP) for Highly Specific Detection of microRNAs.},
journal = {Analytical chemistry},
volume = {93},
number = {22},
pages = {7942-7948},
doi = {10.1021/acs.analchem.1c00686},
pmid = {34038095},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *MicroRNAs/genetics ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; },
abstract = {Loop-mediated isothermal amplification (LAMP) has been increasingly applied in nucleic acid detection for clinical diagnosis and monitoring pathogenic microorganisms due to its isothermal nature and high sensitivity. However, the false-positive signal resulting from the non-specific amplification and the complexity of primer design are still technically challenging for wide applications. In this paper, we developed the CRISPR/Cas12a-assisted sequence-specific detection of LAMP products to eliminate the effect of non-specific amplification from primer dimers and spurious amplicons. Moreover, by designing a pair of target-specific stem-loop DNA probes, we greatly simplified the primer design for LAMP. The DNA probes could be ligated to form a double-stem-loop DNA template by the detected target, which initiated LAMP reaction and achieved one-nucleotide resolution due to the highly specific ligase reaction. Using microRNAs (miRNAs) as the model targets, the CRISPR/Cas12a-assisted ligation-initiated loop-mediated isothermal amplification (CAL-LAMP) can sensitively detect as low as 0.1 fM miRNAs with high specificity.},
}
@article {pmid34037380,
year = {2021},
author = {Palaz, F and Kalkan, AK and Can, &#xd6; and Demir, AN and Tozluyurt, A and Özcan, A and Ozsoz, M},
title = {CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1245-1267},
doi = {10.1021/acssynbio.1c00107},
pmid = {34037380},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; Early Detection of Cancer/*methods ; Gene Editing/*methods ; Genome, Human ; Humans ; Liquid Biopsy ; Molecular Targeted Therapy/methods ; Neoplasms/*diagnosis/genetics/pathology/*therapy ; Precision Medicine/methods ; RNA/genetics ; Transcriptome ; },
abstract = {Over the past decades, significant progress has been made in targeted cancer therapy. In precision oncology, molecular profiling of cancer patients enables the use of targeted cancer therapeutics. However, current diagnostic methods for molecular analysis of cancer are costly and require sophisticated equipment. Moreover, targeted cancer therapeutics such as monoclonal antibodies and small-molecule drugs may cause off-target effects and they are available for only a minority of cancer driver proteins. Therefore, there is still a need for versatile, efficient, and precise tools for cancer diagnostics and targeted cancer treatment. In recent years, the CRISPR-based genome and transcriptome engineering toolbox has expanded rapidly. Particularly, the RNA-targeting CRISPR-Cas13 system has unique biochemical properties, making Cas13 a promising tool for cancer diagnosis, therapy, and research. Cas13-based diagnostic methods allow early detection and monitoring of cancer markers from liquid biopsy samples without the need for complex instrumentation. In addition, Cas13 can be used for targeted cancer therapy through degrading and manipulating cancer-associated transcripts with high efficiency and specificity. Moreover, Cas13-mediated programmable RNA manipulation tools offer invaluable opportunities for cancer research, identification of drug-resistance mechanisms, and discovery of novel therapeutic targets. Here, we review and discuss the current use and potential applications of the CRISPR-Cas13 system in cancer diagnosis, therapy, and research. Thus, researchers will gain a deep understanding of CRISPR-Cas13 technologies, which have the potential to be used as next-generation cancer diagnostics and therapeutics.},
}
@article {pmid34036333,
year = {2021},
author = {O'Hagan, D and Kruger, RE and Gu, B and Ralston, A},
title = {Efficient generation of endogenous protein reporters for mouse development.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {13},
pages = {},
pmid = {34036333},
issn = {1477-9129},
support = {R35 GM131759/GM/NIGMS NIH HHS/United States ; T32 HD087166/HD/NICHD NIH HHS/United States ; R35 GM131759/NH/NIH HHS/United States ; },
mesh = {Animals ; Blastocyst/*physiology ; CRISPR-Cas Systems ; Cell Line ; Fluorescent Antibody Technique ; Gene Knock-In Techniques ; *Genome ; Green Fluorescent Proteins/genetics ; Mice ; Transcription Factors ; },
abstract = {Fluorescent proteins and epitope tags can reveal protein localization in cells and animals, yet the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. Here, we present two strategies for higher throughput production of endogenous protein reporters in mice, focusing on the blastocyst model of development. Our first strategy makes use of a split fluorescent protein, mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest, was highly efficient in embryos, potentially obviating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. Our second strategy achieves in-frame knock-in of a relatively small protein tag, which provides high efficiency and higher sensitivity protein reporting. Together, these two approaches provide complementary advantages and enable broad downstream applications.},
}
@article {pmid34035409,
year = {2021},
author = {Georgiadis, C and Rasaiyaah, J and Gkazi, SA and Preece, R and Etuk, A and Christi, A and Qasim, W},
title = {Base-edited CAR T cells for combinational therapy against T cell malignancies.},
journal = {Leukemia},
volume = {35},
number = {12},
pages = {3466-3481},
pmid = {34035409},
issn = {1476-5551},
support = {/WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {Animals ; Antigens, CD7/chemistry/*genetics/metabolism ; CD3 Complex/antagonists &amp; inhibitors/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Editing ; Humans ; Immunotherapy, Adoptive/*methods ; Leukemia, T-Cell/immunology/pathology/*therapy ; Mice ; Mice, Inbred NOD ; Mice, SCID ; T-Lymphocytes/*immunology ; Xenograft Model Antitumor Assays ; },
abstract = {Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.},
}
@article {pmid34035211,
year = {2021},
author = {Meng, N and Grimm, D},
title = {Membrane-destabilizing ionizable phospholipids: Novel components for organ-selective mRNA delivery and CRISPR-Cas gene editing.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {206},
pmid = {34035211},
issn = {2059-3635},
mesh = {CRISPR-Cas Systems/genetics ; Cell Membrane/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; Humans ; Phospholipids/*genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics ; },
}
@article {pmid34035168,
year = {2021},
author = {Musharova, O and Medvedeva, S and Klimuk, E and Guzman, NM and Titova, D and Zgoda, V and Shiriaeva, A and Semenova, E and Severinov, K and Savitskaya, E},
title = {Prespacers formed during primed adaptation associate with the Cas1-Cas2 adaptation complex and the Cas3 interference nuclease-helicase.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34035168},
issn = {1091-6490},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA Helicases/*metabolism ; Endonucleases/*metabolism ; Escherichia coli/*metabolism ; Escherichia coli Proteins/*metabolism ; },
abstract = {For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease-helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1-Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1-Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.},
}
@article {pmid34035043,
year = {2021},
author = {Dong, SS and Zhu, DL and Zhou, XR and Rong, Y and Zeng, M and Chen, JB and Jiang, F and Tuo, XM and Feng, Z and Yang, TL and Guo, Y},
title = {An Intronic Risk SNP rs12454712 for Central Obesity Acts As an Allele-Specific Enhancer To Regulate BCL2 Expression.},
journal = {Diabetes},
volume = {70},
number = {8},
pages = {1679-1688},
doi = {10.2337/db20-1151},
pmid = {34035043},
issn = {1939-327X},
mesh = {Adipocytes/metabolism ; *Alleles ; Brain/metabolism ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Female ; *Gene Expression Regulation ; Humans ; *Introns ; Male ; Obesity, Abdominal/*genetics/metabolism ; *Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-bcl-2/*genetics ; },
abstract = {Genome-wide association studies (GWAS) have reproducibly associated the single nucleotide polymorphism (SNP) rs12454712 with waist-to-hip ratio adjusted for BMI (WHRadjBMI), but the functional role underlying this intronic variant is unknown. Integrative genomic and epigenomic analyses supported rs12454712 as a functional independent variant. We further demonstrated that rs12454712 acted as an allele-specific enhancer regulating expression of its located gene BCL2 by using dual-luciferase reporter assays and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Specifically, the rs12454712-C allele can bind transcription factor ZNF329, which efficiently elevates the enhancer activity and increases BCL2 expression. Knocking down Bcl2 in 3T3-L1 cells led to the downregulation of adipogenic differentiation marker genes and increased cell apoptosis. A significant negative correlation between BCL2 expression in subcutaneous adipose tissues and obesity was observed. Our findings illustrate the molecular mechanisms behind the intronic SNP rs12454712 for central obesity, which would be a potential and promising target for developing appropriate therapies.},
}
@article {pmid34034859,
year = {2021},
author = {Le Vasseur, M and Friedman, J and Jost, M and Xu, J and Yamada, J and Kampmann, M and Horlbeck, MA and Salemi, MR and Phinney, BS and Weissman, JS and Nunnari, J},
title = {Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {34034859},
issn = {2050-084X},
support = {K99 GM130964/GM/NIGMS NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; R35 GM141800/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; S10 OD021801/OD/NIH HHS/United States ; R00 HL133372/HL/NHLBI NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; R01 GM126081/GM/NIGMS NIH HHS/United States ; R37 GM097432/GM/NIGMS NIH HHS/United States ; K99 HL133372/HL/NHLBI NIH HHS/United States ; },
mesh = {Antimycin A/pharmacology ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electron Transport Complex III/antagonists &amp; inhibitors/genetics/*metabolism ; Endopeptidases/genetics/metabolism ; Genome-Wide Association Study ; Humans ; K562 Cells ; Mitochondria/drug effects/*enzymology/genetics ; Neoplasm Proteins/genetics/*metabolism ; Oxidative Phosphorylation ; Prohibitins ; Proteolysis ; Repressor Proteins/genetics/*metabolism ; },
abstract = {Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.},
}
@article {pmid34034027,
year = {2021},
author = {Wu, X and Tay, JK and Goh, CK and Chan, C and Lee, YH and Springs, SL and Wang, Y and Loh, KS and Lu, TK and Yu, H},
title = {Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids.},
journal = {Biomaterials},
volume = {274},
number = {},
pages = {120876},
doi = {10.1016/j.biomaterials.2021.120876},
pmid = {34034027},
issn = {1878-5905},
mesh = {*COVID-19 ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Epstein-Barr Virus Infections ; Herpesvirus 4, Human/genetics ; Humans ; *Nucleic Acids ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6-2027 copies/μL (R[2] value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA's versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients' serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5-2100 copies/μL (R[2] value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.},
}
@article {pmid34033873,
year = {2021},
author = {Hirakawa, Y},
title = {CLAVATA3, a plant peptide controlling stem cell fate in the meristem.},
journal = {Peptides},
volume = {142},
number = {},
pages = {170579},
doi = {10.1016/j.peptides.2021.170579},
pmid = {34033873},
issn = {1873-5169},
mesh = {Arabidopsis/*metabolism ; Arabidopsis Proteins/chemistry/*metabolism ; Meristem/*cytology/drug effects ; Peptide Fragments/*pharmacology ; Stem Cells/*cytology/drug effects ; },
abstract = {CLAVATA3 (CLV3) is a peptide signal initially identified in the analysis of clv mutants in the model plant Arabidopsis thaliana, as a regulator of meristem homeostasis and floral organ numbers. CLV3 homologs are widely conserved in land plants, collectively called CLV3/ESR-related (CLE) genes. A 12-amino acid CLE peptide with hydroxyproline residues was identified in Zinnia elegans cell culture system, in which cells secrete a CLE peptide called tracheary element differentiation factor (TDIF) into the culture medium. Mature CLV3 peptide is also a post-translationally modified short peptide containing additional triarabinosylation on a hydroxyproline residue. Genetic studies have revealed the involvement of leucin-rich repeat receptor-like kinases (LRR-RLKs) in CLV3 signaling, including CLV1/BAM-CIK, CLV2-CRN and RPK2, although the mechanisms of signal transduction and integration via crosstalk is still largely unknown. Recent studies on bryophyte model species provided a clue to understand evolution and ancestral function of CLV signaling in land plants. Fundamental understanding on CLV signaling provided an opportunity to optimize the crop yield traits using a novel breeding technology with CRISPR/Cas genome editing.},
}
@article {pmid34033692,
year = {2022},
author = {Kaligotla, VSA and Jasti, T and Kandra, P},
title = {CRISPR/Cas9 in cancer: An attempt to the present trends and future prospects.},
journal = {Biotechnology and applied biochemistry},
volume = {69},
number = {3},
pages = {1238-1251},
doi = {10.1002/bab.2200},
pmid = {34033692},
issn = {1470-8744},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy/methods ; Mutation ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer is the second leading cause of death globally. Series of sequential, repeated genetic changes and epigenetic modifications are leading to the formation of tumors. These tumors subsequently causing the infected cells to invade and transform their surrounding cells by metastasis are some hallmarks in cancer. Although tremendous efforts have been extended for structurally characterizing the numerous genomic mutations undergoing in cancer cells, there is a lack of information regarding the functions of many mutated genes. Clustered Regularly Interspaced Short Palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) has become a robust method for building changes in genome of many organisms. Recent reports have suggested that modification of CRISPR/Cas9 can provide plot form to probe the mechanisms in tumorigenesis and in cancer therapies. This review focuses on the historical perspectives of CRISPR/Cas9. The study highlights the applications and also role in cancer cell genome editing, which is helpful to understand the dynamics. Intense research in progress on mechanism of action of CRISPR/Cas9 has been reviewed and critically discussed. Further, relevant literature on animal models focusing on various approaches has been highlighted to emphasize the therapeutics of CRISPR/Cas9 with current trends and future challenges.},
}
@article {pmid34033106,
year = {2021},
author = {Roberts, CM and Ratner, ES},
title = {Two Plasmid-Based Systems for CRISPR/Cas9 Mediated Knockout of Target Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2255},
number = {},
pages = {213-232},
pmid = {34033106},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Gene Editing ; *Gene Knockout Techniques ; Humans ; Plasmids/*genetics ; },
abstract = {CRISPR/Cas9-based gene editing is a recent advance that allows for the knockout or alteration of target genes within mammalian cells. Many variations of the technique exist, but here we describe two systems of plasmid-based CRISPR gene knockout which together allow for the selective knockout of virtually any gene target. Compared with other CRISPR-based systems, these plasmids have the advantages of delivering all the necessary components in one plasmid, choice of multiple selectable markers, and choice of route of administration into target cells. In addition, potential off-target effects from one system (dependent upon selection of target gene) can be overcome through use of the second system. Strategies for optimizing the knockout process and selection of finished cell lines are also presented.},
}
@article {pmid34032267,
year = {2021},
author = {Kaiser, K and Jang, A and Kompanikova, P and Lun, MP and Prochazka, J and Machon, O and Dani, N and Prochazkova, M and Laurent, B and Gyllborg, D and van Amerongen, R and Fame, RM and Gupta, S and Wu, F and Barker, RA and Bukova, I and Sedlacek, R and Kozmik, Z and Arenas, E and Lehtinen, MK and Bryja, V},
title = {MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {10},
pages = {},
pmid = {34032267},
issn = {1477-9129},
support = {/DH_/Department of Health/United Kingdom ; U54 HD090255/HD/NICHD NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; R01 NS088566/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Brain/embryology ; CRISPR-Cas Systems/genetics ; Cell Line ; Choroid Plexus/*embryology ; Epithelial Cells/metabolism ; Epithelium/*metabolism ; Female ; Fourth Ventricle/*embryology ; HEK293 Cells ; Humans ; Mice ; Mice, Knockout ; Myeloid Ecotropic Viral Integration Site 1 Protein/*metabolism ; Promoter Regions, Genetic/genetics ; Receptor Tyrosine Kinase-like Orphan Receptors/metabolism ; Signal Transduction/physiology ; Wnt-5a Protein/genetics/*metabolism ; },
abstract = {The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.},
}
@article {pmid34031549,
year = {2021},
author = {Scholefield, J and Harrison, PT},
title = {Prime editing - an update on the field.},
journal = {Gene therapy},
volume = {28},
number = {7-8},
pages = {396-401},
pmid = {34031549},
issn = {1476-5462},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
}
@article {pmid34031411,
year = {2021},
author = {Dai, M and Yan, G and Wang, N and Daliah, G and Edick, AM and Poulet, S and Boudreault, J and Ali, S and Burgos, SA and Lebrun, JJ},
title = {In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3055},
pmid = {34031411},
issn = {2041-1723},
support = {8117//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Combined Modality Therapy/*methods ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; HEK293 Cells ; Hippo Signaling Pathway ; Humans ; Mechanistic Target of Rapamycin Complex 1 ; Mechanistic Target of Rapamycin Complex 2 ; Mice ; Protein Serine-Threonine Kinases/*genetics ; TOR Serine-Threonine Kinases/*genetics ; Triple Negative Breast Neoplasms/*genetics ; Verteporfin ; Xenograft Model Antitumor Assays ; },
abstract = {Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.},
}
@article {pmid34031406,
year = {2021},
author = {Wang, X and Fu, Y and Beatty, WL and Ma, M and Brown, A and Sibley, LD and Zhang, R},
title = {Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3065},
pmid = {34031406},
issn = {2041-1723},
support = {R01 AI034036/AI/NIAID NIH HHS/United States ; R01 GM141109/GM/NIGMS NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems ; *Cryoelectron Microscopy ; Host-Parasite Interactions/*physiology ; Humans ; Microtubule Proteins/metabolism/*ultrastructure ; Microtubule-Associated Proteins/chemistry/metabolism ; Microtubules/metabolism/*ultrastructure ; Paclitaxel/chemistry ; Toxoplasma/*metabolism ; Tubulin/chemistry/metabolism ; },
abstract = {In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.},
}
@article {pmid34031396,
year = {2021},
author = {Müller, I and Moroni, AS and Shlyueva, D and Sahadevan, S and Schoof, EM and Radzisheuskaya, A and Højfeldt, JW and Tatar, T and Koche, RP and Huang, C and Helin, K},
title = {MPP8 is essential for sustaining self-renewal of ground-state pluripotent stem cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3034},
pmid = {34031396},
issn = {2041-1723},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Proliferation ; DNA Methylation ; *Epigenesis, Genetic ; Gene Knock-In Techniques ; HEK293 Cells ; Histone-Lysine N-Methyltransferase ; Humans ; Long Interspersed Nucleotide Elements/genetics ; Mice ; Mouse Embryonic Stem Cells ; Phosphoproteins/*genetics/*metabolism ; Pluripotent Stem Cells/*metabolism ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together, our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.},
}
@article {pmid34031394,
year = {2021},
author = {Amit, I and Iancu, O and Levy-Jurgenson, A and Kurgan, G and McNeill, MS and Rettig, GR and Allen, D and Breier, D and Ben Haim, N and Wang, Y and Anavy, L and Hendel, A and Yakhini, Z},
title = {CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {3042},
pmid = {34031394},
issn = {2041-1723},
mesh = {Algorithms ; *CRISPR-Cas Systems ; Computational Biology/*methods ; DNA-Binding Proteins/genetics ; Gene Editing/*methods ; HEK293 Cells ; Homeodomain Proteins/genetics ; Humans ; Nuclear Proteins/genetics ; Software ; Transcription Factors/genetics ; },
abstract = {Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity.},
}
@article {pmid34031258,
year = {2021},
author = {Chen, J and Luo, J and Wang, Y and Gurav, AS and Li, M and Akbari, OS and Montell, C},
title = {Suppression of female fertility in Aedes aegypti with a CRISPR-targeted male-sterile mutation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {22},
pages = {},
pmid = {34031258},
issn = {1091-6490},
support = {DP1 AI124453/AI/NIAID NIH HHS/United States ; R56 AI153334/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/*genetics ; Animals ; CRISPR-Cas Systems ; Female ; Gene Editing ; Infertility, Female ; Infertility, Male/*genetics ; Male ; *Pest Control, Biological ; },
abstract = {Aedes aegypti spread devastating viruses such as dengue, which causes disease among 100 to 400 million people annually. A potential approach to control mosquito disease vectors is the sterile insect technique (SIT). The strategy involves repeated release of large numbers of sterile males, which reduces insect populations because the sterile males mate and thereby suppress the fertility of females that would otherwise mate with fertile males. While SIT has been successful in suppressing certain agricultural pests, it has been less effective in depressing populations of Ae. aegypti This limitation is in part because of the fitness effects resulting from mutagenizing the mosquitoes nonspecifically. Here, we introduced and characterized the impact on female fertility of an Ae. aegypti mutation that disrupts a gene that is specifically expressed in testes. We used CRISPR/Cas9 to generate a null mutation in the Ae. aegypti β2-tubulin (B2t) gene, which eliminates male fertility. When we allowed wild-type females to first mate with B2t mutant males, most of the females did not produce progeny even after being subsequently exposed to wild-type males. We also introduced B2t mutant and wild-type males simultaneously with wild-type females and found that a larger number of B2t mutant males relative to the wild-type males was effective in significantly suppressing female fertility. These results raise the possibility of employing B2t sterile males to improve the efficacy of SIT in suppressing populations of Ae. aegypti through repeated releases and thereby reduce the transmission of viruses by these invasive mosquitoes.},
}
@article {pmid34031210,
year = {2022},
author = {Lane, M and Slocum, M and Khokha, MK},
title = {Raising and Maintaining Xenopus tropicalis from Tadpole to Adult.},
journal = {Cold Spring Harbor protocols},
volume = {2022},
number = {4},
pages = {Pdb.prot106369},
doi = {10.1101/pdb.prot106369},
pmid = {34031210},
issn = {1559-6095},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Larva ; Xenopus/genetics ; },
abstract = {Xenopus tropicalis is a powerful model organism for cell and developmental biology research. Recently, precise gene-editing methods such as CRISPR-Cas9 have allowed facile creation of mutants. The ability to raise and maintain lines of wild-type and mutant animals through all life stages is thus critical for researchers using this model organism. The long fertile life (>8-10 yr) and relatively hardy nature of X. tropicalis makes this a straightforward process. Environmental parameters such as water temperature, pH, and conductivity often vary slightly among husbandry protocols. However, the stability of these variables is essential for rearing success. This protocol describes conditions to optimally raise and maintain X. tropicalis from embryos to adulthood.},
}
@article {pmid34030401,
year = {2021},
author = {Sarma, H and Islam, NF and Prasad, R and Prasad, MNV and Ma, LQ and Rinklebe, J},
title = {Enhancing phytoremediation of hazardous metal(loid)s using genome engineering CRISPR-Cas9 technology.},
journal = {Journal of hazardous materials},
volume = {414},
number = {},
pages = {125493},
doi = {10.1016/j.jhazmat.2021.125493},
pmid = {34030401},
issn = {1873-3336},
mesh = {Biodegradation, Environmental ; CRISPR-Cas Systems ; Humans ; *Metals, Heavy/analysis ; Soil ; *Soil Pollutants/analysis/toxicity ; Technology ; },
abstract = {Rapid and drastic changes in the global climate today have given a strong impetus to developing newer climate-resilient phytoremediation approaches. These methods are of great public and scientific importance given the urgency of this environmental crisis. Climate change has adverse effects on the growth, outputs, phenology, and overall productivity of plants. Contamination of soil with metal(loid)s is a major worldwide problem. Some metal(loids) are carcinogenic pollutants that have a long half-life and are non-degradable in the environment. There are many instances of the potential link between chronic heavy metal exposure and human disease. The adaptation of plants in the changing environment is, however, a major concern in phytoremediation practice. The creation of climate-resistant metal hyperaccumulation plants using molecular techniques could provide new opportunities to mitigate these problems. Consequently, advancements in molecular science would accelerate our knowledge of adaptive plant remediation/resistance and plant production in the context of global warming. Genome modification using artificial nucleases has the potential to enhance phytoremediation by modifying genomes for a sustainable future. This review focuses on biotechnology to boost climate change tolerant metallicolous plants and the future prospects of such technology, particularly the CRISPR-Cas9 genome editing system, for enhancing phytoremediation of hazardous pollutants.},
}
@article {pmid34029708,
year = {2021},
author = {Santhanam, P and Labbé, C and Fietto, LG and Bélanger, RR},
title = {A reassessment of flocculosin-mediated biocontrol activity of Pseudozyma flocculosa through CRISPR/Cas9 gene editing.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {153},
number = {},
pages = {103573},
doi = {10.1016/j.fgb.2021.103573},
pmid = {34029708},
issn = {1096-0937},
mesh = {*Antibiosis ; Ascomycota/*physiology ; Basidiomycota/genetics/*physiology ; CRISPR-Cas Systems ; Cellobiose/*analogs &amp; derivatives/biosynthesis/genetics/metabolism ; Gene Editing ; Glycolipids/biosynthesis/genetics/*metabolism ; Homologous Recombination ; Hordeum/microbiology ; Plant Diseases/microbiology ; },
abstract = {Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.},
}
@article {pmid34028208,
year = {2021},
author = {Blomeier, T and Fischbach, P and Koch, LA and Andres, J and Miñambres, M and Beyer, HM and Zurbriggen, MD},
title = {Blue Light-Operated CRISPR/Cas13b-Mediated mRNA Knockdown (Lockdown).},
journal = {Advanced biology},
volume = {5},
number = {5},
pages = {e2000307},
doi = {10.1002/adbi.202000307},
pmid = {34028208},
issn = {2701-0198},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Light ; *Optogenetics ; RNA ; RNA, Messenger/genetics ; },
abstract = {The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introduces optical control of RNA levels utilizing a blue light-dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence-specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene-expression and induce protein destabilization with blue light yields efficient triple-controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin-dependent kinase 1 (hCdk1) leads to blue light-induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.},
}
@article {pmid34027480,
year = {2021},
author = {van Gestel, J and Hawkins, JS and Todor, H and Gross, CA},
title = {Computational pipeline for designing guide RNAs for mismatch-CRISPRi.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100521},
pmid = {34027480},
issn = {2666-1667},
support = {R35 GM118061/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Pair Mismatch/*genetics ; CRISPR-Cas Systems/*genetics ; Computational Biology ; *Genetic Techniques ; Genome, Bacterial/genetics ; RNA, Guide/*genetics ; Transcription, Genetic/*genetics ; },
abstract = {CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020).},
}
@article {pmid34027476,
year = {2021},
author = {Sakurai, T and Shindo, T},
title = {Production of single- and multiple-gene-modified mice via maternal SpCas9-based gene editing.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100509},
pmid = {34027476},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Gene Editing ; Mice ; Mice, Transgenic ; *Zygote ; },
abstract = {Maternally and transiently accumulated SpCas9 (maternal SpCas9) in a zygote derived from a systemically SpCas9-expressing transgenic mouse strain was used to generate single- and multiple-gene-modified mice. Maternal SpCas9-based gene editing allows for high indel and knockin mutation efficiency, low mosaicism, increased pup delivery rate, and simultaneous induction of mutations at multiple loci in contrast to conventional CRISPR/SpCas9-based gene editing. For complete details on the use and execution of this protocol, please refer to Sakurai et al. (2020).},
}
@article {pmid34026427,
year = {2021},
author = {Tian, X and Wang, X and Cui, Z and Liu, J and Huang, X and Shi, C and Zhang, M and Liu, T and Du, X and Li, R and Huang, L and Gong, D and Tian, R and Cao, C and Jin, P and Zeng, Z and Pan, G and Xia, M and Zhang, H and Luo, B and Xie, Y and Li, X and Li, T and Wu, J and Zhang, Q and Chen, G and Hu, Z},
title = {A Fifteen-Gene Classifier to Predict Neoadjuvant Chemotherapy Responses in Patients with Stage IB to IIB Squamous Cervical Cancer.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {8},
number = {10},
pages = {2001978},
pmid = {34026427},
issn = {2198-3844},
mesh = {Antineoplastic Combined Chemotherapy Protocols/*administration &amp; dosage ; Biomarkers, Tumor/metabolism ; CRISPR-Cas Systems ; Carcinoma, Squamous Cell/drug therapy/*genetics/*metabolism/pathology ; Cell Line, Tumor ; Cisplatin/administration &amp; dosage ; Drug Resistance, Neoplasm ; Female ; High-Throughput Nucleotide Sequencing/methods ; Humans ; *Mutation ; Neoadjuvant Therapy ; Neoplasm Staging ; Paclitaxel/administration &amp; dosage ; Proteins/metabolism ; Sialyltransferases/metabolism ; Uterine Cervical Neoplasms/*drug therapy/*genetics/pathology ; },
abstract = {Neoadjuvant chemotherapy (NACT) remains an attractive alternative for controlling locally advanced cervical cancer. However, approximately 15-34% of women do not respond to induction therapy. To develop a risk stratification tool, 56 patients with stage IB-IIB cervical cancer are included in 2 research centers from the discovery cohort. Patient-specific somatic mutations led to NACT non-responsiveness are identified by whole-exome sequencing. Next, CRISPR/Cas9-based library screenings are performed based on these genes to confirm their biological contribution to drug resistance. A 15-gene classifier is developed by generalized linear regression analysis combined with the logistic regression model. In an independent validation cohort of 102 patients, the classifier showed good predictive ability with an area under the curve of 0.80 (95% confidence interval (CI), 0.69-0.91). Furthermore, the 15-gene classifier is significantly associated with patient responsiveness to NACT in both univariate (odds ratio, 10.8; 95% CI, 3.55-32.86; p = 2.8 × 10[-5]) and multivariate analysis (odds ratio, 17.34; 95% CI, 4.04-74.40; p = 1.23 × 10[-4]) in the validation set. In conclusion, the 15-gene classifier can accurately predict the clinical response to NACT before treatment, representing a promising approach for guiding the selection of appropriate treatment strategies for locally advanced cervical cancer.},
}
@article {pmid34024338,
year = {2021},
author = {Shi, H and Smits, JPH and van den Bogaard, EH and Brewer, MG},
title = {Research Techniques Made Simple: Delivery of the CRISPR/Cas9 Components into Epidermal Cells.},
journal = {The Journal of investigative dermatology},
volume = {141},
number = {6},
pages = {1375-1381.e1},
pmid = {34024338},
issn = {1523-1747},
support = {U01 AI152011/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; *Gene Transfer Techniques ; Genetic Vectors ; Humans ; Keratinocytes/pathology ; Mutation ; Primary Cell Culture ; RNA, Guide/genetics ; Skin Diseases/*genetics/pathology ; },
abstract = {CRISPR/Cas9 technology is a powerful tool used to alter the genetic landscape of various hosts. This has been exemplified by its success in the transgenic animal world where it has been utilized to develop novel mouse lines modeling numerous disease states. The technology has helped to develop both in vitro and in vivo systems that simulate diseases within the fields of epithelial biology, skin cancer biology, dermatology, and beyond. Importantly, the delivery of the single-guide RNA/Cas9 editing complex to the host cell is key for its success. In this paper, we discuss the various methods that have been utilized as delivery techniques for CRISPR/Cas9 components, the benefits and pitfalls of each, and how successful they have been at genetically modifying epidermal cells. In addition, we acknowledge recent advances in the field of dermatology that have harnessed these methods to better understand epidermal biology, identify potential therapeutic targets, or serve as novel methods to treat disease states.},
}
@article {pmid34024121,
year = {2021},
author = {Wang, F and Chen, Y and Zou, CJ and Luo, R and Yang, T},
title = {Mutagenesis of the Cleavage Site of Pro Renin Receptor Abrogates Angiotensin II-Induced Hypertension in Mice.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {78},
number = {1},
pages = {115-127},
pmid = {34024121},
issn = {1524-4563},
support = {K99 HL155850/HL/NHLBI NIH HHS/United States ; R01 DK104072/DK/NIDDK NIH HHS/United States ; R01 HL135851/HL/NHLBI NIH HHS/United States ; R01 HL139689/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiotensin II ; Animals ; Base Sequence ; Binding Sites/genetics ; Blood Pressure/drug effects/genetics/physiology ; CRISPR-Cas Systems ; Cells, Cultured ; Hypertension/chemically induced/genetics/*physiopathology ; Mice, Knockout ; Mutagenesis ; Protein Precursors/genetics/*metabolism ; Receptors, Cell Surface/genetics/*metabolism ; Renin/genetics/*metabolism ; Renin-Angiotensin System/drug effects/genetics/physiology ; },
abstract = {[Figure: see text].},
}
@article {pmid34023780,
year = {2021},
author = {Homma, Y and Fukuda, M},
title = {Knockout analysis of Rab6 effector proteins revealed the role of VPS52 in the secretory pathway.},
journal = {Biochemical and biophysical research communications},
volume = {561},
number = {},
pages = {151-157},
doi = {10.1016/j.bbrc.2021.05.009},
pmid = {34023780},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Dogs ; Gene Knockdown Techniques/*methods ; Golgi Apparatus ; Humans ; Intracellular Membranes ; Lysosomes/*metabolism ; Protein Transport ; Secretory Pathway/*physiology ; Vesicular Transport Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; rab GTP-Binding Proteins/*metabolism ; },
abstract = {Rab small GTPases regulate intracellular membrane trafficking by interacting with specific binding proteins called Rab effectors. Although Rab6 is implicated in basement membrane formation and secretory cargo trafficking, its precise regulatory mechanisms have remained largely unknown. In the present study we established five knockout cell lines for candidate Rab6 effectors and discovered that knockout of VPS52, a subunit of the GARP complex, resulted in attenuated secretion and lysosomal accumulation of secretory cargos, the same as Rab6-knockout does. We also evaluated the functional importance of the previously uncharacterized C-terminal region of VPS52 for restoring these phenotypes, as well as for the sorting of lysosomal proteins. Our findings suggest that VPS52 is an effector protein that is responsible for the Rab6-dependent secretory cargo trafficking.},
}
@article {pmid34023776,
year = {2021},
author = {Kang, JY and Park, JW and Hwang, Y and Hahm, JY and Park, J and Park, KS and Seo, SB},
title = {The H3K4 methyltransferase SETD1A is required for proliferation of non-small cell lung cancer cells by promoting S-phase progression.},
journal = {Biochemical and biophysical research communications},
volume = {561},
number = {},
pages = {120-127},
doi = {10.1016/j.bbrc.2021.05.026},
pmid = {34023776},
issn = {1090-2104},
mesh = {CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*enzymology/genetics/metabolism/*pathology ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Computational Biology/methods ; DNA Replication ; Databases, Genetic ; Gene Expression Regulation, Neoplastic ; Histone-Lysine N-Methyltransferase/antagonists &amp; inhibitors/genetics/*metabolism ; Humans ; Lung Neoplasms/*enzymology/genetics/metabolism/*pathology ; Protein Serine-Threonine Kinases/metabolism ; S Phase ; },
abstract = {Epigenetic dysregulation has been strongly implicated in carcinogenesis and is one of the mechanisms that contribute to the development of lung cancer. Using genome-wide CRISPR/Cas9 library screening, we showed SET domain-containing protein 1A (SETD1A) is an essential epigenetic modifier of the proliferation of NSCLC H1299 cells. Depletion of SETD1A strikingly inhibited the proliferation of NSCLC cells. IHC staining and bioinformatics showed that SETD1A is upregulated in lung cancer. Kaplan-Meier survival analysis indicated that high expression of SETD1A is associated with poor prognosis of patients with NSCLC. We revealed that loss of SETD1A inhibits DNA replication and induces replication stress accompanied by impaired fork progression. In addition, transcription of CDC7 and TOP1, which are involved in replication origin activation and fork progression, respectively, was significantly reduced by knockdown of SETD1A. Taken together, these findings demonstrated SETD1A is a critical epigenetic modifier of NSCLC cell proliferation by promoting the transcription of a subset of DNA replication-associated genes.},
}
@article {pmid34023220,
year = {2021},
author = {Okano, K and Sato, Y and Hizume, T and Honda, K},
title = {Genome editing by miniature CRISPR/Cas12f1 enzyme in Escherichia coli.},
journal = {Journal of bioscience and bioengineering},
volume = {132},
number = {2},
pages = {120-124},
doi = {10.1016/j.jbiosc.2021.04.009},
pmid = {34023220},
issn = {1347-4421},
mesh = {CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *Gene Editing ; Streptococcus pyogenes/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%-100%) than the CRISPR/SpCas9 system (50%-79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.},
}
@article {pmid34022616,
year = {2021},
author = {Rani, A and Ravindran, VB and Surapaneni, A and Mantri, N and Ball, AS},
title = {Review: Trends in point-of-care diagnosis for Escherichia coli O157:H7 in food and water.},
journal = {International journal of food microbiology},
volume = {349},
number = {},
pages = {109233},
doi = {10.1016/j.ijfoodmicro.2021.109233},
pmid = {34022616},
issn = {1879-3460},
mesh = {*Bacteriological Techniques ; Biosensing Techniques ; CRISPR-Cas Systems ; Escherichia coli Infections/microbiology/prevention &amp; control ; Escherichia coli O157/*isolation &amp; purification ; *Food Microbiology ; Humans ; *Point-of-Care Systems ; Polymerase Chain Reaction ; *Water Microbiology ; },
abstract = {Escherichia coli O157:H7, a Shiga-producing E. coli is a major pathogenic E. coli strain which since the early 1980s has become a crucial food and water-borne pathogen. Several management strategies can be applied to control the spread of infection; however early diagnosis represents the optimum preventive strategy to minimize the infection. Therefore, it is crucial to detect this pathogen in a fast and efficient manner in order to reduce the morbidity and mortality. Currently used gold standard tests rely on culture and pre-enrichment of E. coli O157:H7 from the contaminated source; they are time consuming and laborious. Molecular methods such as polymerase chain reaction are sensitive; however, they require expensive instrumentation. Therefore, there is a requirement for Accurate, Sensitive, Specific, User friendly, Rapid, Equipment free and Deliverable (ASSURED) detection methods for use in the laboratory and in the field. Emerging technologies such as isothermal amplification methods, biosensors, surface enhanced Raman Spectroscopy, paper-based diagnostics and smartphone-based digital methods are recognized as new approaches in the field of E. coli O157:H7 diagnostics and are discussed in this review. Mobile PCR and CRISPR-Cas diagnostic platforms have been identified as new tools in E. coli O157:H7 POC diagnostics with the potential for implementation by industry. This review describes advances and progress in the field of E. coli O157:H7 diagnosis in the context of food and water industry. The focus is on emerging high throughput point-of-care (POC) E. coli O157:H7 diagnostics and the requirement for the transformation to service routine diagnostics in the food and water industry.},
}
@article {pmid34021472,
year = {2021},
author = {Shukla, A and Jani, N and Polra, M and Kamath, A and Patel, D},
title = {CRISPR: The Multidrug Resistance Endgame?.},
journal = {Molecular biotechnology},
volume = {63},
number = {8},
pages = {676-685},
pmid = {34021472},
issn = {1559-0305},
mesh = {Animals ; *CRISPR-Cas Systems ; Drug Delivery Systems ; Drug Resistance, Multiple/*genetics ; Gene Editing ; Genes, MDR ; Humans ; },
abstract = {The flexibility of microbes to undergo or adapt to the changes in their physiology and genotypical traits has enabled the microbes acquiring resistance to latest or recently discovered drugs which have consequently led to the menace of multidrug resistance (MDR). There is a surge in the discovery of novel antibiotics to counter the rising MDR phenomena, and in such a quest, for investigating an efficient alternative mechanism or compound to combat MDR, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has piqued the interests of the researchers across the globe. CRISPR-Cas9 technology is a genome-editing tool with successful widespread applications in cell lines, plants, animals, and even in human clinical trials, and it is seriously being considered as a potential candidate for countering MDR. This review encompasses the broad scope of CRISPR-Cas9 along with its various variations, underlying principles, mechanisms, as well as applications. Furthermore, the implications of recent advancements in various disciplines are highlighted to enhance the applicability of this technique. Consequently, its research gaps and challenges are also identified so that they can be addressed in the possible future thereby further expanding the lore of CRISPR-Cas9 technique.},
}
@article {pmid34021387,
year = {2021},
author = {Guan, Y and Cui, Y and Qu, X and Jing, K},
title = {Safety and robustness aspects analysis of Lactobacillus delbrueckii ssp. bulgaricus LDB-C1 based on the genome analysis and biological tests.},
journal = {Archives of microbiology},
volume = {203},
number = {7},
pages = {3955-3964},
pmid = {34021387},
issn = {1432-072X},
mesh = {Cultured Milk Products/microbiology ; Fermentation ; *Genome, Bacterial/genetics ; Genomic Instability ; *Lactobacillus delbrueckii/genetics/metabolism ; Streptococcus thermophilus/genetics/metabolism ; Yogurt/microbiology ; },
abstract = {Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a microaerophylic anaerobe, which is widely used in the production of yogurt, cheese, and other fermented dairy products. L. bulgaricus and its partner Streptococcus thermophilus were used as starter cultures of yogurt in the world for thousands of years. In our previous study, L. bulgaricus LDB-C1 was obtained from traditional fermented milk, and possessed some characteristics like high exopolysaccharide yield and good fermentation performance. The analysis of its CRISPR-Cas system, antibiotic resistance, virulence factors, and mobile elements, was performed to reveal the stability of the strain LDB-C1. It was found that LDB-C1 contains a plenty of spacers in the CRISPR region, indicating it might have better performance against the infection of phages and plasmids. Furthermore, the acquired or transmittable antibiotic resistance/virulence factor genes were absent in the tested L. bulgaricus strain LDB-C1.},
}
@article {pmid34021238,
year = {2021},
author = {Thomas, LW and Esposito, C and Morgan, RE and Price, S and Young, J and Williams, SP and Maddalena, LA and McDermott, U and Ashcroft, M},
title = {Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {615},
pmid = {34021238},
issn = {2399-3642},
support = {RG93172/WT_/Wellcome Trust/United Kingdom ; MR/K002201/2/MRC_/Medical Research Council/United Kingdom ; RG91141/CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; C7358/A19442/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Cell Proliferation ; *Genes, Mitochondrial ; *Genome, Mitochondrial ; Glycolysis ; Humans ; Hypoxia/*physiopathology ; Mitochondria/*genetics/pathology ; Neoplasms/genetics/*pathology ; Oxidative Phosphorylation ; Tumor Cells, Cultured ; },
abstract = {Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.},
}
@article {pmid34020727,
year = {2021},
author = {Jones, CE and Tan, WS and Grey, F and Hughes, DJ},
title = {Discovering antiviral restriction factors and pathways using genetic screens.},
journal = {The Journal of general virology},
volume = {102},
number = {5},
pages = {},
pmid = {34020727},
issn = {1465-2099},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/N001796/1/MRC_/Medical Research Council/United Kingdom ; MR/N001796/1/MRC_/Medical Research Council/United Kingdom ; BBS/E/D/20002172/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Antiviral Agents/*immunology ; CRISPR-Cas Systems ; Gene Expression ; *Genetic Testing ; Genome-Wide Association Study ; Humans ; Immunity, Innate/genetics/immunology ; Interferons/genetics/immunology ; RNA Interference ; Signal Transduction/genetics/*immunology ; Virus Replication/immunology ; },
abstract = {Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and since the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response result in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9), ISG expression libraries and RNA interference (RNAi) technologies.},
}
@article {pmid34019786,
year = {2021},
author = {Caron, P and Pobega, E and Polo, SE},
title = {A molecular Rosetta Stone to decipher the impact of chromatin features on the repair of Cas9-mediated DNA double-strand breaks.},
journal = {Molecular cell},
volume = {81},
number = {10},
pages = {2059-2060},
doi = {10.1016/j.molcel.2021.04.024},
pmid = {34019786},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; *Chromatin/genetics ; DNA ; *DNA Breaks, Double-Stranded ; DNA Repair ; Humans ; },
abstract = {Using a barcoded reporter introduced within a thousand different chromatin locations in human cells, (Schep et al., 2021) characterize repair outcomes of Cas9-induced DNA double-strand breaks (DSBs) and the relative use of DSB repair pathways depending on the local chromatin context.},
}
@article {pmid34019632,
year = {2021},
author = {Sun, F and Fang, Y and Zhang, MM and Zhang, RJ and Wu, FY and Yang, RM and Tu, PH and Dong, M and Zhao, SX and Song, HD},
title = {Genetic Manipulation on Zebrafish duox Recapitulate the Clinical Manifestations of Congenital Hypothyroidism.},
journal = {Endocrinology},
volume = {162},
number = {8},
pages = {},
doi = {10.1210/endocr/bqab101},
pmid = {34019632},
issn = {1945-7170},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; *Congenital Hypothyroidism ; *Disease Models, Animal ; Female ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Male ; NADPH Oxidases/*genetics ; Phenotype ; Thyroxine ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {Congenital hypothyroidism (CH) is a highly prevalent but treatable neonatal endocrine disorder. Thyroid dyshormonogenesis is the main cause of congenital hypothyroidism in Chinese CH patients, and DUOX2 is the most frequent mutated gene involved in H2O2 production. In humans, the primary sources for H2O2 production are DUOX1 and DUOX2, while in zebrafish there is only a single orthologue for DUOX1 and DUOX2. In this study, duox mutant zebrafish were generated through knockdown duox by morpholino or knockout duox by CRISPR Cas9. The associated phenotypes were investigated and rescued by thyroxine (T4) treatment. Mutant zebrafish displayed hypothyroid phenotypes including growth retardation, goiter and, infertility. Homozygous mutants in adults also displayed extrathyroidal abnormal phenotypes, including lacking barbels, pigmentation defects, erythema in the opercular region, ragged fins, and delayed scales. All these abnormal phenotypes can be rescued by 10 nM T4 treatment. Strikingly, the fertility of zebrafish was dependent on thyroid hormone; T4 treatment should be continued and cannot be stopped over 2 weeks in hypothyroid zebrafish in order to achieve fertility. Thyroid hormones played a role in the developing and maturing of reproductive cells. Our work indicated that duox mutant zebrafish may provide a model for human congenital hypothyroidism.},
}
@article {pmid34019391,
year = {2021},
author = {Wang, X and Zhou, S and Chu, C and Yang, M and Huo, D and Hou, C},
title = {Dual Methylation-Sensitive Restriction Endonucleases Coupling with an RPA-Assisted CRISPR/Cas13a System (DESCS) for Highly Sensitive Analysis of DNA Methylation and Its Application for Point-of-Care Detection.},
journal = {ACS sensors},
volume = {6},
number = {6},
pages = {2419-2428},
doi = {10.1021/acssensors.1c00674},
pmid = {34019391},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Methylation ; DNA Restriction Enzymes ; Point-of-Care Systems ; *Recombinases ; },
abstract = {High-performance detection of DNA methylation possesses great significance for the diagnosis and therapy of cancer. Herein, for the first time, we present a digestion strategy based on dual methylation-sensitive restriction endonucleases coupling with a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system (DESCS) for accurate and sensitive determination of site-specific DNA methylation. This dual methylation-sensitive restriction endonuclease system selectively digests the unmethylated target but exhibits no response to methylated DNA. Therefore, the intact methylated DNA target triggers the RPA reaction for rapid signal amplification. In contrast, the digested unmethylated target initiates no RPA reaction. RPA products with a T7 promoter can execute the T7 transcription in the presence of T7 RNA polymerase to generate a large number of single-stranded RNA (ssRNA). This ssRNA can be recognized by CRISPR/Cas13a to induce the ssRNase activity of Cas13a, showing the indiscriminate cleavage of the collateral FQ reporter to release the fluorescence signal. With such a design, by combining the unique features of dual methylation-sensitive restriction endonucleases with RPA-assisted CRISPR/Cas13a, the DESCS system not only presents the rapid and powerful signal amplification for the determination of methylated DNA with ultrahigh sensitivity but also effectively eliminates the false positive influences from incomplete digestion of the unmethylated target. More importantly, 0.01% methylation level can be effectively distinguished with the existence of excess unmethylated DNA. In addition, the DESCS assay is integrated into the lateral flow biosensor (LFB) for the point-of-care determination of DNA methylation. In view of the superiorities in high sensitivity, outstanding selectivity, and ease of operation, the DESCS system will provide a reliable assay for site-specific analysis of methylation.},
}
@article {pmid34018332,
year = {2021},
author = {Rahman, M and Billmann, M and Costanzo, M and Aregger, M and Tong, AHY and Chan, K and Ward, HN and Brown, KR and Andrews, BJ and Boone, C and Moffat, J and Myers, CL},
title = {A method for benchmarking genetic screens reveals a predominant mitochondrial bias.},
journal = {Molecular systems biology},
volume = {17},
number = {5},
pages = {e10013},
pmid = {34018332},
issn = {1744-4292},
support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; R21 CA235352/CA/NCI NIH HHS/United States ; MOP-142375//CIHR/Canada ; },
mesh = {Algorithms ; Benchmarking ; Bias ; CRISPR-Cas Systems ; Cell Line ; *Gene Regulatory Networks ; Genetic Testing/*methods ; HEK293 Cells ; Humans ; Mitochondria/*genetics ; Systems Biology/*methods ; },
abstract = {We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.},
}
@article {pmid34018325,
year = {2021},
author = {Li, N and Wang, M and Yu, S and Zhou, J},
title = {Optimization of CRISPR-Cas9 through promoter replacement and efficient production of L-homoserine in Corynebacterium glutamicum.},
journal = {Biotechnology journal},
volume = {16},
number = {8},
pages = {e2100093},
doi = {10.1002/biot.202100093},
pmid = {34018325},
issn = {1860-7314},
mesh = {CRISPR-Cas Systems/genetics ; *Corynebacterium glutamicum/genetics ; Gene Editing ; Homoserine ; Plasmids/genetics ; },
abstract = {BACKGROUND: Corynebacterium glutamicum is an important chassis for industrial applications. The low efficiency of commonly used genome editing methods for C. glutamicum limits the rapid multiple engineering of the bacterium.<br><br>In this study, chromosome-borne expression of cas9 and recET from Escherichia coli K12-MG1655 was achieved to avoid toxicity to the strain, increase the probability of homologous recombination, and reduce loss of viability caused by double-strand breaks. Constitutive strong promoters, such as P45 , Ptrc , and PH36 , were used to replace PglyA and to expand the application of the CRISPR-Cas9 system. By using this system, a C. glutamicum strain producing L-homoserine to 22.1&#xa0;g per L in a 5-L bioreactor after 96 h was obtained.<br><br>CONCLUSIONS AND IMPLICATIONS: Through the application of visualized fluorescent protein, the process of plasmid curing was optimized, obtain a continuous and rapid CRISPR-Cas9 genome editing system. The method described here could be useful to construct C. glutamicum mutant rapidly.},
}
@article {pmid34017121,
year = {2021},
author = {Cheung, P and Schaffert, S and Chang, SE and Dvorak, M and Donato, M and Macaubas, C and Foecke, MH and Li, TM and Zhang, L and Coan, JP and Schulert, GS and Grom, AA and Henderson, LA and Nigrovic, PA and Elias, JE and Gozani, O and Mellins, ED and Khatri, P and Utz, PJ and Kuo, AJ},
title = {Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.},
journal = {Nature immunology},
volume = {22},
number = {6},
pages = {711-722},
pmid = {34017121},
issn = {1529-2916},
support = {K08 AR073339/AR/NIAMS NIH HHS/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; R01 AI125197/AI/NIAID NIH HHS/United States ; R01 AR059049/AR/NIAMS NIH HHS/United States ; U19 AI110491/AI/NIAID NIH HHS/United States ; P30 AR070549/AR/NIAMS NIH HHS/United States ; K08 AR072075/AR/NIAMS NIH HHS/United States ; R01 AR061297/AR/NIAMS NIH HHS/United States ; R01 AR073201/AR/NIAMS NIH HHS/United States ; U19 AI057229/AI/NIAID NIH HHS/United States ; P30 AR070253/AR/NIAMS NIH HHS/United States ; R35 GM139569/GM/NIGMS NIH HHS/United States ; U19 AI109662/AI/NIAID NIH HHS/United States ; },
mesh = {Adolescent ; Arthritis, Juvenile/blood/genetics/*immunology ; CRISPR-Cas Systems/genetics ; Cathepsin G/genetics/metabolism ; Cell Differentiation/genetics/*immunology ; Cell Nucleus/metabolism ; Child ; Child, Preschool ; Chromatin/metabolism ; Enzyme Assays ; Epigenesis, Genetic/*immunology ; Epigenomics ; Female ; Gene Knockout Techniques ; Histones/*metabolism ; Humans ; Jurkat Cells ; Leukocyte Elastase/genetics/metabolism ; Leukocytes, Mononuclear/immunology/*metabolism ; Macrophages/*immunology/metabolism ; Male ; Myeloblastin/genetics/metabolism ; Primary Cell Culture ; Proteolysis ; RNA-Seq ; Recombinant Proteins/genetics/metabolism ; THP-1 Cells ; Young Adult ; },
abstract = {Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.},
}
@article {pmid34017003,
year = {2021},
author = {Feng, X and López Del Amo, V and Mameli, E and Lee, M and Bishop, AL and Perrimon, N and Gantz, VM},
title = {Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2960},
pmid = {34017003},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Culex/*genetics ; Drosophila melanogaster/genetics ; Female ; Gene Drive Technology/*methods ; Insecticide Resistance ; Male ; Mosquito Control/*methods ; Mosquito Vectors/*genetics ; Mutagenesis, Site-Directed/methods ; RNA, Guide/genetics ; },
abstract = {Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.},
}
@article {pmid34016988,
year = {2021},
author = {Pierce, SE and Granja, JM and Greenleaf, WJ},
title = {High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2969},
pmid = {34016988},
issn = {2041-1723},
support = {RM1 HG007735/HG/NHGRI NIH HHS/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; U19 AI057266/AI/NIAID NIH HHS/United States ; P50 HG007735/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; UM1 HG009442/HG/NHGRI NIH HHS/United States ; },
mesh = {Binding Sites/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Chromatin/*metabolism ; *Chromatin Immunoprecipitation Sequencing ; Epigenesis, Genetic ; Epigenomics/methods ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Regulatory Networks ; High-Throughput Screening Assays/*methods ; Humans ; Neoplasms/*genetics ; RNA, Guide/genetics ; Single-Cell Analysis/methods ; Transcription Factors/metabolism ; },
abstract = {Chromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.},
}
@article {pmid34016945,
year = {2021},
author = {He, AT and Liu, J and Li, F and Yang, BB},
title = {Targeting circular RNAs as a therapeutic approach: current strategies and challenges.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {185},
pmid = {34016945},
issn = {2059-3635},
support = {PJT-153105//CIHR/Canada ; PJT-155962//CIHR/Canada ; PJT-166107//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; *Gene Knockdown Techniques ; *Genetic Therapy ; Humans ; *RNA, Circular/biosynthesis/genetics ; },
abstract = {Significant progress has been made in circular RNA (circRNA) research in recent years. Increasing evidence suggests that circRNAs play important roles in many cellular processes, and their dysregulation is implicated in the pathogenesis of various diseases. CircRNAs are highly stable and usually expressed in a tissue- or cell type-specific manner. Therefore, they are currently being explored as potential therapeutic targets. Gain-of-function and loss-of-function approaches are typically performed using circRNA expression plasmids and RNA interference-based strategies, respectively. These strategies have limitations that can be mitigated using nanoparticle and exosome delivery systems. Furthermore, recent developments show that the cre-lox system can be used to knockdown circRNAs in a cell-specific manner. While still in the early stages of development, the CRISPR/Cas13 system has shown promise in knocking down circRNAs with high specificity and efficiency. In this review, we describe circRNA properties and functions and highlight their significance in disease. We summarize strategies that can be used to overexpress or knockdown circRNAs as a therapeutic approach. Lastly, we discuss major challenges and propose future directions for the development of circRNA-based therapeutics.},
}
@article {pmid34015926,
year = {2021},
author = {Gong, G and Zhang, Y and Wang, Z and Liu, L and Shi, S and Siewers, V and Yuan, Q and Nielsen, J and Zhang, X and Liu, Z},
title = {GTR 2.0: gRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1328-1337},
doi = {10.1021/acssynbio.0c00560},
pmid = {34015926},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Engineering/methods ; Microorganisms, Genetically-Modified ; Mutagenesis, Site-Directed/methods ; Mutation ; Nucleotides/*genetics ; RNA, Guide/*genetics ; RNA, Transfer/*genetics ; Saccharomyces cerevisiae/*genetics ; Synthetic Biology/methods ; },
abstract = {Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.},
}
@article {pmid34015913,
year = {2021},
author = {Choi, YN and Shin, YR and Park, JM and Lee, JW},
title = {Cell-Free Transcription-Coupled CRISPR/Cas12a Assay for Prototyping Cyanobacterial Promoters.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1300-1307},
doi = {10.1021/acssynbio.1c00148},
pmid = {34015913},
issn = {2161-5063},
mesh = {Biofuels ; *CRISPR-Cas Systems ; Cell-Free System ; Cyanobacteria/*genetics ; Gene Editing/*methods ; High-Throughput Screening Assays/methods ; Metabolic Engineering/*methods ; Microorganisms, Genetically-Modified ; Mutation ; *Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction/methods ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Transcription, Genetic/*genetics ; },
abstract = {Cyanobacteria are promising microbial hosts for the production of diverse biofuels and biochemicals. However, compared to other model microbial hosts such as Escherichia coli and yeast, it takes a long time to genetically modify cyanobacteria. One way to efficiently engineer cyanobacteria while minimizing genetic engineering would be to develop a fast, high-throughput prototyping tool for cyanobacteria. In this study, we developed a CRISPR/Cas12a-based assay coupled with cyanobacteria cell-free systems to rapidly prototype promoter characteristics. Using this newly developed assay, we demonstrated cyanobacteria cell-free transcription for the first time and confirmed a positive correlation between the in vitro and in vivo transcription performance. Furthermore, we generated a synthetic promoter library and evaluated the characteristics of promoter subregions by using the assay. Varied promoter strength derived from random mutations were rapidly and effectively measured in a high-throughput way. We believe that this study offers an easily applicable and rapid prototyping platform to characterize promoters for cyanobacterial engineering.},
}
@article {pmid34013213,
year = {2021},
author = {Sangaré, LO and Wang, Y and Arranz-Solís, D and Saeij, JPJ},
title = {CRISPR screen to determine the in vivo fitness of Toxoplasma genes.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100520},
pmid = {34013213},
issn = {2666-1667},
support = {R01 AI080621/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Female ; *Genetic Techniques ; Loss of Function Mutation/genetics ; Mice ; *Toxoplasma/genetics/pathogenicity/physiology ; Toxoplasmosis/parasitology ; Virulence/*genetics ; },
abstract = {The virulence of eukaryotic parasites like Toxoplasma gondii depends on their capacity to escape from the host immune response and disseminate throughout the host organism. However, Toxoplasma gene products essential for its in vivo pathogenesis remain uncharacterized. Here, we present the complete workflow of a CRISPR-Cas9 in vivo loss-of-function screen to identify Toxoplasma fitness-conferring genes. This protocol can be used to uncover gene products that play a role in Toxoplasma immune evasion, nutrient acquisition, dissemination, and tissue colonization. For complete details on the use and execution of this protocol, please refer to Sangaré et al. (2019).},
}
@article {pmid34012094,
year = {2021},
author = {Rothgangl, T and Dennis, MK and Lin, PJC and Oka, R and Witzigmann, D and Villiger, L and Qi, W and Hruzova, M and Kissling, L and Lenggenhager, D and Borrelli, C and Egli, S and Frey, N and Bakker, N and Walker, JA and Kadina, AP and Victorov, DV and Pacesa, M and Kreutzer, S and Kontarakis, Z and Moor, A and Jinek, M and Weissman, D and Stoffel, M and van Boxtel, R and Holden, K and Pardi, N and Thöny, B and Häberle, J and Tam, YK and Semple, SC and Schwank, G},
title = {In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.},
journal = {Nature biotechnology},
volume = {39},
number = {8},
pages = {949-957},
pmid = {34012094},
issn = {1546-1696},
support = {R01 HL134839/HL/NHLBI NIH HHS/United States ; R01 AI124429/AI/NIAID NIH HHS/United States ; U19 AI142596/AI/NIAID NIH HHS/United States ; },
mesh = {*Adenine ; Animals ; *Cholesterol, LDL/blood/genetics ; Gene Editing/*methods ; Liver/metabolism ; Macaca ; Male ; Mice ; Mice, Inbred C57BL ; Proprotein Convertase 9/*genetics ; RNA, Guide/genetics ; },
abstract = {Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.},
}
@article {pmid34012082,
year = {2021},
author = {Musunuru, K and Chadwick, AC and Mizoguchi, T and Garcia, SP and DeNizio, JE and Reiss, CW and Wang, K and Iyer, S and Dutta, C and Clendaniel, V and Amaonye, M and Beach, A and Berth, K and Biswas, S and Braun, MC and Chen, HM and Colace, TV and Ganey, JD and Gangopadhyay, SA and Garrity, R and Kasiewicz, LN and Lavoie, J and Madsen, JA and Matsumoto, Y and Mazzola, AM and Nasrullah, YS and Nneji, J and Ren, H and Sanjeev, A and Shay, M and Stahley, MR and Fan, SHY and Tam, YK and Gaudelli, NM and Ciaramella, G and Stolz, LE and Malyala, P and Cheng, CJ and Rajeev, KG and Rohde, E and Bellinger, AM and Kathiresan, S},
title = {In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.},
journal = {Nature},
volume = {593},
number = {7859},
pages = {429-434},
pmid = {34012082},
issn = {1476-4687},
mesh = {Adenine/metabolism ; Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Cholesterol, LDL/*blood ; Female ; *Gene Editing ; Hepatocytes/metabolism ; Humans ; Liver/enzymology ; Loss of Function Mutation ; Macaca fascicularis/blood/genetics ; Male ; Mice ; Mice, Inbred C57BL ; *Models, Animal ; Mutagenesis, Site-Directed ; Proprotein Convertase 9/blood/*genetics/metabolism ; Time Factors ; },
abstract = {Gene-editing technologies, which include the CRISPR-Cas nucleases[1-3] and CRISPR base editors[4,5], have the potential to permanently modify disease-causing genes in patients[6]. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide[7]), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.},
}
@article {pmid34011687,
year = {2021},
author = {Evans, BA and Bernstein, DA},
title = {SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome.},
journal = {mSphere},
volume = {6},
number = {3},
pages = {},
pmid = {34011687},
issn = {2379-5042},
support = {R15 AI130950/AI/NIAID NIH HHS/United States ; },
mesh = {B30.2-SPRY Domain/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Candida albicans/*genetics/pathogenicity ; Gene Editing/*methods ; *Genome, Fungal ; Humans ; Mutation ; },
abstract = {Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicansADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences.IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicansADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.},
}
@article {pmid34011685,
year = {2021},
author = {Mosterd, C and Moineau, S},
title = {Primed CRISPR-Cas Adaptation and Impaired Phage Adsorption in Streptococcus mutans.},
journal = {mSphere},
volume = {6},
number = {3},
pages = {},
pmid = {34011685},
issn = {2379-5042},
mesh = {Adaptation, Physiological ; Adsorption ; Bacteriophages/*genetics/pathogenicity ; CRISPR-Cas Systems/*genetics ; DNA, Intergenic/*genetics ; Genome, Viral ; Phenotype ; Streptococcus mutans/*genetics/*virology ; Virus Attachment ; },
abstract = {Streptococcus mutans strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5' end of the spacer, leaving the seed sequence at the 3' end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect S. mutans P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain.IMPORTANCE Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of Streptococcus mutans P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.},
}
@article {pmid34011551,
year = {2021},
author = {Walter, M and Perrone, R and Verdin, E},
title = {Targeting Conserved Sequences Circumvents the Evolution of Resistance in a Viral Gene Drive against Human Cytomegalovirus.},
journal = {Journal of virology},
volume = {95},
number = {15},
pages = {e0080221},
pmid = {34011551},
issn = {1098-5514},
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; Conserved Sequence/*genetics ; Cytomegalovirus/*genetics/growth &amp; development ; Cytomegalovirus Infections/prevention &amp; control/therapy ; Defective Viruses/genetics ; Drug Resistance, Viral/genetics ; Gene Drive Technology/*methods ; Genes, Viral/genetics ; Humans ; Sequence Alignment ; Viral Interference/*genetics ; Viral Proteins/genetics ; },
abstract = {Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wild-type viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. In this study, we analyzed in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wild-type population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionarily conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. IMPORTANCE The use of defective viruses that interfere with the replication of their infectious parent after coinfecting the same cells-a therapeutic strategy known as viral interference-has recently generated a lot of interest. The CRISPR-based system that we recently reported for herpesviruses represents a novel interfering strategy that causes the conversion of wild-type viruses into new recombinant viruses and drives the native viral population to extinction. In this study, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we showed that after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show, however, that targeting evolutionarily conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.},
}
@article {pmid34011151,
year = {2021},
author = {Li, X and Zhang, D and Gan, X and Liu, P and Zheng, Q and Yang, T and Tian, G and Ding, S and Yan, Y},
title = {A Cascade Signal Amplification Based on Dynamic DNA Nanodevices and CRISPR/Cas12a Trans-cleavage for Highly Sensitive MicroRNA Sensing.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1481-1489},
doi = {10.1021/acssynbio.1c00064},
pmid = {34011151},
issn = {2161-5063},
mesh = {Bacterial Proteins/*genetics ; Biosensing Techniques/methods ; Blood Donors ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/*genetics/metabolism ; Endodeoxyribonucleases/*genetics ; Fluorescence ; Gene Editing/*methods ; HeLa Cells ; Humans ; MCF-7 Cells ; MicroRNAs/*genetics/metabolism ; *Nanostructures ; Nucleic Acid Amplification Techniques/*methods ; Nucleic Acid Hybridization/methods ; Sensitivity and Specificity ; },
abstract = {The variations of microRNA (miRNA) expression can be valuable biomarkers in disease diagnosis and prognosis. However, current miRNA detection techniques mainly rely on reverse transcription and template replication, which suffer from slowness, contamination risk, and sample loss. To address these limitations, here we introduce a cascade toehold-mediated strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a. In this work, the target miRNA hybridizes with the terminal toehold site of a rationally designed probe and subsequently initiates dynamic CTSDR, leading to enzyme-free target recycling and the production of multiple programmable DNA duplexes. The obtained DNA duplex acts as an activator to trigger Cas12a trans-cleavage, generating significantly amplified fluorescence readout for highly sensitive detection of the miRNA target. Under the optimal conditions, the developed sensing method can detect target miRNA down to 70.28 fM with a wide linear range from 100 fM to 100 pM. In particular, by designing a set of probes and crRNAs, we demonstrate its broad applicability for the detection of six kinds of miRNAs with high sequence specificity. Furthermore, the method can be satisfactorily applied to monitor miR-21 in total RNA extracted from cells and clinical serum samples. Considering the high sensitivity, specificity, universality, and ease of handling, this strategy provides a great potential platform for the detection of miRNA biomarkers in molecular diagnostic practice.},
}
@article {pmid34010536,
year = {2021},
author = {Skokowa, J},
title = {Circumventing Mutation to Nix Neutropenia.},
journal = {The New England journal of medicine},
volume = {384},
number = {20},
pages = {1956-1958},
doi = {10.1056/NEJMcibr2102952},
pmid = {34010536},
issn = {1533-4406},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Gain of Function Mutation ; *Gene Editing ; Hematopoietic Stem Cells ; Humans ; Leukocyte Elastase/*genetics ; Mice ; *Mutation ; Neutropenia/congenital/genetics/*therapy ; },
}
@article {pmid34010515,
year = {2021},
author = {Hanly, JJ and Robertson, ECN and Corning, OBWH and Martin, A},
title = {Porcupine/Wntless-dependent trafficking of the conserved WntA ligand in butterflies.},
journal = {Journal of experimental zoology. Part B, Molecular and developmental evolution},
volume = {336},
number = {6},
pages = {470-481},
doi = {10.1002/jez.b.23046},
pmid = {34010515},
issn = {1552-5015},
mesh = {Animals ; *Biological Evolution ; Butterflies/*genetics/growth &amp; development ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Ligands ; Phylogeny ; Wings, Animal/growth &amp; development ; Wnt Proteins/*genetics ; *Wnt Signaling Pathway ; },
abstract = {Wnt ligands are key signaling molecules in animals, but little is known about the evolutionary dynamics and mode of action of the WntA orthologs, which are not present in the vertebrates or in Drosophila. Here we show that the WntA subfamily evolved at the base of the Bilateria + Cnidaria clade, and conserved the thumb region and Ser209 acylation site present in most other Wnts, suggesting WntA requires the core Wnt secretory pathway. WntA proteins are distinguishable from other Wnts by a synapomorphic Iso/Val/Ala216 amino-acid residue that replaces the otherwise ubiquitous Thr216 position. WntA embryonic expression is conserved between beetles and butterflies, suggesting functionality, but the WntA gene was lost three times within arthropods, in podoplean copepods, in the cyclorrhaphan fly radiation, and in ensiferan crickets and katydids. Finally, CRISPR mosaic knockouts (KOs) of porcupine and wntless phenocopied the pattern-specific effects of WntA KOs in the wings of Vanessa cardui butterflies. These results highlight the molecular conservation of the WntA protein across invertebrates, and imply it functions as a typical Wnt ligand that is acylated and secreted through the Porcupine/Wntless secretory pathway.},
}
@article {pmid34010004,
year = {2021},
author = {Ediriweera, GR and Chen, L and Yerbury, JJ and Thurecht, KJ and Vine, KL},
title = {Non-Viral Vector-Mediated Gene Therapy for ALS: Challenges and Future Perspectives.},
journal = {Molecular pharmaceutics},
volume = {18},
number = {6},
pages = {2142-2160},
doi = {10.1021/acs.molpharmaceut.1c00297},
pmid = {34010004},
issn = {1543-8392},
mesh = {Amyotrophic Lateral Sclerosis/genetics/*therapy ; Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Clinical Trials, Phase III as Topic ; Disease Models, Animal ; Gene Transfer Techniques/*adverse effects/trends ; Genetic Therapy/adverse effects/*methods/trends ; Genetic Vectors/*administration &amp; dosage/adverse effects ; Humans ; Nanoparticles/administration &amp; dosage ; Primary Cell Culture ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, for which no effective treatment is yet available to either slow or terminate it. Recent advances in gene therapy renew hope for developing an effective approach to control this disease. Non-viral vectors, such as lipid- and polymer-based nanoparticles, cationic polymers, and exosomes, can effectively transfer genes into primary neurons. The resulting gene expression can be long-term, stable, and without immunological complications, which is essential for the effective management of neurological disorders. This Review will first describe the current research and clinical stage of novel therapies for ALS. It will then touch on the journey of non-viral vector use in ALS, subsequently highlighting the application of non-viral vector-mediated gene therapy. The bottlenecks in the translation of non-viral vectors for ALS treatment are also discussed, including the biological barriers of systemic administration and the issues of "when, where, and how much?" for effective gene delivery. The prospect of employing emerging techniques, such as CRISPR-Cas9 gene editing, stem cell methodology, and low-intensity focused ultrasound for fueling the transport of non-viral vectors to the central nervous system for personalized gene therapy, is briefly discussed in the context of ALS. Despite the challenging road that lies ahead, with the current expansion in interest and technological advancement in non-viral vector-delivered gene therapy for ALS, we hold hope that the field is headed toward a positive future.},
}
@article {pmid34009946,
year = {2021},
author = {Lee, D and Lee, JH and Bang, D},
title = {Accurate Detection of Rare Mutant Alleles by Target Base-Specific Cleavage with the CRISPR/Cas9 System.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1451-1464},
doi = {10.1021/acssynbio.1c00056},
pmid = {34009946},
issn = {2161-5063},
mesh = {*Alleles ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Data Accuracy ; Gene Editing/methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; *Mutation ; Neoplasms/blood/*genetics ; *Polymorphism, Single Nucleotide ; Predictive Value of Tests ; RNA, Guide/genetics ; },
abstract = {The detection of low-frequency somatic mutations enables early diagnosis of disease; however, base-substitution errors that arise during genomic library preparation and high-throughput sequencing can lead to false diagnostic information. To discriminate true genomic alterations from technical errors, we developed spCas9-assisted true variant labeling sequencing (CARVE-seq), which detects low-frequency mutant alleles with high accuracy. CARVE-seq utilizes single-base discrimination during spCas9 cleavage reactions to exclude technical errors. Ten single nucleotide variants that recurrently occur in tumors were assayed by CARVE-seq using 20 ng reference samples, and 100% positive predictive value and specificity was observed, which proved the highly accurate performance of CARVE-seq.},
}
@article {pmid34009715,
year = {2021},
author = {Yoshimachi, S and Shirakawa, R and Cao, M and Trinh, DA and Gao, P and Sakata, N and Miyazaki, K and Goto, K and Miura, T and Ariake, K and Maeda, S and Masuda, K and Ishida, M and Ohtsuka, H and Unno, M and Horiuchi, H},
title = {Ral GTPase-activating protein regulates the malignancy of pancreatic ductal adenocarcinoma.},
journal = {Cancer science},
volume = {112},
number = {8},
pages = {3064-3073},
pmid = {34009715},
issn = {1349-7006},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/genetics/metabolism/*pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; GTPase-Activating Proteins/*genetics ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms/genetics/metabolism/*pathology/*secondary ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Pancreatic Neoplasms/genetics/metabolism/*pathology ; ral GTP-Binding Proteins/*metabolism ; },
abstract = {The small GTPases RalA and RalB are members of the Ras family and activated downstream of Ras. Ral proteins are found in GTP-bound active and GDP-bound inactive forms. The activation process is executed by guanine nucleotide exchange factors, while inactivation is mediated by GTPase-activating proteins (GAPs). RalGAPs are complexes that consist of a catalytic α1 or α2 subunit together with a common β subunit. Several reports implicate the importance of Ral in pancreatic ductal adenocarcinoma (PDAC). However, there are few reports on the relationship between levels of RalGAP expression and malignancy in PDAC. We generated RalGAPβ-deficient PDAC cells by CRISPR-Cas9 genome editing to investigate how increased Ral activity affects malignant phenotypes of PDAC cells. RalGAPβ-deficient PDAC cells exhibited several-fold higher Ral activity relative to control cells. They had a high migratory and invasive capacity. The RalGAPβ-deficient cells grew more rapidly than control cells when injected subcutaneously into nude mice. When injected into the spleen, the RalGAPβ-deficient cells formed larger splenic tumors with more liver metastases, and unlike controls, they disseminated into the abdominal cavity. These results indicate that RalGAPβ deficiency in PDAC cells contributes to high activities of RalA and RalB, leading to enhanced cell migration and invasion in vitro, and tumor growth and metastasis in vivo.},
}
@article {pmid34009615,
year = {2021},
author = {Xu, X and Zeisberg, EM},
title = {High-Fidelity CRISPR/Cas9-Based Gene-Specific Hydroxymethylation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2272},
number = {},
pages = {195-206},
pmid = {34009615},
issn = {1940-6029},
mesh = {5-Methylcytosine/*analogs &amp; derivatives/*chemistry ; *CRISPR-Cas Systems ; Chromatin ; Computational Biology/methods ; DNA/*analysis/chemistry/genetics ; *DNA Methylation ; Epigenesis, Genetic ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; Mixed Function Oxygenases/antagonists &amp; inhibitors/genetics/*metabolism ; Oxidation-Reduction ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Sulfites/chemistry ; },
abstract = {Aberrant promoter hypermethylation leads to gene silencing and is associated with various pathologies including cancer and organ fibrosis. Active DNA demethylation is mediated by TET enzymes: TET1, TET2, and TET3, which convert 5-methylcytosine to 5-hydroxymethylcytosine. Induction of gene-specific hydroxymethylation via CRISPR/Cas9 gene technology provides an opportunity to reactivate a single target gene silenced in pathological conditions. We utilized a spCas9 variant fused with TET3 catalytic domain to mediate gene-specific hydroxymethylation with subsequent gene reactivation which holds promise for gene therapy. Here, we present guidelines for gene-specific hydroxymethylation targeting with a specific focus on designing sgRNA and functional assessments in vitro.},
}
@article {pmid34009614,
year = {2021},
author = {Nguyen, TV and Lister, R},
title = {Genomic Targeting of TET Activity for Targeted Demethylation Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2272},
number = {},
pages = {181-194},
pmid = {34009614},
issn = {1940-6029},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; Chromatin ; Computational Biology/methods ; DNA/*analysis/chemistry/genetics ; *DNA Demethylation ; Epigenesis, Genetic ; *Gene Editing ; *Genome, Human ; High-Throughput Nucleotide Sequencing ; Humans ; Mixed Function Oxygenases/antagonists &amp; inhibitors/genetics/*metabolism ; Oxidation-Reduction ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Sulfites/*chemistry ; },
abstract = {Methylation of DNA at cytosine bases is an important DNA modification underlying normal development and disease states. Despite decades of research into the biological function of DNA methylation, most of the observations so far have relied primarily on associative data between observed changes in DNA methylation states and local changes in transcriptional activity or chromatin state processes. This is primarily due to the lack of molecular tools to precisely modify DNA methylation in the genome. Recent advances in genome editing technologies have allowed repurposing the CRISPR-Cas9 system for epigenome editing by fusing the catalytically dead Cas9 (dCas9) to epigenome modifying enzymes. Moreover, methods of recruiting multiple protein domains, including the SunTag system, have increased the efficacy of epigenome editing at target sites. Here, we describe an end-to-end protocol for efficient targeted removal of DNA methylation by recruiting multiple catalytic domain of TET1 enzymes to the target sites with the dCas9-SunTag system, including sgRNA design, molecular cloning, delivery of plasmid into mammalian cells, and targeted DNA methylation analysis.},
}
@article {pmid34009598,
year = {2021},
author = {Eswaran, N and Parameswaran, S and Johnson, TS},
title = {Biofuels and Sustainability.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2290},
number = {},
pages = {317-342},
pmid = {34009598},
issn = {1940-6029},
mesh = {Agriculture/methods/trends ; Biofuels/*economics/statistics &amp; numerical data/*supply &amp; distribution ; Biomass ; Biotechnology/methods/trends ; Crops, Agricultural/genetics ; Fossil Fuels ; Microalgae ; Renewable Energy/*economics ; },
abstract = {Energy security and climate change have cascading effects on the world's burgeoning population in terms of food security, environment, and sustainability. Due to depletion of fossil fuels and undesirable changes of climatic conditions, increase in air and water pollution, mankind started exploring alternate and sustainable means of meeting growing energy needs. One of the options is to use renewable sources of fuel-biofuel. In this chapter the authors have reviewed and presented sustainability impact on production of biofuels. Authors further reviewed state-of-the-art gene editing technologies toward improvement of biofuel crops. The authors recommend a phased transition from first-generation biofuel, and an acceleration toward use of technology to drive adoption of second-generation biofuels. Key aspects of technology and application of resource management models will enable these crops to bridge the global energy demand before we can completely transition to a more sustainable biofuel economy.},
}
@article {pmid34009379,
year = {2021},
author = {Wörle, E and Jakob, L and Schmidbauer, A and Zinner, G and Grohmann, D},
title = {Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions.},
journal = {Nucleic acids research},
volume = {49},
number = {9},
pages = {5278-5293},
pmid = {34009379},
issn = {1362-4962},
mesh = {Bacterial Proteins/*chemistry/genetics/*metabolism ; Base Pair Mismatch ; CRISPR-Associated Proteins/*chemistry/genetics/*metabolism ; Endodeoxyribonucleases/*chemistry/genetics/*metabolism ; Francisella/enzymology ; Mutation ; Protein Conformation ; },
abstract = {The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.},
}
@article {pmid34008219,
year = {2021},
author = {Tian, Q and Shen, L and Luan, J and Zhou, Z and Guo, D and Shen, Y and Jing, W and Zhang, B and Zhang, Q and Zhang, W},
title = {Rice shaker potassium channel OsAKT2 positively regulates salt tolerance and grain yield by mediating K[+] redistribution.},
journal = {Plant, cell &amp; environment},
volume = {44},
number = {9},
pages = {2951-2965},
doi = {10.1111/pce.14101},
pmid = {34008219},
issn = {1365-3040},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Edible Grain/*genetics ; Gene Editing ; Gene Knockdown Techniques ; Oryza/genetics/growth &amp; development/*metabolism/physiology ; Phloem/metabolism ; Plant Leaves/metabolism ; Plant Proteins/genetics/*metabolism/physiology ; Plant Roots/metabolism ; Plant Shoots/metabolism ; Potassium/*metabolism ; Potassium Channels/genetics/*metabolism/physiology ; *Salt Tolerance/genetics/physiology ; },
abstract = {Maintaining Na[+] /K[+] homeostasis is a critical feature for plant survival under salt stress, which depends on the operation of Na[+] and K[+] transporters. Although some K[+] transporters mediating root K[+] uptake have been reported to be essential to the maintenance of Na[+] /K[+] homeostasis, the effect of K[+] long-distance translocation via phloem on plant salt tolerance remains unclear. Here, we provide physiological and genetic evidence of the involvement of phloem-localized OsAKT2 in rice salt tolerance. OsAKT2 is a K[+] channel permeable to K[+] but not to Na[+] . Under salt stress, a T-DNA knock-out mutant, osakt2 and two CRISPR lines showed a more sensitive phenotype and higher Na[+] accumulation than wild type. They also contained more K[+] in shoots but less K[+] in roots, showing higher Na[+] /K[+] ratios. Disruption of OsAKT2 decreases K[+] concentration in phloem sap and inhibits shoot-to-root redistribution of K[+] . In addition, OsAKT2 also regulates the translocation of K[+] and sucrose from old leaves to young leaves, and affects grain shape and yield. These results indicate that OsAKT2-mediated K[+] redistribution from shoots to roots contributes to maintenance of Na[+] /K[+] homeostasis and inhibition of root Na[+] uptake, providing novel insights into the roles of K[+] transporters in plant salt tolerance.},
}
@article {pmid34006489,
year = {2021},
author = {Sun, C and Fang, YC and Li, H and Chen, J and Ye, YL and Ni, LF and Xu, L and Han, BN and Wu, M and Wang, CS and Xu, XW},
title = {Complete genome sequence of marine Roseobacter lineage member Monaibacterium sp. ALG8 with six plasmids isolated from seawater around brown algae.},
journal = {Marine genomics},
volume = {60},
number = {},
pages = {100878},
doi = {10.1016/j.margen.2021.100878},
pmid = {34006489},
issn = {1876-7478},
mesh = {*Phaeophyta ; Plasmids/genetics ; *Rhodobacteraceae/genetics ; *Roseobacter/genetics ; Seawater ; },
abstract = {Monaibacterium sp. ALG8 (=MCCC 1 K04733) was isolated from seawater around brown algae. The genome of Monaibacterium sp. ALG8 was sequenced, one circular 3,036,380 bp chromosome and six circular plasmids ranging from 12,229 to 151,263 bp were found after assembly. The results of genomic annotation showed that Monaibacterium sp. ALG8 lacks the ability to degrade alginate, indicating its ecological role may not be directly related to the degradation of brown algae. The comparison of genomic features in the plasmids showed that almost all of these plasmids, except pALG4, were horizontally recruited from donors, not ancestors. Based on predicted functions, the existence of plasmids may provide strain ALG8 with advantages including nitrate reduction, tolerance of osmotic stress via glycine betaine, resistance to heavy metal stress such as mercury and cobalt, degradation of benzoate metabolites such as p-cumate, transformation of the swim-or-stick lifestyle and improvement of the immune system with two CRISPR-Cas systems. This study provides evidence for the carbon metabolic patterns of Monaibacterium sp. ALG8 and predicts the functions and donors of six plasmids in this strain, broadening our understanding of the ecological roles of bacteria in the environment around brown algae and the functions and evolutionary patterns of plasmids in marine Roseobacter lineage members.},
}
@article {pmid34006179,
year = {2021},
author = {Lin, X and Fonseca, MAS and Breunig, JJ and Corona, RI and Lawrenson, K},
title = {In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation.},
journal = {RNA biology},
volume = {18},
number = {12},
pages = {2203-2217},
pmid = {34006179},
issn = {1555-8584},
support = {K99 CA184415/CA/NCI NIH HHS/United States ; R00 CA184415/CA/NCI NIH HHS/United States ; R01 CA207456/CA/NCI NIH HHS/United States ; },
mesh = {Ascorbate Peroxidases/*genetics ; Biotinylation ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Poly A ; RNA/chemistry/*metabolism ; RNA, Guide/genetics ; RNA, Small Nuclear/genetics ; RNA-Binding Proteins/*metabolism ; Recombinant Fusion Proteins/genetics ; Staining and Labeling ; },
abstract = {RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5'-3' proximity and unexplored subcellular localizations of poly(A)[+] RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.},
}
@article {pmid34006094,
year = {2021},
author = {Jain, S and Xun, G and Abesteh, S and Ho, S and Lingamaneni, M and Martin, TA and Tasan, I and Yang, C and Zhao, H},
title = {Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers.},
journal = {ACS synthetic biology},
volume = {10},
number = {6},
pages = {1320-1327},
doi = {10.1021/acssynbio.0c00548},
pmid = {34006094},
issn = {2161-5063},
support = {U54 DK107965/DK/NIDDK NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Containment of Biohazards/methods ; DNA/metabolism ; DNA-Binding Proteins/genetics ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Ligands ; Protein Binding ; RNA, Guide/genetics ; Streptococcus pyogenes/*enzymology ; Transfection ; Trimethoprim/*metabolism ; },
abstract = {CRISPR/Cas9 is a powerful genome editing tool, but its off-target cleavage activity can result in unintended adverse outcomes for therapeutic applications. Here we report the design of a simple tunable CRISPR controller in which a chemically inducible anti-CRISPR protein AcrIIA4 is engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Dose-dependent control over Cas9 editing and dCas9 induction was achieved, which drastically improved the specificity and biosafety of the CRISPR/Cas9 system. We utilized the anti-CRISPR protein AcrIIA4 as a means to interfere with Cas9 DNA binding activity. By fusing AcrIIA4 to a ligand-inducible destabilization domain DHFR(DD), we show significantly reduced off-target activity in mammalian cells. Furthermore, we describe a new inducible promoter system Acr-OFF based on CRISPR controllers, which is regulated by an FDA-approved ligand trimethoprim.},
}
@article {pmid34004455,
year = {2021},
author = {Li, Y and Adur, MK and Wang, W and Schultz, RB and Hale, B and Wierson, W and Charley, SE and McGrail, M and Essner, J and Tuggle, CK and Ross, JW},
title = {Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system.},
journal = {Theriogenology},
volume = {170},
number = {},
pages = {107-116},
pmid = {34004455},
issn = {1879-3231},
support = {R24 OD019813/OD/NIH HHS/United States ; R24 OD028748/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Fertilization in Vitro/veterinary ; Gene Editing/veterinary ; Male ; Microinjections/veterinary ; Swine ; },
abstract = {The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system. CRISPR/Cas9 is a powerful tool used to generate genetic modifications by causing double-strand breaks (DSBs) in DNA. Artemis (ART; also known as DCLRE1C), is a nuclear protein and is essential for DSB end joining in DNA repair via the canonical non-homologous end joining (c-NHEJ) pathway. In this work, we tested whether ART deficiency affects DNA repair following CRISPR/Cas9 induced DSBs in somatic cells. We also demonstrated the effect of microinjection timing on embryo developmental ability and gene targeting efficiency of CRISPR/Cas9 system to disrupt the interleukin 2 receptor subunit gamma (IL2RG) locus using porcine in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryos. In comparison to non-injected controls, CRISPR/Cas9 injection of IVF derived zygotes at 4 h and 8 h after fertilization did not impact cleavage and blastocyst rate. Gene modification rate was observed to be higher, 53.3% (9/16) in blastocysts injected 4 h post-fertilization as compared to 11.1% (1/9) in blastocysts injected 8 h post-fertilization. Microinjection 8 h after chemical activation of SCNT derived embryos decreased blastocyst development rate compared to non-injected controls but showed a higher gene modification efficiency of 66.7% as compared to 25% in the 4 h post-activation injection group. Furthermore, we observed that male ART[-/-] and ART[+/-] porcine fetal fibroblast (pFF) cells showed lower modification rates (2.5% and 1.9%, respectively) as compared to the ART intact cell line (8.3%). Interestingly, the female ART[-/-] and ART[+/-] pFF cells had modification rates (4.2% and 10.1%, respectively) similar to those seen in the ART intact cells. This study demonstrates the complex effect of various parameters such as microinjection timing and ART deficiency on gene editing efficiency in in vitro derived porcine embryos.},
}
@article {pmid34004353,
year = {2021},
author = {Gross-Cohen, M and Yanku, Y and Kessler, O and Barash, U and Boyango, I and Cid-Arregui, A and Neufeld, G and Ilan, N and Vlodavsky, I},
title = {Heparanase 2 (Hpa2) attenuates tumor growth by inducing Sox2 expression.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {99},
number = {},
pages = {58-71},
doi = {10.1016/j.matbio.2021.05.001},
pmid = {34004353},
issn = {1569-1802},
mesh = {*Glucuronidase/genetics ; *Head and Neck Neoplasms/genetics ; Heparitin Sulfate ; Humans ; SOXB1 Transcription Factors/genetics ; },
abstract = {The pro-tumorigenic properties of heparanase are well documented, and heparanase inhibitors are being evaluated clinically as anti-cancer therapeutics. In contrast, the role of heparanase 2 (Hpa2), a close homolog of heparanase, in cancer is largely unknown. Previously, we have reported that in head and neck cancer, high levels of Hpa2 are associated with prolonged patient survival and decreased tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions to restrain tumorigenesis. Also, patients with high levels of Hpa2 were diagnosed as low grade and exhibited increased expression of cytokeratins, an indication that Hpa2 promotes or maintains epithelial cell differentiation and identity. To reveal the molecular mechanism underlying the tumor suppressor properties of Hpa2, and its ability to induce the expression of cytokeratin, we employed overexpression as well as gene editing (Crispr) approaches, combined with gene array and RNAseq methodologies. At the top of the list of many genes found to be affected by Hpa2 was Sox2. Here we provide evidence that silencing of Sox2 resulted in bigger tumors endowed with reduced cytokeratin levels, whereas smaller tumors were developed by cells overexpressing Sox2, suggesting that in head and neck carcinoma, Sox2 functions to inhibit tumor growth. Notably, Hpa2-null cells engineered by Crispr/Cas 9, produced bigger tumors vs control cells, and rescue of Hpa2 attenuated tumor growth. These results strongly imply that Hpa2 functions as a tumor suppressor in head and neck cancer, involving Sox2 upregulation mediated, in part, by the high-affinity interaction of Hpa2 with heparan sulfate.},
}
@article {pmid34003741,
year = {2021},
author = {Goldsmith, C and Cohen, D and Dubois, A and Martinez, MG and Petitjean, K and Corlu, A and Testoni, B and Hernandez-Vargas, H and Chemin, I},
title = {Cas9-targeted nanopore sequencing reveals epigenetic heterogeneity after de novo assembly of native full-length hepatitis B virus genomes.},
journal = {Microbial genomics},
volume = {7},
number = {5},
pages = {},
pmid = {34003741},
issn = {2057-5858},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/metabolism/virology ; DNA Methylation ; DNA, Viral/genetics ; *Epigenomics ; *Genetic Heterogeneity ; *Genome, Viral ; Genotype ; Hepatitis B virus/*genetics/metabolism ; Humans ; Liver Neoplasms/genetics/metabolism/pathology ; Nanopore Sequencing/*methods ; Nanopores ; Sequence Analysis, DNA ; Sulfites ; },
abstract = {Hepatitis B virus (HBV) contains a 3.2 kb DNA genome and causes acute and chronic hepatitis. HBV infection is a global health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues as to why certain infections are cleared or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With Cas9-guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from primary human hepatocytes (PHHs) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue, followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by nanopore sequencing was benchmarked with bisulfite-quantitative methyl-specific qPCR (BS-qMSP). The 5mCpG levels in HBV determined by BS-qMSP and nanopore sequencing were highly correlated. Our nanopore sequencing approach achieved a coverage of ~2000× of HBV depending on infection efficiency, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified four epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.},
}
@article {pmid34003582,
year = {2021},
author = {Goulding, J and Mistry, SJ and Soave, M and Woolard, J and Briddon, SJ and White, CW and Kellam, B and Hill, SJ},
title = {Subtype selective fluorescent ligands based on ICI 118,551 to study the human β2-adrenoceptor in CRISPR/Cas9 genome-edited HEK293T cells at low expression levels.},
journal = {Pharmacology research &amp; perspectives},
volume = {9},
number = {3},
pages = {e00779},
pmid = {34003582},
issn = {2052-1707},
support = {MR/N020081/1/MRC_/Medical Research Council/United Kingdom ; MR/N020081/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adrenergic beta-2 Receptor Antagonists/*pharmacology ; CRISPR-Cas Systems ; Fluorescence ; Gene Editing ; HEK293 Cells ; Humans ; Ligands ; Propanolamines/*pharmacology ; *Receptors, Adrenergic, beta-2/genetics/metabolism ; },
abstract = {Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β2 -adrenoceptor (β2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β2 AR (pKD >7.0) with good selectivity over the β1 AR (pKD <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; β2 AR pKD 7.48), 9c (ICI 118,551-βAla-βAla-BODIPY-FL-X; β2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; β2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; β2 AR pKD 7.42). 9a (ICI 118,551-βAla-βAla-BODIPY-X-630/650) had the highest affinity at recombinant β2 ARs (pKD 7.57), but also exhibited significant binding affinity to the β1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β2 ARs in HEK293 T cell expressing exogenous β2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T β2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.},
}
@article {pmid34003474,
year = {2021},
author = {Weiss, WF},
title = {Cutting Through the "Gray Area": An Analysis of the IBC Regulatory Oversight of Applications of CRISPR Technology in Clinical Research.},
journal = {Therapeutic innovation &amp; regulatory science},
volume = {55},
number = {5},
pages = {979-983},
pmid = {34003474},
issn = {2168-4804},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Technology ; United States ; },
abstract = {The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technology has quickly ushered in a new era of gene editing and offered exciting potential for the development of biomedical products. While the breadth of applications for CRISPR encompasses nearly the entire field of medical science, its utilization to produce next-generation CAR T cells stands to benefit most substantially in the short-term. These novel therapeutics are now beginning to enter the clinical trial phase of the numerous approval pipelines, but does the existing regulatory framework have the capability to adequately review and approve the initiation of these new age research endeavors in the clinical setting? This analysis will illuminate the similarity and differences between CRISPR developed interventions and existing techniques used to produce biomedical products from a regulatory perspective in the United States. Further, the "gray area" surrounding CRISPR regulatory oversight will be explored and recommendations will be made to facilitate the elimination of inconsistencies that currently exist in the assessment of this work prior to the initiation of associated clinical trials.},
}
@article {pmid34001946,
year = {2021},
author = {Fabrick, JA and LeRoy, DM and Mathew, LG and Wu, Y and Unnithan, GC and Yelich, AJ and Carrière, Y and Li, X and Tabashnik, BE},
title = {CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {10377},
pmid = {34001946},
issn = {2045-2322},
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; Bacillus thuringiensis/genetics ; Bacillus thuringiensis Toxins/*genetics ; CRISPR-Cas Systems/genetics ; Gossypium/*parasitology ; Humans ; Insecticide Resistance/*genetics ; Insecticides/*pharmacology ; Larva/drug effects/genetics/pathogenicity ; Lepidoptera/drug effects/genetics/pathogenicity ; Moths/genetics/pathogenicity ; Mutation/genetics ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.},
}
@article {pmid34000301,
year = {2021},
author = {Mimura, K and Sakamaki, JI and Morishita, H and Kawazu, M and Mano, H and Mizushima, N},
title = {Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100780},
pmid = {34000301},
issn = {1083-351X},
mesh = {Amidophosphoribosyltransferase/genetics ; *Autophagy ; *CRISPR-Cas Systems ; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; HEK293 Cells ; Humans ; Mechanistic Target of Rapamycin Complex 1/genetics ; },
abstract = {Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.},
}
@article {pmid33999995,
year = {2021},
author = {Wang, H and Wang, C and Long, Q and Zhang, Y and Wang, M and Liu, J and Qi, X and Cai, D and Lu, G and Sun, J and Yao, YG and Chan, WY and Chan, WY and Deng, Y and Zhao, H},
title = {Kindlin2 regulates neural crest specification via integrin-independent regulation of the FGF signaling pathway.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {10},
pages = {},
doi = {10.1242/dev.199441},
pmid = {33999995},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Embryo, Nonmammalian/metabolism ; Embryonic Development/genetics/physiology ; Fibroblast Growth Factors/*metabolism ; Gene Expression Regulation, Developmental/genetics ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Integrins/metabolism ; Membrane Proteins/genetics/*metabolism ; Morpholinos/genetics ; Neural Crest/*embryology ; Receptor, Fibroblast Growth Factor, Type 1/metabolism ; Signal Transduction/genetics ; Xenopus Proteins/genetics/*metabolism ; Xenopus laevis/*embryology ; },
abstract = {The focal adhesion protein Kindlin2 is essential for integrin activation, a process that is fundamental to cell-extracellular matrix adhesion. Kindlin 2 (Fermt2) is widely expressed in mouse embryos, and its absence causes lethality at the peri-implantation stage due to the failure to trigger integrin activation. The function of kindlin2 during embryogenesis has not yet been fully elucidated as a result of this early embryonic lethality. Here, we showed that kindlin2 is essential for neural crest (NC) formation in Xenopus embryos. Loss-of-function assays performed with kindlin2-specific morpholino antisense oligos (MOs) or with CRISPR/Cas9 techniques in Xenopus embryos severely inhibit the specification of the NC. Moreover, integrin-binding-deficient mutants of Kindlin2 rescued the phenotype caused by loss of kindlin2, suggesting that the function of kindlin2 during NC specification is independent of integrins. Mechanistically, we found that Kindlin2 regulates the fibroblast growth factor (FGF) pathway, and promotes the stability of FGF receptor 1. Our study reveals a novel function of Kindlin2 in regulating the FGF signaling pathway and provides mechanistic insights into the function of Kindlin2 during NC specification.},
}
@article {pmid33999508,
year = {2021},
author = {Chen, L and Gao, H and Zhou, B and Wang, Y},
title = {Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.},
journal = {FEBS open bio},
volume = {11},
number = {7},
pages = {1965-1980},
pmid = {33999508},
issn = {2211-5463},
mesh = {*CRISPR-Cas Systems/genetics ; DNA End-Joining Repair ; DNA Repair ; Endonucleases/genetics/metabolism ; *Gene Editing ; },
abstract = {The clustered, regularly interspaced, short palindromic repeats-associated DNA nuclease (CRISPR-Cas) protein system allows programmable gene editing through inducing double-strand breaks. Reporter assays for DNA cleavage and DNA repair events play an important role in advancing the CRISPR technology and improving our understanding of the underlying molecular mechanisms. Here, we developed a series of reporter assays to probe mechanisms of action of various editing processes, including nonhomologous DNA end joining, homology-directed repair and single-strand annealing. With special target design, the reporter assays as an optimized toolbox can be used to take advantage of three distinct CRISPR-Cas systems (Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9 and Francisella novicida U112 Cpf1) and two different reporters (GFP and Gaussia luciferase). We further validated the Gaussia reporter assays using a series of small molecules, including NU7441, RI-1 and Mirin, and showcased the use of a GFP reporter assay as an effective tool for enrichment of cells with edited genome.},
}
@article {pmid33999303,
year = {2021},
author = {Ar, E and Demiroğlu, A and Yılmaz, MS and Yılmazer, B and Aslan, ES and Binay, B},
title = {Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology.},
journal = {The protein journal},
volume = {40},
number = {4},
pages = {504-511},
pmid = {33999303},
issn = {1875-8355},
mesh = {*CRISPR-Cas Systems ; Chaetomium/enzymology/*genetics ; Escherichia coli K12/genetics/metabolism ; *Formate Dehydrogenases/biosynthesis/genetics ; *Fungal Proteins/biosynthesis/genetics ; *Gene Expression ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the ω subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the ω subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the ω subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the ω subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO2 reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.},
}
@article {pmid33999055,
year = {2021},
author = {Liu, R and He, Y and Lan, T and Zhang, J},
title = {Installing CRISPR-Cas12a sensors in a portable glucose meter for point-of-care detection of analytes.},
journal = {The Analyst},
volume = {146},
number = {10},
pages = {3114-3120},
doi = {10.1039/d1an00008j},
pmid = {33999055},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Glucose ; Point-of-Care Systems ; },
abstract = {Integrating CRISPR-Cas12a sensors with a portable glucose meter (PGM) was developed based on the target-induced activation of the collateral cleavage activity of Cas12a. Considering the portability, low cost and facile incorporation of the PGM system with suitable Cas12a sensors to recognize many targets, the CRISPR/Cas12a-PGM system demonstrated here paves a way to further broaden the POC applications of CRISPR-based diagnostics.},
}
@article {pmid33997893,
year = {2021},
author = {Wappett, M and Harris, A and Lubbock, ALR and Lobb, I and McDade, S and Overton, IM},
title = {SynLeGG: analysis and visualization of multiomics data for discovery of cancer 'Achilles Heels' and gene function relationships.},
journal = {Nucleic acids research},
volume = {49},
number = {W1},
pages = {W613-W618},
pmid = {33997893},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression ; Gene Expression Profiling ; *Genes, Neoplasm ; Humans ; Mutation ; Neoplasms/*genetics ; Proteomics ; *Software ; *Synthetic Lethal Mutations ; },
abstract = {Achilles' heel relationships arise when the status of one gene exposes a cell's vulnerability to perturbation of a second gene, such as chemical inhibition, providing therapeutic opportunities for precision oncology. SynLeGG (www.overton-lab.uk/synlegg) identifies and visualizes mutually exclusive loss signatures in 'omics data to enable discovery of genetic dependency relationships (GDRs) across 783 cancer cell lines and 30 tissues. While there is significant focus on genetic approaches, transcriptome data has advantages for investigation of GDRs and remains relatively underexplored. SynLeGG depends upon the MultiSEp algorithm for unsupervised assignment of cell lines into gene expression clusters, which provide the basis for analysis of CRISPR scores and mutational status in order to propose candidate GDRs. Benchmarking against SynLethDB demonstrates favourable performance for MultiSEp against competing approaches, finding significantly higher area under the Receiver Operator Characteristic curve and between 2.8-fold to 8.5-fold greater coverage. In addition to pan-cancer analysis, SynLeGG offers investigation of tissue-specific GDRs and recovers established relationships, including synthetic lethality for SMARCA2 with SMARCA4. Proteomics, Gene Ontology, protein-protein interactions and paralogue information are provided to assist interpretation and candidate drug target prioritization. SynLeGG predictions are significantly enriched in dependencies validated by a recently published CRISPR screen.},
}
@article {pmid33997361,
year = {2021},
author = {Gao, J and Jiang, L and Lian, J},
title = {Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products.},
journal = {Synthetic and systems biotechnology},
volume = {6},
number = {2},
pages = {110-119},
pmid = {33997361},
issn = {2405-805X},
abstract = {The methylotrophic yeast Pichia pastoris (a.k.a. Komagataella phaffii) is one of the most commonly used hosts for industrial production of recombinant proteins. As a non-conventional yeast, P. pastoris has unique biological characteristics and its expression system has been well developed. With the advances in synthetic biology, more efforts have been devoted to developing P. pastoris into a chassis for the production of various high-value compounds, such as natural products. This review begins with the introduction of synthetic biology tools for the engineering of P. pastoris, including vectors, promoters, and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System (CRISPR/Cas) for genome editing. This review is then followed by examples of the production of value-added natural products in metabolically engineered P. pastoris strains. Finally, challenges and outlooks in developing P. pastoris as a synthetic biology chassis are prospected.},
}
@article {pmid33996635,
year = {2021},
author = {Ganbaatar, U and Liu, C},
title = {CRISPR-Based COVID-19 Testing: Toward Next-Generation Point-of-Care Diagnostics.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {663949},
pmid = {33996635},
issn = {2235-2988},
support = {R01 EB023607/EB/NIBIB NIH HHS/United States ; R61 AI154642/AI/NIAID NIH HHS/United States ; R01 CA214072/CA/NCI NIH HHS/United States ; },
mesh = {*COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Pandemics ; Point-of-Care Testing ; SARS-CoV-2 ; },
abstract = {As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is an immediate need for a simple, rapid, early and sensitive point-of-care testing for COVID-19 disease. However, current testing approaches do not meet such need. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.},
}
@article {pmid33995459,
year = {2021},
author = {Sánchez-León, S and Giménez, MJ and Barro, F},
title = {The α-Gliadins in Bread Wheat: Effect of Nitrogen Treatment on the Expression of the Major Celiac Disease Immunogenic Complex in Two RNAi Low-Gliadin Lines.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {663653},
pmid = {33995459},
issn = {1664-462X},
abstract = {Celiac Disease (CD) is an autoimmune disorder that affects approximately 1% of the worldwide population. The α-gliadins of wheat contain the 33-mer peptide, the most active peptide in CD both in adults and pediatric patients. In this study, we have characterized the variants and expression profile of an α-gliadins amplicon, harboring the 33-mer peptide, in two low-gliadin RNAi wheat lines, under two different Nitrogen (N) treatments. We estimated that the amplicon expands 45 different α-gliadin variants with high variability due to length, randomly distributed SNPs, and the presence of encoded CD epitopes. Expression of this amplicon is reduced in both RNAi lines in comparison to the wild type. High N treatment significantly increases transcripts of the amplicon in the wild type, but not in the transgenic lines. Classification of α-gliadin variants, considering the number of epitopes, revealed that amplicon variants containing the full complement of 33-mer peptide were affected by N treatment, increasing their expression when N was increased. Line D793 provided higher and more stable silencing through different N fertilization regimes, expressing fewer CD epitopes than D783. Results of this study are important for better understanding of RNAi α-gliadin silencing in response to N treatments, and for undertaking new strategies by RNAi or CRISPR/Cas toward obtaining new varieties suitable for people suffering gluten intolerances.},
}
@article {pmid33994114,
year = {2021},
author = {Cai, MZ and Chen, PT},
title = {Novel combined Cre-Cas system for improved chromosome editing in Bacillus subtilis.},
journal = {Journal of bioscience and bioengineering},
volume = {132},
number = {2},
pages = {113-119},
doi = {10.1016/j.jbiosc.2021.04.005},
pmid = {33994114},
issn = {1347-4421},
mesh = {*Bacillus subtilis/genetics ; *CRISPR-Cas Systems/genetics ; Chromosomes ; Gene Editing ; Integrases ; },
abstract = {To improve the stability and expand applications of genome editing in Bacillus subtilis, we propose a new concept of the Cre-Cas system, which combines Cre-lox72 and CRISPR-Cas9 into an effective and convenient method. Single homologous recombination is used to introduce the integration vector into the chromosome via appropriate guide DNA to inactivate and/or insert genes of interest. The Cre recombinase then removes the region of a selection marker that is no longer needed, and the Escherichia coli replicon between the lox66 and lox71 sites are recombined to a single lox72 site. The CRISPR-Cas9 system can then be applied to remove the inserted foreign gene by targeted cutting. After Cas9 cutting, B. subtilis self-repairs the broken region to its original state without the aid of additional DNA templates. To validate this system, we used T7 and keratinase expression cassettes; self-repair efficiency was evaluated based on the loss or maintenance of the antibiotic resistance gene, as analyzed on selective media. Our results demonstrated that the insertion position in the chromosome is a more critical factor than the insertion length of the gene for efficient self-repair in the B. subtilis genome. This concept can provide the applicability of chromosomal editing in B. subtilis.},
}
@article {pmid33993306,
year = {2021},
author = {Sabat, AJ and Bathoorn, E and Becker, K and Akkerboom, V and Miskoski, M and Durfee, T and Friedrich, AW},
title = {Staphylococcal cassette chromosome mec containing a novel mec gene complex, B4.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {76},
number = {8},
pages = {1986-1990},
pmid = {33993306},
issn = {1460-2091},
support = {R44 GM122120/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics ; Chromosomes, Bacterial/genetics ; Humans ; *Staphylococcal Infections ; Staphylococcus/genetics ; Staphylococcus epidermidis/genetics ; },
abstract = {OBJECTIVES: To describe a new subclass of mec class B complex identified in Staphylococcus epidermidis.<br><br>METHODS: Four S. epidermidis isolates obtained from bloodstream infections in patients at University Medical Center Groningen (UMCG) were analysed by phenotypic antibiotic susceptibility testing and WGS.<br><br>RESULTS: Sequence analysis revealed a new staphylococcal cassette chromosome mec (SCCmec) structure in isolate UMCG335. In this structure, plasmid pUB110 was found to be integrated into SCCmec IVc, creating a new SCCmec subtype, IVUMCG335. SCCmec IVc and a copy of plasmid pUB110 were found in other isolates, UMCG364 and UMCG341, respectively, indicating a probability that SCCmec IVUMCG335 could have evolved at the UMCG. SCCmec of UMCG337 contained a new genetic organization of the mec complex (IS431-&#x394;mecR1-mecA-IS431-pUB110-IS431-&#x3c8;IS1272) that we have named B4. This new subclass of mec class B complex originated by IS431-mediated inversion of the DNA segment encompassing the plasmid and most of the genes of the mec complex with the exception of IS1272. As the SCCmec organization in UMCG337 differed by the inversion of an &#x223c;10&#x2009;kb sequence compared with SCCmec IVUMCG335, we have named it SCCmec subtype IVUMCG337. Isolates UMCG335 and UMCG337 carrying SCCmec IVUMCG335 and IVUMCG337, respectively, were associated with a restriction-modification system and a CRISPR-Cas system, creating a composite island of almost 70&#x2009;kb.<br><br>CONCLUSIONS: Our findings highlight the importance of IS431 in the evolution of the SCCmec region. The increasing genetic diversity identified in the SCCmec elements imposes a great challenge for SCCmec typing methods and highlights possible difficulties with the SCCmec nomenclature.},
}
@article {pmid33991961,
year = {2021},
author = {Li, T and Hu, R and Xia, J and Xu, Z and Chen, D and Xi, J and Liu, BF and Zhu, J and Li, Y and Yang, Y and Liu, M},
title = {G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {187},
number = {},
pages = {113292},
doi = {10.1016/j.bios.2021.113292},
pmid = {33991961},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA, Single-Stranded ; Humans ; },
abstract = {CRISPR-Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed G-CRISPR. We proved that Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics.},
}
@article {pmid33991533,
year = {2021},
author = {Mardick, JI and Rasmussen, NR and Wightman, B and Reiner, DJ},
title = {Parallel Rap1&gt;RalGEF&gt;Ral and Ras signals sculpt the C. elegans nervous system.},
journal = {Developmental biology},
volume = {477},
number = {},
pages = {37-48},
pmid = {33991533},
issn = {1095-564X},
support = {P40 OD010440/OD/NIH HHS/United States ; R01 GM121625/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/embryology/*physiology ; Caenorhabditis elegans Proteins/*physiology ; Embryonic Induction ; Genes, ras ; Guanine Nucleotide Exchange Factors/*physiology ; Nervous System/embryology/*physiopathology ; Neurons/physiology ; *Signal Transduction ; ral GTP-Binding Proteins/*physiology ; ras Proteins/genetics/*physiology ; },
abstract = {Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.},
}
@article {pmid33990913,
year = {2021},
author = {Li, Z and Song, Q and Wang, M and Ren, J and Liu, S and Zhao, S},
title = {Comparative genomics analysis of Pediococcus acidilactici species.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {59},
number = {6},
pages = {573-583},
pmid = {33990913},
issn = {1976-3794},
mesh = {Anti-Bacterial Agents/biosynthesis ; Bacteriocins/biosynthesis ; *Genome, Bacterial ; Genomics ; Pediococcus acidilactici/classification/*genetics/metabolism ; },
abstract = {Pediococcus acidilactici is a reliable bacteriocin producer and a promising probiotic species with wide application in the food and health industry. However, the underlying genetic features of this species have not been analyzed. In this study, we performed a comprehensive comparative genomic analysis of 41 P. acidilactici strains from various ecological niches. The bacteriocin production of 41 strains were predicted and three kinds of bacteriocin encoding genes were identified in 11 P. acidilactici strains, namely pediocin PA-1, enterolysin A, and colicin-B. Moreover, whole-genome analysis showed a high genetic diversity within the population, mainly related to a large proportion of variable genomes, mobile elements, and hypothetical genes obtained through horizontal gene transfer. In addition, comparative genomics also facilitated the genetic explanation of the adaptation for host environment, which specify the protection mechanism against the invasion of foreign DNA (i.e. CRISPR/Cas locus), as well as carbohydrate fermentation. The 41 strains of P. acidilactici can metabolize a variety of carbon sources, which enhances the adaptability of this species and survival in different environments. This study evaluated the antibacterial ability, genome evolution, and ecological flexibility of P. acidilactici from the perspective of genetics and provides strong supporting evidence for its industrial development and application.},
}
@article {pmid33990626,
year = {2021},
author = {Yasutake, H and Lee, JK and Hashimoto, A and Masuyama, K and Li, J and Kuramoto, Y and Higo, S and Hikoso, S and Hidaka, K and Naito, AT and Miyagawa, S and Sawa, Y and Komuro, I and Sakata, Y},
title = {Decreased YAP activity reduces proliferative ability in human induced pluripotent stem cell of duchenne muscular dystrophy derived cardiomyocytes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {10351},
pmid = {33990626},
issn = {2045-2322},
mesh = {Adaptor Proteins, Signal Transducing/*deficiency/genetics ; Adult ; CRISPR-Cas Systems/genetics ; Cardiomyopathy, Dilated/genetics/*pathology ; Cell Proliferation ; Cells, Cultured ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Mechanotransduction, Cellular ; Muscular Dystrophy, Duchenne/*complications/genetics/pathology ; Myocytes, Cardiac/*pathology ; Primary Cell Culture ; Transcription Factors/*deficiency/genetics ; YAP-Signaling Proteins ; },
abstract = {Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48-54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction were disorganized in DMD-iPSC-CMs. Lysophosphatidic acid (LPA), a known lipid mediator on induction of actin polymerization, significantly increased YAP activity and actin dynamics in DMD-iPSC-CMs using live cell imaging. These results suggested that altered YAP activity due to impaired actin dynamics reduced proliferative ability in DMD-iPSC-CMs. Hence, decreased YAP activity in dystrophic heart may contribute to DMD-cardiomyopathy pathogenesis.},
}
@article {pmid33989978,
year = {2021},
author = {Rawat, A and Roy, M and Jyoti, A and Kaushik, S and Verma, K and Srivastava, VK},
title = {Cysteine proteases: Battling pathogenic parasitic protozoans with omnipresent enzymes.},
journal = {Microbiological research},
volume = {249},
number = {},
pages = {126784},
doi = {10.1016/j.micres.2021.126784},
pmid = {33989978},
issn = {1618-0623},
mesh = {Animals ; Antiprotozoal Agents/*pharmacology ; CRISPR-Cas Systems ; Cysteine Endopeptidases/metabolism ; Cysteine Proteases/*metabolism ; Cysteine Proteinase Inhibitors/*pharmacology ; Entamoeba histolytica/drug effects/*enzymology/genetics ; Entamoebiasis/drug therapy/parasitology ; Humans ; Leishmania/drug effects/*enzymology/genetics ; Leishmaniasis/drug therapy/parasitology ; Malaria/drug therapy/parasitology ; Plasmodium/drug effects/*enzymology/genetics ; },
abstract = {Millions of people worldwide lie at the risk of parasitic protozoic infections that kill over a million people each year. The rising inefficacy of conventional therapeutics to combat these diseases, mainly due to the development of drug resistance to a handful of available licensed options contributes substantially to the rising burden of these ailments. Cysteine proteases are omnipresent enzymes that are critically implicated in the pathogenesis of protozoic infections. Despite their significance and druggability, cysteine proteases as therapeutic targets have not yet been translated into the clinic. The review presents the significance of cysteine proteases of members of the genera Plasmodium, Entamoeba, and Leishmania, known to cause Malaria, Amoebiasis, and Leishmaniasis, respectively, the protozoic diseases with the highest morbidity and mortality. Further, projecting them as targets for molecular tools like the CRISPR-Cas technology for favorable manipulation, exploration of obscure genomes, and achieving a better insight into protozoic functioning. Overcoming the hurdles that prevent us from gaining a better insight into the functioning of these enzymes in protozoic systems is a necessity. Managing the burden of parasitic protozoic infections pivotally depends upon the betterment of molecular tools and therapeutic concepts that will pave the path to an array of diagnostic and therapeutic applications.},
}
@article {pmid33989385,
year = {2021},
author = {Bandyopadhyay, S and Douglass, J and Kapell, S and Khan, N and Feitosa-Suntheimer, F and Klein, JA and Temple, J and Brown-Culbertson, J and Tavares, AH and Saeed, M and Lau, NC},
title = {DNA templates with blocked long 3' end single-stranded overhangs (BL3SSO) promote bona fide Cas9-stimulated homology-directed repair of long transgenes into endogenous gene loci.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {8},
pages = {},
pmid = {33989385},
issn = {2160-1836},
support = {R01 AG052465/AG/NIA NIH HHS/United States ; R01 GM135215/GM/NIGMS NIH HHS/United States ; R21 HD088792/HD/NICHD NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA ; Gene Editing ; HEK293 Cells ; Humans ; *Recombinational DNA Repair ; Transgenes ; },
abstract = {Knock-in of large transgenes by Cas9-mediated homology-directed repair (HDR) is an extremely inefficient process. Although the use of single-stranded oligonucleotides (ssODN) as an HDR donor has improved the integration of smaller transgenes, they do not support efficient insertion of large DNA sequences. In an effort to gain insights into the mechanism(s) governing the HDR-mediated integration of larger transgenes and to improve the technology, we conducted knock-in experiments targeting the human EMX1 locus and applied rigorous genomic PCR analyses in the human HEK293 cell line. This exercise revealed an unexpected molecular complication arising from the transgene HDR being initiated at the single homology arm and the subsequent genomic integration of plasmid backbone sequences. To pivot around this problem, we devised a novel PCR-constructed template containing blocked long 3' single-stranded overhangs (BL3SSO) that greatly improved the efficiency of bona fide Cas9-stimulated HDR at the EMX1 locus. We further refined BL3SSO technology and successfully used it to insert GFP transgenes into two important interferon-stimulated genes (ISGs) loci, Viperin/RSAD2, and ISG15. This study demonstrates the utility of the BL3SSO platform for inserting long DNA sequences into both constitutive and inducible endogenous loci to generate novel human cell lines for the study of important biological processes.},
}
@article {pmid33986266,
year = {2021},
author = {Hunt, C and Hartford, SA and White, D and Pefanis, E and Hanna, T and Herman, C and Wiley, J and Brown, H and Su, Q and Xin, Y and Voronin, D and Nguyen, H and Altarejos, J and Crosby, K and Haines, J and Cancelarich, S and Drummond, M and Moller-Tank, S and Malpass, R and Buckley, J and Del Pilar Molina-Portela, M and Droguett, G and Frendewey, D and Chiao, E and Zambrowicz, B and Gong, G},
title = {Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2770},
pmid = {33986266},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Expression/genetics ; Gene Expression Regulation/genetics ; Genetic Engineering/methods ; HEK293 Cells ; Humans ; Hypercholesterolemia/*genetics/pathology ; Liposomes/*pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Nanoparticles ; Prealbumin/genetics/*metabolism ; RNA, Guide/genetics/metabolism ; Transcriptional Activation/*genetics ; },
abstract = {CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.},
}
@article {pmid33986148,
year = {2021},
author = {Athukoralage, JS and White, MF},
title = {Cyclic oligoadenylate signalling and regulation by ring nucleases during type III CRISPR defence.},
journal = {RNA (New York, N.Y.)},
volume = {27},
number = {8},
pages = {855-867},
pmid = {33986148},
issn = {1469-9001},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {In prokaryotes, CRISPR-Cas immune systems recognise and cleave foreign nucleic acids to defend against Mobile Genetic Elements (MGEs). Type III CRISPR-Cas complexes also synthesise cyclic oligoadenylate (cOA) second messengers, which activate CRISPR ancillary proteins involved in antiviral defence. In particular, cOA-stimulated nucleases degrade RNA and DNA non-specifically, which slows MGE replication but also impedes cell growth, necessitating mechanisms to eliminate cOA in order to mitigate collateral damage. Extant cOA is degraded by a new class of enzyme termed a 'ring nuclease', which cleaves cOA specifically and switches off CRISPR ancillary enzymes. Several ring nuclease families have been characterised to date, including a family used by MGEs to circumvent CRISPR immunity, and encompass diverse protein folds and distinct cOA cleavage mechanisms. In this review we outline cOA signalling, discuss how different ring nucleases regulate the cOA signalling pathway, and reflect on parallels between cyclic nucleotide-based immune systems to reveal new areas for exploration.},
}
@article {pmid33984274,
year = {2021},
author = {Watson, BNJ and Steens, JA and Staals, RHJ and Westra, ER and van Houte, S},
title = {Coevolution between bacterial CRISPR-Cas systems and their bacteriophages.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {5},
pages = {715-725},
doi = {10.1016/j.chom.2021.03.018},
pmid = {33984274},
issn = {1934-6069},
mesh = {Bacteria/*genetics/immunology/*virology ; Bacteriophages/genetics/immunology/*physiology ; *Biological Evolution ; *CRISPR-Cas Systems ; Host-Pathogen Interactions ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive, heritable immunity against their viruses (bacteriophages and phages) and other parasitic genetic elements. CRISPR-Cas systems are highly diverse, and we are only beginning to understand their relative importance in phage defense. In this review, we will discuss when and why CRISPR-Cas immunity against phages evolves, and how this, in turn, selects for the evolution of immune evasion by phages. Finally, we will discuss our current understanding of if, and when, we observe coevolution between CRISPR-Cas systems and phages, and how this may be influenced by the mechanism of CRISPR-Cas immunity.},
}
@article {pmid33984272,
year = {2021},
author = {Mougiakos, I and Beisel, CL},
title = {CRISPR transposons on the move.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {5},
pages = {675-677},
doi = {10.1016/j.chom.2021.04.012},
pmid = {33984272},
issn = {1934-6069},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA Transposable Elements ; Recombination, Genetic ; },
abstract = {CRISPR transposons (CASTs) represent unique mobile genetic elements that co-opted CRISPR-Cas immune systems for RNA-guided DNA transposition. However, CAST-encoded CRISPR arrays rarely match the CAST's chromosomal location. A recent publication in Cell helps resolve this paradox by revealing CRISPR-array-independent mechanisms of chromosomal homing unique to different CAST types.},
}
@article {pmid33983378,
year = {2021},
author = {González-Delgado, A and Mestre, MR and Martínez-Abarca, F and Toro, N},
title = {Prokaryotic reverse transcriptases: from retroelements to specialized defense systems.},
journal = {FEMS microbiology reviews},
volume = {45},
number = {6},
pages = {},
pmid = {33983378},
issn = {1574-6976},
mesh = {*Bacteriophages/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Introns ; Prokaryotic Cells/metabolism ; RNA-Directed DNA Polymerase/genetics/metabolism ; *Retroelements/genetics ; },
abstract = {Reverse transcriptases (RTs) catalyze the polymerization of DNA from an RNA template. These enzymes were first discovered in RNA tumor viruses in 1970, but it was not until 1989 that they were found in prokaryotes as a key component of retrons. Apart from RTs encoded by the 'selfish' mobile retroelements known as group II introns, prokaryotic RTs are extraordinarily diverse, but their function has remained elusive. However, recent studies have revealed that different lineages of prokaryotic RTs, including retrons, those associated with CRISPR-Cas systems, Abi-like RTs and other yet uncharacterized RTs, are key components of different lines of defense against phages and other mobile genetic elements. Prokaryotic RTs participate in various antiviral strategies, including abortive infection (Abi), in which the infected cell is induced to commit suicide to protect the host population, adaptive immunity, in which a memory of previous infection is used to build an efficient defense, and other as yet unidentified mechanisms. These prokaryotic enzymes are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, we discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems.},
}
@article {pmid33982688,
year = {2021},
author = {Abe, K and Sugiyama, H and Endo, M},
title = {Construction of an optically controllable CRISPR-Cas9 system using a DNA origami nanostructure.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {45},
pages = {5594-5596},
doi = {10.1039/d1cc00876e},
pmid = {33982688},
issn = {1364-548X},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Cloning, Molecular/*methods ; DNA/*chemistry ; DNA Cleavage ; Enzyme Activation ; Nanostructures/*chemistry ; Photochemical Processes ; },
abstract = {We demonstrated the photo-controlled sequence-selective dsDNA cleavage using a DNA origami structure with Cas9 nuclease. The activity of Cas9 incorporated inside the ring-shaped DNA origami was completely suppressed. After photoirradiation, Cas9 was released, and its activity for selective dsDNA cleavage was restored.},
}
@article {pmid33982289,
year = {2021},
author = {Velíšková, J and Marra, C and Liu, Y and Shekhar, A and Park, DS and Iatckova, V and Xie, Y and Fishman, GI and Velíšek, L and Goldfarb, M},
title = {Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation.},
journal = {Epilepsia},
volume = {62},
number = {7},
pages = {1546-1558},
pmid = {33982289},
issn = {1528-1167},
support = {R01 NS092786/NS/NINDS NIH HHS/United States ; R01 HL142498/HL/NHLBI NIH HHS/United States ; R01 HL105983/HL/NHLBI NIH HHS/United States ; P30 CA196521/CA/NCI NIH HHS/United States ; F31 HL132438/HL/NHLBI NIH HHS/United States ; },
mesh = {Age of Onset ; Animals ; Animals, Newborn ; Arrhythmias, Cardiac/etiology/*genetics ; CRISPR-Cas Systems ; Electrocardiography ; Electroencephalography ; Epilepsy, Tonic-Clonic/genetics ; Fibroblast Growth Factors/*genetics ; Genotype ; Humans ; Mice ; Mice, Transgenic ; Mutation, Missense/genetics ; Oligonucleotides ; Seizures/etiology/genetics ; Spasms, Infantile/*genetics ; *Sudden Unexpected Death in Epilepsy ; Voltage-Gated Sodium Channels/metabolism ; },
abstract = {OBJECTIVE: Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice.<br><br>METHODS: The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1[R52H][/F+] mice. Spontaneous epileptiform events in Fhf1[R52H][/+] mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein.<br><br>RESULTS: All Fhf1[R52H][/+] mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26&#xa0;days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53&#xa0;s after lethal seizure onset, heart rate abruptly declined from 572&#xa0;&#xb1;&#xa0;16&#xa0;bpm to 108&#xa0;&#xb1;&#xa0;15&#xa0;bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1[R52H][/+] mice prior to seizure.<br><br>SIGNIFICANCE: The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.},
}
@article {pmid33981295,
year = {2021},
author = {Ramirez-Chamorro, L and Boulanger, P and Rossier, O},
title = {Strategies for Bacteriophage T5 Mutagenesis: Expanding the Toolbox for Phage Genome Engineering.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {667332},
pmid = {33981295},
issn = {1664-302X},
abstract = {Phage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli. We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5. Here, we present alternative strategies for the construction of mutants in T5. Bacterial retroelements (retrons) proved to be efficient for T5 gene editing by introducing point mutations in the essential gene A1. We set up a protocol based on dilution-amplification-screening (DAS) of phage pools for mutant enrichment that was used to introduce a conditional mutation in another essential gene (A2), insert a new gene (lacZα), and construct a translational fusion of a late phage gene with a fluorescent protein coding gene (pb10-mCherry). The method should be applicable to other virulent phages that are naturally resistant to CRISPR/Cas nucleases.},
}
@article {pmid33981145,
year = {2021},
author = {Izzah, SN and Setyanto, D and Hasanatuludhhiyah, N and Indiastuti, DN and Nasution, Z and d'Arqom, A},
title = {Attitudes of Indonesian Medical Doctors and Medical Students Toward Genome Editing.},
journal = {Journal of multidisciplinary healthcare},
volume = {14},
number = {},
pages = {1017-1027},
pmid = {33981145},
issn = {1178-2390},
abstract = {PURPOSE: This study aimed to measure the attitudes of Indonesian medical doctors and students toward new technology in genome editing.<br><br>MATERIALS AND METHODS: Online questionnaires regarding attitudes toward genome editing on health and non-health conditions, both in somatic cells and embryo, were distributed through researcher networks, email and social media specific to medical doctors and students. The data of 1055 valid questionnaires were processed; descriptive and association analyses between sociodemographic factors and attitudes toward genome editing were performed. Email in-depth interview was performed to explore the respondents' answers.<br><br>RESULTS: The results showed that Indonesian medical doctors' and students' knowledge of genome editing was limited and correlated with gender, place of residence, religion, education, marital status, childbearing and experience abroad. More than half of respondents supported genome editing for the treatment of fatal and debilitating diseases both in somatic cells and embryos, implying their consent to edited gene inheritance. However, this approval decreased when applied to non-health-related aspects, such as physical appearance, intelligence and strength. Factors affecting their attitudes toward genome editing included their status as medical doctors or students, gender, age, education, religion, economic status and place of residence.<br><br>CONCLUSION: Increasing knowledge and awareness of Indonesian medical doctors and students regarding genome editing is important. Even though its application in health-related matter was supported by a majority of the respondents, discussion from ethical and religious perspectives is necessary to ensure the acceptance.},
}
@article {pmid33981077,
year = {2021},
author = {DeFrancesco, L},
title = {Drug pipeline 1Q21-the old and the new.},
journal = {Nature biotechnology},
volume = {39},
number = {5},
pages = {536-537},
doi = {10.1038/s41587-021-00917-4},
pmid = {33981077},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Copy Number Variations/genetics ; Drug Approval ; Graft vs Host Disease/*drug therapy/genetics/pathology ; Humans ; Muromonab-CD3/*therapeutic use ; United States ; United States Food and Drug Administration ; },
}
@article {pmid33980985,
year = {2021},
author = {Stuart, WD and Fink-Baldauf, IM and Tomoshige, K and Guo, M and Maeda, Y},
title = {CRISPRi-mediated functional analysis of NKX2-1-binding sites in the lung.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {568},
pmid = {33980985},
issn = {2399-3642},
support = {U01 HL134745/HL/NHLBI NIH HHS/United States ; R01 CA240317/CA/NCI NIH HHS/United States ; },
mesh = {Binding Sites/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Transformation, Neoplastic/genetics ; Chromatin Immunoprecipitation Sequencing/methods ; Gene Expression Regulation, Neoplastic/genetics ; Genetic Engineering/methods ; Humans ; Lung/*pathology ; Lung Neoplasms/genetics ; Nuclear Proteins/genetics ; Promoter Regions, Genetic/genetics ; Protein Binding/genetics ; Thyroid Nuclear Factor 1/*genetics/*physiology ; Transcription Factors/genetics ; },
abstract = {The transcription factor NKX2-1/TTF-1 is involved in lung pathophysiology, including breathing, innate defense and tumorigenesis. To understand the mechanism by which NKX2-1 regulates genes involved in such pathophysiology, we have previously performed ChIP-seq and identified genome-wide NKX2-1-binding sites, which revealed that NKX2-1 binds to not only proximal promoter regions but also multiple intra- and inter-genic regions of the genes regulated by NKX2-1. However, the roles of such regions, especially non-proximal ones, bound by NKX2-1 have not yet been determined. Here, using CRISPRi (CRISPR/dCas9-KRAB), we scrutinize the functional roles of 19 regions/sites bound by NKX2-1, which are located in genes involved in breathing and innate defense (SFTPB, LAMP3, SFTPA1, SFTPA2) and lung tumorigenesis (MYBPH, LMO3, CD274/PD-L1). Notably, the CRISPRi approach reveals that a portion of NKX2-1-binding sites are functionally indispensable while the rest are dispensable for the expression of the genes, indicating that functional roles of NKX2-1-binding sites are unequally yoked.},
}
@article {pmid33980595,
year = {2021},
author = {Ibrahim, MK and Abdelhafez, TH and Takeuchi, JS and Wakae, K and Sugiyama, M and Tsuge, M and Ito, M and Watashi, K and El Kassas, M and Kato, T and Murayama, A and Suzuki, T and Chayama, K and Shimotohno, K and Muramatsu, M and Aly, HH and Wakita, T},
title = {MafF Is an Antiviral Host Factor That Suppresses Transcription from Hepatitis B Virus Core Promoter.},
journal = {Journal of virology},
volume = {95},
number = {15},
pages = {e0076721},
pmid = {33980595},
issn = {1098-5514},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Hep G2 Cells ; Hepatitis B virus/genetics/physiology ; Hepatitis B, Chronic/*pathology ; Humans ; Immunity, Innate/*immunology ; Interleukin-1beta/immunology ; MafF Transcription Factor/genetics/*metabolism ; Nuclear Proteins/genetics/*metabolism ; Promoter Regions, Genetic/genetics ; RNA Interference ; RNA, Small Interfering/genetics ; Transcription, Genetic/*genetics ; Tumor Necrosis Factor-alpha/immunology ; },
abstract = {Hepatitis B virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system, which is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Here, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using a small interfering RNA (siRNA) library and an HBV/NanoLuc (NL) reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of MafF led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutation of the putative MafF binding region. Loss of MafF expression through CRISPR/Cas9 editing (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB cells]) induced HBV core RNA and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to the HBV core promoter and competitively inhibits HNF-4α binding to an overlapping sequence in the HBV enhancer II sequence (EnhII), as seen by chromatin immunoprecipitation (ChIP) analysis. MafF expression was induced by interleukin-1β (IL-1β) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-dependent manner. Consistently, MafF expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase. IMPORTANCE HBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Therefore, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis and developing anti-HBV strategies. MafF was shown to suppress transcription from the HBV core promoter, leading to significant suppression of the HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB cells). This induction correlated with the levels of inflammatory cytokines (IL-1β and TNF-α). These data suggest that the induction of MafF contributes to the host's antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as an anti-HBV host restriction factor.},
}
@article {pmid33980455,
year = {2021},
author = {Durán-Vinet, B and Araya-Castro, K and Chao, TC and Wood, SA and Gallardo, V and Godoy, K and Abanto, M},
title = {Potential applications of CRISPR/Cas for next-generation biomonitoring of harmful algae blooms: A review.},
journal = {Harmful algae},
volume = {103},
number = {},
pages = {102027},
doi = {10.1016/j.hal.2021.102027},
pmid = {33980455},
issn = {1878-1470},
mesh = {Biological Monitoring ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ecosystem ; *Harmful Algal Bloom ; In Situ Hybridization, Fluorescence ; },
abstract = {Research on harmful algal and cyanobacterial blooms (HABs and CHABs) has risen dramatically due to their increasing global distribution, frequency, and intensity. These blooms jeopardize public health, ecosystem function, sustainability and can have negative economic impacts. Numerous monitoring programs have been established using light microscopy, liquid chromatography coupled to mass spectrometry (LC-MS), ELISA, and spectrophotometry to monitor HABs/CHABs outbreaks. Recently, DNA/RNA-based molecular methods have been integrated into these programs to replace or complement traditional methods through analyzing environmental DNA and RNA (eDNA/eRNA) with techniques such as quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH), sandwich hybridization assay (SHA), isothermal amplification methods, and microarrays. These have enabled the detection of rare or cryptic species, enhanced sample throughput, and reduced costs and the need for visual taxonomic expertise. However, these methods have limitations, such as the need for high capital investment in equipment or detection uncertainties, including determining whether organisms are viable. In this review, we discuss the potential of newly developed molecular diagnosis technology based on Clustered Regularly Interspaced Short Palindromic Repeats/Cas proteins (CRISPR/Cas), which utilizes the prokaryotic adaptative immune systems of bacteria and archaea. Cas12 and Cas13-based platforms can detect both DNA and RNA with attomolar sensitivity within an hour. CRISPR/Cas diagnostic is a rapid, inexpensive, specific, and ultrasensitive technology that, with some further development, will provide many new platforms that can be used for HABs/CHABs biomonitoring and research.},
}
@article {pmid33980009,
year = {2021},
author = {Qing, M and Chen, SL and Sun, Z and Fan, Y and Luo, HQ and Li, NB},
title = {Universal and Programmable Rolling Circle Amplification-CRISPR/Cas12a-Mediated Immobilization-Free Electrochemical Biosensor.},
journal = {Analytical chemistry},
volume = {93},
number = {20},
pages = {7499-7507},
doi = {10.1021/acs.analchem.1c00805},
pmid = {33980009},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; *Nucleic Acids ; },
abstract = {The development of a sensing platform with high sensitivity and specificity, especially programmability and universal applicability, for the detection of clinically relevant molecules is highly valuable for disease monitoring and confirmation but remains a challenge. Here, for the first time, we introduce the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system into an immobilization-free electrochemical biosensing platform for sensitively and specifically detecting the disease-related nucleic acids and small molecules. In this strategy, a modular rolling circle amplification (RCA) is designed to transform and amplify the target recognition event into the universal trigger DNA strand that is used as the trigger to activate the deoxyribonuclease activity of CRISPR/Cas12a for further signal amplification. The cleavage of the target-activated blocker probe allows the methylene blue-labeled reporter probes to be captured by the reduced graphene oxide-modified electrode, leading to an obviously increased electrochemical signal. We only need to simply tune the sequence for target recognition in RCA components, and this strategy can be flexibly applied to the highly sensitive and specific detection of microRNAs, Parvovirus B19 DNA, and adenosine-5'-triphosphate and the calculated limit of detection is 0.83 aM, 0.52 aM, and 0.46 pM, respectively. In addition, we construct DNA logic circuits (YES, NOT, OR, AND) of DNA inputs to experimentally demonstrate the modularity and programmability of the stimuli-responsive RCA-CRISPR/Cas12a system. This work broadens the application of the CRISPR/Cas12a system to the immobilization-free electrochemical biosensing platform and provides a new thinking for developing a robust tool for clinical diagnosis.},
}
@article {pmid33979618,
year = {2021},
author = {Hiatt, J and Cavero, DA and McGregor, MJ and Zheng, W and Budzik, JM and Roth, TL and Haas, KM and Wu, D and Rathore, U and Meyer-Franke, A and Bouzidi, MS and Shifrut, E and Lee, Y and Kumar, VE and Dang, EV and Gordon, DE and Wojcechowskyj, JA and Hultquist, JF and Fontaine, KA and Pillai, SK and Cox, JS and Ernst, JD and Krogan, NJ and Marson, A},
title = {Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins.},
journal = {Cell reports},
volume = {35},
number = {6},
pages = {109105},
pmid = {33979618},
issn = {2211-1247},
support = {T32 AI060537/AI/NIAID NIH HHS/United States ; T32 AI007334/AI/NIAID NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; U19 AI135990/AI/NIAID NIH HHS/United States ; R01 AI150449/AI/NIAID NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; P01 AI063302/AI/NIAID NIH HHS/United States ; R01 AI124471/AI/NIAID NIH HHS/United States ; P50 AI150476/AI/NIAID NIH HHS/United States ; S10 OD010786/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Genome/*genetics ; Humans ; Mice ; Myeloid Cells/*metabolism ; Ribonucleoproteins/*metabolism ; },
abstract = {Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14[+] human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.},
}
@article {pmid33979289,
year = {2022},
author = {Niu, M and Zou, Q},
title = {SgRNA-RF: Identification of SgRNA On-Target Activity With Imbalanced Datasets.},
journal = {IEEE/ACM transactions on computational biology and bioinformatics},
volume = {19},
number = {4},
pages = {2442-2453},
doi = {10.1109/TCBB.2021.3079116},
pmid = {33979289},
issn = {1557-9964},
mesh = {Algorithms ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *RNA, Guide/genetics ; },
abstract = {Single-guide RNA is a guide RNA (gRNA), which guides the insertion or deletion of uridine residues into kinetoplastid during RNA editing. It is a small non-coding RNA that can be combined with pre -mRNA pairing. SgRNA is a critical component of the CRISPR/Cas9 gene knockout system and play an important role in gene editing and gene regulation. It is important to accurately and quickly identify highly on-target activity sgRNAs. Due to its importance, several computational predictors have been proposed to predict sgRNAs on-target activity. All these methods have clearly contributed to the development of this very important field. However, they also have certain limitations. In the paper, we developed a new classifier SgRNA-RF, which extracts the features of nucleic acid composition and structure of on-target activity sgRNA sequence and identified by random forest algorithm. In addition to solving an imbalanced dataset, this paper proposed a new method called CS-Smote. We compared sgRNA-RF with state-of-the-art predictors on the five datasets, and found SgRNA-RF significantly improved the identification accuracy, with accuracies of 0.8636,0.9161,0.894,0.938,0.965,0.77,0.979,0.973, respectively. The user-friendly web server that implements sgRNA-RF is freely available at http://server.malab.cn/sgRNA-RF/.},
}
@article {pmid33979199,
year = {2021},
author = {Yeung, TK and Lau, HW and Ma, HT and Poon, RYC},
title = {One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines.},
journal = {Molecular biology of the cell},
volume = {32},
number = {14},
pages = {1320-1330},
pmid = {33979199},
issn = {1939-4586},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Expression/genetics ; Gene Expression Regulation/*genetics/physiology ; Gene Silencing/*physiology ; Genes, Essential/genetics ; Genes, Reporter/genetics ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; Humans ; Indoleacetic Acids/metabolism ; Loss of Function Mutation/genetics ; Promoter Regions, Genetic/genetics ; Transfection ; Transposases/genetics/metabolism ; },
abstract = {Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.},
}
@article {pmid33979119,
year = {2021},
author = {Ramachandran, A and Santiago, JG},
title = {CRISPR Enzyme Kinetics for Molecular Diagnostics.},
journal = {Analytical chemistry},
volume = {93},
number = {20},
pages = {7456-7464},
doi = {10.1021/acs.analchem.1c00525},
pmid = {33979119},
issn = {1520-6882},
mesh = {*COVID-19 ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Kinetics ; Pandemics ; Pathology, Molecular ; SARS-CoV-2 ; },
abstract = {CRISPR-diagnostic assays have gained significant interest in the last few years. This interest has grown rapidly during the current COVID-19 pandemic, where CRISPR-diagnostics have been frontline contenders for rapid testing solutions. This surge in CRISPR-diagnostic research prompts the following question: what exactly are the achievable limits of detection and associated assay times enabled by the kinetics of enzymes such as Cas12 and Cas13? To explore this question, we here present a model based on Michaelis-Menten enzyme kinetics theory applied to CRISPR enzymes. We use the model to develop analytical solutions for reaction kinetics and develop back-of-the-envelope criteria to validate and check for consistency in reported enzyme kinetic parameters. We applied our analyses to all studies known to us, which report Michaelis-Menten-type kinetic data for CRISPR-associated enzymes. These studies include all subtypes of Cas12 and Cas13 and orthologs. We found all but one study clearly violate at least two of our three rules and therefore present data that violate basic physical limits. We performed an experimental study of reaction kinetics of LbCas12a with both ssDNA and dsDNA activators and use these data to validate our model and its predicted scaling. The validated model is used to explore CRISPR reaction time scales and the degree of reaction completion for practically relevant target concentrations applicable to CRISPR-diagnostic assays. The results have broad implications for achievable limits of detection and assay times of emerging, amplification-free CRISPR-detection methods.},
}
@article {pmid33978171,
year = {2021},
author = {Miura, K and Ogura, A and Kobatake, K and Honda, H and Kaminuma, O},
title = {Progress of genome editing technology and developmental biology useful for radiation research.},
journal = {Journal of radiation research},
volume = {62},
number = {Supplement_1},
pages = {i53-i63},
pmid = {33978171},
issn = {1349-9157},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Developmental Biology ; *Gene Editing ; Genome ; Humans ; *Radiation ; *Research ; },
abstract = {Following the development of genome editing technology, it has become more feasible to create genetically modified animals such as knockout (KO), knock-in, and point-mutated animals. The genome-edited animals are useful to investigate the roles of various functional genes in many fields of biological science including radiation research. Nevertheless, some researchers may experience difficulty in generating genome-edited animals, probably due to the requirement for equipment and techniques for embryo manipulation and handling. Furthermore, after obtaining F0 generation, genome-edited animals generally need to be expanded and maintained for analyzing the target gene function. To investigate genes essential for normal birth and growth, the generation of conditional KO (cKO) animals in which a tissue- or stage-specific gene mutation can be introduced is often required. Here, we describe the basic principle and application of genome editing technology including zinc-finger nuclease, transcription-activator-like effector nuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas) systems. Recently advanced developmental biology methods have enabled application of the technology, especially CRISPR/Cas, to zygotes, leading to the prompt production of genome-edited animals. For pre-implantation embryos, genome editing via oviductal nucleic acid delivery has been developed as an embryo manipulation- or handling-free method. Examining the gene function at F0 generation is becoming possible by employing triple-target CRISPR technology. This technology, in combination with a blastocyst complementation method enables investigation of even birth- and growth-responsible genes without establishing cKO strains. We hope that this review is helpful for understanding and expanding genome editing-related technology and for progressing radiation research.},
}
@article {pmid33977487,
year = {2021},
author = {Budagyan, K and Chernoff, J},
title = {A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2262},
number = {},
pages = {323-334},
pmid = {33977487},
issn = {1940-6029},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Colon/*metabolism ; Epithelial Cells/*metabolism ; *Gene Editing ; Genetic Engineering/*methods ; Mice ; *Mutation ; Proto-Oncogene Proteins p21(ras)/antagonists &amp; inhibitors/*genetics ; },
abstract = {Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS mutations observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. Although clinical evidence suggests biological differences between mutant KRAS alleles, these differences and the mechanisms underlying them are not well understood, and further exploration of allele-specific differences may provide evidence for individualized therapeutics. One approach to study allelic variation involves the use of isogenic cell lines that express different endogenous KRAS mutants. Here we developed an assay using fluorescent co-selection for CRISPR-driven gene editing to generate various Kras mutations in an isogenic murine colon epithelial cell line background. This assay involves generation of a cell line stably expressing Cas9 linked to BFP and simultaneous introduction of single-guide RNAs (sgRNAs) to two different gene loci resulting in double-editing events. Single-stranded donor oligonucleotides are introduced for a GFP gene and a Kras mutant allele of our choice as templates for homologous recombination (HDR). Cells that successfully undergo HDR are GFP-positive and have a higher probability of containing the desired Kras mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent Kras edits. Ultimately, this method allows us to toggle between different mutant alleles and preserve the wild-type allele while maintaining an isogenic background.},
}
@article {pmid33977326,
year = {2021},
author = {Biswas, S and Zhang, D and Shi, J},
title = {CRISPR/Cas systems: opportunities and challenges for crop breeding.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {979-998},
pmid = {33977326},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Gene Editing/legislation &amp; jurisprudence/*methods ; Genome, Plant ; Haploidy ; Hybrid Vigor/genetics ; Mutation ; Plant Breeding/legislation &amp; jurisprudence/*methods ; Plants, Genetically Modified/*genetics ; Synthetic Biology ; },
abstract = {Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.},
}
@article {pmid33976199,
year = {2021},
author = {Kim, T and Weinberg, B and Wong, W and Lu, TK},
title = {Scalable recombinase-based gene expression cascades.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2711},
pmid = {33976199},
issn = {2041-1723},
support = {R01 GM129011/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Cell Differentiation ; Gene Editing/*methods ; *Gene Regulatory Networks ; Genetic Engineering/*methods ; Genetic Loci ; *Genome, Human ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Plasmids/chemistry/*metabolism ; RNA, Guide/genetics/metabolism ; Transcription, Genetic ; Transposases/*genetics/metabolism ; },
abstract = {Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.},
}
@article {pmid33976171,
year = {2021},
author = {Li, Z and Marcel, N and Devkota, S and Auradkar, A and Hedrick, SM and Gantz, VM and Bier, E},
title = {CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2625},
pmid = {33976171},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; *DNA End-Joining Repair ; Drosophila/genetics ; Feasibility Studies ; Female ; *Gene Conversion ; Gene Editing/*methods ; Genetic Loci ; Genetic Therapy/methods ; HEK293 Cells ; Humans ; Male ; Models, Animal ; Proto-Oncogene Proteins c-myc/genetics ; *Recombinational DNA Repair ; },
abstract = {CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.},
}
@article {pmid33975753,
year = {2021},
author = {Gold, A and Levanon, EY and Eisenberg, E},
title = {The New RNA-Editing Era - Ethical Considerations.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {8},
pages = {685-687},
doi = {10.1016/j.tig.2021.04.013},
pmid = {33975753},
issn = {0168-9525},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/ethics ; Genetic Engineering/*ethics ; Genome, Human/genetics ; Humans ; RNA Editing/*ethics ; },
abstract = {The characteristics of RNA editing, including the lower risk compared with genome editing, may loosen the ethical barriers that are currently imposed on genetic engineering, thus opening new possibilities for research, therapy, and human enhancement. We should start considering the future ethical and social implications of this new and promising technology.},
}
@article {pmid33975701,
year = {2021},
author = {Li, B and Shao, Z and Chen, Y},
title = {An exonuclease protection and CRISPR/Cas12a integrated biosensor for the turn-on detection of transcription factors in cancer cells.},
journal = {Analytica chimica acta},
volume = {1165},
number = {},
pages = {338478},
doi = {10.1016/j.aca.2021.338478},
pmid = {33975701},
issn = {1873-4324},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Humans ; *Neoplasms ; Transcription Factors/genetics/metabolism ; },
abstract = {Transcription factors (TFs) are critical proteins that regulate the expression of genes, and the abnormal change of TFs levels is directly related to physical dysfunctions. Herein, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensor for the measurement of TFs level with the assistance of exonuclease protection assay. A dsDNA (activator) with the ability to activate Cas12a was engineered to contain TFs binding domain, and the binding between TFs and the activator can protect the dsDNA from being digested by exonuclease III (Exo III). The reserved activator then triggered a CRISPR/Cas12a reporting reaction to produce fluorescent signal for detection. In the detection of nuclear factor-kappa B (NF-κB) p50 subunit, the limit of detection of 0.2 pM and limit of quantification of 0.6 pM were obtained respectively, and the performance of this biosensor has been challenged by cell nucleoprotein extracts. Additionally, this method can be applied in the screening and evaluation of TFs inhibitors, calculating the IC50 of oridonin. Integrating merits including high sensitivity, low cost, and good portability, this method may enrich the arsenal for TFs-related applications.},
}
@article {pmid33975697,
year = {2021},
author = {Abnous, K and Danesh, NM and Ramezani, M and Alibolandi, M and Nameghi, MA and Zavvar, TS and Taghdisi, SM},
title = {A novel colorimetric aptasensor for ultrasensitive detection of aflatoxin M1 based on the combination of CRISPR-Cas12a, rolling circle amplification and catalytic activity of gold nanoparticles.},
journal = {Analytica chimica acta},
volume = {1165},
number = {},
pages = {338549},
doi = {10.1016/j.aca.2021.338549},
pmid = {33975697},
issn = {1873-4324},
mesh = {Aflatoxin M1/analysis ; *Biosensing Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colorimetry ; Gold ; Limit of Detection ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques ; },
abstract = {Colorimetric approaches have received noticeable attention among sensing methods in view of simplicity and watching the color change of sample by the naked eyes. However, developing colorimetric sensing methods which show high sensitivity is still problematic. Herein, based on CRISPR-Cas12a, rolling circle amplification (RCA) and catalytic activity of gold nanoparticles (AuNPs), a colorimetric aptasensor was introduced for highly sensitive detection of aflatoxin M1 (AFM1). In the presence of AFM1, the CRISPR-Cas12a is inactivated and large single-stranded DNA (ssDNA) structures are formed on the surface of AuNPs following the addition of T4 DNA ligase and phi29 DNA polymerase. So, the sample color remains yellow after addition of 4-nitrophenol. However, no huge DNA structure is observed on the surface of AuNPs in the absence of target because of activation of CRISPR-Cas12a and digestion of primer. So, the color of sample switches to colorless. The results indicated that the biosensor had high selectivity toward AFM1 and the approach achieved a detection limit as low as 0.05 ng/L. In addition, it could sensitively identify AFM1 in the spiked milk samples. Overall, this approach is highly sensitive and does not require sophisticated equipment. Therefore, it maintains promising potential for other mycotoxins detection in real samples by simply replacing the applied sequences.},
}
@article {pmid33974980,
year = {2021},
author = {Süntar, I and Çetinkaya, S and Haydaroğlu, &#xdc;S and Habtemariam, S},
title = {Bioproduction process of natural products and biopharmaceuticals: Biotechnological aspects.},
journal = {Biotechnology advances},
volume = {50},
number = {},
pages = {107768},
doi = {10.1016/j.biotechadv.2021.107768},
pmid = {33974980},
issn = {1873-1899},
mesh = {*Biological Products ; Biotechnology ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {Decades of research have been put in place for developing sustainable routes of bioproduction of high commercial value natural products (NPs) on the global market. In the last few years alone, we have witnessed significant advances in the biotechnological production of NPs. The development of new methodologies has resulted in a better understanding of the metabolic flux within the organisms, which have driven manipulations to improve production of the target product. This was further realised due to the recent advances in the omics technologies such as genomics, transcriptomics, proteomics, metabolomics and secretomics, as well as systems and synthetic biology. Additionally, the combined application of novel engineering strategies has made possible avenues for enhancing the yield of these products in an efficient and economical way. Invention of high-throughput technologies such as next generation sequencing (NGS) and toolkits for genome editing Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) have been the game changers and provided unprecedented opportunities to generate rationally designed synthetic circuits which can produce complex molecules. This review covers recent advances in the engineering of various hosts for the production of bioactive NPs and biopharmaceuticals. It also highlights general approaches and strategies to improve their biosynthesis with higher yields in a perspective of plants and microbes (bacteria, yeast and filamentous fungi). Although there are numerous reviews covering this topic on a selected species at a time, our approach herein is to give a comprehensive understanding about state-of-art technologies in different platforms of organisms.},
}
@article {pmid33973766,
year = {2021},
author = {Chen, Y and Wu, S and Wu, H and Cheng, P and Wang, X and Qian, S and Zhang, M and Xu, J and Ji, F and Wu, J},
title = {CRISPR/Cas12a-Based Versatile Method for Checking Quantitative Polymerase Chain Reaction Samples with Cycles of Threshold Values in the Gray Zone.},
journal = {ACS sensors},
volume = {6},
number = {5},
pages = {1963-1970},
doi = {10.1021/acssensors.1c00515},
pmid = {33973766},
issn = {2379-3694},
mesh = {*African Swine Fever Virus ; Animals ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Polymerase Chain Reaction ; SARS-CoV-2 ; Swine ; },
abstract = {Quantitative polymerase chain reaction (qPCR) is widely applied in foodborne pathogen detection and diagnosis. According to the cycles of threshold (Ct) values of qPCR testing, samples are judged as positive or negative. However, samples with Ct values in the gray zone are classified as "possibly positive" and required to be tested again. Repetitive qPCR may not eliminate the uncertain results but increase the workload of detection. CRISPR/Cas12a can specifically recognize the nucleic acid of the nM level and then indiscriminately slash the single-strand DNA with multiple turnovers. In this way, the detection signals can be greatly amplified. Here, we propose a CRISPR-based checking method to solve gray zone problems. After qPCR testing, the screening gray zone samples can be successfully checked by the CRISPR/Cas12a method. Furthermore, to conduct CRISPR reaction assay more conveniently and prevent possible aerosol contamination in the operational process, a gray zone checking cassette is designed. African swine fever virus (ASFV) is selected as an example to demonstrate the feasibility of the CRISPR-based checking method. Of 28 real swine blood samples, 6 ASFV qPCR gray zone samples are successfully checked. The CRISPR-based checking method provides a novel solution to eliminate gray zone sample problems with no additional effects on the PCR, which is operable and applicable in practical detection. The entire process can be completed within 10-15 min. This method will be a good supplementary and assistance for qPCR-based detection, especially in the diagnosis of diseases such as COVID-19.},
}
@article {pmid33973451,
year = {2021},
author = {Xue, D and Zhu, H and Du, W and Tang, H and Huang, Q},
title = {[Structure-based optimization and design of CRISPR protein xCas9].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {4},
pages = {1385-1395},
doi = {10.13345/j.cjb.200396},
pmid = {33973451},
issn = {1872-2075},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.},
}
@article {pmid33973436,
year = {2021},
author = {Shi, M and Shen, Z and Zhang, N and Wang, L and Yu, C and Yang, Z},
title = {[CRISPR/Cas9 technology in disease research and therapy: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {4},
pages = {1205-1228},
doi = {10.13345/j.cjb.200401},
pmid = {33973436},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Technology ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; },
abstract = {Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.},
}
@article {pmid33973295,
year = {2021},
author = {Dhakal, P and Spencer, TE},
title = {Generation and analysis of Prss28 and Prss29 deficient mice using CRISPR-Cas9 genome-editing.},
journal = {Molecular reproduction and development},
volume = {88},
number = {7},
pages = {482-489},
pmid = {33973295},
issn = {1098-2795},
support = {R01 HD096266/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Endometrium/metabolism ; Female ; Gene Editing/methods ; Gene Knockout Techniques/*methods ; Male ; Mice ; Mice, Knockout ; Pregnancy ; Sequence Deletion/physiology ; Serine Endopeptidases/*genetics/metabolism ; Uterus/metabolism ; },
abstract = {Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands and a critical regulator of their differentiation, development and function. Progesterone and FOXA2 regulate members of a serine proteinase gene family (Prss28 and Prss29). Here, CRISPR-Cas9 genome-editing was used to create mice with a heterozygous or homozygous deletion of Prss28 or/and Prss29 to determine their biological roles in uterine function. Female mice lacking Prss28 and Prss29 or both developed normally and were fertile without alterations in uterine histoarchitecture, uterine gland number, or and gene expression. Thus, Prss28 and Prss29 are dispensable for female fertility and do not impact endometrial gland development or uterine function mice.},
}
@article {pmid33972728,
year = {2021},
author = {Wang, L and Xue, W and Zhang, H and Gao, R and Qiu, H and Wei, J and Zhou, L and Lei, YN and Wu, X and Li, X and Liu, C and Wu, J and Chen, Q and Ma, H and Huang, X and Cai, C and Zhang, Y and Yang, B and Yin, H and Yang, L and Chen, J},
title = {Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations.},
journal = {Nature cell biology},
volume = {23},
number = {5},
pages = {552-563},
pmid = {33972728},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics/metabolism ; *Gene Editing/methods ; HEK293 Cells ; Humans ; Mice ; Mutation/*genetics ; Proprotein Convertase 9/*genetics/metabolism ; },
abstract = {The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30-40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.},
}
@article {pmid33972655,
year = {2021},
author = {Li, PP and Margolis, RL},
title = {Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {9865},
pmid = {33972655},
issn = {2045-2322},
support = {R01 NS100783/NS/NINDS NIH HHS/United States ; NS112687/NS/NINDS NIH HHS/United States ; NS100783/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; DNA Breaks, Single-Stranded ; DNA End-Joining Repair ; Deoxyribonuclease I/genetics/*metabolism ; Electroporation/methods ; Exons/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genome, Human ; Humans ; Induced Pluripotent Stem Cells/*transplantation ; Karyotyping ; Mutation ; Nerve Tissue Proteins/genetics ; Protein Phosphatase 2/genetics ; RNA, Guide/genetics/metabolism ; Spinocerebellar Ataxias/genetics/*therapy ; },
abstract = {Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable "on-target" indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without "on target" indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.},
}
@article {pmid33972524,
year = {2021},
author = {Catlett, TS and Onesto, MM and McCann, AJ and Rempel, SK and Glass, J and Franz, DN and Gómez, TM},
title = {RHOA signaling defects result in impaired axon guidance in iPSC-derived neurons from patients with tuberous sclerosis complex.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2589},
pmid = {33972524},
issn = {2041-1723},
support = {R01 NS099405/NS/NINDS NIH HHS/United States ; R01 NS113314/NS/NINDS NIH HHS/United States ; T32 EY027721/EY/NEI NIH HHS/United States ; T32 GM007215/GM/NIGMS NIH HHS/United States ; },
mesh = {Adolescent ; Axon Guidance/drug effects/*genetics ; Biopsy ; CRISPR-Cas Systems ; Cell Line ; Ephrins/pharmacology ; Fluorescence Resonance Energy Transfer ; Haploinsufficiency ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Myosins/metabolism ; Nerve Net/*metabolism/pathology ; Neurogenesis/drug effects/*genetics ; Neurons/drug effects/*metabolism ; Organoids/cytology/metabolism ; Protein Biosynthesis/drug effects/genetics ; Signal Transduction/drug effects/genetics ; TOR Serine-Threonine Kinases/metabolism ; Tuberous Sclerosis/genetics/*metabolism ; Tuberous Sclerosis Complex 2 Protein/genetics/*metabolism ; rhoA GTP-Binding Protein/*metabolism ; },
abstract = {Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.},
}
@article {pmid33972106,
year = {2021},
author = {Goell, JH and Hilton, IB},
title = {CRISPR/Cas-Based Epigenome Editing: Advances, Applications, and Clinical Utility.},
journal = {Trends in biotechnology},
volume = {39},
number = {7},
pages = {678-691},
doi = {10.1016/j.tibtech.2020.10.012},
pmid = {33972106},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; Epigenome/genetics ; Epigenomics/trends ; *Gene Editing/trends ; Humans ; },
abstract = {The epigenome dynamically regulates gene expression and guides cellular differentiation throughout the lifespan of eukaryotic organisms. Recent advances in clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based epigenome editing technologies have enabled researchers to site-specifically program epigenetic modifications to endogenous DNA and histones and to manipulate the architecture of native chromatin. As a result, epigenome editing has helped to uncover the causal relationships between epigenetic marks and gene expression. As epigenome editing tools have continued to develop, researchers have applied them in new ways to explore the function of the epigenome in human health and disease. In this review, we discuss the recent technical improvements in CRISPR/Cas-based epigenome editing that have advanced clinical research and examine how these technologies could be improved for greater future utility.},
}
@article {pmid33971367,
year = {2021},
author = {Wei, Z and Abdelrahman, M and Gao, Y and Ji, Z and Mishra, R and Sun, H and Sui, Y and Wu, C and Wang, C and Zhao, K},
title = {Engineering broad-spectrum resistance to bacterial blight by CRISPR-Cas9-mediated precise homology directed repair in rice.},
journal = {Molecular plant},
volume = {14},
number = {8},
pages = {1215-1218},
doi = {10.1016/j.molp.2021.05.012},
pmid = {33971367},
issn = {1752-9867},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/*genetics ; Disease Susceptibility ; Gene Expression Regulation, Plant ; Monosaccharide Transport Proteins/*genetics/metabolism ; Oryza/*genetics/growth &amp; development/microbiology ; Plant Diseases/microbiology ; Plant Proteins/genetics/metabolism ; Xanthomonas/*physiology ; },
}
@article {pmid33970941,
year = {2021},
author = {MacLeod, RS and Meyer, MB and Xiong, J and Cawley, KM and Liu, Y and Onal, M and Benkusky, NA and Thostenson, JD and Pike, JW and O'Brien, CA},
title = {Deletion of a putative promoter-proximal Tnfsf11 regulatory region in mice does not alter bone mass or Tnfsf11 expression in vivo.},
journal = {PloS one},
volume = {16},
number = {5},
pages = {e0250974},
pmid = {33970941},
issn = {1932-6203},
support = {I01 BX000294/BX/BLRD VA/United States ; P20 GM125503/GM/NIGMS NIH HHS/United States ; R01 AR049794/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Bone Density/*physiology ; CRISPR-Cas Systems ; Cells, Cultured ; Female ; Lymphocytes/physiology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; Osteoclasts/cytology/*physiology ; Parathyroid Hormone/metabolism ; Promoter Regions, Genetic ; RANK Ligand/*genetics/metabolism ; Regulatory Sequences, Nucleic Acid ; },
abstract = {The cytokine RANKL is essential for osteoclast formation during physiological and pathological bone resorption. RANKL also contributes to lymphocyte production, development of lymph nodes and mammary glands, as well as other biological activities. Transcriptional control of the Tnfsf11 gene, which encodes RANKL, is complex and involves distant regulatory regions. Nevertheless, cell culture studies suggest that an enhancer region near the transcription start site is involved in the control of Tnfsf11 expression by hormones such as 1,25-(OH)2 vitamin D3 and parathyroid hormone, as well as the sympathetic nervous system. To address the significance of this region in vivo, we deleted the sequence between -510 to -1413 bp, relative to Tnfsf11 exon 1, from mice using CRISPR-based gene editing. MicroCT analysis of the femur and fourth lumbar vertebra of enhancer knockout mice showed no differences in bone mass compared to wild type littermates at 5 weeks and 6 months of age, suggesting no changes in osteoclast formation. RNA extracted from the tibia, fifth lumbar vertebra, thymus, and spleen at 6 months of age also showed no reduction in Tnfsf11 mRNA abundance between these groups. However, maximal stimulation of Tnfsf11 mRNA abundance in cultured stromal cells by PTH was reduced approximately 40% by enhancer deletion, while stimulation by 1,25-(OH)2 vitamin D3 was unaffected. The abundance of B and T lymphocytes in the bone marrow did not differ between genotypes. These results demonstrate that the region between -510 and -1413 does not contribute to Tnfsf11 expression, osteoclast support, or lymphocyte production in mice under normal physiological conditions but may be involved in situations of elevated parathyroid hormone.},
}
@article {pmid33970916,
year = {2021},
author = {Hibara, KI and Miya, M and Benvenuto, SA and Hibara-Matsuo, N and Mimura, M and Yoshikawa, T and Suzuki, M and Kusaba, M and Taketa, S and Itoh, JI},
title = {Regulation of the plastochron by three many-noded dwarf genes in barley.},
journal = {PLoS genetics},
volume = {17},
number = {5},
pages = {e1009292},
pmid = {33970916},
issn = {1553-7404},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Cell Division ; *Gene Expression Regulation, Plant ; *Genes, Plant ; Hordeum/cytology/*genetics/*growth &amp; development ; Mutation ; Oryza/genetics ; Phenotype ; Plant Cells ; Plant Leaves/cytology/*genetics/*growth &amp; development ; Time Factors ; },
abstract = {The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.},
}
@article {pmid33970666,
year = {2021},
author = {Akbaba, H and Erel-Akbaba, G and Senturk, S},
title = {Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing.},
journal = {Nanomedicine (London, England)},
volume = {16},
number = {12},
pages = {963-978},
doi = {10.2217/nnm-2020-0412},
pmid = {33970666},
issn = {1748-6963},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Lipids ; *Nanoparticles ; Transfection ; },
abstract = {Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials &amp; methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.},
}
@article {pmid33970608,
year = {2021},
author = {Hoque, ME and Mustafa, G and Basu, S and Balci, H},
title = {Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.},
journal = {ACS synthetic biology},
volume = {10},
number = {5},
pages = {972-978},
pmid = {33970608},
issn = {2161-5063},
support = {R15 GM123443/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA Cleavage ; *G-Quadruplexes ; Gene Expression Regulation ; Humans ; Mutation ; Neuroblastoma/genetics/pathology ; Promoter Regions, Genetic ; Transcription, Genetic/*genetics ; Transfection ; Tyrosine 3-Monooxygenase/genetics ; },
abstract = {Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.},
}
@article {pmid33969526,
year = {2021},
author = {Attenello, FJ and Tsung, K and Bishara, I and Loh, YE and Chen, TC},
title = {In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models.},
journal = {Journal of neuroscience research},
volume = {99},
number = {9},
pages = {2029-2045},
doi = {10.1002/jnr.24850},
pmid = {33969526},
issn = {1097-4547},
mesh = {Animals ; Brain Neoplasms/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Knockout Techniques/methods ; Glioblastoma/*genetics/pathology ; HEK293 Cells ; Humans ; Male ; Mice ; Mice, Nude ; RNA, Untranslated/*genetics ; Tumor Burden/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat)-based genetic screens offer unbiased and powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR screens have been utilized heavily in vitro to identify functional coding and noncoding genes in a large number of cell types, including glioblastoma (GB), though no prior study has described the evaluation of CRISPR screening in GB in vivo. Here, we describe a protocol for targeting and transcriptionally repressing GB-specific long noncoding RNAs (lncRNAs) by CRISPR interference (CRISPRi) system in vivo, with tumor growth in the mouse cerebral cortex. Given the target-specific parameters of each individual screen, we list general steps involved in transducing guide RNA libraries into GB tumor lines, maintaining sufficient coverage, as well as cortically injecting and subsequently isolating transduced screen tumor cell populations for analysis. Finally, in order to demonstrate the use of this technique to discern an essential lncRNA, HOTAIR, from a nonessential lncRNA, we injected a 1:1 (HOTAIR:control nonessential lncRNA knockdown) mixture of fluorescently tagged U87 GB cells into the cortex of eight mice, evaluating selective depletion of HOTAIR-tagged cells at 2 weeks of growth. Fluorescently tagged populations were analyzed via flow cytometry for hiBFP (control knockdown) and green fluorescent protein (HOTAIR knockdown), revealing 17% (p = 0.007) decrease in fluorescence associated with HOTAIR knockdown relative to control. The described in vivo CRISPR screening methodology thus appears to be an effective option for identifying noncoding (and coding) genes affecting GB growth within the mouse cortex.},
}
@article {pmid33969146,
year = {2021},
author = {Singsuksawat, E and Onnome, S and Posiri, P and Suphatrakul, A and Srisuk, N and Nantachokchawapan, R and Praneechit, H and Sae-Kow, C and Chidpratum, P and Sa-Ngiamsuntorn, K and Hongeng, S and Avirutnan, P and Duangchinda, T and Siridechadilok, B},
title = {Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by VLP.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {21},
number = {},
pages = {729-740},
pmid = {33969146},
issn = {2329-0501},
abstract = {With sequencing as a standard frontline protocol to identify emerging viruses such Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), direct utilization of sequence data to program antivirals against the viruses could accelerate drug development to treat their infections. CRISPR-Cas effectors are promising candidates that could be programmed to inactivate viral genetic material based on sequence data, but several challenges such as delivery and design of effective CRISPR RNA (crRNA) need to be addressed to realize practical use. Here, we showed that virus-like particle (VLP) could deliver PspCas13b-crRNA ribonucleoprotein (RNP) in nanomolar range to efficiently suppress dengue virus infection in primary human target cells. Shortening spacer length could significantly enhance RNA-targeting efficiency of PspCas13b in mammalian cells compared to the natural length of 30 nucleotides without compromising multiplex targeting by a crRNA array. Our results demonstrate the potentials of applying PspCas13b RNP to suppress RNA virus infection, with implications in targeting host RNA as well.},
}
@article {pmid33968799,
year = {2021},
author = {Ding, R and Long, J and Yuan, M and Jin, Y and Yang, H and Chen, M and Chen, S and Duan, G},
title = {CRISPR/Cas System: A Potential Technology for the Prevention and Control of COVID-19 and Emerging Infectious Diseases.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {639108},
pmid = {33968799},
issn = {2235-2988},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Communicable Diseases, Emerging/prevention &amp; control ; Humans ; SARS-CoV-2 ; Technology ; },
abstract = {The continued global pandemic of coronavirus disease 2019 (COVID-19) poses a serious threat to global public health and social stability and it has become a serious global public health problem. Unfortunately, existing diagnostic and therapeutic approaches for the prevention and control of COVID-19 have many shortcomings. In recent years, the emerging CRISPR/Cas technology can complement the problems of traditional methods. Biological tools based on CRISPR/Cas systems have been widely used in biomedicine. In particular, they are advantageous in pathogen detection, clinical antiviral therapy, drug, and vaccine development. Therefore, CRISPR/Cas technology may have great potential for application in the prevention and control of COVID-19 and emerging infectious diseases in the future. This article summarizes the existing applications of CRISPR/Cas technology in infectious diseases with the aim of providing effective strategies for the prevention and control of COVID-19 and other emerging infectious diseases in the future.},
}
@article {pmid33964942,
year = {2021},
author = {Goedegebuure, RSA and Kleibeuker, EA and Buffa, FM and Castricum, KCM and Haider, S and Schulkens, IA and Ten Kroode, L and van den Berg, J and Jacobs, MAJM and van Berkel, AM and van Grieken, NCT and Derks, S and Slotman, BJ and Verheul, HMW and Harris, AL and Thijssen, VL},
title = {Interferon- and STING-independent induction of type I interferon stimulated genes during fractionated irradiation.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {40},
number = {1},
pages = {161},
pmid = {33964942},
issn = {1756-9966},
mesh = {Animals ; Astrocytoma/genetics/immunology/metabolism/radiotherapy ; Cell Line, Tumor ; Colonic Neoplasms/genetics/immunology/metabolism/radiotherapy ; Dose Fractionation, Radiation ; Esophageal Neoplasms/genetics/immunology/metabolism/*radiotherapy ; Female ; Gene Expression Regulation, Neoplastic/*radiation effects ; HT29 Cells ; Humans ; Immunity/radiation effects ; Interferon Type I/*genetics/immunology/metabolism ; Membrane Proteins/*genetics/immunology/metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Random Allocation ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Improvement of radiotherapy efficacy requires better insight in the dynamic responses that occur during irradiation. Here, we aimed to identify the molecular responses that are triggered during clinically applied fractionated irradiation.<br><br>METHODS: Gene expression analysis was performed by RNAseq or microarray analysis of cancer cells or xenograft tumors, respectively, subjected to 3-5&#x2009;weeks of 5&#x2009;&#xd7;&#x2009;2&#x2009;Gy/week. Validation of altered gene expression was performed by qPCR and/or ELISA in multiple cancer cell lines as well as in pre- and on-treatment biopsies from esophageal cancer patients (NCT02072720). Targeted protein inhibition and CRISPR/Cas-induced gene knockout was used to analyze the role of type I interferons and cGAS/STING signaling pathway in the molecular and cellular response to fractionated irradiation.<br><br>RESULTS: Gene expression analysis identified type I interferon signaling as the most significantly enriched biological process induced during fractionated irradiation. The commonality of this response was confirmed in all irradiated cell lines, the xenograft tumors and in biopsies from esophageal cancer patients. Time-course analyses demonstrated a peak in interferon-stimulated gene (ISG) expression within 2-3&#x2009;weeks of treatment. The response was accompanied by a variable induction of predominantly interferon-beta and/or -lambda, but blocking these interferons did not affect ISG expression induction. The same was true for targeted inhibition of the upstream regulatory STING protein while knockout of STING expression only delayed the ISG expression induction.<br><br>CONCLUSIONS: Collectively, the presented data show that clinically applied fractionated low-dose irradiation can induce a delayed type I interferon response that occurs independently of interferon expression or STING signaling. These findings have implications for current efforts that aim to target the type I interferon response for cancer treatment.},
}
@article {pmid33964631,
year = {2021},
author = {Zhao, Y and Chen, Y and Li, X and Sun, Y and Shao, Y and Zhang, Y and Liu, Z},
title = {RIPK1 regulates cell function and death mediated by UVB radiation and TNF-α.},
journal = {Molecular immunology},
volume = {135},
number = {},
pages = {304-311},
doi = {10.1016/j.molimm.2021.04.024},
pmid = {33964631},
issn = {1872-9142},
mesh = {Apoptosis/*physiology ; CRISPR-Cas Systems/genetics ; Cell Line, Transformed ; Cell Proliferation/physiology ; Cell Survival/physiology ; Epidermal Cells/*pathology ; G2 Phase Cell Cycle Checkpoints/physiology ; Gene Knockout Techniques ; HaCaT Cells ; Humans ; Interleukin-1alpha/metabolism ; NF-kappa B/metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/genetics/*metabolism ; Signal Transduction/physiology ; Skin/injuries ; Tumor Necrosis Factor-alpha/*metabolism ; Ultraviolet Rays/*adverse effects ; p38 Mitogen-Activated Protein Kinases/metabolism ; },
abstract = {The RIP family plays a key role in mediating cell inflammation, oxidative stress and death. Among them, RIPK1, as an important regulatory factor in the upstream of the NF-κB pathway, is involved in multiple pathways of cell inflammation and death. Epidermal cells constitute the outermost barrier of the human body. Radiation can induce epidermal cell death, inflammation and oxidative stress to cause damage. Therefore, this paper selected HaCaT cell and used CRISPR/Cas technology to construct a cell model of stable knockout of RIPK1 gene, to analyze the effect and regulation of RIPK1 knockout on the function and death of HaCaT cells induced by UVB or TNF-α. The results showed that knockout of RIPK1 had no significant effect on the morphology of HaCaT cells at rest, but it led to slowing cell proliferation and blocking the G2M phase of cell cycle. Compared with HaCaT[WT], HaCaT[RIP1KO] was abnormally sensitive to TNF-α-induced cell death and apoptosis, and may be associated with inhibition of NF-κB pathway. Knocking out RIPK1 led to a more significant inhibition of cell growth by UVB, and up-regulation of the expression of the inflammatory factor IL-1α. P38 MAPK and NF-κB pathways may be involved this process. This study further found that RIPK1 in epidermal cell has a regulatory function on pro-survival signals.},
}
@article {pmid33964614,
year = {2021},
author = {Slaymaker, IM and Gaudelli, NM},
title = {Engineering Cas9 for human genome editing.},
journal = {Current opinion in structural biology},
volume = {69},
number = {},
pages = {86-98},
doi = {10.1016/j.sbi.2021.03.004},
pmid = {33964614},
issn = {1879-033X},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
abstract = {Since the initial reports describing CRISPR-Cas9, labs across the globe have leveraged this valuable gene editing tool to alter the genomes of living cells. With the goal of generating more precise and efficient genome changes, scientists and engineers have mutated, evolved, and covalently altered Cas9 in order to predictably edit the genetic code. Here, we highlight recent advancements and contributions to the growing field of Cas9 engineering. We present key aspects of Cas9 engineering efforts focused on sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements of this important gene-modifying tool.},
}
@article {pmid33964295,
year = {2021},
author = {Zhang, L and Ding, L and Li, Y and Zhang, F and Xu, Y and Pan, H and Wan, X and Yan, G and Yu, F and Li, R},
title = {EHD3 positively regulated by NR5A1 participates in testosterone synthesis via endocytosis.},
journal = {Life sciences},
volume = {278},
number = {},
pages = {119570},
doi = {10.1016/j.lfs.2021.119570},
pmid = {33964295},
issn = {1879-0631},
mesh = {Animals ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*metabolism ; Chromatin Immunoprecipitation ; *Endocytosis ; Exosomes/*metabolism ; Female ; *Gene Expression Regulation ; Leydig Cells/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plasmids/metabolism ; Promoter Regions, Genetic ; RNA, Small Interfering/metabolism ; Steroidogenic Factor 1/genetics/*metabolism ; Testosterone/*metabolism/pharmacology ; },
abstract = {AIMS: Increasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis.<br><br>MAIN METHODS: Testosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology.<br><br>KEY FINDINGS: EHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence "AGGTCA" in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1[f/f] mice.<br><br>SIGNIFICANCE: This study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health.},
}
@article {pmid33964175,
year = {2021},
author = {Qiao, X and Gao, Y and Li, J and Wang, Z and Qiao, H and Qi, H},
title = {Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {8},
pages = {3037-3045},
doi = {10.1002/bit.27813},
pmid = {33964175},
issn = {1097-0290},
mesh = {CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Eubacterium/*chemistry ; *Polymorphism, Single Nucleotide ; RNA/*chemistry/*genetics ; Ruminococcus/*chemistry ; },
abstract = {RNA-guided CRISPR (RNA-targeting clustered regularly interspaced short palindromic repeats) effector Cas13d is the smallest Class II subtype VI proteins identified so far. Here, two recently identified Cas13d effectors from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) were characterized and applied for sensitive nucleic acid detection. We demonstrated that the special target triggered collateral cleavage of these two Cas13d orthologs could provide rapid target RNA detection in picomolar range and then the tolerance for mismatch between crRNA and target RNA was characterized as well. Finally, an additional single mismatch was introduced into crRNA to enhance the two Cas13d orthologs mediated detection of low variant allele fraction, 0.1% T790M. Overall, this study demonstrated that both EsCas13d and RspCas13d could robustly detect target RNA carrying special single-nucleotide variation with high specificity and sensitivity, thereby providing newly qualified machinery in toolbox for efficient molecular diagnostics.},
}
@article {pmid33963853,
year = {2021},
author = {Mariyappa, D and Luhur, A and Overton, D and Zelhof, AC},
title = {Generation of Drosophila attP containing cell lines using CRISPR-Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {8},
pages = {},
pmid = {33963853},
issn = {2160-1836},
support = {P40 OD010949/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Drosophila/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism ; Integrases/genetics/metabolism ; },
abstract = {The generation of Drosophila stable cell lines has become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here, we describe a toolkit and procedure that combines CRISPR/Cas9 and theaaa PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.},
}
@article {pmid33963717,
year = {2021},
author = {Zhang, T and Li, W and Qiu, X and Liu, B and Li, G and Feng, C and Liao, J and Lin, K},
title = {[CRISPR/Cas9-mediated TEAD1 knockout induces phenotypic modulation of corpus cavernosum smooth muscle cells in diabetic rats with erectile dysfunction].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {41},
number = {4},
pages = {567-573},
pmid = {33963717},
issn = {1673-4254},
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; *Diabetes Mellitus, Experimental/complications ; *Erectile Dysfunction/genetics ; Male ; Myocytes, Smooth Muscle ; Nuclear Proteins ; Penis ; Phenotype ; Rats ; Rats, Sprague-Dawley ; TEA Domain Transcription Factors ; Transcription Factors/genetics ; },
abstract = {OBJECTIVE: To construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED.<br><br>OBJECTIVE: Models of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively.<br><br>OBJECTIVE: The primarily cultured CCSMCs from diabetic rats with ED showed a high &#x3b1;-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED.<br><br>OBJECTIVE: We successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.},
}
@article {pmid33963661,
year = {2021},
author = {Nadeem, M and Chen, A and Hong, H and Li, D and Li, J and Zhao, D and Wang, W and Wang, X and Qiu, L},
title = {GmMs1 encodes a kinesin-like protein essential for male fertility in soybean (Glycine max L.).},
journal = {Journal of integrative plant biology},
volume = {63},
number = {6},
pages = {1054-1064},
doi = {10.1111/jipb.13110},
pmid = {33963661},
issn = {1744-7909},
mesh = {Plant Breeding ; Plant Infertility/genetics/physiology ; Plant Proteins/genetics/*metabolism ; Soybeans/genetics/*metabolism ; Transcriptome/genetics ; },
abstract = {The application of heterosis is a promising approach for greatly increasing yield in soybean (Glycine max L.). Nuclear male sterility is essential for hybrid seed production and the utilization of heterosis. Here we report the cloning of the gene underlying the soybean male-sterile mutant ms-1, which has been widely used for recurrent selection in soybean breeding programs. We initially delimited the ms1 locus to a 16.15 kb region on chromosome 13, based on SLAF_BSA sequencing followed by genotyping of an F2 population segregating for the locus. Compared with the same region in fertile plants, the mutant region lacks a sequence of approximately 38.7 kb containing five protein-coding genes, including an ortholog of the kinesin-like protein gene NACK2, named GmMs1. The GmMs1 knockout plants generated via CRISPR/Cas-mediated gene editing displayed a complete male-sterile phenotype. Metabolic profiling showed that fertile anthers accumulated starch and sucrose normally, whereas sterile anthers had higher anthocyanin levels and lower flavonoid levels and lower antioxidant enzyme activities. These results provide insights into the molecular mechanisms governing male sterility and demonstrate that GmMs1 could be used to create male-sterile lines through targeted mutagenesis. These findings pave the way for designing seed production technology and an intelligent male-sterile line system to utilize heterosis in soybean.},
}
@article {pmid33963452,
year = {2021},
author = {Das, AK and Chichghare, SA and Sharma, SK and Kumar, JPT and Singh, S and Baranwal, VK and Kumar, A and Nerkar, S},
title = {Genetic diversity and population structure of 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing in India based on the prophage types.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {37},
number = {6},
pages = {95},
pmid = {33963452},
issn = {1573-0972},
mesh = {CRISPR-Cas Systems ; Citrus/*microbiology ; DNA, Viral/genetics ; Genetic Variation ; India ; Liberibacter/*classification/isolation &amp; purification/virology ; Molecular Typing ; Phylogeny ; Plant Diseases/microbiology ; Prophages/classification/*genetics ; Sequence Analysis, DNA/*methods ; },
abstract = {Huanglongbing (HLB), also known as 'citrus greening', is an extremely destructive disease of citrus worldwide. HLB is associated with three species of the fastidious proteobacterium, Candidatus Liberibacter asiaticus (CaLas), Ca. L. africanus and Ca. L. americanus with CaLas being the most widely distributed around the world and the only species detected and described so far in India, one of the major global citrus fruit producers. Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. Three types of prophages, Type 1, Type 2 and Type 3 have been identified and described in CaLas so far. In the present study, 441 CaLas isolates sampled across 18 Indian states were used for prophage typing. Based on detection of three prophage types by PCR, all the eight probable combinations of CaLas prophages were identified, including single Type 1 (26.5%), single Type 2 (18.8%), single Type 3 (1.4%), Type 1 + Type 2 (20.4%), Type 1 + Type 3 (12.5%), Type 2 + Type 3 (4.8%), Type 1 + Type 2 + Type 3 (11.3%) and None type (4.3%). Prophage types were confirmed by PCR amplicon sequencing and subsequent phylogenetic analysis. By discovery of all 3 prophages and based on genetic identity and genetic distance, CaLas populations from eighteen citrus growing states were separated into two major Prophage Typing Groups (PTGs): PTG1 and PTG2. The PTG1 comprised of CaLas from North-West India and PTG2 from rest of the country (North-East, Central and South India), and both major groups were further divided into two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) subgroups respectively. The findings of CaLas population patterns provide evidence for independent origins of HLB-associated CaLas. CRISPR (clustered regularly interspaced short palindromic repeats) array was also detected in CaLas isolates. This is the first report evaluating the genetic variation of a large population of CaLas bacterium in India using the PCR markers from the prophage regions which would certainly assist the ongoing HLB management efforts in India.},
}
@article {pmid33963188,
year = {2021},
author = {Mathyer, ME and Brettmann, EA and Schmidt, AD and Goodwin, ZA and Oh, IY and Quiggle, AM and Tycksen, E and Ramakrishnan, N and Matkovich, SJ and Guttman-Yassky, E and Edwards, JR and de Guzman Strong, C},
title = {Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2557},
pmid = {33963188},
issn = {2041-1723},
support = {R25 GM103757/GM/NIGMS NIH HHS/United States ; R00 AR055948/AR/NIAMS NIH HHS/United States ; C06 RR015502/RR/NCRR NIH HHS/United States ; R01 AR065523/AR/NIAMS NIH HHS/United States ; UL1 TR002345/TR/NCATS NIH HHS/United States ; R56 AR075427/AR/NIAMS NIH HHS/United States ; T32 GM007067/GM/NIGMS NIH HHS/United States ; T32 HG000045/HG/NHGRI NIH HHS/United States ; R01 LM013096/LM/NLM NIH HHS/United States ; },
mesh = {Africa ; Alleles ; Animals ; CRISPR-Cas Systems ; Chromatin/genetics/metabolism ; Chromatin Immunoprecipitation Sequencing ; Databases, Genetic ; *Enhancer Elements, Genetic ; Gene Expression Regulation/*genetics ; Gene Frequency ; Haplotypes ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Protein Precursors/*genetics/metabolism ; Quantitative Trait Loci ; RNA-Seq ; Regulatory Sequences, Nucleic Acid ; Skin/*metabolism ; },
abstract = {The genetic modules that contribute to human evolution are poorly understood. Here we investigate positive selection in the Epidermal Differentiation Complex locus for skin barrier adaptation in diverse HapMap human populations (CEU, JPT/CHB, and YRI). Using Composite of Multiple Signals and iSAFE, we identify selective sweeps for LCE1A-SMCP and involucrin (IVL) haplotypes associated with human migration out-of-Africa, reaching near fixation in European populations. CEU-IVL is associated with increased IVL expression and a known epidermis-specific enhancer. CRISPR/Cas9 deletion of the orthologous mouse enhancer in vivo reveals a functional requirement for the enhancer to regulate Ivl expression in cis. Reporter assays confirm increased regulatory and additive enhancer effects of CEU-specific polymorphisms identified at predicted IRF1 and NFIC binding sites in the IVL enhancer (rs4845327) and its promoter (rs1854779). Together, our results identify a selective sweep for a cis regulatory module for CEU-IVL, highlighting human skin barrier evolution for increased IVL expression out-of-Africa.},
}
@article {pmid33961975,
year = {2021},
author = {Lin, Z and Li, H and He, L and Jing, Y and Pistolozzi, M and Wang, T and Ye, Y},
title = {Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a.},
journal = {Gene},
volume = {790},
number = {},
pages = {145693},
doi = {10.1016/j.gene.2021.145693},
pmid = {33961975},
issn = {1879-0038},
mesh = {Bacterial Proteins/*antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome, Bacterial ; Plasmids ; Pseudomonas aeruginosa/*genetics/growth &amp; development/metabolism ; },
abstract = {The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.},
}
@article {pmid33961623,
year = {2021},
author = {Oura, S and Koyano, T and Kodera, C and Horisawa-Takada, Y and Matsuyama, M and Ishiguro, KI and Ikawa, M},
title = {KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice.},
journal = {PLoS genetics},
volume = {17},
number = {5},
pages = {e1009412},
pmid = {33961623},
issn = {1553-7404},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; },
mesh = {Anaphase ; Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/deficiency/genetics/*metabolism ; Cell Nucleus/metabolism ; Chromosomal Proteins, Non-Histone/deficiency/genetics/*metabolism ; Chromosome Pairing ; Conserved Sequence ; DNA Damage ; Evolution, Molecular ; Fertility/genetics ; *Genes, Essential ; Histone Deacetylase 1/metabolism ; Male ; *Meiosis ; Meiotic Prophase I ; Metaphase ; Mice ; Nuclear Proteins/deficiency/genetics/*metabolism ; Pachytene Stage ; Phenotype ; Spermatids/cytology ; Spermatocytes/cytology/metabolism ; Testis/metabolism ; Transcription Factors/deficiency/genetics/*metabolism ; Transgenes ; },
abstract = {Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.},
}
@article {pmid33961493,
year = {2021},
author = {Huang, K and Yu, H and Chen, Z and Lin, G and Zhang, Z and Zhang, X and Dong, Y and Chen, H and Zhang, Z and Ma, W and Wu, Y and Liu, T},
title = {CRISPR-Cas13a-based diagnostic method for Chlamydia trachomatis from nongonococcal urethritis.},
journal = {Bioanalysis},
volume = {13},
number = {11},
pages = {901-912},
doi = {10.4155/bio-2021-0022},
pmid = {33961493},
issn = {1757-6199},
mesh = {CRISPR-Cas Systems/*genetics ; Chlamydia Infections/*diagnosis ; Chlamydia trachomatis/*isolation &amp; purification ; Humans ; Polymerase Chain Reaction ; Urethritis/*diagnosis ; },
abstract = {Aim: Development of a routine screening technique for Chlamydia trachomatis infection. The proposed approach involves the CRISPR RNA (crRNA). In the presence of the target sequence, the RNase activity of the Cas13a protein is activated, and it cleaves RNA fluorescent probe so that fluorescence will be emitted. Results: The sensitivity of the detection based on CRISPR-Cas13a was 10 fM. The results obtained by CRISPR-Cas13a and quantitative polymerase chain reaction were closely correlated: χ[2] = 81.798 (p < 0.001). Conclusion: The method can be carried out at room temperature and yields results within 2 h. The developed technique does not require expensive instruments and, thus, can meet the needs of community hospitals and other institutions for screening.},
}
@article {pmid33961413,
year = {2021},
author = {Yue, H and Huang, M and Tian, T and Xiong, E and Zhou, X},
title = {Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies.},
journal = {ACS nano},
volume = {15},
number = {5},
pages = {7848-7859},
doi = {10.1021/acsnano.1c02372},
pmid = {33961413},
issn = {1936-086X},
mesh = {Biological Assay ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Nanotechnology ; },
abstract = {Clustered, regularly interspaced short palindromic repeats (CRISPR)-based diagnoses, derived from gene-editing technology, have been exploited for less than 5 years and are now reaching the stage of precommercial use. CRISPR tools have some notable features, such as recognition at physiological temperature, excellent specificity, and high-efficiency signal amplification capabilities. These characteristics are promising for the development of next-generation diagnostic technologies. In this Perspective, we present a detailed summary of which micro/nanotechnologies play roles in the advancement of CRISPR diagnosis and how they are involved. The use of nanoprobes, nanochips, and nanodevices, microfluidic technology, lateral flow strips, etc. in CRISPR detection systems has led to new opportunities for CRISPR-based diagnosis assay development, such as achieving equipment-free detection, providing more compact detection systems, and improving sensitivity and quantitative capabilities. Although tremendous progress has been made, CRISPR diagnosis has not yet reached its full potential. We discuss upcoming opportunities and improvements and how micro/nanotechnologies will continue to play key roles.},
}
@article {pmid33961220,
year = {2021},
author = {Zoledowska, S and Motyka-Pomagruk, A and Misztak, A and Lojkowska, E},
title = {Comparative Genomics, from the Annotated Genome to Valuable Biological Information: A Case Study.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2242},
number = {},
pages = {91-112},
pmid = {33961220},
issn = {1940-6029},
mesh = {DNA, Bacterial/*genetics ; Databases, Genetic ; Dickeya/*genetics ; *Genome, Bacterial ; *Genomics ; *High-Throughput Nucleotide Sequencing ; Pectobacterium/*genetics ; Research Design ; *Sequence Analysis, DNA ; Software Design ; Workflow ; },
abstract = {High availability of fast, cheap, and high-throughput next generation sequencing techniques resulted in acquisition of numerous de novo sequenced and assembled bacterial genomes. It rapidly became clear that digging out useful biological information from such a huge amount of data presents a considerable challenge. In this chapter we share our experience with utilization of several handy open source comparative genomic tools. All of them were applied in the studies focused on revealing inter- and intraspecies variation in pectinolytic plant pathogenic bacteria classified to Dickeya solani and Pectobacterium parmentieri. As the described software performed well on the species within the Pectobacteriaceae family, it presumably may be readily utilized on some closely related taxa from the Enterobacteriaceae family. First of all, implementation of various annotation software is discussed and compared. Then, tools computing whole genome comparisons including generation of circular juxtapositions of multiple sequences, revealing the order of synteny blocks or calculation of ANI or Tetra values are presented. Besides, web servers intended either for functional annotation of the genes of interest or for detection of genomic islands, plasmids, prophages, CRISPR/Cas are described. Last but not least, utilization of the software designed for pangenome studies and the further downstream analyses is explained. The presented work not only summarizes broad possibilities assured by the comparative genomic approach but also provides a user-friendly guide that might be easily followed by nonbioinformaticians interested in undertaking similar studies.},
}
@article {pmid33960612,
year = {2021},
author = {Zhang, X and Xu, G and Cheng, C and Lei, L and Sun, J and Xu, Y and Deng, C and Dai, Z and Yang, Z and Chen, X and Liu, C and Tang, Q and Su, J},
title = {Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Hemp (Cannabis Sativa L.).},
journal = {Plant biotechnology journal},
volume = {19},
number = {10},
pages = {1979-1987},
pmid = {33960612},
issn = {1467-7652},
mesh = {*Agrobacterium/genetics ; CRISPR-Cas Systems/genetics ; *Cannabis/genetics ; Gene Editing ; Mutagenesis ; Plants, Genetically Modified/genetics ; Transformation, Genetic ; },
abstract = {Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.},
}
@article {pmid33960379,
year = {2021},
author = {Triozzi, PM and Schmidt, HW and Dervinis, C and Kirst, M and Conde, D},
title = {Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.},
journal = {Tree physiology},
volume = {41},
number = {11},
pages = {2216-2227},
pmid = {33960379},
issn = {1758-4469},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; *Populus/genetics ; RNA, Guide/genetics ; },
abstract = {Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.},
}
@article {pmid33959107,
year = {2021},
author = {Steczkiewicz, K and Prestel, E and Bidnenko, E and Szczepankowska, AK},
title = {Expanding Diversity of Firmicutes Single-Strand Annealing Proteins: A Putative Role of Bacteriophage-Host Arms Race.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {644622},
pmid = {33959107},
issn = {1664-302X},
abstract = {Bacteriophage-encoded single strand annealing proteins (SSAPs) are recombinases which can substitute the classical, bacterial RecA and manage the DNA metabolism at different steps of phage propagation. SSAPs have been shown to efficiently promote recombination between short and rather divergent DNA sequences and were exploited for in vivo genetic engineering mainly in Gram-negative bacteria. In opposition to the conserved and almost universal bacterial RecA protein, SSAPs display great sequence diversity. The importance for SSAPs in phage biology and phage-bacteria evolution is underlined by their role as key players in events of horizontal gene transfer (HGT). All of the above provoke a constant interest for the identification and study of new phage recombinase proteins in vivo, in vitro as well as in silico. Despite this, a huge body of putative ssap genes escapes conventional classification, as they are not properly annotated. In this work, we performed a wide-scale identification, classification and analysis of SSAPs encoded by the Firmicutes bacteria and their phages. By using sequence similarity network and gene context analyses, we created a new high quality dataset of phage-related SSAPs, substantially increasing the number of annotated SSAPs. We classified the identified SSAPs into seven distinct families, namely RecA, Gp2.5, RecT/Redβ, Erf, Rad52/22, Sak3, and Sak4, organized into three superfamilies. Analysis of the relationships between the revealed protein clusters led us to recognize Sak3-like proteins as a new distinct SSAP family. Our analysis showed an irregular phylogenetic distribution of ssap genes among different bacterial phyla and specific phages, which can be explained by the high rates of ssap HGT. We propose that the evolution of phage recombinases could be tightly linked to the dissemination of bacterial phage-resistance mechanisms (e.g., abortive infection and CRISPR/Cas systems) targeting ssap genes and be a part of the constant phage-bacteria arms race.},
}
@article {pmid33958776,
year = {2021},
author = {Rönspies, M and Dorn, A and Schindele, P and Puchta, H},
title = {CRISPR-Cas-mediated chromosome engineering for crop improvement and synthetic biology.},
journal = {Nature plants},
volume = {7},
number = {5},
pages = {566-573},
pmid = {33958776},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Plant/*genetics ; *Crop Production/methods ; Crops, Agricultural/genetics ; Genetic Engineering/*methods ; Plants, Genetically Modified/*genetics ; *Synthetic Biology/methods ; },
abstract = {Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR-Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR-Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant chromosome, but also of novel plant species.},
}
@article {pmid33958590,
year = {2021},
author = {Wang, JY and Hoel, CM and Al-Shayeb, B and Banfield, JF and Brohawn, SG and Doudna, JA},
title = {Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2571},
pmid = {33958590},
issn = {2041-1723},
support = {DP2 GM123496/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Proteins/*chemistry/metabolism ; *CRISPR-Cas Systems ; Catalytic Domain ; Cryoelectron Microscopy ; Endonucleases/*chemistry ; Escherichia coli/metabolism ; Integrases/*chemistry ; Piscirickettsiaceae/*chemistry/enzymology/metabolism ; RNA-Directed DNA Polymerase/*chemistry ; Recombinant Proteins ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.},
}
@article {pmid33958317,
year = {2021},
author = {Wang, X and Wu, B and Sui, X and Zhang, Z and Liu, T and Li, Y and Hu, G and He, M and Peng, N},
title = {CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {2},
pages = {115-122},
doi = {10.1016/j.jgg.2021.02.012},
pmid = {33958317},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Damage/*genetics ; DNA End-Joining Repair/*genetics ; DNA, Bacterial/genetics ; Gene Dosage ; Genome, Bacterial/*genetics ; Plasmids/genetics ; Zymomonas/*genetics ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements (MGEs) through uptake of invader-derived spacers. De novo adaptation samples spacers from both invaders and hosts, whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis. Self-derived spacers will lead to CRISPR self-interference. However, our in vivo study demonstrated that this species used the microhomology-mediated end joining (MMEJ) pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers. MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers. Importantly, CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA. Moreover, CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway, probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA in Z. mobilis. Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element (MGE) DNA, and maintains genome stability post CRISPR immunity in Z. mobilis.},
}
@article {pmid33958293,
year = {2021},
author = {Bonaccorsi-Riani, E and Gillooly, A and Brüggenwirth, IMA and Martins, PN},
title = {Delivery of genetic load during ex situ liver machine perfusion with potential for CRISPR-Cas9 gene editing: An innovative strategy for graft treatment.},
journal = {Hepatobiliary &amp; pancreatic diseases international : HBPD INT},
volume = {20},
number = {5},
pages = {503-505},
doi = {10.1016/j.hbpd.2021.04.006},
pmid = {33958293},
issn = {1499-3872},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Load ; Humans ; Liver/surgery ; Perfusion ; },
}
@article {pmid33957124,
year = {2021},
author = {Lora, J and Weskamp, G and Li, TM and Maretzky, T and Shola, DTN and Monette, S and Lichtenthaler, SF and Lu, TT and Yang, C and Blobel, CP},
title = {Targeted truncation of the ADAM17 cytoplasmic domain in mice results in protein destabilization and a hypomorphic phenotype.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100733},
pmid = {33957124},
issn = {1083-351X},
support = {R01 AI079178/AI/NIAID NIH HHS/United States ; F31 GM136144/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; T32 GM008539/GM/NIGMS NIH HHS/United States ; R35 GM134907/GM/NIGMS NIH HHS/United States ; S10 OD019986/OD/NIH HHS/United States ; },
mesh = {ADAM17 Protein/*chemistry/genetics/*metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cytoplasm/*metabolism ; Female ; Fibroblasts/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Phenotype ; Protein Domains ; Protein Stability ; Sequence Deletion ; },
abstract = {A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17-/- mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function.},
}
@article {pmid33957108,
year = {2021},
author = {Dong, Y and Bai, H and Dong, F and Zhang, XB and Ema, H},
title = {Gene knockout in highly purified mouse hematopoietic stem cells by CRISPR/Cas9 technology.},
journal = {Journal of immunological methods},
volume = {495},
number = {},
pages = {113070},
doi = {10.1016/j.jim.2021.113070},
pmid = {33957108},
issn = {1872-7905},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Gene Knockout Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Hematopoietic Stem Cells/immunology/*metabolism ; Mice, Inbred C57BL ; Mice, Transgenic ; PAX5 Transcription Factor/deficiency/*genetics ; Phenotype ; RNA, Guide/genetics/metabolism ; Transduction, Genetic ; },
abstract = {The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201[+]CD150[+]CD48[-]c-Kit[+]Sca-1[+]Lin[-] cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201[+]CD150[-]CD48[-] c-Kit[+]Sca-1[+]Lin[-] cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.},
}
@article {pmid33956991,
year = {2021},
author = {Singroha, G and Sharma, P and Sunkur, R},
title = {Current status of microRNA-mediated regulation of drought stress responses in cereals.},
journal = {Physiologia plantarum},
volume = {172},
number = {3},
pages = {1808-1821},
doi = {10.1111/ppl.13451},
pmid = {33956991},
issn = {1399-3054},
mesh = {Abscisic Acid ; *Droughts ; Edible Grain/genetics ; Gene Expression Regulation, Plant ; *MicroRNAs/genetics ; Stress, Physiological/genetics ; },
abstract = {Drought is one of the most important abiotic stress factors impeding crop productivity. With the uncovering of their role as potential regulators of gene expression, microRNAs (miRNAs) have been recognized as new targets for developing stress resistance. MicroRNAs are small noncoding RNAs whose abundance is significantly altered under stress conditions. Interestingly, plant miRNAs predominantly targets transcription factors (TFs), and some of which are also the most critical drought-responsive genes that in turn could regulate the expression of numerous loci with drought-adaptive potential. The phytohormone ABA plays important roles in regulating stomatal conductance and in initiating an adaptive response to drought stress. miRNAs are implicated in regulating ABA-(abscisic acid) and non-ABA-mediated drought resistance pathways. For instance, miR159-MYB module and miR169-NFYA module participates in an ABA-dependent pathway, whereas several other ABA-independent miRNA-target modules (miR156-SPL; miR393-TIR1; miR160-ARF10, ARF16, ARF17; miR167-ARF6 and ARF8; miR390/TAS3siRNA-ARF2, ARF3, ARF4) collectively regulate drought responses in plants. Overall, miRNA-mediated drought response manifests diverse molecular, biochemical and physiological processes. Because of their immense role in controlling gene expression, miRNA manipulation has significant potential to augment plant tolerance to drought stress. This review compiles the current understanding of drought-responsive miRNAs in major cereals. Also, potential miRNA manipulation strategies currently in use along with the challenges and future perspectives are discussed.},
}
@article {pmid33956155,
year = {2021},
author = {Diehl, V and Wegner, M and Grumati, P and Husnjak, K and Schaubeck, S and Gubas, A and Shah, VJ and Polat, IH and Langschied, F and Prieto-Garcia, C and Müller, K and Kalousi, A and Ebersberger, I and Brandts, CH and Dikic, I and Kaulich, M},
title = {Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.},
journal = {Nucleic acids research},
volume = {49},
number = {10},
pages = {5684-5704},
pmid = {33956155},
issn = {1362-4962},
mesh = {Autophagy/*genetics ; Autophagy-Related Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Carcinoma, Squamous Cell/mortality ; Cell Proliferation/genetics ; Cell Survival/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Databases, Genetic ; Gene Knockout Techniques/*methods ; Gene Library ; Gene Regulatory Networks/*genetics ; Genes, Essential ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Kaplan-Meier Estimate ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Lung Neoplasms/mortality ; Models, Genetic ; RNA, Guide ; RNA-Seq ; Vesicular Transport Proteins/genetics/metabolism ; },
abstract = {Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.},
}
@article {pmid33955811,
year = {2021},
author = {Klitzman, R},
title = {Preparing for the Next Generation of Ethical Challenges Concerning Heritable Human Genome Editing.},
journal = {The American journal of bioethics : AJOB},
volume = {21},
number = {6},
pages = {1-4},
doi = {10.1080/15265161.2021.1913894},
pmid = {33955811},
issn = {1536-0075},
support = {UL1 RR024156/RR/NCRR NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human/genetics ; Humans ; Morals ; },
}
@article {pmid33955355,
year = {2021},
author = {Mitschka, S and Mayr, C},
title = {Endogenous p53 expression in human and mouse is not regulated by its 3'UTR.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33955355},
issn = {2050-084X},
support = {DP1 GM123454/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {3' Untranslated Regions/*genetics ; Animals ; CRISPR-Cas Systems ; Cell Line ; Female ; *Gene Expression Regulation ; Humans ; Mice ; Mice, Inbred C57BL ; Protein Processing, Post-Translational/*genetics ; Proteomics ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {The TP53 gene encodes the tumor suppressor p53 which is functionally inactivated in many human cancers. Numerous studies suggested that 3'UTR-mediated p53 expression regulation plays a role in tumorigenesis and could be exploited for therapeutic purposes. However, these studies did not investigate post-transcriptional regulation of the native TP53 gene. Here, we used CRISPR/Cas9 to delete the human and mouse TP53/Trp53 3'UTRs while preserving endogenous mRNA processing. This revealed that the endogenous 3'UTR is not involved in regulating p53 mRNA or protein expression neither in steady state nor after genotoxic stress. Using reporter assays, we confirmed the previously observed repressive effects of the isolated 3'UTR. However, addition of the TP53 coding region to the reporter had a dominant negative impact on expression as its repressive effect was stronger and abrogated the contribution of the 3'UTR. Our data highlight the importance of genetic models in the validation of post-transcriptional gene regulatory effects.},
}
@article {pmid33953401,
year = {2021},
author = {Griffin, GK and Wu, J and Iracheta-Vellve, A and Patti, JC and Hsu, J and Davis, T and Dele-Oni, D and Du, PP and Halawi, AG and Ishizuka, JJ and Kim, SY and Klaeger, S and Knudsen, NH and Miller, BC and Nguyen, TH and Olander, KE and Papanastasiou, M and Rachimi, S and Robitschek, EJ and Schneider, EM and Yeary, MD and Zimmer, MD and Jaffe, JD and Carr, SA and Doench, JG and Haining, WN and Yates, KB and Manguso, RT and Bernstein, BE},
title = {Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.},
journal = {Nature},
volume = {595},
number = {7866},
pages = {309-314},
pmid = {33953401},
issn = {1476-4687},
support = {DP1 CA216873/CA/NCI NIH HHS/United States ; U01 CA214125/CA/NCI NIH HHS/United States ; U24 CA210986/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antigens, Viral/immunology ; CRISPR-Cas Systems/genetics ; Chromatin/genetics/metabolism ; DNA Transposable Elements/genetics ; Disease Models, Animal ; Female ; *Gene Silencing ; Histocompatibility Antigens Class I/genetics/immunology ; Histone-Lysine N-Methyltransferase/*metabolism ; Humans ; Mice ; Neoplasms/drug therapy/*genetics/*immunology ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors ; T-Lymphocytes, Cytotoxic/cytology/immunology ; },
abstract = {Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape[1,2]. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape[3-5]. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution[6]. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.},
}
@article {pmid33953310,
year = {2021},
author = {Adalsteinsson, BT and Kristjansdottir, T and Merre, W and Helleux, A and Dusaucy, J and Tourigny, M and Fridjonsson, O and Hreggvidsson, GO},
title = {Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {9586},
pmid = {33953310},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Genome, Bacterial ; *Mutation ; Temperature ; Thermus thermophilus/*genetics ; },
abstract = {Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5'-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.},
}
@article {pmid33951625,
year = {2021},
author = {Niu, CM and Xia, MM and Zhong, YN and Zheng, Y},
title = {Mus musculus Barrier-To-Autointegration Factor 2 (Banf2) is Not Essential for Spermatogenesis or Fertility.},
journal = {Cytogenetic and genome research},
volume = {161},
number = {3-4},
pages = {167-177},
doi = {10.1159/000513850},
pmid = {33951625},
issn = {1424-859X},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Gene Expression Regulation, Developmental ; Genes, Essential ; Intracellular Signaling Peptides and Proteins/*genetics/metabolism ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Nuclear Proteins/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogenesis/*genetics ; Spermatozoa/metabolism ; Testis/cytology/metabolism ; },
abstract = {The barrier-to-autointegration factor (BAF) is widely expressed in most human tissues and plays a critical role in chromatin organization, nuclear envelope assembly, gonadal development, and embryonic stem cell self-renewal. Complete loss of BAF has been shown to lead to embryonic lethality and gonadal defects. The BAF paralog, namely, barrier-to-autointegration factor 2 (BANF2), exhibits a testis-predominant expression pattern in both humans and mice. Unlike BAF, it may cause isolated male infertility. Therefore, we used the CRISPR/Cas9 system to generate Banf2-knockout mice to further study its function in spermatogenesis. Unexpectedly, knockout mice did not show any detectable abnormalities in histological structure of the testis, epididymis, ovary, and other tissues, and exhibited normal fertility, indicating that Banf2 is not essential for mouse spermatogenesis and fertility.},
}
@article {pmid33950834,
year = {2021},
author = {Xia, SL and Li, M and Chen, B and Wang, C and Yan, YH and Dong, MQ and Qi, YB},
title = {The LRR-TM protein PAN-1 interacts with MYRF to promote its nuclear translocation in synaptic remodeling.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33950834},
issn = {2050-084X},
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*chemistry/genetics/physiology ; Caenorhabditis elegans Proteins/genetics/*metabolism ; Cell Membrane/metabolism ; HEK293 Cells ; Humans ; Protein Domains ; Protein Transport ; Synapses/physiology ; Transcription Factors/genetics/*metabolism ; },
abstract = {Neural circuits develop through a plastic phase orchestrated by genetic programs and environmental signals. We have identified a leucine-rich-repeat domain transmembrane protein PAN-1 as a factor required for synaptic rewiring in C. elegans. PAN-1 localizes on cell membrane and binds with MYRF, a membrane-bound transcription factor indispensable for promoting synaptic rewiring. Full-length MYRF was known to undergo self-cleavage on ER membrane and release its transcriptional N-terminal fragment in cultured cells. We surprisingly find that MYRF trafficking to cell membrane before cleavage is pivotal for C. elegans development and the timing of N-MYRF release coincides with the onset of synaptic rewiring. On cell membrane PAN-1 and MYRF interact with each other via their extracellular regions. Loss of PAN-1 abolishes MYRF cell membrane localization, consequently blocking myrf-dependent neuronal rewiring process. Thus, through interactions with a cooperating factor on the cell membrane, MYRF may link cell surface activities to transcriptional cascades required for development.},
}
@article {pmid33950408,
year = {2021},
author = {Khan, I and Khan, S and Zhang, Y and Zhou, J and Akhoundian, M and Jan, SA},
title = {CRISPR-Cas technology based genome editing for modification of salinity stress tolerance responses in rice (Oryza sativa L.).},
journal = {Molecular biology reports},
volume = {48},
number = {4},
pages = {3605-3615},
pmid = {33950408},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Oryza/*genetics ; Plant Breeding/*methods ; *Salt Tolerance ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) technology is an effective tool for site-specific genome editing, used to precisely induce mutagenesis in different plant species including rice. Salinity is one of the most stressful environmental constraints affecting agricultural productivity worldwide. As plant adaptation to salinity stress is under polygenic control therefore, 51 rice genes have been identified that play crucial role in response to salinity. This review offers an exclusive overview of genes identified in rice genome for salinity stress tolerance. This will provide an idea to produce rice varieties with enhanced salt tolerance using the potentially efficient CRISPR-Cas technology. Several undesirable off-target effects of CRISPR-Cas technology and their possible solutions have also been highlighted.},
}
@article {pmid33950389,
year = {2021},
author = {Geny, S and Pichard, S and Poterszman, A and Concordet, JP},
title = {Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2305},
number = {},
pages = {153-174},
pmid = {33950389},
issn = {1940-6029},
mesh = {Biotechnology/*methods ; *CRISPR-Cas Systems ; Cell Line ; DNA Helicases/biosynthesis/genetics ; DNA-Binding Proteins/biosynthesis/genetics ; Gene Editing/*methods ; Gene Expression ; Gene Targeting/*methods ; Humans ; K562 Cells ; Oligonucleotides/genetics ; RNA, Guide/metabolism ; Transcription Factor TFIIH/biosynthesis/genetics ; Transfection ; },
abstract = {The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells.},
}
@article {pmid33950175,
year = {2021},
author = {Bohl, SR and Schmalbrock, LK and Bauhuf, I and Meyer, T and Dolnik, A and Szyska, M and Blätte, TJ and Knödler, S and Röhner, L and Miller, D and Kull, M and Langer, C and Döhner, H and Letai, A and Damm, F and Heckl, D and Bullinger, L and Krönke, J},
title = {Comprehensive CRISPR-Cas9 screens identify genetic determinants of drug responsiveness in multiple myeloma.},
journal = {Blood advances},
volume = {5},
number = {9},
pages = {2391-2402},
pmid = {33950175},
issn = {2473-9537},
mesh = {CRISPR-Cas Systems ; Humans ; Lenalidomide ; *Multiple Myeloma/drug therapy/genetics ; Neoplasm Recurrence, Local ; *Pharmaceutical Preparations ; },
abstract = {The introduction of new drugs in the past years has substantially improved outcome in multiple myeloma (MM). However, the majority of patients eventually relapse and become resistant to one or multiple drugs. While the genetic landscape of relapsed/ resistant multiple myeloma has been elucidated, the causal relationship between relapse-specific gene mutations and the sensitivity to a given drug in MM has not systematically been evaluated. To determine the functional impact of gene mutations, we performed combined whole-exome sequencing (WES) of longitudinal patient samples with CRISPR-Cas9 drug resistance screens for lenalidomide, bortezomib, dexamethasone, and melphalan. WES of longitudinal samples from 16 MM patients identified a large number of mutations in each patient that were newly acquired or evolved from a small subclone (median 9, range 1-55), including recurrent mutations in TP53, DNAH5, and WSCD2. Focused CRISPR-Cas9 resistance screens against 170 relapse-specific mutations functionally linked 15 of them to drug resistance. These included cereblon E3 ligase complex members for lenalidomide, structural genes PCDHA5 and ANKMY2 for dexamethasone, RB1 and CDK2NC for bortezomib, and TP53 for melphalan. In contrast, inactivation of genes involved in the DNA damage repair pathway, including ATM, FANCA, RAD54B, and BRCC3, enhanced susceptibility to cytotoxic chemotherapy. Resistance patterns were highly drug specific with low overlap and highly correlated with the treatment-dependent clonal evolution in patients. The functional association of specific genetic alterations with drug sensitivity will help to personalize treatment of MM in the future.},
}
@article {pmid33949481,
year = {2021},
author = {Liu, Y and Wang, Y and Lin, J and Xu, L},
title = {Theophylline-induced synergic activation of guide RNA to control CRISPR/Cas9 function.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {44},
pages = {5418-5421},
doi = {10.1039/d1cc01260f},
pmid = {33949481},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide/chemistry/*metabolism ; Theophylline/chemistry/*metabolism ; },
abstract = {Ligand-induced activation of CRISPR/Cas9 function is achieved based on a synergic approach through the integration of the theophylline aptamer into protein-unrecognized regions of guide RNA. This design of allosteric regulation opens a new window towards the broad involvement of RNA aptamers for conditional control of CRISPR/Cas9 function.},
}
@article {pmid33949306,
year = {2021},
author = {Brown, EB and Shah, KD and Palermo, J and Dey, M and Dahanukar, A and Keene, AC},
title = {Ir56d-dependent fatty acid responses in Drosophila uncover taste discrimination between different classes of fatty acids.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33949306},
issn = {2050-084X},
support = {R01 DC017390/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila/genetics/*physiology ; Fatty Acids/*classification/*metabolism ; Female ; Gene Deletion ; Neurons/*physiology ; Odorants ; Sensory Receptor Cells/physiology ; Taste Perception/*physiology ; },
abstract = {Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food. While animals can discriminate between thousands of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila, taste neurons expressing the ionotropic receptor 56d (IR56d) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Here, we tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. Our results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. While IR56d neurons are broadly responsive to short-, medium-, and long-chain fatty acids, genetic deletion of IR56d selectively disrupts response to medium-chain fatty acids. Further, IR56d+ GR64f+ neurons are necessary for proboscis extension response (PER) to medium-chain fatty acids, but both IR56d and GR64f neurons are dispensable for PER to short- and long-chain fatty acids, indicating the involvement of one or more other classes of neurons. Together, these findings reveal that IR56d is selectively required for medium-chain fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation in shared populations of neurons. Our study uncovers a capacity for the taste system to encode tastant identity within a taste category.},
}
@article {pmid33948565,
year = {2021},
author = {Zocher, S and Kempermann, G},
title = {Generation of mouse hippocampal neural precursor cell lines with CRISPR/Cas9-mediated gene knockouts.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100472},
pmid = {33948565},
issn = {2666-1667},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Knockout Techniques/*methods ; Hippocampus/*cytology ; Mice ; Neural Stem Cells/*cytology ; Neurogenesis/genetics ; },
abstract = {Genetic manipulation of neural precursor cells is an important tool to study mechanisms underlying proliferation, fate specification, and neuron formation. The CRISPR/Cas9 system enables efficient genome editing but requires the clonal expansion of cells containing the desired mutation. Here, we describe a protocol for the effective generation of clonal mouse hippocampal neural precursor lines with CRISPR/Cas9-based gene knockouts. Edited cell lines can be used to investigate gene regulatory networks driving neuronal differentiation and for modeling of diseases that involve hippocampal neurogenesis. For complete details on the use and execution of this protocol, please refer to Pötzsch et al. (2021).},
}
@article {pmid33947214,
year = {2021},
author = {Lu, Q and Liu, R and Sherchan, P and Ren, R and He, W and Fang, Y and Huang, Y and Shi, H and Tang, L and Yang, S and Zhang, JH and Tang, J},
title = {TREM (Triggering Receptor Expressed on Myeloid Cells)-1 Inhibition Attenuates Neuroinflammation via PKC (Protein Kinase C) δ/CARD9 (Caspase Recruitment Domain Family Member 9) Signaling Pathway After Intracerebral Hemorrhage in Mice.},
journal = {Stroke},
volume = {52},
number = {6},
pages = {2162-2173},
pmid = {33947214},
issn = {1524-4628},
support = {R01 NS091042/NS/NINDS NIH HHS/United States ; R21 NS101284/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CARD Signaling Adaptor Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Cerebral Hemorrhage/genetics/*metabolism ; Gene Knockdown Techniques ; Inflammation/genetics/metabolism ; Male ; Mice ; Protein Kinase C-delta/genetics/*metabolism ; *Signal Transduction ; Triggering Receptor Expressed on Myeloid Cells-1/genetics/*metabolism ; },
abstract = {BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high mortality and disability. Inflammatory response promotes secondary brain injury after ICH. TREM (triggering receptor expressed on myeloid cells)-1 is a key regulator of inflammation. The aim of this study was to evaluate the role of TREM-1 in neuroinflammatory response after ICH in mice.<br><br>METHODS: CD1 mice (n=275) were used in this study. Mice were subjected to ICH by autologous blood injection. TREM-1 knockout CRISPR was administered intracerebroventricularly to evaluate the role of TREM-1 after ICH. A selective TREM-1 inhibitor, LP17, was administered intranasally 2 hours after ICH. To elucidate TREM-1 signaling pathway, CARD9 (caspase recruitment domain family member 9) activation CRISPR was administered with LP17 and TREM-1 activating anti-mouse TREM-1 monoclonal antibody (mAb) was administered with Rottlerin, a specific PKC (protein kinase C) &#x3b4; inhibitor. Lastly, to evaluate the role of HMGB1 (high-mobility group box 1) in TREM-1 mediated microglia activation, glycyrrhizin, an inhibitor of HMBG1 was administered with TREM-1 activating mAb. Neurobehavioral test, brain water content, Western blot, immunofluorescence staining, and coimmunoprecipitation was performed.<br><br>RESULTS: TREM-1 knockout reduced ICH-induced neurobehavioral deficits and neuroinflammatory response. The temporal expression of HMGB1, TREM-1, PKC &#x3b4;, and CARD9 increased after ICH. TREM-1 was expressed on microglia. Intranasal administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 and 72 hours after ICH. LP17 promoted M2 microglia polarization and reduced proinflammatory cytokines after ICH, which was reversed with CARD9 activation CRISPR. TREM-1 mAb increased neurobehavior deficits, proinflammatory cytokines, and reduced M2 microglia after ICH, which was reversed with Rottlerin. HMBG1 interaction with TREM-1 increased after ICH, and glycyrrhizin reduced neuroinflammation and promoted M2 microglia which was reversed with TREM-1 mAb.<br><br>CONCLUSIONS: This study demonstrated that TREM-1 enhanced neuroinflammation by modulating microglia polarization after ICH, and this regulation was partly mediated via PKC &#x3b4;/CARD9 signaling pathway and increased HMGB1 activation of TREM-1.},
}
@article {pmid33946570,
year = {2021},
author = {Inoue, YU and Morimoto, Y and Yamada, M and Kaneko, R and Shimaoka, K and Oki, S and Hotta, M and Asami, J and Koike, E and Hori, K and Hoshino, M and Imayoshi, I and Inoue, T},
title = {An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation.},
journal = {Cells},
volume = {10},
number = {5},
pages = {},
pmid = {33946570},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry/genetics/*standards ; Electroporation/methods ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Mice ; Mice, Transgenic ; Microinjections/methods ; Polymerase Chain Reaction/methods ; Zygote/metabolism ; },
abstract = {Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5'-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.},
}
@article {pmid33945505,
year = {2021},
author = {Hohmann, MS and Habiel, DM and Espindola, MS and Huang, G and Jones, I and Narayanan, R and Coelho, AL and Oldham, JM and Noth, I and Ma, SF and Kurkciyan, A and McQualter, JL and Carraro, G and Stripp, B and Chen, P and Jiang, D and Noble, PW and Parks, W and Woronicz, J and Yarranton, G and Murray, LA and Hogaboam, CM},
title = {Antibody-mediated depletion of CCR10+EphA3+ cells ameliorates fibrosis in IPF.},
journal = {JCI insight},
volume = {6},
number = {11},
pages = {},
pmid = {33945505},
issn = {2379-3708},
support = {K23 HL138190/HL/NHLBI NIH HHS/United States ; R01 HL123899/HL/NHLBI NIH HHS/United States ; R43 HL150986/HL/NHLBI NIH HHS/United States ; T32 HL007517/HL/NHLBI NIH HHS/United States ; },
mesh = {Alveolar Epithelial Cells/metabolism ; Animals ; Antibodies, Monoclonal/pharmacology ; CRISPR-Cas Systems ; Chemokines, CC/metabolism ; Fibroblasts/metabolism ; Gene Knockout Techniques ; Humans ; Idiopathic Pulmonary Fibrosis/*metabolism/pathology ; Mesenchymal Stem Cells/drug effects/*metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Receptor, EphA3/*metabolism ; Receptors, CCR10/*metabolism ; },
abstract = {Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.},
}
@article {pmid33945142,
year = {2022},
author = {Thongsin, N and Wattanapanitch, M},
title = {CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {607-624},
pmid = {33945142},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing ; HLA Antigens/genetics ; Histocompatibility Antigens Class I/metabolism ; Humans ; *Induced Pluripotent Stem Cells ; Ribonucleoproteins/metabolism ; },
abstract = {Advances in induced pluripotent stem cell (iPSC) technology provide a renewable source of cells for tissue regeneration and therefore hold great promise for cell replacement therapy. However, immune rejection of allograft due to human leukocyte antigen (HLA) mismatching remains a major challenge. Considerable efforts have been devoted to overcoming the immunogenicity of allograft transplantation. One of the approaches is an elimination of HLA molecules on the surface of allogeneic cells using genome editing technology to generate universal stem cells. Here, we present a simple and effective genome editing approach to knockout the β-2-immunoglobulin (B2M) gene, which encodes B2M protein that forms a heterodimer with HLA class I proteins, in induced pluripotent stem cells (iPSCs) leading to HLA class I (HLA-I) depletion. We also describe detailed procedures for validation of the B2M-knockout iPSCs using flow cytometry, and genotypic analysis for potential off-target regions. Our protocol is also applicable for knocking out other genes in iPSCs and other cell types.},
}
@article {pmid33945139,
year = {2022},
author = {Zhang, X and Li, T and Ou, J and Huang, J and Liang, P},
title = {Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing.},
journal = {Protein &amp; cell},
volume = {13},
number = {5},
pages = {316-335},
pmid = {33945139},
issn = {1674-8018},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Embryo, Mammalian/metabolism ; Endonucleases/genetics/metabolism ; *Gene Editing ; Mammals/genetics/metabolism ; },
abstract = {Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.},
}
@article {pmid33944912,
year = {2021},
author = {Stevenson, NL and Bergen, DJM and Lu, Y and Prada-Sanchez, ME and Kadler, KE and Hammond, CL and Stephens, DJ},
title = {Giantin is required for intracellular N-terminal processing of type I procollagen.},
journal = {The Journal of cell biology},
volume = {220},
number = {6},
pages = {},
pmid = {33944912},
issn = {1540-8140},
support = {21937/VAC_/Versus Arthritis/United Kingdom ; MR/P000177/1/MRC_/Medical Research Council/United Kingdom ; BB/T001984/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 110126/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; BB/L014181/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 22044/VAC_/Versus Arthritis/United Kingdom ; },
mesh = {Animals ; Bone and Bones/*metabolism ; CRISPR-Cas Systems ; Collagen Type I/*metabolism ; Extracellular Matrix/*metabolism ; Golgi Matrix Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Humans ; Procollagen/*metabolism ; Zebrafish ; },
abstract = {Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.},
}
@article {pmid33944602,
year = {2021},
author = {Wheatley, MS and Duan, YP and Yang, Y},
title = {Highly Sensitive and Rapid Detection of Citrus Huanglongbing Pathogen ('Candidatus Liberibacter asiaticus') Using Cas12a-Based Methods.},
journal = {Phytopathology},
volume = {111},
number = {12},
pages = {2375-2382},
doi = {10.1094/PHYTO-09-20-0443-R},
pmid = {33944602},
issn = {0031-949X},
mesh = {Animals ; CRISPR-Cas Systems ; *Citrus ; *Hemiptera ; Liberibacter ; Plant Diseases ; *Rhizobiaceae/genetics ; },
abstract = {Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, 'Candidatus Liberibacter asiaticus' (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.},
}
@article {pmid33942822,
year = {2021},
author = {Huang, RY and Liu, ZH and Weng, WH and Chang, CW},
title = {Magnetic nanocomplexes for gene delivery applications.},
journal = {Journal of materials chemistry. B},
volume = {9},
number = {21},
pages = {4267-4286},
doi = {10.1039/d0tb02713h},
pmid = {33942822},
issn = {2050-7518},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Transfer Techniques ; Genetic Vectors ; Humans ; *Magnetics ; *Nanostructures ; Neoplasms/therapy ; Regenerative Medicine ; Viruses/genetics ; },
abstract = {Gene delivery is an indispensable technique for various biomedical applications such as gene therapy, stem cell engineering and gene editing. Recently, magnetic nanoparticles (MNPs) have received increasing attention for their use in promoting gene delivery efficiency. Under magnetic attraction, gene delivery efficiency using viral or nonviral gene carriers could be universally enhanced. Besides, magnetic nanoparticles could be utilized in magnetic resonance imaging or magnetic hyperthermia therapy, providing extra theranostic opportunities. In this review, recent research integrating MNPs with a viral or nonviral gene vector is summarized from both technical and application perspectives. Applications of MNPs in cutting-edge research technologies, such as biomimetic cell membrane nano-gene carriers, exosome-based gene delivery, cell-based drug delivery systems or CRISPR/Cas9 gene editing, are also discussed.},
}
@article {pmid33942464,
year = {2021},
author = {Jiang, B and Chen, L and Yang, C and Wu, T and Yuan, S and Wu, C and Zhang, M and Gai, J and Han, T and Hou, W and Sun, S},
title = {The cloning and CRISPR/Cas9-mediated mutagenesis of a male sterility gene MS1 of soybean.},
journal = {Plant biotechnology journal},
volume = {19},
number = {6},
pages = {1098-1100},
pmid = {33942464},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Gene Editing ; Humans ; *Infertility, Male ; Male ; Mutagenesis/genetics ; *Soybeans/genetics ; },
}
@article {pmid33942339,
year = {2021},
author = {Ju, E and Li, T and Ramos da Silva, S and Markazi, A and Gao, SJ},
title = {Reversible switching of primary cells between normal and malignant state by oncogenic virus KSHV and CRISPR/Cas9-mediated targeting of a major viral latent protein.},
journal = {Journal of medical virology},
volume = {93},
number = {8},
pages = {5065-5075},
pmid = {33942339},
issn = {1096-9071},
support = {R01 CA197153/CA/NCI NIH HHS/United States ; R01 CA096512/CA/NCI NIH HHS/United States ; CA124332/GF/NIH HHS/United States ; CA132637/GF/NIH HHS/United States ; R01 DE025465/DE/NIDCR NIH HHS/United States ; DE025465/GF/NIH HHS/United States ; CA177377/GF/NIH HHS/United States ; CA197153/GF/NIH HHS/United States ; R01 CA177377/CA/NCI NIH HHS/United States ; R01 CA132637/CA/NCI NIH HHS/United States ; CA096512/GF/NIH HHS/United States ; R01 CA124332/CA/NCI NIH HHS/United States ; R01 CA213275/CA/NCI NIH HHS/United States ; CA213275/GF/NIH HHS/United States ; R01 DE017333/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Antigens, Viral/*genetics/metabolism ; Apoptosis ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Cycle ; Cell Proliferation ; Cell Transformation, Neoplastic/*genetics ; Gene Knockout Techniques ; Genome, Viral/genetics ; Herpesvirus 8, Human/*genetics/pathogenicity ; Humans ; Lymphoma, Primary Effusion/pathology ; Mesenchymal Stem Cells ; Nuclear Proteins/*genetics/metabolism ; Oncogenic Viruses/*genetics/pathogenicity ; RNA, Guide/genetics ; Rats ; Virus Latency/genetics ; },
abstract = {Viral infection has been implicated in the pathogenesis of a plethora of human diseases. Although antiviral therapies effectively confront the viral spread and infection, how to completely eradicate the viral genome from infected cells remains a challenge. In this study, we demonstrated the reversible switching of primary cells between normal and malignant states by an oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) and CRISPR/Cas9-mediated targeting of a major viral latent protein. Primary cells can be transformed into malignant status by infection of KSHV, while elimination of the KSHV genome from latent KSHV-infected cells reverses KSHV-transformed primary cells back to a "normal state" by CRISPR/Cas-mediated knockout of viral major latent gene LANA. As a proof of concept, we demonstrated efficient elimination of KSHV episome in KSHV-associated primary effusion lymphoma cells resulting in the induction of apoptosis by liposome-encapsulated CRISPR/Cas9 ribonucleoprotein complexes (Lipo/Cas9-LANAsgRNA). Our work illustrates CRISPR/Cas as a promising technology for eliminating oncogenic viruses from persistently infected cells by taking advantage of the genetic differences between viral and cellular genomes. Compared to traditional antiviral therapy, our study offer an approach for antagonizing human oncogenic virus-related cancers by directly targeting as well as clearing viral genomes.},
}
@article {pmid33941966,
year = {2021},
author = {Akhtar, M and Jamal, T and Khan, M and Khan, SR and Haider, S and Jalil, F},
title = {CRISPR Cas System: An efficient tool for cancer modelling.},
journal = {JPMA. The Journal of the Pakistan Medical Association},
volume = {71},
number = {2(B)},
pages = {718-724},
doi = {10.47391/JPMA.801},
pmid = {33941966},
issn = {0030-9982},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats-Cas-9 (CRISPR-Cas9) system has been a revolutionising tool in the field of molecular genetics, which provides a versatile range of editing potentials. Researchers can produce breaks or alter genomes with ease using the system. Cancer is one of the multi-gene diseases whose genes need to be studied in detail. The CRISPR-Cas9 technology may also provide a promising potential in the field of cancer genetics. The current narrative review comprised 50 research articles which were keenly analysed and the applications and outcomes of CRISPR-Cas9 system in cancer genetics were comprehensively and critically discussed. It was concluded that application of the system had great potential to help understand cancer biology of various types and could be used for its genetic modelling. However, much work is still needed to be done to apply the technology for understanding the mechanism of cancers and to help in the designing of appropriate therapies.},
}
@article {pmid33941935,
year = {2021},
author = {Xu, C and Zhou, Y and Xiao, Q and He, B and Geng, G and Wang, Z and Cao, B and Dong, X and Bai, W and Wang, Y and Wang, X and Zhou, D and Yuan, T and Huo, X and Lai, J and Yang, H},
title = {Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes.},
journal = {Nature methods},
volume = {18},
number = {5},
pages = {499-506},
pmid = {33941935},
issn = {1548-7105},
mesh = {Animals ; Bacterial Proteins ; *CRISPR-Cas Systems ; Cell Line ; Cloning, Molecular ; Databases, Nucleic Acid ; Dogs ; Humans ; Mice ; *RNA Editing ; RNA Interference ; *RNA, Bacterial ; },
abstract = {Competitive coevolution between microbes and viruses has led to the diversification of CRISPR-Cas defense systems against infectious agents. By analyzing metagenomic terabase datasets, we identified two compact families (775 to 803 amino acids (aa)) of CRISPR-Cas ribonucleases from hypersaline samples, named Cas13X and Cas13Y. We engineered Cas13X.1 (775 aa) for RNA interference experiments in mammalian cell lines. We found Cas13X.1 could tolerate single-nucleotide mismatches in RNA recognition, facilitating prophylactic RNA virus inhibition. Moreover, a minimal RNA base editor, composed of engineered deaminase (385 aa) and truncated Cas13X.1 (445 aa), exhibited robust editing efficiency and high specificity to induce RNA base conversions. Our results suggest that there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, and thus potentially are useful for RNA-editing-based research.},
}
@article {pmid33941066,
year = {2021},
author = {Isaev, AB and Musharova, OS and Severinov, KV},
title = {Microbial Arsenal of Antiviral Defenses. Part II.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {4},
pages = {449-470},
doi = {10.1134/S0006297921040064},
pmid = {33941066},
issn = {1608-3040},
mesh = {Archaea/genetics/physiology/*virology ; Bacteria/genetics/*virology ; Bacterial Physiological Phenomena ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; *Host-Pathogen Interactions ; },
abstract = {Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). The constant threat of phage infection is a major force that shapes evolution of microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering had been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection. In the first part defense associated with cell surface, roles of small molecules, and innate immunity systems relying on DNA modification were discussed. The second part focuses on adaptive immunity systems, abortive infection mechanisms, defenses associated with mobile genetic elements, and novel systems discovered in recent years through metagenomic mining.},
}
@article {pmid33940579,
year = {2021},
author = {Ricci, R and Colasante, G},
title = {CRISPR/dCas9 as a Therapeutic Approach for Neurodevelopmental Disorders: Innovations and Limitations Compared to Traditional Strategies.},
journal = {Developmental neuroscience},
volume = {43},
number = {3-4},
pages = {253-261},
doi = {10.1159/000515845},
pmid = {33940579},
issn = {1421-9859},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Humans ; Mutation ; *Neurodevelopmental Disorders/genetics/therapy ; },
abstract = {Brain development is a complex process that requires a series of precise and coordinated events to take place. When alterations in some of those events occur, neurodevelopmental disorders (NDDs) may appear, with their characteristic symptoms, including cognitive, social motor deficits, and epilepsy. While pharmacologic treatments have been the only therapeutic options for many years, more recently the research is turning to the direct removal of the underlying genetic cause of each specific NDD. This is possible thanks to the increased knowledge of genetic basis of those diseases and the enormous advances in genome-editing tools. Together with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategies, there is a great development also of nuclease defective Cas9 (dCas9) tools that, with an extreme flexibility, allow the recruitment of specific protein functions to the desired genomic sites. In this work, we review dCas9-based tools and discuss all the published applications in the setting of therapeutic approaches for NDDs at the preclinical level. In particular, dCas9-based therapeutic strategies for Dravet syndrome, transcallosal dysconnectivity caused by mutations in C11orf46 gene, and Fragile X syndrome are presented and discussed. A direct comparison with other possible therapeutic strategies, such as classic gene replacement or CRISPR/Cas9-based strategies, is provided. We also highlight not only those aspects that constitute a clear advantage compared to previous strategies but also the main technical hurdles related to their applications that need to be overcome.},
}
@article {pmid33940494,
year = {2021},
author = {Wang, X and Zhou, S and Chu, C and Yang, M and Huo, D and Hou, C},
title = {Target-induced transcription amplification to trigger the trans-cleavage activity of CRISPR/Cas13a (TITAC-Cas) for detection of alkaline phosphatase.},
journal = {Biosensors &amp; bioelectronics},
volume = {185},
number = {},
pages = {113281},
doi = {10.1016/j.bios.2021.113281},
pmid = {33940494},
issn = {1873-4235},
mesh = {Alkaline Phosphatase/genetics/metabolism ; *Biosensing Techniques ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; },
abstract = {Herein, an ultra-sensitive alkaline phosphatase (ALP) sensing strategy is developed by target-induced transcription amplification to trigger the trans-cleavage activity of Cas13a (TITAC-Cas). A double-stranded DNA duplex integrating a T7 promoter with 5'-phosphate and a transcription template (5'P-dsDNA) serves as the ALP substrate. In the absence of ALP, 5'P-dsDNA can be degraded by the λexo, leading to the subsequent transcription failure. In the presence of ALP, dephosphorylation reaction converts the 5'P-dsDNA to 5'OH-dsDNA and provides the protection for T7 promoter against the λexo-digestion. The intact T7 promoter of 5'OH-dsDNA can activate T7 transcription to produce a mass of single-stranded RNA (ssRNA). The ssRNA products possess a full complementarity to the spacer of crRNA and activate the ssRNase activity of CRISPR/Cas13a. As a result, Cas13a exhibits the indiscriminate cleavage of collateral FQ-reporter to release significant fluorescence signal, realizing the ultra-sensitive detection of ALP. Due to the triple signal amplification (ALP self-catalysis, T7 transcription amplification, and trans-cleavage of CRISPR/Cas13a), TITAC-Cas assay shows the ultra-sensitive detection of ALP activity with a wide linear range from 0.008 to 250 U∙L[-1]). The LOD is calculated to be 6 ± 0.52 mU∙L[-1]. TITAC-Cas assay is also successfully applied for analysis of ALP activity in HepG2 cell lysate with high fidelity. In addition, this method is employed to screen ALP inhibitor.},
}
@article {pmid33939982,
year = {2021},
author = {Green, CD and Weigel, C and Oyeniran, C and James, BN and Davis, D and Mahawar, U and Newton, J and Wattenberg, BW and Maceyka, M and Spiegel, S},
title = {CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism.},
journal = {Journal of lipid research},
volume = {62},
number = {},
pages = {100082},
pmid = {33939982},
issn = {1539-7262},
support = {R01 AI125433/AI/NIAID NIH HHS/United States ; R01 HL131340/HL/NHLBI NIH HHS/United States ; P30 CA016059/CA/NCI NIH HHS/United States ; R25 GM090084/GM/NIGMS NIH HHS/United States ; K99 HD096117/HD/NICHD NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; },
abstract = {The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.},
}
@article {pmid33939828,
year = {2021},
author = {Hwang, GH and Jeong, YK and Habib, O and Hong, SA and Lim, K and Kim, JS and Bae, S},
title = {PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.},
journal = {Nucleic acids research},
volume = {49},
number = {W1},
pages = {W499-W504},
pmid = {33939828},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing ; Humans ; Internet ; Mutation ; RNA/chemistry ; Sequence Alignment ; *Software ; },
abstract = {Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.},
}
@article {pmid33939241,
year = {2021},
author = {Hanaki, S and Habara, M and Masaki, T and Maeda, K and Sato, Y and Nakanishi, M and Shimada, M},
title = {PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.},
journal = {Cancer science},
volume = {112},
number = {7},
pages = {2739-2752},
pmid = {33939241},
issn = {1349-7006},
mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Checkpoint Kinase 1/metabolism ; Cyclic AMP-Dependent Protein Kinases/antagonists &amp; inhibitors/*metabolism ; *DNA Damage ; E2F1 Transcription Factor/*genetics ; Endoribonucleases/deficiency/isolation &amp; purification/*metabolism ; Epigenetic Repression ; Gene Expression Regulation ; Histones/*metabolism ; Humans ; Phosphoprotein Phosphatases/deficiency/isolation &amp; purification/*metabolism ; Phosphorylation ; Promoter Regions, Genetic ; Protein Phosphatase 1/*metabolism ; Protein Processing, Post-Translational ; RNA, Messenger/metabolism ; RNA-Binding Proteins/isolation &amp; purification/*metabolism ; Receptors, Neuropeptide Y/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic ; Ultraviolet Rays ; },
abstract = {DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr[11] phosphorylation (H3-pThr[11]) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr[11] by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr[11] both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser[199] residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.},
}
@article {pmid33937256,
year = {2021},
author = {Cui, D and Franz, A and Fillon, SA and Jannetti, L and Isambert, T and Fundel-Clemens, K and Huber, HJ and Viollet, C and Ghanem, A and Niwa, A and Weigle, B and Pflanz, S},
title = {High-Yield Human Induced Pluripotent Stem Cell-Derived Monocytes and Macrophages Are Functionally Comparable With Primary Cells.},
journal = {Frontiers in cell and developmental biology},
volume = {9},
number = {},
pages = {656867},
pmid = {33937256},
issn = {2296-634X},
abstract = {Macrophages are pivotal effectors of host immunity and regulators of tissue homeostasis. Understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (hiPSC)-derived monocytes and macrophages, as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of macrophage biology and implication in human diseases. In this study, we present a fully optimized differentiation protocol of hiPSC-derived monocytes and granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). We present characterization of iPSC-derived myeloid lineage cells at phenotypic, functional, and transcriptomic levels, in comparison with corresponding subsets of peripheral blood-derived cells. We also highlight the application of hiPSC-derived monocytes and macrophages as a gene-editing platform for functional validation in research and drug screening, and the study also provides a reference for cell therapies.},
}
@article {pmid33934839,
year = {2021},
author = {Trivedi, D},
title = {Using CRISPR-Cas9-based genome engineering tools in Drosophila melanogaster.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {85-121},
doi = {10.1016/bs.pmbts.2021.01.006},
pmid = {33934839},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; Genetic Engineering ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
abstract = {Drosophila melanogaster has been used as a model organism for over a century. Mutant-based analyses have been used extensively to understand the genetic basis of different cellular processes, including development, neuronal function and diseases. Most of the earlier genetic mutants and specific tools were generated by random insertions and deletion strategies and then mapped to specific genomic loci. Since all genomic regions are not equally accessible to random mutations and insertions, many genes still remain uncharacterized. Low efficiency of targeted genomic manipulation approaches that rely on homologous recombination, and difficulty in generating resources for sequence-specific endonucleases, such as ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases), could not make these gene targeting techniques very popular. However, recently RNA directed DNA endonucleases, such as CRISPR-Cas, have transformed genome engineering owing to their comparative ease, versatility, and low expense. With the added advantage of preexisting genetic tools, CRISPR-Cas-based manipulations are being extensively used in Drosophila melanogaster and simultaneously being fine-tuned for specific experimental requirements. In this chapter, I will discuss various uses of CRISPR-Cas-based genetic engineering and specific design methods in Drosophila melanogaster. I will summarize various already available tools that are being utilized in conjunction with CRISPR-Cas technology to generate specific genetic manipulation and are being optimized to address specific questions. Finally, I will discuss the future directions of Drosophila genetics research and how CRISPR-Cas can be utilized to target specific questions, addressing which has not been possible thus far.},
}
@article {pmid33934838,
year = {2021},
author = {Sharma, P and Sharma, BS and Verma, RJ},
title = {CRISPR-based genome editing of zebrafish.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {69-84},
doi = {10.1016/bs.pmbts.2021.01.005},
pmid = {33934838},
issn = {1878-0814},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Engineering ; Genetic Therapy ; *Zebrafish/genetics ; },
abstract = {CRISPR/Cas9, once discovered as an adaptive immune system in bacteria, has emerged as a disruptive technology in the field of genetic engineering. Technological advancements in the recent past has enhanced the applicability of CRISPR/Cas9 tool for gene editing, gene therapies, developmental studies and mutational analysis in various model organisms. Zebrafish, one of the excellent animal models, is preferred for conducting CRISPR/Cas9 studies to assess the functional implication of specific genes of interest. CRISPR/Cas9 mediated gene editing techniques, such as, knock-out and knock-in approaches, provide evidences to identify the role of different genes through loss-of-function studies. Also, CRISPR/Cas9 has been proved to be an efficient tool for designing disease models for gene expression studies based on phenotypic screening. The present chapter provides an overview of CRISPR/Cas9 mechanism, different strategies for DNA modifications and gene function analysis, highlighting the translational applications for future prospects, such as screening of drug toxicity and efficacy.},
}
@article {pmid33934837,
year = {2021},
author = {Kirti, A and Sharma, M and Rani, K and Bansal, A},
title = {CRISPRing protozoan parasites to better understand the biology of diseases.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {21-68},
doi = {10.1016/bs.pmbts.2021.01.004},
pmid = {33934837},
issn = {1878-0814},
mesh = {Animals ; Biology ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Protozoan ; Humans ; *Parasites/genetics ; },
abstract = {Precise gene editing techniques are paramount to gain deeper insights into the biological processes such as host-parasite interactions, drug resistance mechanisms, and gene-function relationships. Discovery of CRISPR-Cas9 system has spearheaded mechanistic understanding of protozoan parasite biology as evident from the number of reports in the last decade. Here, we have described the use of CRISPR-Cas9 in understanding the biology of medically important protozoan parasites such as Plasmodium, Leishmania, Trypanosoma, Babesia and Trichomonas. In spite of intrinsic difficulties in genome editing in these protozoan parasites, CRISPR-Cas9 has acted as a catalyst for faster generation of desired transgenic parasites. Modifications in the CRISPR-Cas9 system for improving the efficiency have been useful in better understanding the molecular mechanisms associated with repair of double strand breaks in the parasites. Moreover, improvement in reagents used for CRISPR mediated gene editing have been instrumental in addressing the issue of non-specificity and toxicity for therapeutic use. These application-based modifications may help in further increasing the efficiency of gene editing in protozoan parasites.},
}
@article {pmid33934836,
year = {2021},
author = {Grobler, L and Suleman, E and Thimiri Govinda Raj, DB},
title = {Patents and technology transfer in CRISPR technology.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {153-182},
doi = {10.1016/bs.pmbts.2021.01.009},
pmid = {33934836},
issn = {1878-0814},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Genetic Techniques ; Licensure ; *Patents as Topic ; SARS-CoV-2/isolation &amp; purification ; *Technology Transfer ; },
abstract = {CRISPR technology has revolutionized biological research in the last decade and many academic institutions and companies have patented CRISPR systems and applications. Several patents have been filed for various applications of CRISPR in different industries such as agriculture, synthetic biology, bio-nanotechnology and precision medicine. Despite tremendous pressure on the technology transfer teams, several startups and spin-out companies are already using CRISPR technologies for commercial applications. In this chapter, we discuss the different CRISPR nucleases and their applications. Secondly, we detail our current opinion and perspective on the CRISPR patent and technology landscape for non-mammalian systems. We present two case-studies on CRISPR diagnostics companies, SHERLOCK and Mammoth Biosciences, who are currently at the forefront of establishing diagnostics platforms for coronavirus (SARS-CoV-2) detection. Finally, our chapter identifies future advancements and possible challenges that CRISPR technology might face in non-mammalian systems.},
}
@article {pmid33934835,
year = {2021},
author = {Gohil, N and Bhattacharjee, G and Lam, NL and Perli, SD and Singh, V},
title = {CRISPR-Cas systems: Challenges and future prospects.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {141-151},
doi = {10.1016/bs.pmbts.2021.01.008},
pmid = {33934835},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Synthetic Biology ; Transcription Activator-Like Effector Nucleases ; },
abstract = {The advancement gained over the past couple of decades in clustered regularly interspaced short palindromic repeats and CRISPR associated proteins (CRISPR-Cas) systems have revolutionized the field of synthetic biology, therapeutics, diagnostics and metabolic engineering. The technique has enabled the process of genome editing to be very precise, rapid, cost-effective and highly efficient which were the downfalls for the previously debuted zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) technologies. However, despite its great potential, challenges including off-target activity, method of delivery, ethical and regulatory issues still remain unresolved for the CRISPR-Cas systems. In this chapter, we present and point out the obstacles faced in implementation of the CRISPR-Cas system along with its future prospects.},
}
@article {pmid33934833,
year = {2021},
author = {Nweke, EE and Thimiri Govinda Raj, DB},
title = {Development of insect cell line using CRISPR technology.},
journal = {Progress in molecular biology and translational science},
volume = {180},
number = {},
pages = {1-20},
doi = {10.1016/bs.pmbts.2021.01.003},
pmid = {33934833},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Drosophila melanogaster ; Insecta ; Technology ; },
abstract = {In this chapter, we delineated the methods of CRISPR technology that has been used for the development of engineered insect cell line. We elaborated on how CRISPR/Cas9 genome editing in Drosophila melanogaster, Bombyx mori, Spodoptera frugiperda (Sf9 and Sf21), and Mosquitoes enabled the use of model or non-model insect system in various biological and medical applications. Also, the application of synthetic baculovirus genome along with CRISPR/Cas9 vector system to enable genome editing of insect cell systems for treatment of communicable and non-communicable diseases.},
}
@article {pmid33934500,
year = {2021},
author = {Wang, Y and Liu, X and Zheng, X and Wang, W and Yin, X and Liu, H and Ma, C and Niu, X and Zhu, JK and Wang, F},
title = {Creation of aromatic maize by CRISPR/Cas.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {9},
pages = {1664-1670},
doi = {10.1111/jipb.13105},
pmid = {33934500},
issn = {1744-7909},
mesh = {Amino Acid Sequence ; Betaine-Aldehyde Dehydrogenase/chemistry/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Mutation ; *Odorants ; Zea mays/enzymology/*genetics ; },
abstract = {Aroma is an important quality parameter for breeding in rice (Oryza sativa). For example, the aromatic rice varieties basmati and jasmine rice, with a popcorn-like scent, are popular worldwide and routinely command a price premium. 2-acetyl-1-pyrroline (2AP) is a key flavor compound among over 200 volatiles identified in fragrant rice. A naturally fragrant germplasm exists in multiple plant species besides rice, which all exhibit lower activity of BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2). However, no equivalent aromatic germplasm has been described in maize (Zea mays). Here, we characterized the two maize BADH2 homologs, ZmBADH2a and ZmBADH2b. We generated zmbadh2a and zmbadh2b single mutants and the zmbadh2a-zmbadh2b double mutant by CRISPR/Cas in four inbred lines. A popcorn-like scent was only noticeable in seeds from the double mutant, but not from either single mutant or in wild type. In agreement, we only detected 2AP in fresh kernels and dried mature seeds from the double mutant, which accumulated between 0.028 and 0.723 mg/kg 2AP. These results suggest that ZmBADH2a and ZmBADH2b redundantly participate in 2AP biosynthesis in maize, and represent the creation of the world's first aromatic maize by simultaneous genome editing of the two BADH2 genes.},
}
@article {pmid33934470,
year = {2021},
author = {Peterson, D and Barone, P and Lenderts, B and Schwartz, C and Feigenbutz, L and St Clair, G and Jones, S and Svitashev, S},
title = {Advances in Agrobacterium transformation and vector design result in high-frequency targeted gene insertion in maize.},
journal = {Plant biotechnology journal},
volume = {19},
number = {10},
pages = {2000-2010},
pmid = {33934470},
issn = {1467-7652},
mesh = {*Agrobacterium/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; Mutagenesis, Insertional ; *Zea mays/genetics ; },
abstract = {CRISPR-Cas is a powerful DNA double-strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here, we report an efficient, selectable marker-free site-specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium-mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.},
}
@article {pmid33934462,
year = {2021},
author = {Tripathi, JN and Ntui, VO and Shah, T and Tripathi, L},
title = {CRISPR/Cas9-mediated editing of DMR6 orthologue in banana (Musa spp.) confers enhanced resistance to bacterial disease.},
journal = {Plant biotechnology journal},
volume = {19},
number = {7},
pages = {1291-1293},
pmid = {33934462},
issn = {1467-7652},
mesh = {*Bacterial Infections ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Musa/genetics ; },
}
@article {pmid33934180,
year = {2021},
author = {Kim, SG},
title = {CRISPR innovations in plant breeding.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {913-914},
pmid = {33934180},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genome, Plant/genetics ; *Plant Breeding ; Plants/genetics ; },
}
@article {pmid33933604,
year = {2021},
author = {Zhang, S and Chen, L and Zhang, Y and Fang, D},
title = {Alleviation of neurological disease by RNA editing.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {94-99},
doi = {10.1016/j.ymeth.2021.04.023},
pmid = {33933604},
issn = {1095-9130},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Mice ; *Nervous System Diseases/genetics/therapy ; RNA ; *RNA Editing ; RNA, Guide ; },
abstract = {The development of CRISPR/Cas genome editing tools has revolutionized the life sciences by providing transformative applications in many biological fields, including the field of neurological disorders. Compared with previous CRISPR-Cas systems targeting DNA, a new field of RNA editing using CRISPR-Cas13 systems is gaining immense popularity. CRISPR-Cas13 is a robust, precise, versatile and safe RNA guided RNA-targeting system, which uniquely targets single-strand RNA. Recently, RNA-targeted gene editing tools have been refined by the introduction of an AAV (adeno-associated virus)-based CRISPR-Cas13 system for in vivo therapeutic cell fate conversion, which has been used to treat animal models of Parkinson's disease. This flavor of gene editing showed promising effects on glia-to-neuron conversion in both intact and damaged mature retinas in a mouse model. Herein, we summarize the CRISPR-Cas13 system and its potential for applications in neurological diseases, focusing on the method of applying the AAV-mediated CRISPR-Cas13 system to the conversion of glia-to-neuron.},
}
@article {pmid33932721,
year = {2021},
author = {Li, L and Li, R and Qi, C and Gao, H and Wei, Q and Tan, L and Sun, F},
title = {Mechanisms of polymyxin resistance induced by Salmonella typhimurium in vitro.},
journal = {Veterinary microbiology},
volume = {257},
number = {},
pages = {109063},
doi = {10.1016/j.vetmic.2021.109063},
pmid = {33932721},
issn = {1873-2542},
mesh = {Animals ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics ; CRISPR-Cas Systems ; Drug Resistance, Bacterial/*genetics ; Gene Deletion ; Gene Editing ; Genome, Bacterial ; Microbial Sensitivity Tests ; Polymyxins/*pharmacology ; Salmonella typhimurium/*drug effects/*genetics ; Virulence ; Whole Genome Sequencing ; },
abstract = {The increase incidence of multi-drug resistant (MDR) Salmonella has become a major global health concern. Polymyxin, an ancient polypeptide antibiotic, has been given renewed attention over recent years, resulting in resistance of Gram-negative bacteria to polymyxin, but its resistance mechanism is not completely clear. Thus, it is important to study its resistance mechanisms. In this study, an in vitro induced polymyxin-resistant strain of Salmonella typhimurium in the laboratory were constructed to investigate the mechanism of resistance of Salmonella to polymyxin. Gradual induction of Salmonella typhimurium ATCC13311 (AT) by concentration increment was used to screen for a highly polymyxin-resistant strain AT-P128. The broth dilution technique was used to compare the sensitivity of the two strains to different antimicrobial drugs. Single nucleotide polymorphisms (SNPs) were then identified by whole genome sequencing, and differences in gene expression between the two strains were compared by transcriptome sequencing and reverse transcription-quantitative PCR (RT-qPCR). Finally, for the first time, the CRISPR/Cas9 gene-editing system was used to construct gene deletion mutants in Salmonella to knock out the phoP gene of AT-P128. The results showed that strain AT-P128 was significantly more resistant to amoxicillin, ceftiofur, ampicillin, fluphenazine, and chloramphenicol and significantly less resistant to sulfamethoxazole than the parental strain AT. The growth curve results showed no significant change in the growth rate between AT-P128 and AT. Motility and biofilm formation assays showed a significant decrease in AT-P128. Additionally, the WGS results showed that AT-P128 had mutations in 9 genes involving 14 SNPs. RNA-seq and RT-qPCR results showed increased expression of phoPQ. The loss of the phoP gene decreased AT-P128ΔphoP resistance to polymyxin by 32-fold. These results suggested that polymyxin resistance affected the biology, genome components, and gene expression levels of Salmonella and that the PhoPQ two-component system played a key role in polymyxin resistance in Salmonella, providing insights into the diversity and complexity of polymyxin resistance in Salmonella.},
}
@article {pmid33931895,
year = {2021},
author = {Wang, C and Liu, K and Cao, J and Wang, L and Zhao, Q and Li, Z and Zhang, H and Chen, Q and Zhao, T},
title = {PINK1-mediated mitophagy maintains pluripotency through optineurin.},
journal = {Cell proliferation},
volume = {54},
number = {5},
pages = {e13034},
pmid = {33931895},
issn = {1365-2184},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/deficiency/genetics/*metabolism ; Cellular Reprogramming ; Gene Editing ; Membrane Transport Proteins/deficiency/genetics/*metabolism ; Mice ; Mice, Knockout ; Mitochondria/metabolism ; *Mitophagy ; Mouse Embryonic Stem Cells/cytology/metabolism ; Mutagenesis, Site-Directed ; Phosphorylation ; Protein Kinases/chemistry/genetics/*metabolism ; Protein Serine-Threonine Kinases/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Ubiquitin/metabolism ; },
abstract = {OBJECTIVES: Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.<br><br>MATERIALS AND METHODS: Parkin[+/+] and parkin[-/-] ESCs were established from E3.5 blastocysts of parkin[+/-] x parkin[+/-] mating mice. The pink1[-/-] , optn[-/-] and ndp52[-/-] ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.<br><br>RESULTS: PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1[-/-] ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N).<br><br>CONCLUSIONS: The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.},
}
@article {pmid33931459,
year = {2021},
author = {Chemello, F and Chai, AC and Li, H and Rodriguez-Caycedo, C and Sanchez-Ortiz, E and Atmanli, A and Mireault, AA and Liu, N and Bassel-Duby, R and Olson, EN},
title = {Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.},
journal = {Science advances},
volume = {7},
number = {18},
pages = {},
pmid = {33931459},
issn = {2375-2548},
support = {R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 AR071980/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Dystrophin/genetics/metabolism ; Exons ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Sequence Deletion ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.},
}
@article {pmid33931338,
year = {2021},
author = {Zhu, L and Yang, X and Li, J and Jia, X and Bai, X and Zhao, Y and Cheng, W and Shu, M and Zhu, Y and Jin, S},
title = {Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {2},
pages = {134-146},
doi = {10.1016/j.jgg.2021.01.008},
pmid = {33931338},
issn = {1673-8527},
mesh = {Adenoviridae/genetics ; Adipocytes/cytology/metabolism ; Adipose Tissue/*metabolism ; Animals ; CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Leptin/*genetics/metabolism ; Mice ; Mice, Obese ; Obesity/genetics/metabolism/*therapy ; Point Mutation ; Recombinational DNA Repair ; Treatment Outcome ; },
abstract = {Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.},
}
@article {pmid33930753,
year = {2021},
author = {Song, F and Wei, Y and Wang, P and Ge, X and Li, C and Wang, A and Yang, Z and Wan, Y and Li, J},
title = {Combining tag-specific primer extension and magneto-DNA system for Cas14a-based universal bacterial diagnostic platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {185},
number = {},
pages = {113262},
doi = {10.1016/j.bios.2021.113262},
pmid = {33930753},
issn = {1873-4235},
mesh = {Bacteria/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; },
abstract = {Nucleic acid-based diagnosis using CRISPR-Cas associated enzymes is essential for rapid infectious disease diagnosis and treatment strategies during a global pandemic. The obstacle has been blossomed CRIPSR-Cas based tools that can monitor wide range of pathogens in clinical samples with ultralow concentrations. Here, a universal nucleic acid magneto-DNA nanoparticle system was exploited for the detection of pathogenic bacteria, based on the collateral cleavage activity of CRISPR-Cas14a and tag-specific primer extension. In the system, the target nucleic acids were amplificated and be separated from mixtures by streptavidin-coated magnetic bead. The collateral cleavage activity of CRISPR-Cas14a can be activated via the tag sequence on the target product. Consequently, the fluorophore quencher reporter can be activated by CRISPR-Cas14a, leading to the increasing response. The exploited universal bacterial diagnostic can distinguish six different bacteria strains with 1 cfu/mL or 1 aM sensitivity, which may provide new strategies to construct fast, accurate, cost-effective and sensitive diagnostic tools in environments with limited resources.},
}
@article {pmid33930627,
year = {2021},
author = {Liu, H and Rauch, S and Dickinson, BC},
title = {Programmable technologies to manipulate gene expression at the RNA level.},
journal = {Current opinion in chemical biology},
volume = {64},
number = {},
pages = {27-37},
pmid = {33930627},
issn = {1879-0402},
support = {R01 MH122142/MH/NIMH NIH HHS/United States ; R35 GM119840/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Gene Expression ; *RNA/genetics/metabolism ; RNA Interference ; },
abstract = {RNA has long been an enticing therapeutic target, but is now garnering increased attention, largely driven by clinical successes of RNA interference-based drugs. While gene knockdown by well-established RNA interference- and other oligonucleotide-based strategies continues to advance in the clinic, the repertoire of targetable effectors capable of altering gene expression at the RNA level is also rapidly expanding. In this review, we focus on several recently developed bifunctional molecular technologies that both interact with and act upon a target RNA. These new approaches for programmable RNA knockdown, editing, splicing, translation, and chemical modifications stand to provide impactful new modalities for therapeutic development in the coming decades.},
}
@article {pmid33930546,
year = {2021},
author = {Kiattisewee, C and Dong, C and Fontana, J and Sugianto, W and Peralta-Yahya, P and Carothers, JM and Zalatan, JG},
title = {Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida.},
journal = {Metabolic engineering},
volume = {66},
number = {},
pages = {283-295},
doi = {10.1016/j.ymben.2021.04.002},
pmid = {33930546},
issn = {1096-7184},
mesh = {CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *Metabolic Engineering ; *Pseudomonas putida/genetics ; Transcriptional Activation/genetics ; },
abstract = {CRISPR-Cas transcriptional programming in bacteria is an emerging tool to regulate gene expression for metabolic pathway engineering. Here we implement CRISPR-Cas transcriptional activation (CRISPRa) in P. putida using a system previously developed in E. coli. We provide a methodology to transfer CRISPRa to a new host by first optimizing expression levels for the CRISPRa system components, and then applying rules for effective CRISPRa based on a systematic characterization of promoter features. Using this optimized system, we regulate biosynthesis in the biopterin and mevalonate pathways. We demonstrate that multiple genes can be activated simultaneously by targeting multiple promoters or by targeting a single promoter in a multi-gene operon. This work will enable new metabolic engineering strategies in P. putida and pave the way for CRISPR-Cas transcriptional programming in other bacterial species.},
}
@article {pmid33930295,
year = {2021},
author = {Moussa, HF and Angstman, JF and Khalil, AS},
title = {Here to stay: Writing lasting epigenetic memories.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2281-2283},
doi = {10.1016/j.cell.2021.04.007},
pmid = {33930295},
issn = {1097-4172},
mesh = {*CRISPR-Cas Systems ; Epigenomics ; *Gene Editing ; Promoter Regions, Genetic ; Writing ; },
abstract = {In this issue of Cell, Nuñez et al. develop CRISPRoff, a programmable epigenetic memory writer capable of establishing specific gene silencing programs that are stably maintained across cell division and differentiation. The singular dCas9 fusion offers a simple, reliable, and general tool for genome-wide screens, multiplexed editing, and potential therapeutics.},
}
@article {pmid33930275,
year = {2021},
author = {Hochrein, LM and Li, H and Pierce, NA},
title = {High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas.},
journal = {ACS synthetic biology},
volume = {10},
number = {5},
pages = {964-971},
doi = {10.1021/acssynbio.1c00037},
pmid = {33930275},
issn = {2161-5063},
mesh = {Allosteric Regulation ; Binding Sites ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Flavivirus/genetics/metabolism ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Nanotechnology/methods ; Plasmids/genetics ; RNA Stability/genetics ; RNA, Guide/*genetics/metabolism ; Synthetic Biology/methods ; },
abstract = {The activity of a conditional guide RNA (cgRNA) is dependent on the presence or absence of an RNA trigger, enabling cell-selective regulation of CRISPR/Cas function. cgRNAs are programmable at two levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, or induce a gene of choice) and the trigger-binding sequence controlling the scope of Cas activity (subset of cells expressing the trigger RNA). Allosteric cgRNA mechanisms enable independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Building on prior advances in dynamic RNA nanotechnology that demonstrated the cgRNA concept, here we set the goal of engineering high-performance allosteric cgRNA mechanisms for the mammalian setting, pursuing both ON → OFF logic (conditional inactivation by an RNA trigger) and OFF → ON logic (conditional activation by an RNA trigger). For each mechanism, libraries of orthogonal cgRNA/trigger pairs were designed using NUPACK. In HEK 293T cells expressing cgRNAs, triggers, and inducing dCas9: (1) a library of four ON → OFF "terminator switch" cgRNAs exhibit a median fold-change of ≈50×, a median fractional dynamic range of ≈20%, and a median crosstalk modulus of ≈9%; (2) a library of three OFF → ON "split-terminator switch" cgRNAs exhibit a median fold-change of ≈150×, a median fractional dynamic range of ≈50%, and a median crosstalk modulus of ≈4%. Further, we demonstrate that xrRNA elements that protect viral RNAs from degradation by exoribonucleases can dramatically enhance the performance of RNA synthetic biology. The high-performance allosteric cgRNAs demonstrated here for ON → OFF and OFF → ON logic in mammalian cells provide a foundation for pursuing applications of programmable cell-selective regulation.},
}
@article {pmid33928629,
year = {2021},
author = {Xie, Z and Jiang, J and Cao, L and Jiang, M and Yang, F and Ma, Z and Wang, Z and Ruan, C and Liu, H and Zhou, L},
title = {Nonsense-mediated mRNA decay efficiency influences bleeding severity in ITGA2B c.2659C &gt; T (p.Q887X) knock-in mice.},
journal = {Clinical genetics},
volume = {100},
number = {2},
pages = {213-218},
doi = {10.1111/cge.13975},
pmid = {33928629},
issn = {1399-0004},
mesh = {Animals ; Bleeding Time ; CRISPR-Cas Systems ; Caffeine ; Gene Expression Regulation/drug effects ; Humans ; Integrin alpha2/*genetics ; Mice, Mutant Strains ; *Mutation ; *Nonsense Mediated mRNA Decay/drug effects ; Thrombasthenia/*genetics ; },
abstract = {Glanzmann's thrombasthenia (GT) is a severe hemorrhagic disease. It is caused by mutations in ITGA2B or ITGB3, which are the respective genes encoding integrin αIIb and β3. Despite widespread mutational analysis, the mechanisms underlying the extensive variability in bleeding severity observed among affected individuals remains poorly understood. In order to explore the mechanisms conferring for bleeding heterogeneity, three GT patients with ITGA2B c.2671C > T (p.Q891X) who possessed different bleeding scores were studied. Analysis showed that there was significant difference in nonsense-mediated mRNA decay (NMD) efficiency among the three patients. These differences positively correlated with their bleeding score. Next, a knock-in mouse model (KI mice) with the ITGA2B c.2659C > T (p.Q887X) was generated using CRISPR/Cas9. Importantly, this mutation is homologous to ITGA2B c.2671C > T (p.Q891X) in humans. The bleeding time of KI mice was significantly in comparison to the wide-type mice. Interestingly, bleeding was stopped after treatment with caffeine, which is a known NMD inhibitor. This suggests that NMD efficiency potentially influences bleeding severity in ITGA2B c.2659C > T (p.Q887X) KI mice.},
}
@article {pmid33928566,
year = {2021},
author = {Sioud, M},
title = {RNA Interference: Story and Mechanisms.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2282},
number = {},
pages = {1-15},
pmid = {33928566},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; MicroRNAs/genetics/metabolism ; *RNA Interference ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/*genetics/metabolism/therapeutic use ; RNAi Therapeutics ; },
abstract = {The discovery that gene expression can be silenced by exogenously introduced double-stranded RNAs into cells unveiled a hidden level of gene regulation by a variety of small RNA pathways, which are involved in regulating endogenous gene expression, defending against virus infections, and protecting the genome from invading transposons, both at the posttranscriptional and epigenetic levels. All endogenous RNA interference pathways share a conserved effector complex, which contains at least an argonaute protein and a short single-stranded RNA. Such argonaute-RNA complexes can repress the transcription of genes, target mRNA for site-specific cleavage, or block mRNA translation into proteins. This review outlines the history of RNAi discovery, function, and mechanisms of action. For comparison, it also touches on CRISPR interference.},
}
@article {pmid33928558,
year = {2021},
author = {Dölz, M and Marone, R and Jeker, LT},
title = {Plasmid- or Ribonucleoprotein-Mediated CRISPR/Cas Gene Editing in Primary Murine T Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2285},
number = {},
pages = {255-264},
pmid = {33928558},
issn = {1940-6029},
mesh = {Animals ; CD4-Positive T-Lymphocytes/immunology/*metabolism ; CD8-Positive T-Lymphocytes/immunology/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Electroporation ; *Gene Editing ; Lymphocyte Activation ; Mice ; Phenotype ; Plasmids/*genetics/metabolism ; Primary Cell Culture ; Research Design ; Ribonucleoproteins/*genetics/metabolism ; Workflow ; },
abstract = {The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4[+] or CD8[+] T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.},
}
@article {pmid33927418,
year = {2022},
author = {Petri, K and Zhang, W and Ma, J and Schmidts, A and Lee, H and Horng, JE and Kim, DY and Kurt, IC and Clement, K and Hsu, JY and Pinello, L and Maus, MV and Joung, JK and Yeh, JJ},
title = {CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells.},
journal = {Nature biotechnology},
volume = {40},
number = {2},
pages = {189-193},
pmid = {33927418},
issn = {1546-1696},
support = {R35 HG010717/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 GM134069/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Ribonucleoproteins/genetics ; *Zebrafish/genetics ; },
abstract = {Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.},
}
@article {pmid33927381,
year = {2021},
author = {Batinovic, S and Rose, JJA and Ratcliffe, J and Seviour, RJ and Petrovski, S},
title = {Cocultivation of an ultrasmall environmental parasitic bacterium with lytic ability against bacteria associated with wastewater foams.},
journal = {Nature microbiology},
volume = {6},
number = {6},
pages = {703-711},
pmid = {33927381},
issn = {2058-5276},
mesh = {Actinobacteria/*physiology/virology ; Bacteria/classification/genetics/*growth &amp; development/*isolation &amp; purification ; Bacterial Physiological Phenomena ; Bacteriophages/physiology ; Genome, Bacterial ; Phylogeny ; Waste Water/chemistry/*microbiology ; },
abstract = {Many wastewater treatment plants around the world suffer from the operational problem of foaming. This is characterized by a persistent stable foam that forms on the aeration basin, which reduces effluent quality. The foam is often stabilized by a highly hydrophobic group of Actinobacteria known as the Mycolata[1]. Gordonia amarae is one of the most frequently reported foaming members[1]. With no currently reliable method for treating foams, phage biocontrol has been suggested as an attractive treatment strategy[2]. Phages isolated from related foaming bacteria can destabilize foams at the laboratory scale[3,4]; however, no phage has been isolated that lyses G. amarae. Here, we assemble the complete genomes of G. amarae and a previously undescribed species, Gordonia pseudoamarae, to examine mechanisms that encourage stable foam production. We show that both of these species are recalcitrant to phage infection via a number of antiviral mechanisms including restriction, CRISPR-Cas and bacteriophage exclusion. Instead, we isolate and cocultivate an environmental ultrasmall epiparasitic bacterium from the phylum Saccharibacteria that lyses G. amarae and G. pseudoamarae and several other Mycolata commonly associated with wastewater foams. The application of this parasitic bacterium, 'Candidatus Mycosynbacter amalyticus', may represent a promising strategy for the biocontrol of bacteria responsible for stabilizing wastewater foams.},
}
@article {pmid33926924,
year = {2021},
author = {Li, M and Gong, L and Cheng, F and Yu, H and Zhao, D and Wang, R and Wang, T and Zhang, S and Zhou, J and Shmakov, SA and Koonin, EV and Xiang, H},
title = {Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.},
journal = {Science (New York, N.Y.)},
volume = {372},
number = {6541},
pages = {},
doi = {10.1126/science.abe5601},
pmid = {33926924},
issn = {1095-9203},
mesh = {CRISPR-Associated Proteins/genetics/*physiology ; CRISPR-Cas Systems/genetics/*physiology ; DNA Mutational Analysis ; Gene Expression Regulation, Archaeal ; Haloarcula/genetics/*physiology ; Operon ; RNA, Archaeal/*physiology ; RNA, Transfer, Arg/metabolism ; Toxin-Antitoxin Systems/genetics/*physiology ; },
abstract = {CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNA[UCU] (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.},
}
@article {pmid33925968,
year = {2021},
author = {Grote, S and Ureña-Bailén, G and Chan, KC and Baden, C and Mezger, M and Handgretinger, R and Schleicher, S},
title = {In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment.},
journal = {Cells},
volume = {10},
number = {5},
pages = {},
pmid = {33925968},
issn = {2073-4409},
mesh = {Antigens, Neoplasm/immunology ; B7 Antigens/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cytotoxicity, Immunologic ; Fibroblasts/metabolism ; *Gene Expression Regulation, Neoplastic ; Humans ; Hydrogen-Ion Concentration ; Hypoxia ; Immune System ; Immunosuppression Therapy ; Immunosuppressive Agents ; Immunotherapy, Adoptive/methods ; In Vitro Techniques ; Killer Cells, Natural/*cytology ; Lactic Acid/metabolism ; Lentivirus/genetics ; Melanoma/*immunology/metabolism ; Neoplasm Invasiveness ; Receptors, Chimeric Antigen/*genetics ; Skin Neoplasms/*immunology/metabolism ; *Tumor Microenvironment ; },
abstract = {BACKGROUND: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME).<br><br>METHODS: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity.<br><br>RESULTS: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity.<br><br>CONCLUSIONS: The strong cytotoxic effect of a CD276-specific CAR in combination with an "off-the-shelf" NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.},
}
@article {pmid33925887,
year = {2021},
author = {Matsuzawa, A and Lee, J and Nakagawa, S and Itoh, J and Takahashi Ueda, M and Mitsuhashi, S and Kochi, Y and Kaneko-Ishino, T and Ishino, F},
title = {HERV-Derived Ervpb1 Is Conserved in Simiiformes, Exhibiting Expression in Hematopoietic Cell Lineages Including Macrophages.},
journal = {International journal of molecular sciences},
volume = {22},
number = {9},
pages = {},
pmid = {33925887},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; *Endogenous Retroviruses/genetics/isolation &amp; purification/metabolism ; Fluorescent Dyes ; Gene Editing/methods ; Genes, Viral ; Haplorhini/*virology ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Macrophages/metabolism/*virology ; Viral Fusion Proteins/genetics/metabolism ; Viral Proteins/genetics/metabolism ; },
abstract = {(1) Background: The ERVPb1 gene in humans is derived from an envelope (Env) gene of a human endogenous retrovirus group, HERV-P(b). The ERVPb1 gene reportedly has a conserved open reading frame (ORF) in Old World monkeys. Although its forced expression led to cell-fusion in an ex vivo cell culture system, like other Env-derived genes such as syncytin-1 and -2, its mRNA expression is not placenta-specific, but almost ubiquitous, albeit being quite low in human tissues and organs, implying a distinct role for ERVPb1. (2) Methods: To elucidate the cell lineage(s) in which the ERVPb1 protein is translated in human development, we developed a novel, highly sensitive system for detecting HERV-derived proteins/peptides expressed in the tissue differentiation process of human induced pluripotent stem cells (iPSCs). (3) Results: We first determined that ERVPb1 is also conserved in New World monkeys. Then, we showed that the ERVPb1 protein is translated from a uniquely spliced ERVPb1 transcript in hematopoietic cell lineages, including a subset of macrophages, and further showed that its mRNA expression is upregulated by lipopolysaccharide (LPS) stimulation in primary human monocytes. (4) Conclusions: ERVPb1 is unique to Simiiformes and actually translated in hematopoietic cell lineages, including a subset of macrophages.},
}
@article {pmid33925396,
year = {2021},
author = {Han, JP and Lee, JH and Lee, GS and Koo, OJ and Yeom, SC},
title = {Positive Correlation between nNOS and Stress-Activated Bowel Motility Is Confirmed by In Vivo HiBiT System.},
journal = {Cells},
volume = {10},
number = {5},
pages = {},
pmid = {33925396},
issn = {2073-4409},
mesh = {Animals ; Biochemistry/*methods ; Brain/enzymology ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; *Gastrointestinal Motility ; Gastrointestinal Transit ; Hypothalamo-Hypophyseal System/enzymology/physiopathology ; Intestine, Large/enzymology ; Maternal Deprivation ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type I/*metabolism ; Oligodeoxyribonucleotides/metabolism ; Pituitary-Adrenal System/enzymology/physiopathology ; Stress, Psychological/*enzymology/*physiopathology ; },
abstract = {Neuronal nitric oxide synthase (nNOS) has various roles as a neurotransmitter. However, studies to date have produced insufficient data to fully support the correlation between nNOS and bowel motility. This study aimed to investigate the correlation between nNOS expression and gastrointestinal (GI) tract motility using a stress-induced neonatal maternal separation (NMS) mouse model. In this study, we generated a genetically modified mouse with the HiBiT sequence knock-in into the nNOS gene using CRISPR/Cas9 for analyzing accurate nNOS expression. nNOS expression was measured in the stomach, small intestine, large intestine, adrenal gland, and hypothalamus tissues after establishing the NMS model. The NMS model exhibited a significant increase in nNOS expression in large intestine, adrenal gland, and hypothalamus. Moreover, a significant positive correlation was observed between whole gastrointestinal transit time and the expression level of nNOS. We reasoned that NMS induced chronic stress and consequent nNOS activation in the hypothalamic-pituitary-adrenal (HPA) axis, and led to an excessive increase in intestinal motility in the lower GI tract. These results demonstrated that HiBiT is a sensitive and valuable tool for analyzing in vivo gene activation, and nNOS could be a biomarker of the HPA axis-linked lower intestinal tract dysfunction.},
}
@article {pmid33925088,
year = {2021},
author = {Lin, W and Gupta, SK and Arazi, T and Spitzer-Rimon, B},
title = {MIR172d Is Required for Floral Organ Identity and Number in Tomato.},
journal = {International journal of molecular sciences},
volume = {22},
number = {9},
pages = {},
pmid = {33925088},
issn = {1422-0067},
mesh = {Arabidopsis/genetics/growth &amp; development ; Base Sequence ; CRISPR-Cas Systems ; Flowers/genetics/growth &amp; development ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Genes, Plant ; Lycopersicon esculentum/*genetics/*growth &amp; development ; MicroRNAs/chemistry/*genetics ; Mutation ; Nucleic Acid Conformation ; Phenotype ; Phylogeny ; Plant Leaves/genetics/growth &amp; development ; Plants, Genetically Modified ; RNA, Plant/chemistry/*genetics ; },
abstract = {MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-d[CR]). The slmir172c-d[CR] plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172c[CR] loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172d[CR] alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets.},
}
@article {pmid33924895,
year = {2021},
author = {Jung, H and Lee, A and Jo, SH and Park, HJ and Jung, WY and Kim, HS and Lee, HJ and Jeong, SG and Kim, YS and Cho, HS},
title = {Nitrogen Signaling Genes and SOC1 Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage (Brassica rapa L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple BrSOC1 Homologs.},
journal = {International journal of molecular sciences},
volume = {22},
number = {9},
pages = {},
pmid = {33924895},
issn = {1422-0067},
mesh = {Brassica rapa/*physiology ; CRISPR-Cas Systems ; Feedback, Physiological ; Flowers/*physiology ; Gene Regulatory Networks ; MADS Domain Proteins/*physiology ; Nitrate Reductase/genetics/metabolism ; Nitrogen/*metabolism ; Plant Proteins/*physiology ; Sequence Analysis, RNA ; Transcriptome ; },
abstract = {Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage (Brassica rapa L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, "4004" (early bolting) and "50" (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), were up-regulated in line "50" with or without vernalization. In agreement with the transcription analysis, the shoots in line "50" had substantially higher nitrogen levels than those in "4004". Upon vernalization, the flowering repressor gene Circadian 1 (BrCIR1) was significantly up-regulated in line "50", whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line "4004". CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.},
}
@article {pmid33924851,
year = {2021},
author = {Teng, M and Yao, Y and Nair, V and Luo, J},
title = {Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development.},
journal = {Viruses},
volume = {13},
number = {5},
pages = {},
pmid = {33924851},
issn = {1999-4915},
mesh = {Animals ; *Biomedical Research/methods ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy/methods ; Humans ; Vaccinology/*methods ; Viral Vaccines/*genetics/immunology ; Viruses/*genetics/immunology ; },
abstract = {In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.},
}
@article {pmid33921600,
year = {2021},
author = {Liu, Q and Yang, F and Zhang, J and Liu, H and Rahman, S and Islam, S and Ma, W and She, M},
title = {Application of CRISPR/Cas9 in Crop Quality Improvement.},
journal = {International journal of molecular sciences},
volume = {22},
number = {8},
pages = {},
pmid = {33921600},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/*physiology ; Gene Editing ; Genome, Plant/*genetics ; Humans ; },
abstract = {The various crop species are major agricultural products and play an indispensable role in sustaining human life. Over a long period, breeders strove to increase crop yield and improve quality through traditional breeding strategies. Today, many breeders have achieved remarkable results using modern molecular technologies. Recently, a new gene-editing system, named the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, has also succeeded in improving crop quality. It has become the most popular tool for crop improvement due to its versatility. It has accelerated crop breeding progress by virtue of its precision in specific gene editing. This review summarizes the current application of CRISPR/Cas9 technology in crop quality improvement. It includes the modulation in appearance, palatability, nutritional components and other preferred traits of various crops. In addition, the challenge in its future application is also discussed.},
}
@article {pmid33920749,
year = {2021},
author = {Arroyo-Olarte, RD and Bravo Rodríguez, R and Morales-Ríos, E},
title = {Genome Editing in Bacteria: CRISPR-Cas and Beyond.},
journal = {Microorganisms},
volume = {9},
number = {4},
pages = {},
pmid = {33920749},
issn = {2076-2607},
abstract = {Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.},
}
@article {pmid33920513,
year = {2021},
author = {Shaffaf, T and Ghafar-Zadeh, E},
title = {COVID-19 Diagnostic Strategies. Part I: Nucleic Acid-Based Technologies.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {8},
number = {4},
pages = {},
pmid = {33920513},
issn = {2306-5354},
abstract = {The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused respiratory infection, resulting in more than two million deaths globally and hospitalizing thousands of people by March 2021. A considerable percentage of the SARS-CoV-2 positive patients are asymptomatic or pre-symptomatic carriers, facilitating the viral spread in the community by their social activities. Hence, it is critical to have access to commercialized diagnostic tests to detect the infection in the earliest stages, monitor the disease, and follow up the patients. Various technologies have been proposed to develop more promising assays and move toward the mass production of fast, reliable, cost-effective, and portable PoC diagnostic tests for COVID-19 detection. Not only COVID-19 but also many other pathogens will be able to spread and attach to human bodies in the future. These technologies enable the fast identification of high-risk individuals during future hazards to support the public in such outbreaks. This paper provides a comprehensive review of current technologies, the progress in the development of molecular diagnostic tests, and the potential strategies to facilitate innovative developments in unprecedented pandemics.},
}
@article {pmid33919194,
year = {2021},
author = {Piergentili, R and Del Rio, A and Signore, F and Umani Ronchi, F and Marinelli, E and Zaami, S},
title = {CRISPR-Cas and Its Wide-Ranging Applications: From Human Genome Editing to Environmental Implications, Technical Limitations, Hazards and Bioethical Issues.},
journal = {Cells},
volume = {10},
number = {5},
pages = {},
pmid = {33919194},
issn = {2073-4409},
mesh = {*Bioethical Issues ; CRISPR-Cas Systems/*genetics ; *Embryo, Mammalian ; Gene Editing/methods ; *Genome, Human ; Genome, Plant ; Humans ; },
abstract = {The CRISPR-Cas system is a powerful tool for in vivo editing the genome of most organisms, including man. During the years this technique has been applied in several fields, such as agriculture for crop upgrade and breeding including the creation of allergy-free foods, for eradicating pests, for the improvement of animal breeds, in the industry of bio-fuels and it can even be used as a basis for a cell-based recording apparatus. Possible applications in human health include the making of new medicines through the creation of genetically modified organisms, the treatment of viral infections, the control of pathogens, applications in clinical diagnostics and the cure of human genetic diseases, either caused by somatic (e.g., cancer) or inherited (mendelian disorders) mutations. One of the most divisive, possible uses of this system is the modification of human embryos, for the purpose of preventing or curing a human being before birth. However, the technology in this field is evolving faster than regulations and several concerns are raised by its enormous yet controversial potential. In this scenario, appropriate laws need to be issued and ethical guidelines must be developed, in order to properly assess advantages as well as risks of this approach. In this review, we summarize the potential of these genome editing techniques and their applications in human embryo treatment. We will analyze CRISPR-Cas limitations and the possible genome damage caused in the treated embryo. Finally, we will discuss how all this impacts the law, ethics and common sense.},
}
@article {pmid33918544,
year = {2021},
author = {Ma, J and Sun, S and Whelan, J and Shou, H},
title = {CRISPR/Cas9-Mediated Knockout of GmFATB1 Significantly Reduced the Amount of Saturated Fatty Acids in Soybean Seeds.},
journal = {International journal of molecular sciences},
volume = {22},
number = {8},
pages = {},
pmid = {33918544},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Fatty Acids/*metabolism ; Gene Expression ; *Gene Knockout Techniques ; *Gene Targeting ; Genetic Association Studies ; Genetic Engineering ; Humans ; Mutation ; Phenotype ; Plant Proteins/genetics ; Soybean Oil/genetics/metabolism ; Soybeans/*genetics/*metabolism ; Thiolester Hydrolases/*deficiency ; },
abstract = {Soybean (Glycine max) oil is one of the most widely used vegetable oils across the world. Breeding of soybean to reduce the saturated fatty acid (FA) content, which is linked to cardiovascular disease, would be of great significance for nutritional improvement. Acyl-acyl carrier protein thioesterases (FATs) can release free FAs and acyl-ACP, which ultimately affects the FA profile. In this study, we identified a pair of soybean FATB coding genes, GmFATB1a and GmFATB1b. Mutants that knock out either or both of the GmFATB1 genes were obtained via CRISPR/Cas9. Single mutants, fatb1a and fatb1b, showed a decrease in leaf palmitic and stearic acid contents, ranging from 11% to 21%. The double mutant, fatb1a:1b, had a 42% and 35% decrease in palmitic and stearic acid content, displayed growth defects, and were male sterility. Analysis of the seed oil profile revealed that fatb1a and fatb1b had significant lower palmitic and stearic acid contents, 39-53% and 17-37%, respectively, while that of the unsaturated FAs were the same. The relative content of the beneficial FA, linoleic acid, was increased by 1.3-3.6%. The oil profile changes in these mutants were confirmed for four generations. Overall, our data illustrate that GmFATB1 knockout mutants have great potential in improving the soybean oil quality for human health.},
}
@article {pmid33918348,
year = {2021},
author = {Beerens, D and Franch-Arroyo, S and Sullivan, TJ and Goosmann, C and Brinkmann, V and Charpentier, E},
title = {Survival Strategies of Streptococcus pyogenes in Response to Phage Infection.},
journal = {Viruses},
volume = {13},
number = {4},
pages = {},
pmid = {33918348},
issn = {1999-4915},
mesh = {CRISPR-Cas Systems ; Genome, Viral ; Lysogeny ; *Microbial Viability ; Prophages/genetics ; Streptococcus Phages/*genetics/*pathogenicity ; Streptococcus pyogenes/*physiology/*virology ; Virulence ; },
abstract = {Bacteriophages exert strong evolutionary pressure on their microbial hosts. In their lytic lifecycle, complete bacterial subpopulations are utilized as hosts for bacteriophage replication. However, during their lysogenic lifecycle, bacteriophages can integrate into the host chromosome and alter the host's genomic make-up, possibly resulting in evolutionary important adjustments. Not surprisingly, bacteria have evolved sophisticated immune systems to protect against phage infection. Streptococcus pyogenes isolates are frequently lysogenic and their prophages have been shown to be major contributors to the virulence of this pathogen. Most S. pyogenes phage research has focused on genomic prophages in relation to virulence, but little is known about the defensive arsenal of S. pyogenes against lytic phage infection. Here, we characterized Phage A1, an S. pyogenes bacteriophage, and investigated several mechanisms that S. pyogenes utilizes to protect itself against phage predation. We show that Phage A1 belongs to the Siphoviridae family and contains a circular double-stranded DNA genome that follows a modular organization described for other streptococcal phages. After infection, the Phage A1 genome can be detected in isolated S. pyogenes survivor strains, which enables the survival of the bacterial host and Phage A1 resistance. Furthermore, we demonstrate that the type II-A CRISPR-Cas system of S. pyogenes acquires new spacers upon phage infection, which are increasingly detectable in the absence of a capsule. Lastly, we show that S. pyogenes produces membrane vesicles that bind to phages, thereby limiting the pool of phages available for infection. Altogether, this work provides novel insight into survival strategies employed by S. pyogenes to combat phage predation.},
}
@article {pmid33918190,
year = {2021},
author = {Wilding-Steele, T and Ramette, Q and Jacottin, P and Soucaille, P},
title = {Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33918190},
issn = {1422-0067},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Clostridium acetobutylicum/*genetics/metabolism ; Gene Editing/*methods ; Metabolic Engineering/*methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology-several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.},
}
@article {pmid33917183,
year = {2021},
author = {Antiochia, R},
title = {Paper-Based Biosensors: Frontiers in Point-of-Care Detection of COVID-19 Disease.},
journal = {Biosensors},
volume = {11},
number = {4},
pages = {},
pmid = {33917183},
issn = {2079-6374},
mesh = {Antigens, Viral/analysis ; Biosensing Techniques/instrumentation/*methods ; COVID-19/*diagnosis/virology ; CRISPR-Cas Systems/genetics ; Humans ; Immunoassay ; *Paper ; Point-of-Care Systems ; RNA, Viral/analysis ; SARS-CoV-2/genetics/isolation &amp; purification/metabolism ; },
abstract = {This review summarizes the state of the art of paper-based biosensors (PBBs) for coronavirus disease 2019 (COVID-19) detection. Three categories of PBB are currently being been used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics, namely for viral gene, viral antigen and antibody detection. The characteristics, the analytical performance, the advantages and drawbacks of each type of biosensor are highlighted and compared with traditional methods. It is hoped that this review will be useful for scientists for the development of novel PBB platforms with enhanced performance for helping to contain the COVID-19 outbreak, by allowing early diagnosis at the point of care (POC).},
}
@article {pmid33917142,
year = {2021},
author = {Bloh, K and Rivera-Torres, N},
title = {A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.},
journal = {International journal of molecular sciences},
volume = {22},
number = {8},
pages = {},
pmid = {33917142},
issn = {1422-0067},
support = {PP20GM109021 and P20GM103446/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Repair ; *Gene Editing ; INDEL Mutation ; Models, Biological ; RNA, Guide ; *Recombinational DNA Repair ; },
abstract = {The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.},
}
@article {pmid33916905,
year = {2021},
author = {Wu, QW and Kapfhammer, JP},
title = {The Bacterial Enzyme Cas13 Interferes with Neurite Outgrowth from Cultured Cortical Neurons.},
journal = {Toxins},
volume = {13},
number = {4},
pages = {},
pmid = {33916905},
issn = {2072-6651},
mesh = {Animals ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Cerebral Cortex/*enzymology/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Leptotrichia/*enzymology/genetics ; Mice ; *Neuronal Outgrowth ; Neurons/*enzymology/pathology ; },
abstract = {The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.},
}
@article {pmid33916842,
year = {2021},
author = {Tenea, GN and Ortega, C},
title = {Genome Characterization of Lactiplantibacillus plantarum Strain UTNGt2 Originated from Theobroma grandiflorum (White Cacao) of Ecuadorian Amazon: Antimicrobial Peptides from Safety to Potential Applications.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {4},
pages = {},
pmid = {33916842},
issn = {2079-6382},
abstract = {The genome characterization of the Lactiplantibacillus plantarum strain UTNGt2, isolated from wild copoazu or white cacao (Theobroma grandiflorum), is described. A total of 31 contigs is assembled with a total length of 3,264,448 bases, with all contigs matching the core genome of different groups in the database. The genome size is 3,540,752 bases with GC content of 44.53% and the genome repeat sequences constitute around 457,386 bases of the assembly. The UTNGt2 matches the Lactiplantibacillus plantarum genome with 99% identity. The genome contains 3115 genes, 3052 protein-coding genes, assigned with the EggNOG database. On the basis of the results, 745 proteins are classified with an unknown function, from which 128 proteins have no match in the BLASTN database. It also contains 57 tRNAs, 5 copies of 5S rRNA, and 1 copy of tmRNA. Based on gene prediction and annotation results, 9.4% of proteins are involved in carbohydrate transport and metabolism and 8.46% in transcription, 2.36% are responsible for defense mechanisms, 0.5% are responsible for the biosynthesis of secondary metabolites, transport, and catabolism, while 25.11% have an unknown function. The genome revealed the presence of genes involved in riboflavin and folate production, the presence of CRISPR/Cas genes, phage sequences, the absence of acquired antibiotics resistance genes, virulence, and pathogenic factors, suggesting that UTNGt2 is a safe strain. Its highly antimicrobial capacity is related to the presence of two bacteriocin clusters (class IIc) of the sactipeptide class (contig 4) and plantaricin E class (contig 22), as detected by the BAGEL 4 webserver. Several RiPP-like peptides (non-bactericidal ribosomally produced and post-translationally modified peptides), polyketides (PKs), and terpenes were predicted. Whole-genome sequencing analysis revealed that the UTNGt2 strain has diverse bacteriocins with a high inhibitory capacity, thus it is a bacteriocinogenic strain. Considering the safety profile, UTNGt2 is a nonpathogenic, nonvirulent strain with valuable biotechnological traits and can be further exploited for its probiotic and antimicrobial potential in the food industry or as a potential producer strain of antimicrobial peptides as an alternative to conventional antibiotics.},
}
@article {pmid33916772,
year = {2021},
author = {Um, T and Park, T and Shim, JS and Kim, YS and Lee, GS and Choi, IY and Kim, JK and Seo, JS and Park, SC},
title = {Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33916772},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; *Open Reading Frames ; Plants, Genetically Modified/*genetics ; },
abstract = {Global population growth and climate change are posing increasing challenges to the production of a stable crop supply using current agricultural practices. The generation of genetically modified (GM) crops has contributed to improving crop stress tolerance and productivity; however, many regulations are still in place that limit their commercialization. Recently, alternative biotechnology-based strategies, such as gene-edited (GE) crops, have been in the spotlight. Gene-editing technology, based on the clustered regularly interspaced short palindromic repeats (CRISPR) platform, has emerged as a revolutionary tool for targeted gene mutation, and has received attention as a game changer in the global biotechnology market. Here, we briefly introduce the concept of upstream open reading frames (uORFs) editing, which allows for control of the translation of downstream ORFs, and outline the potential for enhancing target gene expression by mutating uORFs. We discuss the current status of developing stress-tolerant crops, and discuss uORF targets associated with salt stress-responsive genes in rice that have already been verified by transgenic research. Finally, we overview the strategy for developing GE crops using uORF editing via the CRISPR-Cas9 system. A case is therefore made that the mutation of uORFs represents an efficient method for developing GE crops and an expansion of the scope of application of genome editing technology.},
}
@article {pmid33916763,
year = {2021},
author = {Reuven, N and Adler, J and Myers, N and Shaul, Y},
title = {CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33916763},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; HEK293 Cells ; HeLa Cells ; Histone Acetyltransferases/*genetics ; Humans ; *Mutagenesis ; *Mutation ; Proteasome Endopeptidase Complex/*genetics ; TATA-Binding Protein Associated Factors/*genetics ; Transcription Factor TFIID/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is "scarless" since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system's utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.},
}
@article {pmid33916717,
year = {2021},
author = {Hesami, M and Yoosefzadeh Najafabadi, M and Adamek, K and Torkamaneh, D and Jones, AMP},
title = {Synergizing Off-Target Predictions for In Silico Insights of CENH3 Knockout in Cannabis through CRISPR/Cas.},
journal = {Molecules (Basel, Switzerland)},
volume = {26},
number = {7},
pages = {},
pmid = {33916717},
issn = {1420-3049},
mesh = {Area Under Curve ; CRISPR-Cas Systems/*genetics ; Cannabis/*genetics ; Centromere/*metabolism ; *Computer Simulation ; *Gene Knockout Techniques ; Histones/*genetics ; ROC Curve ; Support Vector Machine ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing system has recently been used for haploid production in plants. Haploid induction using the CRISPR/Cas system represents an attractive approach in cannabis, an economically important industrial, recreational, and medicinal plant. However, the CRISPR system requires the design of precise (on-target) single-guide RNA (sgRNA). Therefore, it is essential to predict off-target activity of the designed sgRNAs to avoid unexpected outcomes. The current study is aimed to assess the predictive ability of three machine learning (ML) algorithms (radial basis function (RBF), support vector machine (SVM), and random forest (RF)) alongside the ensemble-bagging (E-B) strategy by synergizing MIT and cutting frequency determination (CFD) scores to predict sgRNA off-target activity through in silico targeting a histone H3-like centromeric protein, HTR12, in cannabis. The RF algorithm exhibited the highest precision, recall, and F-measure compared to all the tested individual algorithms with values of 0.61, 0.64, and 0.62, respectively. We then used the RF algorithm as a meta-classifier for the E-B method, which led to an increased precision with an F-measure of 0.62 and 0.66, respectively. The E-B algorithm had the highest area under the precision recall curves (AUC-PRC; 0.74) and area under the receiver operating characteristic (ROC) curves (AUC-ROC; 0.71), displaying the success of using E-B as one of the common ensemble strategies. This study constitutes a foundational resource of utilizing ML models to predict gRNA off-target activities in cannabis.},
}
@article {pmid33915117,
year = {2021},
author = {Chen, K and Wang, M and Zhang, R and Li, J},
title = {Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH.},
journal = {Analytical biochemistry},
volume = {625},
number = {},
pages = {114211},
doi = {10.1016/j.ab.2021.114211},
pmid = {33915117},
issn = {1096-0309},
mesh = {*CRISPR-Cas Systems ; Epstein-Barr Virus Infections/*diagnosis/virology ; Herpesvirus 4, Human/*genetics ; Humans ; In Situ Hybridization, Fluorescence/*methods ; RNA, Viral/*analysis ; Tissue Fixation/*methods ; },
abstract = {Identification of Epstein-Barr virus (EBV)-infected cells is critical for the diagnosis and clinical management of EBV-associated diseases. EBV-encoded RNA (EBER) located in the nucleus is a reliable marker due to its high levels of expression and inherent stability in tissue specimens. EBER in situ hybridization has long been the gold standard for detecting tumor-associated latent EBV infection and is valuable in determining the primary site and radiation fields of EBV-related malignancies. However, reliable detection is somewhat restricted by diffused signal and time-consuming procedure of this method, especially when proteins and RNA needed to be labeled simultaneously. Here, we optimized and validated our CRISPR-dCas9 mediated in situ RNA imaging tool-RCasFISH that enabled us to detect EBER rapidly and was compatible with IHC methods in fixed cells and tissue sections. Our approach could provide an attractive alternative for the molecular diagnosis of latent EBV infection.},
}
@article {pmid33915112,
year = {2021},
author = {Freije, CA and Sabeti, PC},
title = {Detect and destroy: CRISPR-based technologies for the response against viruses.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {5},
pages = {689-703},
pmid = {33915112},
issn = {1934-6069},
mesh = {COVID-19/*diagnosis/therapy ; *CRISPR-Cas Systems ; Humans ; Mutation ; RNA, Circular/genetics ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {Despite numerous viral outbreaks in the last decade, including a devastating global pandemic, diagnostic and therapeutic technologies remain severely lacking. CRISPR-Cas systems have the potential to address these critical needs in the response against infectious disease. Initially discovered as the bacterial adaptive immune system, these systems provide a unique opportunity to create programmable, sequence-specific technologies for detection of viral nucleic acids and inhibition of viral replication. This review summarizes how CRISPR-Cas systems-in particular the recently discovered DNA-targeting Cas12 and RNA-targeting Cas13, both possessing a unique trans-cleavage activity-are being harnessed for viral diagnostics and therapies. We further highlight the numerous technologies whose development has accelerated in response to the COVID-19 pandemic.},
}
@article {pmid33914777,
year = {2021},
author = {Mok, PL and Anandasayanam, ANK and Oscar David, HM and Tong, J and Farhana, A and Khan, MSA and Sivaprakasam, G and Koh, AE and Alzahrani, B},
title = {Lung development, repair and cancer: A study on the role of MMP20 gene in adenocarcinoma.},
journal = {PloS one},
volume = {16},
number = {4},
pages = {e0250552},
pmid = {33914777},
issn = {1932-6203},
mesh = {A549 Cells ; Adenocarcinoma of Lung/*genetics/pathology ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Humans ; Lung/*growth &amp; development/metabolism/pathology ; Matrix Metalloproteinase 20/*genetics ; Organogenesis/genetics ; Survivin/*genetics ; Transfection ; },
abstract = {Multiple matrix metalloproteinases have significant roles in tissue organization during lung development, and repair. Imbalance of proteinases may lead to chronic inflammation, changes in tissue structure, and are also highly associated to cancer development. The role of MMP20 is not well studied in lung organogenesis, however, it was previously shown to be present at high level in lung adenocarcinoma. The current study aimed to identify the functional properties of MMP20 on cell proliferation and motility in a lung adenocarcinoma in vitro cell model, and relate the interaction of MMP20 with other molecular signalling pathways in the lung cells after gaining tumoral properties. In this study, two different single guide RNA (sgRNAs) that specifically targeted on MMP20 sites were transfected into human lung adenocarcinoma A549 cells by using CRISPR-Cas method. Following that, the changes of PI3-K, survivin, and MAP-K mRNA gene expression were determined by Real-Time Polymerase Chain Reaction (RT-PCR). The occurrence of cell death was also examined by Acridine Orange/Propidium Iodide double staining. Meanwhile, the motility of the transfected cells was evaluated by wound healing assay. All the data were compared with non-transfected cells as a control group. Our results demonstrated that the transfection of the individual sgRNAs significantly disrupted the proliferation of the A549 cell line through suppression in the gene expression of PI3-K, survivin, and MAP-K. When compared to non-transfected cells, both experimental cell groups showed reduction in the migration rate, as reflected by the wider gaps in the wound healing assay. The current study provided preliminary evidence that MMP20 could have regulatory role on stemness and proliferative genes in the lung tissues and affect the cell motility. It also supports the notion that targeting MMP20 could be a potential treatment mode for halting cancer progression.},
}
@article {pmid33913600,
year = {2021},
author = {Zhang, L and Wang, T and Wang, G and Bi, A and Wassie, M and Xie, Y and Cao, L and Xu, H and Fu, J and Chen, L and Zhao, Y and Hu, T},
title = {Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {8},
pages = {1410-1415},
doi = {10.1111/jipb.13101},
pmid = {33913600},
issn = {1744-7909},
mesh = {*Alleles ; Base Sequence ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Heat-Shock Response ; Mutagenesis/genetics ; Mutation/genetics ; Poaceae/*genetics ; *Polyploidy ; Self-Incompatibility in Flowering Plants/*genetics ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.},
}
@article {pmid33913568,
year = {2021},
author = {Liu, Z and Liang, Z and Zhou, Z and Li, L and Meng, D and Li, X and Tao, J and Jiang, Z and Gu, Y and Huang, Y and Liu, X and Yang, Z and Drewniak, L and Liu, T and Liu, Y and Liu, S and Wang, J and Jiang, C and Yin, H},
title = {Mobile genetic elements mediate the mixotrophic evolution of novel Alicyclobacillus species for acid mine drainage adaptation.},
journal = {Environmental microbiology},
volume = {23},
number = {7},
pages = {3896-3912},
doi = {10.1111/1462-2920.15543},
pmid = {33913568},
issn = {1462-2920},
mesh = {*Alicyclobacillus/genetics ; DNA Transposable Elements/genetics ; Genomic Islands ; Mining ; Phylogeny ; },
abstract = {Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments and the important role of MGEs in rapid environment niche expansions.},
}
@article {pmid33913466,
year = {2021},
author = {Akinci, E and Hamilton, MC and Khowpinitchai, B and Sherwood, RI},
title = {Using CRISPR to understand and manipulate gene regulation.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {9},
pages = {},
pmid = {33913466},
issn = {1477-9129},
support = {R01 HG008754/HG/NHGRI NIH HHS/United States ; R21 HG010391/HG/NHGRI NIH HHS/United States ; R21 OD025309/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Epigenomics ; Gene Editing/*methods ; *Gene Expression Regulation ; Gene Regulatory Networks ; Humans ; Mutation ; },
abstract = {Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.},
}
@article {pmid33912919,
year = {2021},
author = {Husser, MC and Skaik, N and Martin, VJJ and Piekny, A},
title = {CRISPR-Cas tools to study gene function in cytokinesis.},
journal = {Journal of cell science},
volume = {134},
number = {8},
pages = {},
doi = {10.1242/jcs.254409},
pmid = {33912919},
issn = {1477-9137},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytokinesis/genetics ; Gene Editing ; Phenotype ; },
abstract = {Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Finally, we address the need for gene editing to study cytokinesis in more physiologically relevant contexts. Therefore, this Review provides a roadmap for gene editing to be used in the study of cytokinesis and other cellular processes.},
}
@article {pmid33911254,
year = {2021},
author = {Le Bras, A},
title = {A new construct for CRISPR delivery in chicken.},
journal = {Lab animal},
volume = {50},
number = {5},
pages = {120},
doi = {10.1038/s41684-021-00765-4},
pmid = {33911254},
issn = {1548-4475},
mesh = {Animals ; CRISPR-Cas Systems ; Chick Embryo ; *Chickens/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plasmids ; },
}
@article {pmid33911248,
year = {2021},
author = {Neff, E},
title = {Interactions in the sea urchin gut.},
journal = {Lab animal},
volume = {50},
number = {5},
pages = {121},
doi = {10.1038/s41684-021-00770-7},
pmid = {33911248},
issn = {1548-4475},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome ; Immunity ; Larva ; Mutagenesis ; Sea Urchins ; Vibrio ; },
}
@article {pmid33910311,
year = {2021},
author = {Zhang, Y and Wang, LY and Li, JZ and Jiang, PF and Hu, JD and Chen, BY},
title = {[CRISPR/Cas9-mediated microRNA-21 knockout increased imatinib sensitivity in chronic myeloid leukemia cells].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {42},
number = {3},
pages = {243-249},
pmid = {33910311},
issn = {0253-2727},
mesh = {*Antineoplastic Agents/pharmacology ; Apoptosis ; CRISPR-Cas Systems ; Cell Proliferation ; Drug Resistance, Neoplasm/genetics ; Fusion Proteins, bcr-abl/genetics ; Humans ; Imatinib Mesylate/pharmacology ; K562 Cells ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics ; *MicroRNAs/genetics ; Phosphatidylinositol 3-Kinases ; },
abstract = {Objective: To observe the effects of miR-21 knockout on proliferation and drug resistance in K562/G01 cells, and to preliminarily explore the mechanism of imatinib sensitivity by knocking out miR-21 in K562/G01 cells. Methods: Using CRISPR/Cas9 to knock out the miR-21 gene in K562/G01 cells, and single-cell-derived clones of miR-21 knockout were obtained by genomic DNA PCR screening, Sanger sequencing, and real-time PCR. We used MTT and cell colony formation assays to assess the cell proliferation, and determined imatinib sensitivity by MTT assay and Annexin-Ⅴ-APC/7-AAD double staining flow cytometry. Using western blot, we examined the potential mechanisms affecting imatinib sensitivity by knocking out miR-21 in K562/G01 cells. Results: Three miR-21 knockout K562/G01 single-cell-derived clones were successfully constructed. The mutation efficiency mediated by CRISPR/Cas9 was 7.12%-8.11%. MiR-21 knockout inhibited the proliferation of K562/G01 cells; the clone formation rates of WT and 1#, 2#, 6# K562/G01 single-cell clones were (57.67±8.25) %, (26.94± 5.36) %, (7.17±2.11) %, (31.50±3.65) %, respectively. MiR-21 knockout increased the sensitivity of K562/G01 cells to imatinib, IC(50) of imatinib in WT, and 1#, 2#, 6# K562/G01 single-cell clones were (21.92±1.36) µmol/ml, (3.98±0.39) µmol/ml, (5.38±1.01) µmol/ml, (9.24±1.36) µmol/ml. After the knockout of miR-21, the activation of PI3K/Akt signaling molecules was inhibited, while the expression of P210(B)CR-ABL and p-P210(BCR-ABL) was downregulated; however, the expression of PTEN was not affected. Conclusion: The knockout of miR-21 can suppress cell proliferation and improve sensitivity to imatinib in K562/G01 cells, which may be achieved by inhibiting the PI3K/AKT signaling pathway and BCR-ABL expression.},
}
@article {pmid33909475,
year = {2021},
author = {Abdeen, AA and Cosgrove, BD and Gersbach, CA and Saha, K},
title = {Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science.},
journal = {Annual review of biomedical engineering},
volume = {23},
number = {},
pages = {493-516},
doi = {10.1146/annurev-bioeng-122019-121602},
pmid = {33909475},
issn = {1545-4274},
support = {U01 EB028901/EB/NIBIB NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; R21 AR072265/AR/NIAMS NIH HHS/United States ; U01 HL156348/HL/NHLBI NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; },
mesh = {Biocompatible Materials ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
abstract = {The recent discovery and subsequent development of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease.},
}
@article {pmid33909408,
year = {2021},
author = {Tang, W and Han, L and Lu, X and Wang, Z and Liu, F and Li, Y and Liu, S and Liu, S and Tian, R and Liu, J and Ding, B},
title = {A Nucleic Acid/Gold Nanorod-Based Nanoplatform for Targeted Gene Editing and Combined Tumor Therapy.},
journal = {ACS applied materials &amp; interfaces},
volume = {13},
number = {18},
pages = {20974-20981},
doi = {10.1021/acsami.1c02122},
pmid = {33909408},
issn = {1944-8252},
mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Combined Modality Therapy ; *Gene Editing ; Gold/*chemistry ; Humans ; MCF-7 Cells ; Microscopy, Confocal ; Nanotubes/*chemistry ; Neoplasms/pathology/*therapy ; Nucleic Acids/*chemistry ; },
abstract = {The CRISPR/Cas9 gene-editing system has become a promising strategy for tumor therapy with its powerful oncogene-editing ability. However, the efficient delivery of sgRNA/Cas9 complex into target tumor cells remains a challenge. Herein, we report a facile strategy for the construction of an sgRNA/Cas9 complex co-assembled nanoplatform for targeted gene editing and combined tumor therapy. In our design, the TAT peptide and thiolated DNA linker functionalized gold nanorod can efficiently load the sgRNA/Cas9 complex through the hybridization between the 3' overhang of sgRNA and the DNA linker. Due to the integration of an active cell targeting group (aptamer) and nuclear targeting peptide (TAT), the multifunctional nanoplatform can elicit the targeted cellular internalization and efficient nuclear targeting transportation to realize endogenous RNase H activated gene editing of the tumor-associated gene polo-like kinase 1 (PLK1). With mild photothermal treatment, this sgRNA/Cas9 complex loaded nanoplatform achieved efficient inhibition of tumor cell proliferation. This multifunctional nanocarrier provides a new strategy for the development of combined tumor therapy.},
}
@article {pmid33907890,
year = {2021},
author = {Zhang, ZX and Wang, LR and Xu, YS and Jiang, WT and Shi, TQ and Sun, XM and Huang, H},
title = {Recent advances in the application of multiplex genome editing in Saccharomyces cerevisiae.},
journal = {Applied microbiology and biotechnology},
volume = {105},
number = {10},
pages = {3873-3882},
pmid = {33907890},
issn = {1432-0614},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Saccharomyces cerevisiae is a widely used microorganism and a greatly popular cell factory for the production of various chemicals. In order to improve the yield of target chemicals, it is often necessary to increase the copy numbers of key genes or engineer the related metabolic pathways, which traditionally required time-consuming repetitive rounds of gene editing. With the development of gene-editing technologies such as meganucleases, TALENs, and the CRISPR/Cas system, multiplex genome editing has entered a period of rapid development to speed up cell factory optimization. Multi-copy insertion and removing bottlenecks in biosynthetic pathways can be achieved through gene integration and knockout, for which multiplexing can be accomplished by targeting repetitive sequences and multiple sites, respectively. Importantly, the development of the CRISPR/Cas system has greatly increased the speed and efficiency of multiplex editing. In this review, the various multiplex genome editing technologies in S. cerevisiae were summarized, and the principles, advantages, and the disadvantages were analyzed and discussed. Finally, the practical applications and future prospects of multiplex genome editing were discussed. KEY POINTS: • The development of multiplex genome editing in S. cerevisiae was summarized. • The pros and cons of various multiplex genome editing technologies are discussed. • Further prospects on the improvement of multiplex genome editing are proposed.},
}
@article {pmid33906967,
year = {2021},
author = {Jiao, C and Sharma, S and Dugar, G and Peeck, NL and Bischler, T and Wimmer, F and Yu, Y and Barquist, L and Schoen, C and Kurzai, O and Sharma, CM and Beisel, CL},
title = {Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9.},
journal = {Science (New York, N.Y.)},
volume = {372},
number = {6545},
pages = {941-948},
pmid = {33906967},
issn = {1095-9203},
mesh = {Base Sequence ; COVID-19/diagnosis/virology ; COVID-19 Nucleic Acid Testing ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Campylobacter jejuni ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Hybridization ; RNA/*analysis/*genetics ; RNA, Bacterial/genetics ; RNA, Guide/*genetics ; RNA, Messenger/genetics ; RNA, Viral/*analysis/genetics ; SARS-CoV-2/*genetics ; Spike Glycoprotein, Coronavirus/genetics ; },
abstract = {CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp[614]→Gly) variant with single-base resolution in patient samples.},
}
@article {pmid33906944,
year = {2021},
author = {Schubert, MG and Goodman, DB and Wannier, TM and Kaur, D and Farzadfard, F and Lu, TK and Shipman, SL and Church, GM},
title = {High-throughput functional variant screens via in vivo production of single-stranded DNA.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {18},
pages = {},
pmid = {33906944},
issn = {1091-6490},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; DNA, Single-Stranded/biosynthesis/*genetics ; Drug Resistance, Microbial/*genetics ; Escherichia coli/genetics ; Gene Library ; *Genetic Engineering ; Genome, Bacterial/*genetics ; Genomics ; High-Throughput Nucleotide Sequencing ; High-Throughput Screening Assays ; Saccharomyces cerevisiae/genetics ; Synthetic Biology ; },
abstract = {Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.},
}
@article {pmid33906919,
year = {2021},
author = {Lu, B and Liu, M and Gu, L and Li, Y and Shen, S and Guo, G and Wang, F and He, X and Zhao, Y and Shang, X and Wang, L and Yang, G and Zhu, Q and Cao, J and Jiang, C and Culleton, R and Wei, G and Zhang, Q},
title = {The Architectural Factor HMGB1 Is Involved in Genome Organization in the Human Malaria Parasite Plasmodium falciparum.},
journal = {mBio},
volume = {12},
number = {2},
pages = {},
pmid = {33906919},
issn = {2150-7511},
mesh = {CRISPR-Cas Systems ; Epigenesis, Genetic ; Gene Expression ; *Gene Expression Regulation ; *Genome, Protozoan ; HMGB1 Protein/*genetics ; Humans ; Plasmodium falciparum/*genetics/pathogenicity ; Protozoan Proteins/*genetics ; },
abstract = {The three-dimensional (3D) genome organization plays a critical role in the regulation of gene expression in eukaryotic organisms. In the unicellular malaria parasite Plasmodium falciparum, the high-order chromosome organization has emerged as an important epigenetic pathway mediating gene expression, particularly for virulence genes, but the related architectural factors and underlying mechanism remain elusive. Herein, we have identified the high-mobility-group protein HMGB1 as a critical architectural factor for maintenance of genome organization in P. falciparum Genome-wide occupancy analysis (chromatin immunoprecipitation sequencing [ChIP-seq]) shows that the HMGB1 protein is recruited mainly to centromeric regions likely via a DNA-binding-independent pathway. Chromosome conformation capture coupled with next-generation sequencing (Hi-C-seq) and 3D modeling analysis show that the loss of HMGB1 disrupts the integrity of centromere/telomere-based chromosome organization accompanied with diminished interaction frequency among centromere clusters. This triggers local chromatin alteration and dysregulated gene expression. Notably, the entire repertoire of the primary virulence genes (var) was completely silenced in the absence of P. falciparum HMGB1 (PfHMGB1). Furthermore, the disrupted nuclear organization was reconstituted by complementation of HMGB1, thereby rescuing the mutually exclusive expression of the var gene family. Collectively, these data demonstrate that the architectural factor HMGB1 is associated with gene expression via mediating the high-order structure of genome organization. This finding not only contributes better understanding of the epigenetic regulation of gene expression but may also provide novel targets for antimalarial strategies.IMPORTANCE Malaria remains a major public health and economic burden currently. The mutually exclusive expression of the virulence genes is associated with the pathogenesis and immune evasion of human malaria parasites in the host. The nuclear architecture provides a well-organized environment for differential gene expression in the nucleus, but the underlying mechanism remains largely unknown. In this study, we have identified the highly conserved high-mobility-group protein HMGB1 as a key architecture regulator involved in virulence gene expression by establishing high-order genome organization in the nucleus of P. falciparum Mechanistic investigation revealed that the specific interaction of HMGB1 and centromeres constructed the precisely organized nuclear architecture, which coordinated with local chromatin structure to control the singular expression of virulence genes. Hence, this protein appears to be a critical architectural regulator for the pathogenesis of malaria infection and may be a new target for the development of an intervention strategy against malaria.},
}
@article {pmid33905883,
year = {2021},
author = {Fallah, MS and Mohebbi, A and Yasaghi, M and Ghaemi, EA},
title = {CRISPR-Cas systems in Proteus mirabilis.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {92},
number = {},
pages = {104881},
doi = {10.1016/j.meegid.2021.104881},
pmid = {33905883},
issn = {1567-7257},
mesh = {Bacteriophages/physiology ; *CRISPR-Cas Systems ; Prophages/physiology ; Proteus mirabilis/*genetics/virology ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a bacterial defense mechanism against bacteriophages composed of two different parts: the CRISPR array and the Cas genes. The spacer acquisition is done by the adaptation module consisting of the hallmark Cas1 Cas2 proteins, which inserts new spacers into the CRISPR array. Here we aimed to describe the CRISPR-Cas system in Proteus mirabilis (P. mirabilis) isolates. CRISPR loci was observed in 30 genomic contents of 109 P. mirabilis isolates that each locus was consisted of two CRISPR arrays and each array had a different preserved leader sequences. Only the type I-E CRISPR-Cas system was common in these isolates. The source of the spacers was identified, including phages and prophages. CRISPR spacer origin analysis also identified a conserved PAM sequence of 5'-AAG-3' nucleotide stretch. Through collecting spacers, CRISPR arrays of P. mirabilis isolates were expanded mostly by integration of bacteriophageal source of spacers. This study shows novel findings in the area of the P-mirabilis CRISPR-Cas system. In this regard, among analyzed genome of P. mirabilis isolates, Class I CRISR-Cas systems were dominant, and all belonged to type I-E. In the flanks of the CRISPR, some other elements with regulatory role were also found. A motif of 11 nt size was found to be preserved among the analyzed genome. We believe that it might has a CRISPR-Cas system transcription facilitator by targeting the Rho element.},
}
@article {pmid33905612,
year = {2021},
author = {Labun, K and Krause, M and Torres Cleuren, Y and Valen, E},
title = {CRISPR Genome Editing Made Easy Through the CHOPCHOP Website.},
journal = {Current protocols},
volume = {1},
number = {4},
pages = {e46},
doi = {10.1002/cpz1.46},
pmid = {33905612},
issn = {2691-1299},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome ; RNA, Guide/genetics ; },
abstract = {The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock-out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock-in Alternate Protocol 2: Design of gRNAs for knock-in using non-homologous end joining Basic Protocol 4: Design of gRNAs for knock-in using Cas9 nickases.},
}
@article {pmid33904395,
year = {2021},
author = {Jost, M and Jacobson, AN and Hussmann, JA and Cirolia, G and Fischbach, MA and Weissman, JS},
title = {CRISPR-based functional genomics in human dendritic cells.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33904395},
issn = {2050-084X},
support = {U01 CA217882/CA/NCI NIH HHS/United States ; U54 CA224081/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; K99 GM130964/GM/NIGMS NIH HHS/United States ; R01 DK110174/DK/NIDDK NIH HHS/United States ; },
mesh = {Bacteroides thetaiotaomicron/immunology ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytokines/genetics/metabolism ; Dendritic Cells/drug effects/*immunology/metabolism ; *Gene Editing ; Gene Expression Regulation ; *Genomics ; Humans ; Immunity, Innate/drug effects/*genetics ; Lipopolysaccharides/pharmacology ; Phenotype ; Signal Transduction ; Toll-Like Receptor 4/agonists/genetics/metabolism ; },
abstract = {Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.},
}
@article {pmid33903218,
year = {2021},
author = {Ramachandran, H and Martins, S and Kontarakis, Z and Krutmann, J and Rossi, A},
title = {Fast but not furious: a streamlined selection method for genome-edited cells.},
journal = {Life science alliance},
volume = {4},
number = {6},
pages = {},
pmid = {33903218},
issn = {2575-1077},
mesh = {Animals ; Biomarkers/analysis ; CRISPR-Cas Systems ; Cell Line ; Cell Separation/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome/genetics ; HEK293 Cells ; Humans ; Magnetics ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods.},
}
@article {pmid33902573,
year = {2021},
author = {Schuijff, M and De Jong, MDT and Dijkstra, AM},
title = {A Q methodology study on divergent perspectives on CRISPR-Cas9 in the Netherlands.},
journal = {BMC medical ethics},
volume = {22},
number = {1},
pages = {48},
pmid = {33902573},
issn = {1472-6939},
mesh = {Biomedical Research/*ethics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*ethics ; Genetic Enhancement/*ethics ; Genome, Human ; Humans ; Netherlands ; *Public Opinion ; },
abstract = {BACKGROUND: CRISPR-Cas9, a technology enabling modification of the human genome, is developing rapidly. There have been calls for public debate to discuss its ethics, societal implications, and governance. So far, however, little is known about public attitudes on CRISPR-Cas9. This study contributes to a better understanding of public perspectives by exploring the various holistic perspectives Dutch citizens have on CRISPR-Cas9.<br><br>METHODS: This study used Q methodology to identify different perspectives of Dutch citizens (N&#x2009;=&#x2009;30) on the use of CRISPR-Cas9. The Q-sort method aims at segmenting audiences based on the structural characteristics of their perspectives. Participants individually ranked 32 statements about CRISPR-Cas9 and discussed their rankings in small groups. By-person factor analysis was performed using PQMethod. Participants' contributions to the discussions were used to further make sense of the audience segments identified.<br><br>RESULTS: Five perspectives on CRISPR-Cas9 were identified: (1) pragmatic optimism (2) concerned scepticism; (3) normative optimism; (4) enthusiastic support; and (5) benevolent generalism. Each perspective represents a unique position motivated by different ranking rationales. Sorting rationales included improving health, preventing negative impacts on society, and fear of a slippery slope. Overall, there is broad, but not universal support for medical uses of CRISPR-Cas9.<br><br>CONCLUSIONS: Research on CRISPR-Cas9 should prioritise the broadly supported applications of the technology. Research and public debates on CRISPR-Cas9, its uses, its broader implications, and the governance of CRISPR-Cas9 are recommended. A discourse that includes all perspectives can contribute to the embedding of future uses of CRISPR-Cas9 in society. This study shows that Q methodology followed by group discussions enables citizens to contribute meaningfully to discourses about research.},
}
@article {pmid33900846,
year = {2021},
author = {Randazzo, P and Bennis, NX and Daran, JM and Daran-Lapujade, P},
title = {gEL DNA: A Cloning- and Polymerase Chain Reaction-Free Method for CRISPR-Based Multiplexed Genome Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {896-913},
pmid = {33900846},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Cloning, Molecular ; DNA/metabolism ; *Gene Editing/methods ; Saccharomyces cerevisiae/genetics ; },
abstract = {Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the literature by means of SpCas9 or FnCas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III RNA polymerases. These gRNA expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in Saccharomyces cerevisiae. Using FnCas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex DNA editing. These DNA fragments can be custom ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other less genetically accessible yeast species.},
}
@article {pmid33900064,
year = {2021},
author = {Montagud-Martínez, R and Heras-Hernández, M and Goiriz, L and Daròs, JA and Rodrigo, G},
title = {CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA.},
journal = {ACS synthetic biology},
volume = {10},
number = {5},
pages = {950-956},
pmid = {33900064},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Computers, Molecular ; DNA, Single-Stranded/*genetics ; Endopeptidase K/genetics ; *Gene Regulatory Networks ; Genetic Engineering/*methods ; Nanotechnology/methods ; RNA, Guide/*genetics ; Ribonuclease H/genetics ; Ribonuclease, Pancreatic/genetics ; Streptococcus pyogenes/genetics ; Transcription, Genetic/genetics ; },
abstract = {DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of substrates and reactions (hardware) for DNA computing.},
}
@article {pmid33900014,
year = {2021},
author = {Lalanne, JB and Parker, DJ and Li, GW},
title = {Spurious regulatory connections dictate the expression-fitness landscape of translation factors.},
journal = {Molecular systems biology},
volume = {17},
number = {4},
pages = {e10302},
pmid = {33900014},
issn = {1744-4292},
support = {R35 GM124732/GM/NIGMS NIH HHS/United States ; T32 GM007287/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacillus subtilis/*genetics ; Bacterial Proteins/metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Peptide Termination Factors/*metabolism ; *Protein Biosynthesis ; Proteome/metabolism ; Stress, Physiological/genetics ; Transcription, Genetic ; },
abstract = {During steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here, we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in the bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization of parts.},
}
@article {pmid33899643,
year = {2021},
author = {Li, P and Wang, L and Yang, J and Di, LJ and Li, J},
title = {Applications of the CRISPR-Cas system for infectious disease diagnostics.},
journal = {Expert review of molecular diagnostics},
volume = {21},
number = {7},
pages = {723-732},
doi = {10.1080/14737159.2021.1922080},
pmid = {33899643},
issn = {1744-8352},
mesh = {*COVID-19/diagnosis/genetics ; *COVID-19 Nucleic Acid Testing ; *CRISPR-Cas Systems ; Humans ; SARS-CoV-2/*genetics ; },
abstract = {INTRODUCTION: Rapid and accurate diagnostic approaches are essential for impeding the spread of infectious diseases. This review aims to summarize current progress of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems in the applications for diagnostics of infectious diseases including the ongoing COVID-19 epidemic.<br><br>AREAS COVERED: In this review, we discuss class 2 CRISPR-Cas biosensing systems-based diagnostics in various emerging and reemerging infectious diseases, CRISPR-Cas systems have created a new era for early diagnostics of infectious diseases, especially with the discovery of the collateral cleavage activity of Cas12 and Cas13. We mainly focus on different CRISPR-Cas effectors for the detection of pathogenic microorganisms as well as provide a detailed explanation of the pros and cons of CRISPR-Cas biosensing systems. In addition, we also introduce future research perspectives.<br><br>EXPERT COMMENTARY: However, further improvement of newly discovered systems and engineering existing ones should be developed to increase the specificity, sensitivity or stability of the diagnostic tools. It may be a long journey to finish the clinical transition from research use. CRISPR-Cas approaches will emerge as more promising and robust tools for infectious disease diagnosis in the future.},
}
@article {pmid33899542,
year = {2021},
author = {Banerjee, A and Mukherjee, S and Maji, BK},
title = {Manipulation of genes could inhibit SARS-CoV-2 infection that causes COVID-19 pandemics.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {246},
number = {14},
pages = {1643-1649},
pmid = {33899542},
issn = {1535-3699},
mesh = {Antiviral Agents/administration &amp; dosage/*pharmacology ; COVID-19/epidemiology/genetics/*virology ; CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Transfer Techniques ; Host-Pathogen Interactions/genetics/*physiology ; Humans ; RNA, Guide/administration &amp; dosage/pharmacology ; SARS-CoV-2/*genetics/pathogenicity ; },
abstract = {The year 2020 witnessed an unpredictable pandemic situation due to novel coronavirus (COVID-19) outbreaks. This condition can be more severe if the patient has comorbidities. Failure of viable treatment for such viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to lack of identification. Thus, modern and productive biotechnology-based tools are being used to manipulate target genes by introducing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system. Moreover, it has now been used as a tool to inhibit viral replication. Hence, it can be hypothesized that the CRISPR/Cas system can be a viable tool to target both the SARS-CoV-2 genome with specific target RNA sequence and host factors to destroy the SARS-CoV-2 community via inhibition of viral replication and infection. Moreover, comorbidities and COVID-19 escalate the rate of mortality globally, and as a result, we have faced this pandemic. CRISPR/Cas-mediated genetic manipulation to knockdown viral sequences may be a preventive strategy against such pandemic caused by SARS-CoV-2. Furthermore, prophylactic antiviral CRISPR in human cells (PAC-MAN) along with CRISPR/Cas13d efficiently degrades the specific RNA sequence to inhibit viral replication. Therefore, we suggest that CRISPR/Cas system with PAC-MAN could be a useful tool to fight against such a global pandemic caused by SARS-CoV-2. This is an alternative preventive approach of management against the pandemic to destroy the target sequence of RNA in SARS-CoV-2 by viral inhibition.},
}
@article {pmid33899435,
year = {2021},
author = {Gao, M and Zhu, X and Wang, S and Zhang, B and Zhang, Y and He, Y and Zhou, Y and Li, S and Yang, G and Liao, G and Bao, J and Bu, H},
title = {[Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal].},
journal = {Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi},
volume = {38},
number = {1},
pages = {111-121},
doi = {10.7507/1001-5515.202006032},
pmid = {33899435},
issn = {1001-5515},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *RNA, Guide/genetics ; Swine ; },
abstract = {The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.},
}
@article {pmid33899015,
year = {2021},
author = {Li, HH and Li, JC and Su, MP and Liu, KL and Chen, CH},
title = {Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100432},
pmid = {33899015},
issn = {2666-1667},
mesh = {Aedes/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*methods ; Larva/genetics ; Male ; Polymerase Chain Reaction ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; },
abstract = {Implementation of CRISPR/Cas9 methodologies for mosquito gene editing has not yet become widespread. This protocol details the procedure for Aedes aegypti mosquito gene editing using homology-directed repair and fluorescent marker insertion, which facilitates the generation and screening of mutant mosquito lines for gene function testing. We describe optimized methods for single guide RNA plasmid preparation, homologous recombination donor plasmid construction, embryo microinjection, and precise gene knock-in confirmation. We also provide general guidance for establishing mutant mosquito lines. For details on the practical use and execution of this protocol, please refer to Li et al. (2020).},
}
@article {pmid33899013,
year = {2021},
author = {Alda-Catalinas, C and Eckersley-Maslin, MA and Reik, W},
title = {Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100426},
pmid = {33899013},
issn = {2666-1667},
support = {MR/M008975/1/MRC_/Medical Research Council/United Kingdom ; BB/T009713/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/B/000C0422/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 105031/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; 210754/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Genomics ; Mice ; Mouse Embryonic Stem Cells/*cytology/metabolism ; RNA-Seq/*methods ; Single-Cell Analysis/*methods ; Transcriptome/*genetics ; },
abstract = {CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with single-cell RNA-sequencing readout, individual perturbations can be assessed in parallel both comprehensively and at scale. Importantly, this allows gene function and regulation to be interrogated at a cellular level in an unbiased manner. Here, we present a protocol to perform pooled CRISPR-activation screens in mouse embryonic stem cells using 10× Genomics scRNA-seq as a readout. For complete information on the generation and use of this protocol, please refer to Alda-Catalinas et al. (2020).},
}
@article {pmid33898975,
year = {2021},
author = {Grützner, R and Martin, P and Horn, C and Mortensen, S and Cram, EJ and Lee-Parsons, CWT and Stuttmann, J and Marillonnet, S},
title = {High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns.},
journal = {Plant communications},
volume = {2},
number = {2},
pages = {100135},
pmid = {33898975},
issn = {2590-3462},
mesh = {Arabidopsis/*genetics/metabolism ; Arabidopsis Proteins/*analysis ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/instrumentation/*methods ; *Genome, Plant ; *Introns ; },
abstract = {The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.},
}
@article {pmid33898973,
year = {2021},
author = {Sretenovic, S and Yin, D and Levav, A and Selengut, JD and Mount, SM and Qi, Y},
title = {Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences.},
journal = {Plant communications},
volume = {2},
number = {2},
pages = {100101},
pmid = {33898973},
issn = {2590-3462},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Plant ; Oryza/*genetics ; Triticum/*genetics ; Zea mays/*genetics ; },
abstract = {The most popular CRISPR-SpCas9 system recognizes canonical NGG protospacer adjacent motifs (PAMs). Previously engineered SpCas9 variants, such as Cas9-NG, favor G-rich PAMs in genome editing. In this manuscript, we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAMs. This study fills a major technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, which greatly expands the targeting scope of CRISPR-Cas9. Finally, our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems, facilitating easy adoption of the systems by others. We anticipate that more tools, such as prime editing, homology-directed repair, CRISPR interference, and CRISPR activation, will be further developed based on our promising iSpyMacCas9 platform.},
}
@article {pmid33897668,
year = {2021},
author = {Wang, Q and Zhang, J and Al Makishah, NH and Sun, X and Wen, Z and Jiang, Y and Yang, S},
title = {Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {654058},
pmid = {33897668},
issn = {1664-302X},
abstract = {Corynebacterium glutamicum has been considered a promising synthetic biological platform for biomanufacturing and bioremediation. However, there are still some challenges in genetic manipulation of C. glutamicum. Recently, more and more genetic parts or elements (replicons, promoters, reporter genes, and selectable markers) have been mined, characterized, and applied. In addition, continuous improvement of classic molecular genetic manipulation techniques, such as allelic exchange via single/double-crossover, nuclease-mediated site-specific recombination, RecT-mediated single-chain recombination, actinophages integrase-mediated integration, and transposition mutation, has accelerated the molecular study of C. glutamicum. More importantly, emerging gene editing tools based on the CRISPR/Cas system is revolutionarily rewriting the pattern of genetic manipulation technology development for C. glutamicum, which made gene reprogramming, such as insertion, deletion, replacement, and point mutation, much more efficient and simpler. This review summarized the recent progress in molecular genetic manipulation technology development of C. glutamicum and discussed the bottlenecks and perspectives for future research of C. glutamicum as a distinctive microbial chassis.},
}
@article {pmid33896583,
year = {2021},
author = {Xue, C and Greene, EC},
title = {DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {7},
pages = {639-656},
pmid = {33896583},
issn = {0168-9525},
support = {R01 CA221858/CA/NCI NIH HHS/United States ; R01 CA236606/CA/NCI NIH HHS/United States ; R35 GM118026/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA Repair/*genetics ; Gene Editing/*trends ; Genome, Human/*genetics ; Homologous Recombination/genetics ; Humans ; Mutagenesis, Insertional/genetics ; Signal Transduction/genetics ; },
abstract = {Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.},
}
@article {pmid33895911,
year = {2021},
author = {Bordoloi, D and Padmavathi, G and Banik, K and Devi, KA and Harsha, C and Girisa, S and Buhrmann, C and Shakibaei, M and Kunnumakkara, AB},
title = {Human tumor necrosis factor alpha-induced protein eight-like 1 exhibited potent anti-tumor effect through modulation of proliferation, survival, migration and invasion of lung cancer cells.},
journal = {Molecular and cellular biochemistry},
volume = {476},
number = {9},
pages = {3303-3318},
pmid = {33895911},
issn = {1573-4919},
mesh = {Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; *Cell Movement ; *Cell Proliferation ; *Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Lung Neoplasms/genetics/metabolism/*pathology ; Neoplasm Invasiveness ; Prognosis ; Tumor Cells, Cultured ; },
abstract = {Lung cancer represents one of the most prevalent neoplasms across the globe. Tobacco smoking, exposure to different occupational and environmental carcinogens, and various dietary factors are strongly implicated in the development of lung cancer. The 5-year survival rate of lung cancer is extremely poor which can be attributed to its propensity for early spread, lack of appropriate biomarkers and proper therapeutic strategies for this aggressive neoplasm. Emerging evidence suggests tumor necrosis factor-α-induced protein eight like 1 (TIPE1 or TNFAIP8L1), which functions as a cell death regulator, to hold high prospect as an important biomarker. Interestingly, this protein was found to be significantly downregulated in human lung cancer tissues compared to normal lung tissues. In addition, this protein exerted marked downregulation in different stages and grades of lung tumor. Further knockout of TIPE1 led to the enhancement in proliferation, survival, migration and invasion of NCIH460 human lung cancer cells through modulation of Akt/mTOR/STAT-3 signaling cascade. In addition, TIPE1 was found to be involved in nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosonornicotine and benzo[a]pyrene-mediated lung cancer through enhanced proliferation, survival and migration of lung cancer cells. Altogether, this newly identified protein plays a critical role in lung cancer pathogenesis and possess enormous prospect to serve as an important tool in the effective management of this aggressive neoplasm.},
}
@article {pmid33895320,
year = {2021},
author = {Namgung, J and Mizuta, H and Yamaguchi, Y and Nagata, J and Todo, T and Yilmaz, O and Hiramatsu, N},
title = {Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system.},
journal = {Comparative biochemistry and physiology. Part A, Molecular &amp; integrative physiology},
volume = {257},
number = {},
pages = {110967},
doi = {10.1016/j.cbpa.2021.110967},
pmid = {33895320},
issn = {1531-4332},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromatography, Liquid ; Egg Proteins/*metabolism ; Female ; Ligands ; Male ; Oocytes/*metabolism ; Oryzias/*genetics/*physiology ; Phylogeny ; RNA, Guide/metabolism ; Receptors, Cell Surface/*metabolism ; Receptors, LDL/metabolism ; Tandem Mass Spectrometry ; Treatment Outcome ; Vitellogenins/metabolism ; },
abstract = {Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8-KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8-KO group. Offspring of lr8-KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8-KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8-KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8-KO is possibly one cause of the low offspring survival.},
}
@article {pmid33894328,
year = {2021},
author = {de Voogt, WS and Tanenbaum, ME and Vader, P},
title = {Illuminating RNA trafficking and functional delivery by extracellular vesicles.},
journal = {Advanced drug delivery reviews},
volume = {174},
number = {},
pages = {250-264},
doi = {10.1016/j.addr.2021.04.017},
pmid = {33894328},
issn = {1872-8294},
mesh = {Animals ; Biological Transport ; Extracellular Vesicles/*metabolism ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Humans ; Nanoparticles ; RNA/*administration &amp; dosage/metabolism ; },
abstract = {RNA-based therapeutics are highly promising for the treatment of numerous diseases, by their ability to tackle the genetic origin in multiple possible ways. RNA molecules are, however, incapable of crossing cell membranes, hence a safe and efficient delivery vehicle is pivotal. Extracellular vesicles (EVs) are endogenously derived nano-sized particles and possess several characteristics which make them excellent candidates as therapeutic RNA delivery agent. This includes the inherent capability to functionally transfer RNAs in a selective manner and an enhanced safety profile compared to synthetic particles. Nonetheless, the fundamental mechanisms underlying this selective inter- and intracellular trafficking and functional transfer of RNAs by EVs are poorly understood. Improving our understanding of these systems is a key element of working towards an EV-based or EV-mimicking system for the functional delivery of therapeutic RNA. In this review, state-of-the-art approaches to detect and visualize RNA in situ and in live cells are discussed, as well as strategies to assess functional RNA transfer, highlighting their potential in studying EV-RNA trafficking mechanisms.},
}
@article {pmid33894004,
year = {2021},
author = {Shumate, KM and Tas, ST and Kavalali, ET and Emeson, RB},
title = {RNA editing-mediated regulation of calcium-dependent activator protein for secretion (CAPS1) localization and its impact on synaptic transmission.},
journal = {Journal of neurochemistry},
volume = {158},
number = {2},
pages = {182-196},
pmid = {33894004},
issn = {1471-4159},
support = {P50 HD103537/HD/NICHD NIH HHS/United States ; R01 DK119508/DK/NIDDK NIH HHS/United States ; R01 MH066198/MH/NIMH NIH HHS/United States ; },
mesh = {Adenosine Deaminase/metabolism ; Animals ; CRISPR-Cas Systems ; Calcium-Binding Proteins/*genetics/*physiology ; Electrophysiological Phenomena ; Glutamic Acid/physiology ; High-Throughput Nucleotide Sequencing ; Hippocampus/cytology/metabolism ; Mice ; Mutation ; Nerve Tissue Proteins/*genetics/*physiology ; Neuronal Plasticity/genetics/physiology ; Primary Cell Culture ; RNA Editing/*genetics/physiology ; Synaptic Transmission/*genetics/*physiology ; gamma-Aminobutyric Acid/physiology ; },
abstract = {Calcium-dependent activator protein for secretion 1 (CAPS1) is a SNARE accessory protein that facilitates formation of the SNARE complex to enable neurotransmitter release. Messenger RNAs encoding CAPS1 are subject to a site-specific adenosine-to-inosine (A-to-I) editing event resulting in a glutamate-to-glycine (E-to-G) substitution in the C-terminal domain of the encoded protein product. The C-terminal domain of CAPS1 is necessary for its synaptic enrichment and Cadps RNA editing has been shown previously to enhance the release of neuromodulatory transmitters. Using mutant mouse lines engineered to solely express CAPS1 protein isoforms encoded by either the non-edited or edited Cadps transcript, primary neuronal cultures from mouse hippocampus were used to explore the effect of Cadps editing on neurotransmission and CAPS1 synaptic localization at both glutamatergic and GABAergic synapses. While the editing of Cadps does not alter baseline evoked neurotransmission, it enhances short-term synaptic plasticity, specifically short-term depression, at inhibitory synapses. Cadps editing also alters spontaneous inhibitory neurotransmission. Neurons that solely express edited Cadps have a greater proportion of synapses that contain CAPS1 than neurons that solely express non-edited Cadps for both glutamatergic and GABAergic synapses. Editing of Cadps transcripts is regulated by neuronal activity, as global network stimulation increases the extent of transcripts edited in wild-type hippocampal neurons, whereas chronic network silencing decreases the level of Cadps editing. Taken together, these results provide key insights into the importance of Cadps editing in modulating its own synaptic localization, as well as the modulation of neurotransmission at inhibitory synapses in hippocampal neurons.},
}
@article {pmid33893996,
year = {2021},
author = {Iqbal, A and Khan, RS and Khan, MA and Gul, K and Jalil, F and Shah, DA and Rahman, H and Ahmed, T},
title = {Genetic Engineering Approaches for Enhanced Insect Pest Resistance in Sugarcane.},
journal = {Molecular biotechnology},
volume = {63},
number = {7},
pages = {557-568},
pmid = {33893996},
issn = {1559-0305},
mesh = {CRISPR-Cas Systems ; *Disease Resistance ; Genetic Engineering/*methods ; Insecticides/*metabolism ; Lectins/genetics/metabolism ; Plant Breeding ; Plants, Genetically Modified/growth &amp; development/parasitology ; Saccharum/genetics/*growth &amp; development/parasitology ; },
abstract = {Sugarcane (Saccharum officinarum), a sugar crop commonly grown for sugar production all over the world, is susceptible to several insect pests attack in addition to bacterial, fungal and viral infections leading to substantial reductions in its yield. The complex genetic makeup and lack of resistant genes in genome of sugarcane have made the conventional breeding a difficult and challenging task for breeders. Using pesticides for control of the attacking insects can harm beneficial insects, human and other animals and the environment as well. As alternative and effective strategy for control of insect pests, genetic engineering has been applied for overexpression of cry proteins, vegetative insecticidal proteins (vip), lectins and proteinase inhibitors (PI). In addition, the latest biotechnological tools such as host-induced gene silencing (HIGS) and CRISPR/Cas9 can be employed for sustainable control of insect pests in sugarcane. In this review overexpression of the cry, vip, lectins and PI genes in transgenic sugarcane and their disease resistance potential is described.},
}
@article {pmid33893795,
year = {2021},
author = {Babaei, M and Sartori, L and Karpukhin, A and Abashkin, D and Matrosova, E and Borodina, I},
title = {Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites.},
journal = {FEMS yeast research},
volume = {21},
number = {4},
pages = {},
pmid = {33893795},
issn = {1567-1364},
mesh = {CRISPR-Cas Systems ; Chromosomes, Fungal/genetics ; Industrial Microbiology ; *Metabolic Engineering ; Plasmids ; RNA, Guide ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.},
}
@article {pmid33893286,
year = {2021},
author = {Kluesner, MG and Lahr, WS and Lonetree, CL and Smeester, BA and Qiu, X and Slipek, NJ and Claudio Vázquez, PN and Pitzen, SP and Pomeroy, EJ and Vignes, MJ and Lee, SC and Bingea, SP and Andrew, AA and Webber, BR and Moriarity, BS},
title = {CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2437},
pmid = {33893286},
issn = {2041-1723},
support = {R03 AI144840/AI/NIAID NIH HHS/United States ; },
mesh = {Adenosine/chemistry/*metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Cells, Cultured ; Computational Biology/*methods ; Cytidine/chemistry/*metabolism ; Gene Editing/*methods ; Humans ; Internet ; K562 Cells ; Reproducibility of Results ; T-Lymphocytes/cytology/metabolism ; },
abstract = {CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR (http://z.umn.edu/spliceR) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.},
}
@article {pmid33893274,
year = {2021},
author = {Bevacqua, RJ and Dai, X and Lam, JY and Gu, X and Friedlander, MSH and Tellez, K and Miguel-Escalada, I and Bonàs-Guarch, S and Atla, G and Zhao, W and Kim, SH and Dominguez, AA and Qi, LS and Ferrer, J and MacDonald, PE and Kim, SK},
title = {CRISPR-based genome editing in primary human pancreatic islet cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2397},
pmid = {33893274},
issn = {2041-1723},
support = {R01 DK107507/DK/NIDDK NIH HHS/United States ; R01 DK108817/DK/NIDDK NIH HHS/United States ; UC4 DK098085/DK/NIDDK NIH HHS/United States ; U01 DK123743/DK/NIDDK NIH HHS/United States ; MR/L02036X/1/MRC_/Medical Research Council/United Kingdom ; P30 DK116074/DK/NIDDK NIH HHS/United States ; 148451//CIHR/Canada ; R01 DK126482/DK/NIDDK NIH HHS/United States ; R01 DK128932/DK/NIDDK NIH HHS/United States ; WT101033/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Diabetes Mellitus, Type 2/genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Homeodomain Proteins/genetics ; Humans ; Insulin-Secreting Cells/cytology/*metabolism ; Islets of Langerhans/cytology/*metabolism ; *Models, Genetic ; Potassium Channels, Inwardly Rectifying/genetics ; Trans-Activators/genetics ; },
abstract = {Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.},
}
@article {pmid33892188,
year = {2021},
author = {Hu, J and Bourne, RA and McGrath, BC and Lin, A and Pei, Z and Cavener, DR},
title = {Co-opting regulation bypass repair as a gene-correction strategy for monogenic diseases.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3274-3292},
pmid = {33892188},
issn = {1525-0024},
support = {R01 DK088140/DK/NIDDK NIH HHS/United States ; UC4 DK098085/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Female ; Gene Editing/*methods ; Gene Expression ; Gene Knockdown Techniques ; Gene Order ; Gene Targeting ; Genes, Reporter ; Genetic Diseases, Inborn/*genetics/*therapy ; Genetic Markers ; *Genetic Therapy/methods ; Genetic Vectors/genetics ; Humans ; Male ; Mice ; Mutation ; RNA, Guide ; eIF-2 Kinase/genetics ; },
abstract = {With the development of CRISPR-Cas9-mediated gene-editing technologies, correction of disease-causing mutations has become possible. However, current gene-correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene-correction strategy, co-opting regulation bypass repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans, which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair.},
}
@article {pmid33891288,
year = {2021},
author = {Selma, S and Orzáez, D},
title = {Perspectives for epigenetic editing in crops.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {381-400},
pmid = {33891288},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Epigenomics ; *Gene Editing ; *Genome, Plant ; Plant Breeding/*methods ; Plants, Genetically Modified/*genetics ; },
abstract = {Site-specific nucleases (SSNs) have drawn much attention in plant biotechnology due to their ability to drive precision mutagenesis, gene targeting or allele replacement. However, when devoid of its nuclease activity, the underlying DNA-binding activity of SSNs can be used to bring other protein functional domains close to specific genomic sites, thus expanding further the range of applications of the technology. In particular, the addition of functional domains encoding epigenetic effectors and chromatin modifiers to the CRISPR/Cas ribonucleoprotein complex opens the possibility to introduce targeted epigenomic modifications in plants in an easily programmable manner. Here we examine some of the most important agronomic traits known to be controlled epigenetically and review the best studied epigenetic catalytic effectors in plants, such as DNA methylases/demethylases or histone acetylases/deacetylases and their associated marks. We also review the most efficient strategies developed to date to functionalize Cas proteins with both catalytic and non-catalytic epigenetic effectors, and the ability of these domains to influence the expression of endogenous genes in a regulatable manner. Based on these new technical developments, we discuss the possibilities offered by epigenetic editing tools in plant biotechnology and their implications in crop breeding.},
}
@article {pmid33885789,
year = {2021},
author = {Cheng, R and Huang, F and Wu, H and Lu, X and Yan, Y and Yu, B and Wang, X and Zhu, B},
title = {A nucleotide-sensing endonuclease from the Gabija bacterial defense system.},
journal = {Nucleic acids research},
volume = {49},
number = {9},
pages = {5216-5229},
pmid = {33885789},
issn = {1362-4962},
mesh = {Adenosine Triphosphate/metabolism ; Adenylyl Imidodiphosphate/metabolism ; Bacillus cereus/enzymology ; Bacterial Proteins/chemistry/*metabolism ; Bacteriophages/genetics ; DNA/metabolism ; DNA Cleavage ; Endodeoxyribonucleases/chemistry/*metabolism ; Nucleotides/metabolism ; Protein Domains ; },
abstract = {The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.},
}
@article {pmid33885199,
year = {2021},
author = {Ni, XY and Lu, WJ and Qiao, X and Huang, J},
title = {Genome editing efficiency of four Drosophila suzukii endogenous U6 promoters.},
journal = {Insect molecular biology},
volume = {30},
number = {4},
pages = {420-426},
doi = {10.1111/imb.12707},
pmid = {33885199},
issn = {1365-2583},
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila/*genetics ; Drosophila Proteins/genetics ; Gene Editing/*methods ; Genetic Engineering ; Insect Control ; Mutagenesis ; Promoter Regions, Genetic/*genetics ; },
abstract = {The invasive spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) has caused serious economic losses to the fruit industry. The conventional control methods have many limitations and genetic engineering technologies such as CRISPR/Cas9-mediated gene drive are promising approaches. In the CRISPR/Cas9 system, the transcriptional regulatory elements play an important role in the activities of gRNA. Thus, in order to improve the genome editing efficiency of the CRISPR/Cas9 system in D. suzukii, we cloned and tested four endogenous U6 promoters to drive mutagenesis of the white gene. Our results showed that all the four promoters could be used with variable efficiency. The promoter DsU6-3 had the highest genome editing efficiency among the four DsU6 promoters. Compared with the DsU6-3 promoter, the DmU6:3 promoter showed lower efficiency to drive mutagenesis in D. suzukii. These findings expand the range of promoters available to express gRNAs in D. suzukii, facilitating the basic and applied research on this important pest.},
}
@article {pmid33883226,
year = {2021},
author = {Krump, NA and Wang, R and Liu, W and Yang, JF and Ma, T and You, J},
title = {Merkel Cell Polyomavirus Infection Induces an Antiviral Innate Immune Response in Human Dermal Fibroblasts.},
journal = {Journal of virology},
volume = {95},
number = {13},
pages = {e0221120},
pmid = {33883226},
issn = {1098-5514},
support = {R21 AR074073/AR/NIAMS NIH HHS/United States ; R01 CA187718/CA/NCI NIH HHS/United States ; P30 AI045008/AI/NIAID NIH HHS/United States ; P30 CA016520/CA/NCI NIH HHS/United States ; R21 AI149761/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Carcinoma, Merkel Cell/pathology ; Cells, Cultured ; Cytokines/biosynthesis/*immunology ; Fibroblasts/*immunology/virology ; HEK293 Cells ; Humans ; Immunity, Innate/genetics/*immunology ; Interferons/biosynthesis/immunology ; Membrane Proteins/genetics ; Merkel cell polyomavirus/growth &amp; development/*immunology ; NF-kappa B/genetics ; Nuclear Proteins/genetics/metabolism ; Nucleotidyltransferases/genetics ; Phosphoproteins/genetics/metabolism ; Polyomavirus Infections/immunology ; Skin/cytology/*immunology ; Tumor Virus Infections/immunology ; },
abstract = {Merkel cell polyomavirus (MCPyV) infects most of the human population asymptomatically, but in rare cases it leads to a highly aggressive skin cancer called Merkel cell carcinoma (MCC). MCC incidence is much higher in aging and immunocompromised populations. The epidemiology of MCC suggests that dysbiosis between the host immune response and the MCPyV infectious cycle could contribute to the development of MCPyV-associated MCC. Insufficient restriction of MCPyV by normal cellular processes, for example, could promote the incidental oncogenic MCPyV integration events and/or entry into the original cell of MCC. Progress toward understanding MCPyV biology has been hindered by its narrow cellular tropism. Our discovery that primary human dermal fibroblasts (HDFs) support MCPyV infection has made it possible to closely model cellular responses to different stages of the infectious cycle. The present study reveals that the onset of MCPyV replication and early gene expression induces an inflammatory cytokine and interferon-stimulated gene (ISG) response. The cGAS-STING pathway, in coordination with NF-κB, mediates induction of this innate immune gene expression program. Further, silencing of cGAS or NF-κB pathway factors led to elevated MCPyV replication. We also discovered that the PYHIN protein IFI16 localizes to MCPyV replication centers but does not contribute to the induction of ISGs. Instead, IFI16 upregulates inflammatory cytokines in response to MCPyV infection by an alternative mechanism. The work described herein establishes a foundation for exploring how changes to the skin microenvironment induced by aging or immunodeficiency might alter the fate of MCPyV and its host cell to encourage carcinogenesis. IMPORTANCE MCC has a high rate of mortality and an increasing incidence. Immune-checkpoint therapies have improved the prognosis of patients with metastatic MCC. Still, a significant proportion of the patients fail to respond to immune-checkpoint therapies or have a medical need for iatrogenic immune-suppression. A greater understanding of MCPyV biology could inform targeted therapies for MCPyV-associated MCC. Moreover, cellular events preceding MCC oncogenesis remain largely unknown. The present study aims to explore how MCPyV interfaces with innate immunity during its infectious cycle. We describe how MCPyV replication and/or transcription elicit an innate immune response via cGAS-STING, NF-κB, and IFI16. We also explore the effects of this response on MCPyV replication. Our findings illustrate how healthy cellular conditions may allow low-level infection that evades immune destruction until highly active replication is restricted by host responses. Conversely, pathological conditions could result in unbridled MCPyV replication that licenses MCC tumorigenesis.},
}
@article {pmid33882988,
year = {2021},
author = {Novikova, G and Andrews, SJ and Renton, AE and Marcora, E},
title = {Beyond association: successes and challenges in linking non-coding genetic variation to functional consequences that modulate Alzheimer's disease risk.},
journal = {Molecular neurodegeneration},
volume = {16},
number = {1},
pages = {27},
pmid = {33882988},
issn = {1750-1326},
mesh = {Age of Onset ; Alzheimer Disease/epidemiology/*genetics ; Bayes Theorem ; CRISPR-Cas Systems ; Causality ; Chromatin/genetics ; Epigenomics ; Gene Editing ; Gene Expression Regulation ; *Genome-Wide Association Study ; Humans ; Models, Genetic ; Molecular Sequence Annotation ; Polymorphism, Single Nucleotide ; Risk ; Transcriptome ; },
abstract = {Alzheimer's disease (AD) is the most common type of dementia, affecting millions of people worldwide; however, no disease-modifying treatments are currently available. Genome-wide association studies (GWASs) have identified more than 40 loci associated with AD risk. However, most of the disease-associated variants reside in non-coding regions of the genome, making it difficult to elucidate how they affect disease susceptibility. Nonetheless, identification of the regulatory elements, genes, pathways and cell type/tissue(s) impacted by these variants to modulate AD risk is critical to our understanding of disease pathogenesis and ability to develop effective therapeutics. In this review, we provide an overview of the methods and approaches used in the field to identify the functional effects of AD risk variants in the causal path to disease risk modification as well as describe the most recent findings. We first discuss efforts in cell type/tissue prioritization followed by recent progress in candidate causal variant and gene nomination. We discuss statistical methods for fine-mapping as well as approaches that integrate multiple levels of evidence, such as epigenomic and transcriptomic data, to identify causal variants and risk mechanisms of AD-associated loci. Additionally, we discuss experimental approaches and data resources that will be needed to validate and further elucidate the effects of these variants and genes on biological pathways, cellular phenotypes and disease risk. Finally, we discuss future steps that need to be taken to ensure that AD GWAS functional mapping efforts lead to novel findings and bring us closer to finding effective treatments for this devastating disease.},
}
@article {pmid33882309,
year = {2021},
author = {D'Angelo, L and Astro, E and De Luise, M and Kurelac, I and Umesh-Ganesh, N and Ding, S and Fearnley, IM and Gasparre, G and Zeviani, M and Porcelli, AM and Fernandez-Vizarra, E and Iommarini, L},
title = {NDUFS3 depletion permits complex I maturation and reveals TMEM126A/OPA7 as an assembly factor binding the ND4-module intermediate.},
journal = {Cell reports},
volume = {35},
number = {3},
pages = {109002},
pmid = {33882309},
issn = {2211-1247},
support = {MC_UP_1002/1/MRC_/Medical Research Council/United Kingdom ; MRF_MRF-155-0002-RG-ZEVIA/MRF/MRF/United Kingdom ; },
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Cell Line, Tumor ; Electron Transport Complex I/deficiency/*genetics ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; Melanocytes/metabolism/pathology ; Membrane Proteins/*genetics/metabolism ; Mice ; Mitochondria/*genetics/metabolism/pathology ; Mitochondrial Membranes/chemistry/metabolism ; Models, Molecular ; NADH Dehydrogenase/deficiency/*genetics ; Optic Atrophy/*genetics/metabolism/pathology ; Osteoblasts/metabolism/pathology ; Protein Binding ; Protein Conformation ; Proteomics ; },
abstract = {Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain, and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergetic machine, we analyze the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We show that, in diverse mammalian cell types, a small amount of functional CI can still be detected in the complete absence of NDUFS3. In addition, we determine the dynamics of CI disassembly when the amount of NDUFS3 is gradually decreased. The process of degradation of the complex occurs in a hierarchical and modular fashion in which the ND4 module remains stable and bound to TMEM126A. We, thus, uncover the function of TMEM126A, the product of a disease gene causing recessive optic atrophy as a factor necessary for the correct assembly and function of CI.},
}
@article {pmid33882189,
year = {2021},
author = {Wang, Y and Zhao, Y and Su, W and Guo, X and Li, S},
title = {Development of a CRISPR-Cas9 Based Luciferase Turn-On System as Nonhomologous End Joining Pathway Reporter.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {22},
number = {12},
pages = {2177-2181},
doi = {10.1002/cbic.202100128},
pmid = {33882189},
issn = {1439-7633},
mesh = {CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair/*genetics ; HEK293 Cells ; High-Throughput Screening Assays ; Humans ; Luciferases/*genetics/metabolism ; },
abstract = {There is a need of a non-homologous end joining (NHEJ) pathway reporter system that facilitates screening and discovery of NHEJ chemical inhibitors. In this study, we developed a CRISPR-Cas9 based luciferase turn-on system as a NHEJ pathway reporter. By substituting nucleotide 205C with ATC, we introduced a reading-frame shift and a pre-stop codon into the luciferase coding region and thereby generated a bioluminescent signal mute HEK293T reporter cell line. Then, a CRISPR-Cas9 plasmid expressing a guide RNA targeting luciferase coding region was introduced into the reporter cell line to generate NHEJ-associated indel to restore the reading frame and subsequently turn on the bioluminescent signal. We observed over three-thousand fold increase in signal after CRISPR-Cas9 vector transfection. Different known chemical inhibitors of the NHEJ pathway, such as NU7441, KU0060648, and KU55933, could significantly inhibit the bioluminescent signal generated by CRISPR-Cas9 targeting. In addition, we validated our system by high throughput sequencing.},
}
@article {pmid33880702,
year = {2021},
author = {Wang, Z and Sun, X and Zhang, X and Dong, B and Yu, H},
title = {Development of a miRNA Sensor by an Inducible CRISPR-Cas9 Construct in Ciona Embryogenesis.},
journal = {Molecular biotechnology},
volume = {63},
number = {7},
pages = {613-620},
pmid = {33880702},
issn = {1559-0305},
mesh = {Animals ; CRISPR-Cas Systems ; Ciona/*embryology/genetics ; Gene Editing/*methods ; Gene Silencing ; MicroRNAs/*genetics ; RNA, Guide/genetics ; },
abstract = {MicroRNAs (miRNAs) regulate multicellular processes and diverse signaling pathways in organisms. The detection of the spatiotemporal expression of miRNA in vivo is crucial for uncovering the function of miRNA. However, most of the current detecting techniques cannot reflect the dynamics of miRNA sensitively in vivo. Here, we constructed a miRNA-induced CRISPR-Cas9 platform (MICR) used in marine chordate Ciona. The key component of MICR is a pre-single guide RNA (sgRNA) flanked by miRNA-binding sites that can be released by RNA-induced silencing complex (RISC) cleavage to form functional sgRNA in the presence of complementary miRNA. By using the miRNA-inducible CRISPR-on system (MICR-ON), we successfully detected the dynamic expression of a miRNA csa-miR-4018a during development of Ciona embryo. The detected patterns were validated to be consistent with the results by in situ hybridization. It is worth noting that the expression of csa-miR-4018a was examined by MICR-ON to be present in additional tissues, where no obvious signaling was detected by in situ hybridization, suggesting that the MICR-ON might be a more sensitive approach to detect miRNA signal in living animal. Thus, MICR-ON was demonstrated to be a sensitive and highly efficient approach for monitoring the dynamics of expression of miRNA in vivo and will facilitate the exploration of miRNA functions in biological systems.},
}
@article {pmid33879980,
year = {2021},
author = {Gadwal, A and Roy, D and Khokhar, M and Modi, A and Sharma, P and Purohit, P},
title = {CRISPR/Cas-New Molecular Scissors in Diagnostics and Therapeutics of COVID-19.},
journal = {Indian journal of clinical biochemistry : IJCB},
volume = {36},
number = {4},
pages = {459-467},
pmid = {33879980},
issn = {0970-1915},
abstract = {The current pandemic of COVID-19, with its climbing number of cases and deaths, has us searching for tools for rapid, reliable, and affordable methods of detection on one hand, and novel, improved therapeutic strategies on the other. The currently employed RT-PCR method, despite its all-encompassing utility, has its shortcomings. Newer diagnostic tools, based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas(CRISPR-Cas) system, with its better diagnostic accuracy measures, have come up to fill that void. These assay platforms are expected to slowly take up the place of COVID-19 diagnostics. Further, the current therapeutic options focus mainly on counteracting the viral proteins and components and their entry into host cells. The CRISPR-based system, especially through the RNA-guided Cas13 approach, can identify the genomic characteristics of SARS-CoV-2 and provide a novel inhibition strategy for coronaviruses. In this mini-review, we have discussed the available and upcoming CRISPR-based diagnostic assays and the potential of the CRISPR/Cas system as a therapeutic or prevention strategy in COVID-19. CRISPR-Cas system shows promise in both diagnostics as well as therapeutics and may as well change the face of molecular diagnosis and precision medicine.},
}
@article {pmid33879884,
year = {2021},
author = {Madhusoodanan, J},
title = {Elusive cancer cells dissected using developmental-biology toolkit.},
journal = {Nature},
volume = {592},
number = {7855},
pages = {647-649},
pmid = {33879884},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Lineage ; Cell Movement ; DNA Barcoding, Taxonomic ; Developmental Biology/*methods ; Heterografts ; Humans ; Mice ; Neoplasms/genetics/immunology/*pathology ; Neoplastic Stem Cells/immunology/*pathology ; Organoids/immunology/metabolism/pathology ; Rats ; },
}
@article {pmid33879582,
year = {2021},
author = {Hu, Z and Wang, Y and Liu, Q and Qiu, Y and Zhong, Z and Li, K and Li, W and Deng, Z and Sun, Y},
title = {Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA.},
journal = {mBio},
volume = {12},
number = {2},
pages = {},
pmid = {33879582},
issn = {2150-7511},
mesh = {Adenine ; CRISPR-Cas Systems ; Cytosine ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mutation ; RNA, Guide/*genetics ; },
abstract = {Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5' end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing.IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.},
}
@article {pmid33879230,
year = {2021},
author = {Yu, F and Zhang, K and Wang, Y and Li, D and Cui, Z and Huang, J and Zhang, S and Li, X and Zhang, L},
title = {CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples.},
journal = {Parasites &amp; vectors},
volume = {14},
number = {1},
pages = {208},
pmid = {33879230},
issn = {1756-3305},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Cattle Diseases/diagnosis/*parasitology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cryptosporidiosis/diagnosis/*parasitology ; Cryptosporidium parvum/classification/genetics/*isolation &amp; purification ; Feces/*parasitology ; Humans ; Polymerase Chain Reaction/*methods ; },
abstract = {BACKGROUND: Cryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity.<br><br>METHODS: By integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor.<br><br>RESULTS: Our ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa.<br><br>CONCLUSIONS: This study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment.},
}
@article {pmid33878708,
year = {2021},
author = {Jung, J and Hwang, S and Seol, H and Kim, AH and Lee, HM and Sung, JJ and Jeong, SM and Choi, YM and Jun, JK and Kim, DW and Jang, J},
title = {Generation of Brachyury-mCherry knock-in reporter human pluripotent stem cell line (SNUe003-A-2) using CRISPR/CAS9 nuclease.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102321},
doi = {10.1016/j.scr.2021.102321},
pmid = {33878708},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Fetal Proteins ; Humans ; *Induced Pluripotent Stem Cells ; *Pluripotent Stem Cells ; T-Box Domain Proteins ; },
abstract = {Brachyury is an embryonic nuclear transcription factor required for mesoderm formation and differentiation. Here, we introduced an mCherry reporter into the C-terminus of Brachyury in the human pluripotent stem cell line SNUhES3 using the CRISPR/Cas9 nuclease approach. Successful gene editing was verified by DNA sequencing. SNUhES3-Brachyury-mCherry cells expressed pluripotent stem cell markers, exhibited a normal karyotype, and could generate all three germ layers. This cell line expressed the red fluorescence protein mCherry upon the induction of mesoderm differentiation. This reporter cell line could be used to monitor mesodermal population enrichment during mesodermal differentiation.},
}
@article {pmid33878707,
year = {2021},
author = {You, J and Cheng, Y and Yang, XJ and Chen, L},
title = {Generation of a homozygous LRP2 knockout human embryonic stem cell line (FDCHDPe010-A-56) by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102342},
pmid = {33878707},
issn = {1876-7753},
support = {P30 EY000331/EY/NEI NIH HHS/United States ; R01 EY026319/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells/metabolism ; *Human Embryonic Stem Cells/metabolism ; Humans ; Low Density Lipoprotein Receptor-Related Protein-2/genetics/metabolism ; },
abstract = {LRP2 is mainly expressed in the cell membrane of epithelia, maintaining normal endocytosis of nutrients from the extracellular microenvironment and mediating growth factor signals. The deficiency of LRP2 can result in abnormal lysosomal and mitochondrial function as well as insufficient resistance to oxidative stress. LRP2-KO animals show enlarged eyes and malfunction of the retinal pigment epithelium (RPE). We were able to generate an LRP2-KO human embryonic stem (ES) cell line using CRISPR/Cas9 gene editing and differentiate the mutant ES cells into RPE cells. Thus, this LRP2-KO human ES line will facilitate studying cellular mechanisms of eye disease due to LRP2 deficiency.},
}
@article {pmid33878591,
year = {2021},
author = {Ding, X and Yin, K and Li, Z and Sfeir, MM and Liu, C},
title = {Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {184},
number = {},
pages = {113218},
pmid = {33878591},
issn = {1873-4235},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R61 AI154642/AI/NIAID NIH HHS/United States ; },
mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral ; SARS-CoV-2 ; },
abstract = {Quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for early diagnosis and timely medical treatment of coronavirus disease 2019. Here, we describe a digital warm-start CRISPR (dWS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The dWS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate and reliable digital quantification of SARS-CoV-2. By targeting SARS-CoV-2's nucleoprotein gene, the dWS-CRISPR assay is able to detect down to 5 copies/μl SARS-CoV-2 RNA in the chip. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples. Moreover, it has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction. Thus, the dWS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.},
}
@article {pmid33878391,
year = {2021},
author = {Park, YR and Yang, JW and Sunwoo, IY and Jang, BK and Kim, SR and Jeong, GT and Kim, SK},
title = {Enhancement of catabolite regulatory genes in Saccharomyces cerevisiae to increase ethanol production using hydrolysate from red seaweed Gloiopeltis furcata.},
journal = {Journal of biotechnology},
volume = {333},
number = {},
pages = {1-9},
doi = {10.1016/j.jbiotec.2021.04.004},
pmid = {33878391},
issn = {1873-4863},
mesh = {Ethanol ; Galactose ; Genes, Regulator ; Repressor Proteins/genetics ; *Rhodophyta ; Saccharomyces cerevisiae/genetics ; *Saccharomyces cerevisiae Proteins/genetics ; *Seaweed ; Transcription Factors/genetics ; },
abstract = {Glucose and galactose are monosaccharides obtained from Gloiopeltis furcata (Red algae). A total monosaccharide yield of 62.3 g/L was obtained from G. furcata using thermal acid hydrolysis and enzymatic saccharification. Activated carbon was used to eliminate hydroxymethylfurfural (HMF) from the hydrolysate. Previously obtained monosaccharides are used for ethanol production by Saccharomyces cerevisiae. S. cerevisiae consumes glucose first, then galactose. The methods for reducing fermentation time and increasing the ethanol yield coefficient using the simultaneous consumption of glucose and galactose have been evaluated. Gal3p and Gal80p of S. cerevisiae act as signal transducers that govern the galactose inducer Gal4p mediated transcriptional activation of the Gal gene family. Gal80p binds to Gal4p for transcription deactivation. Therefore, Gal80p was deleted for Gal4p expression without interruption.},
}
@article {pmid33877851,
year = {2021},
author = {Song, Y and He, S and Abdallah, II and Jopkiewicz, A and Setroikromo, R and van Merkerk, R and Tepper, PG and Quax, WJ},
title = {Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {16},
pages = {4785-4794},
pmid = {33877851},
issn = {1520-5118},
mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Metabolic Engineering ; Polycyclic Sesquiterpenes ; },
abstract = {Engineering strategies to improve terpenoids' production in Bacillus subtilis mainly focus on 2C-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in B. subtilis. Overall, this study provides a universal strategy for further increasing terpenoids' production in B. subtilis by comprehensive and systematic metabolic engineering.},
}
@article {pmid33877687,
year = {2021},
author = {Del Rio, A and Negro, F and Umani Ronchi, F},
title = {Comment on the article by Zaami S, et al. "CRISPR-based techniques: Cas9, Cas13 and their applications in the era of COVID-19". Eur Rev Med Pharmacol Sci 2021; 25 (3): 1752-1761.},
journal = {European review for medical and pharmacological sciences},
volume = {25},
number = {7},
pages = {2830-2833},
doi = {10.26355/eurrev_202104_25535},
pmid = {33877687},
issn = {2284-0729},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; SARS-CoV-2 ; },
}
@article {pmid33876962,
year = {2021},
author = {Fennell, T and Zhang, D and Isik, M and Wang, T and Gotta, G and Wilson, CJ and Marco, E},
title = {CALITAS: A CRISPR-Cas-aware ALigner for In silico off-TArget Search.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {264-274},
doi = {10.1089/crispr.2020.0036},
pmid = {33876962},
issn = {2573-1602},
mesh = {Algorithms ; Bacterial Proteins ; CRISPR-Associated Protein 9 ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Computer Simulation ; Endodeoxyribonucleases ; Endonucleases/*genetics ; Gene Editing ; Genome ; RNA, Guide/genetics ; Software ; },
abstract = {We describe CALITAS, a CRISPR-Cas-aware aligner and integrated off-target search algorithm. CALITAS uses a modified and CRISPR-tuned version of the Needleman-Wunsch algorithm. It supports an unlimited number of mismatches and gaps and allows protospacer adjacent motif (PAM) mismatches or PAMless searches. CALITAS also includes an exhaustive search routine to scan genomes and genome variants provided with a standard Variant Call Format file. By default, CALITAS returns a single best alignment for a given off-target site, which is a significant improvement compared to other off-target algorithms, and it enables off-targets to be referenced directly using alignment coordinates. We validate and compare CALITAS using a selected set of target sites, as well as experimentally derived specificity data sets. In summary, CALITAS is a new tool for precise and relevant alignments and identification of candidate off-target sites across a genome. We believe it is the state of the art for CRISPR-Cas specificity assessments.},
}
@article {pmid33876961,
year = {2021},
author = {Hackley, CR},
title = {A Novel Set of Cas9 Fusion Proteins to Stimulate Homologous Recombination: Cas9-HRs.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {253-263},
doi = {10.1089/crispr.2020.0034},
pmid = {33876961},
issn = {2573-1602},
mesh = {A549 Cells ; Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Repair ; Gene Editing ; *Homologous Recombination ; Humans ; K562 Cells ; Recombinational DNA Repair/*genetics ; },
abstract = {CRISPR- Cas9 has revolutionized genetic engineering. However, the inability to control double-strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice. However, particularly in the case of larger edits, none have been able to bypass the rate-limiting step of homologous recombination (HR): long-range 5' end resection. Here, we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Here, we demonstrate that Cas9-HRs can increase the rate of homology directed repair (HDR) by 2- to 2.5-fold and decrease p53 mediated cellular toxicity by two- to fourfold compared to Cas9 and are functional in multiple mammalian cell lines with minimal apparent editing site bias. These properties should make Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.},
}
@article {pmid33876960,
year = {2021},
author = {Eberherr, AC and Maaske, A and Wolf, C and Giesert, F and Berutti, R and Rusha, E and Pertek, A and Kastlmeier, MT and Voss, C and Plummer, M and Sayed, A and Graf, E and Effner, R and Volz, T and Drukker, M and Strom, TM and Meitinger, T and Stoeger, T and Buyx, AM and Hagl, B and Renner, ED},
title = {Rescue of STAT3 Function in Hyper-IgE Syndrome Using Adenine Base Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {178-190},
doi = {10.1089/crispr.2020.0111},
pmid = {33876960},
issn = {2573-1602},
mesh = {Adenine ; CRISPR-Cas Systems ; Cell Differentiation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblasts ; Gene Editing/*methods ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Immunoglobulin E/genetics ; Induced Pluripotent Stem Cells ; Job Syndrome/*genetics/*therapy ; Mutation ; STAT3 Transcription Factor/*genetics ; Whole Genome Sequencing ; },
abstract = {STAT3-hyper IgE syndrome (STAT3-HIES) is a primary immunodeficiency presenting with destructive lung disease along with other symptoms. CRISPR-Cas9-mediated adenine base editors (ABEs) have the potential to correct one of the most common STAT3-HIES causing heterozygous STAT3 mutations (c.1144C>T/p.R382W). As a proof-of-concept, we successfully applied ABEs to correct STAT3 p.R382W in patient fibroblasts and induced pluripotent stem cells (iPSCs). Treated primary STAT3-HIES patient fibroblasts showed a correction efficiency of 29% ± 7% without detectable off-target effects evaluated through whole-genome and high-throughput sequencing. Compared with untreated patient fibroblasts, corrected single-cell clones showed functional rescue of STAT3 signaling with significantly increased STAT3 DNA-binding activity and target gene expression of CCL2 and SOCS3. Patient-derived iPSCs were corrected with an efficiency of 30% ± 6% and differentiated to alveolar organoids showing preserved plasticity in treated cells. In conclusion, our results are supportive for ABE-based gene correction as a potential causative treatment of STAT3-HIES.},
}
@article {pmid33876959,
year = {2021},
author = {Chu, SH and Packer, M and Rees, H and Lam, D and Yu, Y and Marshall, J and Cheng, LI and Lam, D and Olins, J and Ran, FA and Liquori, A and Gantzer, B and Decker, J and Born, D and Barrera, L and Hartigan, A and Gaudelli, N and Ciaramella, G and Slaymaker, IM},
title = {Rationally Designed Base Editors for Precise Editing of the Sickle Cell Disease Mutation.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {169-177},
doi = {10.1089/crispr.2020.0144},
pmid = {33876959},
issn = {2573-1602},
mesh = {Anemia, Sickle Cell/*genetics/*therapy ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA Breaks, Double-Stranded ; *Gene Editing ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; *Mutation ; RNA ; },
abstract = {Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.},
}
@article {pmid33876957,
year = {2021},
author = {Saifaldeen, M and Al-Ansari, DE and Ramotar, D and Aouida, M},
title = {Dead Cas9-sgRNA Complex Shelters Vulnerable DNA Restriction Enzyme Sites from Cleavage for Cloning Applications.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {275-289},
doi = {10.1089/crispr.2020.0134},
pmid = {33876957},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems ; Chromatin Immunoprecipitation ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; DNA Damage ; DNA Restriction Enzymes/*genetics/*metabolism ; Deoxyribonuclease BamHI ; Epigenomics ; Gene Editing ; Genome ; RNA, Guide/*genetics ; Ribonucleoproteins ; },
abstract = {The creation of the nuclease-dead Cas protein (dCas9) offers a new platform for a plethora of new discoveries. Diverse dCas9 tools have been developed for transcription regulation, epigenetic engineering, base editing, genome imaging, genetic screens, and chromatin immunoprecipitation. Here, we show that dCas9 and single-guide RNA preassembled to form ribonucleoprotein dCas9-sgRNA (referred to as dRNP) is capable of specifically and reversibly blocking the activity of DNA cleavage by restriction enzymes (REs). We show that the inhibition of RE activities occurs when the recognition or the cleavage site of the DNA is overlapped by the sgRNA or the protospacer adjacent motif sequence. Furthermore, we show that multiple dRNPs can be used simultaneously to inhibit more than one RE sites. As such, we exploited this novel finding as a method to demonstrate that inserts can be ligated into vectors, and vice versa, whereby selective RE sites are temporarily sheltered to allow the desired cloning.},
}
@article {pmid33876956,
year = {2021},
author = {Pastuszka, A and Beauruelle, C and Camiade, E and Rousseau, GM and Moineau, S and Mereghetti, L and Horvath, P and Lanotte, P},
title = {Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {233-242},
doi = {10.1089/crispr.2020.0145},
pmid = {33876956},
issn = {2573-1602},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Cloning, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; Humans ; Plasmids/genetics ; RNA ; Streptococcus agalactiae/*enzymology/*genetics ; },
abstract = {Nearly all strains of Streptococcus agalactiae, the leading cause of invasive infections in neonates, encode a type II-A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Interestingly, S. agalactiae strains belonging to the hypervirulent Sequence Type 17 (ST17) contain significantly fewer spacers in their CRISPR locus than other lineages, which could be the result of a less functional CRISPR-Cas system. Here, we revealed one large deletion in the ST17 cas promoter region and we evaluated its impact on the transcription of cas genes as well as the functionalities of the CRISPR-Cas system. We demonstrated that Cas9 interference is functional and that the CRISPR-Cas system of ST17 strains can still acquire new spacers, despite the absence of a regular cas promoter. We demonstrated that a promoter sequence upstream of srn036, a small RNA partially overlapping the antisense tracrRNA, is responsible for the ST17 CRISPR-Cas adaptation and interference activities.},
}
@article {pmid33876955,
year = {2021},
author = {Reti, D and O'Brien, A and Wetzel, P and Tay, A and Bauer, DC and Wilson, LOW},
title = {GOANA: A Universal High-Throughput Web Service for Assessing and Comparing the Outcome and Efficiency of Genome Editing Experiments.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {243-252},
doi = {10.1089/crispr.2020.0068},
pmid = {33876955},
issn = {2573-1602},
mesh = {Bacterial Proteins ; CRISPR-Associated Protein 9 ; CRISPR-Associated Proteins ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases ; Gene Editing/*methods ; Genome ; High-Throughput Nucleotide Sequencing/methods ; High-Throughput Screening Assays/*methods ; Mutation ; Mutation Rate ; Software ; Whole Genome Sequencing ; },
abstract = {The increased development of functionally diverse and highly specialized genome editors has created the need for comparative analytics tools that are able to profile the mutational outcomes, particularly rare and complex outcomes, to assess the editor's applicability to different domains. To address this need, we have developed Generalizable On-target activity ANAlyzer (GOANA), a high-throughput web-based software for determining editing efficiency and cataloguing rare outcomes from next-generation sequencing data. GOANA calculates mutation frequency and outcomes relative to a supplied control sample. It is scalable to thousands of target sites across the entire genome and is 4,000% faster than CRISPResso2. Mutations are reported on a "per-read" level rather than individually, enabling the identification of co-occurring mutations. GOANA is editor agnostic and can be applied to data generated from any targeted editing experiment, including base editors. Requiring only that control and treated reads are aligned to the same reference, GOANA can handle data from any library preparation method, including pooled amplicon and whole-genome sequencing. As a proof of principle, we analyze two large data sets of CRISPR-Cas9 and CRISPR-Cas12a editing, demonstrating the power of GOANA and highlighting several key differences between the two enzymes. GOANA is available for use at https://gt-scan.csiro.au/goana/ and as a command line tool from https://github.com/BauerLab/GOANA.},
}
@article {pmid33876954,
year = {2021},
author = {Hughes, NW and Cong, L},
title = {A CRISPR Landing for Genome Rewriting at Locus-Scale.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {163-166},
pmid = {33876954},
issn = {2573-1602},
support = {R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Genome ; Genome, Human ; Humans ; },
}
@article {pmid33876953,
year = {2021},
author = {Gray, DH and Villegas, I and Long, J and Santos, J and Keir, A and Abele, A and Kuo, CY and Kohn, DB},
title = {Optimizing Integration and Expression of Transgenic Bruton's Tyrosine Kinase for CRISPR-Cas9-Mediated Gene Editing of X-Linked Agammaglobulinemia.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {191-206},
pmid = {33876953},
issn = {2573-1602},
support = {K12 GM102778/GM/NIGMS NIH HHS/United States ; T32 GM007185/GM/NIGMS NIH HHS/United States ; },
mesh = {Agammaglobulinaemia Tyrosine Kinase/deficiency/*genetics/metabolism ; Agammaglobulinemia/*genetics/*therapy ; B-Lymphocytes ; *CRISPR-Cas Systems ; Codon ; DNA, Complementary/genetics ; Gene Editing/*methods ; Genetic Diseases, X-Linked/*genetics/*therapy ; Genetic Loci ; *Genetic Therapy ; Humans ; Introns ; K562 Cells ; Mutation ; Organisms, Genetically Modified ; },
abstract = {X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase (BTK) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the BTK gene. Successful integration of the intron-containing BTK donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in BTK transgene expression. Combining these modifications led to a BTK donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels in vitro. The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.},
}
@article {pmid33876951,
year = {2021},
author = {Lamsfus-Calle, A and Daniel-Moreno, A and Ureña-Bailén, G and Rottenberger, J and Raju, J and Epting, T and Marciano, S and Heumos, L and Baskaran, P and S Antony, J and Handgretinger, R and Mezger, M},
title = {Universal Gene Correction Approaches for β-hemoglobinopathies Using CRISPR-Cas9 and Adeno-Associated Virus Serotype 6 Donor Templates.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {207-222},
doi = {10.1089/crispr.2020.0141},
pmid = {33876951},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Gene Editing ; *Genetic Therapy ; Hematopoietic Stem Cells ; Hemoglobinopathies/*genetics/*therapy ; Humans ; beta-Globins/genetics/metabolism ; beta-Thalassemia/genetics/metabolism/therapy ; },
abstract = {Mutations in the human β-globin gene are the cause of β-hemoglobinopathies, one of the most common inherited single-gene blood disorders in the world. Novel therapeutic approaches are based on lentiviral vectors (LVs) or CRISPR-Cas9-mediated gene disruption to express adult hemoglobin (HbA), or to reactivate the completely functional fetal hemoglobin, respectively. Nonetheless, LVs present a risk of insertional mutagenesis, while gene-disrupting transcription factors (BCL11A, KLF1) involved in the fetal-to-adult hemoglobin switch might generate dysregulation of other cellular processes. Therefore, universal gene addition/correction approaches combining CRISPR-Cas9 and homology directed repair (HDR) by delivering a DNA repair template through adeno-associated virus could mitigate the limitations of both lentiviral gene transfer and gene disruption strategies, ensuring targeted integration and controlled transgene expression. In this study, we attained high rates of gene addition (up to 12%) and gene correction (up to 38%) in hematopoietic stem and progenitor cells from healthy donors without any cell sorting/enrichment or the application of HDR enhancers. Furthermore, these approaches were tested in heterozygous (β[0]/β[+]) and homozygous (β[0]/β[0], β[+]/β[+]) β-thalassemia patients, achieving a significant increase in HbA and demonstrating the universal therapeutic potential of this study for the treatment of β-hemoglobinopathies.},
}
@article {pmid33876950,
year = {2021},
author = {Cereseto, A and Cradick, TJ and Davies, K},
title = {Base Editors Flex Sights on Sickle-Cell Disease.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {166-168},
doi = {10.1089/crispr.2021.29125.kda},
pmid = {33876950},
issn = {2573-1602},
mesh = {*Anemia, Sickle Cell/epidemiology/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid33876949,
year = {2021},
author = {Bodle, JC and Gersbach, CA},
title = {CRISPR Clocks: The Times They Are a-Changin'.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {160-163},
doi = {10.1089/crispr.2021.29123.ger},
pmid = {33876949},
issn = {2573-1602},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Humans ; RNA, Guide ; },
}
@article {pmid33876948,
year = {2021},
author = {Hand, TH and Roth, MO and Smith, CL and Shiel, E and Klein, KN and Gilbert, DM and Li, H},
title = {Catalytically Enhanced Cas9 through Directed Protein Evolution.},
journal = {The CRISPR journal},
volume = {4},
number = {2},
pages = {223-232},
pmid = {33876948},
issn = {2573-1602},
support = {R01 GM083337/GM/NIGMS NIH HHS/United States ; R01 GM099604/GM/NIGMS NIH HHS/United States ; },
mesh = {Actinobacteria/enzymology/genetics ; CRISPR-Associated Protein 9/*genetics/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colonic Neoplasms ; Gene Editing/methods ; HCT116 Cells ; Humans ; *Protein Engineering ; Streptococcus pyogenes/enzymology/genetics ; },
abstract = {Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.},
}
@article {pmid33875803,
year = {2021},
author = {Shinoda, H and Taguchi, Y and Nakagawa, R and Makino, A and Okazaki, S and Nakano, M and Muramoto, Y and Takahashi, C and Takahashi, I and Ando, J and Noda, T and Nureki, O and Nishimasu, H and Watanabe, R},
title = {Amplification-free RNA detection with CRISPR-Cas13.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {476},
pmid = {33875803},
issn = {2399-3642},
mesh = {COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/methods ; *CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/*methods ; RNA/*genetics/metabolism ; RNA, Viral/*genetics/metabolism ; Reproducibility of Results ; SARS-CoV-2/*genetics/physiology ; Sensitivity and Specificity ; },
abstract = {CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows "CRISPR-based amplification-free digital RNA detection (SATORI)", by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.},
}
@article {pmid33875777,
year = {2021},
author = {Guo, HF and Bota-Rabassedas, N and Terajima, M and Leticia Rodriguez, B and Gibbons, DL and Chen, Y and Banerjee, P and Tsai, CL and Tan, X and Liu, X and Yu, J and Tokmina-Roszyk, M and Stawikowska, R and Fields, GB and Miller, MD and Wang, X and Lee, J and Dalby, KN and Creighton, CJ and Phillips, GN and Tainer, JA and Yamauchi, M and Kurie, JM},
title = {A collagen glucosyltransferase drives lung adenocarcinoma progression in mice.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {482},
pmid = {33875777},
issn = {2399-3642},
support = {R01 CA251067/CA/NCI NIH HHS/United States ; R01 CA105155/CA/NCI NIH HHS/United States ; R00 CA225633/CA/NCI NIH HHS/United States ; P50 CA070907/CA/NCI NIH HHS/United States ; K99 CA225633/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma of Lung/*physiopathology ; Animals ; *Disease Progression ; Glucosyltransferases/*metabolism ; Lung Neoplasms/*physiopathology ; Mice ; },
abstract = {Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.},
}
@article {pmid33875479,
year = {2021},
author = {Ouellette, SP and Blay, EA and Hatch, ND and Fisher-Marvin, LA},
title = {CRISPR Interference To Inducibly Repress Gene Expression in Chlamydia trachomatis.},
journal = {Infection and immunity},
volume = {89},
number = {7},
pages = {e0010821},
pmid = {33875479},
issn = {1098-5522},
support = {R21 AI141933/AI/NIAID NIH HHS/United States ; },
mesh = {Binding Sites ; *CRISPR-Cas Systems ; Chlamydia Infections/microbiology ; Chlamydia trachomatis/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Gene Expression Regulation, Bacterial ; Gene Targeting ; Plasmids/genetics ; RNA, Guide ; Ribosomes/metabolism ; },
abstract = {The ability to inducibly repress gene expression is critical to the study of organisms, like Chlamydia, with reduced genomes in which the majority of genes are likely to be essential. We recently described the feasibility of a CRISPR interference (CRISPRi) system to inducibly repress gene expression in Chlamydia trachomatis. However, the initial system suffered from some drawbacks, primarily leaky expression of the anhydrotetracycline (aTc)-inducible dCas9 ortholog and plasmid instability, which prevented population-wide studies (e.g., transcript analyses) of the effects of knockdown. Here, we describe various modifications to the original system that have allowed us to measure gene expression changes within a transformed population of C. trachomatis serovar L2. These modifications include (i) a change in the vector backbone, (ii) the introduction of a weaker ribosome binding site driving dCas9 translation, and (iii) the addition of a degradation tag to dCas9 itself. With these changes, we demonstrate the ability to inducibly repress a target gene sequence, as measured by the absence of protein by immunofluorescence analysis and by decreased transcript levels. Importantly, the expression of dCas9 alone (i.e., without a guide RNA [gRNA]) had minimal impact on chlamydial growth or development. We also describe complementation of the knockdown effect by introducing a transcriptional fusion of the target gene 3' to dCas9. Finally, we demonstrate the functionality of a second CRISPRi system based on a dCas12 system that expands the number of potential chromosomal targets. These tools should provide the ability to study essential gene function in Chlamydia.},
}
@article {pmid33875070,
year = {2021},
author = {Niazvand, F and Fathinezhad, Z and Alfuraiji, N and Etajuri, EA and Amini-Chermahini, F and Chehelgerdi, M and Ranjbar, R},
title = {Clustered Regularly Interspaced Short Palindromic Repeats System of Genome Engineering in Embryos to Repair Genes.},
journal = {Journal of biomedical nanotechnology},
volume = {17},
number = {3},
pages = {331-356},
doi = {10.1166/jbn.2021.3045},
pmid = {33875070},
issn = {1550-7033},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering ; Genome ; Humans ; },
abstract = {CRISPR is a powerful gene editing tool for correcting disease-causing mutations. It is becoming more and more evident that CRISPR is a promising approach to treating human genetic diseases. The technologies for adding or removing genes have made significant advances over the past few years and have shown promising potential outcomes. In the current study, we mainly introduce the CRISPR/Cas system and there are several applications in the treatment of genetic diseases, particularly during embryo development.},
}
@article {pmid33874877,
year = {2021},
author = {Morowvat, MH},
title = {CRISPeering: Bioengineering the Host Cells through CRISPRCas9 Genome Editing System as the Next-generation of Cell Factories.},
journal = {Recent patents on biotechnology},
volume = {15},
number = {2},
pages = {137-147},
doi = {10.2174/1872208315666210419102117},
pmid = {33874877},
issn = {2212-4012},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; *Gene Editing ; Metabolic Engineering ; Patents as Topic ; },
abstract = {BACKGROUND: Nowadays, the CRISPR-Cas9 genome editing system has become a popular bioengineering-based tool for various applications. Owing to its high-target specificity, efficiency, versatility, and simplicity, it has gained attention as a robust tool for molecular biology research, which unveils the biological functions of unexplored genes and engineers the metabolic pathways. Chinese hamster ovary (CHO) cells and Escherichia coli are regarded as the most commonly used expression platforms for industrial- scale production of recombinant proteins. The emergence of the CRISPR-Cas9 genome editing system promotes the current status of expression hosts towards controllable and predictable strains.<br><br>OBJECTIVE: This paper presents the current status of expression hosts for biopharmaceutical production. Some major accomplishments in the utilization of the CRISPR-Cas9 genome editing tool in the different prokaryotic and eukaryotic systems are discussed, and more importantly, the future directions of this newly arrived technology to make the next-generation cell factories with improved or novel properties are suggested. Moreover, the challenges faced in recent patents in this field are also discussed.<br><br>RESULTS AND CONCLUSION: The CRISPR-Cas9 genome-editing tool has been adopted to be utilized in some major expression platforms. CRISPeering has been successfully employed for genome editing in different prokaryotic and eukaryotic host cells. The emergence of systems metabolic engineering, systems biology, and synthetic biology fortify the current situation of the CRISPR-Cas9 genome editing system.},
}
@article {pmid33872346,
year = {2021},
author = {Wang, Z and Wan, L and Xin, Q and Zhang, X and Song, Y and Wang, P and Hong, D and Fan, Z and Yang, G},
title = {Optimizing glyphosate tolerance in rapeseed by CRISPR/Cas9-based geminiviral donor DNA replicon system with Csy4-based single-guide RNA processing.},
journal = {Journal of experimental botany},
volume = {72},
number = {13},
pages = {4796-4808},
doi = {10.1093/jxb/erab167},
pmid = {33872346},
issn = {1460-2431},
mesh = {*Brassica napus/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Glycine/analogs &amp; derivatives ; RNA Processing, Post-Transcriptional ; *RNA, Guide ; Replicon ; },
abstract = {Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.},
}
@article {pmid33871447,
year = {2021},
author = {Mangeot, PE and Guiguettaz, L and Sohier, TJM and Ricci, EP},
title = {Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {169},
pages = {},
doi = {10.3791/62245},
pmid = {33871447},
issn = {1940-087X},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Humans ; Ribonucleoproteins/*genetics ; Transfection ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells. To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named "Nanoblades") were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for "transfection-free" homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.},
}
@article {pmid33871353,
year = {2021},
author = {Ma, P and He, LL and Pironti, A and Laibinis, HH and Ernst, CM and Manson, AL and Bhattacharyya, RP and Earl, AM and Livny, J and Hung, DT},
title = {Genetic determinants facilitating the evolution of resistance to carbapenem antibiotics.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33871353},
issn = {2050-084X},
support = {U19 AI110818/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/*pharmacology ; Carbapenems/*pharmacology ; Drug Resistance, Bacterial/*genetics ; *Evolution, Molecular ; Klebsiella pneumoniae/drug effects/*genetics ; },
abstract = {In this era of rising antibiotic resistance, in contrast to our increasing understanding of mechanisms that cause resistance, our understanding of mechanisms that influence the propensity to evolve resistance remains limited. Here, we identified genetic factors that facilitate the evolution of resistance to carbapenems, the antibiotic of 'last resort', in Klebsiella pneumoniae, the major carbapenem-resistant species. In clinical isolates, we found that high-level transposon insertional mutagenesis plays an important role in contributing to high-level resistance frequencies in several major and emerging carbapenem-resistant lineages. A broader spectrum of resistance-conferring mutations for select carbapenems such as ertapenem also enables higher resistance frequencies and, importantly, creates stepping-stones to achieve high-level resistance to all carbapenems. These mutational mechanisms can contribute to the evolution of resistance, in conjunction with the loss of systems that restrict horizontal resistance gene uptake, such as the CRISPR-Cas system. Given the need for greater antibiotic stewardship, these findings argue that in addition to considering the current efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future efficacy are also important. The genetic background of a clinical isolate and the exact antibiotic identity can and should also be considered as they are determinants of a strain's propensity to become resistant. Together, these findings thus provide a molecular framework for understanding acquisition of carbapenem resistance in K. pneumoniae with important implications for diagnosing and treating this important class of pathogens.},
}
@article {pmid33871049,
year = {2021},
author = {Shenkman, M and Geva, M and Gershoni-Emek, N and Hayden, MR and Lederkremer, GZ},
title = {Pridopidine reduces mutant huntingtin-induced endoplasmic reticulum stress by modulation of the Sigma-1 receptor.},
journal = {Journal of neurochemistry},
volume = {158},
number = {2},
pages = {467-481},
doi = {10.1111/jnc.15366},
pmid = {33871049},
issn = {1471-4159},
mesh = {3T3 Cells ; Animals ; CRISPR-Cas Systems ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress/*drug effects ; Gene Knockout Techniques ; HEK293 Cells ; Heat-Shock Proteins ; Humans ; Huntingtin Protein/*genetics/*toxicity ; Mice ; Mitochondrial Membranes/drug effects ; Piperidines/*pharmacology ; Receptors, sigma/*drug effects ; Unfolded Protein Response/drug effects ; },
abstract = {The endoplasmic reticulum (ER)-localized Sigma-1 receptor (S1R) is neuroprotective in models of neurodegenerative diseases, among them Huntington disease (HD). Recent clinical trials in HD patients and preclinical studies in cellular and mouse HD models suggest a therapeutic potential for the high-affinity S1R agonist pridopidine. However, the molecular mechanisms of the cytoprotective effect are unclear. We have previously reported strong induction of ER stress by toxic mutant huntingtin (mHtt) oligomers, which is reduced upon sequestration of these mHtt oligomers into large aggregates. Here, we show that pridopidine significantly ameliorates mHtt-induced ER stress in cellular HD models, starting at low nanomolar concentrations. Pridopidine reduced the levels of markers of the three branches of the unfolded protein response (UPR), showing the strongest effects on the PKR-like endoplasmic reticulum kinase (PERK) branch. The effect is S1R-dependent, as it is abolished in cells expressing mHtt in which the S1R was deleted using CRISPR/Cas9 technology. mHtt increased the level of the detergent-insoluble fraction of S1R, suggesting a compensatory cellular mechanism that responds to increased ER stress. Pridopidine further enhanced the levels of insoluble S1R, suggesting the stabilization of activated S1R oligomers. These S1R oligomeric species appeared in ER-localized patches, and not in the mitochondria-associated membranes nor the ER-derived quality control compartment. The colocalization of S1R with the chaperone BiP was significantly reduced by mHtt, and pridopidine restored this colocalization to normal, unstressed levels. Pridopidine increased toxic oligomeric mHtt recruitment into less toxic large sodium dodecyl sulfate-insoluble aggregates, suggesting that this in turn reduces ER stress and cytotoxicity.},
}
@article {pmid33870225,
year = {2021},
author = {Gao, P and Dong, X and Wang, Y and Wei, GH},
title = {Optimized CRISPR/Cas9-mediated single nucleotide mutation in adherent cancer cell lines.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100419},
pmid = {33870225},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Gene Editing/*methods ; Humans ; MCF-7 Cells ; Neoplasms/*genetics ; Point Mutation/*genetics ; },
abstract = {CRISPR/Cas9 is an efficient, accurate, and optimizable genome-editing tool. Here, we present a modified CRISPR/Cas9 genome-editing protocol for single nucleotide mutation in adherent cell lines. The protocol was adapted to focus on ease of use and efficiency. The protocol here describes how to generate a single nucleotide mutation in cultured 22Rv1 cells. We have also used the protocol in other adherent cell types. Thus, the protocol can be applied to assessing the effect of non-coding single nucleotide polymorphisms (SNPs) in a variety of cell types. For complete details on the use and execution of this protocol, please refer to Gao et al. (2018).},
}
@article {pmid33868646,
year = {2020},
author = {Vats, N and Sanal, MG and Venugopal, SK and Taneja, P and Sarin, SK},
title = {Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it's Insertion into genomic safe harbor AAVS1 site using CRISPR-Cas9.},
journal = {F1000Research},
volume = {9},
number = {},
pages = {1498},
pmid = {33868646},
issn = {2046-1402},
mesh = {ATP Binding Cassette Transporter, Subfamily B, Member 11 ; *CRISPR-Cas Systems/genetics ; Child ; Cloning, Molecular ; *Escherichia coli/genetics ; Genomics ; Humans ; },
abstract = {Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn't cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a 'safe-harbor' in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.},
}
@article {pmid33868219,
year = {2021},
author = {Garrett, SC},
title = {Pruning and Tending Immune Memories: Spacer Dynamics in the CRISPR Array.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {664299},
pmid = {33868219},
issn = {1664-302X},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) is a type of prokaryotic immune system that is unique in its ability to provide sequence-specific adaptive protection, which can be updated in response to new threats. CRISPR-Cas does this by storing fragments of DNA from invading genetic elements in an array interspersed with short repeats. The CRISPR array can be continuously updated through integration of new DNA fragments (termed spacers) at one end, but over time existing spacers become obsolete. To optimize immunity, spacer uptake, residency, and loss must be regulated. This mini-review summarizes what is known about how spacers are organized, maintained, and lost from CRISPR arrays.},
}
@article {pmid33868193,
year = {2021},
author = {Zhang, R and Xu, W and Shao, S and Wang, Q},
title = {Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {635227},
pmid = {33868193},
issn = {1664-302X},
abstract = {Functional genetic screening is an important method that has been widely used to explore the biological processes and functional annotation of genetic elements. CRISPR/Cas (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein) is the newest tool in the geneticist's toolbox, allowing researchers to edit a genome with unprecedented ease, accuracy, and high-throughput. Most recently, CRISPR interference (CRISPRi) has been developed as an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single-guide RNA (sgRNA) to repress sequence-specific genes. In this review, we summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific. Moreover, we demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis. The recent development of the CRISPRi system will provide a high-throughput, practical, and efficient tool for the discovery of functionally important genes in bacteria.},
}
@article {pmid33867826,
year = {2021},
author = {Hu, X and Hao, F and Li, X and Xun, Z and Gao, Y and Ren, B and Cang, M and Liang, H and Liu, D},
title = {Generation of VEGF knock-in Cashmere goat via the CRISPR/Cas9 system.},
journal = {International journal of biological sciences},
volume = {17},
number = {4},
pages = {1026-1040},
pmid = {33867826},
issn = {1449-2288},
mesh = {Animal Fur/*growth &amp; development ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Fibroblast Growth Factor 5/*genetics ; *Gene Knock-In Techniques ; Goats/*genetics/metabolism ; *Nuclear Transfer Techniques ; Skin/metabolism ; Textiles ; Transcriptome ; Vascular Endothelial Growth Factor A/*genetics ; },
abstract = {Cashmere is a rare and specialised animal fibre, which grows on the outer skin of goats. Owing its low yield and soft, light, and warm properties, it has a high economic value. Here, we attempted to improve existing cashmere goat breeds by simultaneously increasing their fibre length and cashmere yield. We attempted this by knocking in the vascular endothelial growth factor (VEGF) at the fibroblast growth factor 5(FGF5) site using a gene editing technology and then studying its hair growth-promoting mechanisms. We show that a combination of RS-1 and NU7441 significantly improve the efficiency of CRISPR/Cas9-mediated, homologous-directed repair without affecting the embryo cleavage rate or the percentages of embryos at different stages. In addition, we obtained a cashmere goat, which integrated the VEGF gene at the FGF5 site, and the cashmere yield and fibre length of this gene-edited goat were improved. Through next-generation sequencing, we found that the up-regulation of VEGF and the down-regulation of FGF5 affected the cell cycle, proliferation, and vascular tone through the PI3K-AKT signalling pathway and at extracellular matrix-receptor interactions. Owing to this, the gene-edited cashmere goat showed impressive cashmere performance. Overall, in this study, we generated a gene-edited cashmere goat by integrating VEGF at the FGF5 site and provided an animal model for follow-up research on hair growth mechanisms.},
}
@article {pmid33867038,
year = {2021},
author = {Yu, P and Yang, T and Zhang, D and Xu, L and Cheng, X and Ding, S and Cheng, W},
title = {An all-in-one telomerase assay based on CRISPR-Cas12a trans-cleavage while telomere synthesis.},
journal = {Analytica chimica acta},
volume = {1159},
number = {},
pages = {338404},
doi = {10.1016/j.aca.2021.338404},
pmid = {33867038},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HeLa Cells ; Humans ; *Telomerase/genetics ; Telomere/genetics ; },
abstract = {As one of the crucial factors associated with human life span and cancer progression, telomerase is regarded as an emerging biomarker for cancer diagnosis. Therefore, a facile, rapid and sensitive approach for telomerase activity detection with point-of-care (POC) diagnosis potential is in great demands. Herein, an all-in-one telomerase activity detection assay was established based on the telomere synthesis activated CRISPR-Cas12a system. A telomerase extension reaction generated telomere repeats sequences (TTAGGG)n, which was recognized by a customized CRISPR-guided RNA (crRNA) simultaneously, and finally activated a typical trans-cleavage based CRISPR-Cas12a detection assay. With the inherent sensitivity of CRISPR-Cas12a, this approach achieved a great linear regression ranging from 100 to 2000 HeLa cells and a limitation of detection down to 26 HeLa cells. Moreover, by using the proposed method, telomerase can be detected in one pot under isothermal condition (37 °C) by a simple and fast workflow (one step within 1 h). Due to its excellent performance, this all-in-one method shows great potential in POC detection of the telomerase activity.},
}
@article {pmid33866563,
year = {2021},
author = {Kumar, A and Harloff, HJ and Melzer, S and Leineweber, J and Defant, B and Jung, C},
title = {A rhomboid-like protease gene from an interspecies translocation confers resistance to cyst nematodes.},
journal = {The New phytologist},
volume = {231},
number = {2},
pages = {801-813},
doi = {10.1111/nph.17394},
pmid = {33866563},
issn = {1469-8137},
mesh = {Animals ; *Beta vulgaris ; *Cysts ; *Nematoda ; Peptide Hydrolases ; Plant Breeding ; Plant Diseases/genetics ; },
abstract = {Plant-parasitic nematodes are severe pests in crop production worldwide. Chemical control of nematodes has been continuously reduced in recent decades owing to environmental and health concerns. Therefore, breeding nematode-resistant crops is an important aim if we are to secure harvests. The beet cyst nematode impairs root development and causes severe losses in sugar beet production. The only sources for resistance are distantly related wild species of the genus Patellifolia. Nematode resistance had been introduced into the beet genome via translocations from P. procumbens. We sequenced three translocations and identified the translocation breakpoints. By comparative sequence analysis of three translocations, we localized the resistance gene Hs4 within a region c. 230 kb in size. A candidate gene was characterized by CRISPR-Cas-mediated knockout and overexpression in susceptible roots. The gene encodes a rhomboid-like protease, which is predicted to be bound to the endoplasmic reticulum. Gene knockout resulted in complete loss of resistance, while overexpression caused resistance. The data confirm that the Hs4 gene alone protects against the pest. Thus, it constitutes a previously unknown mechanism of plants to combat parasitic nematodes. Its function in a nonrelated species suggests that the gene can confer resistance in crop species from different plant families.},
}
@article {pmid33865853,
year = {2021},
author = {Yasuda, K and Nishikawa, M and Okamoto, K and Horibe, K and Mano, H and Yamaguchi, M and Okon, R and Nakagawa, K and Tsugawa, N and Okano, T and Kawagoe, F and Kittaka, A and Ikushiro, S and Sakaki, T},
title = {Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100668},
pmid = {33865853},
issn = {1083-351X},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Calcifediol/administration &amp; dosage/*metabolism ; Cytochrome P-450 CYP3A/*metabolism ; Metabolome/*drug effects ; Rats ; Vitamin D3 24-Hydroxylase/*antagonists &amp; inhibitors/genetics/metabolism ; Vitamins/administration &amp; dosage/*metabolism ; },
abstract = {CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.},
}
@article {pmid33864868,
year = {2021},
author = {Tang, D and Li, H and Wu, C and Jia, T and He, H and Yao, S and Yu, Y and Chen, Q},
title = {A distinct structure of Cas1-Cas2 complex provides insights into the mechanism for the longer spacer acquisition in Pyrococcus furiosus.},
journal = {International journal of biological macromolecules},
volume = {183},
number = {},
pages = {379-386},
doi = {10.1016/j.ijbiomac.2021.04.074},
pmid = {33864868},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/*genetics ; Pyrococcus furiosus/*genetics ; },
abstract = {In the adaptation stage of CRISPR-Cas systems, the Cas1-Cas2 integrase captures and integrates new invader-derived spacers into the CRISPR locus, serving as a molecular memory of prior infection. As of yet, the structural information of Cas1-Cas2 complex is available only for two species. Here we present the crystal structure of Cas1-Cas2 complex of Pyrococcus furiosus, which showed a distinct architecture from the known Cas1-Cas2 complexes. The shorter C-terminal tail of Pfu Cas2 directs the Cas1 dimers go in the opposite direction, resulting in a different prespacer binding mode. Based on our structural and mutagenesis results, we modeled a prespacer with a shorter duplex and longer 3' overhangs to bind Pfu Cas1-Cas2 complex. The prespacer preference was confirmed by EMSA, fluorescence polarization, and in vitro integration assays. This model provides a potential explanation for the longer spacer acquisition observed in P. furiosus when deleting both cas4 genes. Our study highlights the diversity of the CRISPR adaptation module.},
}
@article {pmid33864508,
year = {2021},
author = {Kujur, S and Senthil-Kumar, M and Kumar, R},
title = {Plant viral vectors: expanding the possibilities of precise gene editing in plant genomes.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {931-934},
pmid = {33864508},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Vectors/genetics ; *Genome, Plant/genetics ; Plants/genetics ; },
}
@article {pmid33864024,
year = {2022},
author = {Marayati, R and Stafman, LL and Williams, AP and Bownes, LV and Quinn, CH and Markert, HR and Easlick, JL and Stewart, JE and Crossman, DK and Mroczek-Musulman, E and Beierle, EA},
title = {CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.},
journal = {Cancer gene therapy},
volume = {29},
number = {5},
pages = {558-572},
pmid = {33864024},
issn = {1476-5500},
support = {T32 CA183926/CA/NCI NIH HHS/United States ; P30 CA013148/CA/NCI NIH HHS/United States ; P30 AR048311/AR/NIAMS NIH HHS/United States ; T32 CA091078/CA/NCI NIH HHS/United States ; T32 GM008361/GM/NIGMS NIH HHS/United States ; P30 AI027767/AI/NIAID NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Transformation, Neoplastic/genetics ; *Hepatoblastoma/genetics/pathology ; Humans ; *Liver Neoplasms/genetics/pathology ; Mice ; *Protein Serine-Threonine Kinases/genetics ; *Proto-Oncogene Proteins/genetics ; },
abstract = {Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.},
}
@article {pmid33863871,
year = {2021},
author = {Lin, P and Jiang, J and Wu, M},
title = {CRISPR base editor treats premature-aging syndrome.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {158},
pmid = {33863871},
issn = {2059-3635},
support = {R01 AI109317/AI/NIAID NIH HHS/United States ; R01 AI138203/AI/NIAID NIH HHS/United States ; },
mesh = {*Aging, Premature ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; },
}
@article {pmid33863334,
year = {2021},
author = {Meccariello, A and Krsticevic, F and Colonna, R and Del Corsano, G and Fasulo, B and Papathanos, PA and Windbichler, N},
title = {Engineered sex ratio distortion by X-shredding in the global agricultural pest Ceratitis capitata.},
journal = {BMC biology},
volume = {19},
number = {1},
pages = {78},
pmid = {33863334},
issn = {1741-7007},
support = {BB/P000843/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Ceratitis capitata/genetics ; Female ; Male ; RNA, Guide ; Sex Ratio ; X Chromosome/genetics ; },
abstract = {BACKGROUND: Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits.<br><br>RESULTS: Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~&#x2009;80%).<br><br>CONCLUSION: We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.},
}
@article {pmid33862567,
year = {2021},
author = {Wang, M and Han, D and Zhang, J and Zhang, R and Li, J},
title = {High-fidelity detection of DNA combining the CRISPR/Cas9 system and hairpin probe.},
journal = {Biosensors &amp; bioelectronics},
volume = {184},
number = {},
pages = {113212},
doi = {10.1016/j.bios.2021.113212},
pmid = {33862567},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Gene Editing ; Limit of Detection ; },
abstract = {Methods that enable specific and sensitive detection of DNA are greatly required for high-fidelity sequence measurement and single-nucleotide variations (SNVs) genotyping. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems have provided revolutionary tools for detecting nucleic acids. However, most of the current CRISPR/Cas-based DNA biosensing platforms suffer from inherent off-target effects of Cas proteins and require pre-amplification processes, which compromise the analytical fidelity. In this work, a CRISPR/Cas9-triggered hairpin probe-mediated biosensing method (namely CHP) was used to directly read the original DNA sequences, while effectively neutralizing the off-target effect and achieving high sensitivity. This technique can quantify DNA targets with a limit of detection (LOD) at the attomole level and identify SNVs with allelic fractions as low as 0.01%~0.1%. Moreover, we show that the CHP system is applicable in detecting mutations in serum samples without DNA isolation steps. Collectively, the CHP system is a sensitive and high-fidelity platform, which promises a great potential for providing robust tool for DNA sequence analysis and SNVs genotyping.},
}
@article {pmid33862076,
year = {2021},
author = {Prakash, A and Kumar, M},
title = {Characterizing the transcripts of Leptospira CRISPR I-B array and its processing with endoribonuclease LinCas6.},
journal = {International journal of biological macromolecules},
volume = {182},
number = {},
pages = {785-795},
doi = {10.1016/j.ijbiomac.2021.04.066},
pmid = {33862076},
issn = {1879-0003},
mesh = {Bacterial Proteins/chemistry/genetics/*metabolism ; Catalytic Domain ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endoribonucleases/chemistry/genetics/*metabolism ; Leptospira interrogans/*genetics/metabolism ; RNA, Messenger/genetics/*metabolism ; },
abstract = {In Leptospira interrogans serovar Copenhageni, the CRISPR-Cas I-B locus possesses a CRISPR array between the two independent cas-operons. Using the reverse transcription-PCR and the in vitro endoribonuclease assay with Cas6 of Leptospira (LinCas6), we account that the CRISPR is transcriptionally active and is conventionally processed. The LinCas6 specifically excises at one site within the synthetic cognate repeat RNA or the repeats of precursor-CRISPR RNA (pre-crRNA) in the sense direction. In contrast, the antisense repeat RNA is cleaved at multiple sites. LinCas6 functions as a single turnover endoribonuclease on its repeat RNA substrate, where substitution of one of predicted active site residues (His38) resulted in reduced activity. This study highlights the comprehensive understanding of the Leptospira CRISPR array transcription and its processing by LinCas6 that is central to RNA-mediated CRISPR-Cas I-B adaptive immunity.},
}
@article {pmid33861513,
year = {2021},
author = {Verhage, L},
title = {Twelve genes at one blow: multiplex genome editing with CRISPR/Cas.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {1},
pages = {6-7},
doi = {10.1111/tpj.15228},
pmid = {33861513},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome, Plant/genetics ; },
}
@article {pmid33859416,
year = {2021},
author = {Vinjamur, DS and Yao, Q and Cole, MA and McGuckin, C and Ren, C and Zeng, J and Hossain, M and Luk, K and Wolfe, SA and Pinello, L and Bauer, DE},
title = {ZNF410 represses fetal globin by singular control of CHD4.},
journal = {Nature genetics},
volume = {53},
number = {5},
pages = {719-728},
pmid = {33859416},
issn = {1546-1718},
support = {R35 HG010717/HG/NHGRI NIH HHS/United States ; DP2 HL137300/HL/NHLBI NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; U54 DK110805/DK/NIDDK NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; },
mesh = {Adult ; Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Chromatin/metabolism ; DNA/metabolism ; Erythroid Cells/metabolism ; Erythropoiesis ; Fetal Hemoglobin/*metabolism ; Gene Editing ; Gene Expression Regulation ; Hematopoiesis ; Hematopoietic Stem Cells/metabolism ; Humans ; Mi-2 Nucleosome Remodeling and Deacetylase Complex/*metabolism ; Mice ; Mutagenesis/genetics ; Protein Binding ; Reproducibility of Results ; Transcription Factors/*metabolism ; },
abstract = {Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.},
}
@article {pmid33859403,
year = {2021},
author = {Jin, S and Lin, Q and Luo, Y and Zhu, Z and Liu, G and Li, Y and Chen, K and Qiu, JL and Gao, C},
title = {Genome-wide specificity of prime editors in plants.},
journal = {Nature biotechnology},
volume = {39},
number = {10},
pages = {1292-1299},
pmid = {33859403},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant/*genetics ; Moloney murine leukemia virus/genetics ; Mutation ; Oryza/genetics ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; Reverse Transcription/genetics ; Whole Genome Sequencing ; },
abstract = {Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome.},
}
@article {pmid33859386,
year = {2021},
author = {Mason, DM and Friedensohn, S and Weber, CR and Jordi, C and Wagner, B and Meng, SM and Ehling, RA and Bonati, L and Dahinden, J and Gainza, P and Correia, BE and Reddy, ST},
title = {Optimization of therapeutic antibodies by predicting antigen specificity from antibody sequence via deep learning.},
journal = {Nature biomedical engineering},
volume = {5},
number = {6},
pages = {600-612},
pmid = {33859386},
issn = {2157-846X},
mesh = {Amino Acid Sequence ; Animals ; Antibody Affinity ; Antibody Specificity ; Antigens/*chemistry/genetics/immunology ; CRISPR-Cas Systems ; *Deep Learning ; Humans ; Hybridomas/chemistry/immunology ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Engineering/*methods ; Receptor, ErbB-2/*chemistry/genetics/immunology ; Recombinational DNA Repair ; Sequence Analysis, Protein ; Trastuzumab/*chemistry/genetics/immunology ; },
abstract = {The optimization of therapeutic antibodies is time-intensive and resource-demanding, largely because of the low-throughput screening of full-length antibodies (approximately 1 × 10[3] variants) expressed in mammalian cells, which typically results in few optimized leads. Here we show that optimized antibody variants can be identified by predicting antigen specificity via deep learning from a massively diverse space of antibody sequences. To produce data for training deep neural networks, we deep-sequenced libraries of the therapeutic antibody trastuzumab (about 1 × 10[4] variants), expressed in a mammalian cell line through site-directed mutagenesis via CRISPR-Cas9-mediated homology-directed repair, and screened the libraries for specificity to human epidermal growth factor receptor 2 (HER2). We then used the trained neural networks to screen a computational library of approximately 1 × 10[8] trastuzumab variants and predict the HER2-specific subset (approximately 1 × 10[6] variants), which can then be filtered for viscosity, clearance, solubility and immunogenicity to generate thousands of highly optimized lead candidates. Recombinant expression and experimental testing of 30 randomly selected variants from the unfiltered library showed that all 30 retained specificity for HER2. Deep learning may facilitate antibody engineering and optimization.},
}
@article {pmid33859300,
year = {2021},
author = {Kishimoto, Y and Nishiura, I and Hirata, W and Yuri, S and Yamamoto, N and Ikawa, M and Isotani, A},
title = {A novel tissue specific alternative splicing variant mitigates phenotypes in Ets2 frame-shift mutant models.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {8297},
pmid = {33859300},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Embryo Loss/genetics/pathology ; Embryonic Development/genetics ; Exons/genetics ; Female ; Frameshift Mutation/*genetics ; Gene Editing/methods ; Humans ; Male ; *Phenotype ; Proto-Oncogene Protein c-ets-2/*genetics ; RNA Splicing/*genetics ; RNA, Messenger/genetics/metabolism ; Skin/metabolism ; Trophoblasts/pathology ; },
abstract = {E26 avian leukemia oncogene 2, 3' domain (Ets2) has been implicated in various biological processes. An Ets2 mutant model (Ets2[db1/db1]), which lacks the DNA-binding domain, was previously reported to exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair and curly whiskers. Here, we generated new Ets2 mutant models with a frame-shift mutation in exon 8 using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as embryonic development stopped before E8.5, as previously reported. When we rescued them by tetraploid complementation, these mice did not exhibit wavy hair or curly whisker phenotypes. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 Ets2[em1/em1] embryos. This exon 8-skipped Ets2 mRNA was translated into protein, suggesting that this Ets2 mutant protein complemented the Ets2 function in the skin. Our data implies that novel splicing variants incidentally generated after genome editing may complicate the phenotypic analysis but may also give insight into the new mechanisms related to biological gene functions.},
}
@article {pmid33857832,
year = {2021},
author = {Cai, J and Kropf, E and Hou, YM and Iacovitti, L},
title = {A stress-free strategy to correct point mutations in patient iPS cells.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102332},
pmid = {33857832},
issn = {1876-7753},
support = {R01 AI125650/AI/NIAID NIH HHS/United States ; R01 NS075839/NS/NINDS NIH HHS/United States ; R03 NS107751/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Homologous Recombination ; Humans ; *Induced Pluripotent Stem Cells ; Mutation ; Point Mutation ; },
abstract = {When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-Puro[R] cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-Puro[R] cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.},
}
@article {pmid33857616,
year = {2021},
author = {Harbottle, JA},
title = {Immunotherapy to get on point with base editing.},
journal = {Drug discovery today},
volume = {26},
number = {10},
pages = {2350-2357},
doi = {10.1016/j.drudis.2021.04.003},
pmid = {33857616},
issn = {1878-5832},
mesh = {CRISPR-Cas Systems/genetics ; Cell- and Tissue-Based Therapy/methods ; *Gene Editing ; Hematologic Neoplasms/genetics/immunology/therapy ; Humans ; Immunotherapy/*methods ; Immunotherapy, Adoptive/methods ; Neoplasms/genetics/immunology/*therapy ; Receptors, Chimeric Antigen/immunology ; },
abstract = {Engineered immune cell therapy is revolutionising the field of cancer therapeutics. US Food and Drug Administration (FDA) approval of two chimeric antigen receptor (CAR)-T cell products for the treatment of haematological malignancies paved the way for individualised cancer treatment. However, multiple genetic edits will be required to improve the efficacy of CAR-T cell therapies if they are to treat refractory malignancies successfully, particularly solid tumours. Off-target effects of CRISPR-Cas9-mediated multiplex editing are likely to hinder its safety and application in the clinic. Novel base editing technologies offer a promising and safer alternative for simultaneous editing that could enhance allogeneic engineered immunotherapies for targeting solid tumours and other complex human diseases.},
}
@article {pmid33857265,
year = {2021},
author = {Akidil, E and Albanese, M and Buschle, A and Ruhle, A and Pich, D and Keppler, OT and Hammerschmidt, W},
title = {Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection.},
journal = {PLoS pathogens},
volume = {17},
number = {4},
pages = {e1009117},
pmid = {33857265},
issn = {1553-7374},
mesh = {B-Lymphocytes/*virology ; CRISPR-Cas Systems/*genetics ; Epstein-Barr Virus Infections/*genetics ; Epstein-Barr Virus Nuclear Antigens/genetics ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; Herpesvirus 4, Human/genetics ; Humans ; Lymphocyte Activation/*genetics/immunology ; Viral Proteins/genetics/metabolism ; Virus Latency/genetics ; },
abstract = {Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.},
}
@article {pmid33856034,
year = {2021},
author = {Liu, X and Wang, Q and Shao, Z and Zhang, S and Hou, M and Jiang, M and Du, M and Li, J and Yuan, H},
title = {Proteomic analysis of aged and OPTN E50K retina in the development of normal tension glaucoma.},
journal = {Human molecular genetics},
volume = {30},
number = {11},
pages = {1030-1044},
doi = {10.1093/hmg/ddab099},
pmid = {33856034},
issn = {1460-2083},
mesh = {Animals ; Autophagy/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/*genetics ; Disease Models, Animal ; Humans ; Low Tension Glaucoma/*genetics/metabolism/pathology ; Membrane Transport Proteins/*genetics ; Mice ; Mutation/genetics ; Phenotype ; Proteomics ; Retina/*metabolism/pathology ; Retinal Ganglion Cells/metabolism/pathology ; Transcription Factor TFIIIA ; },
abstract = {Progressive degeneration of retinal ganglion cells (RGCs) is a major characteristic of glaucoma, whose underlying mechanisms are still largely unknown. An E50K mutation in the Optineurin (OPTN) gene is a leading cause of normal tension glaucoma (NTG), directly affecting RGCs without high intraocular pressure and causing severe glaucomatous symptoms in clinical settings. A systematic analysis of the NTG mouse model is crucial for better understanding of the underlying pathological mechanisms for glaucoma. To elucidate proteomic and biochemical pathway alterations during NTG development, we established an OPTN E50K mutant mouse model through CRISPR/Cas9. Retinal proteins from resulting mice exhibiting glaucomatous phenotypes were subject to tandem mass tag-labeled quantitative proteomics and then analyzed through bioinformatics methods to characterize the molecular and functional signatures of NTG. We identified 6364 quantitative proteins in our proteomic analysis. Bioinformatics analysis revealed that OPTN E50K mice experienced protein synthesis dysregulation, age-dependent energy defects and autophagy-lysosome pathway dysfunction. Certain biological features, including amyloid deposition, RNA splicing, microglia activation and reduction of crystallin production, were similar to Alzheimer's disease. Our study is the first to describe proteomic and biochemical pathway alterations in NTG pathogenesis during disease advancement. Several proteomic signatures overlapped with retinal changes found in the ad mice model, suggesting the presence of common mechanisms between age-related degenerative disorders, as well as prospective new targets for diagnostic and therapeutic strategies.},
}
@article {pmid33856033,
year = {2021},
author = {Tusi, SK and Nguyen, L and Thangaraju, K and Li, J and Cleary, JD and Zu, T and Ranum, LPW},
title = {The alternative initiation factor eIF2A plays key role in RAN translation of myotonic dystrophy type 2 CCUG•CAGG repeats.},
journal = {Human molecular genetics},
volume = {30},
number = {11},
pages = {1020-1029},
pmid = {33856033},
issn = {1460-2083},
support = {R01 NS098819/NS/NINDS NIH HHS/United States ; R37 NS040389/NS/NINDS NIH HHS/United States ; P01 NS058901/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA Repeat Expansion/genetics ; Eukaryotic Initiation Factor-2/*genetics ; HEK293 Cells ; Humans ; Microsatellite Repeats/genetics ; Myotonic Dystrophy/*genetics/physiopathology ; Protein Biosynthesis/genetics ; eIF-2 Kinase/*genetics ; },
abstract = {Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)-/- and PKR-like endoplasmic reticulum kinase (PERK)-/- cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.},
}
@article {pmid33855309,
year = {2021},
author = {Ghetti, S and Burigotto, M and Mattivi, A and Magnani, G and Casini, A and Bianchi, A and Cereseto, A and Fava, LL},
title = {CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells.},
journal = {STAR protocols},
volume = {2},
number = {2},
pages = {100407},
pmid = {33855309},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing ; Gene Knock-In Techniques/*methods ; Humans ; Retinal Pigment Epithelium/*cytology ; Ribonucleoproteins/*genetics ; },
abstract = {hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).},
}
@article {pmid33854845,
year = {2021},
author = {Oliveira de Almeida, M and Carvalho, R and Figueira Aburjaile, F and Malcher Miranda, F and Canário Cerqueira, J and Brenig, B and Ghosh, P and Ramos, R and Kato, RB and de Castro Soares, S and Silva, A and Azevedo, V and Canário Viana, MV},
title = {Characterization of the first vaginal Lactobacillus crispatus genomes isolated in Brazil.},
journal = {PeerJ},
volume = {9},
number = {},
pages = {e11079},
pmid = {33854845},
issn = {2167-8359},
abstract = {BACKGROUND: Lactobacillus crispatus is the dominant species in the vaginal microbiota associated with health and considered a homeostasis biomarker. Interestingly, some strains are even used as probiotics. However, the genetic mechanisms of L. crispatus involved in the control of the vaginal microbiome and protection against bacterial vaginosis (BV) are not entirely known. To further investigate these mechanisms, we sequenced and characterized the first four L. crispatus genomes from vaginal samples from Brazilian women and used genome-wide association study (GWAS) and comparative analyses to identify genetic mechanisms involved in healthy or BV conditions and selective pressures acting in the vaginal microbiome.<br><br>METHODS: The four genomes were sequenced, assembled using ten different strategies and automatically annotated. The functional characterization was performed by bioinformatics tools comparing with known probiotic strains. Moreover, it was selected one representative strain (L. crispatus CRI4) for in vitro detection of phages by electron microscopy. Evolutionary analysis, including phylogeny, GWAS and positive selection were performed using 46 public genomes strains representing health and BV conditions.<br><br>RESULTS: Genes involved in probiotic effects such as lactic acid production, hydrogen peroxide, bacteriocins, and adhesin were identified. Three hemolysins and putrescine production were predicted, although these features are also present in other probiotic strains. The four genomes presented no plasmids, but 14 known families insertion sequences and several prophages were detected. However, none of the mobile genetic elements contained antimicrobial resistance genes. The genomes harbor a CRISPR-Cas subtype II-A system that is probably inactivated due to fragmentation of the genes csn2 and cas9. No genomic feature was associated with a health condition, perhaps due to its multifactorial characteristic. Five genes were identified as under positive selection, but the selective pressure remains to be discovered. In conclusion, the Brazilian strains investigated in this study present potential protective properties, although in vitro and in vivo studies are required to confirm their efficacy and safety to be considered for human use.},
}
@article {pmid33854491,
year = {2021},
author = {Medina-Aparicio, L and Rodriguez-Gutierrez, S and Rebollar-Flores, JE and Martínez-Batallar, &#xc1;G and Mendoza-Mejía, BD and Aguirre-Partida, ED and Vázquez, A and Encarnación, S and Calva, E and Hernández-Lucas, I},
title = {The CRISPR-Cas System Is Involved in OmpR Genetic Regulation for Outer Membrane Protein Synthesis in Salmonella Typhi.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {657404},
pmid = {33854491},
issn = {1664-302X},
abstract = {The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi (S. Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2, and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2, the amount of the OmpC porin decreased substantially, whereas in individual cse2, cas6e, cas1, or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S. Typhi cse2, cas5, cas6e, cas1, cas2, and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S. Typhi.},
}
@article {pmid33854235,
year = {2021},
author = {Simoneschi, D and Rona, G and Zhou, N and Jeong, YT and Jiang, S and Milletti, G and Arbini, AA and O'Sullivan, A and Wang, AA and Nithikasem, S and Keegan, S and Siu, Y and Cianfanelli, V and Maiani, E and Nazio, F and Cecconi, F and Boccalatte, F and Fenyö, D and Jones, DR and Busino, L and Pagano, M},
title = {CRL4[AMBRA1] is a master regulator of D-type cyclins.},
journal = {Nature},
volume = {592},
number = {7856},
pages = {789-793},
pmid = {33854235},
issn = {1476-4687},
support = {P30 CA016087/CA/NCI NIH HHS/United States ; R35 GM136250/GM/NIGMS NIH HHS/United States ; S10 OD018338/OD/NIH HHS/United States ; R01 CA207513/CA/NCI NIH HHS/United States ; R01 CA076584/CA/NCI NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; *Cell Division ; Cyclin D1/*metabolism ; Cyclin D2/metabolism ; Cyclin D3/metabolism ; Cyclin-Dependent Kinase 2/metabolism ; Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors ; Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors ; Female ; Gene Knockout Techniques ; Genes, Tumor Suppressor ; HCT116 Cells ; HEK293 Cells ; Humans ; Male ; Mice ; Neoplasms/genetics ; Ubiquitin/metabolism ; },
abstract = {D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer[1], but the mechanisms that regulate their turnover are still being debated[2,3]. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4[AMBRA1] (also known as CRL4[DCAF3]) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.},
}
@article {pmid33852864,
year = {2021},
author = {Studniarek, C and Tellier, M and Martin, PGP and Murphy, S and Kiss, T and Egloff, S},
title = {The 7SK/P-TEFb snRNP controls ultraviolet radiation-induced transcriptional reprogramming.},
journal = {Cell reports},
volume = {35},
number = {2},
pages = {108965},
doi = {10.1016/j.celrep.2021.108965},
pmid = {33852864},
issn = {2211-1247},
support = {G0400653/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/radiation effects ; Cell Survival ; Chromatin/chemistry/metabolism/radiation effects ; DNA Damage ; Gene Deletion ; Gene Expression Regulation ; Humans ; Leukocytes/cytology/metabolism/*radiation effects ; Positive Transcriptional Elongation Factor B/*genetics/metabolism ; Promoter Regions, Genetic ; Protein Binding ; RNA Polymerase II/*genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; Ribonucleoproteins, Small Nuclear/deficiency/*genetics ; Stress, Physiological/genetics ; Transcription Factors/genetics/metabolism ; Transcription, Genetic/*radiation effects ; Ultraviolet Rays ; },
abstract = {Conversion of promoter-proximally paused RNA polymerase II (RNAPII) into elongating polymerase by the positive transcription elongation factor b (P-TEFb) is a central regulatory step of mRNA synthesis. The activity of P-TEFb is controlled mainly by the 7SK small nuclear ribonucleoprotein (snRNP), which sequesters active P-TEFb into inactive 7SK/P-TEFb snRNP. Here we demonstrate that under normal culture conditions, the lack of 7SK snRNP has only minor impacts on global RNAPII transcription without detectable consequences on cell proliferation. However, upon ultraviolet (UV)-light-induced DNA damage, cells lacking 7SK have a defective transcriptional response and reduced viability. Both UV-induced release of "lesion-scanning" polymerases and activation of key early-responsive genes are compromised in the absence of 7SK. Proper induction of 7SK-dependent UV-responsive genes requires P-TEFb activity directly mobilized from the nucleoplasmic 7SK/P-TEFb snRNP. Our data demonstrate that the primary function of the 7SK/P-TEFb snRNP is to orchestrate the proper transcriptional response to stress.},
}
@article {pmid33852857,
year = {2021},
author = {Froehlich, JJ and Uyar, B and Herzog, M and Theil, K and Glažar, P and Akalin, A and Rajewsky, N},
title = {Parallel genetics of regulatory sequences using scalable genome editing in vivo.},
journal = {Cell reports},
volume = {35},
number = {2},
pages = {108988},
doi = {10.1016/j.celrep.2021.108988},
pmid = {33852857},
issn = {2211-1247},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/growth &amp; development/metabolism ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Gene Editing/methods ; Gene Expression Regulation, Developmental ; *Genetic Association Studies ; *Genome, Helminth ; Genotype ; *INDEL Mutation ; MicroRNAs/*genetics/metabolism ; Phenotype ; RNA, Guide/genetics/metabolism ; Regulatory Sequences, Nucleic Acid ; Transcription Factors/*genetics/metabolism ; },
abstract = {How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3' UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals.},
}
@article {pmid33850477,
year = {2021},
author = {Shi, B and Ding, J and Qi, J and Gu, Z},
title = {Characteristics and prognostic value of potential dependency genes in clear cell renal cell carcinoma based on a large-scale CRISPR-Cas9 and RNAi screening database DepMap.},
journal = {International journal of medical sciences},
volume = {18},
number = {9},
pages = {2063-2075},
pmid = {33850477},
issn = {1449-1907},
mesh = {Biomarkers, Tumor/*genetics ; CRISPR-Cas Systems/genetics ; Carcinoma, Renal Cell/*genetics/mortality/pathology ; Cell Line, Tumor ; Cohort Studies ; DNA Copy Number Variations ; DNA Methylation ; DNA Mutational Analysis/statistics &amp; numerical data ; Databases, Genetic/statistics &amp; numerical data ; Datasets as Topic ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Kidney/*pathology ; Kidney Neoplasms/*genetics/mortality/pathology ; Loss of Function Mutation ; Male ; Membrane Glycoproteins/genetics ; Molecular Chaperones/genetics ; Prognosis ; RNA Interference ; },
abstract = {BACKGROUND: Large-scale loss-of-function screening database such as Cancer Dependency Map (Depmap) provide abundant resources. Investigation of these potential dependency genes from human cancer cell lines in the real-world patients cohort would evaluate their prognostic value thus facilitate their clinical application and guide drug development.<br><br>METHODS: A few genes were selected from top clear cell renal cell carcinoma (ccRCC) lineage preferential dependency candidates from Depmap. Their characteristic including expression levels both in normal and tumor tissues and correlations with methylation or copy number, genetic alterations, functional enrichment, immune-associated interactions, prognostic value were evaluated in KIRC cohort from TCGA, GTEx, and multiple other open databases and platforms.<br><br>RESULTS: 16 genes were collected from 106 ccRCC preferential candidates and further analyzed including B4GALT4, BCL2L1, CDH2, COPG1, CRB3, FERMT2, GET4, GPX4, HNF1B, ITGAV, MDM2, NFE2L2, PAX8, RUVBL1, TFRC, and TNFSF10. The normalized gene effect scores of these genes varied from different ccRCC cell lines and principal component analysis (PCA) showed their tissue specificity expression profiles. Genetic alteration rates of them were low to moderate (0.7%-13%) in KIRC cohort. CDH2, MDM2, TNFSF10 showed a statistically significant higher level in tumors than normal tissues while PAX8 and FERMT2 were significantly downregulated. Moderate positive or negative correlations were observed in several genes between their expression and relative gene copy number or methylation levels, respectively. Based on the multivariable COX regression model adjusted by critical clinical variables revealed the expression of GET4 (p=0.002, HR=1.023 95%CI 1.009-1.038) and CRB3 (p<0.001, HR=0.969 95%CI 0.960-0.980) were independent predictive factors for overall survival in KIRC cohort.<br><br>CONCLUSIONS: A dependency gene validated in cell lines didn't directly represent its role in corresponding patients with same histological type and their prognostic value might be determined by multiple factors including dependency driven types, genetic alteration rates and expression levels. GET4 and CRB3 were the independent prognostic factors for ccRCC patients. CRB3 seemed like a potential broad tumor suppressor gene while GET4 might be a ccRCC preferential dependency gene with a ligandable structure.},
}
@article {pmid33848728,
year = {2021},
author = {Qiao, B and Xu, J and Yin, W and Xin, W and Ma, L and Qiao, J and Liu, Y},
title = {"Aptamer-locker" DNA coupling with CRISPR/Cas12a-guided biosensing for high-efficiency melamine analysis.},
journal = {Biosensors &amp; bioelectronics},
volume = {183},
number = {},
pages = {113233},
doi = {10.1016/j.bios.2021.113233},
pmid = {33848728},
issn = {1873-4235},
mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Humans ; Milk/chemistry ; Triazines/analysis ; },
abstract = {Herein, we report a method that combined "aptamer-locker" DNA with CRISPR/Cas12a-based biosensing for sensitive and rapid melamine analysis. Three strategies were harnessed for designing the DNA sensors that were well characterized by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC) in the absence and presence of melamine. The detection parameters were optimized to achieve good analytic performance. As a result, a limit of detection (LOD) as low as 38 nM was achieved, which is below the threshold (1.0 mg/kg) of allowable melamine in infant milk products. In addition, the sensors show high selectivity for melamine against other analogues such as cyanuric acid, ammeline and ammelide. Moreover, our method was effective for rapid melamine analysis in whole milk samples, with or without sample pretreatment, in less than 20 min. Adopting a commercially available portable fluorimeter, on-site analysis of melamine in milk was accomplished. The strategies demonstrated here can expand to detect other non-nucleic-acid targets by simply replacing the aptamers.},
}
@article {pmid33848270,
year = {2021},
author = {Escobar, H and Krause, A and Keiper, S and Kieshauer, J and Müthel, S and de Paredes, MG and Metzler, E and Kühn, R and Heyd, F and Spuler, S},
title = {Base editing repairs an SGCA mutation in human primary muscle stem cells.},
journal = {JCI insight},
volume = {6},
number = {10},
pages = {},
pmid = {33848270},
issn = {2379-3708},
mesh = {Adolescent ; Animals ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Child ; Female ; Gene Editing/*methods ; Heterografts ; Humans ; Male ; Mice ; Muscle Development/genetics ; Muscle, Skeletal/*cytology ; Muscular Dystrophies, Limb-Girdle/genetics/therapy ; Mutation/*genetics ; Myoblasts/*cytology/metabolism ; Sarcoglycans/*genetics/metabolism ; },
abstract = {Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.},
}
@article {pmid33847986,
year = {2021},
author = {Ramesh, A and Wheeldon, I},
title = {Guide RNA Design for Genome-Wide CRISPR Screens in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2307},
number = {},
pages = {123-137},
pmid = {33847986},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Library ; Genome, Fungal ; Metabolic Engineering ; RNA, Guide/*genetics ; Synthetic Biology ; Yarrowia/*genetics ; },
abstract = {Genome-wide functional genomic screens are essential to determining the genetic underpinning of a biological process. Novel and powerful tools for perturbing gene function, with the help of genetic and epigenetic information, have made it possible to systematically investigate the contribution of every gene to evolved and engineered phenotypes. Functional genomics and screening for enhanced phenotypes become ever more important when dealing with nonconventional hosts. Non-model organisms are valuable to metabolic engineering as they present a range of desirable phenotypes and can help in avoiding complex and intensive engineering of less suitable hosts that do not possess the desired phenotype(s). Domestication of such hosts however requires a suite of synthetic biology tools that allow for targeted genome engineering, regulation of gene expression, and genome-wide mutational screens. The widespread adoption of CRISPR-Cas9 and CRISPR-Cpf1 based systems has allowed for such screens in many organisms. Key considerations in any genome-wide CRISPR screen are the design of a set of unique guide RNAs targeting the required set of genes in the genome and the design of nontargeting guide RNAs that function as appropriate negative controls for the experiment. In this methods chapter, we present protocols for the design of guides for a CRISPR screen, targeting every gene in the genome of the industrially relevant oleaginous yeast Yarrowia lipolytica. The first set of protocols describes the algorithm for the design of genome targeting and nontargeting guides for a genome-wide CRISPR-Cpf1 screen. The second set of protocols describes modifications to the first for the design of guides for a CRISPR-Cas9 screen. The strategies described here should serve as an efficient guide to design a library of gRNAs for most genome-wide CRISPR screens.},
}
@article {pmid33847985,
year = {2021},
author = {Yang, Z and Xu, P},
title = {Implementing CRISPR-Cas12a for Efficient Genome Editing in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2307},
number = {},
pages = {111-121},
pmid = {33847985},
issn = {1940-6029},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; Endodeoxyribonucleases/*metabolism ; Gene Editing/*methods ; Genome, Fungal ; Metabolic Engineering ; Plasmids/genetics ; Yarrowia/*genetics ; },
abstract = {The oleaginous yeast Yarrowia lipolytica has emerged as an industrially relevant chassis to produce various valuable chemicals. Metabolic engineering of Y. lipolytica relies on the availability of genetic engineering tools. Existing engineering strategies for this yeast include homologous recombination, random integration, and episomal plasmid-based gene expression. CRISPR-Cas9 based genome-editing toolbox has also been developed to facilitate multiplexed gene disruption and regulation. Alternative to Cas9, the CRISPR effector Cas12a has also been adopted to perform genome engineering in multiple species. Due to its distinctive features such as short and simple crRNA structure, the ability to process its own crRNA and T-rich PAM sequence (TTTN), Cas12a holds promising potential to be developed as an efficient genome-editing tool. In this chapter, we describe the protocol to implement multiplexed genome editing in Y. lipolytica. The delivery of AsCas12a and crRNA expression via a single plasmid was described. CRISPR-Cas12a-based genome editing could expand the genetic toolbox of Y. lipolytica, whihc is complementary to the classical Cas9-based tools.},
}
@article {pmid33847984,
year = {2021},
author = {Misa, J and Schwartz, C},
title = {CRISPR Interference and Activation to Modulate Transcription in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2307},
number = {},
pages = {95-109},
pmid = {33847984},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Fungal Proteins/*genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; RNA, Guide/genetics ; Transcriptional Activation ; Yarrowia/*genetics ; },
abstract = {Recent developments in RNA-guided nuclease technologies have advanced the engineering of a wide range of organisms, including the nonconventional yeast Yarrowia lipolytica. Y. lipolytica has been the focus of a range of synthetic biology and metabolic engineering studies due to its high capacity to synthesize and accumulate intracellular lipids. The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully adapted and used for genome editing in Y. lipolytica. However, as engineered strains are moved closer to industrialization, the need for finer control of transcription is still present. To overcome this challenge, we have developed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems to allow modulating the transcription of endogenous genes. We begin this protocol chapter by describing how to use the CRISPRi system to repress expression of any gene in Y. lipolytica. A second method describes how to use the CRISPRa system to increase expression of native Y. lipolytica genes. Finally, we describe how CRISPRi or CRISPRa vectors can be combined to enable multiplexed activation or repression of more than one gene. The implementation of CRISPRi and CRISPRa systems improves our ability to control gene expression in Y. lipolytica and promises to enable more advanced synthetic biology and metabolic engineering studies in this host.},
}
@article {pmid33847983,
year = {2021},
author = {Spagnuolo, M and Blenner, M},
title = {Gene Excision by Dual-Guide CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2307},
number = {},
pages = {85-94},
pmid = {33847983},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genes, Fungal ; Metabolic Engineering ; RNA, Guide/*genetics ; Synthetic Biology ; Yarrowia/*genetics ; },
abstract = {CRISPR-Cas9 is frequently used for creating double-strand DNA breaks that result in indels through non-homologous end joining. Indels can revert to wild-type sequence and require sequencing or complex assays to measure. Cutting by two guide RNAs can lead to single indels at either cut site or simultaneous cutting at both sites and repair leading to gene excision.},
}
@article {pmid33847982,
year = {2021},
author = {Spagnuolo, M and Blenner, M},
title = {Simultaneous Gene Excision and Integration by Dual-Guide CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2307},
number = {},
pages = {69-83},
pmid = {33847982},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; INDEL Mutation ; Metabolic Engineering ; RNA, Guide/*genetics ; Synthetic Biology ; Yarrowia/*genetics ; },
abstract = {Metabolic engineering frequently requires both gene knockouts and gene integration. CRISPR-Cas9 has been extensively used to create double-stranded DNA breaks that result in indel mutations; however, such mutations can revert or create toxic product. Gene integration can also be accomplished by CRISPR-Cas9 introduced double-stranded DNA breaks and a donor DNA cassette. Here we describe our protocol for combining an efficient gene knockout created by introducing DNA cuts with two guide RNAs with a gene to be integrated at the knockout site. Including guide RNA target sites flanking the homology regions around the gene to be integrated enables both homology-directed repair and homology-mediated end joining, resulting in few deletions and a significant proportion of correctly knocked out and integrated genes.},
}
@article {pmid33847981,
year = {2021},
author = {Dahlin, J and Holkenbrink, C and Borodina, I},
title = {EasyCloneYALI: Toolbox for CRISPR-Mediated Integrations and Deletions in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2307},
number = {},
pages = {41-68},
pmid = {33847981},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome, Fungal ; INDEL Mutation ; Yarrowia/*genetics ; },
abstract = {In order to unlock the full potential of Yarrowia lipolytica, as model organism and production host, simple and reliable tools for genome engineering are essential. In this chapter, the practical details of working with the EasyCloneYALI Toolbox are described.Highlights of the EasyCloneYALI Toolbox are high genome editing efficiencies, multiplexed Cas9-mediated knockouts, targeted genomic integrations into characterized intergenic loci, as well as streamlined and convenient cloning for both marker-based and marker-free integrative expression vectors.},
}
@article {pmid33847907,
year = {2021},
author = {Riva, A and Striano, P},
title = {Reversing Accumulation of Polyglucosan Bodies by Virally Delivered CRISPR/Cas9 Genome Editing.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {18},
number = {2},
pages = {866-867},
pmid = {33847907},
issn = {1878-7479},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Glucans ; },
}
@article {pmid33847177,
year = {2022},
author = {Hart-Johnson, S and Mankelow, K},
title = {Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals.},
journal = {Laboratory animals},
volume = {56},
number = {1},
pages = {26-34},
doi = {10.1177/00236772211007306},
pmid = {33847177},
issn = {1758-1117},
mesh = {Animals ; *Cryopreservation/methods ; Fertilization in Vitro/methods ; Mice ; Rats ; *Zebrafish ; },
abstract = {With the ever-expanding numbers of genetically altered (GA) animals created in this new age of CRISPR/Cas, tools for helping the management of this vast and valuable resource are essential. Cryopreservation of embryos and germplasm of GA animals has been a widely used tool for many years now, allowing for the archiving, distribution and colony management of stock. However, each year brings an array of advances, improving survival rates of embryos, success rates of in-vitro fertilisation and the ability to better share lines and refine the methods to preserve them. This article will focus on the mouse field, referencing the latest developments and assessing their efficacy and ease of implementation, with a brief note on other common genetically altered species (rat, zebrafish, Xenopus, avian species and non-human Primates).},
}
@article {pmid33846956,
year = {2021},
author = {Schaart, JG and van de Wiel, CCM and Smulders, MJM},
title = {Genome editing of polyploid crops: prospects, achievements and bottlenecks.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {337-351},
pmid = {33846956},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; *Genome, Plant ; Plant Breeding/*methods ; Plants, Genetically Modified/*genetics ; *Polyploidy ; },
abstract = {Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.},
}
@article {pmid33846767,
year = {2021},
author = {Falzone, L and Gattuso, G and Tsatsakis, A and Spandidos, DA and Libra, M},
title = {Current and innovative methods for the diagnosis of COVID‑19 infection (Review).},
journal = {International journal of molecular medicine},
volume = {47},
number = {6},
pages = {},
pmid = {33846767},
issn = {1791-244X},
mesh = {Animals ; Biosensing Techniques/methods ; COVID-19/*diagnosis ; COVID-19 Testing/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Inventions ; Microscopy, Electron/methods ; Nucleic Acid Amplification Techniques/methods ; SARS-CoV-2/genetics/isolation &amp; purification ; Virus Cultivation/methods ; },
abstract = {The Coronavirus Disease 2019 (COVID‑19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID‑19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT‑PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly interspaced short palindromic repeats/Cas (CRISPR/Cas)‑based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID‑19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID‑19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.},
}
@article {pmid33846636,
year = {2021},
author = {Leibowitz, ML and Papathanasiou, S and Doerfler, PA and Blaine, LJ and Sun, L and Yao, Y and Zhang, CZ and Weiss, MJ and Pellman, D},
title = {Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.},
journal = {Nature genetics},
volume = {53},
number = {6},
pages = {895-905},
pmid = {33846636},
issn = {1546-1718},
support = {F32 DK118822/DK/NIDDK NIH HHS/United States ; 2020154/DDCF_/Doris Duke Charitable Foundation/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; R01 CA213404/CA/NCI NIH HHS/United States ; K22 CA216319/CA/NCI NIH HHS/United States ; 2017093/DDCF_/Doris Duke Charitable Foundation/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {Anemia, Sickle Cell/genetics ; Antigens, CD34/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Division ; Chromosomes, Human/genetics ; *Chromothripsis ; DNA Cleavage ; *Gene Editing ; Genome, Human ; Humans ; Micronucleus, Germline/genetics ; Tumor Suppressor Protein p53/metabolism ; },
abstract = {Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.},
}
@article {pmid33846634,
year = {2021},
author = {Inoue, D and Polaski, JT and Taylor, J and Castel, P and Chen, S and Kobayashi, S and Hogg, SJ and Hayashi, Y and Pineda, JMB and El Marabti, E and Erickson, C and Knorr, K and Fukumoto, M and Yamazaki, H and Tanaka, A and Fukui, C and Lu, SX and Durham, BH and Liu, B and Wang, E and Mehta, S and Zakheim, D and Garippa, R and Penson, A and Chew, GL and McCormick, F and Bradley, RK and Abdel-Wahab, O},
title = {Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition.},
journal = {Nature genetics},
volume = {53},
number = {5},
pages = {707-718},
pmid = {33846634},
issn = {1546-1718},
support = {K08 CA230319/CA/NCI NIH HHS/United States ; R35 CA197709/CA/NCI NIH HHS/United States ; R01 HL128239/HL/NHLBI NIH HHS/United States ; R01 HL151651/HL/NHLBI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA251138/CA/NCI NIH HHS/United States ; T32 CA009657/CA/NCI NIH HHS/United States ; R01 DK103854/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Self Renewal ; Cell Transformation, Neoplastic/pathology ; Clone Cells ; Female ; *Genetic Predisposition to Disease ; Genome, Human ; Hematologic Diseases/*genetics/pathology ; Hematopoietic Stem Cells/metabolism ; Humans ; Introns/*genetics ; Male ; Mice, Knockout ; Neoplasms/*genetics ; Noonan Syndrome/genetics ; Pedigree ; RNA/metabolism ; RNA Splicing/genetics ; Ribonucleoproteins/genetics/metabolism ; Spleen/pathology ; Transcription Factors/genetics ; },
abstract = {Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.},
}
@article {pmid33846619,
year = {2021},
author = {Weiss, RJ and Spahn, PN and Chiang, AWT and Liu, Q and Li, J and Hamill, KM and Rother, S and Clausen, TM and Hoeksema, MA and Timm, BM and Godula, K and Glass, CK and Tor, Y and Gordts, PLSM and Lewis, NE and Esko, JD},
title = {Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.},
journal = {Nature chemical biology},
volume = {17},
number = {6},
pages = {684-692},
pmid = {33846619},
issn = {1552-4469},
support = {R01 GM033063/GM/NIGMS NIH HHS/United States ; P30 CA023100/CA/NCI NIH HHS/United States ; R35 GM119850/GM/NIGMS NIH HHS/United States ; K12 HL141956/HL/NHLBI NIH HHS/United States ; T32 GM008326/GM/NIGMS NIH HHS/United States ; DP2 HD087954/HD/NICHD NIH HHS/United States ; R37 GM033063/GM/NIGMS NIH HHS/United States ; R21 CA199292/CA/NCI NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Drug Discovery ; Extracellular Matrix/genetics ; F-Box Proteins/*antagonists &amp; inhibitors ; *Genome-Wide Association Study ; Heparitin Sulfate/*metabolism ; High-Throughput Screening Assays ; Humans ; Jumonji Domain-Containing Histone Demethylases/*antagonists &amp; inhibitors ; Protein Binding/genetics ; RNA-Seq ; Sulfotransferases/antagonists &amp; inhibitors ; },
abstract = {Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.},
}
@article {pmid33846535,
year = {2021},
author = {Young, AN and Perlas, E and Ruiz-Blanes, N and Hierholzer, A and Pomella, N and Martin-Martin, B and Liverziani, A and Jachowicz, JW and Giannakouros, T and Cerase, A},
title = {Deletion of LBR N-terminal domains recapitulates Pelger-Huet anomaly phenotypes in mouse without disrupting X chromosome inactivation.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {478},
pmid = {33846535},
issn = {2399-3642},
mesh = {Animals ; Mice ; Mice, Knockout ; Pelger-Huet Anomaly/*genetics ; Phenotype ; Receptors, Cytoplasmic and Nuclear/*genetics/metabolism ; X Chromosome Inactivation/*genetics ; },
abstract = {Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia (GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes, hindering efforts to understand the molecular etiology of this disorder. Here we show, using CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr gene, generating a protein missing the N-terminal domains of LBR, presents a superior model of human PHA. Further, we address recent reports of a link between Lbr and defects in X chromosome inactivation (XCI) and show that our mouse mutant displays minor X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We suggest that our N-terminal deletion model provides a valuable pre-clinical tool to the research community and will aid in further understanding the etiology of PHA and the diverse functions of LBR.},
}
@article {pmid33846411,
year = {2021},
author = {Pastor-Arroyo, EM and Rodriguez, JMM and Pellegrini, G and Bettoni, C and Levi, M and Hernando, N and Wagner, CA},
title = {Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7943},
pmid = {33846411},
issn = {2045-2322},
mesh = {Animals ; Animals, Newborn ; Body Weight ; Calcium/blood/urine ; Creatinine/urine ; Embryo, Mammalian/pathology ; Feces/chemistry ; Female ; Fibroblast Growth Factor-23 ; Fibroblast Growth Factors/blood ; Heterozygote ; Homozygote ; Male ; Organ Specificity ; Phosphates/blood/metabolism/urine ; Rats ; Sodium-Phosphate Cotransporter Proteins, Type IIb/*metabolism ; Survival Analysis ; },
abstract = {Absorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na[+]/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.},
}
@article {pmid33845891,
year = {2021},
author = {Chen, M and Shi, H and Gou, S and Wang, X and Li, L and Jin, Q and Wu, H and Zhang, H and Li, Y and Wang, L and Li, H and Lin, J and Guo, W and Jiang, Z and Yang, X and Xu, A and Zhu, Y and Zhang, C and Lai, L and Li, X},
title = {In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers.},
journal = {Genome medicine},
volume = {13},
number = {1},
pages = {57},
pmid = {33845891},
issn = {1756-994X},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Child, Preschool ; Disease Models, Animal ; Dystrophin/chemistry/*genetics ; *Gene Editing ; Genome ; HEK293 Cells ; Humans ; Male ; Mice ; Muscle Fibers, Skeletal/*pathology ; Muscular Dystrophy, Duchenne/*genetics ; Mutation/genetics ; Transcriptome/genetics ; },
abstract = {BACKGROUND: Mutations in the DMD gene encoding dystrophin-a critical structural element in muscle cells-cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD.<br><br>METHODS: In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46-54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors.<br><br>RESULTS: Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member &#x3b2;-dystroglycan.<br><br>CONCLUSIONS: We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.},
}
@article {pmid33845443,
year = {2021},
author = {Uygun, ZO and Atay, S},
title = {Label-free highly sensitive detection of DNA approximate length and concentration by impedimetric CRISPR-dCas9 based biosensor technology.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {140},
number = {},
pages = {107812},
doi = {10.1016/j.bioelechem.2021.107812},
pmid = {33845443},
issn = {1878-562X},
mesh = {Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; DNA/*analysis/*genetics ; Dielectric Spectroscopy ; Humans ; *Limit of Detection ; },
abstract = {In this study, we designed a CRISPR-dCas9-based biosensor with potential clinical use in glioblastoma subtype discrimination through detection of isocitrate dehydrogenase R132H (IDH) mutation status. The electrode was modified to detect mutant DNA cysteamine (Cys), PAMAM, dCas9 and sgRNA for R132H mutations, respectively. The biosensor system we proposed was able not only to detect mutant DNA, but also to measure the approximate length of DNA. Therefore, it can be considered that the biosensor technology that we developed is novel in the field of DNA biosensors. Another superior capability of the biosensor system is that it can simultaneously measure DNA concentration by electrochemical impedance spectroscopy and DNA length by capacitive detection, which lowers the concentration-based false-positive signals. The calibration range was obtained between 100 fM and 1000 fM, LOD and LOQ were also calculated as 33.96 fM and 102.91 fM respectively. Moreover, thanks to the sensitivity of the capacitive detection, the biosensor was able to discriminate the same EIS signals of the 200 bp and 250 fM concentration data and 1000 bp and 50 fM concentration data. In conclusion, the biosensor was capable of detect target DNA and DNA length, simultaneously in minutes.},
}
@article {pmid33845135,
year = {2021},
author = {Huan, P and Cui, M and Wang, Q and Liu, B},
title = {CRISPR/Cas9-mediated mutagenesis reveals the roles of calaxin in gastropod larval cilia.},
journal = {Gene},
volume = {787},
number = {},
pages = {145640},
doi = {10.1016/j.gene.2021.145640},
pmid = {33845135},
issn = {1879-0038},
mesh = {Animals ; *CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics/physiology ; Cilia/genetics/*physiology ; Gastropoda/genetics/*physiology ; Gene Knockout Techniques ; Membrane Proteins/genetics/*physiology ; Mutagenesis ; Polymorphism, Single Nucleotide ; },
abstract = {Obtaining detectable knockout phenotypes in the G0 generation is essential for gene function studies. Although CRISPR/Cas9-mediated gene editing has been employed to knock out molluscan genes, detectable phenotypes in the G0 generation have not been reported in these animals. In this study, we determined the knockout phenotype of a cilium-related gene, calaxin, using CRISPR/Cas9 technology in the gastropod mollusk Lottia goshimai. Injections with the Cas9-sgRNA complex caused approximately 30-80% of the injected larvae to exhibit a short-cilia phenotype characteristic of shortened cilia and decreased motility in the larvae. This phenotype was detectable in the G0 generation and was consistent for two independent sgRNAs. Genotyping of the injected larvae revealed various types of deletions and insertions in the target gene, which occurred in all sequences from the short-cilia larvae. This result indicated that the short-cilia phenotype was indeed caused by calaxin knockout. This possibility was supported by an RNAi assay targeting calaxin, which produced a highly similar short-cilia phenotype. We observed that a single SNP in the target sequences of the sgRNAs could show varied effects on the efficiency of mutagenesis. These results help to establish a foundation for future studies on molluscan gene editing using the CRISPR/Cas9 technique and contribute to the body of knowledge on molluscan ciliary functions.},
}
@article {pmid33844549,
year = {2021},
author = {Marsic, T and Ali, Z and Tehseen, M and Mahas, A and Hamdan, S and Mahfouz, M},
title = {Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2.},
journal = {Nano letters},
volume = {21},
number = {8},
pages = {3596-3603},
pmid = {33844549},
issn = {1530-6992},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; *RNA, Viral ; Reverse Transcription ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.},
}
@article {pmid33843934,
year = {2021},
author = {Klobasa, W and Chu, FC and Huot, O and Grubbs, N and Rotenberg, D and Whitfield, AE and Lorenzen, MD},
title = {Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {169},
pages = {},
doi = {10.3791/62417},
pmid = {33843934},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Endonucleases/genetics ; Female ; Gene Editing/*methods ; Zea mays/*chemistry ; },
abstract = {The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.},
}
@article {pmid33843197,
year = {2021},
author = {Durmusoglu, D and Al'Abri, IS and Collins, SP and Cheng, J and Eroglu, A and Beisel, CL and Crook, N},
title = {In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii.},
journal = {ACS synthetic biology},
volume = {10},
number = {5},
pages = {1039-1052},
doi = {10.1021/acssynbio.0c00562},
pmid = {33843197},
issn = {2161-5063},
support = {P30 DK034987/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*metabolism ; CRISPR-Cas Systems ; Feces/chemistry ; Female ; Gastrointestinal Microbiome ; Gastrointestinal Tract/*metabolism/*microbiology ; Gene Editing/methods ; Gene Expression ; Indoles/*metabolism ; Male ; Metabolic Engineering/*methods ; Mice ; Mice, Inbred C57BL ; Microorganisms, Genetically-Modified ; Multigene Family ; Plasmids/genetics ; Probiotics/*metabolism ; Promoter Regions, Genetic ; Provitamins/*biosynthesis ; Saccharomyces boulardii/genetics/*metabolism ; Saccharomyces cerevisiae/genetics ; beta Carotene/*biosynthesis ; },
abstract = {Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (β-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled β-carotene synthesis (194 μg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional β-carotene recovered in feces was 56-fold higher than the β-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.},
}
@article {pmid33843019,
year = {2021},
author = {Men, H and Hankins, MA and Bock, AS and Beaton, BP and Davis, DJ and Chesney, KL and Bryda, EC},
title = {Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {32},
number = {3},
pages = {173-182},
pmid = {33843019},
issn = {1432-1777},
support = {P40 OD011062/OD/NIH HHS/United States ; T32 OD011126/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Genetic Predisposition to Disease ; Genotype ; Humans ; Inflammatory Bowel Diseases/*genetics/pathology ; Mutation/genetics ; Nod2 Signaling Adaptor Protein/*genetics ; Polymorphism, Single Nucleotide/genetics ; Rats ; Receptors, Interleukin/*genetics ; Vesicular Transport Proteins/genetics ; },
abstract = {Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.},
}
@article {pmid33842501,
year = {2021},
author = {Yeh, KB and Scullion, M and Michelotti, JM and Olinger, G},
title = {First Movers in Molecular Detection: Case Comparison on Harnessing Research and Development, Industry, and Entrepreneurship.},
journal = {Frontiers in medicine},
volume = {8},
number = {},
pages = {639440},
pmid = {33842501},
issn = {2296-858X},
abstract = {The current unprecedented COVID-19 pandemic underscores the importance of diagnostic assays in health security preparedness and readiness. Advancing new technologies for rapid molecular detection of high consequence infectious pathogens is an ongoing challenge that requires ingenuity and vision. Sustainment of a robust supply chain for materials and the logistics of timely product delivery further challenge diagnostic kit and device manufacturers. Business economists often characterize technology companies that discover unique breakthroughs in their field and are first to bring related products to market as first movers. From a market perspective, three first mover characteristics include: having the knowledge and capability to address a unique breakthrough, excellent technological leadership, and the ability to capitalize on the opportunity. Current mainstays for molecular detection include using Taq DNA Polymerase enzyme and fluorescent chemistry for quantitative PCR (qPCR). A newer and promising technology uses CRISPR-Cas proteins for nucleic acid detection. Our panel discussion from the 2020 ASM Biothreats conference, which included members from two prototypical first mover companies, explored their respective corporate experiences. Both companies were selected for the discussion based on their revolutionary innovations and similarities in their research and development, corporate culture and trajectory. One company, established over 20 years ago, became a market leader in the biothreat detection market by advancing air thermocycling qPCR across multiple product families. The second company is a rapidly growing start-up and a scientific pioneer in establishing next generation CRISPR technologies. Here we discuss their technology development, product deployment, and customer markets to draw lessons learned for researchers, end users, and funders.},
}
@article {pmid33842017,
year = {2021},
author = {Zhang, D and Zhang, Z and Unver, T and Zhang, B},
title = {CRISPR/Cas: A powerful tool for gene function study and crop improvement.},
journal = {Journal of advanced research},
volume = {29},
number = {},
pages = {207-221},
pmid = {33842017},
issn = {2090-1224},
mesh = {Agriculture/methods ; Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Crops, Agricultural/*genetics/growth &amp; development ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Targeting/methods ; Genome, Plant ; Humans ; Phenotype ; Plant Breeding/methods ; Plant Development ; Plants, Genetically Modified ; Stress, Physiological ; },
abstract = {BACKGROUND: It is a long-standing goal of scientists and breeders to precisely control a gene for studying its function as well as improving crop yield, quality, and tolerance to various environmental stresses. The discovery and modification of CRISPR/Cas system, a nature-occurred gene editing tool, opens an era for studying gene function and precision crop breeding.<br><br>AIM OF REVIEW: In this review, we first introduce the brief history of CRISPR/Cas discovery followed the mechanism and application of CRISPR/Cas system on gene function study and crop improvement. Currently, CRISPR/Cas genome editing has been becoming a mature cutting-edge biotechnological tool for crop improvement that already used in many different traits in crops, including pathogen resistance, abiotic tolerance, plant development and morphology and even secondary metabolism and fiber development. Finally, we point out the major issues associating with CRISPR/Cas system and the future research directions.Key Scientific Concepts of Review: CRISPR/Cas9 system is a robust and powerful biotechnological tool for targeting an individual DNA and RNA sequence in the genome. It can be used to target a sequence for gene knockin, knockout and replacement as well as monitoring and regulating gene expression at the genome and epigenome levels by binding a specific sequence. Agrobacterium-mediated method is still the major and efficient method for delivering CRISPR/Cas regents into targeted plant cells. However, other delivery methods, such as virus-mediated method, have been developed and enhanced the application potentials of CRISPR/Cas9-based crop improvement. PAM requirement offers the CRISPR/Cas9-targted genetic loci and also limits the application of CRISPR/Cas9. Discovering new Cas proteins and modifying current Cas enzymes play an important role in CRISPR/Cas9-based genome editing. Developing a better CRISPR/Cas9 system, including the delivery system and the methods eliminating off-target effects, and finding key/master genes for controlling crop growth and development is two major directions for CRISPR/Cas9-based crop improvement.},
}
@article {pmid33841487,
year = {2021},
author = {Kumar, S and Rymarquis, LA and Ezura, H and Nekrasov, V},
title = {Editorial: CRISPR-Cas in Agriculture: Opportunities and Challenges.},
journal = {Frontiers in plant science},
volume = {12},
number = {},
pages = {672329},
pmid = {33841487},
issn = {1664-462X},
}
@article {pmid33840678,
year = {2021},
author = {Tanihara, F and Hirata, M and Otoi, T},
title = {Current status of the application of gene editing in pigs.},
journal = {The Journal of reproduction and development},
volume = {67},
number = {3},
pages = {177-187},
pmid = {33840678},
issn = {1348-4400},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Gene Editing/*veterinary ; Genetic Engineering ; Swine ; },
abstract = {Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.},
}
@article {pmid33839534,
year = {2021},
author = {Niu, C and Wang, C and Li, F and Zheng, X and Xing, X and Zhang, C},
title = {Aptamer assisted CRISPR-Cas12a strategy for small molecule diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {183},
number = {},
pages = {113196},
doi = {10.1016/j.bios.2021.113196},
pmid = {33839534},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded ; Oligonucleotides ; },
abstract = {Molecular diagnostics are vital for the identification, prevention, and treatment of numerous diseases and are of particular demand in point-of-care (POC) settings. Nevertheless, most reported biosensors based on the CRISPR-Cas system have focused on nucleic-acid targets. Here, we report a versatile diagnostic strategy for small molecules called Molecular Radar (Random Molecular Aptamer-Dependent CRISPR-Assist Reporter), The workflow is simple, convenient, and rapid (conducted at 37 °C in under 25 min), indicating the substantial potential of the proposed assay could be adapted into a biosensor for POC settings and on-site molecular diagnostics. This strategy is based on the CRISPR Cas12a-assisted fluorescence reporter system that consists of Cas12a, CRISPR RNA (crRNA), a single-stranded DNA (ssDNA) probe labeled with a fluorophore at the 5' end and a quencher at the 3' end (F-Q probe), and a single-stranded DNA aptamer for the target molecule. In the presence of a target molecule, the aptamer binds to this small molecule with high specificity and affinity, resulting in a decrease of aptamer hybridized to the crRNA-Cas12a duplex. This decrease in activated Cas12a leads to a significant reduction in fluorescence signal. In this study, adenosine-5'-triphosphate (ATP) was selected as model target molecule and an ATP detect method was developed with high specificity and sensitivity with a linear range from 25 to 500 μM and a detection limit of 104 nM. Moreover, the particular characteristics of CRISPR-Cas12a that we report here for the first time have enriched our understanding of Cas12a and provided guidance for further research on CRISPR-Cas12a-based biosensors.},
}
@article {pmid33839407,
year = {2021},
author = {Watanabe, M and Takahashi, Y and Hiura, K and Nakano, K and Okamura, T and Sasaki, H and Sasaki, N},
title = {A single amino acid substitution in PRKDC is a determinant of sensitivity to Adriamycin-induced renal injury in mouse.},
journal = {Biochemical and biophysical research communications},
volume = {556},
number = {},
pages = {121-126},
doi = {10.1016/j.bbrc.2021.03.150},
pmid = {33839407},
issn = {1090-2104},
mesh = {Albuminuria/chemically induced/complications ; *Amino Acid Substitution ; Animals ; Base Sequence ; Biomarkers ; CRISPR-Cas Systems ; DNA-Activated Protein Kinase/*genetics/metabolism ; DNA-Binding Proteins/*genetics/metabolism ; *Disease Models, Animal ; Doxorubicin/*pharmacology ; Female ; Kidney Diseases/*chemically induced/complications/metabolism/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mutation ; Renal Insufficiency/chemically induced/complications/metabolism/pathology ; },
abstract = {Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-Prkdc[R2140C], that possess the R2140C mutation. After administration of ADR, B6-Prkdc[R2140C] mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-Prkdc[R2140C] and BALB/c. These findings demonstrated that B6-Prkdc[R2140C] show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-Prkdc[R2140C].},
}
@article {pmid33839288,
year = {2022},
author = {Kostyusheva, A and Brezgin, S and Babin, Y and Vasilyeva, I and Glebe, D and Kostyushev, D and Chulanov, V},
title = {CRISPR-Cas systems for diagnosing infectious diseases.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {431-446},
pmid = {33839288},
issn = {1095-9130},
mesh = {*African Swine Fever Virus ; Animals ; *COVID-19/diagnosis/epidemiology ; CRISPR-Cas Systems/genetics ; *Communicable Diseases/diagnosis/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Swine ; },
abstract = {Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.},
}
@article {pmid33838632,
year = {2021},
author = {Isaev, AB and Musharova, OS and Severinov, KV},
title = {Microbial Arsenal of Antiviral Defenses - Part I.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {3},
pages = {319-337},
doi = {10.1134/S0006297921030081},
pmid = {33838632},
issn = {1608-3040},
mesh = {Archaea/genetics/metabolism/physiology/*virology ; Bacteria/genetics/metabolism/*virology ; Bacterial Physiological Phenomena ; *Bacteriophages ; CRISPR-Cas Systems ; Host Microbial Interactions ; },
abstract = {Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). Constant threat of phage infection is a major force that shapes evolution of the microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering have been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection, with a focus on novel systems discovered in recent years. First chapter covers defense associated with cell surface, role of small molecules, and innate immunity systems relying on DNA modification.},
}
@article {pmid33838271,
year = {2021},
author = {Lindner, L and Cayrou, P and Rosahl, TW and Zhou, HH and Birling, MC and Herault, Y and Pavlovic, G},
title = {Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents.},
journal = {Methods (San Diego, Calif.)},
volume = {191},
number = {},
pages = {107-119},
doi = {10.1016/j.ymeth.2021.04.001},
pmid = {33838271},
issn = {1095-9130},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; DNA ; Genomics ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Rodentia/genetics ; },
abstract = {Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. In-depth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knock-out, FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knock-out lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreER[T2] deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.},
}
@article {pmid33838111,
year = {2021},
author = {Nuñez, JK and Chen, J and Pommier, GC and Cogan, JZ and Replogle, JM and Adriaens, C and Ramadoss, GN and Shi, Q and Hung, KL and Samelson, AJ and Pogson, AN and Kim, JYS and Chung, A and Leonetti, MD and Chang, HY and Kampmann, M and Bernstein, BE and Hovestadt, V and Gilbert, LA and Weissman, JS},
title = {Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2503-2519.e17},
pmid = {33838111},
issn = {1097-4172},
support = {RM1 HG007735/HG/NHGRI NIH HHS/United States ; DP1 CA216873/CA/NCI NIH HHS/United States ; F32 AG063487/AG/NIA NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R00 GM134154/GM/NIGMS NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; F31 NS115380/NS/NINDS NIH HHS/United States ; K99 GM134154/GM/NIGMS NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Differentiation ; *Cellular Reprogramming ; CpG Islands ; DNA Methylation ; *Epigenesis, Genetic ; *Epigenome ; *Gene Editing ; Gene Silencing ; Histone Code ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Neurons/*cytology/metabolism ; Protein Processing, Post-Translational ; },
abstract = {A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.},
}
@article {pmid33837633,
year = {2021},
author = {Chen, SP and Liu, ZX and Chen, YT and Wang, Y and Chen, JZ and Fu, S and Ma, WF and Xia, S and Liu, D and Wu, T and Yang, G},
title = {CRISPR/Cas9-mediated knockout of LW-opsin reduces the efficiency of phototaxis in the diamondback moth Plutella xylostella.},
journal = {Pest management science},
volume = {77},
number = {7},
pages = {3519-3528},
doi = {10.1002/ps.6405},
pmid = {33837633},
issn = {1526-4998},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Knockout Techniques ; Male ; *Moths/genetics ; *Opsins/genetics ; *Phototaxis ; },
abstract = {BACKGROUND: Opsins are crucial for animal vision. The identity and function of opsins in Plutella xylostella remain unknown. The aim of the research is to confirm which opsin gene(s) contribute to phototaxis of P. xylostella.<br><br>RESULTS: LW-opsin, BL-opsin and UV-opsin, were identified in the P. xylostella genome. LW-opsin was more highly expressed than the other two opsin genes, and all three genes were specifically expressed in the head. Three P. xylostella strains, LW-13 with a 13-bp deletion in LW-opsin, BL&#x2009;+&#xa0;2 with a 2-bp insertion in BL-opsin, and UV-29 with a 5-bp insertion and a 34-bp deletion in UV-opsin, were established from the strain G88 using the CRISPR/Cas9 system. Among the three opsin-knockout strains, only male and female LW-13 exhibited weaker phototaxis to lights of different wavelengths and white light than G88 at 2.5&#xa0;lx due to defective locomotion, and LW-13 was defective to sense white, green and infrared lights. The locomotion of LW-13 was reduced compared with G88 at 2.5, 10, 20, 60, 80, 100, and 200&#x2009;lx under the green light, but the locomotion of LW-13 female was recovered at 80, 100 and 200&#x2009;lx. The defective phototaxis to the green light of male LW-13 was not affected by light intensity, while the defective phototaxis to the green light of female LW-13 was recovered at 10, 20, 60, 80, 100, and 200&#x2009;lx.<br><br>CONCLUSION: LW-opsin is involved in light sensing and locomotion of P. xylostella, providing a potential target gene for controlling the pest. &#xa9; 2021 Society of Chemical Industry.},
}
@article {pmid33837536,
year = {2021},
author = {Jain, M and Garg, R},
title = {Enhancers as potential targets for engineering salinity stress tolerance in crop plants.},
journal = {Physiologia plantarum},
volume = {173},
number = {4},
pages = {1382-1391},
doi = {10.1111/ppl.13421},
pmid = {33837536},
issn = {1399-3054},
mesh = {*Chromatin ; Crops, Agricultural/genetics/*physiology ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; Promoter Regions, Genetic ; Salinity ; *Salt Tolerance ; },
abstract = {Enhancers represent noncoding regulatory regions of the genome located distantly from their target genes. They regulate gene expression programs in a context-specific manner via interacting with promoters of one or more target genes and are generally associated with transcription factor binding sites and epi(genomic)/chromatin features, such as regions of chromatin accessibility and histone modifications. The enhancers are difficult to identify due to the modularity of their associated features. Although enhancers have been studied extensively in human and animals, only a handful of them has been identified in few plant species till date due to nonavailability of plant-specific experimental and computational approaches for their discovery. Being an important regulatory component of the genome, enhancers represent potential targets for engineering agronomic traits, including salinity stress tolerance in plants. Here, we provide a review of the available experimental and computational approaches along with the associated sequence and chromatin/epigenetic features for the discovery of enhancers in plants. In addition, we provide insights into the challenges and future prospects of enhancer research in plant biology with emphasis on potential applications in engineering salinity stress tolerance in crop plants.},
}
@article {pmid33837440,
year = {2021},
author = {Ou, L and Long, J and Teng, Y and Yang, H and Xi, Y and Duan, G and Chen, S},
title = {Diversity of the type I-U CRISPR-Cas system in Bifidobacterium.},
journal = {Archives of microbiology},
volume = {203},
number = {6},
pages = {3235-3243},
pmid = {33837440},
issn = {1432-072X},
mesh = {*Bifidobacterium/genetics ; *CRISPR-Cas Systems/genetics ; Gene Transfer, Horizontal ; *Genetic Variation ; Humans ; Multilocus Sequence Typing ; },
abstract = {The CRISPR-Cas system is widely distributed in prokaryotes and plays an important role in the adaptive immunity of bacteria and archaea. Bifidobacterium is an important component of the intestinal flora of humans and animals, and some species of this bacterium can be employed as food additives. However, the Bifidobacterium CRISPR-Cas system has not been fully elucidated to date. In this study, the genomes of 110 strains of Bifidobacterium were employed to research the diversity of the type I-U system. The 110 strains were divided into five groups according to the genes adjacent to the CRISPR locus, including group A, B, C, D and E. Strains in the intergroup had unique species classifications and MLST types. An evolutionary tree was constructed based on the conserved cas4/cas1 fusion gene. The results showed that group A had a different evolutionary branch compared with the other groups and had a relatively low spacer number. Notably, group B, C and E had exhibited ABC transporter regulators in the genes adjacent to the CRISPR locus. ABC transporters play important roles in the exocytosis of many antibiotics and are involved in horizontal gene transfer. This mechanism may have promoted the evolution of Bifidobacterium and the horizontal gene transfer of the type I-U system, which may have promoted the generation of system diversity. In summary, our results help to elucidate the role of the type I-U system in the evolution of Bifidobacterium.},
}
@article {pmid33837304,
year = {2021},
author = {Caeser, R and Gao, J and Di Re, M and Gong, C and Hodson, DJ},
title = {Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells.},
journal = {Nature protocols},
volume = {16},
number = {5},
pages = {2499-2519},
pmid = {33837304},
issn = {1750-2799},
support = {MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; 203151/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; A25117/CRUK_/Cancer Research UK/United Kingdom ; /DH_/Department of Health/United Kingdom ; MR/M008584/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {B-Lymphocytes/cytology/*metabolism ; CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Cell Proliferation/genetics ; Gene Deletion ; *Genetic Techniques ; Genomics ; Germinal Center/*cytology ; Humans ; },
abstract = {Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR-Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.},
}
@article {pmid33837189,
year = {2021},
author = {Liu, P and Liang, SQ and Zheng, C and Mintzer, E and Zhao, YG and Ponnienselvan, K and Mir, A and Sontheimer, EJ and Gao, G and Flotte, TR and Wolfe, SA and Xue, W},
title = {Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {2121},
pmid = {33837189},
issn = {2041-1723},
support = {R01 GM115911/GM/NIGMS NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; UG3 TR002668/TR/NCATS NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; UH3 TR002668/TR/NCATS NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Mice ; Neoplasms/*genetics/pathology ; RNA, Guide/*genetics ; Transfection ; },
abstract = {Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.},
}
@article {pmid33836291,
year = {2021},
author = {Lee, S and Kim, YY and Ahn, HJ},
title = {Systemic delivery of CRISPR/Cas9 to hepatic tumors for cancer treatment using altered tropism of lentiviral vector.},
journal = {Biomaterials},
volume = {272},
number = {},
pages = {120793},
doi = {10.1016/j.biomaterials.2021.120793},
pmid = {33836291},
issn = {1878-5905},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Genetic Vectors ; Lentivirus/genetics ; *Liver Neoplasms/genetics/therapy ; Mice ; Transduction, Genetic ; Tropism ; Viral Envelope Proteins/genetics ; },
abstract = {Therapeutic application of CRISPR/Cas9 nucleases remains a challenge due to the lack of efficient in vivo delivery carriers. Here, we examine the ability of lentiviral vectors pseudotyped with hepatitis C virus (HCV)/E1E2 envelope glycoproteins to systemically deliver CRISPR/Cas9 to hepatic tumors in vivo. We demonstrated that systemic administration of E1E2-pseudotyped lentiviral vectors can selectively deliver Cas9 and sgRNA specific for kinesin spindle protein (KSP) to Huh7 tumors in the orthotopic Huh7 mice due to the specific interactions between E1E2 and their cellular receptors. This specific delivery leads to effective KSP gene disruption, potently inhibiting tumor growth. Furthermore, we demonstrated that E1E2-pseudotyping is more suitable for systemic delivery of CRISPR/Cas9 in cancer therapy than vesicular stomatitis virus-pseudotyping, the most widely used pseudotyping, because of stability in human serum, little transduction to DCs, low innate immune response, and cell-specific targeting ability. This study suggests that E1E2-pseudotyped lentivirus carrying CRISPR/Cas9 can substantially benefit the treatment of Huh7 tumors.},
}
@article {pmid33836165,
year = {2021},
author = {Kwon, AT and Mohri, K and Takizawa, S and Arakawa, T and Takahashi, M and Kaczkowski, B and Furuno, M and Suzuki, H and Tagami, S and Mukai, H and Arner, E},
title = {Development of p53 knockout U87MG cell line for unbiased drug delivery testing system using CRISPR-Cas9 and transcriptomic analysis.},
journal = {Journal of biotechnology},
volume = {332},
number = {},
pages = {72-82},
doi = {10.1016/j.jbiotec.2021.03.017},
pmid = {33836165},
issn = {1873-4863},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Genes, p53 ; *Pharmaceutical Preparations ; Transcriptome ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.},
}
@article {pmid33836052,
year = {2021},
author = {Sizova, I and Kelterborn, S and Verbenko, V and Kateriya, S and Hegemann, P},
title = {Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {7},
pages = {},
pmid = {33836052},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems/genetics ; *Chlamydomonas/genetics/metabolism ; DNA-Directed DNA Polymerase/metabolism ; Gene Targeting/methods ; Gene Editing/methods ; DNA End-Joining Repair ; },
abstract = {The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent nonhomologous end-joining (cNHEJ) and DNA polymerase theta (POLQ)-mediated end joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair (HDR)/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect HDR of the inactivated paromomycin-resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate HDR/gene inactivation. Our data suggest that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced HDR and random integration of transgenes, whereas KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in C. reinhardtii and can be used for future development of algal biotechnology.},
}
@article {pmid33835761,
year = {2021},
author = {Li, C and Brant, E and Budak, H and Zhang, B},
title = {CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement.},
journal = {Journal of Zhejiang University. Science. B},
volume = {22},
number = {4},
pages = {253-284},
pmid = {33835761},
issn = {1862-1783},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Gene Editing/*methods ; *Genetic Therapy ; Humans ; Nobel Prize ; *Plant Breeding ; },
abstract = {Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.},
}
@article {pmid33834450,
year = {2021},
author = {Kim, GN and Sung, YH},
title = {The Genetic Basis of Reporter Mouse Strains.},
journal = {Advances in experimental medicine and biology},
volume = {1310},
number = {},
pages = {551-564},
pmid = {33834450},
issn = {0065-2598},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Endonucleases/genetics ; Gene Editing ; Gene Targeting ; Genetic Engineering ; Mice ; Transcription Activator-Like Effector Nucleases/genetics ; Zygote ; },
abstract = {Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.},
}
@article {pmid33834266,
year = {2022},
author = {Kratzer, K and Getz, LJ and Peterlini, T and Masson, JY and Dellaire, G},
title = {Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.},
journal = {Human genetics},
volume = {141},
number = {6},
pages = {1175-1193},
pmid = {33834266},
issn = {1432-1203},
support = {PJT-156017/CAPMC/CIHR/Canada ; PJT-156017/CAPMC/CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genetic Therapy/methods ; Germ Cells ; Humans ; },
abstract = {Gene therapies for genetic diseases have been sought for decades, and the relatively recent development of the CRISPR/Cas9 gene-editing system has encouraged a new wave of interest in the field. There have nonetheless been significant setbacks to gene therapy, including unintended biological consequences, ethical scandals, and death. The major focus of research has been on technological problems such as delivery, potential immune responses, and both on and off-target effects in an effort to avoid negative clinical outcomes. While the field has concentrated on how we can better achieve gene therapies and gene editing techniques, there has been less focus on when and why we should use such technology. Here we combine discussion of both the technical and ethical barriers to the widespread clinical application of gene therapy and gene editing, providing a resource for gene therapy experts and novices alike. We discuss ethical problems and solutions, using cystic fibrosis and beta-thalassemia as case studies where gene therapy might be suitable, and provide examples of situations where human germline gene editing may be ethically permissible. Using such examples, we propose criteria to guide researchers and clinicians in deciding whether or not to pursue gene therapy as a treatment. Finally, we summarize how current progress in the field adheres to principles of biomedical ethics and highlight how this approach might fall short of ethical rigour using examples in the bioethics literature. Ultimately by addressing both the technical and ethical aspects of gene therapy and editing, new frameworks can be developed for the fair application of these potentially life-saving treatments.},
}
@article {pmid33833453,
year = {2021},
author = {Gu, Z and Liu, Y and Zhang, Y and Cao, H and Lyu, J and Wang, X and Wylie, A and Newkirk, SJ and Jones, AE and Lee, M and Botten, GA and Deng, M and Dickerson, KE and Zhang, CC and An, W and Abrams, JM and Xu, J},
title = {Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia.},
journal = {Nature genetics},
volume = {53},
number = {5},
pages = {672-682},
pmid = {33833453},
issn = {1546-1718},
support = {R15 GM131263/GM/NIGMS NIH HHS/United States ; R01 GM115682/GM/NIGMS NIH HHS/United States ; R21 OD017965/OD/NIH HHS/United States ; R01 CA222579/CA/NCI NIH HHS/United States ; R01 CA230631/CA/NCI NIH HHS/United States ; R01 CA248736/CA/NCI NIH HHS/United States ; R01 DK111430/DK/NIDDK NIH HHS/United States ; T32 CA124334/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics/pathology ; Cell Line, Tumor ; Epigenesis, Genetic ; Gene Expression Regulation, Leukemic ; *Gene Silencing ; Genome, Human ; Genomic Instability ; Hematopoiesis/genetics ; Humans ; Leukemia, Myeloid/*genetics ; Long Interspersed Nucleotide Elements/*genetics ; Mice, Inbred C57BL ; Mice, Knockout ; Phosphoproteins/genetics ; },
abstract = {Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.},
}
@article {pmid33833286,
year = {2021},
author = {Wong, TH and Khater, IM and Joshi, B and Shahsavari, M and Hamarneh, G and Nabi, IR},
title = {Single molecule network analysis identifies structural changes to caveolae and scaffolds due to mutation of the caveolin-1 scaffolding domain.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7810},
pmid = {33833286},
issn = {2045-2322},
support = {PJT-159845//CIHR/Canada ; PJT-156424//CIHR/Canada ; },
mesh = {Caveolin 1/*chemistry ; Cell Line ; Humans ; Mutation ; Protein Conformation ; Protein Domains/*genetics ; },
abstract = {Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.},
}
@article {pmid33833247,
year = {2021},
author = {Miller, K and Eggenberger, AL and Lee, K and Liu, F and Kang, M and Drent, M and Ruba, A and Kirscht, T and Wang, K and Jiang, S},
title = {An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7695},
pmid = {33833247},
issn = {2045-2322},
mesh = {*Biolistics ; *CRISPR-Cas Systems ; DNA, Plant/*genetics ; Gene Editing/methods ; Plants/*genetics ; },
abstract = {Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.},
}
@article {pmid33833118,
year = {2021},
author = {Fugger, K and Bajrami, I and Silva Dos Santos, M and Young, SJ and Kunzelmann, S and Kelly, G and Hewitt, G and Patel, H and Goldstone, R and Carell, T and Boulton, SJ and MacRae, J and Taylor, IA and West, SC},
title = {Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.},
journal = {Science (New York, N.Y.)},
volume = {372},
number = {6538},
pages = {156-165},
pmid = {33833118},
issn = {1095-9203},
support = {//Wellcome Trust/United Kingdom ; FC001212/CRUK_/Cancer Research UK/United Kingdom ; FC001212/MRC_/Medical Research Council/United Kingdom ; FC001212//Wellcome Trust/United Kingdom ; },
mesh = {Antineoplastic Agents/*pharmacology ; Apoptosis ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; DNA Replication ; DNA, Neoplasm/metabolism ; Deoxycytidine Monophosphate/analogs &amp; derivatives/metabolism/pharmacology ; Deoxyuracil Nucleotides/metabolism ; Drug Resistance, Neoplasm ; Genes, BRCA1 ; Humans ; Hydrolysis ; N-Glycosyl Hydrolases/*antagonists &amp; inhibitors/genetics/*metabolism ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Poly(ADP-ribose) Polymerases/metabolism ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors/genetics/*metabolism ; Synthetic Lethal Mutations ; Thymidine/analogs &amp; derivatives/antagonists &amp; inhibitors/metabolism/pharmacology ; Uracil-DNA Glycosidase/metabolism ; },
abstract = {Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.},
}
@article {pmid33833093,
year = {2021},
author = {Dixon, G and Pan, H and Yang, D and Rosen, BP and Jashari, T and Verma, N and Pulecio, J and Caspi, I and Lee, K and Stransky, S and Glezer, A and Liu, C and Rivas, M and Kumar, R and Lan, Y and Torregroza, I and He, C and Sidoli, S and Evans, T and Elemento, O and Huangfu, D},
title = {QSER1 protects DNA methylation valleys from de novo methylation.},
journal = {Science (New York, N.Y.)},
volume = {372},
number = {6538},
pages = {},
pmid = {33833093},
issn = {1095-9203},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; T32 GM008539/GM/NIGMS NIH HHS/United States ; T32 HD060600/HD/NICHD NIH HHS/United States ; R01 HG006827/HG/NHGRI NIH HHS/United States ; R01 DK096239/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA/*metabolism ; DNA (Cytosine-5-)-Methyltransferases/metabolism ; *DNA Methylation ; DNA Methyltransferase 3A ; Enhancer Elements, Genetic ; Gene Expression Regulation, Developmental ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genome, Human ; Human Embryonic Stem Cells/*metabolism ; Humans ; Mixed Function Oxygenases/genetics/metabolism ; Promoter Regions, Genetic ; Protein Binding ; Proto-Oncogene Proteins/genetics/metabolism ; Transcription, Genetic ; },
abstract = {DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.},
}
@article {pmid33831647,
year = {2021},
author = {Sun, C and Li, B and Yang, M and Guo, R and Yuan, S and Wang, J and Hu, H},
title = {Generation of GPAM knockout human embryonic stem cell line SYSUe-008-A using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102303},
doi = {10.1016/j.scr.2021.102303},
pmid = {33831647},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {GPAM (glycerol-3-phosphateacyltransferase1) is a mitochondrial enzyme that catalyze an essential step in glycerolphospholipids and triacylglycerol biosynthesis process. Loss-of-function mutation of GPAM has been shown to lead to hypomyelination of corticospinal tract in cerebral palsy patient. To model this rare disease with human brain organoid, we generated a GPAM knockout human embryonic stem cell line SYSUe-008-A by CRISPR/cas9. The GPAM knockout cell line maintains a normal karyotype and shows comparable level of pluripotent stem cell marker expression and differentiation potential as wild-type human embryonic stem cells.},
}
@article {pmid33830997,
year = {2021},
author = {Wallace, AD and Sasani, TA and Swanier, J and Gates, BL and Greenland, J and Pedersen, BS and Varley, KE and Quinlan, AR},
title = {CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.},
journal = {PloS one},
volume = {16},
number = {4},
pages = {e0241253},
pmid = {33830997},
issn = {1932-6203},
support = {R01 HG006693/HG/NHGRI NIH HHS/United States ; R25 HG009886/HG/NHGRI NIH HHS/United States ; T32 HG008962/HG/NHGRI NIH HHS/United States ; T32 GM007464/GM/NIGMS NIH HHS/United States ; R01 GM124355/GM/NIGMS NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics ; C9orf72 Protein/*genetics ; *CRISPR-Cas Systems ; *DNA Repeat Expansion ; *Genome, Human ; *High-Throughput Nucleotide Sequencing ; Humans ; *Microsatellite Repeats ; *Nanopores ; },
abstract = {A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.},
}
@article {pmid33830476,
year = {2021},
author = {Gumusgoz, E and Guisso, DR and Kasiri, S and Wu, J and Dear, M and Verhalen, B and Nitschke, S and Mitra, S and Nitschke, F and Minassian, BA},
title = {Targeting Gys1 with AAV-SaCas9 Decreases Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {18},
number = {2},
pages = {1414-1425},
pmid = {33830476},
issn = {1878-7479},
support = {P01 NS097197/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Brain/*metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing ; Glucans/*metabolism ; Glycogen/*metabolism ; Glycogen Storage Disease/*genetics/metabolism/therapy ; Glycogen Synthase/*genetics ; Lafora Disease/*genetics/metabolism/therapy ; Mice ; Nervous System Diseases/*genetics/metabolism/therapy ; Neuroinflammatory Diseases/*genetics/metabolism/therapy ; Proof of Concept Study ; RNA, Messenger/*metabolism ; },
abstract = {Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases.},
}
@article {pmid33830454,
year = {2022},
author = {Thamodaran, V and Rani, S and Velayudhan, SR},
title = {Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {755-773},
pmid = {33830454},
issn = {1940-6029},
support = {/WT_/Wellcome Trust/United Kingdom ; IA/S/17/1/503118/WTDBT_/DBT-Wellcome Trust India Alliance/India ; },
mesh = {CRISPR-Cas Systems/genetics ; Doxycycline/pharmacology ; *Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. Due to the ease of programming, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-based gene editing tools have gained pace in gene manipulation studies, including investigating complex diseases like cancer. An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies.},
}
@article {pmid33830423,
year = {2021},
author = {Ghorbani, A and Hadifar, S and Salari, R and Izadpanah, K and Burmistrz, M and Afsharifar, A and Eskandari, MH and Niazi, A and Denes, CE and Neely, GG},
title = {A short overview of CRISPR-Cas technology and its application in viral disease control.},
journal = {Transgenic research},
volume = {30},
number = {3},
pages = {221-238},
pmid = {33830423},
issn = {1573-9368},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing ; *Genetic Engineering ; Genome/*genetics ; Humans ; Mammals ; Virus Diseases/*genetics/therapy/virology ; Viruses/genetics/pathogenicity ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea where they provide an adaptive immune response through sequence-specific degradation of an invading pathogen's genome. This system has been reconfigured for use in genome editing, drug development, gene expression regulation, diagnostics, the prevention and treatment of cancers, and the treatment of genetic and infectious diseases. In recent years, CRISPR-Cas systems have been used in the diagnosis and control of viral diseases, for example, CRISPR-Cas12/13 coupled with new amplification techniques to improve the specificity of sequence-specific fluorescent probe detection. Importantly, CRISPR applications are both sensitive and specific and usually only require commonly available lab equipment. Unlike the canonical Cas9 which is guided to double-stranded DNA sites of interest, Cas13 systems target RNA sequences and thus can be employed in strategies directed against RNA viruses or for transcriptional silencing. Many challenges remain for these approach, including issues with specificity and the requirement for better mammalian delivery systems. In this review, we summarize the applications of CRISPR-Cas systems in controlling mammalian viral infections. Following necessary improvements, it is expected that CRISPR-Cas systems will be used effectively for such applications in the future.},
}
@article {pmid33828531,
year = {2021},
author = {Balboa, D and Iworima, DG and Kieffer, TJ},
title = {Human Pluripotent Stem Cells to Model Islet Defects in Diabetes.},
journal = {Frontiers in endocrinology},
volume = {12},
number = {},
pages = {642152},
pmid = {33828531},
issn = {1664-2392},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Differentiation ; Diabetes Mellitus/*metabolism/physiopathology ; Disease Models, Animal ; Endocrine Cells/metabolism ; Genetic Variation ; Genomics ; Glucose/metabolism ; Humans ; Induced Pluripotent Stem Cells ; Insulin/metabolism ; Insulin-Secreting Cells/*cytology ; Islets of Langerhans/*cytology/metabolism ; Phosphorylation ; Pluripotent Stem Cells/*cytology ; Proteomics ; Transcriptome ; },
abstract = {Diabetes mellitus is characterized by elevated levels of blood glucose and is ultimately caused by insufficient insulin production from pancreatic beta cells. Different research models have been utilized to unravel the molecular mechanisms leading to the onset of diabetes. The generation of pancreatic endocrine cells from human pluripotent stem cells constitutes an approach to study genetic defects leading to impaired beta cell development and function. Here, we review the recent progress in generating and characterizing functional stem cell-derived beta cells. We summarize the diabetes disease modeling possibilities that stem cells offer and the challenges that lie ahead to further improve these models.},
}
@article {pmid33828299,
year = {2021},
author = {Mo, CY and Mathai, J and Rostøl, JT and Varble, A and Banh, DV and Marraffini, LA},
title = {Type III-A CRISPR immunity promotes mutagenesis of staphylococci.},
journal = {Nature},
volume = {592},
number = {7855},
pages = {611-615},
pmid = {33828299},
issn = {1476-4687},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; F30 AI157535/AI/NIAID NIH HHS/United States ; F32 GM128271/GM/NIGMS NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Bacteriophages/classification/physiology ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics/*immunology ; DNA, Single-Stranded/genetics/metabolism ; Deoxyribonucleases/metabolism ; Drug Resistance, Microbial/drug effects ; *Mutagenesis ; *Mutation ; SOS Response, Genetics/drug effects ; Staphylococcus/drug effects/*genetics/immunology/virology ; Staphylococcus aureus/drug effects/genetics/virology ; Staphylococcus epidermidis/drug effects/genetics/virology ; Time Factors ; },
abstract = {Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors[1]. Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids[2] and bacteriophages[3], thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA[4,5]; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.},
}
@article {pmid33828203,
year = {2021},
author = {Hendricks-Wenger, A and Aycock, KN and Nagai-Singer, MA and Coutermarsh-Ott, S and Lorenzo, MF and Gannon, J and Uh, K and Farrell, K and Beitel-White, N and Brock, RM and Simon, A and Morrison, HA and Tuohy, J and Clark-Deener, S and Vlaisavljevich, E and Davalos, RV and Lee, K and Allen, IC},
title = {Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7584},
pmid = {33828203},
issn = {2045-2322},
support = {P01 CA207206/CA/NCI NIH HHS/United States ; R21 EB028429/EB/NIBIB NIH HHS/United States ; R01 CA213423/CA/NCI NIH HHS/United States ; R21 OD027062/OD/NIH HHS/United States ; R01 CA240476/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma/pathology/physiopathology/*therapy ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA-Binding Proteins/deficiency/genetics/immunology ; Electric Conductivity ; Electroporation/*methods ; Female ; Gene Knockout Techniques ; Humans ; Immunocompromised Host ; Interleukin Receptor Common gamma Subunit/deficiency/genetics/immunology ; Male ; Mice ; Pancreatic Neoplasms/pathology/physiopathology/*therapy ; Proof of Concept Study ; Swine ; Translational Research, Biomedical ; Xenograft Model Antitumor Assays ; },
abstract = {New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.},
}
@article {pmid33827648,
year = {2021},
author = {Gurumurthy, CB and O'Brien, AR and Quadros, RM and Adams, J and Alcaide, P and Ayabe, S and Ballard, J and Batra, SK and Beauchamp, MC and Becker, KA and Bernas, G and Brough, D and Carrillo-Salinas, F and Chan, W and Chen, H and Dawson, R and DeMambro, V and D'Hont, J and Dibb, K and Eudy, JD and Gan, L and Gao, J and Gonzales, A and Guntur, A and Guo, H and Harms, DW and Harrington, A and Hentges, KE and Humphreys, N and Imai, S and Ishii, H and Iwama, M and Jonasch, E and Karolak, M and Keavney, B and Khin, NC and Konno, M and Kotani, Y and Kunihiro, Y and Lakshmanan, I and Larochelle, C and Lawrence, CB and Li, L and Lindner, V and Liu, XD and Lopez-Castejon, G and Loudon, A and Lowe, J and Jerome-Majeweska, L and Matsusaka, T and Miura, H and Miyasaka, Y and Morpurgo, B and Motyl, K and Nabeshima, YI and Nakade, K and Nakashiba, T and Nakashima, K and Obata, Y and Ogiwara, S and Ouellet, M and Oxburgh, L and Piltz, S and Pinz, I and Ponnusamy, MP and Ray, D and Redder, RJ and Rosen, CJ and Ross, N and Ruhe, MT and Ryzhova, L and Salvador, AM and Alam, SS and Sedlacek, R and Sharma, K and Smith, C and Staes, K and Starrs, L and Sugiyama, F and Takahashi, S and Tanaka, T and Trafford, A and Uno, Y and Vanhoutte, L and Vanrockeghem, F and Willis, BJ and Wright, CS and Yamauchi, Y and Yi, X and Yoshimi, K and Zhang, X and Zhang, Y and Ohtsuka, M and Das, S and Garry, DJ and Hochepied, T and Thomas, P and Parker-Thornburg, J and Adamson, AD and Yoshiki, A and Schmouth, JF and Golovko, A and Thompson, WR and Lloyd, KCK and Wood, JA and Cowan, M and Mashimo, T and Mizuno, S and Zhu, H and Kasparek, P and Liaw, L and Miano, JM and Burgio, G},
title = {Response to correspondence on "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation".},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {99},
doi = {10.1186/s13059-021-02320-3},
pmid = {33827648},
issn = {1474-760X},
support = {107851/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; MR/M008908/1/MRC_/Medical Research Council/United Kingdom ; MR/P023576/2/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; Animals ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Mice ; Reproducibility of Results ; },
}
@article {pmid33827646,
year = {2021},
author = {Yang, H and Wang, H and Jaenisch, R},
title = {Response to "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation".},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {98},
pmid = {33827646},
issn = {1474-760X},
support = {R01 MH104610/MH/NIMH NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Mice ; Reproducibility of Results ; },
}
@article {pmid33827404,
year = {2021},
author = {Rozhkova, AM and Kislitsin, VY},
title = {CRISPR/Cas Genome Editing in Filamentous Fungi.},
journal = {Biochemistry. Biokhimiia},
volume = {86},
number = {Suppl 1},
pages = {S120-S139},
doi = {10.1134/S0006297921140091},
pmid = {33827404},
issn = {1608-3040},
mesh = {Aspergillus/genetics ; *CRISPR-Cas Systems ; Fungi/*genetics ; Gene Editing/*methods ; Penicillium/genetics ; Trichoderma/genetics ; },
abstract = {The review describes the CRISPR/CAS system and its adaptation for the genome editing in filamentous fungi commonly used for production of enzyme complexes, enzymes, secondary metabolites, and other compounds used in industrial biotechnology and agriculture. In the second part of this review, examples of the CRISPR/CAS technology application for improving properties of the industrial strains of fungi from the Trichoderma, Aspergillus, Penicillium, and other genera are presented. Particular attention is given to the efficiency of genome editing, as well as system optimization for specific industrial producers.},
}
@article {pmid33826163,
year = {2021},
author = {Li, R and Zhang, R and Tan, P and Wang, M and Chen, Y and Zhang, J and Han, D and Han, Y and Li, J and Zhang, R},
title = {Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing.},
journal = {Journal of clinical laboratory analysis},
volume = {35},
number = {5},
pages = {e23746},
pmid = {33826163},
issn = {1098-2825},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing/*standards ; Humans ; Mice, Inbred NOD ; Mice, SCID ; MutL Protein Homolog 1/*deficiency/*genetics ; Mutation/genetics ; Quality Control ; *Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Mismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision-making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories.<br><br>METHODS: CRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein-deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next-generation sequencing (NGS) and were then used to prepare formalin-fixed, paraffin-embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&amp;E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing.<br><br>RESULTS: We successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&amp;E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples.<br><br>CONCLUSIONS: We successfully established MLH1 protein-deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.},
}
@article {pmid33825445,
year = {2021},
author = {Matsumoto, D and Nomura, W},
title = {Molecular Switch Engineering for Precise Genome Editing.},
journal = {Bioconjugate chemistry},
volume = {32},
number = {4},
pages = {639-648},
doi = {10.1021/acs.bioconjchem.1c00088},
pmid = {33825445},
issn = {1520-4812},
mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing ; *Genetic Engineering ; Nucleic Acid Hybridization ; Proteolysis ; RNA/genetics ; },
abstract = {Genome editing technology commenced in 1996 with the discovery of the first zinc-finger nuclease. Application of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (Cas9) technology to genome editing of mammalian cells allowed researchers to use genome editing more easily and cost-effectively. However, one of the technological problems that remains to be solved is "off-target effects", which are unexpected mutations in nontarget DNA. One significant improvement in genome editing technology has been achieved with molecular/protein engineering. The key to this engineering is a "switch" to control function. In this review, we discuss recent efforts to design novel "switching" systems for precise editing using genome editing tools.},
}
@article {pmid33825100,
year = {2021},
author = {Ramirez-Torres, F and Ghogare, R and Stowe, E and Cerdá-Bennasser, P and Lobato-Gómez, M and Williamson-Benavides, BA and Giron-Calva, PS and Hewitt, S and Christou, P and Dhingra, A},
title = {Genome editing in fruit, ornamental, and industrial crops.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {499-528},
pmid = {33825100},
issn = {1573-9368},
support = {T32 GM008336/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Fruit/*genetics ; *Gene Editing ; Gene Targeting ; *Genome, Plant ; Plant Breeding/*methods ; Plants, Genetically Modified/*genetics ; },
abstract = {The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies.},
}
@article {pmid33824975,
year = {2021},
author = {Thijssen, R and Diepstraten, ST and Moujalled, D and Chew, E and Flensburg, C and Shi, MX and Dengler, MA and Litalien, V and MacRaild, S and Chen, M and Anstee, NS and Reljić, B and Gabriel, SS and Djajawi, TM and Riffkin, CD and Aubrey, BJ and Chang, C and Tai, L and Xu, Z and Morley, T and Pomilio, G and Bruedigam, C and Kallies, A and Stroud, DA and Bajel, A and Kluck, RM and Lane, SW and Schoumacher, M and Banquet, S and Majewski, IJ and Strasser, A and Roberts, AW and Huang, DCS and Brown, FC and Kelly, GL and Wei, AH},
title = {Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.},
journal = {Blood},
volume = {137},
number = {20},
pages = {2721-2735},
pmid = {33824975},
issn = {1528-0020},
mesh = {Animals ; Antineoplastic Agents/administration &amp; dosage/*pharmacology/therapeutic use ; Apoptosis/*drug effects/physiology ; Apoptosis Regulatory Proteins/*antagonists &amp; inhibitors/physiology ; Bridged Bicyclo Compounds, Heterocyclic/administration &amp; dosage/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Damage ; Genes, p53 ; Humans ; Indolizines/*pharmacology/therapeutic use ; Interleukin-2 Receptor alpha Subunit/deficiency ; Isoquinolines/*pharmacology/therapeutic use ; Leukemia, Lymphocytic, Chronic, B-Cell/*drug therapy ; Leukemia, Myeloid, Acute/*drug therapy/pathology/therapy ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Morpholines/*pharmacology/therapeutic use ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists &amp; inhibitors ; Neoplasm Proteins/antagonists &amp; inhibitors/*physiology ; Oxidative Phosphorylation/drug effects ; Peptide Fragments/*antagonists &amp; inhibitors ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors ; Proto-Oncogene Proteins c-bcl-2/antagonists &amp; inhibitors ; Sulfonamides/administration &amp; dosage/*pharmacology/therapeutic use ; Tumor Suppressor Protein p53/deficiency/*physiology ; Xenograft Model Antitumor Assays ; },
abstract = {Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.},
}
@article {pmid33824390,
year = {2021},
author = {Ueda, T and Iwayama, T and Tomita, K and Matsumoto, S and Iwashita, M and Bhongsatiern, P and Sakashita, H and Fujihara, C and Takedachi, M and Murakami, S},
title = {Zbp1-positive cells are osteogenic progenitors in periodontal ligament.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7514},
pmid = {33824390},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Clone Cells/cytology ; Humans ; Mesenchymal Stem Cells/cytology ; Mice ; Osteogenesis/*genetics ; Periodontal Ligament/*growth &amp; development ; Periodontium/*growth &amp; development ; RNA-Binding Proteins/*genetics ; RNA-Seq ; Stem Cells/cytology ; },
abstract = {Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.},
}
@article {pmid33824204,
year = {2021},
author = {Negreiros, RS and Lander, N and Chiurillo, MA and Vercesi, AE and Docampo, R},
title = {Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.},
journal = {mBio},
volume = {12},
number = {2},
pages = {},
pmid = {33824204},
issn = {2150-7511},
support = {K99 AI137322/AI/NIAID NIH HHS/United States ; },
mesh = {Anion Transport Proteins/*genetics/metabolism ; Biological Transport ; CRISPR-Cas Systems ; DNA Replication ; Gene Knockout Techniques ; Mitochondrial Membrane Transport Proteins/*genetics ; Protozoan Proteins/*genetics/metabolism ; Pyruvic Acid/*metabolism ; Trypanosoma cruzi/*genetics/*metabolism/pathogenicity ; },
abstract = {Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruziMPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca[2+] uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.IMPORTANCETrypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruziMPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.},
}
@article {pmid33823546,
year = {2021},
author = {Villegas Kcam, MC and Tsong, AJ and Chappell, J},
title = {Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range.},
journal = {Nucleic acids research},
volume = {49},
number = {8},
pages = {4793-4802},
pmid = {33823546},
issn = {1362-4962},
mesh = {Bacteria/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Phylogeny ; Protein Binding ; Protein Domains/genetics ; Protein Engineering/*methods ; RNA, Guide/*genetics ; Transcriptional Activation ; },
abstract = {CRISPR-Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR-Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range.},
}
@article {pmid33823541,
year = {2021},
author = {Netter, Z and Boyd, CM and Silvas, TV and Seed, KD},
title = {A phage satellite tunes inducing phage gene expression using a domesticated endonuclease to balance inhibition and virion hijacking.},
journal = {Nucleic acids research},
volume = {49},
number = {8},
pages = {4386-4401},
pmid = {33823541},
issn = {1362-4962},
support = {R01 AI127652/AI/NIAID NIH HHS/United States ; R01 AI153303/AI/NIAID NIH HHS/United States ; T32 GM132022/GM/NIGMS NIH HHS/United States ; R01AI127652-S1/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/genetics/*metabolism ; Bacteriophages/genetics/*growth &amp; development ; Binding Sites ; CRISPR-Cas Systems ; Capsid Proteins/genetics ; DNA, Satellite/*genetics ; DNA-Binding Proteins/chemistry/genetics/*metabolism ; Endonucleases/chemistry/genetics/*metabolism ; *Gene Expression Regulation, Viral ; *Interspersed Repetitive Sequences ; Operon/genetics ; Protein Domains ; Transduction, Genetic ; Vibrio cholerae/enzymology/genetics/*virology ; Virion/genetics/growth &amp; development ; },
abstract = {Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1's capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.},
}
@article {pmid33823301,
year = {2021},
author = {Choi, E and Koo, T},
title = {CRISPR technologies for the treatment of Duchenne muscular dystrophy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {11},
pages = {3179-3191},
pmid = {33823301},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; Clinical Studies as Topic ; Disease Management ; Disease Models, Animal ; Dystrophin/genetics ; Exons ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Muscular Dystrophy, Duchenne/*genetics/*therapy ; Mutation ; Treatment Outcome ; },
abstract = {The emerging clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing technologies have progressed remarkably in recent years, opening up the potential of precise genome editing as a therapeutic approach to treat various diseases. The CRISPR-CRISPR-associated (Cas) system is an attractive platform for the treatment of Duchenne muscular dystrophy (DMD), which is a neuromuscular disease caused by mutations in the DMD gene. CRISPR-Cas can be used to permanently repair the mutated DMD gene, leading to the expression of the encoded protein, dystrophin, in systems ranging from cells derived from DMD patients to animal models of DMD. However, the development of more efficient therapeutic approaches and delivery methods remains a great challenge for DMD. Here, we review various therapeutic strategies that use CRISPR-Cas to correct or bypass DMD mutations and discuss their therapeutic potential, as well as obstacles that lie ahead.},
}
@article {pmid33823269,
year = {2021},
author = {Liang, Y and Yu, H},
title = {Genetic toolkits for engineering Rhodococcus species with versatile applications.},
journal = {Biotechnology advances},
volume = {49},
number = {},
pages = {107748},
doi = {10.1016/j.biotechadv.2021.107748},
pmid = {33823269},
issn = {1873-1899},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genetic Engineering ; Homologous Recombination ; Promoter Regions, Genetic ; *Rhodococcus/genetics ; },
abstract = {Rhodococcus spp. are a group of non-model gram-positive bacteria with diverse catabolic activities and strong adaptive capabilities, which enable their wide application in whole-cell biocatalysis, environmental bioremediation, and lignocellulosic biomass conversion. Compared with model microorganisms, the engineering of Rhodococcus is challenging because of the lack of universal molecular tools, high genome GC content (61% ~ 71%), and low transformation and recombination efficiencies. Nevertheless, because of the high interest in Rhodococcus species for bioproduction, various genetic elements and engineering tools have been recently developed for Rhodococcus spp., including R. opacus, R. jostii, R. ruber, and R. erythropolis, leading to the expansion of the genetic toolkits for Rhodococcus engineering. In this article, we provide a comprehensive review of the important developed genetic elements for Rhodococcus, including shuttle vectors, promoters, antibiotic markers, ribosome binding sites, and reporter genes. In addition, we also summarize gene transfer techniques and strategies to improve transformation efficiency, as well as random and precise genome editing tools available for Rhodococcus, including transposition, homologous recombination, recombineering, and CRISPR/Cas9. We conclude by discussing future trends in Rhodococcus engineering. We expect that more synthetic and systems biology tools (such as multiplex genome editing, dynamic regulation, and genome-scale metabolic models) will be adapted and optimized for Rhodococcus.},
}
@article {pmid33823021,
year = {2021},
author = {Gaillochet, C and Develtere, W and Jacobs, TB},
title = {CRISPR screens in plants: approaches, guidelines, and future prospects.},
journal = {The Plant cell},
volume = {33},
number = {4},
pages = {794-813},
pmid = {33823021},
issn = {1532-298X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; High-Throughput Screening Assays/methods ; Mutation ; Plants/*genetics ; Plants, Genetically Modified ; Population Density ; RNA, Guide ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in the development of powerful new screens to test gene functions at the genomic scale. While there is tremendous potential to map and interrogate gene regulatory networks at unprecedented speed and scale using CRISPR screens, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools, and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports describing the use of this strategy to generate mutant knockout collections or to diversify DNA sequences. In addition, we provide insight into how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene functions in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the numerous genomic profiles that have been generated over the past two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will lead to great advances in functional and synthetic biology.},
}
@article {pmid33820795,
year = {2021},
author = {McAllister, KN and Martinez Aguirre, A and Sorg, JA},
title = {The Selenophosphate Synthetase Gene, selD, Is Important for Clostridioides difficile Physiology.},
journal = {Journal of bacteriology},
volume = {203},
number = {12},
pages = {e0000821},
pmid = {33820795},
issn = {1098-5530},
support = {P01 AI152999/AI/NIAID NIH HHS/United States ; R01 AI116895/AI/NIAID NIH HHS/United States ; U01 AI124290/AI/NIAID NIH HHS/United States ; 5R01AI116895/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Clostridioides difficile/*enzymology/*genetics ; Gene Deletion ; Gene Editing ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Genome, Bacterial ; Phosphotransferases/*genetics/*metabolism ; RNA, Bacterial/genetics ; RNA-Seq ; },
abstract = {The endospore-forming pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and health care. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis, and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using transcriptome sequencing (RNA-seq), we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD[+] can be regenerated by other pathways. IMPORTANCE C. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile life cycle, other nonantibiotic treatment options are in urgent need of development. Toward this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology: Stickland metabolism. Here, we use our CRISPR-Cas9 system to "knock in" a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology, from vegetative growth to spore formation and outgrowth postgermination.},
}
@article {pmid33819020,
year = {2021},
author = {Yu, L and Marchisio, MA},
title = {Saccharomyces cerevisiae Synthetic Transcriptional Networks Harnessing dCas12a and Type V-A anti-CRISPR Proteins.},
journal = {ACS synthetic biology},
volume = {10},
number = {4},
pages = {870-883},
doi = {10.1021/acssynbio.1c00006},
pmid = {33819020},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fungal Proteins/genetics/metabolism ; Gene Regulatory Networks ; Saccharomyces cerevisiae/genetics/*metabolism ; },
abstract = {Type V-A anti-CRISPR proteins (AcrVAs) represent the response from phages to the CRISPR-Cas12a prokaryotic immune system. CRISPR-Cas12a was repurposed, in high eukaryotes, to carry out gene editing and transcription regulation, the latter via a nuclease-dead Cas12a (dCas12a). Consequently, AcrVAs were adopted to regulate (d)Cas12a activity. However, the usage of both dCas12a-based transcription factors and AcrVAs in the yeast Saccharomyces cerevisiae has not been explored. In this work, we show that, in the baker's yeast, two dCas12a proteins (denAsCas12a and dLbCas12a) work both as activators (upon fusion to a strong activation domain) and repressors, whereas dMbCa12a is nonfunctional. The activation efficiency of dCas12a-ADs manifests a dependence on the number of crRNA binding sites, whereas it is not directly correlated to the amount of crRNA in the cells. Moreover, AcrVA1, AcrVA4, and AcrVA5 are able to inhibit dLbCa12a in yeast, and denAsCas12a is only inhibited by AcrVA1. However, AcrVA1 performs well at high concentration only. Coexpression of two or three AcrVAs does not enhance inhibition of dCas12a(-AD), suggesting a competition between different AcrVAs. Further, AcrVA4 significantly limits gene editing by LbCas12a. Overall, our results indicate that dCas12a:crRNA and AcrVA proteins are highly performant components in S. cerevisiae synthetic transcriptional networks.},
}
@article {pmid33817273,
year = {2020},
author = {Schenke, D},
title = {CRISPR/Cas9 or prime editing? - It depends on….},
journal = {Open life sciences},
volume = {15},
number = {1},
pages = {868-870},
pmid = {33817273},
issn = {2391-5412},
}
@article {pmid33816560,
year = {2021},
author = {Zhuang, C and Zhuang, C and Zhou, Q and Huang, X and Gui, Y and Lai, Y and Yang, S},
title = {Engineered CRISPR/Cas13d Sensing hTERT Selectively Inhibits the Progression of Bladder Cancer In Vitro.},
journal = {Frontiers in molecular biosciences},
volume = {8},
number = {},
pages = {646412},
pmid = {33816560},
issn = {2296-889X},
abstract = {Aptazyme and CRISPR/Cas gene editing system were widely used for regulating gene expression in various diseases, including cancer. This work aimed to reconstruct CRISPR/Cas13d tool for sensing hTERT exclusively based on the new device OFF-switch hTERT aptazyme that was inserted into the 3' UTR of the Cas13d. In bladder cancer cells, hTERT ligand bound to aptamer in OFF-switch hTERT aptazyme to inhibit the degradation of Cas13d. Results showed that engineered CRISPR/Cas13d sensing hTERT suppressed cell proliferation, migration, invasion and induced cell apoptosis in bladder cancer 5637 and T24 cells without affecting normal HFF cells. In short, we constructed engineered CRISPR/Cas13d sensing hTERT selectively inhibited the progression of bladder cancer cells significantly. It may serve as a promising specifically effective therapy for bladder cancer cells.},
}
@article {pmid33813884,
year = {2021},
author = {Potts, RWA and Gutierrez, AP and Penaloza, CS and Regan, T and Bean, TP and Houston, RD},
title = {Potential of genomic technologies to improve disease resistance in molluscan aquaculture.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {376},
number = {1825},
pages = {20200168},
pmid = {33813884},
issn = {1471-2970},
support = {BB/M010996/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/20002172/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/30002275/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Aquaculture/*instrumentation ; Genomics/*methods ; Mollusca/*genetics ; },
abstract = {Molluscan aquaculture is a major contributor to global seafood production, but is hampered by infectious disease outbreaks that can cause serious economic losses. Selective breeding has been widely used to improve disease resistance in major agricultural and aquaculture species, and has clear potential in molluscs, albeit its commercial application remains at a formative stage. Advances in genomic technologies, especially the development of cost-efficient genomic selection, have the potential to accelerate genetic improvement. However, tailored approaches are required owing to the distinctive reproductive and life cycle characteristics of molluscan species. Transgenesis and genome editing, in particular CRISPR/Cas systems, have been successfully trialled in molluscs and may further understanding and improvement of genetic resistance to disease through targeted changes to the host genome. Whole-organism genome editing is achievable on a much greater scale compared to other farmed species, making genome-wide CRISPR screening approaches plausible. This review discusses the current state and future potential of selective breeding, genomic tools and genome editing approaches to understand and improve host resistance to infectious disease in molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.},
}
@article {pmid33813849,
year = {2021},
author = {Sun, H and Hodgkinson, CP and Pratt, RE and Dzau, VJ},
title = {CRISPR/Cas9 Mediated Deletion of the Angiotensinogen Gene Reduces Hypertension: A Potential for Cure?.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {77},
number = {6},
pages = {1990-2000},
doi = {10.1161/HYPERTENSIONAHA.120.16870},
pmid = {33813849},
issn = {1524-4563},
support = {R01 HL131814/HL/NHLBI NIH HHS/United States ; R01 HL139718/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiotensinogen/*genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line ; *Gene Deletion ; Hypertension/*genetics/metabolism ; Liver/*metabolism ; Rats ; Rats, Wistar ; },
abstract = {[Figure: see text].},
}
@article {pmid33813641,
year = {2021},
author = {Baliga, P and Shekar, M and Kallappa, GS},
title = {Genome-Wide Identification and Analysis of Chromosomally Integrated Putative Prophages Associated with Clinical Klebsiella pneumoniae Strains.},
journal = {Current microbiology},
volume = {78},
number = {5},
pages = {2015-2024},
pmid = {33813641},
issn = {1432-0991},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; Humans ; *Klebsiella pneumoniae/genetics ; *Prophages/genetics ; },
abstract = {Klebsiella pneumoniae, an opportunistic pathogen found in the environment and human mucosal surfaces, is a leading cause of nosocomial infections. K. pneumoniae is now considered a global threat owing to the emergence of multidrug-resistant strains making its infections untreatable. In this study, 254 strains of K. pneumoniae were screened for the presence of prophages using the PHASTER tool. Very few strains lacked prophages (3.1%), while the remaining harboured both intact (811) and defective prophages (709). A subset of 42 unique strains of K. pneumoniae was chosen for further analysis. Our analysis revealed the presence of 110 complete prophages which were further classified as belonging to Myoviridae (67.3%), Siphoviridae (28.2%) and Podoviridae family (4.5%). An alignment of the 110 complete, prophage genome sequences clustered the prophages into 16 groups and 3 singletons. While none of the prophages encoded for virulence factors, 2 (1.8%) prophages were seen to encode for the antibiotic resistance-related genes. The CRISPR-Cas system was prevalent in 10 (23.8%) out of the 42 strains. Further analysis of the CRISPR spacers revealed 11.42% of the total spacers integrated in K. pneumoniae chromosome to match prophage protein sequences.},
}
@article {pmid33813271,
year = {2021},
author = {Ibayashi, M and Aizawa, R and Tsukamoto, S},
title = {mRNA decapping factor Dcp1a is essential for embryonic growth in mice.},
journal = {Biochemical and biophysical research communications},
volume = {555},
number = {},
pages = {128-133},
doi = {10.1016/j.bbrc.2021.03.117},
pmid = {33813271},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Endoribonucleases/*genetics/*metabolism ; Female ; *Gene Expression Regulation, Developmental ; Heart/embryology ; Heart Defects, Congenital/genetics ; Male ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Mice, Transgenic ; Trans-Activators/*genetics/*metabolism ; },
abstract = {mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo, we employed a transgenic rescue strategy to produce Dcp1a-deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a-deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth.},
}
@article {pmid33813174,
year = {2021},
author = {Yao, M and Su, P and Li, Z and Cui, X and Yang, Q and Xing, X and Lei, Y and Lian, M and Ren, T and Wang, X and Zheng, Y and Wu, C},
title = {Knockout of Dip2c in murine ES cell line IBMSe001-B-1 by CRISPR/Cas9 genome editing technology.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102236},
doi = {10.1016/j.scr.2021.102236},
pmid = {33813174},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Female ; *Gene Editing ; Mice ; Mice, Knockout ; Pregnancy ; Technology ; },
abstract = {DIP2 protein contains three members: DIP2A, DIP2B and DIP2C, and are broadly expressed in the nervous system from Drosophila to human during embryonic development. Dip2c gene-associated mutations have been reported in tumors and neuronal diseases. However, the role ofDip2cin the context of mouse embryonic stem (mES) cells has not been explored.To investigate the biological function of Dip2c during early embryo development, we generated Dip2c[-/-] mES line using a CRISPR/Cas9 system. This cell line has contributed to further investigation of molecular mechanism of Dip2c during cell differentiation, as well as a cell model for screening for neurogenic drug and cancer clinical cure.},
}
@article {pmid33813034,
year = {2021},
author = {Karottki, KJC and Hefzi, H and Li, S and Pedersen, LE and Spahn, PN and Joshi, C and Ruckerbauer, D and Bort, JAH and Thomas, A and Lee, JS and Borth, N and Lee, GM and Kildegaard, HF and Lewis, NE},
title = {A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.},
journal = {Metabolic engineering},
volume = {66},
number = {},
pages = {114-122},
pmid = {33813034},
issn = {1096-7184},
support = {R35 GM119850/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Gene Editing ; *Glutamine/genetics/metabolism ; },
abstract = {Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.},
}
@article {pmid33812982,
year = {2021},
author = {Luo, J and Li, S and Xu, J and Yan, L and Ma, Y and Xia, L},
title = {Pyramiding favorable alleles in an elite wheat variety in one generation by CRISPR-Cas9-mediated multiplex gene editing.},
journal = {Molecular plant},
volume = {14},
number = {6},
pages = {847-850},
doi = {10.1016/j.molp.2021.03.024},
pmid = {33812982},
issn = {1752-9867},
mesh = {*Alleles ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Plants, Genetically Modified ; Triticum/*genetics ; },
}
@article {pmid33812776,
year = {2021},
author = {Buquicchio, FA and Satpathy, AT},
title = {Interrogating immune cells and cancer with CRISPR-Cas9.},
journal = {Trends in immunology},
volume = {42},
number = {5},
pages = {432-446},
pmid = {33812776},
issn = {1471-4981},
support = {K08 CA230188/CA/NCI NIH HHS/United States ; U01 CA260852/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR-Cas9 technologies have transformed the study of genetic pathways governing cellular differentiation and function. Recent advances have adapted these methods to immune cells, which has accelerated the pace of functional genomics in immunology and enabled new avenues for the design of cellular immunotherapies for cancer. In this review, we summarize recent developments in CRISPR-Cas9 technology and discuss how they have been leveraged to discover and manipulate novel genetic regulators of the immune system. We envision that these results will provide a valuable resource to aid in the design, implementation, and interpretation of CRISPR-Cas9-based screens in immunology and immuno-oncology.},
}
@article {pmid33812397,
year = {2021},
author = {Wang, LY and Lin, RZ and Jiang, PF and Zhang, Y and Li, JZ and Chen, YW and Hu, JD},
title = {[Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {29},
number = {2},
pages = {339-347},
doi = {10.19746/j.cnki.issn.1009-2137.2021.02.006},
pmid = {33812397},
issn = {1009-2137},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; *Gene Editing ; *MicroRNAs/genetics ; Sequence Analysis, RNA ; Tretinoin ; },
abstract = {OBJECTIVE: To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation.<br><br>METHODS: The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry.<br><br>RESULTS: A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased.<br><br>CONCLUSION: CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.},
}
@article {pmid33812335,
year = {2021},
author = {Tang, X and Fu, J and Chen, Z and Luo, L and Li, DW and Liu, Y},
title = {Generation of a homozygous CRISPR/Cas9-mediated knockout H9 hESC subline for the MERTK locus.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102310},
doi = {10.1016/j.scr.2021.102310},
pmid = {33812335},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Human Embryonic Stem Cells ; Humans ; c-Mer Tyrosine Kinase/genetics ; },
abstract = {MERTK mutations are associate with rod-cone dystrophies. To enable investigations into the mechanism of this disease, we generated a cell line resource of H9 human embryonic stem cells harboring large fragment deletion mutation in a homozygous state in exon 19 of the MERTK gene. This subline expressed pluripotent stem cell markers, presented a normal karyotype, and preserved the ability to differentiate into endodermal, mesodermal, and ectodermal lineages.},
}
@article {pmid33811466,
year = {2021},
author = {Crotchett, BLM and Ceresa, BP},
title = {Knockout of c-Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation.},
journal = {Pharmacology research &amp; perspectives},
volume = {9},
number = {2},
pages = {e00756},
pmid = {33811466},
issn = {2052-1707},
support = {R01 EY028911/EY/NEI NIH HHS/United States ; EY028911/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Endocytosis/drug effects/genetics ; Epidermal Growth Factor/*metabolism ; Epithelium, Corneal/drug effects/injuries/*metabolism ; ErbB Receptors/metabolism ; Gene Knockout Techniques ; Humans ; Phosphorylation ; Proteolysis/drug effects ; Proto-Oncogene Proteins c-cbl/antagonists &amp; inhibitors/genetics/*metabolism ; Ubiquitination/drug effects/genetics ; },
abstract = {Epidermal growth factor receptor (EGFR) activity is necessary and sufficient for corneal epithelial homeostasis. However, the addition of exogenous Epidermal Growth Factor (EGF) does not reliably restore the corneal epithelium when wounded. This is likely due to high levels of endogenous EGF in tear fluid as well as desensitization of the EGFR following ligand stimulation. We hypothesize that preventing receptor downregulation is an alternative mechanism to enhance EGFR signaling and promote the restoration of compromised corneas. Ligand-dependent EGFR ubiquitylation is associated with the targeted degradation of the receptor. In this manuscript, we determine whether knockout of c-Cbl, an E3 ubiquitin ligase that ubiquitylates the EGFR, is sufficient to prolong EGFR phosphorylation and sustain signaling. Using CRISPR/Cas9 gene editing, we generated immortalized human corneal epithelial (hTCEpi) cells lacking c-Cbl. Knockout (KO) cells expressed the other E3 ligases at the same levels as the control cells, indicating other E3 ligases were not up-regulated. As compared to the control cells, EGF-stimulated EGFR ubiquitylation was reduced in KO cells, but not completely abolished. Similarly, EGF:EGFR trafficking was slowed, with a 35% decrease in the rate of endocytosis and a twofold increase in the receptor half-life. This resulted in a twofold increase in the magnitude of EGFR phosphorylation, with no change in duration. Conversely, Mitogen Activating Protein Kinase (MAPK) phosphorylation did not increase in magnitude but was sustained for 2-3 h as compared to control cells. We propose antagonizing c-Cbl will partially alter receptor ubiquitylation and endocytic trafficking but this is sufficient to enhance downstream signaling.},
}
@article {pmid33810044,
year = {2021},
author = {Usman, B and Zhao, N and Nawaz, G and Qin, B and Liu, F and Liu, Y and Li, R},
title = {CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.},
journal = {International journal of molecular sciences},
volume = {22},
number = {6},
pages = {},
pmid = {33810044},
issn = {1422-0067},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Cysteine Proteinase Inhibitors ; DNA, Bacterial/genetics ; Edible Grain/genetics/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Gene Order ; Gene Regulatory Networks ; *Genes, Plant ; Genetic Association Studies ; Genetic Vectors/genetics ; Genome, Plant ; Genotyping Techniques ; *Mutagenesis ; Mutation ; Oryza/classification/*genetics/metabolism ; Plant Breeding ; Plant Proteins/*genetics/metabolism ; Proteomics ; *Quantitative Trait, Heritable ; Signal Transduction ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.},
}
@article {pmid33809410,
year = {2021},
author = {Adiguzel, MC and Goulart, DB and Wu, Z and Pang, J and Cengiz, S and Zhang, Q and Sahin, O},
title = {Distribution of CRISPR Types in Fluoroquinolone-Resistant Campylobacter jejuni Isolates.},
journal = {Pathogens (Basel, Switzerland)},
volume = {10},
number = {3},
pages = {},
pmid = {33809410},
issn = {2076-0817},
abstract = {To aid development of phage therapy against Campylobacter, we investigated the distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) systems in fluoroquinolone (FQ)-resistant Campylobacter jejuni. A total of 100 FQ-resistant C. jejuni strains from different sources were analyzed by PCR and DNA sequencing to determine resistance-conferring mutation in the gyrA gene and the presence of various CRISPR systems. All but one isolate harbored 1-5 point mutations in gyrA, and the most common mutation was the Thr86Ile change. Ninety-five isolates were positive with the CRISPR PCR, and spacer sequences were found in 86 of them. Among the 292 spacer sequences identified in this study, 204 shared 93-100% nucleotide homology to Campylobacter phage D10, 44 showed 100% homology to Campylobacter phage CP39, and 3 had 100% homology with Campylobacter phage CJIE4-5. The remaining 41 spacer sequences did not match with any phages in the database. Based on the results, it was inferred that the FQ-resistant C. jejuni isolates analyzed in this study were potentially resistant to Campylobacter phages D10, CP39, and CJIE4-5 as well as some unidentified phages. These phages should be excluded from cocktails of phages that may be utilized to treat FQ-resistant Campylobacter.},
}
@article {pmid33808947,
year = {2021},
author = {Mahas, A and Hassan, N and Aman, R and Marsic, T and Wang, Q and Ali, Z and Mahfouz, MM},
title = {LAMP-Coupled CRISPR-Cas12a Module for Rapid and Sensitive Detection of Plant DNA Viruses.},
journal = {Viruses},
volume = {13},
number = {3},
pages = {},
pmid = {33808947},
issn = {1999-4915},
mesh = {Begomovirus/*genetics/isolation &amp; purification ; Biosensing Techniques/methods ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Plant/genetics ; Genome, Viral/genetics ; Lycopersicon esculentum/virology ; Molecular Diagnostic Techniques/methods ; Plant Diseases/*virology ; },
abstract = {One important factor for successful disease management is the ability to rapidly and accurately identify the causal agent. Plant viruses cause severe economic losses and pose a serious threat to sustainable agriculture. Therefore, optimization of the speed, sensitivity, feasibility, portability, and accuracy of virus detection is urgently needed. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid diagnostic method utilizing the CRISPR-Cas12a system for detecting two geminiviruses, tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV), which have single-stranded DNA genomes. Our assay detected TYLCV and ToLCNDV in infected plants with high sensitivity and specificity. Our newly developed assay can be performed in ~1 h and provides easy-to-interpret visual readouts using a simple, low-cost fluorescence visualizer, making it suitable for point-of-use applications.},
}
@article {pmid33808752,
year = {2021},
author = {Park, BJ and Park, MS and Lee, JM and Song, YJ},
title = {Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay.},
journal = {Biosensors},
volume = {11},
number = {3},
pages = {},
pmid = {33808752},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Herpesvirus 1, Cercopithecine ; Humans ; Influenza A virus/*isolation &amp; purification ; Influenza B virus/*isolation &amp; purification ; Influenza, Human/diagnosis ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; Pandemics ; Reverse Transcription ; Sensitivity and Specificity ; },
abstract = {A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 10[0] plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.},
}
@article {pmid33807610,
year = {2021},
author = {Diakatou, M and Dubois, G and Erkilic, N and Sanjurjo-Soriano, C and Meunier, I and Kalatzis, V},
title = {Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3.},
journal = {International journal of molecular sciences},
volume = {22},
number = {5},
pages = {},
pmid = {33807610},
issn = {1422-0067},
mesh = {Alleles ; Animals ; Base Sequence ; COS Cells ; Cell Line ; Chlorocebus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genes, Dominant/*genetics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/physiology ; Mutation/*genetics ; Orphan Nuclear Receptors/genetics ; Retina/physiology ; Retinitis Pigmentosa/*genetics ; },
abstract = {Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.},
}
@article {pmid33806233,
year = {2021},
author = {Numan, M and Khan, AL and Asaf, S and Salehin, M and Beyene, G and Tadele, Z and Ligaba-Osena, A},
title = {From Traditional Breeding to Genome Editing for Boosting Productivity of the Ancient Grain Tef [Eragrostis tef (Zucc.) Trotter].},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {4},
pages = {},
pmid = {33806233},
issn = {2223-7747},
abstract = {Tef (Eragrostis tef (Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population and is currently cultivated in several countries for grain and forage production. It is one of the most nutritious grains, and is also more resilient to marginal soil and climate conditions than major cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during the growing season. Climate change is triggering several biotic and abiotic stresses which are expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative and robust approach in order to improve resilience to diverse types of stresses and increase crop yields. Traditional breeding has been extensively implemented to develop crop varieties with traits of interest, although the technique has several limitations. Currently, genome editing technologies are receiving increased interest among plant biologists as a means of improving key agronomic traits. In this review, the potential application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system. This is expected to improve stress resilience and boost productivity, thereby ensuring food and nutrition security in the region where it is needed the most.},
}
@article {pmid33806186,
year = {2021},
author = {Gu, D and Xue, H and Yuan, X and Yu, J and Xu, X and Huang, Y and Li, M and Zhai, X and Pan, Z and Zhang, Y and Jiao, X},
title = {Genome-Wide Identification of Genes Involved in Acid Stress Resistance of Salmonella Derby.},
journal = {Genes},
volume = {12},
number = {4},
pages = {},
pmid = {33806186},
issn = {2073-4425},
mesh = {Acids/*pharmacology ; Adaptation, Physiological ; Animals ; Bacterial Proteins/genetics/*metabolism ; Gene Expression Regulation, Bacterial/*drug effects ; *Genome, Bacterial ; Genome-Wide Association Study ; Humans ; Salmonella/drug effects/*genetics/growth &amp; development/metabolism ; Salmonella Infections/genetics/metabolism/*microbiology ; *Stress, Physiological ; Swine ; },
abstract = {Resistance to and survival under acidic conditions are critical for Salmonella to infect the host. As one of the most prevalent serotypes identified in pigs and humans, how S. Derby overcomes acid stress remains unclear. Here, we de novo sequenced the genome of a representative S. Derby strain 14T from our S. Derby strain stock and identified its acid resistance-associated genes using Tn-seq analysis. A total of 35 genes, including those belonging to two-component systems (TCS) (cpxAR), the CRISPR-Cas system (casCE), and other systems, were identified as essential for 14T to survive under acid stress. The results demonstrated that the growth curve and survival ability of ΔcpxA and ΔcpxR were decreased under acid stress, and the adhesion and invasion abilities to the mouse colon cancer epithelial cells (MC38) of ΔcpxR were also decreased compared with the wild type strain, suggesting that the TCS CpxAR plays an essential role in the acid resistance and virulence of S. Derby. Also, CasC and CasE were found to be responsible for acid resistance in S. Derby. Our results indicate that acid stress induces multiple genes' expression to mediate the acid resistance of S. Derby and enhance its pathogenesis during an infection.},
}
@article {pmid33805897,
year = {2021},
author = {Sansbury, BM and Kmiec, EB},
title = {On the Origins of Homology Directed Repair in Mammalian Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33805897},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Cell Cycle ; DNA/*chemistry ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA, Single-Stranded ; Escherichia coli ; *Gene Editing ; Genetic Engineering ; Genome, Human ; Humans ; Mutagenesis ; *Mutation ; Oligonucleotides ; *Recombinational DNA Repair ; Saccharomyces cerevisiae ; },
abstract = {Over the course of the last five years, expectations surrounding our capacity to selectively modify the human genome have never been higher. The reduction to practice site-specific nucleases designed to cleave at a unique site within the DNA is now centerstage in the development of effective molecular therapies. Once viewed as being impossible, this technology now has great potential and, while cellular and molecular barriers persist to clinical implementations, there is little doubt that these barriers will be crossed, and human beings will soon be treated with gene editing tools. The most ambitious of these desires is the correction of genetic mutations resident within the human genome that are responsible for oncogenesis and a wide range of inherited diseases. The process by which gene editing activity could act to reverse these mutations to wild-type and restore normal protein function has been generally categorized as homology directed repair. This is a catch-all basket term that includes the insertion of short fragments of DNA, the replacement of long fragments of DNA, and the surgical exchange of single bases in the correction of point mutations. The foundation of homology directed repair lies in pioneering work that unravel the mystery surrounding genetic exchange using single-stranded DNA oligonucleotides as the sole gene editing agent. Single agent gene editing has provided guidance on how to build combinatorial approaches to human gene editing using the remarkable programmable nuclease complexes known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their closely associated (Cas) nucleases. In this manuscript, we outline the historical pathway that has helped evolve the current molecular toolbox being utilized for the genetic re-engineering of the human genome.},
}
@article {pmid33805879,
year = {2021},
author = {Chen, M and Zhu, X and Liu, X and Wu, C and Yu, C and Hu, G and Chen, L and Chen, R and Bouzayen, M and Zouine, M and Hao, Y},
title = {Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33805879},
issn = {1422-0067},
mesh = {Abscisic Acid/chemistry/metabolism ; Arabidopsis Proteins/genetics ; CRISPR-Cas Systems ; Chlorophyll/chemistry ; *Droughts ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Glucuronidase/metabolism ; Indoleacetic Acids/*metabolism ; Lycopersicon esculentum/*genetics ; Malondialdehyde/chemistry ; Mutation ; Plant Leaves/metabolism ; Plant Proteins/*genetics/metabolism ; Plant Stomata/metabolism ; Plants, Genetically Modified/metabolism ; RNA-Seq ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcription Factors/*genetics/metabolism ; Transcriptome ; Water/metabolism ; },
abstract = {Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and β-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes' promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.},
}
@article {pmid33805827,
year = {2021},
author = {Halat, M and Klimek-Chodacka, M and Orleanska, J and Baranska, M and Baranski, R},
title = {Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex.},
journal = {International journal of molecular sciences},
volume = {22},
number = {6},
pages = {},
pmid = {33805827},
issn = {1422-0067},
mesh = {Base Pairing ; Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Circular Dichroism ; DNA/*chemistry/genetics/metabolism ; Gene Editing/methods ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; RNA, Guide/*chemistry/genetics/metabolism ; Ribonucleoproteins/*chemistry/genetics/metabolism ; Streptococcus pyogenes/*chemistry/enzymology ; },
abstract = {The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.},
}
@article {pmid33805113,
year = {2021},
author = {Nidhi, S and Anand, U and Oleksak, P and Tripathi, P and Lal, JA and Thomas, G and Kuca, K and Tripathi, V},
title = {Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33805113},
issn = {1422-0067},
mesh = {Animals ; Archaea/metabolism ; Bacteria/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Genetic Engineering/history/*methods ; Genome ; History, 20th Century ; History, 21st Century ; Humans ; Livestock ; },
abstract = {According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.},
}
@article {pmid33804420,
year = {2021},
author = {Achigar, R and Scarrone, M and Rousseau, GM and Philippe, C and Machado, F and Duvós, V and Campot, MP and Dion, MB and Shao, Y and Pianzzola, MJ and Moineau, S},
title = {Ectopic Spacer Acquisition in Streptococcus thermophilus CRISPR3 Array.},
journal = {Microorganisms},
volume = {9},
number = {3},
pages = {},
pmid = {33804420},
issn = {2076-2607},
abstract = {Streptococcus thermophilus relies heavily on two type II-A CRISPR-Cas systems, CRISPR1 and CRISPR3, to resist siphophage infections. One hallmark of these systems is the integration of a new spacer at the 5' end of the CRISPR arrays following phage infection. However, we have previously shown that ectopic acquisition of spacers can occur within the CRISPR1 array. Here, we present evidence of the acquisition of new spacers within the array of CRISPR3 of S. thermophilus. The analysis of randomly selected bacteriophage-insensitive mutants of the strain Uy01 obtained after phage infection, as well as the comparison with other S. thermophilus strains with similar CRISPR3 content, showed that a specific spacer within the array could be responsible for misguiding the adaptation complex. These results also indicate that while the vast majority of new spacers are added at the 5' end of the CRISPR array, ectopic spacer acquisition is a common feature of both CRISPR1 and CRISPR3 systems in S. thermophilus, and it can still provide phage resistance. Ectopic spacer acquisition also appears to have occurred naturally in some strains of Streptococcus pyogenes, suggesting that it is a general phenomenon, at least in type II-A systems.},
}
@article {pmid33803852,
year = {2021},
author = {Liu, Y and Tian, S and Thaker, H and Dong, M},
title = {Shiga Toxins: An Update on Host Factors and Biomedical Applications.},
journal = {Toxins},
volume = {13},
number = {3},
pages = {},
pmid = {33803852},
issn = {2072-6651},
support = {T32 DK060442/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Escherichia coli Infections/genetics/*metabolism/microbiology ; Hemolytic-Uremic Syndrome/genetics/*metabolism/microbiology ; Host-Pathogen Interactions ; Humans ; Immunotoxins/therapeutic use ; Models, Molecular ; Neoplasms/drug therapy/immunology/metabolism/pathology ; Protein Conformation ; Shiga Toxins/chemistry/genetics/*metabolism/therapeutic use ; Shiga-Toxigenic Escherichia coli/genetics/*metabolism ; Structure-Activity Relationship ; Trihexosylceramides/*metabolism ; },
abstract = {Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.},
}
@article {pmid33802904,
year = {2021},
author = {Parsons, C and Brown, P and Kathariou, S},
title = {Use of Bacteriophage Amended with CRISPR-Cas Systems to Combat Antimicrobial Resistance in the Bacterial Foodborne Pathogen Listeria monocytogenes.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {3},
pages = {},
pmid = {33802904},
issn = {2079-6382},
abstract = {Listeria monocytogenes is a bacterial foodborne pathogen and the causative agent of the disease listeriosis, which though uncommon can result in severe symptoms such as meningitis, septicemia, stillbirths, and abortions and has a high case fatality rate. This pathogen can infect humans and other animals, resulting in massive health and economic impacts in the United States and globally. Listeriosis is treated with antimicrobials, typically a combination of a beta-lactam and an aminoglycoside, and L. monocytogenes has remained largely susceptible to the drugs of choice. However, there are several reports of antimicrobial resistance (AMR) in both L. monocytogenes and other Listeria species. Given the dire health outcomes associated with listeriosis, the prospect of antimicrobial-resistant L. monocytogenes is highly problematic for human and animal health. Developing effective tools for the control and elimination of L. monocytogenes, including strains with antimicrobial resistance, is of the utmost importance to prevent further dissemination of AMR in this pathogen. One tool that has shown great promise in combating antibiotic-resistant pathogens is the use of bacteriophages (phages), which are natural bacterial predators and horizontal gene transfer agents. Although native phages can be effective at killing antibiotic-resistant pathogens, limited host ranges and evolved resistance to phages can compromise their use in the efforts to mitigate the global AMR challenge. However, recent advances can allow the use of CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) to selectively target pathogens and their AMR determinants. Employment of CRISPR-Cas systems for phage amendment can overcome previous limitations in using phages as biocontrol and allow for the effective control of L. monocytogenes and its AMR determinants.},
}
@article {pmid33802772,
year = {2021},
author = {Ge, H and Marchisio, MA},
title = {Aptamers, Riboswitches, and Ribozymes in S. cerevisiae Synthetic Biology.},
journal = {Life (Basel, Switzerland)},
volume = {11},
number = {3},
pages = {},
pmid = {33802772},
issn = {2075-1729},
abstract = {Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at the mRNA level without the need for transcription factors. Potentially, the use of chemical-responsive ribozymes/riboswitches would drastically simplify the design of genetic circuits. In this review, we describe synthetic RNA structures that have been used so far in the yeast Saccharomyces cerevisiae. We present their interaction mode with different chemicals (e.g., theophylline and antibiotics) or proteins (such as the RNase III) and their recent employment into clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas) systems. Particular attention is paid, throughout the whole paper, to their usage and performance into synthetic gene circuits.},
}
@article {pmid33801907,
year = {2021},
author = {Zakaria, MM and Schemmerling, B and Ober, D},
title = {CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.},
journal = {Molecules (Basel, Switzerland)},
volume = {26},
number = {6},
pages = {},
pmid = {33801907},
issn = {1420-3049},
mesh = {Alkyl and Aryl Transferases/*genetics/metabolism ; Boraginaceae/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Comfrey/*genetics/metabolism ; Gene Editing/methods ; Gene Expression Regulation, Plant/genetics ; Plant Roots/genetics ; Plants, Medicinal/genetics ; Pyrrolizidine Alkaloids/chemistry/*metabolism ; },
abstract = {Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.},
}
@article {pmid33801686,
year = {2021},
author = {Salanga, CM and Salanga, MC},
title = {Genotype to Phenotype: CRISPR Gene Editing Reveals Genetic Compensation as a Mechanism for Phenotypic Disjunction of Morphants and Mutants.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33801686},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Codon, Nonsense ; Gene Editing/*methods ; *Genetic Association Studies ; Genetic Techniques ; Genomics ; Genotype ; INDEL Mutation ; Mutagenesis ; *Mutation ; Nonsense Mediated mRNA Decay/*genetics ; Phenotype ; Transcription Activator-Like Effector Nucleases/*genetics ; Zebrafish ; },
abstract = {Forward genetic screens have shown the consequences of deleterious mutations; however, they are best suited for model organisms with fast reproductive rates and large broods. Furthermore, investigators must faithfully identify changes in phenotype, even if subtle, to realize the full benefit of the screen. Reverse genetic approaches also probe genotype to phenotype relationships, except that the genetic targets are predefined. Until recently, reverse genetic approaches relied on non-genomic gene silencing or the relatively inefficient, homology-dependent gene targeting for loss-of-function generation. Fortunately, the flexibility and simplicity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has revolutionized reverse genetics, allowing for the precise mutagenesis of virtually any gene in any organism at will. The successful integration of insertions/deletions (INDELs) and nonsense mutations that would, at face value, produce the expected loss-of-function phenotype, have been shown to have little to no effect, even if other methods of gene silencing demonstrate robust loss-of-function consequences. The disjunction between outcomes has raised important questions about our understanding of genotype to phenotype and highlights the capacity for compensation in the central dogma. This review describes recent studies in which genomic compensation appears to be at play, discusses the possible compensation mechanisms, and considers elements important for robust gene loss-of-function studies.},
}
@article {pmid33801499,
year = {2021},
author = {Bæksted Holme, I and Dionisio, G and Brinch-Pedersen, H},
title = {A Roadmap to Modulated Anthocyanin Compositions in Carrots.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {3},
pages = {},
pmid = {33801499},
issn = {2223-7747},
abstract = {Anthocyanins extracted from black carrots have received increased interest as natural colorants in recent years. The reason is mainly their high content of acylated anthocyanins that stabilizes the color and thereby increases the shelf-life of products colored with black carrot anthocyanins. Still, the main type of anthocyanins synthesized in all black carrot cultivars is cyanidin limiting their use as colorants due to the narrow color variation. Additionally, in order to be competitive against synthetic colors, a higher percentage of acylated anthocyanins and an increased anthocyanin content in black carrots are needed. However, along with the increased interest in black carrots there has also been an interest in identifying the structural and regulatory genes associated with anthocyanin biosynthesis in black carrots. Thus, huge progress in the identification of genes involved in anthocyanin biosynthesis has recently been achieved. Given this information it is now possible to attempt to modulate anthocyanin compositions in black carrots through genetic modifications. In this review we look into genetic modification opportunities for generating taproots of black carrots with extended color palettes, with a higher percentage of acylated anthocyanins or a higher total content of anthocyanins.},
}
@article {pmid33801123,
year = {2021},
author = {Ryczek, N and Hryhorowicz, M and Zeyland, J and Lipiński, D and Słomski, R},
title = {CRISPR/Cas Technology in Pig-to-Human Xenotransplantation Research.},
journal = {International journal of molecular sciences},
volume = {22},
number = {6},
pages = {},
pmid = {33801123},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA Repair ; *Gene Editing ; *Genetic Engineering ; Graft Rejection ; Graft Survival ; Heterografts ; Humans ; Models, Animal ; *Organ Transplantation/adverse effects/methods ; Swine ; *Transplantation, Heterologous ; },
abstract = {CRISPR/Cas (clustered regularly interspaced short palindromic repeats linked to Cas nuclease) technology has revolutionized many aspects of genetic engineering research. Thanks to it, it became possible to study the functions and mechanisms of biology with greater precision, as well as to obtain genetically modified organisms, both prokaryotic and eukaryotic. The changes introduced by the CRISPR/Cas system are based on the repair paths of the single or double strand DNA breaks that cause insertions, deletions, or precise integrations of donor DNA. These changes are crucial for many fields of science, one of which is the use of animals (pigs) as a reservoir of tissues and organs for xenotransplantation into humans. Non-genetically modified animals cannot be used to save human life and health due to acute immunological reactions resulting from the phylogenetic distance of these two species. This review is intended to collect and summarize the advantages as well as achievements of the CRISPR/Cas system in pig-to-human xenotransplantation research. In addition, it demonstrates barriers and limitations that require careful evaluation before attempting to experiment with this technology.},
}
@article {pmid33800815,
year = {2021},
author = {Costamagna, G and Comi, GP and Corti, S},
title = {Advancing Drug Discovery for Neurological Disorders Using iPSC-Derived Neural Organoids.},
journal = {International journal of molecular sciences},
volume = {22},
number = {5},
pages = {},
pmid = {33800815},
issn = {1422-0067},
mesh = {Animals ; Automation ; Brain/cytology ; CRISPR-Cas Systems ; Cell Culture Techniques ; Collagen ; Drug Combinations ; Drug Discovery/*methods ; Drug Evaluation, Preclinical/methods ; Drug Industry/organization &amp; administration ; Forecasting ; High-Throughput Screening Assays ; Humans ; Induced Pluripotent Stem Cells/cytology/*drug effects ; Laminin ; Machine Learning ; Microscopy/methods ; Nervous System Diseases/*drug therapy/pathology ; Organoids/*drug effects ; Proteoglycans ; RNA-Seq ; Reproducibility of Results ; Single-Cell Analysis ; },
abstract = {In the last decade, different research groups in the academic setting have developed induced pluripotent stem cell-based protocols to generate three-dimensional, multicellular, neural organoids. Their use to model brain biology, early neural development, and human diseases has provided new insights into the pathophysiology of neuropsychiatric and neurological disorders, including microcephaly, autism, Parkinson's disease, and Alzheimer's disease. However, the adoption of organoid technology for large-scale drug screening in the industry has been hampered by challenges with reproducibility, scalability, and translatability to human disease. Potential technical solutions to expand their use in drug discovery pipelines include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create isogenic models, single-cell RNA sequencing to characterize the model at a cellular level, and machine learning to analyze complex data sets. In addition, high-content imaging, automated liquid handling, and standardized assays represent other valuable tools toward this goal. Though several open issues still hamper the full implementation of the organoid technology outside academia, rapid progress in this field will help to prompt its translation toward large-scale drug screening for neurological disorders.},
}
@article {pmid33800561,
year = {2021},
author = {Morimoto, T and Nakazawa, T and Matsuda, R and Nishimura, F and Nakamura, M and Yamada, S and Nakagawa, I and Park, YS and Tsujimura, T and Nakase, H},
title = {CRISPR-Cas9-Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {7},
pages = {},
pmid = {33800561},
issn = {1422-0067},
mesh = {Brain Neoplasms/*genetics/metabolism/therapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Genome, Human ; Glioma/*genetics/metabolism/therapy ; Hepatitis A Virus Cellular Receptor 2/*genetics ; Humans ; Immunotherapy/methods ; Kaplan-Meier Estimate ; Killer Cells, Natural/metabolism ; Ligands ; Oligonucleotide Array Sequence Analysis ; RNA, Guide/metabolism ; Transgenes ; },
abstract = {Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell-mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.},
}
@article {pmid33799666,
year = {2021},
author = {Stamereilers, C and Wong, S and Tsourkas, PK},
title = {Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages.},
journal = {Viruses},
volume = {13},
number = {3},
pages = {},
pmid = {33799666},
issn = {1999-4915},
mesh = {Animals ; Bacteriophages/*genetics ; Bees/microbiology ; CRISPR-Cas Systems/*genetics ; DNA, Intergenic/analysis/*genetics ; Genome, Bacterial ; Genome, Viral ; Paenibacillus larvae/*genetics/immunology/*virology ; Prophages/genetics ; Sequence Analysis, DNA ; },
abstract = {The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage-host systems.},
}
@article {pmid33799639,
year = {2021},
author = {He, L and Matošević, ZJ and Mitić, D and Markulin, D and Killelea, T and Matković, M and Bertoša, B and Ivančić-Baće, I and Bolt, EL},
title = {A Tryptophan 'Gate' in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity.},
journal = {International journal of molecular sciences},
volume = {22},
number = {6},
pages = {},
pmid = {33799639},
issn = {1422-0067},
mesh = {Adenosine Triphosphate/chemistry/genetics/metabolism ; Alanine/chemistry/genetics/metabolism ; Amino Acid Sequence ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Catalytic Domain/genetics ; Circular Dichroism ; DNA/chemistry/genetics/*metabolism ; DNA Helicases/chemistry/genetics/*metabolism ; DNA, Single-Stranded/chemistry/genetics/*metabolism ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/chemistry/genetics/*metabolism ; Gene Editing/methods ; Humans ; Mutation, Missense ; Protein Conformation ; Sequence Homology, Amino Acid ; Temperature ; Tryptophan/chemistry/genetics/*metabolism ; },
abstract = {Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3[W406A] protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3[W406A] protein may offer improved Cas3-based genetic editing in human cells.},
}
@article {pmid33799339,
year = {2021},
author = {Murtazina, RZ and Zhukov, IS and Korenkova, OM and Popova, EA and Kuvarzin, SR and Efimova, EV and Kubarskaya, LG and Batotsyrenova, EG and Zolotoverkhaya, EA and Vaganova, AN and Apryatin, SA and Alenina, NV and Gainetdinov, RR},
title = {Genetic Deletion of Trace-Amine Associated Receptor 9 (TAAR9) in Rats Leads to Decreased Blood Cholesterol Levels.},
journal = {International journal of molecular sciences},
volume = {22},
number = {6},
pages = {},
pmid = {33799339},
issn = {1422-0067},
mesh = {Animals ; Atherosclerosis/blood/*genetics/pathology ; CRISPR-Cas Systems ; Central Nervous System/metabolism/pathology ; Cholesterol/*blood/genetics ; Disease Models, Animal ; Erythrocytes/metabolism/pathology ; Gene Knockout Techniques ; Humans ; Ligands ; Osmotic Fragility/genetics ; Rats ; Receptors, G-Protein-Coupled/*genetics ; },
abstract = {In the last two decades, interest has grown significantly in the investigation of the role of trace amines and their receptors in mammalian physiology and pathology. Trace amine-associated receptor 9 (TAAR9) is one of the least studied members of this receptor family with unidentified endogenous ligands and an unknown role in the central nervous system and periphery. In this study, we generated two new TAAR9 knockout (TAAR9-KO) rat strains by CRISPR-Cas9 technology as in vivo models to evaluate the role of TAAR9 in mammalian physiology. In these mutant rats, we performed a comparative analysis of a number of hematological and biochemical parameters in the blood. Particularly, we carried out a complete blood count, erythrocyte osmotic fragility test, and screening of a panel of basic biochemical parameters. No significant alterations in any of the hematological and most biochemical parameters were found between mutant and WT rats. However, biochemical studies revealed a significant decrease in total and low-density lipoprotein cholesterol levels in the blood of both strains of TAAR9-KO rats. Such role of TAAR9 in cholesterol regulation not only brings a new understanding of mechanisms and biological pathways of lipid exchange but also provides a new potential drug target for disorders involving cholesterol-related pathology, such as atherosclerosis.},
}
@article {pmid33799274,
year = {2021},
author = {Xiao, X and Chen, Y and Peng, L and Zhang, T},
title = {Generation of a homozygous ALX1 knockout human embryonic stem cell line (WAe001-A-060) by a CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102309},
doi = {10.1016/j.scr.2021.102309},
pmid = {33799274},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells ; Female ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {Human ALX1 gene (ALX Homeobox 1) is a protein coding gene and gene ontology annotations related to this gene include DNA-binding transcription factor activity and protein heterdimerization activity. It is necessary for survival of forebrain mesenchyme and may be involved in development of cervix. However, the function of the gene has yet to be determined in humans. Here we generated an ALX1 homozygous human embryonic stem cell line (WAe001-A-060) by a CRISPR/Cas9 system. The WAe001-A-060 has a normal undifferentiated morphology and karyotype, pluripotency and three germ layers differentiation potential in vivo.},
}
@article {pmid33798831,
year = {2021},
author = {Ezawa, M and Kouno, F and Kubo, H and Sakuma, T and Yamamoto, T and Kinoshita, T},
title = {Pou5f3.3 is involved in establishment and maintenance of hematopoietic cells during Xenopus development.},
journal = {Tissue &amp; cell},
volume = {72},
number = {},
pages = {101531},
doi = {10.1016/j.tice.2021.101531},
pmid = {33798831},
issn = {1532-3072},
mesh = {Anemia/pathology ; Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Movement ; *Embryonic Development ; Gene Expression Regulation, Developmental ; Hematopoiesis ; Hematopoietic Stem Cells/*metabolism ; Mutagenesis/genetics ; POU Domain Factors/blood/genetics/*metabolism ; Xenopus Proteins/blood/genetics/*metabolism ; Xenopus laevis/*embryology/genetics ; },
abstract = {Three POU family class V gene homologues are expressed in the development of Xenopus. In contrast to the expression of Pou5f3.1 and Pou5f3.2 in organogenesis, Pou5f3.3 is expressed during oogenesis in ovary. We investigated the expression and function of Pou5f3.3 in organogenesis of Xenopus laevis. RT-PCR and immunohistochemical analysis indicated that Pou5f3.3 was expressed in a small number of adult liver cells and blood cells. Immunocytochemical investigation proved that Bmi1, a marker for hematopoietic progenitor cells, was co-expressed in Pou5f3.3-expressing small spherical cells in the peripheral blood. In anemic induction by intraperitoneal injection of phenyl hydrazine, the number of Pou5f3.3-expressing cells significantly increased within 3 days after phenyl hydrazine injection. In CRISPR/Cas mutagenesis of Pou5f3.3, Bmi1-positive hematopoietic progenitor cell count decreased in the hematopoietic dorsal-lateral plate (DLP) region, resulting in a considerable reduction in peripheral blood cells. CRISPR/Cas-induced hematopoietic deficiency was completely rescued by Pou5f3.3 supplementation, but not by Pou5f3.1 or Pou5f3.2. Transplantation experiments using the H2B-GFP transgenic line demonstrated that DLP-derived Pou5f3.3-positive and Bmi1-positive cells were translocated into the liver and bone through the bloodstream. These results suggest that Pou5f3.3 plays an essential role in the establishment and maintenance of hematopoietic progenitor cells during Xenopus development.},
}
@article {pmid33797713,
year = {2021},
author = {Carrijo, J and Illa-Berenguer, E and LaFayette, P and Torres, N and Aragão, FJL and Parrott, W and Vianna, GR},
title = {Two efficient CRISPR/Cas9 systems for gene editing in soybean.},
journal = {Transgenic research},
volume = {30},
number = {3},
pages = {239-249},
pmid = {33797713},
issn = {1573-9368},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genetic Engineering ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/genetics ; Soybeans/*genetics/growth &amp; development ; },
abstract = {Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.},
}
@article {pmid33797415,
year = {2021},
author = {Mckay, A and Burgio, G},
title = {Harnessing CRISPR-Cas system diversity for gene editing technologies.},
journal = {Journal of biomedical research},
volume = {35},
number = {2},
pages = {91-106},
pmid = {33797415},
issn = {1674-8301},
abstract = {The discovery and utilization of RNA-guided surveillance complexes, such as CRISPR-Cas9, for sequence-specific DNA or RNA cleavage, has revolutionised the process of gene modification or knockdown. To optimise the use of this technology, an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements, coupled with high cleavage efficacy and specificity. Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.},
}
@article {pmid33797032,
year = {2021},
author = {Wei, Y and Zhou, Y and Liu, Y and Ying, W and Lv, R and Zhao, Q and Zhou, H and Zuo, E and Sun, Y and Yang, H and Zhou, C},
title = {Indiscriminate ssDNA cleavage activity of CRISPR-Cas12a induces no detectable off-target effects in mouse embryos.},
journal = {Protein &amp; cell},
volume = {12},
number = {9},
pages = {741-745},
pmid = {33797032},
issn = {1674-8018},
mesh = {Animals ; Bacterial Proteins/*genetics/metabolism ; Blastomeres/cytology/*metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; DNA Cleavage ; DNA, Single-Stranded/chemistry/*genetics/metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endodeoxyribonucleases/*genetics/metabolism ; Gene Editing/*methods ; Genes, Reporter ; Genetic Therapy/methods/trends ; *Genome ; Green Fluorescent Proteins/genetics/metabolism ; INDEL Mutation ; Mice ; Polymorphism, Single Nucleotide ; Whole Genome Sequencing ; },
}
@article {pmid33796478,
year = {2021},
author = {Azmi, I and Faizan, MI and Kumar, R and Raj Yadav, S and Chaudhary, N and Kumar Singh, D and Butola, R and Ganotra, A and Datt Joshi, G and Deep Jhingan, G and Iqbal, J and Joshi, MC and Ahmad, T},
title = {A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {632646},
pmid = {33796478},
issn = {2235-2988},
support = {IA/I/15/2/502086/WTDBT_/DBT-Wellcome Trust India Alliance/India ; },
mesh = {COVID-19/*diagnosis ; COVID-19 Testing/*methods ; *CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; RNA, Viral/*isolation &amp; purification ; Real-Time Polymerase Chain Reaction ; SARS-CoV-2/genetics/*isolation &amp; purification ; Saliva/*chemistry ; Sensitivity and Specificity ; Smartphone ; Specimen Handling/methods ; Workflow ; },
abstract = {A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based &amp; Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.},
}
@article {pmid33795753,
year = {2021},
author = {Nguyen, ND and Matsuura, T and Kato, Y and Watanabe, H},
title = {DNMT3.1 controls trade-offs between growth, reproduction, and life span under starved conditions in Daphnia magna.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7326},
pmid = {33795753},
issn = {2045-2322},
mesh = {Adaptation, Physiological ; Alleles ; Animals ; Body Size ; CRISPR-Cas Systems ; DNA (Cytosine-5-)-Methyltransferases/*genetics ; DNA Methyltransferase 3A ; Daphnia/*metabolism ; *Food Deprivation ; Gene Expression Regulation ; Life History Traits ; *Longevity ; Mitosis ; Molecular Biology ; Mutation ; Phenotype ; RNA/metabolism ; RNA-Seq ; Reproduction ; Transcriptome ; Vitellogenins/metabolism ; },
abstract = {The cladoceran crustacean Daphnia has long been a model of energy allocation studies due to its important position in the trophic cascade of freshwater ecosystems. However, the loci for controlling energy allocation between life history traits still remain unknown. Here, we report CRISPR/Cas-mediated target mutagenesis of DNA methyltransferase 3.1 (DNMT3.1) that is upregulated in response to caloric restriction in Daphnia magna. The resulting biallelic mutant is viable and did not show any change in growth rate, reproduction, and longevity under nutrient rich conditions. In contrast, under starved conditions, the growth rate of this DNMT3.1 mutant was increased but its reproduction was reciprocally reduced compared to the wild type when the growth and reproduction activities competed during a period from instar 4 to 8. The life span of this mutant was significantly shorter than that of the wild type. We also compared transcriptomes between DNMT3.1 mutant and wild type under nutrient-rich and starved conditions. Consistent with the DNMT3.1 mutant phenotypes, the starved condition led to changes in the transcriptomes of the mutant including differential expression of vitellogenin genes. In addition, we found upregulation of the I am not dead yet (INDY) ortholog, which has been known to shorten the life span in Drosophila, explaining the shorter life span of the DNMT3.1 mutant. These results establish DNMT3.1 as a key regulator for life span and energy allocation between growth and reproduction during caloric restriction. Our findings reveal how energy allocation is implemented by selective expression of a DNMT3 ortholog that is widely distributed among animals. We also infer a previously unidentified adaptation of Daphnia that invests more energy for reproduction than growth under starved conditions.},
}
@article {pmid33795333,
year = {2021},
author = {Lu, H and Liu, J and Feng, T and Guo, Z and Yin, Y and Gao, F and Cao, G and Du, X and Wu, S},
title = {A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor.},
journal = {Genome research},
volume = {31},
number = {5},
pages = {900-909},
pmid = {33795333},
issn = {1549-5469},
mesh = {*Alleles ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mutagenesis, Insertional ; Mutation ; Swine ; },
abstract = {Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.},
}
@article {pmid33794364,
year = {2022},
author = {Vaidyanathan, S and Baik, R and Chen, L and Bravo, DT and Suarez, CJ and Abazari, SM and Salahudeen, AA and Dudek, AM and Teran, CA and Davis, TH and Lee, CM and Bao, G and Randell, SH and Artandi, SE and Wine, JJ and Kuo, CJ and Desai, TJ and Nayak, JV and Sellers, ZM and Porteus, MH},
title = {Targeted replacement of full-length CFTR in human airway stem cells by CRISPR-Cas9 for pan-mutation correction in the endogenous locus.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {30},
number = {1},
pages = {223-237},
pmid = {33794364},
issn = {1525-0024},
support = {K08 DK124684/DK/NIDDK NIH HHS/United States ; U01 CA176299/CA/NCI NIH HHS/United States ; R01 HL151677/HL/NHLBI NIH HHS/United States ; R35 CA197563/CA/NCI NIH HHS/United States ; R01 AG056575/AG/NIA NIH HHS/United States ; P30 DK065988/DK/NIDDK NIH HHS/United States ; K08 DE027730/DE/NIDCR NIH HHS/United States ; U01 DK085527/DK/NIDDK NIH HHS/United States ; U01 CA217851/CA/NCI NIH HHS/United States ; U19 AI116484/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cystic Fibrosis/genetics/metabolism/therapy ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Epithelial Cells/metabolism ; Humans ; Mutation ; Stem Cells/metabolism ; },
abstract = {Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19[+] UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.},
}
@article {pmid33793824,
year = {2021},
author = {Riediger, M and Spät, P and Bilger, R and Voigt, K and Maček, B and Hess, WR},
title = {Analysis of a photosynthetic cyanobacterium rich in internal membrane systems via gradient profiling by sequencing (Grad-seq).},
journal = {The Plant cell},
volume = {33},
number = {2},
pages = {248-269},
pmid = {33793824},
issn = {1532-298X},
mesh = {Bacterial Proteins/metabolism ; Gene Expression Regulation, Bacterial ; Intracellular Membranes/*metabolism ; Multiprotein Complexes/metabolism ; *Photosynthesis/genetics ; Phylogeny ; Protein Binding ; Protein Biosynthesis ; Proteome/metabolism ; RNA, Bacterial/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Ribonucleoproteins/metabolism ; *Sequence Analysis, RNA ; Synechocystis/*genetics ; Thylakoids/metabolism ; Toxin-Antitoxin Systems ; Transcriptome/genetics ; },
abstract = {Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.},
}
@article {pmid33793816,
year = {2021},
author = {Lopez, FB and Fort, A and Tadini, L and Probst, AV and McHale, M and Friel, J and Ryder, P and Pontvianne, FDR and Pesaresi, P and Sulpice, R and McKeown, P and Brychkova, G and Spillane, C},
title = {Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number.},
journal = {The Plant cell},
volume = {33},
number = {4},
pages = {1135-1150},
pmid = {33793816},
issn = {1532-298X},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems ; Chromatin/genetics ; DNA, Ribosomal/genetics ; *Dosage Compensation, Genetic ; *Gene Dosage ; Gene Expression Regulation, Plant ; Genomic Instability ; Plants, Genetically Modified ; RNA, Ribosomal/metabolism ; },
abstract = {The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as 'back-up' copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.},
}
@article {pmid33792878,
year = {2021},
author = {Yi, B and Larter, K and Xi, Y},
title = {CRISPR/Cas9 System to Knockdown MicroRNA In Vitro and In Vivo.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2300},
number = {},
pages = {133-139},
pmid = {33792878},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; DEAD-box RNA Helicases/genetics ; Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques/*methods ; Mice ; MicroRNAs/*genetics ; Plasmids/genetics ; RNA, Messenger/*genetics ; Ribonuclease III/genetics ; Transfection ; },
abstract = {MicroRNAs (miRNAs) are a class of small noncoding single-stranded RNA molecules containing 18-22 nucleotides that play an important role in the regulation of gene expression at the post-transcriptional and translational levels. Loss-of-function studies are the fundamental strategy to examine miRNA function and target genes in cellular and molecular biology. Traditional methods for miRNA loss-of-function studies include miRNA-specific antisense inhibitors, miRNA sponges, and genetic knockout. However, efficiency, specificity, and stability of these methods are not adequate. Our study suggests that CRISPR/Cas9 is an economic, convenient, and innovative strategy with high efficiency, specificity, and stability for the modulation of miRNA expression. Herein, we describe a detailed protocol for knocking out miRNA genes in vitro and in vivo with the CRISPR/Cas9 system.},
}
@article {pmid33791773,
year = {2021},
author = {Mizuguchi, T and Toyota, T and Miyatake, S and Mitsuhashi, S and Doi, H and Kudo, Y and Kishida, H and Hayashi, N and Tsuburaya, RS and Kinoshita, M and Fukuyama, T and Fukuda, H and Koshimizu, E and Tsuchida, N and Uchiyama, Y and Fujita, A and Takata, A and Miyake, N and Kato, M and Tanaka, F and Adachi, H and Matsumoto, N},
title = {Complete sequencing of expanded SAMD12 repeats by long-read sequencing and Cas9-mediated enrichment.},
journal = {Brain : a journal of neurology},
volume = {144},
number = {4},
pages = {1103-1117},
doi = {10.1093/brain/awab021},
pmid = {33791773},
issn = {1460-2156},
mesh = {Adult ; Aged ; Aged, 80 and over ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA Repeat Expansion/*genetics ; Epilepsies, Myoclonic/*genetics ; Female ; Genetic Association Studies ; Humans ; Male ; Microsatellite Repeats ; Middle Aged ; Nerve Tissue Proteins/*genetics ; Sequence Analysis, DNA/*methods ; },
abstract = {A pentanucleotide TTTCA repeat insertion into a polymorphic TTTTA repeat element in SAMD12 causes benign adult familial myoclonic epilepsy. Although the precise determination of the entire SAMD12 repeat sequence is important for molecular diagnosis and research, obtaining this sequence remains challenging when using conventional genomic/genetic methods, and even short-read and long-read next-generation sequencing technologies have been insufficient. Incomplete information regarding expanded repeat sequences may hamper our understanding of the pathogenic roles played by varying numbers of repeat units, genotype-phenotype correlations, and mutational mechanisms. Here, we report a new approach for the precise determination of the entire expanded repeat sequence and present a workflow designed to improve the diagnostic rates in various repeat expansion diseases. We examined 34 clinically diagnosed benign adult familial myoclonic epilepsy patients, from 29 families using repeat-primed PCR, Southern blot, and long-read sequencing with Cas9-mediated enrichment. Two cases with questionable results from repeat-primed PCR and/or Southern blot were confirmed as pathogenic using long-read sequencing with Cas9-mediated enrichment, resulting in the identification of pathogenic SAMD12 repeat expansions in 76% of examined families (22/29). Importantly, long-read sequencing with Cas9-mediated enrichment was able to provide detailed information regarding the sizes, configurations, and compositions of the expanded repeats. The inserted TTTCA repeat size and the proportion of TTTCA sequences among the overall repeat sequences were highly variable, and a novel repeat configuration was identified. A genotype-phenotype correlation study suggested that the insertion of even short (TTTCA)14 repeats contributed to the development of benign adult familial myoclonic epilepsy. However, the sizes of the overall TTTTA and TTTCA repeat units are also likely to be involved in the pathology of benign adult familial myoclonic epilepsy. Seven unsolved SAMD12-negative cases were investigated using whole-genome long-read sequencing, and infrequent, disease-associated, repeat expansions were identified in two cases. The strategic workflow resolved two questionable SAMD12-positive cases and two previously SAMD12-negative cases, increasing the diagnostic yield from 69% (20/29 families) to 83% (24/29 families). This study indicates the significant utility of long-read sequencing technologies to explore the pathogenic contributions made by various repeat units in complex repeat expansions and to improve the overall diagnostic rate.},
}
@article {pmid33791736,
year = {2021},
author = {Liu, TY and Knott, GJ and Smock, DCJ and Desmarais, JJ and Son, S and Bhuiya, A and Jakhanwal, S and Prywes, N and Agrawal, S and de León Derby, MD and Switz, NA and Armstrong, M and Harris, AR and Charles, EJ and Thornton, BW and Fozouni, P and Shu, J and Stephens, SI and Kumar, GR and Zhao, C and Mok, A and Iavarone, AT and Escajeda, AM and McIntosh, R and Kim, SE and Dugan, EJ and , and Pollard, KS and Tan, MX and Ott, M and Fletcher, DA and Lareau, LF and Hsu, PD and Savage, DF and Doudna, JA},
title = {Accelerated RNA detection using tandem CRISPR nucleases.},
journal = {medRxiv : the preprint server for health sciences},
volume = {},
number = {},
pages = {},
pmid = {33791736},
support = {T32 GM007618/GM/NIGMS NIH HHS/United States ; },
abstract = {Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule [1,2] , but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ∼30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.},
}
@article {pmid33791325,
year = {2021},
author = {Silveira, MC and Rocha-de-Souza, CM and de Oliveira Santos, IC and Pontes, LDS and Oliveira, TRTE and Tavares-Teixeira, CB and Cossatis, NA and Pereira, NF and da Conceição-Neto, OC and da Costa, BS and Rodrigues, DCS and Albano, RM and da Silva, FAB and Marques, EA and Leão, RS and Carvalho-Assef, APD},
title = {Genetic Basis of Antimicrobial Resistant Gram-Negative Bacteria Isolated From Bloodstream in Brazil.},
journal = {Frontiers in medicine},
volume = {8},
number = {},
pages = {635206},
pmid = {33791325},
issn = {2296-858X},
abstract = {Multidrug-resistant microorganisms are a well-known global problem, and gram-negative bacilli are top-ranking. When these pathogens are associated with bloodstream infections (BSI), outcomes become even worse. Here we applied whole-genome sequencing to access information about clonal distribution, resistance mechanism diversity and other molecular aspects of gram-negative bacilli (GNB) isolated from bloodstream infections in Brazil. It was possible to highlight international high-risk clones circulating in the Brazilian territory, such as CC258 for Klebsiella pneumoniae, ST79 for Acinetobacter baumannii and ST233 for Pseudomonas aeruginosa. Important associations can be made such as a negative correlation between CRISPR-Cas and K. pneumoniae CC258, while the genes bla TEM, bla KPC and bla CTX-M are highly associated with this clone. Specific relationships between A. baumannii clones and bla OXA-51 variants were also observed. All P. aeruginosa ST233 isolates showed the genes bla VIM and bla OXA486. In addition, some trends could be identified, where a new P. aeruginosa MDR clone (ST3079), a novel A. baumannii clonal profile circulating in Brazil (ST848), and important resistance associations in the form of bla VIM-2 and bla IMP-56 being found together in one ST233 strain, stand out. Such findings may help to develop approaches to deal with BSI and even other nosocomial infections caused by these important GNB.},
}
@article {pmid33790381,
year = {2021},
author = {Singh, K and Cornell, CS and Jackson, R and Kabiri, M and Phipps, M and Desai, M and Fogle, R and Ying, X and Anarat-Cappillino, G and Geller, S and Johnson, J and Roberts, E and Malley, K and Devlin, T and DeRiso, M and Berthelette, P and Zhang, YV and Ryan, S and Rao, S and Thurberg, BL and Bangari, DS and Kyostio-Moore, S},
title = {CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {7254},
pmid = {33790381},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; *Gene Knockout Techniques ; Male ; Mice ; Mice, Knockout ; Phenylalanine Hydroxylase/*genetics ; Phenylketonurias/*genetics ; },
abstract = {Phenylketonuria (PKU) is an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development.},
}
@article {pmid33789675,
year = {2021},
author = {Salekdeh, PR and Ma'mani, L and Tavakkoly-Bazzaz, J and Mousavi, H and Modarressi, MH and Salekdeh, GH},
title = {Bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica nanoparticles: a promising nanocarrier for delivery of Cas9-sgRNA ribonucleoproteine.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {95},
pmid = {33789675},
issn = {1477-3155},
mesh = {Adsorption ; CRISPR-Cas Systems ; Cell Survival ; Cloning, Molecular ; Drug Liberation ; Dynamic Light Scattering ; Gene Editing/methods ; Guanidines/*chemistry ; Nanoparticles/*chemistry ; Polyethylene Glycols/*chemistry ; Polymers/chemistry ; RNA, Guide/genetics ; Ribonucleoproteins/*chemistry ; Silanes ; Streptococcus pyogenes/genetics ; },
abstract = {BACKGROUND: There is a great interest in the efficient intracellular delivery of Cas9-sgRNA ribonucleoprotein complex (RNP) and its possible applications for in vivo CRISPR-based gene editing. In this study, a nanoporous mediated gene-editing approach has been successfully performed using a bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica (PMO) nanoparticles (RNP@AGu@PEG1500-PMO) as a potent and biocompatible nanocarrier for RNP delivery.<br><br>RESULTS: The bi-functionalized MSN-based nanomaterials have been fully characterized using electron microscopy (TEM and SEM), nitrogen adsorption measurements, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and dynamic light scattering (DLS). The results confirm that AGu@PEG1500-PMO can be applied for gene-editing with an efficiency of about 40% as measured by GFP gene knockdown of HT1080-GFP cells with no notable change in the morphology of the cells.<br><br>CONCLUSIONS: Due to the high stability and biocompatibility, simple synthesis, and cost-effectiveness, the developed bi-functionalized PMO-based nano-network introduces a tailored nanocarrier that has remarkable potential as a promising trajectory for biomedical and RNP delivery applications.},
}
@article {pmid33789420,
year = {2021},
author = {V, K and Chandrashekar, BK and K, K and Ag, S and Makarla, U and Ramu, VS},
title = {Disruption in the DNA Mismatch Repair Gene MSH2 by CRISPR-Cas9 in Indica Rice Can Create Genetic Variability.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {14},
pages = {4144-4152},
doi = {10.1021/acs.jafc.1c00328},
pmid = {33789420},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; DNA Mismatch Repair/genetics ; Gene Editing ; Genome, Plant ; *Oryza/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {Genetic variation is crucial for crop improvement. We adopted a gene editing approach to create variations in the rice genome by targeting the mutator locus homolog 2 (MSH2), a DNA mismatch repair gene. The hypothesis is that disruption of the MSH2 gene leads to a reduced DNA mismatch repair that creates INDELs, resulting in altered phenotypes. The Indica rice (IR-64) genotype was transformed with a guide RNA targeted to the MSH2 gene using an Agrobacterium-mediated in planta method. Many plants showed integration of Cas9 and gRNA constructs in rice plants. One of the msh2 mutants showed a superior phenotype due to editing and possible INDELs in the whole genome. The stable integration of the transgene and its flanking sequence analysis confirms no disruption of any gene, and the observed phenotype is due to the mutations in the MSH2 gene. Few transgenic plants showed disruption of genes due to T-DNA integration that led to altered phenotypes. The plants with altered phenotypes having more tiller number, early flowering, and robust growth with a high biomass were identified. These genetically reprogrammed rice plants could be a potential resource to create more segregating population or act as donor lines to stabilize the important agronomic traits that may help in a speed breeding process.},
}
@article {pmid33788831,
year = {2021},
author = {Biayna, J and Garcia-Cao, I and Álvarez, MM and Salvadores, M and Espinosa-Carrasco, J and McCullough, M and Supek, F and Stracker, TH},
title = {Loss of the abasic site sensor HMCES is synthetic lethal with the activity of the APOBEC3A cytosine deaminase in cancer cells.},
journal = {PLoS biology},
volume = {19},
number = {3},
pages = {e3001176},
pmid = {33788831},
issn = {1545-7885},
mesh = {Adenocarcinoma of Lung/*genetics/metabolism ; Ataxia Telangiectasia Mutated Proteins/metabolism ; Carcinoma, Non-Small-Cell Lung/genetics/metabolism ; Cell Line, Tumor ; Checkpoint Kinase 1/metabolism ; Cytidine Deaminase/*genetics/metabolism ; Cytosine Deaminase/genetics/metabolism ; DNA/genetics/metabolism ; DNA Damage/genetics/physiology ; DNA Replication/genetics/physiology ; DNA-Binding Proteins/*genetics/metabolism ; Humans ; Proteins/*genetics/metabolism ; },
abstract = {Analysis of cancer mutagenic signatures provides information about the origin of mutations and can inform the use of clinical therapies, including immunotherapy. In particular, APOBEC3A (A3A) has emerged as a major driver of mutagenesis in cancer cells, and its expression results in DNA damage and susceptibility to treatment with inhibitors of the ATR and CHK1 checkpoint kinases. Here, we report the implementation of CRISPR/Cas-9 genetic screening to identify susceptibilities of multiple A3A-expressing lung adenocarcinoma (LUAD) cell lines. We identify HMCES, a protein recently linked to the protection of abasic sites, as a central protein for the tolerance of A3A expression. HMCES depletion results in synthetic lethality with A3A expression preferentially in a TP53-mutant background. Analysis of previous screening data reveals a strong association between A3A mutational signatures and sensitivity to HMCES loss and indicates that HMCES is specialized in protecting against a narrow spectrum of DNA damaging agents in addition to A3A. We experimentally show that both HMCES disruption and A3A expression increase susceptibility of cancer cells to ionizing radiation (IR), oxidative stress, and ATR inhibition, strategies that are often applied in tumor therapies. Overall, our results suggest that HMCES is an attractive target for selective treatment of A3A-expressing tumors.},
}
@article {pmid33786987,
year = {2021},
author = {Hoesl, C and Fröhlich, T and Posch, C and Kneitz, H and Goebeler, M and Schneider, MR and Dahlhoff, M},
title = {The transmembrane protein LRIG1 triggers melanocytic tumor development following chemically induced skin carcinogenesis.},
journal = {Molecular oncology},
volume = {15},
number = {8},
pages = {2140-2155},
pmid = {33786987},
issn = {1878-0261},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Heterografts ; Humans ; Melanoma/chemically induced/enzymology/*pathology ; Membrane Glycoproteins/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Phosphorylation ; Receptor Protein-Tyrosine Kinases/metabolism ; Skin Neoplasms/chemically induced/enzymology/*pathology ; },
abstract = {The incidence of melanoma and nonmelanoma skin cancer has increased tremendously in recent years. Although novel treatment options have significantly improved patient outcomes, the prognosis for most patients with an advanced disease remains dismal. It is, thus, imperative to understand the molecular mechanisms involved in skin carcinogenesis in order to develop new targeted treatment strategies. Receptor tyrosine kinases (RTK) like the ERBB receptor family, including EGFR/ERBB1, ERBB2/NEU, ERBB3, and ERBB4, are important regulators of skin homeostasis and their dysregulation often results in cancer, which makes them attractive therapeutic targets. Members of the leucine-rich repeats and immunoglobulin-like domains protein family (LRIG1-3) are ERBB regulators and thus potential therapeutic targets to manipulate ERBB receptors. Here, we analyzed the function of LRIG1 during chemically induced skin carcinogenesis in transgenic mice expressing LRIG1 in the skin under the control of the keratin 5 promoter (LRIG1-TG mice). We observed a significant induction of melanocytic tumor formation in LRIG1-TG mice and no difference in papilloma incidence between LRIG1-TG and control mice. Our findings also revealed that LRIG1 affects ERBB signaling via decreased phosphorylation of EGFR and increased activation of the oncoprotein ERBB2 during skin carcinogenesis. The epidermal proliferation rate was significantly decreased during epidermal tumorigenesis under LRIG1 overexpression, and the apoptosis marker cleaved caspase 3 was significantly activated in the epidermis of transgenic LRIG1 mice. Additionally, we detected LRIG1 expression in human cutaneous squamous cell carcinoma and melanoma samples. Therefore, we depleted LRIG1 in human melanoma cells (A375) by CRISPR/Cas9 technology and found that this caused EGFR and ERBB3 downregulation in A375 LRIG1 knockout cells 6 h following stimulation with EGF. In conclusion, our study demonstrated that LRIG1-TG mice develop melanocytic skin tumors during chemical skin carcinogenesis and a deletion of LRIG1 in human melanoma cells reduces EGFR and ERBB3 expression after EGF stimulation.},
}
@article {pmid33786461,
year = {2021},
author = {Hoppe, C and Ashe, HL},
title = {CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos.},
journal = {STAR protocols},
volume = {2},
number = {1},
pages = {100380},
pmid = {33786461},
issn = {2666-1667},
support = {/WT_/Wellcome Trust/United Kingdom ; 204832/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila/*embryology ; Gene Editing/*methods ; Gene Expression Profiling/*methods ; Genome ; Inverted Repeat Sequences/genetics ; RNA, Messenger/genetics ; },
abstract = {CRISPR-Cas9 genome editing has transformed biology by enabling site-specific genome modifications to be simply engineered. Here, we describe two CRISPR-Cas9 approaches to introduce MS2 stem-loop sequences into endogenous gene loci in Drosophila. This can facilitate live imaging of nascent transcription in Drosophila. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).},
}
@article {pmid33785958,
year = {2021},
author = {Miranda, AV and Wiyono, L and Nurachman, LA},
title = {Clustered regularly interspaced palindromic repeats-cas9-based strategies towards HIV eradication: A literature review.},
journal = {JPMA. The Journal of the Pakistan Medical Association},
volume = {71(Suppl 2)},
number = {2},
pages = {S134-S139},
pmid = {33785958},
issn = {0030-9982},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *HIV Infections/drug therapy/prevention &amp; control ; *HIV-1/genetics ; Humans ; Virus Latency ; },
abstract = {OBJECTIVE: Despite Human Immunodeficiency Virus (HIV) being a major global health burden, no currently available therapy can eliminate it. One of the major challenges in developing treatment is the presence of latent HIV reservoirs. On the other hand, development of Clustered Regularly Interspaced Palindromic Repeats-Cas9 (CRISPR-Cas9) has made genome editing possible and thus can be used to address HIV latency and successfully treat HIV. This literature review aims to identify and appraise existing CRISPR-Cas9 strategies that address HIV treatment, particularly during latency.<br><br>METHODS: The PubMed Database was used to retrieve relevant articles. This review included articles that mentioned the use of CRISPR-Cas9 as a treatment for HIV and are written in English and/or Indonesian language.<br><br>RESULTS: The included studies (n = 17) showed that the CRISPR-Cas9 system can be utilized to disrupt the HIV-1 genome to inhibit viral reproduction and virulence. This system can be further optimized by combining several CRISPR-Cas9 systems. However, the use of CRISPR-Cas9 may cause HIV resistance, particularly to its guide RNA. This technique has also never been applied in vivo, thus more research is needed before wider implementation. A limitation of this review is the lack of data regarding CRISPR-Cas9 systems quality in some studies, thus limiting appraisal.<br><br>CONCLUSIONS: While the use of CRISPR-Cas9 to cure HIV seems promising, further studies regarding CRISPR-Cas9 quality, potential for development of gRNA-resistant HIV-1 strains and in vivo demonstration of the techniques are needed to progress this concept toward HIV eradication.},
}
@article {pmid33785624,
year = {2021},
author = {Hoikkala, V and Ravantti, J and Díez-Villaseñor, C and Tiirola, M and Conrad, RA and McBride, MJ and Moineau, S and Sundberg, LR},
title = {Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System.},
journal = {mBio},
volume = {12},
number = {2},
pages = {},
pmid = {33785624},
issn = {2150-7511},
mesh = {Adaptation, Physiological ; Bacteriophages/*genetics/physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Flavobacterium/*genetics/physiology/*virology ; Genome, Bacterial ; RNA, Viral/genetics/*metabolism ; },
abstract = {CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules.IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader's genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system "borrows" acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.},
}
@article {pmid33785418,
year = {2021},
author = {Nie, JJ and Liu, Y and Qi, Y and Zhang, N and Yu, B and Chen, DF and Yang, M and Xu, FJ},
title = {Charge-reversal nanocomolexes-based CRISPR/Cas9 delivery system for loss-of-function oncogene editing in hepatocellular carcinoma.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {333},
number = {},
pages = {362-373},
doi = {10.1016/j.jconrel.2021.03.030},
pmid = {33785418},
issn = {1873-4995},
mesh = {Animals ; CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/drug therapy/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Liver Neoplasms/drug therapy/genetics ; Mice ; Oncogenes ; },
abstract = {Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. There are still challenges for HCC treatments, especially high resistance of the cancer cells to chemotherapy and/or target therapy. In this study, a responsive charge-reversal vehicle consists of negatively charged heparin core and positively charged ethanolamine (EA)-modified poly(glycidyl methacrylate) (PGEA) shell (named Hep@PGEA) with self-accelerating release for condensed nucleic acids was proposed to deliver the pCas9 plasmid encoding clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and the sgRNA targeting oncogene survivin to treat HCC. The Hep@PGEA/pCas9 system showed high anti-tumor efficiency via inducing apoptosis and inhibiting proliferation, migration and invasion capability of HCC cells. The Hep@PGEA/pCas9 system was further utilized to treat orthotopic HCC in mice via tail vein injection. The system exhibited an evident accumulation in the liver of mice and achieved obvious anti-tumor effects. The Hep@PGEA/pCas9 system also showed marked improvement of HCC therapy with sorafenib and provided promising combination HCC treatment potentials. Moreover, enrichment of the Hep@PGEA-based delivery system in liver highlights its possibilities for treatments of other liver diseases.},
}
@article {pmid33785374,
year = {2021},
author = {Sledzinski, P and Dabrowska, M and Nowaczyk, M and Olejniczak, M},
title = {Paving the way towards precise and safe CRISPR genome editing.},
journal = {Biotechnology advances},
volume = {49},
number = {},
pages = {107737},
doi = {10.1016/j.biotechadv.2021.107737},
pmid = {33785374},
issn = {1873-1899},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; *Gene Editing ; RNA, Guide/genetics ; },
abstract = {As the possibilities of CRISPR-Cas9 technology have been revealed, we have entered a new era of research aimed at increasing its specificity and safety. This stage of technology development is necessary not only for its wider application in the clinic but also in basic research to better control the process of genome editing. Research during the past eight years has identified some factors influencing editing outcomes and led to the development of highly specific endonucleases, modified guide RNAs and computational tools supporting experiments. More recently, large-scale experiments revealed a previously overlooked feature: Cas9 can generate reproducible mutation patterns. As a result, it has become apparent that Cas9-induced double-strand break (DSB) repair is nonrandom and can be predicted to some extent. Here, we review the present state of knowledge regarding the specificity and safety of CRISPR-Cas9 technology to define gRNA, protein and target-related problems and solutions. These issues include sequence-specific off-target effects, immune responses, genetic variation and chromatin accessibility. We present new insights into the role of DNA repair in genome editing and define factors influencing editing outcomes. In addition, we propose practical guidelines for increasing the specificity of editing and discuss novel perspectives in improvement of this technology.},
}
@article {pmid33785179,
year = {2021},
author = {Chen, YC},
title = {CRISPR based genome editing and removal of human viruses.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {93-116},
doi = {10.1016/bs.pmbts.2020.12.014},
pmid = {33785179},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Humans ; *Virus Diseases ; Viruses/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins 9 (Cas9), a gene-editing technology, has been extensively applied as a tool for genetic engineering in basic research. Efficient genome engineering has been performed in viruses, human cells, bacteria, fungi, plants and animals, etc. Currently, it has been employed to edit human viruses for studying viral molecular biology, pathogenesis and oncogenesis, and facilitate the development of antiviral agents and vaccine. The virus is ubiquitous worldwide and elicits global health problems, many human diseases are associated with virus infections. Although traditional drugs can be used to treat or prevent productive viral infections, their efficacy is limited because of toxicity, side effects and other problems. Additionally, no current drugs are approved to be indicated for latent infections. Therefore, the next highlight is to develop antiviral approaches to against both productive and latent infections. Fortunately, CRISPR has been successfully applied in the removal of human viruses ex vivo and/or in vivo, and has the potential to be used to manufacture antiviral agents for clinical application. CRISPR/Cas9 is promising in applications, even though some technical challenges, safety concerns, ethic concerns need to be improved. In this article, the discovery and application of genome editing and removal of human viruses based on CRISPR are explored. Additionally, we evaluate the prospects and limitations of this novel antiviral strategies.},
}
@article {pmid33785178,
year = {2021},
author = {Jothi, R and Karthika, C and Kamaladevi, A and Satish, L and Pandian, SK and Gowrishankar, S},
title = {CRISPR based bacterial genome editing and removal of pathogens.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {77-92},
doi = {10.1016/bs.pmbts.2020.12.013},
pmid = {33785178},
issn = {1878-0814},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome, Bacterial ; Humans ; RNA, Guide/genetics ; },
abstract = {Engineering nucleases to achieve targeted genome editing has turned out to be a revolutionary means for manipulating the genetic content in diversified living organisms. For targeted genome editing, till to date, only three engineered nucleases exist viz. zinc finger nucleases, transcription activator-like effector nucleases and RNA-mediated nucleases (RGNs) (Cas nucleases) from the clustered regularly interspaced short palindromic repeat (CRISPR). Among, Cas9 nuclease has been considered as a simplest tool for efficient modification of endogenous genes in an extensive stretch of organisms, owing to its amenability to design guide RNA compatible to the sequence of new targets. Moreover, CRISPR/Cas system delivers a multipurpose RNA-guided DNA-targeting platform called as CRISPR interference (CRISPRi), as well as epigenetic modifications and high throughput screening in diverse organism including bacteria, all in a sequence explicit way. With these entire advancements, the present chapter illustrates the deployment of CRISPR/Cas9 in bacterial genome editing and removal of pathogens.},
}
@article {pmid33785177,
year = {2021},
author = {Hillary, VE and Ceasar, SA},
title = {Genome engineering in insects for the control of vector borne diseases.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {197-223},
doi = {10.1016/bs.pmbts.2020.12.017},
pmid = {33785177},
issn = {1878-0814},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Genome, Insect ; Humans ; Insecta/genetics ; *Vector Borne Diseases ; Zinc Finger Nucleases ; },
abstract = {Insects cause many vector-borne infectious diseases and have become a major threat to human health. Although many control measures are undertaken, some insects are resistant to it, exacerbated by environmental changes which is a major challenge for control measures. Genetic studies by targeting the genomes of insects may offer an alternative strategy. Developments with novel genome engineering technologies have stretched our ability to target and modify any genomic sequence in Eukaryotes including insects. Genome engineering tools such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently discovered, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) systems hold the potential to control the vector-borne diseases. In this chapter, we review the vector control strategy undertaken by employing three major genome engineering tools (ZFNs, TALENs, and CRISPR/Cas9) and discuss the future prospects of this system to control insect vectors. Finally, we also discuss the CRISPR-based gene drive system and its concerns due to ecological impacts.},
}
@article {pmid33785176,
year = {2021},
author = {Arazoe, T},
title = {CRISPR-based pathogenic fungal genome editing for control of infection and disease.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {161-196},
doi = {10.1016/bs.pmbts.2020.12.016},
pmid = {33785176},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Ecosystem ; Fungi/genetics ; *Gene Editing ; Genome, Fungal/genetics ; Humans ; },
abstract = {Fungi play important roles in many aspects of human life, such as in various food, beverage, agricultural, chemical, and pharmaceutical industries. Meanwhile, some fungal species cause several severe diseases in plants, humans and animals. Fungal and fungal-like diseases pose a severe threat to human health, food security, and ecosystem health worldwide. This chapter introduces CRISPR-based genome editing technologies for pathogenic fungi and their application in controlling fungal diseases.},
}
@article {pmid33785175,
year = {2021},
author = {Satish, L and Lavanya, G and Kasthuri, T and Kalaivaani, A and Shamili, S and Muthuramalingam, P and Gowrishankar, S and Pandian, SK and Singh, V and Sitrit, Y and Kushmaro, A},
title = {CRISPR based development of RNA editing and the diagnostic platform.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {117-159},
doi = {10.1016/bs.pmbts.2020.12.015},
pmid = {33785175},
issn = {1878-0814},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; DNA ; Humans ; *RNA Editing/genetics ; },
abstract = {Clustered Regularly Interspersed Short Palindromic Repeat-CRISPR-Associated (CRISPR-Cas) system has improved the ability to edit and control gene expression as desired. Genome editing approaches are currently leading the biomedical research with improved focus on direct nuclease dependent editing. So far, the research was predominantly intended on genome editing over the DNA level, recent adapted techniques are initiating to secure momentum through their proficiency to provoke modifications in RNA sequence. Integration of this system besides to lateral flow method allows reliable, quick, sensitive, precise and inexpensive diagnostic. These interesting methods illustrate only a small proportion of what is technically possible for this novel technology, but several technological obstacles need to be overcome prior to the CRISPR-Cas genome editing system can meet its full ability. This chapter covers the particulars on recent advances in CRISPR-Cas9 genome editing technology including diagnosis and technical advancements, followed by molecular mechanism of CRISPR-based RNA editing and diagnostic tools and types, and CRISPR-Cas-based biosensors.},
}
@article {pmid33785174,
year = {2021},
author = {Agarwal, N and Gupta, R},
title = {History, evolution and classification of CRISPR-Cas associated systems.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {11-76},
doi = {10.1016/bs.pmbts.2020.12.012},
pmid = {33785174},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
abstract = {This chapter provides a detailed description of the history of CRISPR-Cas and its evolution into one of the most efficient genome-editing strategies. The chapter begins by providing information on early findings that were critical in deciphering the role of CRISPR-Cas associated systems in prokaryotes. It then describes how CRISPR-Cas had been evolved into an efficient genome-editing strategy. In the subsequent section, latest developments in the genome-editing approaches based on CRISPR-Cas are discussed. The chapter ends with the recent classification and possible evolution of CRISPR-Cas systems.},
}
@article {pmid33785173,
year = {2021},
author = {Singh, V},
title = {An introduction and use of the CRISPR-Cas systems.},
journal = {Progress in molecular biology and translational science},
volume = {179},
number = {},
pages = {1-10},
doi = {10.1016/bs.pmbts.2020.12.011},
pmid = {33785173},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genome/genetics ; RNA, Guide ; },
abstract = {Clusters of regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) is a rapidly evolving field of targeted genome engineering. The type II CRISPR-Cas9 is used for genome editing of many organisms. Single guide RNA (sgRNA) can bind to Cas9 protein that can target desired sequences in presence of protospacer adjacent motif (PAM) sequences. This complex binds and generate a DSB that is repaired by NHEJ or HDR pathways, subsequently gene insertion/deletion (Indels) is generated that leads to change in the organism's genotype followed by its phenotype. In this chapter, CRISPR-mediated targeted genome editing in different lower organisms has been highlighted to promote its basic understanding to be applied for biotechnological, biomedical and therapeutic applications.},
}
@article {pmid33784525,
year = {2021},
author = {Griesbeck, O},
title = {CRISPR/Cas9-based directed evolution in mammalian cells.},
journal = {Current opinion in structural biology},
volume = {69},
number = {},
pages = {35-40},
doi = {10.1016/j.sbi.2021.02.005},
pmid = {33784525},
issn = {1879-033X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mutagenesis ; },
abstract = {An increasingly powerful set of new CRISPR/Cas-based methods is becoming available for directed evolution of proteins in mammalian cells. Although in vitro techniques or microbial expression systems have been dominating directed evolution, there are now promising approaches to diversify proteins in mammalian cells in situ. This can be achieved by simple indel mutagenesis or more sophisticated homology repair mechanisms for cassette mutagenesis of coding sequences. Cas9 variant fusions to base editors and other effectors pose another promising way to introduce diversity into proteins. CRISPR/Cas9-based directed evolution in mammalian cells opens a new exciting era of discovery for the many classes of proteins for which a mammalian cellular context is preferable.},
}
@article {pmid33784496,
year = {2021},
author = {Pelletier, JF and Sun, L and Wise, KS and Assad-Garcia, N and Karas, BJ and Deerinck, TJ and Ellisman, MH and Mershin, A and Gershenfeld, N and Chuang, RY and Glass, JI and Strychalski, EA},
title = {Genetic requirements for cell division in a genomically minimal cell.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2430-2440.e16},
doi = {10.1016/j.cell.2021.03.008},
pmid = {33784496},
issn = {1097-4172},
support = {P41 GM103412/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems ; Chromosomes, Bacterial/*genetics ; DNA, Bacterial/*genetics ; Genetic Engineering ; *Genome, Bacterial ; Mycoplasma/*genetics ; Synthetic Biology/*methods ; },
abstract = {Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.},
}
@article {pmid33784178,
year = {2021},
author = {English, MA and Gayet, RV and Collins, JJ},
title = {Designing Biological Circuits: Synthetic Biology Within the Operon Model and Beyond.},
journal = {Annual review of biochemistry},
volume = {90},
number = {},
pages = {221-244},
doi = {10.1146/annurev-biochem-013118-111914},
pmid = {33784178},
issn = {1545-4509},
mesh = {CRISPR-Cas Systems ; Epigenomics/*methods ; Feedback, Physiological ; Gene Expression Regulation ; Molecular Biology/methods ; *Operon ; Proteins/*genetics/metabolism ; RNA, Messenger/genetics ; Synthetic Biology/*methods ; Transcription, Genetic ; },
abstract = {In 1961, Jacob and Monod proposed the operon model of gene regulation. At the model's core was the modular assembly of regulators, operators, and structural genes. To illustrate the composability of these elements, Jacob and Monod linked phenotypic diversity to the architectures of regulatory circuits. In this review, we examine how the circuit blueprints imagined by Jacob and Monod laid the foundation for the first synthetic gene networks that launched the field of synthetic biology in 2000. We discuss the influences of the operon model and its broader theoretical framework on the first generation of synthetic biological circuits, which were predominantly transcriptional and posttranscriptional circuits. We also describe how recent advances in molecular biology beyond the operon model-namely, programmable DNA- and RNA-binding molecules as well as models of epigenetic and posttranslational regulation-are expanding the synthetic biology toolkit and enabling the design of more complex biological circuits.},
}
@article {pmid33783997,
year = {2021},
author = {Huang, W and Zeng, ZC and Wang, WT and Sun, YM and Chen, YQ and Luo, XQ and Fang, K},
title = {A CRISPR/CAS9-based strategy targets the personalized chimeric neosequence in fusion-driven cancer genome for precision medicine.},
journal = {Clinical and translational medicine},
volume = {11},
number = {3},
pages = {e355},
pmid = {33783997},
issn = {2001-1326},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Fusion/*genetics ; Genome/*genetics ; Humans ; Neoplasms/*genetics ; Precision Medicine/*methods ; },
}
@article {pmid33783162,
year = {2021},
author = {Zheng, X and Zheng, P and Sun, J},
title = {[CRISPR/Cas-based genome editing in Aspergillus niger].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {3},
pages = {980-990},
doi = {10.13345/j.cjb.200613},
pmid = {33783162},
issn = {1872-2075},
mesh = {Aspergillus niger/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome ; },
abstract = {Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.},
}
@article {pmid33783160,
year = {2021},
author = {Li, H and Liang, X and Zhou, J},
title = {[Progress in gene editing technologies for Saccharomyces cerevisiae].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {37},
number = {3},
pages = {950-965},
doi = {10.13345/j.cjb.200542},
pmid = {33783160},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; *Gene Editing ; *Saccharomyces cerevisiae/genetics ; Technology ; },
abstract = {Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.},
}
@article {pmid33782689,
year = {2021},
author = {Paraskevopoulos, M and McGuigan, AP},
title = {Application of CRISPR screens to investigate mammalian cell competition.},
journal = {Briefings in functional genomics},
volume = {20},
number = {3},
pages = {135-147},
doi = {10.1093/bfgp/elab020},
pmid = {33782689},
issn = {2041-2657},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Cell Competition ; Genetic Testing ; Genome ; Mammals/genetics ; },
abstract = {Cell competition is defined as the context-dependent elimination of cells that is mediated by intercellular communication, such as paracrine or contact-dependent cell signaling, and/or mechanical stresses. It is considered to be a quality control mechanism that facilitates the removal of suboptimal cells from both adult and embryonic tissues. Cell competition, however, can also be hijacked by transformed cells to acquire a 'super-competitor' status and outcompete the normal epithelium to establish a precancerous field. To date, many genetic drivers of cell competition have been identified predominately through studies in Drosophila. Especially during the last couple of years, ethylmethanesulfonate-based genetic screens have been instrumental to our understanding of the molecular regulators behind some of the most common competition mechanisms in Drosophila, namely competition due to impaired ribosomal function (or anabolism) and mechanical sensitivity. Despite recent findings in Drosophila and in mammalian models of cell competition, the drivers of mammalian cell competition remain largely elusive. Since the discovery of CRISPR/Cas9, its use in functional genomics has been indispensable to uncover novel cancer vulnerabilities. We envision that CRISPR/Cas9 screens will enable systematic, genome-scale probing of mammalian cell competition to discover novel mutations that not only trigger cell competition but also identify novel molecular components that are essential for the recognition and elimination of less fit cells. In this review, we summarize recent contributions that further our understanding of the molecular mechanisms of cell competition by genetic screening in Drosophila, and provide our perspective on how similar and novel screening strategies made possible by whole-genome CRISPR/Cas9 screening can advance our understanding of mammalian cell competition in the future.},
}
@article {pmid33782402,
year = {2021},
author = {Zhang, Y and Ren, Q and Tang, X and Liu, S and Malzahn, AA and Zhou, J and Wang, J and Yin, D and Pan, C and Yuan, M and Huang, L and Yang, H and Zhao, Y and Fang, Q and Zheng, X and Tian, L and Cheng, Y and Le, Y and McCoy, B and Franklin, L and Selengut, JD and Mount, SM and Que, Q and Zhang, Y and Qi, Y},
title = {Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1944},
pmid = {33782402},
issn = {2041-1723},
mesh = {Agrobacterium tumefaciens ; Alleles ; Arabidopsis/*genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural ; Endodeoxyribonucleases/*genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/*methods ; *Genome, Plant ; Humans ; Isoenzymes/genetics/metabolism ; Oryza/*genetics/metabolism ; Plants, Genetically Modified ; RNA, Guide/genetics/metabolism ; Sequence Alignment ; },
abstract = {CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.},
}
@article {pmid33782125,
year = {2021},
author = {France, MG and Enderle, J and Röhrig, S and Puchta, H and Franklin, FCH and Higgins, JD},
title = {ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {14},
pages = {},
pmid = {33782125},
issn = {1091-6490},
mesh = {Arabidopsis ; Arabidopsis Proteins/genetics/*metabolism ; *Crossing Over, Genetic ; Meiosis ; Synaptonemal Complex/genetics/metabolism ; },
abstract = {The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5 These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.},
}
@article {pmid33782117,
year = {2021},
author = {Koreman, GT and Xu, Y and Hu, Q and Zhang, Z and Allen, SE and Wolfner, MF and Wang, B and Han, C},
title = {Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {14},
pages = {},
pmid = {33782117},
issn = {1091-6490},
support = {R01 NS099125/NS/NINDS NIH HHS/United States ; R21 HD088744/HD/NICHD NIH HHS/United States ; R21 OD023824/OD/NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Female ; *Genetic Techniques ; Male ; *Mutagenesis ; RNA, Guide ; },
abstract = {CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila These improvements may be also applied to other model systems.},
}
@article {pmid33782110,
year = {2021},
author = {Liu, J and Cvirkaite-Krupovic, V and Baquero, DP and Yang, Y and Zhang, Q and Shen, Y and Krupovic, M},
title = {Virus-induced cell gigantism and asymmetric cell division in archaea.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {15},
pages = {},
pmid = {33782110},
issn = {1091-6490},
mesh = {Archaeal Proteins/metabolism ; Archaeal Viruses/*pathogenicity ; *Asymmetric Cell Division ; CRISPR-Cas Systems ; Endosomal Sorting Complexes Required for Transport/metabolism ; Giant Cells/*metabolism/virology ; Sulfolobales/genetics/physiology/*virology ; },
abstract = {Archaeal viruses represent one of the most mysterious parts of the global virosphere, with many virus groups sharing no evolutionary relationship to viruses of bacteria or eukaryotes. How these viruses interact with their hosts remains largely unexplored. Here we show that nonlytic lemon-shaped virus STSV2 interferes with the cell cycle control of its host, hyperthermophilic and acidophilic archaeon Sulfolobus islandicus, arresting the cell cycle in the S phase. STSV2 infection leads to transcriptional repression of the cell division machinery, which is homologous to the eukaryotic endosomal sorting complexes required for transport (ESCRT) system. The infected cells grow up to 20-fold larger in size, have 8,000-fold larger volume compared to noninfected cells, and accumulate massive amounts of viral and cellular DNA. Whereas noninfected Sulfolobus cells divide symmetrically by binary fission, the STSV2-infected cells undergo asymmetric division, whereby giant cells release normal-sized cells by budding, resembling the division of budding yeast. Reinfection of the normal-sized cells produces a new generation of giant cells. If the CRISPR-Cas system is present, the giant cells acquire virus-derived spacers and terminate the virus spread, whereas in its absence, the cycle continues, suggesting that CRISPR-Cas is the primary defense system in Sulfolobus against STSV2. Collectively, our results show how an archaeal virus manipulates the cell cycle, transforming the cell into a giant virion-producing factory.},
}
@article {pmid33779839,
year = {2021},
author = {Bouchaut, B and Asveld, L},
title = {Responsible Learning About Risks Arising from Emerging Biotechnologies.},
journal = {Science and engineering ethics},
volume = {27},
number = {2},
pages = {22},
pmid = {33779839},
issn = {1471-5546},
mesh = {*Biotechnology ; European Union ; Humans ; Risk Assessment ; *Risk Management ; },
abstract = {Genetic engineering techniques (e.g., CRISPR-Cas) have led to an increase in biotechnological developments, possibly leading to uncertain risks. The European Union aims to anticipate these by embedding the Precautionary Principle in its regulation for risk management. This principle revolves around taking preventive action in the face of uncertainty and provides guidelines to take precautionary measures when dealing with important values such as health or environmental safety. However, when dealing with 'new' technologies, it can be hard for risk managers to estimate the societal or environmental consequences of a biotechnology that might arise once introduced or embedded in society due to that these sometimes do not comply with the established norms within risk assessment. When there is insufficient knowledge, stakeholders active in early developmental stages (e.g., researchers) could provide necessary knowledge by conducting research specifically devoted to what these unknown risks could entail. In theory, the Safe-by-Design (SbD) approach could enable such a controlled learning environment to gradually identify what these uncertain risks are, to which we refer as responsible learning. In this paper, we argue that three conditions need to be present to enable such an environment: (1) regulatory flexibility, (2) co-responsibility between researchers and regulators, and (3) openness towards all stakeholders. If one of these conditions would not be present, the SbD approach cannot be implemented to its fullest potential, thereby limiting an environment for responsible learning and possibly leaving current policy behind to anticipate uncertain risks.},
}
@article {pmid33778788,
year = {2021},
author = {Lin, X and Liu, Y and Chemparathy, A and Pande, T and La Russa, M and Qi, LS},
title = {A comprehensive analysis and resource to use CRISPR-Cas13 for broad-spectrum targeting of RNA viruses.},
journal = {Cell reports. Medicine},
volume = {2},
number = {4},
pages = {100245},
pmid = {33778788},
issn = {2666-3791},
mesh = {COVID-19/pathology/virology ; CRISPR-Cas Systems/*genetics ; Humans ; RNA Virus Infections/pathology/virology ; RNA Viruses/*genetics/isolation &amp; purification ; RNA, Guide/*metabolism ; RNA, Viral/metabolism ; SARS-CoV-2/genetics/isolation &amp; purification ; Species Specificity ; },
abstract = {The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and variants has led to significant mortality. We recently reported that an RNA-targeting CRISPR-Cas13 system, called prophylactic antiviral CRISPR in human cells (PAC-MAN), offered an antiviral strategy against SARS-CoV-2 and influenza A virus. Here, we expand in silico analysis to use PAC-MAN to target a broad spectrum of human- or livestock-infectious RNA viruses with high specificity, coverage, and predicted efficiency. Our analysis reveals that a minimal set of 14 CRISPR RNAs (crRNAs) is able to target >90% of human-infectious viruses across 10 RNA virus families. We predict that a set of 5 experimentally validated crRNAs can target new SARS-CoV-2 variant sequences with zero mismatches. We also build an online resource (crispr-pacman.stanford.edu) to support community use of CRISPR-Cas13 for broad-spectrum RNA virus targeting. Our work provides a new bioinformatic resource for using CRISPR-Cas13 to target diverse RNA viruses to facilitate the development of CRISPR-based antivirals.},
}
@article {pmid33775851,
year = {2021},
author = {Rautela, I and Uniyal, P and Thapliyal, P and Chauhan, N and Bhushan Sinha, V and Dev Sharma, M},
title = {An extensive review to facilitate understanding of CRISPR technology as a gene editing possibility for enhanced therapeutic applications.},
journal = {Gene},
volume = {785},
number = {},
pages = {145615},
doi = {10.1016/j.gene.2021.145615},
pmid = {33775851},
issn = {1879-0038},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/trends ; *Genetic Therapy ; Humans ; },
abstract = {CRISPR are the sequences in bacterial and archaeal genome which provide resistance against viral infections. They might be the natural part of bacterial genomes for providing protection against viruses like bacteriophages but science has successfully achieved their use in the benefit of man-kind by using them for the treatment of deadly diseases like cancer, AIDS or genetic disorders like sickle cell disease and Leber congenital amaurosis. CRISPR system is majorly divided into two classes i.e class I and class II, of which the class II CRISPR/Cas9 system performs site specific cleavage of DNA with a guide RNA Cas12 (Cpf1). With the new emerging discoveries it is being found that CRISPR not only works on double stranded DNA but can also be useful to induce any sort of site specific cleavage in RNA too by Cas13 earlier known as C2c2, which is a protein found in CRISPR system and has ability to cure viral infections in plants. CRISPR is being used in the field of gene manipulation and various animals models are available to serve this purpose with short lifespan, rapid reproducibility and lower maintenance cost. Many successful studies and experiments performed using CRISPR, reveals their potency and utility to bring revolution in the areas which were previously believed to be out of scope of science and medicine.},
}
@article {pmid33775846,
year = {2021},
author = {Li, S and Toya, M and Sato, M},
title = {Simplification of nutritional conditions in transformation procedures for genome editing with the CRISPR/Cas9 system for fission yeast.},
journal = {Gene},
volume = {784},
number = {},
pages = {145595},
doi = {10.1016/j.gene.2021.145595},
pmid = {33775846},
issn = {1879-0038},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Culture Media/*chemistry ; Gene Editing/methods ; Genetic Vectors/genetics ; Nitrogen/chemistry ; Point Mutation ; RNA, Guide/*genetics ; Schizosaccharomyces/genetics/*growth &amp; development ; Schizosaccharomyces pombe Proteins/*genetics ; },
abstract = {CRISPR/Cas9 is a powerful tool for genome editing. Several studies have been conducted to take the benefit of the versatile tool in the fission yeast Schizosaccharomyces pombe. However, the protocols for the CRISPR/Cas9 system proposed in previous studies are complicated in culture conditions compared to traditional genome editing methods. In this study, we introduced vectors for expression of sgRNA as well as Cas9, which employ natMX6 and bsdMX6 dominant selection markers. Using these materials, we examined nutritional conditions of cell cultures and found that nitrogen depletion introduced in previous methods does not affect the efficiency of genome editing. We found that bsdMX6-based plasmids enable us to skip any recovery steps before plating onto medium containing blasticidin S, unlike other antibiotic resistance selection markers. We thus propose easier transformation procedures with natMX6 and particularly bsdMX6 markers. We also simulate prescreening of mutants by genotyping with DNA endonucleases or proofreading PCR instead of relying on existing knowledge of mutant phenotypes. These materials and methods assist easy construction of S. pombe strains using CRISPR/Cas9, thereby accelerating seamless introduction of CRISPR/Cas9 to S. pombe researchers.},
}
@article {pmid33774733,
year = {2021},
author = {Singh, R and Chandel, S and Ghosh, A and Dey, D and Chakravarti, R and Roy, S and Ravichandiran, V and Ghosh, D},
title = {Application of CRISPR/Cas System in the Metabolic Engineering of Small Molecules.},
journal = {Molecular biotechnology},
volume = {63},
number = {6},
pages = {459-476},
pmid = {33774733},
issn = {1559-0305},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Microbial/genetics ; Metabolic Engineering/*methods ; Small Molecule Libraries/*metabolism ; Yeasts/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas protein technology area is rapidly growing technique for genome editing and modulation of transcription of several microbes. Successful engineering in microbes requires an emphasis on the aspect of efficiency and targeted aiming, which can be employed using CRISPR/Cas system. Hence, this type of system is used to modify the genome of several microbes such as yeast and bacteria. In recent years, CRISPR/Cas systems have been chosen for metabolic engineering in microbes due to their specificity, orthogonality, and efficacy. Therefore, we need to review the scheme which was acquired for the execution of the CRISPR/Cas system for the modification and metabolic engineering in yeast and bacteria. In this review, we highlighted the application of the CRISPR/Cas system which has been used for the production of small molecules in the microbial system that is chemically and biologically important.},
}
@article {pmid33774312,
year = {2021},
author = {El Ouar, I and Djekoun, A},
title = {Therapeutic and diagnostic relevance of Crispr technology.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {138},
number = {},
pages = {111487},
doi = {10.1016/j.biopha.2021.111487},
pmid = {33774312},
issn = {1950-6007},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; Gene Editing/*methods/trends ; Genetic Engineering/*methods/trends ; Genetic Therapy/*methods/trends ; Humans ; },
abstract = {CRISPR is a family of DNA repeats providing immunity against viral and plasmid invading DNA in bacteria and archaea. The system consist of an endonuclease Cas, guided by a RNA sequence, able to cleave the DNA double strand at a specific site. The discovery of Crispr function in 2007 has revolutionized genetic engineering by giving to the world the most powerful and precise tool for targeted genome editing. The aim of this review is to synthesize the current knowledge on Crispr/cas system and its application in biomedical field. In particular, we focus on the relevance of this new tool in progressing our comprehension for biological mechanisms and improving our ability to treat and prevent genetic diseases, to control microbial virulence and to generate animal models for basic and clinical research. We discuss also the ethical issues that may prevent the application of Crispr technology in living beings.},
}
@article {pmid33774158,
year = {2021},
author = {Hao, L and Pu, X and Song, J},
title = {Introduction of mutations in plants with prime editing.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {83-93},
doi = {10.1016/j.ymeth.2021.03.014},
pmid = {33774158},
issn = {1095-9130},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; DNA ; *Gene Editing ; *Mutation ; },
abstract = {The emergence of a clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) system has had a revolutionary impact on plant biology. However, this system and further developed base editing are limited by their inherent imperfection. Prime editing, a just arrival technology based on CRISPR/Cas, can directly and precisely edit a specified DNA site without double strand breaks and donor DNA by integrating an engineered reverse transcriptase (RT) with a catalytically impaired Cas9 endonuclease and introducing genetic information into prime editing guide RNA (pegRNA). In addition, it has a wider range of editing types than base editing and can install all types of editing theoretically. Prime editing was originally developed in mammalian cells and has recently been applied to plants. Here, we describe the origin of prime editing and compare it with traditional CRISPR/Cas9 and base editing; then, we exemplify it in plants, including strategies and methods. Accordingly, we generate the overall procedures of prime editing to provide instructions for its application. Furthermore, we summarize its improvements in the approach, such as optimizing the length of a primer binding site and RT template, as well as pursuing an optimal nicking site in the unedited sequence. Finally, we discuss the potential impact on domestication and improvement of agricultural crops, sustainable utilization of medicinal plants, cultivation of varieties of horticultural plants, and revelation of the genetic code, in order to offer a reference for the further study and development of prime editing.},
}
@article {pmid33774157,
year = {2021},
author = {Kuang, J and Lyu, Q and Wang, J and Cui, Y and Zhao, J},
title = {Advances in base editing with an emphasis on an AAV-based strategy.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {56-64},
doi = {10.1016/j.ymeth.2021.03.015},
pmid = {33774157},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; Cytosine ; DNA ; Dependovirus/genetics ; *Gene Editing ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based base editors have been developed for precisely installing point mutations in genomes with high efficiency. Two editing systems of cytosine base editors (CBEs) and adenine base editors (ABEs) have been developed for conversion of C.G-to-T.A and A.T-to-G.C, respectively, showing the prominence in genomic DNA correction and mutation. Here, we summarize recent optimized approaches in improving base editors, including the evolution of Cas proteins, the choice of deamination enzymes, modification on linker length, base-editor expression, and addition of functional domains. Specifically, in this paper we highlight a strategy of split-intein mediated base-editor reconstitution for its adeno-associated virus (AAV) delivery. The purpose of this article is to offer readers with a better understanding of AAV-mediated base editors, and facilitate them to use this tool in in vivo experiments and potential clinical applications.},
}
@article {pmid33774156,
year = {2021},
author = {Yang, L and Tang, J and Ma, X and Lin, Y and Ma, G and Shan, M and Wang, L and Yang, Y},
title = {Progression and application of CRISPR-Cas genomic editors.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {65-74},
doi = {10.1016/j.ymeth.2021.03.013},
pmid = {33774156},
issn = {1095-9130},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genomics ; Mutation ; },
abstract = {Base editing technology is an efficient tool for genome editing, particularly in the correction of base mutations. Diverse base editing systems were developed according to the dCas9 or nCas9 linked with different deaminase or reverse transcriptase in the editors, including ABEs, CBEs, PEs and dual-functional of base editor (such as CGBE1, A&amp;C-BEmax, ACBE, etc.). Currently, Base editing technology has been widely applied to various fields such as microorganisms, plants, animals and medicine for basic research and therapeutics. Here, we reviewed the advancement of base editing technology. We also discussed the application of base editors in different areas in the future.},
}
@article {pmid33774131,
year = {2021},
author = {Umemura, K and Ohtsuki, S and Nagaoka, M and Kusamori, K and Inoue, T and Takahashi, Y and Takakura, Y and Nishikawa, M},
title = {Critical contribution of macrophage scavenger receptor 1 to the uptake of nanostructured DNA by immune cells.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {34},
number = {},
pages = {102386},
doi = {10.1016/j.nano.2021.102386},
pmid = {33774131},
issn = {1549-9642},
mesh = {Animals ; CRISPR-Cas Systems ; DNA/chemistry/*metabolism ; Dextran Sulfate/pharmacology ; Female ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Macrophages/drug effects/*metabolism ; Mice ; Mice, Inbred BALB C ; *Nanostructures ; RAW 264.7 Cells ; Scavenger Receptors, Class A/genetics/*metabolism ; Transfection ; },
abstract = {Despite the efficient uptake of polypod-like nanostructured DNA, or polypodna, by macrophage-like RAW264.7 and other immune cells, the detailed mechanism has not been fully elucidated. Our previous study using HEK-Blue hTLR9 cells showed that transfection of macrophage scavenger receptor 1 (MSR1) increased the uptake of tetrapod-like structured DNA. Here, we investigated the involvement of MSR1 in the structure-dependent uptake of polypodna. Transfection of MSR1 to HEK-Blue hTLR9 cells pod number-dependently increased the uptake of polypodna, and its knockout in RAW264.7 cells reduced the uptake and subsequent cytokine release. To examine the binding of DNA with MSR1, biotinylated DNA added to RAW264.7 cells was cross-linked with cell surface proteins. Then, MSR1 cross-linked with polypodna, but not with single-stranded DNA. Similar results were obtained with murine primary immune cells. Taken together, MSR1 discriminates between simple and nanostructured DNAs and plays a dominant role in the efficient uptake of polypodna by immune cells.},
}
@article {pmid33773911,
year = {2021},
author = {Ferreira, TR and Couñago, RM and Moretti, NS},
title = {Raising the Bar(-seq) in Leishmania Genetic Screens.},
journal = {Trends in parasitology},
volume = {37},
number = {5},
pages = {367-369},
doi = {10.1016/j.pt.2021.03.002},
pmid = {33773911},
issn = {1471-5007},
mesh = {Animals ; CRISPR-Cas Systems ; *Leishmania/genetics ; Life Cycle Stages ; },
abstract = {Our understanding of regulatory factors in Leishmania differentiation has long been restricted by the available genetic tools, but the availability of CRISPR/Cas9 has changed the landscape forever. Recently, Baker and Catta-Preta et al. applied Cas9 editing and kinome-wide bar-seq to dissect the function of 204 kinases in the Leishmania mexicana life cycle.},
}
@article {pmid33773050,
year = {2021},
author = {Zhang, Y and Yuan, J},
title = {CRISPR/Cas12a-mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus.},
journal = {Microbial biotechnology},
volume = {14},
number = {6},
pages = {2700-2710},
pmid = {33773050},
issn = {1751-7915},
mesh = {Bacteria ; *CRISPR-Cas Systems ; Gene Editing ; Genome, Bacterial ; *Rhodobacter capsulatus/genetics ; },
abstract = {Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Template-free disruption method mediated by CRISPR/Cas12a reached ˜ 90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ˜ 80%. In summary, our work represents the first report to develop CRISPR/Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches.},
}
@article {pmid33772993,
year = {2021},
author = {Jiang, Y and An, X and Li, Z and Yan, T and Zhu, T and Xie, K and Liu, S and Hou, Q and Zhao, L and Wu, S and Liu, X and Zhang, S and He, W and Li, F and Li, J and Wan, X},
title = {CRISPR/Cas9-based discovery of maize transcription factors regulating male sterility and their functional conservation in plants.},
journal = {Plant biotechnology journal},
volume = {19},
number = {9},
pages = {1769-1784},
pmid = {33772993},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Plant Breeding ; *Plant Infertility/genetics ; *Transcription Factors/genetics ; *Zea mays/genetics ; },
abstract = {Identifying genic male-sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with Arabidopsis and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9-mediated gene mutagenesis, and then, 12 genes in eight types, including ZmbHLH51, ZmbHLH122, ZmTGA9-1/-2/-3, ZmTGA10, ZmMYB84, ZmMYB33-1/-2, ZmPHD11 and ZmLBD10/27, were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably, ZmTGA9-1/-2/-3 triple-gene mutants and ZmMYB33-1/-2 double-gene mutants displayed complete male sterility, but their double- or single-gene mutants showed male fertility. Similarly, ZmLBD10/27 double-gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition, ZmbHLH51 was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co-segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14-type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and Arabidopsis. These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male-sterility lines in maize.},
}
@article {pmid33771254,
year = {2021},
author = {Shi, L and Li, W and Dong, Y and Shi, Y and Zhou, Y and Liao, X},
title = {NADPH-cytochrome P450 reductase potentially involved in indoxacarb resistance in Spodoptera litura.},
journal = {Pesticide biochemistry and physiology},
volume = {173},
number = {},
pages = {104775},
doi = {10.1016/j.pestbp.2021.104775},
pmid = {33771254},
issn = {1095-9939},
mesh = {Animals ; *Insecticide Resistance/genetics ; *NADPH-Ferrihemoprotein Reductase/genetics ; Oxazines ; Spodoptera/genetics ; },
abstract = {NADPH-cytochrome P450 reductase (CPR) plays a central role in the metabolism of insecticides. Numerous studies have shown that CPR is associated with insecticide resistance in insect. In this study, two transcripts of Spodoptera litura CPR (SlCPR-X1 and SlCPR-X2) were identified and cloned, and the deduced protein of SlCPR-X1 contains all the conserved CPR structural features (N-terminal membrane anchor, FMN, FAD and NADP binding domains, FAD binding motif, and catalytic residues). However, no N-terminal member anchor and a shorter FMN binding region have been identified in the deduced protein of SlCPR-X2. The specific expression patterns showed that SlCPR-X1 and SlCPR-X2 were detected in all tested developmental stages and tissues, but highly expressed in third-, fourth-, and fifth-instar larvae, and in midgut and fat body. In addition, compared with the susceptible strain, SlCPR-X1 and SlCPR-X2 were up-regulated and more inducible when treated with indoxacarb in the indoxacarb-resistant strain. However, the relative expression, up-regulation and induction of SlCPR-X1 were all higher than those of SlCPR-X2 in the indoxacarb-resistant strain. Furthermore, RNA interference and baculovirus expression system combined with MTT cytotoxicity assay demonstrated that only SlCPR-X1 with the N-terminal membrane anchor as the major CPR potentially involved in S. litura indoxacarb resistance. The outcome of this study further expands our understanding of the important role of insect CPR in xenobiotics detoxification and resistance development, and CPR could be a potential target for insecticide resistance management mediated by RNAi or CRISPR/Cas.},
}
@article {pmid33770501,
year = {2021},
author = {Saito, M and Ladha, A and Strecker, J and Faure, G and Neumann, E and Altae-Tran, H and Macrae, RK and Zhang, F},
title = {Dual modes of CRISPR-associated transposon homing.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2441-2453.e18},
pmid = {33770501},
issn = {1097-4172},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteria/*genetics/metabolism ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements/*physiology ; DNA, Bacterial/genetics/*metabolism ; Gene Editing ; *RNA, Guide ; Recombination, Genetic ; Transposases/genetics/*metabolism ; },
abstract = {Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing.},
}
@article {pmid33770071,
year = {2021},
author = {Pavlova, YS and Paez-Espino, D and Morozov, AY and Belalov, IS},
title = {Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling.},
journal = {PLoS computational biology},
volume = {17},
number = {3},
pages = {e1008841},
pmid = {33770071},
issn = {1553-7358},
mesh = {Archaea/genetics ; Bacteria/genetics ; Bacteriophages/genetics ; CRISPR-Cas Systems/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics/immunology ; DNA, Intergenic/genetics ; DNA, Viral/genetics ; Metagenome/*genetics ; Metagenomics ; *Models, Genetic ; },
abstract = {Understanding CRISPR-Cas systems-the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks-is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical 'rich-get-richer' mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.},
}
@article {pmid33769784,
year = {2021},
author = {Balci, H and Globyte, V and Joo, C},
title = {Targeting G-quadruplex Forming Sequences with Cas9.},
journal = {ACS chemical biology},
volume = {16},
number = {4},
pages = {596-603},
pmid = {33769784},
issn = {1554-8937},
mesh = {CRISPR-Associated Protein 9/*chemistry ; CRISPR-Cas Systems ; *G-Quadruplexes ; Nucleic Acid Conformation ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into noncanonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of the CRISPR-Cas9 complex may be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the nontarget strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity.},
}
@article {pmid33768011,
year = {2021},
author = {Chung, CH and Allen, AG and Atkins, A and Link, RW and Nonnemacher, MR and Dampier, W and Wigdahl, B},
title = {Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy.},
journal = {Frontiers in cellular and infection microbiology},
volume = {11},
number = {},
pages = {593077},
pmid = {33768011},
issn = {2235-2988},
support = {R01 MH110360/MH/NIMH NIH HHS/United States ; T32 MH079785/MH/NIMH NIH HHS/United States ; P30 MH092177/MH/NIMH NIH HHS/United States ; },
mesh = {Antiviral Agents ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Viral ; *HIV-1/genetics ; Humans ; *RNA, Guide/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.},
}
@article {pmid33767677,
year = {2021},
author = {Wang, Y and Ji, Q and Li, S and Liu, M},
title = {Prevalence and Genetic Diversity of Listeria monocytogenes Isolated From Retail Pork in Wuhan, China.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {620482},
pmid = {33767677},
issn = {1664-302X},
abstract = {Listeria monocytogenes is a ubiquitous bacteria and causative agent of zoonotic listeriosis with high mortality. The consumption of contaminated animal-derived foods has been linked with both epidemic and sporadic listeriosis. In this work, a total of 64 L. monocytogenes isolates from 259 pork samples sold in 11 supermarket chains were identified and characterized by comparative whole-genome analysis. All isolates were delineated into eight clonal complexes (CCs), namely CC2, CC8, CC9, CC11, CC155, CC121, CC204, and CC619, spanning two lineages (I and II) and carrying 3-5 antibiotic-resistant genes (fosX, lnu, mprF, tetM, and dhfR). It is noted that Listeria pathogenicity island (LIPI)-1, LIPI-3, and LIPI-4 were distributed in all ST619 isolates from two supermarket chains that were closely related with clinical isolates (<40 SNP). Some of the isolates from different supermarket chains with 0 SNP difference indicated a common pork supply source. Notably, 57.81% of the strains carried types IB, IIA, or IIIB CRISPR-Cas system, CC121 isolates carried both types IB and IIA CRISPR-Cas systems, Cas proteins of CC155 isolates located between two CRISPR loci, each CC has unique organization of Cas proteins as well as CRISPR loci. CRISPR-Cas system-based subtyping improved discrimination of pork-derived L. monocytogenes isolates. Comparisons at the genome level contributed to understand the genetic diversities and variations among the isolates and provided insights into the genetic makeup and relatedness of these pathogens.},
}
@article {pmid33767395,
year = {2021},
author = {Lin, Q and Jin, S and Zong, Y and Yu, H and Zhu, Z and Liu, G and Kou, L and Wang, Y and Qiu, JL and Li, J and Gao, C},
title = {High-efficiency prime editing with optimized, paired pegRNAs in plants.},
journal = {Nature biotechnology},
volume = {39},
number = {8},
pages = {923-927},
pmid = {33767395},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Oryza/*genetics ; RNA, Guide/*genetics ; RNA, Plant/*genetics ; },
abstract = {Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner.},
}
@article {pmid33767386,
year = {2021},
author = {Deng, HX and Zhai, H and Shi, Y and Liu, G and Lowry, J and Liu, B and Ryan, &#xc9;B and Yan, J and Yang, Y and Zhang, N and Yang, Z and Liu, E and Ma, YC and Siddique, T},
title = {Efficacy and long-term safety of CRISPR/Cas9 genome editing in the SOD1-linked mouse models of ALS.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {396},
pmid = {33767386},
issn = {2399-3642},
support = {R01 NS118928/NS/NINDS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Amyotrophic Lateral Sclerosis/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Editing/*statistics &amp; numerical data ; Mice ; Mice, Transgenic ; Superoxide Dismutase-1/chemistry/*genetics/metabolism ; },
abstract = {CRISPR/Cas9-mediated genome editing provides potential for therapeutic development. Efficacy and long-term safety represent major concerns that remain to be adequately addressed in preclinical studies. Here we show that CRISPR/Cas9-mediated genome editing in two distinct SOD1-amyotrophic lateral sclerosis (ALS) transgenic mouse models prevented the development of ALS-like disease and pathology. The disease-linked transgene was effectively edited, with rare off-target editing events. We observed frequent large DNA deletions, ranging from a few hundred to several thousand base pairs. We determined that these large deletions were mediated by proximate identical sequences in Alu elements. No evidence of other diseases was observed beyond 2 years of age in these genome edited mice. Our data provide preclinical evidence of the efficacy and long-term safety of the CRISPR/Cas9 therapeutic approach. Moreover, the molecular mechanism of proximate identical sequences-mediated recombination provides mechanistic information to optimize therapeutic targeting design, and to avoid or minimize unintended and potentially deleterious recombination events.},
}
@article {pmid33767353,
year = {2021},
author = {Gilad, Y and Eliaz, Y and Yu, Y and Dean, AM and Han, SJ and Qin, L and O'Malley, BW and Lonard, DM},
title = {A genome-scale CRISPR Cas9 dropout screen identifies synthetically lethal targets in SRC-3 inhibited cancer cells.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {399},
pmid = {33767353},
issn = {2399-3642},
support = {S10 OD018033/OD/NIH HHS/United States ; R01 HD007857/HD/NICHD NIH HHS/United States ; R01 HD008188/HD/NICHD NIH HHS/United States ; S10 OD023469/OD/NIH HHS/United States ; F32 HD008188/HD/NICHD NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Humans ; MCF-7 Cells ; Nuclear Receptor Coactivator 3/*genetics/metabolism ; },
abstract = {Steroid receptor coactivator 3 (SRC-3/NCoA3/AIB1), is a key regulator of gene transcription and it plays a central role in breast cancer (BC) tumorigenesis, making it a potential therapeutic target. Beyond its function as an important regulator of estrogen receptor transcriptional activity, SRC-3 also functions as a coactivator for a wide range of other transcription factors, suggesting SRC-3 inhibition can be beneficial in hormone-independent cancers as well. The recent discovery of a potent SRC-3 small molecule inhibitor, SI-2, enabled the further development of additional related compounds. SI-12 is an improved version of SI-2 that like SI-2 has anti-proliferative activity in various cancer types, including BC. Here, we sought to identify gene targets, that when inhibited in the presence of SI-12, would lead to enhanced BC cell cytotoxicity. We performed a genome-scale CRISPR-Cas9 screen in MCF-7 BC cells under conditions of pharmacological pressure with SI-12. A parallel screen was performed with an ER inhibitor, fulvestrant, to shed light on both common and distinct activities between SRC-3 and ERα inhibition. Bearing in mind the key role of SRC-3 in tumorigenesis of other types of cancer, we extended our study by validating potential hits identified from the MCF-7 screen in other cancer cell lines.},
}
@article {pmid33766732,
year = {2021},
author = {Stincic, TL and Bosch, MA and Hunker, AC and Juarez, B and Connors, AM and Zweifel, LS and Rønnekleiv, OK and Kelly, MJ},
title = {CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance.},
journal = {Molecular metabolism},
volume = {49},
number = {},
pages = {101218},
pmid = {33766732},
issn = {2212-8778},
support = {R01 DK068098/DK/NIDDK NIH HHS/United States ; R56 DK068098/DK/NIDDK NIH HHS/United States ; T32 GM007270/GM/NIGMS NIH HHS/United States ; },
mesh = {Action Potentials ; Agouti-Related Protein/*metabolism ; Animals ; Arcuate Nucleus of Hypothalamus/*metabolism ; Body Weight ; CRISPR-Cas Systems ; Diet, High-Fat ; Energy Metabolism/*physiology ; Estradiol/metabolism ; Fasting ; Feeding Behavior ; Female ; KCNQ3 Potassium Channel/*genetics/*metabolism ; Male ; Mice ; Neurons/*metabolism ; Neuropeptide Y/genetics/*metabolism ; Neuropeptides/*metabolism ; },
abstract = {OBJECTIVE: Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17β-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons.<br><br>METHODS: We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance.<br><br>RESULTS: The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals.<br><br>CONCLUSIONS: The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.},
}
@article {pmid33766595,
year = {2021},
author = {Pan, S and Su, Y and Sun, B and Hao, R and Gao, X and Han, B},
title = {Knockout of CD147 inhibits the proliferation, invasion, and drug resistance of human oral cancer CAL27 cells in Vitro and in Vivo.},
journal = {International journal of biological macromolecules},
volume = {181},
number = {},
pages = {378-389},
doi = {10.1016/j.ijbiomac.2021.03.102},
pmid = {33766595},
issn = {1879-0003},
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Apoptosis/drug effects ; Basigin/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Disease Models, Animal ; Doxorubicin/pharmacology/therapeutic use ; *Drug Resistance, Neoplasm/drug effects ; *Gene Knockout Techniques ; Humans ; Matrix Metalloproteinases/metabolism ; Mice, Nude ; Mouth Neoplasms/*drug therapy/*pathology ; Neoplasm Invasiveness ; },
abstract = {With the development of modern biomedicine, research on the molecular mechanism of tumors has developed gradually. The CD147 gene has been applied to tumor molecular targeted therapy, and significant differences were found in the expression of the CD147 gene in different tumor tissues and normal tissues. Many previous studies have also shown that the expression of the CD147 gene plays a crucial role in the development of tumors. To understand whether CD147 can be used as a therapeutic target for oral cancer, CRISPR/Cas9 gene-editing technology was used to knock out the CD147 gene in cal27 cells to obtain knockout cell lines. Using CCK-8, Transwell, RT-PCR, and Western blotting, the proliferation and invasion abilities of the knockout cell lines were decreased significantly, and the expression of matrix metalloproteinase was also inhibited. Next, a subcutaneously transplanted tumor model in nude mice was constructed to detect the effect of the CD147 gene on tumors. Subcutaneous tumor growth and immunohistochemistry results showed that the proliferation and doxorubicin resistance of knockout cell line were significantly inhibited compared with those in the wild-type group. These results indicated that knocking out CD147 significantly reduced the proliferation and invasion of cal27 cells, and CD147 may be a potential therapeutic target for oral cancer.},
}
@article {pmid33766108,
year = {2021},
author = {Keller-Costa, T and Lago-Lestón, A and Saraiva, JP and Toscan, R and Silva, SG and Gonçalves, J and Cox, CJ and Kyrpides, N and Nunes da Rocha, U and Costa, R},
title = {Metagenomic insights into the taxonomy, function, and dysbiosis of prokaryotic communities in octocorals.},
journal = {Microbiome},
volume = {9},
number = {1},
pages = {72},
pmid = {33766108},
issn = {2049-2618},
mesh = {Animals ; Anthozoa/*microbiology ; Bacteria/*classification/*genetics ; Dysbiosis ; *Host-Pathogen Interactions ; Metagenome/*genetics ; *Metagenomics ; *Phylogeny ; RNA, Ribosomal, 16S/genetics ; },
abstract = {BACKGROUND: In octocorals (Cnidaria Octocorallia), the functional relationship between host health and its symbiotic consortium has yet to be determined. Here, we employed comparative metagenomics to uncover the distinct functional and phylogenetic features of the microbiomes of healthy Eunicella gazella, Eunicella verrucosa, and Leptogorgia sarmentosa tissues, in contrast with the microbiomes found in seawater and sediments. We further explored how the octocoral microbiome shifts to a pathobiome state in E. gazella.<br><br>RESULTS: Multivariate analyses based on 16S rRNA genes, Clusters of Orthologous Groups of proteins (COGs), Protein families (Pfams), and secondary metabolite-biosynthetic gene clusters annotated from 20 Illumina-sequenced metagenomes each revealed separate clustering of the prokaryotic communities of healthy tissue samples of the three octocoral species from those of necrotic E. gazella tissue and surrounding environments. While the healthy octocoral microbiome was distinguished by so-far uncultivated Endozoicomonadaceae, Oceanospirillales, and Alteromonadales phylotypes in all host species, a pronounced increase of Flavobacteriaceae and Alphaproteobacteria, originating from seawater, was observed in necrotic E. gazella tissue. Increased abundances of eukaryotic-like proteins, exonucleases, restriction endonucleases, CRISPR/Cas proteins, and genes encoding for heat-shock proteins, inorganic ion transport, and iron storage distinguished the prokaryotic communities of healthy octocoral tissue regardless of the host species. An increase of arginase and nitric oxide reductase genes, observed in necrotic E. gazella tissues, suggests the existence of a mechanism for suppression of nitrite oxide production by which octocoral pathogens may overcome the host's immune system.<br><br>CONCLUSIONS: This is the first study to employ primer-less, shotgun metagenome sequencing to unveil the taxonomic, functional, and secondary metabolism features of prokaryotic communities in octocorals. Our analyses reveal that the octocoral microbiome is distinct from those of the environmental surroundings, is host genus (but not species) specific, and undergoes large, complex structural changes in the transition to the dysbiotic state. Host-symbiont recognition, abiotic-stress response, micronutrient acquisition, and an antiviral defense arsenal comprising multiple restriction endonucleases, CRISPR/Cas systems, and phage lysogenization regulators are signatures of prokaryotic communities in octocorals. We argue that these features collectively contribute to the stabilization of symbiosis in the octocoral holobiont and constitute beneficial traits that can guide future studies on coral reef conservation and microbiome therapy. Video Abstract.},
}
@article {pmid33766097,
year = {2021},
author = {Bhattacherjee, A and Jung, J and Zia, S and Ho, M and Eskandari-Sedighi, G and St Laurent, CD and McCord, KA and Bains, A and Sidhu, G and Sarkar, S and Plemel, JR and Macauley, MS},
title = {The CD33 short isoform is a gain-of-function variant that enhances Aβ1-42 phagocytosis in microglia.},
journal = {Molecular neurodegeneration},
volume = {16},
number = {1},
pages = {19},
pmid = {33766097},
issn = {1750-1326},
support = {//CIHR/Canada ; },
mesh = {Alleles ; Amyloid beta-Peptides/*metabolism ; Animals ; CRISPR-Cas Systems ; Crosses, Genetic ; Female ; Gain of Function Mutation ; Gene Editing ; Gene Regulatory Networks ; Genes, Immediate-Early ; Humans ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Microglia/*physiology ; Peptide Fragments/*metabolism ; Phagocytosis/*genetics ; Polysaccharides/metabolism ; Protein Isoforms/genetics/physiology ; RNA-Seq ; Sialic Acid Binding Ig-like Lectin 3/antagonists &amp; inhibitors/*genetics/physiology ; Single-Cell Analysis ; U937 Cells ; },
abstract = {BACKGROUND: CD33 is genetically linked to Alzheimer's disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant.<br><br>METHODS: We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently.<br><br>RESULTS: In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m[+] microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding.<br><br>CONCLUSIONS: These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.},
}
@article {pmid33765408,
year = {2021},
author = {Chakraborty, D and Agrawal, A and Maiti, S},
title = {Rapid identification and tracking of SARS-CoV-2 variants of concern.},
journal = {Lancet (London, England)},
volume = {397},
number = {10282},
pages = {1346-1347},
pmid = {33765408},
issn = {1474-547X},
mesh = {*COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Humans ; *SARS-CoV-2 ; },
}
@article {pmid33764482,
year = {2021},
author = {Wang, H and Calvisi, DF and Chen, X},
title = {Organoids for the Study of Liver Cancer.},
journal = {Seminars in liver disease},
volume = {41},
number = {1},
pages = {19-27},
pmid = {33764482},
issn = {1098-8971},
support = {R01 CA239251/CA/NCI NIH HHS/United States ; P30 DK026743/DK/NIDDK NIH HHS/United States ; R01 CA190606/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Biological Specimen Banks ; CRISPR-Cas Systems ; Humans ; *Induced Pluripotent Stem Cells ; *Liver Neoplasms ; Mice ; Organoids ; },
abstract = {Liver cancer is the second most lethal malignancy worldwide. Cell lines and murine models are the most common tools for modeling human liver carcinogenesis. Most recently, organoids with a three-dimensional structure derived from primary tissues or cells have been applied to liver cancer research. Organoids can be generated from induced pluripotent stem cells, embryonic or adult, healthy or diseased tissues. In particular, liver organoids have been widely employed in mechanistic studies aimed at delineating the molecular pathways responsible for hepatocarcinogenesis. The introduction of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) and microengineered miniorganoid technologies into liver organoids for cancer study has significantly accelerated these investigations. Translational advances have been made by utilizing liver tumor organoids for anticancer drug screening, biobanking, omics profiling, and biomarker discovery. This review summarizes the latest advances and the remaining challenges in the use of organoid models for the study of liver cancer.},
}
@article {pmid33764459,
year = {2021},
author = {Meng, F and Zhao, H and Zhu, B and Zhang, T and Yang, M and Li, Y and Han, Y and Jiang, J},
title = {Genomic editing of intronic enhancers unveils their role in fine-tuning tissue-specific gene expression in Arabidopsis thaliana.},
journal = {The Plant cell},
volume = {33},
number = {6},
pages = {1997-2014},
pmid = {33764459},
issn = {1532-298X},
mesh = {Arabidopsis/*genetics/growth &amp; development ; Arabidopsis Proteins/genetics ; Chromatin/genetics ; *Enhancer Elements, Genetic ; Flowers/genetics ; Gene Editing/*methods ; *Gene Expression Regulation, Plant ; Genes, Reporter ; Glucuronidase/genetics ; *Introns ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Transcription Factors/genetics ; },
abstract = {Enhancers located in introns are abundant and play a major role in the regulation of gene expression in mammalian species. By contrast, the functions of intronic enhancers in plants have largely been unexplored and only a handful of plant intronic enhancers have been reported. We performed a genome-wide prediction of intronic enhancers in Arabidopsis thaliana using open chromatin signatures based on DNase I sequencing. We identified 941 candidate intronic enhancers associated with 806 genes in seedling tissue and 1,271 intronic enhancers associated with 1,069 genes in floral tissue. We validated the function of 15 of 21 (71%) of the predicted intronic enhancers in transgenic assays using a reporter gene. We also created deletion lines of three intronic enhancers associated with two different genes using CRISPR/Cas. Deletion of these enhancers, which span key transcription factor binding sites, did not abolish gene expression but caused varying levels of transcriptional repression of their cognate genes. Remarkably, the transcriptional repression of the deletion lines occurred at specific developmental stages and resulted in distinct phenotypic effects on plant morphology and development. Clearly, these three intronic enhancers are important in fine-tuning tissue- and development-specific expression of their cognate genes.},
}
@article {pmid33764415,
year = {2021},
author = {Xiao, R and Li, Z and Wang, S and Han, R and Chang, L},
title = {Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease.},
journal = {Nucleic acids research},
volume = {49},
number = {7},
pages = {4120-4128},
pmid = {33764415},
issn = {1362-4962},
support = {R01 GM138675/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/*metabolism ; *CRISPR-Associated Proteins/chemistry/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; DNA Cleavage ; Endodeoxyribonucleases/chemistry/*metabolism/pharmacokinetics ; Gene Editing ; Models, Molecular ; Nucleic Acid Heteroduplexes/*metabolism ; Protein Binding ; },
abstract = {Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.},
}
@article {pmid33764372,
year = {2021},
author = {Li, Q and Sun, B and Chen, J and Zhang, Y and Jiang, Y and Yang, S},
title = {A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli.},
journal = {Acta biochimica et biophysica Sinica},
volume = {53},
number = {5},
pages = {620-627},
doi = {10.1093/abbs/gmab036},
pmid = {33764372},
issn = {1745-7270},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics ; *Gene Editing ; *Genome, Bacterial ; Plasmids/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.},
}
@article {pmid33764336,
year = {2021},
author = {Chen, L and Ouyang, J and Li, X and Xiao, X and Sun, W and Li, S and Zhou, L and Liao, Y and Zhang, Q},
title = {DNAH17 is essential for rat spermatogenesis and fertility.},
journal = {Journal of genetics},
volume = {100},
number = {},
pages = {},
pmid = {33764336},
issn = {0973-7731},
mesh = {Animals ; Axonemal Dyneins/antagonists &amp; inhibitors/*physiology ; *CRISPR-Cas Systems ; Homozygote ; Infertility, Male/etiology/*pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Sperm Motility ; *Spermatogenesis ; Testis/metabolism/*pathology ; },
abstract = {Knockout Dnah17 rats were generated due to its potential involvement in myopia. Subsequent study suggested that the homozygous truncation variants in DNAH17 is associated with male fertility but not myopia. Sperm count and sperm motility were measured in male rats. HE staining, immunofluorescence staining and TUNEL staining were used to observe the gross and histopathology of testis in homozygous and wild rats. Dnah17 knockout rats were generated by CRISPR/Cas9 gene editing. In the process of breeding rats, homozygous male rats were noted to be infertile, with significantly decreased number of sperm suggesting cryptozoospermia that was further confirmed by histologic studies. TUNEL assay showed an increased apoptosis in homozygous testes compared with wild type (P<0.001). A significant reduction of spermatocytes was observed in homozygotes compared with wild type (P=0.025) by immunofluorescence. These results suggest that DNAH17 is critical for spermatogenesis in male rats.},
}
@article {pmid33763075,
year = {2021},
author = {Luff, DH and Wojdyla, K and Oxley, D and Chessa, T and Hudson, K and Hawkins, PT and Stephens, LR and Barry, ST and Okkenhaug, K},
title = {PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4[+] T Cells.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {631271},
pmid = {33763075},
issn = {1664-3224},
support = {BBS/E/B/000C0409/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; BBS/E/B/000L0799/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/R000409/1/MRC_/Medical Research Council/United Kingdom ; BBS/E/B/0000H235/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CD4-Positive T-Lymphocytes/classification/drug effects/*immunology ; CRISPR-Cas Systems ; Cell Differentiation/*immunology ; Class I Phosphatidylinositol 3-Kinases/*genetics/*immunology ; Gene Editing ; Gene Knockout Techniques ; Interleukin-2/pharmacology ; Lymphocyte Activation/immunology ; Mice ; Mice, Inbred C57BL ; Multiprotein Complexes/*biosynthesis/*immunology ; Receptors, Antigen, T-Cell/genetics/*immunology ; Signal Transduction ; Specific Pathogen-Free Organisms ; },
abstract = {Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4[+] T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4[+] T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4[+] T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4[+] T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells.},
}
@article {pmid33762831,
year = {2021},
author = {Oishee, MJ and Ali, T and Jahan, N and Khandker, SS and Haq, MA and Khondoker, MU and Sil, BK and Lugova, H and Krishnapillai, A and Abubakar, AR and Kumar, S and Haque, M and Jamiruddin, MR and Adnan, N},
title = {COVID-19 Pandemic: Review of Contemporary and Forthcoming Detection Tools.},
journal = {Infection and drug resistance},
volume = {14},
number = {},
pages = {1049-1082},
pmid = {33762831},
issn = {1178-6973},
abstract = {Recent severe acute respiratory syndrome 2 (SARS-CoV-2) known as COVID-19, presents a deadly challenge to the global healthcare system of developing and developed countries, exposing the limitations of health facilities preparedness for emerging infectious disease pandemic. Opportune detection, confinement, and early treatment of infected cases present the first step in combating COVID-19. In this review, we elaborate on various COVID-19 diagnostic tools that are available or under investigation. Consequently, cell culture, followed by an indirect fluorescent antibody, is one of the most accurate methods for detecting SARS-CoV-2 infection. However, restrictions imposed by the regulatory authorities prevented its general use and implementation. Diagnosis via radiologic imaging and reverse transcriptase PCR assay is frequently employed, considered as standard procedures, whereas isothermal amplification methods are currently on the verge of clinical introduction. Notably, techniques such as CRISPR-Cas and microfluidics have added new dimensions to the SARS-CoV-2 diagnosis. Furthermore, commonly used immunoassays such as enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), neutralization assay, and the chemiluminescent assay can also be used for early detection and surveillance of SARS-CoV-2 infection. Finally, advancement in the next generation sequencing (NGS) and metagenomic analysis are smoothing the viral detection further in this global challenge.},
}
@article {pmid33762639,
year = {2021},
author = {Wetzel, KS and Guerrero-Bustamante, CA and Dedrick, RM and Ko, CC and Freeman, KG and Aull, HG and Divens, AM and Rock, JM and Zack, KM and Hatfull, GF},
title = {CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {6796},
pmid = {33762639},
issn = {2045-2322},
support = {R35 GM131729/GM/NIGMS NIH HHS/United States ; GT12053/HHMI/Howard Hughes Medical Institute/United States ; R21 AI151264/AI/NIAID NIH HHS/United States ; 54308198/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Electroporation ; Genetic Engineering/*methods ; RNA, Guide/metabolism ; },
abstract = {Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.},
}
@article {pmid33762584,
year = {2021},
author = {Chiong, KT and Cody, WB and Scholthof, HB},
title = {RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {6769},
pmid = {33762584},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Expression ; Gene Order ; *Gene Silencing ; Gene Transfer Techniques ; Genetic Engineering ; Genetic Vectors/*genetics ; Plants, Genetically Modified ; Plasmids/genetics ; *RNA Interference ; *RNA, Guide ; Tobacco Mosaic Virus/*genetics ; Viral Proteins/genetics/metabolism ; },
abstract = {We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3'gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.},
}
@article {pmid33760809,
year = {2021},
author = {Baron, N and Tupperwar, N and Dahan, I and Hadad, U and Davidov, G and Zarivach, R and Shapira, M},
title = {Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion.},
journal = {PLoS neglected tropical diseases},
volume = {15},
number = {3},
pages = {e0008352},
pmid = {33760809},
issn = {1935-2735},
mesh = {Adaptation, Physiological/*genetics ; Amino Acid Sequence/genetics ; Animals ; Antigens, Surface/biosynthesis/genetics ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Cytoskeletal Proteins/biosynthesis/genetics ; Eukaryotic Initiation Factor-4E/*genetics/*metabolism ; Eukaryotic Initiation Factor-4G/*metabolism ; Gene Expression Regulation/genetics ; Humans ; Leishmania/*genetics ; Macrophages/parasitology ; Psychodidae/parasitology ; Sequence Alignment ; },
abstract = {Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.},
}
@article {pmid33760341,
year = {2021},
author = {Jungblut, AD and Raymond, F and Dion, MB and Moineau, S and Mohit, V and Nguyen, GQ and Déraspe, M and Francovic-Fontaine, &#xc9; and Lovejoy, C and Culley, AI and Corbeil, J and Vincent, WF},
title = {Genomic diversity and CRISPR-Cas systems in the cyanobacterium Nostoc in the High Arctic.},
journal = {Environmental microbiology},
volume = {23},
number = {6},
pages = {2955-2968},
doi = {10.1111/1462-2920.15481},
pmid = {33760341},
issn = {1462-2920},
mesh = {*CRISPR-Cas Systems ; Genomics ; Multigene Family ; *Nostoc/genetics ; Phylogeny ; },
abstract = {Nostoc (Nostocales, Cyanobacteria) has a global distribution in the Polar Regions. However, the genomic diversity of Nostoc is little known and there are no genomes available for polar Nostoc. Here we carried out the first genomic analysis of the Nostoc commune morphotype with a recent sample from the High Arctic and a herbarium specimen collected during the British Arctic Expedition (1875-76). Comparisons of the polar genomes with 26 present-day non-polar members of the Nostocales family highlighted that there are pronounced genetic variations among Nostoc strains and species. Osmoprotection and other stress genes were found in all Nostoc strains, but the two Arctic strains had markedly higher numbers of biosynthetic gene clusters for uncharacterised non-ribosomal peptide synthetases, suggesting a high diversity of secondary metabolites. Since viral-host interactions contribute to microbial diversity, we analysed the CRISPR-Cas systems in the Arctic and two temperate Nostoc species. There were a large number of unique repeat-spacer arrays in each genome, indicating diverse histories of viral attack. All Nostoc strains had a subtype I-D system, but the polar specimens also showed evidence of a subtype I-B system that has not been previously reported in cyanobacteria, suggesting diverse cyanobacteria-virus interactions in the Arctic.},
}
@article {pmid33759566,
year = {2021},
author = {Hirshorn, ST and Steele, N and Zavros, Y},
title = {Modeling pancreatic pathophysiology using genome editing of adult stem cell-derived and induced pluripotent stem cell (iPSC)-derived organoids.},
journal = {American journal of physiology. Gastrointestinal and liver physiology},
volume = {320},
number = {6},
pages = {G1142-G1150},
pmid = {33759566},
issn = {1522-1547},
support = {P30 CA023074/CA/NCI NIH HHS/United States ; R01 DK083402/DK/NIDDK NIH HHS/United States ; },
mesh = {Adult Stem Cells/*cytology ; Animals ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Organoids/*cytology ; Pancreas/*cytology ; },
abstract = {In recent years, organoids have become a novel in vitro method to study gastrointestinal organ development, physiology, and disease. An organoid, in short, may be defined as a miniaturized organ that can be grown from adult stem cells in vitro and studied at the microscopic level. Organoids have been used in multitudes of different ways to study the physiology of different human diseases including gastrointestinal cancers such as pancreatic cancer. The development of genome editing based on the bacterial defense mechanism clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a laboratory tool that provides the opportunity to study the effects of specific genetic changes on organ development, physiology, and disease. The CRISPR/Cas9 approach can be combined with organoid technology including the use of induced pluripotent stem cell (iPSC)-derived and tissue-derived organoids. The goal of this review is to provide highlights on the development of organoid technology, and the use of this culture system to study the pathophysiology of specific mutations in the development of pancreatic and gastric cancers.NEW &amp; NOTEWORTHY The goal of this review is not only to provide highlights on the development of organoid technology but also to subsequently use this information to study the pathophysiology of those specific mutations in the formation of malignant pancreatic and gastric cancer.},
}
@article {pmid33759354,
year = {2021},
author = {Sharma, AK and Nymark, M and Flo, S and Sparstad, T and Bones, AM and Winge, P},
title = {Simultaneous knockout of multiple LHCF genes using single sgRNAs and engineering of a high-fidelity Cas9 for precise genome editing in marine algae.},
journal = {Plant biotechnology journal},
volume = {19},
number = {8},
pages = {1658-1669},
pmid = {33759354},
issn = {1467-7652},
mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; Diatoms/*genetics ; Endonucleases ; *Gene Editing ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system is an RNA-guided sequence-specific genome editing tool, which has been adopted for single or multiple gene editing in a wide range of organisms. When working with gene families with functional redundancy, knocking out multiple genes within the same family may be required to generate a phenotype. In this study, we tested the possibility of exploiting the known tolerance of Cas9 for mismatches between the single-guide RNA (sgRNA) and target site to simultaneously introduce indels in multiple homologous genes in the marine diatom Phaeodactylum tricornutum. As a proof of concept, we designed two sgRNAs that could potentially target the same six light-harvesting complex (LHC) genes belonging to the LHCF subgroup. Mutations in up to five genes were achieved simultaneously using a previously established CRISPR/Cas9 system for P. tricornutum. A visible colour change was observed in knockout mutants with multiple LHCF lesions. A combination of pigment, LHCF protein and growth analyses was used to further investigate the phenotypic differences between the multiple LHCF mutants and WT. Furthermore, we used the two same sgRNAs in combination with a variant of the existing Cas9 where four amino acids substitutions had been introduced that previously have been shown to increase Cas9 specificity. A significant reduction of off-target editing events was observed, indicating that the altered Cas9 functioned as a high-fidelity (HiFi) Cas9 nuclease.},
}
@article {pmid33759347,
year = {2021},
author = {Barkovskaya, A and Goodwin, CM and Seip, K and Hilmarsdottir, B and Pettersen, S and Stalnecker, C and Engebraaten, O and Briem, E and Der, CJ and Moestue, SA and Gudjonsson, T and Maelandsmo, GM and Prasmickaite, L},
title = {Detection of phenotype-specific therapeutic vulnerabilities in breast cells using a CRISPR loss-of-function screen.},
journal = {Molecular oncology},
volume = {15},
number = {8},
pages = {2026-2045},
pmid = {33759347},
issn = {1878-0261},
support = {F32 CA221005/CA/NCI NIH HHS/United States ; T32 CA009156/CA/NCI NIH HHS/United States ; R35 CA232113/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/therapeutic use ; *CRISPR-Cas Systems ; Cell Proliferation ; Drug Screening Assays, Antitumor ; Epithelial-Mesenchymal Transition ; Everolimus/therapeutic use ; Female ; Fluorouracil/therapeutic use ; Humans ; *Loss of Function Mutation ; *Phenotype ; Signal Transduction/genetics ; Triple Negative Breast Neoplasms/drug therapy/genetics/*pathology ; },
abstract = {Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple-negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR-Cas9 loss-of-function screen targeting a 2240-gene 'druggable genome' to identify phenotype-specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR-RAS-MAPK signaling, while the mesenchymal-like cells had increased sensitivity to knockout of G2 -M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype-specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype-associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2 -M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR-Cas9 loss-of-function screen enables the identification of phenotype-specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.},
}
@article {pmid33759340,
year = {2021},
author = {Lyu, Y and Yang, C and Lyu, X and Pu, K},
title = {Active Delivery of CRISPR System Using Targetable or Controllable Nanocarriers.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {17},
number = {24},
pages = {e2005222},
doi = {10.1002/smll.202005222},
pmid = {33759340},
issn = {1613-6829},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Therapy ; },
abstract = {Among programmable nuclease-based genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR) system with accuracy and the convenient operation is most promising to be applied in gene therapy. The development of effective delivery carriers for the CRISPR system is the major premise to achieve practical applications. Although many nanocarrier-mediated deliveries have been reported to be safer and cheaper over the physical and viral delivery, the accumulation at disease sites or controllability with the spatial or temporal resolution are still desired on nanocarriers to reduce side effects and off-target from the CRISPR system. Therefore, the targetable and controllable nanocarriers to actively deliver the CRISPR system are summarized. The cell or even organ selective nanocarriers are introduced first, followed by the discussion of nanocarriers controlled by biochemical or physical signals. At last, the potential challenges faced by existing nanocarriers are discussed.},
}
@article {pmid33758365,
year = {2021},
author = {van der Weyden, L and Harle, V and Turner, G and Offord, V and Iyer, V and Droop, A and Swiatkowska, A and Rabbie, R and Campbell, AD and Sansom, OJ and Pardo, M and Choudhary, JS and Ferreira, I and Tullett, M and Arends, MJ and Speak, AO and Adams, DJ},
title = {CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {395},
pmid = {33758365},
issn = {2399-3642},
support = {MR/S00386X/2/MRC_/Medical Research Council/United Kingdom ; C20510/A13031/CRUK_/Cancer Research UK/United Kingdom ; WT098051/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Biomarkers, Tumor/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/genetics/*metabolism/secondary ; Male ; Melanoma, Experimental/genetics/*metabolism/secondary ; Membrane Proteins/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Knockout ; Neoplasm Invasiveness ; Skin Neoplasms/genetics/*metabolism/pathology ; Up-Regulation ; },
abstract = {Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.},
}
@article {pmid33758082,
year = {2021},
author = {Wise, LM and Xi, Y and Purdy, JG},
title = {Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.},
journal = {mBio},
volume = {12},
number = {2},
pages = {},
pmid = {33758082},
issn = {2150-7511},
mesh = {Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Cytomegalovirus/*pathogenicity ; Cytomegalovirus Infections/*virology ; Host Microbial Interactions ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/metabolism ; Kynurenine/analysis/*antagonists &amp; inhibitors/metabolism ; Metabolomics/methods ; Receptors, Aryl Hydrocarbon/genetics/metabolism ; Signal Transduction ; Virus Replication/*genetics ; },
abstract = {Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling.IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.},
}
@article {pmid33757554,
year = {2021},
author = {Yokota, M and Kakuta, S and Shiga, T and Ishikawa, KI and Okano, H and Hattori, N and Akamatsu, W and Koike, M},
title = {Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons.},
journal = {Molecular brain},
volume = {14},
number = {1},
pages = {58},
pmid = {33757554},
issn = {1756-6606},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cells, Cultured ; Dopaminergic Neurons/*ultrastructure ; Gene Editing ; Gene Knock-In Techniques ; Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/*cytology/drug effects ; Microscopy, Electron/methods ; Microscopy, Fluorescence ; Mitochondria/*ultrastructure ; Neurogenesis ; RNA, Guide/genetics ; Spheroids, Cellular ; Tyrosine 3-Monooxygenase/genetics ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson's disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.},
}
@article {pmid33757429,
year = {2021},
author = {McGlincy, NJ and Meacham, ZA and Reynaud, KK and Muller, R and Baum, R and Ingolia, NT},
title = {A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {205},
pmid = {33757429},
issn = {1471-2164},
support = {R01GM130996/GM/NIGMS NIH HHS/United States ; R01 GM130996/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; DP2 CA195768/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Phenotype ; *RNA, Guide/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis.<br><br>RESULTS: We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription.<br><br>CONCLUSIONS: Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.},
}
@article {pmid33756158,
year = {2021},
author = {Zhou, W and Wang, X},
title = {Human gene therapy: A scientometric analysis.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {138},
number = {},
pages = {111510},
doi = {10.1016/j.biopha.2021.111510},
pmid = {33756158},
issn = {1950-6007},
mesh = {Academic Medical Centers/statistics &amp; numerical data/trends ; *Bibliometrics ; Data Mining/*methods/statistics &amp; numerical data ; Genetic Therapy/statistics &amp; numerical data/*trends ; Humans ; Periodicals as Topic/statistics &amp; numerical data/*trends ; },
abstract = {To provide a clear landscape, trends, and research frontiers of gene therapy, we systematically retrieved a total of 62,961 peer-viewed studies published between 1996 and 2020 from the Scopus, Web of Science, and 42,120 Inpadoc patent families from Derwent Innovation databases. Multiple bibliometric approaches suggest that gene therapy began to recover in 2013 after a period of significant decline. However, metrics in terms of authors and scholarly output growth, FWCI, annual citations, percentage of high-impact journal literature, and patent-citations per scholarly output are still weak at this stage, indicating a lack of research momentum. We also visualized gene therapy's knowledge structure by employing citation analysis, co-citation analysis, and co-word analysis, revealing its research hotspots and trends by text mining with Natural Language Processing. For the current predicament, we propose that the future success of gene therapy may depend on breakthroughs in more advanced and exhilarating technologies such as the CRISPR-Cas system, CAR-T cell therapies, and gene delivery vector technology. The results show that evidence-based bibliometrics allows the dissection of gene therapy to inform scientific planning and decision-making.},
}
@article {pmid33755904,
year = {2022},
author = {Brandão, KO and Grandela, C and Yiangou, L and Mummery, CL and Davis, RP},
title = {CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {531-557},
pmid = {33755904},
issn = {1940-6029},
support = {638030/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.},
}
@article {pmid33755903,
year = {2022},
author = {Benetó, N and Grinberg, D and Vilageliu, L and Canals, I},
title = {Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2549},
number = {},
pages = {409-425},
pmid = {33755903},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; Genome, Human ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Pluripotent Stem Cells/metabolism ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions.Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.},
}
@article {pmid33755901,
year = {2022},
author = {Sanjurjo-Soriano, C and Erkilic, N and Mamaeva, D and Kalatzis, V},
title = {CRISPR/Cas9-Mediated Genome Editing to Generate Clonal iPSC Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {589-606},
pmid = {33755901},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mammals/genetics ; },
abstract = {The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian cells. The coupling of both human (h)iPSCs and CRISPR/Cas9 technology offers great promise for cell therapy and regenerative medicine. However, several limitations including time and labor consumption, efficiency and efficacy of the system, and the potential off-targets effects induced by the Cas9 nuclease still need to be addressed. Here, we describe a detailed method for easily engineering genetic changes in hiPSCs, using a nucleofection-mediated protocol to deliver the CRISPR/Cas9 components into the cells, and discuss key points to be considered when designing your experiment. The clonal, genome-edited hiPSC line generated via our method can be directly used for downstream applications.},
}
@article {pmid33755093,
year = {2021},
author = {Ueda, N and Cahen, M and Danger, Y and Moreaux, J and Sirac, C and Cogné, M},
title = {Immunotherapy perspectives in the new era of B-cell editing.},
journal = {Blood advances},
volume = {5},
number = {6},
pages = {1770-1779},
pmid = {33755093},
issn = {2473-9537},
mesh = {B-Lymphocytes/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Humans ; Immunotherapy ; *Receptors, Chimeric Antigen/metabolism ; },
abstract = {Since the early days of vaccination, targeted immunotherapy has gone through multiple conceptual changes and challenges. It now provides the most efficient and up-to-date strategies for either preventing or treating infections and cancer. Its most recent and successful weapons are autologous T cells carrying chimeric antigen receptors, engineered purposely for binding cancer-specific antigens and therefore used for so-called adoptive immunotherapy. We now face the merger of such achievements in cell therapy: using lymphocytes redirected on purpose to bind specific antigens and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) revolution, which conferred genome-editing methodologies with both safety and efficacy. This unique affiliation will soon and considerably expand the scope of diseases susceptible to adoptive immunotherapy and of immune cells available for being reshaped as therapeutic tools, including B cells. Following the monumental success story of passive immunotherapy with monoclonal antibodies (mAbs), we are thus entering into a new era, where a combination of gene therapy/cell therapy will enable reprogramming of the patient's immune system and notably endow his B cells with the ability to produce therapeutic mAbs on their own.},
}
@article {pmid33754804,
year = {2021},
author = {Dailey, KM and Allgood, JE and Johnson, PR and Ostlie, MA and Schaner, KC and Brooks, BD and Brooks, AE},
title = {The next frontier of oncotherapy: accomplishing clinical translation of oncolytic bacteria through genetic engineering.},
journal = {Future microbiology},
volume = {16},
number = {5},
pages = {341-368},
pmid = {33754804},
issn = {1746-0921},
support = {P20 GM109024/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Bacteria/classification/genetics ; Bacterial Physiological Phenomena ; *Biological Therapy ; *Genetic Engineering ; Genome, Bacterial/genetics ; Host Microbial Interactions ; Humans ; Neoplasms/microbiology/*therapy ; Tumor Microenvironment ; },
abstract = {The development of a 'smart' drug capable of distinguishing tumor from host cells has been sought for centuries, but the microenvironment of solid tumors continues to confound therapeutics. Solid tumors present several challenges for current oncotherapeutics, including aberrant vascularization, hypoxia, necrosis, abnormally high pH and local immune suppression. While traditional chemotherapeutics are limited by such an environment, oncolytic microbes are drawn to it - having an innate ability to selectively infect, colonize and eradicate solid tumors. Development of an oncolytic species would represent a shift in the cancer therapeutic paradigm, with ramifications reaching from the medical into the socio-economic. Modern genetic engineering techniques could be implemented to customize 'Frankenstein' bacteria with advantageous characteristics from several species.},
}
@article {pmid33754479,
year = {2021},
author = {Miao, Y and Xia, Y and Kong, Y and Zhu, H and Mei, H and Li, P and Feng, H and Xun, W and Xu, Z and Zhang, N and Liu, D and Shen, Q and Zhang, R},
title = {Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi.},
journal = {Environmental microbiology},
volume = {23},
number = {6},
pages = {2937-2954},
doi = {10.1111/1462-2920.15477},
pmid = {33754479},
issn = {1462-2920},
mesh = {*CRISPR-Cas Systems/genetics ; Fungi/genetics ; Gene Editing ; Homologous Recombination ; *RNA, Guide/genetics ; },
abstract = {Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.},
}
@article {pmid33754170,
year = {2021},
author = {Meliawati, M and Schilling, C and Schmid, J},
title = {Recent advances of Cas12a applications in bacteria.},
journal = {Applied microbiology and biotechnology},
volume = {105},
number = {8},
pages = {2981-2990},
pmid = {33754170},
issn = {1432-0614},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; RNA, Guide ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. KEY POINTS: • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches.},
}
@article {pmid33754021,
year = {2021},
author = {Huang, X and Wang, M and Liu, Y and Gui, Y},
title = {Synthesis of RNA-based gene regulatory devices for redirecting cellular signaling events mediated by p53.},
journal = {Theranostics},
volume = {11},
number = {10},
pages = {4688-4698},
pmid = {33754021},
issn = {1838-7640},
mesh = {*Aptamers, Nucleotide ; CRISPR-Cas Systems ; Fibroblasts ; Genes, p53/*genetics ; *Genetic Therapy ; HCT116 Cells ; HEK293 Cells ; Humans ; In Vitro Techniques ; Integrases ; RNA ; *RNA, Catalytic ; Synthetic Biology ; },
abstract = {Rationale: The p53 gene is a well-known tumor suppressor, and its mutation often contributes to the occurrence and development of tumors. Due to the diversity and complexity of p53 mutations, there is still no effective p53 gene therapy. In this study, we designed and constructed an aptazyme switch that could effectively sense cellular wild-type p53 protein and regulate downstream gene function flexibly. The application of this artificial device in combination with Cre-LoxP and dCas9-VP64 tools achieved a precisely targeted killing effect on tumor cells. Methods: The affinity of the aptamer to p53 protein was verified by SPR. p53 aptazyme and gene circuits were chemically synthesized. The function of the gene circuit was detected by cell proliferation assay, apoptosis assay and Western blot. The nude mouse transplantation tumor experiment was used to evaluate the inhibitory effect of gene circuits on tumor cells in vivo. Results: The results of the SPR experiment showed that the p53 aptamer RNA sequence had a robust binding effect with p53 protein. The p53 aptazyme could efficiently sense wild-type p53 protein and initiate self-cleavage in cells. The Cre-p53 aptazyme gene circuit and dCas9-VP64/sgRNA mediated gene circuit designed based on p53 aptazyme significantly inhibited the growth and promoted the apoptosis of wild-type p53-deficient cancer cells in vitro. In addition, the gene circuits also had a significant inhibitory effect on tumors in vivo. Conclusion: The study developed a novel and efficient ribozyme switch for p53-specific recognition and provided a modular strategy for aptazyme binding to cellular proteins. In addition, the p53 aptazyme successfully inhibited tumor growth through a combined application with other synthetic biological tools, providing a new perspective for cancer therapy.},
}
@article {pmid33754016,
year = {2021},
author = {Gao, YY and Ling, ZY and Zhu, YR and Shi, C and Wang, Y and Zhang, XY and Zhang, ZQ and Jiang, Q and Chen, MB and Yang, S and Cao, C},
title = {The histone acetyltransferase HBO1 functions as a novel oncogenic gene in osteosarcoma.},
journal = {Theranostics},
volume = {11},
number = {10},
pages = {4599-4615},
pmid = {33754016},
issn = {1838-7640},
mesh = {Animals ; Apoptosis/drug effects/*genetics ; Bone Neoplasms/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/drug effects/genetics ; Cell Proliferation/drug effects/*genetics ; Cell Survival/drug effects/genetics ; Enzyme Inhibitors/pharmacology ; Female ; Gene Knockout Techniques ; Histone Acetyltransferases/antagonists &amp; inhibitors/*genetics ; Humans ; Male ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Oncogenes ; Osteosarcoma/*genetics ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; RNA, Small Interfering ; Trans-Activators/metabolism ; },
abstract = {HBO1 (KAT7 or MYST2) is a histone acetyltransferase that acetylates H3 and H4 histones. Methods: HBO1 expression was tested in human OS tissues and cells. Genetic strategies, including shRNA, CRISPR/Cas9 and overexpression constructs, were applied to exogenously alter HBO1 expression in OS cells. The HBO1 inhibitor WM-3835 was utilized to block HBO1 activation. Results:HBO1 mRNA and protein expression is significantly elevated in OS tissues and cells. In established (MG63/U2OS lines) and primary human OS cells, shRNA-mediated HBO1 silencing and CRISPR/Cas9-induced HBO1 knockout were able to potently inhibit cell viability, growth, proliferation, as well as cell migration and invasion. Significant increase of apoptosis was detected in HBO1-silenced/knockout OS cells. Conversely, ectopic HBO1 overexpression promoted OS cell proliferation and migration. We identified ZNF384 (zinc finger protein 384) as a potential transcription factor of HBO1. Increased binding between ZNF384 and HBO1 promoter was detected in OS cell and tissues, whereas ZNF384 silencing via shRNA downregulated HBO1 and produced significant anti-OS cell activity. In vivo, intratumoral injection of HBO1 shRNA lentivirus silenced HBO1 and inhibited OS xenograft growth in mice. Furthermore, growth of HBO1-knockout OS xenografts was significantly slower than the control xenografts. WM-3835, a novel and high-specific small molecule HBO1 inhibitor, was able to potently suppressed OS cell proliferation and migration, and led to apoptosis activation. Furthermore, intraperitoneal injection of a single dose of WM-3835 potently inhibited OS xenograft growth in SCID mice. Conclusion: HBO1 overexpression promotes OS cell growth in vitro and in vivo.},
}
@article {pmid33753930,
year = {2021},
author = {Dharia, NV and Kugener, G and Guenther, LM and Malone, CF and Durbin, AD and Hong, AL and Howard, TP and Bandopadhayay, P and Wechsler, CS and Fung, I and Warren, AC and Dempster, JM and Krill-Burger, JM and Paolella, BR and Moh, P and Jha, N and Tang, A and Montgomery, P and Boehm, JS and Hahn, WC and Roberts, CWM and McFarland, JM and Tsherniak, A and Golub, TR and Vazquez, F and Stegmaier, K},
title = {A first-generation pediatric cancer dependency map.},
journal = {Nature genetics},
volume = {53},
number = {4},
pages = {529-538},
pmid = {33753930},
issn = {1546-1718},
support = {T32 GM007226/GM/NIGMS NIH HHS/United States ; P01 CA217959/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; F32 CA243266/CA/NCI NIH HHS/United States ; R00 CA201592/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; K08 CA245251/CA/NCI NIH HHS/United States ; R35 CA210030/CA/NCI NIH HHS/United States ; R01 CA204915/CA/NCI NIH HHS/United States ; },
mesh = {Adult ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Child ; Chromosome Mapping/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease ; *Genome, Human ; Humans ; *Mutation ; Neoplasm Proteins/classification/*genetics/metabolism ; Neoplasms/*genetics/metabolism/pathology ; RNA, Guide/genetics/metabolism ; },
abstract = {Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.},
}
@article {pmid33753928,
year = {2021},
author = {Loveless, TB and Grotts, JH and Schechter, MW and Forouzmand, E and Carlson, CK and Agahi, BS and Liang, G and Ficht, M and Liu, B and Xie, X and Liu, CC},
title = {Lineage tracing and analog recording in mammalian cells by single-site DNA writing.},
journal = {Nature chemical biology},
volume = {17},
number = {6},
pages = {739-747},
pmid = {33753928},
issn = {1552-4469},
support = {R21 GM126287/GM/NIGMS NIH HHS/United States ; S10 OD021718/OD/NIH HHS/United States ; S10 OD010794/OD/NIH HHS/United States ; P30 CA062203/CA/NCI NIH HHS/United States ; S10 RR025496/RR/NCRR NIH HHS/United States ; DP2 GM119163/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Lineage/*genetics ; Cells, Cultured ; DNA/*chemistry ; DNA-Directed DNA Polymerase/chemistry ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mutagenesis, Insertional ; Mutation/genetics ; Nucleotides ; RNA Editing ; RNA, Guide/chemistry ; },
abstract = {Studying cellular and developmental processes in complex multicellular organisms can require the non-destructive observation of thousands to billions of cells deep within an animal. DNA recorders address the staggering difficulty of this task by converting transient cellular experiences into mutations at defined genomic sites that can be sequenced later in high throughput. However, existing recorders act primarily by erasing DNA. This is problematic because, in the limit of progressive erasure, no record remains. We present a DNA recorder called CHYRON (Cell History Recording by Ordered Insertion) that acts primarily by writing new DNA through the repeated insertion of random nucleotides at a single locus in temporal order. To achieve in vivo DNA writing, CHYRON combines Cas9, a homing guide RNA and the template-independent DNA polymerase terminal deoxynucleotidyl transferase. We successfully applied CHYRON as an evolving lineage tracer and as a recorder of user-selected cellular stimuli.},
}
@article {pmid33753848,
year = {2021},
author = {Warwick, T and Schulz, MH and Günther, S and Gilsbach, R and Neme, A and Carlberg, C and Brandes, RP and Seuter, S},
title = {A hierarchical regulatory network analysis of the vitamin D induced transcriptome reveals novel regulators and complete VDR dependency in monocytes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {6518},
pmid = {33753848},
issn = {2045-2322},
mesh = {CCAAT-Enhancer-Binding Proteins/genetics ; CRISPR-Cas Systems/genetics ; Cholecalciferol/genetics/*pharmacology ; Gene Expression Regulation/drug effects ; Gene Knockout Techniques ; Gene Regulatory Networks/drug effects ; Genome, Human/genetics ; Humans ; Monocytes/drug effects/metabolism ; Proto-Oncogene Protein c-ets-1/genetics ; RNA-Seq ; Receptors, Calcitriol/*genetics ; Transcriptome/drug effects/*genetics ; Vitamin D/*genetics/metabolism ; },
abstract = {The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D3 (1,25(OH)2D3). In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.},
}
@article {pmid33753124,
year = {2021},
author = {Waldt, N and Kesseler, C and Fala, P and John, P and Kirches, E and Angenstein, F and Mawrin, C},
title = {Crispr/Cas-based modeling of NF2 loss in meningioma cells.},
journal = {Journal of neuroscience methods},
volume = {356},
number = {},
pages = {109141},
doi = {10.1016/j.jneumeth.2021.109141},
pmid = {33753124},
issn = {1872-678X},
mesh = {Animals ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Meningeal Neoplasms/genetics ; *Meningioma/genetics ; Mice ; Neurofibromin 2/genetics/metabolism ; },
abstract = {BACKGROUND: Alterations of the neurofibromatosis type 2 gene (NF2) occur in more than fifty percent of sporadic meningiomas. Meningiomas develop frequently in the setting of the hereditary tumor syndrome NF2. Investigation of potential drug-based treatment options has been limited by the lack of appropriate in vitro and in vivo models.<br><br>NEW METHODS: Using Crispr/Cas gene editing, of the malignant meningioma cell line IOMM-Lee, we generated a pair of cell clones characterized by either stable knockout of NF2 and loss of the protein product merlin or retained merlin protein (transfected control without gRNA).<br><br>RESULTS: IOMM-Lee cells lacking NF2 showed reduced apoptosis and formed bigger colonies compared to control IOMM-Lee cells. Treatment of non-transfected IOMM-Lee cells with the focal adhesion kinase (FAK) inhibitor GSK2256098 resulted in reduced colony sizes. Orthotopic mouse xenografts showed the formation of convexity tumors typical for meningiomas with NF2-depleted and control cells.<br><br>No orthotopic meningioma models with genetically-engineered cell pairs are available so far.<br><br>CONCLUSION: Our model based on Crispr/Cas-based gene editing provides paired meningioma cells suitable to study functional consequences and therapeutic accessibility of NF2/merlin loss.},
}
@article {pmid33752742,
year = {2021},
author = {Gu, X and Wang, D and Xu, Z and Wang, J and Guo, L and Chai, R and Li, G and Shu, Y and Li, H},
title = {Prevention of acquired sensorineural hearing loss in mice by in vivo Htra2 gene editing.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {86},
pmid = {33752742},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cell Survival/genetics ; Dependovirus/genetics ; Disease Models, Animal ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Engineering ; Genetic Therapy/methods ; Genetic Vectors/administration &amp; dosage/genetics ; Hair Cells, Auditory/metabolism ; Hearing Loss, Sensorineural/chemically induced/etiology/*genetics/therapy ; Hearing Tests ; High-Temperature Requirement A Serine Peptidase 2/*genetics ; Mice ; RNA, Guide ; Transduction, Genetic ; Transgenes ; Treatment Outcome ; },
abstract = {BACKGROUND: Aging, noise, infection, and ototoxic drugs are the major causes of human acquired sensorineural hearing loss, but treatment options are limited. CRISPR/Cas9 technology has tremendous potential to become a new therapeutic modality for acquired non-inherited sensorineural hearing loss. Here, we develop CRISPR/Cas9 strategies to prevent aminoglycoside-induced deafness, a common type of acquired non-inherited sensorineural hearing loss, via disrupting the Htra2 gene in the inner ear which is involved in apoptosis but has not been investigated in cochlear hair cell protection.<br><br>RESULTS: The results indicate that adeno-associated virus (AAV)-mediated delivery of CRISPR/SpCas9 system ameliorates neomycin-induced apoptosis, promotes hair cell survival, and significantly improves hearing function in neomycin-treated mice. The protective effect of the AAV-CRISPR/Cas9 system in vivo is sustained up to 8&#x2009;weeks after neomycin exposure. For more efficient delivery of the whole CRISPR/Cas9 system, we also explore the AAV-CRISPR/SaCas9 system to prevent neomycin-induced deafness. The in vivo editing efficiency of the SaCas9 system is 1.73% on average. We observed significant improvement in auditory brainstem response thresholds in the injected ears compared with the non-injected ears. At 4&#x2009;weeks after neomycin exposure, the protective effect of the AAV-CRISPR/SaCas9 system is still obvious, with the improvement in auditory brainstem response threshold up to 50&#x2009;dB at 8&#x2009;kHz.<br><br>CONCLUSIONS: These findings demonstrate the safe and effective prevention of aminoglycoside-induced deafness via Htra2 gene editing and support further development of the CRISPR/Cas9 technology in the treatment of non-inherited hearing loss as well as other non-inherited diseases.},
}
@article {pmid33751237,
year = {2021},
author = {Amritha, PP and Shah, JM},
title = {Can genetic engineering-based methods for gene function identification be eclipsed by genome editing in plants? A comparison of methodologies.},
journal = {Molecular genetics and genomics : MGG},
volume = {296},
number = {3},
pages = {485-500},
pmid = {33751237},
issn = {1617-4623},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Plant/genetics ; Mutagenesis/genetics ; Mutation/genetics ; Plants/*genetics ; },
abstract = {Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.},
}
@article {pmid33751147,
year = {2021},
author = {de Oliveira Luz, AC and da Silva Junior, WJ and do Nascimento Junior, JB and da Silva, JMA and de Queiroz Balbino, V and Leal-Balbino, TC},
title = {Genetic characteristics and phylogenetic analysis of Brazilian clinical strains of Pseudomonas aeruginosa harboring CRISPR/Cas systems.},
journal = {Current genetics},
volume = {67},
number = {4},
pages = {663-672},
pmid = {33751147},
issn = {1432-0983},
mesh = {Bacteria/genetics ; Brazil ; CRISPR-Cas Systems/*genetics ; Genome, Bacterial/*genetics ; *Genomics ; Humans ; Phylogeny ; Pseudomonas aeruginosa/*genetics/pathogenicity ; },
abstract = {The CRISPR-Cas are adaptive immune systems found in archaea and bacteria, responsible for providing sequence-specific resistance against foreign DNA. Strains of Pseudomonas aeruginosa may carry CRISPR/Cas system types I-F, I-E and/or I-C; however, several aspects related to the epidemiology and functionality of these systems have not yet been revealed. Here, we report 13 genomes of clinical strains of P. aeruginosa from Brazil that were positive for CRISPR/Cas system types I-F and I-E, a rare feature in this species. The phylogenetic tree, which was constructed with 161 other publicly available genomes, suggested no direct relationship between positive strains, and the various types of CRISPR/Cas systems were spread throughout the tree. Comparative analysis of the genetic locations of type I-F and a specific orphan CRISPR array (without cas genes), named the LES locus, showed sequence similarities between this orphan locus and type I-F, but these LES loci were inserted in a different genomic location. We also report the presence of prophages, the presence of anti-CRISPR genes, and possibly the presence of self-targeting spacers. Here, we conclude that CRISPR/Cas is highly associated with certain lineages and is spread throughout the phylogenetic tree, showing no clear pattern of evolutionary distribution. Moreover, the LES locus might be a CRISPR1 locus related to type I-F that may have been misplaced and maintained over time. Furthermore, strains carrying I-F and I-E are rare, and not all of them are closely related. Further functional work is needed to better comprehend if aspects reported in this study are functional, including the LES locus, self-targeting spacers, anti-CRISPR protection, and I-F/I-E-carrying lineages.},
}
@article {pmid33751124,
year = {2021},
author = {Zhang, X and Lv, S and Luo, Z and Hu, Y and Peng, X and Lv, J and Zhao, S and Feng, J and Huang, G and Wan, QL and Liu, J and Huang, H and Luan, B and Wang, D and Zhao, X and Lin, Y and Zhou, Q and Zhang, ZN and Rong, Z},
title = {MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo.},
journal = {Nucleic acids research},
volume = {49},
number = {7},
pages = {4171-4185},
pmid = {33751124},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Caenorhabditis elegans ; Campylobacter jejuni ; Fibroblast Growth Factors/*metabolism ; *Gene Editing ; HEK293 Cells ; Humans ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; RNA, Guide/*metabolism ; },
abstract = {CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.},
}
@article {pmid33750926,
year = {2022},
author = {Chen, Y and Wen, R and Yang, Z and Chen, Z},
title = {Genome editing using CRISPR/Cas9 to treat hereditary hematological disorders.},
journal = {Gene therapy},
volume = {29},
number = {5},
pages = {207-216},
pmid = {33750926},
issn = {1476-5462},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Hematologic Diseases/genetics/therapy ; Humans ; RNA, Guide/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile and convenient genome-editing tool with prospects in gene therapy. This technique is based on customized site-specific nucleases with programmable guiding RNAs that cleave and introduce double-strand breaks (DSBs) at the target locus and achieve precise genome modification by triggering DNA repair mechanisms. Human hematopoietic stem/progenitor cells (HSPCs) are conventional cell targets for gene therapy in hematological diseases and have been widely used in most studies. Induced pluripotent stem cells (iPSCs) can be generated from a variety of somatic cells and hold great promise for personalized cell-based therapies. CRISPR/Cas9-mediated genome editing in autologous HSPCs and iPSCs is an ideal therapeutic solution for treating hereditary hematological disorders. Here, we review and summarize the latest studies about CRISPR/Cas9-mediated genome editing in patient-derived HSPCs and iPSCs to treat hereditary hematological disorders. Current challenges and prospects are also discussed.},
}
@article {pmid33750221,
year = {2021},
author = {Dasgupta, I and Flotte, TR and Keeler, AM},
title = {CRISPR/Cas-Dependent and Nuclease-Free In Vivo Therapeutic Gene Editing.},
journal = {Human gene therapy},
volume = {32},
number = {5-6},
pages = {275-293},
pmid = {33750221},
issn = {1557-7422},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 DK098252/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing ; Gene Targeting ; Humans ; Prospective Studies ; },
abstract = {Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered nucleases induces a double-stranded break (DSB) in the target genomic locus, thereby activating DNA repair mechanisms. DSBs triggered by nucleases are repaired either by the nonhomologous end-joining or the homology-directed repair pathway, enabling efficient gene editing. While there are several ongoing ex vivo genome editing clinical trials, current research underscores the therapeutic potential of CRISPR/Cas-based (clustered regularly interspaced short palindrome repeats-associated Cas nuclease) in vivo gene editing. In this review, we provide an overview of the CRISPR/Cas-mediated in vivo genome therapy applications and explore their prospective clinical translatability to treat human monogenic disorders. In addition, we discuss the various challenges associated with in vivo genome editing technologies and strategies used to circumvent them. Despite the robust and precise nuclease-mediated gene editing, a promoterless, nuclease-independent gene targeting strategy has been utilized to evade the drawbacks of the nuclease-dependent system, such as off-target effects, immunogenicity, and cytotoxicity. Thus, the rapidly evolving paradigm of gene editing technologies will continue to foster the progress of gene therapy applications.},
}
@article {pmid33750218,
year = {2021},
author = {Flotte, TR},
title = {CRISPR Keeps Things Fresh: Next-Generation Tools for Gene Editing.},
journal = {Human gene therapy},
volume = {32},
number = {5-6},
pages = {235-236},
doi = {10.1089/hum.2021.29152.trf},
pmid = {33750218},
issn = {1557-7422},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; },
}
@article {pmid33749720,
year = {2021},
author = {Xie, J and Jusuf, PR and Bui, BV and Dudczig, S and Sztal, TE and Goodbourn, PT},
title = {Altered Visual Function in a Larval Zebrafish Knockout of Neurodevelopmental Risk Gene pdzk1.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {62},
number = {3},
pages = {29},
pmid = {33749720},
issn = {1552-5783},
mesh = {Animals ; CRISPR-Associated Protein 9 ; Contrast Sensitivity/physiology ; Electroretinography ; *Gene Knockout Techniques ; Genotyping Techniques ; Larva ; PDZ Domains/*genetics ; Psychomotor Performance/*physiology ; Real-Time Polymerase Chain Reaction ; Receptors, Corticotropin-Releasing Hormone/metabolism ; Receptors, N-Methyl-D-Aspartate/metabolism ; Retina/metabolism/*physiopathology ; Serotonin/metabolism ; Vision Disorders/*genetics/metabolism/physiopathology ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {PURPOSE: The human PDZK1 gene is located in a genomic susceptibility region for neurodevelopmental disorders. A genome-wide association study identified links between PDZK1 polymorphisms and altered visual contrast sensitivity, an endophenotype for schizophrenia and autism spectrum disorder. The PDZK1 protein is implicated in neurological functioning, interacting with synaptic molecules including postsynaptic density 95 (PSD-95), N-methyl-d-aspartate receptors (NMDARs), corticotropin-releasing factor receptor 1 (CRFR1), and serotonin 2A receptors. The purpose of the present study was to elucidate the role of PDZK1.<br><br>METHODS: We generated pdzk1-knockout (pdzk1-KO) zebrafish using CRISPR/Cas-9 genome editing. Visual function of 7-day-old fish was assessed at behavioral and functional levels using the optomotor response and scotopic electroretinogram (ERG). We also quantified retinal morphology and densities of PSD-95, NMDAR1, CRFR1, and serotonin in the synaptic inner plexiform layer at 7 days, 4 weeks, and 8 weeks of age. Standard RT-PCR and nonsense-mediated decay interference treatment were also performed to assess genetic compensation in mutants.<br><br>RESULTS: Relative to wild-type, pdzk1-KO larvae showed spatial frequency tuning functions with increased amplitude (likely due to abnormal gain control) and reduced ERG b-waves (suggestive of inner retinal dysfunction). No synaptic phenotypes, but possible morphological retinal phenotypes, were identified. We confirmed that the absence of major histological phenotypes was not attributable to genetic compensatory mechanisms.<br><br>CONCLUSIONS: Our findings point to a role for pdzk1 in zebrafish visual function, and our model system provides a platform for investigating other genes associated with abnormal visual behavior.},
}
@article {pmid33748164,
year = {2021},
author = {Yang, Y and Xu, J and Ge, S and Lai, L},
title = {CRISPR/Cas: Advances, Limitations, and Applications for Precision Cancer Research.},
journal = {Frontiers in medicine},
volume = {8},
number = {},
pages = {649896},
pmid = {33748164},
issn = {2296-858X},
abstract = {Cancer is one of the most leading causes of mortalities worldwide. It is caused by the accumulation of genetic and epigenetic alterations in 2 types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. In recent years, development of the clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome engineering for different cancer research ranging for research ranging from fundamental science to translational medicine and precise cancer treatment. The CRISPR/CRISPR associated proteins (CRISPR/Cas) are prokaryote-derived genome editing systems that have enabled researchers to detect, image, manipulate and annotate specific DNA and RNA sequences in various types of living cells. The CRISPR/Cas systems have significant contributions to discovery of proto-oncogenes and TSGs, tumor cell epigenome normalization, targeted delivery, identification of drug resistance mechanisms, development of high-throughput genetic screening, tumor models establishment, and cancer immunotherapy and gene therapy in clinics. Robust technical improvements in CRISPR/Cas systems have shown a considerable degree of efficacy, specificity, and flexibility to target the specific locus in the genome for the desired applications. Recent developments in CRISPRs technology offers a significant hope of medical cure against cancer and other deadly diseases. Despite significant improvements in this field, several technical challenges need to be addressed, such as off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency, in vivo delivery of the Cas system components, and immune responses. This study aims to overview the recent technological advancements, preclinical and perspectives on clinical applications of CRISPR along with their advantages and limitations. Moreover, the potential applications of CRISPR/Cas in precise cancer tumor research, genetic, and other precise cancer treatments discussed.},
}
@article {pmid33745750,
year = {2021},
author = {Bier, E and Nizet, V},
title = {Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {8},
pages = {745-757},
doi = {10.1016/j.tig.2021.02.007},
pmid = {33745750},
issn = {0168-9525},
support = {R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/adverse effects/*therapeutic use ; CRISPR-Cas Systems/*genetics ; Drug Resistance, Microbial/*genetics ; Gene Editing/methods ; Genome, Bacterial/drug effects/*genetics ; Humans ; },
abstract = {Bacterial resistance to antibiotics has reached critical levels, skyrocketing in hospitals and the environment and posing a major threat to global public health. The complex and challenging problem of reducing antibiotic resistance (AR) requires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics.},
}
@article {pmid33744974,
year = {2021},
author = {Murugan, K and Suresh, SK and Seetharam, AS and Severin, AJ and Sashital, DG},
title = {Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR-Cas9 variants.},
journal = {Nucleic acids research},
volume = {49},
number = {7},
pages = {4037-4053},
pmid = {33744974},
issn = {1362-4962},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Staphylococcus aureus/*genetics ; Substrate Specificity ; },
abstract = {Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.},
}
@article {pmid33744818,
year = {2021},
author = {Wang, L and Wang, L and Liu, Y and Wang, Z and Chen, Q and Liu, Z and Hu, J},
title = {Characterization of type I-F CRISPR-Cas system in Laribacter hongkongensis isolates from animals, the environment and diarrhea patients.},
journal = {International journal of food microbiology},
volume = {346},
number = {},
pages = {109153},
doi = {10.1016/j.ijfoodmicro.2021.109153},
pmid = {33744818},
issn = {1879-3460},
mesh = {Alleles ; Animals ; Anura/*microbiology ; Betaproteobacteria/classification/*genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Diarrhea/*microbiology ; Fishes/*microbiology ; Foodborne Diseases/*microbiology ; Gastroenteritis/*microbiology ; Genomics ; Genotype ; Humans ; Plasmids/genetics ; },
abstract = {Laribacter hongkongensis is a foodborne organism that is associated with gastroenteritis and diarrhea in humans. Here we describe the structural characteristics and potential function of CRISPR systems to obtain insight into the genotypic diversity of L. hongkongensis. Specifically, we analyzed the genomic content of six L. hongkongensis genomes and identified two CRISPR loci (CRISPR1 and CRISPR2) belonging to the I-F subtype of CRISPR systems. CRISPR1 was flanked on one side by cas genes and a 170 bp-long putative leader sequence, while CRISPR2 arrays located further and processed by the same cas genes. Then a combination of PCR and sequencing was used to determine the prevalence and distribution of the two CRISPR arrays in 112 L. hongkongensis strains isolated from patients, animals, and water reservoirs. In total, the CRISPR1-Cas system of complete subtype I-F was detected in 91.5% (108/118) of the isolates, whereas CRISPR2 locus existed in 72.0% (85/118). Ten strains only possessed part of the cas genes of subtype I-F and four of them with CRISPR2 array. The two loci contained highly conserved and identical direct repeat sequences which were stable in their RNA secondary structure. Additionally, 2564 total spacers including 980 unique spacers arranged in 59 alleles were identified. Homology analysis showed only 1.8% (18/980) of the spacers matched with plasmid or phage. CRISPR polymorphism present in human isolates and frog isolates was more closely related and more extensive than that of fish isolates based on spacer polymorphism. The elucidation of the structural characteristics of the CRISPR-Cas system may be helpful for further studying the specific mechanism of adaptive immunity and other biological functions mediated by CRISPR in L. hongkongensis. The conservation of CRISPR loci and hypervariable repeat-spacer arrays imply the potential for molecular typing of L. hongkongensis.},
}
@article {pmid33744462,
year = {2021},
author = {Bandara, AB and Drake, JC and James, CC and Smyth, JW and Brown, DA},
title = {Complex I protein NDUFS2 is vital for growth, ROS generation, membrane integrity, apoptosis, and mitochondrial energetics.},
journal = {Mitochondrion},
volume = {58},
number = {},
pages = {160-168},
pmid = {33744462},
issn = {1872-8278},
support = {F31 HL140909/HL/NHLBI NIH HHS/United States ; R00 AG057825/AG/NIA NIH HHS/United States ; R01 HL132236/HL/NHLBI NIH HHS/United States ; },
mesh = {Adenosine Triphosphate/biosynthesis ; Apoptosis/*physiology ; CRISPR-Cas Systems ; Energy Metabolism/*physiology ; Glycolysis ; HEK293 Cells ; Humans ; Mitochondria/*metabolism ; NADH Dehydrogenase/*physiology ; Oxygen Consumption ; Reactive Oxygen Species/*metabolism ; },
abstract = {Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.},
}
@article {pmid33743211,
year = {2021},
author = {Flynn, RA and Belk, JA and Qi, Y and Yasumoto, Y and Wei, J and Alfajaro, MM and Shi, Q and Mumbach, MR and Limaye, A and DeWeirdt, PC and Schmitz, CO and Parker, KR and Woo, E and Chang, HY and Horvath, TL and Carette, JE and Bertozzi, CR and Wilen, CB and Satpathy, AT},
title = {Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.},
journal = {Cell},
volume = {184},
number = {9},
pages = {2394-2411.e16},
pmid = {33743211},
issn = {1097-4172},
support = {RM1 HG007735/HG/NHGRI NIH HHS/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; U54 CA260517/CA/NCI NIH HHS/United States ; R01 AI141970/AI/NIAID NIH HHS/United States ; S10 OD021763/OD/NIH HHS/United States ; P50 HG007735/HG/NHGRI NIH HHS/United States ; K08 CA230188/CA/NCI NIH HHS/United States ; K08 AI128043/AI/NIAID NIH HHS/United States ; R01 AI140186/AI/NIAID NIH HHS/United States ; S10 OD018220/OD/NIH HHS/United States ; },
mesh = {Animals ; COVID-19/virology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chlorocebus aethiops ; Female ; Genome, Viral ; *Host-Pathogen Interactions ; Humans ; Lung/virology ; Male ; Mass Spectrometry ; Mitochondria/metabolism/ultrastructure ; Proteome/metabolism ; RNA, Viral/*genetics ; RNA-Binding Proteins/metabolism ; SARS-CoV-2/*genetics/ultrastructure ; Vero Cells ; },
abstract = {SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.},
}
@article {pmid33742755,
year = {2021},
author = {Li, G and Sretenovic, S and Eisenstein, E and Coleman, G and Qi, Y},
title = {Highly efficient C-to-T and A-to-G base editing in a Populus hybrid.},
journal = {Plant biotechnology journal},
volume = {19},
number = {6},
pages = {1086-1088},
pmid = {33742755},
issn = {1467-7652},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Gene Editing ; *Populus/genetics/metabolism ; },
}
@article {pmid33742663,
year = {2021},
author = {Klatt Shaw, D and Mokalled, MH},
title = {Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {8},
pages = {},
pmid = {33742663},
issn = {2160-1836},
support = {R01 NS113915/NS/NINDS NIH HHS/United States ; T32 EB028092/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; INDEL Mutation ; Mutagenesis ; Phenotype ; *Zebrafish/genetics ; },
abstract = {Adult zebrafish are widely used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 "crispants". We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.},
}
@article {pmid33742256,
year = {2022},
author = {Chennakesavulu, K and Singh, H and Trivedi, PK and Jain, M and Yadav, SR},
title = {State-of-the-Art in CRISPR Technology and Engineering Drought, Salinity, and Thermo-tolerant crop plants.},
journal = {Plant cell reports},
volume = {41},
number = {3},
pages = {815-831},
pmid = {33742256},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems/genetics ; Droughts ; Gene Editing/methods ; Genome, Plant/genetics ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Salinity ; Technology ; },
abstract = {Our review has described principles and functional importance of CRISPR-Cas9 with emphasis on the recent advancements, such as CRISPR-Cpf1, base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing and their potential applications in generating stress-tolerant plants. Improved agricultural practices and enhanced food crop production using innovative crop breeding technology is essential for increasing access to nutritious foods across the planet. The crop plants play a pivotal role in energy and nutrient supply to humans. The abiotic stress factors, such as drought, heat, and salinity cause a substantial yield loss in crop plants and threaten food security. The most sustainable and eco-friendly way to overcome these challenges are the breeding of crop cultivars with improved tolerance against abiotic stress factors. The conventional plant breeding methods have been highly successful in developing abiotic stress-tolerant crop varieties, but usually cumbersome and time-consuming. Alternatively, the CRISPR/Cas genome editing has emerged as a revolutionary tool for making efficient and precise genetic manipulations in plant genomes. Here, we provide a comprehensive review of the CRISPR/Cas genome editing (GE) technology with an emphasis on recent advances in the plant genome editing, including base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing (CRISPR-IGE), which can be used for obtaining cultivars with enhanced tolerance to various abiotic stress factors. We also describe tissue culture-free, DNA-free GE technology, and some of the CRISPR-based tools that can be modified for their use in crop plants.},
}
@article {pmid33742119,
year = {2021},
author = {Cheng, C and Pei, X and Li, SW and Yang, J and Li, C and Tang, J and Hu, K and Huang, G and Min, WP and Sang, Y},
title = {CRISPR/Cas9 library screening uncovered methylated PKP2 as a critical driver of lung cancer radioresistance by stabilizing β-catenin.},
journal = {Oncogene},
volume = {40},
number = {16},
pages = {2842-2857},
pmid = {33742119},
issn = {1476-5594},
mesh = {CRISPR-Cas Systems/*genetics ; Humans ; Lung Neoplasms/*genetics/pathology ; Plakophilins/*metabolism ; Radiation-Sensitizing Agents/*therapeutic use ; beta Catenin/*metabolism ; },
abstract = {Radiation resistance is a major cause of lung cancer treatment failure. Armadillo (ARM) superfamily proteins participate in various fundamental cellular processes; however, whether ARM proteins regulate radiation resistance is not fully understood. Here, we used an unbiased CRISPR/Cas9 library screen and identified plakophilin 2 (PKP2), a member of the ARM superfamily of proteins, as a critical driver of radiation resistance in lung cancer. The PKP2 level was significantly higher after radiotherapy than before radiotherapy, and high PKP2 expression after radiotherapy predicted poor overall survival (OS) and postprogression survival (PPS). Mechanistically, mass spectrometry analysis identified that PKP2 was methylated at the arginine site and interacted with protein arginine methyltransferase 1 (PRMT1). Methylation of PKP2 by PRMT1 stabilized β-catenin by recruiting USP7, further inducing LIG4, a key DNA ligase in nonhomologous end-joining (NHEJ) repair. Concomitantly, PKP2-induced radioresistance depended on facilitating LIG4-mediated NHEJ repair in lung cancer. More strikingly, after exposure to irradiation, treatment with the PRMT1 inhibitor C-7280948 abolished PKP2-induced radioresistance, and C-7280948 is a potential radiosensitizer in lung cancer. In summary, our results demonstrate that targeting the PRMT1/PKP2/β-catenin/LIG4 pathway is an effective approach to overcome radiation resistance in lung cancer.},
}
@article {pmid33741959,
year = {2021},
author = {Ooi, KH and Liu, MM and Tay, JWD and Teo, SY and Kaewsapsak, P and Jin, S and Lee, CK and Hou, J and Maurer-Stroh, S and Lin, W and Yan, B and Yan, G and Gao, YG and Tan, MH},
title = {An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1739},
pmid = {33741959},
issn = {2041-1723},
mesh = {Bacterial Proteins/genetics ; COVID-19/*diagnosis/virology ; COVID-19 Testing/*methods ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/genetics ; Humans ; Molecular Diagnostic Techniques/methods ; Mutation ; Nasopharynx/virology ; Nucleic Acid Amplification Techniques/methods ; *RNA, Guide ; RNA, Viral/genetics ; SARS-CoV-2/*genetics ; Sensitivity and Specificity ; },
abstract = {Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.},
}
@article {pmid33741908,
year = {2021},
author = {Ahmed, M and Soares, F and Xia, JH and Yang, Y and Li, J and Guo, H and Su, P and Tian, Y and Lee, HJ and Wang, M and Akhtar, N and Houlahan, KE and Bosch, A and Zhou, S and Mazrooei, P and Hua, JT and Chen, S and Petricca, J and Zeng, Y and Davies, A and Fraser, M and Quigley, DA and Feng, FY and Boutros, PC and Lupien, M and Zoubeidi, A and Wang, L and Walsh, MJ and Wang, T and Ren, S and Wei, GH and He, HH},
title = {CRISPRi screens reveal a DNA methylation-mediated 3D genome dependent causal mechanism in prostate cancer.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1781},
pmid = {33741908},
issn = {2041-1723},
support = {153234//CIHR/Canada ; 152864//CIHR/Canada ; 152863//CIHR/Canada ; R01 DK118946/DK/NIDDK NIH HHS/United States ; P50 CA211015/CA/NCI NIH HHS/United States ; R01 GM119189/GM/NIGMS NIH HHS/United States ; 159567//CIHR/Canada ; 142246//CIHR/Canada ; P30 CA016042/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CCCTC-Binding Factor/genetics/metabolism ; *CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; *DNA Methylation ; Gene Editing/*methods ; Genetic Predisposition to Disease/*genetics ; Genome-Wide Association Study/*methods ; Humans ; Male ; Mice, Inbred NOD ; Mice, SCID ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic/genetics ; Prostatic Neoplasms/*genetics ; Proto-Oncogene Proteins c-myc/genetics ; Quantitative Trait Loci/genetics ; Regulatory Elements, Transcriptional/genetics ; Risk Factors ; },
abstract = {Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.},
}
@article {pmid33741731,
year = {2021},
author = {Lee, MY and Robbins, Y and Sievers, C and Friedman, J and Abdul Sater, H and Clavijo, PE and Judd, N and Tsong, E and Silvin, C and Soon-Shiong, P and Padget, MR and Schlom, J and Hodge, J and Hinrichs, C and Allen, C},
title = {Chimeric antigen receptor engineered NK cellular immunotherapy overcomes the selection of T-cell escape variant cancer cells.},
journal = {Journal for immunotherapy of cancer},
volume = {9},
number = {3},
pages = {},
pmid = {33741731},
issn = {2051-1426},
support = {T32 DC000008/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; B7-H1 Antigen/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Databases, Genetic ; *Gene Editing ; HLA Antigens/metabolism ; Head and Neck Neoplasms/genetics/immunology/metabolism/*therapy ; Humans ; *Immunotherapy, Adoptive ; Interferon-gamma/metabolism ; Killer Cells, Natural/immunology/metabolism/*transplantation ; Lymphocyte Activation ; Lymphocytes, Tumor-Infiltrating/*immunology/metabolism ; Mice, Inbred NOD ; Mice, SCID ; Receptors, Chimeric Antigen/*genetics/immunology ; Squamous Cell Carcinoma of Head and Neck/genetics/immunology/metabolism/*therapy ; T-Lymphocytes/immunology/metabolism/*transplantation ; Tumor Burden ; *Tumor Escape ; Tumor Microenvironment ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: As heterogeneous tumors develop in the face of intact immunity, tumor cells harboring genomic or expression defects that favor evasion from T-cell detection or elimination are selected. For patients with such tumors, T cell-based immunotherapy alone infrequently results in durable tumor control.<br><br>METHODS: Here, we developed experimental models to study mechanisms of T-cell escape and demonstrated that resistance to T-cell killing can be overcome by the addition of natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) targeting programmed death ligand-1 (PD-L1).<br><br>RESULTS: In engineered models of tumor heterogeneity, PD-L1 CAR-engineered NK cells (PD-L1 t-haNKs) prevented the clonal selection of T cell-resistant tumor cells observed with T-cell treatment alone in multiple models. Treatment of heterogenous cancer cell populations with T cells resulted in interferon gamma (IFN-&#x3b3;) release and subsequent upregulation of PD-L1 on tumor cells that escaped T-cell killing through defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs in vitro and in vivo.<br><br>CONCLUSIONS: These results describe the underlying mechanisms governing synergistic antitumor activity between T cell-based immunotherapy that results in IFN-&#x3b3; production, upregulation of PD-L1 on T-cell escape cells, and the use of PD-L1 CAR-engineered NK cells to target and eliminate resistant tumor cell populations.},
}
@article {pmid33741730,
year = {2021},
author = {Guo, X and Mahlakõiv, T and Ye, Q and Somanchi, S and He, S and Rana, H and DiFiglia, A and Gleason, J and van der Touw, W and Hariri, R and Zhang, X},
title = {CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy.},
journal = {Journal for immunotherapy of cancer},
volume = {9},
number = {3},
pages = {},
pmid = {33741730},
issn = {2051-1426},
mesh = {Adaptor Proteins, Signal Transducing/*deficiency/genetics ; Animals ; Antigens, CD34/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coculture Techniques ; *Cytotoxicity, Immunologic ; Female ; GPI-Linked Proteins/metabolism ; Gene Knockout Techniques ; HL-60 Cells ; Humans ; *Immunotherapy, Adoptive ; K562 Cells ; Killer Cells, Natural/immunology/metabolism/*transplantation ; Mice, Inbred NOD ; Mice, SCID ; NK Cell Lectin-Like Receptor Subfamily C/metabolism ; Neoplasms/immunology/metabolism/*therapy ; Phenotype ; Placenta/cytology ; Pregnancy ; Proto-Oncogene Proteins c-cbl/*deficiency/genetics ; Receptors, IgG/metabolism ; *Stem Cells/immunology/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Tumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization. Here, we evaluated the effect of the genetic deletion of ubiquitin ligase Casitas B-lineage lymphoma pro-oncogene-b (CBLB), a negative regulator of lymphocyte activity, on placental CD34[+] cell-derived NK (PNK) cell cytotoxicity against tumor cells.<br><br>METHODS: Using CRISPR/Cas9 technology, CBLB was knocked out in placenta-derived CD34[+] hematopoietic stem cells, followed by differentiation into PNK cells. Cell expansion, phenotype and cytotoxicity against tumor cells were characterized in vitro. The antitumor efficacy of CBLB knockout (KO) PNK cells was tested in an acute myeloid leukemia (HL-60) tumor model in NOD-scid IL2R gamma[null] (NSG) mice. PNK cell persistence, biodistribution, proliferation, phenotype and antitumor activity were evaluated.<br><br>RESULTS: 94% of CBLB KO efficacy was achieved using CRISPR/Cas9 gene editing technology. CBLB KO placental CD34[+] cells differentiated into PNK cells with high cell yield and >90% purity determined by CD56[+] CD3[-] cell identity. Ablation of CBLB did not impact cell proliferation, NK cell differentiation or phenotypical characteristics of PNK cells. When compared with the unmodified PNK control, CBLB KO PNK cells exhibited higher cytotoxicity against a range of liquid and solid tumor cell lines in vitro. On infusion into busulfan-conditioned NSG mice, CBLB KO PNK cells showed in vivo proliferation and maturation as evidenced by increased expression of CD16, killer Ig-like receptors and NKG2A over 3 weeks. Additionally, CBLB KO PNK cells showed greater antitumor activity in a disseminated HL60-luciferase mouse model compared with unmodified PNK cells.<br><br>CONCLUSION: CBLB ablation increased PNK cell effector function and proliferative capacity compared with non-modified PNK cells. These data suggest that targeting CBLB may offer therapeutic advantages via enhancing antitumor activities of NK cell therapies.},
}
@article {pmid33740642,
year = {2021},
author = {Li, Y and Li, J and Zhou, T and Pan, G and Huang, K},
title = {Generation of PARP1 gene knockout human embryonic stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102288},
doi = {10.1016/j.scr.2021.102288},
pmid = {33740642},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; DNA Repair ; Embryonic Stem Cells ; Gene Knockout Techniques ; *Human Embryonic Stem Cells ; Humans ; Poly (ADP-Ribose) Polymerase-1/genetics ; },
abstract = {PARP1 encodes a chromatin-associated enzyme which responsible for post-translational poly(ADP-ribosyl)ation modification (Hsieh et al., 2017). It plays an important role in nucleotide excision repair, non-homologous end joining, DNA mismatch repair and many other DNA repair process. Also, PARP1 participates in inflammation and aging. However, its role in human embryonic stem cell biology has not been fully resolved. To clarify the function of PARP1 in human embryonic stem cells, we reported a PARP1 knockout human embryonic stem cell line, generated by CRISPR/Cas9 mediated gene targeting. This cell line shows normal karyotype, pluripotent stem cell marker expression and differentiation potential in vitro.},
}
@article {pmid33740450,
year = {2021},
author = {Zoloth, L},
title = {The ethical scientist in a time of uncertainty.},
journal = {Cell},
volume = {184},
number = {6},
pages = {1430-1439},
doi = {10.1016/j.cell.2021.02.051},
pmid = {33740450},
issn = {1097-4172},
mesh = {CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; *Ethics, Research ; Genetics ; Humans ; Malaria/genetics/prevention &amp; control ; *Research Personnel ; Risk ; *Uncertainty ; },
abstract = {All of science takes place amidst a world shaken by uncertainty, social and political upheaval, and challenges to truthful testimony. Just at the moment in which increasing control over biology has been theorized, our social world has become increasingly contentious and its values more divisive. Using the example of gene drives for malaria control to explore the problem of deep uncertainty in biomedical research, I argue that profound uncertainty is an essential feature. Applying the language and presumptions of the discipline of philosophical ethics, I describe three types of uncertainty that raise ethical challenges in scientific research. Rather than mitigate these challenges with excessive precautions and limits on progress, I suggest that researchers can cultivate classic values of veracity, courage, humility, and fidelity in their research allowing science to proceed ethically under conditions of deep uncertainty.},
}
@article {pmid33740170,
year = {2021},
author = {Béziat, V and Meyts, I},
title = {The "Editors" Take to RAG: Promise of CRISPR/Cas9/rAAV6-Based Gene Therapy for RAG2 Deficiency.},
journal = {Journal of clinical immunology},
volume = {41},
number = {5},
pages = {849-851},
pmid = {33740170},
issn = {1573-2592},
mesh = {*CRISPR-Cas Systems ; DNA-Binding Proteins ; *Genetic Therapy ; Humans ; Nuclear Proteins ; },
}
@article {pmid33739640,
year = {2020},
author = {Miccio, A and Munnich, A},
title = {[CRISPR/Cas9: a patch on the double helix ?].},
journal = {La Revue du praticien},
volume = {70},
number = {10},
pages = {1051-1052},
pmid = {33739640},
issn = {2101-017X},
mesh = {*Awards and Prizes ; *CRISPR-Cas Systems ; Humans ; Nobel Prize ; },
}
@article {pmid33739246,
year = {2021},
author = {Singh, DD and Verma, R and Parimoo, P and Sahu, A and Kumar, V and Upadhyay, E and Yadav, DK},
title = {Potential Therapeutic Relevance of CRISPR/Cas9 Guided Epigenetic Regulations for Neuropsychiatric Disorders.},
journal = {Current topics in medicinal chemistry},
volume = {21},
number = {10},
pages = {878-894},
doi = {10.2174/1568026621666210317154502},
pmid = {33739246},
issn = {1873-4294},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Methylation ; Epigenesis, Genetic/*genetics ; Gene Editing/methods ; Gene Expression Regulation ; Genetic Therapy/methods ; Histones/genetics ; Humans ; Mental Disorders/*therapy ; Neurocognitive Disorders/*therapy ; Signal Transduction ; Transcription Factors/metabolism ; },
abstract = {Brain function activity is regulated by several mechanisms of genetic and epigenetic factors such as histone modelling, DNA methylation, and non-coding RNA. Alterations in these regulatory mechanisms affect the normal development of neurons that causes Neuropsychiatric Disorders (ND). However, it is required to analyse the functional significance of neuropsychiatric disorders associated with a molecular mechanism to bring about therapeutic advances in early diagnosis and treatment of the patients. The CRISPR/Cas 9 (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing tools have revolutionized multiple genome and epigenome manipulation targets the same time. This review discussed the possibilities of using CRISPR/Cas 9 tools during molecular mechanism in the ND as a therapeutic approach to overcome ND that is caused due to genetic and epigenetic abnormalities.},
}
@article {pmid33738922,
year = {2021},
author = {Pan, Y and Fang, G and Wang, Z and Cao, Y and Liu, Y and Li, G and Liu, X and Xiao, Q and Zhan, S},
title = {Chromosome-level genome reference and genome editing of the tea geometrid.},
journal = {Molecular ecology resources},
volume = {21},
number = {6},
pages = {2034-2049},
doi = {10.1111/1755-0998.13385},
pmid = {33738922},
issn = {1755-0998},
mesh = {Adaptation, Physiological ; Animals ; CRISPR-Cas Systems ; Chromosomes, Insect ; *Gene Editing ; *Genome, Insect ; Insecta/*genetics ; },
abstract = {The tea geometrid is a destructive insect pest on tea plants, which seriously affects tea production in terms of both yield and quality and causes severe economic losses. The tea geometrid also provides an important study system to address the ecological adaptive mechanisms underlying its unique host plant adaptation and protective resemblance. In this study, we fully sequenced and de novo assembled the reference genome of the tea geometrid, Ectropis grisescens, using long sequencing reads. We presented a highly continuous, near-complete genome reference (787.4 Mb; scaffold N50: 26.9 Mb), along with the annotation of 18,746 protein-coding genes and 53.3% repeat contents. Importantly, we successfully placed 97.8% of the assembly in 31 chromosomes based on Hi-C interactions and characterized the sex chromosome based on sex-biased sequencing coverage. Multiple quality-control assays and chromosome-scale synteny with the model species all supported the high quality of the presented genome reference. We focused biological annotations on gene families related to the host plant adaptation and camouflage in the tea geometrid and performed comparisons with other representative lepidopteran species. Important findings include the E. grisescens-specific expansion of CYP6 P450 genes that might be involved in metabolism of tea defensive chemicals and unexpected massive expansion of gustatory receptor gene families that suggests potential polyphagy for this tea pest. Furthermore, we developed an efficient genome editing system based on CRISPR/Cas9 technology and successfully implement mutagenesis of a Hox gene in the tea geometrid. Our study provides key genomic resources both for exploring unique mechanisms underlying the ecological adaptation of tea geometrids and for developing environment-friendly strategies for tea pest management.},
}
@article {pmid33738807,
year = {2021},
author = {Ke, C and Ma, Y and Pan, D and Wan, Z and Feng, T and Yu, D and Liu, X and Wang, H and Du, M and Huang, L and Zhang, Y and Du, L and Wang, X and Li, K and Yu, D and Zhang, M and Huang, J and Qu, J and Ren, L and Hu, Y and Cao, G and Hu, X and Wu, S and Han, H and Zhao, Y},
title = {FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs.},
journal = {Immunology},
volume = {163},
number = {4},
pages = {448-459},
pmid = {33738807},
issn = {1365-2567},
mesh = {Animals ; Animals, Genetically Modified ; Animals, Newborn ; Breast Feeding ; CRISPR-Cas Systems ; Cattle ; Colostrum/*metabolism ; Female ; Gene Knockout Techniques ; Histocompatibility Antigens Class I/genetics/*metabolism ; Homeostasis ; Humans ; Immunity, Maternally-Acquired ; Immunoglobulin G/metabolism ; Intestine, Small/*metabolism ; Placenta/*metabolism ; Pregnancy ; Rabbits ; Receptors, Fc/genetics/*metabolism ; Sheep ; Swine/*immunology ; },
abstract = {In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.},
}
@article {pmid33738531,
year = {2021},
author = {Sarjit, A and Ravensdale, JT and Coorey, R and Fegan, N and Dykes, GA},
title = {Survival of Salmonella Under Heat Stress is Associated with the Presence/Absence of CRISPR Cas Genes and Iron Levels.},
journal = {Current microbiology},
volume = {78},
number = {5},
pages = {1741-1751},
pmid = {33738531},
issn = {1432-0991},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Bacterial ; Heat-Shock Response ; Iron ; Salmonella/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) cas genes have been linked to stress response in Salmonella. Our aim was to identify the presence of CRISPR cas in Salmonella and its response to heat in the presence of iron. Whole genomes of Salmonella (n = 50) of seven serovars were compared to identify the presence of CRISPR cas genes, direct-repeats and spacers. All Salmonella genomes had all cas genes present except S. Newport 2393 which lacked these genes. Gene-specific primers were used to confirm the absence of these genes in S. Newport 2393. The presence/absence of CRISPR cas genes was further investigated among 469 S. Newport genomes from PATRIC with 283 genomes selected for pan-genome analysis. The response of eleven Salmonella strains of various serovars to gradual heat in ferrous and ferric forms of iron was investigated. A total of 32/283 S. Newport genomes that lacked all CRISPR cas genes clustered together. S. Newport 2393 was the most heat-sensitive strain at higher iron levels (200 and 220 pm) in ferrous and ferric forms of iron. The absence of CRISPR cas genes in S. Newport 2393 may contribute to its increase in heat sensitivity and iron may play a role in this. The high reduction in numbers of most Salmonella strains exposed to heat makes it unfeasible to extract RNA and conduct transcription studies. Further studies should be conducted to validate the survival of Salmonella when exposed to heat in the presence/absence of CRISPR cas genes and different iron levels.},
}
@article {pmid33737921,
year = {2021},
author = {Agius, JE and Phalen, DN and Rose, K and Eden, JS},
title = {Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {635208},
pmid = {33737921},
issn = {1664-302X},
abstract = {Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.},
}
@article {pmid33737502,
year = {2021},
author = {Bivalkar-Mehla, S and Puri, D and Singh, SB and Subramanyam, D},
title = {Understanding the role of Beclin1 in mouse embryonic stem cell differentiation through CRISPR-Cas9-mediated gene editing.},
journal = {Journal of biosciences},
volume = {46},
number = {},
pages = {},
pmid = {33737502},
issn = {0973-7138},
mesh = {Animals ; Autophagy/genetics ; Beclin-1/genetics/*physiology ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Endoderm/cytology ; Mesoderm/cytology ; Mice ; Mice, Knockout ; Mice, SCID ; Mouse Embryonic Stem Cells/*cytology/*physiology ; },
abstract = {Autophagy is a vacuolar pathway for the regulated degradation and recycling of cellular components. Beclin1, a Bcl2-interacting protein, is a well-studied autophagy regulator. Homozygous loss of Beclin1 in mice leads to early embryonic lethality. However, the role of Beclin1 in regulating the pluripotency of embryonic stem cells and their differentiation remains poorly explored. To study this, we generated Beclin1-Knockout (KO) mouse embryonic stem cells (mESCs) using the CRISPR-Cas9 genome-editing tool. Interestingly, Beclin1-KO mESCs did not show any change in the expression of pluripotency marker genes. Beclin1-KO mESCs also displayed active autophagy, suggesting the presence of Beclin1-independent autophagy in mESCs. However, loss of Beclin1 resulted in compromised differentiation of mESCs in vitro and in vivo due to misregulated expression of transcription factors. Our results suggest that Beclin1 may play an autophagy-independent role in regulating the differentiation of mESCs.},
}
@article {pmid33737370,
year = {2021},
author = {Mulkerrins, KB and Lyons, C and Shiaris, MP},
title = {Draft Genome Sequence of Enterococcus faecalis AS003, a Strain Possessing All Three Type II-a CRISPR Loci.},
journal = {Microbiology resource announcements},
volume = {10},
number = {11},
pages = {},
pmid = {33737370},
issn = {2576-098X},
abstract = {Enterococcus faecalis is a clinically significant member of the human microbiome. Three CRISPR-Cas loci are located in conserved locations. Previous studies provide evidence that E. faecalis strains with functional CRISPR-Cas genes are negatively correlated with antibiotic resistance. Here, we report the genome sequence of an unusual strain possessing all three CRISPR-Cas loci.},
}
@article {pmid33737367,
year = {2021},
author = {Muntyan, VS and Afonin, AM and Vladimirova, ME and Saksaganskaya, AS and Gribchenko, ES and Baturina, O and Roumiantseva, ML},
title = {Complete Genome Sequence of Sinorhizobium meliloti S35m, a Salt-Tolerant Isolate from Alfalfa Rhizosphere in Soil Native to the Caucasus Region.},
journal = {Microbiology resource announcements},
volume = {10},
number = {11},
pages = {},
pmid = {33737367},
issn = {2576-098X},
abstract = {The genome of a symbiotically effective salt-tolerant strain, Sinorhizobium meliloti S35m, isolated from alfalfa rhizosphere in soil native to the Caucasus region, was sequenced. Genomic islands, prophages, and elements of a potential CRISPR/Cas I type (Cas3_0_I) system were identified in the genome.},
}
@article {pmid33735615,
year = {2021},
author = {Bao, X and Zhang, X and Wang, L and Wang, Z and Huang, J and Zhang, Q and Ye, Y and Liu, Y and Chen, D and Zuo, Y and Liu, Q and Xu, P and Huang, B and Fang, J and Lao, J and Feng, X and Li, Y and Kurita, R and Nakamura, Y and Yu, W and Ju, C and Huang, C and Mohandas, N and Li, D and Zhao, C and Xu, X},
title = {Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia.},
journal = {American journal of human genetics},
volume = {108},
number = {4},
pages = {709-721},
pmid = {33735615},
issn = {1537-6605},
support = {P01 DK032094/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD34/metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Child ; DNA (Cytosine-5-)-Methyltransferases/genetics ; DNA Methylation ; DNA Methyltransferase 3A ; *Epigenesis, Genetic ; Erythroid Precursor Cells/cytology/metabolism ; Female ; Fetal Hemoglobin/genetics ; Gene Editing ; *Gene Expression Profiling ; Humans ; Male ; Mice ; Promoter Regions, Genetic/genetics ; Repressor Proteins/*deficiency/*genetics ; Reproducibility of Results ; Sulfites ; Whole Genome Sequencing ; beta-Thalassemia/*genetics/pathology ; gamma-Globins/*genetics ; },
abstract = {The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA[+] erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34[+] erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.},
}
@article {pmid33734711,
year = {2021},
author = {Wang, T and Zhang, C and Zhang, H and Zhu, H},
title = {CRISPR/Cas9-Mediated Gene Editing Revolutionizes the Improvement of Horticulture Food Crops.},
journal = {Journal of agricultural and food chemistry},
volume = {69},
number = {45},
pages = {13260-13269},
doi = {10.1021/acs.jafc.1c00104},
pmid = {33734711},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant ; Horticulture ; Humans ; },
abstract = {Horticultural food crops are important sources of nutrients for humans. With the increase of the global population, enhanced horticulture food crop production has become a new challenge, and enriching their nutritional content has also been required. Gene editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have accelerated crop improvement through the modification of targeted genomes precisely. Here, we review the development of various gene editors and compare their advantages and shortcomings, especially the newly emerging CRISPR/Cas systems, such as base editing and prime editing. We also summarize their practical applications in crop trait improvement, including yield, nutritional quality, and other consumer traits.},
}
@article {pmid33733999,
year = {2021},
author = {Ratner, HK and Weiss, DS},
title = {crRNA complementarity shifts endogenous CRISPR-Cas systems between transcriptional repression and DNA defense.},
journal = {RNA biology},
volume = {18},
number = {11},
pages = {1560-1573},
pmid = {33733999},
issn = {1555-8584},
support = {R01 AI110701/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics/*metabolism ; Francisella/*genetics/growth &amp; development/metabolism ; RNA, Bacterial/*genetics ; *Transcription, Genetic ; *Virulence ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that recognize and cleave nucleic acid targets using small RNAs called CRISPR RNAs (crRNAs) to guide Cas protein(s). There is increasing evidence for the broader endogenous roles of these systems. The CRISPR-Cas9 system of Francisella novicida also represses endogenous transcription using a non-canonical small RNA (scaRNA). We examined whether the crRNAs of the native F. novicida CRISPR-Cas systems, Cas12a and Cas9, can guide transcriptional repression. Both systems repressed mRNA transcript levels when crRNA-target complementarity was limited, and led to target cleavage with extended complementarity. Using these parameters we engineered the CRISPR array of Cas12a to guide the transcriptional repression of a new and endogenous target. Since the majority of crRNA targets remain unidentified, this work suggests that a re-analysis of crRNAs for endogenous targets with limited complementarity could reveal new, diverse regulatory roles for CRISPR-Cas systems in prokaryotic biology.},
}
@article {pmid33733873,
year = {2021},
author = {Lu, Z and Yao, X and Lyu, P and Yadav, M and Yoo, K and Atala, A and Lu, B},
title = {Lentiviral Capsid-Mediated Streptococcus pyogenes Cas9 Ribonucleoprotein Delivery for Efficient and Safe Multiplex Genome Editing.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {914-928},
doi = {10.1089/crispr.2020.0106},
pmid = {33733873},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Capsid/metabolism ; *Gene Editing ; *Ribonucleoproteins/genetics/metabolism ; Streptococcus pyogenes/genetics ; },
abstract = {Transient expression of the CRISPR-Cas9 machinery is desirable to reduce the risks of off-targets and immune responses. Electroporation of Cas9 ribonucleoproteins (RNPs) is the most common delivery method to achieve transient Cas9 expression. Recently, retroviral capsids have been used for delivering Streptococcus pyogenes Cas9 RNPs, in which Cas9 was fused to the viral proteins. The fusion strategy may cause relative low capsid assembly efficiency. We recently developed virus-like particles (VLPs) consisting of lentiviral capsid and Staphylococcus aureus Cas9 RNPs using the specific interactions between aptamer and aptamer-binding protein (ABP), and obtained near-normal capsid assembly efficiency. Here we test whether highly active Streptococcus pyogenes Cas9 (SpCas9) RNP VLPs can be generated with high efficiency by aptamer/ABP interaction. We found that by optimizing the locations and types of aptamer used for single guide RNA modification, highly active SpCas9 RNP VLPs can be generated efficiently. VLP-delivered SpCas9 generated lower off-target insertions and deletions than SpCas9 RNPs delivered by electroporation. VLPs containing Cas9 from different species and targeting multiple genes can be efficiently prepared in single-particle preparation. Multiple-target VLPs were more efficient than the combination of single-target VLPs for simultaneous targeting of multiple genes. Thus, in addition to better safety features, the Cas9 VLPs are especially suited for multiplex genome editing. In summary, our VLPs offer safe, efficient, and flexible multiplex genome editing.},
}
@article {pmid33733242,
year = {2021},
author = {McConville, TH and Giddins, MJ and Uhlemann, AC},
title = {An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant Klebsiella pneumoniae.},
journal = {STAR protocols},
volume = {2},
number = {1},
pages = {100373},
pmid = {33733242},
issn = {2666-1667},
support = {K08 AI146284/AI/NIAID NIH HHS/United States ; R01 AI116939/AI/NIAID NIH HHS/United States ; T32 AI100852/AI/NIAID NIH HHS/United States ; U54 DK104309/DK/NIDDK NIH HHS/United States ; },
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Techniques ; Humans ; Klebsiella pneumoniae/*genetics ; Recombination, Genetic/genetics ; Whole Genome Sequencing ; },
abstract = {Multi-drug resistant (MDR) Klebsiella pneumoniae remains an urgent public health threat. While whole-genome sequencing has helped identify genetic changes underlying resistance, functional validation remains difficult due to a lack of genetic manipulation systems for MDR K. pneumoniae. CRISPR-Cas9 has revolutionized molecular biology, but its use was only recently adapted in bacteria by overcoming the lack of genetic repair systems. We describe a CRISPR-Cas9/lambda recombineering system utilizing a zeocin resistance cassette allowing efficient and versatile genetic manipulation of K. pneumoniae. For complete details on the use and execution of this protocol, please refer to McConville et al. (2020).},
}
@article {pmid33733066,
year = {2021},
author = {Zhou, Y and Bravo, JPK and Taylor, HN and Steens, JA and Jackson, RN and Staals, RHJ and Taylor, DW},
title = {Structure of a type IV CRISPR-Cas ribonucleoprotein complex.},
journal = {iScience},
volume = {24},
number = {3},
pages = {102201},
pmid = {33733066},
issn = {2589-0042},
support = {R35 GM138080/GM/NIGMS NIH HHS/United States ; R35 GM138348/GM/NIGMS NIH HHS/United States ; },
abstract = {We reveal the cryo-electron microscopy structure of a type IV-B CRISPR ribonucleoprotein (RNP) complex (Csf) at 3.9-Å resolution. The complex best resembles the type III-A CRISPR Csm effector complex, consisting of a Cas7-like (Csf2) filament intertwined with a small subunit (Cas11) filament, but the complex lacks subunits for RNA processing and target DNA cleavage. Surprisingly, instead of assembling around a CRISPR-derived RNA (crRNA), the complex assembles upon heterogeneous RNA of a regular length arranged in a pseudo-A-form configuration. These findings provide a high-resolution glimpse into the assembly and function of enigmatic type IV CRISPR systems, expanding our understanding of class I CRISPR-Cas system architecture, and suggesting a function for type IV-B RNPs that may be distinct from other class 1 CRISPR-associated systems.},
}
@article {pmid33732804,
year = {2021},
author = {Volke, DC and Wirth, NT and Nikel, PI},
title = {Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids.},
journal = {Bio-protocol},
volume = {11},
number = {4},
pages = {e3917},
pmid = {33732804},
issn = {2331-8325},
abstract = {Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present a fast and versatile protocol of fluorescent marker-assisted genome editing in Pseudomonas putida, followed by clean curing of auxiliary plasmids through user-controlled plasmid replication. One fluorescent marker facilitates identification of genome-edited colonies, while the second reporter enables detection of plasmid-free bacterial clones. Not only is this protocol the fastest available for Pseudomonas species, but it can be easily adapted to any type of genome modifications, including sequence deletions, insertions, and replacements. Graphical abstract: Rapid genome engineering of Pseudomonas with curable plasmids.},
}
@article {pmid33731154,
year = {2021},
author = {Haddad, CO and Kalt, I and Shovman, Y and Xia, L and Schlesinger, Y and Sarid, R and Parnas, O},
title = {Targeting the Kaposi's sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells.},
journal = {Virology journal},
volume = {18},
number = {1},
pages = {56},
pmid = {33731154},
issn = {1743-422X},
mesh = {Antigens, Viral ; *CRISPR-Cas Systems ; Genes, Reporter ; *Genome, Viral ; Green Fluorescent Proteins ; *Herpesviridae Infections ; *Herpesvirus 8, Human/genetics ; Humans ; Nuclear Proteins/metabolism ; Virus Latency ; },
abstract = {BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpesvirus. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure patients from the virus. In addition, there is an urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well as for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV.<br><br>METHODS: We have used the recombinat KSHV&#xa0;BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp, which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP, viral DNA levels and LANA expression were monitored and viral genomes were sequenced.<br><br>RESULTS: We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting.<br><br>CONCLUSIONS: Our study provides insights into the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.},
}
@article {pmid33730414,
year = {2021},
author = {Sukegawa, S and Saika, H and Toki, S},
title = {Plant genome editing: ever more precise and wide reaching.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {5},
pages = {1208-1218},
doi = {10.1111/tpj.15233},
pmid = {33730414},
issn = {1365-313X},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Gene Editing ; Gene Targeting ; *Genetic Engineering ; Genome, Plant/*genetics ; Plant Breeding ; },
abstract = {Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.},
}
@article {pmid33729479,
year = {2021},
author = {Hannan, FM and Stevenson, M and Bayliss, AL and Stokes, VJ and Stewart, M and Kooblall, KG and Gorvin, CM and Codner, G and Teboul, L and Wells, S and Thakker, RV},
title = {Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2.},
journal = {Human molecular genetics},
volume = {30},
number = {10},
pages = {880-892},
pmid = {33729479},
issn = {1460-2083},
support = {/WT_/Wellcome Trust/United Kingdom ; MC_UP_1502/1/MRC_/Medical Research Council/United Kingdom ; 106995/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; 205011/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adaptor Protein Complex 2/*genetics ; Adaptor Protein Complex sigma Subunits/*genetics ; Animals ; Bone Density/genetics ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Cinacalcet/pharmacology ; Disease Models, Animal ; Fibroblast Growth Factor-23/*genetics ; Gene Editing ; Humans ; Hypercalcemia/drug therapy/*genetics/metabolism/pathology ; Mice ; Mutation/genetics ; Phenotype ; Receptors, Calcium-Sensing/*genetics ; },
abstract = {Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2β2, AP2μ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1-3 (FHH1-3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.},
}
@article {pmid33728594,
year = {2021},
author = {Ghogare, R and Ludwig, Y and Bueno, GM and Slamet-Loedin, IH and Dhingra, A},
title = {Genome editing reagent delivery in plants.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {321-335},
pmid = {33728594},
issn = {1573-9368},
mesh = {Agrobacterium ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Vectors/*administration &amp; dosage ; *Genome, Plant ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; Protoplasts ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants.},
}
@article {pmid33727726,
year = {2021},
author = {},
title = {Revamp of UK CRISPR regulation will require public trust.},
journal = {Nature},
volume = {591},
number = {7850},
pages = {345},
pmid = {33727726},
issn = {1476-4687},
mesh = {Agriculture/*legislation &amp; jurisprudence/*methods/standards ; Animals ; Asparagine/biosynthesis ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/genetics/standards ; Food, Genetically Modified/*classification/standards ; Gene Editing/classification/*legislation &amp; jurisprudence/standards ; Government Regulation ; *Public Opinion ; Triticum/genetics/metabolism ; *Trust ; United Kingdom ; },
}
@article {pmid33727557,
year = {2021},
author = {Huang, HH and Bellato, M and Qian, Y and Cárdenas, P and Pasotti, L and Magni, P and Del Vecchio, D},
title = {dCas9 regulator to neutralize competition in CRISPRi circuits.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1692},
pmid = {33727557},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Regulatory Networks ; Genetic Techniques ; RNA, Guide/genetics ; },
abstract = {CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits.},
}
@article {pmid33727171,
year = {2021},
author = {Zhang, X and Hu, X and Jan, S and Rasheed, SM and Zhang, Y and Du, M and Yang, E},
title = {Development of CRISPR-Cas9 genome editing system in Talaromyces marneffei.},
journal = {Microbial pathogenesis},
volume = {154},
number = {},
pages = {104822},
doi = {10.1016/j.micpath.2021.104822},
pmid = {33727171},
issn = {1096-1208},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Mycoses ; *Talaromyces/genetics ; },
abstract = {Talaromyces marneffei is an important pathogenic thermally dimorphic fungus causing systemic talaromycosis mainly prevalent in Southeast Asia. The dimorphic transition between mycelium and yeast is considered crucial for the pathogenicity of T. marneffei. However, the lack of genetic toolbox has been a major impediment for understanding its pathogenicity. Here a CRISPR-Cas9 system was developed to facilitate genetic manipulations in this organism. In this study, the CRISPR-Cas9 gene editing system uses a native U6 snRNA promoter from T. marneffei to drive the expression of sgRNA. Employing this system and PEG-mediated protoplast transformation, the sakA gene was mutated. Sanger sequencing confirmed nearly 40% site-directed mutation rate. The phenotype analysis confirmed the sakA gene function in T. marneffei dimorphic transition. Our study provided a powerful genome-manipulating tool, which could accelerate studies on T. marneffei for further revealing the mechanisms of its pathogenicity.},
}
@article {pmid33727070,
year = {2021},
author = {Fernández-Vizarra, E and López-Calcerrada, S and Formosa, LE and Pérez-Pérez, R and Ding, S and Fearnley, IM and Arenas, J and Martín, MA and Zeviani, M and Ryan, MT and Ugalde, C},
title = {SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFI[KO] cells.},
journal = {Biochimica et biophysica acta. Bioenergetics},
volume = {1862},
number = {7},
pages = {148414},
doi = {10.1016/j.bbabio.2021.148414},
pmid = {33727070},
issn = {1879-2650},
support = {MC_UP_1002/1/MRC_/Medical Research Council/United Kingdom ; MRF_MRF-155-0002-RG-ZEVIA/MRF/MRF/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Electron Transport ; Electron Transport Complex IV/antagonists &amp; inhibitors/genetics/*metabolism ; HEK293 Cells ; Humans ; Isotope Labeling/*methods ; Mass Spectrometry ; Mitochondria/*metabolism ; *Oxidative Phosphorylation ; },
abstract = {The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFI[KO]) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFI[KO] cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.},
}
@article {pmid33726812,
year = {2021},
author = {Shiraishi, Y and Koga, K and Yamagata, R and Hatada, I and Shiratori-Hayashi, M and Tsuda, M},
title = {α1A-adrenaline receptors in dorsal horn inhibitory neurons have an inhibitory role in the regulation of chloroquine-induced itch in mice.},
journal = {Molecular brain},
volume = {14},
number = {1},
pages = {55},
pmid = {33726812},
issn = {1756-6606},
mesh = {Animals ; CRISPR-Cas Systems ; Chloroquine/*toxicity ; Female ; Gene Editing ; Gene Knockout Techniques ; Interneurons/*physiology ; Male ; Mice ; Nerve Tissue Proteins/biosynthesis/deficiency/genetics/*physiology ; Posterior Horn Cells/*physiology ; Pruritus/chemically induced/*physiopathology ; RNA, Messenger/biosynthesis/genetics ; Receptors, Adrenergic, alpha-1/biosynthesis/deficiency/genetics/*physiology ; Vesicular Inhibitory Amino Acid Transport Proteins/biosynthesis/genetics ; p-Methoxy-N-methylphenethylamine/pharmacology ; },
abstract = {Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1a[flox/flox] mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.},
}
@article {pmid33725650,
year = {2021},
author = {Su, Q and Zhou, M and Cheng, C and Niu, J},
title = {Harnessing the power of directed evolution to improve genome editing systems.},
journal = {Current opinion in chemical biology},
volume = {64},
number = {},
pages = {10-19},
pmid = {33725650},
issn = {1879-0402},
support = {DP2 HG011027/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome ; },
abstract = {The recent development of genome editing systems, such as zinc-finger nucleases, transcription activator-like effectors, CRISPR-Cas nucleases, and base editors has enabled the unprecedented capability to engineer the genomes of living cells. The ever-increasing demand for genome editors with improved accuracy, activity, and functionality has stimulated significant efforts to further engineer the genome editing systems. Directed evolution represents a promising strategy to improve the existing genome editing systems and enable new editing functions. Here, we review recent representative strategies to harness the power of directed evolution to improve genome editing systems, which have led to state-of-the-art genome editors that have significant implications for diverse applications in both laboratories and clinics.},
}
@article {pmid33725575,
year = {2021},
author = {Meng, H and Shang, Y and Cheng, Y and Wang, K and Yu, J and Cao, P and Fan, S and Li, Y and Cui, J},
title = {Knockout of zebrafish colony-stimulating factor 1 receptor by CRISPR/Cas9 affects metabolism and locomotion capacity.},
journal = {Biochemical and biophysical research communications},
volume = {551},
number = {},
pages = {93-99},
doi = {10.1016/j.bbrc.2021.02.122},
pmid = {33725575},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques ; Larva/genetics/metabolism ; Locomotion/*genetics ; Mutation ; Receptor, Macrophage Colony-Stimulating Factor/*deficiency/genetics ; Zebrafish/*genetics/*metabolism ; },
abstract = {Colony-stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor and a key regulator of proliferation, differentiation, migration, and colonization in macrophage lineage cells. CSF1R was found to be involved in the pathogenesis of immune disorders, hematopoietic diseases, tissue damage, tumor growth and metastasis, and so on. Hence, understanding the role of CSF1R is important. CSF1R is highly conserved among vertebrates. In zebrafish, it is encoded by the colony-stimulating factor 1 receptor a (csf1ra) gene. In this study, a csf1ra[-/-] zebrafish mutant line was generated using clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) technology. csf1ra[-/-] larvae lacked the yellow cast on their heads and over their flanks, while adult mutants had poorly formed stripes. RNA-sequence analysis revealed that genes related to bile acid secretion, fat digestion and absorption, and pancreatic secretion were differentially expressed in csf1ra[-/-] mutants, which led to fatty changes in the liver. In addition, genes related to locomotion were also significantly changed, with the more active movement observed in csf1ra[-/-] larvae. Our study demonstrated that csf1ra participates in the metabolic process and behavior. This study provides new insights into csf1ra function during zebrafish development.},
}
@article {pmid33725516,
year = {2021},
author = {Xu, Q and Huang, L and Xing, J and Zhang, J and Li, H and Liu, L and Hu, C and Liao, M and Yue, J and Qi, W},
title = {Japanese encephalitis virus manipulates lysosomes membrane for RNA replication and utilizes autophagy components for intracellular growth.},
journal = {Veterinary microbiology},
volume = {255},
number = {},
pages = {109025},
doi = {10.1016/j.vetmic.2021.109025},
pmid = {33725516},
issn = {1873-2542},
mesh = {Animals ; Antimalarials/pharmacology ; Autophagy ; CRISPR-Cas Systems ; Cell Line ; Cell Membrane/*physiology ; Chloroquine/pharmacology ; Cricetinae ; Encephalitis Virus, Japanese/*physiology ; Gene Deletion ; Gene Expression Regulation, Viral/drug effects ; Humans ; Lysosomes/*physiology ; Protein Transport ; RNA, Viral/genetics/*metabolism ; Viral Proteins/genetics/metabolism ; Virus Replication/*physiology ; },
abstract = {Japanese encephalitis virus is absolutely dependent on their host cells and has evolved various strategies to manipulate the cellular secretory pathways for viral replication. However, how cellular secretory pathways are hijacked, and the origin of the viral vesicles remains elusive during JEV replication. Here we show how JEV manipulates multiple components of the cellular secretory pathway, including autophagic machinery, to generate a superior environment for genome replication. We utilized double-strand RNA antibodies to label JEV RNA complex seeking the viral replication compartments and found that JEV genome replication takes place in lysosomes (LAMP1), not in autophagosomes (LC3). Subsequently, in situ hybridization results showed that viral RNAs (vRNAs) of JEV strongly colocalized with LAMP1. What surprised us was that JEV vRNAs markedly colocalized with LC3, indicating that autophagy plays an active role in JEV replication. Interestingly, we found that JEV utilized autophagic components for intracellular growth in an autophagy-dependent manner and the fusion of autophagosome-lysosome plays a positive role in JEV post-RNA replication processes. Collectively, our findings demonstrate that JEV can manipulate cellular secretory pathway to form genome replication organelles and exploit autophagy components for intracellular growth, providing new insights into the life cycle of JEV and uncovering an attractive target for antiviral drugs.},
}
@article {pmid33724663,
year = {2021},
author = {Ah-Fong, AMV and Boyd, AM and Matson, MEH and Judelson, HS},
title = {A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.},
journal = {Molecular plant pathology},
volume = {22},
number = {6},
pages = {737-752},
pmid = {33724663},
issn = {1364-3703},
mesh = {Alleles ; CRISPR-Cas Systems ; Chromatin/genetics ; *Gene Editing ; Genomics ; Phytophthora infestans/*genetics/physiology ; Plant Diseases/*parasitology ; Solanum tuberosum/*parasitology ; },
abstract = {Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported. We discovered that it is difficult to express CRISPR/Cas9 but not a catalytically inactive form in transformants, suggesting that the active nuclease is toxic. We were able to achieve editing with CRISPR/Cas12a using vectors in which the nuclease and its guide RNA were expressed from a single transcript. Using the elicitor gene Inf1 as a target, we observed editing of one or both alleles in up to 13% of transformants. Editing was more efficient when guide RNA processing relied on the Cas12a direct repeat instead of ribozyme sequences. INF1 protein was not made when both alleles were edited in the same transformant, but surprisingly also when only one allele was altered. We discovered that the isolate used for editing, 1306, exhibited monoallelic expression of Inf1 due to insertion of a copia-like element in the promoter of one allele. The element exhibits features of active retrotransposons, including a target site duplication, long terminal repeats, and an intact polyprotein reading frame. Editing occurred more often on the transcribed allele, presumably due to differences in chromatin structure. The Cas12a system not only provides a tool for modifying genes in P. infestans, but also for other members of the genus by expanding the number of editable sites. Our work also highlights a natural mechanism that remodels oomycete genomes.},
}
@article {pmid33724658,
year = {2021},
author = {Maino, E and Wojtal, D and Evagelou, SL and Farheen, A and Wong, TWY and Lindsay, K and Scott, O and Rizvi, SZ and Hyatt, E and Rok, M and Visuvanathan, S and Chiodo, A and Schneeweiss, M and Ivakine, EA and Cohn, RD},
title = {Targeted genome editing in vivo corrects a Dmd duplication restoring wild-type dystrophin expression.},
journal = {EMBO molecular medicine},
volume = {13},
number = {5},
pages = {e13228},
pmid = {33724658},
issn = {1757-4684},
mesh = {Animals ; CRISPR-Cas Systems ; *Dystrophin/genetics ; Gene Editing ; Mice ; *Muscular Dystrophy, Duchenne/genetics/therapy ; RNA, Guide ; },
abstract = {Tandem duplication mutations are increasingly found to be the direct cause of many rare heritable diseases, accounting for up to 10% of cases. Unfortunately, animal models recapitulating such mutations are scarce, limiting our ability to study them and develop genome editing therapies. Here, we describe the generation of a novel duplication mouse model, harboring a multi-exonic tandem duplication in the Dmd gene which recapitulates a human mutation. Duplication correction of this mouse was achieved by implementing a single-guide RNA (sgRNA) CRISPR/Cas9 approach. This strategy precisely removed a duplication mutation in vivo, restored full-length dystrophin expression, and was accompanied by improvements in both histopathological and clinical phenotypes. We conclude that CRISPR/Cas9 represents a powerful tool to accurately model and treat tandem duplication mutations. Our findings will open new avenues of research for exploring the study and therapeutics of duplication disorders.},
}
@article {pmid33724509,
year = {2021},
author = {Policarpi, C and Dabin, J and Hackett, JA},
title = {Epigenetic editing: Dissecting chromatin function in context.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {43},
number = {5},
pages = {e2000316},
doi = {10.1002/bies.202000316},
pmid = {33724509},
issn = {1521-1878},
mesh = {*CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Epigenesis, Genetic/genetics ; Epigenomics ; *Gene Editing ; },
abstract = {How epigenetic mechanisms regulate genome output and response to stimuli is a fundamental question in development and disease. Past decades have made tremendous progress in deciphering the regulatory relationships involved by correlating aggregated (epi)genomics profiles with global perturbations. However, the recent development of epigenetic editing technologies now enables researchers to move beyond inferred conclusions, towards explicit causal reasoning, through 'programing' precise chromatin perturbations in single cells. Here, we first discuss the major unresolved questions in the epigenetics field that can be addressed by programable epigenome editing, including the context-dependent function and memory of chromatin states. We then describe the epigenetic editing toolkit focusing on CRISPR-based technologies, and highlight its achievements, drawbacks and promise. Finally, we consider the potential future application of epigenetic editing to the study and treatment of specific disease conditions.},
}
@article {pmid33724420,
year = {2021},
author = {Gomulkiewicz, R and Thies, ML and Bull, JJ},
title = {Evading resistance to gene drives.},
journal = {Genetics},
volume = {217},
number = {2},
pages = {},
pmid = {33724420},
issn = {1943-2631},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Drive Technology/*methods ; Gene Frequency ; Genetic Fitness ; Linkage Disequilibrium ; *Models, Genetic ; Plants/genetics ; *Selection, Genetic ; },
abstract = {Gene drives offer the possibility of altering and even suppressing wild populations of countless plant and animal species, and CRISPR technology now provides the technical feasibility of engineering them. However, population-suppression gene drives are prone to select resistance, should it arise. Here, we develop mathematical and computational models to identify conditions under which suppression drives will evade resistance, even if resistance is present initially. Previous models assumed resistance is allelic to the drive. We relax this assumption and show that linkage between the resistance and drive loci is critical to the evolution of resistance and that evolution of resistance requires (negative) linkage disequilibrium between the two loci. When the two loci are unlinked or only partially so, a suppression drive that causes limited inviability can evolve to fixation while causing only a minor increase in resistance frequency. Once fixed, the drive allele no longer selects resistance. Our analyses suggest that among gene drives that cause moderate suppression, toxin-antidote systems are less apt to select for resistance than homing drives. Single drives of moderate effect might cause only moderate population suppression, but multiple drives (perhaps delivered sequentially) would allow arbitrary levels of suppression. The most favorable case for evolution of resistance appears to be with suppression homing drives in which resistance is dominant and fully suppresses transmission distortion; partial suppression by resistance heterozygotes or recessive resistance are less prone to resistance evolution. Given that it is now possible to engineer CRISPR-based gene drives capable of circumventing allelic resistance, this design may allow for the engineering of suppression gene drives that are effectively resistance-proof.},
}
@article {pmid33724210,
year = {2021},
author = {Dingwei, P and Ruiqiang, L and Wu, Z and Min, W and Xuan, S and Jianhua, Z and Xiaohong, L and Yaoshen, C and Zuyong, H},
title = {Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs.},
journal = {Yi chuan = Hereditas},
volume = {43},
number = {3},
pages = {261-270},
doi = {10.16288/j.yczz.20-222},
pmid = {33724210},
issn = {0253-9772},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Cystine Knot Motifs ; Female ; Muscle Development/genetics ; *Myostatin/genetics ; Swine ; },
abstract = {Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) family, and functions as an inhibitor of muscle growth. Disrupting the inhibitory effect of MSTN on growth can provide an effective way to increase the muscle yield of livestock and poultry. The cysteine knot motif of TGF-β can stabilize the structure of MSTN protein and plays an important regulatory role in the biological function of MSTN. Accordingly, in this study, we used the CRISRP/Cas9 to edit the exon 3 of MSTN in the kidney cells of Liang Guang Small Spotted pig (LPKCs), in order to disrupt the cysteine knot motif of MSTN and remove the inhibitory effect of MSTN on its target genes.MSTN-edited LPKCs were obtained through fluorescence-activated cell sorting (FACS) and used as donor cells for somatic cell nuclear transfer (SCNT) to generate cloned embryos, which were then transferred to surrogate sows to finally obtain eight MSTN-edited Liang Guang Small Spotted piglets. Among them, two survived to 10 days old. Genotyping revealed that these two piglets were gene edited heterozygotes with base deletion and substitution occurred within the coding sequence of C106 and C108 at the cystine knot motif of MSTN. These changes resulted in frameshift mutations, and conversion of C106 and C108 to other amino acids. More developments of muscles were observed at the shoulders and hips of the heterozygotes of MSTN-edited Liang Guang Small Spotted pigs. H&amp;E analysis showed that the cross-sectional area (CSA) of myofiber inMSTN-edited pigs was significantly decreased, and the number of myofiber were significantly increased. Western blot analysis showed that the disruption of C106 and C108 did not affect the expression of MSTN protein, but significantly up-regulated the expression of its target genes such as Myf5, MyoD, Myogenin and other myogenic regulatory factors. In summary, the gene-edited pig model obtained in this study did not cause complete loss of MSTN expression, and could retain other biological functions of MSTN, thereby promoting muscle growth while minimizing the potential adverse effects on complete loss of MSTN in the Liang Guang Small Spotted pigs.},
}
@article {pmid33723654,
year = {2021},
author = {Garrigues, S and Kun, RS and de Vries, RP},
title = {Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness.},
journal = {Current genetics},
volume = {67},
number = {4},
pages = {673-684},
pmid = {33723654},
issn = {1432-0983},
mesh = {Antigens, Fungal/*genetics ; Aspergillus niger/*genetics ; CRISPR-Cas Systems/*genetics ; *DNA Barcoding, Taxonomic ; Gene Editing ; Gene Expression Regulation, Fungal/genetics ; },
abstract = {Safe use of genetically modified organisms (GMOs) in biotechnology requires the ability to track the presence of these strains in any environment in which they are applied. For this, introduction of genetic barcodes within the editing site represents a valuable tool for the identification of microbial strains that have undergone genetic modifications. However, it is not known whether these barcodes would have any unexpected effect in the resulting strains or affect the efficiency of the genetic modification. CRISPR/Cas9 has become one of the fastest-growing technologies for genome editing in a range of organisms, including fungi. However, this technology enables the generation of scarless GMOs that are very difficult to distinguish from naturally occurring mutants or other modified organisms. In this study, we address this issue using the industrial workhorse Aspergillus niger as a test case. We applied CRISPR/Cas9 technology to delete the genes encoding the transcriptional regulators XlnR and AraR, involved in the production of plant biomass-degrading enzymes. We generated 20-bp barcoded and non-barcoded ΔxlnR and ΔaraR mutants and analyzed the traceability and fitness of the resulting strains, as well as the efficiency of the genetic modification. Results showed that both barcoded and non-barcoded mutants can be traced by routine PCR reactions when the specific CRISPR/Cas9 modification is known. Additionally, barcodes neither affected the efficiency of the genetic modification nor the growth or protein production of the resulting strains. These results confirm the suitability of genetic barcodes to trace CRISPR-derived GMOs without affecting the performance of the resulting strains.},
}
@article {pmid33723072,
year = {2021},
author = {Capilla-Pérez, L and Durand, S and Hurel, A and Lian, Q and Chambon, A and Taochy, C and Solier, V and Grelon, M and Mercier, R},
title = {The synaptonemal complex imposes crossover interference and heterochiasmy in Arabidopsis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {12},
pages = {},
pmid = {33723072},
issn = {1091-6490},
mesh = {Arabidopsis/*physiology ; Arabidopsis Proteins/genetics/metabolism ; Biomarkers ; CRISPR-Cas Systems ; Chromosomes, Plant ; *Crossing Over, Genetic ; Gene Editing ; Meiosis/genetics ; Mutagenesis ; Synaptonemal Complex/*metabolism ; },
abstract = {Meiotic crossovers (COs) have intriguing patterning properties, including CO interference, the tendency of COs to be well-spaced along chromosomes, and heterochiasmy, the marked difference in male and female CO rates. During meiosis, transverse filaments transiently associate the axes of homologous chromosomes, a process called synapsis that is essential for CO formation in many eukaryotes. Here, we describe the spatial organization of the transverse filaments in Arabidopsis (ZYP1) and show it to be evolutionary conserved. We show that in the absence of ZYP1 (zyp1azyp1b null mutants), chromosomes associate in pairs but do not synapse. Unexpectedly, in absence of ZYP1, CO formation is not prevented but increased. Furthermore, genome-wide analysis of recombination revealed that CO interference is abolished, with the frequent observation of close COs. In addition, heterochiasmy was erased, with identical CO rates in males and females. This shows that the tripartite synaptonemal complex is dispensable for CO formation and has a key role in regulating their number and distribution, imposing CO interference and heterochiasmy.},
}
@article {pmid33722979,
year = {2021},
author = {Wang, Y and Hsu, AY and Walton, EM and Park, SJ and Syahirah, R and Wang, T and Zhou, W and Ding, C and Lemke, AP and Zhang, G and Tobin, DM and Deng, Q},
title = {A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.},
journal = {Journal of cell science},
volume = {134},
number = {8},
pages = {},
pmid = {33722979},
issn = {1477-9137},
support = {R01 AI125517/AI/NIAID NIH HHS/United States ; R35 GM119787/GM/NIGMS NIH HHS/United States ; R01 AI127715/AI/NIAID NIH HHS/United States ; R01 AI130236/AI/NIAID NIH HHS/United States ; R35 GM124913/GM/NIGMS NIH HHS/United States ; P30 CA023168/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neutrophils/metabolism ; *Zebrafish/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; rac GTP-Binding Proteins/metabolism ; },
abstract = {CRISPR/Cas9-based tissue-specific knockout techniques are essential for probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of single guide (sg)RNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 genes restores neutrophil motility in the corresponding knockout background. Moreover, active Rac and force-bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.},
}
@article {pmid33722289,
year = {2021},
author = {Gao, P and Lyu, Q and Ghanam, AR and Lazzarotto, CR and Newby, GA and Zhang, W and Choi, M and Slivano, OJ and Holden, K and Walker, JA and Kadina, AP and Munroe, RJ and Abratte, CM and Schimenti, JC and Liu, DR and Tsai, SQ and Long, X and Miano, JM},
title = {Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {83},
pmid = {33722289},
issn = {1474-760X},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 HL139794/HL/NHLBI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 HL138987/HL/NHLBI NIH HHS/United States ; R01 HL147476/HL/NHLBI NIH HHS/United States ; 15055101/AG/NIA NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 HL122686/HL/NHLBI NIH HHS/United States ; R01 HL136224/HL/NHLBI NIH HHS/United States ; UG3TR002636/NH/NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Binding Sites ; *CRISPR-Cas Systems ; Fluorescent Antibody Technique/methods ; *Gene Editing/methods ; *Gene Expression Regulation ; Mice ; Mice, Transgenic ; Nerve Tissue Proteins/genetics ; Organ Specificity/genetics ; *Point Mutation ; Promoter Regions, Genetic ; Protein Binding ; Recombinational DNA Repair ; Tetraspanins/genetics ; },
abstract = {BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter.<br><br>RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.<br><br>CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.},
}
@article {pmid33721902,
year = {2021},
author = {Huang, WRH and Schol, C and Villanueva, SL and Heidstra, R and Joosten, MHAJ},
title = {Knocking out SOBIR1 in Nicotiana benthamiana abolishes functionality of transgenic receptor-like protein Cf-4.},
journal = {Plant physiology},
volume = {185},
number = {2},
pages = {290-294},
pmid = {33721902},
issn = {1532-2548},
mesh = {Ascomycota/*physiology ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Lycopersicon esculentum/*genetics/immunology/microbiology ; Plant Diseases/*immunology/microbiology ; Plant Proteins/genetics/*metabolism ; Tobacco/*genetics/microbiology ; Transgenes ; },
abstract = {Knocking out SOBIR1 in Nicotiana benthamiana by CRISPR/Cas9, abolishes the functionality of the transgenic receptor-like protein Cf-4, recognizing the Avr4 effector of the fungus Cladosporium fulvum.},
}
@article {pmid33721140,
year = {2021},
author = {Jefferson, OA and Lang, S and Williams, K and Koellhofer, D and Ballagh, A and Warren, B and Schellberg, B and Sharma, R and Jefferson, R},
title = {Mapping CRISPR-Cas9 public and commercial innovation using The Lens institutional toolkit.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {585-599},
doi = {10.1007/s11248-021-00237-y},
pmid = {33721140},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; Commerce ; Crops, Agricultural/*genetics ; *Gene Editing ; Gene Targeting ; *Genome, Plant ; Humans ; Inventions/*statistics &amp; numerical data ; *Private Sector ; *Public Sector ; },
abstract = {CRISPR-Cas9 is a revolutionary technology because it is precise, fast and easy to implement, cheap and components are readily accessible. This versatility means that the technology can deliver a timely end product and can be used by many stakeholders. In plant cells, the technology can be applied to knockout genes by using CRISPR-Cas nucleases that can alter coding gene regions or regulatory elements, alter precisely a genome by base editing to delete or regulate gene expression, edit precisely a genome by homology-directed repair mechanism (cellular DNA), or regulate transcriptional machinery by using dead Cas proteins to recruit regulators to the promoter region of a gene. All these applications can be for: 1) Research use (Non commercial), 2) Uses related product components for the technology itself (reagents, equipment, toolkits, vectors etc), and 3) Uses related to the development and sale of derived end products based on this technology. In this contribution, we present a prototype report that can engage the community in open, inclusive and collaborative innovation mapping. Using the open data at the Lens.org platform and other relevant sources, we tracked, analyzed, organized, and assembled contextual and bridged patent and scholarly knowledge about CRISPR-Cas9 and with the assistance of a new Lens institutional capability, The Lens Report Builder, currently in beta release, mapped the public and commercial innovation pathways of the technology. When scaled, this capability will also enable coordinated editing and curation by credentialed experts to inform policy makers, businesses and private or public investment.},
}
@article {pmid33721016,
year = {2021},
author = {Hu, Z and Zhang, C and Wang, S and Gao, S and Wei, J and Li, M and Hou, L and Mao, H and Wei, Y and Qi, T and Liu, H and Liu, D and Lan, F and Lu, D and Wang, H and Li, J and Wang, Y},
title = {Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity.},
journal = {Nucleic acids research},
volume = {49},
number = {7},
pages = {4008-4019},
pmid = {33721016},
issn = {1362-4962},
mesh = {*Bacterial Proteins/genetics ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Fibroblasts ; Gene Editing ; HEK293 Cells ; HeLa Cells ; Humans ; *Staphylococcus/genetics ; },
abstract = {The compact CRISPR/Cas9 system, which can be delivered with their gRNA and a full-length promoter for expression by a single adeno-associated virus (AAV), is a promising platform for therapeutic applications. We previously identified a compact SauriCas9 that displays high activity and requires a simple NNGG PAM, but the specificity is moderate. Here, we identified three compact Cas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Of these three Cas9 orthologs, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. Importantly, we generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We finally engineered SlugCas9 with mutations to generate a high-fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.},
}
@article {pmid33720688,
year = {2021},
author = {Zhang, Y and Yun, K and Huang, H and Tu, R and Hua, E and Wang, M},
title = {Antisense RNA Interference-Enhanced CRISPR/Cas9 Base Editing Method for Improving Base Editing Efficiency in Streptomyces lividans 66.},
journal = {ACS synthetic biology},
volume = {10},
number = {5},
pages = {1053-1063},
doi = {10.1021/acssynbio.0c00563},
pmid = {33720688},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Repair/genetics ; Down-Regulation/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genetic Loci ; Genome, Bacterial ; Genomic Instability/genetics ; Microorganisms, Genetically-Modified ; *RNA Interference ; RNA, Antisense/*genetics ; Streptomyces coelicolor/genetics/metabolism ; Streptomyces lividans/*enzymology/*genetics ; Uracil/metabolism ; Uracil-DNA Glycosidase/genetics/metabolism ; },
abstract = {CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of Streptomyces, a base editing method has been successfully applied in several model and non-model species, such as Streptomyces coelicolor and Streptomyces griseofuscus. In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in Streptomyces lividans 66, albeit with a much lower editing efficiency compared to that of S. coelicolor. Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4-67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed antisense RNA interference-enhanced CRISPR/Cas9 Base Editing method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8-65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in S. lividans 66 and might be beneficial for improving the base editing efficiency and genome stability in other Streptomyces strains.},
}
@article {pmid33720266,
year = {2021},
author = {Song, J and Kim, S and Kim, HY and Hur, KH and Kim, Y and Park, HG},
title = {A novel method to detect mutation in DNA by utilizing exponential amplification reaction triggered by the CRISPR-Cas9 system.},
journal = {Nanoscale},
volume = {13},
number = {15},
pages = {7193-7201},
doi = {10.1039/d1nr00438g},
pmid = {33720266},
issn = {2040-3372},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Humans ; Mutation ; RNA, Guide ; },
abstract = {We, herein, describe a novel method to detect mutation in DNA by utilizing exponential amplification reaction (EXPAR) triggered by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, called CRISPR-EXPAR. The CRISPR system consisting of two Cas9/sgRNA complexes was designed to cut out a specific mutation region within the target DNA, which would consequently promote EXPAR by continuously repeated extension and nicking reactions. As a consequence, a large number of final EXPAR products, which can be monitored through duplex-specific fluorescent staining, are produced. Based on this design principle, we successfully identified a model target mutation within the human epidermal growth factor receptor 2 (HER2) gene down to 437 aM with excellent specificity. The practical capability of this method was verified by reliably identifying the target mutation directly from the genomic DNA (gDNA) extracted from the lung cancer cell line, NCI-H1781 (H1781), and its universal applicability was further confirmed by identifying another EFGF L858R mutation. This technique could serve as a new isothermal platform to identify various mutations by rationally redesigning single guide RNA (sgRNA) according to the target mutation site.},
}
@article {pmid33720132,
year = {2021},
author = {Ma, J and van der Zon, G and Sanchez-Duffhues, G and Ten Dijke, P},
title = {TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {168},
pages = {},
doi = {10.3791/62198},
pmid = {33720132},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Endothelial Cells/*metabolism ; Endothelial Growth Factors/genetics/metabolism ; Fluorescent Antibody Technique ; *Gene Editing ; Mesoderm/*metabolism ; Mice ; Snail Family Transcription Factors/metabolism ; Transforming Growth Factor beta/genetics/*metabolism ; },
abstract = {In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-β), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-β-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-β-induced EndMT. Using these techniques, we provide evidence that TGF-β2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.},
}
@article {pmid33719095,
year = {2021},
author = {Wang, Q and Gong, Y and He, Y and Xin, Y and Lv, N and Du, X and Li, Y and Jeong, BR and Xu, J},
title = {Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {4},
pages = {1148-1162},
doi = {10.1111/tpj.15227},
pmid = {33719095},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; Genetic Engineering ; Genome/*genetics ; Microalgae/*genetics ; Phenotype ; Plasmids/*genetics ; Stramenopiles/*genetics ; },
abstract = {Industrial microalgae are promising photosynthetic cell factories, yet tools for large-scale targeted genome engineering are limited. Here for the model industrial oleaginous microalga Nannochloropsis oceanica, we established a method to precisely and serially delete large genome fragments of ~100 kb from its 30.01 Mb nuclear genome. We started by identifying the 'non-essential' chromosomal regions (i.e. low expression region or LER) based on minimal gene expression under N-replete and N-depleted conditions. The largest such LER (LER1) is ~98 kb in size, located near the telomere of the 502.09-kb-long Chromosome 30 (Chr 30). We deleted 81 kb and further distal and proximal deletions of up to 110 kb (21.9% of Chr 30) in LER1 by dual targeting the boundaries with the episome-based CRISPR/Cas9 system. The telomere-deletion mutants showed normal telomeres consisting of CCCTAA repeats, revealing telomere regeneration capability after losing the distal part of Chr 30. Interestingly, the deletions caused no significant alteration in growth, lipid production or photosynthesis (transcript-abundance change for < 3% genes under N depletion). We also achieved double-deletion of both LER1 and LER2 (from Chr 9) that total ~214 kb at maximum, which can result in slightly higher growth rate and biomass productivity than the wild-type. Therefore, loss of the large, yet 'non-essential' regions does not necessarily sacrifice important traits. Such serial targeted deletions of large genomic regions had not been previously reported in microalgae, and will accelerate crafting minimal genomes as chassis for photosynthetic production.},
}
@article {pmid33719068,
year = {2021},
author = {Zhou, X and Wang, X and Luo, H and Wang, Y and Wang, Y and Tu, T and Qin, X and Su, X and Bai, Y and Yao, B and Huang, H and Zhang, J},
title = {Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {7},
pages = {2448-2459},
doi = {10.1002/bit.27753},
pmid = {33719068},
issn = {1097-0290},
mesh = {*CRISPR-Cas Systems ; Clostridium butyricum/*genetics ; *Gene Editing ; *Probiotics ; },
abstract = {Clostridium butyricum has been widely used as a probiotic for humans and food animals. However, the mechanisms of beneficial effects of C. butyricum on the host remain poorly understood, largely due to the lack of high-throughput genome engineering tools. Here, we report the exploitation of heterologous Type II CRISPR-Cas9 system and endogenous Type I-B CRISPR-Cas system in probiotic C. butyricum for seamless genome engineering. Although successful genome editing was achieved in C. butyricum when CRISPR-Cas9 system was employed, the expression of toxic cas9 gene result in really poor transformation, spurring us to develop an easy-applicable and high-efficient genome editing tool. Therefore, the endogenous Type I-B CRISPR-Cas machinery located on the megaplasmid of C. butyricum was co-opted for genome editing. In vivo plasmid interference assays identified that ACA and TAA were functional protospacer adjacent motif sequences needed for site-specific CRISPR attacking. Using the customized endogenous CRISPR-Cas system, we successfully deleted spo0A and aldh genes in C. butyricum, yielding an efficiency of up to 100%. Moreover, the conjugation efficiency of endogenous CRISPR-Cas system was dramatically enhanced due to the precluding expression of cas9. Altogether, the two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C. butyricum.},
}
@article {pmid33718690,
year = {2020},
author = {Hernandez-Gordillo, V and Casolaro, TC and Ebrahimkhani, MR and Kiani, S},
title = {Multicellular Systems to Translate Somatic Cell Genome Editors to Humans.},
journal = {Current opinion in biomedical engineering},
volume = {16},
number = {},
pages = {72-81},
pmid = {33718690},
issn = {2468-4511},
support = {R01 HL141805/HL/NHLBI NIH HHS/United States ; U01 EB029372/EB/NIBIB NIH HHS/United States ; U01 DK127713/DK/NIDDK NIH HHS/United States ; R01 EB024562/EB/NIBIB NIH HHS/United States ; U01 HL145794/HL/NHLBI NIH HHS/United States ; R01 EB028532/EB/NIBIB NIH HHS/United States ; P30 DK120531/DK/NIDDK NIH HHS/United States ; },
abstract = {As genome editors move into clinical trials, there is a need to establish ex vivo multicellular systems to rapidly assess and predict toxic effects of genome editors in physiologically relevant human models. Advancements in organoid and organs-on-chip technologies offer the possibility to create multicellular systems that replicate the cellular composition and metabolic function of native tissues. Some multicellular systems have been validated in multiple applications for drug discovery and could be easily adapted to test genome editors; other models, especially those of the adaptive immune system, will require validation before being used as benchmarks for testing genome editors. Likewise, protocols to assess immunogenicity, to detect off-target effects, and to predict ex vivo to in vivo translation will need to be established and validated. This review will discuss key aspects to consider when designing, building, and/or adopting in vitro human multicellular systems for testing genome editors.},
}
@article {pmid33717855,
year = {2021},
author = {Park, JS and Hsieh, K and Chen, L and Kaushik, A and Trick, AY and Wang, TH},
title = {Digital CRISPR/Cas-Assisted Assay for Rapid and Sensitive Detection of SARS-CoV-2.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {8},
number = {5},
pages = {2003564},
pmid = {33717855},
issn = {2198-3844},
support = {R01 AI137272/AI/NIAID NIH HHS/United States ; },
abstract = {The unprecedented demand for rapid diagnostics in response to the COVID-19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)-assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital-based detection. Thus motivated, the first digital CRISPR/Cas-assisted assay-coined digitization-enhanced CRISPR/Cas-assisted one-pot virus detection (deCOViD)-is developed and applied toward SARS-CoV-2 detection. deCOViD is realized through tuning and discretizing a one-step, fluorescence-based, CRISPR/Cas12a-assisted reverse transcription recombinase polymerase amplification assay into sub-nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal-to-background ratio, broad dynamic range, and high sensitivity-down to 1 genome equivalent (GE) µL[-1] of SARS-CoV-2 RNA and 20 GE µL[-1] of heat-inactivated SARS-CoV-2, which outstrips its benchtop-based counterpart and represents one of the fastest and most sensitive CRISPR/Cas-assisted SARS-CoV-2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas-assisted assays and combating the COVID-19 pandemic and beyond.},
}
@article {pmid33716172,
year = {2021},
author = {Fan, HH and Lee, KH and Chen, YT and Lin, LJ and Yang, TL and Lin, SW and Yu, IS},
title = {Wdhd1 is essential for early mouse embryogenesis.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1868},
number = {6},
pages = {119011},
doi = {10.1016/j.bbamcr.2021.119011},
pmid = {33716172},
issn = {1879-2596},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Line ; Cell Proliferation ; DNA-Binding Proteins/*genetics/*metabolism ; *Embryonic Development ; Fertility ; Gastrulation ; Gene Editing ; Loss of Function Mutation ; Mice ; },
abstract = {WD repeat and HMG-box DNA binding protein 1 (Wdhd1) is the mouse ortholog of budding yeast Chromosome Transmission Fidelity 4 (CTF4), the protein product of which integrates the MCM2-7 helicase and DNA polymerase α/primase complex to initiate DNA replication. Previous work in fruit flies, Xenopus egg extracts, and human cell lines suggest that Wdhd1 is required for efficient DNA synthesis. However, rigorous in vivo functional studies on Wdhd1 in mammals are unavailable. In the present study, we have successfully generated a Wdhd1 null allele in mice through CRISPR/Cas9-mediated genome editing to investigate the role of Wdhd1 in embryogenesis in vivo. We characterized Wdhd1 expression using quantitative reverse-transcription polymerase chain reaction, and assessed embryonic cell proliferation by histology in both pre- and peri-implantation embryos. While Wdhd1 heterozygous mutant mice were grossly normal and fertile, we observed a reduction in cell proliferation by the gastrulation stage in Wdhd1 homozygous null mutant embryos which severely hampered their growth and viability. These results indicate that Wdhd1 plays a major role in cell proliferation during embryogenesis in mice.},
}
@article {pmid33715215,
year = {2021},
author = {Liu, X and Lin, L and Tang, L and Xie, H and Gu, L and Lv, X and Liu, C and Zhao, J and Deng, R and Liu, Y and Qu, J and Gu, F},
title = {Lb2Cas12a and its engineered variants mediate genome editing in human cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {4},
pages = {e21270},
doi = {10.1096/fj.202001013RR},
pmid = {33715215},
issn = {1530-6860},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/*genetics ; *Gene Editing ; Gene Expression Regulation ; Genome, Human ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; *Protein Engineering ; },
abstract = {Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5'-BYYV-3') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.},
}
@article {pmid33715060,
year = {2021},
author = {Li, H and Yu, H and Du, S and Li, Q},
title = {CRISPR/Cas9 Mediated High Efficiency Knockout of Myosin Essential Light Chain Gene in the Pacific Oyster (Crassostrea Gigas).},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {23},
number = {2},
pages = {215-224},
pmid = {33715060},
issn = {1436-2236},
mesh = {Animals ; CRISPR-Cas Systems ; Crassostrea/*genetics/growth &amp; development ; INDEL Mutation ; Larva/genetics/growth &amp; development ; Muscle Development/*genetics ; Myosin Heavy Chains/metabolism ; Myosin Light Chains/*genetics ; },
abstract = {Pacific oyster (Crassostrea gigas) is one of the most widely cultivated shellfish species in the world. Because of its economic value and complex life cycle involving drastic changes from a free-swimming larva to a sessile juvenile, C. gigas has been used as a model for developmental, environmental, and aquaculture research. However, due to the lack of genetic tools for functional analysis, gene functions associated with biological or economic traits cannot be easily determined. Here, we reported a successful application of CRISPR/Cas9 technology for knockout of myosin essential light chain gene (CgMELC) in C. gigas. C. gigas embryos injected with sgRNAs/Cas9 contained extensive indel mutations at the target sites. The mutant larvae showed defective musculature and reduced motility. In addition, knockout of CgMELC disrupted the expression and patterning of myosin heavy chain positive myofibers in larvae. Together, these data indicate that CgMELC is involved in larval muscle contraction and myogenesis in oyster larvae.},
}
@article {pmid33714068,
year = {2021},
author = {Gao, X and Qiu, SW and Wang, WQ and Kang, DY and Su, N and Dai, P and Yuan, YY},
title = {Generation of a gene corrected human isogenic iPSC line (CPGHi002-A-1) from a DDOD patient with heterozygous c.1516 C&gt;T mutation in the ATP6V1B2 gene.},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102271},
doi = {10.1016/j.scr.2021.102271},
pmid = {33714068},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells ; Mutation ; *Vacuolar Proton-Translocating ATPases/genetics ; },
abstract = {Dominant deafness-onychodystrophy (DDOD) syndrome is a rare autosomal dominant disorder caused by mutations in ATP6V1B2 gene. We previously generated an induced pluripotent stem cell (iPSC) line (CPGHi002-A) from a DDOD patient with a heterozygous c.1516 C>T mutation in the ATP6V1B2 gene. Here we genetically corrected the c.1516 C>T mutation in the ATP6V1B2 gene using CRISPR/Cas9 technology to generate an isogenic control, CPGHi002-A-1. The characterization of CPGHi002-A-1 demonstrates normal karyotype, pluripotent state, and potential to differentiate in vitro towards endoderm, mesoderm, and ectoderm.},
}
@article {pmid33713445,
year = {2021},
author = {Xu, W and Cheng, H and Zhu, S and Cheng, J and Ji, H and Zhang, B and Cao, S and Wang, C and Tong, G and Zhen, C and Mu, L and Zhou, Y and Cheng, Y},
title = {Functional understanding of secondary cell wall cellulose synthases in Populus trichocarpa via the Cas9/gRNA-induced gene knockouts.},
journal = {The New phytologist},
volume = {231},
number = {4},
pages = {1478-1495},
pmid = {33713445},
issn = {1469-8137},
mesh = {CRISPR-Cas Systems ; Cell Wall/*enzymology ; Cellulose/metabolism ; Gene Knockout Techniques ; Glucosyltransferases/*metabolism ; *Populus/enzymology/genetics ; RNA, Guide ; Wood/metabolism ; },
abstract = {Plant cellulose is synthesized by a large plasma membrane-localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts. Here, the Cas9/gRNA-induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level. Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one-layer-walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G-fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)- and G-layers and retained the thicker S-layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs-synthesized cellulose in wood and phloem fibre wall structures. Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G-fibre and phloem fibre wall structures in the tree.},
}
@article {pmid33712802,
year = {2021},
author = {Sweeney, CL and Pavel-Dinu, M and Choi, U and Brault, J and Liu, T and Koontz, S and Li, L and Theobald, N and Lee, J and Bello, EA and Wu, X and Meis, RJ and Dahl, GA and Porteus, MH and Malech, HL and De Ravin, SS},
title = {Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair.},
journal = {Gene therapy},
volume = {28},
number = {6},
pages = {373-390},
pmid = {33712802},
issn = {1476-5462},
support = {ZIA AI000644/ImNIH/Intramural NIH HHS/United States ; R01 AI097320/AI/NIAID NIH HHS/United States ; Z01 AI000645/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA, Complementary ; Exons ; *Granulomatous Disease, Chronic/genetics/therapy ; Hematopoietic Stem Cells ; Humans ; Mice ; NADPH Oxidase 2/genetics ; NADPH Oxidases/genetics ; },
abstract = {X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91[phox] protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1-13 or 2-13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1-13 cDNA did not restore physiologic gp91[phox] levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2-13 cDNA fully restored gp91[phox] and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91[phox] expression in exon 2-13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91[phox] and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.},
}
@article {pmid33712750,
year = {2021},
author = {Remmel, A},
title = {CRISPR-based gene therapy dampens pain in mice.},
journal = {Nature},
volume = {591},
number = {7850},
pages = {359},
pmid = {33712750},
issn = {1476-4687},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; Mice ; Pain/genetics ; },
}
@article {pmid33712738,
year = {2021},
author = {Demirer, GS and Silva, TN and Jackson, CT and Thomas, JB and W Ehrhardt, D and Rhee, SY and Mortimer, JC and Landry, MP},
title = {Nanotechnology to advance CRISPR-Cas genetic engineering of plants.},
journal = {Nature nanotechnology},
volume = {16},
number = {3},
pages = {243-250},
pmid = {33712738},
issn = {1748-3395},
support = {R35 GM128922/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Plant/genetics ; Nanotechnology/*trends ; Plants, Genetically Modified/*genetics ; },
abstract = {CRISPR-Cas genetic engineering of plants holds tremendous potential for providing food security, battling biotic and abiotic crop stresses caused by climate change, and for environmental remediation and sustainability. Since the discovery of CRISPR-Cas technology, its usefulness has been demonstrated widely, including for genome editing in plants. Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant genetic engineering, there remain many challenges for CRISPR applications in plant biotechnology. Nanomaterials could address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. This Perspective identifies major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discusses ways that nanoparticle technologies can lower or eliminate these barriers. We also describe advances that are needed in nanotechnology to facilitate and accelerate plant genome editing. Timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under a changing global climate.},
}
@article {pmid33712726,
year = {2021},
author = {Hitz, E and Grüninger, O and Passecker, A and Wyss, M and Scheurer, C and Wittlin, S and Beck, HP and Brancucci, NMB and Voss, TS},
title = {The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {336},
pmid = {33712726},
issn = {2399-3642},
mesh = {Antimalarials/pharmacology ; CRISPR-Cas Systems ; Casein Kinase II/antagonists &amp; inhibitors/genetics/*metabolism ; Catalytic Domain ; Erythrocytes/*parasitology ; *Gametogenesis ; Gene Editing ; Humans ; Life Cycle Stages ; Plasmodium falciparum/drug effects/genetics/growth &amp; development/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Protozoan Proteins/genetics/*metabolism ; Reproduction, Asexual ; },
abstract = {Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2α catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2α localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2α expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2α is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2α as a potential target for dual-active antimalarial drugs.},
}
@article {pmid33712601,
year = {2021},
author = {Pacini, C and Dempster, JM and Boyle, I and Gonçalves, E and Najgebauer, H and Karakoc, E and van der Meer, D and Barthorpe, A and Lightfoot, H and Jaaks, P and McFarland, JM and Garnett, MJ and Tsherniak, A and Iorio, F},
title = {Integrated cross-study datasets of genetic dependencies in cancer.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1661},
pmid = {33712601},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Biomarkers, Tumor ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Copy Number Variations ; Genes, Essential/genetics ; Genomics/methods ; Humans ; Neoplasms/*genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 viability screens are increasingly performed at a genome-wide scale across large panels of cell lines to identify new therapeutic targets for precision cancer therapy. Integrating the datasets resulting from these studies is necessary to adequately represent the heterogeneity of human cancers and to assemble a comprehensive map of cancer genetic vulnerabilities. Here, we integrated the two largest public independent CRISPR-Cas9 screens performed to date (at the Broad and Sanger institutes) by assessing, comparing, and selecting methods for correcting biases due to heterogeneous single-guide RNA efficiency, gene-independent responses to CRISPR-Cas9 targeting originated from copy number alterations, and experimental batch effects. Our integrated datasets recapitulate findings from the individual datasets, provide greater statistical power to cancer- and subtype-specific analyses, unveil additional biomarkers of gene dependency, and improve the detection of common essential genes. We provide the largest integrated resources of CRISPR-Cas9 screens to date and the basis for harmonizing existing and future functional genetics datasets.},
}
@article {pmid33712038,
year = {2021},
author = {Sugimoto, H and Horii, T and Hirota, JN and Sano, Y and Shinoda, Y and Konno, A and Hirai, H and Ishizaki, Y and Hirase, H and Hatada, I and Furuichi, T and Sadakata, T},
title = {The Ser19Stop single nucleotide polymorphism (SNP) of human PHYHIPL affects the cerebellum in mice.},
journal = {Molecular brain},
volume = {14},
number = {1},
pages = {52},
pmid = {33712038},
issn = {1756-6606},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Cell Shape ; Cerebellum/*cytology ; Codon, Terminator ; Female ; Gene Knock-In Techniques ; HapMap Project ; Humans ; Interneurons/physiology ; Intracellular Signaling Peptides and Proteins/*genetics/physiology ; Learning ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Motor Activity ; Nerve Fibers/physiology ; *Polymorphism, Single Nucleotide ; Purkinje Cells/metabolism/*ultrastructure ; Rotarod Performance Test ; Sequence Alignment ; Sequence Homology, Amino Acid ; },
abstract = {The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases.},
}
@article {pmid33711567,
year = {2021},
author = {Wu, X and Wu, H and Wang, H and Luo, L and Wang, J and Wu, B and He, Q and Cao, G and Lei, Y and Chen, X and Dai, J},
title = {A new strategy to develop pseudorabies virus-based bivalent vaccine with high immunogenicity of porcine circovirus type 2.},
journal = {Veterinary microbiology},
volume = {255},
number = {},
pages = {109022},
doi = {10.1016/j.vetmic.2021.109022},
pmid = {33711567},
issn = {1873-2542},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Circovirus/*immunology ; Female ; HEK293 Cells ; *Herpesvirus 1, Suid ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; Pseudorabies/*prevention &amp; control ; Pseudorabies Vaccines/*immunology ; Swine ; Swine Diseases/blood/*prevention &amp; control ; Viral Proteins/immunology ; },
abstract = {Herpesvirus based multivalent vaccines have been extensively studied, whereas few of them have been successfully used in clinic and animal husbandry industry due to the low expression of foreign immunogens in herpesvirus. In this study, we developed a new strategy to construct herpesvirus based bivalent vaccine with high-level expression of foreign immunogen, by which the ORF2 gene encoding the major antigen protein Cap of porcine circovirus type 2 (PCV2), was highly expressed in pseudorabies virus (PRV). To obtain the high expression of PCV2 immunogen, tandem repeats of PCV2 ORF2 gene were firstly linked by protein quantitation ratioing (PQR) linker to reach equal expression of each ORF2 gene. Then, the multiple copies of ORF2 gene were respectively inserted into the gE and gG sites of PRV using CRISPR/Cas9 system, in which the expression of ORF2 gene was driven by endogenous strong promoters of PRV. Through this way, the highest yield of Cap protein was achieved in two copies of quadruple ORF2 gene insertion. Finally, in mice and pigs immunized with the bivalent vaccine candidate, we detected high titer of specific antibodies for PRV and neutralized antibodies for PCV2, and observed protective effect of the bivalent vaccine candidate against PRV challenge in immunized pigs, suggesting a potential clinical application of the bivalent vaccine candidate we constructed. Together, our strategy could be extensively applied to the generation of other multivalent vaccines, and will pave the way to construct herpesvirus based multivalent vaccines to effectively reduce the cost of vaccine.},
}
@article {pmid33711268,
year = {2021},
author = {Kagita, A and Lung, MSY and Xu, H and Kita, Y and Sasakawa, N and Iguchi, T and Ono, M and Wang, XH and Gee, P and Hotta, A},
title = {Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.},
journal = {Stem cell reports},
volume = {16},
number = {4},
pages = {985-996},
pmid = {33711268},
issn = {2213-6711},
mesh = {Alleles ; Anti-Bacterial Agents/pharmacology ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Distal Myopathies/genetics/therapy ; Dysferlin/genetics/metabolism ; Exons/genetics ; Gene Editing ; HEK293 Cells ; Haplotypes/genetics ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Muscular Atrophy/genetics/therapy ; Muscular Dystrophy, Duchenne/genetics ; Mutagenesis, Insertional/genetics ; Mutation/genetics ; Oligodeoxyribonucleotides/*metabolism ; RNA/*metabolism ; RNA Splicing/genetics ; RNA, Guide/metabolism ; Ribonucleases/metabolism ; Ribonucleoproteins/*metabolism ; },
abstract = {Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.},
}
@article {pmid33711017,
year = {2021},
author = {Yeo, GHT and Juez, O and Chen, Q and Banerjee, B and Chu, L and Shen, MW and Sabry, M and Logister, I and Sherwood, RI and Gifford, DK},
title = {Detection of gene cis-regulatory element perturbations in single-cell transcriptomes.},
journal = {PLoS computational biology},
volume = {17},
number = {3},
pages = {e1008789},
pmid = {33711017},
issn = {1553-7358},
support = {R21 OD025309/OD/NIH HHS/United States ; K01 DK101684/DK/NIDDK NIH HHS/United States ; R01 HG008754/HG/NHGRI NIH HHS/United States ; R01 HG008363/HG/NHGRI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; },
mesh = {Algorithms ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology ; Databases, Factual ; Humans ; Mice ; RNA, Guide/genetics ; Regulatory Elements, Transcriptional/*genetics ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/*methods ; Transcriptome/*genetics ; },
abstract = {We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss of target gene expression occurs in only ~5% of cells. This low rate of biallelic loss significantly increases the number of cells required to detect the consequences of changes to the regulatory genome, but can be ameliorated by transcript-targeted sequencing. Based on our experimental results we model the power to detect regulatory genome induced transcriptomic effects based on the rate of mono/biallelic loss, baseline gene expression, and the number of cells per target gRNA.},
}
@article {pmid33710783,
year = {2021},
author = {Zhao, Y and Karan, R and Altpeter, F},
title = {Error-free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre-recombinase.},
journal = {Biotechnology journal},
volume = {16},
number = {6},
pages = {e2000650},
doi = {10.1002/biot.202000650},
pmid = {33710783},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Integrases/genetics ; Nucleotides ; *Saccharum/genetics ; },
abstract = {Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9-mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon-optimized Cre-mediated site-specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter-less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon-optimized Cre, or the combination of sgRNA and Cas9 targeting the 5' loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)-mediated error-free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.},
}
@article {pmid33710778,
year = {2021},
author = {Koyanagi, T and Saga, Y and Takahashi, Y and Tamura, K and Yoshiba, T and Takahashi, S and Taneichi, A and Takei, Y and Urabe, M and Mizukami, H and Fujiwara, H},
title = {Knockout of vasohibin-2 reduces tubulin carboxypeptidase activity and increases paclitaxel sensitivity in ovarian cancer.},
journal = {Cancer medicine},
volume = {10},
number = {8},
pages = {2732-2739},
pmid = {33710778},
issn = {2045-7634},
mesh = {Angiogenic Proteins/*genetics ; Antineoplastic Agents, Phytogenic/pharmacology ; CRISPR-Cas Systems ; Carboxypeptidases/genetics/*metabolism ; Cell Culture Techniques/methods ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Cyclin B1/metabolism ; Female ; Gene Knockdown Techniques ; Humans ; Ovarian Neoplasms/*drug therapy/*genetics/metabolism/pathology ; Paclitaxel/*pharmacology ; Tubulin/metabolism ; Tyrosine/genetics/metabolism ; },
abstract = {Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor, and vasohibin-2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere-forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2.},
}
@article {pmid33710766,
year = {2021},
author = {Jervis, AJ and Hanko, EKR and Dunstan, MS and Robinson, CJ and Takano, E and Scrutton, NS},
title = {A plasmid toolset for CRISPR-mediated genome editing and CRISPRi gene regulation in Escherichia coli.},
journal = {Microbial biotechnology},
volume = {14},
number = {3},
pages = {1120-1129},
pmid = {33710766},
issn = {1751-7915},
support = {BB/M017702/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; *Gene Editing ; Plasmids/genetics ; },
abstract = {CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid-based systems for genome editing (gene knockouts/knock-ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR-Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR-Cas12 genome editing, genome integration and CRISPRi implementation. These simple-to-use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory.},
}
@article {pmid33710467,
year = {2021},
author = {Kim, D and Hager, M and Brant, E and Budak, H},
title = {Efficient genome editing in wheat using Cas9 and Cpf1 (AsCpf1 and LbCpf1) nucleases.},
journal = {Functional &amp; integrative genomics},
volume = {21},
number = {3-4},
pages = {355-366},
pmid = {33710467},
issn = {1438-7948},
mesh = {*CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; *Gene Editing/methods ; *Genome, Plant ; *Triticum/genetics/metabolism ; },
abstract = {Genome editing can be used to create new wheat varieties with enhanced performance. Clustered regularly interspaced short palindromic repeat (CRISPR) is a powerful tool for knockout generation, precise modification, multiplex engineering, and the activation and repression of target genes. Targeted mutagenesis via RNA-guided genome editing using type II CRISPR-Cas9 is highly efficient in some plant species, but not in others. One possible solution is to use newly discovered variants of genome editing enzymes such as the class 2 system component Cpf1 (CRISPR from Prevotella and Francisella 1) in place of the more commonly used Cas9. We compared the editing efficiency of Cas9 and two Cpf1 orthologs, AsCpf1 (Acidaminococcus spp. BV3L6) and LbCpf1 (Lachnospiraceae bacterium ND2006) in wheat (Triticum aestivum). LbCpf1 had a higher editing efficiency for the target gene TaPDS than AsCpf1 and Cas9, and Cas9 induced more off-target mutations than AsCpf1 and LbCpf1, suggesting that CRISPR-LbCpf1 is a powerful genome editing tool for polyploid plants such as wheat.},
}
@article {pmid33710258,
year = {2021},
author = {Maguin, P and Marraffini, LA},
title = {From the discovery of DNA to current tools for DNA editing.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {4},
pages = {},
pmid = {33710258},
issn = {1540-9538},
mesh = {Animals ; CRISPR-Associated Protein 9/history ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Codon/history ; DNA/*history ; Gene Editing/*history ; History, 20th Century ; History, 21st Century ; Humans ; },
abstract = {In 1944, the Journal of Experimental Medicine published the groundbreaking discovery that DNA is the molecule holding genetic information (1944. J. Exp. Med.https://doi.org/10.1084/jem.79.2.137). This seminal finding was the genesis of molecular biology and the beginning of an incredible journey to understand, read, and manipulate the genetic code.},
}
@article {pmid33706382,
year = {2021},
author = {Diao, Z and Ji, Q and Wu, Z and Zhang, W and Cai, Y and Wang, Z and Hu, J and Liu, Z and Wang, Q and Bi, S and Huang, D and Ji, Z and Liu, GH and Wang, S and Song, M and Qu, J},
title = {SIRT3 consolidates heterochromatin and counteracts senescence.},
journal = {Nucleic acids research},
volume = {49},
number = {8},
pages = {4203-4219},
pmid = {33706382},
issn = {1362-4962},
mesh = {Aging/genetics/*metabolism ; Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cells, Cultured ; Cellular Senescence/genetics/physiology ; Gene Knockout Techniques ; HEK293 Cells ; Heterochromatin/genetics/*metabolism ; Humans ; Male ; Mesenchymal Stem Cells/metabolism/physiology ; Mice ; Mice, Nude ; Mice, SCID ; Nuclear Envelope/metabolism ; Protein Domains ; Sirtuin 3/chemistry/genetics/*physiology ; },
abstract = {Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase linked to a broad range of physiological and pathological processes, including aging and aging-related diseases. However, the role of SIRT3 in regulating human stem cell homeostasis remains unclear. Here we found that SIRT3 expression was downregulated in senescent human mesenchymal stem cells (hMSCs). CRISPR/Cas9-mediated depletion of SIRT3 led to compromised nuclear integrity, loss of heterochromatin and accelerated senescence in hMSCs. Further analysis indicated that SIRT3 interacted with nuclear envelope proteins and heterochromatin-associated proteins. SIRT3 deficiency resulted in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, increased chromatin accessibility and aberrant repetitive sequence transcription. The re-introduction of SIRT3 rescued the disorganized heterochromatin and the senescence phenotypes. Taken together, our study reveals a novel role for SIRT3 in stabilizing heterochromatin and counteracting hMSC senescence, providing new potential therapeutic targets to ameliorate aging-related diseases.},
}
@article {pmid33705914,
year = {2021},
author = {Tiwari, P and Khare, T and Shriram, V and Bae, H and Kumar, V},
title = {Plant synthetic biology for producing potent phyto-antimicrobials to combat antimicrobial resistance.},
journal = {Biotechnology advances},
volume = {48},
number = {},
pages = {107729},
doi = {10.1016/j.biotechadv.2021.107729},
pmid = {33705914},
issn = {1873-1899},
mesh = {Animals ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; *Anti-Infective Agents/pharmacology ; Drug Resistance, Bacterial ; Humans ; Prospective Studies ; Synthetic Biology ; },
abstract = {Inappropriate and injudicious use of antimicrobial drugs in human health, hygiene, agriculture, animal husbandry and food industries has contributed significantly to rapid emergence and persistence of antimicrobial resistance (AMR), one of the serious global public health threats. The crisis of AMR versus slower discovery of newer antibiotics put forth a daunting task to control these drug-resistant superbugs. Several phyto-antimicrobials have been identified in recent years with direct-killing (bactericidal) and/or drug-resistance reversal (re-sensitization of AMR phenotypes) potencies. Phyto-antimicrobials may hold the key in combating AMR owing to their abilities to target major microbial drug-resistance determinants including cell membrane, drug-efflux pumps, cell communication and biofilms. However, limited distribution, low intracellular concentrations, eco-geographical variations, beside other considerations like dynamic environments, climate change and over-exploitation of plant-resources are major blockades in full potential exploration phyto-antimicrobials. Synthetic biology (SynBio) strategies integrating metabolic engineering, RNA-interference, genome editing/engineering and/or systems biology approaches using plant chassis (as engineerable platforms) offer prospective tools for production of phyto-antimicrobials. With expanding SynBio toolkit, successful attempts towards introduction of entire gene cluster, reconstituting the metabolic pathway or transferring an entire metabolic (or synthetic) pathway into heterologous plant systems highlight the potential of this field. Through this perspective review, we are presenting herein the current situation and options for addressing AMR, emphasizing on the significance of phyto-antimicrobials in this apparently post-antibiotic era, and effective use of plant chassis for phyto-antimicrobial production at industrial scales along with major SynBio tools and useful databases. Current knowledge, recent success stories, associated challenges and prospects of translational success are also discussed.},
}
@article {pmid33705859,
year = {2021},
author = {Yang, Y and Wu, W and Liu, T and Dong, L and Lei, H},
title = {A robust method for protein depletion based on gene editing.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {3-11},
doi = {10.1016/j.ymeth.2021.03.001},
pmid = {33705859},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases ; *Gene Editing ; Genome ; Humans ; RNA, Guide/genetics ; },
abstract = {The technology of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 (CRISPR-Cas9) is a powerful system for protein depletion resulting from insertions and deletions following Cas9 cleavage of genome at specific site in vitro and in vivo. We herein present a relatively standard protocol for protein depletion in a step-by-step procedure, including guide RNA designation and vector construction, lentivirus production, cell selection, and experimentally validate the function of targeted protein. We exemplified this approach by editing PDGFRβ in human epithelial cells, and expected that this simplified and detailed protocol will be more broadly applied on specific genes to aid understanding gene functions.},
}
@article {pmid33705684,
year = {2021},
author = {Wang, G and Wen, B and Ren, X and Li, E and Zhang, Y and Guo, M and Xu, Y and Whitsett, JA and Kalin, TV and Kalinichenko, VV},
title = {Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.},
journal = {American journal of respiratory and critical care medicine},
volume = {204},
number = {3},
pages = {326-338},
pmid = {33705684},
issn = {1535-4970},
support = {R01 HL149631/HL/NHLBI NIH HHS/United States ; R01 HL152973/HL/NHLBI NIH HHS/United States ; U01 HL148856/HL/NHLBI NIH HHS/United States ; U01 HL134745/HL/NHLBI NIH HHS/United States ; R01 HL132849/HL/NHLBI NIH HHS/United States ; R01 HL158659/HL/NHLBI NIH HHS/United States ; R01 HL084151/HL/NHLBI NIH HHS/United States ; R01 HL123490/HL/NHLBI NIH HHS/United States ; R01 HL141174/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; Chimera ; Disease Models, Animal ; Embryonic Stem Cells/*cytology/metabolism ; Endothelial Progenitor Cells/*cytology/metabolism/transplantation ; Forkhead Transcription Factors/genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Infant, Newborn ; Mice ; Persistent Fetal Circulation Syndrome/metabolism/pathology/*therapy ; Pluripotent Stem Cells ; RNA-Seq ; Rats ; Single-Cell Analysis ; *Stem Cell Transplantation ; },
abstract = {Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1[+]cKIT[+] EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1[+]cKIT[+] gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1[+]cKIT[+] gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1[+]cKIT[+] gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1[+]cKIT[+] endothelial progenitors may be considered for treatment of human ACDMPV.},
}
@article {pmid33705438,
year = {2021},
author = {Ortolano, NA and Romero-Morales, AI and Rasmussen, ML and Bodnya, C and Kline, LA and Joshi, P and Connelly, JP and Rose, KL and Pruett-Miller, SM and Gama, V},
title = {A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.},
journal = {PloS one},
volume = {16},
number = {3},
pages = {e0248000},
pmid = {33705438},
issn = {1932-6203},
support = {P50 HD103537/HD/NICHD NIH HHS/United States ; R35 GM128915/GM/NIGMS NIH HHS/United States ; T32 HD007502/HD/NICHD NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytochromes c/metabolism ; Gene Editing ; Humans ; Pluripotent Stem Cells/*metabolism ; Proteomics/methods ; *Signal Transduction ; Transferases/*metabolism ; },
abstract = {CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.},
}
@article {pmid33705349,
year = {2021},
author = {He, J and Ding, Y and Nowik, N and Jager, C and Eeza, MNH and Alia, A and Baelde, HJ and Spaink, HP},
title = {Leptin deficiency affects glucose homeostasis and results in adiposity in zebrafish.},
journal = {The Journal of endocrinology},
volume = {249},
number = {2},
pages = {125-134},
doi = {10.1530/JOE-20-0437},
pmid = {33705349},
issn = {1479-6805},
mesh = {Adiposity/*genetics/physiology ; Animals ; Blood Glucose ; Body Weight ; CRISPR-Cas Systems ; Gene Deletion ; Glucose/*metabolism ; Homeostasis/genetics/*physiology ; Hypertrophy/etiology ; Kidney Glomerulus/pathology ; Leptin/*deficiency/*genetics/metabolism ; Zebrafish ; },
abstract = {Leptin is a hormone which functions in the regulation of energy homeostasis via suppression of appetite. In zebrafish, there are two paralogous genes encoding leptin, called lepa and lepb. In a gene expression study, we found that the lepb gene, not the lepa gene, was significantly downregulated under the state of insulin-resistance in zebrafish larvae, suggesting that the lepb plays a role in glucose homeostasis. In the current study, we characterised lepb-deficient (lepb-/-) adult zebrafish generated via a CRISPR-CAS9 gene editing approach by investigating whether the disruption of the lepb gene would result in the development of type 2 diabetes mellitus (T2DM) and diabetic complications. We observed that lepb-/- adult zebrafish had an increase in body weight, length and visceral fat accumulation, compared to age-matched control zebrafish. In addition, lepb-/- zebrafish had significantly higher blood glucose levels compared to control zebrafish. These data collectively indicate that lepb-/- adult zebrafish display the features of T2DM. Furthermore, we showed that lepb-/- adult zebrafish had glomerular hypertrophy and thickening of the glomerular basement membrane, compared to control zebrafish, suggesting that lepb-/- adult zebrafish develop early signs of diabetic nephropathy. In conclusion, our results demonstrate that lepb regulates glucose homeostasis and adiposity in zebrafish, and suggest that lepb-/- mutant zebrafish are a promising model to investigate the role of leptin in the development of T2DM and are an attractive model to perform mechanistic and therapeutic research in T2DM and its complications.},
}
@article {pmid33704703,
year = {2021},
author = {Hargadon, KM and Bushhouse, DZ and Johnson, CE and Williams, CJ},
title = {Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2265},
number = {},
pages = {25-46},
pmid = {33704703},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Forkhead Transcription Factors/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genetic Vectors ; Melanoma/*genetics ; Mice ; Transfection ; },
abstract = {Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.},
}
@article {pmid33704523,
year = {2021},
author = {Nagy, ED and Stevens, JL and Yu, N and Hubmeier, CS and LaFaver, N and Gillespie, M and Gardunia, B and Cheng, Q and Johnson, S and Vaughn, AL and Vega-Sanchez, ME and Deng, M and Rymarquis, L and Lawrence, RJ and Garvey, GS and Gaeta, RT},
title = {Novel disease resistance gene paralogs created by CRISPR/Cas9 in soy.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {1047-1058},
pmid = {33704523},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/*genetics ; Gene Dosage ; Gene Editing/methods ; Plant Diseases/genetics ; Plant Proteins/genetics ; Plants, Genetically Modified/*genetics ; Soybeans/*genetics ; },
abstract = {Novel disease resistance gene paralogues are generated by targeted chromosome cleavage of tandem duplicated NBS-LRR gene complexes and subsequent DNA repair in soybean. This study demonstrates accelerated diversification of innate immunity of plants using CRISPR. Nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) gene families are key components of effector-triggered immunity. They are often arranged in tandem duplicated arrays in the genome, a configuration that is conducive to recombinations that will lead to new, chimeric genes. These rearrangements have been recognized as major sources of novel disease resistance phenotypes. Targeted chromosome cleavage by CRISPR/Cas9 can conceivably induce rearrangements and thus emergence of new resistance gene paralogues. Two NBS-LRR families of soy have been selected to demonstrate this concept: a four-copy family in the Rpp1 region (Rpp1L) and a large, complex locus, Rps1 with 22 copies. Copy-number variations suggesting large-scale, CRISPR/Cas9-mediated chromosome rearrangements in the Rpp1L and Rps1 complexes were detected in up to 58.8% of progenies of primary transformants using droplet-digital PCR. Sequencing confirmed development of novel, chimeric paralogs with intact open reading frames. These novel paralogs may confer new disease resistance specificities. This method to diversify innate immunity of plants by genome editing is readily applicable to other disease resistance genes or other repetitive loci.},
}
@article {pmid33703873,
year = {2021},
author = {Fu, X and Shi, Y and Peng, F and Zhou, M and Yin, Y and Tan, Y and Chen, M and Yin, X and Ke, G and Zhang, XB},
title = {Exploring the Trans-Cleavage Activity of CRISPR/Cas12a on Gold Nanoparticles for Stable and Sensitive Biosensing.},
journal = {Analytical chemistry},
volume = {93},
number = {11},
pages = {4967-4974},
doi = {10.1021/acs.analchem.1c00027},
pmid = {33703873},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/genetics ; Gold ; *Metal Nanoparticles ; },
abstract = {Taking advantage of the excellent trans-cleavage activity, CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics. However, the single-stranded DNA reporter of the current CRISPR-Dx suffers from poor stability and limited sensitivity, which make their application in complex biological environments difficult. Herein, we, for the first time, explore the trans-cleavage activity of CRISPR/Cas12a toward the substrate on gold nanoparticles and apply the new phenomenon to develop a spherical nucleic acid (SNA) reporter for stable and sensitive CRISPR-Dx biosensing. By anchoring the DNA substrate on gold nanoparticles, we discovered different trans-cleavage activities of different types of the Cas12a system (e.g., LbCas12a and AsCas12a) on a nanoparticle surface. The further study suggests that the trans-cleavage activity of LbCas12a on the nanoparticle surface is highly dependent on the density and length of DNA strands. Based on these interesting discoveries, we furthermore develop SNA reporter-based fluorescent CRISPR-Dx for stable and sensitive biosensing application. Compared to traditional ssDNA reporters, the SNA reporter exhibits improved stability, which enables the stable application in a complex serum environment. In addition, the SNA reporter system with tunable density exhibits high sensitivity with a detection limit of 10 fM, which is about 2 orders of magnitude lower than that of the ssDNA reporter system. Finally, the practical application of SNA reporter-based CRISPR-Dx in clinical serum was successfully achieved. These results indicate their significant potential in future research on biology science and medical diagnoses.},
}
@article {pmid33693715,
year = {2021},
author = {Jakhanwal, S and Cress, BF and Maguin, P and Lobba, MJ and Marraffini, LA and Doudna, JA},
title = {A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.},
journal = {Nucleic acids research},
volume = {49},
number = {6},
pages = {3546-3556},
pmid = {33693715},
issn = {1362-4962},
support = {U24 HG010423/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F32 GM131654/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/chemistry/*metabolism ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/metabolism ; Genome ; Integrases/*metabolism ; Protein Domains ; RNA/chemistry/metabolism ; },
abstract = {CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9's activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.},
}
@article {pmid33693649,
year = {2021},
author = {Madhangi, M and Dutta, D and Show, S and Bhat, VK and Rather, MI and Tiwari, A and Singh, N and Duvvari, MR and Murthy, GJ and Kumar, A and Nongthomba, U},
title = {Exome sequencing and functional studies in zebrafish identify WDR8 as the causative gene for isolated Microspherophakia in Indian families.},
journal = {Human molecular genetics},
volume = {30},
number = {6},
pages = {467-484},
doi = {10.1093/hmg/ddab061},
pmid = {33693649},
issn = {1460-2083},
mesh = {Adult ; Animals ; Child ; Corneal Diseases/etiology/metabolism/*pathology ; Ectopia Lentis/etiology/metabolism/*pathology ; *Exome ; Female ; Glaucoma/etiology/metabolism/*pathology ; HeLa Cells ; Humans ; India ; Iris/*abnormalities/metabolism/pathology ; Male ; *Mutation ; Pedigree ; Proteins/*genetics/metabolism ; Whole Exome Sequencing/*methods ; Young Adult ; Zebrafish ; },
abstract = {Isolated Microspherophakia (MSP) is an autosomal recessive disorder characterized by a smaller than normal spherical lens. Till date, LTBP2 is the only gene shown to cause MSP. We used homozygosity mapping and whole-exome sequencing and identified a homozygous mutation, c.1148C > T (p.Pro383Leu), in the WDR8 (or WRAP73) gene in two Indian MSP families. In vitro experiments showed that the missense mutation renders the protein unstable. WDR8 is a centriolar protein that has important roles in centrosomal assembly, spindle pole formation and ciliogenesis. Co-immunoprecipitation experiments from HeLa cells indicated that the mutation interferes with the interaction of WDR8 with its binding partners. In zebrafish, both morpholino-mediated knockdown and CRISPR/Cas knockout of wdr8 resulted in decreased eye and lens size. The lack of wdr8 affected cell cycle progression in the retinal cells, causing a reduction in cell numbers in the retina and lens. The reduction in eye size and the cell cycle defects were rescued by exogenous expression of the human wild-type WDR8. However, the human mutant WDR8 (p.Pro383Leu) was unable to rescue the eye defects, indicating that the missense mutation abrogates WDR8 protein function. Thus, our zebrafish results suggested that WDR8 is the causative gene for MSP in these Indian families.},
}
@article {pmid33692551,
year = {2021},
author = {Leeper, K and Kalhor, K and Vernet, A and Graveline, A and Church, GM and Mali, P and Kalhor, R},
title = {Lineage barcoding in mice with homing CRISPR.},
journal = {Nature protocols},
volume = {16},
number = {4},
pages = {2088-2108},
pmid = {33692551},
issn = {1750-2799},
support = {R01 GM123313/GM/NIGMS NIH HHS/United States ; R01 MH103910/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Barcoding, Taxonomic/*methods ; Mice ; Mutation/genetics ; *Phylogeny ; RNA, Guide/genetics ; },
abstract = {Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.},
}
@article {pmid33692485,
year = {2021},
author = {Broniewski, JM and Chisnall, MAW and Høyland-Kroghsbo, NM and Buckling, A and Westra, ER},
title = {The effect of Quorum sensing inhibitors on the evolution of CRISPR-based phage immunity in Pseudomonas aeruginosa.},
journal = {The ISME journal},
volume = {15},
number = {8},
pages = {2465-2473},
pmid = {33692485},
issn = {1751-7370},
support = {BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Pseudomonas aeruginosa/genetics ; Quorum Sensing ; },
abstract = {Quorum sensing controls the expression of a wide range of important traits in the opportunistic pathogen Pseudomonas aeruginosa, including the expression of virulence genes and its CRISPR-cas immune system, which protects from bacteriophage (phage) infection. This finding has led to the speculation that synthetic quorum sensing inhibitors could be used to limit the evolution of CRISPR immunity during phage therapy. Here we use experimental evolution to explore if and how a quorum sensing inhibitor influences the population and evolutionary dynamics of P. aeruginosa upon phage DMS3vir infection. We find that chemical inhibition of quorum sensing decreases phage adsorption rates due to downregulation of the Type IV pilus, which causes delayed lysis of bacterial cultures and favours the evolution of CRISPR immunity. Our data therefore suggest that inhibiting quorum sensing may reduce rather than improve the therapeutic efficacy of pilus-specific phage, and this is likely a general feature when phage receptors are positively regulated by quorum sensing.},
}
@article {pmid33692197,
year = {2021},
author = {Jin, Y and Sun, T and Zhou, G and Li, D and Chen, S and Zhang, W and Li, X and Zhang, R and Yang, H and Duan, G},
title = {Pathogenesis Study of Enterovirus 71 Using a Novel Human SCARB2 Knock-In Mouse Model.},
journal = {mSphere},
volume = {6},
number = {2},
pages = {},
pmid = {33692197},
issn = {2379-5042},
mesh = {Animals ; Astrocytes/virology ; CRISPR-Cas Systems ; Disease Models, Animal ; Enterovirus A, Human/*genetics/*pathogenicity ; Enterovirus Infections/immunology/*pathology ; Female ; Gene Knock-In Techniques ; Hand, Foot and Mouth Disease/complications/virology ; Humans ; Lung/pathology/virology ; Lysosome-Associated Membrane Glycoproteins/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Nervous System Diseases/virology ; Receptors, Scavenger/*genetics ; },
abstract = {Enterovirus 71 (EV71) can cause a severe hand-foot-mouth disease in children. However, the precise mechanism of EV71-associated disease, particularly the neuropathogenesis and pulmonary disorder, is still not fully understood because no suitable animal models are available. The human scavenger receptor class B, member 2 (hSCARB2), is a cellular receptor for EV71. Here, we generated a novel knock-in (KI) mouse model using the CRISPR/Cas9 system to insert the hSCARB2 gene into the mouse Rosa26 locus to study the pathogenesis of EV71. The hSCARB2 KI mice infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia, paralysis, and death. Viral replication was detected in mainly astrocytes and a limited number of neurons and microglia, accompanied by gliosis. Vascular leakage and alveoli filled with erythrocytes were detected, suggesting that edema and hemorrhage, which are observed in human patients, also occurred in EV71-infected KI mice. In addition, proinflammatory cytokines and chemokines were significantly increased in the serum of infected KI mice. These pathological features of the KI mice after infection resembled those of EV71 encephalomyelitis in humans. Therefore, our KI mouse model is suitable to study the pathogenesis of EV71 and is of great significance for development of antiviral drugs and vaccines to treat or prevent EV71 infection.IMPORTANCE Enterovirus 71 (EV71) is associated with severe hand-foot-mouth disease. Recently, outbreaks of EV71 infection with high mortality have been reported in the Asia-Pacific region, posing a great challenge for global public health. To date, the precise mechanism of EV71-induced disease, particularly the neuropathogenesis and respiratory disorders, is still not fully understood because no suitable animal models are available. Human scavenger receptor class B, member 2 (hSCARB2), has been identified as a cellular receptor for EV71. Here, we introduce a novel CRISPR/Cas9-mediated hSCARB2 knock-in (KI) mouse model for the study of EV71 pathogenesis, which is of great significance for the development of antiviral drugs and vaccines.},
}
@article {pmid33692106,
year = {2021},
author = {Wan, T and Pan, Q and Ping, Y},
title = {Microneedle-assisted genome editing: A transdermal strategy of targeting NLRP3 by CRISPR-Cas9 for synergistic therapy of inflammatory skin disorders.},
journal = {Science advances},
volume = {7},
number = {11},
pages = {},
pmid = {33692106},
issn = {2375-2548},
mesh = {Administration, Cutaneous ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics ; Polymers ; Ribonucleoproteins/genetics ; Skin/metabolism ; },
abstract = {We report a dissolvable microneedle (MN) patch that can mediate transdermal codelivery of CRISPR-Cas9-based genome-editing agents and glucocorticoids for the effective treatment of inflammatory skin disorders (ISDs). The MN is loaded with polymer-encapsulated Cas9 ribonucleoprotein (RNP) targeting NLRP3 and dexamethasone (Dex)-containing polymeric nanoparticles. Upon insertion into the skin, the MN can be dissolved quickly to release two types of nanoformulations, which are subsequently internalized by keratinocytes and surrounding immune cells to exert their therapeutic effects in the inflammatory subcutaneous layers. Thus, the MN-enabled transdermal codelivery of Cas9 RNP nanocomplexes and Dex nanoparticles result in the disruption of subcutaneous intracellular NLRP3 inflammasomes, which is demonstrated to be critical to alleviate skin inflammations and contributes to glucocorticoid therapy in mouse models of ISDs, including psoriasis and atopic dermatitis. Our study offers innovative insights into the rational design of transdermal delivery systems and defines an effective therapeutic option for the treatment of ISDs.},
}
@article {pmid33692102,
year = {2021},
author = {Strohkendl, I and Saifuddin, FA and Gibson, BA and Rosen, MK and Russell, R and Finkelstein, IJ},
title = {Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin.},
journal = {Science advances},
volume = {7},
number = {11},
pages = {},
pmid = {33692102},
issn = {2375-2548},
support = {P01 GM066275/GM/NIGMS NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; R35 GM131777/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Chromatin/genetics ; DNA/genetics ; Endonucleases/metabolism ; Humans ; *Nucleosomes/genetics ; },
abstract = {Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.},
}
@article {pmid33691767,
year = {2021},
author = {Rao, S and Yao, Y and Bauer, DE},
title = {Editing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation.},
journal = {Genome medicine},
volume = {13},
number = {1},
pages = {41},
pmid = {33691767},
issn = {1756-994X},
support = {DP2 HL137300/HL/NHLBI NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; },
mesh = {Epigenesis, Genetic ; Gene Editing ; *Genetic Predisposition to Disease ; *Genetic Variation ; *Genome-Wide Association Study ; Humans ; Quantitative Trait Loci ; },
abstract = {Genome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.},
}
@article {pmid33691754,
year = {2021},
author = {Huang, C and Li, G and Wu, J and Liang, J and Wang, X},
title = {Identification of pathogenic variants in cancer genes using base editing screens with editing efficiency correction.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {80},
pmid = {33691754},
issn = {1474-760X},
mesh = {Algorithms ; Base Sequence ; *Biomarkers, Tumor ; CRISPR-Cas Systems ; Chromosome Mapping ; Computational Biology/*methods ; Databases, Genetic ; *Gene Editing ; Genes, BRCA1 ; Genes, BRCA2 ; *Genetic Variation ; Humans ; Loss of Function Mutation ; Models, Molecular ; Neoplasms/*genetics ; *Oncogenes ; RNA, Guide ; Structure-Activity Relationship ; },
abstract = {BACKGROUND: Millions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens.<br><br>RESULTS: To improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons of BRCA1 and BRCA2 genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants for BRCA1/2, including 151 variants in the noncoding part of the genes such as the 5' untranslated regions. Many of them are identified in cancer patients and are reported as "benign/likely benign" or "variants of uncertain significance" by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer.<br><br>CONCLUSIONS: Our results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.},
}
@article {pmid33691522,
year = {2022},
author = {Panda, SK and McGrew, MJ},
title = {Genome editing of avian species: implications for animal use and welfare.},
journal = {Laboratory animals},
volume = {56},
number = {1},
pages = {50-59},
pmid = {33691522},
issn = {1758-1117},
support = {BBS/E/D/20320000/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; NC/V001124/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chickens/genetics ; *Gene Editing/methods ; Genome ; Germ Cells/metabolism ; },
abstract = {Avian species are used as model systems in research and have contributed to ground-breaking concepts in developmental biology, immunology, genetics, virology, cancer and cell biology. The chicken in particular is an important research model and an agricultural animal as a major contributor to animal protein resources for the global population. The development of genome editing methods, including CRISPR/Cas9, to mediate germline engineering of the avian genome will have important applications in biomedical, agricultural and biotechnological activities. Notably, these precise genome editing tools have the potential to enhance avian health and productivity by identifying and validating beneficial genetic variants in bird populations. Here, we present a concise description of the existing methods and current applications of the genome editing tools in bird species, focused on chickens, with attention on animal use and welfare issues for each of the techniques presented.},
}
@article {pmid33691131,
year = {2021},
author = {Yi, F and Klebanoff, CA},
title = {Erasing iatrogenic neoantigens from in vivo CRISPR screens.},
journal = {Immunity},
volume = {54},
number = {3},
pages = {406-408},
doi = {10.1016/j.immuni.2021.02.017},
pmid = {33691131},
issn = {1097-4180},
support = {R37 CA259177/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Antigens ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy ; Humans ; Iatrogenic Disease ; Immunotherapy ; },
abstract = {In vivo genetic screens using CRISPR-Cas9 are a powerful tool to resolve the molecular determinants of response and resistance to cancer immunotherapies; however, vector immunogenicity can introduce artifact. In this issue of Immunity, Dubrot et al. report a strategy to "erase" vector-associated neoantigens, enabling a more physiologic assessment of tumor-immune cell interactions in immunocompetent hosts.},
}
@article {pmid33691101,
year = {2021},
author = {Orlando, L and Tanasijevic, B and Nakanishi, M and Reid, JC and García-Rodríguez, JL and Chauhan, KD and Porras, DP and Aslostovar, L and Lu, JD and Shapovalova, Z and Mitchell, RR and Boyd, AL and Bhatia, M},
title = {Phosphorylation state of the histone variant H2A.X controls human stem and progenitor cell fate decisions.},
journal = {Cell reports},
volume = {34},
number = {10},
pages = {108818},
doi = {10.1016/j.celrep.2021.108818},
pmid = {33691101},
issn = {2211-1247},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage ; Cell Self Renewal/*physiology ; Ectoderm/metabolism ; Hematopoietic Stem Cells/cytology/metabolism ; Histones/deficiency/genetics/*metabolism ; Humans ; Leukemia, Myeloid, Acute/metabolism/pathology ; Mesoderm/metabolism ; Neoplastic Stem Cells/*cytology/metabolism ; Neurons/cytology/metabolism ; Nucleosomes/metabolism ; Phosphorylation ; Pluripotent Stem Cells/*cytology/metabolism ; },
abstract = {Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.},
}
@article {pmid33689695,
year = {2021},
author = {Li, G and Wang, H and Zhang, X and Wu, Z and Yang, H},
title = {A Cas9-transcription factor fusion protein enhances homology-directed repair efficiency.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100525},
pmid = {33689695},
issn = {1083-351X},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; *Gene Editing ; HEK293 Cells ; Humans ; *Recombinational DNA Repair ; Repressor Proteins/genetics/*metabolism ; },
abstract = {Precise gene insertion or replacement in cells and animals that requires incorporation of a foreign DNA template into the genome target site by homology-directed repair (HDR) remains an inefficient process. One of the limiting factors for the inefficiency of HDR lies in the limited chance for colocalization of the donor template and target in the huge genome space. We here present a strategy to enhance HDR efficiency in animal cells by spatial and temporal colocalization of the donor and Cas9 by coupling the CRISPR system with a transcription factor (TF). We first identified that THAP domain-containing 11 (THAP11) can coordinate with CRISPR/Cas9 to increase HDR stably through screening multiple TFs from different species. We next designed donor structures with different fusion patterns with TF-specific DNA-binding motifs and found that appending two copies of THAP11-specific DNA binding motifs to both ends of the double-stranded donor DNA has an optimal effect to promote HDR. The THAP11-fused CRISPR system achieved more than twofold increase in HDR-mediated knock-in efficiency for enhanced green fluorescent protein (EGFP) tagging of endogenous genes in 293T cells. We also demonstrated up to 6-fold increases of knock-in through the combinational use of the TF-fused CRISPR and valnemulin, a recently discovered small-molecule HDR enhancer. This modified CRISPR system provides a simple but highly efficient platform to facilitate CRISPR-mediated KI manipulations.},
}
@article {pmid33689678,
year = {2021},
author = {Fang, H and Bygrave, AM and Roth, RH and Johnson, RC and Huganir, RL},
title = {An optimized CRISPR/Cas9 approach for precise genome editing in neurons.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33689678},
issn = {2050-084X},
support = {P30 NS050274/NS/NINDS NIH HHS/United States ; RF1 MH123212/MH/NIMH NIH HHS/United States ; R01 NS036715/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Electroporation ; Female ; Gene Editing/*instrumentation ; *Genome ; INDEL Mutation ; Male ; Mice ; Rats ; },
abstract = {The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.},
}
@article {pmid33689075,
year = {2021},
author = {Singina, GN and Sergiev, PV and Lopukhov, AV and Rubtsova, MP and Taradajnic, NP and Ravin, NV and Shedova, EN and Taradajnic, TE and Polejaeva, IA and Dozev, AV and Brem, G and Dontsova, OA and Zinovieva, NA},
title = {Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle.},
journal = {Doklady. Biochemistry and biophysics},
volume = {496},
number = {1},
pages = {48-51},
pmid = {33689075},
issn = {1608-3091},
mesh = {Animals ; Animals, Genetically Modified/embryology/*genetics ; *CRISPR-Cas Systems ; Cattle/embryology/*genetics ; Cloning, Organism/*methods ; Embryo, Mammalian/*cytology/metabolism ; Fibroblasts/*cytology/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Nuclear Transfer Techniques ; },
abstract = {Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.},
}
@article {pmid33688075,
year = {2021},
author = {Gandhi, S and Li, Y and Tang, W and Christensen, JB and Urrutia, HA and Vieceli, FM and Piacentino, ML and Bronner, ME},
title = {A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {7},
pages = {},
pmid = {33688075},
issn = {1477-9129},
support = {K99 DE029240/DE/NIDCR NIH HHS/United States ; R01 DE027568/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chick Embryo ; Gene Editing/*methods ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques/methods ; Neural Crest/metabolism ; PAX6 Transcription Factor/genetics ; PAX7 Transcription Factor ; Plasmids/*genetics ; RNA, Guide/genetics ; SOXE Transcription Factors/genetics ; },
abstract = {An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing β-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.},
}
@article {pmid33687760,
year = {2021},
author = {Smedley, MA and Hayta, S and Clarke, M and Harwood, WA},
title = {CRISPR-Cas9 Based Genome Editing in Wheat.},
journal = {Current protocols},
volume = {1},
number = {3},
pages = {e65},
doi = {10.1002/cpz1.65},
pmid = {33687760},
issn = {2691-1299},
support = {BB/P013511/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Plant Breeding ; *Triticum/genetics ; },
abstract = {The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR-associated protein (Cas), is a programmable RNA-guided nuclease enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9 Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly Basic Protocol 3: Screening for CRISPR-Cas9 genome edits Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits.},
}
@article {pmid33687685,
year = {2021},
author = {Rot, S and Kappler, M},
title = {Modulation of a Stem Cell Gene: LGR4 Knockout in a Human Cell Line by CRISPR/Cas Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2269},
number = {},
pages = {255-268},
pmid = {33687685},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Receptors, G-Protein-Coupled/*genetics/metabolism ; Stem Cells/*metabolism ; },
abstract = {The modulation of gene expression is essential for the investigation of function or involved pathway of a single gene of interest, in particular in the developmental/stem cell biology. The temporary knock down of gene expression via siRNA is a well-established but with a residual expression connected modulation method. The chapter describes the complete knockout of a defined target and allows a comprehensive study of different gene like the stem cell gene LGR4 (Leucine-rich repeat-containing G-protein-coupled receptor 4) using the new developed CRISPR/Cas method (clustered regularly interspaced short palindromic repeats).},
}
@article {pmid33686787,
year = {2021},
author = {Fathi, M and Pustokhina, I and Kuznetsov, SV and Khayrullin, M and Hojjat-Farsangi, M and Karpisheh, V and Jalili, A and Jadidi-Niaragh, F},
title = {T-cell immunoglobulin and ITIM domain, as a potential immune checkpoint target for immunotherapy of colorectal cancer.},
journal = {IUBMB life},
volume = {73},
number = {5},
pages = {726-738},
doi = {10.1002/iub.2461},
pmid = {33686787},
issn = {1521-6551},
mesh = {Adenocarcinoma/immunology/pathology/*therapy ; Amino Acid Motifs ; Animals ; Antigens, CD/immunology ; CRISPR-Cas Systems ; Colorectal Neoplasms/immunology/pathology/radiotherapy/*therapy ; Combined Modality Therapy ; Gastrointestinal Microbiome ; Gene Expression Regulation, Neoplastic ; Humans ; *Immune Checkpoint Inhibitors ; Immune Checkpoint Proteins/*immunology ; Immunotherapy/*methods ; Mice ; Prognosis ; Protein Domains ; Receptors, Immunologic/*antagonists &amp; inhibitors/biosynthesis/deficiency/genetics/immunology ; Tumor Microenvironment ; },
abstract = {The importance of the tumor microenvironment in cancer progression has been well studied for many years. Immune checkpoint inhibitors (ICIs) are regarded as potential strategies in enhancing the immune responses in patients with cancer, particularly colorectal cancer (CRC). Notably, CRCs are extraordinarily heterogeneous and mostly are microsatellite-stable (MSS) or cold tumors, which means that the immune response is not usually as strong as that of foreign cells. T-cell immunoglobulin and ITIM domain (TIGIT) is a new immune checkpoint receptor overexpressed inside the CRC tumor-immune microenvironments. Moreover, several studies have shown that TIGIT in combination with other ICIs and/or conventional treatments, can lead to a robust anti-tumor response in CRC. This review looks deep inside TIGIT expression patterns, their various functions, and possible immunotherapy strategies to increase survival rates and decrease immune-related adverse events.},
}
@article {pmid33686287,
year = {2021},
author = {Baggen, J and Persoons, L and Vanstreels, E and Jansen, S and Van Looveren, D and Boeckx, B and Geudens, V and De Man, J and Jochmans, D and Wauters, J and Wauters, E and Vanaudenaerde, BM and Lambrechts, D and Neyts, J and Dallmeier, K and Thibaut, HJ and Jacquemyn, M and Maes, P and Daelemans, D},
title = {Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2.},
journal = {Nature genetics},
volume = {53},
number = {4},
pages = {435-444},
pmid = {33686287},
issn = {1546-1718},
mesh = {Bronchoalveolar Lavage Fluid/cytology ; COVID-19/epidemiology/*genetics/virology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Coronavirus 229E, Human/genetics ; Epidemics ; Epithelial Cells/virology ; Gene Expression ; Genome, Human/*genetics ; Genome-Wide Association Study/*methods ; Host-Pathogen Interactions ; Humans ; Membrane Proteins/*genetics ; Nerve Tissue Proteins/*genetics ; Proviruses/physiology ; SARS-CoV-2/physiology ; Virus Internalization ; },
abstract = {The ongoing COVID-19 pandemic has caused a global economic and health crisis. To identify host factors essential for coronavirus infection, we performed genome-wide functional genetic screens with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E. These screens uncovered virus-specific as well as shared host factors, including TMEM41B and PI3K type 3. We discovered that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells. TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, suggesting a role in viral entry. Furthermore, single-cell RNA-sequencing of airway cells from patients with COVID-19 demonstrated that TMEM106B expression correlates with SARS-CoV-2 infection. The present study uncovered a collection of coronavirus host factors that may be exploited to develop drugs against SARS-CoV-2 infection or future zoonotic coronavirus outbreaks.},
}
@article {pmid33686254,
year = {2021},
author = {Freitas, GP and Lopes, HB and Souza, ATP and Gomes, MPO and Quiles, GK and Gordon, J and Tye, C and Stein, JL and Stein, GS and Lian, JB and Beloti, MM and Rosa, AL},
title = {Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation.},
journal = {Gene therapy},
volume = {28},
number = {12},
pages = {748-759},
pmid = {33686254},
issn = {1476-5462},
support = {R01 AR039588/AR/NIAMS NIH HHS/United States ; R01 DE029311/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; *Growth Differentiation Factor 2/genetics ; *Mesenchymal Stem Cells/cytology ; *Osteogenesis/genetics ; Rats ; },
abstract = {Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPR[BMP-9+], followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPR[BMP-9+] displayed significant changes in the expression of a panel of genes involved in TGF-β/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPR[BMP-9+] exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.},
}
@article {pmid33685992,
year = {2021},
author = {Yang, Z and Liang, SQ and Yang, H and Xu, D and Bruggmann, R and Gao, Y and Deng, H and Berezowska, S and Hall, SRR and Marti, TM and Kocher, GJ and Zhou, Q and Schmid, RA and Peng, RW},
title = {CRISPR-Mediated Kinome Editing Prioritizes a Synergistic Combination Therapy for FGFR1-Amplified Lung Cancer.},
journal = {Cancer research},
volume = {81},
number = {11},
pages = {3121-3133},
doi = {10.1158/0008-5472.CAN-20-2276},
pmid = {33685992},
issn = {1538-7445},
mesh = {Aged ; Animals ; Apoptosis ; Benzamides/*pharmacology ; *CRISPR-Cas Systems ; Cell Cycle ; Cell Cycle Proteins/*antagonists &amp; inhibitors/genetics ; Cell Proliferation ; Combined Modality Therapy ; *Gene Amplification ; *Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/genetics/metabolism/pathology/*therapy ; Male ; Mice ; Piperazines/*pharmacology ; Protein Kinase Inhibitors/pharmacology ; Protein Serine-Threonine Kinases/*antagonists &amp; inhibitors/genetics ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors/genetics ; Pyrazoles/*pharmacology ; Receptor, Fibroblast Growth Factor, Type 1/*antagonists &amp; inhibitors/genetics ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Oncogenic activation of the FGFR pathway is frequent in lung and other cancers. However, due to drug resistance, pharmacological blockage of aberrant FGFR signaling has provided little clinical benefit in patients with FGFR-amplified tumors. The determining factors for the limited efficacy of FGFR-targeted therapy remain incompletely understood. In this study, we performed kinome-wide CRISPR/Cas9 loss-of-function screens in FGFR1-amplified lung cancer cells treated with an FGFR inhibitor. These screens identified PLK1 as a potent synthetic lethal target that mediates a resistance mechanism by overriding DNA damage and cell-cycle arrest upon FGFR1 inhibition. Genetic and pharmacological antagonism of PLK1 in combination with FGFR inhibitor therapy synergized to enhance antiproliferative effects and drove cancer cell death in vitro and in vivo through activation of the γH2AX-CHK-E2F1 axis. These findings suggest a previously unappreciated role for PLK1 in modulating FGFR1 inhibitor sensitivity and demonstrate a synergistic drug combination for treating FGFR1-amplified lung cancer. SIGNIFICANCE: The identification of PLK1 as a potent synthetic lethal target for FGFR-targeted therapy provides an innovative rationale for the treatment of lung and other FGFR1-amplified cancers.},
}
@article {pmid33685602,
year = {2021},
author = {Kazi, TA and Biswas, SR},
title = {CRISPR/dCas system as the modulator of gene expression.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {99-122},
doi = {10.1016/bs.pmbts.2020.12.002},
pmid = {33685602},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Expression ; Genome ; Genomics ; Humans ; *RNA, Guide ; },
abstract = {CRISPR/Cas has been a very exciting field of research because of its multifaceted applications in biological science for editing genome. This tool can be programmed to target any region of DNA of choice by designing gRNA. The potential of gRNA to recruit a CRISPR-associated protein at a specific genomic site allowed scientists to engineer genome of diverse species for research and development. The application of Cas9 has been further expanded with a recently developed catalytically inactive protein (dead Cas9). CRISPR/dCas system is widely used as a programmable vector to deliver functional cargo (transcriptional effectors) to the desired sites at the genome for targeted transcriptional repression (CRISPR interference, CRISPRi) or activation (CRISPR activation, CRISPRa). It is now possible to regulate gene expression in cells without altering the DNA sequence. These CRISPRi/a toolboxes have explored many unsolved biological issues. Further research on CRISPR system could help diagnose and treat various human diseases.},
}
@article {pmid33685601,
year = {2021},
author = {Gupta, R and Gupta, D and Ahmed, KT and Dey, D and Singh, R and Swarnakar, S and Ravichandiran, V and Roy, S and Ghosh, D},
title = {Modification of Cas9, gRNA and PAM: Key to further regulate genome editing and its applications.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {85-98},
doi = {10.1016/bs.pmbts.2020.12.001},
pmid = {33685601},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome/genetics ; Humans ; *RNA, Guide/genetics ; },
abstract = {The discovery of CRISPR-Cas9 system has revolutionized the genome engineering research and has been established as a gold standard genome editing platform. This system has found its application in biochemical researches as well as in medical fields including disease diagnosis, development of therapeutics, etc. The enormous versatility of the CRISPR-Cas9 as a high throughput genome engineering platform, is derailed by its off-target activity. To overcome this, researchers from all over the globe have explored the system structurally and functionally and postulated several strategies to upgrade the system components including redesigning of Cas9 Nuclease and modification of guide RNA(gRNA) structure and customization of the protospacer adjacent motif. Here in this review, we portray the comprehensive overview of the strategies that has been adopted for redesigning the CRISPR-Cas9 system to enhance the efficiency and fidelity of the technology.},
}
@article {pmid33685600,
year = {2021},
author = {Hossain, MA},
title = {CRISPR-Cas9: A fascinating journey from bacterial immune system to human gene editing.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {63-83},
doi = {10.1016/bs.pmbts.2021.01.001},
pmid = {33685600},
issn = {1878-0814},
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Immune System ; RNA, Guide ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system has been discovered as an adaptive-immune system in prokaryotes. Microbes like bacteria and archaea use CRISPR-Cas9 as a part of their defense mechanism to ward off the virus and cleave their DNA. Over the past decades, researchers have identified that this simple CRISPR-Cas9 system of bacteria can be utilized to cut any DNA. It is also possible to make precise editing in the genome of almost any organism. This discovery has revolutionized the CRISPR-Cas9 tools and made it one of the most precise gene editing technology known till date. The simple, versatile and programmable nature of CRISPR-Cas9 system 5wthat contains a single guide RNA and Cas9 enzyme, made it an attractive choice for genome editing application. Scientists in the field of molecular biology, genetics and medicine extensively use this transformative technology to study gene regulation and also for treatment of several incurable genetic diseases. Today, CRISPR-Cas9 is the most powerful breakthrough of the century for its immense potential to modulate gene expression in living cells and its application to medicine and human health. Recently, ethical challenges associated with the application of this technology to human health become a hot debate in the scientific community. In this chapter the brief history of development of CRISPR-Cas9 tools and its immense application potential have been discussed.},
}
@article {pmid33685599,
year = {2021},
author = {Bonnerjee, D and Bagh, S},
title = {Application of CRISPR-Cas systems in neuroscience.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {231-264},
doi = {10.1016/bs.pmbts.2020.12.010},
pmid = {33685599},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; *Nervous System Diseases/genetics/therapy ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
abstract = {CRISPR-Cas systems have, over the years, emerged as indispensable tools for Genetic interrogation in contexts of clinical interventions, elucidation of genetic pathways and metabolic engineering and have pervaded almost every aspect of modern biology. Within this repertoire, the nervous system comes with its own set of perplexities and mysteries. Scientists have, over the years, tried to draw up a clearer genetic picture of the neuron and how it functions in a network, mainly in an endeavor to mitigate diseases of the human nervous system like Alzheimer's, Parkinson's, Huntington's, Autism Spectrum Disorder (ASD), etc. With most being progressive in nature, these diseases have plagued mankind for centuries. In spite of our immense progress in modern biology, we are yet to get a grasp over these diseases and unraveling their mechanisms is of utmost importance. Before CRISPR-Cas systems came along, the elucidation of the complex interactome of the mammalian nervous system was attempted with erstwhile existing electrophysiological, histological and pharmacological techniques coupled with Next Generation Sequencing and cell-specific targeting technologies. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), imparted excellent sequence specific DNA targeting capabilities but came with their huge baggage of extensive protein engineering requirements, which practically rendered them unsuitable for high throughput exercises. With the discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR Associated proteins(CAS) systems by Ishino (1987)[1], the era of extensive custom made endonuclease targeting was ushered in. For the first time in 2012, Jinek et al. (2012)[2] repurposed the CRISPR-Cas mediated bacterial immune system for customizable mammalian gene editing. The CRISPR-Cas technology made it possible to easily customize Cas9 endonucleases to cleave near specifically targeted sequences, thereby facilitating knock-ins or knock-outs, silencing or activating or editing any gene, at any locus of the genome, both at the base-pair level or at the epigenetic level. With this enhanced degree of freedom, decrypting the nervous system and therapeutic interventions for neuropathies became significantly less cumbersome an exercise. Here we take a brisk walk through the several endeavors of research that show how the humble bacteria's CRISPR-Cas system gave us the "nerves" to "talk" to our nerves with ease.},
}
@article {pmid33685598,
year = {2021},
author = {Roy, S},
title = {Immune responses to CRISPR-Cas protein.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {213-229},
doi = {10.1016/bs.pmbts.2020.12.003},
pmid = {33685598},
issn = {1878-0814},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Genome ; Immunity ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) protein technologies have evolved as promising, cost-effective, and efficient methods for editing genomes. Editing genomes with high specificity and precision is a daunting task, where errors can lead to undesirable outcomes. Many elegant studies have successfully shown that the CRISPR-Cas9 system can modify, disrupt, and add new DNA sequences directly into the genomes of the cells or animals being studied. As such, the CRISPR-Cas9 technology holds immense potential for biomedical research as well as agricultural and therapeutic applications, further emphasized by its unprecedented movement into the clinical setting. Throughout every stage of life, missense mutations can lead to highly unfavorable outcomes, syndromes, and diseases. Many of these mutations are transferred directly through the fertilization process and, thereby, acquired at birth and propagated to the next generation. As such, it has been of great interest to develop techniques to repair these mutations using genetic manipulation, prior to or following birth. CRISPR-Cas9 has many advantages in this regard over numerous other existing technologies. Regardless, editing bases within a genome can be associated with numerous challenges that were previously unrecognized and lead to unforeseen consequences. While the CRISPR-Cas9 method is perfectly suitable for editing cells outside the body with limited risk to the normal functioning of the cell, recent publications have illustrated a number of challenging conditions resulting from its use. One of them is directed to the host immune response toward CRISPR-Cas9. With this in mind, this review will discuss recent observations on the host immune response to CRISPR-Cas9 and the associated challenges that arise as a result.},
}
@article {pmid33685597,
year = {2021},
author = {Prakash, R and Kannan, A},
title = {Mitochondrial DNA modification by CRISPR/Cas system: Challenges and future direction.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {193-211},
doi = {10.1016/bs.pmbts.2020.12.009},
pmid = {33685597},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; DNA, Mitochondrial/genetics ; Gene Editing ; Humans ; *Virus Diseases ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR associated endonuclease), a hotshot genome editing tool which is originally known to be the form of prokaryotic adaptive immune system against viral infections has gained all the attention of scientific community as a promising genome editing platform. This review encompasses a brief description of mitochondrial disease conditions associated with the alteration in mitochondrial genome (mtDNA) and highlights the key role of the CRISPR/Cas system pertaining to its working mechanism and its involvement in gene-based therapeutics in treating the foresaid mitochondrial diseases. Here, we also extend the perception related to the detailed mechanism of CRISPR/Cas system in mtDNA modification.},
}
@article {pmid33685596,
year = {2021},
author = {Mukhopadhyay, S and Bhutia, SK},
title = {Trends in CRISPR-Cas9 technology application in cancer.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {175-192},
doi = {10.1016/bs.pmbts.2020.12.004},
pmid = {33685596},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; Technology ; },
abstract = {The evolution of the CRISPR-Cas9 technology in cancer research has tremendous potential to shape the future of oncology. Although this gene-editing tool's pre-clinical progress is into its nascent stage, there are many unanswered questions regarding health benefits and therapy precision using CRISPR. The application of CRISPR is highly specific, economically sustainable, and is a high throughput technique, but on the other hand, its application involves measured risk of countering the toxic immune response of Cas protein, off-target effects, limitation of delivering the edited cells back into cancer patients. The current chapter highlights the possibilities and perils of the present-day CRISPR engineering in cancer that should highlight CRISPR translation to therapy.},
}
@article {pmid33685595,
year = {2021},
author = {Roy, S and Naha, S and Rao, A and Basu, S},
title = {CRISPR-Cas system, antibiotic resistance and virulence in bacteria: Through a common lens.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {123-174},
doi = {10.1016/bs.pmbts.2020.12.005},
pmid = {33685595},
issn = {1878-0814},
mesh = {Anti-Bacterial Agents/pharmacology ; *Bacteria/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; Drug Resistance, Microbial/drug effects/genetics ; Humans ; Virulence/drug effects/genetics ; },
abstract = {CRISPR-Cas system, antibiotic resistance and virulence are different modes of survival for the bacteria. CRISPR-Cas is an adaptive immune system that can degrade foreign DNA, antibiotic resistance helps bacteria to evade drugs that can threaten their existence and virulence determinants are offensive tools that can facilitate the establishment of infection by pathogens. This chapter focuses on these three aspects, providing insights about the CRISPR system and resistance mechanisms in brief, followed by understanding the synergistic or antagonistic relationship of resistance and virulence determinants in connection to the CRISPR system. We have addressed the discussion of this evolving topic through specific examples and studies. Different approaches for successful detection of this unique defense system in bacteria and various applications of the CRISPR-Cas systems to show how it can be harnessed to tackle the increasing problem of antibiotic resistance have been put forth. World Health Organization has declared antibiotic resistance as a serious global problem of the 21st century. As antibiotic-resistant bacteria increase their footprint across the globe, newer tools such as the CRISPR-Cas system hold immense promise to tackle this problem.},
}
@article {pmid33685594,
year = {2021},
author = {Randhawa, S and Sengar, S},
title = {The evolution and history of gene editing technologies.},
journal = {Progress in molecular biology and translational science},
volume = {178},
number = {},
pages = {1-62},
doi = {10.1016/bs.pmbts.2021.01.002},
pmid = {33685594},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Technology ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases ; },
abstract = {Scientific enquiry must be the driving force of research. This sentiment is manifested as the profound impact gene editing technologies are having in our current world. There exist three main gene editing technologies today: Zinc Finger Nucleases, TALENs and the CRISPR-Cas system. When these systems were being uncovered, none of the scientists set out to design tools to engineer genomes. They were simply trying to understand the mechanisms existing in nature. If it was not for this simple sense of wonder, we probably would not have these breakthrough technologies. In this chapter, we will discuss the history, applications and ethical issues surrounding these technologies, focusing on the now predominant CRISPR-Cas technology. Gene editing technologies, as we know them now, are poised to have an overwhelming impact on our world. However, it is impossible to predict the route they will take in the future or to comprehend the full impact of its repercussions.},
}
@article {pmid33685382,
year = {2021},
author = {S Zibitt, M and Hartford, CCR and Lal, A},
title = {Interrogating lncRNA functions via CRISPR/Cas systems.},
journal = {RNA biology},
volume = {18},
number = {12},
pages = {2097-2106},
pmid = {33685382},
issn = {1555-8584},
support = {ZIA BC011646/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Knockdown Techniques/*methods ; Humans ; RNA, Long Noncoding/*genetics ; },
abstract = {Long noncoding RNAs (lncRNAs) are an increasing focus of investigation due to their implications in diverse biological processes and disease. Nevertheless, the majority of lncRNAs are low in abundance and poorly conserved, posing challenges to functional studies. The CRISPR/Cas system, an innovative technology that has emerged over the last decade, can be utilized to further understand lncRNA function. The system targets specific DNA and/or RNA sequences via a guide RNA (gRNA) and Cas nuclease complex. We and others have utilized this technology in various applications such as lncRNA knockout, knockdown, overexpression, and imaging. In this review, we summarize how the CRISPR/Cas technology provides new tools to investigate the roles and therapeutic implications of lncRNAs.},
}
@article {pmid33685363,
year = {2021},
author = {De Leo, MG and Berger, P and Mayer, A},
title = {WIPI1 promotes fission of endosomal transport carriers and formation of autophagosomes through distinct mechanisms.},
journal = {Autophagy},
volume = {17},
number = {11},
pages = {3644-3670},
pmid = {33685363},
issn = {1554-8635},
mesh = {Autophagosomes/*metabolism ; Autophagy-Related Proteins/*metabolism/physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Endocytosis ; Gene Editing ; Humans ; Membrane Proteins/*metabolism/physiology ; Microscopy, Confocal ; Multivesicular Bodies/*metabolism/physiology ; Mutagenesis, Site-Directed ; },
abstract = {Autophagosome formation requires PROPPIN/WIPI proteins and monophosphorylated phosphoinositides, such as phosphatidylinositol-3-phosphate (PtdIns3P) or PtdIns5P. This process occurs in association with mammalian endosomes, where the PROPPIN WIPI1 has additional, undefined roles in vesicular traffic. To explore whether these functions are interconnected, we dissected routes and subreactions of endosomal trafficking requiring WIPI1. WIPI1 specifically acts in the formation and fission of tubulo-vesicular endosomal transport carriers. This activity supports the PtdIns(3,5)P2-dependent transport of endosomal cargo toward the plasma membrane, Golgi, and lysosomes, suggesting a general role of WIPI1 in endosomal protein exit. Three features differentiate the endosomal and macroautophagic/autophagic activities of WIPI1: phosphoinositide binding site II, the requirement for PtdIns(3,5)P2, and bilayer deformation through a conserved amphipathic α-helix. Their inactivation preserves autophagy but leads to a strong enlargement of endosomes, which accumulate micrometer-long endosomal membrane tubules carrying cargo proteins. WIPI1 thus supports autophagy and protein exit from endosomes by different modes of action. We propose that the type of phosphoinositides occupying its two lipid binding sites, the most unusual feature of PROPPIN/WIPI family proteins, switches between these effector functions.Abbreviations: EGF: epidermal growth factorEGFR: epidermal growth factor receptorKD: knockdownKO: knockoutPtdIns3P: phosphatidylinositol-3-phosphatePtdIns5P: phosphatidylinositol-5-phosphatePtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphateTF: transferrinTFRC: transferrin receptorWT: wildtype.},
}
@article {pmid33683104,
year = {2021},
author = {Jiang, Y and Hu, M and Liu, AA and Lin, Y and Liu, L and Yu, B and Zhou, X and Pang, DW},
title = {Detection of SARS-CoV-2 by CRISPR/Cas12a-Enhanced Colorimetry.},
journal = {ACS sensors},
volume = {6},
number = {3},
pages = {1086-1093},
doi = {10.1021/acssensors.0c02365},
pmid = {33683104},
issn = {2379-3694},
mesh = {Bacterial Proteins ; COVID-19/*diagnosis ; COVID-19 Testing/*methods ; CRISPR-Associated Proteins ; CRISPR-Cas Systems ; Cell Line, Tumor ; Colorimetry ; DNA/chemistry ; DNA Probes ; Endodeoxyribonucleases ; Gold/chemistry ; Humans ; Metal Nanoparticles/chemistry ; RNA, Viral/*analysis ; SARS-CoV-2/*genetics ; },
abstract = {The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription-polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.},
}
@article {pmid33682991,
year = {2021},
author = {Qiao, SP and Liu, ZN and Li, HC and He, X and Pan, H and Gao, YZ},
title = {Construction of a CRISPR-Biolayer Interferometry Platform for Real-Time, Sensitive, and Specific DNA Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {22},
number = {11},
pages = {1974-1984},
doi = {10.1002/cbic.202100054},
pmid = {33682991},
issn = {1439-7633},
mesh = {Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/*analysis ; Time Factors ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA-recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real-time, sensitive, and rapid digital system called CRISPR-BLI was established for the detection of double-stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR-BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR-BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.},
}
@article {pmid33679662,
year = {2021},
author = {Chimukuche, NM and Williams, MJ},
title = {Genetic Manipulation of Non-tuberculosis Mycobacteria.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {633510},
pmid = {33679662},
issn = {1664-302X},
abstract = {Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms.},
}
@article {pmid33679633,
year = {2021},
author = {Pereira, HS and Tagliaferri, TL and Mendes, TAO},
title = {Enlarging the Toolbox Against Antimicrobial Resistance: Aptamers and CRISPR-Cas.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {606360},
pmid = {33679633},
issn = {1664-302X},
abstract = {In the post-genomic era, molecular treatments and diagnostics have been envisioned as powerful techniques to tackle the antimicrobial resistance (AMR) crisis. Among the molecular approaches, aptamers and CRISPR-Cas have gained support due to their practicality, sensibility, and flexibility to interact with a variety of extra- and intracellular targets. Those characteristics enabled the development of quick and onsite diagnostic tools as well as alternative treatments for pan-resistant bacterial infections. Even with such potential, more studies are necessary to pave the way for their successful use against AMR. In this review, we highlight those two robust techniques and encourage researchers to refine them toward AMR. Also, we describe how aptamers and CRISPR-Cas can work together with the current diagnostic and treatment toolbox.},
}
@article {pmid33678249,
year = {2021},
author = {de Alencastro, G and Puzzo, F and Pavel-Dinu, M and Zhang, F and Pillay, S and Majzoub, K and Tiffany, M and Jang, H and Sheikali, A and Cromer, MK and Meetei, R and Carette, JE and Porteus, MH and Pekrun, K and Kay, MA},
title = {Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {3},
pages = {1016-1027},
pmid = {33678249},
issn = {1525-0024},
support = {R01 AI130123/AI/NIAID NIH HHS/United States ; R01 HL064274/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Helicases/*antagonists &amp; inhibitors/genetics/metabolism ; DNA-Binding Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Dependovirus/*genetics ; *Gene Editing ; Genetic Vectors ; HeLa Cells ; Hematopoietic Stem Cells/cytology/*metabolism ; Homologous Recombination ; Humans ; RecQ Helicases/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to ∼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by ∼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34[+] hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by ∼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.},
}
@article {pmid33677572,
year = {2021},
author = {Dion, MB and Plante, PL and Zufferey, E and Shah, SA and Corbeil, J and Moineau, S},
title = {Streamlining CRISPR spacer-based bacterial host predictions to decipher the viral dark matter.},
journal = {Nucleic acids research},
volume = {49},
number = {6},
pages = {3127-3138},
pmid = {33677572},
issn = {1362-4962},
mesh = {*Bacteriophages ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Databases, Nucleic Acid ; Gastrointestinal Tract/microbiology ; *Genome, Bacterial ; Internet ; Metagenomics/*methods ; Software ; },
abstract = {Thousands of new phages have recently been discovered thanks to viral metagenomics. These phages are extremely diverse and their genome sequences often do not resemble any known phages. To appreciate their ecological impact, it is important to determine their bacterial hosts. CRISPR spacers can be used to predict hosts of unknown phages, as spacers represent biological records of past phage-bacteria interactions. However, no guidelines have been established to standardize host prediction based on CRISPR spacers. Additionally, there are no tools that use spacers to perform host predictions on large viral datasets. Here, we developed a set of tools that includes all the necessary steps for predicting the hosts of uncharacterized phages. We created a database of >11 million spacers and a program to execute host predictions on large viral datasets. Our host prediction approach uses biological criteria inspired by how CRISPR-Cas naturally work as adaptive immune systems, which make the results easy to interpret. We evaluated the performance using 9484 phages with known hosts and obtained a recall of 49% and a precision of 69%. We also found that this host prediction method yielded higher performance for phages that infect gut-associated bacteria, suggesting it is well suited for gut-virome characterization.},
}
@article {pmid33677541,
year = {2021},
author = {Ashley, GE and Duong, T and Levenson, MT and Martinez, MAQ and Johnson, LC and Hibshman, JD and Saeger, HN and Palmisano, NJ and Doonan, R and Martinez-Mendez, R and Davidson, BR and Zhang, W and Ragle, JM and Medwig-Kinney, TN and Sirota, SS and Goldstein, B and Matus, DQ and Dickinson, DJ and Reiner, DJ and Ward, JD},
title = {An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.},
journal = {Genetics},
volume = {217},
number = {3},
pages = {},
pmid = {33677541},
issn = {1943-2631},
support = {R35 GM134838/GM/NIGMS NIH HHS/United States ; K99 GM107345/GM/NIGMS NIH HHS/United States ; F31 HD100091/HD/NICHD NIH HHS/United States ; F32 GM131577/GM/NIGMS NIH HHS/United States ; T32 GM133391/GM/NIGMS NIH HHS/United States ; R01 GM138701/GM/NIGMS NIH HHS/United States ; R00 GM107345/GM/NIGMS NIH HHS/United States ; R01 GM121597/GM/NIGMS NIH HHS/United States ; R01 GM121625/GM/NIGMS NIH HHS/United States ; T32 GM008444/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R00 GM115964/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Arabidopsis Proteins/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/metabolism ; F-Box Proteins/chemistry/*genetics/metabolism ; Genes, Reporter ; Genetic Engineering/*methods ; Indoleacetic Acids/*metabolism ; Luminescent Proteins/genetics/metabolism ; Organ Specificity ; *Proteolysis ; Receptors, Cell Surface/chemistry/*genetics/metabolism ; Transgenes ; },
abstract = {The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.},
}
@article {pmid33676891,
year = {2021},
author = {Tian, X and Zhou, B},
title = {Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100509},
pmid = {33676891},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems ; Catalysis ; DNA Nucleotidyltransferases/*metabolism ; Enzyme Activation ; Integrases/metabolism ; Light ; Plants/enzymology ; Promoter Regions, Genetic ; *Recombination, Genetic ; },
abstract = {Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.},
}
@article {pmid33676427,
year = {2021},
author = {Du, R and Sullivan, DK and Azizian, NG and Liu, Y and Li, Y},
title = {Inhibition of ERAD synergizes with FTS to eradicate pancreatic cancer cells.},
journal = {BMC cancer},
volume = {21},
number = {1},
pages = {237},
pmid = {33676427},
issn = {1471-2407},
support = {K22 CA207598/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology/therapeutic use ; Apoptosis/drug effects/genetics ; CRISPR-Cas Systems/genetics ; Carcinoma, Pancreatic Ductal/*drug therapy/pathology ; Cell Line, Tumor ; Drug Screening Assays, Antitumor ; Endoplasmic Reticulum-Associated Degradation/*drug effects/genetics ; Farnesol/analogs &amp; derivatives/pharmacology/therapeutic use ; Gene Knockout Techniques ; Humans ; Hydrazones/pharmacology/therapeutic use ; Hydroxyurea/analogs &amp; derivatives/pharmacology/therapeutic use ; Mice ; Pancreatic Neoplasms/*drug therapy/pathology ; Proteins/genetics ; Salicylates/pharmacology/therapeutic use ; Synthetic Lethal Mutations ; Ubiquitin-Protein Ligases/genetics ; Unfolded Protein Response/drug effects ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is driven by oncogenic KRAS mutations. Farnesyl thiosalicylic acid (FTS), also known as salirasib, is a RAS inhibitor that selectively dislodges active RAS proteins from cell membrane, inhibiting downstream signaling. FTS has demonstrated limited therapeutic efficacy in PDAC patients despite being well tolerated.<br><br>METHODS: To improve the efficacy of FTS in PDAC, we performed a genome-wide CRISPR synthetic lethality screen to identify genetic targets that synergize with FTS treatment. Among the top candidates, multiple genes in the endoplasmic reticulum-associated protein degradation (ERAD) pathway were identified. The role of ERAD inhibition in enhancing the therapeutic efficacy of FTS was further investigated in pancreatic cancer cells using pharmaceutical and genetic approaches.<br><br>RESULTS: In murine and human PDAC cells, FTS induced unfolded protein response (UPR), which was further augmented upon treatment with a chemical inhibitor of ERAD, Eeyarestatin I (EerI). Combined treatment with FTS and EerI significantly upregulated the expression of UPR marker genes and induced apoptosis in pancreatic cancer cells. Furthermore, CRISPR-based genetic ablation of the key ERAD components, HRD1 and SEL1L, sensitized PDAC cells to FTS treatment.<br><br>CONCLUSION: Our study reveals a critical role for ERAD in therapeutic response of FTS and points to the modulation of UPR as a novel approach to improve the efficacy of FTS in PDAC treatment.},
}
@article {pmid33675788,
year = {2021},
author = {Tikoo, S and Jain, R and Tomasetig, F and On, K and Martinez, B and Heu, C and Stehle, D and Obeidy, P and Guo, D and Vincent, JN and Cook, AJL and Roediger, B and Feil, R and Whan, RM and Weninger, W},
title = {Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities.},
journal = {The Journal of investigative dermatology},
volume = {141},
number = {8},
pages = {2090-2094.e6},
doi = {10.1016/j.jid.2021.01.025},
pmid = {33675788},
issn = {1523-1747},
mesh = {Animals ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Imaging, Three-Dimensional ; Melanins/biosynthesis ; Melanoma, Experimental/*diagnostic imaging/pathology ; Metabolic Engineering/*methods ; Mice ; Skin Neoplasms/*diagnostic imaging/pathology ; Tumor Microenvironment ; },
}
@article {pmid33674748,
year = {2021},
author = {Riedel, M and Berthelsen, MF and Cai, H and Haldrup, J and Borre, M and Paludan, SR and Hager, H and Vendelbo, MH and Wagner, EF and Bakiri, L and Thomsen, MK},
title = {In vivo CRISPR inactivation of Fos promotes prostate cancer progression by altering the associated AP-1 subunit Jun.},
journal = {Oncogene},
volume = {40},
number = {13},
pages = {2437-2447},
pmid = {33674748},
issn = {1476-5594},
support = {741888/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Proliferation ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice ; PTEN Phosphohydrolase/*genetics ; Prostate/pathology ; Prostatic Intraepithelial Neoplasia/*genetics/pathology ; Prostatic Neoplasms/*genetics/pathology ; Proto-Oncogene Proteins c-fos/*genetics ; Transcription Factor AP-1/*genetics ; },
abstract = {Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.},
}
@article {pmid33674604,
year = {2021},
author = {Terradas, G and Buchman, AB and Bennett, JB and Shriner, I and Marshall, JM and Akbari, OS and Bier, E},
title = {Inherently confinable split-drive systems in Drosophila.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1480},
pmid = {33674604},
issn = {2041-1723},
support = {DP2 AI152071/AI/NIAID NIH HHS/United States ; R01 AI151004/AI/NIAID NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; R21 AI149161/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; Drosophila/*genetics ; Drosophila melanogaster/genetics ; Female ; Gene Drive Technology/*methods ; Gene Editing/*methods ; Male ; Recombinational DNA Repair ; },
abstract = {CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.},
}
@article {pmid33674593,
year = {2021},
author = {Hung, MH and Lee, JS and Ma, C and Diggs, LP and Heinrich, S and Chang, CW and Ma, L and Forgues, M and Budhu, A and Chaisaingmongkol, J and Ruchirawat, M and Ruppin, E and Greten, TF and Wang, XW},
title = {Tumor methionine metabolism drives T-cell exhaustion in hepatocellular carcinoma.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1455},
pmid = {33674593},
issn = {2041-1723},
support = {ZIA BC010313/ImNIH/Intramural NIH HHS/United States ; ZIA BC010876/ImNIH/Intramural NIH HHS/United States ; ZIA BC010877/ImNIH/Intramural NIH HHS/United States ; ZIA BC011870/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; Biomarkers, Tumor ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Carcinoma, Hepatocellular/genetics/immunology/*metabolism/pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Liver/metabolism/pathology ; Liver Neoplasms/genetics/immunology/*metabolism ; Methionine/*metabolism ; Methionine Adenosyltransferase/blood/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; S-Adenosylmethionine/metabolism ; T-Lymphocytes/*metabolism ; Transcriptome ; },
abstract = {T-cell exhaustion denotes a hypofunctional state of T lymphocytes commonly found in cancer, but how tumor cells drive T-cell exhaustion remains elusive. Here, we find T-cell exhaustion linked to overall survival in 675 hepatocellular carcinoma (HCC) patients with diverse ethnicities and etiologies. Integrative omics analyses uncover oncogenic reprograming of HCC methionine recycling with elevated 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM) to be tightly linked to T-cell exhaustion. SAM and MTA induce T-cell dysfunction in vitro. Moreover, CRISPR-Cas9-mediated deletion of MAT2A, a key SAM producing enzyme, results in an inhibition of T-cell dysfunction and HCC growth in mice. Thus, reprogramming of tumor methionine metabolism may be a viable therapeutic strategy to improve HCC immunity.},
}
@article {pmid33673701,
year = {2021},
author = {Shin, SW and Kim, D and Lee, JS},
title = {Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {5},
pages = {},
pmid = {33673701},
issn = {1422-0067},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; *Gene Editing ; Gene Targeting/*methods ; Plasmids/*genetics/metabolism ; Recombinant Proteins/*genetics ; Transgenes/*physiology ; },
abstract = {Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.},
}
@article {pmid33673381,
year = {2021},
author = {Shabbir, W and Topcagic, N and Aufy, M and Oz, M},
title = {CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {4},
pages = {},
pmid = {33673381},
issn = {1422-0067},
mesh = {A549 Cells ; *CRISPR-Cas Systems ; *Epithelial Sodium Channels/genetics/metabolism ; Humans ; Tumor Necrosis Factor-alpha/genetics/*metabolism ; },
abstract = {Tumor necrosis factor (TNF) is known to activate the epithelial Na[+] channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na[+] current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na[+] current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.},
}
@article {pmid33673107,
year = {2021},
author = {Osborn, MJ and Bhardwaj, A and Bingea, SP and Knipping, F and Feser, CJ and Lees, CJ and Collins, DP and Steer, CJ and Blazar, BR and Tolar, J},
title = {CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {8},
number = {2},
pages = {},
pmid = {33673107},
issn = {2306-5354},
support = {P01 CA065493/CA/NCI NIH HHS/United States ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.},
}
@article {pmid33672015,
year = {2021},
author = {Emmanouilidis, I and Fili, N and Cook, AW and Hari-Gupta, Y and Dos Santos, &#xc1; and Wang, L and Martin-Fernandez, ML and Ellis, PJI and Toseland, CP},
title = {A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.},
journal = {Biomolecules},
volume = {11},
number = {2},
pages = {},
pmid = {33672015},
issn = {2218-273X},
support = {MC_EX_MR/K015591/1/MRC_/Medical Research Council/United Kingdom ; MR/M020606/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Survival ; Cisplatin/pharmacology ; Computer Simulation ; *DNA Breaks, Double-Stranded ; *DNA Damage ; DNA Repair ; Electroporation ; Endonucleases/genetics ; Escherichia coli/metabolism ; Gene Editing/methods ; Genome, Human ; Genomic Instability ; Genomics ; Humans ; Microscopy, Confocal ; Microscopy, Fluorescence ; Mutagens ; RNA, Guide ; Stochastic Processes ; },
abstract = {Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.},
}
@article {pmid33671852,
year = {2021},
author = {Price, E and Gianfrancesco, O and Harrison, PT and Frank, B and Bubb, VJ and Quinn, JP},
title = {CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region.},
journal = {International journal of molecular sciences},
volume = {22},
number = {4},
pages = {},
pmid = {33671852},
issn = {1422-0067},
mesh = {Alu Elements/*genetics ; Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Intergenic/*genetics ; Gene Expression ; Gene Expression Regulation ; Genes, Reporter ; HEK293 Cells ; Humans ; Mice ; Minisatellite Repeats/*genetics ; Primates/genetics ; Promoter Regions, Genetic/*genetics ; Short Interspersed Nucleotide Elements/*genetics ; TRPV Cation Channels/*genetics ; },
abstract = {SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.},
}
@article {pmid33671590,
year = {2021},
author = {Zhang, I and Hsiao, Z and Liu, F},
title = {Development of Genome Editing Approaches against Herpes Simplex Virus Infections.},
journal = {Viruses},
volume = {13},
number = {2},
pages = {},
pmid = {33671590},
issn = {1999-4915},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Viral ; Herpesviridae Infections/*virology ; Herpesvirus 1, Human/*genetics/physiology ; Humans ; },
abstract = {Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 can engage in lytic infection as well as establish latent infection. Current anti-HSV-1 therapies effectively block viral replication and infection. However, they have little effect on viral latency and cannot completely eliminate viral infection. These issues, along with the emergence of drug-resistant viral strains, pose a need to develop new compounds and novel strategies for the treatment of HSV-1 infection. Genome editing methods represent a promising approach against viral infection by modifying or destroying the genetic material of human viruses. These editing methods include homing endonucleases (HE) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) RNA-guided nuclease system. Recent studies have showed that both HE and CRISPR/Cas systems are effective in inhibiting HSV-1 infection in cultured cells in vitro and in mice in vivo. This review, which focuses on recently published progress, suggests that genome editing approaches could be used for eliminating HSV-1 latent and lytic infection and for treating HSV-1 associated diseases.},
}
@article {pmid33671263,
year = {2021},
author = {Seroussi, E},
title = {Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing.},
journal = {Genes},
volume = {12},
number = {2},
pages = {},
pmid = {33671263},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/genetics ; DNA Copy Number Variations/*genetics ; Gene Dosage/*genetics ; INDEL Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA Editing/genetics ; Sequence Analysis, DNA/*trends ; Software ; },
abstract = {Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.},
}
@article {pmid33670702,
year = {2021},
author = {Galano, M and Li, Y and Li, L and Sottas, C and Papadopoulos, V},
title = {Role of Constitutive STAR in Leydig Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {4},
pages = {},
pmid = {33670702},
issn = {1422-0067},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Diglycerides/metabolism ; Gene Deletion ; Leydig Cells/*metabolism/ultrastructure ; Ligands ; Lipid Droplets/metabolism/ultrastructure ; Male ; Mice, Inbred C57BL ; Models, Biological ; Phosphoproteins/*metabolism ; Progesterone/metabolism ; Rats, Sprague-Dawley ; Receptors, GABA/metabolism ; Signal Transduction ; Steroids/biosynthesis ; },
abstract = {Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography-mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.},
}
@article {pmid33669513,
year = {2021},
author = {Guo, D and Chen, J and Zhao, X and Luo, Y and Jin, M and Fan, F and Park, C and Yang, X and Sun, C and Yan, J and Chen, W and Liu, Z},
title = {Genetic and Chemical Engineering of Phages for Controlling Multidrug-Resistant Bacteria.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {10},
number = {2},
pages = {},
pmid = {33669513},
issn = {2079-6382},
abstract = {Along with the excessive use of antibiotics, the emergence and spread of multidrug-resistant bacteria has become a public health problem and a great challenge vis-à-vis the control and treatment of bacterial infections. As the natural predators of bacteria, phages have reattracted researchers' attentions. Phage therapy is regarded as one of the most promising alternative strategies to fight pathogens in the post-antibiotic era. Recently, genetic and chemical engineering methods have been applied in phage modification. Among them, genetic engineering includes the expression of toxin proteins, modification of host recognition receptors, and interference of bacterial phage-resistant pathways. Chemical engineering, meanwhile, involves crosslinking phage coats with antibiotics, antimicrobial peptides, heavy metal ions, and photothermic matters. Those advances greatly expand the host range of phages and increase their bactericidal efficiency, which sheds light on the application of phage therapy in the control of multidrug-resistant pathogens. This review reports on engineered phages through genetic and chemical approaches. Further, we present the obstacles that this novel antimicrobial has incurred.},
}
@article {pmid33669425,
year = {2021},
author = {Girault, MS and Dupuis, S and Ialy-Radio, C and Stouvenel, L and Viollet, C and Pierre, R and Favier, M and Ziyyat, A and Barbaux, S},
title = {Deletion of the Spata3 Gene Induces Sperm Alterations and In Vitro Hypofertility in Mice.},
journal = {International journal of molecular sciences},
volume = {22},
number = {4},
pages = {},
pmid = {33669425},
issn = {1422-0067},
mesh = {Acrosome/metabolism/*pathology/ultrastructure ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Fertilization in Vitro/*methods ; *Gene Deletion ; Infertility, Male/*genetics/metabolism ; Lipid Droplets/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Phenotype ; Pregnancy ; Proteins/*genetics/metabolism ; Sperm Motility/genetics ; Spermatogenesis/genetics ; Testis/metabolism ; },
abstract = {Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.},
}
@article {pmid33669346,
year = {2021},
author = {Brakebusch, C},
title = {CRISPR Genome Editing: How to Make a Fantastic Method Even Better.},
journal = {Cells},
volume = {10},
number = {2},
pages = {},
pmid = {33669346},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Humans ; },
abstract = {CRISPR genome editing describes targeted mutagenesis involving a programmable DNA scissor consisting of a protein (Cas9) bound to a short RNA [...].},
}
@article {pmid33669319,
year = {2021},
author = {Florea, S and Jaromczyk, J and Schardl, CL},
title = {Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi.},
journal = {Toxins},
volume = {13},
number = {2},
pages = {},
pmid = {33669319},
issn = {2072-6651},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endophytes/*genetics/growth &amp; development/metabolism ; Epichloe/*genetics/growth &amp; development/metabolism ; Ergot Alkaloids/biosynthesis/*genetics/toxicity ; *Gene Editing ; Gene Expression Regulation, Fungal ; *Gene Knockout Techniques ; *Multigene Family ; Reproduction, Asexual ; Secondary Metabolism ; },
abstract = {The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala-a triploid hybrid species-were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.},
}
@article {pmid33668636,
year = {2021},
author = {Pramanik, D and Shelake, RM and Park, J and Kim, MJ and Hwang, I and Park, Y and Kim, JY},
title = {CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew.},
journal = {International journal of molecular sciences},
volume = {22},
number = {4},
pages = {},
pmid = {33668636},
issn = {1422-0067},
mesh = {Begomovirus/*metabolism ; *CRISPR-Cas Systems ; Disease Resistance/*genetics ; *Gene Editing ; Genome, Plant ; *Lycopersicon esculentum/genetics/growth &amp; development/microbiology/virology ; *Plant Diseases/genetics/microbiology/virology ; *Plants, Genetically Modified/genetics/growth &amp; development/microbiology/virology ; },
abstract = {Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.},
}
@article {pmid33668622,
year = {2021},
author = {Tyumentseva, M and Mikhaylova, Y and Prelovskaya, A and Tyumentsev, A and Petrova, L and Fomina, V and Zamyatin, M and Shelenkov, A and Akimkin, V},
title = {Genomic and Phenotypic Analysis of Multidrug-Resistant Acinetobacter baumannii Clinical Isolates Carrying Different Types of CRISPR/Cas Systems.},
journal = {Pathogens (Basel, Switzerland)},
volume = {10},
number = {2},
pages = {},
pmid = {33668622},
issn = {2076-0817},
abstract = {Acinetobacter baumannii is an opportunistic pathogen being one of the most important causative agents of a wide range of nosocomial infections associated with multidrug resistance and high mortality rate. This study presents a multiparametric and correlation analyses of clinical multidrug-resistant A. baumannii isolates using short- and long-read whole-genome sequencing, which allowed us to reveal specific characteristics of the isolates with different CRISPR/Cas systems. We also compared antibiotic resistance and virulence gene acquisition for the groups of the isolates having functional CRISPR/Cas systems, just CRISPR arrays without cas genes, and without detectable CRISPR spacers. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as phylogenetic analysis of full-length cas gene sequences, predicted prophage sequences and CRISPR array type determination. For the first time the differences between the isolates carrying Type I-F1 and Type I-F2 CRISPR/Cas systems were investigated. A. baumannii isolates with Type I-F1 system were shown to have smaller number of reliably detected CRISPR arrays, and thus they could more easily adapt to environmental conditions through acquisition of antibiotic resistance genes, while Type I-F2 A. baumannii might have stronger "immunity" and use CRISPR/Cas system to block the dissemination of these genes. In addition, virulence factors abaI, abaR, bap and bauA were overrepresented in A. baumannii isolates lacking CRISPR/Cas system. This indicates the role of CRISPR/Cas in fighting against phage infections and preventing horizontal gene transfer. We believe that the data presented will contribute to further investigations in the field of antimicrobial resistance and CRISPR/Cas studies.},
}
@article {pmid33668187,
year = {2021},
author = {Tanihara, F and Hirata, M and Nguyen, NT and Sawamoto, O and Kikuchi, T and Otoi, T},
title = {One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {5},
pages = {},
pmid = {33668187},
issn = {1422-0067},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Antigens, Heterophile/*biosynthesis ; *CRISPR-Cas Systems ; Female ; Galactosyltransferases/*antagonists &amp; inhibitors ; Gene Editing ; Mixed Function Oxygenases/*antagonists &amp; inhibitors ; N-Acetylgalactosaminyltransferases/*antagonists &amp; inhibitors ; Swine ; Zygote/*physiology ; },
abstract = {Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.},
}
@article {pmid33667951,
year = {2021},
author = {Sarkar, E and Khan, A},
title = {Erratic journey of CRISPR/Cas9 in oncology from bench-work to successful-clinical therapy.},
journal = {Cancer treatment and research communications},
volume = {27},
number = {},
pages = {100289},
doi = {10.1016/j.ctarc.2020.100289},
pmid = {33667951},
issn = {2468-2942},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clinical Trials as Topic ; Disease Models, Animal ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; Humans ; Immunotherapy, Adoptive/*methods ; Medical Oncology/methods/trends ; Neoplasms/genetics/immunology/mortality/*therapy ; Programmed Cell Death 1 Receptor/genetics ; Progression-Free Survival ; Receptors, Chimeric Antigen/genetics/immunology ; },
abstract = {CRISPR is a customized molecular scissor, comprising genetic guide made of RNA and an enzyme, Cas9 which snips DNA in simpler, cheaper and more precise way than any other gene editing tools. In recent years CRISPR/Cas has taken the research world by storm being go-to genome editor for potential gene therapy to fix cancer as well as several hereditary disorders. This review explores the literature around the mechanism of Nobel winning CRISPR/Cas9 and its journey from its discovery to various pre-clinical and clinical trials in oncology, focusing mostly on PD-1 knockout CAR-T cell therapy. It also discusses the hurdles and ethical dispute associated with CRISPR, such as unintended on-target and off-target cuts, embryonic germ-line editing. Despite the controversies regarding the safety of this technique, many studies reported promising results on targeting cancer and other diseases using CRISPR/Cas9. Outcomes from the first successful clinical trial showed the beneficial long term effect on genetically modified T-cells in targeting cancer cells which opens the door for CRISPR to be the most preferred technique to help treating cancer and other diseases in future. As far as germ-line editing is concerned, further studies are needed to support the safety of this technique in humans fixing genetic disorders and mutations. Therefore till date only somatic cell editing is ethically approved.},
}
@article {pmid33667348,
year = {2021},
author = {Hendelman, A and Zebell, S and Rodriguez-Leal, D and Dukler, N and Robitaille, G and Wu, X and Kostyun, J and Tal, L and Wang, P and Bartlett, ME and Eshed, Y and Efroni, I and Lippman, ZB},
title = {Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.},
journal = {Cell},
volume = {184},
number = {7},
pages = {1724-1739.e16},
doi = {10.1016/j.cell.2021.02.001},
pmid = {33667348},
issn = {1097-4172},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Alleles ; Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Chromatin/metabolism ; Gene Expression Regulation, Plant ; *Genes, Plant ; Genetic Pleiotropy/*genetics ; Homeodomain Proteins/*genetics ; Inflorescence/genetics ; Lycopersicon esculentum/genetics ; Mutagenesis ; Plant Development/genetics ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics/growth &amp; development/metabolism ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid/*genetics ; Solanaceae/genetics/growth &amp; development ; },
abstract = {Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.},
}
@article {pmid33667229,
year = {2021},
author = {Di Stazio, M and Foschi, N and Athanasakis, E and Gasparini, P and d'Adamo, AP},
title = {Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique.},
journal = {PloS one},
volume = {16},
number = {3},
pages = {e0247603},
pmid = {33667229},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA, Single-Stranded/genetics ; Gene Editing/*methods ; *Genome, Human ; HEK293 Cells ; Humans ; Mutation ; Nucleotides/genetics ; RNA, Guide/genetics ; Recombinational DNA Repair/*genetics ; Transfection ; Trinucleotide Repeats/genetics ; Tumor Necrosis Factor-alpha/*genetics ; },
abstract = {The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.},
}
@article {pmid33667117,
year = {2021},
author = {Marchant, GE},
title = {Global Governance of Human Genome Editing: What Are the Rules?.},
journal = {Annual review of genomics and human genetics},
volume = {22},
number = {},
pages = {385-405},
doi = {10.1146/annurev-genom-111320-091930},
pmid = {33667117},
issn = {1545-293X},
mesh = {CRISPR-Cas Systems ; Ecosystem ; *Gene Editing ; *Genome, Human ; Humans ; },
abstract = {Human gene editing, particularly using the new CRISPR/Cas9 technology, will greatly increase the capability to make precise changes to human genomes. Human gene editing can be broken into four major categories: somatic therapy, heritable gene editing, genetic enhancement, and basic and applied research. Somatic therapy is generally well governed by national regulatory systems, so the need for global governance is less urgent. All nations are in agreement that heritable gene editing should not proceed at this time, but there is likely to be divergence if and when such procedures are shown to be safe and effective. Gene editing for enhancement purposes is not feasible today but is more controversial with the public, and many nations do not have well-developed regulatory systems for addressing genetic enhancement. Finally, different nations treat research with human embryos very differently based on deeply embedded social, cultural, ethical, and legal traditions. Several international governance mechanisms are currently in operation for human gene editing, and several other governance mechanisms have been proposed. It is unlikely that any single mechanism will alone be effective for governing human gene editing; rather, a polycentric or ecosystem approach that includes several overlapping and interacting components is likely to be necessary.},
}
@article {pmid33666363,
year = {2021},
author = {Maganti, HB and Bailey, AJM and Kirkham, AM and Shorr, R and Pineault, N and Allan, DS},
title = {Persistence of CRISPR/Cas9 gene edited hematopoietic stem cells following transplantation: A systematic review and meta-analysis of preclinical studies.},
journal = {Stem cells translational medicine},
volume = {10},
number = {7},
pages = {996-1007},
pmid = {33666363},
issn = {2157-6580},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Hemoglobins ; },
abstract = {Gene editing blood-derived cells is an attractive approach to cure selected monogenic diseases but remains experimental. A systematic search of preclinical controlled studies is needed to determine the persistence of edited cells following reinfusion. All studies identified in our systematic search (to 20 October 2020) examining the use of CRISPR/Cas9 gene editing in blood-derived cells for transplantation were included. Meta-analysis was performed to determine the engraftment and persistence of gene edited cells. A total of 3538 preclinical studies were identified with 15 published articles meeting eligibility for meta-analysis. These in vivo animal studies examined editing of hemoglobin to correct sickle cell disease (eight studies), inducing resistance to acquired immunodeficiency syndrome (two studies), and six other monogenic disorders (single studies). CRISPR-Cas9 edited hematopoietic stem and progenitor cells demonstrated equivalent early engraftment compared to controls in meta-analysis but persistence of gene-edited cells was reduced at later time points and in secondary transplant recipients. Subgroup analysis in studies targeting the hemoglobin gene revealed a significant reduction in the persistence of gene-edited cells whether homology-directed repair or nonhomologous end-joining were used. No adverse side effects were reported. Significant heterogeneity in study design and outcome reporting was observed and the potential for bias was identified in all studies. CRISPR-Cas9 gene edited cells engraft similarly to unedited hematopoietic cells. Persistence of gene edited cells, however, remains a challenge and improved methods of targeting hematopoietic stem cells are needed. Reducing heterogeneity and potential risk of bias will hasten the development of informative clinical trials.},
}
@article {pmid33666335,
year = {2021},
author = {Li, S and Garay, JP and Tubbs, CA and Franco, HL},
title = {CRISPR-based knock-in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi-allelic proteins in cancer cells.},
journal = {FEBS open bio},
volume = {11},
number = {6},
pages = {1537-1551},
pmid = {33666335},
issn = {2211-5463},
support = {K99 CA204628/CA/NCI NIH HHS/United States ; P50 CA058223/CA/NCI NIH HHS/United States ; R00 CA204628/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Hepatocyte Nuclear Factor 3-alpha/*genetics ; Humans ; Mutagenesis ; Mutation ; Neoplasms/*genetics/pathology ; },
abstract = {Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of the clustered regularly interspaced short palindromic repeats (CRISPR) technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate or knockout specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knock-in strategies of genes in typical diploid cells (i.e., cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein, we detail our methodology for replacing endogenous proteins with intended knock-in mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF-7 breast cancer cells. We confirm mutant FOXA1 protein expression and intended amino acid substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele frequency estimation is easily achieved by topoisomerase-based cloning combined with allele-specific PCR, which we later confirmed by next-generation RNA-sequencing. Typically, there are 4 - 5 copies (alleles) of FOXA1 in breast cancer cells, making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wild-type FOXA1 with precise knock-in mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.},
}
@article {pmid33666174,
year = {2021},
author = {Kandul, NP and Liu, J and Bennett, JB and Marshall, JM and Akbari, OS},
title = {A confinable home-and-rescue gene drive for population modification.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33666174},
issn = {2050-084X},
support = {R01 AI151004/AI/NIAID NIH HHS/United States ; R21 AI149161/AI/NIAID NIH HHS/United States ; R21RAI149161A/NH/NIH HHS/United States ; DP2 AI152071/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila melanogaster/*genetics ; Gene Drive Technology/*methods ; Genetic Engineering/methods ; Genetic Fitness ; Genetics, Population ; Models, Theoretical ; },
abstract = {Homing-based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, we engineer a confinable population modification home-and-rescue (HomeR) drive in Drosophila targeting an essential gene. In our experiments, resistant alleles that disrupt the target gene function were recessive lethal and therefore disadvantaged. We demonstrate that HomeR can achieve an increase in frequency in population cage experiments, but that fitness costs due to the Cas9 insertion limit drive efficacy. Finally, we conduct mathematical modeling comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.},
}
@article {pmid33665030,
year = {2021},
author = {Portet, A and Galinier, R and Lassalle, D and Faille, A and Gourbal, B and Duval, D},
title = {Hemocyte siRNA uptake is increased by 5' cholesterol-TEG addition in Biomphalaria glabrata, snail vector of schistosome.},
journal = {PeerJ},
volume = {9},
number = {},
pages = {e10895},
pmid = {33665030},
issn = {2167-8359},
abstract = {Biomphalaria glabrata is one of the snail intermediate hosts of Schistosoma mansoni, the causative agent of intestinal schistosomiasis disease. Numerous molecular studies using comparative approaches between susceptible and resistant snails to S. mansoni infection have helped identify numerous snail key candidates supporting such susceptible/resistant status. The functional approach using RNA interference (RNAi) remains crucial to validate the function of such candidates. CRISPR-Cas systems are still under development in many laboratories, and RNA interference remains the best tool to study B. glabrata snail genetics. Herein, we describe the use of modified small interfering RNA (siRNA) molecules to enhance cell delivery, especially into hemocytes, the snail immune cells. Modification of siRNA with 5' Cholesteryl TriEthylene Glycol (Chol-TEG) promotes cellular uptake by hemocytes, nearly eightfold over that of unmodified siRNA. FACS analysis reveals that more than 50% of hemocytes have internalized Chol-TEG siRNA conjugated to Cy3 fluorophores, 2 hours only after in vivo injection into snails. Chol-TEG siRNA targeting BgTEP1 (ThioEster-containing Protein), a parasite binding protein, reduced BgTEP1 transcript expression by 70-80% compared to control. The level of BgTEP1 protein secreted in the hemolymph was also decreased. However, despite the BgTEP1 knock-down at both RNA and protein levels, snail compatibility with its sympatric parasite is not affected suggesting functional redundancy among the BgTEP genes family in snail-schistosoma interaction.},
}
@article {pmid33664852,
year = {2021},
author = {Chu, CY and Lee, YC and Hsieh, CH and Yeh, CT and Chao, TY and Chen, PH and Lin, IH and Hsieh, TH and Shih, JW and Cheng, CH and Chang, CC and Lin, PS and Huang, YL and Chen, TM and Yen, Y and Ann, DK and Kung, HJ},
title = {Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.},
journal = {Theranostics},
volume = {11},
number = {8},
pages = {3624-3641},
pmid = {33664852},
issn = {1838-7640},
mesh = {Animals ; Arginine/*deficiency ; Argininosuccinate Synthase/deficiency/genetics/metabolism ; Breast Neoplasms/genetics/metabolism/therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chemokine CCL2/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; Female ; Gene Knockout Techniques ; Humans ; Hydrolases/*pharmacology ; Inflammation/genetics/metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Models, Biological ; Molecular Targeted Therapy ; Polyethylene Glycols/*pharmacology ; Precision Medicine ; Prostatic Neoplasms/genetics/metabolism/therapy ; Signal Transduction ; Triggering Receptor Expressed on Myeloid Cells-1/antagonists &amp; inhibitors/genetics/metabolism ; Up-Regulation ; Xenograft Model Antitumor Assays ; },
abstract = {Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.},
}
@article {pmid33664716,
year = {2021},
author = {Tittes, C and Schwarzer, S and Pfeiffer, F and Dyall-Smith, M and Rodriguez-Franco, M and Oksanen, HM and Quax, TEF},
title = {Cellular and Genomic Properties of Haloferax gibbonsii LR2-5, the Host of Euryarchaeal Virus HFTV1.},
journal = {Frontiers in microbiology},
volume = {12},
number = {},
pages = {625599},
pmid = {33664716},
issn = {1664-302X},
abstract = {Hypersaline environments are the source of many viruses infecting different species of halophilic euryarchaea. Information on infection mechanisms of archaeal viruses is scarce, due to the lack of genetically accessible virus-host models. Recently, a new archaeal siphovirus, Haloferax tailed virus 1 (HFTV1), was isolated together with its host belonging to the genus Haloferax, but it is not infectious on the widely used model euryarcheon Haloferax volcanii. To gain more insight into the biology of HFTV1 host strain LR2-5, we studied characteristics that might play a role in its virus susceptibility: growth-dependent motility, surface layer, filamentous surface structures, and cell shape. Its genome sequence showed that LR2-5 is a new strain of Haloferax gibbonsii. LR2-5 lacks obvious viral defense systems, such as CRISPR-Cas, and the composition of its cell surface is different from Hfx. volcanii, which might explain the different viral host range. This work provides first deep insights into the relationship between the host of halovirus HFTV1 and other members of the genus Haloferax. Given the close relationship to the genetically accessible Hfx. volcanii, LR2-5 has high potential as a new model for virus-host studies in euryarchaea.},
}
@article {pmid33664418,
year = {2021},
author = {Azami, Y and Tsuyama, N and Abe, Y and Sugai-Takahashi, M and Kudo, KI and Ota, A and Sivasundaram, K and Muramatsu, M and Shigemura, T and Sasatani, M and Hashimoto, Y and Saji, S and Kamiya, K and Hanamura, I and Ikezoe, T and Onodera, M and Sakai, A},
title = {Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {5216},
pmid = {33664418},
issn = {2045-2322},
mesh = {B-Lymphocytes/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line, Tumor ; Chromosome Aberrations ; Chromosomes, Human, Pair 11/genetics ; Chromosomes, Human, Pair 14/genetics ; Cyclin D1/*genetics ; Hematopoietic Stem Cells ; Humans ; Immunoglobulin Heavy Chains/*genetics ; In Situ Hybridization, Fluorescence ; Induced Pluripotent Stem Cells/metabolism/pathology ; Multiple Myeloma/*genetics ; Translocation, Genetic/genetics ; Tumor Suppressor Protein p53/*genetics ; VDJ Exons/genetics ; },
abstract = {Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain potential to transform by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we have already established normal B cell-derived induced pluripotent stem cells (BiPSCs). Here we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5'-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14). These BiPSCs differentiated into hematopoietic progenitor cells (HPCs). However, unlike cord blood, those HPCs did not differentiated into B lymphocytes by co-culture with BM stromal cell. Therefore, further ingenuity is required to differentiate those BiPSCs-derived HPCs into B lymphocytes.},
}
@article {pmid33664368,
year = {2021},
author = {Shaw, TI and Dong, L and Tian, L and Qian, C and Liu, Y and Ju, B and High, A and Kavdia, K and Pagala, VR and Shaner, B and Pei, D and Easton, J and Janke, LJ and Porter, SN and Ma, X and Cheng, C and Pruett-Miller, SM and Choi, J and Yu, J and Peng, J and Gu, W and Look, AT and Downing, JR and Zhang, J},
title = {Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL).},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {5154},
pmid = {33664368},
issn = {2045-2322},
support = {R01 GM134382/GM/NIGMS NIH HHS/United States ; R35 CA210064/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Lineage/genetics ; Cell Proliferation/genetics ; Gene Expression Regulation, Leukemic/genetics ; Haploinsufficiency/genetics ; Humans ; Oncogenes/*genetics ; Pediatrics ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/*genetics/pathology ; T-Cell Acute Lymphocytic Leukemia Protein 1/*genetics ; Transcriptional Activation/genetics ; Ubiquitin-Specific Peptidase 7/*genetics ; },
abstract = {USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.},
}
@article {pmid33664305,
year = {2021},
author = {Faber, NR and McFarlane, GR and Gaynor, RC and Pocrnic, I and Whitelaw, CBA and Gorjanc, G},
title = {Novel combination of CRISPR-based gene drives eliminates resistance and localises spread.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {3719},
pmid = {33664305},
issn = {2045-2322},
mesh = {Alleles ; Animals ; *Biodiversity ; CRISPR-Cas Systems/*genetics ; Gene Drive Technology/methods ; Genes, Essential/genetics ; Humans ; Introduced Species ; Pest Control/*methods ; *Pest Control, Biological ; },
abstract = {Invasive species are among the major driving forces behind biodiversity loss. Gene drive technology may offer a humane, efficient and cost-effective method of control. For safe and effective deployment it is vital that a gene drive is both self-limiting and can overcome evolutionary resistance. We present HD-ClvR in this modelling study, a novel combination of CRISPR-based gene drives that eliminates resistance and localises spread. As a case study, we model HD-ClvR in the grey squirrel (Sciurus carolinensis), which is an invasive pest in the UK and responsible for both biodiversity and economic losses. HD-ClvR combats resistance allele formation by combining a homing gene drive with a cleave-and-rescue gene drive. The inclusion of a self-limiting daisyfield gene drive allows for controllable localisation based on animal supplementation. We use both randomly mating and spatial models to simulate this strategy. Our findings show that HD-ClvR could effectively control a targeted grey squirrel population, with little risk to other populations. HD-ClvR offers an efficient, self-limiting and controllable gene drive for managing invasive pests.},
}
@article {pmid33664298,
year = {2021},
author = {Shekoohi, S and Rajasekaran, S and Patel, D and Yang, S and Liu, W and Huang, S and Yu, X and Witt, SN},
title = {Knocking out alpha-synuclein in melanoma cells dysregulates cellular iron metabolism and suppresses tumor growth.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {5267},
pmid = {33664298},
issn = {2045-2322},
mesh = {Animals ; Antigens, CD/*genetics ; CRISPR-Cas Systems/genetics ; Cation Transport Proteins/*genetics ; Cell Proliferation/genetics ; Cell Survival/genetics ; Ferritins/genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Heterografts ; Humans ; Iron/*metabolism ; Melanoma/*genetics/metabolism/pathology ; Mice ; Receptors, Transferrin/*genetics ; alpha-Synuclein/antagonists &amp; inhibitors/*genetics ; },
abstract = {The protein alpha-synuclein (α-syn) is unusual because, depending on its conformation and the type of cell in which it is expressed, it is pro-death or pro-survival, triggering neurodegeneration in Parkinson's disease and enhancing cell survival of some melanomas. To probe the function of α-syn in melanoma, we used CRISPR/Cas9 to knockout SNCA, the gene that codes for α-syn, in SK-Mel-28 melanoma cells. The SNCA-knockout clones in culture exhibited a decrease in the transferrin receptor 1 (TfR1), an increase in ferritin, an increase of reactive oxygen species and proliferated slower than control cells. These SNCA-knockout clones grafted into SCID mice grew significantly slower than the SK-Mel-28 control cells that expressed α-syn. In the excised SNCA-knockout xenografts, TfR1 decreased 3.3-fold, ferritin increased 6.2-fold, the divalent metal ion transporter 1 (DMT1) increased threefold, and the iron exporter ferroportin (FPN1) decreased twofold relative to control xenografts. The excised SNCA-KO tumors exhibited significantly more ferric iron and TUNEL staining relative to the control melanoma xenografts. Collectively, depletion of α-syn in SK-Mel-28 cells dysregulates cellular iron metabolism, especially in xenografts, yielding melanoma cells that are deficient in TfR1 and FPN1, that accumulate ferric iron and ferritin, and that undergo apoptosis relative to control cells expressing α-syn.},
}
@article {pmid33663564,
year = {2021},
author = {Ten, KE and Md Zoqratt, MZH and Ayub, Q and Tan, HS},
title = {Characterization of multidrug-resistant Acinetobacter baumannii strain ATCC BAA1605 using whole-genome sequencing.},
journal = {BMC research notes},
volume = {14},
number = {1},
pages = {83},
pmid = {33663564},
issn = {1756-0500},
mesh = {*Acinetobacter baumannii/genetics ; Anti-Bacterial Agents/pharmacology ; Genome, Bacterial/genetics ; Plasmids/genetics ; Whole Genome Sequencing ; },
abstract = {OBJECTIVE: The nosocomial pathogen, Acinetobacter baumannii, has acquired clinical significance due to its ability to persist in hospital settings and survive antibiotic treatment, which eventually resulted in the rapid spread of this bacterium with antimicrobial resistance (AMR) phenotypes. This study used a multidrug-resistant A. baumannii (strain ATCC BAA1605) as a model to study the genomic features of this pathogen.<br><br>RESULTS: One circular chromosome and one circular plasmid were discovered in the complete genome of A. baumannii ATCC BAA1605 using whole-genome sequencing. The chromosome is 4,039,171&#xa0;bp long with a GC content of 39.24%. Many AMR genes, which confer resistance to major classes of antibiotics (beta-lactams, aminoglycosides, tetracycline, sulphonamides), were found on the chromosome. Two genomic islands were predicted on the chromosome, one of which (Genomic Island 1) contains a cluster of AMR genes and mobile elements, suggesting the possibility of horizontal gene transfer. A subtype I-F CRISPR-Cas system was also identified on the chromosome of A. baumannii ATCC BAA1605. This study provides valuable genome data that can be used as a reference for future studies on A. baumannii. The genome of A. baumannii ATCC BAA1605 has been deposited at GenBank under accession no. CP058625 and CP058626.},
}
@article {pmid33662563,
year = {2022},
author = {Leung, RK and Cheng, QX and Wu, ZL and Khan, G and Liu, Y and Xia, HY and Wang, J},
title = {CRISPR-Cas12-based nucleic acids detection systems.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {276-281},
doi = {10.1016/j.ymeth.2021.02.018},
pmid = {33662563},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA, Single-Stranded ; *Gene Editing/methods ; },
abstract = {Because of the outstanding contribution in genome editing, CRISPR has undoubtedly become the most popular technology around the world and two pioneers are awarded the Nobel Prize in Chemistry this year. Besides, along with the discovery of nonspecific trans-cleavage activities of several Cas proteins such as Cas12 and Cas13, many CRISPR-based molecular diagnostic systems have been successfully created, showing advantages in sensitivity, specificity and operation convenience. Among them, systems with Cas12, which targets DNA and trans-cleaves single-stranded DNA probes, are both simple and highly efficient. Here in this review, we mainly focus on the Cas12-based methods and briefly discuss their applications in nucleic acids detection and beyond.},
}
@article {pmid33662274,
year = {2021},
author = {Zou, RS and Liu, Y and Wu, B and Ha, T},
title = {Cas9 deactivation with photocleavable guide RNAs.},
journal = {Molecular cell},
volume = {81},
number = {7},
pages = {1553-1565.e8},
pmid = {33662274},
issn = {1097-4164},
support = {T32 GM136577/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U01 DK127432/DK/NIDDK NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; F30 CA254160/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/metabolism ; *CRISPR-Cas Systems ; *DNA Damage ; *Gene Editing ; RNA, Guide/*chemistry/metabolism ; },
abstract = {Precise control of CRISPR-Cas9 would improve its safety and applicability. Controlled CRISPR inhibition is a promising approach but is complicated by separate inhibitor delivery, incomplete deactivation, and slow kinetics. To overcome these obstacles, we engineered photocleavable guide RNAs (pcRNAs) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1 min) and most complete (<1% residual indels) approach for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild-type Cas9. Time-resolved deactivation revealed that 12-36 h of Cas9 activity or 2-4 h of base editor activity was sufficient to achieve high editing efficiency. pcRNA is useful for studies of the cellular response to DNA damage by abolishing sustained cycles of damage and repair that would otherwise desynchronize response trajectories. Together, pcRNA expands the CRISPR toolbox for precision genome editing and studies of DNA damage and repair.},
}
@article {pmid33661998,
year = {2021},
author = {Baik, R and Wyman, SK and Kabir, S and Corn, JE},
title = {Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms.},
journal = {PloS one},
volume = {16},
number = {3},
pages = {e0247858},
pmid = {33661998},
issn = {1932-6203},
support = {DP2 HL141006/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Coculture Techniques ; Erythroid Precursor Cells/cytology/metabolism ; *Gene Editing ; Hematopoietic Stem Cells/cytology/metabolism ; Humans ; Janus Kinase 2/genetics ; Myeloproliferative Disorders/*genetics ; },
abstract = {Myeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop "scarless" Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.},
}
@article {pmid33661950,
year = {2021},
author = {Gross, T and Jeney, C and Halm, D and Finkenzeller, G and Stark, GB and Zengerle, R and Koltay, P and Zimmermann, S},
title = {Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and Emulsion Coupling assay as a low-cellularity workflow for single-cell cloning.},
journal = {PloS one},
volume = {16},
number = {3},
pages = {e0238330},
pmid = {33661950},
issn = {1932-6203},
mesh = {Bioprinting/*methods ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Cloning, Molecular/*methods ; Gene Knockout Techniques/*methods ; Humans ; Mesenchymal Stem Cells/cytology/*metabolism ; Osteoblasts/cytology/metabolism ; RANK Ligand/*genetics ; Single-Cell Analysis/*methods ; Workflow ; },
abstract = {The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.},
}
@article {pmid33661348,
year = {2021},
author = {Zhang, B and Zhou, J and Li, M and Wei, Y and Wang, J and Wang, Y and Shi, P and Li, X and Huang, Z and Tang, H and Song, Z},
title = {Evaluation of CRISPR/Cas9 site-specific function and validation of sgRNA sequence by a Cas9/sgRNA-assisted reverse PCR technique.},
journal = {Analytical and bioanalytical chemistry},
volume = {413},
number = {9},
pages = {2447-2456},
pmid = {33661348},
issn = {1618-2650},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; DNA Mutational Analysis/*methods ; Gene Editing/*methods ; Mutation ; Polymerase Chain Reaction/methods ; RNA, Guide/*genetics ; },
abstract = {The effective application of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system in biology, medicine and other fields is hindered by the off-target effects and loci-affinity of Cas9-sgRNA, especially at a genome-wide scale. In order to eliminate the occurrence of off-target effects and evaluate loci-affinity by CRISPR/Cas9 site-specific detection and screening of high-affinity sgRNA sequences, respectively, we develop a CRISPR/Cas9-assisted reverse PCR method for site-specific detection and sgRNA sequence validation. The detection method based on PCR can be used directly in the laboratory with PCR reaction conditions, without the need for an additional detection system, and the whole process of detection can be completed within 2 h. Therefore, it can be easily popularized with a PCR instrument. Finally, this method is fully verified by detecting multiple forms of site mutations and evaluating the affinity of a variety of sgRNA sequences for the CRISPR/Cas9 system. In sum, it provides an effective new analysis tool for CRISPR/Cas9 genome editing-related research. A CRISPR/Cas9-assisted reverse PCR method was developed for Cas9/sgRNA site-specific detection and sgRNA sequence validation. The technique detects target DNA in three steps: (1) target DNA is specifically cut by a pair of Cas9/sgRNA complexes; (2) the cleaved DNA is rapidly linked by T4 DNA ligase; (3) the ligated DNA is efficiently amplified by PCR (PCR or qPCR).},
}
@article {pmid33660777,
year = {2021},
author = {Lu, WT and Trost, CN and Müller-Esparza, H and Randau, L and Davidson, AR},
title = {Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.},
journal = {Nucleic acids research},
volume = {49},
number = {6},
pages = {3381-3393},
pmid = {33660777},
issn = {1362-4962},
support = {MOP-130482//CIHR/Canada ; FDN-15427//CIHR/Canada ; },
mesh = {CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/chemistry/*metabolism ; DNA, Single-Stranded/metabolism ; Mutagenesis ; Protein Binding ; Proteins/chemistry/genetics/metabolism ; },
abstract = {Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.},
}
@article {pmid33660775,
year = {2021},
author = {Reuter, A and Hilpert, C and Dedieu-Berne, A and Lematre, S and Gueguen, E and Launay, G and Bigot, S and Lesterlin, C},
title = {Targeted-antibacterial-plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity.},
journal = {Nucleic acids research},
volume = {49},
number = {6},
pages = {3584-3598},
pmid = {33660775},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Carbapenem-Resistant Enterobacteriaceae/genetics ; Conjugation, Genetic ; Enterobacteriaceae/*genetics ; Escherichia coli/genetics ; Plasmids/*genetics ; RNA/chemistry ; Software ; Species Specificity ; },
abstract = {The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on targeted-antibacterial-plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step toward the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.},
}
@article {pmid33660393,
year = {2021},
author = {Xu, J and Yin, Y and Jian, L and Han, B and Chen, Z and Yan, J and Liu, X},
title = {Seeing is believing: a visualization toolbox to enhance selection efficiency in maize genome editing.},
journal = {Plant biotechnology journal},
volume = {19},
number = {5},
pages = {872-874},
pmid = {33660393},
issn = {1467-7652},
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Zea mays/genetics/metabolism ; },
}
@article {pmid33660219,
year = {2021},
author = {Zhang, ZX and Wang, YZ and Xu, YS and Sun, XM and Huang, H},
title = {Developing GDi-CRISPR System for Multi-copy Integration in Saccharomyces cerevisiae.},
journal = {Applied biochemistry and biotechnology},
volume = {193},
number = {7},
pages = {2379-2388},
pmid = {33660219},
issn = {1559-0291},
mesh = {*CRISPR-Cas Systems ; *Genetic Engineering ; Recombinant Proteins/biosynthesis/genetics ; Saccharomyces cerevisiae/*genetics/metabolism ; },
abstract = {In recent years, Saccharomyces cerevisiae has been widely used in the production of biofuels and value-added chemicals. To stably express the target products, it is necessary to integrate multiple target genes into the chromosome of S. cerevisiae. CRISPR multi-copy integration technology relying on delta sites has been developed, but it often requires the help of high-throughput screening or resistance markers, resulting in non-replicability and high cost. This study aims to develop a low-cost and easy-to-use multi-copy integration tool in S. cerevisiae. Firstly, twenty-one Cas proteins from different microorganisms were tested in S. cerevisiae to find the functional Cas proteins with optimal cleavage ability. Results showed that eight Cas proteins can complete gene editing. However, most of the transformants have low copy numbers, which may be caused by high cutting efficiency exceeding the repair rate. Therefore, the effect of donor translocation order was further investigated. Results showed that 4 copies were obtained by donor first translocation. Then, the gene drive delta site integration system by the CRISPR system (GDi-CRISPR) was developed by combining gene drive principle and CRISPR system. To be clear, the gRNA was put into donor fragments. Then, both of them were integrated into the genome, which can drive further cutting and repair due to increasing number of gRNA. Instead of high-throughput screening or resistance pressure, 6 copies were obtained in only 5-6 days using the GDi-CRISPR system. It is expected to further advance the development of S. cerevisiae multi-copy integration tools.},
}
@article {pmid33659470,
year = {2020},
author = {Tovar, EA and Sheridan, R and Essenburg, CJ and Dischinger, PS and Arumugam, M and Callaghan, ME and Graveel, CR and Steensma, MR},
title = {Dissecting the Rat Mammary Gland: Isolation, Characterization, and Culture of Purified Mammary Epithelial Cells and Fibroblasts.},
journal = {Bio-protocol},
volume = {10},
number = {22},
pages = {e3818},
pmid = {33659470},
issn = {2331-8325},
abstract = {With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.},
}
@article {pmid33658378,
year = {2021},
author = {Rieblinger, B and Sid, H and Duda, D and Bozoglu, T and Klinger, R and Schlickenrieder, A and Lengyel, K and Flisikowski, K and Flisikowska, T and Simm, N and Grodziecki, A and Perleberg, C and Bähr, A and Carrier, L and Kurome, M and Zakhartchenko, V and Kessler, B and Wolf, E and Kettler, L and Luksch, H and Hagag, IT and Wise, D and Kaufman, J and Kaufer, BB and Kupatt, C and Schnieke, A and Schusser, B},
title = {Cas9-expressing chickens and pigs as resources for genome editing in livestock.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {10},
pages = {},
pmid = {33658378},
issn = {1091-6490},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; *CRISPR-Cas Systems ; Chickens/*genetics ; *Gene Editing ; Livestock/*genetics ; Swine/*genetics ; },
abstract = {Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.},
}
@article {pmid33657682,
year = {2021},
author = {Lyzenga, WJ and Pozniak, CJ and Kagale, S},
title = {Advanced domestication: harnessing the precision of gene editing in crop breeding.},
journal = {Plant biotechnology journal},
volume = {19},
number = {4},
pages = {660-670},
pmid = {33657682},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Domestication ; *Gene Editing ; Genome, Plant/genetics ; Plant Breeding ; },
abstract = {Human population growth has increased the demand for food crops, animal feed, biofuel and biomaterials, all the while climate change is impacting environmental growth conditions. There is an urgent need to develop crop varieties which tolerate adverse growth conditions while requiring fewer inputs. Plant breeding is critical to global food security and, while it has benefited from modern technologies, it remains constrained by a lack of valuable genetic diversity, linkage drag, and an effective way to combine multiple favourable alleles for complex traits. CRISPR/Cas technology has transformed genome editing across biological systems and promises to transform agriculture with its high precision, ease of design, multiplexing ability and low cost. We discuss the integration of CRISPR/Cas-based gene editing into crop breeding to advance domestication and refine inbred crop varieties for various applications and growth environments. We highlight the use of CRISPR/Cas-based gene editing to fix desirable allelic variants, generate novel alleles, break deleterious genetic linkages, support pre-breeding and for introgression of favourable loci into elite lines.},
}
@article {pmid33657669,
year = {2021},
author = {Dou, S and Zhang, T and Tu, J and Shen, J and Yi, B and Wen, J and Fu, T and Dai, C and Ma, C},
title = {Generation of novel self-incompatible Brassica napus by CRISPR/Cas9.},
journal = {Plant biotechnology journal},
volume = {19},
number = {5},
pages = {875-877},
pmid = {33657669},
issn = {1467-7652},
mesh = {*Brassica napus/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
}
@article {pmid33657402,
year = {2021},
author = {Wang, Y and Cottle, WT and Wang, H and Feng, XA and Mallon, J and Gavrilov, M and Bailey, S and Ha, T},
title = {Genome oligopaint via local denaturation fluorescence in situ hybridization.},
journal = {Molecular cell},
volume = {81},
number = {7},
pages = {1566-1577.e8},
pmid = {33657402},
issn = {1097-4164},
support = {T32 GM080189/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM097330/GM/NIGMS NIH HHS/United States ; U01 DK127432/DK/NIDDK NIH HHS/United States ; T32 GM007445/GM/NIGMS NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry ; *CRISPR-Cas Systems ; Cell Line ; Female ; Fibroblasts/chemistry/metabolism ; *Hot Temperature ; Humans ; *In Situ Hybridization, Fluorescence ; *Nucleic Acid Denaturation ; RNA, Guide/*chemistry ; },
abstract = {Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.},
}
@article {pmid33657370,
year = {2021},
author = {Lindborg, JA and Tran, NM and Chenette, DM and DeLuca, K and Foli, Y and Kannan, R and Sekine, Y and Wang, X and Wollan, M and Kim, IJ and Sanes, JR and Strittmatter, SM},
title = {Optic nerve regeneration screen identifies multiple genes restricting adult neural repair.},
journal = {Cell reports},
volume = {34},
number = {9},
pages = {108777},
pmid = {33657370},
issn = {2211-1247},
support = {P30 EY026878/EY/NEI NIH HHS/United States ; R00 EY029360/EY/NEI NIH HHS/United States ; F32 EY028448/EY/NEI NIH HHS/United States ; UL1 TR001863/TR/NCATS NIH HHS/United States ; U01 EY027256/EY/NEI NIH HHS/United States ; R21 EY029820/EY/NEI NIH HHS/United States ; R01 EY031751/EY/NEI NIH HHS/United States ; T32 EY022312/EY/NEI NIH HHS/United States ; R35 NS097283/NS/NINDS NIH HHS/United States ; K99 EY029360/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Axons/metabolism/pathology ; CRISPR-Cas Systems ; Dependovirus/genetics ; Female ; Gene Editing ; Gene Expression Regulation ; Genetic Association Studies ; HEK293 Cells ; Humans ; Interleukins/genetics/metabolism ; MAP Kinase Kinase Kinases/genetics/metabolism ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Nerve Regeneration/*genetics ; Neurogenesis/*genetics ; Optic Nerve/*metabolism/pathology/physiopathology ; Optic Nerve Injuries/*genetics/metabolism/pathology/physiopathology ; Retinal Ganglion Cells/metabolism/pathology ; STAT3 Transcription Factor/genetics/metabolism ; Signal Transduction ; },
abstract = {Adult mammalian central nervous system (CNS) trauma interrupts neural networks and, because axonal regeneration is minimal, neurological deficits persist. Repair via axonal growth is limited by extracellular inhibitors and cell-autonomous factors. Based on results from a screen in vitro, we evaluate nearly 400 genes through a large-scale in vivo regeneration screen. Suppression of 40 genes using viral-driven short hairpin RNAs (shRNAs) promotes retinal ganglion cell (RGC) axon regeneration after optic nerve crush (ONC), and most are validated by separate CRISPR-Cas9 editing experiments. Expression of these axon-regeneration-suppressing genes is not significantly altered by axotomy. Among regeneration-limiting genes, loss of the interleukin 22 (IL-22) cytokine allows an early, yet transient, inflammatory response in the retina after injury. Reduced IL-22 drives concurrent activation of signal transducer and activator of transcription 3 (Stat3) and dual leucine zipper kinase (DLK) pathways and upregulation of multiple neuron-intrinsic regeneration-associated genes (RAGs). Including IL-22, our screen identifies dozens of genes that limit CNS regeneration. Suppression of these genes in the context of axonal damage could support improved neural repair.},
}
@article {pmid33656289,
year = {2021},
author = {Hayta, S and Smedley, MA and Clarke, M and Forner, M and Harwood, WA},
title = {An Efficient Agrobacterium-Mediated Transformation Protocol for Hexaploid and Tetraploid Wheat.},
journal = {Current protocols},
volume = {1},
number = {3},
pages = {e58},
doi = {10.1002/cpz1.58},
pmid = {33656289},
issn = {2691-1299},
support = {BB/P013511/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Agrobacterium tumefaciens/genetics ; Plant Breeding ; *Tetraploidy ; Transformation, Genetic ; *Triticum/genetics ; },
abstract = {Wheat, though a key crop plant with considerable influence on world food security, has nonetheless trailed behind other major cereals in the advancement of gene transformation technology for its improvement. New breeding technologies such as genome editing allow precise DNA manipulation, but their potential is limited by low regeneration efficiencies in tissue culture and the lack of transformable genotypes. We developed, in the hexaploid spring wheat cultivar "Fielder," a robust, reproducible Agrobacterium tumefaciens-mediated transformation system with transformation efficiencies of up to 33%. The system requires immature embryos as starting material and includes a centrifugation pretreatment before the inoculation with Agrobacterium. This high-throughput, highly efficient, and repeatable transformation system has been used effectively to introduce genes of interest for overexpression, RNA interference, and CRISPR-Cas-based genome editing. With slight modifications reported here, the standard protocol can be applied to the hexaploid wheat "Cadenza" and the tetraploid durum wheat "Kronos" with efficiencies of up to 4% and 10%, respectively. The system has also been employed to assess the developmental gene fusion GRF-GIF with outstanding results. In our hands, this technology combined with our transformation system improved transformation efficiency to 77.5% in Fielder. This combination should help alleviate the genotype dependence of wheat transformation, allowing new genome-editing tools to be used directly in more elite wheat varieties. © 2021 The Authors. Basic Protocol 1: Growing of donor plants Basic Protocol 2: Transformation of Agrobacterium with vector by electroporation Basic Protocol 3: Starting material collection, sterilization, and embryo inoculation Basic Protocol 4: Selection, regeneration, rooting, and acclimatization of transformants.},
}
@article {pmid33656042,
year = {2021},
author = {Fu, X and Yin, Y and Zhang, M and Peng, F and Shi, Y and Liu, Y and Tan, Y and Zhao, Z and Yin, X and Song, J and Ke, G and Zhang, XB},
title = {Size-selective DNA nanocage-based activatable CRISPR-Cas12a for sensitive and accurate detection of mature microRNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {26},
pages = {3291-3294},
doi = {10.1039/d1cc00178g},
pmid = {33656042},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*chemistry/genetics ; Humans ; MicroRNAs/*analysis/blood/genetics ; Nanostructures/chemistry ; Nucleic Acid Hybridization ; },
abstract = {The sensitive and accurate detection of mature miRNA without the signal interference by pre-miRNAs is highly important. Herein, a size-selective DNA nanocage-based activatable CRISPR/Cas12a system was developed to achieve this goal.},
}
@article {pmid33655280,
year = {2021},
author = {Nemudryi, A and Nemudraia, A and Wiegand, T and Nichols, J and Snyder, DT and Hedges, JF and Cicha, C and Lee, H and Vanderwood, KK and Bimczok, D and Jutila, M and Wiedenheft, B},
title = {SARS-CoV-2 genomic surveillance identifies naturally occurring truncations of ORF7a that limit immune suppression.},
journal = {medRxiv : the preprint server for health sciences},
volume = {},
number = {},
pages = {},
pmid = {33655280},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
abstract = {Over 200,000 whole genome sequences of SARS-CoV-2 have been determined for viruses isolated from around the world. These sequences have been critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We have isolated one of these mutant viruses from a patient sample and used viral challenge experiments to demonstrate that Δ115 mutation results in a growth defect. ORF7a has been implicated in immune modulation, and we show that the C-terminal truncation results in distinct changes in interferon stimulated gene expression. Collectively, this work indicates that ORF7a mutations occur frequently and that these changes affect viral mechanisms responsible for suppressing the immune response.},
}
@article {pmid33654077,
year = {2021},
author = {Chen, L and Park, JE and Paa, P and Rajakumar, PD and Prekop, HT and Chew, YT and Manivannan, SN and Chew, WL},
title = {Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1384},
pmid = {33654077},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Repair/*genetics ; *Gene Editing ; HEK293 Cells ; Humans ; Nucleotide Motifs/genetics ; },
abstract = {Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.},
}
@article {pmid33653719,
year = {2021},
author = {Fleming, TJ and Schrankel, CS and Vyas, H and Rosenblatt, HD and Hamdoun, A},
title = {CRISPR/Cas9 mutagenesis reveals a role for ABCB1 in gut immune responses to Vibrio diazotrophicus in sea urchin larvae.},
journal = {The Journal of experimental biology},
volume = {224},
number = {7},
pages = {},
pmid = {33653719},
issn = {1477-9145},
support = {F32 ES029843/ES/NIEHS NIH HHS/United States ; R01 ES027921/ES/NIEHS NIH HHS/United States ; R01 ES030318/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Immunity ; Larva/genetics ; Mutagenesis ; *Sea Urchins ; Vibrio ; },
abstract = {The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host-microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment.},
}
@article {pmid33653259,
year = {2021},
author = {Siegner, SM and Karasu, ME and Schröder, MS and Kontarakis, Z and Corn, JE},
title = {PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants.},
journal = {BMC bioinformatics},
volume = {22},
number = {1},
pages = {101},
pmid = {33653259},
issn = {1471-2105},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine ; Gene Editing ; *RNA, Guide/genetics ; },
abstract = {BACKGROUND: The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents.<br><br>RESULTS: We developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ CONCLUSION: With PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.},
}
@article {pmid33651980,
year = {2021},
author = {Rossiter, NJ and Huggler, KS and Adelmann, CH and Keys, HR and Soens, RW and Sabatini, DM and Cantor, JR},
title = {CRISPR screens in physiologic medium reveal conditionally essential genes in human cells.},
journal = {Cell metabolism},
volume = {33},
number = {6},
pages = {1248-1263.e9},
pmid = {33651980},
issn = {1932-7420},
support = {T32 GM007287/GM/NIGMS NIH HHS/United States ; F31 CA228241/CA/NCI NIH HHS/United States ; T32 GM008349/GM/NIGMS NIH HHS/United States ; K22 CA225864/CA/NCI NIH HHS/United States ; R01 AI047389/AI/NIAID NIH HHS/United States ; R01 CA103866/CA/NCI NIH HHS/United States ; R37 AI047389/AI/NIAID NIH HHS/United States ; T32 HG002760/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Culture Media ; Humans ; },
abstract = {Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly reflect metabolite availability in human blood. Here, we performed CRISPR-based screens in traditional versus human plasma-like medium (HPLM). Sets of conditionally essential genes in human cancer cell lines span several cellular processes and vary with both natural cell-intrinsic diversity and the combination of basal and serum components that comprise typical media. Notably, we traced the causes for each of three conditional CRISPR phenotypes to the availability of metabolites uniquely defined in HPLM versus conventional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.},
}
@article {pmid33651945,
year = {2021},
author = {Sheng, A and Wang, P and Yang, J and Tang, L and Chen, F and Zhang, J},
title = {MXene Coupled with CRISPR-Cas12a for Analysis of Endotoxin and Bacteria.},
journal = {Analytical chemistry},
volume = {93},
number = {10},
pages = {4676-4681},
doi = {10.1021/acs.analchem.1c00371},
pmid = {33651945},
issn = {1520-6882},
mesh = {Bacteria ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/genetics ; Endotoxins ; },
abstract = {With hydrophilic surface and high density of functional groups, MXene can efficiently adsorb single-stranded DNA to enhance target-induced strand release and quench the fluorescence. Herein, MXene is coupled with CRISPR-Cas12a to sensitively detect LPS and bacteria. Specifically, the aptamer is well designed to initiate the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave single-stranded DNA, resulting it to be far away from MXene and the recovery of fluorescence. The target can effectually induce the release of the aptamer strand from the hybrid duplex with the assistance of MXene. The formed aptamer/target complex will inhibit the activation of CRISPR-Cas12a and its trans-cleavage on single-stranded DNA. The established method can selectively and sensitively quantify LPS and Gram-negative bacteria in different samples with detection limits of 11 pg/mL and 23 CFU/mL, respectively. Our study provides a new insight for exploration of universal analytical methods based on MXene coupled with CRISPR-Cas12a.},
}
@article {pmid33651591,
year = {2021},
author = {Jarczynska, ZD and Rendsvig, JKH and Pagels, N and Viana, VR and Nødvig, CS and Kirchner, FH and Strucko, T and Nielsen, ML and Mortensen, UH},
title = {DIVERSIFY: A Fungal Multispecies Gene Expression Platform.},
journal = {ACS synthetic biology},
volume = {10},
number = {3},
pages = {579-588},
doi = {10.1021/acssynbio.0c00587},
pmid = {33651591},
issn = {2161-5063},
mesh = {Aspergillus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cellulose 1,4-beta-Cellobiosidase/genetics/metabolism ; Fungi/genetics/*metabolism ; Gene Editing/*methods ; Glucuronidase/genetics/metabolism ; RNA, Guide/metabolism ; Salicylates/metabolism ; },
abstract = {Recent sequencing of numerous fungal species revealed large repertoires of putative biotechnologically relevant genes and secondary metabolite gene clusters. However, often the commercial potential of these species is impeded by difficulties to predict host physiological and metabolic compatibility with a given product, and lack of adequate genetic tools. Consequently, most heterologous production is performed in standard hosts where genetic tools and experience are in place. However, these species may not be suitable for all products. To increase chances of successful heterologous production, we have created a flexible platform, DIVERSIFY, for multispecies heterologous gene expression. This reduces the workload to construction of a single gene expression cassette, used to transform all DIVERSIFY strains in order to identify the optimal cell factory host. As proof of principle of the DIVERSIFY concept, we present the first version of our platform, DIVERSIFY 1.0, which we have successfully used for the production of three proteins and a metabolite in four different Aspergilli species, and for the identification of the best producer for each of the products. Moreover, we show that DIVERSIFY 1.0 is compatible with marker-free gene targeting induced by the CRISPR nucleases Cas9 and MAD7.},
}
@article {pmid33650757,
year = {2021},
author = {Yan, Y and Zhu, J and Qi, X and Cheng, B and Liu, C and Xie, C},
title = {Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {9},
pages = {1671-1680},
doi = {10.1111/jipb.13086},
pmid = {33650757},
issn = {1744-7909},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genes, Reporter ; Luminescent Proteins/*genetics ; Zea mays/*genetics ; },
abstract = {Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)-assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En-SFR/Cas9), embryos (Em-SFR/Cas9), or both tissues (Em/En-SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En-SFR/Cas9, Em-SFR/Cas9, and Em/En- SFR/Cas9 to identify plants not harboring the genome-editing cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries, and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9, Em-SFR/Cas9, and Em/En-SFR/Cas9. SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.},
}
@article {pmid33650465,
year = {2022},
author = {Challagulla, A and Shi, S and Nair, K and O'Neil, TE and Morris, KR and Wise, TG and Cahill, DM and Tizard, ML and Doran, TJ and Jenkins, KA},
title = {Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells.},
journal = {Animal biotechnology},
volume = {33},
number = {6},
pages = {1235-1245},
doi = {10.1080/10495398.2021.1885428},
pmid = {33650465},
issn = {1532-2378},
mesh = {Chick Embryo ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; Germ Cells/metabolism ; Chickens/genetics ; Cell Line ; },
abstract = {Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.},
}
@article {pmid33649592,
year = {2021},
author = {Frangieh, CJ and Melms, JC and Thakore, PI and Geiger-Schuller, KR and Ho, P and Luoma, AM and Cleary, B and Jerby-Arnon, L and Malu, S and Cuoco, MS and Zhao, M and Ager, CR and Rogava, M and Hovey, L and Rotem, A and Bernatchez, C and Wucherpfennig, KW and Johnson, BE and Rozenblatt-Rosen, O and Schadendorf, D and Regev, A and Izar, B},
title = {Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion.},
journal = {Nature genetics},
volume = {53},
number = {3},
pages = {332-341},
pmid = {33649592},
issn = {1546-1718},
support = {U54 CA225088/CA/NCI NIH HHS/United States ; R01 CA238039/CA/NCI NIH HHS/United States ; T32 GM007367/GM/NIGMS NIH HHS/United States ; K08 CA222663/CA/NCI NIH HHS/United States ; F32 AI138458/AI/NIAID NIH HHS/United States ; U19 AI133524/AI/NIAID NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P01 CA163222/CA/NCI NIH HHS/United States ; },
mesh = {CD58 Antigens/genetics/*immunology/metabolism ; CRISPR-Cas Systems ; Coculture Techniques ; Computational Biology/methods ; Drug Resistance, Neoplasm/drug effects/genetics/*immunology ; Epitopes/genetics ; Gene Knockout Techniques ; Humans ; Immune Checkpoint Inhibitors/pharmacology ; Interferon-gamma/immunology/metabolism ; Lymphocytes, Tumor-Infiltrating/pathology ; Melanoma/drug therapy/immunology/*pathology ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; *Tumor Escape/genetics ; },
abstract = {Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.},
}
@article {pmid33649239,
year = {2021},
author = {Brosh, R and Laurent, JM and Ordoñez, R and Huang, E and Hogan, MS and Hitchcock, AM and Mitchell, LA and Pinglay, S and Cadley, JA and Luther, RD and Truong, DM and Boeke, JD and Maurano, MT},
title = {A versatile platform for locus-scale genome rewriting and verification.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {10},
pages = {},
pmid = {33649239},
issn = {1091-6490},
support = {R35 GM119703/GM/NIGMS NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Genetic Loci ; *Genome, Human ; *Human Embryonic Stem Cells ; Humans ; Mice ; *Mouse Embryonic Stem Cells ; },
abstract = {Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.},
}
@article {pmid33647468,
year = {2021},
author = {Zhao, Y and Schuhmacher, LN and Roberts, M and Kakugawa, S and Bineva-Todd, G and Howell, S and O'Reilly, N and Perret, C and Snijders, AP and Vincent, JP and Jones, EY},
title = {Notum deacylates octanoylated ghrelin.},
journal = {Molecular metabolism},
volume = {49},
number = {},
pages = {101201},
pmid = {33647468},
issn = {2212-8778},
support = {C375/A17721/CRUK_/Cancer Research UK/United Kingdom ; MR/M000141/1/MRC_/Medical Research Council/United Kingdom ; 203141/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Acylation ; Animals ; Butyrylcholinesterase/metabolism ; Esterases/chemistry/genetics/*metabolism ; Ghrelin/*genetics/*metabolism ; Humans ; Ligands ; Male ; Mice ; Mice, Knockout ; },
abstract = {OBJECTIVES: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined.<br><br>METHODS: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation.<br><br>RESULTS: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2&#xa0;&#xc5; resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet.<br><br>CONCLUSIONS: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.},
}
@article {pmid33647431,
year = {2021},
author = {Abbasi, S and Uchida, S and Toh, K and Tockary, TA and Dirisala, A and Hayashi, K and Fukushima, S and Kataoka, K},
title = {Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {332},
number = {},
pages = {260-268},
doi = {10.1016/j.jconrel.2021.02.026},
pmid = {33647431},
issn = {1873-4995},
mesh = {Animals ; Brain ; CRISPR-Cas Systems ; *Gene Editing ; Mice ; Micelles ; *RNA, Guide/genetics ; RNA, Messenger/genetics ; },
abstract = {Genome editing using CRISPR/Cas9 has attracted considerable attention for the treatment of genetic disorders and viral infections. Co-delivery of Cas9 mRNA and single guide (sg)RNA is a promising strategy to efficiently edit the genome of various cell types, including non-dividing cells, with minimal safety concerns. However, co-delivery of two RNA species with significantly different sizes, such as Cas9 mRNA (4.5 kb) and sgRNA (0.1 kb), is still challenging, especially in vivo. Here, we addressed this issue by using a PEGylated polyplex micelle (PM) condensing the RNA in its core. PM loading sgRNA alone released sgRNA at minimal dilution in buffer, while PM loading Cas9 mRNA alone was stable even at higher dilutions. Interestingly, co-encapsulating sgRNA with Cas9 mRNA in a single PM prevented sgRNA release upon dilution, which led to the enhanced tolerability of sgRNA against enzymatic degradation. Subsequently, PM with co-encapsulated RNA widely induced genome editing in parenchymal cells in the mouse brain, including neurons, astrocytes, and microglia, following intraparenchymal injection, at higher efficiency than that by co-delivery of PMs loaded with either Cas9 mRNA or sgRNA separately. To the best of our knowledge, this is the first report demonstrating the utility of RNA-based delivery of CRISPR/Cas9 in inducing genome editing in the brain parenchymal cells. Furthermore, the efficiency of genome editing using PMs was higher than using a non-PEGylated polyplex, due to the enhanced diffusion of PMs in the brain tissue. The results reported herein demonstrate the potential of using PMs to co-encapsulate Cas9 mRNA and sgRNA for in vivo genome editing.},
}
@article {pmid33647260,
year = {2021},
author = {Barkau, CL and O'Reilly, D and Eddington, SB and Damha, MJ and Gagnon, KT},
title = {Small nucleic acids and the path to the clinic for anti-CRISPR.},
journal = {Biochemical pharmacology},
volume = {189},
number = {},
pages = {114492},
pmid = {33647260},
issn = {1873-2968},
support = {R01 GM135646/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*drug effects/physiology ; Gene Editing/*methods ; Gene Targeting/*methods ; Humans ; Nucleic Acids/*administration &amp; dosage/genetics/metabolism ; },
abstract = {CRISPR-based therapeutics have entered clinical trials but no methods to inhibit Cas enzymes have been demonstrated in a clinical setting. The ability to inhibit CRISPR-based gene editing or gene targeting drugs should be considered a critical step in establishing safety standards for many CRISPR-Cas therapeutics. Inhibitors can act as a failsafe or as an adjuvant to reduce off-target effects in patients. In this review we discuss the need for clinical inhibition of CRISPR-Cas systems and three existing inhibitor technologies: anti-CRISPR (Acr) proteins, small molecule Cas inhibitors, and small nucleic acid-based CRISPR inhibitors, CRISPR SNuBs. Due to their unique properties and the recent successes of other nucleic acid-based therapeutics, CRISPR SNuBs appear poised for clinical application in the near-term.},
}
@article {pmid33646511,
year = {2021},
author = {Huang, TK and Puchta, H},
title = {Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {529-549},
pmid = {33646511},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Plant/*genetics ; *Gene Editing ; *Genetic Techniques ; *Genome, Plant ; Homologous Recombination ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.},
}
@article {pmid33646510,
year = {2021},
author = {Buyel, JF and Stöger, E and Bortesi, L},
title = {Targeted genome editing of plants and plant cells for biomanufacturing.},
journal = {Transgenic research},
volume = {30},
number = {4},
pages = {401-426},
pmid = {33646510},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting ; Genetic Engineering/*methods ; *Genome, Plant ; Plant Breeding/*methods ; Plants/chemistry/*genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as "plant molecular farming" (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose "chassis" for PMF.},
}
@article {pmid33646289,
year = {2021},
author = {Yu, X and Sun, N and Yang, X and Zhao, Z and Su, X and Zhang, J and He, Y and Lin, Y and Ge, J and Fan, Z},
title = {Nanophthalmos-Associated MYRF Gene Mutation Causes Ciliary Zonule Defects in Mice.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {62},
number = {3},
pages = {1},
pmid = {33646289},
issn = {1552-5783},
mesh = {Animals ; Anterior Chamber/pathology ; Blotting, Western ; CRISPR-Cas Systems/genetics ; Ciliary Body/*pathology ; Disease Models, Animal ; Female ; Fibrillin-1/genetics ; Fibrillin-2/genetics ; *Frameshift Mutation ; Gene Expression Regulation/physiology ; Genotyping Techniques ; Glaucoma, Angle-Closure/*genetics ; Humans ; Hyperopia/*genetics ; Immunohistochemistry ; Ligaments/metabolism/*pathology ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Microphthalmos/*genetics ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; Transcription Factors/*genetics ; Uveal Diseases/*genetics/metabolism/pathology ; },
abstract = {PURPOSE: Patients with nanophthalmos who undergo intraocular surgery often present with abnormal ciliary zonules. In a previous study, we reported mutation in MYRF that is implicated in the pathogenesis of nanophthalmos. The aim of this study was to model the mutation in mice to explore the role of MYRF on zonule structure and its major molecular composition, including FBN1 and FBN2.<br><br>METHODS: Human MYRF nanophthalmos frameshift mutation was generated in mouse using the CRISPR-Cas9 system. PCR and Sanger sequencing were used for genotype analysis of the mice model. Anterior chamber depth (ACD) was measured using hematoxylin and eosin-stained histology samples. Morphologic analysis of ciliary zonules was carried out using silver staining and immunofluorescence. Transcript and protein expression levels of MYRF, FBN1, and FBN2 in ciliary bodies were quantified using quantitative real-time PCR (qRT-PCR) and Western blot.<br><br>RESULTS: A nanophthalmos frameshift mutation (c.789delC, p.N264fs) of MYRF in mice showed ocular phenotypes similar to those reported in patients with nanophthalmos. ACD was reduced in MYRF mutant mice (MYRFmut/+) compared with that in littermate control mice (MYRF+/+). In addition, the morphology of ciliary zonules showed reduced zonular fiber density and detectable structural dehiscence of zonular fibers. Furthermore, qRT-PCR analysis and Western blot showed a significant decrease in mRNA expression levels of MYRF, FBN1, and FBN2 in MYRFmut/+ mice.<br><br>CONCLUSIONS: Changes in the structure and major molecular composition of ciliary zonules accompanied with shallowing anterior chamber were detected in MYRFmut/+ mice. Therefore, MYRF mutant mice strain is a useful model for exploring pathogenesis of zonulopathy, which is almost elusive for basic researches due to lack of appropriate animal models.},
}
@article {pmid33645561,
year = {2021},
author = {Sieber, K and Saar, M and Opachaloemphan, C and Gallitto, M and Yang, H and Yan, H},
title = {Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {168},
pages = {},
doi = {10.3791/61930},
pmid = {33645561},
issn = {1940-087X},
mesh = {Animals ; Ants/genetics/*physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Larva/genetics/*physiology ; *Mutagenesis ; Phenotype ; *Reproduction ; *Social Behavior ; },
abstract = {The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.},
}
@article {pmid33645226,
year = {2021},
author = {Zhu, X and Wang, X and Li, S and Luo, W and Zhang, X and Wang, C and Chen, Q and Yu, S and Tai, J and Wang, Y},
title = {Rapid, Ultrasensitive, and Highly Specific Diagnosis of COVID-19 by CRISPR-Based Detection.},
journal = {ACS sensors},
volume = {6},
number = {3},
pages = {881-888},
pmid = {33645226},
issn = {2379-3694},
mesh = {*Bacterial Proteins ; Biosensing Techniques ; COVID-19/*diagnosis ; *COVID-19 Testing ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases ; Humans ; *Nucleic Acid Amplification Techniques ; *RNA, Viral ; SARS-CoV-2/*genetics ; Sensitivity and Specificity ; },
abstract = {Coronavirus Disease 2019 (COVID-19), which is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has rapidly spread leading to a global pandemic. Here, we combined multiple cross displacement amplification (MCDA) with CRISPR-Cas12a-based detection to develop a novel diagnostic test (MCCD) and applied for the diagnosis of COVID-19, called COVID-19 MCCD. The MCCD protocol conducts reverse transcription MCDA (RT-MCDA) reaction for RNA templates followed by CRISPR-Cas12a/CrRNA complex detection of predefined target sequences after which degradation of a single-strand DNA (ssDNA) molecule confirms detection of the target sequence. Two MCDA primer sets and two CrRNAs were designed targeting the opening reading frame 1a/b (ORF1ab) and nucleoprotein (N) of SARS-CoV-2. The optimal conditions include two RT-MCDA reactions at 63 °C for 35 min and a CRISPR-Cas12a/CrRNA detection reaction at 37 °C for 5 min. The COVID-19 MCCD assay can be visualized on a lateral flow biosensor (LFB) and completed within 1 h including RNA extraction (15 min), RT-MCDA reaction (35 min), CRISPR-Cas12a/CrRNA detection reaction (5 min), and reporting of result (within 2 min). The COVID-19 MCCD assay is very sensitive and detects the target gene with as low as seven copies per test and does not cross-react with non-SARS-CoV-2 templates. SARS-CoV-2 was detected in 37 of 37 COVID-19 patient samples, and nonpositive results were detected from 77 non-COVID-19 patients. Therefore, the COVID-19 MCCD assay is a useful tool for the reliable and quick diagnosis of SARS-CoV-2 infection.},
}
@article {pmid33644903,
year = {2021},
author = {Jönsson, ME and Garza, R and Sharma, Y and Petri, R and Södersten, E and Johansson, JG and Johansson, PA and Atacho, DA and Pircs, K and Madsen, S and Yudovich, D and Ramakrishnan, R and Holmberg, J and Larsson, J and Jern, P and Jakobsson, J},
title = {Activation of endogenous retroviruses during brain development causes an inflammatory response.},
journal = {The EMBO journal},
volume = {40},
number = {9},
pages = {e106423},
pmid = {33644903},
issn = {1460-2075},
mesh = {Animals ; Brain/*growth &amp; development/immunology/virology ; CRISPR-Cas Systems ; Cells, Cultured ; Encephalitis/*genetics/immunology/virology ; Endogenous Retroviruses/*genetics/immunology ; Epigenesis, Genetic ; *Gene Deletion ; Gene Expression Regulation ; Histones/metabolism ; Mice ; Transcriptional Activation ; Tripartite Motif-Containing Protein 28/*genetics ; },
abstract = {Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.},
}
@article {pmid33642503,
year = {2021},
author = {Kawasaki, H},
title = {[Investigation of the Mechanisms Underlying Development and Diseases of the Cerebral Cortex Using Mice and Ferrets].},
journal = {Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan},
volume = {141},
number = {3},
pages = {349-357},
doi = {10.1248/yakushi.20-00198-3},
pmid = {33642503},
issn = {1347-5231},
mesh = {Animals ; Brain Diseases/*etiology/genetics/pathology ; CRISPR-Cas Systems ; *Cerebral Cortex/cytology/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation ; Ferrets ; Fibroblast Growth Factors/metabolism ; Hedgehog Proteins/metabolism ; Mice ; Polymicrogyria/etiology ; Signal Transduction ; },
abstract = {Folds of the cerebral cortex, which are called gyri and sulci, are one of the most prominent features of the mammalian brain. However, the mechanisms underlying the development and malformation of cortical folds are largely unknown, mainly because they are difficult to investigate in mice, whose brain do not have cortical folds. To investigate the mechanisms underlying the development and malformation of cortical folds, we developed a genetic manipulation technique for the cerebral cortex of gyrencephalic carnivore ferrets. Genes-of-interest can be expressed in the ferret cortex rapidly and efficiently. We also demonstrated that genes-of-interest can be knocked out in the ferret cortex by combining in utero electroporation and the CRISPR/Cas9 system. Using our technique, we found that fibroblast growth factor (FGF) signaling and sonic hedgehog (Shh) signaling are crucial for cortical folding. In addition, we found that FGF signaling and Shh signaling preferentially increased outer radial glial cells and the thickness of upper layers of the cerebral cortex. Furthermore, over-activation of FGF signaling and Shh signaling resulted in polymicrogyria. Our findings provide in vivo data about the mechanisms of cortical folding in gyrencephalic mammals. Our technique for the ferret cerebral cortex should be useful for investigating the mechanisms underlying the development and diseases of the cerebral cortex that cannot be investigated using mice.},
}
@article {pmid33640690,
year = {2021},
author = {Janz, A and Zink, M and Cirnu, A and Hartleb, A and Albrecht, C and Rost, S and Klopocki, E and Günther, K and Edenhofer, F and Ergün, S and Gerull, B},
title = {CRISPR/Cas9-edited PKP2 knock-out (JMUi001-A-2) and DSG2 knock-out (JMUi001-A-3) iPSC lines as an isogenic human model system for arrhythmogenic cardiomyopathy (ACM).},
journal = {Stem cell research},
volume = {53},
number = {},
pages = {102256},
doi = {10.1016/j.scr.2021.102256},
pmid = {33640690},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Cardiomyopathies/genetics ; Desmoglein 2/genetics/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Myocytes, Cardiac/metabolism ; Plakophilins/genetics ; },
abstract = {Arrhythmogenic cardiomyopathy (ACM) is characterized by fibro-fatty replacement of the myocardium, heart failure and life-threatening ventricular arrhythmias. Causal mutations were identified in genes encoding for proteins of the desmosomes, predominantly plakophilin-2 (PKP2) and desmoglein-2 (DSG2). We generated gene-edited knock-out iPSC lines for PKP2 (JMUi001-A-2) and DSG2 (JMUi001-A-3) using the CRISPR/Cas9 system in a healthy control iPSC background (JMUi001-A). Stem cell-like morphology, robust expression of pluripotency markers, embryoid body formation and normal karyotypes confirmed the generation of high quality iPSCs to provide a novel isogenic human in vitro model system mimicking ACM when differentiated into cardiomyocytes.},
}
@article {pmid33640033,
year = {2021},
author = {Xie, Q and Cao, S and Zhang, W and Wang, W and Li, L and Kan, Q and Fu, H and Geng, T and Li, T and Wan, Z and Gao, W and Shao, H and Qin, A and Ye, J},
title = {A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus.},
journal = {Veterinary research},
volume = {52},
number = {1},
pages = {35},
pmid = {33640033},
issn = {1297-9716},
mesh = {Adenoviridae Infections/prevention &amp; control/*veterinary/virology ; Animals ; Aviadenovirus/*physiology ; CRISPR-Cas Systems ; *Chickens ; Gene Editing ; Genes, Viral ; Poultry Diseases/*prevention &amp; control/virology ; Serogroup ; Vaccines, Attenuated/administration &amp; dosage ; Viral Vaccines/*administration &amp; dosage ; },
abstract = {Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.},
}
@article {pmid33902727,
year = {2020},
author = {Bhatnagar, S and Cowley, ES and Kopf, SH and Pérez Castro, S and Kearney, S and Dawson, SC and Hanselmann, K and Ruff, SE},
title = {Microbial community dynamics and coexistence in a sulfide-driven phototrophic bloom.},
journal = {Environmental microbiome},
volume = {15},
number = {1},
pages = {3},
pmid = {33902727},
issn = {2524-6372},
abstract = {BACKGROUND: Lagoons are common along coastlines worldwide and are important for biogeochemical element cycling, coastal biodiversity, coastal erosion protection and blue carbon sequestration. These ecosystems are frequently disturbed by weather, tides, and human activities. Here, we investigated a shallow lagoon in New England. The brackish ecosystem releases hydrogen sulfide particularly upon physical disturbance, causing blooms of anoxygenic sulfur-oxidizing phototrophs. To study the habitat, microbial community structure, assembly and function we carried out in situ experiments investigating the bloom dynamics over time.<br><br>RESULTS: Phototrophic microbial mats and permanently or seasonally stratified water columns commonly contain multiple phototrophic lineages that coexist based on their light, oxygen and nutrient preferences. We describe similar coexistence patterns and ecological niches in estuarine&#xa0;planktonic blooms of phototrophs. The water column showed steep gradients of oxygen, pH, sulfate, sulfide, and salinity. The upper part of the bloom was dominated by aerobic phototrophic Cyanobacteria, the middle and lower parts by anoxygenic purple sulfur bacteria (Chromatiales) and green sulfur bacteria (Chlorobiales), respectively. We show stable coexistence of phototrophic lineages from five bacterial phyla and present metagenome-assembled genomes (MAGs) of two uncultured Chlorobaculum and Prosthecochloris species. In addition to genes involved in sulfur oxidation and photopigment biosynthesis the MAGs contained complete operons encoding for terminal oxidases. The metagenomes also contained numerous contigs affiliating with Microviridae viruses, potentially affecting Chlorobi. Our data suggest a short sulfur cycle within the bloom in which elemental sulfur produced by sulfide-oxidizing phototrophs is most likely reduced back to sulfide by Desulfuromonas sp.<br><br>CONCLUSIONS: The release of sulfide creates a habitat selecting for anoxygenic sulfur-oxidizing phototrophs, which in turn create a niche for sulfur reducers. Strong syntrophism between these guilds apparently drives a short sulfur cycle that may explain the rapid development of the bloom. The fast growth and high biomass yield of Chlorobi-affiliated organisms implies that the studied lineages of green sulfur bacteria can thrive in hypoxic habitats. This oxygen tolerance is corroborated by oxidases found in MAGs of uncultured Chlorobi. The findings improve our understanding of the ecology and ecophysiology of anoxygenic phototrophs and their impact on the coupled biogeochemical cycles of sulfur and carbon.},
}
@article {pmid33654707,
year = {2020},
author = {Wegner, M and Husnjak, K and Kaulich, M},
title = {Unbiased and Tailored CRISPR/Cas gRNA Libraries by SynthesizingCovalently-closed-circular (3Cs) DNA.},
journal = {Bio-protocol},
volume = {10},
number = {1},
pages = {e3472},
pmid = {33654707},
issn = {2331-8325},
abstract = {Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning techniques, thus coupling library diversity with distribution. Here, we provide additional technical expertise to apply our covalently-closed-circular synthesized (3Cs) gRNA library generation technology for the generation of high-quality CRISPR/Cas gRNA libraries. F1-origin of replication-containing plasmid DNA is transformed into CJ236 bacteria for single colony outgrow followed by M13KO7 bacteriophage superinfection for the production and preparation of circular dU-containing ssDNA. dU-ssDNA is annealed with homology- and gRNA-encoding DNA oligonucleotides for their T7 DNA polymerase-mediated extension to form hetero-duplexed CCC-dsDNA (3Cs-dsDNA). 3Cs-dsDNA is electroporated for the selected amplification of the newly synthesized, gRNA-containing strand. To remove wild-type plasmid remnants, the purified plasmid DNA is digested with restriction enzymes targeting the gRNA-placeholder sequence in the template DNA. Undigested plasmid is electroporated for the extraction of the final 3Cs gRNA library. Due to the absence of PCR amplification and conventional cloning steps, the 3Cs technology uncouples sequence diversity from sequence distribution, thereby generating gRNA libraries with near-uniform distribution in diversities being only limited by electroporation efficiencies.},
}
@article {pmid33654892,
year = {2019},
author = {Jia, Y and Shen, D and Wang, X and Sun, J and Peng, P and Xu, RG and Xu, B and Ni, JQ},
title = {flySAM Transgenic CRISPRa System Manual.},
journal = {Bio-protocol},
volume = {9},
number = {2},
pages = {e3147},
pmid = {33654892},
issn = {2331-8325},
abstract = {Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.},
}
@article {pmid33717282,
year = {2018},
author = {Rubeis, G and Steger, F},
title = {Risks and benefits of human germline genome editing: An ethical analysis.},
journal = {Asian bioethics review},
volume = {10},
number = {2},
pages = {133-141},
pmid = {33717282},
issn = {1793-9453},
abstract = {With the arrival of new methods of genome editing, especially CRISPR/Cas 9, new perspectives on germline interventions have arisen. Supporters of germ line genome editing (GGE) claim that the procedure could be used as a means of disease prevention. As a possible life-saving therapy, it provides benefits that outweigh its risks. Opponents of GGE claim that the medical and societal risks, especially the use of GGE for genetic enhancement, are too high. In our paper, we analyze the risks and benefits of GGE. We show that the medical risk on an individual level might be reduced by further research in the near future so that they may be outweighed by the benefits. We also show that the societal risks of the procedure, i.e. genetic enhancement, are manageable by establishing a regulative framework before the GGE is implemented. Since the effects of modifying genes for the genepool of a given population are extremely difficult to model, the medical risks on the population level might be too high.},
}
@article {pmid33639215,
year = {2021},
author = {Laudermilch, E and Chandran, K},
title = {MAVERICC: Marker-free Vaccinia Virus Engineering of Recombinants through in vitro CRISPR/Cas9 Cleavage.},
journal = {Journal of molecular biology},
volume = {433},
number = {9},
pages = {166896},
pmid = {33639215},
issn = {1089-8638},
support = {R01 AI132633/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chlorocebus aethiops ; DNA, Recombinant/*genetics ; Epitopes/genetics/immunology ; Gene Editing/*methods ; Genes, Viral/genetics ; Genetic Markers/genetics ; Genetic Vectors/genetics ; Genome, Viral/genetics ; Helper Viruses/genetics ; Membrane Fusion ; Vaccinia virus/*genetics/*metabolism ; Virion/genetics ; Virus Internalization ; },
abstract = {Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.},
}
@article {pmid33639162,
year = {2021},
author = {Yang, Y and Kang, X and Hu, S and Chen, B and Xie, Y and Song, B and Zhang, Q and Wu, H and Ou, Z and Xian, Y and Fan, Y and Li, X and Lai, L and Sun, X},
title = {CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100464},
pmid = {33639162},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Disease Models, Animal ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Genetic Engineering/methods ; Hematopoietic Stem Cells/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Rabbits ; beta-Globins/*genetics/metabolism ; beta-Thalassemia/*genetics/metabolism ; },
abstract = {β-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent β-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, β-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit β-globin gene HBB2 with high efficiency and generated a β-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of β-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a β-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of β-thalassemia and associated complications.},
}
@article {pmid33638992,
year = {2021},
author = {Miki, D and Wang, R and Li, J and Kong, D and Zhang, L and Zhu, JK},
title = {Gene Targeting Facilitated by Engineered Sequence-Specific Nucleases: Potential Applications for Crop Improvement.},
journal = {Plant &amp; cell physiology},
volume = {62},
number = {5},
pages = {752-765},
pmid = {33638992},
issn = {1471-9053},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; DNA Breaks, Double-Stranded ; Endonucleases/*genetics ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Gene Editing ; Gene Targeting/*methods ; Plants/*genetics ; Promoter Regions, Genetic ; Protein Engineering/*methods ; },
abstract = {Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining and are only rarely repaired via error-free homology-directed repair if an appropriate donor template is provided. Gene targeting (GT), i.e. the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.},
}
@article {pmid33638865,
year = {2021},
author = {Manriquez-Roman, C and Siegler, EL and Kenderian, SS},
title = {CRISPR Takes the Front Seat in CART-Cell Development.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {35},
number = {2},
pages = {113-124},
pmid = {33638865},
issn = {1179-190X},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Immunotherapy, Adoptive ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; },
abstract = {Chimeric antigen receptor T (CART)-cell immunotherapies have opened a door in the development of specialized gene therapies for hematological and solid cancers. Impressive response rates in pivotal trials led to the FDA approval of CART-cell therapy for certain hematological malignancies. However, autologous CART products are costly and time-intensive to manufacture, and most patients experience disease relapse within 1 year of CART administration. Additionally, CART-cell efficacy in solid tumors is extremely limited. CART-cell therapy is also associated with serious toxicities. Manufacturing difficulties, intrinsic T-cell defects, CART exhaustion, and treatment-associated toxicities are some of the current barriers to widespread adoption of CART-cell therapy. Genome editing tools such as CRISPR/Cas systems have demonstrated efficacy in further engineering CART cells to overcome these limitations. In this review, we will summarize the current approaches that use CRISPR to facilitate off-the-shelf CART products, increase CART-cell efficacy, and minimize CART-associated toxicities.},
}
@article {pmid33638281,
year = {2021},
author = {Raffan, S and Sparks, C and Huttly, A and Hyde, L and Martignago, D and Mead, A and Hanley, SJ and Wilkinson, PA and Barker, G and Edwards, KJ and Curtis, TY and Usher, S and Kosik, O and Halford, NG},
title = {Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.},
journal = {Plant biotechnology journal},
volume = {19},
number = {8},
pages = {1602-1613},
pmid = {33638281},
issn = {1467-7652},
support = {BB/E000126/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/E01268X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/F010370/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EGA17701/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Asparagine/metabolism ; *Aspartate-Ammonia Ligase/genetics ; CRISPR-Cas Systems/genetics ; Edible Grain/metabolism ; Gene Editing ; *Triticum/genetics/metabolism ; },
abstract = {Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.},
}
@article {pmid33638271,
year = {2021},
author = {Shi, Y and Fu, X and Yin, Y and Peng, F and Yin, X and Ke, G and Zhang, X},
title = {CRISPR-Cas12a System for Biosensing and Gene Regulation.},
journal = {Chemistry, an Asian journal},
volume = {16},
number = {8},
pages = {857-867},
doi = {10.1002/asia.202100043},
pmid = {33638271},
issn = {1861-471X},
mesh = {Bacterial Proteins/*genetics ; *Biosensing Techniques ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endodeoxyribonucleases/*genetics ; Gene Editing ; Gene Expression Regulation/genetics ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR-associated (Cas) proteins, Cas12a is an RNA-guided nuclease in the type V CRISPR-Cas system, which has been a robust tool for gene editing. In addition, due to the discovery of target-binding-induced indiscriminate single-stranded DNase activity of Cas12a, CRISPR-Cas12a also exhibits great promise in biosensing. This minireview not only gives a brief introduction to the mechanism of CRISPR-Cas12a but also highlights the recent developments and applications in biosensing and gene regulation. Finally, future prospects of the CRISPR-Cas12a system are also discussed. We expect this minireview will inspire innovative work on the CRISPR-Cas12a system by making full use of its features and advantages.},
}
@article {pmid33638256,
year = {2021},
author = {Wittayarat, M and Hirata, M and Namula, Z and Sato, Y and Nguyen, NT and Le, QA and Lin, Q and Takebayashi, K and Tanihara, F and Otoi, T},
title = {Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides.},
journal = {Animal science journal = Nihon chikusan Gakkaiho},
volume = {92},
number = {1},
pages = {e13534},
doi = {10.1111/asj.13534},
pmid = {33638256},
issn = {1740-0929},
mesh = {Animals ; Blastocyst ; *CRISPR-Cas Systems ; Electroporation/*methods ; Fertilization in Vitro/*veterinary ; Gene Editing/*methods/*veterinary ; *Oligodeoxyribonucleotides ; *Point Mutation ; Proto-Oncogene Proteins p21(ras)/*genetics ; Swine/*embryology/*genetics ; *Zygote ; },
abstract = {This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.},
}
@article {pmid33637962,
year = {2021},
author = {Gregg, JR and Thompson, TC},
title = {Considering the potential for gene-based therapy in prostate cancer.},
journal = {Nature reviews. Urology},
volume = {18},
number = {3},
pages = {170-184},
pmid = {33637962},
issn = {1759-4820},
support = {P50 CA140388/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Transfer Techniques ; *Genetic Therapy ; *Genetic Vectors ; Humans ; *Immunotherapy, Adoptive ; Male ; Prostatic Neoplasms/genetics/*therapy ; *Receptors, Chimeric Antigen ; },
abstract = {Therapeutic gene manipulation has been at the forefront of popular scientific discussion and basic and clinical research for decades. Basic and clinical research applications of CRISPR-Cas9-based technologies and ongoing clinical trials in this area have demonstrated the potential of genome editing to cure human disease. Evaluation of research and clinical trials in gene therapy reveals a concentration of activity in prostate cancer research and practice. Multiple aspects of prostate cancer care - including anatomical considerations that enable direct tumour injections and sampling, the availability of preclinical immune-competent models and the delineation of tumour-related antigens that might provide targets for an induced immune system - make gene therapy an appealing treatment option for this common malignancy. Vaccine-based therapies that induce an immune response and new technologies exploiting CRISPR-Cas9-assisted approaches, including chimeric antigen receptor (CAR) T cell therapies, are very promising and are currently under investigation both in the laboratory and in the clinic. Although laboratory and preclinical advances have, thus far, not led to oncologically relevant outcomes in the clinic, future studies offer great promise for gene therapy to become established in prostate cancer care.},
}
@article {pmid33637726,
year = {2021},
author = {Thompson, NA and Ranzani, M and van der Weyden, L and Iyer, V and Offord, V and Droop, A and Behan, F and Gonçalves, E and Speak, A and Iorio, F and Hewinson, J and Harle, V and Robertson, H and Anderson, E and Fu, B and Yang, F and Zagnoli-Vieira, G and Chapman, P and Del Castillo Velasco-Herrera, M and Garnett, MJ and Jackson, SP and Adams, DJ},
title = {Combinatorial CRISPR screen identifies fitness effects of gene paralogues.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1302},
pmid = {33637726},
issn = {2041-1723},
support = {206194/WT_/Wellcome Trust/United Kingdom ; C6/A18796/CRUK_/Cancer Research UK/United Kingdom ; 206388/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; C6946/A24843/CRUK_/Cancer Research UK/United Kingdom ; WT203144/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Apoptosis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics ; Gene Knockout Techniques ; *Genome ; Heterografts ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Proteins/*genetics ; RNA-Binding Proteins/genetics ; Transcriptome ; },
abstract = {Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.},
}
@article {pmid33637689,
year = {2021},
author = {Baxter, PS and Márkus, NM and Dando, O and He, X and Al-Mubarak, BR and Qiu, J and Hardingham, GE},
title = {Targeted de-repression of neuronal Nrf2 inhibits α-synuclein accumulation.},
journal = {Cell death &amp; disease},
volume = {12},
number = {2},
pages = {218},
pmid = {33637689},
issn = {2041-4889},
support = {/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Astrocytes/metabolism/pathology ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Death/drug effects ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coculture Techniques ; Epigenetic Repression ; Female ; *Gene Targeting ; Hydroquinones/*pharmacology ; Lewy Body Disease/genetics/metabolism/pathology/*therapy ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; NF-E2-Related Factor 2/*agonists/genetics/metabolism ; Neurons/drug effects/*metabolism/pathology ; Neuroprotective Agents/*pharmacology ; Prosencephalon/*drug effects/metabolism/pathology ; Proteostasis/drug effects ; alpha-Synuclein/genetics/*metabolism ; },
abstract = {Many neurodegenerative diseases are associated with neuronal misfolded protein accumulation, indicating a need for proteostasis-promoting strategies. Here we show that de-repressing the transcription factor Nrf2, epigenetically shut-off in early neuronal development, can prevent protein aggregate accumulation. Using a paradigm of α-synuclein accumulation and clearance, we find that the classical electrophilic Nrf2 activator tBHQ promotes endogenous Nrf2-dependent α-synuclein clearance in astrocytes, but not cortical neurons, which mount no Nrf2-dependent transcriptional response. Moreover, due to neuronal Nrf2 shut-off and consequent weak antioxidant defences, electrophilic tBHQ actually induces oxidative neurotoxicity, via Nrf2-independent Jun induction. However, we find that epigenetic de-repression of neuronal Nrf2 enables them to respond to Nrf2 activators to drive α-synuclein clearance. Moreover, activation of neuronal Nrf2 expression using gRNA-targeted dCas9-based transcriptional activation complexes is sufficient to trigger Nrf2-dependent α-synuclein clearance. Thus, targeting reversal of the developmental shut-off of Nrf2 in forebrain neurons may alter neurodegenerative disease trajectory by boosting proteostasis.},
}
@article {pmid33636398,
year = {2021},
author = {Kweon, J and Yoon, JK and Jang, AH and Shin, HR and See, JE and Jang, G and Kim, JI and Kim, Y},
title = {Engineered prime editors with PAM flexibility.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {6},
pages = {2001-2007},
pmid = {33636398},
issn = {1525-0024},
mesh = {Alleles ; Amino Acid Substitution ; Binding Sites ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Genetic Engineering/methods ; HEK293 Cells ; Humans ; Mutation ; *Nucleotide Motifs ; Proto-Oncogene Proteins B-raf/genetics ; },
abstract = {Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.},
}
@article {pmid33636132,
year = {2021},
author = {Huang, H and Zhou, P and Wei, J and Long, L and Shi, H and Dhungana, Y and Chapman, NM and Fu, G and Saravia, J and Raynor, JL and Liu, S and Palacios, G and Wang, YD and Qian, C and Yu, J and Chi, H},
title = {In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8[+] T cell fate decisions.},
journal = {Cell},
volume = {184},
number = {5},
pages = {1245-1261.e21},
pmid = {33636132},
issn = {1097-4172},
support = {R01 AI131703/AI/NIAID NIH HHS/United States ; R01 CA250533/CA/NCI NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; R01 CA176624/CA/NCI NIH HHS/United States ; R01 CA221290/CA/NCI NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; R01 AI105887/AI/NIAID NIH HHS/United States ; R01 GM134382/GM/NIGMS NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Transport Systems/metabolism ; Amino Acids/*metabolism ; Animals ; CD8-Positive T-Lymphocytes/*cytology/immunology ; CRISPR-Cas Systems ; Cell Cycle ; Cell Differentiation ; Disease Models, Animal ; Female ; Gene Knock-In Techniques ; *Immunologic Memory ; Lymphocytic Choriomeningitis/immunology ; Male ; Mice ; Mice, Transgenic ; Precursor Cells, T-Lymphoid/cytology ; *Signal Transduction ; },
abstract = {How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.},
}
@article {pmid33636129,
year = {2021},
author = {Chen, Z and Arai, E and Khan, O and Zhang, Z and Ngiow, SF and He, Y and Huang, H and Manne, S and Cao, Z and Baxter, AE and Cai, Z and Freilich, E and Ali, MA and Giles, JR and Wu, JE and Greenplate, AR and Hakeem, MA and Chen, Q and Kurachi, M and Nzingha, K and Ekshyyan, V and Mathew, D and Wen, Z and Speck, NA and Battle, A and Berger, SL and Wherry, EJ and Shi, J},
title = {In vivo CD8[+] T cell CRISPR screening reveals control by Fli1 in infection and cancer.},
journal = {Cell},
volume = {184},
number = {5},
pages = {1262-1280.e22},
pmid = {33636129},
issn = {1097-4172},
support = {P01 CA210944/CA/NCI NIH HHS/United States ; R01 HG010480/HG/NHGRI NIH HHS/United States ; T32 CA009140/CA/NCI NIH HHS/United States ; U19 AI082630/AI/NIAID NIH HHS/United States ; P01 AI112521/AI/NIAID NIH HHS/United States ; R01 AI115712/AI/NIAID NIH HHS/United States ; K00 CA234842/CA/NCI NIH HHS/United States ; P01 AI108545/AI/NIAID NIH HHS/United States ; R01 AI105343/AI/NIAID NIH HHS/United States ; R01 MH109905/MH/NIMH NIH HHS/United States ; U19 AI117950/AI/NIAID NIH HHS/United States ; R01 AI155577/AI/NIAID NIH HHS/United States ; F99 CA234842/CA/NCI NIH HHS/United States ; U19 AI149680/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CD8-Positive T-Lymphocytes/*cytology/immunology/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Chronic Disease ; Core Binding Factor Alpha 3 Subunit/metabolism ; Epigenesis, Genetic ; Gene Regulatory Networks ; Infections/immunology ; Mice ; Neoplasms/immunology ; Proto-Oncogene Protein c-fli-1/*metabolism ; },
abstract = {Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8[+] T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8[+] T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.},
}
@article {pmid33635925,
year = {2021},
author = {Bruce, JW and Bracken, M and Evans, E and Sherer, N and Ahlquist, P},
title = {ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses.},
journal = {PLoS pathogens},
volume = {17},
number = {2},
pages = {e1009364},
pmid = {33635925},
issn = {1553-7374},
support = {P01 CA022443/CA/NCI NIH HHS/United States ; R01 AI110221/AI/NIAID NIH HHS/United States ; T32 CA009135/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Acetylation ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *DNA Damage ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; HIV Infections/*genetics/metabolism/pathology/virology ; HIV-1/*genetics ; Histone Deacetylases/genetics/metabolism ; Humans ; Promoter Regions, Genetic ; Repressor Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Transcription, Genetic ; Virus Replication ; tat Gene Products, Human Immunodeficiency Virus/genetics/*metabolism ; vpr Gene Products, Human Immunodeficiency Virus/genetics/*metabolism ; },
abstract = {Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1's ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.},
}
@article {pmid33635334,
year = {2021},
author = {Pavani, G and Fabiano, A and Laurent, M and Amor, F and Cantelli, E and Chalumeau, A and Maule, G and Tachtsidi, A and Concordet, JP and Cereseto, A and Mavilio, F and Ferrari, G and Miccio, A and Amendola, M},
title = {Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells.},
journal = {Blood advances},
volume = {5},
number = {5},
pages = {1137-1153},
pmid = {33635334},
issn = {2473-9537},
mesh = {Animals ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; Humans ; Mice ; alpha-Globins/genetics ; beta-Globins/genetics ; *beta-Thalassemia/genetics/therapy ; },
abstract = {β-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. β-globin associates with α-globin to form adult hemoglobin (HbA, α2β2), the main oxygen-carrier in erythrocytes. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of β-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat β-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) β-globin expression, by targeted integration of a β-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of β-thalassemia (human erythroid progenitor cell [HUDEP-2] β0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited β-thal HSPCs and achieved correction of α/β globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/β globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for β-thal.},
}
@article {pmid33632302,
year = {2021},
author = {Macken, WL and Godwin, A and Wheway, G and Stals, K and Nazlamova, L and Ellard, S and Alfares, A and Aloraini, T and AlSubaie, L and Alfadhel, M and Alajaji, S and Wai, HA and Self, J and Douglas, AGL and Kao, AP and Guille, M and Baralle, D},
title = {Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly.},
journal = {Genome medicine},
volume = {13},
number = {1},
pages = {34},
pmid = {33632302},
issn = {1756-994X},
support = {101480Z/WT_/Wellcome Trust/United Kingdom ; BB/K019988/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; RP-2016-07-011/DH_/Department of Health/United Kingdom ; 204378/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adolescent ; *Alleles ; Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; Cataract/*genetics ; Child ; Coatomer Protein/chemistry/*genetics ; Family ; Female ; *Genetic Variation ; Humans ; Intellectual Disability/*genetics ; Male ; Microcephaly/*genetics ; Mutation, Missense/genetics ; Pedigree ; Syndrome ; Xenopus ; },
abstract = {BACKGROUND: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies".<br><br>METHODS: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (&#x3b2;-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation.<br><br>RESULTS: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between &#x3b2;-COP and &#x3b2;'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant &#x3b2;-COP, with the mutant protein being retarded in the Golgi.<br><br>CONCLUSIONS: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.},
}
@article {pmid33631677,
year = {2021},
author = {Wen, Z and Liu, D and Zhu, H and Sun, X and Xiao, Y and Lin, Z and Zhang, A and Ye, C and Gao, J},
title = {Deficiency for Lcn8 causes epididymal sperm maturation defects in mice.},
journal = {Biochemical and biophysical research communications},
volume = {548},
number = {},
pages = {7-13},
doi = {10.1016/j.bbrc.2021.02.052},
pmid = {33631677},
issn = {1090-2104},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Epididymis/*pathology ; Fertility ; Lipocalins/*metabolism ; Male ; Mice, Inbred C57BL ; Sperm Maturation/*physiology ; Spermatogenesis ; Spermatozoa ; },
abstract = {Lipocalin family members, LCN8 and LCN9, are specifically expressed in the initial segment of mouse caput epididymis. However, the biological functions of the molecules in vivo are yet to be clarified. In this study, CRISPR/Cas9 technology was used to generate Lcn8 and Lcn9 knockout mice, respectively. Lcn8[-/-] and Lcn9[-/-] male mice showed normal spermatogenesis and fertility. In the cauda epididymis of Lcn8[-/-] male mice, morphologically abnormal sperm was increased significantly, the proportion of progressive motility sperm was decreased, the proportion of immobilized sperm was elevated, and the sperm spontaneous acrosome reaction (AR) frequency was increased. Conversely, the knockout of Lcn9 did not have any effect on the ratio of morphologically abnormal sperm, sperm motility, and sperm spontaneous AR frequencies. These results demonstrated the role of LCN8 in maintaining the sperm quality in the epididymis, and suggested that the deficiency of LCN8 leads to epididymal sperm maturation defects.},
}
@article {pmid33631556,
year = {2021},
author = {Shi, J and Zheng, S and Wu, X and Peng, Z and Li, C and Wang, S and Xin, C and Xu, S and Li, J},
title = {Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome.},
journal = {Molecular immunology},
volume = {133},
number = {},
pages = {63-66},
doi = {10.1016/j.molimm.2021.01.024},
pmid = {33631556},
issn = {1872-9142},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Circoviridae Infections/*therapy/veterinary/virology ; Circovirus/*genetics ; Genes, Essential/genetics ; Genome, Viral/genetics ; Glycosylation ; Porcine Postweaning Multisystemic Wasting Syndrome/*therapy/virology ; RNA, Guide/*genetics ; Swine ; Swine Diseases/therapy/virology ; Virus Replication/genetics ; },
abstract = {Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.},
}
@article {pmid33630838,
year = {2021},
author = {Greenbaum, G and Feldman, MW and Rosenberg, NA and Kim, J},
title = {Designing gene drives to limit spillover to non-target populations.},
journal = {PLoS genetics},
volume = {17},
number = {2},
pages = {e1009278},
pmid = {33630838},
issn = {1553-7404},
support = {R01 HG005855/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Ecosystem ; Gene Drive Technology/*methods ; Gene Frequency ; Gene Targeting/*methods ; Introduced Species/statistics &amp; numerical data ; Malaria/*transmission ; Models, Genetic ; Models, Theoretical ; Rodentia ; },
abstract = {The prospect of utilizing CRISPR-based gene-drive technology for controlling populations has generated much excitement. However, the potential for spillovers of gene-drive alleles from the target population to non-target populations has raised concerns. Here, using mathematical models, we investigate the possibility of limiting spillovers to non-target populations by designing differential-targeting gene drives, in which the expected equilibrium gene-drive allele frequencies are high in the target population but low in the non-target population. We find that achieving differential targeting is possible with certain configurations of gene-drive parameters, but, in most cases, only under relatively low migration rates between populations. Under high migration, differential targeting is possible only in a narrow region of the parameter space. Because fixation of the gene drive in the non-target population could severely disrupt ecosystems, we outline possible ways to avoid this outcome. We apply our model to two potential applications of gene drives-field trials for malaria-vector gene drives and control of invasive species on islands. We discuss theoretical predictions of key requirements for differential targeting and their practical implications.},
}
@article {pmid33629465,
year = {2021},
author = {Gadalla, MR and Morrison, E and Serebryakova, MV and Han, X and Wolff, T and Freund, C and Kordyukova, L and Veit, M},
title = {NS1-mediated upregulation of ZDHHC22 acyltransferase in influenza a virus infected cells.},
journal = {Cellular microbiology},
volume = {23},
number = {6},
pages = {e13322},
doi = {10.1111/cmi.13322},
pmid = {33629465},
issn = {1462-5822},
mesh = {A549 Cells ; Acylation ; Animals ; CRISPR-Cas Systems ; Carnitine O-Palmitoyltransferase/*genetics ; Cell Line ; Dogs ; Gene Knockout Techniques ; Humans ; Influenza A virus/*physiology ; Madin Darby Canine Kidney Cells ; Membrane Proteins/*genetics ; *Up-Regulation ; Viral Nonstructural Proteins/*genetics ; Virus Replication ; },
abstract = {Influenza A viruses contain two S-acylated proteins, the ion channel M2 and the glycoprotein hemagglutinin (HA). Acylation of the latter is essential for virus replication. Here we analysed the expression of each of the 23 members of the family of ZDHHC acyltransferases in human airway cells, the site of virus replication. RT-PCR revealed that every ZDHHC acyltransferase (except ZDHHC19) is expressed in A549 and Calu cells. Interestingly, expression of one ZDHHC, ZDHHC22, is upregulated in virus-infected cells; this effect is more pronounced after infection with an avian compared to a human virus strain. The viral protein NS1 triggers ZDHHC22 expression in transfected cells, whereas recombinant viruses lacking a functional NS1 gene did not cause ZDHHC22 upregulation. CRISPR/Cas9 technology was then used to knock-out the ZDHHC22 gene in A549 cells. However, acylation of M2 and HA was not reduced, as analysed for intracellular HA and M2 and the stoichiometry of S-acylation of HA incorporated into virus particles did not change according to MALDI-TOF mass spectrometry analysis. Comparative mass spectrometry of palmitoylated proteins in wt and ΔZDHHC22 cells identified 25 potential substrates of ZDHHC22 which might be involved in virus replication.},
}
@article {pmid33629456,
year = {2021},
author = {Ordon, J and Martin, P and Erickson, JL and Ferik, F and Balcke, G and Bonas, U and Stuttmann, J},
title = {Disentangling cause and consequence: genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {4},
pages = {1008-1023},
doi = {10.1111/tpj.15215},
pmid = {33629456},
issn = {1365-313X},
mesh = {Arabidopsis/*genetics/immunology/physiology ; Arabidopsis Proteins/genetics/metabolism ; Autoimmunity/genetics ; Gene Fusion ; Genes, Reporter ; Genetic Loci ; Immunity, Innate/*genetics ; NLR Proteins/genetics/*metabolism ; Plant Diseases/*immunology ; Plant Immunity/*genetics ; Tobacco/genetics/immunology ; },
abstract = {Nucleotide-binding domain-leucine-rich repeat-type immune receptors (NLRs) protect plants against pathogenic microbes through intracellular detection of effector proteins. However, this comes at a cost, as NLRs can also induce detrimental autoimmunity in genetic interactions with foreign alleles. This may occur when independently evolved genomes are combined in inter- or intraspecific crosses, or when foreign alleles are introduced by mutagenesis or transgenesis. Most autoimmunity-inducing NLRs are encoded within highly variable NLR gene clusters with no known immune functions, which were termed autoimmune risk loci. Whether risk NLRs differ from sensor NLRs operating in natural pathogen resistance and how risk NLRs are activated in autoimmunity is unknown. Here, we analyzed the DANGEROUS MIX2 risk locus, a major autoimmunity hotspot in Arabidopsis thaliana. By gene editing and heterologous expression, we show that a single gene, DM2h, is necessary and sufficient for autoimmune induction in three independent cases of autoimmunity in accession Landsberg erecta. We focus on autoimmunity provoked by an EDS1-yellow fluorescent protein (YFP)[NLS] fusion protein to characterize DM2h functionally and determine features of EDS1-YFP[NLS] activating the immune receptor. Our data suggest that risk NLRs function in a manner reminiscent of sensor NLRs, while autoimmunity-inducing properties of EDS1-YFP[NLS] in this context are unrelated to the protein's functions as an immune regulator. We propose that autoimmunity, at least in some cases, may be caused by spurious, stochastic interactions of foreign alleles with coincidentally matching risk NLRs.},
}
@article {pmid33629345,
year = {2021},
author = {Zaami, S and Piergentili, R and Marinelli, E and Montanari Vergallo, G},
title = {Commentary - CRISPR-based techniques: Cas9, Cas13 and their applications in the era of COVID-19.},
journal = {European review for medical and pharmacological sciences},
volume = {25},
number = {3},
pages = {1752-1761},
doi = {10.26355/eurrev_202102_24886},
pmid = {33629345},
issn = {2284-0729},
mesh = {*COVID-19/therapy/virology ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Therapy ; Humans ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system enables scientists to edit diverse genome types with relative ease, with the aim - in the near future - to prevent future human beings from developing genetic diseases. The new opportunities arising from the system are broad-ranging and revolutionary, but such prospects have also been the cause for alarm throughout the international scientific community. The authors have laid out a review of the trials carried out so far in terms of genome editing, for the ultimate purpose of weighing implications and criticisms. We feel that possible valuable alternatives, such as induced pluripotent stem cells should not be overlooked.},
}
@article {pmid33628946,
year = {2021},
author = {Bogers, JFM and Berghuis, NF and Busker, RW and van Booma, A and Paauw, A and van Leeuwen, HC},
title = {Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles.},
journal = {Biology methods &amp; protocols},
volume = {6},
number = {1},
pages = {bpaa020},
pmid = {33628946},
issn = {2396-8923},
abstract = {Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specific functions of two enzymes, CRISPR-Cas12a and terminal deoxynucleotidyl transferase (TdT), we developed a DNA detection assay that generates copper nanoparticles (CuNPs) that are easily visible to the naked eye under UV-light; we named this detection assay Cas12a Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP). Upon specific target DNA recognition by Cas12a, single-stranded DNA (ssDNA) reporter oligos with blocked 3'-ends are cut into smaller ssDNA fragments, thereby generating neo 3'-hydroxyl moieties. TdT subsequently elongates these newly formed ssDNA fragments, incorporating only dTTP nucleotides, and these poly(thymine)-tails subsequently function as scaffolds for the formation of CuNPs. These CuNPs produce a bright fluorescent signal upon UV excitation, and thus, this bright orange signal indicates the presence of target DNA, which in this proof-of-concept study consisted of anthrax lethal factor plasmid DNA. CANTRIP, which combines two detection platforms consisting of CRISPR-Cas12a and fluorescent CuNPs into a single reaction, appears to be a robust, low-cost and simple diagnostic tool.},
}
@article {pmid33627728,
year = {2021},
author = {Kieu, NP and Lenman, M and Wang, ES and Petersen, BL and Andreasson, E},
title = {Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {4487},
pmid = {33627728},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; Citrus/genetics ; Disease Resistance/*genetics ; Gene Editing/methods ; Lycopersicon esculentum/genetics ; Mutation/*genetics ; Oryza/genetics ; Phenotype ; Plant Breeding/methods ; Plant Diseases/genetics ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; Solanum tuberosum/*genetics ; },
abstract = {The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.},
}
@article {pmid33627663,
year = {2021},
author = {Kolb, R and De, U and Khan, S and Luo, Y and Kim, MC and Yu, H and Wu, C and Mo, J and Zhang, X and Zhang, P and Zhang, X and Borcherding, N and Koppel, D and Fu, YX and Zheng, SG and Avram, D and Zheng, G and Zhou, D and Zhang, W},
title = {Proteolysis-targeting chimera against BCL-XL destroys tumor-infiltrating regulatory T cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1281},
pmid = {33627663},
issn = {2041-1723},
support = {F30 CA206255/CA/NCI NIH HHS/United States ; R01 CA260239/CA/NCI NIH HHS/United States ; R01 CA219836/CA/NCI NIH HHS/United States ; R01 CA242003/CA/NCI NIH HHS/United States ; R01 CA200673/CA/NCI NIH HHS/United States ; R01 AI067846/AI/NIAID NIH HHS/United States ; R01 CA203834/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Cell Survival/genetics/physiology ; Cells, Cultured ; Female ; Flow Cytometry ; Immunoblotting ; Lymphocytes, Tumor-Infiltrating/*metabolism ; Mice ; Mice, Inbred C57BL ; Proteolysis ; T-Lymphocytes, Regulatory/*metabolism ; bcl-X Protein/genetics/metabolism ; },
abstract = {Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis and, within tumors, their upregulation is common and promotes an immunosuppressive microenvironment. Therapeutic strategies that can eliminate Tregs in the tumor (i.e., therapies that do not run the risk of affecting normal tissues), are urgently needed for the development of cancer immunotherapies. Here we report our discovery of B-cell lymphoma extra-large (BCL-XL) as a potential molecular target of tumor-infiltrating (TI) Tregs. We show that pharmacological degradation of BCL-XL using a newly developed platelet-sparing BCL-XL Proteolysis-targeting chimera (PROTAC) induces the apoptosis of TI-Tregs and the activation of TI-CD8[+] T cells. Moreover, these activities result in an effective suppression of syngeneic tumor growth in immunocompetent, but not in immunodeficient or CD8[+] T cell-depleted mice. Notably, treatment with BCL-XL PROTAC does not cause detectable damage within several normal tissues or thrombocytopenia. These findings identify BCL-XL as a target in the elimination of TI-Tregs as a component of cancer immunotherapies, and that the BCL-XL-specific PROTAC has the potential to be developed as a therapeutic for cancer immunotherapy.},
}
@article {pmid33627649,
year = {2021},
author = {Mattsson, J and Ekdahl, L and Junghus, F and Ajore, R and Erlandsson, E and Niroula, A and Pertesi, M and Frendéus, B and Teige, I and Nilsson, B},
title = {Accelerating target deconvolution for therapeutic antibody candidates using highly parallelized genome editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1277},
pmid = {33627649},
issn = {2041-1723},
mesh = {Antibodies/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; },
abstract = {Therapeutic antibodies are transforming the treatment of cancer and autoimmune diseases. Today, a key challenge is finding antibodies against new targets. Phenotypic discovery promises to achieve this by enabling discovery of antibodies with therapeutic potential without specifying the molecular target a priori. Yet, deconvoluting the targets of phenotypically discovered antibodies remains a bottleneck; efficient deconvolution methods are needed for phenotypic discovery to reach its full potential. Here, we report a comprehensive investigation of a target deconvolution approach based on pooled CRISPR/Cas9. Applying this approach within three real-world phenotypic discovery programs, we rapidly deconvolute the targets of 38 of 39 test antibodies (97%), a success rate far higher than with existing approaches. Moreover, the approach scales well, requires much less work, and robustly identifies antibodies against the major histocompatibility complex. Our data establish CRISPR/Cas9 as a highly efficient target deconvolution approach, with immediate implications for the development of antibody-based drugs.},
}
@article {pmid33627635,
year = {2021},
author = {Mo, F and Duan, S and Jiang, X and Yang, X and Hou, X and Shi, W and Carlos, CJJ and Liu, A and Yin, S and Wang, W and Yao, H and Yu, Z and Tang, Z and Xie, S and Ding, Z and Zhao, X and Hammock, BD and Lu, X},
title = {Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy.},
journal = {Signal transduction and targeted therapy},
volume = {6},
number = {1},
pages = {80},
pmid = {33627635},
issn = {2059-3635},
support = {P42 ES004699/ES/NIEHS NIH HHS/United States ; R35 ES030443/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Endoglin/*immunology/therapeutic use ; Humans ; *Immunotherapy ; Immunotherapy, Adoptive/methods ; Male ; Mice ; Neoplasms/immunology/*therapy ; Receptors, Antigen, T-Cell/genetics/immunology ; Receptors, Chimeric Antigen/genetics/*immunology ; Single-Domain Antibodies/immunology/pharmacology ; T-Lymphocytes, Cytotoxic/immunology ; Xenograft Model Antitumor Assays ; },
abstract = {Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105[+] target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105[+] target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105[+] tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.},
}
@article {pmid33627493,
year = {2021},
author = {Hu, Y and Zhou, Y and Zhang, M and Ge, W and Li, Y and Yang, L and Wei, G and Han, L and Wang, H and Yu, S and Chen, Y and Wang, Y and He, X and Zhang, X and Gao, M and Yang, J and Li, X and Ren, J and Huang, H},
title = {CRISPR/Cas9-Engineered Universal CD19/CD22 Dual-Targeted CAR-T Cell Therapy for Relapsed/Refractory B-cell Acute Lymphoblastic Leukemia.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {27},
number = {10},
pages = {2764-2772},
doi = {10.1158/1078-0432.CCR-20-3863},
pmid = {33627493},
issn = {1557-3265},
mesh = {Adult ; Antigens, CD19/*immunology ; *CRISPR-Cas Systems ; Drug Resistance, Neoplasm ; Female ; Gene Editing ; *Genetic Engineering ; Humans ; *Immunotherapy, Adoptive/methods ; Male ; Middle Aged ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis/etiology/metabolism/*therapy ; Receptors, Chimeric Antigen/genetics/*immunology ; Recurrence ; Sialic Acid Binding Ig-like Lectin 2/*immunology ; T-Lymphocytes/immunology/metabolism ; Treatment Outcome ; },
abstract = {PURPOSE: Autologous chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed/refractory acute lymphoblastic leukemia (r/r ALL). However, certain characteristics of autologous CAR-T cells can delay treatment availability. Relapse caused by antigen escape after single-targeted CAR-T therapy is another issue. Therefore, we aim to develop CRISPR-edited universal off-the-shelf CD19/CD22 dual-targeted CAR-T cells as a novel therapy for r/r ALL.<br><br>PATIENTS AND METHODS: In this open-label dose-escalation phase I study, universal CD19/CD22-targeting CAR-T cells (CTA101) with a CRISPR/Cas9-disrupted TRAC region and CD52 gene to avoid host immune-mediated rejection were infused in patients with r/r ALL. Safety, efficacy, and CTA101 cellular kinetics were evaluated.<br><br>RESULTS: CRISPR/Cas9 technology mediated highly efficient, high-fidelity gene editing and production of universal CAR-T cells. No gene editing-associated genotoxicity or chromosomal translocation was observed. Six patients received CTA101 infusions at doses of 1 (3 patients) and 3 (3 patients) &#xd7; 10[6] CAR[+] T cells/kg body weight. Cytokine release syndrome occurred in all patients. No dose-limiting toxicity, GvHD, neurotoxicity, or genome editing-associated adverse events have occurred to date. The complete remission (CR) rate was 83.3% on day 28 after CTA101 infusion. With a median follow-up of 4.3 months, 3 of the 5 patients who achieved CR or CR with incomplete hematologic recovery (CR/CRi) remained minimal residual disease (MRD) negative.<br><br>CONCLUSIONS: CRISPR/Cas9-engineered universal CD19/CD22 CAR-T cells exhibited a manageable safety profile and prominent antileukemia activity. Universal dual-targeted CAR-T cell therapy may offer an alternative therapy for patients with r/r ALL.},
}
@article {pmid33627402,
year = {2021},
author = {Xie, L and Yin, Y and Benowitz, L},
title = {Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {9},
pages = {},
pmid = {33627402},
issn = {1091-6490},
support = {P30 EY012196/EY/NEI NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 NS072030/NS/NINDS NIH HHS/United States ; U54 HD090255/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chemokine CCL5/genetics/*metabolism ; Ciliary Neurotrophic Factor/*genetics/metabolism ; Ciliary Neurotrophic Factor Receptor alpha Subunit/genetics/metabolism ; Gene Editing ; Genetic Therapy ; Genetic Vectors/genetics ; *Nerve Regeneration/genetics ; Optic Nerve/*metabolism ; Optic Nerve Injuries/etiology/therapy ; Retinal Ganglion Cells/metabolism ; },
abstract = {Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.},
}
@article {pmid33627123,
year = {2021},
author = {Duan, A and Wang, H and Zhu, Y and Wang, Q and Zhang, J and Hou, Q and Xing, Y and Shi, J and Hou, J and Qin, Z and Chen, Z and Liu, Z and Yang, J},
title = {Chromatin architecture reveals cell type-specific target genes for kidney disease risk variants.},
journal = {BMC biology},
volume = {19},
number = {1},
pages = {38},
pmid = {33627123},
issn = {1741-7007},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromatin/*metabolism ; *Epigenome ; Gene Editing ; Humans ; Kidney Diseases/*genetics ; Zebrafish ; },
abstract = {BACKGROUND: Cell type-specific transcriptional programming results from the combinatorial interplay between the repertoire of active regulatory elements. Disease-associated variants disrupt such programming, leading to altered expression of downstream regulated genes and the onset of pathological states. However, due to the non-linear regulatory properties of non-coding elements such as enhancers, which can activate transcription at long distances and in a non-directional way, the identification of causal variants and their target genes remains challenging. Here, we provide a multi-omics analysis to identify regulatory elements associated with functional kidney disease variants, and downstream regulated genes.<br><br>RESULTS: In order to understand the genetic risk of kidney diseases, we generated a comprehensive dataset of the chromatin landscape of human kidney tubule cells, including transcription-centered 3D chromatin organization, histone modifications distribution and transcriptome with HiChIP, ChIP-seq and RNA-seq. We identified genome-wide functional elements and thousands of interactions between the distal elements and target genes. The results revealed that risk variants for renal tumor and chronic kidney disease were enriched in kidney tubule cells. We further pinpointed the target genes for the variants and validated two target genes by CRISPR/Cas9 genome editing techniques in zebrafish, demonstrating that SLC34A1 and MTX1 were indispensable genes to maintain kidney function.<br><br>CONCLUSIONS: Our results provide a valuable multi-omics resource on the chromatin landscape of human kidney tubule cells and establish a bioinformatic pipeline in dissecting functions of kidney disease-associated variants based on cell type-specific epigenome.},
}
@article {pmid33626581,
year = {2021},
author = {Marín-Quílez, A and García-Tuñón, I and Fernández-Infante, C and Hernández-Cano, L and Palma-Barqueros, V and Vuelta, E and Sánchez-Martín, M and González-Porras, JR and Guerrero, C and Benito, R and Rivera, J and Hernández-Rivas, JM and Bastida, JM},
title = {Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model.},
journal = {Thrombosis and haemostasis},
volume = {121},
number = {9},
pages = {1193-1205},
doi = {10.1055/s-0041-1723987},
pmid = {33626581},
issn = {2567-689X},
mesh = {Animals ; Blood Platelet Disorders/blood/*genetics ; Blood Platelets/*metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Core Binding Factor Alpha 2 Subunit/blood/*genetics ; Cytoplasmic Granules/genetics/metabolism ; Disease Models, Animal ; Gene Knock-In Techniques ; Genetic Predisposition to Disease ; Hemostasis ; Mice, Inbred C57BL ; Mice, Transgenic ; *Mutation ; Phenotype ; Platelet Activation/*genetics ; Platelet Glycoprotein GPIIb-IIIa Complex/metabolism ; Secretory Pathway ; Thrombopoiesis ; },
abstract = {RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1[WT/WT], RUNX1[WT/L43S], and RUNX1[L43S/L43S]). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1[L43S/L43S] and RUNX1[WT/L43S] mice had a significantly longer tail-bleeding time than RUNX1[WT/WT] mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1[L43S/L43S] and RUNX1[WT/L43S] displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1[L43S/L43S] and RUNX1[WT/L43S] using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1[L43S/L43S] and RUNX1[WT/L43S] suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.},
}
@article {pmid33626495,
year = {2021},
author = {Fogerty, J and Perkins, BD},
title = {Knockout of the CEP290 gene in human induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102243},
pmid = {33626495},
issn = {1876-7753},
support = {P30 EY025585/EY/NEI NIH HHS/United States ; R01 EY017037/EY/NEI NIH HHS/United States ; R01 EY030574/EY/NEI NIH HHS/United States ; },
mesh = {Antigens, Neoplasm/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics ; Cytoskeletal Proteins ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Neoplasm Proteins/genetics ; },
abstract = {CEP290 is a principal component of the primary cilium and is important for the proper function of ciliated cells. CEP290 mutations have been linked to numerous ciliopathies, with a wide range of phenotypic severities, but with poor genotype:phenotype correlation. Here we have used CRISPR/Cas9 technology to target the CEP290 gene and generate a line of induced pluripotent stem cells that lack detectable CEP290 expression, but retain a normal karyotype and differentiation potential. This line of cells will be useful for the study of disorders resulting from CEP290 mutations.},
}
@article {pmid33626494,
year = {2021},
author = {Laverde-Paz, MJ and Nuytemans, K and Wang, L and Vance, JM and Pericak-Vance, MA and Dykxhoorn, DM and Cukier, HN},
title = {Derivation of stem cell line UMi028-A-2 containing a CRISPR/Cas9 induced Alzheimer's disease risk variant p.S1038C in the TTC3 gene.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102258},
doi = {10.1016/j.scr.2021.102258},
pmid = {33626494},
issn = {1876-7753},
mesh = {*Alzheimer Disease/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Male ; Ubiquitin-Protein Ligases ; },
abstract = {The UMi028-A-2 human induced pluripotent stem cell line carries a homozygous mutation (rs377155188, C>G, p.S1038C) in the tetratricopeptide repeat domain 3 (TTC3) gene that was introduced via CRISPR/Cas9 genome editing. The line was originally derived from a neurologically normal male and has been thoroughly characterized following editing. The p.S1038C variant has been shown to potentially contribute to the risk of late onset Alzheimer's disease and is a resource to further investigate the consequences of TTC3 and this alteration in disease pathology.},
}
@article {pmid33626344,
year = {2021},
author = {Luan, J and Xiang, G and Gómez-García, PA and Tome, JM and Zhang, Z and Vermunt, MW and Zhang, H and Huang, A and Keller, CA and Giardine, BM and Zhang, Y and Lan, Y and Lis, JT and Lakadamyali, M and Hardison, RC and Blobel, GA},
title = {Distinct properties and functions of CTCF revealed by a rapidly inducible degron system.},
journal = {Cell reports},
volume = {34},
number = {8},
pages = {108783},
pmid = {33626344},
issn = {2211-1247},
support = {R01 DK054937/DK/NIDDK NIH HHS/United States ; R01 GM121613/GM/NIGMS NIH HHS/United States ; R24 DK106766/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CCCTC-Binding Factor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Chromatin/genetics/*metabolism ; Chromatin Assembly and Disassembly ; *Chromatin Immunoprecipitation Sequencing ; Erythroblasts/*metabolism ; Gene Editing ; Kinetics ; Mice ; Molecular Dynamics Simulation ; Protein Binding ; Proteolysis ; RNA Polymerase II/metabolism ; *Single Molecule Imaging ; Transcription, Genetic ; },
abstract = {CCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor implicated in a wide range of functions, including genome organization, transcription activation, and elongation. To explore the basis for CTCF functional diversity, we coupled an auxin-induced degron system with precision nuclear run-on. Unexpectedly, oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner independent of bound CTCF. Moreover, CTCF at different binding sites (CBSs) displays highly variable resistance to degradation. Motif sequence does not significantly predict degradation behavior, but location at chromatin boundaries and chromatin loop anchors, as well as co-occupancy with cohesin, are associated with delayed degradation. Single-molecule tracking experiments link chromatin residence time to CTCF degradation kinetics, which has ramifications regarding architectural CTCF functions. Our study highlights the heterogeneity of CBSs, uncovers properties specific to architecturally important CBSs, and provides insights into the basic processes of genome organization and transcription regulation.},
}
@article {pmid33626339,
year = {2021},
author = {Gonçalves, OS and Souza, FO and Bruckner, FP and Santana, MF and Alfenas-Zerbini, P},
title = {Widespread distribution of prophages signaling the potential for adaptability and pathogenicity evolution of Ralstonia solanacearum species complex.},
journal = {Genomics},
volume = {113},
number = {3},
pages = {992-1000},
doi = {10.1016/j.ygeno.2021.02.011},
pmid = {33626339},
issn = {1089-8646},
mesh = {Genome, Bacterial ; Humans ; *Prophages/genetics ; *Ralstonia solanacearum/genetics ; Virulence ; Virulence Factors/genetics ; },
abstract = {Integrated bacteriophages (prophages) can impact host cells, affecting their lifestyle, genomic diversity, and fitness. However, many basic aspects of how these organisms affect the host cell remain poorly understood. Ralstonia solanacearum is a gram-negative plant pathogenic bacterium that encompasses a great diversity of ecotypes regarded as a species complex (R. solanacearum Species Complex - RSSC). RSSC genomes have a mosaic structure containing numerous elements, signaling the potential for its evolution through horizontal gene transfer. Here, we analyzed 120 Ralstonia spp. genomes from the public database to identify prophage sequences. In total, 379 prophage-like elements were found in the chromosome and megaplasmid of Ralstonia spp. These elements encode genes related to host fitness, virulence factors, antibiotic resistance, and niche adaptation, which might contribute to RSSC adaptability. Prophage-like elements are widespread into the complex in different species and geographic origins, suggesting that the RSSC phages are ancestrally acquired. Complete prophages belonging to the families Inoviridae, Myoviridae, and Siphoviridae were found, being the members of Inoviridae the most abundant. Analysis of CRISPR-Cas spacer sequences demonstrated the presence of prophages sequences that indicate successive infection events during bacterial evolution. Besides complete prophages, we also demonstrated 14 novel putative prophages integrated into Ralstonia spp. genomes. Altogether, our results provide insights into the diversity of prophages in RSSC genomes and suggest that these elements may deeply affect the virulence and host adaptation and shaping the genomes among the strains of this important pathogen.},
}
@article {pmid33626327,
year = {2021},
author = {Turchiano, G and Andrieux, G and Klermund, J and Blattner, G and Pennucci, V and El Gaz, M and Monaco, G and Poddar, S and Mussolino, C and Cornu, TI and Boerries, M and Cathomen, T},
title = {Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq.},
journal = {Cell stem cell},
volume = {28},
number = {6},
pages = {1136-1147.e5},
doi = {10.1016/j.stem.2021.02.002},
pmid = {33626327},
issn = {1875-9777},
mesh = {*CRISPR-Cas Systems/genetics ; Chromosome Aberrations ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Stem Cells ; },
abstract = {Genome editing has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by off-target effects of programmable nucleases. Here we describe chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrations derived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptional activator-like effector nucleases (TALENs), respectively, in human hematopoietic stem cells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocations in 0%-0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harbored gross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations, such as homology-mediated translocations, that are mediated by homologous recombination and not off-target activity. CAST-Seq is a sensitive assay able to identify and quantify unintended chromosomal rearrangements in addition to the more typical mutations at off-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genome editing to enable thorough risk assessment before clinical application of gene-edited products.},
}
@article {pmid33623155,
year = {2021},
author = {Koch, L},
title = {Finding function with base editing screens.},
journal = {Nature reviews. Genetics},
volume = {22},
number = {4},
pages = {200-201},
pmid = {33623155},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
}
@article {pmid33623142,
year = {2021},
author = {Heyes, E and Schmidt, L and Manhart, G and Eder, T and Proietti, L and Grebien, F},
title = {Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML.},
journal = {Leukemia},
volume = {35},
number = {9},
pages = {2526-2538},
pmid = {33623142},
issn = {1476-5551},
support = {636855/ERC_/European Research Council/International ; },
mesh = {Animals ; Biomarkers, Tumor/*genetics ; CCAAT-Enhancer-Binding Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; *Gene Expression Regulation, Neoplastic ; Hematopoiesis ; Humans ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; Mice ; *Mutation ; Prognosis ; RNA, Small Interfering/*genetics ; RNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10-15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.},
}
@article {pmid33623024,
year = {2021},
author = {Baker, N and Catta-Preta, CMC and Neish, R and Sadlova, J and Powell, B and Alves-Ferreira, EVC and Geoghegan, V and Carnielli, JBT and Newling, K and Hughes, C and Vojtkova, B and Anand, J and Mihut, A and Walrad, PB and Wilson, LG and Pitchford, JW and Volf, P and Mottram, JC},
title = {Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1244},
pmid = {33623024},
issn = {2041-1723},
support = {MR/L00092X/1/MRC_/Medical Research Council/United Kingdom ; MR/P027989/1/MRC_/Medical Research Council/United Kingdom ; 200807/Z/16/Z//Wellcome Trust/United Kingdom ; //Wellcome Trust/United Kingdom ; 200807/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Survival ; Female ; Flagella/enzymology ; Gene Deletion ; Leishmania mexicana/*cytology/*enzymology ; Leishmaniasis/parasitology/pathology ; Mice, Inbred BALB C ; Models, Biological ; Mutation/genetics ; Protein Kinases/genetics/metabolism ; Proteome/metabolism ; Psychodidae/parasitology ; },
abstract = {Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.},
}
@article {pmid33622708,
year = {2021},
author = {Chan, W and Gottschalk, RA and Yao, Y and Pomerantz, JL and Germain, RN},
title = {Efficient Immune Cell Genome Engineering with Enhanced CRISPR Editing Tools.},
journal = {ImmunoHorizons},
volume = {5},
number = {2},
pages = {117-132},
doi = {10.4049/immunohorizons.2000082},
pmid = {33622708},
issn = {2573-7732},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Humans ; *Immune System ; Lymphocytes/metabolism ; Macrophages/metabolism ; Mice ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and-run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian cell types of interest.},
}
@article {pmid33622064,
year = {2022},
author = {Tröder, SE and Zevnik, B},
title = {History of genome editing: From meganucleases to CRISPR.},
journal = {Laboratory animals},
volume = {56},
number = {1},
pages = {60-68},
doi = {10.1177/0023677221994613},
pmid = {33622064},
issn = {1758-1117},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering ; },
abstract = {CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this ground-breaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double-strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants.},
}
@article {pmid33621144,
year = {2021},
author = {Ballios, BG and Pierce, EA and Huckfeldt, RM},
title = {Gene editing technology: Towards precision medicine in inherited retinal diseases.},
journal = {Seminars in ophthalmology},
volume = {36},
number = {4},
pages = {176-184},
doi = {10.1080/08820538.2021.1887903},
pmid = {33621144},
issn = {1744-5205},
support = {R01 EY012910/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Humans ; Precision Medicine ; *Retinal Diseases/genetics/therapy ; },
abstract = {Purpose: To review preclinical and clinical advances in gene therapy, with a focus on gene editing technologies, and application to inherited retinal disease.Methods: A narrative overview of the literature, summarizing the state-of-the-art in clinical gene therapy for inherited retinal disease, as well as the science and application of new gene editing technology.Results: The last three years has seen the first FDA approval of an in vivo gene replacement therapy for a hereditary blinding eye disease and, recently, the first clinical application of an in vivo gene editing technique. Limitations and challenges in this evolving field are highlighted, as well as new technologies developed to address the multitude of molecular mechanisms of disease.Conclusion: Genetic therapy for the treatment of inherited retinal disease is a rapidly expanding area of ophthalmology. New technologies have revolutionized the field of genome engineering and rekindled an interest in precision medicines for these conditions.},
}
@article {pmid33621063,
year = {2021},
author = {Rabiee, N and Bagherzadeh, M and Heidarian Haris, M and Ghadiri, AM and Matloubi Moghaddam, F and Fatahi, Y and Dinarvand, R and Jarahiyan, A and Ahmadi, S and Shokouhimehr, M},
title = {Polymer-Coated NH2-UiO-66 for the Codelivery of DOX/pCRISPR.},
journal = {ACS applied materials &amp; interfaces},
volume = {13},
number = {9},
pages = {10796-10811},
doi = {10.1021/acsami.1c01460},
pmid = {33621063},
issn = {1944-8252},
mesh = {Acrylic Resins/*chemistry/toxicity ; Adsorption ; *CRISPR-Cas Systems ; Doxorubicin/chemistry/*metabolism ; Drug Carriers/*chemistry/toxicity ; Drug Liberation ; Gene Transfer Techniques ; Green Chemistry Technology ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Metal-Organic Frameworks/*chemistry/toxicity ; Polyhydroxyethyl Methacrylate/*chemistry/toxicity ; Porosity ; },
abstract = {Herein, the NH2-UiO-66 metal organic framework (MOF) has been green synthesized with the assistance of high gravity to provide a suitable and safe platform for drug loading. The NH2-UiO-66 MOF was characterized using a field-emission scanning electron microscope, transmission electron microscope (TEM), X-ray diffraction, and zeta potential analysis. Doxorubicin was then encapsulated physically on the porosity of the green MOF. Two different stimulus polymers, p(HEMA) and p(NIPAM), were used as the coating agents of the MOFs. Doxorubicin was loaded onto the polymer-coated MOFs as well, and a drug payload of more than 51% was obtained, which is a record by itself. In the next step, pCRISPR was successfully tagged on the surface of the modified MOFs, and the performance of the final nanosystems were evaluated by the GFP expression. In addition, successful loadings and internalizations of doxorubicin were investigated via confocal laser scanning microscopy. Cellular images from the HeLa cell line for the UiO-66@DOX@pCRISPR and GMA-UiO-66@DOX@pCRISPR do not show any promising and successful gene transfections, with a maximum EGFP of 1.6%; however, the results for the p(HEMA)-GMA-UiO-66@DOX@pCRISPR show up to 4.3% transfection efficiency. Also, the results for the p(NIPAM)-GMA-UiO-66@DOX@pCRISPR showed up to 6.4% transfection efficiency, which is the first and superior report of a MOF-based nanocarrier for the delivery of pCRISPR. Furthermore, the MTT assay does not shown any critical cytotoxicity, which is a promising result for further biomedical applications. At the end of the study, the morphologies of all of the nanomaterials were screened after drug and gene delivery procedures and showed partial degradation of the nanomaterial. However, the cubic structure of the MOFs has been shown in TEM, and this is further proof of the stability of these green MOFs for biomedical applications.},
}
@article {pmid33620655,
year = {2021},
author = {Golz, JC and Stingl, K},
title = {Natural Competence and Horizontal Gene Transfer in Campylobacter.},
journal = {Current topics in microbiology and immunology},
volume = {431},
number = {},
pages = {265-292},
pmid = {33620655},
issn = {0070-217X},
mesh = {*Bacteriophages/genetics ; *Campylobacter/genetics ; *Campylobacter jejuni/genetics ; Drug Resistance, Microbial ; Gene Transfer, Horizontal ; Humans ; },
abstract = {Thermophilic Campylobacter, in particular Campylobacter jejuni, C. coli and C. lari are the main relevant Campylobacter species for human infections. Due to their high capacity of genetic exchange by horizontal gene transfer (HGT), rapid adaptation to changing environmental and host conditions contribute to successful spreading and persistence of these foodborne pathogens. However, extensive HGT can exert dangerous side effects for the bacterium, such as the incorporation of gene fragments leading to disturbed gene functions. Here we discuss mechanisms of HGT, notably natural transformation, conjugation and bacteriophage transduction and limiting regulatory strategies of gene transfer. In particular, we summarize the current knowledge on how the DNA macromolecule is exchanged between single cells. Mechanisms to stimulate and to limit HGT obviously coevolved and maintained an optimal balance. Chromosomal rearrangements and incorporation of harmful mutations are risk factors for survival and can result in drastic loss of fitness. In Campylobacter, the restricted recognition and preferential uptake of free DNA from relatives are mediated by a short methylated DNA pattern and not by a classical DNA uptake sequence as found in other bacteria. A class two CRISPR-Cas system is present but also other DNases and restriction-modification systems appear to be important for Campylobacter genome integrity. Several lytic and integrated bacteriophages have been identified, which contribute to genome diversity. Furthermore, we focus on the impact of gene transfer on the spread of antibiotic resistance genes (resistome) and persistence factors. We discuss remaining open questions in the HGT field, supposed to be answered in the future by current technologies like whole-genome sequencing and single-cell approaches.},
}
@article {pmid33619799,
year = {2021},
author = {Whitbread, AL and Dorn, A and Röhrig, S and Puchta, H},
title = {Different functional roles of RTR complex factors in DNA repair and meiosis in Arabidopsis and tomato.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {4},
pages = {965-977},
doi = {10.1111/tpj.15211},
pmid = {33619799},
issn = {1365-313X},
mesh = {Arabidopsis/enzymology/*genetics/physiology ; Arabidopsis Proteins/genetics/*metabolism ; Carrier Proteins/genetics/*metabolism ; DNA Repair/*genetics ; DNA Topoisomerases, Type I/genetics/metabolism ; Genomic Instability ; Homologous Recombination ; Lycopersicon esculentum/enzymology/*genetics/physiology ; Meiosis/*genetics ; Mutation ; Phenotype ; Plant Proteins/genetics/*metabolism ; RecQ Helicases/genetics/metabolism ; },
abstract = {The RTR (RecQ/Top3/Rmi1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyper-recombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: the top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom.},
}
@article {pmid33619565,
year = {2021},
author = {Wu, C and Tang, D and Cheng, J and Hu, D and Yang, Z and Ma, X and He, H and Yao, S and Fu, TM and Yu, Y and Chen, Q},
title = {Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.},
journal = {Nucleic acids research},
volume = {49},
number = {5},
pages = {2973-2984},
pmid = {33619565},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; DNA/chemistry ; Models, Molecular ; Synechocystis/*genetics ; },
abstract = {CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR-Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR-Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1-Cas2-prespacer complexes, and negative stained electron microscopy structure of the Cas4-Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1-Cas2-prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4-Cas1 and Cas1-Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.},
}
@article {pmid33619539,
year = {2021},
author = {Collins, SP and Rostain, W and Liao, C and Beisel, CL},
title = {Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.},
journal = {Nucleic acids research},
volume = {49},
number = {5},
pages = {2985-2999},
pmid = {33619539},
issn = {1362-4962},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; DNA/chemistry ; Endodeoxyribonucleases/*metabolism ; Escherichia coli/genetics ; Phosphotransferases (Alcohol Group Acceptor)/genetics ; Protein Biosynthesis ; RNA/*chemistry/metabolism ; RNA, Messenger/metabolism ; Transcription, Genetic ; },
abstract = {CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a 'trigger' RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5' end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.},
}
@article {pmid33619356,
year = {2021},
author = {Liu, L and Gallagher, J and Arevalo, ED and Chen, R and Skopelitis, T and Wu, Q and Bartlett, M and Jackson, D},
title = {Enhancing grain-yield-related traits by CRISPR-Cas9 promoter editing of maize CLE genes.},
journal = {Nature plants},
volume = {7},
number = {3},
pages = {287-294},
pmid = {33619356},
issn = {2055-0278},
mesh = {CRISPR-Cas Systems ; Edible Grain/*genetics/growth &amp; development ; Gene Editing ; *Genes, Plant ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; *Promoter Regions, Genetic ; Signal Transduction ; Zea mays/*genetics/growth &amp; development ; },
abstract = {Several yield-related traits selected during crop domestication and improvement[1,2] are associated with increases in meristem size[3], which is controlled by CLE peptide signals in the CLAVATA-WUSCHEL pathway[4-13]. Here, we engineered quantitative variation for yield-related traits in maize by making weak promoter alleles of CLE genes, and a null allele of a newly identified partially redundant compensating CLE gene, using CRISPR-Cas9 genome editing. These strategies increased multiple maize grain-yield-related traits, supporting the enormous potential for genomic editing in crop enhancement.},
}
@article {pmid33619355,
year = {2021},
author = {Chen, Q and Tian, F},
title = {Towards knowledge-driven breeding.},
journal = {Nature plants},
volume = {7},
number = {3},
pages = {242-243},
pmid = {33619355},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Phenotype ; Plant Breeding ; *Zea mays ; },
}
@article {pmid33619312,
year = {2021},
author = {Zhao, X and Jayarathna, S and Turesson, H and Fält, AS and Nestor, G and González, MN and Olsson, N and Beganovic, M and Hofvander, P and Andersson, R and Andersson, M},
title = {Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {4311},
pmid = {33619312},
issn = {2045-2322},
mesh = {1,4-alpha-Glucan Branching Enzyme/*genetics/*metabolism ; Alleles ; Amylose/chemistry/*metabolism ; Biomass ; CRISPR-Cas Systems ; Gene Editing ; Genotype ; Magnetic Resonance Spectroscopy ; *Mutagenesis ; Mutation ; Phenotype ; Plant Proteins/genetics ; Plants, Genetically Modified ; Polymerization ; Solanum tuberosum/*enzymology/*genetics ; Starch/*metabolism ; },
abstract = {DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.},
}
@article {pmid33619101,
year = {2021},
author = {Rajagopalan, KN and Chen, X and Weinberg, DN and Chen, H and Majewski, J and Allis, CD and Lu, C},
title = {Depletion of H3K36me2 recapitulates epigenomic and phenotypic changes induced by the H3.3K36M oncohistone mutation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {9},
pages = {},
pmid = {33619101},
issn = {1091-6490},
support = {F30 CA224971/CA/NCI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; T32 HL105323/HL/NHLBI NIH HHS/United States ; TL1 TR002386/TR/NCATS NIH HHS/United States ; P01 CA196539/CA/NCI NIH HHS/United States ; R35 GM138181/GM/NIGMS NIH HHS/United States ; //CIHR/Canada ; },
mesh = {Animals ; Antimetabolites, Antineoplastic/pharmacology ; CRISPR-Cas Systems ; Carcinogenesis/*genetics ; Cell Line ; Chromatin/chemistry/metabolism ; Cytarabine/pharmacology ; Decitabine/pharmacology ; *Epigenesis, Genetic ; Gene Editing ; Histone-Lysine N-Methyltransferase/deficiency/genetics ; Histones/genetics/*metabolism ; Humans ; Lysine/metabolism ; Mesenchymal Stem Cells/cytology/drug effects/metabolism ; Methylation/drug effects ; Mice ; Mutation ; Neoplasm Proteins/genetics/*metabolism ; Neoplasms/genetics/*metabolism/pathology ; Phenotype ; Protein Isoforms/genetics/metabolism ; *Protein Processing, Post-Translational ; Repressor Proteins/deficiency/genetics ; Transcriptome/drug effects ; },
abstract = {Hotspot histone H3 mutations have emerged as drivers of oncogenesis in cancers of multiple lineages. Specifically, H3 lysine 36 to methionine (H3K36M) mutations are recurrently identified in chondroblastomas, undifferentiated sarcomas, and head and neck cancers. While the mutation reduces global levels of both H3K36 dimethylation (H3K36me2) and trimethylation (H3K36me3) by dominantly inhibiting their respective specific methyltransferases, the relative contribution of these methylation states to the chromatin and phenotypic changes associated with H3K36M remains unclear. Here, we specifically deplete H3K36me2 or H3K36me3 in mesenchymal cells, using CRISPR-Cas9 to separately knock out the corresponding methyltransferases NSD1/2 or SETD2. By profiling and comparing the epigenomic and transcriptomic landscapes of these cells with cells expressing the H3.3K36M oncohistone, we find that the loss of H3K36me2 could largely recapitulate H3.3K36M's effect on redistribution of H3K27 trimethylation (H3K27me3) and gene expression. Consistently, knockout of Nsd1/2, but not Setd2, phenocopies the differentiation blockade and hypersensitivity to the DNA-hypomethylating agent induced by H3K36M. Together, our results support a functional divergence between H3K36me2 and H3K36me3 and their nonredundant roles in H3K36M-driven oncogenesis.},
}
@article {pmid33617832,
year = {2021},
author = {Spoelstra, WK and Jacques, JM and Gonzalez-Linares, R and Nobrega, FL and Haagsma, AC and Dogterom, M and Meijer, DH and Idema, T and Brouns, SJJ and Reese, L},
title = {CRISPR-based DNA and RNA detection with liquid-liquid phase separation.},
journal = {Biophysical journal},
volume = {120},
number = {7},
pages = {1198-1209},
pmid = {33617832},
issn = {1542-0086},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Endonucleases ; *RNA/genetics ; },
abstract = {The ability to detect specific nucleic acid sequences allows for a wide range of applications such as the identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence, both enzymes activate indiscriminate nucleic acid cleavage, which has been exploited for sequence-specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We furthermore demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction that hints towards possible improvements of an LLPS-based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.},
}
@article {pmid33617628,
year = {2021},
author = {Ishikawa, K and Soejima, S and Masuda, F and Saitoh, S},
title = {Implementation of dCas9-mediated CRISPRi in the fission yeast Schizosaccharomyces pombe.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {4},
pages = {},
pmid = {33617628},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Expression ; Plasmids ; RNA, Guide/genetics ; *Schizosaccharomyces/genetics ; },
abstract = {Controllable and reversible transcriptional repression is an essential method to study gene functions. A systematic knock-down method using catalytically inactive Cas9 (dCas9) was originally established in bacteria. dCas9 forms a ribonucleoprotein with a small guide RNA and uses it to recognize a specific DNA sequence via Watson-Crick base-pairing. When specifically bound to a targeted DNA, dCas9 impairs RNA polymerase activity and represses transcription of that target gene. This technology, CRISPRi, has been implemented in several organisms, but not in Schizosaccharomyces pombe using dCas9. Here, we provide a plasmid that expresses dCas9 and sgRNA in fission yeast. With this plasmid, CRISPRi repressed endogenous gene transcription by as much as 87%. This transcriptional repression method is controllable, reversible, and efficient enough to alter cellular phenotypes. Here, we offer a CRISPRi method to choose proper targeting sequences for transcriptional repression in fission yeast. Implementation of CRISPRi will help to reveal gene functions and to develop tools based on dCas9 technology in S. pombe.},
}
@article {pmid33616448,
year = {2021},
author = {Lin, D and Scheller, SH and Robinson, MM and Izadpanah, R and Alt, EU and Braun, SE},
title = {Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {92-103},
pmid = {33616448},
issn = {2573-1602},
support = {P51 OD011104/OD/NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; HEK293 Cells ; HIV Infections/*therapy/virology ; HIV-1/genetics ; Hematopoietic Stem Cells ; Humans ; Lentivirus ; *Mutation ; RNA, Guide/*genetics ; Receptors, CCR5/*genetics ; Sequence Analysis, DNA ; Virus Replication ; },
abstract = {CCR5 is a coreceptor of human immunodeficiency virus type 1 (HIV-1). Transplantation of hematopoietic stem cells homozygous for a 32-bp deletion in CCR5 resulted in a loss of detectable HIV-1 in two patients, suggesting that genetic strategies to knockout CCR5 expression would be a promising gene therapy approach for HIV-1-infected patients. In this study, we targeted CCR5 by CRISPR-Cas9 with a single-guide (sgRNA) and observed 35% indel frequency. When we expressed hCas9 and two gRNAs, the Surveyor assay showed that Cas9-mediated cleavage was increased by 10% with two sgRNAs. Genotype analysis on individual clones showed 11 of 13 carried biallelic mutations, where 4 clones had frameshift (FS) mutations. Taken together, these results indicate that the efficiency of biallelic FS mutations and the knockout of the CCR5 necessary to prevent viral replication were significantly increased with two sgRNAs. These studies demonstrate the knockout of CCR5 and the potential for translational development.},
}
@article {pmid33616447,
year = {2021},
author = {Navarro-Serna, S and Hachem, A and Canha-Gouveia, A and Hanbashi, A and Garrappa, G and Lopes, JS and París-Oller, E and Sarrías-Gil, L and Flores-Flores, C and Bassett, A and Sánchez, R and Bermejo-Álvarez, P and Matás, C and Romar, R and Parrington, J and Gadea, J},
title = {Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {132-146},
doi = {10.1089/crispr.2020.0078},
pmid = {33616447},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems ; Calcium Channels/genetics ; Embryo Transfer ; Embryo, Mammalian ; Female ; Fertilization ; Fetus ; Gene Knockout Techniques/*methods ; Germ Cells ; *Insemination ; Karyotype ; Male ; Mice ; Mice, Knockout ; Microinjections/*methods ; Models, Animal ; Mosaicism ; Mutation ; *Oocytes ; Phenotype ; RNA, Guide ; Swine/*genetics ; Zygote ; },
abstract = {Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.},
}
@article {pmid33616446,
year = {2021},
author = {Urnov, FD},
title = {The Cas9 Hammer-and Sickle: A Challenge for Genome Editors.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {6-13},
doi = {10.1089/crispr.2021.29120.fur},
pmid = {33616446},
issn = {2573-1602},
mesh = {Anemia, Sickle Cell/*genetics/*therapy ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Gene Editing/*methods ; Genetic Therapy ; Genome-Wide Association Study ; Hemoglobinopathies/genetics ; Humans ; Mutation ; United States ; United States Food and Drug Administration ; beta-Thalassemia/genetics/therapy ; },
abstract = {Genome editing using CRISPR-Cas9 has produced a functional cure for a small number of patients with sickle cell disease and beta-thalassemia. Rather than repairing the causative mutation, this striking outcome was attained by the knockout of a lineage-specific regulatory element for a gene, BCL11A, that controls fetal hemoglobin levels: a first example of clinical success in targeting a locus initially identified in a genome-wide association study, and formal proof of the "in the age of CRISPR, the entire genome is a druggable target" notion. This remarkable development, along with advancement to the clinic of several additional editing-based approaches to the hemoglobinopathies, highlights a sense of urgency in accelerating scientific, regulatory, and public health innovation that will allow broad and equitable access to editing-based cures.},
}
@article {pmid33616445,
year = {2021},
author = {Collantes, JC and Tan, VM and Xu, H and Ruiz-Urigüen, M and Alasadi, A and Guo, J and Tao, H and Su, C and Tyc, KM and Selmi, T and Lambourne, JJ and Harbottle, JA and Stombaugh, J and Xing, J and Wiggins, CM and Jin, S},
title = {Development and Characterization of a Modular CRISPR and RNA Aptamer Mediated Base Editing System.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {58-68},
pmid = {33616445},
issn = {2573-1602},
support = {R21 AA027050/AA/NIAAA NIH HHS/United States ; R21 CA216604/CA/NCI NIH HHS/United States ; T32 GM135141/GM/NIGMS NIH HHS/United States ; T32 GM008339/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Aptamers, Nucleotide ; Bacteria/genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; Humans ; INDEL Mutation ; *RNA Editing ; RNA, Guide/genetics ; Recombinational DNA Repair ; Whole Exome Sequencing ; },
abstract = {Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point[TM] that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.},
}
@article {pmid33616444,
year = {2021},
author = {Boob, A and Zhao, H},
title = {Can Deep Learning Solve the Cas9 Dilemma?.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {13-15},
doi = {10.1089/crispr.2020.29117.hzh},
pmid = {33616444},
issn = {2573-1602},
mesh = {Algorithms ; *CRISPR-Cas Systems ; Computational Biology ; *Deep Learning ; },
}
@article {pmid33616442,
year = {2021},
author = {Martella, A and Fisher, DI},
title = {Regulation of Gene Expression and the Elucidative Role of CRISPR-Based Epigenetic Modifiers and CRISPR-Induced Chromosome Conformational Changes.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {43-57},
doi = {10.1089/crispr.2020.0108},
pmid = {33616442},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Chromatin ; Chromosomes/*chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Demethylation ; DNA Methylation ; Epigenesis, Genetic ; Epigenomics/*methods ; Gene Editing/methods ; *Gene Expression Regulation ; Genome ; Histone Code ; Humans ; Protein Processing, Post-Translational ; },
abstract = {In complex multicellular systems, gene expression is regulated at multiple stages through interconnected complex molecular pathways and regulatory networks. Transcription is the first step in gene expression and is subject to multiple layers of regulation in which epigenetic mechanisms such as DNA methylation, histone tail modifications, and chromosomal conformation play an essential role. In recent years, CRISPR-Cas9 systems have been employed to unearth this complexity and provide new insights on the contribution of chromatin dysregulation in the development of genetic diseases, as well as new tools to prevent or reverse this dysregulation. In this review, we outline the recent development of a variety of CRISPR-based epigenetic editors for targeted DNA methylation/demethylation, histone modification, and three-dimensional DNA conformational change, highlighting their relative performance and impact on gene regulation. Finally, we provide insights on the future developments aimed to accelerate our understanding of the causal relationship between epigenetic marks, genome organization, and gene regulation.},
}
@article {pmid33616441,
year = {2021},
author = {Borges, AL},
title = {The Art of Being Single.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {16-17},
doi = {10.1089/crispr.2021.29118.abo},
pmid = {33616441},
issn = {2573-1602},
mesh = {Bacteria/*genetics ; Bacterial Physiological Phenomena/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA ; },
}
@article {pmid33616439,
year = {2021},
author = {Bloemberg, D and Sosa-Miranda, CD and Nguyen, T and Weeratna, RD and McComb, S},
title = {Self-Cutting and Integrating CRISPR Plasmids Enable Targeted Genomic Integration of Genetic Payloads for Rapid Cell Engineering.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {104-119},
doi = {10.1089/crispr.2020.0090},
pmid = {33616439},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Engineering/*methods ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Knockout Techniques ; *Genome ; Humans ; Mice ; *Plasmids ; Recombinational DNA Repair ; Transfection ; Transgenes ; },
abstract = {Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited insertion or deletion formation. Interestingly, including longer homology arms (HAs; 500 bp) in varying orientations only modestly increased knock-in efficiency (by around twofold). Using a SCIP-payload design (SCIPpay) that liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs of concept, we used SCIPpay to insert (1) a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T-cell activation, and (2) a chimeric antigen receptor gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines, and we suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.},
}
@article {pmid33616121,
year = {2021},
author = {Mukherjee, M and Caroll, E and Wang, ZQ},
title = {Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {168},
pages = {},
doi = {10.3791/61993},
pmid = {33616121},
issn = {1940-087X},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular/*methods ; DNA Primers/metabolism ; DNA Replication/genetics ; Escherichia coli/genetics ; Gene Expression ; *Genes ; *Genetic Engineering ; Genetic Vectors/genetics ; Plasmids/genetics ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/methods ; Transcription, Genetic ; },
abstract = {The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.},
}
@article {pmid33616113,
year = {2021},
author = {Sun, R and Brogan, D and Buchman, A and Yang, T and Akbari, OS},
title = {Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {168},
pages = {},
doi = {10.3791/62154},
pmid = {33616113},
issn = {1940-087X},
support = {R21 AI149161/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila melanogaster/*genetics/growth &amp; development ; Female ; Gene Editing/*methods ; Male ; Organ Specificity ; RNA/*genetics ; *RNA Interference ; *Transcriptome ; },
abstract = {CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.},
}
@article {pmid33615649,
year = {2021},
author = {Germani, F and Wäscher, S and Biller-Andorno, N},
title = {A CRISPR response to pandemics?: Exploring the ethics of genetically engineering the human immune system.},
journal = {EMBO reports},
volume = {22},
number = {3},
pages = {e52319},
pmid = {33615649},
issn = {1469-3178},
support = {/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Immune System ; *Pandemics ; },
abstract = {Ethical challenges should be addressed before gene editing is made available to improve the immune response against emerging viruses.},
}
@article {pmid33615437,
year = {2021},
author = {Lin, W and Tian, T and Jiang, Y and Xiong, E and Zhu, D and Zhou, X},
title = {A CRISPR/Cas9 eraser strategy for contamination-free PCR end-point detection.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {5},
pages = {2053-2066},
pmid = {33615437},
issn = {1097-0290},
mesh = {African Swine Fever Virus/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Humans ; Polymerase Chain Reaction/*methods ; RNA, Guide ; RNA, Viral/isolation &amp; purification ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; SARS-CoV-2/isolation &amp; purification ; Swine ; },
abstract = {Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.},
}
@article {pmid33614630,
year = {2020},
author = {Tong, B and Dong, H and Cui, Y and Jiang, P and Jin, Z and Zhang, D},
title = {The Versatile Type V CRISPR Effectors and Their Application Prospects.},
journal = {Frontiers in cell and developmental biology},
volume = {8},
number = {},
pages = {622103},
pmid = {33614630},
issn = {2296-634X},
abstract = {The class II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR-Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.},
}
@article {pmid33612827,
year = {2021},
author = {Torregrosa, T and Lehman, S and Hana, S and Marsh, G and Xu, S and Koszka, K and Mastrangelo, N and McCampbell, A and Henderson, CE and Lo, SC},
title = {Use of CRISPR/Cas9-mediated disruption of CNS cell type genes to profile transduction of AAV by neonatal intracerebroventricular delivery in mice.},
journal = {Gene therapy},
volume = {28},
number = {7-8},
pages = {456-468},
pmid = {33612827},
issn = {1476-5462},
mesh = {Animals ; CRISPR-Cas Systems ; Central Nervous System ; *Dependovirus/genetics ; Gene Transfer Techniques ; *Genetic Vectors/genetics ; Mice ; Neurons ; Transduction, Genetic ; },
abstract = {Adeno-associated virus (AAV) transduction efficiency and tropism are conventionally determined by high expression of a fluorescent reporter gene. Emerging data has suggested that such conventional methods may underestimate AAV transduction for cells in which reporter expression from AAV vectors is undetectable. To explore an alternative method that captures AAV transduction in cells in which low expression of a cargo is sufficient for the intended activity, we sought after CRISPR/Cas9-mediated gene disruption. In this study, we use AAV to deliver CRISPR/guide RNA designed to abolish the genes NeuN, GFAP, or MOG expressed specifically in neurons, astrocytes, or oligodendrocytes respectively in the central nervous system (CNS) of mice. Abrogated expression of these cell-type-specific genes can be measured biochemically in CNS subregions and provides quantitative assessment of AAV transduction in these CNS cell types. By using this method, we compared CNS transduction of AAV9, AAV-PHP.B, and AAV-PHP.eB delivered via intracerebroventricular injection (ICV) in neonatal mice. We found both AAV-PHP.B and AAV-PHP.eB resulted in marked disruption of the NeuN gene by CRISPR/Cas9, significantly greater than AAV9 in several brain regions and spinal cord. In contrast, only modest disruption of the GFAP gene and the MOG gene was observed by all three AAV variants. Since the procedure of ICV circumvents the blood-brain barrier, our data suggests that, independent of their ability to cross the blood-brain barrier, AAV-PHP.B variants also exhibit remarkably improved neuronal transduction in the CNS. We anticipate this approach will facilitate profiling of AAV cellular tropism in murine CNS.},
}
@article {pmid33612802,
year = {2020},
author = {De Silva, JR and Ching, XT and Lau, YL},
title = {Investigative study on the role of the Toxo 5699 gene in the Toxoplasma gondii lytic cycle using the CRISPR/Cas9 system.},
journal = {Tropical biomedicine},
volume = {37},
number = {2},
pages = {324-332},
pmid = {33612802},
issn = {2521-9855},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Line ; *Genes, Protozoan ; Humans ; Plasmids ; RNA, Guide/genetics ; Toxoplasma/*genetics/*pathogenicity ; },
abstract = {The focus of the current study was to disrupt the Toxo 5699 gene via CRISPR/Cas9 to evaluate the effects of gene disruption on the parasite lytic cycle. In the present work, a single plasmid expressing both the guide RNA and Cas9 nuclease together with a selectable marker of human dihydrofolate reductase (DHFR) was introduced into Toxoplasma gondii. Targeted disruption of the Toxo 5699 gene was carried out via the CRISPR/Cas9 system and confirmed by PCR, sequencing, and immunofluorescence microscopy. Disrupted and nondisrupted control parasites were allowed to invade HS27 cell monolayers and plaques were counted. The average number of plaques from three replicates per group was obtained between the disrupted and non-disrupted T. gondii RH strain and was compared using a onetailed t-test. It was observed that there was a significant decrease in number and size of plaque formation in the Toxo 5699 gene disrupted parasite line. This is an indication that the Toxo 5699 gene may play a role in the lytic cycle of the parasite, particularly during the replication phase and thus would be a novel target for disruption or silencing. The Toxo 5699 gene presented in the current work is an important part of the T. gondii lytic cycle, therefore meriting further inquiry into its potential as a target for further genetic-silencing or disruption studies.},
}
@article {pmid33612456,
year = {2020},
author = {Li, X and Hu, T and Liu, J and Fang, B and Geng, X and Xiong, Q and Zhang, L and Jin, Y and Liu, X and Li, L and Wang, Y and Li, R and Bai, X and Yang, H and Dai, Y},
title = {A Bama miniature pig model of monoallelic TSC1 mutation for human tuberous sclerosis complex.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {12},
pages = {735-742},
doi = {10.1016/j.jgg.2020.11.005},
pmid = {33612456},
issn = {1673-8527},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Heart Neoplasms/complications/*genetics/pathology ; Humans ; Mice ; Mutation/genetics ; Nuclear Transfer Techniques ; Rhabdomyoma/complications/*genetics/pathology ; Ribosomal Protein S6/*genetics ; Swine/genetics ; Swine, Miniature/genetics ; TOR Serine-Threonine Kinases/genetics ; Tuberous Sclerosis/complications/*genetics/pathology ; Tuberous Sclerosis Complex 1 Protein/*genetics ; Tuberous Sclerosis Complex 2 Protein/genetics ; },
abstract = {Tuberous sclerosis complex (TSC) is a dominant genetic neurocutaneous syndrome characterized by multiple organ hamartomas. Although rodent models bearing a germline mutation in either TSC1 or TSC2 gene have been generated, they do not develop pathogenic lesions matching those seen in patients with TSC because of the significant differences between mice and humans, highlighting the need for an improved large animal model of TSC. Here, we successfully generate monoallelic TSC1-modified Bama miniature pigs using the CRISPR/Cas9 system along with somatic cell nuclear transfer (SCNT) technology. The expression of phosphorylated target ribosomal protein S6 is significantly enhanced in the piglets, indicating that disruption of a TSC1 allele activate the mechanistic target of rapamycin (mTOR) signaling pathway. Notably, differing from the mouse TSC models reported previously, the TSC1[+/-] Bama miniature pig developed cardiac rhabdomyoma and subependymal nodules, resembling the major clinical features that occur in patients with TSC. These TSC1[+/-] Bama miniature pigs could serve as valuable large animal models for further elucidation of the pathogenesis of TSC and the development of therapeutic strategies for TSC disease.},
}
@article {pmid33612423,
year = {2021},
author = {Yoshioka, I and Kirimura, K},
title = {Rapid and marker-free gene replacement in citric acid-producing Aspergillus tubingensis (A. niger) WU-2223L by the CRISPR/Cas9 system-based genome editing technique using DNA fragments encoding sgRNAs.},
journal = {Journal of bioscience and bioengineering},
volume = {131},
number = {6},
pages = {579-588},
doi = {10.1016/j.jbiosc.2021.01.011},
pmid = {33612423},
issn = {1347-4421},
mesh = {*Aspergillus/genetics/metabolism ; Aspergillus niger/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Citric Acid/*metabolism ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Bacterial/chemistry/genetics ; Gene Editing/*methods ; Metabolic Engineering/methods ; Organisms, Genetically Modified ; RNA, Guide/*genetics ; },
abstract = {Strains belonging to Aspergillus section Nigri, including Aspergillus niger, are used for industrial production of citric acid from carbohydrates such as molasses and starch. The objective of this study was to construct the genome editing system that could enable rapid and efficient gene replacement in citric acid-producing fungi for genetic breeding. Using the citric acid-hyperproducer A. tubingensis (formerly A. niger) WU-2223L as a model strain, we developed a CRISPR/Cas9 system-based genome editing technique involving co-transformation of Cas9 and the DNA fragment encoding single guide RNA (sgRNA). Using this system, ATP-sulfurylase gene (sC) knock-out strain derived from WU-2223L was generated; the knock-out efficiency was 29 transformants when 5 μg Cas9 was added to 5 × 10[5] protoplasts. In the gene replacement method based on this system, a DNA fragment encoding sgRNAs that target both the gene of interest and marker gene was used, and replacement of nitrate reductase gene (niaD) using sC gene as a marker gene was attempted. More than 90% of the sC-knock-out transformants exhibited replaced niaD, indicating efficient gene replacement. Moreover, one-step marker rescue of the sC marker gene was accomplished by excising the knock-in donor via intramolecular homologous recombination, enabling marker-free genome editing and drastically shortening the gene replacement period by circumventing the transformation procedure to recover the sC gene. Thus, we succeeded in constructing a CRISPR/Cas9 system-based rapid and marker-free gene replacement system for the citric acid-hyperproducer strain WU-2223L.},
}
@article {pmid33612399,
year = {2021},
author = {Ma, W and Xu, YS and Sun, XM and Huang, H},
title = {Transposon-Associated CRISPR-Cas System: A Powerful DNA Insertion Tool.},
journal = {Trends in microbiology},
volume = {29},
number = {7},
pages = {565-568},
doi = {10.1016/j.tim.2021.01.017},
pmid = {33612399},
issn = {1878-4380},
mesh = {*CRISPR-Cas Systems ; *DNA Transposable Elements ; Gene Editing/*methods ; *Genome ; Humans ; },
abstract = {The transposon-associated CRISPR-Cas system is emerging as a powerful tool for targeted genetic manipulation. Using this system, targeted genomic knockins and knockouts can be achieved, independently of the host-cell repair machinery, thereby significantly improving our ability to perform genome editing in a wider variety of cell types.},
}
@article {pmid33611045,
year = {2021},
author = {Huang, Y and Wu, H and Han, X and Wu, J and Yu, M and Zhao, ZA and Shen, Z and Hu, S and Lei, W},
title = {Generation of an EFNB2-2A-mCherry reporter human embryonic stem cell line using CRISPR/Cas9-mediated site-specific homologous recombination.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102241},
doi = {10.1016/j.scr.2021.102241},
pmid = {33611045},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Endothelial Cells ; *Ephrin-B2 ; Homologous Recombination ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {Ephrin B2 (EFNB2) is the first identified and most widely used marker for arterial endothelial cells (AECs). We generated a heterozygous EFNB2-2A-mCherry reporter H1 cell line, H1-EFNB2-2A-mCherry[+/-] (WAe001-A-57), by CRISPR/Cas9-mediated insertion of 2A-mCherry cassette into the EFNB2 gene locus, immediately before the translation stop codon. The H1-EFNB2-2A-mCherry reporter cells were pluripotent and could differentiate into all three germ layer lineages. Simultaneous expression of mCherry was observed when expression of EFNB2 was increased during endothelial cell differentiation. Thus, the generated reporter cells enable live identification of EFNB2-positive AECs, and screening of small molecule compound and target genes that promote AEC differentiation.},
}
@article {pmid33611044,
year = {2021},
author = {Sims, CHC and Autio, MI and Buskin, A and Cheung, C and Heer, R and Foo, RSY and Wang, X},
title = {Modified CRISPR/Cas9 mediated generation of two MKK7 knockout human embryonic stem cell lines.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102238},
doi = {10.1016/j.scr.2021.102238},
pmid = {33611044},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {Two cell lines were generated by CRISPR/Cas9 mediated knockout of MKK7 (MAP2K7) by removal of exon 1 or exons 4 through 7. These knockouts were confirmed at the transcript and protein levels. These hESCs are pluripotent and maintain tri-lineage differentiation capacity. These cell lines are a useful resource for studying MKK7 function in humans.},
}
@article {pmid33611043,
year = {2021},
author = {Hong, W and Yun, W and Choi, W and Son, D and Song, G and You, S},
title = {Generation of a WA14 hESC sub-line carrying a hemizygous ABCD1 (C.1696_1710 del) mutation introduced by CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102244},
doi = {10.1016/j.scr.2021.102244},
pmid = {33611043},
issn = {1876-7753},
mesh = {ATP Binding Cassette Transporter, Subfamily D, Member 1 ; *Adrenoleukodystrophy/genetics ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells ; Humans ; Mutation/genetics ; Technology ; },
abstract = {ATP-binding cassette transporter subfamily D member 1 (ABCD1) gene is a member of ABC transporter super family, which conduct peroxisomal import of very long chain fatty acid and crucial underlying factor that induces X-linked adrenoleukodystrophy (X-ALD) when the gene is defected. Here, we report the generation of a human embryonic stem cell sub-line harboring a hemizygous ABCD1 mutation (C.1696_1710 del) using CRISPR/Cas9 system. Established line expresses pluripotency marker genes, can be differentiated to three germ layers, and maintains a normal karyotype.},
}
@article {pmid33610762,
year = {2021},
author = {Chen, JX and Li, WX and Lyu, J and Hu, YT and Huang, G and Zhang, WQ},
title = {CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae).},
journal = {Comparative biochemistry and physiology. Part A, Molecular &amp; integrative physiology},
volume = {256},
number = {},
pages = {110921},
doi = {10.1016/j.cbpa.2021.110921},
pmid = {33610762},
issn = {1531-4332},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Fertility ; Gene Expression Regulation, Developmental ; *Gene Knockdown Techniques ; Hemiptera/*physiology ; Mutagenesis ; Phylogeny ; Pigmentation/*genetics ; RNA Interference ; },
abstract = {In insects, cuticular pigmentation genes have been exploited as potential visible markers for constructing genetic manipulation systems. Here, we cloned cysteine sulfinic acid decarboxylase (CSAD), an orthologue of melanin metabolism pathway genes, and performed RNAi experiments in the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae). The results showed that a decrease in the level of transcription of NlCSAD increased melanin deposition in the body compared to the control group, resulting in darker cuticle pigmentation. Female adults treated with dsNlCSAD and mated with wild-type males laid significantly fewer eggs than the dsGFP-treated group, and lower hatchability of the eggs was also observed. In addition, two melanic mutant N. lugens strains (NlCSAD[-/+] and NlCSAD[-/-]) constructed by the CRISPR/Cas9 genome editing system showed darker cuticular melanisation and a reduced oviposition and hatching rate, but the homozygotes had a darker body colour, fewer eggs and lower hatchability than heterozygotes or individuals after RNAi. Thus, we have provided the first evidence that NlCSAD is required for normal body pigmentation in adults and has a role in the fecundity of females and hatchability of eggs in N. lugens via a combination of RNAi and knockout of target genes based on the CRISPR/Cas9 genome editing system. Our results suggest that NlCSAD is a candidate visual reference gene for genetic manipulation of this important crop pest.},
}
@article {pmid33610547,
year = {2021},
author = {Jin, X and Zhang, Z and Nie, Z and Wang, C and Meng, F and Yi, Q and Chen, M and Sun, J and Zou, J and Jiang, P and Guan, MX},
title = {An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100437},
pmid = {33610547},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Electron Transport Complex I/genetics/metabolism ; Electron Transport Complex IV/genetics/metabolism ; Gene Knockout Techniques ; HeLa Cells ; Humans ; *Mitochondrial Proteins/genetics/metabolism ; *Oxidative Phosphorylation ; Retina/*enzymology ; Tyrosine-tRNA Ligase/*deficiency/metabolism ; Zebrafish/genetics/*metabolism ; *Zebrafish Proteins/genetics/metabolism ; },
abstract = {Mitochondria maintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNA synthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2 mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNA[Tyr] aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system (OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitation assays showed that YARS2 bound to specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2 cDNA in the YARS2[KO] cells. In zebrafish, the yars2[KO] larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2[KO] zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments.},
}
@article {pmid33610018,
year = {2021},
author = {Fontaine, V and Duboscq-Bidot, L and Jouve, C and Hamlin, M and Curjol, A and Briand, V and Janiak, P and Hulot, JS and Pruniaux-Harnist, MP and Charron, P and Villard, E},
title = {Generation of iPSC line from MYH7 R403L mutation carrier with severe hypertrophic cardiomyopathy and isogenic CRISPR/Cas9 corrected control.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102245},
doi = {10.1016/j.scr.2021.102245},
pmid = {33610018},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cardiac Myosins/genetics ; *Cardiomyopathy, Hypertrophic/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Mutation ; Myocytes, Cardiac ; Myosin Heavy Chains/genetics ; },
abstract = {MYH7 is a major gene responsible for hypertrophic cardiomyopathy (HCM). From patient's skin fibroblasts, we derived an iPSC line (CDGEN1.16) harboring the heterozygous MYH7 R403L mutation, a hot-spot codon in HCM. We subsequently corrected the mutated codon using CRISPR/Cas9 editing and obtained the isogenic control line (CDGEN1.16.40.5) preserving the genomic background of the patient. Both lines were pluripotent and could be efficiently committed to beating cardiomyocytes (CM) suitable for subsequent cell or pseudo-tissue study of HCM pathology.},
}
@article {pmid33609734,
year = {2021},
author = {Bonafont, J and Mencía, A and Chacón-Solano, E and Srifa, W and Vaidyanathan, S and Romano, R and Garcia, M and Hervás-Salcedo, R and Ugalde, L and Duarte, B and Porteus, MH and Del Rio, M and Larcher, F and Murillas, R},
title = {Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {6},
pages = {2008-2018},
pmid = {33609734},
issn = {1525-0024},
mesh = {CRISPR-Cas Systems ; Cell Line ; Collagen Type VII/genetics ; Dependovirus/genetics ; Epidermolysis Bullosa Dystrophica/*genetics/therapy ; Gene Editing/*methods ; Gene Expression ; Gene Transfer Techniques ; *Genes, Recessive ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Humans ; Keratinocytes/metabolism ; *Mutation ; *Recombinational DNA Repair ; },
abstract = {Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34[+] cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.},
}
@article {pmid33609733,
year = {2021},
author = {Wang, L and Breton, C and Warzecha, CC and Bell, P and Yan, H and He, Z and White, J and Zhu, Y and Li, M and Buza, EL and Jantz, D and Wilson, JM},
title = {Long-term stable reduction of low-density lipoprotein in nonhuman primates following in vivo genome editing of PCSK9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {6},
pages = {2019-2029},
pmid = {33609733},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; *Gene Editing ; Gene Expression ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors/genetics ; Lipoproteins, LDL/genetics/*metabolism ; Liver/metabolism ; Mice ; Mice, Knockout ; Primates ; Proprotein Convertase 9/*genetics/metabolism ; },
abstract = {Gene disruption via programmable, sequence-specific nucleases represents a promising gene therapy strategy in which the reduction of specific protein levels provides a therapeutic benefit. Proprotein convertase subtilisin/kexin type 9 (PCSK9), an antagonist of the low-density lipoprotein (LDL) receptor, is a suitable target for nuclease-mediated gene disruption as an approach to treat hypercholesterolemia. We sought to determine the long-term durability and safety of PCSK9 knockdown in non-human primate (NHP) liver by adeno-associated virus (AAV)-delivered meganuclease following our initial report on the feasibility of this strategy. Six previously treated NHPs and additional NHPs administered AAV-meganuclease in combination with corticosteroid treatment or an alternative AAV serotype were monitored for a period of up to 3 years. The treated NHPs exhibited a sustained reduction in circulating PCSK9 and LDL cholesterol (LDL-c) through the course of the study concomitant with stable gene editing of the PCSK9 locus. Low-frequency off-target editing remained stable, and no obvious adverse changes in histopathology of the liver were detected. We demonstrate similar on-target nuclease activity in primary human hepatocytes using a chimeric liver-humanized mouse model. These studies demonstrate that targeted in vivo gene disruption exerts a lasting therapeutic effect and provide pivotal data for safety considerations, which support clinical translation.},
}
@article {pmid33609360,
year = {2021},
author = {Stamatiadis, P and Boel, A and Cosemans, G and Popovic, M and Bekaert, B and Guggilla, R and Tang, M and De Sutter, P and Van Nieuwerburgh, F and Menten, B and Stoop, D and Chuva de Sousa Lopes, SM and Coucke, P and Heindryckx, B},
title = {Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences.},
journal = {Human reproduction (Oxford, England)},
volume = {36},
number = {5},
pages = {1242-1252},
doi = {10.1093/humrep/deab027},
pmid = {33609360},
issn = {1460-2350},
mesh = {Animals ; Blastocyst ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Development/genetics ; Female ; Genes, Homeobox ; Humans ; *In Vitro Oocyte Maturation Techniques ; Male ; Mice ; Octamer Transcription Factor-3/genetics ; Pregnancy ; },
abstract = {STUDY QUESTION: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?<br><br>SUMMARY ANSWER: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation.<br><br>WHAT IS KNOWN ALREADY: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage.<br><br>STUDY DESIGN, SIZE, DURATION: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n&#x2009;=&#x2009;135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n&#x2009;=&#x2009;23). Four control groups were included: non-injected media-control zygotes (n&#x2009;=&#x2009;43)/oocytes (n&#x2009;=&#x2009;48); sham-injected zygotes (n&#x2009;=&#x2009;45)/oocytes (n&#x2009;=&#x2009;47); Cas9-protein injected zygotes (n&#x2009;=&#x2009;23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n&#x2009;=&#x2009;27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n&#x2009;=&#x2009;37), media control zygotes (n&#x2009;=&#x2009;19), and sham-injected zygotes (n&#x2009;=&#x2009;15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n&#x2009;=&#x2009;29) and media control zygotes (n&#x2009;=&#x2009;30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n&#x2009;=&#x2009;52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n&#x2009;=&#x2009;101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n&#x2009;=&#x2009;33). Immunofluorescence analysis was performed in human CRISPR-injected (n&#x2009;=&#x2009;10) and media control (n&#x2009;=&#x2009;9) human embryos.<br><br>A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4&#x2009;days (mouse) or 6.5&#x2009;days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing.<br><br>Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n&#x2009;=&#x2009;101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts.<br><br>One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes.<br><br>Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis.<br><br>The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests.<br><br>TRIAL REGISTRATION NUMBER: N/A.},
}
@article {pmid33609253,
year = {2021},
author = {Elliott, EK and Haupt, LM and Griffiths, LR},
title = {Mini review: genome and transcriptome editing using CRISPR-cas systems for haematological malignancy gene therapy.},
journal = {Transgenic research},
volume = {30},
number = {2},
pages = {129-141},
pmid = {33609253},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering/*methods ; Genetic Therapy/*methods ; *Genome, Human ; Hematologic Neoplasms/genetics/pathology/*therapy ; Humans ; *Transcriptome ; },
abstract = {The recent introduction of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas) systems, offer an array of genome and transcriptome editing tools for clinical repair strategies. These include Cas9, Cas12a, dCas9 and more recently Cas13 effectors. RNA targeting CRISPR-Cas13 complexes show unique characteristics with the capability to engineer transcriptomes and modify gene expression, providing a potential clinical cancer therapy tool across various tissue types. Cas13 effectors such as RNA base editing for A to I replacement allows for precise transcript modification. Further applications of Cas13a highlights its capability of producing rapid diagnostic results in a mobile platform. This review will focus on the adaptions of existing CRISPR-Cas systems, along with new Cas effectors for transcriptome or RNA modifications used in disease modelling and gene therapy for haematological malignancy. We also address the current diagnostic and therapeutic potential of CRISPR-Cas systems for personalised haematological malignancy.},
}
@article {pmid33608974,
year = {2021},
author = {Kilambi, HV and Dindu, A and Sharma, K and Nizampatnam, NR and Gupta, N and Thazath, NP and Dhanya, AJ and Tyagi, K and Sharma, S and Kumar, S and Sharma, R and Sreelakshmi, Y},
title = {The new kid on the block: a dominant-negative mutation of phototropin1 enhances carotenoid content in tomato fruits.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {3},
pages = {844-861},
doi = {10.1111/tpj.15206},
pmid = {33608974},
issn = {1365-313X},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carotenoids/*metabolism ; Fruit/*genetics/metabolism ; Gene Editing ; Loss of Function Mutation ; Lycopersicon esculentum/*genetics/metabolism ; Metabolic Networks and Pathways/genetics ; Mutation/genetics ; Phototropins/*genetics/metabolism ; },
abstract = {Phototropins, the UVA-blue light photoreceptors, endow plants to detect the direction of light and optimize photosynthesis by regulating positioning of chloroplasts and stomatal gas exchange. Little is known about their functions in other developmental responses. A tomato Non-phototropic seedling1 (Nps1) mutant, bearing an Arg495His substitution in the vicinity of LOV2 domain in phototropin1, dominant-negatively blocks phototropin1 responses. The fruits of Nps1 mutant were enriched in carotenoids, particularly lycopene, compared with its parent, Ailsa Craig. On the contrary, CRISPR/CAS9-edited loss of function phototropin1 mutants displayed subdued carotenoids compared with the parent. The enrichment of carotenoids in Nps1 fruits is genetically linked with the mutation and exerted in a dominant-negative fashion. Nps1 also altered volatile profiles with high levels of lycopene-derived 6-methyl 5-hepten2-one. The transcript levels of several MEP and carotenogenesis pathway genes were upregulated in Nps1. Nps1 fruits showed altered hormonal profiles with subdued ethylene emission and reduced respiration. Proteome profiles showed a causal link between higher carotenogenesis and increased levels of protein protection machinery, which may stabilize proteins contributing to MEP and carotenogenesis pathways. The enhancement of carotenoid content by Nps1 in a dominant-negative fashion offers a potential tool for high lycopene-bearing hybrid tomatoes.},
}
@article {pmid33608618,
year = {2021},
author = {Manger, S and Ermel, UH and Frangakis, AS},
title = {Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {234},
pmid = {33608618},
issn = {2399-3642},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Expression Regulation, Fungal ; *Microscopy, Electron, Transmission ; Proof of Concept Study ; RNA Polymerase III/genetics/*metabolism/ultrastructure ; Saccharomyces cerevisiae/enzymology/*genetics ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; *Transcription, Genetic ; },
abstract = {The direct study of transcription or DNA-protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.},
}
@article {pmid33608581,
year = {2021},
author = {Navarro-Guerrero, E and Tay, C and Whalley, JP and Cowley, SA and Davies, B and Knight, JC and Ebner, D},
title = {Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {4245},
pmid = {33608581},
issn = {2045-2322},
support = {090532/Z/09/Z/WT_/Wellcome Trust/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; 204969/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Gene Knockdown Techniques ; Genome-Wide Association Study ; Genomic Library ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Macrophages/*cytology/*metabolism ; },
abstract = {Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes-HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.},
}
@article {pmid33608071,
year = {2021},
author = {Li, F and Ye, Q and Chen, M and Xiang, X and Zhang, J and Pang, R and Xue, L and Wang, J and Gu, Q and Lei, T and Wei, X and Ding, Y and Wu, Q},
title = {Cas12aFDet: A CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeria monocytogenes serotype 4c.},
journal = {Analytica chimica acta},
volume = {1151},
number = {},
pages = {338248},
doi = {10.1016/j.aca.2021.338248},
pmid = {33608071},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Listeria monocytogenes/genetics ; Nucleic Acid Amplification Techniques ; Serogroup ; },
abstract = {The CRISPR/Cas12a system has displayed remarkable potential in the development of new methods for nucleic acid detection owing to the trans-cleavage activity of Cas12a. Despite the tremendous development in recent years, existing CRISPR/Cas12a-based methods have several limitations such as the time-consuming process, which takes up to 2 h, and the risk of aerosol contamination during DNA amplicon transfer. Herein, we propose a CRISPR/Cas12a-based fluorescence detection platform named "Cas12aFDet" for rapid nucleic acid detection that overcomes these limitations. By integrating PCR or recombinase-aided amplification (RAA) methods with Cas12a-mediated cleavage in a sealed reaction tube, Cas12aFDet-based detection of amplified products could be accomplished within 15 min, while avoiding amplicon contamination. The detection limits of PCR-based Cas12aFDet and RAA-based Cas12aFDet were determined to be 3.37 × 10[1] cfu/mL and 1.35 × 10[2] cfu/mL of Listeria monocytogenes serotype 4c in pure culture, respectively. Most importantly, RAA-based Cas12aFDet exhibited 0.64 aM sensitivity for DNA detection, and showed high specificity for detection of other serotypes of Listeria and non-Listeria strains. Furthermore, the feasibility of the RAA-based Cas12aFDet method was evaluated in spiked and natural samples, enabling the quantitative detection of 1.35 × 10[8]-1.35 × 10[3] cfu/g fresh grass carp of the target L. monocytogenes serotype 4c, and the results obtained for 22 natural aquatic samples were highly consistent with those of the culture-based serotyping method. The established Cas12aFDet platform is expected to provide a new paradigm for the sensitive and specific detection of pathogens in food safety and clinical diagnosis.},
}
@article {pmid33607867,
year = {2021},
author = {Liu, Y and Zhan, L and Qin, Z and Sackrison, J and Bischof, JC},
title = {Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis.},
journal = {ACS nano},
volume = {15},
number = {3},
pages = {3593-3611},
doi = {10.1021/acsnano.0c10035},
pmid = {33607867},
issn = {1936-086X},
mesh = {Biological Assay ; Enzyme-Linked Immunosorbent Assay ; *Nucleic Acids ; *Point-of-Care Systems ; Point-of-Care Testing ; },
abstract = {Lateral flow assays (LFAs) are paper-based point-of-care (POC) diagnostic tools that are widely used because of their low cost, ease of use, and rapid format. Unfortunately, traditional commercial LFAs have significantly poorer sensitivities (μM) and specificities than standard laboratory tests (enzyme-linked immunosorbent assay, ELISA: pM-fM; polymerase chain reaction, PCR: aM), thus limiting their impact in disease control. In this Perspective, we review the evolving efforts to increase the sensitivity and specificity of LFAs. Recent work to improve the sensitivity through assay improvement includes optimization of the assay kinetics and signal amplification by either reader systems or additional reagents. Together, these efforts have produced LFAs with ELISA-level sensitivities (pM-fM). In addition, sample preamplification can be applied to both nucleic acids (direct amplification) and other analytes (indirect amplification) prior to LFA testing, which can lead to PCR-level (aM) sensitivity. However, these amplification strategies also increase the detection time and assay complexity, which inhibits the large-scale POC use of LFAs. Perspectives to achieve future rapid (<30 min), ultrasensitive (PCR-level), and "sample-to-answer" POC diagnostics are also provided. In the case of LFA specificity, recent research efforts have focused on high-affinity molecules and assay optimization to reduce nonspecific binding. Furthermore, novel highly specific molecules, such as CRISPR/Cas systems, can be integrated into diagnosis with LFAs to produce not only ultrasensitive but also highly specific POC diagnostics. In summary, with continuing improvements, LFAs may soon offer performance at the POC that is competitive with laboratory techniques while retaining a rapid format.},
}
@article {pmid33607472,
year = {2021},
author = {Zhang, C and Wei, Y and Zhang, Y and Zhang, J and Xing, Q and Zhou, M and Shan, Y},
title = {Establishment of a CRISPR/Cas9-mediated ANP32A homozygous knockout human embryonic stem cell line.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102234},
doi = {10.1016/j.scr.2021.102234},
pmid = {33607472},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; Nuclear Proteins/genetics ; RNA-Binding Proteins ; },
abstract = {ANP32A is a member of acidic leucine-rich nuclear phosphoprotein 32 family, which is involved in diverse biochemical processes, including chromatin modification and remodeling. Here, we established the CRISPR/Cas9-mediated ANP32A homozygous knockout human embryonic stem cell (ESC) line to investigate the roles of ANP32A in pluripotency maintenance and differentiation process of human ESCs. This cell line shows the normal karyotype and typical stem cell morphology, in accordance with high expression of pluripotent genes and the differentiation potential in vitro. Consequently, the ANP32A knockout cell line provides a promising approach for investigating the roles of ANP32A in human ESC cell fate decisions.},
}
@article {pmid33607266,
year = {2021},
author = {Wang, Q and Chear, S and Wing, K and Stellon, D and Nguyen Tran, MT and Talbot, J and Pébay, A and Hewitt, AW and Cook, AL},
title = {Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {18-29},
doi = {10.1016/j.ymeth.2021.02.009},
pmid = {33607266},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.},
}
@article {pmid33606978,
year = {2021},
author = {Cuella-Martin, R and Hayward, SB and Fan, X and Chen, X and Huang, JW and Taglialatela, A and Leuzzi, G and Zhao, J and Rabadan, R and Lu, C and Shen, Y and Ciccia, A},
title = {Functional interrogation of DNA damage response variants with base editing screens.},
journal = {Cell},
volume = {184},
number = {4},
pages = {1081-1097.e19},
pmid = {33606978},
issn = {1097-4172},
support = {P01 CA174653/CA/NCI NIH HHS/United States ; R01 CA197774/CA/NCI NIH HHS/United States ; R01 CA227450/CA/NCI NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Ataxia Telangiectasia Mutated Proteins/metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; Camptothecin/pharmacology ; Cell Line ; *DNA Damage/genetics ; DNA Repair/genetics ; Female ; *Gene Editing ; *Genetic Testing ; Humans ; Mutation/genetics ; Phenotype ; Protein Binding ; Protein Domains ; RNA, Guide/genetics ; Topoisomerase Inhibitors/pharmacology ; Tumor Suppressor p53-Binding Protein 1/chemistry/genetics ; Ubiquitin Thiolesterase/metabolism ; Ubiquitin-Protein Ligases/chemistry/genetics/metabolism ; },
abstract = {Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.},
}
@article {pmid33606973,
year = {2021},
author = {Parrish, PCR and Berger, AH},
title = {CRISPR base editor screens identify variant function at scale.},
journal = {Molecular cell},
volume = {81},
number = {4},
pages = {647-648},
pmid = {33606973},
issn = {1097-4164},
support = {P50 CA228944/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; },
abstract = {Cuella-Martin et al. (2021) and Hanna et al. (2021) showcase CRISPR base editing in large-scale pooled screens in human cells to discover both loss- and gain-of-function variants, enabling protein structure/function insights and clinical variant interpretation.},
}
@article {pmid33606203,
year = {2021},
author = {Shola, DTN and Yang, C and Han, C and Norinsky, R and Peraza, RD},
title = {Generation of Mouse Model (KI and CKO) via Easi-CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2224},
number = {},
pages = {1-27},
pmid = {33606203},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Single-Stranded/*genetics ; Exons/genetics ; Female ; Gene Editing/methods ; Gene Knock-In Techniques ; Genes, Reporter/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; RNA, Guide/genetics ; Zygote/physiology ; },
abstract = {Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109-122, 2020), published by Mary Ann Libert, Inc).},
}
@article {pmid33604667,
year = {2021},
author = {Goh, CJH and Wong, JH and El Farran, C and Tan, BX and Coffill, CR and Loh, YH and Lane, D and Arumugam, P},
title = {Identification of pathways modulating vemurafenib resistance in melanoma cells via a genome-wide CRISPR/Cas9 screen.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {2},
pages = {},
pmid = {33604667},
issn = {2160-1836},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Neoplasm ; Humans ; *Melanoma/genetics ; Mutation ; Vemurafenib ; },
abstract = {Vemurafenib is a BRAF kinase inhibitor (BRAFi) that is used to treat melanoma patients harboring the constitutively active BRAF-V600E mutation. However, after a few months of treatment patients often develop resistance to vemurafenib leading to disease progression. Sequence analysis of drug-resistant tumor cells and functional genomic screens has identified several genes that regulate vemurafenib resistance. Reactivation of mitogen-activated protein kinase (MAPK) pathway is a recurrent feature of cells that develop resistance to vemurafenib. We performed a genome-scale CRISPR-based knockout screen to identify modulators of vemurafenib resistance in melanoma cells with a highly improved CRISPR sgRNA library called Brunello. We identified 33 genes that regulate resistance to vemurafenib out of which 14 genes have not been reported before. Gene ontology enrichment analysis showed that the hit genes regulate histone modification, transcription and cell cycle. We discuss how inactivation of hit genes might confer resistance to vemurafenib and provide a framework for follow-up investigations.},
}
@article {pmid33603089,
year = {2021},
author = {Yu, L and Wang, L and Wu, X and Yi, H},
title = {RSPO4-CRISPR alleviates liver injury and restores gut microbiota in a rat model of liver fibrosis.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {230},
pmid = {33603089},
issn = {2399-3642},
mesh = {Animals ; *CRISPR-Cas Systems ; Carbon Tetrachloride ; Cells, Cultured ; Chemical and Drug Induced Liver Injury/genetics/metabolism/microbiology/*therapy ; Dysbiosis ; *Gastrointestinal Microbiome ; *Genetic Therapy ; Hepatic Stellate Cells/metabolism/pathology ; Intestines/*microbiology ; Liver/*metabolism/pathology ; Liver Cirrhosis, Experimental/genetics/metabolism/microbiology/*therapy ; Male ; Rats, Sprague-Dawley ; Thrombospondins/genetics/*metabolism ; Wnt Signaling Pathway ; },
abstract = {Wnt signaling dysfunction and gut dysbiosis may lead to liver fibrosis, yet the underlying mechanisms are not well elucidated. This study demonstrated the role of RSPO4, a Wnt signaling agonist, in liver fibrogenesis and its impact on the gut microbiome. RSPO4 gene in CCl4-induced fibrotic-liver rats was knockout by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, with healthy rats served as the control. Tissue samples and hepatic stellate cells (HSCs) isolated from rats were examined for curative effect of RSPO4-CRISPR treatment. Fecal sample were collected and analyzed with 16 S rRNA sequencing. We found RSPO4-CRISPR relieved liver fibrosis in rats and reversed HSC activation. Further, results showed RSPO4-CRISPR tended to restore the microflora composition. Significance species between groups were identified. Bacteroides and Escherichia-Shigella were the key microbes in the model and negative group, whereas Lactobacillus, Romboutsia, and Lachnospiraceae NK4A136 group were abundant in the control. Notably, Bacteroidales S24-7 group and Ruminococcaceae UCG-005 were the significantly enriched in CRISPR group. We show that the microbiome of rats treated with RSPO4-CRISPR presents a trend towards the restoration of the original condition. Our findings pave a new way to evaluate the curative effect of liver fibrosis treatment.},
}
@article {pmid33602820,
year = {2021},
author = {Cheong, MC and Wang, Z and Jaleta, TG and Li, X and Lok, JB and Kliewer, SA and Mangelsdorf, DJ},
title = {Identification of a nuclear receptor/coactivator developmental signaling pathway in the nematode parasite Strongyloides stercoralis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {8},
pages = {},
pmid = {33602820},
issn = {1091-6490},
support = {R01 AI050668/AI/NIAID NIH HHS/United States ; R33 AI105856/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/*growth &amp; development/metabolism ; Caenorhabditis elegans Proteins/genetics/*metabolism ; *Gene Expression Regulation, Developmental ; Larva/genetics/growth &amp; development/*metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Strongyloides stercoralis/genetics/*parasitology ; Transcription Factors/genetics/*metabolism ; },
abstract = {DAF-12 is nematode-specific nuclear receptor that has been proposed to govern development of the infectious stage of parasitic species, including Strongyloides stercoralis Here, we identified a parasite-specific coactivator, called DAF-12 interacting protein-1 (DIP-1), that is required for DAF-12 ligand-dependent transcriptional activity. DIP-1 is found only in Strongyloides spp. and selectively interacts with DAF-12 through an atypical receptor binding motif. Using CRISPR/Cas9-directed mutagenesis, we demonstrate that DAF-12 is required for the requisite developmental arrest and the ligand-dependent reactivation of infectious S. stercoralis infective third-stage larvae, and that these effects require the DIP-1 coactivator. These studies reveal the existence of a distinct nuclear receptor/coactivator signaling pathway that governs parasite development.},
}
@article {pmid33602788,
year = {2021},
author = {Assi, M and Achouri, Y and Loriot, A and Dauguet, N and Dahou, H and Baldan, J and Libert, M and Fain, JS and Guerra, C and Bouwens, L and Barbacid, M and Lemaigre, FP and Jacquemin, P},
title = {Dynamic Regulation of Expression of KRAS and Its Effectors Determines the Ability to Initiate Tumorigenesis in Pancreatic Acinar Cells.},
journal = {Cancer research},
volume = {81},
number = {10},
pages = {2679-2689},
doi = {10.1158/0008-5472.CAN-20-2976},
pmid = {33602788},
issn = {1538-7445},
mesh = {Acinar Cells/metabolism/*pathology ; Animals ; Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Disease Models, Animal ; ErbB Receptors/genetics/metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice ; *Mutation ; Pancreatic Neoplasms/etiology/metabolism/*pathology ; Pancreatitis/etiology/metabolism/*pathology ; Proto-Oncogene Proteins p21(ras)/antagonists &amp; inhibitors/genetics/*metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. SIGNIFICANCE: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.},
}
@article {pmid33602235,
year = {2021},
author = {Yu, W and Li, J and Huang, S and Li, X and Li, P and Li, G and Liang, A and Chi, T and Huang, X},
title = {Harnessing A3G for efficient and selective C-to-T conversion at C-rich sequences.},
journal = {BMC biology},
volume = {19},
number = {1},
pages = {34},
pmid = {33602235},
issn = {1741-7007},
mesh = {APOBEC-3G Deaminase/*genetics ; *CRISPR-Cas Systems ; Cytidine Deaminase/*metabolism ; *Gene Editing ; *RNA, Guide ; },
abstract = {BACKGROUND: Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising rat APOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C's within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C's in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3.<br><br>RESULTS: By replacing rA1 with an optimized&#xa0;human A3G&#xa0;(oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome.<br><br>CONCLUSIONS: oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.},
}
@article {pmid33602066,
year = {2021},
author = {Ahmed, T and Noman, M and Shahid, M and Muhammad, S and Tahir Ul Qamar, M and Ali, MA and Maqsood, A and Hafeez, R and Ogunyemi, SO and Li, B},
title = {Potential Application of CRISPR/Cas9 System to Engineer Abiotic Stress Tolerance in Plants.},
journal = {Protein and peptide letters},
volume = {28},
number = {8},
pages = {861-877},
doi = {10.2174/0929866528666210218220138},
pmid = {33602066},
issn = {1875-5305},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; *Genome, Plant ; Plants/*genetics ; Stress, Physiological/*genetics ; },
abstract = {Abiotic stresses in plants such as salinity, drought, heavy metal toxicity, heat, and nutrients limitations significantly reduce agricultural production worldwide. The genome editing techniques such as transcriptional activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have been used for genome manipulations in plants. However, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technique has recently emerged as a promising tool for genome editing in plants to acquire desirable traits. The CRISPR/Cas9 system has a great potential to develop crop varieties with improved tolerance against abiotic stresses. This review is centered on the biology and potential application of the CRISPR/Cas9 system to improve abiotic stress tolerance in plants. Furthermore, this review highlighted the recent advancements of CRISPR/Cas9-mediated genome editing for sustainable agriculture.},
}
@article {pmid33601053,
year = {2021},
author = {Li, M and Xu, Y and Liang, J and Lin, H and Qi, X and Li, F and Han, P and Gao, Y and Yang, X},
title = {USP22 deficiency in melanoma mediates resistance to T cells through IFNγ-JAK1-STAT1 signal axis.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {6},
pages = {2108-2120},
pmid = {33601053},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytotoxicity, Immunologic/genetics ; Gene Expression Profiling ; Genome-Wide Association Study ; Humans ; Interferon-gamma/*metabolism ; Janus Kinase 1/*metabolism ; Melanoma ; Mice ; Protein Stability ; STAT1 Transcription Factor/*metabolism ; *Signal Transduction ; T-Lymphocytes/*immunology/*metabolism ; Ubiquitin Thiolesterase/*deficiency ; Ubiquitination ; },
abstract = {Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9)-mediated loss-of-function screens are powerful tools for identifying genes responsible for diverse phenotypes. Here, we perturbed genes in melanoma cells to screen for genes involved in tumor escape from T cell-mediated killing. Multiple interferon gamma (IFNγ) signaling-related genes were enriched in melanoma cells resistant to T cell killing. In addition, deletion of the deubiquitinating protease ubiquitin specific peptidase 22 (USP22) in mouse melanoma (B16-OVA) cells decreased the efficacy of T cell-mediated killing, both in vitro and in vivo, while overexpression enhanced tumor-cell sensitivity to T (OT-I) cell-mediated killing. USP22 deficiency in both mouse and human melanoma cells showed impaired sensitivity to interferon pathway and USP22 was positively correlated with key molecules of interferon pathway in clinical melanoma samples. Mechanistically, USP22 may directly interact with signal transducer and activator of transcription 1 (STAT1), deubiquitinate it, and improve its stability in both human and mouse melanoma cells. Our findings identified a previously unknown function of USP22 and linked the loss of genes in tumor cells that are essential for escaping the effector function of CD8[+] T cells during immunotherapy.},
}
@article {pmid33600891,
year = {2021},
author = {Hartz, P and Gehl, M and König, L and Bernhardt, R and Hannemann, F},
title = {Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium.},
journal = {Journal of biotechnology},
volume = {329},
number = {},
pages = {170-179},
doi = {10.1016/j.jbiotec.2021.02.006},
pmid = {33600891},
issn = {1873-4863},
mesh = {*Bacillus megaterium/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Plasmids/genetics ; Promoter Regions, Genetic ; },
abstract = {Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous β-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.},
}
@article {pmid33600562,
year = {2021},
author = {Muñoz, SS and Garner, B and Ooi, L},
title = {Generation of APOE knock-down SK-N-SH human neuroblastoma cells using CRISPR/Cas9: a novel cellular model relevant to Alzheimer's disease research.},
journal = {Bioscience reports},
volume = {41},
number = {2},
pages = {},
pmid = {33600562},
issn = {1573-4935},
mesh = {Alzheimer Disease/*genetics ; Apolipoproteins E/*genetics ; *CRISPR-Cas Systems ; Cell Differentiation ; Cell Line, Tumor ; Humans ; Neuroblastoma/pathology ; },
abstract = {APOE ε4 is the major genetic risk factor for Alzheimer's disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells.},
}
@article {pmid33600503,
year = {2021},
author = {Leoni, C and Bianchi, N and Vincenzetti, L and Monticelli, S},
title = {An optimized workflow for CRISPR-Cas9 deletion of surface and intracellular factors in primary human T lymphocytes.},
journal = {PloS one},
volume = {16},
number = {2},
pages = {e0247232},
pmid = {33600503},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/deficiency/genetics ; Cells, Cultured ; Endoribonucleases/deficiency/genetics ; Gene Editing/*methods ; Humans ; Membrane Proteins/deficiency/*genetics ; RNA, Guide/genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/deficiency/genetics ; T-Box Domain Proteins/deficiency/genetics ; T-Lymphocytes/cytology/metabolism ; Transcription Factors/deficiency/*genetics ; },
abstract = {The appropriate regulation of T lymphocyte functions is key to achieve protective immune responses, while at the same time limiting the risks of tissue damage and chronic inflammation. Deciphering the mechanisms underpinning T cell responses in humans may therefore be beneficial for a range of infectious and chronic diseases. Recently, the development of methods based on CRISPR-Cas9 gene-editing has greatly expanded the available tool-box for the mechanistic studies of primary human T cell responses. While the deletion of a surface protein has become a relatively straightforward task, as long as an antibody for detection is available, the identification and selection of cells lacking an intracellular protein, a non-coding RNA or a protein for which no antibody is available, remain more problematic. Here, we discuss the options currently available to scientists interested in performing gene-editing in primary human T lymphocytes and we describe the optimization of a workflow for the screening and analysis of lymphocytes following gene-editing with CRISPR-Cas9 based on T cell cloning and T7 endonuclease I cleavage assay.},
}
@article {pmid33600432,
year = {2021},
author = {Oberhofer, G and Ivy, T and Hay, BA},
title = {Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive.},
journal = {PLoS genetics},
volume = {17},
number = {2},
pages = {e1009385},
pmid = {33600432},
issn = {1553-7404},
support = {T32 GM007616/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Drosophila melanogaster/*genetics ; Female ; Gene Drive Technology/*methods ; Gene Editing/*methods ; Genes, Essential/*genetics ; Male ; Models, Genetic ; Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Drive mechanisms that are self-limiting are attractive because they allow control over the duration and extent of trait spread in time and space, and are reversible through natural selection as drive wanes. Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. We use modeling to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner-which is still self-limiting-by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement split ClvR in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make up ClvR elements-Cas9 and gRNAs and a Rescue/Cargo.},
}
@article {pmid33599746,
year = {2021},
author = {Critcher, M and O'Leary, T and Huang, ML},
title = {Glycoengineering: scratching the surface.},
journal = {The Biochemical journal},
volume = {478},
number = {4},
pages = {703-719},
doi = {10.1042/BCJ20200612},
pmid = {33599746},
issn = {1470-8728},
mesh = {Animals ; CRISPR-Cas Systems ; Click Chemistry ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Glycocalyx/chemistry/*physiology ; Glycoconjugates/chemical synthesis/*chemistry ; Glycoproteins/metabolism ; Glycosylation ; Glycosyltransferases/genetics ; Humans ; Monosaccharides/chemistry ; Mucins/metabolism ; Oligosaccharides/chemistry ; Polysaccharides/metabolism ; Protein Engineering/methods ; Protein Processing, Post-Translational ; RNA, Small Interfering/genetics ; Recombinant Proteins/metabolism ; Surface Properties ; },
abstract = {At the surface of many cells is a compendium of glycoconjugates that form an interface between the cell and its surroundings; the glycocalyx. The glycocalyx serves several functions that have captivated the interest of many groups. Given its privileged residence, this meshwork of sugar-rich biomolecules is poised to transmit signals across the cellular membrane, facilitating communication with the extracellular matrix and mediating important signalling cascades. As a product of the glycan biosynthetic machinery, the glycocalyx can serve as a partial mirror that reports on the cell's glycosylation status. The glycocalyx can also serve as an information-rich barrier, withholding the entry of pathogens into the underlying plasma membrane through glycan-rich molecular messages. In this review, we provide an overview of the different approaches devised to engineer glycans at the cell surface, highlighting considerations of each, as well as illuminating the grand challenges that face the next era of 'glyco-engineers'. While we have learned much from these techniques, it is evident that much is left to be unearthed.},
}
@article {pmid33599292,
year = {2021},
author = {Shihong Gao, D and Zhu, X and Lu, B},
title = {Development and application of sensitive, specific, and rapid CRISPR-Cas13-based diagnosis.},
journal = {Journal of medical virology},
volume = {93},
number = {7},
pages = {4198-4204},
pmid = {33599292},
issn = {1096-9071},
mesh = {COVID-19/*diagnosis ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; RNA, Viral/*genetics ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {Nucleic acid detection is a necessary part of medical treatment and fieldwork. However, the current detection technologies are far from ideal. A lack of timely and accessible testing for identifying cases and close contacts has allowed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative virus of the ongoing coronavirus disease-2019 (COVID-19) pandemic, to spread uncontrollably. The slow and expensive detection of mutations-predictors for chronic diseases such as cancer-form a barrier to personalized treatment. A recently developed diagnostic assay is ideal and field-ready-it relies on CRISPR-Cas13. CRISPR-Cas13 works similarly to other CRISPR systems: Cas13 is guided by a crRNA to cleave next to a specific RNA target sequence. Additionally, Cas13 boasts a unique collateral cleavage activity; collateral cleavage of a fluorescent reporter detects the presence of the target sequence in sample RNA. This system forms the basis of CRISPR-Cas13 diagnostic assays. CRISPR-Cas13 assays have >95% sensitivity and >99% specificity. Detection is rapid (<2 h), inexpensive ($0.05 per test), and portable-a test using lateral flow strips is akin to a pregnancy test. The recent adaptation of micro-well chips facilitates high-level multiplexing and is high-throughput. In this review, we cover the development of CRISPR-Cas13 assays for medical diagnosis, discuss the advantages of CRISPR-Cas13-based diagnosis over the traditional reverse transcription polymerase chain reaction (RT-PCR), and present examples of detection from real patient samples.},
}
@article {pmid33598715,
year = {2021},
author = {Germic, N and Hosseini, A and Stojkov, D and Oberson, K and Claus, M and Benarafa, C and Calzavarini, S and Angelillo-Scherrer, A and Arnold, IC and Müller, A and Riether, C and Yousefi, S and Simon, HU},
title = {ATG5 promotes eosinopoiesis but inhibits eosinophil effector functions.},
journal = {Blood},
volume = {137},
number = {21},
pages = {2958-2969},
pmid = {33598715},
issn = {1528-0020},
mesh = {Animals ; Autophagy-Related Protein 5/biosynthesis/deficiency/genetics/*physiology ; Bone Marrow/pathology ; CRISPR-Cas Systems ; Cell Degranulation ; Cell Line, Tumor ; Cells, Cultured ; Citrobacter rodentium ; Colony-Forming Units Assay ; Enterobacteriaceae Infections/immunology ; Eosinophils/cytology/immunology/*physiology ; Humans ; Hypereosinophilic Syndrome/blood/pathology ; Interleukin-5/genetics ; Leukocyte Count ; MAP Kinase Signaling System/genetics ; Mice ; Mice, Knockout ; Mice, Transgenic ; Myelopoiesis/*physiology ; Oncogene Proteins, Fusion/genetics ; Receptor, Platelet-Derived Growth Factor alpha/genetics ; mRNA Cleavage and Polyadenylation Factors/genetics ; },
abstract = {Eosinophils are white blood cells that contribute to the regulation of immunity and are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available regarding the role of autophagy in eosinophil differentiation and functions. To study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also observed in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils exhibited augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5, which controls the amplitude of overall antibacterial eosinophil immune responses.},
}
@article {pmid33598657,
year = {2021},
author = {Olivieri, M and Durocher, D},
title = {Genome-scale chemogenomic CRISPR screens in human cells using the TKOv3 library.},
journal = {STAR protocols},
volume = {2},
number = {1},
pages = {100321},
pmid = {33598657},
issn = {2666-1667},
support = {FDN143343//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Library ; *Genome, Human ; Humans ; },
abstract = {CRISPR-based genetic screens revolutionized our ability to genetically probe cell biology. We present a protocol to conduct genome-scale chemogenomic dropout CRISPR screens in the human RPE1-hTERT p53[-/-] cell line. We use the TKOv3 library, which contains 70,948 sgRNAs targeting 18,053 genes. Here, we describe how to set up the screen, the reagents required, and how to sequence and analyze the results. This protocol can be customized for other libraries, cell lines, and sequencing instruments. For complete details on the use and execution of this protocol, please refer to Olivieri et al. (2020).},
}
@article {pmid33597771,
year = {2021},
author = {Weisheit, I and Kroeger, JA and Malik, R and Wefers, B and Lichtner, P and Wurst, W and Dichgans, M and Paquet, D},
title = {Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping.},
journal = {Nature protocols},
volume = {16},
number = {3},
pages = {1714-1739},
pmid = {33597771},
issn = {1750-2799},
mesh = {Animals ; Base Sequence/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genotype ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Polymorphism, Single Nucleotide/*genetics ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 d.},
}
@article {pmid33597562,
year = {2021},
author = {Nakanishi, T and Maekawa, A and Suzuki, M and Tabata, H and Sato, K and Mori, M and Saito, I},
title = {Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {3961},
pmid = {33597562},
issn = {2045-2322},
mesh = {Adenoviridae/*genetics ; Animals ; CRISPR-Cas Systems ; Cosmids ; Fibroblasts/metabolism ; Gene Editing/methods ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Hep G2 Cells ; Humans ; INDEL Mutation/genetics ; Mice, Inbred C57BL ; Plasmids/genetics ; RNA, Guide/*genetics/metabolism ; },
abstract = {Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.},
}
@article {pmid33596732,
year = {2022},
author = {Galichet, C and Lovell-Badge, R},
title = {Applications of genome editing on laboratory animals.},
journal = {Laboratory animals},
volume = {56},
number = {1},
pages = {13-25},
doi = {10.1177/0023677221993141},
pmid = {33596732},
issn = {1758-1117},
support = {13-1270/AICR_/Worldwide Cancer Research/United Kingdom ; MC_U117562207/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Animals, Laboratory ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide/genetics/metabolism ; },
abstract = {For four decades, genetically altered laboratory animals have provided invaluable information. Originally, genetic modifications were performed on only a few animal species, often chosen because of the ready accessibility of embryonic materials and short generation times. The methods were often slow, inefficient and expensive. In 2013, a new, extremely efficient technology, namely CRISPR/Cas9, not only made the production of genetically altered organisms faster and cheaper, but also opened it up to non-conventional laboratory animal species. CRISPR/Cas9 relies on a guide RNA as a 'location finder' to target DNA double strand breaks induced by the Cas9 enzyme. This is a prerequisite for non-homologous end joining repair to occur, an error prone mechanism often generating insertion or deletion of genetic material. If a DNA template is also provided, this can lead to homology directed repair, allowing precise insertions, deletions or substitutions. Due to its high efficiency in targeting DNA, CRISPR/Cas9-mediated genetic modification is now possible in virtually all animal species for which we have genome sequence data. Furthermore, modifications of Cas9 have led to more refined genetic alterations from targeted single base-pair mutations to epigenetic modifications. The latter offer altered gene expression without genome alteration. With this ever growing genetic toolbox, the number and range of genetically altered conventional and non-conventional laboratory animals with simple or complex genetic modifications is growing exponentially.},
}
@article {pmid33596731,
year = {2022},
author = {Caso, F and Davies, B},
title = {Base editing and prime editing in laboratory animals.},
journal = {Laboratory animals},
volume = {56},
number = {1},
pages = {35-49},
pmid = {33596731},
issn = {1758-1117},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adenine ; Animals ; Animals, Laboratory ; *CRISPR-Cas Systems ; Cytosine ; *Gene Editing/methods ; },
abstract = {Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple 'search and replace' editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.},
}
@article {pmid33596056,
year = {2021},
author = {Zhang, M and Yang, C and Tasan, I and Zhao, H},
title = {Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration.},
journal = {ACS synthetic biology},
volume = {10},
number = {3},
pages = {429-446},
doi = {10.1021/acssynbio.0c00576},
pmid = {33596056},
issn = {2161-5063},
support = {U54 DK107965/DK/NIDDK NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics/*metabolism ; DNA Repair ; Gene Editing/*methods ; Humans ; Microsatellite Repeats/genetics ; Protein Engineering ; RNA, Guide/genetics/metabolism ; },
abstract = {Inserting custom designed DNA sequences into the mammalian genome plays an essential role in synthetic biology. In particular, the ability to introduce foreign DNA in a site-specific manner offers numerous advantages over random DNA integration. In this review, we focus on two mechanistically distinct systems that have been widely adopted for targeted DNA insertion in mammalian cells, the CRISPR/Cas9 system and site-specific recombinases. The CRISPR/Cas9 system has revolutionized the genome engineering field thanks to its high programmability and ease of use. However, due to its dependence on linearized DNA donor and endogenous cellular pathways to repair the induced double-strand break, CRISPR/Cas9-mediated DNA insertion still faces limitations such as small insert size, and undesired editing outcomes via error-prone repair pathways. In contrast, site-specific recombinases, in particular the Serine integrases, demonstrate large-cargo capability and no dependence on cellular repair pathways for DNA integration. Here we first describe recent advances in improving the overall efficacy of CRISPR/Cas9-based methods for DNA insertion. Moreover, we highlight the advantages of site-specific recombinases over CRISPR/Cas9 in the context of targeted DNA integration, with a special focus on the recent development of programmable recombinases. We conclude by discussing the importance of protein engineering to further expand the current toolkit for targeted DNA insertion in mammalian cells.},
}
@article {pmid33595922,
year = {2021},
author = {Meng, Q and Wang, X and Wang, Y and Dang, L and Liu, X and Ma, X and Chi, T and Wang, X and Zhao, Q and Yang, G and Liu, M and Huang, X and Ma, P},
title = {Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA.},
journal = {Biotechnology journal},
volume = {16},
number = {6},
pages = {e2100040},
doi = {10.1002/biot.202100040},
pmid = {33595922},
issn = {1860-7314},
mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; Mutation ; *RNA, Guide ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus ; },
abstract = {Detection of pathogens with single-nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS-CoV-2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called "synthetic mismatch integrated crRNA guided Cas12a detection" (symRNA-Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the -2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13-fold over the ancestral crRNA. With this selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA-Cas12a method to specifically, sensitively and rapidly detect the SARS-CoV-2 D614G mutation.},
}
@article {pmid33595147,
year = {2021},
author = {Che, P and Chang, S and Simon, MK and Zhang, Z and Shaharyar, A and Ourada, J and O'Neill, D and Torres-Mendoza, M and Guo, Y and Marasigan, KM and Vielle-Calzada, JP and Ozias-Akins, P and Albertsen, MC and Jones, TJ},
title = {Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {3},
pages = {817-830},
pmid = {33595147},
issn = {1365-313X},
mesh = {Agrobacterium/genetics ; Cotyledon/genetics/growth &amp; development/metabolism ; Gene Editing/*methods ; Gene Transfer Techniques ; Genome, Plant/genetics ; Plant Shoots/growth &amp; development ; Plants, Genetically Modified ; Seeds/*genetics/growth &amp; development/metabolism ; Transformation, Genetic ; Vigna/*genetics/growth &amp; development/metabolism ; },
abstract = {Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.},
}
@article {pmid33594101,
year = {2021},
author = {Bari, VK and Nassar, JA and Aly, R},
title = {CRISPR/Cas9 mediated mutagenesis of MORE AXILLARY GROWTH 1 in tomato confers resistance to root parasitic weed Phelipanche aegyptiaca.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {3905},
pmid = {33594101},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Gene Editing ; Host-Parasite Interactions/*genetics ; Lycopersicon esculentum/genetics/metabolism/*parasitology ; *Orobanchaceae ; Plant Weeds ; },
abstract = {Root parasitic weeds infect numerous economically important crops, affecting total yield quantity and quality. A lack of an efficient control method limits our ability to manage newly developing and more virulent races of root parasitic weeds. To control the parasite induced damage in most host crops, an innovative biotechnological approach is urgently required. Strigolactones (SLs) are plant hormones derived from carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase (CCD) 7, CCD8 and More Axillary Growth 1 (MAX1) genes. SLs act as branching inhibitory hormones and strictly required for the germination of root parasitic weeds. Here, we demonstrate that CRISPR/Cas9-mediated targted editing of SL biosynthetic gene MAX1, in tomato confers resistance against root parasitic weed Phelipanche aegyptiaca. We designed sgRNA to target the third exon of MAX1 in tomato plants using the CRISPR/Cas9 system. The T0 plants were edited very efficiently at the MAX1 target site without any non-specific off-target effects. Genotype analysis of T1 plants revealed that the introduced mutations were stably passed on to the next generation. Notably, MAX1-Cas9 heterozygous and homozygous T1 plants had similar morphological changes that include excessive growth of axillary bud, reduced plant height and adventitious root formation relative to wild type. Our results demonstrated that, MAX1-Cas9 mutant lines exhibit resistance against root parasitic weed P. aegyptiaca due to reduced SL (orobanchol) level. Moreover, the expression of carotenoid biosynthetic pathway gene PDS1 and total carotenoid level was altered, as compared to wild type plants. Taking into consideration, the impact of root parasitic weeds on the agricultural economy and the obstacle to prevent and eradicate them, the current study provides new aspects into the development of an efficient control method that could be used to avoid germination of root parasitic weeds.},
}
@article {pmid33593370,
year = {2021},
author = {Sun, Y and Yu, L and Liu, C and Ye, S and Chen, W and Li, D and Huang, W},
title = {One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a.},
journal = {Journal of translational medicine},
volume = {19},
number = {1},
pages = {74},
pmid = {33593370},
issn = {1479-5876},
mesh = {Base Sequence ; COVID-19/*diagnosis/*virology ; COVID-19 Testing/*methods ; CRISPR-Cas Systems/*genetics ; Humans ; Limit of Detection ; RNA, Viral/genetics ; Real-Time Polymerase Chain Reaction/*methods ; Reference Standards ; Reverse Transcription/*genetics ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {BACKGROUND: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.<br><br>METHODS: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.<br><br>RESULTS: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50&#xa0;min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/&#xb5;l input (RNA standard) and 1 copy/&#xb5;l input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/&#xb5;l input.<br><br>CONCLUSIONS: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.},
}
@article {pmid33593219,
year = {2021},
author = {El Jaddaoui, I and Allali, M and Raoui, S and Sehli, S and Habib, N and Chaouni, B and Al Idrissi, N and Benslima, N and Maher, W and Benrahma, H and Hamamouch, N and El Bissati, K and El Kasmi, S and Hamdi, S and Bakri, Y and Nejjari, C and Amzazi, S and Ghazal, H},
title = {A review on current diagnostic techniques for COVID-19.},
journal = {Expert review of molecular diagnostics},
volume = {21},
number = {2},
pages = {141-160},
doi = {10.1080/14737159.2021.1886927},
pmid = {33593219},
issn = {1744-8352},
mesh = {Antibodies, Viral/immunology ; Biosensing Techniques ; COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*methods/*trends ; COVID-19 Serological Testing/*methods/*trends ; CRISPR-Cas Systems ; Clinical Laboratory Techniques ; Humans ; Immunoassay ; Immunoglobulin G/immunology ; Immunoglobulin M/immunology ; Laboratories ; Radiography, Thoracic ; Reagent Kits, Diagnostic ; Reverse Transcriptase Polymerase Chain Reaction ; Tomography, X-Ray Computed ; },
abstract = {INTRODUCTION: SARS-Cov-2 first appeared in Wuhan, China, in December 2019 and spread all over the world soon after that. Given the infectious nature ofSARS-CoV-2, fast and accurate diagnosis tools are important to detect the virus. In this review, we discuss the different diagnostic tests that are currently being implemented in laboratories and provide a description of various COVID-19 kits.<br><br>AREAS COVERED: We summarize molecular techniques that target the viral load, serological methods used for SARS-CoV-2 specific antibodies detection as well as newly developed faster assays for the detection of SARS-COV 2 in various biological samples.<br><br>EXPERT OPINION: In the light of the widespread pandemic, the massive diagnosis of COVID-19, using various detection techniques, appears to be the most effective strategy for monitoring and containing its propagation.},
}
@article {pmid33592566,
year = {2021},
author = {Sun, X and Zhou, X and Dong, B and Wang, C and Xiao, X and Wang, Y},
title = {Generation of a gene-corrected isogenic iPSC line (AHQUi001-A-1) from a patient with familial hypertriglyceridemia (FHTG) carrying a heterozygous p.C310R (c.928 T &gt; C) mutation in LPL gene using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102230},
doi = {10.1016/j.scr.2021.102230},
pmid = {33592566},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Heterozygote ; Humans ; *Hyperlipoproteinemia Type IV ; *Induced Pluripotent Stem Cells ; Mutation ; },
abstract = {Mutations in the LPL gene lead to familial hypertriglyceridemia (FHTG) . We have previously generated an iPSC line (AHQUi001-A) from a FHTG patient with a heterozygous p.C310R (c.928 T > C) mutation in the LPL gene. Here we genetically corrected the C310R mutation in the LPL gene using CRISPR/Cas9 technology to generate AHQUi001-A-1, which demonstrates normal karyotype, morphology, pluripotency, and potential to differentiate towards three germ layers.},
}
@article {pmid33592378,
year = {2021},
author = {Yin, JH and Elumalai, P and Kim, SY and Zhang, SZ and Shin, S and Lee, M and Chung, YJ},
title = {TNNC1 knockout reverses metastatic potential of ovarian cancer cells by inactivating epithelial-mesenchymal transition and suppressing F-actin polymerization.},
journal = {Biochemical and biophysical research communications},
volume = {547},
number = {},
pages = {44-51},
doi = {10.1016/j.bbrc.2021.02.021},
pmid = {33592378},
issn = {1090-2104},
mesh = {Actins/*metabolism ; Antigens, CD/metabolism ; CRISPR-Cas Systems ; Cadherins/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Epithelial-Mesenchymal Transition ; Female ; Gene Knockdown Techniques/methods ; Glycogen Synthase Kinase 3 beta/metabolism ; Humans ; Neoplasm Metastasis ; Ovarian Neoplasms/genetics/metabolism/pathology/*prevention &amp; control ; Proto-Oncogene Proteins c-akt/metabolism ; Troponin I/genetics/*metabolism ; Vimentin/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Troponin C type 1 (TNNC1) is commonly overexpressed in ovarian cancer. However, the biological implications of TNNC1 overexpression on ovarian cancer malignization and its related mechanism remain unknown. To elucidate these implications, we knocked out the TNNC1 gene in TNNC1-overexpressing SKOV-3-13 ovarian cancer cells using CRISPR/Cas-9 technology and observed the changes in metastatic phenotypes and related molecular pathways. TNNC1-knockout (KO) cells showed significantly reduced proliferation and colony formation when compared with TNNC1 wild-type cells (P < 0.01). In TNNC1-KO cells, wound healing, migration, and invasive phenotypes decreased. Upon observation of upstream regulators of epithelial-mesenchymal transition (EMT), levels of phosphorylated AKT (Ser-473 and Thr-308) and GSK-3β (inactive form) were found to be decreased in TNNC1-KO cells. Accordingly, SNAIL and SLUG expression decreased and were almost completely localized in the cytoplasm following TNNC1 silencing. Regarding downstream EMT markers, N-cadherin and vimentin expression decreased, but E-cadherin expression increased. Related matrix metalloproteinase and chemokine expression generally decreased. TNNC1 deficiency also suppressed F-actin polymerization. In conclusion, TNNC1 overexpression contributes to the metastatic behavior of ovarian cancer by perturbation of EMT and actin microfilaments. Our results provide a better understanding of the detailed molecular mechanism of ovarian cancer metastasis associated with TNNC1 overexpression.},
}
@article {pmid33591941,
year = {2021},
author = {Chen, F and Fang, S and Du, Y and Ghosh, A and Reed, MN and Long, Y and Suppiramaniam, V and Tang, S and Hong, H},
title = {CRISPR/Cas9-mediated CysLT1R deletion reverses synaptic failure, amyloidosis and cognitive impairment in APP/PS1 mice.},
journal = {Aging},
volume = {13},
number = {5},
pages = {6634-6661},
pmid = {33591941},
issn = {1945-4589},
mesh = {Amyloid beta-Protein Precursor ; Amyloidosis/*prevention &amp; control ; Animals ; *CRISPR-Cas Systems ; Cognitive Dysfunction/*prevention &amp; control ; Disease Models, Animal ; *Gene Deletion ; Mice, Knockout ; Mice, Transgenic ; Neuronal Plasticity ; Receptors, Leukotriene/*genetics ; *Synaptic Transmission ; },
abstract = {As a major pathological hallmark of Alzheimer's disease (AD), amyloid-β (Aβ) is regarded as a causative factor for cognitive impairment. Extensive studies have found Aβ induces a series of pathophysiological responses, finally leading to memory loss in AD. Our previous results demonstrated that cysteinyl leukotrienes receptor 1 (CysLT1R) antagonists improved exogenous Aβ-induced memory impairment. But the role of CysLT1R in AD and its underlying mechanisms still remain elusive. In this study, we investigated CysLT1R levels in AD patients and APP/PS1 mice. We also generated APP/PS1-CysLT1R[-/-] mice by clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated CysLT1R deletion in APP/PS1 mice and studied the effect of CysLT1R knockout on amyloidogenesis, synapse structure and plasticity, cognition, neuroinflammation, and kynurenine pathway. These attributes were also studied after lentivirus-mediated knockdown of CysLT1R gene in APP/PS1 mice. We found that CysLT1R knockout or knockdown could conserve synaptic structure and plasticity, and improve cognition in APP/PS1 mice. These effects were associated with concurrent decreases in amyloid processing, reduced neuroinflammation and suppression of the kynurenine pathway. Our study demonstrates that CysLT1R deficiency can mediate several beneficial effects against AD pathogenesis, and genetic/pharmacological ablation of this protein could be a potential therapeutic option for AD.},
}
@article {pmid33591555,
year = {2021},
author = {Saha, O and Rakhi, NN and Hoque, MN and Sultana, M and Hossain, MA},
title = {Genome-wide genetic marker analysis and genotyping of Escherichia fergusonii strain OTSVEF-60.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {52},
number = {2},
pages = {989-1004},
pmid = {33591555},
issn = {1678-4405},
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Drug Resistance, Bacterial/drug effects/genetics ; Escherichia/drug effects/*genetics/isolation &amp; purification ; Genome, Bacterial/*genetics ; Genotype ; Microbial Sensitivity Tests ; Poultry/microbiology ; Prophages/genetics ; Virulence/genetics ; },
abstract = {Poultry originated Escherichia fergusonii (POEF), an emerging bacterial pathogen, causes a wide range of intestinal and extra-intestinal infections in the poultry industry which incurred significant economic losses worldwide. Chromosomal co-existence of antibiotics and metal resistance genes has recently been the focal point of POEF isolates besides its pathogenic potentials. This study reports the complete genome analysis of POEF strain OTSVEF-60 from the poultry originated samples of Bangladesh. The assembled draft genome of the strain was 4.2 Mbp containing 4503 coding sequences, 120 RNA (rRNA = 34, tRNA = 79, ncRNA = 7), and three intact phage signature regions. Forty-one broad range antibiotic resistance genes (ARGs) including dfrA12, qnrS1, blaTEM-1, aadA2, tet(A), and sul-2 along with multiple efflux pump genes were detected, which translated to phenotypic resistant patterns of the pathogen to trimethoprim, fluoroquinolones, β-lactams, aminoglycoside, tetracycline, and sulfonamides. Moreover, 22 metal resistance genes were found co-existing within the genome of the POEF strain, and numerous virulence genes (VGs) coding for cit (AB), feo (AB), fep (ABCG), csg (ABCDEFG), fliC, ompA, gadA, ecpD, etc. were also identified throughout the genome. In addition, we detected a class I integron gene cassette harboring dfrA12, ant (3″)-I, and qacEΔ-sul2 genes; 42 copies of insertion sequence (IS) elements; and two CRISPR arrays. The genomic functional analysis predicted several metabolic pathways related to motility, flagellar assembly, epithelial cell invasion, quorum sensing, biofilm formation, and biosynthesis of vitamin, co-factors, and secondary metabolites. We herein for the first time detected multiple ARGs, VGs, mobile genetic elements, and some metabolic functional genes in the complete genome of POEF strain OTSVEF-60, which might be associated with the pathogenesis, spreading of ARGs and VGs, and subsequent treatment failure against this emerging avian pathogen with currently available antimicrobials.},
}
@article {pmid33591308,
year = {2021},
author = {Li, D and Sun, X and Yu, F and Perle, MA and Araten, D and Boeke, JD},
title = {Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency.},
journal = {Nucleic acids research},
volume = {49},
number = {5},
pages = {2642-2654},
pmid = {33591308},
issn = {1362-4962},
support = {P01 AG051449/AG/NIA NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; P50 GM107632/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Toxins/toxicity ; *CRISPR-Cas Systems ; *Cell Engineering ; Cell Line ; Chromosomes, Human, X ; Genetic Markers ; Heterozygote ; Humans ; Membrane Proteins/*genetics ; Mutation ; N-Acetylglucosaminyltransferases/genetics ; Pore Forming Cytotoxic Proteins/toxicity ; RNA/genetics ; *Sequence Deletion ; },
abstract = {The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).},
}
@article {pmid33590868,
year = {2021},
author = {Li, X and Xu, Y and Zhang, H and Yin, H and Zhou, D and Sun, Y and Ma, L and Shen, B and Zhu, C},
title = {ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae).},
journal = {Journal of medical entomology},
volume = {58},
number = {3},
pages = {1202-1209},
doi = {10.1093/jme/tjab016},
pmid = {33590868},
issn = {1938-2928},
mesh = {Animals ; *CRISPR-Cas Systems ; Culex/*genetics ; Female ; Gene Editing/*methods ; *Germ-Line Mutation ; Mosquito Vectors/genetics ; *Mutagenesis ; Ribonucleoproteins/genetics ; },
abstract = {The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes-14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G0 progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G0 progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control.},
}
@article {pmid33590814,
year = {2021},
author = {Watters, KE and Kirkpatrick, J and Palmer, MJ and Koblentz, GD},
title = {The CRISPR revolution and its potential impact on global health security.},
journal = {Pathogens and global health},
volume = {115},
number = {2},
pages = {80-92},
pmid = {33590814},
issn = {2047-7732},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Global Health ; },
abstract = {Global health security is constantly under threat from infectious diseases. Despite advances in biotechnology that have improved diagnosis and treatment of such diseases, delays in detecting outbreaks and the lack of countermeasures for some biological agents continue to pose severe challenges to global health security. In this review, we describe some of the challenges facing global health security and how genome editing technologies can help overcome them. We provide specific examples of how the genome-editing tool CRISPR is being used to develop new tools to characterize pathogenic agents, diagnose infectious disease, and develop vaccines and therapeutics to mitigate the effects of an outbreak. The article also discusses some of the challenges associated with genome-editing technologies and the efforts that scientists are undertaking to mitigate them. Overall, CRISPR and genome-editing technologies are poised to have a significant positive influence on global health security over the years to come.},
}
@article {pmid33590098,
year = {2021},
author = {Zhu, W and McQuarrie, S and Grüschow, S and McMahon, SA and Graham, S and Gloster, TM and White, MF},
title = {The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence.},
journal = {Nucleic acids research},
volume = {49},
number = {5},
pages = {2777-2789},
pmid = {33590098},
issn = {1362-4962},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 204821/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Clostridiales/enzymology ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry ; Deoxyribonuclease I/*chemistry/metabolism ; Enzyme Activation ; Escherichia coli/virology ; Interspersed Repetitive Sequences ; Metals/chemistry ; Models, Molecular ; Protein Domains ; Ribonucleases/*chemistry/metabolism ; },
abstract = {Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.},
}
@article {pmid33589617,
year = {2021},
author = {Hsu, JY and Grünewald, J and Szalay, R and Shih, J and Anzalone, AV and Lam, KC and Shen, MW and Petri, K and Liu, DR and Joung, JK and Pinello, L},
title = {PrimeDesign software for rapid and simplified design of prime editing guide RNAs.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {1034},
pmid = {33589617},
issn = {2041-1723},
support = {R35 HG010717/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; },
mesh = {Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Databases, Genetic ; Fabry Disease/genetics/metabolism/pathology ; Gene Editing/*methods ; *Genome, Human ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Hemophilia A/genetics/metabolism/pathology ; Humans ; Models, Biological ; Muscular Dystrophy, Duchenne/genetics/metabolism/pathology ; Mutation ; Nucleic Acid Conformation ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.},
}
@article {pmid33589527,
year = {2021},
author = {Hou, J and Wang, Y and Shi, L and Chen, Y and Xu, C and Saeedi, A and Pan, K and Bohat, R and Egan, NA and McKenzie, JA and Mbofung, RM and Williams, LJ and Yang, Z and Sun, M and Liang, X and Rodon Ahnert, J and Varadarajan, N and Yee, C and Chen, Y and Hwu, P and Peng, W},
title = {Integrating genome-wide CRISPR immune screen with multi-omic clinical data reveals distinct classes of tumor intrinsic immune regulators.},
journal = {Journal for immunotherapy of cancer},
volume = {9},
number = {2},
pages = {},
pmid = {33589527},
issn = {2051-1426},
support = {P50 CA221703/CA/NCI NIH HHS/United States ; R01 GM130838/GM/NIGMS NIH HHS/United States ; R01 CA187076/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; S10 OD024977/OD/NIH HHS/United States ; P50 CA093459/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cytotoxicity, Immunologic/genetics ; *Genomics ; Humans ; Immune Checkpoint Inhibitors/therapeutic use ; Immunotherapy ; Lymphocytes, Tumor-Infiltrating/immunology ; Mice, Inbred C57BL ; Mice, Transgenic ; Neoplasms/*genetics/immunology/therapy ; Protein-Arginine N-Methyltransferases/genetics ; Receptor-Interacting Protein Serine-Threonine Kinases/genetics ; Repressor Proteins/genetics ; T-Lymphocytes/immunology ; Tumor Escape/*genetics ; Tumor Microenvironment/*genetics/immunology ; },
abstract = {BACKGROUND: Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer.<br><br>METHODS: To identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in an unbiased manner, we performed genome-wide CRISPR immune screens and integrated our results with multi-omics clinical data to evaluate the role of tumor intrinsic factors in regulating two rate-limiting steps of cancer immunotherapy, namely, T cell tumor infiltration and T cell-mediated tumor killing.<br><br>RESULTS: Our studies revealed two distinct types of immune resistance regulators and demonstrated their potential as therapeutic targets to improve the efficacy of immunotherapy. Among them, PRMT1 and RIPK1 were identified as a dual immune resistance regulator and a cytotoxicity resistance regulator, respectively. Although the magnitude varied between different types of immunotherapy, genetically targeting PRMT1 and RIPK1 sensitized tumors to T-cell killing and anti-PD-1/OX40 treatment. Interestingly, a RIPK1-specific inhibitor enhanced the antitumor activity of T cell-based and anti-OX40 therapy, despite limited impact on T cell tumor infiltration.<br><br>CONCLUSIONS: Collectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations.},
}
@article {pmid33586431,
year = {2021},
author = {Wang, Y and Liu, D and Lin, H and Chen, D and Sun, J and Xie, Y and Wang, X and Ma, P and Nie, Y and Mei, H and Zhao, B and Huang, X and Jiang, G and Jiang, X and Qu, J and Zhao, J and Liu, J},
title = {Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2.},
journal = {ACS chemical biology},
volume = {16},
number = {3},
pages = {491-500},
pmid = {33586431},
issn = {1554-8937},
mesh = {Adenoviridae/chemistry ; Animals ; Bacterial Proteins/*genetics ; COVID-19/*diagnosis/genetics ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/*genetics ; Fluorescent Dyes/chemistry ; Humans ; Limit of Detection ; Mice, Inbred BALB C ; Nucleic Acid Amplification Techniques ; RNA Probes/genetics ; RNA, Viral/*analysis/genetics ; SARS-CoV-2/*chemistry ; Specimen Handling ; Spectrometry, Fluorescence ; },
abstract = {The outbreak of novel coronavirus SARS-CoV-2 has caused a worldwide threat to public health. COVID-19 patients with SARS-CoV-2 infection can develop clinical symptoms that are often confused with the infections of other respiratory pathogens. Sensitive and specific detection of SARS-CoV-2 with the ability to discriminate from other viruses is urgently needed for COVID-19 diagnosis. Herein, we streamlined a highly efficient CRISPR-Cas12a-based nucleic acid detection platform, termed Cas12a-linked beam unlocking reaction (CALIBURN). We show that CALIBURN could detect SARS-CoV-2 and other coronaviruses and influenza viruses with little cross-reactivity. Importantly, CALIBURN allowed accurate diagnosis of clinical samples with extremely low viral loads, which is a major obstacle for the clinical applications of existing CRISPR diagnostic platforms. When tested on the specimens from SARS-CoV-2-positive and negative donors, CALIBURN exhibited 73.0% positive and 19.0% presumptive positive rates and 100% specificity. Moreover, unlike existing CRISPR detection methods that were mainly restricted to respiratory specimens, CALIBURN displayed consistent performance across both respiratory and nonrespiratory specimens, suggesting its broad specimen compatibility. Finally, using a mouse model of SARS-CoV-2 infection, we demonstrated that CALIBURN allowed detection of coexisting pathogens without cross-reactivity from a single tissue specimen. Our results suggest that CALIBURN can serve as a versatile platform for the diagnosis of COVID-19 and other respiratory infectious diseases.},
}
@article {pmid33586253,
year = {2021},
author = {Stojkovic, M and Han, D and Jeong, M and Stojkovic, P and Stankovic, KM},
title = {Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss.},
journal = {Stem cells (Dayton, Ohio)},
volume = {39},
number = {6},
pages = {673-696},
doi = {10.1002/stem.3353},
pmid = {33586253},
issn = {1549-4918},
support = {R01DC015824/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Hearing Loss, Sensorineural/genetics/*therapy ; Humans ; Induced Pluripotent Stem Cells/*cytology ; },
abstract = {Hearing loss (HL) is a major global health problem of pandemic proportions. The most common type of HL is sensorineural hearing loss (SNHL) which typically occurs when cells within the inner ear are damaged. Human induced pluripotent stem cells (hiPSCs) can be generated from any individual including those who suffer from different types of HL. The development of new differentiation protocols to obtain cells of the inner ear including hair cells (HCs) and spiral ganglion neurons (SGNs) promises to expedite cell-based therapy and screening of potential pharmacologic and genetic therapies using human models. Considering age-related, acoustic, ototoxic, and genetic insults which are the most frequent causes of irreversible damage of HCs and SGNs, new methods of genome editing (GE), especially the CRISPR/Cas9 technology, could bring additional opportunities to understand the pathogenesis of human SNHL and identify novel therapies. However, important challenges associated with both hiPSCs and GE need to be overcome before scientific discoveries are correctly translated to effective and patient-safe applications. The purpose of the present review is (a) to summarize the findings from published reports utilizing hiPSCs for studies of SNHL, hence complementing recent reviews focused on animal studies, and (b) to outline promising future directions for deciphering SNHL using disruptive molecular and genomic technologies.},
}
@article {pmid33586084,
year = {2022},
author = {Tang, Y and Liao, S and Liu, G and Xiong, X and Liu, H and Li, F and Tan, Z and Kong, X and Yin, Y and Tan, B},
title = {Advanced single-cell pooled CRISPR screening identifies C19orf53 required for cell proliferation based on mTORC1 regulators.},
journal = {Cell biology and toxicology},
volume = {38},
number = {1},
pages = {43-68},
pmid = {33586084},
issn = {1573-6822},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Genomics ; Mechanistic Target of Rapamycin Complex 1/genetics ; *Transcriptome ; },
abstract = {Multiplexed single-cell CRISPR screening has accelerated the systematic dissection of biological discoveries; however, the efficiency of CRISPR-based gene knockout has inherent limitations. Here, we present DoNick-seq, an advanced method for facilitating gene knockout and reducing off-target activity. We re-engineered two popular plasmid constructs suitable for use in pooled CRISPR screening of the single-cell transcriptome. We then used DoNick-seq to probe mTORC1 regulators and obtain genomic perturbation and transcriptome profiles from the same cell. Thus, DoNick-seq enabled us to simultaneously evaluate multiple gene interactions and the effect of amino acid depletion. By analyzing more than 20,000 cells from two cell lines, DoNick-seq efficiently identified gene targets, cell numbers, and cellular profiles. Our data also revealed the characteristics of mTORC1 negative and positive regulators, thereby shedding new insights into the mechanisms regulating cell growth and inhibition. We demonstrate that mTORC1 hyperactivation exhausts cellular free amino acids via increased proliferation ability. Furthermore, DoNick-seq identified the gene C19orf53, which mediates excessive cell proliferation, resulting in metabolic imbalance, and greatly enhances oxidative stress in response to toxins. Thus, our findings suggest that DoNick-seq facilitates high-throughput functional dissection of complex cellular responses at the single-cell level and increases the accuracy of CRISPR single-cell transcriptomics.},
}
@article {pmid33585286,
year = {2020},
author = {Newsom, S and Parameshwaran, HP and Martin, L and Rajan, R},
title = {The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {619763},
pmid = {33585286},
issn = {2235-2988},
mesh = {Archaea/genetics ; Biotechnology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine.},
}
@article {pmid33584573,
year = {2020},
author = {Tripathi, L and Ntui, VO and Tripathi, JN and Kumar, PL},
title = {Application of CRISPR/Cas for Diagnosis and Management of Viral Diseases of Banana.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {609784},
pmid = {33584573},
issn = {1664-302X},
abstract = {Viral diseases are significant biotic constraints for banana (Musa spp.) production as they affect the yield and limit the international movement of germplasm. Among all the viruses known to infect banana, the banana bunchy top virus and banana streak viruses are widespread and economically damaging. The use of virus-resistant bananas is the most cost-effective option to minimize the negative impacts of viral-diseases on banana production. CRISPR/Cas-based genome editing is emerging as the most powerful tool for developing virus-resistant crop varieties in several crops, including the banana. The availability of a vigorous genetic transformation and regeneration system and a well-annotated whole-genome sequence of banana makes it a compelling candidate for genome editing. A robust CRISPR/Cas9-based genome editing of the banana has recently been established, which can be applied in developing disease-resistant varieties. Recently, the CRISPR system was exploited to detect target gene sequences using Cas9, Cas12, Cas13, and Cas14 enzymes, thereby unveiling the use of this technology for virus diagnosis. This article presents a synopsis of recent advancements and perspectives on the application of CRISPR/Cas-based genome editing for diagnosing and developing resistance against banana viruses and challenges in genome-editing of banana.},
}
@article {pmid33584572,
year = {2020},
author = {Shahid, MS and Sattar, MN and Iqbal, Z and Raza, A and Al-Sadi, AM},
title = {Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {609376},
pmid = {33584572},
issn = {1664-302X},
abstract = {In recent years, next-generation sequencing (NGS) and contemporary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have revolutionized the life sciences and the field of plant virology. Both these technologies offer an unparalleled platform for sequencing and deciphering viral metagenomes promptly. Over the past two decades, NGS technologies have improved enormously and have impacted plant virology. NGS has enabled the detection of plant viruses that were previously undetectable by conventional approaches, such as quarantine and archeological plant samples, and has helped to track the evolutionary footprints of viral pathogens. The CRISPR-Cas-based genome editing (GE) and detection techniques have enabled the development of effective approaches to virus resistance. Different versions of CRISPR-Cas have been employed to successfully confer resistance against diverse plant viruses by directly targeting the virus genome or indirectly editing certain host susceptibility factors. Applications of CRISPR-Cas systems include targeted insertion and/or deletion, site-directed mutagenesis, induction/expression/repression of the gene(s), epigenome re-modeling, and SNPs detection. The CRISPR-Cas toolbox has been equipped with precision GE tools to engineer the target genome with and without double-stranded (ds) breaks or donor templates. This technique has also enabled the generation of transgene-free genetically engineered plants, DNA repair, base substitution, prime editing, detection of small molecules, and biosensing in plant virology. This review discusses the utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology.},
}
@article {pmid33583210,
year = {2021},
author = {Liu, S and Feng, Y and Huang, Y and Jiang, X and Tang, C and Tang, F and Zeng, C and Liu, L},
title = {A GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {246},
number = {11},
pages = {1330-1341},
pmid = {33583210},
issn = {1535-3699},
mesh = {Animals ; Autophagy/*physiology ; Behavior, Animal/physiology ; Brain/metabolism/pathology ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; G(M1) Ganglioside/*metabolism ; Gangliosidosis, GM1/etiology/*genetics/*pathology ; Gene Editing/methods ; Male ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Mutant Strains ; Microglia/*pathology ; Phenotype ; beta-Galactosidase/genetics ; },
abstract = {GM1 gangliosidosis is a rare lysosomal storage disease caused by a deficiency of β-galactosidase due to mutations in the GLB1 gene. We established a C57BL/6 mouse model with Glb1[G455R] mutation using CRISPR/Cas9 genome editing. The β-galactosidase enzyme activity of Glb1[G455R] mice measured by fluorometric assay was negligible throughout the whole body. Mutant mice displayed no marked phenotype at eight weeks. After 16 weeks, GM1 ganglioside accumulation in the brain of mutant mice was observed by immunohistochemical staining. Meanwhile, a declining performance in behavioral tests was observed among mutant mice from 16 to 32 weeks. As the disease progressed, the neurological symptoms of mutant mice worsened, and they then succumbed to the disease by 47 weeks of age. We also observed microglia activation and proliferation in the cerebral cortex of mutant mice at 16 and 32 weeks. In these activated microglia, the level of autophagy regulator LC3 was up-regulated but the mRNA level of LC3 was normal. In conclusion, we developed a novel murine model that mimicked the chronic phenotype of human GM1. This Glb1[G455R] murine model is a practical in vivo model for studying the pathogenesis of GM1 gangliosidosis and exploring potential therapies.},
}
@article {pmid33582559,
year = {2021},
author = {Zhou, CM and Wu, Q and Wang, B and Lin, P and Wu, M and Yu, XJ},
title = {Calcium-responsive kinase LadS modulates type I-F CRISPR-Cas adaptive immunity.},
journal = {Biochemical and biophysical research communications},
volume = {546},
number = {},
pages = {155-161},
doi = {10.1016/j.bbrc.2021.01.100},
pmid = {33582559},
issn = {1090-2104},
mesh = {Bacterial Proteins/*metabolism ; Base Sequence ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*physiology ; Calcium/*metabolism ; Protein Kinases/*metabolism ; Pseudomonas aeruginosa/*enzymology/genetics/metabolism ; RNA, Bacterial/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; },
abstract = {The CRISPR-Cas systems are recently discovered adaptive immune strategies in bacteria and archaea against foreign genetic elements. Although gene-editing enabled by CRISPR-Cas9 has shown great promise for clinical application, little is known about potential mechanisms of CRISPR-Cas systems for regulating their own gene expression and altering the virulence within bacteria. Here, Gram-negative bacterium Pseudomonas aeruginosa PA14 that contains a Type I-F CRISPR-Cas system was used to study the mechanism endogenous CRISPR-Cas of regulation mechanism. We delineated the role of calcium as a positive regulator of the transcription of cas/csy complex and CRISPR-Cas immunity through the two-component system (TCS) protein kinase LadS. Furthermore, we identified a LadS downstream post-transcriptional regulator, RsmA, which targeted translation region of cas mRNA via A(N)GGA motif. Importantly, calcium-mediated influencing of CRISPR-Cas system was dependent on LadS and RsmA. Altogether, our findings uncover the previously unrecognized role of LadS/RsmA in modulating Type I-F CRISPR-Cas system via sensing calcium.},
}
@article {pmid33582546,
year = {2021},
author = {Han, HJ and Kim, JH},
title = {Establishment of a TLR3 homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {52},
number = {},
pages = {102187},
doi = {10.1016/j.scr.2021.102187},
pmid = {33582546},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; RNA, Double-Stranded ; *Toll-Like Receptor 3/genetics/metabolism ; },
abstract = {The Toll-like receptor (TLR) family plays an important role in the recognition of pathogens, including bacteria, viruses, fungi, and parasites, followed by the activation of innate immunity. TLR3 recognizes double-stranded RNA, a form of genetic material produced by positive-strand RNA viruses and DNA viruses, and is activated by viral infection. Upon recognition, TLR3 promotes the activation of interferon regulatory factor 3 to enhance the expression and secretion of type I interferons that signal other cells to enhance their antiviral defenses. We generated biallelic mutants of the TLR3 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). TLR3 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.},
}
@article {pmid33582407,
year = {2021},
author = {Tian, X and Ahsan, N and Lulla, A and Lev, A and Abbosh, P and Dicker, DT and Zhang, S and El-Deiry, WS},
title = {P53-independent partial restoration of the p53 pathway in tumors with mutated p53 through ATF4 transcriptional modulation by ERK1/2 and CDK9.},
journal = {Neoplasia (New York, N.Y.)},
volume = {23},
number = {3},
pages = {304-325},
pmid = {33582407},
issn = {1476-5586},
mesh = {Activating Transcription Factor 4/*genetics/*metabolism ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; Cyclin-Dependent Kinase 9/*metabolism ; Endoplasmic Reticulum Stress ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genes, myc ; Humans ; Inhibitory Concentration 50 ; MAP Kinase Signaling System ; Models, Biological ; *Mutation ; *Signal Transduction ; Tumor Suppressor Protein p53/*genetics/*metabolism ; },
abstract = {A long-term goal in the cancer-field has been to develop strategies for treating p53-mutated tumors. A novel small-molecule, PG3-Oc, restores p53 pathway-signaling in tumor cells with mutant-p53, independently of p53/p73. PG3-Oc partially upregulates the p53-transcriptome (13.7% of public p53 target-gene dataset; 15.2% of in-house dataset) and p53-proteome (18%, HT29; 16%, HCT116-p53[-/-]). Bioinformatic analysis indicates critical p53-effectors of growth-arrest (p21), apoptosis (PUMA, DR5, Noxa), autophagy (DRAM1), and metastasis-suppression (NDRG1) are induced by PG3-Oc. ERK1/2- and CDK9-kinases are required to upregulate ATF4 by PG3-Oc which restores p53 transcriptomic-targets in cells without functional-p53. PG3-Oc represses MYC (ATF4-independent), and upregulates PUMA (ATF4-dependent) in mediating cell death. With largely nonoverlapping transcriptomes, induced-ATF4 restores p53 transcriptomic targets in drug-treated cells including functionally important mediators such as PUMA and DR5. Our results demonstrate novel p53-independent drug-induced molecular reprogramming involving ERK1/2, CDK9, and ATF4 to restore upregulation of p53 effector genes required for cell death and tumor suppression.},
}
@article {pmid33582298,
year = {2021},
author = {Blondal, T and Gamba, C and Møller Jagd, L and Su, L and Demirov, D and Guo, S and Johnston, CM and Riising, EM and Wu, X and Mikkelsen, MJ and Szabova, L and Mouritzen, P},
title = {Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing.},
journal = {Methods (San Diego, Calif.)},
volume = {191},
number = {},
pages = {68-77},
doi = {10.1016/j.ymeth.2021.02.003},
pmid = {33582298},
issn = {1095-9130},
support = {HHSN261200800001E/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; },
abstract = {Validation of CRISPR-Cas9 editing typically explores the immediate vicinity of the gene editing site and distal off-target sequences, which has led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene-edited region in four cell lines on long- and short-read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kilobases-large insertions in three of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.},
}
@article {pmid33582214,
year = {2021},
author = {Hua, R and Cheng, J and Yang, L and Zhang, Z and Xu, D and Chen, H and Li, Y and Duan, J and Li, X and Geng, G and Liu, J and Li, Q},
title = {Development and characterization of in vitro self-assembled recombinant human follicle stimulating hormone originated from goat mammary epithelial cells.},
journal = {Molecular and cellular endocrinology},
volume = {526},
number = {},
pages = {111211},
doi = {10.1016/j.mce.2021.111211},
pmid = {33582214},
issn = {1872-8057},
mesh = {Animals ; Aromatase/genetics/metabolism ; Base Sequence ; CHO Cells ; CRISPR-Cas Systems/genetics ; Cricetulus ; Cyclic AMP/metabolism ; Endocytosis/drug effects ; Epithelial Cells/drug effects/*metabolism ; Estradiol/blood ; Female ; Follicle Stimulating Hormone, beta Subunit/*biosynthesis/pharmacology ; Gene Expression Regulation/drug effects ; Glycoprotein Hormones, alpha Subunit/*biosynthesis/pharmacology ; Glycosylation ; Goats/*physiology ; Granulosa Cells/drug effects/metabolism ; Lactoglobulins/genetics ; Ligands ; Mammary Glands, Animal/*cytology ; Mice ; N-Acetylneuraminic Acid/metabolism ; Ovulation/drug effects ; Protein Subunits/pharmacology ; RNA, Guide/metabolism ; Rats ; Recombinant Proteins/*biosynthesis/pharmacology ; Weight Gain/drug effects ; },
abstract = {Follicle stimulating hormone (FSH), composed of FSHα and FSHβ subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHβ stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHβ expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHβ gene separately into goat β-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHβ subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHβ can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.},
}
@article {pmid33581720,
year = {2021},
author = {Owen, JR and Hennig, SL and McNabb, BR and Mansour, TA and Smith, JM and Lin, JC and Young, AE and Trott, JF and Murray, JD and Delany, ME and Ross, PJ and Van Eenennaam, AL},
title = {One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.},
journal = {BMC genomics},
volume = {22},
number = {1},
pages = {118},
pmid = {33581720},
issn = {1471-2164},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; Female ; Gene Editing ; Gene Knock-In Techniques ; Male ; *Zygote ; },
abstract = {BACKGROUND: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.<br><br>RESULTS: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1&#x2009;kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1&#x2009;kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26&#x2009;bp insertion allele, and a complex 38&#x2009;kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18&#x2009;kb allele was detected that looks to be a derivative of the 38&#x2009;kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.<br><br>CONCLUSION: The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.},
}
@article {pmid33581654,
year = {2021},
author = {Kwarteng, A and Sylverken, A and Asiedu, E and Ahuno, ST},
title = {Genome editing as control tool for filarial infections.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {137},
number = {},
pages = {111292},
doi = {10.1016/j.biopha.2021.111292},
pmid = {33581654},
issn = {1950-6007},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Filariasis/genetics/parasitology/*therapy ; Filaricides/therapeutic use ; Filarioidea/drug effects/*genetics/pathogenicity ; *Gene Editing ; *Genetic Therapy ; Humans ; Protozoan Vaccines/therapeutic use ; },
abstract = {Human filarial infections are vector-borne nematode infections, which include lymphatic filariasis, onchocerciasis, loiasis, and mansonella filariasis. With a high prevalence in developing countries, filarial infections are responsible for some of the most debilitating morbidities and a vicious cycle of poverty and disease. Global initiatives set to eradicate these infections include community mass treatments, vector control, provision of care for morbidity, and search for vaccines. However, there are growing challenges associated with mass treatments, vector control, and antifilarial vaccine development. With the emergence of genome editing tools and successful applications in other infectious diseases, the integration of genetic editing techniques in future control strategies for filarial infections would offer the best option for eliminating filarial infections. In this review, we briefly discuss the mechanisms of the three main genetic editing techniques and explore the potential applications of these powerful tools to control filarial infections.},
}
@article {pmid33581428,
year = {2021},
author = {Li, F and Ye, Q and Chen, M and Zhou, B and Zhang, J and Pang, R and Xue, L and Wang, J and Zeng, H and Wu, S and Zhang, Y and Ding, Y and Wu, Q},
title = {An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {179},
number = {},
pages = {113073},
doi = {10.1016/j.bios.2021.113073},
pmid = {33581428},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Humans ; *Listeria monocytogenes/genetics ; },
abstract = {Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high patient mortality. Accordingly, it is necessary to develop a L. monocytogenes detection platform with high specificity, sensitivity, and exploitability. CRISPR/Cas systems have shown great potential in the development of next-generation biosensors for nucleic acid detection, owing to the trans-cleavage capabilities of the Cas effector proteins. Herein, we introduce the trans-cleavage activity of CRISPR/Cas12a into an electrochemical biosensor (E-CRISPR), combined with recombinase-assisted amplification (RAA), to establish a cost-effective, specific and ultrasensitive method; namely RAA-based E-CRISPR. The concept behind this approach is that the target will induce the number change of the surface signaling probe (containing an electrochemical tag), which leads to a variation in the electron transfer of the electrochemical tag. The introduction of an RAA-based Cas12a system into the E-CRISPR sensor achieves a more prominent signal change between the presence and absence of the target. Under optimized conditions, RAA-based E-CRISPR can detect as low as 0.68 aM of genomic DNA and 26 cfu/mL of L. monocytogenes in pure cultures. More importantly, the RAA-based E-CRISPR enables rapid and ultrasensitive detection of L. monocytogenes in spiked and natural Flammulina velutipes samples. Moreover, no cross-reactivity with other non-target bacteria was observed. This system thus demonstrates to be a simple, high-sensitivity, and high-accuracy platform for L. monocytogenes detection.},
}
@article {pmid33581252,
year = {2021},
author = {Wu, QW and Kapfhammer, JP},
title = {Conditional gene silencing via a CRISPR system in cerebellar Purkinje cells.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1865},
number = {6},
pages = {129869},
doi = {10.1016/j.bbagen.2021.129869},
pmid = {33581252},
issn = {1872-8006},
mesh = {Animals ; *CRISPR-Cas Systems ; Cerebellum/metabolism/*pathology ; *Gene Silencing ; Mice ; Mice, Knockout ; Protein Kinase C/*antagonists &amp; inhibitors/genetics ; Purkinje Cells/metabolism/*pathology ; },
}
@article {pmid33581168,
year = {2021},
author = {Wei, J},
title = {Accurate and sensitive analysis of Staphylococcus aureus through CRISPR-Cas12a based recycling signal amplification cascades for early diagnosis of skin and soft tissue infections.},
journal = {Journal of microbiological methods},
volume = {183},
number = {},
pages = {106167},
doi = {10.1016/j.mimet.2021.106167},
pmid = {33581168},
issn = {1872-8359},
mesh = {CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/*methods ; Sensitivity and Specificity ; Soft Tissue Infections/diagnosis/*microbiology ; Staphylococcal Infections/diagnosis/*microbiology ; Staphylococcal Skin Infections/diagnosis/*microbiology ; Staphylococcus aureus/classification/*genetics/growth &amp; development/*isolation &amp; purification ; },
abstract = {Early-diagnosis and treatments of skin and soft tissue infections remain a huge challenge due to the difficulties in the detection of trace amounts of bacteria. We develop here a novel method through CRISPR-Cas12a based recycling signal amplification cascades and demonstrated its feasibility of Staphylococcus aureus detection in a sensitive and accurate way. The highlights of the proposed method are calculated as: i) the designed allosteric probe is responsible for accurate identification of SA through PBP2a-specific aptamer; ii) high sensitivity from three processes, including DNA polymerase-based target SA release, Nb.BbvCI enzyme induced ssDNA generation and attached CRISPR-Cas12a. As a result, the proposed method exhibited a detection range from 10[6] to 10[2] CFU/ml. Eventually, we believe that the proposed method could be expanded for the construction of diverse sensing platforms for detecting different trace bacteria.},
}
@article {pmid33580825,
year = {2021},
author = {Chumduri, C and Turco, MY},
title = {Organoids of the female reproductive tract.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {99},
number = {4},
pages = {531-553},
pmid = {33580825},
issn = {1432-1440},
mesh = {Animals ; CRISPR-Cas Systems ; Drug Discovery/methods ; Estrous Cycle/physiology ; Extracellular Matrix/physiology ; Female ; Fertility ; Gene Editing/methods/trends ; Genital Diseases, Female/pathology/therapy ; Genitalia, Female/anatomy &amp; histology/*cytology/physiology ; Gestational Age ; Gonadal Steroid Hormones/physiology ; Humans ; Maternal-Fetal Exchange ; Mice ; *Organoids/cytology ; Pituitary Hormones, Anterior/physiology ; Placenta/cytology ; Precision Medicine/methods/trends ; Pregnancy ; Stem Cells/cytology ; Tissue Engineering/methods/trends ; },
abstract = {Healthy functioning of the female reproductive tract (FRT) depends on balanced and dynamic regulation by hormones during the menstrual cycle, pregnancy and childbirth. The mucosal epithelial lining of different regions of the FRT-ovaries, fallopian tubes, uterus, cervix and vagina-facilitates the selective transport of gametes and successful transfer of the zygote to the uterus where it implants and pregnancy takes place. It also prevents pathogen entry. Recent developments in three-dimensional (3D) organoid systems from the FRT now provide crucial experimental models that recapitulate the cellular heterogeneity and physiological, anatomical and functional properties of the organ in vitro. In this review, we summarise the state of the art on organoids generated from different regions of the FRT. We discuss the potential applications of these powerful in vitro models to study normal physiology, fertility, infections, diseases, drug discovery and personalised medicine.},
}
@article {pmid33580373,
year = {2021},
author = {Wan, ZY and Lin, VCL and Hua, YG},
title = {Pomc Plays an Important Role in Sexual Size Dimorphism in Tilapia.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {23},
number = {2},
pages = {201-214},
pmid = {33580373},
issn = {1436-2236},
mesh = {Animals ; Brain/metabolism ; Estrogens ; Feeding Behavior/drug effects ; Female ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Male ; Pro-Opiomelanocortin/*genetics ; Sequence Analysis, DNA ; *Sex Characteristics ; Tilapia/*genetics/growth &amp; development ; Zebrafish/*genetics/growth &amp; development ; },
abstract = {Sexual dimorphism is common across the animal kingdom. Knowledge of the mechanisms of sexual size dimorphism is limited although it is important in biology and aquaculture. Tilapia is the common name for ~ 100 species of cichlid fish. Some are important aquaculture species and males outgrow females. To gain novel insights into the mechanisms underlying sexual size dimorphism, we analyzed the differences of brain transcriptomes between males and females in Mozambique tilapia and studied the function of the pro-opiomelanocortin (Pomc) gene in tilapia and zebrafish. The transcriptome analysis identified 123, 55, and 2706 sex-biased genes at 5, 30, and 90 dph (days post-hatch), respectively, indicating sexual dimorphism of gene expressions in the brain. The expression of Pomc in the tilapia brain was a female-biased at 30, 90, and 120 dph. An analysis of the DNA sequence located upstream of the tilapia Pomc transcriptional start site identified two estrogenic response elements. In vitro luciferase assay of the two elements revealed that β-estradiol significantly enhanced the expression of luciferase activity, suggesting that the expression of Pomc is mediated by estrogen. We knocked out Pomc in zebrafish using Crispr/Cas-9. The Pomc-knockout zebrafish showed faster growth and higher sensitivity to feeding as compared to the wild-type fish. Taken together, our results indicate that Pomc contributes to sexual size dimorphism and suggest that the high estrogen level in females promotes the expression of Pomc and suppresses feeding in female tilapias, which leads to the slower growth of female tilapias.},
}
@article {pmid33580230,
year = {2021},
author = {},
title = {Transient editing catches the eye.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {127},
doi = {10.1038/s41551-021-00695-z},
pmid = {33580230},
issn = {2157-846X},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Eye Diseases/genetics/*therapy ; Gene Editing/*methods ; Gene Expression ; Humans ; },
}
@article {pmid33580229,
year = {2021},
author = {Bubeck, F and Grimm, D},
title = {'Hit and run' therapy averts macular degeneration.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {132-133},
pmid = {33580229},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Cas Systems ; *Macular Degeneration/therapy ; Mice ; *RNA, Guide ; RNA, Messenger ; },
}
@article {pmid33580228,
year = {2021},
author = {Furling, D},
title = {Cas9 targeting of toxic foci of RNA repeats.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {130-131},
pmid = {33580228},
issn = {2157-846X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; *Myotonic Dystrophy/genetics ; Phenotype ; RNA/genetics ; },
}
@article {pmid33580208,
year = {2021},
author = {Ye, T and Duan, Y and Tsang, HWS and Xu, H and Chen, Y and Cao, H and Chen, Y and Fu, AKY and Ip, NY},
title = {Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {195},
pmid = {33580208},
issn = {2399-3642},
mesh = {Amyloid beta-Peptides/metabolism ; Amyloid beta-Protein Precursor/genetics/metabolism ; Apoptosis ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Copy Number Variations ; *Gene Dosage ; *Gene Editing ; Gene Expression Regulation ; Humans ; Induced Pluripotent Stem Cells/*metabolism/pathology ; Neurogenesis ; Neurons/*metabolism/pathology ; Phosphorylation ; Proof of Concept Study ; tau Proteins/metabolism ; },
abstract = {The dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause of autosomal dominant diseases such as Alzheimer's disease. However, there is currently no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we demonstrate a simple and precise method to simultaneously generate iPSC lines with different gene dosages using paired Cas9 nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motif specificity to expand the targetability of double-nicking-mediated genome editing. As a proof-of-concept study, we examine the gene dosage effects on an Alzheimer's disease patient-derived iPSC line that carries three copies of APP (amyloid precursor protein). This method enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically corrected iPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes of amyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCs with different gene dosages using our method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.},
}
@article {pmid33579329,
year = {2021},
author = {Zhou, T and Yuan, Z and Weng, J and Pei, D and Du, X and He, C and Lai, P},
title = {Challenges and advances in clinical applications of mesenchymal stromal cells.},
journal = {Journal of hematology &amp; oncology},
volume = {14},
number = {1},
pages = {24},
pmid = {33579329},
issn = {1756-8722},
mesh = {Animals ; Artificial Intelligence ; COVID-19/therapy ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Movement ; Clinical Trials as Topic ; Extracellular Vesicles/genetics/immunology/transplantation ; Genetic Engineering/methods ; Humans ; *Mesenchymal Stem Cell Transplantation/methods ; Mesenchymal Stem Cells/*cytology/immunology/metabolism ; },
abstract = {Mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have been intensely investigated for clinical applications within the last decades. However, the majority of registered clinical trials applying MSC therapy for diverse human diseases have fallen short of expectations, despite the encouraging pre-clinical outcomes in varied animal disease models. This can be attributable to inconsistent criteria for MSCs identity across studies and their inherited heterogeneity. Nowadays, with the emergence of advanced biological techniques and substantial improvements in bio-engineered materials, strategies have been developed to overcome clinical challenges in MSC application. Here in this review, we will discuss the major challenges of MSC therapies in clinical application, the factors impacting the diversity of MSCs, the potential approaches that modify MSC products with the highest therapeutic potential, and finally the usage of MSCs for COVID-19 pandemic disease.},
}
@article {pmid33577982,
year = {2022},
author = {Nguyen, LT and Gurijala, J and Rananaware, SR and Pizzano, BLM and Stone, BT and Jain, PK},
title = {CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a.},
journal = {Methods (San Diego, Calif.)},
volume = {203},
number = {},
pages = {116-124},
doi = {10.1016/j.ymeth.2021.02.001},
pmid = {33577982},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; *SARS-CoV-2 ; },
abstract = {Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification. Using the CRISPR-ENHANCE platform and following the methodology detailed in this paper, nucleic acid detection for low copy numbers can be achieved in less than an hour through either a fluorescence-based detection or a lateral flow assay. The step-by-step instructions provided, in addition to describing how to perform both assays, incorporate details on a LAMP/RT-LAMP-based target amplification step to enable detection of RNA, ssDNA and dsDNA. Furthermore, a protocol for in-house expression and purification of LbCas12a using CL7/lm7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided.},
}
@article {pmid33577797,
year = {2021},
author = {Jia, R and Bonifacino, JS},
title = {The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100405},
pmid = {33577797},
issn = {1083-351X},
support = {ZIA HD001607/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Autophagy/physiology ; Cell Line ; Cell Line, Tumor ; Endopeptidases/metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; Intracellular Signaling Peptides and Proteins ; Microtubule-Associated Proteins/*metabolism/physiology ; Protein Processing, Post-Translational ; Sequestosome-1 Protein ; Ubiquitin Thiolesterase/genetics/*metabolism/physiology ; Ubiquitin-Activating Enzymes/metabolism ; Ubiquitination ; },
abstract = {Components of the autophagy machinery are subject to regulation by various posttranslational modifications. Previous studies showed that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination.},
}
@article {pmid33577603,
year = {2021},
author = {Drabavicius, G and Daelemans, D},
title = {Intermedilysin cytolytic activity depends on heparan sulfates and membrane composition.},
journal = {PLoS genetics},
volume = {17},
number = {2},
pages = {e1009387},
pmid = {33577603},
issn = {1553-7404},
mesh = {Bacterial Proteins/genetics/*metabolism ; Bacteriocins/genetics/*metabolism ; CD59 Antigens/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Membrane/*metabolism ; Cholesterol/*metabolism ; Cytotoxins/genetics/*metabolism ; HEK293 Cells ; Heparitin Sulfate/*metabolism ; Humans ; Porosity ; },
abstract = {Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin's cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism.},
}
@article {pmid33577114,
year = {2021},
author = {Stuttmann, J and Barthel, K and Martin, P and Ordon, J and Erickson, JL and Herr, R and Ferik, F and Kretschmer, C and Berner, T and Keilwagen, J and Marillonnet, S and Bonas, U},
title = {Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {1},
pages = {8-22},
doi = {10.1111/tpj.15197},
pmid = {33577114},
issn = {1365-313X},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant/genetics ; Mutation/genetics ; Plants, Genetically Modified/*genetics ; Tobacco/*genetics ; },
abstract = {Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2 , respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1 , we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.},
}
@article {pmid33576013,
year = {2021},
author = {Bhat, MA and Mir, RA and Kumar, V and Shah, AA and Zargar, SM and Rahman, S and Jan, AT},
title = {Mechanistic insights of CRISPR/Cas-mediated genome editing towards enhancing abiotic stress tolerance in plants.},
journal = {Physiologia plantarum},
volume = {172},
number = {2},
pages = {1255-1268},
doi = {10.1111/ppl.13359},
pmid = {33576013},
issn = {1399-3054},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Stress, Physiological/genetics ; },
abstract = {Abiotic stresses such as temperature (high/low), drought, salinity, and others make the environment hostile to plants. Abiotic stressors adversely affect plant growth and development; and thereby makes a direct impact on overall plant productivity. Plants confront stress by developing an internal defense system orchestrated by compatible solutes, reactive oxygen species scavengers and phytohormones. However, routine exposure to unpredictable environmental stressors makes it essential to equip plants with a system that contributes to sustainable agricultural productivity, besides imparting multi-stress tolerance. The sustainable approach against abiotic stress is accomplished through breeding of tolerant cultivars. Though eco-friendly, tedious screening and crossing protocol limits its usage to overcome stress and in attaining the goal of global food security. Advancement on the technological front has enabled adoption of genomic engineering approaches to perform site-specific modification in the plant genome for improving adaptability, increasing the yield and in attributing resilience against different stressors. Of the different genome editing approaches, CRISPR/Cas has revolutionized biological research with wider applicability to crop plants. CRISPR/Cas emerged as a versatile tool in editing genomes for desired traits in highly accurate and precise manner. The present study summarizes advancement of the CRISPR/Cas genome editing tool in its adoption to manipulate plant genomes for novel traits towards developing high-yielding and climate-resilient crop varieties.},
}
@article {pmid33575613,
year = {2020},
author = {Podlevsky, JD and Hudson, CM and Timlin, JA and Williams, KP},
title = {CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data.},
journal = {NAR genomics and bioinformatics},
volume = {2},
number = {3},
pages = {lqaa063},
pmid = {33575613},
issn = {2631-9268},
abstract = {CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays-even isolated arrays-from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.},
}
@article {pmid33575551,
year = {2020},
author = {Pan, M and Hidalgo-Cantabrana, C and Barrangou, R},
title = {Host and body site-specific adaptation of Lactobacillus crispatus genomes.},
journal = {NAR genomics and bioinformatics},
volume = {2},
number = {1},
pages = {lqaa001},
pmid = {33575551},
issn = {2631-9268},
abstract = {Lactobacillus crispatus is a common inhabitant of both healthy poultry gut and human vaginal tract, and the absence of this species has been associated with a higher risk of developing infectious diseases. In this study, we analyzed 105 L. crispatus genomes isolated from a variety of ecological niches, including the human vaginal tract, human gut, chicken gut and turkey gut, to shed light on the genetic and functional features that drive evolution and adaptation of this important species. We performed in silico analyses to identify the pan and core genomes of L. crispatus, and to reveal the genomic differences and similarities associated with their origins of isolation. Our results demonstrated that, although a significant portion of the genomic content is conserved, human and poultry L. crispatus isolates evolved to encompass different genomic features (e.g. carbohydrate usage, CRISPR-Cas immune systems, prophage occurrence) in order to thrive in different environmental niches. We also observed that chicken and turkey L. crispatus isolates can be differentiated based on their genomic information, suggesting significant differences may exist between these two poultry gut niches. These results provide insights into host and niche-specific adaptation patterns in species of human and animal importance.},
}
@article {pmid33574580,
year = {2021},
author = {Wagner, DL and Peter, L and Schmueck-Henneresse, M},
title = {Cas9-directed immune tolerance in humans-a model to evaluate regulatory T cells in gene therapy?.},
journal = {Gene therapy},
volume = {28},
number = {9},
pages = {549-559},
pmid = {33574580},
issn = {1476-5462},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; Humans ; Immune Tolerance ; *T-Lymphocytes, Regulatory ; },
abstract = {The dichotomic nature of the adaptive immune response governs the outcome of clinical gene therapy. On the one hand, neutralizing antibodies and cytotoxic T cells can have a dramatic impact on the efficacy and safety of human gene therapies. On the other hand, regulatory T cells (Treg) can promote tolerance toward transgenes thereby enabling long-term benefits of in vivo gene therapy after a single administration. Pre-existing antibodies and T cell immunity has been a major obstacle for in vivo gene therapies with viral vectors. As CRISPR-Cas9 gene editing advances toward the clinics, the technology's inherent immunogenicity must be addressed in order to guide clinical treatment decisions. This review summarizes the recent evidence on Cas9-specific immunity in humans-including early results from clinical trials-and discusses the risks for in vivo gene therapies. Finally, we focus on solutions and highlight the potential role of Cas9-specific Treg cells to promote immune tolerance. As a "beneficial alliance" beyond Cas9-immunity, antigen-specific Treg cells may serve as a living and targeted immunosuppressant to increase safety and efficacy of gene therapy.},
}
@article {pmid33574281,
year = {2021},
author = {Zhu, Y and Feng, F and Hu, G and Wang, Y and Yu, Y and Zhu, Y and Xu, W and Cai, X and Sun, Z and Han, W and Ye, R and Qu, D and Ding, Q and Huang, X and Chen, H and Xu, W and Xie, Y and Cai, Q and Yuan, Z and Zhang, R},
title = {A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {961},
pmid = {33574281},
issn = {2041-1723},
mesh = {A549 Cells ; Angiotensin-Converting Enzyme 2/genetics/metabolism ; Animals ; COVID-19/genetics/*virology ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; Disease Models, Animal ; Endosomes/virology ; *Genome-Wide Association Study ; HeLa Cells ; *Host-Pathogen Interactions ; Humans ; Mesocricetus ; SARS-CoV-2/*physiology ; Serine Endopeptidases ; Spike Glycoprotein, Coronavirus/metabolism ; Vero Cells ; *Virus Internalization ; },
abstract = {The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.},
}
@article {pmid33574194,
year = {2021},
author = {Leslie, M},
title = {A revealing flaw.},
journal = {Science (New York, N.Y.)},
volume = {371},
number = {6530},
pages = {663-665},
doi = {10.1126/science.371.6530.663},
pmid = {33574194},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; Calcium/metabolism ; Clinical Trials as Topic ; Endoplasmic Reticulum/metabolism ; *Endoplasmic Reticulum Stress/drug effects ; Gene Editing ; Genetic Therapy ; Humans ; Membrane Proteins/*genetics/metabolism ; Rare Diseases/genetics/physiopathology/therapy ; Unfolded Protein Response ; Wolfram Syndrome/genetics/*physiopathology/*therapy ; },
}
@article {pmid33574182,
year = {2021},
author = {Trujillo, CA and Rice, ES and Schaefer, NK and Chaim, IA and Wheeler, EC and Madrigal, AA and Buchanan, J and Preissl, S and Wang, A and Negraes, PD and Szeto, RA and Herai, RH and Huseynov, A and Ferraz, MSA and Borges, FS and Kihara, AH and Byrne, A and Marin, M and Vollmers, C and Brooks, AN and Lautz, JD and Semendeferi, K and Shapiro, B and Yeo, GW and Smith, SEP and Green, RE and Muotri, AR},
title = {Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment.},
journal = {Science (New York, N.Y.)},
volume = {371},
number = {6530},
pages = {},
pmid = {33574182},
issn = {1095-9203},
support = {R35 GM133569/GM/NIGMS NIH HHS/United States ; R01 HL137223/HL/NHLBI NIH HHS/United States ; K12 GM068524/GM/NIGMS NIH HHS/United States ; R01 MH121487/MH/NIMH NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; R01 MH113545/MH/NIMH NIH HHS/United States ; K01 AA026911/AA/NIAAA NIH HHS/United States ; U19 MH107367/MH/NIMH NIH HHS/United States ; T32 HG008345/HG/NHGRI NIH HHS/United States ; U41 HG009889/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Alternative Splicing ; Amino Acid Substitution ; Animals ; Binding Sites ; Biological Evolution ; CRISPR-Cas Systems ; Cell Proliferation ; Cerebral Cortex/cytology/*growth &amp; development/*physiology ; Gene Expression Regulation, Developmental ; Genetic Variation ; Genome ; Genome, Human ; Haplotypes ; Hominidae/genetics ; Humans ; Induced Pluripotent Stem Cells ; Neanderthals/*genetics ; Nerve Net/physiology ; Nerve Tissue Proteins/genetics/metabolism ; Neuro-Oncological Ventral Antigen ; Neurons/*physiology ; Organoids ; RNA-Binding Proteins/*genetics/*metabolism ; Synapses/physiology ; },
abstract = {The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.},
}
@article {pmid33574136,
year = {2021},
author = {Bosch-Guiteras, N and Uroda, T and Guillen-Ramirez, HA and Riedo, R and Gazdhar, A and Esposito, R and Pulido-Quetglas, C and Zimmer, Y and Medová, M and Johnson, R},
title = {Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.},
journal = {Genome research},
volume = {31},
number = {3},
pages = {461-471},
pmid = {33574136},
issn = {1549-5469},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics/metabolism ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA-Activated Protein Kinase/*antagonists &amp; inhibitors/metabolism ; DNA-Binding Proteins/antagonists &amp; inhibitors/metabolism ; *Gene Editing ; *Sequence Deletion ; },
abstract = {CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.},
}
@article {pmid33574039,
year = {2021},
author = {Feng, CW and Burnet, G and Spiller, CM and Cheung, FKM and Chawengsaksophak, K and Koopman, P and Bowles, J},
title = {Identification of regulatory elements required for Stra8 expression in fetal ovarian germ cells of the mouse.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {5},
pages = {},
doi = {10.1242/dev.194977},
pmid = {33574039},
issn = {1477-9129},
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; Binding Sites ; CRISPR-Cas Systems/genetics ; Female ; Fetal Development/genetics ; Fetus/cytology/metabolism ; Gene Expression Regulation/drug effects ; Germ Cells/cytology/*metabolism ; Meiosis ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutagenesis ; Ovary/cytology/metabolism ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid/*genetics ; Retinoid X Receptors/genetics/metabolism ; Transcription Factors/genetics/metabolism/pharmacology ; Tretinoin/pharmacology ; },
abstract = {In mice, the entry of germ cells into meiosis crucially depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages, respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cutdown and mutant constructs, we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels in vivo.},
}
@article {pmid33573639,
year = {2021},
author = {Abdulrachman, D and Eurwilaichitr, L and Champreda, V and Chantasingh, D and Pootanakit, K},
title = {Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {15},
pmid = {33573639},
issn = {1472-6750},
mesh = {Aspergillus/*genetics ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Francisella ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; RNA, Guide ; },
abstract = {BACKGROUND: CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi.<br><br>RESULTS: In this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci.<br><br>CONCLUSIONS: The use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi.},
}
@article {pmid33572970,
year = {2021},
author = {Kim, J and Cho, JY and Kim, JW and Kim, DG and Nam, BH and Kim, BS and Kim, WJ and Kim, YO and Cheong, J and Kong, HJ},
title = {Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {3},
pages = {},
pmid = {33572970},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Fish Diseases/*genetics/immunology ; Fish Proteins/*genetics/immunology ; Flounder/*genetics/immunology/physiology ; Hemorrhagic Septicemia/genetics/immunology/*veterinary ; Host-Pathogen Interactions ; Novirhabdovirus/immunology/*physiology ; Phylogeny ; Repressor Proteins/*genetics/immunology ; Transcription, Genetic ; },
abstract = {MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.},
}
@article {pmid33572577,
year = {2021},
author = {Majchrzak-Celińska, A and Warych, A and Szoszkiewicz, M},
title = {Novel Approaches to Epigenetic Therapies: From Drug Combinations to Epigenetic Editing.},
journal = {Genes},
volume = {12},
number = {2},
pages = {},
pmid = {33572577},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Methylation/genetics ; Drug Combinations ; Epigenesis, Genetic/*genetics ; Epigenome/*genetics ; Gene Editing/trends ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {Cancer development involves both genetic and epigenetic alterations. Aberrant epigenetic modifications are reversible, allowing excellent opportunities for therapeutic intervention. Nowadays, several epigenetic drugs are used worldwide to treat, e.g., myelodysplastic syndromes and leukemias. However, overcoming resistance and widening the therapeutic profiles are the most important challenges faced by traditional epigenetic drugs. Recently, novel approaches to epigenetic therapies have been proposed. Next-generation epigenetic drugs, with longer half-life and better bioavailability, are being developed and tested. Since epigenetic phenomena are interdependent, treatment modalities include co-administration of two different epigenetic drugs. In order to sensitize cancer cells to chemotherapy, epigenetic drugs are administered prior to chemotherapy, or both epigenetic drug and chemotherapy are used together to achieve synergistic effects and maximize treatment efficacy. The combinations of epigenetic drug with immunotherapy are being tested, because they have proved to enhance antitumor immune responses. The next approach involves targeting the metabolic causes of epigenetic changes, i.e., enzymes which, when mutated, produce oncometabolites. Finally, epigenome editing makes it possible to modify individual chromatin marks at a defined region with unprecedented specificity and efficiency. This review summarizes the above attempts in fulfilling the promise of epigenetic drugs in the effective cancer treatment.},
}
@article {pmid33572199,
year = {2021},
author = {Zeng, L and Zhang, Q and Jiang, C and Zheng, Y and Zuo, Y and Qin, J and Liao, Z and Deng, H},
title = {Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {22},
number = {4},
pages = {},
pmid = {33572199},
issn = {1422-0067},
mesh = {Atropa belladonna/genetics/*metabolism ; Atropine/biosynthesis ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Hyoscyamine/*biosynthesis/isolation &amp; purification ; Metabolic Engineering/*methods ; Mixed Function Oxygenases/*genetics/metabolism ; Mutagenesis ; Plant Proteins/genetics/*metabolism ; Plants, Genetically Modified/genetics/metabolism ; Scopolamine/metabolism ; Seeds/genetics ; Solanaceous Alkaloids/biosynthesis ; },
abstract = {Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6β-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.},
}
@article {pmid33571426,
year = {2021},
author = {Tan, X and Letendre, JH and Collins, JJ and Wong, WW},
title = {Synthetic biology in the clinic: engineering vaccines, diagnostics, and therapeutics.},
journal = {Cell},
volume = {184},
number = {4},
pages = {881-898},
pmid = {33571426},
issn = {1097-4172},
support = {R01 GM129011/GM/NIGMS NIH HHS/United States ; R56 EB027729/EB/NIBIB NIH HHS/United States ; DP2 CA186574/CA/NCI NIH HHS/United States ; F31 HL149334/HL/NHLBI NIH HHS/United States ; R01 EB029483/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; *Biomedical Research ; CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Humans ; RNA/genetics ; *Synthetic Biology ; Vaccines/*immunology ; },
abstract = {Synthetic biology is a design-driven discipline centered on engineering novel biological functions through the discovery, characterization, and repurposing of molecular parts. Several synthetic biological solutions to critical biomedical problems are on the verge of widespread adoption and demonstrate the burgeoning maturation of the field. Here, we highlight applications of synthetic biology in vaccine development, molecular diagnostics, and cell-based therapeutics, emphasizing technologies approved for clinical use or in active clinical trials. We conclude by drawing attention to recent innovations in synthetic biology that are likely to have a significant impact on future applications in biomedicine.},
}
@article {pmid33571129,
year = {2021},
author = {Wang, Y and Wu, J and Chen, H and Yang, Y and Xiao, C and Yi, X and Shi, C and Zhong, K and He, H and Li, Y and Wu, Z and Zhou, G and Rao, Q and Wang, X and Zhou, X and Lomberk, G and Liu, B and Zhao, J and Ge, J and Zhou, W and Chu, X and Chen, C and Zhou, X and Wang, L and Guan, K and Qu, L},
title = {Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells.},
journal = {Science advances},
volume = {7},
number = {5},
pages = {},
pmid = {33571129},
issn = {2375-2548},
mesh = {Apoptosis Regulatory Proteins/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Neoplastic Stem Cells/metabolism ; Repressor Proteins/metabolism ; *Sarcoma/drug therapy/genetics ; },
abstract = {Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.},
}
@article {pmid33571114,
year = {2021},
author = {Shi, K and Xie, S and Tian, R and Wang, S and Lu, Q and Gao, D and Lei, C and Zhu, H and Nie, Z},
title = {A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics.},
journal = {Science advances},
volume = {7},
number = {5},
pages = {},
pmid = {33571114},
issn = {2375-2548},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA/genetics ; Feedback ; Humans ; *Nucleic Acids ; },
abstract = {Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.},
}
@article {pmid33571043,
year = {2021},
author = {Bloh, K and Kanchana, R and Bialk, P and Banas, K and Zhang, Z and Yoo, BC and Kmiec, EB},
title = {Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger Sequencing Data.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {120-131},
pmid = {33571043},
issn = {2573-1602},
support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; },
mesh = {*Algorithms ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *DNA Repair ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; INDEL Mutation ; Software ; },
abstract = {During CRISPR-directed gene editing, multiple gene repair mechanisms interact to produce a wide and largely unpredictable variety of sequence changes across an edited population of cells. Shortcomings inherent to previously available proposal-based insertion and deletion (indel) analysis software necessitated the development of a more comprehensive tool that could detect a larger range and variety of indels while maintaining the ease of use of tools currently available. To that end, we developed Deconvolution of Complex DNA Repair (DECODR). DECODR can detect indels formed from single or multi-guide CRISPR experiments without a limit on indel size. The software is accurate in determining the identities and positions of inserted and deleted bases in DNA extracts from both clonally expanded and bulk cell populations. The accurate identification and output of any potential indel allows for DECODR analysis to be executed in experiments utilizing potentially any configuration of donor DNA sequences, CRISPR-Cas, and endogenous DNA repair pathways.},
}
@article {pmid33570802,
year = {2021},
author = {Schurack, S and Depotter, JRL and Gupta, D and Thines, M and Doehlemann, G},
title = {Comparative transcriptome profiling identifies maize line specificity of fungal effectors in the maize-Ustilago maydis interaction.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {3},
pages = {733-752},
doi = {10.1111/tpj.15195},
pmid = {33570802},
issn = {1365-313X},
mesh = {Basidiomycota/genetics/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Disease Resistance ; Gene Editing ; *Gene Expression Profiling/methods ; Genes, Plant/genetics ; *Host-Pathogen Interactions ; Plant Diseases/*microbiology ; Transcriptome/genetics ; Zea mays/genetics/*microbiology ; },
abstract = {The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-for-gene interactions have been identified in the maize-U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA-Seq) analysis of six U. maydis-infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock-out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA-Seq analysis identified effectors with quantitative, maize line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize-U. maydis interaction.},
}
@article {pmid33570455,
year = {2021},
author = {Kuiken, T and Barrangou, R and Grieger, K},
title = {(Broken) Promises of Sustainable Food and Agriculture through New Biotechnologies: The CRISPR Case.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {25-31},
doi = {10.1089/crispr.2020.0098},
pmid = {33570455},
issn = {2573-1602},
mesh = {*Agriculture ; Animals ; *Biotechnology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/*genetics ; *Food ; Food Supply ; Gene Editing ; Livestock/genetics ; },
abstract = {In recent years, the development of diverse CRISPR-based technologies has revolutionized genome manipulation and enabled a broad scientific community in industry, academia, and beyond to redefine research and development for biotechnology products encompassing food, agriculture, and medicine. CRISPR-based genome editing affords tremendous opportunities in agriculture for the breeding of crops and livestock across the food supply chain that could benefit larger portions of the population compared to CRISPR applications in medicine, for example by helping to feed a growing global population, reach sustainability goals, and possibly mitigate the effects of climate change. These promises come alongside concerns of risks and adverse impacts associated with CRISPR-based genome editing and concerns that governance systems that are ill equipped or not well suited to evaluate these risks. The international community will continue to gather, in multiple venues, in the coming years to discuss these concerns. At the same time, responsible research and innovation paradigms also promise to evaluate the risks and benefits better while incorporating broad stakeholder engagement across the research and development process. The CRISPR community therefore must actively engage with these international deliberations, society, and national governance systems that have promised to build better agricultural systems and provide better food products to achieve equitable outcomes while protecting the environment. Without this active engagement, the promises discussed in this paper are sure to be broken.},
}
@article {pmid33570401,
year = {2021},
author = {Zhang, WS and Pan, J and Li, F and Zhu, M and Xu, M and Zhu, H and Yu, Y and Su, G},
title = {Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.},
journal = {Analytical chemistry},
volume = {93},
number = {8},
pages = {4126-4133},
pmid = {33570401},
issn = {1520-6882},
mesh = {Bacterial Proteins ; Base Sequence ; COVID-19/diagnosis ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Colorimetry/*methods ; Coronavirus Nucleocapsid Proteins/genetics ; DNA/*chemistry/genetics ; Endodeoxyribonucleases ; Gold/chemistry ; Humans ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques/*methods ; Phosphoproteins/genetics ; Polyproteins/genetics ; RNA, Viral/*analysis/genetics ; Reverse Transcription ; SARS-CoV-2/chemistry/*isolation &amp; purification ; Surface Plasmon Resonance ; Viral Proteins/genetics ; },
abstract = {The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.},
}
@article {pmid33569817,
year = {2021},
author = {Escalona-Noguero, C and López-Valls, M and Sot, B},
title = {CRISPR/Cas technology as a promising weapon to combat viral infections.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {43},
number = {4},
pages = {e2000315},
pmid = {33569817},
issn = {1521-1878},
mesh = {Biosensing Techniques/*methods ; COVID-19/diagnosis/genetics/therapy ; *CRISPR-Cas Systems ; Gene Knock-In Techniques ; Genome, Viral ; Humans ; RNA, Guide/genetics ; Virus Diseases/*diagnosis/*therapy ; Viruses/*genetics ; },
abstract = {The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA- and RNA-targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral nucleic acids in "Point of Care" sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS-CoV-2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR-based viral detectors. Finally, we discuss future prospects to improve both applications. Also see the video abstract here: https://www.youtube.com/watch?v=C0z1dLpJWl4.},
}
@article {pmid33568756,
year = {2021},
author = {Naser, AA and Miyazaki, T and Wang, J and Takabayashi, S and Pachoensuk, T and Tokumoto, T},
title = {MC4R mutant mice develop ovarian teratomas.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {3483},
pmid = {33568756},
issn = {2045-2322},
mesh = {Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Female ; Gene Editing ; Male ; Mice ; Mutation ; Mutation, Missense ; Neoplasm Proteins/genetics ; Oocytes/metabolism ; Ovarian Neoplasms/*genetics/pathology ; Point Mutation ; Receptor, Melanocortin, Type 4/*genetics/metabolism ; Teratoma/*genetics/pathology ; Testicular Neoplasms/genetics/pathology ; },
abstract = {Teratomas in mice, composed of different tissue types, are derived from primordial germ cells (PGCs) in the foetal gonads. The strongest candidate gene in the testicular teratoma locus (Ter) responsible for testicular teratoma formation was identified as mutation in Dnd1, Dnd1R178*. However, the phenotype of mice with a mutated Dnd1 gene was germ cell loss. This suggests that other genes are involved in teratoma formation. Testicular teratomas can also be induced experimentally (experimentally testicular teratomas: ETTs) in 129/Sv mice by transplanting E12.5 foetal testes into adult testes. Previously, we mapped the ett1 locus, which is the locus responsible for ETT formation on chromosome 18. By exome sequence analysis of the 129 and LTXBJ (LT) strains, we identified a missense mutation in the melanocortin 4 receptor (MC4R) gene among 8 genes in the ett1 region. The missense mutation causes a substitution of glycine 25 by serine. Thus, this gene is a candidate for ETT formation. We established the LT-ett1 congenic strain, which introduced the locus responsible for ETT formation genetically into the genomes of a testicular teratoma non-susceptible strain. In this study, we crossed LT-ett1 and a previously established LT-Ter strain to establish the double congenic strain LT-Ter-ett1. Also, we established a strain with a point mutation in the MC4R gene of the LT strain by genome editing, LT-MC4R[G25S]. Furthermore, double genetically modified strain LT-Ter-MC4R[G25S] was established to address the relation between Ter and MC4R. Surprisingly, highly developed ovarian teratomas (OTs), instead of testicular teratomas, appeared not only in the LT-Ter-MC4R[G25S] and LT-MC4R[G25S] strains but also in the LT-ett1 and LT-Ter-ett1 strains. The incidence of OT formation was high in double genetically modified strains. The results demonstrated that MC4R is one of the genes responsible for OT formation. It was suggested that the effect of the missense mutation in MC4R on teratoma formation was promoted by abnormal germ cell formation by the mutation in DND1.},
}
@article {pmid33567342,
year = {2021},
author = {Liu, X and Liu, X and Zhou, C and Lv, J and He, X and Liu, Y and Xie, H and Wang, B and Lv, X and Tang, L and Li, M and Liu, C and Zhao, J and Liu, Y and Song, Z and Gu, F},
title = {Engineered FnCas12a with enhanced activity through directional evolution in human cells.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100394},
pmid = {33567342},
issn = {1083-351X},
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Endodeoxyribonucleases/genetics/*metabolism ; Evolution, Molecular ; Eye Proteins/*genetics ; Francisella/*enzymology/genetics/isolation &amp; purification ; Gene Editing/methods ; Humans ; *Mutation ; Protein Engineering/methods ; Retinoschisis/*genetics/metabolism/pathology ; Selection, Genetic ; Structure-Activity Relationship ; },
abstract = {Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.},
}
@article {pmid33567216,
year = {2021},
author = {Qin, Y and Wong, B and Zhong, L and Geng, F and Parada, LF and Wen, D},
title = {Generation of Sex-Reversed Female Clonal Mice via CRISPR-Cas9-Mediated Y Chromosome Deletion in Male Embryonic Stem Cells.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {147-154},
pmid = {33567216},
issn = {2573-1602},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 GM129380/GM/NIGMS NIH HHS/United States ; R35 CA210100/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Chromosome Deletion ; Chromosomes, Human, Y ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Mammalian ; *Embryonic Stem Cells ; Female ; Genotyping Techniques ; *Infertility, Male ; Karyotyping ; Male ; Mice ; *Sex Chromosome Aberrations ; *Sex Chromosome Disorders of Sex Development ; *Y Chromosome ; },
abstract = {Mice derived entirely from embryonic stem (ES) cells can be generated through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR-Cas9-mediated deletion of the Y chromosome, allowing generation of female clonal mice by tetraploid complementation. The monosomic XO female mice are viable and able to produce normal male and female offspring. Direct generation of clonal mice in both sexes can significantly accelerate the production of complex genetically modified mouse models.},
}
@article {pmid33566028,
year = {2021},
author = {Wang, Y and Xie, Y and Dong, ZC and Jiang, XJ and Gong, P and Lu, J and Wan, F},
title = {[Levels of sgRNA as a Major Factor Affecting CRISPRi Knockdown Efficiency in K562 Cells].},
journal = {Molekuliarnaia biologiia},
volume = {55},
number = {1},
pages = {86-95},
doi = {10.31857/S0026898421010146},
pmid = {33566028},
issn = {0026-8984},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; DNA-Binding Proteins ; Humans ; K562 Cells ; Promoter Regions, Genetic ; *RNA, Guide ; Transcription Factors ; },
abstract = {To determine how nuclease deactivated Cas9 (dCas9) or single-guide RNA (sgRNA) expression levels affect the knockdown efficiency of CRISPRi, we created K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor. Single clones were selected by fluorescence-activated cell sorting (FACS) for further study. Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries in four cell clones with various KRAB-dCas9 expression levels. The expression level of dCas9 protein/sgRNA levels and the knockdown efficiency were determined by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown. The data describing this clone were statistically different from that on other clones, indicating the strong KRAB-dCas9 expression might be a prerequisite for CRISPRi. By adopting different multiplicity of infection (MOI) in lentiviral transduction of this clone, we modified the expression level of sgRNA and found that the knockdown efficiency was neither affected by the target gene expression level nor correlated with KRAB-dCas9 levels, which remained relatively constant across all knockdown experiments (coefficient of variation = 2.2%). As an example, the following levels of the knockdowns: 74.72, 72.28 and 39.08% for mmadhc, rpia and znf148 genes, respectively, were achieved. These knockdown efficiencies correlated well with the respective sgRNA expression levels. Linear regression models built using this data indicate that the knockdown efficiency may be significantly affected by the levels of both KRAB-dCas9 and sgRNA. Notably, the sgRNA levels have greater impact, being a major factor affecting CRISPRi efficiency.},
}
@article {pmid33563994,
year = {2021},
author = {Li, J and Mahata, B and Escobar, M and Goell, J and Wang, K and Khemka, P and Hilton, IB},
title = {Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {896},
pmid = {33563994},
issn = {2041-1723},
mesh = {Acetylation ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin/genetics ; Drug Resistance, Neoplasm/genetics ; Epigenomics/*methods ; Histones/*metabolism ; Humans ; Indoles/pharmacology ; Phosphorylation ; Promoter Regions, Genetic/genetics ; Ribosomal Protein S6 Kinases, 90-kDa/genetics/*metabolism ; Sulfonamides/pharmacology ; Transcriptional Activation ; },
abstract = {Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.},
}
@article {pmid33563726,
year = {2021},
author = {Ryan, BJ and Bengoa-Vergniory, N and Williamson, M and Kirkiz, E and Roberts, R and Corda, G and Sloan, M and Saqlain, S and Cherubini, M and Poppinga, J and Bogtofte, H and Cioroch, M and Hester, S and Wade-Martins, R},
title = {REST Protects Dopaminergic Neurons from Mitochondrial and α-Synuclein Oligomer Pathology in an Alpha Synuclein Overexpressing BAC-Transgenic Mouse Model.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {41},
number = {16},
pages = {3731-3746},
pmid = {33563726},
issn = {1529-2401},
support = {MR/N029453/1/MRC_/Medical Research Council/United Kingdom ; MR/P007058/1/MRC_/Medical Research Council/United Kingdom ; MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; J-0901/PUK_/Parkinson's UK/United Kingdom ; MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial ; Dopaminergic Neurons/*pathology ; Female ; GABAergic Neurons/pathology ; Gene Expression Regulation ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Mitochondria/*pathology ; Oxidative Stress ; Parkinson Disease/pathology ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics ; Repressor Proteins/*genetics ; Synucleinopathies/*genetics/*pathology ; alpha-Synuclein/*genetics ; },
abstract = {Alpha-synuclein pathology is associated with dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson's patients. Working across human and mouse models, we investigated mechanisms by which the accumulation of soluble α-synuclein oligomers leads to neurodegeneration. Biochemical analysis of the midbrain of α-synuclein overexpressing BAC-transgenic male and female mice revealed age- and region-dependent mitochondrial dysfunction and accumulation of damaged proteins downstream of the RE1 Silencing Transcription Factor (REST). Vulnerable SN dopaminergic neurons displayed low REST levels compared with neighboring protected SN GABAergic neurons, which correlated with the accumulation of α-synuclein oligomers and disrupted mitochondrial morphology. Consistent with a protective role, REST levels were reduced in patient induced pluripotent stem cell-derived dopaminergic neurons carrying the SNCA-Triplication mutation, which accumulated α-synuclein oligomers and mitochondrial damage, and displayed REST target gene dysregulation. Furthermore, CRISPR-mediated REST KO induced mitochondrial dysfunction and impaired mitophagy in vitro Conversely, REST overexpression attenuated mitochondrial toxicity and mitochondrial morphology disruption through the transcription factor PGC-1α. Finally, decreased α-synuclein oligomer accumulation and mitochondrial dysfunction in mice correlated with nuclear REST and PGC-1α in protected SN GABAergic neurons compared with vulnerable dopaminergic neurons. Our findings show that increased levels of α-synuclein oligomers cause dopaminergic neuronal-specific dysfunction through mitochondrial toxicity, which can be attenuated by REST in an early model of Parkinsonian pathology. These findings highlight REST as a mediator of dopaminergic vulnerability in PD.SIGNIFICANCE STATEMENT Understanding early Parkinsonian pathophysiology through studies of advanced preclinical models is fundamental to the translation of disease-modifying therapies. Here we show disease-relevant levels of α-synuclein expression in mice leads to accumulation of α-synuclein oligomers in the absence of overt aggregation, and mitochondrial dysfunction in dopaminergic neurons lacking the RE1 Silencing Transcription Factor. Our findings identify the mechanism of action of RE1 Silencing Transcription Factor and PGC-1α as mediators of dopaminergic vulnerability in α-synuclein BAC-transgenic mice and induced pluripotent stem cell-derived dopaminergic cultures, highlighting their potential as therapeutic targets.},
}
@article {pmid33563662,
year = {2021},
author = {Fix, SM and Jazaeri, AA and Hwu, P},
title = {Applications of CRISPR Genome Editing to Advance the Next Generation of Adoptive Cell Therapies for Cancer.},
journal = {Cancer discovery},
volume = {11},
number = {3},
pages = {560-574},
pmid = {33563662},
issn = {2159-8290},
support = {F32 CA253968/CA/NCI NIH HHS/United States ; TL1 TR003169/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Antigens, Neoplasm/genetics/metabolism ; *CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/adverse effects/*methods ; Clinical Trials as Topic ; Combined Modality Therapy ; Disease Management ; Drug Evaluation, Preclinical ; Gene Editing/*methods ; Genetic Engineering ; Genetic Therapy ; Humans ; Immunotherapy, Adoptive/adverse effects/*methods ; Neoplasms/*therapy ; Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Receptors, Chimeric Antigen/genetics/immunology/metabolism ; T-Lymphocytes/immunology/metabolism ; Treatment Outcome ; },
abstract = {Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. SIGNIFICANCE: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.},
}
@article {pmid33561291,
year = {2021},
author = {Zhu, M and Gao, J and Lin, XJ and Gong, YY and Qi, YC and Ma, YL and Song, YX and Tan, W and Li, FY and Ye, M and Gong, J and Cui, QH and Huang, ZH and Zhang, YY and Wang, XJ and Lan, F and Wang, SQ and Yuan, G and Feng, Y and Xu, M},
title = {Novel roles of an intragenic G-quadruplex in controlling microRNA expression and cardiac function.},
journal = {Nucleic acids research},
volume = {49},
number = {5},
pages = {2522-2536},
pmid = {33561291},
issn = {1362-4962},
mesh = {Animals ; Benzylisoquinolines/pharmacology ; CRISPR-Cas Systems ; Cells, Cultured ; *G-Quadruplexes/drug effects ; Gene Expression Regulation ; MicroRNAs/*chemistry/*metabolism ; Myocardial Contraction/*genetics ; Myocardium/metabolism ; Myocytes, Cardiac/*metabolism/physiology ; RNA Processing, Post-Transcriptional ; RNA-Binding Proteins/metabolism ; Rats ; Rats, Sprague-Dawley ; Ribonuclease III/metabolism ; Ryanodine Receptor Calcium Release Channel/metabolism ; },
abstract = {Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.},
}
@article {pmid33561010,
year = {2021},
author = {Huang, Z and Ning, B and Yang, HS and Youngquist, BM and Niu, A and Lyon, CJ and Beddingfield, BJ and Fears, AC and Monk, CH and Murrell, AE and Bilton, SJ and Linhuber, JP and Norton, EB and Dietrich, ML and Yee, J and Lai, W and Scott, JW and Yin, XM and Rappaport, J and Robinson, JE and Saba, NS and Roy, CJ and Zwezdaryk, KJ and Zhao, Z and Hu, TY},
title = {Sensitive tracking of circulating viral RNA through all stages of SARS-CoV-2 infection.},
journal = {The Journal of clinical investigation},
volume = {131},
number = {7},
pages = {},
pmid = {33561010},
issn = {1558-8238},
support = {P51 OD011104/OD/NIH HHS/United States ; T32 EB027632/EB/NIBIB NIH HHS/United States ; R01 HD090927/HD/NICHD NIH HHS/United States ; U54 CA260581/CA/NCI NIH HHS/United States ; U54 GM104940/GM/NIGMS NIH HHS/United States ; },
mesh = {Adolescent ; Adult ; Aged ; Animals ; COVID-19/*blood/diagnosis/*virology ; COVID-19 Nucleic Acid Testing/methods/statistics &amp; numerical data ; CRISPR-Cas Systems ; Cell-Free Nucleic Acids/*blood/*genetics ; Child ; Child, Preschool ; Disease Models, Animal ; Female ; Humans ; Infant ; Longitudinal Studies ; Macaca mulatta ; Male ; Middle Aged ; Pandemics ; RNA, Viral/*blood/*genetics ; *SARS-CoV-2/genetics ; Sensitivity and Specificity ; Time Factors ; },
abstract = {BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.},
}
@article {pmid33560917,
year = {2020},
author = {Johnston, AD and Abdulrazak, A and Sato, H and Maqbool, SB and Suzuki, M and Greally, JM and Simões-Pires, CA},
title = {A Cellular Stress Response Induced by the CRISPR-dCas9 Activation System Is Not Heritable Through Cell Divisions.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {188-197},
doi = {10.1089/crispr.2019.0077},
pmid = {33560917},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Cell Division/*genetics/*physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenomics ; Gene Editing/methods ; Gene Expression ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Transcription Factors ; },
abstract = {The CRISPR-Cas9 system can be modified to perform "epigenetic editing" by utilizing the catalytically inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions-an epigenetic regulatory property. Here, we show how distinct components of this CRISPR-dCas9 activation system, particularly dCas9 with untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR-dCas9 components in human cells, which is transient and not maintained through cell divisions.},
}
@article {pmid33560911,
year = {2020},
author = {Qi, S},
title = {Special Issue: Expanding the CRISPR Toolbox.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {135},
doi = {10.1089/crispr.2020.29086.cfp3},
pmid = {33560911},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Pathology, Molecular/methods ; Social Media ; },
}
@article {pmid33560910,
year = {2020},
author = {Barrangou, R},
title = {Finding SECURE Ground: USDA Edits the Biotechnology Regulatory Framework.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {136-137},
doi = {10.1089/crispr.2020.29096.rba},
pmid = {33560910},
issn = {2573-1602},
mesh = {Access to Information ; Animals ; Biodiversity ; Biotechnology/*standards ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Food ; Gene Editing ; Humans ; Plants ; Trust ; United States ; United States Department of Agriculture/*standards ; },
}
@article {pmid33560305,
year = {2021},
author = {Abbasi, J},
title = {CRISPR-Based COVID-19 Smartphone Test in Development.},
journal = {JAMA},
volume = {325},
number = {6},
pages = {522},
doi = {10.1001/jama.2021.0493},
pmid = {33560305},
issn = {1538-3598},
mesh = {COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*methods ; *CRISPR-Cas Systems ; Humans ; Microscopy, Fluorescence ; Nose/virology ; RNA, Viral/isolation &amp; purification ; SARS-CoV-2/*genetics/isolation &amp; purification ; *Smartphone ; Viral Load ; },
}
@article {pmid33560267,
year = {2021},
author = {Geisert, RD and Meyer, AE and Pfeiffer, CA and Johns, DN and Lee, K and Wells, KD and Spencer, TE and Prather, RS},
title = {Gene editing to investigate the role of conceptus factors in the establishment of pregnancy in the pig.},
journal = {Reproduction (Cambridge, England)},
volume = {161},
number = {4},
pages = {R79-R88},
doi = {10.1530/REP-20-0604},
pmid = {33560267},
issn = {1741-7899},
mesh = {Animals ; Blastocyst/cytology/*metabolism ; *CRISPR-Cas Systems ; Embryo, Mammalian/cytology/*metabolism ; *Embryonic Development ; Endometrium/cytology/*metabolism ; Female ; *Gene Expression Regulation, Developmental ; Pregnancy ; Swine ; },
abstract = {Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.},
}
@article {pmid33560108,
year = {2021},
author = {Tan, SI and Ng, IS},
title = {CRISPRi-Mediated NIMPLY Logic Gate for Fine-Tuning the Whole-Cell Sensing toward Simple Urine Glucose Detection.},
journal = {ACS synthetic biology},
volume = {10},
number = {2},
pages = {412-421},
doi = {10.1021/acssynbio.1c00014},
pmid = {33560108},
issn = {2161-5063},
mesh = {Biosensing Techniques/*methods ; *CRISPR-Cas Systems ; Cellular Reprogramming Techniques/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA-Directed RNA Polymerases/genetics ; Escherichia coli/*genetics ; Glucose/*analysis ; Glycosuria/*diagnosis ; Plasmids/genetics ; Promoter Regions, Genetic ; RNA, Catalytic/genetics ; },
abstract = {Whole-cell biosensors have been regarded as a prominent alternative to chemical and physical biosensors due to their renewability, environmental friendliness, and biocompatibility. However, there is still a lack of noninvasive measurements of urine glucose, which plays a vital role in monitoring the risk of diabetes in the healthcare system, via whole-cell biosensors. In this study, we characterized a glucose-inducible promoter and further enhanced the sensing performance using three genetic effectors, which encompassed ribozyme regulator (RiboJ), clustered regularly interspaced short palindromic repeat interference (CRISPRi), and plasmid-based T7RNA polymerase (PDT7), to develop the noninvasive glucose biosensor by fluorescent signal. As a result, RiboJ increased dynamic range to 2989 au, but declined signal-to-noise (S/N) to 1.59, while CRISPRi-mediated NIMPLY gate intensified both dynamic range to 5720 au and S/N to 4.58. The use of single PDT7 orthogonal with T7 promoter in cells (i.e., P strain) achieved a 44 180 au of dynamic range with S/N at 3.08. By coupling the PDT7 and NIMPLY-mediated CRISPRi, we constructed an optimum PIGAS strain with the highest S/N value of 4.95. Finally, we adopted the synthetic bacteria into a microdevice to afford an integrative and portable system for daily urine glucose inspection, which would be an alternative approach for medical diagnosis in the future.},
}
@article {pmid33559722,
year = {2021},
author = {Huang, X and Hilscher, J and Stoger, E and Christou, P and Zhu, C},
title = {Modification of cereal plant architecture by genome editing to improve yields.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {953-978},
pmid = {33559722},
issn = {1432-203X},
mesh = {Edible Grain/*anatomy &amp; histology/genetics/growth &amp; development/*physiology ; Gene Editing/*methods ; Plant Breeding/methods ; Plant Leaves/anatomy &amp; histology/genetics ; Plant Proteins/*genetics ; Plant Roots/anatomy &amp; histology/genetics ; Seeds/genetics/growth &amp; development ; },
abstract = {We summarize recent genome editing studies that have focused on the examination (or reexamination) of plant architectural phenotypes in cereals and the modification of these traits for crop improvement. Plant architecture is defined as the three-dimensional organization of the entire plant. Shoot architecture refers to the structure and organization of the aboveground components of a plant, reflecting the developmental patterning of stems, branches, leaves and inflorescences/flowers. Root system architecture is essentially determined by four major shape parameters-growth, branching, surface area and angle. Interest in plant architecture has arisen from the profound impact of many architectural traits on agronomic performance, and the genetic and hormonal regulation of these traits which makes them sensitive to both selective breeding and agronomic practices. This is particularly important in staple crops, and a large body of literature has, therefore, accumulated on the control of architectural phenotypes in cereals, particularly rice due to its twin role as one of the world's most important food crops as well as a model organism in plant biology and biotechnology. These studies have revealed many of the molecular mechanisms involved in the regulation of tiller/axillary branching, stem height, leaf and flower development, root architecture and the grain characteristics that ultimately help to determine yield. The advent of genome editing has made it possible, for the first time, to introduce precise mutations into cereal crops to optimize their architecture and close in on the concept of the ideotype. In this review, we consider recent genome editing studies that have focused on the examination (or reexamination) of plant architectural phenotypes in cereals and the modification of these traits for crop improvement.},
}
@article {pmid33558716,
year = {2021},
author = {Wang, H and Han, M and Qi, LS},
title = {Engineering 3D genome organization.},
journal = {Nature reviews. Genetics},
volume = {22},
number = {6},
pages = {343-360},
pmid = {33558716},
issn = {1471-0064},
support = {U01 EB021240/EB/NIBIB NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Nucleus/*genetics ; *Gene Editing ; Genetic Engineering/*methods ; *Genome ; Genomics/*methods ; Humans ; },
abstract = {Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR-Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR-Cas and related technologies.},
}
@article {pmid33558704,
year = {2021},
author = {Lakshmanan, VK and Jindal, S and Packirisamy, G and Ojha, S and Lian, S and Kaushik, A and Alzarooni, AIMA and Metwally, YAF and Thyagarajan, SP and Do Jung, Y and Chouaib, S},
title = {Nanomedicine-based cancer immunotherapy: recent trends and future perspectives.},
journal = {Cancer gene therapy},
volume = {28},
number = {9},
pages = {911-923},
pmid = {33558704},
issn = {1476-5500},
mesh = {Humans ; Immunotherapy/*methods ; Nanomedicine/*methods ; Nanoparticles/*metabolism ; Neoplasms/*therapy ; },
abstract = {The combination of cancer immunotherapy with efficient functionalized nanosystems has emerged as a beneficial treatment strategy and its use has increased rapidly. The roles of stimuli-responsive nanosystems and nanomedicine-based cancer immunotherapy, a subsidiary discipline in the field of immunology, are pivotal. The present era is witnessing rapid advancements in the use of nanomedicine as a platform for investigating novel therapeutic applications and modern intelligent healthcare management strategies. The development of cancer nanomedicine has posthaste ratified the outcomes of immunotherapy to the subsequent stage in the current era of medical research. This review focuses on key findings with respect to the effectiveness of nanomedicine-based cancer immunotherapies and their applications, which include i) immune checkpoint inhibitors and nanomedicine, ii) CRISPR-Cas nanoparticles (NPs) in cancer immunotherapy, iii) combination cancer immunotherapy with core-shell nanoparticles, iv) biomimetic NPs for cancer immunotherapy, and v) CAR-T cells and cancer nanoimmunotherapy. By evaluating the state-of-the-art tools and taking the challenges involved into consideration, various aspects of the proposed nano-enabled therapeutic approaches have been discussed in this review.},
}
@article {pmid33558692,
year = {2021},
author = {Hana, S and Peterson, M and McLaughlin, H and Marshall, E and Fabian, AJ and McKissick, O and Koszka, K and Marsh, G and Craft, M and Xu, S and Sorets, A and Torregrosa, T and Sun, C and Henderson, CE and Lo, SC},
title = {Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.},
journal = {Gene therapy},
volume = {28},
number = {10-11},
pages = {646-658},
pmid = {33558692},
issn = {1476-5462},
mesh = {Animals ; *CRISPR-Cas Systems ; Central Nervous System ; Gene Editing/methods ; Gene Knockout Techniques ; Mice ; Neurons/metabolism ; *RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.},
}
@article {pmid33558622,
year = {2021},
author = {Fu, Y and Zhu, Z and Meng, G and Zhang, R and Zhang, Y},
title = {A CRISPR-Cas9 based shuffle system for endogenous histone H3 and H4 combinatorial mutagenesis.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {3298},
pmid = {33558622},
issn = {2045-2322},
mesh = {Amino Acid Substitution ; *CRISPR-Cas Systems ; Histones/*genetics ; *Mutagenesis ; *Mutation, Missense ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/*genetics ; },
abstract = {Post-translational modifications of histone proteins greatly impact gene expression and cell fate decisions in eukaryotes. To study these, it is important to develop a convenient, multiplex, and efficient method to precisely introduce mutations to histones. Because eukaryotic cells usually contain multiple copies of histone genes, it is a challenge to mutate all histones at the same time by the traditional homologous recombination method. Here, we developed a CRISPR-Cas9 based shuffle system in Saccharomyces cerevisiae, to generate point mutations on both endogenous histone H3 and H4 genes in a rapid, seamless and multiplex fashion. Using this method, we generated yeast strains containing histone triple H3-K4R-K36R-K79R mutants and histone combinatorial H3-K56Q-H4-K59A double mutants with high efficiencies (70-80%). This CRISPR-Cas9 based mutagenesis system could be an invaluable tool to the epigenetics field.},
}
@article {pmid33558388,
year = {2021},
author = {Wang, G and Li, C and He, S and Liu, Z},
title = {Mosaic CRISPR-stop enables rapid phenotyping of nonsense mutations in essential genes.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {5},
pages = {},
doi = {10.1242/dev.196899},
pmid = {33558388},
issn = {1477-9129},
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/deficiency/genetics ; Blastomeres/cytology/metabolism ; CRISPR-Cas Systems/*genetics ; Codon, Nonsense ; Codon, Terminator ; Genes, Essential/*genetics ; Hair Cells, Auditory, Outer/cytology/metabolism ; Mice ; Mice, Knockout ; RNA, Guide/*metabolism ; RNA-Binding Proteins/genetics/metabolism ; SOXE Transcription Factors/deficiency/genetics ; Zygote/cytology/metabolism ; },
abstract = {CRISPR-stop converts protein-coding sequences into stop codons, which, in the appropriate location, results in a null allele. CRISPR-stop induction in one-cell-stage zygotes generates Founder 0 (F0) mice that are homozygous mutants; this avoids mouse breeding and serves as a rapid screening approach for nonlethal genes. However, loss of function of 25% of mammalian genes causes early lethality. Here, we induced CRISPR-stop in one of the two blastomeres of the zygote, a method we name mosaic CRISPR-stop, to produce mosaic Atoh1 and Sox10 F0 mice; these mice not only survived longer than regular Atoh1/Sox10 knockout mice but also displayed their recognized cochlear phenotypes. Moreover, by using mosaic CRISPR-stop, we uncovered a previously unknown role of another lethal gene, Rbm24, in the survival of cochlear outer hair cells (OHCs), and we further validated the importance of Rbm24 in OHCs by using our Rbm24 conditional knockout model. Together, our results demonstrated that mosaic CRISPR-stop is reliable and rapid, and we believe this method will facilitate rapid genetic screening of developmentally lethal genes in the mouse inner ear and also in other organs.},
}
@article {pmid33557342,
year = {2021},
author = {Deutsch, M and Günther, A and Lerchundi, R and Rose, CR and Balfanz, S and Baumann, A},
title = {AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons.},
journal = {Cells},
volume = {10},
number = {2},
pages = {},
pmid = {33557342},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Dependovirus/*metabolism ; Electrophysiological Phenomena ; Gene Expression Regulation ; Gene Knockdown Techniques ; HEK293 Cells ; Hippocampus/*cytology ; Humans ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics/metabolism ; Mice, Inbred C57BL ; Neurons/*metabolism ; Protein Subunits/metabolism ; *RNA Interference ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/metabolism ; },
abstract = {Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.},
}
@article {pmid33555975,
year = {2021},
author = {LaBelle, J and Ramos-Martinez, A and Shen, K and Motta-Mena, LB and Gardner, KH and Materna, SC and Woo, S},
title = {TAEL 2.0: An Improved Optogenetic Expression System for Zebrafish.},
journal = {Zebrafish},
volume = {18},
number = {1},
pages = {20-28},
pmid = {33555975},
issn = {1557-8542},
support = {R01 GM106239/GM/NIGMS NIH HHS/United States ; R03 DK106358/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Embryo, Nonmammalian ; Gene Expression Regulation, Developmental/*radiation effects ; Genes, Reporter/radiation effects ; *Light ; Optogenetics/*methods ; Signal Transduction/genetics/radiation effects ; *Zebrafish/embryology/genetics ; },
abstract = {Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.},
}
@article {pmid33555973,
year = {2020},
author = {Makarova, KS and Wolf, YI and Shmakov, SA and Liu, Y and Li, M and Koonin, EV},
title = {Unprecedented Diversity of Unique CRISPR-Cas-Related Systems and Cas1 Homologs in Asgard Archaea.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {156-163},
pmid = {33555973},
issn = {2573-1602},
mesh = {Archaea/classification/*genetics/metabolism ; Archaeal Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Genome, Archaeal ; Metagenome ; Phylogeny ; },
abstract = {The principal function of archaeal and bacterial CRISPR-Cas systems is antivirus adaptive immunity. However, recent genome analyses identified a variety of derived CRISPR-Cas variants at least some of which appear to perform different functions. Here, we describe a unique repertoire of CRISPR-Cas-related systems that we discovered by searching archaeal metagenome-assemble genomes of the Asgard superphylum. Several of these variants contain extremely diverged homologs of Cas1, the integrase involved in CRISPR adaptation as well as casposon transposition. Strikingly, the diversity of Cas1 in Asgard archaea alone is greater than that detected so far among the rest of archaea and bacteria. The Asgard CRISPR-Cas derivatives also encode distinct forms of Cas4, Cas5, and Cas7 proteins, and/or additional nucleases. Some of these systems are predicted to perform defense functions, but possibly not programmable ones, whereas others are likely to represent previously unknown mobile genetic elements.},
}
@article {pmid33555591,
year = {2021},
author = {E, X and Kowalik, TF},
title = {A Generally Applicable CRISPR/Cas9 Screening Technique to Identify Host Genes Required for Virus Infection as Applied to Human Cytomegalovirus (HCMV) Infection of Epithelial Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2244},
number = {},
pages = {247-264},
pmid = {33555591},
issn = {1940-6029},
support = {P51 OD011104/OD/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytomegalovirus/*genetics ; Epithelial Cells ; Gene Editing/methods ; Genetic Engineering/*methods ; Genetic Testing/methods ; Host Microbial Interactions/*genetics ; Humans ; Primary Cell Culture ; RNA, Guide/genetics ; Virus Diseases/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens enable virus-host genetic screens to be undertaken in a more robust manner than previously possible and has had a tremendous impact in the field of virus study. Researchers can take advantage of the power of CRISPR genetic screens to discover virus-host interaction genes including host receptors and signaling molecules (Bazzone et al., mBio 10 (1): e02734-18, 2019; E et al., Proc Natl Acad Sci U S A 116(14):7043-7052, 2019; McDougall et al., Curr Opin Virol 29:87-100, 2018; Savidis et al., Cell Rep 16(1):232-246, 2016). In principle, lysis of cells late in the virus infection cycle allows one to screen for essential genes using pooled single-guide RNAs (sgRNAs) that collective target an entire host cell genome simply by identifying mutant cells that are resistant to virus-induced cell death. Here we focus on using this technique on epithelial cells to identify host targets required for human cytomegalovirus (HCMV) infection.},
}
@article {pmid33554142,
year = {2021},
author = {Qiu, Y and Ding, Q},
title = {Optimized protocol for gene editing in adipocytes using CRISPR-Cas9 technology.},
journal = {STAR protocols},
volume = {2},
number = {1},
pages = {100307},
pmid = {33554142},
issn = {2666-1667},
mesh = {Adipocytes/*metabolism/physiology ; Animals ; CRISPR-Cas Systems/*genetics/physiology ; Gene Editing/*methods ; Genetic Vectors/genetics ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; },
abstract = {We present a detailed protocol for gene editing in adipocytes using the CRISPR-Cas technology. This protocol describes sgRNA design, preparation of lentiCRISPR-sgRNA vectors, functional validation of sgRNAs, preparation of lentiviruses, and lentiviruses transduction in adipocytes. Moreover, an optimized method of gene editing using the lentiCRISPRv2 vector expressing two sgRNAs targeting two different genes has also been described. For complete details on the use and execution of this protocol, please refer to Qiu et al. (2020).},
}
@article {pmid33552016,
year = {2020},
author = {Huang, F and Zhu, B},
title = {The Cyclic Oligoadenylate Signaling Pathway of Type III CRISPR-Cas Systems.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {602789},
pmid = {33552016},
issn = {1664-302X},
abstract = {Type III CRISPR-Cas systems, which are widespread in both bacteria and archaea, provide immunity against DNA viruses and plasmids in a transcription-dependent manner. Since an unprecedented cyclic oligoadenylate (cOA) signaling pathway was discovered in type III systems in 2017, the cOA signaling has been extensively studied in recent 3 years, which has expanded our understanding of type III systems immune defense and also its counteraction by viruses. In this review, we summarized recent advances in cOA synthesis, cOA-activated effector protein, cOA signaling-mediated immunoprotection, and cOA signaling inhibition, and highlighted the crosstalk between cOA signaling and other cyclic oligonucleotide-mediated immunity discovered very recently.},
}
@article {pmid33550455,
year = {2021},
author = {Yuan, S and Kawasaki, S and Abdellatif, IMY and Nishida, K and Kondo, A and Ariizumi, T and Ezura, H and Miura, K},
title = {Efficient base editing in tomato using a highly expressed transient system.},
journal = {Plant cell reports},
volume = {40},
number = {4},
pages = {667-676},
pmid = {33550455},
issn = {1432-203X},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genome, Plant ; Homozygote ; Lycopersicon esculentum/*genetics ; *Mutation ; Plants, Genetically Modified ; RNA, Guide ; Transgenes ; },
abstract = {Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.},
}
@article {pmid33549837,
year = {2021},
author = {Baek, S and Utomo, JC and Lee, JY and Dalal, K and Yoon, YJ and Ro, DK},
title = {The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system.},
journal = {Metabolic engineering},
volume = {64},
number = {},
pages = {111-121},
doi = {10.1016/j.ymben.2021.01.011},
pmid = {33549837},
issn = {1096-7184},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Metabolic Networks and Pathways ; *RNA, Guide/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {Saccharomyces cerevisiae is a versatile microbial platform to build synthetic metabolic pathways for production of diverse chemicals. To expedite the construction of complex metabolic pathways by multiplex CRISPR-Cas9 genome-edit, eight desirable intergenic loci, located adjacent to highly expressed genes selected from top 100 expressers, were identified and fully characterized for three criteria after integrating green fluorescent protein (GFP) gene - CRISPR-mediated GFP integration efficiency, expression competency assessed by levels of GFP fluorescence, and assessing growth rates of GFP integrated strains. Five best performing intergenic loci were selected to build a multiplex CRISPR platform, and a synthetic 23-bp DNA comprised of 20-bp synthetic DNA with a protospacer adjacent motif (PAM) was integrated into the five loci using CRISPR-Cas9 in a sequential manner. This process resulted in five different yeast strains harbouring 1-5 synthetic gRNA-binding sites in their genomes. Using these pre-engineered yeast strains, simultaneous integrations of 2-, 3-, 4-, or 5-genes to the targeted loci were demonstrated with efficiencies from 85% to 98% using beet pigment betalain (3-gene pathway), hygromycin and geneticin resistance markers. Integrations of the multiple, foreign genes in the targeted loci with 100% precision were validated by genotyping. Finally, we further developed the strain to have 6th synthetic gRNA-binding site, and the resulting yeast strain was used to generate a yeast strain producing a sesquiterpene lactone, kauniolide by simultaneous 6-gene integrations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in yeast.},
}
@article {pmid33549479,
year = {2020},
author = {Li, L and Meng, H and Zhang, J and Liu, Y and Zou, Q and Gao, Y and Yang, H and Lai, L},
title = {A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {11},
pages = {705-712},
doi = {10.1016/j.jgg.2020.07.009},
pmid = {33549479},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/drug effects/*genetics ; *Drug and Narcotic Control ; Gene Editing ; Gene Expression Regulation/drug effects ; Humans ; *Proteomics ; Transcriptional Activation/*drug effects/genetics ; },
abstract = {Rapid, precise, and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications. Here, we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells. We construct the system by coupling Tet-on 3G and small molecule-assisted shutoff systems, which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules. This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression. The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence, allowing gene function study in a comprehensive expression profile. Our results reveal that the bidirectional control system enables sensitive dosage- and time-dependent regulation for either turn-on or shutoff of gene expression. We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats. The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.},
}
@article {pmid33547931,
year = {2021},
author = {Jung, JW and Shin, JH and Lee, WK and Begum, H and Min, CH and Jang, MH and Oh, HB and Yang, MS and Kim, SR},
title = {Inactivation of the β (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {1025-1035},
pmid = {33547931},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems ; Epitopes/genetics ; Fucosyltransferases/*genetics ; Gene Editing/methods ; Gene Silencing ; Mutation ; Oryza/*genetics ; Pentosyltransferases/*genetics ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; Polysaccharides/genetics/immunology/*metabolism ; },
abstract = {CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific β1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, β1,2-xylose and α1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by β1,2-xylosyltransferase (β1,2-XylT) and α1,3-fucosyltransferase (α1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), β1,2-xylT (OsXylT) and α1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both β1,2-xylose and α1,3-fucose residues in the lines were not detected in Western blot analysis. Consistently, there was no peak corresponding to the N-glycans in MALDI-TOF/MS analysis. Although α1,3-fucose and β1,2-xylose residues were not detected in the line, other plant-specific residues of β1,3-galactose and α1,4-fucose were detected. Thus, we suggest that each enzymes working on the process for complex N-glycan biosynthesis might independently act in rice, hence the double knock-out of both OsXylT and OsFucT might be not enough to humanize N-glycan structure in rice.},
}
@article {pmid33547443,
year = {2021},
author = {Walton, RT and Hsu, JY and Joung, JK and Kleinstiver, BP},
title = {Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.},
journal = {Nature protocols},
volume = {16},
number = {3},
pages = {1511-1547},
pmid = {33547443},
issn = {1750-2799},
support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Nucleotide Motifs/genetics ; RNA, Guide/genetics ; Research Design ; },
abstract = {The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.},
}
@article {pmid33547076,
year = {2021},
author = {Koduri, V and Duplaquet, L and Lampson, BL and Wang, AC and Sabet, AH and Ishoey, M and Paulk, J and Teng, M and Harris, IS and Endress, JE and Liu, X and Dasilva, E and Paulo, JA and Briggs, KJ and Doench, JG and Ott, CJ and Zhang, T and Donovan, KA and Fischer, ES and Gygi, SP and Gray, NS and Bradner, J and Medin, JA and Buhrlage, SJ and Oser, MG and Kaelin, WG},
title = {Targeting oncoproteins with a positive selection assay for protein degraders.},
journal = {Science advances},
volume = {7},
number = {6},
pages = {},
pmid = {33547076},
issn = {2375-2548},
support = {R35 CA210068/CA/NCI NIH HHS/United States ; T32 CA009172/CA/NCI NIH HHS/United States ; K08 CA222657/CA/NCI NIH HHS/United States ; K08 CA252611/CA/NCI NIH HHS/United States ; R00 CA190861/CA/NCI NIH HHS/United States ; R01 GM132129/GM/NIGMS NIH HHS/United States ; R01 GM067945/GM/NIGMS NIH HHS/United States ; R01 CA214608/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics ; Basic Helix-Loop-Helix Transcription Factors/metabolism ; Benzylamines ; CRISPR-Cas Systems ; Humans ; Ikaros Transcription Factor/metabolism ; *Oncogene Proteins/chemistry/metabolism ; *Proteolysis/drug effects ; Quinazolines ; Thalidomide/analysis/pharmacology ; Transcription Factors ; },
abstract = {Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.},
}
@article {pmid33546657,
year = {2021},
author = {Liu, W and Wang, S and Lin, B and Zhang, W and Ji, G},
title = {Applications of CRISPR/Cas9 in the research of malignant musculoskeletal tumors.},
journal = {BMC musculoskeletal disorders},
volume = {22},
number = {1},
pages = {149},
pmid = {33546657},
issn = {1471-2474},
mesh = {Adolescent ; *Bone Neoplasms/genetics/therapy ; CRISPR-Cas Systems/genetics ; Child ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Osteosarcoma/genetics/therapy ; },
abstract = {BACKGROUND: Malignant tumors of the musculoskeletal system, especially osteosarcoma, Ewing sarcoma and rhabdomyosarcoma, pose a major threat to the lives and health of adolescents and children. Current treatments for musculoskeletal tumors mainly include surgery, chemotherapy, and radiotherapy. The problems of chemotherapy resistance, poor long-term outcome of radiotherapy, and the inherent toxicity and side effects of chemical drugs make it extremely urgent to seek new treatment strategies.<br><br>MAIN TEXT: As a potent gene editing tool, the rapid development of CRISPR/Cas9 technology in recent years has prompted scientists to apply it to the study of musculoskeletal tumors. This review summarizes the application of CRISPR/Cas9 technology for the treatment of malignant musculoskeletal tumors, focusing on its essential role in the field of basic research.<br><br>CONCLUSION: CRISPR, has demonstrated strong efficacy in targeting tumor-related genes, and its future application in the clinical treatment of musculoskeletal tumors is promising.},
}
@article {pmid33545636,
year = {2021},
author = {Singh, HD and Ma, JX and Takahashi, Y},
title = {Distinct roles of LRP5 and LRP6 in Wnt signaling regulation in the retina.},
journal = {Biochemical and biophysical research communications},
volume = {545},
number = {},
pages = {8-13},
pmid = {33545636},
issn = {1090-2104},
support = {R01 EY018659/EY/NEI NIH HHS/United States ; P30 GM122744/GM/NIGMS NIH HHS/United States ; R01 EY012231/EY/NEI NIH HHS/United States ; R01 EY028949/EY/NEI NIH HHS/United States ; R01 EY019309/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Culture Media, Conditioned ; Frizzled Receptors/metabolism ; Gene Knockout Techniques ; Humans ; Ligands ; Low Density Lipoprotein Receptor-Related Protein-5/deficiency/genetics/*metabolism ; Low Density Lipoprotein Receptor-Related Protein-6/deficiency/genetics/*metabolism ; Mice ; Retinal Pigment Epithelium/cytology/*metabolism ; Retinoids/metabolism/pharmacology ; *Wnt Signaling Pathway/drug effects ; Wnt3A Protein/metabolism ; },
abstract = {Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.},
}
@article {pmid33545196,
year = {2021},
author = {Rother, M and Dimmler, C and Weege, F and Mollenkopf, HJ and Meyer, TF and Naumann, M},
title = {Discovery of Zika virus host dependency factors in trophoblasts using CRISPR/Cas9 screening.},
journal = {Journal of virological methods},
volume = {290},
number = {},
pages = {114085},
doi = {10.1016/j.jviromet.2021.114085},
pmid = {33545196},
issn = {1879-0984},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Humans ; Placenta/virology ; Pregnancy ; Trophoblasts ; Virus Replication ; *Zika Virus/genetics ; *Zika Virus Infection/diagnosis ; },
abstract = {Emerging mosquito-borne RNA viruses cause massive health complications worldwide. The Zika virus (ZIKV), in particular, has spread dramatically since 2007 and has provoked epidemics in the Americas and the South Pacific. The lack of antiviral therapy and vaccination has focused research on the investigation of ZIKV-host interactions, in order to understand underlying molecular infection mechanisms. We have established an approach for the analysis of ZIKV host dependency factors in a human trophoblast cell line and applied genome-wide CRISPR/Cas9 knockout mutagenesis. The presented method is especially of value for the identification of factors that are essential for placental infection with the potential to serve as targets for antiviral treatment.},
}
@article {pmid33545089,
year = {2021},
author = {Hawkins, MB and Henke, K and Harris, MP},
title = {Latent developmental potential to form limb-like skeletal structures in zebrafish.},
journal = {Cell},
volume = {184},
number = {4},
pages = {899-911.e13},
doi = {10.1016/j.cell.2021.01.003},
pmid = {33545089},
issn = {1097-4172},
mesh = {Actins/metabolism ; Animal Fins/embryology ; Animals ; Base Sequence ; Body Patterning ; Bone and Bones/*embryology ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Epistasis, Genetic ; Extremities/*embryology ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Genes, Reporter ; HeLa Cells ; Homeodomain Proteins/genetics/metabolism ; Humans ; Mice ; Mutation/genetics ; Phenotype ; Phylogeny ; Signal Transduction/genetics ; Zebrafish/*embryology/genetics ; Zebrafish Proteins/genetics/metabolism ; },
abstract = {Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation.},
}
@article {pmid33545079,
year = {2021},
author = {Drysdale, CM and Nassehi, T and Gamer, J and Yapundich, M and Tisdale, JF and Uchida, N},
title = {Hematopoietic-Stem-Cell-Targeted Gene-Addition and Gene-Editing Strategies for β-hemoglobinopathies.},
journal = {Cell stem cell},
volume = {28},
number = {2},
pages = {191-208},
doi = {10.1016/j.stem.2021.01.001},
pmid = {33545079},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; *Hemoglobinopathies/genetics/therapy ; Humans ; },
abstract = {Sickle cell disease (SCD) is caused by a well-defined point mutation in the β-globin gene and therefore is an optimal target for hematopoietic stem cell (HSC) gene-addition/editing therapy. In HSC gene-addition therapy, a therapeutic β-globin gene is integrated into patient HSCs via lentiviral transduction, resulting in long-term phenotypic correction. State-of-the-art gene-editing technology has made it possible to repair the β-globin mutation in patient HSCs or target genetic loci associated with reactivation of endogenous γ-globin expression. With both approaches showing signs of therapeutic efficacy in patients, we discuss current genetic treatments, challenges, and technical advances in this field.},
}
@article {pmid33544854,
year = {2021},
author = {Shin, HR and See, JE and Kweon, J and Kim, HS and Sung, GJ and Park, S and Jang, AH and Jang, G and Choi, KC and Kim, I and Kim, JS and Kim, Y},
title = {Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing.},
journal = {Nucleic acids research},
volume = {49},
number = {4},
pages = {2390-2399},
pmid = {33544854},
issn = {1362-4962},
mesh = {*Adenine ; *CRISPR-Cas Systems ; Depsipeptides/pharmacology ; Doxycycline/pharmacology ; *Gene Editing ; Green Fluorescent Proteins/analysis/genetics ; HEK293 Cells ; HeLa Cells ; Histone Deacetylase Inhibitors/*pharmacology ; Humans ; Luminescent Agents/analysis ; Protein Biosynthesis ; RNA/biosynthesis ; },
abstract = {CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.},
}
@article {pmid33544853,
year = {2021},
author = {León, LM and Park, AE and Borges, AL and Zhang, JY and Bondy-Denomy, J},
title = {Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa.},
journal = {Nucleic acids research},
volume = {49},
number = {4},
pages = {2114-2125},
pmid = {33544853},
issn = {1362-4962},
support = {T32 GM007810/GM/NIGMS NIH HHS/United States ; T32 AI060537/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/metabolism ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; DNA Cleavage ; *Interspersed Repetitive Sequences ; Pseudomonas aeruginosa/*genetics/immunology/virology ; Viral Proteins/metabolism ; },
abstract = {Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR-Cas immune systems use RNA-guided nucleases to target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR-Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR-Cas system encoded on a conjugative pKLC102 island, and its Acr-mediated inhibition by distinct MGEs. Seven genes with anti-Type I-C function (acrIC genes) were identified, many with highly acidic amino acid content, including previously described DNA mimic AcrIF2. Four of the acr genes were broad spectrum, also inhibiting I-E or I-F P. aeruginosa CRISPR-Cas subtypes. Dual inhibition comes at a cost, however, as simultaneous expression of Type I-C and I-F systems renders phages expressing the dual inhibitor AcrIF2 more sensitive to targeting. Mutagenesis of numerous acidic residues in AcrIF2 did not impair anti-I-C or anti-I-F function per se but did exacerbate inhibition defects during competition, suggesting that excess negative charge may buffer DNA mimics against competition. Like AcrIF2, five of the Acr proteins block Cascade from binding DNA, while two function downstream, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found in an 'anti-Cas3' cluster within conjugative elements, encoded alongside bona fide Cas3 inhibitors AcrIF3 and AcrIE1. Our findings demonstrate an active battle between an MGE-encoded CRISPR-Cas system and its diverse MGE targets.},
}
@article {pmid33544493,
year = {2020},
author = {Ryan, D and Prezza, G and Westermann, AJ},
title = {An RNA-centric view on gut Bacteroidetes.},
journal = {Biological chemistry},
volume = {402},
number = {1},
pages = {55-72},
doi = {10.1515/hsz-2020-0230},
pmid = {33544493},
issn = {1437-4315},
mesh = {Bacteroidetes/*genetics ; Gastrointestinal Microbiome/*genetics ; Humans ; RNA/*genetics/metabolism ; },
abstract = {Bacteria employ noncoding RNAs to maintain cellular physiology, adapt global gene expression to fluctuating environments, sense nutrients, coordinate their interaction with companion microbes and host cells, and protect themselves against bacteriophages. While bacterial RNA research has made fundamental contributions to biomedicine and biotechnology, the bulk of our knowledge of RNA biology stems from the study of a handful of aerobic model species. In comparison, RNA research is lagging in many medically relevant obligate anaerobic species, in particular the numerous commensal bacteria comprising our gut microbiota. This review presents a guide to RNA-based regulatory mechanisms in the phylum Bacteroidetes, focusing on the most abundant bacterial genus in the human gut, Bacteroides spp. This includes recent case reports on riboswitches, an mRNA leader, cis- and trans-encoded small RNAs (sRNAs) in Bacteroides spp., and a survey of CRISPR-Cas systems across Bacteroidetes. Recent work from our laboratory now suggests the existence of hundreds of noncoding RNA candidates in Bacteroides thetaiotaomicron, the emerging model organism for functional microbiota research. Based on these collective observations, we predict mechanistic and functional commonalities and differences between Bacteroides sRNAs and those of other model bacteria, and outline open questions and tools needed to boost Bacteroidetes RNA research.},
}
@article {pmid33544464,
year = {2021},
author = {Halm, D and Leibig, N and Martens, J and Stark, GB and Groß, T and Zimmermann, S and Finkenzeller, G and Lampert, F},
title = {Direct comparison of the immunogenicity of major histocompatibility complex-I and -II deficient mesenchymal stem cells in vivo.},
journal = {Biological chemistry},
volume = {402},
number = {6},
pages = {693-702},
doi = {10.1515/hsz-2020-0306},
pmid = {33544464},
issn = {1437-4315},
mesh = {CRISPR-Cas Systems/genetics/immunology ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Flow Cytometry ; Gene Editing ; Humans ; Major Histocompatibility Complex/genetics/*immunology ; Mesenchymal Stem Cells/cytology/*immunology ; },
abstract = {Mesenchymal stem cells (MSCs) play an important role in tissue engineering applications aiming at the regeneration or substitution of damaged tissues. In this context, off-the-shelf allogeneic MSCs would represent an attractive universal cell source. However, immune rejection is a major limitation for the clinical use of allogeneic MSCs. Immune rejection is mediated by the expression of major histocompatibility complexes (MHC)-I and -II on the donor cells. In this study, we eliminated MHC-I and/or MHC-II expression in human MSCs by using the CRISPR/Cas9 technology and investigated the effect of the individual or combined knockout of MHC-I and MHC-II on MSC survival after transplantation into immunocompetent mice. Elimination of MHC-I and/or MHC-II expression did not affect mesenchymal marker gene expression, viability, proliferation and the differentiation potential of MSCs in vitro. However, cell survival of transplanted MSCs was significantly elevated in MHC-I and MHC-II deficient MSCs. A direct side-by-side comparison does not reveal any significant difference in the immunogenicity of MHC-I and MHC-II knockout MSCs. Moreover, double knockout of MHC-I and MHC-II did not further increase in vivo cell survival of transplanted MSCs. Our results demonstrate that knockout of MHC-I and/or MHC-II represents an effective strategy to prevent immune rejection of allogeneic MSCs.},
}
@article {pmid33544462,
year = {2021},
author = {Eissenberg, JC},
title = {In Our Image: The Ethics of CRISPR Genome Editing.},
journal = {Biomolecular concepts},
volume = {12},
number = {1},
pages = {1-7},
doi = {10.1515/bmc-2021-0001},
pmid = {33544462},
issn = {1868-503X},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*ethics ; Humans ; },
abstract = {The advent of genome editing technology promises to transform human health, livestock and agriculture, and to eradicate pest species. This transformative power demands urgent scrutiny and resolution of the ethical conflicts attached to the creation and release of engineered genomes. Here, I discuss the ethics surrounding the transformative CRISPR/Cas9-mediated genome editing technology in the contexts of human genome editing to eradicate genetic disease and of gene drive technology to eradicate animal vectors of human disease.},
}
@article {pmid33544254,
year = {2021},
author = {Ren, M and Mei, H and Zhou, J and Zhou, M and Han, H and Zhao, L},
title = {Early diagnosis of rabies virus infection by RPA-CRISPR techniques in a rat model.},
journal = {Archives of virology},
volume = {166},
number = {4},
pages = {1083-1092},
pmid = {33544254},
issn = {1432-8798},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Early Diagnosis ; Molecular Diagnostic Techniques/*methods ; *Nucleic Acid Amplification Techniques ; RNA, Viral/cerebrospinal fluid/genetics ; Rabies/cerebrospinal fluid/*diagnosis ; Rabies virus/genetics/*isolation &amp; purification ; Rats ; Rats, Sprague-Dawley ; Sensitivity and Specificity ; },
abstract = {Rabies, which is caused by rabies virus (RABV), poses an ever-present threat to public health in most countries of the world. Once clinical signs appear, the mortality of rabies approaches 100%. To date, no effective method for early rabies diagnosis has been developed. In this study, an RPA-CRISPR nucleic-acid-based assay was developed for early rabies diagnosis by detecting viral RNA shedding in the cerebrospinal fluid (CSF) of rats. This method can detect a single copy of RABV genomic RNA in 1 μL of liquid. RABV genomic RNA released from viral particles in the CSF could be detected via RPA-CRISPR as early as 3 days postinfection in a rat model. This study provides an RPA-CRISPR technique for early detection of RABV with potential application in the clinical diagnosis of human rabies.},
}
@article {pmid33542471,
year = {2021},
author = {Liu, S and Cheng, Q and Wei, T and Yu, X and Johnson, LT and Farbiak, L and Siegwart, DJ},
title = {Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing.},
journal = {Nature materials},
volume = {20},
number = {5},
pages = {701-710},
pmid = {33542471},
issn = {1476-4660},
support = {P30 CA142543/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; },
mesh = {Administration, Intravenous ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Cell Membrane/*metabolism ; *Drug Delivery Systems ; Female ; *Gene Editing ; Mice ; Organ Specificity ; *Phospholipids/chemistry/pharmacology ; *RNA, Messenger/chemistry/pharmacology ; },
abstract = {Endosomal escape remains a fundamental barrier hindering the advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering messenger RNA or mRNA/single-guide RNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environments to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure-activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (called iPLNPs) for selective organ targeting. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates substantial value for gene editing research and therapeutic applications.},
}
@article {pmid33542442,
year = {2021},
author = {Ellis, NA and Kim, B and Tung, J and Machner, MP},
title = {A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {157},
pmid = {33542442},
issn = {2399-3642},
support = {ZIA HD008893/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Bacterial ; *Gene Silencing ; Humans ; Legionella pneumophila/*genetics/growth &amp; development/metabolism/pathogenicity ; Proof of Concept Study ; U937 Cells ; Virulence/genetics ; Virulence Factors/*genetics/metabolism ; },
abstract = {Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.},
}
@article {pmid33542270,
year = {2021},
author = {Chirikian, O and Goodyer, WR and Dzilic, E and Serpooshan, V and Buikema, JW and McKeithan, W and Wu, H and Li, G and Lee, S and Merk, M and Galdos, F and Beck, A and Ribeiro, AJS and Paige, S and Mercola, M and Wu, JC and Pruitt, BL and Wu, SM},
title = {CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {3026},
pmid = {33542270},
issn = {2045-2322},
support = {DP1 LM012179/LM/NLM NIH HHS/United States ; P01 HL141084/HL/NHLBI NIH HHS/United States ; R01 HL130840/HL/NHLBI NIH HHS/United States ; T32 HL094274/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cardiac Myosins/genetics ; Cell Differentiation/*genetics ; Green Fluorescent Proteins ; Heart Atria/cytology/*growth &amp; development/metabolism ; Heart Ventricles/cytology/growth &amp; development/metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism/pathology ; Myocytes, Cardiac/cytology/*metabolism ; Myosin Light Chains/genetics ; },
abstract = {Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.},
}
@article {pmid33541329,
year = {2021},
author = {Minami, SA and Shah, PS},
title = {Transient light-activated gene expression in Chinese hamster ovary cells.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {13},
pmid = {33541329},
issn = {1472-6750},
support = {P30 CA093373/CA/NCI NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Female ; Gene Expression/*radiation effects ; Genetic Techniques ; *Light ; *Ovary ; Transfection ; },
abstract = {BACKGROUND: Chinese hamster ovary (CHO) cells are widely used for industrial production of biopharmaceuticals. Many genetic, chemical, and environmental approaches have been developed to modulate cellular pathways to improve titers. However, these methods are often irreversible or have off-target effects. Development of techniques which are precise, tunable, and reversible will facilitate temporal regulation of target pathways to maximize titers. In this study, we investigate the use of optogenetics in CHO cells. The light-activated CRISPR-dCas9 effector (LACE) system was first transiently transfected to express eGFP in a light-inducible manner. Then, a stable system was tested using lentiviral transduction.<br><br>RESULTS: Transient transfections resulted in increasing eGFP expression as a function of LED intensity, and activation for 48&#x2009;h yielded up to 4-fold increased eGFP expression compared to cells kept in the dark. Fluorescence decreased once the LACE system was deactivated, and a protein half-life of 14.9&#x2009;h was calculated, which is in agreement with values reported in the literature. In cells stably expressing the LACE system, eGFP expression was confirmed, but there was no significant increase in expression following light activation.<br><br>CONCLUSIONS: Taken together, these results suggest that optogenetics can regulate CHO cell cultures, but development of stable cell lines requires optimized expression levels of the LACE components to maintain high dynamic range.},
}
@article {pmid33540323,
year = {2021},
author = {Lee, CY and Degani, I and Cheong, J and Lee, JH and Choi, HJ and Cheon, J and Lee, H},
title = {Fluorescence polarization system for rapid COVID-19 diagnosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {178},
number = {},
pages = {113049},
pmid = {33540323},
issn = {1873-4235},
support = {R01 CA229777/CA/NCI NIH HHS/United States ; },
mesh = {Biosensing Techniques/instrumentation/*methods/statistics &amp; numerical data ; COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/instrumentation/*methods/statistics &amp; numerical data ; CRISPR-Cas Systems ; Equipment Design ; Fluorescence Polarization/instrumentation/*methods/statistics &amp; numerical data ; Humans ; Molecular Diagnostic Techniques/instrumentation/methods/statistics &amp; numerical data ; Nucleic Acid Amplification Techniques/instrumentation/methods/statistics &amp; numerical data ; Pandemics ; Point-of-Care Systems/statistics &amp; numerical data ; SARS-CoV-2/*genetics/*isolation &amp; purification ; Signal Processing, Computer-Assisted ; Signal-To-Noise Ratio ; },
abstract = {Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/μL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.},
}
@article {pmid33539846,
year = {2021},
author = {Nimsamer, P and Mayuramart, O and Rattanaburi, S and Chantaravisoot, N and Saengchoowong, S and Puenpa, J and Poovorawan, Y and Payungporn, S},
title = {Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens.},
journal = {Journal of virological methods},
volume = {290},
number = {},
pages = {114092},
pmid = {33539846},
issn = {1879-0984},
mesh = {Bacterial Proteins ; COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*methods/standards ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; SARS-CoV-2/genetics/*isolation &amp; purification ; Sensitivity and Specificity ; Viral Proteins/genetics ; },
abstract = {COVID-19 pandemic caused by SARS-CoV-2 infection continue to cause the morbidity and mortality in many countries. Limitations of the gold standard qRT-PCR for diagnosis of this infection includes need for expensive equipment, specialized molecular laboratory, and experienced staff. Currently, CRISPR-based diagnostic method was approved by the U.S. FDA for rapid detection. Several studies developed SARS-CoV-2 detection based on CRISPR-Cas12a platform; however, the validations with RNA extracted from clinical specimens were limited. Therefore, this study evaluated the clinical performance of previously described CRISPR-Cas12a based diagnostic assays for SARS-CoV-2. According to the results, the CRISPR-Cas12a assays on N1 and S genes provided diagnostic accuracy (≥ 95 %) comparable to the qRT-PCR results. The assays with E, N2 and S genes yielded acceptable sensitivity of detection (≥ 95 %) whereas N1 and S genes provided outstanding specificity of detection (100 %). Preferably, multiple target genes should be detected by using CRISPR-Cas12a to ensure the most effective SARS-CoV-2 detection. Therefore, the N1 and S genes would be attractive target genes for SARS-CoV-2 detection based on CRISPR-Cas12a.},
}
@article {pmid33539788,
year = {2021},
author = {Hundley, FV and Sanvisens Delgado, N and Marin, HC and Carr, KL and Tian, R and Toczyski, DP},
title = {A comprehensive phenotypic CRISPR-Cas9 screen of the ubiquitin pathway uncovers roles of ubiquitin ligases in mitosis.},
journal = {Molecular cell},
volume = {81},
number = {6},
pages = {1319-1336.e9},
doi = {10.1016/j.molcel.2021.01.014},
pmid = {33539788},
issn = {1097-4164},
support = {R35 GM118104/GM/NIGMS NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Humans ; *Mitosis ; *Signal Transduction ; *Ubiquitin/genetics/metabolism ; *Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {The human ubiquitin proteasome system, composed of over 700 ubiquitin ligases (E3s) and deubiquitinases (DUBs), has been difficult to characterize systematically and phenotypically. We performed chemical-genetic CRISPR-Cas9 screens to identify E3s/DUBs whose loss renders cells sensitive or resistant to 41 compounds targeting a broad range of biological processes, including cell cycle progression, genome stability, metabolism, and vesicular transport. Genes and compounds clustered functionally, with inhibitors of related pathways interacting similarly with E3s/DUBs. Some genes, such as FBXW7, showed interactions with many of the compounds. Others, such as RNF25 and FBXO42, showed interactions primarily with a single compound (methyl methanesulfonate for RNF25) or a set of related compounds (the mitotic cluster for FBXO42). Mutation of several E3s with sensitivity to mitotic inhibitors led to increased aberrant mitoses, suggesting a role for these genes in cell cycle regulation. Our comprehensive CRISPR-Cas9 screen uncovered 466 gene-compound interactions covering 25% of the interrogated E3s/DUBs.},
}
@article {pmid33539781,
year = {2021},
author = {Yu, H and Lin, T and Meng, X and Du, H and Zhang, J and Liu, G and Chen, M and Jing, Y and Kou, L and Li, X and Gao, Q and Liang, Y and Liu, X and Fan, Z and Liang, Y and Cheng, Z and Chen, M and Tian, Z and Wang, Y and Chu, C and Zuo, J and Wan, J and Qian, Q and Han, B and Zuccolo, A and Wing, RA and Gao, C and Liang, C and Li, J},
title = {A route to de novo domestication of wild allotetraploid rice.},
journal = {Cell},
volume = {184},
number = {5},
pages = {1156-1170.e14},
doi = {10.1016/j.cell.2021.01.013},
pmid = {33539781},
issn = {1097-4172},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Domestication ; Food Security ; Gene Editing ; Genetic Variation ; Genome, Plant ; Oryza/classification/*genetics ; Polyploidy ; },
abstract = {Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.},
}
@article {pmid33538637,
year = {2021},
author = {Vicencio, J and Cerón, J},
title = {A Living Organism in your CRISPR Toolbox: Caenorhabditis elegans Is a Rapid and Efficient Model for Developing CRISPR-Cas Technologies.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {32-42},
doi = {10.1089/crispr.2020.0103},
pmid = {33538637},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; *Gene Editing ; Gene Knock-In Techniques ; Models, Animal ; RNA, Guide ; Streptococcus pyogenes/enzymology ; },
abstract = {The Cas9 nuclease from Streptococcus pyogenes (SpCas9) is the most popular enzyme for CRISPR technologies. However, considering the wide diversity of microorganisms (discovered and still unknown), a massive number of CRISPR effectors are being and will be identified and characterized in the search of optimal Cas variants for each of the many applications of CRISPR. In this context, a versatile and efficient multicellular system for CRISPR editing such as Caenorhabditis elegans would be of great help in the development of these effectors. Here, we highlight the benefits of using C. elegans for the rapid evaluation of new CRISPR effectors, and for optimizing CRISPR efficiency in animals in several ways such as by modulating the balance between repair pathways, modifying chromatin accessibility, or controlling the expression and activity of nucleases and guide RNAs.},
}
@article {pmid33538626,
year = {2021},
author = {Mohanraju, P and Mougiakos, I and Albers, J and Mabuchi, M and Fuchs, RT and Curcuru, JL and van Kranenburg, R and Robb, GB and van der Oost, J},
title = {Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles.},
journal = {The CRISPR journal},
volume = {4},
number = {1},
pages = {82-91},
pmid = {33538626},
issn = {2573-1602},
mesh = {Bacillus/genetics ; Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/*genetics ; Endonucleases/genetics ; Escherichia coli ; Francisella/genetics ; *Gene Editing ; Genome, Bacterial ; Plasmids ; Recombination, Genetic ; },
abstract = {The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Francisella novicida Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium Bacillus smithii as our experimental platform, we demonstrate that FnCas12a is active in vivo at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for B. smithii, combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of B. smithii cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.},
}
@article {pmid33537681,
year = {2021},
author = {Zhao, R and Yang, Y and Yang, K and Han, W},
title = {Expression, purification, and characterization of a membrane-associated cyclic oligo-adenylate degrader from Sulfolobus islandicus.},
journal = {STAR protocols},
volume = {2},
number = {1},
pages = {100299},
pmid = {33537681},
issn = {2666-1667},
mesh = {*Archaeal Proteins/biosynthesis/chemistry/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Cell Membrane/*enzymology/genetics ; *Gene Expression ; Recombinant Proteins/biosynthesis/chemistry/genetics/isolation &amp; purification ; Sulfolobus/*enzymology/genetics ; },
abstract = {Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus, membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).},
}
@article {pmid33537251,
year = {2021},
author = {Lee, H},
title = {Rapid Way to Generate Mouse Models for In Vivo Studies of the Endothelium.},
journal = {Journal of lipid and atherosclerosis},
volume = {10},
number = {1},
pages = {24-41},
pmid = {33537251},
issn = {2287-2892},
abstract = {A single layer of squamous endothelial cells (ECs), the endothelium, regulates the flow of substance and fluid into and out of a tissue. The endothelium is also involved in vasculogenesis, the formation of new blood vessels, which is a crucial process for organ development in the embryo and fetus. Because most murine mutations of genes involved in EC development cause early embryo lethality, EC-specific conditional knockout (cKO) mouse models are indispensable for in vivo studies. cKO mice including the floxed allele can be generated through advanced approaches including embryonic stem cell-mediated gene targeting or the CRISPR/Cas system. EC-specific mouse models can be generated through further breeding of floxed mice with a Cre driver line, the latest information of which is available in the Jackson Cre Repository or the EUCOMMTOOLS project. Because it takes a long time (generally 1-2 years) to generate EC-specific mouse models, researchers must thoroughly design and plan a breeding strategy before full-scale mouse experiments, which saves time and money for in vivo study. In summary, revolutionary technical advances in embryo manipulation and assisted reproduction technologies have made it easier to generate EC-specific mouse models, which have been used as essential resources for in vivo studies of the endothelium.},
}
@article {pmid33536629,
year = {2021},
author = {Blanchard, EL and Vanover, D and Bawage, SS and Tiwari, PM and Rotolo, L and Beyersdorf, J and Peck, HE and Bruno, NC and Hincapie, R and Michel, F and Murray, J and Sadhwani, H and Vanderheyden, B and Finn, MG and Brinton, MA and Lafontaine, ER and Hogan, RJ and Zurla, C and Santangelo, PJ},
title = {Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents.},
journal = {Nature biotechnology},
volume = {39},
number = {6},
pages = {717-726},
pmid = {33536629},
issn = {1546-1696},
mesh = {Animals ; COVID-19/genetics/*therapy/virology ; CRISPR-Cas Systems/genetics ; Cricetinae ; Disease Models, Animal ; Humans ; Influenza, Human/genetics/*therapy/virology ; Mice ; Orthomyxoviridae/drug effects/genetics/pathogenicity ; RNA, Messenger/genetics/*pharmacology ; RNA, Viral/genetics ; Respiratory System/drug effects/metabolism ; SARS-CoV-2/*genetics/pathogenicity ; },
abstract = {Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.},
}
@article {pmid33536571,
year = {2021},
author = {Duan, L and Hu, M and Tamm, JA and Grinberg, YY and Shen, F and Chai, Y and Xi, H and Gibilisco, L and Ravikumar, B and Gautam, V and Karran, E and Townsend, M and Talanian, RV},
title = {Arrayed CRISPR reveals genetic regulators of tau aggregation, autophagy and mitochondria in Alzheimer's disease model.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {2879},
pmid = {33536571},
issn = {2045-2322},
mesh = {Alzheimer Disease/*genetics/pathology ; Autophagy/*genetics ; Brain/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Expression Regulation ; Genetic Engineering ; Humans ; Machine Learning ; Mitochondria/genetics/pathology ; *Models, Genetic ; Neurons ; Protein Aggregation, Pathological/*genetics/pathology ; Signal Transduction/genetics ; tau Proteins/*metabolism ; },
abstract = {Alzheimer's disease (AD) is a common neurodegenerative disease with poor prognosis. New options for drug discovery targets are needed. We developed an imaging based arrayed CRISPR method to interrogate the human genome for modulation of in vitro correlates of AD features, and used this to assess 1525 human genes related to tau aggregation, autophagy and mitochondria. This work revealed (I) a network of tau aggregation modulators including the NF-κB pathway and inflammatory signaling, (II) a correlation between mitochondrial morphology, respiratory function and transcriptomics, (III) machine learning predicted novel roles of genes and pathways in autophagic processes and (IV) individual gene function inferences and interactions among biological processes via multi-feature clustering. These studies provide a platform to interrogate underexplored aspects of AD biology and offer several specific hypotheses for future drug discovery efforts.},
}
@article {pmid33536556,
year = {2021},
author = {Mehta, D and Vanderschuren, H},
title = {Towards responsible communication of agricultural biotechnology research for the common good.},
journal = {Nature reviews. Molecular cell biology},
volume = {22},
number = {5},
pages = {301-302},
pmid = {33536556},
issn = {1471-0080},
mesh = {Agriculture/*trends ; Biotechnology/*trends ; CRISPR-Cas Systems/*genetics ; Humans ; },
}
@article {pmid33536327,
year = {2021},
author = {Bui, HTN and Passecker, A and Brancucci, NMB and Voss, TS},
title = {Investigation of Heterochromatin Protein 1 Function in the Malaria Parasite Plasmodium falciparum Using a Conditional Domain Deletion and Swapping Approach.},
journal = {mSphere},
volume = {6},
number = {1},
pages = {},
pmid = {33536327},
issn = {2379-5042},
mesh = {Antigenic Variation ; CRISPR-Cas Systems ; Cell Line ; Chromobox Protein Homolog 5 ; Chromosomal Proteins, Non-Histone/*genetics/*metabolism ; Erythrocytes/parasitology ; Gene Expression Regulation ; *Gene Silencing ; Humans ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/*genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; },
abstract = {The human malaria parasite Plasmodium falciparum encodes a single ortholog of heterochromatin protein 1 (PfHP1) that plays a crucial role in the epigenetic regulation of various survival-related processes. PfHP1 is essential for parasite proliferation and the heritable silencing of genes linked to antigenic variation, host cell invasion, and sexual conversion. Here, we employed CRISPR/Cas9-mediated genome editing combined with the DiCre/loxP system to investigate how the PfHP1 chromodomain (CD), hinge domain, and chromoshadow domain (CSD) contribute to overall PfHP1 function. We show that the 76 C-terminal residues are responsible for targeting PfHP1 to the nucleus. Furthermore, we reveal that each of the three functional domains of PfHP1 are required for heterochromatin formation, gene silencing, and mitotic parasite proliferation. Finally, we discovered that the hinge domain and CSD of HP1 are functionally conserved between P. falciparum and P. berghei, a related malaria parasite infecting rodents. In summary, our study provides new insights into PfHP1 function and offers a tool for further studies on epigenetic regulation and life cycle decision in malaria parasites.IMPORTANCE Malaria is caused by unicellular Plasmodium species parasites that repeatedly invade and replicate inside red blood cells. Some blood-stage parasites exit the cell cycle and differentiate into gametocytes that are essential for malaria transmission via the mosquito vector. Epigenetic control mechanisms allow the parasites to alter the expression of surface antigens and to balance the switch between parasite multiplication and gametocyte production. These processes are crucial to establish chronic infection and optimize parasite transmission. Here, we performed a mutational analysis of heterochromatin protein 1 (HP1) in P. falciparum We demonstrate that all three domains of this protein are indispensable for the proper function of HP1 in parasite multiplication, heterochromatin formation, and gene silencing. Moreover, expression of chimeric proteins revealed the functional conservation of HP1 proteins between different Plasmodium species. These results provide new insight into the function and evolution of HP1 as an essential epigenetic regulator of parasite survival.},
}
@article {pmid33534660,
year = {2021},
author = {Philippe, C and Moineau, S},
title = {The endless battle between phages and CRISPR-Cas systems in Streptococcus thermophilus.},
journal = {Biochemistry and cell biology = Biochimie et biologie cellulaire},
volume = {99},
number = {4},
pages = {397-402},
doi = {10.1139/bcb-2020-0593},
pmid = {33534660},
issn = {1208-6002},
mesh = {*Adaptation, Physiological ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; *Gene Editing ; Streptococcus thermophilus/*physiology ; },
abstract = {This review describes the contribution of basic research on phage-bacteria interactions to the understanding of CRISPR-Cas systems and their various applications. It focuses on the natural function of CRISPR-Cas systems as adaptive defense mechanisms against mobile genetic elements such as bacteriophage genomes and plasmids. Some of the advances in the characterization of the type II-A CRISPR-Cas system of Streptococcus thermophilus and Streptococcus pyogenes led to the development of the CRISPR-Cas9 genome-editing technology. We mostly discuss the 3 stages of the CRISPR-Cas system in S. thermophilus, namely the adaptation stage, which is unique to this resistance mechanism; the CRISPR RNA biogenesis; and the DNA-cutting activity in the interference stage to protect bacteria against phages. Finally, we look into applications of CRISPR-Cas in microbiology, including overcoming limitations in genome editing.},
}
@article {pmid33531685,
year = {2021},
author = {Kupatt, C and Windisch, A and Moretti, A and Wolf, E and Wurst, W and Walter, MC},
title = {Genome editing for Duchenne muscular dystrophy: a glimpse of the future?.},
journal = {Gene therapy},
volume = {28},
number = {9},
pages = {542-548},
pmid = {33531685},
issn = {1476-5462},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Dogs ; Gene Editing ; Genetic Therapy ; Mice ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Swine ; },
abstract = {Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45-52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro- and minidystrophins as well as utrophin, a protein with partially overlapping functions. With the advent of the CRISPR-Cas9-nuclease, genome editing offers a novel option of correction of the disease-cuasing mutations. Full restoration of the healthy gene by homology directed repair is a rare event. However, non-homologous end-joining (NHEJ) may restore the reading frame by causing exon excision. This approach has first been demonstrated in mice and then translated to large animals (dogs, pigs). This review discusses the potential opportunities and limitations of genome editing in DMD, including the generation of appropriate animal models as well as new developments in genome editing tools.},
}
@article {pmid33531004,
year = {2021},
author = {Li, Y and Xian, H and Xu, Y and Zhu, Y and Sun, Z and Wang, Q and Qi, Q},
title = {Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli.},
journal = {Microbial cell factories},
volume = {20},
number = {1},
pages = {32},
pmid = {33531004},
issn = {1475-2859},
mesh = {Adenosine Triphosphate/metabolism ; CRISPR-Cas Systems/*genetics ; Carbon Isotopes ; Down-Regulation ; Energy Metabolism ; Escherichia coli/*enzymology ; Fermentation ; Glucosephosphate Dehydrogenase/*metabolism ; *Glycolysis ; Metabolic Flux Analysis ; Mevalonic Acid/*metabolism ; NADP/metabolism ; Pentose Phosphate Pathway ; Phosphofructokinases/*metabolism ; Promoter Regions, Genetic/genetics ; },
abstract = {BACKGROUND: Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and "bifid shunt", to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA).<br><br>RESULT: Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2&#xa0;g/L of MVA after 72&#xa0;h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53&#xa0;g/L, and the conversion rate from glucose reached 68.7%.<br><br>CONCLUSION: This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.},
}
@article {pmid33530834,
year = {2021},
author = {Bastiaanssen, C and Joo, C},
title = {Small RNA-directed DNA elimination: the molecular mechanism and its potential for genome editing.},
journal = {RNA biology},
volume = {18},
number = {11},
pages = {1540-1545},
pmid = {33530834},
issn = {1555-8584},
support = {819299/ERC_/European Research Council/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Transposable Elements ; *Gene Editing ; Genetic Therapy/*methods ; *Genome, Protozoan ; Humans ; Tetrahymena/*genetics ; },
abstract = {Transposable elements have both detrimental and beneficial effects on their host genome. Tetrahymena is a unicellular eukaryote that deals with transposable elements in a unique way. It has a separate somatic and germline genome in two nuclei in a single cell. During sexual reproduction, a small RNA directed system compares the germline and somatic genome to identify transposable elements and related sequences. These are subsequently marked by heterochromatin and excised. In this Review, current knowledge of this system and the gaps therein are discussed. Additionally, the possibility to exploit the Tetrahymena machinery for genome editing and its advantages over the widely used CRISPR-Cas9 system will be explored. While the bacterial derived CRISPR-Cas9 has difficulty to access eukaryotic chromatin, Tetrahymena proteins are adept at acting in a chromatin context. Furthermore, Tetrahymena based gene therapy in humans might be a safer alternative to Cas9 because the latter can trigger an immune response.},
}
@article {pmid33530582,
year = {2021},
author = {Zabulica, M and Jakobsson, T and Ravaioli, F and Vosough, M and Gramignoli, R and Ellis, E and Rooyackers, O and Strom, SC},
title = {Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {3},
pages = {},
pmid = {33530582},
issn = {1422-0067},
mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Disease Susceptibility ; *Gene Editing ; Gene Expression Profiling ; Genetic Association Studies ; Genetic Variation ; Hepatocytes/cytology/*metabolism ; Humans ; Immunohistochemistry ; Induced Pluripotent Stem Cells/cytology/metabolism ; Metabolic Networks and Pathways/*genetics ; Models, Biological ; Organoids/*cytology ; Stem Cells/cytology/*metabolism ; Urea/*metabolism ; },
abstract = {Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy.},
}
@article {pmid33529861,
year = {2021},
author = {Sheng, Y and Zhang, T and Zhang, S and Johnston, M and Zheng, X and Shan, Y and Liu, T and Huang, Z and Qian, F and Xie, Z and Ai, Y and Zhong, H and Kuang, T and Dincer, C and Urban, GA and Hu, J},
title = {A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {178},
number = {},
pages = {113027},
doi = {10.1016/j.bios.2021.113027},
pmid = {33529861},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *Carcinoma, Non-Small-Cell Lung ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA, Catalytic ; Humans ; *Lung Neoplasms/diagnosis/genetics ; },
abstract = {Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 μL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.},
}
@article {pmid33529763,
year = {2021},
author = {Zou, Z and Wang, R and Go, EP and Desaire, H and Sun, PD},
title = {High level stable expression of recombinant HIV gp120 in glutamine synthetase gene deficient HEK293T cells.},
journal = {Protein expression and purification},
volume = {181},
number = {},
pages = {105837},
doi = {10.1016/j.pep.2021.105837},
pmid = {33529763},
issn = {1096-0279},
support = {R01 AI125093/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems ; Codon ; Cricetulus ; Gene Knockdown Techniques ; Glutamate-Ammonia Ligase/*genetics ; HEK293 Cells ; HIV Envelope Protein gp120/*metabolism ; HIV-1/*metabolism ; Humans ; Protein Sorting Signals ; Recombinant Proteins/metabolism ; },
abstract = {Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.},
}
@article {pmid33529430,
year = {2021},
author = {Nishizawa-Yokoi, A and Toki, S},
title = {A piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice.},
journal = {Plant biotechnology journal},
volume = {19},
number = {7},
pages = {1386-1395},
pmid = {33529430},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Transfer Techniques ; *Oryza/genetics ; Transgenes/genetics ; },
abstract = {Targeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double-stranded transfer DNA (dsT-DNA), with subsequent excision of the transgenes by the re-transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T-DNA works to deliver transgenes in rice. Following positive-negative selection to exclude transgenic cells randomly transformed with T-DNA, piggyBac-mediated transgenesis from the extrachromosomal dsT-DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof-of-concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9-induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac-mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/Cas9-mediated targeted mutagenesis in vegetatively propagated crops.},
}
@article {pmid33529193,
year = {2021},
author = {Lian, H and Wang, L and Ma, N and Zhou, CM and Han, L and Zhang, TQ and Wang, JW},
title = {Redundant and specific roles of individual MIR172 genes in plant development.},
journal = {PLoS biology},
volume = {19},
number = {2},
pages = {e3001044},
pmid = {33529193},
issn = {1545-7885},
mesh = {Arabidopsis/*genetics/*growth &amp; development/metabolism ; CRISPR-Cas Systems ; Flowers/genetics/growth &amp; development ; Gene Editing ; Gene Expression Regulation, Plant ; Genes, Plant ; MicroRNAs/*genetics ; Plant Development/genetics ; },
abstract = {Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.},
}
@article {pmid33528277,
year = {2021},
author = {Suryaletha, K and Chandrika, SK and Thomas, S},
title = {Comprehensive genomics depict accessory genes encoding pathogenicity and biofilm determinants in Enterococcus faecalis.},
journal = {Future microbiology},
volume = {16},
number = {3},
pages = {175-184},
doi = {10.2217/fmb-2020-0111},
pmid = {33528277},
issn = {1746-0921},
mesh = {Bacterial Proteins/*genetics/metabolism ; *Biofilms ; CRISPR-Cas Systems ; Enterococcus faecalis/*genetics/*pathogenicity/physiology ; Genome, Bacterial ; Genomic Islands ; Gram-Positive Bacterial Infections/microbiology ; Humans ; Plasmids/genetics/metabolism ; Virulence ; },
abstract = {Aim:Enterococcus faecalis is a leading nosocomial pathogen in biofilm-associated polymicrobial infections. The study aims to understand pathogenicity and biofilm determinants of the pathogen by genome analysis. Methodology: Genome sequencing of a strong biofilm forming clinical isolate Enterococcus faecalis SK460 devoid of Fsr quorum-signaling system, was performed and comparative genomics was carried out among a set of pathogenic biofilm formers and nonpathogenic weak biofilm formers. Results: Analysis revealed a pool of virulence and adhesion related factors associated with pathogenicity. Absence of CRISPR-Cas system facilitated acquisition of pheromone responsive plasmid, pathogenicity island and phages. Comprehensive analysis identified a subset of accessory genes encoding polysaccharide lyase, sugar phosphotransferase system, phage proteins and transcriptional regulators exclusively in pathogenic biofilm formers. Conclusion: The study identified a set of genes specific to pathogenic biofilm formers and these can act as targets which in turn help to develop future treatment endeavors against enterococcal infections.},
}
@article {pmid33527896,
year = {2021},
author = {Fazio, M and van Rooijen, E and Dang, M and van de Hoek, G and Ablain, J and Mito, JK and Yang, S and Thomas, A and Michael, J and Fabo, T and Modhurima, R and Pessina, P and Kaufman, CK and Zhou, Y and White, RM and Zon, LI},
title = {SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33527896},
issn = {2050-084X},
support = {P01 CA163222/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA103846/CA/NCI NIH HHS/United States ; T32 HL007627/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Models, Animal ; Drug Resistance, Neoplasm/*genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Matrix Attachment Region Binding Proteins/genetics/*metabolism ; Melanoma/drug therapy/*genetics/metabolism ; Neoplasm Invasiveness/*genetics ; Neural Crest/cytology ; Transcription Factors/genetics/*metabolism ; Zebrafish ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITF[low]AXL[high] and AQP1[+]NGFR1[high] drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.},
}
@article {pmid33526930,
year = {2021},
author = {Dunbar, CE},
title = {A plethora of gene therapies for hemoglobinopathies.},
journal = {Nature medicine},
volume = {27},
number = {2},
pages = {202-204},
pmid = {33526930},
issn = {1546-170X},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing ; *Hemoglobinopathies/genetics/therapy ; Humans ; *beta-Thalassemia ; },
}
@article {pmid33526582,
year = {2021},
author = {Shoesmith, JR and Solomon, CU and Yang, X and Wilkinson, LG and Sheldrick, S and van Eijden, E and Couwenberg, S and Pugh, LM and Eskan, M and Stephens, J and Barakate, A and Drea, S and Houston, K and Tucker, MR and McKim, SM},
title = {APETALA2 functions as a temporal factor together with BLADE-ON-PETIOLE2 and MADS29 to control flower and grain development in barley.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {5},
pages = {},
doi = {10.1242/dev.194894},
pmid = {33526582},
issn = {1477-9129},
support = {669182/ERC_/European Research Council/International ; BB/L001934/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/J01446X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Alleles ; Base Sequence ; CRISPR-Cas Systems/genetics ; Edible Grain/anatomy &amp; histology/metabolism ; Flowers/growth &amp; development/metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Genotype ; Homeodomain Proteins/chemistry/genetics/*metabolism ; Hordeum/growth &amp; development/*metabolism ; MADS Domain Proteins/genetics/*metabolism ; Mutagenesis ; Phenotype ; Plant Proteins/chemistry/genetics/*metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Cereal grain develops from fertilised florets. Alterations in floret and grain development greatly influence grain yield and quality. Despite this, little is known about the underlying genetic control of these processes, especially in key temperate cereals such as barley and wheat. Using a combination of near-isogenic mutant comparisons, gene editing and genetic analyses, we reveal that HvAPETALA2 (HvAP2) controls floret organ identity, floret boundaries, and maternal tissue differentiation and elimination during grain development. These new roles of HvAP2 correlate with changes in grain size and HvAP2-dependent expression of specific HvMADS-box genes, including the B-sister gene, HvMADS29 Consistent with this, gene editing demonstrates that HvMADS29 shares roles with HvAP2 in maternal tissue differentiation. We also discovered that a gain-of-function HvAP2 allele masks changes in floret organ identity and grain size due to loss of barley LAXATUM.A/BLADE-ON-PETIOLE2 (HvBOP2) gene function. Taken together, we reveal novel pleiotropic roles and regulatory interactions for an AP2-like gene controlling floret and grain development in a temperate cereal.},
}
@article {pmid33525876,
year = {2021},
author = {Chi, J and Zhao, J and Wei, S and Li, Y and Zhi, J and Wang, H and Hou, X and Hu, L and Zheng, X and Gao, M},
title = {A CRISPR-Cas9-Based Near-Infrared Upconversion-Activated DNA Methylation Editing System.},
journal = {ACS applied materials &amp; interfaces},
volume = {13},
number = {5},
pages = {6043-6052},
doi = {10.1021/acsami.0c21223},
pmid = {33525876},
issn = {1944-8252},
mesh = {Adaptor Proteins, Signal Transducing/genetics ; Animals ; Apoptosis/genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Survival ; Cells, Cultured ; DNA Methylation/genetics ; Female ; *Gene Editing ; *Genetic Techniques ; HEK293 Cells ; Humans ; *Infrared Rays ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasms, Experimental/genetics/pathology/therapy ; Particle Size ; Surface Properties ; Thyroid Neoplasms/*genetics/pathology/therapy ; },
abstract = {DNA methylation is a kind of a crucial epigenetic marker orchestrating gene expression, molecular function, and cellular phenotype. However, manipulating the methylation status of specific genes remains challenging. Here, a clustered regularly interspaced palindromic repeats-Cas9-based near-infrared upconversion-activated DNA methylation editing system (CNAMS) was designed for the optogenetic editing of DNA methylation. The fusion proteins of photosensitive CRY2PHR, the catalytic domain of DNMT3A or TET1, and the fusion proteins for CIBN and catalytically inactive Cas9 (dCas9) were engineered. The CNAMS could control DNA methylation editing in response to blue light, thus allowing methylation editing in a spatiotemporal manner. Furthermore, after combination with upconversion nanoparticles, the spectral sensitivity of DNA methylation editing was extended from the blue light to near-infrared (NIR) light, providing the possibility for remote DNA methylation editing. These results demonstrated a meaningful step forward toward realizing the specific editing of DNA methylation, suggesting the wide utility of our CNAMS for functional studies on epigenetic regulation and potential therapeutic strategies for related diseases.},
}
@article {pmid33525637,
year = {2021},
author = {Cetin, R and Quandt, E and Kaulich, M},
title = {Functional Genomics Approaches to Elucidate Vulnerabilities of Intrinsic and Acquired Chemotherapy Resistance.},
journal = {Cells},
volume = {10},
number = {2},
pages = {},
pmid = {33525637},
issn = {2073-4409},
mesh = {Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/*genetics ; Gene Targeting ; *Genomics ; Humans ; Translational Research, Biomedical ; },
abstract = {Drug resistance is a commonly unavoidable consequence of cancer treatment that results in therapy failure and disease relapse. Intrinsic (pre-existing) or acquired resistance mechanisms can be drug-specific or be applicable to multiple drugs, resulting in multidrug resistance. The presence of drug resistance is, however, tightly coupled to changes in cellular homeostasis, which can lead to resistance-coupled vulnerabilities. Unbiased gene perturbations through RNAi and CRISPR technologies are invaluable tools to establish genotype-to-phenotype relationships at the genome scale. Moreover, their application to cancer cell lines can uncover new vulnerabilities that are associated with resistance mechanisms. Here, we discuss targeted and unbiased RNAi and CRISPR efforts in the discovery of drug resistance mechanisms by focusing on first-in-line chemotherapy and their enforced vulnerabilities, and we present a view forward on which measures should be taken to accelerate their clinical translation.},
}
@article {pmid33525517,
year = {2021},
author = {Razzaq, MK and Aleem, M and Mansoor, S and Khan, MA and Rauf, S and Iqbal, S and Siddique, KHM},
title = {Omics and CRISPR-Cas9 Approaches for Molecular Insight, Functional Gene Analysis, and Stress Tolerance Development in Crops.},
journal = {International journal of molecular sciences},
volume = {22},
number = {3},
pages = {},
pmid = {33525517},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/genetics/*growth &amp; development ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genomics ; Metabolomics ; Plant Proteins/*genetics ; Reverse Genetics ; Stress, Physiological ; },
abstract = {Plants are regularly exposed to biotic and abiotic stresses that adversely affect agricultural production. Omics has gained momentum in the last two decades, fueled by statistical methodologies, computational capabilities, mass spectrometry, nucleic-acid sequencing, and peptide-sequencing platforms. Functional genomics-especially metabolomics, transcriptomics, and proteomics-have contributed substantially to plant molecular responses to stress. Recent progress in reverse and forward genetics approaches have mediated high-throughput techniques for identifying stress-related genes. Furthermore, web-based genetic databases have mediated bioinformatics techniques for detecting families of stress-tolerant genes. Gene ontology (GO) databases provide information on the gene product's functional features and help with the computational estimation of gene function. Functional omics data from multiple platforms are useful for positional cloning. Stress-tolerant plants have been engineered using stress response genes, regulatory networks, and pathways. The genome-editing tool, CRISPR-Cas9, reveals the functional features of several parts of the plant genome. Current developments in CRISPR, such as de novo meristem induction genome-engineering in dicots and temperature-tolerant LbCas12a/CRISPR, enable greater DNA insertion precision. This review discusses functional omics for molecular insight and CRISPR-Cas9-based validation of gene function in crop plants. Omics and CRISPR-Cas9 are expected to garner knowledge on molecular systems and gene function and stress-tolerant crop production.},
}
@article {pmid33524674,
year = {2021},
author = {Dong, T and Zhang, S and Chang, Y and Bai, R and Jiang, Y and Ma, S and Li, Y and Jiang, HF and Lu, WJ},
title = {The establishment of a homozygous SNTA1 knockout human embryonic stem cell line (WAe009-A-50) using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {51},
number = {},
pages = {102196},
doi = {10.1016/j.scr.2021.102196},
pmid = {33524674},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; Homozygote ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {SNTA1 encodes α1-syntrophin, a scaffold protein, which is a component of the dystrophin-associated protein complex. Additionally, α1-syntrophin interacts with SCN5A and nNOS-PMCA4b complex in cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmia and cardiomyopathy. We generated a homozygous SNTA1 knockout human embryonic stem cell (H9SNTA1KO) using the CRISPR/Cas9 system. H9SNTA1KO maintained pluripotency and a normal karyotype and differentiated into three germ layers in vivo.},
}
@article {pmid33524499,
year = {2021},
author = {Shrestha, P and Han, SR and Lee, JH and Park, H and Oh, TJ},
title = {A computational approach to identify CRISPR-Cas loci in the complete genomes of the lichen-associated Burkholderia sp. PAMC28687 and PAMC26561.},
journal = {Genomics},
volume = {113},
number = {3},
pages = {881-888},
doi = {10.1016/j.ygeno.2021.01.019},
pmid = {33524499},
issn = {1089-8646},
mesh = {Animals ; *Burkholderia/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; *Lichens/genetics ; Sequence Analysis, DNA ; },
abstract = {The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.},
}
@article {pmid33524495,
year = {2021},
author = {Gertsenstein, M and Nutter, LMJ},
title = {Production of knockout mouse lines with Cas9.},
journal = {Methods (San Diego, Calif.)},
volume = {191},
number = {},
pages = {32-43},
doi = {10.1016/j.ymeth.2021.01.005},
pmid = {33524495},
issn = {1095-9130},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Electroporation ; Endonucleases/genetics/metabolism ; *Gene Editing ; Gene Knockout Techniques ; Mice ; Mice, Knockout ; RNA, Guide/genetics ; Zygote/metabolism ; },
abstract = {Knockout mice are used extensively to explore the phenotypic effects of mammalian gene dysfunction. With the application of RNA-guided Cas9 nuclease technology for the production of knockout mouse lines, the time, as well as the resources needed, to progress from identification of a gene of interest to production of a knockout line is significantly reduced. Here we present our standard methodology to produce knockout mouse lines by the electroporation of Cas9 ribonucleoprotein (RNP) into mouse zygotes. Using this protocol, we have obtained an 80% success rate in the generation of founders for null alleles with a subsequent 93% germline transmission rate. These methods rely on equipment already present in the majority of transgenic facilities and should be straightforward to implement where appropriate embryo handling expertise exists.},
}
@article {pmid33524138,
year = {2021},
author = {Xiao, Y and Uh, K and Negrón-Pérez, VM and Haines, H and Lee, K and Hansen, PJ},
title = {Regulation of gene expression in the bovine blastocyst by colony-stimulating factor 2 is disrupted by CRISPR/Cas9-mediated deletion of CSF2RA.},
journal = {Biology of reproduction},
volume = {104},
number = {5},
pages = {995-1007},
pmid = {33524138},
issn = {1529-7268},
support = {R01 HD088352/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Blastocyst/*metabolism ; CRISPR-Cas Systems ; Cattle ; *Gene Deletion ; *Gene Expression Regulation, Developmental ; Granulocyte-Macrophage Colony-Stimulating Factor/*genetics/metabolism ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/*deficiency/metabolism ; },
abstract = {Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The β subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and β subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.},
}
@article {pmid33523917,
year = {2021},
author = {Kelly, JJ and Saee-Marand, M and Nyström, NN and Evans, MM and Chen, Y and Martinez, FM and Hamilton, AM and Ronald, JA},
title = {Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking.},
journal = {Science advances},
volume = {7},
number = {4},
pages = {},
pmid = {33523917},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; *Cell Tracking ; Gene Editing/methods ; Gene Knock-In Techniques ; Genes, Reporter ; Recombinational DNA Repair ; Tissue Distribution ; },
abstract = {Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.},
}
@article {pmid33523494,
year = {2021},
author = {Parameshwaran, HP and Babu, K and Tran, C and Guan, K and Allen, A and Kathiresan, V and Qin, PZ and Rajan, R},
title = {The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.},
journal = {FEBS letters},
volume = {595},
number = {7},
pages = {892-912},
pmid = {33523494},
issn = {1873-3468},
support = {P20 GM103640/GM/NIGMS NIH HHS/United States ; R01 GM124413/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA Cleavage ; DNA, Single-Stranded/genetics ; Deoxyribonuclease I/*genetics ; Endodeoxyribonucleases/*genetics ; Francisella/genetics ; Gene Editing ; Nucleic Acid Conformation ; RNA, Guide/genetics/*ultrastructure ; },
abstract = {Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12a[KD2P]) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12a[KD2P] accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a[KD2P] also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.},
}
@article {pmid33522487,
year = {2021},
author = {Chou, HC and Bhalla, K and Demerdesh, OE and Klingbeil, O and Hanington, K and Aganezov, S and Andrews, P and Alsudani, H and Chang, K and Vakoc, CR and Schatz, MC and McCombie, WR and Stillman, B},
title = {The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33522487},
issn = {2050-084X},
support = {P30 CA045508/CA/NCI NIH HHS/United States ; P01 CA013106/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Cycle Proteins/genetics ; Cell Line ; DNA Replication/*genetics ; Gene Knockout Techniques ; Humans ; Minichromosome Maintenance Proteins/genetics ; Mitosis/*genetics ; Origin Recognition Complex/*genetics ; },
abstract = {The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.},
}
@article {pmid33521928,
year = {2021},
author = {Lee, Z and Raabe, M and Hu, WS},
title = {Epigenomic features revealed by ATAC-seq impact transgene expression in CHO cells.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {5},
pages = {1851-1861},
doi = {10.1002/bit.27701},
pmid = {33521928},
issn = {1097-0290},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/genetics ; Chromatin Immunoprecipitation Sequencing/*methods ; Cricetinae ; Cricetulus ; Enhancer Elements, Genetic/*genetics ; Epigenesis, Genetic/*genetics ; Transgenes/*genetics ; },
abstract = {Different regions of a mammalian genome have different accessibilities to transcriptional machinery. The integration site of a transgene affects how actively it is transcribed. Highly accessible genomic regions called super-enhancers have been recently described as strong regulatory elements that shape cell identity. Super-enhancers have been identified in Chinese hamster ovary (CHO) cells using the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq). Genes near super-enhancer regions had high transcript levels and were enriched for oncogenic signaling and proliferation functions, consistent with an immortalized phenotype. Inaccessible regions in the genome with low ATAC signal also had low transcriptional activity. Genes in inaccessible regions were enriched for remote tissue functions such as taste, smell, and neuronal activation. A lentiviral reporter integration assay showed integration into super-enhancer regions conferred higher reporter expression than insertion into inaccessible regions. Targeted integration of an IgG vector into the Plec super-enhancer region yielded clones that expressed the immunoglobulin light chain gene mostly in the top 20% of all transcripts with the majority in the top 5%. The results suggest the epigenomic landscape of CHO cells can guide the selection of integration sites in the development of cell lines for therapeutic protein production.},
}
@article {pmid33521855,
year = {2021},
author = {Liang, Z and Wu, Y and Guo, Y and Liu, Y and Ma, L and Wu, Y},
title = {Bi-functional selection markers assist segregation of transgene-free, genome-edited mutants.},
journal = {Science China. Life sciences},
volume = {64},
number = {9},
pages = {1567-1570},
pmid = {33521855},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genes, Plant ; Mutation/*genetics ; *Transgenes ; },
}
@article {pmid33519884,
year = {2020},
author = {Nadakuduti, SS and Enciso-Rodríguez, F},
title = {Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {637159},
pmid = {33519884},
issn = {1664-462X},
abstract = {The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with "The Nobel Prize in Chemistry, 2020." CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.},
}
@article {pmid33517231,
year = {2021},
author = {Zhang, K and Fan, Z and Yao, B and Ding, Y and Zhao, J and Xie, M and Pan, J},
title = {Exploring the trans-cleavage activity of CRISPR-Cas12a for the development of a Mxene based electrochemiluminescence biosensor for the detection of Siglec-5.},
journal = {Biosensors &amp; bioelectronics},
volume = {178},
number = {},
pages = {113019},
doi = {10.1016/j.bios.2021.113019},
pmid = {33517231},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; RNA, Guide ; Sialic Acid Binding Immunoglobulin-like Lectins ; },
abstract = {Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a's side-cutting feature to build a 2D ultra-thin Ti3C2Tx (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.},
}
@article {pmid33515912,
year = {2021},
author = {Kato, S and Fukazawa, T and Kubo, T},
title = {Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis.},
journal = {Biochemical and biophysical research communications},
volume = {543},
number = {},
pages = {50-55},
doi = {10.1016/j.bbrc.2020.11.038},
pmid = {33515912},
issn = {1090-2104},
mesh = {Animals ; *CRISPR-Cas Systems ; Cold Temperature ; Flow Cytometry/methods ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockdown Techniques/*methods ; MicroRNAs/genetics ; Microinjections/methods ; Xenopus laevis/*genetics/growth &amp; development/metabolism ; },
abstract = {The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.},
}
@article {pmid33514840,
year = {2021},
author = {Wang, Z and Maluenda, J and Giraut, L and Vieille, T and Lefevre, A and Salthouse, D and Radou, G and Moulinas, R and Astete, S and D'Avezac, P and Smith, G and André, C and Allemand, JF and Bensimon, D and Croquette, V and Ouellet, J and Hamilton, G},
title = {Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA.},
journal = {Communications biology},
volume = {4},
number = {1},
pages = {128},
pmid = {33514840},
issn = {2399-3642},
mesh = {5' Untranslated Regions ; *Base Pairing ; CRISPR-Cas Systems ; DNA/*genetics/metabolism ; DNA Methylation ; DNA, Bacterial/genetics/metabolism ; *Epigenesis, Genetic ; Escherichia coli/genetics/metabolism ; Fragile X Mental Retardation Protein/*genetics/metabolism ; Fragile X Syndrome/*genetics/metabolism ; *Genetic Variation ; Humans ; Magnets ; RNA/*genetics/metabolism ; *Single Molecule Imaging/instrumentation ; Trinucleotide Repeats ; },
abstract = {Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.},
}
@article {pmid33514753,
year = {2021},
author = {Wang, Y and Cheng, H and Liu, Y and Liu, Y and Wen, X and Zhang, K and Ni, X and Gao, N and Fan, L and Zhang, Z and Liu, J and Chen, J and Wang, L and Guo, Y and Zheng, P and Wang, M and Sun, J and Ma, Y},
title = {In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {678},
pmid = {33514753},
issn = {2041-1723},
mesh = {Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Corynebacterium glutamicum/genetics/metabolism ; DNA, Bacterial/genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Genes, Bacterial/genetics ; Glycerol/metabolism ; Industrial Microbiology/*methods ; Lycopene/metabolism ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Multigene Family/*genetics ; Transformation, Bacterial ; Xylose/metabolism ; },
abstract = {Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, we describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in-situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5' untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. We apply BETTER to simultaneously regulate expression of up to ten genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis. Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms.},
}
@article {pmid33514718,
year = {2021},
author = {Wilkinson, AC and Dever, DP and Baik, R and Camarena, J and Hsu, I and Charlesworth, CT and Morita, C and Nakauchi, H and Porteus, MH},
title = {Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {686},
pmid = {33514718},
issn = {2041-1723},
support = {K99 HL150218/HL/NHLBI NIH HHS/United States ; R01 HL147124/HL/NHLBI NIH HHS/United States ; R01 HL135607/HL/NHLBI NIH HHS/United States ; R01 DK116944/DK/NIDDK NIH HHS/United States ; R01 AI120766/AI/NIAID NIH HHS/United States ; R01 AI097320/AI/NIAID NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/blood/diagnosis/genetics/*therapy ; Animals ; CRISPR-Cas Systems/genetics ; Combined Modality Therapy/methods ; Dependovirus ; Disease Models, Animal ; Erythropoiesis/*genetics ; Female ; Gene Editing/methods ; Gene Knock-In Techniques ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Hematopoietic Stem Cell Transplantation/*methods ; Humans ; Mice ; Mice, Transgenic ; Parvovirinae/genetics ; Transplantation, Autologous/methods ; beta-Globins/*genetics ; },
abstract = {CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.},
}
@article {pmid33514392,
year = {2021},
author = {Kamali, E and Rahbarizadeh, F and Hojati, Z and Frödin, M},
title = {CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {9},
pmid = {33514392},
issn = {1472-6750},
mesh = {CD52 Antigen/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation ; Gene Editing/methods ; *Gene Knockout Techniques ; Genomics ; HEK293 Cells ; Humans ; INDEL Mutation ; Isoantigens/*genetics ; Plasmids ; T-Lymphocytes/*metabolism ; },
abstract = {BACKGROUND: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient.<br><br>RESULTS: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293&#x2009;T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis.<br><br>CONCLUSION: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.},
}
@article {pmid33513265,
year = {2021},
author = {Chen, X and Xu, Y and Tu, W and Huang, F and Zuo, Y and Zhang, HG and Jin, L and Feng, Q and Ren, T and He, J and Miao, Y and Yuan, Y and Zhao, Q and Liu, J and Zhang, R and Zhu, L and Qian, F and Zhu, C and Zheng, H and Wang, J},
title = {Ubiquitin E3 ligase MID1 inhibits the innate immune response by ubiquitinating IRF3.},
journal = {Immunology},
volume = {163},
number = {3},
pages = {278-292},
pmid = {33513265},
issn = {1365-2567},
mesh = {Animals ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Immunity, Innate ; Influenza A Virus, H1N1 Subtype/*physiology ; Influenza, Human/*immunology ; Interferon Regulatory Factor-3/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Orthomyxoviridae Infections/*immunology ; Proteolysis ; RNA, Small Interfering/genetics ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Ubiquitination ; },
abstract = {Interferon regulatory factor 3 (IRF3) is a critical transcription factor for inducing production of type I interferons (IFN-I) and regulating host antiviral response. Although IRF3 activation during viral infection has been extensively studied, the inhibitory regulation of IRF3 remains largely unexplored. Here, we revealed that Midline-1 (MID1) is a ubiquitin E3 ligase of IRF3 that plays essential roles in regulating the production of IFN-I. We found that MID1 physically interacts with IRF3 and downregulates IRF3 protein levels. Next, we demonstrated that MID1 can induce K48-linked polyubiquitination of IRF3, thus lowing the protein stability of IRF3. Our further studies identified Lys313 as a major ubiquitin acceptor lysine of IRF3 induced by MID1. Finally, MID1-mediated ubiquitination and degradation of IRF3 restrict IFN-I production and cellular antiviral response. This study uncovers a role of MID1 in regulating innate antiviral immunity and may provide a potential target for enhancing host antiviral activity.},
}
@article {pmid33513160,
year = {2021},
author = {Emmer, BT and Sherman, EJ and Lascuna, PJ and Graham, SE and Willer, CJ and Ginsburg, D},
title = {Genome-scale CRISPR screening for modifiers of cellular LDL uptake.},
journal = {PLoS genetics},
volume = {17},
number = {1},
pages = {e1009285},
pmid = {33513160},
issn = {1553-7404},
support = {K08 HL148552/HL/NHLBI NIH HHS/United States ; R35 HL135793/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Atherosclerosis/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Cholesterol/*genetics/metabolism ; Endocytosis/genetics ; Gene Expression Regulation/genetics ; Genome, Human/genetics ; Hep G2 Cells ; Hepatocytes/metabolism/pathology ; Humans ; Hypercholesterolemia/genetics/pathology ; Lipoproteins, LDL/*genetics/metabolism ; RNA, Guide/genetics ; Receptors, LDL/*genetics ; },
abstract = {Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.},
}
@article {pmid33513149,
year = {2021},
author = {Hammond, A and Karlsson, X and Morianou, I and Kyrou, K and Beaghton, A and Gribble, M and Kranjc, N and Galizi, R and Burt, A and Crisanti, A and Nolan, T},
title = {Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance.},
journal = {PLoS genetics},
volume = {17},
number = {1},
pages = {e1009321},
pmid = {33513149},
issn = {1553-7404},
support = {P30 ES019776/ES/NIEHS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Culicidae/*genetics/parasitology ; DNA End-Joining Repair/genetics ; Drosophila melanogaster/genetics ; Eggs/parasitology ; Endonucleases/*genetics ; Fertility/genetics ; Genetic Fitness/*genetics ; Germ-Line Mutation/genetics ; Heterozygote ; Humans ; Larva/genetics/parasitology ; Malaria/epidemiology/*genetics/parasitology/transmission ; },
abstract = {Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive's potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression.},
}
@article {pmid33512458,
year = {2021},
author = {Iacobucci, I and Qu, C and Varotto, E and Janke, LJ and Yang, X and Seth, A and Shelat, A and Friske, JD and Fukano, R and Yu, J and Freeman, BB and Kennedy, JA and Sperling, AS and Zheng, R and Wang, Y and Jogiraju, H and Dickerson, KM and Payne-Turner, D and Morris, SM and Hollis, ES and Ghosn, N and Haggard, GE and Lindsley, RC and Ebert, BL and Mullighan, CG},
title = {Modeling and targeting of erythroleukemia by hematopoietic genome editing.},
journal = {Blood},
volume = {137},
number = {12},
pages = {1628-1640},
pmid = {33512458},
issn = {1528-0020},
support = {R35 CA253125/CA/NCI NIH HHS/United States ; R25 CA023944/CA/NCI NIH HHS/United States ; R01 GM134382/GM/NIGMS NIH HHS/United States ; K08 CA204734/CA/NCI NIH HHS/United States ; K08 CA252174/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clonal Evolution ; Epigenesis, Genetic ; *Gene Editing ; Hematopoiesis ; Humans ; Leukemia, Erythroblastic, Acute/*genetics ; Mice ; Mutation ; Transcriptome ; },
abstract = {Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor-mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a-mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.},
}
@article {pmid33512438,
year = {2021},
author = {Mu, A and Hira, A and Niwa, A and Osawa, M and Yoshida, K and Mori, M and Okamoto, Y and Inoue, K and Kondo, K and Kanemaki, MT and Matsuda, T and Ito, E and Kojima, S and Nakahata, T and Ogawa, S and Tanaka, K and Matsuo, K and Saito, MK and Takata, M},
title = {Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency.},
journal = {Blood},
volume = {137},
number = {15},
pages = {2021-2032},
doi = {10.1182/blood.2020009111},
pmid = {33512438},
issn = {1528-0020},
mesh = {Aldehyde Dehydrogenase, Mitochondrial/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Congenital Bone Marrow Failure Syndromes/diagnosis/*genetics/pathology ; DNA Damage ; Fanconi Anemia/diagnosis/*genetics/pathology ; Gene Deletion ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Mutation ; },
abstract = {We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.},
}
@article {pmid33511840,
year = {2021},
author = {Wang, Y and Zhang, Y and Chen, J and Wang, M and Zhang, T and Luo, W and Li, Y and Wu, Y and Zeng, B and Zhang, K and Deng, R and Li, W},
title = {Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification.},
journal = {Analytical chemistry},
volume = {93},
number = {7},
pages = {3393-3402},
pmid = {33511840},
issn = {1520-6882},
mesh = {COVID-19/*virology ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; Molecular Diagnostic Techniques ; Mutation ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global health emergency, and its gene mutation and evolution further posed uncertainty of epidemic risk. Herein, we reported a light-up CRISPR-Cas13 transcription amplification method, which enables the detection of SARS-CoV-2 and its mutated variants. Sequence specificity was ensured by both the ligation process and Cas13a/crRNA recognition, allowing us to identify viral RNA mutation. Light-up RNA aptamer allows sensitive output of amplification signals via target-activated ribonuclease activity of CRISPR-Cas13a. The RNA virus assay has been designed to detect coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS), and SARS, as well as the influenza viruses such as, H1N1, H7N9, and H9N2. It was accommodated to sense as low as 82 copies of SARS-CoV-2. Particularly, it allowed us to strictly discriminate key mutation of the SARS-CoV-2 variant, D614G, which may induce higher epidemic and pathogenetic risk. The proposed RNA virus assays are promising for point-of-care monitoring of SARS-CoV-2 and its risking variants.},
}
@article {pmid33511745,
year = {2021},
author = {Huang, TK and Armstrong, B and Schindele, P and Puchta, H},
title = {Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a.},
journal = {Plant biotechnology journal},
volume = {19},
number = {7},
pages = {1314-1324},
pmid = {33511745},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene Targeting ; Temperature ; *Tobacco/genetics ; },
abstract = {Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.},
}
@article {pmid33511075,
year = {2020},
author = {Laugi, H},
title = {Discovery of Hepatitis C Virus: 2020 Nobel Prize in Medicine.},
journal = {Euroasian journal of hepato-gastroenterology},
volume = {10},
number = {2},
pages = {105-108},
pmid = {33511075},
issn = {2231-5047},
abstract = {Hepatitis C virus (HCV) accounts for hepatitis, liver cirrhosis, hepatocellular carcinoma, and liver transplantation. This virus is a single-stranded RNA virus that belongs to the Flaviviridae family. According to the WHO, about 71 million people have chronic HCV infections around the globe in 2020, and hence, it is a plague of humankind. The credit of discovery of HCV goes to Michael Houghton, Harvey Alter, and Charles Rice for which they are awarded 2020 Nobel Prize in Medicine. Their contribution has given better hope to mankind to cure HCV for the first time in the history. With the use of pegylated interferon and ribavirin jointly, higher SVR has been found comparatively, even in patients with chronic liver diseases. However, due to excessive pain tolerated by patients, interferon (IFN)-based therapy is rapidly being replaced with IFN-free DAA regimens. With the onset of resistance to DAA drugs, CRISPR-Cas system can be used to modify the viral genome to impair their ability to develop resistance. How to cite this article: Laugi H. Discovery of Hepatitis C Virus: 2020 Nobel Prize in Medicine. Euroasian J Hepato-Gastroenterol 2020;10(2): 105-108.},
}
@article {pmid33510440,
year = {2021},
author = {Arnold, C},
title = {What's new in clinical CRISPR?.},
journal = {Nature medicine},
volume = {27},
number = {2},
pages = {184-185},
doi = {10.1038/s41591-020-01222-4},
pmid = {33510440},
issn = {1546-170X},
mesh = {CRISPR-Cas Systems/*genetics ; Genetics, Medical/*trends ; Humans ; },
}
@article {pmid33510438,
year = {2021},
author = {Kim, B and Kim, J and Chun, M and Park, I and Kwak, D and Choi, M and Kim, K and Choe, HK},
title = {Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {2575},
pmid = {33510438},
issn = {2045-2322},
mesh = {Animals ; Body Temperature ; CRISPR-Cas Systems/genetics/*physiology ; Circadian Clocks/genetics/physiology ; Circadian Rhythm/genetics/physiology ; Dependovirus/genetics ; Locomotion/genetics/physiology ; Mice ; Neurosciences ; },
abstract = {The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.},
}
@article {pmid33509781,
year = {2020},
author = {Cao, JX and Wang, YL and Wang, ZX},
title = {Advances in precise regulation of CRISPR/Cas9 gene editing technology.},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {12},
pages = {1168-1177},
doi = {10.16288/j.yczz.20-069},
pmid = {33509781},
issn = {0253-9772},
mesh = {Animals ; Biotechnology ; *CRISPR-Cas Systems ; Crops, Agricultural ; *Gene Editing ; Humans ; Livestock ; RNA, Guide/*genetics ; },
abstract = {Gene editing is a genetic engineering technology that can modify, delete, or insert a small piece of DNA at a specific point in the genome of cells and organisms. Gene editing technology holds great promises in the fields of disease treatment, gene function regulation, gene detection, drug research and development, and crop breeding. However, side effects, such as off-target editing, genotoxicity and other issues, have gradually emerged in the application. In the CRISPR (clustered regularly interspaced short palindromic repeats) system, the Cas9 nuclease can specifically recognize the target DNA by the base pairing of a guide RNA (gRNA) with the target DNA. Upon target recognition, the two DNA strands are cleaved by distinct domains of the Cas9 nuclease. Since both Cas9 nuclease and gRNA possess different characteristics in their own activities, recognition sites and binding ability to specific target, it is essential to precisely regulate the activity of Cas9 nuclease and gRNA in both time and space manners, thus preventing the risk of side effects and enhancing the precise regulation of the CRISPR/Cas9 gene editing technology. In this review, we summarize the advances in the precise control of gene editing, especially CRISPR/cas9 over several dimensions using fusion Cas9 proteins regulated by light, temperature and drugs, exploiting and screening anti-CRISPRs proteins, synthesizing and identifying small molecules- inhibitors, and developing other therapeutic agents, thereby providing a reference and research ideas for human disease treatment, crop and livestock improvement and prevention of biotechnology misuse.},
}
@article {pmid33509763,
year = {2021},
author = {Zhou, L and Ye, Y and Yuan, H and Wu, C and Wu, S},
title = {[Construction of macrophage RAW 264.7 cells with gsdmd gene knockout by CRISPR/Cas9 system].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {41},
number = {1},
pages = {116-122},
pmid = {33509763},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Mice ; Plasmids ; RAW 264.7 Cells ; *RNA, Guide ; },
abstract = {OBJECTIVE: To construct a cell model of gsdmd gene knockout in macrophage RAW 264.7 cells using CRISPR/Cas9 system.<br><br>METHODS: Four specific single guide RNAs (sgRNAs) targeting gsdmd were designed to construct pGL3-sgRNA recombinant plasmids, which were identified by PCR amplification and sequencing.Cas9 and the recombinant plasmids were transfected into RAW 264.7 cells in two steps, and the cellular expression of cas9 was detected with real-time quantitative PCR (qPCR).The positive cell clones with gsdmd gene knockout were screened using puromycin and verified by sequencing and Western blotting.Annexin &#x2164;/PI staining and LDH release assay were performed in gsdmd-/-RAW 264.7 cells after being co-cultured with Salmonella Typhimurium.<br><br>RESULTS: qPCR results showed that cas9 gene was stably expressed in RAW 264.7-Cas9 cells (P< 0.01).PCR and sequencing results demonstrated successful construction of the recombinant plasmid pGL3-sgRNA. The results of PCR, sequencing and Western blotting all confirmed that gsdmd[-/-]RAW 264.7 cells were successfully constructed. Annexin &#x2164;/PI staining and LDH release assay showed that gsdmd gene knockout significantly inhibited macrophage death caused by S.Typhimurium infection (P < 0.01).<br><br>CONCLUSIONS: gsdmd[-/-]RAW 264.7 cells provide a cell model for studying the mechanisms underlying GSDMD-mediated macrophage death.},
}
@article {pmid33508235,
year = {2021},
author = {Patrizi, C and Llado, M and Benati, D and Iodice, C and Marrocco, E and Guarascio, R and Surace, EM and Cheetham, ME and Auricchio, A and Recchia, A},
title = {Allele-specific editing ameliorates dominant retinitis pigmentosa in a transgenic mouse model.},
journal = {American journal of human genetics},
volume = {108},
number = {2},
pages = {295-308},
pmid = {33508235},
issn = {1537-6605},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Line ; Dependovirus/genetics ; Disease Models, Animal ; Electroretinography ; *Gene Editing ; Genetic Therapy ; Humans ; INDEL Mutation ; Mice ; Mice, Transgenic ; Mutation, Missense ; Photoreceptor Cells, Vertebrate/metabolism ; Retina/metabolism/physiopathology ; Retinitis Pigmentosa/*genetics/*therapy ; Rhodopsin/*genetics/metabolism ; },
abstract = {Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.},
}
@article {pmid33507915,
year = {2021},
author = {Béal, J and Pantolini, L and Noël, V and Barillot, E and Calzone, L},
title = {Personalized logical models to investigate cancer response to BRAF treatments in melanomas and colorectal cancers.},
journal = {PLoS computational biology},
volume = {17},
number = {1},
pages = {e1007900},
pmid = {33507915},
issn = {1553-7358},
mesh = {Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Colorectal Neoplasms/genetics/metabolism/therapy ; Computational Biology ; Genetic Therapy ; Humans ; Machine Learning ; *Melanoma/genetics/metabolism/therapy ; *Patient-Specific Modeling ; Proto-Oncogene Proteins B-raf/*antagonists &amp; inhibitors ; Signal Transduction/drug effects ; Transcriptome/drug effects ; },
abstract = {The study of response to cancer treatments has benefited greatly from the contribution of different omics data but their interpretation is sometimes difficult. Some mathematical models based on prior biological knowledge of signaling pathways facilitate this interpretation but often require fitting of their parameters using perturbation data. We propose a more qualitative mechanistic approach, based on logical formalism and on the sole mapping and interpretation of omics data, and able to recover differences in sensitivity to gene inhibition without model training. This approach is showcased by the study of BRAF inhibition in patients with melanomas and colorectal cancers who experience significant differences in sensitivity despite similar omics profiles. We first gather information from literature and build a logical model summarizing the regulatory network of the mitogen-activated protein kinase (MAPK) pathway surrounding BRAF, with factors involved in the BRAF inhibition resistance mechanisms. The relevance of this model is verified by automatically assessing that it qualitatively reproduces response or resistance behaviors identified in the literature. Data from over 100 melanoma and colorectal cancer cell lines are then used to validate the model's ability to explain differences in sensitivity. This generic model is transformed into personalized cell line-specific logical models by integrating the omics information of the cell lines as constraints of the model. The use of mutations alone allows personalized models to correlate significantly with experimental sensitivities to BRAF inhibition, both from drug and CRISPR targeting, and even better with the joint use of mutations and RNA, supporting multi-omics mechanistic models. A comparison of these untrained models with learning approaches highlights similarities in interpretation and complementarity depending on the size of the datasets. This parsimonious pipeline, which can easily be extended to other biological questions, makes it possible to explore the mechanistic causes of the response to treatment, on an individualized basis.},
}
@article {pmid33506343,
year = {2021},
author = {Liao, X and Wu, C and Shao, Z and Zhang, S and Zou, Y and Wang, K and Ha, Y and Xing, J and Zheng, A and Shen, Z and Zheng, S and Guo, J and Jie, W},
title = {SETD4 in the Proliferation, Migration, Angiogenesis, Myogenic Differentiation and Genomic Methylation of Bone Marrow Mesenchymal Stem Cells.},
journal = {Stem cell reviews and reports},
volume = {17},
number = {4},
pages = {1374-1389},
pmid = {33506343},
issn = {2629-3277},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Proliferation/genetics ; *DNA Methylation ; Gene Knockout Techniques ; *Mesenchymal Stem Cells/cytology ; Methyltransferases/*physiology ; Mice ; Myocytes, Cardiac/cytology ; Myocytes, Smooth Muscle/cytology ; Neovascularization, Physiologic ; },
abstract = {Epigenetic modification is a crucial mechanism affecting the biological function of stem cells. SETD4 is a histone methyltransferase, and its biological role in bone marrow mesenchymal stem cells (BMSCs) is currently unknown. In this study, we employed CRISPR/Cas9 technology edited mouse model and found that SETD4 knockout significantly promoted the proliferation of BMSCs, impaired BMSCs migration and differentiation potentials of lineages of cardiacmyocyte and smooth muscle cell, and even the angiogenesis via paracrine of VEGF. Through Reduced Representation Bisulfite Sequencing (RRBS) method, we verified that the overall genomic methylation of BMSCs in the SETD4 knockout group only was decreased by 0.47 % compared with wild type. However, the changed genomic methylation covers a total of 96,331 differential methylated CpG sites and 8,692 differential methylation regions (DMRs), with part of them settled in promoter regions. Bioinformatic analysis revealed that differential CpG islands and DMRs in promoter impacted 270 GO functions and 34 KEGG signaling pathways, with some closely related to stem cell biology. Mechanismly, SETD4 knockout inhibited sets of monomethylases and dimethylases for histone lysine, along with significant changes in some factors including Nkx2.5, Gata4, Gli2, Grem2, E2f7, Map7, Nr2f2 and Shox2 that associated with stem cell biology. These results are the first to reveal that even though SETD4 changes the genome's overall methylation to a limited extent in BMSCs, it still affects the numerous cellular functions and signaling pathways, implying SETD4-altered genomic methylation serves a crucial molecular role in BMSCs' biological functions.},
}
@article {pmid33505962,
year = {2020},
author = {Nguyen, T and Urrutia-Cabrera, D and Liou, RH and Luu, CD and Guymer, R and Wong, RC},
title = {New Technologies to Study Functional Genomics of Age-Related Macular Degeneration.},
journal = {Frontiers in cell and developmental biology},
volume = {8},
number = {},
pages = {604220},
pmid = {33505962},
issn = {2296-634X},
abstract = {Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in people over 50 years old in developed countries. Currently, we still lack a comprehensive understanding of the genetic factors contributing to AMD, which is critical to identify effective therapeutic targets to improve treatment outcomes for AMD patients. Here we discuss the latest technologies that can facilitate the identification and functional study of putative genes in AMD pathology. We review improved genomic methods to identify novel AMD genes, advances in single cell transcriptomics to profile gene expression in specific retinal cell types, and summarize recent development of in vitro models for studying AMD using induced pluripotent stem cells, organoids and biomaterials, as well as new molecular technologies using CRISPR/Cas that could facilitate functional studies of AMD-associated genes.},
}
@article {pmid33504736,
year = {2021},
author = {Ijaz, F and Ikegami, K},
title = {Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.},
journal = {Cell structure and function},
volume = {46},
number = {1},
pages = {21-35},
doi = {10.1247/csf.21002},
pmid = {33504736},
issn = {1347-3700},
mesh = {*5' Untranslated Regions ; CRISPR-Cas Systems ; Cell Line ; Gene Knock-In Techniques ; Humans ; },
abstract = {Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5'UTR of the endogenous gene loci. With this 5'UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5'UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells.Key words: CRISPR/Cas9, knock-in, primary cilium, UTR, tubulin.},
}
@article {pmid33504469,
year = {2021},
author = {Lian, M and Chen, F and Huang, X and Zhao, X and Gou, S and Li, N and Jin, Q and Shi, H and Liang, Y and Xie, J and Ge, W and Zhuang, Z and Wang, J and Ye, Y and Yang, Y and Wang, K and Lai, L and Wu, H},
title = {Improving the Cpf1-mediated base editing system by combining dCas9/dead sgRNA with human APOBEC3A variants.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {1},
pages = {92-95},
doi = {10.1016/j.jgg.2020.07.010},
pmid = {33504469},
issn = {1673-8527},
mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Cytidine Deaminase/genetics ; Gene Editing ; Humans ; Proteins ; },
}
@article {pmid33503428,
year = {2021},
author = {Bao, MH and Yang, C and Tse, AP and Wei, L and Lee, D and Zhang, MS and Goh, CC and Chiu, DK and Yuen, VW and Law, CT and Chin, WC and Chui, NN and Wong, BP and Chan, CY and Ng, IO and Chung, CY and Wong, CM and Wong, CC},
title = {Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer.},
journal = {Cell reports},
volume = {34},
number = {4},
pages = {108676},
doi = {10.1016/j.celrep.2020.108676},
pmid = {33503428},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems ; Cardiolipins/*biosynthesis/genetics ; Cell Hypoxia/physiology ; HCT116 Cells ; Hep G2 Cells ; Heterografts ; Humans ; Liver Neoplasms/genetics/*metabolism/pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; PC-3 Cells ; PTEN Phosphohydrolase/genetics/*metabolism ; },
abstract = {Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1β as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.},
}
@article {pmid33503407,
year = {2021},
author = {Huang, Z and Liang, N and Goñi, S and Damdimopoulos, A and Wang, C and Ballaire, R and Jager, J and Niskanen, H and Han, H and Jakobsson, T and Bracken, AP and Aouadi, M and Venteclef, N and Kaikkonen, MU and Fan, R and Treuter, E},
title = {The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages.},
journal = {Molecular cell},
volume = {81},
number = {5},
pages = {953-968.e9},
doi = {10.1016/j.molcel.2020.12.040},
pmid = {33503407},
issn = {1097-4164},
mesh = {Adipose Tissue/immunology/pathology ; Animals ; CRISPR-Cas Systems ; Chemokine CCL2/*genetics/immunology ; Co-Repressor Proteins/*genetics/immunology ; *Enhancer Elements, Genetic ; Gene Editing ; Gene Expression Regulation/drug effects ; HEK293 Cells ; Histone Acetyltransferases/genetics/immunology ; Histones/genetics/immunology ; Humans ; Intracellular Signaling Peptides and Proteins/*genetics/immunology ; Lipopolysaccharides/pharmacology ; Macrophage Activation/drug effects ; Male ; Mediator Complex Subunit 1/genetics/immunology ; Mice ; Mice, Obese ; Nuclear Receptor Co-Repressor 2/*genetics/immunology ; Obesity/*genetics/immunology/pathology ; RAW 264.7 Cells ; RNA, Untranslated/genetics/immunology ; Signal Transduction ; *Silencer Elements, Transcriptional ; },
abstract = {While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.},
}
@article {pmid33503261,
year = {2021},
author = {Feng, H and Guo, J and Wang, T and Zhang, C and Xing, XH},
title = {Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.},
journal = {Nucleic acids research},
volume = {49},
number = {3},
pages = {1263-1277},
pmid = {33503261},
issn = {1362-4962},
mesh = {Base Pair Mismatch ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Escherichia coli/genetics ; Models, Genetic ; Protein Binding ; RNA/chemistry/*metabolism ; Thermodynamics ; },
abstract = {As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.},
}
@article {pmid33502175,
year = {2021},
author = {Rahimi, H and Salehiabar, M and Barsbay, M and Ghaffarlou, M and Kavetskyy, T and Sharafi, A and Davaran, S and Chauhan, SC and Danafar, H and Kaboli, S and Nosrati, H and Yallapu, MM and Conde, J},
title = {CRISPR Systems for COVID-19 Diagnosis.},
journal = {ACS sensors},
volume = {6},
number = {4},
pages = {1430-1445},
pmid = {33502175},
issn = {2379-3694},
mesh = {*COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; SARS-CoV-2 ; },
abstract = {The emergence of the new coronavirus 2019 (COVID-19) was first seen in December 2019, which has spread rapidly and become a global pandemic. The number of cases of COVID-19 and its associated mortality have raised serious concerns worldwide. Early diagnosis of viral infection undoubtedly allows rapid intervention, disease management, and substantial control of the rapid spread of the disease. Currently, the standard approach for COVID-19 diagnosis globally is the RT-qPCR test; however, the limited access to kits and associated reagents, the need for specialized lab equipment, and the need for highly skilled personnel has led to a detection slowdown. Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems has reshaped molecular diagnosis. The benefits of the CRISPR system such as speed, precision, specificity, strength, efficiency, and versatility have inspired researchers to develop CRISPR-based diagnostic and therapeutic methods. With the global COVID-19 outbreak, different groups have begun to design and develop diagnostic and therapeutic programs based on the efficient CRISPR system. CRISPR-based COVID-19 diagnostic systems have advantages such as a high detection speed (i.e., 30 min from raw sample to reach a result), high sensitivity and precision, portability, and no need for specialized laboratory equipment. Here, we review contemporary studies on the detection of COVID-19 based on the CRISPR system.},
}
@article {pmid33501828,
year = {2021},
author = {Spasskaya, DS and Kotlov, MI and Lekanov, DS and Tutyaeva, VV and Snezhkina, AV and Kudryavtseva, AV and Karpov, VL and Karpov, DS},
title = {CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.},
journal = {ACS synthetic biology},
volume = {10},
number = {2},
pages = {297-308},
doi = {10.1021/acssynbio.0c00426},
pmid = {33501828},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA-Binding Proteins/genetics/metabolism ; Debaryomyces/*enzymology/*genetics ; Extremophiles/enzymology/genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Genome, Fungal ; Organisms, Genetically Modified ; Osmoregulation/genetics ; Oxidative Stress/genetics ; Proteasome Endopeptidase Complex/*genetics/metabolism ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Salt Stress/genetics ; Transcription Factors/genetics/metabolism ; Transcription, Genetic ; },
abstract = {The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.},
}
@article {pmid33500573,
year = {2021},
author = {Boehm, JS and Garnett, MJ and Adams, DJ and Francies, HE and Golub, TR and Hahn, WC and Iorio, F and McFarland, JM and Parts, L and Vazquez, F},
title = {Cancer research needs a better map.},
journal = {Nature},
volume = {589},
number = {7843},
pages = {514-516},
pmid = {33500573},
issn = {1476-4687},
mesh = {Antineoplastic Agents, Immunological/pharmacology/therapeutic use ; Biomedical Research/economics/*organization &amp; administration/*trends ; CRISPR-Cas Systems/genetics ; Cell Survival/drug effects ; DNA Mutational Analysis ; Datasets as Topic ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; *Goals ; Humans ; Information Dissemination ; *International Cooperation ; Machine Learning ; Molecular Targeted Therapy/*trends ; Mutation ; Neoplasms/*drug therapy/*genetics/metabolism/pathology ; Pilot Projects ; Precision Medicine ; Reproducibility of Results ; },
}
@article {pmid33500419,
year = {2021},
author = {Shorrocks, AK and Jones, SE and Tsukada, K and Morrow, CA and Belblidia, Z and Shen, J and Vendrell, I and Fischer, R and Kessler, BM and Blackford, AN},
title = {The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {585},
pmid = {33500419},
issn = {2041-1723},
support = {C29215/A20772/CRUK_/Cancer Research UK/United Kingdom ; MC_UU_12009/MRC_/Medical Research Council/United Kingdom ; MC_UU_12010/MRC_/Medical Research Council/United Kingdom ; 107457/Z/15Z/WT_/Wellcome Trust/United Kingdom ; IS-BRC-1215-20008/DH_/Department of Health/United Kingdom ; G0902418/MRC_/Medical Research Council/United Kingdom ; MC_UU_12025/MRC_/Medical Research Council/United Kingdom ; MR/N013468/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Amino Acid Motifs/genetics ; Bloom Syndrome/*genetics ; CRISPR-Cas Systems/genetics ; DNA Damage ; *DNA Replication ; DNA Topoisomerases, Type I/metabolism ; DNA, Single-Stranded/genetics/*metabolism ; DNA-Binding Proteins/genetics/metabolism ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Mitosis/genetics ; Mutation ; Protein Binding/genetics ; Protein Domains/genetics ; RecQ Helicases/genetics/*metabolism ; Recombinant Proteins/genetics/metabolism ; Recombinational DNA Repair/genetics ; Replication Protein A/*metabolism ; },
abstract = {The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.},
}
@article {pmid33500247,
year = {2021},
author = {Barnabas, GD and Lee, JS and Shami, T and Harel, M and Beck, L and Selitrennik, M and Jerby-Arnon, L and Erez, N and Ruppin, E and Geiger, T},
title = {Serine Biosynthesis Is a Metabolic Vulnerability in IDH2-Driven Breast Cancer Progression.},
journal = {Cancer research},
volume = {81},
number = {6},
pages = {1443-1456},
pmid = {33500247},
issn = {1538-7445},
support = {ZIA BC011802/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; Breast/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Datasets as Topic ; Disease Models, Animal ; Energy Metabolism/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Isocitrate Dehydrogenase/genetics/*metabolism ; Kaplan-Meier Estimate ; Metabolomics ; Mice ; Phosphoglycerate Dehydrogenase/antagonists &amp; inhibitors/genetics/*metabolism ; Proteomics ; Serine/*biosynthesis ; Synthetic Lethal Mutations ; Transaminases/genetics/metabolism ; Triple Negative Breast Neoplasms/genetics/mortality/*pathology ; Warburg Effect, Oncologic ; },
abstract = {Cancer-specific metabolic phenotypes and their vulnerabilities represent a viable area of cancer research. In this study, we explored the association of breast cancer subtypes with different metabolic phenotypes and identified isocitrate dehydrogenase 2 (IDH2) as a key player in triple-negative breast cancer (TNBC) and HER2. Functional assays combined with mass spectrometry-based analyses revealed the oncogenic role of IDH2 in cell proliferation, anchorage-independent growth, glycolysis, mitochondrial respiration, and antioxidant defense. Genome-scale metabolic modeling identified phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) as the synthetic dosage lethal (SDL) partners of IDH2. In agreement, CRISPR-Cas9 knockout of PHGDH and PSAT1 showed the essentiality of serine biosynthesis proteins in IDH2-high cells. The clinical significance of the SDL interaction was supported by patients with IDH2-high/PHGDH-low tumors, who exhibited longer survival than patients with IDH2-high/PHGDH-high tumors. Furthermore, PHGDH inhibitors were effective in treating IDH2-high cells in vitro and in vivo. Altogether, our study creates a new link between two known cancer regulators and emphasizes PHGDH as a promising target for TNBC with IDH2 overexpression. SIGNIFICANCE: These findings highlight the metabolic dependence of IDH2 on the serine biosynthesis pathway, adding an important layer to the connection between TCA cycle and glycolysis, which can be translated into novel targeted therapies.},
}
@article {pmid33499763,
year = {2021},
author = {Danner, E and Lebedin, M and de la Rosa, K and Kühn, R},
title = {A homology independent sequence replacement strategy in human cells using a CRISPR nuclease.},
journal = {Open biology},
volume = {11},
number = {1},
pages = {200283},
pmid = {33499763},
issn = {2046-2441},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA End-Joining Repair/genetics ; DNA Polymerase beta/genetics ; Exons ; Gene Editing/*methods ; Genes, Reporter ; Genetic Loci ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Guide/metabolism ; },
abstract = {Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method 'Replace' editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16-54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.},
}
@article {pmid33498106,
year = {2021},
author = {Tian, T and Shu, B and Jiang, Y and Ye, M and Liu, L and Guo, Z and Han, Z and Wang, Z and Zhou, X},
title = {An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.},
journal = {ACS nano},
volume = {15},
number = {1},
pages = {1167-1178},
doi = {10.1021/acsnano.0c08165},
pmid = {33498106},
issn = {1936-086X},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Enterococcus faecalis ; Escherichia coli ; Humans ; Klebsiella pneumoniae ; MCF-7 Cells ; MicroRNAs/analysis ; *Microfluidics ; Pseudomonas aeruginosa ; RNA/*analysis ; RNA, Ribosomal, 16S/analysis ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics ; Staphylococcus aureus ; },
abstract = {Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.},
}
@article {pmid33497883,
year = {2021},
author = {Zhao, T and Bai, R and Wu, F and Lu, WJ and Zhang, J},
title = {Generation of a TBX5 homozygous knockout embryonic stem cell line (WAe009-A-45) by CRISPR/Cas9 genome editing.},
journal = {Stem cell research},
volume = {51},
number = {},
pages = {102156},
doi = {10.1016/j.scr.2021.102156},
pmid = {33497883},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells ; *Gene Editing ; Humans ; T-Box Domain Proteins/genetics ; *Upper Extremity Deformities, Congenital/genetics ; },
abstract = {Holt-Oram syndrome (HOS), which is caused by genetic changes in the TBX5 gene, affects the hands and heart. HOS patients have heart defects, including atrial septal defects (ASD), ventricular septal defects (VSD) and heart conduction disease. Here, we generated a homozygous TBX5 knockout human embryonic stem cell (hESC) line (TBX5-KO) using a CRISPR/Cas9 system. The TBX5-KO maintained stem cell like morphology, pluripotency markers, normal karyotype, and could differentiate into all three germ layers in vivo. This cell line can provide an in vitro platform for studying the pathogenic mechanisms and biological function of TBX5 in the heart development.},
}
@article {pmid33497879,
year = {2021},
author = {Nouri, R and Tang, Z and Dong, M and Liu, T and Kshirsagar, A and Guan, W},
title = {CRISPR-based detection of SARS-CoV-2: A review from sample to result.},
journal = {Biosensors &amp; bioelectronics},
volume = {178},
number = {},
pages = {113012},
pmid = {33497879},
issn = {1873-4235},
mesh = {Bacterial Proteins/genetics ; Biosensing Techniques/methods/trends ; COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/*methods/trends ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics ; Humans ; Molecular Diagnostic Techniques/methods/trends ; Pandemics ; Point-of-Care Testing/trends ; RNA, Viral/genetics/isolation &amp; purification ; SARS-CoV-2/genetics/isolation &amp; purification ; Workflow ; },
abstract = {The current pandemic of the 2019 novel coronavirus (COVID-19) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concern. Rapid, affordable, and accurate diagnostics of SARS-CoV-2 is essential for early treatment and control of the disease spread. In the past few years, CRISPR technology has shown great potential for highly sensitive and specific molecular diagnostics. Amid the ongoing COVID-19 pandemic, there is an increasing interest in implementing CRISPR-based diagnostic principles to develop fast and precise methods for detecting SARS-CoV-2. In this work, we reviewed and summarized these CRISPR-based diagnostic systems as well as their characteristics and challenges. We also provided future perspectives of CRISPR-based sensing towards point-of-care molecular diagnosis applications.},
}
@article {pmid33497498,
year = {2021},
author = {Galat, Y and Gu, H and Perepitchka, M and Taylor, R and Yoon, JW and Glukhova, XA and Li, XN and Beletsky, IP and Walterhouse, DO and Galat, V and Iannaccone, PM},
title = {CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.},
journal = {Stem cells (Dayton, Ohio)},
volume = {39},
number = {5},
pages = {564-580},
pmid = {33497498},
issn = {1549-4918},
support = {P01 ES010549/ES/NIEHS NIH HHS/United States ; P01ES010549/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Cell Lineage/drug effects/*genetics ; Gene Expression Regulation, Developmental/drug effects ; Human Embryonic Stem Cells/*cytology/metabolism ; Humans ; Introns/genetics ; Pyridines/pharmacology ; Pyrimidines/pharmacology ; Signal Transduction/genetics ; Zinc Finger Protein GLI1/antagonists &amp; inhibitors/*genetics ; },
abstract = {GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.},
}
@article {pmid33497205,
year = {2021},
author = {Li, H and Li, M and Yang, Y and Wang, F and Wang, F and Li, C},
title = {Aptamer-Linked CRISPR/Cas12a-Based Immunoassay.},
journal = {Analytical chemistry},
volume = {93},
number = {6},
pages = {3209-3216},
doi = {10.1021/acs.analchem.0c04687},
pmid = {33497205},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Immunoassay ; Oligonucleotides ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has shown great promising applications in the area of nucleic acid biosensing. However, because of the dearth of versatile signal transduction strategies, this system is usually compromised to low versatility, moderate sensitivity, and complex operation for non-nucleic acid targets, limiting its clinical transition. Herein, we describe a direct method to establish the correlation between non-nucleic acid analytes and the CRISPR/Cas12a system using a series of rationally designed, aptamer-flanked activator DNA strands, which enable ultrasensitive detection of biomarkers from different species, greatly broadening the possibility of the CRISPR/Cas system in bioanalysis. Meanwhile, the signal output is highly optional and the sensing principle is akin to the traditional enzyme-linked immunosorbent assay (ELISA), so it can be directly imposed on the currently available ELISA platform, further facilitating its application in medical diagnostics.},
}
@article {pmid33496064,
year = {2021},
author = {Manjunath, M and Choudhary, B and Raghavan, SC},
title = {SCR7, a potent cancer therapeutic agent and a biochemical inhibitor of nonhomologous DNA end-joining.},
journal = {Cancer reports (Hoboken, N.J.)},
volume = {4},
number = {3},
pages = {e1341},
pmid = {33496064},
issn = {2573-8348},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*drug effects ; DNA Ligase ATP/antagonists &amp; inhibitors/metabolism ; Disease Models, Animal ; Gene Editing/methods ; Humans ; Mice ; Neoplasms/*drug therapy/genetics ; Pyrimidines/*pharmacology/therapeutic use ; Schiff Bases/*pharmacology/therapeutic use ; },
abstract = {BACKGROUND: DNA double-strand breaks (DSBs) are harmful to the cell as it could lead to genomic instability and cell death when left unrepaired. Homologous recombination and nonhomologous end-joining (NHEJ) are two major DSB repair pathways, responsible for ensuring genome integrity in mammals. There have been multiple efforts using small molecule inhibitors to target these DNA repair pathways in cancers. SCR7 is a very well-studied anticancer molecule that blocks NHEJ by targeting one of the critical enzymes, Ligase IV.<br><br>RECENT FINDINGS: In this review, we have highlighted the anticancer effects of SCR7 as a single agent and in combination with other chemotherapeutic agents and radiation. SCR7 blocked NHEJ effectively both in vitro and ex vivo. SCR7 has been used for biochemical studies like chromosomal territory resetting and in understanding the role of repair proteins in cell cycle phases. Various forms of SCR7 and its derivatives are discussed. SCR7 is also used as a potent biochemical inhibitor of NHEJ, which has found its application in improving genome editing using a CRISPR-Cas system.<br><br>CONCLUSION: SCR7 is a potent NHEJ inhibitor with unique properties and wide applications as an anticancer agent. Most importantly, SCR7 has become a handy aid for improving genome editing across different model systems.},
}
@article {pmid33496003,
year = {2021},
author = {Goshayeshi, L and Yousefi Taemeh, S and Dehdilani, N and Nasiri, M and Ghahramani Seno, MM and Dehghani, H},
title = {CRISPR/dCas9-mediated transposition with specificity and efficiency of site-directed genomic insertions.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {2},
pages = {e21359},
doi = {10.1096/fj.202001830RR},
pmid = {33496003},
issn = {1530-6860},
mesh = {CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Gene Knock-In Techniques/*methods ; Genes, Reporter ; HEK293 Cells ; Humans ; Mutagenesis, Insertional ; RNA, Guide/metabolism ; Transposases/genetics/metabolism ; },
abstract = {The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboring-specific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them.},
}
@article {pmid33495647,
year = {2021},
author = {Li, Z and Zhang, H and Xiao, R and Han, R and Chang, L},
title = {Cryo-EM structure of the RNA-guided ribonuclease Cas12g.},
journal = {Nature chemical biology},
volume = {17},
number = {4},
pages = {387-393},
pmid = {33495647},
issn = {1552-4469},
support = {R01 GM138675/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics/*ultrastructure ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy/methods ; RNA, Bacterial/chemistry ; RNA, Guide/genetics/*ultrastructure ; Ribonucleases/genetics/metabolism/ultrastructure ; },
abstract = {Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.},
}
@article {pmid33495338,
year = {2021},
author = {Wijshake, T and Zou, Z and Chen, B and Zhong, L and Xiao, G and Xie, Y and Doench, JG and Bennett, L and Levine, B},
title = {Tumor-suppressor function of Beclin 1 in breast cancer cells requires E-cadherin.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {5},
pages = {},
pmid = {33495338},
issn = {1091-6490},
support = {U19 AI109725/AI/NIAID NIH HHS/United States ; U19 AI142784/AI/NIAID NIH HHS/United States ; },
mesh = {Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Autophagy-Related Proteins/metabolism ; Beclin-1/*metabolism ; Breast Neoplasms/*metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cadherins/*metabolism ; Cell Membrane/metabolism ; Cell Proliferation/genetics ; Female ; *Genes, Tumor Suppressor ; Genome, Human ; Humans ; Interferons/metabolism ; MCF-7 Cells ; Mice, Inbred NOD ; Mice, SCID ; Protein Transport ; Signal Transduction ; Tumor Suppressor Proteins/metabolism ; Xenograft Model Antitumor Assays ; alpha Catenin/metabolism ; },
abstract = {Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting CDH1 and CTNNA1, tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of CDH1 or CTNNA1 reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of CDH1 or CTNNA1 inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of BECN1 and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, UVRAG, but not PI3KC3-C1-specific ATG14 or other autophagy genes ATG13, ATG5, or ATG7, resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin-mediated tumor suppression in breast cancer cells.},
}
@article {pmid33493900,
year = {2021},
author = {Wu, H and Chen, Y and Shi, Y and Wang, L and Zhang, M and Wu, J and Chen, H},
title = {Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR/Cas12a.},
journal = {Biosensors &amp; bioelectronics},
volume = {178},
number = {},
pages = {113001},
doi = {10.1016/j.bios.2021.113001},
pmid = {33493900},
issn = {1873-4235},
mesh = {Biosensing Techniques/instrumentation/methods ; COVID-19/*diagnosis/*virology ; COVID-19 Nucleic Acid Testing/instrumentation/*methods ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics/isolation &amp; purification ; Food Microbiology ; Humans ; Magnetics ; Molecular Diagnostic Techniques/instrumentation/*methods ; Nucleic Acid Amplification Techniques/instrumentation/*methods ; Polypropylenes ; RNA, Viral/genetics/isolation &amp; purification ; SARS-CoV-2/*genetics/*isolation &amp; purification ; Salmonella typhimurium/genetics/isolation &amp; purification ; Self-Testing ; },
abstract = {Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.},
}
@article {pmid33493854,
year = {2021},
author = {Bruch, R and Johnston, M and Kling, A and Mattmüller, T and Baaske, J and Partel, S and Madlener, S and Weber, W and Urban, GA and Dincer, C},
title = {CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {177},
number = {},
pages = {112887},
doi = {10.1016/j.bios.2020.112887},
pmid = {33493854},
issn = {1873-4235},
support = {I 3194/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {*Biosensing Techniques ; Child ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *MicroRNAs/genetics ; Microfluidics ; },
abstract = {Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient's sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X ('X' highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-min-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17-92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.},
}
@article {pmid33493343,
year = {2021},
author = {Butbul Aviel, Y and Ofir, A and Ben-Izhak, O and Vlodavsky, E and Karbian, N and Brik, R and Mevorach, D and Magen, D},
title = {A novel loss-of-function mutation in LACC1 underlies hereditary juvenile arthritis with extended intra-familial phenotypic heterogeneity.},
journal = {Rheumatology (Oxford, England)},
volume = {60},
number = {10},
pages = {4888-4898},
doi = {10.1093/rheumatology/keab017},
pmid = {33493343},
issn = {1462-0332},
mesh = {Adolescent ; Adult ; Arthritis, Juvenile/*genetics/pathology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytokines/blood ; Female ; Flow Cytometry ; Gene Editing ; Humans ; Immunoblotting ; Intracellular Signaling Peptides and Proteins/*genetics ; Loss of Function Mutation/*genetics ; Male ; Pedigree ; Whole Exome Sequencing ; Young Adult ; },
abstract = {OBJECTIVE: To investigate phenotypic and molecular characteristics of a consanguineous family with autosomal-recessive, polyarticular, juvenile isiopathic arthriris (JIA) with extra-articular manifestations, including renal amyloidosis and Crohn's disease, associated with a novel homozygous truncating variant in LACC1.<br><br>METHODS: Whole exome sequencing (WES) or targeted Sanger verification were performed in 15 participants. LACC1 expression and cytokine array were analysed in patient-derived and CRISPR/Cas9-generated LACC1-knockout macrophages (M&#x3d5;).<br><br>RESULTS: A homozygous truncating variant (p.Glu348Ter) in LACC1 was identified in three affected and one asymptomatic family member, and predicted harmful by causing premature stop of the LACC1 protein sequences, and by absence from ethnically-matched controls and public variation databases. Expression studies in patient-derived macrophages (M&#x3d5;) showed no endogenous p.Glu348Ter-LACC1 RNA transcription or protein expression, compatible with nonsense-mediated mRNA decay. WES analysis in the asymptomatic homozygous subject for p. Glu348Ter-LACC1 detected an exclusive heterozygous variant (p.Arg928Gln) in complement component C5. Further complement activity analysis suggested a protective role for the p.Arg928Gln-C5 variant as a phenotypic modifier of LACC1-associated disease. Finally, cytokine profile analysis indicated increased levels of pro-inflammatory cytokines in LACC1-disrupted as compared with wild-type M&#x3d5;.<br><br>CONCLUSIONS: Our findings reinforce the role of LACC1 disruption in autosomal-recessive JIA, extend the clinical spectrum and intra-familial heterogeneity of the disease-associated phenotype, indicate a modulatory effect of complement factor C5 on phenotypic severity, and suggest an inhibitory role for wild-type LACC1 on pro-inflammatory pathways.},
}
@article {pmid33493182,
year = {2021},
author = {Papa, G and Mallery, DL and Albecka, A and Welch, LG and Cattin-Ortolá, J and Luptak, J and Paul, D and McMahon, HT and Goodfellow, IG and Carter, A and Munro, S and James, LC},
title = {Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.},
journal = {PLoS pathogens},
volume = {17},
number = {1},
pages = {e1009246},
pmid = {33493182},
issn = {1553-7374},
support = {MC_U105178783/MRC_/Medical Research Council/United Kingdom ; MC_U105181010/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; COVID-19 ; CRISPR-Cas Systems ; *Cell Fusion ; Chlorocebus aethiops ; Furin/*genetics ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Protein Structure, Tertiary ; SARS-CoV-2 ; Serine Endopeptidases ; Spike Glycoprotein, Coronavirus/*chemistry ; Vero Cells ; *Virus Internalization ; },
abstract = {Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.},
}
@article {pmid33493091,
year = {2021},
author = {Tan, Q and Link, PA and Meridew, JA and Pham, TX and Caporarello, N and Ligresti, G and Tschumperlin, DJ},
title = {Spontaneous Lung Fibrosis Resolution Reveals Novel Antifibrotic Regulators.},
journal = {American journal of respiratory cell and molecular biology},
volume = {64},
number = {4},
pages = {453-464},
pmid = {33493091},
issn = {1535-4989},
support = {R01 HL142596/HL/NHLBI NIH HHS/United States ; R01 HL153026/HL/NHLBI NIH HHS/United States ; R01 HL092961/HL/NHLBI NIH HHS/United States ; R01 HL133320/HL/NHLBI NIH HHS/United States ; T32 HL007035/HL/NHLBI NIH HHS/United States ; T32 HL105355/HL/NHLBI NIH HHS/United States ; },
mesh = {Aldehyde Dehydrogenase, Mitochondrial/genetics/metabolism ; Animals ; Bleomycin ; CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Fibroblasts/*metabolism/pathology ; Gene Editing ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Idiopathic Pulmonary Fibrosis/chemically induced/genetics/*metabolism/pathology ; Lung/*metabolism/pathology ; Mice, Transgenic ; RNA-Seq ; Receptors, Glucocorticoid/genetics/metabolism ; Remission, Spontaneous ; Signal Transduction ; Time Factors ; Transcriptome ; },
abstract = {Fibroblast activation is transient in successful wound repair but persistent in fibrotic pathologies. Understanding fibroblast deactivation during successful wound healing may provide new approaches to therapeutically reverse fibroblast activation. To characterize the gene programs that accompany fibroblast activation and reversal during lung fibrosis resolution, we used RNA sequencing analysis of flow sorted Col1α1-GFP-positive and CD45-, CD31-, and CD326-negative cells isolated from the lungs of young mice exposed to bleomycin. We compared fibroblasts isolated from control mice with those isolated at Days 14 and 30 after bleomycin exposure, representing the peak of extracellular matrix deposition and an early stage of fibrosis resolution, respectively. Bleomycin exposure dramatically altered fibroblast gene programs at Day 14. Principal component and differential gene expression analyses demonstrated the predominant reversal of these trends at Day 30. Upstream regulator and pathway analyses of reversing "resolution" genes identified novel candidate antifibrotic genes and pathways. Two genes from these analyses that were decreased in expression at Day 14 and reversed at Day 30, Aldh2 and Nr3c1, were selected for further analysis. Enhancement of endogenous expression of either gene by CRISPR activation in cultured human idiopathic pulmonary fibrosis fibroblasts was sufficient to reduce profibrotic gene expression, fibronectin deposition, and collagen gel compaction, consistent with roles for these genes in fibroblast deactivation. This combination of RNA sequencing analysis of freshly sorted fibroblasts and hypothesis testing in cultured idiopathic pulmonary fibrosis fibroblasts offers a path toward identification of novel regulators of lung fibroblast deactivation, with potential relevance to understanding fibrosis resolution and its failure in human disease.},
}
@article {pmid33491919,
year = {2021},
author = {Huang, MY and Cravener, MC and Mitchell, AP},
title = {Targeted Genetic Changes in Candida albicans Using Transient CRISPR-Cas9 Expression.},
journal = {Current protocols},
volume = {1},
number = {1},
pages = {e19},
pmid = {33491919},
issn = {2691-1299},
support = {R01 AI146103/AI/NIAID NIH HHS/United States ; /NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Candida albicans/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Homozygote ; RNA, Guide/genetics ; },
abstract = {Candida albicans is an opportunistic fungal pathogen responsible for significant disease and mortality. Absent complete mating and other convenient methods, dissection of its virulence factors relies on robust tools to delete, complement, and otherwise modify genes of interest in this diploid organism. Here we describe the design principles and use of CRISPR associated nuclease 9 (Cas9) and single-guide RNAs transiently expressed from PCR cassettes to modify genes of interest, generating homozygous mutants in a single transformation step. © 2021 Wiley Periodicals LLC. Basic Protocol 1: PCR amplification of CRISPR components Basic Protocol 2: Transformation of Candida albicans Basic Protocol 3: Selecting and genotyping transformants Alternate Protocol 1: Deletion with recyclable markers by CRISPR induced marker excision (CRIME) Alternate Protocol 2: Knock-in and combining multiple cassettes with overlapping homology.},
}
@article {pmid33490975,
year = {2021},
author = {Zhou, P and Wan, YK and Chan, BKC and Choi, GCG and Wong, ASL},
title = {Extensible combinatorial CRISPR screening in mammalian cells.},
journal = {STAR protocols},
volume = {2},
number = {1},
pages = {100255},
pmid = {33490975},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Genetic Testing ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {The CRISPR-Cas system coupled with Combinatorial Genetics En Masse (CombiGEM) enables systematic analysis of high-order genetic perturbations that are important for understanding biological processes and discovering therapeutic target combinations. Here, we present detailed steps and technical considerations for building multiplexed guide RNA libraries and carrying out a combinatorial CRISPR screen in mammalian cells. We also present an analytical pipeline, CombiPIPE, for mapping two- and three-way genetic interactions. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2020).},
}
@article {pmid33488814,
year = {2020},
author = {Padayachee, J and Singh, M},
title = {Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials.},
journal = {Nanobiomedicine},
volume = {7},
number = {},
pages = {1849543520983196},
pmid = {33488814},
issn = {1849-5435},
abstract = {Globally, approximately 1 in 4 cancers in women are diagnosed as breast cancer (BC). Despite significant advances in the diagnosis and therapy BCs, many patients develop metastases or relapses. Hence, novel therapeutic strategies are required, that can selectively and efficiently kill malignant cells. Direct targeting of the genetic and epigenetic aberrations that occur in BC development is a promising strategy to overcome the limitations of current therapies, which target the tumour phenotype. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, composed of only an easily modifiable single guide RNA (sgRNA) sequence bound to a Cas9 nuclease, has revolutionised genome editing due to its simplicity and efficiency compared to earlier systems. CRISPR/Cas9 and its associated catalytically inactivated dCas9 variants facilitate the knockout of overexpressed genes, correction of mutations in inactivated genes, and reprogramming of the epigenetic landscape to impair BC growth. To achieve efficient genome editing in vivo, a vector is required to deliver the components to target cells. Gold nanomaterials, including gold nanoparticles and nanoclusters, display many advantageous characteristics that have facilitated their widespread use in theranostics, as delivery vehicles, and imaging and photothermal agents. This review highlights the therapeutic applications of CRISPR/Cas9 in treating BCs, and briefly describes gold nanomaterials and their potential in CRISPR/Cas9 delivery.},
}
@article {pmid33488667,
year = {2020},
author = {Shin, U and Brondani, V},
title = {Analysis of Wild Type LbCpf1 Protein, and PAM Recognition Variants, in a Cellular Context.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {571591},
pmid = {33488667},
issn = {1664-8021},
abstract = {Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition selectivity with the NGS analysis. The heterologous marker system has revealed that LbCpf1 is a more specific nuclease than AsCpf1 in a cellular context. We controlled the in vitro activity of the Cpf1 nuclease complexes expressed in mammalian cells and demonstrated that they are responsible of the DNA cleavage at the target site. In addition, we generated and tested LbCpf1 variants with several combinations of mutations at the PAM-recognition positions G532, K538 and Y542. Finally, we showed that the results of the in vitro DNA cleavage assay with the wild type and mutants LbCpf1 corroborate with the selection of 6TG resistant cells associated to the genomic disruption of hprt gene.},
}
@article {pmid33488615,
year = {2020},
author = {Kressler, C and Gasparoni, G and Nordström, K and Hamo, D and Salhab, A and Dimitropoulos, C and Tierling, S and Reinke, P and Volk, HD and Walter, J and Hamann, A and Polansky, JK},
title = {Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Treg Phenotype.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {609891},
pmid = {33488615},
issn = {1664-3224},
mesh = {Animals ; CD4-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems/immunology ; Cell Proliferation/physiology ; Cells, Cultured ; DNA Methylation/*immunology ; Forkhead Transcription Factors/*immunology ; Gene Editing/methods ; Gene Expression Regulation/immunology ; Humans ; Jurkat Cells ; Mice ; Mice, Inbred C57BL ; Phenotype ; T-Lymphocytes, Regulatory/*immunology ; Th1 Cells ; },
abstract = {CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.},
}
@article {pmid33488567,
year = {2020},
author = {Zhao, Y and Wang, F and Wang, C and Zhang, X and Jiang, C and Ding, F and Shen, L and Zhang, Q},
title = {Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {625862},
pmid = {33488567},
issn = {1664-302X},
abstract = {Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.},
}
@article {pmid33487569,
year = {2021},
author = {Dan, J and Memczak, S and Izpisua Belmonte, JC},
title = {Expanding the Toolbox and Targets for Gene Editing.},
journal = {Trends in molecular medicine},
volume = {27},
number = {3},
pages = {203-206},
doi = {10.1016/j.molmed.2020.12.005},
pmid = {33487569},
issn = {1471-499X},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods/trends ; Genetic Therapy/methods/trends ; Genome ; Genome, Mitochondrial ; Humans ; Translational Research, Biomedical ; },
abstract = {Genome editing holds great promise for treating a range of human genetic diseases. While emerging clustered regularly interspaced short-palindromic repeats (CRISPR) technologies allow editing of the nuclear genome, it is still not possible to precisely manipulate mitochondrial DNA (mtDNA). Here, we summarize past developments and recent advances in nuclear and mitochondrial genome editing.},
}
@article {pmid33486346,
year = {2021},
author = {Vitale, MR and Zöller, JEM and Jansch, C and Janz, A and Edenhofer, F and Klopocki, E and van den Hove, D and Vanmierlo, T and Rivero, O and Nadif Kasri, N and Ziegler, GC and Lesch, KP},
title = {Generation of induced pluripotent stem cell (iPSC) lines carrying a heterozygous (UKWMPi002-A-1) and null mutant knockout (UKWMPi002-A-2) of Cadherin 13 associated with neurodevelopmental disorders using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {51},
number = {},
pages = {102169},
doi = {10.1016/j.scr.2021.102169},
pmid = {33486346},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems ; Cadherins ; Female ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells ; Middle Aged ; *Neurodevelopmental Disorders/genetics ; },
abstract = {Fibroblasts isolated from a skin biopsy of a healthy 46-year-old female were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate isogenic cell lines with a gene dose-dependent deficiency of CDH13, a risk gene associated with neurodevelopmental and psychiatric disorders. Thereby, a heterozygous CDH13 knockout (CDH13[+/-]) and a CDH13 null mutant (CDH13[-/-]) iPSC line was obtained. All three lines showed expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal female karyotype.},
}
@article {pmid33484494,
year = {2021},
author = {Qiu, M and Li, Y and Ye, W and Zheng, X and Wang, Y},
title = {A CRISPR/Cas9-mediated in situ complementation method for Phytophthora sojae mutants.},
journal = {Molecular plant pathology},
volume = {22},
number = {3},
pages = {373-381},
pmid = {33484494},
issn = {1364-3703},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Mutation ; Phytophthora/*genetics ; Plant Diseases/*parasitology ; Soybeans/*parasitology ; },
abstract = {Phytophthora sojae is an important model species for oomycete functional genomics research. Recently, a CRISPR/Cas9-mediated genome-editing technology has been successfully established in P. sojae, which has been rapidly and widely applied in oomycete research. However, there is an emerging consensus in the biological community that a complete functional gene research system is needed such as developed in the investigations in functional complementation carried out in this study. We report the development of an in situ complementation method for accurate restoration of the mutated gene. We targeted a regulatory B-subunit of protein phosphatase 2A (PsPP2Ab1) to verify this knockout and subsequent complementation system. We found that the deletion of PsPP2Ab1 in P. sojae leads to severe defects in vegetative hyphal growth, soybean infection, and loss of the ability to produce sporangia. Subsequently, the reintroduction of PsPP2Ab1 into the knockout mutant remedied all of the deficiencies. This study demonstrates the successful implementation of an in situ complementation system by CRISPR/Cas9, which will greatly accelerate functional genomics research of oomycetes in the post-genomic era.},
}
@article {pmid33484441,
year = {2021},
author = {Lopes, AMM and Martins, M and Goldbeck, R},
title = {Heterologous Expression of Lignocellulose-Modifying Enzymes in Microorganisms: Current Status.},
journal = {Molecular biotechnology},
volume = {63},
number = {3},
pages = {184-199},
pmid = {33484441},
issn = {1559-0305},
mesh = {Bacteria/genetics/*growth &amp; development ; Biological Products/metabolism ; Esterases/genetics/metabolism ; Fungi/genetics/*growth &amp; development ; Glycoside Hydrolases/genetics/metabolism ; Lignin/*metabolism ; Protein Engineering/*methods ; Synthetic Biology ; },
abstract = {Heterologous expression of the carbohydrate-active enzymes in microorganisms is a promising approach to produce bio-based compounds, such as fuels, nutraceuticals and other value-added products from sustainable lignocellulosic sources. Several microorganisms, including Saccharomyces cerevisiae, Escherichia coli, and the filamentous fungi Aspergillus nidulans, have unique characteristics desirable for a biorefinery production approach like well-known genetic tools, thermotolerance, high fermentative capacity and product tolerance, and high amount of recombinant enzyme secretion. These microbial factories are already stablished in the heterologous production of the carbohydrate-active enzymes to produce, among others, ethanol, xylooligosaccharides and the valuable coniferol. A complete biocatalyst able to heterologous express the CAZymes of glycoside hydrolases, carbohydrate esterases and auxiliary activities families could release these compounds faster, with higher yield and specificity. Recent advances in the synthetic biology tools could expand the number and diversity of enzymes integrated in these microorganisms, and also modify those already integrated. This review outlines the heterologous expression of carbohydrate-active enzymes in microorganisms, as well as recent updates in synthetic biology.},
}
@article {pmid33484202,
year = {2021},
author = {Uranga, M and Aragonés, V and Selma, S and Vázquez-Vilar, M and Orzáez, D and Daròs, JA},
title = {Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector.},
journal = {The Plant journal : for cell and molecular biology},
volume = {106},
number = {2},
pages = {555-565},
pmid = {33484202},
issn = {1365-313X},
mesh = {Agrobacterium tumefaciens/genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genes, Plant/genetics ; Genetic Vectors/*genetics ; Plants/genetics ; Potexvirus/*genetics ; RNA, Guide/*genetics ; Tobacco ; },
abstract = {Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.},
}
@article {pmid33483498,
year = {2021},
author = {Collias, D and Beisel, CL},
title = {CRISPR technologies and the search for the PAM-free nuclease.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {555},
pmid = {33483498},
issn = {2041-1723},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {Binding Sites/genetics ; CRISPR-Associated Protein 9/classification/genetics/*metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Models, Genetic ; *Nucleotide Motifs ; Phylogeny ; Protein Engineering/methods ; RNA, Guide/genetics/*metabolism ; },
abstract = {The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.},
}
@article {pmid33483422,
year = {2021},
author = {Condon, KJ and Orozco, JM and Adelmann, CH and Spinelli, JB and van der Helm, PW and Roberts, JM and Kunchok, T and Sabatini, DM},
title = {Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {4},
pages = {},
pmid = {33483422},
issn = {1091-6490},
support = {T32 GM007287/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F31 CA228241/CA/NCI NIH HHS/United States ; K00 CA234839/CA/NCI NIH HHS/United States ; F31 CA232355/CA/NCI NIH HHS/United States ; R01 CA129105/CA/NCI NIH HHS/United States ; R01 AI047389/AI/NIAID NIH HHS/United States ; R01 CA103866/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; F30 CA210373/CA/NCI NIH HHS/United States ; },
mesh = {3-Hydroxyacyl CoA Dehydrogenases/genetics/metabolism ; Activating Transcription Factor 4/*genetics/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Amino Acids/deficiency/pharmacology ; Antimycin A/analogs &amp; derivatives/pharmacology ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Culture Media/chemistry/pharmacology ; Gene Editing/*methods ; Gene Expression Regulation ; Genome, Human ; Glucose/deficiency/pharmacology ; HEK293 Cells ; Humans ; Insulin Receptor Substrate Proteins/genetics/metabolism ; Mechanistic Target of Rapamycin Complex 1/*genetics/metabolism ; Mitochondria/drug effects/*genetics/metabolism/pathology ; Neoplasm Proteins/genetics/metabolism ; Oligomycins/pharmacology ; Protein Serine-Threonine Kinases/*genetics/metabolism ; RNA, Guide/genetics/metabolism ; Serine-Arginine Splicing Factors/genetics/metabolism ; Signal Transduction ; eIF-2 Kinase/genetics/metabolism ; },
abstract = {In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.},
}
@article {pmid33482978,
year = {2021},
author = {Ping, Y and Church, GM},
title = {Delivery of biomacromolecules for therapeutic genome editing.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {1-2},
doi = {10.1016/j.addr.2020.12.013},
pmid = {33482978},
issn = {1872-8294},
mesh = {CRISPR-Cas Systems ; Drug Delivery Systems/*methods ; Gene Editing/*methods ; Genetic Vectors ; Humans ; Macromolecular Substances/*administration &amp; dosage ; },
}
@article {pmid33482972,
year = {2020},
author = {Boland, J and Nedelcu, E},
title = {CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology.},
journal = {The Permanente journal},
volume = {24},
number = {},
pages = {1-3},
pmid = {33482972},
issn = {1552-5775},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {Precise genomic editing has given rise to treatments in previously untreatable genetic diseases and has led to revolutions in treatment for cancer. In the past decade, the discovery and development of clustered regularly interspaced short palindromic repeats (CRISPR) technologies has led to advances across medicine and biotechnology. Specifically, the CRISPR/Cas9 system has improved translational discovery and therapeutics for oncology across tumor types. In this review, we briefly summarize the history and development of CRISPR, explain CRISPR-Cas systems and CRISPR gene editing tools, highlight the development and application of CRISPR technologies for translational and therapeutic purposes in different oncologic tumors, and review novel treatment paradigms using CRISPR in immuno-oncology, including checkpoint inhibitors and chimeric antigen receptor T cell therapy.},
}
@article {pmid33482281,
year = {2021},
author = {Wang, S and Xu, C and Shi, J and Wang, H and Wu, S and Bao, W},
title = {Regulatory effect and mechanism of APN gene on porcine epidemic diarrhea virus resistance.},
journal = {Gene},
volume = {775},
number = {},
pages = {145448},
doi = {10.1016/j.gene.2021.145448},
pmid = {33482281},
issn = {1879-0038},
mesh = {Animals ; CD13 Antigens/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line ; Cell Proliferation ; DNA Methylation ; *Disease Resistance ; Gene Knockout Techniques ; *Polymorphism, Single Nucleotide ; Porcine epidemic diarrhea virus/genetics/*pathogenicity ; Promoter Regions, Genetic ; RNA, Viral/genetics ; Swine ; *Up-Regulation ; },
abstract = {PURPOSE: The expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulating APN expression level in response to PEDV infection.<br><br>METHODS: Quantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate a APN-knockout IPEC-J2 cell line, exploring the effects of APN on cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in the APN promoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay.<br><br>RESULTS: APN expression was significantly upregulated within 4-24&#xa0;h post-infection. The cytoactivity of the APN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed in APN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24&#xa0;h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in the APN promoter region. However, there was no evident correlation between the methylation status of APN promoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity of APN.<br><br>CONCLUSIONS: These results suggested that APN knockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in the APN promoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulating APN and may contribute to the application of APN gene in resistance breeding of swine epidemic diarrhea.},
}
@article {pmid33482199,
year = {2021},
author = {Papke, CM and Smolen, KA and Swingle, MR and Cressey, L and Heng, RA and Toporsian, M and Deng, L and Hagen, J and Shen, Y and Chung, WK and Kettenbach, AN and Honkanen, RE},
title = {A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100313},
pmid = {33482199},
issn = {1083-351X},
support = {R35 GM119455/GM/NIGMS NIH HHS/United States ; },
mesh = {Autistic Disorder/genetics/pathology ; CRISPR-Cas Systems/genetics ; Genetic Diseases, Inborn/*genetics/pathology ; *Genetic Predisposition to Disease ; Humans ; Intellectual Disability/genetics/pathology ; Megalencephaly/genetics/pathology ; Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; Protein Phosphatase 2/*genetics ; *Proteomics ; Proto-Oncogene Proteins c-akt/genetics ; TOR Serine-Threonine Kinases/*genetics ; },
abstract = {Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS[3]), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.},
}
@article {pmid33482173,
year = {2021},
author = {Pieplow, A and Dastaw, M and Sakuma, T and Sakamoto, N and Yamamoto, T and Yajima, M and Oulhen, N and Wessel, GM},
title = {CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin.},
journal = {Developmental biology},
volume = {472},
number = {},
pages = {85-97},
pmid = {33482173},
issn = {1095-564X},
support = {R01 GM126043/GM/NIGMS NIH HHS/United States ; P20 GM119943/GM/NIGMS NIH HHS/United States ; R01 GM125071/GM/NIGMS NIH HHS/United States ; R35 GM140897/GM/NIGMS NIH HHS/United States ; R01 GM132222/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; Embryonic Development/genetics ; Gene Editing/*methods ; Gene Expression ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; *Genetic Loci ; Germ Cells/metabolism ; Homeodomain Proteins/genetics/metabolism ; Nodal Protein/genetics/metabolism ; Promoter Regions, Genetic/*genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics ; RNA-Binding Proteins/genetics/metabolism ; Strongylocentrotus purpuratus/*embryology/*genetics ; Transcription, Genetic/genetics ; },
abstract = {We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-β signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.},
}
@article {pmid33481791,
year = {2021},
author = {Azlan, A and Obeidat, SM and Theva Das, K and Yunus, MA and Azzam, G},
title = {Genome-wide identification of Aedes albopictus long noncoding RNAs and their association with dengue and Zika virus infection.},
journal = {PLoS neglected tropical diseases},
volume = {15},
number = {1},
pages = {e0008351},
pmid = {33481791},
issn = {1935-2735},
mesh = {Aedes/*genetics/metabolism/*virology ; Animals ; CRISPR-Cas Systems ; Cell Line ; Dengue/virology ; Dengue Virus/genetics/*physiology ; Gene Expression Regulation ; Genome ; Host Microbial Interactions/genetics/physiology ; Mosquito Vectors/genetics/virology ; RNA, Long Noncoding/*genetics/*metabolism ; Transcriptome ; Zika Virus/genetics/*physiology ; Zika Virus Infection/virology ; },
abstract = {The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). Long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae. albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae. albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutations in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus.},
}
@article {pmid33481304,
year = {2021},
author = {Gu, H and Zhou, Y and Yang, J and Li, J and Peng, Y and Zhang, X and Miao, Y and Jiang, W and Bu, G and Hou, L and Li, T and Zhang, L and Xia, X and Ma, Z and Xiong, Y and Zuo, B},
title = {Targeted overexpression of PPARγ in skeletal muscle by random insertion and CRISPR/Cas9 transgenic pig cloning enhances oxidative fiber formation and intramuscular fat deposition.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {2},
pages = {e21308},
doi = {10.1096/fj.202001812RR},
pmid = {33481304},
issn = {1530-6860},
mesh = {Adipocytes/metabolism ; Adipogenesis/genetics/physiology ; Animals ; Blotting, Southern ; Blotting, Western ; CCAAT-Enhancer-Binding Protein-alpha ; CRISPR-Cas Systems/genetics/physiology ; Cell Differentiation/genetics/physiology ; Cells, Cultured ; DNA Copy Number Variations/genetics ; DNA, Mitochondrial/*metabolism ; Fatty Acid-Binding Proteins/genetics/metabolism ; Lipid Metabolism/genetics/physiology ; Muscle, Skeletal/*metabolism ; Oxidation-Reduction ; Oxidative Stress/genetics/physiology ; PPAR gamma/*metabolism ; Perilipin-1/genetics/metabolism ; Proteomics ; Real-Time Polymerase Chain Reaction ; Swine ; },
abstract = {Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca[2+] in PPARγ pigs. Raising of Ca[2+] could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.},
}
@article {pmid33481207,
year = {2021},
author = {Zhang, H and Zheng, Q and Chen-Tsai, RY},
title = {Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases.},
journal = {Transgenic research},
volume = {30},
number = {1},
pages = {91-104},
pmid = {33481207},
issn = {1573-9368},
support = {R44GM108071/NH/NIH HHS/United States ; R43GM108071/NH/NIH HHS/United States ; R44GM108071/NH/NIH HHS/United States ; R43GM108071/NH/NIH HHS/United States ; },
mesh = {Aging/genetics ; Animals ; Autoimmunity/genetics ; CRISPR-Cas Systems/*genetics ; Cardiovascular Diseases/genetics ; Communicable Diseases/genetics ; *Disease Models, Animal ; Genetic Diseases, Inborn/*genetics/therapy ; Humans ; Inflammation/genetics ; Integrases/*genetics ; Neoplasms/genetics ; Rats ; Transplantation ; },
abstract = {The goal of this study is to establish a Cre/loxP rat resource for conditional and physiologically predictive rat models of human diseases. The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Up till recently, the ability of creating genetically modified rats has been limited compared to that in the mouse mainly due to lack of genetic manipulation tools and technologies in the rat. Recent advances in nucleases, such as CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9), as well as TARGATT™ integrase system enables fast, efficient and site-specific introduction of exogenous genetic elements into the rat genome. Here, we report the generation of a collection of tissue-specific, inducible transgenic Cre rats as tool models using TARGATT™, CRISPR/Cas9 and random transgenic approach. More specifically, we generated Cre driver rat models that allow controlled gene expression or knockout (conditional models) both temporally and spatially through the Cre-ERT2/loxP system. A total of 10 Cre rat lines and one Cre reporter/test line were generated, including eight (8) Cre lines for neural specific and two (2) lines for cardiovascular specific Cre expression. All of these lines have been deposited with the Rat Resource and Research Center and provide a much-needed resource for the bio-medical community who employ rat models for their studies of human diseases.},
}
@article {pmid33481178,
year = {2021},
author = {Gasanov, EV and Jędrychowska, J and Pastor, M and Wiweger, M and Methner, A and Korzh, VP},
title = {An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique.},
journal = {Molecular biology reports},
volume = {48},
number = {2},
pages = {1951-1957},
pmid = {33481178},
issn = {1573-4978},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Deletion ; Gene Editing/*methods ; Heterozygote ; Nerve Tissue Proteins/genetics ; Nucleic Acid Denaturation ; RNA, Guide/*genetics ; Voltage-Dependent Anion Channels/genetics ; Zebrafish/*genetics ; Zebrafish Proteins/genetics ; },
abstract = {Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.},
}
@article {pmid33480022,
year = {2021},
author = {Zhu, J and Xu, H and Song, H and Li, X and Wang, N and Zhao, J and Zheng, X and Kim, KY and Zhang, H and Mao, Q and Xia, H},
title = {CRISPR/Cas9-mediated grna gene knockout leads to neurodevelopmental defects and motor behavior changes in zebrafish.},
journal = {Journal of neurochemistry},
volume = {157},
number = {3},
pages = {520-531},
doi = {10.1111/jnc.15307},
pmid = {33480022},
issn = {1471-4159},
mesh = {Animals ; Axons/pathology/ultrastructure ; Behavior, Animal ; Body Weight/genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Exons/genetics ; Frameshift Mutation ; Gene Knockout Techniques/*methods ; Genotype ; High-Throughput Screening Assays/methods ; Intercellular Signaling Peptides and Proteins/*genetics ; Mice, Transgenic ; Motor Activity/*genetics ; Motor Neurons/pathology/ultrastructure ; Neurodevelopmental Disorders/*genetics/physiopathology/psychology ; RNA, Guide/*genetics ; *Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions in early embryogenesis, anti-inflammation, and neurodegeneration. A good model for the functional study of PGRN is the zebrafish with knockdown or knockout of grn, the gene encoding PGRN. Morpholino oligonucleotides (MOs) and zinc finger nucleases have been used to generate zebrafish grn models, yet they have shown inconsistent phenotypes due to either the neurotoxicity of the MOs or possible genetic compensation responses during gene editing. In this study, we generated stable grna (one of the major grn homologues of zebrafish) knockout zebrafish by using CRISPR/Cas9-mediated genome editing. A grna sgRNA was designed to target the similar repeated sequence shared by exon 13, exon 15, and exon 19 in zebrafish. The F1 generation with the frameshift mutation of + 4 bp (the addition of 4 bp to exon15), which causes a premature termination, was obtained and subjected to morphological and behavioral evaluation. The grna knockout zebrafish showed neurodevelopmental defects, including spinal motor neurons with shorter axons, decreased sensory hair cells, thinning of the outer nuclear layer and thickening of the inner nuclear layer of the retina, decreased expression of rhodopsin in the cone cells, and motor behavior changes. Moreover, the phenotypes of grna knockout zebrafish could be rescued with the Tol2 system carrying the grna gene. The grna knockout zebrafish model generated in this study provides a useful tool to study PGRN function and has potential for high-throughput drug screening for disease therapy.},
}
@article {pmid33479502,
year = {2021},
author = {Aban, CE and Lombardi, A and Neiman, G and Biani, MC and La Greca, A and Waisman, A and Moro, LN and Sevlever, G and Miriuka, S and Luzzani, C},
title = {Downregulation of E-cadherin in pluripotent stem cells triggers partial EMT.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {2048},
pmid = {33479502},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Cadherins/*genetics ; Cell Differentiation/*genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Connexin 43/genetics ; Embryoid Bodies/metabolism ; Epithelial-Mesenchymal Transition/*genetics ; Gene Expression Regulation, Developmental/genetics ; Humans ; Occludin/genetics ; Pluripotent Stem Cells/cytology/*metabolism ; RNA, Long Noncoding/genetics ; Snail Family Transcription Factors/genetics ; beta Catenin/genetics ; },
abstract = {Epithelial to mesenchymal transition (EMT) is a critical cellular process that has been well characterized during embryonic development and cancer metastasis and it also is implicated in several physiological and pathological events including embryonic stem cell differentiation. During early stages of differentiation, human embryonic stem cells pass through EMT where deeper morphological, molecular and biochemical changes occur. Though initially considered as a decision between two states, EMT process is now regarded as a fluid transition where cells exist on a spectrum of intermediate states. In this work, using a CRISPR interference system in human embryonic stem cells, we describe a molecular characterization of the effects of downregulation of E-cadherin, one of the main initiation events of EMT, as a unique start signal. Our results suggest that the decrease and delocalization of E-cadherin causes an incomplete EMT where cells retain their undifferentiated state while expressing several characteristics of a mesenchymal-like phenotype. Namely, we found that E-cadherin downregulation induces SNAI1 and SNAI2 upregulation, promotes MALAT1 and LINC-ROR downregulation, modulates the expression of tight junction occludin 1 and gap junction connexin 43, increases human embryonic stem cells migratory capacity and delocalize β-catenin. Altogether, we believe our results provide a useful tool to model the molecular events of an unstable intermediate state and further identify multiple layers of molecular changes that occur during partial EMT.},
}
@article {pmid33479380,
year = {2021},
author = {Hoang Trung, H and Yoshihara, T and Nakao, A and Hayashida, K and Hirata, Y and Shirasuna, K and Kuwamura, M and Nakagawa, Y and Kaneko, T and Mori, Y and Asano, M and Kuramoto, T},
title = {Deficiency of the RIβ subunit of protein kinase A causes body tremor and impaired fear conditioning memory in rats.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {2039},
pmid = {33479380},
issn = {2045-2322},
mesh = {Animals ; Behavior, Animal/physiology ; CRISPR-Cas Systems/genetics ; Cyclic AMP/metabolism ; Cyclic AMP-Dependent Protein Kinase RIbeta Subunit/*genetics ; Cyclic AMP-Dependent Protein Kinases/genetics ; Disease Models, Animal ; Fear/physiology ; Hippocampus/*metabolism/pathology ; Humans ; Memory/physiology ; Memory Disorders/*genetics/physiopathology ; Mice ; Mutation/genetics ; Neuronal Plasticity/genetics ; Neurons/metabolism/pathology ; Rats ; Tremor/*genetics/physiopathology ; },
abstract = {The RIβ subunit of cAMP-dependent protein kinase (PKA), encoded by Prkar1b, is a neuronal isoform of the type I regulatory subunit of PKA. Mice lacking the RIβ subunit exhibit normal long-term potentiation (LTP) in the Schaffer collateral pathway of the hippocampus and normal behavior in the open-field and fear conditioning tests. Here, we combined genetic, electrophysiological, and behavioral approaches to demonstrate that the RIβ subunit was involved in body tremor, LTP in the Schaffer collateral pathway, and fear conditioning memory in rats. Genetic analysis of WTC-furue, a mutant strain with spontaneous tremors, revealed a deletion in the Prkar1b gene of the WTC-furue genome. Prkar1b-deficient rats created by the CRISPR/Cas9 system exhibited body tremor. Hippocampal slices from mutant rats showed deficient LTP in the Schaffer collateral-CA1 synapse. Mutant rats also exhibited decreased freezing time following contextual and cued fear conditioning, as well as increased exploratory behavior in the open field. These findings indicate the roles of the RIβ subunit in tremor pathogenesis and contextual and cued fear memory, and suggest that the hippocampal and amygdala roles of this subunit differ between mice and rats and that rats are therefore beneficial for exploring RIβ function.},
}
@article {pmid33479218,
year = {2021},
author = {Ward, CM and Aumann, RA and Whitehead, MA and Nikolouli, K and Leveque, G and Gouvi, G and Fung, E and Reiling, SJ and Djambazian, H and Hughes, MA and Whiteford, S and Caceres-Barrios, C and Nguyen, TNM and Choo, A and Crisp, P and Sim, SB and Geib, SM and Marec, F and Häcker, I and Ragoussis, J and Darby, AC and Bourtzis, K and Baxter, SW and Schetelig, MF},
title = {White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {491},
pmid = {33479218},
issn = {2041-1723},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Ceratitis capitata/genetics ; Female ; Fertility/genetics ; Genome, Insect/genetics ; Insect Proteins/*genetics ; Male ; *Mutation ; Pest Control, Biological/*methods ; Phenotype ; Pupa/*genetics ; Reproduction/genetics ; Tephritidae/classification/*genetics ; },
abstract = {Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp[-] mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp[-] mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.},
}
@article {pmid33479216,
year = {2021},
author = {Li, S and Akrap, N and Cerboni, S and Porritt, MJ and Wimberger, S and Lundin, A and Möller, C and Firth, M and Gordon, E and Lazovic, B and Sieńska, A and Pane, LS and Coelho, MA and Ciotta, G and Pellegrini, G and Sini, M and Xu, X and Mitra, S and Bohlooly-Y, M and Taylor, BJM and Sienski, G and Maresca, M},
title = {Universal toxin-based selection for precise genome engineering in human cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {497},
pmid = {33479216},
issn = {2041-1723},
mesh = {Animals ; CD4-Positive T-Lymphocytes/cytology/metabolism ; *CRISPR-Cas Systems ; Cell Proliferation/genetics ; Cell Survival/genetics ; Cells, Cultured ; Gene Editing/*methods ; Genetic Engineering/*methods ; HCT116 Cells ; HEK293 Cells ; Heparin-binding EGF-like Growth Factor/*genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Mice ; *Mutation ; },
abstract = {Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.},
}
@article {pmid33479180,
year = {2021},
author = {Murakami, K and Terakado, Y and Saito, K and Jomen, Y and Takeda, H and Oshima, M and Barker, N},
title = {A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {4},
pages = {},
pmid = {33479180},
issn = {1091-6490},
mesh = {Adenomatous Polyposis Coli Protein/genetics/metabolism ; Adult Stem Cells/cytology/*metabolism ; Anaplastic Lymphoma Kinase/*genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Cell Proliferation ; Epithelial Cells/cytology/*metabolism ; Gastric Mucosa/cytology/metabolism ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Library ; Glycogen Synthase Kinase 3 beta/genetics/metabolism ; HEK293 Cells ; Humans ; Interleukins/genetics/metabolism ; Mice ; Organoids/cytology/*metabolism ; RNA-Binding Proteins/*genetics/metabolism ; Receptors, G-Protein-Coupled/genetics/metabolism ; Stomach/cytology ; Wnt Signaling Pathway/*genetics ; },
abstract = {An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3β, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.},
}
@article {pmid33479121,
year = {2021},
author = {Quinn, JJ and Jones, MG and Okimoto, RA and Nanjo, S and Chan, MM and Yosef, N and Bivona, TG and Weissman, JS},
title = {Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts.},
journal = {Science (New York, N.Y.)},
volume = {371},
number = {6532},
pages = {},
pmid = {33479121},
issn = {1095-9203},
support = {U19 AI090023/AI/NIAID NIH HHS/United States ; K08 CA222625/CA/NCI NIH HHS/United States ; T32 GM067547/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; F32 GM125247/GM/NIGMS NIH HHS/United States ; R01 CA204302/CA/NCI NIH HHS/United States ; U54 CA224081/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 CA211052/CA/NCI NIH HHS/United States ; R01 CA231300/CA/NCI NIH HHS/United States ; R01 CA169338/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Lineage ; Clone Cells ; Gene Expression Regulation, Neoplastic ; Humans ; Keratin-17/genetics ; Lung Neoplasms/genetics/*pathology ; Mice ; Neoplasm Invasiveness/genetics ; *Neoplasm Metastasis/genetics/pathology ; Neoplasm Seeding ; Neoplasm Transplantation ; Phenotype ; RNA-Seq ; Single-Cell Analysis ; Transcriptome ; Transplantation, Heterologous ; },
abstract = {Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.},
}
@article {pmid33478937,
year = {2021},
author = {Nakato, M and Shiranaga, N and Tomioka, M and Watanabe, H and Kurisu, J and Kengaku, M and Komura, N and Ando, H and Kimura, Y and Kioka, N and Ueda, K},
title = {ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100166},
pmid = {33478937},
issn = {1083-351X},
mesh = {ATP-Binding Cassette Transporters/deficiency/*genetics ; Adenosine Triphosphate/metabolism ; Animals ; Bipolar Disorder/genetics/metabolism/pathology ; Cell Membrane/metabolism ; Cerebral Cortex/metabolism/pathology ; Cholesterol/*metabolism ; Depressive Disorder, Major/genetics/metabolism/pathology ; Disease Models, Animal ; Endocytosis/*genetics ; Gangliosides/metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrolysis ; Mice ; Mice, Knockout ; Mutation ; Neurons/*metabolism/pathology ; *Prepulse Inhibition ; Primary Cell Culture ; Protein Transport ; Schizophrenia/genetics/metabolism/pathology ; Synaptic Vesicles/metabolism/pathology ; Transgenes ; },
abstract = {ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.},
}
@article {pmid33478580,
year = {2021},
author = {Gonçalves, E and Thomas, M and Behan, FM and Picco, G and Pacini, C and Allen, F and Vinceti, A and Sharma, M and Jackson, DA and Price, S and Beaver, CM and Dovey, O and Parry-Smith, D and Iorio, F and Parts, L and Yusa, K and Garnett, MJ},
title = {Minimal genome-wide human CRISPR-Cas9 library.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {40},
pmid = {33478580},
issn = {1474-760X},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Gene Library ; *Genome, Human ; Genome-Wide Association Study ; *Genomic Library ; Humans ; Organoids ; RNA, Guide/genetics ; },
abstract = {CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.},
}
@article {pmid33478577,
year = {2021},
author = {Zhang, Y and Nguyen, TM and Zhang, XO and Wang, L and Phan, T and Clohessy, JG and Pandolfi, PP},
title = {Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {41},
pmid = {33478577},
issn = {1474-760X},
support = {R35 NCI (CA197529-01)/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Screening Assays ; Humans ; RNA/*genetics ; RNA Splicing ; RNA, Circular/*genetics ; RNA, Guide/genetics ; RNA, Small Interfering ; },
abstract = {Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.},
}
@article {pmid33478513,
year = {2021},
author = {Asemoloye, MD and Marchisio, MA and Gupta, VK and Pecoraro, L},
title = {Genome-based engineering of ligninolytic enzymes in fungi.},
journal = {Microbial cell factories},
volume = {20},
number = {1},
pages = {20},
pmid = {33478513},
issn = {1475-2859},
mesh = {Cell Wall/genetics/metabolism ; Fungi/enzymology/*genetics ; Genetic Engineering/*methods ; Genome, Fungal/*genetics ; Laccase/*genetics/metabolism ; Lignin/metabolism ; Metabolic Networks and Pathways/genetics ; Peroxidases/*genetics/metabolism ; Synthetic Biology/methods ; },
abstract = {BACKGROUND: Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes.<br><br>SHORT CONCLUSION: Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.},
}
@article {pmid33478128,
year = {2021},
author = {Varanda, CM and Félix, MDR and Campos, MD and Patanita, M and Materatski, P},
title = {Plant Viruses: From Targets to Tools for CRISPR.},
journal = {Viruses},
volume = {13},
number = {1},
pages = {},
pmid = {33478128},
issn = {1999-4915},
mesh = {Agriculture/methods ; CRISPR-Cas Systems ; Crops, Agricultural/virology ; Disease Resistance/genetics ; Gene Editing ; Gene Expression ; Gene Transfer Techniques ; *Genetic Engineering ; Genetic Vectors/genetics ; Genome, Viral ; Host-Pathogen Interactions/genetics ; Plant Diseases/genetics/*virology ; Plant Viruses/*physiology ; },
abstract = {Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for 'clustered regularly interspaced short palindromic repeats' and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.},
}
@article {pmid33476633,
year = {2021},
author = {Yahya, EB and Alqadhi, AM},
title = {Recent trends in cancer therapy: A review on the current state of gene delivery.},
journal = {Life sciences},
volume = {269},
number = {},
pages = {119087},
doi = {10.1016/j.lfs.2021.119087},
pmid = {33476633},
issn = {1879-0631},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Transfer Techniques ; *Genetic Therapy ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {Cancer treatment has been always considered one of the most critical and vital themes of clinical issues. Many approaches have been developed, depending on the type and the stage of tumor. Gene therapy has the potential to revolutionize different cancer therapy. With the advent of recent bioinformatics technologies and genetic science, it become possible to identify, diagnose and determine the potential treatment using the technology of gene delivery. Several approaches have been developed and experimented in vitro and vivo for cancer therapy including: naked nucleic acids based therapy, targeting micro RNAs, oncolytic virotherapy, suicide gene based therapy, targeting telomerase, cell mediated gene therapy, and CRISPR/Cas9 based therapy. In this review, we present a straightforward introduction to cancer biology and occurrence, highlighting different viral and non-viral gene delivery systems for gene therapy and critically discussed the current and various strategies for cancer gene therapy.},
}
@article {pmid33476437,
year = {2021},
author = {Richard, C and Viret, S and Cantero Aguilar, L and Lefevre, C and Leduc, M and Faouzi, EH and Azar, N and Lavazec, C and Mayeux, P and Verdier, F},
title = {Myotonic dystrophy kinase-related CDC42-binding kinase α, a new transferrin receptor type 2-binding partner, is a regulator of erythropoiesis.},
journal = {American journal of hematology},
volume = {96},
number = {4},
pages = {480-492},
doi = {10.1002/ajh.26104},
pmid = {33476437},
issn = {1096-8652},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Endocytosis ; Erythroblasts/cytology/metabolism ; Erythropoiesis/*physiology ; Gene Knockout Techniques ; Humans ; Iron/metabolism ; Mice ; Myotonin-Protein Kinase/isolation &amp; purification/*physiology ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Receptors, Erythropoietin/metabolism ; Receptors, Transferrin/metabolism ; cdc42 GTP-Binding Protein/metabolism ; },
abstract = {Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.},
}
@article {pmid33476374,
year = {2021},
author = {Li, J and Liu, F and Lv, Y and Sun, K and Zhao, Y and Reilly, J and Zhang, Y and Tu, J and Yu, S and Liu, X and Qin, Y and Huang, Y and Gao, P and Jia, D and Chen, X and Han, Y and Shu, X and Luo, D and Tang, Z and Liu, M},
title = {Prpf31 is essential for the survival and differentiation of retinal progenitor cells by modulating alternative splicing.},
journal = {Nucleic acids research},
volume = {49},
number = {4},
pages = {2027-2043},
pmid = {33476374},
issn = {1362-4962},
mesh = {*Alternative Splicing ; Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Survival ; DNA Damage ; DNA Repair ; Exons ; Gene Knockout Techniques ; M Phase Cell Cycle Checkpoints ; Neural Stem Cells/cytology/*metabolism ; Neurogenesis/*genetics ; Retina/*embryology ; Retinal Neurons/cytology/metabolism ; Spindle Apparatus/ultrastructure ; Tumor Suppressor Protein p53/metabolism ; Zebrafish/embryology/genetics ; Zebrafish Proteins/genetics/metabolism/*physiology ; },
abstract = {Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.},
}
@article {pmid33476303,
year = {2021},
author = {Barghout, SH and Aman, A and Nouri, K and Blatman, Z and Arevalo, K and Thomas, GE and MacLean, N and Hurren, R and Ketela, T and Saini, M and Abohawya, M and Kiyota, T and Al-Awar, R and Schimmer, AD},
title = {A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity.},
journal = {JCI insight},
volume = {6},
number = {5},
pages = {},
pmid = {33476303},
issn = {2379-3708},
support = {//CIHR/Canada ; },
mesh = {ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/*metabolism ; ATP-Binding Cassette Transporters ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/genetics ; *Enzyme Inhibitors/pharmacology/therapeutic use ; *Gene Expression Regulation, Neoplastic ; Genome ; Humans ; *Leukemia, Myeloid, Acute/drug therapy ; Male ; Mice ; Neoplasm Proteins/genetics/*metabolism ; *Pyrazoles/pharmacology/therapeutic use ; *Pyrimidines/pharmacology/therapeutic use ; Repressor Proteins/genetics/*metabolism ; *Sulfides/pharmacology/therapeutic use ; *Sulfonamides/pharmacology/therapeutic use ; },
abstract = {TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.},
}
@article {pmid33475781,
year = {2021},
author = {Miladinovic, D and Antunes, D and Yildirim, K and Bakhsh, A and Cvejić, S and Kondić-Špika, A and Marjanovic Jeromela, A and Opsahl-Sorteberg, HG and Zambounis, A and Hilioti, Z},
title = {Targeted plant improvement through genome editing: from laboratory to field.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {935-951},
pmid = {33475781},
issn = {1432-203X},
mesh = {Animal Feed ; CRISPR-Cas Systems ; Crops, Agricultural/genetics/*growth &amp; development ; Disease Resistance ; Food Quality ; Gene Editing/*methods ; Laboratories ; Lignin/genetics ; Plant Breeding/*methods ; Plant Diseases ; Plants, Genetically Modified/*genetics ; },
abstract = {This review illustrates how far we have come since the emergence of GE technologies and how they could be applied to obtain superior and sustainable crop production. The main challenges of today's agriculture are maintaining and raising productivity, reducing its negative impact on the environment, and adapting to climate change. Efficient plant breeding can generate elite varieties that will rapidly replace obsolete ones and address ongoing challenges in an efficient and sustainable manner. Site-specific genome editing in plants is a rapidly evolving field with tangible results. The technology is equipped with a powerful toolbox of molecular scissors to cut DNA at a pre-determined site with different efficiencies for designing an approach that best suits the objectives of each plant breeding strategy. Genome editing (GE) not only revolutionizes plant biology, but provides the means to solve challenges related to plant architecture, food security, nutrient content, adaptation to the environment, resistance to diseases and production of plant-based materials. This review illustrates how far we have come since the emergence of these technologies and how these technologies could be applied to obtain superior, safe and sustainable crop production. Synergies of genome editing with other technological platforms that are gaining significance in plants lead to an exciting new, post-genomic era for plant research and production. In previous months, we have seen what global changes might arise from one new virus, reminding us of what drastic effects such events could have on food production. This demonstrates how important science, technology, and tools are to meet the current time and the future. Plant GE can make a real difference to future sustainable food production to the benefit of both mankind and our environment.},
}
@article {pmid33475257,
year = {2021},
author = {Vavassori, V and Mercuri, E and Marcovecchio, GE and Castiello, MC and Schiroli, G and Albano, L and Margulies, C and Buquicchio, F and Fontana, E and Beretta, S and Merelli, I and Cappelleri, A and Rancoita, PM and Lougaris, V and Plebani, A and Kanariou, M and Lankester, A and Ferrua, F and Scanziani, E and Cotta-Ramusino, C and Villa, A and Naldini, L and Genovese, P},
title = {Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.},
journal = {EMBO molecular medicine},
volume = {13},
number = {3},
pages = {e13545},
pmid = {33475257},
issn = {1757-4684},
mesh = {Animals ; Gene Editing ; Hematopoietic Stem Cells ; Humans ; *Hyper-IgM Immunodeficiency Syndrome ; *Hyper-IgM Immunodeficiency Syndrome, Type 1 ; Mice ; T-Lymphocytes ; },
abstract = {Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.},
}
@article {pmid33474893,
year = {2021},
author = {He, XY and Zhang, AQ and Gong, T and Li, YQ},
title = {[Transcriptomic Analysis of csn2 Gene Mutant Strains of Streptococcus mutans CRISPR-Cas9 System].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {52},
number = {1},
pages = {76-81},
doi = {10.12182/20210160505},
pmid = {33474893},
issn = {1672-173X},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Profiling ; *Streptococcus mutans/genetics ; *Transcriptome ; },
abstract = {OBJECTIVE: To explore the differences in transcriptional levels between mutant strains of csn2 gene of CRISPR-Cas9 system of Streptococcus mutans(S. mutans) and wild-type strains.<br><br>METHODS: The S. mutans UA159, csn2-gene-deleted strains (&#x394; csn2) and csn2-gene-covering strains (&#x394; csn2/pDL278- csn2) of S. mutans were cultivated. Total RNA was extracted, and high-throughput sequencing technology was used for transcriptome sequencing. Based on the GO analysis and the KEGG analysis of the differentially expressed genes, the biological processes involved were thoroughly examined. The qRT-PCR method was used to verify the transcriptome sequencing results.<br><br>RESULTS: The transcriptome results showed that, compared with UA159, there were 176 genes in &#x394; csn2 whose gene expression changed more than one fold (P<0.05), of which 72 were up-regulated and 104 were down-regulated. The GO enrichment analysis and the KEGG enrichment analysis revealed that both the up-regulated and down-regulated differentially expressed genes (DEG) were involved in amino acid transport and metabolism. In addition, the biological processes that up-regulated DEGs participated in were mainly related to carbohydrate metabolism, energy production and conversion, and transcription; down-regulated DEGs were mainly related to lipid metabolism, DNA replication, recombination and repair, signal transduction mechanisms, nucleotide transport and metabolism. The functions of some DEGs were still unclear. Results of qRT-PCR verified that the expressions of leuA, leuC and leuD(genes related to the formation of branched-chain amino acids) were significantly down-regulated in &#x394; csn2 when compared with UA159 and &#x394; csn2/pDL278- csn2.<br><br>CONCLUSION: Through transcriptome sequencing and qRT-PCR verification, it was found that the expression of genes related to branched-chain amino acid synthesis and cell membrane permeability in &#x394; csn2 changed significantly.},
}
@article {pmid33473139,
year = {2021},
author = {Sharma, R and Dever, DP and Lee, CM and Azizi, A and Pan, Y and Camarena, J and Köhnke, T and Bao, G and Porteus, MH and Majeti, R},
title = {The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {472},
pmid = {33473139},
issn = {2041-1723},
support = {R01 HL135607/HL/NHLBI NIH HHS/United States ; R01 CA251331/CA/NCI NIH HHS/United States ; R01 CA188055/CA/NCI NIH HHS/United States ; R01 AI120766/AI/NIAID NIH HHS/United States ; R01 AI097320/AI/NIAID NIH HHS/United States ; T32 HL120824/HL/NHLBI NIH HHS/United States ; R01 HL142637/HL/NHLBI NIH HHS/United States ; },
mesh = {*Alleles ; Animals ; CRISPR-Cas Systems ; *Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/methods ; Gene Targeting/*methods ; Genetic Therapy/methods ; *Hematopoietic Stem Cells ; Humans ; Mice ; Mutation ; *Recombination, Genetic ; Targeted Gene Repair/methods ; },
abstract = {Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.},
}
@article {pmid33473114,
year = {2021},
author = {Butterfield, NC and Curry, KF and Steinberg, J and Dewhurst, H and Komla-Ebri, D and Mannan, NS and Adoum, AT and Leitch, VD and Logan, JG and Waung, JA and Ghirardello, E and Southam, L and Youlten, SE and Wilkinson, JM and McAninch, EA and Vancollie, VE and Kussy, F and White, JK and Lelliott, CJ and Adams, DJ and Jacques, R and Bianco, AC and Boyde, A and Zeggini, E and Croucher, PI and Williams, GR and Bassett, JHD},
title = {Accelerating functional gene discovery in osteoarthritis.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {467},
pmid = {33473114},
issn = {2041-1723},
support = {110141/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; 110140/WT_/Wellcome Trust/United Kingdom ; 110141/WT_/Wellcome Trust/United Kingdom ; R01 DK077148/DK/NIDDK NIH HHS/United States ; 101123/WT_/Wellcome Trust/United Kingdom ; R01 DK065055/DK/NIDDK NIH HHS/United States ; 206194/WT_/Wellcome Trust/United Kingdom ; 098051/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Bone and Bones/pathology ; CRISPR-Cas Systems ; Cartilage/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; Drug Discovery ; Gene Editing ; *Genetic Association Studies ; Genetic Predisposition to Disease/*genetics ; Gonadotropin-Releasing Hormone/genetics ; Iodide Peroxidase ; Mice ; Mice, Knockout ; Osteoarthritis/*genetics/pathology/surgery ; Paired Box Transcription Factors/genetics ; Phenotype ; },
abstract = {Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease.},
}
@article {pmid33472516,
year = {2021},
author = {Hanson, B and Wood, MJA and Roberts, TC},
title = {Molecular correction of Duchenne muscular dystrophy by splice modulation and gene editing.},
journal = {RNA biology},
volume = {18},
number = {7},
pages = {1048-1062},
pmid = {33472516},
issn = {1555-8584},
mesh = {Animals ; CRISPR-Cas Systems ; Dystrophin/deficiency/*genetics ; Exons ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/*metabolism/pathology ; Muscular Dystrophy, Duchenne/genetics/metabolism/pathology/*therapy ; Mutation ; Myocardium/metabolism/pathology ; Neuromuscular Agents/therapeutic use ; Oligonucleotides, Antisense/genetics/metabolism/*therapeutic use ; *RNA Splicing ; },
abstract = {Duchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame. Indeed, three exon skipping oligonucleotides have received FDA approval for use in DMD patients. Second-generation exon skipping drugs (i.e. peptide-antisense oligonucleotide conjugates) exhibit enhanced potency, and also induce dystrophin restoration in the heart. Similarly, multiple additional antisense oligonucleotide drugs targeting various exons are in clinical development in order to treat a greater proportion of DMD patient mutations. Relatively recent advances in the field of genome engineering (specifically, the development of the CRISPR/Cas system) have provided multiple promising therapeutic approaches for the RNA-directed genetic correction of DMD, including exon excision, exon reframing via the introduction of insertion/deletion mutations, disruption of splice signals to promote exon skipping, and the templated correction of point mutations by seamless homology directed repair or base editing technology. Potential limitations to the clinical translation of the splice modulation and gene editing approaches are discussed, including drug delivery, the importance of uniform dystrophin expression in corrected myofibres, safety issues (e.g. renal toxicity, viral vector immunogenicity, and off-target gene editing), and the high cost of therapy.},
}
@article {pmid33472179,
year = {2021},
author = {Lee, MH and Lin, CC and Thomas, JL and Chan, CK and Lin, HY},
title = {Epitope recognition of magnetic peptide-imprinted chitosan composite nanoparticles for the extraction of CRISPR/dCas9a proteins from transfected cells.},
journal = {Nanotechnology},
volume = {32},
number = {18},
pages = {18LT02},
doi = {10.1088/1361-6528/abde00},
pmid = {33472179},
issn = {1361-6528},
mesh = {CRISPR-Associated Protein 9/*isolation &amp; purification ; CRISPR-Cas Systems ; Chitosan/*chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epitopes/isolation &amp; purification ; Gene Editing ; HEK293 Cells ; Humans ; Magnets/chemistry ; *Molecular Imprinting ; Nanoparticles/*chemistry ; Peptides/*chemistry ; Transfection ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) technology is a powerful method for genetic modification (and regulation) that is of great current interest. The development of new, economical methods of detecting and extracting Cas9 (and/or dCas9) from transfected cells is thus an important advance. In this work, we employed molecular imprinting, using two peptides from the Cas9 protein, to make magnetic peptide-imprinted chitosan nanoparticles. dCas9 was encoded in a plasmid which was then transfected into human embryonic kidney (HEK293T) cells. The expression of dCas9 protein was measured by using total protein kits. Finally, the imprinted nanoparticles were used to extract dCas9 from transfected cell homogenates.},
}
@article {pmid33472057,
year = {2021},
author = {Wang, B and Zhang, T and Yin, J and Yu, Y and Xu, W and Ding, J and Patel, DJ and Yang, H},
title = {Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.},
journal = {Molecular cell},
volume = {81},
number = {5},
pages = {1100-1115.e5},
pmid = {33472057},
issn = {1097-4164},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/*chemistry/genetics/metabolism ; Base Pairing ; Base Sequence ; Binding Sites ; CRISPR-Associated Proteins/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; Cryoelectron Microscopy ; Endodeoxyribonucleases/*chemistry/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; Leptotrichia/*genetics/metabolism ; Models, Molecular ; Mutation ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Interaction Domains and Motifs ; RNA Cleavage ; RNA, Guide/*chemistry/genetics/metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Substrate Specificity ; },
abstract = {Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13a[crRNA] in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.},
}
@article {pmid33471982,
year = {2021},
author = {Malech, HL},
title = {Treatment by CRISPR-Cas9 Gene Editing - A Proof of Principle.},
journal = {The New England journal of medicine},
volume = {384},
number = {3},
pages = {286-287},
doi = {10.1056/NEJMe2034624},
pmid = {33471982},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *beta-Thalassemia ; },
}
@article {pmid33471981,
year = {2021},
author = {Walters, MC},
title = {Induction of Fetal Hemoglobin by Gene Therapy.},
journal = {The New England journal of medicine},
volume = {384},
number = {3},
pages = {284-285},
doi = {10.1056/NEJMe2034338},
pmid = {33471981},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Fetal Hemoglobin/genetics ; Gene Editing ; Genetic Therapy ; Humans ; *beta-Thalassemia/genetics ; },
}
@article {pmid33471959,
year = {2021},
author = {Chen, A and Wen, S and Liu, F and Zhang, Z and Liu, M and Wu, Y and He, B and Yan, M and Kang, T and Lam, EW and Wang, Z and Liu, Q},
title = {CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A inhibitor resistance in breast cancer.},
journal = {Cancer communications (London, England)},
volume = {41},
number = {2},
pages = {121-139},
pmid = {33471959},
issn = {2523-3548},
mesh = {Animals ; *Aurora Kinase A ; *Breast Neoplasms/drug therapy/metabolism ; *CRISPR-Cas Systems ; Cell Cycle Proteins ; Early Detection of Cancer ; Female ; Humans ; Kinesins/metabolism ; Kinetochores/metabolism ; Mice ; Microtubules/metabolism ; },
abstract = {BACKGROUND: Overexpression of Aurora-A (AURKA) is a feature of breast cancer and associates with adverse prognosis. The selective Aurora-A inhibitor alisertib (MLN8237) has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients. Thus, identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome.<br><br>METHODS: Here, we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237. Then, we performed competitive growth assays, colony formation assays, cell viability assays, apoptosis assays, and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin (GSG2) depletion or inhibition with MLN8237. For mechanistic studies, immunofluorescence was used to detect the state of microtubules and the localization of Aurora-B and mitotic centromere-associated kinesin (MCAK).<br><br>RESULTS: Among the hits, we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237. Mechanistic studies showed that co-treatment with Aurora-A and Haspin inhibitors abolished the recruitment of Aurora-B and mitotic centromere-associated kinesin (MCAK) to centromeres which were associated with excessive microtubule depolymerization, kinetochore-microtubule (KT-MT) attachment failure, and severe mitotic catastrophe. We further showed that the combination of MLN8237 and the Haspin inhibitor CHR-6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth.<br><br>CONCLUSIONS: These findings establish Haspin as a synthetic lethal target and demonstrate CHR-6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.},
}
@article {pmid33471334,
year = {2021},
author = {Liu, J and Liang, D and Yao, L and Zhang, Y and Liu, C and Liu, Y and Wang, Y and Zhou, H and Kelliher, T and Zhang, X and Bandyopadhyay, A},
title = {Rice Haploid Inducer Development by Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {221-230},
pmid = {33471334},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/genetics ; *Genome, Plant ; *Haploidy ; Oryza/genetics/*growth &amp; development ; Plant Breeding ; Plant Proteins/*genetics ; },
abstract = {The current method to induce haploids in rice is anther culture, which is time-consuming and labor intensive and only works for some varieties. Here we describe a seed-based haploid induction system created by CRISPR/Cas9 technology. By editing OsMATL, we generate rice haploid inducer lines with a 2-5% haploid induction rate in different germplasms.},
}
@article {pmid33471333,
year = {2021},
author = {Yimam, YT and Zhou, J and Akher, SA and Zheng, X and Qi, Y and Zhang, Y},
title = {Improving a Quantitative Trait in Rice by Multigene Editing with CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {205-219},
pmid = {33471333},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Plant/*genetics ; *Gene Editing ; Genetic Vectors/genetics ; *Genome, Plant ; Multigene Family ; Oryza/genetics/*growth &amp; development ; Plant Proteins/*genetics ; *Quantitative Trait Loci ; },
abstract = {CRISPR-Cas9 system is one sequence-specific nuclease (SSN) that has several advantages over zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), such as multiplex genome editing. With multiplex genome editing, CRISPR-Cas9 becomes a preferred SSN to edit many quantitative trait loci (QTL) simultaneously for trait improvement in major crops. A multiplexed CRISPR system is also important for deletion of a large fragment within a chromosome, analysis of the function of gene families, exon exchange, gene activation, and repression. Therefore, assembly of several single guide RNAs (sgRNAs) into one binary vector is the main step in multigene editing by CRISPR-Cas9. Different vector construction methods have been practiced including Golden Gate assembly. This chapter provides a detailed protocol for the construction of a T-DNA binary vector for expressing Cas9 and three sgRNAs for simultaneous targeting of three QTL genes for improving seed trait in rice.},
}
@article {pmid33471332,
year = {2021},
author = {Tang, X and Qi, Y and Zhang, Y},
title = {Single Transcript Unit CRISPR 2.0 Systems for Genome Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {193-204},
pmid = {33471332},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/genetics ; *Genome, Plant ; Oryza/genetics/*growth &amp; development ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems in plants. Recently, we developed a single transcript unit (STU) CRISPR 2.0 toolbox for applications in plants, which contains two STU-Cas9 systems and one STU-Cas12a system. Here, we describe a detailed protocol about using the STU CRISPR 2.0 systems to achieve single and multiplex genome editing in rice.},
}
@article {pmid33471331,
year = {2021},
author = {Bes, M and Herbert, L and Mounier, T and Meunier, AC and Durandet, F and Guiderdoni, E and Périn, C},
title = {Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {173-191},
pmid = {33471331},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/genetics ; *Genome, Plant ; Oryza/genetics/*growth &amp; development ; Plant Breeding ; Plants, Genetically Modified/genetics/*growth &amp; development ; Protoplasts/*physiology ; Transformation, Genetic ; Transgenes/physiology ; },
abstract = {Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.},
}
@article {pmid33471330,
year = {2021},
author = {Liu, G and Qi, Y and Zhang, T},
title = {Analysis of Off-Target Mutations in CRISPR-Edited Rice Plants Using Whole-Genome Sequencing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {145-172},
pmid = {33471330},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/genetics ; *Genome, Plant ; *Mutation ; Oryza/genetics/*growth &amp; development ; Plants, Genetically Modified/genetics/*growth &amp; development ; Transformation, Genetic ; Transgenes/physiology ; Whole Genome Sequencing/*methods ; },
abstract = {The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq[*], Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.},
}
@article {pmid33471329,
year = {2021},
author = {Tra, MVT and Yin, X and Bajal, I and Balahadia, CP and Quick, WP and Bandyopadhyay, A},
title = {Single Base Editing Using Cytidine Deaminase to Change Grain Size and Seed Coat Color in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {135-143},
pmid = {33471329},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cytidine Deaminase/*antagonists &amp; inhibitors/genetics ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Genome, Plant ; Oryza/genetics/*growth &amp; development ; *Plant Breeding ; Plants, Genetically Modified/genetics/growth &amp; development ; *Polymorphism, Single Nucleotide ; Seeds/genetics/*growth &amp; development ; Transformation, Genetic ; Transgenes/physiology ; },
abstract = {The fast-moving CRISPR technology has allowed plant scientists to manipulate plant genomes in a targeted manner. So far, most of the applications were focused on gene knocking out by creating indels. However, more precise genome editing tools are demanded to assist the introduction of functional single nucleotide polymorphisms (SNPs) in breeding programs. The CRISPR base editing tools were developed to meet this need. In this chapter, we present a cytidine deaminase base editing method for editing the point mutations that control the grain size and seed coat color in rice.},
}
@article {pmid33471328,
year = {2021},
author = {Das, A and Ghana, P and Rudrappa, B and Gandhi, R and Tavva, VS and Mohanty, A},
title = {Genome Editing of Rice by CRISPR-Cas: End-to-End Pipeline for Crop Improvement.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {115-134},
pmid = {33471328},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/*growth &amp; development ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/*genetics ; Genome, Plant ; Oryza/genetics/*growth &amp; development ; Plant Breeding ; Plants, Genetically Modified/genetics/*growth &amp; development ; *Transformation, Genetic ; Transgenes/physiology ; },
abstract = {CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.},
}
@article {pmid33471327,
year = {2021},
author = {Sretenovic, S and Pan, C and Tang, X and Zhang, Y and Qi, Y},
title = {Rapid Vector Construction and Assessment of BE3 and Target-AID C to T Base Editing Systems in Rice Protoplasts.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {95-113},
pmid = {33471327},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cytidine Deaminase/*antagonists &amp; inhibitors/genetics ; *Gene Editing ; Gene Transfer Techniques ; Genetic Vectors/*genetics ; Genome, Plant ; Oryza/genetics/*growth &amp; development ; Plants, Genetically Modified/genetics/*growth &amp; development ; Protoplasts/physiology ; *Transformation, Genetic ; Transgenes/physiology ; },
abstract = {CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.},
}
@article {pmid33471325,
year = {2021},
author = {Majumder, S and Datta, K and Datta, SK},
title = {Agrobacterium tumefaciens-Mediated Transformation of Rice by Hygromycin Phosphotransferase (hptII) Gene Containing CRISPR/Cas9 Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {69-79},
pmid = {33471325},
issn = {1940-6029},
mesh = {Agrobacterium tumefaciens/*genetics ; CRISPR-Cas Systems ; Gene Editing ; *Gene Transfer Techniques ; Genetic Vectors ; Genome, Plant ; Oryza/genetics/*growth &amp; development ; Phosphotransferases (Alcohol Group Acceptor)/*antagonists &amp; inhibitors/genetics ; Plants, Genetically Modified/genetics/*growth &amp; development ; *Transformation, Genetic ; Transgenes/*physiology ; },
abstract = {The CRISPR/Cas9 technique for rice genome engineering is gaining momentum and requires a precise gene delivery system. For rice and other crop plants, Agrobacterium tumefaciens-mediated transformation (AMT) is considered a suitable gene transformation method. The AMT for indica-type rice is a challenge because it is less efficient in tissue culture response than japonica-type rice. Here is a protocol of the AMT method that we developed for IR64 variety which has been successfully tested in other popular indica-type rice varieties. We used embryogenic calli as explant and an empty gRNA-containing CRISPR/Cas9 vector with hptII (hygromycin phosphotransferase) gene for the transformation. This technique would speed up rice genome editing via CRISPR/Cas9 technology and facilitate to achieve varied application in the future.},
}
@article {pmid33470989,
year = {2021},
author = {Cañedo, EC and Totten, S and Ahn, R and Savage, P and MacNeil, D and Hudson, J and Autexier, C and Deblois, G and Park, M and Witcher, M and Ursini-Siegel, J},
title = {p66ShcA potentiates the cytotoxic response of triple-negative breast cancers to PARP inhibitors.},
journal = {JCI insight},
volume = {6},
number = {4},
pages = {},
pmid = {33470989},
issn = {2379-3708},
support = {PJT-168832//CIHR/Canada ; PJT-159663//CIHR/Canada ; MOP133449//CIHR/Canada ; },
mesh = {Antineoplastic Agents/*pharmacology ; Apoptosis ; BRCA1 Protein/genetics ; BRCA2 Protein/genetics ; Breast Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival ; DNA Damage ; Genomic Instability ; Humans ; MCF-7 Cells ; Poly (ADP-Ribose) Polymerase-1/*drug effects ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use ; Src Homology 2 Domain-Containing, Transforming Protein 1 ; Triple Negative Breast Neoplasms/*drug therapy/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.},
}
@article {pmid33469138,
year = {2021},
author = {Fernandes, LGV and Hornsby, RL and Nascimento, ALTO and Nally, JE},
title = {Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1768},
pmid = {33469138},
issn = {2045-2322},
mesh = {Antigens, Bacterial/*genetics ; Bacterial Outer Membrane Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Silencing ; Humans ; Leptospira interrogans/*genetics ; Leptospirosis/microbiology ; Lipoproteins/*genetics ; Phenotype ; RNA, Guide/genetics ; },
abstract = {Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.},
}
@article {pmid33469032,
year = {2021},
author = {Hörnblad, A and Bastide, S and Langenfeld, K and Langa, F and Spitz, F},
title = {Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {439},
pmid = {33469032},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Ectoderm/embryology ; Embryo, Mammalian ; Embryonic Development/*genetics ; Enhancer Elements, Genetic/*genetics ; Extremities/embryology ; Feasibility Studies ; Female ; Fibroblast Growth Factor 8/*genetics/metabolism ; *Gene Expression Regulation, Developmental ; Gene Regulatory Networks ; Genetic Engineering/*methods ; Male ; Mesencephalon/embryology ; Mice ; Mice, Transgenic ; Rhombencephalon/embryology ; },
abstract = {Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.},
}
@article {pmid33469008,
year = {2021},
author = {Chen, X and Lloyd, SM and Kweon, J and Gamalong, GM and Bao, X},
title = {Epidermal progenitors suppress GRHL3-mediated differentiation through intronic polyadenylation promoted by CPSF-HNRNPA3 collaboration.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {448},
pmid = {33469008},
issn = {2041-1723},
support = {T32 GM008061/GM/NIGMS NIH HHS/United States ; UL1 TR001422/TR/NCATS NIH HHS/United States ; R01 AR075015/AR/NIAMS NIH HHS/United States ; R00 AR065480/AR/NIAMS NIH HHS/United States ; P30 AR075049/AR/NIAMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Self Renewal/genetics ; Cleavage And Polyadenylation Specificity Factor/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation ; Gene Knockdown Techniques ; Gene Knockout Techniques ; HEK293 Cells ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics/*metabolism ; Humans ; Introns/genetics ; Keratinocytes/*physiology ; Polyadenylation/genetics ; Primary Cell Culture ; Re-Epithelialization/*genetics ; Stem Cells/*physiology ; Transcription Factors/genetics/*metabolism ; },
abstract = {In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.},
}
@article {pmid33468705,
year = {2021},
author = {Singh, A and Gaur, M and Sharma, V and Khanna, P and Bothra, A and Bhaduri, A and Mondal, AK and Dash, D and Singh, Y and Misra, R},
title = {Comparative Genomic Analysis of Mycobacteriaceae Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex.},
journal = {mSystems},
volume = {6},
number = {1},
pages = {},
pmid = {33468705},
issn = {2379-5077},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus-like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS6110 The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5' end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii This study retracing the evolutionary events of HGT and IS6110-driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages.IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis, the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii, by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.},
}
@article {pmid33468685,
year = {2021},
author = {Xiao, H and Wyler, E and Milek, M and Grewe, B and Kirchner, P and Ekici, A and Silva, ABOV and Jungnickl, D and Full, F and Thomas, M and Landthaler, M and Ensser, A and Überla, K},
title = {CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.},
journal = {mBio},
volume = {12},
number = {1},
pages = {},
pmid = {33468685},
issn = {2150-7511},
mesh = {Cell Nucleus/genetics/virology ; Cytosol/metabolism/virology ; DEAD-box RNA Helicases/genetics/metabolism ; Exons ; Gene Regulatory Networks ; HIV-1/*genetics/metabolism ; Host-Pathogen Interactions/genetics ; Humans ; Introns ; Jurkat Cells ; Nuclear Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; Protein Interaction Mapping ; *RNA Splicing ; RNA Splicing Factors/genetics/metabolism ; RNA, Messenger/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; RNA, Viral/*genetics/metabolism ; Spliceosomes/genetics/metabolism ; Transcriptome ; rev Gene Products, Human Immunodeficiency Virus/*genetics/metabolism ; },
abstract = {The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.},
}
@article {pmid33467721,
year = {2021},
author = {Kamara, N and Jiao, Y and Lu, Z and Aloryi, KD and Wu, J and Liu, X and Shahid, MQ},
title = {Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33467721},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Plant/genetics ; Gene Expression Profiling ; *Gene Expression Regulation, Plant ; *Genome, Plant ; Meiosis ; Mutation ; Oryza/*genetics/physiology ; Plant Breeding ; Plant Infertility/genetics ; Pollen/*genetics ; RNA-Seq ; *Tetraploidy ; },
abstract = {Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F2-3 generations and RNA-seq of F4 generation detected a common fragment, i.e., 18,915,234-19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.},
}
@article {pmid33467049,
year = {2021},
author = {Iaffaldano, B and Reiser, J},
title = {Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33467049},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Directed Molecular Evolution/*methods ; Gene Editing/*methods ; Gene Targeting/*methods ; *Mutagenesis ; Plants/genetics ; },
abstract = {Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.},
}
@article {pmid33466489,
year = {2021},
author = {M Iyer, L and Anantharaman, V and Krishnan, A and Burroughs, AM and Aravind, L},
title = {Jumbo Phages: A Comparative Genomic Overview of Core Functions and Adaptions for Biological Conflicts.},
journal = {Viruses},
volume = {13},
number = {1},
pages = {},
pmid = {33466489},
issn = {1999-4915},
mesh = {Bacteriophages/*genetics/*physiology/ultrastructure ; CRISPR-Cas Systems ; *Genome, Viral ; Genomics ; Host Microbial Interactions ; Phylogeny ; Principal Component Analysis ; },
abstract = {Jumbo phages have attracted much attention by virtue of their extraordinary genome size and unusual aspects of biology. By performing a comparative genomics analysis of 224 jumbo phages, we suggest an objective inclusion criterion based on genome size distributions and present a synthetic overview of their manifold adaptations across major biological systems. By means of clustering and principal component analysis of the phyletic patterns of conserved genes, all known jumbo phages can be classified into three higher-order groups, which include both myoviral and siphoviral morphologies indicating multiple independent origins from smaller predecessors. Our study uncovers several under-appreciated or unreported aspects of the DNA replication, recombination, transcription and virion maturation systems. Leveraging sensitive sequence analysis methods, we identify novel protein-modifying enzymes that might help hijack the host-machinery. Focusing on host-virus conflicts, we detect strategies used to counter different wings of the bacterial immune system, such as cyclic nucleotide- and NAD[+]-dependent effector-activation, and prevention of superinfection during pseudolysogeny. We reconstruct the RNA-repair systems of jumbo phages that counter the consequences of RNA-targeting host effectors. These findings also suggest that several jumbo phage proteins provide a snapshot of the systems found in ancient replicons preceding the last universal ancestor of cellular life.},
}
@article {pmid33465779,
year = {2021},
author = {Yan, X and Stuurman, N and Ribeiro, SA and Tanenbaum, ME and Horlbeck, MA and Liem, CR and Jost, M and Weissman, JS and Vale, RD},
title = {High-content imaging-based pooled CRISPR screens in mammalian cells.},
journal = {The Journal of cell biology},
volume = {220},
number = {2},
pages = {},
pmid = {33465779},
issn = {1540-8140},
support = {R35 GM118106/GM/NIGMS NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Nucleus/genetics ; Cell Nucleus Size/genetics ; Flow Cytometry ; *Genetic Testing ; Green Fluorescent Proteins/metabolism ; Humans ; *Imaging, Three-Dimensional ; Optics and Photonics ; Phenotype ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.},
}
@article {pmid33464298,
year = {2021},
author = {Kanfer, G and Sarraf, SA and Maman, Y and Baldwin, H and Dominguez-Martin, E and Johnson, KR and Ward, ME and Kampmann, M and Lippincott-Schwartz, J and Youle, RJ},
title = {Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.},
journal = {The Journal of cell biology},
volume = {220},
number = {2},
pages = {},
pmid = {33464298},
issn = {1540-8140},
support = {DP2 GM119139/GM/NIGMS NIH HHS/United States ; },
mesh = {Artificial Intelligence ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Cyclic AMP Response Element-Binding Protein A/metabolism ; Deep Learning ; *Genetic Testing ; *Genome ; Green Fluorescent Proteins ; HEK293 Cells ; Humans ; *Imaging, Three-Dimensional ; Models, Biological ; Neural Networks, Computer ; Phenotype ; RNA, Guide/metabolism ; Reproducibility of Results ; Single-Cell Analysis ; Support Vector Machine ; Ubiquitin-Protein Ligases/metabolism ; },
abstract = {Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.},
}
@article {pmid33464043,
year = {2021},
author = {Standage-Beier, K and Tekel, SJ and Brafman, DA and Wang, X},
title = {Prime Editing Guide RNA Design Automation Using PINE-CONE.},
journal = {ACS synthetic biology},
volume = {10},
number = {2},
pages = {422-427},
pmid = {33464043},
issn = {2161-5063},
support = {R01 GM106081/GM/NIGMS NIH HHS/United States ; R01 GM121698/GM/NIGMS NIH HHS/United States ; R01 GM131405/GM/NIGMS NIH HHS/United States ; R21 AG056706/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Automation ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; DNA Breaks, Double-Stranded ; Drosophila melanogaster/genetics ; Gene Editing/*methods ; Genome ; Humans ; Mice ; Oligonucleotides/genetics ; Polymorphism, Single Nucleotide ; RNA Editing/*genetics ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/genetics ; *Software ; Zebrafish/genetics ; },
abstract = {CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE's utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer's Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.},
}
@article {pmid33463756,
year = {2020},
author = {Rautela, J and Surgenor, E and Huntington, ND},
title = {Drug target validation in primary human natural killer cells using CRISPR RNP.},
journal = {Journal of leukocyte biology},
volume = {108},
number = {4},
pages = {1397-1408},
doi = {10.1002/JLB.2MA0620-074R},
pmid = {33463756},
issn = {1938-3673},
mesh = {Animals ; *CRISPR-Cas Systems ; *Drug Discovery ; *Electroporation ; Humans ; Killer Cells, Natural/cytology/*immunology ; Mice ; },
abstract = {The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric Ag receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting NK cells have stalled in part by their resistance to traditional viral gene delivery systems. Here, an efficient approach is described to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. Electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods are detailed. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, NCR1 and CISH are deleted in primary human NK cells and murine findings are validated on their key roles in regulating NK cell antitumor function.},
}
@article {pmid33462442,
year = {2021},
author = {Huang, TP and Newby, GA and Liu, DR},
title = {Precision genome editing using cytosine and adenine base editors in mammalian cells.},
journal = {Nature protocols},
volume = {16},
number = {2},
pages = {1089-1128},
pmid = {33462442},
issn = {1750-2799},
support = {U01 AI142756/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Adenine/chemistry ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Cytosine/chemistry ; DNA/genetics ; DNA Breaks, Double-Stranded ; *Gene Editing/methods ; HEK293 Cells ; Polymorphism, Single Nucleotide ; },
abstract = {Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies-for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1-3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.},
}
@article {pmid33462439,
year = {2021},
author = {Kim, D and Kang, BC and Kim, JS},
title = {Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq.},
journal = {Nature protocols},
volume = {16},
number = {2},
pages = {1170-1192},
pmid = {33462439},
issn = {1750-2799},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Chromatin ; Chromosome Mapping/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Endonucleases/metabolism ; Gene Editing/*methods ; Genome, Human ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Nucleotide Deaminases/genetics/metabolism ; RNA, Guide/genetics ; Ribonucleases/genetics/metabolism ; Sequence Analysis, DNA/*methods ; Whole Genome Sequencing/methods ; },
abstract = {Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1-2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.},
}
@article {pmid33462394,
year = {2021},
author = {Verma, P and Zhou, Y and Cao, Z and Deraska, PV and Deb, M and Arai, E and Li, W and Shao, Y and Puentes, L and Li, Y and Patankar, S and Mach, RH and Faryabi, RB and Shi, J and Greenberg, RA},
title = {ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells.},
journal = {Nature cell biology},
volume = {23},
number = {2},
pages = {160-171},
pmid = {33462394},
issn = {1476-4679},
support = {R01 CA174904/CA/NCI NIH HHS/United States ; R01 GM101149/GM/NIGMS NIH HHS/United States ; },
mesh = {BRCA1 Protein/genetics ; BRCA2 Protein/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chromatin/*metabolism ; Chromatin Assembly and Disassembly/drug effects ; Chromosome Aberrations ; DNA Helicases/chemistry/*metabolism ; DNA Repair/drug effects ; DNA-Binding Proteins/chemistry/*metabolism ; Epistasis, Genetic/drug effects ; Genomic Instability ; Green Fluorescent Proteins/metabolism ; Homologous Recombination/drug effects/*genetics ; Humans ; Methyl Methanesulfonate ; Mutation/genetics ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Poly Adenosine Diphosphate Ribose/metabolism ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Poly(ADP-ribose) Polymerases/metabolism ; Protein Domains ; },
abstract = {The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.},
}
@article {pmid33462297,
year = {2021},
author = {Almeida, MP and Welker, JM and Siddiqui, S and Luiken, J and Ekker, SC and Clark, KJ and Essner, JJ and McGrail, M},
title = {Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1732},
pmid = {33462297},
issn = {2045-2322},
support = {R24 OD020166/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Knock-In Techniques/*methods ; Homologous Recombination ; Integrases/genetics/*metabolism ; Promoter Regions, Genetic ; Zebrafish/*genetics/growth &amp; development/metabolism ; },
abstract = {We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)[is75], Tg(olig2-2A-Cre)[is76], and Tg(neurod1-2A-Cre)[is77] expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.},
}
@article {pmid33461211,
year = {2021},
author = {Rostøl, JT and Xie, W and Kuryavyi, V and Maguin, P and Kao, K and Froom, R and Patel, DJ and Marraffini, LA},
title = {The Card1 nuclease provides defence during type III CRISPR immunity.},
journal = {Nature},
volume = {590},
number = {7847},
pages = {624-629},
pmid = {33461211},
issn = {1476-4687},
support = {S10 RR029205/RR/NCRR NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine Nucleotides/immunology/*metabolism ; Adenosine Triphosphate/metabolism ; Bacteriophages/immunology/physiology ; Biocatalysis ; CRISPR-Cas Systems/*immunology ; Catalytic Domain ; DNA, Single-Stranded/*metabolism ; Deoxyribonucleases/chemistry/genetics/*metabolism ; Endoribonucleases/chemistry/genetics/*metabolism ; Enzyme Activation ; Ligands ; Manganese/chemistry/metabolism ; Models, Molecular ; Oligoribonucleotides/immunology/*metabolism ; Plasmids/genetics/metabolism ; Protein Multimerization ; RNA/*metabolism ; Rotation ; Staphylococcus/*enzymology/growth &amp; development/*immunology/virology ; Substrate Specificity ; },
abstract = {In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain[1,2]. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction endonuclease-like domain[3,4]. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases[5,6], whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.},
}
@article {pmid33461039,
year = {2021},
author = {Yang, Z and Yuan, F and Wang, N and Liu, Y and Zhang, J and Wei, R and Wu, F and Wu, Y and You, K and Chen, Y and Yang, F and Li, YX},
title = {Generation of a DKK1 homozygous knockout human embryonic stem cell line WAe001-A-21 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {51},
number = {},
pages = {102161},
doi = {10.1016/j.scr.2021.102161},
pmid = {33461039},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; Intercellular Signaling Peptides and Proteins/genetics ; },
abstract = {Dickkopf1 (DKK1) is a secreted inhibitor for the Wnt signalling, which is involved in cell proliferation, tissue regeneration and embryonic development. Using CRISPR/Cas9 editing, we established a homozygous mutant DKK1 human embryonic stem cell line (WAe001-A-21). It has a 41 bp deletion in exon 2 of DKK1, leading to its coding frame shift. The WAe001-A-21 cell line maintains a normal karyotype, pluripotency markers, typical stem cell morphology and the ability to differentiate into three germ layers.},
}
@article {pmid33460640,
year = {2021},
author = {Nechipurenko, I and Lavrentyeva, S and Sengupta, P},
title = {GRDN-1/Girdin regulates dendrite morphogenesis and cilium position in two specialized sensory neuron types in C. elegans.},
journal = {Developmental biology},
volume = {472},
number = {},
pages = {38-51},
pmid = {33460640},
issn = {1095-564X},
support = {R35 GM122463/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Axons/metabolism ; Basal Bodies/metabolism ; CRISPR-Cas Systems ; Cadherins/genetics/metabolism ; Caenorhabditis elegans/*genetics/metabolism ; Caenorhabditis elegans Proteins/*genetics/*metabolism ; Cell Cycle Proteins/metabolism ; Cilia/*metabolism ; Dendrites/*metabolism ; Gene Knockdown Techniques ; Glycoproteins/metabolism ; Kinesins/metabolism ; Microfilament Proteins/*genetics/*metabolism ; Mutation ; Neurogenesis/*genetics ; Sensory Receptor Cells/*metabolism ; Wnt Signaling Pathway/genetics ; },
abstract = {Primary cilia are located at the dendritic tips of sensory neurons and house the molecular machinery necessary for detection and transduction of sensory stimuli. The mechanisms that coordinate dendrite extension with cilium position during sensory neuron development are not well understood. Here, we show that GRDN-1, the Caenorhabditis elegans ortholog of the highly conserved scaffold and signaling protein Girdin/GIV, regulates both cilium position and dendrite extension in the postembryonic AQR and PQR gas-sensing neurons. Mutations in grdn-1 disrupt dendrite outgrowth and mislocalize cilia to the soma or proximal axonal segments in AQR, and to a lesser extent, in PQR. GRDN-1 is localized to the basal body and regulates localization of HMR-1/Cadherin to the distal AQR dendrite. However, knockdown of HMR-1 and/or loss of SAX-7/LICAM, molecules previously implicated in sensory dendrite development in C. elegans, do not alter AQR dendrite morphology or cilium position. We find that GRDN-1 localization in AQR is regulated by UNC-116/Kinesin-1, and that correspondingly, unc-116 mutants exhibit severe AQR dendrite outgrowth and cilium positioning defects. In contrast, GRDN-1 and cilium localization in PQR is modulated by LIN-44/Wnt signaling. Together, these findings identify upstream regulators of GRDN-1, and describe new cell-specific roles for this multifunctional protein in sensory neuron development.},
}
@article {pmid33459978,
year = {2021},
author = {Cao, Y and Zhou, H and Zhou, X and Li, F},
title = {Conferring Resistance to Plant RNA Viruses with the CRISPR/CasRx System.},
journal = {Virologica Sinica},
volume = {36},
number = {4},
pages = {814-817},
pmid = {33459978},
issn = {1995-820X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Diseases ; *Plant Viruses/genetics ; *RNA Viruses/genetics ; RNA, Plant ; },
}
@article {pmid33459801,
year = {2021},
author = {Teriyapirom, I and Batista-Rocha, AS and Koo, BK},
title = {Genetic engineering in organoids.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {99},
number = {4},
pages = {555-568},
pmid = {33459801},
issn = {1432-1440},
mesh = {Adult Stem Cells/cytology/drug effects ; Animals ; Animals, Genetically Modified ; Brain/cytology ; CRISPR-Cas Systems ; Cellular Reprogramming Techniques ; Gastrointestinal Tract/cytology ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods/trends ; Humans ; Induced Pluripotent Stem Cells/cytology/drug effects ; Kidney/cytology ; Liver/cytology ; Mammary Glands, Animal/cytology ; Mice ; *Organoids/cytology ; },
abstract = {Three-dimensional organoids have been widely used for developmental and disease modeling. Organoids are derived from both adult and pluripotent stem cells. Various types are available for mimicking almost all major organs and tissues in the mouse and human. While culture protocols for stepwise differentiation and long-term expansion are well established, methods for genetic manipulation in organoids still need further standardization. In this review, we summarized different methods for organoid genetics and provide the pros and cons of each method for designing an optimal strategy.},
}
@article {pmid33455878,
year = {2021},
author = {Park, SH and Bao, G},
title = {CRISPR/Cas9 gene editing for curing sickle cell disease.},
journal = {Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis},
volume = {60},
number = {1},
pages = {103060},
pmid = {33455878},
issn = {1473-0502},
support = {OT2 HL154977/HL/NHLBI NIH HHS/United States ; R01 HL152314/HL/NHLBI NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*therapy ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; },
abstract = {Sickle cell disease (SCD) is the most common monogenic blood disorder marked by severe pain, end-organ damage, and early mortality. Treatment options for SCD remain very limited. There are only four FDA approved drugs to reduce acute complications. The only curative therapy for SCD is hematopoietic stem cell transplantation, typically from a matched, related donor. Ex vivo engineering of autologous hematopoietic stem and progenitor cells followed by transplantation of genetically modified cells potentially provides a permanent cure applicable to all patients regardless of the availability of suitable donors and graft-vs-host disease. In this review, we focus on the use of CRISPR/Cas9 gene-editing for curing SCD, including the curative correction of SCD mutation in β-globin (HBB) and the induction of fetal hemoglobin to reverse sickling. We summarize the major achievements and challenges, aiming to provide a clearer perspective on the potential of gene-editing based approaches in curing SCD.},
}
@article {pmid33454599,
year = {2021},
author = {Ashraf, MU and Salman, HM and Khalid, MF and Khan, MHF and Anwar, S and Afzal, S and Idrees, M and Chaudhary, SU},
title = {CRISPR-Cas13a mediated targeting of hepatitis C virus internal-ribosomal entry site (IRES) as an effective antiviral strategy.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {136},
number = {},
pages = {111239},
doi = {10.1016/j.biopha.2021.111239},
pmid = {33454599},
issn = {1950-6007},
mesh = {CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Targeting ; Hepacivirus/*genetics/growth &amp; development/metabolism ; Hepatitis C/genetics/*therapy/virology ; Humans ; *Internal Ribosome Entry Sites ; RNA Stability ; RNA, Viral/*genetics/metabolism ; Virus Replication/drug effects ; },
abstract = {Hepatitis C is an inflammatory liver disease caused by the single-stranded RNA (ssRNA) hepatitis C virus (HCV). The genetic diversity of the virus and quasispecies produced during replication have resulted in viral resistance to direct-acting antivirals (DAAs) as well as impediments in vaccine development. The recent adaptation of CRISPR-Cas as an alternative antiviral approach has demonstrated degradation of viral nucleic acids in eukaryotes. In particular, the CRISPR-effector Cas13 enzyme has been shown to target ssRNA viruses effectively. In this work, we have employed Cas13a to knockdown HCV in mammalian cells. Using a computational screen, we identified several potential Cas13a target sites within highly conserved regions of the HCV internal ribosomal entry site (IRES). Our results demonstrate significant inhibition of HCV replication as well as translation in huh-7.5 cells with minimal effects on cell viability. These findings were validated using a multi-modality approach involving qRT-PCR, luciferase assay, and MTT cell viability assay. In conclusion, the CRISPR-Cas13a system efficiently targets HCV in vitro, suggesting its potential as a programmable therapeutic antiviral strategy.},
}
@article {pmid33454424,
year = {2021},
author = {Nandadasa, S and O'Donnell, A and Murao, A and Yamaguchi, Y and Midura, RJ and Olson, L and Apte, SS},
title = {The versican-hyaluronan complex provides an essential extracellular matrix niche for Flk1[+] hematoendothelial progenitors.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {97},
number = {},
pages = {40-57},
pmid = {33454424},
issn = {1569-1802},
support = {P01 HL107147/HL/NHLBI NIH HHS/United States ; RF1 AG057579/AG/NIA NIH HHS/United States ; S10 RR026820/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Extracellular Matrix/*metabolism ; Hedgehog Proteins/metabolism ; Hematopoiesis ; Hyaluronic Acid/*metabolism ; Membrane Proteins/metabolism ; Mice ; Mouse Embryonic Stem Cells/*cytology/metabolism ; Stem Cell Niche ; Up-Regulation ; Vascular Endothelial Growth Factor Receptor-2/*metabolism ; Versicans/*genetics/metabolism ; },
abstract = {Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1[+] hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcan[hdf] lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation within embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. Hyaluronan was severely depleted in Vcan[hdf] embryos, with corresponding upregulation of the hyaluronan-depolymerase TMEM2. Conversely, hyaluronan-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-hyaluronan matrix, specifically versican chondroitin sulfate chains, for binding. Versican-hyaluronan ECM is thus an obligate requirement for vasculogenesis and primitive hematopoiesis, providing a vasculogenic factor-enriching microniche for Flk1[+] progenitors from their origin at gastrulation.},
}
@article {pmid33453719,
year = {2021},
author = {Lipták, N and Gál, Z and Biró, B and Hiripi, L and Hoffmann, OI},
title = {Rescuing lethal phenotypes induced by disruption of genes in mice: a review of novel strategies.},
journal = {Physiological research},
volume = {70},
number = {1},
pages = {3-12},
pmid = {33453719},
issn = {1802-9973},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Embryo Loss/genetics/*prevention &amp; control ; Gene Editing/*methods ; Mice ; Mice, Knockout ; Phenotype ; Transcription Activator-Like Effector Nucleases/*genetics ; Zinc Finger Nucleases/*genetics ; },
abstract = {Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: "lethality", "mice", "knock-out", "deficient", "embryonic", "perinatal", "rescue". Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human disease-relevant homozygous KO mice lethal phenotypes were summarized.},
}
@article {pmid33453283,
year = {2021},
author = {Koay, TW and Osterhof, C and Orlando, IMC and Keppner, A and Andre, D and Yousefian, S and Suárez Alonso, M and Correia, M and Markworth, R and Schödel, J and Hankeln, T and Hoogewijs, D},
title = {Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100291},
pmid = {33453283},
issn = {1083-351X},
mesh = {Animals ; Binding Sites ; Brain/cytology/growth &amp; development/metabolism ; Calmodulin-Binding Proteins/*genetics/metabolism ; Cattle ; Cilia/*genetics/metabolism ; Enhancer Elements, Genetic ; Female ; Forkhead Transcription Factors/*genetics/metabolism ; Gene Expression Regulation, Developmental ; Gene Ontology ; Globins/*genetics/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Lung/cytology/growth &amp; development/metabolism ; MCF-7 Cells ; Male ; Molecular Sequence Annotation ; Ovary/cytology/growth &amp; development/metabolism ; Promoter Regions, Genetic ; Protein Binding ; Protein Isoforms/genetics/metabolism ; Regulatory Factor X Transcription Factors/*genetics/metabolism ; Sequence Analysis, RNA ; Testis/cytology/growth &amp; development/metabolism ; *Transcriptome ; },
abstract = {Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that-in addition to the testes-ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.},
}
@article {pmid33452819,
year = {2021},
author = {Barbour, A and Glogauer, J and Grinfeld, L and Ostadsharif Memar, R and Fine, N and Tenenbaum, H and Glogauer, M},
title = {The role of CRISPR-Cas in advancing precision periodontics.},
journal = {Journal of periodontal research},
volume = {56},
number = {3},
pages = {454-461},
doi = {10.1111/jre.12846},
pmid = {33452819},
issn = {1600-0765},
mesh = {Biofilms ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Periodontics ; },
abstract = {The significant advancement of molecular biology has revolutionized medicine and provided important technologies to further clinical research development. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are DNA sequences derived from bacteriophages which have previously infected the bacterial species. The CRISPR-Cas system plays a key role in bacterial defense by detecting and destroying DNA fragments during subsequent bacteriophage invasions. The Cas9 enzyme recognizes and cleaves new invading CRISPR-complementary DNA sequences. Researchers have taken advantage of this biological device to manipulate microbes' genes and develop novel therapeutics to tackle systemic disease. In this review, we discuss the potential of utilizing CRISPR-Cas systems in the periodontal field to develop personalized periodontal care. We summarize promising attempts to bring this technology to the clinical setting. Finally, we provide insights regarding future developments to best utilize the CRISPR-Cas systems to advance precision periodontics. Although further research is imperative to evaluate the safety and potential of using CRISPR-Cas to develop precision periodontics approaches, few studies showed promising data to support the investment into this important technology in the dental sector. CRISPR-Cas9 can be a useful tool to create knockouts in vitro and in vivo as a screening tool to identify cellular pathways involved in the pathogenesis of periodontitis. Alternative CRISPR systems such as CRISPRa, CRISPRi, and Cas13 can be used to modify the transcriptome and gene expression of genes involved in periodontitis progression. CRISPR systems such as Cas3 can be used to target the periodontal biofilm and to develop new strategies to reduce or eliminate periodontal pathogens. Currently, the utility of CRISPR-Cas applications in clinical settings is limited. Through this review, we hope to foster further discussion in the periodontal research and clinical communities with respect to the potential clinical application of novel, CRISPR-Cas based, therapeutics for periodontitis.},
}
@article {pmid33452424,
year = {2021},
author = {Aprilyanto, V and Aditama, R and Tanjung, ZA and Utomo, C and Liwang, T},
title = {CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1504},
pmid = {33452424},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Computational Biology/*methods ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Variation/genetics ; Genome/genetics ; Models, Theoretical ; RNA, Guide/genetics ; Software ; },
abstract = {The off-target effect, in which DNA cleavage was conducted outside the targeted region, is a major problem which limits the applications of CRISPR/Cas9 genome editing system. CRISPR Off-target Predictor (CROP) is standalone program developed to address this problem by predicting off-target propensity of guide RNAs and thereby allowing the user to select the optimum guides. The approach used by CROP involves generating substitution, deletion and insertion combinations which are then mapped into the reference genome. Based on these mapped variants, scoring and alignment are conducted and then reported as a table comprising the off-target propensity of all guide RNAs from a given gene sequence. The Python script for this program is freely available from: https://github.com/vaprilyanto/crop .},
}
@article {pmid33452022,
year = {2021},
author = {Cui, Z and Zheng, H and Zhang, J and Jiang, Z and Zhu, Z and Liu, X and Qi, Q and Hou, J},
title = {A CRISPR/Cas9-Mediated, Homology-Independent Tool Developed for Targeted Genome Integration in Yarrowia lipolytica.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {6},
pages = {},
pmid = {33452022},
issn = {1098-5336},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Canthaxanthin/metabolism ; DNA End-Joining Repair ; Gene Editing ; Genome, Fungal ; Yarrowia/*genetics/metabolism ; beta Carotene/metabolism ; },
abstract = {Yarrowia lipolytica has been extensively used to produce essential chemicals and enzymes. As in most other eukaryotes, nonhomologous end joining (NHEJ) is the major repair pathway for DNA double-strand breaks in Y. lipolytica Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica Through optimization of the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with 12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, the cell cycle, and the integration sites. Thus, the targeted integration rate reached 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes in the pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers and achieves one-step targeted genome integration of the 8,417-bp DNA fragment, potentially replacing current HR-dependent genome-editing methods for Y. lipolyticaIMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica This tool does not require the construction of homologous templates and can be used to rapidly verify genetic elements and to iteratively integrate multiple-gene pathways in Y. lipolytica This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.},
}
@article {pmid33451982,
year = {2021},
author = {Lo, YH and Kolahi, KS and Du, Y and Chang, CY and Krokhotin, A and Nair, A and Sobba, WD and Karlsson, K and Jones, SJ and Longacre, TA and Mah, AT and Tercan, B and Sockell, A and Xu, H and Seoane, JA and Chen, J and Shmulevich, I and Weissman, JS and Curtis, C and Califano, A and Fu, H and Crabtree, GR and Kuo, CJ},
title = {A CRISPR/Cas9-Engineered ARID1A-Deficient Human Gastric Cancer Organoid Model Reveals Essential and Nonessential Modes of Oncogenic Transformation.},
journal = {Cancer discovery},
volume = {11},
number = {6},
pages = {1562-1581},
pmid = {33451982},
issn = {2159-8290},
support = {K00 CA212433/CA/NCI NIH HHS/United States ; U54 CA224081/CA/NCI NIH HHS/United States ; U01 CA199241/CA/NCI NIH HHS/United States ; U01 CA217875/CA/NCI NIH HHS/United States ; R00 GM134154/GM/NIGMS NIH HHS/United States ; S10 OD012351/OD/NIH HHS/United States ; K99 GM134154/GM/NIGMS NIH HHS/United States ; R35 CA197745/CA/NCI NIH HHS/United States ; R01 CA163915/CA/NCI NIH HHS/United States ; U01 CA217858/CA/NCI NIH HHS/United States ; U01 CA217851/CA/NCI NIH HHS/United States ; U19 AI116484/AI/NIAID NIH HHS/United States ; S10 OD021764/OD/NIH HHS/United States ; U01 CA217883/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Cell Transformation, Neoplastic ; DNA-Binding Proteins/*genetics ; Humans ; Models, Biological ; Mutation ; Stomach Neoplasms/*genetics ; Transcription Factors/*genetics ; },
abstract = {Mutations in ARID1A rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout (KO) in primary TP53[-/-] human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/β-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein-Barr virus-associated subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFICANCE: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of ARID1A-deficient gastric cancers.See related commentary by Zafra and Dow, p. 1327.This article is highlighted in the In This Issue feature, p. 1307.},
}
@article {pmid33451939,
year = {2020},
author = {Liu, X and Yang, Y and Qiu, Y and Reyad-Ul-Ferdous, M and Ding, Q and Wang, Y},
title = {SeqCor: correct the effect of guide RNA sequences in clustered regularly interspaced short palindromic repeats/Cas9 screening by machine learning algorithm.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {11},
pages = {672-680},
doi = {10.1016/j.jgg.2020.10.007},
pmid = {33451939},
issn = {1673-8527},
mesh = {Algorithms ; Base Sequence/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Library ; Humans ; Machine Learning ; RNA, Guide/*genetics ; *Software ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screening using various guide RNA (gRNA) libraries has been executed to identify functional components for a wide range of phenotypes with regard to numerous cell types and organisms. Using data from public CRISPR/Cas9-based screening experiments, we found that the sequences of gRNAs in the library influence CRISPR/Cas9-based screening. As building a standard strategy for correcting results of all gRNA libraries is impractical, we developed SeqCor, an open-source programming bundle that enables researchers to address the result bias potentially triggered by the composition of gRNA sequences via the organization of gRNA in the library used in CRISPR/Cas9-based screening. Furthermore, SeqCor completely computerizes the extraction of sequence features that may influence single-guide RNA knockout efficiency using a machine learning approach. Taken together, we have developed a software program bundle that ought to be beneficial to the CRISPR/Cas9-based screening platform.},
}
@article {pmid33451103,
year = {2021},
author = {Ghatak, S and Hascall, VC and Markwald, RR and Misra, S},
title = {FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33451103},
issn = {1422-0067},
mesh = {ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Biomarkers ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Self Renewal/genetics ; Colonic Neoplasms/drug therapy/*genetics/*metabolism/pathology ; Drug Resistance, Neoplasm/genetics ; Fluorouracil/therapeutic use ; Gene Expression Profiling ; Gene Knockout Techniques ; Humans ; Hyaluronan Receptors/*genetics/metabolism ; Immunophenotyping ; Leucovorin/therapeutic use ; Neoplastic Stem Cells/*drug effects/*metabolism ; Organoplatinum Compounds/therapeutic use ; Signal Transduction ; Y-Box-Binding Protein 1/*metabolism ; },
abstract = {Cancer initiating cells (CICs) drive tumor formation and drug-resistance, but how they develop drug-resistance characteristics is not well understood. In this study, we demonstrate that chemotherapeutic agent FOLFOX, commonly used for drug-resistant/metastatic colorectal cancer (CRC) treatment, induces overexpression of CD44v6, MDR1, and oncogenic transcription/translation factor Y-box-binding protein-1 (YB-1). Our study revealed that CD44v6, a receptor for hyaluronan, increased the YB-1 expression through PGE2/EP1-mTOR pathway. Deleting CD44v6, and YB-1 by the CRISPR/Cas9 system attenuates the in vitro and in vivo tumor growth of CICs from FOLFOX resistant cells. The results of DNA:CD44v6 immunoprecipitated complexes by ChIP (chromatin-immunoprecipitation) assay showed that CD44v6 maintained the stemness traits by promoting several antiapoptotic and stemness genes, including cyclin-D1, BCL2, FZD1, GINS-1, and MMP9. Further, computer-based analysis of the clones obtained from the DNA:CD44v6 complex revealed the presence of various consensus binding sites for core stemness-associated transcription factors "CTOS" (c-Myc, TWIST1, OCT4, and SOX2). Simultaneous expressions of CD44v6 and CTOS in CD44v6 knockout CICs reverted differentiated CD44v6-knockout CICs into CICs. Finally, this study for the first time describes a positive feedback loop that couples YB-1 induction and CD44 alternative splicing to sustain the MDR1 and CD44v6 expressions, and CD44v6 is required for the reversion of differentiated tumor cells into CICs.},
}
@article {pmid33450609,
year = {2021},
author = {Oh, Y and Kim, H and Kim, SG},
title = {Virus-induced plant genome editing.},
journal = {Current opinion in plant biology},
volume = {60},
number = {},
pages = {101992},
doi = {10.1016/j.pbi.2020.101992},
pmid = {33450609},
issn = {1879-0356},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; *Plant Viruses/genetics ; RNA, Guide ; },
abstract = {Plant viruses have been engineered to express heterologous proteins and RNAs in plants for several decades. This viral system can now be applied to editing plant genomes. Virus vectors can deliver Cas proteins and guide RNAs, two key components of the CRISPR gene-editing system, into a plant cell without a complicated experimental procedure. In some cases, plant viruses move to meristematic cells and express gene-editing components in the cell, which results in the production of mutant seeds. Here, we focus on three main issues of the virus-induced genome editing (VIGE) technology in plants: (1) how to express the relatively large size of Cas proteins, (2) how to express guide RNA, and (3) how to increase the efficiency with which viruses are delivered into meristematic cells. We highlight recent advances in how plant virus vectors can be used efficiently in plant-genome editing.},
}
@article {pmid33450190,
year = {2021},
author = {Hayashi, H and Osaka, S and Sakabe, K and Fukami, A and Kishimoto, E and Aihara, E and Sabu, Y and Mizutani, A and Kusuhara, H and Naritaka, N and Zhang, W and Huppert, SS and Sakabe, M and Nakamura, T and Hu, YC and Mayhew, C and Setchell, K and Takebe, T and Asai, A},
title = {Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.},
journal = {Stem cell reports},
volume = {16},
number = {2},
pages = {309-323},
pmid = {33450190},
issn = {2213-6711},
support = {R01 DK107553/DK/NIDDK NIH HHS/United States ; R01 DK120765/DK/NIDDK NIH HHS/United States ; P30 DK078392/DK/NIDDK NIH HHS/United States ; R01 DK107530/DK/NIDDK NIH HHS/United States ; R01 DK123181/DK/NIDDK NIH HHS/United States ; },
mesh = {ATP Binding Cassette Transporter, Subfamily B, Member 11/*physiology ; Bile Acids and Salts/*metabolism ; Biological Transport ; CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; Hepatocytes/*cytology/*metabolism ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Models, Biological ; Mutation ; },
abstract = {The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.},
}
@article {pmid33450159,
year = {2021},
author = {Zhu, GH and Albishi, NM and Chen, X and Brown, RL and Palli, SR},
title = {Expanding the Toolkit for Genome Editing in a Disease Vector, Aedes aegypti: Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.},
journal = {The CRISPR journal},
volume = {4},
number = {6},
pages = {846-853},
pmid = {33450159},
issn = {2573-1602},
support = {R21 AI131427/AI/NIAID NIH HHS/United States ; R01 GM070559/GM/NIGMS NIH HHS/United States ; },
mesh = {*Aedes/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Disease Vectors ; *Gene Editing/methods ; Mosquito Vectors/genetics/metabolism ; Mutagenesis ; RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and the juvenile hormone receptor, Methoprene-tolerant (Met). PiggyBac transformation vectors were prepared to express sgRNAs targeting kmo and Met under the control of the U6 promoter. Transgenic Ae. aegypti expressing kmo-sgRNA or Met-sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in kmo-sgRNA and Met-sgRNA groups, respectively, showed mutations in their target genes.},
}
@article {pmid33449167,
year = {2021},
author = {Brandes, RP and Dueck, A and Engelhardt, S and Kaulich, M and Kupatt, C and De Angelis, MT and Leisegang, MS and le Noble, F and Moretti, A and Müller, OJ and Skryabin, BV and Thum, T and Wurst, W},
title = {DGK and DZHK position paper on genome editing: basic science applications and future perspective.},
journal = {Basic research in cardiology},
volume = {116},
number = {1},
pages = {2},
pmid = {33449167},
issn = {1435-1803},
mesh = {Animals ; *CRISPR-Cas Systems ; Congresses as Topic ; Diffusion of Innovation ; Disease Models, Animal ; Forecasting ; Gene Editing/*trends ; Genetic Predisposition to Disease ; Genetic Therapy/*trends ; Humans ; Phenotype ; Translational Research, Biomedical/*trends ; },
abstract = {For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.},
}
@article {pmid33449100,
year = {2021},
author = {Pal, M and Herold, MJ},
title = {CRISPR base editing applications for identifying cancer-driving mutations.},
journal = {Biochemical Society transactions},
volume = {49},
number = {1},
pages = {269-280},
pmid = {33449100},
issn = {1470-8752},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Transformation, Neoplastic/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Mutational Analysis/methods ; Gene Editing/*methods ; Genes, Neoplasm ; Humans ; Mutation ; Neoplasms/genetics ; Oncogenes/genetics ; },
abstract = {CRISPR base editing technology is a promising genome editing tool as (i) it does not require a DNA template to introduce mutations and (ii) it avoids creating DNA double-strand breaks, which can lead to unintended chromosomal alterations or elicit an unwanted DNA damage response. Given many cancers originate from point mutations in cancer-driving genes, the application of base editing for either modelling tumour development, therapeutic editing, or functional screening is of great promise. In this review, we summarise current DNA base editing technologies and will discuss recent advancements and existing hurdles for its usage in cancer research.},
}
@article {pmid33448579,
year = {2020},
author = {Zhang, Y and Ling, X and Su, X and Zhang, S and Wang, J and Zhang, P and Feng, W and Zhu, YY and Liu, T and Tang, X},
title = {Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {47},
pages = {20895-20899},
doi = {10.1002/anie.202009890},
pmid = {33448579},
issn = {1521-3773},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics/metabolism ; Gene Editing/*methods ; HEK293 Cells ; Humans ; RNA/chemistry/*genetics/radiation effects ; Ribonucleoproteins/genetics/metabolism ; Ultraviolet Rays ; Vitamin E/analogs &amp; derivatives/radiation effects ; },
abstract = {Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5' terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.},
}
@article {pmid33448189,
year = {2021},
author = {Feng, Y and Liu, S and Chen, R and Xie, A},
title = {Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing.},
journal = {Journal of Zhejiang University. Science. B},
volume = {22},
number = {1},
pages = {73-86},
pmid = {33448189},
issn = {1862-1783},
mesh = {Base Pairing ; Binding Sites/genetics ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA Repair/genetics/*physiology ; Gene Editing/*methods ; Humans ; Models, Biological ; RNA, Guide/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.},
}
@article {pmid33446553,
year = {2021},
author = {Harding, AT and Goff, MA and Froggatt, HM and Lim, JK and Heaton, NS},
title = {GPER1 is required to protect fetal health from maternal inflammation.},
journal = {Science (New York, N.Y.)},
volume = {371},
number = {6526},
pages = {271-276},
pmid = {33446553},
issn = {1095-9203},
support = {R21 AI139593/AI/NIAID NIH HHS/United States ; R01 AI150837/AI/NIAID NIH HHS/United States ; R01 HL142985/HL/NHLBI NIH HHS/United States ; T32 AI007647/AI/NIAID NIH HHS/United States ; R01 AI137031/AI/NIAID NIH HHS/United States ; T32 CA009111/CA/NCI NIH HHS/United States ; R21 AI144844/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Benzodioxoles/pharmacology ; CRISPR-Cas Systems ; Female ; Fetal Diseases/*immunology/virology ; Fetus/immunology/virology ; Humans ; Inflammation/*immunology ; Influenza A virus/immunology ; Influenza, Human/immunology ; Interferon Type I/immunology ; Maternal-Fetal Exchange/*immunology ; Mice ; Mice, Inbred C57BL ; Placenta/immunology/virology ; Pregnancy ; Pregnancy Complications, Infectious/*immunology ; Quinolines/pharmacology ; Receptors, Estrogen/antagonists &amp; inhibitors/*metabolism ; Receptors, G-Protein-Coupled/antagonists &amp; inhibitors/*metabolism ; },
abstract = {Type I interferon (IFN) signaling in fetal tissues causes developmental abnormalities and fetal demise. Although pathogens that infect fetal tissues can induce birth defects through the local production of type I IFN, it remains unknown why systemic IFN generated during maternal infections only rarely causes fetal developmental defects. Here, we report that activation of the guanine nucleotide-binding protein-coupled estrogen receptor 1 (GPER1) during pregnancy is both necessary and sufficient to suppress IFN signaling and does so disproportionately in reproductive and fetal tissues. Inactivation of GPER1 in mice halted fetal development and promoted fetal demise, but only in the context of maternal inflammation. Thus, GPER1 is a central regulator of IFN signaling during pregnancy that allows dynamic antiviral responses in maternal tissues while also preserving fetal health.},
}
@article {pmid33446509,
year = {2021},
author = {Hernandez, VA and Carvajal-Moreno, J and Papa, JL and Shkolnikov, N and Li, J and Ozer, HG and Yalowich, JC and Elton, TS},
title = {CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells.},
journal = {Molecular pharmacology},
volume = {99},
number = {3},
pages = {226-241},
pmid = {33446509},
issn = {1521-0111},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; R01 CA226906/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Survival ; DNA Topoisomerases, Type II/*genetics ; *Down-Regulation ; Drug Resistance, Neoplasm ; Etoposide/*pharmacology ; Gene Editing/*methods ; Humans ; Introns ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy/enzymology/*genetics ; Poly-ADP-Ribose Binding Proteins/*genetics ; RNA Splice Sites ; },
abstract = {An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.},
}
@article {pmid33446040,
year = {2021},
author = {Lek, A and Ma, K and Woodman, KG and Lek, M},
title = {Nuclease-Deficient Clustered Regularly Interspaced Short Palindromic Repeat-Based Approaches for In Vitro and In Vivo Gene Activation.},
journal = {Human gene therapy},
volume = {32},
number = {5-6},
pages = {260-274},
doi = {10.1089/hum.2020.241},
pmid = {33446040},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases ; *Gene Editing ; Transcriptional Activation ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been adapted to achieve a wide range of genome modifications, including transcription regulation. The focus of this review is on the application of CRISPR-based platforms such as nuclease-deficient Cas9 and Cas12a, to achieve targeted gene activation. We review studies to date that have used CRISPR-based activation technology for the elucidation of biological mechanism and disease correction, as well as its application in genetic screens as a powerful tool for high-throughput genotype-phenotype mapping. In addition to our synthesis and critical analysis of published studies, we explore key considerations for the potential clinical translation of CRISPR-based activation technology.},
}
@article {pmid33445555,
year = {2021},
author = {Salava, H and Thula, S and Mohan, V and Kumar, R and Maghuly, F},
title = {Application of Genome Editing in Tomato Breeding: Mechanisms, Advances, and Prospects.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33445555},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Epigenomics/methods ; *Gene Editing ; Gene Knockdown Techniques ; *Genome, Plant ; *Genomics/methods ; Lycopersicon esculentum/*genetics ; Mutagenesis ; Oxidative Stress ; *Plant Breeding ; Plants, Genetically Modified ; },
abstract = {Plants regularly face the changing climatic conditions that cause biotic and abiotic stress responses. The abiotic stresses are the primary constraints affecting crop yield and nutritional quality in many crop plants. The advances in genome sequencing and high-throughput approaches have enabled the researchers to use genome editing tools for the functional characterization of many genes useful for crop improvement. The present review focuses on the genome editing tools for improving many traits such as disease resistance, abiotic stress tolerance, yield, quality, and nutritional aspects of tomato. Many candidate genes conferring tolerance to abiotic stresses such as heat, cold, drought, and salinity stress have been successfully manipulated by gene modification and editing techniques such as RNA interference, insertional mutagenesis, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9). In this regard, the genome editing tools such as CRISPR/Cas9, which is a fast and efficient technology that can be exploited to explore the genetic resources for the improvement of tomato and other crop plants in terms of stress tolerance and nutritional quality. The review presents examples of gene editing responsible for conferring both biotic and abiotic stresses in tomato simultaneously. The literature on using this powerful technology to improve fruit quality, yield, and nutritional aspects in tomato is highlighted. Finally, the prospects and challenges of genome editing, public and political acceptance in tomato are discussed.},
}
@article {pmid33445135,
year = {2021},
author = {Zuo, F and Marcotte, H},
title = {Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing.},
journal = {Current opinion in biotechnology},
volume = {70},
number = {},
pages = {75-82},
doi = {10.1016/j.copbio.2020.12.015},
pmid = {33445135},
issn = {1879-0429},
mesh = {Bifidobacterium ; Bioengineering ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Lactobacillus/genetics ; *Probiotics ; },
abstract = {Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.},
}
@article {pmid33445042,
year = {2021},
author = {Awasthi, P and Kocábek, T and Mishra, AK and Nath, VS and Shrestha, A and Matoušek, J},
title = {Establishment of CRISPR/Cas9 mediated targeted mutagenesis in hop (Humulus lupulus).},
journal = {Plant physiology and biochemistry : PPB},
volume = {160},
number = {},
pages = {1-7},
doi = {10.1016/j.plaphy.2021.01.006},
pmid = {33445042},
issn = {1873-2690},
mesh = {Agrobacterium tumefaciens ; *CRISPR-Cas Systems ; Chlorophyll ; Chlorophyll A ; Gene Editing ; Genome, Plant/genetics ; Humulus/enzymology/*genetics ; Mutagenesis ; Oxidoreductases/*genetics ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9-based targeted genome editing has emerged as a versatile technique, widely employed in plant genome engineering, both to decipher gene function and as an alternative to classical breeding technique for traits improvement in plants. However, to date, no such platform has been developed for hop (Humulus lupulus L.), which is an economically important crop producing valuable secondary metabolites utilized in the brewing and pharmaceutical industries. Here, we present the first report on the successful establishment of efficient CRISPR/Cas9-based genome editing using the visible endogenous marker gene phytoene desaturase (PDS) involved in carotenoid biosynthesis to demonstrate successful genome editing in hop. Agrobacterium tumefaciens-mediated transformation of in vitro generated internodal explants was used for the stable integration of constructs expressing plant codon-optimized Cas9 and a pair of co-expressed guide RNAs to target the distinct genomic sites of the PDS gene of hop. Analysis of RNA-guided genome-editing events, including mutant lines screening and homozygosity assessment using the T7 endonuclease assay showed that 33.3% of transformed plants were successfully edited at the target site, displaying albino and mosaic regenerants. Intriguingly, the detected mutations were ranges of deletions (16 bp to 39 bp) which led to disruption of the exon-intron boundary, few base substitutions, and a 1 bp insertion at 3 bp upstream of the PAM region of the target site. The decrease in chlorophyll a/b, and carotenoid content in the mutant lines further confirmed the functional disruption of the HlPDS gene. Taken together, our results demonstrate that the CRISPR/Cas9 system can precisely edit the targeted genome sequences, which may revolutionize our way to overcome some of the obstacles that have plagued the traits improvement in hop.},
}
@article {pmid33444739,
year = {2021},
author = {Long, Y and Cech, TR},
title = {Targeted mutagenesis in human iPSCs using CRISPR genome-editing tools.},
journal = {Methods (San Diego, Calif.)},
volume = {191},
number = {},
pages = {44-58},
pmid = {33444739},
issn = {1095-9130},
support = {K99 GM132546/GM/NIGMS NIH HHS/United States ; S10 OD021601/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells ; Mutagenesis ; },
abstract = {Mutagenesis studies have rapidly evolved in the era of CRISPR genome editing. Precise manipulation of genes in human induced pluripotent stem cells (iPSCs) allows biomedical researchers to study the physiological functions of individual genes during development. Furthermore, such genetic manipulation applied to patient-specific iPSCs allows disease modeling, drug screening and development of therapeutics. Although various genome-editing methods have been developed to introduce or remove mutations in human iPSCs, comprehensive strategic designs taking account of the potential side effects of CRISPR editing are needed. Here we present several novel and highly efficient strategies to introduce point mutations, insertions and deletions in human iPSCs, including step-by-step experimental protocols. These approaches involve the application of drug selection for effortless clone screening and the generation of a wild type control strain along with the mutant. We also present several examples of application of these strategies in human iPSCs and show that they are highly efficient and could be applied to other cell types.},
}
@article {pmid33444684,
year = {2021},
author = {Hosoi, H and Niibori-Nambu, A and Nah, GSS and Bahirvani, AG and Mok, MMH and Sanda, T and Kumar, AP and Tenen, DG and Ito, Y and Sonoki, T and Osato, M},
title = {Super-enhancers for RUNX3 are required for cell proliferation in EBV-infected B cell lines.},
journal = {Gene},
volume = {774},
number = {},
pages = {145421},
pmid = {33444684},
issn = {1879-0038},
support = {P01 HL131477/HL/NHLBI NIH HHS/United States ; R35 CA197697/CA/NCI NIH HHS/United States ; },
mesh = {Azepines/pharmacology ; B-Lymphocytes/cytology/*virology ; Burkitt Lymphoma/genetics/virology ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Cell Proliferation/drug effects/*genetics ; Core Binding Factor Alpha 3 Subunit/*genetics ; *Enhancer Elements, Genetic ; G1 Phase/drug effects ; Gene Expression Regulation/drug effects ; Genes, myc ; Herpesvirus 4, Human/*physiology ; Humans ; Protein Domains ; Proteins/antagonists &amp; inhibitors/metabolism ; Triazoles/pharmacology ; },
abstract = {Epstein-Barr virus nuclear antigens 2 (EBNA2) mediated super-enhancers, defined by in silico data, localize near genes associated with B cell transcription factors including RUNX3. However, the biological function of super-enhancer for RUNX3 gene (seR3) remains unclear. Here, we show that two seR3s, tandemly-located at 59- and 70-kb upstream of RUNX3 transcription start site, named seR3 -59h and seR3 -70h, are required for RUNX3 expression and cell proliferation in Epstein-Barr virus (EBV)-positive malignant B cells. A BET bromodomain inhibitor, JQ1, potently suppressed EBV-positive B cell growth through the reduction of RUNX3 and MYC expression. Excision of either or both seR3s by employing CRISPR/Cas9 system resulted in the decrease in RUNX3 expression and the subsequent suppression of cell proliferation and colony forming capability. The expression of MYC was also reduced when seR3s were deleted, probably due to the loss of trans effect of seR3s on the super-enhancers for MYC. These findings suggest that seR3s play a pivotal role in expression and biological function of both RUNX3 and MYC. seR3s would serve as a potential therapeutic target in EBV-related widespread tumors.},
}
@article {pmid33444542,
year = {2021},
author = {Li, Y and Bondy-Denomy, J},
title = {Anti-CRISPRs go viral: The infection biology of CRISPR-Cas inhibitors.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {5},
pages = {704-714},
pmid = {33444542},
issn = {1934-6069},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*genetics/immunology/*virology ; Bacteriophages/genetics/*immunology ; *CRISPR-Cas Systems ; Host-Pathogen Interactions ; Viral Proteins/genetics/*immunology ; },
abstract = {Bacteriophages encode diverse anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas immunity during infection of their bacterial hosts. Although detailed mechanisms have been characterized for multiple Acr proteins, an understanding of their role in phage infection biology is just emerging. Here, we review recent work in this area and propose a framework of "phage autonomy" to evaluate CRISPR-immune evasion strategies. During phage infection, Acr proteins are deployed by a tightly regulated "fast on-fast off" transcriptional burst, which is necessary, but insufficient, for CRISPR-Cas inactivation. Instead of a single phage shutting down CRISPR-Cas immunity, a community of acr-carrying phages cooperate to suppress bacterial immunity, displaying low phage autonomy. Enzymatic Acr proteins with novel mechanisms have been recently revealed and are predicted to enhance phage autonomy, while phage DNA protective measures offer the highest phage autonomy observed. These varied Acr mechanisms and strengths also have unexpected impacts on the bacterial populations and competing phages.},
}
@article {pmid33444446,
year = {2021},
author = {Roliński, M and Montaldo, NP and Aksu, ME and Fordyce Martin, SL and Brambilla, A and Kunath, N and Johansen, J and Erlandsen, SE and Liabbak, NB and Rian, K and Bjørås, M and Sætrom, P and van Loon, B},
title = {Loss of Mediator complex subunit 13 (MED13) promotes resistance to alkylation through cyclin D1 upregulation.},
journal = {Nucleic acids research},
volume = {49},
number = {3},
pages = {1470-1484},
pmid = {33444446},
issn = {1362-4962},
mesh = {Antineoplastic Agents, Alkylating/*pharmacology ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Cyclin D1/*genetics/metabolism ; Cyclin-Dependent Kinase 8/antagonists &amp; inhibitors ; Cyclin-Dependent Kinases/antagonists &amp; inhibitors ; DNA Damage ; Drug Resistance, Neoplasm ; Gene Expression Regulation ; Humans ; Mediator Complex/metabolism/*physiology ; Up-Regulation ; },
abstract = {Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.},
}
@article {pmid33444382,
year = {2021},
author = {Chon, C and Chon, G and Matsui, Y and Zeng, H and Lai, ZC and Liu, A},
title = {Efficient multiplexed genome engineering with a polycistronic tRNA and CRISPR guide-RNA reveals an important role of detonator in reproduction of Drosophila melanogaster.},
journal = {PloS one},
volume = {16},
number = {1},
pages = {e0245454},
pmid = {33444382},
issn = {1932-6203},
support = {R03 HD101765/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/genetics/physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila Proteins/genetics ; Drosophila melanogaster/*genetics/physiology ; Female ; Fertility ; *Gene Editing/methods ; Loss of Function Mutation ; Male ; RNA, Guide/*genetics ; RNA, Transfer/*genetics ; Reproduction ; },
abstract = {Genome association studies in human and genetic studies in mouse implicated members of the transmembrane protein 132 (TMEM132) family in multiple conditions including panic disorder, hearing loss, limb and kidney malformation. However, the presence of five TMEM132 paralogs in mammalian genomes makes it extremely challenging to reveal the full requirement for these proteins in vivo. In contrast, there is only one TMEM132 homolog, detonator (dtn), in the genome of fruit fly Drosophila melanogaster, enabling straightforward research into its in vivo function. In the current study, we generate multiple loss-of-function dtn mutant fly strains through a polycistronic tRNA-gRNA approach, and show that most embryos lacking both maternal and paternal dtn fail to hatch into larvae, indicating an essential role of dtn in Drosophila reproduction.},
}
@article {pmid33444322,
year = {2021},
author = {Allen, SE and Koreman, GT and Sarkar, A and Wang, B and Wolfner, MF and Han, C},
title = {Versatile CRISPR/Cas9-mediated mosaic analysis by gRNA-induced crossing-over for unmodified genomes.},
journal = {PLoS biology},
volume = {19},
number = {1},
pages = {e3001061},
pmid = {33444322},
issn = {1545-7885},
support = {R21 OD023824/OD/NIH HHS/United States ; R01 NS099125/NS/NINDS NIH HHS/United States ; R37 HD038921/HD/NICHD NIH HHS/United States ; T32 GM007273/GM/NIGMS NIH HHS/United States ; R01 HD038921/HD/NICHD NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; R03 HD101732/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular/methods ; Crossing Over, Genetic/*genetics ; Drosophila melanogaster/embryology/genetics ; Embryo, Nonmammalian ; Female ; Gene Editing/methods ; Gene Targeting/methods ; Genetic Vectors ; Genome, Insect ; Male ; Mosaicism/*embryology ; Phenotype ; RNA, Guide/*physiology ; },
abstract = {Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for 1 chromosome arm, we demonstrate the method's application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites into the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can in principle be applied in any organism that is compatible with CRISPR/Cas9.},
}
@article {pmid33443621,
year = {2021},
author = {Ren, J and Meng, X and Hu, F and Liu, Q and Cao, Y and Li, H and Yan, C and Li, J and Wang, K and Yu, H and Wang, C},
title = {Expanding the scope of genome editing with SpG and SpRY variants in rice.},
journal = {Science China. Life sciences},
volume = {64},
number = {10},
pages = {1784-1787},
pmid = {33443621},
issn = {1869-1889},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting ; Genome, Plant/*genetics ; Mutation ; Oryza/*genetics ; Plants, Genetically Modified ; Streptococcus pyogenes/enzymology/genetics ; Substrate Specificity ; },
}
@article {pmid33443212,
year = {2021},
author = {Rodrigues, SD and Karimi, M and Impens, L and Van Lerberge, E and Coussens, G and Aesaert, S and Rombaut, D and Holtappels, D and Ibrahim, HMM and Van Montagu, M and Wagemans, J and Jacobs, TB and De Coninck, B and Pauwels, L},
title = {Efficient CRISPR-mediated base editing in Agrobacterium spp.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {2},
pages = {},
pmid = {33443212},
issn = {1091-6490},
mesh = {Agrobacterium/*genetics ; Agrobacterium tumefaciens/genetics ; CRISPR-Associated Proteins/*genetics/metabolism ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Plant/genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; Genome, Plant/genetics ; Mutagenesis/genetics ; Mutation/genetics ; Zea mays/genetics ; },
abstract = {Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.},
}
@article {pmid33443210,
year = {2021},
author = {Bosch, JA and Birchak, G and Perrimon, N},
title = {Precise genome engineering in Drosophila using prime editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {1},
pages = {},
pmid = {33443210},
issn = {1091-6490},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; R24 OD030002/OD/NIH HHS/United States ; T32 GM007748/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {ATP-Binding Cassette Transporters/genetics ; Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Codon, Terminator ; Crosses, Genetic ; DNA-Binding Proteins/genetics ; Drosophila Proteins/genetics ; Drosophila melanogaster/*genetics ; Eye Proteins/genetics ; Female ; Gene Editing/*methods ; Gene Targeting/*methods ; Genome ; Germ Cells ; Male ; Microfilament Proteins/genetics ; Recombination, Genetic ; },
abstract = {Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.},
}
@article {pmid33443205,
year = {2021},
author = {Stolle, AS and Meader, BT and Toska, J and Mekalanos, JJ},
title = {Endogenous membrane stress induces T6SS activity in Pseudomonas aeruginosa.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {1},
pages = {},
pmid = {33443205},
issn = {1091-6490},
support = {R01 AI026289/AI/NIAID NIH HHS/United States ; R37 AI018045/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Outer Membrane Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Membrane/genetics/*metabolism/pathology ; Cell Survival/genetics ; Gene Knockdown Techniques ; Gene Silencing ; Genes, Essential/genetics/*physiology ; Genotype ; Periplasmic Proteins/genetics/*metabolism ; Phenotype ; Pseudomonas aeruginosa/cytology/*genetics/growth &amp; development/metabolism ; RNA-Seq ; Signal Transduction/genetics ; Stress, Physiological ; Type VI Secretion Systems/*metabolism ; Vibrio cholerae/genetics/growth &amp; development ; },
abstract = {The type 6 secretion system (T6SS) is a dynamic organelle encoded by many gram-negative bacteria that can be used to kill competing bacterial prey species in densely occupied niches. Some predatory species, such as Vibrio cholerae, use their T6SS in an untargeted fashion while in contrast, Pseudomonas aeruginosa assembles and fires its T6SS apparatus only after detecting initial attacks by other bacterial prey cells; this targeted attack strategy has been termed the T6SS tit-for-tat response. Molecules that interact with the P. aeruginosa outer membrane such as polymyxin B can also trigger assembly of T6SS organelles via a signal transduction pathway that involves protein phosphorylation. Recent work suggests that a phospholipase T6SS effector (TseL) of V. cholerae can induce T6SS dynamic activity in P. aeruginosa when delivered to or expressed in the periplasmic space of this organism. Here, we report that inhibiting expression of essential genes involved in outer membrane biogenesis can also trigger T6SS activation in P. aeruginosa Specifically, we developed a CRISPR interference (CRISPRi) system to knock down expression of bamA, tolB, and lptD and found that these knockdowns activated T6SS activity. This increase in T6SS activity was dependent on the same signal transduction pathway that was previously shown to be required for the tit-for-tat response. We conclude that outer membrane perturbation can be sensed by P. aeruginosa to activate the T6SS even when the disruption is generated by aberrant cell envelope biogenesis.},
}
@article {pmid33443184,
year = {2021},
author = {Wang, Y and Mallon, J and Wang, H and Singh, D and Hyun Jo, M and Hua, B and Bailey, S and Ha, T},
title = {Real-time observation of Cas9 postcatalytic domain motions.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {2},
pages = {},
pmid = {33443184},
issn = {1091-6490},
support = {R01 GM097330/GM/NIGMS NIH HHS/United States ; R35 GM122569/GM/NIGMS NIH HHS/United States ; T32 GM080189/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism/physiology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/metabolism ; DNA Cleavage ; Fluorescence Resonance Energy Transfer/methods ; Gene Editing/*methods ; Molecular Conformation ; Molecular Dynamics Simulation ; Protein Domains/genetics ; RNA, Guide/metabolism ; Single Molecule Imaging/*methods ; },
abstract = {CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3' flap generated by NTS cleavage can be rapidly digested by a 3' to 5' single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3' flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9's conformation and suggest possible biotechnological applications.},
}
@article {pmid33443157,
year = {2021},
author = {Kurtz, S and Lucas-Hahn, A and Schlegelberger, B and Göhring, G and Niemann, H and Mettenleiter, TC and Petersen, B},
title = {Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {2},
pages = {},
pmid = {33443157},
issn = {1091-6490},
mesh = {Amino Acid Sequence/genetics ; Animals ; DNA-Binding Proteins/genetics ; Disorders of Sex Development/genetics ; Frameshift Mutation/genetics ; Genes, sry/genetics ; HMG-Box Domains/genetics ; Male ; Mutation/genetics ; Nuclear Proteins/genetics ; Proof of Concept Study ; Protein Domains/genetics ; Sex Determination Processes/*genetics ; Sex-Determining Region Y Protein/*genetics/*metabolism ; Swine/genetics ; Transcription Factors/genetics ; Y Chromosome/genetics ; },
abstract = {The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.},
}
@article {pmid33443122,
year = {2020},
author = {Ahmed, UKB and Shadid, TM and Larabee, JL and Ballard, JD},
title = {Combined and Distinct Roles of Agr Proteins in Clostridioides difficile 630 Sporulation, Motility, and Toxin Production.},
journal = {mBio},
volume = {11},
number = {6},
pages = {},
pmid = {33443122},
issn = {2150-7511},
support = {R01 AI119048/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; Bacterial Toxins/biosynthesis/*genetics ; CRISPR-Cas Systems ; Clostridioides difficile/*genetics/*physiology ; *Gene Expression Regulation, Bacterial ; Movement ; Mutation ; Phenotype ; Phylogeny ; Signal Transduction ; Spores, Bacterial/genetics/*growth &amp; development ; },
abstract = {The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action.IMPORTANCEC. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr's mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.},
}
@article {pmid33442060,
year = {2021},
author = {Chelmicki, T and Roger, E and Teissandier, A and Dura, M and Bonneville, L and Rucli, S and Dossin, F and Fouassier, C and Lameiras, S and Bourc'his, D},
title = {m[6]A RNA methylation regulates the fate of endogenous retroviruses.},
journal = {Nature},
volume = {591},
number = {7849},
pages = {312-316},
pmid = {33442060},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/metabolism ; Chromatin/chemistry/genetics/metabolism ; Endogenous Retroviruses/*genetics ; Gene Knockout Techniques ; Genes, Intracisternal A-Particle/*genetics ; Half-Life ; *Methylation ; Methyltransferases/metabolism ; Mice ; Mouse Embryonic Stem Cells ; Nuclear Proteins/metabolism ; RNA Splicing Factors/metabolism ; RNA Stability ; RNA, Messenger/chemistry/*genetics/*metabolism ; RNA-Binding Proteins/metabolism ; },
abstract = {Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that affect genome regulation and cell physiology throughout their RNA-centred life cycle[1]. Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases[2,3]. Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m[6]A RNA methylation as a way to restrict ERVs. Methylation of ERV mRNAs is catalysed by the complex of methyltransferase-like METTL3-METTL14[4] proteins, and we found that depletion of METTL3-METTL14, along with their accessory subunits WTAP and ZC3H13, led to increased mRNA abundance of intracisternal A-particles (IAPs) and related ERVK elements specifically, by targeting their 5' untranslated region. Using controlled auxin-dependent degradation of the METTL3-METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m[6]A catalysis. By monitoring chromatin states and mRNA stability upon METTL3-METTL14 double depletion, we found that m[6]A methylation mainly acts by reducing the half-life of IAP mRNA, and this occurs by the recruitment of the YTHDF family of m[6]A reader proteins[5]. Together, our results indicate that RNA methylation provides a protective effect in maintaining cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent.},
}
@article {pmid33441979,
year = {2021},
author = {Mózsik, L and Hoekzema, M and de Kok, NAW and Bovenberg, RAL and Nygård, Y and Driessen, AJM},
title = {CRISPR-based transcriptional activation tool for silent genes in filamentous fungi.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1118},
pmid = {33441979},
issn = {2045-2322},
mesh = {Anti-Infective Agents/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fungi/*genetics/metabolism ; Gene Expression Regulation, Fungal ; *Genes, Fungal ; Genes, Reporter ; Multigene Family ; Penicillium/*genetics/metabolism ; Promoter Regions, Genetic ; Sesquiterpenes/metabolism ; *Transcriptional Activation ; },
abstract = {Filamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA "plug-and-play" module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.},
}
@article {pmid33441796,
year = {2021},
author = {Vieira, AA and Vianna, GR and Carrijo, J and Aragão, FJL and Vieira, PM},
title = {Generation of Trichoderma harzianum with pyr4 auxotrophic marker by using the CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1085},
pmid = {33441796},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genes, Fungal ; Hypocreales/*genetics ; },
abstract = {Trichoderma harzianum is a filamentous fungus used as a biological control agent for agricultural pests. Genes of this microorganism have been studied, and their applications are patented for use in biofungicides and plant breeding strategies. Gene editing technologies would be of great importance for genetic characterization of this species, but have not yet been reported. This work describes mutants obtained with an auxotrophic marker in this species using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas (CRISPR-associated) system. For this, sequences for a guide RNA and Cas9 overexpression were inserted via biolistics, and the sequencing approach confirmed deletions and insertions at the pyr4 gene. Phenotypic characterization demonstrated a reduction in the growth of mutants in the absence of uridine, as well as resistance to 5-fluorotic acid. In addition, the gene disruption did not reduce mycoparasitc activity against phytopathogens. Thus, target disruption of the pyr4 gene in T. harzianum using the CRISPR/Cas9 system was demonstrated, and it was also shown that endogenous expression of the system did not interfere with the biological control activity of pathogens. This work is the first report of CRISPR Cas9-based editing in this biocontrol species, and the mutants expressing Cas9 have potential for the generation of useful technologies in agricultural biotechnology.},
}
@article {pmid33441761,
year = {2021},
author = {Heintze, T and Klein, K and Hofmann, U and Zanger, UM},
title = {Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1000},
pmid = {33441761},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Cytochrome P-450 CYP1A2/genetics ; Cytochrome P-450 Enzyme System/*genetics ; Cytochrome-B(5) Reductase/*genetics ; Cytochromes b5/*genetics ; Down-Regulation/genetics ; Gene Knockout Techniques/methods ; HEK293 Cells ; Hepatocytes/metabolism ; Humans ; Metabolic Clearance Rate/genetics ; NADPH-Ferrihemoprotein Reductase/genetics ; },
abstract = {HepaRG cells are increasingly accepted as model for human drug metabolism and other hepatic functions. We used lentiviral transduction of undifferentiated HepaRG cells to deliver Cas9 and two alternative sgRNAs targeted at NADPH:cytochrome P450 oxidoreductase (POR), the obligate electron donor for microsomal cytochromes P450 (CYP). Cas9-expressing HepaRG[VC] (vector control) cells were phenotypically similar to wild type HepaRG cells and could be differentiated into hepatocyte-like cells by DMSO. Genetic POR-knockout resulted in phenotypic POR knockdown of up to 90% at mRNA, protein, and activity levels. LC-MS/MS measurement of seven CYP-activities showed differential effects of POR-knockdown with CYP2C8 being least and CYP2C9 being most affected. Further studies on cytochrome b5 (CYB5), an alternative NADH-dependent electron donor indicated particularly strong support of CYP2C8-dependent amodiaquine N-deethylation by CYB5 and this was confirmed by genetic CYB5 single- and POR/CYB5 double-knockout. POR-knockdown also affected CYP expression on mRNA and protein level, with CYP1A2 being induced severalfold, while CYP2C9 was strongly downregulated. In summary our results show that POR/NADPH- and CYB5/NADH-electron transport systems influence human drug metabolizing CYPs differentially and differently than mouse Cyps. Our Cas9-expressing HepaRG[VC] cells should be suitable to study the influence of diverse genes on drug metabolism and other hepatic functions.},
}
@article {pmid33441553,
year = {2021},
author = {Goldberg, GW and Spencer, JM and Giganti, DO and Camellato, BR and Agmon, N and Ichikawa, DM and Boeke, JD and Noyes, MB},
title = {Engineered dual selection for directed evolution of SpCas9 PAM specificity.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {349},
pmid = {33441553},
issn = {2041-1723},
support = {F32 GM137482/GM/NIGMS NIH HHS/United States ; R01 GM118851/GM/NIGMS NIH HHS/United States ; R01 GM133936/GM/NIGMS NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; DNA/chemistry/genetics/*metabolism ; Directed Molecular Evolution/methods ; Gene Editing/*methods ; Humans ; Mutation ; Nucleic Acid Conformation ; Nucleotide Motifs/genetics ; Protein Engineering/methods ; RNA, Guide/genetics/*metabolism ; Streptococcus pyogenes/genetics/*metabolism ; Substrate Specificity ; },
abstract = {The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.},
}
@article {pmid33441535,
year = {2021},
author = {Song, Y and Du, T and Ray, A and Chauhan, K and Samur, M and Munshi, N and Chauhan, D and Anderson, KC},
title = {Identification of novel anti-tumor therapeutic target via proteomic characterization of ubiquitin receptor ADRM1/Rpn13.},
journal = {Blood cancer journal},
volume = {11},
number = {1},
pages = {13},
pmid = {33441535},
issn = {2044-5385},
support = {P01 CA155258/CA/NCI NIH HHS/United States ; R01 CA050947/CA/NCI NIH HHS/United States ; R01 CA207237/CA/NCI NIH HHS/United States ; P50 CA100707/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; HCT116 Cells ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Multiple Myeloma/genetics/metabolism ; Neoplasms/genetics/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; Proteolysis ; Proteomics ; Ubiquitin/metabolism ; },
}
@article {pmid33441159,
year = {2021},
author = {Scott, TA and Morris, KV},
title = {Designer nucleases to treat malignant cancers driven by viral oncogenes.},
journal = {Virology journal},
volume = {18},
number = {1},
pages = {18},
pmid = {33441159},
issn = {1743-422X},
support = {R01 113407-01/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Carcinogenesis/*drug effects ; Cell Line ; Cell Transformation, Neoplastic/drug effects/genetics ; Cell Transformation, Viral/drug effects/genetics ; Humans ; Mice ; Neoplasms/*drug therapy/*virology ; Oncogenes ; Oncogenic Viruses/*genetics/pathogenicity ; },
abstract = {Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.},
}
@article {pmid33440775,
year = {2021},
author = {Zhang, X and Zhou, W and Zhang, P and Gao, F and Zhao, X and Shum, WW and Zeng, X},
title = {Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33440775},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics/*metabolism ; Cell Line ; Epididymis/*cytology ; Gene Expression Profiling ; Male ; Mice ; Mice, Knockout ; Sperm Tail/*metabolism ; *Spermatogenesis/genetics ; Spermatozoa/cytology/*metabolism ; Transcriptome ; },
abstract = {The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece-principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1[-/-] mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.},
}
@article {pmid33440180,
year = {2021},
author = {Zhang, G and Li, X and Sun, Y and Wang, X and Liu, G and Huang, Y},
title = {A Genetic Screen Identifies Etl4-Deficiency Capable of Stabilizing the Haploidy in Embryonic Stem Cells.},
journal = {Stem cell reports},
volume = {16},
number = {1},
pages = {29-38},
pmid = {33440180},
issn = {2213-6711},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Energy Metabolism ; Gene Editing ; Glycolysis ; Haploidy ; Mice ; Mitochondria/metabolism ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Oxidative Phosphorylation ; Proteins/*genetics/metabolism ; Transcriptome ; },
abstract = {Mammalian haploid embryonic stem cells (haESCs) hold great promise for functional genetic studies and forward screening. However, all established haploid cells are prone to spontaneous diploidization during long-term culture, rendering application challenging. Here, we report a genome-wide loss-of-function screening that identified gene mutations that could significantly reduce the rate of self-diploidization in haESCs. We further demonstrated that CRISPR/Cas9-mediated Etl4 knockout (KO) stabilizes the haploid state in different haESC lines. More interestingly, Etl4 deficiency increases mitochondrial oxidative phosphorylation (OXPHOS) capacity and decreases glycolysis in haESCs. Mimicking this effect by regulating the energy metabolism with drugs decreased the rate of self-diploidization. Collectively, our study identified Etl4 as a novel haploidy-related factor linked to an energy metabolism transition occurring during self-diploidization of haESCs.},
}
@article {pmid33440058,
year = {2021},
author = {Zhou, J and Yuan, M and Zhao, Y and Quan, Q and Yu, D and Yang, H and Tang, X and Xin, X and Cai, G and Qian, Q and Qi, Y and Zhang, Y},
title = {Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice.},
journal = {Plant biotechnology journal},
volume = {19},
number = {6},
pages = {1240-1252},
pmid = {33440058},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Chlorophyll A ; Gene Editing ; *Oryza/genetics ; Plants, Genetically Modified/genetics ; RNA, Circular ; },
abstract = {CRISPR-Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non-coding RNAs in plants is still at its infancy. Despite being a major class of non-coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR-Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR-Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs' participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA-miRNA-mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops.},
}
@article {pmid33438302,
year = {2021},
author = {Yadav, SK and Mishra, PK},
title = {Intracellular matrix metalloproteinase-9 mediates epigenetic modifications and autophagy to regulate differentiation in human cardiac stem cells.},
journal = {Stem cells (Dayton, Ohio)},
volume = {39},
number = {4},
pages = {497-506},
pmid = {33438302},
issn = {1549-4918},
support = {R01 HL113281/HL/NHLBI NIH HHS/United States ; U54 GM115458/GM/NIGMS NIH HHS/United States ; },
mesh = {Autophagosomes/drug effects/metabolism ; Autophagy/drug effects/*genetics ; Azacitidine/pharmacology ; CRISPR-Cas Systems ; Cell Differentiation/drug effects ; DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; DNA Methylation/drug effects ; DNA Methyltransferase 3A/genetics/metabolism ; *Epigenesis, Genetic ; Gene Deletion ; Homeodomain Proteins/genetics/metabolism ; Humans ; MEF2 Transcription Factors/genetics/metabolism ; Macrolides/pharmacology ; Matrix Metalloproteinase 9/deficiency/*genetics ; Myocytes, Cardiac/cytology/drug effects/*metabolism ; Paracrine Communication/drug effects ; Signal Transduction ; Sirtuin 1/genetics/metabolism ; Stem Cells/cytology/drug effects/*metabolism ; },
abstract = {Epigenetic reprogramming and autophagy have critical roles in differentiation of stem cells. However, very little is known about how epigenetic modifications are mediated and how they contribute to autophagy and differentiation in human cardiac stem cells (hCSCs). Previously, we have reported that intracellular matrix metalloproteinase-9 (MMP9), a collagenase, mediates cell death in hCSCs. Here, we investigated whether intracellular MMP9 mediates epigenetic modifications and autophagy in hCSCs. We created MMP9KO hCSCs and treated them with 5-azacytidine, an inhibitor of DNA methylation, and bafilomycin A1, an inhibitor of autophagosome degradation, and evaluated epigenetic modifications, autophagic flux, and differentiation. Our results showed compromised epigenetic modifications, reduced autophagy, and impaired differentiation in MMP9KO hCSCs. Remarkably, paracrine MMP9 supplementation restored epigenetic modifications but further reduced autophagy in MMP9KO hCSCs. We conclude that intracellular MMP9 is a critical mediator of epigenetic modifications and autophagy in hCSCs. Furthermore, the endocrine and paracrine effects of MMP9 vary for regulating autophagy in hCSCs. These novel roles of MMP9 are valuable for stem cell therapy.},
}
@article {pmid33436886,
year = {2021},
author = {Rozen-Gagnon, K and Yi, S and Jacobson, E and Novack, S and Rice, CM},
title = {A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {736},
pmid = {33436886},
issn = {2045-2322},
support = {R01 AI116943/AI/NIAID NIH HHS/United States ; F32 AI120579/AI/NIAID NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; },
mesh = {Aedes/*genetics ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Eukaryotic Initiation Factors/antagonists &amp; inhibitors/*genetics ; Gene Editing/*methods ; Genome, Insect ; Mosquito Vectors/*genetics ; Plasmids/*genetics ; },
abstract = {Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.},
}
@article {pmid33434526,
year = {2021},
author = {Wu, H and Voeltz, GK},
title = {Reticulon-3 Promotes Endosome Maturation at ER Membrane Contact Sites.},
journal = {Developmental cell},
volume = {56},
number = {1},
pages = {52-66.e7},
pmid = {33434526},
issn = {1878-1551},
support = {R01 GM120998/GM/NIGMS NIH HHS/United States ; T32 GM008759/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Motifs ; Autophagosomes/genetics/metabolism ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*metabolism ; Cell Line, Tumor ; Endoplasmic Reticulum/genetics/*metabolism ; Endosomes/genetics/*metabolism ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Lysosomes/genetics/metabolism ; Membrane Proteins/genetics/*metabolism ; Microtubules/metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Nogo Proteins/genetics/metabolism ; Protein Transport/*genetics ; RNA, Small Interfering ; rab GTP-Binding Proteins/genetics/metabolism ; },
abstract = {ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.},
}
@article {pmid33433941,
year = {2021},
author = {Wu, H and Wang, F and Jiang, JH},
title = {Inducible CRISPR-dCas9 Transcriptional Systems for Sensing and Genome Regulation.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {22},
number = {11},
pages = {1894-1900},
doi = {10.1002/cbic.202000723},
pmid = {33433941},
issn = {1439-7633},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Humans ; Transcription, Genetic/genetics ; Transcriptional Activation ; },
abstract = {The clustered, regularly interspaced short palindromic repeats-associated protein 9 endonuclease (CRISPR-Cas9) and the nuclease-deactivated Cas9 (dCas9) systems have revolutionized our ability to precisely engineer and regulate genomes. Inducible CRISPR-dCas9-based transcriptional systems have been rapidly developed to conditionally control genetic manipulation. Current strategies mainly focus on conditional control of gRNA function and dCas9 protein using exogenous and endogenous triggers, including external light, small molecules, synthetic and intracellular oligonucleotides. These strategies have established novel platforms for the spatiotemporal regulation of genome activation and repression, epigenome editing, and so on. Herein, we summarize the recent progress in conditionally controlling CRISPR-dCas9 transcriptional systems through gRNA modulation and dCas9 protein engineering.},
}
@article {pmid33433564,
year = {2021},
author = {Abbasi, J},
title = {Promising Strategies for Sickle Cell Disease and β-Thalassemia.},
journal = {JAMA},
volume = {325},
number = {2},
pages = {121},
doi = {10.1001/jama.2020.26232},
pmid = {33433564},
issn = {1538-3598},
mesh = {Anemia, Sickle Cell/genetics/*therapy ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Repressor Proteins/genetics ; beta-Thalassemia/genetics/*therapy ; },
}
@article {pmid33432236,
year = {2021},
author = {Yim, SS and McBee, RM and Song, AM and Huang, Y and Sheth, RU and Wang, HH},
title = {Robust direct digital-to-biological data storage in living cells.},
journal = {Nature chemical biology},
volume = {17},
number = {3},
pages = {246-253},
pmid = {33432236},
issn = {1552-4469},
support = {R01 AI132403/AI/NIAID NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; Carbon/chemistry ; Cell Engineering/*methods ; DNA/classification/*genetics/metabolism ; Electricity ; *Electrochemical Techniques ; *Electrons ; Escherichia coli/*genetics/metabolism ; Ferrocyanides/chemistry ; Humans ; Information Storage and Retrieval/*methods ; Oligonucleotide Array Sequence Analysis ; Oxidation-Reduction ; Sequence Analysis, DNA ; Silicon/chemistry ; },
abstract = {DNA has been the predominant information storage medium for biology and holds great promise as a next-generation high-density data medium in the digital era. Currently, the vast majority of DNA-based data storage approaches rely on in vitro DNA synthesis. As such, there are limited methods to encode digital data into the chromosomes of living cells in a single step. Here, we describe a new electrogenetic framework for direct storage of digital data in living cells. Using an engineered redox-responsive CRISPR adaptation system, we encoded binary data in 3-bit units into CRISPR arrays of bacterial cells by electrical stimulation. We demonstrate multiplex data encoding into barcoded cell populations to yield meaningful information storage and capacity up to 72 bits, which can be maintained over many generations in natural open environments. This work establishes a direct digital-to-biological data storage framework and advances our capacity for information exchange between silicon- and carbon-based entities.},
}
@article {pmid33432198,
year = {2021},
author = {Yin, D and Ling, S and Wang, D and Dai, Y and Jiang, H and Zhou, X and Paludan, SR and Hong, J and Cai, Y},
title = {Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice.},
journal = {Nature biotechnology},
volume = {39},
number = {5},
pages = {567-577},
pmid = {33432198},
issn = {1546-1696},
support = {786602/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Herpesvirus 1, Human/genetics/pathogenicity ; Humans ; Keratitis, Herpetic/*genetics/therapy/virology ; Mice ; Simplexvirus/*genetics/pathogenicity ; Virus Replication/*genetics ; },
abstract = {Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.},
}
@article {pmid33431240,
year = {2021},
author = {Ding, X and Schimenti, JC},
title = {Strategies to Identify Genetic Variants Causing Infertility.},
journal = {Trends in molecular medicine},
volume = {27},
number = {8},
pages = {792-806},
pmid = {33431240},
issn = {1471-499X},
support = {P50 HD096723/HD/NICHD NIH HHS/United States ; R01 HD082568/HD/NICHD NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Computational Biology/methods ; Disease Management ; Gene Editing/methods ; Genetic Association Studies/*methods ; *Genetic Predisposition to Disease ; Genetic Testing/methods ; *Genetic Variation ; Genome-Wide Association Study/methods ; Genomics/methods ; High-Throughput Screening Assays ; Humans ; Infertility/*diagnosis/*genetics/therapy ; Reproductive Techniques, Assisted ; },
abstract = {Genetic causes are thought to underlie about half of infertility cases, but understanding the genetic bases has been a major challenge. Modern genomics tools allow more sophisticated exploration of genetic causes of infertility through population, family-based, and individual studies. Nevertheless, potential therapies based on genetic diagnostics will be limited until there is certainty regarding the causality of genetic variants identified in an individual. Genome modulation and editing technologies have revolutionized our ability to functionally test such variants, and also provide a potential means for clinical correction of infertility variants. This review addresses strategies being used to identify causative variants of infertility.},
}
@article {pmid33429883,
year = {2021},
author = {Kivrak, E and Pauzaite, T and Copeland, NA and Hardy, JG and Kara, P and Firlak, M and Yardimci, AI and Yilmaz, S and Palaz, F and Ozsoz, M},
title = {Detection of CRISPR-Cas9-Mediated Mutations Using a Carbon Nanotube-Modified Electrochemical Genosensor.},
journal = {Biosensors},
volume = {11},
number = {1},
pages = {},
pmid = {33429883},
issn = {2079-6374},
mesh = {3T3 Cells ; Animals ; Biosensing Techniques/*instrumentation ; CRISPR-Cas Systems ; Carbodiimides/chemistry ; Electrodes ; Genetic Engineering/*instrumentation ; Graphite/chemistry ; Limit of Detection ; Mice ; Mutagenesis, Site-Directed ; Nanotubes, Carbon/*chemistry ; Nuclear Proteins/*genetics ; *Point Mutation ; },
abstract = {The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5'-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.},
}
@article {pmid33429291,
year = {2021},
author = {van der Weyden, L and Jonkers, J and Adams, DJ},
title = {The use of CRISPR/Cas9-based gene editing strategies to explore cancer gene function in mice.},
journal = {Current opinion in genetics &amp; development},
volume = {66},
number = {},
pages = {57-62},
doi = {10.1016/j.gde.2020.12.005},
pmid = {33429291},
issn = {1879-0380},
support = {/WT_/Wellcome Trust/United Kingdom ; /CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Editing/*trends ; Humans ; Mice ; Neoplasms/*genetics/pathology ; Oncogenes/genetics ; Phenotype ; },
abstract = {CRISPR/Cas9 systems have revolutionised the field of gene editing, allowing for precise modifications to be generated in vivo to mimic the genetic events found in human cancer cells. These systems may be used to generate germline or somatic loss-of-function of events, and also chromosomal rearrangements, either constitutively or in a spatiotemporally controlled manner. Forward genetic screens have also been performed using CRISPR/Cas9 systems to identify new driver genes and approaches using catalytically inactive Cas9 fused to base editors have enabled genome editing with single-base precision. Here we discuss the many 'flavours' of the CRISPR/Cas9 system and give examples of their use for the generation of clinically-relevant mouse models of cancer.},
}
@article {pmid33428981,
year = {2021},
author = {Safari, F and Afarid, M and Rastegari, B and Borhani-Haghighi, A and Barekati-Mowahed, M and Behzad-Behbahani, A},
title = {CRISPR systems: Novel approaches for detection and combating COVID-19.},
journal = {Virus research},
volume = {294},
number = {},
pages = {198282},
pmid = {33428981},
issn = {1872-7492},
mesh = {Antiviral Agents/therapeutic use ; COVID-19/diagnosis/*drug therapy/therapy ; COVID-19 Nucleic Acid Testing ; CRISPR-Associated Proteins/therapeutic use ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Viral/genetics/metabolism ; SARS-CoV-2/*drug effects/genetics/*isolation &amp; purification ; },
abstract = {Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.},
}
@article {pmid33428900,
year = {2021},
author = {Palaz, F and Kalkan, AK and Tozluyurt, A and Ozsoz, M},
title = {CRISPR-based tools: Alternative methods for the diagnosis of COVID-19.},
journal = {Clinical biochemistry},
volume = {89},
number = {},
pages = {1-13},
pmid = {33428900},
issn = {1873-2933},
mesh = {COVID-19/*diagnosis/virology ; COVID-19 Testing/*methods ; *CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/*methods ; SARS-CoV-2/genetics/*isolation &amp; purification ; },
abstract = {The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread all over the world rapidly and caused a global pandemic. To prevent the virus from spreading to more individuals, it is of great importance to identify and isolate infected individuals through testing. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of coronavirus disease (COVID-19) worldwide. However, performing RT-qPCR is limited to centralized laboratories because of the need for sophisticated laboratory equipment and skilled personnel. Further, it can sometimes give false negative or uncertain results. Recently, new methods have been developed for nucleic acid detection and pathogen diagnosis using CRISPR-Cas systems. These methods present rapid and cost-effective diagnostic platforms that provide high sensitivity and specificity without the need for complex instrumentation. Using the CRISPR-based SARS-CoV-2 detection methods, it is possible to increase the number of daily tests in existing laboratories, reduce false negative or uncertain result rates obtained with RT-qPCR, and perform testing in resource-limited settings or at points of need where performing RT-qPCR is not feasible. Here, we briefly describe the RT-qPCR method, and discuss its limitations in meeting the current diagnostic needs. We explain how the unique properties of various CRISPR-associated enzymes are utilized for nucleic acid detection and pathogen diagnosis. Then, we highlight the important features of CRISPR-based diagnostic methods developed for SARS-CoV-2 detection. Finally, we examine the advantages and limitations of these methods, and discuss how they can contribute to improving the efficiency of the current testing systems for combating SARS-CoV-2.},
}
@article {pmid33428725,
year = {2021},
author = {Peng, H and Zheng, Y and Zhao, Z and Li, J},
title = {Multigene editing: current approaches and beyond.},
journal = {Briefings in bioinformatics},
volume = {22},
number = {5},
pages = {},
doi = {10.1093/bib/bbaa396},
pmid = {33428725},
issn = {1477-4054},
mesh = {Animals ; BRCA1 Protein/genetics/metabolism ; BRCA2 Protein/genetics/metabolism ; Bacteria/genetics/metabolism ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Humans ; Multigene Family ; Neoplasm Proteins/genetics/metabolism ; Neoplasms/*genetics/metabolism/pathology ; Neurofibromin 1/genetics/metabolism ; PTEN Phosphohydrolase/genetics/metabolism ; Plants/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; RNA, Guide/*genetics/metabolism ; Repressor Proteins/genetics/metabolism ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {CRISPR/Cas9 multigene editing is an active and widely studied topic in the fields of biomedicine and biology. It involves a simultaneous participation of multiple single-guide RNAs (sgRNAs) to edit multiple target genes in a way that each gene is edited by one of these sgRNAs. There are possibly numerous sgRNA candidates capable of on-target editing on each of these genes with various efficiencies. Meanwhile, each of these sgRNA candidates may cause unwanted off-target editing at many other genes. Therefore, selection optimization of these multiple sgRNAs is demanded so as to minimize the number of sgRNAs and thus reduce the collective negative effects caused by the off-target editing. This survey reviews wet-laboratory approaches to the implementation of multigene editing and their needs of computational tools for better design. We found that though off-target editing is unavoidable during the gene editing, those disfavored cuttings by some target genes' sgRNAs can potentially become on-target editing sites for some other genes of interests. This off-to-on role conversion is beneficial to optimize the sgRNA selection in multigene editing. We present a preference cutting score to assess those beneficial off-target cutting sites, which have a few mismatches with their host genes' on-target editing sites. These potential sgRNAs can be prioritized for recommendation via ranking their on-target average cutting efficiency, the total off-target site number and their average preference cutting score. We also present case studies on cancer-associated genes to demonstrate tremendous usefulness of the new method.},
}
@article {pmid33428399,
year = {2021},
author = {Li, CY and Zheng, B and Li, JT and Gao, JL and Liu, YH and Pang, DW and Tang, HW},
title = {Holographic Optical Tweezers and Boosting Upconversion Luminescent Resonance Energy Transfer Combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a Biosensors.},
journal = {ACS nano},
volume = {15},
number = {5},
pages = {8142-8154},
doi = {10.1021/acsnano.0c09986},
pmid = {33428399},
issn = {1936-086X},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Humans ; Optical Tweezers ; },
abstract = {Taking advantage of outstanding precision in target recognition and trans-cleavage ability, the recently discovered CRISPR/Cas12a system provides an alternative opportunity for designing fluorescence biosensors. To fully exploit the analytical potential, we introduce here some meaningful concepts. First, the collateral cleavage of CRISPR/Cas12a is efficiently activated in a functional DNA regulation manner and the bottleneck which largely applicable to nucleic acids detection is broken. After selection of a representative aptamer and DNAzyme as the transduction pathways, the sensing coverage is extended to a small organic compound (ATP) and a metal ion (Na[+]). The assay sensitivity is significantly improved by utilizing a bead-supported enrichment strategy wherein emerging holographic optical tweezers are used to enhance imaging stability and simultaneously achieve multiflux analysis. Last, a sandwich-structured energy-concentrating upconversion nanoparticle triggered boosting luminescent resonance energy transfer mode is comined to face with complicated biological samples by skillfully confining the emitters into a very limited inner shell. Following the above attempts, the developed CRISPR/Cas12a biosensors not only present an ultrasensitive assay behavior toward these model non-nucleic acid analytes but also can serve as a formidable toolbox for determining real samples including single cell lysates and human plasma, proving a good practical application capacity.},
}
@article {pmid33427630,
year = {2020},
author = {Guillermin, C and Bouchet, M and Garcès, A and Enriquez, J},
title = {[Development and maintenance of the neuromuscular system].},
journal = {Medecine sciences : M/S},
volume = {36 Hors s&#xe9;rie n&#xb0; 2},
number = {},
pages = {13-16},
doi = {10.1051/medsci/2020207},
pmid = {33427630},
issn = {1958-5381},
mesh = {Adult ; Animals ; Axons/physiology ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Genomics/methods ; Humans ; Motor Neurons/physiology ; Muscle, Skeletal/*growth &amp; development/innervation/*physiology ; RNA-Seq ; Regeneration/genetics/*physiology ; },
abstract = {Diseases of the locomotor system are at the origin of disabilities with severe social and economic consequences. The study of the neuromuscular system development and maintenance has become a key challenge for the scientific community in order to design efficient therapies. My thesis project aims to elucidate the mechanisms at the origin of the communication between motoneuron axons and their muscle targets in order to understand how specific innervations are generated during development and maintained during adulthood. The first part of the project will address the understanding of the mechanisms controlling the specific muscle-axon recognition during development. I will perform live imaging and fixed tissues experiments to visualize and understand the development of myoblasts and motoneurons at the same time. Then, I will perform transcriptomic experiments to discover molecules playing a role in the specific axon-muscle recognition. The second part of the project is meant to elucidate the mechanism controlling the system maintenance in the adult. To answer this question I will study the function of morphological transcription factors in adulthood, which are known as transcription factors controlling the morphology of motoneurons during development. To conclude, this project will lead to novel biological concepts that will increase our fundamental knowledge on developmental biology. Understanding the mechanisms that specify the muscle innervation will allow to find efficient ways to tackle neuromuscular diseases.},
}
@article {pmid33425987,
year = {2020},
author = {Srivastava, S and Upadhyay, DJ and Srivastava, A},
title = {Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {582499},
pmid = {33425987},
issn = {2296-889X},
abstract = {Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.},
}
@article {pmid33425776,
year = {2020},
author = {Panfil, AR and Green, PL and Yoder, KE},
title = {CRISPR Genome Editing Applied to the Pathogenic Retrovirus HTLV-1.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {580371},
pmid = {33425776},
issn = {2235-2988},
support = {P01 CA100730/CA/NCI NIH HHS/United States ; R21 AI142794/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/genetics ; CRISPR-Cas Systems ; *Gene Editing ; Gene Products, tax/genetics/metabolism ; *Human T-lymphotropic virus 1/genetics ; Humans ; Retroviridae Proteins/genetics ; },
abstract = {CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukemia virus type 1 (HTLV-1) as an excellent model to advance CRISPR/Cas9 genome editing technologies against actively expressing and latent retroviral proviruses. HTLV-1 is a tumorigenic human retrovirus responsible for the development of both leukemia/lymphoma (ATL) and a neurological disease (HAM/TSP). The virus immortalizes and persists in CD4[+] T lymphocytes that survive for the lifetime of the host. The most important drivers of HTLV-1-mediated transformation and proliferation are the tax and hbz viral genes. Tax, transcribed from the plus-sense or genome strand, is essential for de novo infection and cellular immortalization. Hbz, transcribed from the minus-strand, supports proliferation and survival of infected cells in both its protein and mRNA forms. Abrogating the function or expression of tax and/or hbz by genome editing and mutagenic double-strand break repair may disable HTLV-1-infected cell growth/survival and prevent immune modulatory effects and ultimately HTLV-1-associated disease. In addition, the HTLV-1 viral genome is highly conserved with remarkable sequence homogeneity, both within the same host and even among different HTLV isolates. This offers more focused guide RNA targeting. In addition, there are several well-established animal models for studying HTLV-1 infection in vivo as well as cell immortalization in vitro. Therefore, studies with HTLV-1 may provide a better basis to assess and advance in vivo genome editing against retroviral infections.},
}
@article {pmid33423074,
year = {2021},
author = {Yarra, R and Sahoo, L},
title = {Base editing in rice: current progress, advances, limitations, and future perspectives.},
journal = {Plant cell reports},
volume = {40},
number = {4},
pages = {595-604},
pmid = {33423074},
issn = {1432-203X},
mesh = {Adenine ; CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Cytidine/genetics ; Gene Editing/*methods ; Genome, Plant ; Oryza/*genetics ; Plant Breeding/*methods ; Plants, Genetically Modified ; },
abstract = {Base editing is one of the promising genome editing tools for generating single-nucleotide changes in rice genome. Rice (Oryza sativa L.) is an important staple food crop, feeding half of the population around the globe. Developing new rice varieties with desirable agronomic traits is necessary for sustaining global food security. The use of genome editing technologies for developing rice varieties is pre-requisite in the present scenario. Among the genome editing technologies developed for rice crop improvement, base editing technology has emerged as an efficient and reliable tool for precise genome editing in rice plants. Base editing technology utilizes either adenosine or cytidine base editor for precise editing at the target region. A base editor (adenosine or cytidine) is a fusion of catalytically inactive CRISPR/Cas9 domain and adenosine or cytidine deaminase domain. In this review, authors have discussed the different adenine and cytosine base editors developed so far for precise genome editing of rice via base editing technology. We address the current progress, advances, limitations, as well as future perspectives of the base editing technology for rice crop improvement.},
}
@article {pmid33422709,
year = {2021},
author = {Karabiyik, G},
title = {Milestones of CRISPR/Cas9 system, promises and roadblocks.},
journal = {Cancer treatment and research communications},
volume = {26},
number = {},
pages = {100304},
doi = {10.1016/j.ctarc.2021.100304},
pmid = {33422709},
issn = {2468-2942},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA ; *Gene Editing ; Humans ; Translational Science, Biomedical ; },
abstract = {In this issue of Cancer Treatment and Research Communications, Sarkar et al. describe CRISPR/Cas9-based gene-editing from its discovery to current views. Originally a bacterial adaptive immune system, CRISPR/Cas9 was adapted to mammalian cells as a tool to perform a wide range of actions given its capability of accurately targeting specific DNA loci. While the CRISPR/Cas9 system is readily used in laboratories as a research tool for a few years now, the first clinical trials have recently started utilizing this system in translational medicine. Although several obstacles have been resolved related to the usage of CRISPR/Cas9 on mammalian cells, the scientific community is facing new challenges with this technology.},
}
@article {pmid33422676,
year = {2021},
author = {Wang, R and Graham, S and Gao, L and Tam, J and Levesque, MC},
title = {Editing the immune system in vivo in mice using CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing of transplanted hematopoietic stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {30-36},
doi = {10.1016/j.ymeth.2021.01.001},
pmid = {33422676},
issn = {1095-9130},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Hematopoietic Stem Cells/metabolism ; Immune System ; Mice ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {CRISPR/Cas9-based genome editing has been widely used to evaluate target gene function in biomedical research. The CRISPR/Cas9 system can introduce gene knockout, knock-in and mutations with more ease than earlier generations of genome editing tools. Using CRISPR/Cas9-based genome editing, researchers have successfully modified the DNA of different immune components, including primary T cells, B cells, macrophages, and immune system progenitors, i.e. hematopoietic stem cells (HSCs), which are also known as Lin-Sca1 + Kit + cells (LSKs) in mice. We previously reported that the transplantation of HSCs with lentivirus-mediated CRISPR/Cas9-based genetic modifications into lethally irradiated congenic mice repopulated the ablated recipient immune system with the donor immune system. In this report, we efficiently manipulated CD40 expression in LSK cells using Cas9 RNP and demonstrated the functional impact in a colitis model. Compared to a virus-based strategy, the RNP approach has the potential to enable investigation of target gene biology in any mouse strain and eliminates the time and effort associated with virus production and infection. Therefore, in vivo RNP-based CRISPR/Cas9 gene editing of transplanted HSCs represents a promising new strategy for exploring gene function in the immune system of mice.},
}
@article {pmid33422414,
year = {2021},
author = {Behbahani, RG and Danyaei, A and Teimoori, A and Neisi, N and Tahmasbi, MJ},
title = {Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique.},
journal = {Cancer radiotherapie : journal de la Societe francaise de radiotherapie oncologique},
volume = {25},
number = {3},
pages = {222-228},
doi = {10.1016/j.canrad.2020.08.048},
pmid = {33422414},
issn = {1769-6658},
mesh = {Apoptosis/radiation effects ; Breast Neoplasms/*genetics/*radiotherapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/radiation effects ; Female ; Gene Expression ; Gene Knockout Techniques/*methods ; Humans ; Osteopontin/*genetics/metabolism ; Plasmids/genetics ; RNA, Messenger/metabolism ; Radiation Tolerance/*genetics ; Real-Time Polymerase Chain Reaction ; Transfection/methods ; },
abstract = {PURPOSE: Osteopontin (OPN) is a phosphoglycoprotein, with a wide range of physiological and pathological roles. High expression of OPN promotes aggressive behavior, causes poor prognosis in tumor cells, and reduces the survival of patients. Since overexpression of OPN gives rise to radioresistance, the effects of the gene knock out using the CRISPR/Cas9 system in combination with radiation are emphasized.<br><br>MATERIAL AND METHODS: We used the CRISPR/Cas9 technique to knock out the OPN gene in the MDA-MB-231 cell line. After transfection, the cells were irradiated. The changes of the OPN mRNA levels, the apoptosis, and the differences in cell viability were assessed.<br><br>RESULTS: A significant reduction in the OPN expression was observed alone or along with irradiation. The knocked out gene alone increased apoptosis rate. The cell viability decreased to after knocking out of the OPN gene. The gene knocking-out combined with irradiation led to more decline of cell viability.<br><br>CONCLUSION: Our results demonstrated that after knocking out the OPN gene, the MDA-MB-231 cells showed a significant radiosensitivity. Therefore, the OPN knock out in combination with conventional radiotherapy, may become an efficient therapeutic target in the future.},
}
@article {pmid33421768,
year = {2021},
author = {Oh, SH and Lee, HJ and Ahn, MK and Jeon, MY and Yoon, JS and Jung, YJ and Kim, GN and Baek, IJ and Kim, I and Kim, KM and Sung, YH},
title = {Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA.},
journal = {Biochemical and biophysical research communications},
volume = {539},
number = {},
pages = {48-55},
doi = {10.1016/j.bbrc.2020.12.072},
pmid = {33421768},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; Cell Line ; Embryo, Mammalian/*metabolism ; Endonucleases/*metabolism ; *Gene Editing ; Gene Targeting/*methods ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; RNA, Guide/genetics/*metabolism ; },
abstract = {CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.},
}
@article {pmid33421620,
year = {2022},
author = {Ansari, I and Chaturvedi, A and Chitkara, D and Singh, S},
title = {CRISPR/Cas mediated epigenome editing for cancer therapy.},
journal = {Seminars in cancer biology},
volume = {83},
number = {},
pages = {570-583},
doi = {10.1016/j.semcancer.2020.12.018},
pmid = {33421620},
issn = {1096-3650},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Methylation ; Epigenesis, Genetic ; Epigenome/genetics ; Epigenomics ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {The understanding of the relationship between epigenetic alterations, their effects on gene expression and the knowledge that these epigenetic alterations are reversible, have opened up new therapeutic pathways for treating various diseases, including cancer. This has led the research for a better understanding of the mechanism and pathways of carcinogenesis and provided the opportunity to develop the therapeutic approaches by targeting such pathways. Epi-drugs, DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are the best examples of epigenetic therapies with clinical applicability. Moreover, precise genome editing technologies such as CRISPR/Cas has proven their efficacy in epigenome editing, including the alteration of epigenetic markers, such as DNA methylation or histone modification. The main disadvantage with DNA gene editing technologies is off-target DNA sequence alteration, which is not an issue with epigenetic editing. It is known that cancer is linked with epigenetic alteration, and thus CRISPR/Cas system shows potential for cancer therapy via epigenome editing. This review outlines the epigenetic therapeutic approach for cancer therapy using CRISPR/Cas, from the basic understanding of cancer epigenetics to potential applications of CRISPR/Cas in treating cancer.},
}
@article {pmid33421369,
year = {2021},
author = {Workman, RE and Pammi, T and Nguyen, BTK and Graeff, LW and Smith, E and Sebald, SM and Stoltzfus, MJ and Euler, CW and Modell, JW},
title = {A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.},
journal = {Cell},
volume = {184},
number = {3},
pages = {675-688.e19},
doi = {10.1016/j.cell.2020.12.017},
pmid = {33421369},
issn = {1097-4172},
mesh = {Autoimmunity/genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Conserved Sequence ; Down-Regulation/genetics ; *Gene Expression ; Homeostasis/*genetics ; Models, Genetic ; Mutation/genetics ; Operon/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; Streptococcus pyogenes/genetics ; Stress, Physiological/genetics ; Transcription, Genetic ; Transcriptional Activation/genetics ; },
abstract = {CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.},
}
@article {pmid33421132,
year = {2021},
author = {Guyon-Debast, A and Alboresi, A and Terret, Z and Charlot, F and Berthier, F and Vendrell-Mir, P and Casacuberta, JM and Veillet, F and Morosinotto, T and Gallois, JL and Nogué, F},
title = {A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens.},
journal = {The New phytologist},
volume = {230},
number = {3},
pages = {1258-1272},
pmid = {33421132},
issn = {1469-8137},
mesh = {*Bryopsida/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Mutagenesis, Site-Directed ; },
abstract = {CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.},
}
@article {pmid33420926,
year = {2021},
author = {Zhang, JP and Yang, ZX and Zhang, F and Fu, YW and Dai, XY and Wen, W and Zhang, B and Choi, H and Chen, W and Brown, M and Baylink, D and Zhang, L and Qiu, H and Wang, C and Cheng, T and Zhang, XB},
title = {HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs.},
journal = {Science China. Life sciences},
volume = {64},
number = {9},
pages = {1449-1462},
pmid = {33420926},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems ; DNA End-Joining Repair/*drug effects ; Gene Editing/*methods ; Histone Deacetylase Inhibitors/*pharmacology ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Vorinostat/*pharmacology ; },
abstract = {Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA. Among the six HDAC inhibitors we examined, vorinostat, or suberoylanilide hydroxamic acid (SAHA), led to the highest HDR efficiency at both open and closed loci, with acceptable toxicity. HDAC inhibitors equally increased non-homologous end joining (NHEJ) editing efficiencies (∼50%) at both open and closed loci, due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression. However, we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin (2.8 vs. 1.7-fold change). These studies provide a new strategy for HDR-editing of silent genes in iPSCs.},
}
@article {pmid33420919,
year = {2021},
author = {Wang, B and Lv, X and Wang, Y and Wang, Z and Liu, Q and Lu, B and Liu, Y and Gu, F},
title = {CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome.},
journal = {Science China. Life sciences},
volume = {64},
number = {9},
pages = {1463-1472},
pmid = {33420919},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems ; DNA Repair ; DNA, Mitochondrial/*genetics ; Gene Editing/*methods ; Genome, Human ; *Genome, Mitochondrial ; Humans ; INDEL Mutation ; Mitochondria/metabolism ; Mutagenesis/*genetics ; },
abstract = {Genetic manipulation of mitochondrial DNA (mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion (InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break (DSB) lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor, iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.},
}
@article {pmid33420656,
year = {2021},
author = {Yuza, K and Nagahashi, M and Ichikawa, H and Hanyu, T and Nakajima, M and Shimada, Y and Ishikawa, T and Sakata, J and Takeuchi, S and Okuda, S and Matsuda, Y and Abe, M and Sakimura, K and Takabe, K and Wakai, T},
title = {Activin a Receptor Type 2A Mutation Affects the Tumor Biology of Microsatellite Instability-High Gastric Cancer.},
journal = {Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract},
volume = {25},
number = {9},
pages = {2231-2241},
pmid = {33420656},
issn = {1873-4626},
support = {R01 CA160688/CA/NCI NIH HHS/United States ; R01CA160688/CA/NCI NIH HHS/United States ; },
mesh = {Activin Receptors, Type II ; Activins ; Aged ; Biology ; Humans ; *Microsatellite Instability ; Mutation ; Retrospective Studies ; *Stomach Neoplasms/genetics ; },
abstract = {BACKGROUND: Activin A receptor type 2A (ACVR2A) is one of the most frequently mutated genes in microsatellite instability-high (MSI-H) gastric cancer. However, the clinical relevance of the ACVR2A mutation in MSI-H gastric cancer patients remains unclear. The aims of this study were to explore the effect of ACVR2A mutation on the tumor behavior and to identify the clinicopathological characteristics of gastric cancer patients with ACVR2A mutations.<br><br>METHODS: An in vitro study was performed to investigate the biological role of ACVR2A via CRISPR/Cas9-mediated ACVR2A knockout MKN74 human gastric cancer cells. One hundred twenty-four patients with gastric cancer were retrospectively analyzed, and relations between MSI status, ACVR2A mutations, and clinicopathological factors were evaluated.<br><br>RESULTS: ACVR2A knockout cells showed less aggressive tumor biology than mock-transfected cells, displaying reduced proliferation, migration, and invasion (P&#x2009;<&#x2009;0.05). MSI mutations were found in 10% (13/124) of gastric cancer patients, and ACVR2A mutations were found in 8.1% (10/124) of patients. All ACVR2A mutations were accompanied by MSI. The 5-year overall survival rates of ACVR2A wild-type patients and ACVR2A-mutated patients were 57% and 90%, respectively (P&#x2009;=&#x2009;0.048). Multivariate analysis revealed that older age (P&#x2009;=&#x2009;0.015), distant metastasis (P&#x2009;<&#x2009;0.001), and ACVR2A wild-type status (P&#x2009;=&#x2009;0.040) were independent prognostic factors for overall survival.<br><br>CONCLUSIONS: Our study demonstrated that gastric cancer patients with ACVR2A mutation have a significantly better prognosis than those without. Dysfunction of ACVR2A in MKN74 human gastric cancer cells caused less aggressive tumor biology, indicating the importance of ACVR2A in the progression of MSI-H tumors.},
}
@article {pmid33420514,
year = {2021},
author = {Massel, K and Lam, Y and Wong, ACS and Hickey, LT and Borrell, AK and Godwin, ID},
title = {Hotter, drier, CRISPR: the latest edit on climate change.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {134},
number = {6},
pages = {1691-1709},
pmid = {33420514},
issn = {1432-2242},
mesh = {Agriculture ; *CRISPR-Cas Systems ; *Climate Change ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/*genetics ; Edible Grain/*genetics ; *Gene Editing ; Hot Temperature ; Phenotype ; Plant Breeding ; },
abstract = {Integrating CRISPR/Cas9 genome editing into modern breeding programs for crop improvement in cereals. Global climate trends in many agricultural regions have been rapidly changing over the past decades, and major advances in global food systems are required to ensure food security in the face of these emerging challenges. With increasing climate instability due to warmer temperatures and rising CO2 levels, the productivity of global agriculture will continue to be negatively impacted. To combat these growing concerns, creative approaches will be required, utilising all the tools available to produce more robust and tolerant crops with increased quality and yields under more extreme conditions. The integration of genome editing and transgenics into current breeding strategies is one promising solution to accelerate genetic gains through targeted genetic modifications, producing crops that can overcome the shifting climate realities. This review focuses on how revolutionary genome editing tools can be directly implemented into breeding programs for cereal crop improvement to rapidly counteract many of the issues affecting agriculture production in the years to come.},
}
@article {pmid33420513,
year = {2021},
author = {Houzelstein, D and Simon-Chazottes, D and Batista, L and Tokuda, S and Langa Vives, F and Flamand, M and Montagutelli, X and Panthier, JJ},
title = {The ring finger protein 213 gene (Rnf213) contributes to Rift Valley fever resistance in mice.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {32},
number = {1},
pages = {30-37},
pmid = {33420513},
issn = {1432-1777},
mesh = {Adenosine Triphosphatases/*genetics ; Animals ; CRISPR-Cas Systems ; Chromosome Mapping ; Disease Models, Animal ; Disease Resistance/*genetics ; Host-Pathogen Interactions/*genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Rift Valley Fever/*genetics/*virology ; Rift Valley fever virus/*physiology ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Rift Valley fever (RVF) is an emerging viral zoonosis that primarily affects ruminants and humans. We have previously shown that wild-derived MBT/Pas mice are highly susceptible to RVF virus and that part of this phenotype is controlled by a locus located on distal Chromosome 11. Using congenic strains, we narrowed down the critical interval to a 530 kb region containing five protein-coding genes among which Rnf213 emerged as a potential candidate. We generated Rnf213-deficient mice by CRISPR/CAS9 on the C57BL/6 J background and showed that they were significantly more susceptible to RVF than control mice, with an average survival time post-infection reduced from 7 to 4 days. The human RNF213 gene had been associated with the cerebrovascular Moyamoya disease (MMD or MYMY) but the inactivation of this gene in the mouse resulted only in mild anomalies of the neovascularization. This study provides the first evidence that the Rnf213 gene may also impact the resistance to infectious diseases such as RVF.},
}
@article {pmid33420494,
year = {2021},
author = {Nakamura, M and Gao, Y and Dominguez, AA and Qi, LS},
title = {CRISPR technologies for precise epigenome editing.},
journal = {Nature cell biology},
volume = {23},
number = {1},
pages = {11-22},
pmid = {33420494},
issn = {1476-4679},
support = {U01 DK127405/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Epigenome ; *Gene Editing ; *Gene Expression Regulation ; Humans ; },
abstract = {The epigenome involves a complex set of cellular processes governing genomic activity. Dissecting this complexity necessitates the development of tools capable of specifically manipulating these processes. The repurposing of prokaryotic CRISPR systems has allowed for the development of diverse technologies for epigenome engineering. Here, we review the state of currently achievable epigenetic manipulations along with corresponding applications. With future optimization, CRISPR-based epigenomic editing stands as a set of powerful tools for understanding and controlling biological function.},
}
@article {pmid33420369,
year = {2021},
author = {Jung, H and Lee, KS and Choi, JK},
title = {Comprehensive characterisation of intronic mis-splicing mutations in human cancers.},
journal = {Oncogene},
volume = {40},
number = {7},
pages = {1347-1361},
pmid = {33420369},
issn = {1476-5594},
mesh = {CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cell Line, Tumor ; Exons/genetics ; Hematologic Neoplasms/*genetics/pathology ; Humans ; Introns/genetics ; Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA Splice Sites/genetics ; RNA Splicing/*genetics ; Shelterin Complex ; Telomere-Binding Proteins/*genetics ; },
abstract = {Previous studies studying mis-splicing mutations were based on exome data and thus our current knowledge is largely limited to exons and the canonical splice sites. To comprehensively characterise intronic mis-splicing mutations, we analysed 1134 pan-cancer whole genomes and transcriptomes together with 3022 normal control samples. The ratio-based splicing analysis resulted in 678 somatic intronic mutations, with 46% residing in deep introns. Among the 309 deep intronic single nucleotide variants, 245 altered core splicing codes, with 38% activating cryptic splice sites, 12% activating cryptic polypyrimidine tracts, and 36% and 12% disrupting authentic polypyrimidine tracts and branchpoints, respectively. All the intronic cryptic splice sites were created at pre-existing GT/AG dinucleotides or by GC-to-GT conversion. Notably, 85 deep intronic mutations indicated gain of splicing enhancers or loss of splicing silencers. We found that 64 tumour suppressors were affected by intronic mutations and blood cancers showed higher proportion of deep intronic mutations. In particular, a telomere maintenance gene, POT1, was recurrently mis-spliced by deep intronic mutations in blood cancers. We validated a pseudoexon activation involving a splicing silencer in POT1 by CRISPR/Cas9. Our results shed light on previously unappreciated mechanisms by which noncoding mutations acting on splicing codes in deep introns contribute to tumourigenesis.},
}
@article {pmid33420081,
year = {2021},
author = {Hou, G and Harley, ITW and Lu, X and Zhou, T and Xu, N and Yao, C and Qin, Y and Ouyang, Y and Ma, J and Zhu, X and Yu, X and Xu, H and Dai, D and Ding, H and Yin, Z and Ye, Z and Deng, J and Zhou, M and Tang, Y and Namjou, B and Guo, Y and Weirauch, MT and Kottyan, LC and Harley, JB and Shen, N},
title = {SLE non-coding genetic risk variant determines the epigenetic dysfunction of an immune cell specific enhancer that controls disease-critical microRNA expression.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {135},
pmid = {33420081},
issn = {2041-1723},
support = {R01 AI024717/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Line, Tumor ; Epigenesis, Genetic/*immunology ; Female ; Genetic Predisposition to Disease ; Genotyping Techniques ; HEK293 Cells ; Healthy Volunteers ; Humans ; Interferon Type I/metabolism ; Leukocytes, Mononuclear/transplantation ; Lupus Erythematosus, Systemic/blood/*genetics/immunology ; Mice ; MicroRNAs/*genetics/metabolism ; NF-kappa B/metabolism ; Polymorphism, Single Nucleotide ; Primary Cell Culture ; Promoter Regions, Genetic ; RNA-Seq ; Regulatory Sequences, Nucleic Acid/*genetics ; Signal Transduction/genetics/immunology ; },
abstract = {Since most variants that impact polygenic disease phenotypes localize to non-coding genomic regions, understanding the consequences of regulatory element variants will advance understanding of human disease mechanisms. Here, we report that the systemic lupus erythematosus (SLE) risk variant rs2431697 as likely causal for SLE through disruption of a regulatory element, modulating miR-146a expression. Using epigenomic analysis, genome-editing and 3D chromatin structure analysis, we show that rs2431697 tags a cell-type dependent distal enhancer specific for miR-146a that physically interacts with the miR-146a promoter. NF-kB binds the disease protective allele in a sequence-specific manner, increasing expression of this immunoregulatory microRNA. Finally, CRISPR activation-based modulation of this enhancer in the PBMCs of SLE patients attenuates type I interferon pathway activation by increasing miR-146a expression. Our work provides a strategy to define non-coding RNA functional regulatory elements using disease-associated variants and provides mechanistic links between autoimmune disease risk genetic variation and disease etiology.},
}
@article {pmid33418923,
year = {2021},
author = {Kim, J and Chang, KS and Lee, S and Jin, E},
title = {Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395.},
journal = {International journal of molecular sciences},
volume = {22},
number = {2},
pages = {},
pmid = {33418923},
issn = {1422-0067},
mesh = {Adenine Phosphoribosyltransferase/genetics/metabolism ; Algal Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Chlorella vulgaris/*genetics ; Gene Editing/*methods ; Nitrate Reductase/genetics/metabolism ; RNA, Guide/metabolism ; },
abstract = {To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.},
}
@article {pmid33418558,
year = {2021},
author = {Khaled, M and Moustafa, AS and El-Khazragy, N and Ahmed, MI and Abd Elkhalek, MA and El Salahy, EM},
title = {CRISPR/Cas9 mediated knock-out of VPREB1 gene induces a cytotoxic effect in myeloma cells.},
journal = {PloS one},
volume = {16},
number = {1},
pages = {e0245349},
pmid = {33418558},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; Cell Proliferation ; Gene Editing ; Genetic Therapy ; Humans ; Immunoglobulin Light Chains, Surrogate/*genetics ; Multiple Myeloma/*genetics/pathology/therapy ; RNA, Messenger/genetics ; Tumor Cells, Cultured ; },
abstract = {BACKGROUND: Multiple Myeloma (MM) is a heterogeneous, hematological neoplasm that accounts 2% of all cancers. Although, autologous stem cell transplantation and chemotherapy are currently the most effective therapy, it carries a notable hazards, in addition for being non curative. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has been successfully tried at the experimental level, for the treatment of several hematological malignancies.<br><br>OBJECTIVES: We aimed to investigate the in-vitro effect of CRISPR-cas9-mediated knock-out of V-set pre B-cell surrogate light chain 1"VPREB1" gene on the malignant proliferation of primary cultured myeloma cells.<br><br>METHODS: Bioinformatics' analysis was performed to explore the gene expression profile of MM, and the VPREB1 gene was selected as a target gene for this study. We knocked-out the VPREB1 gene in primary cultured myeloma cells using CRISPR-cas9, the VPREB1 gene editing efficacy was verified by determining VPREB1 gene expression at both the mRNA and protein levels by qPCR and immunofluorescence, respectively. Furthermore, the cytotoxic effect on primary myeloma cells proliferation was evaluated using cytotoxicity assay.<br><br>RESULTS: There was a statistically significant reduction of both VPREB1 mRNA and protein expression levels (p<0.01). knock-out of VPREB1 gene in myeloma cell line resulted in a statistically significant reduction of myeloma cell proliferation.<br><br>CONCLUSION: CRISPR-cas9-mediated knock-out of VPREB1 gene is effective for inhibiting the proliferation of primary myeloma cells. This would provide a basis for a promising therapeutic strategy for patients with multiple myeloma.},
}
@article {pmid33418105,
year = {2021},
author = {Das Mukhopadhyay, C and Sharma, P and Sinha, K and Rajarshi, K},
title = {Recent trends in analytical and digital techniques for the detection of the SARS-Cov-2.},
journal = {Biophysical chemistry},
volume = {270},
number = {},
pages = {106538},
pmid = {33418105},
issn = {1873-4200},
mesh = {Antigens, Viral/analysis ; Biosensing Techniques/*methods ; COVID-19/*diagnosis/virology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Immunoassay ; Nucleic Acid Amplification Techniques ; RNA, Viral/analysis ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {The current global outbreak of COVID-19 due to SARS-CoV-2 is an unprecedented humanitarian crisis. Considering the gravity of its impact there is an immediate need to develop a detection technique that is sensitive, specific, fast, and affordable for the clinical diagnosis of the disease. Real time Polymerase Chain Reaction (RT-PCR)-based detection platforms are contemplated to be the gold standard to detect viral RNA. However, that may be susceptible to errors, and there is a risk of obtaining false results, which ultimately compromises the strategy of efficient disease management. Several modern techniques exhibiting assured results with enhanced sensitivity and specificity against the SARS-CoV-2 associated viral components or immune response against it have been developed and may be implemented. The review deals with the conventional RT-PCR detection techniques and compares them to other detection platforms viz., biosensor based detection of antigens, fluorescent or colorimetric detection systems including CRISPR-Cas 13 based SHERLOCK kit, CRISPR Cas-9 based FELUDA test kit, CRISPR DETECTR kit, Next Generation Sequencing or microarray-based kits. These modern techniques are great as a point of care detection methods but should be followed by RT PCR based detection for the confirmation of COVID-19 status.},
}
@article {pmid33418061,
year = {2021},
author = {Zhao, D and Jones, JL and Gasperini, RJ and Charlesworth, JC and Liu, GS and Burdon, KP},
title = {Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {37-47},
doi = {10.1016/j.ymeth.2020.12.004},
pmid = {33418061},
issn = {1095-9130},
mesh = {Animals ; Blindness ; CRISPR-Cas Systems ; *Cataract/genetics ; Gene Editing ; Humans ; Ribonucleoproteins/genetics/metabolism ; *Zebrafish/genetics/metabolism ; },
abstract = {Cataract is the leading cause of blindness worldwide. Congenital or paediatric cataract can result in permanent visual impairment or blindness even with best attempts at treatment. A significant proportion of paediatric cataract has a genetic cause. Therefore, identifying the genes that lead to cataract formation is essential for understanding the pathological process of inherited paediatric cataract as well as to the development of new therapies. Despite clear progress in genomics technologies, verification of the biological effects of newly identified candidate genes and variants is still challenging. Here, we provide a step-by-step pipeline to evaluate cataract candidate genes in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes (RNP). Detailed descriptions of CRISPR-Cas9 RNP design and formulation, microinjection, optimization of CRISPR-Cas9 RNP reagent dose and delivery route, editing efficacy analysis as well as cataract formation evaluation are included. Following this protocol, any cataract candidates can be readily and efficiently evaluated within 2 weeks using basic laboratory supplies.},
}
@article {pmid33417623,
year = {2021},
author = {Louie, W and Shen, MW and Tahiry, Z and Zhang, S and Worstell, D and Cassa, CA and Sherwood, RI and Gifford, DK},
title = {Machine learning based CRISPR gRNA design for therapeutic exon skipping.},
journal = {PLoS computational biology},
volume = {17},
number = {1},
pages = {e1008605},
pmid = {33417623},
issn = {1553-7358},
support = {R21 HG010391/HG/NHGRI NIH HHS/United States ; R01 HG008754/HG/NHGRI NIH HHS/United States ; R01 HG010372/HG/NHGRI NIH HHS/United States ; R01 HG008363/HG/NHGRI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Embryonic Stem Cells ; Exons ; Gene Editing/*methods ; Gene Library ; Genetic Therapy/*methods ; Humans ; *Machine Learning ; Mice ; RNA, Guide/*genetics ; },
abstract = {Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent alleviation of genetic disease. We show that machine learning can select Cas9 guide RNAs that disrupt splice acceptors and cause the skipping of targeted exons. We experimentally measured the exon skipping frequencies of a diverse genome-integrated library of 791 splice sequences targeted by 1,063 guide RNAs in mouse embryonic stem cells. We found that our method, SkipGuide, is able to identify effective guide RNAs with a precision of 0.68 (50% threshold predicted exon skipping frequency) and 0.93 (70% threshold predicted exon skipping frequency). We anticipate that SkipGuide will be useful for selecting guide RNA candidates for evaluation of CRISPR-Cas9-mediated exon skipping therapy.},
}
@article {pmid33416493,
year = {2021},
author = {Kroll, F and Powell, GT and Ghosh, M and Gestri, G and Antinucci, P and Hearn, TJ and Tunbak, H and Lim, S and Dennis, HW and Fernandez, JM and Whitmore, D and Dreosti, E and Wilson, SW and Hoffman, EJ and Rihel, J},
title = {A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.},
journal = {eLife},
volume = {10},
number = {},
pages = {},
pmid = {33416493},
issn = {2050-084X},
support = {217150/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; MR/L003775/1/MRC_/Medical Research Council/United Kingdom ; BB/T001844/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 204708/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; G0501487/MRC_/Medical Research Council/United Kingdom ; MR/T020164/1/MRC_/Medical Research Council/United Kingdom ; G0900994/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 095722/Z/11/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Behavior, Animal ; *CRISPR-Cas Systems ; Embryo, Nonmammalian ; *Gene Knockout Techniques ; Genetic Testing/*methods ; Phenotype ; RNA, Guide/*analysis ; Zebrafish/embryology/*genetics ; },
abstract = {Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.},
}
@article {pmid33414358,
year = {2020},
author = {Xue, G and Chen, JP and Li, Y and Zhang, ZQ and Zhu, JL and Dong, W},
title = {MicroRNA-6862 inhibition elevates sphingosine kinase 1 and protects neuronal cells from MPP[+]-induced apoptosis.},
journal = {Aging},
volume = {13},
number = {1},
pages = {1369-1382},
pmid = {33414358},
issn = {1945-4589},
mesh = {1-Methyl-4-phenylpyridinium/toxicity ; Antagomirs/pharmacology ; Apoptosis/drug effects/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival ; Gene Knockout Techniques ; Humans ; MicroRNAs/genetics/*metabolism ; Neurons/drug effects/*metabolism ; Parkinson Disease/genetics/metabolism ; Parkinsonian Disorders/genetics/metabolism ; Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism ; RNA, Messenger ; },
abstract = {MPP[+] (1-methyl-4-phenylpyridinium)-induced dopaminergic neuronal cell apoptosis is associated with sphingosine kinase 1 (SphK1) inhibition. We here tested the potential effect of microRNA-6862 (miR-6862), a novel SphK1-targeting miRNA, on MPP[+]-induced cytotoxicity in neuronal cells. MiR-6862 locates in the cytoplasm of SH-SY5Y neuronal cells. It directly binds to SphK1 mRNA. In SH-SY5Y cells and HCN-2 cells, ectopic overexpression of miR-6862 decreased SphK13'-untranslated region luciferase reporter activity and downregulated its expression. miR-6862 inhibition exerted opposite activity and elevated SphK1 expression. In neuronal cells, MPP[+]-induced cell death was significantly inhibited through miR-6862 inhibition. Conversely, ectopic overexpression of miR-6862 or CRISPR/Cas9-induced SphK1 knockout augmented MPP[+]-induced apoptosis in the neuronal cells. Importantly, antagomiR-6862 failed to inhibit MPP[+]-induced apoptosis in SphK1-knockout SH-SY5Y cells. These results suggest that inhibition of miR-6862 induces SphK1 elevation and protects neuronal cells from MPP[+]-induced cell death.},
}
@article {pmid33414202,
year = {2021},
author = {Kaiser, J},
title = {Gene therapy beats premature-aging syndrome in mice.},
journal = {Science (New York, N.Y.)},
volume = {371},
number = {6525},
pages = {114},
doi = {10.1126/science.371.6525.114},
pmid = {33414202},
issn = {1095-9203},
mesh = {Adolescent ; Animals ; CRISPR-Cas Systems ; Child ; Child, Preschool ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Mice ; Progeria/*therapy ; },
}
@article {pmid33413301,
year = {2021},
author = {Lee, S and Jung, MH and Song, K and Jin, JX and Taweechaipaisankul, A and Kim, GA and Oh, HJ and Koo, OJ and Park, SC and Lee, BC},
title = {Failure to maintain full-term pregnancies in pig carrying klotho monoallelic knockout fetuses.},
journal = {BMC biotechnology},
volume = {21},
number = {1},
pages = {1},
pmid = {33413301},
issn = {1472-6750},
mesh = {Aging/physiology ; Animals ; Blastocyst ; CRISPR-Cas Systems ; Cell Line ; Cloning, Organism ; Female ; Fetal Development ; Fetus/*metabolism ; Fibroblasts/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Glucuronidase/*genetics/*metabolism ; Klotho Proteins ; Nuclear Transfer Techniques ; Placenta ; Pregnancy ; Swine ; },
abstract = {BACKGROUND: Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs.<br><br>RESULTS: In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7&#x2009;days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes.<br><br>CONCLUSIONS: Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.},
}
@article {pmid33412935,
year = {2021},
author = {Graham, C and Hart, S},
title = {CRISPR/Cas9 gene editing therapies for cystic fibrosis.},
journal = {Expert opinion on biological therapy},
volume = {21},
number = {6},
pages = {767-780},
doi = {10.1080/14712598.2021.1869208},
pmid = {33412935},
issn = {1744-7682},
mesh = {CRISPR-Cas Systems/genetics ; *Cystic Fibrosis/genetics/therapy ; DNA End-Joining Repair ; *Gene Editing ; Genetic Therapy ; Humans ; },
abstract = {Cystic fibrosis (CF) is a life-limiting genetic disorder affecting approximately 70,000 people worldwide. Current burden of treatment is high. While the latest pharmaceutical innovation has benefitted many, patients with certain genotypes remain excluded. Gene editing has the potential to correct the underlying cause of disease for all patients, representing a permanent cure.Areas covered: Various DNA editing-based strategies for treatment are currently being developed. Different strategies are called for based upon location of mutations (intronic vs. exonic), delivery mechanism of editing machinery, and cell type being targeted. Furthermore, the unique physiology of the CF lung presents a variety of barriers to delivery of CRISPR-Cas9 machinery.Expert opinion: The most significant obstacle to the use of CRISPR-Cas9 in vivo is the fact that the most clinically relevant and accessible CF tissue, the airway epithelium, is made up of non-dividing cells where precise editing via homology-directed repair (HDR) does not occur; rather, potentially deleterious imprecise editing via non-homologous end joining (NHEJ) dominates. Future research should focus on the development of either more precise NHEJ-based approaches, access to airway basal cells, editing approaches that do not involve introducing genomic double-strand breaks, and strategies with ex vivo edited cells.},
}
@article {pmid33412762,
year = {2020},
author = {Park, YK and Yoon, BH and Park, SJ and Kim, BK and Kim, SY},
title = {BaSDAS: a web-based pooled CRISPR-Cas9 knockout screening data analysis system.},
journal = {Genomics &amp; informatics},
volume = {18},
number = {4},
pages = {e46},
pmid = {33412762},
issn = {1598-866X},
abstract = {We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.},
}
@article {pmid33412428,
year = {2021},
author = {Liu, PF and Zhao, KR and Liu, ZJ and Wang, L and Ye, SY and Liang, GX},
title = {Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {176},
number = {},
pages = {112954},
doi = {10.1016/j.bios.2020.112954},
pmid = {33412428},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA/genetics ; Gold ; Humans ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.},
}
@article {pmid33411765,
year = {2021},
author = {Jubair, L and Lam, AK and Fallaha, S and McMillan, NAJ},
title = {CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice.},
journal = {PloS one},
volume = {16},
number = {1},
pages = {e0223288},
pmid = {33411765},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Death/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Gene Editing/methods ; Genetic Therapy/methods ; HeLa Cells ; Human papillomavirus 16/*genetics ; Humans ; Jurkat Cells ; Liposomes/*metabolism ; Mice ; Mice, Inbred C57BL ; Neoplasms/*genetics/virology ; Oncogene Proteins, Viral/genetics ; Papillomavirus E7 Proteins/genetics ; Papillomavirus Infections/*genetics/virology ; Uterine Cervical Neoplasms/*genetics/virology ; },
abstract = {Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.},
}
@article {pmid33411694,
year = {2021},
author = {Schmid, RS and Deng, X and Panikker, P and Msackyi, M and Breton, C and Wilson, JM},
title = {CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.},
journal = {The Journal of clinical investigation},
volume = {131},
number = {5},
pages = {},
pmid = {33411694},
issn = {1558-8238},
mesh = {Angelman Syndrome/genetics/*metabolism/*therapy ; Animals ; Brain/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Mice ; RNA, Antisense/genetics/*metabolism ; Ubiquitin-Protein Ligases/genetics/*metabolism ; },
abstract = {Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.},
}
@article {pmid33411517,
year = {2021},
author = {Zhou, T and Huang, M and Lin, J and Huang, R and Xing, D},
title = {High-Fidelity CRISPR/Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA.},
journal = {Analytical chemistry},
volume = {93},
number = {4},
pages = {2038-2044},
doi = {10.1021/acs.analchem.0c03708},
pmid = {33411517},
issn = {1520-6882},
mesh = {Biosensing Techniques ; *CRISPR-Cas Systems ; Cell Line ; DNA, Catalytic/genetics/*metabolism ; Humans ; MicroRNAs/*chemistry ; Protein Conformation ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.},
}
@article {pmid33410425,
year = {2020},
author = {Sreepadmanabh, M and Sahu, AK and Chande, A},
title = {COVID-19: Advances in diagnostic tools, treatment strategies, and vaccine development.},
journal = {Journal of biosciences},
volume = {45},
number = {1},
pages = {},
pmid = {33410425},
issn = {0973-7138},
mesh = {Antiviral Agents/chemical synthesis/*therapeutic use ; Artificial Intelligence ; COVID-19/immunology/*prevention &amp; control/therapy/virology ; COVID-19 Nucleic Acid Testing/*methods ; COVID-19 Vaccines/*biosynthesis/genetics ; CRISPR-Cas Systems ; Disease Management ; Drug Discovery/methods ; Drug Repositioning/methods ; Humans ; Immunization, Passive/methods ; Molecular Diagnostic Techniques ; Molecular Docking Simulation ; Nucleic Acid Amplification Techniques ; Pandemics/*prevention &amp; control ; Protein Engineering/methods ; SARS-CoV-2/drug effects/immunology/*pathogenicity ; },
abstract = {An unprecedented worldwide spread of the SARS-CoV-2 has imposed severe challenges on healthcare facilities and medical infrastructure. The global research community faces urgent calls for the development of rapid diagnostic tools, effective treatment protocols, and most importantly, vaccines against the pathogen. Pooling together expertise across broad domains to innovate effective solutions is the need of the hour. With these requirements in mind, in this review, we provide detailed critical accounts on the leading efforts at developing diagnostics tools, therapeutic agents, and vaccine candidates. Importantly, we furnish the reader with a multidisciplinary perspective on how conventional methods like serology and RT-PCR, as well as cutting-edge technologies like CRISPR/Cas and artificial intelligence/machine learning, are being employed to inform and guide such investigations. We expect this narrative to serve a broad audience of both active and aspiring researchers in the field of biomedical sciences and engineering and help inspire radical new approaches towards effective detection, treatment, and prevention of this global pandemic.},
}
@article {pmid33410130,
year = {2021},
author = {Huang, D and Shi, Z and Qian, J and Bi, K and Fang, M and Xu, Z},
title = {A CRISPR-Cas12a-derived biosensor enabling portable personal glucose meter readout for quantitative detection of SARS-CoV-2.},
journal = {Biotechnology and bioengineering},
volume = {118},
number = {4},
pages = {1587-1596},
doi = {10.1002/bit.27673},
pmid = {33410130},
issn = {1097-0290},
mesh = {*Biosensing Techniques/instrumentation ; COVID-19/*diagnosis ; COVID-19 Testing ; *CRISPR-Cas Systems ; Glucose/*analysis ; Humans ; Nucleic Acid Amplification Techniques ; SARS-CoV-2/*isolation &amp; purification ; },
abstract = {Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the whole world and caused significant difficulties in the prevention and control of the epidemic. In this case, several detection methods have been established based on nucleic acid diagnostic techniques and immunoassays to achieve sensitive and specific detection of SARS-CoV-2. However, most methods are still largely dependent on professional instruments, highly trained operators, and centralized laboratories. These limitations gravely diminish their practicality and portability. Herein, a clustered regularly interspaced short palindromic repeats (CRISPR) Cas12a based assay was developed for portable, rapid and sensitive of SARS-CoV-2. In this assay, samples were quickly pretreated and amplified by reverse transcription recombinase-aided amplification under mild conditions. Then, by combining the CRISPR Cas12a system and a glucose-producing reaction, the signal of the virus was converted to that of glucose, which can be quantitatively read by a personal glucose meter in a few seconds. Nucleocapsid protein gene was tested as a model target, and the sensitivity for quantitative detection was as low as 10 copies/μl, which basically meet the needs of clinical diagnosis. In addition, with the advantages of lower material cost, shorter detection time, and no requirement for professional instrument in comparison with quantitative reverse transcription-polymerase chain reaction, this assay is expected to provide a powerful technical support for the early diagnosis and intervention during epidemic prevention and control.},
}
@article {pmid33408386,
year = {2021},
author = {Tang, L},
title = {Versatile genome editing.},
journal = {Nature methods},
volume = {18},
number = {1},
pages = {27},
doi = {10.1038/s41592-020-01025-y},
pmid = {33408386},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Epigenomics/*methods ; *Gene Editing ; Genetic Engineering/*methods ; *Genome, Human ; Humans ; },
}
@article {pmid33407863,
year = {2021},
author = {Le, QA and Tanihara, F and Wittayarat, M and Namula, Z and Sato, Y and Lin, Q and Takebayashi, K and Hirata, M and Otoi, T},
title = {Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages.},
journal = {BMC research notes},
volume = {14},
number = {1},
pages = {7},
pmid = {33407863},
issn = {1756-0500},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation ; Microinjections ; Swine ; },
abstract = {OBJECTIVE: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos.<br><br>RESULTS: First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p&#x2009;<&#x2009;0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p&#x2009;<&#x2009;0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.},
}
@article {pmid33407829,
year = {2021},
author = {Kim, E and Hart, T},
title = {Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier.},
journal = {Genome medicine},
volume = {13},
number = {1},
pages = {2},
pmid = {33407829},
issn = {1756-994X},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; },
mesh = {*Algorithms ; Bayes Theorem ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Data Accuracy ; *Genes, Essential ; *Genetic Testing ; Humans ; Likelihood Functions ; Regression Analysis ; },
abstract = {BACKGROUND: Identifying essential genes in genome-wide loss-of-function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome-wide genetic screens.<br><br>RESULTS: We introduce an updated version, BAGEL2, which employs an improved model that offers a greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes; a multi-target correction that reduces false positives from off-target CRISPR guide RNA; and the implementation of a cross-validation strategy that improves performance ~&#x2009;10&#xd7; over the prior bootstrap resampling approach. We also describe a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower quality data in substantially different ways.<br><br>CONCLUSIONS: BAGEL2 substantially improves the sensitivity, specificity, and performance over BAGEL and establishes the new state of the art in the analysis of CRISPR knockout fitness screens. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).},
}
@article {pmid33407116,
year = {2021},
author = {Zhang, Y and Held, MA and Kaur, D and Showalter, AM},
title = {CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis.},
journal = {BMC plant biology},
volume = {21},
number = {1},
pages = {16},
pmid = {33407116},
issn = {1471-2229},
mesh = {Arabidopsis/*genetics/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Galactans/genetics/*metabolism ; Galactosyltransferases/*genetics/*metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Genes, Plant ; Genetic Variation ; Genome, Plant ; Genotype ; Glycosylation ; Mucoproteins/genetics/*metabolism ; Mutation ; Plant Breeding/methods ; },
abstract = {BACKGROUND: Arabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich proteins (HRGPs) that are heavily glycosylated (> 90%) with type II arabinogalactans (AGs). AGPs are implicated in various plant growth and development processes including cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, hormone signaling, male and female gametophyte development, and defense. To date, eight Hyp-O-galactosyltransferases (GALT2-6, HPGT1-3) have been identified; these enzymes are responsible for adding the first sugar, galactose, onto AGPs. Due to gene redundancy among the GALTs, single or double galt genetic knockout mutants are often not sufficient to fully reveal their biological functions.<br><br>RESULTS: Here, we report the successful application of CRISPR-Cas9 gene editing/multiplexing technology to generate higher-order knockout mutants of five members of the GALT gene family (GALT2-6). AGPs analysis of higher-order galt mutants (galt2 galt5, galt3 galt4 galt6, and galt2 galt3 galt4 galt5 gal6) demonstrated significantly less glycosylated AGPs in rosette leaves, stems, and siliques compared to the corresponding wild-type organs. Monosaccharide composition analysis of AGPs isolated from rosette leaves revealed significant decreases in arabinose and galactose in all the higher-order galt mutants. Phenotypic analyses revealed that mutation of two or more GALT genes was able to overcome the growth inhibitory effect of &#x3b2;-D-Gal-Yariv reagent, which specifically binds to &#x3b2;-1,3-galactan backbones on AGPs. In addition, the galt2 galt3 galt4 galt5 gal6 mutant exhibited reduced overall growth, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Reciprocal crossing experiments demonstrated that galt2 galt3 galt4 galt5 gal6 mutants had defects in the female gametophyte which were responsible for reduced seed set.<br><br>CONCLUSIONS: Our CRISPR/Cas9 gene editing/multiplexing approach provides a simpler and faster way to generate higher-order mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. Higher-order galt mutants produced and characterized in this study provide insight into the relationship between sugar decorations and the various biological functions attributed to AGPs in plants.},
}
@article {pmid33406420,
year = {2021},
author = {Shirasaki, R and Matthews, GM and Gandolfi, S and de Matos Simoes, R and Buckley, DL and Raja Vora, J and Sievers, QL and Brüggenthies, JB and Dashevsky, O and Poarch, H and Tang, H and Bariteau, MA and Sheffer, M and Hu, Y and Downey-Kopyscinski, SL and Hengeveld, PJ and Glassner, BJ and Dhimolea, E and Ott, CJ and Zhang, T and Kwiatkowski, NP and Laubach, JP and Schlossman, RL and Richardson, PG and Culhane, AC and Groen, RWJ and Fischer, ES and Vazquez, F and Tsherniak, A and Hahn, WC and Levy, J and Auclair, D and Licht, JD and Keats, JJ and Boise, LH and Ebert, BL and Bradner, JE and Gray, NS and Mitsiades, CS},
title = {Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.},
journal = {Cell reports},
volume = {34},
number = {1},
pages = {108532},
pmid = {33406420},
issn = {2211-1247},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA179483/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; R01 CA180475/CA/NCI NIH HHS/United States ; R00 CA190861/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; R01 CA196664/CA/NCI NIH HHS/United States ; U01 CA225730/CA/NCI NIH HHS/United States ; R01 CA050947/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*metabolism ; Animals ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9/metabolism ; Drug Resistance, Neoplasm ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genes, Overlapping ; Genome-Wide Association Study ; Genomics/methods ; Humans ; Mice ; Multiple Myeloma/drug therapy/*genetics/*metabolism ; Oncogene Proteins/metabolism ; Proteins/antagonists &amp; inhibitors/metabolism ; Proteolysis ; Tumor Cells, Cultured ; Ubiquitin-Protein Ligases/*metabolism ; Von Hippel-Lindau Tumor Suppressor Protein/*metabolism ; },
abstract = {Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.},
}
@article {pmid33406233,
year = {2021},
author = {Ceccato-Antonini, SR and Covre, EA},
title = {From baker's yeast to genetically modified budding yeasts: the scientific evolution of bioethanol industry from sugarcane.},
journal = {FEMS yeast research},
volume = {20},
number = {8},
pages = {},
doi = {10.1093/femsyr/foaa065},
pmid = {33406233},
issn = {1567-1364},
mesh = {*Biofuels ; CRISPR-Cas Systems ; *Ethanol ; Fermentation ; Gene Editing ; *Industrial Microbiology/trends ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomycetales/genetics/*metabolism ; *Saccharum ; },
abstract = {The peculiarities of Brazilian fuel ethanol fermentation allow the entry of native yeasts that may dominate over the starter strains of Saccharomyces cerevisiae and persist throughout the sugarcane harvest. The switch from the use of baker's yeast as starter to selected budding yeasts obtained by a selective pressure strategy was followed by a wealth of genomic information that enabled the understanding of the superiority of selected yeast strains. This review describes how the process of yeast selection evolved in the sugarcane-based bioethanol industry, the selection criteria and recent advances in genomics that could advance the fermentation process. The prospective use of genetically modified yeast strains, specially designed for increased robustness and product yield, with special emphasis on those obtained by the CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome-editing approach, is discussed as a possible solution to confer higher performance and stability to the fermentation process for fuel ethanol production.},
}
@article {pmid33405030,
year = {2021},
author = {Gago, S and Overton, NLD and Bowyer, P},
title = {CRISPR-Cas9-Mediated Gene Silencing in Cultured Human Epithelia.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2260},
number = {},
pages = {37-47},
pmid = {33405030},
issn = {1940-6029},
mesh = {Bronchi/*metabolism/microbiology ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epithelial Cells/*metabolism/microbiology ; Fungi/*pathogenicity ; *Gene Editing ; Gene Expression Regulation ; *Gene Silencing ; Host-Parasite Interactions ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {CRISPR/Cas9 technology enables rapid and efficient genome editing in a variety of experimental systems. Genome editing using CRISPR/Cas9 has become an increasingly popular genetic engineering tool due to (1) an extensive array of commercial ready-to-use CRIPSR/Cas9 systems, (2) improved efficiency of cell delivery, and (3) the possibility to do multigene editing. Here, we describe a method to introduce single gene disruption in lung bronchial epithelial cells. This approach can be used to study host factors important for pathogen interaction or to identify and study genetic markers determining susceptibility to fungal disease.},
}
@article {pmid33405027,
year = {2021},
author = {Woolford, CA and Mitchell, AP},
title = {Diminished Expression Alleles for Analysis of Virulence Traits and Genetic Interactions in Candida albicans.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2260},
number = {},
pages = {1-13},
pmid = {33405027},
issn = {1940-6029},
support = {R21 AI100270/AI/NIAID NIH HHS/United States ; R01 AI067703/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Candida albicans/*genetics/metabolism/pathogenicity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fungal Proteins/*genetics/metabolism ; *Gene Editing ; Gene Expression Regulation, Fungal ; Genes, Essential ; Mutation ; Saccharomyces cerevisiae/genetics/metabolism ; Virulence/genetics ; Virulence Factors/*genetics/metabolism ; },
abstract = {Here we present a protocol of interest to those who want to look at the functional consequences of decreasing expression of essential genes constitutively as well as being able to study the regulatory pathways of that essential gene by also deleting one or two additional genes. This allows epistasis relationships to be determined.},
}
@article {pmid33404234,
year = {2021},
author = {Kachwala, MJ and Smith, CW and Nandu, N and Yigit, MV},
title = {Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction.},
journal = {Analytical chemistry},
volume = {93},
number = {4},
pages = {1934-1938},
pmid = {33404234},
issn = {1520-6882},
support = {R15 GM128115/GM/NIGMS NIH HHS/United States ; },
mesh = {Biosensing Techniques/methods ; *CRISPR-Cas Systems ; DNA/chemistry ; Electrophoresis, Gel, Two-Dimensional/*methods ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acid Hybridization/methods ; },
abstract = {Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.},
}
@article {pmid33402545,
year = {2020},
author = {Konishi, CT and Long, C},
title = {Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases.},
journal = {Journal of biomedical research},
volume = {35},
number = {2},
pages = {148-162},
pmid = {33402545},
issn = {1674-8301},
abstract = {There are an estimated 10 000 monogenic diseases affecting tens of millions of individuals worldwide. The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients. CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA. The complexity of genomic insults resulting in heritable disease requires patient-specific genome editing strategies with consideration of DNA repair pathways, and CRISPR/Cas systems of different types, species, and those with additional enzymatic capacity and/or delivery methods. In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair, non-homologous end joining, microhomology-mediated end joining, and base editing to permanently correct diverse monogenic diseases.},
}
@article {pmid33402532,
year = {2021},
author = {Rodríguez de Los Santos, M and Rivalan, M and David, FS and Stumpf, A and Pitsch, J and Tsortouktzidis, D and Velasquez, LM and Voigt, A and Schilling, K and Mattei, D and Long, M and Vogt, G and Knaus, A and Fischer-Zirnsak, B and Wittler, L and Timmermann, B and Robinson, PN and Horn, D and Mundlos, S and Kornak, U and Becker, AJ and Schmitz, D and Winter, Y and Krawitz, PM},
title = {A CRISPR-Cas9-engineered mouse model for GPI-anchor deficiency mirrors human phenotypes and exhibits hippocampal synaptic dysfunctions.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {2},
pages = {},
pmid = {33402532},
issn = {1091-6490},
mesh = {Abnormalities, Multiple/genetics ; Amino Acid Sequence ; Amino Acids/genetics ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Epilepsy/genetics ; Glycosylphosphatidylinositols/deficiency/*genetics/*metabolism ; Hippocampus/metabolism ; Intellectual Disability/genetics ; Mannosyltransferases/*metabolism/physiology ; Mice ; Mice, Inbred C57BL ; Mutation ; Mutation, Missense ; Phenotype ; Protein Engineering/methods ; Seizures/genetics/physiopathology ; },
abstract = {Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI)-anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C > A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv[341E] mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv[341E] mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv[341E]-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor-related pathways in brain development.},
}
@article {pmid33402270,
year = {2021},
author = {Zhang, Y and Xi, H and Juhas, M},
title = {Biosensing Detection of the SARS-CoV-2 D614G Mutation.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {4},
pages = {299-302},
pmid = {33402270},
issn = {0168-9525},
mesh = {Amino Acid Substitution ; *Biosensing Techniques ; *Mutation ; SARS-CoV-2/*genetics ; Spike Glycoprotein, Coronavirus/*genetics ; },
abstract = {The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.},
}
@article {pmid33401973,
year = {2021},
author = {Aslesh, T and Erkut, E and Yokota, T},
title = {Restoration of dystrophin expression and correction of Duchenne muscular dystrophy by genome editing.},
journal = {Expert opinion on biological therapy},
volume = {21},
number = {8},
pages = {1049-1061},
doi = {10.1080/14712598.2021.1872539},
pmid = {33401973},
issn = {1744-7682},
support = {//CIHR/Canada ; },
mesh = {Adult ; Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Dystrophin/genetics ; Exons ; Gene Editing ; Humans ; Male ; *Muscular Dystrophy, Duchenne/genetics/therapy ; },
abstract = {Introduction: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder that affects approximately one in 3500-5000 male births. Patients experience muscle degeneration, loss of ambulation, and eventual death from cardiac or respiratory failure in early adulthood due to a lack of functional dystrophin protein, which is required to maintain the integrity of muscle cell membranes. Out-of-frame mutations in the DMD gene generally lead to no dystrophin protein expression and a more severe phenotype (DMD). Conversely, in-frame mutations are often associated with milder Becker muscular dystrophy (BMD) with a truncated dystrophin expression.Areas covered: Genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system can induce permanent corrections of the DMD gene, thus becoming an increasingly popular potential therapeutic method. In this review, we outline recent developments in CRISPR/Cas9 genome editing for the correction of DMD, both in vitro and in vivo, as well as novel delivery methods.Expert opinion: Despite recent advances, many limitations to CRISPR/Cas9 therapy are still prevalent such as off-target editing and immunogenicity. Specifically, for DMD, intervention time and efficient delivery to cardiac and skeletal muscles also present inherent challenges. Research needs to focus on the therapeutic safety and efficacy of this approach.},
}
@article {pmid33401751,
year = {2021},
author = {Taliansky, M and Samarskaya, V and Zavriev, SK and Fesenko, I and Kalinina, NO and Love, AJ},
title = {RNA-Based Technologies for Engineering Plant Virus Resistance.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {1},
pages = {},
pmid = {33401751},
issn = {2223-7747},
abstract = {In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.},
}
@article {pmid33401227,
year = {2021},
author = {Pan, C and Sretenovic, S and Qi, Y},
title = {CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants.},
journal = {Current opinion in plant biology},
volume = {60},
number = {},
pages = {101980},
doi = {10.1016/j.pbi.2020.101980},
pmid = {33401227},
issn = {1879-0356},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Epigenesis, Genetic/genetics ; Gene Editing ; Plants/genetics ; },
abstract = {The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants.},
}
@article {pmid33400990,
year = {2021},
author = {Wang, T and Yue, S and Jin, Y and Wei, H and Lu, L},
title = {Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {147},
number = {},
pages = {103509},
doi = {10.1016/j.fgb.2020.103509},
pmid = {33400990},
issn = {1096-0937},
mesh = {CRISPR-Cas Systems ; Fungal Proteins/*genetics ; Gene Editing/*methods ; Pleurotus/*genetics ; },
abstract = {For decades, the edible mushroom Pleurotus eryngii (P. eryngii) has been cultivated as important raw materials for food and pharmaceutical industries in most of Asian countries, especially in China. Unfortunately, the generation and improvement of new cultivars are very difficult since there are many barriers which have not been solved thoroughly by gene editing tools, even though the CRISPR-Cas9 technique has been widely applied in other species. In this study, we identified the point-mutated variant of the endogenous sdhB gene (cbx[r]) as a more stable selection marker than hygromycin B resistance gene (hph) in P. eryngii. Furthermore, using a codon-optimized Cas9, a predicted native U6 promoter-guided sgRNA, as well as an optimized protoplast transformation system, a highly efficient pyrG gene editing system was established in P. eryngii, that incorporated varied insertions and deletions (indels) by non-homologous end joining (NHEJ) and homology-directed repair (HDR). Findings for a successful targeted gene editing strategy in the edible mushroom P. eryngii may open a new chapter for the improvement of edible mushroom cultivars.},
}
@article {pmid33400844,
year = {2021},
author = {Cuevas, E and Holder, DL and Alshehri, AH and Tréguier, J and Lakowski, J and Sowden, JC},
title = {NRL[-/-] gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors.},
journal = {Stem cells (Dayton, Ohio)},
volume = {39},
number = {4},
pages = {414-428},
pmid = {33400844},
issn = {1549-4918},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/M015688/1/MRC_/Medical Research Council/United Kingdom ; 109053/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Base Sequence ; Basic-Leucine Zipper Transcription Factors/deficiency/*genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Exons ; Eye Proteins/*genetics ; Gene Editing/methods ; Gene Expression ; Homeodomain Proteins/genetics/metabolism ; Human Embryonic Stem Cells/cytology/*metabolism ; Humans ; Opsins/genetics/metabolism ; Organoids/*metabolism/pathology ; Recoverin/genetics/metabolism ; Retinal Cone Photoreceptor Cells/*metabolism/pathology ; Retinoid X Receptor gamma/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Zonula Occludens-1 Protein/genetics/metabolism ; },
abstract = {Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL[-/-]), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL[-/-] OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL[-/-] OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.},
}
@article {pmid33399467,
year = {2021},
author = {Qin, Z and Yang, Y and Yu, S and Liu, L and Chen, Y and Chen, J and Zhou, J},
title = {Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003.},
journal = {ACS synthetic biology},
volume = {10},
number = {1},
pages = {84-93},
doi = {10.1021/acssynbio.0c00456},
pmid = {33399467},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Carbon Cycle/genetics ; Gene Editing/*methods ; Gene Expression ; Gluconobacter oxydans/genetics/*metabolism ; Luminescent Proteins/genetics/metabolism ; Metabolic Engineering ; Pentose Phosphate Pathway/genetics ; },
abstract = {Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.},
}
@article {pmid33398349,
year = {2021},
author = {Wang, Q and Liu, J and Janssen, JM and Le Bouteiller, M and Frock, RL and Gonçalves, MAFV},
title = {Precise and broad scope genome editing based on high-specificity Cas9 nickases.},
journal = {Nucleic acids research},
volume = {49},
number = {2},
pages = {1173-1198},
pmid = {33398349},
issn = {1362-4962},
mesh = {Bacterial Proteins/genetics/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Clone Cells ; Deoxyribonuclease I/genetics/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genotyping Techniques ; HEK293 Cells ; HeLa Cells ; Heterochromatin/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Induced Pluripotent Stem Cells ; Polymorphism, Genetic ; RNA, Guide/genetics ; Recombinant Proteins/metabolism ; Streptococcus pyogenes/enzymology ; Substrate Specificity ; Transfection ; },
abstract = {RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.},
}
@article {pmid33398345,
year = {2021},
author = {Doi, G and Okada, S and Yasukawa, T and Sugiyama, Y and Bala, S and Miyazaki, S and Kang, D and Ito, T},
title = {Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability.},
journal = {Nucleic acids research},
volume = {49},
number = {2},
pages = {954-968},
pmid = {33398345},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/chemistry/*metabolism ; *CRISPR-Cas Systems ; DNA Copy Number Variations ; *DNA Replication ; DNA, Fungal/*genetics/metabolism ; DNA, Single-Stranded/genetics ; Gene Editing ; Genes, Fungal ; *Genomic Instability ; Metallothionein/genetics ; Mutagenesis ; Niacinamide/pharmacology ; Rad52 DNA Repair and Recombination Protein/metabolism ; Saccharomyces cerevisiae/enzymology/*genetics ; Saccharomyces cerevisiae Proteins/chemistry/genetics/*metabolism ; Tandem Repeat Sequences ; },
abstract = {Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.},
}
@article {pmid33398341,
year = {2021},
author = {Fu, YW and Dai, XY and Wang, WT and Yang, ZX and Zhao, JJ and Zhang, JP and Wen, W and Zhang, F and Oberg, KC and Zhang, L and Cheng, T and Zhang, XB},
title = {Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.},
journal = {Nucleic acids research},
volume = {49},
number = {2},
pages = {969-985},
pmid = {33398341},
issn = {1362-4962},
mesh = {Adult ; *CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Copy Number Variations ; *DNA End-Joining Repair/drug effects ; Dependovirus ; *Gene Editing ; Genetic Vectors/*genetics ; HEK293 Cells ; Humans ; Hydroxamic Acids/pharmacology ; INDEL Mutation ; Induced Pluripotent Stem Cells ; Kinetics ; Parvovirinae/*genetics ; RNA, Guide/genetics ; *Recombinational DNA Repair/drug effects ; Ribonucleoproteins/*metabolism ; T-Lymphocytes ; Transduction, Genetic ; },
abstract = {Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.},
}
@article {pmid33398196,
year = {2021},
author = {Torre, EA and Arai, E and Bayatpour, S and Jiang, CL and Beck, LE and Emert, BL and Shaffer, SM and Mellis, IA and Fane, ME and Alicea, GM and Budinich, KA and Weeraratna, AT and Shi, J and Raj, A},
title = {Genetic screening for single-cell variability modulators driving therapy resistance.},
journal = {Nature genetics},
volume = {53},
number = {1},
pages = {76-85},
pmid = {33398196},
issn = {1546-1718},
support = {T32 DK007780/DK/NIDDK NIH HHS/United States ; T32 HL007439/HL/NHLBI NIH HHS/United States ; DP5 OD028144/OD/NIH HHS/United States ; P30 CA006973/CA/NCI NIH HHS/United States ; R01 CA238237/CA/NCI NIH HHS/United States ; P30 CA010815/CA/NCI NIH HHS/United States ; F30 CA236129/CA/NCI NIH HHS/United States ; P01 CA114046/CA/NCI NIH HHS/United States ; T32 GM007170/GM/NIGMS NIH HHS/United States ; P50 CA174523/CA/NCI NIH HHS/United States ; R01 CA207935/CA/NCI NIH HHS/United States ; U01 CA227550/CA/NCI NIH HHS/United States ; F30 HG010822/HG/NHGRI NIH HHS/United States ; T32 EB009384/EB/NIBIB NIH HHS/United States ; R01 CA174746/CA/NCI NIH HHS/United States ; R01 CA232256/CA/NCI NIH HHS/United States ; U54 CA193417/CA/NCI NIH HHS/United States ; RM1 HG007743/HG/NHGRI NIH HHS/United States ; P30 CA016520/CA/NCI NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; R01 GM137425/GM/NIGMS NIH HHS/United States ; F30 NS100595/NS/NINDS NIH HHS/United States ; U01 HL129998/HL/NHLBI NIH HHS/United States ; T32 HG000046/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line, Tumor ; Cell Plasticity/*genetics ; Cell Proliferation/genetics ; Drug Resistance, Neoplasm/*drug effects/*genetics ; *Genetic Testing ; Histone-Lysine N-Methyltransferase/genetics ; Humans ; Melanoma/drug therapy/genetics/pathology ; Mice, Inbred NOD ; Mice, SCID ; Models, Biological ; Molecular Targeted Therapy ; Neoplasms/drug therapy/*genetics/*pathology ; Proto-Oncogene Proteins B-raf/genetics ; Transcription, Genetic ; },
abstract = {Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.},
}
@article {pmid33398178,
year = {2021},
author = {Gao, N and Hu, J and He, B and Ji, Z and Hu, X and Huang, J and Wei, Y and Peng, J and Wei, Y and Zhou, Y and Shen, X and Li, H and Feng, X and Xiao, Q and Shi, L and Sun, Y and Zhou, C and Zhou, H and Yang, H},
title = {Endogenous promoter-driven sgRNA for monitoring the expression of low-abundance transcripts and lncRNAs.},
journal = {Nature cell biology},
volume = {23},
number = {1},
pages = {99-108},
pmid = {33398178},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Neuroblastoma/*genetics/*metabolism/pathology ; *Promoter Regions, Genetic ; RNA, Guide/genetics/*metabolism ; RNA, Long Noncoding/genetics/*metabolism ; RNA, Messenger/genetics/*metabolism ; },
abstract = {Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.},
}
@article {pmid33398158,
year = {2021},
author = {Ren, Q and Sretenovic, S and Liu, S and Tang, X and Huang, L and He, Y and Liu, L and Guo, Y and Zhong, Z and Liu, G and Cheng, Y and Zheng, X and Pan, C and Yin, D and Zhang, Y and Li, W and Qi, L and Li, C and Qi, Y and Zhang, Y},
title = {PAM-less plant genome editing using a CRISPR-SpRY toolbox.},
journal = {Nature plants},
volume = {7},
number = {1},
pages = {25-33},
pmid = {33398158},
issn = {2055-0278},
mesh = {B30.2-SPRY Domain/genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant/*genetics ; Larix/genetics ; Oryza/genetics ; Protoplasts ; },
abstract = {The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research[1-6]. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)[7], limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant[8]. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. [9]) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.},
}
@article {pmid33398131,
year = {2021},
author = {Ling, S and Yang, S and Hu, X and Yin, D and Dai, Y and Qian, X and Wang, D and Pan, X and Hong, J and Sun, X and Yang, H and Paludan, SR and Cai, Y},
title = {Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {144-156},
pmid = {33398131},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Vectors/physiology ; HEK293 Cells ; Humans ; Lentivirus/*physiology ; Macular Degeneration/*genetics ; Mice ; Mice, Inbred C57BL ; RNA, Messenger/genetics ; Vascular Endothelial Growth Factor A/*genetics ; },
abstract = {Therapeutic genome editing requires effective and targeted delivery methods. The delivery of Cas9 mRNA using adeno-associated viruses has led to potent in vivo therapeutic efficacy, but can cause sustained Cas9 expression, anti-Cas9 immune responses and off-target edits. Lentiviral vectors have been engineered to deliver nucleases that are expressed transiently, but in vivo evidence of their biomedical efficacy is lacking. Here, we show that the lentiviral codelivery of Streptococcus pyogenes Cas9 mRNA and expression cassettes that encode a guide RNA that targets vascular endothelial growth factor A (Vegfa) is efficacious in a mouse model of wet age-related macular degeneration induced by Vegfa. A single subretinal injection of engineered lentiviruses knocked out 44% of Vegfa in retinal pigment epithelium and reduced the area of choroidal neovascularization by 63% without inducing off-target edits or anti-Cas9 immune responses. Engineered lentiviruses for the transient expression of nucleases may form the basis of new treatments for retinal neovascular diseases.},
}
@article {pmid33398095,
year = {2021},
author = {Smith, LM and Jackson, SA and Malone, LM and Ussher, JE and Gardner, PP and Fineran, PC},
title = {The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.},
journal = {Nature microbiology},
volume = {6},
number = {2},
pages = {162-172},
pmid = {33398095},
issn = {2058-5276},
mesh = {Bacterial Proteins/genetics/*physiology ; Bacteriophages/genetics/*physiology ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology ; Flow Cytometry ; Gene Expression Regulation, Bacterial ; High-Throughput Nucleotide Sequencing ; Mutagenesis ; Plasmids/genetics/physiology ; Serratia/*physiology/*virology ; Stress, Physiological/genetics ; },
abstract = {Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.},
}
@article {pmid33397707,
year = {2021},
author = {Goh, YJ and Barrangou, R},
title = {Portable CRISPR-Cas9[N] System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {6},
pages = {},
pmid = {33397707},
issn = {1098-5336},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Deoxyribonuclease I ; Gene Editing/*methods ; Genome, Bacterial ; Lactobacillus acidophilus/*genetics ; Lactobacillus gasseri/*genetics ; Lactobacillus paracasei/*genetics ; },
abstract = {Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplement industries, and specific strains, such as Lactobacillus acidophilus NCFM, have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR (clustered regularly interspaced palindromic repeats)-SpyCas9[D10A] nickase (Cas9[N])-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9[N] vector harboring cas9[N] under the regulation of a Lactobacillus promoter and a cloning region for a customized single guide RNA (sgRNA) and editing template. The functionality of the pLbCas9[N] system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS, and ltaS), yielding 35 to 100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9[N] system. To showcase the versatility of this system, we also inserted an mCherry fluorescent-protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9[N] system was further deployed in other species to generate a concurrent single-base substitution and gene deletion in Lactobacillus gasseri ATCC 33323 and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered with by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9[N] system for genome manipulations in diverse lactobacilli and open new avenues for the engineering of health-promoting lactic acid bacteria.IMPORTANCE This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three Lactobacillus species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.},
}
@article {pmid33397704,
year = {2021},
author = {Synefiaridou, D and Veening, JW},
title = {Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {6},
pages = {},
pmid = {33397704},
issn = {1098-5336},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Bacterial ; Streptococcus pneumoniae/*genetics ; Virulence Factors/genetics ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by the detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae Specifically, we demonstrate an approach for making targeted markerless gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) is exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid, which is temperature sensitive for replication, by growing it at the nonpermissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The approaches developed here could potentially be adapted for use with other Gram-positive bacteria.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing more than 1 million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts to identify new vaccine candidates. Here, we introduced an easy-to-use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The approaches used here will advance genome editing projects in this important human pathogen.},
}
@article {pmid33397431,
year = {2021},
author = {Xu, Z and Kuang, Y and Ren, B and Yan, D and Yan, F and Spetz, C and Sun, W and Wang, G and Zhou, X and Zhou, H},
title = {SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.},
journal = {Genome biology},
volume = {22},
number = {1},
pages = {6},
pmid = {33397431},
issn = {1474-760X},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA, Plant ; *Gene Editing ; Genes, Plant/*genetics ; Genome, Plant ; Genotype ; Oryza/*genetics ; Plants, Genetically Modified ; },
abstract = {BACKGROUND: Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome.<br><br>RESULTS: In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*&#x394; and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines.<br><br>CONCLUSIONS: The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.},
}
@article {pmid33396822,
year = {2020},
author = {Malabarba, J and Chevreau, E and Dousset, N and Veillet, F and Moizan, J and Vergne, E},
title = {New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {1},
pages = {},
pmid = {33396822},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Chimerism ; Cytidine Deaminase/genetics ; *Gene Editing ; Gene Targeting ; Genome, Plant ; Malus/*genetics ; Phenotype ; Plants, Genetically Modified ; Pyrus/*genetics ; Research Design ; },
abstract = {Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for "regenerated T0" lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase-ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.},
}
@article {pmid33396746,
year = {2020},
author = {Szulc, B and Zadorozhna, Y and Olczak, M and Wiertelak, W and Maszczak-Seneczko, D},
title = {Novel Insights into Selected Disease-Causing Mutations within the SLC35A1 Gene Encoding the CMP-Sialic Acid Transporter.},
journal = {International journal of molecular sciences},
volume = {22},
number = {1},
pages = {},
pmid = {33396746},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Cell Membrane/metabolism ; Chromatography, High Pressure Liquid ; Cytidine Monophosphate/metabolism ; Flow Cytometry ; Gene Knockdown Techniques ; Genetic Association Studies ; Genetic Predisposition to Disease ; Glycoconjugates/metabolism ; Glycosylation ; HEK293 Cells ; Humans ; Lectins/metabolism ; *Mutation ; Nucleotide Transport Proteins/*genetics/*metabolism ; Organic Anion Transporters/*genetics/*metabolism ; Symporters/*genetics/*metabolism ; },
abstract = {Congenital disorders of glycosylation (CDG) are a group of rare genetic and metabolic diseases caused by alterations in glycosylation pathways. Five patients bearing CDG-causing mutations in the SLC35A1 gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the SLC35A1 gene affect the protein's properties in two previously described SLC35A1-CDG cases: one caused by a substitution (Q101H) and another involving a compound heterozygous mutation (T156R/E196K). The effects of single mutations and the combination of T156R and E196K mutations on the CST's functionality was examined separately in CST-deficient HEK293T cells. As shown by microscopic studies, none of the CDG-causing mutations affected the protein's proper localization in the Golgi apparatus. Cellular glycophenotypes were characterized using lectins, structural assignment of N- and O-glycans and analysis of glycolipids. Single Q101H, T156R and E196K mutants were able to partially restore sialylation in CST-deficient cells, and the deleterious effect of a single T156R or E196K mutation on the CST functionality was strongly enhanced upon their combination. We also revealed differences in the ability of CST variants to form dimers. The results of this study improve our understanding of the molecular background of SLC35A1-CDG cases.},
}
@article {pmid33396657,
year = {2020},
author = {Mukae, T and Okumura, S and Watanobe, T and Yoshii, K and Tagami, T and Oishi, I},
title = {Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens.},
journal = {Genes},
volume = {12},
number = {1},
pages = {},
pmid = {33396657},
issn = {2073-4425},
mesh = {Animals ; Animals, Genetically Modified ; Antibodies, Monoclonal/*biosynthesis/isolation &amp; purification ; Bioreactors ; *CRISPR-Cas Systems ; Chickens/*genetics ; Egg White/*chemistry ; Female ; Gene Editing/methods ; Humans ; Plasmids/chemistry/metabolism ; Receptor, ErbB-2/*antagonists &amp; inhibitors/genetics/immunology ; Trastuzumab/biosynthesis/isolation &amp; purification ; Zygote/chemistry/metabolism ; },
abstract = {Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.},
}
@article {pmid33396568,
year = {2020},
author = {Ben Shlush, I and Samach, A and Melamed-Bessudo, C and Ben-Tov, D and Dahan-Meir, T and Filler-Hayut, S and Levy, AA},
title = {CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato.},
journal = {Genes},
volume = {12},
number = {1},
pages = {},
pmid = {33396568},
issn = {2073-4425},
mesh = {Alleles ; Biological Assay ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Crosses, Genetic ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA, Plant/chemistry/*genetics/metabolism ; Gene Editing/methods ; Genetic Loci ; *Genome, Plant ; Lycopersicon esculentum/*genetics/metabolism ; Plant Breeding/methods ; *Recombinational DNA Repair ; },
abstract = {Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.},
}
@article {pmid33396075,
year = {2021},
author = {Rai, KK and Pandey, N and Meena, RP and Rai, SP},
title = {Biotechnological strategies for enhancing heavy metal tolerance in neglected and underutilized legume crops: A comprehensive review.},
journal = {Ecotoxicology and environmental safety},
volume = {208},
number = {},
pages = {111750},
doi = {10.1016/j.ecoenv.2020.111750},
pmid = {33396075},
issn = {1090-2414},
mesh = {Biodegradation, Environmental ; Biotechnology ; Crops, Agricultural/metabolism/*physiology ; Fabaceae/metabolism/*physiology ; Metals, Heavy/analysis/metabolism/*toxicity ; Soil Pollutants/metabolism/*toxicity ; Stress, Physiological ; Vegetables/metabolism ; },
abstract = {Contamination of agricultural land and water by heavy metals due to rapid industrialization and urbanization including various natural processes have become one of the major constraints to crop growth and productivity. Several studies have reported that to counteract heavy metal stress, plants should be able to maneuver various physiological, biochemical and molecular processes to improve their growth and development under heavy metal stress. With the advent of modern biotechnological tools and techniques it is now possible to tailor legume and other plants overexpressing stress-induced genes, transcription factors, proteins, and metabolites that are directly involved in heavy metal stress tolerance. This review provides an in-depth overview of various biotechnological approaches and/or strategies that can be used for enhancing detoxification of the heavy metals by stimulating phytoremediation processes. Synthetic biology tools involved in the engineering of legume and other crop plants against heavy metal stress tolerance are also discussed herewith some pioneering examples where synthetic biology tools that have been used to modify plants for specific traits. Also, CRISPR based genetic engineering of plants, including their role in modulating the expression of several genes/ transcription factors in the improvement of abiotic stress tolerance and phytoremediation ability using knockdown and knockout strategies has also been critically discussed.},
}
@article {pmid33395371,
year = {2021},
author = {Liu, J and Ge, Y and Wang, N and Wen, J and Wang, W and Zeng, B and Chen, GL},
title = {A Simple and Efficient Method to Generate Gene-Knockout and Transgenic Cell Lines.},
journal = {DNA and cell biology},
volume = {40},
number = {2},
pages = {239-246},
doi = {10.1089/dna.2020.6183},
pmid = {33395371},
issn = {1557-7430},
mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Knockout Techniques/*methods ; *Gene Transfer Techniques ; Humans ; Ion Channels/deficiency/genetics ; TRPC Cation Channels/genetics ; },
abstract = {Knockout (KO) or exogenous expression of a gene of interest in cultured cells is one of the most important ways to study the function of the gene. Compared with transient transfection, stable cell lines possess great advantages such as excellent cell homogeneity and feasibility for long-term use. However, technical challenges in generating stable cell lines still exist in many laboratories using conventional techniques like limiting dilution or cloning cylinders. In this study we describe an optimized method to efficiently create stable cell lines for functional studies. This method was successfully used to generate a PIEZO1 gene-KO cell line with the CRISPR/Cas9 technology, and TRPC5/GCaMP6f-mCherry-coexpressing cell lines without antibiotic selection. Monoclonal cell lines can be obtained in 2-4 weeks after transfection. This method does not require any special equipment or consumables and can be conducted in all laboratories with general cell-culture facility.},
}
@article {pmid33393280,
year = {2021},
author = {Wang, W and Liang, Z and Ma, P and Zhao, Q and Dai, M and Zhu, J and Han, X and Xu, H and Chang, Q and Zhen, Y},
title = {Application of CRISPR/Cas9 System to Reverse ABC-Mediated Multidrug Resistance.},
journal = {Bioconjugate chemistry},
volume = {32},
number = {1},
pages = {73-81},
doi = {10.1021/acs.bioconjchem.0c00627},
pmid = {33393280},
issn = {1520-4812},
mesh = {ATP-Binding Cassette Transporters/*physiology ; Animals ; Antineoplastic Agents/pharmacology ; *CRISPR-Cas Systems ; Drug Resistance, Multiple/drug effects/genetics/*physiology ; Gene Editing ; Humans ; },
abstract = {Multidrug resistance (MDR) is the main obstacle in cancer chemotherapy. ATP-binding cassette (ABC) transporters can transport a wide range of antitumor drugs out of cells, which is the most common reason in the development of resistance to drugs. Currently, various therapeutic strategies are used to reverse MDR, among which CRISPR/Cas9 gene editing technique is expected to be an effective way. Here, we reviewed the research progress of reversing ABC-mediated drug resistance by CRISPR/Cas9 system.},
}
@article {pmid33393066,
year = {2021},
author = {Binnie, A and Fernandes, E and Almeida-Lousada, H and de Mello, RA and Castelo-Branco, P},
title = {CRISPR-based strategies in infectious disease diagnosis and therapy.},
journal = {Infection},
volume = {49},
number = {3},
pages = {377-385},
pmid = {33393066},
issn = {1439-0973},
mesh = {Animals ; Bacteria/genetics/isolation &amp; purification/pathogenicity ; *CRISPR-Cas Systems ; Communicable Diseases/*diagnosis/*therapy ; Gene Editing ; Humans ; Molecular Diagnostic Techniques ; Molecular Targeted Therapy ; Viruses/genetics/isolation &amp; purification/pathogenicity ; },
abstract = {PURPOSE: CRISPR gene-editing technology has the potential to transform the diagnosis and treatment of infectious diseases, but most clinicians are unaware of its broad applicability. Derived from an ancient microbial defence system, these so-called "molecular scissors" enable precise gene editing with a low error rate. However, CRISPR systems can also be targeted against pathogenic DNA or RNA sequences. This potential is being combined with innovative delivery systems to develop new therapeutic approaches to infectious diseases.<br><br>METHODS: We searched Pubmed and Google Scholar for CRISPR-based strategies in the diagnosis and treatment of infectious diseases. Reference lists were reviewed and synthesized for narrative review.<br><br>RESULTS: CRISPR-based strategies represent a novel approach to many challenging infectious diseases. CRISPR technologies can be harnessed to create rapid, low-cost diagnostic systems, as well as to identify drug-resistance genes. Therapeutic strategies, such as CRISPR systems that cleave integrated viral genomes or that target&#xa0;resistant bacteria, are in development. CRISPR-based therapies for emerging viruses, such as SARS-CoV-2, have also&#xa0;been proposed. Finally, CRISPR systems can be used to reprogram human B cells to produce neutralizing antibodies. The risks of CRISPR-based therapies include off-target and on-target modifications. Strategies to control these risks are being&#xa0;developed and a&#xa0;phase 1 clinical trials of CRISPR-based therapies for cancer&#xa0;and monogenic diseases&#xa0;are already underway.<br><br>CONCLUSIONS: CRISPR systems have broad applicability in the field of infectious diseases and may offer solutions to many of the most challenging human&#xa0;infections.},
}
@article {pmid33391497,
year = {2021},
author = {Wang, L and Zhou, J and Wang, Q and Wang, Y and Kang, C},
title = {Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.},
journal = {Theranostics},
volume = {11},
number = {2},
pages = {649-664},
pmid = {33391497},
issn = {1838-7640},
mesh = {Antiviral Agents/*administration &amp; dosage ; COVID-19/*drug therapy/virology ; CRISPR-Cas Systems/genetics ; Computational Biology ; Drug Evaluation, Preclinical ; Genetic Vectors/administration &amp; dosage/genetics ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; RNA, Guide/*genetics ; SARS-CoV-2/genetics ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus/*genetics ; },
abstract = {The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.},
}
@article {pmid33391496,
year = {2021},
author = {Zhang, S and Shen, J and Li, D and Cheng, Y},
title = {Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing.},
journal = {Theranostics},
volume = {11},
number = {2},
pages = {614-648},
pmid = {33391496},
issn = {1838-7640},
mesh = {Animals ; CRISPR-Associated Protein 9/*administration &amp; dosage/genetics/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; Humans ; *RNA, Guide ; },
abstract = {CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.},
}
@article {pmid33389894,
year = {2020},
author = {Chinnapaiyan, S and Dutta, RK and Unwalla, HJ},
title = {Novel Approaches of CRISPR-Cas Technology in Airway Diseases.},
journal = {Critical reviews in biomedical engineering},
volume = {48},
number = {3},
pages = {169-176},
doi = {10.1615/CritRevBiomedEng.2020034594},
pmid = {33389894},
issn = {1943-619X},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Technology ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR) technique plays a vital role in preclinical modelling of many respiratory diseases. Diseases such as chronic obstructive pulmonary disease (COPD), asthma, acute tracheal bronchitis, pneumonia, tuberculosis, lung cancer, and influenza infection continue to significantly impact human health. CRISPR associated (Cas) proteins, isolated from the immune system of prokaryotes, are one component of a very useful technique to manipulate gene sequences or editing and gene expression with significant implications for respiratory research in the field of molecular biology. CRISPR technology is a promising tool that is easily adaptable for specific editing of DNA sequences of interest with a goal towards modifying or eliminating gene function. Among its many potential applications, CRISPR can be applied to correcting genetic defects as well as for therapeutic approaches for treatment. This review elucidates recent advances in CRISPR-Cas technology in airway diseases.},
}
@article {pmid33389353,
year = {2021},
author = {Zhang, DF and Cui, XW and Li, WJ and Zhang, XM and Xue, HP and Huang, JK and Zhang, AH},
title = {Description of Salinimonas profundi sp. nov., a deep-sea bacterium harboring a transposon Tn6333.},
journal = {Antonie van Leeuwenhoek},
volume = {114},
number = {1},
pages = {69-81},
pmid = {33389353},
issn = {1572-9699},
mesh = {Bacterial Typing Techniques ; Base Composition ; DNA, Bacterial/genetics ; Fatty Acids ; *Phospholipids ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; *Seawater ; Sequence Analysis, DNA ; Ubiquinone ; },
abstract = {A Gram-staining-negative bacterium, strain HHU 13199[T], was isolated from a marine sediment sample collected from South China Sea (119°19.896'E, 19°41.569'N) at a depth of 2918 m. The 16S rRNA gene sequence analysis indicated that strain HHU 13199[T] represents a member of the genus Salinimonas with the highest sequence similarity (99.8%) to the type strain S. iocasae KX18D6[T]. However, the average nucleotide identity values and digital DNA-DNA hybridization between strain HHU 13199[T] and closely related members of the genus Salinimonas were all below the cut-off level (95-96 % and 70%, respectively) for species delineation. This strain grew with sea salt of 0.5-18% (w/v) (optimum, 2-5%), but no growth observed when using NaCl instead. The major fatty acids are C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The predominant isoprenoid quinone is ubiquinone-8. The polar lipids mainly consist of phosphatidylethanolamine, and phosphatidylglycerol. Genomic characterization revealed that strain HHU 13199[T] harbors a distinct type I-F CRISPR-Cas system and plenty of genes associated with heavy metal resistance, including a transposon (Tn6333) conferring mercury resistance. In addition, a phylogenetic tree based on the bac120 core genes suggested that the genus Salinimonas should be a subclade within Alteromonas. On the basis of the phenotypic, phylogenetic and chemotaxonomic characterizations, strain HHU 13199[T] represents a novel species of the genus Salinimonas, for which the name Salinimonas profundi sp. nov. is proposed. The type strain is HHU 13199[T] (= KCTC 72837[T] = MCCC 1K04127[T]).},
}
@article {pmid33389047,
year = {2021},
author = {Dinkins, RD and Hancock, J and Coe, BL and May, JB and Goodman, JP and Bass, WT and Liu, J and Fan, Y and Zheng, Q and Zhu, H},
title = {Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover.},
journal = {Plant cell reports},
volume = {40},
number = {3},
pages = {517-528},
pmid = {33389047},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation, Plant ; Genistein/metabolism ; Isoflavones/genetics/*metabolism ; Oxygenases/*genetics/metabolism ; Plant Proteins/*genetics/metabolism ; Plant Root Nodulation/genetics ; Plants, Genetically Modified ; Rhizobium/physiology ; Rhizosphere ; Trifolium/*genetics/*metabolism ; },
abstract = {Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.},
}
@article {pmid33388855,
year = {2021},
author = {Chuang, YF and Phipps, AJ and Lin, FL and Hecht, V and Hewitt, AW and Wang, PY and Liu, GS},
title = {Approach for in vivo delivery of CRISPR/Cas system: a recent update and future prospect.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {78},
number = {6},
pages = {2683-2708},
pmid = {33388855},
issn = {1420-9071},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Vectors/genetics/metabolism ; Humans ; Immunity ; Lentivirus/genetics ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system provides a groundbreaking genetic technology that allows scientists to modify genes by targeting specific genomic sites. Due to the relative simplicity and versatility of the CRISPR/Cas system, it has been extensively applied in human genetic research as well as in agricultural applications, such as improving crops. Since the gene editing activity of the CRISPR/Cas system largely depends on the efficiency of introducing the system into cells or tissues, an efficient and specific delivery system is critical for applying CRISPR/Cas technology. However, there are still some hurdles remaining for the translatability of CRISPR/Cas system. In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that can be improved to elevate the potential for CRISPR/Cas translatability.},
}
@article {pmid33387638,
year = {2021},
author = {Shirai, Y and Ohde, T and Daimon, T},
title = {Functional conservation and diversification of yellow-y in lepidopteran insects.},
journal = {Insect biochemistry and molecular biology},
volume = {128},
number = {},
pages = {103515},
doi = {10.1016/j.ibmb.2020.103515},
pmid = {33387638},
issn = {1879-0240},
mesh = {Animals ; Biological Evolution ; Bombyx/genetics/metabolism ; CRISPR-Cas Systems ; Genes, Insect ; Insect Proteins/genetics/metabolism ; *Lepidoptera/genetics/metabolism ; Melanins/*metabolism ; Mutagenesis ; Pigmentation/*genetics ; *Spodoptera/genetics/metabolism ; },
abstract = {The diverse colors and patterns found in Lepidoptera are important for success of these species. Similar to the wings of adult butterflies, lepidopteran larvae exhibit diverse color variations to adapt to their habitats. Compared with butterfly wings, however, less attention has been paid to larval body colorations and patterns. In the present study, we focus on the yellow-y gene, which participates in the melanin synthesis pathway. We conducted CRISPR/Cas9-mediated targeted mutagenesis of yellow-y in the tobacco cutworm Spodoptera litura. We analyzed the role of S. litura yellow-y in pigmentation by morphological observation and discovered that yellow-y is necessary for normal black pigmentation in S. litura. We also showed species- and tissue-specific requirements of yellow-y in pigmentation in comparison with those of Bombyx mori yellow-y mutants. Furthermore, we found that almost none of the yellow-y mutant embryos hatched unaided. We provide evidence that S. litura yellow-y has a novel important function in egg hatching, in addition to pigmentation. The present study will enable a greater understanding of the functions and diversification of the yellow-y gene in insects.},
}
@article {pmid33387103,
year = {2021},
author = {Detsika, MG and Goudevenou, K and Geurts, AM and Gakiopoulou, H and Grapsa, E and Lianos, EA},
title = {Generation of a novel decay accelerating factor (DAF) knock-out rat model using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9), genome editing.},
journal = {Transgenic research},
volume = {30},
number = {1},
pages = {11-21},
pmid = {33387103},
issn = {1573-9368},
support = {I01 BX004333/BX/BLRD VA/United States ; },
mesh = {Acute Kidney Injury/*genetics/pathology ; Albuminuria ; Animals ; Antibodies, Anti-Idiotypic/pharmacology ; CD55 Antigens/deficiency/*genetics/immunology ; CRISPR-Cas Systems/genetics ; Complement Activation/*genetics/immunology ; Complement C3-C5 Convertases/genetics ; Complement C5/genetics ; Gene Knockout Techniques ; Heymann Nephritis Antigenic Complex/genetics/immunology ; Humans ; Podocytes/*metabolism/pathology ; Rats ; },
abstract = {Decay accelerating factor (DAF), a key complement activation control protein, is a 70 kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf[-/-]) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf[+/+] rats. Renal histology in Daf[-/-] rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf[+/+] rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf[-/-] rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.},
}
@article {pmid33387102,
year = {2021},
author = {Zhang, L and Wang, Y and Li, T and Qiu, H and Xia, Z and Dong, Y},
title = {Target-specific mutations efficiency at multiple loci of CRISPR/Cas9 system using one sgRNA in soybean.},
journal = {Transgenic research},
volume = {30},
number = {1},
pages = {51-62},
pmid = {33387102},
issn = {1573-9368},
mesh = {CRISPR-Cas Systems/genetics ; Fatty Acid Desaturases/*genetics ; Gene Editing ; Genome, Plant ; Mutagenesis, Site-Directed ; Mutation/genetics ; Oxidoreductases/*genetics ; Phenotype ; Plants, Genetically Modified/*genetics/growth &amp; development ; RNA, Guide/genetics ; Soybeans/*genetics/growth &amp; development ; },
abstract = {Soybean has a palaeopolyploid genome with nearly 75% of the genes present in multiple copies. Although the CRISPR/Cas9 system has been employed in soybean to generate site-directed mutagenesis, a systematical assessment of mutation efficiency of the CRISPR/Cas9 system for the multiple-copy genes is still urgently needed. Here, we successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS. The results showed that simultaneous site-directed mutagenesis of two homoeologous loci by one sgRNA, the mutation frequency in the T0 generation were 64.71% for GmPDS, 60.0% for GmFAD2 and 42.86% for GmALS, respectively. The chimeric and heterozygous mutations were dominant types. Moreover, association of phenotypes with mutation pattern at target loci of GmPDS11 and GmPDS18 could help us further demonstrate that the CRISPR/Cas9 system can efficiently generate target specific mutations at multiple loci using one sgRNA in soybean, albeit with a relatively low transformation efficiency.},
}
@article {pmid33386580,
year = {2021},
author = {Kim, DH and Lee, J and Suh, Y and Lee, K},
title = {Necessity for Validation of Effectiveness of Selected Guide RNA In Silico for Application of CRISPR/Cas9.},
journal = {Molecular biotechnology},
volume = {63},
number = {2},
pages = {140-149},
pmid = {33386580},
issn = {1559-0305},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; *Computer Simulation ; Genetic Vectors/metabolism ; Green Fluorescent Proteins/metabolism ; INDEL Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; Quail ; RNA, Guide/*genetics ; Retinol-Binding Proteins/genetics ; },
abstract = {Selection of guide RNA (gRNA) is important to increase the efficiency of gene editing in the CRISPR/Cas9 system. Due to the variation in actual efficiency of insertion/deletion (indel) mutation among selected gRNAs in silico, reliable methods for validation of efficiency of gRNA need to be developed. Three gRNAs with high on-target scores (72.0 for target 1, 65.4 for target 2, and 62.9 for target 3) were designed to target the quail retinol binding protein 7 (qRbp7) gene, and indel efficiencies were predicted by the Sanger sequencing and Inference of CRISPR Edits (ICE) analysis of sorted cell populations receiving the CRISPR/Cas9 vector. Unlike the order of on-target scores among 3 gRNAs, predicted rates of indel mutations were highest in gRNA2, intermediate in gRNA1, and lowest in gRNA3. This was confirmed by actual indel mutation rates, 51.8% in gRNA2, 31% in gRNA1, and 12.9% in gRNA3, which were calculated by sequencing individual allele cloned into a vector. These data showed a rapid and reliable method for estimation of the efficiency of selected gRNAs, providing a critical necessary step for successful gene editing for further applications.},
}
@article {pmid33386579,
year = {2021},
author = {Daisy, PS and Shreyas, KS and Anitha, TS},
title = {Will CRISPR-Cas9 Have Cards to Play Against Cancer? An Update on its Applications.},
journal = {Molecular biotechnology},
volume = {63},
number = {2},
pages = {93-108},
pmid = {33386579},
issn = {1559-0305},
mesh = {Adaptive Immunity ; Animals ; Biomedical Research ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Humans ; Neoplasms/diagnosis/immunology/*therapy ; T-Lymphocytes/immunology ; },
abstract = {Genome editing employs targeted nucleases as powerful tools to precisely alter the genome of target cells and regulate functional genes. Various strategies have been risen so far as the molecular scissors-mediated genome editing that includes zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-related protein 9. These tools allow researchers to understand the basics of manipulating the genome, create animal models to study human diseases, understand host-pathogen interactions and design disease targets. Targeted genome modification utilizing RNA-guided nucleases are of recent curiosity, as it is a fast and effective strategy that enables the researchers to manipulate the gene of interest, carry out functional studies, understand the molecular basis of the disease and design targeted therapies. CRISPR-Cas9, a bacterial defense system employed against viruses, consists of a single-strand RNA-guided Cas9 nuclease connected to the corresponding complementary target sequence. This powerful and versatile tool has gained tremendous attention among the researchers, owing to its ability to correct genetic disorders. To help illustrate the potential of this gene editor in unexplored corners of oncology, we describe the history of CRISPR-Cas9, its rapid progression in cancer research as well as future perspectives.},
}
@article {pmid33386504,
year = {2021},
author = {Adachi, K and Hirose, A and Kanazashi, Y and Hibara, M and Hirata, T and Mikami, M and Endo, M and Hirose, S and Maruyama, N and Ishimoto, M and Abe, J and Yamada, T},
title = {Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.},
journal = {Transgenic research},
volume = {30},
number = {1},
pages = {77-89},
pmid = {33386504},
issn = {1573-9368},
mesh = {Antigens, Plant/adverse effects/*genetics/immunology ; Biolistics ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/genetics ; Gene Editing ; Genome, Plant ; Humans ; Mutagenesis, Site-Directed ; Mutation/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics/growth &amp; development/immunology ; Soybean Proteins/adverse effects/*genetics/immunology ; Soybeans/*genetics/growth &amp; development/immunology ; Transgenes/*genetics/immunology ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.},
}
@article {pmid33386277,
year = {2021},
author = {Watanabe, K and Perez, CMT and Kitahori, T and Hata, K and Aoi, M and Takahashi, H and Sakuma, T and Okamura, Y and Nakashimada, Y and Yamamoto, T and Matsuyama, K and Mayuzumi, S and Aki, T},
title = {Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing.},
journal = {Journal of bioscience and bioengineering},
volume = {131},
number = {4},
pages = {373-380},
doi = {10.1016/j.jbiosc.2020.11.013},
pmid = {33386277},
issn = {1347-4421},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fatty Acids/*biosynthesis ; Gene Editing ; Genome ; Metabolic Engineering ; Mutagenesis ; Stramenopiles/genetics/*metabolism ; },
abstract = {Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with β-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.},
}
@article {pmid33385410,
year = {2021},
author = {Ghaemi, A and Bagheri, E and Abnous, K and Taghdisi, SM and Ramezani, M and Alibolandi, M},
title = {CRISPR-cas9 genome editing delivery systems for targeted cancer therapy.},
journal = {Life sciences},
volume = {267},
number = {},
pages = {118969},
doi = {10.1016/j.lfs.2020.118969},
pmid = {33385410},
issn = {1879-0631},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Gene Editing/methods ; Genetic Therapy/methods/*trends ; Genome ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {The prokaryotic CRISPR-Cas systems could be applied as revolutionized genome editing tool in live cells of various species to modify, visualize and identify definite sequences of DNA and RNA. CRISPR-Cas could edit the genome by homology-directed repair and non-homologous end joining mechanisms. Furthermore, DNA-targeting modification by CRISPR-Cas methodology provides opportunity for diagnosis, therapy and the genetic disorders investigation. Here, we summarized delivery systems employed for CRISPR-Cas9 for genome editing. Then preclinical studies of the CRISPR-Cas9-based therapeutics will be discussed considering the associated challenges and developments in its translation to clinic for cancer therapy.},
}
@article {pmid33385273,
year = {2021},
author = {Niu, M and Lin, Y and Zou, Q},
title = {sgRNACNN: identifying sgRNA on-target activity in four crops using ensembles of convolutional neural networks.},
journal = {Plant molecular biology},
volume = {105},
number = {4-5},
pages = {483-495},
pmid = {33385273},
issn = {1573-5028},
mesh = {*Algorithms ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Crops, Agricultural/classification/*genetics ; Gene Editing/*methods ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Humans ; Internet ; *Neural Networks, Computer ; RNA, Guide/*genetics ; Sorghum/genetics ; Soybeans/genetics ; Triticum/genetics ; Zea mays/genetics ; },
abstract = {We proposed an ensemble convolutional neural network model to identify sgRNA high on-target activity in four crops and we used one-hot encoding and k-mers for sequence encoding. As an important component of the CRISPR/Cas9 system, single-guide RNA (sgRNA) plays an important role in gene redirection and editing. sgRNA has played an important role in the improvement of agronomic species, but there is a lack of effective bioinformatics tools to identify the activity of sgRNA in agronomic species. Therefore, it is necessary to develop a method based on machine learning to identify sgRNA high on-target activity. In this work, we proposed a simple convolutional neural network method to identify sgRNA high on-target activity. Our study used one-hot encoding and k-mers for sequence data conversion and a voting algorithm for constructing the convolutional neural network ensemble model sgRNACNN for the prediction of sgRNA activity. The ensemble model sgRNACNN was used for predictions in four crops: Glycine max, Zea mays, Sorghum bicolor and Triticum aestivum. The accuracy rates of the four crops in the sgRNACNN model were 82.43%, 80.33%, 78.25% and 87.49%, respectively. The experimental results showed that sgRNACNN realizes the identification of high on-target activity sgRNA of agronomic data and can meet the demands of sgRNA activity prediction in agronomy to a certain extent. These results have certain significance for guiding crop gene editing and academic research. The source code and relevant dataset can be found in the following link: https://github.com/nmt315320/sgRNACNN.git .},
}
@article {pmid33384170,
year = {2021},
author = {Zheng, X and Kuijer, HNJ and Al-Babili, S},
title = {Carotenoid Biofortification of Crops in the CRISPR Era.},
journal = {Trends in biotechnology},
volume = {39},
number = {9},
pages = {857-860},
doi = {10.1016/j.tibtech.2020.12.003},
pmid = {33384170},
issn = {1879-3096},
mesh = {*Biofortification/trends ; *CRISPR-Cas Systems ; *Carotenoids ; Gene Editing/trends ; *Plants, Genetically Modified/genetics ; },
abstract = {Carotenoids are micronutrients important for human health. The continuous improvements in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing techniques make rapid, DNA/transgene-free and targeted multiplex genetic modification a reality, thus promising to accelerate the breeding and generation of 'golden' staple crops. We discuss here the progress and future prospects of CRISPR/Cas9 applications for carotenoid biofortification.},
}
@article {pmid33383772,
year = {2020},
author = {Fan, J and Papadopoulos, V},
title = {Mitochondrial TSPO Deficiency Triggers Retrograde Signaling in MA-10 Mouse Tumor Leydig Cells.},
journal = {International journal of molecular sciences},
volume = {22},
number = {1},
pages = {},
pmid = {33383772},
issn = {1422-0067},
support = {MOP125983/CAPMC/CIHR/Canada ; PJT148659/CAPMC/CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosome Mapping ; Extracellular Matrix/metabolism ; Gene Editing ; Gene Expression Profiling ; Gene Expression Regulation ; INDEL Mutation ; Leydig Cells/*metabolism ; Male ; Mice ; Mitochondria/*genetics/*metabolism ; NF-kappa B/metabolism ; Receptors, GABA/*deficiency ; Sertoli-Leydig Cell Tumor/*etiology/*metabolism/pathology ; *Signal Transduction ; Transcriptome ; },
abstract = {The mitochondrial translocator protein (TSPO) has been shown to bind cholesterol with high affinity and is involved in mediating its availability for steroidogenesis. We recently reported that targeted Tspo gene deletion in MA-10 mouse tumor Leydig cells resulted in reduced cAMP-stimulated steroid formation and significant reduction in the mitochondrial membrane potential (ΔΨm) compared to control cells. We hypothesized that ΔΨm reduction in the absence of TSPO probably reflects the dysregulation and/or maintenance failure of some basic mitochondrial function(s). To explore the consequences of TSPO depletion via CRISPR-Cas9-mediated deletion (indel) mutation in MA-10 cells, we assessed the transcriptome changes in TSPO-mutant versus wild-type (Wt) cells using RNA-seq. Gene expression profiles were validated using real-time PCR. We report herein that there are significant changes in nuclear gene expression in Tspo mutant versus Wt cells. The identified transcriptome changes were mapped to several signaling pathways including the regulation of membrane potential, calcium signaling, extracellular matrix, and phagocytosis. This is a retrograde signaling pathway from the mitochondria to the nucleus and is probably the result of changes in expression of several transcription factors, including key members of the NF-κB pathway. In conclusion, TSPO regulates nuclear gene expression through intracellular signaling. This is the first evidence of a compensatory response to the loss of TSPO with transcriptome changes at the cellular level.},
}
@article {pmid33383688,
year = {2020},
author = {Usman, B and Nawaz, G and Zhao, N and Liao, S and Qin, B and Liu, F and Liu, Y and Li, R},
title = {Programmed Editing of Rice (Oryza sativa L.) OsSPL16 Gene Using CRISPR/Cas9 Improves Grain Yield by Modulating the Expression of Pyruvate Enzymes and Cell Cycle Proteins.},
journal = {International journal of molecular sciences},
volume = {22},
number = {1},
pages = {},
pmid = {33383688},
issn = {1422-0067},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics/metabolism ; Computational Biology/methods ; Edible Grain/*genetics ; *Gene Editing ; *Gene Expression Regulation, Enzymologic ; *Gene Expression Regulation, Plant ; Genotype ; High-Throughput Nucleotide Sequencing ; Oryza/*genetics/metabolism ; Phenotype ; Plants, Genetically Modified ; Proteome ; Proteomics ; Pyruvic Acid/metabolism ; Quantitative Trait Loci ; RNA, Guide/genetics ; Sequence Analysis, DNA ; },
abstract = {Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0, T1, and T2 generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.},
}
@article {pmid33383400,
year = {2021},
author = {Capdeville, N and Merker, L and Schindele, P and Puchta, H},
title = {Sophisticated CRISPR/Cas tools for fine-tuning plant performance.},
journal = {Journal of plant physiology},
volume = {257},
number = {},
pages = {153332},
doi = {10.1016/j.jplph.2020.153332},
pmid = {33383400},
issn = {1618-1328},
mesh = {*CRISPR-Cas Systems ; *Genetic Engineering ; *Genome, Plant ; *Plant Breeding ; },
abstract = {Over the last years, the discovery of various natural and the development of a row of engineered CRISPR/Cas nucleases have made almost every site of plant genomes accessible for the induction of specific changes. Newly developed tools open up a wide range of possibilities for the induction of genetic variability, from changing a single bp to Mbps, and thus to fine-tune plant performance. Whereas early approaches focused on targeted mutagenesis, recently developed tools enable the induction of precise and predefined genomic modifications. The use of base editors allows the substitution of single nucleotides, whereas the use of prime editors and gene targeting methods enables the induction of larger sequence modifications from a few bases to several kbp. Recently, through CRISPR/Cas-mediated chromosome engineering, it became possible to induce heritable inversions and translocations in the Mbp range. Thus, a novel way of breaking and fixing genetic linkages has come into reach for breeders. In addition, sequence-specific recruitment of various factors involved in transcriptional and post-transcriptional regulation has been shown to provide an additional class of methods for the fine tuning of plant performance. In this review, we provide an overview of the most recent progress in the field of CRISPR/Cas-based tool development for plant genome engineering and try to evaluate the importance of these developments for breeding and biotechnological applications.},
}
@article {pmid33383202,
year = {2021},
author = {Zhang, C and Wang, Y and Wang, F and Zhao, S and Song, J and Feng, F and Zhao, J and Yang, J},
title = {Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants.},
journal = {Molecular plant},
volume = {14},
number = {2},
pages = {191-194},
doi = {10.1016/j.molp.2020.12.016},
pmid = {33383202},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; DNA, Bacterial/genetics ; *Gene Editing ; *Genetic Engineering ; Nucleotide Motifs/genetics ; Oryza/genetics ; Plants/*genetics ; },
}
@article {pmid33383157,
year = {2021},
author = {Markiewicz, &#x141; and Uśpieński, T and Baran, B and Niedziółka, SM and Niewiadomski, P},
title = {Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus.},
journal = {Cellular signalling},
volume = {80},
number = {},
pages = {109907},
doi = {10.1016/j.cellsig.2020.109907},
pmid = {33383157},
issn = {1873-3913},
mesh = {Active Transport, Cell Nucleus ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Nucleus/*metabolism ; Hedgehog Proteins/metabolism ; Humans ; Karyopherins/antagonists &amp; inhibitors/genetics/metabolism ; Mice ; RNA Interference ; RNA, Guide/metabolism ; RNA, Small Interfering/metabolism ; Receptors, Cytoplasmic and Nuclear/antagonists &amp; inhibitors/genetics/metabolism ; *Signal Transduction ; Transcription, Genetic ; Zinc Finger Protein Gli2/genetics/*metabolism ; ran GTP-Binding Protein/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Dynamic bidirectional transport between the nucleus and the cytoplasm is critical for the regulation of many transcription factors, whose levels inside the nucleus must be tightly controlled. Efficient shuttling across the nuclear membrane is especially crucial with regard to the Hedgehog (Hh) pathway, where the transcriptional signal depends on the fine balance between the amounts of Gli protein activator and repressor forms in the nucleus. The nuclear export machinery prevents the unchecked nuclear accumulation of Gli proteins, but the mechanistic insight into this process is limited. We show that the atypical exportin Xpo7 functions as a major nuclear export receptor that actively excludes Gli2 from the nucleus and controls the outcome of Hh signaling. We show that Xpo7 interacts with several domains of Gli2 and that this interaction is modulated by SuFu, a key negative regulator of Hh signaling. Our data pave the way for a more complete understanding of the nuclear shuttling of Gli proteins and the regulation of their transcriptional activity.},
}
@article {pmid33382779,
year = {2020},
author = {Zhang, Y and Nunoura, T and Nishiura, D and Hirai, M and Shimamura, S and Kurosawa, K and Ishiwata, C and Deguchi, S},
title = {A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0244464},
pmid = {33382779},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Cell-Free System/enzymology ; DNA/*analysis/metabolism ; DNA Restriction Enzymes/*metabolism ; Genetic Engineering/*methods ; Microscopy, Fluorescence/methods ; Single Molecule Imaging/*methods ; },
abstract = {Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases' behaviors and functions in daily genetic engineering experiments.},
}
@article {pmid33381141,
year = {2020},
author = {Peng, C and Zheng, M and Ding, L and Chen, X and Wang, X and Feng, X and Wang, J and Xu, J},
title = {Accurate Detection and Evaluation of the Gene-Editing Frequency in Plants Using Droplet Digital PCR.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {610790},
pmid = {33381141},
issn = {1664-462X},
abstract = {Gene-editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been reported that detect mutations at targeted loci induced by the CRISPR/Cas system in different organisms, they are semiquantitative and have difficulty in the detection of mutants in processed food samples containing low initial concentrations of DNA and may not accurately quantify editing frequency, especially at very low frequencies in a complex polyploid plant genome. In this study, we developed a duplexed dPCR-based method for the detection and evaluation of gene-editing frequencies in plants. We described the design, performance, accurate quantification, and comparison with other detection systems. The results show that the dPCR-based method is sensitive to different kinds of gene-editing mutations induced by gene-editing. Moreover, the method is applicable to polyploid plants and processed food samples containing low initial concentrations of DNA. Compared with qPCR and NGS-based methods, the dPCR method has a lower limit of detection (LOD) of the editing frequency and a better relationship with the expected editing frequency in detecting the edited region of gene-edited rice samples. Taken together, the duplexed dPCR assay is accurate and precise, and it will be a powerful tool for the detection and evaluation of gene-editing frequencies in plants in gene-editing technology.},
}
@article {pmid33380298,
year = {2022},
author = {Zhen, S and Qiang, R and Lu, J and Tuo, X and Yang, X and Li, X},
title = {TGF-β1-based CRISPR/Cas9 Gene Therapy Attenuates Radiation-induced Lung Injury.},
journal = {Current gene therapy},
volume = {22},
number = {1},
pages = {59-65},
doi = {10.2174/1566523220666201230100523},
pmid = {33380298},
issn = {1875-5631},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cytokines/genetics/metabolism ; Fibrosis ; Genetic Therapy ; Lung/metabolism ; *Lung Injury/genetics/therapy ; Mice ; Mice, Inbred C57BL ; Transforming Growth Factor beta1/genetics/metabolism ; },
abstract = {BACKGROUND: Radiation-induced lung injury (RILI) is lacking effective therapeutic strategies. In this study, we conducted TGF-β1-based CRISPR/Cas9 gene therapy for RILI.<br><br>OBJECTIVE: Mouse lungs were irradiated with a single-dose of 20-Gy gamma rays followed by intravenous administration of Ad-CRISPR-TGF-&#x3b2;1 or Ad- CRISPR-Null.<br><br>METHODS: Haematoxylin and eosin staining, as well as Masson staining, were performed to observe lung morphology. Albumin and IgM concentrations in bronchoalveolar lavage fluid were measured by ELISA. Cytokine levels were measured using ELISA and/or real-time PCR with terminal deoxynucleotidyl transferase-mediated nick-end labelling.<br><br>RESULTS: Ad-CRISPR-TTGF-&#x3b2;1 improved histopathological and biochemical markers of lung injury, reduced secretion and expression of inflammatory cytokines, and inhibited progression of fibrosis. Importantly, the SK1/S1P axis, which is known to play a key role via S1P1 in TGF-&#x3b2;1-dependent S1PR pattern remodelling, is responsible for promoting fibrosis.<br><br>CONCLUSION: Our results indicate novel insights for RILI therapy.},
}
@article {pmid33380218,
year = {2021},
author = {Seastedt, KP and Pruett, N and Hoang, CD},
title = {Mouse models for mesothelioma drug discovery and development.},
journal = {Expert opinion on drug discovery},
volume = {16},
number = {6},
pages = {697-708},
pmid = {33380218},
issn = {1746-045X},
support = {ZIA BC011657/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; *Asbestos ; Carcinogenesis ; Drug Discovery ; *Lung Neoplasms ; *Mesothelioma/drug therapy ; *Mesothelioma, Malignant ; Mice ; Tumor Microenvironment ; },
abstract = {INTRODUCTION: Mesothelioma is an aggressive mesothelial lining tumor. Available drug therapies include chemotherapeutic agents, targeted molecular therapies, and immune system modulators. Mouse models were instrumental in the discovery and evaluation of such therapies, but there is need for improved understanding of the role of inflammation, tumor heterogeneity, mechanisms of carcinogenesis, and the tumor microenvironment. Novel mouse models may provide new insights and drive drug therapy discovery that improves efficacy.<br><br>AREAS COVERED: This review concerns available mouse models for mesothelioma drug discovery and development including the advantages and disadvantages of each. Gaps in current knowledge of mesothelioma are highlighted, and future directions for mouse model research are considered.<br><br>EXPERT OPINION: Soon, CRISPR-Cas gene-editing will improve understanding of mesothelioma mechanisms foundational to the discovery and testing of efficacious therapeutic targets. There are at least two likely areas of upcoming methodology development. One is concerned with precise modeling of inflammation - is it a causal process whereby inflammatory signals contribute to tumor initiation, or is it a secondary passenger process driven by asbestos exposure effects? The other area of methods improvement regards the availability of humanized immunocompromised mice harboring patient-derived xenografts. Combining human tumors in an environment with human immune cells will enable rapid innovation in immuno-oncology therapeutics.},
}
@article {pmid33378675,
year = {2020},
author = {Covarrubias, S and Vollmers, AC and Capili, A and Boettcher, M and Shulkin, A and Correa, MR and Halasz, H and Robinson, EK and O'Briain, L and Vollmers, C and Blau, J and Katzman, S and McManus, MT and Carpenter, S},
title = {High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.},
journal = {Cell reports},
volume = {33},
number = {13},
pages = {108541},
pmid = {33378675},
issn = {2211-1247},
support = {R03 AI131019/AI/NIAID NIH HHS/United States ; R21 AI142165/AI/NIAID NIH HHS/United States ; T32 GM008646/GM/NIGMS NIH HHS/United States ; U01 CA217882/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {3' Untranslated Regions ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Survival ; Clustered Regularly Interspaced Short Palindromic Repeats ; Flow Cytometry/*methods ; Gene Expression Regulation ; HEK293 Cells ; High-Throughput Screening Assays/*methods ; Humans ; Inflammation/*genetics/*metabolism ; Macrophages/*physiology ; Mice ; NF-kappa B/*physiology ; RNA, Guide/genetics ; Signal Transduction ; Tumor Necrosis Factor-alpha/*physiology ; },
abstract = {Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.},
}
@article {pmid33378644,
year = {2021},
author = {Clarke, R and Terry, AR and Pennington, H and Hasty, C and MacDougall, MS and Regan, M and Merrill, BJ},
title = {Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities.},
journal = {Molecular cell},
volume = {81},
number = {2},
pages = {226-238.e5},
pmid = {33378644},
issn = {1097-4164},
support = {F30 CA225058/CA/NCI NIH HHS/United States ; F30 HD090938/HD/NICHD NIH HHS/United States ; R21 OD027080/OD/NIH HHS/United States ; },
mesh = {Animals ; Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Nucleic Acid Conformation ; Plasmids/chemistry/metabolism ; Promoter Regions, Genetic ; RNA, Guide/chemistry/*genetics/metabolism ; Streptococcus pyogenes/chemistry/enzymology ; },
abstract = {Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.},
}
@article {pmid33377102,
year = {2020},
author = {Amorim, JP and Bordeira-Carriço, R and Gali-Macedo, A and Perrod, C and Bessa, J},
title = {CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish.},
journal = {STAR protocols},
volume = {1},
number = {3},
pages = {100208},
pmid = {33377102},
issn = {2666-1667},
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Gene Editing/*methods ; Genetic Engineering/methods ; Genome ; Genomics ; Microinjections/*methods ; Mutagenesis, Site-Directed/*methods ; RNA, Guide/genetics ; Sequence Deletion/genetics ; Zebrafish/genetics ; },
abstract = {Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).},
}
@article {pmid33377075,
year = {2020},
author = {Pelletier, S and Tummers, B and Green, DR},
title = {Generation of Casp8[FL122/123GG] Mice Using CRISPR-Cas9 Technology.},
journal = {STAR protocols},
volume = {1},
number = {3},
pages = {100181},
pmid = {33377075},
issn = {2666-1667},
support = {R01 AI044828/AI/NIAID NIH HHS/United States ; R35 CA231620/CA/NCI NIH HHS/United States ; R37 AI044828/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Caspase 8/*genetics ; Female ; Genotype ; Mice, Inbred C57BL ; Mice, Transgenic ; Microinjections ; Polymerase Chain Reaction ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/metabolism ; Zygote/metabolism ; },
abstract = {The purpose of this protocol is to describe the generation of missense mutations in mice using CRISPR-Cas9 technology. The current protocol focuses on the generation of a Casp8[FL122/123GG] missense mutation, but it can be adapted to introduce any missense or nonsense mutation. For complete details on the use and execution of this protocol, please refer to Tummers et al. (2020).},
}
@article {pmid33377062,
year = {2020},
author = {Mito, M and Mishima, Y and Iwasaki, S},
title = {Protocol for Disome Profiling to Survey Ribosome Collision in Humans and Zebrafish.},
journal = {STAR protocols},
volume = {1},
number = {3},
pages = {100168},
pmid = {33377062},
issn = {2666-1667},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Polymerase Chain Reaction ; RNA, Guide/metabolism ; RNA, Ribosomal/metabolism ; Ribosomes/*metabolism ; Zebrafish/*metabolism ; },
abstract = {Ribosomes often encounter obstacles during translation elongation and thus collide with each other. Disome profiling, an optimized ribosome profiling method, specifically sequences the long ribosome footprints generated from collided ribosomes produced by the ribosome pause and thus allows the survey of sites in a genome-wide manner. This protocol details the procedure from lysate preparation of human tissue cultures and zebrafish embryos to sequencing library construction. For complete details on the use and execution of this protocol, please refer to Han et al. (2020).},
}
@article {pmid33377052,
year = {2020},
author = {Xi, H and Young, CS and Pyle, AD},
title = {Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells.},
journal = {STAR protocols},
volume = {1},
number = {3},
pages = {100158},
pmid = {33377052},
issn = {2666-1667},
support = {R01 AR064327/AR/NIAMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions/genetics ; Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Count ; Cell Differentiation ; Conserved Sequence ; Drug Resistance, Microbial ; *Genes, Reporter ; Genotype ; Humans ; Mammals ; Mesoderm/embryology ; MicroRNAs/genetics/metabolism ; *Muscle Development ; PAX7 Transcription Factor/chemistry/*metabolism ; Plasmids/genetics ; Pluripotent Stem Cells/*metabolism ; Protein Isoforms/chemistry/metabolism ; RNA, Guide/genetics ; Reproducibility of Results ; Somites/embryology ; },
abstract = {This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020).},
}
@article {pmid33377009,
year = {2020},
author = {Hildreth, AD and Riggan, L and O'Sullivan, TE},
title = {CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Primary Innate Immune Cells.},
journal = {STAR protocols},
volume = {1},
number = {3},
pages = {100113},
pmid = {33377009},
issn = {2666-1667},
support = {P30 DK063491/DK/NIDDK NIH HHS/United States ; R01 AI145997/AI/NIAID NIH HHS/United States ; T32 AI007323/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Electroporation/*methods ; Gene Editing/*methods ; Genomics ; Humans ; Immunity, Innate/*genetics/physiology ; Lymphocytes/metabolism ; Myeloid Cells/metabolism ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {CRISPR-Cas9 genome engineering can be used to functionally investigate the complex mechanisms of immune system regulation. Decades of work have aimed to genetically reprogram innate immunity, but current approaches are inefficient or nonspecific, limiting their use. Here, we detail an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells, resulting in high-efficiency editing of target gene expression from a single electroporation. For complete details on the use and execution of this protocol, please refer to Riggan et al. (2020).},
}
@article {pmid33376220,
year = {2020},
author = {Wang, R and Lenoir, WF and Wang, C and Su, D and McLaughlin, M and Hu, Q and Shen, X and Tian, Y and Klages-Mundt, N and Lynn, E and Wood, RD and Chen, J and Hart, T and Li, L},
title = {DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {52},
pages = {33436-33445},
pmid = {33376220},
issn = {1091-6490},
support = {P01 CA193124/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA190635/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cyclin-Dependent Kinase 4 ; DNA Damage ; *DNA Replication ; DNA-Directed DNA Polymerase/*metabolism ; Fanconi Anemia/*enzymology ; Genome, Human ; HCT116 Cells ; Humans ; Mutation/genetics ; *Stress, Physiological ; Synthetic Lethal Mutations/genetics ; },
abstract = {Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.},
}
@article {pmid33376219,
year = {2020},
author = {Marshall, JL and Doughty, BR and Subramanian, V and Guckelberger, P and Wang, Q and Chen, LM and Rodriques, SG and Zhang, K and Fulco, CP and Nasser, J and Grinkevich, EJ and Noel, T and Mangiameli, S and Bergman, DT and Greka, A and Lander, ES and Chen, F and Engreitz, JM},
title = {HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {52},
pages = {33404-33413},
pmid = {33376219},
issn = {1091-6490},
support = {DP5 OD024583/OD/NIH HHS/United States ; K99 HG009917/HG/NHGRI NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Probes/*genetics ; Gene Expression ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Introns/genetics ; K562 Cells ; Kidney/cytology ; Mice ; *Nucleic Acid Hybridization ; Polyadenylation ; RNA/*metabolism ; RNA, Messenger/genetics/metabolism ; *Single-Cell Analysis ; THP-1 Cells ; Time Factors ; },
abstract = {Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.},
}
@article {pmid33375805,
year = {2020},
author = {Jouve de la Barreda, N},
title = {[From transgenesis to gene edition. Bioethical and applied considerations].},
journal = {Cuadernos de bioetica : revista oficial de la Asociacion Espanola de Bioetica y Etica Medica},
volume = {31},
number = {103},
pages = {387-401},
doi = {10.30444/CB.78},
pmid = {33375805},
issn = {1132-1989},
mesh = {Agriculture/methods ; Animal Husbandry/methods ; Animals ; Bacteriological Techniques ; CRISPR-Cas Systems ; Environment ; Food Safety ; Food Security ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Gene Transfer Techniques/*ethics ; Genetic Enhancement/ethics/legislation &amp; jurisprudence ; Global Health ; Humans ; *Organisms, Genetically Modified ; Risk Assessment ; },
abstract = {Transgenesis is a parcel of biotechnology that allows the introduction of genetic information not proper to the genome of living beings, apart from the mechanisms of natural genetic exchange. This made possible to address important applications in bacteria, animals and plants with significant benefits in health, food and environmental aspects. Since its origin, the production of genetically modified organisms (GMOs) caused some controversy due to the possible negative influence of these organisms or their derived products on health and the environment. Over time, genetic modification techniques have renewed, giving way to others of greater precision, simplicity and safety. Currently the CRISPR-Cas9 technique is widely used, which allows to edit, modify or eliminate specific DNA sequences, with multiple applications in the same fields of transgenesis, but adding greater simplicity, security and lower cost. This work presents the main techniques, applications and ethical implications of using these methods and their perspectives in an ever-evolving world. The bacteria for obtaining products of pharmacological interest, new varieties of cultivated plants of higher production, more resistance to growth limiting agents and better nutritional quality and domestic animals modified genetically, offer a set of advantages needed to address the global challenges that affect the lives of many people around the world.},
}
@article {pmid33375801,
year = {2020},
author = {Santa María D Angelo, R and Quiceno Osorio, JD and Torres Flor, A and Perochena Escalante, AC},
title = {[The crispr / cas9 techniques applied to human genetic enhancement: a biotechnological, anthropological-philosophical and legal dialogue].},
journal = {Cuadernos de bioetica : revista oficial de la Asociacion Espanola de Bioetica y Etica Medica},
volume = {31},
number = {103},
pages = {343-355},
doi = {10.30444/CB.74},
pmid = {33375801},
issn = {1132-1989},
mesh = {Anthropology ; Biotechnology/ethics/legislation &amp; jurisprudence/methods ; *CRISPR-Cas Systems ; Congenital Abnormalities/genetics ; Eugenics/legislation &amp; jurisprudence/methods ; Gene Editing ; Genetic Enhancement/*ethics/legislation &amp; jurisprudence/methods ; Genetic Therapy ; Genome, Human ; Human Characteristics ; Human Rights ; Humans ; *Interdisciplinary Communication ; Internationality ; Mutation ; Philosophy ; Respect ; },
abstract = {The CRISPR editing method is revolutionary. This technique opens the possibility of countless operations in the genome of living beings. However, the risks are high and, in some cases, unpredictable. Therefore, based on an anthropology that recognizes the human person with an inherent dignity that includes the body, this article intends to propose bases for a regulation capable of facing the challenge of CRISPR, especially, given the possibility of confusing its therapeutic resource with the eugenics, also before the imminent risk of unleashing unforeseen consequences such as mutations, malformations and side effects that could be devastating for human life.},
}
@article {pmid33375604,
year = {2020},
author = {Désaulniers, K and Ortiz, L and Dufour, C and Claudel, A and Plourde, MB and Merindol, N and Berthoux, L},
title = {Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.},
journal = {Viruses},
volume = {13},
number = {1},
pages = {},
pmid = {33375604},
issn = {1999-4915},
mesh = {Antiviral Restriction Factors ; CRISPR-Cas Systems ; *Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques ; HIV Infections/*genetics/*virology ; HIV-1/*physiology ; Host-Pathogen Interactions/genetics ; Humans ; Jurkat Cells ; RNA, Guide ; T-Lymphocytes/immunology/*metabolism/*virology ; Tripartite Motif Proteins/*genetics ; Ubiquitin-Protein Ligases/*genetics ; Viral Tropism/*genetics ; },
abstract = {Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.},
}
@article {pmid33374633,
year = {2020},
author = {Holý, O and Parra-Flores, J and Lepuschitz, S and Alarcón-Lavín, MP and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Ruppitsch, W and Forsythe, S},
title = {Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk.},
journal = {Foods (Basel, Switzerland)},
volume = {10},
number = {1},
pages = {},
pmid = {33374633},
issn = {2304-8158},
abstract = {Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in vitro putative virulence factors of six Cronobacter sakazakii strains isolated from powdered milk (PM) in the Czech Republic. Strains were identified by MALDI-TOF MS and whole-genome sequencing (WGS). Virulence and resistance genes were detected with the Ridom SeqSphere+ software task template and the Comprehensive Antibiotic Resistance Database (CARD) platform. Adherence and invasion ability were performed using the mouse neuroblastoma (N1E-115 ATCCCRL-2263) cell line. The CRISPR-Cas system was searched with CRISPRCasFinder. Core genome MLST identified four different sequence types (ST1, ST145, ST245, and ST297) in six isolates. Strains 13755-1B and 1847 were able to adhere in 2.2 and 3.2 × 10[6] CFU/mL, while 0.00073% invasion frequency was detected only in strain 1847. Both strains 13755-1B and 1847 were positive for three (50.0%) and four virulence genes, respectively. The cpa gene was not detected. Twenty-eight genes were detected by WGS and grouped as flagellar or outer membrane proteins, chemotaxis, hemolysins, and invasion, plasminogen activator, colonization, transcriptional regulator, and survival in macrophages. The colistin-resistance-encoding mcr-9.1 and cephalothin-resis-encoding blaCSA genes and IncFII(pECLA) and IncFIB(pCTU3) plasmids were detected. All strains exhibited CRISPR matrices and four of them two type I-E and I-F matrices. Combined molecular methodologies improve Cronobacter spp. decision-making for health authorities to protect the population.},
}
@article {pmid33374143,
year = {2020},
author = {Zhao, S and Jiang, D and Wang, F and Yang, Y and Tabashnik, BE and Wu, Y},
title = {Independent and Synergistic Effects of Knocking out Two ABC Transporter Genes on Resistance to Bacillus thuringiensis Toxins Cry1Ac and Cry1Fa in Diamondback Moth.},
journal = {Toxins},
volume = {13},
number = {1},
pages = {},
pmid = {33374143},
issn = {2072-6651},
support = {2018FYD0201201-3//National Key Research and Development Program of China/International ; },
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; Bacillus thuringiensis ; Bacillus thuringiensis Toxins/*chemistry/*pharmacology ; CRISPR-Cas Systems ; Endotoxins/*chemistry/*pharmacology ; Gene Editing ; Hemolysin Proteins/*chemistry/*pharmacology ; Insecticide Resistance/*genetics ; Larva ; Moths/*drug effects/*genetics ; Multidrug Resistance-Associated Protein 2 ; Neoplasm Proteins ; },
abstract = {Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic crops to control insect pests. However, evolution of resistance by pests can reduce the efficacy of Bt toxins. Here we analyzed resistance to Bt toxins Cry1Ac and Cry1Fa in the diamondback moth (Plutella xylostella), one of the world's most destructive pests of vegetable crops. We used CRISPR/Cas9 gene editing to create strains with knockouts of the ATP-binding cassette (ABC) transporter genes PxABCC2, PxABCC3, or both. Bioassay results show that knocking out either gene alone caused at most 2.9-fold resistance but knocking out both caused >10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa. Cry1Ac resistance in the double knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci. The results provide insight into the mechanism of cross-resistance to Cry1Fa in diamondback moth. They also confirm previous work with this pest showing that mutations disrupting both genes cause higher resistance to Cry1Ac than mutations affecting either PxABCC2 or PxABCC3 alone. Together with previous work, the results here highlight the value of using single and multiple gene knockouts to better understand the independent and synergistic effects of putative Bt toxin receptors on resistance to Bt toxins.},
}
@article {pmid33373406,
year = {2020},
author = {Li, G and Zhou, Z and Liang, L and Song, Z and Hu, Y and Cui, J and Chen, W and Hu, K and Cheng, J},
title = {Genome-wide identification and analysis of highly specific CRISPR/Cas9 editing sites in pepper (Capsicum annuum L.).},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0244515},
pmid = {33373406},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/*genetics ; Capsicum/*genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Plant Breeding ; Sequence Alignment ; },
abstract = {The CRISPR/Cas9 system is an efficient genome editing tool that possesses the outstanding advantages of simplicity and high efficiency. Genome-wide identification and specificity analysis of editing sites is an effective approach for mitigating the risk of off-target effects of CRISPR/Cas9 and has been applied in several plant species but has not yet been reported in pepper. In present study, we first identified genome-wide CRISPR/Cas9 editing sites based on the 'Zunla-1' reference genome and then evaluated the specificity of CRISPR/Cas9 editing sites through whole-genome alignment. Results showed that a total of 603,202,314 CRISPR/Cas9 editing sites, including 229,909,837 (~38.11%) NGG-PAM sites and 373,292,477 (~61.89%) NAG-PAM sites, were detectable in the pepper genome, and the systematic characterization of their composition and distribution was performed. Furthermore, 29,623,855 highly specific NGG-PAM sites were identified through whole-genome alignment analysis. There were 26,699,38 (~90.13%) highly specific NGG-PAM sites located in intergenic regions, which was 9.13 times of the number in genic regions, but the average density in genic regions was higher than that in intergenic regions. More importantly, 34,251 (~96.93%) out of 35,336 annotated genes exhibited at least one highly specific NGG-PAM site in their exons, and 90.50% of the annotated genes exhibited at least 4 highly specific NGG- PAM sites, indicating that the set of highly specific CRISPR/Cas9 editing sites identified in this study was widely applicable and conducive to the minimization of the off-target effects of CRISPR/Cas9 in pepper.},
}
@article {pmid33372184,
year = {2020},
author = {Yudovich, D and Bäckström, A and Schmiderer, L and Žemaitis, K and Subramaniam, A and Larsson, J},
title = {Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22393},
pmid = {33372184},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell Tracking ; *Gene Editing ; *Gene Transfer Techniques ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology/*metabolism ; Heterografts ; Humans ; Hyaluronan Receptors/genetics/metabolism ; K562 Cells ; *Lentivirus ; Leukocyte Common Antigens/genetics/metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; RNA/*genetics ; },
abstract = {The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34[+] HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.},
}
@article {pmid33372066,
year = {2021},
author = {Qiu, H and Fujisawa, S and Nozaki, S and Katoh, Y and Nakayama, K},
title = {Interaction of INPP5E with ARL13B is essential for its ciliary membrane retention but dispensable for its ciliary entry.},
journal = {Biology open},
volume = {10},
number = {1},
pages = {},
pmid = {33372066},
issn = {2046-6390},
mesh = {ADP-Ribosylation Factors/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Membrane/*metabolism ; Cilia/*physiology ; Fluorescent Antibody Technique ; Gene Expression ; Gene Knockout Techniques ; Humans ; Mice ; Models, Biological ; Mutation ; Phenotype ; Phosphoric Monoester Hydrolases/genetics/*metabolism ; Protein Binding ; Protein Transport ; },
abstract = {Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E-ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia.},
}
@article {pmid33371847,
year = {2021},
author = {Qadri, H and Shah, AH and Mir, M},
title = {Novel Strategies to Combat the Emerging Drug Resistance in Human Pathogenic Microbes.},
journal = {Current drug targets},
volume = {22},
number = {12},
pages = {1424-1436},
doi = {10.2174/1389450121666201228123212},
pmid = {33371847},
issn = {1873-5592},
mesh = {*Anti-Bacterial Agents/pharmacology ; Drug Discovery ; *Drug Resistance, Multiple, Bacterial/drug effects ; Drug Therapy, Combination ; Humans ; },
abstract = {The major health-care burden for the developing world are infectious diseases where antimicrobial agents prove to be the magical drugs to combat this. But the phenomenon of antimicrobial resistance (AMR) represents a challenging global issue, which requires to be addressed effectively. The antimicrobial treatment for the emerging multidrug-resistant bacterial (e.g. TB, Cholera) and fungal (e.g. Candidiasis) infections is very limited, and there are multiple causes and reasons responsible for the evolution of such resistance. Considering the critical issues of increasing AMR, there is an urgent requirement of the identification, development, validation, and progression of novel strategies and approaches that can easily be utilized for overcoming this serious issue. Immunotherapy represents a significant way to improve host defenses and combat the issue of antimicrobial drug resistance. Similarly, drug combination therapy represents another promising approach for reducing the evolution of resistance and enhancing the longevity of the antimicrobial agents. Bacteriophage therapy also acts as a novel therapeutic option to control the development of the multidrug resistance (MDR) phenomenon. Besides, CRISPR, an innovative genome editing technology, offers multiple applications to safeguard host defenses to overcome different resistance challenges. The novel approaches/ strategies like combination therapy, bacteriophage therapy, immunotherapy, and CRISPR/Cas discussed here presents an overview of some of the novel strategies/approaches to be adopted against the pathogenic microbes/microbial invasions along with advanced knowledge of different drug resistance mechanisms adopted by the microbial pathogens to gain resistance against different antimicrobial agents. Therefore, understanding the novel control plans/approaches and different drug resistance mechanisms will help achieve the goals of the successful development of potential antimicrobial drugs and their respective targets and eventually help curtail the problem of increasing antimicrobial drug resistance menace in various human pathogenic microbes.},
}
@article {pmid33371215,
year = {2020},
author = {Lyu, P and Wang, L and Lu, B},
title = {Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing.},
journal = {Life (Basel, Switzerland)},
volume = {10},
number = {12},
pages = {},
pmid = {33371215},
issn = {2075-1729},
abstract = {The discovery of designer nucleases has made genome editing much more efficient than before. The designer nucleases have been widely used for mechanistic studies, animal model generation and gene therapy development. However, potential off-targets and host immune responses are issues still need to be addressed for in vivo uses, especially clinical applications. Short term expression of the designer nucleases is necessary to reduce both risks. Currently, various delivery methods are being developed for transient expression of designer nucleases including Zinc Finger Nuclease (ZNF), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas). Recently, virus-like particles are being used for gene editing. In this review, we will talk through commonly used genome editing nucleases, discuss gene editing delivery tools and review the latest literature using virus-like particles to deliver gene editing effectors.},
}
@article {pmid33370873,
year = {2020},
author = {Wang, Z and Cui, Y and Shan, Y and Kang, B and Shi, L and Geng, K and Han, J},
title = {Generation of a MCPH1 knockout human embryonic stem cell line by CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102105},
doi = {10.1016/j.scr.2020.102105},
pmid = {33370873},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle Proteins ; *Cell Line ; Cytoskeletal Proteins ; *Human Embryonic Stem Cells ; Humans ; Microcephaly ; Technology ; },
abstract = {Human MCPH1 (Microcephalin 1) encodes a DNA damage response protein. Mutations in this gene have been associated with Primary Autosomal Recessive Microcephaly and premature chromosome condensation syndrome. To further understand the roles of MCPH1 in neural differentiation and brain development, here we generated a MCPH1 knockout human embryonic stem cell line by CRISPR/Cas9 genome editing technology. This cell line maintained a normal karyotype and typical undifferentiated state in terms of morphology, pluripotent gene expression, and had differentiation potential in vitro. This cell line provides a good resource to study the role of MCPH1 gene in neurogenesis and regulation of the size of the cerebral cortex in vitro.},
}
@article {pmid33369835,
year = {2021},
author = {Hu, C and Sheng, O and Deng, G and He, W and Dong, T and Yang, Q and Dou, T and Li, C and Gao, H and Liu, S and Yi, G and Bi, F},
title = {CRISPR/Cas9-mediated genome editing of MaACO1 (aminocyclopropane-1-carboxylate oxidase 1) promotes the shelf life of banana fruit.},
journal = {Plant biotechnology journal},
volume = {19},
number = {4},
pages = {654-656},
pmid = {33369835},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Fruit/genetics ; *Gene Editing ; *Musa/genetics ; Oxidoreductases/genetics ; },
}
@article {pmid33369608,
year = {2020},
author = {Smith, A and Bergwell, M and Smith, E and Mathew, D and Iyer, J},
title = {CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {166},
pages = {},
doi = {10.3791/62001},
pmid = {33369608},
issn = {1940-087X},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/*genetics ; Collagen/*genetics ; DNA Primers/metabolism ; *Genetic Testing ; Gonads/metabolism ; Homozygote ; Microinjections ; RNA Editing/genetics ; RNA, Ribosomal/genetics/metabolism ; Restriction Mapping ; Ribonucleoproteins/genetics/*metabolism ; Streptococcus pyogenes/genetics ; },
abstract = {The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing in less than two weeks.},
}
@article {pmid33369120,
year = {2021},
author = {Zhan, X and Lu, Y and Zhu, JK and Botella, JR},
title = {Genome editing for plant research and crop improvement.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {1},
pages = {3-33},
doi = {10.1111/jipb.13063},
pmid = {33369120},
issn = {1744-7909},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genome, Plant/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.},
}
@article {pmid33369118,
year = {2021},
author = {Schweickert, PG and Wang, N and Sandefur, SL and Lloyd, ME and Konieczny, SF and Frye, CC and Cheng, Z},
title = {CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.},
journal = {Biotechnology journal},
volume = {16},
number = {4},
pages = {e2000308},
doi = {10.1002/biot.202000308},
pmid = {33369118},
issn = {1860-7314},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetinae ; Cricetulus ; Gene Editing ; },
abstract = {Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.},
}
@article {pmid33368291,
year = {2021},
author = {Mitra, D and Vega-Rubin-de-Celis, S and Royla, N and Bernhardt, S and Wilhelm, H and Tarade, N and Poschet, G and Buettner, M and Binenbaum, I and Borgoni, S and Vetter, M and Kantelhardt, EJ and Thomssen, C and Chatziioannou, A and Hell, R and Kempa, S and Müller-Decker, K and Wiemann, S},
title = {Abrogating GPT2 in triple-negative breast cancer inhibits tumor growth and promotes autophagy.},
journal = {International journal of cancer},
volume = {148},
number = {8},
pages = {1993-2009},
doi = {10.1002/ijc.33456},
pmid = {33368291},
issn = {1097-0215},
mesh = {Animals ; Autophagy/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MCF-7 Cells ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; RNA Interference ; Survival Analysis ; Transaminases/antagonists &amp; inhibitors/*genetics/metabolism ; Triple Negative Breast Neoplasms/*genetics/metabolism/therapy ; Tumor Burden/*genetics ; Xenograft Model Antitumor Assays/methods ; },
abstract = {Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.},
}
@article {pmid33367825,
year = {2021},
author = {Cha, S and Hong, CP and Kang, HA and Hahn, JS},
title = {Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae.},
journal = {Nucleic acids research},
volume = {49},
number = {2},
pages = {745-759},
pmid = {33367825},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Cell Respiration ; Chromatin Immunoprecipitation ; DNA-Binding Proteins/chemistry/*genetics ; Enzyme Activation ; Ethanol/metabolism ; Glycerol/metabolism ; Glycolysis ; Protein Binding ; Protein Domains ; Protein Isoforms/chemistry/genetics ; RNA-Seq ; Reverse Transcriptase Polymerase Chain Reaction ; Saccharomyces cerevisiae/*enzymology/genetics/growth &amp; development ; Saccharomyces cerevisiae Proteins/chemistry/*genetics ; Transcription Factors/chemistry/deficiency/*genetics ; },
abstract = {Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.},
}
@article {pmid33367730,
year = {2020},
author = {Li, HL and Wang, XY and Zheng, XL and Lu, W},
title = {Research Progress on Oviposition-Related Genes in Insects.},
journal = {Journal of insect science (Online)},
volume = {20},
number = {6},
pages = {},
pmid = {33367730},
issn = {1536-2442},
mesh = {Animals ; CRISPR-Cas Systems ; Egg Proteins/genetics ; Female ; Gene Expression ; Genes, Insect ; Insect Control/methods ; Insecta/*genetics ; Oogenesis/genetics ; Oviposition/*genetics ; RNA Interference ; Receptors, Cell Surface/genetics ; Vitellogenins/genetics ; },
abstract = {Oviposition-related genes have remained a consistent focus of insect molecular biology. Previous research has gradually clarified our mechanistic understanding of oviposition-related genes, including those related to oviposition-gland-related genes, oogenesis-related genes, oviposition-site-selection-related genes, and genes related to ovulation and hatching. Moreover, some of this research has revealed how the expression of single oviposition-related genes affects the expression of related genes, and more importantly, how individual node genes function to link the expression of upstream and downstream genes. However, the research to date is not sufficient to completely explain the overall interactions among the genes of the insect oviposition system. Through a literature review of a large number of studies, this review provides references for future research on oviposition-related genes in insects and the use of RNAi or CRISPR/Cas9 technology to verify the functions of oviposition-related genes and to prevent and control harmful insects.},
}
@article {pmid33367260,
year = {2020},
author = {Veillet, F and Durand, M and Kroj, T and Cesari, S and Gallois, JL},
title = {Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens.},
journal = {Plant communications},
volume = {1},
number = {5},
pages = {100102},
pmid = {33367260},
issn = {2590-3462},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Disease Resistance/*genetics ; *Gene Editing ; Gene Knockdown Techniques ; Genome, Plant/genetics ; Host-Pathogen Interactions/genetics ; Plant Breeding/*methods ; Plant Diseases/*genetics/microbiology ; },
abstract = {Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes, the CRISPR technology has revolutionized plant research and precision crop breeding. The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health. In this review, we provide an extensive overview on recent breakthroughs in CRISPR technology, including the newly developed prime editing system that allows precision gene editing in plants. We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations, and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.},
}
@article {pmid33367259,
year = {2020},
author = {Zhang, C and Zhang, B and Mu, B and Zheng, X and Zhao, F and Lan, W and Fu, A and Luan, S},
title = {A Thylakoid Membrane Protein Functions Synergistically with GUN5 in Chlorophyll Biosynthesis.},
journal = {Plant communications},
volume = {1},
number = {5},
pages = {100094},
pmid = {33367259},
issn = {2590-3462},
mesh = {Arabidopsis/*metabolism ; Arabidopsis Proteins/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chlorophyll/*biosynthesis ; Chloroplasts/metabolism/ultrastructure ; Gene Editing ; Gene Knockdown Techniques ; Homeostasis ; Lyases/*metabolism ; Magnesium/metabolism ; Microscopy, Electron, Transmission ; Thylakoid Membrane Proteins/*metabolism ; Thylakoids/metabolism ; },
abstract = {Chlorophyll (Chl) is essential for photosynthetic reactions and chloroplast development. While the enzymatic pathway for Chl biosynthesis is well established, the regulatory mechanism underlying the homeostasis of Chl levels remains largely unknown. In this study, we identified CBD1 (Chlorophyll Biosynthetic Defect1), which functions in the regulation of chlorophyll biosynthesis. The CBD1 gene was expressed specifically in green tissues and its protein product was embedded in the thylakoid membrane. Furthermore, CBD1 was precisely co-expressed and functionally correlated with GUN5 (Genome Uncoupled 5). Analysis of chlorophyll metabolic intermediates indicated that cbd1 and cbd1gun5 mutants over-accumulated magnesium protoporphyrin IX (Mg-Proto IX). In addition, the cbd1 mutant thylakoid contained less Mg than the wild type not only as a result of lower Chl content, but also implicating CBD1 in Mg transport. This was supported by the finding that CBD1 complemented a Mg[2+] uptake-deficient Salmonella strain under low Mg conditions. Taken together, these results indicate that CBD1 functions synergistically with CHLH/GUN5 in Mg-Proto IX processing, and may serve as a Mg-transport protein to maintain Mg homeostasis in the chloroplast.},
}
@article {pmid33367239,
year = {2020},
author = {Xu, R and Li, J and Liu, X and Shan, T and Qin, R and Wei, P},
title = {Development of Plant Prime-Editing Systems for Precise Genome Editing.},
journal = {Plant communications},
volume = {1},
number = {3},
pages = {100043},
pmid = {33367239},
issn = {2590-3462},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Plant ; *Genotyping Techniques ; *Mutation ; Oryza/*genetics ; Plants, Genetically Modified ; },
abstract = {Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT[-ATG] reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T0 plants, pPE2-generated mutants occurred with 0%-31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT[-ATG] reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.},
}
@article {pmid33365307,
year = {2020},
author = {Lian, J and Wang, Y and Luo, Y and Li, C},
title = {Editorial: Development and Application of Novel Genome Engineering Tools in Microbial Biotechnology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {621851},
doi = {10.3389/fbioe.2020.621851},
pmid = {33365307},
issn = {2296-4185},
}
@article {pmid33363552,
year = {2020},
author = {Deguchi, M and Kane, S and Potlakayala, S and George, H and Proano, R and Sheri, V and Curtis, WR and Rudrabhatla, S},
title = {Metabolic Engineering Strategies of Industrial Hemp (Cannabis sativa L.): A Brief Review of the Advances and Challenges.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {580621},
pmid = {33363552},
issn = {1664-462X},
abstract = {Industrial hemp (Cannabis sativa L.) is a diploid (2n = 20), dioecious plant that is grown for fiber, seed, and oil. Recently, there has been a renewed interest in this crop because of its panoply of cannabinoids, terpenes, and other phenolic compounds. Specifically, hemp contains terpenophenolic compounds such as cannabidiol (CBD) and cannabigerol (CBG), which act on cannabinoid receptors and positively regulate various human metabolic, immunological, and physiological functions. CBD and CBG have an effect on the cytokine metabolism, which has led to the examination of cannabinoids on the treatment of viral diseases, including COVID-19. Based on genomic, transcriptomic, and metabolomic studies, several synthetic pathways of hemp secondary metabolite production have been elucidated. Nevertheless, there are few reports on hemp metabolic engineering despite obvious impact on scientific and industrial sectors. In this article, recent status and current perspectives on hemp metabolic engineering are reviewed. Three distinct approaches to expedite phytochemical yield are discussed. Special emphasis has been placed on transgenic and transient gene delivery systems, which are critical for successful metabolic engineering of hemp. The advent of new tools in synthetic biology, particularly the CRISPR/Cas systems, enables environment-friendly metabolic engineering to increase the production of desirable hemp phytochemicals while eliminating the psychoactive compounds, such as tetrahydrocannabinol (THC).},
}
@article {pmid33363214,
year = {2020},
author = {Yao, L and Zhang, Q and Li, A and Ma, B and Zhang, Z and Liu, J and Liang, L and Zhu, S and Gan, Y and Zhang, Q},
title = {Synthetic Artificial Long Non-coding RNA Shows Higher Efficiency in Specific Malignant Phenotype Inhibition Compared to the CRISPR/Cas Systems.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {617600},
pmid = {33363214},
issn = {2296-889X},
abstract = {Objective: Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play an important regulator in human cancer by transcriptional and post-transcriptional regulation, respectively. These phenomena raise questions about the ability of artificial device to regulate miRNAs and TFs simultaneously. In this study, we aimed to construct an artificial long non-coding RNA, "alncRNA," which imitated CRISPR/Cas systems and to illuminate its therapeutic effects in bladder cancer cell lines. At the same time, we also compared the efficiency of alncRNA and CRISPR/Cas systems in regulating gene expression. Study Design and Methods: Based on engineering principles of synthetic biology, we combined tandem arrayed cDNA sequences of aptamer for TFs with tandem arrayed cDNA copies of binding sites for the miRNAs to construct alncRNA. In order to prove the utility of this platform, we chose β -catenin, NF-κB, miR-940, and miR-495 as the functional targets and used the bladder cancer cell lines 5637 and T24 as the test models. Real-time Quantitative PCR (qPCR), dual-luciferase assay and relative phenotypic experiments were applied to severally test the expression of relative gene and therapeutic effects of our devices. Result: Dual-luciferase assay indicated alncRNA could inhibit transcriptional activity of TFs. What's more, the result of qPCR showed that expression levels of the relative TFs target genes and miRNAs were reduced by corresponding alncRNA and the inhibitory effect was better than CRIPSR dCas9-KRAB. By functional experiments, decreased cell proliferation, increased apoptosis, and motility inhibition were observed in alncRNA-infected bladder cells. Conclusion: In summary, our synthetic devices indeed function as anti-tumor regulator, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cell and show higher efficiency in specific malignant phenotype inhibition compared to the CRISPR/Cas systems. Most importantly, Anti-cancer effects were induced by the synthetic alncRNA in the bladder cancer lines. Our devices, therefore, provides a novel strategy for cancer therapy and could be a useful "weapon" for cancer cell.},
}
@article {pmid33362853,
year = {2020},
author = {Zhang, N and Bewick, B and Xia, G and Furling, D and Ashizawa, T},
title = {A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {594576},
pmid = {33362853},
issn = {1664-8021},
abstract = {Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) - a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that LshCas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, LshCas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated LshCas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of LshCas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases.},
}
@article {pmid33361628,
year = {2020},
author = {Kaul, T and Sony, SK and Verma, R and Motelb, KFA and Prakash, AT and Eswaran, M and Bharti, J and Nehra, M and Kaul, R},
title = {Revisiting CRISPR/Cas-mediated crop improvement: Special focus on nutrition.},
journal = {Journal of biosciences},
volume = {45},
number = {},
pages = {},
pmid = {33361628},
issn = {0973-7138},
mesh = {*Artificial Intelligence ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; DNA Breaks, Double-Stranded ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; },
abstract = {Genome editing (GE) technology has emerged as a multifaceted strategy that instantaneously popularised the mechanism to modify the genetic constitution of an organism. The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein-based genome editing (CRISPR/Cas) approach has huge potential for efficacious editing of genomes of numerous organisms. This framework has demonstrated to be more economical in contrast to mega-nucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) for its flexibility, versatility, and potency. The advent of sequence-specific nucleases (SSNs) allowed the precise induction of double-strand breaks (DSBs) into the genome, ensuring desired alterations through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. Researchers have utilized CRISPR/Cas-mediated genome alterations across crop varieties to generate desirable characteristics for yield enhancement, enriched nutritional quality, and stressresistance. Here, we highlighted the recent progress in the area of nutritional improvement of crops via the CRISPR/Cas-based tools for fundamental plant research and crop genetic advancements. Application of this genome editing aids in unraveling the basic biology facts in plants supplemented by the incorporation of genome-wide association studies, artificial intelligence, and various bioinformatic frameworks, thereby providing futuristic model studies and their affirmations. Strategies for reducing the 'off-target' effects and the societal approval of genome-modified crops developed via this modern biotechnological approach have been reviewed.},
}
@article {pmid33361431,
year = {2021},
author = {Van Cleemput, J and Koyuncu, OO and Laval, K and Engel, EA and Enquist, LW},
title = {CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing.},
journal = {Journal of virology},
volume = {95},
number = {6},
pages = {},
pmid = {33361431},
issn = {1098-5514},
support = {R01 NS033506/NS/NINDS NIH HHS/United States ; R01 NS060699/NS/NINDS NIH HHS/United States ; },
mesh = {Alphaherpesvirinae/physiology ; Animals ; Axonal Transport ; CRISPR-Cas Systems ; Capsid/metabolism ; Cells, Cultured ; *Gene Silencing ; Genome, Viral/*genetics ; Herpesvirus 1, Suid/*physiology ; Mutation ; Neurons/metabolism/virology ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Swine ; Viral Proteins/genetics/*metabolism ; Virus Latency ; },
abstract = {Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins.IMPORTANCE Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.},
}
@article {pmid33361125,
year = {2020},
author = {Burda, PC and Bisio, H and Marq, JB and Soldati-Favre, D and Heussler, VT},
title = {CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites.},
journal = {mSphere},
volume = {5},
number = {6},
pages = {},
pmid = {33361125},
issn = {2379-5042},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Membrane/*chemistry ; GTP-Binding Protein alpha Subunits, Gq-G11/*genetics ; HeLa Cells ; Hepatocytes/*parasitology ; Humans ; Malaria/parasitology ; Plasmodium berghei/genetics/*physiology ; Toxoplasma/metabolism ; },
abstract = {Toxoplasma gondii and members of the genus Plasmodium are obligate intracellular parasites that leave their infected host cell upon a tightly controlled process of egress. Intracellular replication of the parasites occurs within a parasitophorous vacuole, and its membrane as well as the host plasma membrane need to be disrupted during egress, leading to host cell lysis. While several parasite-derived factors governing egress have been identified, much less is known about host cell factors involved in this process. Previously, RNA interference (RNAi)-based knockdown and antibody-mediated depletion identified a host signaling cascade dependent on guanine nucleotide-binding protein subunit alpha q (GNAQ) to be required for the egress of Toxoplasma tachyzoites and Plasmodium blood stage merozoites. Here, we used CRISPR/Cas9 technology to generate HeLa cells deficient in GNAQ and tested their capacity to support the egress of T. gondii tachyzoites and Plasmodium berghei liver stage parasites. While we were able to confirm the importance of GNAQ for the egress of T. gondii, we found that the egress of P. berghei liver stages was unaffected in the absence of GNAQ. These results may reflect differences between the lytic egress process in apicomplexans and the formation of host cell-derived vesicles termed merosomes by P. berghei liver stages.IMPORTANCE The coordinated release of apicomplexan parasites from infected host cells prior to reinvasion is a critical process for parasite survival and the spread of infection. While Toxoplasma tachyzoites and Plasmodium blood stages induce a fast disruption of their surrounding membranes during their egress from host cells, Plasmodium liver stages keep the host cell membrane intact and leave their host cell in host cell-derived vesicles called merosomes. The knockout of GNAQ, a protein involved in G-protein-coupled receptor signaling, demonstrates the importance of this host factor for the lytic egress of T. gondii tachyzoites. Contrastingly, the egress of P. berghei is independent of GNAQ at the liver stage, indicating the existence of a mechanistically distinct strategy to exit the host cell.},
}
@article {pmid33360715,
year = {2021},
author = {Fage, C and Lemire, N and Moineau, S},
title = {Delivery of CRISPR-Cas systems using phage-based vectors.},
journal = {Current opinion in biotechnology},
volume = {68},
number = {},
pages = {174-180},
doi = {10.1016/j.copbio.2020.11.012},
pmid = {33360715},
issn = {1879-0429},
support = {/DH_/Department of Health/United Kingdom ; },
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Antimicrobial resistance has spread quickly on a worldwide scale, reducing therapeutic options for bacterial infections. CRISPR-Cas is an adaptive immune system found in many prokaryotes that can be designed to target bacterial genomes, leading to cell death. Repurposing the CRISPR-Cas system as a therapeutic strategy offers an attractive way to overcome antimicrobial resistance. However, this strategy requires efficient vectors for the CRISPR-Cas system to reach the bacterial genomes. Engineered phages offer an attractive option as cargo delivery vectors. In this review, we discuss the production of phage-based vectors and the relevance of using repurposed CRISPR-Cas systems as antimicrobials. We also discuss recent progress in phage engineering that can potentially overcome the limitations and increase the efficiency of CRISPR-Cas delivery.},
}
@article {pmid33360689,
year = {2021},
author = {Jandova, J and Wondrak, GT},
title = {Genomic GLO1 deletion modulates TXNIP expression, glucose metabolism, and redox homeostasis while accelerating human A375 malignant melanoma tumor growth.},
journal = {Redox biology},
volume = {39},
number = {},
pages = {101838},
pmid = {33360689},
issn = {2213-2317},
support = {P30 CA023074/CA/NCI NIH HHS/United States ; P30 ES006694/ES/NIEHS NIH HHS/United States ; P01 CA229112/CA/NCI NIH HHS/United States ; R03 CA230949/CA/NCI NIH HHS/United States ; R01 CA229418/CA/NCI NIH HHS/United States ; R21 ES029579/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; Carrier Proteins ; Genomics ; Glucose ; Homeostasis ; Humans ; *Lactoylglutathione Lyase/genetics ; Male ; *Melanoma/genetics ; Mice ; Mice, SCID ; Oxidation-Reduction ; Thioredoxins ; },
abstract = {Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human malignant melanoma cells and patient tumors and substantiated a novel role of GLO1 as a molecular determinant of invasion and metastasis in melanoma. Here, employing NanoString™ gene expression profiling (nCounter™ 'PanCancer Progression Panel'), we report that CRISPR/Cas 9-based GLO1 deletion from human A375 malignant melanoma cells alters glucose metabolism and redox homeostasis, observable together with acceleration of tumorigenesis. Nanostring™ analysis identified TXNIP (encoding thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis, displaying the most pronounced expression change in response to GLO1 elimination, confirmed by RT-qPCR and immunoblot analysis. TXNIP was also upregulated in CRISPR/Cas9-engineered DU145 prostate carcinoma cells lacking GLO1, and treatment with MG or a pharmacological GLO1 inhibitor (TLSC702) mimicked GLO1_KO status, suggesting that GLO1 controls TXNIP expression through regulation of MG. GLO1_KO status was characterized by (i) altered oxidative stress response gene expression, (ii) attenuation of glucose uptake and metabolism with downregulation of gene expression (GLUT1, GFAT1, GFAT2, LDHA) and depletion of related key metabolites (glucose-6-phosphate, UDP-N-acetylglucosamine), and (iii) immune checkpoint modulation (PDL1). While confirming our earlier finding that GLO1 deletion limits invasion and metastasis with modulation of EMT-related genes (e.g. TGFBI, MMP9, ANGPTL4, TLR4, SERPINF1), we observed that GLO1_KO melanoma cells displayed a shortened population doubling time, cell cycle alteration with increased M-phase population, and enhanced anchorage-independent growth, a phenotype supported by expression analysis (CXCL8, CD24, IL1A, CDKN1A). Concordantly, an accelerated growth rate of GLO1_KO tumors, accompanied by TXNIP overexpression and metabolic reprogramming, was observable in a SCID mouse melanoma xenograft model, demonstrating that A375 melanoma tumor growth and metastasis can be dysregulated in opposing ways as a consequence of GLO1 elimination.},
}
@article {pmid33360431,
year = {2021},
author = {Schultzhaus, Z and Wang, Z and Stenger, D},
title = {Systematic analysis, identification, and use of CRISPR/Cas13a-associated crRNAs for sensitive and specific detection of the lcrV gene of Yersinia pestis.},
journal = {Diagnostic microbiology and infectious disease},
volume = {99},
number = {3},
pages = {115275},
doi = {10.1016/j.diagmicrobio.2020.115275},
pmid = {33360431},
issn = {1879-0070},
mesh = {Antigens, Bacterial/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Screening Assays/*methods ; Humans ; Pathology, Molecular/*methods ; Pore Forming Cytotoxic Proteins/*genetics ; RNA, Bacterial/*genetics ; Virulence ; Yersinia pestis/*genetics/pathogenicity ; },
abstract = {CRISPR-associated proteins that produce a signal in the presence of a target nucleic acid represent potentially powerful tools for diagnostics, but they also exhibit shortfalls that plague many CRISPR systems. For instance, not all targets elicit robust activity, which challenges the timely development of sensitive assays, and though many such tests have been reported, they often avoid discussion of the crRNA design and screening process. Here, motivated by the desire to detect the Yersinia pestis lcrV virulence gene, we detail the process involved in developing components for a CRISPR-based test that provides sensitive and specific identification of this sequence using Cas13a. This includes detailing the diversity of crRNA performance, identifying sequence that enable detection with attomolar sensitivity and species-level specificity, and presenting a method for simple streamlining of the crRNA screening process to allow for the high-throughput testing required for developing assay design rules in the future.},
}
@article {pmid33360156,
year = {2021},
author = {Ghafouri-Fard, S and Shoorei, H and Abak, A and Abbas Raza, SH and Pichler, M and Taheri, M},
title = {Role of non-coding RNAs in modulating the response of cancer cells to paclitaxel treatment.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {134},
number = {},
pages = {111172},
doi = {10.1016/j.biopha.2020.111172},
pmid = {33360156},
issn = {1950-6007},
mesh = {Animals ; Antineoplastic Agents/adverse effects/chemistry/*therapeutic use ; CRISPR-Cas Systems ; Drug Carriers ; Drug Compounding ; *Drug Resistance, Neoplasm/drug effects ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/genetics/*metabolism ; Neoplasms/*drug therapy/genetics/metabolism/pathology ; Oligonucleotides/therapeutic use ; Paclitaxel/adverse effects/chemistry/*therapeutic use ; RNA, Long Noncoding/genetics/*metabolism ; Signal Transduction ; },
abstract = {Paclitaxel is a chemotherapeutic substance that is administered for treatment of an extensive spectrum of human malignancies. In spite of its potent short-term effects against tumor cells, resistance to paclitaxel occurs in a number of patients precluding its long-term application in these patients. Non-coding RNAs have been shown to influence response of cancer cells to this chemotherapeutic agent via different mechanisms. Mechanistically, these transcripts regulate expression of several genes particularly those being involved in the apoptotic processes. Lots of in vivo and in vitro assays have demonstrated the efficacy of oligonucleotide-mediated microRNAs (miRNA)/ long non-coding RNAs (lncRNA) silencing in enhancement of response of cancer cells to paclitaxel. Therefore, targeted therapies against non-coding RNAs have been suggested as applicable modalities for combatting resistance to this agent. In the present review, we provide a summary of studies which assessed the role of miRNAs and lncRNAs in conferring resistance to paclitaxel.},
}
@article {pmid33359667,
year = {2021},
author = {Arnaoutova, I and Zhang, L and Chen, HD and Mansfield, BC and Chou, JY},
title = {Correction of metabolic abnormalities in a mouse model of glycogen storage disease type Ia by CRISPR/Cas9-based gene editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {4},
pages = {1602-1610},
pmid = {33359667},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Disease Models, Animal ; *Gene Editing ; *Genetic Therapy ; Genetic Vectors/genetics ; Glucose/genetics/metabolism ; Glucose-6-Phosphatase/*genetics ; Glycogen Storage Disease Type I/genetics/metabolism/pathology/*therapy ; Humans ; Liver/metabolism/pathology ; Mice ; },
abstract = {Glycogen storage disease type Ia (GSD-Ia), deficient in glucose-6-phosphatase-α (G6PC), is characterized by impaired glucose homeostasis and a hallmark of fasting hypoglycemia. We have developed a recombinant adeno-associated virus (rAAV) vector-mediated gene therapy for GSD-Ia that is currently in a phase I/II clinical trial. While therapeutic expression of the episomal rAAV-G6PC clinical vector is stable in mice, the long-term durability of expression in humans is currently being established. Here we evaluated CRISPR/Cas9-based in vivo genome editing technology to correct a prevalent pathogenic human variant, G6PC-p.R83C. We have generated a homozygous G6pc-R83C mouse strain and shown that the G6pc-R83C mice manifest impaired glucose homeostasis and frequent hypoglycemic seizures, mimicking the pathophysiology of GSD-Ia patients. We then used a CRISPR/Cas9-based gene editing system to treat newborn G6pc-R83C mice and showed that the treated mice grew normally to age 16 weeks without hypoglycemia seizures. The treated G6pc-R83C mice, expressing ≥ 3% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, displayed normalized blood metabolites, and could sustain 24 h of fasting. Taken together, we have developed a second-generation therapy in which in vivo correction of a pathogenic G6PC-p.R83C variant in its native genetic locus could lead to potentially permanent, durable, long-term correction of the GSD-Ia phenotype.},
}
@article {pmid33358912,
year = {2021},
author = {Takahashi, K and Ito, Y and Yoshimura, M and Nikaido, M and Yuikawa, T and Kawamura, A and Tsuda, S and Kage, D and Yamasu, K},
title = {A globin-family protein, Cytoglobin 1, is involved in the development of neural crest-derived tissues and organs in zebrafish.},
journal = {Developmental biology},
volume = {472},
number = {},
pages = {1-17},
doi = {10.1016/j.ydbio.2020.12.016},
pmid = {33358912},
issn = {1095-564X},
mesh = {Animals ; Animals, Genetically Modified ; Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Chromosomes/genetics ; Cytoglobin/*genetics/metabolism ; Embryonic Development/genetics ; Gene Expression ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Mutation ; Neural Crest/*cytology/*embryology/metabolism ; Phenotype ; Zebrafish/*embryology/*genetics/metabolism ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.},
}
@article {pmid33358778,
year = {2020},
author = {Feng, Y and Yu, P and Li, J and Cao, Y and Zhang, J},
title = {Phosphatidylinositol 4-kinase β is required for the ciliogenesis of zebrafish otic vesicle.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {10},
pages = {627-636},
doi = {10.1016/j.jgg.2020.07.007},
pmid = {33358778},
issn = {1673-8527},
mesh = {1-Phosphatidylinositol 4-Kinase/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Cell Movement/genetics ; Cilia/*genetics/physiology ; Embryonic Development/*genetics ; Gene Expression Regulation, Developmental/genetics ; Humans ; Phosphatidylinositol Phosphates/genetics ; Sequence Deletion/genetics ; Signal Transduction/genetics ; Zebrafish/genetics ; Zebrafish Proteins/*genetics ; },
abstract = {The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.},
}
@article {pmid33358112,
year = {2021},
author = {Peng, R and Zhang, B},
title = {Foxtail Millet: A New Model for C4 Plants.},
journal = {Trends in plant science},
volume = {26},
number = {3},
pages = {199-201},
doi = {10.1016/j.tplants.2020.12.003},
pmid = {33358112},
issn = {1878-4372},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Oryza/genetics ; *Setaria Plant/genetics ; },
abstract = {Arabidopsis and rice are major models for C3 plants, but we still lack a model for C4 plants. Recently, Yang and coworkers developed foxtail millet as a C4 plant model; with the rapid development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technology, this will open a new era for plant functional studies and crop improvement.},
}
@article {pmid33357766,
year = {2019},
author = {Moscoso, CG and Steer, CJ},
title = {Liver targeted gene therapy: Insights into emerging therapies.},
journal = {Drug discovery today. Technologies},
volume = {34},
number = {},
pages = {9-19},
doi = {10.1016/j.ddtec.2020.11.001},
pmid = {33357766},
issn = {1740-6749},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Disease Models, Animal ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; Genetic Vectors/administration &amp; dosage/genetics ; Humans ; Liver/metabolism/pathology ; Liver Diseases/genetics/metabolism/pathology/*therapy ; Metabolic Diseases/genetics/metabolism/pathology/*therapy ; Transposases/genetics ; },
abstract = {The large number of monogenic metabolic disorders originating in the liver poses a unique opportunity for development of gene therapy modalities to pursue curative approaches. Various disorders have been successfully treated via liver-directed gene therapy, though most of the advances have been in animal models, with only limited success in clinical trials. Pre-clinical data in animals using non-viral approaches, including the Sleeping Beauty transposon system, are discussed. The various advances with viral vectors for liver-directed gene therapy are also a focus of this review, including retroviral, adenoviral, recombinant adeno-associated viral, and SV40 vectors. Genome editing techniques, including zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR), are also described. Further, the various controversies in the field with regards to somatic vs. germline editing using CRISPR in humans are explored, while also highlighting the myriad of preclinical advances. Lastly, newer technologies are reviewed, including base editing and prime editing, which use CRISPR with exciting adjunctive properties to avoid double-stranded breaks and thus the recruitment of endogenous repair mechanisms. While encouraging results have been achieved recently, there are still significant challenges to overcome prior to the broad use of vector-based and genome editing techniques in the clinical arena. As these technologies mature, the promise of a cure for many disabling inherited metabolic disorders is within reach, and urgently needed.},
}
@article {pmid33357464,
year = {2021},
author = {Hoffmann, HH and Sánchez-Rivera, FJ and Schneider, WM and Luna, JM and Soto-Feliciano, YM and Ashbrook, AW and Le Pen, J and Leal, AA and Ricardo-Lax, I and Michailidis, E and Hao, Y and Stenzel, AF and Peace, A and Zuber, J and Allis, CD and Lowe, SW and MacDonald, MR and Poirier, JT and Rice, CM},
title = {Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {2},
pages = {267-280.e5},
pmid = {33357464},
issn = {1934-6069},
support = {R01 AI150275/AI/NIAID NIH HHS/United States ; R01 AI116943/AI/NIAID NIH HHS/United States ; P30 CA016087/CA/NCI NIH HHS/United States ; R01 AI091707/AI/NIAID NIH HHS/United States ; U01 CA213359/CA/NCI NIH HHS/United States ; R01 CA190261/CA/NCI NIH HHS/United States ; R01 AI124690/AI/NIAID NIH HHS/United States ; P01 CA196539/CA/NCI NIH HHS/United States ; F32 AI133910/AI/NIAID NIH HHS/United States ; P01 AI138938/AI/NIAID NIH HHS/United States ; R01 CA204639/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA213448/CA/NCI NIH HHS/United States ; U19 AI111825/AI/NIAID NIH HHS/United States ; R03 AI141855/AI/NIAID NIH HHS/United States ; T32 CA160001/CA/NCI NIH HHS/United States ; R21 AI161212/AI/NIAID NIH HHS/United States ; R01 AI143295/AI/NIAID NIH HHS/United States ; R21 AI142010/AI/NIAID NIH HHS/United States ; },
mesh = {COVID-19/*virology ; CRISPR-Cas Systems ; Coronavirus 229E, Human/genetics/metabolism ; Coronavirus NL63, Human/genetics/metabolism ; Coronavirus OC43, Human ; Genes, Viral ; Host-Pathogen Interactions ; Humans ; SARS-CoV-2/genetics/*metabolism ; Viral Proteins/genetics/metabolism ; },
abstract = {The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.},
}
@article {pmid33357381,
year = {2020},
author = {Aharon-Hefetz, N and Frumkin, I and Mayshar, Y and Dahan, O and Pilpel, Y and Rak, R},
title = {Manipulation of the human tRNA pool reveals distinct tRNA sets that act in cellular proliferation or cell cycle arrest.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33357381},
issn = {2050-084X},
support = {1332/14//Israel Science Foundation/International ; 616622/ERC_/European Research Council/International ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Cycle/genetics ; *Cell Cycle Checkpoints/genetics ; Cell Line ; *Cell Proliferation/genetics ; Cloning, Molecular ; Gene Editing ; Genomic Library ; HeLa Cells ; Humans ; RNA, Transfer/genetics/*metabolism ; },
abstract = {Different subsets of the tRNA pool in human cells are expressed in different cellular conditions. The 'proliferation-tRNAs' are induced upon normal and cancerous cell division, while the 'differentiation-tRNAs' are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. We measured tRNA essentiality for cellular growth and found that most proliferation-tRNAs are essential compared to differentiation- tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation-tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states.},
}
@article {pmid33356428,
year = {2021},
author = {Cui, X and Liu, K and Atta, S and Zeng, C and Zhou, C and Wang, X},
title = {Two Unique Prophages of 'Candidatus Liberibacter asiaticus' Strains from Pakistan.},
journal = {Phytopathology},
volume = {111},
number = {5},
pages = {784-788},
doi = {10.1094/PHYTO-10-20-0454-SC},
pmid = {33356428},
issn = {0031-949X},
mesh = {*Citrus ; Liberibacter ; Pakistan ; Plant Diseases ; Prophages/genetics ; *Rhizobiaceae/genetics ; },
abstract = {'Candidatus Liberibacter asiaticus' (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.},
}
@article {pmid33356427,
year = {2021},
author = {Wheatley, MS and Yang, Y},
title = {Versatile Applications of the CRISPR/Cas Toolkit in Plant Pathology and Disease Management.},
journal = {Phytopathology},
volume = {111},
number = {7},
pages = {1080-1090},
doi = {10.1094/PHYTO-08-20-0322-IA},
pmid = {33356427},
issn = {0031-949X},
mesh = {*CRISPR-Cas Systems/genetics ; Disease Management ; Genome, Plant ; Plant Breeding ; Plant Diseases ; *Plant Pathology ; },
abstract = {New tools and advanced technologies have played key roles in facilitating basic research in plant pathology and practical approaches for disease management and crop health. Recently, the CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR-associated) system has emerged as a powerful and versatile tool for genome editing and other molecular applications. This review aims to introduce and highlight the CRISPR/Cas toolkit and its current and future impact on plant pathology and disease management. We will cover the rapidly expanding horizon of various CRISPR/Cas applications in the basic study of plant-pathogen interactions, genome engineering of plant disease resistance, and molecular diagnosis of diverse pathogens. Using the citrus greening disease as an example, various CRISPR/Cas-enabled strategies are presented to precisely edit the host genome for disease resistance, to rapidly detect the pathogen for disease management, and to potentially use gene drive for insect population control. At the cutting edge of nucleic acid manipulation and detection, the CRISPR/Cas toolkit will accelerate plant breeding and reshape crop production and disease management as we face the challenges of 21st century agriculture.},
}
@article {pmid33354860,
year = {2021},
author = {Wu, J and Peng, H and Lu, X and Lai, M and Zhang, H and Le, XC},
title = {Binding-Mediated Formation of Ribonucleoprotein Corona for Efficient Delivery and Control of CRISPR/Cas9.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {20},
pages = {11104-11109},
pmid = {33354860},
issn = {1521-3773},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/metabolism ; CRISPR-Cas Systems ; Humans ; Particle Size ; Protein Binding ; Protein Corona/*chemistry ; Ribonucleoproteins/*chemistry ; },
abstract = {Protein coronae formed with nanoparticles confer several useful properties. However, the non-specific nature of protein corona formation makes it difficult to deliver specific proteins for therapeutic applications. Herein, we report on the construction of a new type of protein corona, termed binding-mediated protein corona. This new corona enables the efficient and controllable delivery of functional proteins, which is otherwise challenging for conventional protein coronae. We show the design and delivery of the ribonucleoprotein corona for the CRISPR/Cas9 system. Successful gene editing in human cell lines (Hela and HEK293) demonstrates the efficient delivery, high stability, low cytotoxicity, and well-controlled activity of the Cas9-guide RNA ribonucleoprotein. The binding-mediated protein corona strategy opens up new opportunities for therapeutic protein delivery.},
}
@article {pmid33353186,
year = {2020},
author = {Galow, AM and Goldammer, T and Hoeflich, A},
title = {Xenogeneic and Stem Cell-Based Therapy for Cardiovascular Diseases: Genetic Engineering of Porcine Cells and Their Applications in Heart Regeneration.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33353186},
issn = {1422-0067},
mesh = {Animals ; Cardiovascular Diseases/*therapy ; *Genetic Engineering ; Humans ; Myocytes, Cardiac/*cytology ; *Regenerative Medicine ; Stem Cell Transplantation/*methods ; Stem Cells/*cytology ; Swine ; *Transplantation, Heterologous ; },
abstract = {Cardiovascular diseases represent a major health concern worldwide with few therapy options for ischemic injuries due to the limited regeneration potential of affected cardiomyocytes. Innovative cell replacement approaches could facilitate efficient regenerative therapy. However, despite extensive attempts to expand primary human cells in vitro, present technological limitations and the lack of human donors have so far prevented their broad clinical use. Cell xenotransplantation might provide an ethically acceptable unlimited source for cell replacement therapies and bridge the gap between waiting recipients and available donors. Pigs are considered the most suitable candidates as a source for xenogeneic cells and tissues due to their anatomical and physiological similarities with humans. The potential of porcine cells in the field of stem cell-based therapy and regenerative medicine is under intensive investigation. This review outlines the current progress and highlights the most promising approaches in xenogeneic cell therapy with a focus on the cardiovascular system.},
}
@article {pmid33353099,
year = {2020},
author = {Kim, D and Le, QV and Wu, Y and Park, J and Oh, YK},
title = {Nanovesicle-Mediated Delivery Systems for CRISPR/Cas Genome Editing.},
journal = {Pharmaceutics},
volume = {12},
number = {12},
pages = {},
pmid = {33353099},
issn = {1999-4923},
abstract = {Genome-editing technology has emerged as a potential tool for treating incurable diseases for which few therapeutic modalities are available. In particular, discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system together with the design of single-guide RNAs (sgRNAs) has sparked medical applications of genome editing. Despite the great promise of the CRISPR/Cas system, its clinical application is limited, in large part, by the lack of adequate delivery technology. To overcome this limitation, researchers have investigated various systems, including viral and nonviral vectors, for delivery of CRISPR/Cas and sgRNA into cells. Among nonviral delivery systems that have been studied are nanovesicles based on lipids, polymers, peptides, and extracellular vesicles. These nanovesicles have been designed to increase the delivery of CRISPR/Cas and sgRNA through endosome escape or using various stimuli such as light, pH, and environmental features. This review covers the latest research trends in nonviral, nanovesicle-based delivery systems that are being applied to genome-editing technology and suggests directions for future progress.},
}
@article {pmid33352158,
year = {2021},
author = {Zhang, S and Li, T and Huo, Y and Yang, J and Fleming, J and Shi, M and Wang, Y and Wei, W and Gu, S and Bi, L and Jiang, T and Zhang, H},
title = {Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity.},
journal = {International journal of biological macromolecules},
volume = {170},
number = {},
pages = {140-149},
doi = {10.1016/j.ijbiomac.2020.12.014},
pmid = {33352158},
issn = {1879-0003},
mesh = {Adenylyl Cyclases/genetics ; Amino Acid Sequence ; Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Conserved Sequence ; DNA-Directed DNA Polymerase/genetics ; Evolution, Molecular ; Models, Molecular ; Mutagenesis ; Mycobacterium tuberculosis/*enzymology/genetics ; Oligonucleotides/metabolism ; Protein Conformation ; Protein Domains ; Recombinant Proteins/metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Structure-Activity Relationship ; Substrate Specificity ; Thermococcus/enzymology/genetics ; },
abstract = {Prokaryotic CRISPR/Cas systems confer immunity against invading nucleic acids through effector complexes. Csm1, the signature protein of Type III effector complexes, catalyses cyclic oligoadenylate synthesis when in the effector complex, but not when alone, activating the Csm6 nuclease and switching on the antiviral response. Here, we provide biochemical evidence that M. tuberculosis Csm1 (MtbCsm1) has ion-dependent polymerase activity when independent of the effector complex. Structural studies provide supporting evidence that the catalytic core of the MtbCsm1 palm2 domain is almost identical to that of classical DNA polymerase Pol IV, and that the palm1 and B domains function as the other structural elements required (thumb and fingers) for DNA polymerase activity. MtbCsm1 polymerase activity is relatively weak in vitro and its functional relevance in vivo is unknown. Our structural and mutagenesis data suggest that residue K692 in the palm2 domain has been significant in the evolution of Csm1 from a polymerase to a cyclase, and support the notion that the cyclase activity of Csm1 requires the presence of other elements provided by the CRISPR/Cas effector complex. This structural rationale for Csm1 polymerase (alone) and cyclase (within the effector complex) activity should benefit future functional investigations and engineering.},
}
@article {pmid33349703,
year = {2021},
author = {Jin, S and Gao, Q and Gao, C},
title = {An unbiased method for evaluating the genome-wide specificity of base editors in rice.},
journal = {Nature protocols},
volume = {16},
number = {1},
pages = {431-457},
pmid = {33349703},
issn = {1750-2799},
mesh = {Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant ; Oryza/*genetics ; Polymorphism, Single Nucleotide ; Transformation, Genetic ; Whole Genome Sequencing ; },
abstract = {Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12-15 weeks.},
}
@article {pmid33349652,
year = {2021},
author = {Wheatley, RM and MacLean, RC},
title = {CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa.},
journal = {The ISME journal},
volume = {15},
number = {5},
pages = {1420-1433},
pmid = {33349652},
issn = {1751-7370},
support = {/WT_/Wellcome Trust/United Kingdom ; 106918/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Transfer, Horizontal ; Pseudomonas aeruginosa/genetics ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic pathogen Pseudomonas aeruginosa, which has emerged as an important model system for understanding CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are associated with smaller genomes and higher GC content, suggesting that CRISPR-Cas inhibits the acquisition of foreign DNA. Although phage is the major target of CRISPR-Cas spacers, more than 80% of isolates with an active CRISPR-Cas system have spacers that target integrative conjugative elements (ICE) or the conserved conjugative transfer machinery used by plasmids and ICE. Consistent with these results, genomes containing active CRISPR-Cas systems harbour a lower abundance of both prophage and ICE. Crucially, spacers in genomes with active CRISPR-Cas systems map to ICE and phage that are integrated into the chromosomes of closely related genomes lacking CRISPR-Cas immunity. We propose that CRISPR-Cas acts as an important constraint to horizontal gene transfer, and the evolutionary mechanisms that ensure its maintenance or drive its loss are key to the ability of this pathogen to adapt to new niches and stressors.},
}
@article {pmid33349126,
year = {2021},
author = {Cornu, TI and Mussolino, C and Müller, MC and Wehr, C and Kern, WV and Cathomen, T},
title = {HIV Gene Therapy: An Update.},
journal = {Human gene therapy},
volume = {32},
number = {1-2},
pages = {52-65},
doi = {10.1089/hum.2020.159},
pmid = {33349126},
issn = {1557-7422},
mesh = {Gene Editing ; Genetic Therapy ; *HIV Infections/genetics/therapy ; Humans ; Quality of Life ; },
abstract = {Progress in antiretroviral therapy has considerably reduced mortality and notably improved the quality of life of individuals infected with HIV since the pandemic began some 40 years ago. However, drug resistance, treatment-associated toxicity, adherence to medication, and the need for lifelong therapy have remained major challenges. While the development of an HIV vaccine has remained elusive, considerable progress in developing innovative cell and gene therapies to treat HIV infection has been made. This includes immune cell therapies, such as chimeric antigen receptor T cells to target HIV infected cells, as well as gene therapies and genome editing strategies to render the patient's immune system resistant to HIV. Nonetheless, all of these attempts to achieve a functional cure in HIV patients have failed thus far. This review introduces the clinical as well as the technical challenges of treating HIV infection, and summarizes the most promising cell and gene therapy concepts that have aspired to bring about functional cure for people living with HIV. It further discusses socioeconomic aspects as well as future directions for developing cell and gene therapies with a potential to be an effective one-time treatment with minimal toxicity.},
}
@article {pmid33349060,
year = {2021},
author = {Pillon, MC and Gordon, J and Frazier, MN and Stanley, RE},
title = {HEPN RNases - an emerging class of functionally distinct RNA processing and degradation enzymes.},
journal = {Critical reviews in biochemistry and molecular biology},
volume = {56},
number = {1},
pages = {88-108},
pmid = {33349060},
issn = {1549-7798},
support = {R00 ES030735/ES/NIEHS NIH HHS/United States ; ZIA ES103247/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Catalytic Domain ; Endoribonucleases/chemistry/genetics/*metabolism ; Humans ; Protein Conformation, alpha-Helical ; Protein Multimerization ; *Proteolysis ; *RNA Processing, Post-Transcriptional ; RNA Stability ; RNA-Binding Proteins/chemistry/genetics/*metabolism ; Toxin-Antitoxin Systems ; },
abstract = {HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) RNases are an emerging class of functionally diverse RNA processing and degradation enzymes. Members are defined by a small α-helical bundle encompassing a short consensus RNase motif. HEPN dimerization is a universal requirement for RNase activation as the conserved RNase motifs are precisely positioned at the dimer interface to form a composite catalytic center. While the core HEPN fold is conserved, the organization surrounding the HEPN dimer can support large structural deviations that contribute to their specialized functions. HEPN RNases are conserved throughout evolution and include bacterial HEPN RNases such as CRISPR-Cas and toxin-antitoxin associated nucleases, as well as eukaryotic HEPN RNases that adopt large multi-component machines. Here we summarize the canonical elements of the growing HEPN RNase family and identify molecular features that influence RNase function and regulation. We explore similarities and differences between members of the HEPN RNase family and describe the current mechanisms for HEPN RNase activation and inhibition.},
}
@article {pmid33347431,
year = {2020},
author = {Lu, L and Hu, J and Chao, T and Chen, Z and Liu, Z and Luo, X and Liang, Y and He, P and Zhang, L},
title = {Loss of natural resistance to schistosome in T cell deficient rat.},
journal = {PLoS neglected tropical diseases},
volume = {14},
number = {12},
pages = {e0008909},
pmid = {33347431},
issn = {1935-2735},
mesh = {Animals ; CD3 Complex/genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation ; Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics/metabolism ; Mice, Inbred C57BL ; Mice, Knockout ; Rats ; Rats, Sprague-Dawley ; Schistosoma japonicum/physiology ; Schistosomiasis japonica/*immunology ; T-Lymphocytes/*physiology ; },
abstract = {Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis.},
}
@article {pmid33346897,
year = {2021},
author = {Trogu, S and Ermert, AL and Stahl, F and Nogué, F and Gans, T and Hughes, J},
title = {Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens.},
journal = {Plant molecular biology},
volume = {107},
number = {4-5},
pages = {327-336},
pmid = {33346897},
issn = {1573-5028},
mesh = {Bryopsida/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gravitropism/genetics/radiation effects ; Light ; *Multigene Family ; *Mutagenesis ; Mutation ; Phenotype ; Phytochrome/*genetics ; },
abstract = {We mutated all seven Physcomitrium (Physcomitrella) patens phytochrome genes using highly-efficient CRISPR-Cas9 procedures. We thereby identified phy5a as the phytochrome primarily responsible for inhibiting gravitropism, proving the utility of the mutant library. The CRISPR-Cas9 system is a powerful tool for genome editing. Here we report highly-efficient multiplex CRISPR-Cas9 editing of the seven-member phytochrome gene family in the model bryophyte Physcomitrium (Physcomitrella) patens. Based on the co-delivery of an improved Cas9 plasmid with multiple sgRNA plasmids and an efficient screening procedure to identify high-order multiple mutants prior to sequencing, we demonstrate successful targeting of all seven PHY genes in a single transfection. We investigated further aspects of the CRISPR methodology in Physcomitrella, including the significance of spacing between paired sgRNA targets and the efficacy of NHEJ and HDR in repairing the chromosome when excising a complete locus. As proof-of-principle, we show that the septuple phy[-] mutant remains gravitropic in light, in line with expectations, and on the basis of data from lower order multiplex knockouts conclude that phy5a is the principal phytochrome responsible for inhibiting gravitropism in light. We expect, therefore, that this mutant collection will be valuable for further studies of phytochrome function and that the methods we describe will allow similar approaches to revealing specific functions in other gene families.},
}
@article {pmid33346718,
year = {2020},
author = {Adashi, EY and Burgess, MM and Burall, S and Cohen, IG and Fleck, LM and Harris, J and Holm, S and Lafont, C and Moreno, JD and Neblo, MA and Niemeyer, SJ and Rowe, EJ and Scheufele, DA and Tetsa, PF and Vayena, E and Watermeyer, RP and Fung, A},
title = {Heritable Human Genome Editing: The Public Engagement Imperative.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {434-439},
doi = {10.1089/crispr.2020.0049},
pmid = {33346718},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*ethics/*trends ; Genome, Human ; Germ Cells ; Humans ; Public Opinion ; },
abstract = {In the view of many, heritable human genome editing (HHGE) harbors the remedial potential of ridding the world of deadly genetic diseases. A Hippocratic obligation, if there ever was one, HHGE is widely viewed as a life-sustaining proposition. The national go/no-go decision regarding the implementation of HHGE, however, must not, in the collective view of the authors, proceed absent thorough public engagement. A comparable call for an "extensive societal dialogue" was recently issued by the International Commission on the Clinical Use of Human Germline Genome Editing. In this communication, the authors lay out the foundational principles undergirding the formation, modification, and evaluation of public opinion. It is against this backdrop that the societal decision to warrant or enjoin the clinical conduct of HHGE will doubtlessly transpire.},
}
@article {pmid33346717,
year = {2020},
author = {Gao, C and Chen, J},
title = {Special Issue: CRISPR in CHINA.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {419},
doi = {10.1089/crispr.2020.29103.cfp2},
pmid = {33346717},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; China ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods/*trends ; Humans ; },
}
@article {pmid33346716,
year = {2020},
author = {Barrangou, R},
title = {Sharpening the CRISPR Toolbox.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {421},
doi = {10.1089/crispr.2020.29114.rba},
pmid = {33346716},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods/*trends ; Humans ; },
}
@article {pmid33346715,
year = {2020},
author = {Pruett-Miller, SM},
title = {Assessing Off-Target Editing of CRISPR-Cas9 Systems.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {430-432},
doi = {10.1089/crispr.2020.29116.smi},
pmid = {33346715},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
}
@article {pmid33346714,
year = {2020},
author = {Lambert, LJ},
title = {CRISPR to the Core.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {433},
doi = {10.1089/crispr.2020.29113.lle},
pmid = {33346714},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods/*trends ; History, 21st Century ; Humans ; },
}
@article {pmid33346713,
year = {2020},
author = {Moreb, EA and Hutmacher, M and Lynch, MD},
title = {CRISPR-Cas "Non-Target" Sites Inhibit On-Target Cutting Rates.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {550-561},
doi = {10.1089/crispr.2020.0065},
pmid = {33346713},
issn = {2573-1602},
mesh = {Artifacts ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/physiology ; Computational Biology/*methods ; DNA/genetics ; Gene Editing/*methods ; Genome/genetics ; Genomics/methods ; Nucleotide Motifs/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.},
}
@article {pmid33346712,
year = {2020},
author = {Yuan, H and Ruan, Y and Tan, Y and Reed-Maldonado, AB and Chen, Y and Zhao, D and Wang, Z and Zhou, F and Peng, D and Banie, L and Wang, G and Liu, J and Lin, G and Qi, LS and Lue, TF},
title = {Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {562-572},
pmid = {33346712},
issn = {2573-1602},
support = {R56 DK105097/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology ; Computational Biology/methods ; Female ; Gene Editing/methods ; Gene Silencing/physiology ; Genomics/methods ; Guided Tissue Regeneration/*methods ; Muscle, Skeletal/metabolism ; Muscle, Striated/*metabolism/physiology ; Myoblasts/metabolism ; Myostatin/*genetics/metabolism ; Obesity/complications ; Pelvic Floor ; RNA, Guide ; Rats ; Rats, Zucker ; Regeneration/physiology ; Urethra/metabolism/physiology ; Urinary Bladder ; Urinary Incontinence, Stress/etiology ; },
abstract = {Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Lepr[fa] 186 and 14 fatty ZUC-Lepr[fa] 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.},
}
@article {pmid33346711,
year = {2020},
author = {Xu, X and Luo, T and Gao, J and Lin, N and Li, W and Xia, X and Wang, J},
title = {CRISPR-Assisted DNA Detection: A Novel dCas9-Based DNA Detection Technique.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {487-502},
doi = {10.1089/crispr.2020.0041},
pmid = {33346711},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; DNA, Viral/genetics ; Genetic Engineering/methods ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/*methods ; Nucleic Acid Hybridization/*methods ; Papillomavirus Infections/genetics ; RNA, Guide/genetics/metabolism ; },
abstract = {Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.},
}
@article {pmid33346710,
year = {2020},
author = {Chaudhari, HG and Penterman, J and Whitton, HJ and Spencer, SJ and Flanagan, N and Lei Zhang, MC and Huang, E and Khedkar, AS and Toomey, JM and Shearer, CA and Needham, AW and Ho, TW and Kulman, JD and Cradick, TJ and Kernytsky, A},
title = {Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {440-453},
pmid = {33346710},
issn = {2573-1602},
mesh = {Artifacts ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*ethics/*methods/*trends ; Genome, Human/genetics ; Genomic Instability/genetics ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics/pathogenicity ; },
abstract = {The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.},
}
@article {pmid33346709,
year = {2020},
author = {Pourcel, C},
title = {New Insights into CRISPR Arrays.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {422-424},
doi = {10.1089/crispr.2020.29111.cpo},
pmid = {33346709},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
}
@article {pmid33346708,
year = {2020},
author = {Wang, E and Hsu, PD},
title = {A Catalogue of Cas9 Orthologs to Advance Genome Engineering.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {427-430},
doi = {10.1089/crispr.2020.29115.ewa},
pmid = {33346708},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genome/genetics ; },
}
@article {pmid33346707,
year = {2020},
author = {Shmakov, SA and Utkina, I and Wolf, YI and Makarova, KS and Severinov, KV and Koonin, EV},
title = {CRISPR Arrays Away from cas Genes.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {535-549},
pmid = {33346707},
issn = {2573-1602},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/physiology ; Computational Biology/*methods ; Gene Editing/*methods ; Genome, Archaeal/genetics ; Genome, Bacterial/genetics ; Genome, Viral/genetics ; Genomics/methods ; Phylogeny ; },
abstract = {CRISPR-Cas systems typically consist of a CRISPR array and cas genes that are organized in one or more operons. However, a substantial fraction of CRISPR arrays are not adjacent to cas genes. Definitive identification of such isolated CRISPR arrays runs into the problem of false-positives, with unrelated types of repetitive sequences mimicking CRISPR. We developed a computational pipeline to eliminate false CRISPR predictions and found that up to 25% of the CRISPR arrays in complete bacterial and archaeal genomes are located away from cas genes. Most of the repeats in these isolated arrays are identical to repeats in cas-adjacent CRISPR arrays in the same or closely related genomes, indicating an evolutionary relationship between isolated arrays and arrays in typical CRISPR-cas loci. The spacers in isolated CRISPR arrays show nearly as many matches to viral genomes as spacers from complete CRISPR-cas loci, suggesting that the isolated arrays were either functionally active recently or continue to function. Reconstruction of evolutionary events in closely related bacterial genomes suggests three routes of evolution of isolated CRISPR arrays: (1) loss of cas genes in a CRISPR-cas locus, (2) de novo generation of arrays from off-target spacer integration into sequences resembling the corresponding repeats, and (3) transfer by mobile genetic elements. Both combination of de novo emerging arrays with cas genes and regain of cas genes by isolated arrays via recombination likely contribute to functional diversification in CRISPR-Cas evolution.},
}
@article {pmid33346706,
year = {2020},
author = {Wu, Y and Yuan, Q and Zhu, Y and Gao, X and Song, J and Yin, Z},
title = {Improving FnCas12a Genome Editing by Exonuclease Fusion.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {503-511},
doi = {10.1089/crispr.2020.0073},
pmid = {33346706},
issn = {2573-1602},
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endodeoxyribonucleases/genetics/*metabolism ; Endonucleases/genetics ; Exonucleases/genetics/metabolism ; Francisella/genetics/*metabolism ; Gene Editing/*methods ; },
abstract = {Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a)-a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells at multiple genomic loci in three different cell lines. TEXT results in higher insertion and deletion efficiency than FnCas12a under different spacer lengths from 18 nt to 23 nt. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. Compared to other Cas12a orthologs, including AsCas12a and LbCas12a, TEXT achieves the highest on-targeting efficiency and shows minimal off-targeting effects at all tested sites. TEXT enhances the activity of FnCas12a nuclease and expands its targeting scope and efficiency in human cell genome engineering.},
}
@article {pmid33346704,
year = {2020},
author = {Zhao, G and Li, J and Tang, Y},
title = {AsCRISPR: A Web Server for Allele-Specific Single Guide RNA Design in Precision Medicine.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {512-522},
doi = {10.1089/crispr.2020.0071},
pmid = {33346704},
issn = {2573-1602},
mesh = {Algorithms ; Alleles ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; Computers ; Gene Editing/*methods ; Genomics/methods ; Humans ; Polymorphism, Single Nucleotide/genetics ; Precision Medicine/*methods ; RNA, Guide/*genetics/pharmacology ; Software ; },
abstract = {Allele-specific genomic targeting by CRISPR is a versatile strategy that has been increasingly exploited not only in treating inherited dominant diseases and mutation-driven cancers, but also in other important fields such as genome imprinting, haploinsufficiency, and genome loci imaging. Despite its tremendous utilities, few bioinformatic tools have been implemented for the allele-specific purpose of CRISPR. We thus developed AsCRISPR (Allele-specific CRISPR), a comprehensive web tool to aid the design of short-guide RNA (sgRNA) sequences that can discriminate between alleles. AsCRISPR allows users to analyze both their own identified variants and heterozygous single nucleotide polymorphisms and, importantly, output the candidate sgRNAs and their quality control information. To facilitate targeting dominant diseases, AsCRISPR analyzed dominant single nucleotide variants (SNVs) retrieved from ClinVar and OMIM databases, and generated a dominant database of candidate-discriminating sgRNAs that may specifically target the alternative allele for each dominant SNV site. Moreover, a validated database was established, which manually curated the discriminating sgRNAs that were experimentally validated in the mounting literature for multiple allele-specific purposes.},
}
@article {pmid33346665,
year = {2021},
author = {Choi, JH and Lim, J and Shin, M and Paek, SH and Choi, JW},
title = {CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis.},
journal = {Nano letters},
volume = {21},
number = {1},
pages = {693-699},
doi = {10.1021/acs.nanolett.0c04303},
pmid = {33346665},
issn = {1530-6992},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colorimetry ; DNA/genetics ; Gold ; *Metal Nanoparticles ; *Nucleic Acids ; },
abstract = {Cell-free DNA (cfDNA) has attracted significant attention due to its high potential to diagnose diseases, such as cancer. Still, its detection by amplification method has limitations because of false-positive signals and difficulty in designing target-specific primers. CRISPR-Cas-based fluorescent biosensors have been developed but also need the amplification step for the detection. In this study, for the first time CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence (MEF) using DNA-functionalized Au nanoparticle (AuNP). Upon activating the CRISPR-Cas12a complex by the target cfDNA and subsequent single-strand DNA (ssDNA) degradation between AuNP and fluorophore, MEF occurred with color changes from purple to red-purple. Using this system, breast cancer gene-1 (BRCA-1) can be detected with very high sensitivity in 30 min. This rapid and highly selective sensor can be applied to measure other nucleic acid biomarkers such as viral DNA in field-deployable and point-of-care testing (POCT) platform.},
}
@article {pmid33346327,
year = {2021},
author = {Napoletano, S and Piersanti, V and Rallo, G},
title = {CRISPR -Cas9: a groundbreaking new technique which ushers in new prospects and just as many doubts.},
journal = {La Clinica terapeutica},
volume = {171},
number = {1},
pages = {e52-e54},
doi = {10.7417/CT.2021.2281},
pmid = {33346327},
issn = {1972-6007},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Mammalian ; Gene Editing/*ethics/*instrumentation ; Humans ; },
abstract = {Human germline engineering arguably constitutes one of the most promising and at the same time controversial prospects in the realm of gene editing overall, and particularly in the context of the current state of research. The issues raised by such techniques have sparked heated debate worldwide: the scientific and industrial establishments have been strongly supporting CRISPR-Cas9 research, but a well-balanced approach needs to be adopted in order to reconcile the needs of scientific research with the life and dignity of human embryos.},
}
@article {pmid33346188,
year = {2020},
author = {Li, H and Qin, H and Zhang, N and Zhao, J and Xin, J and Perez-Campo, FM and Liu, H},
title = {Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {166},
pages = {},
doi = {10.3791/59639},
pmid = {33346188},
issn = {1940-087X},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA/metabolism ; DNA Repair ; *Gene Knockout Techniques ; *Genes, Reporter ; Genetic Vectors/metabolism ; Luciferases/genetics/*metabolism ; Mammals/*metabolism ; Oligonucleotides/metabolism ; Plasmids/*genetics ; RNA, Guide/genetics ; Reproducibility of Results ; Sheep ; Transformation, Genetic ; },
abstract = {Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).},
}
@article {pmid33345196,
year = {2020},
author = {Hulton, CH and Costa, EA and Shah, NS and Quintanal-Villalonga, A and Heller, G and de Stanchina, E and Rudin, CM and Poirier, JT},
title = {Direct genome editing of patient-derived xenografts using CRISPR-Cas9 enables rapid in vivo functional genomics.},
journal = {Nature cancer},
volume = {1},
number = {3},
pages = {359-369},
pmid = {33345196},
issn = {2662-1347},
support = {R01 CA197936/CA/NCI NIH HHS/United States ; U24 CA213274/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 EY012196/EY/NEI NIH HHS/United States ; U01 CA199215/CA/NCI NIH HHS/United States ; P01 CA129243/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genomics ; Heterografts ; Humans ; Mice ; },
abstract = {Patient-derived xenografts are high fidelity in vivo tumor models that accurately reflect many key aspects of human cancer. In contrast to either cancer cell lines or genetically engineered mouse models, the utility of PDXs has been limited by the inability to perform targeted genome editing of these tumors. To address this limitation, we have developed methods for CRISPR-Cas9 editing of PDXs using a tightly regulated, inducible Cas9 vector that does not require in vitro culture for selection of transduced cells. We demonstrate the utility of this platform in PDXs (1) to analyze genetic dependencies by targeted gene disruption and (2) to analyze mechanisms of acquired drug resistance by site-specific gene editing using templated homology-directed repair. This flexible system has broad application to other explant models and substantially augments the utility of PDXs as genetically programmable models of human cancer.},
}
@article {pmid33343607,
year = {2020},
author = {Maioli, A and Gianoglio, S and Moglia, A and Acquadro, A and Valentino, D and Milani, AM and Prohens, J and Orzaez, D and Granell, A and Lanteri, S and Comino, C},
title = {Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {607161},
pmid = {33343607},
issn = {1664-462X},
abstract = {Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.},
}
@article {pmid33342694,
year = {2021},
author = {Zhang, T and Zhou, W and Lin, X and Khan, MR and Deng, S and Zhou, M and He, G and Wu, C and Deng, R and He, Q},
title = {Light-up RNA aptamer signaling-CRISPR-Cas13a-based mix-and-read assays for profiling viable pathogenic bacteria.},
journal = {Biosensors &amp; bioelectronics},
volume = {176},
number = {},
pages = {112906},
doi = {10.1016/j.bios.2020.112906},
pmid = {33342694},
issn = {1873-4235},
mesh = {*Aptamers, Nucleotide ; Bacteria/genetics ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; },
abstract = {Viable pathogenic bacteria cause serious human diseases via systemic infections and food poisoning. Herein, we constructed a light-up RNA aptamer signaling-CRISPR-Cas13a assay enabling mix-and-read detection of viable pathogenic bacteria. Directly targeting pathogen RNAs via CRISPR-Cas13a allows precisely discriminating viable bacteria from dead bacteria. We introduced a light-up RNA aptamer, Broccoli, serving as the substate of activated CRISPR-Cas13a to monitor the presence of pathogen RNAs, eliminating the need to use chemically labeled RNA substrate. Sequentially, the assay allows a reverse transcription-free, nucleic acid amplification-free, and label-free quantification of RNA targets and viable pathogenic bacteria. It could detect as low as 10 CFU of Bacillus cereus and precisely quantify viable bacteria with a content ranging from 0% to 100% in 10[5] CFU total bacteria. The quantification of viable bacteria allows more accurately estimating the ability of B. cereus to spoil food. The RNA assay promises its use in point-of-use detection of viable pathogens and biosafety control.},
}
@article {pmid33340931,
year = {2021},
author = {Zhen, S and Qiang, R and Lu, J and Tuo, X and Yang, X and Li, X},
title = {Enhanced antiviral benefit of combination therapy with anti-HBV and anti-PD1 gRNA/cas9 produces a synergistic antiviral effect in HBV infection.},
journal = {Molecular immunology},
volume = {130},
number = {},
pages = {7-13},
doi = {10.1016/j.molimm.2020.12.004},
pmid = {33340931},
issn = {1872-9142},
mesh = {Animals ; Antiviral Agents/metabolism ; CRISPR-Cas Systems/*genetics ; Combined Modality Therapy/methods ; Drug Synergism ; Female ; Gene Editing/*methods ; Genetic Therapy/methods ; Hep G2 Cells ; Hepatitis B/genetics/*therapy ; Hepatitis B virus/*genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/*genetics ; RNA, Guide/genetics/*therapeutic use ; },
abstract = {Targeted therapy for patients with hepatitis B virus (HBV) infection can lead to objective responses, although response times may be short. At the same time, the response rate to programmed cell death-1 (PD-1) treatment was more durable. It is speculated that HBV targeted therapy can synergistically enhance the antitumor activity with PD-1 blockade. To test this hypothesis, we evaluated the effect of crispr-cas9 on HBV and PD-1 in vitro and in vivo. We found that HBV targeting gRNA/cas9 induced a decrease in the expression of HBsAg, while the PD-1 gene could be knocked out by electroporation targeting gRNA / cas9 by polymerase chain reaction. In HBV transgenic mice, the immunophenotype and cytokine expression of human dendritic cells (DCS) were detected by crispr-cas9 system stimulation, flow cytometry and polymerase chain reaction. These results indicate that gRNA/cas9 treatment upregulates the expression of CD80, CD83 and CD86, and significantly increases the mRNA levels of IL-6, IL-12, IL-23 and tumor necrosis factor alpha. The combination of anti HBV and anti PD-1 therapy can inhibit HBV expression and significantly improve the survival of HBV transgenic mice. In addition, the combination therapy increased the production of interferon by T cells, and then enhanced the expression of Th1 related immunostimulatory genes, thereby reducing the transcription of regulatory / inhibitory immune genes. In general, this response can reshape the tumor microenvironment from immunosuppression to immune stimulation. Finally, anti HBV therapy can induce the expression of interferon dependent programmed cell death ligand-1 in HBV transgenic mice in vivo. To sum up, these results demonstrate that the combination of HBV targeted therapy and PD-1 immune checkpoint block has a strong synergistic effect, thus supporting the transformation potential of this combined therapy strategy in clinical treatment of HBV infection.},
}
@article {pmid33340357,
year = {2021},
author = {Libby, ARG and Joy, DA and McDevitt, TC},
title = {Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2258},
number = {},
pages = {105-116},
pmid = {33340357},
issn = {1940-6029},
mesh = {*Body Patterning ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Expression Regulation, Developmental ; Microscopy, Fluorescence ; Microscopy, Video ; Pluripotent Stem Cells/*physiology ; RNA, Guide/genetics/metabolism ; Signal Transduction ; Time Factors ; Time-Lapse Imaging ; },
abstract = {Pluripotent stem cells (PSCs) possess the ability to self-organize into complex tissue-like structures; however, the genetic mechanisms and multicellular dynamics that direct such patterning are difficult to control. Here, we pair live imaging with controlled induction of gene knockdown by CRISPR interference (CRISPRi) to generate changes within subpopulations of human PSCs, allowing for control over organization and analysis of emergent behaviors. Specifically, we use forced aggregation of mixtures of cells with and without an inducible CRISPRi system to knockdown molecular regulators of tissue symmetry. We then track the resulting multicellular organization through fluorescence live imaging concurrent with the induction of knockdown. Overall, this technique allows for controlled initiation of symmetry breaking by CRISPRi to produce changes in cellular behavior that can be tracked over time within high-density pluripotent stem cell colonies.},
}
@article {pmid33340356,
year = {2021},
author = {Nanos, V and Levin, M},
title = {Rewiring Endogenous Bioelectric Circuits in the Xenopus laevis Embryo Model.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2258},
number = {},
pages = {93-103},
pmid = {33340356},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Connexins/genetics/*metabolism ; Electrical Synapses/genetics/*metabolism ; Embryo, Nonmammalian/metabolism ; Embryonic Development ; *Gene Editing ; Gene Expression Regulation, Developmental ; Ion Channels/genetics/*metabolism ; Membrane Potentials ; Microscopy, Fluorescence ; RNA, Guide/genetics/metabolism ; Time Factors ; Xenopus laevis/embryology/genetics/*metabolism ; },
abstract = {Embryogenesis, as well as regeneration, is increasingly recognized to be orchestrated by an interplay of transcriptional and bioelectric networks. Spatiotemporal patterns of resting potentials direct the size, shape, and locations of numerous organ primordia during patterning. These bioelectrical properties are established by the function of ion channels and pumps that set voltage potentials of individual cells, and gap junctions (electrical synapses) that enable physiological states to propagate across tissue networks. Functional experiments to probe the roles of bioelectrical states can be carried out by targeting endogenous ion channels during development. Here, we describe protocols, optimized for the highly tractable Xenopus laevis embryo, for molecular genetic targeting of ion channels and connexins based on CRISPR, and monitoring of resting potential states using voltage-sensing fluorescent dye. Similar strategies can be adapted to other model species.},
}
@article {pmid33339985,
year = {2020},
author = {Ishibashi, A and Saga, K and Hisatomi, Y and Li, Y and Kaneda, Y and Nimura, K},
title = {A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22345},
pmid = {33339985},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genetic Vectors/genetics ; Homozygote ; Humans ; Mice ; RNA, Guide/*genetics ; Sequence Deletion/genetics ; },
abstract = {CRISPR-Cas9 system can be used to generate knock-out cancer cell lines. An insertion or deletion induced by a single guide RNA (gRNA) is often used to generate knock-out cells, however, some cells express the target gene by skipping the disrupted exon, or by using a splicing variant, thus losing the target exon. To overcome this unexpected expression of the target gene, almost the entire gene can be swapped with a selection marker. However, it is time-consuming to create a targeting vector which contains 5' and 3' homology arms flanked by a selection marker. Here, we developed a simple and easy method called SUCCESS (Single-strand oligodeoxynucleotides, Universal Cassette, and CRISPR/Cas9 produce Easy Simple knock-out System), to knock-out a target gene without constructing a targeting vector. Our method removed the targeted large genomic region by using two pX330 plasmids encoding Cas9 and gRNA, two 80mer single strand oligodeoxynucleotides (ssODN), and a blunt-ended universal selection maker sequence in B16F10 murine cancer cell and ID8 murine ovarian cancer cell. SUCCESS generated knock-out clones in two murine cancer cell lines by homozygous deletion of the target genomic region, and without constructing targeting vectors. Thus, our method can be widely applied to generate homozygous knock-out cell lines, as well as knock-in cell lines.},
}
@article {pmid33339870,
year = {2020},
author = {Hennig, SL and Owen, JR and Lin, JC and Young, AE and Ross, PJ and Van Eenennaam, AL and Murray, JD},
title = {Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22309},
pmid = {33339870},
issn = {2045-2322},
support = {S10 OD010786/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cattle ; Embryo, Mammalian ; *Gene Editing ; Genome/genetics ; Mosaicism ; Mutation/genetics ; Mutation Rate ; RNA, Messenger/genetics ; },
abstract = {The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.},
}
@article {pmid33339510,
year = {2020},
author = {Choo, A and Fung, E and Chen, IY and Saint, R and Crisp, P and Baxter, SW},
title = {Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni.},
journal = {BMC genetics},
volume = {21},
number = {Suppl 2},
pages = {127},
pmid = {33339510},
issn = {1471-2156},
mesh = {Alleles ; Amino Acid Sequence ; Animals ; Australia ; *CRISPR-Cas Systems ; Genetic Fitness ; Genotype ; Insect Control ; Phenotype ; *Point Mutation ; Sequence Alignment ; *Synthetic Lethal Mutations ; Temperature ; Tephritidae/*genetics ; },
abstract = {BACKGROUND: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required.<br><br>RESULTS: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29&#x2009;&#xb0;C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shi[ts1]) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shi[ts1] mutant allele and shi[ts1] homozygotes were not viable at 21&#x2009;&#xb0;C, which is a more severe phenotype than that documented in D. melanogaster.<br><br>CONCLUSIONS: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.},
}
@article {pmid33339496,
year = {2020},
author = {Primo, P and Meccariello, A and Inghilterra, MG and Gravina, A and Del Corsano, G and Volpe, G and Sollazzo, G and Aceto, S and Robinson, MD and Salvemini, M and Saccone, G},
title = {Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations.},
journal = {BMC genetics},
volume = {21},
number = {Suppl 2},
pages = {150},
pmid = {33339496},
issn = {1471-2156},
mesh = {Alternative Splicing ; Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Ceratitis capitata/*genetics ; Female ; Genes, Insect ; Male ; RNA, Guide/genetics ; *Sex Determination Processes ; },
abstract = {BACKGROUND: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs.<br><br>RESULTS: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults.<br><br>CONCLUSIONS: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.},
}
@article {pmid33339449,
year = {2020},
author = {Chung, PJ and Chung, H and Oh, N and Choi, J and Bang, SW and Jung, SE and Jung, H and Shim, JS and Kim, JK},
title = {Efficiency of Recombinant CRISPR/rCas9-Mediated miRNA Gene Editing in Rice.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33339449},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Droughts ; Gene Editing/*methods ; MicroRNAs/*genetics ; Oryza/*genetics/physiology ; Plant Breeding/*methods ; Stress, Physiological ; },
abstract = {Drought is one of the major environmental stresses adversely affecting crop productivity worldwide. Precise characterization of genes involved in drought response is necessary to develop new crop varieties with enhanced drought tolerance. Previously, we identified 66 drought-induced miRNAs in rice plants. For the further functional investigation of the miRNAs, we applied recombinant codon-optimized Cas9 (rCas9) for rice with single-guide RNAs specifically targeting mature miRNA sequences or sites required for the biogenesis of mature miRNA. A total of 458 T0 transgenic plants were analyzed to determine the frequency and type of mutations induced by CRISPR/rCas9 on 13 independent target miRNAs. The average mutation frequency for 13 genes targeted by single guide RNAs (sgRNAs) in T0 generation was 59.4%, including mono-allelic (8.54%), bi-allelic (11.1%), and hetero-allelic combination (39.7%) mutations. The mutation frequency showed a positive correlation with Tm temperature of sgRNAs. For base insertion, one base insertion (99%) was predominantly detected in transgenic plants. Similarly, one base deletion accounted for the highest percentage, but there was also a significant percentage of cases in which more than one base was deleted. The deletion of more than two bases in OsmiR171f and OsmiR818b significantly reduced the level of corresponding mature miRNAs. Further functional analysis using CRISPR/Cas9-mediated mutagenesis confirmed that OsmiR818b is involved in drought response in rice plants. Overall, this study suggests that the CRISPR/rCas9 system is a powerful tool for loss-of-function analysis of miRNA in rice.},
}
@article {pmid33339441,
year = {2020},
author = {Janik, E and Niemcewicz, M and Ceremuga, M and Krzowski, L and Saluk-Bijak, J and Bijak, M},
title = {Various Aspects of a Gene Editing System-CRISPR-Cas9.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33339441},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Epigenesis, Genetic ; Gene Editing/ethics/*methods/standards ; Genetic Therapy/ethics/methods/standards ; Humans ; },
abstract = {The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR-Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR-Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR-Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR-Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR-Cas9.},
}
@article {pmid33339321,
year = {2020},
author = {Chiu, W and Hsun, YH and Chang, KJ and Yarmishyn, AA and Hsiao, YJ and Chien, Y and Chien, CS and Ma, C and Yang, YP and Tsai, PH and Chiou, SH and Lin, TY and Cheng, HM},
title = {Current Genetic Survey and Potential Gene-Targeting Therapeutics for Neuromuscular Diseases.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33339321},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Clinical Trials as Topic ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Neuromuscular Diseases/*genetics/therapy ; },
abstract = {Neuromuscular diseases (NMDs) belong to a class of functional impairments that cause dysfunctions of the motor neuron-muscle functional axis components. Inherited monogenic neuromuscular disorders encompass both muscular dystrophies and motor neuron diseases. Understanding of their causative genetic defects and pathological genetic mechanisms has led to the unprecedented clinical translation of genetic therapies. Challenged by a broad range of gene defect types, researchers have developed different approaches to tackle mutations by hijacking the cellular gene expression machinery to minimize the mutational damage and produce the functional target proteins. Such manipulations may be directed to any point of the gene expression axis, such as classical gene augmentation, modulating premature termination codon ribosomal bypass, splicing modification of pre-mRNA, etc. With the soar of the CRISPR-based gene editing systems, researchers now gravitate toward genome surgery in tackling NMDs by directly correcting the mutational defects at the genome level and expanding the scope of targetable NMDs. In this article, we will review the current development of gene therapy and focus on NMDs that are available in published reports, including Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked myotubular myopathy (XLMTM), Spinal Muscular Atrophy (SMA), and Limb-girdle muscular dystrophy Type 2C (LGMD2C).},
}
@article {pmid33338753,
year = {2021},
author = {Askjaer, P and Harr, JC},
title = {Genetic approaches to revealing the principles of nuclear architecture.},
journal = {Current opinion in genetics &amp; development},
volume = {67},
number = {},
pages = {52-60},
doi = {10.1016/j.gde.2020.11.001},
pmid = {33338753},
issn = {1879-0380},
mesh = {Adenine/metabolism ; CRISPR-Cas Systems/genetics ; Cell Nucleus/genetics/ultrastructure ; Chromatin/genetics/*ultrastructure ; Chromosomes/genetics/*ultrastructure ; DNA Methylation/*genetics ; DNA Repair/genetics ; Gene Expression Regulation/genetics ; Genome/*genetics ; Humans ; In Situ Hybridization, Fluorescence ; RNA Interference ; },
abstract = {The spatial organization of chromosomes inside the eukaryotic nucleus is important for DNA replication, repair and gene expression. During development of multicellular organisms, different compendiums of genes are either repressed or activated to produce specific cell types. Genetic manipulation of tractable organisms is invaluable to elucidate chromosome configuration and the underlying mechanisms. Systematic inhibition of genes through RNA interference and, more recently, CRISPR/Cas9-based screens have identified new proteins with significant roles in nuclear organization. Coupling this with advances in imaging techniques, such as multiplexed DNA fluorescence in situ hybridization, and with tissue-specific genome profiling by DNA adenine methylation identification has increased our knowledge about the immense complexity and dynamics of the nucleus.},
}
@article {pmid33338493,
year = {2021},
author = {Mejdani, M and Pawluk, A and Maxwell, KL and Davidson, AR},
title = {Anti-CRISPR AcrIE2 Binds the Type I-E CRISPR-Cas Complex But Does Not Block DNA Binding.},
journal = {Journal of molecular biology},
volume = {433},
number = {3},
pages = {166759},
doi = {10.1016/j.jmb.2020.166759},
pmid = {33338493},
issn = {1089-8638},
support = {MOP-130482//CIHR/Canada ; FDN-15427//CIHR/Canada ; PJT-152918//CIHR/Canada ; },
mesh = {Amino Acid Sequence ; CRISPR-Associated Proteins/*chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics/*metabolism ; DNA-Binding Proteins/chemistry/genetics/metabolism ; Magnetic Resonance Spectroscopy ; Models, Molecular ; Promoter Regions, Genetic ; Protein Binding ; Protein Conformation ; Solubility ; Structure-Activity Relationship ; },
abstract = {Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage.},
}
@article {pmid33338433,
year = {2021},
author = {Park, CH and Jeoung, YH and Uh, KJ and Park, KE and Bridge, J and Powell, A and Li, J and Pence, L and Zhang, L and Liu, T and Sun, HX and Gu, Y and Shen, Y and Wu, J and Izpisua Belmonte, JC and Telugu, BP},
title = {Extraembryonic Endoderm (XEN) Cells Capable of Contributing to Embryonic Chimeras Established from Pig Embryos.},
journal = {Stem cell reports},
volume = {16},
number = {1},
pages = {212-223},
pmid = {33338433},
issn = {2213-6711},
support = {R01 HD092304/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Blastocyst/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Chimera ; Collagen Type I, alpha 1 Chain/genetics ; Embryo, Mammalian/*cytology/metabolism ; Endoderm/*cytology/metabolism ; Gene Editing ; Mice ; Swine ; },
abstract = {Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.},
}
@article {pmid33338421,
year = {2021},
author = {Hoffmann, HH and Schneider, WM and Rozen-Gagnon, K and Miles, LA and Schuster, F and Razooky, B and Jacobson, E and Wu, X and Yi, S and Rudin, CM and MacDonald, MR and McMullan, LK and Poirier, JT and Rice, CM},
title = {TMEM41B Is a Pan-flavivirus Host Factor.},
journal = {Cell},
volume = {184},
number = {1},
pages = {133-148.e20},
pmid = {33338421},
issn = {1097-4172},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 AI124690/AI/NIAID NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Asians/genetics ; Autophagy ; COVID-19/genetics/metabolism/virology ; CRISPR-Cas Systems ; Cell Line ; Flavivirus/*physiology ; Flavivirus Infections/*genetics/immunology/metabolism/virology ; Gene Knockout Techniques ; Genome-Wide Association Study ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Membrane Proteins/genetics/*metabolism ; Polymorphism, Single Nucleotide ; SARS-CoV-2/physiology ; Virus Replication ; Yellow fever virus/physiology ; Zika Virus/physiology ; },
abstract = {Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.},
}
@article {pmid33338046,
year = {2020},
author = {O'Leary, S and Adelman, ZN},
title = {CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes.},
journal = {PLoS neglected tropical diseases},
volume = {14},
number = {12},
pages = {e0008971},
pmid = {33338046},
issn = {1935-2735},
mesh = {Aedes/*genetics/physiology/virology ; Animals ; *CRISPR-Cas Systems ; Female ; Flight, Animal ; Gene Knockout Techniques ; Humans ; Insecticides/*pharmacology ; Male ; *Mosquito Control ; Mosquito Vectors/*genetics/physiology/virology ; Phenotype ; Zika Virus/*physiology ; Zika Virus Infection/*prevention &amp; control/transmission/virology ; },
abstract = {Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito.},
}
@article {pmid33337558,
year = {2021},
author = {You, H and Mayer, JU and Johnston, RL and Sivakumaran, H and Ranasinghe, S and Rivera, V and Kondrashova, O and Koufariotis, LT and Du, X and Driguez, P and French, JD and Waddell, N and Duke, MG and Ittiprasert, W and Mann, VH and Brindley, PJ and Jones, MK and McManus, DP},
title = {CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {1},
pages = {e21205},
doi = {10.1096/fj.202001745RR},
pmid = {33337558},
issn = {1530-6860},
support = {107475/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Acetylcholinesterase/genetics/*metabolism ; Animals ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; Helminth Proteins/genetics/*metabolism ; Mice ; Schistosoma mansoni/*enzymology/genetics ; Schistosomiasis mansoni/genetics/*metabolism ; },
abstract = {CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.},
}
@article {pmid33337270,
year = {2020},
author = {Flotte, TR},
title = {Writing the Story of Gene Editing and CRISPR: An Interview with Kevin Davies, PhD.},
journal = {Human gene therapy},
volume = {31},
number = {23-24},
pages = {1217-1220},
doi = {10.1089/hum.2020.29144.int},
pmid = {33337270},
issn = {1557-7422},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Genetic Therapy/*trends ; Humans ; Peer Review, Research/*trends ; },
}
@article {pmid33336604,
year = {2021},
author = {Luo, N and Li, J and Chen, Y and Xu, Y and Wei, Y and Lu, J and Dong, R},
title = {Hepatic stellate cell reprogramming via exosome-mediated CRISPR/dCas9-VP64 delivery.},
journal = {Drug delivery},
volume = {28},
number = {1},
pages = {10-18},
pmid = {33336604},
issn = {1521-0464},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Drug Carriers ; Exosomes/*physiology ; Gene Editing/*methods ; Hepatic Stellate Cells/*physiology ; Humans ; Mice ; Mice, Inbred C57BL ; },
abstract = {Hepatic stellate cells (HSCs) play a crucial role in the progression of liver fibrosis, which can be considered as the specific therapeutic target of anti-fibrotic treatment. Targeted induction of HSCs to hepatocytes via delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (dCas9) system holds promise for hepatic fibrosis treatment. Our study here revealed that CRISPR/dCas9-VP64 system encapsulated in AML12 cell-derived exosomes could efficiently and successfully be delivered into the HSCs. In turn, the CRISPR/dCas9-VP64 system loaded in the exosomes can be efficiently released into the HSCs. As a proof-of-concept study, gRNA against hepatocyte nuclear factor 4α (HNF4α) together with the delivery of CRISPR/dCas9-VP64 system induced the HSCs to hepatocyte-like phenotype. In conclusion, our study here revealed that CRISPR/dCas9-VP64 system encapsulated in AML12 cell-derived exosomes could be functional in HSCs, emerging as a gene therapy strategy for hepatic fibrosis.},
}
@article {pmid33336280,
year = {2021},
author = {Thongsin, N and Wattanapanitch, M},
title = {CRISPR/Cas9-Mediated GFP Reporter Knock-in in K562 and Raji Cell Lines for Tracking Immune Cell Killing Assay.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2211},
number = {},
pages = {213-229},
pmid = {33336280},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Clonal Evolution/genetics ; Cytotoxicity, Immunologic/*genetics ; Flow Cytometry ; *Gene Editing ; Gene Expression ; Gene Knock-In Techniques ; *Genes, Reporter ; Green Fluorescent Proteins/genetics ; Humans ; K562 Cells ; Plasmids/genetics ; },
abstract = {Cell-mediated cytotoxicity plays an important role in several fundamental immunological processes and is crucial for biological evaluation in in vitro studies. In order to determine the immunological activities of the cells, an assay should be safe, reproducible, and cost-effective. Here, we present a simple and cost-effective approach for evaluation of natural killer (NK) cell-mediated cytotoxicity by generating a CRISPR/Cas9-mediated GFP reporter knock-in in the target cell line, K562, and the non-target cell line, Raji, using a plasmid-based transfection method. The GFP[+] target cells facilitate tracking of the immune cell killing assay, which avoids the need for multiple cell labeling with fluorescent dyes. Our approach is also applicable to the genome editing of other target cell types for functional analysis of effector cells.},
}
@article {pmid33336279,
year = {2021},
author = {Wattanapanitch, M},
title = {Correction of Hemoglobin E/Beta-Thalassemia Patient-Derived iPSCs Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2211},
number = {},
pages = {193-211},
pmid = {33336279},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cloning, Molecular ; *Gene Editing/methods ; Gene Expression ; Gene Targeting ; Genetic Vectors/genetics ; Hematopoietic Stem Cells/cytology/metabolism ; Hemoglobin E/*genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Mutation ; RNA, Guide/genetics ; Recombinational DNA Repair ; beta-Globins/genetics ; beta-Thalassemia/*genetics ; },
abstract = {HbE/β-thalassemia is one of the most common thalassemic syndromes in Southeast Asia and Thailand. Patients have mutations in β hemoglobin (HBB) gene resulting in decreased and/or abnormal production of β hemoglobin. Here, we describe a protocol for CRISPR/Cas9-mediated gene correction of the mutated hemoglobin E from one allele of the HBB gene by homology-directed repair (HDR) in HbE/β-thalassemia patient-derived induced pluripotent stem cells (iPSCs) using a CRISPR/Cas9 plasmid-based transfection method and a single-stranded DNA oligonucleotide (ssODN) repair template harboring the correct nucleotides. Our strategy allows the seamless HbE gene correction with the editing efficiency (HDR) up to 3%, as confirmed by Sanger sequencing. This protocol provides a simple one-step genetic correction of HbE mutation in the patient-derived iPSCs. Further differentiation of the corrected iPSCs into hematopoietic stem/progenitor cells will provide an alternative renewable source of cells for the application in autologous transplantation in the future.},
}
@article {pmid33336278,
year = {2021},
author = {Chooi, WH and Chin, JS and Chew, SY},
title = {Scaffold-Based Delivery of CRISPR/Cas9 Ribonucleoproteins for Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2211},
number = {},
pages = {183-191},
pmid = {33336278},
issn = {1940-6029},
mesh = {Adhesives/chemistry ; Animals ; CRISPR-Associated Protein 9/chemistry ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Expression ; Gene Transfer Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Lipids/chemistry ; Macromolecular Substances/chemistry/metabolism ; Nanofibers/chemistry ; RNA, Guide/chemistry/genetics ; Ribonucleoproteins/genetics/*metabolism ; Tissue Engineering ; *Tissue Scaffolds/chemistry ; },
abstract = {The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.},
}
@article {pmid33335523,
year = {2020},
author = {Ahmad, S and Huang, Q and Ni, J and Xiao, Y and Yang, Y and Shen, Y},
title = {Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {607431},
pmid = {33335523},
issn = {1664-302X},
abstract = {EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn[2+] was used as the co-factor instead of Mg[2+], SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10[-3]) than that of the wild type (4.6 × 10[-6]). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.},
}
@article {pmid33335280,
year = {2020},
author = {Simora, RMC and Xing, D and Bangs, MR and Wang, W and Ma, X and Su, B and Khan, MGQ and Qin, Z and Lu, C and Alston, V and Hettiarachchi, D and Johnson, A and Li, S and Coogan, M and Gurbatow, J and Terhune, JS and Wang, X and Dunham, RA},
title = {CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22271},
pmid = {33335280},
issn = {2045-2322},
mesh = {Alligators and Crocodiles/*genetics ; Animals ; Antimicrobial Cationic Peptides/*genetics ; CRISPR-Cas Systems/*genetics ; Catfishes/*genetics/growth &amp; development ; Gene Editing ; Gene Knock-In Techniques ; Gene Targeting/methods ; Genome/genetics ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; },
abstract = {CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.},
}
@article {pmid33335226,
year = {2020},
author = {Honda, T and Nishikawa, Y and Nishimura, K and Teng, D and Takemoto, K and Ueda, K},
title = {Effects of activation of the LINE-1 antisense promoter on the growth of cultured cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22136},
pmid = {33335226},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Line ; Cell Proliferation ; Gene Expression Profiling ; Humans ; *Long Interspersed Nucleotide Elements ; Open Reading Frames ; *Promoter Regions, Genetic ; Retroelements ; Transcriptional Activation ; Transcriptome ; },
abstract = {Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~ 17% of the human genome. Although ~ 6000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. The L1 5' untranslated region has bidirectional promoter activity with a sense promoter driving L1 mRNA production and an antisense promoter (ASP) driving the production of L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using CRISPR-Cas9 technology to evaluate its biological impacts. Activation of the L1 ASP upregulated the expression of L1 ASP-driven ORF0 and enhanced cell growth. Furthermore, the exogenous expression of ORF0 also enhanced cell growth. These results indicate that activation of L1 ASP activity fuels cell growth at least through ORF0 expression. To our knowledge, this is the first report demonstrating the role of the L1 ASP in a biological context. Considering that L1 sequences are desilenced in various tumor cells, our results indicate that activation of the L1 ASP may be a cause of tumor growth; therefore, interfering with L1 ASP activity may be a potential strategy to suppress the growth.},
}
@article {pmid33335164,
year = {2020},
author = {Kang, S and Jeon, S and Kim, S and Chang, YK and Kim, YC},
title = {Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22158},
pmid = {33335164},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Chlamydomonas reinhardtii/*genetics ; DNA, Chloroplast ; *Gene Editing ; Gene Targeting ; *Gene Transfer Techniques ; Genetic Engineering ; Mutagenesis ; *Peptides/chemistry ; RNA, Guide/genetics ; Ribonucleoproteins/*genetics ; },
abstract = {Recent technical advances related to the CRISPR/Cas9-based genome editing system have enabled sophisticated genome editing in microalgae. Although the demand for research on genome editing in microalgae has increased over time, methodological research has not been established to date for the delivery of a ribonucleoprotein (Cas9/sgRNA complex) using a cell penetrating peptide into microalgal cell lines. Here, we present a ribonucleoprotein delivery system for Chlamydomonas reinhardtii mediated by the cell penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) which is in a non-covalent form. Using this technically simple method, the ribonucleoprotein was successfully delivered into C. reinhardtii. Gene Maa7 and FKB12 were disrupted, and their distinguishing patterns of Indel mutations were analyzed with the observation of several insertions of sequences not originating from the genome DNA, such as chloroplast DNA, into the expected loci. In addition, the cytotoxicity of Cas9 and the ribonucleoprotein was investigated according to the concentration and time in the algal cells. It was observed that Cas9 alone without the sgRNA induces a more severe cytotoxicity compared to the ribonucleoprotein. Our study will not only contribute to algal cell biology and its genetic engineering for further applications involving various organisms but will also provide a deeper understating of the basic science of the CRISPR/Cas9 system.},
}
@article {pmid33334884,
year = {2021},
author = {Agrawal, P and Blinka, S and Pulakanti, K and Reimer, MH and Stelloh, C and Meyer, AE and Rao, S},
title = {Genome editing demonstrates that the -5 kb Nanog enhancer regulates Nanog expression by modulating RNAPII initiation and/or recruitment.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100189},
pmid = {33334884},
issn = {1083-351X},
support = {F30 DK120152/DK/NIDDK NIH HHS/United States ; R01 CA204231/CA/NCI NIH HHS/United States ; T32 GM080202/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Enhancer Elements, Genetic ; Gene Editing ; Gene Expression Regulation ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Nanog Homeobox Protein/*genetics ; RNA Polymerase II/*genetics ; Transcriptional Activation ; },
abstract = {Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmid-based reporter assays of gene expression. At present, histone marks are used to identify and define enhancers but do not consider the endogenous role of an enhancer in the context of native chromatin. We employed a combination of genomic editing, single cell analyses, and sequencing approaches to investigate a Nanog-associated cis-regulatory element, which has been reported by others to be either an alternative promoter or a super-enhancer. We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We next demonstrate that the dominant super-enhancer modulates Nanog globally and operates by recruiting and/or initiating RNA Polymerase II. Our studies have important implications to how transcriptional enhancers are defined and how they regulate gene expression.},
}
@article {pmid33334730,
year = {2021},
author = {Upadhyay, R and Boiarsky, JA and Pantsulaia, G and Svensson-Arvelund, J and Lin, MJ and Wroblewska, A and Bhalla, S and Scholler, N and Bot, A and Rossi, JM and Sadek, N and Parekh, S and Lagana, A and Baccarini, A and Merad, M and Brown, BD and Brody, JD},
title = {A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy.},
journal = {Cancer discovery},
volume = {11},
number = {3},
pages = {599-613},
pmid = {33334730},
issn = {2159-8290},
support = {R37 CA246239/CA/NCI NIH HHS/United States ; U19 AI128949/AI/NIAID NIH HHS/United States ; R01 AI113221/AI/NIAID NIH HHS/United States ; R33 CA223947/CA/NCI NIH HHS/United States ; R01 CA154947/CA/NCI NIH HHS/United States ; R01 CA190400/CA/NCI NIH HHS/United States ; T32 AI007605/AI/NIAID NIH HHS/United States ; P30 CA196521/CA/NCI NIH HHS/United States ; CI-76-14/DRCRF_/Damon Runyon Cancer Research Foundation/United States ; R01 AT011326/AT/NCCIH NIH HHS/United States ; T32 GM007280/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Antigens, Neoplasm/immunology ; Bystander Effect/immunology ; CD8-Positive T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; *Cytotoxicity, Immunologic ; Disease Models, Animal ; Gene Editing ; Genetic Engineering ; Humans ; *Immunotherapy/adverse effects/methods ; Immunotherapy, Adoptive/adverse effects/methods ; Mice ; Mice, Knockout ; Neoplasms/etiology/therapy ; Receptors, Chimeric Antigen ; T-Cell Antigen Receptor Specificity ; T-Lymphocytes/*immunology/*metabolism ; Treatment Outcome ; Xenograft Model Antitumor Assays ; fas Receptor/*metabolism ; },
abstract = {T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants. SIGNIFICANCE: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse.This article is highlighted in the In This Issue feature, p. 521.},
}
@article {pmid33334019,
year = {2020},
author = {Moranguinho, I and Valente, ST},
title = {Block-And-Lock: New Horizons for a Cure for HIV-1.},
journal = {Viruses},
volume = {12},
number = {12},
pages = {},
pmid = {33334019},
issn = {1999-4915},
support = {R01 AI097012/AI/NIAID NIH HHS/United States ; R01 AI118432/AI/NIAID NIH HHS/United States ; R33 AI116226/AI/NIAID NIH HHS/United States ; R61 AI140439/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-HIV Agents/*pharmacology/therapeutic use ; Antiretroviral Therapy, Highly Active ; CD4-Positive T-Lymphocytes/drug effects/immunology/metabolism ; Combined Modality Therapy/methods ; Gene Expression Regulation, Viral ; Genetic Therapy/methods ; HIV Infections/drug therapy/immunology/*virology ; HIV-1/*drug effects/*physiology ; Host-Pathogen Interactions ; Humans ; Proviruses/genetics ; Transcription, Genetic ; Treatment Outcome ; Virus Activation/drug effects ; Virus Latency/drug effects ; Virus Replication/drug effects ; },
abstract = {HIV-1/AIDS remains a global public health problem. The world health organization (WHO) reported at the end of 2019 that 38 million people were living with HIV-1 worldwide, of which only 67% were accessing antiretroviral therapy (ART). Despite great success in the clinical management of HIV-1 infection, ART does not eliminate the virus from the host genome. Instead, HIV-1 remains latent as a viral reservoir in any tissue containing resting memory CD4[+] T cells. The elimination of these residual proviruses that can reseed full-blown infection upon treatment interruption remains the major barrier towards curing HIV-1. Novel approaches have recently been developed to excise or disrupt the virus from the host cells (e.g., gene editing with the CRISPR-Cas system) to permanently shut off transcription of the virus (block-and-lock and RNA interference strategies), or to reactivate the virus from cell reservoirs so that it can be eliminated by the immune system or cytopathic effects (shock-and-kill strategy). Here, we will review each of these approaches, with the major focus placed on the block-and-lock strategy.},
}
@article {pmid33334013,
year = {2020},
author = {Clifford, RE and Govindarajah, N and Bowden, D and Sutton, P and Glenn, M and Darvish-Damavandi, M and Buczacki, S and McDermott, U and Szulc, Z and Ogretmen, B and Parsons, JL and Vimalachandran, D},
title = {Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer.},
journal = {Cells},
volume = {9},
number = {12},
pages = {},
pmid = {33334013},
issn = {2073-4409},
support = {MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Acid Ceramidase/*metabolism ; Apoptosis/radiation effects ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/radiation effects ; Gene Editing ; Humans ; Models, Biological ; Organoids/pathology/radiation effects ; *Radiation Tolerance ; Rectal Neoplasms/*enzymology/*radiotherapy ; Tumor Suppressor Protein p53/metabolism ; X-Rays ; },
abstract = {Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer.},
}
@article {pmid33333712,
year = {2020},
author = {Yatsuka, H and Hada, K and Shiraishi, H and Umeda, R and Morisaki, I and Urushibata, H and Shimizu, N and Sebastian, WA and Hikida, T and Ishitani, T and Hanada, R and Shimada, T and Kimoto, K and Kubota, T and Hanada, T},
title = {Exosc2 deficiency leads to developmental disorders by causing a nucleotide pool imbalance in zebrafish.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {4},
pages = {1470-1476},
doi = {10.1016/j.bbrc.2020.10.044},
pmid = {33333712},
issn = {1090-2104},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Embryo, Nonmammalian/abnormalities ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Larva/genetics/physiology ; Motor Neurons/drug effects/pathology ; Myelin Basic Protein/genetics ; Nucleotides/genetics/*metabolism ; Sirolimus/pharmacology ; Zebrafish/embryology/*genetics ; },
abstract = {Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2[-/-]) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2[-/-] zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.},
}
@article {pmid33333349,
year = {2021},
author = {Chu, W and Prodromou, R and Day, KN and Schneible, JD and Bacon, KB and Bowen, JD and Kilgore, RE and Catella, CM and Moore, BD and Mabe, MD and Alashoor, K and Xu, Y and Xiao, Y and Menegatti, S},
title = {Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics.},
journal = {Journal of chromatography. A},
volume = {1635},
number = {},
pages = {461632},
doi = {10.1016/j.chroma.2020.461632},
pmid = {33333349},
issn = {1873-3778},
mesh = {Antibodies/isolation &amp; purification ; Biological Products/*isolation &amp; purification ; Chemistry, Pharmaceutical/*methods ; *Chromatography, Affinity ; Family Characteristics ; Humans ; *Ligands ; Peptides/isolation &amp; purification ; Peptoids/chemistry ; Proteins/isolation &amp; purification ; },
abstract = {Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as β-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.},
}
@article {pmid33333262,
year = {2021},
author = {Krasnopolsky, S and Novikov, A and Kuzmina, A and Taube, R},
title = {CRISPRi-mediated depletion of Spt4 and Spt5 reveals a role for DSIF in the control of HIV latency.},
journal = {Biochimica et biophysica acta. Gene regulatory mechanisms},
volume = {1864},
number = {1},
pages = {194656},
doi = {10.1016/j.bbagrm.2020.194656},
pmid = {33333262},
issn = {1876-4320},
mesh = {*CRISPR-Cas Systems ; *Gene Expression Regulation, Viral ; HIV-1/*physiology ; Humans ; Jurkat Cells ; Nuclear Proteins/genetics/*metabolism ; *RNA Interference ; Repressor Proteins/genetics/*metabolism ; *Transcriptional Activation ; Transcriptional Elongation Factors/genetics/*metabolism ; *Virus Latency ; tat Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; },
abstract = {Pivotal studies on the control of HIV transcription has laid the foundations for our understanding of how metazoan transcription is executed, and what are the factors that control this step. Part of this work established a role for DRB Sensitivity Inducing Factor (DSIF), consisting of Spt4 and Spt5, in promoting pause-release of RNA Polymerase II (Pol II) for optimal elongation. However, while there has been substantial progress in understanding the role of DSIF in mediating HIV gene transcription, its involvement in establishing viral latency has not been explored. Moreover, the effects of depleting Spt4 or Spt5, or simultaneously knocking down both subunits of DSIF have not been examined. In this study, we employed CRISPR interference (CRIPSRi) to knockdown the expression of Spt4, Spt5 or the entire DSIF complex, and monitored effects on HIV transcription and viral latency. Knocking down DSIF, or each of its subunits, inhibited HIV transcription, primarily at the step of Tat transactivation. This was accompanied by a decrease in promoter occupancy of Pol II and Cdk9, and to a lesser extent, AFF4. Interestingly, targeting the expression of one subunit of DSIF, reduced the protein stability of its counterpart partner. Moreover, depletion of Spt4, Spt5 or DSIF complex impaired cell growth, but did not cause cell death. Finally, knockdown of Spt4, Spt5 or DSIF, facilitated entry of HIV into latency. We conclude that each DSIF subunit plays a role in maintaining the stability of its other partner, achieving optimal function of the DSIF to enhance viral gene transcription.},
}
@article {pmid33333121,
year = {2021},
author = {Suzuki, Y and Onuma, H and Sato, R and Sato, Y and Hashiba, A and Maeki, M and Tokeshi, M and Kayesh, MEH and Kohara, M and Tsukiyama-Kohara, K and Harashima, H},
title = {Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {330},
number = {},
pages = {61-71},
doi = {10.1016/j.jconrel.2020.12.013},
pmid = {33333121},
issn = {1873-4995},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Hepatitis B virus/genetics ; Humans ; Lab-On-A-Chip Devices ; Lipids ; *Nanoparticles ; Oligonucleotides ; Ribonucleoproteins/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.},
}
@article {pmid33333018,
year = {2021},
author = {Takeda, SN and Nakagawa, R and Okazaki, S and Hirano, H and Kobayashi, K and Kusakizako, T and Nishizawa, T and Yamashita, K and Nishimasu, H and Nureki, O},
title = {Structure of the miniature type V-F CRISPR-Cas effector enzyme.},
journal = {Molecular cell},
volume = {81},
number = {3},
pages = {558-570.e3},
doi = {10.1016/j.molcel.2020.11.035},
pmid = {33333018},
issn = {1097-4164},
mesh = {CRISPR-Associated Proteins/genetics/metabolism/*ultrastructure ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy ; DNA/genetics/metabolism/*ultrastructure ; *Gene Editing ; Models, Molecular ; Nucleotide Motifs ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Subunits ; RNA, Guide/genetics/metabolism/*ultrastructure ; Structure-Activity Relationship ; },
abstract = {RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.},
}
@article {pmid33332762,
year = {2020},
author = {Banta, AB and Ward, RD and Tran, JS and Bacon, EE and Peters, JM},
title = {Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.},
journal = {Current protocols in microbiology},
volume = {59},
number = {1},
pages = {e130},
pmid = {33332762},
issn = {1934-8533},
support = {K22 AI137122/AI/NIAID NIH HHS/United States ; T32 GM007133/GM/NIGMS NIH HHS/United States ; T32 GM008349/GM/NIGMS NIH HHS/United States ; T32 GM135066/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*genetics ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Gene Knockdown Techniques/*methods ; Genome, Bacterial ; Gram-Negative Bacteria/genetics ; RNA, Bacterial ; },
abstract = {Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene-knockdown tool that uses an RNA-protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile-CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile-CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile-CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile-CRISPRi vectors, Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria, and ICEBs1 transfer of Mobile-CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile-CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile-CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl2 -competent E. coli.},
}
@article {pmid33332748,
year = {2020},
author = {Caine, EA and Mahan, SD and Johnson, RL and Nieman, AN and Lam, N and Warren, CR and Riching, KM and Urh, M and Daniels, DL},
title = {Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.},
journal = {Current protocols in pharmacology},
volume = {91},
number = {1},
pages = {e81},
pmid = {33332748},
issn = {1934-8290},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Electroporation ; Humans ; *Proteolysis ; Recombinant Fusion Proteins/*chemistry ; },
abstract = {To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors. Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag Basic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions.},
}
@article {pmid33332569,
year = {2021},
author = {Gabel, C and Li, Z and Zhang, H and Chang, L},
title = {Structural basis for inhibition of the type I-F CRISPR-Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14.},
journal = {Nucleic acids research},
volume = {49},
number = {1},
pages = {584-594},
pmid = {33332569},
issn = {1362-4962},
support = {T32 GM132024/GM/NIGMS NIH HHS/United States ; R01 GM138675/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/metabolism ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA, Bacterial/metabolism ; Datasets as Topic ; Electrophoretic Mobility Shift Assay ; Image Processing, Computer-Assisted ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Domains ; Protein Interaction Mapping ; Pseudomonas Phages/*chemistry/genetics ; Pseudomonas aeruginosa/*metabolism ; R-Loop Structures ; Recombinant Proteins/chemistry/metabolism ; Viral Proteins/*chemistry/metabolism ; },
abstract = {CRISPR-Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR-Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR-Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.},
}
@article {pmid33332554,
year = {2021},
author = {Pae, J and Ersching, J and Castro, TBR and Schips, M and Mesin, L and Allon, SJ and Ordovas-Montanes, J and Mlynarczyk, C and Melnick, A and Efeyan, A and Shalek, AK and Meyer-Hermann, M and Victora, GD},
title = {Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {4},
pages = {},
pmid = {33332554},
issn = {1540-9538},
support = {R35 CA220499/CA/NCI NIH HHS/United States ; R01 AI139117/AI/NIAID NIH HHS/United States ; DP2 GM119419/GM/NIGMS NIH HHS/United States ; R01 AI119006/AI/NIAID NIH HHS/United States ; U24 AI118672/AI/NIAID NIH HHS/United States ; DP1 AI144248/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/*immunology ; Burkitt Lymphoma/genetics ; CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Proliferation/*genetics ; Cells, Cultured ; Chimera/immunology ; Cyclin D3/genetics/*physiology ; Female ; Gain of Function Mutation ; Gene Editing/methods ; Germinal Center/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Somatic Hypermutation, Immunoglobulin/*genetics ; T Follicular Helper Cells/immunology ; },
abstract = {During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by "inertia." We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.},
}
@article {pmid33332350,
year = {2020},
author = {House, NCM and Parasuram, R and Layer, JV and Price, BD},
title = {Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0237759},
pmid = {33332350},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics ; DNA Breaks, Double-Stranded ; DNA Damage/*genetics ; DNA Repair/*genetics ; Endonucleases/genetics ; Genome/genetics ; HEK293 Cells ; Humans ; Light ; Oxidative Stress/*genetics ; RNA, Guide/genetics ; },
abstract = {DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in "clustered" oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in "real time" by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.},
}
@article {pmid33331764,
year = {2021},
author = {Ye, Z and Moreb, EA and Li, S and Lebeau, J and Menacho-Melgar, R and Munson, M and Lynch, MD},
title = {Escherichia coli Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability.},
journal = {ACS synthetic biology},
volume = {10},
number = {1},
pages = {29-37},
doi = {10.1021/acssynbio.0c00398},
pmid = {33331764},
issn = {2161-5063},
mesh = {CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics/*metabolism ; Endonucleases/genetics/*metabolism ; Escherichia coli/*genetics/metabolism ; Escherichia coli Proteins/genetics/*metabolism ; Gene Editing/*methods ; Oligonucleotide Array Sequence Analysis ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; RNA Stability ; RNA, Guide/*metabolism ; },
abstract = {CRISPR-based interference has become common in various applications from genetic circuits to dynamic metabolic control. In E. coli, the native CRISPR Cascade system can be utilized for silencing by deletion of the cas3 nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of spacer sequences from guide arrays utilized for dynamic silencing. We report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of spacers from guide arrays. Subsequently, mutant guide arrays can be amplified through selection. Replacing a constitutive promoter driving Cascade complex expression with a tightly controlled inducible promoter improves guide array stability, while minimizing leaky gene silencing. Additionally, these results demonstrate the potential of Cas1/2 mediated guide deletion as a mechanism to avoid CRISPR based autoimmunity.},
}
@article {pmid33331178,
year = {2021},
author = {Atkins, AJ and Allen, AG and Dampier, W and Haddad, EK and Nonnemacher, MR and Wigdahl, B},
title = {HIV-1 cure strategies: why CRISPR?.},
journal = {Expert opinion on biological therapy},
volume = {21},
number = {6},
pages = {781-793},
doi = {10.1080/14712598.2021.1865302},
pmid = {33331178},
issn = {1744-7682},
support = {R01 MH110360/MH/NIMH NIH HHS/United States ; T32 MH079785/MH/NIMH NIH HHS/United States ; P30 MH092177/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *HIV Infections/drug therapy ; *HIV-1/genetics ; Humans ; Proviruses ; Virus Activation ; },
abstract = {INTRODUCTION: Antiretroviral therapy (ART) has transformed prognoses for HIV-1-infected individuals but requires lifelong adherence to prevent viral resurgence. Targeted elimination or permanent deactivation of the latently infected reservoir harboring integrated proviral DNA, which drives viral rebound, is a major focus of HIV-1 research.<br><br>AREAS COVERED: This review covers the current approaches to developing curative strategies for HIV-1 that target the latent reservoir. Discussed herein are shock and kill, broadly neutralizing antibodies (bNAbs), block and lock, Chimeric antigen receptor (CAR) T cells, immune checkpoint modulation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) coreceptor ablation, and CRISPR/Cas9 proviral excision. Emphasis is placed on CRISPR/Cas9 proviral excision/inactivation. Recent advances and future directions toward discovery and translation of HIV-1 therapeutics are discussed.<br><br>EXPERT OPINION: CRISPR/Cas9 proviral targeting fills a niche amongst HIV-1 cure strategies by directly targeting the integrated provirus without the necessity of an innate or adaptive immune response. Each strategy discussed in this review has shown promising results with the potential to yield curative or adjuvant therapies. CRISPR/Cas9 is singular among these in that it addresses the root of the problem, integrated proviral DNA, with the capacity to permanently remove or deactivate the source of HIV-1 recrudescence.},
}
@article {pmid33331117,
year = {2021},
author = {},
title = {Crispr Developers Win 2020 Nobel Prize for Chemistry.},
journal = {American journal of medical genetics. Part A},
volume = {185},
number = {1},
pages = {8-9},
doi = {10.1002/ajmg.a.61645},
pmid = {33331117},
issn = {1552-4833},
mesh = {CRISPR-Cas Systems/*genetics ; History, 20th Century ; Humans ; *Nobel Prize ; },
}
@article {pmid33330940,
year = {2021},
author = {Hoffmann, MD and Mathony, J and Upmeier Zu Belzen, J and Harteveld, Z and Aschenbrenner, S and Stengl, C and Grimm, D and Correia, BE and Eils, R and Niopek, D},
title = {Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein.},
journal = {Nucleic acids research},
volume = {49},
number = {5},
pages = {e29},
pmid = {33330940},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/*chemistry ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Light ; Models, Molecular ; Neisseria meningitidis/*enzymology ; Optogenetics/*methods ; Protein Engineering ; Proteins/chemistry/radiation effects ; },
abstract = {Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.},
}
@article {pmid33330425,
year = {2020},
author = {Meng, J and Qiu, Y and Shi, S},
title = {CRISPR/Cas9 Systems for the Development of Saccharomyces cerevisiae Cell Factories.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {594347},
pmid = {33330425},
issn = {2296-4185},
abstract = {Synthetic yeast cell factories provide a remarkable solution for the sustainable supply of a range of products, ranging from large-scale industrial chemicals to high-value pharmaceutical compounds. Synthetic biology is a field in which metabolic pathways are intensively studied and engineered. The clustered, regularly interspaced, short, palindromic repeat-associated (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has emerged as the state-of-the-art gene editing technique for synthetic biology. Recently, the use of different CRISPR/Cas9 systems has been extended to the field of yeast engineering for single-nucleotide resolution editing, multiple-gene editing, transcriptional regulation, and genome-scale modifications. Such advancing systems have led to accelerated microbial engineering involving less labor and time and also enhanced the understanding of cellular genetics and physiology. This review provides a brief overview of the latest research progress and the use of CRISPR/Cas9 systems in genetic manipulation, with a focus on the applications of Saccharomyces cerevisiae cell factory engineering.},
}
@article {pmid33330126,
year = {2020},
author = {Vilela, J and Rohaim, MA and Munir, M},
title = {Application of CRISPR/Cas9 in Understanding Avian Viruses and Developing Poultry Vaccines.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {581504},
pmid = {33330126},
issn = {2235-2988},
support = {BB/M008681/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00001852/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Poultry ; *Viral Vaccines/genetics ; *Viruses/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats associated protein nuclease 9 (CRISPR-Cas9) technology offers novel approaches to precisely, cost-effectively, and user-friendly edit genomes for a wide array of applications and across multiple disciplines. This methodology can be leveraged to underpin host-virus interactions, elucidate viral gene functions, and to develop recombinant vaccines. The successful utilization of CRISPR/Cas9 in editing viral genomes has paved the way of developing novel and multiplex viral vectored poultry vaccines. Furthermore, CRISPR/Cas9 can be exploited to rectify major limitations of conventional approaches including reversion to virulent form, recombination with field viruses and transgene, and genome instability. This review provides comprehensive analysis of the potential of CRISPR/Cas9 genome editing technique in understanding avian virus-host interactions and developing novel poultry vaccines. Finally, we discuss the simplest and practical aspects of genome editing approaches in generating multivalent recombinant poultry vaccines that conform simultaneous protection against major avian diseases.},
}
@article {pmid33329659,
year = {2020},
author = {An, Y and Geng, Y and Yao, J and Fu, C and Lu, M and Wang, C and Du, J},
title = {Efficient Genome Editing in Populus Using CRISPR/Cas12a.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {593938},
pmid = {33329659},
issn = {1664-462X},
abstract = {The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.},
}
@article {pmid33329634,
year = {2020},
author = {Modrzejewski, D and Hartung, F and Lehnert, H and Sprink, T and Kohl, C and Keilwagen, J and Wilhelm, R},
title = {Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {574959},
pmid = {33329634},
issn = {1664-462X},
abstract = {CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.},
}
@article {pmid33329523,
year = {2020},
author = {Kuo, CJ and Hsu, YC and Wang, ST and Liou, BY and Lim, SB and Chen, YW and Chen, CS},
title = {IGLR-2, a Leucine-Rich Repeat Domain Containing Protein, Is Required for the Host Defense in Caenorhabditis elegans.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {561337},
pmid = {33329523},
issn = {1664-3224},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*immunology/metabolism ; Caenorhabditis elegans Proteins/genetics/*immunology ; Enterohemorrhagic Escherichia coli/*pathogenicity ; Escherichia coli Infections/*immunology/microbiology ; Gene Knockdown Techniques ; Host Microbial Interactions/*immunology ; Immunity, Innate ; Membrane Proteins/genetics/*immunology ; p38 Mitogen-Activated Protein Kinases/metabolism ; },
abstract = {Enterohemorrhagic Escherichia coli (EHEC), a human pathogen, also infects Caenorhabditis elegans. We demonstrated previously that C. elegans activates the p38 MAPK innate immune pathway to defend against EHEC infection. However, whether a C. elegans pattern recognition receptor (PRR) exists to regulate the immune pathway remains unknown. PRRs identified in other metazoans contain several conserved domains, including the leucine-rich repeat (LRR). By screening a focused RNAi library, we identified the IGLR-2, a transmembrane protein containing the LRR domain, as a potential immune regulator in C. elegans. Our data showed that iglr-2 regulates the host susceptibility to EHEC infection. Moreover, iglr-2 is required for pathogen avoidance to EHEC. The iglr-2 overexpressed strain, which was more resistant to EHEC originally, showed hypersusceptibility to EHEC upon knockdown of the p38 MAPK pathway. Together, our data suggested that iglr-2 plays an important role in C. elegans to defend EHEC by regulating pathogen-avoidance behavior and the p38 MAPK pathway.},
}
@article {pmid33328654,
year = {2021},
author = {Shivram, H and Cress, BF and Knott, GJ and Doudna, JA},
title = {Controlling and enhancing CRISPR systems.},
journal = {Nature chemical biology},
volume = {17},
number = {1},
pages = {10-19},
pmid = {33328654},
issn = {1552-4469},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; F32 GM131654/GM/NIGMS NIH HHS/United States ; U19 AI135990/AI/NIAID NIH HHS/United States ; },
mesh = {Antibiosis/genetics ; Archaea/*genetics/metabolism/virology ; Bacteria/*genetics/metabolism/virology ; Bacteriophages/genetics/growth &amp; development/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; *Gene Expression Regulation, Archaeal ; *Gene Expression Regulation, Bacterial ; Genetic Engineering/methods ; Humans ; Interspersed Repetitive Sequences ; RNA, Guide/genetics/metabolism ; },
abstract = {Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) systems to defend themselves from mobile genetic elements. These CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature.},
}
@article {pmid33328531,
year = {2020},
author = {Voigt, O and Knabe, N and Nitsche, S and Erdmann, EA and Schumacher, J and Gorbushina, AA},
title = {An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {22021},
pmid = {33328531},
issn = {2045-2322},
mesh = {Ascomycota/*genetics ; CRISPR-Cas Systems ; Fluorescence ; Gene Editing ; Gene Silencing ; Genes, Essential ; *Genetic Techniques ; Geologic Sediments/*microbiology ; Homologous Recombination ; Luminescent Proteins/metabolism ; Mutation/genetics ; Pigmentation/genetics ; },
abstract = {Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi's phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.},
}
@article {pmid33328249,
year = {2021},
author = {Amici, DR and Jackson, JM and Truica, MI and Smith, RS and Abdulkadir, SA and Mendillo, ML},
title = {FIREWORKS: a bottom-up approach to integrative coessentiality network analysis.},
journal = {Life science alliance},
volume = {4},
number = {2},
pages = {},
pmid = {33328249},
issn = {2575-1077},
support = {F30 CA264513/CA/NCI NIH HHS/United States ; PPO 16-135/HX/HSRD VA/United States ; R00 CA175293/CA/NCI NIH HHS/United States ; T32 GM008152/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; *Gene Expression Regulation ; *Gene Regulatory Networks ; Gene Targeting ; Genetic Loci ; *Genomics/methods ; Humans ; Models, Biological ; *Software ; },
abstract = {Genetic coessentiality analysis, a computational approach which identifies genes sharing a common effect on cell fitness across large-scale screening datasets, has emerged as a powerful tool to identify functional relationships between human genes. However, widespread implementation of coessentiality to study individual genes and pathways is limited by systematic biases in existing coessentiality approaches and accessibility barriers for investigators without computational expertise. We created FIREWORKS, a method and interactive tool for the construction and statistical analysis of coessentiality networks centered around gene(s) provided by the user. FIREWORKS incorporates a novel bias reduction approach to reduce false discoveries, enables restriction of coessentiality analyses to custom subsets of cell lines, and integrates multiomic and drug-gene interaction datasets to investigate and target contextual gene essentiality. We demonstrate the broad utility of FIREWORKS through case vignettes investigating gene function and specialization, indirect therapeutic targeting of "undruggable" proteins, and context-specific rewiring of genetic networks.},
}
@article {pmid33326752,
year = {2021},
author = {Lee, WD and Pirona, AC and Sarvin, B and Stern, A and Nevo-Dinur, K and Besser, E and Sarvin, N and Lagziel, S and Mukha, D and Raz, S and Aizenshtein, E and Shlomi, T},
title = {Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability.},
journal = {Cell metabolism},
volume = {33},
number = {1},
pages = {190-198.e6},
doi = {10.1016/j.cmet.2020.12.002},
pmid = {33326752},
issn = {1932-7420},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carbon Cycle/genetics ; Cell Line ; Cytosol/*metabolism ; Folic Acid/genetics/*metabolism ; Glycine Hydroxymethyltransferase/deficiency/*genetics/metabolism ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Mitochondria/*metabolism ; Neoplasms/*metabolism/pathology ; Reduced Folate Carrier Protein/*genetics/metabolism ; },
abstract = {Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.},
}
@article {pmid33326746,
year = {2020},
author = {Tycko, J and DelRosso, N and Hess, GT and Aradhana, and Banerjee, A and Mukund, A and Van, MV and Ego, BK and Yao, D and Spees, K and Suzuki, P and Marinov, GK and Kundaje, A and Bassik, MC and Bintu, L},
title = {High-Throughput Discovery and Characterization of Human Transcriptional Effectors.},
journal = {Cell},
volume = {183},
number = {7},
pages = {2020-2035.e16},
pmid = {33326746},
issn = {1097-4172},
support = {T32 GM007365/GM/NIGMS NIH HHS/United States ; R35 GM128947/GM/NIGMS NIH HHS/United States ; T32 GM007276/GM/NIGMS NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; R01 HG011866/HG/NHGRI NIH HHS/United States ; F99 DK126120/DK/NIDDK NIH HHS/United States ; T32 GM008294/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems/genetics ; Female ; Gene Silencing ; Genes, Reporter ; HEK293 Cells ; *High-Throughput Screening Assays ; Homeodomain Proteins/genetics/metabolism ; Humans ; K562 Cells ; Lentivirus/physiology ; Molecular Sequence Annotation ; Mutation/genetics ; Nuclear Proteins/metabolism ; Promoter Regions, Genetic/genetics ; Protein Domains ; Repressor Proteins/chemistry/metabolism ; Reproducibility of Results ; Transcription Factors/*metabolism ; Transcription, Genetic ; Zinc Fingers ; },
abstract = {Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.},
}
@article {pmid33326085,
year = {2021},
author = {Liu, SJ and Horlbeck, MA and Weissman, JS and Lim, DA},
title = {Genome-Scale Perturbation of Long Noncoding RNA Expression Using CRISPR Interference.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2254},
number = {},
pages = {323-338},
pmid = {33326085},
issn = {1940-6029},
support = {T32 GM007618/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; P50 CA097257/CA/NCI NIH HHS/United States ; R01 NS091544/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Gene Knockdown Techniques/*methods ; HEK293 Cells ; Humans ; Lentivirus/genetics/*physiology ; Promoter Regions, Genetic ; RNA, Long Noncoding/*genetics ; Transcription, Genetic ; },
abstract = {CRISPR-mediated interference (CRISPRi), a robust and specific system for programmably repressing transcription, provides a versatile tool for systematically characterizing the function of long noncoding RNAs (lncRNAs). When used with highly parallel, lentiviral pooled screening approaches, CRISPRi enables the targeted knockdown of tens of thousands of lncRNA-expressing loci in a single screen. Here we describe the use of CRISPRi to target lncRNA loci in a pooled screen, using cell growth and proliferation as an example of a phenotypic readout. Considerations for custom lncRNA-targeting libraries, alternative phenotypic readouts, and orthogonal validation approaches are also discussed.},
}
@article {pmid33326084,
year = {2021},
author = {Cheng, X and Peters, ST and Pruett-Miller, SM and Saunders, TL and Joe, B},
title = {In Vivo CRISPR/Cas9-Based Targeted Disruption and Knockin of a Long Noncoding RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2254},
number = {},
pages = {305-321},
pmid = {33326084},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development ; Gene Editing/*methods ; Gene Knock-In Techniques ; RNA, Long Noncoding/*genetics ; Rats ; Rats, Inbred Dahl ; Sequence Deletion ; },
abstract = {The CRISPR/Cas9 system has been widely used as an efficient genome-editing tool for studying physiological functions of long noncoding RNAs (lncRNAs). In this chapter, we describe the experimental procedures for using the CRISPR/Cas9 system to genetically modify a long noncoding RNA in vivo through the targeted disruption and knockin approaches.},
}
@article {pmid33326083,
year = {2021},
author = {Yamazaki, T and Hirose, T},
title = {CRISPR-Mediated Mutagenesis of Long Noncoding RNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2254},
number = {},
pages = {283-303},
pmid = {33326083},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Haploidy ; Humans ; Mutagenesis, Site-Directed/*methods ; RNA, Long Noncoding/*genetics/metabolism ; RNA-Binding Proteins/metabolism ; Sequence Deletion ; },
abstract = {Functional characterizations and molecular dissections of long noncoding RNAs (lncRNAs) are critical to understand their involvement in the cellular regulatory network. LncRNAs exert their effects through functional RNA domains that interact with other molecules, including proteins, DNA, and RNA. Here, we describe experimental procedures for generating genomic deletions in a human haploid cell line using the CRISPR/Cas9 system. This method can be applied to examine functions of lncRNAs and their domains by establishing knockout and partial deletion mutant cell lines. In addition, we describe a CRISPR-mediated knockin method for artificial tethering of partner RNA-binding proteins to lncRNAs and its use to validate lncRNA-mediated functions.},
}
@article {pmid33323971,
year = {2021},
author = {Periyasamy, M and Singh, AK and Gemma, C and Farzan, R and Allsopp, RC and Shaw, JA and Charmsaz, S and Young, LS and Cunnea, P and Coombes, RC and Győrffy, B and Buluwela, L and Ali, S},
title = {Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK-directed activation of NF-κB.},
journal = {Oncogene},
volume = {40},
number = {6},
pages = {1077-1090},
pmid = {33323971},
issn = {1476-5594},
support = {12011/CRUK_/Cancer Research UK/United Kingdom ; A18784/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Cisplatin/pharmacology ; Cytidine Deaminase/*genetics ; DNA Repair/drug effects ; Etoposide/pharmacology ; Fluorouracil/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Genetic Heterogeneity ; HCT116 Cells ; Humans ; MCF-7 Cells ; Minor Histocompatibility Antigens/*genetics ; Mutation/genetics ; NF-kappa B/genetics ; Neoplasms/*genetics/pathology ; Transcription Factor RelA/*genetics ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.},
}
@article {pmid33323021,
year = {2021},
author = {Li, F and Zhou, C and Tu, T and Liu, Y and Lv, X and Wang, B and Song, Z and Zhao, Q and Liu, C and Gu, F and Zhao, J},
title = {Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells.},
journal = {Human gene therapy},
volume = {32},
number = {5-6},
pages = {302-309},
doi = {10.1089/hum.2020.247},
pmid = {33323021},
issn = {1557-7422},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing ; Genome ; Humans ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.},
}
@article {pmid33322954,
year = {2021},
author = {Grand Moursel, L and Visser, M and Servant, G and Durmus, S and Zuurmond, AM},
title = {CRISPRing future medicines.},
journal = {Expert opinion on drug discovery},
volume = {16},
number = {4},
pages = {463-473},
doi = {10.1080/17460441.2021.1850687},
pmid = {33322954},
issn = {1746-045X},
mesh = {Animals ; Biomedical Research/methods ; CRISPR-Cas Systems/*genetics ; Cell- and Tissue-Based Therapy ; Cost-Benefit Analysis ; Drug Development/methods ; Drug Discovery/*methods ; Genetic Engineering ; Genetic Therapy/*methods ; Humans ; },
abstract = {Introduction: The ability to engineer mammalian genomes in a quick and cost-effective way has led to rapid adaptation of CRISPR technology in biomedical research. CRISPR-based engineering has the potential to accelerate drug discovery, to support the reduction of high attrition rate in drug development and to enhance development of cell and gene-based therapies.Areas covered: How CRISPR technology is transforming drug discovery is discussed in this review. From target identification to target validation in both in vitro and in vivo models, CRISPR technology is positively impacting the early stages of drug development by providing a straightforward way to genome engineering. This property also attracted attention for CRISPR application in the cell and gene therapy area.Expert opinion: CRISPR technology is rapidly becoming the preferred tool for genome engineering and nowadays it is hard to imagine the drug discovery pipeline without this technology. With the years to come, CRISPR technology will undoubtedly be further refined and will flourish into a mature technology that will play a key role in supporting genome engineering requirements in the drug discovery pipeline as well as in cell and gene therapy development.},
}
@article {pmid33322729,
year = {2020},
author = {Kim, JE and Choi, YJ and Lee, SJ and Gong, JE and Lim, Y and Hong, JT and Hwang, DY},
title = {Molecular Characterization of Constipation Disease as Novel Phenotypes in CRISPR-Cas9-Generated Leptin Knockout Mice with Obesity.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33322729},
issn = {1422-0067},
mesh = {Adipogenesis/genetics ; Animals ; Aquaporin 3/metabolism ; Aquaporins/metabolism ; CRISPR-Cas Systems ; Colon/cytology/*metabolism/pathology/ultrastructure ; Constipation/complications/genetics/*metabolism/pathology ; Disease Models, Animal ; Female ; Gastrointestinal Hormones/metabolism ; *Gastrointestinal Motility/genetics/physiology ; Interstitial Cells of Cajal/metabolism ; Leptin/genetics/*metabolism ; Lipid Metabolism/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Microscopy, Electron, Transmission ; Mucins/metabolism ; Myocytes, Smooth Muscle/*metabolism ; Neurons/metabolism ; Obesity/complications/genetics ; Receptors, Muscarinic/*metabolism ; Signal Transduction/genetics ; },
abstract = {(1) Background: We characterized a novel animal model with obesity-induced constipation because constipation is rarely known in genetically engineered mice (GEM); (2) Methods: The changes in the constipation parameters and mechanisms were analyzed in CRISPR-Cas9-mediated leptin (Lep) knockout (KO) mice from eight to 24 weeks; (3) Results: Significant constipation phenotypes were observed in the Lep KO mice since 16 weeks old. These mice showed a significant decrease in the gastrointestinal motility, mucosal layer thickness and ability for mucin secretion as well as the abnormal ultrastructure of Lieberkühn crypts in the transverse colon. The density or function of the enteric neurons, intestinal Cajal cells (ICC), smooth muscle cells, and the concentration of gastrointestinal (GI) hormones for the GI motility were remarkably changed in Lep KO mice. The downstream signaling pathway of muscarinic acetylcholine receptors (mAChRs) were activated in Lep KO mice, while the expression of adipogenesis-regulating genes were alternatively reduced in the transverse colon of the same mice; (4) Conclusions: These results provide the first strong evidence that Lep KO mice can represent constipation successfully through dysregulation of the GI motility mediated by myenteric neurons, ICC, and smooth muscle cells in the transverse colon during an abnormal function of the lipid metabolism.},
}
@article {pmid33322084,
year = {2020},
author = {Smith, RH and Chen, YC and Seifuddin, F and Hupalo, D and Alba, C and Reger, R and Tian, X and Araki, D and Dalgard, CL and Childs, RW and Pirooznia, M and Larochelle, A},
title = {Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.},
journal = {Genes},
volume = {11},
number = {12},
pages = {},
pmid = {33322084},
issn = {2073-4425},
support = {Z99 HL999999/HL/NHLBI NIH HHS/United States ; },
mesh = {Adult ; *CRISPR-Cas Systems ; Chromosomes, Human, Pair 19/*genetics ; Chromosomes, Human, Pair 2/*genetics ; *Clone Cells ; Female ; *Gene Editing ; Genetic Loci ; *Hematopoietic Stem Cells ; Humans ; Receptors, CXCR4/genetics ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.},
}
@article {pmid33321968,
year = {2020},
author = {Alayande, KA and Aiyegoro, OA and Ateba, CN},
title = {Distribution of Important Probiotic Genes and Identification of the Biogenic Amines Produced by Lactobacillus acidophilus PNW3.},
journal = {Foods (Basel, Switzerland)},
volume = {9},
number = {12},
pages = {},
pmid = {33321968},
issn = {2304-8158},
abstract = {The genome of Lactobacillus acidophilus PNW3 was assessed for probiotic and safety potentials. The genome was completely sequenced, assembled using SPAdes, and thereafter annotated with NCBI prokaryotic genome annotation pipeline (PGAP) and rapid annotation using subsystem technology (RAST). Further downstream assessment was determined using appropriate bioinformatics tools. The production of biogenic amines was confirmed through HPLC analysis and the evolutionary trend of the strain was determined through the Codon Tree pipeline. The strain was predicted as a non-human pathogen. A plethora of encoding genes for lactic acids and bioactive peptides production, adhesion molecules, resistance to the harsh gut environmental conditions, and improvement of the host metabolism, which are putative for important probiotic functionalities, were located at different loci within the genome. A bacteriocin predicted to be helveticin J was identified as one of the secondary metabolites. The maximum zone of inhibition exhibited by the crude bacteriocin against STEC E. coli O177 was 21.7 ± 0.58 mm and 24.3 ± 1.15 mm after partial purification (250 µg/mL). Three coding sequences were identified for insertion sequences and one for the CRISPR-Cas fragment. The protein-encoding sequence for Ornithine decarboxylase was found within the genome. L. acidophilus PNW3 presents important features categorizing it as a viable and safe probiotic candidate, though further safety investigations are necessary. The application of probiotics in livestock-keeping would ensure improved public health and food security.},
}
@article {pmid33321741,
year = {2020},
author = {Wu, J and Mukama, O and Wu, W and Li, Z and Habimana, JD and Zhang, Y and Zeng, R and Nie, C and Zeng, L},
title = {A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood.},
journal = {Biosensors},
volume = {10},
number = {12},
pages = {},
pmid = {33321741},
issn = {2079-6374},
mesh = {African Swine Fever Virus/*isolation &amp; purification ; Animals ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded ; Fluorescence ; Polymerase Chain Reaction ; Swine ; },
abstract = {Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR-Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10[-15] M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.},
}
@article {pmid33321328,
year = {2021},
author = {Abboodi, F and Buckhaults, P and Altomare, D and Liu, C and Hosseinipour, M and Banister, CE and Creek, KE and Pirisi, L},
title = {HPV-inactive cell populations arise from HPV16-transformed human keratinocytes after p53 knockout.},
journal = {Virology},
volume = {554},
number = {},
pages = {9-16},
doi = {10.1016/j.virol.2020.12.005},
pmid = {33321328},
issn = {1096-0341},
support = {R21 CA201853/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Transformed ; Cell Proliferation ; Cell Survival ; *Cell Transformation, Viral ; Gene Expression ; Gene Knockout Techniques ; Genes, p53 ; Human papillomavirus 16/genetics/*physiology ; Humans ; Keratinocytes/*physiology/*virology ; Loss of Function Mutation ; Oncogene Proteins, Viral/*genetics/metabolism ; Papillomavirus E7 Proteins/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; Transfection ; Tumor Suppressor Protein p53/*genetics/physiology ; },
abstract = {HPV-inactive head and neck and cervical cancers contain HPV DNA but do not express HPV E6/E7. HPV-positive primary head and neck tumors usually express E6/E7, however they may produce HPV-inactive metastases. These observations led to our hypothesis that HPV-inactive cancers begin as HPV-active lesions, losing dependence on E6/E7 expression during progression. Because HPV-inactive cervical cancers often have mutated p53, we investigated whether p53 loss may play a role in the genesis of HPV-inactive cancers. p53 knockout (p53-KO) by CRISPR-Cas9 resulted in a 5-fold reduction of E7 mRNA in differentiation-resistant HPV16 immortalized human keratinocytes (HKc/DR). E7 expression was restored by 5-Aza-2 deoxycytidine in p53 KO lines, suggesting a role of DNA methylation in this process. In-situ hybridization showed that p53 KO lines consist of mixed populations of E6/E7-positive and negative cells. Hence, loss of p53 predisposes HPV16 transformed cells to losing dependence on the continuous expression of HPV oncogenes for proliferation.},
}
@article {pmid33321292,
year = {2021},
author = {Cruz, LJ and van Dijk, T and Vepris, O and Li, TMWY and Schomann, T and Baldazzi, F and Kurita, R and Nakamura, Y and Grosveld, F and Philipsen, S and Eich, C},
title = {PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells.},
journal = {Biomaterials},
volume = {268},
number = {},
pages = {120580},
doi = {10.1016/j.biomaterials.2020.120580},
pmid = {33321292},
issn = {1878-5905},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Erythroid Cells ; Gene Editing ; Humans ; *Nanoparticles ; },
abstract = {Ex vivo gene editing of CD34[+] hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial 'burst' of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the γ-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9-PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases.},
}
@article {pmid33320883,
year = {2020},
author = {Xiong, D and Dai, W and Gong, J and Li, G and Liu, N and Wu, W and Pan, J and Chen, C and Jiao, Y and Deng, H and Ye, J and Zhang, X and Huang, H and Li, Q and Xue, L and Zhang, X and Tang, G},
title = {Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.},
journal = {PLoS biology},
volume = {18},
number = {12},
pages = {e3000978},
pmid = {33320883},
issn = {1545-7885},
mesh = {Base Sequence ; COVID-19 Testing/*methods ; CRISPR-Cas Systems/*genetics ; Humans ; Reproducibility of Results ; SARS-CoV-2/*genetics/*isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.},
}
@article {pmid33320095,
year = {2020},
author = {Adams, BM and Canniff, NP and Guay, KP and Larsen, ISB and Hebert, DN},
title = {Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33320095},
issn = {2050-084X},
support = {R01 GM086874/GM/NIGMS NIH HHS/United States ; T32 GM008515/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Calnexin/metabolism ; Calreticulin/metabolism ; Cell Line ; Endoplasmic Reticulum/*metabolism ; Gene Knockdown Techniques ; Glucosyltransferases/*metabolism ; Glycoproteins/*metabolism ; Glycosylation ; HEK293 Cells ; Humans ; Molecular Chaperones/metabolism ; Protein Transport/*physiology ; Proteome/metabolism ; },
abstract = {UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway, yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from the ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells. The 71 UGGT substrates identified were mainly large multidomain and heavily glycosylated proteins when compared to the general N-glycoproteome. UGGT1 was the dominant glucosyltransferase with a preference toward large plasma membrane proteins whereas UGGT2 favored the modification of smaller, soluble lysosomal proteins. This study sheds light on differential specificities and roles of UGGT1 and UGGT2 and provides insight into the cellular reliance on the carbohydrate-dependent chaperone system to facilitate proper folding and maturation of the cellular N-glycoproteome.},
}
@article {pmid33320092,
year = {2020},
author = {Wang, H and Wan, X and Pilch, PF and Ellisen, LW and Fried, SK and Liu, L},
title = {An AMPK-dependent, non-canonical p53 pathway plays a key role in adipocyte metabolic reprogramming.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33320092},
issn = {2050-084X},
support = {R01 DK112945/DK/NIDDK NIH HHS/United States ; DK-112945/DK/NIDDK NIH HHS/United States ; },
mesh = {3T3-L1 Cells ; AMP-Activated Protein Kinases/*metabolism ; Adipocytes/*metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cellular Reprogramming ; Energy Metabolism/*physiology ; Gene Editing/methods ; Glucose/metabolism ; Lipid Metabolism/physiology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Obesity/*pathology ; RNA Interference ; RNA, Small Interfering/genetics ; Starvation/metabolism ; Sterol Esterase/metabolism ; Tumor Suppressor Protein p53/genetics/*metabolism ; },
abstract = {It has been known adipocytes increase p53 expression and activity in obesity, however, only canonical p53 functions (i.e. senescence and apoptosis) are attributed to inflammation-associated metabolic phenotypes. Whether or not p53 is directly involved in mature adipocyte metabolic regulation remains unclear. Here we show p53 protein expression can be up-regulated in adipocytes by nutrient starvation without activating cell senescence, apoptosis, or a death-related p53 canonical pathway. Inducing the loss of p53 in mature adipocytes significantly reprograms energy metabolism and this effect is primarily mediated through a AMP-activated protein kinase (AMPK) pathway and a novel downstream transcriptional target, lysosomal acid lipase (LAL). The pathophysiological relevance is further demonstrated in a conditional and adipocyte-specific p53 knockout mouse model. Overall, these data support a non-canonical p53 function in the regulation of adipocyte energy homeostasis and indicate that the dysregulation of this pathway may be involved in developing metabolic dysfunction in obesity.},
}
@article {pmid33320085,
year = {2020},
author = {Trivedi, D and Cm, V and Bisht, K and Janardan, V and Pandit, A and Basak, B and H, S and Ramesh, N and Raghu, P},
title = {A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33320085},
issn = {2050-084X},
support = {BT/PRJ3748/GET/l 19/27/2015//Department of Biotechnology, Ministry of Science and Technology, India/International ; IA/S/14/2/501540//Wellcome Trust DBt India Alliance/International ; 12R-D-TFR-5.04-0900//Department of Atomic Energy, Government of India/International ; /WT_/Wellcome Trust/United Kingdom ; 12R-D-TFR-5.04-08002//Department of Atomic Energy, Government of India/International ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drosophila melanogaster/embryology/*genetics ; Eye/*embryology/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Genome, Insect/genetics ; Lipid Metabolism ; Phosphatidylinositols/*metabolism ; RNA, Guide/biosynthesis/genetics ; Sequence Deletion/genetics ; Signal Transduction/physiology ; },
abstract = {Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.},
}
@article {pmid33319680,
year = {2021},
author = {Barazesh, M and Mohammadi, S and Bahrami, Y and Mokarram, P and Morowvat, MH and Saidijam, M and Karimipoor, M and Kavousipour, S and Vosoughi, AR and Khanaki, K},
title = {CRISPR/Cas9 Technology as a Modern Genetic Manipulation Tool for Recapitulating of Neurodegenerative Disorders in Large Animal Models.},
journal = {Current gene therapy},
volume = {21},
number = {2},
pages = {130-148},
doi = {10.2174/1566523220666201214115024},
pmid = {33319680},
issn = {1875-5631},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Central Nervous System/metabolism/pathology ; Disease Models, Animal ; *Gene Targeting ; Genetic Engineering/trends ; *Genetic Therapy ; Genomics/trends ; Humans ; Neurodegenerative Diseases/genetics/*therapy ; Primates/genetics ; },
abstract = {BACKGROUND: Neurodegenerative diseases are often the consequence of alterations in structures and functions of the Central Nervous System (CNS) in patients. Despite obtaining massive genomic information concerning the molecular basis of these diseases and since the neurological disorders are multifactorial, causal connections between pathological pathways at the molecular level and CNS disorders development have remained obscure and need to be elucidated to a great extent.<br><br>OBJECTIVE: Animal models serve as accessible and valuable tools for understanding and discovering the roles of causative factors in the development of neurodegenerative disorders and finding appropriate treatments. Contrary to rodents and other small animals, large animals, especially non-human primates (NHPs), are remarkably similar to humans; hence, they establish suitable models for recapitulating the main human's neuropathological manifestations that may not be seen in rodent models. In addition, they serve as useful models to discover effective therapeutic targets for neurodegenerative disorders due to their similarity to humans in terms of physiology, evolutionary distance, anatomy, and behavior.<br><br>METHODS: In this review, we recommend different strategies based on the CRISPR-Cas9 system for generating animal models of human neurodegenerative disorders and explaining in vivo CRISPR-Cas9 delivery procedures that are applied to disease models for therapeutic purposes.<br><br>RESULTS: With the emergence of CRISPR/Cas9 as a modern specific gene-editing technology in the field of genetic engineering, genetic modification procedures such as gene knock-in and knock-out have become increasingly easier compared to traditional gene targeting techniques. Unlike the old techniques, this versatile technology can efficiently generate transgenic large animal models without the need to complicate lab instruments. Hence, these animals can accurately replicate the signs of neurodegenerative disorders.<br><br>CONCLUSION: Preclinical applications of CRISPR/Cas9 gene-editing technology supply a unique opportunity to establish animal models of neurodegenerative disorders with high accuracy and facilitate perspectives for breakthroughs in the research on the nervous system disease therapy and drug discovery. Furthermore, the useful outcomes of CRISPR applications in various clinical phases are hopeful for their translation to the clinic in a short time.},
}
@article {pmid33318646,
year = {2021},
author = {Berber, B and Aydin, C and Kocabas, F and Guney-Esken, G and Yilancioglu, K and Karadag-Alpaslan, M and Caliseki, M and Yuce, M and Demir, S and Tastan, C},
title = {Gene editing and RNAi approaches for COVID-19 diagnostics and therapeutics.},
journal = {Gene therapy},
volume = {28},
number = {6},
pages = {290-305},
pmid = {33318646},
issn = {1476-5462},
mesh = {*COVID-19/diagnosis/therapy ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Oligonucleotides, Antisense ; *RNA Interference ; },
abstract = {The novel coronavirus pneumonia (COVID-19) is a highly infectious acute respiratory disease caused by Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) (Prec Clin Med 2020;3:9-13, Lancet 2020;395:497-506, N. Engl J Med 2020a;382:1199-207, Nature 2020;579:270-3). SARS-CoV-2 surveillance is essential to controlling widespread transmission. However, there are several challenges associated with the diagnostic of the COVID-19 during the current outbreak (Liu and Li (2019), Nature 2020;579:265-9, N. Engl J Med 2020;382:727-33). Firstly, the high number of cases overwhelms diagnostic test capacity and proposes the need for a rapid solution for sample processing (Science 2018;360:444-8). Secondly, SARS-CoV-2 is closely related to other important coronavirus species and subspecies, so detection assays can give false-positive results if they are not efficiently specific to SARS-CoV-2. Thirdly, patients with suspected SARS-CoV-2 infection sometimes have a different respiratory viral infection or co-infections with SARS-CoV-2 and other respiratory viruses (MedRxiv 2020a;1-18). Confirmation of the COVID-19 is performed mainly by virus isolation followed by RT-PCR and sequencing (N. Engl J Med 2020;382:727-33, MedRxiv 2020a, Turkish J Biol 2020;44:192-202). The emergence and outbreak of the novel coronavirus highlighted the urgent need for new therapeutic technologies that are fast, precise, stable, easy to manufacture, and target-specific for surveillance and treatment. Molecular biology tools that include gene-editing approaches such as CRISPR-Cas12/13-based SHERLOCK, DETECTR, CARVER and PAC-MAN, antisense oligonucleotides, antisense peptide nucleic acids, ribozymes, aptamers, and RNAi silencing approaches produced with cutting-edge scientific advances compared to conventional diagnostic or treatment methods could be vital in COVID-19 and other future outbreaks. Thus, in this review, we will discuss potent the molecular biology approaches that can revolutionize diagnostic of viral infections and therapies to fight COVID-19 in a highly specific, stable, and efficient way.},
}
@article {pmid33318207,
year = {2020},
author = {Patel, AK and Broyer, RM and Lee, CD and Lu, T and Louie, MJ and La Torre, A and Al-Ali, H and Vu, MT and Mitchell, KL and Wahlin, KJ and Berlinicke, CA and Jaskula-Ranga, V and Hu, Y and Duan, X and Vilar, S and Bixby, JL and Weinreb, RN and Lemmon, VP and Zack, DJ and Welsbie, DS},
title = {Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {52},
pages = {33597-33607},
pmid = {33318207},
issn = {1091-6490},
support = {P30 EY026877/EY/NEI NIH HHS/United States ; R01 EY023295/EY/NEI NIH HHS/United States ; P30 EY022589/EY/NEI NIH HHS/United States ; R01 EY029342/EY/NEI NIH HHS/United States ; R01 EY030138/EY/NEI NIH HHS/United States ; R01 EY031318/EY/NEI NIH HHS/United States ; R01 EY024932/EY/NEI NIH HHS/United States ; R01 EY028106/EY/NEI NIH HHS/United States ; R00 EY024648/EY/NEI NIH HHS/United States ; R21 EY031122/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Axons/*enzymology/*pathology ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Death/drug effects ; Cell Survival/drug effects ; Central Nervous System/*pathology ; Dependovirus/metabolism ; Disease Models, Animal ; Germinal Center Kinases/*metabolism ; Humans ; Mice, Inbred C57BL ; *Nerve Regeneration/drug effects ; Neuronal Outgrowth/drug effects ; Optic Nerve Injuries/metabolism/pathology ; Organoids/metabolism ; Protein Kinase Inhibitors/pharmacology ; Retinal Ganglion Cells/drug effects/metabolism ; Signal Transduction/drug effects ; },
abstract = {Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.},
}
@article {pmid33317552,
year = {2020},
author = {Xie, Q and Li, S and Zhao, D and Ye, L and Li, Q and Zhang, X and Zhu, L and Bi, C},
title = {Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9-Lag) to improve metabolic pathway efficiency.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {229},
pmid = {33317552},
issn = {1475-2859},
mesh = {Aquaporins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Carotenoids/*metabolism ; Cell Membrane/enzymology ; Chromosomes, Bacterial/genetics/ultrastructure ; DNA, Bacterial/genetics ; Escherichia coli/*genetics/metabolism/ultrastructure ; Escherichia coli Proteins/genetics/metabolism ; *Metabolic Engineering ; *Metabolic Networks and Pathways ; Mixed Function Oxygenases/genetics/metabolism ; Oxygenases/genetics/metabolism ; Plasmids/genetics ; Recombinant Fusion Proteins/metabolism ; Xanthophylls/metabolism ; Zeaxanthins/*biosynthesis ; },
abstract = {BACKGROUND: Deactivated Cas9 (dCas9) led to significant improvement of CRISPR/Cas9-based techniques because it can be fused with a variety of functional groups to form diverse molecular devices, which can manipulate or modify target DNA cassettes. One important metabolic engineering strategy is to localize the enzymes in proximity of their substrates for improved catalytic efficiency. In this work, we developed a novel molecular device to manipulate the cellular location of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas9 (Cas9-Lag), and applied the technique for synthetic biology applications. Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane compartment.<br><br>RESULTS: Carotenoids like &#x3b2;-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane components. To improve the functional expression of membrane-bound enzymes and localize them in proximity to the substrates, Cas9-Lag was used to pull plasmids or chromosomal DNA expressing carotenoid enzymes onto the cell membrane. For this purpose, dCas9 was fused to the E. coli membrane docking tag GlpF, and gRNA was designed to direct this fusion protein to the DNA expression cassettes. With Cas9-Lag, the zeaxanthin and astaxanthin titer increased by 29.0% and 26.7% respectively. Due to experimental limitations, the electron microscopy images of cells expressing Cas9-Lag vaguely indicated that GlpF-Cas9 might have pulled the target DNA cassettes in close proximity to membrane. Similarly, protein mass spectrometry analysis of membrane proteins suggested an increased expression of carotenoid-converting enzymes in the membrane components.<br><br>CONCLUSION: This work therefore provides a novel molecular device, Cas9-Lag, which was proved to increase zeaxanthin and astaxanthin production and might be used to manipulate DNA cassette location.},
}
@article {pmid33317042,
year = {2020},
author = {Lucere, KM and O'Malley, MMR and Diermeier, SD},
title = {Functional Screening Techniques to Identify Long Non-Coding RNAs as Therapeutic Targets in Cancer.},
journal = {Cancers},
volume = {12},
number = {12},
pages = {},
pmid = {33317042},
issn = {2072-6694},
abstract = {Recent technological advancements such as CRISPR/Cas-based systems enable multiplexed, high-throughput screening for new therapeutic targets in cancer. While numerous functional screens have been performed on protein-coding genes to date, long non-coding RNAs (lncRNAs) represent an emerging class of potential oncogenes and tumor suppressors, with only a handful of large-scale screens performed thus far. Here, we review in detail currently available screening approaches to identify new lncRNA drivers of tumorigenesis and tumor progression. We discuss the various approaches of genomic and transcriptional targeting using CRISPR/Cas9, as well as methods to post-transcriptionally target lncRNAs via RNA interference (RNAi), antisense oligonucleotides (ASOs) and CRISPR/Cas13. We discuss potential advantages, caveats and future applications of each method to provide an overview and guide on investigating lncRNAs as new therapeutic targets in cancer.},
}
@article {pmid33315895,
year = {2020},
author = {Ghoshal, B and Vong, B and Picard, CL and Feng, S and Tam, JM and Jacobsen, SE},
title = {A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana.},
journal = {PLoS genetics},
volume = {16},
number = {12},
pages = {e1008983},
pmid = {33315895},
issn = {1553-7404},
support = {F32 GM136115/GM/NIGMS NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R35 GM130272/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Arabidopsis ; Arabidopsis Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *DNA Methylation ; Epigenome ; Gene Editing/*methods ; Gene Targeting/*methods ; Plant Viruses/*genetics ; RNA, Guide/*genetics ; RNA, Transfer/genetics ; Transcriptional Activation ; },
abstract = {Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.},
}
@article {pmid33315879,
year = {2020},
author = {Petrozziello, T and Dios, AM and Mueller, KA and Vaine, CA and Hendriks, WT and Glajch, KE and Mills, AN and Mangkalaphiban, K and Penney, EB and Ito, N and Fernandez-Cerado, C and Legarda, GPA and Velasco-Andrada, MS and Acuña, PJ and Ang, MA and Muñoz, EL and Diesta, CCE and Macalintal-Canlas, R and Acuña, G and Sharma, N and Ozelius, LJ and Bragg, DC and Sadri-Vakili, G},
title = {SVA insertion in X-linked Dystonia Parkinsonism alters histone H3 acetylation associated with TAF1 gene.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0243655},
pmid = {33315879},
issn = {1932-6203},
support = {P01 NS087997/NS/NINDS NIH HHS/United States ; },
mesh = {Acetylation ; Cells, Cultured ; Dystonic Disorders/*genetics/metabolism ; Fibroblasts/metabolism ; Genetic Diseases, X-Linked/*genetics/metabolism ; Histone Acetyltransferases/*genetics ; Histones/*metabolism ; Humans ; Introns ; Retroelements ; TATA-Binding Protein Associated Factors/*genetics ; Transcription Factor TFIID/*genetics ; },
abstract = {X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.},
}
@article {pmid33315547,
year = {2020},
author = {Banks, J},
title = {Spellchecking for the Story of Life With CRISPR-Cas9 and Base, Prime Editors.},
journal = {IEEE pulse},
volume = {11},
number = {6},
pages = {6-9},
doi = {10.1109/MPULS.2020.3038019},
pmid = {33315547},
issn = {2154-2317},
mesh = {*CRISPR-Cas Systems ; DNA/chemistry/genetics/metabolism ; *Gene Editing ; Genetic Therapy ; Humans ; Mutation ; },
abstract = {Just four letters-A, G, T, and C-make up the alphabet of the genome. It may seem simple, but a small difference in spelling can create mutations that result in life-threatening diseases. Gene variants that cause genetic diseases come in many varieties. Transition point mutations cause conditions such as progeria, the rapid aging disease. Transversion point mutations cause sickle-cell disease and other major disorders. Small insertions can cause Tay-Sachs, which stops nerves working properly and is usually fatal, and deletions can result in cystic fibrosis.},
}
@article {pmid33315302,
year = {2020},
author = {Kujoth, GC and Sullivan, TD and Klein, BS},
title = {Gene Editing in Dimorphic Fungi Using CRISPR/Cas9.},
journal = {Current protocols in microbiology},
volume = {59},
number = {1},
pages = {e132},
pmid = {33315302},
issn = {1934-8533},
support = {R01 AI035681/AI/NIAID NIH HHS/United States ; R01 AI040996/AI/NIAID NIH HHS/United States ; R01 AI130411/AI/NIAID NIH HHS/United States ; R37 AI035681/AI/NIAID NIH HHS/United States ; },
mesh = {Agrobacterium ; Base Sequence ; Blastomyces/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coculture Techniques ; Fungi/*genetics ; Gene Editing/*methods ; Gene Targeting/methods ; Microbiological Techniques/methods ; Polymerase Chain Reaction ; RNA, Guide/genetics ; },
abstract = {Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks.},
}
@article {pmid33315259,
year = {2020},
author = {Nie, JB},
title = {Human Genome Editing and a Global Socio-bioethics Approach.},
journal = {The Hastings Center report},
volume = {50},
number = {6},
pages = {44-45},
doi = {10.1002/hast.1200},
pmid = {33315259},
issn = {1552-146X},
mesh = {*Bioethics ; CRISPR-Cas Systems ; Female ; *Gene Editing ; Genome, Human ; Humans ; Morals ; },
abstract = {A global socio-bioethics is called upon to address the ethical challenges arising from the revolutionary gene editing technologies such as CRISPR-Cas9, which offers the capability to rewrite the human genome. The ethical inquiry Françoise Baylis has undertaken in the book Altered Inheritance: CRISPR and the Ethics of Human Genome Editing (Harvard University Press, 2019) operates at individual, societal and global levels. Baylis has not only presented insights on how to practice "slow science" and achieve broad societal consensus through empowering the public, but she also shown what a global socio-bioethics approach can offer for the further development of bioethics.},
}
@article {pmid33311584,
year = {2020},
author = {Vyas, P and Wood, MB and Zhang, Y and Goldring, AC and Chakir, FZ and Fuchs, PA and Hiel, H},
title = {Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {21814},
pmid = {33311584},
issn = {2045-2322},
support = {P30 NS050274/NS/NINDS NIH HHS/United States ; T32DC000023/NH/NIH HHS/United States ; R01 DC015309/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Editing ; Hair Cells, Auditory, Outer/cytology/*metabolism ; Male ; Mice ; Mice, Knockout ; Receptors, Nicotinic/*biosynthesis/genetics ; },
abstract = {Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.},
}
@article {pmid33311465,
year = {2020},
author = {Das, A and Hand, TH and Smith, CL and Wickline, E and Zawrotny, M and Li, H},
title = {The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6346},
pmid = {33311465},
issn = {2041-1723},
support = {S10 OD021527/OD/NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; R01 GM099604/GM/NIGMS NIH HHS/United States ; P41 GM111244/GM/NIGMS NIH HHS/United States ; S10 RR029205/RR/NCRR NIH HHS/United States ; },
mesh = {Actinobacteria/*enzymology/genetics ; Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Protein 9/*chemistry/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; Cytosine ; DNA/chemistry/genetics/metabolism ; Gene Editing/*methods ; Genome ; High-Throughput Nucleotide Sequencing ; Methylation ; Models, Molecular ; Protein Conformation ; RNA, Guide/chemistry ; },
abstract = {Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.},
}
@article {pmid33310733,
year = {2021},
author = {Ning, B and Yu, T and Zhang, S and Huang, Z and Tian, D and Lin, Z and Niu, A and Golden, N and Hensley, K and Threeton, B and Lyon, CJ and Yin, XM and Roy, CJ and Saba, NS and Rappaport, J and Wei, Q and Hu, TY},
title = {A smartphone-read ultrasensitive and quantitative saliva test for COVID-19.},
journal = {Science advances},
volume = {7},
number = {2},
pages = {},
pmid = {33310733},
issn = {2375-2548},
support = {R01 AI122932/AI/NIAID NIH HHS/United States ; P51 OD011104/OD/NIH HHS/United States ; U54 GM104940/GM/NIGMS NIH HHS/United States ; R01 AI113725/AI/NIAID NIH HHS/United States ; R01 HD090927/HD/NICHD NIH HHS/United States ; R21 AI126361/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; COVID-19/*diagnosis ; *COVID-19 Nucleic Acid Testing ; CRISPR-Cas Systems ; Chlorocebus aethiops ; Computer Simulation ; Female ; Humans ; Limit of Detection ; Macaca mulatta ; Male ; Molecular Diagnostic Techniques/instrumentation ; *Point-of-Care Testing ; Reverse Transcriptase Polymerase Chain Reaction ; Saliva/*virology ; Sensitivity and Specificity ; *Smartphone ; Vero Cells ; Viral Load ; },
abstract = {Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 10[5] copies/μl) and exhibited a limit of detection (0.38 copies/μl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.},
}
@article {pmid33309880,
year = {2021},
author = {Bloomer, H and Smith, RH and Hakami, W and Larochelle, A},
title = {Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {4},
pages = {1611-1624},
pmid = {33309880},
issn = {1525-0024},
support = {Z99 HL999999/ImNIH/Intramural NIH HHS/United States ; Z99 HL999999/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair/genetics ; DNA Repair/genetics ; Dependovirus/genetics ; Gene Editing ; *Genetic Therapy ; Genetic Vectors/genetics ; Genome, Human/genetics ; Hematologic Diseases/*genetics/pathology/therapy ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology/metabolism ; Humans ; Mice ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; Stem Cells/cytology/metabolism ; },
abstract = {Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated site-specific integration utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in HSPCs, where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Recently, a novel NHEJ-based approach to CRISPR-Cas9-mediated transgene knockin, known as homology-independent targeted integration (HITI), has demonstrated improved site-specific integration frequencies in non-dividing cells. Here we utilize a HITI-based approach to achieve robust site-specific transgene integration in human mobilized peripheral blood CD34+ HSPCs. As proof of concept, a reporter gene was targeted to a clinically relevant genetic locus using a recombinant adeno-associated virus serotype 6 vector and single guide RNA/Cas9 ribonucleoprotein complexes. We demonstrate high levels of stable HITI-mediated genome editing (∼21%) in repopulating HSPCs after transplantation into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs.},
}
@article {pmid33309782,
year = {2021},
author = {Wu, W and Yang, Y and Yao, F and Dong, L and Xia, X and Zhang, S and Lei, H},
title = {AAV-mediated in vivo genome editing in vascular endothelial cells.},
journal = {Methods (San Diego, Calif.)},
volume = {194},
number = {},
pages = {12-17},
doi = {10.1016/j.ymeth.2020.12.001},
pmid = {33309782},
issn = {1095-9130},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; *Endothelial Cells ; *Gene Editing ; Mice ; Promoter Regions, Genetic ; Retina ; },
abstract = {In vivo genome editing meets numerous challenges including efficiency and safety. Here we describe an efficient in vivo genome editing method of delivering CRISPR-Cas9 into vascular endothelial cells with adeno-associated viruses (AAVs). In this system, expression of SpCas9 is driven by a specific endothelial promoter of intercellular adhesion molecule 2 (pICAM2) to restrict this foreign enzyme in vascular endothelial cells, which can be efficiently infected by AAV1. We exemplify this approach by editing VEGFR2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy, and expect that this simplified protocol can be expanded to other researches on editing endothelial genome in vivo.},
}
@article {pmid33309323,
year = {2021},
author = {Le, TK and Paris, C and Khan, KS and Robson, F and Ng, WL and Rocchi, P},
title = {Nucleic Acid-Based Technologies Targeting Coronaviruses.},
journal = {Trends in biochemical sciences},
volume = {46},
number = {5},
pages = {351-365},
pmid = {33309323},
issn = {0968-0004},
mesh = {COVID-19/genetics/immunology/metabolism/therapy ; *COVID-19 Vaccines/genetics/immunology/therapeutic use ; *CRISPR-Cas Systems ; Humans ; *RNA, Messenger/genetics/immunology/metabolism ; *RNA, Viral/genetics/immunology/metabolism ; *SARS-CoV-2/genetics/immunology/metabolism ; },
abstract = {The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.},
}
@article {pmid33309104,
year = {2021},
author = {Brest, P and Mograbi, B and Hofman, P and Milano, G},
title = {Using Genetics To Dissect SARS-CoV-2 Infection.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {3},
pages = {203-204},
pmid = {33309104},
issn = {0168-9525},
mesh = {Angiotensin-Converting Enzyme 2/*genetics/metabolism ; COVID-19/*genetics/metabolism/virology ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression ; Genome, Viral/*genetics ; Genome-Wide Association Study/*methods ; Humans ; RNA Interference ; SARS-CoV-2/*genetics/metabolism/physiology ; },
abstract = {To uncover the key cellular pathways associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity, Daniloski and coworkers used CRISPR-based whole-genome screening. Their results could propose new or repositioned drugs for the ongoing fight against COVID-19.},
}
@article {pmid33308024,
year = {2021},
author = {Corrado, A and Aceto, R and Silvestri, R and Dell'Anno, I and Ricci, B and Miglietta, S and Romei, C and Giovannoni, R and Poliseno, L and Evangelista, M and Vitiello, M and Cipollini, M and Garritano, S and Giusti, L and Zallocco, L and Elisei, R and Landi, S and Gemignani, F},
title = {Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System.},
journal = {Thyroid : official journal of the American Thyroid Association},
volume = {31},
number = {7},
pages = {1056-1066},
doi = {10.1089/thy.2020.0366},
pmid = {33308024},
issn = {1557-9077},
mesh = {Adult ; Alleles ; CRISPR-Cas Systems ; Case-Control Studies ; Female ; Galectin 3/*genetics ; Genetic Association Studies ; *Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; *Polymorphism, Single Nucleotide ; Thyroid Epithelial Cells/*metabolism/pathology ; Thyroid Neoplasms/*genetics/metabolism/pathology ; Transcriptome ; },
abstract = {Background: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46-0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.},
}
@article {pmid33307189,
year = {2021},
author = {Nishida, K and Kondo, A},
title = {CRISPR-derived genome editing technologies for metabolic engineering.},
journal = {Metabolic engineering},
volume = {63},
number = {},
pages = {141-147},
doi = {10.1016/j.ymben.2020.12.002},
pmid = {33307189},
issn = {1096-7184},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; *Metabolic Engineering ; Metabolic Networks and Pathways ; },
abstract = {In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.},
}
@article {pmid33307135,
year = {2021},
author = {Liu, C and Yang, Z and Cai, M and Shi, Y and Cui, H and Yuan, J},
title = {Generation of Plasmodium yoelii malaria parasite for conditional degradation of proteins.},
journal = {Molecular and biochemical parasitology},
volume = {241},
number = {},
pages = {111346},
doi = {10.1016/j.molbiopara.2020.111346},
pmid = {33307135},
issn = {1872-9428},
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression ; Gene Targeting ; *Genetic Engineering ; Malaria/parasitology/transmission ; Mice ; Plasmodium yoelii/*genetics/*metabolism ; Proteolysis ; Protozoan Proteins/genetics/metabolism ; },
abstract = {The auxin-inducible degron (AID) system is a robust chemical-genetic method for manipulating endogenous protein level by conditional proteasomal degradation via a small molecule. So far, this system has not been adapted in the P. yoelii, an important and widely used Plasmodium rodent parasite model for malaria biology. Here, using the CRISPR/Cas9 genome editing method, we generated two marker-free transgenic P. yoelii parasite lines (eef1a-Tir1 and soap-Tir1) stably expressing the Oryza sativa gene tir1 under the promoters of eef1a and soap respectively. These two lines develop normally during the parasite life cycle. In these backgrounds, we used the CRISPR/Cas9 method to tag two genes (cdc50c and fbxo1) with the AID motif and interrogate the expression of these two proteins with auxin. The eef1a-Tir1 line allows efficient degradation of the AID-tagged endogenous protein in the asexual schizont and sexual gametocyte stages, while the soap-Tir1 line allows protein degradation in the ookinetes. These two lines will be a useful resource for studying the Plasmodium parasite biology based on the P. yoelii.},
}
@article {pmid33306959,
year = {2021},
author = {Fozouni, P and Son, S and Díaz de León Derby, M and Knott, GJ and Gray, CN and D'Ambrosio, MV and Zhao, C and Switz, NA and Kumar, GR and Stephens, SI and Boehm, D and Tsou, CL and Shu, J and Bhuiya, A and Armstrong, M and Harris, AR and Chen, PY and Osterloh, JM and Meyer-Franke, A and Joehnk, B and Walcott, K and Sil, A and Langelier, C and Pollard, KS and Crawford, ED and Puschnik, AS and Phelps, M and Kistler, A and DeRisi, JL and Doudna, JA and Fletcher, DA and Ott, M},
title = {Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.},
journal = {Cell},
volume = {184},
number = {2},
pages = {323-333.e9},
pmid = {33306959},
issn = {1097-4172},
support = {R61 DA048444/DA/NIDA NIH HHS/United States ; F30 AI143401/AI/NIAID NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; R61 AI140465/AI/NIAID NIH HHS/United States ; U54 HL143541/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; COVID-19 Nucleic Acid Testing/economics/instrumentation/*methods ; CRISPR-Cas Systems ; Cell Line ; Cell Phone/*instrumentation ; Coronavirus Nucleocapsid Proteins/genetics ; Humans ; Nasopharynx/virology ; Optical Imaging/instrumentation/*methods ; Phosphoproteins/genetics ; Point-of-Care Testing ; RNA Interference ; RNA, Viral/*analysis/genetics ; Sensitivity and Specificity ; Viral Load/economics/instrumentation/*methods ; },
abstract = {The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.},
}
@article {pmid33306952,
year = {2020},
author = {Hoffmann, ER and Roig, I},
title = {Cas9 in Human Embryos: On Target but No Repair.},
journal = {Cell},
volume = {183},
number = {6},
pages = {1464-1466},
doi = {10.1016/j.cell.2020.11.022},
pmid = {33306952},
issn = {1097-4172},
mesh = {Alleles ; CRISPR-Cas Systems ; Chromosomes ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Humans ; },
abstract = {In this issue of Cell, Zuccaro and colleagues show that on-target Cas9-mediated double-strand breaks cause chromosome loss or mis-repair of the disease allele in > 90% of human embryos. End joining repair pathways dominate, causing small insertions or deletions, which raises serious questions about using double-strand breaks for "gene surgery".},
}
@article {pmid33306075,
year = {2021},
author = {Sha, Y and Huang, R and Huang, M and Yue, H and Shan, Y and Hu, J and Xing, D},
title = {Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {2},
pages = {247-250},
doi = {10.1039/d0cc06412b},
pmid = {33306075},
issn = {1364-548X},
mesh = {Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Limit of Detection ; MicroRNAs/*analysis/genetics/metabolism ; Reproducibility of Results ; },
abstract = {Existing CRISPR/cas-based biosensors usually improve sensitivity by target amplification, which is time-consuming and susceptible to impurities in complex biofluid. Herein, this is the first time a cascade CRISPR/cas (casCRISPR) system has been developed, which can provide a detection limit of 1.33 fM (∼1000 times lower than direct Cas13a-based miRNA detection) and single-base resolution for miR-17 detection without resorting to target amplification. casCRISPR can also be applied to detect miRNA in complicated cell extracts and serum samples. Overall, casCRISPR will provide a heuristic idea for CRISPR/cas based biosensing, and could be a promising tool for miRNA diagnostics.},
}
@article {pmid33305496,
year = {2021},
author = {Bollier, N and Andrade Buono, R and Jacobs, TB and Nowack, MK},
title = {Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR.},
journal = {Plant biotechnology journal},
volume = {19},
number = {4},
pages = {651-653},
pmid = {33305496},
issn = {1467-7652},
support = {639234/ERC_/European Research Council/International ; 864952/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Mutagenesis ; Plants/*genetics ; },
}
@article {pmid33305456,
year = {2021},
author = {Mohamadian, M and Chiti, H and Shoghli, A and Biglari, S and Parsamanesh, N and Esmaeilzadeh, A},
title = {COVID-19: Virology, biology and novel laboratory diagnosis.},
journal = {The journal of gene medicine},
volume = {23},
number = {2},
pages = {e3303},
pmid = {33305456},
issn = {1521-2254},
mesh = {COVID-19/*diagnosis/physiopathology/prevention &amp; control/virology ; COVID-19 Nucleic Acid Testing/methods ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Microarray Analysis ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics/metabolism/*pathogenicity ; },
abstract = {BACKGROUND: At the end of December 2019, a novel coronavirus tentatively named SARS-CoV-2 in Wuhan, a central city in China, was announced by the World Health Organization. SARS-CoV-2 is an RNA virus that has become a major public health concern after the outbreak of the Middle East Respiratory Syndrome-CoV (MERS-CoV) and Severe Acute Respiratory Syndrome-CoV (SARS-CoV) in 2002 and 2012, respectively. As of 29 October 2020, the total number of COVID-19 cases had reached over 44 million worldwide, with more than 1.17 million confirmed deaths.<br><br>DISCUSSION: SARS-CoV-2 infected patients usually present with severe viral pneumonia. Similar to SARS-CoV, the virus enters respiratory tract cells via the angiotensin-converting enzyme receptor 2. The structural proteins play an essential role in budding the virus particles released from different host cells. To date, an approved vaccine or treatment option of a preventive character to avoid severe courses of COVID-19 is still not available.<br><br>CONCLUSIONS: In the present study, we provide a brief review of the general biological features of CoVs and explain the pathogenesis, clinical symptoms and diagnostic approaches regarding monitoring future infectivity and prevent emerging COVID-19 infections.},
}
@article {pmid33305314,
year = {2021},
author = {Levi, O and Arava, YS},
title = {Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.},
journal = {Nucleic acids research},
volume = {49},
number = {1},
pages = {432-443},
pmid = {33305314},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Methionine/metabolism ; Methionine-tRNA Ligase/*metabolism ; Peptide Elongation Factors/*biosynthesis/genetics ; Polyribosomes/metabolism ; Protein Binding ; *Protein Biosynthesis ; Pseudouridine/*physiology ; RNA Processing, Post-Transcriptional ; RNA, Fungal/*genetics ; RNA, Messenger/*genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism ; },
abstract = {Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.},
}
@article {pmid33304462,
year = {2020},
author = {Antao, AM and Karapurkar, JK and Lee, DR and Kim, KS and Ramakrishna, S},
title = {Disease modeling and stem cell immunoengineering in regenerative medicine using CRISPR/Cas9 systems.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {3649-3665},
pmid = {33304462},
issn = {2001-0370},
abstract = {CRISPR/Cas systems are popular genome editing tools that belong to a class of programmable nucleases and have enabled tremendous progress in the field of regenerative medicine. We here outline the structural and molecular frameworks of the well-characterized type II CRISPR system and several computational tools intended to facilitate experimental designs. The use of CRISPR tools to generate disease models has advanced research into the molecular aspects of disease conditions, including unraveling the molecular basis of immune rejection. Advances in regenerative medicine have been hindered by major histocompatibility complex-human leukocyte antigen (HLA) genes, which pose a major barrier to cell- or tissue-based transplantation. Based on progress in CRISPR, including in recent clinical trials, we hypothesize that the generation of universal donor immune-engineered stem cells is now a realistic approach to tackling a multitude of disease conditions.},
}
@article {pmid33303992,
year = {2021},
author = {Ferrari, G and Thrasher, AJ and Aiuti, A},
title = {Gene therapy using haematopoietic stem and progenitor cells.},
journal = {Nature reviews. Genetics},
volume = {22},
number = {4},
pages = {216-234},
pmid = {33303992},
issn = {1471-0064},
support = {/WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Genetic Therapy ; Hematopoietic Stem Cell Transplantation/*methods ; Hematopoietic Stem Cells/cytology ; Humans ; Primary Immunodeficiency Diseases/genetics/*therapy ; beta-Thalassemia/genetics/*therapy ; },
abstract = {Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.},
}
@article {pmid33303593,
year = {2020},
author = {Kaiser, J},
title = {Tweaking genes with CRISPR or viruses fixes blood disorders.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6522},
pages = {1254-1255},
doi = {10.1126/science.370.6522.1254},
pmid = {33303593},
issn = {1095-9203},
mesh = {Anemia, Sickle Cell/genetics/*therapy ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Repressor Proteins/*genetics ; *Viruses/genetics ; beta-Thalassemia/genetics/*therapy ; },
}
@article {pmid33303588,
year = {2020},
author = {Muller, R and Meacham, ZA and Ferguson, L and Ingolia, NT},
title = {CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6522},
pages = {},
pmid = {33303588},
issn = {1095-9203},
support = {DP2 CA195768/CA/NCI NIH HHS/United States ; R01 GM130996/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; },
mesh = {Alcohol Oxidoreductases/genetics ; Aminohydrolases/genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Eukaryotic Initiation Factor-2/metabolism ; *Gene Expression ; Gene Expression Profiling/*methods ; *Gene Regulatory Networks ; Phenotype ; Phosphorylation ; Protein Serine-Threonine Kinases/metabolism ; Pyrophosphatases/genetics ; RNA, Guide/genetics ; RNA, Transfer/genetics/metabolism ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {To realize the promise of CRISPR-Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.},
}
@article {pmid33303323,
year = {2021},
author = {Dronina, J and Bubniene, US and Ramanavicius, A},
title = {The application of DNA polymerases and Cas9 as representative of DNA-modifying enzymes group in DNA sensor design (review).},
journal = {Biosensors &amp; bioelectronics},
volume = {175},
number = {},
pages = {112867},
doi = {10.1016/j.bios.2020.112867},
pmid = {33303323},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; COVID-19/*diagnosis/genetics/virology ; CRISPR-Cas Systems/genetics ; Diagnostic Tests, Routine ; Humans ; RNA, Viral/genetics/*isolation &amp; purification ; SARS-CoV-2/*isolation &amp; purification/pathogenicity ; },
abstract = {Rapid detection of nucleic acids (DNA or RNA) by inexpensive, selective, accurate, and highly sensitive methods is very important for biosensors. DNA-sensors based on DNA-modifying enzymes for fast determination and monitoring of pathogenic (Zika, Dengue, SARS-Cov-2 (inducer of COVID-19), human papillomavirus, HIV, etc.) viruses and diagnosis of virus-induced diseases is a key factor of this overview. Recently, DNA-modifying enzymes (Taq DNA polymerase, Phi29 DNA polymerase) have been widely used for the diagnosis of virus or pathogenic disease by gold standard (PCR, qPCR, RT-qPCR) methods, therefore, alternative methods have been reviewed. The main mechanisms of DNA metabolism (replication cycle, amplification) and the genomeediting tool CRISPR-Cas9 are purposefully discussed in order to address strategic possibility to design DNA-sensors based on immobilized DNA-enzymes. However, the immobilization of biologically active proteins on a gold carrier technique with the ability to detect viral or bacterial nucleic acids is individual for each DNA-modifying enzyme group, due to a different number of active sites, C and N terminal locations and arrangement, therefore, individual protocols based on the 'masking' of active sites should be elaborated for each enzyme.},
}
@article {pmid33301730,
year = {2021},
author = {Lan, X and Ren, R and Feng, R and Ly, LC and Lan, Y and Zhang, Z and Aboreden, N and Qin, K and Horton, JR and Grevet, JD and Mayuranathan, T and Abdulmalik, O and Keller, CA and Giardine, B and Hardison, RC and Crossley, M and Weiss, MJ and Cheng, X and Shi, J and Blobel, GA},
title = {ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.},
journal = {Molecular cell},
volume = {81},
number = {2},
pages = {239-254.e8},
pmid = {33301730},
issn = {1097-4164},
support = {R01 HL119479/HL/NHLBI NIH HHS/United States ; R24 DK106766/DK/NIDDK NIH HHS/United States ; R35 GM134744/GM/NIGMS NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; COS Cells ; CRISPR-Cas Systems ; Chlorocebus aethiops ; DNA/*genetics/metabolism ; Erythroid Precursor Cells/cytology/*metabolism/transplantation ; Fetal Blood/cytology/metabolism ; Fetal Hemoglobin/*genetics/metabolism ; Fetus ; Gene Editing ; HEK293 Cells ; Heterografts ; Humans ; Mi-2 Nucleosome Remodeling and Deacetylase Complex/chemistry/*genetics/metabolism ; Mice ; Models, Molecular ; Mouse Embryonic Stem Cells/cytology ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Transcription Factors/chemistry/*genetics/metabolism ; Transcriptional Activation ; },
abstract = {Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.},
}
@article {pmid33301298,
year = {2021},
author = {Kan, A and Gelfat, I and Emani, S and Praveschotinunt, P and Joshi, NS},
title = {Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917.},
journal = {ACS synthetic biology},
volume = {10},
number = {1},
pages = {94-106},
pmid = {33301298},
issn = {2161-5063},
support = {R01 DK110770/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli/*genetics ; Gastrointestinal Tract/metabolism ; Gene Editing ; Gene Expression ; Genetic Engineering/methods ; Mice ; Mice, Inbred C57BL ; Plasmids/genetics/*metabolism ; Promoter Regions, Genetic ; Temperature ; },
abstract = {Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.},
}
@article {pmid33301289,
year = {2020},
author = {Zhang, BC and Luo, BY and Zou, JJ and Wu, PY and Jiang, JL and Le, JQ and Zhao, RR and Chen, L and Shao, JW},
title = {Co-delivery of Sorafenib and CRISPR/Cas9 Based on Targeted Core-Shell Hollow Mesoporous Organosilica Nanoparticles for Synergistic HCC Therapy.},
journal = {ACS applied materials &amp; interfaces},
volume = {12},
number = {51},
pages = {57362-57372},
doi = {10.1021/acsami.0c17660},
pmid = {33301289},
issn = {1944-8252},
mesh = {Animals ; Aptamers, Nucleotide/chemistry/toxicity ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*drug therapy ; Cell Line, Tumor ; Drug Carriers/chemistry/toxicity ; Drug Liberation ; Epithelial Cell Adhesion Molecule/chemistry ; ErbB Receptors/genetics/metabolism ; Gene Editing ; Genes, erbB-1 ; Humans ; Liver Neoplasms/*drug therapy ; Mice ; Nanoparticles/*chemistry/toxicity ; Polyamines/chemistry/toxicity ; Porosity ; Signal Transduction/drug effects ; Silicon Dioxide/*chemistry/toxicity ; Sorafenib/*therapeutic use ; },
abstract = {The rapid development of CRISPR/Cas9 systems has opened up tantalizing prospects to sensitize cancers to chemotherapy using efficient targeted genome editing, but safety concerns and possible off-target effects of viral vectors remain a major obstacle for clinical application. Thus, the construction of novel nonviral tumor-targeting nanodelivery systems has great potential for the safe application of CRISPR/Cas9 systems for gene-chemo-combination therapy. Here, we report a polyamidoamine-aptamer-coated hollow mesoporous silica nanoparticle for the co-delivery of sorafenib and CRISPR/Cas9. The core-shell nanoparticles had good stability, enabled ultrahigh drug loading, targeted delivery, and controlled-release of the gene-drug combination. The nanocomplex showed >60% EGFR-editing efficiency without off-target effects in all nine similar sites, regulating the EGFR-PI3K-Akt pathway to inhibit angiogenesis, and exhibited a synergistic effect on cell proliferation. Importantly, the co-delivery nanosystem achieved efficient EGFR gene therapy and caused 85% tumor inhibition in a mouse model. Furthermore, the nanocomplex showed high accumulation at the tumor site in vivo and exhibited good safety with no damage to major organs. Due to these properties, the nanocomplex provides a versatile delivery approach for efficient co-loading of gene-drug combinations, allowing for precise gene editing and synergistic inhibition of tumor growth without apparent side effects on normal tissues.},
}
@article {pmid33301111,
year = {2021},
author = {Geny, S and Pichard, S and Brion, A and Renaud, JB and Jacquemin, S and Concordet, JP and Poterszman, A},
title = {Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2247},
number = {},
pages = {39-57},
pmid = {33301111},
issn = {1940-6029},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Cloning, Molecular ; DNA/*genetics ; Flow Cytometry ; *Gene Editing ; Gene Expression ; Gene Targeting ; Green Fluorescent Proteins/*genetics ; HEK293 Cells ; Humans ; Microscopy, Fluorescence ; Models, Molecular ; Plasmids/genetics ; Protein Conformation ; RNA, Guide ; Recombinant Fusion Proteins/chemistry/*genetics ; Structure-Activity Relationship ; },
abstract = {Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.},
}
@article {pmid33300874,
year = {2020},
author = {Xu, N and Oltmanns, A and Zhao, L and Girot, A and Karimi, M and Hoepfner, L and Kelterborn, S and Scholz, M and Beißel, J and Hegemann, P and Bäumchen, O and Liu, LN and Huang, K and Hippler, M},
title = {Altered N-glycan composition impacts flagella-mediated adhesion in Chlamydomonas reinhardtii.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33300874},
issn = {2050-084X},
support = {HI737/12-1//Deutsche Forschungsgemeinschaft/International ; 31671399//National Natural Science Foundation of China/International ; UF120411//Royal Society/International ; BB/R003890/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M012441/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; URF\R\180030//Royal Society/International ; RGF\EA\181061//Royal Society/International ; RGF\EA\180233//Royal Society/International ; 2019M662335//China Postdoctoral Science Foundation/International ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Adhesion ; Chlamydomonas reinhardtii/metabolism ; Flagella/metabolism ; Gene Editing ; Gene Knockout Techniques ; Glycosylation ; Microscopy, Atomic Force ; Polysaccharides/*metabolism ; },
abstract = {For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRISPR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. This results in impaired polystyrene bead binding and transport but not gliding of cells on solid surfaces. Notably, assembly, intraflagellar transport, and protein import into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.},
}
@article {pmid33300275,
year = {2021},
author = {Derry, WB},
title = {CRISPR: development of a technology and its applications.},
journal = {The FEBS journal},
volume = {288},
number = {2},
pages = {358-359},
doi = {10.1111/febs.15621},
pmid = {33300275},
issn = {1742-4658},
mesh = {Animals ; Bacteria/genetics/immunology/virology ; Bacteriophages/genetics/metabolism/pathogenicity ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Molecular Biology/methods ; RNA, Guide/*genetics/metabolism ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) is a prokaryotic immune surveillance system that is used by bacteria to recognize genetic material of infectious organisms, such as phage viruses. Using CRISPR-associated (Cas) proteins, this system cleaves foreign nucleic acid into fragments, thus defending the bacterium against the attacker. The 2020 Nobel Prize in Chemistry was awarded to CRISPR-Cas pioneers Emmanuelle Charpentier and Jennifer Doudna, who developed the CRISPR-Cas system to precisely edit genomic DNA. This technology has exploded at a breathtaking pace and is now used by almost every molecular biology laboratory around the world in a myriad of organisms. In this Virtual Issue, the FEBS Journal features articles reviewing the development of CRISPR/Cas9 technology and its applications to understand the functions of proteins in vivo.},
}
@article {pmid33299166,
year = {2020},
author = {Ledford, H},
title = {CRISPR gene therapy shows promise against blood diseases.},
journal = {Nature},
volume = {588},
number = {7838},
pages = {383},
pmid = {33299166},
issn = {1476-4687},
mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genetic Therapy ; *Hematologic Diseases ; Humans ; *beta-Thalassemia ; },
}
@article {pmid33299151,
year = {2020},
author = {Schwartz, C and Lenderts, B and Feigenbutz, L and Barone, P and Llaca, V and Fengler, K and Svitashev, S},
title = {CRISPR-Cas9-mediated 75.5-Mb inversion in maize.},
journal = {Nature plants},
volume = {6},
number = {12},
pages = {1427-1431},
pmid = {33299151},
issn = {2055-0278},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genes, Plant ; Mutagenesis, Insertional/*methods ; Plant Breeding/*methods ; Sequence Inversion ; Zea mays/*genetics ; },
abstract = {CRISPR-Cas is a powerful double-strand-break technology with wide-ranging applications from gene discovery to commercial product development. Thus far, this tool has been almost exclusively used for gene knockouts and deletions, with a few examples of gene edits and targeted gene insertions. Here, we demonstrate the application of CRISPR-Cas9 technology to mediate targeted 75.5-Mb pericentric inversion in chromosome 2 in one of the elite maize inbred lines from Corteva Agriscience. This inversion unlocks a large chromosomal region containing substantial genetic variance for recombination, thus providing opportunities for the development of new maize varieties with improved phenotypes.},
}
@article {pmid33298570,
year = {2021},
author = {Zhang, X and Zhao, X and Li, G and Zhang, M and Xing, P and Li, Z and Chen, B and Yang, H and Wu, Z},
title = {Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation.},
journal = {Biology open},
volume = {10},
number = {1},
pages = {},
pmid = {33298570},
issn = {2046-6390},
mesh = {Adult Germline Stem Cells/*cytology/*metabolism ; Alleles ; Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/deficiency/*genetics ; Gene Editing ; Genotype ; Mice ; *Mice, Knockout ; *Models, Animal ; *Stem Cell Transplantation ; Transcription Factors/deficiency/*genetics ; Transplantation, Homologous ; },
abstract = {Spermatogonial stem cell (SSC) transplantation is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5[-/-] mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5[-/-] mice. At 12 weeks of age, the tubules of Etv5[-/-] mice lacked almost all spermatogenic cells with a Sertoli cell-only phenotype, and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3- (immature) or 7-week-old (mature) Etv5[-/-] mice. Partial restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5[-/-] mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5[-/-] mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.},
}
@article {pmid33298251,
year = {2021},
author = {Kim, S and Hupperetz, C and Lim, S and Kim, CH},
title = {Genome editing of immune cells using CRISPR/Cas9.},
journal = {BMB reports},
volume = {54},
number = {1},
pages = {59-69},
pmid = {33298251},
issn = {1976-670X},
mesh = {CRISPR-Cas Systems/*genetics/immunology ; *Gene Editing ; Humans ; T-Lymphocytes/*immunology ; },
abstract = {The ability to read, write, and edit genomic information in living organisms can have a profound impact on research, health, economic, and environmental issues. The CRISPR/Cas system, recently discovered as an adaptive immune system in prokaryotes, has revolutionized the ease and throughput of genome editing in mammalian cells and has proved itself indispensable to the engineering of immune cells and identification of novel immune mechanisms. In this review, we summarize the CRISPR/ Cas9 system and the history of its discovery and optimization. We then focus on engineering T cells and other types of immune cells, with emphasis on therapeutic applications. Last, we describe the different modifications of Cas9 and their recent applications in the genome-wide screening of immune cells. [BMB Reports 2021; 54(1): 59-69].},
}
@article {pmid33298245,
year = {2021},
author = {Lee, HK and Oh, Y and Hong, J and Lee, SH and Hur, JK},
title = {Development of CRISPR technology for precise single-base genome editing: a brief review.},
journal = {BMB reports},
volume = {54},
number = {2},
pages = {98-105},
pmid = {33298245},
issn = {1976-670X},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPRmediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications. [BMB Reports 2021; 54(2): 98-105].},
}
@article {pmid33298142,
year = {2020},
author = {Yang, Y and Tang, T and Feng, B and Li, S and Hou, N and Ma, X and Jiang, L and Xin, X and Chen, Q},
title = {Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage.},
journal = {Parasites &amp; vectors},
volume = {13},
number = {1},
pages = {611},
pmid = {33298142},
issn = {1756-3305},
mesh = {Animals ; Antigens, Protozoan/*genetics/*metabolism ; Antimalarials/metabolism ; Base Sequence ; Binding Sites ; CRISPR-Cas Systems ; Diagnostic Tests, Routine ; Gene Targeting ; Heme/*metabolism ; Hemoglobins ; Humans ; Life Cycle Stages ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/genetics/*metabolism ; Polymerase Chain Reaction ; Protozoan Proteins/*genetics/*metabolism ; Transcriptome ; },
abstract = {BACKGROUND: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.<br><br>METHODS: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2[-]-3D7 parasites.<br><br>RESULTS: Pfhrp2[-] transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1).<br><br>CONCLUSION: Our data revealed changes in the transcriptome expression profile of the Pfhrp2[-]-3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.},
}
@article {pmid33297464,
year = {2020},
author = {Sobiak, B and Leśniak, W},
title = {Effect of SUV39H1 Histone Methyltransferase Knockout on Expression of Differentiation-Associated Genes in HaCaT Keratinocytes.},
journal = {Cells},
volume = {9},
number = {12},
pages = {},
pmid = {33297464},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems ; Cell Adhesion ; Cell Differentiation ; Chromatin Immunoprecipitation ; DNA Methylation ; Epigenesis, Genetic ; Gene Expression Profiling ; Gene Expression Regulation ; HaCaT Cells ; Histone Methyltransferases/*genetics ; Histones/metabolism ; Humans ; Keratinocytes/*metabolism ; Lysine/metabolism ; Methyltransferases/*genetics/*physiology ; Protein Processing, Post-Translational ; Repressor Proteins/*genetics/*physiology ; Skin Diseases/metabolism ; },
abstract = {Keratinocytes undergo a complex differentiation process, coupled with extensive changes in gene expression through which they acquire distinctive features indispensable for cells that form the external body barrier-epidermis. Disturbed epidermal differentiation gives rise to multiple skin diseases. The involvement of epigenetic factors, such as DNA methylation or histone modifications, in the regulation of epidermal gene expression and differentiation has not been fully recognized yet. In this work we performed a CRISPR/Cas9-mediated knockout of SUV39H1, a gene-encoding H3K9 histone methyltransferase, in HaCaT cells that originate from spontaneously immortalized human keratinocytes and examined changes in the expression of selected differentiation-specific genes located in the epidermal differentiation complex (EDC) and other genomic locations by RT-qPCR. The studied genes revealed a diverse differentiation state-dependent or -independent response to a lower level of H3K9 methylation. We also show, by means of chromatin immunoprecipitation, that the expression of genes in the LCE1 subcluster of EDC was regulated by the extent of trimethylation of lysine 9 in histone H3 bound to their promoters. Changes in gene expression were accompanied by changes in HaCaT cell morphology and adhesion.},
}
@article {pmid33295625,
year = {2021},
author = {Liu, X and Qiu, S and Mei, L and Jing, H and Lin, X and Wang, Q},
title = {A High-Resolution Melting Analysis with an Unlabeled Probe for CRISPR/Cas9-Induced ZBED6 Knockout Pigs Detection.},
journal = {Journal of AOAC International},
volume = {104},
number = {3},
pages = {541-545},
doi = {10.1093/jaoacint/qsaa161},
pmid = {33295625},
issn = {1944-7922},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Swine/genetics ; },
abstract = {BACKGROUND: The zinc finger BED-type containing six knockout (ZBED6-KO) pigs were created to improve economic traits by increasing the expression of insulin-like growth factor 2. They were generated by CRISPR/CRISPR-associated protein 9 (Cas9) technology and a single-base deletion of ZBED6 was found. An efficient and rapid method was needed to detect this type of pig.<br><br>OBJECTIVE: This study aimed to develop a high-resolution melting (HRM) method to detect ZBED6-KO pigs.<br><br>METHODS: An unlabeled probe and two primers were designed to develop the HRM method. The limit of detection, specificity, and accuracy of the established method were tested by the constructed plasmid and DNA extracts of tissue specimens.<br><br>RESULTS: The limit of detection by the established method was 102 copies/&#xb5;L. The HRM method with an unlabeled probe showed good specificity and high accuracy.<br><br>CONCLUSIONS: The established HRM analysis with an unlabeled probe showed it to be a highly effective, rapid, and reliable method to distinguish ZBED6-KO pigs from wild-type pigs.<br><br>HIGHLIGHTS: It is the first time that HRM analysis with an unlabeled probe has been used in the detection of genome editing pigs by the CRISPR/Cas9 technology.},
}
@article {pmid33295064,
year = {2021},
author = {Xiong, E and Jiang, L and Tian, T and Hu, M and Yue, H and Huang, M and Lin, W and Jiang, Y and Zhu, D and Zhou, X},
title = {Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {10},
pages = {5307-5315},
doi = {10.1002/anie.202014506},
pmid = {33295064},
issn = {1521-3773},
mesh = {COVID-19/*diagnosis/virology ; *CRISPR-Cas Systems ; *Genes, Viral ; Humans ; Nasopharynx/virology ; RNA, Viral ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; SARS-CoV-2/genetics/*isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.},
}
@article {pmid33294131,
year = {2020},
author = {Shibata, T and Iwasaki, W and Hirota, K},
title = {The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {3350-3360},
pmid = {33294131},
issn = {2001-0370},
abstract = {Double-stranded (ds)DNA, not dsRNA, has an ability to form a homologous complex with single-stranded (ss)DNA or ssRNA of homologous sequence. D-loops and homologous triplexes are homologous complexes formed with ssDNA by RecA/Rad51-family homologous-pairing proteins, and are a key intermediate of homologous (genetic/DNA) recombination. R-loop formation independent of transcription (R-loop formation in trans) was recently found to play roles in gene regulation and development of mammals and plants. In addition, the crRNA-Cas effector complex in CRISPR-Cas systems also relies on R-loop formation to recognize specific target. In homologous complex formation, ssDNA/ssRNA finds a homologous sequence in dsDNA by Watson-Crick base-pairing. crRNA-Cas effector complexes appear to actively melt dsDNA to make its bases available for annealing to crRNA. On the other hand, in D-loop formation and homologous-triplex formation, it is likely that dsDNA recognizes the homologous sequence before the melting of its double helix by using its intrinsic molecular function depending on CH2 at the 2'-position of the deoxyribose, and that the major role of RecA is the extension of ssDNA and the holding dsDNA at a position suitable for homology search. This intrinsic dsDNA function would also play a role in R-loop formation. The dependency of homologous-complex formation on 2'-CH2 of the deoxyribose would explain the absence of homologous complex formation by dsRNA, and dsDNA as sole genome molecule in all cellular organisms.},
}
@article {pmid33293588,
year = {2020},
author = {Hayashi, H and Kubo, Y and Izumida, M and Matsuyama, T},
title = {Efficient viral delivery of Cas9 into human safe harbor.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {21474},
pmid = {33293588},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Dependovirus/*genetics ; Gene Editing ; Gene Transfer Techniques ; Genetic Loci ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/*genetics ; RNA, Guide/genetics ; Streptococcus pyogenes/*genetics ; *Transduction, Genetic/methods ; },
abstract = {Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.},
}
@article {pmid33293555,
year = {2020},
author = {Carlson-Stevermer, J and Das, A and Abdeen, AA and Fiflis, D and Grindel, BI and Saxena, S and Akcan, T and Alam, T and Kletzien, H and Kohlenberg, L and Goedland, M and Dombroe, MJ and Saha, K},
title = {Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6277},
pmid = {33293555},
issn = {2041-1723},
support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Computer Simulation ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Glycogen Storage Disease Type II/genetics/*therapy ; Humans ; Induced Pluripotent Stem Cells ; Infant ; Inheritance Patterns ; Liver/cytology ; Male ; Models, Genetic ; Mutation ; Primary Cell Culture ; },
abstract = {Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.},
}
@article {pmid33293518,
year = {2020},
author = {Fujihara, K and Yamada, K and Ichitani, Y and Kakizaki, T and Jiang, W and Miyata, S and Suto, T and Kato, D and Saito, S and Watanabe, M and Kajita, Y and Ohshiro, T and Mushiake, H and Miyasaka, Y and Mashimo, T and Yasuda, H and Yanagawa, Y},
title = {CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.},
journal = {Translational psychiatry},
volume = {10},
number = {1},
pages = {426},
pmid = {33293518},
issn = {2158-3188},
support = {JP17K17628//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP15H05879//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP26290002//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP15H05872//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP16H06276//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP17H05550//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP19K06881//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; JP19dm0207001//Japan Agency for Medical Research and Development (AMED)/International ; },
mesh = {Adult ; Animals ; Brain/metabolism ; CRISPR-Cas Systems ; Glutamate Decarboxylase/genetics/metabolism ; Hippocampus/metabolism ; Humans ; Rats ; *Schizophrenia/genetics ; },
abstract = {GABAergic dysfunctions have been implicated in the pathogenesis of schizophrenia, especially the associated cognitive impairments. The GABA synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) encoded by the GAD1 gene is downregulated in the brains of patients with schizophrenia. Furthermore, a patient with schizophrenia harboring a homozygous mutation of GAD1 has recently been discovered. However, it remains unclear whether loss of function of GAD1 leads to the symptoms observed in schizophrenia, including cognitive impairment. One of the obstacles faced in experimental studies to address this issue is the perinatal lethality of Gad1 knockout (KO) mice, which precluded characterization at the adult stage. In the present study, we successfully generated Gad1 KO rats using CRISPR/Cas9 genome editing technology. Surprisingly, 33% of Gad1 KO rats survived to adulthood and could be subjected to further characterization. The GABA concentration in the Gad1 KO cerebrum was reduced to ~52% of the level in wild-type rats. Gad1 KO rats exhibited impairments in both spatial reference and working memory without affecting adult neurogenesis in the hippocampus. In addition, Gad1 KO rats showed a wide range of behavioral alterations, such as enhanced sensitivity to an NMDA receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty. Taken together, the results suggest that Gad1 KO rats could provide a novel model covering not only cognitive deficits but also other aspects of the disorder. Furthermore, the present study teaches an important lesson: differences between species should be considered when developing animal models of human diseases.},
}
@article {pmid33293370,
year = {2021},
author = {Park, H and Kim, H and Hassebroek, V and Azuma, Y and Slawson, C and Azuma, M},
title = {Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100164},
pmid = {33293370},
issn = {1083-351X},
support = {P30 CA168524/CA/NCI NIH HHS/United States ; R03 CA223949/CA/NCI NIH HHS/United States ; },
mesh = {Alanine/metabolism ; Amino Acid Substitution ; Anaphase ; *Aneuploidy ; Aspartic Acid/metabolism ; Aurora Kinase B/*genetics/metabolism ; Bone Neoplasms/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosome Segregation ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Humans ; Metaphase ; Models, Biological ; Mutation ; Oncogene Proteins, Fusion/*genetics/metabolism ; Phosphorylation ; Prophase ; *Protein Processing, Post-Translational ; Recombinant Proteins/genetics/metabolism ; Sarcoma, Ewing/genetics/metabolism/pathology ; Signal Transduction ; Threonine/*metabolism ; Transgenes ; },
abstract = {Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.},
}
@article {pmid33292386,
year = {2020},
author = {Hoser, SM and Hoffmann, A and Meindl, A and Gamper, M and Fallmann, J and Bernhart, SH and Müller, L and Ploner, M and Misslinger, M and Kremser, L and Lindner, H and Geley, S and Schaal, H and Stadler, PF and Huettenhofer, A},
title = {Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {299},
pmid = {33292386},
issn = {1474-760X},
mesh = {Adaptor Proteins, Signal Transducing/genetics ; *Alternative Splicing ; CRISPR-Cas Systems ; DNA Mutational Analysis ; DNA-Binding Proteins/genetics ; Exons ; Humans ; *Introns ; Mitochondria/*genetics ; RNA Splice Sites ; RNA Splicing ; RNA, Messenger ; RNA, Transfer/genetics/*metabolism ; RNA-Binding Proteins/genetics ; },
abstract = {BACKGROUND: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive.<br><br>RESULTS: Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNA[Lys] within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3'-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNA[Tyr] by an electrophoretic mobility shift assay.<br><br>CONCLUSIONS: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.},
}
@article {pmid33292120,
year = {2021},
author = {Holl, NJ and Lee, HJ and Huang, YW},
title = {Evolutionary Timeline of Genetic Delivery and Gene Therapy.},
journal = {Current gene therapy},
volume = {21},
number = {2},
pages = {89-111},
doi = {10.2174/1566523220666201208092517},
pmid = {33292120},
issn = {1875-5631},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; *Gene Transfer Techniques ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy/*trends ; Humans ; },
abstract = {There are more than 3,500 genes that are being linked to hereditary diseases or correlated with an elevated risk of certain illnesses. As an alternative to conventional treatments with small molecule drugs, gene therapy has arisen as an effective treatment with the potential to not just alleviate disease conditions but also cure them completely. In order for these treatment regimens to work, genes or editing tools intended to correct diseased genetic material must be efficiently delivered to target sites. There have been many techniques developed to achieve such a goal. In this article, we systematically review a variety of gene delivery and therapy methods that include physical methods, chemical and biochemical methods, viral methods, and genome editing. We discuss their historical discovery, mechanisms, advantages, limitations, safety, and perspectives.},
}
@article {pmid33291479,
year = {2020},
author = {Zeng, F and Beck, V and Schuierer, S and Garnier, I and Manneville, C and Agarinis, C and Morelli, L and Quinn, L and Knehr, J and Roma, G and Bassilana, F and Nash, M},
title = {A Simple and Efficient CRISPR Technique for Protein Tagging.},
journal = {Cells},
volume = {9},
number = {12},
pages = {},
pmid = {33291479},
issn = {2073-4409},
mesh = {A549 Cells ; Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; DNA/*genetics ; *DNA End-Joining Repair ; Electroporation ; Fibroblasts/metabolism ; Genome, Human ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism ; Humans ; RNA, Guide/metabolism ; Rats ; Skin/metabolism ; },
abstract = {Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.},
}
@article {pmid33291011,
year = {2020},
author = {Ovchinnikov, DA},
title = {Genetically-modified cell lines: categorisation and considerations for characterisation.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102103},
doi = {10.1016/j.scr.2020.102103},
pmid = {33291011},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Endonucleases/genetics ; Genome, Human ; Humans ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Stem Cell Research is pleased to introduce into its publication portfolio a new article type: a template-driven short report on the generation of a novel Genetically Modified Cell Line. This resource type is typically derived from human pluripotent stem cell lines via the introduction of nucleases and/or foreign genetic material leading to stable genomic alterations, maintained in a single cell-derived clonal cell line. Interest in, and demand for, genetically modified cell lines has grown exponentially in the last few years. This overview provides a brief introduction to this incredibly versatile lab resource and marks the beginning of a new and exciting addition to the publication portfolio of Stem Cell Research. A dramatic increase in the accessibility of the human genome in the last decade has given a long-anticipated boost to advanced biomedical studies in human in vitro systems. Pluripotent stem cells represent a particularly attractive gateway into this line of experimentation due to their unique suitability for the isolation of clonal genetically modified cell lines (GMCLs), and the ability to be differentiated into essentially any cell type upon the lines' virtually limitless expansion.},
}
@article {pmid33290744,
year = {2020},
author = {Songailiene, I and Juozapaitis, J and Tamulaitiene, G and Ruksenaite, A and Šulčius, S and Sasnauskas, G and Venclovas, &#x10c; and Siksnys, V},
title = {HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.},
journal = {Molecular cell},
volume = {80},
number = {6},
pages = {955-970.e7},
doi = {10.1016/j.molcel.2020.11.034},
pmid = {33290744},
issn = {1097-4164},
mesh = {Adenosine Monophosphate/genetics ; Antidotes/chemistry ; Antitoxins/*genetics/metabolism ; Aphanizomenon/chemistry/genetics ; Bacterial Toxins/*genetics ; CRISPR-Cas Systems/genetics ; Nucleotidyltransferases/genetics/metabolism ; Ribonucleases/*genetics/metabolism ; Toxin-Antitoxin Systems/*genetics ; Tyrosine/genetics ; },
abstract = {Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.},
}
@article {pmid33290505,
year = {2021},
author = {Mitrofanov, A and Alkhnbashi, OS and Shmakov, SA and Makarova, KS and Koonin, EV and Backofen, R},
title = {CRISPRidentify: identification of CRISPR arrays using machine learning approach.},
journal = {Nucleic acids research},
volume = {49},
number = {4},
pages = {e20},
pmid = {33290505},
issn = {1362-4962},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Archaeal ; Genome, Bacterial ; *Machine Learning ; *Software ; },
abstract = {CRISPR-Cas are adaptive immune systems that degrade foreign genetic elements in archaea and bacteria. In carrying out their immune functions, CRISPR-Cas systems heavily rely on RNA components. These CRISPR (cr) RNAs are repeat-spacer units that are produced by processing of pre-crRNA, the transcript of CRISPR arrays, and guide Cas protein(s) to the cognate invading nucleic acids, enabling their destruction. Several bioinformatics tools have been developed to detect CRISPR arrays based solely on DNA sequences, but all these tools employ the same strategy of looking for repetitive patterns, which might correspond to CRISPR array repeats. The identified patterns are evaluated using a fixed, built-in scoring function, and arrays exceeding a cut-off value are reported. Here, we instead introduce a data-driven approach that uses machine learning to detect and differentiate true CRISPR arrays from false ones based on several features. Our CRISPR detection tool, CRISPRidentify, performs three steps: detection, feature extraction and classification based on manually curated sets of positive and negative examples of CRISPR arrays. The identified CRISPR arrays are then reported to the user accompanied by detailed annotation. We demonstrate that our approach identifies not only previously detected CRISPR arrays, but also CRISPR array candidates not detected by other tools. Compared to other methods, our tool has a drastically reduced false positive rate. In contrast to the existing tools, our approach not only provides the user with the basic statistics on the identified CRISPR arrays but also produces a certainty score as a practical measure of the likelihood that a given genomic region is a CRISPR array.},
}
@article {pmid33289517,
year = {2021},
author = {Lau, CH and Bolt, EL},
title = {Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9.},
journal = {Bioscience reports},
volume = {41},
number = {1},
pages = {},
pmid = {33289517},
issn = {1573-4935},
support = {BB/M020541/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T006625/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; DNA/*metabolism ; *DNA Transposable Elements ; Escherichia coli/genetics ; In Vitro Techniques ; Methanosarcina/genetics ; *R-Loop Structures ; Substrate Specificity ; },
abstract = {CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1-Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase-Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase-dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.},
}
@article {pmid33289251,
year = {2020},
author = {Schneider, A},
title = {A short history of guide RNAs: The intricate path that led to the discovery of a basic biological concept.},
journal = {EMBO reports},
volume = {21},
number = {12},
pages = {e51918},
pmid = {33289251},
issn = {1469-3178},
mesh = {History, 20th Century ; Homeostasis ; *Nobel Prize ; *RNA, Guide/genetics ; },
abstract = {This year's Nobel prize for the CRISPR/Cas system is an illustrative example of how scientific breakthroughs rests on preceding work: the discovery of guide RNAs in the 1990s.},
}
@article {pmid33288962,
year = {2021},
author = {Kling, J},
title = {CRISPR gets crunchy.},
journal = {Lab animal},
volume = {50},
number = {1},
pages = {9-11},
pmid = {33288962},
issn = {1548-4475},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
}
@article {pmid33288960,
year = {2021},
author = {Li, S and Li, X and Xue, W and Zhang, L and Yang, LZ and Cao, SM and Lei, YN and Liu, CX and Guo, SK and Shan, L and Wu, M and Tao, X and Zhang, JL and Gao, X and Zhang, J and Wei, J and Li, J and Yang, L and Chen, LL},
title = {Screening for functional circular RNAs using the CRISPR-Cas13 system.},
journal = {Nature methods},
volume = {18},
number = {1},
pages = {51-59},
pmid = {33288960},
issn = {1548-7105},
support = {55008728/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Alternative Splicing ; Animals ; Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; Colonic Neoplasms/genetics/metabolism/*pathology ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; RNA, Circular/*genetics ; RNA, Messenger/genetics/*metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR-RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d-BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.},
}
@article {pmid33288953,
year = {2021},
author = {Bao, XR and Pan, Y and Lee, CM and Davis, TH and Bao, G},
title = {Tools for experimental and computational analyses of off-target editing by programmable nucleases.},
journal = {Nature protocols},
volume = {16},
number = {1},
pages = {10-26},
pmid = {33288953},
issn = {1750-2799},
support = {R01 HL152314/HL/NHLBI NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Gene Editing/*methods ; Genomics/methods ; High-Throughput Nucleotide Sequencing/methods ; Humans ; },
abstract = {Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms; performing analysis using the particular genome of the cells in question rather than the reference genome; and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification.},
}
@article {pmid33287895,
year = {2020},
author = {Li, ZQ and Li, CH},
title = {CRISPR/Cas9 from bench to bedside: what clinicians need to know before application?.},
journal = {Military Medical Research},
volume = {7},
number = {1},
pages = {61},
pmid = {33287895},
issn = {2054-9369},
mesh = {*CRISPR-Cas Systems ; Humans ; Point-of-Care Systems/*trends ; },
abstract = {In October 2020, Dr. Emmanuelle Charpentier and Dr. Jennifer Doudna won the Nobel Prize in Chemistry for their pioneering work in precise genome editing using the CRISPR technology. Although CRISPR technology has developed rapidly in the last decade, there are still many uncertainties before eventual use in clinical settings. In this mini review, we summarize the current efforts in addressing the limitations of CRISPR technology and future directions.},
}
@article {pmid33287207,
year = {2020},
author = {Shaidullina, E and Shelenkov, A and Yanushevich, Y and Mikhaylova, Y and Shagin, D and Alexandrova, I and Ershova, O and Akimkin, V and Kozlov, R and Edelstein, M},
title = {Antimicrobial Resistance and Genomic Characterization of OXA-48- and CTX-M-15-Co-Producing Hypervirulent Klebsiella pneumoniae ST23 Recovered from Nosocomial Outbreak.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {9},
number = {12},
pages = {},
pmid = {33287207},
issn = {2079-6382},
abstract = {Multidrug resistance (MDR) and hypervirulence (hv) have been long considered distinct evolutionary traits for Klebsiella pneumoniae (Kp), a versatile human pathogen. The recent emergence of Kp strains combining these traits poses a serious global threat. In this article, we describe the phenotypic and genomic characteristics of an MDR hvKp isolate, MAR14-456, representative of a nosocomial outbreak in Moscow, Russia, that was recovered from a postoperative wound in a patient who later developed multiple abscesses, fatal sepsis, and septic shock. Broth microdilution testing revealed decreased susceptibility of MAR14-456 to carbapenems (MICs 0.5-2 mg/L) and a high-level resistance to most β-lactams, β-lactam-β-lactamase-inhibitor combinations, and non-β-lactam antibiotics, except ceftazidime-avibactam, amikacin, tigecycline, and colistin. Whole-genome sequencing using Illumina MiSeq and ONT MinION systems allowed to identify and completely assemble two conjugative resistance plasmids, a typical 'European' epidemic IncL/M plasmid that carries the gene of OXA-48 carbapenemase, and an IncFIIK plasmid that carries the gene of CTX-M-15 ESBL and other resistance genes. MLST profile, capsular, lipopolysaccharide, virulence genes encoded on chromosome and IncHI1B/FIB plasmid, and the presence of apparently functional type I-E* CRISPR-Cas system were all characteristic of hvKp ST23, serotype K1-O1v2. Phylogenetic analysis showed the closest relatedness of MAR14-456 to ST23 isolates from China. This report highlights the threat of multiple resistance acquisition by hvKp strain and its spread as a nosocomial pathogen.},
}
@article {pmid33285253,
year = {2021},
author = {Chen, G and Zhou, Y and Kishchenko, O and Stepanenko, A and Jatayev, S and Zhang, D and Borisjuk, N},
title = {Gene editing to facilitate hybrid crop production.},
journal = {Biotechnology advances},
volume = {46},
number = {},
pages = {107676},
doi = {10.1016/j.biotechadv.2020.107676},
pmid = {33285253},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crop Production ; Crops, Agricultural/genetics ; *Gene Editing ; },
abstract = {Capturing heterosis (hybrid vigor) is a promising way to increase productivity in many crops; hybrid crops often have superior yields, disease resistance, and stress tolerance compared with their parental inbred lines. The full utilization of heterosis faces a number of technical problems related to the specifics of crop reproductive biology, such as difficulties with generating and maintaining male-sterile lines and the low efficiency of natural cross-pollination for some genetic combinations. Innovative technologies, such as development of artificial in vitro systems for hybrid production and apomixis-based systems for maintenance of the resulting heterotic progeny, may substantially facilitate the production of hybrids. Genome editing using specifically targeted nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (CRISPR/Cas9) systems, which recognize targets by RNA:DNA complementarity, has recently become an integral part of research and development in life science. In this review, we summarize the progress of genome editing technologies for facilitating the generation of mutant male sterile lines, applications of haploids for hybrid production, and the use of apomixis for the clonal propagation of elite hybrid lines.},
}
@article {pmid33285226,
year = {2021},
author = {Dey, A},
title = {CRISPR/Cas genome editing to optimize pharmacologically active plant natural products.},
journal = {Pharmacological research},
volume = {164},
number = {},
pages = {105359},
doi = {10.1016/j.phrs.2020.105359},
pmid = {33285226},
issn = {1096-1186},
mesh = {*Biological Products ; *CRISPR-Cas Systems ; *Gene Editing/ethics/legislation &amp; jurisprudence ; Government Regulation ; Metabolic Engineering ; *Plant Preparations ; Plants/genetics/metabolism ; },
abstract = {Since time immemorial, human use medicinal plants as sources of food, therapy and industrial purpose. Classical biotechnology and recent next-generation sequencing (NGS) techniques have been successfully used to optimize plant-derived natural-products of biomedical significance. Earlier, protein based editing tools viz. zinc-finger nucleases (ZFNs) and transcription activator-like endonucleases (TALENs) have been popularized for transcriptional level genome manipulation. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is an efficient, robust and selective site-directed mutagenesis strategy for RNA-guided genome-editing. CRISPR/Cas9 genome-editing tool employs designed guide-RNAs that identifies a 3 base-pair protospacer adjacent motif (PAM) sequence occurring downstream of the target-DNA. The present review comprehensively complies the recent literature (2010-2020) retrieved from scientific-databases on the application of CRISPR/Cas9-editing-tools as potent genome-editing strategies in medicinal-plants discussing the recent developments, challenges and future-perspectives with notes on broader applicability of the technique in plants and lower-organisms. In plants, CRISPR/Cas-editing has been implemented successfully in relation to crop-yield and stress-tolerance. However, very few medicinal plants have been edited using CRISPR/Cas genome tool owing to the lack of whole-genome and mRNA-sequences and shortfall of suitable transformation and regeneration strategies. However, recently a number of plant secondary metabolic-pathways (viz. alkaloid, terpenoid, flavonoids, phenolic, saponin etc.) have been engineered employing CRISPR/Cas-editing via knock-out, knock-in, point-mutation, fine-tuning of gene-expression and targeted-mutagenesis. This genome-editing tool further extends its applicability incorporating the tools of synthetic- and systems-biology, functional-genomics and NGS to produce genetically-engineered medicinal-crops with advanced-traits facilitating the production of pharmaceuticals and nutraceuticals.},
}
@article {pmid33283989,
year = {2021},
author = {Frangoul, H and Altshuler, D and Cappellini, MD and Chen, YS and Domm, J and Eustace, BK and Foell, J and de la Fuente, J and Grupp, S and Handgretinger, R and Ho, TW and Kattamis, A and Kernytsky, A and Lekstrom-Himes, J and Li, AM and Locatelli, F and Mapara, MY and de Montalembert, M and Rondelli, D and Sharma, A and Sheth, S and Soni, S and Steinberg, MH and Wall, D and Yen, A and Corbacioglu, S},
title = {CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.},
journal = {The New England journal of medicine},
volume = {384},
number = {3},
pages = {252-260},
doi = {10.1056/NEJMoa2031054},
pmid = {33283989},
issn = {1533-4406},
mesh = {Adult ; Anemia, Sickle Cell/genetics/*therapy ; *CRISPR-Cas Systems ; Female ; Fetal Hemoglobin/*biosynthesis/genetics ; Gene Editing/*methods ; *Genetic Therapy ; Humans ; Repressor Proteins/*genetics/metabolism ; Young Adult ; beta-Thalassemia/genetics/*therapy ; },
abstract = {Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).},
}
@article {pmid33283887,
year = {2021},
author = {Navarro, IC and Tuorto, F and Jordan, D and Legrand, C and Price, J and Braukmann, F and Hendrick, AG and Akay, A and Kotter, A and Helm, M and Lyko, F and Miska, EA},
title = {Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.},
journal = {The EMBO journal},
volume = {40},
number = {6},
pages = {e105496},
pmid = {33283887},
issn = {1460-2075},
support = {092096/Z/10/Z/WT_/Wellcome Trust/United Kingdom ; C6946/A24843/CRUK_/Cancer Research UK/United Kingdom ; 203144/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; C13474/A27826/CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 104640/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; 11832/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {5-Methylcytosine/*metabolism ; Adaptation, Physiological/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/*genetics/physiology ; Cytosine/chemistry ; Gene Editing ; Heat-Shock Response/*genetics ; Hot Temperature ; Leucine/chemistry ; Methyltransferases/metabolism ; Mitochondria/genetics/metabolism ; Proline/chemistry ; Protein Biosynthesis/genetics/physiology ; RNA/chemistry/genetics ; RNA Processing, Post-Transcriptional/*genetics ; Ribosomes/metabolism ; },
abstract = {Methylation of carbon-5 of cytosines (m[5] C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m[5] C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m[5] C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m[5] C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m[5] C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m[5] C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m[5] C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m[5] C tRNA wobble methylation in the adaptation to higher temperatures.},
}
@article {pmid33283798,
year = {2021},
author = {Bukasov, R and Dossym, D and Filchakova, O},
title = {Detection of RNA viruses from influenza and HIV to Ebola and SARS-CoV-2: a review.},
journal = {Analytical methods : advancing methods and applications},
volume = {13},
number = {1},
pages = {34-55},
doi = {10.1039/d0ay01886d},
pmid = {33283798},
issn = {1759-9679},
mesh = {Antibodies, Viral/analysis ; Chemistry Techniques, Analytical/*methods ; Data Accuracy ; Humans ; Immunoassay/*methods ; Limit of Detection ; RNA Viruses/*isolation &amp; purification ; SARS-CoV-2/isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {RNA-based viruses likely make up the highest pandemic threat among all known pathogens in about the last 100 years, since the Spanish Flu of 1918 with 50 M deaths up to COVID-19. Nowadays, an efficient and affordable testing strategy for such viruses have become the paramount target for the fields of virology and bioanalytical chemistry. The detection of the viruses (influenza, hepatitis, HIV, Zika, SARS, Ebola, SARS-CoV-2, etc.) and human antibodies to these viruses is described and tabulated in terms of the reported methods of detection, time to results, accuracy and specificity, if they are reported. The review is focused, but not limited to publications in the last decade. Finally, the limits of detection for each representative publication are tabulated by detection methods and discussed. These methods include PCR, lateral flow immunoassays, LAMP-based methods, ELISA, electrochemical methods (e.g., amperometry, voltammetry), fluorescence spectroscopy, AFM, SPR and SERS spectroscopy, silver staining and CRISPR-Cas based methods, bio-barcode detection, and resonance light scattering. The review is likely to be interesting for various scientists, and particularly helpful with information for establishing interdisciplinary research.},
}
@article {pmid33283789,
year = {2020},
author = {Louradour, I and Ghosh, K and Inbar, E and Sacks, DL and Aluvihare, C and Harrell, RA},
title = {Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {165},
pages = {},
doi = {10.3791/61924},
pmid = {33283789},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Embryo, Nonmammalian/*metabolism ; Female ; Male ; Mice ; *Microinjections ; Microtechnology ; Mutagenesis/*genetics ; Mutation/genetics ; Needles ; Phlebotomus/*embryology/genetics/immunology/parasitology ; },
abstract = {Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.},
}
@article {pmid33283409,
year = {2021},
author = {Kim, HM and Lee, SH and Lim, J and Yoo, J and Hwang, DY},
title = {The epidermal growth factor receptor variant type III mutation frequently found in gliomas induces astrogenesis in human cerebral organoids.},
journal = {Cell proliferation},
volume = {54},
number = {2},
pages = {e12965},
pmid = {33283409},
issn = {1365-2184},
mesh = {Apoptosis/drug effects ; Astrocytes/*cytology/metabolism ; Brain/cytology/drug effects/metabolism/pathology ; Brain Neoplasms/genetics/*pathology ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Cell Proliferation ; ErbB Receptors/genetics/*metabolism ; Gene Editing ; Glioblastoma/genetics/*pathology ; Human Embryonic Stem Cells/cytology/metabolism ; Humans ; Karyotype ; Models, Biological ; Mutation ; Organoids/cytology/drug effects/metabolism/*pathology ; Receptor, ErbB-3 ; Temozolomide/pharmacology ; },
abstract = {OBJECTIVES: The epidermal growth factor receptor variant type III (EGFRvIII) is the most common mutation of EGFR in glioblastoma multiforme (GBM) and is found in approximately 25% of all GBMs. Intriguingly, EGFRvIII is mostly found in GFAP[+] astrocytic tumour cells in the brain, suggesting connection of EGFRvIII to astrogenesis. In this study, we explored whether EGFRvIII mutation facilitates astrogenesis in human development setting.<br><br>MATERIALS AND METHODS: Using CRISPR-Cas9, we generated EGFRvIII mutations in H9-hESCs. Wild type (wt) H9-hESCs were used as an isogenic control. Next, we generated cerebral organoids using the wt and EGFRvIII-hESCs and examined the astrogenic differentiation of the brain organoids.<br><br>RESULTS: EGFRvIII-organoids showed abundant astrocytes (GFAP[+] , S100&#x3b2;[+]), while no astrocytes were detected in wt hESC-derived organoids at day 49. On the contrary, TUJ1[+] neurons were more abundant in the wt-organoids than the EGFRvIII-organoids. This result suggested that constitutively active EGFRvIII promoted astrogenesis at the expense of neurogenesis. In addition, the EGFRvIII-organoids were larger in size and retained more Ki67[+] cells than wt-organoids, indicating enhanced cell proliferation by the mutation. The EGFRvIII-organoids displayed massive apoptotic cell death after treatment with temozolomide and hence, could be used for evaluation of anti-GBM drugs.<br><br>CONCLUSIONS: EGFRvIII mutation-induced astrogenesis and massive cell proliferation in a human brain development model. These results provide us new insights into the mechanisms relating EGFRvIII mutation-mediated gliogenesis and gliomagenesis.},
}
@article {pmid33283228,
year = {2021},
author = {Cipullo, M and Pearce, SF and Lopez Sanchez, IG and Gopalakrishna, S and Krüger, A and Schober, F and Busch, JD and Li, X and Wredenberg, A and Atanassov, I and Rorbach, J},
title = {Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.},
journal = {Nucleic acids research},
volume = {49},
number = {1},
pages = {354-370},
pmid = {33283228},
issn = {1362-4962},
mesh = {Bone Neoplasms/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation ; Gene Knockout Techniques ; Guanosine Triphosphate/metabolism ; HEK293 Cells ; Humans ; Mitochondrial Proteins/*metabolism ; Mitochondrial Ribosomes/*metabolism ; Monomeric GTP-Binding Proteins/*physiology ; Osteosarcoma/pathology ; Oxidative Phosphorylation ; Protein Interaction Mapping ; Ribosomal Proteins/*metabolism ; Ribosome Subunits, Large, Eukaryotic/*metabolism ; },
abstract = {Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.},
}
@article {pmid33282821,
year = {2020},
author = {Minarini, LADR and de Andrade, LN and De Gregorio, E and Grosso, F and Naas, T and Zarrilli, R and Camargo, ILBC},
title = {Editorial: Antimicrobial Resistance as a Global Public Health Problem: How Can We Address It?.},
journal = {Frontiers in public health},
volume = {8},
number = {},
pages = {612844},
pmid = {33282821},
issn = {2296-2565},
mesh = {*Anti-Bacterial Agents/therapeutic use ; *Drug Resistance, Bacterial ; Drug Resistance, Multiple, Bacterial ; Microbial Sensitivity Tests ; Public Health ; },
}
@article {pmid33280580,
year = {2020},
author = {Shemyakin, IG and Firstova, VV and Fursova, NK and Abaev, IV and Filippovich, SY and Ignatov, SG and Dyatlov, IA},
title = {Next-Generation Antibiotics, Bacteriophage Endolysins, and Nanomaterials for Combating Pathogens.},
journal = {Biochemistry. Biokhimiia},
volume = {85},
number = {11},
pages = {1374-1388},
doi = {10.1134/S0006297920110085},
pmid = {33280580},
issn = {1608-3040},
mesh = {*Anti-Bacterial Agents/chemistry/therapeutic use ; Bacteria/*growth &amp; development ; Bacterial Infections/*drug therapy/metabolism ; Bacteriophages/*chemistry ; Endopeptidases/chemistry/therapeutic use ; *Nanostructures/chemistry/therapeutic use ; *Viral Proteins/chemistry/therapeutic use ; },
abstract = {This review presents various strategies to fight causative agents of infectious diseases. Species-specific programmable RNA-containing antibiotics open up new possibilities for creating next-generation of personalized drugs based on microbiome editing and can serve as a new tool for selective elimination of pathogenic bacterial species while keeping intact the rest of microbiota. Another promising approach in combating bacterial infections is genome editing using the CRISPR-Cas systems. Expanding knowledge on the molecular mechanisms of innate immunity has been actively used for developing new antimicrobials. However, obvious risks of using antibiotic adjuvants aimed at activation of the host immune system include development of the autoimmune response with subsequent organ damage. To avoid these risks, it is essential to elucidate action mechanisms of the specific ligands and signal molecules used as components of the hybrid antibiotics. Bacteriophage endolysins are also considered as effective antimicrobials against antibiotic-resistant bacteria, metabolically inactive persisters, and microbial biofilms. Despite significant advances in the design of implants with antibacterial properties, the problem of postoperative infections still remains. Different nanomodifications of the implant surface have been designed to reduce bacterial contamination. Here, we review bactericidal, fungicidal, and immunomodulating properties of compounds used for the implant surface nanomodifications, such as silver, boron nitride nanomaterials, nanofibers, and nanogalvanic materials.},
}
@article {pmid33280575,
year = {2020},
author = {Bobrovsky, PA and Moroz, VD and Lavrenova, VN and Manuvera, VA and Lazarev, VN},
title = {Inhibition of Chlamydial Infection by CRISPR/Cas9-SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins Gene Expression in HeLa Cells.},
journal = {Biochemistry. Biokhimiia},
volume = {85},
number = {11},
pages = {1310-1318},
doi = {10.1134/S0006297920110036},
pmid = {33280575},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; *Chlamydia/genetics/metabolism ; *Chlamydia Infections/genetics/metabolism ; *Cytokines/biosynthesis/genetics ; *Gene Editing ; *Gene Expression Regulation ; HeLa Cells ; Humans ; },
abstract = {The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural substances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram-positive and Gram-negative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the components of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genome-editing technology with synergistic activation mediators (CRISPR/Cas9-SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial two-component gene system (ctcB-ctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9-SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Gram-positive and Gram-negative bacteria.},
}
@article {pmid33279608,
year = {2021},
author = {Xie, Z and Sun, R and Qi, C and Jiao, S and Jiang, Y and Liu, Z and Zhao, D and Liu, R and Li, Q and Yang, K and Hu, L and Wang, X and Tang, X and Ouyang, H and Pang, D},
title = {Generation of a pHSPA6 gene-based multifunctional live cell sensor.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1868},
number = {2},
pages = {118919},
doi = {10.1016/j.bbamcr.2020.118919},
pmid = {33279608},
issn = {1879-2596},
mesh = {Animals ; Biosensing Techniques/*methods ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Fluorescent Dyes ; Gene Expression ; Gene Knock-In Techniques/*methods ; Genes, Reporter ; Green Fluorescent Proteins/*genetics ; HSP70 Heat-Shock Proteins/*genetics ; Plasmids/genetics ; Smart Materials ; Swine ; },
abstract = {Biosensors utilizing intact live cells can report responses to certain stimuli rapidly and sensitively and have attracted a great deal of attention. The expression pattern of HSPA6, a little studied HSPA family member, has contributed to the development of multifunctional and intelligent whole-cell sensors. Herein, a new pHSPA6-based EGFP fluorescent reporter cell line was designed and developed via a CRISPR/Cas9-mediated knock-in strategy. The fluorescent reporter cell line has a precise EGFP integration site and gene copy number, and no selectable marker genes were introduced during the selection processes. Stimulation experiments with HSPA6-specific stressors indicated that EGFP fluorescent reporter cells could rapidly and effectively convert stress signals into EGFP fluorescent signals. Furthermore, cell proliferation and gene expression pattern analysis showed that the fluorescent reporter cells grew well and that both the integrated EGFP gene and the pHSPA6 gene were expressed rapidly and sensitively in response to stimulation. This study provides a new strategy for the construction of a cell model for HSPA6 expression/interaction and an intelligent live cell sensor, which can potentially be applied to numerous fields, such as those focusing on cellular models of HSPA6 signaling cascades, biomaterials, food security, environmental assessment, and drug screening.},
}
@article {pmid33279320,
year = {2021},
author = {Liu, Y and Chang, J and Yang, C and Zhang, T and Chen, X and Shi, R and Liang, Y and Xia, Q and Ma, S},
title = {Genome-wide CRISPR-Cas9 screening in Bombyx mori reveals the toxicological mechanisms of environmental pollutants, fluoride and cadmium.},
journal = {Journal of hazardous materials},
volume = {410},
number = {},
pages = {124666},
doi = {10.1016/j.jhazmat.2020.124666},
pmid = {33279320},
issn = {1873-3336},
mesh = {Animals ; *Bombyx/genetics ; CRISPR-Cas Systems ; Cadmium/toxicity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Ecosystem ; *Environmental Pollutants/toxicity ; Fluorides/toxicity ; Humans ; },
abstract = {Fluoride and cadmium, two typical environmental pollutants, have been extensively existed in the ecosystem and severely injured various organisms including humans. To explore the toxicological properties and the toxicological mechanism of fluoride and cadmium in silkworm, we perform a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -based functional genomic screen, which can directly measure the genetic requirement of genes in response to the pollutants. Our screen identifies 751 NaF-resistance genes, 753 NaF-sensitive genes, 757 CdCl2-resistance genes, and 725 CdCl2-sensitive genes. The top-ranked resistant genes are experimentally verified and the results show that their loss conferred resistance to fluoride or cadmium. Functional analysis of the resistant- and sensitive-genes demonstrates enrichment of multiple signaling pathways, among which the MAPK signaling pathway and DNA damage and repair are both required for fluoride- or cadmium-induced cell death, whereas the Toll and Imd signaling pathway and Autophagy are fluoride- or cadmium-specific. Moreover, we confirm that these pathways are truly involved in the toxicological mechanism in both cultured cells and individual tissues. Our results supply potential targets for rescuing the biohazards of fluoride and cadmium in silkworm, and reveal the feasible toxicological mechanism, which highlights the role of functional genomic screens in elucidating the toxicity mechanisms of environmental pollutants.},
}
@article {pmid33277595,
year = {2020},
author = {Stratman, AN and Burns, MC and Farrelly, OM and Davis, AE and Li, W and Pham, VN and Castranova, D and Yano, JJ and Goddard, LM and Nguyen, O and Galanternik, MV and Bolan, TJ and Kahn, ML and Mukouyama, YS and Weinstein, BM},
title = {Chemokine mediated signalling within arteries promotes vascular smooth muscle cell recruitment.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {734},
pmid = {33277595},
issn = {2399-3642},
support = {ZIA HD001011/ImNIH/Intramural NIH HHS/United States ; ZIA HD008915/ImNIH/Intramural NIH HHS/United States ; R00 HL125683/HL/NHLBI NIH HHS/United States ; ZIA HL005702/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Chemokines/*metabolism ; Mice ; Muscle, Smooth, Vascular/*cytology ; Mutation ; Myocytes, Smooth Muscle ; Receptors, CXCR4/genetics/metabolism ; Signal Transduction ; Zebrafish ; },
abstract = {The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.},
}
@article {pmid33277326,
year = {2020},
author = {Mukherjee, A},
title = {Mutagenomics: The Future of Genetic Screens.},
journal = {Plant physiology},
volume = {184},
number = {4},
pages = {1616-1617},
pmid = {33277326},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; *Genetic Testing ; Mutation ; },
}
@article {pmid33277045,
year = {2021},
author = {de Miguel Beriain, &#xcd;},
title = {The Geneva Statement on Heritable Human Genome Editing: A Criticism.},
journal = {Trends in biotechnology},
volume = {39},
number = {3},
pages = {219-220},
doi = {10.1016/j.tibtech.2020.11.004},
pmid = {33277045},
issn = {1879-3096},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human ; Humans ; },
}
@article {pmid33277043,
year = {2021},
author = {Zeballos C, MA and Gaj, T},
title = {Next-Generation CRISPR Technologies and Their Applications in Gene and Cell Therapy.},
journal = {Trends in biotechnology},
volume = {39},
number = {7},
pages = {692-705},
pmid = {33277043},
issn = {1879-3096},
support = {T32 EB019944/EB/NIBIB NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/trends ; Endonucleases/genetics ; *Gene Editing ; Humans ; },
abstract = {The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) nucleases has transformed biotechnology by providing an easy, efficient, and versatile platform for editing DNA. However, traditional CRISPR-based technologies initiate editing by activating DNA double-strand break (DSB) repair pathways, which can cause adverse effects in cells and restrict certain therapeutic applications of the technology. To this end, several new CRISPR-based modalities have been developed that are capable of catalyzing editing without the requirement for a DSB. Here, we review three of these technologies: base editors, prime editors, and RNA-targeting CRISPR-associated protein (Cas)13 effectors. We discuss their strengths compared to traditional gene-modifying systems, we highlight their emerging therapeutic applications, and we examine challenges facing their safe and effective clinical implementation.},
}
@article {pmid33276371,
year = {2021},
author = {Koh, A and Tao, S and Goh, YJ and Chaganty, V and See, K and Purushothaman, K and Orbán, L and Mathuru, AS and Wohland, T and Winkler, C},
title = {A Neurexin2aa deficiency results in axon pathfinding defects and increased anxiety in zebrafish.},
journal = {Human molecular genetics},
volume = {29},
number = {23},
pages = {3765-3780},
doi = {10.1093/hmg/ddaa260},
pmid = {33276371},
issn = {1460-2083},
mesh = {Animals ; Anxiety/etiology/metabolism/*pathology ; *Axon Guidance ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; Motor Neurons/metabolism/*pathology ; Nerve Tissue Proteins/*deficiency/genetics ; *Neurogenesis ; Zebrafish ; Zebrafish Proteins/*deficiency/genetics ; },
abstract = {Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.},
}
@article {pmid33275853,
year = {2020},
author = {Russel, J and Pinilla-Redondo, R and Mayo-Muñoz, D and Shah, SA and Sørensen, SJ},
title = {CRISPRCasTyper: Automated Identification, Annotation, and Classification of CRISPR-Cas Loci.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {462-469},
doi = {10.1089/crispr.2020.0059},
pmid = {33275853},
issn = {2573-1602},
mesh = {Archaea/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*classification/*methods/trends ; Genome, Bacterial/genetics ; Metagenome/genetics ; Phylogeny ; RNA, Guide/genetics ; Software ; },
abstract = {Automated classification of CRISPR-Cas systems has been challenged by their dynamic nature and expanding classification. Here, we developed CRISPRCasTyper, an automated tool with improved capabilities for identifying and typing CRISPR arrays and cas loci based on the latest nomenclature (44 subtypes/variants). As a novel feature, CRISPRCasTyper uses a machine learning approach to subtype CRISPR arrays based on the sequences of the repeats, which allows the typing of orphan and distant arrays. CRISPRCasTyper provides a graphical output, where CRISPRs and cas operons are visualized as gene maps, thus aiding annotation of partial and novel systems through synteny. CRISPRCasTyper was benchmarked against a manually curated set of 31 subtypes with a median accuracy of 98.6% and used to explore CRISPR-Cas diversity across >3,000 metagenomes. Altogether, we present an up-to-date software for improved automated prediction of CRISPR-Cas loci. CRISPRCasTyper is available through conda and as a web server (cctyper.crispr.dk).},
}
@article {pmid33275752,
year = {2020},
author = {Basar, R and Daher, M and Rezvani, K},
title = {Next-generation cell therapies: the emerging role of CAR-NK cells.},
journal = {Hematology. American Society of Hematology. Education Program},
volume = {2020},
number = {1},
pages = {570-578},
pmid = {33275752},
issn = {1520-4383},
mesh = {*CRISPR-Cas Systems ; *Cell- and Tissue-Based Therapy ; Female ; *Gene Editing ; Humans ; Killer Cells, Natural/*transplantation ; Lymphoma, Follicular/*therapy ; Middle Aged ; *Receptors, Chimeric Antigen/genetics/therapeutic use ; },
abstract = {T cells engineered with chimeric antigen receptors (CARs) have revolutionized the field of cell therapy and changed the paradigm of treatment for many patients with relapsed or refractory B-cell malignancies. Despite this progress, there are limitations to CAR-T cell therapy in both the autologous and allogeneic settings, including practical, logistical, and toxicity issues. Given these concerns, there is a rapidly growing interest in natural killer cells as alternative vehicles for CAR engineering, given their unique biological features and their established safety profile in the allogeneic setting. Other immune effector cells, such as invariant natural killer T cells, γδ T cells, and macrophages, are attracting interest as well and eventually may be added to the repertoire of engineered cell therapies against cancer. The pace of these developments will undoubtedly benefit from multiple innovative technologies, such as the CRISPR-Cas gene editing system, which offers great potential to enhance the natural ability of immune effector cells to eliminate refractory cancers.},
}
@article {pmid33274276,
year = {2020},
author = {Rauch, S and Jones, KA and Dickinson, BC},
title = {Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation.},
journal = {ACS central science},
volume = {6},
number = {11},
pages = {1987-1996},
pmid = {33274276},
issn = {2374-7943},
support = {R01 MH122142/MH/NIMH NIH HHS/United States ; R35 GM119840/GM/NIGMS NIH HHS/United States ; },
abstract = {All aspects of mRNA lifetime and function, including its stability, translation into protein, and trafficking through the cell, are tightly regulated through coordinated post-transcriptional modifications and interactions with a multitude of RNA effector proteins. Despite the increasing recognition of RNA regulation as a critical layer of mammalian gene expression control and its increasing excitement as a therapeutic target, tools to study and control RNA regulatory mechanisms with temporal precision in their endogenous environment are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS). The CIRTS biosensor platform is based on guide RNA (gRNA)-dependent RNA binding domains that interact with a target transcript using Watson-Crick-Franklin base pair interactions. Addition of a small molecule recruits an RNA effector to the target transcript, thereby eliciting a local effect on the transcript. In this work, we showcase that these CIRTS biosensors can trigger inducible RNA editing, degradation, or translation on target transcripts in a small molecule-dependent manner. We further go on to show that the CIRTS RNA base editor biosensor can induce RNA base editing in a small molecule-controllable manner in vivo. Collectively this work provides a new set of tools to probe the dynamics of RNA regulatory systems and control gene expression at the RNA level.},
}
@article {pmid33273577,
year = {2020},
author = {Terzioglu, M and Saralahti, A and Piippo, H and Rämet, M and Andressoo, JO},
title = {Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {21023},
pmid = {33273577},
issn = {2045-2322},
support = {724922/ERC_/European Research Council/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Follicular Fluid/metabolism ; Gene Editing/*methods ; Hydrogen Sulfide/pharmacology ; *Mutagenesis ; Oocytes/drug effects/metabolism ; Temperature ; Zebrafish/*genetics/growth &amp; development ; Zygote/cytology/*metabolism ; },
abstract = {CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish-extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H2S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 °C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.},
}
@article {pmid33273523,
year = {2020},
author = {Kushwaha, SK and Bhavesh, NLS and Abdella, B and Lahiri, C and Marathe, SA},
title = {The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {21156},
pmid = {33273523},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; Conserved Sequence/genetics ; DNA, Intergenic/genetics ; Evolution, Molecular ; Genetic Loci ; Interspersed Repetitive Sequences/genetics ; Operon/genetics ; *Phylogeny ; Salmonella/*genetics ; },
abstract = {Salmonellae display intricate evolutionary patterns comprising over 2500 serovars having diverse pathogenic profiles. The acquisition and/or exchange of various virulence factors influences the evolutionary framework. To gain insights into evolution of Salmonella in association with the CRISPR-Cas genes we performed phylogenetic surveillance across strains of 22 Salmonella serovars. The strains differed in their CRISPR1-leader and cas operon features assorting into two main clades, CRISPR1-STY/cas-STY and CRISPR1-STM/cas-STM, comprising majorly typhoidal and non-typhoidal Salmonella serovars respectively. Serovars of these two clades displayed better relatedness, concerning CRISPR1-leader and cas operon, across genera than between themselves. This signifies the acquisition of CRISPR1/Cas region could be through a horizontal gene transfer event owing to the presence of mobile genetic elements flanking CRISPR1 array. Comparison of CRISPR and cas phenograms with that of multilocus sequence typing (MLST) suggests differential evolution of CRISPR/Cas system. As opposed to broad-host-range, the host-specific serovars harbor fewer spacers. Mapping of protospacer sources suggested a partial correlation of spacer content with habitat diversity of the serovars. Some serovars like serovar Enteritidis and Typhimurium that inhabit similar environment/infect similar hosts hardly shared their protospacer sources.},
}
@article {pmid33271878,
year = {2020},
author = {Kim, U and Kim, N and Shin, HY},
title = {Modeling Non-Alcoholic Fatty Liver Disease (NAFLD) Using "Good-Fit" Genome-Editing Tools.},
journal = {Cells},
volume = {9},
number = {12},
pages = {},
pmid = {33271878},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Variation/genetics ; Genome/*genetics ; Humans ; Non-alcoholic Fatty Liver Disease/*genetics ; },
abstract = {Non-alcoholic fatty liver disease (NAFLD), which affects both adults and children, is the most common liver disorder worldwide. NAFLD is characterized by excess fat accumulation in the liver in the absence of significant alcohol use. NAFLD is strongly associated with obesity, insulin resistance, metabolic syndrome, as well as specific genetic polymorphisms. Severe NAFLD cases can further progress to cirrhosis, hepatocellular carcinoma (HCC), or cardiovascular complications. Here, we describe the pathophysiological features and critical genetic variants associated with NAFLD. Recent advances in genome-engineering technology have provided a new opportunity to generate in vitro and in vivo models that reflect the genetic abnormalities of NAFLD. We review the currently developed NAFLD models generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. We further discuss unique features of CRISPR/Cas9 and Cas9 variants, including base editors and prime editor, that are useful for replicating genetic features specific to NAFLD. We also compare advantages and limitations of currently available methods for delivering genome-editing tools necessary for optimal genome editing. This review should provide helpful guidance for selecting "good fit" genome-editing tools and appropriate gene-delivery methods for the successful development of NAFLD models and clinical therapeutics.},
}
@article {pmid33271061,
year = {2020},
author = {Petassi, MT and Hsieh, SC and Peters, JE},
title = {Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons.},
journal = {Cell},
volume = {183},
number = {7},
pages = {1757-1771.e18},
pmid = {33271061},
issn = {1097-4172},
support = {R01 GM129118/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; Base Sequence ; CRISPR-Cas Systems/*genetics ; DNA Transposable Elements/*genetics ; Gammaproteobacteria/metabolism ; Phylogeny ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; Transcription Factors/metabolism ; Zygote/metabolism ; },
abstract = {CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways: one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Multiple mechanisms allow functionally distinct guide RNAs for transposition: a conventional system capable of acquiring guide RNAs to new plasmid and phage targets and a second providing long-term memory for access to chromosomal sites upon entry into a new host. Guide RNAs are privatized to be recognized only by the transposon-adapted system via sequence specialization, mismatch tolerance, and selective regulation to avoid toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals promising avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.},
}
@article {pmid33270888,
year = {2021},
author = {Wang, J and Sui, X and Ding, Y and Fu, Y and Feng, X and Liu, M and Zhang, Y and Xian, M and Zhao, G},
title = {A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy.},
journal = {Nucleic acids research},
volume = {49},
number = {2},
pages = {e12},
pmid = {33270888},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Chloramphenicol/pharmacology ; Clone Cells ; Drug Resistance, Microbial/*genetics ; Escherichia coli/drug effects/genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Gene Knockout Techniques ; Genes, Bacterial ; Kanamycin/pharmacology ; Kanamycin Resistance/genetics ; Klebsiella pneumoniae/drug effects/genetics/metabolism ; Lactates/metabolism ; Mutation ; Nucleotide Motifs ; Plasmids/*genetics ; Promoter Regions, Genetic/genetics ; Pyruvic Acid/metabolism ; RNA, Guide/*genetics ; Selection, Genetic ; Tetracycline/pharmacology ; Tetracycline Resistance/genetics ; Time Factors ; Transformation, Bacterial ; },
abstract = {The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.},
}
@article {pmid33270841,
year = {2021},
author = {Soares, F and Chen, B and Lee, JB and Ahmed, M and Ly, D and Tin, E and Kang, H and Zeng, Y and Akhtar, N and Minden, MD and He, HH and Zhang, L},
title = {CRISPR screen identifies genes that sensitize AML cells to double-negative T-cell therapy.},
journal = {Blood},
volume = {137},
number = {16},
pages = {2171-2181},
doi = {10.1182/blood.2019004108},
pmid = {33270841},
issn = {1528-0020},
support = {//CIHR/Canada ; },
mesh = {Adoptive Transfer ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Female ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute/*genetics/*therapy ; Mice, Inbred NOD ; Receptors, IgG/genetics ; T-Lymphocytes/*transplantation ; },
abstract = {Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.},
}
@article {pmid33270810,
year = {2020},
author = {Ogata, T and Ishizaki, T and Fujita, M and Fujita, Y},
title = {CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0243376},
pmid = {33270810},
issn = {1932-6203},
mesh = {Abscisic Acid/*metabolism ; CRISPR-Cas Systems/genetics ; *Droughts ; Frameshift Mutation ; Mutagenesis ; Oryza/*physiology ; Plant Proteins/genetics/*metabolism ; Plant Roots/growth &amp; development ; Plant Stomata/physiology ; Plants, Genetically Modified/physiology ; *Stress, Physiological ; },
abstract = {Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants. Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the β-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.},
}
@article {pmid33270627,
year = {2020},
author = {Navarro-Payá, D and Flis, I and Anderson, MAE and Hawes, P and Li, M and Akbari, OS and Basu, S and Alphey, L},
title = {Targeting female flight for genetic control of mosquitoes.},
journal = {PLoS neglected tropical diseases},
volume = {14},
number = {12},
pages = {e0008876},
pmid = {33270627},
issn = {1935-2735},
support = {BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 200171/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Aedes/*genetics ; Animals ; CRISPR-Cas Systems ; Culex/*genetics/*physiology ; Female ; *Flight, Animal ; Gene Deletion ; Gene Expression Regulation ; Insect Proteins/genetics/*metabolism ; Male ; *Mosquito Control ; Mutation ; },
abstract = {Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.},
}
@article {pmid33270431,
year = {2020},
author = {Moon, J and Kwon, HJ and Yong, D and Lee, IC and Kim, H and Kang, H and Lim, EK and Lee, KS and Jung, J and Park, HG and Kang, T},
title = {Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9.},
journal = {ACS sensors},
volume = {5},
number = {12},
pages = {4017-4026},
pmid = {33270431},
issn = {2379-3694},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Colorimetry ; Drug Resistance, Viral/*genetics ; Humans ; Influenza A Virus, H1N1 Subtype/*genetics/*isolation &amp; purification ; SARS-CoV-2/drug effects/*genetics/*isolation &amp; purification ; },
abstract = {Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3',5,5'-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.},
}
@article {pmid33268788,
year = {2020},
author = {Coradini, ALV and Hull, CB and Ehrenreich, IM},
title = {Building genomes to understand biology.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6177},
pmid = {33268788},
issn = {2041-1723},
support = {R35 GM130381/GM/NIGMS NIH HHS/United States ; T32 GM118289/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chimerism ; Chromosomes, Artificial/*chemistry/metabolism ; DNA/*genetics/metabolism ; Escherichia coli/genetics/metabolism ; Genetic Code ; *Genome ; Genomics/methods/*trends ; Humans ; Plasmids/chemistry/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Synthetic Biology/methods/*trends ; },
abstract = {Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.},
}
@article {pmid33268787,
year = {2020},
author = {Lu, Z and Guo, JK and Wei, Y and Dou, DR and Zarnegar, B and Ma, Q and Li, R and Zhao, Y and Liu, F and Choudhry, H and Khavari, PA and Chang, HY},
title = {Structural modularity of the XIST ribonucleoprotein complex.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6163},
pmid = {33268787},
issn = {2041-1723},
support = {RM1 HG007735/HG/NHGRI NIH HHS/United States ; R01 HG004361/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P50 HG007735/HG/NHGRI NIH HHS/United States ; P30 CA014089/CA/NCI NIH HHS/United States ; R00 HG009662/HG/NHGRI NIH HHS/United States ; },
mesh = {Adenine/*analogs &amp; derivatives/metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Conserved Sequence ; Cross-Linking Reagents ; Female ; Ficusin/chemistry ; Formaldehyde/chemistry ; Gene Knock-In Techniques ; Humans ; K562 Cells ; Male ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Nucleic Acid Conformation ; Pregnancy ; RNA, Long Noncoding/*chemistry/genetics/metabolism ; Ribonucleoproteins/*chemistry/genetics/metabolism ; Sequence Analysis, RNA ; },
abstract = {Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m[6]A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m[6]A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.},
}
@article {pmid33268478,
year = {2020},
author = {Alberti, F and Kaleem, S and Weaver, JA},
title = {Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research.},
journal = {Biology open},
volume = {9},
number = {12},
pages = {},
pmid = {33268478},
issn = {2046-6390},
mesh = {Basidiomycota/*genetics/*metabolism ; Biological Products/chemistry/*metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; Genetic Engineering/methods ; *Genomics/methods ; *Metabolomics/methods ; Research ; Secondary Metabolism ; Structure-Activity Relationship ; },
abstract = {Basidiomycota are a large and diverse phylum of fungi. They can make bioactive metabolites that are used or have inspired the synthesis of antibiotics and agrochemicals. Terpenoids are the most abundant class of natural products encountered in this taxon. Other natural product classes have been described, including polyketides, peptides, and indole alkaloids. The discovery and study of natural products made by basidiomycete fungi has so far been hampered by several factors, which include their slow growth and complex genome architecture. Recent developments of tools for genome and metabolome studies are allowing researchers to more easily tackle the secondary metabolome of basidiomycete fungi. Inexpensive long-read whole-genome sequencing enables the assembly of high-quality genomes, improving the scaffold upon which natural product gene clusters can be predicted. CRISPR/Cas9-based engineering of basidiomycete fungi has been described and will have an important role in linking natural products to their genetic determinants. Platforms for the heterologous expression of basidiomycete genes and gene clusters have been developed, enabling natural product biosynthesis studies. Molecular network analyses and publicly available natural product databases facilitate data dereplication and natural product characterisation. These technological advances combined are prompting a revived interest in natural product discovery from basidiomycete fungi.This article has an associated Future Leader to Watch interview with the first author of the paper.},
}
@article {pmid33267838,
year = {2020},
author = {Ying, X and Jiang, X and Zhang, H and Liu, B and Huang, Y and Zhu, X and Qi, D and Yuan, G and Luo, J and Ji, W},
title = {Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development.},
journal = {Molecular cancer},
volume = {19},
number = {1},
pages = {169},
pmid = {33267838},
issn = {1476-4598},
mesh = {Adenosine/*analogs &amp; derivatives/metabolism ; Antigens, Neoplasm/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics/*pathology ; Cell Adhesion Molecules/*genetics/metabolism ; Cell Line, Tumor ; Humans ; Methyltransferases/*metabolism ; Protein Biosynthesis ; RNA, Messenger/genetics/metabolism ; Urinary Bladder Neoplasms/*genetics/*pathology ; },
abstract = {Accumulating evidence has revealed significant roles for N6-methyladenosine (m 6 A) modification in the development of various cancers. We previously demonstrated an oncogenic role of m 6 A-modified CUB domain containing protein 1 (CDCP1) in bladder cancer (BC) progression. However, the biological functions and underlying molecular mechanisms of engineered programmable m 6 A modification of CDCP1 mRNA in BC remain obscure. Here, we established a targeted m 6 A RNA methylation system by fusing the catalytic domain of methyltransferase like 3 (METTL3CD) to RCas9 as the RNA-targeting module. The constructed RCas9- METTL3 retained methylation activity and mediated efficient site-specific m 6 A installation in the presence of a cognate single guide RNA and short protospacer adjacent motif-containing ssDNA molecule . Subsequently, targeting m 6 A installation onto the 3' untranslated region of CDCP1 promoted CDCP1 mRNA translation and facilitated BC development in vitro and in vivo. Our findings demonstrate that the RCas9-METTL3 system mediates efficient sitespecific m 6 A installation on CDCP1 mRNA and promotes BC development. Thus, the RCas9-METTL3 system provides a new tool for studying m 6 A function and a potential strategy for BC epitranscriptome-modulating therapies.},
}
@article {pmid33266100,
year = {2020},
author = {Welter, H and Herrmann, C and Fröhlich, T and Flenkenthaler, F and Eubler, K and Schorle, H and Nettersheim, D and Mayerhofer, A and Müller-Taubenberger, A},
title = {Filamin A Orchestrates Cytoskeletal Structure, Cell Migration and Stem Cell Characteristics in Human Seminoma TCam-2 Cells.},
journal = {Cells},
volume = {9},
number = {12},
pages = {},
pmid = {33266100},
issn = {2073-4409},
mesh = {ATPases Associated with Diverse Cellular Activities/metabolism ; Actin Cytoskeleton/metabolism ; Actins/metabolism ; Adult ; Aged ; Autoantigens/metabolism ; CRISPR-Cas Systems/physiology ; Cell Line, Tumor ; Cell Movement/*physiology ; Cytoskeletal Proteins/metabolism ; Cytoskeleton/*metabolism ; Filamins/*metabolism ; Humans ; Male ; Middle Aged ; Non-Fibrillar Collagens/metabolism ; RNA-Binding Proteins/metabolism ; Seminoma/*metabolism ; Stem Cells/*metabolism ; Testicular Neoplasms/metabolism ; Testis/metabolism ; Transcription, Genetic/physiology ; },
abstract = {Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.},
}
@article {pmid33264648,
year = {2021},
author = {Kangussu-Marcolino, MM and Morgado, P and Manna, D and Yee, H and Singh, U},
title = {Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept.},
journal = {International journal for parasitology},
volume = {51},
number = {2-3},
pages = {193-200},
pmid = {33264648},
issn = {1879-0135},
support = {K12 GM088033/GM/NIGMS NIH HHS/United States ; R21 AI102277/AI/NIAID NIH HHS/United States ; R21 AI149268/AI/NIAID NIH HHS/United States ; T35 OD010989/OD/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Entamoeba histolytica/genetics ; Gene Editing ; Humans ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.},
}
@article {pmid33264623,
year = {2020},
author = {Black, JB and McCutcheon, SR and Dube, S and Barrera, A and Klann, TS and Rice, GA and Adkar, SS and Soderling, SH and Reddy, TE and Gersbach, CA},
title = {Master Regulators and Cofactors of Human Neuronal Cell Fate Specification Identified by CRISPR Gene Activation Screens.},
journal = {Cell reports},
volume = {33},
number = {9},
pages = {108460},
pmid = {33264623},
issn = {2211-1247},
support = {T32 GM008555/GM/NIGMS NIH HHS/United States ; DP2 OD008586/OD/NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; R01 HG010741/HG/NHGRI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; F31 NS105419/NS/NINDS NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Gene Expression Regulation/*genetics ; Humans ; Neurons/*metabolism ; Transcriptional Activation/*genetics ; },
abstract = {Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators. We utilize deactivated Cas9 (dCas9)-based gene activation to target 1,496 putative transcription factors (TFs) in the human genome. Using a reporter of neuronal commitment, we profile the neurogenic activity of these factors in human pluripotent stem cells (PSCs), leading to a curated set of pro-neuronal factors. Activation of pairs of TFs reveals neuronal cofactors, including E2F7, RUNX3, and LHX8, that improve conversion efficiency, subtype specificity, and maturation of neuronal cell types. Finally, using multiplexed gene regulation with orthogonal CRISPR systems, we demonstrate improved neuronal differentiation with concurrent activation and repression of target genes, underscoring the power of CRISPR-based gene regulation for programming complex cellular phenotypes.},
}
@article {pmid33263998,
year = {2020},
author = {Lee, M and Woo, HM},
title = {A Logic NAND Gate for Controlling Gene Expression in a Circadian Rhythm in Cyanobacteria.},
journal = {ACS synthetic biology},
volume = {9},
number = {12},
pages = {3210-3216},
doi = {10.1021/acssynbio.0c00455},
pmid = {33263998},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Circadian Rhythm/*genetics ; Gene Expression Regulation, Bacterial ; *Logic ; Luminescent Proteins/genetics/metabolism ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Spectrometry, Fluorescence ; Synechococcus/genetics/*metabolism ; },
abstract = {To enable circadian control of gene expression in cyanobacteria, we constructed a genetic logic gate (NAND) using orthogonal promoters via modular CRISPR interference. The NAND gates were tested in Synechococcus elongatus PCC 7942 using a fluorescent reporter. The NAND gate dynamics were characterized based on the affinity of the dCas9 complex to the output element. Upon connecting tight gene repressions with the circadian promoter (the purF gene; peak expression at dawn), inversed peak expressions were obtained as an output of the NAND gate although the retroactivities were shown in the ON and OFF states. A dark-responsive genetic element of the NAND gate was also expanded to an AND gate in S. elongatus PCC 7942. These cyanobacterial NAND and AND gates could facilitate the control of gene expressions in dynamic metabolic engineering technologies, thereby enabling the cyanobacteria to serve as biosolar cell factories.},
}
@article {pmid33263914,
year = {2021},
author = {Čermák, T and Gasparini, K and Kevei, Z and Zsögön, A},
title = {Genome Editing to Achieve the Crop Ideotype in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2264},
number = {},
pages = {219-244},
doi = {10.1007/978-1-0716-1201-9_16},
pmid = {33263914},
issn = {1940-6029},
mesh = {Agrobacterium/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Targeting ; Genetic Vectors ; *Genome, Plant ; Lycopersicon esculentum/*genetics/growth &amp; development ; *Transformation, Genetic ; },
abstract = {For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type ("ideotype"). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.},
}
@article {pmid33263910,
year = {2021},
author = {Nicolia, A and Fält, AS and Hofvander, P and Andersson, M},
title = {Protoplast-Based Method for Genome Editing in Tetraploid Potato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2264},
number = {},
pages = {177-186},
doi = {10.1007/978-1-0716-1201-9_12},
pmid = {33263910},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Plant ; Mutation ; Plant Proteins/*antagonists &amp; inhibitors/genetics ; Protoplasts/*physiology ; Solanum tuberosum/*genetics/growth &amp; development ; *Tetraploidy ; },
abstract = {The cultivated potato is tetraploid with four probably equivalent loci for each gene. A potato variety is furthermore commonly genetically heterogeneous and selected based on a beneficial genetic context which is maintained by clonal propagation. When introducing genetic changes by genome editing it is then desirable to achieve edits in all four loci for a certain gene target. This is in order to avoid crosses to achieve homozygosity for edited gene loci and at the same time reduce risk of inbreeding depression. In such a context transient transfection of protoplasts for the introduction of mutations, avoiding stable insertion of foreign DNA, would be very attractive. The protocol of this chapter has been shown to be applicable for the introduction of mutations by DNA vectors containing expression cassettes of TALEN, Cas9, and Cas9 deaminase fusions together with sgRNA expression cassettes on either single or separate vectors. Furthermore, the protoplast-based system has been shown to work very efficiently for mutations introduced by in vitro-produced and transfected RNP (ribonucleoprotein) complexes.},
}
@article {pmid33262250,
year = {2021},
author = {Miller, SR and Zhang, X and Hau, RK and Jilek, JL and Jennings, EQ and Galligan, JJ and Foil, DH and Zorn, KM and Ekins, S and Wright, SH and Cherrington, NJ},
title = {Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.},
journal = {Molecular pharmacology},
volume = {99},
number = {2},
pages = {147-162},
pmid = {33262250},
issn = {1521-0111},
support = {T32 ES007091/ES/NIEHS NIH HHS/United States ; R01 GM123643/GM/NIGMS NIH HHS/United States ; P30 ES006694/ES/NIEHS NIH HHS/United States ; R41 GM131433/GM/NIGMS NIH HHS/United States ; R44 GM122196/GM/NIGMS NIH HHS/United States ; },
mesh = {Acetates/*pharmacology ; Bayes Theorem ; Biological Transport ; CRISPR-Cas Systems ; Cell Line ; Dideoxynucleosides/*pharmacology ; Drug Interactions ; Equilibrative Nucleoside Transporter 1/*genetics/metabolism ; Equilibrative-Nucleoside Transporter 2/*genetics/metabolism ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Machine Learning ; Nevirapine/*pharmacology ; Thioinosine/analogs &amp; derivatives/pharmacology ; Ticagrelor/*pharmacology ; Uridine/*analogs &amp; derivatives/*metabolism/pharmacology ; },
abstract = {Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [[3]H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.},
}
@article {pmid33261648,
year = {2020},
author = {Höijer, I and Johansson, J and Gudmundsson, S and Chin, CS and Bunikis, I and Häggqvist, S and Emmanouilidou, A and Wilbe, M and den Hoed, M and Bondeson, ML and Feuk, L and Gyllensten, U and Ameur, A},
title = {Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {290},
pmid = {33261648},
issn = {1474-760X},
mesh = {*Base Sequence ; *CRISPR-Cas Systems ; Computational Biology/*methods ; DNA ; Gene Editing/*methods ; Genetic Variation ; Genomics ; HEK293 Cells ; Humans ; Mutation ; Nanopore Sequencing ; RNA, Guide ; Sequence Analysis, DNA ; Software ; },
abstract = {BACKGROUND: One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro.<br><br>RESULTS: The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites.<br><br>CONCLUSIONS: Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.},
}
@article {pmid33260774,
year = {2020},
author = {Papagiannitsis, CC and Verra, A and Galani, V and Xitsas, S and Bitar, I and Hrabak, J and Petinaki, E},
title = {Unravelling the Features of Success of VIM-Producing ST111 and ST235 Pseudomonas aeruginosa in a Greek Hospital.},
journal = {Microorganisms},
volume = {8},
number = {12},
pages = {},
pmid = {33260774},
issn = {2076-2607},
abstract = {The objective of this study was to analyze the characteristics that contribute to the successful dissemination of VIM-producing Pseudomonas aeruginosa (P. aeruginosa), belonging to ST111 and ST235, in a Greek hospital. A total of 120 non-repetitive P. aeruginosa, which had meropenem minimal inhibitory concentrations (MICs) greater than 2 mg/L, were studied. VIM-encoding genes were amplified and sequenced within their integrons. Isolates were typed by multilocus sequence typing (MLST). Six VIM-producers, representative of different integron structures and sequence types (STs), were completely sequenced using Illumina platform. Sixty-one P. aeruginosa were confirmed to produce VIM-type carbapenemases. ST111 dominated (n = 34) among VIM-producers, while 15 VIM-producers belonged to ST235. The blaVIM-like genes were located in three integron types, including In59, In595 and In1760, which were integrated into P. aeruginosa chromosomes. Whole genome sequencing (WGS) data demonstrated that ST111 and ST235 MBL producers carried several resistance and virulence genes. Additionally, the presence of type I-C and type I-E clustered regularly interspaced short palindromic repeats (CRISPR)/Cas locus was observed in ST235 and ST395 isolates, respectively. In conclusion, our findings confirmed the clonal spread of ST111 P. aeruginosa, carrying the VIM-2-encoding integron In59, in the University Hospital of Larissa (UHL). In addition, they highlighted the important role of high-risk clones, ST111 and ST235, in the successful dissemination and establishment into hospital settings of clinically important pathogens carrying resistance determinants.},
}
@article {pmid33260366,
year = {2020},
author = {Ungefroren, H and Otterbein, H and Wellner, UF and Keck, T and Lehnert, H and Marquardt, JU},
title = {RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility.},
journal = {Cancers},
volume = {12},
number = {12},
pages = {},
pmid = {33260366},
issn = {2072-6694},
abstract = {Autocrine transforming growth factor (TGF)β has been implicated in epithelial-mesenchymal transition (EMT) and invasion of several cancers including pancreatic ductal adenocarcinoma (PDAC) as well as triple-negative breast cancer (TNBC). However, the precise mechanism and the upstream inducers or downstream effectors of endogenous TGFB1 remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGFβ1 via downregulation of the TGFβ type I receptor, ALK5, but whether RAC1B also impacts autocrine TGFβ signaling has not yet been studied. Intriguingly, RNA interference-mediated knockdown (RNAi-KD) or CRISPR/Cas-mediated knockout of RAC1B in TGFβ1-secreting PDAC-derived Panc1 cells resulted in a dramatic decrease in secreted bioactive TGFβ1 in the culture supernatants and TGFB1 mRNA expression, while the reverse was true for TNBC-derived MDA-MB-231 cells ectopically expressing RAC1B. Surprisingly, the antibody-mediated neutralization of secreted bioactive TGFβ or RNAi-KD of the endogenous TGFB1 gene, was associated with increased rather than decreased migratory activities of Panc1 and MDA-MB-231 cells, upregulation of the promigratory genes SNAI1, SNAI2 and RAC1, and downregulation of the invasion suppressor genes CDH1 (encoding E-cadherin) and SMAD3. Intriguingly, ectopic re-expression of SMAD3 was able to rescue Panc1 and MDA-MB-231 cells from the TGFB1 KD-induced rise in migratory activity. Together, these data suggest that RAC1B favors synthesis and secretion of autocrine TGFβ1 which in a SMAD3-dependent manner blocks EMT-associated gene expression and cell motility.},
}
@article {pmid33259127,
year = {2020},
author = {Karayel, &#xd6; and Xu, P and Bludau, I and Velan Bhoopalan, S and Yao, Y and Ana Rita, FC and Santos, A and Schulman, BA and Alpi, AF and Weiss, MJ and Mann, M},
title = {Integrative proteomics reveals principles of dynamic phosphosignaling networks in human erythropoiesis.},
journal = {Molecular systems biology},
volume = {16},
number = {12},
pages = {e9813},
pmid = {33259127},
issn = {1744-4292},
support = {P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {Biomarkers/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; *Erythropoiesis ; Gene Ontology ; Humans ; Membrane Proteins/metabolism ; Phosphorylation ; Protein Kinases/metabolism ; Proteome/metabolism ; *Proteomics ; Reproducibility of Results ; *Signal Transduction ; },
abstract = {Human erythropoiesis is an exquisitely controlled multistep developmental process, and its dysregulation leads to numerous human diseases. Transcriptome and epigenome studies provided insights into system-wide regulation, but we currently lack a global mechanistic view on the dynamics of proteome and post-translational regulation coordinating erythroid maturation. We established a mass spectrometry (MS)-based proteomics workflow to quantify and dynamically track 7,400 proteins and 27,000 phosphorylation sites of five distinct maturation stages of in vitro reconstituted erythropoiesis of CD34[+] HSPCs. Our data reveal developmental regulation through drastic proteome remodeling across stages of erythroid maturation encompassing most protein classes. This includes various orchestrated changes in solute carriers indicating adjustments to altered metabolic requirements. To define the distinct proteome of each maturation stage, we developed a computational deconvolution approach which revealed stage-specific marker proteins. The dynamic phosphoproteomes combined with a kinome-targeted CRISPR/Cas9 screen uncovered coordinated networks of erythropoietic kinases and pinpointed downregulation of c-Kit/MAPK signaling axis as key driver of maturation. Our system-wide view establishes the functional dynamic of complex phosphosignaling networks and regulation through proteome remodeling in erythropoiesis.},
}
@article {pmid33258500,
year = {2021},
author = {Wang, X and Ray, R and Kratochvil, S and Melzi, E and Lin, YC and Giguere, S and Xu, L and Warner, J and Cheon, D and Liguori, A and Groschel, B and Phelps, N and Adachi, Y and Tingle, R and Wu, L and Crotty, S and Kirsch, KH and Nair, U and Schief, WR and Batista, FD},
title = {Multiplexed CRISPR/CAS9-mediated engineering of pre-clinical mouse models bearing native human B cell receptors.},
journal = {The EMBO journal},
volume = {40},
number = {2},
pages = {e105926},
pmid = {33258500},
issn = {1460-2075},
support = {UM1 AI144462/AI/NIAID NIH HHS/United States ; OPP1214085/GATES/Bill &amp; Melinda Gates Foundation/United States ; },
mesh = {Animals ; B-Lymphocytes/immunology/*metabolism ; Broadly Neutralizing Antibodies/immunology ; CRISPR-Cas Systems/*genetics/immunology ; Cell Line ; Gene Knock-In Techniques/methods ; Germinal Center/immunology/metabolism ; HEK293 Cells ; HIV-1/immunology ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Receptors, Antigen, B-Cell/immunology/*metabolism ; },
abstract = {B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.},
}
@article {pmid33258473,
year = {2021},
author = {Rönspies, M and Schindele, P and Puchta, H},
title = {CRISPR/Cas-mediated chromosome engineering: opening up a new avenue for plant breeding.},
journal = {Journal of experimental botany},
volume = {72},
number = {2},
pages = {177-183},
doi = {10.1093/jxb/eraa463},
pmid = {33258473},
issn = {1460-2431},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering ; Genome, Plant ; Plant Breeding ; Plants/genetics ; },
abstract = {The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs.},
}
@article {pmid33258411,
year = {2021},
author = {Hong, Y and Meng, J and He, X and Zhang, Y and Liu, Y and Zhang, C and Qi, H and Luan, Y},
title = {Editing miR482b and miR482c Simultaneously by CRISPR/Cas9 Enhanced Tomato Resistance to Phytophthora infestans.},
journal = {Phytopathology},
volume = {111},
number = {6},
pages = {1008-1016},
doi = {10.1094/PHYTO-08-20-0360-R},
pmid = {33258411},
issn = {0031-949X},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; *Lycopersicon esculentum/genetics ; *Phytophthora infestans ; Plant Diseases ; },
abstract = {Late blight, caused by Phytophthora infestans, is severely damaging to the global tomato industry. Micro-RNAs (miRNAs) have been widely demonstrated to play vital roles in plant resistance by repressing their target genes. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) method has been continuously improved and extensively applied to edit plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9 in tomato has not been studied yet. We knocked out miR482b and miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 system. Two transgenic plants with silenced miR482b and miR482c simultaneously and one transgenic line with silenced miR482b alone were obtained. Compared with wild-type plants, the disease symptoms of three transgenic plants upon infection were reduced, accompanied by increased expression of their common target nucleotide binding site-leucine-rich repeat genes and decreased levels of reactive oxygen species. Furthermore, silencing miR482b and miR482c simultaneously was more resistant than silencing miR482b alone in tomato. More importantly, we found that knocking out miR482b and miR482c can elicit expression perturbation of other miRNAs, suggesting cross-regulation between miRNAs. Our study demonstrated that editing miR482b and miR482c simultaneously with CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant tomatoes, and cross-regulation between miRNAs may reveal the novel mechanism in tomato-P. infestans interactions.},
}
@article {pmid33258314,
year = {2021},
author = {Dhar, BC and Steimberg, N and Mazzoleni, G},
title = {Point-of-Care Pathogen Detection with CRISPR-based Programmable Nucleic Acid Binding Proteins.},
journal = {ChemMedChem},
volume = {16},
number = {10},
pages = {1566-1575},
doi = {10.1002/cmdc.202000782},
pmid = {33258314},
issn = {1860-7187},
mesh = {CRISPR-Cas Systems/genetics ; DNA-Binding Proteins/*genetics ; Humans ; Mycobacterium tuberculosis/genetics/*isolation &amp; purification ; *Point-of-Care Systems ; Polymorphism, Single Nucleotide/genetics ; RNA-Binding Proteins/*genetics ; },
abstract = {The contemporary discovery of extremely versatile engineered nucleic acid-binding proteins has transformed a brave new world in the genome-editing scientific area. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated programmable nucleic acid-binding proteins have brought about a revolution in diagnostic platforms. The groundbreaking finding that bacteria and archaea that harbored prokaryotes have transmitted adaptive immunity through CRISPR and CRISPR-associated (Cas) proteins has driven revolutionary advances in molecular biology. Importantly, advances in gene editing focus how expanding visions in CRISPR-Cas biology are revolutionizing the area of molecular diagnostics for identifying DNA and RNA in emerging microbiological pathogens, for single nucleotide polymorphism (SNP) identifications, and for cell-free mutation. Recent advances, such as improvements in multiplexing and quantitative capabilities as well as instrument-free detection of nucleic acids, will potentially leverage the introduction of these novel technologies to detecting bacteria and viruses at the point of care (POC). In this review, we highlight the fundamental features of CRISPR/Cas-based molecular diagnostic technologies and summarize a vision of the next applications for identifying pathogens in POC settings.},
}
@article {pmid33258137,
year = {2021},
author = {Tian, Z and Wang, JW and Li, J and Han, B},
title = {Designing future crops: challenges and strategies for sustainable agriculture.},
journal = {The Plant journal : for cell and molecular biology},
volume = {105},
number = {5},
pages = {1165-1178},
doi = {10.1111/tpj.15107},
pmid = {33258137},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/*genetics ; Ecosystem ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plant Breeding ; },
abstract = {Crop production is facing unprecedented challenges. Despite the fact that the food supply has significantly increased over the past half-century, ~8.9 and 14.3% people are still suffering from hunger and malnutrition, respectively. Agricultural environments are continuously threatened by a booming world population, a shortage of arable land, and rapid changes in climate. To ensure food and ecosystem security, there is a need to design future crops for sustainable agriculture development by maximizing net production and minimalizing undesirable effects on the environment. The future crops design projects, recently launched by the National Natural Science Foundation of China and Chinese Academy of Sciences (CAS), aim to develop a roadmap for rapid design of customized future crops using cutting-edge technologies in the Breeding 4.0 era. In this perspective, we first introduce the background and missions of these projects. We then outline strategies to design future crops, such as improvement of current well-cultivated crops, de novo domestication of wild species and redomestication of current cultivated crops. We further discuss how these ambitious goals can be achieved by the recent development of new integrative omics tools, advanced genome-editing tools and synthetic biology approaches. Finally, we summarize related opportunities and challenges in these projects.},
}
@article {pmid33257571,
year = {2020},
author = {Zhou, M and Kuruvilla, L and Shi, X and Viviano, S and Ahearn, IM and Amendola, CR and Su, W and Badri, S and Mahaffey, J and Fehrenbacher, N and Skok, J and Schlessinger, J and Turk, BE and Calderwood, DA and Philips, MR},
title = {Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {50},
pages = {31914-31922},
pmid = {33257571},
issn = {1091-6490},
support = {R01 CA116034/CA/NCI NIH HHS/United States ; R01 CA163489/CA/NCI NIH HHS/United States ; R35 GM122515/GM/NIGMS NIH HHS/United States ; T32 AR064184/AR/NIAMS NIH HHS/United States ; R35 CA253178/CA/NCI NIH HHS/United States ; P50 CA196530/CA/NCI NIH HHS/United States ; },
mesh = {Alkyl and Aryl Transferases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Membrane/*metabolism ; Computational Biology ; Datasets as Topic ; Dimethylallyltranstransferase/*metabolism ; Gene Knockdown Techniques ; Humans ; Matrix Attachment Region Binding Proteins/genetics/*metabolism ; Neoplasms/genetics/*pathology ; Nuclear Matrix-Associated Proteins/genetics/*metabolism ; Protein Prenylation ; Protein Subunits/metabolism ; Proto-Oncogene Proteins p21(ras)/genetics/*metabolism ; Receptors, Estrogen/genetics/*metabolism ; },
abstract = {Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.},
}
@article {pmid33257546,
year = {2020},
author = {Bhave, M and Mino, RE and Wang, X and Lee, J and Grossman, HM and Lakoduk, AM and Danuser, G and Schmid, SL and Mettlen, M},
title = {Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {50},
pages = {31591-31602},
pmid = {33257546},
issn = {1091-6490},
support = {R37 MH061345/MH/NIMH NIH HHS/United States ; R35 GM136428/GM/NIGMS NIH HHS/United States ; R01 MH061345/MH/NIMH NIH HHS/United States ; R01 GM042455/GM/NIGMS NIH HHS/United States ; R01 GM073165/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Vesicular Transport/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Membrane/*metabolism ; Clathrin/*metabolism ; Cluster Analysis ; Coated Pits, Cell-Membrane/*metabolism ; Endocytosis/*physiology ; Gene Knockdown Techniques ; Green Fluorescent Proteins/chemistry/genetics ; Humans ; Intravital Microscopy/methods ; Luminescent Agents/chemistry ; Microscopy, Fluorescence/methods ; Molecular Imaging/methods ; RNA, Small Interfering/metabolism ; },
abstract = {Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.},
}
@article {pmid33256983,
year = {2021},
author = {Pak, B and Schmitt, CE and Oh, S and Kim, JD and Choi, W and Han, O and Kim, M and Kim, MJ and Ham, HJ and Kim, S and Huh, TL and Kim, JI and Jin, SW},
title = {Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.},
journal = {Biochemical and biophysical research communications},
volume = {534},
number = {},
pages = {359-366},
doi = {10.1016/j.bbrc.2020.11.077},
pmid = {33256983},
issn = {1090-2104},
mesh = {Animals ; Animals, Genetically Modified ; Bacterial Infections/immunology ; CRISPR-Cas Systems ; Erythropoiesis/genetics/*immunology ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Granulocytes/immunology ; Immunity, Innate/genetics/physiology ; Myelopoiesis/genetics/*immunology ; PAX9 Transcription Factor/deficiency/genetics/*immunology ; Zebrafish/embryology/genetics/*immunology ; Zebrafish Proteins/deficiency/genetics/*immunology ; },
abstract = {Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.},
}
@article {pmid33256828,
year = {2020},
author = {Zaidi, SS and Mahas, A and Vanderschuren, H and Mahfouz, MM},
title = {Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {289},
pmid = {33256828},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/*genetics ; Disease Resistance/*genetics ; Gene Editing ; Genome ; Genome, Plant ; Mutagenesis, Insertional ; Plant Diseases/*genetics/microbiology/virology ; Plants, Genetically Modified/genetics ; },
abstract = {To meet increasing global food demand, breeders and scientists aim to improve the yield and quality of major food crops. Plant diseases threaten food security and are expected to increase because of climate change. CRISPR genome-editing technology opens new opportunities to engineer disease resistance traits. With precise genome engineering and transgene-free applications, CRISPR is expected to resolve the major challenges to crop improvement. Here, we discuss the latest developments in CRISPR technologies for engineering resistance to viruses, bacteria, fungi, and pests. We conclude by highlighting current concerns and gaps in technology, as well as outstanding questions for future research.},
}
@article {pmid33256757,
year = {2020},
author = {Song, I and Kim, J and Baek, K and Choi, Y and Shin, B and Jin, E},
title = {The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {220},
pmid = {33256757},
issn = {1475-2859},
mesh = {Algal Proteins/genetics ; Biosynthetic Pathways ; CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics/*metabolism ; Gene Knockout Techniques ; Industrial Microbiology ; Intramolecular Lyases/*genetics ; Metabolic Engineering ; Oxidoreductases/genetics ; Zeaxanthins/*biosynthesis ; },
abstract = {BACKGROUND: Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin.<br><br>RESULTS: In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of &#x3b1;-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24&#xa0;mg L[-&#x2009;1]) and content (7.28&#xa0;mg g[-&#x2009;1]) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84&#xa0;mg L[-&#x2009;1].<br><br>CONCLUSIONS: A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.},
}
@article {pmid33256602,
year = {2020},
author = {Refrégier, G and Sola, C and Guyeux, C},
title = {Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {841},
pmid = {33256602},
issn = {1471-2164},
mesh = {Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Transposable Elements ; Molecular Epidemiology ; *Mycobacterium tuberculosis/genetics ; },
abstract = {BACKGROUND: Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes.<br><br>RESULTS: We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats.<br><br>CONCLUSION: This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.},
}
@article {pmid33255340,
year = {2020},
author = {Saifullah, and Sakari, M and Suzuki, T and Yano, S and Tsukahara, T},
title = {Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells.},
journal = {International journal of molecular sciences},
volume = {21},
number = {23},
pages = {},
pmid = {33255340},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Caspases/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Survival/genetics ; Gene Knockdown Techniques ; Humans ; Lung Neoplasms/*genetics/pathology ; *Molecular Targeted Therapy ; Oncogene Proteins, Fusion/*genetics ; RNA/genetics ; RNA Interference ; },
abstract = {RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA-guide RNA complex residing at the 5' of the crRNA. The activity of Cas13a was maximal for guide RNAs 24-30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70-76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.},
}
@article {pmid33254128,
year = {2020},
author = {Lüthy, IA and Lamb, CA},
title = {[Nobel Prize in Chemistry 2020 for gene editing with CRISPR / Cas9 technology].},
journal = {Medicina},
volume = {80},
number = {6},
pages = {738-740},
pmid = {33254128},
issn = {1669-9106},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Nobel Prize ; Technology ; },
}
@article {pmid33253976,
year = {2021},
author = {Modell, AE and Siriwardena, SU and Shoba, VM and Li, X and Choudhary, A},
title = {Chemical and optical control of CRISPR-associated nucleases.},
journal = {Current opinion in chemical biology},
volume = {60},
number = {},
pages = {113-121},
doi = {10.1016/j.cbpa.2020.10.003},
pmid = {33253976},
issn = {1879-0402},
support = {R01 GM132825/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/*metabolism ; Gene Editing ; Humans ; *Optical Phenomena ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system of bacteria has furnished programmable nucleases (e.g., Cas9) that are transforming the field of genome editing with applications in basic and biomedical research, biotechnology, and agriculture. However, broader real-world applications of Cas9 require precision control of its activity over dose, time, and space as off-target effects, embryonic mosaicism, chromosomal translocations, and genotoxicity have been observed with elevated and/or prolonged nuclease activity. Here, we review chemical and optical methods for precision control of Cas9's activity.},
}
@article {pmid33253759,
year = {2021},
author = {Ka, Y and Katano, I and Nishinaka, E and Welcker, J and Mochizuki, M and Kawai, K and Goto, M and Tomiyama, K and Ogura, T and Yamamoto, T and Ito, M and Ito, R and Takahashi, R},
title = {Improved engraftment of human peripheral blood mononuclear cells in NOG MHC double knockout mice generated using CRISPR/Cas9.},
journal = {Immunology letters},
volume = {229},
number = {},
pages = {55-61},
doi = {10.1016/j.imlet.2020.11.011},
pmid = {33253759},
issn = {1879-0542},
mesh = {Animals ; Biomarkers ; *CRISPR-Cas Systems ; *Cell Transplantation/adverse effects/methods ; Gene Editing ; *Gene Knockout Techniques ; Gene Targeting ; Genetic Loci ; Graft Survival ; Graft vs Host Disease/diagnosis/etiology ; Histocompatibility Antigens/*genetics ; Humans ; Immunohistochemistry ; Immunophenotyping ; Interleukin Receptor Common gamma Subunit/*deficiency ; Leukocytes, Mononuclear/*immunology/*metabolism ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Models, Animal ; Severity of Illness Index ; Spleen/immunology/metabolism ; },
abstract = {Humanized mice are widely used to study the human immune system in vivo and develop therapies for various human diseases. Human peripheral blood mononuclear cells (PBMC)-engrafted NOD/Shi-scid IL2rγ[null] (NOG) mice are useful models for characterization of human T cells. However, the development of graft-versus-host disease (GVHD) limits the use of NOG PBMC models. We previously established a NOG-major histocompatibility complex class I/II double knockout (dKO) mouse model. Although humanized dKO mice do not develop severe GVHD, they have impaired reproductive performance and reduced chimerism of human cells. In this study, we established a novel beta-2 microglobulin (B2m) KO mouse model using CRISPR/Cas9. By crossing B2m KO mice with I-Ab KO mice, we established a modified dKO (dKO-em) mouse model. Reproductivity was slightly improved in dKO-em mice, compared with conventional dKO (dKO-tm) mice. dKO-em mice showed no signs of GVHD after the transfer of human PBMCs; they also exhibited high engraftment efficiency. Engrafted human PBMCs survived significantly longer in the peripheral blood and spleens of dKO-em mice, compared with dKO-tm mice. In conclusion, dKO-em mice might constitute a promising PBMC-based humanized mouse model for the development and preclinical testing of novel therapeutics for human diseases.},
}
@article {pmid33253175,
year = {2020},
author = {Li, R and Xia, X and Wang, X and Sun, X and Dai, Z and Huo, D and Zheng, H and Xiong, H and He, A and Wu, X},
title = {Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model.},
journal = {PLoS biology},
volume = {18},
number = {11},
pages = {e3000749},
pmid = {33253175},
issn = {1545-7885},
support = {P51 OD011132/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Doxycycline/metabolism ; Embryonic Stem Cells/metabolism/physiology ; Gene Editing/*methods ; Gene Expression/genetics ; Gene Silencing/physiology ; Kruppel-Like Transcription Factors/*genetics/metabolism ; Loss of Function Mutation/genetics ; Mice ; Mice, Knockout ; Models, Biological ; RNA, Guide/genetics ; Reproducibility of Results ; Transgenes/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.},
}
@article {pmid33252245,
year = {2020},
author = {Ameur, A},
title = {CRISPR and Long-Read Sequencing: A Perfect Match.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {425-427},
doi = {10.1089/crispr.2020.29110.aam},
pmid = {33252245},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Tumor Suppressor ; Mutation ; Sequence Analysis, DNA ; },
}
@article {pmid33252243,
year = {2020},
author = {Park, KE and Frey, JF and Waters, J and Simpson, SG and Coutu, C and Plummer, S and Campbell, M and Donovan, DM and Telugu, BP},
title = {One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {523-534},
pmid = {33252243},
issn = {2573-1602},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Cattle ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryo, Mammalian ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genomics/methods ; Selective Breeding/*genetics ; Zygote/metabolism ; },
abstract = {Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless introgression of novel alleles is required, especially in cattle with long generation intervals. We report for the first time the use of CRISPR-Cas genome editors to introduce novel PRNP allelic variants that have been shown to provide resilience towards human prion pandemics. From one round of embryo injections, we have established six pregnancies and birth of seven edited offspring, with two founders showing >90% targeted homology-directed repair modifications. This study lays out the framework for in vitro optimization, unbiased deep-sequencing to identify editing outcomes, and generation of high frequency homology-directed repair-edited calves.},
}
@article {pmid33251765,
year = {2020},
author = {Fang, L and Wang, W and Li, G and Zhang, L and Li, J and Gan, D and Yang, J and Tang, Y and Ding, Z and Zhang, M and Zhang, W and Deng, D and Song, Z and Zhu, Q and Cui, H and Hu, Y and Chen, W},
title = {CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.},
journal = {Molecular systems biology},
volume = {16},
number = {11},
pages = {e10025},
pmid = {33251765},
issn = {1744-4292},
mesh = {CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; Gene Regulatory Networks ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Methylation ; Methyltransferases/*genetics/metabolism ; RNA Processing, Post-Transcriptional/*genetics ; RNA, Guide/genetics ; RNA, Messenger/analysis/*metabolism ; Sequence Analysis, RNA/*methods ; tRNA Methyltransferases/genetics ; },
abstract = {Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m[6] A and m[5] C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m[5] C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m[5] C sites and together contributed to almost all the m[5] C modification in mRNA. Finally, using m[1] A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.},
}
@article {pmid33251190,
year = {2020},
author = {Tavares, LM and de Jesus, LCL and da Silva, TF and Barroso, FAL and Batista, VL and Coelho-Rocha, ND and Azevedo, V and Drumond, MM and Mancha-Agresti, P},
title = {Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {517166},
pmid = {33251190},
issn = {2296-4185},
abstract = {Lactic acid bacteria (LAB) are traditionally used in fermentation and food preservation processes and are recognized as safe for consumption. Recently, they have attracted attention due to their health-promoting properties; many species are already widely used as probiotics for treatment or prevention of various medical conditions, including inflammatory bowel diseases, infections, and autoimmune disorders. Some LAB, especially Lactococcus lactis, have been engineered as live vehicles for delivery of DNA vaccines and for production of therapeutic biomolecules. Here, we summarize work on engineering of LAB, with emphasis on the model LAB, L. lactis. We review the various expression systems for the production of heterologous proteins in Lactococcus spp. and its use as a live delivery system of DNA vaccines and for expression of biotherapeutics using the eukaryotic cell machinery. We have included examples of molecules produced by these expression platforms and their application in clinical disorders. We also present the CRISPR-Cas approach as a novel methodology for the development and optimization of food-grade expression of useful substances, and detail methods to improve DNA delivery by LAB to the gastrointestinal tract. Finally, we discuss perspectives for the development of medical applications of recombinant LABs involving animal model studies and human clinical trials, and we touch on the main safety issues that need to be taken into account so that bioengineered versions of these generally recognized as safe organisms will be considered acceptable for medical use.},
}
@article {pmid33251158,
year = {2020},
author = {Kumar, P and Malik, YS and Ganesh, B and Rahangdale, S and Saurabh, S and Natesan, S and Srivastava, A and Sharun, K and Yatoo, MI and Tiwari, R and Singh, RK and Dhama, K},
title = {CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {576875},
pmid = {33251158},
issn = {2235-2988},
mesh = {Animals ; COVID-19/diagnosis/therapy/*virology ; *CRISPR-Cas Systems ; Humans ; SARS-CoV-2/*genetics/metabolism ; },
abstract = {COVID-19, the human coronavirus disease caused by SARS-CoV-2, was reported for the first time in Wuhan, China in late 2019. COVID-19 has no preventive vaccine or proven standard pharmacological treatment, and consequently, the outbreak swiftly became a pandemic affecting more than 215 countries around the world. For the diagnosis of COVID-19, the only reliable diagnostics is a qPCR assay. Among other diagnostic tools, the CRISPR-Cas system is being investigated for rapid and specific diagnosis of COVID-19. The CRISPR-Cas-based methods diagnose the SARS-CoV-2 infections within an hour. Apart from its diagnostic ability, CRISPR-Cas system is also being assessed for antiviral therapy development; however, till date, no CRISPR-based therapy has been approved for human use. The Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN), which is Cas 13 based strategy, has been developed against coronavirus. Although this strategy has the potential to be developed as a therapeutic modality, it may face significant challenges for approval in human clinical trials. This review is focused on describing potential use and challenges of CRISPR-Cas based approaches for the development of rapid and accurate diagnostic technique and/or a possible therapeutic alternative for combating COVID-19. The assessment of potential risks associated with use of CRISPR will be important for future clinical advancements.},
}
@article {pmid33250920,
year = {2020},
author = {Alcorta, J and Alarcón-Schumacher, T and Salgado, O and Díez, B},
title = {Taxonomic Novelty and Distinctive Genomic Features of Hot Spring Cyanobacteria.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {568223},
pmid = {33250920},
issn = {1664-8021},
abstract = {Several cyanobacterial species are dominant primary producers in hot spring microbial mats. To date, hot spring cyanobacterial taxonomy, as well as the evolution of their genomic adaptations to high temperatures, are poorly understood, with genomic information currently available for only a few dominant genera, including Fischerella and Synechococcus. To address this knowledge gap, the present study expands the genomic landscape of hot spring cyanobacteria and traces the phylum-wide genomic consequences of evolution in high temperature environments. From 21 globally distributed hot spring metagenomes, with temperatures between 32 and 75°C, 57 medium- and high-quality cyanobacterial metagenome-assembled genomes were recovered, representing taxonomic novelty for 1 order, 3 families, 15 genera and 36 species. Comparative genomics of 93 hot spring genomes (including the 57 metagenome-assembled genomes) and 66 non-thermal genomes, showed that the former have smaller genomes and a higher GC content, as well as shorter proteins that are more hydrophilic and basic, when compared to the non-thermal genomes. Additionally, the core accessory orthogroups from the hot spring genomes of some genera had a greater abundance of functional categories, such as inorganic ion metabolism, translation and post-translational modifications. Moreover, hot spring genomes showed increased abundances of inorganic ion transport and amino acid metabolism, as well as less replication and transcription functions in the protein coding sequences. Furthermore, they showed a higher dependence on the CRISPR-Cas defense system against exogenous nucleic acids, and a reduction in secondary metabolism biosynthetic gene clusters. This suggests differences in the cyanobacterial response to environment-specific microbial communities. This phylum-wide study provides new insights into cyanobacterial genomic adaptations to a specific niche where they are dominant, which could be essential to trace bacterial evolution pathways in a warmer world, such as the current global warming scenario.},
}
@article {pmid33250580,
year = {2020},
author = {Marchfelder, A},
title = {[Not Available].},
journal = {Biospektrum : Zeitschrift der Gesellschaft fur Biologishe Chemie (GBCH) und der Vereinigung fur Allgemeine und Angewandte Mikrobiologie (VAAM)},
volume = {26},
number = {7},
pages = {806},
doi = {10.1007/s12268-020-1500-5},
pmid = {33250580},
issn = {0947-0867},
}
@article {pmid33249867,
year = {2022},
author = {Kumar, P and Sinha, R and Shukla, P},
title = {Artificial intelligence and synthetic biology approaches for human gut microbiome.},
journal = {Critical reviews in food science and nutrition},
volume = {62},
number = {8},
pages = {2103-2121},
doi = {10.1080/10408398.2020.1850415},
pmid = {33249867},
issn = {1549-7852},
mesh = {Artificial Intelligence ; *Gastrointestinal Microbiome/genetics ; Gene Editing/methods ; Humans ; *Microbiota ; Synthetic Biology ; },
abstract = {The gut microbiome comprises a variety of microorganisms whose genes encode proteins to carry out crucial metabolic functions that are responsible for the majority of health-related issues in human beings. The advent of the technological revolution in artificial intelligence (AI) assisted synthetic biology (SB) approaches will play a vital role in the modulating the therapeutic and nutritive potential of probiotics. This can turn human gut as a reservoir of beneficial bacterial colonies having an immense role in immunity, digestion, brain function, and other health benefits. Hence, in the present review, we have discussed the role of several gene editing tools and approaches in synthetic biology that have equipped us with novel tools like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) systems to precisely engineer probiotics for diagnostic, therapeutic and nutritive value. A brief discussion over the AI techniques to understand the metagenomic data from the healthy and diseased gut microbiome is also presented. Further, the role of AI in potentially impacting the pace of developments in SB and its current challenges is also discussed. The review also describes the health benefits conferred by engineered microbes through the production of biochemicals, nutraceuticals, drugs or biotherapeutics molecules etc. Finally, the review concludes with the challenges and regulatory concerns in adopting synthetic biology engineered microbes for clinical applications. Thus, the review presents a synergistic approach of AI and SB toward human gut microbiome for better health which will provide interesting clues to researchers working in the area of rapidly evolving food and nutrition science.},
}
@article {pmid33248248,
year = {2021},
author = {Rabinowitz, R and Offen, D},
title = {Single-Base Resolution: Increasing the Specificity of the CRISPR-Cas System in Gene Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {3},
pages = {937-948},
pmid = {33248248},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Endonucleases/*metabolism ; *Gene Editing ; *Genome, Human ; Genomics ; Humans ; *Polymorphism, Single Nucleotide ; },
abstract = {The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, the use of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a subregion within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity and compare the standard gRNA design methodology to the single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach, an alternative method for allele-specific targeting. The combination of the two methods may be advantageous in designing CRISPR-based therapeutics and diagnostics for heterozygous patients.},
}
@article {pmid33248026,
year = {2020},
author = {McBride, TM and Schwartz, EA and Kumar, A and Taylor, DW and Fineran, PC and Fagerlund, RD},
title = {Diverse CRISPR-Cas Complexes Require Independent Translation of Small and Large Subunits from a Single Gene.},
journal = {Molecular cell},
volume = {80},
number = {6},
pages = {971-979.e7},
doi = {10.1016/j.molcel.2020.11.003},
pmid = {33248026},
issn = {1097-4164},
mesh = {Adaptive Immunity/*genetics ; CRISPR-Associated Proteins/*genetics/immunology ; CRISPR-Cas Systems/*genetics ; *Evolution, Molecular ; Prokaryotic Cells/immunology/virology ; Protein Biosynthesis ; Viruses/immunology ; },
abstract = {CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.},
}
@article {pmid33247284,
year = {2021},
author = {Hendriks, D and Artegiani, B and Hu, H and Chuva de Sousa Lopes, S and Clevers, H},
title = {Establishment of human fetal hepatocyte organoids and CRISPR-Cas9-based gene knockin and knockout in organoid cultures from human liver.},
journal = {Nature protocols},
volume = {16},
number = {1},
pages = {182-217},
pmid = {33247284},
issn = {1750-2799},
mesh = {*CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Cells, Cultured ; Fetus/cytology/metabolism ; Gene Editing/methods ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques/methods ; Hepatocytes/*cytology/metabolism ; Humans ; Liver/*cytology/metabolism ; Organoids/*cytology/metabolism ; },
abstract = {The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR-Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR-Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1-2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2-3 months.},
}
@article {pmid33247197,
year = {2020},
author = {Momen-Roknabadi, A and Oikonomou, P and Zegans, M and Tavazoie, S},
title = {An inducible CRISPR interference library for genetic interrogation of Saccharomyces cerevisiae biology.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {723},
pmid = {33247197},
issn = {2399-3642},
support = {R01 GM139215/GM/NIGMS NIH HHS/United States ; R01-AI077562/AO/NIAID NIH HHS/United States ; },
mesh = {Adenine/biosynthesis ; Arginine/biosynthesis ; *CRISPR-Cas Systems ; *Gene Library ; Genes, Fungal ; Plasmids ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite the dominance of Saccharomyces cerevisiae as a model eukaryote, an inducible genome-wide CRISPRi library in yeast has not yet been presented. Here, we present a genome-wide, inducible CRISPRi library, based on spacer design rules optimized for S. cerevisiae. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and a low false discovery rate across a variety of experimental conditions, enabling rapid and reliable assessment of genetic function and interactions in S. cerevisiae.},
}
@article {pmid33247091,
year = {2020},
author = {Mancuso, P and Chen, C and Kaminski, R and Gordon, J and Liao, S and Robinson, JA and Smith, MD and Liu, H and Sariyer, IK and Sariyer, R and Peterson, TA and Donadoni, M and Williams, JB and Siddiqui, S and Bunnell, BA and Ling, B and MacLean, AG and Burdo, TH and Khalili, K},
title = {CRISPR based editing of SIV proviral DNA in ART treated non-human primates.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6065},
pmid = {33247091},
issn = {2041-1723},
support = {P51 OD011104/OD/NIH HHS/United States ; R01 MH116844/MH/NIMH NIH HHS/United States ; R01 NS104016/NS/NINDS NIH HHS/United States ; T32 OD011124/OD/NIH HHS/United States ; },
mesh = {Animals ; Anti-Retroviral Agents/*pharmacology ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA, Viral/blood/*genetics ; *Gene Editing ; Genome, Viral ; Humans ; Lung/drug effects/virology ; Lymph Nodes/drug effects/virology ; Macaca mulatta ; Proviruses/drug effects/*genetics ; Simian Acquired Immunodeficiency Syndrome/blood/virology ; Simian Immunodeficiency Virus/*genetics ; Spleen/pathology/virology ; Tissue Distribution ; Transgenes ; },
abstract = {Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.},
}
@article {pmid33245685,
year = {2021},
author = {Vatankhah, M and Azizi, A and Sanajouyan Langeroudi, A and Ataei Azimi, S and Khorsand, I and Kerachian, MA and Motaei, J},
title = {CRISPR-based biosensing systems: a way to rapidly diagnose COVID-19.},
journal = {Critical reviews in clinical laboratory sciences},
volume = {58},
number = {4},
pages = {225-241},
doi = {10.1080/10408363.2020.1849010},
pmid = {33245685},
issn = {1549-781X},
mesh = {Biosensing Techniques/*methods ; COVID-19/*diagnosis ; COVID-19 Nucleic Acid Testing/*methods ; CRISPR-Cas Systems/*genetics ; Humans ; Real-Time Polymerase Chain Reaction ; *SARS-CoV-2/genetics/isolation &amp; purification ; },
abstract = {The outbreak of the emerging SARS-CoV-2 virus has highlighted the challenges of detecting viral infections, especially in resource-limited settings. The SARS-CoV-2 virus transmission chain is interrupted when screening and diagnosis can be performed on a large scale by identifying asymptomatic or moderately symptomatic patients. Diagnosis of COVID-19 with reverse transcription polymerase chain reaction (RT-PCR) has been limited due to inadequate access to complex, expensive equipment and reagents, which has impeded efforts to reduce the spread of virus transmission. Recently, the development of several diagnostic platforms based on the CRISPR-Cas system has reduced the dependence on RT-PCR. The first CRISPR-based diagnostic test for SARS-CoV-2 was recently approved by the U.S. Food and Drug Administration. The biosensing systems have several important features that make them suitable for point-of-care tests, including the speed of design and synthesis of each platform in less than a few days, an assay time of 1-2 h, and the cost of materials and reagents less than one dollar per test. The HUDSON-SHERLOCK and STOPCovid biosensing systems, as field-deployable and rapid diagnostic tests, can detect low-copy viruses in body fluids without nucleic acid extraction and with minimal equipment. In addition, Cas13-based treatment strategies could potentially be an effective antiviral strategy for the prevention and treatment of emerging pandemic viruses such as SARS-CoV-2. In this review, we describe recent advances in CRISPR-based diagnostic platforms with an emphasis on their use in the rapid diagnosis and potential treatment of COVID-19.},
}
@article {pmid33245213,
year = {2020},
author = {Li, Z and Fei, T},
title = {Improving Cancer Immunotherapy with CRISPR-Based Technology.},
journal = {Advanced biosystems},
volume = {4},
number = {11},
pages = {e1900253},
doi = {10.1002/adbi.201900253},
pmid = {33245213},
issn = {2366-7478},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Immunotherapy ; Mice ; *Neoplasms/genetics/therapy ; },
abstract = {The rapidly evolving field of immunotherapy has attracted great attention in the field of cancer research and already revolutionized the clinical practice standard for treating cancer. Genetically engineered T cells expressing either T cell receptors or chimeric antigen receptors represent novel treatment modalities and are considered powerful weapons to fight cancer. The immune checkpoint blockade, which harnesses the negative control signaling behind the anti-tumor immune response with therapeutic antibodies by blocking cytotoxic T lymphocyte-associated protein 4 or the programmed cell death 1 pathways are another mainstream direction for cancer immunotherapy. In addition to cytotoxic T cells, other immune cell types such as nature killer cells and macrophages also possess the ability to eradicate cancer cells, which may serve as the basis to develop novel cancer immunotherapies. The advent of cutting-edge genome editing technology, especially clustered regularly interspaced palindromic repeats (CRISPR)-based tools, has greatly expedited many biomedical research areas, including cancer immunology and immunotherapy. In this review, the contribution of current CRISPR techniques to basic and translational cancer immunology research is discussed, and the future for cancer immunotherapy in the age of CRISPR is predicted.},
}
@article {pmid33244929,
year = {2020},
author = {Zhang, D and Luo, Y and Chen, W},
title = {[Current development of gene editing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {11},
pages = {2345-2356},
doi = {10.13345/j.cjb.190557},
pmid = {33244929},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; China ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plants/genetics ; },
abstract = {As the breakthrough in gene editing, represented by CRISPR/Cas9, gene manipulations now are more maneuverable, economically feasible and time saving. It is possible for China to catch an overtaking in researching and industrializing of downside sections (especially the application of plant gene editing), also the incubation of professional companies in gene editing fields. For this consideration, it is necessary and urgent to find the key demands and potential application for gene editing in China. Questionnaire and statistic analysis were carried out to find the key demands and the most potential application fields of the development for gene editing. Firstly, an ordered multi-classification Logistic regression model was established following with dependent variable analysis. Eight out of 24 questionnaires questions in 4 categories were regarded as independent variables with significance test. Then, regression model based on ordered multi-classification logistic method was established to analyze the specific impact of different options on the development of gene editing. The results showed that most researchers in the field of gene editing take the view that development of potential competitive advantages lies in the field of plant science. The results also showed that major gene editing experts believe more attention should be paid on how to carry out technology industrialization while focusing on basic technology development, as well as the development of potential competitive advantages of gene editing technology in plant field. To promote the development of gene editing in China, not only the participation of scientific research institution was needed, but also the synergy of various forces both universities and governments. It is urgent both properly guiding public opinion on gene editing and establishing a national safety standard system. At the same time, the key point of technology risk avoidance should be put on biological weapons and bioterrorism, gene editing related infectious disease, and the potential risk of species genetic change on the ecological environment, etc.},
}
@article {pmid33244639,
year = {2021},
author = {Westermann, L and Neubauer, B and Köttgen, M},
title = {Nobel Prize 2020 in Chemistry honors CRISPR: a tool for rewriting the code of life.},
journal = {Pflugers Archiv : European journal of physiology},
volume = {473},
number = {1},
pages = {1-2},
pmid = {33244639},
issn = {1432-2013},
mesh = {*CRISPR-Cas Systems ; Chemistry/*history ; Gene Editing/*methods ; History, 20th Century ; *Nobel Prize ; },
}
@article {pmid33244160,
year = {2020},
author = {Rabiee, N and Bagherzadeh, M and Ghadiri, AM and Salehi, G and Fatahi, Y and Dinarvand, R},
title = {ZnAl nano layered double hydroxides for dual functional CRISPR/Cas9 delivery and enhanced green fluorescence protein biosensor.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20672},
pmid = {33244160},
issn = {2045-2322},
mesh = {Biosensing Techniques/methods ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Survival/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; Electrophoresis/methods ; Fluorescence ; Gene Transfer Techniques ; Green Fluorescent Proteins/*genetics ; HEK293 Cells ; Humans ; Hydroxides/*chemistry ; Nanoparticles/*chemistry ; Transfection/methods ; },
abstract = {Evaluation of the effect of different parameters for designing a non-viral vector in gene delivery systems has great importance. In this manner, 2D crystals, precisely layered double hydroxides, have attracted the attention of scientists due to their significant adjustability and low-toxicity and low-cost preparation procedure. In this work, the relationship between different physicochemical properties of LDH, including pH, size, zeta potential, and synthesis procedure, was investigated and optimized for CRISPR/Cas9 delivery and reverse fluorescence response to the EGFP. In this manner, ZnAl LDH and ZnAl HMTA LDH were synthesized and characterized and applied in the HEK-293 cell line to deliver CRISPR/Cas9. The results were optimized by different characterizations as well as Gel Electrophoresis and showed acceptable binding ability with the DNA that could be considered as a promising and also new gold-standard for the delivery of CRISPR/Cas9. Also, the relationship of the presence of tertiary amines (in this case, hexamethylenetetramine (HMTA) as the templates) in the structure of the ZnAl LDH, as well as the gene delivery application, was evaluated. The results showed more than 79% of relative cell viability in most of the weight ratios of LDH to CRISPR/Cas9; fully quenching the fluorescence intensity of the EGFP/LDH in the presence of 15 µg mL[-1] of the protoporphyrins along with the detection limit of below 2.1 µg mL[-1], the transfection efficiency of around 33% of the GFP positive cell for ZnAl LDH and more than 38% for the ZnAl LDH in the presence of its tertiary amine template.},
}
@article {pmid33243861,
year = {2020},
author = {Jin, X and Simmons, SK and Guo, A and Shetty, AS and Ko, M and Nguyen, L and Jokhi, V and Robinson, E and Oyler, P and Curry, N and Deangeli, G and Lodato, S and Levin, JZ and Regev, A and Zhang, F and Arlotta, P},
title = {In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6520},
pages = {},
pmid = {33243861},
issn = {1095-9203},
support = {P50 MH094271/MH/NIMH NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; //Howard Hughes Medical Insititue/International ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 MH096066/MH/NIMH NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; U01 MH115727/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Ankyrins/genetics/metabolism ; Autistic Disorder/*genetics/*pathology ; Brain/*abnormalities ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Frameshift Mutation ; Gene Expression Profiling ; Genetic Loci ; Humans ; Mice ; Neuroglia/metabolism/*pathology ; Neurons/metabolism/*pathology ; Repressor Proteins/genetics ; Risk ; Transcription Factors/genetics ; },
abstract = {The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.},
}
@article {pmid33242354,
year = {2022},
author = {Sharma, B and Shukla, P},
title = {Futuristic avenues of metabolic engineering techniques in bioremediation.},
journal = {Biotechnology and applied biochemistry},
volume = {69},
number = {1},
pages = {51-60},
doi = {10.1002/bab.2080},
pmid = {33242354},
issn = {1470-8744},
mesh = {Biodegradation, Environmental ; CRISPR-Cas Systems ; *Environmental Pollutants ; Gene Editing ; *Metabolic Engineering ; },
abstract = {Bioremediation is a promising technology for the treatment of environmental contaminants and paving new avenues for the betterment of the environment. Over the last some years, several approaches have been employed to optimize the genetic machinery of microorganisms relevant to bioremediation. Metabolic engineering is one of them that provides a new insight for bioremediation. This review envisages the critical role of these techniques toward exploring the possibilities of the creation of a new pathway, leading to pathway expansion to new substrates by assembling of catabolic modules from different origins in the same microbial cell. The recombinant DNA technology and gene editing tools were also explored for the construction of metabolically engineered microbial strains for the degradation of complex pollutants. Moreover, the importance of CRISPR-Cas system for knock-in and knock-out of genes was described by using recent studies. Further, the idea of the cocultivation of more than one metabolic engineered microbial communities is also discussed, which can be crucial in the bioremediation of multiple and complex pollutants. Finally, this review also elucidates the effective application of metabolic engineering in bioremediation through these techniques and tools.},
}
@article {pmid33242073,
year = {2021},
author = {Franck, S and Barbé, L and Ardui, S and De Vlaeminck, Y and Allemeersch, J and Dziedzicka, D and Spits, C and Vanroye, F and Hilven, P and Duqué, G and Vermeesch, JR and Gheldof, A and Sermon, K},
title = {MSH2 knock-down shows CTG repeat stability and concomitant upstream demethylation at the DMPK locus in myotonic dystrophy type 1 human embryonic stem cells.},
journal = {Human molecular genetics},
volume = {29},
number = {21},
pages = {3566-3577},
doi = {10.1093/hmg/ddaa250},
pmid = {33242073},
issn = {1460-2083},
mesh = {CRISPR-Cas Systems ; DNA Methylation ; DNA Repair ; *Demethylation ; *Genomic Instability ; Human Embryonic Stem Cells/metabolism/*pathology ; Humans ; MutS Homolog 2 Protein/*antagonists &amp; inhibitors/genetics/metabolism ; Myotonic Dystrophy/genetics/*pathology ; Myotonin-Protein Kinase/*genetics ; *Trinucleotide Repeat Expansion ; },
abstract = {Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.},
}
@article {pmid33239638,
year = {2020},
author = {Manav, MC and Van, LB and Lin, J and Fuglsang, A and Peng, X and Brodersen, DE},
title = {Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5993},
pmid = {33239638},
issn = {2041-1723},
mesh = {Archaeal Proteins/*antagonists &amp; inhibitors/metabolism/ultrastructure ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/metabolism/ultrastructure ; CRISPR-Cas Systems/genetics ; DNA Cleavage ; Host-Pathogen Interactions/genetics ; Protein Domains/genetics ; Repressor Proteins/genetics/*metabolism ; Rudiviridae/genetics/metabolism/pathogenicity ; Sulfolobus/*genetics/virology ; Viral Proteins/genetics/*metabolism/ultrastructure ; },
abstract = {A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.},
}
@article {pmid33238709,
year = {2020},
author = {Pang, B and Xu, J and Liu, Y and Peng, H and Feng, W and Cao, Y and Wu, J and Xiao, H and Pabbaraju, K and Tipples, G and Joyce, MA and Saffran, HA and Tyrrell, DL and Zhang, H and Le, XC},
title = {Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology.},
journal = {Analytical chemistry},
volume = {92},
number = {24},
pages = {16204-16212},
pmid = {33238709},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; *Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/*genetics ; },
abstract = {We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.},
}
@article {pmid33238421,
year = {2020},
author = {Wang, M and Gao, S and Zeng, W and Yang, Y and Ma, J and Wang, Y},
title = {Plant Virology Delivers Diverse Toolsets for Biotechnology.},
journal = {Viruses},
volume = {12},
number = {11},
pages = {},
pmid = {33238421},
issn = {1999-4915},
mesh = {Biotechnology/*methods ; CRISPR-Cas Systems ; Crops, Agricultural/genetics/*virology ; Gene Editing ; Gene Silencing ; Genetic Vectors ; Host Microbial Interactions/*genetics ; Plant Pathology ; Plant Viruses/*genetics ; Plants/genetics/*virology ; Vaccines ; },
abstract = {Over a hundred years of research on plant viruses has led to a detailed understanding of viral replication, movement, and host-virus interactions. The functions of vast viral genes have also been annotated. With an increased understanding of plant viruses and plant-virus interactions, various viruses have been developed as vectors to modulate gene expressions for functional studies as well as for fulfilling the needs in biotechnology. These approaches are invaluable not only for molecular breeding and functional genomics studies related to pivotal agronomic traits, but also for the production of vaccines and health-promoting carotenoids. This review summarizes the latest progress in these forefronts as well as the available viral vectors for economically important crops and beyond.},
}
@article {pmid33238136,
year = {2021},
author = {Sharma, G and Sharma, AR and Bhattacharya, M and Lee, SS and Chakraborty, C},
title = {CRISPR-Cas9: A Preclinical and Clinical Perspective for the Treatment of Human Diseases.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {2},
pages = {571-586},
pmid = {33238136},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Development ; *Gene Editing/methods ; Genetic Diseases, Inborn/*genetics/*therapy ; *Genetic Therapy/methods ; Humans ; },
abstract = {At present, the idea of genome modification has revolutionized the modern therapeutic research era. Genome modification studies have traveled a long way from gene modifications in primary cells to genetic modifications in animals. The targeted genetic modification may result in the modulation (i.e., either upregulation or downregulation) of the predefined gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) is a promising genome-editing tool that has therapeutic potential against incurable genetic disorders by modifying their DNA sequences. In comparison with other genome-editing techniques, CRISPR-Cas9 is simple, efficient, and very specific. This enabled CRISPR-Cas9 genome-editing technology to enter into clinical trials against cancer. Besides therapeutic potential, the CRISPR-Cas9 tool can also be applied to generate genetically inhibited animal models for drug discovery and development. This comprehensive review paper discusses the origin of CRISPR-Cas9 systems and their therapeutic potential against various genetic disorders, including cancer, allergy, immunological disorders, Duchenne muscular dystrophy, cardiovascular disorders, neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-related disorders, and viral infection. Finally, we discuss the different challenges, safety concerns, and strategies that can be applied to overcome the obstacles during CRISPR-Cas9-mediated therapeutic approaches.},
}
@article {pmid33238093,
year = {2020},
author = {Schilling, C and Koffas, MAG and Sieber, V and Schmid, J},
title = {Novel Prokaryotic CRISPR-Cas12a-Based Tool for Programmable Transcriptional Activation and Repression.},
journal = {ACS synthetic biology},
volume = {9},
number = {12},
pages = {3353-3363},
doi = {10.1021/acssynbio.0c00424},
pmid = {33238093},
issn = {2161-5063},
mesh = {Alcohol Oxidoreductases/genetics/metabolism ; Butylene Glycols/metabolism ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics/*metabolism ; Gene Editing/*methods ; *Gene Expression Regulation ; Lactate Dehydrogenases/genetics/metabolism ; Paenibacillus polymyxa/genetics/*metabolism ; RNA, Guide/metabolism ; *Transcriptional Activation ; },
abstract = {Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9-based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable the programmable activation of gene expression. A proof of principle of transcriptional regulation was demonstrated on the basis of fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli. Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knockouts. The simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.},
}
@article {pmid33237723,
year = {2020},
author = {Paßreiter, A and Thomas, A and Grogna, N and Delahaut, P and Thevis, M},
title = {First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS.},
journal = {Analytical chemistry},
volume = {92},
number = {24},
pages = {16322-16328},
doi = {10.1021/acs.analchem.0c04445},
pmid = {33237723},
issn = {1520-6882},
mesh = {CRISPR-Associated Protein 9/*blood ; CRISPR-Cas Systems/*genetics ; Doping in Sports/*prevention &amp; control ; Genomics ; Humans ; Streptococcus pyogenes/*enzymology ; Time Factors ; },
abstract = {The discovery of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system as a programmable, RNA-guided endonuclease has revolutionized the utilization of gene technology. Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools for genome editing. However, these advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to achieve performance-enhancing effects in sporting competitions. This abuse is classified as gene doping, which is banned in sports according to the Prohibited List of the World Anti-Doping Agency (WADA). Therefore, there is a pressing need for an adequate analytical method to detect the misuse of the CRISPR/Cas system by athletes. Hence, the first aim accomplished with this study was the identification of the exogenous protein Cas9 from the bacterium Streptococcus pyogenes (SpCas9) in plasma samples by means of a bottom-up analytical approach via immunoaffinity purification, tryptic digestion, and subsequent detection by HPLC-HRMS/MS. A qualitative method validation was conducted with three specific peptides allowing for a limit of detection of 25 ng/mL. Additionally, it was shown that the developed method is also applicable to the detection of (illicit) gene regulation through the identification of catalytically inactive Cas9. A proof-of-concept administration study employing an in vivo mouse model revealed a detection window of SpCas9 for up to 8 h post administration, confirming the suitability of the test strategy for the analysis of authentic doping control samples.},
}
@article {pmid33237352,
year = {2021},
author = {Biagioni, A and Laurenzana, A and Menicacci, B and Peppicelli, S and Andreucci, E and Bianchini, F and Guasti, D and Paoli, P and Serratì, S and Mocali, A and Calorini, L and Del Rosso, M and Fibbi, G and Chillà, A and Margheri, F},
title = {uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {78},
number = {6},
pages = {3057-3072},
pmid = {33237352},
issn = {1420-9071},
mesh = {Animals ; Antigens, CD/genetics/*metabolism ; Cadherins/genetics/*metabolism ; Cell Line ; Endothelial Cells/cytology/metabolism ; ErbB Receptors/antagonists &amp; inhibitors/genetics/metabolism ; Exosomes/*metabolism ; Gefitinib/pharmacology ; Gene Editing ; Humans ; Melanoma/metabolism/pathology ; Mice ; Mice, SCID ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Neovascularization, Physiologic ; Phosphorylation/drug effects ; RNA Interference ; RNA, Small Interfering ; Receptors, Urokinase Plasminogen Activator/antagonists &amp; inhibitors/genetics/*metabolism ; *Signal Transduction ; },
abstract = {Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.},
}
@article {pmid33236982,
year = {2020},
author = {Lupanga, U and Röhrich, R and Askani, J and Hilmer, S and Kiefer, C and Krebs, M and Kanazawa, T and Ueda, T and Schumacher, K},
title = {The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant-specific motif.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33236982},
issn = {2050-084X},
support = {SFB1101 TPA02//DFG/International ; 19H05675//Ministry of Education, Culture, Sports, Science and Technology/International ; 18H02470//Ministry of Education, Culture, Sports, Science and Technology/International ; 18K14738//Ministry of Education, Culture, Sports, Science and Technology/International ; },
mesh = {Arabidopsis/*enzymology/*genetics ; Arabidopsis Proteins/*genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Enzymologic/*physiology ; Gene Expression Regulation, Plant/*physiology ; Genotype ; Mutagenesis, Site-Directed ; Phylogeny ; Plant Roots/enzymology ; Pollen ; Seeds ; Vacuolar Proton-Translocating ATPases/*genetics ; },
abstract = {The V-ATPase is a versatile proton-pump found in a range of endomembrane compartments yet the mechanisms governing its differential targeting remain to be determined. In Arabidopsis, VHA-a1 targets the V-ATPase to the TGN/EE whereas VHA-a2 and VHA-a3 are localized to the tonoplast. We report here that the VHA-a1 targeting domain serves as both an ER-exit and as a TGN/EE-retention motif and is conserved among seed plants. In contrast, Marchantia encodes a single VHA-isoform that localizes to the TGN/EE and the tonoplast in Arabidopsis. Analysis of CRISPR/Cas9 generated null alleles revealed that VHA-a1 has an essential function for male gametophyte development but acts redundantly with the tonoplast isoforms during vegetative growth. We propose that in the absence of VHA-a1, VHA-a3 is partially re-routed to the TGN/EE. Our findings contribute to understanding the evolutionary origin of V-ATPase targeting and provide a striking example that differential localization does not preclude functional redundancy.},
}
@article {pmid33236499,
year = {2021},
author = {Li, J and Jiao, G and Sun, Y and Chen, J and Zhong, Y and Yan, L and Jiang, D and Ma, Y and Xia, L},
title = {Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9.},
journal = {Plant biotechnology journal},
volume = {19},
number = {5},
pages = {937-951},
pmid = {33236499},
issn = {1467-7652},
mesh = {Amylose ; CRISPR-Cas Systems/genetics ; Plant Breeding ; *Starch/metabolism ; *Triticum/genetics/metabolism ; },
abstract = {Foods high in amylose content and resistant starch (RS) offer great potential to improve human health and lower the risk of serious noninfectious diseases. Common wheat (Triticum aestivum L.) is a major staple food crop globally. However, the RS contents in the grains of modern wheat varieties are low. Here, we report the generation of high-amylose wheat through targeted mutagenesis of TaSBEIIa in a modern winter wheat cv Zhengmai 7698 (ZM) and a spring wheat cv Bobwhite by CRISPR/Cas9, respectively. We generated a series of transgene-free mutant lines either with partial or triple-null TasbeIIa alleles in ZM and Bobwhite, respectively. Analyses of starch composition, structure and properties revealed that the effects of partial or triple-null alleles were dosage dependent with triple-null lines demonstrated more profound impacts on starch composition, fine structures of amylopectin and physiochemical and nutritional properties. The flours of triple-null lines possessed significantly increased amylose, RS, protein and soluble pentosan contents which benefit human health. Baking quality analyses indicated that the high-amylose flours may be used as additives or for making cookies. Collectively, we successfully modified the starch composition, structure and properties through targeted mutagenesis of TaSBEIIa by CRISPR/Cas9 in both winter and spring wheat varieties and generated transgene-free high-amylose wheat. Our finding provides deep insights on the role of TaSBEIIa in determining starch composition, structure, properties and end-use quality in different genetic backgrounds and improving RS content with multiple breeding and end-use applications in cereal crop species through genome editing for health benefits.},
}
@article {pmid33236397,
year = {2021},
author = {Zhou, J and Yan, Q and Tang, C and Liao, Y and Zhang, Q and Wang, X and Zhou, X and Lai, L and Zou, Q},
title = {Development of a rabbit model of Wiskott-Aldrich syndrome.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {2},
pages = {e21226},
doi = {10.1096/fj.202002118RR},
pmid = {33236397},
issn = {1530-6860},
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Knockout Techniques/methods ; Phenotype ; *Rabbits ; Wiskott-Aldrich Syndrome/*genetics/pathology ; Wiskott-Aldrich Syndrome Protein/*genetics ; },
abstract = {The Wiskott-Aldrich syndrome (WAS) is a severe recessive X-linked immunodeficiency resulting from loss-of-function mutations in the WAS gene. Mouse is the only mammalian model used for investigation of WAS pathogenesis. However, the mouse model does not accurately recapitulate WAS clinical phenotypes, thus, limiting its application in WAS clinical research. Herein, we report the generation of WAS knockout (KO) rabbits via embryo co-injection of Cas9 mRNA and a pair of sgRNAs targeting exons 2 and 7. WAS KO rabbits exhibited many symptoms similar to those of WAS patients, including thrombocytopenia, bleeding tendency, infections, and reduced numbers of T cell in the spleen and peripheral blood. The WAS KO rabbit model provides a new valuable tool for preclinical trials of WAS treatment.},
}
@article {pmid33235312,
year = {2020},
author = {Hunziker, J and Nishida, K and Kondo, A and Kishimoto, S and Ariizumi, T and Ezura, H},
title = {Multiple gene substitution by Target-AID base-editing technology in tomato.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20471},
pmid = {33235312},
issn = {2045-2322},
mesh = {Alleles ; Base Pairing ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Cytidine Deaminase/*metabolism ; Gene Editing/*methods ; Lycopersicon esculentum/genetics/*growth &amp; development/metabolism ; Plant Proteins/*genetics ; },
abstract = {The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.},
}
@article {pmid33235250,
year = {2020},
author = {Kumar, S and Punetha, M and Jose, B and Bharati, J and Khanna, S and Sonwane, A and Green, JA and Whitworth, K and Sarkar, M},
title = {Modulation of granulosa cell function via CRISPR-Cas fuelled editing of BMPR-IB gene in goats (Capra hircus).},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20446},
pmid = {33235250},
issn = {2045-2322},
mesh = {Animals ; Bone Morphogenetic Protein 4/*pharmacology ; Bone Morphogenetic Protein Receptors, Type I/*genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Cell Survival/drug effects ; Cells, Cultured ; Female ; Gene Knockout Techniques/*methods ; Goats ; Granulosa Cells/*cytology/drug effects/metabolism ; Loss of Function Mutation ; Signal Transduction/drug effects ; },
abstract = {BMPs are multifunctional growth factors implicated in regulating the ovarian function as key intra-ovarian factors. Biological effects of BMPs are mediated through binding with membrane bound receptors like BMPR-IB and initiating downstream Smad signaling pathway. FecB mutation, regarded as a loss of function mutation in the BMPR-IB gene was identified in certain sheep breeds having high fecundity. Similar type of fecundity genes in goats have not been discovered so far. Hence, the current study was designed to investigate the effects of BMPR-IB gene modulation on granulosa cell function in goats. The BMPR-IB gene was knocked out using CRISPR-Cas technology in granulosa cells and cultured in vitro with BMP-4 stimulation for three different durations In addition, the FecB mutation was introduced in the BMPR-IB gene applying Easi-CRISPR followed by BMP-4/7 stimulation for 72 h. Steroidogenesis and cell viability were studied to explore the granulosa cell function on BMPR-IB gene modulation. BMPRs were found to be expressed stage specifically in granulosa cells of goats. Higher transcriptional abundance of R-Smads, LHR and FSHR indicating sensitisation of Smad signaling and increased gonadotropin sensitivity along with a significant reduction in the cell proliferation and viability was observed in granulosa cells upon BMPR-IB modulation. The inhibitory action of BMP-4/7 on P4 secretion was abolished in both KO and KI cells. Altogether, the study has revealed an altered Smad signaling, steroidogenesis and cell viability upon modulation of BMPR-IB gene in granulosa cells similar to that are documented in sheep breeds carrying the FecB mutation.},
}
@article {pmid33234550,
year = {2021},
author = {Lange, KI and Tsiropoulou, S and Kucharska, K and Blacque, OE},
title = {Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans.},
journal = {Disease models &amp; mechanisms},
volume = {14},
number = {1},
pages = {},
pmid = {33234550},
issn = {1754-8411},
support = {P40 OD010440/OD/NIH HHS/United States ; 16/BBSRC/3394/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Abnormalities, Multiple/*genetics/physiopathology ; Alleles ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/metabolism ; Cerebellum/*abnormalities/physiopathology ; Cilia/*metabolism ; Disease Models, Animal ; Eye Abnormalities/*genetics/physiopathology ; Gene Editing ; Genetic Association Studies ; Genotype ; Humans ; Kidney Diseases, Cystic/*genetics/physiopathology ; Membrane Proteins/metabolism ; Mutation ; *Mutation, Missense ; Phenotype ; Retina/*abnormalities/physiopathology ; },
abstract = {Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have used a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S and G155S) in mksr-2/B9D2 associated with Joubert syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment and regulates the molecular composition and sensory/signalling functions of the cilium. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm that both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and MKS-2/TMEM216. Together, these data establish C. elegans as a model for interpreting JBTS mutations and provide further insight into MKS module organisation. This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid33233548,
year = {2020},
author = {Ghribi, M and Nouemssi, SB and Meddeb-Mouelhi, F and Desgagné-Penix, I},
title = {Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas.},
journal = {Life (Basel, Switzerland)},
volume = {10},
number = {11},
pages = {},
pmid = {33233548},
issn = {2075-1729},
abstract = {Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.},
}
@article {pmid33233344,
year = {2020},
author = {Saifaldeen, M and Al-Ansari, DE and Ramotar, D and Aouida, M},
title = {CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering.},
journal = {Cells},
volume = {9},
number = {11},
pages = {},
pmid = {33233344},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/*physiology ; Deoxyribonucleases, Type II Site-Specific/*metabolism ; Gene Editing/*methods ; Genomics/*methods ; Humans ; Tissue Engineering/*methods ; },
abstract = {The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR-FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.},
}
@article {pmid33232900,
year = {2020},
author = {Li, J and Li, Y and Zhan, XY and Ran, X and Tse, HF and Dang, S and Nie, Y and Huang, K},
title = {Generation of GADD45A gene knockout human embryonic stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102090},
doi = {10.1016/j.scr.2020.102090},
pmid = {33232900},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle Proteins ; *Cell Line ; Gene Knockout Techniques ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {GADD45A is a DNA damage and stressful growth arrest inducible protein, also it is shown to a be tumor suppressor gene and a chromatin relaxer associated with opening chromatin during the somatic reprogramming. However, its role in human embryonic stem cells and human embryonic stem cell modeled development has been merely documented. To illustrate the function of GADD45A in the human embryonic stem cell biology, we reported a GADD45A knockout human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting. This cell line displayed normal karyotype, pluripotent stem cell marker expression and differentiation potential both in vivo and vitro.},
}
@article {pmid33232480,
year = {2020},
author = {Basar, R and Daher, M and Rezvani, K},
title = {Next-generation cell therapies: the emerging role of CAR-NK cells.},
journal = {Blood advances},
volume = {4},
number = {22},
pages = {5868-5876},
pmid = {33232480},
issn = {2473-9537},
support = {P01 CA148600/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; P50 CA100632/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; },
mesh = {Cell- and Tissue-Based Therapy ; Gene Editing ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; *Receptors, Chimeric Antigen/genetics ; },
abstract = {T cells engineered with chimeric antigen receptors (CARs) have revolutionized the field of cell therapy and changed the paradigm of treatment for many patients with relapsed or refractory B-cell malignancies. Despite this progress, there are limitations to CAR-T cell therapy in both the autologous and allogeneic settings, including practical, logistical, and toxicity issues. Given these concerns, there is a rapidly growing interest in natural killer cells as alternative vehicles for CAR engineering, given their unique biological features and their established safety profile in the allogeneic setting. Other immune effector cells, such as invariant natural killer T cells, γδ T cells, and macrophages, are attracting interest as well and eventually may be added to the repertoire of engineered cell therapies against cancer. The pace of these developments will undoubtedly benefit from multiple innovative technologies, such as the CRISPR-Cas gene editing system, which offers great potential to enhance the natural ability of immune effector cells to eliminate refractory cancers.},
}
@article {pmid33232302,
year = {2021},
author = {Xu, J and Livraghi-Butrico, A and Hou, X and Rajagopalan, C and Zhang, J and Song, J and Jiang, H and Wei, HG and Wang, H and Bouhamdan, M and Ruan, J and Yang, D and Qiu, Y and Xie, Y and Barrett, R and McClellan, S and Mou, H and Wu, Q and Chen, X and Rogers, TD and Wilkinson, KJ and Gilmore, RC and Esther, CR and Zaman, K and Liang, X and Sobolic, M and Hazlett, L and Zhang, K and Frizzell, RA and Gentzsch, M and O'Neal, WK and Grubb, BR and Chen, YE and Boucher, RC and Sun, F},
title = {Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.},
journal = {JCI insight},
volume = {6},
number = {1},
pages = {},
pmid = {33232302},
issn = {2379-3708},
support = {R01 DK068196/DK/NIDDK NIH HHS/United States ; R21 OD020187/OD/NIH HHS/United States ; UH3 HL123645/HL/NHLBI NIH HHS/United States ; P30 DK020572/DK/NIDDK NIH HHS/United States ; UH2 HL123645/HL/NHLBI NIH HHS/United States ; P01 HL108808/HL/NHLBI NIH HHS/United States ; P30 DK065988/DK/NIDDK NIH HHS/United States ; P30 ES010126/ES/NIEHS NIH HHS/United States ; R01 HL136961/HL/NHLBI NIH HHS/United States ; R01 HL133162/HL/NHLBI NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cystic Fibrosis/*genetics/pathology/physiopathology ; Cystic Fibrosis Transmembrane Conductance Regulator/*antagonists &amp; inhibitors/*genetics/metabolism ; Disease Models, Animal ; Female ; Gastrointestinal Tract/pathology/physiopathology ; Gene Knockout Techniques ; Humans ; Male ; Phenotype ; RNA, Messenger/genetics/metabolism ; Rabbits ; Respiratory System/pathology/physiopathology ; Tissue Distribution ; Transcriptome ; },
abstract = {Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.},
}
@article {pmid33231685,
year = {2021},
author = {Abid, HZ and Young, E and McCaffrey, J and Raseley, K and Varapula, D and Wang, HY and Piazza, D and Mell, J and Xiao, M},
title = {Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.},
journal = {Nucleic acids research},
volume = {49},
number = {2},
pages = {e8},
pmid = {33231685},
issn = {1362-4962},
support = {R01 HG005946/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Base Sequence ; Benzoxazoles/analysis ; *CRISPR-Cas Systems ; Chromosome Mapping/*methods ; Chromosomes, Bacterial/*genetics ; Computer Simulation ; Conserved Sequence/genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial/genetics ; Fluorescent Dyes/analysis ; Gene Editing/methods ; Genome, Bacterial ; Genome, Human ; Haemophilus influenzae/drug effects/*genetics ; Haplotypes/genetics ; Humans ; Lab-On-A-Chip Devices ; Nalidixic Acid/pharmacology ; Novobiocin/pharmacology ; Nucleotide Motifs/genetics ; Polymorphism, Single Nucleotide ; Quinolinium Compounds/analysis ; RNA, Guide/chemical synthesis/*genetics ; Repetitive Sequences, Nucleic Acid/genetics ; Sequence Alignment ; Staining and Labeling/methods ; Viral Proteins ; },
abstract = {Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.},
}
@article {pmid33230293,
year = {2021},
author = {Vo, PLH and Ronda, C and Klompe, SE and Chen, EE and Acree, C and Wang, HH and Sternberg, SH},
title = {CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering.},
journal = {Nature biotechnology},
volume = {39},
number = {4},
pages = {480-489},
pmid = {33230293},
issn = {1546-1696},
support = {R01 AI132403/AI/NIAID NIH HHS/United States ; U01 GM110714/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA Transposable Elements ; Gene Editing/*methods ; Genome, Bacterial ; Plasmids/genetics ; RNA, Guide/*genetics ; Vibrio cholerae/*genetics ; },
abstract = {Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.},
}
@article {pmid33230133,
year = {2020},
author = {O'Brien, RE and Santos, IC and Wrapp, D and Bravo, JPK and Schwartz, EA and Brodbelt, JS and Taylor, DW},
title = {Structural basis for assembly of non-canonical small subunits into type I-C Cascade.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5931},
pmid = {33230133},
issn = {2041-1723},
support = {R01 GM121714/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/metabolism ; Binding Sites ; CRISPR-Associated Proteins/*chemistry/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism ; Desulfovibrio vulgaris/chemistry/genetics ; Models, Molecular ; Multiprotein Complexes/chemistry/metabolism ; Nucleotide Motifs ; Protein Conformation ; Protein Subunits/chemistry/metabolism ; RNA, Bacterial/chemistry/metabolism ; },
abstract = {Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.},
}
@article {pmid33229326,
year = {2020},
author = {Lin, MT and Lai, LL and Zhao, M and Lin, BW and Yao, XP},
title = {[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {10},
pages = {1017-1027},
doi = {10.16288/j.yczz.20-138},
pmid = {33229326},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Mice ; Mice, Knockout ; *Models, Animal ; RNA, Guide/genetics ; *Sodium-Phosphate Cotransporter Proteins, Type III/genetics ; },
abstract = {Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.},
}
@article {pmid33229321,
year = {2020},
author = {Bao, LW and Zhou, YY and Zeng, FY},
title = {[Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {10},
pages = {949-964},
doi = {10.16288/j.yczz.20-110},
pmid = {33229321},
issn = {0253-9772},
mesh = {CRISPR-Cas Systems/genetics ; Clinical Trials as Topic ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genetic Therapy ; *Hemophilia A/genetics/therapy ; Humans ; Research/trends ; Technology ; *beta-Thalassemia/genetics/therapy ; },
abstract = {Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for β-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.},
}
@article {pmid33228790,
year = {2020},
author = {Takadera, M and Satomi, K and Szulzewsky, F and Cimino, PJ and Holland, EC and Yamamoto, T and Ichimura, K and Ozawa, T},
title = {Phenotypic characterization with somatic genome editing and gene transfer reveals the diverse oncogenicity of ependymoma fusion genes.},
journal = {Acta neuropathologica communications},
volume = {8},
number = {1},
pages = {203},
pmid = {33228790},
issn = {2051-5960},
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Animals ; Brain Neoplasms/*genetics ; CRISPR-Cas Systems ; Carcinogenesis/*genetics ; Cells, Cultured ; Chromothripsis ; DNA Copy Number Variations ; DNA-Binding Proteins/*genetics ; Ependymoma/*genetics ; Gene Editing ; Gene Transfer Techniques ; Mice ; Oncogene Fusion/*genetics ; Phenotype ; Transcription Factor RelA/*genetics ; Transcription Factors/*genetics ; YAP-Signaling Proteins ; },
abstract = {Recurrent RELA and YAP1 fusions are intimately associated with tumorigenesis in supratentorial ependymomas. Chromothripsis and focal copy number alterations involving 11q are hallmarks of these tumors. However, it is unknown whether the chromosomal alterations are a direct causal event resulting in fusion transcripts. In addition, the biological significance of the RELA fusion variants and YAP1 fusions is not yet fully characterized. In this study, we generated gene rearrangements on 11q with the CRISPR/Cas9 system and investigated the formation of oncogenic ependymoma fusion genes. Further, we examined the oncogenic potential of RELA fusion variants and YAP1 fusions in a lentiviral gene transfer model. We observed that endogenous RELA fusion events were successfully induced by CRISPR/Cas9-mediated genome rearrangement in cultured cells. In vivo genome editing in mouse brain resulted in the development of ependymoma-like brain tumors that harbored the Rela fusion gene. All RELA fusion variants tested, except a variant lacking the Rel homology domain, were able to induce tumor formation, albeit with different efficacy. Furthermore, expression of YAP1-FAM118B and YAP1-MAMLD1 fusions induced the formation of spindle-cell-like tumors at varying efficacy. Our results indicate that chromosomal rearrangements involving the Rela locus are the causal event for the formation of Rela fusion-driven ependymomas in mice. Furthermore, the type of RELA. fusion might affect the aggressiveness of tumors and that the Rel homology domain is essential for the oncogenic functions of RELA. fusions. The YAP1 fusion genes are also oncogenic when expressed in mice.},
}
@article {pmid33228647,
year = {2020},
author = {Henkel, L and Rauscher, B and Schmitt, B and Winter, J and Boutros, M},
title = {Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {174},
pmid = {33228647},
issn = {1741-7007},
mesh = {*CRISPR-Cas Systems ; Genes, Essential ; *Genome, Human ; Humans ; Single-Cell Analysis/*methods ; },
abstract = {BACKGROUND: In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria.<br><br>RESULTS: We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes.<br><br>CONCLUSION: We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.},
}
@article {pmid33227699,
year = {2021},
author = {Zhang, M and Eshraghian, EA and Jammal, OA and Zhang, Z and Zhu, X},
title = {CRISPR technology: The engine that drives cancer therapy.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {133},
number = {},
pages = {111007},
doi = {10.1016/j.biopha.2020.111007},
pmid = {33227699},
issn = {1950-6007},
mesh = {Animals ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Diffusion of Innovation ; *Gene Editing ; Gene Expression Regulation, Neoplastic ; Gene Transfer Techniques ; *Genetic Therapy/adverse effects ; Humans ; *Immunotherapy/adverse effects ; Molecular Diagnostic Techniques ; *Molecular Targeted Therapy/adverse effects ; Neoplasms/genetics/immunology/metabolism/*therapy ; Predictive Value of Tests ; },
abstract = {CRISPR gene editing technology belongs to the third generation of gene editing technology. Since its discovery, it has attracted the attention of a large number of researchers. Investigators have published a series of academic articles and obtained breakthrough research results through in-depth research. In recent years, this technology has developed rapidly and been widely applied in many fields, especially in medicine. This review focuses on concepts of CRISPR gene editing technology, its application in cancer treatments, its existing limitations, and the new progress in recent years for detailed analysis and sharing.},
}
@article {pmid33226787,
year = {2021},
author = {Shokr, A and Pacheco, LGC and Thirumalaraju, P and Kanakasabapathy, MK and Gandhi, J and Kartik, D and Silva, FSR and Erdogmus, E and Kandula, H and Luo, S and Yu, XG and Chung, RT and Li, JZ and Kuritzkes, DR and Shafiee, H},
title = {Mobile Health (mHealth) Viral Diagnostics Enabled with Adaptive Adversarial Learning.},
journal = {ACS nano},
volume = {15},
number = {1},
pages = {665-673},
pmid = {33226787},
issn = {1936-086X},
support = {R01 AI118502/AI/NIAID NIH HHS/United States ; R01 AI138800/AI/NIAID NIH HHS/United States ; R61 AI140489/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, Viral/isolation &amp; purification ; COVID-19/*diagnosis ; COVID-19 Testing/*instrumentation/*methods ; CRISPR-Cas Systems ; Communicable Disease Control ; *Deep Learning ; Disaster Planning ; Humans ; Image Processing, Computer-Assisted/methods ; Metal Nanoparticles/chemistry ; Neural Networks, Computer ; Platinum ; Point-of-Care Testing ; Public Health ; Reproducibility of Results ; *Signal Processing, Computer-Assisted ; Smartphone ; Telemedicine/*methods ; },
abstract = {Deep-learning (DL)-based image processing has potential to revolutionize the use of smartphones in mobile health (mHealth) diagnostics of infectious diseases. However, the high variability in cellphone image data acquisition and the common need for large amounts of specialist-annotated images for traditional DL model training may preclude generalizability of smartphone-based diagnostics. Here, we employed adversarial neural networks with conditioning to develop an easily reconfigurable virus diagnostic platform that leverages a dataset of smartphone-taken microfluidic chip photos to rapidly generate image classifiers for different target pathogens on-demand. Adversarial learning was also used to augment this real image dataset by generating 16,000 realistic synthetic microchip images, through style generative adversarial networks (StyleGAN). We used this platform, termed smartphone-based pathogen detection resource multiplier using adversarial networks (SPyDERMAN), to accurately detect different intact viruses in clinical samples and to detect viral nucleic acids through integration with CRISPR diagnostics. We evaluated the performance of the system in detecting five different virus targets using 179 patient samples. The generalizability of the system was confirmed by rapid reconfiguration to detect SARS-CoV-2 antigens in nasal swab samples (n = 62) with 100% accuracy. Overall, the SPyDERMAN system may contribute to epidemic preparedness strategies by providing a platform for smartphone-based diagnostics that can be adapted to a given emerging viral agent within days of work.},
}
@article {pmid33226620,
year = {2021},
author = {Weltner, J and Trokovic, R},
title = {Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2239},
number = {},
pages = {175-198},
doi = {10.1007/978-1-0716-1084-8_12},
pmid = {33226620},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Cellular Reprogramming/*genetics ; Electroporation/methods ; Fibroblasts/cytology/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Immunohistochemistry ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Plasmids/genetics/isolation &amp; purification/metabolism ; Polymerase Chain Reaction ; RNA, Guide/genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; SOXB1 Transcription Factors/genetics/metabolism ; Transcription Factors/genetics/*metabolism ; },
abstract = {CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs.},
}
@article {pmid33226616,
year = {2021},
author = {Wen, W and Cheng, T and Zhang, XB},
title = {Episomal Reprogramming of Human Peripheral Blood Mononuclear Cells into Pluripotency.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2239},
number = {},
pages = {117-133},
doi = {10.1007/978-1-0716-1084-8_8},
pmid = {33226616},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Cells, Cultured ; Cellular Reprogramming/*genetics ; Cryopreservation/methods ; Electroporation/instrumentation/methods ; Feeder Cells ; Flow Cytometry ; Gene Editing/*methods ; Genetic Vectors ; Humans ; Immunohistochemistry ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Karyotyping ; Kruppel-Like Factor 4 ; Kruppel-Like Transcription Factors/genetics/metabolism ; Leukocytes, Mononuclear/*cytology/metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Plasmids/*genetics/metabolism ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; bcl-X Protein/genetics/metabolism ; },
abstract = {Peripheral blood is an easily accessible cell resource for reprogramming into pluripotency by episomal vectors. Here, we describe an approach for efficient generation of integration-free induced pluripotent stem cells (iPSCs) under feeder or feeder-free conditions. Additionally, in combination with the CRISPR-Cas9 genome-editing system, we can directly generate edited iPSCs from blood cells. With this protocol, one can easily generate either integration-free iPSCs or genetically edited iPSCs from peripheral blood at high efficiency.},
}
@article {pmid33226067,
year = {2021},
author = {Padilha, VA and Alkhnbashi, OS and Tran, VD and Shah, SA and Carvalho, ACPLF and Backofen, R},
title = {Casboundary: automated definition of integral Cas cassettes.},
journal = {Bioinformatics (Oxford, England)},
volume = {37},
number = {10},
pages = {1352-1359},
pmid = {33226067},
issn = {1367-4811},
mesh = {*Archaea/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; },
abstract = {MOTIVATION: CRISPR-Cas are important systems found in most archaeal and many bacterial genomes, providing adaptive immunity against mobile genetic elements in prokaryotes. The CRISPR-Cas systems are encoded by a set of consecutive cas genes, here termed cassette. The identification of cassette boundaries is key for finding cassettes in CRISPR research field. This is often carried out by using Hidden Markov Models and manual annotation. In this article, we propose the first method able to automatically define the cassette boundaries. In addition, we present a Cas-type predictive model used by the method to assign each gene located in the region defined by a cassette's boundaries a Cas label from a set of pre-defined Cas types. Furthermore, the proposed method can detect potentially new cas genes and decompose a cassette into its modules.<br><br>RESULTS: We evaluate the predictive performance of our proposed method on data collected from the two most recent CRISPR classification studies. In our experiments, we obtain an average similarity of 0.86 between the predicted and expected cassettes. Besides, we achieve F-scores above 0.9 for the classification of cas genes of known types and 0.73 for the unknown ones. Finally, we conduct two additional study cases, where we investigate the occurrence of potentially new cas genes and the occurrence of module exchange between different genomes.<br><br>https://github.com/BackofenLab/Casboundary.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid33226023,
year = {2020},
author = {Laoharawee, K and Johnson, MJ and Lahr, WS and Peterson, JJ and Webber, BR and Moriarity, BS},
title = {Genome Engineering of Primary Human B Cells Using CRISPR/Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {165},
pages = {},
pmid = {33226023},
issn = {1940-087X},
support = {R01 AI146009/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD19/metabolism ; B-Lymphocytes/cytology/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Proliferation ; Cells, Cultured ; Dependovirus/metabolism ; Genetic Engineering/*methods ; *Genome, Human ; Green Fluorescent Proteins/metabolism ; Humans ; INDEL Mutation/genetics ; },
abstract = {B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.},
}
@article {pmid33225386,
year = {2020},
author = {Park, JW and Lee, JH and Han, JS and Shin, SP and Park, TS},
title = {Muscle differentiation induced by p53 signaling pathway-related genes in myostatin-knockout quail myoblasts.},
journal = {Molecular biology reports},
volume = {47},
number = {12},
pages = {9531-9540},
doi = {10.1007/s11033-020-05935-0},
pmid = {33225386},
issn = {1573-4978},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Frameshift Mutation ; Gene Editing ; Gene Expression Regulation ; High-Throughput Nucleotide Sequencing ; Leukemia Inhibitory Factor/genetics/metabolism ; Muscle Development/*genetics ; Muscle, Skeletal/cytology/metabolism ; Myoblasts/cytology/*metabolism ; Myostatin/deficiency/*genetics ; Quail/*genetics/metabolism ; Signal Transduction ; Tumor Suppressor Protein p53/*genetics/metabolism ; },
abstract = {The myostatin (MSTN) gene is of interest in the livestock industry because mutations in this gene are closely related to growth performance and muscle differentiation. Thus, in this study, we established MSTN knockout (KO) quail myoblasts (QM7) and investigated the regulatory pathway of the myogenic differentiation process. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate MSTN KO QM7 cells and subsequently isolated a single cell-derived MSTN KO QM7 subline with 10- and 16-nucleotide deletions that induced translational frameshift mutations. The differentiation capacity and proliferation rate of MSTN KO QM7 cells were enhanced. We conducted next-generation-sequencing (NGS) analysis to compare the global gene expression profiles of wild-type (WT) QM7 and MSTN KO QM7 cells. Intriguingly, NGS expression profiles showed different expression patterns of p21 and p53 in MSTN KO QM7 cells. Moreover, we identified downregulated expression patterns of leukemia inhibitory factor and DNA Damage Inducible Transcript 4, which are genes in the p53 signaling pathway. Using quantitative RT-PCR (qRT-PCR) analysis and western blotting, we concluded that p53-related genes promote the cell cycle by upregulating p21 and enhancing muscle differentiation in MSTN KO QM7 cells. These results could be applied to improve economic traits in commercial poultry by regulating MSTN-related networks.},
}
@article {pmid33225338,
year = {2021},
author = {Pan, MM and Wang, YF and Wang, L and Yu, X and Xu, L},
title = {Recent advances in visual detection for cancer biomarkers and infectious pathogens.},
journal = {Journal of materials chemistry. B},
volume = {9},
number = {1},
pages = {35-52},
doi = {10.1039/d0tb01883j},
pmid = {33225338},
issn = {2050-7518},
mesh = {Animals ; Bioluminescence Resonance Energy Transfer Techniques/*methods ; Biomarkers, Tumor/genetics/*metabolism ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/physiology ; Colorimetry/methods ; Humans ; Neoplasms/diagnosis/*metabolism ; Surface Plasmon Resonance/methods ; },
abstract = {It is an urgency to detect infectious pathogens or cancer biomarkers using rapid, simple, convenient and cost-effective methods in complex biological samples. Many existing approaches (traditional virus culture, ELISA or PCR) for the pathogen and biomarker assays face several challenges in the clinical applications that require lengthy time, sophisticated sample pre-treatment and expensive instruments. Due to the simple and rapid detection manner as well as no requirement of expensive equipment, many visual detection methods have been considered to resolve the aforementioned problems. Meanwhile, various new materials and colorimetric/fluorescent methods have been tried to construct new biosensors for infectious pathogens and biomarkers. However, the recent progress of these aspects is rarely reviewed, especially in terms of integration of new materials, microdevice and detection mechanism into the visual detection systems. Herein, we provide a broad field of view to discuss the recent progress in the visual detection of infectious pathogens and cancer biomarkers along with the detection mechanism, new materials, novel detection methods, special targets as well as multi-functional microdevices and systems. The novel visual approaches for the infectious pathogens and biomarkers, such as bioluminescence resonance energy transfer (BRET), metal-induced metallization and clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors, are discussed. Additionally, recent advancements in visual assays utilizing various new materials for proteins, nucleic acids, viruses, exosomes and small molecules are comprehensively reviewed. Future perspectives on the visual sensing systems for infectious pathogens and cancers are also proposed.},
}
@article {pmid33222517,
year = {2021},
author = {Elkhadragy, L and Regan, MR and M Totura, W and Goli, KD and Patel, S and Garcia, K and Stewart, M and Schook, LB and Gaba, RC and Schachtschneider, KM},
title = {Generation of genetically tailored porcine liver cancer cells by CRISPR/Cas9 editing.},
journal = {BioTechniques},
volume = {70},
number = {1},
pages = {37-48},
pmid = {33222517},
issn = {1940-9818},
support = {R21 CA219461/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/genetics ; Cell Line ; Gene Editing ; *Liver Neoplasms/genetics ; Swine ; },
abstract = {Pigs provide a valuable large animal model for several diseases due to their similarity with humans in anatomy, physiology, genetics and drug metabolism. We recently generated a porcine model for TP53[R167H] and KRAS[G12D] driven hepatocellular carcinoma (HCC) by autologous liver implantation. Here we describe a streamlined approach for developing genetically tailored porcine HCC cells by CRISPR/Cas9 gene editing and isolation of homogenous genetically validated cell clones. The combination of CRISPR/Cas9 editing of HCC cells described herein with the orthotopic HCC model enables development of various porcine HCC models, each with a specific mutational profile. This allows modeling the effect of different driver mutation combinations on tumor progression and in vivo testing of novel targeted therapeutic approaches in a clinically relevant large animal model.},
}
@article {pmid33222361,
year = {2021},
author = {Syombua, ED and Zhang, Z and Tripathi, JN and Ntui, VO and Kang, M and George, OO and Edward, NK and Wang, K and Yang, B and Tripathi, L},
title = {A CRISPR/Cas9-based genome-editing system for yam (Dioscorea spp.).},
journal = {Plant biotechnology journal},
volume = {19},
number = {4},
pages = {645-647},
pmid = {33222361},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Dioscorea/genetics ; Gene Editing ; },
}
@article {pmid33221881,
year = {2021},
author = {Mathis, AD and Otto, RM and Reynolds, KA},
title = {A simplified strategy for titrating gene expression reveals new relationships between genotype, environment, and bacterial growth.},
journal = {Nucleic acids research},
volume = {49},
number = {1},
pages = {e6},
pmid = {33221881},
issn = {1362-4962},
support = {R01 GM136842/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Division/genetics ; Computational Biology/*methods ; Escherichia coli/*genetics/growth &amp; development ; *Gene Expression Regulation, Bacterial ; Gene-Environment Interaction ; Genetic Techniques ; Genotype ; Mutation ; RNA, Guide/*genetics ; },
abstract = {A lack of high-throughput techniques for making titrated, gene-specific changes in expression limits our understanding of the relationship between gene expression and cell phenotype. Here, we present a generalizable approach for quantifying growth rate as a function of titrated changes in gene expression level. The approach works by performing CRISPRi with a series of mutated single guide RNAs (sgRNAs) that modulate gene expression. To evaluate sgRNA mutation strategies, we constructed a library of 5927 sgRNAs targeting 88 genes in Escherichia coli MG1655 and measured the effects on growth rate. We found that a compounding mutational strategy, through which mutations are incrementally added to the sgRNA, presented a straightforward way to generate a monotonic and gradated relationship between mutation number and growth rate effect. We also implemented molecular barcoding to detect and correct for mutations that 'escape' the CRISPRi targeting machinery; this strategy unmasked deleterious growth rate effects obscured by the standard approach of ignoring escapers. Finally, we performed controlled environmental variations and observed that many gene-by-environment interactions go completely undetected at the limit of maximum knockdown, but instead manifest at intermediate expression perturbation strengths. Overall, our work provides an experimental platform for quantifying the phenotypic response to gene expression variation.},
}
@article {pmid33221434,
year = {2021},
author = {Li, N and Gou, S and Wang, J and Zhang, Q and Huang, X and Xie, J and Li, L and Jin, Q and Ouyang, Z and Chen, F and Ge, W and Shi, H and Liang, Y and Zhuang, Z and Zhao, X and Lian, M and Ye, Y and Quan, L and Wu, H and Lai, L and Wang, K},
title = {CRISPR/Cas9-Mediated Gene Correction in Newborn Rabbits with Hereditary Tyrosinemia Type I.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {3},
pages = {1001-1015},
pmid = {33221434},
issn = {1525-0024},
mesh = {Animals ; Animals, Newborn ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Dependovirus/*genetics ; Disease Models, Animal ; Female ; *Gene Editing ; Gene Expression Regulation ; Genetic Therapy ; Genetic Vectors/*administration &amp; dosage ; Hydrolases/*genetics ; Kidney/metabolism ; Liver/metabolism ; Male ; RNA-Seq ; Rabbits ; Tyrosinemias/genetics/pathology/*therapy ; },
abstract = {Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.},
}
@article {pmid33220661,
year = {2021},
author = {Tao, Y and Yi, K and Hu, H and Shao, D and Li, M},
title = {Coassembly of nucleus-targeting gold nanoclusters with CRISPR/Cas9 for simultaneous bioimaging and therapeutic genome editing.},
journal = {Journal of materials chemistry. B},
volume = {9},
number = {1},
pages = {94-100},
doi = {10.1039/d0tb01925a},
pmid = {33220661},
issn = {2050-7518},
mesh = {CRISPR-Associated Protein 9/administration &amp; dosage/genetics/*metabolism ; CRISPR-Cas Systems/drug effects/*physiology ; Cell Nucleus/drug effects/genetics/*metabolism ; Cell Survival/drug effects/physiology ; Gene Editing/*methods ; Gold/administration &amp; dosage/*metabolism ; Humans ; *Metal Nanoparticles/administration &amp; dosage ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology enables genome editing with high precision and versatility and has been widely utilized to combat viruses, bacteria, cancers, and genetic diseases. Nonviral nanocarriers can overcome several limitations of viral vehicles, including immunogenicity, inflammation, carcinogenicity, and low versatility, and thus represent promising platforms for CRISPR/Cas9 delivery. Herein, we for the first time develop the application of protamine-capped gold nanoclusters (protamine-AuNCs) as an effective nanocarrier for Cas9-sgRNA plasmid transport and release to achieve efficient genome editing. The protamine-AuNCs integrate the merits of AuNCs and protamine: AuNCs are able to promptly assemble with Cas9-sgRNA plasmids to allow efficient cellular delivery, while the cationic protamine facilitates the effective release of Cas9-sgRNA plasmids into the cellular nucleus. The AuNCs/Cas9-gRNA plasmid nanocomplexes can not only achieve successful gene editing in cells but also knock out the oncogenic gene for cancer therapy. Moreover, the AuNCs with excellent photoluminescence characteristics endow our nanoplatform with the functionality of bioimaging. Overall, our study provides strong evidence that demonstrates protamine-AuNCs as an effective CRISPR/Cas9 delivery tool for gene therapy.},
}
@article {pmid33220342,
year = {2021},
author = {Katsuma, S and Shoji, K and Suzuki, Y and Kiuchi, T},
title = {CRISPR/Cas9-mediated mutagenesis of Ago2 and Siwi in silkworm cultured cells.},
journal = {Gene},
volume = {768},
number = {},
pages = {145314},
doi = {10.1016/j.gene.2020.145314},
pmid = {33220342},
issn = {1879-0038},
mesh = {Animals ; Argonaute Proteins/*genetics ; Bombyx/*cytology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Gene Editing ; Insect Proteins/genetics ; Loss of Function Mutation ; Mutagenesis, Site-Directed/*methods ; RNA, Small Interfering/genetics ; RNA, Viral/genetics ; Signal Transduction ; Tymoviridae/*genetics ; },
abstract = {The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.},
}
@article {pmid33219687,
year = {2020},
author = {Nobrega, FL and Walinga, H and Dutilh, BE and Brouns, SJJ},
title = {Prophages are associated with extensive CRISPR-Cas auto-immunity.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12074-12084},
pmid = {33219687},
issn = {1362-4962},
support = {639707/ERC_/European Research Council/International ; },
mesh = {Autoimmunity/genetics ; Bacteria/*genetics/immunology/virology ; Base Sequence ; CRISPR-Associated Protein 9/genetics/immunology ; CRISPR-Associated Proteins/*genetics/immunology ; CRISPR-Cas Systems/*immunology ; Chromosome Mapping/statistics &amp; numerical data ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; *Genome, Bacterial ; Prophages/*genetics ; Software ; },
abstract = {CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.},
}
@article {pmid33219380,
year = {2021},
author = {Cao, Y and Liu, X and Zhao, J and Du, M},
title = {AMPKα1 regulates Idh2 transcription through H2B O-GlcNAcylation during brown adipogenesis.},
journal = {Acta biochimica et biophysica Sinica},
volume = {53},
number = {1},
pages = {112-118},
doi = {10.1093/abbs/gmaa136},
pmid = {33219380},
issn = {1745-7270},
mesh = {AMP-Activated Protein Kinases/*genetics/*metabolism ; Acylation ; Adipogenesis/physiology ; Adipose Tissue, Brown/cytology/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line ; Diet, High-Fat ; Female ; Glucose/metabolism ; Histones/*metabolism ; Isocitrate Dehydrogenase/*genetics/metabolism ; Mesenchymal Stem Cells ; Mice ; Mice, Inbred C57BL ; Obesity/metabolism ; Promoter Regions, Genetic ; },
abstract = {AMP-activated protein kinase (AMPK) is indispensable for the development and maintenance of brown adipose tissue (BAT), and its activity is inhibited due to obesity. The isocitrate dehydrogenase 2 (IDH2) is a mitochondrial enzyme responsible for the production of α-ketoglutarate, a key intermediate metabolite integrating multiple metabolic processes. We previously found that AMPKα1 ablation reduced cellular α-ketoglutarate concentration during brown adipocyte differentiation, but the effect of AMPKα1 on Idh2 expression remains undefined. In the present study, mouse C3H10T1/2 cells were transfected with Idh2-CRISPR/Cas9, and induced to brown adipogenesis. Our data suggested that brown adipogenesis was compromised due to IDH2 deficiency in vitro, which was accompanied by down-regulation of PR-domain containing 16. Importantly, the IDH2 content was reduced in brown stromal vascular cells (BSVs) separated from AMPKα1 knockout (KO) BAT, which was associated with lower contents of histone 2B (H2B) O-GlcNAcylation and monoubiquitination. Furthermore, both GlcNAcylated-H2B (S112) and ubiquityl-histone 2B (K120) contents in the Idh2 promoter were decreased in AMPKα1 KO BSVs. Meanwhile, ectopic O-linked N-acetylglucosamine transferase (OGT) expression was positively correlated with Idh2 expression, while OGT (T444A) mutation abolished the regulatory effect of AMPKα1 on Idh2. In vivo, reduced AMPKα1 activity and lower IDH2 abundance were observed in BAT of obese mice when compared with those in control mice. Taken together, our data demonstrated that IDH2 is necessary for brown adipogenesis and that AMPKα1 deficiency attenuates Idh2 expression, which might be by suppressing H2B O-GlcNAcylation modification.},
}
@article {pmid33219320,
year = {2020},
author = {Akhtar, A},
title = {Pac-Man like DNA cut-and-paste tool allows larger gene edits.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {709},
pmid = {33219320},
issn = {2399-3642},
mesh = {*CRISPR-Associated Proteins ; DNA/genetics ; *Gene Editing ; Humans ; },
abstract = {The CRISPR-Cas toolbox allows genetic manipulation of cultured cells, plants and animals on the basis of simpler RNA-guided DNA recognition. It has provided breakthrough scientific opportunities to engineer desirable traits, cure genetic diseases and enable point-of-care diagnostics. A recent study by Joseph Bondy-Denomy and colleagues further equips this toolbox to cut larger chunks of DNA from a cell’s genome.},
}
@article {pmid33218644,
year = {2020},
author = {Liu, L and Zhang, J and Xu, J and Li, Y and Guo, L and Wang, Z and Zhang, X and Zhao, B and Guo, YD and Zhang, N},
title = {CRISPR/Cas9 targeted mutagenesis of SlLBD40, a lateral organ boundaries domain transcription factor, enhances drought tolerance in tomato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {301},
number = {},
pages = {110683},
doi = {10.1016/j.plantsci.2020.110683},
pmid = {33218644},
issn = {1873-2259},
mesh = {Acetates/metabolism ; CRISPR-Cas Systems ; Cyclopentanes/metabolism ; Droughts ; Fruit/genetics/physiology ; Lycopersicon esculentum/*genetics/physiology ; Mutagenesis ; Oxylipins/metabolism ; Plant Growth Regulators/*metabolism ; Plant Leaves/genetics/physiology ; Plant Proteins/genetics/*metabolism ; Plant Roots/genetics/physiology ; Plants, Genetically Modified ; Transcription Factors/genetics/metabolism ; },
abstract = {The LATERAL ORGAN BOUNDARIES DOMAIN (LBD)-containing genes are plant-specific genes that play important roles in lateral organ development. In this study, we identified LBD40 (Solyc02g085910), which belongs to subfamily II of the LBD family of genes in tomato. LBD40 was highly expressed in roots and fruit. LBD40 expression was significantly induced by PEG and salt. Moreover, SlLBD40 expression was induced by methyl jasmonate treatment, while SlLBD40 expression could not be induced in the jasmonic acid-insensitive1 (jai1) mutant or MYC2-silenced plants, in which jasmonic acid (JA) signaling was disrupted. These findings demonstrate that SlLBD40 expression was dependent on JA signaling and that it might be downstream of SlMYC2, which is the master transcription factor in the JA signal transduction pathway. Overexpressing and CRISPR/Cas9 mediated knockout transgenic tomato plants were generated to explore SlLBD40 function. The drought tolerance test showed that two SlLBD40 knockout lines wilted slightly, while SlLBD40 overexpressing plants suffered severe wilting. The statistical water loss rate and midday leaf water potential also confirmed that knockout of SlLBD40 improved the water-holding ability of tomato under drought conditions. Taken together, our study demonstrates that SlLBD40, involved in JA signaling, was a negative regulator of drought tolerance and that knockout of SlLBD40 enhanced drought tolerance in tomato. This study also provides a novel function of SlLBD40, which belongs to subfamily II of LBD genes.},
}
@article {pmid33218076,
year = {2020},
author = {Tanmoy, AM and Saha, C and Sajib, MSI and Saha, S and Komurian-Pradel, F and van Belkum, A and Louwen, R and Saha, SK and Endtz, HP},
title = {CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries.},
journal = {Genes},
volume = {11},
number = {11},
pages = {},
pmid = {33218076},
issn = {2073-4425},
mesh = {Bangladesh ; CRISPR-Cas Systems/*genetics ; Drug Resistance, Bacterial/*genetics ; Genetic Variation ; Genome, Bacterial ; Humans ; India ; Nepal ; Pakistan ; Salmonella typhi/*genetics/isolation &amp; purification ; Typhoid Fever/*microbiology ; },
abstract = {Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a global health concern and its treatment is problematic due to the rise in antimicrobial resistance (AMR). Rapid detection of patients infected with AMR positive S. Typhi is, therefore, crucial to prevent further spreading. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas), is an adaptive immune system that initially was used for typing purposes. Later, it was discovered to play a role in defense against phages and plasmids, including ones that carry AMR genes, and, at present, it is being explored for its usage in diagnostics. Despite the availability of whole-genome sequences (WGS), very few studied the CRISPR-Cas system of S. Typhi, let alone in typing purposes or relation to AMR. In the present study, we analyzed the CRISPR-Cas system of S. Typhi using WGS data of 1059 isolates obtained from Bangladesh, India, Nepal, and Pakistan in combination with demographic data and AMR status. Our results reveal that the S. Typhi CRISPR loci can be classified into two groups: A (evidence level >2) and B (evidence level ≤2), in which we identified a total of 47 unique spacers and 15 unique direct repeats. Further analysis of the identified spacers and repeats demonstrated specific patterns that harbored significant associations with genotype, demographic characteristics, and AMR status, thus raising the possibility of their usage as biomarkers. Potential spacer targets were identified and, interestingly, the phage-targeting spacers belonged to the group-A and plasmid-targeting spacers to the group-B CRISPR loci. Further analyses of the spacer targets led to the identification of an S. Typhi protospacer adjacent motif (PAM) sequence, TTTCA/T. New cas-genes known as DinG, DEDDh, and WYL were also discovered in the S. Typhi genome. However, a specific variant of the WYL gene was only identified in the extensively drug-resistant (XDR) lineage from Pakistan and ciprofloxacin-resistant lineage from Bangladesh. From this work, we conclude that there are strong correlations between variations identified in the S. Typhi CRISPR-Cas system and endemic AMR positive S. Typhi isolates.},
}
@article {pmid33217332,
year = {2021},
author = {Münch, PC and Franzosa, EA and Stecher, B and McHardy, AC and Huttenhower, C},
title = {Identification of Natural CRISPR Systems and Targets in the Human Microbiome.},
journal = {Cell host &amp; microbe},
volume = {29},
number = {1},
pages = {94-106.e4},
pmid = {33217332},
issn = {1934-6069},
support = {R24 DK110499/DK/NIDDK NIH HHS/United States ; U54 DK102557/DK/NIDDK NIH HHS/United States ; },
mesh = {Bacteria/*genetics/metabolism ; Bacteriophages/genetics/physiology ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gastrointestinal Microbiome/genetics ; Gene Ontology ; Genes, Bacterial ; Genome, Bacterial ; Humans ; Metagenome ; Methylation ; Microbiota/*genetics ; Mouth/microbiology ; Viral Proteins/genetics/metabolism ; Virus Physiological Phenomena ; },
abstract = {Many bacteria resist invasive DNA by incorporating sequences into CRISPR loci, which enable sequence-specific degradation. CRISPR systems have been well studied from isolate genomes, but culture-independent metagenomics provide a new window into their diversity. We profiled CRISPR loci and cas genes in the body-wide human microbiome using 2,355 metagenomes, yielding functional and taxonomic profiles for 2.9 million spacers by aligning the spacer content to each sample's metagenome and corresponding gene families. Spacer and repeat profiles agree qualitatively with those from isolate genomes but expand their diversity by approximately 13-fold, with the highest spacer load present in the oral microbiome. The taxonomy of spacer sequences parallels that of their source community, with functional targets enriched for viral elements. When coupled with cas gene systems, CRISPR-Cas subtypes are highly site and taxon specific. Our analysis provides a comprehensive collection of natural CRISPR-cas loci and targets in the human microbiome.},
}
@article {pmid33217139,
year = {2021},
author = {Tang, Y and Abdelrahman, M and Li, J and Wang, F and Ji, Z and Qi, H and Wang, C and Zhao, K},
title = {CRISPR/Cas9 induces exon skipping that facilitates development of fragrant rice.},
journal = {Plant biotechnology journal},
volume = {19},
number = {4},
pages = {642-644},
pmid = {33217139},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exons/genetics ; *Oryza/genetics ; },
}
@article {pmid33216968,
year = {2021},
author = {Mingoia, M and Caria, CA and Ye, L and Asunis, I and Marongiu, MF and Manunza, L and Sollaino, MC and Wang, J and Cabriolu, A and Kurita, R and Nakamura, Y and Cucca, F and Kan, YW and Marini, MG and Moi, P},
title = {Induction of therapeutic levels of HbF in genome-edited primary β[0] 39-thalassaemia haematopoietic stem and progenitor cells.},
journal = {British journal of haematology},
volume = {192},
number = {2},
pages = {395-404},
doi = {10.1111/bjh.17167},
pmid = {33216968},
issn = {1365-2141},
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Fetal Hemoglobin/*genetics ; Gene Editing/*methods ; HEK293 Cells ; Hematopoietic Stem Cells/*metabolism ; Humans ; K562 Cells ; Up-Regulation ; beta-Thalassemia/*genetics ; },
abstract = {Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of β-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δβ[0] -thalassaemia, is able to completely rescue the β-major thalassaemia phenotype caused by the β[0] 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from β[0] -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous β[0] 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of β-thalassaemia irrespective of the β-globin gene (HBB) mutations.},
}
@article {pmid33216845,
year = {2020},
author = {Wang, L and Jiang, J and Li, X and Li, K and He, R and Li, J and Duan, L and Luo, W and Hu, Z and Luo, D},
title = {Improved EGFR mutation detection sensitivity after enrichment by Cas9/sgRNA digestion and PCR amplification.},
journal = {Acta biochimica et biophysica Sinica},
volume = {52},
number = {12},
pages = {1316-1324},
doi = {10.1093/abbs/gmaa123},
pmid = {33216845},
issn = {1745-7270},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/blood/diagnosis/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/metabolism ; ErbB Receptors/blood/genetics ; Exons/genetics ; Genetic Loci ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing ; Humans ; Limit of Detection ; Lung Neoplasms/blood/diagnosis/*genetics ; Mutation/*genetics ; Polymerase Chain Reaction ; RNA, Guide/*metabolism ; Sensitivity and Specificity ; },
abstract = {The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.},
}
@article {pmid33216642,
year = {2021},
author = {Filipek-Górniok, B and Habicher, J and Ledin, J and Kjellén, L},
title = {Heparan Sulfate Biosynthesis in Zebrafish.},
journal = {The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society},
volume = {69},
number = {1},
pages = {49-60},
pmid = {33216642},
issn = {1551-5044},
mesh = {Animals ; *Biosynthetic Pathways ; CRISPR-Cas Systems ; Glycosyltransferases/genetics/metabolism ; Heparitin Sulfate/genetics/*metabolism ; Mutation ; Sulfotransferases/genetics/metabolism ; Zebrafish/genetics/*metabolism ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {The biosynthesis of heparan sulfate (HS) proteoglycans occurs in the Golgi compartment of cells and will determine the sulfation pattern of HS chains, which in turn will have a large impact on the biological activity of the proteoglycans. Earlier studies in mice have demonstrated the importance of HS for embryonic development. In this review, the enzymes participating in zebrafish HS biosynthesis, along with a description of enzyme mutants available for functional studies, are presented. The consequences of the zebrafish genome duplication and maternal transcript contribution are briefly discussed as are the possibilities of CRISPR/Cas9 methodologies to use the zebrafish model system for studies of biosynthesis as well as proteoglycan biology.},
}
@article {pmid33216174,
year = {2021},
author = {Gao, Y and Wu, S and Pan, J and Zhang, K and Li, X and Xu, Y and Jin, C and He, X and Shi, J and Ma, L and Wu, F and Yao, Y and Wang, P and He, Q and Lan, F and Zhang, H and Tian, M},
title = {CRISPR/Cas9-edited triple-fusion reporter gene imaging of dynamics and function of transplanted human urinary-induced pluripotent stem cell-derived cardiomyocytes.},
journal = {European journal of nuclear medicine and molecular imaging},
volume = {48},
number = {3},
pages = {708-720},
pmid = {33216174},
issn = {1619-7089},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Reporter ; Humans ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac ; },
abstract = {PURPOSE: To investigate the post-transplantation behaviour and therapeutic efficacy of human urinary-induced pluripotent stem cell-derived cardiomyocytes (hUiCMs) in infarcted heart.<br><br>METHODS: We used clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology to integrate a triple-fusion (TF) reporter gene into the AAVS1 locus in human urine-derived hiPSCs (hUiPSCs) to generate TF-hUiPSCs that stably expressed monomeric red fluorescent protein for fluorescence imaging, firefly luciferase for bioluminescence imaging (BLI) and herpes simplex virus thymidine kinase for positron emission tomography (PET) imaging.<br><br>RESULTS: Transplanted cardiomyocytes derived from TF-hUiPSCs (TF-hUiCMs) engrafted and proliferated in the infarcted heart as monitored by both BLI and PET imaging and significantly improved cardiac function. Under ischaemic conditions, TF-hUiCMs enhanced cardiomyocyte (CM) glucose metabolism and promoted angiogenic activity.<br><br>CONCLUSION: This study established a CRISPR/Cas9-mediated multimodality reporter gene imaging system that can determine the dynamics and function of TF-hUiCMs in myocardial infarction, which is helpful for investigating the application of stem cell therapy.},
}
@article {pmid33215320,
year = {2021},
author = {Chiti, A},
title = {Reporter gene imaging "visualized" the integration of two growing technologies: CRISPR/Cas9-based genome editing and induced pluripotent stem cell therapy.},
journal = {European journal of nuclear medicine and molecular imaging},
volume = {48},
number = {3},
pages = {664-665},
pmid = {33215320},
issn = {1619-7089},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genes, Reporter ; Humans ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac ; },
}
@article {pmid33214661,
year = {2020},
author = {Dalla Costa, L and Piazza, S and Pompili, V and Salvagnin, U and Cestaro, A and Moffa, L and Vittani, L and Moser, C and Malnoy, M},
title = {Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20155},
pmid = {33214661},
issn = {2045-2322},
mesh = {Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; DNA, Bacterial ; Gene Editing/methods ; *Gene Transfer Techniques ; Genes, Plant ; Genetic Vectors/*genetics ; Genome, Plant ; Malus/*genetics ; Plants, Genetically Modified/*genetics ; Vitis/*genetics ; },
abstract = {Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.},
}
@article {pmid33214258,
year = {2020},
author = {Pennisi, E},
title = {Like CRISPR, mystery gene editor began as a virus fighter.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6519},
pages = {898-899},
doi = {10.1126/science.370.6519.898},
pmid = {33214258},
issn = {1095-9203},
mesh = {Bacteria/*virology ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; *Gene Editing ; RNA, Bacterial/genetics ; RNA-Directed DNA Polymerase/physiology ; },
}
@article {pmid33213345,
year = {2021},
author = {Ray, SK and Mukherjee, S},
title = {Genome Editing with CRISPR-Cas9: A Budding Biological Contrivance for Colorectal Carcinoma Research and its Perspective in Molecular Medicine.},
journal = {Current molecular medicine},
volume = {21},
number = {6},
pages = {462-475},
doi = {10.2174/1566524020666201119143943},
pmid = {33213345},
issn = {1875-5666},
mesh = {*CRISPR-Cas Systems ; *Colorectal Neoplasms/genetics/metabolism/therapy ; *Gene Editing ; *Genetic Therapy ; Humans ; },
abstract = {Genome editing is an addition, deletion, or replacement of a gene to remove or initiating explicit and preferred characters in the genome. Utilizing gene-editing tools like CRISPR-Cas9 technology could be accomplished either by gene-based methodology or protein-based technology that has been under scrutiny for the protracted time wherein physical techniques, viral and non-viral strategies have been utilized together. Transplanting ex vivo CRISPR edited cells empowers screening of single guide RNAs with high-throughput and CRISPR based screening in organoids transplantation to validate cancer cells including colorectal carcinoma in various phases of its development and treatment.CRISPR knockout screens have recognized genes that drive interest in colon cancer to develop hallmarks, especially in some cancer cell lines with single guide RNA, to disclose drug resistance mechanisms. One advantage of this method is to deal with CRISPR knockout genomic screening, which disrupts gene expression, rather than the partial knockdown that is mostly done with RNA interference and CRISPR/Cas technology. This technique is used to treat different forms of cancer because of its proficient editing of the target gene, along with the CRISPR/Cas system. Latest research has shown that the CRISPR/Cas gene-editing technique has theoretically reformed the expression in colorectal carcinoma of long non-coding RNA. For the next decade CRISPR/Cas9 technology will positively fuel the development of more in vivo gene editing clinical trials in colon cancer and will have an enormous impact on molecular medicine.},
}
@article {pmid33208938,
year = {2020},
author = {Ma, H and Kim, AS and Kafai, NM and Earnest, JT and Shah, AP and Case, JB and Basore, K and Gilliland, TC and Sun, C and Nelson, CA and Thackray, LB and Klimstra, WB and Fremont, DH and Diamond, MS},
title = {LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.},
journal = {Nature},
volume = {588},
number = {7837},
pages = {308-314},
pmid = {33208938},
issn = {1476-4687},
support = {R01 AI095436/AI/NIAID NIH HHS/United States ; U19 AI142759/AI/NIAID NIH HHS/United States ; T32 AI007172/AI/NIAID NIH HHS/United States ; HHSN272201700060C/AI/NIAID NIH HHS/United States ; R01 AI153209/AI/NIAID NIH HHS/United States ; R01 AI143673/AI/NIAID NIH HHS/United States ; U19 AI142790/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Encephalitis Virus, Venezuelan Equine/*metabolism/pathogenicity ; Encephalomyelitis, Venezuelan Equine/metabolism/prevention &amp; control/virology ; Female ; Genetic Complementation Test ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Protein Binding ; Receptors, LDL/deficiency/genetics/*metabolism ; Receptors, Virus/genetics/*metabolism ; Virus Attachment ; Virus Internalization ; },
abstract = {Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans[1]. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.},
}
@article {pmid33208877,
year = {2020},
author = {Schukur, L and Zimmermann, T and Niewoehner, O and Kerr, G and Gleim, S and Bauer-Probst, B and Knapp, B and Galli, GG and Liang, X and Mendiola, A and Reece-Hoyes, J and Rapti, M and Barbosa, I and Reschke, M and Radimerski, T and Thoma, CR},
title = {Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20044},
pmid = {33208877},
issn = {2045-2322},
mesh = {Apoptosis ; *CRISPR-Cas Systems ; Humans ; Neoplasms/genetics/metabolism/*pathology ; Protein Binding ; *Proteolysis ; Proto-Oncogene Proteins c-myc/genetics/*metabolism ; Tumor Cells, Cultured ; Ubiquitin-Protein Ligases/genetics/*metabolism ; *Ubiquitination ; },
abstract = {MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.},
}
@article {pmid33208534,
year = {2020},
author = {Alphey, LS and Crisanti, A and Randazzo, FF and Akbari, OS},
title = {Opinion: Standardizing the definition of gene drive.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {49},
pages = {30864-30867},
pmid = {33208534},
issn = {1091-6490},
support = {DP2 AI152071/AI/NIAID NIH HHS/United States ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007034/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Drive Technology/*standards ; Gene Frequency/genetics ; Humans ; Reference Standards ; },
}
@article {pmid33208517,
year = {2020},
author = {Bourgeois, J and Lazinski, DW and Camilli, A},
title = {Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System.},
journal = {mSphere},
volume = {5},
number = {6},
pages = {},
pmid = {33208517},
issn = {2379-5042},
support = {T32 GM007310/GM/NIGMS NIH HHS/United States ; R37 AI055058/AI/NIAID NIH HHS/United States ; R01 AI055058/AI/NIAID NIH HHS/United States ; T32 GM008448/GM/NIGMS NIH HHS/United States ; R21 AI147658/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Bacterial/genetics ; DNA, Intergenic/*genetics ; Vibrio cholerae/*genetics ; },
abstract = {The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5'-TT-3' protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5'-TT-3' PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5' position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5'-RTT-3' PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5'-RTT-3' PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5' spacer pyrimidine.IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.},
}
@article {pmid33208369,
year = {2020},
author = {Rosenblum, D and Gutkin, A and Kedmi, R and Ramishetti, S and Veiga, N and Jacobi, AM and Schubert, MS and Friedmann-Morvinski, D and Cohen, ZR and Behlke, MA and Lieberman, J and Peer, D},
title = {CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy.},
journal = {Science advances},
volume = {6},
number = {47},
pages = {},
pmid = {33208369},
issn = {2375-2548},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Gene Transfer Techniques ; Liposomes ; *Nanoparticles ; *Neoplasms/genetics/therapy ; },
abstract = {Harnessing CRISPR-Cas9 technology for cancer therapeutics has been hampered by low editing efficiency in tumors and potential toxicity of existing delivery systems. Here, we describe a safe and efficient lipid nanoparticle (LNP) for the delivery of Cas9 mRNA and sgRNAs that use a novel amino-ionizable lipid. A single intracerebral injection of CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) into aggressive orthotopic glioblastoma enabled up to ~70% gene editing in vivo, which caused tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30%. To reach disseminated tumors, cLNPs were also engineered for antibody-targeted delivery. Intraperitoneal injections of EGFR-targeted sgPLK1-cLNPs caused their selective uptake into disseminated ovarian tumors, enabled up to ~80% gene editing in vivo, inhibited tumor growth, and increased survival by 80%. The ability to disrupt gene expression in vivo in tumors opens new avenues for cancer treatment and research and potential applications for targeted gene editing of noncancerous tissues.},
}
@article {pmid33208059,
year = {2021},
author = {Song, Y and Ke, X and Chen, L},
title = {The Potential Use of RNA-based Therapeutics for Breast Cancer Treatment.},
journal = {Current medicinal chemistry},
volume = {28},
number = {25},
pages = {5110-5136},
doi = {10.2174/0929867327666201117100336},
pmid = {33208059},
issn = {1875-533X},
mesh = {Animals ; *Breast Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems ; Female ; Genetic Therapy ; Humans ; *RNA ; },
abstract = {Breast cancer is one of the most lethal cancers in women worldwide, and the development of efficient treatments faces several challenges. Breast cancer is characterized by histological and functional heterogeneity in aspects such as tumorigenesis, metastasis, and drug resistance. RNA therapy has emerged as a highly attractive class of drugs for the treatment and prevention of breast cancer. It might play remarkable regulatory roles in the treatment of targeted cells by either increasing or silencing expressions of specific proteins, and such features of RNA-based drugs cause high selectivity and low risk of off-target effect in breast cancer. RNA therapy exerts anti-proliferative and pro-apoptotic effects upon cell culture systems, animal models, and in clinical trials in most studies. In this mini-review, we outline the classifications, mechanisms, advantages, and challenges of RNA therapy and highlight its application in breast cancer treatment. Additionally, we summarize the clinical trials of RNA-targeting therapies and the development of anti-tumor RNA drugs and provide future directions for RNA therapeutics in breast cancer.},
}
@article {pmid33207306,
year = {2020},
author = {Ding, Y and Wang, L and Ji, W and Chen, Z and Wang, D and Chen, C and Tong, H and Han, Z and Niu, C and Chu, M and Huang, J and Guo, X},
title = {Generation of a human induced pluripotent stem cell line with Cas9 driven by Tet-on operator via AAVS1 safe harbor gene-editing.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102064},
doi = {10.1016/j.scr.2020.102064},
pmid = {33207306},
issn = {1876-7753},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Cell Line ; *Gene Editing ; Germ Layers ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Human induced pluripotent stem (iPS) cells expressing Cas9 protein are valuable for the pathogenic mechanism study and drug discovery. These cells can be efficiently induced to differentiate into disease cell models with specific mutations through adding designed sgRNAs. Here, we generated a human gene-editable iPS cell line by gene editing method that Cas9 gene driven by Tet-on operator was perfectly integrated into the human AAVS1 safe harbor locus. The established Cas9 expression iPS cell line named as WMUi013-A can express endogenous pluripotent markers, has the ability to differentiate into the three germ layers, and possesses a normal karyotype.},
}
@article {pmid33203764,
year = {2020},
author = {Reicher, A and Koren, A and Kubicek, S},
title = {Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time.},
journal = {Genome research},
volume = {30},
number = {12},
pages = {1846-1855},
pmid = {33203764},
issn = {1549-5469},
mesh = {CRISPR-Cas Systems ; Clone Cells/*drug effects/metabolism ; Gene Editing ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Molecular Imaging ; Pharmaceutical Preparations ; Protein Transport/drug effects ; Proteins/drug effects/*metabolism ; Sequence Analysis, DNA/*methods ; Time-Lapse Imaging/*methods ; },
abstract = {The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein-protein interactions, condensate formation, and chemical degradation.},
}
@article {pmid33203452,
year = {2020},
author = {Huynh, N and Depner, N and Larson, R and King-Jones, K},
title = {A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {279},
pmid = {33203452},
issn = {1474-760X},
support = {P40 OD018537/OD/NIH HHS/United States ; CIHR #PS 169196//CIHR/Canada ; },
mesh = {Adenosine Deaminase/metabolism ; Animals ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Endonucleases/metabolism ; Gene Expression ; RNA/*genetics ; *RNA Processing, Post-Transcriptional ; RNA, Mitochondrial ; RNA-Binding Proteins/metabolism ; },
abstract = {Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.},
}
@article {pmid33202909,
year = {2020},
author = {Yolcu, S and Alavilli, H and Lee, BH},
title = {Natural Genetic Resources from Diverse Plants to Improve Abiotic Stress Tolerance in Plants.},
journal = {International journal of molecular sciences},
volume = {21},
number = {22},
pages = {},
pmid = {33202909},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Crops, Agricultural/genetics/growth &amp; development ; *Gene Editing ; *Genome, Plant ; *Plants, Genetically Modified/genetics/growth &amp; development ; },
abstract = {The current agricultural system is biased for the yield increase at the cost of biodiversity. However, due to the loss of precious genetic diversity during domestication and artificial selection, modern cultivars have lost the adaptability to cope with unfavorable environments. There are many reports on variations such as single nucleotide polymorphisms (SNPs) and indels in the stress-tolerant gene alleles that are associated with higher stress tolerance in wild progenitors, natural accessions, and extremophiles in comparison with domesticated crops or model plants. Therefore, to gain a better understanding of stress-tolerant traits in naturally stress-resistant plants, more comparative studies between the modern crops/model plants and crop progenitors/natural accessions/extremophiles are required. In this review, we discussed and summarized recent progress on natural variations associated with enhanced abiotic stress tolerance in various plants. By applying the recent biotechniques such as the CRISPR/Cas9 gene editing tool, natural genetic resources (i.e., stress-tolerant gene alleles) from diverse plants could be introduced to the modern crop in a non-genetically modified way to improve stress-tolerant traits.},
}
@article {pmid33202255,
year = {2021},
author = {Liu, J and Sun, M and Cho, KB and Gao, X and Guo, B},
title = {A CRISPR-Cas9 repressor for epigenetic silencing of KRAS.},
journal = {Pharmacological research},
volume = {164},
number = {},
pages = {105304},
pmid = {33202255},
issn = {1096-1186},
support = {R01 CA186100/CA/NCI NIH HHS/United States ; },
mesh = {Acetylation ; Apoptosis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Gene Editing ; Histone Deacetylase 1/*genetics ; Histones/metabolism ; Humans ; Neoplasms/therapy ; Proto-Oncogene Proteins p21(ras)/*genetics ; },
abstract = {KRAS is one of the most frequently mutated oncogenes in cancers. Currently no direct and effective anti-KRAS therapies are available. Using the powerful CRISPR-Cas9 technology to target the mutant KRAS promoter, we designed an epigenetic repressor to silence KRAS through epigenome editing. Catalytically dead Cas9 (dCas9) functioned as a DNA binding device, which was fused with a transcriptional repressor histone deacetylase 1 (HDAC1). We designed a panel of three CRISPR RNAs (crRNAs) covering 1500-bp range of the KRAS promoter and identified that crRNA1 and crRNA2 efficiently silenced KRAS. The suppression of K-Ras resulted in a significant inhibition of cell growth, suppression of colony formation in soft agar and induction of cell death in cancer cells with KRAS mutations. In addition, the chromatin immunoprecipitation (ChIP) assay demonstrated dCas9-HDAC1 modified histone acetylation on the KRAS promoter. Furthermore, transfection of dCas9-HDAC1 protein and gRNA ribonucleoprotein complex also inhibited K-Ras and suppressed cell proliferation. In summary, we have developed a new strategy that combines CRISPR-Cas9 technology with HDAC1 epigenetic silencing to target cancers driven by KRAS mutations.},
}
@article {pmid33202035,
year = {2020},
author = {Potts, MA and McDonald, JA and Sutherland, KD and Herold, MJ},
title = {Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy.},
journal = {European journal of immunology},
volume = {50},
number = {12},
pages = {1871-1884},
doi = {10.1002/eji.202048712},
pmid = {33202035},
issn = {1521-4141},
mesh = {Animals ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/immunology ; Gene Editing/methods ; Humans ; Immunotherapy/methods ; Neoplasms/*genetics/*immunology/therapy ; },
abstract = {The mutational landscape of human cancers is highly complex. While next generation sequencing aims to comprehensively catalogue somatic alterations in tumor cells, it fails to delineate driver from passenger mutations. Functional genomic approaches, particularly CRISPR/Cas9, enable both gene discovery, and annotation of gene function. Indeed, recent CRISPR/Cas9 technologies have flourished with the development of more sophisticated and versatile platforms capable of gene knockouts to high throughput genome wide editing of a single nucleotide base. With new platforms constantly emerging, it can be challenging to navigate what CRISPR tools are available and how they can be effectively applied to understand cancer biology. This review provides an overview of current and emerging CRISPR technologies and their power to model cancer and identify novel treatments. Specifically, how CRISPR screening approaches have been exploited to enhance immunotherapies through the identification of tumor intrinsic and extrinsic mechanisms to escape immune recognition will be discussed.},
}
@article {pmid33201998,
year = {2020},
author = {Yang, BX and Xie, CY and Xia, ZY and Wu, YJ and Gou, M and Tang, YQ},
title = {Improving xylitol yield by deletion of endogenous xylitol-assimilating genes: a study of industrial Saccharomyces cerevisiae in fermentation of glucose and xylose.},
journal = {FEMS yeast research},
volume = {20},
number = {8},
pages = {},
doi = {10.1093/femsyr/foaa061},
pmid = {33201998},
issn = {1567-1364},
mesh = {Aldehyde Reductase/genetics ; CRISPR-Cas Systems ; D-Xylulose Reductase/genetics ; *Fermentation ; Gene Deletion ; Glucose/metabolism ; *Metabolic Engineering ; Phosphotransferases (Alcohol Group Acceptor)/genetics ; Saccharomyces cerevisiae/*enzymology/genetics ; Xylose/*metabolism ; },
abstract = {Engineered Saccharomyces cerevisiae can reduce xylose to xylitol. However, in S.cerevisiae, there are several endogenous enzymes including xylitol dehydrogenase encoded by XYL2, sorbitol dehydrogenases encoded by SOR1/SOR2 and xylulokinase encoded by XKS1 may lead to the assimilation of xylitol. In this study, to increase xylitol accumulation, these genes were separately deleted through CRISPR/Cas9 system. Their effects on xylitol yield of an industrial S. cerevisiae CK17 overexpressing Candida tropicalis XYL1 (encoding xylose reductase) were investigated. Deletion of SOR1/SOR2 or XKS1 increased the xylitol yield in both batch and fed-batch fermentation with different concentrations of glucose and xylose. The analysis of the transcription level of key genes in the mutants during fed-batch fermentation suggests that SOR1/SOR2 are more crucially responsible for xylitol oxidation than XYL2 under the genetic background of S.cerevisiae CK17. The deletion of XKS1 gene could also weaken SOR1/SOR2 expression, thereby increasing the xylitol accumulation. The XKS1-deleted strain CK17ΔXKS1 produced 46.17 g/L of xylitol and reached a xylitol yield of 0.92 g/g during simultaneous saccharification and fermentation (SSF) of pretreated corn stover slurry. Therefore, the deletion of XKS1 gene provides a promising strategy to meet the industrial demands for xylitol production from lignocellulosic biomass.},
}
@article {pmid33201862,
year = {2021},
author = {Uyhazi, KE and Bennett, J},
title = {A CRISPR view of the 2020 Nobel Prize in Chemistry.},
journal = {The Journal of clinical investigation},
volume = {131},
number = {1},
pages = {},
pmid = {33201862},
issn = {1558-8238},
support = {K08 EY031754/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Chemistry ; History, 21st Century ; Humans ; *Nobel Prize ; },
}
@article {pmid33201595,
year = {2021},
author = {Yi, JY and Kim, M and Min, H and Kim, BG and Son, J and Kwon, OS and Sung, C},
title = {New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis.},
journal = {Drug testing and analysis},
volume = {13},
number = {4},
pages = {871-875},
doi = {10.1002/dta.2980},
pmid = {33201595},
issn = {1942-7611},
mesh = {CRISPR-Cas Systems/*genetics ; DNA, Complementary/genetics ; Doping in Sports/*prevention &amp; control ; Erythropoietin/*genetics ; Humans ; In Vitro Techniques ; Limit of Detection ; RNA, Guide/genetics ; },
abstract = {The increased potential for gene doping since the introduction of gene therapy presents the need to develop antidoping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon-exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for antidoping analysis of gene doping.},
}
@article {pmid33201182,
year = {2020},
author = {Li, Z and Wei, J and Di, D and Wang, X and Li, C and Li, B and Qiu, Y and Liu, K and Gu, F and Tong, M and Wang, S and Wu, X and Ma, Z},
title = {Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay.},
journal = {Acta biochimica et biophysica Sinica},
volume = {52},
number = {12},
pages = {1413-1419},
doi = {10.1093/abbs/gmaa135},
pmid = {33201182},
issn = {1745-7270},
mesh = {African Swine Fever/blood/*diagnosis/virology ; African Swine Fever Virus/*genetics ; Animals ; Bacterial Proteins/biosynthesis/isolation &amp; purification ; CRISPR-Associated Proteins/biosynthesis/isolation &amp; purification ; CRISPR-Cas Systems ; China ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Viral/genetics ; Deoxyribonuclease I/genetics ; Endodeoxyribonucleases/biosynthesis/isolation &amp; purification ; Fluorescence ; Limit of Detection ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/*methods ; Real-Time Polymerase Chain Reaction ; Recombinases/metabolism ; Sensitivity and Specificity ; Swine/*blood ; },
abstract = {The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.},
}
@article {pmid33200806,
year = {2021},
author = {Chen, B and Chang, P and Shen, X and Zhang, X and Zhang, J and Wang, X and Yu, J},
title = {Cardiac‑specific deletion of natriuretic peptide receptor A induces differential myocardial expression of circular RNA and mRNA molecules involved in metabolism in mice.},
journal = {Molecular medicine reports},
volume = {23},
number = {1},
pages = {},
pmid = {33200806},
issn = {1791-3004},
mesh = {Animals ; CRISPR-Cas Systems ; Computational Biology ; Gene Expression Profiling ; Gene Ontology ; Gene Regulatory Networks ; Metabolism/*genetics ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/metabolism ; Myocardium/*metabolism ; Oligonucleotide Array Sequence Analysis ; RNA, Circular/*metabolism ; RNA, Messenger/*metabolism ; Receptors, Atrial Natriuretic Factor/*genetics ; Software ; },
abstract = {Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are important biological markers and regulators of cardiac function. The natriuretic peptide receptor A (NPRA), also known as NPR1 or guanylyl cyclase A, binds ANP and BNP to initiate transmembrane signal transduction by elevating the intracellular levels of cyclic guanosine monophosphate. However, the effects and mechanisms downstream of NPRA are largely unknown. The aim of the present study was to evaluate the changes in the global pattern of mRNA and circular RNA (circRNA) expression in NPRA[‑/‑] and NPRA[+/+] myocardium. Differentially expressed mRNA molecules were characterised using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis and were found to be primarily related to metabolic processes. Moreover, circRNA expression was also examined, and a possible competing endogenous RNA network consisting of circRNA, microRNA (miRNA), and mRNA molecules was constructed. The results of this study indicated that NPRA may play a role in cardiac metabolism, which could be mediated by circRNA through endogenous competition mechanisms. These findings may provide insight into future characterisation of various ceRNA network pathways.},
}
@article {pmid33199912,
year = {2021},
author = {Li, L and Williams, P and Ren, W and Wang, MY and Gao, Z and Miao, W and Huang, M and Song, J and Wang, Y},
title = {YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression.},
journal = {Nature chemical biology},
volume = {17},
number = {2},
pages = {161-168},
pmid = {33199912},
issn = {1552-4469},
support = {R35 ES031707/ES/NIEHS NIH HHS/United States ; R35 GM119721/GM/NIGMS NIH HHS/United States ; T32 ES018827/ES/NIEHS NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems ; Chromatin/metabolism ; DNA/*chemistry/*genetics ; *G-Quadruplexes ; Gene Expression/*genetics ; HEK293 Cells ; Humans ; Promoter Regions, Genetic ; Protein Binding ; YY1 Transcription Factor/*genetics ; Zinc Fingers ; },
abstract = {The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.},
}
@article {pmid33198306,
year = {2020},
author = {Vrancianu, CO and Gheorghe, I and Dobre, EG and Barbu, IC and Cristian, RE and Popa, M and Lee, SH and Limban, C and Vlad, IM and Chifiriuc, MC},
title = {Emerging Strategies to Combat β-Lactamase Producing ESKAPE Pathogens.},
journal = {International journal of molecular sciences},
volume = {21},
number = {22},
pages = {},
pmid = {33198306},
issn = {1422-0067},
mesh = {Acinetobacter baumannii/drug effects/enzymology ; Animals ; Anti-Bacterial Agents/*pharmacology ; Bacteriophages ; CRISPR-Cas Systems ; Carbapenems/pharmacology ; *Drug Resistance, Multiple, Bacterial ; Enterobacter/drug effects/enzymology ; Enterococcus/drug effects/enzymology ; Humans ; Klebsiella pneumoniae/drug effects/enzymology ; Metal Nanoparticles/chemistry ; Microbial Sensitivity Tests ; Pore Forming Cytotoxic Proteins/pharmacology ; Pseudomonas aeruginosa/drug effects/enzymology ; Staphylococcus aureus/drug effects/enzymology ; Vaccination ; beta-Lactamase Inhibitors/pharmacology ; beta-Lactamases/*metabolism ; },
abstract = {Since the discovery of penicillin by Alexander Fleming in 1929 as a therapeutic agent against staphylococci, β-lactam antibiotics (BLAs) remained the most successful antibiotic classes against the majority of bacterial strains, reaching a percentage of 65% of all medical prescriptions. Unfortunately, the emergence and diversification of β-lactamases pose indefinite health issues, limiting the clinical effectiveness of all current BLAs. One solution is to develop β-lactamase inhibitors (BLIs) capable of restoring the activity of β-lactam drugs. In this review, we will briefly present the older and new BLAs classes, their mechanisms of action, and an update of the BLIs capable of restoring the activity of β-lactam drugs against ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens. Subsequently, we will discuss several promising alternative approaches such as bacteriophages, antimicrobial peptides, nanoparticles, CRISPR (clustered regularly interspaced short palindromic repeats) cas technology, or vaccination developed to limit antimicrobial resistance in this endless fight against Gram-negative pathogens.},
}
@article {pmid33198099,
year = {2020},
author = {Shelenkov, A and Petrova, L and Fomina, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V},
title = {Multidrug-Resistant Proteus mirabilis Strain with Cointegrate Plasmid.},
journal = {Microorganisms},
volume = {8},
number = {11},
pages = {},
pmid = {33198099},
issn = {2076-2607},
abstract = {Proteus mirabilis is a component of the normal intestinal microflora of humans and animals, but can cause urinary tract infections and even sepsis in hospital settings. In recent years, the number of multidrug-resistant P. mirabilis isolates, including the ones producing extended-spectrum β-lactamases (ESBLs), is increasing worldwide. However, the number of investigations dedicated to this species, especially, whole-genome sequencing, is much lower in comparison to the members of the ESKAPE pathogens group. This study presents a detailed analysis of clinical multidrug-resistant ESBL-producing P. mirabilis isolate using short- and long-read whole-genome sequencing, which allowed us to reveal possible horizontal gene transfer between Klebsiella pneumoniae and P. mirabilis plasmids and to locate the CRISPR-Cas system in the genome together with its probable phage targets, as well as multiple virulence genes. We believe that the data presented will contribute to the understanding of antibiotic resistance acquisition and virulence mechanisms for this important pathogen.},
}
@article {pmid33197925,
year = {2021},
author = {Surka, C and Jin, L and Mbong, N and Lu, CC and Jang, IS and Rychak, E and Mendy, D and Clayton, T and Tindall, E and Hsu, C and Fontanillo, C and Tran, E and Contreras, A and Ng, SWK and Matyskiela, M and Wang, K and Chamberlain, P and Cathers, B and Carmichael, J and Hansen, J and Wang, JCY and Minden, MD and Fan, J and Pierce, DW and Pourdehnad, M and Rolfe, M and Lopez-Girona, A and Dick, JE and Lu, G},
title = {CC-90009, a novel cereblon E3 ligase modulator, targets acute myeloid leukemia blasts and leukemia stem cells.},
journal = {Blood},
volume = {137},
number = {5},
pages = {661-677},
pmid = {33197925},
issn = {1528-0020},
support = {P30 GM124169/GM/NIGMS NIH HHS/United States ; S10 OD021832/OD/NIH HHS/United States ; 154293/CAPMC/CIHR/Canada ; 89932/CAPMC/CIHR/Canada ; },
mesh = {Acetamides/*pharmacology/therapeutic use ; Adaptor Proteins, Signal Transducing/*antagonists &amp; inhibitors ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Humans ; Isoindoles/*pharmacology/therapeutic use ; Leukemia, Myeloid, Acute/*pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Models, Molecular ; *Molecular Targeted Therapy ; Neoplasm Proteins/*antagonists &amp; inhibitors ; Neoplastic Stem Cells/*drug effects/enzymology ; Nuclear Factor 45 Protein/physiology ; Nuclear Factor 90 Proteins/physiology ; Peptide Termination Factors/metabolism ; Piperidones/*pharmacology/therapeutic use ; Proteasome Endopeptidase Complex/metabolism ; Protein Conformation ; Protein Processing, Post-Translational/drug effects ; Proteolysis ; Small Molecule Libraries ; Stress, Physiological ; TOR Serine-Threonine Kinases/physiology ; U937 Cells ; Ubiquitin-Protein Ligases/*antagonists &amp; inhibitors ; Ubiquitination/drug effects ; Xenograft Model Antitumor Assays ; },
abstract = {A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).},
}
@article {pmid33197867,
year = {2020},
author = {Gong, Y and Chen, T and Feng, N and Meng, X and Sun, W and Wang, T and Zhao, Y and Yang, S and Song, X and Li, W and Dong, H and Wang, H and He, H and Wang, J and Zhang, L and Gao, Y and Xia, X},
title = {A highly efficient recombinant canarypox virus-based vaccine against canine distemper virus constructed using the CRISPR/Cas9 gene editing method.},
journal = {Veterinary microbiology},
volume = {251},
number = {},
pages = {108920},
doi = {10.1016/j.vetmic.2020.108920},
pmid = {33197867},
issn = {1873-2542},
mesh = {Animals ; Antibodies, Viral/*blood ; *CRISPR-Cas Systems ; Canarypox virus/*genetics/immunology ; Chick Embryo/cytology ; Chickens ; Chlorocebus aethiops ; Distemper/*prevention &amp; control ; Distemper Virus, Canine/*genetics/*immunology ; Dogs ; Female ; Fibroblasts/virology ; Foxes/immunology ; Gene Editing/*methods ; Glycoproteins/genetics/immunology ; Hemagglutinins, Viral/genetics/immunology ; Male ; Mink/immunology ; Vaccines, Synthetic/administration &amp; dosage/immunology ; Vero Cells ; Viral Fusion Proteins/genetics/immunology ; Viral Vaccines/administration &amp; dosage/genetics/*immunology ; },
abstract = {Canine distemper virus (CDV) is the causative agent of canine distemper (CD), which is one of the most important infectious diseases affecting wild and domestic carnivores. Vaccination represents an effective approach to prevent CDV infection among domestic carnivores. Canarypox-vectored recombinant CD vaccines (such as Recombitek CDV, PureVax Ferret Distemper, and Merial) with the CDV hemagglutinin (H) and fusion (F) genes can induce a potent immune response in dogs and ferrets. However, the vaccine's effectiveness varies with the species. In the current study, we developed a highly efficient recombinant canarypox virus termed as "ALVAC-CDV-M-F-H/C5[-]" that contained CDV virus-like particles (VLPs) by using the CRISPR/Cas9 gene editing method, which enabled concurrent expression of the matrix (M), H, and F genes. The recombinant strain provided faster seroconversion than the parent strain among minks as well as provided higher rates of antibody positivity than the parent strain among foxes and minks even before the administration of a second booster vaccination. We demonstrated, for the first time, that the CRISPR/Cas9 system can be applied for the rapid and efficient modification of the ALVAC-CDV-F-H genome and also that a high-dose new recombinant strain that produces CDV VLPs may present good outcomes in the prevention of CD among foxes and minks.},
}
@article {pmid33197651,
year = {2021},
author = {Tomasich, E and Topakian, T and Heller, G and Udovica, S and Krainer, M and Marhold, M},
title = {Loss of HCRP1 leads to upregulation of PD-L1 via STAT3 activation and is of prognostic significance in EGFR-dependent cancer.},
journal = {Translational research : the journal of laboratory and clinical medicine},
volume = {230},
number = {},
pages = {21-33},
doi = {10.1016/j.trsl.2020.11.005},
pmid = {33197651},
issn = {1878-1810},
mesh = {B7-H1 Antigen/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Endosomal Sorting Complexes Required for Transport/genetics/*metabolism ; ErbB Receptors/genetics/*metabolism ; Gene Deletion ; Humans ; STAT3 Transcription Factor/genetics/*metabolism ; Up-Regulation ; },
abstract = {Loss of hepatocellular carcinoma-related protein 1 (HCRP1) (alias VPS37A) plays a role in endocytosis of receptor tyrosine kinases as a member of the ESCRT complex and has been linked to poor patient outcome in various types of epithelial cancer. To this date, the molecular and biological mechanisms explaining how its absence would contribute to tumor progression remain unknown. Using genomic editing with CRISPR-Cas9, we generated ovarian and breast cancer cell lines with loss-of-function mutations of HCRP1. We hypothesized that pathways downstream of receptor tyrosine kinases such as epidermal growth factor receptor are affected by HCRP1 loss and looked for deregulated signaling using immunoblotting and classical cancer biology assays. In our study, we show that endogenous deletion of HCRP1 leads to elevated phosphorylation of the transcription factor Signal transducer and activator of transcription 3 (STAT3) and induces upregulation of PD-L1, an important regulator of immune checkpoint inhibition. HCRP1 loss further leads to a mesenchymal phenotype switch in cancer cells, leading to increased proliferation and migration. Concludingly, our data emphasize the role of the tumor microenvironment in tumors with low or absent HCRP1 expression and suggest HCRP1 loss as a potential marker for metastatic potential and immunogenicity of epidermal growth factor receptor-driven cancer.},
}
@article {pmid33196851,
year = {2020},
author = {Sgro, A and Blancafort, P},
title = {Epigenome engineering: new technologies for precision medicine.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {12453-12482},
pmid = {33196851},
issn = {1362-4962},
support = {R01 CA170370/CA/NCI NIH HHS/United States ; R01 DA036906/DA/NIDA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/*genetics ; DNA Methylation/*genetics ; Epigenome/*genetics ; *Gene Editing ; Genetic Engineering ; Humans ; Precision Medicine/trends ; },
abstract = {Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.},
}
@article {pmid33196849,
year = {2020},
author = {Chai, P and Yu, J and Jia, R and Wen, X and Ding, T and Zhang, X and Ni, H and Jia, R and Ge, S and Zhang, H and Fan, X},
title = {Generation of onco-enhancer enhances chromosomal remodeling and accelerates tumorigenesis.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12135-12150},
pmid = {33196849},
issn = {1362-4962},
mesh = {Animals ; CCCTC-Binding Factor/*genetics/metabolism ; CRISPR-Cas Systems ; Carcinogenesis/*genetics/metabolism/pathology ; Case-Control Studies ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; E1A-Associated p300 Protein/*genetics/metabolism ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Melanocytes/metabolism/pathology ; Melanoma/*genetics/metabolism/mortality/pathology ; Mice ; Mice, Nude ; Neurotensin/antagonists &amp; inhibitors/*genetics/metabolism ; Promoter Regions, Genetic ; Sequence Deletion ; Survival Analysis ; Tumor Burden ; Uveal Neoplasms/*genetics/metabolism/mortality/pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Chromatin remodeling impacts the structural neighborhoods and regulates gene expression. However, the role of enhancer-guided chromatin remodeling in the gene regulation remains unclear. Here, using RNA-seq and ChIP-seq, we identified for the first time that neurotensin (NTS) serves as a key oncogene in uveal melanoma and that CTCF interacts with the upstream enhancer of NTS and orchestrates an 800 kb chromosomal loop between the promoter and enhancer. Intriguingly, this novel CTCF-guided chromatin loop was ubiquitous in a cohort of tumor patients. In addition, a disruption in this chromosomal interaction prevented the histone acetyltransferase EP300 from embedding in the promoter of NTS and resulted in NTS silencing. Most importantly, in vitro and in vivo experiments showed that the ability of tumor formation was significantly suppressed via deletion of the enhancer by CRISPR-Cas9. These studies delineate a novel onco-enhancer guided epigenetic mechanism and provide a promising therapeutic concept for disease therapy.},
}
@article {pmid33195154,
year = {2020},
author = {Malcı, K and Walls, LE and Rios-Solis, L},
title = {Multiplex Genome Engineering Methods for Yeast Cell Factory Development.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {589468},
pmid = {33195154},
issn = {2296-4185},
abstract = {As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker's yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of pre-placed gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development.},
}
@article {pmid33193271,
year = {2020},
author = {Pickering, AC and Fitzgerald, JR},
title = {The Role of Gram-Positive Surface Proteins in Bacterial Niche- and Host-Specialization.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {594737},
pmid = {33193271},
issn = {1664-302X},
support = {/WT_/Wellcome Trust/United Kingdom ; },
abstract = {Gram-positive bacterial pathogens have an array of proteins on their cell surface that mediate interactions with the host environment. In particular, bacterial cell wall-associated (CWA) proteins play key roles in both colonization and pathogenesis. Furthermore, some CWA proteins promote specialization for host-species or mediate colonization of specific anatomical niches within a host. In this mini review, we provide examples of the many ways by which major pathogens, such as Staphylococci, Streptococci and Listeria monocytogenes, utilize CWA proteins for both host- and niche-specialization. We describe different biological mechanisms mediated by CWA proteins including: the acquisition of iron from hemoglobin in the bloodstream, adherence to and invasion of host cells, and innate immune evasion through binding to the plasma proteins fibrinogen, immunoglobulin G, and complement. We also discuss the limitations of using animal models for understanding the role of specific CWA proteins in host-specialization and how transformative technologies, such as CRISPR-Cas, offer tremendous potential for developing transgenic models that simulate the host environment of interest. Improved understanding of the role of CWA proteins in niche- or host-specificity will allow the design of new therapeutic approaches which target key host-pathogen interactions underpinning Gram-positive bacterial infections.},
}
@article {pmid33193268,
year = {2020},
author = {Cao, Y and Zhou, H and Zhou, X and Li, F},
title = {Control of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {593700},
pmid = {33193268},
issn = {1664-302X},
abstract = {Plant diseases caused by invading plant viruses pose serious threats to agricultural production in the world, and the antiviral engineering initiated by molecular biotechnology has been an effective strategy to prevent and control plant viruses. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-mediated DNA or RNA editing/interference in plants make them very attractive tools applicable to the plant protection field. Here, we review the development of CRISPR/Cas systems and summarize their applications in controlling different plant viruses by targeting viral sequences or host susceptibility genes. We list some potential recessive resistance genes that can be utilized in antiviral breeding and emphasize the importance and promise of recessive resistance gene-based antiviral breeding to generate transgene-free plants without developmental defects. Finally, we discuss the challenges and opportunities for the application of CRISPR/Cas techniques in the prevention and control of plant viruses in the field.},
}
@article {pmid33192264,
year = {2020},
author = {Duarte, F and Déglon, N},
title = {Genome Editing for CNS Disorders.},
journal = {Frontiers in neuroscience},
volume = {14},
number = {},
pages = {579062},
pmid = {33192264},
issn = {1662-4548},
abstract = {Central nervous system (CNS) disorders have a social and economic burden on modern societies, and the development of effective therapies is urgently required. Gene editing may prevent or cure a disease by inducing genetic changes at endogenous loci. Genome editing includes not only the insertion, deletion or replacement of nucleotides, but also the modulation of gene expression and epigenetic editing. Emerging technologies based on ZFs, TALEs, and CRISPR/Cas systems have extended the boundaries of genome manipulation and promoted genome editing approaches to the level of promising strategies for counteracting genetic diseases. The parallel development of efficient delivery systems has also increased our access to the CNS. In this review, we describe the various tools available for genome editing and summarize in vivo preclinical studies of CNS genome editing, whilst considering current limitations and alternative approaches to overcome some bottlenecks.},
}
@article {pmid33191932,
year = {2020},
author = {Loganathan, SK and Malik, A and Langille, E and Luxenburg, C and Schramek, D},
title = {In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {165},
pages = {},
doi = {10.3791/61693},
pmid = {33191932},
issn = {1940-087X},
support = {365252//CIHR/Canada ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Epithelium/metabolism ; *Genetic Testing ; High-Throughput Nucleotide Sequencing ; Integrases/metabolism ; Mice ; Microinjections ; Mouth/*metabolism ; Phenotype ; Skin/*metabolism ; Ultrasonics ; },
abstract = {Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct in vivo CRISPR screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.},
}
@article {pmid33191402,
year = {2021},
author = {Sreedurgalakshmi, K and Srikar, R and Rajkumari, R},
title = {CRISPR-Cas deployment in non-small cell lung cancer for target screening, validations, and discoveries.},
journal = {Cancer gene therapy},
volume = {28},
number = {6},
pages = {566-580},
pmid = {33191402},
issn = {1476-5500},
mesh = {CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/*genetics/pathology/therapy ; Early Detection of Cancer ; *Gene Editing ; Genome, Human/genetics ; Humans ; *Molecular Targeted Therapy ; },
abstract = {Continued advancements in CRISPR-Cas systems have accelerated genome research. Use of CRISPR-Cas in cancer research has been of great interest that is resulting in development of orthogonal methods for drug target validations and discovery of new therapeutic targets through genome-wide screens of cancer cells. CRISPR-based screens have also revealed several new cancer drivers through alterations in tumor suppressor genes (TSGs) and oncogenes inducing resistance to targeted therapies via activation of alternate signaling pathways. Given such dynamic status of cancer, we review the application of CRISPR-Cas in non-small cell lung cancer (NSCLC) for development of mutant models, drug screening, target validation, novel target discoveries, and other emerging potential applications. In addition, CRISPR-based approach for development of novel anticancer combination therapies is also discussed in this review.},
}
@article {pmid33191355,
year = {2020},
author = {Sugihara, H and Kimura, K and Yamanouchi, K and Teramoto, N and Okano, T and Daimon, M and Morita, H and Takenaka, K and Shiga, T and Tanihata, J and Aoki, Y and Inoue-Nagamura, T and Yotsuyanagi, H and Komuro, I},
title = {Age-Dependent Echocardiographic and Pathologic Findings in a Rat Model with Duchenne Muscular Dystrophy Generated by CRISPR/Cas9 Genome Editing.},
journal = {International heart journal},
volume = {61},
number = {6},
pages = {1279-1284},
doi = {10.1536/ihj.20-372},
pmid = {33191355},
issn = {1349-3299},
mesh = {Age Factors ; Animals ; Blood Flow Velocity ; CRISPR-Cas Systems ; Cardiomyopathies/diagnostic imaging/genetics/pathology/*physiopathology ; *Disease Models, Animal ; Dystrophin/*genetics ; Echocardiography ; Frameshift Mutation ; Gene Editing ; Heart/diagnostic imaging/*physiopathology ; Heart Ventricles/diagnostic imaging/pathology/physiopathology ; Male ; Muscular Dystrophy, Duchenne/diagnostic imaging/genetics/pathology/*physiopathology ; Myocardium/*pathology ; *Rats ; },
abstract = {Duchenne muscular dystrophy (DMD) is X-linked recessive myopathy caused by mutations in the dystrophin gene. Although conventional treatments have improved their prognosis, inevitable progressive cardiomyopathy is still the leading cause of death in patients with DMD. To explore novel therapeutic options, a suitable animal model with heart involvement has been warranted.We have generated a rat model with an out-of-frame mutation in the dystrophin gene using CRISPR/Cas9 genome editing (DMD rats). The aim of this study was to evaluate their cardiac functions and pathologies to provide baseline data for future experiments developing treatment options for DMD.In comparison with age-matched wild rats, 6-month-old DMD rats showed no significant differences by echocardiographic evaluations. However, 10-month-old DMD rats showed significant deterioration in left ventricular (LV) fractional shortening (P = 0.024), and in tissue Doppler peak systolic velocity (Sa) at the LV lateral wall (P = 0.041) as well as at the right ventricular (RV) free-wall (P = 0.004). These functional findings were consistent with the fibrotic distributions by histological analysis.Although the cardiac phenotype was milder than anticipated, DMD rats showed similar distributions and progression of heart involvement to those of patients with DMD. This animal may be a useful model with which to develop effective drugs and to understand the underlying mechanisms of progressive heart failure in patients with DMD.},
}
@article {pmid33190579,
year = {2021},
author = {Riching, KM and Schwinn, MK and Vasta, JD and Robers, MB and Machleidt, T and Urh, M and Daniels, DL},
title = {CDK Family PROTAC Profiling Reveals Distinct Kinetic Responses and Cell Cycle-Dependent Degradation of CDK2.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {26},
number = {4},
pages = {560-569},
doi = {10.1177/2472555220973602},
pmid = {33190579},
issn = {2472-5560},
mesh = {Adaptor Proteins, Signal Transducing/genetics/metabolism ; *Biological Assay ; CRISPR-Cas Systems ; Cell Cycle/*drug effects/genetics ; Cyclin-Dependent Kinase 2/genetics/*metabolism ; Cyclin-Dependent Kinase 4/genetics/*metabolism ; HEK293 Cells ; Humans ; Kinetics ; Oxindoles/*pharmacology ; Piperidines/*pharmacology ; Proteasome Endopeptidase Complex/drug effects ; Protein Binding ; *Protein Processing, Post-Translational ; Proteolysis/drug effects ; Recombinant Fusion Proteins/genetics/metabolism ; Staining and Labeling ; Ubiquitin-Protein Ligases/genetics/metabolism ; Ubiquitination/drug effects ; },
abstract = {Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.},
}
@article {pmid33190185,
year = {2022},
author = {Petazzi, P and Menéndez, P and Sevilla, A},
title = {CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {559-574},
pmid = {33190185},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/metabolism ; Endonucleases/genetics/metabolism ; Gene Knockout Techniques ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR/Cas9-mediated editing has become the gold standard for precise genome manipulation as it offers a unique, versatile, and limitless tool for fast, robust, and efficient genome editing. Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.},
}
@article {pmid33188726,
year = {2021},
author = {Lee, J and Choi, A and Cho, SY and Jun, Y and Na, D and Lee, A and Jang, G and Kwon, JY and Kim, J and Lee, S and Lee, C},
title = {Genome-scale CRISPR screening identifies cell cycle and protein ubiquitination processes as druggable targets for erlotinib-resistant lung cancer.},
journal = {Molecular oncology},
volume = {15},
number = {2},
pages = {487-502},
pmid = {33188726},
issn = {1878-0261},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Cycle ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/drug effects/genetics ; Erlotinib Hydrochloride/*pharmacology ; Female ; Genome-Wide Association Study ; Humans ; *Lung Neoplasms/drug therapy/genetics/metabolism ; Mice ; Mice, Nude ; Mice, SCID ; *Neoplasm Proteins/drug effects/genetics/metabolism ; *Ubiquitination ; },
abstract = {Erlotinib is highly effective in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. However, despite initial favorable responses, most patients rapidly develop resistance to erlotinib soon after the initial treatment. This study aims to identify new genes and pathways associated with erlotinib resistance mechanisms in order to develop novel therapeutic strategies. Here, we induced knockout (KO) mutations in erlotinib-resistant human lung cancer cells (NCI-H820) using a genome-scale CRISPR-Cas9 sgRNA library to screen for genes involved in erlotinib susceptibility. The spectrum of sgRNAs incorporated among erlotinib-treated cells was substantially different to that of the untreated cells. Gene set analyses showed a significant depletion of 'cell cycle process' and 'protein ubiquitination pathway' genes among erlotinib-treated cells. Chemical inhibitors targeting genes in these two pathways, such as nutlin-3 and carfilzomib, increased cancer cell death when combined with erlotinib in both in vitro cell line and in vivo patient-derived xenograft experiments. Therefore, we propose that targeting cell cycle processes or protein ubiquitination pathways are promising treatment strategies for overcoming resistance to EGFR inhibitors in lung cancer.},
}
@article {pmid33187879,
year = {2020},
author = {Liu, H and Wang, K and Tang, H and Gong, Q and Du, L and Pei, X and Ye, X},
title = {CRISPR/Cas9 editing of wheat TaQ genes alters spike morphogenesis and grain threshability.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {9},
pages = {563-575},
doi = {10.1016/j.jgg.2020.08.004},
pmid = {33187879},
issn = {1673-8527},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Domestication ; Edible Grain/*genetics/growth &amp; development ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Morphogenesis/*genetics ; Plant Proteins/*genetics ; Triticum/*genetics/growth &amp; development ; },
abstract = {The TaQ alleles as one of the AP2-like transcription factors in common wheat (Triticum aestivum) play an important role in the evolution of spike characteristics from wild and domesticated emmer to modern wheat cultivars. Its loss-of-function mutant not only changed threshability and spike architecture but also affected plant height, flowering time, and floret structure. However, the comprehensive functions of TaAQ and TaDq genes in wheat have not been fully elucidated yet. Here, CRISPR/SpCas9 was used to edit wheat TaAQ and TaDq. We obtained homozygous plants in the T1 generation with loss of function of only TaAQ or TaDq and simultaneous loss of function of TaAQ and TaDq to analyze the effect of these genes on wheat spikes and floret shapes. The results demonstrated that the TaAQ-edited plants and the TaAQ and TaDq simultaneously-edited plants were nearly similar in spike architecture, whereas the TaDq-edited plants were different from the wild-type ones only in plant height. Moreover, the TaAQ-edited plants or the TaAQ and TaDq simultaneously-edited plants were more brittle than the wild-type and the TaDq-edited plants. Based on the expression profiling, we postulated that the VRN1, FUL2, SEP2, SEP5, and SEP6 genes might affect the number of spikelets and florets per spike in wheat by regulating the expression of TaQ. Combining the results of this report and previous reports, we conceived a regulatory network of wheat traits, including plant height, spike shape, and floral organs, which were influenced by AP2-like family genes. The results achieved in this study will help us to understand the regulating mechanisms of TaAQ and TaDq alleles on wheat floral organs and inflorescence development.},
}
@article {pmid33185863,
year = {2021},
author = {Zhang, Z and Yuan, S and Xu, S and Guo, D and Chen, L and Hou, W and Wang, M},
title = {Suppression of HIV-1 Integration by Targeting HIV-1 Integrase for Degradation with A Chimeric Ubiquitin Ligase.},
journal = {Virologica Sinica},
volume = {36},
number = {3},
pages = {424-437},
pmid = {33185863},
issn = {1995-820X},
mesh = {*HIV Integrase/genetics ; *HIV-1/genetics ; Humans ; Ubiquitin/genetics ; Virus Replication ; },
abstract = {Human immunodeficiency virus (HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome (AIDS). Treatment with combination antiretroviral therapy (cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway (UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases (E3s) based on known human E3s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation. Herein, a series of prototypical chimeric E3s have been designed and constructed, and original substrate-binding domains of these E3s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146LI was then further optimized to generate 146LIS (146LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair (HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection.},
}
@article {pmid33185860,
year = {2021},
author = {Batool, A and Malik, F and Andrabi, KI},
title = {Expansion of the CRISPR/Cas Genome-Sculpting Toolbox: Innovations, Applications and Challenges.},
journal = {Molecular diagnosis &amp; therapy},
volume = {25},
number = {1},
pages = {41-57},
pmid = {33185860},
issn = {1179-2000},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Repair ; Epigenesis, Genetic ; Gene Expression ; Genetic Engineering/*methods ; Humans ; },
abstract = {The emergence of the versatile gene-editing technology using programmable sequence-specific endonuclease system (CRISPR-Cas9) has instigated a major upheaval in biomedical research. In a brief span of time, CRISPR/Cas has been adopted by research labs around the globe because of its potential for significant progress and applicability in terms of efficiency, versatility and simplicity. It is a breakthrough technique for systematic genetic engineering, genome labelling, epigenetic and transcriptional modulation, and multiplexed gene editing, amongst others. This review provides an illustrative overview of the current research trends using CRISPR/Cas technology. We highlight the latest developments in CRISPR/Cas technique including CRISPR imaging, discovery of novel CRISPR systems, and applications in altering the genome, epigenome or RNA in different organisms. Finally, we address the potential challenges of this technique for its future use. Development of new CRISPR/Cas systems.},
}
@article {pmid33185489,
year = {2020},
author = {Maroufi, F and Maali, A and Abdollahpour-Alitappeh, M and Ahmadi, MH and Azad, M},
title = {CRISPR-mediated modification of DNA methylation pattern in the new era of cancer therapy.},
journal = {Epigenomics},
volume = {12},
number = {20},
pages = {1845-1859},
doi = {10.2217/epi-2020-0110},
pmid = {33185489},
issn = {1750-192X},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *DNA Methylation ; Epigenesis, Genetic ; Epigenome ; *Gene Editing ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {In the last 2 decades, a wide variety of studies have been conducted on epigenetics and its role in various cancers. A major mechanism of epigenetic regulation is DNA methylation, including aberrant DNA methylation variations such as hypermethylation and hypomethylation in the promoters of critical genes, which are commonly detected in tumors and mark the early stages of cancer development. Therefore, epigenetic therapy has been of special importance in the last decade for cancer treatment. In epigenetic therapy, all efforts are made to modulate gene expression to the normal status. Importantly, recent studies have shown that epigenetic therapy is focusing on the new gene editing technology, CRISPR-Cas9. This tool was found to be able to effectively modulate gene expression and alter almost any sequence in the genome of cells, resulting in events such as a change in acetylation, methylation, or histone modifications. Of note, the CRISPR-Cas9 system can be used for the treatment of cancers caused by epigenetic alterations. The CRISPR-Cas9 system has greater advantages than other available methods, including potent activity, easy design and high velocity as well as the ability to target any DNA or RNA site. In this review, we described epigenetic modulators, which can be used in the CRISPR-Cas9 system, as well as their functions in gene expression alterations that lead to cancer initiation and progression. In addition, we surveyed various species of CRISPR-dead Cas9 (dCas9) systems, a mutant version of Cas9 with no endonuclease activity. Such systems are applicable in epigenetic therapy for gene expression modulation through chemical group editing on nucleosomes and chromatin remodeling, which finally return the cell to the normal status and prevent cancer progression.},
}
@article {pmid33184267,
year = {2020},
author = {Luo, B and Zhan, Y and Luo, M and Dong, H and Liu, J and Lin, Y and Zhang, J and Wang, G and Verhoeyen, E and Zhang, Y and Zhang, H},
title = {Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration.},
journal = {Cell death &amp; disease},
volume = {11},
number = {11},
pages = {973},
pmid = {33184267},
issn = {2041-4889},
mesh = {Animals ; Antibodies, Monoclonal/biosynthesis/genetics/*immunology ; B-Lymphocytes/cytology/immunology ; *CRISPR-Cas Systems ; Cell Differentiation/immunology ; Cell Line, Tumor ; HEK293 Cells ; Humans ; Immunotherapy/*methods ; Mice ; Plasma Cells/cytology/*immunology ; Programmed Cell Death 1 Receptor/*genetics/*immunology ; T-Lymphocytes/immunology ; Transgenes ; },
abstract = {Long-lived plasma cells (LLPCs) are robust specialized antibody-secreting cells that mainly stay in the bone marrow and can persist a lifetime. As they can be generated by inducing the differentiation of B-lymphocytes, we investigated the possibility that human LLPCs might be engineered to express α-PD-1 monoclonal antibody to substitute recombinant α-PD-1 antitumor immunotherapy. To this end, we inserted an α-PD-1 cassette into the GAPDH locus through Cas9/sgRNA-guided specific integration in B-lymphocytes, which was mediated by an integrase-defective lentiviral vector. The edited B cells were capable of differentiating into LLPCs both in vitro and in vivo. Transcriptional profiling analysis confirmed that these cells were typical LLPCs. Importantly, these cells secreted de novo antibodies persistently, which were able to inhibit human melanoma growth via an antibody-mediated checkpoint blockade in xenograft-tumor mice. Our work suggests that the engineered LLPCs may be utilized as a vehicle to constantly produce special antibodies for long-term cellular immunotherapy to eradicate tumors and cellular reservoirs for various pathogens including human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV).},
}
@article {pmid33184093,
year = {2020},
author = {Beigl, TB and Kjosås, I and Seljeseth, E and Glomnes, N and Aksnes, H},
title = {Efficient and crucial quality control of HAP1 cell ploidy status.},
journal = {Biology open},
volume = {9},
number = {11},
pages = {},
pmid = {33184093},
issn = {2046-6390},
mesh = {CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Diploidy ; Flow Cytometry ; Gene Editing ; Gene Knockdown Techniques ; Haploidy ; Humans ; Nerve Tissue Proteins/*genetics/metabolism ; *Ploidies ; },
abstract = {The near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.},
}
@article {pmid33182769,
year = {2020},
author = {Storey, N and Rabiey, M and Neuman, BW and Jackson, RW and Mulley, G},
title = {Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages.},
journal = {Viruses},
volume = {12},
number = {11},
pages = {},
pmid = {33182769},
issn = {1999-4915},
mesh = {Agaricales/*metabolism/virology ; Agaricus/metabolism/virology ; Amino Acid Sequence ; Culture Media/chemistry ; DNA, Bacterial/genetics/isolation &amp; purification ; Genome, Bacterial ; *Genome, Viral ; Multigene Family ; Peptide Synthases/genetics/metabolism ; Pseudomonas/*isolation &amp; purification/metabolism/virology ; Pseudomonas Phages/*genetics/*isolation &amp; purification/metabolism ; Sequence Analysis, DNA ; Type III Secretion Systems/genetics/metabolism ; },
abstract = {Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonastolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonasagarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.},
}
@article {pmid33181064,
year = {2020},
author = {Gaudelli, NM and Komor, AC},
title = {Celebrating Rosalind Franklin's Centennial with a Nobel Win for Doudna and Charpentier.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2519-2520},
pmid = {33181064},
issn = {1525-0024},
mesh = {COVID-19/*epidemiology/virology ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; *Gene Editing ; Humans ; *Nobel Prize ; *Pandemics ; },
}
@article {pmid33180479,
year = {2020},
author = {Greisch, JF and van der Laarse, SAM and Heck, AJR},
title = {Enhancing Top-Down Analysis Using Chromophore-Assisted Infrared Multiphoton Dissociation from (Phospho)peptides to Protein Assemblies.},
journal = {Analytical chemistry},
volume = {92},
number = {23},
pages = {15506-15516},
pmid = {33180479},
issn = {1520-6882},
mesh = {*Infrared Rays ; Mass Spectrometry/*methods ; Phosphopeptides/*chemistry ; Phosphoproteins/*chemistry ; *Photons ; Protein Aggregates ; },
abstract = {Infrared multiphoton dissociation (IRMPD) has been used in mass spectrometry to fragment peptides and proteins, providing fragments mostly similar to collisional activation. Using the 10.6 μm wavelength of a CO2 laser, IRMPD suffers from the relative low absorption cross-section of peptides and small proteins. Focusing on top-down analysis, we investigate different means to tackle this issue. We first reassess efficient sorting of phosphopeptides from nonphosphopeptides based on IR-absorption cross-sectional enhancement by phosphate moieties. We subsequently demonstrate that a myo-inositol hexakisphosphate (IP6) noncovalent adduct can substantially enhance IRMPD for nonphosphopeptides and that this strategy can be extended to proteins. As a natural next step, we show that native phospho-proteoforms of proteins display a distinct and enhanced fragmentation, compared to their unmodified counterparts, facilitating phospho-group site localization. We then evaluate the impact of size on the IRMPD of proteins and their complexes. When applied to protein complexes ranging from a 365 kDa CRISPR-Cas Csy ribonucleoprotein hetero-decamer, a 800 kDa GroEL homo-tetradecamer in its apo-form or loaded with its ATP cofactor, to a 1 MDa capsid-like homo-hexacontamer, we conclude that while phosphate moieties present in crRNA and ATP molecules enhance IRMPD, an increase in the IR cross-section with the size of the protein assembly also favorably accrues dissociation yields. Overall, our work showcases the versatility of IRMPD in the top-down analysis of peptides, phosphopeptides, proteins, phosphoproteins, ribonucleoprotein assemblies, and large protein complexes.},
}
@article {pmid33180466,
year = {2020},
author = {Baumschabl, M and Prielhofer, R and Mattanovich, D and Steiger, MG},
title = {Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds.},
journal = {ACS synthetic biology},
volume = {9},
number = {12},
pages = {3202-3209},
pmid = {33180466},
issn = {2161-5063},
support = {W 1224/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {CRISPR-Cas Systems/*genetics ; Genes, Reporter ; Pichia/*genetics ; Plasmids/genetics/metabolism ; RNA, Guide/*metabolism ; Riboflavin/biosynthesis ; Thiamine/pharmacology ; Transcription Factors/genetics/metabolism ; Transcription, Genetic/drug effects ; },
abstract = {For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris. It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.},
}
@article {pmid33180295,
year = {2021},
author = {Hwang, GH and Bae, S},
title = {Web-Based Base Editing Toolkits: BE-Designer and BE-Analyzer.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2189},
number = {},
pages = {81-88},
doi = {10.1007/978-1-0716-0822-7_7},
pmid = {33180295},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Internet ; *Sequence Analysis, DNA ; *Software ; },
abstract = {The CRISPR-Cas system is broadly used for genome editing because of its convenience and relatively low cost. However, the use of CRISPR nucleases to induce specific nucleotide changes in target DNA requires complex procedures and additional donor DNAs. Furthermore, CRISPR nuclease-mediated DNA cleavage at target sites frequently causes large deletions or genomic rearrangements. In contrast, base editors that consist of catalytically dead Cas9 (dCas9) or Cas9 nickase (nCas9) connected to a cytidine or a guanine deaminase can correct point mutations in the absence of additional donor DNA and without generating double-strand breaks (DSBs) in the target region. To design target sites and assess mutation ratios for cytosine and adenine base editors (CBEs and ABEs), we have developed web tools, named BE-Designer and BE-Analyzer. These tools are easy to use (such that tasks are accomplished by clicking on relevant buttons) and do not require a deep knowledge of bioinformatics.},
}
@article {pmid33180294,
year = {2021},
author = {Nobles, CL},
title = {iGUIDE Method for CRISPR Off-Target Detection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2189},
number = {},
pages = {71-80},
doi = {10.1007/978-1-0716-0822-7_6},
pmid = {33180294},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *Gene Editing ; Humans ; RNA, Guide/*genetics ; *Sequence Analysis, DNA ; },
abstract = {With the advent of genome editing technologies, scientists have recognized that these technologies can be prone to nonspecific or off-target activity. As many areas of the genome are sensitive and can give rise to abnormalities if mutated, it is imperative that scientists identify regions of off-target activity in order to utilize these new technologies for medical benefits. GUIDE-seq and iGUIDE both use an oligo-based marker method to identify regions of DNA double-strand breaks in an unbiased manner. The repeated observation of these double-strand breaks across the genome in comparison with target sequences (such as guide RNAs) has allowed researchers to identify on- and off-target sites related to their targeted-nuclease technologies.},
}
@article {pmid33180293,
year = {2021},
author = {Liu, H and Wang, X},
title = {CRISPR-ERA: A Webserver for Guide RNA Design of Gene Editing and Regulation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2189},
number = {},
pages = {65-69},
doi = {10.1007/978-1-0716-0822-7_5},
pmid = {33180293},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Library ; *Internet ; RNA, Guide/*genetics ; *Software ; },
abstract = {The CRISPR/Cas9 system has been developed as a powerful technology for both targeted genome editing and gene regulation. However, the design of efficient single-guide RNAs (sgRNAs) remains challenging with the consideration of many criteria. In this section, we introduce how to design sgRNA sequences and build genome-wide sgRNA library using CRISPR-ERA, which is one of the state-of-the-art designer webserver tools for sgRNA design based on a set of sgRNA design rules summarized from published reports.},
}
@article {pmid33179869,
year = {2020},
author = {Mao, M and Chang, CC and Pickar-Oliver, A and Cervia, LD and Wang, L and Ji, J and Liton, PB and Gersbach, CA and Yuan, F},
title = {Redirecting Vesicular Transport to Improve Nonviral Delivery of Molecular Cargo.},
journal = {Advanced biosystems},
volume = {4},
number = {8},
pages = {e2000059},
pmid = {33179869},
issn = {2366-7478},
support = {R01 GM098520/GM/NIGMS NIH HHS/United States ; R01 EY027733/EY/NEI NIH HHS/United States ; U01EB028901/NH/NIH HHS/United States ; R01 EY026885/EY/NEI NIH HHS/United States ; R01 GM130830/GM/NIGMS NIH HHS/United States ; },
mesh = {Biological Transport ; CRISPR-Cas Systems ; DNA Transposable Elements ; Drug Delivery Systems/*methods ; Electroporation ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Lysosomes/drug effects/metabolism ; Microtubule-Associated Proteins/genetics/metabolism ; Plasmids/chemistry/metabolism ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Raffinose/*pharmacology ; Ribonucleoproteins/genetics/metabolism ; Sucrose/*pharmacology ; Trehalose/*pharmacology ; },
abstract = {Cell engineering relies heavily on viral vectors for the delivery of molecular cargo into cells due to their superior efficiency compared to nonviral ones. However, viruses are immunogenic and expensive to manufacture, and have limited delivery capacity. Nonviral delivery approaches avoid these limitations but are currently inefficient for clinical applications. This work demonstrates that the efficiency of nonviral delivery of plasmid DNA, mRNA, Sleeping Beauty transposon, and ribonucleoprotein can be significantly enhanced through pretreatment of cells with the nondegradable sugars (NDS), such as sucrose, trehalose, and raffinose. The enhancement is mediated by the incorporation of the NDS into cell membranes, causing enlargement of lysosomes and formation of large (>500 nm) amphisome-like bodies (ALBs). The changes in subcellular structures redirect transport of cargo to ALBs rather than to lysosomes, reducing cargo degradation in cells. The data indicate that pretreatment of cells with NDS is a promising approach to improve nonviral cargo delivery in biomedical applications.},
}
@article {pmid33179815,
year = {2020},
author = {Ullah, M and Xia, L and Xie, S and Sun, S},
title = {CRISPR/Cas9-based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi.},
journal = {Biotechnology and applied biochemistry},
volume = {67},
number = {6},
pages = {835-851},
doi = {10.1002/bab.2077},
pmid = {33179815},
issn = {1470-8744},
mesh = {*CRISPR-Cas Systems ; Fungi/*genetics ; *Gene Editing ; *Genome, Fungal ; },
abstract = {Filamentous fungi have several industrial, environmental, and medical applications. However, they are rarely utilized owing to the limited availability of full-genome sequences and genetic manipulation tools. Since the recent discovery of the full-genome sequences for certain industrially important filamentous fungi, CRISPR/Cas9 technology has drawn attention for the efficient development of engineered strains of filamentous fungi. CRISPR/Cas9 genome editing has been successfully applied to diverse filamentous fungi. In this review, we briefly discuss the use of common genetic transformation techniques as well as CRISPR/Cas9-based systems in filamentous fungi. Furthermore, we describe potential limitations and challenges in the practical application of genome engineering of filamentous fungi. Finally, we provide suggestions and highlight future research prospects in the area.},
}
@article {pmid33179250,
year = {2020},
author = {Benler, S and Koonin, EV},
title = {Phage lysis-lysogeny switches and programmed cell death: Danse macabre.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {42},
number = {12},
pages = {e2000114},
doi = {10.1002/bies.202000114},
pmid = {33179250},
issn = {1521-1878},
mesh = {Apoptosis ; Bacteriophage lambda/genetics ; *Bacteriophages/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Lysogeny ; Virus Latency ; },
abstract = {Exploration of immune systems in prokaryotes, such as restriction-modification or CRISPR-Cas, shows that both innate and adaptive systems possess programmed cell death (PCD) potential. The key outstanding question is how the immune systems sense and "predict" infection outcomes to "decide" whether to fight the pathogen or induce PCD. There is a striking parallel between this life-or-death decision faced by the cell and the decision by temperate viruses to protect or kill their hosts, epitomized by the lysis-lysogeny switch of bacteriophage Lambda. Immune systems and temperate phages sense the same molecular inputs, primarily, DNA damage, that determine whether the cell lives or dies. Because temperate (pro)phages are themselves components of prokaryotic genomes, their shared "interests" with the hosts result in coregulation of the lysis-lysogeny switch and immune systems that jointly provide the cell with the decision machinery to probe and predict infection outcomes, answering the life-or-death question.},
}
@article {pmid33178158,
year = {2020},
author = {Mancilla-Rojano, J and Ochoa, SA and Reyes-Grajeda, JP and Flores, V and Medina-Contreras, O and Espinosa-Mazariego, K and Parra-Ortega, I and Rosa-Zamboni, D and Castellanos-Cruz, MDC and Arellano-Galindo, J and Cevallos, MA and Hernández-Castro, R and Xicohtencatl-Cortes, J and Cruz-Córdova, A},
title = {Molecular Epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex Isolated From Children at the Hospital Infantil de México Federico Gómez.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {576673},
pmid = {33178158},
issn = {1664-302X},
abstract = {The Acinetobacter calcoaceticus-baumannii (Acb) complex is regarded as a group of phenotypically indistinguishable opportunistic pathogens responsible for mainly causing hospital-acquired pneumonia and bacteremia. The aim of this study was to determine the frequency of isolation of the species that constitute the Acb complex, as well as their susceptibility to antibiotics, and their distribution at the Hospital Infantil de Mexico Federico Gomez (HIMFG). A total of 88 strains previously identified by Vitek 2®, 40 as Acinetobacter baumannii and 48 as Acb complex were isolated from 52 children from 07, January 2015 to 28, September 2017. A. baumannii accounted for 89.77% (79/88) of the strains; Acinetobacter pittii, 6.82% (6/88); and Acinetobacter nosocomialis, 3.40% (3/88). Most strains were recovered mainly from patients in the intensive care unit (ICU) and emergency wards. Blood cultures (BC) provided 44.32% (39/88) of strains. The 13.63% (12/88) of strains were associated with primary bacteremia, 3.4% (3/88) with secondary bacteremia, and 2.3% (2/88) with pneumonia. In addition, 44.32% (39/88) were multidrug-resistant (MDR) strains and, 11.36% (10/88) were extensively drug-resistant (XDR). All strains amplified the bla OXA-51 gene; 51.13% (45/88), the bla OXA-23 gene; 4.54% (4/88), the bla OXA-24 gene; and 2.27% (2/88), the bla OXA-58 gene. Plasmid profiles showed that the strains had 1-6 plasmids. The strains were distributed in 52 pulsotypes, and 24 showed identical restriction patterns, with a correlation coefficient of 1.0. Notably, some strains with the same pulsotype were isolated from different patients, wards, or years, suggesting the persistence of more than one clone. Twenty-seven sequence types (STs) were determined for the strains based on a Pasteur multilocus sequence typing (MLST) scheme using massive sequencing; the most prevalent was ST 156 (27.27%, 24/88). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas I-Fb system provided amplification in A. baumannii and A. pittii strains (22.73%, 20/88). This study identified an increased number of MDR strains and the relationship among strains through molecular typing. The data suggest that more than one strain could be causing an infection in some patient. The implementation of molecular epidemiology allowed the characterization of a set of strains and identification of different attributes associated with its distribution in a specific environment.},
}
@article {pmid33177521,
year = {2020},
author = {Du, K and Luo, Q and Yin, L and Wu, J and Liu, Y and Gan, J and Dong, A and Shen, WH},
title = {OsChz1 acts as a histone chaperone in modulating chromatin organization and genome function in rice.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5717},
pmid = {33177521},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Chromatin/genetics/*metabolism ; DNA Methylation ; Flowers/metabolism ; Gene Expression Regulation, Plant ; Genome, Plant ; Histones/genetics/*metabolism ; Molecular Chaperones/genetics/metabolism ; Mutation ; Nucleosomes/genetics ; Oryza/*genetics/growth &amp; development/metabolism ; Phylogeny ; Plant Proteins/genetics/*metabolism ; Plants, Genetically Modified ; Protein Multimerization ; },
abstract = {While the yeast Chz1 acts as a specific histone-chaperone for H2A.Z, functions of CHZ-domain proteins in multicellular eukaryotes remain obscure. Here, we report on the functional characterization of OsChz1, a sole CHZ-domain protein identified in rice. OsChz1 interacts with both the canonical H2A-H2B dimer and the variant H2A.Z-H2B dimer. Within crystal structure the C-terminal region of OsChz1 binds H2A-H2B via an acidic region, pointing to a previously unknown recognition mechanism. Knockout of OsChz1 leads to multiple plant developmental defects. At genome-wide level, loss of OsChz1 causes mis-regulations of thousands of genes and broad alterations of nucleosome occupancy as well as reductions of H2A.Z-enrichment. While OsChz1 associates with chromatin regions enriched of repressive histone marks (H3K27me3 and H3K4me2), its loss does not affect the genome landscape of DNA methylation. Taken together, it is emerging that OsChz1 functions as an important H2A/H2A.Z-H2B chaperone in dynamic regulation of chromatin for higher eukaryote development.},
}
@article {pmid33177215,
year = {2020},
author = {Petitjean, O and Girardi, E and Ngondo, RP and Lupashin, V and Pfeffer, S},
title = {Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection.},
journal = {mSphere},
volume = {5},
number = {6},
pages = {},
pmid = {33177215},
issn = {2379-5042},
support = {R01 GM083144/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Viruses/*metabolism ; Golgi Apparatus/metabolism ; HCT116 Cells ; Heparitin Sulfate/metabolism ; Humans ; Immunity, Innate ; RNA Viruses/*metabolism ; RNA, Double-Stranded/*metabolism ; Virus Diseases/*metabolism/pathology ; },
abstract = {Double-stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss-of-function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry, and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the conserved oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knockout led to a decrease of extracellular HS that specifically affected dsRNA transfection efficiency and reduced viral production, which explains the increased cell survival of these mutants.IMPORTANCE When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.},
}
@article {pmid33176267,
year = {2020},
author = {Sun, L and Li, J and Li, E and Niu, S and Qin, Z and Zhi, Q and Zhao, J and Xiong, H and Li, Y and Jian, L and Zhang, L},
title = {CRISPR/Cas9 mediated establishment of a human CSRP3 compound heterozygous knockout hESC line to model cardiomyopathy and heart failure.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102077},
doi = {10.1016/j.scr.2020.102077},
pmid = {33176267},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Cardiomyopathies/genetics ; Cell Line ; Female ; *Heart Failure/genetics ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {The role of muscle LIM protein (MLP), encoded by CSRP3, is not well understood in humans. CSRP3 knockout mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Using CRISPR/Cas9, we generated an MLP deficient human ESC line WAe009-A-41 carrying a compound heterozygous 13 bp deletion/1 bp insertion in the CSRP3 gene. The WAe009-A-41 line exhibited a normal female karyotype (46, XX), expressed pluripotency markers and exhibited capability to differentiate into the three germ layers in vitro. MLP expression was not detectable in WAe009-A-41 line. This cell line can be a useful tool for studying the role of CSRP3 in cardiomyopathy and heart failure.},
}
@article {pmid33176167,
year = {2020},
author = {Mehta, HM and Corey, SJ},
title = {Getting Back to Normal: Correcting SCN by Universal or Precision Strikes.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2525-2526},
pmid = {33176167},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Congenital Bone Marrow Failure Syndromes ; *Hematopoietic Stem Cell Transplantation ; Mutation ; Neutropenia/congenital ; },
}
@article {pmid33175893,
year = {2020},
author = {Smirnikhina, SA and Kondrateva, EV and Adilgereeva, EP and Anuchina, AA and Zaynitdinova, MI and Slesarenko, YS and Ershova, AS and Ustinov, KD and Yasinovsky, MI and Amelina, EL and Voronina, ES and Yakushina, VD and Tabakov, VY and Lavrov, AV},
title = {P.F508del editing in cells from cystic fibrosis patients.},
journal = {PloS one},
volume = {15},
number = {11},
pages = {e0242094},
pmid = {33175893},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Cystic Fibrosis/*genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; DNA Repair ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out.},
}
@article {pmid33175375,
year = {2021},
author = {Miki, D and Zinta, G and Zhang, W and Peng, F and Feng, Z and Zhu, JK},
title = {CRISPR/Cas9-Based Genome Editing Toolbox for Arabidopsis thaliana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2200},
number = {},
pages = {121-146},
doi = {10.1007/978-1-0716-0880-7_5},
pmid = {33175375},
issn = {1940-6029},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome, Plant ; },
abstract = {CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system. Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing in other plant species such as rice.},
}
@article {pmid33174655,
year = {2020},
author = {Ye, Q and Sung, TC and Yang, JM and Ling, QD and He, Y and Higuchi, A},
title = {Generation of universal and hypoimmunogenic human pluripotent stem cells.},
journal = {Cell proliferation},
volume = {53},
number = {12},
pages = {e12946},
pmid = {33174655},
issn = {1365-2184},
mesh = {CRISPR-Cas Systems/*genetics ; Family Characteristics ; *Gene Editing ; Humans ; Killer Cells, Natural/*cytology ; Pluripotent Stem Cells/*cytology ; Stem Cell Transplantation/methods ; },
abstract = {There is a need to store very large numbers of conventional human pluripotent stem cell (hPSC) lines for their off-the-shelf usage in stem cell therapy. Therefore, it is valuable to generate "universal" or "hypoimmunogenic" hPSCs with gene-editing technology by knocking out or in immune-related genes. A few universal or hypoimmunogenic hPSC lines should be enough to store for their off-the-shelf usage. Here, we overview and discuss how to prepare universal or hypoimmunogenic hPSCs and their disadvantages. β2-Microglobulin-knockout hPSCs did not harbour human leukocyte antigen (HLA)-expressing class I cells but rather activated natural killer (NK) cells. To avoid NK cell and macrophage activities, homozygous hPSCs expressing a single allele of an HLA class I molecule, such as HLA-C, were developed. Major HLA class I molecules were knocked out, and PD-L1, HLA-G and CD47 were knocked in hPSCs using CRISPR/Cas9 gene editing. These cells escaped activation of not only T cells but also NK cells and macrophages, generating universal hPSCs.},
}
@article {pmid33174413,
year = {2020},
author = {Aksoy, YA and Yang, B and Chen, W and Hung, T and Kuchel, RP and Zammit, NW and Grey, ST and Goldys, EM and Deng, W},
title = {Spatial and Temporal Control of CRISPR-Cas9-Mediated Gene Editing Delivered via a Light-Triggered Liposome System.},
journal = {ACS applied materials &amp; interfaces},
volume = {12},
number = {47},
pages = {52433-52444},
doi = {10.1021/acsami.0c16380},
pmid = {33174413},
issn = {1944-8252},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Embryo, Nonmammalian/metabolism ; Gene Editing/*methods ; Gene Expression/drug effects ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Light ; Liposomes/*chemistry ; Singlet Oxygen/metabolism ; Tumor Necrosis Factor alpha-Induced Protein 3/genetics/metabolism ; Tumor Necrosis Factor-alpha/pharmacology ; Zebrafish/growth &amp; development/metabolism ; },
abstract = {The CRISPR-Cas9 and related systems offer a unique genome-editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with the CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with the CRISPR-Cas9 system. We demonstrated its efficient protein release by respectively assessing the targeted knockout of the eGFP gene in human HEK293/GFP cells and the TNFAIP3 gene in TNFα-induced HEK293 cells. We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). These findings indicate that light-triggered liposomes may have new options for precise control of CRISPR-Cas9 release and editing.},
}
@article {pmid33172989,
year = {2020},
author = {Zhang, H and Zoued, A and Liu, X and Sit, B and Waldor, MK},
title = {Type I interferon remodels lysosome function and modifies intestinal epithelial defense.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29862-29871},
pmid = {33172989},
issn = {1091-6490},
support = {R01 AI042347/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Epithelial Cells/chemistry/cytology/*immunology/metabolism ; Gene Expression Regulation, Bacterial/immunology ; HT29 Cells ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Hydrogen-Ion Concentration ; Immunity, Innate ; Interferon Type I/*metabolism ; Intestinal Mucosa/cytology/*immunology/microbiology ; Lysosomes/chemistry/immunology/*metabolism ; Mice ; Mice, Knockout ; Necroptosis/immunology ; Peptide Hydrolases/metabolism ; Proteomics ; Receptor, Interferon alpha-beta/genetics/metabolism ; Salmonella Infections/*immunology/microbiology ; Salmonella typhimurium/immunology/pathogenicity ; Signal Transduction/immunology ; Virulence/immunology ; Virulence Factors/genetics/metabolism ; },
abstract = {Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.},
}
@article {pmid33172906,
year = {2020},
author = {Lach, FP and Singh, S and Rickman, KA and Ruiz, PD and Noonan, RJ and Hymes, KB and DeLacure, MD and Kennedy, JA and Chandrasekharappa, SC and Smogorzewska, A},
title = {Esophageal cancer as initial presentation of Fanconi anemia in patients with a hypomorphic FANCA variant.},
journal = {Cold Spring Harbor molecular case studies},
volume = {6},
number = {6},
pages = {},
pmid = {33172906},
issn = {2373-2873},
support = {UL1 TR000043/TR/NCATS NIH HHS/United States ; TL1 TR002386/TR/NCATS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA204127/CA/NCI NIH HHS/United States ; UL1 TR001866/TR/NCATS NIH HHS/United States ; R01 HL120922/HL/NHLBI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chromosome Breakage ; DNA ; DNA Repair ; Esophageal Neoplasms/diagnosis/*genetics/therapy ; Esophageal Squamous Cell Carcinoma/diagnosis/genetics/metabolism ; Fanconi Anemia/diagnosis/*genetics/therapy ; Fanconi Anemia Complementation Group A Protein/*genetics ; Fibroblasts/metabolism ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Mutation ; },
abstract = {Fanconi anemia (FA) is a clinically heterogenous and genetically diverse disease with 22 known complementation groups (FA-A to FA-W), resulting from the inability to repair DNA interstrand cross-links. This rare disorder is characterized by congenital defects, bone marrow failure, and cancer predisposition. FANCA is the most commonly mutated gene in FA and a variety of mostly private mutations have been documented, including small and large indels and point and splicing variants. Genotype-phenotype associations in FA are complex, and a relationship between particular FANCA variants and the observed cellular phenotype or illness severity remains unclear. In this study, we describe two siblings with compound heterozygous FANCA variants (c.3788_3790delTCT and c.4199G > A) who both presented with esophageal squamous cell carcinoma at the age of 51. The proband came to medical attention when he developed pancytopenia after a single cycle of low-dose chemotherapy including platinum-based therapy. Other than a minor thumb abnormality, neither patient had prior findings to suggest FA, including normal blood counts and intact fertility. Patient fibroblasts from both siblings display increased chromosomal breakage and hypersensitivity to interstrand cross-linking agents as seen in typical FA. Based on our functional data demonstrating that the c.4199G > A/p.R1400H variant represents a hypomorphic FANCA allele, we conclude that the residual activity of the Fanconi anemia repair pathway accounts for lack of spontaneous bone marrow failure or infertility with the late presentation of malignancy as the initial disease manifestation. This and similar cases of adult-onset esophageal cancer stress the need for chromosome breakage testing in patients with early onset of aerodigestive tract squamous cell carcinomas before platinum-based therapy is initiated.},
}
@article {pmid33172594,
year = {2021},
author = {Bennett, H and Aguilar-Martinez, E and Adamson, AD},
title = {CRISPR-mediated knock-in in the mouse embryo using long single stranded DNA donors synthesised by biotinylated PCR.},
journal = {Methods (San Diego, Calif.)},
volume = {191},
number = {},
pages = {3-14},
doi = {10.1016/j.ymeth.2020.10.012},
pmid = {33172594},
issn = {1095-9130},
support = {097820/Z/11/B/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA/genetics ; DNA, Single-Stranded/genetics ; *Gene Editing ; Gene Knock-In Techniques ; Mice ; Polymerase Chain Reaction ; },
abstract = {Successful gene knock-in by CRISPR-Cas9 in the mouse zygote requires three components; guideRNA, Cas9 protein and a suitable donor template, which usually comprises homology flanked insert sequence. Recently, long single stranded DNA (lssDNA) donors have emerged as a popular choice of DNA donor, outperforming dsDNA templates in terms of knock-in efficiency for gene tagging and generating conditional alleles. The generation of these donors can be achieved through several methods that may introduce errors in the sequence, result in poor yields, and contain dsDNA contamination. We have developed our own cost-effective lssDNA synthesis methodology that results in high purity, sequence verified, low contamination lssDNA donors. We provide a detailed methodology on the design and generation of such donors for gene tagging experiments and generating conditional alleles.},
}
@article {pmid33172134,
year = {2020},
author = {Zink, IA and Wimmer, E and Schleper, C},
title = {Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales.},
journal = {Biomolecules},
volume = {10},
number = {11},
pages = {},
pmid = {33172134},
issn = {2218-273X},
support = {695192/ERC_/European Research Council/International ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; RNA/genetics ; Species Specificity ; Sulfolobales/*genetics/*immunology ; },
abstract = {Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.},
}
@article {pmid33171139,
year = {2020},
author = {Marsh, S and Hanson, B and Wood, MJA and Varela, MA and Roberts, TC},
title = {Application of CRISPR-Cas9-Mediated Genome Editing for the Treatment of Myotonic Dystrophy Type 1.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2527-2539},
pmid = {33171139},
issn = {1525-0024},
support = {MR/P01741X/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/administration &amp; dosage ; Humans ; Mice ; Myotonic Dystrophy/genetics/*therapy ; Myotonin-Protein Kinase/genetics ; RNA, Guide/genetics ; Treatment Outcome ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder, caused by expansion of a CTG microsatellite repeat in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. To date, novel therapeutic approaches have focused on transient suppression of the mutant, repeat-expanded RNA. However, recent developments in the field of genome editing have raised the exciting possibility of inducing permanent correction of the DM1 genetic defect. Specifically, repurposing of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system has enabled programmable, site-specific, and multiplex genome editing. CRISPR-based strategies for the treatment of DM1 can be applied either directly to patients, or indirectly through the ex vivo modification of patient-derived cells, and they include excision of the repeat expansion, insertion of synthetic polyadenylation signals upstream of the repeat, steric interference with RNA polymerase II procession through the repeat leading to transcriptional downregulation of DMPK, and direct RNA targeting of the mutant RNA species. Potential obstacles to such therapies are discussed, including the major challenge of Cas9 and guide RNA transgene/ribonuclear protein delivery, off-target gene editing, vector genome insertion at cut sites, on-target unintended mutagenesis (e.g., repeat inversion), pre-existing immunity to Cas9 or AAV antigens, immunogenicity, and Cas9 persistence.},
}
@article {pmid33171122,
year = {2020},
author = {Lahey, LJ and Mardjuki, RE and Wen, X and Hess, GT and Ritchie, C and Carozza, JA and Böhnert, V and Maduke, M and Bassik, MC and Li, L},
title = {LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP.},
journal = {Molecular cell},
volume = {80},
number = {4},
pages = {578-591.e5},
doi = {10.1016/j.molcel.2020.10.021},
pmid = {33171122},
issn = {1097-4164},
support = {DP2 CA228044/CA/NCI NIH HHS/United States ; T32 GM120007/GM/NIGMS NIH HHS/United States ; DP2 HD084069/HD/NICHD NIH HHS/United States ; },
mesh = {Biological Transport ; *CRISPR-Cas Systems ; Cyclopentanes/pharmacology ; Humans ; Indans/pharmacology ; Lysophospholipids/pharmacology ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Nucleotides, Cyclic/*metabolism ; Sphingosine/analogs &amp; derivatives/pharmacology ; U937 Cells ; },
abstract = {Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.},
}
@article {pmid33170892,
year = {2020},
author = {Alghamdi, A and Aldossary, W and Albahkali, S and Alotaibi, B and Alrfaei, BM},
title = {The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu).},
journal = {PloS one},
volume = {15},
number = {11},
pages = {e0241902},
pmid = {33170892},
issn = {1932-6203},
mesh = {Amino Acid Substitution ; Animals ; Apoptosis ; Astrocytes/*cytology/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Cytochrome P-450 CYP1B1/*genetics ; Cytokines/metabolism ; Glaucoma/*congenital/genetics ; Humans ; Male ; Mesenchymal Stem Cells/*cytology/metabolism ; Microglia/*cytology/metabolism ; Models, Animal ; NADP/metabolism ; *Point Mutation ; Rats ; },
abstract = {BACKGROUND: Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation.<br><br>METHODS AND RESULTS: CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control.<br><br>CONCLUSIONS: The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia's loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).},
}
@article {pmid33170439,
year = {2020},
author = {Xie, F and Zhou, X and Lin, T and Wang, L and Liu, C and Luo, X and Luo, L and Chen, H and Guo, K and Wei, H and Wang, Y},
title = {Production of gene-edited pigs harboring orthologous human mutations via double cutting by CRISPR/Cas9 with long single-stranded DNAs as homology-directed repair templates by zygote injection.},
journal = {Transgenic research},
volume = {29},
number = {5-6},
pages = {587-598},
doi = {10.1007/s11248-020-00218-7},
pmid = {33170439},
issn = {1573-9368},
support = {National Major Project of China for Transgenic Organisms (No. 2018ZX08010-10B; No. 2016ZX08009-003-006)//Ministry of Agriculture and Rural Affairs/International ; },
mesh = {Alleles ; Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems ; Connexin 26/*genetics ; DNA, Single-Stranded ; Female ; Gene Editing/methods ; Humans ; Male ; *Mutation ; Swine ; Swine, Miniature/*genetics ; Zygote ; },
abstract = {Precise gene editing of model organisms is required for accurately modeling human diseases and deciphering gene functions. In this study, we used a pair of guide RNAs (sgRNAs), which in vitro transcribed along with other CRISPR RNA components, to generate two cleavage sites flanking pig GJB2 (pGJB2) CDS. By using long single-stranded DNAs (lssDNA) as homology-directed repair (HDR) templates, we efficiently obtained two gene-edited pigs, of which GJB2 CDS replaced with CDSs containing human GJB2 c.235delC mutation and orthologous human p.V37I mutation, respectively. These mutations were commonly observed in patients with hearing loss. Genetic analysis of the two gene-edited pigs showed that the HDR-derived gene-editing efficiency were as high as 80% (4/5) and 50% (2/4), respectively. While no mutation was observed in the group of single cutting with one sgRNA covering the 235th nucleotide C in pGJB2 CDS, using a short single-stranded oligo DNA containing c.235delC mutation as HDR template. Extra experiments proved that the intended mutations were successfully transmitted to offspring or extensively integrated into various tissues including gonad of founder pigs. Our work indicated that the new "double cutting with lssDNA template" gene editing method can expand sgRNA selection scope and avoids direct cutting of gene CDS. Additionally, can introduce precise mutations into mammalian genomic sites, especially those with unavailable proper protospacer sequence or being resistant to gene editing. Moreover, this method can be performed with CRISPR RNA reagents instead of CRISPR ribonucleoproteins applied in previous reports.},
}
@article {pmid33170255,
year = {2020},
author = {Bennett, EP and Petersen, BL and Johansen, IE and Niu, Y and Yang, Z and Chamberlain, CA and Met, &#xd6; and Wandall, HH and Frödin, M},
title = {INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {11958-11981},
pmid = {33170255},
issn = {1362-4962},
mesh = {Animals ; *CRISPR-Cas Systems ; Cloning, Organism/methods ; DNA/*genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; Gene Editing/*methods ; Gene Knockout Techniques ; *Genome ; Humans ; *INDEL Mutation ; Mice ; Sheep/genetics ; Solanum tuberosum/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.},
}
@article {pmid33168908,
year = {2020},
author = {Siegrist, CM and Kinahan, SM and Settecerri, T and Greene, AC and Santarpia, JL},
title = {CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {19307},
pmid = {33168908},
issn = {2045-2322},
mesh = {Antiviral Agents ; Bacterial Proteins/metabolism ; *CRISPR-Cas Systems ; Dependovirus/*genetics ; Gene Editing/methods ; Genetic Vectors ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; Monkeypox/*therapy/virology ; Orthopoxvirus/*metabolism ; Plasmids/metabolism ; RNA, Guide/*metabolism ; Smallpox/*therapy/virology ; Transfection ; Vaccinia virus ; },
abstract = {A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR-Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.},
}
@article {pmid33168840,
year = {2020},
author = {Kadrmas, JL and Beckerle, MC and Yoshigi, M},
title = {Genetic analyses in mouse fibroblast and melanoma cells demonstrate novel roles for PDGF-AB ligand and PDGF receptor alpha.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {19303},
pmid = {33168840},
issn = {2045-2322},
support = {GM50867/NH/NIH HHS/United States ; R01 GM084103/GM/NIGMS NIH HHS/United States ; GM084103/NH/NIH HHS/United States ; R01 GM050877/GM/NIGMS NIH HHS/United States ; P30 CA042014/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Becaplermin/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Fibroblasts/*metabolism ; Gene Expression Regulation, Neoplastic ; Ligands ; Melanoma/genetics/*metabolism ; Mice ; Platelet-Derived Growth Factor/*metabolism ; Protein Binding ; Protein Multimerization ; Receptor, Platelet-Derived Growth Factor alpha/*metabolism ; Signal Transduction ; },
abstract = {Platelet Derived Growth Factor Receptor (PDGFR) signaling is a central mitogenic pathway in development, as well as tissue repair and homeostasis. The rules governing the binding of PDGF ligand to the receptor to produce activation and downstream signaling have been well defined over the last several decades. In cultured cells after a period of serum deprivation, treatment with PDGF leads to the rapid formation of dramatic, actin-rich Circular Dorsal Ruffles (CDRs). Using CDRs as a robust visual readout of early PDGFR signaling, we have identified several contradictory elements in the widely accepted model of PDGF activity. Employing CRISPR/Cas9 gene editing to disrupt the Pdgfra gene in two different murine cell lines, we show that in addition to the widely accepted function for PDGFR-beta in CDR formation, PDGFR-alpha is also clearly capable of eliciting CDRs. Moreover, we demonstrate activity for heterodimeric PDGF-AB ligand in the vigorous activation of PDGFR-beta homodimers to produce CDRs. These findings are key to a more complete understanding of PDGF ligand-receptor interactions and their downstream signaling consequences. This knowledge will allow for more rigorous experimental design in future studies of PDGFR signaling and its contributions to development and disease.},
}
@article {pmid33168709,
year = {2020},
author = {Yu, X and Zhao, Q and Li, X and Chen, Y and Tian, Y and Liu, S and Xiong, W and Huang, P},
title = {Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29894-29903},
pmid = {33168709},
issn = {1091-6490},
mesh = {Animals ; COS Cells ; CRISPR-Cas Systems/genetics ; Chlorocebus aethiops ; Deafness/*genetics/pathology ; Disease Models, Animal ; Gene Knock-In Techniques ; HEK293 Cells ; Hair Cells, Auditory, Inner/metabolism/*pathology ; Humans ; Mechanotransduction, Cellular/*genetics ; Membrane Proteins/*genetics/isolation &amp; purification/*metabolism ; Mice ; Mice, Transgenic ; Point Mutation ; Protein Binding/genetics ; Two-Hybrid System Techniques ; },
abstract = {Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.},
}
@article {pmid33168193,
year = {2021},
author = {Han, S and Fei, F and Sun, S and Zhang, D and Dong, Q and Wang, X and Wang, L},
title = {Increased anxiety was found in serpini1 knockout zebrafish larval.},
journal = {Biochemical and biophysical research communications},
volume = {534},
number = {},
pages = {1013-1019},
doi = {10.1016/j.bbrc.2020.10.048},
pmid = {33168193},
issn = {1090-2104},
mesh = {Animals ; Anxiety/*genetics ; Anxiety Disorders/*genetics ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Knockout Techniques ; Humans ; Larva/genetics ; Neuropeptides/*genetics ; Serpins/*genetics ; Transcriptome ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {Serpini1, which encodes neuroserpin, has been implicated in the development and normal function of the nervous system. Mutations in serpini1 cause familial encephalopathy, a rare neurodegenerative disorder characterized with neuroserpin inclusion bodies. However, function of neuroserpin in the nervous system is not fully understood. In this study, we generated a novel serpini1 mutant zebrafish model to investigate the loss of function of neuroserpin. Serpini1- deficient mutation was created with the CRISPR/Cas9 technique. No severe morphological characteristics were found in serpini1- deficient zebrafish. Serpini1[-/-] zebrafish larvae did not cause locomotor defects but displayed anxiety-like behavior. Extension of motoneurons axon defect was observed in serpini1[-/-] zebrafish. Furthermore, RNA-sequencing analysis revealed that loss of serpini1 resulted in affected expression of neurodegeneration-related genes.},
}
@article {pmid33167938,
year = {2020},
author = {Dutt, M and Mou, Z and Zhang, X and Tanwir, SE and Grosser, JW},
title = {Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {58},
pmid = {33167938},
issn = {1472-6750},
support = {FLA-MCS-005676//SCRI/International ; },
mesh = {Agrobacterium/genetics ; Base Sequence ; *CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Citrus/embryology/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Plant ; Mutation ; Oxidoreductases/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; },
abstract = {BACKGROUND: Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study.<br><br>RESULTS: Citrus sinensis 'EV2' embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6-26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1-2&#x2009;bp), substitution (1&#x2009;bp), or deletion (1-3&#x2009;bp) mutations that occurred close to the protospacer adjacent motif.<br><br>CONCLUSIONS: Both the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.},
}
@article {pmid33167309,
year = {2020},
author = {Su, L and Shi, C and Huang, X and Wang, Y and Li, G},
title = {Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing.},
journal = {Genes},
volume = {11},
number = {11},
pages = {},
pmid = {33167309},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/physiology ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Genotype ; Lancelets/*genetics ; Phenotype ; Phylogeny ; },
abstract = {The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.},
}
@article {pmid33167031,
year = {2021},
author = {Galperin, MY and Wolf, YI and Makarova, KS and Vera Alvarez, R and Landsman, D and Koonin, EV},
title = {COG database update: focus on microbial diversity, model organisms, and widespread pathogens.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D274-D281},
pmid = {33167031},
issn = {1362-4962},
support = {/LM/NLM NIH HHS/United States ; },
mesh = {Archaea/*genetics/metabolism ; Archaeal Proteins/classification/genetics/metabolism ; Bacteria/*genetics/immunology/metabolism ; Bacterial Proteins/classification/genetics/metabolism ; CRISPR-Cas Systems ; *Databases, Genetic ; Gene Ontology ; *Genome, Archaeal ; *Genome, Bacterial ; Humans ; Molecular Sequence Annotation ; Spores, Bacterial/genetics/growth &amp; development ; },
abstract = {The Clusters of Orthologous Genes (COG) database, also referred to as the Clusters of Orthologous Groups of proteins, was created in 1997 and went through several rounds of updates, most recently, in 2014. The current update, available at https://www.ncbi.nlm.nih.gov/research/COG, substantially expands the scope of the database to include complete genomes of 1187 bacteria and 122 archaea, typically, with a single genome per genus. In addition, the current version of the COGs includes the following new features: (i) the recently deprecated NCBI's gene index (gi) numbers for the encoded proteins are replaced with stable RefSeq or GenBank\ENA\DDBJ coding sequence (CDS) accession numbers; (ii) COG annotations are updated for >200 newly characterized protein families with corresponding references and PDB links, where available; (iii) lists of COGs grouped by pathways and functional systems are added; (iv) 266 new COGs for proteins involved in CRISPR-Cas immunity, sporulation in Firmicutes and photosynthesis in cyanobacteria are included; and (v) the database is made available as a web page, in addition to FTP. The current release includes 4877 COGs. Future plans include further expansion of the COG collection by adding archaeal COGs (arCOGs), splitting the COGs containing multiple paralogs, and continued refinement of COG annotations.},
}
@article {pmid33166572,
year = {2021},
author = {Turra, GL and Schneider, L and Liedgens, L and Deponte, M},
title = {Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae.},
journal = {Molecular and biochemical parasitology},
volume = {241},
number = {},
pages = {111336},
doi = {10.1016/j.molbiopara.2020.111336},
pmid = {33166572},
issn = {1872-9428},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Drug Resistance ; *Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques ; Gene Targeting ; Genes, Reporter ; Genetic Vectors/genetics ; Leishmania/drug effects/*genetics/metabolism ; Plasmids/genetics ; Protozoan Proteins/genetics ; RNA Interference ; *RNA, Catalytic ; },
abstract = {Leishmania parasites include important pathogens and model organisms and are even used for the production of recombinant proteins. However, functional genomics and the characterization of essential genes are often limited in Leishmania because of low-throughput technologies for gene disruption or tagging and the absence of components for RNA interference. Here, we tested the T7 RNA polymerase-dependent CRISPR-Cas9 system by Beneke et al. and the glmS ribozyme-based knock-down system in the model parasite Leishmania tarentolae. We successfully deleted two reference genes encoding the flagellar motility factor Pf16 and the salvage-pathway enzyme adenine phosphoribosyltransferase, resulting in immotile and drug-resistant parasites, respectively. In contrast, we were unable to disrupt the gene encoding the mitochondrial flavoprotein Erv. Cultivation of L. tarentolae in standard BHI medium resulted in a constitutive down-regulation of an episomal mCherry-glmS reporter by 40 to 60%. For inducible knock-downs, we evaluated the growth of L. tarentolae in alternative media and identified supplemented MEM, IMDM and McCoy's 5A medium as candidates. Cultivation in supplemented MEM allowed an inducible, glucosamine concentration-dependent down-regulation of the episomal mCherry-glmS reporter by more than 70%. However, chromosomal glmS-tagging of the genes encoding Pf16, adenine phosphoribosyltransferase or Erv did not reveal a knock-down phenotype. Our data demonstrate the suitability of the CRISPR-Cas9 system for the disruption and tagging of genes in L. tarentolae as well as the limitations of the glmS system, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.},
}
@article {pmid33166385,
year = {2020},
author = {Guo, R and Xu, Y and Leu, NA and Zhang, L and Fuchs, SY and Ye, L and Wang, PJ},
title = {The ssDNA-binding protein MEIOB acts as a dosage-sensitive regulator of meiotic recombination.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12219-12233},
pmid = {33166385},
issn = {1362-4962},
support = {R35 GM118052/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Chromosome Pairing ; DNA, Single-Stranded/*genetics/metabolism ; DNA-Binding Proteins/deficiency/*genetics/metabolism ; Female ; Gene Dosage ; Gene Editing ; HEK293 Cells ; Homologous Recombination ; Humans ; Infertility, Male/genetics/metabolism/pathology ; Male ; Mice ; Mice, Transgenic ; *Mutation, Missense ; Ovary/metabolism ; RNA, Guide/genetics/metabolism ; Species Specificity ; Testis/*metabolism/pathology ; },
abstract = {Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner.},
}
@article {pmid33166100,
year = {2020},
author = {Becker, A and Mannebach, S and Mathar, I and Weissgerber, P and Freichel, M and Loodin, AP and Fecher-Trost, C and Belkacemi, A and Beck, A and Philipp, SE},
title = {Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {54},
number = {6},
pages = {1115-1131},
doi = {10.33594/000000304},
pmid = {33166100},
issn = {1421-9778},
support = {CRC 894 A3, CRC 894 A14, CRC 894 P2, CRC 1188 B02//DFG (Deutsche Forschungsgemeinschaft)/Germany ; },
mesh = {Animals ; *Calcium Signaling ; Cell Line ; *Insulin Secretion ; Insulin-Secreting Cells/*metabolism ; Mice ; Mice, Mutant Strains ; Rats ; TRPM Cation Channels/genetics/*metabolism ; },
abstract = {BACKGROUND/AIMS: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca[2+] influx into pancreatic beta cells by the opening of voltage-gated Ca[2+] channels. Transient Receptor Potential Melastatin 3 proteins build Ca[2+] permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca[2+] influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release.<br><br>METHODS: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca[2+] channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests.<br><br>RESULTS: TRPM3 channels triggered the activity of voltage-gated Ca[2+] channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca[2+] signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca[2+] signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose.<br><br>CONCLUSION: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.},
}
@article {pmid33165862,
year = {2021},
author = {Frati, G and Miccio, A},
title = {A Genome Editing System for Therapeutical Targeting of Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2185},
number = {},
pages = {383-398},
doi = {10.1007/978-1-0716-0810-4_24},
pmid = {33165862},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Differentiation/*genetics ; *Gene Editing ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism ; Heterografts ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; },
abstract = {The CRISPR/Cas9 system can be exploited to disrupt genes or cis-regulatory elements in the genome of human hematopoietic stem cells. Here, we describe a protocol to deliver the CRISPR/Cas9 ribonucleoprotein complexes into primary human hematopoietic stem cells and to evaluate the engraftment and multilineage differentiation of edited cells in immunodeficient mice. This procedure allows the editing of a high proportion of long-term repopulating hematopoietic stem cells.},
}
@article {pmid33165782,
year = {2021},
author = {Zou, G and Zhou, Z},
title = {CRISPR/Cas9-Mediated Genome Editing of Trichoderma reesei.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2234},
number = {},
pages = {87-98},
doi = {10.1007/978-1-0716-1048-0_8},
pmid = {33165782},
issn = {1940-6029},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; *Genome, Fungal ; Hypocreales/*genetics ; Protoplasts/metabolism ; RNA, Guide/genetics ; Transcription, Genetic ; Transformation, Genetic ; },
abstract = {In this protocol, we describe the establishment of a CRISPR/Cas9 system in Trichoderma reesei by generating a specific, codon-optimized Cas9-expressing strain and by in vitro transcription of a gRNA. This system induces mutagenesis or introduces a gene in a targeted way based on PEG-mediated protoplast transformation. Up to three targets, multiplexed genome editing can be obtained in one transformation.},
}
@article {pmid33162936,
year = {2020},
author = {Hu, M and Cherkaoui, I and Misra, S and Rutter, GA},
title = {Functional Genomics in Pancreatic β Cells: Recent Advances in Gene Deletion and Genome Editing Technologies for Diabetes Research.},
journal = {Frontiers in endocrinology},
volume = {11},
number = {},
pages = {576632},
pmid = {33162936},
issn = {1664-2392},
support = {MR/N020472/1/MRC_/Medical Research Council/United Kingdom ; MR/M012646/1/MRC_/Medical Research Council/United Kingdom ; MR/L02036X/1/MRC_/Medical Research Council/United Kingdom ; MR/K001981/1/MRC_/Medical Research Council/United Kingdom ; MR/R010676/1/MRC_/Medical Research Council/United Kingdom ; MR/N00275X/1/MRC_/Medical Research Council/United Kingdom ; MR/R022259/1/MRC_/Medical Research Council/United Kingdom ; WT098424AIA/WT_/Wellcome Trust/United Kingdom ; 212625/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; MR/J0003042/1/MRC_/Medical Research Council/United Kingdom ; MR/L020149/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Diabetes Mellitus, Type 2/genetics/pathology/*therapy ; *Gene Deletion ; *Gene Editing ; *Genetic Therapy ; Genomics/*methods ; Humans ; Insulin-Secreting Cells/*metabolism ; },
abstract = {The inheritance of variants that lead to coding changes in, or the mis-expression of, genes critical to pancreatic beta cell function can lead to alterations in insulin secretion and increase the risk of both type 1 and type 2 diabetes. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene editing tools provide a powerful means of understanding the impact of identified variants on cell function, growth, and survival and might ultimately provide a means, most likely after the transplantation of genetically "corrected" cells, of treating the disease. Here, we review some of the disease-associated genes and variants whose roles have been probed up to now. Next, we survey recent exciting developments in CRISPR/Cas9 technology and their possible exploitation for β cell functional genomics. Finally, we will provide a perspective as to how CRISPR/Cas9 technology may find clinical application in patients with diabetes.},
}
@article {pmid33162553,
year = {2020},
author = {Ha, DI and Lee, JM and Lee, NE and Kim, D and Ko, JH and Kim, YS},
title = {Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2'-O-methylated uridinylate-rich 3'-overhang in mouse zygotes.},
journal = {Experimental &amp; molecular medicine},
volume = {52},
number = {11},
pages = {1823-1830},
pmid = {33162553},
issn = {2092-6413},
mesh = {Animals ; Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; Endodeoxyribonucleases/*metabolism ; *Gene Editing/methods ; Genetic Engineering ; Mice ; *RNA, Guide ; Ribose/analogs &amp; derivatives/metabolism ; Substrate Specificity ; Zygote/*metabolism ; },
abstract = {The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3'-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2'-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.},
}
@article {pmid33162067,
year = {2021},
author = {Edick, AM and Audette, J and Burgos, SA},
title = {CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells.},
journal = {Journal of dairy science},
volume = {104},
number = {1},
pages = {1123-1135},
doi = {10.3168/jds.2020-18700},
pmid = {33162067},
issn = {1525-3198},
mesh = {Amino Acids/*metabolism ; Animals ; CRISPR-Cas Systems ; Cattle/genetics/*metabolism ; Epithelial Cells/metabolism ; Eukaryotic Initiation Factor-2/genetics/metabolism ; Female ; Gene Knockout Techniques ; Lactation ; Mammary Glands, Animal/*metabolism ; Methionine/metabolism ; Phosphorylation ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Up-Regulation ; },
abstract = {The objective of this study was to determine the role of GCN2 in the response to AA deprivation of primary bovine mammary epithelial cells (BMEC). Cells were isolated from the mammary tissue of 2 lactating Holstein cows by enzymatic digestion, expanded, and induced to differentiate for 5 to 7 d. Relative mRNA expression was measured by real-time quantitative PCR. Protein abundance and site-specific phosphorylation were measured by immunoblotting. Knockout of GCN2 in BMEC was accomplished by lentiviral delivery of a targeted single guide RNA and endonuclease Cas9. To investigate the role of GCN2, we treated lactogenic differentiated BMEC with either culture medium lacking Arg, Leu, and Lys combined or lacking only one of the 3 AA of interest, in comparison to a control with a full complement of AA. Activation of GCN2 was inferred by the phosphorylation status of its downstream target eIF2α Ser51. We found that GCN2 was activated by both the deprivation of Arg, Leu, and Lys combined and of Arg alone, as shown by a 2.73- and 2.82-fold increase in phosphorylated eIF2α Ser51 after 1 h of deprivation, respectively. In addition, activation of GCN2 as measured by increased phosphorylation of eIF2α Ser51 during the deprivation of Arg, Leu, and Lys combined and of Arg alone was sustained for up to 8 h of deprivation. Phosphorylated eIF2α selectively upregulates translation of transcription factor ATF4, among others, during AA deprivation, which then targets genes necessary for restoring AA homeostasis. Therefore, we investigated the expression of ATF4 transcriptional targets, AA enzyme ASNS and AA transporters SLC7A1 and SLC38A2. We found that ASNS was upregulated in response to combined AA deprivation and by Arg deprivation alone by 3.6- and 4.51-fold, respectively, at 24 h of treatment. We found that SLC7A1 was upregulated in response to combined AA deprivation and deprivation of Arg alone by 2.0- and 2.36-fold, respectively, at 8 h of treatment. To establish the role of GCN2 (encoded by EIF2AK4) in the response to AA deprivation, we ablated GCN2 in BMEC using clustered regularly interspaced short palindromic repeats-Cas9. We showed that BMEC transduced with single guide RNA targeting EIF2AK4 were not as responsive to combined AA deprivation, compared with BMEC transduced with nontargeting single guide RNA. Taken together, our results demonstrate a critical role for GCN2 in sensing AA deprivation in BMEC.},
}
@article {pmid33161548,
year = {2021},
author = {Figueredo, EF and Quecine, MC},
title = {Gene Knockout of Beneficial Plant-associated Bacillus spp. Using the CRISPR-Cas9 Double Plasmid System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2232},
number = {},
pages = {173-191},
doi = {10.1007/978-1-0716-1040-4_15},
pmid = {33161548},
issn = {1940-6029},
mesh = {Bacillus/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques/*methods ; Plants/genetics/*microbiology ; Plasmids/genetics ; },
abstract = {Bacillus spp. have great agricultural potential as a plant growth promoter and biocontrol agent. However, little is known concerning the bacterial molecular basis for the improvement of plant fitness. Thus, it is highly desirable to develop techniques that can contribute to the elucidation of the genetic basis for the mechanisms involved in beneficial bacterium-plant interactions. In this context, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is a powerful tool based on programmable molecular scissors that perform precise incisions in any DNA sequence. CRISPR-Cas9 can alter gene sequences and constitutes a cutting-edge tool to elucidate the role and function of bacterial genes associated with the benefits of plant interactions. The method described here uses a feasible CRISPR-Cas9 system in a double plasmid, one plasmid harboring the Cas9 endonuclease and the other the sgRNA, to promote gene knockout/editing in the Bacillus genus. This approach favors high efficiency in generating mutants for one or more genes in continuous or multiplex editing. Additionally, due to its universality, it can be applied to genera other than Bacillus.},
}
@article {pmid33161238,
year = {2020},
author = {Nagel, M and Müßig, S and Höflinger, P and Schöls, L and Hauser, S and Schüle, R},
title = {Generation of the CRISPR/Cas9-mediated KIF1C knock-out human iPSC line HIHRSi003-A-1.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102059},
doi = {10.1016/j.scr.2020.102059},
pmid = {33161238},
issn = {1876-7753},
support = {R01 NS072248/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; Kinesins/genetics ; *Spastic Paraplegia, Hereditary/genetics ; },
abstract = {Bi-allelic loss-of-function mutations in the gene encoding the motor protein KIF1C are associated with Hereditary Spastic Paraplegia (HSP) type SPG58, a slowly progressive neurodegenerative motoneuron disease. The biological role of KIF1C is incompletely understood. We used a protein-based CRISPR/Cas9 genome editing approach to generate a homozygous KIF1C knock-out iPSC line (HIHRSi003-A-1) from a healthy control. This iPSC-KIF1C[-/-] line and the corresponding isogenic control are a useful model to study the physiological function of KIF1C and the pathophysiological consequences of KIF1C dysfunction in human disease.},
}
@article {pmid33161094,
year = {2021},
author = {de Assis Lima, M and da Silva, SV and Serrano-Garrido, O and Hülsemann, M and Santos-Neres, L and Rodríguez-Manzaneque, JC and Hodgson, L and Freitas, VM},
title = {Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity.},
journal = {Cellular signalling},
volume = {77},
number = {},
pages = {109827},
pmid = {33161094},
issn = {1873-3913},
support = {R35 GM136226/GM/NIGMS NIH HHS/United States ; },
mesh = {ADAMTS1 Protein/deficiency/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cell Survival ; Female ; Focal Adhesion Kinase 1/metabolism ; Hepatocyte Growth Factor/pharmacology ; Humans ; Phosphorylation ; RNA, Guide/metabolism ; Signal Transduction ; cdc42 GTP-Binding Protein/*metabolism ; src-Family Kinases/metabolism ; },
abstract = {ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn[2+]/Ca[2+], involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.},
}
@article {pmid33160457,
year = {2021},
author = {Tatiossian, KJ and Clark, RDE and Huang, C and Thornton, ME and Grubbs, BH and Cannon, PM},
title = {Rational Selection of CRISPR-Cas9 Guide RNAs for Homology-Directed Genome Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {3},
pages = {1057-1069},
pmid = {33160457},
issn = {1525-0024},
support = {U19 HL129902/HL/NHLBI NIH HHS/United States ; U19 HL156247/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Gene Editing ; HEK293 Cells ; Humans ; *INDEL Mutation ; K562 Cells ; RNA, Guide/*genetics ; *Recombinational DNA Repair ; },
abstract = {Homology-directed repair (HDR) of a DNA break allows copying of genetic material from an exogenous DNA template and is frequently exploited in CRISPR-Cas9 genome editing. However, HDR is in competition with other DNA repair pathways, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), and the efficiency of HDR outcomes is not predictable. Consequently, to optimize HDR editing, panels of CRISPR-Cas9 guide RNAs (gRNAs) and matched homology templates must be evaluated. We report here that CRISPR-Cas9 indel signatures can instead be used to identify gRNAs that maximize HDR outcomes. Specifically, we show that the frequency of deletions resulting from MMEJ repair, characterized as deletions greater than or equal to 3 bp, better predicts HDR frequency than consideration of total indel frequency. We further demonstrate that tools that predict gRNA indel signatures can be repurposed to identify gRNAs to promote HDR. Finally, by comparing indels generated by S. aureus and S. pyogenes Cas9 targeted to the same site, we add to the growing body of data that the targeted DNA sequence is a major factor governing genome editing outcomes.},
}
@article {pmid33160075,
year = {2021},
author = {Bengtsson, NE and Tasfaout, H and Hauschka, SD and Chamberlain, JS},
title = {Dystrophin Gene-Editing Stability Is Dependent on Dystrophin Levels in Skeletal but Not Cardiac Muscles.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {3},
pages = {1070-1085},
pmid = {33160075},
issn = {1525-0024},
support = {P30 DK017047/DK/NIDDK NIH HHS/United States ; P50 AR065139/AR/NIAMS NIH HHS/United States ; R01 AR044533/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; *Gene Editing ; Genetic Therapy/methods ; Genetic Vectors/*administration &amp; dosage/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Skeletal/*metabolism ; Muscular Dystrophy, Animal/genetics/pathology/*prevention &amp; control ; Muscular Dystrophy, Duchenne/genetics/pathology/*prevention &amp; control ; Mutation ; Myocardium/*metabolism ; Myocytes, Cardiac/metabolism/pathology ; RNA, Guide ; },
abstract = {Gene editing is often touted as a permanent method for correcting mutations, but its long-term benefits in Duchenne muscular dystrophy (DMD) may depend on sufficiently high editing efficiencies to halt muscle degeneration. Here, we explored the persistence of dystrophin expression following recombinant adeno-associated virus serotype 6 (rAAV6):CRISPR-Cas9-mediated multi-exon deletion/reframing in systemically injected 2- and 11-week-old dystrophic mice and show that induction of low dystrophin levels persists for several months in cardiomyocytes but not in skeletal muscles, where myofibers remain susceptible to necrosis and regeneration. Whereas gene-correction efficiency in both muscle types was enhanced with increased ratios of guide RNA (gRNA)-to-nuclease vectors, obtaining high dystrophin levels in skeletal muscles via multi-exon deletion remained challenging. In contrast, when AAV-microdystrophin was codelivered with editing components, long-term gene-edited dystrophins persisted in both muscle types. These results suggest that the high rate of necrosis and regeneration in skeletal muscles, compared with the relative stability of dystrophic cardiomyocytes, caused the rapid loss of edited genomes. Consequently, stable dystrophin expression in DMD skeletal muscles will require either highly efficient gene editing or the use of cotreatments that decrease skeletal muscle degeneration.},
}
@article {pmid33159392,
year = {2021},
author = {Li, H and Cui, X and Sun, L and Deng, X and Liu, S and Zou, X and Li, B and Wang, C and Wang, Y and Liu, Y and Lu, B and Cao, B},
title = {High concentration of Cas12a effector tolerates more mismatches on ssDNA.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {1},
pages = {e21153},
doi = {10.1096/fj.202001475R},
pmid = {33159392},
issn = {1530-6860},
mesh = {Bacterial Proteins/*chemistry ; CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; DNA, Single-Stranded/*chemistry ; Endodeoxyribonucleases/*chemistry ; Humans ; Mycobacterium tuberculosis/*chemistry ; },
abstract = {Rapid pathogen detection is critical for prompt treatment, interrupting transmission routes, and decreasing morbidity and mortality. The V-type CRISPR system had been used for rapid pathogen detection. However, whether single-stranded DNA in CRISPR system can cause false positives remains undetermined. Herein, we show that high molar concentration of Cas12a effector tolerated more mismatches on ssDNA and activated its trans-cleavage activity at six base matches. Reducing Cas12a and crRNA molar concentration increased the minimal base-match number required for Cas12a ssDNA activation to 11, which reducing nonspecific activation. We then established a Cas12a-based M tuberculosis detection system with a primer having an 8 bp overlap with crRNA. This system did not exhibit primer-induced false positives, and minimum detection copy reached 1 copy/uL (inputting 1-μL sample) in standard strains. The Cas12a-based M tuberculosis detection system showed 80.0% sensitivity and 100.0% specificity in verification using clinical specimens, compared with Xpert MTB/RIF, which showed 72.0% sensitivity and 90.9% specificity. All these results prove that appropriate concentration of cas12a effector can effectively perform nucleic acid detection.},
}
@article {pmid33159233,
year = {2020},
author = {Zhu, K and Liu, Y and Fan, C and Zhang, M and Cao, H and He, X and Li, N and Chu, D and Li, F and Zou, M and Hua, J and Wang, H and Wang, Y and Fan, G and Zhang, S},
title = {Etv5 safeguards trophoblast stem cells differentiation from mouse EPSCs by regulating fibroblast growth factor receptor 2.},
journal = {Molecular biology reports},
volume = {47},
number = {12},
pages = {9259-9269},
doi = {10.1007/s11033-020-05969-4},
pmid = {33159233},
issn = {1573-4978},
mesh = {Animals ; Benzamides/pharmacology ; CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Cells, Cultured ; Culture Media ; DNA-Binding Proteins/genetics/*metabolism ; Dimethindene/pharmacology ; Diphenylamine/analogs &amp; derivatives/pharmacology ; Embryonic Development/genetics ; Gene Knockout Techniques ; MAP Kinase Signaling System/drug effects/*genetics ; Mice ; Minocycline/pharmacology ; Mouse Embryonic Stem Cells/drug effects/*metabolism ; Receptor, Fibroblast Growth Factor, Type 2/*metabolism ; Transcription Factors/genetics/*metabolism ; Transfection ; Trophoblasts/*cytology/*metabolism ; },
abstract = {Previous studies have demonstrated that transcription factor Etv5 plays an important role in the segregation between epiblast and primitive endoderm at the second fate decision of early embryo. However, it remains elusive whether Etv5 functions in the segregation between inner cell mass and trophectoderm at the first cell fate decision. In this study, we firstly generated Etv5 knockout mouse embryonic stem cells (mESCs) by CRISPR/Cas9, then converted them into extended potential stem cells (EPSCs) by culturing the cells in small molecule cocktail medium LCDM (LIF, CHIR99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride), and finally investigated their differentiation efficiency of trophoblast stem cells (TSCs). The results showed that Etv5 knockout significantly decreased the efficiency of TSCs (CDX2[+]) differentiated from EPSCs. In addition, Etv5 knockout resulted in higher incidence of the differentiated cells with tetraploid and octoploid than that from wild type. Mechanistically, Etv5 was activated by extracellular-signal-regulated kinase (ERK) signaling pathway; in turn, Etv5 had a positive feedback on the expression of fibroblast growth factor receptor 2 (FGFR2) which lies upstream of ERK. Etv5 knockout decreased the expression of FGFR2, whose binding with fibroblast growth factor 4 was essentially needed for TSCs differentiation. Collectively, the findings in this study suggest that Etv5 is required to safeguard the TSCs differentiation by regulating FGFR2 and provide new clues to understand the specification of trophectoderm in vivo.},
}
@article {pmid33159140,
year = {2020},
author = {Osakabe, K and Wada, N and Miyaji, T and Murakami, E and Marui, K and Ueta, R and Hashimoto, R and Abe-Hara, C and Kong, B and Yano, K and Osakabe, Y},
title = {Genome editing in plants using CRISPR type I-D nuclease.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {648},
pmid = {33159140},
issn = {2399-3642},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Deoxyribonucleases/*genetics ; *Gene Editing ; *Genetic Engineering ; *Genome, Plant ; Lycopersicon esculentum/*genetics ; },
abstract = {Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I-the most abundant CRISPR system in bacteria-has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.},
}
@article {pmid33159058,
year = {2020},
author = {Pinilla-Redondo, R and Shehreen, S and Marino, ND and Fagerlund, RD and Brown, CM and Sørensen, SJ and Fineran, PC and Bondy-Denomy, J},
title = {Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5652},
pmid = {33159058},
issn = {2041-1723},
support = {DP5 OD021344/OD/NIH HHS/United States ; F32 GM133127/GM/NIGMS NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*genetics/immunology/virology ; Bacterial Proteins/genetics/*immunology ; Bacteriophages/genetics/physiology ; *CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; Multigene Family ; },
abstract = {Many prokaryotes employ CRISPR-Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I-F (Pseudomonas, Pectobacterium, and Serratia), I-E (Pseudomonas and Serratia), and I-C (Pseudomonas). We uncover 11 type I-F and/or I-E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.},
}
@article {pmid33158848,
year = {2021},
author = {Li, J and Yuan, S and Norgard, RJ and Yan, F and Sun, YH and Kim, IK and Merrell, AJ and Sela, Y and Jiang, Y and Bhanu, NV and Garcia, BA and Vonderheide, RH and Blanco, A and Stanger, BZ},
title = {Epigenetic and Transcriptional Control of the Epidermal Growth Factor Receptor Regulates the Tumor Immune Microenvironment in Pancreatic Cancer.},
journal = {Cancer discovery},
volume = {11},
number = {3},
pages = {736-753},
pmid = {33158848},
issn = {2159-8290},
support = {F30 CA224970/CA/NCI NIH HHS/United States ; P01 CA196539/CA/NCI NIH HHS/United States ; U54 CA232568/CA/NCI NIH HHS/United States ; R01 CA229803/CA/NCI NIH HHS/United States ; R01 CA169123/CA/NCI NIH HHS/United States ; R01 AI118891/AI/NIAID NIH HHS/United States ; R01 DK083355/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Combined Modality Therapy ; *Epigenesis, Genetic ; ErbB Receptors/genetics/metabolism ; *Gene Expression Regulation, Neoplastic ; Genomics/methods ; Humans ; Immunity/genetics ; Jumonji Domain-Containing Histone Demethylases/genetics/metabolism ; Lymphocytes, Tumor-Infiltrating/immunology/metabolism/pathology ; Mice ; Mutation ; Pancreatic Neoplasms/*etiology/*metabolism/mortality/pathology ; Prognosis ; T-Lymphocytes/immunology/metabolism/pathology ; Transcriptome ; Treatment Outcome ; Tumor Microenvironment/*genetics ; },
abstract = {Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.},
}
@article {pmid33157390,
year = {2020},
author = {Zhang, C and Yu, Y and Li, F and Lan, X and Wang, L},
title = {Generating an MEIS1 homozygous knockout human embryonic stem cell line using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102069},
doi = {10.1016/j.scr.2020.102069},
pmid = {33157390},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; },
abstract = {Myeloid ecotropic viral integration site 1 (MEIS1) plays an essential role in the development of several embryonic organs, such as the central nervous system and eyes. To further investigate the role of MEIS1 in embryonic development, herein, we generated a MEIS1 homozygous knockout human embryonic stem cell (hESC) line using the CRISPR/Cas9 genome-editing technology. We believe that this cell line will be a good resource for exploring the function of the MEIS1 gene in embryonic development in vitro. Furthermore, the gene-knockout method reported in this study is efficient and labor-saving, which may provide an effective strategy for hESC gene deletion.},
}
@article {pmid33157388,
year = {2020},
author = {Feng, Y and Li, X and Zhang, L and Sun, Y and He, Y and Wen, R and Hu, Y and Wang, X and Liu, J and Long, B},
title = {Generation of a RGS18 gene knockout cell line from a human embryonic stem cell line by CRISPR/Cas9.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102072},
doi = {10.1016/j.scr.2020.102072},
pmid = {33157388},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Stem Cells/metabolism ; Gene Knockout Techniques ; *Human Embryonic Stem Cells/metabolism ; Humans ; *RGS Proteins/genetics/metabolism ; },
abstract = {RGS18 is a member of the RGS (Regulators of G-protein signaling) protein family, involved in megakaryopoiesis, megakaryocyte differentiation and chemotaxis. Here, we created a RGS18 knockout cell line from a human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, to further understand roles of RGS18 in these processes. The cell line maintains stem cell morphology and normal karyotype, and retains expression of pluripotent marker genes and differentiation potential in vivo. The RGS18[-/-] cell line will facilitate investigation of the role of RGS18 during multiple cellular processes in human pluripotent stem cell modeled hematopoiesis.},
}
@article {pmid33157047,
year = {2020},
author = {Hendriks, D and Clevers, H and Artegiani, B},
title = {CRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids.},
journal = {Cell stem cell},
volume = {27},
number = {5},
pages = {705-731},
doi = {10.1016/j.stem.2020.10.014},
pmid = {33157047},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genetic Engineering ; Humans ; *Organoids ; },
abstract = {CRISPR-Cas technology has revolutionized biological research and holds great therapeutic potential. Here, we review CRISPR-Cas systems and their latest developments with an emphasis on application to human cells. We also discuss how different CRISPR-based strategies can be used to accomplish a particular genome engineering goal. We then review how different CRISPR tools have been used in genome engineering of human stem cells in vitro, covering both the pluripotent (iPSC/ESC) and somatic adult stem cell fields and, in particular, 3D organoid cultures. Finally, we discuss the progress and challenges associated with CRISPR-based genome editing of human stem cells for therapeutic use.},
}
@article {pmid33523177,
year = {2019},
author = {Green, AA},
title = {Synthetic bionanotechnology: synthetic biology finds a toehold in nanotechnology.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {5},
pages = {507-516},
pmid = {33523177},
issn = {2397-8554},
support = {DP2 GM126892/GM/NIGMS NIH HHS/United States ; R21 AI136571/AI/NIAID NIH HHS/United States ; },
abstract = {Enabled by its central role in the molecular networks that govern cell function, RNA has been widely used for constructing components used in biological circuits for synthetic biology. Nucleic acid nanotechnology, which exploits predictable nucleic acid interactions to implement programmable molecular systems, has seen remarkable advances in in vitro nanoscale self-assembly and molecular computation, enabling the production of complex nanostructures and DNA-based neural networks. Living cells genetically engineered to execute nucleic acid nanotechnology programs thus have outstanding potential to significantly extend the current limits of synthetic biology. This perspective discusses the recent developments and future challenges in the field of synthetic bionanotechnology. Thus far, researchers in this emerging area have implemented dozens of programmable RNA nanodevices that provide precise control over gene expression at the transcriptional and translational levels and through CRISPR/Cas effectors. Moreover, they have employed synthetic self-assembling RNA networks in engineered bacteria to carry out computations featuring up to a dozen inputs and to substantially enhance the rate of chemical synthesis. Continued advancement of the field will benefit from improved in vivo strategies for streamlining nucleic acid network synthesis and new approaches for enhancing network function. As the field matures and the complexity gap between in vitro and in vivo systems narrows, synthetic bionanotechnology promises to have diverse potential applications ranging from intracellular circuits that detect and treat disease to synthetic enzymatic pathways that efficiently produce novel drug molecules.},
}
@article {pmid33523169,
year = {2019},
author = {Berger, I and Tölzer, C and Gupta, K},
title = {The MultiBac system: a perspective.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {5},
pages = {477-482},
doi = {10.1042/ETLS20190084},
pmid = {33523169},
issn = {2397-8554},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Baculovirus expression is a time-tested technique to produce proteins in insect cells, in high quality and quantity for a range of applications. MultiBac is a baculovirus expression system we developed originally for producing multiprotein complexes comprising many subunits, for structural and mechanistic studies. First introduced in 2004, MultiBac is now in use in many laboratories worldwide, accelerating research programmes in academia and industry. We have continuously optimized our MultiBac system, providing customized reagents and standard operating protocols to facilitate its use also by non-specialists. More recently, we have generated MultiBac genomes tailored for specific purposes, for example, to produce humanized glycoproteins, high-value pharmaceutical targets including kinases, viral polymerases, and virus-like particles (VLPs) as promising vaccine candidates. By altering the host tropism of the baculovirion, we created MultiBacMam, a heterologous DNA delivery toolkit to target mammalian cells, tissues and organisms. Introducing CRISPR/Cas modalities, we set the stage for large-scale genomic engineering applications utilizing this high-capacity DNA delivery tool. Exploiting synthetic biology approaches and bottom-up design, we engage in optimizing the properties of our baculoviral genome, also to improve manufacturing at scale. Here we provide a perspective of our MultiBac system and its developments, past, present and future.},
}
@article {pmid33523137,
year = {2019},
author = {Romito, M and Rai, R and Thrasher, AJ and Cavazza, A},
title = {Genome editing for blood disorders: state of the art and recent advances.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {3},
pages = {289-299},
pmid = {33523137},
issn = {2397-8554},
abstract = {In recent years, tremendous advances have been made in the use of gene editing to precisely engineer the genome. This technology relies on the activity of a wide range of nuclease platforms - such as zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR-Cas system - that can cleave and repair specific DNA regions, providing a unique and flexible tool to study gene function and correct disease-causing mutations. Preclinical studies using gene editing to tackle genetic and infectious diseases have highlighted the therapeutic potential of this technology. This review summarizes the progresses made towards the development of gene editing tools for the treatment of haematological disorders and the hurdles that need to be overcome to achieve clinical success.},
}
@article {pmid33525825,
year = {2018},
author = {Zhang, Y and Lin, J and Feng, M and She, Q},
title = {Molecular mechanisms of III-B CRISPR-Cas systems in archaea.},
journal = {Emerging topics in life sciences},
volume = {2},
number = {4},
pages = {483-491},
doi = {10.1042/ETLS20180023},
pmid = {33525825},
issn = {2397-8554},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems provide the adaptive antiviral immunity against invasive genetic elements in archaea and bacteria. These immune systems are divided into at least six different types, among which Type III CRISPR-Cas systems show several distinct antiviral activities as demonstrated from the investigation of bacterial III-A and archaeal III-B systems in the past decade. First, although initial experiments suggested that III-A systems provided DNA interference activity, whereas III-B system was active only in RNA interference, these immune systems were subsequently found to mediate the transcription-dependent DNA interference and the dual DNA/RNA interference. Second, their ribonucleoprotein (RNP) complexes show target RNA (tgRNA) cleavage by a ruler mechanism and RNA-activated indiscriminate single-stranded DNA cleavage, the latter of which is subjected to spatiotemporal regulation such that the DNase activity occurs only at the right place in the right time. Third, RNPs of Type III systems catalyse the synthesis of cyclic oligoadenylates (cOAs) that function as second messengers to activate Csm6 and Csx1, both of which are potent Cas accessory RNases after activation. To date, Type III CRISPR systems are the only known antiviral immunity that utilizes multiple interference mechanisms for antiviral defence.},
}
@article {pmid33265669,
year = {2018},
author = {Goethe, M and Fita, I and Rubi, JM},
title = {Entropic Stabilization of Cas4 Protein SSO0001 Predicted with Popcoen.},
journal = {Entropy (Basel, Switzerland)},
volume = {20},
number = {8},
pages = {},
pmid = {33265669},
issn = {1099-4300},
abstract = {Popcoen is a method for configurational entropy estimation of proteins based on machine-learning. Entropy is predicted with an artificial neural network which was trained on simulation trajectories of a large set of representative proteins. Popcoen is extremely fast compared to other approaches based on the sampling of a multitude of microstates. Consequently, Popcoen can be incorporated into a large class of protein software which currently neglects configurational entropy for performance reasons. Here, we apply Popcoen to various conformations of the Cas4 protein SSO0001 of Sulfolobus solfataricus, a protein that assembles to a decamer of known toroidal shape. We provide numerical evidence that the native state (NAT) of a SSO0001 monomer has a similar structure to the protomers of the oligomer, where NAT of the monomer is stabilized mainly entropically. Due to its large amount of configurational entropy, NAT has lower free energy than alternative conformations of very low enthalpy and solvation free-energy. Hence, SSO0001 serves as an example case where neglecting configurational entropy leads to incorrect conclusion. Our results imply that no refolding of the subunits is required during oligomerization which suggests that configurational entropy is employed by nature to largely enhance the rate of assembly.},
}
@article {pmid33250120,
year = {2018},
author = {Shtratnikova, VY and Belalov, I and Kasianov, AS and Schelkunov, MI and Logacheva Maria, DA and Novikov, AD and Shatalov, AA and Gerasimova, TV and Yanenko, AS and Makeev, VJ},
title = {The complete genome of the oil emulsifying strain Thalassolituus oleivorans K-188 from the Barents Sea.},
journal = {Marine genomics},
volume = {37},
number = {},
pages = {18-20},
doi = {10.1016/j.margen.2017.08.005},
pmid = {33250120},
issn = {1876-7478},
abstract = {Gammaproteobacterium Thalassolituus oleivorans plays an important role in oil degradation in sea water through emulsifying crude oil and alkanes at low temperatures in polar sea environment. Here we report the complete genome sequence of K-188 strain (VKPM B-9394) isolated in the Barents Sea and compare it with other known Thalassolituus oleivorans strains. The Thalassolituus strains are differed in orthologs number of the genes of alkane degradation, transport proteins, genes of sugar utilization, endonucleases, signaling proteins, transcriptional regulators and presence of CRISPR/Cas locus. Also only the genome of K-188 contains the 3-hydroxyalkanoate synthetase.},
}
@article {pmid33525759,
year = {2017},
author = {Van Eck, J},
title = {Gene editing in tomatoes.},
journal = {Emerging topics in life sciences},
volume = {1},
number = {2},
pages = {183-191},
doi = {10.1042/ETLS20170056},
pmid = {33525759},
issn = {2397-8554},
abstract = {Tomato is an effective model plant species because it possesses the qualities necessary for genetic and functional studies, but is also a food crop making what is learned more translatable for crop improvement when compared with other non-food crop models. The availability of genome sequences for many genotypes and amenability to transformation methodologies (Agrobacterium-mediated, direct DNA uptake via protoplasts, biolistics) make tomato the perfect platform to study the application of gene-editing technologies. This review includes information related to tomato transformation methodology, one of the necessary requirements for gene editing, along with the status of site-directed mutagenesis by TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated Proteins). In addition to the reports on proof-of-concept experiments to demonstrate the feasibility of gene editing in tomato, there are many reports that show the power of these technologies for modification of traits, such as fruit characteristics (ripening, size, and parthenocarpy), pathogen susceptibility, architecture (plant and inflorescence), and metabolic engineering. Also highlighted in this review are reports on the application of a recent CRISPR technology called base editing that allows the modification of one base pair in a gene sequence and a strategy that takes advantage of a geminivirus replicon for delivery of DNA repair template.},
}
@article {pmid32789192,
year = {2020},
author = {Sankaranarayanan, G and Coghlan, A and Driguez, P and Lotkowska, ME and Sanders, M and Holroyd, N and Tracey, A and Berriman, M and Rinaldi, G},
title = {Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni.},
journal = {Wellcome open research},
volume = {5},
number = {},
pages = {178},
pmid = {32789192},
issn = {2398-502X},
support = {/WT_/Wellcome Trust/United Kingdom ; },
abstract = {Background. At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 (ω1) gene. Methods. We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. Results. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Conclusions. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.},
}
@article {pmid33155979,
year = {2020},
author = {de Wet, TJ and Winkler, KR and Mhlanga, M and Mizrahi, V and Warner, DF},
title = {Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33155979},
issn = {2050-084X},
support = {U01 HD085531/HD/NICHD NIH HHS/United States ; 261669//Norges Forskningsr&#xe5;d/International ; },
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; Gene Knockdown Techniques ; Gene Library ; Genes, Bacterial/*genetics/physiology ; Genome, Bacterial/genetics/physiology ; Image Processing, Computer-Assisted/*methods ; Metabolic Networks and Pathways/genetics ; Multigene Family/genetics ; Mycobacterium smegmatis/drug effects/*genetics/metabolism ; Mycobacterium tuberculosis/genetics ; Mycolic Acids/metabolism ; },
abstract = {Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.},
}
@article {pmid33155547,
year = {2020},
author = {Zhao, Z and Zhang, Z and Li, J and Dong, Q and Xiong, J and Li, Y and Lan, M and Li, G and Zhu, B},
title = {Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33155547},
issn = {2050-084X},
support = {2018YFE0203300//the Chinese ministry of Science and technology/International ; 31530047//the national natural science foundation of China/International ; 31761163001//the national natural science foundation of China/International ; XDB 39000000//Chinese Academy of Sciences/International ; QYZDY-SSW-SMC031//Chinese Academy of Sciences/International ; 2017133//Youth Innovation Promotion Association of the Chinese Academy of Sciences/International ; 2020097//Youth Innovation Promotion Association of the Chinese Academy of Sciences/International ; 033/2017/AFJ//the NSFC-FDCT joint grant/International ; },
mesh = {CRISPR-Cas Systems ; Calcitonin Gene-Related Peptide/genetics/metabolism ; DNA Demethylation ; *Epigenesis, Genetic ; Gene Deletion ; Genes, Reporter ; Genetic Engineering ; HEK293 Cells ; Humans ; Inflammation/metabolism ; Mixed Function Oxygenases/genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; Transcription Factor RelA/genetics/metabolism ; *Transcription, Genetic ; Tumor Necrosis Factor-alpha/*pharmacology ; },
abstract = {Transcriptional memory allows certain genes to respond to previously experienced signals more robustly. However, whether and how the key proinflammatory cytokine TNF-α mediates transcriptional memory are poorly understood. Using HEK293F cells as a model system, we report that sustained TNF-α stimulation induces transcriptional memory dependent on TET enzymes. The hypomethylated status of transcriptional regulatory regions can be inherited, facilitating NF-κB binding and more robust subsequent activation. A high initial methylation level and CpG density around κB sites are correlated with the functional potential of transcriptional memory modules. Interestingly, the CALCB gene, encoding the proven migraine therapeutic target CGRP, exhibits the best transcriptional memory. A neighboring primate-specific endogenous retrovirus stimulates more rapid, more strong, and at least 100-fold more sensitive CALCB induction in subsequent TNF-α stimulation. Our study reveals that TNF-α-mediated transcriptional memory is governed by active DNA demethylation and greatly sensitizes memory genes to much lower doses of inflammatory cues.},
}
@article {pmid33155277,
year = {2020},
author = {Murakami, Y and Futamata, R and Horibe, T and Ueda, K and Kinoshita, M},
title = {CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.},
journal = {Development, growth &amp; differentiation},
volume = {62},
number = {9},
pages = {554-567},
doi = {10.1111/dgd.12700},
pmid = {33155277},
issn = {1440-169X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; Deoxyribonuclease I/*genetics/metabolism ; Genes, Lethal/*genetics ; Oryzias/genetics ; },
abstract = {The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.},
}
@article {pmid33154568,
year = {2020},
author = {Marx, V},
title = {Guide RNAs: it's good to be choosy.},
journal = {Nature methods},
volume = {17},
number = {12},
pages = {1179-1182},
pmid = {33154568},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; RNA, Guide/*genetics ; },
}
@article {pmid33154487,
year = {2020},
author = {Ingvarsen, SZ and Gårdsvoll, H and van Putten, S and Nørregaard, KS and Krigslund, O and Meilstrup, JA and Tran, C and Jürgensen, HJ and Melander, MC and Nielsen, CH and Kjaer, A and Bugge, TH and Engelholm, LH and Behrendt, N},
title = {Tumor cell MT1-MMP is dispensable for osteosarcoma tumor growth, bone degradation and lung metastasis.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {19138},
pmid = {33154487},
issn = {2045-2322},
mesh = {Animals ; Bone Neoplasms/*genetics/metabolism/secondary ; Bone and Bones/metabolism/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/*genetics ; Disease Progression ; Gene Knockdown Techniques ; Humans ; Lung Neoplasms/*genetics/metabolism/secondary ; Matrix Metalloproteinase 14/*genetics/metabolism ; Mice ; Neoplasm Invasiveness/genetics/pathology ; Osteosarcoma/*genetics/metabolism/secondary ; },
abstract = {The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.},
}
@article {pmid33154469,
year = {2020},
author = {Oo, JA and Irmer, B and Günther, S and Warwick, T and Pálfi, K and Izquierdo Ponce, J and Teichmann, T and Pflüger-Müller, B and Gilsbach, R and Brandes, RP and Leisegang, MS},
title = {ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {19079},
pmid = {33154469},
issn = {2045-2322},
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems ; Cells, Cultured ; Endothelial Cells/*cytology/*metabolism ; Gene Expression Profiling ; Gene Knockout Techniques ; Histones/metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Mutagenesis, Site-Directed ; Myocytes, Smooth Muscle/cytology/metabolism ; *Neovascularization, Physiologic/genetics ; Promoter Regions, Genetic ; RNA, Small Interfering/genetics ; Repressor Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Tripartite Motif-Containing Protein 28/metabolism ; Zinc Fingers/genetics ; },
abstract = {Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.},
}
@article {pmid33153299,
year = {2021},
author = {Mayuramart, O and Nimsamer, P and Rattanaburi, S and Chantaravisoot, N and Khongnomnan, K and Chansaenroj, J and Puenpa, J and Suntronwong, N and Vichaiwattana, P and Poovorawan, Y and Payungporn, S},
title = {Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {246},
number = {4},
pages = {400-405},
pmid = {33153299},
issn = {1535-3699},
mesh = {COVID-19/*diagnosis ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Influenza A virus/*genetics/isolation &amp; purification ; Influenza B virus/*genetics/isolation &amp; purification ; Influenza, Human/*diagnosis ; Limit of Detection ; *Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques ; RNA, Viral/analysis/genetics ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {Due to the common symptoms of COVID-19, patients are similar to influenza-like illness. Therefore, the detection method would be crucial to discriminate between SARS-CoV-2 and influenza virus-infected patients. In this study, CRISPR-Cas12a-based detection was applied for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus which would be a practical and attractive application for screening of patients with COVID-19 and influenza in areas with limited resources. The limit of detection for SARS-CoV-2, influenza A, and influenza B detection was 10, 10[3], and 10[3] copies/reaction, respectively. Moreover, the assays yielded no cross-reactivity against other respiratory viruses. The results revealed that the detection of influenza virus and SARS-CoV-2 by using RT-RPA and CRISPR-Cas12a technology reaches 96.23% sensitivity and 100% specificity for SARS-CoV-2 detection. The sensitivity for influenza virus A and B detections was 85.07% and 94.87%, respectively. In addition, the specificity for influenza virus A and B detections was approximately 96%. In conclusion, the RT-RPA with CRISPR-Cas12a assay was an effective method for the screening of influenza viruses and SARS-CoV-2 which could be applied to detect other infectious diseases in the future.},
}
@article {pmid33152994,
year = {2020},
author = {Lugli, GA and Tarracchini, C and Alessandri, G and Milani, C and Mancabelli, L and Turroni, F and Neuzil-Bunesova, V and Ruiz, L and Margolles, A and Ventura, M},
title = {Decoding the Genomic Variability among Members of the Bifidobacterium dentium Species.},
journal = {Microorganisms},
volume = {8},
number = {11},
pages = {},
pmid = {33152994},
issn = {2076-2607},
abstract = {Members of the Bifidobacterium dentium species are usually identified in the oral cavity of humans and associated with the development of plaque and dental caries. Nevertheless, they have also been detected from fecal samples, highlighting a widespread distribution among mammals. To explore the genetic variability of this species, we isolated and sequenced the genomes of 18 different B. dentium strains collected from fecal samples of several primate species and an Ursus arctos. Thus, we investigated the genomic variability and metabolic abilities of the new B. dentium isolates together with 20 public genome sequences. Comparative genomic analyses provided insights into the vast metabolic repertoire of the species, highlighting 19 glycosyl hydrolases families shared between each analyzed strain. Phylogenetic analysis of the B. dentium taxon, involving 1140 conserved genes, revealed a very close phylogenetic relatedness among members of this species. Furthermore, low genomic variability between strains was also confirmed by an average nucleotide identity analysis showing values higher than 98.2%. Investigating the genetic features of each strain, few putative functional mobile elements were identified. Besides, a consistent occurrence of defense mechanisms such as CRISPR-Cas and restriction-modification systems may be responsible for the high genome synteny identified among members of this taxon.},
}
@article {pmid33152914,
year = {2020},
author = {Lotfi, M and Rezaei, N},
title = {CRISPR/Cas13: A potential therapeutic option of COVID-19.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {131},
number = {},
pages = {110738},
pmid = {33152914},
issn = {1950-6007},
mesh = {Betacoronavirus/drug effects/*genetics ; COVID-19 ; CRISPR-Associated Proteins/pharmacology ; *CRISPR-Cas Systems ; Conserved Sequence ; Coronavirus Infections/drug therapy/genetics/*therapy ; *Gene Editing ; Genetic Therapy/*methods ; Genome, Viral ; Humans ; Pandemics ; Pneumonia, Viral/genetics/*therapy ; RNA, Guide/genetics ; RNA, Viral/antagonists &amp; inhibitors/*genetics ; SARS-CoV-2 ; },
abstract = {The novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be considered as the most important current global issue, as it has caused the novel coronavirus disease (COVID-19) pandemic, which has resulted in high mortality and morbidity rates all around the world. Although scientists are trying to discover novel therapies and develop and evaluate various previous treatments, at the time of writing this paper, there was no definite therapy and vaccine for COVID-19. So, as COVID-19 has called ideas for treatment, controlling, and diagnosis, we discussed the application of Clustered Regularly Interspaced Short Palindromic Repeats/Cas13 (CRISPR/Cas13) as a treatment of COVID-19, which received less attention compared with other potential therapeutic options.},
}
@article {pmid33152605,
year = {2021},
author = {Fenster, JA and Eckert, CA},
title = {High-Throughput Functional Genomics for Energy Production.},
journal = {Current opinion in biotechnology},
volume = {67},
number = {},
pages = {7-14},
doi = {10.1016/j.copbio.2020.09.010},
pmid = {33152605},
issn = {1879-0429},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genomics ; *Metabolic Engineering ; Mutagenesis ; },
abstract = {Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Additionally, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.},
}
@article {pmid33152519,
year = {2021},
author = {Pramanik, D and Shelake, RM and Kim, MJ and Kim, JY},
title = {CRISPR-Mediated Engineering across the Central Dogma in Plant Biology for Basic Research and Crop Improvement.},
journal = {Molecular plant},
volume = {14},
number = {1},
pages = {127-150},
doi = {10.1016/j.molp.2020.11.002},
pmid = {33152519},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Epigenesis, Genetic ; Gene Editing ; *Genetic Engineering ; *Research ; },
abstract = {The central dogma (CD) of molecular biology is the transfer of genetic information from DNA to RNA to protein. Major CD processes governing genetic flow include the cell cycle, DNA replication, chromosome packaging, epigenetic changes, transcription, posttranscriptional alterations, translation, and posttranslational modifications. The CD processes are tightly regulated in plants to maintain genetic integrity throughout the life cycle and to pass genetic materials to next generation. Engineering of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world population. CRISPR technology enables programmable editing of CD processes to alter DNA, RNA, or protein, which would have been impossible in the past. Here, an overview of recent advancements in CRISPR tool development and CRISPR-based CD modulations that expedite basic and applied plant research is provided. Furthermore, CRISPR applications in major thriving areas of research, such as gene discovery (allele mining and cryptic gene activation), introgression (de novo domestication and haploid induction), and application of desired traits beneficial to farmers or consumers (biotic/abiotic stress-resilient crops, plant cell factories, and delayed senescence), are described. Finally, the global regulatory policies, challenges, and prospects for CRISPR-mediated crop improvement are discussed.},
}
@article {pmid33152517,
year = {2020},
author = {Zeng, D and Liu, T and Tan, J and Zhang, Y and Zheng, Z and Wang, B and Zhou, D and Xie, X and Guo, M and Liu, YG and Zhu, Q},
title = {PhieCBEs: Plant High-Efficiency Cytidine Base Editors with Expanded Target Range.},
journal = {Molecular plant},
volume = {13},
number = {12},
pages = {1666-1669},
doi = {10.1016/j.molp.2020.11.001},
pmid = {33152517},
issn = {1752-9867},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cytidine/*genetics ; Cytidine Deaminase/metabolism ; *Gene Editing ; RNA, Guide/genetics ; },
}
@article {pmid33152516,
year = {2021},
author = {Zhao, D and Zhu, X and Zhou, H and Sun, N and Wang, T and Bi, C and Zhang, X},
title = {CRISPR-based metabolic pathway engineering.},
journal = {Metabolic engineering},
volume = {63},
number = {},
pages = {148-159},
doi = {10.1016/j.ymben.2020.10.004},
pmid = {33152516},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; *Metabolic Engineering ; Metabolic Networks and Pathways/genetics ; },
abstract = {A highly effective metabolic pathway is the key for an efficient cell factory. However, the engineered homologous or heterologous multi-gene pathway may be unbalanced, inefficient and causing the accumulation of potentially toxic intermediates. Therefore, pathways must be constructed optimally to minimize these negative effects and maximize catalytic efficiency. With the development of CRISPR technology, some of the problems of previous pathway engineering and genome editing techniques were resolved, providing higher efficiency, lower cost, and easily customizable targets. Moreover, CRISPR was demonstrated as robust and effective in various organisms including both prokaryotes and eukaryotes. In recent years, researchers in the field of metabolic engineering and synthetic biology have exploited various CRISPR-based pathway engineering approaches, which are both effective and convenient, as well as valuable from a theoretical standpoint. In this review, we systematically summarize novel pathway engineering techniques and strategies based on CRISPR nucleases system, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa), including figures and descriptions for easy understanding, with the aim to facilitate their broader application among fellow researchers.},
}
@article {pmid33152324,
year = {2021},
author = {Zhu, XG and Chudnovskiy, A and Baudrier, L and Prizer, B and Liu, Y and Ostendorf, BN and Yamaguchi, N and Arab, A and Tavora, B and Timson, R and Heissel, S and de Stanchina, E and Molina, H and Victora, GD and Goodarzi, H and Birsoy, K},
title = {Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells.},
journal = {Cell metabolism},
volume = {33},
number = {1},
pages = {211-221.e6},
pmid = {33152324},
issn = {1932-7420},
support = {DP2 CA228042/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Carcinoma, Pancreatic Ductal/genetics/*metabolism/pathology ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Knockout ; Mice, Transgenic ; Neoplasms, Experimental/genetics/metabolism/pathology ; Pancreatic Neoplasms/genetics/*metabolism/pathology ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) cells require substantial metabolic rewiring to overcome nutrient limitations and immune surveillance. However, the metabolic pathways necessary for pancreatic tumor growth in vivo are poorly understood. To address this, we performed metabolism-focused CRISPR screens in PDAC cells grown in culture or engrafted in immunocompetent mice. While most metabolic gene essentialities are unexpectedly similar under these conditions, a small fraction of metabolic genes are differentially required for tumor progression. Among these, loss of heme synthesis reduces tumor growth due to a limiting role of heme in vivo, an effect independent of tissue origin or immune system. Our screens also identify autophagy as a metabolic requirement for pancreatic tumor immune evasion. Mechanistically, autophagy protects cancer cells from CD8+ T cell killing through TNFα-induced cell death in vitro. Altogether, this resource provides metabolic dependencies arising from microenvironmental limitations and the immune system, nominating potential anti-cancer targets.},
}
@article {pmid33152323,
year = {2021},
author = {Biancur, DE and Kapner, KS and Yamamoto, K and Banh, RS and Neggers, JE and Sohn, ASW and Wu, W and Manguso, RT and Brown, A and Root, DE and Aguirre, AJ and Kimmelman, AC},
title = {Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer.},
journal = {Cell metabolism},
volume = {33},
number = {1},
pages = {199-210.e8},
pmid = {33152323},
issn = {1932-7420},
support = {U01 CA224146/CA/NCI NIH HHS/United States ; T32 CA009161/CA/NCI NIH HHS/United States ; P30 CA016087/CA/NCI NIH HHS/United States ; R35 CA232124/CA/NCI NIH HHS/United States ; K08 CA218420/CA/NCI NIH HHS/United States ; R01 CA157490/CA/NCI NIH HHS/United States ; R01 GM095567/GM/NIGMS NIH HHS/United States ; U54 DK110858/DK/NIDDK NIH HHS/United States ; R01 CA188048/CA/NCI NIH HHS/United States ; P50 CA127003/CA/NCI NIH HHS/United States ; F99 CA245822/CA/NCI NIH HHS/United States ; P01 CA117969/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Carcinoma, Pancreatic Ductal/genetics/*metabolism/pathology ; Cell Proliferation ; Humans ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental/genetics/metabolism/pathology ; Pancreatic Neoplasms/genetics/*metabolism/pathology ; Tumor Cells, Cultured ; Tumor Microenvironment/genetics ; },
abstract = {Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.},
}
@article {pmid33152077,
year = {2020},
author = {Fedorova, I and Vasileva, A and Selkova, P and Abramova, M and Arseniev, A and Pobegalov, G and Kazalov, M and Musharova, O and Goryanin, I and Artamonova, D and Zyubko, T and Shmakov, S and Artamonova, T and Khodorkovskii, M and Severinov, K},
title = {PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12297-12309},
pmid = {33152077},
issn = {1362-4962},
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Associated Protein 9/chemistry/*genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics/metabolism ; Gene Editing/methods ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; *Genome, Bacterial ; HEK293 Cells ; Humans ; Nucleic Acid Conformation ; Pasteurella pneumotropica/enzymology/*genetics ; RNA, Guide/chemistry/*genetics/metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Rhodobacteraceae/enzymology/genetics ; Sequence Alignment ; Sequence Homology, Amino Acid ; },
abstract = {CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.},
}
@article {pmid33152068,
year = {2020},
author = {Cooper, SE and Schwartzentruber, J and Bello, E and Coomber, EL and Bassett, AR},
title = {Screening for functional transcriptional and splicing regulatory variants with GenIE.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {e131},
pmid = {33152068},
issn = {1362-4962},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alleles ; Alternative Splicing/genetics ; Alzheimer Disease/*genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; Clusterin/*genetics ; Enhancer Elements, Genetic/*genetics ; Gene Editing ; Genetic Variation/genetics ; Genome-Wide Association Study ; Humans ; Induced Pluripotent Stem Cells/metabolism/transplantation ; Mutation ; Polymorphism, Single Nucleotide/genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; },
abstract = {Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.},
}
@article {pmid33151638,
year = {2020},
author = {Vasquez, CA and Cowan, QT and Komor, AC},
title = {Base Editing in Human Cells to Produce Single-Nucleotide-Variant Clonal Cell Lines.},
journal = {Current protocols in molecular biology},
volume = {133},
number = {1},
pages = {e129},
pmid = {33151638},
issn = {1934-3647},
support = {T32 GM008326/GM/NIGMS NIH HHS/United States ; R21 GM135736/GM/NIGMS NIH HHS/United States ; T32 CA009523/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; *Clone Cells ; *Gene Editing ; Humans ; *Point Mutation ; },
abstract = {Base-editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome-editing methods that use DNA double-strand breaks, such as zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system. This allows the generation of single-nucleotide-variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time- and resource-effective manner. These single-nucleotide-variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype-to-phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base-editing constructs, transfect adherent cells, quantify base-editing efficiencies in bulk, and generate single-nucleotide-variant clonal cell lines. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Design and production of plasmids for base-editing experiments Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing Alternate Protocol 1: Next-generation sequencing to quantify base editing Basic Protocol 4: Single-cell isolation of base-edited cells using FACS Alternate Protocol 2: Single-cell isolation of base-edited cells using dilution plating Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones.},
}
@article {pmid33151576,
year = {2021},
author = {Wu, W and Xia, X and Tang, L and Yao, F and Xu, H and Lei, H},
title = {Normal vitreous promotes angiogenesis via activation of Axl.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {1},
pages = {e21152},
doi = {10.1096/fj.201903105R},
pmid = {33151576},
issn = {1530-6860},
support = {R01EY01250/GF/NIH HHS/United States ; },
mesh = {Animals ; Benzocycloheptenes/pharmacology ; CRISPR-Cas Systems ; Diabetic Retinopathy/enzymology/genetics/pathology ; Enzyme Activation/drug effects/genetics ; HEK293 Cells ; Humans ; Mice ; Neovascularization, Pathologic/*enzymology/genetics/pathology ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Receptor Protein-Tyrosine Kinases/antagonists &amp; inhibitors/genetics/*metabolism ; Retinal Vessels/*enzymology/pathology ; Triazoles/pharmacology ; Vitreoretinopathy, Proliferative/enzymology/genetics/pathology ; Vitreous Body/*enzymology/pathology ; },
abstract = {Vitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.},
}
@article {pmid33151389,
year = {2021},
author = {Figueres-Oñate, M and Sánchez-González, R and López-Mascaraque, L},
title = {Deciphering neural heterogeneity through cell lineage tracing.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {78},
number = {5},
pages = {1971-1982},
pmid = {33151389},
issn = {1420-9071},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Cell Lineage/*genetics ; Gene Editing/methods ; Gene Expression Profiling/*methods ; High-Throughput Nucleotide Sequencing/methods ; Nervous System/cytology/*metabolism ; Neural Stem Cells/cytology/*metabolism ; Single-Cell Analysis/methods ; },
abstract = {Understanding how an adult brain reaches an appropriate size and cell composition from a pool of progenitors that proliferates and differentiates is a key question in Developmental Neurobiology. Not only the control of final size but also, the proper arrangement of cells of different embryonic origins is fundamental in this process. Each neural progenitor has to produce a precise number of sibling cells that establish clones, and all these clones will come together to form the functional adult nervous system. Lineage cell tracing is a complex and challenging process that aims to reconstruct the offspring that arise from a single progenitor cell. This tracing can be achieved through strategies based on genetically modified organisms, using either genetic tracers, transfected viral vectors or DNA constructs, and even single-cell sequencing. Combining different reporter proteins and the use of transgenic mice revolutionized clonal analysis more than a decade ago and now, the availability of novel genome editing tools and single-cell sequencing techniques has vastly improved the capacity of lineage tracing to decipher progenitor potential. This review brings together the strategies used to study cell lineages in the brain and the role they have played in our understanding of the functional clonal relationships among neural cells. In addition, future perspectives regarding the study of cell heterogeneity and the ontogeny of different cell lineages will also be addressed.},
}
@article {pmid33150679,
year = {2021},
author = {Li, J and Zhang, S and Zhang, R and Gao, J and Qi, Y and Song, G and Li, W and Li, Y and Li, G},
title = {Efficient multiplex genome editing by CRISPR/Cas9 in common wheat.},
journal = {Plant biotechnology journal},
volume = {19},
number = {3},
pages = {427-429},
pmid = {33150679},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; RNA, Guide/genetics ; Triticum/genetics ; },
}
@article {pmid33150374,
year = {2021},
author = {Shen, YJ and Mishima, Y and Shi, J and Sklavenitis-Pistofidis, R and Redd, RA and Moschetta, M and Manier, S and Roccaro, AM and Sacco, A and Tai, YT and Mercier, F and Kawano, Y and Su, NK and Berrios, B and Doench, JG and Root, DE and Michor, F and Scadden, DT and Ghobrial, IM},
title = {Progression signature underlies clonal evolution and dissemination of multiple myeloma.},
journal = {Blood},
volume = {137},
number = {17},
pages = {2360-2372},
pmid = {33150374},
issn = {1528-0020},
mesh = {Adaptor Proteins, Signal Transducing/antagonists &amp; inhibitors/genetics/*metabolism ; Animals ; Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; Bone Marrow/metabolism/pathology ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Clonal Evolution ; *Disease Models, Animal ; Disease Progression ; Female ; *Gene Expression Regulation, Neoplastic ; HMGA1a Protein/antagonists &amp; inhibitors/genetics/*metabolism ; Humans ; Mice ; Mice, SCID ; Multiple Myeloma/genetics/metabolism/*pathology ; Neoplasm Recurrence, Local/genetics/metabolism/*pathology ; Prognosis ; RNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Survival Rate ; Tumor Cells, Cultured ; },
abstract = {Clonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.},
}
@article {pmid33149284,
year = {2020},
author = {Zhu, H and Li, C and Gao, C},
title = {Author Correction: Applications of CRISPR-Cas in agriculture and plant biotechnology.},
journal = {Nature reviews. Molecular cell biology},
volume = {21},
number = {12},
pages = {782},
doi = {10.1038/s41580-020-00312-y},
pmid = {33149284},
issn = {1471-0080},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid33149206,
year = {2020},
author = {Fliedner, A and Gregor, A and Ferrazzi, F and Ekici, AB and Sticht, H and Zweier, C},
title = {Loss of PHF6 leads to aberrant development of human neuron-like cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {19030},
pmid = {33149206},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Epilepsy/genetics/pathology ; Face/abnormalities/pathology ; Female ; *Gene Knockdown Techniques ; Growth Disorders/genetics/pathology ; Humans ; Hypogonadism/genetics/pathology ; Intellectual Disability/genetics ; Male ; Mental Retardation, X-Linked/genetics/pathology ; Mutation, Missense ; Neurons/*cytology/metabolism ; Obesity/genetics/pathology ; Repressor Proteins/*genetics ; },
abstract = {Pathogenic variants in PHD finger protein 6 (PHF6) cause Borjeson-Forssman-Lehmann syndrome (BFLS), a rare X-linked neurodevelopmental disorder, which manifests variably in both males and females. To investigate the mechanisms behind overlapping but distinct clinical aspects between genders, we assessed the consequences of individual variants with structural modelling and molecular techniques. We found evidence that de novo variants occurring in females are more severe and result in loss of PHF6, while inherited variants identified in males might be hypomorph or have weaker effects on protein stability. This might contribute to the different phenotypes in male versus female individuals with BFLS. Furthermore, we used CRISPR/Cas9 to induce knockout of PHF6 in SK-N-BE (2) cells which were then differentiated to neuron-like cells in order to model nervous system related consequences of PHF6 loss. Transcriptome analysis revealed a broad deregulation of genes involved in chromatin and transcriptional regulation as well as in axon and neuron development. Subsequently, we could demonstrate that PHF6 is indeed required for proper neuron proliferation, neurite outgrowth and migration. Impairment of these processes might therefore contribute to the neurodevelopmental and cognitive dysfunction in BFLS.},
}
@article {pmid33148808,
year = {2020},
author = {Ramachandran, A and Huyke, DA and Sharma, E and Sahoo, MK and Huang, C and Banaei, N and Pinsky, BA and Santiago, JG},
title = {Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29518-29525},
pmid = {33148808},
issn = {1091-6490},
mesh = {COVID-19 Nucleic Acid Testing/*methods ; *CRISPR-Cas Systems ; Humans ; Isotachophoresis/*methods ; Microfluidics/*methods ; Nasal Mucosa/virology ; SARS-CoV-2/genetics/isolation &amp; purification ; },
abstract = {The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.},
}
@article {pmid33148705,
year = {2021},
author = {Mustafa, MI and Makhawi, AM},
title = {SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases.},
journal = {Journal of clinical microbiology},
volume = {59},
number = {3},
pages = {},
pmid = {33148705},
issn = {1098-660X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Communicable Diseases/diagnosis ; *Communicable Diseases, Emerging ; Humans ; Prospective Studies ; },
abstract = {Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases offers a better understanding of their pathogenesis. According to the World Health Organization, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free, and cost-effective. Nucleic acid pathogen detection methods, typically PCR, have numerous limitations, such as highly sophisticated equipment requirements, reagents, and trained personnel relying on well-established laboratories, besides being time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools that are rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit unpredicted in vitro properties of CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic acid binding event. These effectors can be attached to a diversity of reporters and utilized in tandem with isothermal amplification approaches to create sensitive identification in multiple deployable field formats. Although still in their beginning, SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious diseases, with ultrasensitive tests that do not require complicated processing. This review describes SHERLOCK and DETECTR technologies and assesses their properties, functions, and prospective to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.},
}
@article {pmid33147779,
year = {2020},
author = {van Rijn, JM and Werner, L and Aydemir, Y and Spronck, JMA and Pode-Shakked, B and van Hoesel, M and Shimshoni, E and Polak-Charcon, S and Talmi, L and Eren, M and Weiss, B and H J Houwen, R and Barshack, I and Somech, R and Nieuwenhuis, EES and Sagi, I and Raas-Rothschild, A and Middendorp, S and Shouval, DS},
title = {Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33147779},
issn = {1422-0067},
mesh = {Antigens, Bacterial/chemistry ; Bacterial Toxins/chemistry ; CRISPR-Cas Systems ; Collagen/*metabolism ; Consanguinity ; Diarrhea/congenital ; Duodenum/*metabolism ; Extracellular Matrix/metabolism ; Humans ; Hyalinosis, Systemic/genetics/*metabolism ; Infant ; Male ; Microscopy, Electron ; Mutation ; Phenotype ; Protein-Losing Enteropathies/genetics/*metabolism ; Receptors, Peptide/deficiency/*genetics ; Signal Transduction ; },
abstract = {Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2[KO] organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.},
}
@article {pmid33147453,
year = {2020},
author = {Marina, RJ and Brannan, KW and Dong, KD and Yee, BA and Yeo, GW},
title = {Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing.},
journal = {Cell reports},
volume = {33},
number = {5},
pages = {108350},
pmid = {33147453},
issn = {2211-1247},
support = {R01 EY029166/EY/NEI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; T32 GM008666/GM/NIGMS NIH HHS/United States ; K22 NS112678/NS/NINDS NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; F31 NS111859/NS/NINDS NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; },
mesh = {Adenosine Deaminase/metabolism ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; *Genetic Engineering ; HEK293 Cells ; Humans ; RNA Editing/*genetics ; RNA, Guide/metabolism ; RNA-Binding Proteins/metabolism ; RNA-Seq ; Transcriptome/genetics ; },
abstract = {Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.},
}
@article {pmid33147260,
year = {2020},
author = {Liu, H and Robinson, DS and Wu, ZY and Kuo, R and Yoshikuni, Y and Blaby, IK and Cheng, JF},
title = {Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.},
journal = {PloS one},
volume = {15},
number = {11},
pages = {e0241867},
pmid = {33147260},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Bacterial ; Integrases/*metabolism ; Photorhabdus/*genetics ; },
abstract = {The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.},
}
@article {pmid33146573,
year = {2020},
author = {Aliaga Goltsman, DS and Alexander, LM and Devoto, AE and Albers, JB and Liu, J and Butterfield, CN and Brown, CT and Thomas, BC},
title = {Novel Type V-A CRISPR Effectors Are Active Nucleases with Expanded Targeting Capabilities.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {454-461},
pmid = {33146573},
issn = {2573-1602},
support = {S10 OD018174/OD/NIH HHS/United States ; },
mesh = {Bacteria/genetics ; Bacterial Proteins/genetics/*isolation &amp; purification/*metabolism ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/genetics/*isolation &amp; purification/*metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/genetics/*isolation &amp; purification/*metabolism ; Endonucleases/genetics ; Gene Editing/*methods/trends ; Humans ; Metagenomics/methods ; Phylogeny ; RNA, Guide/genetics ; },
abstract = {Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed representatives of these systems into gene-editing platforms. The nucleases display extensive protein variation and can be programmed by a single-guide RNA with specific motifs. The majority of these enzymes are part of systems recovered from uncultivated organisms, some of which also encode a divergent Type V effector. Biochemical analysis uncovered unexpected protospacer adjacent motif diversity, indicating that these systems will facilitate a variety of genome-engineering applications. The simplicity of guide sequences and activity in human cell lines suggest utility in gene and cell therapies.},
}
@article {pmid33146562,
year = {2020},
author = {Li, J and Li, Y and Pawlik, KM and Napierala, JS and Napierala, M},
title = {A CRISPR-Cas9, Cre-lox, and Flp-FRT Cascade Strategy for the Precise and Efficient Integration of Exogenous DNA into Cellular Genomes.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {470-486},
pmid = {33146562},
issn = {2573-1602},
support = {R01 NS081366/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; Gene Editing/*methods/trends ; Genetic Techniques ; Genetic Vectors/genetics ; Genome/genetics ; Integrases/genetics ; Promoter Regions, Genetic/genetics ; Recombination, Genetic/genetics ; },
abstract = {We describe a protocol for the precise integration of exogenous DNA into user-defined genomic loci in cultured cells. This strategy first introduces a promoter and a lox site to a specific location via a Cas9-induced double-strand break. Second, a gene of interest (GOI) is inserted into the lox site via Cre-lox recombination. Upon correct insertion, a cis-linked antibiotic resistance gene will be expressed from a promoter introduced into the genome in the first step assuring selection for correct integrants. Last, the selection cassette is excised via a Flp-FRT recombination event, leaving a precisely targeted GOI. This method is broadly applicable to any exogenous DNA to be integrated, choice of integration site, and choice of cell type. The most remarkable aspect of this versatile approach, termed "CasPi" (cascaded precise integration), is that it allows for precise genome targeting with large, frequently complex, and repetitive DNA sequences that do not integrate efficiently or at all with current genome targeting methods.},
}
@article {pmid33145822,
year = {2021},
author = {Du, X and McManus, DP and French, JD and Jones, MK and You, H},
title = {CRISPR/Cas9: A new tool for the study and control of helminth parasites.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {43},
number = {1},
pages = {e2000185},
doi = {10.1002/bies.202000185},
pmid = {33145822},
issn = {1521-1878},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics ; Gene Editing ; Mutagenesis ; *Parasites ; },
abstract = {Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9-targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9-mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.},
}
@article {pmid33144677,
year = {2020},
author = {Okitsu, Y and Nagano, M and Yamagata, T and Ito, C and Toshimori, K and Dohra, H and Fujii, W and Yogo, K},
title = {Dlec1 is required for spermatogenesis and male fertility in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18883},
pmid = {33144677},
issn = {2045-2322},
mesh = {A549 Cells ; Animals ; CRISPR-Cas Systems ; Gene Deletion ; HEK293 Cells ; Humans ; Infertility, Male/*genetics/metabolism ; Ion Channels/metabolism ; Male ; Mice ; Organ Specificity ; Spermatogenesis ; Spermatozoa/*metabolism ; Tubulin/metabolism ; Tumor Suppressor Proteins/*genetics/*metabolism ; },
abstract = {Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1[-/-] mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet-Biedl Syndrome (BBS) protein complex subunits, as well as α- and β-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.},
}
@article {pmid33144673,
year = {2020},
author = {Gowripalan, A and Smith, S and Stefanovic, T and Tscharke, DC},
title = {Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {643},
pmid = {33144673},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation, Viral ; *Genetic Engineering ; Humans ; Vaccinia virus/*genetics ; Virus Replication ; },
abstract = {In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient. As a result, Cas9 targeting makes only modest, if any, improvements to basal rates of homologous recombination between repair constructs and poxvirus genomes. Instead, Cas9 cleavage leads to inhibition of poxvirus DNA replication thereby suppressing virus spread in culture. This unexpected outcome allows Cas9 to be used as a powerful tool for selecting conventionally generated poxvirus recombinants, which are otherwise impossible to separate from a large background of parental virus without the use of marker genes. This application of CRISPR/Cas9 greatly speeds up the generation of poxvirus-based vaccines, making this platform considerably more attractive in the context of personalised cancer vaccines and emerging disease outbreaks.},
}
@article {pmid33144657,
year = {2020},
author = {Job, A and Tatura, M and Schäfer, C and Lutz, V and Schneider, H and Lankat-Buttgereit, B and Zielinski, A and Borgmann, K and Bauer, C and Gress, TM and Buchholz, M and Gallmeier, E},
title = {The POLD1[R689W] variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18924},
pmid = {33144657},
issn = {2045-2322},
mesh = {*Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; Checkpoint Kinase 1/antagonists &amp; inhibitors ; Colorectal Neoplasms/*drug therapy/genetics ; DNA Polymerase III/*genetics ; DNA Replication/drug effects ; Gene Knockout Techniques ; Humans ; Indoles ; Mice ; Morpholines ; Pyrimidines/*administration &amp; dosage/pharmacology ; Sulfonamides ; Sulfoxides/*administration &amp; dosage/pharmacology ; Xenograft Model Antitumor Assays ; },
abstract = {Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1[R689W] affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1[R689W] strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1[R689W] variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.},
}
@article {pmid33144569,
year = {2020},
author = {van den Boomen, DJH and Sienkiewicz, A and Berlin, I and Jongsma, MLM and van Elsland, DM and Luzio, JP and Neefjes, JJC and Lehner, PJ},
title = {A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5559},
pmid = {33144569},
issn = {2041-1723},
support = {MR/R0009015/1/MRC_/Medical Research Council/United Kingdom ; 084957/Z/08/Z/WT_/Wellcome Trust/United Kingdom ; MR/R009015/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 100140/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Biological Transport ; CRISPR-Cas Systems/genetics ; Cholesterol/*metabolism ; Cholesterol, LDL/metabolism ; Endosomes/metabolism/ultrastructure ; Fluorescent Dyes/metabolism ; Genome, Human ; Guanine Nucleotide Exchange Factors/metabolism ; HEK293 Cells ; HeLa Cells ; Homeostasis ; Humans ; Hydroxymethylglutaryl-CoA Synthase/metabolism ; Intracellular Signaling Peptides and Proteins/*metabolism ; Lysosomes/*metabolism/ultrastructure ; Models, Biological ; Multiprotein Complexes/metabolism ; Niemann-Pick C1 Protein ; Protein Binding ; *Protein Multimerization ; rab GTP-Binding Proteins/*metabolism ; rab7 GTP-Binding Proteins ; },
abstract = {Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.},
}
@article {pmid33144546,
year = {2021},
author = {Xu, X and Feng, J and Zhang, P and Fan, J and Yin, WB},
title = {A CRISPR/Cas9 Cleavage System for Capturing Fungal Secondary Metabolite Gene Clusters.},
journal = {Journal of microbiology and biotechnology},
volume = {31},
number = {1},
pages = {8-15},
doi = {10.4014/jmb.2008.08040},
pmid = {33144546},
issn = {1738-8872},
mesh = {Aspergillus/genetics/metabolism ; Biological Products/metabolism ; Biosynthetic Pathways/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Fungal ; Fungi/*genetics/*metabolism ; Gene Editing ; Genome, Fungal ; *Multigene Family ; Plasmids ; Saccharomyces cerevisiae/genetics ; Secondary Metabolism/*genetics ; },
abstract = {More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.},
}
@article {pmid33143019,
year = {2020},
author = {Spasskaya, DS and Nadolinskaia, NI and Tutyaeva, VV and Lysov, YP and Karpov, VL and Karpov, DS},
title = {Yeast Rpn4 Links the Proteasome and DNA Repair via RAD52 Regulation.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33143019},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; DNA Damage ; *DNA Repair ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; *Gene Expression Regulation, Fungal ; Mutation ; Proteasome Endopeptidase Complex/*metabolism ; Rad52 DNA Repair and Recombination Protein/antagonists &amp; inhibitors/genetics/*metabolism ; Saccharomyces cerevisiae/genetics/growth &amp; development/*metabolism ; Saccharomyces cerevisiae Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Transcription Factors/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Environmental and intracellular factors often damage DNA, but multiple DNA repair pathways maintain genome integrity. In yeast, the 26S proteasome and its transcriptional regulator and substrate Rpn4 are involved in DNA damage resistance. Paradoxically, while proteasome dysfunction may induce hyper-resistance to DNA-damaging agents, Rpn4 malfunction sensitizes yeasts to these agents. Previously, we proposed that proteasome inhibition causes Rpn4 stabilization followed by the upregulation of Rpn4-dependent DNA repair genes and pathways. Here, we aimed to elucidate the key Rpn4 targets responsible for DNA damage hyper-resistance in proteasome mutants. We impaired the Rpn4-mediated regulation of candidate genes using the CRISPR/Cas9 system and tested the sensitivity of mutant strains to 4-NQO, MMS and zeocin. We found that the separate or simultaneous deregulation of 19S or 20S proteasome subcomplexes induced MAG1, DDI1, RAD23 and RAD52 in an Rpn4-dependent manner. Deregulation of RAD23, DDI1 and RAD52 sensitized yeast to DNA damage. Genetic, epigenetic or dihydrocoumarin-mediated RAD52 repression restored the sensitivity of the proteasome mutants to DNA damage. Our results suggest that the Rpn4-mediated overexpression of DNA repair genes, especially RAD52, defines the DNA damage hyper-resistant phenotype of proteasome mutants. The developed yeast model is useful for characterizing drugs that reverse the DNA damage hyper-resistance phenotypes of cancers.},
}
@article {pmid33142991,
year = {2020},
author = {Williams, AE and Sanchez-Vargas, I and Reid, WR and Lin, J and Franz, AWE and Olson, KE},
title = {The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti.},
journal = {Viruses},
volume = {12},
number = {11},
pages = {},
pmid = {33142991},
issn = {1999-4915},
support = {R01 AI130085/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/genetics/*virology ; Animals ; Animals, Genetically Modified/virology ; CRISPR-Cas Systems ; Disease Resistance/*genetics ; Disease Susceptibility/virology ; Female ; *Genome, Viral ; Male ; Mosquito Vectors/genetics/virology ; RNA, Small Interfering/genetics/*metabolism ; Saliva/virology ; Viral Load ; Virus Replication ; Zika Virus/*genetics/physiology ; Zika Virus Infection/virology ; },
abstract = {The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015-2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.},
}
@article {pmid33142942,
year = {2020},
author = {Wang, H and Song, S and Cheng, H and Tan, YW},
title = {State-of-the-Art Technologies for Understanding Brassinosteroid Signaling Networks.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33142942},
issn = {1422-0067},
mesh = {Brassinosteroids/*metabolism ; CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; Plant Proteins/genetics/*metabolism ; Plants/*genetics/*metabolism ; Proteomics/*methods ; Signal Transduction ; },
abstract = {Brassinosteroids, the steroid hormones of plants, control physiological and developmental processes through its signaling pathway. The major brassinosteroid signaling network components, from the receptor to transcription factors, have been identified in the past two decades. The development of biotechnologies has driven the identification of novel brassinosteroid signaling components, even revealing several crosstalks between brassinosteroid and other plant signaling pathways. Herein, we would like to summarize the identification and improvement of several representative brassinosteroid signaling components through the development of new technologies, including brassinosteroid-insensitive 1 (BRI1), BRI1-associated kinase 1 (BAK1), BR-insensitive 2 (BIN2), BRI1 kinase inhibitor 1 (BKI1), BRI1-suppressor 1 (BSU1), BR signaling kinases (BSKs), BRI1 ethyl methanesulfonate suppressor 1 (BES1), and brassinazole resistant 1 (BZR1). Furthermore, improvement of BR signaling knowledge, such as the function of BKI1, BES1 and its homologous through clustered regularly interspaced short palindromic repeats (CRISPR), the regulation of BIN2 through single-molecule methods, and the new in vivo interactors of BIN2 identified by proximity labeling are described. Among these technologies, recent advanced methods proximity labeling and single-molecule methods will be reviewed in detail to provide insights to brassinosteroid and other phytohormone signaling pathway studies.},
}
@article {pmid33140167,
year = {2021},
author = {Yu, Z and Jiang, S and Wang, Y and Tian, X and Zhao, P and Xu, J and Feng, M and She, Q},
title = {CRISPR-Cas adaptive immune systems in Sulfolobales: genetic studies and molecular mechanisms.},
journal = {Science China. Life sciences},
volume = {64},
number = {5},
pages = {678-696},
pmid = {33140167},
issn = {1869-1889},
mesh = {Adaptive Immunity/*genetics ; *CRISPR-Cas Systems ; DNA, Archaeal/genetics ; Genes, Archaeal ; Models, Biological ; RNA, Archaeal/genetics ; Sulfolobales/*genetics/*immunology ; Transcription, Genetic ; },
abstract = {CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria. Organisms of Sulfolobales, an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum, usually contain both type I and type III CRISPR-Cas systems. Two species, Saccharolobus solfataricus and Sulfolobus islandicus, have been important models for CRISPR study in archaea, and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps: adaptation, expression and crRNA processing, and interference. Four subtypes of CRISPR-Cas systems are common in these organisms, including I-A, I-D, III-B, and III-D. These cas genes form functional modules, e.g., all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and iCas modules. Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea, and these methodologies are useful in demonstration of the protospacer-adjacent motif (PAM)-dependent DNA interference by I-A interference modules and multiple interference activities by III-B Cmr systems. Ribonucleoprotein effector complexes have been isolated for Sulfolobales III-B and III-D systems, and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.},
}
@article {pmid33139946,
year = {2020},
author = {Dance, A},
title = {Studying life at the extremes.},
journal = {Nature},
volume = {587},
number = {7832},
pages = {165-166},
pmid = {33139946},
issn = {1476-4687},
mesh = {Bacteria/*cytology/*genetics/isolation &amp; purification ; CRISPR-Cas Systems ; Cell Division ; Clostridium thermocellum/genetics ; DNA Methylation ; *Extreme Environments ; Gene Editing/methods ; Genes, Bacterial/genetics ; Halobacterium salinarum/cytology ; Haloferax volcanii/cytology/genetics/metabolism ; *Laboratories ; Microbiological Techniques/*methods ; Microscopy/instrumentation/methods ; *Research Design ; *Research Personnel ; Sulfolobus acidocaldarius/cytology ; },
}
@article {pmid33139880,
year = {2021},
author = {Martínez Arbas, S and Narayanasamy, S and Herold, M and Lebrun, LA and Hoopmann, MR and Li, S and Lam, TJ and Kunath, BJ and Hicks, ND and Liu, CM and Price, LB and Laczny, CC and Gillece, JD and Schupp, JM and Keim, PS and Moritz, RL and Faust, K and Tang, H and Ye, Y and Skupin, A and May, P and Muller, EEL and Wilmes, P},
title = {Roles of bacteriophages, plasmids and CRISPR immunity in microbial community dynamics revealed using time-series integrated meta-omics.},
journal = {Nature microbiology},
volume = {6},
number = {1},
pages = {123-135},
pmid = {33139880},
issn = {2058-5276},
support = {R01 AI108888/AI/NIAID NIH HHS/United States ; R01 AI143254/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteria/*genetics/virology ; Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; Metagenome/genetics ; Microbial Consortia/genetics ; Microbial Interactions/*genetics/physiology ; Plasmids/*genetics ; Sewage/microbiology ; Water Purification ; },
abstract = {Viruses and plasmids (invasive mobile genetic elements (iMGEs)) have important roles in shaping microbial communities, but their dynamic interactions with CRISPR-based immunity remain unresolved. We analysed generation-resolved iMGE-host dynamics spanning one and a half years in a microbial consortium from a biological wastewater treatment plant using integrated meta-omics. We identified 31 bacterial metagenome-assembled genomes encoding complete CRISPR-Cas systems and their corresponding iMGEs. CRISPR-targeted plasmids outnumbered their bacteriophage counterparts by at least fivefold, highlighting the importance of CRISPR-mediated defence against plasmids. Linear modelling of our time-series data revealed that the variation in plasmid abundance over time explained more of the observed community dynamics than phages. Community-scale CRISPR-based plasmid-host and phage-host interaction networks revealed an increase in CRISPR-mediated interactions coinciding with a decrease in the dominant 'Candidatus Microthrix parvicella' population. Protospacers were enriched in sequences targeting genes involved in the transmission of iMGEs. Understanding the factors shaping the fitness of specific populations is necessary to devise control strategies for undesirable species and to predict or explain community-wide phenotypes.},
}
@article {pmid33139742,
year = {2020},
author = {Gasiunas, G and Young, JK and Karvelis, T and Kazlauskas, D and Urbaitis, T and Jasnauskaite, M and Grusyte, MM and Paulraj, S and Wang, PH and Hou, Z and Dooley, SK and Cigan, M and Alarcon, C and Chilcoat, ND and Bigelyte, G and Curcuru, JL and Mabuchi, M and Sun, Z and Fuchs, RT and Schildkraut, E and Weigele, PR and Jack, WE and Robb, GB and Venclovas, &#x10c; and Siksnys, V},
title = {A catalogue of biochemically diverse CRISPR-Cas9 orthologs.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5512},
pmid = {33139742},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Computational Biology ; DNA Cleavage ; Gene Editing/*methods ; RNA, Guide/*genetics/metabolism ; Sequence Homology, Nucleic Acid ; },
abstract = {Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.},
}
@article {pmid33139725,
year = {2020},
author = {Dougherty, GW and Mizuno, K and Nöthe-Menchen, T and Ikawa, Y and Boldt, K and Ta-Shma, A and Aprea, I and Minegishi, K and Pang, YP and Pennekamp, P and Loges, NT and Raidt, J and Hjeij, R and Wallmeier, J and Mussaffi, H and Perles, Z and Elpeleg, O and Rabert, F and Shiratori, H and Letteboer, SJ and Horn, N and Young, S and Strünker, T and Stumme, F and Werner, C and Olbrich, H and Takaoka, K and Ide, T and Twan, WK and Biebach, L and Große-Onnebrink, J and Klinkenbusch, JA and Praveen, K and Bracht, DC and Höben, IM and Junger, K and Gützlaff, J and Cindrić, S and Aviram, M and Kaiser, T and Memari, Y and Dzeja, PP and Dworniczak, B and Ueffing, M and Roepman, R and Bartscherer, K and Katsanis, N and Davis, EE and Amirav, I and Hamada, H and Omran, H},
title = {CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5520},
pmid = {33139725},
issn = {2041-1723},
support = {R01 DK072301/DK/NIDDK NIH HHS/United States ; },
mesh = {Adenine Nucleotides/*metabolism ; Adolescent ; Adult ; Animals ; Asthenozoospermia/*genetics/pathology ; Axoneme/ultrastructure ; CRISPR-Cas Systems/genetics ; Cilia/metabolism/ultrastructure ; Cytoskeletal Proteins/*deficiency/genetics ; DNA Mutational Analysis ; Disease Models, Animal ; Epididymis/pathology ; Female ; Flagella/metabolism/ultrastructure ; Humans ; Loss of Function Mutation ; Male ; Mice ; Mice, Knockout ; Middle Aged ; Planarians/cytology/genetics/metabolism ; Respiratory Mucosa/cytology/pathology ; Situs Inversus/diagnostic imaging/*genetics/pathology ; Sperm Motility/genetics ; Tomography, X-Ray Computed ; Whole Exome Sequencing ; },
abstract = {Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45[-/-] mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.},
}
@article {pmid33139551,
year = {2020},
author = {Takao, T and Sato, M and Maruyama, T},
title = {Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {46},
pages = {28579-28581},
pmid = {33139551},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Embryo Implantation ; Fertility ; Leukemia Inhibitory Factor/genetics/*metabolism ; Mice, Inbred ICR ; *Optogenetics ; },
abstract = {Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.},
}
@article {pmid33138773,
year = {2020},
author = {Mallikarjunappa, S and Shandilya, UK and Sharma, A and Lamers, K and Bissonnette, N and Karrow, NA and Meade, KG},
title = {Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9.},
journal = {BMC genetics},
volume = {21},
number = {1},
pages = {121},
pmid = {33138773},
issn = {1471-2156},
support = {NA//Teagasc/International ; NA//Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle/*genetics ; Cell Line ; Cytokines/genetics ; Epithelial Cells/*microbiology ; Gene Expression ; Gene Knockout Techniques ; *Mycobacterium avium subsp. paratuberculosis ; Paratuberculosis/immunology ; Receptors, Interleukin-10/*genetics ; },
abstract = {BACKGROUND: The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne's disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate.<br><br>RESULTS: An IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/- IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level&#xa0;knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-&#x3b1; and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative&#xa0;to unedited cells.<br><br>CONCLUSIONS: The findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.},
}
@article {pmid33137817,
year = {2020},
author = {Yan, J and Xue, D and Chuai, G and Gao, Y and Zhang, G and Liu, Q},
title = {Benchmarking and integrating genome-wide CRISPR off-target detection and prediction.},
journal = {Nucleic acids research},
volume = {48},
number = {20},
pages = {11370-11379},
pmid = {33137817},
issn = {1362-4962},
mesh = {Algorithms ; Benchmarking ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Computer Simulation ; Databases, Genetic ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome ; Genomics/*methods ; Humans ; Models, Molecular ; RNA, Guide ; Whole Genome Sequencing ; },
abstract = {Systematic evaluation of genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) off-target profiles is a fundamental step for the successful application of the CRISPR system to clinical therapies. Many experimental techniques and in silico tools have been proposed for detecting and predicting genome-wide CRISPR off-target profiles. These techniques and tools, however, have not been systematically benchmarked. A comprehensive benchmark study and an integrated strategy that takes advantage of the currently available tools to improve predictions of genome-wide CRISPR off-target profiles are needed. We focused on the specificity of the traditional CRISPR SpCas9 system for gene knockout. First, we benchmarked 10 available genome-wide off-target cleavage site (OTS) detection techniques with the published OTS detection datasets. Second, taking the datasets generated from OTS detection techniques as the benchmark datasets, we benchmarked 17 available in silico genome-wide OTS prediction tools to evaluate their genome-wide CRISPR off-target prediction performances. Finally, we present the first one-stop integrated Genome-Wide Off-target cleavage Search platform (iGWOS) that was specifically designed for the optimal genome-wide OTS prediction by integrating the available OTS prediction algorithms with an AdaBoost ensemble framework.},
}
@article {pmid33137193,
year = {2021},
author = {Wang, J and Dai, W and Li, J and Li, Q and Xie, R and Zhang, Y and Stubenrauch, C and Lithgow, T},
title = {AcrHub: an integrative hub for investigating, predicting and mapping anti-CRISPR proteins.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D630-D638},
pmid = {33137193},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; Data Analysis ; *Databases, Protein ; Internet ; },
abstract = {Anti-CRISPR (Acr) proteins naturally inhibit CRISPR-Cas adaptive immune systems across bacterial and archaeal domains of life. This emerging field has caused a paradigm shift in the way we think about the CRISPR-Cas system, and promises a number of useful applications from gene editing to phage therapy. As the number of verified and predicted Acrs rapidly expands, few online resources have been developed to deal with this wealth of information. To overcome this shortcoming, we developed AcrHub, an integrative database to provide an all-in-one solution for investigating, predicting and mapping Acr proteins. AcrHub catalogs 339 non-redundant experimentally validated Acrs and over 70 000 predicted Acrs extracted from genome sequence data from a diverse range of prokaryotic organisms and their viruses. It integrates state-of-the-art predictors to predict potential Acrs, and incorporates three analytical modules: similarity analysis, phylogenetic analysis and homology network analysis, to analyze their relationships with known Acrs. By interconnecting all modules as a platform, AcrHub presents enriched and in-depth analysis of known and potential Acrs and therefore provides new and exciting insights into the future of Acr discovery and validation. AcrHub is freely available at http://pacrispr.erc.monash.edu/AcrHub/.},
}
@article {pmid33137185,
year = {2021},
author = {Choi, A and Jang, I and Han, H and Kim, MS and Choi, J and Lee, J and Cho, SY and Jun, Y and Lee, C and Kim, J and Lee, B and Lee, S},
title = {iCSDB: an integrated database of CRISPR screens.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D956-D961},
pmid = {33137185},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *Databases, Genetic ; Gene Editing/*methods ; Gene Targeting/*methods ; Genome, Human/*genetics ; Genome-Wide Association Study/*methods ; Genomics/*methods ; Humans ; Internet ; Web Browser ; },
abstract = {High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.},
}
@article {pmid33137164,
year = {2020},
author = {Schleicher, EM and Dhoonmoon, A and Jackson, LM and Clements, KE and Stump, CL and Nicolae, CM and Moldovan, GL},
title = {Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.},
journal = {PLoS genetics},
volume = {16},
number = {11},
pages = {e1009176},
pmid = {33137164},
issn = {1553-7404},
support = {F31 CA243301/CA/NCI NIH HHS/United States ; R01 ES026184/ES/NIEHS NIH HHS/United States ; R01 GM134681/GM/NIGMS NIH HHS/United States ; },
mesh = {Apoptosis/drug effects/genetics ; Ataxia Telangiectasia Mutated Proteins/*antagonists &amp; inhibitors ; Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; DNA Replication/drug effects/genetics ; Drug Resistance, Neoplasm/*genetics ; Drug Screening Assays, Antitumor ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Indoles ; Mediator Complex/genetics/metabolism ; Morpholines ; Neoplasms/*drug therapy/genetics/pathology ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Pyrimidines/pharmacology/therapeutic use ; Signal Transduction/drug effects/genetics ; Sulfonamides ; Sulfoxides/pharmacology/therapeutic use ; Transforming Growth Factor beta/metabolism ; },
abstract = {The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.},
}
@article {pmid33137136,
year = {2020},
author = {Chen, K and Yu, Y and Yang, D and Yang, X and Tang, L and Liu, Y and Luo, X and Walters, JR and Liu, Z and Xu, J and Huang, Y},
title = {Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.},
journal = {PLoS genetics},
volume = {16},
number = {11},
pages = {e1009194},
pmid = {33137136},
issn = {1553-7404},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*physiology ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/*genetics ; Female ; Gene Expression Regulation, Developmental ; Insect Proteins/genetics/*metabolism ; Male ; Mutation ; Nuclear Proteins/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Sex Determination Processes/*genetics ; },
abstract = {Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.},
}
@article {pmid33135430,
year = {2020},
author = {Straiton, J},
title = {CRISPR vs COVID-19: how can gene editing help beat a virus?.},
journal = {BioTechniques},
volume = {69},
number = {5},
pages = {327-329},
doi = {10.2144/btn-2020-0145},
pmid = {33135430},
issn = {1940-9818},
mesh = {Antiviral Agents/therapeutic use ; Betacoronavirus/genetics ; COVID-19 ; *CRISPR-Cas Systems ; *Coronavirus Infections/diagnosis/drug therapy/therapy ; *Gene Editing ; Gene Targeting ; Humans ; Molecular Diagnostic Techniques/methods ; *Molecular Targeted Therapy ; *Pandemics ; *Pneumonia, Viral/diagnosis/therapy ; SARS-CoV-2 ; },
abstract = {[Formula: see text] Known to be a sturdy weapon in a scientist's arsenal, how has the gene editing tool CRISPR been applied in the fight against COVID-19?},
}
@article {pmid33134311,
year = {2020},
author = {Konwarh, R},
title = {Can CRISPR/Cas Technology Be a Felicitous Stratagem Against the COVID-19 Fiasco? Prospects and Hitches.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {557377},
pmid = {33134311},
issn = {2296-889X},
abstract = {The current global debacle of COVID-19, spelled by SARS-CoV-2 needs no elaboration. With incessant and constantly clambering number of deaths across various nations, the need of the hour is to develop readily deployable, fast, affordable detection assays and kits, yielding precise and consistent results as well as timely availability of efficacious anti-SARS-CoV-2 strategies to contain it. Conventionally employed real time PCR based technique for detection of the virus suffers from a couple of handicaps. Amongst other approaches, CRISPR based technology has ushered in new hopes. Recent efforts have been directed toward developing CRISPR/Cas based low-cost, rapid detection methods as well as development of one-pot assay platforms. The plausible application of CRISPR-Cas system to counteract the viral assault has also been assessed. The write up in this article mirrors the current status, the prospects and the practical snags of CRISPR/Cas technology for the detection and inactivation of the novel corona virus, SARS-CoV-2.},
}
@article {pmid33134100,
year = {2020},
author = {Lomov, NA and Viushkov, VS and Zamalutdinov, AV and Sboeva, MD and Rubtsov, MA},
title = {Direct ENIT: An easy and reliable tool for gRNA efficacy verification by tracking induced chromosomal translocation.},
journal = {MethodsX},
volume = {7},
number = {},
pages = {101104},
pmid = {33134100},
issn = {2215-0161},
abstract = {CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment.•The approach requires only routinely used enzymes and equipment.•Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification.•High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.},
}
@article {pmid33132845,
year = {2020},
author = {Li, F and Wing, K and Wang, JH and Luu, CD and Bender, JA and Chen, J and Wang, Q and Lu, Q and Nguyen Tran, MT and Young, KM and Wong, RCB and Pébay, A and Cook, AL and Hung, SSC and Liu, GS and Hewitt, AW},
title = {Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing.},
journal = {Frontiers in cellular neuroscience},
volume = {14},
number = {},
pages = {570917},
pmid = {33132845},
issn = {1662-5102},
abstract = {CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.},
}
@article {pmid33132758,
year = {2020},
author = {Miri, SM and Tafsiri, E and Cho, WCS and Ghaemi, A},
title = {Correction to: CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.},
journal = {Cancer cell international},
volume = {20},
number = {},
pages = {521},
pmid = {33132758},
issn = {1475-2867},
abstract = {[This corrects the article DOI: 10.1186/s12935-020-01546-8.].},
}
@article {pmid33131175,
year = {2021},
author = {Chen, Y and Fu, M and Li, H and Wang, L and Liu, R and Liu, Z and Zhang, X and Jin, S},
title = {High-oleic acid content, nontransgenic allotetraploid cotton (Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {19},
number = {3},
pages = {424-426},
pmid = {33131175},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant ; *Gossypium/genetics ; Oleic Acid ; Plants, Genetically Modified/genetics ; },
}
@article {pmid33130469,
year = {2020},
author = {Bu, Q and Zhang, H and Liu, Q and Dai, Y and Wei, Q and Xue, A and Huang, Y and Zhong, K and Huang, Y and Gao, H and Cen, X},
title = {Generation of an NANS homozygous knockout human induced pluripotent stem cell line by the insertion of GFP-P2A-Puro via CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102052},
doi = {10.1016/j.scr.2020.102052},
pmid = {33130469},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Oxo-Acid-Lyases ; },
abstract = {N-acetylneuraminic acid synthase (NANS), the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), is closely associated with infantile-onset severe developmental delay and skeletal dysplasia. However, the role and the involved mechanisms of NANS functioning have not been fully understood to date. Here, we generated a homozygous NANS-knockout human induced pluripotent stem cell (iPSC) line, NCCSEDi001-A-1, via the CRISPR/Cas9-based gene editing method. The NCCSEDi001-A-1 cell line does not express NANS protein, but maintains a normal karyotype, pluripotency, and trilineage differentiation potential.},
}
@article {pmid33130330,
year = {2020},
author = {Kauffman, MR and Nazemidashtarjandi, S and Ghazanfari, D and Allen, AE and Reynolds, NM and Faik, A and Burdick, MM and McCall, KD and Goetz, DJ},
title = {Evidence that knock down of GSK-3β in Chronic Myelogenous Leukemia cells augments IFN-γ-induced apoptosis.},
journal = {Leukemia research},
volume = {99},
number = {},
pages = {106464},
pmid = {33130330},
issn = {1873-5835},
support = {R15 GM110602/GM/NIGMS NIH HHS/United States ; T32 HL076124/HL/NHLBI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; CRISPR-Cas Systems ; Cell Adhesion/drug effects ; Cell Line, Tumor/drug effects ; Codon, Nonsense ; Drug Interactions ; Flow Cytometry ; Frameshift Mutation ; Fusion Proteins, bcr-abl/antagonists &amp; inhibitors ; Gene Knockdown Techniques ; Glycogen Synthase Kinase 3 beta/*antagonists &amp; inhibitors/genetics ; Humans ; Interferon-gamma/*pharmacology/therapeutic use ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/enzymology/pathology ; Male ; Neoplasm Proteins/*antagonists &amp; inhibitors ; Protein Kinase Inhibitors/*pharmacology ; RNA, Guide/genetics/pharmacology ; Spectrometry, Fluorescence ; },
abstract = {The role of interferon-gamma (IFN-γ) in Chronic Myelogenous/Myeloid Leukemia (CML) and in the treatment of CML remains unclear; specifically, the effect of IFN-γ on apoptosis. There is reported interplay between IFN-γ and glycogen synthase kinase-3 (GSK-3), a kinase which has been implicated in both cell death and, conversely, cell survival. Thus, we utilized the CML-derived HAP1 cell line and a mutant HAP1 GSK-3β knocked-down cell line (GSK-3β 31bp) to investigate whether GSK-3 modulates IFN-γ's action on CML cells. Significantly less GSK-3β 31bp cells, relative to HAP1 cells, were present after 48 h treatment with IFN-γ. IFN-γ treatment significantly decreased GSK-3β 31bp substrate adhesiveness (relative to HAP1 cells); an observation often correlated with cell death. Fluorescence microscopy revealed that IFN-γ induces a modest level of apoptosis in the HAP1 cells and that IFN-γ induced apoptosis is significantly enhanced in GSK-3β 31bp cells. Utilizing a complementary GSK-3β knocked-down cell line (8bp) we found, via flow cytometric analysis, that IFN-γ induced apoptosis is significantly enhanced in GSK-3β 8bp cells relative to HAP1 cells. Combined, our findings suggest that IFN-γ induces apoptosis of CML cells and that loss of GSK-3β significantly augments IFN-γ-induced apoptosis.},
}
@article {pmid33128956,
year = {2020},
author = {Fulgencio-Covián, A and Álvarez, M and Pepers, BA and López-Márquez, A and Ugarte, M and Pérez, B and van Roon-Mom, WMC and Desviat, LR and Richard, E},
title = {Generation of a gene-corrected human isogenic line (UAMi006-A) from propionic acidemia patient iPSC with an homozygous mutation in the PCCB gene using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102055},
doi = {10.1016/j.scr.2020.102055},
pmid = {33128956},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Methylmalonyl-CoA Decarboxylase/genetics ; Mutation/genetics ; *Propionic Acidemia/genetics ; Technology ; },
abstract = {Propionic acidemia (PA) is an inherited metabolic disease caused by mutations in the PCCA and PCCB genes. We have previously generated an induced pluripotent stem cell (iPSC) line (UAMi004-A) from a PA patient with the c.1218_1231del14ins12 (p.Gly407Argfs*14) homozygous mutation in the PCCB gene. Here, we report the generation of the isogenic control in which the mutation was genetically corrected using CRISPR/Cas9 technology. Off-target editing presence was excluded and the iPSCs had typical embryonic stem cell-like morphology and normal karyotype that expressed pluripotency markers and maintained their in vitro differentiation potential.},
}
@article {pmid33128952,
year = {2020},
author = {Tang, X and Chen, Z and Tan, X and Luo, L and Liu, X and Gong, L and Li, DW and Liu, Y},
title = {Generation of a homozygous CRISPR/Cas9-mediated knockout H9 hESC subline for the CRB1 locus.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102057},
doi = {10.1016/j.scr.2020.102057},
pmid = {33128952},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Eye Proteins/genetics ; *Human Embryonic Stem Cells/metabolism ; Humans ; Membrane Proteins/genetics/metabolism ; Mutation ; Nerve Tissue Proteins/metabolism ; },
abstract = {Mutations in the CRB1 gene reportedly cause early-onset autosomal recessive retinitis pigmentosa (RP), which can result in severe loss of vision at an early age. To investigate the mechanism of CRB1-knockout (CRB1[-/-]) induced RP, we generated a subline of H9 human embryonic stem cells harboring frame shift mutations in a homozygous state in exon 2 of the CRB1 gene. This subline expressed pluripotent stem cell markers, presented a normal karyotype, and preserved the ability to differentiate into endodermal, mesodermal, and ectodermal lineages.},
}
@article {pmid33128871,
year = {2020},
author = {Takahashi, N and Cho, P and Selfors, LM and Kuiken, HJ and Kaul, R and Fujiwara, T and Harris, IS and Zhang, T and Gygi, SP and Brugge, JS},
title = {3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis.},
journal = {Molecular cell},
volume = {80},
number = {5},
pages = {828-844.e6},
pmid = {33128871},
issn = {1097-4164},
support = {R01 GM067945/GM/NIGMS NIH HHS/United States ; },
mesh = {A549 Cells ; *CRISPR-Cas Systems ; *Cell Culture Techniques ; *Cell Proliferation ; *Ferroptosis ; Humans ; NF-E2-Related Factor 2/genetics/*metabolism ; Neoplasm Proteins/genetics/*metabolism ; Neoplasms/genetics/*metabolism/pathology ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics/metabolism ; Spheroids, Cellular/*metabolism/pathology ; TOR Serine-Threonine Kinases/genetics/metabolism ; },
abstract = {Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.},
}
@article {pmid33128663,
year = {2021},
author = {Yang, Q and Wu, M and Zhu, YL and Yang, YQ and Mei, YZ and Dai, CC},
title = {The disruption of the MAPKK gene triggering the synthesis of flavonoids in endophytic fungus Phomopsis liquidambaris.},
journal = {Biotechnology letters},
volume = {43},
number = {1},
pages = {119-132},
pmid = {33128663},
issn = {1573-6776},
mesh = {Batch Cell Culture Techniques ; CRISPR-Cas Systems ; Culture Media/chemistry/metabolism ; Fermentation ; Flavonoids/analysis/*biosynthesis/genetics ; Fungal Proteins/*genetics/metabolism ; Gene Editing ; Glucose/metabolism ; Mitogen-Activated Protein Kinase Kinases/*genetics/metabolism ; *Phomopsis/genetics/metabolism ; Xylose/metabolism ; },
abstract = {Flavonoids, which are mainly extracted from plants, are important antioxidants and play an important role in human diseases. However, the growing market demand is limited by low productivity and complex production processes. Herein, the flavonoids biosynthesis pathway of the endophytic fungus Phomopsis liquidambaris was revealed. The mitogen-activated protein kinase kinase (MAPKK) of the strain was disrupted using a newly constructed CRISPR-Cas9 system mediated by two gRNAs which was conducive to cause plasmid loss. The disruption of the MAPKK gene triggered the biosynthesis of flavonoids against stress and resulted in the precipitation of flavonoids from fermentation broth. Naringenin, kaempferol and quercetin were detected in fed-batch fermentation with yields of 5.65 mg/L, 1.96 mg/L and 2.37 mg/L from P. liquidambaris for dry cell weigh using the mixture of glucose and xylose and corn steep powder as carbon source and nitrogen source for 72 h, respectively. The biosynthesis of flavonoids was triggered by disruption of MAPKK gene in P. liquidambaris and the mutant could utilize xylose.},
}
@article {pmid33127914,
year = {2020},
author = {Liu, Y and Huang, W and Cai, Z},
title = {Synthesizing AND gate minigene circuits based on CRISPReader for identification of bladder cancer cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5486},
pmid = {33127914},
issn = {2041-1723},
mesh = {Animals ; Apoptosis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epithelial Cells ; Female ; Gene Editing/methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks/*genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Synthetic Biology ; Urinary Bladder ; Urinary Bladder Neoplasms/*diagnosis/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {The logical AND gate gene circuit based on the CRISPR-Cas9 system can distinguish bladder cancer cells from normal bladder epithelial cells. However, the layered artificial gene circuits have the problems of high complexity, difficulty in accurately predicting the behavior, and excessive redundancy, which cannot be applied to clinical translation. Here, we construct minigene circuits based on the CRISPReader, a technology used to control promoter-less gene expression in a robust manner. The minigene circuits significantly induce robust gene expression output in bladder cancer cells, but have nearly undetectable gene expression in normal bladder epithelial cells. The minigene circuits show a higher capability for cancer identification and intervention when compared with traditional gene circuits, and could be used for in vivo cancer gene therapy using the all-in-one AAV vector. This approach expands the design ideas and concepts of gene circuits in medical synthetic biology.},
}
@article {pmid33127524,
year = {2021},
author = {Gao, J and Wu, L and Yang, D and Gong, W and Wang, J},
title = {A One-Pot CRISPR/Cas9-Typing PCR for DNA Detection and Genotyping.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {23},
number = {1},
pages = {46-60},
doi = {10.1016/j.jmoldx.2020.10.004},
pmid = {33127524},
issn = {1943-7811},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Viral/*genetics ; Gene Editing/methods ; Genotype ; Genotyping Techniques/*methods ; HeLa Cells ; Human papillomavirus 16/*genetics ; Human papillomavirus 18/*genetics ; Humans ; Oligonucleotides/genetics ; Papillomavirus Infections/*diagnosis/pathology/virology ; Polymerase Chain Reaction/*methods ; RNA, Guide/genetics ; Sensitivity and Specificity ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) has high specificity to its target DNA as a gene editing tool. This characteristic makes it useful for DNA detection. Combining the advantages of CRISPR/Cas9 and PCR, this study establishes a novel CRISPR/Cas9-based DNA detection method, named CRISPR/Cas9-typing PCR version 4.0 (ctPCR4.0). This method can detect target DNA in one pot with high specificity and sensitivity. In a homogenous reaction, the target DNA is first cleaved by a pair of Cas9- single-guide RNA complexes and thus releases two single strands with free 3' ends, allowing a pair of oligonucleotides to anneal with the strands. The annealed oligonucleotides provide templates for DNA polymerization from the free 3' ends. A universal primer annealing site is thus produced at the end of two single strands. The target DNA is then amplified by PCR using a universal primer. This method was first verified by accurately detecting the cloned L1 fragments of 10 genotypes of high-risk human papilloma viruses (HPVs). This method was then validated by detecting the L1 fragments of two highest-risk HPVs, HPV 16 and HPV 18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, this method was further validated by accurately detecting 10 high-risk HPVs in 30 clinical samples.},
}
@article {pmid33126728,
year = {2020},
author = {Ophinni, Y and Miki, S and Hayashi, Y and Kameoka, M},
title = {Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape.},
journal = {Viruses},
volume = {12},
number = {11},
pages = {},
pmid = {33126728},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Genetic Vectors ; Genome, Viral ; HIV-1/*immunology/physiology ; Humans ; Lentivirus/genetics ; Mutation ; Proviruses/genetics ; RNA, Guide/*genetics ; T-Lymphocytes/immunology/*virology ; Virus Replication/genetics ; tat Gene Products, Human Immunodeficiency Virus/antagonists &amp; inhibitors/*genetics ; },
abstract = {HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 tat and rev using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1NL4-3 challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three tat-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed tat gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting tat and rev. A single-vector tat-targeting construct shows non-inferiority to the tat-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in tat are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of tat further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.},
}
@article {pmid33126177,
year = {2021},
author = {Wang, R and Qian, C and Pang, Y and Li, M and Yang, Y and Ma, H and Zhao, M and Qian, F and Yu, H and Liu, Z and Ni, T and Zheng, Y and Wang, Y},
title = {opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {172},
number = {},
pages = {112766},
pmid = {33126177},
issn = {1873-4235},
mesh = {Base Sequence ; COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/instrumentation/*methods/statistics &amp; numerical data ; *CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/instrumentation/*methods/statistics &amp; numerical data ; Nucleic Acid Amplification Techniques/instrumentation/*methods/statistics &amp; numerical data ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2/*genetics/isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.},
}
@article {pmid33125875,
year = {2020},
author = {Gupta, MK and Vethe, H and Softic, S and Rao, TN and Wagh, V and Shirakawa, J and Barsnes, H and Vaudel, M and Takatani, T and Kahraman, S and Sakaguchi, M and Martinez, R and Hu, J and Bjørlykke, Y and Raeder, H and Kulkarni, RN},
title = {Leptin Receptor Signaling Regulates Protein Synthesis Pathways and Neuronal Differentiation in Pluripotent Stem Cells.},
journal = {Stem cell reports},
volume = {15},
number = {5},
pages = {1067-1079},
pmid = {33125875},
issn = {2213-6711},
support = {P30 DK040561/DK/NIDDK NIH HHS/United States ; P30 DK036836/DK/NIDDK NIH HHS/United States ; K12 HD000850/HD/NICHD NIH HHS/United States ; R01 DK067536/DK/NIDDK NIH HHS/United States ; R01 DK103215/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage ; Eukaryotic Initiation Factor-4E/genetics/*metabolism ; Fibroblasts/metabolism ; Gene Editing ; Gene Expression Regulation, Developmental ; Induced Pluripotent Stem Cells/*metabolism ; Metabolome ; Mice ; Mice, Knockout ; Neurogenesis ; *Protein Biosynthesis ; Proteins ; Proteomics ; Receptors, Leptin/genetics/*metabolism ; STAT3 Transcription Factor/*metabolism ; *Signal Transduction ; },
abstract = {The role of leptin receptor (OB-R) signaling in linking pluripotency with growth and development and the consequences of dysfunctional leptin signaling on progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying the db/db mutation exhibit metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) show altered expression of proteins involved in embryonic development. An upregulation in expression of eukaryotic translation initiation factor 4e (Eif4e) and Stat3 binding to the Eif4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs toward the neuronal lineage showed defects. Gene editing to correct the point mutation in db/db iPSCs using CRISPR-Cas9, restored expression of neuronal markers and protein synthesis while reversing the metabolic defects. These data imply a direct role for OB-R in regulating metabolism in embryonic fibroblasts and key developmental pathways in iPSCs.},
}
@article {pmid33125070,
year = {2021},
author = {Wu, Q and Shou, J},
title = {Toward precise CRISPR DNA fragment editing and predictable 3D genome engineering.},
journal = {Journal of molecular cell biology},
volume = {12},
number = {11},
pages = {828-856},
pmid = {33125070},
issn = {1759-4685},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; *Gene Editing ; *Genetic Engineering ; *Genome ; Humans ; *Imaging, Three-Dimensional ; },
abstract = {Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s, the reverse genetic approach of precise editing of any genomic locus has greatly accelerated biomedical research and biotechnology development. In particular, the recent development of the CRISPR/Cas9 system has greatly expedited genetic dissection of 3D genomes. CRISPR gene-editing outcomes result from targeted genome cleavage by ectopic bacterial Cas9 nuclease followed by presumed random ligations via the host double-strand break repair machineries. Recent studies revealed, however, that the CRISPR genome-editing system is precise and predictable because of cohesive Cas9 cleavage of targeting DNA. Here, we synthesize the current understanding of CRISPR DNA fragment-editing mechanisms and recent progress in predictable outcomes from precise genetic engineering of 3D genomes. Specifically, we first briefly describe historical genetic studies leading to CRISPR and 3D genome engineering. We then summarize different types of chromosomal rearrangements by DNA fragment editing. Finally, we review significant progress from precise 1D gene editing toward predictable 3D genome engineering and synthetic biology. The exciting and rapid advances in this emerging field provide new opportunities and challenges to understand or digest 3D genomes.},
}
@article {pmid33125053,
year = {2021},
author = {Zhang, L and Jiang, X and Pfau, D and Ling, Y and Nathan, CF},
title = {Type I interferon signaling mediates Mycobacterium tuberculosis-induced macrophage death.},
journal = {The Journal of experimental medicine},
volume = {218},
number = {2},
pages = {},
pmid = {33125053},
issn = {1540-9538},
support = {R01 AI138940/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Autocrine Communication/drug effects/physiology ; CRISPR-Cas Systems/drug effects/physiology ; Cell Death/drug effects/*physiology ; Cell Line ; HEK293 Cells ; Humans ; Interferon Type I/*metabolism ; Macrophages/drug effects/*metabolism ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis/drug effects ; Paracrine Communication/drug effects/physiology ; RAW 264.7 Cells ; Rifampin/pharmacology ; Signal Transduction/drug effects/*physiology ; Tuberculosis/drug therapy/*metabolism/microbiology ; },
abstract = {Macrophages help defend the host against Mycobacterium tuberculosis (Mtb), the major cause of tuberculosis (TB). Once phagocytized, Mtb resists killing by macrophages, replicates inside them, and leads to their death, releasing Mtb that can infect other cells. We found that the death of Mtb-infected mouse macrophages in vitro does not appear to proceed by a currently known pathway. Through genome-wide CRISPR-Cas9 screening, we identified a critical role for autocrine or paracrine signaling by macrophage-derived type I IFNs in the death of Mtb-infected macrophages in vitro, and blockade of type I IFN signaling augmented the effect of rifampin, a first-line TB drug, in Mtb-infected mice. Further definition of the pathway of type I IFN-mediated macrophage death may allow for host-directed therapy of TB that is more selective than systemic blockade of type I IFN signaling.},
}
@article {pmid33124980,
year = {2020},
author = {Stringer, AM and Baniulyte, G and Lasek-Nesselquist, E and Seed, KD and Wade, JT},
title = {Transcription termination and antitermination of bacterial CRISPR arrays.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33124980},
issn = {2050-084X},
support = {R01 GM122836/GM/NIGMS NIH HHS/United States ; R21 AI126416/AI/NIAID NIH HHS/United States ; R01 AI127652/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteria/*genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Codon, Terminator/genetics ; RNA, Bacterial/genetics/metabolism ; *Transcription Termination, Genetic ; },
abstract = {A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences ('repeats') interspersed with short, variable sequences ('spacers'). CRISPR arrays are transcribed and processed into individual CRISPR RNAs that each include a single spacer, and direct Cas proteins to complementary sequences in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho can prematurely terminate transcription of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of transcription termination and antitermination in the evolution of bacterial CRISPR-Cas systems.},
}
@article {pmid33123984,
year = {2020},
author = {Yang, LQ and Chen, M and Ren, DL and Hu, B},
title = {Dual Oxidase Mutant Retards Mauthner-Cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish.},
journal = {Neuroscience bulletin},
volume = {36},
number = {12},
pages = {1500-1512},
pmid = {33123984},
issn = {1995-8218},
mesh = {Animals ; Axons/*physiology ; CRISPR-Cas Systems ; Dual Oxidases/*genetics ; *Mitochondrial Dynamics ; *Nerve Regeneration ; Transcriptome ; *Zebrafish ; },
abstract = {Dual oxidase (duox)-derived reactive oxygen species (ROS) have been correlated with neuronal polarity, cerebellar development, and neuroplasticity. However, there have not been many comprehensive studies of the effect of individual duox isoforms on central-axon regeneration in vivo. Here, we explored this question in zebrafish, an excellent model organism for central-axon regeneration studies. In our research, mutation of the duox gene with CRISPR/Cas9 significantly retarded the single-axon regeneration of the zebrafish Mauthner cell in vivo. Using deep transcriptome sequencing, we found that the expression levels of related functional enzymes in mitochondria were down-regulated in duox mutant fish. In vivo imaging showed that duox mutants had significantly disrupted mitochondrial transport and redox state in single Mauthner-cell axon. Our research data provide insights into how duox is involved in central-axon regeneration by changing mitochondrial transport.},
}
@article {pmid33123803,
year = {2020},
author = {Khalil, AM},
title = {The genome editing revolution: review.},
journal = {Journal, genetic engineering &amp; biotechnology},
volume = {18},
number = {1},
pages = {68},
pmid = {33123803},
issn = {2090-5920},
abstract = {BACKGROUND: Development of efficient strategies has always been one of the great perspectives for biotechnologists. During the last decade, genome editing of different organisms has been a fast advancing field and therefore has received a lot of attention from various researchers comprehensively reviewing latest achievements and offering opinions on future directions. This review presents a brief history, basic principles, advantages and disadvantages, as well as various aspects of each genome editing technology including the modes, applications, and challenges that face delivery of gene editing components.<br><br>MAIN BODY: Genetic modification techniques cover a wide range of studies, including the generation of transgenic animals, functional analysis of genes, model development for diseases, or drug development. The delivery of certain proteins such as monoclonal antibodies, enzymes, and growth hormones has been suffering from several obstacles because of their large size. These difficulties encouraged scientists to explore alternative approaches, leading to the progress in gene editing. The distinguished efforts and enormous experimentation have now been able to introduce methodologies that can change the genetic constitution of the living cell. The genome editing strategies have evolved during the last three decades, and nowadays, four types of "programmable" nucleases are available in this field: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system. Each group has its own characteristics necessary for researchers to select the most suitable method for gene editing tool for a range of applications. Genome engineering/editing technology will revolutionize the creation of precisely manipulated genomes of cells or organisms in order to modify a specific characteristic. Of the potential applications are those in human health and agriculture. Introducing constructs into target cells or organisms is the key step in genome engineering.<br><br>CONCLUSIONS: Despite the success already achieved, the genome editing techniques are still suffering certain difficulties. Challenges must be overcome before the full potential of genome editing can be realized.},
}
@article {pmid33123724,
year = {2021},
author = {Zhao, J and Inomata, R and Kato, Y and Miyagishi, M},
title = {Development of aptamer-based inhibitors for CRISPR/Cas system.},
journal = {Nucleic acids research},
volume = {49},
number = {3},
pages = {1330-1344},
pmid = {33123724},
issn = {1362-4962},
mesh = {*Aptamers, Nucleotide/chemistry ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; *Gene Editing ; HEK293 Cells ; Humans ; Oligonucleotides ; RNA/antagonists &amp; inhibitors ; SELEX Aptamer Technique ; Transcriptional Activation ; },
abstract = {The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.},
}
@article {pmid33123486,
year = {2020},
author = {Zhang, Y and Zhao, G and Ahmed, FYH and Yi, T and Hu, S and Cai, T and Liao, Q},
title = {In silico Method in CRISPR/Cas System: An Expedite and Powerful Booster.},
journal = {Frontiers in oncology},
volume = {10},
number = {},
pages = {584404},
pmid = {33123486},
issn = {2234-943X},
abstract = {The CRISPR/Cas system has stood in the center of attention in the last few years as a revolutionary gene editing tool with a wide application to investigate gene functions. However, the labor-intensive workflow requires a sophisticated pre-experimental and post-experimental analysis, thus becoming one of the hindrances for the further popularization of practical applications. Recently, the increasing emergence and advancement of the in silico methods play a formidable role to support and boost experimental work. However, various tools based on distinctive design principles and frameworks harbor unique characteristics that are likely to confuse users about how to choose the most appropriate one for their purpose. In this review, we will present a comprehensive overview and comparisons on the in silico methods from the aspects of CRISPR/Cas system identification, guide RNA design, and post-experimental assistance. Furthermore, we establish the hypotheses in light of the new trends around the technical optimization and hope to provide significant clues for future tools development.},
}
@article {pmid33122768,
year = {2020},
author = {Aumann, RA and Häcker, I and Schetelig, MF},
title = {Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18611},
pmid = {33122768},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Ceratitis capitata/*genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drosophila Proteins/*genetics ; Drosophila melanogaster/genetics/physiology ; Female ; Infertility, Male/genetics ; Male ; Pest Control, Biological/methods ; Point Mutation/*genetics ; Reproduction/genetics ; Ribonucleoproteins/*genetics ; Sex Determination Processes/*genetics ; },
abstract = {The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2[ts]) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2[ts] SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2[ts2] mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2[ts2] homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.},
}
@article {pmid33122441,
year = {2020},
author = {Holmes, DL and Vogt, DT and Lagunoff, M},
title = {A CRISPR-Cas9 screen identifies mitochondrial translation as an essential process in latent KSHV infection of human endothelial cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {28384-28392},
pmid = {33122441},
issn = {1091-6490},
support = {R01 CA189986/CA/NCI NIH HHS/United States ; R01 CA217788/CA/NCI NIH HHS/United States ; R21 CA240479/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Herpesviridae Infections/*genetics ; Herpesvirus 8, Human/*genetics/physiology ; Humans ; Lymphoma, Primary Effusion/genetics ; Mitochondria/*metabolism ; Sarcoma, Kaposi ; Virus Latency/*genetics ; Virus Replication ; },
abstract = {Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.},
}
@article {pmid33122438,
year = {2020},
author = {Westra, ER and Levin, BR},
title = {It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {27777-27785},
pmid = {33122438},
issn = {1091-6490},
support = {R01 GM091875/GM/NIGMS NIH HHS/United States ; R35 GM136407/GM/NIGMS NIH HHS/United States ; BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Archaea/genetics ; Bacteria/*genetics ; Bacteriophages/genetics ; CRISPR-Cas Systems/*physiology ; Computer Simulation ; Evolution, Molecular ; Gene Transfer, Horizontal ; Interspersed Repetitive Sequences ; Models, Theoretical ; Plasmids/*genetics ; Viruses/genetics ; },
abstract = {Articles on CRISPR commonly open with some variant of the phrase "these short palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other mobile genetic elements." There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro. Not at all clear are the ubiquity, magnitude, and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical-computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea, and the mobile genetic elements that infect them.},
}
@article {pmid33122427,
year = {2020},
author = {Esk, C and Lindenhofer, D and Haendeler, S and Wester, RA and Pflug, F and Schroeder, B and Bagley, JA and Elling, U and Zuber, J and von Haeseler, A and Knoblich, JA},
title = {A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6519},
pages = {935-941},
doi = {10.1126/science.abb5390},
pmid = {33122427},
issn = {1095-9203},
mesh = {Brain/*growth &amp; development/metabolism ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*physiology ; Cell Line ; Cell Lineage ; Endoplasmic Reticulum/*metabolism ; Extracellular Matrix Proteins/*metabolism ; Gene Knockout Techniques ; Genetic Testing/*methods ; Humans ; Membrane Proteins/genetics/*physiology ; Microcephaly/*genetics ; Organ Size ; Organoids/growth &amp; development/metabolism ; },
abstract = {Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized IER3IP1, which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.},
}
@article {pmid33122116,
year = {2020},
author = {van Rhijn, N and Furukawa, T and Zhao, C and McCann, BL and Bignell, E and Bromley, MJ},
title = {Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {145},
number = {},
pages = {103479},
pmid = {33122116},
issn = {1096-0937},
support = {208396/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 1640253/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Wellcome Trust/United Kingdom ; MR/M02010X/1/MRC_/Medical Research Council/United Kingdom ; MR/N006364/2/MRC_/Medical Research Council/United Kingdom ; MR/S001824/1/MRC_/Medical Research Council/United Kingdom ; R01 AI143198/AI/NIAID NIH HHS/United States ; },
mesh = {Aspergillus fumigatus/*genetics/pathogenicity ; CRISPR-Cas Systems/genetics ; Epitopes/*genetics ; Fungal Proteins/genetics ; Gene Editing/trends ; Humans ; *Mutagenesis, Site-Directed ; Mycoses/*genetics/microbiology ; },
abstract = {Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.},
}
@article {pmid33122083,
year = {2021},
author = {Wang, X and Hayes, JE and Xu, X and Gao, X and Mehta, D and Lilja, HG and Klein, RJ},
title = {Validation of prostate cancer risk variants rs10993994 and rs7098889 by CRISPR/Cas9 mediated genome editing.},
journal = {Gene},
volume = {768},
number = {},
pages = {145265},
pmid = {33122083},
issn = {1879-0038},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA092629/CA/NCI NIH HHS/United States ; R01 CA175491/CA/NCI NIH HHS/United States ; U01 HG007033/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Deletion ; Gene Editing/methods ; Genetic Predisposition to Disease/*genetics ; *Genome-Wide Association Study ; Histone Code/genetics ; Humans ; Linkage Disequilibrium/genetics ; Male ; Nuclear Receptor Coactivators/*biosynthesis/genetics ; Polymorphism, Single Nucleotide/genetics ; Promoter Regions, Genetic/genetics ; Prostatic Neoplasms/*genetics ; Prostatic Secretory Proteins/*genetics ; RNA, Messenger/biosynthesis ; },
abstract = {GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the β-microseminoprotein (MSMB) promoter region, mediates MSMB prostate secretion levels, and is linked to mRNA expression changes in both MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in positive linkage disequilibrium with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation studies to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available. AUTHOR SUMMARY: In pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in the human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and cell-type specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded β-MSP in prostate cancer pathogenesis.},
}
@article {pmid33121685,
year = {2021},
author = {Bandara, TAMK and Otsuka, K and Matsubara, S and Shiraishi, A and Satake, H and Kimura, AP},
title = {A dual enhancer-silencer element, DES-K16, in mouse spermatocyte-derived GC-2spd(ts) cells.},
journal = {Biochemical and biophysical research communications},
volume = {534},
number = {},
pages = {1007-1012},
doi = {10.1016/j.bbrc.2020.10.049},
pmid = {33121685},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Histone Code ; Male ; Mice/*genetics ; Mice, Inbred C57BL ; *Silencer Elements, Transcriptional ; Spermatocytes/*metabolism ; },
abstract = {The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16, respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type.},
}
@article {pmid33121109,
year = {2020},
author = {Dolskiy, AA and Grishchenko, IV and Yudkin, DV},
title = {Cell Cultures for Virology: Usability, Advantages, and Prospects.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33121109},
issn = {1422-0067},
mesh = {Animals ; Cell Culture Techniques/*methods ; Genes, Reporter ; Genetic Engineering ; Humans ; Synthetic Biology/methods ; Virology/methods ; Viruses/*growth &amp; development ; },
abstract = {Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.},
}
@article {pmid33121045,
year = {2020},
author = {Zamai, L},
title = {Unveiling Human Non-Random Genome Editing Mechanisms Activated in Response to Chronic Environmental Changes: I. Where Might These Mechanisms Come from and What Might They Have Led To?.},
journal = {Cells},
volume = {9},
number = {11},
pages = {},
pmid = {33121045},
issn = {2073-4409},
mesh = {APOBEC Deaminases/metabolism ; CRISPR-Cas Systems/genetics ; *Environment ; *Gene Editing ; *Genome, Human ; Humans ; Mutation/genetics ; },
abstract = {This article challenges the notion of the randomness of mutations in eukaryotic cells by unveiling stress-induced human non-random genome editing mechanisms. To account for the existence of such mechanisms, I have developed molecular concepts of the cell environment and cell environmental stressors and, making use of a large quantity of published data, hypothesised the origin of some crucial biological leaps along the evolutionary path of life on Earth under the pressure of natural selection, in particular, (1) virus-cell mating as a primordial form of sexual recombination and symbiosis; (2) Lamarckian CRISPR-Cas systems; (3) eukaryotic gene development; (4) antiviral activity of retrotransposon-guided mutagenic enzymes; and finally, (5) the exaptation of antiviral mutagenic mechanisms to stress-induced genome editing mechanisms directed at "hyper-transcribed" endogenous genes. Genes transcribed at their maximum rate (hyper-transcribed), yet still unable to meet new chronic environmental demands generated by "pollution", are inadequate and generate more and more intronic retrotransposon transcripts. In this scenario, RNA-guided mutagenic enzymes (e.g., Apolipoprotein B mRNA editing catalytic polypeptide-like enzymes, APOBECs), which have been shown to bind to retrotransposon RNA-repetitive sequences, would be surgically targeted by intronic retrotransposons on opened chromatin regions of the same "hyper-transcribed" genes. RNA-guided mutagenic enzymes may therefore "Lamarkianly" generate single nucleotide polymorphisms (SNP) and gene copy number variations (CNV), as well as transposon transposition and chromosomal translocations in the restricted areas of hyper-functional and inadequate genes, leaving intact the rest of the genome. CNV and SNP of hyper-transcribed genes may allow cells to surgically explore a new fitness scenario, which increases their adaptability to stressful environmental conditions. Like the mechanisms of immunoglobulin somatic hypermutation, non-random genome editing mechanisms may generate several cell mutants, and those codifying for the most environmentally adequate proteins would have a survival advantage and would therefore be Darwinianly selected. Non-random genome editing mechanisms represent tools of evolvability leading to organismal adaptation including transgenerational non-Mendelian gene transmission or to death of environmentally inadequate genomes. They are a link between environmental changes and biological novelty and plasticity, finally providing a molecular basis to reconcile gene-centred and "ecological" views of evolution.},
}
@article {pmid33117361,
year = {2020},
author = {Pavlovic, K and Tristán-Manzano, M and Maldonado-Pérez, N and Cortijo-Gutierrez, M and Sánchez-Hernández, S and Justicia-Lirio, P and Carmona, MD and Herrera, C and Martin, F and Benabdellah, K},
title = {Using Gene Editing Approaches to Fine-Tune the Immune System.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {570672},
pmid = {33117361},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Cancer Vaccines/*immunology ; Gene Editing/*methods ; Genetic Therapy ; Graft Rejection/*immunology ; Graft vs Host Disease/*therapy ; Humans ; Immunotherapy, Adoptive/*methods ; Neoplasms/*therapy ; Receptors, Chimeric Antigen/*genetics ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases/metabolism ; },
abstract = {Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.},
}
@article {pmid33117331,
year = {2020},
author = {Azangou-Khyavy, M and Ghasemi, M and Khanali, J and Boroomand-Saboor, M and Jamalkhah, M and Soleimani, M and Kiani, J},
title = {CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {2062},
pmid = {33117331},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Genome-Wide Association Study ; Humans ; Immune Checkpoint Inhibitors/*therapeutic use ; Immunotherapy, Adoptive/*methods ; Neoplasms/genetics/*therapy ; Oncolytic Virotherapy ; T-Lymphocytes/*immunology/transplantation ; },
abstract = {The clustered regularly interspaced short palindromic repeats system has demonstrated considerable advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Given that cancer is caused by an excessive accumulation of mutations that lead to the activation of oncogenes and inactivation of tumor suppressor genes, the CRISPR/Cas9 system is a therapy of choice for tumor genome editing and treatment. In defining its superior use, we have reviewed the novel applications of the CRISPR genome editing tool in discovering, sorting, and prioritizing targets for subsequent interventions, and passing different hurdles of cancer treatment such as epigenetic alterations and drug resistance. Moreover, we have reviewed the breakthroughs precipitated by the CRISPR system in the field of cancer immunotherapy, such as identification of immune system-tumor interplay, production of universal Chimeric Antigen Receptor T cells, inhibition of immune checkpoint inhibitors, and Oncolytic Virotherapy. The existing challenges and limitations, as well as the prospects of CRISPR based systems, are also discussed.},
}
@article {pmid33116147,
year = {2020},
author = {Schmidt, JK and Strelchenko, N and Park, MA and Kim, YH and Mean, KD and Schotzko, ML and Kang, HJ and Golos, TG and Slukvin, II},
title = {Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18457},
pmid = {33116147},
issn = {2045-2322},
support = {P51 OD011106/OD/NIH HHS/United States ; R24 OD021322/OD/NIH HHS/United States ; R24OD021322/NH/NIH HHS/United States ; HD099154-01/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *Animals, Genetically Modified/embryology/genetics ; *CRISPR-Cas Systems ; Embryo, Mammalian/*metabolism ; *Gene Editing ; Macaca fascicularis ; *Receptors, CCR5/genetics/metabolism ; },
abstract = {The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23-37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.},
}
@article {pmid33115806,
year = {2020},
author = {Liu, Y and Guo, B and Aguilera-Jimenez, E and Chu, VS and Zhou, J and Wu, Z and Francis, JM and Yang, X and Choi, PS and Bailey, SD and Zhang, X},
title = {Chromatin Looping Shapes KLF5-Dependent Transcriptional Programs in Human Epithelial Cancers.},
journal = {Cancer research},
volume = {80},
number = {24},
pages = {5464-5477},
pmid = {33115806},
issn = {1538-7445},
support = {R00 CA215244/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Lineage/genetics ; Cell Proliferation/genetics ; Chromatin/*metabolism ; Epithelial Cells/*metabolism ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Humans ; Kruppel-Like Transcription Factors/genetics/*metabolism ; Neoplasms, Glandular and Epithelial/*metabolism/pathology ; Phenotype ; Promoter Regions, Genetic ; Protein Binding/genetics ; Transcription, Genetic/*genetics ; },
abstract = {Activation of transcription factors is a key driver event in cancer. We and others have recently reported that the Krüppel-like transcription factor KLF5 is activated in multiple epithelial cancer types including squamous cancer and gastrointestinal adenocarcinoma, yet the functional consequences and the underlying mechanisms of this activation remain largely unknown. Here we demonstrate that activation of KLF5 results in strongly selective KLF5 dependency for these cancer types. KLF5 bound lineage-specific regulatory elements and activated gene expression programs essential to cancer cells. HiChIP analysis revealed that multiple distal KLF5 binding events cluster and synergize to activate individual target genes. Immunoprecipitation-mass spectrometry assays showed that KLF5 interacts with other transcription factors such as TP63 and YAP1, as well as the CBP/EP300 acetyltransferase complex. Furthermore, KLF5 guided the CBP/EP300 complex to increase acetylation of H3K27, which in turn enhanced recruitment of the bromodomain protein BRD4 to chromatin. The 3D chromatin architecture aggregated KLF5-dependent BRD4 binding to activate polymerase II elongation at KLF5 target genes, which conferred a transcriptional vulnerability to proteolysis-targeting chimera-induced degradation of BRD4. Our study demonstrates that KLF5 plays an essential role in multiple epithelial cancers by activating cancer-related genes through 3D chromatin loops, providing an evidence-based rationale for targeting the KLF5 pathway. SIGNIFICANCE: An integrative 3D genomics methodology delineates mechanisms underlying the function of KLF5 in multiple epithelial cancers and suggests potential strategies to target cancers with aberrantly activated KLF5.},
}
@article {pmid33114405,
year = {2020},
author = {Dahlhaus, M and Roos, J and Engel, D and Tews, D and Halbgebauer, D and Funcke, JB and Kiener, S and Schuler, PJ and Döscher, J and Hoffmann, TK and Zinngrebe, J and Rojewski, M and Schrezenmeier, H and Debatin, KM and Wabitsch, M and Fischer-Posovszky, P},
title = {CD90 Is Dispensable for White and Beige/Brown Adipocyte Differentiation.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33114405},
issn = {1422-0067},
mesh = {Adipose Tissue, Beige/*cytology/metabolism ; Adipose Tissue, Brown/*cytology/metabolism ; Adipose Tissue, White/cytology/metabolism ; Adult ; Arrhythmias, Cardiac/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Female ; Flow Cytometry ; Gene Knockout Techniques ; Genetic Diseases, X-Linked/*genetics/metabolism ; Gigantism/*genetics/metabolism ; Heart Defects, Congenital/*genetics/metabolism ; Humans ; Intellectual Disability/*genetics/metabolism ; Male ; Middle Aged ; Stromal Cells/metabolism ; Thermogenesis ; Thy-1 Antigens/*genetics/*metabolism ; Up-Regulation ; },
abstract = {Brown adipose tissue (BAT) is a thermogenic organ in rodents and humans. In mice, the transplantation of BAT has been successfully used to combat obesity and its comorbidities. While such beneficial properties of BAT are now evident, the developmental and cellular origins of brown, beige, and white adipocytes have remained only poorly understood, especially in humans. We recently discovered that CD90 is highly expressed in stromal cells isolated from human white adipose tissue (WAT) compared to BAT. Here, we studied whether CD90 interferes with brown or white adipogenesis or white adipocyte beiging. We applied flow cytometric sorting of human adipose tissue stromal cells (ASCs), a CRISPR/Cas9 knockout strategy in the human Simpson-Golabi-Behmel syndrome (SGBS) adipocyte model system, as well as a siRNA approach in human approaches supports the hypothesis that CD90 affects brown or white adipogenesis or white adipocyte beiging in humans. Taken together, our findings call the conclusions drawn from previous studies, which claimed a central role of CD90 in adipocyte differentiation, into question.},
}
@article {pmid33114339,
year = {2020},
author = {Bröker, JN and Müller, B and Prüfer, D and Schulze Gronover, C},
title = {Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {7},
number = {4},
pages = {},
pmid = {33114339},
issn = {2306-5354},
abstract = {Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.},
}
@article {pmid33113937,
year = {2020},
author = {Usman, B and Nawaz, G and Zhao, N and Liao, S and Liu, Y and Li, R},
title = {Precise Editing of the OsPYL9 Gene by RNA-Guided Cas9 Nuclease Confers Enhanced Drought Tolerance and Grain Yield in Rice (Oryza sativa L.) by Regulating Circadian Rhythm and Abiotic Stress Responsive Proteins.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33113937},
issn = {1422-0067},
mesh = {Abscisic Acid/*metabolism ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Circadian Rhythm ; Droughts ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Intracellular Signaling Peptides and Proteins/*genetics ; Oryza/genetics/*growth &amp; development/metabolism ; Plant Proteins/genetics/*metabolism ; Plants, Genetically Modified/growth &amp; development ; Proteomics ; Reactive Oxygen Species/metabolism ; Stress, Physiological ; },
abstract = {Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein-protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance.},
}
@article {pmid33113496,
year = {2021},
author = {Malone, LM and Birkholz, N and Fineran, PC},
title = {Conquering CRISPR: how phages overcome bacterial adaptive immunity.},
journal = {Current opinion in biotechnology},
volume = {68},
number = {},
pages = {30-36},
doi = {10.1016/j.copbio.2020.09.008},
pmid = {33113496},
issn = {1879-0429},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The rise of antibiotic-resistant bacteria has led to renewed interest in the use of their natural enemies, phages, for the prevention and treatment of infections. However, phage therapy requires detailed knowledge of the interactions between these entities. Bacteria defend themselves against phage predation with a large repertoire of defences. Among these, CRISPR-Cas systems stand out due to their adaptive character, mechanistic complexity and diversity, and present a significant hurdle for phage infection. Here, we provide an overview of how phages can circumvent CRISPR-Cas defence, ranging from target sequence mutations and DNA modifications to anti-CRISPR proteins and nucleus-like protective structures. An in-depth understanding of these phage evasion strategies is crucial for the successful development of phage therapy applications.},
}
@article {pmid33113365,
year = {2020},
author = {Wang, HF and Warrier, T and Farran, CA and Zheng, ZH and Xing, QR and Fullwood, MJ and Zhang, LF and Li, H and Xu, J and Lim, TM and Loh, YH},
title = {Defining Essential Enhancers for Pluripotent Stem Cells Using a Features-Oriented CRISPR-Cas9 Screen.},
journal = {Cell reports},
volume = {33},
number = {4},
pages = {108309},
doi = {10.1016/j.celrep.2020.108309},
pmid = {33113365},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Enhancer Elements, Genetic/*genetics ; Pluripotent Stem Cells/*metabolism ; },
abstract = {cis-regulatory elements (CREs) regulate the expression of genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of their target genes in the cellular native environment. In this study, we perform a features-oriented CRISPR-utilized systematic (FOCUS) screen of OCT4-bound CREs using CRISPR-Cas9 to identify functional enhancers important for pluripotency maintenance in mESCs. From the initial 235 candidates tested, 16 CREs are identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncover a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.},
}
@article {pmid33111098,
year = {2020},
author = {Abe, F and Ishida, Y and Hisano, H and Endo, M and Komari, T and Toki, S and Sato, K},
title = {Protocol for Genome Editing to Produce Multiple Mutants in Wheat.},
journal = {STAR protocols},
volume = {1},
number = {2},
pages = {100053},
pmid = {33111098},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Mutation/*genetics ; Plant Breeding/*methods ; Triticum/*genetics ; },
abstract = {Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019).},
}
@article {pmid33111087,
year = {2020},
author = {Xu, Z and Li, Y and Yan, A},
title = {Repurposing the Native Type I-F CRISPR-Cas System in Pseudomonas aeruginosa for Genome Editing.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100039},
pmid = {33111087},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; Drug Resistance, Multiple, Bacterial ; Gene Editing/*methods ; Humans ; Pseudomonas Infections/microbiology ; Pseudomonas aeruginosa/*genetics ; },
abstract = {Repurposing the broadly distributed native CRISPR-Cas systems in prokaryotes for genome editing is emerging as a new strategy for genetic manipulations. We recently reported the establishment of a single plasmid-mediated, one-step genome-editing technique in a multidrug-resistant genotype of the opportunistic pathogen Pseudomonas aeruginosa by harnessing its endogenous type I-F CRISPR-Cas system. The platform is readily applicable in additional type I-F CRISPR-containing clinical and environmental P. aeruginosa isolates. Herein, we provide the detailed protocol for the methodology. For complete details on the establishment and exploitation of this protocol, please refer to Xu et al. (2019).},
}
@article {pmid33111085,
year = {2020},
author = {Wang, Y and Yang, LZ and Chen, LL},
title = {Protocol for Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 System.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100037},
pmid = {33111085},
issn = {2666-1667},
support = {55008728/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; HeLa Cells ; Humans ; *RNA/analysis/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Ribonucleases/*metabolism ; Single Molecule Imaging/*methods ; Single-Cell Analysis ; },
abstract = {This protocol uses endonuclease-dead, programmable RNA-guided RNA-targeting Cas13 RNases (d)Cas13 proteins fused with fluorescent proteins to visualize and track RNA dynamics in live cells. This protocol details several aspects of the procedure, including gRNA design, fluorescent protein selection, nuclear localization signal adjustment, raw data analysis, operation steps, and extended optional applications that have been successfully applied in the visualization of NEAT1, SatIII, MUC4, and GCN4 RNAs. For complete information on the use and execution of this protocol, please refer to Yang et al. (2019).},
}
@article {pmid33111078,
year = {2020},
author = {Wang, Y and Wang, Z and Ji, Q},
title = {CRISPR-Cas9-Based Genome Editing and Cytidine Base Editing in Acinetobacter baumannii.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100025},
pmid = {33111078},
issn = {2666-1667},
mesh = {Acinetobacter baumannii/*genetics ; CRISPR-Cas Systems/*genetics ; Cytidine/*genetics ; Electroporation ; Gene Editing/*methods ; Point Mutation/genetics ; },
abstract = {Infections caused by drug-resistant Acinetobacter baumannii have posed a serious threat to global public health. However, genetic manipulation methods, the primary way to study pathogenesis and drug-resistance mechanisms, remain time consuming and inefficient. Here, we provide a detailed protocol for genetic manipulation, including gene deletion, insertion, and point mutation in A. baumannii using the platform. For complete details on the use and execution of this protocol, please refer to Wang et al. (2019).},
}
@article {pmid33111067,
year = {2020},
author = {Ma, Y and Yuan, J and Chang, X},
title = {Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100005},
pmid = {33111067},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Cytidine Deaminase/*genetics/metabolism ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Muscular Dystrophy, Duchenne/genetics ; RNA Splice Sites/genetics ; RNA Splicing/*genetics ; },
abstract = {This protocol uses lipofectamine to deliver base editors (i.e., dCas9 and AIDx fusion protein) and sgRNA expression vectors into Duchenne Muscular Dystrophy (DMD) patient-derived human induced pluripotent stem cells (hiPSCs). This protocol details mutation of the 5' splice site of DMD exon50 with TAM (targeted AID-induced mutagenesis) followed by amplicon-based NGS library preparation for high-throughput sequencing analysis. This protocol can be generalized for base editing in other hIPSCs and for correcting aberrant splicing associated with other genetic diseases. For complete information on the generation and use of this protocol, please refer to Yuan et al. (2018).},
}
@article {pmid33111065,
year = {2020},
author = {Marshall, R and Beisel, CL and Noireaux, V},
title = {Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100003},
pmid = {33111065},
issn = {2666-1667},
mesh = {CRISPR-Cas Systems/*genetics ; Cell-Free System/*metabolism ; DNA/metabolism ; *Endonucleases/genetics/metabolism ; *Escherichia coli/genetics/metabolism ; *RNA, Guide/genetics/metabolism ; },
abstract = {We present a protocol to rapidly test DNA binding and cleavage activity by CRISPR nucleases using cell-free transcription-translation (TXTL). Nuclease activity is assessed by adding DNA encoding a nuclease, a guide RNA, and a targeted reporter to a TXTL reaction and by measuring the fluorescence for several h. The reactions, performed in a few microliters, allow for parallel testing of many nucleases and guide RNAs. The protocol includes representative results for (d)Cas9 from Streptococcus pyogenes targeting a GFP reporter gene. For complete information on the generation and use of this protocol, please refer to the paper by Marshall et al. (2018).},
}
@article {pmid33110234,
year = {2021},
author = {Li, H and Zhao, L and Lau, YS and Zhang, C and Han, R},
title = {Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth.},
journal = {Oncogene},
volume = {40},
number = {1},
pages = {177-188},
pmid = {33110234},
issn = {1476-5594},
support = {R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Azoxymethane/*adverse effects ; CRISPR-Cas Systems ; Cell Survival/drug effects ; Colitis/chemically induced/complications/*genetics/metabolism ; Colorectal Neoplasms/chemically induced/*genetics/metabolism ; Dextran Sulfate/*adverse effects ; Disease Models, Animal ; Female ; Galectin 2/*genetics ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Hydrogen Peroxide/adverse effects ; Male ; Mice ; Oxidative Stress ; Phosphorylation ; Reactive Oxygen Species/metabolism ; STAT3 Transcription Factor/metabolism ; Whole Genome Sequencing/*methods ; },
abstract = {Colorectal cancer is the third leading cause of cancer-related deaths in the United States and the third most common cancer in men and women. Around 20% colon cancer cases are closely linked with colitis. Both environmental and genetic factors are thought to contribute to colon inflammation and tumor development. However, the genetic factors regulating colitis and colon tumorigenesis remain elusive. Since reactive oxygen species (ROS) is vitally involved in tissue inflammation and tumorigenesis, here we employed a genome-wide CRISPR knockout screening approach to systemically identify the genetic factors involved in the regulation of oxidative stress. Next generation sequencing (NGS) showed that over 600 gRNAs including the ones targeting LGALS2 were highly enriched in cells survived after sublethal H2O2 challenge. LGALS2 encodes the glycan-binding protein Galectin 2 (Gal2), which is predominantly expressed in the gastrointestinal tract and downregulated in human colon tumors. To examine the role of Gal2 in colitis, we employed the dextran sodium sulfate (DSS)-induced acute colitis model in mice with (WT) or without Lgals2 (Gal2-KO) and showed that Gal2 deficiency ameliorated DSS-induced colitis. We further demonstrated that Gal2-KO mice developed significantly larger tumors than WT mice using Azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal cancer model. We found that STAT3 phosphorylation was significantly increased in Gal2-deficient tumors as compared to those in WT mice. Gal2 overexpression decreased the proliferation of human colon tumor epithelial cells and blunted H2O2-induced STAT3 phosphorylation. Overall, our results demonstrate that Gal2 plays a suppressive role in colon tumor growth and highlights the therapeutic potential of Gal2 in colon cancer.},
}
@article {pmid33109771,
year = {2020},
author = {Sun, H and Li, Q and Yin, G and Ding, X and Xie, J},
title = {Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes.},
journal = {Aging},
volume = {12},
number = {20},
pages = {20432-20444},
pmid = {33109771},
issn = {1945-4589},
mesh = {Adult ; Humans ; Interleukin-1beta/*biosynthesis/drug effects ; Interleukin-6/*biosynthesis ; Ku Autoantigen/*physiology ; Lipopolysaccharides/pharmacology ; Macrophages/*immunology ; Male ; Monocytes/*immunology ; Tumor Necrosis Factor-alpha/*biosynthesis/drug effects ; },
abstract = {In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.},
}
@article {pmid33109226,
year = {2020},
author = {Koga, K and Shiraishi, Y and Yamagata, R and Tozaki-Saitoh, H and Shiratori-Hayashi, M and Tsuda, M},
title = {Intrinsic braking role of descending locus coeruleus noradrenergic neurons in acute and chronic itch in mice.},
journal = {Molecular brain},
volume = {13},
number = {1},
pages = {144},
pmid = {33109226},
issn = {1756-6606},
mesh = {Acute Disease ; Adrenergic Neurons/metabolism/*pathology ; Animals ; CRISPR-Cas Systems/genetics ; Chronic Disease ; Gene Silencing ; Locus Coeruleus/*pathology ; Mice, Inbred C57BL ; Pruritus/*pathology ; Receptors, Adrenergic, alpha-1/metabolism ; Spinal Cord Dorsal Horn/metabolism ; Synaptic Transmission/physiology ; },
abstract = {Itch is defined as an unpleasant sensation that provokes a desire to scratch. Our understanding of neuronal circuits for itch information transmission and processing in the spinal dorsal horn (SDH) has progressively advanced following the identification of SDH neuron subsets that are crucial for scratching behavior in models of itch. However, little is known about the control of acute and chronic itch by descending signals from the brain to the SDH. In this study, using genetic approaches that enable cell-type and circuit-specific functional manipulation, we reveal an intrinsic potential of locus coeruleus (LC)-noradrenergic (NAergic) neurons that project to the SDH to control acute and chronic itch. Activation and silencing of SDH-projecting LC-NAergic neurons reduced and enhanced scratching behavior, respectively, in models of histamine-dependent and -independent acute itch. Furthermore, in a model of chronic itch associated with contact dermatitis, repetitive scratching behavior was suppressed by the activation of the descending LC-NAergic pathway and by knocking out NA transporters specific to descending LC-NAergic neurons using a CRISPR-Cas9 system. Moreover, patch-clamp recording using spinal slices showed that noradrenaline facilitated inhibitory synaptic inputs onto gastrin-releasing peptide receptor-expressing SDH neurons, a neuronal subset known to be essential for itch transmission. Our findings suggest that descending LC-NAergic signaling intrinsically controls acute and chronic itch and provide potential therapeutic strategies for the treatment of acute and chronic itch.},
}
@article {pmid33107387,
year = {2021},
author = {Man, JCK and van Duijvenboden, K and Krijger, PHL and Hooijkaas, IB and van der Made, I and de Gier-de Vries, C and Wakker, V and Creemers, EE and de Laat, W and Boukens, BJ and Christoffels, VM},
title = {Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart.},
journal = {Circulation research},
volume = {128},
number = {1},
pages = {115-129},
doi = {10.1161/CIRCRESAHA.120.317045},
pmid = {33107387},
issn = {1524-4571},
mesh = {Animals ; Atrial Natriuretic Factor/*genetics/metabolism ; Binding Sites ; Binding, Competitive ; CRISPR-Cas Systems ; Cell Line ; Disease Models, Animal ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; Gene Expression Regulation, Developmental ; Humans ; Hypertrophy, Left Ventricular/*genetics/metabolism/pathology ; Mice, Knockout ; *Multigene Family ; Myocardial Infarction/*genetics/metabolism/pathology ; Myocytes, Cardiac/*metabolism/pathology ; Natriuretic Peptide, Brain/*genetics/metabolism ; Promoter Regions, Genetic ; },
abstract = {RATIONALE: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster.<br><br>OBJECTIVE: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb coregulation in vivo.<br><br>METHODS AND RESULTS: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa.<br><br>CONCLUSIONS: Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.},
}
@article {pmid33107304,
year = {2020},
author = {Saha, A and Arantes, PR and Hsu, RV and Narkhede, YB and Jinek, M and Palermo, G},
title = {Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a.},
journal = {Journal of chemical information and modeling},
volume = {60},
number = {12},
pages = {6427-6437},
pmid = {33107304},
issn = {1549-960X},
support = {R01 EY027440/EY/NEI NIH HHS/United States ; },
mesh = {COVID-19/*diagnosis ; CRISPR-Cas Systems ; Catalytic Domain ; DNA Cleavage ; DNA, Viral/*analysis/*genetics ; Gene Editing ; Humans ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; Phase Transition ; SARS-CoV-2/*genetics ; Substrate Specificity ; },
abstract = {CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system's specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.},
}
@article {pmid33106923,
year = {2020},
author = {Wrenn, ED and Moore, BM and Greenwood, E and McBirney, M and Cheung, KJ},
title = {Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids.},
journal = {Journal of mammary gland biology and neoplasia},
volume = {25},
number = {4},
pages = {337-350},
pmid = {33106923},
issn = {1573-7039},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; R37CA234488/NH/NIH HHS/United States ; R37 CA234488/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/genetics/*pathology ; CRISPR-Cas Systems/genetics ; Cell Culture Techniques/*methods ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Humans ; Intravital Microscopy ; Mice ; Organoids/*pathology ; Spheroids, Cellular/*pathology ; Time-Lapse Imaging ; Tumor Microenvironment/genetics ; },
abstract = {Tumor organoids mimic the architecture and heterogeneity of in vivo tumors and enable studies of collective interactions between tumor cells as well as with their surrounding microenvironment. Although tumor organoids hold significant promise as cancer models, they are also more costly and labor-intensive to cultivate than traditional 2D cell culture. We sought to identify critical factors regulating organoid growth ex vivo, and to use these observations to develop a more efficient organoid expansion method. Using time-lapse imaging of mouse mammary tumor organoids in 3D culture, we observed that outgrowth potential varies non-linearly with initial organoid size. Maximal outgrowth occurred in organoids with a starting size between ~10 to 1000 cells. Based on these observations, we developed a suspension culture method that maintains organoids in the ideal size range, enabling expansion from 1 million to over 100 million cells in less than 2 weeks and less than 3 hours of hands-on time. Our method facilitates the rapid, cost-effective expansion of organoids for CRISPR based studies and other assays requiring a large amount of organoid starting material.},
}
@article {pmid33106643,
year = {2020},
author = {Crunkhorn, S},
title = {Restoring vision loss with base editing.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {12},
pages = {835},
doi = {10.1038/d41573-020-00186-x},
pmid = {33106643},
issn = {1474-1784},
mesh = {*Adenine ; Animals ; CRISPR-Cas Systems ; Gene Editing ; Mice ; *Retinal Diseases ; },
}
@article {pmid33106580,
year = {2020},
author = {Duong, MM and Carmody, CM and Ma, Q and Peters, JE and Nugen, SR},
title = {Optimization of T4 phage engineering via CRISPR/Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18229},
pmid = {33106580},
issn = {2045-2322},
support = {R01 EB027895/EB/NIBIB NIH HHS/United States ; R21 EB024623/EB/NIBIB NIH HHS/United States ; R01EB027895/EB/NIBIB NIH HHS/United States ; R21EB024623/EB/NIBIB NIH HHS/United States ; },
mesh = {Bacteria/metabolism/*virology ; Bacteriophage T4/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods/standards ; Genetic Engineering/methods/*standards ; Viral Plaque Assay/*methods ; },
abstract = {A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03-3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.},
}
@article {pmid33106426,
year = {2020},
author = {Alward, BA and Laud, VA and Skalnik, CJ and York, RA and Juntti, SA and Fernald, RD},
title = {Modular genetic control of social status in a cichlid fish.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {28167-28174},
pmid = {33106426},
issn = {1091-6490},
support = {R37 NS034950/NS/NINDS NIH HHS/United States ; R01 NS034950/NS/NINDS NIH HHS/United States ; R21 MH096220/MH/NIMH NIH HHS/United States ; R56 NS034950/NS/NINDS NIH HHS/United States ; R03 MH101373/MH/NIMH NIH HHS/United States ; },
mesh = {Androgens/metabolism ; Animals ; CRISPR-Cas Systems ; *Cichlids/genetics/physiology ; Female ; Gene Editing ; *Gene Expression Regulation/genetics/physiology ; Male ; Mutation ; Receptors, Androgen/genetics/physiology ; Social Behavior ; *Social Dominance ; },
abstract = {Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARβ, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARβ, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARβ, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.},
}
@article {pmid33106324,
year = {2020},
author = {Kagaya, K and Noma-Takayasu, N and Yamamoto, I and Tashiro, S and Ishikawa, F and Hayashi, MT},
title = {Chromosome instability induced by a single defined sister chromatid fusion.},
journal = {Life science alliance},
volume = {3},
number = {12},
pages = {},
pmid = {33106324},
issn = {2575-1077},
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Cell Division/genetics ; Chromatids/genetics/pathology/physiology ; Chromosomal Instability/*genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/methods ; HCT116 Cells ; Humans ; Microscopy, Fluorescence/methods ; Neoplasms/genetics ; Single-Cell Analysis/*methods ; Sister Chromatid Exchange/genetics/*physiology ; },
abstract = {Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.},
}
@article {pmid33104788,
year = {2020},
author = {Xu, H and Wang, J and Liang, Y and Fu, Y and Li, S and Huang, J and Xu, H and Zou, W and Chen, B},
title = {TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {e127},
pmid = {33104788},
issn = {1362-4962},
mesh = {Alleles ; Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Chromatin/*genetics ; Fluorescent Antibody Technique/methods ; Gene Expression Regulation/genetics ; Gene Regulatory Networks/*genetics ; Humans ; *Molecular Imaging ; *Single-Cell Analysis ; Transcription, Genetic ; },
abstract = {A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.},
}
@article {pmid33104071,
year = {2020},
author = {Xu, D and Mondol, PC and Uzair, M and Tucker, MR and Zhang, D},
title = {Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {164},
pages = {},
doi = {10.3791/61665},
pmid = {33104071},
issn = {1940-087X},
mesh = {Agrobacterium/*genetics ; Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genotype ; Mutagenesis/genetics ; Mutation/genetics ; Oryza/*genetics/*physiology ; Phenotype ; Plants, Genetically Modified/genetics ; Pollen/*genetics/*growth &amp; development ; Reproduction/physiology ; Seedlings/genetics ; Tissue Culture Techniques ; *Transformation, Genetic ; },
abstract = {Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.},
}
@article {pmid33102460,
year = {2020},
author = {Yu, L and Marchisio, MA},
title = {Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {575393},
pmid = {33102460},
issn = {2296-4185},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins), a prokaryotic RNA-mediated adaptive immune system, has been repurposed for gene editing and synthetic gene circuit construction both in bacterial and eukaryotic cells. In the last years, the emergence of the anti-CRISPR proteins (Acrs), which are natural OFF-switches for CRISPR-Cas, has provided a new means to control CRISPR-Cas activity and promoted a further development of CRISPR-Cas-based biotechnological toolkits. In this review, we focus on type I and type V-A anti-CRISPR proteins. We first narrate Acrs discovery and analyze their inhibitory mechanisms from a structural perspective. Then, we describe their applications in gene editing and transcription regulation. Finally, we discuss the potential future usage-and corresponding possible challenges-of these two kinds of anti-CRISPR proteins in eukaryotic synthetic gene circuits.},
}
@article {pmid33102417,
year = {2020},
author = {Almeida, LM and Gaca, A and Bispo, PM and Lebreton, F and Saavedra, JT and Silva, RA and Basílio-Júnior, ID and Zorzi, FM and Filsner, PH and Moreno, AM and Gilmore, MS},
title = {Coexistence of the Oxazolidinone Resistance-Associated Genes cfr and optrA in Enterococcus faecalis From a Healthy Piglet in Brazil.},
journal = {Frontiers in public health},
volume = {8},
number = {},
pages = {518},
pmid = {33102417},
issn = {2296-2565},
support = {P01 AI083214/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Brazil ; Drug Resistance, Bacterial/genetics ; Enterococcus faecalis/genetics ; *Enterococcus faecium ; *Gram-Positive Bacterial Infections/drug therapy ; *Oxazolidinones/pharmacology ; Swine ; United States ; },
abstract = {Oxazolidinones are one of the most important antimicrobials potentially active against glycopeptide- and β-lactam-resistant Gram-positive pathogens. Linezolid-the first oxazolidinone to be approved for clinical use in 2000 by the US Food and Drug Administration-and the newer molecule in the class, tedizolid, inhibit protein synthesis by suppressing the formation of the 70S ribosomal complex in bacteria. Over the past two decades, transferable oxazolidinone resistance genes, in particular cfr and optrA, have been identified in Firmicutes isolated from healthcare-related infections, livestock, and the environment. Our goals in this study were to investigate the genetic contexts and the transferability of the cfr and optrA genes and examine genomic features, such as antimicrobial resistance genes, plasmid incompatibility types, and CRISPR-Cas defenses of a linezolid-resistant Enterococcus faecalis isolated in feces from a healthy pig during an antimicrobial surveillance program for animal production in Brazil. The cfr gene was found to be integrated into a transposon-like structure of 7,759 nt flanked by IS1216E and capable of excising and circularizing, distinguishing it from known genetic contexts for cfr in Enterococcus spp., while optrA was inserted into an Inc18 broad host-range plasmid of >58 kb. Conjugal transfer of cfr and optrA was shown by filter mating. The coexistence of cfr and optrA in an E. faecalis isolated from a healthy nursery pig highlights the need for monitoring the use of antibiotics in the Brazilian swine production system for controlling spread and proliferation of antibiotic resistance.},
}
@article {pmid33100228,
year = {2020},
author = {Navas-Pérez, E and Vicente-García, C and Mirra, S and Burguera, D and Fernàndez-Castillo, N and Ferrán, JL and López-Mayorga, M and Alaiz-Noya, M and Suárez-Pereira, I and Antón-Galindo, E and Ulloa, F and Herrera-Úbeda, C and Cuscó, P and Falcón-Moya, R and Rodríguez-Moreno, A and D'Aniello, S and Cormand, B and Marfany, G and Soriano, E and Carrión, &#xc1;M and Carvajal, JJ and Garcia-Fernàndez, J},
title = {Characterization of an eutherian gene cluster generated after transposon domestication identifies Bex3 as relevant for advanced neurological functions.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {267},
pmid = {33100228},
issn = {1474-760X},
mesh = {Animals ; Apoptosis Regulatory Proteins/*genetics ; Autism Spectrum Disorder/genetics ; Brain ; CRISPR-Cas Systems ; *DNA Transposable Elements ; DNA-Binding Proteins/genetics ; *Domestication ; Eutheria/*genetics ; Evolution, Molecular ; Female ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; *Multigene Family ; Nerve Tissue Proteins/genetics ; Neurodevelopmental Disorders/genetics ; Nuclear Proteins/genetics ; Phylogeny ; Placenta ; Pregnancy ; TOR Serine-Threonine Kinases/genetics ; Transcription Factors/genetics ; },
abstract = {BACKGROUND: One of the most unusual sources of phylogenetically restricted genes is the molecular domestication of transposable elements into a host genome as functional genes. Although these kinds of events are sometimes at the core of key macroevolutionary changes, their origin and organismal function are generally poorly understood.<br><br>RESULTS: Here, we identify several previously unreported transposable element domestication events in the human and mouse genomes. Among them, we find a remarkable molecular domestication that gave rise to a multigenic family in placental mammals, the Bex/Tceal gene cluster. These genes, which act as hub proteins within diverse signaling pathways, have been associated with neurological features of human patients carrying genomic microdeletions in chromosome X. The Bex/Tceal genes display neural-enriched patterns and are differentially expressed in human neurological disorders, such as autism and schizophrenia. Two different murine alleles of the cluster member Bex3 display morphological and physiopathological brain modifications, such as reduced interneuron number and hippocampal electrophysiological imbalance, alterations that translate into distinct behavioral phenotypes.<br><br>CONCLUSIONS: We provide an in-depth understanding of the emergence of a gene cluster that originated by transposon domestication and gene duplication at the origin of placental mammals, an evolutionary process that transformed a non-functional transposon sequence into novel components of the eutherian genome. These genes were integrated into existing signaling pathways involved in the development, maintenance, and function of the CNS in eutherians. At least one of its members, Bex3, is relevant for higher brain functions in placental mammals and may be involved in human neurological disorders.},
}
@article {pmid33099790,
year = {2021},
author = {Li, W and Wang, L and Zhou, F and Li, C and Ma, W and Chen, H and Wang, G and Pickett, JA and Zhou, JJ and Lin, Y},
title = {Overexpression of the homoterpene synthase gene, OsCYP92C21, increases emissions of volatiles mediating tritrophic interactions in rice.},
journal = {Plant, cell &amp; environment},
volume = {44},
number = {3},
pages = {948-963},
doi = {10.1111/pce.13924},
pmid = {33099790},
issn = {1365-3040},
support = {BB/J020281/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L001683/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Alkyl and Aryl Transferases/*metabolism ; Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Oryza/genetics/*metabolism ; *Plant Defense Against Herbivory ; Plant Proteins/*metabolism ; Plants, Genetically Modified ; Real-Time Polymerase Chain Reaction ; Terpenes/metabolism ; Volatile Organic Compounds/*metabolism ; Wasps ; },
abstract = {Plant defence homoterpenes can be used to attract pest natural enemies. However, the biosynthetic pathway of homoterpenes is still unknown in rice, and the practical application of such indirect defence systems suffers from inherent limitations due to their low emissions from plants. Here, we demonstrated that the protein OsCYP92C21 is responsible for homoterpene biosynthesis in rice. We also revealed that the ability of rice to produce homoterpenes is dependent on the subcellular precursor pools. By increasing the precursor pools through specifically subcellular targeting expression, genetic transformation and genetic introgression, we significantly enhanced homoterpene biosynthesis in rice. The final introgressed GM rice plants exhibited higher homoterpene emissions than the wild type rice and the highest homoterpene emission reported so far for such GM plants even without the induction of herbivore attack. As a result, these GM rice plants demonstrated strong attractiveness to the parasitic wasp Cotesia chilonis. This study discovered the homoterpene biosynthesis pathway in rice, and lays the foundation for the utilisation of plant indirect defence mechanism in the "push-pull" strategy of integrated pest management through increasing precursor pools in the subcellular compartments and overexpressing homoterpene synthase by genetic transformation.},
}
@article {pmid33099727,
year = {2021},
author = {Sun, H and Zheng, J and Yi, M and Wan, Y},
title = {Conditional Genome Editing in the Mammalian Brain Using CRISPR-Cas9.},
journal = {Neuroscience bulletin},
volume = {37},
number = {3},
pages = {423-426},
pmid = {33099727},
issn = {1995-8218},
mesh = {Animals ; Brain ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; },
}
@article {pmid33098376,
year = {2020},
author = {Chen, HX and Yang, ZY and Hou, HT and Wang, J and Wang, XL and Yang, Q and Liu, L and He, GW},
title = {Novel mutations of TCTN3/LTBP2 with cellular function changes in congenital heart disease associated with polydactyly.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {23},
pages = {13751-13762},
pmid = {33098376},
issn = {1582-4934},
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Alleles ; Apoptosis Regulatory Proteins/*genetics ; Biomarkers ; CRISPR-Cas Systems ; Computational Biology/methods ; DNA Mutational Analysis ; Gene Editing ; Gene Expression Profiling ; Genetic Association Studies ; *Genetic Predisposition to Disease ; Heart Defects, Congenital/*diagnosis/*genetics ; Humans ; Latent TGF-beta Binding Proteins/*genetics ; *Mutation ; Myocytes, Cardiac/metabolism ; Phenotype ; Pluripotent Stem Cells/metabolism ; Polydactyly/*genetics ; Radiography ; Ultrasonography ; Whole Exome Sequencing ; },
abstract = {Congenital heart disease (CHD) associated with polydactyly involves various genes. We aimed to identify variations from genes related to complex CHD with polydactyly and to investigate the cellular functions related to the mutations. Blood was collected from a complex CHD case with polydactyly, and whole exome sequencing (WES) was performed. The CRISPR/Cas9 system was used to generate human pluripotent stem cell with mutations (hPSCs-Mut) that were differentiated into cardiomyocytes (hPSC-CMs-Mut) and analysed by transcriptomics on day 0, 9 and 13. Two heterozygous mutations, LTBP2 (c.2206G>A, p.Asp736Asn, RefSeq NM_000428.2) and TCTN3 (c.1268G>A, p.Gly423Glu, RefSeq NM_015631.5), were identified via WES but no TBX5 mutations were found. The stable cell lines of hPSCs-LTBP2[mu] /TCTN3[mu] were constructed and differentiated into hPSC-CMs-LTBP2[mu] /TCTN3[mu] . Compared to the wild type, LTBP2 mutation delayed the development of CMs. The TCTN3 mutation consistently presented lower rate and weaker force of the contraction of CMs. For gene expression pattern of persistent up-regulation, pathways in cardiac development and congenital heart disease were enriched in hPSCs-CM-LTBP2[mu] , compared with hPSCs-CM-WT. Thus, the heterozygous mutations in TCTN3 and LTBP2 affect contractility (rate and force) of cardiac myocytes and may affect the development of the heart. These findings provide new insights into the pathogenesis of complex CHD with polydactyly.},
}
@article {pmid33098074,
year = {2021},
author = {Yue, P and Xia, S and Wu, G and Liu, L and Zhou, K and Liao, H and Li, J and Zheng, X and Guo, Y and Hua, Y and Zhang, D and Li, Y},
title = {Attenuation of Cardiomyocyte Hypertrophy via Depletion Myh7 using CASAAV.},
journal = {Cardiovascular toxicology},
volume = {21},
number = {3},
pages = {255-264},
pmid = {33098074},
issn = {1559-0259},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cardiomegaly/genetics/metabolism/pathology/*prevention &amp; control ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics/metabolism ; Disease Models, Animal ; Down-Regulation ; *Gene Knock-In Techniques ; Mice, Transgenic ; Myocytes, Cardiac/*metabolism/pathology ; Myosin Heavy Chains/*deficiency/genetics ; },
abstract = {Myh7 is a classic biomarker for cardiac remodeling and a potential target to attenuate cardiomyocyte (CM) hypertrophy. This study aimed to identify the dominant function of Myh7 after birth and determine whether its removal would affect CM maturation or contribute to reversal of pathological hypertrophy phenotypes. The CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis) technique was used to deplete Myh6 and Myh7, and an AAV dosage of 5 × 10[9] vg/g was used to generate a mosaic CM depletion model to explore the function of Myh7 in adulthood. CM hypertrophy was induced by transverse aortic constriction (TAC) in Rosa26[Cas9-P2A-GFP] mice at postnatal day 28 (PND28). Heart function was measured by echocardiography. Isolated CMs and in situ imaging were used to analyze the structure and morphology of CM. We discovered that CASAAV successfully silenced Myh6 and Myh7 in CMs, and early depletion of Myh7 led to mild adulthood lethality. However, the Myh7 PND28-knockout mice had normal heart phenotype and function, with normal cellular size and normal organization of sarcomeres and T-tubules. The TAC mice also received AAV-Myh7-Cre to produce Myh7-knockout CMs, which were also of normal size, and echocardiography demonstrated a reversal of cardiac hypertrophy. In conclusion, Myh7 has a role during the maturation period but rarely functions in adulthood. Thus, the therapeutic time should exceed the period of maturation. These results confirm Myh7 as a potential therapeutic target and indicate that its inhibition could help reverse CM hypertrophy.},
}
@article {pmid33097833,
year = {2021},
author = {Wang, K and Zhang, Z and Tsai, HI and Liu, Y and Gao, J and Wang, M and Song, L and Cao, X and Xu, Z and Chen, H and Gong, A and Wang, D and Cheng, F and Zhu, H},
title = {Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells.},
journal = {Cell death and differentiation},
volume = {28},
number = {4},
pages = {1222-1236},
pmid = {33097833},
issn = {1476-5403},
mesh = {AMP-Activated Protein Kinases/metabolism ; Animals ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/drug therapy/*genetics/metabolism ; Female ; Ferroptosis/*drug effects ; Hep G2 Cells ; Humans ; Iron/metabolism ; Lipid Peroxidation ; Liver Neoplasms/drug therapy/*genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Minor Histocompatibility Antigens/*genetics/metabolism ; Pregnancy Proteins/*genetics/metabolism ; Signal Transduction/drug effects ; Sorafenib/*pharmacology ; Transaminases/*genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Ferroptosis, a form of iron-dependent cell death driven by cellular metabolism and iron-dependent lipid peroxidation, has been implicated as a tumor-suppressor function for cancer therapy. Recent advance revealed that the sensitivity to ferroptosis is tightly linked to numerous biological processes, including metabolism of amino acid and the biosynthesis of glutathione. Here, by using a high-throughput CRISPR/Cas9-based genetic screen in HepG2 hepatocellular carcinoma cells to search for metabolic proteins inhibiting ferroptosis, we identified a branched-chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib, and sulfasalazine) activated AMPK/SREBP1 signaling pathway through iron-dependent ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 as the key enzyme mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc[-] inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. On the contrary, direct inhibition of BCAT2 by RNA interference, or indirect inhibition by blocking system Xc[-] activity, triggers ferroptosis. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.},
}
@article {pmid33097793,
year = {2020},
author = {Matsumoto, D and Tamamura, H and Nomura, W},
title = {A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {601},
pmid = {33097793},
issn = {2399-3642},
mesh = {Binding Sites ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; *Cell Cycle ; Cell Line ; *Gene Editing ; Gene Expression Regulation ; Humans ; Mutation ; Plasmids/genetics ; *RNA, Guide ; Recombinational DNA Repair ; },
abstract = {The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.},
}
@article {pmid33097693,
year = {2020},
author = {Schene, IF and Joore, IP and Oka, R and Mokry, M and van Vugt, AHM and van Boxtel, R and van der Doef, HPJ and van der Laan, LJW and Verstegen, MMA and van Hasselt, PM and Nieuwenhuis, EES and Fuchs, SA},
title = {Prime editing for functional repair in patient-derived disease models.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5352},
pmid = {33097693},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Copper-Transporting ATPases/genetics ; Deoxyribonuclease I/metabolism ; Diacylglycerol O-Acyltransferase/genetics ; Gene Editing/*methods ; HEK293 Cells ; Hepatolenticular Degeneration/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Organoids/*metabolism ; Recombinational DNA Repair ; Stem Cells ; Targeted Gene Repair/methods ; beta Catenin/*genetics ; },
abstract = {Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.},
}
@article {pmid33097661,
year = {2020},
author = {Wei, X and Yang, J and Adair, SJ and Ozturk, H and Kuscu, C and Lee, KY and Kane, WJ and O'Hara, PE and Liu, D and Demirlenk, YM and Habieb, AH and Yilmaz, E and Dutta, A and Bauer, TW and Adli, M},
title = {Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {28068-28079},
pmid = {33097661},
issn = {1091-6490},
support = {R01 CA060499/CA/NCI NIH HHS/United States ; L30 CA124093/CA/NCI NIH HHS/United States ; P30 CA044579/CA/NCI NIH HHS/United States ; R25 CA206972/CA/NCI NIH HHS/United States ; R01 CA211648/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Deoxycytidine/*analogs &amp; derivatives/pharmacology ; Drug Development ; Gene Knockout Techniques ; Humans ; Mice, Nude ; Pancreatic Neoplasms/*metabolism ; *Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.},
}
@article {pmid33097539,
year = {2020},
author = {Yang, P and Chou, SJ and Li, J and Hui, W and Liu, W and Sun, N and Zhang, RY and Zhu, Y and Tsai, ML and Lai, HI and Smalley, M and Zhang, X and Chen, J and Romero, Z and Liu, D and Ke, Z and Zou, C and Lee, CF and Jonas, SJ and Ban, Q and Weiss, PS and Kohn, DB and Chen, K and Chiou, SH and Tseng, HR},
title = {Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.},
journal = {Science advances},
volume = {6},
number = {43},
pages = {},
pmid = {33097539},
issn = {2375-2548},
support = {DP5 OD028181/OD/NIH HHS/United States ; R21 EB016270/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Vectors/genetics ; *Hemoglobinopathies/genetics/therapy ; Hemoglobins/genetics ; Mice ; },
abstract = {Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.},
}
@article {pmid33097498,
year = {2020},
author = {García-Moyano, A and Larsen, &#xd8; and Gaykawad, S and Christakou, E and Boccadoro, C and Puntervoll, P and Bjerga, GEK},
title = {Fragment Exchange Plasmid Tools for CRISPR/Cas9-Mediated Gene Integration and Protease Production in Bacillus subtilis.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {1},
pages = {},
pmid = {33097498},
issn = {1098-5336},
mesh = {Bacillus subtilis/enzymology/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Peptide Hydrolases/*genetics/metabolism ; Plasmids/*genetics/metabolism ; },
abstract = {Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains.IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.},
}
@article {pmid33097066,
year = {2020},
author = {Bashir, S and Dang, T and Rossius, J and Wolf, J and Kühn, R},
title = {Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {57},
pmid = {33097066},
issn = {1472-6750},
support = {TAL-CUT 03V0261//Bundesministerium f&#xfc;r Forschung und Technologie (DE)/International ; },
mesh = {Animals ; BRCA1 Protein/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; DNA Repair ; DNA-Binding Proteins/genetics ; Gene Editing/*methods ; Gene Expression ; Gene Knock-In Techniques ; HEK293 Cells ; Histones/*genetics ; Humans ; Lamin Type B/genetics ; Recombinational DNA Repair ; Ubiquitin/chemistry ; Ubiquitin-Protein Ligases/genetics ; *Ubiquitination ; },
abstract = {BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology.<br><br>RESULTS: For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci.<br><br>CONCLUSIONS: Our findings provide a novel efficient approach to promote precise gene editing in human cells.},
}
@article {pmid33096014,
year = {2021},
author = {Bertelsen, MB and Senissar, M and Nielsen, MH and Bisiak, F and Cunha, MV and Molinaro, AL and Daines, DA and Brodersen, DE},
title = {Structural Basis for Toxin Inhibition in the VapXD Toxin-Antitoxin System.},
journal = {Structure (London, England : 1993)},
volume = {29},
number = {2},
pages = {139-150.e3},
pmid = {33096014},
issn = {1878-4186},
support = {U01 DC014756/DC/NIDCD NIH HHS/United States ; },
mesh = {Bacterial Proteins/*chemistry/metabolism ; Bacterial Toxins/*chemistry/metabolism ; Binding Sites ; Haemophilus influenzae/chemistry/enzymology ; Membrane Glycoproteins/*chemistry/metabolism ; Molecular Docking Simulation ; Protein Domains ; *Toxin-Antitoxin Systems ; },
abstract = {Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogenicity; however, the mode of action of VapD and the mechanism of inhibition by the VapX antitoxin remain unknown. Here, we report the structure of the intact H. influenzae VapXD complex, revealing an unusual 2:1 TA molecular stoichiometry where a Cas2-like homodimer of VapD binds a single VapX antitoxin. VapX consists of an oligonucleotide/oligosaccharide-binding domain that docks into an asymmetrical cavity on the toxin dimer. Structures of isolated VapD further reveal how a symmetrical toxin homodimer adapts to interacting with an asymmetrical antitoxin and suggest how a primordial TA system evolved to become part of CRISPR-Cas immunity systems.},
}
@article {pmid33095315,
year = {2021},
author = {Nazlamova, L and Thomas, NS and Cheung, MK and Legebeke, J and Lord, J and Pengelly, RJ and Tapper, WJ and Wheway, G},
title = {A CRISPR and high-content imaging assay compliant with ACMG/AMP guidelines for clinical variant interpretation in ciliopathies.},
journal = {Human genetics},
volume = {140},
number = {4},
pages = {593-607},
pmid = {33095315},
issn = {1432-1203},
support = {/WT_/Wellcome Trust/United Kingdom ; 204378/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Ciliopathies/*diagnostic imaging/*genetics ; Diagnostic Imaging/*methods ; Eye Proteins/*genetics ; Gene Editing ; Gene Knockout Techniques ; Guidelines as Topic ; Image Processing, Computer-Assisted ; Mutation, Missense ; Retina/diagnostic imaging ; Retinal Degeneration/diagnostic imaging/genetics ; Retinitis Pigmentosa/diagnostic imaging/genetics ; },
abstract = {Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24-60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology, it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one-third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here, we present a high-throughput high-content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31[+/-] human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high-content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance, and can be extended to variant classification in other ciliopathies.},
}
@article {pmid33095053,
year = {2020},
author = {Galizi, R and Duncan, JN and Rostain, W and Quinn, CM and Storch, M and Kushwaha, M and Jaramillo, A},
title = {Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {398-408},
doi = {10.1089/crispr.2020.0029},
pmid = {33095053},
issn = {2573-1602},
support = {BB/P020615/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M017982/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Biosensing Techniques ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell-Free System ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; Escherichia coli/genetics ; Genetic Engineering ; RNA, Guide/genetics/*metabolism ; RNA, Messenger/genetics/*metabolism ; Transcription, Genetic ; vif Gene Products, Human Immunodeficiency Virus/analysis ; },
abstract = {CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.},
}
@article {pmid33095052,
year = {2020},
author = {Wiegand, T and Semenova, E and Shiriaeva, A and Fedorov, I and Datsenko, K and Severinov, K and Wiedenheft, B},
title = {Reproducible Antigen Recognition by the Type I-F CRISPR-Cas System.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {378-387},
pmid = {33095052},
issn = {2573-1602},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {Antigens/metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Replication ; DNA, Bacterial/genetics/metabolism ; Endoribonucleases/*metabolism ; Escherichia coli/enzymology/genetics ; Pseudomonas aeruginosa/enzymology/genetics ; RNA, Guide/metabolism ; },
abstract = {CRISPR-associated proteins 1 and 2 (Cas1-2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1-2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided surveillance complex (Csy) improves the efficiency of adaptation and significantly increases the fidelity of protospacer adjacent motif selection. Sequences selected for integration are preferentially derived from specific regions of extrachromosomal DNA, and patterns of spacer selection are highly reproducible between independent biological replicates. This work helps define the role of the Csy complex in I-F adaptation and reveals that actively replicating mobile genetic elements have antigenic signatures that facilitate their integration during CRISPR adaptation.},
}
@article {pmid33095051,
year = {2020},
author = {Polkoff, KM and Chung, J and Simpson, SG and Gleason, K and Piedrahita, JA},
title = {In Vitro Validation of Transgene Expression in Gene-Edited Pigs Using CRISPR Transcriptional Activators.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {409-418},
pmid = {33095051},
issn = {2573-1602},
support = {R21 OD019738/OD/NIH HHS/United States ; R01 OD023138/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Associated Protein 9/genetics/*metabolism ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression ; Nuclear Transfer Techniques ; RNA, Guide/genetics/*metabolism ; Swine ; Trans-Activators/genetics/*metabolism ; *Transgenes ; },
abstract = {The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.},
}
@article {pmid33095046,
year = {2020},
author = {Barrangou, R},
title = {Commissions, Consensus, and CRISPR.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {316-317},
doi = {10.1089/crispr.2020.29107.rba},
pmid = {33095046},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Consensus ; *Embryo Research/ethics/legislation &amp; jurisprudence ; *Gene Editing/ethics/legislation &amp; jurisprudence ; *Genetic Therapy/ethics/legislation &amp; jurisprudence ; *Genome, Human ; Humans ; },
}
@article {pmid33095045,
year = {2020},
author = {Casas-Mollano, JA and Zinselmeier, MH and Erickson, SE and Smanski, MJ},
title = {CRISPR-Cas Activators for Engineering Gene Expression in Higher Eukaryotes.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {350-364},
pmid = {33095045},
issn = {2573-1602},
support = {T32 GM008347/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression ; Humans ; Plants/genetics ; Trans-Activators/*genetics ; *Transcriptional Activation ; Yeasts/genetics ; },
abstract = {CRISPR-Cas-based transcriptional activators allow genetic engineers to specifically induce expression of one or many target genes in trans. Here we review the many design variations of these versatile tools and compare their effectiveness in different eukaryotic systems. Lastly, we highlight several applications of programmable transcriptional activation to interrogate and engineer complex biological processes.},
}
@article {pmid33095042,
year = {2020},
author = {Baylis, F and Darnovsky, M and Hasson, K and Krahn, TM},
title = {Human Germ Line and Heritable Genome Editing: The Global Policy Landscape.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {365-377},
doi = {10.1089/crispr.2020.0082},
pmid = {33095042},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Embryo Research/*legislation &amp; jurisprudence ; Embryonic Stem Cells ; Gene Editing/*legislation &amp; jurisprudence ; *Genome, Human ; *Germ Cells ; Humans ; },
abstract = {Discussions and debates about the governance of human germline and heritable genome editing should be informed by a clear and accurate understanding of the global policy landscape. This policy survey of 106 countries yields significant new data. A large majority of countries (96 out of 106) surveyed have policy documents-legislation, regulations, guidelines, codes, and international treaties-relevant to the use of genome editing to modify early-stage human embryos, gametes, or their precursor cells. Most of these 96 countries do not have policies that specifically address the use of genetically modified in vitro embryos in laboratory research (germline genome editing); of those that do, 23 prohibit this research and 11 explicitly permit it. Seventy-five of the 96 countries prohibit the use of genetically modified in vitro embryos to initiate a pregnancy (heritable genome editing). Five of these 75 countries provide exceptions to their prohibitions. No country explicitly permits heritable human genome editing. These data contrast markedly with previously reported findings.},
}
@article {pmid33095041,
year = {2020},
author = {Barrangou, R},
title = {Nobel Dreams Come True for Doudna and Charpentier.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {317-318},
doi = {10.1089/crispr.2020.29109.rba},
pmid = {33095041},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Chemistry ; Gene Editing/*methods ; *Nobel Prize ; },
}
@article {pmid33094683,
year = {2020},
author = {Park, JJ and Kim, JE and Jeon, Y and Lee, MR and Choi, JY and Song, BR and Park, JW and Kang, MJ and Choi, HJ and Bae, SJ and Lee, H and Kang, BC and Hwang, DY},
title = {Deletion of NKX3.1 via CRISPR/Cas9 Induces Prostatic Intraepithelial Neoplasia in C57BL/6 Mice.},
journal = {Technology in cancer research &amp; treatment},
volume = {19},
number = {},
pages = {1533033820964425},
pmid = {33094683},
issn = {1533-0338},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; ErbB Receptors/genetics ; Homeodomain Proteins/*genetics ; Humans ; Ki-67 Antigen/genetics ; Male ; Mice ; Mice, Knockout ; Morphogenesis/*genetics ; Prostate/growth &amp; development/pathology ; Prostatic Intraepithelial Neoplasia/*genetics/pathology ; Transcription Factors/*genetics ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Several techniques have been employed for deletion of the NKX3.1 gene, resulting in developmental defects of the prostate, including alterations in ductal branching morphogenesis and prostatic secretions as well as epithelial hyperplasia and dysplasia. To investigate whether the CRISPR/Cas9-mediated technique can be applied to study prostate carcinogenesis through exon I deletion of NKX3.1 gene, alterations in the prostatic intraepithelial neoplasia (PIN) and their regulatory mechanism were observed in the prostate of NKX3.1 knockout (KO) mice produced by the CRISPR/Cas9-mediated NKX3.1 mutant gene, at the ages of 16 and 24 weeks. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were observed to be increased in only the 24 weeks KO mice, although morphogenesis was constant in all groups. Obvious PIN 1 and 2 lesions were frequently detected in prostate of the 24 weeks KO mice, as compared with the same age wild type (WT) mice. Ki67, a key indicator for PIN, was densely stained in the epithelium of prostate in the 24 weeks KO mice, while the expression of p53 protein was suppressed in the same group. Also, both the 16 and 24 weeks KO mice reveal inhibition of the PI3K/AKT/mTOR pathway in the prostate. However, prostate specific antigen (PSA) levels and Bax/Bcl-2 expressions were decreased in the prostate of 16 weeks KO mice, and were increased in only the 24 weeks KO mice. Taken together, the results of the present study provide additional evidence that CRISPR/Cas9-mediated exon 1 deletion of the NKX3.1 gene successfully induces PIN lesions, along with significant alterations of Ki67 expression, EGFR signaling pathway, and cancer-regulated proteins.},
}
@article {pmid33094378,
year = {2020},
author = {Golkar, Z},
title = {CRISPR: a journey of gene-editing based medicine.},
journal = {Genes &amp; genomics},
volume = {42},
number = {12},
pages = {1369-1380},
pmid = {33094378},
issn = {2092-9293},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Precision Medicine ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is one of the hallmark of biological tools, contemplated as a valid and hopeful alternatives to genome editing. Advancements in CRISPR-based technologies have empowered scientists with an editing kit that allows them to employ their knowledge for deleting, replacing and lately "Gene Surgery", and provides unique control over genes in broad range of species, and presumably in humans. These fast-growing technologies have high strength and flexibility and are becoming an adaptable tool with implementations that are altering organism's genome and easily used for chromatin manipulation. In addition to the popularity of CRISPR in genome engineering and modern biology, this major tool authorizes breakthrough discoveries and methodological advancements in science. As scientists are developing new types of experiments, some of the applications are raising questions about what CRISPR can enable. The results of evidence-based research strongly suggest that CRISPR is becoming a practical tool for genome-engineering and to create genetically modified eukaryotes, which is needed to establish guidelines on new regulatory concerns for scientific communities.},
}
@article {pmid33093796,
year = {2020},
author = {Sudheer, S and Bai, RG and Usmani, Z and Sharma, M},
title = {Insights on Engineered Microbes in Sustainable Agriculture: Biotechnological Developments and Future Prospects.},
journal = {Current genomics},
volume = {21},
number = {5},
pages = {321-333},
pmid = {33093796},
issn = {1389-2029},
abstract = {BACKGROUND: Enhanced agricultural production is essential for increasing demand of the growing world population. At the same time, to combat the adverse effects caused by conventional agriculture practices to the environment along with the impact on human health and food security, a sustainable and healthy agricultural production needs to be practiced using beneficial microorganisms for enhanced yield. It is quite challenging because these microorganisms have rich biosynthetic repositories to produce biomolecules of interest; however, the intensive research in allied sectors and emerging genetic tools for improved microbial consortia are accepting new approaches that are helpful to farmers and agriculturists to meet the ever-increasing demand of sustainable food production. An important advancement is improved strain development via genetically engineered microbial systems (GEMS) as well as genetically modified microorganisms (GMOs) possessing known and upgraded functional characteristics to promote sustainable agriculture and food security. With the development of novel technologies such as DNA automated synthesis, sequencing and influential computational tools, molecular biology has entered the systems biology and synthetic biology era. More recently, CRISPR/Cas has been engineered to be an important tool in genetic engineering for various applications in the agri sector. The research in sustainable agriculture is progressing tremendously through GMOs/GEMS for their potential use in biofertilizers and as biopesticides.<br><br>CONCLUSION: In this review, we discuss the beneficial effects of engineered microorganisms through integrated sustainable agriculture production practices to improve the soil microbial health in order to increase crop productivity.},
}
@article {pmid33093518,
year = {2020},
author = {Lissandrello, CA and Santos, JA and Hsi, P and Welch, M and Mott, VL and Kim, ES and Chesin, J and Haroutunian, NJ and Stoddard, AG and Czarnecki, A and Coppeta, JR and Freeman, DK and Flusberg, DA and Balestrini, JL and Tandon, V},
title = {High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18045},
pmid = {33093518},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/*methods ; Cells, Cultured ; Electroporation/*methods ; Gene Editing/*methods ; *Gene Transfer Techniques ; Humans ; Microfluidics/*methods ; RNA, Messenger/*genetics ; *T-Lymphocytes ; Transfection/*methods ; },
abstract = {Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10[Formula: see text]-10[Formula: see text] cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.},
}
@article {pmid33093479,
year = {2020},
author = {Güralp, H and Skaftnesmo, KO and Kjærner-Semb, E and Straume, AH and Kleppe, L and Schulz, RW and Edvardsen, RB and Wargelius, A},
title = {Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {18042},
pmid = {33093479},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; *Fisheries ; Genetic Introgression/*genetics ; *Germ Cells ; Infertility/*genetics ; Male ; Oocytes ; Quantitative Trait, Heritable ; RNA-Binding Proteins/*genetics ; Salmo salar/*embryology/*genetics ; Spermatogonia ; Triploidy ; },
abstract = {Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.},
}
@article {pmid33092788,
year = {2020},
author = {Liang, Z and Qin, Z and Riker, AI and Xi, Y},
title = {CRISPR/Cas9 ablating viral microRNA promotes lytic reactivation of Kaposi's sarcoma-associated herpesvirus.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {4},
pages = {1400-1405},
pmid = {33092788},
issn = {1090-2104},
support = {R01 CA192395/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Viral ; Genome, Viral ; Herpesvirus 8, Human/*genetics/pathogenicity ; Host-Pathogen Interactions/genetics ; Humans ; Latent Infection/*genetics ; Lymphoma, Primary Effusion/*virology ; MicroRNAs ; Oxygen/metabolism ; Precision Medicine/methods ; Sarcoma, Kaposi/virology ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system is an RNA-guided, DNA editing method that has been widely used for gene editing, including human viruses. Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), following latent infection in human cells, can cause a variety of malignancies, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), with a high prevalence in immunocompromised patients. Of significant concern, the latent infection with KSHV has been shown to lead to increased resistance to antiviral therapies. MicroRNAs (miRNAs) are a set of non-coding, small RNA molecules that regulate protein-coding genes at the post-transcriptional and translational levels. KSHV has its miRNAs, most of which are expressed in latently infected cells and play a crucial role in maintaining KSHV latency. Notably, by regulating the expression of the downstream target genes in host cells, KSHV miRNAs can interact with the host environment to promote the development of KSHV-related diseases. Although CRISPR/Cas9 has been reported to edit KSHV protein-coding genes, there is no published literature on whether the CRISPR/Cas9 system can regulate the expression of KSHV miRNAs. In this study, we used CRISPR/Cas9 to inhibit the expression of KSHV miRNAs by directly editing the DNA sequences of individual KSHV miRNAs, or the promoter of clustered KHSV miRNAs, in latent KSHV-infected PEL cells. Our results show that CRISPR/Cas9 can ablate KSHV miRNAs expression, which in turn leads to the upregulation of viral lytic genes and alteration of host cellular gene expression. To the best of our knowledge, our study is the first reported demonstration of the CRISPR/Cas9 system editing KSHV miRNAs, further expanding the application of CRISPR/Cas9 as a novel antiviral strategy targeting KSHV latency.},
}
@article {pmid33091225,
year = {2021},
author = {Lu, Y and Tian, Y and Shen, R and Yao, Q and Zhong, D and Zhang, X and Zhu, JK},
title = {Precise genome modification in tomato using an improved prime editing system.},
journal = {Plant biotechnology journal},
volume = {19},
number = {3},
pages = {415-417},
pmid = {33091225},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Lycopersicon esculentum/genetics ; },
}
@article {pmid33087932,
year = {2020},
author = {Wolter, JM and Mao, H and Fragola, G and Simon, JM and Krantz, JL and Bazick, HO and Oztemiz, B and Stein, JL and Zylka, MJ},
title = {Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA.},
journal = {Nature},
volume = {587},
number = {7833},
pages = {281-284},
pmid = {33087932},
issn = {1476-4687},
support = {R01 MH120125/MH/NIMH NIH HHS/United States ; 1R01NS109304-01A1/NH/NIH HHS/United States ; P50 HD103573/HD/NICHD NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; P30 NS045892/NS/NINDS NIH HHS/United States ; R00 MH102357/MH/NIMH NIH HHS/United States ; U54 HD079124/HD/NICHD NIH HHS/United States ; T32 HD040127/HD/NICHD NIH HHS/United States ; T32 NS007431/NS/NINDS NIH HHS/United States ; R01 MH118349/MH/NIMH NIH HHS/United States ; R01 NS109304/NS/NINDS NIH HHS/United States ; },
mesh = {Angelman Syndrome/*genetics/*therapy ; Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Female ; *Gene Editing ; Gene Silencing ; Genetic Therapy/*methods ; Genetic Vectors/genetics ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Nervous System/metabolism ; Paternal Inheritance/genetics ; Phenotype ; RNA, Guide/genetics ; RNA, Long Noncoding/*genetics ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest[1,2]. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.},
}
@article {pmid33087834,
year = {2020},
author = {Alghadban, S and Bouchareb, A and Hinch, R and Hernandez-Pliego, P and Biggs, D and Preece, C and Davies, B},
title = {Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17912},
pmid = {33087834},
issn = {2045-2322},
support = {106130/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; 203141/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Alleles ; Animals ; Animals, Genetically Modified/*embryology/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/*methods ; Embryonic Development/genetics ; Female ; *Gene Knock-In Techniques ; *Gene Transfer Techniques ; Mice, Inbred C57BL/*embryology/*genetics ; Microinjections/*methods ; Mutagenesis/genetics ; Oligodeoxyribonucleotides ; Ribonucleoproteins ; *Zygote ; },
abstract = {CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.},
}
@article {pmid33087788,
year = {2020},
author = {Sangsri, T and Saiprom, N and Tubsuwan, A and Monk, P and Partridge, LJ and Chantratita, N},
title = {Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17972},
pmid = {33087788},
issn = {2045-2322},
support = {U01AI115520/AI/NIAID NIH HHS/United States ; },
mesh = {A549 Cells ; Antibodies, Monoclonal ; Burkholderia pseudomallei/*pathogenicity ; CRISPR-Cas Systems ; *Cell Fusion ; Cells, Cultured ; Giant Cells/microbiology ; Humans ; Melioidosis/*microbiology/therapy ; Phagocytes/*physiology ; Tetraspanin 28 ; Tetraspanin 29 ; Tetraspanins/metabolism/*physiology ; },
abstract = {Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.},
}
@article {pmid33087559,
year = {2020},
author = {Lin, RJ and Kuo, MW and Yang, BC and Tsai, HH and Chen, K and Huang, JR and Lee, YS and Yu, AL and Yu, J},
title = {B3GALT5 knockout alters gycosphingolipid profile and facilitates transition to human naïve pluripotency.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {44},
pages = {27435-27444},
pmid = {33087559},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; Galactosyltransferases/genetics/*metabolism ; Gene Knockdown Techniques ; Glycosphingolipids/*metabolism ; Humans ; Pluripotent Stem Cells/*metabolism ; Stage-Specific Embryonic Antigens/*metabolism ; },
abstract = {Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca[2+], reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca[2+], and conducive for transition to naïve pluripotency.},
}
@article {pmid33086895,
year = {2022},
author = {Vats, S and Bansal, R and Rana, N and Kumawat, S and Bhatt, V and Jadhav, P and Kale, V and Sathe, A and Sonah, H and Jugdaohsingh, R and Sharma, TR and Deshmukh, R},
title = {Unexplored nutritive potential of tomato to combat global malnutrition.},
journal = {Critical reviews in food science and nutrition},
volume = {62},
number = {4},
pages = {1003-1034},
doi = {10.1080/10408398.2020.1832954},
pmid = {33086895},
issn = {1549-7852},
support = {MR/R005699/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Antioxidants ; Carotenoids ; Gene Editing ; Humans ; *Lycopersicon esculentum/genetics ; *Malnutrition ; },
abstract = {Tomato, a widely consumed vegetable crop, offers a real potential to combat human nutritional deficiencies. Tomatoes are rich in micronutrients and other bioactive compounds (including vitamins, carotenoids, and minerals) that are known to be essential or beneficial for human health. This review highlights the current state of the art in the molecular understanding of the nutritional aspects, conventional and molecular breeding efforts, and biofortification studies undertaken to improve the nutritional content and quality of tomato. Transcriptomics and metabolomics studies, which offer a deeper understanding of the molecular regulation of the tomato's nutrients, are discussed. The potential uses of the wastes from the tomato processing industry (i.e., the peels and seed extracts) that are particularly rich in oils and proteins are also discussed. Recent advancements with CRISPR/Cas mediated gene-editing technology provide enormous opportunities to enhance the nutritional content of agricultural produces, including tomatoes. In this regard, genome editing efforts with respect to biofortification in the tomato plant are also discussed. The recent technological advancements and knowledge gaps described herein aim to help explore the unexplored nutritional potential of the tomato.},
}
@article {pmid33086040,
year = {2020},
author = {Vink, JNA and Brouns, SJJ and Hohlbein, J},
title = {Extracting Transition Rates in Particle Tracking Using Analytical Diffusion Distribution Analysis.},
journal = {Biophysical journal},
volume = {119},
number = {10},
pages = {1970-1983},
pmid = {33086040},
issn = {1542-0086},
support = {639707/ERC_/European Research Council/International ; },
mesh = {*Algorithms ; Diffusion ; Escherichia coli ; Kinetics ; *Single Molecule Imaging ; },
abstract = {Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behavior of biomolecules in living cells.},
}
@article {pmid33085911,
year = {2021},
author = {Zimmer, AM and Mandic, M and Yew, HM and Kunert, E and Pan, YK and Ha, J and Kwong, RWM and Gilmour, KM and Perry, SF},
title = {Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio).},
journal = {American journal of physiology. Regulatory, integrative and comparative physiology},
volume = {320},
number = {1},
pages = {R55-R68},
doi = {10.1152/ajpregu.00215.2020},
pmid = {33085911},
issn = {1522-1490},
mesh = {*Acid-Base Equilibrium ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Carbonic Anhydrases/*deficiency/genetics ; Chlorides/*metabolism ; *Gene Knockout Techniques ; Hydrogen-Ion Concentration ; Ion Transport ; Mutation ; Sodium/*metabolism ; Zebrafish/embryology/genetics/*metabolism ; Zebrafish Proteins/*deficiency/genetics ; },
abstract = {In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish (Danio rerio) is expressed abundantly in Na[+]-absorbing/H[+]-secreting H[+]-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a[-/-] mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a[-/-] mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na[+] uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na[+] content remained constant. While Cl[-] uptake was significantly reduced in ca17a[-/-] mutants, Cl[-] content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl[-] uptake, implicating Ca17a in the mechanism of Cl[-] uptake by larval zebrafish. H[+] secretion, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a[-/-] mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl[-] uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.},
}
@article {pmid33085742,
year = {2020},
author = {Li, Q and Wang, M and Zhang, Y and Wang, L and Yu, W and Bao, X and Zhang, B and Xiang, Y and Deng, A},
title = {BIX-01294-enhanced chemosensitivity in nasopharyngeal carcinoma depends on autophagy-induced pyroptosis.},
journal = {Acta biochimica et biophysica Sinica},
volume = {52},
number = {10},
pages = {1131-1139},
doi = {10.1093/abbs/gmaa097},
pmid = {33085742},
issn = {1745-7270},
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/administration &amp; dosage/*pharmacology ; Autophagy/*drug effects ; Autophagy-Related Protein 5/genetics ; Azepines/administration &amp; dosage/*pharmacology ; CRISPR-Cas Systems ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Survival/drug effects ; Chloroquine/administration &amp; dosage/pharmacology ; Cisplatin/administration &amp; dosage/pharmacology ; Gene Knockout Techniques ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microtubule-Associated Proteins/metabolism ; Nasopharyngeal Carcinoma/*drug therapy/metabolism/pathology ; Nasopharyngeal Neoplasms/*drug therapy/metabolism/pathology ; Pyroptosis/*drug effects ; Quinazolines/administration &amp; dosage/*pharmacology ; Receptors, Estrogen/metabolism ; Signal Transduction/drug effects ; bcl-2-Associated X Protein/metabolism ; },
abstract = {Nasopharyngeal carcinoma (NPC) is a common cancer in southern China and Southeast Asia. Nowadays, radiotherapy is the therapy of choice for NPC patients, and chemotherapy has been found as an alternative treatment for advanced NPC patients. However, finding novel drugs and pharmacologically therapeutic targets for NPC patients is still urgent and beneficial. Our study showed that BIX-01294 (BIX) can induce autophagic vacuoles formation and conversion of LC3B-I to LC3B-II in NPC cells in both dose- and time-dependent manners. Notably, the combination of BIX and chemotherapeutic drugs significantly decreased the cell viability and increased the lactate dehydrogenase release. Meanwhile, BIX plus cis-platinum (Cis) treatment induced pyroptosis in NPC cells as featured by cell swelling and bubble blowing from the plasma membrane, the increased frequency of annexin V and propidium iodide (PI) double-positive cells, as well as the cleavage of gasdermin E (GSDME) and caspase-3. Moreover, the deficiency of GSDME completely shifted pyroptosis to apoptosis. Furthermore, the inhibition of autophagy by chloroquine and the knockout of ATG5 gene significantly blocked the BIX-induced autophagy as well as pyroptosis in both in vitro and in vivo studies. Our data demonstrated that BIX-combined chemotherapeutic drugs could induce the Bax/caspase-3/GSDME-mediated pyroptosis through the activation of autophagy to enhance the chemosensitivity in NPC.},
}
@article {pmid33085710,
year = {2020},
author = {Minguet, EG},
title = {Ares-GT: Design of guide RNAs targeting multiple genes for CRISPR-Cas experiments.},
journal = {PloS one},
volume = {15},
number = {10},
pages = {e0241001},
pmid = {33085710},
issn = {1932-6203},
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/*genetics ; *CRISPR-Cas Systems ; Programming Languages ; RNA, Guide/*genetics ; *Software ; },
abstract = {Guide RNA design for CRISPR genome editing of gene families is a challenging task as usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. To address this issues, I have developed ARES-GT, a Python local command line tool compatible with any operative system. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms. ARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).},
}
@article {pmid33084893,
year = {2021},
author = {Störtz, F and Minary, P},
title = {crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D855-D861},
pmid = {33084893},
issn = {1362-4962},
support = {BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S507593/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Biological Assay/*methods ; CRISPR-Cas Systems/*genetics ; *DNA Cleavage ; *Databases, Genetic ; Epigenesis, Genetic ; Streptococcus pyogenes/genetics ; User-Computer Interface ; },
abstract = {With ongoing development of the CRISPR/Cas programmable nuclease system, applications in the area of in vivo therapeutic gene editing are increasingly within reach. However, non-negligible off-target effects remain a major concern for clinical applications. Even though a multitude of off-target cleavage datasets have been published, a comprehensive, transparent overview tool has not yet been established. Here, we present crisprSQL (http://www.crisprsql.com), an interactive and bioinformatically enhanced collection of CRISPR/Cas9 off-target cleavage studies aimed at enriching the fields of cleavage profiling, gene editing safety analysis and transcriptomics. The current version of crisprSQL contains cleavage data from 144 guide RNAs on 25,632 guide-target pairs from human and rodent cell lines, with interaction-specific references to epigenetic markers and gene names. The first curated database of this standard, it promises to enhance safety quantification research, inform experiment design and fuel development of computational off-target prediction algorithms.},
}
@article {pmid33084874,
year = {2021},
author = {Gurumayum, S and Jiang, P and Hao, X and Campos, TL and Young, ND and Korhonen, PK and Gasser, RB and Bork, P and Zhao, XM and He, LJ and Chen, WH},
title = {OGEE v3: Online GEne Essentiality database with increased coverage of organisms and human cell lines.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D998-D1003},
pmid = {33084874},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Computational Biology/*methods ; Data Mining/methods ; *Databases, Genetic ; Genes, Essential/*genetics ; Genetic Predisposition to Disease/genetics ; Genomics/*methods ; Humans ; Internet ; Neoplasms/*genetics/pathology ; Oncogenes/*genetics ; RNA Interference ; },
abstract = {OGEE is an Online GEne Essentiality database. Gene essentiality is not a static and binary property, rather a context-dependent and evolvable property in all forms of life. In OGEE we collect not only experimentally tested essential and non-essential genes, but also associated gene properties that contributes to gene essentiality. We tagged conditionally essential genes that show variable essentiality statuses across datasets to highlight complex interplays between gene functions and environmental/experimental perturbations. OGEE v3 contains gene essentiality datasets for 91 species; almost doubled from 48 species in previous version. To accommodate recent advances on human cancer essential genes (as known as tumor dependency genes) that could serve as targets for cancer treatment and/or drug development, we expanded the collection of human essential genes from 16 cell lines in previous to 581. These human cancer cell lines were tested with high-throughput experiments such as CRISPR-Cas9 and RNAi; in total, 150 of which were tested by both techniques. We also included factors known to contribute to gene essentiality for these cell lines, such as genomic mutation, methylation and gene expression, along with extensive graphical visualizations for ease of understanding of these factors. OGEE v3 can be accessible freely at https://v3.ogee.info.},
}
@article {pmid33084577,
year = {2020},
author = {Little, JC and Garcia-Garcia, E and Sul, A and Kalderon, D},
title = {Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33084577},
issn = {2050-084X},
support = {R01 GM041815/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cloning, Molecular ; Cyclic AMP-Dependent Protein Kinases/genetics/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster ; Female ; Gene Expression Regulation/physiology ; Genotype ; Hedgehog Proteins/genetics/*metabolism ; Imaginal Discs/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Kinesins/genetics/*metabolism ; Male ; Repressor Proteins/genetics/*metabolism ; Signal Transduction ; Transcription Factors/genetics/*metabolism ; },
abstract = {Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.},
}
@article {pmid33083786,
year = {2020},
author = {Shang, Z and Chan, SY and Song, Q and Li, P and Huang, W},
title = {The Strategies of Pathogen-Oriented Therapy on Circumventing Antimicrobial Resistance.},
journal = {Research (Washington, D.C.)},
volume = {2020},
number = {},
pages = {2016201},
pmid = {33083786},
issn = {2639-5274},
abstract = {The emerging antimicrobial resistance (AMR) poses serious threats to the global public health. Conventional antibiotics have been eclipsed in combating with drug-resistant bacteria. Moreover, the developing and deploying of novel antimicrobial drugs have trudged, as few new antibiotics are being developed over time and even fewer of them can hit the market. Alternative therapeutic strategies to resolve the AMR crisis are urgently required. Pathogen-oriented therapy (POT) springs up as a promising approach in circumventing antibiotic resistance. The tactic underling POT is applying antibacterial compounds or materials directly to infected regions to treat specific bacteria species or strains with goals of improving the drug efficacy and reducing nontargeting and the development of drug resistance. This review exemplifies recent trends in the development of POTs for circumventing AMR, including the adoption of antibiotic-antibiotic conjugates, antimicrobial peptides, therapeutic monoclonal antibodies, nanotechnologies, CRISPR-Cas systems, and microbiota modulations. Employing these alternative approaches alone or in combination shows promising advantages for addressing the growing clinical embarrassment of antibiotics in fighting drug-resistant bacteria.},
}
@article {pmid33082952,
year = {2020},
author = {Montenarh, M and Götz, C},
title = {Protein kinase CK2 and ion channels (Review).},
journal = {Biomedical reports},
volume = {13},
number = {6},
pages = {55},
pmid = {33082952},
issn = {2049-9434},
abstract = {Protein kinase CK2 appears as a tetramer or higher molecular weight oligomer composed of catalytic CK2α, CK2α' subunits and non-catalytic regulatory CK2β subunits or as individual subunits. It is implicated in a variety of different regulatory processes, such as Akt signalling, splicing and DNA repair within eukaryotic cells. The present review evaluates the influence of CK2 on ion channels in the plasma membrane. CK2 phosphorylates platform proteins such as calmodulin and ankyrin G, which bind to channel proteins for a physiological transport to and positioning into the membrane. In addition, CK2 directly phosphorylates a variety of channel proteins directly to regulate opening and closing of the channels. Thus, modulation of CK2 activities by specific inhibitors, by siRNA technology or by CRISPR/Cas technology has an influence on intracellular ion concentrations and thereby on cellular signalling. The physiological regulation of the intracellular ion concentration is important for cell survival and correct intracellular signalling. Disturbance of this regulation results in a variety of different diseases including epilepsy, heart failure, cystic fibrosis and diabetes. Therefore, these effects should be considered when using CK2 inhibition as a treatment option for cancer.},
}
@article {pmid33082438,
year = {2020},
author = {Lee, J and Sheen, JH and Lim, O and Lee, Y and Ryu, J and Shin, D and Kim, YY and Kim, M},
title = {Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17753},
pmid = {33082438},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/*methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; HLA Antigens/*genetics ; Humans ; T-Lymphocytes/*metabolism ; },
abstract = {As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.},
}
@article {pmid33082254,
year = {2020},
author = {Afonina, I and Ong, J and Chua, J and Lu, T and Kline, KA},
title = {Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis.},
journal = {mBio},
volume = {11},
number = {5},
pages = {},
pmid = {33082254},
issn = {2150-7511},
mesh = {Bacterial Proteins/genetics/metabolism ; Biofilms/*growth &amp; development ; *CRISPR-Cas Systems ; Enterococcus faecalis/*genetics/*physiology ; Genes, Essential ; },
abstract = {Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication.IMPORTANCEEnterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.},
}
@article {pmid33082141,
year = {2020},
author = {Pinci, F and Gaidt, MM and Jung, C and Kuut, G and Jackson, MA and Bauernfried, S and Hornung, V},
title = {C-tag TNF: a reporter system to study TNF shedding.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {52},
pages = {18065-18075},
pmid = {33082141},
issn = {1083-351X},
mesh = {ADAM Proteins/genetics/*metabolism ; *Genes, Reporter ; HEK293 Cells ; Humans ; Image Processing, Computer-Assisted/*methods ; Molecular Imaging/*methods ; Protein Kinase C/genetics/*metabolism ; Proteolysis ; Tumor Necrosis Factor-alpha/genetics/*metabolism ; },
abstract = {TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.},
}
@article {pmid33081820,
year = {2020},
author = {Ten Hacken, E and Clement, K and Li, S and Hernández-Sánchez, M and Redd, R and Wang, S and Ruff, D and Gruber, M and Baranowski, K and Jacob, J and Flynn, J and Jones, KW and Neuberg, D and Livak, KJ and Pinello, L and Wu, CJ},
title = {High throughput single-cell detection of multiplex CRISPR-edited gene modifications.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {266},
pmid = {33081820},
issn = {1474-760X},
support = {P01 CA081534/CA/NCI NIH HHS/United States ; R35 HG010717/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U10 CA180861/CA/NCI NIH HHS/United States ; R01 CA216273/CA/NCI NIH HHS/United States ; P01 CA206978/CA/NCI NIH HHS/United States ; UG1 CA233338/CA/NCI NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R50 CA251956/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; High-Throughput Screening Assays ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/*genetics ; *Loss of Function Mutation ; Mice ; Single-Cell Analysis/*methods ; },
abstract = {CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.},
}
@article {pmid33081350,
year = {2020},
author = {Fernandez-Garcia, L and Pacios, O and González-Bardanca, M and Blasco, L and Bleriot, I and Ambroa, A and López, M and Bou, G and Tomás, M},
title = {Viral Related Tools against SARS-CoV-2.},
journal = {Viruses},
volume = {12},
number = {10},
pages = {},
pmid = {33081350},
issn = {1999-4915},
mesh = {Antibodies, Viral/immunology/therapeutic use ; Antiviral Agents/therapeutic use ; Bacteriophages ; Betacoronavirus/isolation &amp; purification ; COVID-19 ; CRISPR-Cas Systems ; Coronavirus Infections/*therapy/*virology ; Humans ; Pandemics ; Phage Therapy ; Pneumonia, Viral/*therapy/*virology ; SARS-CoV-2 ; Viral Vaccines/immunology ; },
abstract = {At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure.},
}
@article {pmid33080227,
year = {2020},
author = {Xing, L and Kalebic, N and Namba, T and Vaid, S and Wimberger, P and Huttner, WB},
title = {Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex.},
journal = {Neuron},
volume = {108},
number = {6},
pages = {1113-1129.e6},
doi = {10.1016/j.neuron.2020.09.034},
pmid = {33080227},
issn = {1097-4199},
support = {G0701018/MRC_/Medical Research Council/United Kingdom ; G1100578/MRC_/Medical Research Council/United Kingdom ; MR/N004272/1/MRC_/Medical Research Council/United Kingdom ; MR/R006237/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Proliferation/drug effects/*physiology ; Ferrets ; Gene Expression Regulation, Developmental ; Lateral Ventricles/*cytology/drug effects/metabolism ; Mice ; Neocortex/*cytology/drug effects/metabolism ; Neural Stem Cells/*cytology/drug effects/metabolism ; Neurogenesis/drug effects/physiology ; Receptor, Serotonin, 5-HT2A/genetics/*metabolism ; Serotonin/pharmacology ; },
abstract = {Evolutionary expansion of the mammalian neocortex (Ncx) has been linked to increased abundance and proliferative capacity of basal progenitors (BPs) in the subventricular zone during development. BP proliferation is governed by both intrinsic and extrinsic signals, several of which have been identified. However, a role of neurotransmitters, a canonical class of extrinsic signaling molecules, in BP proliferation remains to be established. Here, we show that serotonin (5-HT), via its receptor HTR2A, promotes BP proliferation in an evolutionarily relevant manner. HTR2A is not expressed in embryonic mouse Ncx; accordingly, 5-HT does not increase mouse BP proliferation. However, ectopic HTR2A expression can increase mouse BP proliferation. Conversely, CRISPR/Cas9-mediated knockout of endogenous HTR2A in embryonic ferret Ncx reduces BP proliferation. Pharmacological activation of endogenous HTR2A in fetal human Ncx ex vivo increases BP proliferation via HER2/ERK signaling. Hence, 5-HT emerges as an important extrinsic pro-proliferative signal for BPs, which may have contributed to evolutionary Ncx expansion.},
}
@article {pmid33080209,
year = {2020},
author = {Hawkins, JS and Silvis, MR and Koo, BM and Peters, JM and Osadnik, H and Jost, M and Hearne, CC and Weissman, JS and Todor, H and Gross, CA},
title = {Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.},
journal = {Cell systems},
volume = {11},
number = {5},
pages = {523-535.e9},
pmid = {33080209},
issn = {2405-4720},
support = {F32 GM108222/GM/NIGMS NIH HHS/United States ; T32 GM007810/GM/NIGMS NIH HHS/United States ; U01 CA168370/CA/NCI NIH HHS/United States ; F32 GM116331/GM/NIGMS NIH HHS/United States ; R35 GM118061/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; K22 AI137122/AI/NIAID NIH HHS/United States ; K99 GM130964/GM/NIGMS NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacillus subtilis/*genetics/metabolism ; Bacterial Proteins/metabolism ; CRISPR-Cas Systems ; Escherichia coli/*genetics/metabolism ; Gene Expression/genetics ; Gene Expression Regulation, Bacterial/genetics ; Genes, Essential/*genetics/physiology ; Genetic Fitness/genetics ; },
abstract = {Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coli and Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coli and B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.},
}
@article {pmid33079628,
year = {2021},
author = {Tyagi, S and Kumar, R and Kumar, V and Won, SY and Shukla, P},
title = {Engineering disease resistant plants through CRISPR-Cas9 technology.},
journal = {GM crops &amp; food},
volume = {12},
number = {1},
pages = {125-144},
pmid = {33079628},
issn = {2164-5701},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Gene Editing ; Plant Diseases/genetics ; },
abstract = {Plants are susceptible to phytopathogens, including bacteria, fungi, and viruses, which cause colossal financial shortfalls (pre- and post-harvest) and threaten global food safety. To combat with these phytopathogens, plant possesses two-layer of defense in the form of PAMP-triggered immunity (PTI), or Effectors-triggered immunity (ETI). The understanding of plant-molecular interactions and revolution of high-throughput molecular techniques have opened the door for innovations in developing pathogen-resistant plants. In this context, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has transformed genome editing (GE) technology and being harnessed for altering the traits. Here we have summarized the complexities of plant immune system and the use of CRISPR-Cas9 to edit the various components of plant immune system to acquire long-lasting resistance in plants against phytopathogens. This review also sheds the light on the limitations of CRISPR-Cas9 system, regulation of CRISPR-Cas9 edited crops and future prospective of this technology.},
}
@article {pmid33079482,
year = {2021},
author = {Lin, PC and Liu, R and Alvin, K and Wahyu, S and Murgolo, N and Ye, J and Du, Z and Song, Z},
title = {Improving Antibody Production in Stably Transfected CHO Cells by CRISPR-Cas9-Mediated Inactivation of Genes Identified in a Large-Scale Screen with Chinese Hamster-Specific siRNAs.},
journal = {Biotechnology journal},
volume = {16},
number = {3},
pages = {e2000267},
doi = {10.1002/biot.202000267},
pmid = {33079482},
issn = {1860-7314},
mesh = {Animals ; *Antibody Formation ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; RNA, Small Interfering/genetics ; Recombinant Proteins/genetics ; },
abstract = {The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.},
}
@article {pmid33078994,
year = {2020},
author = {Lenskaia, T and Boley, D},
title = {Prokaryote autoimmunity in the context of self-targeting by CRISPR-Cas systems.},
journal = {Journal of bioinformatics and computational biology},
volume = {18},
number = {5},
pages = {2050033},
doi = {10.1142/S021972002050033X},
pmid = {33078994},
issn = {1757-6334},
mesh = {Archaea/genetics/*immunology ; Autoimmunity/*genetics ; Bacteria/genetics/*immunology ; *CRISPR-Cas Systems ; Genome, Archaeal ; Genome, Bacterial ; Microorganisms, Genetically-Modified/genetics/*immunology ; Plasmids/genetics ; Prokaryotic Cells/physiology ; },
abstract = {Prokaryote adaptive immunity (CRISPR-Cas systems) can be a threat to its carriers. We analyze the risks of autoimmune reactions related to adaptive immunity in prokaryotes by computational methods. We found important differences between bacteria and archaea with respect to autoimmunity potential. According to the results of our analysis, CRISPR-Cas systems in bacteria are more prone to self-targeting even though they possess fewer spacers per organism on average than archaea. The results of our study provide opportunities to use self-targeting in prokaryotes for biological and medical applications.},
}
@article {pmid33078813,
year = {2020},
author = {Xu, Y and Liu, R and Dai, Z},
title = {Key considerations in designing CRISPR/Cas9-carrying nanoparticles for therapeutic genome editing.},
journal = {Nanoscale},
volume = {12},
number = {41},
pages = {21001-21014},
doi = {10.1039/d0nr05452f},
pmid = {33078813},
issn = {2040-3372},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Transfer Techniques ; *Nanoparticles ; },
abstract = {CRISPR-Cas9, the breakthrough genome-editing technology, has emerged as a promising tool to prevent and cure various diseases. The efficient genome editing technology strongly relies on the specific and effective delivery of CRISPR/Cas9 cargos. However, the lack of a safe, specific, and efficient non-viral delivery system for in vivo genome editing remains a major limit for its clinical translation. In this review, we will first briefly introduce the working mechanism of CRISPR/Cas9 and the patterns of CRISPR/Cas9 delivery. Furthermore, the physiological obstacles for the delivery process in vivo are elaborated. Finally, the key considerations will be deeply discussed in designing non-viral nanovectors for therapeutic CRISPR/Cas9 delivery in vivo, including the effective encapsulation of large-size macromolecules, targeting specific tissues and cells, efficient endosomal escape and safety concerns of the vector systems, in the hope of inviting more comprehensive studies on the development of safe, specific, and efficient non-viral nanovectors for delivering a CRISPR/Cas9 system.},
}
@article {pmid33077967,
year = {2020},
author = {Csörgő, B and León, LM and Chau-Ly, IJ and Vasquez-Rifo, A and Berry, JD and Mahendra, C and Crawford, ED and Lewis, JD and Bondy-Denomy, J},
title = {A compact Cascade-Cas3 system for targeted genome engineering.},
journal = {Nature methods},
volume = {17},
number = {12},
pages = {1183-1190},
pmid = {33077967},
issn = {1548-7105},
support = {DP5 OD021344/OD/NIH HHS/United States ; T32 GM007810/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Base Sequence/genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Helicases/*metabolism ; Escherichia coli/genetics ; Escherichia coli Proteins/*metabolism ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial/genetics ; Klebsiella pneumoniae/genetics ; Pseudomonas aeruginosa/genetics ; Pseudomonas syringae/genetics ; Sequence Deletion/genetics ; },
abstract = {CRISPR-Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7-424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade-Cas3, but not Cas9. A transferable 'all-in-one' vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR-Cas use was enhanced with an 'anti-anti-CRISPR' strategy. P. aeruginosa Type I-C Cascade-Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.},
}
@article {pmid33077937,
year = {2020},
author = {Porto, EM and Komor, AC and Slaymaker, IM and Yeo, GW},
title = {Base editing: advances and therapeutic opportunities.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {12},
pages = {839-859},
pmid = {33077937},
issn = {1474-1784},
support = {R01 EY029166/EY/NEI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; R21 GM135736/GM/NIGMS NIH HHS/United States ; T32 GM008326/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Disease/*genetics ; *Gene Editing ; Hearing Loss/genetics/*therapy ; Humans ; Liver Diseases/genetics/*therapy ; Muscular Dystrophy, Duchenne/genetics/*therapy ; Neoplasms/genetics/*therapy ; Polymorphism, Single Nucleotide ; },
abstract = {Base editing - the introduction of single-nucleotide variants (SNVs) into DNA or RNA in living cells - is one of the most recent advances in the field of genome editing. As around half of known pathogenic genetic variants are due to SNVs, base editing holds great potential for the treatment of numerous genetic diseases, through either temporary RNA or permanent DNA base alterations. Recent advances in the specificity, efficiency, precision and delivery of DNA and RNA base editors are revealing exciting therapeutic opportunities for these technologies. We expect the correction of single point mutations will be a major focus of future precision medicine.},
}
@article {pmid33077929,
year = {2020},
author = {Pilosof, S and Alcalá-Corona, SA and Wang, T and Kim, T and Maslov, S and Whitaker, R and Pascual, M},
title = {The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversification.},
journal = {Nature ecology &amp; evolution},
volume = {4},
number = {12},
pages = {1650-1660},
pmid = {33077929},
issn = {2397-334X},
mesh = {*Biological Evolution ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {As a heritable sequence-specific adaptive immune system, CRISPR-Cas is a powerful force shaping strain diversity in host-virus systems. While the diversity of CRISPR alleles has been explored, the associated structure and dynamics of host-virus interactions have not. We explore the role of CRISPR in mediating the interplay between host-virus interaction structure and eco-evolutionary dynamics in a computational model and compare the results with three empirical datasets from natural systems. We show that the structure of the networks describing who infects whom and the degree to which strains are immune, are respectively modular (containing groups of hosts and viruses that interact strongly) and weighted-nested (specialist hosts are more susceptible to subsets of viruses that in turn also infect the more generalist hosts with many spacers matching many viruses). The dynamic interplay between these networks influences transitions between dynamical regimes of virus diversification and host control. The three empirical systems exhibit weighted-nested immunity networks, a pattern our theory shows is indicative of hosts able to suppress virus diversification. Previously missing from studies of microbial host-pathogen systems, the immunity network plays a key role in the coevolutionary dynamics.},
}
@article {pmid33077249,
year = {2021},
author = {Ng Kwan Lim, E and Sasseville, C and Carrier, MC and Massé, E},
title = {Keeping Up with RNA-Based Regulation in Bacteria: New Roles for RNA Binding Proteins.},
journal = {Trends in genetics : TIG},
volume = {37},
number = {1},
pages = {86-97},
doi = {10.1016/j.tig.2020.09.014},
pmid = {33077249},
issn = {0168-9525},
mesh = {Bacteria/*genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; RNA, Bacterial/*genetics ; RNA, Small Untranslated/*genetics ; RNA-Binding Proteins/genetics/*metabolism ; },
abstract = {RNA binding proteins (RBPs) are ubiquitously found in all kingdoms of life. They are involved in a plethora of regulatory events, ranging from direct regulation of gene expression to guiding modification of RNA molecules. As bacterial regulators, RBPs can act alone or in concert with RNA-based regulators, such as small regulatory RNAs (sRNAs), riboswitches, or clustered regularly interspaced short palindromic repeats (CRISPR) RNAs. Various functions of RBPs, whether dependent or not on an RNA regulator, have been described in the past. However, the past decade has been a fertile ground for the development of novel high-throughput methods. These methods acted as stepping-stones for the discovery of new functions of RBPs and helped in the understanding of the molecular mechanisms behind previously described regulatory events. Here, we present an overview of the recently identified roles of major bacterial RBPs from different model organisms. Moreover, the tight relationship between RBPs and RNA-based regulators will be explored.},
}
@article {pmid33076970,
year = {2020},
author = {Brinton, LT and Zhang, P and Williams, K and Canfield, D and Orwick, S and Sher, S and Wasmuth, R and Beaver, L and Cempre, C and Skinner, J and Cannon, M and Govande, M and Harrington, B and Lehman, A and Byrd, JC and Lapalombella, R and Blachly, JS},
title = {Synergistic effect of BCL2 and FLT3 co-inhibition in acute myeloid leukemia.},
journal = {Journal of hematology &amp; oncology},
volume = {13},
number = {1},
pages = {139},
pmid = {33076970},
issn = {1756-8722},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; R35 CA197734/CA/NCI NIH HHS/United States ; R01 CA223165/CA/NCI NIH HHS/United States ; },
mesh = {Aniline Compounds/therapeutic use ; Animals ; Antineoplastic Agents/therapeutic use ; Bridged Bicyclo Compounds, Heterocyclic/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Knockout Techniques ; Genetic Therapy ; Humans ; Leukemia, Myeloid, Acute/genetics/*therapy ; Mice, SCID ; Protein Kinase Inhibitors/*therapeutic use ; Proto-Oncogene Proteins c-bcl-2/*genetics ; Pyrazines/therapeutic use ; Staurosporine/analogs &amp; derivatives/therapeutic use ; Sulfonamides/therapeutic use ; fms-Like Tyrosine Kinase 3/*antagonists &amp; inhibitors ; },
abstract = {Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.},
}
@article {pmid33076477,
year = {2020},
author = {Jin, S and Bae, J and Song, Y and Pearcy, N and Shin, J and Kang, S and Minton, NP and Soucaille, P and Cho, BK},
title = {Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.},
journal = {International journal of molecular sciences},
volume = {21},
number = {20},
pages = {},
pmid = {33076477},
issn = {1422-0067},
mesh = {Acetates/*metabolism ; Biodegradation, Environmental ; Clostridium/genetics/metabolism ; Genetic Engineering/*methods ; Industrial Microbiology/*methods ; Natural Gas/*microbiology ; Synthetic Biology/methods ; },
abstract = {Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.},
}
@article {pmid33076176,
year = {2021},
author = {Li, H and Xing, S and Xu, J and He, Y and Lai, Y and Wang, Y and Zhang, G and Guo, S and Deng, M and Zeng, M and Liu, W},
title = {Aptamer-based CRISPR/Cas12a assay for the ultrasensitive detection of extracellular vesicle proteins.},
journal = {Talanta},
volume = {221},
number = {},
pages = {121670},
doi = {10.1016/j.talanta.2020.121670},
pmid = {33076176},
issn = {1873-3573},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Extracellular Vesicles ; Oligonucleotides ; Prognosis ; },
abstract = {Tumor-derived extracellular vesicles (TEVs) have emerged as promising sources of diagnostic and prognostic biomarkers for nasopharyngeal carcinoma (NPC). However, the lack of high-sensitivity analytic methods for ultratrace membrane proteins on TEVs hamper their clinical application of TEVs. Herein, by combining aptamers that specifically bind to protein targets on TEVs, PCR-based exponential amplification and CRISPR/Cas12a real-time DNA detection, we developed a novel technique, termed the aptamer-CRISPR/Cas12a assay, to detect CD109[+] and EGFR[+] TEVs from cell lines and complex biofluids. The platform enables highly sensitive detection of CD109[+] and EGFR[+] TEVs at as low as 100 particles/mL with a linear range spanning 6 orders of magnitude (10[2]-10[8] particles/mL), which was found to be sufficient to effectively detect TEV proteins directly in low-volume (50 μl) samples. Furthermore, clinical serum sample analysis verified that the combination of serum CD109[+] and EGFR[+] TEV levels yielded high diagnostic accuracy, with an AUC of 0.934 (95% CI: 0.868-1.000), a sensitivity of 84.1% and a specificity of 85.0%, in discriminating NPC from healthy controls. Moreover, the dramatic decrease in both biomarkers in responders after radiotherapy indicated their potential roles in radiotherapy surveillance. Given that the aptamer-CRISPR/Cas12a assay rapidly and conveniently detects ultralow concentrations of CD109[+] and EGFR[+] TEVs directly in serum, it could be useful in NPC diagnosis and prognosis.},
}
@article {pmid33075468,
year = {2021},
author = {Wang, J and Yang, J and Li, D and Li, J},
title = {Technologies for targeting DNA methylation modifications: Basic mechanism and potential application in cancer.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1875},
number = {1},
pages = {188454},
doi = {10.1016/j.bbcan.2020.188454},
pmid = {33075468},
issn = {1879-2561},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Methylation/*genetics ; Epigenesis, Genetic/genetics ; Genome, Human/genetics ; Humans ; Molecular Biology/*methods ; Neoplasms/*genetics/pathology ; Protein Processing, Post-Translational/genetics ; },
abstract = {DNA methylation abnormalities are regarded as critical event for cancer initiation and development. Tumor-associated genes encompassing aberrant DNA methylation alterations at specific locus are correlated with chromatin remodeling and dysregulation of gene expression in various malignancies. Thus, technologies designed to manipulate DNA methylation at specific loci of genome are necessary for the functional study and therapeutic application in the context of cancer management. Traditionally, the method for DNA methylation modifications demonstrates an unspecific feature, adversely causing global-genome epigenetic alterations and confusing the function of desired gene. Novel approaches for targeted DNA methylation regulation have a great advantage of manipulating gene epigenetic alterations in a more specific and efficient method. In this review, we described different targeting DNA methylation techniques, including both their advantages and limitations. Through a comprehensive understanding of these targeting tools, we hope to open a new perspective for cancer treatment.},
}
@article {pmid33075436,
year = {2020},
author = {Murphy, ZC and Getman, MR and Myers, JA and Burgos Villar, KN and Leshen, E and Kurita, R and Nakamura, Y and Steiner, LA},
title = {Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.},
journal = {Experimental hematology},
volume = {91},
number = {},
pages = {32-38.e6},
pmid = {33075436},
issn = {1873-2399},
support = {R01 DK104920/DK/NIDDK NIH HHS/United States ; },
mesh = {Acetylation ; Anemia, Dyserythropoietic, Congenital/blood/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/ultrastructure ; Cell Survival ; Chromatin/ultrastructure ; Erythroid Cells/*cytology/metabolism ; Erythropoiesis/*genetics/physiology ; Exons/genetics ; Gene Editing ; Glycoproteins/deficiency/*genetics/physiology ; Histone Code ; Humans ; Nuclear Proteins/deficiency/*genetics/physiology ; Phenotype ; Protein Processing, Post-Translational ; },
abstract = {The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.},
}
@article {pmid33074543,
year = {2021},
author = {McDougall, A and Hebras, C and Gomes, I and Dumollard, R},
title = {Gene Editing in the Ascidian Phallusia mammillata and Tail Nerve Cord Formation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2219},
number = {},
pages = {217-230},
pmid = {33074543},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Microinjections ; Urochordata/*embryology/*genetics/ultrastructure ; },
abstract = {Functional approaches for studying embryonic development have greatly advanced thanks to the CRISPR-Cas9 gene editing technique. Previously practiced in just a few organisms, these knockout techniques are now widely applied. Here we describe simple techniques for applying the CRISPR-Cas9 system to study the development of the nerve cord in the ascidian Phallusia mammillata.},
}
@article {pmid33074435,
year = {2021},
author = {Tran, MT and Doan, DTH and Kim, J and Song, YJ and Sung, YW and Das, S and Kim, EJ and Son, GH and Kim, SH and Van Vu, T and Kim, JY},
title = {CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {999-1011},
pmid = {33074435},
issn = {1432-203X},
mesh = {Agrobacterium tumefaciens/genetics ; Alleles ; *CRISPR-Cas Systems ; Cloning, Molecular ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genome, Plant ; Germination/genetics ; Lycopersicon esculentum/*genetics/physiology ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics/physiology ; Protein Domains ; RNA, Guide ; Salt Stress/genetics/*physiology ; Transformation, Bacterial ; },
abstract = {CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.},
}
@article {pmid33074412,
year = {2020},
author = {Dhokane, D and Bhadra, B and Dasgupta, S},
title = {CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein.},
journal = {Molecular biology reports},
volume = {47},
number = {11},
pages = {8747-8755},
pmid = {33074412},
issn = {1573-4978},
mesh = {Biotechnology ; *CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Tryptophan Synthase/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular alga Chlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase β-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.},
}
@article {pmid33074086,
year = {2020},
author = {Hampton, HG and Smith, LM and Ferguson, S and Meaden, S and Jackson, SA and Fineran, PC},
title = {Functional genomics reveals the toxin-antitoxin repertoire and AbiE activity in Serratia.},
journal = {Microbial genomics},
volume = {6},
number = {11},
pages = {},
pmid = {33074086},
issn = {2057-5858},
mesh = {Bacteriophages/*growth &amp; development ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Gene Expression Regulation, Bacterial/genetics ; Genome, Bacterial/genetics ; Nucleotidyltransferases/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Transfer/genetics ; Serratia/*genetics/pathogenicity ; Toxin-Antitoxin Systems/*genetics ; },
abstract = {Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin-antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to 'shut down', limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.},
}
@article {pmid33073517,
year = {2020},
author = {Sharma, S and Dincer, C and Weidemüller, P and Wright, GJ and Petsalaki, E},
title = {CEN-tools: an integrative platform to identify the contexts of essential genes.},
journal = {Molecular systems biology},
volume = {16},
number = {10},
pages = {e9698},
pmid = {33073517},
issn = {1744-4292},
support = {/WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Computational Biology/*methods ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; Gene Regulatory Networks ; *Genes, Essential ; Humans ; Melanoma/*genetics/*metabolism/pathology ; Metabolomics/*methods ; Mutation ; SOXE Transcription Factors/genetics/metabolism ; Serum Response Factor/genetics/metabolism ; Signal Transduction/genetics ; Skin Neoplasms/*genetics/*metabolism/pathology ; Software ; },
abstract = {An emerging theme from large-scale genetic screens that identify genes essential for cell fitness is that essentiality of a given gene is highly context-specific. Identification of such contexts could be the key to defining gene function and also to develop novel therapeutic interventions. Here, we present Context-specific Essentiality Network-tools (CEN-tools), a website and python package, in which users can interrogate the essentiality of a gene from large-scale genome-scale CRISPR screens in a number of biological contexts including tissue of origin, mutation profiles, expression levels and drug responses. We show that CEN-tools is suitable for the systematic identification of genetic dependencies and for more targeted queries. The associations between genes and a given context are represented as dependency networks (CENs), and we demonstrate the utility of these networks in elucidating novel gene functions. In addition, we integrate the dependency networks with existing protein-protein interaction networks to reveal context-dependent essential cellular pathways in cancer cells. Together, we demonstrate the applicability of CEN-tools in aiding the current efforts to define the human cellular dependency map.},
}
@article {pmid33073252,
year = {2020},
author = {Zhong, A and Li, M and Zhou, T},
title = {Protocol for the Generation of Human Pluripotent Reporter Cell Lines Using CRISPR/Cas9.},
journal = {STAR protocols},
volume = {1},
number = {2},
pages = {},
pmid = {33073252},
issn = {2666-1667},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cells, Cultured ; Clone Cells/cytology ; Gene Editing/*methods ; Humans ; Pluripotent Stem Cells/*cytology ; },
abstract = {Reporter cell lines based on human pluripotent stem cells (hPSCs) are highly desirable for studying differentiation, lineage tracing, and target cell selection. However, several technical bottlenecks, such as DNA transduction, low homology recombination rate (HDR), and single-cell cloning, have made this effort an arduous process in hPSCs. Here, we provide a step-by-step protocol and practical guide for generating reporter lines in hPSCs via CRISPR/Cas9-mediated HDR. We also elaborate on the process of generating a TBXT-GFP reporter line as an example.},
}
@article {pmid33072091,
year = {2020},
author = {D'Agostino, G and Artinger, M and Locati, M and Perez, L and Legler, DF and Bianchi, ME and Rüegg, C and Thelen, M and Marchese, A and Rocchi, MBL and Cecchinato, V and Uguccioni, M},
title = {β-Arrestin1 and β-Arrestin2 Are Required to Support the Activity of the CXCL12/HMGB1 Heterocomplex on CXCR4.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {550824},
pmid = {33072091},
issn = {1664-3224},
support = {R01 GM106727/GM/NIGMS NIH HHS/United States ; },
mesh = {Actins/chemistry/metabolism ; CRISPR-Cas Systems ; Chemokine CXCL12/*metabolism ; Chemotaxis ; Gene Editing ; Gene Knockdown Techniques ; HMGB1 Protein/*metabolism ; HeLa Cells ; Humans ; Multiprotein Complexes/metabolism ; Protein Binding ; Protein Multimerization ; Protein Transport ; Receptors, CXCR4/*metabolism ; beta-Arrestin 1/genetics/*metabolism ; beta-Arrestin 2/genetics/*metabolism ; },
abstract = {The chemokine receptor CXCR4 plays a fundamental role in homeostasis and pathology by orchestrating recruitment and positioning of immune cells, under the guidance of a CXCL12 gradient. The ability of chemokines to form heterocomplexes, enhancing their function, represents an additional level of regulation on their cognate receptors. In particular, the multi-faceted activity of the heterocomplex formed between CXCL12 and the alarmin HMGB1 is emerging as an unexpected player able to modulate a variety of cell responses, spanning from tissue regeneration to chronic inflammation. Nowadays, little is known on the selective signaling pathways activated when CXCR4 is triggered by the CXCL12/HMGB1 heterocomplex. In the present work, we demonstrate that this heterocomplex acts as a CXCR4 balanced agonist, activating both G protein and β-arrestins-mediated signaling pathways to sustain chemotaxis. We generated β-arrestins knock out HeLa cells by CRISPR/Cas9 technology and show that the CXCL12/HMGB1 heterocomplex-mediated actin polymerization is primarily β-arrestin1 dependent, while chemotaxis requires both β-arrestin1 and β-arrestin2. Triggering of CXCR4 with the CXCL12/HMGB1 heterocomplex leads to an unexpected receptor retention on the cell surface, which depends on β-arrestin2. In conclusion, the CXCL12/HMGB1 heterocomplex engages the β-arrestin proteins differently from CXCL12, promoting a prompt availability of CXCR4 on the cell surface, and enhancing directional cell migration. These data unveil the signaling induced by the CXCL12/HMGB1 heterocomplex in view of identifying biased CXCR4 antagonists or agonists targeting the variety of functions it exerts.},
}
@article {pmid33072060,
year = {2020},
author = {Zhang, Z and Li, Y and Luo, L and Hao, J and Li, J},
title = {Corrigendum: Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {595238},
doi = {10.3389/fmicb.2020.595238},
pmid = {33072060},
issn = {1664-302X},
abstract = {[This corrects the article DOI: 10.3389/fmicb.2020.01824.].},
}
@article {pmid33072011,
year = {2020},
author = {Yimer, SA and Kalayou, S and Homberset, H and Birhanu, AG and Riaz, T and Zegeye, ED and Lutter, T and Abebe, M and Holm-Hansen, C and Aseffa, A and Tønjum, T},
title = {Lineage-Specific Proteomic Signatures in the Mycobacterium tuberculosis Complex Reveal Differential Abundance of Proteins Involved in Virulence, DNA Repair, CRISPR-Cas, Bioenergetics and Lipid Metabolism.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {550760},
pmid = {33072011},
issn = {1664-302X},
abstract = {Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7.},
}
@article {pmid33070670,
year = {2020},
author = {Mandl, M and Ritthammer, H and Ejaz, A and Wagner, SA and Hatzmann, FM and Baumgarten, S and Viertler, HP and Zwierzina, ME and Mattesich, M and Schiller, V and Rauchenwald, T and Ploner, C and Waldegger, P and Pierer, G and Zwerschke, W},
title = {CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.},
journal = {Adipocyte},
volume = {9},
number = {1},
pages = {626-635},
pmid = {33070670},
issn = {2162-397X},
mesh = {Adipose Tissue/*cytology ; Biomarkers ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; *Gene Editing ; *Gene Knockout Techniques ; Genes, Reporter ; Genetic Vectors/genetics ; Humans ; Mutation ; RNA, Small Interfering/genetics ; Stem Cells/*metabolism ; },
abstract = {The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.},
}
@article {pmid33070614,
year = {2020},
author = {Luo, YL and Liang, LF and Gan, YJ and Liu, J and Zhang, Y and Fan, YN and Zhao, G and Czarna, A and Lu, ZD and Du, XJ and Shen, S and Xu, CF and Lian, ZX and Wang, J},
title = {An All-in-One Nanomedicine Consisting of CRISPR-Cas9 and an Autoantigen Peptide for Restoring Specific Immune Tolerance.},
journal = {ACS applied materials &amp; interfaces},
volume = {12},
number = {43},
pages = {48259-48271},
doi = {10.1021/acsami.0c10885},
pmid = {33070614},
issn = {1944-8252},
mesh = {Animals ; Autoantigens/*immunology ; CRISPR-Cas Systems/*immunology ; Cell Engineering ; Cells, Cultured ; Dendritic Cells ; Humans ; Immune Tolerance ; Mice ; Mice, Inbred NOD ; *Nanomedicine ; Particle Size ; Peptides/*immunology ; Surface Properties ; },
abstract = {Nanotechnology has shown great promise in treating diverse diseases. However, developing nanomedicines that can cure autoimmune diseases without causing systemic immunosuppression is still quite challenging. Herein, we propose an all-in-one nanomedicine comprising an autoantigen peptide and CRISPR-Cas9 to restore specific immune tolerance by engineering dendritic cells (DCs) into a tolerogenic phenotype, which can expand autoantigen-specific regulatory T (Treg) cells. In brief, we utilized cationic lipid-assisted poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles to simultaneously encapsulate an autoimmune diabetes-relevant peptide (2.5mi), a CRISPR-Cas9 plasmid (pCas9), and three guide RNAs (gRNAs) targeting costimulatory molecules (CD80, CD86, and CD40). We demonstrated that the all-in-one nanomedicine was able to effectively codeliver these components into DCs, followed by simultaneous disruption of the three costimulatory molecules and presentation of the 2.5mi peptide on the genome-edited DCs. The resulting tolerogenic DCs triggered the generation and expansion of autoantigen-specific Treg cells by presenting the 2.5mi peptide to CD4[+] T cells in the absence of costimulatory signals. Using autoimmune type 1 diabetes (T1D) as a typical disease model, we demonstrated that our nanomedicine prevented autoimmunity to islet components and inhibited T1D development. Our all-in-one nanomedicine achieved codelivery of CRISPR-Cas9 and the peptide to DCs and could be easily applied to other autoimmune diseases by substitution of different autoantigen peptides.},
}
@article {pmid33069955,
year = {2021},
author = {Du, YC and Wang, SY and Wang, YX and Ma, JY and Wang, DX and Tang, AN and Kong, DM},
title = {Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background.},
journal = {Biosensors &amp; bioelectronics},
volume = {171},
number = {},
pages = {112734},
doi = {10.1016/j.bios.2020.112734},
pmid = {33069955},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Nucleotidylexotransferase ; *Uracil-DNA Glycosidase/genetics/metabolism ; },
abstract = {A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10[-6] U/mL and 1 × 10[-4] U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.},
}
@article {pmid33069188,
year = {2020},
author = {Hameed, A and Hussain, SA and Ijaz, MU and Umer, M},
title = {Deletions of the Idh1, Eco1, Rom2, and Taf10 Genes Differently Control the Hyphal Growth, Drug Tolerance, and Virulence of Candida albicans.},
journal = {Folia biologica},
volume = {66},
number = {3},
pages = {91-103},
pmid = {33069188},
issn = {0015-5500},
mesh = {Acetyltransferases/deficiency/genetics/physiology ; Antifungal Agents/pharmacology ; CRISPR-Cas Systems ; Calcium/physiology ; Candida albicans/drug effects/genetics/pathogenicity/*physiology ; Cations/pharmacology ; Cell Adhesion ; Cell Cycle ; Cell Wall/drug effects ; Chitinases/pharmacology ; DNA Damage ; Fungal Proteins/genetics/*physiology ; Gene Deletion ; *Genes, Fungal ; Glucan Endo-1,3-beta-D-Glucosidase/pharmacology ; Hyphae/growth &amp; development ; Isocitrate Dehydrogenase/deficiency/genetics/physiology ; Open Reading Frames ; Reproduction, Asexual ; TATA-Binding Protein Associated Factors/deficiency/genetics/physiology ; Virulence/genetics ; },
abstract = {The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.},
}
@article {pmid33068435,
year = {2021},
author = {Huang, L and Yang, B and Yi, H and Asif, A and Wang, J and Lithgow, T and Zhang, H and Minhas, FUAA and Yin, Y},
title = {AcrDB: a database of anti-CRISPR operons in prokaryotes and viruses.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D622-D629},
pmid = {33068435},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; *Databases, Genetic ; Internet ; Operon/*genetics ; Prokaryotic Cells/*metabolism ; Viruses/*metabolism ; },
abstract = {CRISPR-Cas is an anti-viral mechanism of prokaryotes that has been widely adopted for genome editing. To make CRISPR-Cas genome editing more controllable and safer to use, anti-CRISPR proteins have been recently exploited to prevent excessive/prolonged Cas nuclease cleavage. Anti-CRISPR (Acr) proteins are encoded by (pro)phages/(pro)viruses, and have the ability to inhibit their host's CRISPR-Cas systems. We have built an online database AcrDB (http://bcb.unl.edu/AcrDB) by scanning ∼19 000 genomes of prokaryotes and viruses with AcrFinder, a recently developed Acr-Aca (Acr-associated regulator) operon prediction program. Proteins in Acr-Aca operons were further processed by two machine learning-based programs (AcRanker and PaCRISPR) to obtain numerical scores/ranks. Compared to other anti-CRISPR databases, AcrDB has the following unique features: (i) It is a genome-scale database with the largest collection of data (39 799 Acr-Aca operons containing Aca or Acr homologs); (ii) It offers a user-friendly web interface with various functions for browsing, graphically viewing, searching, and batch downloading Acr-Aca operons; (iii) It focuses on the genomic context of Acr and Aca candidates instead of individual Acr protein family and (iv) It collects data with three independent programs each having a unique data mining algorithm for cross validation. AcrDB will be a valuable resource to the anti-CRISPR research community.},
}
@article {pmid33068434,
year = {2020},
author = {Lin, J and Liu, Y and Lai, P and Ye, H and Xu, L},
title = {Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions.},
journal = {Nucleic acids research},
volume = {48},
number = {20},
pages = {11773-11784},
pmid = {33068434},
issn = {1362-4962},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; HEK293 Cells ; Humans ; MicroRNAs/metabolism ; RNA/chemistry/*metabolism ; RNA, Small Untranslated/metabolism ; Transcriptional Activation ; },
abstract = {A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.},
}
@article {pmid33068408,
year = {2020},
author = {Eki, R and She, J and Parlak, M and Benamar, M and Du, KP and Kumar, P and Abbas, T},
title = {A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {e126},
pmid = {33068408},
issn = {1362-4962},
support = {R01 GM135376/GM/NIGMS NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/metabolism ; Biological Assay/*methods ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; DNA Ligase ATP/metabolism ; *DNA Repair ; DNA Repair Enzymes/metabolism ; DNA-Binding Proteins/metabolism ; Fluorescent Dyes/*chemistry ; *Genes, Reporter ; HEK293 Cells ; Humans ; },
abstract = {DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.},
}
@article {pmid33068406,
year = {2021},
author = {Dwane, L and Behan, FM and Gonçalves, E and Lightfoot, H and Yang, W and van der Meer, D and Shepherd, R and Pignatelli, M and Iorio, F and Garnett, MJ},
title = {Project Score database: a resource for investigating cancer cell dependencies and prioritizing therapeutic targets.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D1365-D1372},
pmid = {33068406},
issn = {1362-4962},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/*genetics ; CRISPR-Cas Systems ; Carcinogenesis/drug effects/genetics/metabolism/pathology ; Cell Line, Tumor ; *Databases, Factual ; *Gene Expression Regulation, Neoplastic ; Genetic Fitness ; *Genome, Human ; Humans ; Internet ; Molecular Targeted Therapy ; Neoplasms/drug therapy/*genetics/metabolism/pathology ; Oncogenes ; *Software ; },
abstract = {CRISPR genetic screens in cancer cell models are a powerful tool to elucidate oncogenic mechanisms and to identify promising therapeutic targets. The Project Score database (https://score.depmap.sanger.ac.uk/) uses genome-wide CRISPR-Cas9 dropout screening data in hundreds of highly annotated cancer cell models to identify genes required for cell fitness and prioritize novel oncology targets. The Project Score database currently allows users to investigate the fitness effect of 18 009 genes tested across 323 cancer cell models. Through interactive interfaces, users can investigate data by selecting a specific gene, cancer cell model or tissue type, as well as browsing all gene fitness scores. Additionally, users can identify and rank candidate drug targets based on an established oncology target prioritization pipeline, incorporating genetic biomarkers and clinical datasets for each target, and including suitability for drug development based on pharmaceutical tractability. Data are freely available and downloadable. To enhance analyses, links to other key resources including Open Targets, COSMIC, the Cell Model Passports, UniProt and the Genomics of Drug Sensitivity in Cancer are provided. The Project Score database is a valuable new tool for investigating genetic dependencies in cancer cells and the identification of candidate oncology targets.},
}
@article {pmid33068118,
year = {2021},
author = {Chan, AN and Wang, LL and Zhu, YJ and Fan, YY and Zhuang, JY and Zhang, ZH},
title = {Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling grain weight in rice.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {134},
number = {1},
pages = {327-337},
pmid = {33068118},
issn = {1432-2242},
mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; Gene Editing ; Gene Knockout Techniques ; Genes, Plant ; Mutagenesis ; Oryza/*genetics ; *Quantitative Trait Loci ; Seeds/*growth &amp; development ; },
abstract = {A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect. The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)[3]/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9-2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8-9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.},
}
@article {pmid33067443,
year = {2020},
author = {Huang, X and Sun, W and Cheng, Z and Chen, M and Li, X and Wang, J and Sheng, G and Gong, W and Wang, Y},
title = {Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5241},
pmid = {33067443},
issn = {2041-1723},
mesh = {Catalysis ; Catalytic Domain ; DNA Cleavage ; DNA, Single-Stranded/chemistry/genetics/*metabolism ; Deoxyribonuclease I/*chemistry/genetics/*metabolism ; Ions/chemistry/metabolism ; Metals/chemistry/*metabolism ; },
abstract = {To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2-crRNA-DNA complexes and a Cas12i2-crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1-8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9-15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.},
}
@article {pmid33067194,
year = {2020},
author = {Le, Y and Fu, Y and Sun, J},
title = {Genome Editing of the Anaerobic Thermophile Thermoanaerobacter ethanolicus Using Thermostable Cas9.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {1},
pages = {},
pmid = {33067194},
issn = {1098-5336},
mesh = {Anaerobiosis ; Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Ethanol/metabolism ; Fermentation ; *Gene Editing ; Thermoanaerobacter/enzymology/*genetics ; },
abstract = {Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen, and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to the utilization of both pentose and hexose. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9-based system for genome editing of T. ethanolicus As a proof of principle, three genes, including the thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE), and redox sensing protein gene (rsp), were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knockout and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome-editing efficiency was observed. Furthermore, our in vivo results revealed that redox sensing protein (RSP) plays a more important role in regulation of energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us with an effective strategy to identify genes involved in biosynthesis and energy metabolism.IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. On the basis of the biochemical experiments of previous investigation, the formation of ethanol was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here, we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus The thermostable Cas9-based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system represents an important expansion of the genetic tool box of anaerobic thermophile T. ethanolicus strains.},
}
@article {pmid33066557,
year = {2020},
author = {Saha, C and Horst-Kreft, D and Kross, I and van der Spek, PJ and Louwen, R and van Baarlen, P},
title = {Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {33066557},
issn = {2073-4425},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; Caco-2 Cells ; Campylobacter Infections/genetics/metabolism/*microbiology ; Campylobacter jejuni/genetics/*metabolism ; Epithelial Cells/metabolism/microbiology/*pathology ; Gene Expression Profiling ; *Gene Expression Regulation ; Humans ; Intestines/microbiology/*pathology ; *Transcriptome ; },
abstract = {The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.},
}
@article {pmid33065764,
year = {2021},
author = {Baars, MJD and Douma, T and Simeonov, DR and Myers, DR and Kulhanek, K and Banerjee, S and Zwakenberg, S and Baltissen, MP and Amini, M and de Roock, S and van Wijk, F and Vermeulen, M and Marson, A and Roose, JP and Vercoulen, Y},
title = {Dysregulated RASGRP1 expression through RUNX1 mediated transcription promotes autoimmunity.},
journal = {European journal of immunology},
volume = {51},
number = {2},
pages = {471-482},
pmid = {33065764},
issn = {1521-4141},
support = {/CRI/Cancer Research Institute/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; P01 AI091580/AI/NIAID NIH HHS/United States ; R01 DK119979/DK/NIDDK NIH HHS/United States ; R01 AI104789/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity/*genetics/immunology ; CD4-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems/genetics/immunology ; Core Binding Factor Alpha 2 Subunit/*genetics/immunology ; DNA-Binding Proteins/*genetics/immunology ; Gene Expression Regulation/genetics/immunology ; Guanine Nucleotide Exchange Factors/*genetics/immunology ; Histones/genetics/immunology ; Humans ; Inflammation/genetics/immunology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Transcription, Genetic/*genetics/immunology ; },
abstract = {RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4[+] T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4[+] T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.},
}
@article {pmid33060832,
year = {2020},
author = {Hiraga, K and Inoue, YU and Asami, J and Hotta, M and Morimoto, Y and Tatsumoto, S and Hoshino, M and Go, Y and Inoue, T},
title = {Redundant type II cadherins define neuroepithelial cell states for cytoarchitectonic robustness.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {574},
pmid = {33060832},
issn = {2399-3642},
mesh = {Animals ; CRISPR-Cas Systems ; Cadherins/*genetics/*metabolism ; Cell Differentiation/genetics ; Chromosome Mapping ; Embryonic Development/genetics ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression Regulation, Developmental ; Gene Targeting ; Genomics/methods ; Humans ; Immunohistochemistry ; Mice ; Neural Plate/embryology/metabolism ; Neural Tube/embryology/metabolism ; Neuroepithelial Cells/*metabolism ; },
abstract = {Individual cell shape and integrity must precisely be orchestrated during morphogenesis. Here, we determine function of type II cadherins, Cdh6, Cdh8, and Cdh11, whose expression combinatorially demarcates the mouse neural plate/tube. While CRISPR/Cas9-based single type II cadherin mutants show no obvious phenotype, Cdh6/8 double knockout (DKO) mice develop intermingled forebrain/midbrain compartments as these two cadherins' expression opposes at the nascent boundary. Cdh6/8/11 triple, Cdh6/8 or Cdh8/11 DKO mice further cause exencephaly just within the cranial region where mutated cadherins' expression merges. In the Cdh8/11 DKO midbrain, we observe less-constricted apical actin meshwork, ventrally-directed spreading, and occasional hyperproliferation among dorsal neuroepithelial cells as origins for exencephaly. These results provide rigid evidence that, by conferring distinct adhesive codes to each cell, redundant type II cadherins serve essential and shared roles in compartmentalization and neurulation, both of which proceed under the robust control of the number, positioning, constriction, and fluidity of neuroepithelial cells.},
}
@article {pmid33060800,
year = {2020},
author = {},
title = {Henceforth CRISPR.},
journal = {Nature biomedical engineering},
volume = {4},
number = {11},
pages = {1023},
pmid = {33060800},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/history/*methods ; History, 21st Century ; Humans ; Nobel Prize ; },
abstract = {In less than a decade, the genome-editing technology now recognized by the Nobel Prize in Chemistry has impacted the biological and biomedical sciences widely. What’s next for CRISPR in biomedicine?},
}
@article {pmid33060647,
year = {2020},
author = {Møller, TC and Pedersen, MF and van Senten, JR and Seiersen, SD and Mathiesen, JM and Bouvier, M and Bräuner-Osborne, H},
title = {Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17395},
pmid = {33060647},
issn = {2045-2322},
support = {FDN-148431//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; Endocytosis/*physiology ; G-Protein-Coupled Receptor Kinase 2/genetics/*physiology ; G-Protein-Coupled Receptor Kinase 3/genetics/*physiology ; Gene Editing ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Phosphorylation ; Receptors, Opioid, mu/*metabolism ; Reproducibility of Results ; beta-Arrestin 2/*metabolism ; },
abstract = {Most G protein-coupled receptors (GPCRs) recruit β-arrestins and internalize upon agonist stimulation. For the μ-opioid receptor (μ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for μ-OR recruitment of β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in β-arrestin2 recruitment and μ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced μ-OR internalization and β-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of β-arrestin2 recruitment to the plasma membrane upon μ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.},
}
@article {pmid33060338,
year = {2020},
author = {Cohen, J},
title = {A cut above: pair that developed CRISPR earns historic award.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6514},
pages = {271-272},
doi = {10.1126/science.370.6514.271},
pmid = {33060338},
issn = {1095-9203},
mesh = {Animals ; *CRISPR-Cas Systems ; Chemistry/*history ; Embryo Research/ethics/history ; France ; Gene Editing/*history ; History, 21st Century ; Humans ; *Nobel Prize ; Patents as Topic/history ; United States ; },
}
@article {pmid33060287,
year = {2021},
author = {Walsh, T and Casadei, S and Munson, KM and Eng, M and Mandell, JB and Gulsuner, S and King, MC},
title = {CRISPR-Cas9/long-read sequencing approach to identify cryptic mutations in BRCA1 and other tumour suppressor genes.},
journal = {Journal of medical genetics},
volume = {58},
number = {12},
pages = {850-852},
pmid = {33060287},
issn = {1468-6244},
support = {R35 CA197458/CA/NCI NIH HHS/United States ; },
mesh = {BRCA1 Protein/*genetics ; BRCA2 Protein/genetics ; Breast Neoplasms/genetics ; *CRISPR-Cas Systems ; Exons/genetics ; Family Health ; Female ; *Genes, Tumor Suppressor ; Germ-Line Mutation ; Humans ; Introns/genetics ; Mutagenesis, Insertional ; *Mutation ; Promoter Regions, Genetic/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Reproducibility of Results ; Retroelements/genetics ; Sequence Analysis, DNA/*methods ; },
abstract = {Current clinical approaches for mutation discovery are based on short sequence reads (100-300 bp) of exons and flanking splice sites targeted by multigene panels or whole exomes. Short-read sequencing is highly accurate for detection of single nucleotide variants, small indels and simple copy number differences but is of limited use for identifying complex insertions and deletions and other structural rearrangements. We used CRISPR-Cas9 to excise complete BRCA1 and BRCA2 genomic regions from lymphoblast cells of patients with breast cancer, then sequenced these regions with long reads (>10 000 bp) to fully characterise all non-coding regions for structural variation. In a family severely affected with early-onset bilateral breast cancer and with negative (normal) results by gene panel and exome sequencing, we identified an intronic SINE-VNTR-Alu retrotransposon insertion that led to the creation of a pseudoexon in the BRCA1 message and introduced a premature truncation. This combination of CRISPR-Cas9 excision and long-read sequencing reveals a class of complex, damaging and otherwise cryptic mutations that may be particularly frequent in tumour suppressor genes replete with intronic repeats.},
}
@article {pmid33060138,
year = {2020},
author = {Han, Z and Lo, WS and Lightfoot, JW and Witte, H and Sun, S and Sommer, RJ},
title = {Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition in Pristionchus Nematodes.},
journal = {Genetics},
volume = {216},
number = {4},
pages = {947-956},
pmid = {33060138},
issn = {1943-2631},
mesh = {Animals ; *CRISPR-Cas Systems ; *Codon Usage ; Gene Editing/*methods/standards ; Introns ; Rhabditida/*genetics ; *Transgenes ; },
abstract = {A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.},
}
@article {pmid33059726,
year = {2020},
author = {Dede, M and McLaughlin, M and Kim, E and Hart, T},
title = {Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {262},
pmid = {33059726},
issn = {1474-760X},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119//National Institute of General Medical Sciences (US)/International ; },
mesh = {A549 Cells ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Genes, Essential ; HT29 Cells ; Humans ; Neoplasms/*genetics ; },
abstract = {BACKGROUND: Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell.<br><br>RESULTS: Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes.<br><br>CONCLUSIONS: Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.},
}
@article {pmid33059029,
year = {2021},
author = {Kondrateva, E and Demchenko, A and Lavrov, A and Smirnikhina, S},
title = {An overview of currently available molecular Cas-tools for precise genome modification.},
journal = {Gene},
volume = {769},
number = {},
pages = {145225},
doi = {10.1016/j.gene.2020.145225},
pmid = {33059029},
issn = {1879-0038},
mesh = {*CRISPR-Cas Systems ; DNA Damage ; Epigenesis, Genetic ; *Gene Editing ; *Genome ; },
abstract = {CRISPR-Cas system was first mentioned in 1987, and over the years have been studied so active that now it becomes the state-of-the-art tool for genome editing. Its working principle is based on Cas nuclease ability to bind short RNA, which targets it to complementary DNA or RNA sequence for highly precise cleavage. This alone or together with donor DNA allows to modify targeted sequence in different ways. Considering the many limitations of using native CRISPR-Cas systems, scientists around the world are working on creating modified variants to improve their specificity and efficiency in different objects. In addition, the use of the Cas effectors' targeting function in complex systems with other proteins is a promising work direction, as a result of which new tools are created with features such as single base editing, editing DNA without break and donor DNA, activation and repression of transcription, epigenetic regulation, modifying of different repair pathways involvement etc. In this review, we decided to consider in detail exactly this issue of variants of Cas effectors, their modifications and fusion molecules, which improve DNA-targeting and expand the scope of Cas effectors.},
}
@article {pmid33058471,
year = {2021},
author = {Xiong, X and Liang, J and Li, Z and Gong, BQ and Li, JF},
title = {Multiplex and optimization of dCas9-TV-mediated gene activation in plants.},
journal = {Journal of integrative plant biology},
volume = {63},
number = {4},
pages = {634-645},
doi = {10.1111/jipb.13023},
pmid = {33058471},
issn = {1744-7909},
mesh = {Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/genetics ; },
abstract = {Synthetic gene activators consisting of nuclease-dead Cas9 (dCas9) for single-guide RNA (sgRNA)-directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9-TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9-TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA-sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000-fold in rice. Moreover, the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9-TV-refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.},
}
@article {pmid33058410,
year = {2020},
author = {Poretsky, E and Dressano, K and Weckwerth, P and Ruiz, M and Char, SN and Shi, D and Abagyan, R and Yang, B and Huffaker, A},
title = {Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.},
journal = {The Plant journal : for cell and molecular biology},
volume = {104},
number = {6},
pages = {1582-1602},
doi = {10.1111/tpj.15022},
pmid = {33058410},
issn = {1365-313X},
support = {GM131881/NH/NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Genes, Plant/genetics ; Peptides/metabolism/*physiology ; Phylogeny ; *Plant Defense Against Herbivory ; Plant Growth Regulators/metabolism/physiology ; Receptors, Peptide/genetics/metabolism/physiology ; Zea mays/genetics/immunology/metabolism/*physiology ; },
abstract = {Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.},
}
@article {pmid33058300,
year = {2021},
author = {Li, XJ and Li, QL and Ju, LG and Zhao, C and Zhao, LS and Du, JW and Wang, Y and Zheng, L and Song, BL and Li, LY and Li, L and Wu, M},
title = {Deficiency of Histone Methyltransferase SET Domain-Containing 2 in Liver Leads to Abnormal Lipid Metabolism and HCC.},
journal = {Hepatology (Baltimore, Md.)},
volume = {73},
number = {5},
pages = {1797-1815},
doi = {10.1002/hep.31594},
pmid = {33058300},
issn = {1527-3350},
mesh = {Alanine Transaminase/blood ; Animals ; Aspartate Aminotransferases/blood ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*etiology/metabolism ; Cell Line, Tumor ; Cholesterol/blood ; Chromatin Immunoprecipitation ; Gene Editing ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; Hep G2 Cells ; Histone-Lysine N-Methyltransferase/*deficiency/metabolism ; Humans ; *Lipid Metabolism ; Liver/*metabolism ; Liver Neoplasms/*etiology/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Triglycerides/blood ; },
abstract = {BACKGROUND AND AIMS: Trimethylation of Lys36 on histone 3 (H3K36me3) catalyzed by histone methyltransferase SET domain-containing 2 (SETD2) is one of the most conserved epigenetic marks from yeast to mammals. SETD2 is frequently mutated in multiple cancers and acts as a tumor suppressor.<br><br>APPROACH AND RESULTS: Here, using a liver-specific Setd2 depletion model, we found that Setd2 deficiency is sufficient to trigger spontaneous HCC. Meanwhile, Setd2 depletion significantly increased tumor and tumor size of a diethylnitrosamine-induced HCC model. The mechanistic study showed that Setd2 suppresses HCC not only through modulating DNA damage response, but also by regulating lipid metabolism in the liver. Setd2 deficiency down-regulated H3K36me3 enrichment and expression of cholesterol efflux genes and caused lipid accumulation. High-fat diet enhanced lipid accumulation and promoted the development of HCC in Setd2-deficient mice. Chromatin immunoprecipitation sequencing analysis further revealed that Setd2 depletion induced c-Jun/activator protein 1 (AP-1) activation in the liver, which was trigged by accumulated lipid. c-Jun acts as an oncogene in HCC and functions through inhibiting p53 in Setd2-deficient cells.<br><br>CONCLUSIONS: We revealed the roles of Setd2 in HCC and the underlying mechanisms in regulating cholesterol homeostasis and c-Jun/AP-1 signaling.},
}
@article {pmid33057942,
year = {2020},
author = {Zha, J and Lai, Q and Deng, M and Shi, P and Zhao, H and Chen, Q and Wu, H and Xu, B},
title = {Disruption of CTCF Boundary at HOXA Locus Promote BET Inhibitors' Therapeutic Sensitivity in Acute Myeloid Leukemia.},
journal = {Stem cell reviews and reports},
volume = {16},
number = {6},
pages = {1280-1291},
doi = {10.1007/s12015-020-10057-y},
pmid = {33057942},
issn = {2629-3277},
mesh = {Apoptosis/drug effects ; Azepines/pharmacology ; CCCTC-Binding Factor/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/drug effects ; Cell Line, Tumor ; Gene Expression Regulation, Leukemic/drug effects ; *Genetic Loci ; Homeodomain Proteins/*metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics/*metabolism/pathology ; Nucleophosmin ; RNA Polymerase II/metabolism ; Transcription, Genetic/drug effects ; Transcriptional Elongation Factors/metabolism ; Triazoles/pharmacology ; },
abstract = {Both HOX gene expression and CTCF regulation have been well demonstrated to play a critical role in regulating maintenance of leukemic stem cells (LSCs) that are known to be resistant to BET inhibitor (BETi). To investigate the regulatory role of CTCF boundary in aberrant HOX gene expression and the therapeutic sensitivity of BETi in AML, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the CTCF binding motif which is located between HOXA7 and HOXA9 genes (CBS7/9) in different subtypes of AML with either MLL-rearrangement or NPM1 mutation. Our results revealed that HOXA9 is significantly downregulated in response to the CBS7/9 deletion. Moreover, CBS7/9 boundary deletion sensitized the BETi treatment reaction in both MOLM-13 and OCI-AML3 cells. To further examine whether BETi therapeutic sensitivity in AML is depended on the expression level of the HOXA9 gene, we overexpressed the HOXA9 in the CBS7/9 deleted AML cell lines, which can rescue and restore the resistance to BETi treatment of the CBS7/9 KO cells by activating MAPK signaling pathway. Deletion of CBS7/9 specifically decreased the recruitment of BRD4 and RNA pol II to the posterior HOXA genes, in which, a transcription elongation factor ELL3 is the key factor in regulating HOXA gene transcription monitored by CBS7/9 chromatin boundary. Thus, disruption of CBS7/9 boundary perturbs HOXA9 transcription and regulates BETi sensitivity in AML treatment. Moreover, alteration of CTCF boundaries in the oncogene loci may provide a novel strategy to overcome the drug resistance of LSCs. Graphical abstract.},
}
@article {pmid33057691,
year = {2021},
author = {Gu, S and Li, J and Li, S and Cao, J and Bu, J and Ren, Y and Du, W and Chen, Z and Xu, C and Wang, M and Jiang, L and Huang, C and Du, J},
title = {Efficient replacement of long DNA fragments via non-homologous end joining at non-coding regions.},
journal = {Journal of molecular cell biology},
volume = {13},
number = {1},
pages = {75-77},
pmid = {33057691},
issn = {1759-4685},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Gene Editing/*methods ; RNA, Guide ; Tyrosine 3-Monooxygenase/genetics ; Zebrafish/genetics ; Zebrafish Proteins/genetics ; },
}
@article {pmid33057575,
year = {2021},
author = {Schimenti, JC},
title = {Conditional surrender in one generation: determining the reproductive roles of mouse embryo lethal genes by embryo complementation.},
journal = {Biology of reproduction},
volume = {104},
number = {1},
pages = {8-10},
doi = {10.1093/biolre/ioaa193},
pmid = {33057575},
issn = {1529-7268},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Targeting ; *Genes, Lethal ; Mice ; Reproduction ; },
abstract = {The laboratory mouse is the most widely used animal model for studying the genetics and biology of mammalian development and reproduction. Embryonic stem cell (ESC) gene targeting technology, and the sophisticated genomic manipulations it allowed, was unique to this organism for a long period of time; this was a major factor in the mouse's rise to pre-eminence as a model system over the past three decades or so. The recent advent of CRISPR/Cas9 technology has democratized the application of genome editing to essentially all organisms. Nevertheless, the scientific infrastructure behind the mouse still makes it the organism of choice for studying molecular mechanisms of mammalian development, and for modeling human development and disease.},
}
@article {pmid33057400,
year = {2020},
author = {Maroc, L and Zhou-Li, Y and Boisnard, S and Fairhead, C},
title = {A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1008627},
pmid = {33057400},
issn = {1553-7404},
mesh = {CRISPR-Cas Systems/genetics ; Candida glabrata/*genetics ; Cell Death/genetics ; Chromosomes, Fungal/genetics ; DNA Breaks, Double-Stranded ; Deoxyribonucleases, Type II Site-Specific/*genetics ; Endonucleases/*genetics ; Gene Expression Regulation, Fungal/genetics ; Genes, Mating Type, Fungal/*genetics ; Homologous Recombination/genetics ; Rad51 Recombinase/genetics ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/*genetics ; },
abstract = {Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. In the model species Saccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT, repaired by homologous recombination (HR) using one of the two silent mating-type loci, HMLalpha and HMRa. The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the yeast Candida glabrata, phylogenetically close to S. cerevisiae, and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S. cerevisiae's Ho (ScHo) gene triggers mating-type switching in C. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by ScHo in wild-type strains of C. glabrata. To understand the link between mating-type switching and cell death in C. glabrata, we constructed strains mutated precisely at the Ho recognition sites. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. We demonstrate that mating-type switching in C. glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S. cerevisiae, but that donor preference is not conserved in C. glabrata. Altogether, these results suggest that a DSB at MAT can be repaired by HR in C. glabrata, but that repair is prevented by ScHo.},
}
@article {pmid33057349,
year = {2020},
author = {Gallagher, DN and Pham, N and Tsai, AM and Janto, NV and Choi, J and Ira, G and Haber, JE},
title = {A Rad51-independent pathway promotes single-strand template repair in gene editing.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1008689},
pmid = {33057349},
issn = {1553-7404},
support = {R01 GM080600/GM/NIGMS NIH HHS/United States ; R01 GM125650/GM/NIGMS NIH HHS/United States ; R35 GM127029/GM/NIGMS NIH HHS/United States ; T32 GM007122/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA Helicases/genetics ; DNA Repair/*genetics ; DNA, Single-Stranded/*genetics ; DNA-Binding Proteins/genetics ; DNA-Directed DNA Polymerase/genetics ; Deoxyribonucleases, Type II Site-Specific/*genetics ; Endodeoxyribonucleases/genetics ; Endonucleases/*genetics ; Exodeoxyribonucleases/genetics ; Oligonucleotides/genetics ; Rad51 Recombinase/genetics ; Rad52 DNA Repair and Recombination Protein/genetics ; Rec A Recombinases/genetics ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/*genetics ; },
abstract = {The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.},
}
@article {pmid33057331,
year = {2020},
author = {Liu, J and Zuo, Z and Zou, M and Finkel, T and Liu, S},
title = {Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1009068},
pmid = {33057331},
issn = {1553-7404},
support = {P30 AG024827/AG/NIA NIH HHS/United States ; R01 AI145879/AI/NIAID NIH HHS/United States ; R56 AI148134/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; BTB-POZ Domain/genetics ; CRISPR-Cas Systems/genetics ; Elongation Factor 2 Kinase/*genetics ; Gene Expression Regulation/genetics ; Histidine/*analogs &amp; derivatives/biosynthesis/genetics ; Humans ; Kruppel-Like Transcription Factors/*genetics ; Methyltransferases ; Mice ; Minor Histocompatibility Antigens/*genetics ; Promoter Regions, Genetic/genetics ; Protein Binding/genetics ; Protein Processing, Post-Translational/genetics ; RAW 264.7 Cells ; Transcription Factors/genetics ; Tumor Suppressor Proteins/*genetics ; },
abstract = {Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.},
}
@article {pmid33057195,
year = {2020},
author = {Song, M and Pebworth, MP and Yang, X and Abnousi, A and Fan, C and Wen, J and Rosen, JD and Choudhary, MNK and Cui, X and Jones, IR and Bergenholtz, S and Eze, UC and Juric, I and Li, B and Maliskova, L and Lee, J and Liu, W and Pollen, AA and Li, Y and Wang, T and Hu, M and Kriegstein, AR and Shen, Y},
title = {Cell-type-specific 3D epigenomes in the developing human cortex.},
journal = {Nature},
volume = {587},
number = {7835},
pages = {644-649},
pmid = {33057195},
issn = {1476-4687},
support = {R01 AG057497/AG/NIA NIH HHS/United States ; U01 HG009391/HG/NHGRI NIH HHS/United States ; R01 MH106611/MH/NIMH NIH HHS/United States ; U41 HG010972/HG/NHGRI NIH HHS/United States ; R01 HL129132/HL/NHLBI NIH HHS/United States ; U01 MH114825/MH/NIMH NIH HHS/United States ; R35 NS097305/NS/NINDS NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; S10 OD021822/OD/NIH HHS/United States ; U01 CA200060/CA/NCI NIH HHS/United States ; U54 HD079124/HD/NICHD NIH HHS/United States ; R21 HG010065/HG/NHGRI NIH HHS/United States ; R25 DA027995/DA/NIDA NIH HHS/United States ; U24 ES026699/ES/NIEHS NIH HHS/United States ; T32 GM007175/GM/NIGMS NIH HHS/United States ; P30 EY002162/EY/NEI NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; R01 EY027789/EY/NEI NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; R01 HG007175/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Lineage/genetics ; Cells/*classification/*metabolism ; Cells, Cultured ; Cerebral Cortex/*cytology/*embryology ; Chromatin/genetics/metabolism ; DNA Transposable Elements ; *Epigenome ; *Epigenomics ; Histones/chemistry/metabolism ; Humans ; Imaging, Three-Dimensional ; Methylation ; Multifactorial Inheritance/genetics ; Organogenesis/*genetics ; Polymorphism, Single Nucleotide/genetics ; Promoter Regions, Genetic/genetics ; Regulatory Elements, Transcriptional ; Reproducibility of Results ; Transcription, Genetic ; },
abstract = {Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone[1,2]. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.},
}
@article {pmid33057066,
year = {2020},
author = {Zhao, C and Liu, H and Xiao, T and Wang, Z and Nie, X and Li, X and Qian, P and Qin, L and Han, X and Zhang, J and Ruan, J and Zhu, M and Miao, YL and Zuo, B and Yang, K and Xie, S and Zhao, S},
title = {CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5178},
pmid = {33057066},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calreticulin/genetics/metabolism ; Encephalitis Virus, Japanese/metabolism/*pathogenicity ; Encephalitis, Japanese/*pathology/virology ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Gene Library ; HEK293 Cells ; Host-Pathogen Interactions/*genetics ; Humans ; Membrane Proteins/genetics/metabolism ; RNA, Guide/genetics ; RNA, Small Interfering/metabolism ; Sus scrofa ; *Virus Replication ; },
abstract = {Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.},
}
@article {pmid33055427,
year = {2020},
author = {Koppes, EA and Redel, BK and Johnson, MA and Skvorak, KJ and Ghaloul-Gonzalez, L and Yates, ME and Lewis, DW and Gollin, SM and Wu, YL and Christ, SE and Yerle, M and Leshinski, A and Spate, LD and Benne, JA and Murphy, SL and Samuel, MS and Walters, EM and Hansen, SA and Wells, KD and Lichter-Konecki, U and Wagner, RA and Newsome, JT and Dobrowolski, SF and Vockley, J and Prather, RS and Nicholls, RD},
title = {A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.},
journal = {JCI insight},
volume = {5},
number = {20},
pages = {},
pmid = {33055427},
issn = {2379-3708},
support = {R21 NS099450/NS/NINDS NIH HHS/United States ; U42 OD011140/OD/NIH HHS/United States ; },
mesh = {Adolescent ; Adult ; Animals ; CRISPR-Cas Systems/genetics ; Diet ; Disease Models, Animal ; Gene Editing ; Humans ; Liver/drug effects/*metabolism ; Phenotype ; Phenylalanine/*genetics/metabolism/pharmacology ; Phenylalanine Hydroxylase/*genetics ; Phenylketonurias/diet therapy/*genetics/metabolism/pathology ; Swine ; },
abstract = {Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.},
}
@article {pmid33054837,
year = {2020},
author = {Zhang, T and Lu, R and Chen, Y and Yuan, Y and Song, S and Yan, K and Zha, Y and Zhuang, W and Cheng, Y and Liang, J},
title = {Hyperhomocysteinemia and dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9.},
journal = {Lipids in health and disease},
volume = {19},
number = {1},
pages = {224},
pmid = {33054837},
issn = {1476-511X},
mesh = {Animals ; Betaine/pharmacology ; Body Weight/genetics ; *CRISPR-Cas Systems ; Cystathionine beta-Synthase/*genetics ; Disease Models, Animal ; Dyslipidemias/*genetics ; Female ; Gene Knockout Techniques ; Hyperhomocysteinemia/*genetics ; Hyperlipidemias/drug therapy/genetics ; Liver/pathology ; Male ; Point Mutation ; Rabbits ; Vitamin B Complex/pharmacology ; },
abstract = {BACKGROUND: Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS.<br><br>METHODS: CRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed.<br><br>RESULTS: Six CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6&#x2009;weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals.<br><br>CONCLUSION: A CBS[G307S] rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.},
}
@article {pmid33054419,
year = {2020},
author = {Blanck, M and Budnik-Zawilska, MB and Lenger, SR and McGonigle, JE and Martin, GRA and le Sage, C and Lawo, S and Pemberton, HN and Tiwana, GS and Sorrell, DA and Cross, BCS},
title = {A Flexible, Pooled CRISPR Library for Drug Development Screens.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {211-222},
pmid = {33054419},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins ; *Drug Development ; Gene Knockout Techniques ; *Gene Library ; HT29 Cells ; High-Throughput Screening Assays ; Humans ; Pharmaceutical Preparations ; RNA, Guide/genetics ; },
abstract = {Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.},
}
@article {pmid33053801,
year = {2020},
author = {Olson, A and Basukala, B and Lee, S and Gagne, M and Wong, WW and Henderson, AJ},
title = {Targeted Chromatinization and Repression of HIV-1 Provirus Transcription with Repurposed CRISPR/Cas9.},
journal = {Viruses},
volume = {12},
number = {10},
pages = {},
pmid = {33053801},
issn = {1999-4915},
support = {R01 AI138960/AI/NIAID NIH HHS/United States ; R33 DA047032/DA/NIDA NIH HHS/United States ; P30 AI042853/AI/NIAID NIH HHS/United States ; R01 AI138960/NH/NIH HHS/United States ; },
mesh = {Acetylation ; CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenesis, Genetic/genetics ; HEK293 Cells ; HIV Infections/virology ; HIV Long Terminal Repeat/genetics ; HIV-1/*genetics ; Histones/metabolism ; Humans ; Jurkat Cells ; Methylation ; Proviruses/*genetics ; RNA, Guide/*genetics ; Repressor Proteins/metabolism ; Transcription, Genetic/genetics ; Virus Activation/*genetics ; Virus Latency/*genetics ; },
abstract = {The major barrier to HIV-1 cure is the persistence of latent provirus, which is not eradicated by antiretroviral therapy. The "shock and kill" approach entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. CRISPR/Cas9 has been utilized to excise integrated Human Immunodeficiency Virus (HIV) DNA from infected cells in an RNA-guided, sequence-specific manner. Here, we seek to epigenetically silence the proviral DNA by introducing nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB). We show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies.},
}
@article {pmid33053652,
year = {2020},
author = {Park, JS and Lee, KY and Han, JY},
title = {Precise Genome Editing in Poultry and Its Application to Industries.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {33053652},
issn = {2073-4425},
mesh = {Animal Husbandry ; Animals ; Animals, Genetically Modified/*genetics ; Biotechnology/*methods ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; *Genome ; Poultry/*genetics ; },
abstract = {Poultry such as chickens are valuable model animals not only in the food industry, but also in developmental biology and biomedicine. Recently, precise genome-editing technologies mediated by the CRISPR/Cas9 system have developed rapidly, enabling the production of genome-edited poultry models with novel traits that are applicable to basic sciences, agriculture, and biomedical industry. In particular, these techniques have been combined with cultured primordial germ cells (PGCs) and viral vector systems to generate a valuable genome-edited avian model for a variety of purposes. Here, we summarize recent progress in CRISPR/Cas9-based genome-editing technology and its applications to avian species. In addition, we describe further applications of genome-edited poultry in various industries.},
}
@article {pmid33053363,
year = {2021},
author = {Králová, M and Bergougnoux, V and Frébort, I},
title = {CRISPR/Cas9 genome editing in ergot fungus Claviceps purpurea.},
journal = {Journal of biotechnology},
volume = {325},
number = {},
pages = {341-354},
doi = {10.1016/j.jbiotec.2020.09.028},
pmid = {33053363},
issn = {1873-4863},
mesh = {CRISPR-Cas Systems/genetics ; *Claviceps/genetics ; Gene Editing ; Mutagenesis ; Protoplasts ; },
abstract = {Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5'-phosphate decarboxylase involved in pyrimidine biosynthesis and the α-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.},
}
@article {pmid33053221,
year = {2020},
author = {Gong, J and Wang, HX and Lao, YH and Hu, H and Vatan, N and Guo, J and Ho, TC and Huang, D and Li, M and Shao, D and Leong, KW},
title = {A Versatile Nonviral Delivery System for Multiplex Gene-Editing in the Liver.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {32},
number = {46},
pages = {e2003537},
pmid = {33053221},
issn = {1521-4095},
support = {UH3 TR002142/TR/NCATS NIH HHS/United States ; UH3 TR002151/TR/NCATS NIH HHS/United States ; UG3 TR002151/TR/NCATS NIH HHS/United States ; UG3-NS115598/GF/NIH HHS/United States ; UG3 NS115598/NS/NINDS NIH HHS/United States ; UH3-TR002151/GF/NIH HHS/United States ; UH3-TR002142/GF/NIH HHS/United States ; },
mesh = {Angiopoietin-Like Protein 3 ; Angiopoietin-like Proteins/genetics ; CRISPR-Cas Systems/genetics ; Drug Carriers/*chemistry ; *Gene Editing ; Gene Transfer Techniques ; Humans ; Lipids/chemistry ; Liver/*metabolism ; Nanoparticles/chemistry ; Proprotein Convertase 9/genetics ; },
abstract = {Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics.},
}
@article {pmid33052901,
year = {2020},
author = {Semesta, KM and Tian, R and Kampmann, M and von Zastrow, M and Tsvetanova, NG},
title = {A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1009103},
pmid = {33052901},
issn = {1553-7404},
support = {DP2 GM119139/GM/NIGMS NIH HHS/United States ; R01 DA012864/DA/NIDA NIH HHS/United States ; R01 DA010711/DA/NIDA NIH HHS/United States ; R00 MH109633/MH/NIMH NIH HHS/United States ; R29 DA010711/DA/NIDA NIH HHS/United States ; },
mesh = {ATPases Associated with Diverse Cellular Activities/genetics ; CRISPR-Cas Systems/*genetics ; Carrier Proteins/genetics ; Cell Differentiation/genetics ; Cell Proliferation/*genetics ; Cells, Cultured ; Cyclic AMP/*genetics ; DNA Helicases/genetics ; DNA Primase/genetics ; HEK293 Cells ; Humans ; Membrane Proteins/genetics ; Nuclear Pore Complex Proteins/genetics ; Protein Serine-Threonine Kinases/genetics ; Protein-Tyrosine Kinases/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; RNA Splicing Factors/genetics ; Receptors, G-Protein-Coupled/*genetics ; Signal Transduction/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.},
}
@article {pmid33052229,
year = {2020},
author = {Jiang, W and Li, H and Liu, X and Zhang, J and Zhang, W and Li, T and Liu, L and Yu, X},
title = {Precise and efficient silencing of mutant Kras[G12D] by CRISPR-CasRx controls pancreatic cancer progression.},
journal = {Theranostics},
volume = {10},
number = {25},
pages = {11507-11519},
pmid = {33052229},
issn = {1838-7640},
mesh = {Amino Acid Substitution ; Animals ; CRISPR-Cas Systems/*genetics ; Carcinoma, Pancreatic Ductal/genetics/pathology/*therapy ; Cell Line, Tumor ; Deoxycytidine/analogs &amp; derivatives/pharmacology/therapeutic use ; Dependovirus/genetics ; Disease Progression ; Drug Resistance, Neoplasm/genetics ; Female ; Gene Knockdown Techniques ; Genetic Therapy/*methods ; Genetic Vectors/administration &amp; dosage/genetics ; Humans ; Mice ; Mutation ; Pancreatic Neoplasms/genetics/pathology/*therapy ; Proof of Concept Study ; Proto-Oncogene Proteins p21(ras)/*genetics ; RNA, Guide/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Rationale: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with few therapeutic targets and rare effective treatments. Over 90% of PDAC tumors bear a Kras mutation, and the single-site mutation G12D (Kras[G12D]) is most prevalent. Methods: Here, we applied the CRISPR-CasRx system to silence the mutant Kras[G12D] transcript in PDAC cells. We also used a capsid-optimized adenovirus-associated virus 8 vector (AAV8) to deliver the CRISPR-CasRx system into PDAC orthotopic tumors and patient-derived tumor xenografts (PDX). Results: Our data showed that guided by a KrasG12D-specific gRNA, CasRx is able to precisely and efficiently silence the mutant KrasG12D expression in PDAC cells. The knockdown of mutant KrasG12D by CasRx abolishes the aberrant activation of downstream signaling induced by mutant KrasG12D and subsequently suppresses the tumor growth and improves the sensitivity of gemcitabine in PDAC. Additionally, delivering CasRx-gRNA via AAV8 into the orthotopic KrasG12D PDAC tumors substantially improves the survival of mice without obvious toxicity. Furthermore, targeting KrasG12D through CasRx suppresses the growth of PDAC PDXs. In conclusion, our study provides a proof-of-concept that CRISPR-CasRx can be utilized to target and silence mutant KrasG12D transcripts and therefore inhibit PDAC malignancy.},
}
@article {pmid33052227,
year = {2020},
author = {Hu, BC and Wu, GH and Shao, ZQ and Zheng, Y and Liu, JQ and Zhang, R and Hong, J and Yang, XH and Sun, RH and Mo, SJ},
title = {Redox DAPK1 destabilizes Pellino1 to govern inflammation-coupling tubular damage during septic AKI.},
journal = {Theranostics},
volume = {10},
number = {25},
pages = {11479-11496},
pmid = {33052227},
issn = {1838-7640},
mesh = {Acute Kidney Injury/*immunology/pathology/prevention &amp; control ; Animals ; CRISPR-Cas Systems/genetics ; Cell Hypoxia/drug effects/immunology ; Cell Line ; Death-Associated Protein Kinases/antagonists &amp; inhibitors/genetics/*metabolism ; Disease Models, Animal ; Epithelial Cells ; Gene Knockout Techniques ; Heterocyclic Compounds, 2-Ring/pharmacology/therapeutic use ; Humans ; Kidney Tubules/cytology/pathology ; Mice ; Mice, Knockout ; Myeloid Differentiation Factor 88/antagonists &amp; inhibitors/metabolism ; Nuclear Proteins/genetics/*metabolism ; Oxidation-Reduction/drug effects ; Phosphorylation/drug effects/genetics ; RAW 264.7 Cells ; Sepsis/*complications/drug therapy/immunology ; Spiro Compounds/pharmacology/therapeutic use ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Ubiquitination/drug effects/genetics ; },
abstract = {Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1[-/-] mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCF[Fbxw7α]-dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management.},
}
@article {pmid33051367,
year = {2020},
author = {Goldman-Pinkovich, A and Kannan, S and Nitzan-Koren, R and Puri, M and Pawar, H and Bar-Avraham, Y and McDonald, J and Sur, A and Zhang, WW and Matlashewski, G and Madhubala, R and Michaeli, S and Myler, PJ and Zilberstein, D},
title = {Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.},
journal = {mBio},
volume = {11},
number = {5},
pages = {},
pmid = {33051367},
issn = {2150-7511},
mesh = {Animals ; Arginine/*metabolism ; CRISPR-Cas Systems ; Female ; *Host-Parasite Interactions ; Leishmania/*growth &amp; development/*metabolism ; Leishmaniasis/metabolism/parasitology ; Lysosomes/parasitology ; Macrophages/*parasitology/physiology ; Membrane Transport Proteins/genetics ; Mice ; Mice, Inbred BALB C ; Phagosomes/parasitology/physiology ; },
abstract = {Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.},
}
@article {pmid33051130,
year = {2020},
author = {Richards, DY and Winn, SR and Dudley, S and Fedorov, L and Rimann, N and Thöny, B and Harding, CO},
title = {A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency.},
journal = {Molecular genetics and metabolism},
volume = {131},
number = {3},
pages = {306-315},
pmid = {33051130},
issn = {1096-7206},
support = {R01 NS080866/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Exons/genetics ; Gene Editing ; Genetic Vectors/genetics/pharmacology ; Humans ; Liver/drug effects/*metabolism/pathology ; Mice ; Phenylalanine/*genetics/metabolism ; Phenylalanine Hydroxylase/*genetics/pharmacology ; Phenylketonurias/genetics/pathology/*therapy ; },
abstract = {Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.},
}
@article {pmid33050923,
year = {2020},
author = {Coelho, FS and Rodpai, R and Miller, A and Karinshak, SE and Mann, VH and Dos Santos Carvalho, O and Caldeira, RL and de Moraes Mourão, M and Brindley, PJ and Ittiprasert, W},
title = {Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor.},
journal = {Parasites &amp; vectors},
volume = {13},
number = {1},
pages = {511},
pmid = {33050923},
issn = {1756-3305},
support = {HHSN272201000005I/AI/NIAID NIH HHS/United States ; 107475/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Biomphalaria/cytology/genetics/parasitology ; CRISPR-Cas Systems ; Calcium-Binding Proteins/*genetics ; Cell Adhesion/*genetics ; Cell Line/parasitology ; Gene Editing/methods ; Gene Knockout Techniques ; Hemocytes/immunology ; Host-Parasite Interactions ; Humans ; Microfilament Proteins ; Schistosoma mansoni/parasitology/*pathogenicity ; Schistosomiasis/transmission ; },
abstract = {BACKGROUND: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.<br><br>METHODS: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.<br><br>RESULTS: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2&#xa0;bp insertions and/or 8-30&#xa0;bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5&#x2009;&#xb1;&#x2009;20.2% SD, P&#x2009;&#x2264;&#x2009;0.05, n&#x2009;=&#x2009;12). Adherence by BgAIF gene-edited (&#x394;BgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer &#x394;BgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66&#x2009;&#xb1;&#x2009;0.10, &#x394;BgAIF, 2.30&#x2009;&#xb1;&#x2009;0.22 (P&#x2009;&#x2264;&#x2009;0.01).<br><br>CONCLUSIONS: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.},
}
@article {pmid33050853,
year = {2020},
author = {Shuvalova, ML and Kopylov, AT and Mazurov, DV and Pichugin, AV and Bovin, NV and Filatov, AV},
title = {CD44-Associated Tn Antigen as a New Biomarker of Tumor Cells with Aberrant Glycosylation.},
journal = {Biochemistry. Biokhimiia},
volume = {85},
number = {9},
pages = {1064-1081},
doi = {10.1134/S0006297920090060},
pmid = {33050853},
issn = {1608-3040},
mesh = {A549 Cells ; Adenocarcinoma of Lung/*diagnosis/immunology/metabolism ; Antibodies, Monoclonal/*immunology ; Antigens, Tumor-Associated, Carbohydrate/immunology/*metabolism ; Biomarkers, Tumor/*metabolism ; CRISPR-Cas Systems ; Glycosylation ; Humans ; Hyaluronan Receptors/immunology/*metabolism ; Lung Neoplasms/*diagnosis/immunology/metabolism ; Molecular Chaperones/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Tn antigen is a tumor-associated antigen that appears on cancer cells as a result of aberrant O-glycosylation. The most studied form of Tn antigen is found in mucins, in particular, in mucin 1 (MUC1). Antibodies against this form of Tn antigen are used to diagnose tumors, as well as to generate T-killers with a chimeric receptor. Some carcinomas do not carry MUC1 and antibodies of a different specificity are required to detect Tn antigen on these cells. In our work, we searched for anti-Tn antibodies without preliminary assumptions about the proteins that may be carriers of the Tn antigen. For this purpose, we obtained several pairs of isogenic cell lines with the wild type and knockout of the Cosmc gene, which is essential for correct protein O-glycosylation. Using the created lines as immunogens, we generated a monoclonal antibody AKC3, which reacted with the Cosmc-deficient A549 lung adenocarcinoma cells and did not bind to the wild-type cells. Using mass spectrometry, as well as co-immunoprecipitation, it was shown that the AKC3 antibody recognized the Tn antigen in the context of CD44 protein - a protein important for tumor growth. The AKC3 antibody can be used for tumor diagnosis, and to generate T cells with a chimeric receptor for treatment of tumors that do not express mucins.},
}
@article {pmid33050591,
year = {2020},
author = {Lu, Z and Guo, X and Huang, Z and Xia, J and Li, X and Wu, J and Yu, H and Shahid, MQ and Liu, X},
title = {Transcriptome and Gene Editing Analyses Reveal MOF1a Defect Alters the Expression of Genes Associated with Tapetum Development and Chromosome Behavior at Meiosis Stage Resulting in Low Pollen Fertility of Tetraploid Rice.},
journal = {International journal of molecular sciences},
volume = {21},
number = {20},
pages = {},
pmid = {33050591},
issn = {1422-0067},
mesh = {Biomarkers ; CRISPR-Cas Systems ; Chromosomes, Plant ; Fertility ; *Gene Editing ; Gene Expression Profiling ; *Gene Expression Regulation, Plant ; Immunohistochemistry ; Meiosis/*genetics ; Oryza/*physiology ; Plant Development/*genetics ; Pollen ; Tetraploidy ; *Transcriptome ; },
abstract = {Autotetraploid rice is a useful rice germplasm for polyploid rice breeding. However, low fertility limits its commercial production. A neo-tetraploid rice with high fertility was developed from the progenies of crossing between autotetraploid lines by our research group. Our previous study showed that a myeloblastosis (MYB) transcription factor, MOF1, might be associated with the pollen development in tetraploid rice. However, little information is available about its role in pollen development in tetraploid rice. Here, we identified a new haplotype of MOF1 from neo-tetraploid rice and marked it as MOF1a. Transcriptome and qRT-PCR analysis demonstrated that MOF1a highly expressed in anthers, and displayed differential expression in neo-tetraploid rice compared to tetraploid rice line with low pollen fertility. The mutant (mof1a) of MOF1a, which was generated by CRISPR/Cas9 knockout in neo-tetraploid rice, showed low pollen fertility, and also exhibited abnormal tapetum and middle layer development, and defective chromosome behaviors during meiosis. A total of 13 tapetal related genes were found to be up-regulated in meiotic anthers of MOF1a compared with wild type plants by RNA-seq analysis, including CYP703A3, PTC1, and OsABCG26, which had been demonstrated to affect tapetal development. Moreover, 335 meiosis-related genes displayed differential expression patterns at same stage, including nine important meiosis-related genes, such as metallothionein OsMT1a. These results demonstrated that MOF1a plays an important role in pollen development and provides a foundation for understanding the molecular mechanism underlying MOF1a in reproduction of tetraploid rice.},
}
@article {pmid33049308,
year = {2020},
author = {Kayesh, MEH and Amako, Y and Hashem, MA and Murakami, S and Ogawa, S and Yamamoto, N and Hifumi, T and Miyoshi, N and Sugiyama, M and Tanaka, Y and Mizokami, M and Kohara, M and Tsukiyama-Kohara, K},
title = {Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA.},
journal = {Virus research},
volume = {290},
number = {},
pages = {198191},
doi = {10.1016/j.virusres.2020.198191},
pmid = {33049308},
issn = {1872-7492},
mesh = {Adenoviridae/genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/administration &amp; dosage/*genetics ; *CRISPR-Cas Systems ; DNA, Circular/*genetics ; DNA, Viral/genetics ; *Gene Transfer Techniques ; *Genetic Vectors ; Guanine/analogs &amp; derivatives/pharmacology ; HEK293 Cells ; Hep G2 Cells ; Hepatitis B/therapy/virology ; Hepatitis B Surface Antigens/genetics ; Hepatitis B virus/drug effects/*genetics ; Humans ; Male ; Mice ; Plasmids/genetics ; RNA, Guide/*genetics ; },
abstract = {Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.},
}
@article {pmid33049228,
year = {2020},
author = {Niu, Y and Yang, L and Gao, T and Dong, C and Zhang, B and Yin, P and Hopp, AK and Li, D and Gan, R and Wang, H and Liu, X and Cao, X and Xie, Y and Meng, X and Deng, H and Zhang, X and Ren, J and Hottiger, MO and Chen, Z and Zhang, Y and Liu, X and Feng, Y},
title = {A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation.},
journal = {Molecular cell},
volume = {80},
number = {3},
pages = {512-524.e5},
doi = {10.1016/j.molcel.2020.09.015},
pmid = {33049228},
issn = {1097-4164},
mesh = {ADP-Ribosylation/physiology ; Bacterial Proteins/genetics ; Bacteriophages/genetics ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryoelectron Microscopy/methods ; DNA/metabolism ; Models, Molecular ; RNA, Bacterial/metabolism ; Viral Proteins/genetics/*metabolism ; },
abstract = {CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.},
}
@article {pmid33048924,
year = {2020},
author = {Mutalik, VK and Adler, BA and Rishi, HS and Piya, D and Zhong, C and Koskella, B and Kutter, EM and Calendar, R and Novichkov, PS and Price, MN and Deutschbauer, AM and Arkin, AP},
title = {High-throughput mapping of the phage resistance landscape in E. coli.},
journal = {PLoS biology},
volume = {18},
number = {10},
pages = {e3000877},
pmid = {33048924},
issn = {1545-7885},
mesh = {Bacterial Proteins/genetics/metabolism ; Bacteriophages/drug effects/*physiology ; Biosynthetic Pathways/drug effects ; CRISPR-Cas Systems/genetics ; Cyclic GMP/analogs &amp; derivatives/pharmacology ; DNA/genetics ; Down-Regulation/drug effects/genetics ; Escherichia coli/drug effects/genetics/*virology ; Gene Expression Regulation, Bacterial/drug effects ; Genes, Essential ; Genome, Bacterial ; Mutation/genetics ; Phenotype ; Reproducibility of Results ; Suppression, Genetic ; },
abstract = {Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.},
}
@article {pmid33048255,
year = {2021},
author = {Wang, Q and Zhao, Q and Liu, Q and He, X and Zhong, Y and Qin, Y and Gao, L and Liu, G and Qu, Y},
title = {CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoter-driven guide RNAs.},
journal = {Biotechnology letters},
volume = {43},
number = {2},
pages = {495-502},
pmid = {33048255},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Hypocreales/*genetics ; Penicillium/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; RNA, Ribosomal, 5S/genetics ; },
abstract = {OBJECTIVE: To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei.<br><br>RESULTS: Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the &#x3b2;-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%.<br><br>CONCLUSIONS: Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.},
}
@article {pmid33048115,
year = {2020},
author = {Hu, TT and Yang, JW and Yan, Y and Chen, YY and Xue, HB and Xiang, YQ and Ye, LC},
title = {Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9.},
journal = {Bioscience reports},
volume = {40},
number = {10},
pages = {},
pmid = {33048115},
issn = {1573-4935},
mesh = {Antineoplastic Agents, Immunological/*pharmacology/therapeutic use ; Biomarkers, Tumor/*genetics ; CRISPR-Cas Systems/genetics ; Caco-2 Cells ; Cetuximab/*pharmacology/therapeutic use ; Colorectal Neoplasms/*drug therapy/genetics/pathology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/*genetics ; Gene Knockout Techniques ; HT29 Cells ; Humans ; },
abstract = {Colorectal cancer (CRC) is a common malignant tumor in digestive tract with highly invasive and metastatic capacity. Drug sensitivity remains a significant obstacle to successful chemotherapy in CRC patients. The present study aimed to explore genes related to cetuximab (CTX) sensitivity in CRC by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Celigo image cytometer was used to detect suitable cells and optimal dosage of CTX. Inhibition rate of CTX on Caco-2 cells was evaluated by cell counting kit-8 (CCK-8) method before and after transfection. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was performed to explore suitable concentration of puromycin and multiplicity of infection (MOI). CRISPR-Cas9, sequencing data quality analysis and cell viability test were used for the selection of genes related to CTX sensitivity in CRC cells. Finally, the selected genes associated with CTX sensitivity in CRC cells were further validated by colony formation and CCK-8 assays. In the present study, Caco-2 cells had a better prolificacy, and CTX 100 μg/ml exhibited a good inhibition trend on the 7th and 14th days of infection. MTT assay indicated that the minimum lethal concentration of puromycin was 2.5 μg/ml. Forty-six candidate genes were preliminarily screened via sequencing data quality analysis. Subsequently, we found that knockout of any of the four genes (MMP15, MRPL48, CALN1 and HADHB) could enhance CTX sensitivity in Caco-2 cells, which was further confirmed by colony formation assay. In summary, MMP15, MRPL48, CALN1 and HADHB genes are related to the mediation of CTX sensitivity in CRC.},
}
@article {pmid33047952,
year = {2020},
author = {Huang, D and Qian, J and Shi, Z and Zhao, J and Fang, M and Xu, Z},
title = {CRISPR-Cas12a-Assisted Multicolor Biosensor for Semiquantitative Point-of-Use Testing of the Nopaline Synthase Terminator in Genetically Modified Crops by Unaided Eyes.},
journal = {ACS synthetic biology},
volume = {9},
number = {11},
pages = {3114-3123},
doi = {10.1021/acssynbio.0c00365},
pmid = {33047952},
issn = {2161-5063},
mesh = {Amino Acid Oxidoreductases/*genetics ; Bacterial Proteins/*genetics ; Biosensing Techniques/methods ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; DNA/genetics ; DNA Primers/genetics ; Endodeoxyribonucleases/*genetics ; Glucose Oxidase/genetics ; Gold/chemistry ; Nanotubes/chemistry ; Plants, Genetically Modified/*genetics ; Recombinases/genetics ; Terminator Regions, Genetic/*genetics ; },
abstract = {Existing methods of detecting foreign genes and their expression products from genetically modified organisms (GMOs) suffer from the requirement of professional equipment and skillful operators. The same problem stays for the CRISPR-Cas12a system, although it has been emerging as a powerful tool for nucleic acid detection due to its remarkable sensitivity and specificity. In this report, a portable platform for the visible detection of GMOs based on CRISPR-Cas12a was established, which relies on a color change of gold nanorods (GNRs) caused by the invertase-glucose oxidase cascade reaction and the Fenton reaction for signal readout. A nopaline synthase (NOS) terminator was employed as a model target commonly existing in foreign genes of GMOs. With the help of recombinase-aided amplification, this platform achieved comparable sensitivity of DNA targets (1 aM) with that of a fluorescence reporting assay. As low as 0.1 wt % genetically modified (GM) content in Bt-11 maize was visually observed by unaided eyes, and the semiquantitation of GM ingredients can be obtained within the range of 0.1 to 40 wt % through the absorption measurement of GNRs. Furthermore, five real samples were tested by our method, and the results indicated that the GM ingredient percentages of GMO samples were 2.24 and 24.08 wt %, respectively, while the other three samples were GMO-free. With the advantages of a simple procedure, no need for large or professional instruments, high sensitivity, and selectivity, this platform is expected to provide reasonable technical support for the safe supervision of GMOs.},
}
@article {pmid33047833,
year = {2020},
author = {Shahabipour, F and Oskuee, RK and Shokrgozar, MA and Naderi-Meshkin, H and Goshayeshi, L and Bonakdar, S},
title = {CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.},
journal = {The journal of gene medicine},
volume = {22},
number = {12},
pages = {e3288},
doi = {10.1002/jgm.3288},
pmid = {33047833},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems ; *Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Extracellular Matrix Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Mesenchymal Stem Cells/*cytology/metabolism ; Osteoblasts/*cytology/metabolism ; *Osteogenesis ; Phosphoproteins/*antagonists &amp; inhibitors/genetics/metabolism ; Promoter Regions, Genetic ; },
abstract = {BACKGROUND: Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase[1] . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation.<br><br>METHODS: In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays.<br><br>RESULTS: We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs [2] .<br><br>CONCLUSIONS: The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.},
}
@article {pmid33047464,
year = {2021},
author = {Van Vu, T and Thi Hai Doan, D and Kim, J and Sung, YW and Thi Tran, M and Song, YJ and Das, S and Kim, JY},
title = {CRISPR/Cas-based precision genome editing via microhomology-mediated end joining.},
journal = {Plant biotechnology journal},
volume = {19},
number = {2},
pages = {230-239},
pmid = {33047464},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *Gene Editing ; Gene Targeting ; Homologous Recombination ; },
abstract = {Gene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide-directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)-based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single-stranded breaks (SSBs) or double-stranded breaks (DSBs)), a substantial advancement in HR-mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology-mediated end joining (MMEJ) and discuss their potential applications in crop improvement.},
}
@article {pmid33047235,
year = {2021},
author = {Wei, C and Chen, T and Zhang, Y and Wang, Y and Shi, D and Jiang, Z and Li, K and Xiao, L and Shen, J},
title = {A Novel White-to-Blue Colony Formation Assay to Select for Optimized sgRNAs.},
journal = {Molecular biotechnology},
volume = {63},
number = {1},
pages = {1-12},
pmid = {33047235},
issn = {1559-0305},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Mutation ; PC-3 Cells ; RNA, Guide/*genetics ; Receptor, EphA2/*genetics ; Sequence Analysis, DNA ; },
abstract = {CRISPR/Cas9-mediated genome editing technology consists of a single-guide RNA (sgRNA), and the Cas9 endonuclease has the potential to treat genetic diseases in most tissues and organisms. In this system, the Cas9 protein can be directed to target genomic DNA sequences as "molecular scissors" with the guidance of sgRNAs. However, the target-specific activities of different sgRNAs are highly variable; thus, it is crucial to search for a simple, quick and economical method to screen for optimized sgRNAs with high target specificity. We have adopted and verified a newly developed white-to-blue colony formation assay to quickly screen for sgRNAs optimized for the EphA2 gene, which is highly expressed in hormone-resistant prostate cancer (PC-3) cells. This assay promises to screen for optimized sgRNAs more simply, rapidly, and efficiently. Our results suggest that the white-to-blue colony formation assay might be a useful screening strategy to quickly select for optimized sgRNAs.},
}
@article {pmid33046904,
year = {2020},
author = {Zhu, H and Li, C and Gao, C},
title = {Publisher Correction: Applications of CRISPR-Cas in agriculture and plant biotechnology.},
journal = {Nature reviews. Molecular cell biology},
volume = {21},
number = {11},
pages = {712},
doi = {10.1038/s41580-020-00304-y},
pmid = {33046904},
issn = {1471-0080},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid33046744,
year = {2020},
author = {Kang, D and Shin, W and Yoo, H and Kim, S and Lee, S and Rhee, K},
title = {Cep215 is essential for morphological differentiation of astrocytes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17000},
pmid = {33046744},
issn = {2045-2322},
mesh = {Animals ; Astrocytes/*physiology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cell Differentiation ; Cell Line ; Mice ; Microtubules/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neuroglia/*physiology ; RNA, Small Interfering/genetics ; },
abstract = {Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.},
}
@article {pmid33045965,
year = {2020},
author = {Shaikh, SB and Bhandary, YP},
title = {CRISPR/Cas9 Genome Editing Tool: A Promising Tool for Therapeutic Applications on Respiratory Diseases.},
journal = {Current gene therapy},
volume = {20},
number = {5},
pages = {333-346},
doi = {10.2174/1566523220666201012145731},
pmid = {33045965},
issn = {1875-5631},
mesh = {CRISPR-Cas Systems/*genetics ; Cystic Fibrosis/genetics/therapy ; *Gene Editing ; Genetic Therapy/*trends ; Humans ; Lung Diseases/genetics/*therapy ; Lung Neoplasms/genetics/therapy ; Respiratory Distress Syndrome/genetics/therapy ; },
abstract = {Respiratory diseases are one of the prime topics of concern in the current era due to improper diagnostics tools. Gene-editing therapy, like Clustered regularly interspaced palindromic repeats- associated nuclease 9 (CRISPR/Cas9), is gaining popularity in pulmonary research, opening up doors to invaluable insights on underlying mechanisms. CRISPR/Cas9 can be considered as a potential gene-editing tool with a scientific community that is helping in the advancement of knowledge in respiratory health and therapy. As an appealing therapeutic tool, we hereby explore the advanced research on the application of CRISPR/Cas9 tools in chronic respiratory diseases such as lung cancer, Acute respiratory distress syndrome (ARDS) and cystic fibrosis (CF). We also address the urgent need to establish this gene-editing tool in various other lung diseases such as asthma, Chronic obstructive pulmonary disease (COPD) and Idiopathic pulmonary fibrosis (IPF). The present review introduces CRISPR/Cas9 as a worthy application in targeting epithelial-mesenchymal transition and fibrinolytic system via editing specific genes. Thereby, based on the efficiency of CRISPR/Cas9, it can be considered as a promising therapeutic tool in respiratory health research.},
}
@article {pmid33044108,
year = {2020},
author = {Honer, M and Buscemi, K and Barrett, N and Riazati, N and Orlando, G and Nelson, MD},
title = {Orcokinin neuropeptides regulate sleep in Caenorhabditis elegans.},
journal = {Journal of neurogenetics},
volume = {34},
number = {3-4},
pages = {440-452},
pmid = {33044108},
issn = {1563-5260},
support = {P40 OD010440/OD/NIH HHS/United States ; R15 GM122058/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Arthropods/physiology ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/*physiology ; Caenorhabditis elegans Proteins/genetics/*physiology ; Conserved Sequence ; Defecation/physiology ; Gene Editing ; Genes, Helminth ; Hot Temperature ; Loss of Function Mutation ; Motor Activity ; Neurons/metabolism ; Neuropeptides/genetics/*physiology ; Sequence Alignment ; Sleep/genetics/*physiology ; Species Specificity ; Stress, Physiological/physiology ; Transgenes ; Up-Regulation ; },
abstract = {Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans.},
}
@article {pmid33043922,
year = {2020},
author = {Tian, W and Liu, X and Wang, G and Liu, C},
title = {A hyperbranched transcription-activated CRISPR-Cas12a signal amplification strategy for sensitive microRNA sensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {87},
pages = {13445-13448},
doi = {10.1039/d0cc06034h},
pmid = {33043922},
issn = {1364-548X},
mesh = {Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; *Limit of Detection ; MicroRNAs/*analysis ; *Transcription, Genetic ; },
abstract = {A CRISPR-Cas12a-based strategy is developed for sensitive microRNA sensing, in which hyperbranched rolling circle amplification and transcription are integrated for the activation of the trans-cleavage ability of Cas12a. The integrated triplex signal amplification endows the strategy with a low background and high sensitivity. This design expands the scope of the CRISPR-Cas-based sensing toolbox and shows great potential in biological and biomedical studies.},
}
@article {pmid33042266,
year = {2020},
author = {Sun, NH and Chen, DY and Ye, LP and Sheng, G and Gong, JJ and Chen, BH and Lu, YM and Han, F},
title = {CRISPR-Sunspot: Imaging of endogenous low-abundance RNA at the single-molecule level in live cells.},
journal = {Theranostics},
volume = {10},
number = {24},
pages = {10993-11012},
pmid = {33042266},
issn = {1838-7640},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Embryo, Mammalian ; Female ; Fetal Hemoglobin/genetics ; HEK293 Cells ; Humans ; Intravital Microscopy/*methods ; Microscopy, Confocal/methods ; Molecular Imaging/*methods ; Neurons ; Primary Cell Culture ; RNA, Guide/genetics ; RNA, Messenger/genetics/*metabolism ; Rats ; Single Molecule Imaging/*methods ; Transcriptional Activation ; Transfection ; },
abstract = {CRISPR/Cas-based mRNA imaging has been developed to labeling of high-abundance mRNAs. A lack of non-genetically encoded mRNA-tagged imaging tools has limited our ability to explore the functional distributions of endogenous low-abundance mRNAs in cells. Here, we developed a CRISPR-Sunspot method based on the SunTag signal amplification system that allows efficient imaging of low-abundance mRNAs with CRISPR/Cas9. Methods: We created a stable TRE3G-dCas9-EGFP cell line and generated an Inducible dCas9-EGFP imaging system for assessment of two factors, sgRNA and dCas9, which influence imaging quality. Based on SunTag system, we established a CRISPR-Sunspot imaging system for amplifying signals from single-molecule mRNA in live cells. CRISPR-Sunspot was used to track co-localization of Camk2a mRNA with regulatory protein Xlr3b in neurons. CRISPR-Sunspot combined with CRISPRa was used to determine elevated mRNA molecules. Results: Our results showed that manipulating the expression of fluorescent proteins and sgRNA increased the efficiency of RNA imaging in cells. CRISPR-Sunspot could target endogenous mRNAs in the cytoplasm and amplified signals from single-molecule mRNA. Furthermore, CRISPR-Sunspot was also applied to visualize mRNA distributions with its regulating proteins in neurons. CRISPR-Sunspot detected the co-localization of Camk2a mRNA with overexpressed Xlr3b proteins in the neuronal dendrites. Moreover, we also manipulated CRISPR-Sunspot to detect transcriptional activation of target gene such as HBG1 in live cells. Conclusion: Our findings suggest that CRISPR-Sunspot is a novel applicable imaging tool for visualizing the distributions of low-abundance mRNAs in cells. This study provides a novel strategy to unravel the molecular mechanisms of diseases caused by aberrant mRNA molecules.},
}
@article {pmid33039910,
year = {2021},
author = {Bonini, A and Poma, N and Vivaldi, F and Kirchhain, A and Salvo, P and Bottai, D and Tavanti, A and Di Francesco, F},
title = {Advances in biosensing: The CRISPR/Cas system as a new powerful tool for the detection of nucleic acids.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {192},
number = {},
pages = {113645},
pmid = {33039910},
issn = {1873-264X},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids/genetics ; Point-of-Care Systems ; },
abstract = {A main challenge in the development of biosensing devices for the identification and quantification of nucleic acids is to avoid the amplification of the genetic material from the sample by polymerase chain reaction (PCR), which is at present necessary to enhance sensitivity and selectivity of assays. PCR has undoubtedly revolutionized genetic analyses, but it requires careful purification procedures that are not easily implemented in point of care (POC) devices. In recent years, a new strategy for nucleic acid detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein systems (Cas) seems to offer unprecedented possibilities. The coupling of the CRISPR/Cas system with recent isothermal amplification methods is fostering the development of innovative optical and electrochemical POC devices. In this review, the mechanisms of action of several new CRISRP/Cas systems are reported together with their use in biosensing of nucleic acids.},
}
@article {pmid33039728,
year = {2020},
author = {Mahdizadeh, S and Sansom, FM and Lee, SW and Browning, GF and Marenda, MS},
title = {Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system.},
journal = {Veterinary microbiology},
volume = {250},
number = {},
pages = {108868},
doi = {10.1016/j.vetmic.2020.108868},
pmid = {33039728},
issn = {1873-2542},
mesh = {Aminoglycosides/pharmacology ; Anti-Infective Agents/pharmacology ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; *Genome, Bacterial ; Methyltransferases/genetics ; Microbial Sensitivity Tests ; Mutagenesis, Site-Directed/*methods ; Mycoplasma gallisepticum/drug effects/*genetics ; Plasmids/genetics ; },
abstract = {New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen. Three constructs carrying different CRISPR arrays targeting regions in the ksgA gene (pK1-CRISPR, pK-CRISPR-1 and pK-CRISPR-2) were assembled and introduced into M. gallisepticum on an oriC plasmid. The loss of KsgA prevents ribosomal methylation, which in turn confers resistance to the aminoglycoside antimicrobial kasugamycin, enabling selection for ksgA mutants. Analyses of the complete sequence of the ksgA gene in 78 resistant transformants revealed various modifications of the target region, presumably caused by the directed CRISPR/Cas activity of M. gallisepticum. The analyses suggested that M. gallisepticum may utilize a non-homologous end joining (NHEJ) repair system, which can result in deletion or duplication of a short DNA segment in the presence of double-stranded breaks. This study has generated an improved understanding of the M. gallisepticum CRISPR/Cas system, and may also facilitate further development of tools to genetically modify this important pathogen.},
}
@article {pmid33039248,
year = {2020},
author = {Burgio, G and Teboul, L},
title = {Anticipating and Identifying Collateral Damage in Genome Editing.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {12},
pages = {905-914},
pmid = {33039248},
issn = {0168-9525},
support = {MC_UP_1502/1/MRC_/Medical Research Council/United Kingdom ; U42 OD011174/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*standards ; *Genome ; Humans ; Risk Assessment ; },
abstract = {Genome editing has powerful applications in research, healthcare, and agriculture. However, the range of possible molecular events resulting from genome editing has been underestimated and the technology remains unpredictable on, and away from, the target locus. This has considerable impact in providing a safe approach for therapeutic genome editing, agriculture, and other applications. This opinion article discusses how to anticipate and detect those editing events by a combination of assays to capture all possible genomic changes. It also discusses strategies for preventing unwanted effects, critical to appraise the benefit or risk associated with the use of the technology. Anticipating and verifying the result of genome editing are essential for the success for all applications.},
}
@article {pmid33038909,
year = {2021},
author = {Abbaszadeh-Goudarzi, K and Nematollahi, MH and Khanbabaei, H and Nave, HH and Mirzaei, HR and Pourghadamyari, H and Sahebkar, A},
title = {Targeted Delivery of CRISPR/Cas13 as a Promising Therapeutic Approach to Treat SARS-CoV-2.},
journal = {Current pharmaceutical biotechnology},
volume = {22},
number = {9},
pages = {1149-1155},
doi = {10.2174/1389201021666201009154517},
pmid = {33038909},
issn = {1873-4316},
mesh = {Antiviral Agents ; *COVID-19 ; CRISPR-Cas Systems ; Genome, Viral ; Humans ; *SARS-CoV-2 ; },
abstract = {On a worldwide scale, the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to extensive damage to the health system as well as the global economy. Hitherto, there has been no approved drug or vaccine for this disease. Therefore, the use of general antiviral drugs is at the first line of treatment, though complicated with limited effectiveness and systemic side effects. Given the pathophysiology of the disease, researchers have proposed various strategies not only to find a more specific therapeutic way but also to reduce the side effects. One strategy to accomplish these goals is to use CRISPR/Cas13 system. Recently, a group of scientists has used the CRISPR/Cas13 system, which is highly effective in eliminating the genome of RNA viruses. Due to the RNA nature of the coronavirus genome, it seems that this system can be effective against the disease. The main challenge regarding the application of this system is to deliver it to the target cells efficiently. To solve this challenge, it seems that using virosomes with protein S on their membrane surface can be helpful. Studies have shown that protein S interacts with its specific receptor in target cells named Angiotensin-Converting Enzyme 2 (ACE2). Here, we propose if CRISPR/Cas13 gene constructs reach the infected cells efficiently using a virosomal delivery system, the virus genome will be cleaved and inactivated. Considering the pathophysiology of the disease, an important step to implement this hypothesis is to embed protein S on the membrane surface of virosomes to facilitate the delivery of gene constructs to the target cells.},
}
@article {pmid33038743,
year = {2020},
author = {Gosstola, NC and Huang, Z and Tong, X and Nourbakhsh, A and Chen, ZY and Dykxhoorn, DM and Zhong Liu, X},
title = {Characterization of UMi028-A-1 stem cell line that contains a CRISPR/Cas9 induced hearing loss-associated variant (V60L (c.178G &gt; T)) in the P2RX2 gene.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102017},
doi = {10.1016/j.scr.2020.102017},
pmid = {33038743},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Hearing Loss/genetics ; Heterozygote ; Humans ; Male ; Receptors, Purinergic P2X2 ; },
abstract = {UMi028-A-1 hiPSC line contains a CRISPR/Cas9-induced heterozygous, hearing loss-associated variant (V60L (GTA > TTA)) in the Purinergic Receptor P2X2 (P2RX2) gene. This line, derived from an unaffected male iPSC line, has been successfully characterized for its cellular and genetic properties. The c.178G > T variant in P2RX2 is associated with non-syndromic, dominant, progressive hearing loss. Once differentiated into inner ear cell types, UMi028-A-1 will serve as a resource for understanding the molecular mechanisms underlying hearing loss and serve as a potential platform for testing therapeutic approaches to restore inner ear function.},
}
@article {pmid33038615,
year = {2020},
author = {Schoger, E and Argyriou, L and Zimmermann, WH and Cyganek, L and Zelarayán, LC},
title = {Generation of homozygous CRISPRa human induced pluripotent stem cell (hiPSC) lines for sustained endogenous gene activation.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101944},
doi = {10.1016/j.scr.2020.101944},
pmid = {33038615},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; Transcriptional Activation ; },
abstract = {CRISPR/Cas9 technology is a powerful tool, owing to its robust on-target activity and high fidelity. Mutated Cas9 without nuclease activity (dCas9) fused to transcriptional modulators, can function as transcriptional inhibitors or activators (CRISPRa). We generated homozygous human induced pluripotent stem cell (hiPSC) lines with an inserted CRISPRa cassette into the AAVS1 locus whilst maintaining pluripotency and genomic integrity, the ability to differentiate into all three germ layers, generate functional cardiomyocytes, and validated Cas9-mediated induction of endogenous gene expression. Our generated hiPSC-CRISPRa offers a valuable tool for studying endogenous transcriptional modulation with single and multiplexed possibilities in all human cell types.},
}
@article {pmid33037689,
year = {2020},
author = {Curci, L and Brukman, NG and Weigel Muñoz, M and Rojo, D and Carvajal, G and Sulzyk, V and Gonzalez, SN and Rubinstein, M and Da Ros, VG and Cuasnicú, PS},
title = {Functional redundancy and compensation: Deletion of multiple murine Crisp genes reveals their essential role for male fertility.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {12},
pages = {15718-15733},
doi = {10.1096/fj.202001406R},
pmid = {33037689},
issn = {1530-6860},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Female ; Fertility/*genetics ; Infertility, Male/genetics ; Male ; Membrane Glycoproteins/*genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Seminal Plasma Proteins/*genetics ; Sperm-Ovum Interactions/genetics ; Spermatozoa/metabolism ; },
abstract = {Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca[2+] channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2[-/-] .Crisp4[-/-] mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca[2+] dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.},
}
@article {pmid33037407,
year = {2022},
author = {Cring, MR and Sheffield, VC},
title = {Gene therapy and gene correction: targets, progress, and challenges for treating human diseases.},
journal = {Gene therapy},
volume = {29},
number = {1-2},
pages = {3-12},
pmid = {33037407},
issn = {1476-5462},
support = {R01 EY011298/EY/NEI NIH HHS/United States ; R01 EY017168/EY/NEI NIH HHS/United States ; P30 EY025580/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Genetic Therapy/methods ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {The field of gene therapy has made significant strides over the last several decades toward the treatment of previously untreatable genetic disease. Gene therapy techniques have been aimed at mitigating disease features of recessive and dominant disorders, as well as several cancers and other diseases. While there have been numerous disease targets of gene therapy trials, only four therapies have reached FDA and/or EMA approval for clinical use. Gene correction using CRISPR-Cas9 is an extension of gene therapy that has received considerable attention in recent years and boasts many possible uses beyond classical gene therapy approaches. While there is significant therapeutic potential using gene therapy and gene correction strategies, a number of hurdles remain to be overcome before they become more common in clinical use, particularly with regards to safety and efficacy. As research progresses in this exciting field, it is likely that these therapies will become first-line treatments and will have tremendous positive impacts on the lives of patients with genetic disorders.},
}
@article {pmid33037077,
year = {2020},
author = {Martin, SA and Page, SJ and Piccinni, MZ and Guille, MJ},
title = {Confirming Antibody Specificity in Xenopus.},
journal = {Cold Spring Harbor protocols},
volume = {2020},
number = {12},
pages = {},
doi = {10.1101/pdb.prot105601},
pmid = {33037077},
issn = {1559-6095},
mesh = {Animals ; Antibody Specificity/*immunology ; Blotting, Western ; CRISPR-Cas Systems ; Electrophoresis, Polyacrylamide Gel ; Embryo, Nonmammalian/*metabolism ; Female ; Microinjections ; Oocytes/cytology/metabolism ; RNA, Messenger/administration &amp; dosage/genetics/metabolism ; Recombinant Proteins/genetics/*immunology/metabolism ; Xenopus laevis/embryology/genetics/*metabolism ; },
abstract = {Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in Xenopus embryos is either knocked down using CRISPR-Cas9 technology (for zygotic proteins) or enhanced by microinjection of a synthetic mRNA (for maternal proteins). Western blotting analysis is then performed. If the antibody recognizes the target protein, the western blot will show a relatively weak band for CRISPR-injected embryos and a relatively strong band for RNA-injected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in Xenopus.},
}
@article {pmid33034647,
year = {2020},
author = {Adashi, EY and Cohen, IG},
title = {Heritable Human Genome Editing: The International Commission Report.},
journal = {JAMA},
volume = {324},
number = {19},
pages = {1941-1942},
doi = {10.1001/jama.2020.19059},
pmid = {33034647},
issn = {1538-3598},
mesh = {CRISPR-Cas Systems ; Embryo Research ; Gene Editing/ethics/legislation &amp; jurisprudence/*standards ; *Genome, Human ; Humans ; Internationality ; },
}
@article {pmid33034197,
year = {2022},
author = {Wang, Z and Sui, Y and Li, J and Tian, X and Wang, Q},
title = {Biological control of postharvest fungal decays in citrus: a review.},
journal = {Critical reviews in food science and nutrition},
volume = {62},
number = {4},
pages = {861-870},
doi = {10.1080/10408398.2020.1829542},
pmid = {33034197},
issn = {1549-7852},
mesh = {*Citrus ; Fruit ; Fungi ; *Fungicides, Industrial ; Plant Diseases/prevention &amp; control ; },
abstract = {Citrus (Citrus spp.) species produce a variety of fruits that are popular worldwide. Citrus fruits, however, are susceptible to postharvest decays caused by various pathogenic fungi, including Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, Aspergillus niger, and Aspergillus flavus. Decays resulting from infections by these pathogens cause a significant reduction in citrus quality and marketable yield. Biological control of postharvest decay utilizing antagonistic bacteria and fungi has been explored as a promising alternative to synthetic fungicides. In the present article, the isolation of antagonists utilized to manage postharvest decays in citrus is reviewed, and the mechanism of action including recent molecular and genomic studies is discussed as well. Several recently-postulated mechanisms of action, such as biofilm formation and an oxidative burst of reactive oxygen species have been highlighted. Improvements in biocontrol efficacy of antagonists through the use of a combination of microbial antagonists and additives are also reviewed. Biological control utilizing bacterial and yeast antagonists is a critical component of an integrated management approach for the sustainable development of the citrus industry. Further research will be needed, however, to explore and utilize beneficial microbial consortia and novel approaches like CRISPR/Cas technology for management of postharvest decays.},
}
@article {pmid33033974,
year = {2020},
author = {Yan, M and Li, J},
title = {Combined application of CRISPR-Cas and stem cells for clinical and basic research.},
journal = {Cell regeneration (London, England)},
volume = {9},
number = {1},
pages = {19},
pmid = {33033974},
issn = {2045-9769},
}
@article {pmid33033362,
year = {2020},
author = {Yang, L and Geng, T and Yang, G and Ma, J and Wang, L and Ketkar, H and Yang, D and Lin, T and Hwang, J and Zhu, S and Wang, Y and Dai, J and You, F and Cheng, G and Vella, AT and Flavell, RA and Fikrig, E and Wang, P},
title = {Macrophage scavenger receptor 1 controls Chikungunya virus infection through autophagy in mice.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {556},
pmid = {33033362},
issn = {2399-3642},
support = {R01AI132526/AI/NIAID NIH HHS/United States ; UL1 TR001863/TR/NCATS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AI132526/AI/NIAID NIH HHS/United States ; P30 DK034989/DK/NIDDK NIH HHS/United States ; R01 DK095835/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *Autophagy ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chikungunya Fever/*immunology ; Chikungunya virus/*metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class A/metabolism/*physiology ; },
abstract = {Macrophage scavenger receptor 1 (MSR1) mediates the endocytosis of modified low-density lipoproteins and plays an important antiviral role. However, the molecular mechanism underlying MSR1 antiviral actions remains elusive. We report that MSR1 activates autophagy to restrict infection of Chikungunya virus (CHIKV), an arthritogenic alphavirus that causes acute and chronic crippling arthralgia. Msr1 expression was rapidly upregulated after CHIKV infection in mice. Msr1 knockout mice had elevated viral loads and increased susceptibility to CHIKV arthritis along with a normal type I IFN response. Induction of LC3 lipidation by CHIKV, a marker of autophagy, was reduced in Msr1[-/-] cells. Mechanistically, MSR1 interacted with ATG12 through its cytoplasmic tail and this interaction was enhanced by CHIKV nsP1 protein. MSR1 repressed CHIKV replication through ATG5-ATG12-ATG16L1 and this was dependent on the FIP200-and-WIPI2-binding domain, but not the WD40 domain of ATG16L1. Our results elucidate an antiviral role for MSR1 involving the autophagic function of ATG5-ATG12-ATG16L1.},
}
@article {pmid33033246,
year = {2020},
author = {Martinez-Lage, M and Torres-Ruiz, R and Puig-Serra, P and Moreno-Gaona, P and Martin, MC and Moya, FJ and Quintana-Bustamante, O and Garcia-Silva, S and Carcaboso, AM and Petazzi, P and Bueno, C and Mora, J and Peinado, H and Segovia, JC and Menendez, P and Rodriguez-Perales, S},
title = {In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5060},
pmid = {33033246},
issn = {2041-1723},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Doxorubicin/therapeutic use ; Fusion Proteins, bcr-abl/genetics ; Gene Deletion ; Genetic Loci ; Genomic Instability ; HEK293 Cells ; Humans ; Introns/genetics ; Mice, Nude ; Neoplasms/drug therapy/*genetics/pathology ; Oncogene Fusion/*genetics ; Oncogene Proteins, Fusion/genetics ; RNA, Guide/metabolism ; Reproducibility of Results ; Xenograft Model Antitumor Assays ; },
abstract = {Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.},
}
@article {pmid33033131,
year = {2020},
author = {Sieber, RN and Overballe-Petersen, S and Kaya, H and Larsen, AR and Petersen, A},
title = {Complete Genome Sequences of Methicillin-Resistant Staphylococcus aureus Strains 110900 and 128254, Two Representatives of the CRISPR-Cas-Carrying Sequence Type 630/spa Type t4549 Lineage.},
journal = {Microbiology resource announcements},
volume = {9},
number = {41},
pages = {},
pmid = {33033131},
issn = {2576-098X},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) and spa type t4549 is an emerging lineage in Nordic countries, and some representatives carry the CRISPR-Cas system. Here, the complete genome sequences of two isolates from this lineage are presented, comprising chromosomes of 2,918,239 and 2,877,083 nucleotides, respectively, and a 2,473-nucleotide plasmid carrying erm(C).},
}
@article {pmid33033111,
year = {2020},
author = {Rushworth, LK and Harle, V and Repiscak, P and Clark, W and Shaw, R and Hall, H and Bushell, M and Leung, HY and Patel, R},
title = {In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer.},
journal = {Life science alliance},
volume = {3},
number = {12},
pages = {},
pmid = {33033111},
issn = {2575-1077},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA-Binding Proteins/*genetics/metabolism ; Docetaxel/pharmacology ; Drug Resistance, Neoplasm/*genetics ; Gene Expression Regulation, Neoplastic/genetics ; Genetic Engineering/methods ; Male ; Mice ; Mice, Nude ; Prostatic Neoplasms/*genetics/metabolism ; Taxoids/pharmacology ; Transcription Factors/*genetics/metabolism ; Xenograft Model Antitumor Assays/methods ; },
abstract = {Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A-like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.},
}
@article {pmid33031826,
year = {2020},
author = {Hazafa, A and Mumtaz, M and Farooq, MF and Bilal, S and Chaudhry, SN and Firdous, M and Naeem, H and Ullah, MO and Yameen, M and Mukhtiar, MS and Zafar, F},
title = {CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions.},
journal = {Life sciences},
volume = {263},
number = {},
pages = {118525},
pmid = {33031826},
issn = {1879-0631},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; *Genome, Human ; Humans ; Neoplasms/genetics/pathology/*therapy ; },
abstract = {Cancer is one of the most leading causes of death and a major public health problem, universally. According to accumulated data, annually, approximately 8.5 million people died because of the lethality of cancer. Recently, a novel RNA domain-containing endonuclease-based genome engineering technology, namely the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9) have been proved as a powerful technique in the treatment of cancer cells due to its multifunctional properties including high specificity, accuracy, time reducing and cost-effective strategies with minimum off-target effects. The present review investigates the overview of recent studies on the newly developed genome-editing strategy, CRISPR/Cas9, as an excellent pre-clinical therapeutic option in the reduction and identification of new tumor target genes in the solid tumors. Based on accumulated data, we revealed that CRISPR/Cas9 significantly inhibited the robust tumor cell growth (breast, lung, liver, colorectal, and prostate) by targeting the oncogenes, tumor-suppressive genes, genes associated to therapies by inhibitors, genes associated to chemotherapies drug resistance, and suggested that CRISPR/Cas9 could be a potential therapeutic target in inhibiting the tumor cell growth by suppressing the cell-proliferation, metastasis, invasion and inducing the apoptosis during the treatment of malignancies in the near future. The present review also discussed the current challenges and barriers, and proposed future recommendations for a better understanding.},
}
@article {pmid33028993,
year = {2020},
author = {Ledford, H and Callaway, E},
title = {Pioneers of revolutionary CRISPR gene editing win chemistry Nobel.},
journal = {Nature},
volume = {586},
number = {7829},
pages = {346-347},
pmid = {33028993},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Deoxyribonuclease I ; *Gene Editing ; RNA ; },
}
@article {pmid33028827,
year = {2020},
author = {Carlson-Stevermer, J and Kelso, R and Kadina, A and Joshi, S and Rossi, N and Walker, J and Stoner, R and Maures, T},
title = {CRISPRoff enables spatio-temporal control of CRISPR editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5041},
pmid = {33028827},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics/radiation effects ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; Feasibility Studies ; Gene Editing/*methods ; HEK293 Cells ; Humans ; *Light ; RNA Stability/*radiation effects ; RNA, Guide/*metabolism/radiation effects ; Ribonucleoproteins/metabolism ; Translocation, Genetic ; },
abstract = {Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.},
}
@article {pmid33028634,
year = {2020},
author = {Manjunath, LE and Singh, A and Sahoo, S and Mishra, A and Padmarajan, J and Basavaraju, CG and Eswarappa, SM},
title = {Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {50},
pages = {17009-17026},
pmid = {33028634},
issn = {1083-351X},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {3' Untranslated Regions/*genetics ; Animals ; Aorta/cytology/metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cattle ; Codon, Terminator/*genetics ; Endothelium, Vascular/cytology/metabolism ; HEK293 Cells ; Humans ; *Membrane Potential, Mitochondrial ; Mitochondrial Membrane Transport Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; *Protein Biosynthesis ; Protein Isoforms ; RNA, Messenger/*genetics ; Ribosomes/metabolism ; },
abstract = {Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3' UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3' UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t1/2 (<1 h). MTCH2 read-through-deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential.},
}
@article {pmid33028045,
year = {2020},
author = {Kostyushev, D and Kostyusheva, A and Brezgin, S and Smirnov, V and Volchkova, E and Lukashev, A and Chulanov, V},
title = {Gene Editing by Extracellular Vesicles.},
journal = {International journal of molecular sciences},
volume = {21},
number = {19},
pages = {},
pmid = {33028045},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Extracellular Vesicles/*genetics ; Gene Editing/*trends ; *Gene Transfer Techniques ; Humans ; Mutation/genetics ; RNA/*genetics ; },
abstract = {CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.},
}
@article {pmid33027946,
year = {2020},
author = {Shalaby, K and Aouida, M and El-Agnaf, O},
title = {Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors.},
journal = {International journal of molecular sciences},
volume = {21},
number = {19},
pages = {},
pmid = {33027946},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Therapy/*trends ; Genetic Vectors/*genetics/therapeutic use ; Humans ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.},
}
@article {pmid33024076,
year = {2020},
author = {Schieber, M and Marinaccio, C and Bolanos, LC and Haffey, WD and Greis, KD and Starczynowski, DT and Crispino, JD},
title = {FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome.},
journal = {Blood cancer journal},
volume = {10},
number = {10},
pages = {98},
pmid = {33024076},
issn = {2044-5385},
support = {R01 CA237039/CA/NCI NIH HHS/United States ; R35 HL135787/HL/NHLBI NIH HHS/United States ; S10 RR027015/RR/NCRR NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Cell Transformation, Neoplastic/genetics/*metabolism/pathology ; F-Box Proteins/genetics/*metabolism ; Gene Deletion ; Humans ; Leukemia, Myeloid, Acute/genetics/*metabolism/pathology ; Myelodysplastic Syndromes/genetics/*metabolism/pathology ; Protein-Arginine N-Methyltransferases/genetics/*metabolism ; Tumor Suppressor Proteins/genetics/*metabolism ; },
abstract = {Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.},
}
@article {pmid33023639,
year = {2020},
author = {Jiang, YY and Chai, YP and Lu, MH and Han, XL and Lin, Q and Zhang, Y and Zhang, Q and Zhou, Y and Wang, XC and Gao, C and Chen, QJ},
title = {Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {257},
pmid = {33023639},
issn = {1474-760X},
mesh = {Acetolactate Synthase/*genetics ; Gene Editing/*methods/statistics &amp; numerical data ; Genetic Vectors ; Mutagenesis, Site-Directed/*methods ; Plants, Genetically Modified ; RNA, Guide/genetics/*metabolism ; Zea mays/enzymology/*genetics ; },
abstract = {Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.},
}
@article {pmid33022264,
year = {2020},
author = {Dimitriu, T and Szczelkun, MD and Westra, ER},
title = {Evolutionary Ecology and Interplay of Prokaryotic Innate and Adaptive Immune Systems.},
journal = {Current biology : CB},
volume = {30},
number = {19},
pages = {R1189-R1202},
pmid = {33022264},
issn = {1879-0445},
support = {714478/ERC_/European Research Council/International ; 788405/ERC_/European Research Council/International ; BB/L000873/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity/*immunology ; Archaea/genetics ; Bacteria/genetics ; Biological Evolution ; Ecology ; Evolution, Molecular ; Immune System/immunology/metabolism ; Immunity, Innate/*immunology ; Prokaryotic Cells/*immunology ; },
abstract = {Like many organisms, bacteria and archaea have both innate and adaptive immune systems to defend against infection by viruses and other parasites. Innate immunity most commonly relies on the endonuclease-mediated cleavage of any incoming DNA that lacks a specific epigenetic modification, through a system known as restriction-modification. CRISPR-Cas-mediated adaptive immunity relies on the insertion of short DNA sequences from parasite genomes into CRISPR arrays on the host genome to provide sequence-specific protection. The discovery of each of these systems has revolutionised our ability to carry out genetic manipulations, and, as a consequence, the enzymes involved have been characterised in exquisite detail. In comparison, much less is known about the importance of these two arms of the defence for the ecology and evolution of prokaryotes and their parasites. Here, we review our current ecological and evolutionary understanding of these systems in isolation, and discuss the need to study how innate and adaptive immune responses are integrated when they coexist in the same cell.},
}
@article {pmid33022212,
year = {2021},
author = {Holmgaard, AB and Askou, AL and Jensen, EG and Alsing, S and Bak, RO and Mikkelsen, JG and Corydon, TJ},
title = {Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {1},
pages = {191-207},
pmid = {33022212},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Gene Knockout Techniques ; Gene Transfer Techniques ; Genetic Therapy ; Humans ; Mice ; RNA, Guide/*genetics ; Retina/*metabolism ; Ribonucleoproteins/*metabolism ; Transfection ; Vascular Endothelial Growth Factor A/*genetics ; },
abstract = {The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.},
}
@article {pmid33022126,
year = {2020},
author = {Miao, X and Sun, T and Golan, M and Mager, J and Cui, W},
title = {Loss of POLR1D results in embryonic lethality prior to blastocyst formation in mice.},
journal = {Molecular reproduction and development},
volume = {87},
number = {11},
pages = {1152-1158},
pmid = {33022126},
issn = {1098-2795},
support = {R01 HD083311/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Blastocyst ; CRISPR-Cas Systems ; DNA Damage ; DNA-Directed RNA Polymerases/*deficiency/genetics/physiology ; Embryo, Mammalian/*metabolism ; *Embryonic Development ; Exons/genetics ; Female ; Gastrulation ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Genes, Lethal ; Gestational Age ; In Situ Nick-End Labeling ; Mice ; Mice, Inbred C57BL ; Morula/chemistry/ultrastructure ; Organ Culture Techniques ; Organelle Biogenesis ; Pregnancy ; Reactive Oxygen Species/analysis ; Ribosomes ; Sequence Deletion ; },
abstract = {In eukaryotic cells, RNA polymerase (Pol) I and Pol III are dedicated to the synthesis of ribosomal RNA precursors and a variety of small RNAs, respectively. Although RNA Pol I and Pol III complexes are crucial for the regulation of cell growth and cell cycle in all cell types, many of the components of the Pol I and Pol III complexes have not been functionally characterized in mammals. Here, we provide the first in vivo functional characterization of POLR1D, a subunit shared by RNA Pol I and Pol III, during early mammalian embryo development. Our results show that Polr1d mutant embryos cannot be recovered at E7.5 early post-gastrulation stage, suggesting failed implantation. Although Polr1d mutants can be recovered at E3.5, they exhibit delayed/stalled development with morula morphology rather than differentiation into blastocysts. Even with extended time in culture, mutant embryos fail to form blastocysts and eventually die. Analysis of E3.0 embryos revealed severe DNA damage in Polr1d mutants. Additionally, lineage assessment reveals that trophectoderm specification is compromised in the absence of Polr1d. In summary, these findings demonstrate the essential role of POLR1D during early mammalian embryogenesis and highlight cell-lethal phenotype without Polr1d function.},
}
@article {pmid33021880,
year = {2021},
author = {Hahn, M and Scalliet, G},
title = {One Cut to Change Them All: CRISPR/Cas, a Groundbreaking Tool for Genome Editing in Botrytis cinerea and Other Fungal Plant Pathogens.},
journal = {Phytopathology},
volume = {111},
number = {3},
pages = {474-477},
doi = {10.1094/PHYTO-09-20-0379-PER},
pmid = {33021880},
issn = {0031-949X},
mesh = {Botrytis/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Fungal/genetics ; Genome, Plant ; Plant Diseases ; },
abstract = {CRISPR/Cas is a genome editing technology that has opened new dimensions in functional biology. In a recent publication, we presented a highly efficient CRISPR/Cas technique for Botrytis cinerea, which dramatically increases our options to mutagenize and modify single or multiple genes. In this Perspectives article, we describe the essential features of the method and demonstrate with several examples how it opens new avenues for unraveling the virulence mechanisms of Botrytis and other plant pathogenic fungi and can accelerate research for the identification of new antifungal compounds.},
}
@article {pmid33021790,
year = {2020},
author = {Grant, CV and Cai, S and Risinger, AL and Liang, H and O'Keefe, BR and Doench, JG and Cichewicz, RH and Mooberry, SL},
title = {CRISPR-Cas9 Genome-Wide Knockout Screen Identifies Mechanism of Selective Activity of Dehydrofalcarinol in Mesenchymal Stem-like Triple-Negative Breast Cancer Cells.},
journal = {Journal of natural products},
volume = {83},
number = {10},
pages = {3080-3092},
pmid = {33021790},
issn = {1520-6025},
support = {U01 CA182740/CA/NCI NIH HHS/United States ; },
mesh = {17-Hydroxysteroid Dehydrogenases/drug effects/genetics ; Aldehyde Oxidoreductases/drug effects/genetics ; Antineoplastic Agents, Phytogenic/*pharmacology ; Breast Neoplasms/*drug therapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Knockout Techniques/*methods ; Humans ; Molecular Structure ; Neoplastic Stem Cells/*drug effects ; PPAR gamma/agonists ; RNA, Small Interfering/pharmacology ; Triple Negative Breast Neoplasms/*drug therapy ; },
abstract = {There are no targeted therapies available for triple-negative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from Desmanthodium guatemalense was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (1a). This genomic screen identified HSD17B11, the gene encoding the enzyme 17β-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of 1a in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on HSD17B11 expression, and it was found these were also highly sensitive to 1a. This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.},
}
@article {pmid33020664,
year = {2021},
author = {Griffin, ME and Sorum, AW and Miller, GM and Goddard, WA and Hsieh-Wilson, LC},
title = {Sulfated glycans engage the Ang-Tie pathway to regulate vascular development.},
journal = {Nature chemical biology},
volume = {17},
number = {2},
pages = {178-186},
pmid = {33020664},
issn = {1552-4469},
support = {R01 GM093627/GM/NIGMS NIH HHS/United States ; R01 GM127920/GM/NIGMS NIH HHS/United States ; },
mesh = {Angiopoietin-1/*genetics ; Animals ; Blood Vessels/*drug effects/*growth &amp; development ; CRISPR-Cas Systems ; Cell Line ; Female ; Glycosaminoglycans/pharmacology ; Heparitin Sulfate/pharmacology ; Ligands ; Male ; Mice ; Mice, Transgenic ; Polysaccharides/*pharmacology ; Receptors, TIE/*genetics ; Ribonuclease, Pancreatic/genetics ; Signal Transduction/*drug effects/genetics ; Sulfates/*pharmacology ; },
abstract = {The angiopoietin (Ang)-Tie pathway is essential for the proper maturation and remodeling of the vasculature. Despite its importance in disease, the mechanisms that control signal transduction through this pathway are poorly understood. Here, we demonstrate that heparan sulfate glycosaminoglycans (HS GAGs) regulate Ang-Tie signaling through direct interactions with both Ang ligands and Tie1 receptors. HS GAGs formed ternary complexes with Ang1 or Ang4 and Tie2 receptors, resulting in potentiation of endothelial survival signaling. In addition, HS GAGs served as ligands for the orphan receptor Tie1. The HS-Tie1 interaction promoted Tie1-Tie2 heterodimerization and enhanced Tie1 stability within the mature vasculature. Loss of HS-Tie1 binding using CRISPR-Cas9-mediated mutagenesis in vivo led to decreased Tie protein levels, pathway suppression and aberrant retinal vascularization. Together, these results reveal that sulfated glycans use dual mechanisms to regulate Ang-Tie signaling and are important for the development and maintenance of the vasculature.},
}
@article {pmid33020655,
year = {2020},
author = {Alerasool, N and Segal, D and Lee, H and Taipale, M},
title = {An efficient KRAB domain for CRISPRi applications in human cells.},
journal = {Nature methods},
volume = {17},
number = {11},
pages = {1093-1096},
pmid = {33020655},
issn = {1548-7105},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Catalytic Domain ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Reporter ; HEK293 Cells ; Humans ; K562 Cells ; Kruppel-Like Transcription Factors/*genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Repressor Proteins/genetics ; Transcription, Genetic ; },
abstract = {Clustered regularly interspaced short palindromic repeat interference (CRISPRi), based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression. However, it suffers from incomplete silencing of target genes. We assayed 57 KRAB domains for their repressive potency and identified the ZIM3 KRAB domain as an exceptionally potent repressor. We establish that ZIM3 KRAB-dCas9 fusion silences gene expression more efficiently than existing platforms.},
}
@article {pmid33020633,
year = {2020},
author = {Mamidi, S and Healey, A and Huang, P and Grimwood, J and Jenkins, J and Barry, K and Sreedasyam, A and Shu, S and Lovell, JT and Feldman, M and Wu, J and Yu, Y and Chen, C and Johnson, J and Sakakibara, H and Kiba, T and Sakurai, T and Tavares, R and Nusinow, DA and Baxter, I and Schmutz, J and Brutnell, TP and Kellogg, EA},
title = {A genome resource for green millet Setaria viridis enables discovery of agronomically valuable loci.},
journal = {Nature biotechnology},
volume = {38},
number = {10},
pages = {1203-1210},
pmid = {33020633},
issn = {1546-1696},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; DNA Transposable Elements/genetics ; Domestication ; Edible Grain/genetics ; Gene Editing ; Genome, Plant/*genetics ; Genotype ; Millets/*genetics ; Phenotype ; Phylogeny ; Plant Proteins/*genetics ; Setaria Plant/*genetics ; },
abstract = {Wild and weedy relatives of domesticated crops harbor genetic variants that can advance agricultural biotechnology. Here we provide a genome resource for the wild plant green millet (Setaria viridis), a model species for studies of C4 grasses, and use the resource to probe domestication genes in the close crop relative foxtail millet (Setaria italica). We produced a platinum-quality genome assembly of S. viridis and de novo assemblies for 598 wild accessions and exploited these assemblies to identify loci underlying three traits: response to climate, a 'loss of shattering' trait that permits mechanical harvest and leaf angle, a predictor of yield in many grass crops. With CRISPR-Cas9 genome editing, we validated Less Shattering1 (SvLes1) as a gene whose product controls seed shattering. In S. italica, this gene was rendered nonfunctional by a retrotransposon insertion in the domesticated loss-of-shattering allele SiLes1-TE (transposable element). This resource will enhance the utility of S. viridis for dissection of complex traits and biotechnological improvement of panicoid crops.},
}
@article {pmid33020616,
year = {2020},
author = {Matharu, N and Ahituv, N},
title = {Modulating gene regulation to treat genetic disorders.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {11},
pages = {757-775},
pmid = {33020616},
issn = {1474-1784},
mesh = {Animals ; CRISPR-Cas Systems/drug effects/genetics ; Gene Expression Regulation/*drug effects/genetics ; Genetic Diseases, Inborn/*drug therapy/genetics ; Humans ; Mutation/drug effects/genetics ; Pharmaceutical Preparations/*administration &amp; dosage ; },
abstract = {Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.},
}
@article {pmid33020605,
year = {2020},
author = {Leech, R and Sampath, K},
title = {A CRISPR cut for messenger RNAs.},
journal = {Lab animal},
volume = {49},
number = {11},
pages = {317-319},
pmid = {33020605},
issn = {1548-4475},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; RNA, Messenger/genetics ; },
}
@article {pmid33020327,
year = {2020},
author = {Zebell, SG},
title = {Excising the Mystery of Single-Guide RNA Processing.},
journal = {Plant physiology},
volume = {184},
number = {2},
pages = {572-573},
pmid = {33020327},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; Gene Expression ; Nucleic Acid Conformation ; *RNA, Guide ; },
}
@article {pmid33020192,
year = {2020},
author = {Mao, D and Jia, Y and Peng, P and Shen, D and Ren, X and Zhu, R and Qiu, Y and Han, Y and Yu, J and Che, Q and Li, Y and Lu, X and Liu, LP and Wang, Z and Liu, Q and Sun, J and Ni, JQ},
title = {Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {12},
pages = {4483-4488},
pmid = {33020192},
issn = {2160-1836},
mesh = {Animals ; Base Composition ; CRISPR-Cas Systems ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Transcription Initiation Site ; },
abstract = {The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.},
}
@article {pmid33019636,
year = {2020},
author = {Costa-Broseta, &#xc1; and Castillo, M and León, J},
title = {Nitrite Reductase 1 Is a Target of Nitric Oxide-Mediated Post-Translational Modifications and Controls Nitrogen Flux and Growth in Arabidopsis.},
journal = {International journal of molecular sciences},
volume = {21},
number = {19},
pages = {},
pmid = {33019636},
issn = {1422-0067},
mesh = {Ammonium Compounds/metabolism ; Arabidopsis/enzymology/*genetics/growth &amp; development ; Arabidopsis Proteins/chemistry/*genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems ; Gene Editing ; *Gene Expression Regulation, Plant ; Mitochondria/metabolism ; Models, Molecular ; Mutation ; Nitrates/metabolism ; Nitric Oxide/metabolism ; Nitrite Reductases/chemistry/*genetics/metabolism ; Nitrites/*metabolism ; Nitrogen/metabolism ; Nitroso Compounds/metabolism ; Plant Leaves/enzymology/*genetics/growth &amp; development ; Plant Roots/enzymology/genetics/growth &amp; development ; Plants, Genetically Modified ; Plastids/metabolism ; Protein Conformation ; *Protein Processing, Post-Translational ; Spinacia oleracea/enzymology/genetics ; },
abstract = {Plant growth is the result of the coordinated photosynthesis-mediated assimilation of oxidized forms of C, N and S. Nitrate is the predominant N source in soils and its reductive assimilation requires the successive activities of soluble cytosolic NADH-nitrate reductases (NR) and plastid stroma ferredoxin-nitrite reductases (NiR) allowing the conversion of nitrate to nitrite and then to ammonium. However, nitrite, instead of being reduced to ammonium in plastids, can be reduced to nitric oxide (NO) in mitochondria, through a process that is relevant under hypoxic conditions, or in the cytoplasm, through a side-reaction catalyzed by NRs. We use a loss-of-function approach, based on CRISPR/Cas9-mediated genetic edition, and gain-of-function, using transgenic overexpressing HA-tagged Arabidopsis NiR1 to characterize the role of this enzyme in controlling plant growth, and to propose that the NO-related post-translational modifications, by S-nitrosylation of key C residues, might inactivate NiR1 under stress conditions. NiR1 seems to be a key target in regulating nitrogen assimilation and NO homeostasis, being relevant to the control of both plant growth and performance under stress conditions. Because most higher plants including crops have a single NiR, the modulation of its function might represent a relevant target for agrobiotechnological purposes.},
}
@article {pmid33017688,
year = {2020},
author = {Lata, KS and Vaghasia, V and Bhairappanavar, SB and Kumar, S and Ayachit, G and Patel, S and Das, J},
title = {Whole genome sequencing and de novo assembly of three virulent Indian isolates of Leptospira.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {85},
number = {},
pages = {104579},
doi = {10.1016/j.meegid.2020.104579},
pmid = {33017688},
issn = {1567-7257},
mesh = {Computational Biology/methods ; *Genome, Bacterial ; *Genomics/methods ; Humans ; India/epidemiology ; Leptospira/*genetics/isolation &amp; purification/pathogenicity ; Leptospirosis/*epidemiology/*microbiology ; Polymorphism, Single Nucleotide ; Virulence/genetics ; Virulence Factors/genetics ; *Whole Genome Sequencing ; },
abstract = {Leptospirosis is a re-emerging bacterial zoonosis caused by pathogenic Leptospira, with a worldwide distribution and becoming a major public health concern. Prophylaxis of this disease is difficult due to several factors such as non-specific variable clinical manifestation, presence of a large number of serovar, species and asymptomatic reservoir hosts, lack of proper diagnostics and vaccines. Despite its global importance and severity of the disease, knowledge about the molecular mechanism of pathogenesis and evolution of pathogenic species of Leptospira remains limited. In this study, we sequenced and analyzed three highly pathogenic species of Indian isolates of Leptospira (interrogans, santarosai, and kirschneri). Additionally, we identified some virulence-related and CRISPR-Cas genes. The virulent analysis showed 232 potential virulence factors encoding proteins in L. interrogans strain Salinem and L. santarosai strain M-4 genome. While the genome of L. kirschneri strain Wumalasena was predicted to encode 198 virulence factor proteins. The variant calling analysis revealed 1151, 19,786, and 22,996 single nucleotide polymorphisms (SNPs) for L. interrogans strain Salinem, L. kirschneri strain Wumalasena and L. santarosai strain M-4, respectively, with a maximum of 5315 missense and 12,221 synonymous mutations for L. santarosai strain M-4. The structural analyses of genomes indicated potential evidence of inversions and structural rearrangment in all three genomes. The availability of these genome sequences and in silico analysis of Leptospira will provide a basis for a deeper understanding of their molecular diversity and pathogenesis mechanism, and further pave a way towards proper management of the disease.},
}
@article {pmid33017151,
year = {2020},
author = {Li, L and Tan, D and Liu, S and Jiao, R and Yang, X and Li, F and Wu, H and Huang, W},
title = {Optimization of Factor Combinations for Stem Cell Differentiations on a Design-of-Experiment Microfluidic Chip.},
journal = {Analytical chemistry},
volume = {92},
number = {20},
pages = {14228-14235},
doi = {10.1021/acs.analchem.0c03488},
pmid = {33017151},
issn = {1520-6882},
mesh = {Biocompatible Materials/*chemistry ; Biosensing Techniques ; CRISPR-Cas Systems ; Cell Differentiation/*physiology ; Cells, Cultured ; Cloning, Molecular ; Endoderm/cytology/metabolism ; Fluorescent Dyes/chemistry ; Gene Expression ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Microfluidics/*instrumentation ; Myostatin/genetics ; Optical Imaging ; Polymethyl Methacrylate/*chemistry ; Surface Properties ; },
abstract = {Directed differentiation of stem cells plays a vital role in cell replacement therapy. Many activators and inhibitors targeting different signaling pathways have been identified to contribute to each step of differentiation. Most studies relied on empirically optimizing the combinations of the aforementioned factors for each step to optimize the efficiency of differentiation, which are time-consuming and nonsystematic. Design-of-experiment (DOE) is a powerful strategy to identify the critical combinations from multiple factors systematically. However, it is prohibitively complicated for typical laboratories, given a large number of potential combinations. Here, we develop a multilayer polymethyl methacrylate-based, reusable microfluidic chip to directly facilitate the DOE in the differentiation of stem cells. The chip consists of an inlet layer and multiple disperse layers. Different solutions are injected simultaneously to the chip through the inlet layer. Subsequently, the channels in the disperse layers split and recombine the flow streams to generate solution combinations based on hard-wired DOE designs. We demonstrated that it is in quantitative agreement with the designs using fluorescent dyes. Moreover, we constructed a human-induced pluripotent stem reporter cell line to improve the consistency of the cellular state measurements and use the chip to identify critical factors for cell differentiation to definitive endoderm (DE). We found that the differentiation efficiencies under various factor combinations are significantly different, and CHIR99201 and GDF8 are the most critical factors for differentiation to DE. Our method is potentially applicable to the optimization of factor combinations for multi-step stem cell differentiation and combinatorial drug screening.},
}
@article {pmid33016258,
year = {2020},
author = {Hebras, J and Marty, V and Personnaz, J and Mercier, P and Krogh, N and Nielsen, H and Aguirrebengoa, M and Seitz, H and Pradere, JP and Guiard, BP and Cavaille, J},
title = {Reassessment of the involvement of Snord115 in the serotonin 2c receptor pathway in a genetically relevant mouse model.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33016258},
issn = {2050-084X},
support = {DEQ20160334936//Fondation pour la Recherche M&#xe9;dicale/International ; ANR-18-CE12-0008-01//Agence Nationale de la Recherche/International ; },
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Diet, High-Fat ; *Emotions ; Feeding Behavior/*physiology ; Gene Expression Regulation/*physiology ; Mice ; Mice, Knockout ; RNA, Messenger/genetics/metabolism ; RNA, Small Nucleolar/genetics/*metabolism ; Receptor, Serotonin, 5-HT2C/genetics/*metabolism ; },
abstract = {SNORD115 has been proposed to promote the activity of serotonin (HTR2C) receptor via its ability to base pair with its pre-mRNA and regulate alternative RNA splicing and/or A-to-I RNA editing. Because SNORD115 genes are deleted in most patients with the Prader-Willi syndrome (PWS), diminished HTR2C receptor activity could contribute to the impaired emotional response and/or compulsive overeating characteristic of this disease. In order to test this appealing but never demonstrated hypothesis in vivo, we created a CRISPR/Cas9-mediated Snord115 knockout mouse. Surprisingly, we uncovered only modest region-specific alterations in Htr2c RNA editing profiles, while Htr2c alternative RNA splicing was unchanged. These subtle changes, whose functional relevance remains uncertain, were not accompanied by any discernible defects in anxio-depressive-like phenotypes. Energy balance and eating behavior were also normal, even after exposure to high-fat diet. Our study raises questions concerning the physiological role of SNORD115, notably its involvement in behavioural disturbance associated with PWS.},
}
@article {pmid33014683,
year = {2020},
author = {Meena, MR and Kumar, R and Chinnaswamy, A and Karuppaiyan, R and Kulshreshtha, N and Ram, B},
title = {Current breeding and genomic approaches to enhance the cane and sugar productivity under abiotic stress conditions.},
journal = {3 Biotech},
volume = {10},
number = {10},
pages = {440},
pmid = {33014683},
issn = {2190-572X},
abstract = {Sugarcane (Saccharum spp.) crop is vulnerable to many abiotic stresses such as drought, salinity, waterlogging, cold and high temperature due to climate change. Over the past few decades new breeding and genomic approaches have been used to enhance the genotypic performance under abiotic stress conditions. In sugarcane, introgression of genes from wild species and allied genera for abiotic stress tolerance traits plays a significant role in the development of several stress-tolerant varieties. Moreover, the genomics and transcriptomics approaches have helped to elucidate the key genes/TFs and pathways involved in abiotic stress tolerance in sugarcane. Several novel miRNAs families /proteins or regulatory elements that are responsible for drought, salinity, and cold tolerance have been identified through high-throughput sequencing. The existing sugarcane monoploid genome sequence information opens new gateways and opportunities for researchers to improve the desired traits through efficient genome editing tools, such as the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) system. TALEN mediated mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane significantly reduces the lignin content in the cell wall which is amenable for biofuel production from lignocellulosic biomass. In this review, we focus on current breeding with genomic approaches and their substantial role in enhancing cane production under the abiotic stress conditions, which is expected to provide new insights to plant breeders and biotechnologists to modify their strategy in developing stress-tolerant sugarcane varieties, which can highlight the future demand of cane, bio-energy, and viability of sugar industries.},
}
@article {pmid33014011,
year = {2020},
author = {Rabinowitz, R and Almog, S and Darnell, R and Offen, D},
title = {CrisPam: SNP-Derived PAM Analysis Tool for Allele-Specific Targeting of Genetic Variants Using CRISPR-Cas Systems.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {851},
pmid = {33014011},
issn = {1664-8021},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising novel technology that holds the potential of treating genetic diseases. Safety and specificity of the treatment are to be further studied and developed prior to implementation of the technology into the clinic. The guide-RNA (gRNA) allows precise position-specific DNA targeting, although it may tolerate small changes such as point mutations. The permissive nature of the CRISPR-Cas system makes allele-specific targeting a challenging goal. Hence, an allele-specific targeting approach is in need for future treatments of heterozygous patients suffering from diseases caused by dominant negative mutations. The single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach allows highly allele-specific DNA cleavage due to the existence of a novel PAM sequence only at the target allele. Here, we present CrisPam, a computational tool that detects PAMs within the variant allele for allele-specific targeting by CRISPR-Cas systems. The algorithm scans the sequences and attempts to identify the generation of multiple PAMs for a given reference sequence and its variations. A successful result is such that at least a single PAM is generated by the variation nucleotide. Since the PAM is present within the variant allele only, the Cas enzyme will bind the variant allele exclusively. Analyzing a dataset of human pathogenic point mutations revealed that 90% of the analyzed mutations generated at least a single PAM. Thus, the SNP-derived PAM approach is ideal for targeting most of the point mutations in an allele-specific manner. CrisPam simplifies the gRNAs design process to specifically target the allele of interest and scans a wide range of 26 unique PAMs derived from 23 Cas enzymes. CrisPam is freely available at https://www.danioffenlab.com/crispam.},
}
@article {pmid33014001,
year = {2020},
author = {Gramazio, P and Takayama, M and Ezura, H},
title = {Challenges and Prospects of New Plant Breeding Techniques for GABA Improvement in Crops: Tomato as an Example.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {577980},
pmid = {33014001},
issn = {1664-462X},
abstract = {Over the last seven decades, γ-aminobutyric acid (GABA) has attracted great attention from scientists for its ubiquity in plants, animals and microorganisms and for its physiological implications as a signaling molecule involved in multiple pathways and processes. Recently, the food and pharmaceutical industries have also shown significantly increased interest in GABA, because of its great potential benefits for human health and the consumer demand for health-promoting functional compounds, resulting in the release of a plethora of GABA-enriched products. Nevertheless, many crop species accumulate appreciable GABA levels in their edible parts and could help to meet the daily recommended intake of GABA for promoting positive health effects. Therefore, plant breeders are devoting much effort into breeding elite varieties with improved GABA contents. In this regard, tomato (Solanum lycopersicum), the most produced and consumed vegetable worldwide and a fruit-bearing model crop, has received much consideration for its accumulation of remarkable GABA levels. Although many different strategies have been implemented, from classical crossbreeding to induced mutagenesis, new plant breeding techniques (NPBTs) have achieved the best GABA accumulation results in red ripe tomato fruits along with shedding light on GABA metabolism and gene functions. In this review, we summarize, analyze and compare all the studies that have substantially contributed to tomato GABA breeding with further discussion and proposals regarding the most recent NPBTs that could bring this process to the next level of precision and efficiency. This document also provides guidelines with which researchers of other crops might take advantage of the progress achieved in tomato for more efficient GABA breeding programs.},
}
@article {pmid33012992,
year = {2020},
author = {Damerum, A and Chapman, MA and Taylor, G},
title = {Innovative breeding technologies in lettuce for improved post-harvest quality.},
journal = {Postharvest biology and technology},
volume = {168},
number = {},
pages = {111266},
pmid = {33012992},
issn = {0925-5214},
abstract = {Societal awareness of healthy eating is increasing alongside the market for processed bagged salads, which remain as one of the strongest growing food sectors internationally, including most recently from indoor growing systems. Lettuce represents a significant proportion of this ready-to-eat salad market. However, such products typically have a short shelf life, with decay of post-harvest quality occurring through complex biochemical and physiological changes in leaves and resulting in spoilage, food waste and risks to health. We review the functional and quantitative genetic understanding of lettuce post-harvest quality, revealing that few findings have translated into improved cultivar development. We identify (i) phytonutrient status (for enhanced antioxidant and vitamin status, aroma and flavour) (ii) leaf biophysical, cell wall and water relations traits (for longer shelf life) (iii) leaf surface traits (for enhanced food safety and reduced spoilage) and (iv) chlorophyll, other pigments and developmental senescence traits (for appearance and colour), as key targets for future post-harvest breeding. Lettuce is well-placed for rapid future exploitation to address postharvest quality traits with extensive genomic resources including the recent release of the lettuce genome and the development of innovative breeding technologies. Although technologies such as CRISPR/Cas genome editing are paving the way for accelerated crop improvement, other equally important resources available for lettuce include extensive germplasm collections, bi-parental mapping and wide populations with genotyping for genomic selection strategies and extensive multiomic datasets for candidate gene discovery. We discuss current progress towards post-harvest quality breeding for lettuce and how such resources may be utilised for future crop improvement.},
}
@article {pmid33012507,
year = {2020},
author = {Wang, Z and Li, X and Zhou, B},
title = {Drosophila ZnT1 is essential in the intestine for dietary zinc absorption.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {4},
pages = {1004-1011},
doi = {10.1016/j.bbrc.2020.09.077},
pmid = {33012507},
issn = {1090-2104},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cation Transport Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Diet ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Drosophila melanogaster/genetics/growth &amp; development/*metabolism ; Female ; Gene Knockdown Techniques ; Genes, Insect ; Humans ; Intestinal Absorption/genetics/physiology ; Male ; Mutation ; Phylogeny ; Recombinant Proteins/genetics/metabolism ; Trace Elements/administration &amp; dosage/metabolism/pharmacokinetics ; Zinc/administration &amp; dosage/*metabolism/pharmacokinetics ; },
abstract = {Zinc is an essential trace element and participates in a variety of biological processes. ZnT (SLC30) family members are generally responsible for zinc efflux across the membrane regulating zinc homeostasis. In mammals, the only predominantly plasma membrane resident ZnT has been reported to be ZnT1, and ZnT1[-]/ZnT1[-] mice die at the embryonic stage. In Drosophila, knock down of ZnT1 homologue (dZnT1//ZnT63C/CG17723) results in growth arrest under zinc-limiting conditions. To investigate the essentiality of dZnT1 for zinc homeostasis, as well as its role in dietary zinc uptake especially under normal physiological conditions, we generated dZnT1 mutants by the CRISPER/Cas9 method. Homozygous mutant dZnT1 is lethal, with substantial zinc accumulation in the iron cell region, posterior midgut as well as gastric caeca. Expression of human ZnT1 (hZnT1), in the whole body or in the entire midgut, fully rescued the dZnT1 mutant lethality, whereas tissue-specific expression of hZnT1 in the iron cell region and posterior midgut partially rescued the developmental defect of the dZnT1 mutant. Supplementation of zinc together with clioquinol or hinokitiol conferred a limited but observable rescue upon dZnT1 loss. Our work demonstrated the absolute requirement of dZnT1 in Drosophila survival and indicated that the most essential role of dZnT1 is in the gut.},
}
@article {pmid33011993,
year = {2021},
author = {Gonçalves, BC and Lopes Barbosa, MG and Silva Olak, AP and Belebecha Terezo, N and Nishi, L and Watanabe, MA and Marinello, P and Zendrini Rechenchoski, D and Dejato Rocha, SP and Faccin-Galhardi, LC},
title = {Antiviral therapies: advances and perspectives.},
journal = {Fundamental &amp; clinical pharmacology},
volume = {35},
number = {2},
pages = {305-320},
pmid = {33011993},
issn = {1472-8206},
mesh = {Antiviral Agents/*therapeutic use ; COVID-19/*drug therapy ; Humans ; SARS-CoV-2/*drug effects ; },
abstract = {Viral infections cause high morbidity and mortality, threaten public health, and impose a socioeconomic burden. We have seen the recent emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), the causative agent of COVID-19 that has already infected more than 29 million people, with more than 900 000 deaths since its identification in December 2019. Considering the significant impact of viral infections, research and development of new antivirals and control strategies are essential. In this paper, we summarize 96 antivirals approved by the Food and Drug Administration between 1987 and 2019. Of these, 49 (51%) are used in treatments against human immunodeficiency virus (HIV), four against human papillomavirus, six against cytomegalovirus, eight against hepatitis B virus, five against influenza, six against herpes simplex virus, 17 against hepatitis C virus and one against respiratory syncytial virus. This review also describes future perspectives for new antiviral therapies such as nanotechnologies, monoclonal antibodies and the CRISPR-Cas system. These strategies are suggested as inhibitors of viral replication by various means, such as direct binding to the viral particle, blocking the infection, changes in intracellular mechanisms or viral genes, preventing replication and virion formation. We also observed that a large number of viral agents have no therapy available and the majority of those approved in the last 32 years are restricted to some groups, especially anti-HIV. Additionally, the emergence of new viruses and strains resistant to available antivirals has necessitated the formulation of new antivirals.},
}
@article {pmid33011743,
year = {2021},
author = {Meaden, S and Capria, L and Alseth, E and Gandon, S and Biswas, A and Lenzi, L and van Houte, S and Westra, ER},
title = {Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity.},
journal = {The ISME journal},
volume = {15},
number = {2},
pages = {534-544},
pmid = {33011743},
issn = {1751-7370},
support = {/WT_/Wellcome Trust/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; Pseudomonas aeruginosa/genetics ; },
abstract = {CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.},
}
@article {pmid33011454,
year = {2020},
author = {Yang, Z and Blenner, M},
title = {Genome editing systems across yeast species.},
journal = {Current opinion in biotechnology},
volume = {66},
number = {},
pages = {255-266},
pmid = {33011454},
issn = {1879-0429},
support = {R35 GM133803/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Phylogeny ; Saccharomyces cerevisiae/genetics ; },
abstract = {Yeasts are used to produce a myriad of value-added compounds. Engineering yeasts into cost-efficient cell factories is greatly facilitated by the availability of genome editing tools. While traditional engineering techniques such as homologous recombination-based gene knockout and pathway integration continue to be widely used, novel genome editing systems including multiplexed approaches, bacteriophage integrases, CRISPR-Cas systems, and base editors are emerging as more powerful toolsets to accomplish rapid genome scale engineering and phenotype screening. In this review, we summarized the techniques which have been successfully implemented in model yeast Saccharomyces cerevisiae as well as non-conventional yeast species. The mechanisms and applications of various genome engineering systems are discussed and general guidelines to expand genome editing systems from S. cerevisiae to other yeast species are also highlighted.},
}
@article {pmid33010669,
year = {2020},
author = {Seyed Hosseini, E and Riahi Kashani, N and Nikzad, H and Azadbakht, J and Hassani Bafrani, H and Haddad Kashani, H},
title = {The novel coronavirus Disease-2019 (COVID-19): Mechanism of action, detection and recent therapeutic strategies.},
journal = {Virology},
volume = {551},
number = {},
pages = {1-9},
pmid = {33010669},
issn = {1096-0341},
mesh = {Antiviral Agents/therapeutic use ; COVID-19/*diagnosis/*drug therapy/immunology/virology ; COVID-19 Testing ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Humans ; Immunity ; Phylogeny ; SARS-CoV-2/*pathogenicity/*physiology/ultrastructure ; },
abstract = {Novel coronavirus SARS-CoV-2, designated as COVID-19 by the World Health Organization (WHO) on the February 11, 2020, is one of the highly pathogenic β-coronaviruses which infects human. Early diagnosis of COVID-19 is the most critical step to treat infection. The diagnostic tools are generally molecular methods, serology and viral culture. Recently CRISPR-based method has been investigated to diagnose and treat coronavirus infection. The emergence of 2019-nCoV during the influenza season, has led to the extensive use of antibiotics and neuraminidase enzyme inhibitors, taken orally and intravenously. Currently, antiviral inhibitors of SARS and MERS spike proteins, neuraminidase inhibitors, anti-inflammatory drugs and EK1 peptide are the available therapeutic options for SARS-CoV-2 infected individuals. In addition, Chloroquine, which was previously used for malarial and autoimmune disease, has shown efficacy in the 2019-nCoV infection treatment. In severe hypoxaemia, a combination of antibiotics, α-interferon, lopinavir and mechanical ventilation can effectively mitigate the symptoms. Comprehensive knowledge on the innate and adaptive immune responses, will make it possible to propose potent antiviral drugs with their effective therapeutic measures for the prevention of viral infection. This therapeutic strategy will help patients worldwide to protect themselves against severe and fatal viral infections, that potentially can evolve and develop drug resistance, and to reduce mortality rates.},
}
@article {pmid33010275,
year = {2020},
author = {Zhao, X and Zheng, H and Zhen, J and Shu, W and Yang, S and Xu, J and Song, H and Ma, Y},
title = {Multiplex genetic engineering improves endogenous expression of mesophilic α-amylase gene in a wild strain Bacillus amyloliquefaciens 205.},
journal = {International journal of biological macromolecules},
volume = {165},
number = {Pt A},
pages = {609-618},
doi = {10.1016/j.ijbiomac.2020.09.210},
pmid = {33010275},
issn = {1879-0003},
mesh = {*Bacillus amyloliquefaciens/enzymology/genetics ; *Bacterial Proteins/biosynthesis/genetics ; CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation, Bacterial ; *Gene Expression Regulation, Enzymologic ; *alpha-Amylases/biosynthesis/genetics ; },
abstract = {A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo[-] was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo[-] achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.},
}
@article {pmid33010268,
year = {2020},
author = {Deng, H and Liang, W and Fan, TP and Zheng, X and Cai, Y},
title = {Modular engineering of Shiraia bambusicola for hypocrellin production through an efficient CRISPR system.},
journal = {International journal of biological macromolecules},
volume = {165},
number = {Pt A},
pages = {796-803},
doi = {10.1016/j.ijbiomac.2020.09.208},
pmid = {33010268},
issn = {1879-0003},
mesh = {*Ascomycota/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Perylene/*analogs &amp; derivatives/metabolism ; Phenol/*metabolism ; Quinones/*metabolism ; },
abstract = {Shiraia bambusicola exhibits an excellent capability to produce high-value pharmacological drugs, such as hypocrellin. However, less effective molecular tools hamper the processes to discover or exploit these metabolites. To address this issue, the more effective CRISPR/Cas9 system was constructed by optimizing the sgRNA transcription elements and disrupting the endogenous non-homologous end-joining pathway. These tactics prompted the gene-targeting frequency of 100% and simultaneously multiplex genome editing in S. bambusicola. This optimal CRISPR system encouraged us to rewire the entire hypocrellin flux and improve the yield by orchestrating the substrate pool supply, the central hypocrellin pathway, and the antioxidant system. Thus, 8632 mg/L hypocrellin was obtained, resulting in a 12-fold increase than that of the wild-type strain. This engineered S. bambusicola can still endure oxidative stresses from higher target metabolites and sustain an excellent biological activity. This study provides a whole conception to establish the more efficient genome-editing system. Higher conserved transcription elements for sgRNA expressions inspire us to adopt this system for gene modifications of other filamentous fungi. The rational and global biosystems outline will offer guidance to modulate metabolite productivity in other filamentous fungi.},
}
@article {pmid33010154,
year = {2021},
author = {Cui, Y and Cheng, X and Chen, Q and Song, B and Chiu, A and Gao, Y and Dawson, T and Chao, L and Zhang, W and Li, D and Zeng, Z and Yu, J and Li, Z and Fei, T and Peng, S and Li, W},
title = {CRISP-view: a database of functional genetic screens spanning multiple phenotypes.},
journal = {Nucleic acids research},
volume = {49},
number = {D1},
pages = {D848-D854},
pmid = {33010154},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; *Databases, Genetic ; *Genetic Testing ; Metadata ; Molecular Sequence Annotation ; Phenotype ; User-Computer Interface ; },
abstract = {High-throughput genetic screening based on CRISPR/Cas9 or RNA-interference (RNAi) enables the exploration of genes associated with the phenotype of interest on a large scale. The rapid accumulation of public available genetic screening data provides a wealth of knowledge about genotype-to-phenotype relationships and a valuable resource for the systematic analysis of gene functions. Here we present CRISP-view, a comprehensive database of CRISPR/Cas9 and RNAi screening datasets that span multiple phenotypes, including in vitro and in vivo cell proliferation and viability, response to cancer immunotherapy, virus response, protein expression, etc. By 22 September 2020, CRISP-view has collected 10 321 human samples and 825 mouse samples from 167 papers. All the datasets have been curated, annotated, and processed by a standard MAGeCK-VISPR analysis pipeline with quality control (QC) metrics. We also developed a user-friendly webserver to visualize, explore, and search these datasets. The webserver is freely available at http://crispview.weililab.org.},
}
@article {pmid33009787,
year = {2020},
author = {Ushakova, VM and Morozova, AY and Reznik, AM and Kostyuk, GP and Chekhonin, VP},
title = {[Molecular Biological Aspects of Depressive Disorders: A Modern View].},
journal = {Molekuliarnaia biologiia},
volume = {54},
number = {5},
pages = {725-749},
doi = {10.31857/S0026898420050110},
pmid = {33009787},
issn = {0026-8984},
mesh = {Animals ; CRISPR-Cas Systems ; *Depressive Disorder/genetics ; Disease Models, Animal ; Gene Knockdown Techniques ; Humans ; Polymorphism, Genetic ; },
abstract = {Depression is a serious mental disorder that affects more than 300 million people worldwide. Due to the lack of effective treatment methods, the pathogenesis of depression is necessary to study in order to understand its development and find new therapies. The review describes the main mechanisms of depression, including the monoamine hypothesis, impairment of the hipotalamic-pituitary-adrenal axis, decreased production of neurotropic factors, and neuroinflammation. Genetic correlations, gene polymorphisms, and epigenetic mechanisms are also considered. Common and different features of the etiology are analyzed for depression and depressive conditions associated with other pathologies (schizophrenia, Parkinson disease, and Alzheimer's disease). Modern experimental methods used to investigate the molecular mechanisms of depressive conditions are described with a focus on gene knockouts in laboratory animals and the CRISPR/Cas technology. Consideration is given to optogenetic and chemogenetic methods and analyses of genetic polymorphisms and their combinations. The data may provide for a better integral understanding of the modern ideas about the pathogenesis of depression as an isolated or comorbid disorder and the prospects in studying the mechanisms of depressive conditions.},
}
@article {pmid33008045,
year = {2020},
author = {Hewes, AM and Sansbury, BM and Kmiec, EB},
title = {The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {33008045},
issn = {2073-4425},
support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; Cell Line ; DNA/*chemistry/genetics ; Endodeoxyribonucleases/metabolism ; *Gene Editing ; Gene Knockout Techniques ; Gene Targeting ; Humans ; Oligodeoxyribonucleotides/chemistry/genetics ; Recombinational DNA Repair ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.},
}
@article {pmid33007987,
year = {2020},
author = {Adaui, V and Kröber-Boncardo, C and Brinker, C and Zirpel, H and Sellau, J and Arévalo, J and Dujardin, JC and Clos, J},
title = {Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {33007987},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; Endopeptidase Clp/*genetics/metabolism ; Gene Editing ; Gene Targeting ; Genes, Protozoan ; Heat-Shock Proteins/*genetics/metabolism ; Leishmania braziliensis/*genetics/physiology ; Leishmania major/genetics/physiology ; Mutation ; Polymerase Chain Reaction ; Protozoan Proteins/*genetics/metabolism ; *Reverse Genetics ; Thermotolerance ; },
abstract = {The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.},
}
@article {pmid33007277,
year = {2021},
author = {Wegler, C and Gazit, M and Issa, K and Subramaniam, S and Artursson, P and Karlgren, M},
title = {Expanding the Efflux In Vitro Assay Toolbox: A CRISPR-Cas9 Edited MDCK Cell Line with Human BCRP and Completely Lacking Canine MDR1.},
journal = {Journal of pharmaceutical sciences},
volume = {110},
number = {1},
pages = {388-396},
doi = {10.1016/j.xphs.2020.09.039},
pmid = {33007277},
issn = {1520-6017},
mesh = {*ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/metabolism ; Animals ; Biological Transport ; *CRISPR-Cas Systems ; Cell Line ; Dogs ; Humans ; Madin Darby Canine Kidney Cells ; Neoplasm Proteins/genetics/metabolism ; },
abstract = {The Breast Cancer Resistance Protein (BCRP) is a key transporter in drug efflux and drug-drug interactions. However, endogenous expression of Multidrug Resistance Protein 1 (MDR1) confounds the interpretation of BCRP-mediated transport in in vitro models. Here we used a CRISPR-Cas9 edited Madin-Darby canine kidney (MDCK) II cell line (MDCK[cMDR1-KO]) for stable expression of human BCRP (hBCRP) with no endogenous canine MDR1 (cMDR1) expression (MDCK-hBCRP[cMDR1-KO]). Targeted quantitative proteomics verified expression of hBCRP, and global analysis of the entire proteome corroborated no or very low background expression of other drug transport proteins or metabolizing enzymes. This new cell line, had similar proteome like MDCK[cMDR1-KO] and a previously established, corresponding cell line overexpressing human MDR1 (hMDR1), MDCK-hMDR1[cMDR1-KO]. Functional studies with MDCK-hBCRP[cMDR1-KO] confirmed high hBCRP activity. The MDCK-hBCRP[cMDR1-KO] cell line together with the MDCK-hMDR1[cMDR1-KO] easily and accurately identified shared or specific substrates of the hBCRP and the hMDR1 transporters. These cell lines offer new, improved in vitro tools for the assessment of drug efflux and drug-drug interactions in drug development.},
}
@article {pmid33007045,
year = {2020},
author = {Thakur, VS and Welford, SM},
title = {Generation of a conditional mutant knock-in under the control of the natural promoter using CRISPR-Cas9 and Cre-Lox systems.},
journal = {PloS one},
volume = {15},
number = {10},
pages = {e0240256},
pmid = {33007045},
issn = {1932-6203},
support = {R01 CA187053/CA/NCI NIH HHS/United States ; },
mesh = {Blotting, Western ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Genetic Vectors/genetics ; Humans ; Integrases/*genetics ; Promoter Regions, Genetic/genetics ; Recombination, Genetic/genetics ; },
abstract = {Modulation of gene activity by creating mutations has contributed significantly to the understanding of protein functions. Oftentimes, however, mutational analyses use overexpression studies, in which proteins are taken out of their normal contexts and stoichiometries. In the present work, we sought to develop an approach to simultaneously use the CRISPR/Cas9 and Cre-Lox techniques to modify the endogenous SAT1 gene to introduce mutant forms of the protein while still under the control of its natural gene promoter. We cloned the C-terminal portion of wild type (WT) SAT1, through the transcriptional stop elements, and flanked by LoxP sites in front of an identical version of SAT1 containing point mutations in critical binding sites. The construct was inserted into the endogenous SAT1 locus by Non-Homologous End Joining (NHEJ) after a CRISPR/Cas9 induced DNA double strand break. After validating that normal function of SAT1 was not altered by the insertional event, we were then able to assess the impact of point mutations by introduction of Cre recombinase. The system thus enables generation of cells in which endogenous WT SAT1 can be conditionally modified, and allow investigation of the functional consequences of site specific mutations in the context of the normal promoter and chromatin regulation.},
}
@article {pmid33006762,
year = {2021},
author = {Falk, MJ},
title = {The pursuit of precision mitochondrial medicine: Harnessing preclinical cellular and animal models to optimize mitochondrial disease therapeutic discovery.},
journal = {Journal of inherited metabolic disease},
volume = {44},
number = {2},
pages = {312-324},
pmid = {33006762},
issn = {1573-2665},
support = {R35 GM134863/GM/NIGMS NIH HHS/United States ; R01 HD065858/HD/NICHD NIH HHS/United States ; R01 GM120762/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Caenorhabditis elegans ; Cell Line ; Disease Models, Animal ; Fibroblasts ; Humans ; Mice ; Mitochondria/*drug effects/metabolism ; Mitochondrial Diseases/*drug therapy ; Precision Medicine/*methods ; Zebrafish ; },
abstract = {Mitochondria share extensive evolutionary conservation across nearly all living species. This homology allows robust insights to be gained into pathophysiologic mechanisms and therapeutic targets for the heterogeneous class of primary mitochondrial diseases (PMDs) through the study of diverse in vitro cellular and in vivo animal models. Dramatic advances in genetic technologies, ranging from RNA interference to achieve graded knock-down of gene expression to CRISPR/Cas-based gene editing that yields a stable gene knock-out or targeted mutation knock-in, have enabled the ready establishment of mitochondrial disease models for a plethora of individual nuclear gene disorders. These models are complemented and extended by the use of pharmacologic inhibitor-based stressors to characterize variable degrees, onset, duration, and combinations of acute on chronic mitochondrial dysfunction in individual respiratory chain enzyme complexes or distinct biochemical pathways within mitochondria. Herein is described the rationale for, and progress made in, "therapeutic cross-training," a novel approach meant to improve the validity and rigor of experimental conclusions when testing therapies by studying treatment effects in multiple, evolutionarily-distinct species, including Caenorhabditis elegans (invertebrate, worm), Danio rerio (vertebrate, zebrafish), Mus musculus (mammal, mouse), and/or human patient primary fibroblast cell line models of PMD. The goal of these preclinical studies is to identify lead therapies from candidate molecules or library screens that consistently demonstrate efficacy, with minimal toxicity, in specific subtypes of mitochondrial disease. Conservation of in vitro and in vivo therapeutic effects of lead molecules across species has proven extensive, where molar concentrations found to be toxic or efficacious in one species are often consistent with therapeutic effects at similar doses seen in other mitochondrial disease models. Phenotypic outcome studies in all models are prioritized at the level of survival and function, to reflect the ultimate goal of developing highly potent therapies for human mitochondrial disease. Lead compounds that demonstrate significant benefit on gross phenotypes may be further scrutinized in these same models to decipher their cellular targets, mechanism(s), and detailed biochemical effects. High-throughput, automated technologic advances will be discussed that enable efficient, parallel screening in a diverse array of mitochondrial disease disorders and overarching subclasses of compounds, concentrations, libraries, and combinations. Overall, this therapeutic cross-training approach has proven valuable to identify compounds with optimal potency and safety profiles among major biochemical subtypes or specific genetic etiologies of mitochondrial disease. This approach further supports rational prioritization of lead compounds, target concentrations, and specific disease phenotypes, outcomes, and subgroups to optimally inform the design of clinical trials that test their efficacy in human mitochondrial disease subjects.},
}
@article {pmid33006690,
year = {2020},
author = {Schüller, A and Wolansky, L and Berger, H and Studt, L and Gacek-Matthews, A and Sulyok, M and Strauss, J},
title = {A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {22},
pages = {9801-9822},
pmid = {33006690},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; Genes, Fungal ; *Nucleosomes/genetics ; RNA, Guide ; Transcriptional Activation ; },
abstract = {Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled "dead" Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway. Graphical abstract KEYPOINTS: • An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans. • Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning. • The system was successfully applied to activate genes within two silent biosynthetic gene clusters.},
}
@article {pmid33005486,
year = {2020},
author = {Hoque, MN and Chaudhury, A and Akanda, MAM and Hossain, MA and Islam, MT},
title = {Genomic diversity and evolution, diagnosis, prevention, and therapeutics of the pandemic COVID-19 disease.},
journal = {PeerJ},
volume = {8},
number = {},
pages = {e9689},
pmid = {33005486},
issn = {2167-8359},
abstract = {The coronavirus disease 19 (COVID-19) is a highly transmittable and pathogenic viral infection caused by a novel evolutionarily divergent RNA virus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus first emerged in Wuhan, China in December 2019, and subsequently spreaded around the world. Genomic analyses revealed that this zoonotic virus may be evolved naturally but not a purposefully manipulated laboratory construct. However, currently available data are not sufficient to precisely conclude the origin of this fearsome virus. Comprehensive annotations of the whole-genomes revealed hundreds of nucleotides, and amino acids mutations, substitutions and/or deletions at different positions of the ever changing SARS-CoV-2 genome. The spike (S) glycoprotein of SARS-CoV-2 possesses a functional polybasic (furin) cleavage site at the S1-S2 boundary through the insertion of 12 nucleotides. It leads to the predicted acquisition of 3-O-linked glycan around the cleavage site. Although real-time RT-PCR methods targeting specific gene(s) have widely been used to diagnose the COVID-19 patients, however, recently developed more convenient, cheap, rapid, and specific diagnostic tools targeting antigens or CRISPR-Cas-mediated method or a newly developed plug and play method should be available for the resource-poor developing countries. A large number of candidate drugs, vaccines and therapies have shown great promise in early trials, however, these candidates of preventive or therapeutic agents have to pass a long path of trials before being released for the practical application against COVID-19. This review updates current knowledge on origin, genomic evolution, development of the diagnostic tools, and the preventive or therapeutic remedies of the COVID-19. We also discussed the future scopes for research, effective management, and surveillance of the newly emerged COVID-19 disease.},
}
@article {pmid33005303,
year = {2020},
author = {Xu, Y and Li, Z},
title = {CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {2401-2415},
pmid = {33005303},
issn = {2001-0370},
abstract = {Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations, the applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.},
}
@article {pmid33004844,
year = {2020},
author = {de Melo, BP and Lourenço-Tessutti, IT and Paixão, JFR and Noriega, DD and Silva, MCM and de Almeida-Engler, J and Fontes, EPB and Grossi-de-Sa, MF},
title = {Transcriptional modulation of AREB-1 by CRISPRa improves plant physiological performance under severe water deficit.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {16231},
pmid = {33004844},
issn = {2045-2322},
mesh = {Arabidopsis/genetics/physiology ; Arabidopsis Proteins/genetics/*physiology ; Basic-Leucine Zipper Transcription Factors/genetics/*physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Dehydration/genetics ; Gene Editing ; Gene Expression Regulation, Plant ; Plant Physiological Phenomena/*genetics ; Plants, Genetically Modified ; },
abstract = {Plants are sessile organisms, which are vulnerable to environmental stresses. As such, plants have developed multiple molecular, physiological, and cellular mechanisms to cope with natural stressors. However, these environmental adversities, including drought, are sources of the main agribusiness problems since they interfere with plant growth and productivity. Particularly under water deprivation conditions, the abscisic acid-responsive element-binding protein AREB1/ABF2 plays an important role in drought stress response and physiological adaptation. In this investigation, we provide substantial confirmation for the role of AREB1/ABF2 in plant survival under severe water deficit using the CRISPR activation (CRISPRa) technique to enhance the AREB1 gene expression. In our strategy, the inactive nuclease dCas9 was fused with an Arabidopsis histone acetyltransferase 1, which improves gene expression by remodeling chromatin. The AREB1 overexpression promotes an improvement in the physiological performance of the transgenic homozygous plants under drought, which was associated with an increase in chlorophyll content, antioxidant enzyme activity, and soluble sugar accumulation, leading to lower reactive oxygen species accumulation. Finally, we found that the CRISPR-mediated up-regulation of AREB1 changes the abundance of several downstream ABA-inducible genes, allowing us to report that CRISPRa dCas9-HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.},
}
@article {pmid33004521,
year = {2020},
author = {Miyawaki, S and Kuroki, S and Maeda, R and Okashita, N and Koopman, P and Tachibana, M},
title = {The mouse Sry locus harbors a cryptic exon that is essential for male sex determination.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6512},
pages = {121-124},
doi = {10.1126/science.abb6430},
pmid = {33004521},
issn = {1095-9203},
mesh = {Animals ; CRISPR-Cas Systems ; *Exons ; Gene Editing ; *Genes, Essential ; Genetic Loci ; Male ; Mice ; Mice, Transgenic ; RNA, Untranslated/genetics ; Sequence Deletion ; *Sex Determination Processes ; Sex-Determining Region Y Protein/*genetics ; Transcription, Genetic ; },
abstract = {The mammalian sex-determining gene Sry induces male development. Since its discovery 30 years ago, Sry has been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouse Sry and a corresponding two-exon type Sry (Sry-T) transcript. XY mice lacking Sry-T were sex-reversed, and ectopic expression of Sry-T in XX mice induced male development. Sry-T messenger RNA is expressed similarly to that of canonical single-exon type Sry (Sry-S), but SRY-T protein is expressed predominantly because of the absence of a degron in the C terminus of SRY-S. Sry exon2 appears to have evolved recently in mice through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.},
}
@article {pmid33004258,
year = {2021},
author = {Bourgeois, JS and Smith, CM and Ko, DC},
title = {These Are the Genes You're Looking For: Finding Host Resistance Genes.},
journal = {Trends in microbiology},
volume = {29},
number = {4},
pages = {346-362},
pmid = {33004258},
issn = {1878-4380},
support = {R01 AI118903/AI/NIAID NIH HHS/United States ; F31 AI143147/AI/NIAID NIH HHS/United States ; R21 AI133305/AI/NIAID NIH HHS/United States ; U19 AI084044/AI/NIAID NIH HHS/United States ; R21 AI146520/AI/NIAID NIH HHS/United States ; R21 AI144586/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bacteria/genetics/pathogenicity ; CRISPR-Cas Systems ; Gene Editing ; *Genomics ; High-Throughput Nucleotide Sequencing ; Host-Pathogen Interactions/*genetics ; Humans ; Mice ; },
abstract = {Humanity's ongoing struggle with new, re-emerging and endemic infectious diseases serves as a frequent reminder of the need to understand host-pathogen interactions. Recent advances in genomics have dramatically advanced our understanding of how genetics contributes to host resistance or susceptibility to bacterial infection. Here we discuss current trends in defining host-bacterial interactions at the genome-wide level, including screens that harness CRISPR/Cas9 genome editing, natural genetic variation, proteomics, and transcriptomics. We report on the merits, limitations, and findings of these innovative screens and discuss their complementary nature. Finally, we speculate on future innovation as we continue to progress through the postgenomic era and towards deeper mechanistic insight and clinical applications.},
}
@article {pmid33004008,
year = {2020},
author = {Kim, H and Choi, J and Won, KH},
title = {A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {449},
pmid = {33004008},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems/*genetics ; Capsicum/*genetics ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; *Genetic Variation ; Genome, Plant ; Genotype ; Republic of Korea ; Ribonucleoproteins/*genetics ; },
abstract = {BACKGROUND: DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops.<br><br>RESULTS: This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum 'CM334' and C. annuum 'Dempsey'. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs.<br><br>CONCLUSIONS: Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.},
}
@article {pmid33003547,
year = {2020},
author = {Choi, EJ and Palacios-Prado, N and Sáez, JC and Lee, J},
title = {Identification of Cx45 as a Major Component of GJs in HeLa Cells.},
journal = {Biomolecules},
volume = {10},
number = {10},
pages = {},
pmid = {33003547},
issn = {2218-273X},
support = {project no:2018-12-0140//Yonsei University/International ; Iniciativa Cient&#xed;fica Milenio-Econom&#xed;a P09-022-F, grants 3180272, and grant 1191329//Ministerio de Ciencia, Tecnolog&#xed;a, Conocimiento e Innovaci&#xf3;n de Chile/International ; 2016R1D1A1A02937397 and 2018R1A6A1A03023718//Ministry of Education of Korea/International ; },
mesh = {A549 Cells ; Biological Transport/genetics ; CRISPR-Cas Systems/genetics ; Cell Communication/*genetics ; Connexin 43/*genetics ; Connexins/*genetics ; Gap Junctions/*genetics ; HeLa Cells ; Humans ; Neoplasms/genetics/pathology ; },
abstract = {&amp;nbsp;Gap junctions (GJs) are intercellular channels that connect adjacent cells electrically and metabolically. The iodide-yellow fluorescent protein (I-YFP) gap junctional intercellular communication (GJIC) assay is a recently developed method with high sensitivity. HeLa cells have been widely used as GJ-deficient cells for GJ-related research. Herein, we present evidence showing that HeLa cells have functional GJs comprising connexin (Cx) 45 using the I-YFP GJ assay and CRISPR/Cas9 system. We conducted the I-YFP GJIC assay in HeLa cells, which revealed a weak level of GJIC that could not be detected by the Lucifer yellow scrape-loading assay. The mRNA expression of GJB5 (Cx31.1), GJA1 (Cx43), and GJC1 (Cx45) was detected in HeLa cells by RT-PCR analysis. Knocking out GJC1 (Cx45) abolished GJIC, as analyzed by the I-YFP assay and dual whole-cell patch-clamp assay. These results suggest that HeLa cells express Cx45-based GJs and that the I-YFP GJIC assay can be used for cells with weak GJIC, such as Cx45-expressing HeLa cells. Further, GJC1 (Cx45)-knockout HeLa cells are more suitable as a GJ-null cell model for transfection experiments than wild-type HeLa cells. This experimental design was successfully applied to knock out Cx43 expression and GJIC in A549 lung cancer cells and can thus be used to identify major Cxs in other cell types and to establish GJ assay systems for different Cxs.},
}
@article {pmid33003492,
year = {2020},
author = {Luis, A},
title = {The Old and the New: Prospects for Non-Integrating Lentiviral Vector Technology.},
journal = {Viruses},
volume = {12},
number = {10},
pages = {},
pmid = {33003492},
issn = {1999-4915},
mesh = {CRISPR-Cas Systems ; DNA, Viral ; Gene Expression ; Genetic Therapy/methods ; *Genetic Vectors ; Immunotherapy ; Integrases ; Lentivirus/*genetics ; Mutagenesis ; Plasmids ; Transgenes ; },
abstract = {Lentiviral vectors have been developed and used in multiple gene and cell therapy applications. One of their main advantages over other vectors is the ability to integrate the genetic material into the genome of the host. However, this can also be a disadvantage as it may lead to insertional mutagenesis. To address this, non-integrating lentiviral vectors (NILVs) were developed. To generate NILVs, it is possible to introduce mutations in the viral enzyme integrase and/or mutations on the viral DNA recognised by integrase (the attachment sites). NILVs are able to stably express transgenes from episomal DNA in non-dividing cells or transiently if the target cells divide. It has been shown that these vectors are able to transduce multiple cell types and tissues. These characteristics make NILVs ideal vectors to use in vaccination and immunotherapies, among other applications. They also open future prospects for NILVs as tools for the delivery of CRISPR/Cas9 components, a recent revolutionary technology now widely used for gene editing and repair.},
}
@article {pmid33003308,
year = {2020},
author = {Klatt, D and Ha, TC and Schinke, M and Selich, A and Lieske, A and Dahlke, J and Morgan, M and Maetzig, T and Schambach, A},
title = {Competitive sgRNA Screen Identifies p38 MAPK as a Druggable Target to Improve HSPC Engraftment.},
journal = {Cells},
volume = {9},
number = {10},
pages = {},
pmid = {33003308},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Disease Models, Animal ; Genetic Diseases, X-Linked/genetics/pathology/therapy ; Genetic Therapy/methods ; Granulomatous Disease, Chronic/genetics/pathology/*therapy ; Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells/*metabolism/pathology ; Humans ; Inflammation/genetics/pathology/therapy ; Interleukin-1beta/*genetics ; Mice ; RNA/genetics/therapeutic use ; Receptors, CXCR4/*genetics ; Signal Transduction/genetics ; p38 Mitogen-Activated Protein Kinases/*genetics ; },
abstract = {Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified Mapk14 (p38) as a potential target to increase HSPC engraftment. Knockout of p38 prior to transplantation was sufficient to induce a selective advantage. Inhibition of p38 increased expression of the HSC homing factor CXCR4 and reduced apoptosis and proliferation in HSPCs. For potential clinical translation, treatment of IL1B-challenged or X-CGD HSPCs with a p38 inhibitor led to a 1.5-fold increase of donor cell engraftment. In summary, our findings demonstrate that p38 may serve as a potential druggable target to restore engraftment of HSPCs in the context of X-CGD gene therapy.},
}
@article {pmid33003295,
year = {2020},
author = {Bai, X},
title = {Stem Cell-Based Disease Modeling and Cell Therapy.},
journal = {Cells},
volume = {9},
number = {10},
pages = {},
pmid = {33003295},
issn = {2073-4409},
support = {R01 GM112696/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation/*genetics ; *Cell- and Tissue-Based Therapy ; Gene Editing/methods ; Humans ; Induced Pluripotent Stem Cells/*transplantation ; Neurons/pathology/transplantation ; Organoids/transplantation ; Stem Cell Research ; },
abstract = {Stem cell science is among the fastest moving fields in biology, with many highly promising directions for translatability. To centralize and contextualize some of the latest developments, this Special Issue presents state-of-the-art research of adult stem cells, induced pluripotent stem cells (iPSCs), and embryonic stem cells as well as cancer stem cells. The studies we include describe efficient differentiation protocols of generation of chondrocytes, adipocytes, and neurons, maturation of iPSC-derived cardiomyocytes and neurons, dynamic characterization of iPSC-derived 3D cerebral organoids, CRISPR/Cas9 genome editing, and non-viral minicircle vector-based gene modification of stem cells. Different applications of stem cells in disease modeling are described as well. This volume also highlights the most recent developments and applications of stem cells in basic science research and disease treatments.},
}
@article {pmid33002832,
year = {2020},
author = {Kung, LHW and Sampurno, L and Yammine, KM and Graham, A and McDonald, P and Bateman, JF and Shoulders, MD and Lamandé, SR},
title = {CRISPR/Cas9 editing to generate a heterozygous COL2A1 p.G1170S human chondrodysplasia iPSC line, MCRIi019-A-2, in a control iPSC line, MCRIi019-A.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101962},
doi = {10.1016/j.scr.2020.101962},
pmid = {33002832},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Collagen Type II/genetics ; *Gene Editing ; Heterozygote ; Humans ; *Induced Pluripotent Stem Cells ; *Osteochondrodysplasias/genetics ; },
abstract = {To develop an in vitro disease model of a human chondrodysplasia, we used CRISPR/Cas9 gene editing to generate a heterozygous COL2A1 exon 50 c.3508 GGT > TCA (p.G1170S) mutation in a control human iPSC line. Both the control and COL2A1 mutant lines displayed typical iPSC characteristics, including normal cell morphology, expression of pluripotency markers, the ability to differentiate into endoderm, ectoderm and mesoderm lineages and normal karyotype. These chondrodysplasia mutant and isogenic control cell lines can be used to explore disease mechanisms underlying type II collagenopathies and aid in the discovery of new therapeutic strategies.},
}
@article {pmid33002715,
year = {2020},
author = {Ruiz-Hernández, UE and Pelcastre-Rodriguez, LI and Cabrero-Martínez, OA and Hernández-Cortez, C and Castro-Escarpulli, G},
title = {Analysis of CRISPR-Cas systems in Gardnerella suggests its potential role in the mechanisms of bacterial vaginosis.},
journal = {Computational biology and chemistry},
volume = {89},
number = {},
pages = {107381},
doi = {10.1016/j.compbiolchem.2020.107381},
pmid = {33002715},
issn = {1476-928X},
mesh = {*CRISPR-Cas Systems ; Female ; Gardnerella/chemistry/*genetics ; Humans ; Phylogeny ; Vaginosis, Bacterial/*microbiology ; Virulence Factors/genetics ; },
abstract = {Bacterial vaginosis (BV) is the principal cause of vaginal discharge among women, and it can lead to many comorbidities with a negative impact in women's daily activities. Despite the fact that the pathophysiological process of BV remains unclear, great advances had been achieved in determining consequences of the shift in the vaginal community, and it was defined that Gardnerella spp., plays a key role in the pathogenesis of BV. Interactions of vaginal phage communities and bacterial hosts may be relevant in eubiosis/dysbiosis states, so defense mechanisms in Gardnerella spp., against phage infections could be relevant in BV development. In this study, we analyzed CRISPR-Cas systems among the 13 Gardnerella species recently classified, considering that these systems act as prokaryotic immune systems against phages, plasmids, and other mobile genetic elements. In silico analyses for CRISPR-Cas systems mining over the 81 Gardnerella spp., strains genomes analyzed led to the identification of subtypes I-E and II-C. Spacers analyses showed a hypervariable region across species, providing a high resolution level in order to distinguish clonality in strains, which was supported with phylogenomic analyses based on Virtual Genomic Fingerprinting. Moreover, most of the spacers revealed interactions between Gardnerella spp., strains and prophages over the genus. Furthermore, virulence traits of the 13 species showed insights of potential niche specificity in the vaginal microbiome. Overall, our results suggest that the CRISPR-Cas systems in the genus Gardnerella may play an important role in the mechanisms of the development and maintenance of BV, considering that the Gardnerella species occupies different niches in the vaginal community; in addition, spacer sequences can be used for genotyping studies.},
}
@article {pmid33002419,
year = {2021},
author = {Neggers, JE and Jacquemyn, M and Dierckx, T and Kleinstiver, BP and Thibaut, HJ and Daelemans, D},
title = {enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {1},
pages = {208-224},
pmid = {33002419},
issn = {1525-0024},
support = {R00 CA218870/CA/NCI NIH HHS/United States ; },
mesh = {Binding Sites ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance/*genetics ; Endonucleases/*metabolism ; Genetic Testing/*methods ; *Mutation ; Protein Binding ; },
abstract = {While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.},
}
@article {pmid33001567,
year = {2021},
author = {Endo, M and Iwakami, S and Toki, S},
title = {Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing.},
journal = {Plant biotechnology journal},
volume = {19},
number = {3},
pages = {563-574},
pmid = {33001567},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/metabolism ; *Gene Editing ; *Gene Targeting ; Homologous Recombination ; Plants/*genetics ; },
abstract = {Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase-type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)-mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR-mediated GT is extremely rare in higher plants, positive-negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac-transposon-mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I-SceI break and subsequent single-strand annealing (SSA)-mediated DNA repair system.},
}
@article {pmid33001025,
year = {2020},
author = {Zhang, H and Zhang, Y and Zhou, X and Wright, S and Hyle, J and Zhao, L and An, J and Zhao, X and Shao, Y and Xu, B and Lee, HM and Chen, T and Zhou, Y and Chen, X and Lu, R and Li, C},
title = {Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33001025},
issn = {2050-084X},
support = {P30 CA021765/CA/NCI NIH HHS/United States ; R01 HL153220/HL/NHLBI NIH HHS/United States ; IRG15-59-IRG//American Cancer Society/International ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Epigenesis, Genetic ; *Gene Expression Regulation, Leukemic ; Genes, Reporter ; Histone-Lysine N-Methyltransferase/genetics ; Homeodomain Proteins/*metabolism ; Humans ; Leukemia/*metabolism ; Myeloid-Lymphoid Leukemia Protein/genetics ; Upstream Stimulatory Factors/*metabolism ; },
abstract = {Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.},
}
@article {pmid32999445,
year = {2020},
author = {Cheatle Jarvela, AM and Trelstad, CS and Pick, L},
title = {Regulatory gene function handoff allows essential gene loss in mosquitoes.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {540},
pmid = {32999445},
issn = {2399-3642},
support = {R01 GM113230/GM/NIGMS NIH HHS/United States ; R01GM113230/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Conserved Sequence/genetics ; Drosophila/genetics ; Drosophila Proteins/genetics ; Female ; Gene Deletion ; Gene Editing ; Gene Expression Regulation/genetics ; Gene Regulatory Networks/genetics ; Genes, Essential/*genetics ; Genes, Insect/genetics ; Genes, Regulator/*genetics ; Male ; Nuclear Proteins/genetics ; Phylogeny ; Sequence Alignment ; Trans-Activators/genetics ; },
abstract = {Regulatory genes are often multifunctional and constrained, which results in evolutionary conservation. It is difficult to understand how a regulatory gene could be lost from one species' genome when it is essential for viability in closely related species. The gene paired is a classic Drosophila pair-rule gene, required for formation of alternate body segments in diverse insect species. Surprisingly, paired was lost in mosquitoes without disrupting body patterning. Here, we demonstrate that a paired family member, gooseberry, has acquired paired-like expression in the malaria mosquito Anopheles stephensi. Anopheles-gooseberry CRISPR-Cas9 knock-out mutants display pair-rule phenotypes and alteration of target gene expression similar to what is seen in Drosophila and beetle paired mutants. Thus, paired was functionally replaced by the related gene, gooseberry, in mosquitoes. Our findings document a rare example of a functional replacement of an essential regulatory gene and provide a mechanistic explanation of how such loss can occur.},
}
@article {pmid32999368,
year = {2020},
author = {Leungtongkam, U and Thummeepak, R and Kitti, T and Tasanapak, K and Wongwigkarn, J and Styles, KM and Wellington, EMH and Millard, AD and Sagona, AP and Sitthisak, S},
title = {Genomic analysis reveals high virulence and antibiotic resistance amongst phage susceptible Acinetobacter baumannii.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {16154},
pmid = {32999368},
issn = {2045-2322},
mesh = {Acinetobacter Infections/*microbiology ; Acinetobacter baumannii/drug effects/*genetics ; Anti-Bacterial Agents/pharmacology ; Bacteriophages ; Biofilms/drug effects ; Drug Resistance, Bacterial/*genetics ; *Genome, Bacterial ; Virulence ; },
abstract = {In this study, we examined the association between antimicrobial resistance, CRISPR/Cas systems and virulence with phage susceptibility in Acinetobacter baumannii and investigated draft genomes of phage susceptible multidrug resistant A. baumannii strains from Thailand. We investigated 230 A. baumannii strains using 17 lytic A. baumannii phages and the phage susceptibility was 46.5% (107/230). Phage susceptibility was also associated with resistance to numerous antibiotics (p-value < 0.05). We also found association between biofilm formation and the presence of ompA gene among phage susceptible A. baumannii strains (p-value < 0.05). A. baumannii isolates carrying cas5 or combinations of two or three other cas genes, showed a significant increase in phage resistance. Whole-genome sequences of seven phage susceptible A. baumannii isolates revealed that six groups of antibiotic resistance genes were carried by all seven phage susceptible A. baumannii. All strains carried biofilm associated genes and two strains harbored complete prophages, acquired copper tolerance genes, and CRISPR-associated (cas) genes. In conclusion, our data exhibits an association between virulence determinants and biofilm formation among phage susceptible A. baumannii strains. These data help to understand the bacterial co-evolution with phages.},
}
@article {pmid32999292,
year = {2020},
author = {Nguyen, LT and Smith, BM and Jain, PK},
title = {Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4906},
pmid = {32999292},
issn = {2041-1723},
mesh = {Bacterial Proteins/*metabolism ; Betacoronavirus/*genetics/isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; Clinical Laboratory Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/diagnosis/virology ; DNA, Single-Stranded ; Endodeoxyribonucleases/*metabolism ; Nucleic Acid Amplification Techniques/*methods ; Pandemics ; Pneumonia, Viral ; RNA, Guide/genetics ; RNA, Viral/genetics/*isolation &amp; purification ; SARS-CoV-2 ; Trans-Activators/*metabolism ; },
abstract = {The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.},
}
@article {pmid32997714,
year = {2020},
author = {Pöhler, M and Guttmann, S and Nadzemova, O and Lenders, M and Brand, E and Zibert, A and Schmidt, HH and Sandfort, V},
title = {CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0239411},
pmid = {32997714},
issn = {1932-6203},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Copper-Transporting ATPases/deficiency/*genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; *Mutation ; },
abstract = {Wilson's disease (WD) is a monogenetic liver disease that is based on a mutation of the ATP7B gene and leads to a functional deterioration in copper (Cu) excretion in the liver. The excess Cu accumulates in various organs such as the liver and brain. WD patients show clinical heterogeneity, which can range from acute or chronic liver failure to neurological symptoms. The course of the disease can be improved by a life-long treatment with zinc or chelators such as D-penicillamine in a majority of patients, but serious side effects have been observed in a significant portion of patients, e.g. neurological deterioration and nephrotoxicity, so that a liver transplant would be inevitable. An alternative therapy option would be the genetic correction of the ATP7B gene. The novel gene therapy method CRISPR/Cas9, which has recently been used in the clinic, may represent a suitable therapeutic opportunity. In this study, we first initiated an artificial ATP7B point mutation in a human cell line using CRISPR/Cas9 gene editing, and corrected this mutation by the additional use of single-stranded oligo DNA nucleotides (ssODNs), simulating a gene correction of a WD point mutation in vitro. By the addition of 0.5 mM of Cu three days after lipofection, a high yield of CRISPR/Cas9-mediated ATP7B repaired cell clones was achieved (60%). Moreover, the repair efficiency was enhanced using ssODNs that incorporated three blocking mutations. The repaired cell clones showed a high resistance to Cu after exposure to increasing Cu concentrations. Our findings indicate that CRISPR/Cas9-mediated correction of ATP7B point mutations is feasible and may have the potential to be transferred to the clinic.},
}
@article {pmid32996421,
year = {2020},
author = {Watanabe, K and Nakamura, T and Onodera, S and Saito, A and Shibahara, T and Azuma, T},
title = {A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors.},
journal = {Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine},
volume = {42},
number = {9},
pages = {1010428320962588},
doi = {10.1177/1010428320962588},
pmid = {32996421},
issn = {1423-0380},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Chromogranins/antagonists &amp; inhibitors/*genetics ; GTP-Binding Protein alpha Subunits, Gs/antagonists &amp; inhibitors/*genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Male ; Mice ; Mice, SCID ; *Models, Biological ; *Mutation ; Teratoma/genetics/*pathology ; },
abstract = {A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNAS[R201H]-mutated (GNAS[R201H/+]) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNAS[R201H]-mutated induced pluripotent stem cells and GNAS[R201H]-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNAS[R201H]-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.},
}
@article {pmid32996244,
year = {2020},
author = {Jiang, M and He, Y and Chen, X and Zhang, X and Guo, Y and Yang, S and Huang, J and Traw, MB},
title = {CRISPR-based assessment of genomic structure in the conserved SQUAMOSA promoter-binding-like gene clusters in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {104},
number = {5},
pages = {1301-1314},
doi = {10.1111/tpj.15001},
pmid = {32996244},
issn = {1365-313X},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Conserved Sequence ; Evolution, Molecular ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; *Genome, Plant ; *Multigene Family ; Oryza/*genetics ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Synteny ; Transcription Factors/genetics ; },
abstract = {Although SQUAMOSA promoter-binding-like (SPL) transcription factors are important regulators of development in rice (Oryza sativa), prior assessments of the SPL family have been limited to single genes. A functional comparison across the full gene family in standardized genetic backgrounds has not been reported previously. Here, we demonstrate that the SPL gene family in rice is enriched due to the most recent whole genome duplication (WGD). Notably, 10 of 19 rice SPL genes (52%) cluster in four units that have persisted for at least 50 million years. We show that SPL gene grouping and retention following WGD is widespread in angiosperms, suggesting the conservatism and importance of this gene arrangement. We used Cas9 editing to generate transformation lines for all 19 SPL genes in a common set of backgrounds, and found that knockouts of 14 SPL genes exhibited defects in plant height, 10 exhibited defects in panicle size, and nine had altered grain lengths. We observed subfunctionalization of genes in the paleoduplicated pairs, but little evidence of neofunctionalization. Expression of OsSPL3 was negatively correlated with that of its closest neighbor in its synteny group, OsSPL4, and its sister paired gene, OsSPL12, in the opposing group. Nucleotide diversity was lower in eight of the nine singleton genes in domesticated rice, relative to wild rice, whereas the reverse was true for the paired genes. Together, these results provide functional information on eight previously unexamined OsSPL family members and suggest that paleoduplicate pair redundancy benefits plant survival and innovation.},
}
@article {pmid32996045,
year = {2021},
author = {Xie, X and Liu, W and Dong, G and Zhu, Q and Liu, YG},
title = {MMEJ-KO: a web tool for designing paired CRISPR guide RNAs for microhomology-mediated end joining fragment deletion.},
journal = {Science China. Life sciences},
volume = {64},
number = {6},
pages = {1021-1024},
pmid = {32996045},
issn = {1869-1889},
mesh = {Algorithms ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Homologous Recombination ; *Internet ; Models, Genetic ; *RNA, Guide ; },
}
@article {pmid32994506,
year = {2020},
author = {Owen, JR and Hennig, SL and McNabb, BR and Lin, JC and Young, AE and Murray, JD and Ross, PJ and Van Eenennaam, AL},
title = {Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {16031},
pmid = {32994506},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Cattle ; DNA End-Joining Repair/physiology ; DNA Repair/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Genetic Engineering/*methods ; Genome/genetics ; Homologous Recombination/genetics ; Microinjections/methods ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; Zygote/physiology ; },
abstract = {Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.},
}
@article {pmid32994412,
year = {2020},
author = {Lundin, A and Porritt, MJ and Jaiswal, H and Seeliger, F and Johansson, C and Bidar, AW and Badertscher, L and Wimberger, S and Davies, EJ and Hardaker, E and Martins, CP and James, E and Admyre, T and Taheri-Ghahfarokhi, A and Bradley, J and Schantz, A and Alaeimahabadi, B and Clausen, M and Xu, X and Mayr, LM and Nitsch, R and Bohlooly-Y, M and Barry, ST and Maresca, M},
title = {Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4903},
pmid = {32994412},
issn = {2041-1723},
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics ; Cell Line, Tumor ; Doxycycline/pharmacology ; Drug Discovery/*methods ; Drug Screening Assays, Antitumor/methods ; Female ; Gene Editing/*methods ; Gene Expression/drug effects/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Genetic Vectors/genetics ; HEK293 Cells ; High-Throughput Screening Assays/methods ; Humans ; Lung Neoplasms/*drug therapy/genetics ; Male ; Mice ; Mice, Transgenic ; RNA, Guide/genetics ; Recombination, Genetic/drug effects ; Reproducibility of Results ; Transcriptional Activation/drug effects ; Transfection/methods ; Transgenes/genetics ; },
abstract = {The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.},
}
@article {pmid32994292,
year = {2020},
author = {Solbiati, J and Duran-Pinedo, A and Godoy Rocha, F and Gibson, FC and Frias-Lopez, J},
title = {Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.},
journal = {mSystems},
volume = {5},
number = {5},
pages = {},
pmid = {32994292},
issn = {2379-5077},
support = {R01 DE021553/DE/NIDCR NIH HHS/United States ; R01 DE029775/DE/NIDCR NIH HHS/United States ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis, an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalis In vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δcas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δcas3 mutant resulted in a statistically significant increase in mortality of G. mellonella A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δcas3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalis IMPORTANCE Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.},
}
@article {pmid32994224,
year = {2020},
author = {Canver, MC and Tripathi, P and Bullen, MJ and Olshansky, M and Kumar, Y and Wong, LH and Turner, SJ and Lessard, S and Pinello, L and Orkin, SH and Das, PP},
title = {A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies cis- and trans-regulatory elements of Oct4 in murine ESCs.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {47},
pages = {15797-15809},
pmid = {32994224},
issn = {1083-351X},
support = {R00 HG008399/HG/NHGRI NIH HHS/United States ; R35 HG010717/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Genetic Loci ; Genome-Wide Association Study ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Octamer Transcription Factor-3/genetics/*metabolism ; *Regulatory Elements, Transcriptional ; },
abstract = {Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.},
}
@article {pmid32993386,
year = {2021},
author = {Quinn, J and Musa, A and Kantor, A and McClements, ME and Cehajic-Kapetanovic, J and MacLaren, RE and Xue, K},
title = {Genome-Editing Strategies for Treating Human Retinal Degenerations.},
journal = {Human gene therapy},
volume = {32},
number = {5-6},
pages = {247-259},
pmid = {32993386},
issn = {1557-7422},
support = {MC_PC_18059/MRC_/Medical Research Council/United Kingdom ; 216593/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Antigens, Neoplasm ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins ; Cytoskeletal Proteins ; Gene Editing ; Genetic Therapy ; Humans ; *Leber Congenital Amaurosis/genetics/therapy ; Mice ; *Retinal Degeneration/genetics/therapy ; },
abstract = {Inherited retinal degenerations (IRDs) are a leading cause of blindness. Although gene-supplementation therapies have been developed, they are only available for a small proportion of recessive IRD mutations. In contrast, genome editing using clustered-regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated (Cas) systems could provide alternative therapeutic avenues for treating a wide range of genetic retinal diseases through targeted knockdown or correction of mutant alleles. Progress in this rapidly evolving field has been highlighted by recent Food and Drug Administration clinical trial approval for EDIT-101 (Editas Medicine, Inc., Cambridge, MA), which has demonstrated efficacious genome editing in a mouse model of CEP290-associated Leber congenital amaurosis and safety in nonhuman primates. Nonetheless, there remains a significant number of challenges to developing clinically viable retinal genome-editing therapies. In particular, IRD-causing mutations occur in more than 200 known genes, with considerable heterogeneity in mutation type and position within each gene. Additionally, there are remaining safety concerns over long-term expression of Cas9 in vivo. This review highlights (i) the technological advances in gene-editing technology, (ii) major safety concerns associated with retinal genome editing, and (iii) potential strategies for overcoming these challenges to develop clinical therapies.},
}
@article {pmid32991731,
year = {2020},
author = {Ariga, H and Toki, S and Ishibashi, K},
title = {Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants.},
journal = {Plant &amp; cell physiology},
volume = {61},
number = {11},
pages = {1946-1953},
pmid = {32991731},
issn = {1471-9053},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Mutagenesis, Site-Directed/methods ; Potexvirus/*genetics/metabolism ; Tobacco/*genetics ; },
abstract = {Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.},
}
@article {pmid32991234,
year = {2021},
author = {Zhang, Y and Marchisio, MA},
title = {Type II anti-CRISPR proteins as a new tool for synthetic biology.},
journal = {RNA biology},
volume = {18},
number = {8},
pages = {1085-1098},
pmid = {32991234},
issn = {1555-8584},
mesh = {Archaea/*genetics/immunology/virology ; Bacteria/*genetics/immunology/virology ; Bacteriophages/*genetics/metabolism ; Biological Coevolution ; Biotechnology/instrumentation/trends ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Humans ; Prophages/*genetics/metabolism ; RNA, Guide/genetics/metabolism ; Synthetic Biology/instrumentation/trends ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.},
}
@article {pmid32990907,
year = {2021},
author = {Yang, W and Qi, W and Li, Y and Wang, J and Luo, Y and Ding, D and Mo, S and Chen, B and Lu, Y and Li, H and Jiang, L},
title = {Programmed sequential cutting endows Cas9 versatile base substitution capability in plants.},
journal = {Science China. Life sciences},
volume = {64},
number = {6},
pages = {1025-1028},
pmid = {32990907},
issn = {1869-1889},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; Codon ; *Gene Editing ; Gene Expression Regulation, Plant ; Genes, Plant ; Genome, Plant ; Herbicide Resistance/*genetics ; Point Mutation ; RNA, Guide ; },
}
@article {pmid32990416,
year = {2020},
author = {Kim, GE and Lee, SY and Park, HH},
title = {A high-resolution (1.2 Å) crystal structure of the anti-CRISPR protein AcrIF9.},
journal = {FEBS open bio},
volume = {10},
number = {12},
pages = {2532-2540},
pmid = {32990416},
issn = {2211-5463},
mesh = {Bacterial Proteins/*chemistry/isolation &amp; purification/metabolism ; CRISPR-Associated Proteins/*chemistry/isolation &amp; purification/metabolism ; Crystallography, X-Ray ; Models, Molecular ; Protein Conformation ; Pseudomonas aeruginosa/*chemistry ; },
abstract = {Prokaryotic adaptive immunity by CRISPR-Cas systems, which confer resistance to foreign genetic elements, has been used by bacteria to combat viruses. To cope, viruses evolved multiple anti-CRISPR proteins, which can inhibit system function through various mechanisms. Although the structures and mechanisms of several anti-CRISPR proteins have been elucidated, those of the AcrIF9 family have not yet been identified. To understand the molecular basis underlying AcrIF9 anti-CRISPR function, we determined the 1.2 Å crystal structure of AcrIF9. Structural and biochemical studies showed that AcrIF9 exists in monomeric form in solution and can directly interact with DNA using a positively charged cleft. Based on analysis of the structure, we suggest part of the anti-CRISPR molecular mechanism by AcrIF9.},
}
@article {pmid32989329,
year = {2020},
author = {Schumann, K and Raju, SS and Lauber, M and Kolb, S and Shifrut, E and Cortez, JT and Skartsis, N and Nguyen, VQ and Woo, JM and Roth, TL and Yu, R and Nguyen, MLT and Simeonov, DR and Nguyen, DN and Targ, S and Gate, RE and Tang, Q and Bluestone, JA and Spitzer, MH and Ye, CJ and Marson, A},
title = {Functional CRISPR dissection of gene networks controlling human regulatory T cell identity.},
journal = {Nature immunology},
volume = {21},
number = {11},
pages = {1456-1466},
pmid = {32989329},
issn = {1529-2916},
support = {T32 AI007334/AI/NIAID NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; P30 AR070155/AR/NIAMS NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; DP5 OD023056/OD/NIH HHS/United States ; },
mesh = {Biomarkers ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Susceptibility ; *Gene Expression Profiling ; *Gene Expression Regulation ; Gene Knockout Techniques ; *Gene Regulatory Networks ; Gene Targeting ; Graft vs Host Disease/etiology ; High-Throughput Nucleotide Sequencing ; Humans ; T-Lymphocytes, Regulatory/*immunology/*metabolism ; *Transcriptome ; },
abstract = {Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.},
}
@article {pmid32989284,
year = {2021},
author = {Chow, RD and Chen, JS and Shen, J and Chen, S},
title = {A web tool for the design of prime-editing guide RNAs.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {190-194},
pmid = {32989284},
issn = {2157-846X},
support = {U54 CA209992/CA/NCI NIH HHS/United States ; RF1 DA048811/DA/NIDA NIH HHS/United States ; F30 HL149151/HL/NHLBI NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; T32 GM136651/GM/NIGMS NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Internet ; RNA, Guide/*genetics ; *Software ; },
abstract = {Prime editing enables diverse genomic alterations to be written into target sites without requiring double-strand breaks or donor templates. The design of prime-editing guide RNAs (pegRNAs), which must be customized for each edit, can however be complex and time consuming. Compared with single guide RNAs (sgRNAs), pegRNAs have an additional 3' extension composed of a primer binding site and a reverse-transcription template. Here we report a web tool, which we named pegFinder (http://pegfinder.sidichenlab.org), for the rapid design of pegRNAs from reference and edited DNA sequences. pegFinder can incorporate sgRNA on-target and off-target scoring predictions into its ranking system, and nominates secondary nicking sgRNAs for increasing editing efficiency. CRISPR-associated protein 9 variants with expanded targeting ranges are also supported. To facilitate downstream experimentation, pegFinder produces a comprehensive table of candidate pegRNAs, along with oligonucleotide sequences for cloning.},
}
@article {pmid32989016,
year = {2020},
author = {Lin, Z and King, R and Tang, V and Myers, G and Balbin-Cuesta, G and Friedman, A and McGee, B and Desch, K and Ozel, AB and Siemieniak, D and Reddy, P and Emmer, B and Khoriaty, R},
title = {The Endoplasmic Reticulum Cargo Receptor SURF4 Facilitates Efficient Erythropoietin Secretion.},
journal = {Molecular and cellular biology},
volume = {40},
number = {23},
pages = {},
pmid = {32989016},
issn = {1098-5549},
support = {P30 CA046592/CA/NCI NIH HHS/United States ; K08 HL148552/HL/NHLBI NIH HHS/United States ; T32 GM007863/GM/NIGMS NIH HHS/United States ; R25 GM086262/GM/NIGMS NIH HHS/United States ; T32 CA009357/CA/NCI NIH HHS/United States ; K08 HL128794/HL/NHLBI NIH HHS/United States ; R01 HL148333/HL/NHLBI NIH HHS/United States ; R01 HL141399/HL/NHLBI NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Endoplasmic Reticulum/*metabolism ; Erythropoiesis/*physiology ; Erythropoietin/analysis/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Membrane Proteins/genetics/*metabolism ; Protein Transport/physiology ; RNA, Guide/genetics ; },
abstract = {Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.},
}
@article {pmid32988972,
year = {2020},
author = {Wong, TWY and Ahmed, A and Yang, G and Maino, E and Steiman, S and Hyatt, E and Chan, P and Lindsay, K and Wong, N and Golebiowski, D and Schneider, J and Delgado-Olguín, P and Ivakine, EA and Cohn, RD},
title = {A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy.},
journal = {Disease models &amp; mechanisms},
volume = {13},
number = {9},
pages = {},
pmid = {32988972},
issn = {1754-8411},
support = {6210100686/CAPMC/CIHR/Canada ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cardiomegaly/complications/physiopathology ; Cardiomyopathies/complications/*genetics/physiopathology ; Disease Models, Animal ; Dystroglycans/metabolism ; Dystrophin/*genetics ; Exons/*genetics ; Female ; *Gene Deletion ; Mice, Inbred C57BL ; Mice, Transgenic ; Muscle Strength ; Muscle, Skeletal/pathology/physiopathology ; Muscular Dystrophy, Duchenne/complications/*genetics/*pathology/physiopathology ; Sarcolemma/metabolism ; Tachycardia/complications/physiopathology ; },
abstract = {Duchenne muscular dystrophy (DMD) is a life-threatening neuromuscular disease caused by the lack of dystrophin, resulting in progressive muscle wasting and locomotor dysfunctions. By adulthood, almost all patients also develop cardiomyopathy, which is the primary cause of death in DMD. Although there has been extensive effort in creating animal models to study treatment strategies for DMD, most fail to recapitulate the complete skeletal and cardiac disease manifestations that are presented in affected patients. Here, we generated a mouse model mirroring a patient deletion mutation of exons 52-54 (Dmd Δ52-54). The Dmd Δ52-54 mutation led to the absence of dystrophin, resulting in progressive muscle deterioration with weakened muscle strength. Moreover, Dmd Δ52-54 mice present with early-onset hypertrophic cardiomyopathy, which is absent in current pre-clinical dystrophin-deficient mouse models. Therefore, Dmd Δ52-54 presents itself as an excellent pre-clinical model to evaluate the impact on skeletal and cardiac muscles for both mutation-dependent and -independent approaches.},
}
@article {pmid32988631,
year = {2021},
author = {Peng, R and Jones, DC and Liu, F and Zhang, B},
title = {From Sequencing to Genome Editing for Cotton Improvement.},
journal = {Trends in biotechnology},
volume = {39},
number = {3},
pages = {221-224},
doi = {10.1016/j.tibtech.2020.09.001},
pmid = {32988631},
issn = {1879-3096},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genome, Plant/genetics ; *Gossypium/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; Whole Genome Sequencing ; },
abstract = {Traditional breeding techniques are proven, but additional knowledge learned from genome sequencing provides vast new data that might help identify gene targets for improving cotton sustainability. CRISPR/Cas9 provides a powerful tool for precision cotton breeding. Here, we discuss the opportunities and challenges of genome sequencing and editing for cotton improvement.},
}
@article {pmid32988588,
year = {2020},
author = {Lee, SY and Kim, GE and Kim, YG and Park, HH},
title = {A 1.3 Å high-resolution crystal structure of an anti-CRISPR protein, AcrI E2.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {4},
pages = {751-757},
doi = {10.1016/j.bbrc.2020.09.067},
pmid = {32988588},
issn = {1090-2104},
mesh = {CRISPR-Associated Proteins/*chemistry ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; Models, Molecular ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Pseudomonas aeruginosa/*virology ; Viral Proteins/*chemistry/metabolism ; },
abstract = {As a result of bacterial infection with viruses, bacteria have developed CRISPR-Cas as an adaptive immune system, which allows them to destroy the viral genetic material introduced via infection. However, viruses have also evolved to develop multiple anti-CRISPR proteins, which are capable of inactivating the CRISPR-Cas adaptive immune system to combat bacteria. In this study, we aimed to elucidate the molecular mechanisms associated with anti-CRISPR proteins by determining a high-resolution crystal structure (1.3 Å) of Type I-E anti-CRISPR protein called AcrIE2. Our structural analysis revealed that AcrIE2 was composed of unique folds comprising five antiparallel β-sheets (β1∼β5) surrounding one α-helix (α1) in the order, β2β1α1β5β4β3. Structural comparison of AcrIE2 with a structural homolog called AcrIF9 showed that AcrIE2 contained a long and flexible β4-β5 connecting loop and a distinct surface feature. These results indicated that the inhibitory mechanism of AcrIE2 might be different from that of AcrIF9. This unique structure of AcrIE2 indicates its special mode of CRISPR-Cas inhibitory activity. Therefore, this study helps us understand the diversity in the inhibitory mechanisms of Acr family.},
}
@article {pmid32987694,
year = {2020},
author = {Xu, H and Jiang, Y and Li, S and Xie, L and Tao, YX and Li, Y},
title = {Zebrafish Oxr1a Knockout Reveals Its Role in Regulating Antioxidant Defenses and Aging.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {32987694},
issn = {2073-4425},
mesh = {Animals ; Antioxidants/*metabolism ; Apoptosis ; *CRISPR-Cas Systems ; *Longevity ; Mutation ; *Oxidative Stress ; Reactive Oxygen Species/metabolism ; *Reproduction ; Signal Transduction ; *Transcriptome ; Zebrafish ; Zebrafish Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Oxidation resistance gene 1 (OXR1) is essential for protection against oxidative stress in mammals, but its functions in non-mammalian vertebrates, especially in fish, remain uncertain. Here, we created a homozygous oxr1a-knockout zebrafish via the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system. Compared with wild-type (WT) zebrafish, oxr1a[-/-] mutants exhibited higher mortality and more apoptotic cells under oxidative stress, and multiple antioxidant genes (i.e., gpx1b, gpx4a, gpx7 and sod3a) involved in detoxifying cellular reactive oxygen species were downregulated significantly. Based on these observations, we conducted a comparative transcriptome analysis of early oxidative stress response. The results show that oxr1a mutation caused more extensive changes in transcriptional networks compared to WT zebrafish, and several stress response and pro-inflammatory pathways in oxr1a[-/-] mutant zebrafish were strongly induced. More importantly, we only observed the activation of the p53 signaling and apoptosis pathway in oxr1a[-/-] mutant zebrafish, revealing an important role of oxr1a in regulating apoptosis via the p53 signaling pathway. Additionally, we found that oxr1a mutation displayed a shortened lifespan and premature ovarian failure in prolonged observation, which may be caused by the loss of oxr1a impaired antioxidant defenses, thereby increasing pro-apoptotic events. Altogether, our findings demonstrate that oxr1a is vital for antioxidant defenses and anti-aging in zebrafish.},
}
@article {pmid32987327,
year = {2020},
author = {Li, CY and Zheng, B and Liu, YH and Gao, JL and Zheng, MQ and Pang, DW and Tang, HW},
title = {A boosting upconversion luminescent resonance energy transfer and biomimetic periodic chip integrated CRISPR/Cas12a biosensor for functional DNA regulated transduction of non-nucleic acid targets.},
journal = {Biosensors &amp; bioelectronics},
volume = {169},
number = {},
pages = {112650},
doi = {10.1016/j.bios.2020.112650},
pmid = {32987327},
issn = {1873-4235},
mesh = {Biomimetics ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Humans ; },
abstract = {Apart from gene editing capacity, the newly discovered CRISPR/Cas systems offer an exciting option for biosensing field because of their excellent target recognition accuracy. However, the currently constructed sensors are not only limited to nucleic acid analysis but also suffer from poor adaptability in complex samples and unsatisfying sensitivity. We herein introduce some advanced concepts to break through these bottlenecks. First, the sensing targets are extended by skillfully designing a functional DNA such as aptamer (for protein) and DNAzyme (for metal ion) to regulate the transduction of non-nucleic acid species and further activate the trans cleavage of CRISPR/Cas12a. Second, a boosting upconversion luminescent resonance energy is triggered by using a peculiar energy-confining notion, whereby the luminescence domain is intensively restricted in a very narrow space (~2.44 nm) and up to 92.9% of the green emission can be quenched by the approaching BHQ-1 modified reporters. Third, a bio-inspired periodic arrangement biomimetic chip (photonic crystal) is employed to selectively reflect the upconversion luminescence to achieve noteworthy signal enhancement (~35-fold). By utilizing very simple detection devices (a 980 nm portable laser and a smartphone), the CRISPR/Cas12a biosensor shows commendable sensitivity and specificity toward model targets (ATP and Na[+], limits of detection are ~ 18 nM and ~0.37 μM, respectively). More importantly, the analysis of real complex samples demonstrate that the as-proposed platform can work as a powerful toolbox for monitoring the ATP fluctuation in single cell and point-of-care testing Na[+] in human plasma, enabling a broad application prospect.},
}
@article {pmid32987180,
year = {2020},
author = {Lyu, C and Shi, H and Cui, Y and Li, M and Yan, Z and Yan, L and Jiang, Y},
title = {CRISPR-based biosensing is prospective for rapid and sensitive diagnosis of pediatric tuberculosis.},
journal = {International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases},
volume = {101},
number = {},
pages = {183-187},
doi = {10.1016/j.ijid.2020.09.1428},
pmid = {32987180},
issn = {1878-3511},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Diagnostic Tests, Routine/*methods ; Humans ; Mycobacterium tuberculosis/classification/genetics/*isolation &amp; purification ; Prospective Studies ; Sensitivity and Specificity ; Tuberculosis/*diagnosis/*microbiology ; },
abstract = {Pediatric tuberculosis (TB) is an important part of global TB prevention and control. Diagnosis of childhood TB still remains challenging when using conventional tests, due to the non-specific clinical manifestations and paucibacillary nature of the specimens. Thus, a sensitive, rapid and low-cost diagnostic test is of great demand. Benefiting from specific and rapid Cas-protein-based catalytic activities, CRISPR-based biosensing platforms (CRISPR platforms) are showing superiority in detecting pathogen nucleic acid traces in clinical samples. Based on their excellent sensitivity, and time and cost saved in existing research, this study aimed to highlight the potential of CRISPR platforms as a tool for diagnosing pediatric TB, and advocate for studies to evaluate its performance in specimens collected from children, especially noninvasive specimens. These platforms are also promising in identifying drug resistance and genotyping. All of the above will help early diagnosis of pediatric TB, thus guide reasonable treatment, and be significant in achieving the World Health Organization End-TB strategy.},
}
@article {pmid32986839,
year = {2020},
author = {Ma, S and Wang, X and Hu, Y and Lv, J and Liu, C and Liao, K and Guo, X and Wang, D and Lin, Y and Rong, Z},
title = {Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10590-10601},
pmid = {32986839},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Cholangiocarcinoma/*genetics/pathology ; DNA-Binding Proteins/genetics ; Gene Editing ; Gene Knock-In Techniques ; Genetic Therapy ; Humans ; Mice ; Oncogene Proteins, Fusion/genetics ; Protein Domains/genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; RNA, Guide ; RNA, Untranslated/genetics ; Receptors, Antigen, T-Cell/*genetics ; T-Lymphocytes/metabolism/pathology ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.},
}
@article {pmid32986708,
year = {2020},
author = {Gao, Y and Liu, YC and Jia, SZ and Liang, YT and Tang, Y and Xu, YS and Kawasaki, H and Wang, HB},
title = {Imaginal disc growth factor maintains cuticle structure and controls melanization in the spot pattern formation of Bombyx mori.},
journal = {PLoS genetics},
volume = {16},
number = {9},
pages = {e1008980},
pmid = {32986708},
issn = {1553-7404},
mesh = {Animals ; Bombyx/anatomy &amp; histology/*physiology ; CRISPR-Cas Systems ; Gene Expression Regulation ; Gene Knockout Techniques ; Insect Proteins/genetics/*metabolism ; Larva/genetics/physiology ; Melanins/biosynthesis/genetics/*metabolism ; Metabolomics/methods ; Mutation ; Phylogeny ; Pigmentation/*physiology ; Transcription Factors/genetics/metabolism ; },
abstract = {The complex stripes and patterns of insects play key roles in behavior and ecology. However, the fine-scale regulation mechanisms underlying pigment formation and morphological divergence remain largely unelucidated. Here we demonstrated that imaginal disc growth factor (IDGF) maintains cuticle structure and controls melanization in spot pattern formation of Bombyx mori. Moreover, our knockout experiments showed that IDGF is suggested to impact the expression levels of the ecdysone inducible transcription factor E75A and pleiotropic factors apt-like and Toll8/spz3, to further control the melanin metabolism. Furthermore, the untargeted metabolomics analyses revealed that BmIDGF significantly affected critical metabolites involved in phenylalanine, beta-alanine, purine, and tyrosine metabolism pathways. Our findings highlighted not only the universal function of IDGF to the maintenance of normal cuticle structure but also an underexplored space in the gene function affecting melanin formation. Therefore, this study furthers our understanding of insect pigment metabolism and melanin pattern polymorphisms.},
}
@article {pmid32986696,
year = {2020},
author = {Gai, T and Tong, X and Han, M and Li, C and Fang, C and Zou, Y and Hu, H and Xiang, H and Xiang, Z and Lu, C and Dai, F},
title = {Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon.},
journal = {PLoS genetics},
volume = {16},
number = {9},
pages = {e1009004},
pmid = {32986696},
issn = {1553-7404},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*enzymology/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Homozygote ; Life Cycle Stages/*physiology ; Mutation ; Phylogeny ; Selection, Genetic ; Species Specificity ; },
abstract = {Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.},
}
@article {pmid32986041,
year = {2020},
author = {Meuti, ME and Harrell, R},
title = {Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {163},
pages = {},
doi = {10.3791/61651},
pmid = {32986041},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Culex/*embryology/genetics ; Female ; Gene Editing ; Loss of Function Mutation ; Mosquito Vectors ; *Mutation ; West Nile virus ; },
abstract = {Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.},
}
@article {pmid32985711,
year = {2021},
author = {Zhang, X and Lin, JS and Spruyt, K},
title = {Sleep problems in Rett syndrome animal models: A systematic review.},
journal = {Journal of neuroscience research},
volume = {99},
number = {2},
pages = {529-544},
doi = {10.1002/jnr.24730},
pmid = {32985711},
issn = {1097-4547},
mesh = {Animals ; Brain/physiopathology ; CRISPR-Cas Systems ; *Disease Models, Animal ; Drosophila melanogaster/genetics ; Epileptic Syndromes/genetics/physiopathology ; Female ; Gene Editing ; Gene Knock-In Techniques ; Humans ; Macaca fascicularis/genetics ; Male ; Methyl-CpG-Binding Protein 2/genetics ; Mice ; Mice, Mutant Strains/genetics ; Mutation ; Rett Syndrome/*complications/genetics/physiopathology ; Sleep Stages ; Sleep Wake Disorders/*etiology/physiopathology ; Spasms, Infantile/genetics/physiopathology ; Species Specificity ; },
abstract = {Due to the discovery of Rett Syndrome (RTT) genetic mutations, animal models have been developed. Sleep research in RTT animal models may unravel novel neural mechanisms for this severe neurodevelopmental heritable rare disease. In this systematic literature review we summarize the findings on sleep research of 13 studies in animal models of RTT. We found disturbed efficacy and continuity of sleep in all genetically mutated models of mice, cynomolgus monkeys, and Drosophila. Models presented highly fragmented sleep with distinct differences in 24-hr sleep/wake cyclicity and circadian arrhythmicity. Overall, animal models mimic sleep complaints reported in individuals with RTT. However, contrary to human studies, in mutant mice, attenuated sleep delta waves, and sleep apneas in non-rapid eye movement sleep were reported. Future studies may focus on sleep structure and EEG alterations, potential central mechanisms involved in sleep fragmentation and the occurrence of sleep apnea across different sleep stages. Given that locomotor dysfunction is characteristic of individuals with RTT, studies may consider to integrate its potential impact on the behavioral analysis of sleep.},
}
@article {pmid32985619,
year = {2020},
author = {Yuan, T and Mukama, O and Li, Z and Chen, W and Zhang, Y and de Dieu Habimana, J and Zhang, Y and Zeng, R and Nie, C and He, Z and Zeng, L},
title = {A rapid and sensitive CRISPR/Cas12a based lateral flow biosensor for the detection of Epstein-Barr virus.},
journal = {The Analyst},
volume = {145},
number = {19},
pages = {6388-6394},
doi = {10.1039/d0an00663g},
pmid = {32985619},
issn = {1364-5528},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Viral/genetics ; *Epstein-Barr Virus Infections/diagnosis ; Herpesvirus 4, Human/genetics ; Humans ; *Nasopharyngeal Neoplasms/diagnosis ; },
abstract = {Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in the world, and several studies have associated Epstein-Barr virus (EBV) with NPC occurrence and development. EBV-PCR (polymerase chain reaction), in situ hybridization and immunoassays are the most common methods for NPC identification. However, these approaches have drawbacks, which include tedious procedures and false results. Therefore, a rapid, accurate, and sensitive clinical diagnostic method for the prognosis of EBV-related diseases is needed. In this study, we developed a simple and sensitive approach for EBV detection based on the combination of CRISPR-Cas12a and a lateral flow biosensor (LFB). Cas12a exhibits collateral cleavage propensity of both target DNA and any single-stranded(ss) DNA in the vicinity (herein referred to as a reporter). The LFB test line contained an ssDNA probe complementary to the reporter. In the presence of the target, Cas12a trans-cleaved the ssDNA reporter, which resulted in the inability of cleaved sequences to bind the LFB test line. With a PCR pre-amplification of the target (45 min), the assay achieved a sensitivity of 7.1 × 10-14 M (∼42 000 copies per μl) both in plasmid and plasmid-spiked samples. The assay attained a high specificity in the presence of various bacteria and applicability in EBV Burkitt's lymphoma serum samples. This method could be applied for the detection of EBV and other infectious diseases.},
}
@article {pmid32983033,
year = {2020},
author = {Ye, Q and Zhao, X and Liu, J and Zeng, Z and Zhang, Z and Liu, T and Li, Y and Han, W and Peng, N},
title = {CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in Sulfolobus islandicus.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {2038},
pmid = {32983033},
issn = {1664-302X},
abstract = {Acquisition of spacers confers the CRISPR-Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in Sulfolobus islandicus. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5'-CAAAA-3') and mutation at its CARF domain, which reduces the binding affinity. Csa3b also binds the promoter of adaptation cas genes, and the cOA analog enhances their binding probably by allosteric regulation. Deletion of the csa3b gene triggers spacer acquisition from both plasmid and viral DNAs, indicating that Csa3b acted as a repressor for CRISPR adaptation. Moreover, we also find that Csa3b activates the expression of subtype cmr-α and cmr-β genes according to transcriptome data and demonstrate that Csa3b binds the promoters of cmr genes. The deletion of the csa3b gene reduces Cmr-mediated RNA interference activity, indicating that Csa3b acts as a transcriptional activator for Cmr-mediated RNA interference. In summary, our findings reveal a novel pathway for the regulation of CRISPR adaptation and CRISPR-Cmr RNA interference in S. islandicus. Our results also suggest a feedback repression of CRIPSR adaptation by the Csa3b factor and the cOA signal produced by the Cmr complex at the CRISPR interference stage.},
}
@article {pmid32980951,
year = {2020},
author = {Ma, D and Endo, S and Betsuyaku, S and Shimotohno, A and Fukuda, H},
title = {CLE2 regulates light-dependent carbohydrate metabolism in Arabidopsis shoots.},
journal = {Plant molecular biology},
volume = {104},
number = {6},
pages = {561-574},
pmid = {32980951},
issn = {1573-5028},
mesh = {Arabidopsis/genetics/*metabolism/radiation effects ; Arabidopsis Proteins/genetics/*physiology ; CRISPR-Cas Systems ; *Carbohydrate Metabolism ; Darkness ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Gene-Environment Interaction ; Light ; Plant Shoots/*metabolism/radiation effects ; Plants, Genetically Modified ; Protein Domains/genetics ; Signal Transduction ; },
abstract = {This study focused on the role of CLE1-CLE7 peptides as environmental mediators and indicated that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots. Plants sense environmental stimuli and convert them into cellular signals, which are transmitted to distinct cells and tissues to induce adequate responses. Plant hormones and small secretory peptides often function as environmental stress mediators. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED proteins, CLE1-CLE7, which share closely related CLE domains, mediate environmental stimuli in Arabidopsis thaliana. Expression analysis of CLE1-CLE7 revealed that these genes respond to different environmental stimuli, such as nitrogen deprivation, nitrogen replenishment, cold, salt, dark, and sugar starvation, in a sophisticated manner. To further investigate the function of CLE2, we generated transgenic Arabidopsis lines expressing the β-glucuronidase gene under the control of the CLE2 promoter or expressing the CLE2 gene under the control of an estradiol-inducible promoter. We also generated cle2-1 and cle2-2 mutants using the CRISPR/Cas9 technology. In these transgenic lines, dark induced the expression of CLE2 in the root vasculature. Additionally, induction of CLE2 in roots induced the expression of various genes not only in roots but also in shoots, and genes related to light-dependent carbohydrate metabolism were particularly induced in shoots. In addition, cle2 mutant plants showed chlorosis when subjected to a shade treatment. These results suggest that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots.},
}
@article {pmid32980909,
year = {2021},
author = {Jung, YJ and Lee, HJ and Yu, J and Bae, S and Cho, YG and Kang, KK},
title = {Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {1013-1024},
pmid = {32980909},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems ; Chlorophyll/biosynthesis/genetics ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Gene Knockout Techniques/*methods ; Homozygote ; Mutation Rate ; Oryza/genetics/*physiology ; Phenotype ; Plants, Genetically Modified ; },
abstract = {The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.},
}
@article {pmid32980690,
year = {2020},
author = {Sleiman, Y and Souidi, M and Kumar, R and Yang, E and Jaffré, F and Zhou, T and Bernardin, A and Reiken, S and Cazorla, O and Kajava, AV and Moreau, A and Pasquié, JL and Marks, AR and Lerman, BB and Chen, S and Cheung, JW and Evans, T and Lacampagne, A and Meli, AC},
title = {Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes.},
journal = {EBioMedicine},
volume = {60},
number = {},
pages = {103024},
pmid = {32980690},
issn = {2352-3964},
mesh = {Alleles ; CRISPR-Cas Systems ; Calcium/metabolism ; Calcium Signaling ; *Cell Differentiation ; Genotype ; Homeostasis ; Humans ; Immunohistochemistry ; Induced Pluripotent Stem Cells/*cytology ; *Models, Biological ; Mutation ; Myocytes, Cardiac/*cytology/*metabolism ; Protein Processing, Post-Translational ; Stem Cell Transplantation ; Tachycardia, Ventricular/*diagnosis/etiology/*therapy ; },
abstract = {BACKGROUND: While mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2.<br><br>METHODS: Blood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared.<br><br>FINDINGS: RyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca[2+] leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca[2+] release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls.<br><br>INTERPRETATION: To conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca[2+] homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties.<br><br>FUNDING: French Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), "Fondation de la Recherche M&#xe9;dicale" (FRM; SPF20130526710), "Institut National pour la Sant&#xe9; et la Recherche M&#xe9;dicale" (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156).},
}
@article {pmid32980369,
year = {2020},
author = {Bhat, MA and Bhat, MA and Kumar, V and Wani, IA and Bashir, H and Shah, AA and Rahman, S and Jan, AT},
title = {The era of editing plant genomes using CRISPR/Cas: A critical appraisal.},
journal = {Journal of biotechnology},
volume = {324},
number = {},
pages = {34-60},
doi = {10.1016/j.jbiotec.2020.09.013},
pmid = {32980369},
issn = {1873-4863},
mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {The versatility of Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) genome editing tool ushered biologists into an exciting era of editing genomes with great efficiency and at a pace that was never imagined before. Though the CRISPR/Cas genome editing was developed after Zinc Finger Nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs), it is more popular and successful than these genome editing systems. The advent of targetable nucleases such as Cas9 has enabled manipulation of genomes in an accurate and precise manner. The CRISPR/Cas system of editing plant genomes has technical and economical advantages over conventional breeding methods. It has led to the development of traits within plant genomes that fulfill the needs of mankind. Advent of innovative procedures have paved the way for effective and efficient genome editing that has revolutionized genetic aspects and meets the safety regulations toward development of crops. The present review highlights the critical aspects of employing CRISPR/Cas for editing plant genomes in comparison with previously known editing approaches, such as ZFNs and TALENs. The study includes descriptive information on the approaches, procedural programs and applications in editing plant genomes for improving traits such as crop yield, resistance against emerging pathogens, abiotic stresses and herbicide tolerance thereof in the present-day world.},
}
@article {pmid32979593,
year = {2020},
author = {Chen, Y and Shi, Y and Chen, Y and Yang, Z and Wu, H and Zhou, Z and Li, J and Ping, J and He, L and Shen, H and Chen, Z and Wu, J and Yu, Y and Zhang, Y and Chen, H},
title = {Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {169},
number = {},
pages = {112642},
pmid = {32979593},
issn = {1873-4235},
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; Biosensing Techniques/instrumentation/*methods ; COVID-19 ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/*diagnosis/virology ; Equipment Design ; Fluorescence ; Humans ; Molecular Diagnostic Techniques/instrumentation/*methods ; Nucleic Acid Amplification Techniques/instrumentation/*methods ; Pandemics ; Pneumonia, Viral/*diagnosis/virology ; *Point-of-Care Systems ; SARS-CoV-2 ; Sensitivity and Specificity ; Smartphone ; },
abstract = {The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.},
}
@article {pmid32979185,
year = {2020},
author = {Xiong, Y and Han, X and Zhang, J and Zhao, G and Wang, Z and Zhuang, R and Nie, X and Xie, S and Li, C and Li, X and Liu, X and Li, K and Zhao, S and Ruan, J},
title = {Identification of ACTB Gene as a Potential Safe Harbor Locus in Pig Genome.},
journal = {Molecular biotechnology},
volume = {62},
number = {11-12},
pages = {589-597},
doi = {10.1007/s12033-020-00276-6},
pmid = {32979185},
issn = {1559-0305},
mesh = {Actins/*genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cell Line ; Gene Expression ; Gene Knock-In Techniques/*methods ; Gene Targeting ; Green Fluorescent Proteins/genetics/*metabolism ; Homologous Recombination ; Swine ; },
abstract = {Transgenic pigs play an important role in biomedicine and agriculture. The "safe harbor" locus maintains consistent foreign gene expression in cells and is important for transgenic pig generation. However, as only several safe harbor loci(Rosa26, pH11 and Pifs501) have been identified in pigs, meeting the needs of the insertion of various foreign genes is difficult. In this study, we develop a novel strategy for the efficient knock-in of gene-of-interest fragments into endogenous beta-actin(ACTB) gene via CRISPR/Cas9 mediated homologous recombination with normal expression of ACTB. Thus, we provide an alternative strategy to integrate exogenous genes into the pig genome that can be applied to agricultural breeding and biomedical models.},
}
@article {pmid32978771,
year = {2020},
author = {Jamehdor, S and Zaboli, KA and Naserian, S and Thekkiniath, J and Omidy, HA and Teimoori, A and Johari, B and Taromchi, AH and Sasano, Y and Kaboli, S},
title = {An overview of applications of CRISPR-Cas technologies in biomedical engineering.},
journal = {Folia histochemica et cytobiologica},
volume = {58},
number = {3},
pages = {163-173},
doi = {10.5603/FHC.a2020.0023},
pmid = {32978771},
issn = {1897-5631},
mesh = {Animals ; Biomedical Engineering/*methods ; *CRISPR-Cas Systems ; Cell Engineering ; Drug Discovery ; Gene Editing/methods ; Humans ; Models, Biological ; },
abstract = {Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.},
}
@article {pmid32978715,
year = {2020},
author = {Hirata, M and Wittayarat, M and Tanihara, F and Sato, Y and Namula, Z and Le, QA and Lin, Q and Takebayashi, K and Otoi, T},
title = {One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs.},
journal = {In vitro cellular &amp; developmental biology. Animal},
volume = {56},
number = {8},
pages = {614-621},
doi = {10.1007/s11626-020-00507-9},
pmid = {32978715},
issn = {1543-706X},
mesh = {Animals ; Base Sequence ; Blastocyst/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Electroporation ; *Gene Editing ; Interleukin Receptor Common gamma Subunit/genetics ; Mutation/genetics ; RNA, Guide/*metabolism ; Receptors, Somatotropin/genetics ; Swine ; Zygote/*metabolism ; },
abstract = {In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival.},
}
@article {pmid32978399,
year = {2020},
author = {Nguyen Tran, MT and Mohd Khalid, MKN and Wang, Q and Walker, JKR and Lidgerwood, GE and Dilworth, KL and Lisowski, L and Pébay, A and Hewitt, AW},
title = {Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4871},
pmid = {32978399},
issn = {2041-1723},
mesh = {Adenine/*chemistry ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cytosine ; DNA ; Exome ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome ; HEK293 Cells ; Humans ; RNA/*metabolism ; RNA Editing ; },
abstract = {Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.},
}
@article {pmid32978063,
year = {2020},
author = {Baeten, P and Hellings, N and Broux, B},
title = {In Vitro Tailoring of Regulatory T Cells Prior to Cell Therapy.},
journal = {Trends in molecular medicine},
volume = {26},
number = {11},
pages = {1059-1060},
doi = {10.1016/j.molmed.2020.08.008},
pmid = {32978063},
issn = {1471-499X},
mesh = {Autoimmune Diseases/immunology/therapy ; CRISPR-Cas Systems ; *Cell- and Tissue-Based Therapy/methods ; Gene Editing ; Humans ; Immunotherapy, Adoptive ; Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Receptors, Chimeric Antigen ; T-Lymphocytes, Regulatory/*immunology/*metabolism ; },
}
@article {pmid32977396,
year = {2020},
author = {Ates, I and Rathbone, T and Stuart, C and Bridges, PH and Cottle, RN},
title = {Delivery Approaches for Therapeutic Genome Editing and Challenges.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {32977396},
issn = {2073-4425},
support = {P20 GM103499/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques/*standards ; *Genetic Therapy ; Genetic Vectors/*administration &amp; dosage ; Humans ; },
abstract = {Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.},
}
@article {pmid32977294,
year = {2020},
author = {Zhao, H and He, L and Huang, H and Li, S and Cheng, N and Tang, F and Han, X and Lin, Z and Huang, R and Zhou, P and Deng, S and Huang, J and Li, Z},
title = {Generation of a tdTomato-GAD67 reporter human epilepsia mutation induced pluripotent stem cell line, USTCi001-A-2, using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {102003},
doi = {10.1016/j.scr.2020.102003},
pmid = {32977294},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Epilepsies, Myoclonic/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Luminescent Proteins ; Mutation/genetics ; },
abstract = {Dravet syndrome is an epileptic encephalopathy largely due to haploinsufficiency of the voltage-gated sodium channel Nav1.1 that is expressed primarily in GABAergic neurons. In order to distinguish the different subtypes, we used gene editing to introduce tdTomato gene into the genome of iPSCs to label the GABAergic neurons in the differentiated neuronal networks. The gene-edited cell line demonstrates normal karyotype, expresses the main pluripotency markers, and shows the presence of differentiation into the three embryonic germ layers in teratomas.},
}
@article {pmid32977162,
year = {2020},
author = {Cook, A and Bernstein, E},
title = {A strike against indolent neuroblastoma.},
journal = {EBioMedicine},
volume = {60},
number = {},
pages = {103000},
pmid = {32977162},
issn = {2352-3964},
mesh = {Biomarkers, Tumor ; CRISPR-Cas Systems ; Disease Management ; Disease Susceptibility ; Gene Targeting ; Humans ; Molecular Targeted Therapy ; Mutation ; Neuroblastoma/diagnosis/*etiology/therapy ; X-linked Nuclear Protein/antagonists &amp; inhibitors ; },
}
@article {pmid32976930,
year = {2020},
author = {Arya, SS and Rookes, JE and Cahill, DM and Lenka, SK},
title = {Next-generation metabolic engineering approaches towards development of plant cell suspension cultures as specialized metabolite producing biofactories.},
journal = {Biotechnology advances},
volume = {45},
number = {},
pages = {107635},
doi = {10.1016/j.biotechadv.2020.107635},
pmid = {32976930},
issn = {1873-1899},
mesh = {Artificial Intelligence ; CRISPR-Cas Systems ; Family Characteristics ; Gene Editing ; *Metabolic Engineering ; *Plant Cells ; },
abstract = {Plant cell suspension culture (PCSC) has emerged as a viable technology to produce plant specialized metabolites (PSM). While Taxol® and ginsenoside are two examples of successfully commercialized PCSC-derived PSM, widespread utilization of the PCSC platform has yet to be realized primarily due to a lack of understanding of the molecular genetics of PSM biosynthesis. Recent advances in computational, molecular and synthetic biology tools provide the opportunity to rapidly characterize and harness the specialized metabolic potential of plants. Here, we discuss the prospects of integrating computational modeling, artificial intelligence, and precision genome editing (CRISPR/Cas and its variants) toolboxes to discover the genetic regulators of PSM. We also explore how synthetic biology can be applied to develop metabolically optimized PSM-producing native and heterologous PCSC systems. Taken together, this review provides an interdisciplinary approach to realize and link the potential of next-generation computational and molecular tools to convert PCSC into commercially viable PSM-producing biofactories.},
}
@article {pmid32975636,
year = {2021},
author = {Hooghvorst, I and Nogués, S},
title = {Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.},
journal = {Plant cell reports},
volume = {40},
number = {2},
pages = {255-270},
pmid = {32975636},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems ; Chromosomes, Plant/*genetics ; Crops, Agricultural/*genetics ; *Gene Editing ; Haploidy ; Parthenogenesis ; Plant Breeding ; },
abstract = {The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.},
}
@article {pmid32975299,
year = {2020},
author = {Poljak, M},
title = {Simplification of hepatitis C testing: a time to act.},
journal = {Acta dermatovenerologica Alpina, Pannonica, et Adriatica},
volume = {29},
number = {3},
pages = {129-132},
pmid = {32975299},
issn = {1581-2979},
mesh = {Hepacivirus/*isolation &amp; purification ; Hepatitis C/*diagnosis ; Humans ; },
abstract = {Hepatitis C virus (HCV) affects 71 million people worldwide. The development of reliable diagnostic tools in the last 2 decades and recent implementation of highly potent and safe antiviral drug combinations have paved the way to potential elimination of HCV as a public health threat by 2030. This article briefly discusses current and upcoming solutions for simplification of HCV testing taking into account the most recent guidance documents issued by major professional societies. The general consensus is that all patients with suspected HCV infection should be tested for anti-HCV antibodies as a first-line diagnostic test. Some anti-HCV rapid diagnostic tests have already attained the performance of standard anti-HCV enzyme immunoassays. If anti-HCV antibodies are detected, the presence of HCV RNA or HCV core antigen should be determined to identify patients with ongoing infection. Several innovative devices for detecting HCV in serum or plasma are in the late stages of development and are based on loop-mediated isothermal amplification, smartphone-operated instruments, biosensors, lab-on-a-chip solutions, paper-based microfluidics, and CRISPR-Cas. An important solution for low- and middle-income countries is the availability of HCV tests that could perform equally reliably from whole blood and dried blood spots as well as from serum or plasma. Another interesting diagnostic concept for these countries is near-to-patient diagnostics using mobile microbiological laboratories, following either the lab-on-a-drone or rent-a-point-of-care-test concepts. Using current and upcoming diagnostic approaches, the elimination of HCV is plausible, but in several countries this is probably not possible within the timeframe suggested by the World Health Organization. Two different elimination approaches have already been successfully tested in real-life conditions: micro-elimination and macro-elimination. The micro-elimination approach has resulted in successful elimination in specific population subgroups in some high-income countries. In at least two countries, Georgia and Egypt, a macro-elimination approach has shown impressive real-life results relatively quickly.},
}
@article {pmid32975231,
year = {2020},
author = {Sen, P and Ganguly, P and Kulkarni, KK and Budhwar, R and Ganguly, N},
title = {Differential transcriptome analysis in HPV-positive and HPV-negative cervical cancer cells through CRISPR knockout of miR-214.},
journal = {Journal of biosciences},
volume = {45},
number = {},
pages = {},
pmid = {32975231},
issn = {0973-7138},
mesh = {Amino Acid Oxidoreductases/genetics/metabolism ; CD24 Antigen/genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cell Line, Tumor ; Cytochrome P-450 CYP1B1/genetics/metabolism ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; High-Throughput Nucleotide Sequencing ; Humans ; Hyaluronan Receptors/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; MicroRNAs/agonists/antagonists &amp; inhibitors/*genetics/metabolism ; Mismatch Repair Endonuclease PMS2/genetics/metabolism ; Neoplasm Proteins/*genetics/metabolism ; Papillomavirus Infections/*genetics/metabolism/pathology ; Protein Serine-Threonine Kinases/genetics/metabolism ; Proto-Oncogene Proteins c-met/genetics/metabolism ; *Transcriptome ; Tumor Necrosis Factor alpha-Induced Protein 3/genetics/metabolism ; Uterine Cervical Neoplasms/*genetics/metabolism/pathology ; rab GTP-Binding Proteins/genetics/metabolism ; },
abstract = {In this study we have investigated the effects of a tumour suppressor microRNA, miR-214, on gene expression in HPV-positive (CaSki) and HPV-negative cervical cancer cells (C33A) by RNA sequencing using next generation sequencing. The HPV-positive and HPV-negative cervical cancer cells were either miR-214- knocked-out or miR-214-overexpressed. Gene expression analysis showed that a total of 904 genes were upregulated and 365 genes were downregulated between HPV-positive and HPV-negative cervical cancer cells with a fold change of +/- 2. Furthermore, 11 differentially expressed and relevant genes (TNFAIP3, RAB25, MET, CYP1B1, NDRG1, CD24, LOXL2, CD44, PMS2, LATS1 and MDM1) which showed a fold change of +/-5 were selected to confirm by real-time PCR. This study represents the first report of miR-214 on global gene expression in the context of HPV.},
}
@article {pmid32974560,
year = {2019},
author = {Yan, Y and Finnigan, GC},
title = {Analysis of CRISPR gene drive design in budding yeast.},
journal = {Access microbiology},
volume = {1},
number = {9},
pages = {e000059},
pmid = {32974560},
issn = {2516-8290},
abstract = {Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems in vivo, including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in Saccharomyces cerevisiae that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of 'resistant' clones that still harboured both the drive and target selection markers following expression of Streptococcus pyogenes Cas9; de novo mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.},
}
@article {pmid32974171,
year = {2020},
author = {Chasov, V and Mirgayazova, R and Zmievskaya, E and Khadiullina, R and Valiullina, A and Stephenson Clarke, J and Rizvanov, A and Baud, MGJ and Bulatov, E},
title = {Key Players in the Mutant p53 Team: Small Molecules, Gene Editing, Immunotherapy.},
journal = {Frontiers in oncology},
volume = {10},
number = {},
pages = {1460},
pmid = {32974171},
issn = {2234-943X},
abstract = {The transcription factor p53 is a key tumor suppressor that is inactivated in almost all cancers due to either point mutations in the TP53 gene or overexpression of its negative regulators. The p53 protein is known as the "cellular gatekeeper" for its roles in facilitating DNA repair, cell cycle arrest or apoptosis upon DNA damage. Most p53 mutations are missense and result in either structural destabilization of the protein, causing its partial unfolding and deactivation under physiological conditions, or impairment of its DNA-binding properties. Tumor cells with p53 mutations are generally more immunogenic due to "hot spot" neoantigens that instigate the immune system response. In this review, we discuss the key therapeutic strategies targeting mutant p53 tumors, including classical approaches based on small molecule intervention and emerging technologies such as gene editing and T cell immunotherapy.},
}
@article {pmid32973772,
year = {2020},
author = {Zhang, S and Shrestha, CL and Wisniewski, BL and Pham, H and Hou, X and Li, W and Dong, Y and Kopp, BT},
title = {Consequences of CRISPR-Cas9-Mediated CFTR Knockout in Human Macrophages.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {1871},
pmid = {32973772},
issn = {1664-3224},
support = {K08 AI108792/AI/NIAID NIH HHS/United States ; R01 HL148171/HL/NHLBI NIH HHS/United States ; UL1 TR002733/TR/NCATS NIH HHS/United States ; },
mesh = {Adult ; Aged ; CRISPR-Cas Systems ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/*immunology ; Female ; Gene Editing ; Gene Knockout Techniques/*methods ; Humans ; Macrophages/*immunology ; Male ; Middle Aged ; Young Adult ; },
abstract = {Macrophage dysfunction is fundamentally related to altered immunity in cystic fibrosis (CF). How genetic deficits in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to these defects remains unknown. Rapid advances in genomic editing such as the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) system provide new tools for scientific study. We aimed to create a stable CFTR knockout (KO) in human macrophages in order to study how CFTR regulates macrophage function. Peripheral blood monocytes were isolated from non-CF healthy volunteers and differentiated into monocyte-derived macrophages (MDMs). MDMs were transfected with a CRISPR Cas9 CFTR KO plasmid. CFTR KO efficiency was verified and macrophage halide efflux, phagocytosis, oxidative burst, apoptosis, and cytokine functional assays were performed. CFTR KO in human MDMs was efficient and stable after puromycin selection. CFTR KO was confirmed by CFTR mRNA and protein expression. CFTR function was abolished in CFTR KO MDMs. CFTR KO recapitulated known defects in human CF MDM (CFTR class I/II variants) dysfunction including (1) increased apoptosis, (2) decreased phagocytosis, (3) reduced oxidative burst, and (4) increased bacterial load. Activation of the oxidative burst via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assembly was diminished in CFTR KO MDMs (decreased phosphorylated p47[phox]). Cytokine production was unchanged or decreased in response to infection in CFTR KO MDMs. In conclusion, we developed a primary human macrophage CFTR KO system. CFTR KO mimics most pathology observed in macrophages obtained from persons with CF, which suggests that many aspects of CF macrophage dysfunction are CFTR-dependent and not just reflective of the CF inflammatory milieu.},
}
@article {pmid32973430,
year = {2020},
author = {Gallego, C and Gonçalves, MAFV and Wijnholds, J},
title = {Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing.},
journal = {Frontiers in neuroscience},
volume = {14},
number = {},
pages = {838},
pmid = {32973430},
issn = {1662-4548},
abstract = {Inherited retinal diseases encompass a highly heterogenous group of disorders caused by a wide range of genetic variants and with diverse clinical symptoms that converge in the common trait of retinal degeneration. Indeed, mutations in over 270 genes have been associated with some form of retinal degenerative phenotype. Given the immune privileged status of the eye, cell replacement and gene augmentation therapies have been envisioned. While some of these approaches, such as delivery of genes through recombinant adeno-associated viral vectors, have been successfully tested in clinical trials, not all patients will benefit from current advancements due to their underlying genotype or phenotypic traits. Gene editing arises as an alternative therapeutic strategy seeking to correct mutations at the endogenous locus and rescue normal gene expression. Hence, gene editing technologies can in principle be tailored for treating retinal degeneration. Here we provide an overview of the different gene editing strategies that are being developed to overcome the challenges imposed by the post-mitotic nature of retinal cell types. We further discuss their advantages and drawbacks as well as the hurdles for their implementation in treating retinal diseases, which include the broad range of mutations and, in some instances, the size of the affected genes. Although therapeutic gene editing is at an early stage of development, it has the potential of enriching the portfolio of personalized molecular medicines directed at treating genetic diseases.},
}
@article {pmid32973401,
year = {2020},
author = {Miri, SM and Tafsiri, E and Cho, WCS and Ghaemi, A},
title = {CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.},
journal = {Cancer cell international},
volume = {20},
number = {},
pages = {456},
pmid = {32973401},
issn = {1475-2867},
abstract = {Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called "adoptive cell transfer", or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.},
}
@article {pmid32973356,
year = {2020},
author = {Zhu, H and Li, C and Gao, C},
title = {Applications of CRISPR-Cas in agriculture and plant biotechnology.},
journal = {Nature reviews. Molecular cell biology},
volume = {21},
number = {11},
pages = {661-677},
pmid = {32973356},
issn = {1471-0080},
abstract = {The prokaryote-derived CRISPR-Cas genome editing technology has altered plant molecular biology beyond all expectations. Characterized by robustness and high target specificity and programmability, CRISPR-Cas allows precise genetic manipulation of crop species, which provides the opportunity to create germplasms with beneficial traits and to develop novel, more sustainable agricultural systems. Furthermore, the numerous emerging biotechnologies based on CRISPR-Cas platforms have expanded the toolbox of fundamental research and plant synthetic biology. In this Review, we first briefly describe gene editing by CRISPR-Cas, focusing on the newest, precise gene editing technologies such as base editing and prime editing. We then discuss the most important applications of CRISPR-Cas in increasing plant yield, quality, disease resistance and herbicide resistance, breeding and accelerated domestication. We also highlight the most recent breakthroughs in CRISPR-Cas-related plant biotechnologies, including CRISPR-Cas reagent delivery, gene regulation, multiplexed gene editing and mutagenesis and directed evolution technologies. Finally, we discuss prospective applications of this game-changing technology.},
}
@article {pmid32973188,
year = {2020},
author = {Moro, LN and Viale, DL and Bastón, JI and Arnold, V and Suvá, M and Wiedenmann, E and Olguín, M and Miriuka, S and Vichera, G},
title = {Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {15587},
pmid = {32973188},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; Embryo, Mammalian/cytology/*metabolism ; Female ; Fibroblasts/cytology/metabolism ; *Gene Editing ; Gene Knockout Techniques/*veterinary ; Horses ; Mutation ; Myostatin/antagonists &amp; inhibitors/*genetics ; Nuclear Transfer Techniques/*veterinary ; Sequence Homology ; },
abstract = {The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.},
}
@article {pmid32973160,
year = {2020},
author = {He, X and Chen, Y and Beltran, DG and Kelly, M and Ma, B and Lawrie, J and Wang, F and Dodds, E and Zhang, L and Guo, J and Niu, W},
title = {Functional genetic encoding of sulfotyrosine in mammalian cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4820},
pmid = {32973160},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Chemokines/metabolism ; Crystallography, X-Ray ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Ligands ; Models, Molecular ; Protein Conformation ; Receptors, CXCR4/genetics/metabolism ; Tyrosine/*analogs &amp; derivatives/*genetics/*metabolism ; Tyrosine-tRNA Ligase/chemistry/*genetics/*metabolism ; },
abstract = {Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.},
}
@article {pmid32972752,
year = {2020},
author = {Na, HH and Moon, S and Kim, KC},
title = {Knockout of SETDB1 gene using the CRISPR/cas-9 system increases migration and transforming activities via complex regulations of E-cadherin, β-catenin, STAT3, and Akt.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {3},
pages = {486-492},
doi = {10.1016/j.bbrc.2020.09.026},
pmid = {32972752},
issn = {1090-2104},
mesh = {A549 Cells ; Antibiotics, Antineoplastic/pharmacology ; Antigens, CD/metabolism ; CRISPR-Cas Systems ; Cadherins/metabolism ; *Cell Movement ; *Cell Transformation, Neoplastic ; Doxorubicin/pharmacology ; Gene Knockout Techniques ; Histone-Lysine N-Methyltransferase/genetics/*physiology ; Humans ; Proto-Oncogene Proteins c-akt/metabolism ; STAT3 Transcription Factor/metabolism ; beta Catenin/metabolism ; },
abstract = {SETDB1 HMTase participates in various cellular processes via epigenetic transcriptional regulation. SETDB1 expression is downregulated by anticancer drug treatment in cancer cells, but we still need to verify the functional significance on SETDB1 downregulation. CRISPR/cas9 is a useful technology for doing a knockout (KO) of a target gene. It is widely used to examine the function of genes. In this study, we prepared SETDB1-KO from A549 human lung cancer cells using the CRISPR/Cas9 system, and we compared molecular changes between the A549 cells and the SETDB1-KO cells. The SETDB1-KO cell proliferation rate was slightly decreased as compared to the A549 cells, but there was no large difference in sensitivity with doxorubicin treatment. Instead, the migration activity and transforming activity were dramatically increased in SETDB-KO cells. Using a western blot analysis and an immunostaining experiment, we confirmed that SETDB1-KO downregulates the expression of E-cadherin and β-catenin. A qPCR and an RT-PCR analysis suggested that SETDB1 transcriptionally regulates E-cadherin and β-catenin. Moreover, E-cadherin expression was also detected in the cytoplasmic region of SETDB1-KO cells, indicating that functional localization of E-cadherin might be changed in SETDB1-KO cells. On the other hand, total levels of STAT3 and Akt were increased in the SETDB1-KO cells, but activation of STAT3 (pSTAT3) was not induced in doxorubicin-treated SETDB1-KO cells. SETDB1 overexpression into SETDB1-KO cells restores the expression of E-cadherin, β-catenin, STAT3, and Akt, suggesting that those proteins are tightly regulated by SETDB1. Collectively, we suggest that complex regulations on E-cadherin, β-catenin, STAT3, and Akt are correlated with the increased migration and transforming activity of SETDB1-KO cells.},
}
@article {pmid32972746,
year = {2020},
author = {Cao, X and Kouyama-Suzuki, E and Pang, B and Kurihara, T and Mori, T and Yanagawa, T and Shirai, Y and Tabuchi, K},
title = {Inhibition of DNA ligase IV enhances the CRISPR/Cas9-mediated knock-in efficiency in mouse brain neurons.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {3},
pages = {449-457},
doi = {10.1016/j.bbrc.2020.09.053},
pmid = {32972746},
issn = {1090-2104},
mesh = {Animals ; Brain/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; DNA Ligase ATP/*antagonists &amp; inhibitors/genetics ; Electroporation ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics ; Mice ; Mice, Inbred C57BL ; Neurons/cytology/*metabolism/physiology ; Rad51 Recombinase/genetics/metabolism ; Recombinational DNA Repair ; Transfection ; },
abstract = {CRISPR/Cas9-mediated gene knock-in in in vivo neurons using in utero electroporation is a powerful technique, but the knock-in efficiency is generally low. We previously demonstrated that co-transfection with RAD51, a key molecule of the initial step of homology-directed repair (HDR), expression vector increased EGFP knock-in efficiency in the β-actin site up to 2.5-fold in the pyramidal neurons in layer 2/3 of the somatosensory cortex of mouse brain. To further improve the efficiency, we examined the effect of inhibition of DNA ligase IV (LIG4) that is an essential molecule for non-homologous end joining (NHEJ). Co-transfection with small hairpin RNA for LIG4 (shlig4) expression vector increased the EGFP knock-in efficiency in the β-actin site up to 3.6-fold compared to the condition without shlig4. RAD51 and shlig4 expression vector co-transfection further increased the knock-in efficiency up to 4.7-fold of the control condition. These results suggest that the inhibition of LIG4 is more effective than RAD51 overexpression, and it enhances the effect of RAD51 overexpression on HDR-mediated gene knock-in in vivo neurons.},
}
@article {pmid32970905,
year = {2021},
author = {Tang, H and Liu, H and Zhou, Y and Liu, H and Du, L and Wang, K and Ye, X},
title = {Fertility recovery of wheat male sterility controlled by Ms2 using CRISPR/Cas9.},
journal = {Plant biotechnology journal},
volume = {19},
number = {2},
pages = {224-226},
pmid = {32970905},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Fertility ; Humans ; *Infertility, Male ; Male ; Plant Infertility/genetics ; *Triticum/genetics ; },
}
@article {pmid32969481,
year = {2020},
author = {Järås, M},
title = {[Not Available].},
journal = {Lakartidningen},
volume = {117},
number = {},
pages = {},
pmid = {32969481},
issn = {1652-7518},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; },
}
@article {pmid32968780,
year = {2020},
author = {Değirmenci, L and Geiger, D and Rogé Ferreira, FL and Keller, A and Krischke, B and Beye, M and Steffan-Dewenter, I and Scheiner, R},
title = {CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees.},
journal = {Chemical senses},
volume = {45},
number = {8},
pages = {655-666},
doi = {10.1093/chemse/bjaa063},
pmid = {32968780},
issn = {1464-3553},
mesh = {Animals ; Bees/*genetics/*physiology ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; *Mutagenesis ; *Mutation ; Receptors, G-Protein-Coupled/genetics ; Taste/*genetics/*physiology ; },
abstract = {Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.},
}
@article {pmid32968206,
year = {2020},
author = {Makhov, P and Sohn, JA and Serebriiskii, IG and Fazliyeva, R and Khazak, V and Boumber, Y and Uzzo, RG and Kolenko, VM},
title = {CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.},
journal = {British journal of cancer},
volume = {123},
number = {12},
pages = {1749-1756},
pmid = {32968206},
issn = {1532-1827},
support = {R03 CA235060/CA/NCI NIH HHS/United States ; R03 CA216173/CA/NCI NIH HHS/United States ; R03 CA212949/CA/NCI NIH HHS/United States ; R01 CA218802/CA/NCI NIH HHS/United States ; R21 CA223394/CA/NCI NIH HHS/United States ; R03 CA246011/CA/NCI NIH HHS/United States ; R50 CA211479/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacokinetics/*pharmacology ; Apoptosis ; CRISPR-Cas Systems ; Carcinoma, Renal Cell/*drug therapy/genetics/metabolism/pathology ; Cell Line, Tumor ; DNA Fragmentation ; Drug Interactions ; Drug Resistance, Neoplasm/*genetics ; Drug Therapy, Combination ; Enzyme Inhibitors/pharmacology ; Farnesyltranstransferase/*antagonists &amp; inhibitors ; High-Throughput Screening Assays ; Humans ; Kidney Neoplasms/*drug therapy/genetics/metabolism/pathology ; Lysosomes ; Male ; Mechanistic Target of Rapamycin Complex 1/antagonists &amp; inhibitors/metabolism ; Mice ; Molecular Targeted Therapy ; Neoplasm Transplantation ; Piperidines/*pharmacology ; Progression-Free Survival ; Protein Kinase Inhibitors/pharmacology ; Pyridines/*pharmacology ; RNA, Small Interfering ; Random Allocation ; Sunitinib/pharmacokinetics/*pharmacology ; },
abstract = {BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits.<br><br>METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC.<br><br>RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo.<br><br>CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.},
}
@article {pmid32968154,
year = {2020},
author = {Seruggia, D and Fernández, A and Cantero, M and Fernández-Miñán, A and Gomez-Skarmeta, JL and Pelczar, P and Montoliu, L},
title = {Boundary sequences flanking the mouse tyrosinase locus ensure faithful pattern of gene expression.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {15494},
pmid = {32968154},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Chromatin/metabolism/ultrastructure ; Enhancer Elements, Genetic/genetics ; Gene Editing ; Gene Expression Regulation/*genetics ; Genetic Loci/*genetics ; HEK293 Cells ; Humans ; Mice ; Monophenol Monooxygenase/*genetics/metabolism ; Promoter Regions, Genetic/genetics ; Zebrafish/embryology/genetics ; },
abstract = {Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5' and 3' boundaries of this mammalian gene that function as enhancers and insulators. By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their mechanistic role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.},
}
@article {pmid32968060,
year = {2020},
author = {Garbett, D and Bisaria, A and Yang, C and McCarthy, DG and Hayer, A and Moerner, WE and Svitkina, TM and Meyer, T},
title = {T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4818},
pmid = {32968060},
issn = {2041-1723},
support = {F32 GM116328/GM/NIGMS NIH HHS/United States ; R01 GM095977/GM/NIGMS NIH HHS/United States ; R35 GM118067/GM/NIGMS NIH HHS/United States ; R35 GM127026/GM/NIGMS NIH HHS/United States ; },
mesh = {Actin Cytoskeleton/metabolism ; Actins/metabolism ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line ; Cell Movement/*physiology ; Cell Surface Extensions/*metabolism ; Cytoskeleton/metabolism ; Extracellular Matrix/metabolism ; Gene Knockout Techniques ; Humans ; Kinetics ; Membrane Glycoproteins/genetics/*metabolism/ultrastructure ; Microfilament Proteins/genetics/*metabolism/ultrastructure ; Myosins/metabolism ; Pseudopodia/metabolism ; Receptor, EphB2 ; },
abstract = {Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.},
}
@article {pmid32967957,
year = {2020},
author = {Huang, Y and Lin, Q and Huo, Z and Chen, C and Zhou, S and Ma, X and Gao, H and Lin, Y and Li, X and He, J and Zhang, P and Liu, C},
title = {Inositol-Requiring Enzyme 1α Promotes Zika Virus Infection through Regulation of Stearoyl Coenzyme A Desaturase 1-Mediated Lipid Metabolism.},
journal = {Journal of virology},
volume = {94},
number = {23},
pages = {},
pmid = {32967957},
issn = {1098-5514},
mesh = {A549 Cells ; Animals ; Brain/pathology/virology ; CRISPR-Cas Systems ; Cell Line ; Disease Models, Animal ; Endoplasmic Reticulum/metabolism ; Endoribonucleases/genetics/*metabolism ; Gene Editing ; Gene Knockout Techniques ; Humans ; Inositol/*metabolism ; Lipid Metabolism/*physiology ; Mice ; Oleic Acid/metabolism ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Stearoyl-CoA Desaturase/genetics/*metabolism ; Unfolded Protein Response ; Virus Replication/physiology ; Zika Virus/*metabolism ; Zika Virus Infection/*metabolism/pathology ; },
abstract = {Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which has become a global epidemic threat due to its rapid spread and association with serious consequences of infection, including neonatal microcephaly. Inositol-requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-related transmembrane protein that mediates unfolded protein response (UPR) pathway and has been indicated to play an important role in flavivirus replication. However, the mechanism of how IRE1α affects ZIKV replication remains unknown. In this study, we explored the role of IRE1α in ZIKV infection in vitro and in vivo by using CRISPR/Cas9-based gene knockout and RNA interference-based gene knockdown techniques. Both knockout and knockdown of IRE1α dramatically reduced ZIKV replication levels, including viral RNA levels, protein expression, and titers in different human cell lines. Trans-complementation with IRE1α restored viral replication levels decreased by IRE1α depletion. Furthermore, the proviral effect of IRE1α was dependent on its kinase and RNase activities. Importantly, we found that IRE1α promoted the replication of ZIKV through upregulating the accumulation of monounsaturated fatty acid (MUFA) rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1), which further affected the production of oleic acid (OA) and lipid droplet. Finally, our data demonstrated that in the brain tissues of ZIKV-infected mice, the replication levels of ZIKV and virus-related lesions were significantly suppressed by both the kinase and RNase inhibitors of IRE1α. Taken together, our results identified IRE1α as a ZIKV dependency factor which promotes viral replication through affecting SCD1-mediated lipid metabolism, potentially providing a novel molecular target for the development of anti-ZIKV agents.IMPORTANCE Zika virus (ZIKV) has been linked to serious neurologic disorders and causes widespread concern in the field of global public health. Inositol requiring enzyme 1α (IRE1α) is an ER-related transmembrane protein that mediates unfolded protein response (UPR) pathway. Here, we revealed that IRE1α is a proviral factor for ZIKV replication both in culture cells and mice model, which relies on its kinase and RNase activities. Importantly, we further provided evidence that upon ZIKV infection, IRE1α is activated and splices XBP1 mRNA which enhances the expression of monounsaturated fatty acids rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1) and subsequent lipid droplet production. Our data uncover a novel mechanism of IRE1α proviral effect by modulating lipid metabolism, providing the first evidence of a close relationship between IRE1α-mediated UPR, lipid metabolism, and ZIKV replication and indicating IRE1α inhibitors as potentially effective anti-ZIKV agents.},
}
@article {pmid32966912,
year = {2020},
author = {Montaña, S and Vilacoba, E and Fernandez, JS and Traglia, GM and Sucari, A and Pennini, M and Iriarte, A and Centron, D and Melano, RG and Ramírez, MS},
title = {Genomic analysis of two Acinetobacter baumannii strains belonging to two different sequence types (ST172 and ST25).},
journal = {Journal of global antimicrobial resistance},
volume = {23},
number = {},
pages = {154-161},
doi = {10.1016/j.jgar.2020.09.006},
pmid = {32966912},
issn = {2213-7173},
support = {SC3 GM125556/GM/NIGMS NIH HHS/United States ; T37 MD001368/MD/NIMHD NIH HHS/United States ; },
mesh = {*Acinetobacter baumannii/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Genome, Bacterial ; Genomics ; Whole Genome Sequencing ; },
abstract = {OBJECTIVES: Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the main focus of attention in clinical settings owing to its intrinsic ability to persist in the hospital environment and its capacity to acquire determinants of resistance and virulence. Here we present the genomic sequencing, molecular characterisation and genomic comparison of two A. baumannii strains belonging to two different sequence types (STs), one sporadic and one widely distributed in our region.<br><br>METHODS: Whole-genome sequencing (WGS) of Ab42 and Ab376 was performed using Illumina MiSeq-I and the genomes were assembled with SPAdes. ARG-ANNOT, CARD-RGI, ISfinder, PHAST, PlasmidFinder, plasmidSPAdes and IslandViewer were used to analyse both genomes.<br><br>RESULTS: Genome analysis revealed that Ab42 belongs to ST172, an uncommon ST, whilst Ab376 belongs to ST25, a widely distributed ST. Molecular characterisation showed the presence of two antibiotic resistance genes in Ab42 and nine in Ab376. No insertion sequences were detected in Ab42, however 22 were detected in Ab376. Moreover, two prophages were found in Ab42 and three in Ab376. In addition, a CRISPR-cas type I-Fb and two plasmids, one of which harboured an AbGRI1-like island, were found in Ab376.<br><br>CONCLUSIONS: We present WGS analysis of twoA. baumannii strains belonging to two different STs. These findings allowed us to characterise a previously undescribed ST (ST172) and provide new insights to the widely studied ST25.},
}
@article {pmid32966770,
year = {2020},
author = {Wang, PY and Cripe, TP},
title = {Gene Editing Thumbs a Ride with Oncolytic Virotherapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {10},
pages = {2103-2104},
pmid = {32966770},
issn = {1525-0024},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; ErbB Receptors ; Gene Editing ; Genomics ; Humans ; *Neoplasms/genetics/therapy ; *Oncolytic Virotherapy ; *Oncolytic Viruses/genetics ; Thumb ; },
}
@article {pmid32966744,
year = {2020},
author = {Khan, P and Aufdembrink, LM and Engelhart, AE},
title = {Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings.},
journal = {ACS synthetic biology},
volume = {9},
number = {11},
pages = {2861-2880},
pmid = {32966744},
issn = {2161-5063},
mesh = {COVID-19/*diagnosis/*epidemiology/prevention &amp; control/virology ; COVID-19 Testing/*methods ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Polymerase Chain Reaction/methods ; SARS-CoV-2/*genetics/*immunology/isolation &amp; purification ; Self-Sustained Sequence Replication/methods ; Sensitivity and Specificity ; Serologic Tests/methods ; },
abstract = {The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.},
}
@article {pmid32966423,
year = {2020},
author = {Li, Y and Li, T and Liu, BF and Hu, R and Zhu, J and He, T and Zhou, X and Li, C and Yang, Y and Liu, M},
title = {CRISPR-Cas12a trans-cleaves DNA G-quadruplexes.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {83},
pages = {12526-12529},
doi = {10.1039/d0cc05540a},
pmid = {32966423},
issn = {1364-548X},
mesh = {Aptamers, Peptide/metabolism ; Bacterial Proteins/chemistry/*metabolism ; CRISPR-Associated Proteins/chemistry/*metabolism ; CRISPR-Cas Systems/genetics ; Circular Dichroism ; DNA Cleavage ; Endodeoxyribonucleases/chemistry/*metabolism ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes/chemistry ; *G-Quadruplexes ; Humans ; Kinetics ; RNA, Guide/*metabolism ; Telomere/metabolism ; },
abstract = {We for the first time report that the activated CRISPR-Cas12a system trans-cleaves DNA G-quadruplexes (G4). The cleavage activity on human telomere G4 and TBA G4 was investigated and verified by FRET, CD, gel electrophoresis and NMR. We believe that this finding will pave a new avenue for advancing the applications of CRISPR-Cas12a and G4 in biosensing and biochemistry.},
}
@article {pmid32965719,
year = {2020},
author = {Rafia, C and Harly, C and Scotet, E},
title = {Beyond CAR T cells: Engineered Vγ9Vδ2 T cells to fight solid tumors.},
journal = {Immunological reviews},
volume = {298},
number = {1},
pages = {117-133},
doi = {10.1111/imr.12920},
pmid = {32965719},
issn = {1600-065X},
mesh = {Humans ; *Immunotherapy, Adoptive ; *Neoplasms/therapy ; Receptors, Antigen, T-Cell, gamma-delta/genetics ; T-Lymphocyte Subsets ; },
abstract = {Despite recent significant progress in cancer immunotherapies based on adoptive cell transfer(s)(ACT), the eradication of cancers still represents a major clinical challenge. In particular, the efficacy of current ACT-based therapies against solid tumors is dramatically reduced by physical barriers that prevent tumor infiltration of adoptively transferred effectors, and the tumor environment that suppress their anti-tumor functions. Novel immunotherapeutic strategies are thus needed to circumvent these issues. Human peripheral blood Vγ9Vδ2 T cells, a non-alloreactive innate-like T lymphocyte subset, recently proved to be a promising anti-tumor effector subset for ACT-based immunotherapies. Furthermore, new cell engineering tools that leverage the potential of CRISPR/Cas technology open astounding opportunities to optimize their anti-tumor effector functions. In this review, we present the current ACT strategies based on engineered T cells and their limitations. We then discuss the potential of engineered Vγ9Vδ2 T cell to overcome these limitations and improve ACT-based cancer immunotherapies.},
}
@article {pmid32964920,
year = {2020},
author = {Santos-Moreno, J and Schaerli, Y},
title = {CRISPR-based gene expression control for synthetic gene circuits.},
journal = {Biochemical Society transactions},
volume = {48},
number = {5},
pages = {1979-1993},
pmid = {32964920},
issn = {1470-8752},
mesh = {Animals ; Binding Sites ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Expression ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Regulatory Networks ; *Genes, Synthetic ; Genetic Engineering ; Humans ; Mice ; Synthetic Biology ; Transcription Factors/*metabolism ; },
abstract = {Synthetic gene circuits allow us to govern cell behavior in a programmable manner, which is central to almost any application aiming to harness engineered living cells for user-defined tasks. Transcription factors (TFs) constitute the 'classic' tool for synthetic circuit construction but some of their inherent constraints, such as insufficient modularity, orthogonality and programmability, limit progress in such forward-engineering endeavors. Here we review how CRISPR (clustered regularly interspaced short palindromic repeats) technology offers new and powerful possibilities for synthetic circuit design. CRISPR systems offer superior characteristics over TFs in many aspects relevant to a modular, predictable and standardized circuit design. Thus, the choice of CRISPR technology as a framework for synthetic circuit design constitutes a valid alternative to complement or replace TFs in synthetic circuits and promises the realization of more ambitious designs.},
}
@article {pmid32964665,
year = {2021},
author = {Song, CW and Rathnasingh, C and Park, JM and Kwon, M and Song, H},
title = {CRISPR-Cas9 mediated engineering of Bacillus licheniformis for industrial production of (2R,3S)-butanediol.},
journal = {Biotechnology progress},
volume = {37},
number = {1},
pages = {e3072},
doi = {10.1002/btpr.3072},
pmid = {32964665},
issn = {1520-6033},
mesh = {Bacillus licheniformis/genetics/growth &amp; development/*metabolism ; Bacterial Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Butylene Glycols/*metabolism ; CRISPR-Cas Systems ; Fermentation ; GTP-Binding Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Isocitrate Dehydrogenase/antagonists &amp; inhibitors/genetics/metabolism ; Metabolic Engineering ; Mutation ; },
abstract = {Bacillus lichenformis is an industrially promising generally recognized as safe (GRAS) strain that can be used for the production of a valuable chemical, 2,3-butanediol (BDO). Conventional gene deletion vectors and/or methods are time-consuming and have poor efficiency. Therefore, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 mediated homologous recombination was used to engineer a newly isolated and UV-mutagenized B. licheniformis 4071-15 strain. With the help of a CRISPR-Cas9 system, this one-step process could be used for the deletion of ldh gene within 4 days with high-efficiency exceeding 60%. In addition, the sequential deletion of target genes for engineering studies was evaluated, and it was confirmed that a triple mutant strain (ldh, dgp, and acoR) could be obtained by repeated one-step cycles. Furthermore, a practical metabolic engineering study was carried out using a CRISPR-Cas9 system for the stereospecific production of (2R,3S)-BDO. The predicted (2R,3R)-butanediol dehydrogenase encoded by the gdh gene was selected as a target for the production of (2R,3S)-BDO, and the mutant was successfully obtained. The results show that the stereospecific production of (2R,3S)-BDO was possible with the gdh deletion mutant, while the 4071-15 host strain still generated 26% of (2R,3R)-BDO. It was also shown that the 4071-15 Δgdh mutant could produce 115 g/L of (2R,3S)-BDO in 64 hr by two-stage fed-batch fermentation. This study has shown the efficient development of a (2R,3S)-BDO producing B. licheniformis strain based on CRISPR-Cas9 and fermentation technologies.},
}
@article {pmid32963283,
year = {2020},
author = {Krishnan, V and Tallapragada, S and Schaar, B and Kamat, K and Chanana, AM and Zhang, Y and Patel, S and Parkash, V and Rinker-Schaeffer, C and Folkins, AK and Rankin, EB and Dorigo, O},
title = {Omental macrophages secrete chemokine ligands that promote ovarian cancer colonization of the omentum via CCR1.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {524},
pmid = {32963283},
issn = {2399-3642},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chemokines/*metabolism ; Chemokines, CC/metabolism ; Female ; Flow Cytometry ; Gene Editing ; Gene Knockdown Techniques ; Humans ; Macrophages/*metabolism ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Omentum/*pathology ; Ovarian Neoplasms/metabolism/*pathology ; Peritoneal Neoplasms/metabolism/*secondary ; Real-Time Polymerase Chain Reaction ; Receptors, CCR1/*metabolism ; Transcriptome ; },
abstract = {The omentum is the most common site of ovarian cancer metastasis. Immune cell clusters called milky spots are found throughout the omentum. It is however unknown if these immune cells contribute to ovarian cancer metastasis. Here we report that omental macrophages promote the migration and colonization of ovarian cancer cells to the omentum through the secretion of chemokine ligands that interact with chemokine receptor 1 (CCR1). We found that depletion of macrophages reduces ovarian cancer colonization of the omentum. RNA-sequencing of macrophages isolated from mouse omentum and mesenteric adipose tissue revealed a specific enrichment of chemokine ligand CCL6 in omental macrophages. CCL6 and the human homolog CCL23 were both necessary and sufficient to promote ovarian cancer migration by activating ERK1/2 and PI3K pathways. Importantly, inhibition of CCR1 reduced ovarian cancer colonization. These findings demonstrate a critical mechanism of omental macrophage induced colonization by ovarian cancer cells via CCR1 signaling.},
}
@article {pmid32963084,
year = {2020},
author = {Stoneman, HR and Wrobel, RL and Place, M and Graham, M and Krause, DJ and De Chiara, M and Liti, G and Schacherer, J and Landick, R and Gasch, AP and Sato, TK and Hittinger, CT},
title = {CRISpy-Pop: A Web Tool for Designing CRISPR/Cas9-Driven Genetic Modifications in Diverse Populations.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {11},
pages = {4287-4294},
pmid = {32963084},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Human ; Humans ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas9 is a powerful tool for editing genomes, but design decisions are generally made with respect to a single reference genome. With population genomic data becoming available for an increasing number of model organisms, researchers are interested in manipulating multiple strains and lines. CRISpy-pop is a web application that generates and filters guide RNA sequences for CRISPR/Cas9 genome editing for diverse yeast and bacterial strains. The current implementation designs and predicts the activity of guide RNAs against more than 1000 Saccharomyces cerevisiae genomes, including 167 strains frequently used in bioenergy research. Zymomonas mobilis, an increasingly popular bacterial bioenergy research model, is also supported. CRISpy-pop is available as a web application (https://CRISpy-pop.glbrc.org/) with an intuitive graphical user interface. CRISpy-pop also cross-references the human genome to allow users to avoid the selection of guide RNAs with potential biosafety concerns. Additionally, CRISpy-pop predicts the strain coverage of each guide RNA within the supported strain sets, which aids in functional population genetic studies. Finally, we validate how CRISpy-pop can accurately predict the activity of guide RNAs across strains using population genomic data.},
}
@article {pmid32963031,
year = {2020},
author = {Halaburkova, A and Cahais, V and Novoloaca, A and Araujo, MGDS and Khoueiry, R and Ghantous, A and Herceg, Z},
title = {Pan-cancer multi-omics analysis and orthogonal experimental assessment of epigenetic driver genes.},
journal = {Genome research},
volume = {30},
number = {10},
pages = {1517-1532},
pmid = {32963031},
issn = {1549-5469},
mesh = {CRISPR-Cas Systems ; Cell Proliferation/genetics ; Computer Simulation ; DNA Methylation ; *Epigenesis, Genetic ; Epigenomics ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; *Genes, Neoplasm ; Humans ; Neoplasms/*genetics/pathology ; RNA, Neoplasm/metabolism ; },
abstract = {The recent identification of recurrently mutated epigenetic regulator genes (ERGs) supports their critical role in tumorigenesis. We conducted a pan-cancer analysis integrating (epi)genome, transcriptome, and DNA methylome alterations in a curated list of 426 ERGs across 33 cancer types, comprising 10,845 tumor and 730 normal tissues. We found that, in addition to mutations, copy number alterations in ERGs were more frequent than previously anticipated and tightly linked to expression aberrations. Novel bioinformatics approaches, integrating the strengths of various driver prediction and multi-omics algorithms, and an orthogonal in vitro screen (CRISPR-Cas9) targeting all ERGs revealed genes with driver roles within and across malignancies and shared driver mechanisms operating across multiple cancer types and hallmarks. This is the largest and most comprehensive analysis thus far; it is also the first experimental effort to specifically identify ERG drivers (epidrivers) and characterize their deregulation and functional impact in oncogenic processes.},
}
@article {pmid32962129,
year = {2020},
author = {Brezgin, S and Kostyusheva, A and Ponomareva, N and Volia, V and Goptar, I and Nikiforova, A and Shilovskiy, I and Smirnov, V and Kostyushev, D and Chulanov, V},
title = {Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases.},
journal = {International journal of molecular sciences},
volume = {21},
number = {18},
pages = {},
pmid = {32962129},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Cytidine Deaminase/genetics/*metabolism ; DNA/immunology/metabolism ; HEK293 Cells ; Humans ; Immunity, Innate/genetics ; Minor Histocompatibility Antigens/genetics/*metabolism ; Plasmids/*genetics/metabolism ; Proteins/genetics/*metabolism ; Up-Regulation ; p300-CBP Transcription Factors/genetics/metabolism ; },
abstract = {Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6-50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids.},
}
@article {pmid32960926,
year = {2020},
author = {McDiarmid, TA and Au, V and Moerman, DG and Rankin, CH},
title = {Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0238950},
pmid = {32960926},
issn = {1932-6203},
support = {R24 OD023041/OD/NIH HHS/United States ; PJT-148549//CIHR/Canada ; PJT-165947//CIHR/Canada ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Targeting/*methods ; Homozygote ; Phenotype ; RNA, Guide/genetics ; Toxins, Biological/*genetics/metabolism ; Transgenes ; },
abstract = {Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.},
}
@article {pmid32960509,
year = {2020},
author = {Shenoy, A and Belugali Nataraj, N and Perry, G and Loayza Puch, F and Nagel, R and Marin, I and Balint, N and Bossel, N and Pavlovsky, A and Barshack, I and Kaufman, B and Agami, R and Yarden, Y and Dadiani, M and Geiger, T},
title = {Proteomic patterns associated with response to breast cancer neoadjuvant treatment.},
journal = {Molecular systems biology},
volume = {16},
number = {9},
pages = {e9443},
pmid = {32960509},
issn = {1744-4292},
mesh = {Breast Neoplasms/*metabolism/pathology/*therapy ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Citric Acid Cycle ; Female ; Gene Regulatory Networks ; Humans ; *Neoadjuvant Therapy ; Neoplasm Invasiveness ; Neoplasm Proteins/metabolism ; Prognosis ; Protein Interaction Maps ; *Proteomics ; Pyrroline Carboxylate Reductases/metabolism ; Recurrence ; Survival Analysis ; },
abstract = {Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry-based proteomic approach and analyzed a breast cancer cohort of 113 formalin-fixed paraffin-embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor-adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post-treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER-positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.},
}
@article {pmid32960291,
year = {2020},
author = {Mitsui, R and Yamada, R and Matsumoto, T and Yoshihara, S and Tokumoto, H and Ogino, H},
title = {Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {21},
pages = {9147-9158},
doi = {10.1007/s00253-020-10906-3},
pmid = {32960291},
issn = {1432-0614},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Evolution, Molecular ; Fermentation ; L-Lactate Dehydrogenase/genetics/metabolism ; Lactic Acid ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.},
}
@article {pmid32960267,
year = {2020},
author = {Lin, J and Fuglsang, A and Kjeldsen, AL and Sun, K and Bhoobalan-Chitty, Y and Peng, X},
title = {DNA targeting by subtype I-D CRISPR-Cas shows type I and type III features.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10470-10478},
pmid = {32960267},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/classification/*genetics ; CRISPR-Cas Systems/*genetics/immunology ; DNA/*genetics/immunology ; DNA Helicases/genetics ; DNA, Single-Stranded/genetics ; RNA, Bacterial/genetics ; RNA, Double-Stranded/genetics ; },
abstract = {Prokaryotic CRISPR-Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR-Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3' and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR-Cas systems.},
}
@article {pmid32959978,
year = {2020},
author = {Dai, Y},
title = {Chemical Translational Biology-Guided Molecular Diagnostics: The Front Line To Mediate the Current SARS-CoV-2 Pandemic.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {21},
number = {24},
pages = {3492-3494},
pmid = {32959978},
issn = {1439-7633},
mesh = {Biosensing Techniques/methods ; COVID-19/*diagnosis/epidemiology/virology ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/methods ; Pandemics ; Point-of-Care Testing ; RNA, Viral/*analysis ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics ; },
abstract = {The spread of severe respiratory syndrome coronavirus 2 (SARS-CoV-2) has disrupted our global society in unprecedented ways. The very front line in defense against this pandemic is molecular diagnosis, which is an exceptional representation of how chemical translational biology can benefit our lives. In this viewpoint, I emphasize the imperative demand for a simple and rapid point-of-care system in order to mediate the spread of COVID-19. I further describe how the interdisciplinary combination of chemistry and biology advances biosensing systems, which potentially lead to integrated and automated point-of-care systems capable of relieving the current pandemic.},
}
@article {pmid32959897,
year = {2021},
author = {Zhang, B},
title = {CRISPR/Cas gene therapy.},
journal = {Journal of cellular physiology},
volume = {236},
number = {4},
pages = {2459-2481},
doi = {10.1002/jcp.30064},
pmid = {32959897},
issn = {1097-4652},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; *Gene Editing ; *Gene Targeting ; Genetic Diseases, Inborn/diagnosis/genetics/*therapy ; Genetic Predisposition to Disease ; *Genetic Therapy/adverse effects ; Humans ; Mice, Transgenic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated enzyme (Cas) is a naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system. Currently, CRISPR/Cas9-based genome editing has been becoming one of the most promising tools for treating human genetic diseases, including cardiovascular diseases, neuro-disorders, and cancers. As the quick modification of the CRISPR/Cas9 system, including delivery system, CRISPR/Cas9-based gene therapy has been extensively studied in preclinic and clinic treatments. CRISPR/Cas genome editing is also a robust tool to create animal genetic models for studying and treating human genetic disorders, particularly diseases associated with point mutations. However, significant challenges also remain before CRISPR/Cas technology can be routinely employed in the clinic for treating different genetic diseases, which include toxicity and immune response of treated cells to CRISPR/Cas component, highly throughput delivery method, and potential off-target impact. The off-target effect is one of the major concerns for CRISPR/Cas9 gene therapy, more research should be focused on limiting this impact by designing high specific gRNAs and using high specificity of Cas enzymes. Modifying the CRISPR/Cas9 delivery method not only targets a specific tissue/cell but also potentially limits the off-target impact.},
}
@article {pmid32959126,
year = {2021},
author = {Yu, J and Tu, L and Subburaj, S and Bae, S and Lee, GJ},
title = {Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.},
journal = {Plant cell reports},
volume = {40},
number = {6},
pages = {1037-1045},
pmid = {32959126},
issn = {1432-203X},
mesh = {CRISPR-Associated Protein 9/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knockout Techniques/*methods ; *Genes, Duplicate ; Genes, Plant ; Mutagenesis, Site-Directed ; Petunia/*genetics/physiology ; Pigmentation/*genetics ; Plants, Genetically Modified/genetics ; Protoplasts/cytology/physiology ; RNA, Guide ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.},
}
@article {pmid32958931,
year = {2020},
author = {Xia, B and Amador, G and Viswanatha, R and Zirin, J and Mohr, SE and Perrimon, N},
title = {CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells.},
journal = {Nature protocols},
volume = {15},
number = {10},
pages = {3478-3498},
pmid = {32958931},
issn = {1750-2799},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; R24 OD019847/OD/NIH HHS/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Base Sequence/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drosophila/genetics ; Endonucleases/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Homozygote ; Polyploidy ; RNA, Guide/genetics ; },
abstract = {Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.},
}
@article {pmid32958897,
year = {2021},
author = {Yue, Y and Xu, W and Kan, Y and Zhao, HY and Zhou, Y and Song, X and Wu, J and Xiong, J and Goswami, D and Yang, M and Lamriben, L and Xu, M and Zhang, Q and Luo, Y and Guo, J and Mao, S and Jiao, D and Nguyen, TD and Li, Z and Layer, JV and Li, M and Paragas, V and Youd, ME and Sun, Z and Ding, Y and Wang, W and Dou, H and Song, L and Wang, X and Le, L and Fang, X and George, H and Anand, R and Wang, SY and Westlin, WF and Güell, M and Markmann, J and Qin, W and Gao, Y and Wei, HJ and Church, GM and Yang, L},
title = {Extensive germline genome engineering in pigs.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {134-143},
pmid = {32958897},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cells, Cultured ; Galactosyltransferases/genetics ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Germ Cells/*metabolism ; Mixed Function Oxygenases/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Sus scrofa/*genetics/immunology/*virology ; *Transplantation, Heterologous ; },
abstract = {The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.},
}
@article {pmid32958732,
year = {2020},
author = {Kim, B and Kim, HJ and Lee, SJ},
title = {Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference.},
journal = {Journal of microbiology and biotechnology},
volume = {30},
number = {12},
pages = {1919-1926},
doi = {10.4014/jmb.2008.08058},
pmid = {32958732},
issn = {1738-8872},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Escherichia coli/genetics ; Galactose/genetics ; Gene Expression ; Mutation ; Promoter Regions, Genetic ; RNA, Guide ; },
abstract = {CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.},
}
@article {pmid32958637,
year = {2020},
author = {McWilliam, HEG and Mak, JYW and Awad, W and Zorkau, M and Cruz-Gomez, S and Lim, HJ and Yan, Y and Wormald, S and Dagley, LF and Eckle, SBG and Corbett, AJ and Liu, H and Li, S and Reddiex, SJJ and Mintern, JD and Liu, L and McCluskey, J and Rossjohn, J and Fairlie, DP and Villadangos, JA},
title = {Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {40},
pages = {24974-24985},
pmid = {32958637},
issn = {1091-6490},
mesh = {Antigen Presentation/genetics ; Antigens/genetics/immunology ; CRISPR-Cas Systems/genetics ; Endoplasmic Reticulum/*genetics ; Histocompatibility Antigens Class I/*genetics ; Humans ; Ligands ; Lymphocyte Activation/genetics ; Membrane Transport Proteins/genetics ; Metabolome/*genetics ; Minor Histocompatibility Antigens/*genetics ; Molecular Chaperones/genetics/immunology ; Mucosal-Associated Invariant T Cells/immunology ; *Proteomics ; Riboflavin/genetics ; },
abstract = {The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.},
}
@article {pmid32957821,
year = {2021},
author = {Zink, IA and Fouqueau, T and Tarrason Risa, G and Werner, F and Baum, B and Bläsi, U and Schleper, C},
title = {Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing.},
journal = {RNA biology},
volume = {18},
number = {3},
pages = {421-434},
pmid = {32957821},
issn = {1555-8584},
support = {BB/P001440/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MC_UP_1201/27/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Archaeal Proteins/genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems ; Cell Division/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Knockdown Techniques ; Gene Order ; *Gene Silencing ; Gene Targeting ; *Genes, Archaeal ; *Genes, Essential ; *Genes, Lethal ; Genetic Vectors/genetics ; Mutation ; Operon ; Phenotype ; RNA, Guide ; Sulfolobales/*genetics/metabolism ; },
abstract = {CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.},
}
@article {pmid32956624,
year = {2021},
author = {Amini, L and Wagner, DL and Rössler, U and Zarrinrad, G and Wagner, LF and Vollmer, T and Wendering, DJ and Kornak, U and Volk, HD and Reinke, P and Schmueck-Henneresse, M},
title = {CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {29},
number = {1},
pages = {32-46},
pmid = {32956624},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/immunology ; *Drug Resistance ; *Gene Editing ; Genetic Engineering ; Humans ; Immunosuppressive Agents/pharmacology ; *Immunotherapy, Adoptive ; T-Lymphocytes/*drug effects/immunology/*metabolism ; Tacrolimus/*pharmacology ; Transplant Recipients ; },
abstract = {Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because of malfunction in transferred T cells caused by ongoing IS. We developed a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based good manufacturing practice (GMP)-compliant protocol that efficiently targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This was achieved by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation. We confirmed the tacrolimus resistance of our gene-edited T cell products in vitro and demonstrated performance comparable with non-tacrolimus-treated unmodified T cells. The alternative calcineurin inhibitor cyclosporine A can be administered as a safety switch to shut down tacrolimus-resistant T cell activity in case of adverse effects. Furthermore, we performed safety assessments as a prerequisite for translation to first-in-human applications.},
}
@article {pmid32956422,
year = {2020},
author = {Krasnopolsky, S and Kuzmina, A and Taube, R},
title = {Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency.},
journal = {PLoS pathogens},
volume = {16},
number = {9},
pages = {e1008834},
pmid = {32956422},
issn = {1553-7374},
mesh = {CD4-Positive T-Lymphocytes/*metabolism/virology ; CRISPR-Cas Systems ; *Gene Expression Regulation, Viral ; Gene Knockout Techniques ; *Gene Silencing ; Genome-Wide Association Study ; HIV-1/*physiology ; Humans ; Jurkat Cells ; Promoter Regions, Genetic ; *Repressor Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; *Transcription, Genetic ; Tripartite Motif-Containing Protein 28/genetics/metabolism ; *Virus Latency ; },
abstract = {Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.},
}
@article {pmid32954022,
year = {2020},
author = {Abdullah, and Jiang, Z and Hong, X and Zhang, S and Yao, R and Xiao, Y},
title = {CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants.},
journal = {Synthetic and systems biotechnology},
volume = {5},
number = {4},
pages = {277-292},
pmid = {32954022},
issn = {2405-805X},
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.},
}
@article {pmid32951947,
year = {2020},
author = {Molla, KA and Qi, Y and Karmakar, S and Baig, MJ},
title = {Base Editing Landscape Extends to Perform Transversion Mutation.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {12},
pages = {899-901},
doi = {10.1016/j.tig.2020.09.001},
pmid = {32951947},
issn = {0168-9525},
mesh = {Adenine ; *CRISPR-Cas Systems ; Cytosine ; *Gene Editing ; Mutation ; },
abstract = {Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.},
}
@article {pmid32951534,
year = {2020},
author = {Guo, M and Xu, Y and Dong, Z and Zhou, Z and Cong, N and Gao, M and Huang, W and Wang, Y and Liu, G and Xian, X},
title = {Inactivation of ApoC3 by CRISPR/Cas9 Protects Against Atherosclerosis in Hamsters.},
journal = {Circulation research},
volume = {127},
number = {11},
pages = {1456-1458},
doi = {10.1161/CIRCRESAHA.120.317686},
pmid = {32951534},
issn = {1524-4571},
mesh = {Animals ; Apolipoprotein C-III/deficiency/*genetics ; Atherosclerosis/genetics/metabolism/pathology/*prevention &amp; control ; Biomarkers/blood ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Diet, High-Fat ; Disease Models, Animal ; *Gene Deletion ; *Genetic Therapy ; Lipids/blood ; Mesocricetus ; Plaque, Atherosclerotic ; },
}
@article {pmid32948757,
year = {2020},
author = {Ding, X and Yin, K and Li, Z and Lalla, RV and Ballesteros, E and Sfeir, MM and Liu, C},
title = {Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4711},
pmid = {32948757},
issn = {2041-1723},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R21 TW010625/TW/FIC NIH HHS/United States ; },
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; Coronavirus Infections/diagnosis/*virology ; Genes, Viral ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; Pneumonia, Viral/diagnosis/*virology ; Point-of-Care Systems ; RNA, Viral/analysis/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; Viral Proteins/analysis/genetics ; },
abstract = {The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.},
}
@article {pmid32948588,
year = {2020},
author = {Li, Z and Kim, KS},
title = {RELATe enables genome-scale engineering in fungal genomics.},
journal = {Science advances},
volume = {6},
number = {38},
pages = {},
pmid = {32948588},
issn = {2375-2548},
support = {R21 AI147699/AI/NIAID NIH HHS/United States ; R21 AI155009/AI/NIAID NIH HHS/United States ; R21 NS094012/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cryptococcus ; Gene Editing/methods ; Genome, Fungal ; Genomics/methods ; Humans ; *RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9-based screening with single-guide RNA (sgRNA) libraries has emerged as a revolutionary tool for comprehensive analysis of genetic elements. However, genome-scale sgRNA libraries are currently available only in a few model organisms. The traditional approach is to synthesize thousands to tens of thousands of sgRNAs, which is laborious and expensive. We have developed a simple method, RELATe (restriction/ligation coupled with Agrobacterium-mediated transformation), to generate sgRNA libraries from 10 μg of genomic DNA, targeting over 98% of the protein-coding genes in the human fungal pathogen Cryptococcus neoformans Functional screens identified 142 potential C. neoformans genes contributing to blood-brain barrier penetration. We selected two cryptococcal genes, SFP1 and WDR1, for a proof-of-concept demonstration that RELATe-identified genes are relevant to C. neoformans central nervous system infection. Our RELATe method can be used in many other fungal species and is powerful and cost-effective for genome-wide high-throughput screening for elucidating functional genomics.},
}
@article {pmid32948190,
year = {2020},
author = {Lavrov, AV and Varenikov, GG and Skoblov, MY},
title = {Genome scale analysis of pathogenic variants targetable for single base editing.},
journal = {BMC medical genomics},
volume = {13},
number = {Suppl 8},
pages = {80},
pmid = {32948190},
issn = {1755-8794},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Databases, Nucleic Acid ; Disease/genetics ; *Gene Editing ; Genome, Human ; Humans ; *Mutation ; },
abstract = {BACKGROUND: Single nucleotide variants account for approximately 90% of all known pathogenic variants responsible for human diseases. Recently discovered CRISPR/Cas9 base editors can correct individual nucleotides without cutting DNA and inducing double-stranded breaks. We aimed to find all possible pathogenic variants which can be efficiently targeted by any of the currently described base editors and to present them for further selection and development of targeted therapies.<br><br>METHODS: ClinVar database (GRCh37_clinvar_20171203) was used to search and select mutations available for current single-base editing systems. We included only pathogenic and likely pathogenic variants for further analysis. For every potentially editable mutation we checked the presence of PAM. If a PAM was found, we analyzed the sequence to find possibility to edit only one nucleotide without changing neighboring nucleotides. The code of the script to search Clinvar database and to analyze the sequences was written in R and is available in the appendix.<br><br>RESULTS: We analyzed 21 editing system currently reported in 9 publications. Every system has different working characteristics such as the editing window and PAM sequence. C&#x2009;>&#x2009;T base editors can precisely target 3196 mutations (46% of all pathogenic T&#x2009;>&#x2009;C variants), and A&#x2009;>&#x2009;G editors - 6900 mutations (34% of all pathogenic G&#x2009;>&#x2009;A variants).<br><br>CONCLUSIONS: Protein engineering helps to develop new enzymes with a narrower window of base editors as well as using new Cas9 enzymes with different PAM sequences. But, even now the list of mutations which can be targeted with currently available systems is huge enough to choose and develop new targeted therapies.},
}
@article {pmid32947885,
year = {2020},
author = {Song, S and Yan, R and Wang, C and Wang, J and Sun, H},
title = {Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.},
journal = {International journal of molecular sciences},
volume = {21},
number = {18},
pages = {},
pmid = {32947885},
issn = {1422-0067},
mesh = {ATP-Binding Cassette Transporters/genetics/*physiology ; Agrobacterium tumefaciens/genetics ; Base Sequence ; CRISPR-Cas Systems ; Culture Media/pharmacology ; DNA, Plant/genetics ; Gene Expression Regulation, Plant ; Genetic Vectors/genetics ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Indoleacetic Acids/metabolism ; Lilium/embryology/*genetics ; Mutation ; Plant Proteins/genetics/*physiology ; Plant Somatic Embryogenesis Techniques/*methods ; Plants, Genetically Modified ; RNA, Guide/genetics ; Sequence Alignment ; Sequence Homology, Nucleic Acid ; *Transformation, Genetic ; },
abstract = {Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7-13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.},
}
@article {pmid32947034,
year = {2020},
author = {Fan, Y and Lin, X},
title = {An intergenic "safe haven" region in Cryptococcus neoformans serotype D genomes.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {144},
number = {},
pages = {103464},
pmid = {32947034},
issn = {1096-0937},
support = {R01 AI140719/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cryptococcosis/*genetics/microbiology ; Cryptococcus neoformans/*genetics/pathogenicity ; DNA, Intergenic/*genetics ; Genome, Fungal/*genetics ; Humans ; Serogroup ; Virulence/*genetics ; },
abstract = {Cryptococcus neoformans is an opportunistic human fungal pathogen and serves as a model organism for studies of eukaryotic microbiology and microbial pathogenesis. C. neoformans species complex is classified into serotype A, serotype D, and AD hybrids, which are currently considered different subspecies. Different serotype strains display varied phenotypes, virulence, and gene regulation. Genetic investigation of important pathways is often performed in both serotype A and D reference strains in order to identify diversification or conservation of the interrogated signaling network. Many genetic tools have been developed for C. neoformans serotype A reference strain H99, including the gene free "safe haven" (SH) regions for DNA integration identified based on genomic features. However, no such a genomic safe haven region has been identified in serotype D strains. Here, capitalizing on the available genomic, transcriptomic, and chromatin data, we identified an intergenic region named as SH3 for the serotype D reference strains JEC21 and XL280. We also designed a sgRNA and a vector facilitating any alien gene integration into SH3 through a CRISPR-Cas9 system. We found that gene inserted in this region complemented the corresponding gene deletion mutant. Fluorescent reporter gene inserted in SH3 can also be expressed efficiently. Insertion in SH3 itself did not alter the expression of adjacent genes and did not affect the growth or mating of C. neoformans. Thus, SH3 provides a resource for genetic manipulations in serotype D strains and will facilitate comparative analyses of gene functions in this species complex. In addition, the incorporation of the multi-omic data in our selection of the safe haven region could help similar studies in other organisms.},
}
@article {pmid32946490,
year = {2020},
author = {Teixeira, LPR and Lopes, FEM and Antunes, ASLM and Alves, MS and Miranda, AM and Gaudencio Neto, S and Martins, LT and Moreira, ACOM and Tavares, KCS},
title = {Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0239435},
pmid = {32946490},
issn = {1932-6203},
mesh = {Animals ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Cost-Benefit Analysis ; DNA/*genetics/*isolation &amp; purification ; Fibroblasts/cytology/metabolism ; *Gene Editing ; Goats ; Transgenes/*genetics ; },
abstract = {The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.},
}
@article {pmid32946000,
year = {2020},
author = {Gratacap, RL and Jin, YH and Mantsopoulou, M and Houston, RD},
title = {Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {22},
number = {5},
pages = {717-724},
pmid = {32946000},
issn = {1436-2236},
support = {BB/S004343/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/20002172/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/30002275/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002070/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Electroporation/methods ; Gene Editing/*methods ; Oncorhynchus mykiss/genetics ; Ribonucleoproteins/chemistry/*genetics ; Salmo salar/genetics ; Salmon/genetics ; },
abstract = {Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.},
}
@article {pmid32945564,
year = {2020},
author = {Hoffmann, S and Izquierdo, HM and Gamba, R and Chardon, F and Dumont, M and Keizer, V and Hervé, S and McNulty, SM and Sullivan, BA and Manel, N and Fachinetti, D},
title = {A genetic memory initiates the epigenetic loop necessary to preserve centromere position.},
journal = {The EMBO journal},
volume = {39},
number = {20},
pages = {e105505},
pmid = {32945564},
issn = {1460-2075},
support = {R01 GM129263/GM/NIGMS NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; R21 CA238758/CA/NCI NIH HHS/United States ; R01 GM124041/GM/NIGMS NIH HHS/United States ; },
mesh = {CD4-Positive T-Lymphocytes/cytology/*metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Centromere/genetics/*metabolism ; Centromere Protein A/*metabolism ; Centromere Protein B/*metabolism ; Chromosomal Proteins, Non-Histone/*metabolism ; *Chromosome Segregation/genetics ; Computational Biology ; Epigenesis, Genetic ; Gene Targeting ; Humans ; In Situ Hybridization, Fluorescence ; Nucleosomes/*genetics/metabolism ; RNA, Small Interfering ; },
abstract = {Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-A[OFF/ON]). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4[+] T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.},
}
@article {pmid32944907,
year = {2021},
author = {El Marjou, F and Jouhanneau, C and Krndija, D},
title = {Targeted Transgenic Mice Using CRISPR /Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2214},
number = {},
pages = {125-141},
doi = {10.1007/978-1-0716-0958-3_9},
pmid = {32944907},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Electroporation/methods ; Female ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Mice ; Mice, Transgenic/*genetics ; RNA, Guide/genetics ; Zygote ; },
abstract = {CRISPR /Cas9 is a powerful technology that has transformed gene editing of mammalian genomes, being faster and more cost-effective than standard gene targeting techniques. In this chapter, we provide a step-by-step protocol to obtain Knock-Out (KO) or Knock-In (KI) mouse models using CRISPR /Cas9 technology. Detailed instructions for the design of single guide RNAs (sgRNA) for KO approaches and single-strand oligonucleotide (ssODN) matrix for generation of KI animals are included. We also describe two independent CRISPR /Cas9 delivery methods to produce gene-edited animals starting from zygote-stage embryos, based either on cytoplasmic injection or electroporation.},
}
@article {pmid32944905,
year = {2021},
author = {Carlini, V and Gretarsson, KH and Hackett, JA},
title = {Genome-Scale CRISPR Screening for Regulators of Cell Fate Transitions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2214},
number = {},
pages = {91-108},
doi = {10.1007/978-1-0716-0958-3_7},
pmid = {32944905},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Embryonic Germ Cells/*cytology/metabolism ; Gene Regulatory Networks ; HEK293 Cells ; Humans ; Mice ; Mouse Embryonic Stem Cells/*cytology/metabolism ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Knockout CRISPR screening enables the unbiased discovery of genes with a functional role in almost any cellular or molecular process of interest. The approach couples a genome-scale library of guide RNA (gRNA), the Cas9 endonuclease, and a faithful phenotypic read-out to systematically identify candidate genes via their loss-of-function effect. Here we provide a detailed description of the CRISPR screen protocol and outline how to apply it to decipher the gene networks that underlie developmental cell fate decisions. As a paradigm we use the in vitro model of cell state transition(s) from naive pluripotency to primordial germ cell (PGC) fate, exploiting the Stella-GFP:Esg1-tdTomato (SGET) mouse ESC line. The principles in this protocol can be readily adapted to characterize lineage regulators for other cell fate models and/or for other species.},
}
@article {pmid32944406,
year = {2020},
author = {Wu, Y and Zhou, L and Wang, Z and Wang, X and Zhang, R and Zheng, L and Kang, T},
title = {Systematic screening for potential therapeutic targets in osteosarcoma through a kinome-wide CRISPR-Cas9 library.},
journal = {Cancer biology &amp; medicine},
volume = {17},
number = {3},
pages = {782-794},
pmid = {32944406},
issn = {2095-3941},
mesh = {Adaptor Proteins, Signal Transducing/genetics/metabolism ; Bone Neoplasms/genetics/*metabolism/pathology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Knockout Techniques ; Gene Library ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Membrane Proteins/genetics/metabolism ; Nuclear Proteins/genetics/metabolism ; Osteosarcoma/genetics/*metabolism/pathology ; Protein Serine-Threonine Kinases/genetics/metabolism ; Protein-Tyrosine Kinases/genetics/metabolism ; },
abstract = {Objective: Osteosarcoma is the most common primary malignant bone tumor. However, the survival of patients with osteosarcoma has remained unchanged during the past 30 years, owing to a lack of efficient therapeutic targets. Methods: We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma. The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) Python package. The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout. The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2: Genomics Analysis and Visualization Platform. Results: We identified 53 potential kinase targets in osteosarcoma. Among these targets, we analyzed 3 kinases, TRRAP, PKMYT1, and TP53RK, to validate their oncogenic functions in osteosarcoma. PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue, whereas TRRAP showed no significant difference. High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma. Conclusions: Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library, including target design, library construction, screening workflow, data analysis, and functional validation. This method may also be useful in potentially accelerating drug discovery for other cancer types.},
}
@article {pmid32943441,
year = {2020},
author = {Bowen, C and Shibata, M and Zhang, H and Bergren, SK and Shen, MM and Gelmann, EP},
title = {CRISPR/Cas9-Mediated Point Mutation in Nkx3.1 Prolongs Protein Half-Life and Reverses Effects Nkx3.1 Allelic Loss.},
journal = {Cancer research},
volume = {80},
number = {21},
pages = {4805-4814},
pmid = {32943441},
issn = {1538-7445},
support = {K99 CA194287/CA/NCI NIH HHS/United States ; P01 CA154293/CA/NCI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; P30 CA023074/CA/NCI NIH HHS/United States ; R01 CA238005/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Homeodomain Proteins/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Point Mutation ; Prostatic Neoplasms/*genetics/pathology ; Transcription Factors/*genetics ; },
abstract = {NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1[S186A/-], Nkx3.1[+/-] , and Nkx3.1[+/+] mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1[S186A/-] mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1[+/-] mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss.},
}
@article {pmid32941662,
year = {2021},
author = {Zhou Li, Y and Boisnard, S and Enache-Angoulvant, A and Fairhead, C},
title = {Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.},
journal = {Yeast (Chichester, England)},
volume = {38},
number = {1},
pages = {57-71},
doi = {10.1002/yea.3522},
pmid = {32941662},
issn = {1097-0061},
mesh = {CRISPR-Cas Systems ; DNA, Fungal/*genetics ; *Gene Editing ; *Genes, Mating Type, Fungal ; *Genome, Fungal ; *Meiosis ; Phylogeny ; Reproduction/genetics ; Saccharomycetales/*genetics/*physiology ; },
abstract = {The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.},
}
@article {pmid32941652,
year = {2020},
author = {Kim, DY and Moon, SB and Ko, JH and Kim, YS and Kim, D},
title = {Unbiased investigation of specificities of prime editing systems in human cells.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10576-10589},
pmid = {32941652},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *DNA Breaks, Single-Stranded ; Gene Editing/*methods ; Genome, Human/genetics ; Humans ; Whole Genome Sequencing ; },
abstract = {Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.},
}
@article {pmid32941625,
year = {2020},
author = {Nishimura, K and Yamada, R and Hagihara, S and Iwasaki, R and Uchida, N and Kamura, T and Takahashi, K and Torii, KU and Fukagawa, T},
title = {A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {e108},
pmid = {32941625},
issn = {1362-4962},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Chickens ; Gene Knockout Techniques/*methods ; Humans ; Indoleacetic Acids/*chemistry ; Oryza/metabolism ; Plant Proteins/*genetics ; *Proteolysis ; },
abstract = {The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.},
}
@article {pmid32941607,
year = {2020},
author = {Kot, W and Olsen, NS and Nielsen, TK and Hutinet, G and de Crécy-Lagard, V and Cui, L and Dedon, PC and Carstens, AB and Moineau, S and Swairjo, MA and Hansen, LH},
title = {Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10383-10396},
pmid = {32941607},
issn = {1362-4962},
support = {R01 GM110588/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/drug effects/*genetics ; Base Sequence/drug effects ; CRISPR-Cas Systems/genetics ; DNA/drug effects/*genetics ; DNA Restriction-Modification Enzymes/drug effects ; Escherichia coli/virology ; Gene Editing ; Guanine/analogs &amp; derivatives/pharmacology ; Humans ; Nanopore Sequencing ; Nucleotide Motifs/drug effects/*genetics ; Pyrimidinones/*pharmacology ; Pyrroles/*pharmacology ; Siphoviridae/genetics ; },
abstract = {In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.},
}
@article {pmid32940375,
year = {2021},
author = {Niklasson, CU and Fredlund, E and Monni, E and Lindvall, JM and Kokaia, Z and Hammarlund, EU and Bronner, ME and Mohlin, S},
title = {Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {250},
number = {2},
pages = {191-236},
pmid = {32940375},
issn = {1097-0177},
support = {R01 DE027568/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/*physiology ; CDX2 Transcription Factor/metabolism ; CRISPR-Cas Systems ; Chick Embryo ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Developmental ; Hepatocyte Nuclear Factor 1-beta/metabolism ; Humans ; Neural Crest/*embryology/metabolism ; SOX9 Transcription Factor/metabolism ; },
abstract = {BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development.<br><br>RESULTS: Here, we show that HIF-2&#x3b1; is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2&#x3b1; in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2&#x3b1; reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest.<br><br>CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2&#x3b1; must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.},
}
@article {pmid32939873,
year = {2020},
author = {Hong, S and Hu, P and Jang, JH and Carrington, B and Sood, R and Berger, SI and Roessler, E and Muenke, M},
title = {Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.},
journal = {Human mutation},
volume = {41},
number = {12},
pages = {2155-2166},
doi = {10.1002/humu.24119},
pmid = {32939873},
issn = {1098-1004},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Family ; Genetic Association Studies ; *Genetic Variation ; Guidelines as Topic ; Hedgehog Proteins/*genetics ; Holoprosencephaly/*genetics ; Humans ; Loss of Function Mutation/genetics ; Mutation/genetics ; Phenotype ; RNA, Messenger/genetics/metabolism ; Societies, Scientific ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {Genetic variation in the highly conserved Sonic Hedgehog (SHH) gene is one of the most common genetic causes for the malformations of the brain and face in humans described as the holoprosencephaly clinical spectrum. However, only a minor fraction of known SHH variants have been experimentally proven to lead to abnormal function. Employing a phenotypic rescue assay with synthetic human messenger RNA variant constructs in shha[-/-] knockout zebrafish, we evaluated 104 clinically reported in-frame and missense SHH variants. Our data helped us to classify them into loss of function variants (31), hypomorphic variants (33), and nonpathogenic variants (40). We discuss the strengths and weaknesses of currently accepted predictors of variant deleteriousness and the American College of Medical Genetics and Genomics guidelines for variant interpretation in the context of this functional model; furthermore, we demonstrate the robustness of model systems such as zebrafish as a rapid method to resolve variants of uncertain significance.},
}
@article {pmid32939188,
year = {2020},
author = {Jolany Vangah, S and Katalani, C and Booneh, HA and Hajizade, A and Sijercic, A and Ahmadian, G},
title = {CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.},
journal = {Biological procedures online},
volume = {22},
number = {},
pages = {22},
pmid = {32939188},
issn = {1480-9222},
abstract = {Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR's remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR-Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed.},
}
@article {pmid32939090,
year = {2020},
author = {Zou, Y and Henry, WS and Ricq, EL and Graham, ET and Phadnis, VV and Maretich, P and Paradkar, S and Boehnke, N and Deik, AA and Reinhardt, F and Eaton, JK and Ferguson, B and Wang, W and Fairman, J and Keys, HR and Dančík, V and Clish, CB and Clemons, PA and Hammond, PT and Boyer, LA and Weinberg, RA and Schreiber, SL},
title = {Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.},
journal = {Nature},
volume = {585},
number = {7826},
pages = {603-608},
pmid = {32939090},
issn = {1476-4687},
support = {P30 DK040561/DK/NIDDK NIH HHS/United States ; P01 CA080111/CA/NCI NIH HHS/United States ; K99 CA248610/CA/NCI NIH HHS/United States ; R35 CA220487/CA/NCI NIH HHS/United States ; U01 CA217848/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Ethers/chemistry/*metabolism ; Female ; *Ferroptosis ; Gene Editing ; Humans ; Kidney Neoplasms/metabolism/pathology ; Lipid Peroxidation ; Male ; Mice ; Myocytes, Cardiac/cytology/metabolism ; Neurons/cytology/metabolism ; Ovarian Neoplasms/metabolism/pathology ; Peroxisomes/genetics/*metabolism ; Phospholipids/*chemistry/*metabolism ; },
abstract = {Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers[1]. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions[2-5]. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.},
}
@article {pmid32938767,
year = {2020},
author = {Liu, Y and Dai, L and Dong, J and Chen, C and Zhu, J and Rao, VB and Tao, P},
title = {Covalent Modifications of the Bacteriophage Genome Confer a Degree of Resistance to Bacterial CRISPR Systems.},
journal = {Journal of virology},
volume = {94},
number = {23},
pages = {},
pmid = {32938767},
issn = {1098-5514},
support = {R56 AI081726/AI/NIAID NIH HHS/United States ; R01 AI064389/AI/NIAID NIH HHS/United States ; U01 AI056443/AI/NIAID NIH HHS/United States ; R01 AI111538/AI/NIAID NIH HHS/United States ; U01 AI082086/AI/NIAID NIH HHS/United States ; R01 AI081726/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteria ; Bacterial Proteins/genetics ; Bacteriophage T4/genetics ; Bacteriophages/*genetics ; Base Sequence ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine ; Endodeoxyribonucleases/genetics ; Escherichia coli/genetics ; Sequence Analysis, DNA ; },
abstract = {The interplay between defense and counterdefense systems of bacteria and bacteriophages has been driving the evolution of both organisms, leading to their great genetic diversity. Restriction-modification systems are well-studied defense mechanisms of bacteria, while phages have evolved covalent modifications as a counterdefense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant, which contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR escape mutations, allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential countermechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counterdefense mechanisms exhibited by bacteria and phages, while affording protection against one another, also provide evolutionary benefits for both.IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well accepted that T4 and other phages have evolved counterdefense mechanisms to protect their genomes from R-M cleavage by covalent modifications, such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRISPR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and it is also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counterdefense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity.},
}
@article {pmid32938753,
year = {2020},
author = {Wang, J and Nan, N and Li, N and Liu, Y and Wang, TJ and Hwang, I and Liu, B and Xu, ZY},
title = {A DNA Methylation Reader-Chaperone Regulator-Transcription Factor Complex Activates OsHKT1;5 Expression during Salinity Stress.},
journal = {The Plant cell},
volume = {32},
number = {11},
pages = {3535-3558},
pmid = {32938753},
issn = {1532-298X},
mesh = {CRISPR-Cas Systems ; *DNA Methylation ; DNA Transposable Elements ; DNA, Plant/metabolism ; Gene Expression Regulation, Plant ; Molecular Chaperones/genetics/metabolism ; Multiprotein Complexes/genetics/metabolism ; Oryza/genetics/*physiology ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Salt Stress/*genetics ; Transcription Factors/genetics/metabolism ; },
abstract = {Irrigated lands are increasingly salinized, which adversely affects agricultural productivity. To respond to high sodium (Na[+]) concentrations, plants harbor multiple Na[+] transport systems. Rice (Oryza sativa) HIGH-AFFINITY POTASSIUM (K[+]) TRANSPORTER1;5 (OsHKT1;5), a Na[+]-selective transporter, maintains K[+]/Na[+] homeostasis under salt stress. However, the mechanism regulating OsHKT1;5 expression remains unknown. Here, we present evidence that a protein complex consisting of rice BCL-2-ASSOCIATED ATHANOGENE4 (OsBAG4), OsMYB106, and OsSUVH7 regulates OsHKT1;5 expression in response to salt stress. We isolated a salt stress-sensitive mutant, osbag4-1, that showed significantly reduced OsHKT1;5 expression and reduced K[+] and elevated Na[+] levels in shoots. Using comparative interactomics, we isolated two OsBAG4-interacting proteins, OsMYB106 (a MYB transcription factor) and OsSUVH7 (a DNA methylation reader), that were crucial for OsHKT1;5 expression. OsMYB106 and OsSUVH7 bound to the MYB binding cis-element (MYBE) and the miniature inverted-repeat transposable element (MITE) upstream of the MYBE, respectively, in the OsHKT1;5 promoter. OsBAG4 functioned as a bridge between OsSUVH7 and OsMYB106 to facilitate OsMYB106 binding to the consensus MYBE in the OsHKT1;5 promoter, thereby activating the OsHKT1;5 expression. Elimination of the MITE or knockout of OsMYB106 or OsSUVH7 decreased OsHKT1;5 expression and increased salt sensitivity. Our findings reveal a transcriptional complex, consisting of a DNA methylation reader, a chaperone regulator, and a transcription factor, that collaboratively regulate OsHKT1;5 expression during salinity stress.},
}
@article {pmid32938718,
year = {2020},
author = {Szulc, B and Sosicka, P and Maszczak-Seneczko, D and Skurska, E and Shauchuk, A and Olczak, T and Freeze, HH and Olczak, M},
title = {Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {48},
pages = {16445-16463},
pmid = {32938718},
issn = {1083-351X},
support = {R01 DK099551/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; Cricetulus ; Gene Knockdown Techniques ; Glycosyltransferases/genetics/*metabolism ; Golgi Apparatus/genetics/*metabolism ; HEK293 Cells ; Hep G2 Cells ; Humans ; Membrane Transport Proteins/genetics/metabolism ; Monosaccharide Transport Proteins/genetics/metabolism ; Nucleotide Transport Proteins/genetics/*metabolism ; Polysaccharides/*biosynthesis/genetics ; },
abstract = {Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.},
}
@article {pmid32937678,
year = {2020},
author = {Khodadad, N and Fani, M and Jamehdor, S and Nahidsamiei, R and Makvandi, M and Kaboli, S and Teimoori, A and Thekkiniath, J},
title = {A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors.},
journal = {Folia histochemica et cytobiologica},
volume = {58},
number = {3},
pages = {174-181},
doi = {10.5603/FHC.a2020.0020},
pmid = {32937678},
issn = {1897-5631},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Deoxyribonuclease BamHI/genetics ; Deoxyribonucleases, Type II Site-Specific/genetics ; Gene Knockdown Techniques ; *Genes, Viral ; *Genetic Vectors ; Herpesvirus 1, Human/*genetics ; RNA, Guide ; Software ; Viral Envelope Proteins/*genetics ; },
abstract = {INTRODUCTION: Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes.<br><br>MATERIALS AND METHODS: To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes.<br><br>RESULTS: CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR.<br><br>CONCLUSION: The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.},
}
@article {pmid32937141,
year = {2020},
author = {Nagahama, K and Sakoori, K and Watanabe, T and Kishi, Y and Kawaji, K and Koebis, M and Nakao, K and Gotoh, Y and Aiba, A and Uesaka, N and Kano, M},
title = {Setd1a Insufficiency in Mice Attenuates Excitatory Synaptic Function and Recapitulates Schizophrenia-Related Behavioral Abnormalities.},
journal = {Cell reports},
volume = {32},
number = {11},
pages = {108126},
doi = {10.1016/j.celrep.2020.108126},
pmid = {32937141},
issn = {2211-1247},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; *Behavior, Animal ; CRISPR-Cas Systems/genetics ; Excitatory Postsynaptic Potentials/physiology ; Female ; Gene Deletion ; Gene Expression Regulation ; Glutamic Acid/metabolism ; Histone-Lysine N-Methyltransferase/chemistry/*deficiency/genetics/metabolism ; Humans ; Male ; Mice, Inbred ICR ; Mutation/genetics ; Neurodevelopmental Disorders/genetics ; Prefrontal Cortex/metabolism ; Presynaptic Terminals/physiology ; Pyramidal Cells/metabolism ; Schizophrenia/genetics/*physiopathology ; Social Behavior ; Synapses/*physiology ; },
abstract = {SETD1A encodes a histone methyltransferase whose de novo mutations are identified in schizophrenia (SCZ) patients and confer a large increase in disease risk. Here, we generate Setd1a mutant mice carrying the frameshift mutation that closely mimics a loss-of-function variant of SCZ. Our Setd1a (+/-) mice display various behavioral abnormalities relevant to features of SCZ, impaired excitatory synaptic transmission in layer 2/3 (L2/3) pyramidal neurons of the medial prefrontal cortex (mPFC), and altered expression of diverse genes related to neurodevelopmental disorders and synaptic functions in the mPFC. RNAi-mediated Setd1a knockdown (KD) specifically in L2/3 pyramidal neurons of the mPFC only recapitulates impaired sociality among multiple behavioral abnormalities of Setd1a (+/-) mice. Optogenetics-assisted selective stimulation of presynaptic neurons combined with Setd1a KD reveals that Setd1a at postsynaptic site is essential for excitatory synaptic transmission. Our findings suggest that reduced SETD1A may attenuate excitatory synaptic function and contribute to the pathophysiology of SCZ.},
}
@article {pmid32937129,
year = {2020},
author = {Zhao, R and Yang, Y and Zheng, F and Zeng, Z and Feng, W and Jin, X and Wang, J and Yang, K and Liang, YX and She, Q and Han, W},
title = {A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.},
journal = {Cell reports},
volume = {32},
number = {11},
pages = {108133},
doi = {10.1016/j.celrep.2020.108133},
pmid = {32937129},
issn = {2211-1247},
mesh = {Adenine Nucleotides/metabolism ; Archaeal Proteins/isolation &amp; purification/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Membrane/*enzymology ; Endonucleases/isolation &amp; purification/*metabolism ; Metals/metabolism ; Models, Biological ; Oligoribonucleotides/metabolism ; *Second Messenger Systems ; Sulfolobus/*enzymology ; },
abstract = {Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.},
}
@article {pmid32937062,
year = {2020},
author = {Joung, J and Ladha, A and Saito, M and Kim, NG and Woolley, AE and Segel, M and Barretto, RPJ and Ranu, A and Macrae, RK and Faure, G and Ioannidi, EI and Krajeski, RN and Bruneau, R and Huang, MW and Yu, XG and Li, JZ and Walker, BD and Hung, DT and Greninger, AL and Jerome, KR and Gootenberg, JS and Abudayyeh, OO and Zhang, F},
title = {Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing.},
journal = {The New England journal of medicine},
volume = {383},
number = {15},
pages = {1492-1494},
pmid = {32937062},
issn = {1533-4406},
support = {1DP1-HL141201/NH/NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; 1R01- MH110049/NH/NIH HHS/United States ; R21 AI149694/AI/NIAID NIH HHS/United States ; },
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; COVID-19 ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/*diagnosis ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis ; RNA, Viral/*analysis ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2 ; Sensitivity and Specificity ; },
}
@article {pmid32936799,
year = {2020},
author = {Fiaux, PC and Chen, HV and Chen, PB and Chen, AR and McVicker, G},
title = {Discovering functional sequences with RELICS, an analysis method for CRISPR screens.},
journal = {PLoS computational biology},
volume = {16},
number = {9},
pages = {e1008194},
pmid = {32936799},
issn = {1553-7358},
support = {P30 CA014195/CA/NCI NIH HHS/United States ; R01 DK122607/DK/NIDDK NIH HHS/United States ; R01 AI107027/AI/NIAID NIH HHS/United States ; },
mesh = {Bayes Theorem ; CRISPR-Cas Systems/*genetics ; Genomics/*methods ; Humans ; Jurkat Cells ; RNA, Guide/genetics ; Sequence Analysis, DNA/*methods ; *Software ; },
abstract = {CRISPR screens are a powerful technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By targeting non-coding sequences for perturbation, CRISPR screens have the potential to systematically discover novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from CRISPR screens. RELICS specifically addresses many of the challenges of non-coding CRISPR screens such as the unknown locations of functional sequences, overdispersion in the observed single guide RNA counts, and the need to combine information across multiple pools in an experiment. RELICS outperforms existing methods with higher precision, higher recall, and finer-resolution predictions on simulated datasets. We apply RELICS to published CRISPR interference and CRISPR activation screens to predict and experimentally validate novel regulatory sequences that are missed by other analysis methods. In summary, RELICS is a powerful new analysis method for CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.},
}
@article {pmid32936726,
year = {2020},
author = {Wenjing, W and Chen, Q and Singh, PK and Huang, Y and Pei, D},
title = {CRISPR/Cas9 edited HSFA6a and HSFA6b of Arabidopsis thaliana offers ABA and osmotic stress insensitivity by modulation of ROS homeostasis.},
journal = {Plant signaling &amp; behavior},
volume = {15},
number = {12},
pages = {1816321},
pmid = {32936726},
issn = {1559-2324},
mesh = {Abscisic Acid/*metabolism ; Arabidopsis/*physiology ; Arabidopsis Proteins/*genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation, Plant ; Genetic Vectors/metabolism ; Germination ; Heat Shock Transcription Factors/*genetics/metabolism ; Heat-Shock Proteins/*genetics/metabolism ; *Homeostasis ; Mutation/genetics ; *Osmotic Pressure ; Phenotype ; Plant Stomata/physiology ; Plants, Genetically Modified ; Reactive Oxygen Species/*metabolism ; Reproducibility of Results ; Seeds/growth &amp; development ; Stress, Physiological/genetics ; Transcription Factors/*genetics/metabolism ; Transcription, Genetic ; },
abstract = {The role of Heat Shock Transcription Factor 6 (HSFA6a &amp; HSFA6b) in response to abiotic stresses such as ABA, drought, salinity, drought, and osmotic stress is individually well established. Unfortunately, the functional redundancy between the HSFA6a and HSFA6b as well as the consequences of simultaneous editing of both in response to aforementioned stresses remains elusive. Therefore, this study was designed with the aim of addressing whether there is any functional redundancy between HSFA6a and HSFA6b as well as to decipher their role in abiotic stresses tolerance in Arabidopsis thaliana, by using the CRISPR-Cas9. We have generated the single (hsfa6a and hsfa6b) as well as double mutants (hsfa6a/hsfa6b-1 and hsfa6a/hsfa6b-2) of HSFA6a and HSFA6b with higher frequencies of deletion, insertion, and substitution. The phenotypic characterization of generated double and single mutants under abiotic stresses such as ABA, mannitol, and NaCl identified double mutants more tolerant to subjected abiotic stresses than those of their single mutants. It warrants mentioning that we have identified that HSFA6a and HSFA6b also involved in other major ABA responses, including ABA-inhibited seed germination, stomatal movement, and water loss. In addition to the above, the simultaneous editing of HSFA6a and HSFA6b lead to a reduced ROS accumulation, accompanied by increased expression of much abiotic stress and ABA-responsive genes, including involved in regulation of ROS level. In conclusion, these results suggest that HSFA6a and HSFA6b may offer abiotic stress tolerance by regulating the ROS homeostasis in plants.},
}
@article {pmid32935194,
year = {2021},
author = {Skeens, E and East, KW and Lisi, GP},
title = {[1]H, [13]C, [15] N backbone resonance assignment of the recognition lobe subdomain 3 (Rec3) from Streptococcus pyogenes CRISPR-Cas9.},
journal = {Biomolecular NMR assignments},
volume = {15},
number = {1},
pages = {25-28},
pmid = {32935194},
issn = {1874-270X},
support = {P20 GM109035/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Nuclear Magnetic Resonance, Biomolecular ; Streptococcus pyogenes ; },
abstract = {Rec3 is a subdomain of the recognition (Rec) lobe within CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein Cas9 that is involved in nucleic acid binding and is critical to HNH endonuclease activation. Here, we report the backbone resonance assignments of an engineered construct of the Rec3 subdomain from Streptococcus pyogenes Cas9. We also analyze backbone chemical shift data to predict secondary structure and an overall fold that is consistent with that of Rec3 from the full-length S. pyogenes Cas9 protein.},
}
@article {pmid32933033,
year = {2020},
author = {Gutierrez-Guerrero, A and Cosset, FL and Verhoeyen, E},
title = {Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy.},
journal = {Viruses},
volume = {12},
number = {9},
pages = {},
pmid = {32933033},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; Gene Transfer Techniques ; *Genetic Therapy ; *Genetic Vectors ; Hematopoietic Stem Cells/metabolism ; Humans ; Killer Cells, Natural ; Lentivirus/*genetics ; Measles virus/genetics ; Membrane Glycoproteins/metabolism ; Nipah Virus ; Research ; T-Lymphocytes/metabolism ; Vesicular stomatitis Indiana virus/genetics ; Viral Envelope Proteins/genetics ; },
abstract = {Viruses have been repurposed into tools for gene delivery by transforming them into viral vectors. The most frequently used vectors are lentiviral vectors (LVs), derived from the human immune deficiency virus allowing efficient gene transfer in mammalian cells. They represent one of the safest and most efficient treatments for monogenic diseases affecting the hematopoietic system. LVs are modified with different viral envelopes (pseudotyping) to alter and improve their tropism for different primary cell types. The vesicular stomatitis virus glycoprotein (VSV-G) is commonly used for pseudotyping as it enhances gene transfer into multiple hematopoietic cell types. However, VSV-G pseudotyped LVs are not able to confer efficient transduction in quiescent blood cells, such as hematopoietic stem cells (HSC), B and T cells. To solve this problem, VSV-G can be exchanged for other heterologous viral envelopes glycoproteins, such as those from the Measles virus, Baboon endogenous retrovirus, Cocal virus, Nipah virus or Sendai virus. Here, we provide an overview of how these LV pseudotypes improved transduction efficiency of HSC, B, T and natural killer (NK) cells, underlined by multiple in vitro and in vivo studies demonstrating how pseudotyped LVs deliver therapeutic genes or gene editing tools to treat different genetic diseases and efficiently generate CAR T cells for cancer treatment.},
}
@article {pmid32930628,
year = {2020},
author = {Gholizadeh, P and Aghazadeh, M and Ghotaslou, R and Ahangarzadeh Rezaee, M and Pirzadeh, T and Köse, &#x15e; and Ganbarov, K and Yousefi, M and Kafil, HS},
title = {CRISPR-cas system in the acquisition of virulence genes in dental-root canal and hospital-acquired isolates of Enterococcus faecalis.},
journal = {Virulence},
volume = {11},
number = {1},
pages = {1257-1267},
pmid = {32930628},
issn = {2150-5608},
mesh = {Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; Child ; Child, Preschool ; Cross Infection/*microbiology ; Dental Pulp Cavity/*microbiology ; Enterococcus faecalis/*genetics/*pathogenicity ; Female ; Genotype ; Hemolysis ; Humans ; Infant ; Male ; Middle Aged ; Phenotype ; Virulence/genetics ; Virulence Factors/*genetics ; Young Adult ; },
abstract = {Enterococcus faecalis is one of the important causative agents of nosocomial and life-threatening infections in human. Several studies have demonstrated that the presence of CRISPR-cas is associated with antibiotic susceptibility and lack of virulence traits. In this study, we aimed to assess the phenotypic and genotypic virulence determinants in relation to CRISPR elements from the dental-root canals and hospital-acquired isolates of E. faecalis. Eighty-eight hospital-acquired and 73 dental-root canal isolates of E. faecalis were assessed in this study. Phenotypic screening of the isolates included biofilm formation, and gelatinase and hemolysis activities. Genotypical screening using PCR was further used to evaluate the presence of CRISPR elements and different virulence-associated genes such as efaA, esp, cylA, hyl, gelE, ace, ebpR, and asa1. Biofilm formation, gelatinase, and hemolysis activities were detected in 93.8%, 29.2%, and 19.2% of the isolates, respectively. The most prevalent virulence-associated gene was ace, which was followed by efaA, whereas cylA was the least identified. The presence of CRISPR1-cas, orphan CRISPR2, and CRISPR3-cas was determined in 13%, 55.3%, and 17.4% of the isolates, respectively. CRISPR elements were significantly more prevalent in the dental-root canal isolates. An inverse significant correlation was found between CRISPR-cas loci, esp, and gelE, while direct correlations were observed in the case of cylA, hyl, gelE (among CRISPR-loci 1 and 3), asa1, ace, biofilm formation, and hemolysis activity. Findings, therefore, indicate that CRISPR-cas might prevent the acquisition of some respective pathogenicity factors in some isolates, though not all; so selective forces could not influence pathogenic traits. Abbreviations: BHI: brain-heart infusion agar; CRISPRs: Clustered regularly interspaced short palindromic repeats; Esp: Cell wall-associated protein; ENT: ear-nose-throat; ICU: intensive care units; OD: optical densities; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; UTI: urinary tract infection.},
}
@article {pmid32930578,
year = {2020},
author = {Zou, D and Maina, SW and Zhang, F and Yan, Z and Ding, L and Shao, Y and Xin, Z},
title = {Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {41},
pages = {11358-11367},
doi = {10.1021/acs.jafc.0c03694},
pmid = {32930578},
issn = {1520-5118},
mesh = {Bacillus subtilis/chemistry/*genetics/*metabolism ; *CRISPR-Cas Systems ; Chromatography, High Pressure Liquid ; Fatty Acids/*biosynthesis/chemistry/pharmacology ; Fungi/drug effects/growth &amp; development ; Gas Chromatography-Mass Spectrometry ; Genome, Bacterial ; Multigene Family ; Oligopeptides/*biosynthesis/chemistry/pharmacology ; Operon ; Peptides, Cyclic/*biosynthesis/chemistry/pharmacology ; },
abstract = {CRISPR/Cas9 is one of the robust and effective gene manipulation tools which has been widely applied in various organisms. In this study, the plipastatin gene cluster was successfully expressed in genome-modified Bacillus subtilis 1A751 by disrupting the surfactin operon (srf) through CRISPR/Cas9 technology. The presumed plipastatin biosynthetic pathway was proposed based on the analysis of its biosynthetic gene cluster. Two new plipastatins were identified by a combination of ultra-high performance liquid chromatography-coupled electron spray ionization-tandem mass spectrometry and gas chromatography-mass spectrometry analyses, together with nine known plipastatins or their derivatives. The yield of plipastatin was as high as 1600 mg/L which is the highest reported to date. Antimicrobial experiments revealed that its methanolic extracts exhibited powerful inhibitory effects on the growth of the tested pathogens and fungi. The results from this investigation highlight the remarkable utility of CRISPR/Cas9 in mining new plipastatins and increasing the total plipastatin yield, providing a new pipeline for the industrial application of plipastatin.},
}
@article {pmid32929347,
year = {2020},
author = {Xing, S and Lu, Z and Huang, Q and Li, H and Wang, Y and Lai, Y and He, Y and Deng, M and Liu, W},
title = {An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification.},
journal = {Theranostics},
volume = {10},
number = {22},
pages = {10262-10273},
pmid = {32929347},
issn = {1838-7640},
mesh = {Aptamers, Nucleotide/*genetics ; Biomarkers, Tumor/genetics ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Extracellular Vesicles/genetics ; Humans ; Membrane Proteins/*genetics ; Nucleic Acid Hybridization/*genetics ; },
abstract = {Tumor-derived extracellular vesicle (TEV) protein biomarkers facilitate cancer diagnosis and prognostic evaluations. However, the lack of reliable and convenient quantitative methods for evaluating TEV proteins prevents their clinical application. Methods: Here, based on dual amplification of hybridization chain reaction (HCR) and CRISPR-Cas12a, we developed the apta-HCR-CRISPR assay for direct high-sensitivity detection of TEV proteins. The TEV protein-targeted aptamer was amplified by HCR to produce a long-repeated sequence comprising multiple CRISPR RNA (crRNA) targetable barcodes, and the signals were further amplified by CRISPR-Cas12a collateral cleavage activities, resulting in a fluorescence signal. Results: The established strategy was verified by detecting the TEV protein markers nucleolin and programmed death ligand 1 (PD-L1). Both achieved limit of detection (LOD) values as low as 10[2] particles/µL, which is at least 10[4]-fold more sensitive than aptamer-ELISA and 10[2]-fold more sensitive than apta-HCR-ELISA. We directly applied our assay to a clinical analysis of circulating TEVs from 50 µL of serum, revealing potential applications of nucleolin[+] TEVs for nasopharyngeal carcinoma cancer (NPC) diagnosis and PD-L1[+] TEVs for therapeutic monitoring. Conclusion: The platform was simple and easy to operate, and this approach should be useful for the highly sensitive and versatile quantification of TEV proteins in clinical samples.},
}
@article {pmid32929188,
year = {2021},
author = {Batra, R and Nelles, DA and Roth, DM and Krach, F and Nutter, CA and Tadokoro, T and Thomas, JD and Sznajder, &#x141;J and Blue, SM and Gutierrez, HL and Liu, P and Aigner, S and Platoshyn, O and Miyanohara, A and Marsala, M and Swanson, MS and Yeo, GW},
title = {The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1.},
journal = {Nature biomedical engineering},
volume = {5},
number = {2},
pages = {157-168},
pmid = {32929188},
issn = {2157-846X},
support = {P50 NS048843/NS/NINDS NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; },
mesh = {Adenoviridae/physiology ; Animals ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Gene Editing/*methods ; Genetic Vectors/physiology ; Male ; Mice, Transgenic ; Muscle, Skeletal/metabolism ; Myotonic Dystrophy/genetics/*metabolism ; Phenotype ; RNA/*metabolism ; },
abstract = {Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia. The sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.},
}
@article {pmid32929070,
year = {2020},
author = {Li, Y and Weng, Y and Bai, D and Jia, Y and Liu, Y and Zhang, Y and Kou, X and Zhao, Y and Ruan, J and Chen, J and Yin, J and Wang, H and Teng, X and Wang, Z and Liu, W and Gao, S},
title = {Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4593},
pmid = {32929070},
issn = {2041-1723},
mesh = {*Alleles ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Nucleus/*genetics ; Disease Models, Animal ; Dwarfism/genetics ; Embryo Loss/genetics ; Female ; *Gene Editing ; Gene Targeting ; Genes, Dominant ; Genomic Imprinting ; Heterozygote ; Mice, Inbred C57BL ; Mice, Knockout ; *Mitochondrial Replacement Therapy ; Mutation ; Reproducibility of Results ; Time Factors ; },
abstract = {Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.},
}
@article {pmid32929034,
year = {2020},
author = {Champer, J and Yang, E and Lee, E and Liu, J and Clark, AG and Messer, PW},
title = {A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {39},
pages = {24377-24383},
pmid = {32929034},
issn = {1091-6490},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 ES029929/ES/NIEHS NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; Drosophila melanogaster/*genetics/physiology ; Female ; Gene Editing ; Germ Cells/cytology ; Male ; Models, Genetic ; Pedigree ; RNA, Guide/genetics ; },
abstract = {Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.},
}
@article {pmid32928875,
year = {2020},
author = {Brinkley, G and Nam, H and Shim, E and Kirkman, R and Kundu, A and Karki, S and Heidarian, Y and Tennessen, JM and Liu, J and Locasale, JW and Guo, T and Wei, S and Gordetsky, J and Johnson-Pais, TL and Absher, D and Rakheja, D and Challa, AK and Sudarshan, S},
title = {Teleological role of L-2-hydroxyglutarate dehydrogenase in the kidney.},
journal = {Disease models &amp; mechanisms},
volume = {13},
number = {11},
pages = {},
pmid = {32928875},
issn = {1754-8411},
support = {R35 GM119557/GM/NIGMS NIH HHS/United States ; P30 CA013148/CA/NCI NIH HHS/United States ; F30 CA232397/CA/NCI NIH HHS/United States ; P30 CA054174/CA/NCI NIH HHS/United States ; T32 GM008361/GM/NIGMS NIH HHS/United States ; R01 CA200653/CA/NCI NIH HHS/United States ; I01 BX002930/BX/BLRD VA/United States ; },
mesh = {Alcohol Oxidoreductases/genetics/*metabolism ; Animals ; Brain/enzymology/pathology ; CRISPR-Cas Systems/genetics ; Carcinoma, Renal Cell/genetics/pathology ; Citric Acid Cycle ; Fertility ; Gene Expression Regulation, Neoplastic ; Glutarates/metabolism ; Heterozygote ; Kidney/*enzymology/pathology ; Kidney Neoplasms/genetics/pathology ; Male ; Metabolic Flux Analysis ; Metabolome ; Metabolomics ; Mice, Knockout ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism ; Succinic Acid/metabolism ; },
abstract = {L-2-hydroxyglutarate (L-2HG) is an oncometabolite found elevated in renal tumors. However, this molecule might have physiological roles that extend beyond its association with cancer, as L-2HG levels are elevated in response to hypoxia and during Drosophila larval development. L-2HG is known to be metabolized by L-2HG dehydrogenase (L2HGDH), and loss of L2HGDH leads to elevated L-2HG levels. Despite L2HGDH being highly expressed in the kidney, its role in renal metabolism has not been explored. Here, we report our findings utilizing a novel CRISPR/Cas9 murine knockout model, with a specific focus on the role of L2HGDH in the kidney. Histologically, L2hgdh knockout kidneys have no demonstrable histologic abnormalities. However, GC-MS metabolomics demonstrates significantly reduced levels of the TCA cycle intermediate succinate in multiple tissues. Isotope labeling studies with [U-[13]C] glucose demonstrate that restoration of L2HGDH in renal cancer cells (which lowers L-2HG) leads to enhanced incorporation of label into TCA cycle intermediates. Subsequent biochemical studies demonstrate that L-2HG can inhibit the TCA cycle enzyme α-ketoglutarate dehydrogenase. Bioinformatic analysis of mRNA expression data from renal tumors demonstrates that L2HGDH is co-expressed with genes encoding TCA cycle enzymes as well as the gene encoding the transcription factor PGC-1α, which is known to regulate mitochondrial metabolism. Restoration of PGC-1α in renal tumor cells results in increased L2HGDH expression with a concomitant reduction in L-2HG levels. Collectively, our analyses provide new insight into the physiological role of L2HGDH as well as mechanisms that promote L-2HG accumulation in disease states.},
}
@article {pmid32928112,
year = {2020},
author = {Wang, X and He, S and Zhao, N and Liu, X and Cao, Y and Zhang, G and Wang, G and Guo, C},
title = {Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus.},
journal = {BMC microbiology},
volume = {20},
number = {1},
pages = {282},
pmid = {32928112},
issn = {1471-2180},
support = {2019B020211005 and 2019B020211003//Developing methods for the detection of ASFV from the Guangdong Provincial Department of Science and Technology/International ; 201804020039//Science and Technology Planning Project for Guangzhou/International ; },
mesh = {African Swine Fever/*diagnosis/virology ; African Swine Fever Virus/*genetics ; Animals ; Bacterial Proteins/genetics ; Blood/virology ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Endodeoxyribonucleases/genetics ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/*methods ; RNA, Viral/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Swine ; },
abstract = {BACKGROUND: As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV.<br><br>RESULTS: Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency.<br><br>CONCLUSIONS: In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.},
}
@article {pmid32927811,
year = {2020},
author = {Calverley, BC and Kadler, KE and Pickard, A},
title = {Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.},
journal = {Cells},
volume = {9},
number = {9},
pages = {},
pmid = {32927811},
issn = {2073-4409},
support = {110126/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; 203128/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 210062/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Collagen Type I/analysis/metabolism ; Luminescence ; Mice ; NIH 3T3 Cells ; },
abstract = {The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.},
}
@article {pmid32927657,
year = {2020},
author = {Henderson, SW and Henderson, ST and Goetz, M and Koltunow, AMG},
title = {Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification.},
journal = {Genes},
volume = {11},
number = {9},
pages = {},
pmid = {32927657},
issn = {2073-4425},
mesh = {*Apomixis ; Asteraceae/*genetics/growth &amp; development/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Genetic Loci ; Oxidoreductases/*antagonists &amp; inhibitors/genetics ; Phenotype ; Plant Proteins/*antagonists &amp; inhibitors/genetics ; Plants, Genetically Modified/*genetics/growth &amp; development/metabolism ; Seeds/*genetics/growth &amp; development/metabolism ; },
abstract = {Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.},
}
@article {pmid32926388,
year = {2021},
author = {Ballarino, R and Bouwman, BAM and Crosetto, N},
title = {Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {261-281},
doi = {10.1007/978-1-0716-0687-2_15},
pmid = {32926388},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Gene Targeting/*methods ; HEK293 Cells ; Humans ; },
abstract = {Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.},
}
@article {pmid32926387,
year = {2021},
author = {Wang, X and Wu, Y and Yee, JK},
title = {Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {243-260},
doi = {10.1007/978-1-0716-0687-2_14},
pmid = {32926387},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/*genetics ; *Virus Integration ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.},
}
@article {pmid32926386,
year = {2021},
author = {Kim, D and Kim, JS},
title = {Profiling Genome-Wide Specificity of CRISPR-Cas9 Using Digenome-Seq.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {233-242},
doi = {10.1007/978-1-0716-0687-2_13},
pmid = {32926386},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Whole Genome Sequencing ; },
abstract = {Digenome-seq is a highly sensitive method for analyzing the genome-wide specificity of CRISPR-Cas9 nuclease activity. In this procedure, genomic DNA is first subjected to digestion by CRISPR-Cas9 in vitro and then to whole genome sequencing, which results in unusual patterns of straight alignments at on-target and potential off-target sites. Analysis of these data with the Digenome-seq computer program allows for identification of the in vitro cleavage sites associated with the straight alignments. Here, we present a detailed Digenome-seq protocol for genome-wide profiling of in vitro CRISPR-Cas9 nuclease cleavage sites.},
}
@article {pmid32926385,
year = {2021},
author = {Cromwell, CR and Hubbard, BP},
title = {In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {215-232},
doi = {10.1007/978-1-0716-0687-2_12},
pmid = {32926385},
issn = {1940-6029},
support = {PS-408552//CIHR/Canada ; },
mesh = {Base Sequence/genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; DNA Cleavage ; Gene Editing/*methods ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity. These include the development of new protein variants of Cas9, as well as chemically modified guide RNA molecules. Here, we provide detailed protocols for two in vitro methods that enable the specificity of first- and next-generation CRISPR/Cas9 systems to be compared, and we demonstrate their applicability to evaluating chemically modified guide RNAs. One of these assays allows the specificity of different guide RNA/Cas9 complexes to be compared on a set of known off-target DNA sequences, while the second provides a broad specificity profile based on cleavage of a massive library of potential off-target DNA sequences. Collectively, these assays may be used to evaluate the specificity of different CRISPR/Cas9 systems on any DNA target sequence in a time- and cost-effective manner.},
}
@article {pmid32926384,
year = {2021},
author = {Cromwell, CR and Jovel, J and Hubbard, BP},
title = {Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {197-213},
doi = {10.1007/978-1-0716-0687-2_11},
pmid = {32926384},
issn = {1940-6029},
support = {PS-408552//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA Cleavage ; Gene Editing/*methods ; Genome/genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {The CRISPR/Cas9 system has transformed how gene knockout and knock-in studies are performed in the lab, and it is poised to revolutionize medicine. However, one of the present limitations of this technology is its imperfect specificity. While CRISPR/Cas9 can be programmed to cut a specific DNA target sequence with relative precision, off-target sequence cleavage can occur in large genomes. Importantly, several techniques have recently been developed to measure CRISPR/Cas9 on- and off-target DNA cleavage in cells. Here, we present detailed protocols for evaluating the specificity of CRISPR/Cas9 and related systems in cells using both targeted-approaches, in which off-target sites are known a priori, and unbiased approaches which are able to identify off-target cleavage events throughout an entire genome. Together, these techniques can be used to assess the reliability of experimental models generated using CRISPR/Cas9 as well as the safety of therapeutics employing this technology.},
}
@article {pmid32926383,
year = {2021},
author = {Shin, HR and Kweon, J and Kim, Y},
title = {Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {185-193},
doi = {10.1007/978-1-0716-0687-2_10},
pmid = {32926383},
issn = {1940-6029},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/*genetics ; Gene Editing/*methods ; Humans ; Protein Engineering/*methods ; RNA, Guide/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.},
}
@article {pmid32926382,
year = {2021},
author = {Ferry, QRV and Fulga, TA},
title = {Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {153-184},
doi = {10.1007/978-1-0716-0687-2_9},
pmid = {32926382},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Expression Regulation/genetics ; Humans ; RNA, Guide/genetics ; Transcriptional Activation/*genetics ; },
abstract = {The type-II CRISPR-Cas9 system has been repurposed to create synthetic programmable transcriptional regulators (CRISPR-TRs). Subsequent modifications of the system now allow for spatiotemporal control of CRISPR-mediated gene activation and repression. Among these solutions, the development of inducible spacer-blocking hairpin guide RNAs (iSBH-sgRNAs) provide an easy to implement and versatile way to condition the activation of most CRISPR-TRs on the presence of a user defined inducer. In this chapter, I cover the know-how relating to the design and synthesis of iSBH-sgRNAs, as well as the implementation in mammalian cells of inducible CRISPR-TR strategies based on this technology.},
}
@article {pmid32926381,
year = {2021},
author = {Shechner, DM},
title = {Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA-ncRNA Expression Constructs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {115-152},
doi = {10.1007/978-1-0716-0687-2_8},
pmid = {32926381},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Expression Regulation/genetics ; Genome/genetics ; Genomics/*methods ; RNA Editing/genetics ; RNA, Guide/*genetics ; RNA, Untranslated/genetics ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA-ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA "cargo" domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA-ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA-ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of "best practices" for the design and implementation of CRISPR-Display experiments.},
}
@article {pmid32926380,
year = {2021},
author = {Knapp, DJHF and Fulga, TA},
title = {Harnessing tRNA for Processing Ability and Promoter Activity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {89-114},
doi = {10.1007/978-1-0716-0687-2_7},
pmid = {32926380},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Molecular Biology/*methods ; Promoter Regions, Genetic/*genetics ; RNA Polymerase II/genetics ; RNA Processing, Post-Transcriptional/*genetics ; RNA, Guide/genetics ; RNA, Transfer/*genetics ; },
abstract = {Transfer RNA (tRNA) and their associated production and processing machinery can be coopted as a versatile tool for the production of guide RNAs (gRNAs) for Cas9-based genome engineering. Using different tRNA variants enables the production of gRNAs at a variety of steady state levels. Furthermore, engineered tRNAs can be used to process gRNAs from Pol-II transcripts, thus enabling spatial/temporal control of gRNA expression. Here we describe the design, cloning, and testing of tRNA scaffolds for both Pol-III-driven expression of different levels of gRNAs, and for processing gRNAs from Pol-II transcripts.},
}
@article {pmid32926379,
year = {2021},
author = {Lu, S and Zhang, Y and Yin, H},
title = {Chimeric DNA-RNA Guide RNA Designs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {79-85},
doi = {10.1007/978-1-0716-0687-2_6},
pmid = {32926379},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/*genetics ; Gene Editing/*methods ; Genome/genetics ; Genomics/*methods ; Humans ; RNA, Guide/*genetics ; },
abstract = {CRISPR-associated nuclease (Cas) has been widely applied to modify the genomes of various cell types. As RNA-guided endonucleases, Cas enzymes can target different genomic sequences simply by changing the guide sequence of the CRISPR RNA (crRNA) or single guide RNA (sgRNA). Recent studies have demonstrated that DNA-RNA chimeric crRNA or sgRNA can efficiently guide the Cas9 protein for genome editing with reduced off-target effects. This chapter aims to describe a procedure for using chimeric RNA to modify the genomes of mammalian cells.},
}
@article {pmid32926378,
year = {2021},
author = {Taemaitree, L and Shivalingam, A and El-Sagheer, AH and Brown, T},
title = {"Split-and-Click" sgRNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {61-78},
doi = {10.1007/978-1-0716-0687-2_5},
pmid = {32926378},
issn = {1940-6029},
support = {BB/J001694/2/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M025624/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R008655/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Gene Editing/*methods ; Humans ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. Solid-phase RNA synthesis can overcome these issues, but highly pure full-length sgRNA remains at the limits of current synthetic methods. Here, we demonstrate a "split-and-click" approach that separates the sgRNA into its two smaller components - a DNA-targeting ~20-mer RNA and a constant Cas9-binding 79-mer RNA - and chemically ligates them together to generate a biologically active sgRNA. The benefits of our approach lie in the stringent purification of the DNA-targeting 20-mer, the reduced synthesis of the constant 79-mer each time a new sgRNA is required, and the rapid access it provides to custom libraries of sgRNAs.},
}
@article {pmid32926377,
year = {2021},
author = {McMahon, MA and Rahdar, M},
title = {Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {49-60},
doi = {10.1007/978-1-0716-0687-2_4},
pmid = {32926377},
issn = {1940-6029},
mesh = {Bacterial Proteins/genetics ; Base Sequence/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Endodeoxyribonucleases/genetics ; Endonucleases/*genetics ; Gene Editing/*methods ; Genome/*genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {CRISPR-based gene editing in mammalian cells is a powerful research tool which has demonstrated efficient site-specific gene modifications and is showing promise as a therapeutic for patients with genetic diseases. Multiple different CRISPR systems have been identified, each with its own target DNA recognition sequence, expanding the editable mammalian genome. It is also now appreciated that chemically modified nucleic acids can substitute for unmodified nucleotides in guide RNAs, providing protection from exonuclease degradation and improving gene editing efficiency. CRISPR-Cpf1 unlike CRISPR-Cas9, has a substantially lower propensity for off-target genomic cleavage, making it a preferred gene editing system for many applications. Here we provide a detailed protocol for use of CRISPR-Cpf1 and chemically modified guide RNAs in cell lines, outlining the steps from designing guide RNAs to a target gene of interest, delivery and expression in cells, and analysis of gene editing events.},
}
@article {pmid32926376,
year = {2021},
author = {Shapiro, J and Tovin, A and Iancu, O and Allen, D and Hendel, A},
title = {Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {37-48},
doi = {10.1007/978-1-0716-0687-2_3},
pmid = {32926376},
issn = {1940-6029},
mesh = {Antigens, CD34/genetics ; CRISPR-Cas Systems/*genetics ; DNA, Single-Stranded/genetics ; Electroporation/*methods ; Gene Editing/*methods ; Hematopoietic Stem Cells ; Humans ; RNA, Guide/*genetics ; },
abstract = {Human CD34[+] hematopoietic stem and progenitor cells (HSPCs) have the unique ability to repopulate the entire hematopoietic system and thus are at the center of diverse, therapeutically relevant studies. The recent development of the CRISPR/Cas9 tool made the powerful research technique of genome editing highly accessible. Our previous studies demonstrated that high editing efficiency is reached when the CRISPR/Cas9 is introduced to CD34+ HSPCs as a ribonucleoprotein (RNP) complex with chemically modified guide RNAs (gRNAs). The current protocol details a quick 4-day procedure for ex vivo genome editing in CD34+ HSPCs by RNP complexes that are targeted to a specific locus by either a single gRNA (sgRNA) or a 2-part gRNA. The delivery of RNP complexes is performed by electroporation in the presence of a nonspecific, ssDNA electroporation enhancer, which highly improves editing efficiency under the described conditions. This approach is simple and effective with the potential to accelerate many biotechnological and therapeutic applications of the CRISPR/Cas9 technology.},
}
@article {pmid32926375,
year = {2021},
author = {Hwang, GH and Kim, JS and Bae, S},
title = {Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {23-33},
doi = {10.1007/978-1-0716-0687-2_2},
pmid = {32926375},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Computational Biology/*methods ; Gene Editing/*methods ; Humans ; Internet ; RNA, Guide/genetics ; *Software ; },
abstract = {The CRISPR-Cas system facilitates highly efficient genome editing; thus, it has been applied in many research fields such as biological science, medicine, and gene therapy. However, CRISPR nucleases can cleave off-target sites as well as on-target sites, causing unwanted mutations. Furthermore, after CRISPR treatments are delivered into cells or organisms, it is important to estimate the resulting mutation rates and to determine the patterns of mutations, but these tasks can be difficult. To address these issues, we have developed a tool for identifying potential off-target sites (Cas-OFFinder), a tool for designing CRISPR targets (Cas-Designer), and an assessment tool (Cas-Analyzer). These programs are all implemented on our website so that researchers can easily design CRISPR guide RNAs and assess the resulting mutations by simply clicking on the appropriate buttons; no login process is required.},
}
@article {pmid32926374,
year = {2021},
author = {Heigwer, F and Boutros, M},
title = {Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {3-22},
doi = {10.1007/978-1-0716-0687-2_1},
pmid = {32926374},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Cloud Computing ; Computational Biology/methods ; Gene Editing/*methods ; Genome/genetics ; Genomics/*methods ; Humans ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas-based genome editing in any biological application requires the evaluation of suitable genomic target sites to design efficient reagents. Considerations for the design of short guide (sg) RNAs include the assessment of possible off-target activities, the prediction of on-target efficacies and mutational outcome. Manual design of sgRNAs taking into account these parameters, however, remains a difficult task. Thus, computational tools to design sgRNA reagents from small scale to genome-wide libraries have been developed that assist during all steps of the design process. Here, we will describe practical guidance for the sgRNA design process using the web-based tool E-CRISP used in the design of individual sgRNAs. E-CRISP (www.e-crisp.org) has been the first web-based sgRNA design tool and uniquely features simple, yet efficient, scoring schemes in combination with fast evaluation and simple usage. We will also discuss the installation of a dockerized version of CRISPR Library Designer (CLD) that can be deployed locally or in the cloud to support the end-to-end design of sgRNA libraries for more than 50 different organisms. CLD was built upon E-CRISP to further increase the scope of sgRNA design to more experimental modalities (CRISPRa/i, Cas12a, all possible protospacer adjacency motifs) offering the same flexibility as E-CRISP, plus the scalability through local and cloud installation. Together, these tools facilities the design of small and large-scale CRISPR/Cas experiments.},
}
@article {pmid32926267,
year = {2020},
author = {Song, X and Zhang, XY and Xiong, ZQ and Liu, XX and Xia, YJ and Wang, SJ and Ai, LZ},
title = {CRISPR-Cas-mediated gene editing in lactic acid bacteria.},
journal = {Molecular biology reports},
volume = {47},
number = {10},
pages = {8133-8144},
doi = {10.1007/s11033-020-05820-w},
pmid = {32926267},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genes, Bacterial ; Lactobacillales/*genetics ; *Multigene Family ; },
abstract = {The high efficiency, convenience and diversity of clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are driving a technological revolution in the gene editing of lactic acid bacteria (LAB). Cas-RNA cassettes have been adopted as tools to perform gene deletion, insertion and point mutation in several species of LAB. In this article, we describe the basic mechanisms of the CRISPR-Cas system, and the current gene editing methods available, focusing on the CRISPR-Cas models developed for LAB. We also compare the different types of CRISPR-Cas-based genomic manipulations classified according to the different Cas proteins and the type of recombineering, and discuss the rapidly evolving landscape of CRISPR-Cas application in LAB.},
}
@article {pmid32923591,
year = {2020},
author = {Yu, Y and Wu, X and Guan, N and Shao, J and Li, H and Chen, Y and Ping, Y and Li, D and Ye, H},
title = {Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors.},
journal = {Science advances},
volume = {6},
number = {28},
pages = {eabb1777},
pmid = {32923591},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair ; *Gene Editing ; Humans ; Mammals/genetics ; Mice ; *Neoplasms/genetics/therapy ; Optogenetics ; },
abstract = {It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)-activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode-based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.},
}
@article {pmid32922405,
year = {2020},
author = {Bermejo-Jambrina, M and Blatzer, M and Jauregui-Onieva, P and Yordanov, TE and Hörtnagl, P and Valovka, T and Huber, LA and Wilflingseder, D and Posch, W},
title = {CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {2010},
pmid = {32922405},
issn = {1664-3224},
mesh = {CD11b Antigen/genetics ; CD11c Antigen/genetics ; CD18 Antigens/genetics ; CRISPR-Cas Systems ; Complement System Proteins/metabolism ; Dendritic Cells/*immunology ; HIV Infections/*immunology ; HIV-1/*physiology ; Humans ; Immunity ; Integrin alphaXbeta2/genetics/*metabolism ; Macrophage-1 Antigen/genetics/*metabolism ; Sequence Deletion/genetics ; Signal Transduction ; THP-1 Cells ; },
abstract = {Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.},
}
@article {pmid32920019,
year = {2020},
author = {Riedl, A and Gruber, S and Ruzsics, Z},
title = {Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli.},
journal = {Plasmid},
volume = {111},
number = {},
pages = {102531},
doi = {10.1016/j.plasmid.2020.102531},
pmid = {32920019},
issn = {1095-9890},
mesh = {Chromosomes, Artificial, Bacterial ; Chromosomes, Bacterial ; DNA Replication ; Escherichia coli/*genetics ; Gene Editing ; Gene Expression Regulation, Bacterial ; *Genetic Engineering ; Genome, Bacterial ; Plasmids/*genetics ; Recombination, Genetic ; Temperature ; },
abstract = {Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.},
}
@article {pmid32919352,
year = {2020},
author = {Liu, H and Li, S and Ren, C and Liu, W and Zhu, B and Wang, L and Xu, H and Xie, W and Zuo, X and Zhou, Y and Luo, L and Jiang, X},
title = {Generation of an ESRG Pr-tdTomato reporter human embryonic stem cell line, CSUe011-A, using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101983},
doi = {10.1016/j.scr.2020.101983},
pmid = {32919352},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; *Human Embryonic Stem Cells ; Humans ; Luminescent Proteins ; },
abstract = {ESRG was first identified in our previous study. It is highly expressed in human embryonic stem cells (hESCs), whereas it is significantly down-regulated in differentiated cells and is undetectable in adult tissues. To develop an hESC line for monitoring the expression of ESRG for further study of its function, we used gene editing techniques to insert fusion sequences of ESRG promoter and tdTomato fluorescent protein gene into the AAVS1 human safe harbor locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiation potential.},
}
@article {pmid32918936,
year = {2020},
author = {Nguyen, DX and Nakazawa, T and Myo, G and Inoue, C and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {A promoter assay system using gene targeting in agaricomycetes Pleurotus ostreatus and Coprinopsis cinerea.},
journal = {Journal of microbiological methods},
volume = {179},
number = {},
pages = {106053},
doi = {10.1016/j.mimet.2020.106053},
pmid = {32918936},
issn = {1872-8359},
mesh = {Agaricales/*genetics ; CRISPR-Cas Systems/genetics ; Cellulase/genetics ; Gene Expression Regulation, Fungal/genetics ; *Gene Targeting ; Peroxidases/genetics ; Pleurotus/*genetics ; Promoter Regions, Genetic/*genetics ; },
abstract = {A novel promoter assay was developed for Agaricomycetes, using a gene-targeting approach, with or without the CRISPR/Cas9 technique. It enables precise evaluation of promoter activity at the original site of the chromosome without random and multiple integrations in conventional transformation experiments.},
}
@article {pmid32918875,
year = {2020},
author = {Funato, Y and Yoshida, A and Hirata, Y and Hashizume, O and Yamazaki, D and Miki, H},
title = {The Oncogenic PRL Protein Causes Acid Addiction of Cells by Stimulating Lysosomal Exocytosis.},
journal = {Developmental cell},
volume = {55},
number = {4},
pages = {387-397.e8},
doi = {10.1016/j.devcel.2020.08.009},
pmid = {32918875},
issn = {1878-1551},
mesh = {Acids/*metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/metabolism ; Conserved Sequence ; Dogs ; Evolution, Molecular ; *Exocytosis ; HEK293 Cells ; Humans ; Immediate-Early Proteins/*metabolism ; Intracellular Membranes/metabolism ; Lysosomes/*metabolism ; Madin Darby Canine Kidney Cells ; Mice, Inbred C57BL ; Neoplasm Metastasis ; Neoplasm Proteins/*metabolism ; Protein Tyrosine Phosphatases/*metabolism ; },
abstract = {Extracellular pH is usually maintained around 7.4 in multicellular organisms, and cells are optimized to proliferate under this condition. Here, we find cells can adapt to a more acidic pH of 6.5 and become addicted to this acidic microenvironment by expressing phosphatase of regenerating liver (PRL), a driver of cancer malignancy. Genome-scale CRISPR-Cas9 knockout screening and subsequent analyses revealed that PRL promotes H[+] extrusion and acid addiction by stimulating lysosomal exocytosis. Further experiments using cultured cells and Caenorhabditis elegans clarified the molecular link between PRL and lysosomal exocytosis across species, involving activation of lysosomal Ca[2+] channel TRPML by ROS. Indeed, disruption of TRPML in cancer cells abolished PRL-stimulated lysosomal exocytosis, acid addiction, and metastasis. Thus, PRL is the molecular switch turning cells addicted to an acidic condition, which should benefit cancer cells to thrive in an acidic tumor microenvironment.},
}
@article {pmid32918585,
year = {2020},
author = {Feng, S and Hu, L and Zhang, Q and Zhang, F and Du, J and Liang, G and Li, A and Song, G and Liu, Y},
title = {CRISPR/Cas technology promotes the various application of Dunaliella salina system.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {20},
pages = {8621-8630},
doi = {10.1007/s00253-020-10892-6},
pmid = {32918585},
issn = {1432-0614},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering ; Technology ; },
abstract = {Dunaliella salina (D. salina) has been widely applied in various fields because of its inherent advantages, such as the study of halotolerant mechanism, wastewater treatment, recombinant proteins expression, biofuel production, preparation of natural materials, and others. However, owing to the existence of low yield or in the laboratory exploration stage, D. salina system has been greatly restricted for practical production of various components. In past decade, significant progresses have been achieved for research of D. salina in these fields. Among them, D. salina as a novel expression system demonstrated a bright prospect, especially for large-scale production of foreign proteins, like the vaccines, antibodies, and other therapeutic proteins. Due to the low efficiency, application of traditional regulation tools is also greatly limited for exploration of D. salina system. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system offers a precise editing tool to overcome the obstacles of D. salina system. This review not only comprehensively summarizes the recent progresses of D. salina in domain of gene engineering but also gives a deep analysis of problems and deficiencies in different fields of D. salina. Moreover, further prospects of CRISPR/Cas system and its significant challenges have been discussed in various aspects of D. salina. It provides a great referencing value for speeding up the maturity of D. salina system, and also supplies practical guiding significance to expand the new application fields for D. salina. KEY POINTS: • The review provides recent research progresses of various applications of D. salina. • The problems and deficiencies in different fields of D. salina were deeply analyzed. • The further prospects of CRISPR/Cas technology in D. salina system were predicted. • CRISPR/Cas system will promote the new application fields and maturity for D. salina.},
}
@article {pmid32917893,
year = {2020},
author = {Franklin, JM and Ghosh, RP and Shi, Q and Reddick, MP and Liphardt, JT},
title = {Concerted localization-resets precede YAP-dependent transcription.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4581},
pmid = {32917893},
issn = {2041-1723},
support = {U01 EB021237/EB/NIBIB NIH HHS/United States ; U54 CA143836/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Cytoplasm/metabolism ; Gene Expression Regulation ; Gene Knock-In Techniques ; HEK293 Cells ; Humans ; Mechanotransduction, Cellular/physiology ; Oncogenes/genetics ; Transcription Factors/genetics/*metabolism ; YAP-Signaling Proteins ; },
abstract = {Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.},
}
@article {pmid32917647,
year = {2020},
author = {Burkhart, DL and Morel, KL and Wadosky, KM and Labbé, DP and Galbo, PM and Dalimov, Z and Xu, B and Loda, M and Ellis, L},
title = {Evidence that EZH2 Deregulation is an Actionable Therapeutic Target for Prevention of Prostate Cancer.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {13},
number = {12},
pages = {979-988},
pmid = {32917647},
issn = {1940-6215},
support = {R01 CA207757/CA/NCI NIH HHS/United States ; R21 CA205627/CA/NCI NIH HHS/United States ; PJT-162246//CIHR/Canada ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell Proliferation ; *Cellular Senescence ; Enhancer of Zeste Homolog 2 Protein/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Prostatic Intraepithelial Neoplasia/etiology/metabolism/pathology/*prevention &amp; control ; Prostatic Neoplasms/etiology/metabolism/pathology/*prevention &amp; control ; },
abstract = {Chemoprevention trials for prostate cancer by androgen receptor or androgen synthesis inhibition have proven ineffective. Recently, it has been demonstrated that the histone methlytransferase, EZH2 is deregulated in mouse and human high-grade prostatic intraepithelial neoplasia (HG-PIN). Using preclinical mouse and human models of prostate cancer, we demonstrate that genetic and chemical disruption of EZH2 expression and catalytic activity reversed the HG-PIN phenotype. Furthermore, inhibition of EZH2 function was associated with loss of cellular proliferation and induction of Tp53-dependent senescence. Together, these data provide provocative evidence for EZH2 as an actionable therapeutic target toward prevention of prostate cancer.},
}
@article {pmid32917636,
year = {2020},
author = {Weber, L and Frati, G and Felix, T and Hardouin, G and Casini, A and Wollenschlaeger, C and Meneghini, V and Masson, C and De Cian, A and Chalumeau, A and Mavilio, F and Amendola, M and Andre-Schmutz, I and Cereseto, A and El Nemer, W and Concordet, JP and Giovannangeli, C and Cavazzana, M and Miccio, A},
title = {Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype.},
journal = {Science advances},
volume = {6},
number = {7},
pages = {},
pmid = {32917636},
issn = {2375-2548},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; Binding Sites ; CRISPR-Cas Systems ; Fetal Hemoglobin/genetics/metabolism ; Gene Editing/methods ; Humans ; Phenotype ; beta-Globins/genetics/metabolism ; *beta-Thalassemia/genetics/metabolism/therapy ; gamma-Globins/genetics/metabolism ; },
abstract = {Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.},
}
@article {pmid32917362,
year = {2020},
author = {Cheng, YF and Tsai, YH and Huang, CY and Lee, YS and Chang, PC and Lu, YC and Hsu, CJ and Wu, CC},
title = {Generation and pathological characterization of a transgenic mouse model carrying a missense PJVK mutation.},
journal = {Biochemical and biophysical research communications},
volume = {532},
number = {4},
pages = {675-681},
doi = {10.1016/j.bbrc.2020.07.101},
pmid = {32917362},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Knock-In Techniques ; Hair Cells, Auditory/pathology ; Hearing Loss, Sensorineural/*genetics/pathology/physiopathology ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation, Missense ; Proteins/*genetics ; Spiral Ganglion/pathology ; Vestibule, Labyrinth/physiopathology ; },
abstract = {Hearing loss is the most prevalent hereditary sensory disorder in children. Approximately 2 in 1000 infants are affected by genetic hearing loss. The PJVK gene, which encodes the pejvakin protein, has been linked to autosomal recessive non-syndromic hearing loss DFNB59. Previous clinical studies have revealed that PJVK mutations might be associated with a wide spectrum of auditory manifestations, ranging from hearing loss of pure cochlear origin to that involving the retrocochlear central auditory pathway. The phenotypic variety makes the pathogenesis of this disease difficult to determine. Similarly, mouse models carrying different Pjvk defects show phenotypic variability and inconsistency. In this study, we generated a knockin mouse model carrying the c.874G > A (p.G292R) variant to model and investigate the auditory and vestibular phenotypes of DFNB59.},
}
@article {pmid32917146,
year = {2020},
author = {Xu, X and Harvey-Samuel, T and Yang, J and Alphey, L and You, M},
title = {Ommochrome pathway genes kynurenine 3-hydroxylase and cardinal participate in eye pigmentation in Plutella xylostella.},
journal = {BMC molecular and cell biology},
volume = {21},
number = {1},
pages = {63},
pmid = {32917146},
issn = {2661-8850},
support = {BBS/E/I/00007033, BBS/E/I/00007038 and BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S506680/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Compound Eye, Arthropod/*physiology ; Frameshift Mutation/genetics ; Gene Knockout Techniques/methods ; Insect Proteins/*genetics ; Kynurenine 3-Monooxygenase/*genetics ; Larva/genetics ; Moths/*genetics/*metabolism ; Phenothiazines/*metabolism ; Pigmentation/*genetics ; },
abstract = {BACKGROUND: Eye pigmentation genes have been utilized as visible markers for constructing genetic control prototypes in several insect vectors of human disease. Here, orthologs of two ommochrome pathway genes, kynurenine 3-hydroxylase (kmo) and cardinal, were investigated in Plutella xylostella, a globally distributed, economically important pest of Brassica crops.<br><br>RESULTS: Both somatic mosaic and germline mutations were efficiently created using the CRISPR/Cas9 system, and null mutant strains of Pxkmo and Pxcardinal were obtained. A frame-shift mutation in Pxkmo caused yellow compound eyes at adult stage while an in-frame mutation lacking two amino acids resulted in a hypomorphic red eye phenotypes. In contrast, Pxcardinal-deficient moths with a frame-shift mutation exhibited yellow eye pigmentation in newly emerged adults which turned to red as the adults aged. Additionally, differences were observed in the coloration of larval ocelli, brains and testes in Pxkmo and Pxcardinal yellow-eye mutant lines.<br><br>CONCLUSIONS: Our work identifies the important roles of Pxkmo and Pxcardinal in P. xylostella eye pigmentation and provides tools for future genetic manipulation of this important crop pest.},
}
@article {pmid32916055,
year = {2020},
author = {Su, C and Tuan, NQ and Lee, MJ and Zhang, XY and Cheng, JH and Jin, YY and Zhao, XQ and Suh, JW},
title = {Enhanced Production of Active Ecumicin Component with Higher Antituberculosis Activity by the Rare Actinomycete Nonomuraea sp. MJM5123 Using a Novel Promoter-Engineering Strategy.},
journal = {ACS synthetic biology},
volume = {9},
number = {11},
pages = {3019-3029},
doi = {10.1021/acssynbio.0c00248},
pmid = {32916055},
issn = {2161-5063},
mesh = {Actinobacteria/*genetics/*metabolism ; Antitubercular Agents/*metabolism/pharmacology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genes, Bacterial ; Peptides, Cyclic/*genetics/*metabolism ; Promoter Regions, Genetic/*genetics ; },
abstract = {Ecumicins are potent antituberculosis natural compounds produced by the rare actinomycete Nonomuraea sp. MJM5123. Here, we report an efficient genetic manipulation platform of this rare actinomycete. CRISPR/Cas9-based genome editing was achieved based on successful sporulation. Two genes in the ecumicin gene cluster were further investigated, ecuN and ecuE, which potentially encode a pretailoring cytochrome P450 hydroxylase and the core peptide synthase, respectively. Deletion of ecuN led to an enhanced ratio of the ecumicin compound EcuH16 relative to that of EcuH14, indicating that EcuN is indeed a P450 hydroxylase, and there is catalyzed hydroxylation at the C-3 position in unit12 phenylalanine to transform EcuH16 to the compound EcuH14. Furthermore, promoter engineering of ecuE by employing the strong promoter kasO*P was performed and optimized. We found that integrating the endogenous ribosome-binding site (RBS) of ecuE together with the RBS from kasO*P led to improved ecumicin production and resulted in a remarkably high EcuH16/EcuH14 ratio. Importantly, production of the more active component EcuH16 was considerably increased in the double RBSs engineered strain EPR1 compared to that in the wild-type strain, reaching 310 mg/L. At the same time, this production level was 2.3 times higher than that of the control strain EPA1 with only one RBS from kasO*P. To the best of our knowledge, this is the first report of genome editing and promoter engineering on the rare actinomycete Nonomuraea.},
}
@article {pmid32916053,
year = {2020},
author = {Angstenberger, M and de Signori, F and Vecchi, V and Dall'Osto, L and Bassi, R},
title = {Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii.},
journal = {ACS synthetic biology},
volume = {9},
number = {10},
pages = {2840-2850},
pmid = {32916053},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Nucleus/*genetics ; Chlamydomonas reinhardtii/*genetics ; Chloroplast Proteins/genetics ; DNA End-Joining Repair/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; *Genome, Plant ; *Homologous Recombination ; Membrane Proteins/genetics ; Plant Proteins/genetics ; *Transformation, Genetic ; },
abstract = {In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (HR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR. This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.},
}
@article {pmid32915222,
year = {2019},
author = {Mills, P},
title = {Preimplantation genome editing: CCR5 in China.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {6},
pages = {695-700},
doi = {10.1042/ETLS20190114},
pmid = {32915222},
issn = {2397-8554},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Gene Editing ; Genetic Therapy ; HIV Infections/*prevention &amp; control/*therapy ; Humans ; Receptors, CCR5/*genetics ; Targeted Gene Repair ; },
abstract = {Part of the criticism of the one reported case of human preimplantation genome editing (PGE) turned on the inadequacy of the purpose for which it was undertaken (inherent immunity to HIV) and its target (the CCR5 gene). The discussion of CCR5 in this context reveals the different values that inform the idea of acceptable uses of PGE and of the conditions of responsible biomedical innovation among the scientist responsible and his critics. While the use of PGE for any indication remains unacceptable (or, at the very least, premature), neither position offers a satisfactory response to this prospective biotechnology.},
}
@article {pmid32915215,
year = {2019},
author = {Iacomussi, S},
title = {Regulating genome editing technologies: a comparison of expert recommendations in the U.K. and in the U.S.A.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {6},
pages = {701-705},
doi = {10.1042/ETLS20180101},
pmid = {32915215},
issn = {2397-8554},
mesh = {CRISPR-Cas Systems ; England ; Gene Editing/*legislation &amp; jurisprudence/trends ; Genetic Therapy ; Government Regulation ; Humans ; Public Policy ; United States ; },
abstract = {The present paper aims to inform the bioethical debate on the regulation of human genome editing technologies with a specific focus on the role of scientific experts and their interactions with the general public in the formulation of policy. It reviews and compares two of the major contributions to this debate in the U.K. and in the U.S.A., comparing expert approaches towards regulation on genome editing technologies. The results of this analysis offer important lessons that should be appreciated in building an international regulatory framework. On the basis of these results, I conclude that the experts should embrace a socially responsible approach and encourage active public engagement.},
}
@article {pmid32915213,
year = {2019},
author = {Bryant, JA},
title = {From bacterial battles to CRISPR crops; progress towards agricultural applications of genome editing.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {6},
pages = {687-693},
doi = {10.1042/ETLS20190065},
pmid = {32915213},
issn = {2397-8554},
mesh = {Agriculture ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Disease Resistance/genetics ; Gene Editing/*methods ; Genetic Engineering ; Genome, Plant ; Humans ; Plants, Genetically Modified/*genetics ; },
abstract = {Genome editing is the precise alteration of DNA in living cells by the cutting or removal of specific sequences, sometimes followed by insertion of new sequences at the cut site. CRISPR-Cas9 has become firmly established as the genome-editing method of choice, replacing the systems that had been developed and in use since the early 1990s. The CRISPR-Cas9 system has been developed from a mechanism used in prokaryotes as a defence against bacteriophage but actually functions in cells of all types of organisms. It is widely used in research as a gene knockout and editing tool; applications in veterinary medicine (such as increased resistance to disease) and human medicine (such as correction of disease-causing mutations) are under development. In agriculture and horticulture, the potential for various aspects of crop improvement is very large. Selected aspects of this potential are presented here, with particular focus on crop quality and disease resistance. The article ends with a brief discussion of the regulatory 'environment' in the USA and the EU.},
}
@article {pmid32913194,
year = {2020},
author = {Watry, HL and Feliciano, CM and Gjoni, K and Takahashi, G and Miyaoka, Y and Conklin, BR and Judge, LM},
title = {Rapid, precise quantification of large DNA excisions and inversions by ddPCR.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14896},
pmid = {32913194},
issn = {2045-2322},
support = {R01 HL130533/HL/NHLBI NIH HHS/United States ; P01 HL146366/HL/NHLBI NIH HHS/United States ; U01 EB029374/EB/NIBIB NIH HHS/United States ; R01 EY028249/EY/NEI NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; *Chromosome Inversion ; DNA/*genetics/*metabolism ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism/*physiology ; Polymerase Chain Reaction/*methods ; },
abstract = {The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.},
}
@article {pmid32913123,
year = {2020},
author = {Zhang, A and Berardinelli, SJ and Leonhard-Melief, C and Vasudevan, D and Liu, TW and Taibi, A and Giannone, S and Apte, SS and Holdener, BC and Haltiwanger, RS},
title = {O-Fucosylation of ADAMTSL2 is required for secretion and is impacted by geleophysic dysplasia-causing mutations.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {46},
pages = {15742-15753},
pmid = {32913123},
issn = {1083-351X},
support = {R01 CA123071/CA/NCI NIH HHS/United States ; R01 HD090156/HD/NICHD NIH HHS/United States ; R01 HD096030/HD/NICHD NIH HHS/United States ; },
mesh = {ADAMTS Proteins/chemistry/genetics/*metabolism ; Amino Acid Sequence ; Animals ; Bone Diseases, Developmental/genetics/*pathology ; CRISPR-Cas Systems/genetics ; Disaccharides/chemistry ; Extracellular Matrix Proteins/chemistry/genetics/*metabolism ; Fucosyltransferases/deficiency/genetics ; Gene Editing ; Glycosylation ; Glycosyltransferases/deficiency/genetics ; HEK293 Cells ; Humans ; Limb Deformities, Congenital/genetics/*pathology ; Mannose/chemistry ; Mice ; Mutagenesis, Site-Directed ; Protein Domains ; Recombinant Proteins/biosynthesis/chemistry/isolation &amp; purification ; Sequence Alignment ; },
abstract = {ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-β binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose-modified TSRs are subsequently elongated to a glucose β1-3-fucose (GlcFuc) disaccharide by β1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2[-/-] but not in B3GLCT[-/-] cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.},
}
@article {pmid32913081,
year = {2020},
author = {Cohen, J},
title = {Narrow path charted for editing genes of human embryos.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6509},
pages = {1283},
doi = {10.1126/science.369.6509.1283},
pmid = {32913081},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Embryo Research/*ethics ; Gene Editing/*ethics ; Genetic Diseases, Inborn/genetics/therapy ; Humans ; Practice Guidelines as Topic ; Reproduction/genetics ; },
}
@article {pmid32912697,
year = {2020},
author = {Manglekar, RR and Geng, A},
title = {CRISPR-Cas9-mediated seb1 disruption in Talaromyces pinophilus EMU for its enhanced cellulase production.},
journal = {Enzyme and microbial technology},
volume = {140},
number = {},
pages = {109646},
doi = {10.1016/j.enzmictec.2020.109646},
pmid = {32912697},
issn = {1879-0909},
mesh = {Biomass ; CRISPR-Cas Systems ; Cellulase/*metabolism ; Fungal Proteins/*genetics/metabolism ; Gene Editing ; Gene Expression Regulation, Fungal ; Hyphae/genetics/growth &amp; development/metabolism ; Mutation ; Phenotype ; Talaromyces/genetics/growth &amp; development/*metabolism ; Transcription Factors/*genetics/metabolism ; },
abstract = {Filamentous fungi are working horses for industrial enzyme production. Combinatory approaches, such as random mutagenesis and rational genetic engineering, were adopted to improve their enzyme productivity. The filamentous fungus Talaromyces pinophilus EMU is a hyper cellulase-producing filamentous fungus obtained through random mutagenesis. This study further enhanced its cellulase production through the disruption of seb1 gene, which encodes Seb1, a transcription factor that binds to the stress response element (STRE) and regulates a variety of cellular processes. Gene seb1 was cloned from strain T. pinophilus EMU and disrupted using CRISPR-Cas9 technology. The seb1-disruptants (TpΔseb1 strains) showed distinct morphology from its parent strain. They presented a hyphal branching phenotype with decreased transcription levels of rhoA and ras1 genes involved in hyphal branching. Furthermore, TpΔseb1 strains displayed lower cell biomass, higher specific protein content, and 20%-40% enhancement in filter paper cellulase (FPase) activity, however, insignificant changes in the transcription levels of cbh1 and bgl1 genes involved in cellulase production. Through this study, we confirmed that seb1 gene disruption in T. pinophilus EMU caused more hyphal branching, reduced cell growth, increased protein secretion, and enhanced cellulase production. In addition, we successfully established the CRISPR-Cas9 genome-editing platform in T. pinophilus EMU.},
}
@article {pmid32912681,
year = {2020},
author = {de Souza, CP and Ribeiro, BD and Zarur Coelho, MA and Almeida, RV and Nicaud, JM},
title = {Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.},
journal = {Enzyme and microbial technology},
volume = {140},
number = {},
pages = {109621},
doi = {10.1016/j.enzmictec.2020.109621},
pmid = {32912681},
issn = {1879-0909},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Culture Media/metabolism ; Fluorescence ; Fungal Proteins/genetics ; Gene Targeting ; Metabolic Engineering/*methods ; Mutation ; RNA, Guide/genetics ; Uracil/metabolism ; Yarrowia/*genetics/growth &amp; development/metabolism ; beta Carotene/biosynthesis/genetics ; },
abstract = {Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for β-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of β-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.},
}
@article {pmid32912679,
year = {2020},
author = {Pattharaprachayakul, N and Lee, M and Incharoensakdi, A and Woo, HM},
title = {Current understanding of the cyanobacterial CRISPR-Cas systems and development of the synthetic CRISPR-Cas systems for cyanobacteria.},
journal = {Enzyme and microbial technology},
volume = {140},
number = {},
pages = {109619},
doi = {10.1016/j.enzmictec.2020.109619},
pmid = {32912679},
issn = {1879-0909},
mesh = {CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Cyanobacteria/*genetics/metabolism ; Gene Editing ; Gene Expression Regulation, Bacterial ; Metabolic Engineering ; Photosynthesis/genetics ; RNA, Guide/genetics ; },
abstract = {Cyanobacteria are photosynthetic microorganisms that are capable of converting CO2 to value-added chemicals. Engineering of cyanobacteria with synthetic biology tools, including the CRISPR-Cas system, has allowed an opportunity for biological CO2 utilization. Here, we described natural CRISPR-Cas systems for understanding cyanobacterial genomics and synthetic CRISPR-Cas systems for metabolic engineering applications. The natural CRISPR-Cas systems in cyanobacteria have been identified as Class 1, with type I and III, and some Class 2, with type V, as an adaptive immune system against viral invasion. As synthetic tools, CRISPR-Cas9 and -Cas12a have been successfully established in cyanobacteria to delete a target gene without a selection marker. Deactivated Cas9 and Cas12a have also been used to repress genes for metabolic engineering. In addition, a perspective on how advanced CRISPR-Cas systems and a pool of the guide RNAs can be advantageous for precise genome engineering and understanding of unknown functions was discussed for advanced engineering of cyanobacteria.},
}
@article {pmid32912325,
year = {2020},
author = {Gabr, H and El Ghamrawy, MK and Almaeen, AH and Abdelhafiz, AS and Hassan, AOS and El Sissy, MH},
title = {CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation.},
journal = {Stem cell research &amp; therapy},
volume = {11},
number = {1},
pages = {390},
pmid = {32912325},
issn = {1757-6512},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Hematopoietic Stem Cells/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; beta-Globins/genetics/metabolism ; *beta-Thalassemia/genetics/therapy ; },
abstract = {BACKGROUND: β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation.<br><br>METHODS AND RESULTS: We successfully isolated CD34[+] cells from peripheral blood of &#x3b2;-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34[+] cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin.<br><br>CONCLUSION: CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of &#x3b2;-thalassemia.},
}
@article {pmid32912286,
year = {2020},
author = {Brandt, M and Gokden, A and Ziosi, M and Lappalainen, T},
title = {A polyclonal allelic expression assay for detecting regulatory effects of transcript variants.},
journal = {Genome medicine},
volume = {12},
number = {1},
pages = {79},
pmid = {32912286},
issn = {1756-994X},
support = {R01 AG057422/AG/NIA NIH HHS/United States ; T32 GM008224/GM/NIGMS NIH HHS/United States ; R01MH106842/NH/NIH HHS/United States ; R01GM122924/NH/NIH HHS/United States ; },
mesh = {*Alleles ; CRISPR-Cas Systems ; Gene Editing ; *Gene Expression Regulation ; *Genetic Variation ; HEK293 Cells ; Humans ; Nonsense Mediated mRNA Decay ; Quantitative Trait Loci ; *Transcription, Genetic ; },
abstract = {We present an assay to experimentally test the regulatory effects of genetic variants within transcripts using CRISPR/Cas9 followed by targeted sequencing. We applied the assay to 32 premature stop-gained variants across the genome and in two Mendelian disease genes, 33 putative causal variants of eQTLs, and 62 control variants in HEK293T cells, replicating a subset of variants in HeLa cells. We detected significant effects in the expected direction (in 60% of variants), demonstrating the ability of the assay to capture regulatory effects of eQTL variants and nonsense-mediated decay triggered by premature stop-gained variants. The results suggest a utility for validating transcript-level effects of genetic variants.},
}
@article {pmid32912206,
year = {2020},
author = {Schleidgen, S and Dederer, HG and Sgodda, S and Cravcisin, S and Lüneburg, L and Cantz, T and Heinemann, T},
title = {Human germline editing in the era of CRISPR-Cas: risk and uncertainty, inter-generational responsibility, therapeutic legitimacy.},
journal = {BMC medical ethics},
volume = {21},
number = {1},
pages = {87},
pmid = {32912206},
issn = {1472-6939},
support = {01GP1616A-C//Bundesministerium f&#xfc;r Bildung und Forschung/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Germ Cells ; Humans ; Uncertainty ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) technology may allow for efficient and highly targeted gene editing in single-cell embryos. This possibility brings human germline editing into the focus of ethical and legal debates again.<br><br>MAIN BODY: Against this background, we explore essential ethical and legal questions of interventions into the human germline by means of CRISPR-Cas: How should issues of risk and uncertainty be handled? What responsibilities arise regarding future generations? Under which conditions can germline editing measures be therapeutically legitimized? For this purpose, we refer to a scenario anticipating potential further development in CRISPR-Cas technology implying improved accuracy and exclusion of germline transmission to future generations. We show that, if certain concepts regarding germline editing are clarified, under such conditions a categorical prohibition of one-generation germline editing of single-cell embryos appears not to be ethically or legally justifiable.<br><br>CONCLUSION: These findings are important prerequisites for the international debate on the ethical and legal justification of germline interventions in the human embryo as well as for the harmonization of international legal standards.},
}
@article {pmid32911927,
year = {2020},
author = {Shen, CC and Lin, MW and Nguyen, BKT and Chang, CW and Shih, JR and Nguyen, MTT and Chang, YH and Hu, YC},
title = {CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells.},
journal = {ACS synthetic biology},
volume = {9},
number = {10},
pages = {2808-2818},
doi = {10.1021/acssynbio.0c00338},
pmid = {32911927},
issn = {2161-5063},
mesh = {Animals ; Batch Cell Culture Techniques ; CHO Cells ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cricetulus ; Endonucleases/*genetics ; Fucosyltransferases/genetics ; Gene Expression ; Gene Knockdown Techniques/*methods ; Gene Silencing ; Immunoglobulin G/analysis/biosynthesis ; Metabolic Engineering/*methods ; RNA, Guide/genetics ; Transfection ; },
abstract = {Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells is desired but challenging. Here, we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44, and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism, and glycosylation (e.g., GS, BAK, BAX, PDK1, and FUT8) in CHO cells with efficiencies ranging from 60% to 80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the Sleeping Beauty system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ≈80%-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering, and cell engineering of CHO cells.},
}
@article {pmid32908085,
year = {2020},
author = {Tamura, R and Toda, M},
title = {Historic Overview of Genetic Engineering Technologies for Human Gene Therapy.},
journal = {Neurologia medico-chirurgica},
volume = {60},
number = {10},
pages = {483-491},
pmid = {32908085},
issn = {1349-8029},
mesh = {Brain Neoplasms/therapy ; CRISPR-Cas Systems ; Gene Editing/*history/instrumentation/methods ; Genetic Therapy/*history/instrumentation/methods ; History, 20th Century ; History, 21st Century ; Humans ; Transcription Activator-Like Effector Nucleases ; },
abstract = {The concepts of gene therapy were initially introduced during the 1960s. Since the early 1990s, more than 1900 clinical trials have been conducted for the treatment of genetic diseases and cancers mainly using viral vectors. Although a variety of methods have also been performed for the treatment of malignant gliomas, it has been difficult to target invasive glioma cells. To overcome this problem, immortalized neural stem cell (NSC) and a nonlytic, amphotropic retroviral replicating vector (RRV) have attracted attention for gene delivery to invasive glioma. Recently, genome editing technology targeting insertions at site-specific locations has advanced; in particular, the clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) has been developed. Since 2015, more than 30 clinical trials have been conducted using genome editing technologies, and the results have shown the potential to achieve positive patient outcomes. Gene therapy using CRISPR technologies for the treatment of a wide range of diseases is expected to continuously advance well into the future.},
}
@article {pmid32907973,
year = {2020},
author = {Kawagishi, T and Kanai, Y and Nouda, R and Fukui, I and Nurdin, JA and Matsuura, Y and Kobayashi, T},
title = {Generation of Genetically RGD σ1-Modified Oncolytic Reovirus That Enhances JAM-A-Independent Infection of Tumor Cells.},
journal = {Journal of virology},
volume = {94},
number = {23},
pages = {},
pmid = {32907973},
issn = {1098-5514},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Cell Adhesion Molecules ; Cell Line, Tumor ; Gene Knockout Techniques ; Humans ; Junctional Adhesion Molecule A/*genetics/*metabolism ; Mammalian orthoreovirus 3/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Nude ; Oligopeptides/*metabolism ; Oncolytic Virotherapy ; Oncolytic Viruses/genetics ; Orthoreovirus/genetics/metabolism ; Receptors, Cell Surface ; Reoviridae/*genetics/*metabolism ; Virus Replication ; },
abstract = {Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.},
}
@article {pmid32907944,
year = {2020},
author = {Lin, SC and Qu, L and Ettayebi, K and Crawford, SE and Blutt, SE and Robertson, MJ and Zeng, XL and Tenge, VR and Ayyar, BV and Karandikar, UC and Yu, X and Coarfa, C and Atmar, RL and Ramani, S and Estes, MK},
title = {Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {38},
pages = {23782-23793},
pmid = {32907944},
issn = {1091-6490},
support = {U19 AI144297/AI/NIAID NIH HHS/United States ; U19 AI116497/AI/NIAID NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; HHSN272201700081C/AI/NIAID NIH HHS/United States ; P30 ES030285/ES/NIEHS NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; T32 DK007664/DK/NIDDK NIH HHS/United States ; P01 AI057788/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Caliciviridae Infections/immunology/virology ; Host-Pathogen Interactions/*immunology ; Humans ; *Interferons/genetics/metabolism ; *Intestines/immunology/virology ; Models, Biological ; *Norovirus/genetics/immunology/pathogenicity ; Organoids/immunology/virology ; Sequence Analysis, RNA ; Transcriptome/genetics ; Virus Replication ; },
abstract = {Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.},
}
@article {pmid32907859,
year = {2020},
author = {Bhagwat, AM and Graumann, J and Wiegandt, R and Bentsen, M and Welker, J and Kuenne, C and Preussner, J and Braun, T and Looso, M},
title = {multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets.},
journal = {Life science alliance},
volume = {3},
number = {11},
pages = {},
pmid = {32907859},
issn = {2575-1077},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; DNA Primers/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Humans ; RNA, Guide/genetics ; Software ; },
abstract = {Targeting the coding genome to introduce nucleotide deletions/insertions via the CRISPR/Cas9 technology has become a standard procedure. It has quickly spawned a multitude of methods such as prime editing, APEX proximity labeling, or homology directed repair, for which supporting bioinformatics tools are, however, lagging behind. New CRISPR/Cas9 applications often require specific gRNA design functionality, and a generic tool is critically missing. Here, we introduce multicrispr, an R/bioconductor tool, intended to design individual gRNAs and complex gRNA libraries. The package is easy to use; detects, scores, and filters gRNAs on both efficiency and specificity; visualizes and aggregates results per target or CRISPR/Cas9 sequence; and finally returns both genomic ranges and sequences of gRNAs. To be generic, multicrispr defines and implements a genomic arithmetic framework as a basis for facile adaptation to techniques recently introduced such as prime editing or yet to arise. Its performance and design concepts such as target set-specific filtering render multicrispr a tool of choice when dealing with screening-like approaches.},
}
@article {pmid32904687,
year = {2020},
author = {Nemudryi, A and Nemudraia, A and Wiegand, T and Surya, K and Buyukyoruk, M and Cicha, C and Vanderwood, KK and Wilkinson, R and Wiedenheft, B},
title = {Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater.},
journal = {Cell reports. Medicine},
volume = {1},
number = {6},
pages = {100098},
pmid = {32904687},
issn = {2666-3791},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
abstract = {SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community.},
}
@article {pmid32903830,
year = {2020},
author = {Song, S and Wood, TK},
title = {A Primary Physiological Role of Toxin/Antitoxin Systems Is Phage Inhibition.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1895},
pmid = {32903830},
issn = {1664-302X},
abstract = {Toxin/antitoxin (TA) systems are present in most prokaryote genomes. Toxins are almost exclusively proteins that reduce metabolism (but do not cause cell death), and antitoxins are either RNA or proteins that counteract the toxin or the RNA that encodes it. Although TA systems clearly stabilize mobile genetic elements, after four decades of research, the physiological roles of chromosomal TA systems are less clear. For example, recent reports have challenged the notion of TA systems as stress-response elements, including a role in creating the dormant state known as persistence. Here, we present evidence that a primary physiological role of chromosomally encoded TA systems is phage inhibition, a role that is also played by some plasmid-based TA systems. This includes results that show some CRISPR-Cas system elements are derived from TA systems and that some CRISPR-Cas systems mimic the host growth inhibition invoked by TA systems to inhibit phage propagation.},
}
@article {pmid32903718,
year = {2020},
author = {Jesus-Santos, FH and Lobo-Silva, J and Ramos, PIP and Descoteaux, A and Lima, JB and Borges, VM and Farias, LP},
title = {LPG2 Gene Duplication in Leishmania infantum: A Case for CRISPR-Cas9 Gene Editing.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {408},
pmid = {32903718},
issn = {2235-2988},
mesh = {CRISPR-Cas Systems ; Gene Duplication ; Gene Editing ; Glycosphingolipids ; Humans ; *Leishmania infantum/genetics ; *Leishmania major ; Membrane Proteins/metabolism ; Protozoan Proteins/genetics/metabolism ; },
abstract = {On the surface of the Leishmania promastigote, phosphoglycans (PG) such as lipophosphoglycan (LPG), proteophosphoglycan (PPG), free phosphoglycan polymers (PGs), and acid phosphatases (sAP), are dominant and contribute to the invasion and survival of Leishmania within the host cell by modulating macrophage signaling and intracellular trafficking. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by the LPG2 gene. Aiming to investigate the role of PG-containing molecules in Leishmania infantum infection process, herein we describe the generation and characterization of L. infantum LPG2-deficient parasites. This gene was unexpectedly identified as duplicated in the L. infantum genome, which impaired gene targeting using the conventional homologous recombination approach. This limitation was circumvented by the use of CRISPR/Cas9 technology. Knockout parasites were selected by agglutination assays using CA7AE antibodies followed by a lectin (RCA 120). Five clones were isolated and molecularly characterized, all revealing the expected edited genome, as well as the complete absence of LPG and PG-containing molecule expression. Finally, the deletion of LPG2 was found to impair the outcome of infection in human neutrophils, as demonstrated by a pronounced reduction (~83%) in intracellular load compared to wild-type parasite infection. The results obtained herein reinforce the importance of LPG and other PGs as virulence factors in host-parasite interactions.},
}
@article {pmid32903507,
year = {2020},
author = {Rittiner, JE and Moncalvo, M and Chiba-Falek, O and Kantor, B},
title = {Gene-Editing Technologies Paired With Viral Vectors for Translational Research Into Neurodegenerative Diseases.},
journal = {Frontiers in molecular neuroscience},
volume = {13},
number = {},
pages = {148},
pmid = {32903507},
issn = {1662-5099},
support = {R01 AG057522/AG/NIA NIH HHS/United States ; RF1 NS113548/NS/NINDS NIH HHS/United States ; },
abstract = {Diseases of the central nervous system (CNS) have historically been among the most difficult to treat using conventional pharmacological approaches. This is due to a confluence of factors, including the limited regenerative capacity and overall complexity of the brain, problems associated with repeated drug administration, and difficulties delivering drugs across the blood-brain barrier (BBB). Viral-mediated gene transfer represents an attractive alternative for the delivery of therapeutic cargo to the nervous system. Crucially, it usually requires only a single injection, whether that be a gene replacement strategy for an inherited disorder or the delivery of a genome- or epigenome-modifying construct for treatment of CNS diseases and disorders. It is thus understandable that considerable effort has been put towards the development of improved vector systems for gene transfer into the CNS. Different viral vectors are of course tailored to their specific applications, but they generally should share several key properties. The ideal viral vector incorporates a high-packaging capacity, efficient gene transfer paired with robust and sustained expression, lack of oncogenicity, toxicity and pathogenicity, and scalable manufacturing for clinical applications. In this review, we will devote attention to viral vectors derived from human immunodeficiency virus type 1 (lentiviral vectors; LVs) and adeno-associated virus (AAVs). The high interest in these viral delivery systems vectors is due to: (i) robust delivery and long-lasting expression; (ii) efficient transduction into postmitotic cells, including the brain; (iii) low immunogenicity and toxicity; and (iv) compatibility with advanced manufacturing techniques. Here, we will outline basic aspects of LV and AAV biology, particularly focusing on approaches and techniques aiming to enhance viral safety. We will also allocate a significant portion of this review to the development and use of LVs and AAVs for delivery into the CNS, with a focus on the genome and epigenome-editing tools based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) and the development of novel strategies for the treatment of neurodegenerative diseases (NDDs).},
}
@article {pmid32903482,
year = {2020},
author = {Morgan, MA and Büning, H and Sauer, M and Schambach, A},
title = {Use of Cell and Genome Modification Technologies to Generate Improved "Off-the-Shelf" CAR T and CAR NK Cells.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {1965},
pmid = {32903482},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Clinical Trials as Topic ; Combined Modality Therapy ; Gene Editing ; Gene Transfer Techniques ; Genetic Engineering ; Genetic Therapy ; Genetic Vectors/genetics ; Humans ; *Immunotherapy, Adoptive ; Killer Cells, Natural/*immunology/*metabolism ; Neoplasms/immunology/therapy ; Pluripotent Stem Cells ; Receptors, Antigen, T-Cell/genetics/*immunology ; Receptors, Chimeric Antigen/genetics/*immunology ; Stem Cell Transplantation/methods ; T-Lymphocytes/*immunology/*metabolism ; Transplantation, Autologous ; Transplantation, Homologous ; },
abstract = {The broad success of adoptive immunotherapy to treat human cancer has resulted in a paradigm shift in modern medicine. Modification of autologous and allogenic immune cells with chimeric antigen receptors (CAR) designed to target specific antigens on tumor cells has led to production of CAR T and CAR NK cell therapies, which are ever more commonly introduced into cancer patient treatment protocols. While allogenic T cells may offer advantages such as improved anti-tumor activity, they also carry the risk of adverse reactions like graft-versus-host disease. This risk can be mitigated by use of autologous immune cells, however, the time needed for T and/or NK cell isolation, modification and expansion may be too long for some patients. Thus, there is an urgent need for strategies to robustly produce "off-the-shelf" CAR T and CAR NK cells, which could be used as a bridging therapy between cancer diagnosis or relapse and allogeneic transplantation. Advances in genome modification technologies have accelerated the generation of designer cell therapy products, including development of "off-the-shelf" CAR T cells for cancer immunotherapy. The feasibility and safety of such approaches is currently tested in clinical trials. This review will describe cell sources for CAR-based therapies, provide background of current genome editing techniques and the applicability of these approaches for generation of universal "off-the-shelf" CAR T and NK cell therapeutics.},
}
@article {pmid32903441,
year = {2020},
author = {Newire, E and Aydin, A and Juma, S and Enne, VI and Roberts, AP},
title = {Identification of a Type IV-A CRISPR-Cas System Located Exclusively on IncHI1B/IncFIB Plasmids in Enterobacteriaceae.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1937},
pmid = {32903441},
issn = {1664-302X},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are diverse immune systems found in many prokaryotic genomes that target invading foreign DNA such as bacteriophages and plasmids. There are multiple types of CRISPR with arguably the most enigmatic being Type IV. During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a Type IV-A3 CRISPR-Cas system was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the United Kingdom (isolated in 2017). Sequence analysis of all other genomes available in GenBank revealed that this CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on a bacterial chromosome. This system is exclusively located on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Expression of the cas loci was confirmed in the available clinical isolates by RT-PCR. In all cases, the CRISPR-Cas system has a single CRISPR array (CRISPR1) upstream of the cas loci which has several, conserved, spacers which, amongst things, match regions within conjugal transfer genes of IncFIIK/IncFIB(K) plasmids. Our results reveal a Type IV-A3 CRISPR-Cas system exclusively located on IncHI1B/IncFIB plasmids in Enterobacteriaceae that is likely to be able to target IncFIIK/IncFIB(K) plasmids presumably facilitating intracellular, inter-plasmid competition.},
}
@article {pmid32902645,
year = {2021},
author = {Daher, M and Basar, R and Gokdemir, E and Baran, N and Uprety, N and Nunez Cortes, AK and Mendt, M and Kerbauy, LN and Banerjee, PP and Shanley, M and Imahashi, N and Li, L and Lim, FLWI and Fathi, M and Rezvan, A and Mohanty, V and Shen, Y and Shaim, H and Lu, J and Ozcan, G and Ensley, E and Kaplan, M and Nandivada, V and Bdiwi, M and Acharya, S and Xi, Y and Wan, X and Mak, D and Liu, E and Jiang, XR and Ang, S and Muniz-Feliciano, L and Li, Y and Wang, J and Kordasti, S and Petrov, N and Varadarajan, N and Marin, D and Brunetti, L and Skinner, RJ and Lyu, S and Silva, L and Turk, R and Schubert, MS and Rettig, GR and McNeill, MS and Kurgan, G and Behlke, MA and Li, H and Fowlkes, NW and Chen, K and Konopleva, M and Champlin, RE and Shpall, EJ and Rezvani, K},
title = {Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.},
journal = {Blood},
volume = {137},
number = {5},
pages = {624-636},
pmid = {32902645},
issn = {1528-0020},
support = {P01 CA148600/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; },
mesh = {Aerobiosis ; Animals ; Antigens, CD19/immunology ; Burkitt Lymphoma/pathology/therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Fetal Blood/*cytology ; Gene Knockout Techniques ; Glycolysis ; Humans ; Immune Checkpoint Inhibitors/pharmacology ; *Immunotherapy, Adoptive ; Interleukin-15/*genetics/metabolism ; Killer Cells, Natural/*drug effects/immunology/metabolism/transplantation ; Mechanistic Target of Rapamycin Complex 1/physiology ; Mice ; Neoplasm Proteins/*antagonists &amp; inhibitors/genetics/physiology ; Proto-Oncogene Proteins c-akt/physiology ; Receptors, Chimeric Antigen ; Signal Transduction/physiology ; Suppressor of Cytokine Signaling Proteins/*antagonists &amp; inhibitors/genetics/physiology ; Xenograft Model Antitumor Assays ; },
abstract = {Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.},
}
@article {pmid32901864,
year = {2020},
author = {Jiang, FN and Liang, YX and Wei, W and Zou, CY and Chen, GX and Wan, YP and Liu, ZZ and Yang, Y and Han, ZD and Zhu, JG and Zhong, WD},
title = {Functional classification of prostate cancer‑associated miRNAs through CRISPR/Cas9‑mediated gene knockout.},
journal = {Molecular medicine reports},
volume = {22},
number = {5},
pages = {3777-3784},
pmid = {32901864},
issn = {1791-3004},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Disease Progression ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques/*methods ; Glycolysis ; Humans ; Male ; MicroRNAs/*genetics ; Prostatic Neoplasms/*genetics ; },
abstract = {The aim of the present study was to use the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‑associated (Cas) 9‑mediated gene knockout technology for the rapid classification of the differential function of micro (mi)RNAs screened using miRNA expression profiling by microarray. The rational design of single guide RNAs for the CRISPR/Cas9 system was verified to function in human LNCaP cells with rapid and efficient target gene editing. miRNA (miR)‑205, miR‑221, miR‑222, miR‑30c, miR‑224, miR‑455‑3p, miR‑23b and miR‑505 were downregulated in patients with prostate cancer (PCa) and were experimentally validated to function as tumor suppressors in prostate cancer cells, affecting tumor proliferation, invasion and aerobic glycolysis. In addition, the data of the present study suggested that miR‑663a and mfiR‑1225‑5p were upregulated in prostate cancer tissues and cell proliferation of miR‑663a and miR‑1225‑5p knockout PCa cells was significantly lower compared with miR‑NC cells. Furthermore, knockout of miR‑1225‑5p and miR‑663a significantly decreased the lactate production in LNCaP cells in vitro. In conclusion, the present study offered a simple and efficient method for rapidly classifying miRNA function by applying CRISPR/Cas9 in LNCaP cells. The present study suggested, for the first time to the best of the authors' knowledge, that the aberrant expression of miR‑663a and miR‑1225‑5p may be involved with the progression of prostate cancer, implying their potential as candidate markers for this type of cancer. However, the precise role of miR‑663a and miR‑1225‑5p in accelerating the development of prostate cancer and promoting tumor progression remains to be elucidated.},
}
@article {pmid32901070,
year = {2020},
author = {You, Y and Ramachandra, SG and Jin, T},
title = {A CRISPR-based method for testing the essentiality of a gene.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14779},
pmid = {32901070},
issn = {2045-2322},
mesh = {Amino Acid Sequence ; Base Sequence ; *CRISPR-Cas Systems ; Dictyostelium/*genetics/growth &amp; development/metabolism ; *Gene Editing ; *Genes, Essential ; Protozoan Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; Sequence Homology ; },
abstract = {The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations in the targeted region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n + 1 and 3n + 2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.},
}
@article {pmid32900994,
year = {2020},
author = {Crone, MA and Priestman, M and Ciechonska, M and Jensen, K and Sharp, DJ and Anand, A and Randell, P and Storch, M and Freemont, PS},
title = {A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4464},
pmid = {32900994},
issn = {2041-1723},
support = {NIHR-RP-011-048/DH_/Department of Health/United Kingdom ; },
mesh = {Betacoronavirus/*genetics/isolation &amp; purification ; Biological Assay ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/instrumentation/*methods/standards ; Coronavirus Infections/*diagnosis/*virology ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; Pneumonia, Viral/*diagnosis/*virology ; RNA, Viral/analysis/genetics/isolation &amp; purification ; Real-Time Polymerase Chain Reaction ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.},
}
@article {pmid32899704,
year = {2020},
author = {Yang, H and Ren, S and Yu, S and Pan, H and Li, T and Ge, S and Zhang, J and Xia, N},
title = {Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks.},
journal = {International journal of molecular sciences},
volume = {21},
number = {18},
pages = {},
pmid = {32899704},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics/physiology ; DNA Repair/genetics/physiology ; Endonucleases/metabolism ; Gene Editing/*methods/trends ; Humans ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; },
abstract = {Precise gene editing is-or will soon be-in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs-nonhomologous end joining (NHEJ) and homology-directed repair (HDR)-and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.},
}
@article {pmid32897876,
year = {2020},
author = {Sunuwar, L and Frkatović, A and Sharapov, S and Wang, Q and Neu, HM and Wu, X and Haritunians, T and Wan, F and Michel, S and Wu, S and Donowitz, M and McGovern, D and Lauc, G and Sears, C and Melia, J},
title = {Pleiotropic ZIP8 A391T implicates abnormal manganese homeostasis in complex human disease.},
journal = {JCI insight},
volume = {5},
number = {20},
pages = {},
pmid = {32897876},
issn = {2379-3708},
support = {R24 DK099803/DK/NIDDK NIH HHS/United States ; P30 DK089502/DK/NIDDK NIH HHS/United States ; U54 AG062333/AG/NIA NIH HHS/United States ; K08 DK114478/DK/NIDDK NIH HHS/United States ; S10 OD025244/OD/NIH HHS/United States ; R01 DK116352/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cation Transport Proteins/*genetics ; Colitis/chemically induced/genetics/pathology ; Crohn Disease/*genetics/metabolism/pathology ; Dextran Sulfate/toxicity ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Homeostasis/genetics ; Humans ; Kidney/*metabolism/pathology ; Liver/metabolism/pathology ; Male ; Manganese/blood/*metabolism ; Mice ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic SNPs identified in GWAS; A391T has associations with an increased risk of schizophrenia, obesity, Crohn's disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knockin (KI) to generate a mouse model of ZIP8 A391T (Zip8 393T-KI mice). Recapitulating the SNP association with blood Mn, blood Mn was reduced in Zip8 393T-KI mice. There was restricted abnormal tissue Mn homeostasis, with decreases in liver and kidney Mn and a reciprocal increase in biliary Mn, providing in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemically induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of patients with Crohn's disease. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in complex human disease.},
}
@article {pmid32896989,
year = {2020},
author = {Liu, H and Li, D and Zhou, L and Kan, S and He, G and Zhou, K and Wang, L and Chen, M and Shu, W},
title = {LMNA functions as an oncogene in hepatocellular carcinoma by regulating the proliferation and migration ability.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {20},
pages = {12008-12019},
pmid = {32896989},
issn = {1582-4934},
mesh = {Animals ; Apoptosis/genetics ; Base Sequence ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/*genetics/*pathology ; Cell Cycle Checkpoints/genetics ; *Cell Movement/genetics ; Cell Proliferation/genetics ; Extracellular Matrix/metabolism ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; Hep G2 Cells ; Humans ; Lamin Type A/*genetics ; Liver Neoplasms/*genetics/*pathology ; Mice, Inbred BALB C ; Mice, Nude ; Models, Biological ; *Oncogenes ; Signal Transduction ; Tumor Stem Cell Assay ; Up-Regulation/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {The role of the LMNA gene in the development and progression of hepatocellular carcinoma (HCC) and the associated molecular mechanism is not yet clear. Therefore, the purpose of this study was to evaluate the relationship between LMNA and HCC. LMNA gene expression in normal tissues and corresponding tumours was evaluated and the Kaplan-Meier survival analysis was performed. Next, the LMNA gene was knocked out in the 293T and HepG2 cell lines using the CRISPR/Cas9 technique. Subsequently, the proliferation, migration and colony formation rate of the two LMNA knockout cell lines were analysed. Finally, the molecular mechanism affecting the tumorigenesis due to the loss of the LMNA gene was evaluated. The results showed that the LMNA gene was abnormally expressed in many tumours, and the survival rate of the HCC patients with a high expression of the LMNA gene was significantly reduced compared with the rate in patients with a low LMNA expression. The knockout of the LMNA gene in the HCC cell line HepG2 resulted in a decreased tumorigenicity, up-regulation of the P16 expression and down-regulation of the CDK1 expression. These findings suggested that LMNA might function as an oncogene in HCC and provided a potential new target for the diagnosis and treatment of HCC.},
}
@article {pmid32896179,
year = {2020},
author = {Chauhan, N and Soni, S and Gupta, A and Jain, U},
title = {New and developing diagnostic platforms for COVID-19: A systematic review.},
journal = {Expert review of molecular diagnostics},
volume = {20},
number = {9},
pages = {971-983},
doi = {10.1080/14737159.2020.1816466},
pmid = {32896179},
issn = {1744-8352},
mesh = {Betacoronavirus/*genetics ; Biosensing Techniques ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; *Clinical Laboratory Techniques ; Coronavirus Infections/*diagnosis/diagnostic imaging ; Expert Testimony ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis ; Reagent Kits, Diagnostic ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Tomography, X-Ray Computed/methods ; Ultrasonography/methods ; },
abstract = {INTRODUCTION: The starting months of 2020 witnessed a global pandemic of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. The first case of Coronavirus Disease 2019 (COVID-19) was reported in December, 2019 in Wuhan, China and millions of cases and thousands of deaths were reported within five months. Currently, reverse transcription-polymerase chain reaction (RT-PCR) and computed tomography (CT) scanning are clinically prescribed for COVID-19 detection across the globe.<br><br>AREAS COVERED: This systematic review is focused on currently used diagnostic methods for COVID-19 detection and their future prospects. Online searches on Google Scholar, PubMed and online resources were conducted on the period of year 2017 to mid-2020. Studies investigating laboratory examinations, radiographical analysis, and potential sensors for COVID-19 detection were included. Along with this, the current status of commercially available kits for SARS-CoV-2 coronavirus detection is discussed.<br><br>EXPERT OPINION: The search has identified the potential applications of nucleic acid technology, diagnostics radiology examinations, and in-vitro diagnostic kits in detection of COVID-19 infections. Despite having their own limitations of each technology, the emerging diagnostic technologies for COVID-19 detection along with undergoing clinical trials are summarized suggesting more collaborations and funding are required for fast track clinical trials.},
}
@article {pmid32895670,
year = {2020},
author = {Zamolo, SJ and Darbre, T and Reymond, JL},
title = {Transfecting tissue models with CRISPR/Cas9 plasmid DNA using peptide dendrimers.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {80},
pages = {11981-11984},
doi = {10.1039/d0cc04750c},
pmid = {32895670},
issn = {1364-548X},
mesh = {CRISPR-Cas Systems ; Cell Survival/drug effects ; DNA/*chemistry/metabolism ; Dendrimers/*chemistry/metabolism ; Gene Editing ; Genetic Therapy ; HEK293 Cells ; HeLa Cells ; Heparin/chemistry ; Humans ; Peptides/*chemistry ; Phosphatidylethanolamines/chemistry ; Piperidines/chemistry ; Spheroids, Cellular/chemistry ; Transfection/*methods ; },
abstract = {There is currently a lack of efficient reagents to transfect cells with large plasmid DNA, which would be enabling tools for gene editing using CRISPR/Cas9 technology. Herein, we report the discovery of peptide dendrimer Z22 as a non-viral vector for transfecting large CRISPR/Cas9 pDNA into 3D-tumor spheroids with exceptionally high efficiency, low cytotoxicity and low immunogenicity.},
}
@article {pmid32895556,
year = {2020},
author = {Zhang, H and Li, Z and Xiao, R and Chang, L},
title = {Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease.},
journal = {Nature structural &amp; molecular biology},
volume = {27},
number = {11},
pages = {1069-1076},
pmid = {32895556},
issn = {1545-9985},
support = {R01 GM138675/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/chemistry/*metabolism ; CRISPR-Cas Systems ; Clostridiales/chemistry/*metabolism ; DNA/chemistry/metabolism ; Endonucleases/chemistry/*metabolism ; Enzyme Activation ; Models, Molecular ; Protein Conformation ; RNA/chemistry/metabolism ; },
abstract = {Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA-DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications.},
}
@article {pmid32895548,
year = {2021},
author = {Jones, SK and Hawkins, JA and Johnson, NV and Jung, C and Hu, K and Rybarski, JR and Chen, JS and Doudna, JA and Press, WH and Finkelstein, IJ},
title = {Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.},
journal = {Nature biotechnology},
volume = {39},
number = {1},
pages = {84-93},
pmid = {32895548},
issn = {1546-1696},
support = {F32 AG053051/AG/NIA NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Bacterial Proteins/chemistry/genetics/metabolism ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; Gene Editing ; High-Throughput Nucleotide Sequencing/*methods ; Kinetics ; Protein Binding/genetics ; Protein Engineering ; RNA, Guide/chemistry/genetics/metabolism ; Substrate Specificity/genetics ; },
abstract = {Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.},
}
@article {pmid32895531,
year = {2020},
author = {Baker, M},
title = {When antibodies mislead: the quest for validation.},
journal = {Nature},
volume = {585},
number = {7824},
pages = {313-314},
pmid = {32895531},
issn = {1476-4687},
mesh = {Antibodies/*immunology ; Antibody Specificity/*immunology ; *Artifacts ; CRISPR-Cas Systems ; Fluorescent Antibody Technique/standards ; Gene Knockout Techniques ; Immunoblotting/standards ; Immunoprecipitation/standards ; Indicators and Reagents/*standards ; Reproducibility of Results ; *Research Design ; *Validation Studies as Topic ; },
}
@article {pmid32893996,
year = {2020},
author = {Langer, HT and Mossakowski, AA and Willis, BJ and Grimsrud, KN and Wood, JA and Lloyd, KCK and Zbinden-Foncea, H and Baar, K},
title = {Generation of desminopathy in rats using CRISPR-Cas9.},
journal = {Journal of cachexia, sarcopenia and muscle},
volume = {11},
number = {5},
pages = {1364-1376},
pmid = {32893996},
issn = {2190-6009},
support = {K01 OD026608/OD/NIH HHS/United States ; R01 AG045375/AG/NIA NIH HHS/United States ; R01 AG056999/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Desmin/genetics/metabolism ; Dystrophin ; Male ; Mice ; *Muscular Diseases/genetics ; Mutation ; Rats ; },
abstract = {BACKGROUND: Desminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.<br><br>METHODS: Using CRISPR-Cas9 technology, Des c.1045-1046 (AGG&#xa0;>&#xa0;CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n&#xa0;=&#xa0;6) between the age of 120-150&#xa0;days and an equal number of wild type littermates (n&#xa0;=&#xa0;6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.<br><br>RESULTS: We found a difference in the number of central nuclei between desmin mutants (1&#xa0;&#xb1;&#xa0;0.4%) and wild type littermates (0.2&#xa0;&#xb1;&#xa0;0.1%; P&#xa0;<&#xa0;0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P&#xa0;<&#xa0;0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P&#xa0;<&#xa0;0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P&#xa0;<&#xa0;0.05).<br><br>CONCLUSIONS: We created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression.},
}
@article {pmid32893952,
year = {2021},
author = {Wang, Y and Huang, Y and Xu, X and Liu, Z and Li, J and Zhan, X and Yang, G and You, M and You, S},
title = {CRISPR/Cas9-based functional analysis of yellow gene in the diamondback moth, Plutella xylostella.},
journal = {Insect science},
volume = {28},
number = {5},
pages = {1504-1509},
pmid = {32893952},
issn = {1744-7917},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Genes, Insect ; Larva/genetics ; *Moths/genetics ; Oviposition ; *Pigmentation/genetics ; Pupa/genetics ; },
abstract = {The diamondback moth, Plutella xylostella (L.), is an economically important pest of cruciferous crops worldwide. This pest is notorious for rapid evolution of the resistance to different classes of insecticides, making it increasingly difficult to control. Genetics-based control approaches, through manipulation of target genes, have been reported as promising supplements or alternatives to traditional methods of pest management. Here we identified a gene of pigmentation (yellow) in P. xylostella, Pxyellow, which encodes 1674 bp complementary DNA sequence with four exons and three introns. Using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR-associated protein 9 system, we knocked out Pxyellow, targeting two sites in Exon III, to generate 272 chimeric mutants (57% of the CRISPR-treated individuals) with color-changed phenotypes of the 1st to 3rd instar larvae, pupae, and adults, indicating that Pxyellow plays an essential role in the body pigmentation of P. xylostella. Fitness analysis revealed no significant difference in the oviposition of adults, the hatchability of eggs, and the weight of pupae between homozygous mutants and wildtypes, suggesting that Pxyellow is not directly involved in regulation of growth, development, or reproduction. This work advances our understanding of the genetic and insect science molecular basis for body pigmentation of P. xylostella, and opens a wide avenue for development of the genetically based pest control techniques using Pxyellow as a screening marker.},
}
@article {pmid32893711,
year = {2020},
author = {Xue, VW and Wong, SCC and Cho, WCS},
title = {Genome-wide CRISPR screens for the identification of therapeutic targets for cancer treatment.},
journal = {Expert opinion on therapeutic targets},
volume = {24},
number = {11},
pages = {1147-1158},
doi = {10.1080/14728222.2020.1820986},
pmid = {32893711},
issn = {1744-7631},
mesh = {Animals ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems/*genetics ; Drug Development ; Gene Editing ; Humans ; Immunotherapy/methods ; *Molecular Targeted Therapy ; Neoplasms/genetics/pathology/*therapy ; Precision Medicine ; RNA Interference ; },
abstract = {INTRODUCTION: Exploring the function of every gene is a challenging task. There is a paradigm shift of RNA interference with the introduction of clustered regularly interspaced short palindromic repeat (CRISPR)-based genome-wide screening. CRISPR-based screening can detect the loss-of-function and gain-of-function targets. Many DNA-binding proteins are engineered as effective tools for modulating gene expression and for investigating therapeutic targets for a spectrum of diseases. Among them, CRISPR-Cas9 has received extensive attention with its potential for screening cancer treatment targets.<br><br>AREAS COVERED: This article reviews CRISPR toolkit and its applications in screening cancer therapeutic targets, especially genome-wide screens using different CRISPR-Cas9 systems. We compare and summarize the characteristics of CRISPR systems, which would be helpful for understanding and optimizing current CRISPR toolkits, as well as reflecting on the potential future development and clinical applications of CRISPR screens.<br><br>EXPERT OPINION: The application of CRISPR-based therapeutic target screening is broadly used in cancer drug development. Its application in cancer immunotherapy and precision oncology is blooming. Nevertheless, more effective methods of Cas protein delivery and the development of more accurate and efficient genome-editing tools are needed.},
}
@article {pmid32893479,
year = {2021},
author = {Saba, LM and Hoffman, PL and Homanics, GE and Mahaffey, S and Daulatabad, SV and Janga, SC and Tabakoff, B},
title = {A long non-coding RNA (Lrap) modulates brain gene expression and levels of alcohol consumption in rats.},
journal = {Genes, brain, and behavior},
volume = {20},
number = {2},
pages = {e12698},
pmid = {32893479},
issn = {1601-183X},
support = {R37 AA010422/AA/NIAAA NIH HHS/United States ; R24 AA013162/AA/NIAAA NIH HHS/United States ; R01 GM123314/GM/NIGMS NIH HHS/United States ; P30 DA044223/DA/NIDA NIH HHS/United States ; U01 AA020889/AA/NIAAA NIH HHS/United States ; },
mesh = {Alcohol Drinking/*genetics/physiopathology ; Animals ; Brain/*metabolism ; Quantitative Trait Loci ; RNA, Long Noncoding/*genetics/metabolism ; Rats ; Rats, Wistar ; Transcriptome ; },
abstract = {LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The "hub gene" of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of "Response to Ethanol" emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype.},
}
@article {pmid32892813,
year = {2020},
author = {Min, YL and Chemello, F and Li, H and Rodriguez-Caycedo, C and Sanchez-Ortiz, E and Mireault, AA and McAnally, JR and Shelton, JM and Zhang, Y and Bassel-Duby, R and Olson, EN},
title = {Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {9},
pages = {2044-2055},
pmid = {32892813},
issn = {1525-0024},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/metabolism ; *Exons ; *Gene Deletion ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Mice, Inbred C57BL ; Muscle, Skeletal/metabolism ; Muscular Dystrophy, Duchenne/*genetics/metabolism ; Myocytes, Cardiac/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational "hotspot" regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.},
}
@article {pmid32891884,
year = {2020},
author = {Schenke, D and Cai, D},
title = {Applications of CRISPR/Cas to Improve Crop Disease Resistance: Beyond Inactivation of Susceptibility Factors.},
journal = {iScience},
volume = {23},
number = {9},
pages = {101478},
pmid = {32891884},
issn = {2589-0042},
abstract = {Current crop production systems are prone to increasing pathogen pressure. Fundamental understanding of molecular plant-pathogen interactions, the availability of crop and pathogen genomic information, as well as emerging genome editing permits a novel approach for breeding of crop disease resistance. We describe here strategies to identify new targets for resistance breeding with focus on interruption of the compatible plant-pathogen interaction by CRISPR/Cas-mediated genome editing. Basically, crop genome editing can be applied in several ways to achieve this goal. The most common approach focuses on the "simple" knockout by non-homologous end joining repair of plant susceptibility factors required for efficient host colonization. However, genome re-writing via homology-directed repair or base editing can also prevent host manipulation by changing the targets of pathogen-derived effectors or molecules beyond recognition, which also decreases plant susceptibility. We conclude that genome editing by CRISPR/Cas will become increasingly indispensable to generate in relatively short time beneficial resistance traits in crops to meet upcoming challenges.},
}
@article {pmid32891680,
year = {2020},
author = {Boucher, P and Cui, X and Curiel, DT},
title = {Adenoviral vectors for in vivo delivery of CRISPR-Cas gene editors.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {327},
number = {},
pages = {788-800},
pmid = {32891680},
issn = {1873-4995},
support = {R01 CA211096/CA/NCI NIH HHS/United States ; R01 EB026468/EB/NIBIB NIH HHS/United States ; T32 HL007088/HL/NHLBI NIH HHS/United States ; UG3 TR002851/TR/NCATS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Therapy ; Genetic Vectors ; },
abstract = {Harnessing the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing in eukaryotes has revolutionized basic biomedical research and translational sciences. The ability to create targeted alterations of the genome through this easy to design system has presented unprecedented opportunities to treat inherited disorders and other diseases such as cancer through gene therapy. A major hurdle is the lack of an efficient and safe in vivo delivery system, limiting most of the current gene therapy efforts to ex vivo editing of extracted cells. Here we discuss the unique features of adenoviral vectors that enable tissue specific and efficient delivery of the CRISPR-Cas machinery for in vivo genome editing.},
}
@article {pmid32891169,
year = {2020},
author = {Darvish-Ghane, S and Quintana, C and Beaulieu, JM and Martin, LJ},
title = {D1 receptors in the anterior cingulate cortex modulate basal mechanical sensitivity threshold and glutamatergic synaptic transmission.},
journal = {Molecular brain},
volume = {13},
number = {1},
pages = {121},
pmid = {32891169},
issn = {1756-6606},
support = {MOP-13691//CIHR/Canada ; PJT-166171//CIHR/Canada ; },
mesh = {Animals ; Behavior, Animal/drug effects ; Benzazepines/pharmacology ; CRISPR-Cas Systems/genetics ; Excitatory Postsynaptic Potentials/drug effects ; Gene Knockout Techniques ; Glutamic Acid/*metabolism ; Gyrus Cinguli/drug effects/*metabolism/physiopathology ; Ion Channel Gating/drug effects ; Male ; Mice, Inbred C57BL ; Pain/pathology/physiopathology ; Receptors, Dopamine D1/agonists/antagonists &amp; inhibitors/*metabolism ; Receptors, N-Methyl-D-Aspartate/metabolism ; *Sensory Thresholds/drug effects ; *Synaptic Transmission/drug effects ; },
abstract = {The release of dopamine (DA) into target brain areas is considered an essential event for the modulation of many physiological effects. While the anterior cingulate cortex (ACC) has been implicated in pain related behavioral processes, DA modulation of synaptic transmission within the ACC and pain related phenotypes remains unclear. Here we characterized a Crispr/Cas9 mediated somatic knockout of the D1 receptor (D1R) in all neuronal subtypes of the ACC and find reduced mechanical thresholds, without affecting locomotion and anxiety. Further, the D1R high-efficacy agonist SKF 81297 and low efficacy agonist (±)-SKF-38393 inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) currents in the ACC. Paradoxically, the D1R antagonists SCH-23390 and SCH 33961 when co-applied with D1R agonists produced a robust short-term synergistic depression of AMPAR currents in the ACC, demonstrating an overall inhibitory role for D1R ligands. Overall, our data indicate that absence of D1Rs in the ACC enhanced peripheral sensitivity to mechanical stimuli and D1R activation decreased glutamatergic synaptic transmission in ACC neurons.},
}
@article {pmid32890931,
year = {2020},
author = {Wang, DX and Wang, J and Du, YC and Ma, JY and Wang, SY and Tang, AN and Kong, DM},
title = {CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase.},
journal = {Biosensors &amp; bioelectronics},
volume = {168},
number = {},
pages = {112556},
doi = {10.1016/j.bios.2020.112556},
pmid = {32890931},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Phosphoric Monoester Hydrolases ; *Polynucleotide 5'-Hydroxyl-Kinase/genetics ; },
abstract = {We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10[-5] to 2.5 × 10[-2] U/mL T4 PNKP) and a detection limit as low as 3.3 × 10[-6] U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening.},
}
@article {pmid32889721,
year = {2021},
author = {Agier, N and Fleiss, A and Delmas, S and Fischer, G},
title = {A Versatile Protocol to Generate Translocations in Yeast Genomes Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2196},
number = {},
pages = {181-198},
doi = {10.1007/978-1-0716-0868-5_14},
pmid = {32889721},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; DNA Shuffling ; Gene Editing ; Gene Order ; Gene Rearrangement ; Genetic Engineering/methods ; Genetic Vectors/genetics ; *Genome, Fungal ; Plasmids/genetics ; RNA, Guide ; Recombination, Genetic ; Transformation, Genetic ; *Translocation, Genetic ; Yeasts/*genetics ; },
abstract = {Genomic engineering methods represent powerful tools to examine chromosomal modifications and to subsequently study their impacts on cellular phenotypes. However, quantifying the fitness impact of translocations, independently from base substitutions or the insertion of genetic markers, remains a challenge. Here we report a rapid and straightforward protocol for engineering either targeted reciprocal translocations at the base pair level of resolution between two chromosomes or multiple simultaneous rearrangements in the yeast genome, without inserting any marker sequence in the chromosomes. Our CRISPR/Cas9-based method consists of inducing either (1) two double-strand breaks (DSBs) in two different chromosomes with two distinct guide RNAs (gRNAs) while providing specifically designed homologous donor DNA forcing the trans-repair of chromosomal extremities to generate a targeted reciprocal translocation or (2) multiple DSBs with a single gRNA targeting dispersed repeated sequences and leaving endogenous uncut copies of the repeat to be used as donor DNA, thereby generating multiple translocations, often associated with large segmental duplications (Fleiss, et al. PLoS Genet 15:e1008332, 2019).},
}
@article {pmid32889710,
year = {2021},
author = {Kuroda, K and Ueda, M},
title = {CRISPR Nickase-Mediated Base Editing in Yeast.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2196},
number = {},
pages = {27-37},
doi = {10.1007/978-1-0716-0868-5_3},
pmid = {32889710},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Repair ; Deoxyribonuclease I/*metabolism ; *Gene Editing ; Gene Order ; Genetic Vectors/genetics ; Mutagenesis ; RNA, Guide ; Recombinational DNA Repair ; Saccharomyces cerevisiae/genetics/metabolism ; Yeasts/*genetics/*metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5 days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.},
}
@article {pmid32888879,
year = {2020},
author = {Sun, H and Zhi, S and Wu, G and Wu, G and Cao, T and Hao, H and Songyang, Z and Liang, P and Huang, J},
title = {Cost-effective generation of A-to-G mutant mice by zygote electroporation of adenine base editor ribonucleoproteins.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {6},
pages = {337-340},
doi = {10.1016/j.jgg.2020.05.005},
pmid = {32888879},
issn = {1673-8527},
mesh = {Adenine/metabolism ; Animals ; CRISPR-Cas Systems/*genetics ; *Cost-Benefit Analysis ; Electroporation/economics ; Gene Editing/economics ; Humans ; Mice ; RNA, Guide/economics/*genetics ; Ribonucleoproteins/*genetics ; },
}
@article {pmid32888436,
year = {2020},
author = {Bacman, SR and Moraes, CT},
title = {Mitochondrial DNA Base Editing: Good Editing Things Still Come in Small Packages.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {708-709},
doi = {10.1016/j.molcel.2020.08.009},
pmid = {32888436},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Cytidine Deaminase ; *DNA, Mitochondrial ; Mitochondria/genetics ; },
abstract = {The collaborative work of two HHMI groups, one at the University of Washington and the other at the Broad Institute of MIT and Harvard, led to the development of a novel molecular tool to edit single bases in the mtDNA (Mok et al., 2020).},
}
@article {pmid32888434,
year = {2020},
author = {Collins, SP and Beisel, CL},
title = {Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {703-704},
doi = {10.1016/j.molcel.2020.07.030},
pmid = {32888434},
issn = {1097-4164},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine ; DNA, Single-Stranded ; Deamination ; *Gene Editing ; },
abstract = {Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.},
}
@article {pmid32888044,
year = {2020},
author = {Liu, T and Zhu, L and Zhu, Z and Jiang, L},
title = {Genome Sequence Analysis of Clostridium tyrobutyricum, a Promising Microbial Host for Human Health and Industrial Applications.},
journal = {Current microbiology},
volume = {77},
number = {11},
pages = {3685-3694},
pmid = {32888044},
issn = {1432-0991},
mesh = {Butyric Acid ; *Clostridium tyrobutyricum/genetics ; Fermentation ; Humans ; Plasmids ; Sequence Analysis ; },
abstract = {Clostridium tyrobutyricum is a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. At the same time, it also has great potential as a probiotic for the production of short-chain fatty acids in the intestines. However, due to the insufficient knowledge of the genetic characteristics of this organism, there has been little progress in its genetic engineering to date. Here, we present the complete genome sequence of C. tyrobutyricum L319, which consists of a circular chromosome and a plasmid with a G + C content of 31.69%, encompassing approximately 3.09 Mb with 3052 protein-coding genes. Functional gene annotation revealed better results than previous studies based on KEGG pathway classification. Furthermore, we obtained detailed functional characterization of 93 genes previously annotated as putative proteins. Genomic analysis revealed that this organism contains multiple genes encoding enzymes involved in the CRISPR-Cas systems, substrate utilization, isopeptide and ester bonds, transcriptional regulation, and oxidative stress. The safety evaluation at genetic level indicated that this organism does not possess transferable resistance genes, invasive defensive pathogenicity factors, or harmful enzymes. The genome sequence data analyzed in this study will be available for further research and will facilitate the further understanding and development of potential applications of C. tyrobutyricum.},
}
@article {pmid32887975,
year = {2020},
author = {Roman Azcona, MS and Fang, Y and Carusillo, A and Cathomen, T and Mussolino, C},
title = {A versatile reporter system for multiplexed screening of effective epigenome editors.},
journal = {Nature protocols},
volume = {15},
number = {10},
pages = {3410-3440},
pmid = {32887975},
issn = {1750-2799},
mesh = {CRISPR-Cas Systems ; Cell Line ; DNA/genetics ; DNA Methylation/genetics ; Epigenesis, Genetic/genetics ; Epigenome/*genetics ; Epigenomics/*methods ; Gene Editing/*methods ; Genes, Reporter/genetics ; Genome/genetics ; HEK293 Cells ; Humans ; },
abstract = {The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.},
}
@article {pmid32887910,
year = {2020},
author = {Naert, T and Tulkens, D and Edwards, NA and Carron, M and Shaidani, NI and Wlizla, M and Boel, A and Demuynck, S and Horb, ME and Coucke, P and Willaert, A and Zorn, AM and Vleminckx, K},
title = {Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14662},
pmid = {32887910},
issn = {2045-2322},
support = {R01 HD084409/HD/NICHD NIH HHS/United States ; R24 OD030008/OD/NIH HHS/United States ; P01 HD093363/HD/NICHD NIH HHS/United States ; R21 OD023810/OD/NIH HHS/United States ; P40 OD010997/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Frameshift Mutation ; Gene Editing/*methods ; Gene Frequency ; HEK293 Cells ; Humans ; Mice ; Mouse Embryonic Stem Cells/metabolism ; *Penetrance ; RNA, Guide/genetics ; Xenopus laevis/*embryology/*genetics ; Zebrafish/*embryology/*genetics ; },
abstract = {CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.},
}
@article {pmid32887885,
year = {2020},
author = {Schmidt, C and Fransz, P and Rönspies, M and Dreissig, S and Fuchs, J and Heckmann, S and Houben, A and Puchta, H},
title = {Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4418},
pmid = {32887885},
issn = {2041-1723},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems ; Chromosome Inversion ; *Chromosomes, Plant ; Crossing Over, Genetic ; Genetic Engineering/*methods ; Plant Breeding/methods ; Plants ; *Recombination, Genetic ; },
abstract = {Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.},
}
@article {pmid32887747,
year = {2020},
author = {Kurup, SP and Moioffer, SJ and Pewe, LL and Harty, JT},
title = {p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {205},
number = {8},
pages = {2222-2230},
pmid = {32887747},
issn = {1550-6606},
support = {R35 GM134880/GM/NIGMS NIH HHS/United States ; R01 AI100527/AI/NIAID NIH HHS/United States ; R01 AI042767/AI/NIAID NIH HHS/United States ; R01 AI085515/AI/NIAID NIH HHS/United States ; R21 AI042767/AI/NIAID NIH HHS/United States ; R01 AI114543/AI/NIAID NIH HHS/United States ; R37 AI042767/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antigens/immunology ; CD8-Positive T-Lymphocytes/*immunology ; *CRISPR-Cas Systems ; Cell Proliferation/*genetics ; DNA Damage/genetics/immunology ; Immunologic Memory/*genetics ; Mice ; Mice, Transgenic ; *Tumor Suppressor Protein p53/genetics/immunology ; },
abstract = {CRISPR/Cas9 technology has revolutionized rapid and reliable gene editing in cells. Although many cell types have been subjected to CRISPR/Cas9-mediated gene editing, there is no evidence of success in genetic alteration of Ag-experienced memory CD8 T cells. In this study, we show that CRISPR/Cas9-mediated gene editing in memory CD8 T cells precludes their proliferation after Ag re-encounter in vivo. This defect is mediated by the proapoptotic transcription factor p53, a sensor of DNA damage. Temporarily inhibiting p53 function offers a window of opportunity for the memory CD8 T cells to repair the DNA damage, facilitating robust recall responses on Ag re-encounter. We demonstrate this by functionally altering memory CD8 T cells using CRISPR/Cas9-mediated targeted gene disruption under the aegis of p53siRNA in the mouse model. Our approach thus adapts the CRISPR/Cas9 technology for memory CD8 T cells to undertake gene editing in vivo, for the first time, to our knowledge.},
}
@article {pmid32887745,
year = {2020},
author = {Llamosas, N and Arora, V and Vij, R and Kilinc, M and Bijoch, L and Rojas, C and Reich, A and Sridharan, B and Willems, E and Piper, DR and Scampavia, L and Spicer, TP and Miller, CA and Holder, JL and Rumbaugh, G},
title = {SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {40},
number = {41},
pages = {7980-7994},
pmid = {32887745},
issn = {1529-2401},
support = {R01 NS110307/NS/NINDS NIH HHS/United States ; R01 NS064079/NS/NINDS NIH HHS/United States ; S10 OD026857/OD/NIH HHS/United States ; R01 MH113648/MH/NIMH NIH HHS/United States ; R01 MH096847/MH/NIMH NIH HHS/United States ; K08 NS091381/NS/NINDS NIH HHS/United States ; S10 OD025279/OD/NIH HHS/United States ; R01 MH108408/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Size ; Cells, Cultured ; Dendrites/*physiology ; Excitatory Postsynaptic Potentials/genetics ; Female ; Gene Deletion ; Humans ; Nerve Net/*physiology ; Nervous System/*growth &amp; development ; Neurodevelopmental Disorders/genetics ; Pluripotent Stem Cells ; Synapses/*physiology ; ras GTPase-Activating Proteins/*genetics/*physiology ; },
abstract = {SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.},
}
@article {pmid32887690,
year = {2020},
author = {Chaudhary, N and Nho, SH and Cho, H and Gantumur, N and Ra, JS and Myung, K and Kim, H},
title = {Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore.},
journal = {Genome research},
volume = {30},
number = {9},
pages = {1306-1316},
pmid = {32887690},
issn = {1549-5469},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Structures ; Genetic Techniques ; Green Fluorescent Proteins/chemistry/*genetics ; HEK293 Cells ; Humans ; Image Processing, Computer-Assisted ; },
abstract = {The higher-order structural organization and dynamics of the chromosomes play a central role in gene regulation. To explore this structure-function relationship, it is necessary to directly visualize genomic elements in living cells. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. This feature enables the application of CRISPR-based imaging to loci throughout the genome and opens up new possibilities for the study of nuclear processes in living cells.},
}
@article {pmid32887689,
year = {2020},
author = {Lalli, MA and Avey, D and Dougherty, JD and Milbrandt, J and Mitra, RD},
title = {High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation.},
journal = {Genome research},
volume = {30},
number = {9},
pages = {1317-1331},
pmid = {32887689},
issn = {1549-5469},
support = {U01 MH109133/MH/NIMH NIH HHS/United States ; T32 CA009547/CA/NCI NIH HHS/United States ; RF1 MH117070/MH/NIMH NIH HHS/United States ; U54 HD087011/HD/NICHD NIH HHS/United States ; P50 HD103525/HD/NICHD NIH HHS/United States ; UL1 TR002345/TR/NCATS NIH HHS/United States ; R01 GM123203/GM/NIGMS NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; T32 HL125241/HL/NHLBI NIH HHS/United States ; R21 NS087230/NS/NINDS NIH HHS/United States ; UL1 TR000448/TR/NCATS NIH HHS/United States ; },
mesh = {Autism Spectrum Disorder/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques/methods ; HEK293 Cells ; Humans ; Image Processing, Computer-Assisted ; Models, Genetic ; Neurogenesis/*genetics/physiology ; Neuronal Outgrowth/genetics ; Neurons/*metabolism ; Phenotype ; RNA-Seq ; Single-Cell Analysis/*methods ; Transcriptome ; },
abstract = {The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.},
}
@article {pmid32887353,
year = {2020},
author = {Patrulea, V and Borchard, G and Jordan, O},
title = {An Update on Antimicrobial Peptides (AMPs) and Their Delivery Strategies for Wound Infections.},
journal = {Pharmaceutics},
volume = {12},
number = {9},
pages = {},
pmid = {32887353},
issn = {1999-4923},
abstract = {Bacterial infections occur when wound healing fails to reach the final stage of healing, which is usually hindered by the presence of different pathogens. Different topical antimicrobial agents are used to inhibit bacterial growth due to antibiotic failure in reaching the infected site, which is accompanied very often by increased drug resistance and other side effects. In this review, we focus on antimicrobial peptides (AMPs), especially those with a high potential of efficacy against multidrug-resistant and biofilm-forming bacteria and fungi present in wound infections. Currently, different AMPs undergo preclinical and clinical phase to combat infection-related diseases. AMP dendrimers (AMPDs) have been mentioned as potent microbial agents. Various AMP delivery strategies that are used to combat infection and modulate the healing rate-such as polymers, scaffolds, films and wound dressings, and organic and inorganic nanoparticles-have been discussed as well. New technologies such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (CRISPR-Cas) are taken into consideration as potential future tools for AMP delivery in skin therapy.},
}
@article {pmid32887261,
year = {2020},
author = {Sant'Ana, RRA and Caprestano, CA and Nodari, RO and Agapito-Tenfen, SZ},
title = {PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts.},
journal = {Genes},
volume = {11},
number = {9},
pages = {},
pmid = {32887261},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genome, Plant/genetics ; Plant Cells/physiology ; Polyethylene Glycols/*chemistry ; Protoplasts/physiology ; RNA, Guide/genetics ; Ribonucleoproteins/*genetics ; Zea mays/*genetics/physiology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.},
}
@article {pmid32886837,
year = {2021},
author = {Jiao, J and Kong, K and Han, J and Song, S and Bai, T and Song, C and Wang, M and Yan, Z and Zhang, H and Zhang, R and Feng, J and Zheng, X},
title = {Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a-based visual assay.},
journal = {Plant biotechnology journal},
volume = {19},
number = {2},
pages = {394-405},
pmid = {32886837},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gold ; *Metal Nanoparticles ; Plant Diseases ; *RNA Viruses/genetics ; RNA, Viral/genetics ; Sensitivity and Specificity ; *Viroids/genetics ; },
abstract = {Co-infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a-based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide-conjugated gold nanoparticles. The CRISPR/Cas12a-RT-RPA platform exhibited comparable sensitivity to RT-qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT-PCR detection for 52 samples. This novel Cas12a-based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.},
}
@article {pmid32886694,
year = {2020},
author = {Madaha, EL and Mienie, C and Gonsu, HK and Bughe, RN and Fonkoua, MC and Mbacham, WF and Alayande, KA and Bezuidenhout, CC and Ateba, CN},
title = {Whole-genome sequence of multi-drug resistant Pseudomonas aeruginosa strains UY1PSABAL and UY1PSABAL2 isolated from human broncho-alveolar lavage, Yaoundé, Cameroon.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0238390},
pmid = {32886694},
issn = {1932-6203},
mesh = {Base Composition ; Cameroon ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Multiple/*genetics ; Genome, Bacterial/genetics ; Humans ; Phylogeny ; Prophages/genetics ; Pseudomonas aeruginosa/*genetics/isolation &amp; purification ; Sequence Analysis, DNA ; Virulence/genetics ; Whole Genome Sequencing/methods ; },
abstract = {Pseudomonas aeruginosa has been implicated in a wide range of post-operation wound and lung infections. A wide range of acquired resistance and virulence markers indicate surviving strategy of P. aeruginosa. Complete-genome analysis has been identified as efficient approach towards understanding the pathogenicity of this organism. This study was designed to sequence the entire genome of P. aeruginosa UY1PSABAL and UY1PSABAL2; determine drug-resistance profiles and virulence factors of the isolates; assess factors that contribute toward stability of the genomes; and thereafter determine evolutionary relationships between the strains and other isolates from similar sources. The genomes of the MDR P. aeruginosa UY1PSABAL and UY1PSABAL2 were sequenced on the Illumina Miseq platform. The raw sequenced reads were assessed for quality using FastQC v.0.11.5 and filtered for low quality reads and adapter regions using Trimmomatic v.0.36. The de novo genome assembly was made with SPAdes v.3.13 and annotated using Prokka v.2.1.1 annotation pipeline; Rapid Annotation using Subsytems Technology (RAST) server v.2.0; and PATRIC annotation tool v.3.6.2. Antimicrobial resistance genes and virulence determinants were searched through the functional annotation data generated from Prokka, RAST and PATRIC annotation pipelines; In addition to ResFinder and Comprehensive Antibiotic Resistance Database (CARD) which were employed to determine resistance genes. The PHAge Search Tool Enhanced Release (PHASTER) web server was used for the rapid identification and annotation of prophage sequences within bacterial genome. Predictive secondary metabolites were identified with AntiSMASH v.5.0. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and cas genes regions were also investigated with the CRISPRone and CRISPRFinder server. The genome sizes of 7.0 and 6.4 Mb were determined for UY1PSABAL and UY1PSABAL2 strains with G+C contents of 66.1% and 66.48% respectively. β-lactamines resistance genes blaPAO, aminoglycoside phosphorylating enzymes genes aph(3')-IIb, fosfomycine resistance gene fosA, vancomycin vanW and tetracycline tetA were among identified resistance genes harboured in both isolates. UY1PSABAL bore additional aph(6)-Id, aph(3'')-Ib, ciprofloxacin-modifying enzyme crpP and ribosomal methylation enzyme rmtB. Both isolates were found harbouring virulence markers such as flagella and type IV pili; and also present various type III secretion systems such as exoA, exoS, exoU, exoT. Secondary metabolites such as pyochelin and pyoverdine with iron uptake activity were found within the genomes as well as quorum-sensing systems, and various fragments for prophages and insertion sequences. Only the UY1PSABAL2 contains CRISPR-Cas system. The phylogeny revealed a very close evolutionary relationship between UY1PSABAL and the similar strain isolated from Malaysia; the same trend was observed between UY1PSABAL2 and the strain from Chinese origin. Complete analyses of the entire genomes provide a wide range of information towards understanding pathogenicity of the pathogens in question.},
}
@article {pmid32884147,
year = {2020},
author = {Moghadam, F and LeGraw, R and Velazquez, JJ and Yeo, NC and Xu, C and Park, J and Chavez, A and Ebrahimkhani, MR and Kiani, S},
title = {Synthetic immunomodulation with a CRISPR super-repressor in vivo.},
journal = {Nature cell biology},
volume = {22},
number = {9},
pages = {1143-1154},
pmid = {32884147},
issn = {1476-4679},
support = {R01 HL141805/HL/NHLBI NIH HHS/United States ; U01 EB029372/EB/NIBIB NIH HHS/United States ; T32 EB001026/EB/NIBIB NIH HHS/United States ; U01 DK127713/DK/NIDDK NIH HHS/United States ; R01 EB024562/EB/NIBIB NIH HHS/United States ; R01 EB028532/EB/NIBIB NIH HHS/United States ; P30 DK120531/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; Gene Editing/methods ; HEK293 Cells ; Humans ; Immunomodulation/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myeloid Differentiation Factor 88/immunology ; Proprotein Convertase 9 ; RNA, Guide/immunology ; Receptors, Cell Surface/immunology ; },
abstract = {Transient modulation of the genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from the 5' end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors-heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)-are fused to the MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in the induction of humoral immunity against AAV serotypes.},
}
@article {pmid32884115,
year = {2020},
author = {Ray, U and Raghavan, SC},
title = {Modulation of DNA double-strand break repair as a strategy to improve precise genome editing.},
journal = {Oncogene},
volume = {39},
number = {41},
pages = {6393-6405},
pmid = {32884115},
issn = {1476-5594},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*drug effects/genetics ; DNA Repair Enzymes/*antagonists &amp; inhibitors/metabolism ; DNA-Binding Proteins/antagonists &amp; inhibitors/metabolism ; Gene Editing/*methods ; Gene Knockdown Techniques ; Homologous Recombination/*drug effects/genetics ; Humans ; Models, Animal ; Oligonucleotides, Antisense/pharmacology ; Pyrimidines/pharmacology ; Schiff Bases/pharmacology ; },
abstract = {In the present day, it is possible to incorporate targeted mutations or replace a gene using genome editing techniques such as customisable CRISPR/Cas9 system. Although induction of DNA double-strand breaks (DSBs) by genome editing tools can be repaired by both non-homologous end joining (NHEJ) and homologous recombination (HR), the skewness of the former pathway in human and other mammals normally result in imprecise repair. Scientists working at the crossroads of DNA repair and genome editing have devised new strategies for using a specific pathway to their advantage. Refinement in the efficiency of precise gene editing was witnessed upon downregulation of NHEJ by knockdown or using small molecule inhibitors on one hand, and upregulation of HR proteins and addition of HR stimulators, other hand. The exploitation of cell cycle phase differences together with appropriate donor DNA length/sequence and small molecules has provided further improvement in precise genome editing. The present article reviews the mechanisms of improving the efficiency of precise genome editing in several model organisms and in clinics.},
}
@article {pmid32884066,
year = {2020},
author = {Cámara, E and Lenitz, I and Nygård, Y},
title = {A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14605},
pmid = {32884066},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering/*methods ; Genome, Fungal ; Plasmids ; *Promoter Regions, Genetic ; Saccharomyces cerevisiae/genetics/growth &amp; development/*metabolism ; Saccharomyces cerevisiae Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains.},
}
@article {pmid32884006,
year = {2020},
author = {Monazzam, A and Li, SC and Wargelius, H and Razmara, M and Bajic, D and Mi, J and Bergquist, J and Crona, J and Skogseid, B},
title = {Generation and characterization of CRISPR/Cas9-mediated MEN1 knockout BON1 cells: a human pancreatic neuroendocrine cell line.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14572},
pmid = {32884006},
issn = {2045-2322},
mesh = {Biomarkers, Tumor/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; Neuroendocrine Tumors/metabolism/*pathology ; Pancreatic Neoplasms/metabolism/*pathology ; Proteome/*analysis ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors/genetics ; Tumor Cells, Cultured ; },
abstract = {Among patients with the rare diagnosis of pancreatic neuroendocrine tumor (P-NET), a substantial proportion suffer from the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1), which is caused by germline mutations of the MEN1 suppressor gene. Somatic mutations and loss of the MEN1 protein (menin) are frequently also found in sporadic P-NETs. Thus, a human neuroendocrine pancreatic cell line with biallelic inactivation of MEN1 might be of value for studying tumorigenesis. We used the polyclonal human P-NET cell line BON1, which expresses menin, serotonin, chromogranin A and neurotensin, to generate a monoclonal stable MEN1 knockout BON1 cell line (MEN1-KO-BON1) by CRISPR/Cas9 editing. Changes in morphology, hormone secretion, and proliferation were analyzed, and proteomics were assessed using nanoLC-MS/MS and Ingenuity Pathway Analysis (IPA). The menin-lacking MEN1-KO-BON1 cells had increased chromogranin A production and were smaller, more homogenous, rounder and grew faster than their control counterparts. Proteomic analysis revealed 457 significantly altered proteins, and IPA identified biological functions related to cancer, e.g., posttranslational modification and cell death/survival. Among 39 proteins with at least a two-fold difference in expression, twelve are relevant in glucose homeostasis and insulin resistance. The stable monoclonal MEN1-KO-BON1 cell line was found to have preserved neuroendocrine differentiation, increased proliferation, and an altered protein profile.},
}
@article {pmid32883881,
year = {2020},
author = {Ewen-Campen, B and Perrimon, N},
title = {Expanding the horizons of genome editing in the fruit fly with Cas12a.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {39},
pages = {24019-24021},
pmid = {32883881},
issn = {1091-6490},
support = {R24 OD026435/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Drosophila/metabolism ; *Gene Editing ; RNA, Guide ; },
}
@article {pmid32883522,
year = {2020},
author = {Sun, S and Ren, T and Li, X and Cao, X and Gao, J},
title = {Polyunsaturated fatty acids synthesized by freshwater fish: A new insight to the roles of elovl2 and elovl5 in vivo.},
journal = {Biochemical and biophysical research communications},
volume = {532},
number = {3},
pages = {414-419},
doi = {10.1016/j.bbrc.2020.08.074},
pmid = {32883522},
issn = {1090-2104},
mesh = {Acetyltransferases/deficiency/*genetics/*metabolism ; Animals ; Animals, Genetically Modified ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Fatty Acids, Unsaturated/*biosynthesis/chemistry ; Gene Expression Regulation, Enzymologic ; Gene Knockout Techniques ; Humans ; Lipidomics ; Liver/metabolism ; RNA, Messenger/genetics/metabolism ; Zebrafish/*genetics/*metabolism ; Zebrafish Proteins/deficiency/*genetics/*metabolism ; },
abstract = {At present, fish provide an important supply of long-chain polyunsaturated fatty acids (LC-PUFAs) for human consumption. Previous studies have shown that fatty acyl elongase 2 (elovl2) and elovl5 play important roles in fish LC-PUFA synthesis. Generally, freshwater fish have a stronger ability to synthesize LC-PUFAs than marine fish. However, the roles of elovl2, elovl5 and elovl2 + elovl5 in LC-PUFA synthesis of freshwater fish in vivo are not very clear. In this study, the elovl2 knockout zebrafish (elovl2[-/-]), elovl5 knockout zebrafish (elovl5[-/-]) and the double gene knockout zebrafish (DKO) were generated by CRISPR/Cas9 technology for the first time. Compared with wild type zebrafish (WT), elovl5-deletion zebrafish showed a significant increase in C22 PUFA content, which might be due to the up-regulation expressions of elovl4b and elovl2. elovl5 expressed at very low levels in livers of elovl2[-/-] relative to WT, indicating that elovl5 may be an "assistant attacker" of elovl2 in LC-PUFA synthesis of zebrafish. Moreover, there were no significant differences in levels of C18-C22 PUFAs between DKO and WT, indicating that besides elovl2 + elovl5 path, LC-PUFA synthesis in zebrafish could be performed by other paths. In addition, the hepatic lipidomic analysis results revealed that the contents of C22:6n-3 in phosphatidyl ethanolamine (PE-DHA) and PE-C22 PUFAs were more easily affected by the absence of elovl2 and elovl5. Our results suggest that the elovl2+elovl5 path is not the only path for LC-PUFA synthesis in zebrafish, and provide novel insights into the roles of elovl2 and elovl5 in LC-PUFA synthesis of freshwater fish.},
}
@article {pmid32883316,
year = {2020},
author = {Topatana, W and Juengpanich, S and Li, S and Cao, J and Hu, J and Lee, J and Suliyanto, K and Ma, D and Zhang, B and Chen, M and Cai, X},
title = {Advances in synthetic lethality for cancer therapy: cellular mechanism and clinical translation.},
journal = {Journal of hematology &amp; oncology},
volume = {13},
number = {1},
pages = {118},
pmid = {32883316},
issn = {1756-8722},
mesh = {Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; Cell Survival/drug effects ; Cellular Senescence/drug effects ; DNA Damage/drug effects ; DNA Repair ; Drug Design ; Drug Resistance, Neoplasm ; Drug Screening Assays, Antitumor ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Molecular Targeted Therapy/*methods ; Mutation ; Neoplasm Proteins/antagonists &amp; inhibitors/physiology ; Neoplasms/genetics/*therapy ; Patient Selection ; Protein Folding ; RNA Interference ; *Synthetic Lethal Mutations ; Translational Research, Biomedical ; Tumor Microenvironment ; },
abstract = {Synthetic lethality is a lethal phenomenon in which the occurrence of a single genetic event is tolerable for cell survival, whereas the co-occurrence of multiple genetic events results in cell death. The main obstacle for synthetic lethality lies in the tumor biology heterogeneity and complexity, the inadequate understanding of synthetic lethal interactions, drug resistance, and the challenges regarding screening and clinical translation. Recently, DNA damage response inhibitors are being tested in various trials with promising results. This review will describe the current challenges, development, and opportunities for synthetic lethality in cancer therapy. The characterization of potential synthetic lethal interactions and novel technologies to develop a more effective targeted drug for cancer patients will be explored. Furthermore, this review will discuss the clinical development and drug resistance mechanisms of synthetic lethality in cancer therapy. The ultimate goal of this review is to guide clinicians at selecting patients that will receive the maximum benefits of DNA damage response inhibitors for cancer therapy.},
}
@article {pmid32883305,
year = {2020},
author = {Li, Q and Zhao, P and Yin, H and Liu, Z and Zhao, H and Tian, P},
title = {CRISPR interference-guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {174},
pmid = {32883305},
issn = {1475-2859},
mesh = {Aconitic Acid/*metabolism ; Batch Cell Culture Techniques ; CRISPR-Cas Systems ; DNA, Bacterial ; Escherichia coli/*genetics/*metabolism ; Genetic Engineering ; Glucose/metabolism ; Industrial Microbiology ; Isocitrate Dehydrogenase/*genetics ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Pyruvate Kinase/*genetics ; },
abstract = {BACKGROUND: One major mission of microbial breeding is high-level production of desired metabolites. Overproduction of intermediate metabolites in core pathways is challenging as it may impair cell growth and viability.<br><br>RESULTS: Here we report that aconitic acid, an intermediate metabolite in tricarboxylic acid (TCA) cycle, can be overproduced by an engineered CRISPR interference (CRISPRi) system in Escherichia coli. This CRISPRi system was designed to simultaneously target pyruvate kinase (PK) and isocitrate dehydrogenase (IDH), two enzymes in glycolytic pathway and TCA cycle, respectively. Reverse transcription and quantitative PCR and enzyme activity assays showed that this engineered CRISPRi system significantly repressed the genes encoding IDH and PK, resulting in simultaneous reduction in the activities of IDH and PK. In shake-flask and fed-batch cultivation, this CRISPRi strain produced 60-fold (362.80&#x2009;&#xb1;&#x2009;22.05&#xa0;mg/L) and 15-fold (623.80&#x2009;&#xb1;&#x2009;20.05&#xa0;mg/L) of aconitic acid relative to the control strain, respectively. In addition, this two-target CRISPRi strain maintained low levels of acetate and lactate, two problematic byproducts.<br><br>CONCLUSIONS: This work demonstrates that CRISPRi system can improve aconitic acid production by coordinating glycolysis and TCA cycle. This study provides insights for high-level production of the intermediate metabolites in central pathways.},
}
@article {pmid32883277,
year = {2020},
author = {Liu, Z and Dong, H and Cui, Y and Cong, L and Zhang, D},
title = {Application of different types of CRISPR/Cas-based systems in bacteria.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {172},
pmid = {32883277},
issn = {1475-2859},
mesh = {Bacteria/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods/trends ; Gene Expression Regulation, Bacterial ; Genetic Engineering/methods/trends ; *Metabolic Engineering ; },
abstract = {As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.},
}
@article {pmid32882392,
year = {2020},
author = {Matsumura, H and Shiomi, K and Yamamoto, A and Taketani, Y and Kobayashi, N and Yoshizawa, T and Tanaka, SI and Yoshikawa, H and Endo, M and Fukayama, H},
title = {Hybrid Rubisco with Complete Replacement of Rice Rubisco Small Subunits by Sorghum Counterparts Confers C4 Plant-like High Catalytic Activity.},
journal = {Molecular plant},
volume = {13},
number = {11},
pages = {1570-1581},
doi = {10.1016/j.molp.2020.08.012},
pmid = {32882392},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems ; Carbon Dioxide/metabolism ; Catalysis ; Gene Knockout Techniques ; Oryza/*enzymology/genetics ; Photosynthesis ; Plants, Genetically Modified ; Protein Subunits/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Ribulose-Bisphosphate Carboxylase/genetics/*metabolism ; Sorghum/*enzymology/genetics ; Sulfuric Acid Esters/metabolism ; },
abstract = {Photosynthetic rate at the present atmospheric condition is limited by the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate (kcat) and poor affinity for CO2 (Kc) and specificity for CO2 (Sc/o). Rubisco in C4 plants generally shows higher kcat than that in C3 plants. Rubisco consists of eight large subunits and eight small subunits (RbcS). Previously, the chimeric incorporation of sorghum C4-type RbcS significantly increased the kcat of Rubisco in a C3 plant, rice. In this study, we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco. Obtained hybrid Rubisco showed almost C4 plant-like catalytic properties, i.e., higher kcat, higher Kc, and lower Sc/o. Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco, whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO2 condition compared with wild-type rice. High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the βC-βD hairpin in RbcS. We propose that such differences, particularly in the βC-βD hairpin, may impact the flexibility of Rubisco catalytic site and change its catalytic properties.},
}
@article {pmid32882179,
year = {2020},
author = {Cheng, X},
title = {Valproic Acid Thermally Destabilizes and Inhibits SpyCas9 Activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2635-2641},
pmid = {32882179},
issn = {1525-0024},
mesh = {Bacterial Proteins/*chemistry/*metabolism ; CRISPR-Associated Protein 9/*chemistry/*metabolism ; CRISPR-Cas Systems ; Enzyme Activation/drug effects ; Enzyme Inhibitors/chemistry/*metabolism ; Gene Editing ; *Hot Temperature ; Indocyanine Green ; Mutation ; Protein Denaturation/drug effects ; Protein Stability/drug effects ; Streptococcus pyogenes/*enzymology ; Valproic Acid/*metabolism/*pharmacology ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system plays an important role in prokaryotic adaptive immunity. Due to its capacity for sequence-specific gene editing, CRISPR-Cas9 has become one of the most important tools widely used for genome editing in molecular biotechnology. However, its clinical application is currently limited by unwanted mutations at off-target sites. Various strategies have been developed for precise control of Cas9 in order to reduce these off-target effects, including chemical-based approaches. From a chemical screening, I observed that valproic acid (VPA) binds to and destabilizes Streptococcus pyogenes Cas9 (SpyCas9) protein in vitro, as well as in cells, while within its therapeutical concentration range under conditions of hyperthermia as demonstrated. Conditions were generated either by an external heat bag or in combination with the photothermal therapeutic agent indocyanine green activated by a near-infrared laser. Use of other histone deacetylase inhibitors failed, suggesting a histone deacetylase inhibition-independent function of VPA. Thus, this finding provides an uncomplicated thermotherapeutical approach for timely regulation of the activity of the CRISPR-Cas9 system at desired locations.},
}
@article {pmid32880080,
year = {2020},
author = {Liu, C and Ye, D and Wang, H and He, M and Sun, Y},
title = {Elovl2 But Not Elovl5 Is Essential for the Biosynthesis of Docosahexaenoic Acid (DHA) in Zebrafish: Insight from a Comparative Gene Knockout Study.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {22},
number = {5},
pages = {613-619},
doi = {10.1007/s10126-020-09992-1},
pmid = {32880080},
issn = {1436-2236},
mesh = {Animals ; CRISPR-Cas Systems ; Docosahexaenoic Acids/*biosynthesis/genetics ; Embryo, Nonmammalian/metabolism ; Fatty Acid Desaturases/*genetics ; Fatty Acid Elongases/*genetics ; Fish Proteins/genetics ; Gene Knockout Techniques ; Liver/metabolism ; Zebrafish/genetics/*metabolism ; },
abstract = {Teleost fish can synthesize one of the major omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), docosahexaenoic acid (DHA, 22:6n-3), from dietary α-linolenic acid (ALA; 18:3n-3), via elongase of very long-chain fatty acid (Elovl) and fatty acid desaturase (Fads). However, it remains unclear which elongase is primarily responsible for the endogenous synthesis of DHA. Here, in this study, the knockout models of the two major elongases, Elovl2 and Elovl5, were generated by CRISPR/Cas9 approach in zebrafish and comparatively analyzed. The homozygous mutants were validated by Sanger sequencing, mutation-mediated PCR, and whole-mount in situ hybridization analysis of the endogenous target genes. Compared with wild-type (WT) counterparts, the content of DHA was significantly reduced by 67.1% (P < 0.05) in the adult liver and by 91.7% (P < 0.01) in the embryo at 3-day post-fertilization (dpf) of the elovl2 mutant, but not of the elovl5 mutant. Further study revealed that elovl2 and fads2 was upregulated by 9.9-fold (P < 0.01) and 9.7-fold (P < 0.01) in the elovl5 mutant, and elovl5 and fads2 were upregulated by 15.1-fold (P < 0.01) and 21.5-fold (P < 0.01) in the elovl2 mutant. Our study indicates that although both Elovl2 and Elovl5 have the elongase activity toward C20, the upregulation of elovl2 could completely replace the genetic depletion of elovl5, but upregulation of elovl5 could not compensate the endogenous deficiency of elovl2 in mediating DHA synthesis. In conclusion, the endogenous synthesis of DHA in is mediated by Elovl2 but not Elovl5 in zebrafish and a DHA-deficient genetic model of zebrafish has been generated.},
}
@article {pmid32878930,
year = {2020},
author = {Marton, T and Maufrais, C and d'Enfert, C and Legrand, M},
title = {Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in Candida albicans.},
journal = {mSphere},
volume = {5},
number = {5},
pages = {},
pmid = {32878930},
issn = {2379-5042},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Gene Editing/*methods ; *Genome, Fungal ; Genomics ; *Homologous Recombination ; RNA, Guide/genetics ; *Transformation, Genetic ; },
abstract = {Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism's genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes.IMPORTANCE Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen Candida albicans The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of C. albicans is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in C. albicans, since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.},
}
@article {pmid32878767,
year = {2020},
author = {Lu, J and Chen, A and Ma, X and Shang, X and Zhang, Y and Guo, Y and Liu, M and Wang, X},
title = {Generation and Characterization of Cytochrome P450 2J3/10 CRISPR/Cas9 Knockout Rat Model.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {48},
number = {11},
pages = {1129-1136},
doi = {10.1124/dmd.120.000114},
pmid = {32878767},
issn = {1521-009X},
mesh = {Animals ; Astemizole/pharmacokinetics ; Biotransformation ; CRISPR-Cas Systems/genetics ; Cytochrome P-450 CYP2J2 ; Cytochrome P-450 Enzyme System/genetics/*metabolism ; Drug Evaluation, Preclinical/methods ; Feasibility Studies ; Female ; Gene Knockdown Techniques ; Male ; Models, Animal ; Rats ; Rats, Transgenic ; },
abstract = {Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase-muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease. SIGNIFICANCE STATEMENT: Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function in vivo. This study successfully developed a new CYP2J knockout rat model by using CRISPR/Cas9 technology. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.},
}
@article {pmid32877675,
year = {2020},
author = {Shin, JJ and Schröder, MS and Caiado, F and Wyman, SK and Bray, NL and Bordi, M and Dewitt, MA and Vu, JT and Kim, WT and Hockemeyer, D and Manz, MG and Corn, JE},
title = {Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.},
journal = {Cell reports},
volume = {32},
number = {9},
pages = {108093},
pmid = {32877675},
issn = {2211-1247},
support = {DP2 HL141006/HL/NHLBI NIH HHS/United States ; T32 AG000266/AG/NIA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; GATA3 Transcription Factor/*metabolism ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematopoietic Stem Cells/*metabolism ; Humans ; Recombinational DNA Repair/*genetics ; Stem Cells/*metabolism ; },
abstract = {Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.},
}
@article {pmid32877304,
year = {2021},
author = {Fiaz, S and Wang, X and Younas, A and Alharthi, B and Riaz, A and Ali, H},
title = {Apomixis and strategies to induce apomixis to preserve hybrid vigor for multiple generations.},
journal = {GM crops &amp; food},
volume = {12},
number = {1},
pages = {57-70},
pmid = {32877304},
issn = {2164-5701},
mesh = {*Apomixis ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing ; Hybrid Vigor ; Seeds ; },
abstract = {Hybrid seeds of several important crops with supreme qualities including yield, biotic and abiotic stress tolerance have been cultivated for decades. Thus far, a major challenge with hybrid seeds is that they do not have the ability to produce plants with the same qualities over subsequent generations. Apomixis, an asexual mode of reproduction by avoiding meiosis, exists naturally in flowering plants, and ultimately leads to seed production. Apomixis has the potential to preserve hybrid vigor for multiple generations in economically important plant genotypes. The evolution and genetics of asexual seed production are unclear, and much more effort will be required to determine the genetic architecture of this phenomenon. To fix hybrid vigor, synthetic apomixis has been suggested. The development of MiMe (mitosis instead of meiosis) genotypes has been utilized for clonal gamete production. However, the identification and parental origin of genes responsible for synthetic apomixis are little known and need further clarification. Genome modifications utilizing genome editing technologies (GETs), such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (cas), a reverse genetics tool, have paved the way toward the utilization of emerging technologies in plant molecular biology. Over the last decade, several genes in important crops have been successfully edited. The vast availability of GETs has made functional genomics studies easy to conduct in crops important for food security. Disruption in the expression of genes specific to egg cell MATRILINEAL (MTL) through the CRISPR/Cas genome editing system promotes the induction of haploid seed, whereas triple knockout of the Baby Boom (BBM) genes BBM1, BBM2, and BBM3 cause embryo arrest and abortion, which can be fully rescued by male-transmitted BBM1. The establishment of synthetic apomixis by engineering the MiMe genotype by genome editing of BBM1 expression or disruption of MTL leads to clonal seed production and heritability for multiple generations. In the present review, we discuss current developments related to the use of CRISPR/Cas technology in plants and the possibility of promoting apomixis in crops to preserve hybrid vigor. In addition, genetics, evolution, epigenetic modifications, and strategies for MiMe genotype development are discussed in detail.},
}
@article {pmid32876764,
year = {2020},
author = {Zhao, N and Li, L and Luo, G and Xie, S and Lin, Y and Han, S and Huang, Y and Zheng, S},
title = {Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {47},
number = {8},
pages = {599-608},
doi = {10.1007/s10295-020-02304-5},
pmid = {32876764},
issn = {1476-5535},
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/enzymology/*genetics/metabolism ; DNA, Bacterial/*genetics ; *Gene Deletion ; Gene Editing/*methods ; Metabolic Engineering/methods ; },
abstract = {Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.},
}
@article {pmid32875708,
year = {2020},
author = {Kurth, F and Tai, YK and Parate, D and van Oostrum, M and Schmid, YRF and Toh, SJ and Yap, JLY and Wollscheid, B and Othman, A and Dittrich, PS and Franco-Obregón, A},
title = {Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities.},
journal = {Advanced biosystems},
volume = {4},
number = {11},
pages = {e2000146},
doi = {10.1002/adbi.202000146},
pmid = {32875708},
issn = {2366-7478},
support = {681587/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation/*drug effects ; Cell-Derived Microparticles/metabolism ; Drug Delivery Systems/*methods ; Gene Editing ; Mice ; Mitochondria/*drug effects ; *TRPC Cation Channels/genetics/metabolism/pharmacokinetics/pharmacology ; },
abstract = {Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1-mitochondrial axis underlying cell expansion and mitohormetic survival adaptations. This study characterizes cell-derived vesicles (CDVs) generated from C2C12 murine myoblasts and shows that they are equipped with the sufficient molecular machinery to confer mitochondrial respiratory capacity and associated proliferative responses upon their fusion with recipient cells. CDVs derived from wild type C2C12 myoblasts include the cation-permeable transient receptor potential (TRP) channels, TRPC1 and TRPA1, and directly respond to PEMF exposure with TRPC1-mediated calcium entry. By contrast, CDVs derived from C2C12 muscle cells in which TRPC1 has been genetically knocked-down using CRISPR/Cas9 genome editing, do not. Wild type C2C12-derived CDVs are also capable of restoring PEMF-induced proliferative and mitochondrial activation in two C2C12-derived TRPC1 knockdown clonal cell lines in accordance to their endogenous degree of TRPC1 suppression. C2C12 wild type CDVs respond to menthol with calcium entry and accumulation, likewise verifying TRPA1 functional gating and further corroborating compartmental integrity. Proteomic and lipidomic analyses confirm the surface membrane origin of the CDVs providing an initial indication of the minimal cellular machinery required to recover mitochondrial function. CDVs hence possess the potential of restoring respiratory and proliferative capacities to senescent cells and tissues.},
}
@article {pmid32875201,
year = {2020},
author = {Tyagi, S and Kesiraju, K and Saakre, M and Rathinam, M and Raman, V and Pattanayak, D and Sreevathsa, R},
title = {Genome Editing for Resistance to Insect Pests: An Emerging Tool for Crop Improvement.},
journal = {ACS omega},
volume = {5},
number = {33},
pages = {20674-20683},
pmid = {32875201},
issn = {2470-1343},
abstract = {Plants are challenged incessantly by several biotic and abiotic stresses during their entire growth period. As with other biotic stress factors, insect pests have also posed serious concerns related to yield losses due to which agricultural productivity is at stake. In plants, trait modification for crop improvement was initiated with breeding approaches followed by genetic engineering. However, stringent regulatory policies for risk assessment and lack of social acceptance for genetically modified crops worldwide have incited researchers toward alternate strategies. Genome engineering or genome editing has emerged as a new breeding technique with the ability to edit the genomes of plants, animals, microbes, and human beings. Several gene editing strategies are being executed with continuous emergence of variants. The scientific community has unraveled the utility of various editing tools from endonucleases to CRISPR/Cas in several aspects related to plant growth, development, and mitigation of stresses. The categorical focus on the development of tools and techniques including designing of binary vectors to facilitate ease in genome engineering are being pursued. Through this Review, we embark upon the conglomeration of various genome editing strategies that can be and are being used to design insect pest resistance in plants. Case studies and novel crop-based approaches that reiterate the successful use of these tools in insects as well as in plants are highlighted. Further, the Review also provides implications for the requirement of a specific regulatory framework and risk assessment of the edited crops. Genome editing toward insect pest management is here to stay, provided uncompromising efforts are made toward the identification of amiable target genes.},
}
@article {pmid32875106,
year = {2020},
author = {Böhm, S and Splith, V and Riedmayr, LM and Rötzer, RD and Gasparoni, G and Nordström, KJV and Wagner, JE and Hinrichsmeyer, KS and Walter, J and Wahl-Schott, C and Fenske, S and Biel, M and Michalakis, S and Becirovic, E},
title = {A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.},
journal = {Science advances},
volume = {6},
number = {34},
pages = {eaba5614},
pmid = {32875106},
issn = {2375-2548},
mesh = {Animals ; Blindness/genetics/therapy ; *CRISPR-Cas Systems ; *Genetic Therapy ; Mice ; Transcription Factors/genetics ; Transcriptional Activation ; },
abstract = {Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.},
}
@article {pmid32873924,
year = {2021},
author = {Modarai, SR and Kanda, S and Bloh, K and Opdenaker, LM and Kmiec, EB},
title = {Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.},
journal = {Gene therapy},
volume = {28},
number = {1-2},
pages = {105-113},
pmid = {32873924},
issn = {1476-5462},
support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; P20 GM109021/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA ; Endonucleases/genetics ; *Gene Editing ; Humans ; beta-Globins/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, including the position of the cleavage site and the inherent variability among patient samples selected for CRISPR-directed gene editing. Here, we target the beta globin gene in human CD34+ cells to assess the impact of these two variables and find that both contribute to the global diversity of genetic outcomes. Our study demonstrates a unique genetic profile of indels that is generated based on where along the beta globin gene attempts are made to correct the SCD single base mutation. Interestingly, even within the same patient sample, the location of where along the beta globin gene the DNA is cut, HDR activity varies widely. Our data establish a framework upon which realistic protocols inform strategies for gene editing for SCD overcoming the practical hurdles that often impede clinical success.},
}
@article {pmid32873648,
year = {2020},
author = {Davis, L and Khoo, KJ and Zhang, Y and Maizels, N},
title = {POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {37},
pages = {22900-22909},
pmid = {32873648},
issn = {1091-6490},
support = {R01 CA183967/CA/NCI NIH HHS/United States ; R21 CA190675/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; DNA Breaks, Single-Stranded ; DNA End-Joining Repair ; DNA Ligases/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/*metabolism ; Heterozygote ; Humans ; Loss of Heterozygosity ; Mutation ; Recombination, Genetic ; *Recombinational DNA Repair ; },
abstract = {Interhomolog recombination (IHR) occurs spontaneously in somatic human cells at frequencies that are low but sufficient to ameliorate some genetic diseases caused by heterozygous mutations or autosomal dominant mutations. Here we demonstrate that DNA nicks or double-strand breaks (DSBs) targeted by CRISPR-Cas9 to both homologs can stimulate IHR and associated copy-neutral loss of heterozygosity (cnLOH) in human cells. The frequency of IHR is 10-fold lower at nicks than at DSBs, but cnLOH is evident in a greater fraction of recombinants. IHR at DSBs occurs predominantly via reciprocal end joining. At DSBs, depletion of POLQ caused a dramatic increase in IHR and in the fraction of recombinants exhibiting cnLOH, suggesting that POLQ promotes end joining in cis, which limits breaks available for recombination in trans These results define conditions that may produce cnLOH as a mutagenic signature in cancer and may, conversely, promote therapeutic correction of both compound heterozygous and dominant negative mutations associated with genetic disease.},
}
@article {pmid32872311,
year = {2020},
author = {Kantor, A and McClements, ME and MacLaren, RE},
title = {CRISPR-Cas9 DNA Base-Editing and Prime-Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {17},
pages = {},
pmid = {32872311},
issn = {1422-0067},
support = {MC_PC_18059/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Dependovirus/*genetics ; Gene Editing/*methods ; Genetic Predisposition to Disease ; Humans ; Point Mutation ; RNA, Guide/*genetics ; Tropism ; },
abstract = {Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.},
}
@article {pmid32871045,
year = {2020},
author = {Ren, J and Yu, D and Fu, R and An, P and Sun, R and Wang, Z and Guo, R and Li, H and Zhang, Y and Li, Z and Yang, YG and Li, W and Hai, T and Hu, Z},
title = {IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.},
journal = {Cell proliferation},
volume = {53},
number = {10},
pages = {e12863},
pmid = {32871045},
issn = {1365-2184},
mesh = {Animals ; Animals, Genetically Modified/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Editing ; Humans ; Immune System/metabolism ; Interleukin Receptor Common gamma Subunit/antagonists &amp; inhibitors/genetics/*metabolism ; Lymphopenia/pathology ; Melanoma/metabolism/pathology ; RNA Interference ; RNA, Small Interfering/metabolism ; Skin Neoplasms/metabolism/mortality/*pathology ; Survival Rate ; Swine ; Swine, Miniature ; Transplantation, Heterologous ; },
abstract = {OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development.<br><br>MATERIALS AND METHODS: IL2RG[-/Y] pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis.<br><br>RESULTS: Compared to age-matched wild-type pigs, IL2RG[-/Y] pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG[-/Y] pigs but not in wild-type littermates. The human tumours grew faster in IL2RG[-/Y] pigs than in nude mice.<br><br>CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.},
}
@article {pmid32870981,
year = {2020},
author = {Smith, EA and Newton, ILG},
title = {Genomic Signatures of Honey Bee Association in an Acetic Acid Symbiont.},
journal = {Genome biology and evolution},
volume = {12},
number = {10},
pages = {1882-1894},
pmid = {32870981},
issn = {1759-6653},
mesh = {Acetic Acid/metabolism ; Acetobacteraceae/*genetics/metabolism ; Animals ; Bees/*microbiology ; Gene Transfer, Horizontal ; *Genome, Bacterial ; Phylogeny ; Symbiosis/*genetics ; },
abstract = {Recent declines in the health of the honey bee have startled researchers and lay people alike as honey bees are agriculture's most important pollinator. Honey bees are important pollinators of many major crops and add billions of dollars annually to the US economy through their services. One factor that may influence colony health is the microbial community. Indeed, the honey bee worker digestive tract harbors a characteristic community of bee-specific microbes, and the composition of this community is known to impact honey bee health. However, the honey bee is a superorganism, a colony of eusocial insects with overlapping generations where nestmates cooperate, building a hive, gathering and storing food, and raising brood. In contrast to what is known regarding the honey bee worker gut microbiome, less is known of the microbes associated with developing brood, with food stores, and with the rest of the built hive environment. More recently, the microbe Bombella apis was identified as associated with nectar, with developing larvae, and with honey bee queens. This bacterium is related to flower-associated microbes such as Saccharibacter floricola and other species in the genus Saccharibacter, and initial phylogenetic analyses placed it as sister to these environmental bacteria. Here, we used comparative genomics of multiple honey bee-associated strains and the nectar-associated Saccharibacter to identify genomic changes that may be associated with the ecological transition to honey bee association. We identified several genomic differences in the honey bee-associated strains, including a complete CRISPR/Cas system. Many of the changes we note here are predicted to confer upon Bombella the ability to survive in royal jelly and defend themselves against mobile elements, including phages. Our results are a first step toward identifying potential function of this microbe in the honey bee superorganism.},
}
@article {pmid32868807,
year = {2020},
author = {Traenkle, B and Segan, S and Fagbadebo, FO and Kaiser, PD and Rothbauer, U},
title = {A novel epitope tagging system to visualize and monitor antigens in live cells with chromobodies.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14267},
pmid = {32868807},
issn = {2045-2322},
mesh = {Antigens/*immunology ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cells/immunology/ultrastructure ; Electrophoresis, Polyacrylamide Gel ; Epitopes/*immunology ; Fluorescent Antibody Technique ; Gene Editing ; HEK293 Cells ; Humans ; Immunoprecipitation ; Microscopy, Confocal ; Peptides/*immunology ; Recombinant Proteins ; Transfection ; },
abstract = {Epitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifically recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofluorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fluorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.},
}
@article {pmid32868406,
year = {2020},
author = {Burroughs, AM and Aravind, L},
title = {Identification of Uncharacterized Components of Prokaryotic Immune Systems and Their Diverse Eukaryotic Reformulations.},
journal = {Journal of bacteriology},
volume = {202},
number = {24},
pages = {},
pmid = {32868406},
issn = {1098-5530},
mesh = {Amino Acid Sequence ; Bacteria/chemistry/*genetics/*immunology ; Bacterial Proteins/chemistry/genetics/*immunology ; Eukaryota/genetics/*immunology ; Genomics ; Immune System ; Nucleotides/chemistry/immunology ; Sequence Alignment ; },
abstract = {Nucleotide-activated effector deployment, prototyped by interferon-dependent immunity, is a common mechanistic theme shared by immune systems of several animals and prokaryotes. Prokaryotic versions include CRISPR-Cas with the CRISPR polymerase domain, their minimal variants, and systems with second messenger oligonucleotide or dinucleotide synthetase (SMODS). Cyclic or linear oligonucleotide signals in these systems help set a threshold for the activation of potentially deleterious downstream effectors in response to invader detection. We establish such a regulatory mechanism to be a more general principle of immune systems, which can also operate independently of such messengers. Using sensitive sequence analysis and comparative genomics, we identify 12 new prokaryotic immune systems, which we unify by this principle of threshold-dependent effector activation. These display regulatory mechanisms paralleling physiological signaling based on 3'-5' cyclic mononucleotides, NAD[+]-derived messengers, two- and one-component signaling that includes histidine kinase-based signaling, and proteolytic activation. Furthermore, these systems allowed the identification of multiple new sensory signal sensory components, such as a tetratricopeptide repeat (TPR) scaffold predicted to recognize NAD[+]-derived signals, unreported versions of the STING domain, prokaryotic YEATS domains, and a predicted nucleotide sensor related to receiver domains. We also identify previously unrecognized invader detection components and effector components, such as prokaryotic versions of the Wnt domain. Finally, we show that there have been multiple acquisitions of unidentified STING domains in eukaryotes, while the TPR scaffold was incorporated into the animal immunity/apoptosis signal-regulating kinase (ASK) signalosome.IMPORTANCE Both prokaryotic and eukaryotic immune systems face the dangers of premature activation of effectors and degradation of self-molecules in the absence of an invader. To mitigate this, they have evolved threshold-setting regulatory mechanisms for the triggering of effectors only upon the detection of a sufficiently strong invader signal. This work defines general templates for such regulation in effector-based immune systems. Using this, we identify several previously uncharacterized prokaryotic immune mechanisms that accomplish the regulation of downstream effector deployment by using nucleotide, NAD[+]-derived, two-component, and one-component signals paralleling physiological homeostasis. This study has also helped identify several previously unknown sensor and effector modules in these systems. Our findings also augment the growing evidence for the emergence of key animal immunity and chromatin regulatory components from prokaryotic progenitors.},
}
@article {pmid32868402,
year = {2020},
author = {Guan, J and Bondy-Denomy, J},
title = {Intracellular Organization by Jumbo Bacteriophages.},
journal = {Journal of bacteriology},
volume = {203},
number = {2},
pages = {},
pmid = {32868402},
issn = {1098-5530},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/*ultrastructure ; Cytoskeleton/genetics/*physiology ; DNA Replication ; Evolution, Molecular ; Genome, Viral ; },
abstract = {Since their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been "jumbo phages," with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. In this review, we focus on the current knowledge of the cytoskeletal elements and the specialized nuclear compartment derived from jumbo phages, and we highlight their importance in facilitating spatial and temporal organization over the viral life cycle. Additionally, we discuss the evolutionary relationships between jumbo phages and eukaryotic viruses, as well as the therapeutic potential and drawbacks of jumbo phages as antimicrobial agents in phage therapy.},
}
@article {pmid32868079,
year = {2020},
author = {Aoi, Y and Hira, H and Hayakawa, Y and Liu, H and Fukui, K and Dai, X and Tanaka, K and Hayashi, KI and Zhao, Y and Kasahara, H},
title = {UDP-glucosyltransferase UGT84B1 regulates the levels of indole-3-acetic acid and phenylacetic acid in Arabidopsis.},
journal = {Biochemical and biophysical research communications},
volume = {532},
number = {2},
pages = {244-250},
pmid = {32868079},
issn = {1090-2104},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/genetics/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; Gene Expression Regulation, Plant ; Glucosyltransferases/genetics/*metabolism ; Indoleacetic Acids/*metabolism ; Mutation ; Phenylacetates/*metabolism ; Plants, Genetically Modified ; },
abstract = {Auxin is a key plant growth regulator for diverse developmental processes in plants. Indole-3-acetic acid (IAA) is a primary plant auxin that regulates the formation of various organs. Plants also produce phenylacetic acid (PAA), another natural auxin, which occurs more abundantly than IAA in various plant species. Although it has been demonstrated that the two auxins have distinct transport characteristics, the metabolic pathways and physiological roles of PAA in plants remain unsolved. In this study, we investigated the role of Arabidopsis UDP-glucosyltransferase UGT84B1 in IAA and PAA metabolism. We demonstrated that UGT84B1, which converts IAA to IAA-glucoside (IAA-Glc), can also catalyze the conversion of PAA to PAA-glucoside (PAA-Glc), with a higher catalytic activity in vitro. Furthermore, we showed a significant increase in both the IAA and PAA levels in the ugt84b1 null mutants. However, no obvious developmental phenotypes were observed in the ugt84b1 mutants under laboratory growth conditions. Moreover, the overexpression of UGT84B1 resulted in auxin-deficient root phenotypes and changes in the IAA and PAA levels. Our results indicate that UGT84B1 plays an important role in IAA and PAA homeostasis in Arabidopsis.},
}
@article {pmid32867033,
year = {2020},
author = {Bulavaitė, A and Dalgediene, I and Michailoviene, V and Pleckaityte, M},
title = {Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018.},
journal = {Pathogens (Basel, Switzerland)},
volume = {9},
number = {9},
pages = {},
pmid = {32867033},
issn = {2076-0817},
abstract = {Intensive horizontal gene transfer may generate diversity and heterogeneity within the genus Gardnerella. Restriction-modification (R-M) systems and CRISPR-Cas are the principal defense tools against foreign DNA in bacteria. Nearly half of the tested Gardnerella spp. isolates harbored the CRISPR-Cas system. Several putative R-M systems of Gardnerella spp. strains were identified in the REBASE database. However, there was no experimental evidence for restriction endonuclease (REase) activity in the isolates. We showed that G. vaginalis strain ATCC 14018 contains the REase R.Gva14018I, which recognizes GGCC and most probably generates blunt ends on cleavage. Bioinformatics evidence and the activity of recombinant methyltransferase M.Gva14018I in vivo indicate that ATCC 14018 possesses a HaeIII-like R-M system. The truncated R.Gva14018I-4 lacking the C-terminal region was expressed in Escherichia coli and displayed wild-type REase specificity. Polyclonal antibodies against R.Gva14018I-4 detected the wild-type REase in the cell lysate of ATCC 14018. The cofactor requirements for activity and bioinformatics analysis indicated that R.Gva14018I belongs to the PD-(D/E)XK family of REases. The REase-like activity was observed in 5 of 31 tested Gardnerella spp. strains, although none of these matched the DNA digestion pattern of R.Gva14018I.},
}
@article {pmid32866158,
year = {2020},
author = {Liu, C and Grote, A and Ghedin, E and Unnasch, TR},
title = {CRISPR-mediated Transfection of Brugia malayi.},
journal = {PLoS neglected tropical diseases},
volume = {14},
number = {8},
pages = {e0008627},
pmid = {32866158},
issn = {1935-2735},
support = {R21 AI135172/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Base Sequence ; Brugia malayi/*genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Helminth/genetics ; Female ; Gene Editing ; Genome ; Larva/genetics ; Luciferases ; Microfilariae/genetics ; Transfection/*methods ; },
abstract = {The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.},
}
@article {pmid32865887,
year = {2020},
author = {Merker, L and Schindele, P and Puchta, H},
title = {Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana.},
journal = {Current protocols in plant biology},
volume = {5},
number = {3},
pages = {e20117},
doi = {10.1002/cppb.20117},
pmid = {32865887},
issn = {2379-8068},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Targeting ; },
abstract = {CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.},
}
@article {pmid32865499,
year = {2020},
author = {Müller, M and Schneider, M and Salathé, M and Vayena, E},
title = {Assessing Public Opinion on CRISPR-Cas9: Combining Crowdsourcing and Deep Learning.},
journal = {Journal of medical Internet research},
volume = {22},
number = {8},
pages = {e17830},
pmid = {32865499},
issn = {1438-8871},
mesh = {CRISPR-Cas Systems/*physiology ; Crowdsourcing/*methods ; Deep Learning/*standards ; Humans ; *Public Opinion ; },
abstract = {BACKGROUND: The discovery of the CRISPR-Cas9-based gene editing method has opened unprecedented new potential for biological and medical engineering, sparking a growing public debate on both the potential and dangers of CRISPR applications. Given the speed of technology development and the almost instantaneous global spread of news, it is important to follow evolving debates without much delay and in sufficient detail, as certain events may have a major long-term impact on public opinion and later influence policy decisions.<br><br>OBJECTIVE: Social media networks such as Twitter have shown to be major drivers of news dissemination and public discourse. They provide a vast amount of semistructured data in almost real-time and give direct access to the content of the conversations. We can now mine and analyze such data quickly because of recent developments in machine learning and natural language processing.<br><br>METHODS: Here, we used Bidirectional Encoder Representations from Transformers (BERT), an attention-based transformer model, in combination with statistical methods to analyze the entirety of all tweets ever published on CRISPR since the publication of the first gene editing application in 2013.<br><br>RESULTS: We show that the mean sentiment of tweets was initially very positive, but began to decrease over time, and that this decline was driven by rare peaks of strong negative sentiments. Due to the high temporal resolution of the data, we were able to associate these peaks with specific events and to observe how trending topics changed over time.<br><br>CONCLUSIONS: Overall, this type of analysis can provide valuable and complementary insights into ongoing public debates, extending the traditional empirical bioethics toolset.},
}
@article {pmid32865316,
year = {2020},
author = {Renzl, C and Kakoti, A and Mayer, G},
title = {Aptamer-Mediated Reversible Transactivation of Gene Expression by Light.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {50},
pages = {22414-22418},
pmid = {32865316},
issn = {1521-3773},
mesh = {Aptamers, Nucleotide/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Expression ; Humans ; *Light ; Transcriptional Activation/genetics ; },
abstract = {The investigation and manipulation of cellular processes with subcellular resolution requires non-invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light-controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on-switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.},
}
@article {pmid32864285,
year = {2020},
author = {Joshi, RK and Bharat, SS and Mishra, R},
title = {Engineering drought tolerance in plants through CRISPR/Cas genome editing.},
journal = {3 Biotech},
volume = {10},
number = {9},
pages = {400},
pmid = {32864285},
issn = {2190-572X},
abstract = {Drought stress is primarily responsible for heavy yield losses and productivity in major crops and possesses the greatest threat to the global food security. While conventional and molecular breeding approaches along with genetic engineering techniques have been instrumental in developing drought-tolerant crop varieties, these methods are cumbersome, time consuming and the genetically modified varieties are not widely accepted due to regulatory concerns. Plant breeders are now increasingly centring towards the recently available genome-editing tools for improvement of agriculturally important traits. The advent of multiple sequence-specific nucleases has facilitated precise gene modification towards development of novel climate ready crop variants. Amongst the available genome-editing platforms, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) system has emerged as a revolutionary tool for its simplicity, adaptability, flexibility and wide applicability. In this review, we focus on understanding the molecular mechanism of drought response in plants and the application of CRISPR/Cas genome-editing system towards improved tolerance to drought stress.},
}
@article {pmid32861775,
year = {2020},
author = {Fujii, T and Kakino, K and Tanaka, M and Lee, JM and Kusakabe, T and Banno, Y},
title = {A defect in purine nucleotide metabolism in the silkworm, Bombyx mori, causes a translucent larval integument and male infertility.},
journal = {Insect biochemistry and molecular biology},
volume = {126},
number = {},
pages = {103458},
doi = {10.1016/j.ibmb.2020.103458},
pmid = {32861775},
issn = {1879-0240},
mesh = {Animals ; Bombyx/genetics/*metabolism ; CRISPR-Cas Systems ; *Infertility, Male/genetics ; Inosine/metabolism ; Inosine Monophosphate/metabolism ; Integumentary System/*growth &amp; development ; Larva/genetics/metabolism ; Male ; Moths/metabolism ; Mutation ; Nitrogen/metabolism ; Nucleotidases/*genetics ; Purine Nucleotides/metabolism ; RNA-Seq/methods ; Uric Acid/metabolism ; },
abstract = {p-oily (op) is a novel mutant of Bombyx mori exhibiting translucent larval integument and male infertility. Elucidation of the causative gene of the op mutant will help understand the genetic mechanism underlying larval integument coloration and male fertility. Using polymorphisms between B. mori and B. mandarina, the op locus was narrowed down to a 375-kb region. Using RNA-seq analysis, we found that op mutants have a frameshift mutation in the KWMTBOMO13770 gene located in the 375-kb region. A database search indicated that this gene is the human cytosolic 5'-nucleotidase II gene (cN-II) homolog in Bombyx, which mediates the conversion of inosine monophosphate (IMP) to inosine, a precursor of uric acid. CRISPR/Cas9-mediated knockout mutants of the Bm-cN-II gene showed translucent integuments, and there appeared translucent larvae in the crosses between knockout moths and +/op moths. Moreover, the translucent phenotype of, and decreased uric acid content in the larval integument caused by the mutations in the Bm-cN-II gene were rescued by oral administration of inosine. These results indicated that the Bm-cN-II gene is responsible for the op phenotype and that the molecular function of the Bm-cN-II gene is the conversion of IMP to inosine. We also discuss the genetic relationship between the Bm-cN-II gene and male fertility.},
}
@article {pmid32860838,
year = {2020},
author = {Das, S and Chandrasekaran, AP and Jo, KS and Ko, NR and Oh, SJ and Kim, KS and Ramakrishna, S},
title = {HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {12},
pages = {118835},
doi = {10.1016/j.bbamcr.2020.118835},
pmid = {32860838},
issn = {1879-2596},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Damage/genetics ; Drug Resistance, Neoplasm/*genetics ; Female ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Heterografts ; Humans ; Mice ; Ubiquitin-Specific Peptidase 7/*genetics ; Uterine Cervical Neoplasms/drug therapy/*genetics/pathology ; cdc25 Phosphatases/*genetics ; },
abstract = {Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.},
}
@article {pmid32860515,
year = {2020},
author = {Laudermilk, LT and Tovar, A and Homstad, AK and Thomas, JM and McFadden, KM and Tune, MK and Cowley, DO and Mock, JR and Ideraabdullah, F and Kelada, SNP},
title = {Baseline and innate immune response characterization of a Zfp30 knockout mouse strain.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {31},
number = {7-8},
pages = {205-214},
pmid = {32860515},
issn = {1432-1777},
support = {P30 DK034987/DK/NIDDK NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; R01 HL122711/HL/NHLBI NIH HHS/United States ; R01 ES024965/ES/NIEHS NIH HHS/United States ; R03 HL145255/HL/NHLBI NIH HHS/United States ; P30 DK056350/DK/NIDDK NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; K08 HL129075/HL/NHLBI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Biomarkers ; CRISPR-Cas Systems ; Cells, Cultured ; DNA-Binding Proteins/chemistry/*deficiency/genetics/metabolism ; Female ; Gene Editing ; Genetic Association Studies ; Genetic Predisposition to Disease ; Genotype ; Host-Pathogen Interactions/*genetics/*immunology ; Immunity, Innate/*genetics ; Immunomodulation/*genetics ; Male ; Mice ; Mice, Knockout ; Neutrophils/immunology/metabolism ; Phenotype ; Protein Binding ; Protein Interaction Domains and Motifs ; Respiratory Mucosa/immunology/metabolism ; Transcription Factors/chemistry/*deficiency/genetics/metabolism ; },
abstract = {Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.},
}
@article {pmid32860436,
year = {2020},
author = {Lv, Z and Jiang, R and Chen, J and Chen, W},
title = {Nanoparticle-mediated gene transformation strategies for plant genetic engineering.},
journal = {The Plant journal : for cell and molecular biology},
volume = {104},
number = {4},
pages = {880-891},
doi = {10.1111/tpj.14973},
pmid = {32860436},
issn = {1365-313X},
mesh = {Agriculture ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Crops, Agricultural ; Gene Editing ; *Genetic Engineering ; *Nanoparticles ; Plant Breeding ; Plants/*genetics ; Plants, Genetically Modified ; Transformation, Genetic ; },
abstract = {Plant genetic engineering, a recent technological advancement in the field of plant science, is an important tool used to improve crop quality and yield, to enhance secondary metabolite content in medicinal plants or to develop crops for sustainable agriculture. A new approach based on nanoparticle-mediated gene transformation can overcome the obstacle of the plant cell wall and accurately transfer DNA or RNA into plants to produce transient or stable transformation. In this review, several nanoparticle-based approaches are discussed, taking into account recent advances and challenges to hint at potential applications of these approaches in transgenic plant improvement programs. This review also highlights challenges in implementing the nanoparticle-based approaches used in plant genetic engineering. A new technology that improves gene transformation efficiency and overcomes difficulties in plant regeneration has been established and will be used for the de novo production of transgenic plants, and CRISPR/Cas9 genome editing has accelerated crop improvement. Therefore, we outline future perspectives based on combinations of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies to accelerate crop improvement. The information provided here will assist an effective exploration of the technological advances in plant genetic engineering to support plant breeding and important crop improvement programs.},
}
@article {pmid32859923,
year = {2020},
author = {Bennett, NK and Nguyen, MK and Darch, MA and Nakaoka, HJ and Cousineau, D and Ten Hoeve, J and Graeber, TG and Schuelke, M and Maltepe, E and Kampmann, M and Mendelsohn, BA and Nakamura, JL and Nakamura, K},
title = {Defining the ATPome reveals cross-optimization of metabolic pathways.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4319},
pmid = {32859923},
issn = {2041-1723},
support = {R01 HD072455/HD/NICHD NIH HHS/United States ; R01 NS091902/NS/NINDS NIH HHS/United States ; K12 HD000850/HD/NICHD NIH HHS/United States ; S10 RR028962/RR/NCRR NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; P30 AI027763/AI/NIAID NIH HHS/United States ; F32 AG063457/AG/NIA NIH HHS/United States ; U54 CA196519/CA/NCI NIH HHS/United States ; S10 OD016387/OD/NIH HHS/United States ; },
mesh = {Adenosine Triphosphate/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Energy Metabolism ; Female ; Fibroblasts ; Gene Expression Regulation ; Gene Knockdown Techniques ; Glucose/metabolism ; Glycolysis/physiology ; Hexokinase/genetics/metabolism ; Humans ; K562 Cells ; Metabolic Networks and Pathways/*genetics/*physiology ; Metabolomics ; Mitochondria/metabolism ; Pentose Phosphate Pathway ; Point Mutation ; },
abstract = {Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.},
}
@article {pmid32859906,
year = {2020},
author = {Xu, H and Han, M and Zhou, S and Li, BZ and Wu, Y and Yuan, YJ},
title = {Chromosome drives via CRISPR-Cas9 in yeast.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4344},
pmid = {32859906},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Centromere ; Chromosomes/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Indoles ; Metabolic Networks and Pathways/genetics ; Saccharomyces cerevisiae/*genetics/metabolism ; Synthetic Biology/methods ; Thermotolerance/genetics ; Whole Genome Sequencing ; },
abstract = {Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives.},
}
@article {pmid32859627,
year = {2020},
author = {Mazzio, E and Badisa, R and Mack, N and Cassim, S and Zdralevic, M and Pouyssegur, J and Soliman, KFA},
title = {Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.},
journal = {Cancer genomics &amp; proteomics},
volume = {17},
number = {5},
pages = {469-497},
pmid = {32859627},
issn = {1790-6245},
support = {G12 MD007582/MD/NIMHD NIH HHS/United States ; U54 MD007582/MD/NIMHD NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival ; Cytokines/genetics/metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Glucose/metabolism ; Glucose-6-Phosphate Isomerase/genetics/*metabolism ; Humans ; Isoenzymes/antagonists &amp; inhibitors/genetics/metabolism ; L-Lactate Dehydrogenase/antagonists &amp; inhibitors/genetics/*metabolism ; Lactic Acid/metabolism ; Neoplasms/drug therapy/genetics/*pathology ; Oligonucleotide Array Sequence Analysis ; *Warburg Effect, Oncologic ; },
abstract = {BACKGROUND/AIM: Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway.<br><br>MATERIALS AND METHODS: In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays.<br><br>RESULTS: The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPAR&#x3b3; coactivator 1&#x3b1; (PGC-1&#x3b1;), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes.<br><br>CONCLUSION: These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.},
}
@article {pmid32859594,
year = {2020},
author = {Santos, RERS and Batista, BB and da Silva Neto, JF},
title = {Ferric Uptake Regulator Fur Coordinates Siderophore Production and Defense against Iron Toxicity and Oxidative Stress and Contributes to Virulence in Chromobacterium violaceum.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {21},
pages = {},
pmid = {32859594},
issn = {1098-5336},
mesh = {Bacterial Proteins/*genetics/metabolism ; Chromobacterium/*pathogenicity/*physiology ; Iron/*toxicity ; *Oxidative Stress ; Repressor Proteins/*genetics/metabolism ; Siderophores/*metabolism ; Virulence ; },
abstract = {Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the ferric uptake regulator Fur in the physiology and virulence of C. violaceum A Δfur mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR-Cas locus rescued the Δfur mutant growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceumIMPORTANCE Maintenance of iron homeostasis, i.e., avoiding both deficiency and toxicity of this metal, is vital to bacteria and their hosts. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the ferric uptake regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum We determined that fur is a conditionally essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation, and siderophore production. Consistent with these results, a C. violaceumfur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.},
}
@article {pmid32859244,
year = {2020},
author = {Wang, Y and Zhou, L and Tao, R and Liu, N and Long, J and Qin, F and Tang, W and Yang, Y and Chen, Q and Yao, S},
title = {sgBE: a structure-guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion of cytosine and adenosine.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {222},
pmid = {32859244},
issn = {1474-760X},
mesh = {Adenine ; Adenosine/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; Cytosine/*metabolism ; Gene Editing/*methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Nucleotides ; RNA, Guide/*chemistry/genetics ; Software ; },
abstract = {We present a base editing system, in which base editors are attached to different sites of sgRNA scaffold (sgBE). Each independent sgBE has its own specific editing pattern for a given target site. Among tested sgBEs, sgBE-SL4, in which deaminase is attached to the last stem-loop of sgRNA, yields the highest editing efficiency in the window several nucleotides next to the one edited by BE3. sgBE enables the simultaneous editing of adenine and cytosine. Finally, in order to facilitate in vivo base editing, we extend our sgBE system to an AAV-compatible Cas9, SaCas9 (Staphylococcus aureus), and observe robust base editing.},
}
@article {pmid32859098,
year = {2020},
author = {Nawaz, G and Usman, B and Zhao, N and Han, Y and Li, Z and Wang, X and Liu, Y and Li, R},
title = {CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.},
journal = {International journal of molecular sciences},
volume = {21},
number = {17},
pages = {},
pmid = {32859098},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Dioxygenases/*genetics ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Gibberellins/*metabolism ; Glycolysis ; Mutation ; Oryza/genetics/*growth &amp; development/metabolism ; Oxidative Phosphorylation ; Photosynthesis ; Plant Proteins/genetics/metabolism ; Protein Interaction Maps ; Proteomics/*methods ; },
abstract = {In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.},
}
@article {pmid32858938,
year = {2020},
author = {Meile, S and Kilcher, S and Loessner, MJ and Dunne, M},
title = {Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments.},
journal = {Viruses},
volume = {12},
number = {9},
pages = {},
pmid = {32858938},
issn = {1999-4915},
mesh = {Bacteria/genetics/*isolation &amp; purification ; Bacteriolysis ; *Bacteriophages/genetics/physiology ; Biosensing Techniques ; CRISPR-Cas Systems ; Cloning, Molecular ; Colorimetry ; Genes, Reporter ; Genetic Engineering ; Genome, Viral ; Luminescent Measurements ; Microscopy, Fluorescence ; },
abstract = {Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.},
}
@article {pmid32857635,
year = {2020},
author = {Nussenzweig, PM and Marraffini, LA},
title = {Molecular Mechanisms of CRISPR-Cas Immunity in Bacteria.},
journal = {Annual review of genetics},
volume = {54},
number = {},
pages = {93-120},
doi = {10.1146/annurev-genet-022120-112523},
pmid = {32857635},
issn = {1545-2948},
mesh = {Animals ; Bacteria/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Immunity/*genetics ; RNA/genetics ; },
abstract = {Prokaryotes have developed numerous defense strategies to combat the constant threat posed by the diverse genetic parasites that endanger them. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas loci guard their hosts with an adaptive immune system against foreign nucleic acids. Protection starts with an immunization phase, in which short pieces of the invader's genome, known as spacers, are captured and integrated into the CRISPR locus after infection. Next, during the targeting phase, spacers are transcribed into CRISPR RNAs (crRNAs) that guide CRISPR-associated (Cas) nucleases to destroy the invader's DNA or RNA. Here we describe the many different molecular mechanisms of CRISPR targeting and how they are interconnected with the immunization phase through a third phase of the CRISPR-Cas immune response: primed spacer acquisition. In this phase, Cas proteins direct the crRNA-guided acquisition of additional spacers to achieve a more rapid and robust immunization of the population.},
}
@article {pmid32857377,
year = {2021},
author = {Galdos, FX and Darsha, AK and Paige, SL and Wu, SM},
title = {Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent Reporters.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2158},
number = {},
pages = {223-240},
pmid = {32857377},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Cell Differentiation ; Cell Separation/*methods ; Fluorescence ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Luminescent Proteins/genetics/metabolism ; Myocytes, Cardiac/*cytology/metabolism ; *Regeneration ; },
abstract = {Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.},
}
@article {pmid32856292,
year = {2020},
author = {Subedi, U and Jayawardhane, KN and Pan, X and Ozga, J and Chen, G and Foroud, NA and Singer, SD},
title = {The Potential of Genome Editing for Improving Seed Oil Content and Fatty Acid Composition in Oilseed Crops.},
journal = {Lipids},
volume = {55},
number = {5},
pages = {495-512},
doi = {10.1002/lipd.12249},
pmid = {32856292},
issn = {1558-9307},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/trends ; Humans ; Lipids/*genetics ; Plant Breeding ; Plant Oils/chemistry/*metabolism ; Plants, Genetically Modified/*genetics/metabolism ; Seeds/chemistry/metabolism ; Triglycerides/*genetics/metabolism ; },
abstract = {A continuous rise in demand for vegetable oils, which comprise mainly the storage lipid triacylglycerol, is fueling a surge in research efforts to increase seed oil content and improve fatty acid composition in oilseed crops. Progress in this area has been achieved using both conventional breeding and transgenic approaches to date. However, further advancements using traditional breeding methods will be complicated by the polyploid nature of many oilseed crops and associated time constraints, while public perception and the prohibitive cost of regulatory processes hinders the commercialization of transgenic oilseed crops. As such, genome editing using CRISPR/Cas is emerging as a breakthrough breeding tool that could provide a platform to keep pace with escalating demand while potentially minimizing regulatory burden. In this review, we discuss the technology itself and progress that has been made thus far with respect to its use in oilseed crops to improve seed oil content and quality. Furthermore, we examine a number of genes that may provide ideal targets for genome editing in this context, as well as new CRISPR-related tools that have the potential to be applied to oilseed plants and may allow additional gains to be made in the future.},
}
@article {pmid32855962,
year = {2020},
author = {Wang, F and Zeng, Y and Wang, Y and Niu, Y},
title = {The Development and Application of a Base Editor in Biomedicine.},
journal = {BioMed research international},
volume = {2020},
number = {},
pages = {2907623},
pmid = {32855962},
issn = {2314-6141},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; },
abstract = {Using a base editor to generate monogenic disease models and correct pathogenic point mutations is a breakthrough technology for exploration and treatment of human diseases. As a burgeoning approach for genomic modification, the fused CRISPR/Cas9 with various deaminase separately has significantly increased the efficiency of producing a precise point mutation with minimal insertions or deletions (indels). Along with the flexibility and efficiency, a base editor has been widely used in many fields. This review discusses the recent development of a base editor, including evolution and advance, and highlights the applications and challenges in the field of gene therapy. Depending on rapid improvement and optimization of gene editing technology, the prospect of base editor is immeasurable.},
}
@article {pmid32855870,
year = {2020},
author = {Ameri, H and Murat, C and Arbabi, A and Jiang, W and Janga, SR and Qin, PZ and Hamm-Alvarez, SF},
title = {Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption.},
journal = {Translational vision science &amp; technology},
volume = {9},
number = {8},
pages = {23},
pmid = {32855870},
issn = {2164-2591},
support = {K12 EY028873/EY/NEI NIH HHS/United States ; P30 EY029220/EY/NEI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Ependymoglial Cells/metabolism ; Epithelial Cells/metabolism ; Humans ; Retinal Pigments ; Ribonucleoproteins/genetics ; *Vascular Endothelial Growth Factor A/genetics ; },
abstract = {PURPOSE: To evaluate the effects of vascular endothelial growth factor-A (VEGF-A) gene editing in human retinal pigment epithelial (RPE) cells and human Muller cells, which are the main VEGF-A producing cells in the eye.<br><br>METHODS: CRISPR-Cas9 ribonucleoprotein was used to target exon 1 in VEGF-A gene. Lipofectamine CRISPRMAX was used as a vehicle. In vitro gene editing efficiency was assessed on oligonucleotides and genomic DNAs. Sanger sequencing was performed to detect indels. VEGF-A messenger RNA and protein expressions were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.<br><br>RESULTS: In vitro cleavage assay on a 60-nucleotide DNA duplex showed 88% cleavage of the precursor. The cleavage efficiency was 40% in RPE cells and 32% in Muller cells. Sanger sequencing in the CRISPR-Cas9 treated RPE and Muller cells showed indels at the predicted cut site in both cells. After the VEGF-A gene disruption, VEGF-A protein levels decreased 43% in RPE cells (P < 0.0001) and 38% in Muller cells (P < 0.0001).<br><br>CONCLUSIONS: CRISPR-Cas9-mediated gene disruption resulted in a significant decrease in the VEGF-A gene protein expression in human RPE and Muller cells. CRISPR-Cas9 ribonucleoprotein may allow simultaneous targeting of multiple VEGF-A producing cells.<br><br>TRANSLATIONAL RELEVANCE: VEGF-A gene disruption using CRISPR-Cas9 ribonucleoprotein has a potential in treating retinal vascular diseases.},
}
@article {pmid32855333,
year = {2020},
author = {Gao, L and Altae-Tran, H and Böhning, F and Makarova, KS and Segel, M and Schmid-Burgk, JL and Koob, J and Wolf, YI and Koonin, EV and Zhang, F},
title = {Diverse enzymatic activities mediate antiviral immunity in prokaryotes.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6507},
pages = {1077-1084},
pmid = {32855333},
issn = {1095-9203},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenosine Deaminase/*chemistry/classification/genetics ; Archaea/enzymology/*virology ; Archaeal Proteins ; Archaeal Viruses/*immunology ; Bacteria/enzymology/*virology ; Bacterial Proteins ; Bacteriophages/*immunology ; *CRISPR-Cas Systems ; Genes, Archaeal ; Genes, Bacterial ; Protein Domains ; *RNA Editing ; },
abstract = {Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.},
}
@article {pmid32854160,
year = {2021},
author = {Li, B and Liang, S and Alariqi, M and Wang, F and Wang, G and Wang, Q and Xu, Z and Yu, L and Naeem Zafar, M and Sun, L and Si, H and Yuan, D and Guo, W and Wang, Y and Lindsey, K and Zhang, X and Jin, S},
title = {The application of temperature sensitivity CRISPR/LbCpf1 (LbCas12a) mediated genome editing in allotetraploid cotton (G. hirsutum) and creation of nontransgenic, gossypol-free cotton.},
journal = {Plant biotechnology journal},
volume = {19},
number = {2},
pages = {221-223},
pmid = {32854160},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gossypium/genetics ; *Gossypol ; Temperature ; },
}
@article {pmid32853291,
year = {2020},
author = {Hou, T and Zeng, W and Yang, M and Chen, W and Ren, L and Ai, J and Wu, J and Liao, Y and Gou, X and Li, Y and Wang, X and Su, H and Gu, B and Wang, J and Xu, T},
title = {Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19.},
journal = {PLoS pathogens},
volume = {16},
number = {8},
pages = {e1008705},
pmid = {32853291},
issn = {1553-7374},
mesh = {Bacteria/genetics ; Betacoronavirus/*genetics ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems/*genetics ; *Clinical Laboratory Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Coronavirus Infections/*diagnosis/*genetics ; Genes, Viral/genetics ; Genome, Viral/genetics ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Molecular Diagnostic Techniques/economics/methods ; Nucleic Acid Amplification Techniques/economics/methods ; Pandemics ; Pneumonia, Viral/*diagnosis/*genetics ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.},
}
@article {pmid32852759,
year = {2021},
author = {Jostes, S and Nettersheim, D and Schneider, S and Schorle, H},
title = {Cultivation of Testicular Germ Cell Cancer Cell Lines and Establishment of Gene-Edited Subclones Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2195},
number = {},
pages = {85-97},
pmid = {32852759},
issn = {1940-6029},
mesh = {Biomarkers, Tumor ; *CRISPR-Cas Systems ; *Cell Culture Techniques ; Cell Line, Tumor ; Clonal Evolution ; *Gene Editing/methods ; Gene Knockout Techniques ; Humans ; Male ; Neoplasms, Germ Cell and Embryonal/*etiology/*pathology ; Plasmids/genetics ; Testicular Neoplasms/*etiology/*pathology ; Transfection ; },
abstract = {Type II testicular germ cell tumors (GCTs) can be classified as seminoma or embryonal carcinoma. Both subtypes present distinct cellular morphologies and characteristics. Seminomas closely resemble primordial germ cells (PGCs) with respect to their transcriptome and epigenetic signature (DNA hypomethylation). They express the pluripotency markers LIN28, NANOG, and OCT3/4 and the PGC markers SOX17, PRDM1, TFAP2C, DMRT1, and cKIT. Embryonal carcinomas show increased levels of DNA methylation (hypermethylation). They also express the pluripotency markers LIN28, NANOG, and OCT3/4, but additionally DNMT3B and SOX2. In contrast to seminomas, these tumors are pluripotent to totipotent and thus able to differentiate into cells of all three germ layers (teratoma) and extraembryonic tissues (yolk-sac tumor, choriocarcinoma). This protocol summarizes the essential techniques for standard cultivation of seminoma (TCam-2), embryonal carcinoma (NCCIT, NT2/D1, 2102EP), and choriocarcinoma (JEG-3, JAR) cell lines, as well as the methods to establish gene-edited subclones using the CRISPR/Cas9 system.},
}
@article {pmid32852081,
year = {2020},
author = {Sumner, RP and Harrison, L and Touizer, E and Peacock, TP and Spencer, M and Zuliani-Alvarez, L and Towers, GJ},
title = {Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA.},
journal = {The EMBO journal},
volume = {39},
number = {20},
pages = {e103958},
pmid = {32852081},
issn = {1460-2075},
support = {MR/S023380/1/MRC_/Medical Research Council/United Kingdom ; 214344/WT_/Wellcome Trust/United Kingdom ; G0801172/MRC_/Medical Research Council/United Kingdom ; 108183/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Adaptive Immunity ; Antiviral Restriction Factors ; CRISPR-Cas Systems ; Capsid/metabolism ; Cell Line ; DNA, Viral/genetics/*immunology ; Gene Editing ; Gene Products, gag/genetics ; HIV Infections/enzymology/genetics/*immunology/metabolism ; HIV Protease Inhibitors/*pharmacology ; HIV-1/genetics/*immunology/metabolism/pathogenicity ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Immunity, Innate ; Indoles/pharmacology ; Interferons/metabolism/pharmacology ; Macrophages/*metabolism ; Membrane Proteins/genetics/metabolism ; Mutation ; Nucleotidyltransferases/*metabolism ; Phenylalanine/analogs &amp; derivatives/pharmacology ; Signal Transduction/immunology ; Tripartite Motif Proteins/metabolism ; Ubiquitin-Protein Ligases/metabolism ; Virus Replication/*genetics ; },
abstract = {Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV-1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid- and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.},
}
@article {pmid32850723,
year = {2020},
author = {Zhang, K and Zhang, Z and Kang, J and Chen, J and Liu, J and Gao, N and Fan, L and Zheng, P and Wang, Y and Sun, J},
title = {CRISPR/Cas13d-Mediated Microbial RNA Knockdown.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {856},
pmid = {32850723},
issn = {2296-4185},
abstract = {RNA-guided and RNA-targeting type IV-D CRISPR/Cas systems (CRISPR/Cas13d) have recently been identified and employed for efficient and specific RNA knockdown in mammalian and plant cells. Cas13d possesses dual RNase activities and is capable of processing CRISPR arrays and cleaving target RNAs in a protospacer flanking sequence (PFS)-independent manner. These properties make this system a promising tool for multiplex gene expression regulation in microbes. Herein, we aimed to establish a CRISPR/Cas13d-mediated RNA knockdown platform for bacterial chassis. CasRx, Cas13d from Ruminococcus flavefaciens XPD3002, was selected due to its high activity. However, CasRx was found to be highly toxic to both Escherichia coli and Corynebacterium glutamicum, especially when it cooperated with its guide and target RNAs. After employing a low copy number vector, a tightly controlled promoter, and a weakened ribosome binding site, we successfully constructed an inducible expression system for CasRx and applied it for repressing the expression of a green fluorescent protein (GFP) in E. coli. Despite our efforts to optimize inducer usage, guide RNA (gRNA) architecture and combination, and target gene expression level, the highest gene repression efficiency was 30-50% at the protein level and ∼70% at the mRNA level. The moderate RNA knockdown is possibly caused by the collateral cleavage activity toward bystander RNAs, which acts as a mechanism of type IV-D immunity and perturbs microbial metabolism. Further studies on cellular response to CRISPR/Cas13d and improvement in RNA knockdown efficiency are required prior to practical application of this system in microbes.},
}
@article {pmid32849710,
year = {2020},
author = {Kumar, A and Kumar, R and Sengupta, D and Das, SN and Pandey, MK and Bohra, A and Sharma, NK and Sinha, P and Sk, H and Ghazi, IA and Laha, GS and Sundaram, RM},
title = {Deployment of Genetic and Genomic Tools Toward Gaining a Better Understanding of Rice-Xanthomonas oryzae pv. oryzae Interactions for Development of Durable Bacterial Blight Resistant Rice.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {1152},
pmid = {32849710},
issn = {1664-462X},
abstract = {Rice is the most important food crop worldwide and sustainable rice production is important for ensuring global food security. Biotic stresses limit rice production significantly and among them, bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is very important. BB reduces rice yields severely in the highly productive irrigated and rainfed lowland ecosystems and in recent years; the disease is spreading fast to other rice growing ecosystems as well. Being a vascular pathogen, Xoo interferes with a range of physiological and biochemical exchange processes in rice. The response of rice to Xoo involves specific interactions between resistance (R) genes of rice and avirulence (Avr) genes of Xoo, covering most of the resistance genes except the recessive ones. The genetic basis of resistance to BB in rice has been studied intensively, and at least 44 genes conferring resistance to BB have been identified, and many resistant rice cultivars and hybrids have been developed and released worldwide. However, the existence and emergence of new virulent isolates of Xoo in the realm of a rapidly changing climate necessitates identification of novel broad-spectrum resistance genes and intensification of gene-deployment strategies. This review discusses about the origin and occurrence of BB in rice, interactions between Xoo and rice, the important roles of resistance genes in plant's defense response, the contribution of rice resistance genes toward development of disease resistance varieties, identification and characterization of novel, and broad-spectrum BB resistance genes from wild species of Oryza and also presents a perspective on potential strategies to achieve the goal of sustainable disease management.},
}
@article {pmid32849428,
year = {2020},
author = {Zhang, Z and Li, Y and Luo, L and Hao, J and Li, J},
title = {Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1824},
pmid = {32849428},
issn = {1664-302X},
abstract = {Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.},
}
@article {pmid32847597,
year = {2020},
author = {Kanda, M and Shimizu, D and Sawaki, K and Nakamura, S and Umeda, S and Miwa, T and Tanaka, H and Tanaka, C and Hayashi, M and Iguchi, Y and Yamada, S and Katsuno, M and Kodera, Y},
title = {Therapeutic monoclonal antibody targeting of neuronal pentraxin receptor to control metastasis in gastric cancer.},
journal = {Molecular cancer},
volume = {19},
number = {1},
pages = {131},
pmid = {32847597},
issn = {1476-4598},
mesh = {Animals ; Antibodies, Monoclonal/*pharmacology ; Antineoplastic Agents, Immunological/*pharmacology ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Models, Animal ; Gene Expression ; Gene Targeting ; Humans ; Mice ; Mice, Knockout ; Models, Biological ; Neoplasm Metastasis ; Neoplasm Staging ; Phenotype ; Prognosis ; Receptors, Cell Surface/*antagonists &amp; inhibitors/genetics/metabolism ; Signal Transduction/drug effects ; Stomach Neoplasms/drug therapy/genetics/metabolism/pathology ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Controlling metastasis is essential for improving the prognosis of patients with gastric cancer (GC). Here, we aimed to identify a molecule required for GC metastasis and to investigate its potential utility as a target for the development of therapeutic antibodies (Abs).<br><br>METHODS: Transcriptome and bioinformatics analyses of human GC cell lines identified the neuronal pentraxin receptor (NPTXR) as a candidate molecule. NPTXR function was probed by modulating its expression in GC cells and assessing the effects on intracellular signaling and malignant behaviors in vitro and in mouse xenograft models. We also generated anti-NPTXR Abs and Nptxr[-/-] mice, and assessed the clinical significance of NPTXR expression in GC specimens.<br><br>RESULTS: NPTXR mRNA expression in clinical specimens was associated with disease progression and was significantly higher in tissues from GC patients with distant metastasis compared with those without. NPTXR regulated expression of genes involved in metastatic behaviors as well as activation of the PI3K-AKT-mTOR, FAK-JNK, and YAP signaling pathways. NPTXR silencing promoted caspase-mediated apoptosis and attenuated GC cell proliferation, cell cycle progression, migration, invasion, adhesion, stem cell-like properties, and resistance to 5-fluorouracil in vitro, and also inhibited the tumorigenicity of GC cells in vivo. Anti-NPTXR Abs inhibited GC peritoneal metastasis in mice. Nptxr[-/-] mice showed no abnormalities in reproduction, development, metabolism, or motor function.<br><br>CONCLUSIONS: NPTXR plays an essential role in controlling the malignant behavior of GC cells in vitro and in vivo. NPTXR-targeting Abs may thus have utility as novel diagnostic tools and/or treatment modalities for GC.},
}
@article {pmid32846370,
year = {2020},
author = {George, SL and Lorenzi, F and King, D and Hartlieb, S and Campbell, J and Pemberton, H and Toprak, UH and Barker, K and Tall, J and da Costa, BM and van den Boogaard, ML and Dolman, MEM and Molenaar, JJ and Bryant, HE and Westermann, F and Lord, CJ and Chesler, L},
title = {Therapeutic vulnerabilities in the DNA damage response for the treatment of ATRX mutant neuroblastoma.},
journal = {EBioMedicine},
volume = {59},
number = {},
pages = {102971},
pmid = {32846370},
issn = {2352-3964},
support = {MC_PC_16047/MRC_/Medical Research Council/United Kingdom ; MC_PC_18051/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Damage/*drug effects ; DNA Repair/*drug effects ; Disease Models, Animal ; Gene Editing ; Gene Knockout Techniques ; Humans ; Immunohistochemistry ; Mice ; Neuroblastoma/drug therapy/*genetics/mortality/pathology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Prognosis ; X-linked Nuclear Protein/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research.<br><br>METHODS: To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF.<br><br>FINDINGS: In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft.<br><br>INTERPRETATION: ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic.<br><br>FUNDING: This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.},
}
@article {pmid32843756,
year = {2020},
author = {Halpin-Healy, TS and Klompe, SE and Sternberg, SH and Fernández, IS},
title = {Publisher Correction: Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.},
journal = {Nature},
volume = {585},
number = {7825},
pages = {E12},
doi = {10.1038/s41586-020-2662-5},
pmid = {32843756},
issn = {1476-4687},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid32843651,
year = {2020},
author = {Zou, ZV and Gul, N and Lindberg, M and Bokhari, AA and Eklund, EM and Garellick, V and Patel, AAH and Dzanan, JJ and Titmuss, BO and Le Gal, K and Johansson, I and Tivesten, &#xc5; and Forssell-Aronsson, E and Bergö, MO and Staffas, A and Larsson, E and Sayin, VI and Lindahl, P},
title = {Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14156},
pmid = {32843651},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/genetics ; Cell Cycle Proteins/biosynthesis/genetics ; Cell Division ; Cell Line ; Chromatin Immunoprecipitation ; Cisplatin/toxicity ; Cyclin-Dependent Kinase Inhibitor p16/metabolism ; DNA Damage ; DNA-Binding Proteins/deficiency/*genetics/physiology ; Down-Regulation ; E2F Transcription Factors/physiology ; Etoposide/toxicity ; Fibroblasts ; Gene Expression Regulation/*genetics ; Gene Ontology ; Mice ; RNA Interference ; RNA, Small Interfering/genetics ; Signal Transduction/*genetics ; Transcription Factors/deficiency/*genetics/physiology ; Tumor Suppressor Protein p53/*physiology ; },
abstract = {Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.},
}
@article {pmid32843580,
year = {2020},
author = {Antón, Z and Mullally, G and Ford, HC and van der Kamp, MW and Szczelkun, MD and Lane, JD},
title = {Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors.},
journal = {Journal of cell science},
volume = {133},
number = {18},
pages = {},
doi = {10.1242/jcs.248468},
pmid = {32843580},
issn = {1477-9137},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Copy Number Variations ; DNA, Mitochondrial/genetics ; Gene Editing ; HeLa Cells ; Humans ; Mitochondria/genetics ; Reproducibility of Results ; },
abstract = {Current methodologies for targeting the mitochondrial genome for research and/or therapy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. A molecular toolbox for CRISPR-mediated mitochondrial genome editing is desirable, as this could enable targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes. Such 'MitoCRISPR' systems described to date lack reproducibility and independent corroboration. We have explored the requirements for MitoCRISPR in human cells by CRISPR nuclease engineering, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of guide (g)RNA modifications for mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and effects on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Our data link mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary for MitoCRISPR.},
}
@article {pmid32843543,
year = {2020},
author = {Choudhary, HH and Nava, MG and Gartlan, BE and Rose, S and Vinayak, S},
title = {A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum.},
journal = {mBio},
volume = {11},
number = {4},
pages = {},
pmid = {32843543},
issn = {2150-7511},
support = {R01 AI150961/AI/NIAID NIH HHS/United States ; R21 AI142380/AI/NIAID NIH HHS/United States ; T35 OD011145/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cryptosporidium parvum/drug effects/*genetics/metabolism ; Escherichia coli/genetics ; Female ; *Genes, Essential ; Genes, Protozoan ; Genetic Engineering ; Interferon-gamma/genetics ; Male ; Mice ; Mice, Knockout ; Protein Kinases/genetics ; *Proteolysis ; Tetrahydrofolate Dehydrogenase/genetics ; Trimethoprim/pharmacology ; },
abstract = {Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in CryptosporidiumIMPORTANCECryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.},
}
@article {pmid32843437,
year = {2020},
author = {Gaillochet, C and Develtere, W and Jacobs, TB},
title = {CRISPR Screens in Plants: Approaches, Guidelines, and Future Prospects.},
journal = {The Plant cell},
volume = {},
number = {},
pages = {},
doi = {10.1105/tpc.20.00463},
pmid = {32843437},
issn = {1532-298X},
abstract = {CRISPR-Cas systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in new powerful screens to test gene functions at the genomic scale. While there is tremendous potential for CRISPR screens to map and interrogate gene regulatory networks at unprecedented speed and scale, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports using this strategy to generate mutant knockout collections or diversify DNA sequences. In addition, we provide insight on how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene function in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the large resource of genomic profiles that were generated in the last two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will greatly advance plant functional and synthetic biology.},
}
@article {pmid32843348,
year = {2020},
author = {Port, F and Starostecka, M and Boutros, M},
title = {Multiplexed conditional genome editing with Cas12a in Drosophila.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {37},
pages = {22890-22899},
pmid = {32843348},
issn = {1091-6490},
support = {810296/ERC_/European Research Council/International ; },
mesh = {Animals ; Bacterial Proteins/*genetics/*metabolism ; CRISPR-Associated Proteins/*genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology ; Drosophila melanogaster/genetics ; Endodeoxyribonucleases/*genetics/*metabolism ; Endonucleases/metabolism ; Gene Editing/*methods ; RNA/genetics/metabolism ; RNA, Guide/metabolism ; },
abstract = {CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.},
}
@article {pmid32842631,
year = {2020},
author = {Tanikella, AS and Hardy, MJ and Frahs, SM and Cormier, AG and Gibbons, KD and Fitzpatrick, CK and Oxford, JT},
title = {Emerging Gene-Editing Modalities for Osteoarthritis.},
journal = {International journal of molecular sciences},
volume = {21},
number = {17},
pages = {},
pmid = {32842631},
issn = {1422-0067},
support = {P20 GM103408/GM/NIGMS NIH HHS/United States ; P20 GM109095/GM/NIGMS NIH HHS/United States ; P20GM103408/GM/NIGMS NIH HHS/United States ; P20GM109095/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Extracellular Vesicles/transplantation ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Mesenchymal Stem Cells ; Osteoarthritis/*genetics/*therapy ; Regeneration ; },
abstract = {Osteoarthritis (OA) is a pathological degenerative condition of the joints that is widely prevalent worldwide, resulting in significant pain, disability, and impaired quality of life. The diverse etiology and pathogenesis of OA can explain the paucity of viable preventive and disease-modifying strategies to counter it. Advances in genome-editing techniques may improve disease-modifying solutions by addressing inherited predisposing risk factors and the activity of inflammatory modulators. Recent progress on technologies such as CRISPR/Cas9 and cell-based genome-editing therapies targeting the genetic and epigenetic alternations in OA offer promising avenues for early diagnosis and the development of personalized therapies. The purpose of this literature review was to concisely summarize the genome-editing options against chronic degenerative joint conditions such as OA with a focus on the more recently emerging modalities, especially CRISPR/Cas9. Future advancements in novel genome-editing therapies may improve the efficacy of such targeted treatments.},
}
@article {pmid32842577,
year = {2020},
author = {Reddy, P and Vilella, F and Izpisua Belmonte, JC and Simón, C},
title = {Use of Customizable Nucleases for Gene Editing and Other Novel Applications.},
journal = {Genes},
volume = {11},
number = {9},
pages = {},
pmid = {32842577},
issn = {2073-4425},
mesh = {Animals ; *CRISPR-Cas Systems ; Endonucleases/genetics/*metabolism ; Gene Editing/*methods ; *Genetic Engineering ; *Genome ; Humans ; },
abstract = {The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes.},
}
@article {pmid32842152,
year = {2020},
author = {Fayos, I and Meunier, AC and Vernet, A and Navarro-Sanz, S and Portefaix, M and Lartaud, M and Bastianelli, G and Périn, C and Nicolas, A and Guiderdoni, E},
title = {Assessment of the roles of SPO11-2 and SPO11-4 in meiosis in rice using CRISPR/Cas9 mutagenesis.},
journal = {Journal of experimental botany},
volume = {71},
number = {22},
pages = {7046-7058},
doi = {10.1093/jxb/eraa391},
pmid = {32842152},
issn = {1460-2431},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; Meiosis ; Mutagenesis ; *Oryza/genetics ; },
abstract = {In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.},
}
@article {pmid32841560,
year = {2020},
author = {Dong, C and Jiang, L and Xu, S and Huang, L and Cai, J and Lian, J and Xu, Z},
title = {A Single Cas9-VPR Nuclease for Simultaneous Gene Activation, Repression, and Editing in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2252-2257},
doi = {10.1021/acssynbio.0c00218},
pmid = {32841560},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; *Gene Expression Regulation ; Luminescent Proteins/genetics/metabolism ; Metabolic Engineering/*methods ; Saccharomyces cerevisiae/genetics/*metabolism ; },
abstract = {Combinatorial metabolic engineering has been widely established for the development of efficient microbial cell factories to produce the products of interest by precisely regulating the expression levels of multiple genes simultaneously. Here, we report a novel multifunctional CRISPR system that enables simultaneous gene activation, repression, and editing (CRISPR-ARE) with a single Cas9-VPR protein for combinatorial metabolic engineering applications in Saccharomyces cerevisiae. Via gRNA engineering, we achieved orthogonal transcriptional regulations and genome editing using the nuclease active Cas9-VPR fusion protein, individually or in a combinatorial manner. After establishing a system for stable expression of multiple gRNAs on the same plasmid, we first demonstrated CRISPR-ARE for simultaneous mCherry activation, mVenus repression, and ADE2 disruption in a fluorescence reporter strain. Subsequently, we adopted CRISPR-ARE for simple and fast combinatorial metabolic engineering, which improved the production of α-santalene for 2.66-fold in a single step. Because of its simplicity and modularity, the developed CRISPR-ARE system could be applied for facile multifunctional metabolic engineering of microbial cell factories, particularly for which only a few CRISPR proteins have been characterized.},
}
@article {pmid32841542,
year = {2021},
author = {Zou, G and Xiao, M and Chai, S and Zhu, Z and Wang, Y and Zhou, Z},
title = {Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents.},
journal = {Microbial biotechnology},
volume = {14},
number = {6},
pages = {2343-2355},
pmid = {32841542},
issn = {1751-7915},
mesh = {*CRISPR-Cas Systems ; Cordyceps/genetics ; Fungi/*genetics ; *Gene Editing ; Hypocreales/genetics ; Protoplasts/metabolism ; RNA, Guide ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {DNA double-strand break (DSB) repair induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9-CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X-100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP-based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi-target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on in vivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome-edited fungi for the food and medical industries, and has good prospects for commercialization.},
}
@article {pmid32841375,
year = {2021},
author = {Ge, L and Kang, J and Dong, X and Luan, D and Su, G and Li, G and Zhang, Y and Quan, F},
title = {Myostatin site-directed mutation and simultaneous PPARγ site-directed knockin in bovine genome.},
journal = {Journal of cellular physiology},
volume = {236},
number = {4},
pages = {2592-2605},
doi = {10.1002/jcp.30017},
pmid = {32841375},
issn = {1097-4652},
mesh = {Adipogenesis ; Animals ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Cattle ; Cell Line ; Cell Proliferation ; Cell Transdifferentiation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Feasibility Studies ; *Gene Editing ; Gene Expression Regulation, Developmental ; *Gene Knock-In Techniques ; Muscle Development ; *Mutagenesis, Site-Directed ; *Mutation ; Myoblasts/*metabolism ; Myostatin/*genetics/metabolism ; Nuclear Transfer Techniques ; PPAR gamma/*genetics/metabolism ; },
abstract = {Most studies on the acquisition of advantageous traits in transgenic animals only focus on monogenic traits. In practical applications, transgenic animals need to possess multiple advantages. Therefore, multiple genes need to be edited simultaneously. CRISPR/Cas9 technology has been widely used in many research fields. However, few studies on endogenous gene mutation and simultaneous exogenous gene insertion performed via CRISPR/Cas9 technology are available. In this study, the CRISPR/Cas9 technology was used to achieve myostatin (MSTN) point mutation and simultaneous peroxisome proliferator-activated receptor-γ (PPARγ) site-directed knockin in the bovine genome. The feasibility of this gene editing strategy was verified on a myoblast model. The same gene editing strategy was used to construct a mutant myoblast model with MSTN mutation and simultaneous PPARγ knockin. Quantitative reverse-transcription polymerase chain reaction, immunofluorescence staining, and western blot analyses were used to detect the expression levels of MSTN and PPARγ in the mutant myoblast. Results showed that this strategy can inhibit the expression of MSTN and promote the expression of PPARγ. The cell counting kit-8 cell proliferation analysis, 5-ethynyl-2'-deoxyuridine cell proliferation analysis, myotube fusion index statistics, oil red O staining, and triglyceride content detection revealed that the proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities of the mutant myoblasts were higher than those of the wild myoblasts. Finally, transgenic bovine embryos were obtained via somatic cell nuclear transfer. This study provides a breeding material and technical strategy to breed high-quality bovine and a gene editing method to realize the mutation of endogenous genes and simultaneous insertion of exogenous genes in genomes.},
}
@article {pmid32841372,
year = {2021},
author = {Khan, MHU and Hu, L and Zhu, M and Zhai, Y and Khan, SU and Ahmar, S and Amoo, O and Zhang, K and Fan, C and Zhou, Y},
title = {Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.},
journal = {Journal of cellular physiology},
volume = {236},
number = {3},
pages = {1996-2007},
doi = {10.1002/jcp.29986},
pmid = {32841372},
issn = {1097-4652},
mesh = {Base Sequence ; Brassica napus/*genetics/*growth &amp; development ; CRISPR-Cas Systems/genetics ; Cell Size ; Cloning, Molecular ; Cotyledon/anatomy &amp; histology/growth &amp; development ; Gene Editing ; Gene Expression Regulation, Plant ; *Genes, Plant ; Mutagenesis/*genetics ; Mutation/genetics ; Organ Size ; Phenotype ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; RNA, Guide/genetics ; Seeds/*genetics/*growth &amp; development ; Sequence Homology, Amino Acid ; },
abstract = {Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.},
}
@article {pmid32840797,
year = {2021},
author = {Mitrentsi, I and Soutoglou, E},
title = {CRISPR/Cas9-Induced Breaks in Heterochromatin, Visualized by Immunofluorescence.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2153},
number = {},
pages = {439-445},
doi = {10.1007/978-1-0716-0644-5_30},
pmid = {32840797},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA Repair ; Fluorescent Antibody Technique ; Heterochromatin/*genetics ; Mice ; NIH 3T3 Cells ; },
abstract = {CRISPR/Cas9 technology can be used to investigate how double-strand breaks (DSBs) occurring in constitutive heterochromatin are getting repaired. This technology can be used to induce specific breaks on mouse pericentromeric heterochromatin, by using a guide RNA specific for the major satellite repeats and co-expressing it with Cas9. Those clean DSBs can be visualized later by confocal microscopy. More specifically, immunofluorescence can be used to visualize the main factors of each DSB repair pathway and quantify their percentage and pattern of recruitment at the heterochromatic region.},
}
@article {pmid32839482,
year = {2020},
author = {Ishibashi, R and Abe, K and Ido, N and Kitano, S and Miyachi, H and Toyoshima, F},
title = {Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14120},
pmid = {32839482},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Targeting ; Genetic Engineering/methods ; Genome/genetics ; HEK293 Cells ; HeLa Cells ; Humans ; INDEL Mutation/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Transgenic ; Plasmids/*genetics ; RNA, Guide/*genetics ; T-Box Domain Proteins/*genetics ; Transgenes/genetics ; },
abstract = {CRISPR/Cas-mediated genome editing is a powerful tool for generating genetically mutated cells and organisms. Linearisation of donor cassettes with this system has been shown to facilitate both transgene donor insertion and targeted knock-in. Here, we developed a donor plasmid that we name pCriMGET (plasmid of synthetic CRISPR coded RNA target sequence-equipped donor plasmid-mediated gene targeting), in which an off-target free synthetic CRISPR coded RNA-target sequence (syn-crRNA-TS) is incorporated with a multi-cloning site, where a donor cassette can be inserted. With co-expression of Cas9 and the syn-crRNA-TS guide RNA (gRNA), pCriMGET provides a linearised donor cassette in vivo, thereby promoting the transgene donor insertion and targeted knock-in. When co-injected with Cas9 protein and gRNA into murine zygotes, pCriMGET yielded around 20% transgene insertion in embryos. This method also achieved more than 25% in-frame knock-in at the mouse Tbx3 gene locus without predicted insertion-deletion mutations using a transgene donor with 400-bp homology arms. pCriMGET is therefore useful as a versatile CRISPR/Cas9-cleavable donor plasmid for efficient integration and targeted knock-in of exogenous DNA in mice.},
}
@article {pmid32839345,
year = {2020},
author = {Carballar-Lejarazú, R and Ogaugwu, C and Tushar, T and Kelsey, A and Pham, TB and Murphy, J and Schmidt, H and Lee, Y and Lanzaro, GC and James, AA},
title = {Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {37},
pages = {22805-22814},
pmid = {32839345},
issn = {1091-6490},
mesh = {Alleles ; Animals ; Animals, Genetically Modified/genetics ; Anopheles/*genetics ; CRISPR-Cas Systems/genetics ; Gene Drive Technology/*methods ; Genetics, Population/methods ; High-Throughput Nucleotide Sequencing/methods ; Malaria/prevention &amp; control ; Mosquito Control/*methods ; Mosquito Vectors/genetics ; Phenotype ; Transgenes/genetics ; },
abstract = {A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, Anopheles gambiae The drive system targets the cardinal gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro, with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.},
}
@article {pmid32839320,
year = {2020},
author = {Han, S and Zhao, BS and Myers, SA and Carr, SA and He, C and Ting, AY},
title = {RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {36},
pages = {22068-22079},
pmid = {32839320},
issn = {1091-6490},
support = {R01 DK121409/DK/NIDDK NIH HHS/United States ; U01 CA214125/CA/NCI NIH HHS/United States ; U24 CA210986/CA/NCI NIH HHS/United States ; },
mesh = {AlkB Homolog 5, RNA Demethylase/genetics/metabolism ; Biotinylation ; CRISPR-Cas Systems ; DNA Methylation ; DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/*metabolism ; Endonucleases/genetics/*metabolism ; HEK293 Cells ; Humans ; Mass Spectrometry ; Multifunctional Enzymes/genetics/*metabolism ; Protein Binding ; Protein Interaction Mapping/*methods ; RNA/genetics/*metabolism ; Telomerase/genetics/*metabolism ; },
abstract = {RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N[6]-methyladenosine (m[6]A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m[6]A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.},
}
@article {pmid32837247,
year = {2022},
author = {Ibrahim, AU and Al-Turjman, F and Sa'id, Z and Ozsoz, M},
title = {Futuristic CRISPR-based biosensing in the cloud and internet of things era: an overview.},
journal = {Multimedia tools and applications},
volume = {81},
number = {24},
pages = {35143-35171},
pmid = {32837247},
issn = {1380-7501},
abstract = {Biosensors-based devices are transforming medical diagnosis of diseases and monitoring of patient signals. The development of smart and automated molecular diagnostic tools equipped with biomedical big data analysis, cloud computing and medical artificial intelligence can be an ideal approach for the detection and monitoring of diseases, precise therapy, and storage of data over the cloud for supportive decisions. This review focused on the use of machine learning approaches for the development of futuristic CRISPR-biosensors based on microchips and the use of Internet of Things for wireless transmission of signals over the cloud for support decision making. The present review also discussed the discovery of CRISPR, its usage as a gene editing tool, and the CRISPR-based biosensors with high sensitivity of Attomolar (10[-18] M), Femtomolar (10[-15] M) and Picomolar (10[-12] M) in comparison to conventional biosensors with sensitivity of nanomolar 10[-9] M and micromolar 10[-3] M. Additionally, the review also outlines limitations and open research issues in the current state of CRISPR-based biosensing applications.},
}
@article {pmid32835482,
year = {2020},
author = {Sergeeva, D and Lee, GM and Nielsen, LK and Grav, LM},
title = {Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2546-2561},
doi = {10.1021/acssynbio.0c00322},
pmid = {32835482},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; Erythropoietin/genetics/metabolism ; Gene Dosage ; Gene Editing/*methods ; Plasmids/genetics/metabolism ; Recombinant Proteins/*biosynthesis/genetics ; Recombinases/genetics ; },
abstract = {The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on qP. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.},
}
@article {pmid32835412,
year = {2020},
author = {Dai, Y and Xu, W and Somoza, RA and Welter, JF and Caplan, AI and Liu, CC},
title = {An Integrated Multi-Function Heterogeneous Biochemical Circuit for High-Resolution Electrochemistry-Based Genetic Analysis.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {46},
pages = {20545-20551},
pmid = {32835412},
issn = {1521-3773},
support = {P41 EB021911/EB/NIBIB NIH HHS/United States ; 1P41EB021911/NH/NIH HHS/United States ; Wallace R Persons Research Fund//Case Western Reserve University/International ; },
mesh = {Biosensing Techniques/*methods ; COVID-19/pathology/virology ; CRISPR-Cas Systems/genetics ; Cell Line ; Electrochemical Techniques ; *Genes, Viral ; Humans ; Nucleic Acid Amplification Techniques ; RNA, Guide/metabolism ; SARS-CoV-2/*genetics/isolation &amp; purification ; },
abstract = {Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.},
}
@article {pmid32833548,
year = {2020},
author = {Zhu, N and Wong, PK},
title = {Advances in Viral Diagnostic Technologies for Combating COVID-19 and Future Pandemics.},
journal = {SLAS technology},
volume = {25},
number = {6},
pages = {513-521},
pmid = {32833548},
issn = {2472-6311},
mesh = {COVID-19/*diagnosis ; COVID-19 Testing/*methods ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; Pandemics ; SARS-CoV-2/*physiology ; Single-Cell Analysis ; },
abstract = {The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens the health of the global population and challenges our preparedness for pandemic threats. Previous outbreaks of coronaviruses and other viruses have suggested the importance of diagnostic technologies in fighting viral outbreaks. Nucleic acid detection techniques are the gold standard for detecting SARS-CoV-2. Viral antigen tests and serological tests that detect host antibodies have also been developed for studying the epidemiology of COVID-19 and estimating the population that may have immunity to SARS-CoV-2. Nevertheless, the availability, cost, and performance of existing viral diagnostic technologies limit their practicality, and novel approaches are required for improving our readiness for global pandemics. Here, we review the principles and limitations of major viral diagnostic technologies and highlight recent advances of molecular assays for COVID-19. In addition, we discuss emerging technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR) systems, high-throughput sequencing, and single-cell and single-molecule analysis, for improving our ability to understand, trace, and contain viral outbreaks. The prospects of viral diagnostic technologies for combating future pandemic threats are presented.},
}
@article {pmid32833535,
year = {2020},
author = {Tay, LS and Palmer, N and Panwala, R and Chew, WL and Mali, P},
title = {Translating CRISPR-Cas Therapeutics: Approaches and Challenges.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {253-275},
pmid = {32833535},
issn = {2573-1602},
support = {R01 HG009285/HG/NHGRI NIH HHS/United States ; R01 GM123313/GM/NIGMS NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Therapy/*methods ; Humans ; },
abstract = {CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities. Here, we review how CRISPR-Cas therapeutics are translated from technological systems to therapeutic modalities, paying particular attention to the therapeutic cascade from cargo to delivery vector, manufacturing, administration, pipelines, safety, and therapeutic target profiles. We also explore potential solutions to some of the obstacles facing successful CRISPR-Cas translation. We hope to illuminate how CRISPR-Cas is brought from the academic bench toward use in the clinic.},
}
@article {pmid32833534,
year = {2020},
author = {Wallace, DC},
title = {CRISPR-Free Mitochondrial DNA Base Editing.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {228-230},
doi = {10.1089/crispr.2020.29101.dwa},
pmid = {32833534},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA, Mitochondrial ; Gene Editing/*methods ; Humans ; Mitochondrial Diseases/genetics/*therapy ; },
}
@article {pmid32833533,
year = {2020},
author = {Fry, LE and Peddle, CF and Stevanovic, M and Barnard, AR and McClements, ME and MacLaren, RE},
title = {Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {276-283},
pmid = {32833533},
issn = {2573-1602},
support = {MC_PC_18059/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus ; *Gene Editing ; Genetic Vectors ; *Promoter Regions, Genetic ; RNA, Guide/*genetics ; Staphylococcus aureus/enzymology ; },
abstract = {Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.},
}
@article {pmid32833532,
year = {2020},
author = {Fricke, T and Smalakyte, D and Lapinski, M and Pateria, A and Weige, C and Pastor, M and Kolano, A and Winata, C and Siksnys, V and Tamulaitis, G and Bochtler, M},
title = {Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {299-313},
pmid = {32833532},
issn = {2573-1602},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *RNA Stability ; RNA, Messenger/metabolism ; Streptococcus thermophilus/enzymology ; Zebrafish/*genetics ; },
abstract = {RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects.},
}
@article {pmid32833531,
year = {2020},
author = {Barrangou, R},
title = {In Times Like These, We All Need a Moment of Science.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {223},
doi = {10.1089/crispr.2020.29102.rba},
pmid = {32833531},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Journal Impact Factor ; Science ; },
}
@article {pmid32833530,
year = {2020},
author = {LaManna, CM and Pyhtila, B and Barrangou, R},
title = {Sharing the CRISPR Toolbox with an Expanding Community.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {248-252},
doi = {10.1089/crispr.2020.0075},
pmid = {32833530},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*statistics &amp; numerical data/trends ; },
abstract = {Over the past 8 years, the widespread adoption of CRISPR-based technologies has fueled the global genome editing revolution. This platform is based on Cas molecular machines such as Cas9, Cas12, Cas13, as well as other CRISPR effector proteins that are able to alter the genome, transcriptome, and epigenome of virtually any species. Technological improvements have rendered these tools more efficient and precise, and enabled functional diversification and specialization, as recently illustrated by the rise of base editing and the quickly growing demand for prime editing constructs. Here, we discuss the continued adoption of CRISPR tools and constructs distributed by the nonprofit organization Addgene, highlight the trends in the global demand for the CRISPR toolbox, and consider the widespread attitude changes around open sharing that are having a transformative effect on speeding up science.},
}
@article {pmid32833528,
year = {2020},
author = {Taylor, G},
title = {Going Public with Your Research.},
journal = {The CRISPR journal},
volume = {3},
number = {4},
pages = {231-232},
doi = {10.1089/crispr.2020.29098.gta},
pmid = {32833528},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Science/*education ; },
}
@article {pmid32833346,
year = {2020},
author = {Hoellerbauer, P and Kufeld, M and Paddison, PJ},
title = {Efficient Multi-Allelic Genome Editing of Primary Cell Cultures via CRISPR-Cas9 Ribonucleoprotein Nucleofection.},
journal = {Current protocols in stem cell biology},
volume = {54},
number = {1},
pages = {e126},
doi = {10.1002/cpsc.126},
pmid = {32833346},
issn = {1938-8969},
support = {T32 CA080416/NH/NIH HHS/United States ; R01 CA190957/NH/NIH HHS/United States ; U54 DK106829/NH/NIH HHS/United States ; P30 CA015704/NH/NIH HHS/United States ; },
mesh = {*Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Nucleus/*metabolism ; Cells, Cultured ; Gene Editing/*methods ; Humans ; RNA, Guide/genetics ; Ribonucleoproteins/*metabolism ; *Transfection ; },
abstract = {CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR-Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl-3'phosphorothioate-modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion-deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem-like cells. © 2020 Wiley Periodicals LLC Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion-deletion (indel) mutation and genomic deletion generation in primary cell cultures.},
}
@article {pmid32833316,
year = {2021},
author = {Brown, W and Zhou, W and Deiters, A},
title = {Regulating CRISPR/Cas9 Function through Conditional Guide RNA Control.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {22},
number = {1},
pages = {63-72},
pmid = {32833316},
issn = {1439-7633},
support = {R21 HD085206/HD/NICHD NIH HHS/United States ; R01 GM112728/GM/NIGMS NIH HHS/United States ; T32 GM088119/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; RNA, Guide/genetics/*metabolism ; },
abstract = {Conditional control of CRISPR/Cas9 has been developed by using a variety of different approaches, many focusing on manipulation of the Cas9 protein itself. However, more recent strategies for governing CRISPR/Cas9 function are based on guide RNA (gRNA) modifications. They include control of gRNAs by light, small molecules, proteins, and oligonucleotides. These designs have unique advantages compared to other approaches and have allowed precise regulation of gene editing and transcription. Here, we discuss strategies for conditional control of gRNA function and compare effectiveness of these methods.},
}
@article {pmid32833023,
year = {2020},
author = {Liu, Z and Sun, M and Liu, J and Liu, T and Ye, Q and Li, Y and Peng, N},
title = {A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9681-9693},
pmid = {32833023},
issn = {1362-4962},
mesh = {5' Untranslated Regions ; Archaeal Proteins/*genetics/metabolism ; Binding Sites ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Damage ; *DNA Repair ; Gene Expression Profiling ; Gene Expression Regulation, Archaeal ; Genome, Archaeal ; Mutation ; Promoter Regions, Genetic ; Sulfolobus/*genetics/growth &amp; development ; },
abstract = {CRISPR-Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.},
}
@article {pmid32832641,
year = {2020},
author = {Deng, S and Li, X and Liu, S and Chen, J and Li, M and Chew, SY and Leong, KW and Cheng, D},
title = {Codelivery of CRISPR-Cas9 and chlorin e6 for spatially controlled tumor-specific gene editing with synergistic drug effects.},
journal = {Science advances},
volume = {6},
number = {29},
pages = {eabb4005},
pmid = {32832641},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chlorophyllides ; Delayed-Action Preparations ; Gene Editing ; Mice ; NF-E2-Related Factor 2/metabolism ; *Nanoparticles ; *Neoplasms/genetics ; RNA, Guide ; Reactive Oxygen Species/metabolism ; Ribonucleoproteins/genetics ; },
abstract = {Controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and codelivery with other drugs remain a challenge. We demonstrate controlled release of CRISPR-Cas9 RNP and codelivery with antitumor photosensitizer chlorin e6 (Ce6) using near-infrared (NIR)- and reducing agent-responsive nanoparticles in a mouse tumor model. Nitrilotriacetic acid-decorated micelles can bind His-tagged Cas9 RNP. Lysosomal escape of nanoparticles was triggered by NIR-induced reactive oxygen species (ROS) generation by Ce6 in tumor cells. Cytoplasmic release of Cas9/single-guide RNA (sgRNA) was achieved by reduction of disulfide bond. Cas9/sgRNA targeted the antioxidant regulator Nrf2, enhancing tumor cell sensitivity to ROS. Without NIR irradiation, Cas9 was degraded in lysosomes and gene editing failed in normal tissues. The synergistic effects of Ce6 photodynamic therapy and Nrf2 gene editing were confirmed in vivo. Controlled release of CRISPR-Cas9 RNP and codelivery with Ce6 using stimuli-responsive nanoparticles represent a versatile strategy for gene editing with potentially synergistic drug effects.},
}
@article {pmid32831134,
year = {2020},
author = {Bi, C and Wang, L and Yuan, B and Zhou, X and Li, Y and Wang, S and Pang, Y and Gao, X and Huang, Y and Li, M},
title = {Long-read individual-molecule sequencing reveals CRISPR-induced genetic heterogeneity in human ESCs.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {213},
pmid = {32831134},
issn = {1474-760X},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA Repair ; Gene Editing/methods ; Gene Knock-In Techniques ; *Genetic Heterogeneity ; High-Throughput Nucleotide Sequencing/methods ; Human Embryonic Stem Cells ; Humans ; Mutation ; RNA, Guide ; },
abstract = {Quantifying the genetic heterogeneity of a cell population is essential to understanding of biological systems. We develop a universal method to label individual DNA molecules for single-base-resolution haplotype-resolved quantitative characterization of diverse types of rare variants, with frequency as low as 4 × 10[-5], using both short- or long-read sequencing platforms. It provides the first quantitative evidence of persistent nonrandom large structural variants and an increase in single-nucleotide variants at the on-target locus following repair of double-strand breaks induced by CRISPR-Cas9 in human embryonic stem cells.},
}
@article {pmid32830431,
year = {2021},
author = {Nartey, MA and Sun, X and Qin, S and Hou, CX and Li, MW},
title = {CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori.},
journal = {Insect science},
volume = {28},
number = {5},
pages = {1414-1425},
doi = {10.1111/1744-7917.12864},
pmid = {32830431},
issn = {1744-7917},
mesh = {Animals ; *Bombyx/genetics/physiology ; CLOCK Proteins/*genetics ; *CRISPR-Cas Systems ; Circadian Clocks ; *Circadian Rhythm ; Gene Knockout Techniques ; Insect Proteins/*genetics ; },
abstract = {Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.},
}
@article {pmid32829077,
year = {2020},
author = {Meijers, AS and Troost, R and Ummels, R and Maaskant, J and Speer, A and Nejentsev, S and Bitter, W and Kuijl, CP},
title = {Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9.},
journal = {Tuberculosis (Edinburgh, Scotland)},
volume = {124},
number = {},
pages = {101983},
pmid = {32829077},
issn = {1873-281X},
support = {832721/ERC_/European Research Council/International ; MC_PC_16040/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Antitubercular Agents/pharmacology ; Bacterial Proteins/genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Catalase/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Bacterial/drug effects ; Gene Deletion ; *Gene Editing ; Gene Expression Regulation, Bacterial ; INDEL Mutation ; Isoniazid/pharmacology ; Mycobacterium marinum/*genetics ; Mycobacterium tuberculosis/drug effects/*genetics/growth &amp; development ; RNA, Guide/genetics ; Streptococcus thermophilus/enzymology/*genetics ; },
abstract = {The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.},
}
@article {pmid32828714,
year = {2020},
author = {Li, C and Kasinski, AL},
title = {InVivo Cancer-Based Functional Genomics.},
journal = {Trends in cancer},
volume = {6},
number = {12},
pages = {1002-1017},
doi = {10.1016/j.trecan.2020.07.004},
pmid = {32828714},
issn = {2405-8025},
mesh = {Animals ; Biomarkers, Tumor/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA Transposable Elements/genetics ; Disease Models, Animal ; Gene Editing/methods ; Genomics/*methods ; High-Throughput Screening Assays/*methods ; Humans ; Mice ; Mice, Transgenic ; Molecular Targeted Therapy/methods ; Neoplasms/*genetics/therapy ; Precision Medicine/*methods ; RNA, Guide/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Pinpointing the underlying mechanisms that drive tumorigenesis in human patients is a prerequisite for identifying suitable therapeutic targets for precision medicine. In contrast to cell culture systems, mouse models are highly favored for evaluating tumor progression and therapeutic response in a more realistic in vivo context. The past decade has witnessed a dramatic increase in the number of functional genomic studies using diverse mouse models, including in vivo clustered regularly interspaced short palindromic repeats (CRISPR) and RNA interference (RNAi) screens, and these have provided a wealth of knowledge addressing multiple essential questions in translational cancer research. We compare the multiple mouse systems and genomic tools that are commonly used for in vivo screens to illustrate their strengths and limitations. Crucial components of screen design and data analysis are also discussed.},
}
@article {pmid32828559,
year = {2021},
author = {Khouzam, JPS and Tivakaran, VS},
title = {CRISPR-Cas9 Applications in Cardiovascular Disease.},
journal = {Current problems in cardiology},
volume = {46},
number = {3},
pages = {100652},
doi = {10.1016/j.cpcardiol.2020.100652},
pmid = {32828559},
issn = {1535-6280},
mesh = {*CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing ; Humans ; Mutation ; },
abstract = {The CRISPR-Cas9 system is an economical and accessible gene-editing technology first discovered as a naturally occurring bacterial immune system. Since its fairly recent discovery, CRISPR-Cas9 system's efficiency and simplicity have been successfully used to edit genomes of living organisms in many fields, working in vitro and in vivo in germline and somatic cells to knock-out harmful mutated genes or in some cases working to knock-in a beneficial gene. A current application of the gene-editing system works against specific mutations that cause certain cardiovascular diseases. However, there are current technical limitations as well as ethical dilemmas in introducing gene-editing to humans. Here, we explore highlights on the current state of research of the CRISPR-Cas9 system through the lens of cardiovascular disease and examine potential untouched applications of the system in the field of cardiology.},
}
@article {pmid32828556,
year = {2021},
author = {Liu, R and Liang, L and Freed, EF and Gill, RT},
title = {Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing.},
journal = {Trends in biotechnology},
volume = {39},
number = {3},
pages = {262-273},
doi = {10.1016/j.tibtech.2020.07.005},
pmid = {32828556},
issn = {1879-3096},
mesh = {Biotechnology/trends ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome ; Protein Domains ; Protein Engineering/trends ; },
abstract = {CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. In this review, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems.},
}
@article {pmid32828551,
year = {2020},
author = {Liu, H and Zhang, B},
title = {Virus-Based CRISPR/Cas9 Genome Editing in Plants.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {11},
pages = {810-813},
doi = {10.1016/j.tig.2020.08.002},
pmid = {32828551},
issn = {0168-9525},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; Plants/genetics ; },
abstract = {CRISPR/Cas9 is a versatile tool for plant gene function studies and crop improvement. However, traditional CRISPR/Cas9-mediated genome editing requires plant tissue culture that is both time-consuming and genotype-dependent. Ma et al. recently reported a novel virus-based method for delivering CRISPR/Cas9 into plant cells, and this should further expand the application of CRISPR/Cas9-mediated genome editing.},
}
@article {pmid32828318,
year = {2020},
author = {Chen, W and Gao, D and Xie, L and Wang, A and Zhao, H and Guo, C and Sun, Y and Nie, Y and Hong, A and Xiong, S},
title = {SCF-FBXO24 regulates cell proliferation by mediating ubiquitination and degradation of PRMT6.},
journal = {Biochemical and biophysical research communications},
volume = {530},
number = {1},
pages = {75-81},
doi = {10.1016/j.bbrc.2020.06.007},
pmid = {32828318},
issn = {1090-2104},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; F-Box Proteins/genetics/*metabolism ; Humans ; Mutation ; Neoplasm Invasiveness/genetics ; Neoplasms/genetics/metabolism ; Nuclear Proteins/genetics/*metabolism ; Protein-Arginine N-Methyltransferases/genetics/*metabolism ; *Proteolysis ; *Ubiquitination ; Up-Regulation ; },
abstract = {The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression by methylation of the histone H3 on arginine 2 (H3R2), H4R3 and H2AR3 [1,2]. PRMT6 is aberrantly expressed in various types of human cancer, and abnormal methylation in cancers caused by overexpression of PRMT6 is considered to correlate with poor recovery prognosis [3,4]. However, mechanisms that regulate PRMT6 protein stability in cells remain largely unknown. Here we identified that an orphan F-box protein, FBXO24, that binds to 270 to 275 amino acid residues of PRMT6 to cause polyubiquitination of lysine at position 369 of PRMT6, which mediates its degradation via the ubiquitin-proteasome pathway. Overexpression of FBXO24 or knockout of PRMT6 was found to inhibit cell proliferation, migration, and invasion in H1299 cells. PRMT6 K369R mutant became resistant to degradation. Overexpression of PRMT6 K369R caused cell cycle progression, resulting in cell proliferation. Thus, our data confirm that FBXO24 regulates cell proliferation by mediating ubiquitin-dependent proteasomal degradation of PRMT6.},
}
@article {pmid32827549,
year = {2020},
author = {Tan, Y and Du, RL and Su, ZY and Xue, LL and Liu, J and Zhang, BL and Huang, SJ and Li, LJ and Xi-Yang, YB and Xiong, LL},
title = {Vof-16 knockout improves the recovery from hypoxic-ischemic brain damage of neonatal rats.},
journal = {Brain research},
volume = {1748},
number = {},
pages = {147070},
doi = {10.1016/j.brainres.2020.147070},
pmid = {32827549},
issn = {1872-6240},
mesh = {Animals ; Animals, Newborn ; Brain/*metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Hypoxia-Ischemia, Brain/*genetics/metabolism ; Maze Learning/physiology ; Motor Activity/physiology ; Proteins/*genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Recovery of Function/*physiology ; },
abstract = {Hypoxic-ischemic encephalopathy (HIE) results in high neonatal mortality and severe neurological impairments, and its underlying molecular mechanism underwent extensive investigations. Long non-coding RNA (lncRNA) is considered to be an important regulator on brain development and many neurological diseases. Currently, little is known about the role of Vof-16 (lncRNA) in HIE. We detected the relative expression level of Vof-16 in the cortex and hippocampus of hypoxic-ischemic (HI) models whose successful establishment was verified by TTC staining. Then, Vof-16 knockout rats were generated using the CRISPR/Cas engineering technology to search the specific function of the Vof-16 through a series of behavioral evaluations including Neurological severity scores (NSS), Y-maze test, Morris water maze (MWW) test, open field test, and Rotarod test. The results demonstrated the expression of Vof-16 was substantially up-regulated in the cortex and hippocampus of rats with HI injury. Importantly, Vof-16 knockout facilitated the recovery from long-term HI induced nerve damage and neurobehavioral dysfunctions. In conclusion, this study suggests Vof-16 knockout is a promising treatment target for neonatal HIE.},
}
@article {pmid32827460,
year = {2020},
author = {Di Roberto, RB and Castellanos-Rueda, R and Frey, S and Egli, D and Vazquez-Lombardi, R and Kapetanovic, E and Kucharczyk, J and Reddy, ST},
title = {A Functional Screening Strategy for Engineering Chimeric Antigen Receptors with Reduced On-Target, Off-Tumor Activation.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2564-2576},
pmid = {32827460},
issn = {1525-0024},
mesh = {Animals ; Antibody Affinity ; Antigens, Neoplasm/*immunology/metabolism ; Breast Neoplasms/*immunology/pathology ; CRISPR-Cas Systems ; Cell Engineering/*methods ; Coculture Techniques ; Female ; Gene Editing/methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Immunotherapy, Adoptive/*methods ; Interleukin-2/genetics/metabolism ; MCF-7 Cells ; Mice ; Receptor, ErbB-2/*immunology/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/*genetics/*immunology/metabolism ; Receptors, Chimeric Antigen/*genetics/*immunology/metabolism ; Single-Chain Antibodies/immunology ; },
abstract = {In recent years, chimeric antigen receptor (CAR) T cell cancer immunotherapies have advanced substantially in the clinic. However, challenges related to safety persist; one major concern occurs when CARs trigger a response to antigen present on healthy cells (on-target, off-tumor response). A strategy to ameliorate this relies on the complex relationship between receptor affinity and signaling, such that one can engineer a CAR that is only activated by tumor cells expressing high antigen levels. Here, we developed a CAR T cell display platform with stable genomic expression and rapid functional screening based on interleukin-2 signaling. Starting with a CAR with high affinity toward its target antigen, we combined CRISPR-Cas9 genome editing and deep mutational scanning to generate a library of antigen-binding domain variants. This library was subjected to multiple rounds of selection based on either antigen binding or cell signaling. Deep sequencing of the resulting libraries and a comparative analysis revealed the enrichment and depletion of specific variants from which we selected CARs that were selectively activated by tumor cells based on antigen expression levels. Our platform demonstrates how directed evolution based on functional screening and deep sequencing-guided selection can be combined to enhance the selectivity and safety of CARs.},
}
@article {pmid32826895,
year = {2020},
author = {Mazzara, PG and Muggeo, S and Luoni, M and Massimino, L and Zaghi, M and Valverde, PT and Brusco, S and Marzi, MJ and Palma, C and Colasante, G and Iannielli, A and Paulis, M and Cordiglieri, C and Giannelli, SG and Podini, P and Gellera, C and Taroni, F and Nicassio, F and Rasponi, M and Broccoli, V},
title = {Frataxin gene editing rescues Friedreich's ataxia pathology in dorsal root ganglia organoid-derived sensory neurons.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4178},
pmid = {32826895},
issn = {2041-1723},
mesh = {Antioxidants/pharmacology ; CRISPR-Cas Systems ; Cell Differentiation ; Chromatin/metabolism ; Friedreich Ataxia/drug therapy/*genetics/*pathology ; Ganglia, Spinal/drug effects/*metabolism/pathology ; Gene Editing/*methods ; Genetic Predisposition to Disease/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Introns ; Iron-Binding Proteins/*genetics ; Mitochondria/metabolism ; Organoids/drug effects/*metabolism/pathology ; Sensory Receptor Cells/*metabolism/pathology ; Sequence Analysis, RNA ; Transcriptome ; },
abstract = {Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.},
}
@article {pmid32826317,
year = {2020},
author = {Brown, S and Gauvin, CC and Charbonneau, AA and Burman, N and Lawrence, CM},
title = {Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR-Cas that degrades the second messenger cA4.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {44},
pages = {14963-14972},
pmid = {32826317},
issn = {1083-351X},
support = {P20 GM103474/GM/NIGMS NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; S10 OD021527/OD/NIH HHS/United States ; },
mesh = {Adenine Nucleotides/*metabolism ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; Oligoribonucleotides/*metabolism ; Phosphoric Diester Hydrolases/chemistry/*metabolism ; Protein Binding ; Protein Folding ; *Second Messenger Systems ; Signal Transduction ; },
abstract = {Cas10 is the signature gene for type III CRISPR-Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn[2+]-dependent deadenylation exoribonuclease. However, its specific role in CRISPR-Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA4) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA4 signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR-Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.},
}
@article {pmid32826220,
year = {2020},
author = {Dhamad, AE and Lessner, DJ},
title = {A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {21},
pages = {},
pmid = {32826220},
issn = {1098-5336},
mesh = {Archaeal Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression ; Genes, Archaeal/*genetics ; Methanosarcina/*genetics/metabolism ; Nitrogen Fixation/*genetics ; },
abstract = {CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2 Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivoransIMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.},
}
@article {pmid32825374,
year = {2020},
author = {Sgodda, M and Alfken, S and Schambach, A and Eggenschwiler, R and Fidzinski, P and Hummel, M and Cantz, T},
title = {Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing.},
journal = {Cells},
volume = {9},
number = {9},
pages = {},
pmid = {32825374},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Receptors, Notch/*metabolism ; },
abstract = {Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%-45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.},
}
@article {pmid32824735,
year = {2020},
author = {Huang, J and Wang, A and Huang, C and Sun, Y and Song, B and Zhou, R and Li, L},
title = {Generation of Marker-Free pbd-2 Knock-in Pigs Using the CRISPR/Cas9 and Cre/loxP Systems.},
journal = {Genes},
volume = {11},
number = {8},
pages = {},
pmid = {32824735},
issn = {2073-4425},
mesh = {Animals ; *Animals, Genetically Modified ; *CRISPR-Cas Systems ; Cells, Cultured ; Disease Resistance/genetics ; Fibroblasts/metabolism ; Gene Editing ; Gene Expression ; *Gene Knock-In Techniques ; Gene Order ; *Gene Targeting ; *Homologous Recombination ; Integrases/*metabolism ; Phenotype ; Plasmids/genetics ; Swine ; beta-Defensins/*genetics ; },
abstract = {Porcine β-defensin 2 (PBD-2), expressed by different tissues of pigs, is a multifunctional cationic peptide with antimicrobial, immunomodulatory and growth-promoting abilities. As the latest generation of genome-editing tool, CRISPR/Cas9 system makes it possible to enhance the expression of PBD-2 in pigs by site-specific knock-in of pbd-2 gene into the pig genome. In this study, we aimed to generate marker-free pbd-2 knock-in pigs using the CRISPR/Cas9 and Cre/loxP systems. Two copies of pbd-2 gene linked by a T2A sequence were inserted into the porcine Rosa26 locus through CRISPR/Cas9-mediated homology-directed repair. The floxed selectable marker gene neoR, used for G418 screening of positive cell clones, was removed by cell-penetrating Cre recombinase with a recombination efficiency of 48.3%. Cloned piglets were produced via somatic cell nuclear transfer and correct insertion of pbd-2 genes was confirmed by PCR and Southern blot. Immunohistochemistry and immunofluorescence analyses indicated that expression levels of PBD-2 in different tissues of transgenic (TG) piglets were significantly higher than those of their wild-type (WT) littermates. Bactericidal assays demonstrated that there was a significant increase in the antimicrobial properties of the cell culture supernatants of porcine ear fibroblasts from the TG pigs in comparison to those from the WT pigs. Altogether, our study improved the protein expression level of PBD-2 in pigs by site-specific integration of pbd-2 into the pig genome, which not only provided an effective pig model to study the anti-infection mechanisms of PBD-2 but also a promising genetic material for the breeding of disease-resistant pigs.},
}
@article {pmid32824125,
year = {2020},
author = {Spreafico, R and Soriaga, LB and Grosse, J and Virgin, HW and Telenti, A},
title = {Advances in Genomics for Drug Development.},
journal = {Genes},
volume = {11},
number = {8},
pages = {},
pmid = {32824125},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; *Drug Development/methods ; Gene Editing ; Gene Expression Profiling ; Genome-Wide Association Study ; *Genomics/methods ; Genotype ; Humans ; Pharmacogenomic Testing/methods ; Single-Cell Analysis ; Transcriptome ; Whole Genome Sequencing ; },
abstract = {Drug development (target identification, advancing drug leads to candidates for preclinical and clinical studies) can be facilitated by genetic and genomic knowledge. Here, we review the contribution of population genomics to target identification, the value of bulk and single cell gene expression analysis for understanding the biological relevance of a drug target, and genome-wide CRISPR editing for the prioritization of drug targets. In genomics, we discuss the different scope of genome-wide association studies using genotyping arrays, versus exome and whole genome sequencing. In transcriptomics, we discuss the information from drug perturbation and the selection of biomarkers. For CRISPR screens, we discuss target discovery, mechanism of action and the concept of gene to drug mapping. Harnessing genetic support increases the probability of drug developability and approval.},
}
@article {pmid32823735,
year = {2020},
author = {Feng, M and Bai, Y and Chen, Y and Wang, K},
title = {Knockout of the Transducin-Like Enhancer of Split 6 Gene Affects the Proliferation and Cell Cycle Process of Mouse Spermatogonia.},
journal = {International journal of molecular sciences},
volume = {21},
number = {16},
pages = {},
pmid = {32823735},
issn = {1422-0067},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Cell Cycle ; Cell Proliferation ; Co-Repressor Proteins/*genetics/metabolism ; Green Fluorescent Proteins/metabolism ; Male ; Mice, Knockout ; Spermatogonia/*cytology/*metabolism ; },
abstract = {Tle6 (Transducin-like enhancer of split 6) is a member of the Tle co-repressor superfamily, which is expressed in various tissues of invertebrates and vertebrates and participates in the developmental process. However, the current research has only found that the TLE6 mutation is related to infertility, and the key regulatory mechanism of TLE6 remains to be explored. In this study, we combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 and the Tet-on system to construct mouse spermatogonia cell lines that induced TLE6 protein knockout (KO), and studied the effect of Tle6 on mouse spermatogonia proliferation and the cell cycle. The results showed that, after drug induction, the Tle6 gene in mouse spermatogonia was successfully knocked out at the genome and protein levels, and the Tle6 gene knockout efficiency was confirmed to be 87.5% with gene-cloning technology. At the same time, we also found that the mouse spermatogonia proliferated slowly after the Tle6 knockout. Using flow cytometry, we found that the cells did not undergo significant apoptosis, and the number of cells in the S phase decreased. After real-time quantity PCR (qRT-PCR) analysis, we found that the expression of cell-proliferation-related genes, CCAAT enhancer-binding protein α(C/ebp α), granulocyte-colony stimulating factor(G-csf), cyclin-dependent kinases 4(Cdk 4), Cyclin E, proliferating cell nuclear antigen(Pcna), and S-phase kinase-associated protein 2 (Skp2) was significantly reduced, which further affected cell growth. In summary, Tle6 can regulate spermatogonia cell proliferation and the cell cycle and provide a scientific basis for studying the role of TLE6 on spermatogenesis.},
}
@article {pmid32822689,
year = {2020},
author = {Ali, Z and Aman, R and Mahas, A and Rao, GS and Tehseen, M and Marsic, T and Salunke, R and Subudhi, AK and Hala, SM and Hamdan, SM and Pain, A and Alofi, FS and Alsomali, A and Hashem, AM and Khogeer, A and Almontashiri, NAM and Abedalthagafi, M and Hassan, N and Mahfouz, MM},
title = {iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2.},
journal = {Virus research},
volume = {288},
number = {},
pages = {198129},
pmid = {32822689},
issn = {1872-7492},
mesh = {Betacoronavirus/*genetics ; COVID-19 ; COVID-19 Testing ; *CRISPR-Cas Systems ; Clinical Laboratory Techniques/instrumentation/*methods ; Colorimetry/instrumentation/*methods ; Coronavirus Infections/*diagnosis/virology ; Endodeoxyribonucleases/chemistry ; Humans ; Molecular Diagnostic Techniques/instrumentation/*methods ; Nucleic Acid Amplification Techniques/instrumentation/*methods ; Pandemics ; Pneumonia, Viral/*diagnosis/virology ; Point-of-Care Systems ; Rheology ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.},
}
@article {pmid32822592,
year = {2020},
author = {Tran, NT and Graf, R and Wulf-Goldenberg, A and Stecklum, M and Strauß, G and Kühn, R and Kocks, C and Rajewsky, K and Chu, VT},
title = {CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2621-2634},
pmid = {32822592},
issn = {1525-0024},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics ; Congenital Bone Marrow Failure Syndromes/genetics/pathology/*therapy ; Exons ; Genetic Therapy/*methods ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics/metabolism ; HEK293 Cells ; Hematopoietic Stem Cell Transplantation/*methods ; Humans ; Interleukin-3/genetics/metabolism ; Leukocyte Elastase/*genetics ; Mice ; Mice, Transgenic ; *Mutation ; Neutropenia/*congenital/genetics/pathology/therapy ; Neutrophils/metabolism ; RNA, Guide/genetics ; Transfection ; Treatment Outcome ; },
abstract = {Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANE[L172P] mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.},
}
@article {pmid32822589,
year = {2020},
author = {Aulicino, F and Pedone, E and Sottile, F and Lluis, F and Marucci, L and Cosma, MP},
title = {Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via β-Catenin/TCF/LEF Functions.},
journal = {Stem cell reports},
volume = {15},
number = {3},
pages = {646-661},
pmid = {32822589},
issn = {2213-6711},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/N021444/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; Animals ; Biomarkers/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Cell Differentiation/genetics ; *Cell Self Renewal/genetics ; Cells, Cultured ; Ectoderm/metabolism ; Endoderm/metabolism ; Hepatocyte Nuclear Factor 1-alpha/*metabolism ; Lymphoid Enhancer-Binding Factor 1/*metabolism ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells/*cytology/metabolism ; Protein Isoforms/metabolism ; Transcriptome/genetics ; Up-Regulation/genetics ; *Wnt Signaling Pathway ; beta Catenin/*metabolism ; },
abstract = {The Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as β-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a β-catenin-dependent manner. Finally, β-catenin (ΔC) isoforms can rescue β-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.},
}
@article {pmid32822529,
year = {2020},
author = {Tasan, I and Su, CJ and Enghiad, B and Zhang, M and Mishra, S and Zhao, H},
title = {Two-Color Imaging of Nonrepetitive Endogenous Loci in Human Cells.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2502-2514},
doi = {10.1021/acssynbio.0c00295},
pmid = {32822529},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; Carrier Proteins/genetics ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Microscopy, Confocal ; Mutagenesis ; Plasmids/genetics/metabolism ; },
abstract = {Tools for live cell imaging of multiple nonrepetitive genomic loci in mammalian cells are necessary to study chromatin dynamics. Here, we report a new system based on two chromosomally integrated orthogonal irregular repeat arrays and particularly a new general strategy to construct irregular repeat arrays. Briefly, we utilized a "bridge oligonucleotide-mediated ligation" protocol to assemble 8-mer repeats de novo which were then combined into a final 96-mer repeat array using Golden Gate cloning. This strategy was used for assembling a new mutant TetO irregular repeat array, which worked orthogonally to the wild type TetO repeat. Single copy integration of the new repeat array did not cause replication deficiencies at the tagged locus. Moreover, the mutant TetO irregular repeat could also be visualized by CRISPR imaging. Our new irregular repeat assembly method demonstrates a generally applicable strategy that can be used for assembling additional orthogonal repeat arrays for imaging genomic loci and irregular repeats to visualize RNA or proteins via signal amplification.},
}
@article {pmid32822047,
year = {2021},
author = {Kondrashov, A and Karpova, E},
title = {Notes on Functional Modules in the Assembly of CRISPR/Cas9-Mediated Epigenetic Modifiers.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2198},
number = {},
pages = {401-428},
doi = {10.1007/978-1-0716-0876-0_30},
pmid = {32822047},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry ; DNA Methylation/genetics ; Epigenesis, Genetic/genetics ; Epigenomics/*methods ; Gene Editing/*methods ; Humans ; RNA, Guide/genetics ; },
abstract = {CRISPR/cas9 is a popular tool, widely used today for genome editing. However, the modular organization of this tool allows it to be used not only for DNA modifications but also for introducing epigenetic modifications both in DNA (methylation/demethylation) and in histones (acetylation/deacetylation). In these notes we will concentrate on the ways to adapt the CRISPR/cas9 system for epigenetic DNA modification of specific regions of interest. The modular organization represents a universal principal, that allows to create infinite number of functions with a limited number of tools. CRISPR/cas9, in which each subunit can be adapted for a particular task, is an excellent example of this rule. Made of two main subunits, it can be modified for targeted delivery of foreign activity (effector, an epigenetic enzyme in our case) to a selected part of the genome. In doing this the CRISPR/cas9 system represents a unique method that allows the introduction of both genomic and epigenetic modifications. This chapter gives a detailed review of how to prepare DNA for the fully functional CRISPR/cas9 system, able to introduce required modifications in the region of interest. We will discuss specific requirements for each structural component of the system as well as for auxiliary elements (modules), which are needed to ensure efficient expression of the elements of the system within the cell and the needs of selection and visualization.},
}
@article {pmid32821943,
year = {2020},
author = {Artamonova, D and Karneyeva, K and Medvedeva, S and Klimuk, E and Kolesnik, M and Yasinskaya, A and Samolygo, A and Severinov, K},
title = {Spacer acquisition by Type III CRISPR-Cas system during bacteriophage infection of Thermus thermophilus.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9787-9803},
pmid = {32821943},
issn = {1362-4962},
support = {R01 GM10407/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/pathogenicity/*physiology ; *CRISPR-Cas Systems ; Genes, Bacterial ; High-Throughput Nucleotide Sequencing ; Host-Pathogen Interactions/genetics ; RNA-Directed DNA Polymerase/genetics ; Thermus thermophilus/*genetics/*virology ; },
abstract = {Type III CRISPR-Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.},
}
@article {pmid32821942,
year = {2020},
author = {Veeneman, B and Gao, Y and Grant, J and Fruhling, D and Ahn, J and Bosbach, B and Bienkowska, J and Follettie, M and Arndt, K and Myers, J and Zhong, W},
title = {PINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9462-9477},
pmid = {32821942},
issn = {1362-4962},
mesh = {*Algorithms ; Amino Acid Sequence ; Amino Acids/genetics ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Conserved Sequence ; *Databases, Genetic ; Enzymes/genetics/metabolism ; Genome ; Genomic Library ; Humans ; Mice ; Proteins/*genetics/*metabolism ; RNA, Guide/genetics ; Reproducibility of Results ; Thymidine/genetics ; },
abstract = {CRISPR/Cas9 functional genomic screens have emerged as essential tools in drug target discovery. However, the sensitivity of available genome-wide CRISPR libraries is impaired by guides which inefficiently abrogate gene function. While Cas9 cleavage efficiency optimization and essential domain targeting have been developed as independent guide design rationales, no library has yet combined these into a single cohesive strategy to knock out gene function. Here, in a massive reanalysis of CRISPR tiling data using the most comprehensive feature database assembled, we determine which features of guides and their targets best predict activity and how to best combine them into a single guide design algorithm. We present the ProteIN ConsERvation (PINCER) genome-wide CRISPR library, which for the first time combines enzymatic efficiency optimization with conserved length protein region targeting, and also incorporates domains, coding sequence position, U6 termination (TTT), restriction sites, polymorphisms and specificity. Finally, we demonstrate superior performance of the PINCER library compared to alternative genome-wide CRISPR libraries in head-to-head validation. PINCER is available for individual gene knockout and genome-wide screening for both the human and mouse genomes.},
}
@article {pmid32821003,
year = {2020},
author = {Assimos, DG},
title = {Re: CRISPR/Cas9-Mediated Metabolic Pathway Reprogramming in a Novel Humanized Rat Model Ameliorates Primary Hyperoxaluria Type 1.},
journal = {The Journal of urology},
volume = {204},
number = {5},
pages = {1092},
doi = {10.1097/JU.0000000000001258.04},
pmid = {32821003},
issn = {1527-3792},
mesh = {Animals ; *CRISPR-Cas Systems ; *Hyperoxaluria, Primary/genetics ; Metabolic Networks and Pathways ; Rats ; },
}
@article {pmid32820405,
year = {2021},
author = {Fok, ET and Fanucchi, S and Bystricky, K and Mhlanga, MM},
title = {Visualization of Chromatin Dynamics by Live Cell Microscopy Using CRISPR/Cas9 Gene Editing and ANCHOR Labeling.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2157},
number = {},
pages = {197-212},
doi = {10.1007/978-1-0716-0664-3_11},
pmid = {32820405},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Chromatin/*metabolism ; Gene Editing/methods ; Human Umbilical Vein Endothelial Cells ; Humans ; Microscopy/*methods ; Polymerase Chain Reaction ; },
abstract = {The organization of the eukaryotic nucleus facilitates functional chromatin contacts which regulate gene transcription. Despite this being extensively studied through population-based chromatin contact mapping and microscopic observations in single cells, the spatiotemporal dynamics of chromatin behavior have largely remained elusive. The current methods to label and observe specific endogenous genomic loci in living cells have been challenging to implement and too invasive to biological processes. In this protocol, we describe the use of a recently developed DNA labelling strategy (ANCHOR) with CRISPR/Cas9 gene editing, to discreetly label genes for live cell imaging to study chromatin dynamics. Our approach improves on some of the fundamental shortfalls associated with current labelling strategies and has the potential for multiplexed observations.},
}
@article {pmid32820039,
year = {2020},
author = {Yang, B and Schwartz, M and McJunkin, K},
title = {In vivo CRISPR screening for phenotypic targets of the mir-35-42 family in C. elegans.},
journal = {Genes &amp; development},
volume = {34},
number = {17-18},
pages = {1227-1238},
pmid = {32820039},
issn = {1549-5477},
support = {P40 OD010440/OD/NIH HHS/United States ; ZIA DK075147/ImNIH/Intramural NIH HHS/United States ; },
mesh = {3' Untranslated Regions/genetics ; Alleles ; Animals ; Binding Sites/genetics ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Gene Editing ; Genetic Testing ; MicroRNAs/genetics/*metabolism ; Mutation ; Phenotype ; },
abstract = {Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. We devised a novel strategy to test the phenotypic impact of individual microRNA-target interactions by disrupting each predicted miRNA-binding site by CRISPR-Cas9 genome editing in C. elegans We developed a multiplexed negative selection screening approach in which edited loci are deep sequenced, and candidate sites are prioritized based on apparent selection pressure against mutations that disrupt miRNA binding. Importantly, our screen was conducted in vivo on mutant animals, allowing us to interrogate organism-level phenotypes. We used this approach to screen for phenotypic targets of the essential mir-35-42 family. By generating 1130 novel 3'UTR alleles across all predicted targets, we identified egl-1 as a phenotypic target whose derepression partially phenocopies the mir-35-42 mutant phenotype by inducing embryonic lethality and low fecundity. These phenotypes can be rescued by compensatory CRISPR mutations that retarget mir-35 to the mutant egl-1 3'UTR. This study demonstrates that the application of in vivo whole organismal CRISPR screening has great potential to accelerate the discovery of phenotypic negative regulatory elements in the noncoding genome.},
}
@article {pmid32819722,
year = {2020},
author = {Riepsaame, J},
title = {Editing the Mitochondrial Genome: No CRISPR Required.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {11},
pages = {809-810},
doi = {10.1016/j.tig.2020.08.001},
pmid = {32819722},
issn = {0168-9525},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytidine Deaminase ; Gene Editing ; *Genome, Mitochondrial/genetics ; },
abstract = {Precise gene editing of mitochondrial DNA (mtDNA) is essential for the generation of model systems to study rare mitochondrial diseases but was long deemed impossible - until now. A recent publication by Mok et al. describes a gene editing tool capable of installing point mutations in mtDNA, and it does not involve CRISPR.},
}
@article {pmid32818746,
year = {2020},
author = {Jin, J and Jia, B and Yuan, YJ},
title = {Yeast chromosomal engineering to improve industrially-relevant phenotypes.},
journal = {Current opinion in biotechnology},
volume = {66},
number = {},
pages = {165-170},
doi = {10.1016/j.copbio.2020.07.003},
pmid = {32818746},
issn = {1879-0429},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genome, Fungal/genetics ; Phenotype ; *Saccharomyces cerevisiae/genetics ; Synthetic Biology ; },
abstract = {Genome structural variations enable microbes to evolve quickly under environmental stress. Recent efforts in synthetic biology have shown the ability of yeast chromosomal engineering to generate a larger scale of genome structural variations, which require a high efficiency of DNA rearrangement technology. In this review, we summarize the recent development of the SCRaMbLE system, an evolutionary approach, and the CRISPR/Cas9 technology to generate yeast genome rearrangement. Both technologies exhibit the powerful applications of chromosomal engineering to accelerate phenotypic evolution. We highlight several studies where yeast genome rearrangement has successfully improved industrially-relevant phenotypes, including the production of novel medicine, nutrition supplements, anti-tumor molecules, and the tolerance of environmental stress and drug resistance.},
}
@article {pmid32817435,
year = {2020},
author = {Feng, G and Jensen, FE and Greely, HT and Okano, H and Treue, S and Roberts, AC and Fox, JG and Caddick, S and Poo, MM and Newsome, WT and Morrison, JH},
title = {Opportunities and limitations of genetically modified nonhuman primate models for neuroscience research.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {39},
pages = {24022-24031},
pmid = {32817435},
issn = {1091-6490},
support = {P50 MH094271/MH/NIMH NIH HHS/United States ; P51 OD011107/OD/NIH HHS/United States ; U24 OD026638/OD/NIH HHS/United States ; R21 NS105437/NS/NINDS NIH HHS/United States ; U01 MH114819/MH/NIMH NIH HHS/United States ; R21 AG064448/AG/NIA NIH HHS/United States ; },
mesh = {Animal Experimentation/*ethics ; Animals ; *Disease Models, Animal ; Mental Disorders/*genetics/physiopathology ; Nervous System Diseases/*genetics/physiopathology ; Neurosciences/ethics/methods ; Primates/*genetics/physiology ; },
abstract = {The recently developed new genome-editing technologies, such as the CRISPR/Cas system, have opened the door for generating genetically modified nonhuman primate (NHP) models for basic neuroscience and brain disorders research. The complex circuit formation and experience-dependent refinement of the human brain are very difficult to model in vitro, and thus require use of in vivo whole-animal models. For many neurodevelopmental and psychiatric disorders, abnormal circuit formation and refinement might be at the center of their pathophysiology. Importantly, many of the critical circuits and regional cell populations implicated in higher human cognitive function and in many psychiatric disorders are not present in lower mammalian brains, while these analogous areas are replicated in NHP brains. Indeed, neuropsychiatric disorders represent a tremendous health and economic burden globally. The emerging field of genetically modified NHP models has the potential to transform our study of higher brain function and dramatically facilitate the development of effective treatment for human brain disorders. In this paper, we discuss the importance of developing such models, the infrastructure and training needed to maximize the impact of such models, and ethical standards required for using these models.},
}
@article {pmid32817336,
year = {2020},
author = {Liu, TY and Doudna, JA},
title = {Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {42},
pages = {14473-14487},
pmid = {32817336},
issn = {1083-351X},
support = {P01 GM051487/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine Nucleotides/metabolism ; Animals ; CRISPR-Cas Systems/*genetics ; DNA/chemistry/metabolism ; *Gene Editing ; Gene Expression ; Oligoribonucleotides/metabolism ; RNA/antagonists &amp; inhibitors/metabolism ; RNA, Guide/metabolism ; Signal Transduction ; },
abstract = {Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection.},
}
@article {pmid32817219,
year = {2020},
author = {Madigan, VJ and Berry, GE and Tyson, TO and Nardone-White, D and Ark, J and Elmore, ZC and Murlidharan, G and Vincent, HA and Asokan, A},
title = {The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction.},
journal = {Journal of virology},
volume = {94},
number = {21},
pages = {},
pmid = {32817219},
issn = {1098-5514},
support = {R01 GM127708/GM/NIGMS NIH HHS/United States ; R01 HL089221/HL/NHLBI NIH HHS/United States ; R01 NS099371/NS/NINDS NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; Biological Transport/drug effects ; Brain/drug effects/metabolism ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium-Transporting ATPases/deficiency/*genetics ; Cell Line, Tumor ; Chelating Agents/pharmacology ; Dependovirus/drug effects/*genetics/metabolism ; Egtazic Acid/analogs &amp; derivatives/pharmacology ; Gene Deletion ; Genetic Vectors/chemistry/*metabolism ; Golgi Apparatus/drug effects/*metabolism/virology ; HEK293 Cells ; Hepatocytes/drug effects/metabolism/virology ; Humans ; Injections, Intraventricular ; Ionomycin/pharmacology ; Lentivirus/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Stereotaxic Techniques ; Transduction, Genetic ; Vesiculovirus/genetics/metabolism ; },
abstract = {Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription.IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.},
}
@article {pmid32816924,
year = {2020},
author = {Stevenson, ZC and Moerdyk-Schauwecker, MJ and Jamison, B and Phillips, PC},
title = {Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {10},
pages = {3775-3782},
pmid = {32816924},
issn = {2160-1836},
support = {R01 AG056436/AG/NIA NIH HHS/United States ; R35 GM131838/GM/NIGMS NIH HHS/United States ; T32 GM007413/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; *Caenorhabditis elegans/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Transgenes ; },
abstract = {Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert single-copy transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require plate-level screening processes to avoid selecting heritable extrachromosomal arrays or rely on co-CRISPR markers to identify knock-in events. As a result, verification of transgene insertion requires anti-array selection screening methods and PCR genotyping. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native microhomology-based recombination, to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.},
}
@article {pmid32816470,
year = {2020},
author = {Kirkpatrick, RL and Lewis, K and Langan, RA and Lajoie, MJ and Boyken, SE and Eakman, M and Baker, D and Zalatan, JG},
title = {Conditional Recruitment to a DNA-Bound CRISPR-Cas Complex Using a Colocalization-Dependent Protein Switch.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2316-2323},
pmid = {32816470},
issn = {2161-5063},
support = {R35 GM124773/GM/NIGMS NIH HHS/United States ; T32 HG000035/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; DNA/chemistry/*metabolism ; Gene Editing/*methods ; Genes, Reporter ; RNA, Guide/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR-Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.},
}
@article {pmid32816370,
year = {2020},
author = {Bryant, JM and Baumgarten, S and Dingli, F and Loew, D and Sinha, A and Claës, A and Preiser, PR and Dedon, PC and Scherf, A},
title = {Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite.},
journal = {Molecular systems biology},
volume = {16},
number = {8},
pages = {e9569},
pmid = {32816370},
issn = {1744-4292},
support = {P30 ES002109/ES/NIEHS NIH HHS/United States ; },
mesh = {Adenosine Triphosphatases/*metabolism ; Animals ; Antigens, Protozoan/genetics/metabolism ; CRISPR-Cas Systems ; Chromatin Immunoprecipitation Sequencing ; Humans ; Introns ; Mass Spectrometry ; Plasmodium falciparum/genetics/immunology/*pathogenicity ; Promoter Regions, Genetic ; Protein Interaction Maps ; Proteomics/*methods ; Transcription Factors/*metabolism ; Virulence Factors/*genetics/metabolism ; },
abstract = {Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.},
}
@article {pmid32816363,
year = {2020},
author = {Si, Z and Guan, X and Teng, X and Peng, X and Wan, Z and Li, Q and Chen, G and Tan, J and Li, J},
title = {Identification of CYP46A1 as a new regulator of lipid metabolism through CRISPR-based whole-genome screening.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {10},
pages = {13776-13791},
doi = {10.1096/fj.202001067R},
pmid = {32816363},
issn = {1530-6860},
mesh = {Adult ; CRISPR-Cas Systems ; Cholesterol 24-Hydroxylase/genetics/*metabolism ; Female ; Gene Knockout Techniques/methods ; Genomics/methods ; Humans ; *Lipid Metabolism ; Liver/metabolism ; Male ; Middle Aged ; Non-alcoholic Fatty Liver Disease/*genetics/metabolism ; Transcriptome ; },
abstract = {Abnormal lipid droplet (LD) metabolism causes a variety of disorders, especially to nonalcoholic fatty liver disease (NAFLD). But the mechanism of abnormal aggregation of LD is still not fully elucidated. Here, Genome-wide CRISPR-Cas9 knockout (GeCKO) screening was employed to identify candidate genes regulating LD metabolism in L02 cell. We analyzed simultaneously the transcriptomics of liver tissues of NAFLD to find potential genes involved in pathogenesis of NAFLD. After integration these data, we found that the expression of 43 candidate genes from the GeCKO screening was also decreased in tissues of NAFLD patients. Many of these 43 overlapping genes have been reported to play an important role in the formation of LD. Subsequently, we focused on CYP46A1, one of 43 candidate genes and mitochondria-related genes. We confirmed that the protein expression of CYP46A1 is deceased in tissues of NAFLD patients. Downregulation or overexpression of CYP46A1 affected LD accumulation in vitro. Deficiency of CYP46A1 impaired mitochondrial morphology and function, which may be responsible for the accumulation of LD. In summary, this study explored regulatory factors of LD accumulation at the whole-genome level, and demonstrated that CYP46A1 regulated LD formation involving in NAFLD pathogenesis. It provides new clues for studying the molecular mechanisms of diseases related to abnormal lipid metabolism.},
}
@article {pmid32815753,
year = {2020},
author = {Rivera-Torres, N and Banas, K and Kmiec, EB},
title = {Modeling pediatric AML FLT3 mutations using CRISPR/Cas12a- mediated gene editing.},
journal = {Leukemia &amp; lymphoma},
volume = {61},
number = {13},
pages = {3078-3088},
pmid = {32815753},
issn = {1029-2403},
support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; S10 OD016361/OD/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Child ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics/therapy ; Mutation ; fms-Like Tyrosine Kinase 3/genetics ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR) with the associated (Cas) nuclease complexes have democratized genetic engineering through their precision and ease-of-use. We have applied a variation of this technology, known as CRISPR-directed mutagenesis (CDM), to reconstruct genetic profiles within the FLT3 gene of AML patients. We took advantage of the versatility of CDM and built expression vectors that, in combination with a specifically designed donor DNA fragment, recapitulate simple and complex mutations within the FLT3 gene. We generate insertions and point mutations including combinations of these mutations originating from individual patient samples. We then analyze how these complex genetic profiles modulate transformation of Ba/F3 cells. Our results show that FLT3 expression plasmids bearing patient-specific single or multiple mutations recapitulate cellular transformation properties induced by FLT3 ITDs and modify their sensitivity or resistance in response to established AML drugs as a function of these complex mutations.},
}
@article {pmid32814451,
year = {2020},
author = {Vu, A and McCray, PB},
title = {New Directions in Pulmonary Gene Therapy.},
journal = {Human gene therapy},
volume = {31},
number = {17-18},
pages = {921-939},
pmid = {32814451},
issn = {1557-7422},
support = {P01 HL152960/HL/NHLBI NIH HHS/United States ; P01 HL091842/HL/NHLBI NIH HHS/United States ; P30 DK054759/DK/NIDDK NIH HHS/United States ; UH3 HL147366/HL/NHLBI NIH HHS/United States ; UG3 HL147366/HL/NHLBI NIH HHS/United States ; T32 AI007343/AI/NIAID NIH HHS/United States ; },
mesh = {Cystic Fibrosis/genetics/*therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/deficiency/*genetics ; Dependovirus/*genetics ; Gene Editing ; Genetic Therapy/*methods ; Genetic Vectors/*administration &amp; dosage/genetics ; Humans ; Pulmonary Surfactant-Associated Protein A/deficiency/*genetics ; alpha 1-Antitrypsin/*genetics ; },
abstract = {The lung has long been a target for gene therapy, yet efficient delivery and phenotypic disease correction has remained challenging. Although there have been significant advancements in gene therapies of other organs, including the development of several ex vivo therapies, in vivo therapeutics of the lung have been slower to transition to the clinic. Within the past few years, the field has witnessed an explosion in the development of new gene addition and gene editing strategies for the treatment of monogenic disorders. In this review, we will summarize current developments in gene therapy for cystic fibrosis, alpha-1 antitrypsin deficiency, and surfactant protein deficiencies. We will explore the different gene addition and gene editing strategies under investigation and review the challenges of delivery to the lung.},
}
@article {pmid32814147,
year = {2020},
author = {Wang, S and Wang, P},
title = {Functional redundancy of structural proteins of the peritrophic membrane in Trichoplusia ni.},
journal = {Insect biochemistry and molecular biology},
volume = {125},
number = {},
pages = {103456},
doi = {10.1016/j.ibmb.2020.103456},
pmid = {32814147},
issn = {1879-0240},
mesh = {Animals ; Bacillus thuringiensis Toxins/toxicity ; CRISPR-Cas Systems ; Chitin/metabolism ; Digestive System/*metabolism ; Gene Silencing ; Genes, Insect ; Insect Proteins/metabolism ; Insecticides/toxicity ; Larva/metabolism ; Moths/*metabolism ; *Mucins/drug effects/genetics/metabolism ; },
abstract = {The peritrophic membrane (or peritrophic matrix) (PM) in insects is formed by binding of PM proteins with multiple chitin binding domains (CBDs) to chitin fibrils. Multi-CBD chitin binding proteins (CBPs) and the insect intestinal mucin (IIM) are major PM structural proteins. To understand the biochemical and physiological role of IIM in structural formation and physiological function of the PM, Trichoplusia ni mutant strains lacking IIM were generated by CRISPR/Cas9 mutagenesis. The mutant T. ni larvae were confirmed to lack IIM, but PM formation was observed as in wild type larvae and lacking IIM in the PM did not result in changes of protease activities in the larval midgut. Larval growth and development of the mutant strains were similar to the wild type strain on artificial diet and cabbage leaves, but had a decreased survival in the 5th instar. The larvae of the mutant strains with the PM formed without IIM did not have a change of susceptibility to the infection of the baculovirus AcMNPV and the Bacillus thuringiensis (Bt) formulation Dipel, to the toxicity of the Bt toxins Cry1Ac and Cry2Ab and the chemical insecticide sodium aluminofluoride. Treatment of the mutant T. ni larvae with Calcofluor reduced the larval susceptibility to the toxicity of Bt Cry1Ac, as similarly observed in the wild type larvae. Overall, in the mutant T. ni larvae, the PM was formed without IIM and the lacking of IIM in the PM did not drastically impact the performance of larvae on diet or cabbage leaves under the laboratory conditions.},
}
@article {pmid32813255,
year = {2021},
author = {Carmona-Aldana, F and Nuñez-Martinez, HN and Peralta-Alvarez, CA and Tapia-Urzua, G and Recillas-Targa, F},
title = {Generation of Functional Genetic Study Models in Zebrafish Using CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2174},
number = {},
pages = {255-262},
doi = {10.1007/978-1-0716-0759-6_16},
pmid = {32813255},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; DNA/isolation &amp; purification ; Embryo, Nonmammalian ; Gene Editing/*methods ; Genotyping Techniques/methods ; RNA, Guide ; Zebrafish/embryology/*genetics ; },
abstract = {CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.},
}
@article {pmid32811847,
year = {2020},
author = {Yokouchi, Y and Suzuki, S and Ohtsuki, N and Yamamoto, K and Noguchi, S and Soejima, Y and Goto, M and Ishioka, K and Nakamura, I and Suzuki, S and Takenoshita, S and Era, T},
title = {Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13927},
pmid = {32811847},
issn = {2045-2322},
mesh = {Alleles ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Collagen Type VII/genetics/metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Human/genetics ; Humans ; Induced Pluripotent Stem Cells/physiology ; Mutation/genetics ; Nucleotides/genetics ; Pluripotent Stem Cells/physiology ; Polymorphism, Single Nucleotide/*genetics ; Proto-Oncogene Proteins c-ret/genetics/metabolism ; },
abstract = {Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.},
}
@article {pmid32811834,
year = {2020},
author = {Aubert, M and Strongin, DE and Roychoudhury, P and Loprieno, MA and Haick, AK and Klouser, LM and Stensland, L and Huang, ML and Makhsous, N and Tait, A and De Silva Feelixge, HS and Galetto, R and Duchateau, P and Greninger, AL and Stone, D and Jerome, KR},
title = {Gene editing and elimination of latent herpes simplex virus in vivo.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4148},
pmid = {32811834},
issn = {2041-1723},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; R01 AI132599/AI/NIAID NIH HHS/United States ; R21 AI117519/AI/NIAID NIH HHS/United States ; UM1 AI126623/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Chlorocebus aethiops ; Deoxyribonucleases/*genetics ; Dependovirus/*genetics ; Eye Infections/genetics/*therapy/virology ; Female ; Gene Editing/*methods ; HEK293 Cells ; Herpes Simplex/genetics/*therapy ; Herpesvirus 1, Human/*genetics/pathogenicity ; Humans ; Mice ; Neurons/metabolism/virology ; RNA-Seq ; Single-Cell Analysis ; Superior Cervical Ganglion/metabolism/virology ; Vero Cells ; Virus Latency/*genetics ; },
abstract = {We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.},
}
@article {pmid32811807,
year = {2020},
author = {Ma, Y and Zhu, Q and Liang, J and Li, Y and Li, M and Zhang, Y and Wang, X and Zeng, Y and Jiao, Y},
title = {A CRISPR knockout negative screen reveals synergy between CDKs inhibitor and metformin in the treatment of human cancer in vitro and in vivo.},
journal = {Signal transduction and targeted therapy},
volume = {5},
number = {1},
pages = {152},
pmid = {32811807},
issn = {2059-3635},
mesh = {Animals ; Aspartic Acid/metabolism ; CDC2 Protein Kinase/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/genetics ; Citric Acid Cycle ; Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors/*genetics ; Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors/*genetics ; Drug Synergism ; Fatty Acids/metabolism ; Gene Knockdown Techniques ; Genome, Human/drug effects ; High-Throughput Nucleotide Sequencing ; Humans ; MCF-7 Cells ; Metformin/*pharmacology ; Mice ; Neoplasms/*drug therapy/genetics/pathology ; Protein Kinase Inhibitors/pharmacology ; TOR Serine-Threonine Kinases/genetics ; },
abstract = {Laboratory research and pharmacoepidemiology provide support for metformin as a potential antitumor agent. However, the lack of a clear understanding of the indications of metformin limits its efficacy. Here, we performed a genome-wide CRISPR knockout negative screen to identify potential targets that might synergize with metformin. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells after 18 days of metformin treatment (T18) compared to those of the untreated cells at day 0 (T0) yielded candidate genes. Knockdown of a group of cyclin-dependent kinases (CDKs), including CDK1, CDK4, and CDK6, confirmed the results of the screen. Combination treatment of the CDKs inhibitor abemaciclib with metformin profoundly inhibited tumor viability in vitro and in vivo. Although cell cycle parameters were not further altered under the combination treatment, investigation of the metabolome revealed significant changes in cell metabolism, especially with regard to fatty acid oxidation, the tricarboxylic acid cycle and aspartate metabolism. Such changes appeared to be mediated through inhibition of the mTOR pathway. Collectively, our study suggests that the combination of CDKs inhibitor with metformin could be recognized as a potential therapy in future clinical applications.},
}
@article {pmid32811551,
year = {2020},
author = {Noorani, I and Bradley, A and de la Rosa, J},
title = {CRISPR and transposon in vivo screens for cancer drivers and therapeutic targets.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {204},
pmid = {32811551},
issn = {1474-760X},
support = {/WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Transformation, Neoplastic/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements ; *Genes, Neoplasm ; Genetic Testing ; Humans ; Immunotherapy ; Mutagenesis ; Neoplasm Metastasis ; Neoplasms/*genetics ; },
abstract = {Human cancers harbor substantial genetic, epigenetic, and transcriptional changes, only some of which drive oncogenesis at certain times during cancer evolution. Identifying the cancer-driver alterations amongst the vast swathes of "passenger" changes still remains a major challenge. Transposon and CRISPR screens in vivo provide complementary methods for achieving this, and each platform has its own advantages. Here, we review recent major technological breakthroughs made with these two approaches and highlight future directions. We discuss how each genetic screening platform can provide unique insight into cancer evolution, including intra-tumoral heterogeneity, metastasis, and immune evasion, presenting transformative opportunities for targeted therapeutic intervention.},
}
@article {pmid32811500,
year = {2020},
author = {Tanihara, F and Hirata, M and Nguyen, NT and Sawamoto, O and Kikuchi, T and Doi, M and Otoi, T},
title = {Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {40},
pmid = {32811500},
issn = {1472-6750},
mesh = {Animals ; Animals, Genetically Modified ; Blastocyst ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Disaccharides ; Electroporation/*methods ; Female ; Fertilization in Vitro/*methods ; Galactosyltransferases/*deficiency/*genetics ; Gene Editing/*methods ; Heterografts ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Mutation ; RNA, Guide ; Swine ; Transplantation, Heterologous ; Zygote/*metabolism ; },
abstract = {BACKGROUND: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs.<br><br>RESULTS: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets.<br><br>CONCLUSIONS: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.},
}
@article {pmid32811438,
year = {2020},
author = {Shen, L and Zhang, Q and Wang, Z and Wen, H and Hu, G and Ren, D and Hu, J and Zhu, L and Gao, Z and Zhang, G and Guo, L and Zeng, D and Qian, Q},
title = {OsCAF2 contains two CRM domains and is necessary for chloroplast development in rice.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {381},
pmid = {32811438},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems ; Chloroplasts/*physiology ; Gene Expression Profiling ; Introns ; Mutation ; Organelle Biogenesis ; Oryza/genetics/*physiology ; Phenotype ; Plant Proteins/chemistry/genetics/*physiology ; Protein Domains ; RNA Splicing Factors/chemistry/genetics/*physiology ; },
abstract = {BACKGROUND: Chloroplasts play an important role in plant growth and development. The chloroplast genome contains approximately twenty group II introns that are spliced due to proteins encoded by nuclear genes. CAF2 is one of these splicing factors that has been shown to splice group IIB introns in maize and Arabidopsis thaliana. However, the research of the OsCAF2 gene in rice is very little, and the effects of OsCAF2 genes on chloroplasts development are not well characterized.<br><br>RESULTS: In this study, oscaf2 mutants were obtained by editing the OsCAF2 gene in the Nipponbare variety of rice. Phenotypic analysis showed that mutations to OsCAF2 led to albino leaves at the seeding stage that eventually caused plant death, and oscaf2 mutant plants had fewer chloroplasts and damaged chloroplast structure. We speculated that OsCAF2 might participate in the splicing of group IIA and IIB introns, which differs from its orthologs in A. thaliana and maize. Through yeast two-hybrid experiments, we found that the C-terminal region of OsCAF2 interacted with OsCRS2 and formed an OsCAF2-OsCRS2 complex. In addition, the N-terminal region of OsCAF2 interacted with itself to form homodimers.<br><br>CONCLUSION: Taken together, this study improved our understanding of the OsCAF2 protein, and revealed additional information about the molecular mechanism of OsCAF2 in regulating of chloroplast development in rice.},
}
@article {pmid32810562,
year = {2020},
author = {Yokoyama, M and Matsuzawa, T and Yoshikawa, T and Nunomiya, A and Yamaguchi, Y and Yanai, K},
title = {Heparan sulfate controls skeletal muscle differentiation and motor functions.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1864},
number = {12},
pages = {129707},
doi = {10.1016/j.bbagen.2020.129707},
pmid = {32810562},
issn = {1872-8006},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Heparitin Sulfate/antagonists &amp; inhibitors/genetics/*metabolism ; Mice ; Motor Activity ; *Muscle Development ; Muscle, Skeletal/cytology/*physiology ; Myoblasts/*cytology/metabolism ; },
abstract = {BACKGROUND: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear.<br><br>METHODS: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system.<br><br>RESULTS: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells.<br><br>SIGNIFICANCE: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.},
}
@article {pmid32810272,
year = {2020},
author = {Zhuang, J and Tan, J and Wu, C and Zhang, J and Liu, T and Fan, C and Li, J and Zhang, Y},
title = {Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy.},
journal = {Nucleic acids research},
volume = {48},
number = {16},
pages = {8870-8882},
pmid = {32810272},
issn = {1362-4962},
mesh = {Animals ; Aptamers, Nucleotide/*therapeutic use ; *CRISPR-Cas Systems ; *Extracellular Vesicles ; Female ; Genetic Therapy/*methods ; HEK293 Cells ; HeLa Cells ; Hep G2 Cells ; Humans ; Liver/pathology ; Liver Neoplasms/*therapy ; MCF-7 Cells ; Mice, Inbred BALB C ; Mice, Nude ; Nanostructures/*therapeutic use ; Xenograft Model Antitumor Assays ; },
abstract = {Extracellular vesicles (EVs) hold great promise for transporting CRISPR-Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.},
}
@article {pmid32810265,
year = {2020},
author = {Quarton, T and Kang, T and Papakis, V and Nguyen, K and Nowak, C and Li, Y and Bleris, L},
title = {Uncoupling gene expression noise along the central dogma using genome engineered human cell lines.},
journal = {Nucleic acids research},
volume = {48},
number = {16},
pages = {9406-9413},
pmid = {32810265},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; *Gene Editing ; Gene Expression Regulation/genetics ; Genome, Human/*genetics ; Humans ; Models, Genetic ; Protein Biosynthesis/genetics ; Stochastic Processes ; *Transcription, Genetic ; },
abstract = {Eukaryotic protein synthesis is an inherently stochastic process. This stochasticity stems not only from variations in cell content between cells but also from thermodynamic fluctuations in a single cell. Ultimately, these inherently stochastic processes manifest as noise in gene expression, where even genetically identical cells in the same environment exhibit variation in their protein abundances. In order to elucidate the underlying sources that contribute to gene expression noise, we quantify the contribution of each step within the process of protein synthesis along the central dogma. We uncouple gene expression at the transcriptional, translational, and post-translational level using custom engineered circuits stably integrated in human cells using CRISPR. We provide a generalized framework to approximate intrinsic and extrinsic noise in a population of cells expressing an unbalanced two-reporter system. Our decomposition shows that the majority of intrinsic fluctuations stem from transcription and that coupling the two genes along the central dogma forces the fluctuations to propagate and accumulate along the same path, resulting in increased observed global correlation between the products.},
}
@article {pmid32810226,
year = {2020},
author = {Kim, I and Koo, J and An, SY and Hong, S and Ka, D and Kim, EH and Bae, E and Suh, JY},
title = {Structural and mechanistic insights into the CRISPR inhibition of AcrIF7.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9959-9968},
pmid = {32810226},
issn = {1362-4962},
mesh = {Bacteriophages/*chemistry/genetics/pathogenicity ; Binding Sites ; CRISPR-Associated Proteins/chemistry/metabolism ; *CRISPR-Cas Systems ; Molecular Docking Simulation ; Protein Binding ; Pseudomonas aeruginosa/genetics/virology ; Viral Proteins/*chemistry/metabolism ; },
abstract = {The CRISPR-Cas system provides adaptive immunity for bacteria and archaea to combat invading phages and plasmids. Phages evolved anti-CRISPR (Acr) proteins to neutralize the host CRISPR-Cas immune system as a counter-defense mechanism. AcrIF7 in Pseudomonas aeruginosa prophages strongly inhibits the type I-F CRISPR-Cas system. Here, we determined the solution structure of AcrIF7 and identified its target, Cas8f of the Csy complex. AcrIF7 adopts a novel β1β2α1α2β3 fold and interacts with the target DNA binding site of Cas8f. Notably, AcrIF7 competes with AcrIF2 for the same binding interface on Cas8f without common structural motifs. AcrIF7 binding to Cas8f is driven mainly by electrostatic interactions that require position-specific surface charges. Our findings suggest that Acrs of divergent origin may have acquired specificity to a common target through convergent evolution of their surface charge configurations.},
}
@article {pmid32810208,
year = {2020},
author = {Carullo, NVN and Phillips Iii, RA and Simon, RC and Soto, SAR and Hinds, JE and Salisbury, AJ and Revanna, JS and Bunner, KD and Ianov, L and Sultan, FA and Savell, KE and Gersbach, CA and Day, JJ},
title = {Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9550-9570},
pmid = {32810208},
issn = {1362-4962},
support = {DP1 DA039650/DA/NIDA NIH HHS/United States ; R21 DA048348/DA/NIDA NIH HHS/United States ; R00 DA034681/DA/NIDA NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; F31 DA042514/DA/NIDA NIH HHS/United States ; R01 MH114990/MH/NIMH NIH HHS/United States ; R21 DA041878/DA/NIDA NIH HHS/United States ; K99 DA034681/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CREB-Binding Protein/genetics/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Chromatin/metabolism ; *Enhancer Elements, Genetic ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Neurons/cytology/*physiology ; Proto-Oncogene Proteins c-fos/genetics ; RNA/*physiology ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering ; Rats ; Reproducibility of Results ; Sequence Analysis, RNA ; Single Molecule Imaging ; },
abstract = {Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.},
}
@article {pmid32809800,
year = {2020},
author = {Chen, M and Luo, R and Li, S and Li, H and Qin, Y and Zhou, D and Liu, H and Gong, X and Chang, J},
title = {Paper-Based Strip for Ultrasensitive Detection of OSCC-Associated Salivary MicroRNA via CRISPR/Cas12a Coupling with IS-Primer Amplification Reaction.},
journal = {Analytical chemistry},
volume = {92},
number = {19},
pages = {13336-13342},
doi = {10.1021/acs.analchem.0c02642},
pmid = {32809800},
issn = {1520-6882},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics ; Head and Neck Neoplasms/*diagnosis ; Humans ; MicroRNAs/*analysis/genetics ; *Nucleic Acid Amplification Techniques ; *Paper ; Reagent Strips/*chemistry ; Saliva/*chemistry ; Squamous Cell Carcinoma of Head and Neck/*diagnosis ; },
abstract = {As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people's health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of "invading stacking primer" (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5' and 3' termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.},
}
@article {pmid32809205,
year = {2020},
author = {Naseri, G and Mueller-Roeber, B},
title = {A Step-by-Step Protocol for COMPASS, a Synthetic Biology Tool for Combinatorial Gene Assembly.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2205},
number = {},
pages = {277-303},
doi = {10.1007/978-1-0716-0908-8_16},
pmid = {32809205},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Homologous Recombination/genetics ; Metabolic Engineering/methods ; Plants/genetics ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/*methods ; Transcription Factors/genetics ; },
abstract = {For industry-scale production of high-value chemicals in microbial cell factories, the elimination of metabolic flux imbalances is a critical aspect. However, a priori knowledge about the genetic design of optimal production pathways is typically not available. COMPASS, COMbinatorial Pathway ASSembly, is a rapid cloning method for the balanced expression of multiple genes in biochemical pathways. The method generates thousands of individual DNA constructs in modular, parallel, and high-throughput manner. COMPASS employs inducible artificial transcription factors derived from plant (Arabidopsis thaliana) regulators to control the expression of pathway genes in yeast (Saccharomyces cerevisiae). It utilizes homologous recombination for parts assembly and employs a positive selection scheme to identify correctly assembled pathway variants after both in vivo and in vitro recombination. Finally, COMPASS is equipped with a CRISPR/Cas9 genome modification system allowing for the one-step multilocus integration of genes. Although COMPASS was initially developed for pathway engineering, it can equally be employed for balancing gene expression in other synthetic biology projects.},
}
@article {pmid32808670,
year = {2020},
author = {Landberg, J and Wright, NR and Wulff, T and Herrgård, MJ and Nielsen, AT},
title = {CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {12},
pages = {3835-3848},
pmid = {32808670},
issn = {1097-0290},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering ; *Nucleotides/biosynthesis/genetics ; Single-Domain Antibodies/*biosynthesis/genetics ; },
abstract = {Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.},
}
@article {pmid32808386,
year = {2020},
author = {Liu, Y and Ye, S and Yuan, G and Ma, X and Heng, S and Yi, B and Ma, C and Shen, J and Tu, J and Fu, T and Wen, J},
title = {Gene silencing of BnaA09.ZEP and BnaC09.ZEP confers orange color in Brassica napus flowers.},
journal = {The Plant journal : for cell and molecular biology},
volume = {104},
number = {4},
pages = {932-949},
doi = {10.1111/tpj.14970},
pmid = {32808386},
issn = {1365-313X},
mesh = {Brassica napus/enzymology/*genetics/physiology ; CRISPR-Cas Systems ; Carotenoids/*metabolism ; Flavonoids/metabolism ; Flowers/enzymology/genetics/physiology ; *Gene Expression Regulation, Plant ; Gene Silencing ; Lutein/metabolism ; Oxidoreductases/genetics/*metabolism ; Pigmentation/genetics ; Plant Leaves/enzymology/genetics/physiology ; Plant Proteins/genetics/metabolism ; Xanthophylls/metabolism ; },
abstract = {Brassica napus is currently cultivated as an important ornamental crop in China. Flower color has attracted much attention in rapeseed genetics and breeding. Here, we characterize an orange-flowered mutant of B. napus that exhibits an altered carotenoid profile in its petals. As revealed by map-based cloning, the change in color from yellow to orange is attributed to the loss of BnaC09.ZEP (zeaxanthin epoxidase) and a 1695-bp deletion in BnaA09.ZEP. HPLC analysis, genetic complementation and CRISPR/Cas9 experiments demonstrated that BnaA09.ZEP and BnaC09.ZEP have similar functions, and the abolishment of both genes led to a substantial increase in lutein content and a sharp decline in violaxanthin content in petals but not leaves. BnaA09.ZEP and BnaC09.ZEP are predominantly expressed in floral tissues, whereas their homologs, BnaA07.ZEP and BnaC07.ZEP, mainly function in leaves, indicating redundancy and tissue-specific diversification of BnaZEP function. Transcriptome analysis in petals revealed differences in the expression of carotenoid and flavonoid biosynthesis-related genes between the mutant and its complementary lines. Flavonoid profiles in the petals of complementary lines were greatly altered compared to the mutant, indicating potential cross-talk between the regulatory networks underlying the carotenoid and flavonoid pathways. Additionally, our results indicate that there is functional compensation by BnaA07.ZEP and BnaC07.ZEP in the absence of BnaA09.ZEP and BnaC09.ZEP. Cloning and characterization of BnaZEPs provide insights into the molecular mechanisms underlying flower pigmentation in B. napus and would facilitate breeding of B. napus varieties with higher ornamental value.},
}
@article {pmid32807991,
year = {2020},
author = {Luo, EC and Nathanson, JL and Tan, FE and Schwartz, JL and Schmok, JC and Shankar, A and Markmiller, S and Yee, BA and Sathe, S and Pratt, GA and Scaletta, DB and Ha, Y and Hill, DE and Aigner, S and Yeo, GW},
title = {Large-scale tethered function assays identify factors that regulate mRNA stability and translation.},
journal = {Nature structural &amp; molecular biology},
volume = {27},
number = {10},
pages = {989-1000},
pmid = {32807991},
issn = {1545-9985},
support = {R01 HG004659/HG/NHGRI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; U19 MH107367/MH/NIMH NIH HHS/United States ; U41 HG009889/HG/NHGRI NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; Binding Sites ; CRISPR-Cas Systems ; Carrier Proteins/chemistry/genetics/*metabolism ; Cell Line ; Humans ; Luciferases/genetics/metabolism ; Open Reading Frames ; Polyribosomes/genetics/metabolism ; *Protein Biosynthesis ; *RNA Stability ; RNA-Binding Proteins/chemistry/genetics/*metabolism ; Recombinant Proteins/genetics/metabolism ; Ultraviolet Rays ; },
abstract = {The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3'-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR-Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.},
}
@article {pmid32807825,
year = {2020},
author = {Slattery, SS and Wang, H and Giguere, DJ and Kocsis, C and Urquhart, BL and Karas, BJ and Edgell, DR},
title = {Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13879},
pmid = {32807825},
issn = {2045-2322},
mesh = {Ascomycota/*genetics/*metabolism ; *CRISPR-Cas Systems ; Complement System Proteins/*genetics ; Drug Resistance, Bacterial/genetics ; *Gene Deletion ; Gene Editing/*methods ; Genetic Engineering ; Histidine/biosynthesis ; Orotate Phosphoribosyltransferase/metabolism ; Plasmids/*genetics ; Tryptophan/biosynthesis ; Uracil/biosynthesis ; },
abstract = {The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.},
}
@article {pmid32807781,
year = {2020},
author = {Coelho, MA and De Braekeleer, E and Firth, M and Bista, M and Lukasiak, S and Cuomo, ME and Taylor, BJM},
title = {CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4132},
pmid = {32807781},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology ; Gene Editing/*methods ; Humans ; Mutagenesis/genetics/physiology ; RNA, Guide/genetics/*metabolism ; },
abstract = {Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.},
}
@article {pmid32807756,
year = {2020},
author = {Kim, HJ and Oh, SY and Lee, SJ},
title = {Single-Base Genome Editing in Corynebacterium glutamicum with the Help of Negative Selection by Target-Mismatched CRISPR/Cpf1.},
journal = {Journal of microbiology and biotechnology},
volume = {30},
number = {10},
pages = {1583-1591},
doi = {10.4014/jmb.2006.06036},
pmid = {32807756},
issn = {1738-8872},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/*genetics ; Gene Editing/*methods ; Genome, Bacterial ; Mutation ; Oligonucleotides ; Sequence Alignment ; },
abstract = {CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-basemutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various targetmismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate singlemismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.},
}
@article {pmid32807491,
year = {2020},
author = {Ikegami, K and Nakajima, M and Minami, Y and Nagano, M and Masubuchi, S and Shigeyoshi, Y},
title = {cAMP response element induces Per1 in vivo.},
journal = {Biochemical and biophysical research communications},
volume = {531},
number = {4},
pages = {515-521},
doi = {10.1016/j.bbrc.2020.07.105},
pmid = {32807491},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cyclic AMP/*genetics ; Female ; Gene Expression Regulation ; Light ; Locomotion/physiology ; Male ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Period Circadian Proteins/*genetics ; Promoter Regions, Genetic ; Response Elements/*physiology ; Suprachiasmatic Nucleus/*physiology ; },
abstract = {Light is an important cue for resetting the circadian clock. In mammals, light signals are thought to be transmitted to the cAMP response element (CRE) via a binding protein (CREB) to induce the expression of Per1 and Per2 genes in the mammalian circadian pacemaker, the suprachiasmatic nuclei (SCN). Several in vitro studies have suggested candidate CRE sites that contribute to the Per1 and Per2 induction by light, resulting in a phase shift of the circadian rhythm. However, it remains unclear whether the CREs are responsible for the light-induced Per1/2 induction. To address this question, we generated CRE-deleted mice in the Per1 and Per2 promoter regions. Deletion of a cAMP-responsive CRE in the Per1 promoter blunted light-induced Per1 expression in the SCN at night, while deletion of an ATF4 (CREB-2)-associated CRE in the Per2 promoter had no effect on its expression. These results suggested that the CRE in the Per1 promoter works for light induction but not CRE in the Per2 promoter. Behavioral rhythms observed under some light conditions were not affected by the CRE-deletion in Per1 promoter, suggesting that the attenuated Per1 induction did not affect the entrainment in some light conditions.},
}
@article {pmid32806592,
year = {2020},
author = {Liu, Q and Garcia, M and Wang, S and Chen, CW},
title = {Therapeutic Target Discovery Using High-Throughput Genetic Screens in Acute Myeloid Leukemia.},
journal = {Cells},
volume = {9},
number = {8},
pages = {},
pmid = {32806592},
issn = {2073-4409},
support = {R00 CA197498/CA/NCI NIH HHS/United States ; R37 CA233691/CA/NCI NIH HHS/United States ; R01 CA236626/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drug Discovery/*methods ; Drug Resistance, Neoplasm/drug effects ; Energy Metabolism/genetics ; Epigenesis, Genetic ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Leukemia, Myeloid, Acute/*genetics ; Mice ; RNA, Small Interfering/*genetics ; Signal Transduction/genetics ; },
abstract = {The development of high-throughput gene manipulating tools such as short hairpin RNA (shRNA) and CRISPR/Cas9 libraries has enabled robust characterization of novel functional genes contributing to the pathological states of the diseases. In acute myeloid leukemia (AML), these genetic screen approaches have been used to identify effector genes with previously unknown roles in AML. These AML-related genes centralize alongside the cellular pathways mediating epigenetics, signaling transduction, transcriptional regulation, and energy metabolism. The shRNA/CRISPR genetic screens also realized an array of candidate genes amenable to pharmaceutical targeting. This review aims to summarize genes, mechanisms, and potential therapeutic strategies found via high-throughput genetic screens in AML. We also discuss the potential of these findings to instruct novel AML therapies for combating drug resistance in this genetically heterogeneous disease.},
}
@article {pmid32805526,
year = {2020},
author = {Sun, P and Li, X and Yang, M and Zhao, X and Zhang, Z and Wei, D},
title = {Deletion of a small, secreted and cysteine-rich protein Cpl1 leads to increased invasive growth of Cryptococcus neoformans into nutrient agar.},
journal = {Microbiological research},
volume = {241},
number = {},
pages = {126570},
doi = {10.1016/j.micres.2020.126570},
pmid = {32805526},
issn = {1618-0623},
mesh = {Adenosine Triphosphate/biosynthesis ; Agar ; CRISPR-Cas Systems/genetics ; Cryptococcus neoformans/*growth &amp; development/*metabolism ; Cysteine/chemistry ; Fungal Proteins/genetics/*metabolism ; Gene Expression Regulation, Fungal ; Membrane Potential, Mitochondrial/physiology ; Reactive Oxygen Species/metabolism ; Virulence ; Zinc/chemistry ; },
abstract = {Invasive growth of yeast cells into nutrient agar is induced by different stresses and contributes to the survival of yeast cells under several adverse conditions. The mechanism of invasive growth of Saccharomyces cerevisiae has been extensively investigated. However, there is very little information about the mechanism of invasive growth of another human pathogen yeast Cryptococcus neoformans. Here, we report that deletion of a small and secreted cysteine-rich protein Cpl1 in C. neoformans JEC21 leads to increased adhesive and invasive growth into nutrient agar. The increased adhesive and invasive growth does not depend on the only known adhesion protein Cfl1 and its main controller Znf2. Cpl1Δ accumulates significantly higher level of intracellular labile zinc ion, leading to increased glucose uptake, higher level of mitochondrial membrane potential, ATP and Reactive Oxygen Species(ROS) production. Higher level of ROS activates Snf1, leading to invasive growth of Cpl1Δ. Three cysteine residues at the N-terminals of the cysteine-rich domain controls the increased invasive growth under nutrient sufficient conditions. This is the first report that a small and secreted cysteine-rich protein negatively regulates invasive growth of C. neoformans through regulating the intracellular labile zinc ion level. The function of this cysteine-rich domain was systematically investigated by site-directed mutagenensis in C. neoformans. The work contributes to understanding the function of this protein family and the invasive growth mechanism in C. neoformans.},
}
@article {pmid32804988,
year = {2020},
author = {Leisen, T and Bietz, F and Werner, J and Wegner, A and Schaffrath, U and Scheuring, D and Willmund, F and Mosbach, A and Scalliet, G and Hahn, M},
title = {CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.},
journal = {PLoS pathogens},
volume = {16},
number = {8},
pages = {e1008326},
pmid = {32804988},
issn = {1553-7374},
mesh = {Botrytis/*physiology ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Vectors/administration &amp; dosage/genetics ; Oryza/genetics/*microbiology ; Plant Diseases/genetics/*microbiology ; Ribonucleoproteins/*antagonists &amp; inhibitors/genetics ; Telomere/*genetics ; },
abstract = {CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.},
}
@article {pmid32804931,
year = {2020},
author = {Veillet, F and Kermarrec, MP and Chauvin, L and Chauvin, JE and Nogué, F},
title = {CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato.},
journal = {PloS one},
volume = {15},
number = {8},
pages = {e0235942},
pmid = {32804931},
issn = {1932-6203},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Frameshift Mutation ; Gene Editing/methods ; Genome, Plant ; *INDEL Mutation ; Plasmids/genetics ; Solanum tuberosum/*genetics ; Staphylococcus aureus/*enzymology/genetics ; },
abstract = {Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.},
}
@article {pmid32804067,
year = {2020},
author = {Halpern, J and Paolo, D},
title = {Upstream Ethical Mapping of Germline Genome Editing.},
journal = {The American journal of bioethics : AJOB},
volume = {20},
number = {8},
pages = {1-4},
doi = {10.1080/15265161.2020.1792224},
pmid = {32804067},
issn = {1536-0075},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Germ Cells ; Morals ; },
}
@article {pmid32800628,
year = {2020},
author = {Kawamura, A and Ganesan, A},
title = {Editorial overview: Epigenetics equals chemical biology.},
journal = {Current opinion in chemical biology},
volume = {57},
number = {},
pages = {A1-A4},
doi = {10.1016/j.cbpa.2020.07.001},
pmid = {32800628},
issn = {1879-0402},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Methylation ; Drug Discovery ; *Epigenesis, Genetic ; Epigenomics ; Humans ; },
}
@article {pmid32799680,
year = {2020},
author = {Da Silva Sanchez, A and Paunovska, K and Cristian, A and Dahlman, JE},
title = {Treating Cystic Fibrosis with mRNA and CRISPR.},
journal = {Human gene therapy},
volume = {31},
number = {17-18},
pages = {940-955},
pmid = {32799680},
issn = {1557-7422},
support = {T32 GM008433/GM/NIGMS NIH HHS/United States ; R01 GM132985/GM/NIGMS NIH HHS/United States ; UG3 TR002855/TR/NCATS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cystic Fibrosis/genetics/*therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; RNA, Messenger/*administration &amp; dosage/genetics ; },
abstract = {Less than 20% of the protein coding genome is thought to be targetable using small molecules. mRNA therapies are not limited in the same way since in theory, they can silence or edit any gene by encoding CRISPR nucleases, or alternatively, produce any missing protein. Yet not all mRNA therapies are equally likely to succeed. Over the past several years, an increasing number of clinical trials with siRNA- and antisense oligonucleotide-based drugs have revealed three key concepts that will likely extend to mRNA therapies delivered by nonviral systems. First, scientists have come to understand that some genes make better targets for RNA therapies than others. Second, scientists have learned that the type and position of chemical modifications made to an RNA drug can alter its therapeutic window, toxicity, and bioavailability. Third, scientists have found that safe and targeted drug delivery vehicles are required to ferry mRNA therapies into diseased cells. In this study, we apply these learnings to cystic fibrosis (CF). We also describe lessons learned from a subset of CF gene therapies that have already been tested in patients. Finally, we highlight the scientific advances that are still required for nonviral mRNA- or CRISPR-based drugs to treat CF successfully in patients.},
}
@article {pmid32799324,
year = {2020},
author = {Li, S and Zhang, J and Liu, L and Wang, Z and Li, Y and Guo, L and Li, Y and Zhang, X and Ren, S and Zhao, B and Zhang, N and Guo, YD},
title = {SlTLFP8 reduces water loss to improve water-use efficiency by modulating cell size and stomatal density via endoreduplication.},
journal = {Plant, cell &amp; environment},
volume = {43},
number = {11},
pages = {2666-2679},
doi = {10.1111/pce.13867},
pmid = {32799324},
issn = {1365-3040},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Size ; *Endoreduplication ; F-Box Proteins/metabolism/*physiology ; Gene Knockdown Techniques ; Lycopersicon esculentum/genetics/physiology ; Plant Proteins/metabolism/*physiology ; Plant Stomata/*anatomy &amp; histology/physiology ; *Plant Transpiration ; Real-Time Polymerase Chain Reaction ; Water/*metabolism ; },
abstract = {Improving plant water-use efficiency (WUE) is important to plant survival and crop yield in the context of water limitation. In this study, SlTLFP8 (Tubby-like F-box protein 8) was identified as an osmotic-induced gene in tomato. Transgenic tomato with up-regulated expression of SlTLFP8 showed enhanced water-deficient resistance, whereas knockout mutants generated by CRISPR/Cas9 were more sensitive to water deficit. SlTLFP8 overexpression significantly enhanced WUE by suppressing transpiration under both water-sufficient and water-deficient conditions. Further study showed that overexpressing SlTLFP8 significantly increased leaf epidermal cell size and thereby decreased stomatal density 10-20%, conversely SlTLFP8 knockout resulted in decreased cell size and thereby increased stomatal density 20-50%. SlTLFP8 overexpression and knockout modulated ploidy levels in leaf cells. Changes in expression of cell cycle related genes also indicated that SlTLFP8 affected cell size and stomatal density through endocycle transition. Despite changes in stomata density and transpiration, altering the expression of SlTLFP8 did not change photosynthesis. Additionally, biomass was not altered and there was little difference in fruit yield for transgenic and wild type lines under water-sufficient and water-deficient conditions. Our results demonstrate the effect of SlTLFP8 on endoreduplication and the potential of SlTLFP8 for improvement of WUE. BRIEF SUMMERY: This work found a new mechanism of TLP (Tubby like protein) response to water-deficient stress. SlTLFP8, a member of TLP family, regulates water-deficient resistance by modulating water loss via affecting stomatal density. Expression of SlTLFP8 was induced by osmotic stress. Transgenic tomato lines with SlTLFP8 overexpression or SlTLFP8 knockout showed significantly differences in water-use efficiency (WUE) and water-deficient resistance. The difference of leaf water loss caused by transpiration is the main explanation of the difference in WUE and water-deficient resistance. Additionally, overexpressing SlTLFP8 significantly decreased stomatal density, while SlTLFP8 knockout resulted in increased stomatal density, and SlTLFP8 affected stomatal density through endoreduplication and altered epidermal cell size. Despite changes in stomata density, altering the expression of SlTLFP8 did not result in distinct changes in photosynthesis, biomass and yield of tomato.},
}
@article {pmid32798916,
year = {2020},
author = {Sung, JJ and Park, S and Choi, SH and Kim, J and Cho, MS and Kim, DW},
title = {Generation of a gene edited hemophilia A patient-derived iPSC cell line, YCMi001-B-1, by targeted insertion of coagulation factor FVIII using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101948},
doi = {10.1016/j.scr.2020.101948},
pmid = {32798916},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Endothelial Cells/metabolism ; *Factor VIII/genetics/metabolism ; Gene Editing ; *Hemophilia A/genetics/therapy ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Hemophilia A is an ideal target for cell or gene therapy because a mild increase in coagulation factor VIII (FVIII) improves symptoms in patients with severe hemophilia A. In this study, we used CRISPR/Cas9 to insert FVIII cDNA into exon 1 of the mutant FVIII locus in induced pluripotent stem cells (iPSCs) from a hemophilia A patient. This gene-modified YCMi001-B-1 line maintained its pluripotency, formed all three germ layers, and had a normal karyotype. In addition, FVIII expression was confirmed in YCMi001-B-1-derived endothelial cells.},
}
@article {pmid32798742,
year = {2020},
author = {Mashel, TV and Tarakanchikova, YV and Muslimov, AR and Zyuzin, MV and Timin, AS and Lepik, KV and Fehse, B},
title = {Overcoming the delivery problem for therapeutic genome editing: Current status and perspective of non-viral methods.},
journal = {Biomaterials},
volume = {258},
number = {},
pages = {120282},
doi = {10.1016/j.biomaterials.2020.120282},
pmid = {32798742},
issn = {1878-5905},
mesh = {*CRISPR-Cas Systems ; Drug Carriers ; *Gene Editing ; Genetic Vectors ; Ribonucleoproteins ; },
abstract = {Besides its broad application in research and biotechnology, genome editing (GE) has great potential for clinical gene therapy, but delivery of GE tools remains a bottleneck. Whereas significant progress has been made in ex vivo GE delivery (e.g., by electroporation), establishment of efficient and safe in vivo delivery systems is still a challenge. Above and beyond standard vector requirements (safety, minimal/absent toxicity and immunogenicity, sufficient packaging capacity, targeting, straight and low-cost large-scale good manufacturing practice (GMP) production), GE delivery systems ideally use a hit-and-run principle to minimize off-targets as well as display of immunogenic peptides. Since currently used viral vectors do not fulfil all of these requirements, the broad variety of non-viral delivery platforms represents a promising alternative. This review provides a comprehensive analysis of the most relevant aspects of non-viral physical and chemical delivery methods in non-clinical studies and clinical trials, ranging from classic electroporation to advanced drug carriers that can transport GE tools in form of plasmid DNAs (pDNAs), mRNAs, and ribonucleoproteins (RNPs). For comparison, advantages and shortcomings of viral delivery systems are shortly discussed. In summary, we review various delivery approaches and discuss the future perspectives to use drug carriers for in vivo GE in clinical trials.},
}
@article {pmid32798741,
year = {2020},
author = {Liu, Q and Wang, C and Zheng, Y and Zhao, Y and Wang, Y and Hao, J and Zhao, X and Yi, K and Shi, L and Kang, C and Liu, Y},
title = {Virus-like nanoparticle as a co-delivery system to enhance efficacy of CRISPR/Cas9-based cancer immunotherapy.},
journal = {Biomaterials},
volume = {258},
number = {},
pages = {120275},
doi = {10.1016/j.biomaterials.2020.120275},
pmid = {32798741},
issn = {1878-5905},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Immunotherapy ; *Nanoparticles ; *Neoplasms/genetics/therapy ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system holds great promise for the cancer gene therapy. However, due to complicated signal networks and various compensatory mechanisms in tumors, adjusting a single molecular pathway has limited effects on cancer treatments. Herein, a virus-like nanoparticle (VLN) was reported as a versatile nanoplatform to co-deliver CRISPR/Cas9 system and small molecule drugs for effective malignant cancer treatment. VLN has a core-shell structure, in which small molecule drugs and CRISPR/Cas9 system are loaded in the mesoporous silica nanoparticle (MSN)-based core, which is further encapsulated with a lipid shell. This structure allows VLN maintaining stable during blood circulation. As reaching tumors, VLN releases the CRISPR/Cas9 system and small molecule drugs in response to the reductive microenvironment, resulting in the synergistic regulation of multiple cancer-associated pathways. By loading a single guide RNA (sgRNA) targeting programmed death-ligand 1 and axitinib, VLN achieved to disrupt multiple immunosuppressive pathways and suppress the growth of melanoma in vivo. More importantly, VLN can co-deliver almost any combination of sgRNAs and small molecule drugs to tumors, suggesting the great potential of VLN as a general platform for the development of advanced combination therapies against malignant tumors.},
}
@article {pmid32798712,
year = {2020},
author = {Wang, X and Cao, X and Jiang, D and Yang, Y and Wu, Y},
title = {CRISPR/Cas9 mediated ryanodine receptor I4790M knockin confers unequal resistance to diamides in Plutella xylostella.},
journal = {Insect biochemistry and molecular biology},
volume = {125},
number = {},
pages = {103453},
doi = {10.1016/j.ibmb.2020.103453},
pmid = {32798712},
issn = {1879-0240},
mesh = {Animals ; Benzamides/pharmacology ; CRISPR-Cas Systems ; Calcium Signaling/drug effects ; Diamide/*pharmacology ; Gene Silencing ; Genes, Insect ; Insect Control ; Insecticide Resistance/*genetics ; Insecticides/*pharmacology ; Moths/drug effects/*genetics ; Mutation ; Pest Control ; Pyrazoles/pharmacology ; Ryanodine Receptor Calcium Release Channel/drug effects/*genetics ; Sulfones/pharmacology ; ortho-Aminobenzoates/pharmacology ; },
abstract = {The diamondback moth Plutella xylostella is a major destructive pest of Brassica worldwide. P. xylostella has evolved resistance to nearly all commercial insecticides used for its control, including the most recent chemical class, diamide insecticides. Several studies show that the G4946E and I4790M mutations of ryanodine receptor (RyR) are strongly associated with diamide resistance in insects. While the pivotal functional role of G4946E in conferring diamide resistance phenotype has confirmed by several studies in different species, no direct evidence has unambiguously confirmed the functional significance of the single I4790M mutation in diamide resistance. Here, we successfully constructed a knockin homozygous strain (I4790M-KI) of P. xylostella using CRISPR/Cas9 coupled with homology directed repair approach to introduce I4790M into RyR. When compared with the background susceptible IPP-S strain, the manipulated I4790M-KI strain exhibited moderate resistance to the phthalic acid diamide flubendiamide (40.5-fold) and low resistance to anthranilic diamides chlorantraniliprole (6.0-fold) and cyantraniliprole (7.7-fold), with no changes to the toxicities of indoxacarb and β-cypermethrin. Furthermore, the acquired flubendiamide resistance was inherited in an autosomally recessive mode and significantly linked with the I4790M mutation of RyR in this I4790M-KI strain. Our findings provide in vivo functional evidence for the causality of I4790M mutation of PxRyR with moderate levels of resistance to flubendiamide in P. xylostella, and support the hypothesis that the diamide classes have different interactions with RyRs.},
}
@article {pmid32789000,
year = {2019},
author = {Bhagwan, JR and Collins, E and Mosqueira, D and Bakar, M and Johnson, BB and Thompson, A and Smith, JGW and Denning, C},
title = {Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.},
journal = {F1000Research},
volume = {8},
number = {},
pages = {1911},
pmid = {32789000},
issn = {2046-1402},
support = {MR/M017354/1/MRC_/Medical Research Council/United Kingdom ; NC/S001808/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Gene Expression ; Gene Knock-In Techniques ; *Gene Silencing ; *Gene Targeting ; Genes, Reporter ; *Genetic Loci ; Humans ; *Induced Pluripotent Stem Cells ; Myocytes, Cardiac/cytology ; *Transgenes ; },
abstract = {Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca [2+] responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.},
}
@article {pmid32798288,
year = {2020},
author = {Miao, R and Lu, Y and He, X and Liu, X and Chen, Z and Wang, J},
title = {Ubiquitin-specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1-dependent pathway.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {18},
pages = {10946-10957},
pmid = {32798288},
issn = {1582-4934},
mesh = {Angiotensin II/toxicity ; Animals ; Animals, Newborn ; Aortic Valve Stenosis ; CRISPR-Cas Systems ; Cardiomegaly/chemically induced/diagnostic imaging/*physiopathology ; Disease Models, Animal ; Endopeptidases/biosynthesis/deficiency/genetics/*physiology ; Fibrosis ; Inflammation ; MAP Kinase Kinase Kinases/*physiology ; MAP Kinase Signaling System/*physiology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myocytes, Cardiac/drug effects/*enzymology/pathology ; Phenylephrine/pharmacology ; Pressure ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/metabolism ; Ubiquitin-Protein Ligases/physiology ; Ventricular Remodeling/physiology ; },
abstract = {Ubiquitin-specific protease 19 (USP19) belongs to USP family and is involved in promoting skeletal muscle atrophy. Although USP19 is expressed in the heart, the role of USP19 in the heart disease remains unknown. The present study provides in vivo and in vitro data to reveal the role of USP19 in preventing pathological cardiac hypertrophy. We generated USP19-knockout mice and isolated neonatal rat cardiomyocytes (NRCMs) that overexpressed or were deficient in USP19 to investigate the effect of USP19 on transverse aortic constriction (TAC) or phenylephrine (PE)-mediated cardiac hypertrophy. Echocardiography, pathological and molecular analysis were used to determine the extent of cardiac hypertrophy, fibrosis, dysfunction and inflammation. USP19 expression was markedly increased in rodent hypertrophic heart or cardiomyocytes underwent TAC or PE culturing, the increase was mediated by the reduction of Seven In Absentia Homolog-2. The extent of TAC-induced cardiac hypertrophy, fibrosis, dysfunction and inflammation in USP19-knockout mice was exacerbated. Consistently, gain-of-function and loss-of-function approaches that involved USP19 in cardiomyocytes suggested that the down-regulation of USP19 promoted the hypertrophic phenotype, while the up-regulation of USP19 improved the worsened phenotype. Mechanistically, the USP19-elicited cardiac hypertrophy improvement was attributed to the abrogation of the transforming growth factor beta-activated kinase 1 (TAK1)-p38/JNK1/2 transduction. Furthermore, the inhibition of TAK1 abolished the aggravated hypertrophy induced by the loss of USP19. In conclusion, the present study revealed that USP19 and the downstream of TAK1-p38/JNK1/2 signalling pathway might be a potential target to attenuate pathological cardiac hypertrophy.},
}
@article {pmid32797447,
year = {2021},
author = {Sharma, V and Zheng, W and Huang, J and Cook, DE},
title = {CRISPR-Cas RNA Targeting Using Transient Cas13a Expression in Nicotiana benthamiana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2170},
number = {},
pages = {1-18},
doi = {10.1007/978-1-0716-0743-5_1},
pmid = {32797447},
issn = {1940-6029},
mesh = {Biotechnology/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; RNA Editing/*genetics ; RNA Interference/physiology ; Tobacco/genetics/*metabolism ; Transcriptome/genetics ; },
abstract = {Application of the CRISPR-Cas prokaryotic immune system for single-stranded RNA targeting will have significant impacts on RNA analysis and engineering. The class 2 Type VI CRISPR-Cas13 system is an RNA-guided RNA-nuclease system capable of binding and cleaving target single-stranded RNA substrates in a sequence-specific manner. In addition to RNA interference, the Cas13a system has application from manipulating RNA modifications, to editing RNA sequence, to use as a nucleic acid detection tool. This protocol uses the Cas13a ortholog from Leptotrichia buccalis for transient expression in plant cells providing antiviral defense. We cover all the necessary information for cloning the Cas13 protein, crRNA guide cassette, performing transient Agrobacterium-mediated expression of the necessary Cas13a components and target RNA-virus, visualization of virus infection, and molecular quantification of viral accumulation using quantitative PCR.},
}
@article {pmid32797168,
year = {2020},
author = {Chien, JC and Tabet, E and Pinkham, K and da Hora, CC and Chang, JC and Lin, S and Badr, CE and Lai, CP},
title = {A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {e100},
pmid = {32797168},
issn = {1362-4962},
support = {K22 CA197053/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Copepoda/enzymology ; DNA End-Joining Repair ; Female ; *Genes, Reporter ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Luciferases/*genetics/metabolism ; Mice ; Mice, Nude ; Multiplex Polymerase Chain Reaction/methods ; Optical Imaging/methods ; Rad51 Recombinase/genetics/metabolism ; *Recombinational DNA Repair ; Sequence Analysis, DNA/methods ; },
abstract = {Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.},
}
@article {pmid32797156,
year = {2020},
author = {Iwasaki, RS and Batey, RT},
title = {SPRINT: a Cas13a-based platform for detection of small molecules.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {e101},
pmid = {32797156},
issn = {1362-4962},
support = {R01 GM073850/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; Biosensing Techniques/*methods ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/*metabolism ; Enzyme Assays/*methods ; Fluorescent Dyes/chemistry ; Leptotrichia ; Ligands ; Nucleic Acid Synthesis Inhibitors/*analysis/pharmacology ; Riboswitch ; Rifampin/analysis ; Transcription Factors/metabolism ; Transcription, Genetic/drug effects ; },
abstract = {Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.},
}
@article {pmid32796846,
year = {2020},
author = {Roidos, P and Sungalee, S and Benfatto, S and Serçin, &#xd6; and Stütz, AM and Abdollahi, A and Mauer, J and Zenke, FT and Korbel, JO and Mardin, BR},
title = {A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4077},
pmid = {32796846},
issn = {2041-1723},
mesh = {Ataxia Telangiectasia Mutated Proteins/genetics ; *CRISPR-Cas Systems ; Cell Cycle ; Cell Survival ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Breaks, Double-Stranded ; DNA Damage ; DNA End-Joining Repair ; *DNA Repair ; Drug Evaluation, Preclinical ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Poly (ADP-Ribose) Polymerase-1/genetics ; },
abstract = {Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.},
}
@article {pmid32796761,
year = {2020},
author = {Khalaf, K and Janowicz, K and Dyszkiewicz-Konwińska, M and Hutchings, G and Dompe, C and Moncrieff, L and Jankowski, M and Machnik, M and Oleksiewicz, U and Kocherova, I and Petitte, J and Mozdziak, P and Shibli, JA and Iżycki, D and Józkowiak, M and Piotrowska-Kempisty, H and Skowroński, MT and Antosik, P and Kempisty, B},
title = {CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.},
journal = {Genes},
volume = {11},
number = {8},
pages = {},
pmid = {32796761},
issn = {2073-4425},
mesh = {Animals ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Disease ; Disease Models, Animal ; Drug Evaluation, Preclinical ; *Gene Editing ; *Genetic Therapy ; Humans ; *Immunotherapy ; Immunotherapy, Adoptive ; Neoplasms/etiology/*therapy ; Precision Medicine/methods ; },
abstract = {Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.},
}
@article {pmid32796680,
year = {2020},
author = {Aulicino, F and Capin, J and Berger, I},
title = {Synthetic Virus-Derived Nanosystems (SVNs) for Delivery and Precision Docking of Large Multifunctional DNA Circuitry in Mammalian Cells.},
journal = {Pharmaceutics},
volume = {12},
number = {8},
pages = {},
pmid = {32796680},
issn = {1999-4923},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; DNA-DOCK 834631/ERC_/European Research Council/International ; },
abstract = {DNA delivery is at the forefront of current research efforts in gene therapy and synthetic biology. Viral vectors have traditionally dominated the field; however, nonviral delivery systems are increasingly gaining traction. Baculoviruses are arthropod-specific viruses that can be easily engineered and repurposed to accommodate and deliver large sequences of exogenous DNA into mammalian cells, tissues, or ultimately organisms. These synthetic virus-derived nanosystems (SVNs) are safe, readily customized, and can be manufactured at scale. By implementing clustered regularly interspaced palindromic repeats (CRISPR) associated protein (CRISPR/Cas) modalities into this system, we developed SVNs capable of inserting complex DNAs into genomes, at base pair precision. We anticipate a major role for SVNs as an attractive alternative to viral vectors in accelerating genome engineering and gene therapy applications in the future.},
}
@article {pmid32796076,
year = {2020},
author = {Amalfi, S and Molina, GN and Bevacqua, RJ and López, MG and Taboga, O and Alfonso, V},
title = {Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway.},
journal = {Journal of virology},
volume = {94},
number = {21},
pages = {},
pmid = {32796076},
issn = {1098-5514},
mesh = {Animals ; Baculoviridae/*genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems ; DNA, Viral/genetics/immunology ; DNA-Activated Protein Kinase/genetics/immunology ; DNA-Binding Proteins/genetics/immunology ; Gene Expression Regulation ; HEK293 Cells ; Host Specificity ; Humans ; Interferon-beta/*genetics/immunology ; Interferons/*genetics/immunology ; Interleukins/*genetics/immunology ; Membrane Proteins/*genetics/immunology ; Mice ; NIH 3T3 Cells ; Nucleotidyltransferases/*genetics/immunology ; Sf9 Cells ; Signal Transduction ; Spodoptera ; Transduction, Genetic ; },
abstract = {The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-β) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.},
}
@article {pmid32795423,
year = {2020},
author = {Prat, F and Toutain, J and Boutin, J and Amintas, S and Cullot, G and Lalanne, M and Lamrissi-Garcia, I and Moranvillier, I and Richard, E and Blouin, JM and Dabernat, S and Moreau-Gaudry, F and Bedel, A},
title = {Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells.},
journal = {Stem cell reports},
volume = {15},
number = {3},
pages = {677-693},
pmid = {32795423},
issn = {2213-6711},
mesh = {Alleles ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Clone Cells ; Exons/genetics ; *Gene Editing ; Genetic Therapy ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Karyotyping ; Mutation/*genetics ; Porphyrias/*genetics/*therapy ; RNA, Guide/*metabolism ; Stem Cells/*metabolism ; Uroporphyrinogen III Synthetase/genetics ; },
abstract = {CRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations.},
}
@article {pmid32794766,
year = {2020},
author = {Wu, Y and Liu, Y and Lv, X and Li, J and Du, G and Liu, L},
title = {Applications of CRISPR in a Microbial Cell Factory: From Genome Reconstruction to Metabolic Network Reprogramming.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2228-2238},
doi = {10.1021/acssynbio.0c00349},
pmid = {32794766},
issn = {2161-5063},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; *Genome ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/*genetics ; Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {The well-designed microbial cell factory finds wide applications in chemical, pharmaceutical, and food industries due to its sustainable and environmentally friendly features. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) systems have been developed into powerful tools to perform genome editing and transcriptional regulation in prokaryotic and eukaryotic cells. Accordingly, these tools are useful to build microbial cell factories not only by reconstructing the genome but also by reprogramming the metabolic network. In this review, we summarize the recent significant headway and potential uses of the CRISPR technology in the construction of efficient microbial cell factories. Moreover, the future perspectives on the improvement and upgradation of CRISPR-based tools are also discussed.},
}
@article {pmid32794659,
year = {2020},
author = {Iżykowska, K and Rassek, K and Żurawek, M and Nowicka, K and Paczkowska, J and Ziółkowska-Suchanek, I and Podralska, M and Dzikiewicz-Krawczyk, A and Joks, M and Olek-Hrab, K and Giefing, M and Przybylski, GK},
title = {Hypomethylation of the promoter region drives ectopic expression of TMEM244 in Sézary cells.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {18},
pages = {10970-10977},
pmid = {32794659},
issn = {1582-4934},
mesh = {Aged ; Aged, 80 and over ; CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Methylation ; Female ; Gene Expression Regulation/*genetics ; Genetic Vectors ; Hematologic Neoplasms/genetics/metabolism ; Humans ; Lymphoma, Non-Hodgkin/genetics/metabolism ; Male ; Membrane Proteins/biosynthesis/*genetics ; Middle Aged ; Mycosis Fungoides/genetics/metabolism ; Neoplasm Proteins/biosynthesis/*genetics ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/metabolism ; Promoter Regions, Genetic/*genetics ; Sezary Syndrome/*genetics/metabolism ; },
abstract = {Sézary syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by the presence of circulating malignant CD4+ T cells (Sézary cells) with many complex changes in the genome, transcriptome and epigenome. Epigenetic dysregulation seems to have an important role in the development and progression of SS as it was shown that SS cells are characterized by widespread changes in DNA methylation. In this study, we show that the transmembrane protein coding gene TMEM244 is ectopically expressed in all SS patients and SS-derived cell lines and, to a lower extent, in mycosis fungoides and in a fraction of T-cell lymphomas, but not in B-cell malignancies and mononuclear cells of healthy individuals. We show that in patient samples and in the T-cell lines TMEM244 expression is negatively correlated with the methylation level of its promoter. Furthermore, we demonstrate that TMEM244 expression can be activated in vitro by the CRISPR-dCas9-induced specific demethylation of TMEM244 promoter region. Since both, TMEM244 expression and its promoter demethylation, are not detected in normal lymphoid cells, they can be potentially used as markers in Sézary syndrome and some other T-cell lymphomas.},
}
@article {pmid32794605,
year = {2020},
author = {Zhang, A and Chen, J and Gong, T and Lu, M and Tang, B and Zhou, X and Li, Y},
title = {Deletion of csn2 gene affects acid tolerance and exopolysaccharide synthesis in Streptococcus mutans.},
journal = {Molecular oral microbiology},
volume = {35},
number = {5},
pages = {211-221},
doi = {10.1111/omi.12308},
pmid = {32794605},
issn = {2041-1014},
mesh = {Acids/*pharmacology ; Biofilms ; CRISPR-Cas Systems ; Dental Caries ; *Gene Deletion ; *Genes, Bacterial ; Humans ; Polysaccharides, Bacterial/*biosynthesis ; *Streptococcus mutans/genetics/physiology ; Virulence/genetics ; },
abstract = {Csn2 is an important protein of the CRISPR-Cas system. The physiological function of this protein and its regulatory role in Streptococcus mutans, as the primary causative agent of human dental caries, is still unclear. In this study, we investigated whether csn2 deletion would affect S. mutans physiology and virulence gene expression. We used microscopic imaging, acid killing assays, pH drop, biofilm formation, and exopolysaccharide (EPS) production tests to determine whether csn2 deletion influenced S. mutans colony morphology, acid tolerance/production, and glucan formation abilities. Comparisons were made between quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) data from the UA159 and csn2 deletion strain to determine the impact of csn2 knockout on S. mutans gene expression. The results showed that deletion of S. mutans csn2 changed its colony morphotype and made it more sensitive to acid. The expression levels of aciduricity genes, including leuA, leuB, leuC, and leuD, were significantly down-regulated. Acid adaptation restored the aciduricity of csn2 mutant and enhanced the ability to synthesize EPS. The expression levels of EPS synthesis-related genes, including gtfC and gtfD, were significantly up-regulated after acid adaptation. In summary, deletion of S. mutans csn2 exerted multiple effects on the virulence traits of this pathogen, including acid tolerance and EPS formation, and that these alterations could partially be attributed to changes in gene expression upon loss of csn2. Understanding the function of csn2 in S. mutans might lead to novel strategies to prevent or treat imbalances in oral microbiota that may favor diseases.},
}
@article {pmid32794018,
year = {2020},
author = {Huang, C and Guo, L and Wang, J and Wang, N and Huo, YX},
title = {Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {18},
pages = {7943-7956},
doi = {10.1007/s00253-020-10819-1},
pmid = {32794018},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Editing ; Genetic Engineering ; Genome, Bacterial ; Metabolic Engineering ; },
abstract = {Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering. KEY POINTS: • This article reports an efficient genome engineering tool for E. coli. • The tool is advantageous for the manipulations of long DNA fragments. • The tool has been successfully applied for genome simplification. • The tool has been successfully applied for metabolic engineering.},
}
@article {pmid32793540,
year = {2020},
author = {Gupta, P and Singh, MP and Goyal, K},
title = {Diversity of Vaginal Microbiome in Pregnancy: Deciphering the Obscurity.},
journal = {Frontiers in public health},
volume = {8},
number = {},
pages = {326},
pmid = {32793540},
issn = {2296-2565},
mesh = {Eukaryota ; Female ; Gestational Age ; Humans ; Infant, Newborn ; Lactobacillus/genetics ; *Microbiota/genetics ; Pregnancy ; *Vagina ; },
abstract = {Human microbiota plays an indispensable role in physiology, nutrition and most significantly, in imparting immunity. The role of microbiota has remained cryptic for years, until recently meticulous studies revealed the interaction and dynamics of these microbial communities. This diversified state is governed by hormonal, behavioral and physio-chemical changes in the genital tract. Many inclusive studies have revealed "Lactobacillus" to be the most dominant member of vaginal flora in most of the healthy, reproductive age group and pregnant females. A total of five community state types have been described, out of which four are dominated by Lactobacillus while the fifth one by facultative or strict anaerobic species. A variation between species stability and gestational age has also been revealed. Studies have divulged a significant higher stability of vaginal microbiota in early stages of pregnancy and the same increased subsequently. Inter-species and racial variation has shown women belonging to White, Asian, and Caucasian race to harbor more of the anaerobic flora. The vaginal microbiome in pregnancy play a significant role in preterm and spontaneous labor. This Lactobacillus-rich microbiome falls tremendously, becoming more diverse in post-partum period. Apart from these known bacterial communities in human vagina, other microbial communities have also been traced. The major fragment is constituted by vaginal viral virome and very little information exists in relation to vaginal mycobiome. Studies have revealed the abundance of ds DNA viruses in vaginal microbiome, followed by ssDNA, and few unidentified viruses. The eukaryotic viruses detected were very few, with Herpesvirales, and Papillomaviridae being the only pathogenic ones. This flora is transmitted to infants either via maternal gut, vagina or breast milk. Recent studies have given an insight for vaginal microbiome, dissociating the old concept of "healthy" and "diseased." However, more extensive studies are required to study evolution of virome and mycobiome in relation to their association with bacterial communities; to establish and decode full array of vaginal virome under the influence of genotypic and environmental factors, using novel bioinformatic, multi-omic, statistical model, and CRISPR/Cas approaches.},
}
@article {pmid32793272,
year = {2020},
author = {Dort, EN and Tanguay, P and Hamelin, RC},
title = {CRISPR/Cas9 Gene Editing: An Unexplored Frontier for Forest Pathology.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {1126},
pmid = {32793272},
issn = {1664-462X},
abstract = {CRISPR/Cas9 gene editing technology has taken the scientific community by storm since its development in 2012. First discovered in 1987, CRISPR/Cas systems act as an adaptive immune response in archaea and bacteria that defends against invading bacteriophages and plasmids. CRISPR/Cas9 gene editing technology modifies this immune response to function in eukaryotic cells as a highly specific, RNA-guided complex that can edit almost any genetic target. This technology has applications in all biological fields, including plant pathology. However, examples of its use in forest pathology are essentially nonexistent. The aim of this review is to give researchers a deeper understanding of the native CRISPR/Cas systems and how they were adapted into the CRISPR/Cas9 technology used today in plant pathology-this information is crucial for researchers aiming to use this technology in the pathosystems they study. We review the current applications of CRISPR/Cas9 in plant pathology and propose future directions for research in forest pathosystems where this technology is currently underutilized.},
}
@article {pmid32792587,
year = {2020},
author = {Wiechmann, AF and Martin, TA and Horb, ME},
title = {CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13757},
pmid = {32792587},
issn = {2045-2322},
support = {P40 OD010997/OD/NIH HHS/United States ; R01 HD084409/HD/NICHD NIH HHS/United States ; P20 GM104934/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Circadian Rhythm/genetics ; Frameshift Mutation/genetics ; Melatonin/metabolism ; Receptors, Melatonin/*genetics ; Retinal Cone Photoreceptor Cells/pathology ; Retinal Degeneration/*genetics ; Retinal Rod Photoreceptor Cells/*pathology ; Sequence Deletion/genetics ; Signal Transduction/genetics ; Xenopus/*genetics ; Xenopus Proteins/*genetics ; },
abstract = {Nighttime surges in melatonin levels activate melatonin receptors, which synchronize cellular activities with the natural light/dark cycle. Melatonin receptors are expressed in several cell types in the retina, including the photon-sensitive rods and cones. Previous studies suggest that long-term photoreceptor survival and retinal health is in part reliant on melatonin orchestration of circadian homeostatic activities. This scenario would accordingly envisage that disruption of melatonin receptor signaling is detrimental to photoreceptor health. Using in vivo CRISPR/Cas9 genomic editing, we discovered that a small deletion mutation of the Mel1a melatonin receptor (mtnr1a) gene causes a loss of rod photoreceptors in retinas of developing Xenopus tropicalis heterozygous, but not homozygous mutant tadpoles. Cones were relatively spared from degeneration, and the rod loss phenotype was not obvious after metamorphosis. Localization of Mel1a receptor protein appeared to be about the same in wild type and mutant retinas, suggesting that the mutant protein is expressed at some level in mutant retinal cells. The severe impact on early rod photoreceptor viability may signify a previously underestimated critical role in circadian influences on long-term retinal health and preservation of sight. These data offer evidence that disturbance of homeostatic, circadian signaling, conveyed through a mutated melatonin receptor, is incompatible with rod photoreceptor survival.},
}
@article {pmid32792541,
year = {2020},
author = {Foster, DS and Marshall, CD and Gulati, GS and Chinta, MS and Nguyen, A and Salhotra, A and Jones, RE and Burcham, A and Lerbs, T and Cui, L and King, ME and Titan, AL and Ransom, RC and Manjunath, A and Hu, MS and Blackshear, CP and Mascharak, S and Moore, AL and Norton, JA and Kin, CJ and Shelton, AA and Januszyk, M and Gurtner, GC and Wernig, G and Longaker, MT},
title = {Elucidating the fundamental fibrotic processes driving abdominal adhesion formation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4061},
pmid = {32792541},
issn = {2041-1723},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; R01 DE027346/DE/NIDCR NIH HHS/United States ; R01 GM116892/GM/NIGMS NIH HHS/United States ; S10 OD018220/OD/NIH HHS/United States ; },
mesh = {Animals ; Benzophenones/pharmacology ; CRISPR-Cas Systems ; Cells, Cultured ; Doxycycline/pharmacology ; Fibroblasts/drug effects/metabolism ; Fluorescent Antibody Technique ; Gastrointestinal Diseases/metabolism/pathology ; Humans ; Immunohistochemistry ; Isoxazoles/pharmacology ; Liposomes/metabolism ; Mice ; NIH 3T3 Cells ; Parabiosis ; RNA, Messenger/metabolism ; Tamoxifen/pharmacology ; Tissue Adhesions/*metabolism/*pathology ; },
abstract = {Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.},
}
@article {pmid32791795,
year = {2020},
author = {Sun, BK and Liu, SS and Zhang, J and Xuan, SY and Xin, YN},
title = {[Construction of transmembrane 6 superfamily member 2 E167K gene knock-in mouse model by using CRISPR/Cas9 technology].},
journal = {Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology},
volume = {28},
number = {7},
pages = {591-596},
doi = {10.3760/cma.j.cn501113-20191204-00445},
pmid = {32791795},
issn = {1007-3418},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Knock-In Techniques ; Glucose/metabolism ; Male ; Membrane Proteins/*genetics ; Mice ; Mice, Transgenic ; *Non-alcoholic Fatty Liver Disease/genetics ; },
abstract = {Objective: To construct a transmembrane 6 superfamily member 2 (Tm6sf2) E167K gene knock-in mouse model. Methods: The plasmid was constructed to simultaneously express the single-stranded guide RNA Cas9 at a specific site of the mouse Tm6sf2 gene in the donor plasmid carrying the Tm6sf2 E167K fragment. The above two plasmids were injected into the mouse fertilized eggs together. The positive F0 generation mice were validated by PCR detection and sequencing. The number of F2 generation surviving mice in three genotypes of wild (Wt), heterozygous and knock-in (KI) were calculated. Wt and KI male mice (8 mice/ group) of F2 generation littermates were selected and given a normal diet for 8 weeks. The body weight of the mice was recorded every week, and the glucose metabolism and lipid metabolism indexes of the two mice were detected. The comparison between groups was performed with an independent sample t-test. Results: Genotype detection and sequencing results showed that the Tm6sf2 E167K gene knock-in mouse model was successfully established. KI mice had absence of homozygous lethal embryo phenotype. The body weight of KI mice was higher than that of Wt mice during lactation, and the difference between the two groups was statistically significant (P < 0.05).The fasting blood glucose of KI mice (9.50 ± 0.33)mmol/L was higher than that of Wt mice (7.80 ± 0.30)mmol/L, and the difference between the two groups was statistically significant (P < 0.05). During the oral glucose tolerance test, the 2-hour blood glucose level of KI mice (9.20 ± 0.51)mmol/L was higher than that of Wt mice (7.60 ± 0.18)mmol/L, and the difference between the two groups was statistically significant (P < 0.05). The liver triglyceride content of KI mice (8.40 ± 0.55)mg/g was higher than that of Wt mice (7.30 ± 0.63)mg/g, but the difference was not statistically significant (P > 0.05). There was no significant difference in plasma triglyceride levels between the two mice (P > 0.05). The Oil red O staining results showed that KI mice had more lipid accumulation in the centrilobular region of liver than Wt mice. Conclusion: Tm6sf2 E167K gene knock-in mice were successfully constructed. Tm6sf2 E167K gene knock-in can cause abnormal glucose metabolism in mice and promote the occurrence of hepatic steatosis.},
}
@article {pmid32791035,
year = {2020},
author = {He, X and Liu, C and Yang, X and Lv, M and Ni, X and Li, Q and Cheng, H and Liu, W and Tian, S and Wu, H and Gao, Y and Yang, C and Tan, Q and Cong, J and Tang, D and Zhang, J and Song, B and Zhong, Y and Li, H and Zhi, W and Mao, X and Fu, F and Ge, L and Shen, Q and Zhang, M and Saiyin, H and Jin, L and Xu, Y and Zhou, P and Wei, Z and Zhang, F and Cao, Y},
title = {Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.},
journal = {American journal of human genetics},
volume = {107},
number = {3},
pages = {514-526},
pmid = {32791035},
issn = {1537-6605},
mesh = {Abnormalities, Multiple/*genetics/pathology ; Alleles ; Animals ; Asthenozoospermia/*genetics/physiopathology ; Axoneme/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics ; Homozygote ; Humans ; Infertility, Male/*genetics/pathology ; Intercellular Signaling Peptides and Proteins/*genetics ; Loss of Function Mutation/genetics ; Loss of Heterozygosity/genetics ; Male ; Mice ; Mice, Knockout ; Microtubule Proteins/genetics ; Mitochondria/genetics ; Sperm Tail/metabolism/pathology ; Testis/metabolism/pathology ; Whole Exome Sequencing ; },
abstract = {Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.},
}
@article {pmid32790142,
year = {2020},
author = {Zhang, S and Zhang, Q and Hou, XM and Guo, L and Wang, F and Bi, L and Zhang, X and Li, HH and Wen, F and Xi, XG and Huang, X and Shen, B and Sun, B},
title = {Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation.},
journal = {EMBO reports},
volume = {21},
number = {10},
pages = {e50184},
pmid = {32790142},
issn = {1469-3178},
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Dissociative Disorders ; Gene Editing ; Humans ; RNA, Guide/genetics ; *Staphylococcus aureus/genetics ; },
abstract = {Staphylococcus aureus Cas9 (SaCas9) is an RNA-guided endonuclease that targets complementary DNA adjacent to a protospacer adjacent motif (PAM) for cleavage. Its small size facilitates in vivo delivery for genome editing in various organisms. Herein, using single-molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6- and 18-bp of PAM-proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6 bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA-bound SaCas9 a persistent block to DNA-tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM-distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications.},
}
@article {pmid32789179,
year = {2020},
author = {Li, Z and Wang, X and Xu, D and Zhang, D and Wang, D and Dai, X and Wang, Q and Li, Z and Gu, Y and Ouyang, W and Zhao, S and Huang, B and Gong, J and Zhao, J and Chen, A and Shen, Y and Dong, Y and Zhang, W and Xu, X and Xu, C and Jiang, Y},
title = {DNB-based on-chip motif finding: A high-throughput method to profile different types of protein-DNA interactions.},
journal = {Science advances},
volume = {6},
number = {31},
pages = {eabb3350},
pmid = {32789179},
issn = {2375-2548},
mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; DNA/chemistry ; Endonucleases/chemistry ; Gene Editing/methods ; Mutation ; },
abstract = {Here, we report a sensitive DocMF system that uses next-generation sequencing chips to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of different CRISPR systems. DocMF can simultaneously screen both 5' and 3' PAMs with high coverage. For SpCas9, we found noncanonical 5'-NAG-3' (~5%) and 5'-NGA-3' (~1.6%), in addition to its common PAMs, 5'-NGG-3' (~89.9%). More relaxed PAM sequences of two uncharacterized Cas endonucleases, VeCas9 and BvCas12a, were extensively characterized using DocMF. Moreover, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably bound to the previously reported 5'-NGG-3' sequence. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA binding proteins.},
}
@article {pmid32788672,
year = {2020},
author = {Akkaya, M and Bansal, A and Sheehan, PW and Pena, M and Cimperman, CK and Qi, CF and Yazew, T and Otto, TD and Billker, O and Miller, LH and Pierce, SK},
title = {Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13630},
pmid = {32788672},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Extracellular Matrix/*parasitology ; Female ; Malaria, Cerebral/*parasitology ; Mice ; Mice, Inbred C57BL ; Plasmodium berghei/*genetics/growth &amp; development/physiology ; *Polymorphism, Single Nucleotide ; Protozoan Proteins/antagonists &amp; inhibitors/*genetics ; Virulence Factors/antagonists &amp; inhibitors/*genetics ; },
abstract = {Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.},
}
@article {pmid32788576,
year = {2020},
author = {Rai, R and Romito, M and Rivers, E and Turchiano, G and Blattner, G and Vetharoy, W and Ladon, D and Andrieux, G and Zhang, F and Zinicola, M and Leon-Rico, D and Santilli, G and Thrasher, AJ and Cavazza, A},
title = {Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4034},
pmid = {32788576},
issn = {2041-1723},
support = {104807/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; 201250/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Blood Platelets/metabolism ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Codon/genetics ; Female ; *Gene Editing ; Genetic Loci ; *Genetic Therapy ; HEK293 Cells ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Macrophages/metabolism ; Male ; Mice ; Mutagenicity Tests ; Myeloid Cells/metabolism ; T-Lymphocytes/metabolism ; Wiskott-Aldrich Syndrome/*genetics/pathology/*therapy ; Wiskott-Aldrich Syndrome Protein/genetics ; },
abstract = {Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.},
}
@article {pmid32788383,
year = {2020},
author = {Chidawanyika, T and Mark, KMK and Supattapone, S},
title = {A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells.},
journal = {mBio},
volume = {11},
number = {4},
pages = {},
pmid = {32788383},
issn = {2150-7511},
support = {R56 NS094576/NS/NINDS NIH HHS/United States ; R21 NS099928/NS/NINDS NIH HHS/United States ; R01 NS102301/NS/NINDS NIH HHS/United States ; R01 NS118796/NS/NINDS NIH HHS/United States ; R01 NS117276/NS/NINDS NIH HHS/United States ; P20 GM113132/GM/NIGMS NIH HHS/United States ; R21 NS095763/NS/NINDS NIH HHS/United States ; },
mesh = {Biological Transport ; *CRISPR-Cas Systems ; Cell Line ; Cell Survival ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Hydrogen Peroxide/*metabolism ; Leukocytes/drug effects/*metabolism ; Oxidative Stress ; Receptors, G-Protein-Coupled/genetics ; Riboflavin/*metabolism ; },
abstract = {Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H2O2-induced cell death: POR (encoding cytochrome P450 oxidoreductase), RETSAT (retinol saturase), KEAP1 (Kelch-like ECH-associated protein-1), and SLC52A2 (riboflavin transporter). Among these genes, only POR also mediated methyl viologen dichloride hydrate (paraquat)-induced cell death. Because the identification of SLC52A2 as a mediator of H2O2 was both novel and unexpected, we performed additional experiments to characterize the specificity and mechanism of its effect. These experiments showed that paralogs of SLC52A2 with lower riboflavin affinities could not mediate H2O2-induced cell death and that riboflavin depletion protected HAP1 cells from H2O2 toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H2O2 entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H2O2-induced cell death and also show for the first time that a vitamin can regulate membrane transport.IMPORTANCE Using a genetic screen, we discovered that riboflavin controls the entry of hydrogen peroxide into a white blood cell line. To our knowledge, this is the first report of a vitamin playing a role in controlling transport of a small molecule across the cell membrane.},
}
@article {pmid32786921,
year = {2020},
author = {Wei, TY and Wu, YJ and Xie, QP and Tang, JW and Yu, ZT and Yang, SB and Chen, SX},
title = {CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Glarea lozoyensis and Its Application in Manipulating gloF.},
journal = {ACS synthetic biology},
volume = {9},
number = {8},
pages = {1968-1977},
doi = {10.1021/acssynbio.9b00491},
pmid = {32786921},
issn = {2161-5063},
mesh = {Ascomycota/*genetics ; CRISPR-Cas Systems/*genetics ; Echinocandins/biosynthesis/chemistry ; Fungal Proteins/*genetics/metabolism ; Gene Editing/*methods ; Mutagenesis, Site-Directed ; Prolyl Hydroxylases/genetics/metabolism ; RNA, Guide/metabolism ; },
abstract = {Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B0, which is used for the synthesis of antifungal drug caspofungin. However, because of the limitations and complications of traditional genetic tools, G. lozoyensis strain engineering has been hindered. In this study, we established an efficient CRISPR/Cas9-based gene editing tool in G. lozoyensis SIPI1208. With this method, gene mutagenesis efficiency in the target locus can be up to 80%, which enables the rapid gene knockout. According to the reports, GloF and Ap-HtyE, proline hydroxylases involved in pneumocandin and Echinocandin B biosynthesis, respectively, can catalyze the proline to generate different ratios of trans-3-hydroxy-l-proline to trans-4-hydroxy-l-proline. Heterologous expression of Ap-HtyE in G. lozoyensis decreased the ratio of pneumocandin C0 to (pneumocandin B0 + pneumocandin C0) from 33.5% to 11% without the addition of proline to the fermentation medium. Furthermore, the gloF was replaced by ap-htyE to study the production of pneumocandin C0. However, the gene replacement has been hampered by traditional gene tools since gloF and gloG, two contiguous genes indispensable in the biosynthesis of pneumocandins, are cotranscribed into one mRNA. With the CRISPR/Cas9 strategy, ap-htyE was knocked in and successfully replaced gloF, and results showed that the knock-in strain retained the ability to produce pneumocandin B0, but the production of pneumocandin C0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B0. In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis.},
}
@article {pmid32786832,
year = {2020},
author = {Guo, D and Ling, X and Zhou, X and Li, X and Wang, J and Qiu, S and Yang, Y and Zhang, B},
title = {Evaluation of the Quality of a High-Resistant Starch and Low-Glutelin Rice (Oryza sativa L.) Generated through CRISPR/Cas9-Mediated Targeted Mutagenesis.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {36},
pages = {9733-9742},
doi = {10.1021/acs.jafc.0c02995},
pmid = {32786832},
issn = {1520-5118},
mesh = {Alleles ; CRISPR-Cas Systems ; Food, Genetically Modified ; Glutens/*analysis/metabolism ; Mutagenesis ; Oryza/chemistry/*genetics/metabolism ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/*chemistry/genetics/metabolism ; },
abstract = {A high-resistant starch (RS) and low-glutelin diet is beneficial for the health of patients with diabetes and kidney diseases. Rice is an important food crop worldwide. Previous studies have demonstrated that downregulating the expression of rice starch branching enzyme IIb (SBEIIb) affected the composition and the structure of starch. However, there has been no report about generating the loss-of-function mutants of SBEIIb using low-glutelin rice cultivars as recipients. In this study, we adopted a CRISPR/Cas9 system to induce site-specific mutations at the SBEIIb locus in an elite low-glutelin japonica rice cultivar derived from Low Glutelin Content-1 (LGC-1) and successfully obtained two independent transgene-free sbeIIb/Lgc1 mutant lines. In the mutant lines, the apparent amylose content (AAC) was increased by approximately 1.8-fold and the RS content reached approximately 6%. The glutelin content was approximately 2%, maintaining the low-glutelin trait of the recipient cultivar. The formation mechanism of RS was explored by analyzing the fine structures and the properties of starch. According to the X-ray diffraction pattern and the increased lipid content, the high RS content of the sbeIIb/Lgc1 lines was attributed to the increased content of amylose-lipid complex. Further analyses of the nutritional quality revealed that the soluble sugar and lipid contents, especially sucrose and unsaturated fatty acids, increased in the sbeIIb/Lgc1 lines significantly. This research is expected to facilitate the cultivation and the application of functional rice suitable for patients with diabetes and kidney diseases.},
}
@article {pmid32786353,
year = {2020},
author = {Chae, D and Lee, J and Lee, N and Park, K and Moon, SJ and Kim, HH},
title = {Chemical Controllable Gene Drive in Drosophila.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2362-2377},
doi = {10.1021/acssynbio.0c00117},
pmid = {32786353},
issn = {2161-5063},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Drosophila/*genetics/metabolism ; Female ; Gene Drive Technology/*methods ; Gene Expression Regulation/*drug effects ; Male ; Mifepristone/*pharmacology ; Models, Genetic ; RNA, Guide/metabolism ; Recombinases/genetics/metabolism ; },
abstract = {Gene drive systems that propagate transgenes via super-Mendelian inheritance can potentially control insect-borne diseases and agricultural pests. However, concerns have been raised regarding unforeseen ecological consequences, and methods that prevent undesirable gene drive effects have been proposed. Here, we report a chemical-induced control of gene drive. We prepared a CRISPR-based gene drive system that can be removed by a site-specific recombinase, Rippase, the expression of which is induced by the chemical RU486 in fruit flies. Exposure of fruit flies to RU486 resulted in 7-12% removal of gene drive elements at each generation, leading to a significant reduction in gene drive-fly propagation. Mathematical modeling and simulation suggest that our system offers several advantages over a previously reported gene drive control system. Our chemical control system can provide a proof-of-principle for the reversible control of gene drive effects depending on ecological status and human needs.},
}
@article {pmid32786350,
year = {2020},
author = {Brady, JR and Tan, MC and Whittaker, CA and Colant, NA and Dalvie, NC and Love, KR and Love, JC},
title = {Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2515-2524},
pmid = {32786350},
issn = {2161-5063},
support = {P30 CA014051/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin Immunoprecipitation Sequencing/*methods ; DNA, Intergenic/*genetics ; Gene Editing ; Granulocyte Colony-Stimulating Factor/genetics/metabolism ; Human Growth Hormone/genetics/metabolism ; Saccharomycetales/genetics/metabolism ; },
abstract = {Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data.},
}
@article {pmid32786349,
year = {2020},
author = {Borsenberger, V and Croux, C and Daboussi, F and Neuvéglise, C and Bordes, F},
title = {Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2562-2575},
doi = {10.1021/acssynbio.0c00325},
pmid = {32786349},
issn = {2161-5063},
mesh = {Acyl-CoA Oxidase/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Plasmids/genetics/metabolism ; RNA, Guide/metabolism ; Yarrowia/enzymology ; },
abstract = {CRISPR/Cas9 is a powerful tool to edit the genome of the yeast Yarrowia lipolytica. Here, we design a simple and robust method to knockout multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterized acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. We establish a correlation between the high lethality observed upon editing multiple loci and chromosomal translocations resulting from the simultaneous generation of several double-strand breaks (DSBs) and develop multiplex gene editing strategies. Using homologous directed recombination to reduce chromosomal translocations, we demonstrated that simultaneous editing of four genes can be achieved and constructed a strain carrying a sextuple deletion of POX genes. We explore an "excision approach" by simultaneously performing two DSBs in genes and reached 73 to 100% editing efficiency in double disruptions and 41.7% in a triple disruption. This work led to identifying SPS193 as a gene encoding a 2-4 dienoyl-CoA reductase, demonstrating the potential of this method to accelerate knowledge on gene function in expanded gene families.},
}
@article {pmid32786346,
year = {2020},
author = {Tao, D and Liu, J and Nie, X and Xu, B and Tran-Thi, TN and Niu, L and Liu, X and Ruan, J and Lan, X and Peng, G and Sun, L and Ma, Y and Li, X and Li, C and Zhao, S and Xie, S},
title = {Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2339-2350},
doi = {10.1021/acssynbio.0c00057},
pmid = {32786346},
issn = {2161-5063},
mesh = {African Swine Fever Virus/genetics/*isolation &amp; purification ; Animals ; CRISPR-Cas Systems/*genetics ; DNA/chemistry ; DNA, Viral/*analysis/metabolism ; Fluorescent Dyes/chemistry ; Limit of Detection ; Nucleic Acid Amplification Techniques/*methods ; RNA, Guide/genetics/metabolism ; Swine/genetics/*virology ; },
abstract = {African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/μL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.},
}
@article {pmid32786122,
year = {2020},
author = {Weiss, T and Wang, C and Kang, X and Zhao, H and Elena Gamo, M and Starker, CG and Crisp, PA and Zhou, P and Springer, NM and Voytas, DF and Zhang, F},
title = {Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.},
journal = {The Plant journal : for cell and molecular biology},
volume = {104},
number = {3},
pages = {828-838},
doi = {10.1111/tpj.14949},
pmid = {32786122},
issn = {1365-313X},
mesh = {*CRISPR-Cas Systems ; Exodeoxyribonucleases/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome, Plant ; Mutagenesis ; Mutation ; Plant Proteins/genetics ; Plants, Genetically Modified ; Protoplasts/physiology ; Setaria Plant/*genetics ; },
abstract = {In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.},
}
@article {pmid32785241,
year = {2020},
author = {Ashokkumar, S and Jaganathan, D and Ramanathan, V and Rahman, H and Palaniswamy, R and Kambale, R and Muthurajan, R},
title = {Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing.},
journal = {PloS one},
volume = {15},
number = {8},
pages = {e0237018},
pmid = {32785241},
issn = {1932-6203},
mesh = {Alleles ; Betaine-Aldehyde Dehydrogenase/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genes, Plant/genetics ; Genome, Plant/genetics ; Mutation/genetics ; Odorants/analysis ; Oryza/*genetics ; Phenotype ; Plants, Genetically Modified/genetics ; },
abstract = {Fragrance in rice grains is a key quality trait determining its acceptability and marketability. Intensive research on rice aroma identified mutations in betaine aldehyde dehydrogenase (OsBADH2) leading to production of aroma in rice. Gene editing technologies like CRISPR/Cas9 system has opened new avenues for accelerated improvement of rice grain quality through targeted mutagenesis. In this study, we have employed CRISPR/Cas9 tool to create novel alleles of OsBADH2 leading to introduction of aroma into an elite non-aromatic rice variety ASD16. PCR analysis of putative transformants using primers targeting the flanking regions of sgRNA in the 7th exon of OsBADH2 identified 37.5% potential multi-allelic mutations in T0 generation. Sensory evaluation test in the leaves of T0 lines identified thirteen lines belonging to five independent events producing aroma. Sequence analysis of these aromatic T0 lines identified 22 different types of mutations located within -17 bp to +15bp of sgRNA region. The -1/-2 bp deletion in the line # 8-19 and -8/-5 bp deletion in the line # 2-16 produced strong aroma and the phenotype was stably inherited in the T1 generation. Comparative volatile profiling detected novel aromatic compounds viz., pyrrolidine, pyridine, pyrazine, pyradazine and pyrozole in the grains of T1 progenies of line # 8-19. This study has demonstrated the use of CRISPR/Cas9 in creating novel alleles of OsBADH2 to introduce aroma into any non-aromatic rice varieties.},
}
@article {pmid32785089,
year = {2020},
author = {Moscoso, CG and Steer, CJ},
title = {The Evolution of Gene Therapy in the Treatment of Metabolic Liver Diseases.},
journal = {Genes},
volume = {11},
number = {8},
pages = {},
pmid = {32785089},
issn = {2073-4425},
mesh = {Animals ; Biomarkers ; Brain Diseases, Metabolic/*genetics/*therapy ; CRISPR-Cas Systems ; Clinical Studies as Topic ; Disease Management ; Drug Evaluation, Preclinical ; Gene Editing ; Gene Expression ; Gene Transfer Techniques ; Genetic Predisposition to Disease ; *Genetic Therapy/methods ; Genetic Vectors/classification/genetics ; Humans ; Liver Diseases/*genetics/*therapy ; Organ Specificity/genetics ; Transduction, Genetic ; Treatment Outcome ; },
abstract = {Monogenic metabolic disorders of hepatic origin number in the hundreds, and for many, liver transplantation remains the only cure. Liver-targeted gene therapy is an attractive treatment modality for many of these conditions, and there have been significant advances at both the preclinical and clinical stages. Viral vectors, including retroviruses, lentiviruses, adenovirus-based vectors, adeno-associated viruses and simian virus 40, have differing safety, efficacy and immunogenic profiles, and several of these have been used in clinical trials with variable success. In this review, we profile viral vectors and non-viral vectors, together with various payloads, including emerging therapies based on RNA, that are entering clinical trials. Genome editing technologies are explored, from earlier to more recent novel approaches that are more efficient, specific and safe in reaching their target sites. The various curative approaches for the multitude of monogenic hepatic metabolic disorders currently at the clinical development stage portend a favorable outlook for this class of genetic disorders.},
}
@article {pmid32785008,
year = {2020},
author = {Yu, HC and Tung, CH and Huang, KY and Huang, HB and Lu, MC},
title = {The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage.},
journal = {International journal of molecular sciences},
volume = {21},
number = {16},
pages = {},
pmid = {32785008},
issn = {1422-0067},
mesh = {Anti-Citrullinated Protein Antibodies/blood ; Apoptosis/drug effects/*genetics ; Arthritis, Rheumatoid/blood ; Bodily Secretions/*metabolism ; CRISPR-Cas Systems ; Cell Adhesion/*genetics ; Cytokines/genetics/*metabolism ; Gene Knockout Techniques ; Humans ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/*metabolism ; Protein-Arginine Deiminase Type 2/genetics/*metabolism ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; Signal Transduction/drug effects/genetics ; Transcription Factor RelA/metabolism ; U937 Cells ; },
abstract = {OBJECTIVE: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages.<br><br>METHODS: The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1&#x3b2;), IL-6, and tumor necrosis factor-alpha (TNF-&#x3b1;) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate.<br><br>RESULTS: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor &#x3ba;B (NF-&#x3ba;B) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1&#x3b2; and TNF-&#x3b1; in the U937 cells and IL-1&#x3b2; and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1&#x3b2;, IL-6, and TNF-&#x3b1; were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1.<br><br>CONCLUSION: This study showed that PADI2 could promote IL-1&#x3b2;, IL-6, and TNF-&#x3b1; production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.},
}
@article {pmid32784796,
year = {2020},
author = {Ulbricht, A and Nickel, L and Weidenbach, K and Vargas Gebauer, H and Kießling, C and Förstner, KU and Schmitz, RA},
title = {The CARF Protein MM_0565 Affects Transcription of the Casposon-Encoded cas1-solo Gene in Methanosarcina mazei Gö1.},
journal = {Biomolecules},
volume = {10},
number = {8},
pages = {},
pmid = {32784796},
issn = {2218-273X},
support = {SCHM1052/12-2; SCHM1052/21-1//Deutsche Forschungsgemeinschaft/International ; },
mesh = {Amino Acid Motifs/genetics ; CRISPR-Associated Proteins/*chemistry/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression Regulation, Archaeal/genetics ; Methanosarcina/chemistry/*genetics/metabolism ; Open Reading Frames/genetics ; Promoter Regions, Genetic ; Protein Binding ; Protein Folding ; Protein Multimerization/genetics ; RNA-Seq ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 associated to the type I-B CRISPR-locus of Methanosarcina mazei Gö1. We show that purified MM_0565 composed of a CRISPR-Cas Associated Rossmann Fold (CARF) and a winged helix-turn-helix domain forms a dimer in solution; in vivo, the dimeric MM_0565 is strongly stabilized under high salt stress. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, specific binding of MM_0565 to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Moreover, overexpression of MM_0565 strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings, and taking the absence of the expressed CRISPR locus-encoded Cas1 protein into account, we hypothesize that MM_0565 might modulate the activity of the CRISPR systems on different levels.},
}
@article {pmid32784649,
year = {2020},
author = {Ahmar, S and Saeed, S and Khan, MHU and Ullah Khan, S and Mora-Poblete, F and Kamran, M and Faheem, A and Maqsood, A and Rauf, M and Saleem, S and Hong, WJ and Jung, KH},
title = {A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement.},
journal = {International journal of molecular sciences},
volume = {21},
number = {16},
pages = {},
pmid = {32784649},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Endonucleases/metabolism ; Gene Editing/*methods ; Genome, Plant ; Plant Breeding ; },
abstract = {Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist's mind, as it allows genome editing in multiple biological systems.},
}
@article {pmid32783992,
year = {2020},
author = {Ray, U and Vartak, SV and Raghavan, SC},
title = {NHEJ inhibitor SCR7 and its different forms: Promising CRISPR tools for genome engineering.},
journal = {Gene},
volume = {763},
number = {},
pages = {144997},
doi = {10.1016/j.gene.2020.144997},
pmid = {32783992},
issn = {1879-0038},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Ligase ATP/antagonists &amp; inhibitors ; Enzyme Inhibitors/chemistry/*pharmacology ; Gene Editing/*methods ; Humans ; Pyrimidines/chemistry/*pharmacology ; Recombinational DNA Repair/*drug effects ; Schiff Bases/chemistry/*pharmacology ; },
abstract = {The CRISPR-Cas system currently stands as one of the best multifaceted tools for site-specific genome engineering in mammals. An important aspect of research in this field focusses on improving the specificity and efficacy of precise genome editing in multiple model systems. The cornerstone of this mini-review is one of the extensively investigated small molecule inhibitor, SCR7, which abrogates NHEJ, a Ligase IV-dependent DSB repair pathway, thus guiding integration of the foreign DNA fragment via the more precise homology directed repair during genome editing. One of our recent studies sheds light on properties of different forms of SCR7. Here, we give a succinct account on the use of SCR7 and its different forms in CRISPR-Cas system, highlighting their chemical properties and biological relevance as potent efficiency-enhancing CRISPR tools.},
}
@article {pmid32783942,
year = {2020},
author = {Zhou, P and Chan, BKC and Wan, YK and Yuen, CTL and Choi, GCG and Li, X and Tong, CSW and Zhong, SSW and Sun, J and Bao, Y and Mak, SYL and Chow, MZY and Khaw, JV and Leung, SY and Zheng, Z and Cheung, LWT and Tan, K and Wong, KH and Chan, HYE and Wong, ASL},
title = {A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets.},
journal = {Cell reports},
volume = {32},
number = {6},
pages = {108020},
doi = {10.1016/j.celrep.2020.108020},
pmid = {32783942},
issn = {2211-1247},
mesh = {Animals ; Antineoplastic Agents/pharmacology ; *CRISPR-Cas Systems ; Drosophila melanogaster ; *Drug Combinations ; Drug Delivery Systems/*methods ; Gene Knockout Techniques ; HEK293 Cells ; High-Throughput Screening Assays/*methods ; Humans ; Mice ; Neoplasms/drug therapy ; Parkinson Disease/drug therapy ; RNA, Guide ; },
abstract = {We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.},
}
@article {pmid32783656,
year = {2020},
author = {Fredette, NC and Malik, E and Mukhtar, ML and Prossnitz, ER and Terada, N},
title = {A hypertension patient-derived iPSC model demonstrates a role for G protein-coupled estrogen receptor in hypertension risk and development.},
journal = {American journal of physiology. Cell physiology},
volume = {319},
number = {5},
pages = {C825-C838},
pmid = {32783656},
issn = {1522-1563},
support = {GM119977//HHS | National Institutes of Health (NIH)/International ; P30 CA118100/CA/NCI NIH HHS/United States ; R01 CA163890/CA/NCI NIH HHS/United States ; P20 GM121176/GM/NIGMS NIH HHS/United States ; UL1 TR001427/TR/NCATS NIH HHS/United States ; UL1TR001427//Univ of FL Clinical and Translational Sicences Institute/International ; T32 DK074367/DK/NIDDK NIH HHS/United States ; R01 CA194496/CA/NCI NIH HHS/United States ; DK104194//HHS | National Institutes of Health (NIH)/International ; },
mesh = {Adult ; Antigens, CD/genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems ; Cadherins/genetics/metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Engineering/methods ; Endothelial Cells/*metabolism/pathology ; Female ; Gene Editing/methods ; Gene Expression Regulation ; Humans ; Hypertension/*genetics/metabolism/physiopathology ; Induced Pluripotent Stem Cells/*metabolism/pathology ; Models, Biological ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/genetics/metabolism ; Platelet Endothelial Cell Adhesion Molecule-1/genetics/metabolism ; *Polymorphism, Single Nucleotide ; Primary Cell Culture ; Receptors, Estrogen/deficiency/*genetics ; Receptors, G-Protein-Coupled/deficiency/*genetics ; Risk Factors ; THP-1 Cells ; von Willebrand Factor/genetics/metabolism ; },
abstract = {Hypertension (HTN) is a polyfactorial disease that can manifest severe cardiovascular pathologies such as heart failure or stroke. Genome-wide association studies (GWAS) of HTN indicate that single-nucleotide polymorphisms (SNPs) contribute to increased risk for HTN and resistance to some HTN drug regimens (Hiltunen TP et al., J Am Heart Assoc 4: e001521, 2015; Le MT et al., PLoS One 8: e52062, 2013; McDonough CW et al., J Hypertens 31: 698-704, 2013; Vandell AG et al., Hypertension 60: 957-964, 2012). However, cellular mechanistic insights of such SNPs remain largely unknown. Using a bank of induced pluripotent stem cells (iPSCs) derived from patients with HTN and CRISPR/Cas9-mediated gene-editing approach, we investigated the effects of a female HTN risk-associated SNP (rs1154431) of the G protein-coupled estrogen receptor (GPER) (Bassuk SS, Manson JE., Clin Chem 60: 68-77, 2014) in vascular endothelial cells. Although GPER1 deletion reduced endothelial nitric oxide synthase (eNOS) activation in iPSC-derived endothelial cells (iECs), the polymorphism itself did not significantly affect eNOS and NO production in a comparison of isogenic hemizygous iECs expressing either normal (P16) or HTN-associated (L16) GPER. Interestingly, we demonstrate for the first time that GPER plays a role in regulation of adhesion molecule expression and monocyte adhesion to iECs. Moreover, the L16 iECs had higher expression of inflammation genes than P16 iECs, implying that the risk variant may affect carrier individuals through increased inflammatory activity. This study further indicates that iPSCs are a useful platform for exploring mechanistic insights underlying hypertension GWAS endeavors.},
}
@article {pmid32783355,
year = {2020},
author = {Collins, RT and Coxam, B and Fechner, I and Unterweger, I and Szymborska, A and Meier, K and Gerhardt, H},
title = {Intron with transgenic marker (InTraM) facilitates high-throughput screening of endogenous gene reporter lines.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {10-11},
pages = {e23391},
doi = {10.1002/dvg.23391},
pmid = {32783355},
issn = {1526-968X},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; *Genes, Reporter ; High-Throughput Screening Assays/*methods ; Transcription Factors/genetics ; Transgenes ; Zebrafish ; Zebrafish Proteins/genetics ; },
abstract = {The generation and maintenance of genome edited zebrafish lines is typically labor intensive due to the lack of an easy visual read-out for the modification. To facilitate this process, we have developed a novel method that relies on the inclusion of an artificial intron with a transgenic marker (InTraM) within the knock-in sequence of interest, which upon splicing produces a transcript with a precise and seamless modification. We have demonstrated this technology by replacing the stop codon of the zebrafish fli1a gene with a transcriptional activator KALTA4, using an InTraM that enables red fluorescent protein expression in the heart.},
}
@article {pmid32782385,
year = {2020},
author = {Schuster, F and Aldag, P and Frenzel, A and Hadeler, KG and Lucas-Hahn, A and Niemann, H and Petersen, B},
title = {CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13570},
pmid = {32782385},
issn = {2045-2322},
mesh = {Animals ; *Breeding ; CRISPR-Cas Systems/*genetics ; Cattle ; DNA-Binding Proteins/*genetics ; Dairying ; Female ; Fibroblasts/*metabolism ; Genotype ; Horns/*physiology ; Male ; *Mutation ; Phenotype ; *Polymorphism, Single Nucleotide ; Pregnancy ; },
abstract = {In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein-Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.},
}
@article {pmid32782248,
year = {2020},
author = {Kudo, K and Hashimoto, T and Hashimoto, J and Kozone, I and Kagaya, N and Ueoka, R and Nishimura, T and Komatsu, M and Suenaga, H and Ikeda, H and Shin-Ya, K},
title = {In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4022},
pmid = {32782248},
issn = {2041-1723},
mesh = {Biological Products/chemistry/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Molecular Structure ; Multigene Family/genetics ; Polyketide Synthases/*genetics/metabolism ; Sirolimus/chemistry/metabolism ; Stereoisomerism ; Streptomyces/enzymology/genetics/metabolism ; },
abstract = {One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.},
}
@article {pmid32782000,
year = {2020},
author = {North, AR and Burt, A and Godfray, HCJ},
title = {Modelling the suppression of a malaria vector using a CRISPR-Cas9 gene drive to reduce female fertility.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {98},
pmid = {32782000},
issn = {1741-7007},
mesh = {Africa, Western ; Animals ; Animals, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Culicidae/enzymology/*genetics ; Endonucleases/*chemistry ; Female ; Fertility/genetics ; *Gene Drive Technology ; Gene Targeting ; Insect Proteins/chemistry ; Malaria/*prevention &amp; control ; Models, Genetic ; Mosquito Control/*methods ; Mosquito Vectors/*genetics ; },
abstract = {BACKGROUND: Gene drives based on CRISPR-Cas9 technology are increasingly being considered as tools for reducing the capacity of mosquito populations to transmit malaria, and one of the most promising options is driving endonuclease genes that reduce the fertility of female mosquitoes. In particular, there is much interest in constructs that target the conserved mosquito doublesex (dsx) gene such that the emergence of functional drive-resistant alleles is unlikely. Proof of principle that these constructs can lead to substantial population suppression has been obtained in population cages, and they are being evaluated for use in sub-Saharan Africa. Here, we use simulation modelling to understand the factors affecting the spread of this type of gene drive over a one million-square kilometre area of West Africa containing substantial environmental and social heterogeneity.<br><br>RESULTS: We found that a driving endonuclease gene targeting female fertility could lead to substantial reductions in malaria vector populations on a regional scale. The exact level of suppression is influenced by additional fitness costs of the transgene such as the somatic expression of Cas9, and its deposition in sperm or eggs leading to damage to the zygote. In the absence of these costs, or of emergent drive-resistant alleles that restore female fertility, population suppression across the study area is predicted to stabilise at ~&#x2009;95% 4&#xa0;years after releases commence. Small additional fitness costs do not greatly affect levels of suppression, though if the fertility of females whose offspring transmit the construct drops by more than ~&#x2009;40%, then population suppression is much less efficient. We show the suppression potential of a drive allele with high fitness costs can be enhanced by engineering it also to express male bias in the progeny of transgenic males. Irrespective of the strength of the drive allele, the spatial model predicts somewhat less suppression than equivalent non-spatial models, in particular in highly seasonal regions where dry season stochasticity reduces drive efficiency. We explored the robustness of these results to uncertainties in mosquito ecology, in particular their method of surviving the dry season and their dispersal rates.<br><br>CONCLUSIONS: The modelling presented here indicates that considerable suppression of vector populations can be achieved within a few years of using a female sterility gene drive, though the impact is likely to be heterogeneous in space and time.},
}
@article {pmid32781607,
year = {2020},
author = {Szillat, KP and Koethe, S and Wernike, K and Höper, D and Beer, M},
title = {A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line-A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV).},
journal = {Viruses},
volume = {12},
number = {8},
pages = {},
pmid = {32781607},
issn = {1999-4915},
mesh = {Adaptation, Biological ; Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cattle ; Diarrhea Virus 1, Bovine Viral/genetics/*physiology ; Diarrhea Virus 2, Bovine Viral/genetics/*physiology ; Dogs ; Gene Knockout Techniques ; Host-Pathogen Interactions ; Madin Darby Canine Kidney Cells ; Membrane Cofactor Protein/*genetics/metabolism ; Protein Multimerization ; Receptors, Virus/*genetics/metabolism ; Ribonucleoproteins/metabolism ; Viral Envelope Proteins/chemistry/*genetics/metabolism ; Virus Internalization ; Virus Replication ; },
abstract = {Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the E[RNS] in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the E[RNS] resulted in a loss of E[RNS] dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.},
}
@article {pmid32778842,
year = {2020},
author = {Mathiasen, S and Palmisano, T and Perry, NA and Stoveken, HM and Vizurraga, A and McEwen, DP and Okashah, N and Langenhan, T and Inoue, A and Lambert, NA and Tall, GG and Javitch, JA},
title = {G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.},
journal = {Nature chemical biology},
volume = {16},
number = {12},
pages = {1343-1350},
pmid = {32778842},
issn = {1552-4469},
support = {F31 HL152563/HL/NHLBI NIH HHS/United States ; R01 GM120110/GM/NIGMS NIH HHS/United States ; R01 NS103946/NS/NINDS NIH HHS/United States ; F30 GM131672/GM/NIGMS NIH HHS/United States ; R01 GM130142/GM/NIGMS NIH HHS/United States ; R21 MH112156/MH/NIMH NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; },
mesh = {Activating Transcription Factor 6/agonists/chemistry/genetics/*metabolism ; Animals ; Arrestin/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Cell Engineering ; GTP-Binding Protein alpha Subunits, G12-G13/chemistry/genetics/*metabolism ; GTP-Binding Protein alpha Subunits, Gq-G11/chemistry/genetics/metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Kinetics ; Mice ; Mitogen-Activated Protein Kinase 1/chemistry/genetics/metabolism ; Mitogen-Activated Protein Kinase 3/chemistry/genetics/metabolism ; Peptides/chemistry/*metabolism/pharmacology ; Protein Binding ; Receptors, G-Protein-Coupled/chemistry/genetics/*metabolism ; Receptors, Peptide/chemistry/genetics/*metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Signal Transduction ; },
abstract = {The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.},
}
@article {pmid32778571,
year = {2020},
author = {Yang, CH and Li, HC and Ku, TS and Wu, CH and Sim, KC and Lo, SY},
title = {MicroRNA-Independent Modulation of DICER1 Expression by hAgo2.},
journal = {Molecular and cellular biology},
volume = {40},
number = {20},
pages = {},
pmid = {32778571},
issn = {1098-5549},
mesh = {3' Untranslated Regions/genetics ; A549 Cells ; Argonaute Proteins/*genetics/metabolism ; Binding Sites/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; DEAD-box RNA Helicases/*genetics/metabolism ; Gene Expression Regulation, Neoplastic/*genetics ; HEK293 Cells ; HeLa Cells ; Heterogeneous Nuclear Ribonucleoprotein D0/genetics/metabolism ; Humans ; MicroRNAs/*genetics ; Neoplasms/*genetics ; RNA Interference ; RNA, Messenger/genetics ; RNA, Small Interfering/genetics ; Ribonuclease III/*genetics/metabolism ; },
abstract = {Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.},
}
@article {pmid32778162,
year = {2020},
author = {Aslam, B and Rasool, M and Idris, A and Muzammil, S and Alvi, RF and Khurshid, M and Rasool, MH and Zhang, D and Ma, Z and Baloch, Z},
title = {CRISPR-Cas system: a potential alternative tool to cope antibiotic resistance.},
journal = {Antimicrobial resistance and infection control},
volume = {9},
number = {1},
pages = {131},
pmid = {32778162},
issn = {2047-2994},
mesh = {*Anti-Bacterial Agents ; Bacteria/*genetics ; *CRISPR-Cas Systems ; Drug Resistance, Microbial/*genetics ; Humans ; Virulence/genetics ; },
abstract = {Antibiotic exposure leads to massive selective pressures that initiate the emergence and spread of antibiotic resistance in commensal and pathogenic bacteria. The slow process of developing new antibiotics makes this approach counterintuitive for combatting the rapid emergence of new antibiotic resistant pathogens. Therefore, alternative approaches such as, the development of nucleic acid-based anti-bacterial treatments, anti-bacterial peptides, bacteriocins, anti-virulence compounds and bacteriophage therapies should be exploited to cope infections caused by resistant superbugs. In this editorial, we discuss how the newly popular CRISPR-Cas system has been applied to combat antibiotic resistance.},
}
@article {pmid32777770,
year = {2020},
author = {Lee, M and Choi, NY and Park, S and Bang, JS and Lee, Y and Jeong, D and Ham, S and Lim, S and Kim, KH and Ko, K},
title = {Generation of OCT4-EGFP, NANOG-tdTomato dual reporter human induced pluripotent stem cell line, KKUi001-A, using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101943},
doi = {10.1016/j.scr.2020.101943},
pmid = {32777770},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cellular Reprogramming ; Homeodomain Proteins/genetics/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Luminescent Proteins ; Nanog Homeobox Protein/genetics ; Octamer Transcription Factor-3/genetics/metabolism ; },
abstract = {OCT4 and NANOG are core transcription factor genes in self-renewal, differentiation, and reprogramming. Here, we generated an OCT4-EGFP, NANOG-tdTomato dual reporter hiPSC line, KKUi001-A, on the basis of human induced pluripotent stem cells using CRISPR/Cas9 technology. EGFP and tdTomato reporter were inserted into before the stop codon of OCT4 and NANOG, respectively. Simultaneous expression of EGFP and tdTomato was observed when expression of OCT4 and NANOG was changed during differentiation and reprogramming. KKUi001-A hiPSC line will be a useful tool to find initial time point of OCT4 and NANOG expression during reprogramming process and to screen small molecules that promote reprogramming.},
}
@article {pmid32777767,
year = {2020},
author = {Wu, F and Li, X and Bai, R and Li, Y and Gao, J and Lan, F},
title = {Generation of a Junctophilin-2 homozygous knockout human embryonic stem cell line (WAe009-A-36) by an episomal vector-based CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101930},
doi = {10.1016/j.scr.2020.101930},
pmid = {32777767},
issn = {1876-7753},
support = {MOP142265//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Human Embryonic Stem Cells ; Humans ; Membrane Proteins/genetics ; },
abstract = {Mutations in Junctophilin-2(JPH2) gene is the cause of hypertrophic cardiomyopathy (HCM) and leading inherited cause of left ventricular hypertrophy and myofilaments disarray. JPH2 protein, a member of the Junctophilin family, is mainly expressed in heart and plays an important role in E-C coupling. We have generated a homozygous JPH2 knockout (JPH2-KO) human embryonic stem cell (hESC) line using an episomal vector-based CRISPR/Cas9 system. This JPH2-KO hESC line maintained stem cell like morphology, pluripotency, normal karyotype and could differentiate into all three germ layers in vivo.},
}
@article {pmid32777177,
year = {2021},
author = {Pieczynski, JN and Kee, HL},
title = {"Designer babies?!" A CRISPR-based learning module for undergraduates built around the CCR5 gene.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {49},
number = {1},
pages = {80-93},
pmid = {32777177},
issn = {1539-3429},
mesh = {Biotechnology/education ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Curriculum ; DNA/genetics ; Female ; *Gene Editing ; *Genetic Therapy ; Humans ; *Learning ; Mutation ; Receptors, CCR5/*genetics ; Students ; Twins/genetics ; Universities ; },
abstract = {CRISPR-cas technology is being incorporated into undergraduate biology curriculum through lab experiences to immerse students in modern technology that is rapidly changing the landscape of science, medicine and agriculture. We developed and implemented an educational module that introduces students to CRISPR-cas technology in a Genetic course and an Advanced Genetics course. Our primary teaching objective was to immerse students in the design, strategy, conceptual modeling, and application of CRISPR-cas technology using the current research claim of the modification of the CCR5 gene in twin girls. This also allowed us to engage students in an open conversation about the bioethical implications of heritable germline and non-heritable somatic genomic editing. We assessed student-learning outcomes and conclude that this learning module is an effective strategy for teaching undergraduates the fundamentals and application of CRISPR-cas gene editing technology and can be adapted to other genes and diseases that are currently being treated with CRISPR-cas technology.},
}
@article {pmid32776087,
year = {2021},
author = {Zhu, Y and Groth, T and Kelkar, A and Zhou, Y and Neelamegham, S},
title = {A GlycoGene CRISPR-Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways.},
journal = {Glycobiology},
volume = {31},
number = {3},
pages = {173-180},
pmid = {32776087},
issn = {1460-2423},
support = {R01 HL103411/HL/NHLBI NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems/*genetics ; Gene Library ; Glycosylation ; HL-60 Cells ; Humans ; Lectins/chemistry/*metabolism ; Polysaccharides/*biosynthesis ; },
abstract = {Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes and proteins including the following: i. glycosyltransferases that add saccharides; ii. glycosidases that trim glycans; iii. conserved oligomeric golgi complex members that regulate intracellular transport; iv. enzymes aiding the biosynthesis of sugar-nucleotides; and v. sulfotransferases. This manuscript describes a pooled "glycoGene CRISPR" lentiviral library that targets 347 human genes involved in the above processes. Approximately 10 single-guide RNA (sgRNA) are included against each glycogene, with the putative editing site spanning the length of the target. A data analysis scheme is presented in order to determine glycosylation pathways regulating biological processes. As proof of principle, forward genetic screen results are presented to identify penetrating glycogenes that regulate the binding of P-/E-selectin, anti-sialyl Lewis-X monoclonal antibody HECA-452 and selected lectins (phaseolus vulgaris leucoagglutinin, vicia villosa lectin, peanut agglutinin) to HL-60 promyelocytic cells. Besides validating previously established biology, the study identifies three enzymes, PAPSS1, SLC35B2 and TPST2, as key molecules regulating sulfation of the major P-selectin glycoprotein ligand-1 in leukocytes. Approximately 80-90% of the sgRNA used in this study displayed high editing efficiency, and the CRISPR library picked up entire gene sets regulating specific biosynthetic pathways rather than only isolated genes. These data suggest that the glycoGene CRISPR library contains high-efficiency sgRNA. Further, this resource could be useful for the rapid screening of glycosylation-related genes and pathways that control lectin recognition in a variety of contexts.},
}
@article {pmid32775498,
year = {2020},
author = {Tran, NT and Heiner, C and Weber, K and Weiand, M and Wilmot, D and Xie, J and Wang, D and Brown, A and Manokaran, S and Su, Q and Zapp, ML and Gao, G and Tai, PWL},
title = {AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {18},
number = {},
pages = {639-651},
pmid = {32775498},
issn = {2329-0501},
support = {R01 NS076991/NS/NINDS NIH HHS/United States ; },
abstract = {The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.},
}
@article {pmid32772603,
year = {2020},
author = {Liang, JJH and McKinnon, IA and Rankin, CH},
title = {The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases.},
journal = {Journal of neurogenetics},
volume = {34},
number = {3-4},
pages = {527-548},
doi = {10.1080/01677063.2020.1803302},
pmid = {32772603},
issn = {1563-5260},
support = {PJT 165947//CIHR/Canada ; },
mesh = {Animals ; Animals, Genetically Modified ; Apoptosis ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/physiology ; Caenorhabditis elegans Proteins/metabolism ; *Disease Models, Animal ; Genes, Helminth ; Humans ; Mitochondria/metabolism ; Models, Neurological ; Nerve Tissue Proteins/physiology ; Neurodegenerative Diseases/*genetics ; Neurotransmitter Agents/physiology ; Proteostasis ; },
abstract = {Since Caenorhabditis elegans was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using C. elegans to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use C. elegans to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in C. elegans, allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution in vivo. Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease) in C. elegans and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.},
}
@article {pmid32771907,
year = {2020},
author = {Nur Patria, Y and Stenta, T and Lilianty, J and Rowley, L and Stanley, EG and Elefanty, AG and Bateman, JF and Lamandé, SR},
title = {CRISPR/Cas9 gene editing of a SOX9 reporter human iPSC line to produce two TRPV4 patient heterozygous missense mutant iPSC lines, MCRIi001-A-3 (TRPV4 p.F273L) and MCRIi001-A-4 (TRPV4 p.P799L).},
journal = {Stem cell research},
volume = {48},
number = {},
pages = {101942},
doi = {10.1016/j.scr.2020.101942},
pmid = {32771907},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; SOX9 Transcription Factor ; TRPV Cation Channels/genetics ; },
abstract = {To produce in vitro models of human chondrodysplasias caused by dominant missense mutations in TRPV4, we used CRISPR/Cas9 gene editing to introduce two heterozygous patient mutations (p.F273L and p.P799L) into an established control human iPSC line. This control line expressed a fluorescent reporter (tdTomato) at the SOX9 locus to allow real-time monitoring of cartilage differentiation by SOX9 expression. Both TRPV4 mutant iPSC lines had normal karyotypes, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. These iPSC lines, with the parental isogenic control, will be used to study TRPV4 chondrodysplasia mechanisms and explore therapeutic approaches.},
}
@article {pmid32771627,
year = {2020},
author = {Schilling, C and Ciccone, R and Sieber, V and Schmid, J},
title = {Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365.},
journal = {Metabolic engineering},
volume = {61},
number = {},
pages = {381-388},
doi = {10.1016/j.ymben.2020.07.009},
pmid = {32771627},
issn = {1096-7184},
mesh = {Butylene Glycols/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; *Metabolic Engineering ; *Paenibacillus polymyxa/genetics/metabolism ; },
abstract = {Paenibacillus polymyxa is a Gram-positive, non-pathogenic soil bacterium that has been extensively investigated for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers were restricted due to limited genetic accessibility of the organism up to now. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants were generated and compared in batch fermentations for the first time. Downstream processing was facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Spore formation was inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of P. polymyxa. Optimization of the carbon flux towards 2,3-butanediol was achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain showed 45 % increased productivity, reaching a final concentration of 43.8 g L[-1] butanediol. A yield of 0.43 g g[-1] glucose was achieved, accounting for 86 % of the theoretical maximum.},
}
@article {pmid32770110,
year = {2020},
author = {Eghbalsaied, S and Hyder, I and Kues, WA},
title = {A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13332},
pmid = {32770110},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA End-Joining Repair/genetics ; Electroporation/*methods ; Fibroblasts/*physiology ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Genes, Reporter/genetics ; Induced Pluripotent Stem Cells/*physiology ; Mice ; Plasmids/genetics ; RNA, Guide/genetics ; Transfection/*methods ; Transgenes/genetics ; },
abstract = {Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.},
}
@article {pmid32769197,
year = {2020},
author = {Huang, WP and Du, YJ and Yang, Y and He, JN and Lei, Q and Yang, XY and Zhang, KQ and Niu, XM},
title = {Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {20},
pages = {},
pmid = {32769197},
issn = {1098-5336},
mesh = {Adaptation, Physiological/genetics ; *CRISPR-Cas Systems ; Eurotiales/*genetics/metabolism ; Gene Editing ; *Genes, Fungal ; *Homologous Recombination ; RNA, Guide/*genetics ; },
abstract = {Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNA[Gly], were successfully adapted for genome editing in T. dupontii We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.IMPORTANCEThermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.},
}
@article {pmid32769181,
year = {2020},
author = {Davis, KA and Sampson, JK and Panaccione, DG},
title = {Genetic Reprogramming of the Ergot Alkaloid Pathway of Metarhizium brunneum.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {19},
pages = {},
pmid = {32769181},
issn = {1098-5336},
support = {R15 GM114774/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Ergot Alkaloids/*metabolism ; Metabolic Networks and Pathways/*genetics ; Metarhizium/*genetics/metabolism ; Microorganisms, Genetically-Modified/genetics/metabolism ; },
abstract = {Ergot alkaloids are important specialized fungal metabolites that are used to make potent pharmaceuticals for neurological diseases and disorders. Lysergic acid (LA) and dihydrolysergic acid (DHLA) are desirable lead compounds for pharmaceutical semisynthesis but are typically transient intermediates in the ergot alkaloid and dihydroergot alkaloid pathways. Previous work with Neosartorya fumigata demonstrated strategies to produce these compounds as pathway end products, but their percent yield (percentage of molecules in product state as opposed to precursor state) was low. Moreover, ergot alkaloids in N. fumigata are typically retained in the fungus as opposed to being secreted. We used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and heterologous expression approaches to engineer these compounds in Metarhizium brunneum, representing an alternate expression host from a different lineage of fungi. The relative percent yields of LA (86.9%) and DHLA (72.8%) were much higher than those calculated here for previously engineered strains of N. fumigata (2.6% and 2.0%, respectively). Secretion of these alkaloids also was measured, with averages of 98.4% of LA and 87.5% of DHLA being secreted into the growth medium; both values were significantly higher than those measured for the N. fumigata derivatives (both of which were less than 5.6% secreted). We used a similar approach to engineer a novel dihydroergot alkaloid in M. brunneum and, through high-performance liquid chromatography-mass spectrometry (LC-MS) analyses, provisionally identified it as the dihydrogenated form of lysergic acid α-hydroxyethylamide (dihydro-LAH). The engineering of these strains provides a strategy for producing novel and pharmaceutically important chemicals in a fungus more suitable for their production.IMPORTANCE Ergot alkaloids derived from LA or DHLA are the bases for numerous pharmaceuticals with applications in the treatment of dementia, migraines, hyperprolactinemia, and other conditions. However, extraction of ergot alkaloids from natural sources is inefficient, and their chemical synthesis is expensive. The ability to control and redirect ergot alkaloid synthesis in fungi may allow more efficient production of these important chemicals and facilitate research on novel derivatives. Our results show that Metarhizium brunneum can be engineered to efficiently produce and secrete LA and DHLA and, also, to produce a novel derivative of DHLA not previously found in nature. The engineering of dihydroergot alkaloids, including a novel species, is important because very few natural sources of these compounds are known. Our approach establishes a platform with which to use M. brunneum to study the production of other ergot alkaloids, specifically those classified as lysergic acid amides and dihydroergot alkaloids.},
}
@article {pmid32769113,
year = {2020},
author = {Reilly, MM and Rossor, AM},
title = {Humans: the ultimate animal models.},
journal = {Journal of neurology, neurosurgery, and psychiatry},
volume = {91},
number = {11},
pages = {1132-1136},
pmid = {32769113},
issn = {1468-330X},
support = {MR/S005021/1/MRC_/Medical Research Council/United Kingdom ; U54 NS065712/NS/NINDS NIH HHS/United States ; },
mesh = {Amyloid Neuropathies, Familial/genetics/metabolism/therapy ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans ; Charcot-Marie-Tooth Disease/genetics/metabolism/therapy ; Disease Models, Animal ; Drosophila ; Genetic Therapy ; High-Throughput Nucleotide Sequencing ; Human Experimentation ; Humans ; In Vitro Techniques ; Induced Pluripotent Stem Cells ; Longevity ; Mice ; Molecular Targeted Therapy ; Oligonucleotides, Antisense/therapeutic use ; Organ Size ; Peripheral Nerves/growth &amp; development ; Peripheral Nervous System Diseases/*genetics/metabolism/*therapy ; Primates ; RNA, Small Interfering/therapeutic use ; Rats ; Rodentia ; Whole Exome Sequencing ; Whole Genome Sequencing ; Zebrafish ; },
}
@article {pmid32768421,
year = {2020},
author = {Kushawah, G and Hernandez-Huertas, L and Abugattas-Nuñez Del Prado, J and Martinez-Morales, JR and DeVore, ML and Hassan, H and Moreno-Sanchez, I and Tomas-Gallardo, L and Diaz-Moscoso, A and Monges, DE and Guelfo, JR and Theune, WC and Brannan, EO and Wang, W and Corbin, TJ and Moran, AM and Sánchez Alvarado, A and Málaga-Trillo, E and Takacs, CM and Bazzini, AA and Moreno-Mateos, MA},
title = {CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.},
journal = {Developmental cell},
volume = {54},
number = {6},
pages = {805-817.e7},
doi = {10.1016/j.devcel.2020.07.013},
pmid = {32768421},
issn = {1878-1551},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing/methods ; Gene Expression Regulation, Developmental/*genetics ; HEK293 Cells ; Humans ; RNA Interference/physiology ; RNA, Messenger/genetics ; },
abstract = {Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.},
}
@article {pmid32768274,
year = {2020},
author = {Pham, QT and Raad, S and Mangahas, CL and M'Callum, MA and Raggi, C and Paganelli, M},
title = {High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis.},
journal = {Cytotherapy},
volume = {22},
number = {10},
pages = {536-542},
doi = {10.1016/j.jcyt.2020.06.008},
pmid = {32768274},
issn = {1477-2566},
support = {//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA/genetics ; *Gene Editing ; High-Throughput Screening Assays/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mice ; Mutation/*genetics ; *Nucleic Acid Denaturation ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {BACKGROUND AND AIMS: Genome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach.<br><br>METHODS: Here we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays.<br><br>RESULTS AND CONCLUSIONS: Our data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.},
}
@article {pmid32767999,
year = {2020},
author = {Kong, L and Jiang, D and He, C and Xia, J and Wei, H and Zhou, L and Peng, H},
title = {TgROP18 targets IL20RB for host-defense-related-STAT3 activation during Toxoplasma gondii infection.},
journal = {Parasites &amp; vectors},
volume = {13},
number = {1},
pages = {400},
pmid = {32767999},
issn = {1756-3305},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Genes, Protozoan ; Humans ; Inflammation ; Protozoan Proteins/genetics/immunology/metabolism ; Receptors, Interleukin/metabolism ; STAT3 Transcription Factor/*metabolism ; Signal Transduction ; *Toxoplasma/genetics/metabolism/pathogenicity ; Toxoplasmosis/*immunology ; *Virulence Factors/genetics/immunology/metabolism ; },
abstract = {BACKGROUND: Toxoplasma gondii is an opportunistic protozoan infecting almost one-third of the world's population. Toxoplasma gondii rhoptry protein 18 (TgROP18) is a key virulence factor determining the parasite's acute virulence and is secreted into host cells during infection. We previously identified the interaction of TgROP18 and host cell immune-related receptor protein IL20RB, and observed the activation of STAT3 in human keratinocytes (HaCaT) cells infected by the rop16 knockout RH strain, though TgROP16 is regarded as being responsible for host STAT3 activation during T. gondii invasion. Therefore, we hypothesize TgROP18 can activate host STAT3 through binding to IL20RB.<br><br>METHODS: CRISPR-CAS9 technology was used to generate the ROP16 and ROP18 double knockout RH strain, RH-&#x2206;rop16&#x2206;rop18. SDS-PAGE and western blot were used to detect STAT3 activation in different HaCaT cells with high endogenous IL20RB expression treated with T. gondii tachyzoites infection, recombinant ROP18, or IL-20. FRET and co-immunoprecipitation (Co-IP) was used to detect the protein-protein interaction.<br><br>RESULTS: We observed that TgROP18 was involved in a synergic activation of the host JAK/STAT3 pathway together with TgROP16 in human HaCaT cells infected with T. gondii or treated with recombinant TgROP18 protein, stimulating host proinflammatory immune responses such as expression of TNF-&#x3b1;. The effect of recombinant ROP18 on STAT3 phosphorylation was presented in a dose-dependent manner. Additionally, TgROP18 was identified to target IL20RB on its extracellular domain. When we treated different cell lines with the recombinant ROP18, STAT3 phosphorylation could only be observed in the cells with endogenous IL20RB expression, such as HaCaT cells.<br><br>CONCLUSIONS: These findings indicate that TgROP18-IL20RB interaction upon T. gondii invasion was involved in STAT3 activation, which is associated with host cell defense.},
}
@article {pmid32767816,
year = {2021},
author = {Christodoulou, E and Rashid, M and Pacini, C and Droop, A and Robertson, H and Groningen, TV and Teunisse, AFAS and Iorio, F and Jochemsen, AG and Adams, DJ and Doorn, RV},
title = {Analysis of CRISPR-Cas9 screens identifies genetic dependencies in melanoma.},
journal = {Pigment cell &amp; melanoma research},
volume = {34},
number = {1},
pages = {122-131},
pmid = {32767816},
issn = {1755-148X},
support = {MR/V000292/1/MRC_/Medical Research Council/United Kingdom ; /CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; MR/S00386X/1/MRC_/Medical Research Council/United Kingdom ; 641458/MCCC_/Marie Curie/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Proliferation ; Dual-Specificity Phosphatases/*antagonists &amp; inhibitors/genetics/metabolism ; Gene Knockout Techniques/*methods ; *Genome, Human ; Humans ; Melanoma/genetics/metabolism/*pathology ; Mitogen-Activated Protein Kinase Phosphatases/*antagonists &amp; inhibitors/genetics/metabolism ; Protein Phosphatase 2/*antagonists &amp; inhibitors/genetics/metabolism ; Tumor Cells, Cultured ; },
abstract = {Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR-Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type-specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.},
}
@article {pmid32766806,
year = {2020},
author = {Smalakyte, D and Kazlauskiene, M and F Havelund, J and Rukšėnaitė, A and Rimaite, A and Tamulaitiene, G and Færgeman, NJ and Tamulaitis, G and Siksnys, V},
title = {Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains.},
journal = {Nucleic acids research},
volume = {48},
number = {16},
pages = {9204-9217},
pmid = {32766806},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/*genetics ; Chromatography, High Pressure Liquid ; Endonucleases/genetics ; Escherichia coli/genetics/immunology ; Immunity/*genetics ; Protein Domains/genetics ; RNA Stability/genetics/immunology ; Ribonucleases/genetics ; Signal Transduction/genetics ; Streptococcus thermophilus/*genetics/immunology ; Transcription, Genetic/*genetics/immunology ; },
abstract = {The type III CRISPR-Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR-Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.},
}
@article {pmid32766783,
year = {2020},
author = {Steinberger, J and Shen, L and J Kiniry, S and Naineni, SK and Cencic, R and Amiri, M and Aboushawareb, SAE and Chu, J and Maïga, RI and Yachnin, BJ and Robert, F and Sonenberg, N and Baranov, PV and Pelletier, J},
title = {Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9521-9537},
pmid = {32766783},
issn = {1362-4962},
support = {210692/Z/18//WT_/Wellcome Trust/United Kingdom ; FDN-148366//CIHR/Canada ; },
mesh = {*5' Untranslated Regions ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects/*genetics ; Eukaryotic Initiation Factor-4A/antagonists &amp; inhibitors/*genetics/metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy/genetics/pathology ; Mutation ; Ribosomes/genetics/metabolism ; Sterols/*pharmacology ; },
abstract = {Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.},
}
@article {pmid32764465,
year = {2020},
author = {Son, J and Jang, SH and Cha, JW and Jeong, KJ},
title = {Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis.},
journal = {International journal of molecular sciences},
volume = {21},
number = {16},
pages = {},
pmid = {32764465},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Humans ; Lactic Acid/metabolism ; Leuconostoc/*genetics/metabolism ; *Metabolic Engineering ; Plasmids/genetics ; Probiotics/metabolism ; RNA, Guide/genetics ; Riboflavin/biosynthesis/*genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Leuconostoccitreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine-Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.},
}
@article {pmid32764132,
year = {2020},
author = {Orr, RG and Foley, SJ and Sherman, C and Abreu, I and Galotto, G and Liu, B and González-Guerrero, M and Vidali, L},
title = {Robust Survival-Based RNA Interference of Gene Families Using in Tandem Silencing of Adenine Phosphoribosyltransferase.},
journal = {Plant physiology},
volume = {184},
number = {2},
pages = {607-619},
pmid = {32764132},
issn = {1532-2548},
support = {R15 GM134493/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine Phosphoribosyltransferase ; Bryopsida ; Genes, Plant ; *Genetic Techniques ; *Multigene Family ; *RNA Interference ; },
abstract = {RNA interference (RNAi) enables flexible and dynamic interrogation of entire gene families or essential genes without the need for exogenous proteins, unlike CRISPR-Cas technology. Unfortunately, isolation of plants undergoing potent gene silencing requires laborious design, visual screening, and physical separation for downstream characterization. Here, we developed an adenine phosphoribosyltransferase (APT)-based RNAi technology (APTi) in Physcomitrella patens that improves upon the multiple limitations of current RNAi techniques. APTi exploits the prosurvival output of transiently silencing APT in the presence of 2-fluoroadenine, thereby establishing survival itself as a reporter of RNAi. To maximize the silencing efficacy of gene targets, we created vectors that facilitate insertion of any gene target sequence in tandem with the APT silencing motif. We tested the efficacy of APTi with two gene families, the actin-dependent motor, myosin XI (a,b), and the putative chitin receptor Lyk5 (a,b,c). The APTi approach resulted in a homogenous population of transient P. patens mutants specific for our gene targets with zero surviving background plants within 8 d. The observed mutants directly corresponded to a maximal 93% reduction of myosin XI protein and complete loss of chitin-induced calcium spiking in the Lyk5-RNAi background. The positive selection nature of APTi represents a fundamental improvement in RNAi technology and will contribute to the growing demand for technologies amenable to high-throughput phenotyping.},
}
@article {pmid32763403,
year = {2020},
author = {Zhao, B and Liu, N and Chen, L and Geng, S and Fan, Z and Xing, J},
title = {Direct label-free methods for identification of target proteins in agrochemicals.},
journal = {International journal of biological macromolecules},
volume = {164},
number = {},
pages = {1475-1483},
doi = {10.1016/j.ijbiomac.2020.07.237},
pmid = {32763403},
issn = {1879-0003},
mesh = {Agriculture/methods ; Agrochemicals/*chemistry ; Animals ; Biological Products ; Biotechnology/methods ; Bombyx ; CRISPR-Cas Systems ; Chromatography ; Chromatography, Affinity/*methods ; Drug Discovery/methods ; Ecology/methods ; Genome ; Green Chemistry Technology ; Molecular Docking Simulation ; Oxygen/chemistry ; Pesticides/chemistry ; Polymers/chemistry ; Proteome ; Spectrometry, Fluorescence ; Surface Plasmon Resonance ; },
abstract = {Green agrochemicals are important guarantee for food production and security, and target protein identification is the most important basis for development of novel agrochemicals. Affinity chromatography methods for immobilization of agrochemicals have been widely used to identify and confirm new targets. However, this method often requires modification of the active molecules which can affect or damage its biological activity, and biomacromolecules, particularly most natural products, are hard to be modified either. In order to overcome the shortcomings of molecular modification, label-free technology has been developed based on evaluating responses to thermal or proteolytic treatments. Combined with the chemical biology technology and molecular biology technology, it has been used in the development of drugs and agrochemicals. Herein, common methods of label-free technology for identification of direct target of agrochemicals are reviewed, including the principle, advantages, limitations and applications in the research of agrochemicals in the last decade. And the methods for validation of candidate targets obtained by the label-free methods are also reviewed, which are important to obtain the accurate and reliable targets. Combined application of these methods will greatly reduce the experimental costs and shorten the period for the new target identification and validation by improving its accuracy, which will provide a systematic solution for new ecological agrochemicals research and development.},
}
@article {pmid32763224,
year = {2020},
author = {Cosgrove, BD and Gersbach, CA},
title = {Unwinding the Role of FACT in Cas9-based Genome Editing.},
journal = {Molecular cell},
volume = {79},
number = {3},
pages = {365-367},
doi = {10.1016/j.molcel.2020.07.016},
pmid = {32763224},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; DNA-Binding Proteins ; *Gene Editing ; Histones ; *RNA, Long Noncoding ; },
abstract = {In a recent issue of Molecular Cell, Wang et al. (2020) employ unbiased proteomics approaches and live-cell imaging to reveal a key role for the histone chaperone complex FACT (SPT16 and SSRP1) in governing Cas9 turnover at the DNA target site during genome and epigenome editing.},
}
@article {pmid32763143,
year = {2020},
author = {Jeong, YK and Song, B and Bae, S},
title = {Current Status and Challenges of DNA Base Editing Tools.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {9},
pages = {1938-1952},
pmid = {32763143},
issn = {1525-0024},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; DNA/*genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA Replication/genetics ; Deamination ; Gene Editing/*methods ; Humans ; Point Mutation ; RNA, Guide/*genetics/metabolism ; },
abstract = {CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double-strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.},
}
@article {pmid32762648,
year = {2020},
author = {Tasaki, K and Yoshida, M and Nakajima, M and Higuchi, A and Watanabe, A and Nishihara, M},
title = {Molecular characterization of an anthocyanin-related glutathione S-transferase gene in Japanese gentian with the CRISPR/Cas9 system.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {370},
pmid = {32762648},
issn = {1471-2229},
mesh = {Anthocyanins/chemistry/*metabolism ; Biological Transport ; *CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Flavonoids/biosynthesis/genetics ; Flowers/metabolism ; Gene Editing ; Genes, Plant ; Genetic Complementation Test ; Gentiana/*enzymology/*genetics ; Glutathione Transferase/genetics/*metabolism ; High-Throughput Nucleotide Sequencing ; Phenotype ; Plant Leaves/metabolism ; Plant Proteins/genetics/*metabolism ; },
abstract = {BACKGROUND: The blue pigmentation of Japanese gentian flowers is due to a polyacylated anthocyanin, gentiodelphin, and all associated biosynthesis genes and several regulatory genes have been cloned and characterized. However, the final step involving the accumulation of anthocyanins in petal vacuoles remains unclear. We cloned and analyzed the glutathione S-transferases (GSTs) in Japanese gentian that are known to be involved in anthocyanin transport in other plant species.<br><br>RESULTS: We cloned GST1, which is expressed in gentian flower petals. Additionally, this gene belongs to the Phi-type GST clade related to anthocyanin biosynthesis. We used the CRISPR/Cas9-mediated genome editing system to generate loss-of-function GST1 alleles. The edited alleles were confirmed by Sanger and next-generation sequencing analyses. The GST1 genome-edited lines exhibited two types of mutant flower phenotypes, severe (almost white) and mild (pale blue). The phenotypes were associated with decreased anthocyanin accumulation in flower petals. In the GST1 genome-edited lines, sugar-induced stress conditions inhibited the accumulation of anthocyanins in stems and leaves, suggestvhing that GST1 is necessary for stress-related anthocyanin accumulation in organs other than flowers. These observations clearly demonstrate that GST1 is the gene responsible for anthocyanin transport in Japanese gentian, and is necessary for the accumulation of gentiodelphin in flowers.<br><br>CONCLUSIONS: In this study, an anthocyanin-related GST gene in Japanese gentian was functionally characterized. Unlike other biosynthesis genes, the functions of GST genes are difficult to examine in in vitro studies. Thus, the genome-editing strategy described herein may be useful for in vivo investigations of the roles of transport-related genes in gentian plants.},
}
@article {pmid32762627,
year = {2020},
author = {Akatsuka, K and Sasaki-Honda, M and Sawai, T},
title = {Ethical Framework for Next-Generation Genome and Epigenome Editing.},
journal = {The American journal of bioethics : AJOB},
volume = {20},
number = {8},
pages = {32-36},
doi = {10.1080/15265161.2020.1782524},
pmid = {32762627},
issn = {1536-0075},
mesh = {CRISPR-Cas Systems ; *Epigenome ; *Gene Editing ; },
}
@article {pmid32761967,
year = {2020},
author = {Khalili Alashti, S and Fallahi, J and Jokar, A and Fardaei, M},
title = {CRISPR/Cas9 knock-in toward creating a Rett syndrome cell model with a synonymous mutation in the MECP2 gene.},
journal = {The journal of gene medicine},
volume = {22},
number = {11},
pages = {e3258},
doi = {10.1002/jgm.3258},
pmid = {32761967},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems ; *Gene Expression Regulation ; Gene Knock-In Techniques/*methods ; HEK293 Cells ; Humans ; Methyl-CpG-Binding Protein 2/*genetics ; Models, Biological ; *Mutation ; *Phenotype ; Rett Syndrome/genetics/*pathology ; },
abstract = {BACKGROUND: Rett syndrome is an X-linked dominant neurodevelopmental disease caused by mutation in the methyl-CpG-binding protein 2 (MECP2) gene. This gene encodes a methylated DNA-binding protein, which acts as a transcriptional regulatory factor. The present study aimed to establish a cell model of Rett syndrome with the MECP2 synonymous mutation c.354G>T (p.Gly118Gly). In addition, the molecular mechanism of pathogenesis of this mutation was also investigated.<br><br>METHODS: To create a cell line containing the synonymous variant in MECP2 locus, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed repair precise gene editing method was used. In addition, employing the synthesis of cDNA, the effect of this variant on splicing was investigated.<br><br>RESULTS: Using this model and molecular analysis, we found that the c.354G>T synonymous variant created a novel 5' cryptic splice donor site within the exon 3 of MECP2 gene, which resulted in the deletion of 25 nucleotides at the 3' end of exon 3 and presumably protein truncation.<br><br>CONCLUSIONS: The results of the present study show that an apparently neutral synonymous polymorphism, which may be commonly classified as non-pathogenic, may indeed lead to the creation of an aberrant splice site, thereby resulting in disease.},
}
@article {pmid32761584,
year = {2020},
author = {Gao, W and Liu, H and Su, G and Xu, Y and Wang, Y and Cui, L and Huang, R and Yang, H and Gao, M and Xi, S and Shen, G},
title = {[Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1].},
journal = {Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics},
volume = {37},
number = {8},
pages = {811-814},
doi = {10.3760/cma.j.issn.1003-9406.2020.08.002},
pmid = {32761584},
issn = {1003-9406},
mesh = {Base Sequence ; Blood Coagulation Disorders, Inherited/diagnosis/*genetics ; CRISPR-Cas Systems ; Carbon-Carbon Ligases/*genetics ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; *Vitamin K 1 ; },
abstract = {OBJECTIVE: To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1).<br><br>METHODS: In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene.<br><br>RESULTS: Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly.<br><br>CONCLUSION: A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.},
}
@article {pmid32761306,
year = {2020},
author = {Gao, Y and Gao, K and Yang, H},
title = {CRISPR/Cas: a potential gene-editing tool in the nervous system.},
journal = {Cell regeneration (London, England)},
volume = {9},
number = {1},
pages = {12},
pmid = {32761306},
issn = {2045-9769},
abstract = {The rapidly developmental RNA-guided CRISPR/Cas system is a powerful tool for RNA and DNA editing in a variety of cells from different species and makes a great contribution to gene function research, disease model generation and gene therapy development in the past few years. The ease of use, low cost and high efficiency of CRISPR/Cas make it commonly used in various conditions. In this review, we introduce the CRISPR/Cas system and its diverse applications in nervous system briefly, which provides a better understanding for its potential application values.},
}
@article {pmid32761111,
year = {2020},
author = {Shi, X and Tang, T and Lin, Q and Liu, H and Qin, Y and Liang, X and Cong, P and Mo, D and Liu, X and Chen, Y and He, Z},
title = {Efficient generation of bone morphogenetic protein 15-edited Yorkshire pigs using CRISPR/Cas9†.},
journal = {Biology of reproduction},
volume = {103},
number = {5},
pages = {1054-1068},
doi = {10.1093/biolre/ioaa138},
pmid = {32761111},
issn = {1529-7268},
mesh = {Animals ; Bone Morphogenetic Protein 15/*genetics/metabolism ; CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Oocytes/*metabolism ; Ovarian Follicle/*metabolism ; Ovary/*metabolism ; Swine ; },
abstract = {Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.},
}
@article {pmid32760354,
year = {2020},
author = {Ceballos, RM and Drummond, CG and Stacy, CL and Padilla-Crespo, E and Stedman, KM},
title = {Host-Dependent Differences in Replication Strategy of the Sulfolobus Spindle-Shaped Virus Strain SSV9 (a.k.a., SSVK1): Infection Profiles in Hosts of the Family Sulfolobaceae.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1218},
pmid = {32760354},
issn = {1664-302X},
abstract = {The Sulfolobus Spindle-shaped Virus (SSV) system has become a model for studying thermophilic virus biology, including archaeal host-virus interactions and biogeography. Several factors make the SSV system amenable to studying archaeal genetic mechanisms (e.g., CRISPRs) as well as virus-host interactions in high temperature acidic environments. Previously, we reported that SSVs exhibited differential infectivity on allopatric vs. sympatric hosts. We also noticed a wide host range for virus strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as "non-lytic" double-stranded DNA viruses that infect species of the genus Sulfolobus and release virions via budding rather than host lysis. In this study, we show that SSVs infect hosts representing more than one genus of the family Sulfolobaceae in spot-on-lawn "halo" assays and in liquid culture infection assays. Growth curve analyses support the hypothesis that SSV9 virion release causes cell lysis. While SSV9 appears to lyse allopatric hosts, on a single sympatric host, SSV9 exhibits canonical non-lytic viral release historically reported SSVs. Therefore, the nature of SSV9 lytic-like behavior may be driven by allopatric evolution. The SSV9-infected host growth profile does not appear to be driven by multiplicity of infection (MOI). Greater stability of SSV9 vs. other SSVs (i.e., SSV1) in high temperature, low pH environments may contribute to higher transmission rates. However, neither higher transmission rate nor relative virulence in SSV9 infection seems to alter replication profile in susceptible hosts. Although it is known that CRISPR-Cas systems offer protection against viral infection in prokaryotes, CRISPRS are not reported to be a determinant of virus replication strategy. The mechanisms underlying SSV9 lytic-like behavior remain unknown and are the subject of ongoing investigations. These results suggest that genetic elements, potentially resulting from allopatric evolution, mediate distinct virus-host growth profiles of specific SSV-host strain pairings.},
}
@article {pmid32760197,
year = {2020},
author = {Xu, T and Li, L and Liu, YC and Cao, W and Chen, JS and Hu, S and Liu, Y and Li, LY and Zhou, H and Meng, XM and Huang, C and Zhang, L and Li, J and Zhou, H},
title = {CRISPR/Cas9-related technologies in liver diseases: from feasibility to future diversity.},
journal = {International journal of biological sciences},
volume = {16},
number = {13},
pages = {2283-2295},
pmid = {32760197},
issn = {1449-2288},
mesh = {Animals ; CRISPR-Cas Systems ; Genetic Therapy/*methods ; Humans ; Liver Diseases/*therapy ; },
abstract = {Liver diseases are one of the leading causes of mortality in the world, mainly caused by different etiological agents, alcohol consumption, viruses, drug intoxication, and malnutrition. The maturation of gene therapy has heralded new avenues for developing effective interventions for these diseases. Derived from a remarkable microbial defense system, clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9 system (CRISPR/Cas9 system) is driving innovative applications from basic biology to biotechnology and medicine. Recently, the mutagenic function of CRISPR/Cas9 system has been widely adopted for genome and disease research. In this review, we describe the development and applications of CRISPR/Cas9 system on liver diseases for research or translational applications, while highlighting challenges as well as future avenues for innovation.},
}
@article {pmid32759952,
year = {2020},
author = {Shinzawa, N and Nishi, T and Hiyoshi, F and Motooka, D and Yuda, M and Iwanaga, S},
title = {Improvement of CRISPR/Cas9 system by transfecting Cas9-expressing Plasmodium berghei with linear donor template.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {426},
pmid = {32759952},
issn = {2399-3642},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Humans ; Malaria/*genetics/parasitology ; Plasmodium berghei/*genetics/pathogenicity ; Transfection/methods ; },
abstract = {Malaria is caused by infection with Plasmodium parasites and is a major public health concern. The CRISPR/Cas9 system is a promising technology, but still has technical problems, such as low efficiency and unexpected recombination. Here, we solved these problems by transfecting Cas9-expressing parasites with linear donor templates. The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large-scale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the post-genomic era of Plasmodium species.},
}
@article {pmid32759308,
year = {2020},
author = {Prasai, A and Schmidt Cernohorska, M and Ruppova, K and Niederlova, V and Andelova, M and Draber, P and Stepanek, O and Huranova, M},
title = {The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {42},
pages = {14279-14290},
pmid = {32759308},
issn = {1083-351X},
mesh = {Bardet-Biedl Syndrome/genetics/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Line ; Cilia/metabolism ; Cytoplasm/metabolism ; Fluorescence Recovery After Photobleaching ; Gene Editing ; Humans ; Microscopy, Fluorescence ; Microtubule-Associated Proteins/deficiency/genetics/*metabolism ; Mutation ; Protein Subunits/genetics/metabolism ; },
abstract = {Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.},
}
@article {pmid32758911,
year = {2020},
author = {van Dongen, JE and Berendsen, JTW and Steenbergen, RDM and Wolthuis, RMF and Eijkel, JCT and Segerink, LI},
title = {Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities.},
journal = {Biosensors &amp; bioelectronics},
volume = {166},
number = {},
pages = {112445},
pmid = {32758911},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/genetics ; Point-of-Care Systems ; },
abstract = {With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.},
}
@article {pmid32758536,
year = {2020},
author = {Abdel-Mawgoud, AM and Stephanopoulos, G},
title = {Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions.},
journal = {Metabolic engineering},
volume = {62},
number = {},
pages = {106-115},
doi = {10.1016/j.ymben.2020.07.008},
pmid = {32758536},
issn = {1096-7184},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; RNA, Transfer/genetics ; *Yarrowia/genetics ; },
abstract = {Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.},
}
@article {pmid32757937,
year = {2020},
author = {Gray, J},
title = {Revising, Correcting and Transferring Genes: Germline Editing Versus Natural Reproduction.},
journal = {The American journal of bioethics : AJOB},
volume = {20},
number = {8},
pages = {44-46},
doi = {10.1080/15265161.2020.1782518},
pmid = {32757937},
issn = {1536-0075},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Germ Cells ; },
}
@article {pmid32755594,
year = {2020},
author = {Benslimane, Y and Bertomeu, T and Coulombe-Huntington, J and McQuaid, M and Sánchez-Osuna, M and Papadopoli, D and Avizonis, D and Russo, MST and Huard, C and Topisirovic, I and Wurtele, H and Tyers, M and Harrington, L},
title = {Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {846-856.e8},
doi = {10.1016/j.molcel.2020.07.010},
pmid = {32755594},
issn = {1097-4164},
support = {148936//CIHR/Canada ; 163051//CIHR/Canada ; FDN-167277//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Cell Proliferation/*drug effects ; DNA Replication/*drug effects ; Drug Resistance/genetics ; Humans ; Hydroxyurea/pharmacology ; Jurkat Cells ; Nucleotides/metabolism ; Resveratrol/*pharmacology ; S Phase Cell Cycle Checkpoints/drug effects ; Sirtuin 1/metabolism ; Stilbenes/pharmacology ; },
abstract = {Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.},
}
@article {pmid32755585,
year = {2020},
author = {Boontanrart, MY and Schröder, MS and Stehli, GM and Banović, M and Wyman, SK and Lew, RJ and Bordi, M and Gowen, BG and DeWitt, MA and Corn, JE},
title = {ATF4 Regulates MYB to Increase γ-Globin in Response to Loss of β-Globin.},
journal = {Cell reports},
volume = {32},
number = {5},
pages = {107993},
doi = {10.1016/j.celrep.2020.107993},
pmid = {32755585},
issn = {2211-1247},
support = {S10 OD018174/OD/NIH HHS/United States ; },
mesh = {Activating Transcription Factor 4/genetics/*metabolism ; Base Sequence ; Cell Differentiation/genetics ; Cell Line ; DNA, Intergenic/genetics ; Down-Regulation/genetics ; Enhancer Elements, Genetic/genetics ; Fetal Hemoglobin/genetics ; *Gene Expression Regulation ; Hematopoietic Stem Cells/metabolism ; Humans ; Mutation/genetics ; Protein Binding ; Proto-Oncogene Proteins c-myb/*genetics/metabolism ; Repressor Proteins/metabolism ; Time Factors ; Transcription, Genetic ; Transcriptome/genetics ; Up-Regulation/genetics ; beta-Globins/*metabolism ; gamma-Globins/*genetics/metabolism ; },
abstract = {β-Hemoglobinopathies can trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here, we use CRISPR-Cas genome editing to model the stress caused by reduced levels of adult β-globin. We find that decreased β-globin is sufficient to induce robust re-expression of γ-globin, and RNA sequencing (RNA-seq) of differentiating isogenic erythroid precursors implicates ATF4 as a causal regulator of this response. ATF4 binds within the HBS1L-MYB intergenic enhancer and regulates expression of MYB, a known γ-globin regulator. Overall, the reduction of ATF4 upon β-globin knockout decreases the levels of MYB and BCL11A. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could inform strategies to treat hemoglobinopathies.},
}
@article {pmid32755071,
year = {2021},
author = {Parker, E and Hofer, IMJ and Rice, SJ and Earl, L and Anjum, SA and Deehan, DJ and Loughlin, J},
title = {Multi-Tissue Epigenetic and Gene Expression Analysis Combined With Epigenome Modulation Identifies RWDD2B as a Target of Osteoarthritis Susceptibility.},
journal = {Arthritis &amp; rheumatology (Hoboken, N.J.)},
volume = {73},
number = {1},
pages = {100-109},
doi = {10.1002/art.41473},
pmid = {32755071},
issn = {2326-5205},
support = {20679/VAC_/Versus Arthritis/United Kingdom ; 20771/VAC_/Versus Arthritis/United Kingdom ; MR/P020941/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adult ; Aged ; Aged, 80 and over ; CRISPR-Cas Systems ; Cartilage, Articular/*metabolism ; Chondrocytes ; CpG Islands ; DNA Methylation ; *Epigenesis, Genetic ; Female ; Genetic Predisposition to Disease ; Humans ; Male ; Middle Aged ; Osteoarthritis/*genetics/metabolism ; Polymorphism, Single Nucleotide ; Quantitative Trait Loci ; Synovial Membrane/*metabolism ; },
abstract = {OBJECTIVE: Osteoarthritis (OA) is polygenic, with more than 90 risk loci currently mapped, including at the single-nucleotide polymorphism rs6516886. Previous analysis of OA cartilage DNA identified 6 CpG dinucleotides whose methylation levels correlated with the rs6516886 genotype, forming methylation quantitative trait loci (mQTLs). We undertook this study to investigate these mQTLs and to map expression quantitative trait loci (eQTLs) across joint tissues in order to prioritize a particular gene as a target of the rs6516886 association effect.<br><br>METHODS: Nucleic acids were extracted from the cartilage, fat pad, synovium, and peripheral blood from OA patients. Methylation of CpGs and allelic expression imbalance of potential target genes were assessed by pyrosequencing. A chondrocyte cell line expressing deactivated Cas9 (dCas9)-TET1 was used to directly alter CpG methylation levels, with effects on gene expression quantified by polymerase chain reaction.<br><br>RESULTS: Multiple mQTLs were detected, with effects strongest in joint tissues and with methylation at CpG cg20220242 correlating most significantly with the rs6516886 genotype. CpG cg20220242 is located upstream of RWDD2B. Significant rs6516886 eQTLs were observed for this gene, with the OA risk-conferring allele of rs6516886 correlating with reduced expression&#xa0;CpG methylation also correlated with allelic expression of&#xa0;RWDD2B, forming methylation-expression QTLs (meQTLs). Deactivated Cas9-TET1 reduction in the methylation of cg20220242 increased expression of RWDD2B.<br><br>CONCLUSION: The rs6516886 association signal is a multi-tissue meQTL involving cg20220242 and acting on&#xa0;RWDD2B. Modulating CpG methylation reverses the impact of the risk allele. RWDD2B codes for a protein about which little is currently known. Its further analysis as a target of OA genetic risk will provide novel insight into this complex disease.},
}
@article {pmid32755000,
year = {2020},
author = {Zuin Fantoni, N and McGorman, B and Molphy, Z and Singleton, D and Walsh, S and El-Sagheer, AH and McKee, V and Brown, T and Kellett, A},
title = {Development of Gene-Targeted Polypyridyl Triplex-Forming Oligonucleotide Hybrids.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {21},
number = {24},
pages = {3563-3574},
doi = {10.1002/cbic.202000408},
pmid = {32755000},
issn = {1439-7633},
support = {BB/J001694/2/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R008655/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Click Chemistry ; Copper/chemistry/*metabolism ; DNA/chemistry/*metabolism ; Endonucleases/*metabolism ; Metalloproteins/chemical synthesis/chemistry/*metabolism ; Molecular Structure ; Oligonucleotides/chemical synthesis/chemistry/*metabolism ; },
abstract = {In the field of nucleic acid therapy there is major interest in the development of libraries of DNA-reactive small molecules which are tethered to vectors that recognize and bind specific genes. This approach mimics enzymatic gene editors, such as ZFNs, TALENs and CRISPR-Cas, but overcomes the limitations imposed by the delivery of a large protein endonuclease which is required for DNA cleavage. Here, we introduce a chemistry-based DNA-cleavage system comprising an artificial metallo-nuclease (AMN) that oxidatively cuts DNA, and a triplex-forming oligonucleotide (TFO) that sequence-specifically recognises duplex DNA. The AMN-TFO hybrids coordinate Cu[II] ions to form chimeric catalytic complexes that are programmable - based on the TFO sequence employed - to bind and cut specific DNA sequences. Use of the alkyne-azide cycloaddition click reaction allows scalable and high-throughput generation of hybrid libraries that can be tuned for specific reactivity and gene-of-interest knockout. As a first approach, we demonstrate targeted cleavage of purine-rich sequences, optimisation of the hybrid system to enhance stability, and discrimination between target and off-target sequences. Our results highlight the potential of this approach where the cutting unit, which mimics the endonuclease cleavage machinery, is directly bound to a TFO guide by click chemistry.},
}
@article {pmid32753430,
year = {2020},
author = {Musseau, C and Jorly, J and Gadin, S and Sørensen, I and Deborde, C and Bernillon, S and Mauxion, JP and Atienza, I and Moing, A and Lemaire-Chamley, M and Rose, JKC and Chevalier, C and Rothan, C and Fernandez-Lochu, L and Gévaudant, F},
title = {The Tomato Guanylate-Binding Protein SlGBP1 Enables Fruit Tissue Differentiation by Maintaining Endopolyploid Cells in a Non-Proliferative State.},
journal = {The Plant cell},
volume = {32},
number = {10},
pages = {3188-3205},
pmid = {32753430},
issn = {1532-298X},
mesh = {CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Differentiation ; Cell Size ; Cell Wall/genetics/metabolism ; Endoreduplication ; Fruit/*cytology/genetics/*growth &amp; development/metabolism ; GTP-Binding Proteins/genetics/metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/*cytology/genetics/metabolism ; Mutation ; Pectins/genetics/metabolism ; Phenotype ; Plant Cells ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Ploidies ; },
abstract = {Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.},
}
@article {pmid32752068,
year = {2020},
author = {Jung, YJ and Kim, JH and Lee, HJ and Kim, DH and Yu, J and Bae, S and Cho, YG and Kang, KK},
title = {Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of SLR1 Using the CRISPR/Cas9 System in Rice.},
journal = {International journal of molecular sciences},
volume = {21},
number = {15},
pages = {},
pmid = {32752068},
issn = {1422-0067},
mesh = {Alleles ; Amino Acid Sequence/genetics ; CRISPR-Cas Systems/genetics ; Gene Expression Profiling ; Gene Expression Regulation, Plant/genetics ; Gibberellins/metabolism ; Mutation/genetics ; Oryza/*genetics/growth &amp; development ; Phenotype ; Plant Growth Regulators/*genetics/metabolism ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics ; Signal Transduction/genetics ; Transcriptome/*genetics ; },
abstract = {The rice SLR1 gene encodes the DELLA protein, and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.},
}
@article {pmid32751997,
year = {2020},
author = {Il'ina, IV and Dyrkheeva, NS and Zakharenko, AL and Sidorenko, AY and Li-Zhulanov, NS and Korchagina, DV and Chand, R and Ayine-Tora, DM and Chepanova, AA and Zakharova, OD and Ilina, ES and Reynisson, J and Malakhova, AA and Medvedev, SP and Zakian, SM and Volcho, KP and Salakhutdinov, NF and Lavrik, OI},
title = {Design, Synthesis, and Biological Investigation of Novel Classes of 3-Carene-Derived Potent Inhibitors of TDP1.},
journal = {Molecules (Basel, Switzerland)},
volume = {25},
number = {15},
pages = {},
pmid = {32751997},
issn = {1420-3049},
mesh = {Bicyclic Monoterpenes/*chemistry ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; *Drug Design ; Drug Synergism ; Gene Knockout Techniques ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Humans ; Inhibitory Concentration 50 ; Phosphodiesterase Inhibitors/*chemical synthesis/chemistry/*pharmacology ; Phosphoric Diester Hydrolases/genetics/*metabolism ; Signal Transduction/*drug effects ; Topotecan/pharmacology ; },
abstract = {Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC50 value of 0.65 μM. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k, a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 -/- cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.},
}
@article {pmid32751693,
year = {2020},
author = {Mazumdar, A and Urdinez, J and Boro, A and Migliavacca, J and Arlt, MJE and Muff, R and Fuchs, B and Snedeker, JG and Gvozdenovic, A},
title = {Osteosarcoma-Derived Extracellular Vesicles Induce Lung Fibroblast Reprogramming.},
journal = {International journal of molecular sciences},
volume = {21},
number = {15},
pages = {},
pmid = {32751693},
issn = {1422-0067},
mesh = {Actins/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cellular Reprogramming/*genetics ; Extracellular Vesicles/metabolism/pathology ; Fibroblasts/metabolism/pathology ; Gene Expression Regulation, Neoplastic/genetics ; Humans ; Lung/metabolism ; Neoplasm Invasiveness/genetics/pathology ; Neoplasm Metastasis ; Osteosarcoma/*genetics/pathology ; Receptor, Transforming Growth Factor-beta Type I/antagonists &amp; inhibitors/*genetics ; },
abstract = {Tumor-secreted extracellular vesicles (EVs) have been identified as mediators of cancer-host intercellular communication and shown to support pre-metastatic niche formation by modulating stromal cells at future metastatic sites. While osteosarcoma, the most common primary malignant bone tumor in children and adolescents, has a high propensity for pulmonary metastases, the interaction of osteosarcoma cells with resident lung cells remains poorly understood. Here, we deliver foundational in vitro evidence that osteosarcoma cell-derived EVs drive myofibroblast/cancer-associated fibroblast differentiation. Human lung fibroblasts displayed increased invasive competence, in addition to increased α-smooth muscle actin expression and fibronectin production upon EV treatment. Furthermore, we demonstrate, through the use of transforming growth factor beta receptor 1 (TGFBR1) inhibitors and CRISPR-Cas9-mediated knockouts, that TGFβ1 present in osteosarcoma cell-derived EVs is responsible for lung fibroblast differentiation. Overall, our study highlights osteosarcoma-derived EVs as novel regulators of lung fibroblast activation and provides mechanistic insight into how osteosarcoma cells can modulate distant cells to potentially support metastatic progression.},
}
@article {pmid32749657,
year = {2020},
author = {Khan, AH and Tye, GJ and Noordin, R},
title = {CRISPR-Cas9 Genome Editing Tool for the Production of Industrial Biopharmaceuticals.},
journal = {Molecular biotechnology},
volume = {62},
number = {9},
pages = {401-411},
doi = {10.1007/s12033-020-00265-9},
pmid = {32749657},
issn = {1559-0305},
mesh = {Animals ; *Biological Products ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Insecta ; Mammals ; Yeasts ; },
abstract = {A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.},
}
@article {pmid32749114,
year = {2020},
author = {Noor, S and Rasul, A and Iqbal, MS and Ahmed, B and Akash, MSH and Qadir, MI},
title = {Inhibition of Hepatitis B Virus with the Help of CRISPR/Cas9 Technology.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {30},
number = {3},
pages = {273-278},
doi = {10.1615/CritRevEukaryotGeneExpr.2020028453},
pmid = {32749114},
issn = {1045-4403},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Circular/metabolism ; DNA, Viral/metabolism ; Gene Editing ; Genetic Engineering ; Genetic Therapy ; Genome, Viral ; Hepatitis B/*therapy ; Hepatitis B virus/*genetics/physiology ; Humans ; Mutagenesis, Site-Directed ; *Virus Replication ; },
abstract = {Hepatitis B infection caused by hepatitis B virus (HBV) is a serious health issue worldwide. Existing therapeutic strategies hardly eradicate HBV infections, and they fail to attain complete cure. Advanced treatment strategies are urgently needed to successfully terminate further spread of HBV infection and eliminate hidden reservoirs of virus. Recently, a novel RNA-guided gene editing tool, known as the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system, has been established. It facilitates site-specific mutagenesis and reveals a new way to develop applicable techniques for disease treatment, such as extermination of infectious agents like HBV This study highlights the current developments in CRISPR/Cas9 technology and its importance for target-specific inhibition of HBV genome. Benefits, challenges, feasible solutions, and proposed guidelines for forthcoming study in CRISPR/Cas9 are described to highlight the possible cures of and treatments for chronic HBV infection.},
}
@article {pmid32749110,
year = {2020},
author = {Ahmed, B and Arif, M and Qadir, MI},
title = {CRISPR/Cas: A Successful Tool for Genome Editing in Animal Models.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {30},
number = {3},
pages = {239-243},
doi = {10.1615/CritRevEukaryotGeneExpr.2020028791},
pmid = {32749110},
issn = {1045-4403},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome ; Models, Animal ; },
abstract = {CRISPR/Cas9 is an innovative molecular tool that is utilized in advanced biological applications. This review focuses on modifying the genomes of a wide range of animals by CRISPR/Cas9 for greater usability and higher efficiency, providing an overview of the function and mechanism of this system and the utilization of this system for medicinal research. The type II CRISPR-Cas system is found in the Enterobacteriaceae bacteria family, which uses this system as a defense against invading phages and plasmids. This system can be engineered to direct its action toward a targeted site for the modification of a specific genome.},
}
@article {pmid32748599,
year = {2020},
author = {Wang, H and Li, G and Huang, G and Li, Z and Zheng, E and Xu, Z and Yang, H and Wu, Z and Zhang, X and Liu, D},
title = {[Knockdown the expression of ku70 and lig4 in HEK293T cells by CRISPR/Cas13 system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {7},
pages = {1414-1421},
doi = {10.13345/j.cjb.190494},
pmid = {32748599},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems ; *DNA Ligase ATP/genetics ; Gene Expression Regulation/genetics ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; *Ku Autoantigen/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.},
}
@article {pmid32748596,
year = {2020},
author = {Feng, C and Xu, X and Dong, W and Chen, Z and Yan, J},
title = {[CRISPR/Cas9 knockout plin1 enhances lipolysis in 3T3-L1 adipocytes].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {7},
pages = {1386-1394},
doi = {10.13345/j.cjb.190521},
pmid = {32748596},
issn = {1872-2075},
mesh = {3T3-L1 Cells ; Adipocytes/metabolism ; Animals ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; Lipase/metabolism ; *Lipolysis/genetics ; Mice ; *Perilipin-1/genetics/metabolism ; },
abstract = {We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.},
}
@article {pmid32748555,
year = {2021},
author = {Liu, W and Padmashali, R and Monzon, OQ and Lundberg, D and Jin, S and Dwyer, B and Lee, YJ and Korde, A and Park, S and Pan, C and Zhang, B},
title = {Generation of FX[-/-] and Gmds[-/-] CHOZN host cell lines for the production of afucosylated therapeutic antibodies.},
journal = {Biotechnology progress},
volume = {37},
number = {1},
pages = {e3061},
pmid = {32748555},
issn = {1520-6033},
mesh = {Amino Acid Sequence ; Animals ; Antibodies, Monoclonal/*biosynthesis/immunology ; Antibody-Dependent Cell Cytotoxicity ; Base Sequence ; CHO Cells ; CRISPR-Cas Systems ; Carbohydrate Epimerases/*antagonists &amp; inhibitors/genetics/metabolism ; Cricetinae ; Cricetulus ; Fucose/*metabolism ; Glycosylation ; Guanosine Diphosphate/*metabolism ; Humans ; Hydro-Lyases/*antagonists &amp; inhibitors/genetics/metabolism ; Immunoglobulin G/immunology ; Ketone Oxidoreductases/*antagonists &amp; inhibitors/genetics/metabolism ; Receptors, IgG/metabolism ; },
abstract = {Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8[-/-] Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX-/- and Gmds-/-, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX-/- and Gmds-/- host cells could replace Fut8-/- CHO cells for clinical manufacturing of antibody therapeutics.},
}
@article {pmid32748174,
year = {2020},
author = {Zhang, C and Ren, Z and Gong, Z},
title = {Transgenic Expression and Genome Editing by Electroporation of Zebrafish Embryos.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {22},
number = {5},
pages = {644-650},
doi = {10.1007/s10126-020-09985-0},
pmid = {32748174},
issn = {1436-2236},
mesh = {Animals ; CRISPR-Cas Systems ; DNA/genetics ; Electroporation/*methods ; Embryo, Nonmammalian ; Endonucleases/genetics/metabolism ; Gene Editing/*methods ; *Gene Transfer Techniques ; Plasmids/genetics ; RNA, Guide/genetics ; Zebrafish/embryology/*genetics ; },
abstract = {Microinjection is predominantly used to produce genetically modified fish. However, there are certain difficulties involved in some fish species. In this study, we tested an alternative method to produce genetically modified zebrafish by delivering DNA and other materials into embryos by electroporation. We optimized the electroporation conditions of a square wave electroporation system that work efficiently for the introduction of plasmid DNA, recombinant Cas9 nuclease and synthetic dual guide RNAs. Transgenic expression was observed in a wide range of tissues, which is comparable with those obtained by microinjection. We further determined that efficient gene delivery can be achieved during the cleavage stage. This study describes detailed electroporation parameters for gene delivery with high efficiency and low toxicity, providing a novel method to generate transgenic lines and genome editing.},
}
@article {pmid32748081,
year = {2020},
author = {Huang, X and Wang, Y and Xu, J and Wang, N},
title = {Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations.},
journal = {Plant molecular biology},
volume = {104},
number = {3},
pages = {297-307},
doi = {10.1007/s11103-020-01043-6},
pmid = {32748081},
issn = {1573-5028},
mesh = {CRISPR-Cas Systems ; Citrus/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genes, Plant/genetics ; Genome, Plant/*genetics ; *Homozygote ; *Mutation ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Protoplasts ; RNA, Guide/genetics ; RNA, Transfer/genetics ; },
abstract = {We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.},
}
@article {pmid32747749,
year = {2020},
author = {Sun, W and Wang, H},
title = {Recent advances of genome editing and related technologies in China.},
journal = {Gene therapy},
volume = {27},
number = {7-8},
pages = {312-320},
pmid = {32747749},
issn = {1476-5462},
mesh = {*CRISPR-Cas Systems ; China ; Endonucleases/metabolism ; *Gene Editing ; Transcription Activator-Like Effector Nucleases/metabolism ; },
abstract = {Genome editing is a powerful tool, enabling scientists to alter DNA sequence at virtually any genome locus in any species. Different technologies have been developed employing programmable nucleases including meganuclease, zinc-finger nucleases, transcription activator-like effector nucleases, and most recently CRISPR-Cas systems. Chinese research groups are making important contributions at an increasing speed in genome editing field in recent years. In this review, we summarize recent progress made by Chinese scientists on the technological development of genome editing and beyond, focusing on the optimization and expanded application of existing genome editing tools, as well as the exploration of novel proteins as potential genome editing tools.},
}
@article {pmid32747436,
year = {2020},
author = {Wang, X and Kohl, A and Yu, X and Zorio, DAR and Klar, A and Sela-Donenfeld, D and Wang, Y},
title = {Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {21},
pages = {},
pmid = {32747436},
issn = {1477-9129},
support = {R01 DC013074/DC/NIDCD NIH HHS/United States ; R21 DC017267/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; Auditory Pathways/*embryology/*metabolism ; Axons/metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; Chick Embryo ; Chickens ; Dendrites/metabolism ; Fragile X Mental Retardation Protein/*metabolism ; Neural Stem Cells/metabolism ; Presynaptic Terminals/metabolism ; RNA, Small Interfering/metabolism ; Rhombencephalon/*embryology/*metabolism ; Synapses/metabolism ; Time Factors ; },
abstract = {Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1[+] neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1[+] neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.},
}
@article {pmid32745724,
year = {2020},
author = {D'Amore, C and Moro, E and Borgo, C and Itami, K and Hirota, T and Pinna, LA and Salvi, M},
title = {"Janus" efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {11},
pages = {118807},
doi = {10.1016/j.bbamcr.2020.118807},
pmid = {32745724},
issn = {1879-2596},
mesh = {CRISPR-Cas Systems/genetics ; Casein Kinase II/antagonists &amp; inhibitors/*genetics ; Cell Death/drug effects/*genetics ; Gene Editing ; Hep G2 Cells ; Humans ; Indoles/pharmacology ; Neoplasms/*drug therapy ; Pinocytosis/drug effects/genetics ; Pyrimidines/pharmacology ; Quinolines/pharmacology ; Vacuoles/drug effects/genetics ; rac1 GTP-Binding Protein/antagonists &amp; inhibitors/*genetics ; },
abstract = {Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.},
}
@article {pmid32745576,
year = {2020},
author = {Zhang, Z and Zhu, B and Chen, W and Ge, W},
title = {Anti-Müllerian hormone (Amh/amh) plays dual roles in maintaining gonadal homeostasis and gametogenesis in zebrafish.},
journal = {Molecular and cellular endocrinology},
volume = {517},
number = {},
pages = {110963},
doi = {10.1016/j.mce.2020.110963},
pmid = {32745576},
issn = {1872-8057},
mesh = {Activins/physiology ; Animals ; Anti-Mullerian Hormone/deficiency/genetics/*physiology ; Base Sequence ; CRISPR-Cas Systems ; Feedback, Physiological ; Female ; Follicle Stimulating Hormone/biosynthesis/genetics ; Gametogenesis/*physiology ; Gene Knockout Techniques ; Growth Differentiation Factor 9/genetics ; Homeostasis/*physiology ; Hypertrophy ; Infertility, Female/genetics ; Infertility, Male/genetics ; Inhibins/physiology ; Male ; Ovary/metabolism/pathology ; Paracrine Communication ; Pituitary Gland, Anterior/metabolism ; Sexual Maturation/genetics ; Testis/metabolism/pathology ; Zebrafish ; Zebrafish Proteins/*physiology ; },
abstract = {Anti-Müllerian hormone (AMH/Amh) plays a role in gonadal differentiation and function across vertebrates. In zebrafish we demonstrated that Amh deficiency caused severe gonadal dysgenesis and dysfunction. The mutant gonads showed extreme hypertrophy with accumulation of early germ cells in both sexes, namely spermatogonia in the testis and primary growth oocytes in the ovary. In amh mutant females, the folliculogenesis was normal in young fish but receded progressively in adults, which was accompanied by progressive decrease in follicle-stimulating hormone (fshb) expression. Interestingly the expression of fshb increased in the pituitary of juvenile amh mutant males but decreased in adults. The upregulation of fshb in mutant male juveniles was likely one of the mechanisms for triggering gonadal hypergrowth, whereas the downregulation of fshb in adults might involve a negative feedback by gonadal inhibin. Further analysis using mutants of fshb and growth differentiation factor 9 (gdf9) provided evidence for a role of FSH in triggering ovarian hypertrophy in young female amh mutant as well. In summary, the present study provided comprehensive genetic evidence for dual roles of Amh in controlling zebrafish gonadal homeostasis and gametogenesis in both sexes. Amh suppresses proliferation or accumulation of early germ cells (spermatogonia in testis and primary growth oocytes in ovary) while promoting their exit to advanced stages, and its action may involve both endocrine and paracrine pathways.},
}
@article {pmid32745561,
year = {2020},
author = {Yan, Y and Ziemek, J and Schetelig, MF},
title = {CRISPR/Cas9 mediated disruption of the white gene leads to pigmentation deficiency and copulation failure in Drosophila suzukii.},
journal = {Journal of insect physiology},
volume = {126},
number = {},
pages = {104091},
doi = {10.1016/j.jinsphys.2020.104091},
pmid = {32745561},
issn = {1879-1611},
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Copulation ; Drosophila/*genetics/physiology ; Drosophila Proteins/*genetics ; Eye Proteins/*genetics ; Female ; Genes, Insect ; Insect Control/methods ; Male ; Mutation ; Pigmentation/*genetics ; *Sexual Behavior, Animal ; },
abstract = {The Spotted-wing Drosophila (Drosophila suzukii) is a devastating invasive pest of fruit crops. In D. melanogaster, the white (w) gene was associated with pigmentation and mating behavior, which are also important aspects to understand the invasion biology as well as to develop control strategies for D. suzukii. Here, we show that the generation of D. suzukii white-eyed mutants by CRISPR/Cas9 mutagenesis of the w gene resulted in the complete failure of copulation when w[-] males were individually paired with w[-] females in small circular arenas (diameter 0.7 cm) for 24 h. Further analysis showed that the mating defect was associated with w[-] males and could not be rectified by two years of inbreeding by crossing sibling w[-] females with w[+] males, dim red illumination, male-female sexual training, changing to large arenas (diameter 3.5 cm), or different sex ratios. Profound pigmentation deficiency was detected in the compound eyes, ocelli, Malpighian tubules and testis sheaths in the w[-] flies. Specifically, testis imaging showed that w[-] males failed to deposit any pigments into pigment cells of the testis sheath, and produced smaller sperms and less seminal fluid compared to those from wildtype males. Together these observations suggest that the w gene plays an essential role in the regulation of sexual behavior and reproduction in D. suzukii. The similarities and differences in w gene function between D. suzukii and D. melanogaster in the context of pigmentation and mating behavior are discussed.},
}
@article {pmid32745097,
year = {2020},
author = {Singh, SP and Thomason, PA and Lilla, S and Schaks, M and Tang, Q and Goode, BL and Machesky, LM and Rottner, K and Insall, RH},
title = {Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.},
journal = {PLoS biology},
volume = {18},
number = {8},
pages = {e3000774},
pmid = {32745097},
issn = {1545-7885},
support = {A17196/CRUK_/Cancer Research UK/United Kingdom ; A20017/CRUK_/Cancer Research UK/United Kingdom ; A20017/CRUK_/Cancer Research UK/United Kingdom ; R35 GM134895/GM/NIGMS NIH HHS/United States ; R01 GM063691/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line, Tumor ; Chemotaxis/genetics ; Dictyostelium/*genetics/metabolism/ultrastructure ; Gene Editing/methods ; Gene Expression Regulation ; MAP Kinase Kinase Kinase 3/*genetics/metabolism ; Melanocytes/metabolism/ultrastructure ; Mice ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; Mutation ; NIH 3T3 Cells ; Phenotype ; Phosphorylation ; Ploidies ; Protozoan Proteins/*genetics/metabolism ; Pseudopodia/genetics/*metabolism/ultrastructure ; Wiskott-Aldrich Syndrome Protein Family/*genetics/metabolism ; },
abstract = {The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.},
}
@article {pmid32744504,
year = {2020},
author = {Gaertner, B and van Heesch, S and Schneider-Lunitz, V and Schulz, JF and Witte, F and Blachut, S and Nguyen, S and Wong, R and Matta, I and Hübner, N and Sander, M},
title = {A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32744504},
issn = {2050-084X},
support = {R01 DK068471/DK/NIDDK NIH HHS/United States ; P30 DK063491/DK/NIDDK NIH HHS/United States ; 2015-D-021-FEL//Larry L. Hillblom Foundation/International ; R01 DK078803/DK/NIDDK NIH HHS/United States ; AdG788970//Horizon 2020 Framework Programme/International ; ALTF 186-2015//European Molecular Biology Organization/International ; },
mesh = {CRISPR-Cas Systems ; Cell Differentiation/*physiology ; Cells, Cultured ; Gene Deletion ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; HEK293 Cells ; Human Embryonic Stem Cells ; Humans ; Islets of Langerhans/*cytology/embryology ; Male ; Protein Biosynthesis ; RNA, Long Noncoding/genetics/*physiology ; Transcription Factors/metabolism ; },
abstract = {Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin[+] cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.},
}
@article {pmid32744496,
year = {2020},
author = {Najar, IN and Thakur, N},
title = {A systematic review of the genera Geobacillus and Parageobacillus: their evolution, current taxonomic status and major applications.},
journal = {Microbiology (Reading, England)},
volume = {166},
number = {9},
pages = {800-816},
doi = {10.1099/mic.0.000945},
pmid = {32744496},
issn = {1465-2080},
mesh = {Bacillaceae/*classification/enzymology/genetics/*physiology ; Biodegradation, Environmental ; Biofuels ; Biological Evolution ; Biotechnology ; CRISPR-Cas Systems ; Extreme Environments ; Geobacillus/*classification/enzymology/genetics/*physiology ; Industrial Microbiology ; Phylogeny ; Temperature ; },
abstract = {The genus Geobacillus, belonging to the phylum Firmicutes, is one of the most important genera and comprises thermophilic bacteria. The genus Geobacillus was erected with the taxonomic reclassification of various Bacillus species. Taxonomic studies of Geobacillus remain in progress. However, there is no comprehensive review of the characteristic features, taxonomic status and study of various applications of this interesting genus. The main aim of this review is to give a comprehensive account of the genus Geobacillus. At present the genus acomprises 25 taxa, 14 validly published (with correct name), nine validly published (with synonyms) and two not validly published species. We describe only validly published species of the genera Geobacillus and Parageobacillus. Vegetative cells of Geobacillus species are Gram-strain-positive or -variable, rod-shaped, motile, endospore-forming, aerobic or facultatively anaerobic, obligately thermophilic and chemo-organotrophic. Growth occurs in the pH range 6.08.5 and a temperature of 37-75 °C. The major cellular fatty acids are iso-C15:o, iso-C16:0 and iso-C17:o. The main menaquinone type is MK-7. The G-+C content of the DNA ranges between 48.2 and 58 mol%. The genus Geobacillus is widely distributed in nature, being mostly found in many extreme locations such as hot springs, hydrothermal vents, marine trenches, hay composts, etc. Geobacillus species have been widely exploited in various industrial and biotechnological applications, and thus are promising candidates for further studies in the future.},
}
@article {pmid32744433,
year = {2020},
author = {Hao, Y and Li, J and Li, Q and Zhang, L and Shi, J and Zhang, X and Aldalbahi, A and Wang, L and Fan, C and Wang, F},
title = {Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {46},
pages = {20612-20618},
doi = {10.1002/anie.202009062},
pmid = {32744433},
issn = {1521-3773},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Human ; Humans ; Kinetics ; Nucleic Acid Conformation ; RNA, Guide/chemistry/genetics ; Single Molecule Imaging/methods ; },
abstract = {The widespread application of CRISPR-Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold-switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold-switch-mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering.},
}
@article {pmid32744307,
year = {2020},
author = {Russi, M and Martin, E and D'Autréaux, B and Tixier, L and Tricoire, H and Monnier, V},
title = {A Drosophila model of Friedreich ataxia with CRISPR/Cas9 insertion of GAA repeats in the frataxin gene reveals in vivo protection by N-acetyl cysteine.},
journal = {Human molecular genetics},
volume = {29},
number = {17},
pages = {2831-2844},
doi = {10.1093/hmg/ddaa170},
pmid = {32744307},
issn = {1460-2083},
mesh = {Acetylcysteine/*pharmacology ; Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Drosophila melanogaster/genetics ; Friedreich Ataxia/drug therapy/*genetics/pathology ; Humans ; Introns/genetics ; Iron-Binding Proteins/*genetics ; Oxidative Stress/genetics ; RNA-Seq ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Friedreich ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, we generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. We were able to bypass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, we observed a progressive increase of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in our fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, we observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, our model provides the opportunity to elucidate in vivo, the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance.},
}
@article {pmid32741873,
year = {2020},
author = {Sato, M},
title = {[Manipulating Living Systems by Light].},
journal = {Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan},
volume = {140},
number = {8},
pages = {993-1000},
doi = {10.1248/yakushi.20-00012-5},
pmid = {32741873},
issn = {1347-5231},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Differentiation ; DNA/genetics ; Gene Editing ; *Genetic Engineering ; Genome, Human ; Humans ; Induced Pluripotent Stem Cells/physiology ; *Light ; Mice ; *Optogenetics ; Recombination, Genetic ; *Transcriptional Activation ; },
abstract = {The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.},
}
@article {pmid32738651,
year = {2020},
author = {Shi, J and Yang, X and Li, Y and Wang, D and Liu, W and Zhang, Z and Liu, J and Zhang, K},
title = {MicroRNA-responsive release of Cas9/sgRNA from DNA nanoflower for cytosolic protein delivery and enhanced genome editing.},
journal = {Biomaterials},
volume = {256},
number = {},
pages = {120221},
doi = {10.1016/j.biomaterials.2020.120221},
pmid = {32738651},
issn = {1878-5905},
mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; *MicroRNAs/genetics ; RNA, Guide/genetics ; },
abstract = {Nanoparticle-based CRISPR/Cas9 delivery systems hold great promise for specific and precise treatment of genetic disorder diseases. Herein, we developed a DNA nanoflower-based platform for microRNA-responsive cytosolic delivery of Cas9/sgRNA complex into tumor cells. The biocompatible DNA nano-vehicles can efficiently load Cas9/sgRNA by sequence hybridization. Importantly, this hybridization can be replaced by a tumor specific miRNA through toehold-mediated strand displacement process and achieve cell-type-specific release of Cas9/sgRNA from the DNA nanoflowers. We have verified that this miRNA-responsive releasing process can significantly improve the genome editing efficiency comparing with non-responsive control. This strategy suggests a versatile way for designing more specific and efficient CRISPR-based genome therapy system by incorporating stimuli-responsive Cas9/sgRNA release process.},
}
@article {pmid32738650,
year = {2020},
author = {Zhang, K and Chooi, WH and Liu, S and Chin, JS and Murray, A and Nizetic, D and Cheng, D and Chew, SY},
title = {Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering.},
journal = {Biomaterials},
volume = {256},
number = {},
pages = {120225},
doi = {10.1016/j.biomaterials.2020.120225},
pmid = {32738650},
issn = {1878-5905},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nanofibers ; Peptides ; Rats ; Tissue Engineering ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP[-] is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP[+]-RGD are then absorbed via electrostatic interactions. The SAPcoated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.},
}
@article {pmid32738194,
year = {2020},
author = {van de Weijer, ML and Krshnan, L and Liberatori, S and Guerrero, EN and Robson-Tull, J and Hahn, L and Lebbink, RJ and Wiertz, EJHJ and Fischer, R and Ebner, D and Carvalho, P},
title = {Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {768-781.e7},
pmid = {32738194},
issn = {1097-4164},
support = {/WT_/Wellcome Trust/United Kingdom ; 202642/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Cytochrome P-450 Enzyme System/metabolism ; Endoplasmic Reticulum/*metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Membrane Proteins/*metabolism ; Mitochondrial Proteins/*metabolism ; Nerve Tissue Proteins/*metabolism ; Protein Domains ; Protein Folding ; Proteolysis ; Saccharomyces cerevisiae Proteins/metabolism ; Sterol 14-Demethylase/metabolism ; Ubiquitin-Protein Ligases/*metabolism ; },
abstract = {Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.},
}
@article {pmid32737398,
year = {2020},
author = {Tng, PYL and Carabajal Paladino, L and Verkuijl, SAN and Purcell, J and Merits, A and Leftwich, PT and Fragkoudis, R and Noad, R and Alphey, L},
title = {Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {413},
pmid = {32737398},
issn = {2399-3642},
support = {110117/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 200171/Z/15/7/WT_/Wellcome Trust/United Kingdom ; BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00001985/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Aedes/genetics/virology ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chikungunya Fever/*genetics/transmission/virology ; Chikungunya virus/*genetics/pathogenicity ; Culicidae/genetics/virology ; Humans ; Mosquito Vectors/genetics/virology ; Prevotella/genetics ; RNA, Guide/*genetics ; RNA, Viral/genetics ; Virus Replication/genetics ; },
abstract = {Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.},
}
@article {pmid32737393,
year = {2020},
author = {Jedwab, A and Vears, DF and Tse, C and Gyngell, C},
title = {Genetics experience impacts attitudes towards germline gene editing: a survey of over 1500 members of the public.},
journal = {Journal of human genetics},
volume = {65},
number = {12},
pages = {1055-1065},
pmid = {32737393},
issn = {1435-232X},
mesh = {CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*trends ; Genome/*genetics ; Genomics/*trends ; Germ-Line Mutation/genetics ; Humans ; Male ; Surveys and Questionnaires ; },
abstract = {CRISPR-Cas9 has revolutionised genome engineering and has the potential to radically change our approach to genetic disease. However, the potential for genetic modification of embryos has raised significant and complex ethical and social concerns. The scientific community have called for ongoing stakeholder consultation about Germline Gene Editing (GGE), in particular lay publics, to help guide policy, education, research and regulatory priorities. In response, we conducted a survey to gauge public support for GGE and describe the demographic, experiential and contextual factors that influence individual attitudes. Respondent support was measured across nine hypothetical medical and enhancement GGE applications. We received responses from 1537 participants across 67 countries. Respondents were generally supportive of GGE, particularly for medical applications. While the most opposition observed was among religious respondents, those with work experience in genetics or genomics also reported greater resistance to GGE. Personal or family-related experience with genetics or genomics, identifying as female and tertiary education were also associated with less support for GGE. Further research needs to explore a diverse range of community and group attitudes towards GGE; the reasons underlying demographic and experiential differences; and to determine where the public and genetics professionals draw the line on ethical implementation respectively.},
}
@article {pmid32736636,
year = {2020},
author = {Vidgren, V and Halinen, S and Tamminen, A and Olenius, S and Wiebe, MG},
title = {Engineering marine fungi for conversion of D-galacturonic acid to mucic acid.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {156},
pmid = {32736636},
issn = {1475-2859},
mesh = {Aquatic Organisms/genetics/*metabolism ; Ascomycota/genetics/*metabolism ; Biotechnology ; CRISPR-Cas Systems ; Hexuronic Acids/*metabolism ; Metabolic Engineering ; Sugar Acids/*metabolism ; Trichoderma/genetics/*metabolism ; },
abstract = {BACKGROUND: Two marine fungi, a Trichoderma sp. and a Coniochaeta sp., which can grow on D-galacturonic acid and pectin, were selected as hosts to engineer for mucic acid production, assessing the suitability of marine fungi for production of platform chemicals. The pathway for biotechnologcial production of mucic (galactaric) acid from D-galacturonic acid is simple and requires minimal modification of the genome, optimally one deletion and one insertion. D-Galacturonic acid, the main component of pectin, is a potential substrate for bioconversion, since pectin-rich waste is abundant.<br><br>RESULTS: Trichoderma sp. LF328 and Coniochaeta sp. MF729 were engineered using CRISPR-Cas9 to oxidize D-galacturonic acid to mucic acid, disrupting the endogenous pathway for D-galacturonic acid catabolism when inserting a gene encoding bacterial uronate dehydrogenase. The uronate dehydrogenase was expressed under control of a synthetic expression system, which fucntioned in both marine strains. The marine Trichoderma transformants produced 25&#xa0;g L[-1] mucic acid from D-galacturonic acid in equimolar amounts: the yield was 1.0 to 1.1&#xa0;g mucic acid [g D-galacturonic acid utilized][-1]. D-Xylose and lactose were the preferred co-substrates. The engineered marine Trichoderma sp. was more productive than the best Trichoderma reesei strain (D-161646) described in the literature to date, that had been engineered to produce mucic acid. With marine Coniochaeta transformants, D-glucose was the preferred co-substrate, but the highest yield was 0.82&#xa0;g&#xa0;g[-1]: a portion of D-galacturonic acid was still metabolized. Coniochaeta sp. transformants produced adequate pectinases to produce mucic acid from pectin, but Trichoderma sp. transformants did not.<br><br>CONCLUSIONS: Both marine species were successfully engineered using CRISPR-Cas9 and the synthetic expression system was functional in both species. Although Coniochaeta sp. transformants produced mucic acid directly from pectin, the metabolism of D-galacturonic acid was not completely disrupted and mucic acid amounts were low. The D-galacturonic pathway was completely disrupted in the transformants of the marine Trichoderma sp., which produced more mucic acid than a previously constructed T. reesei mucic acid producing strain, when grown under similar conditions. This demonstrated that marine fungi may be useful as production organisms, not only for native enzymes or bioactive compounds, but also for other compounds.},
}
@article {pmid32735884,
year = {2020},
author = {Karimian, A and Gorjizadeh, N and Alemi, F and Asemi, Z and Azizian, K and Soleimanpour, J and Malakouti, F and Targhazeh, N and Majidinia, M and Yousefi, B},
title = {CRISPR/Cas9 novel therapeutic road for the treatment of neurodegenerative diseases.},
journal = {Life sciences},
volume = {259},
number = {},
pages = {118165},
doi = {10.1016/j.lfs.2020.118165},
pmid = {32735884},
issn = {1879-0631},
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Therapy/*methods ; Humans ; Neurodegenerative Diseases/*therapy ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic Repeats)/Cas9 is a new genetic editing technology that can be a beneficial method to advance gene therapy. CRISPR technology is a defense system of some bacteria against invading viruses. Genome editing based on the CRISPR/Cas9 system is an efficient and potential technology that can be a viable alternative to traditional methods. This system is a compound of a short guide RNAs (gRNAs) for identifying the target DNA sequence and Cas9 protein as nuclease for breaking and cutting of DNA. In this review, recent advances in the CRISPR/Cas9-mediated genome editing tools are presented as well as their use in gene therapy strategies for the treatment of neurological disorders including Parkinson's disease, Alzheimer's disease, and Huntington's disease.},
}
@article {pmid32735817,
year = {2020},
author = {Crawford, K and Diaz Quiroz, JF and Koenig, KM and Ahuja, N and Albertin, CB and Rosenthal, JJC},
title = {Highly Efficient Knockout of a Squid Pigmentation Gene.},
journal = {Current biology : CB},
volume = {30},
number = {17},
pages = {3484-3490.e4},
pmid = {32735817},
issn = {1879-0445},
support = {DP5 OD023111/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chromatophores/cytology/*physiology ; Decapodiformes/embryology/enzymology/*genetics ; Embryo, Nonmammalian/cytology/*metabolism ; *Gene Knockout Techniques ; Phenotype ; *Pigmentation ; Tryptophan Oxygenase/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Seminal studies using squid as a model led to breakthroughs in neurobiology. The squid giant axon and synapse, for example, laid the foundation for our current understanding of the action potential [1], ionic gradients across cells [2], voltage-dependent ion channels [3], molecular motors [4-7], and synaptic transmission [8-11]. Despite their anatomical advantages, the use of squid as a model receded over the past several decades as investigators turned to genetically tractable systems. Recently, however, two key advances have made it possible to develop techniques for the genetic manipulation of squid. The first is the CRISPR-Cas9 system for targeted gene disruption, a largely species-agnostic method [12, 13]. The second is the sequencing of genomes for several cephalopod species [14-16]. If made genetically tractable, squid and other cephalopods offer a wealth of biological novelties that could spur discovery. Within invertebrates, not only do they possess by far the largest brains, they also express the most sophisticated behaviors [17]. In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18-20]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.},
}
@article {pmid32735657,
year = {2020},
author = {Makarova, KS and Timinskas, A and Wolf, YI and Gussow, AB and Siksnys, V and Venclovas, &#x10c; and Koonin, EV},
title = {Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense.},
journal = {Nucleic acids research},
volume = {48},
number = {16},
pages = {8828-8847},
pmid = {32735657},
issn = {1362-4962},
mesh = {Archaea/enzymology/*genetics ; Archaeal Proteins/chemistry/*genetics ; Bacteria/enzymology/*genetics ; Bacterial Proteins/chemistry/*genetics ; *CRISPR-Cas Systems ; Evolution, Molecular ; *Protein Domains ; },
abstract = {CRISPR-associated Rossmann Fold (CARF) and SMODS-associated and fused to various effector domains (SAVED) are key components of cyclic oligonucleotide-based antiphage signaling systems (CBASS) that sense cyclic oligonucleotides and transmit the signal to an effector inducing cell dormancy or death. Most of the CARFs are components of a CBASS built into type III CRISPR-Cas systems, where the CARF domain binds cyclic oligoA (cOA) synthesized by Cas10 polymerase-cyclase and allosterically activates the effector, typically a promiscuous ribonuclease. Additionally, this signaling pathway includes a ring nuclease, often also a CARF domain (either the sensor itself or a specialized enzyme) that cleaves cOA and mitigates dormancy or death induction. We present a comprehensive census of CARF and SAVED domains in bacteria and archaea, and their sequence- and structure-based classification. There are 10 major families of CARF domains and multiple smaller groups that differ in structural features, association with distinct effectors, and presence or absence of the ring nuclease activity. By comparative genome analysis, we predict specific functions of CARF and SAVED domains and partition the CARF domains into those with both sensor and ring nuclease functions, and sensor-only ones. Several families of ring nucleases functionally associated with sensor-only CARF domains are also predicted.},
}
@article {pmid32734287,
year = {2020},
author = {Khumsupan, P and Kozlowska, MA and Orr, DJ and Andreou, AI and Nakayama, N and Patron, N and Carmo-Silva, E and McCormick, AJ},
title = {Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.},
journal = {Journal of experimental botany},
volume = {71},
number = {19},
pages = {5963-5975},
doi = {10.1093/jxb/eraa316},
pmid = {32734287},
issn = {1460-2431},
support = {BB/I024488/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014130/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S015531/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M006468/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Mutation ; Phenotype ; *Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; },
abstract = {The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.},
}
@article {pmid32732833,
year = {2020},
author = {Fu, R and Fang, M and Xu, K and Ren, J and Zou, J and Su, L and Chen, X and An, P and Yu, D and Ka, M and Hai, T and Li, Z and Li, W and Yang, Y and Zhou, Q and Hu, Z},
title = {Generation of GGTA1-/-β2M-/-CIITA-/- Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions.},
journal = {Transplantation},
volume = {104},
number = {8},
pages = {1566-1573},
pmid = {32732833},
issn = {1534-6080},
mesh = {Allografts/supply &amp; distribution ; Animals ; Animals, Genetically Modified/immunology ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Female ; Galactosyltransferases/genetics/immunology ; Gene Knockout Techniques/methods ; Genes, MHC Class II/genetics/immunology ; Graft Rejection/immunology/*prevention &amp; control ; Graft Survival/genetics/immunology ; Heterografts/*immunology/transplantation ; Humans ; Male ; Mice ; Organ Transplantation/adverse effects/*methods ; Swine/genetics/immunology ; Transplantation, Heterologous/adverse effects/*methods ; beta 2-Microglobulin/genetics/immunology ; },
abstract = {BACKGROUND: Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II.<br><br>METHODS: We simultaneously depleted GGTA1, &#x3b2;2-microglobulin (&#x3b2;2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1&#x3b2;2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer.<br><br>RESULTS: The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of &#x3b1;-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-&#x3b2;2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts.<br><br>CONCLUSIONS: Taken together, these results demonstrate that elimination of GGTA1, &#x3b2;2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.},
}
@article {pmid32732424,
year = {2020},
author = {Lapinaite, A and Knott, GJ and Palumbo, CM and Lin-Shiao, E and Richter, MF and Zhao, KT and Beal, PA and Liu, DR and Doudna, JA},
title = {DNA capture by a CRISPR-Cas9-guided adenine base editor.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6503},
pages = {566-571},
pmid = {32732424},
issn = {1095-9203},
support = {T32 GM113770/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 GM061115/GM/NIGMS NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; T32 GM095450/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; S10 OD020062/OD/NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine/*chemistry ; Adenosine Deaminase/*chemistry/genetics ; CRISPR-Associated Protein 9/*chemistry/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/*chemistry ; Deamination ; Escherichia coli Proteins/*chemistry/genetics ; *Gene Editing ; },
abstract = {CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.},
}
@article {pmid32732286,
year = {2020},
author = {Nakamura, K and Noguchi, T and Takahara, M and Omori, Y and Furukawa, T and Katoh, Y and Nakayama, K},
title = {Anterograde trafficking of ciliary MAP kinase-like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {38},
pages = {13363-13376},
pmid = {32732286},
issn = {1083-351X},
mesh = {Amino Acid Motifs ; Cilia/genetics/*metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Protein Domains ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Protein Transport ; },
abstract = {ICK (also known as CILK1) is a mitogen-activated protein kinase-like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)-B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein-coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.},
}
@article {pmid32731837,
year = {2020},
author = {Yamaguchi, T and Uchida, E and Okada, T and Ozawa, K and Onodera, M and Kume, A and Shimada, T and Takahashi, S and Tani, K and Nasu, Y and Mashimo, T and Mizuguchi, H and Mitani, K and Maki, K},
title = {Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan.},
journal = {Human gene therapy},
volume = {31},
number = {19-20},
pages = {1043-1053},
pmid = {32731837},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/*administration &amp; dosage/genetics ; Humans ; Japan ; },
abstract = {The development of genome-editing technology could lead to breakthrough gene therapy. Genome editing has made it possible to easily knock out or modify a target gene, while current gene therapy using a virus vector or plasmid hampering modification with respect to gene replacement therapies. Clinical development using these genome-editing tools is progressing rapidly. However, it is also becoming clear that there is a possibility of unintended gene sequence modification or deletion, or the insertion of undesired genes, or the selection of cells with abnormalities in the cancer suppressor gene p53; these unwanted actions are not possible with current gene therapy. The Science Board of the Pharmaceuticals and Medical Devices Agency of Japan has compiled a report on the expected aspects of such genome-editing technology and the risks associated with it. This article summarizes the history of that discussion and compares the key concepts with information provided by other regulatory authorities.},
}
@article {pmid32731256,
year = {2020},
author = {Kuzmenko, A and Oguienko, A and Esyunina, D and Yudin, D and Petrova, M and Kudinova, A and Maslova, O and Ninova, M and Ryazansky, S and Leach, D and Aravin, AA and Kulbachinskiy, A},
title = {DNA targeting and interference by a bacterial Argonaute nuclease.},
journal = {Nature},
volume = {587},
number = {7835},
pages = {632-637},
pmid = {32731256},
issn = {1476-4687},
support = {MR/M019160/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Argonaute Proteins/*metabolism ; Bacteriophages/genetics/physiology ; Biocatalysis ; CRISPR-Cas Systems ; Clostridium butyricum/*enzymology/genetics/virology ; DNA/genetics/*metabolism ; DNA Breaks, Double-Stranded ; DNA Repair ; Exodeoxyribonuclease V/metabolism ; *Gene Silencing ; Plasmids/genetics/metabolism ; Sequence Homology, Nucleic Acid ; },
abstract = {Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes[1,2]. Argonautes are also present in many bacterial and archaeal species[3-5]. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference[6-10]. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.},
}
@article {pmid32730741,
year = {2020},
author = {Sofos, N and Feng, M and Stella, S and Pape, T and Fuglsang, A and Lin, J and Huang, Q and Li, Y and She, Q and Montoya, G},
title = {Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {741-757.e7},
doi = {10.1016/j.molcel.2020.07.008},
pmid = {32730741},
issn = {1097-4164},
mesh = {Adaptive Immunity/*physiology ; Archaeal Proteins/chemistry/physiology/ultrastructure ; CRISPR-Associated Proteins/*chemistry/*physiology/ultrastructure ; *Clustered Regularly Interspaced Short Palindromic Repeats/physiology ; Cryoelectron Microscopy ; DNA, Single-Stranded/metabolism ; Models, Molecular ; Protein Binding ; Protein Conformation ; RNA, Messenger/metabolism ; Structure-Activity Relationship ; Sulfolobus/genetics/physiology ; },
abstract = {Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.},
}
@article {pmid32729961,
year = {2021},
author = {Nan, Y and Ouyang, L and Chu, J},
title = {In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.},
journal = {Biotechnology and applied biochemistry},
volume = {68},
number = {5},
pages = {964-970},
doi = {10.1002/bab.1996},
pmid = {32729961},
issn = {1470-8744},
mesh = {Amidohydrolases/*genetics/metabolism ; Aspergillus niger/enzymology/*genetics ; Biomarkers/analysis/metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; },
abstract = {We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild-type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains.},
}
@article {pmid32729545,
year = {2020},
author = {Wang, M and Zhang, R and Li, J},
title = {CRISPR/cas systems redefine nucleic acid detection: Principles and methods.},
journal = {Biosensors &amp; bioelectronics},
volume = {165},
number = {},
pages = {112430},
pmid = {32729545},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; RNA ; },
abstract = {Methods that enable rapid, sensitive and specific analyses of nucleic acid sequences have positive effects on precise disease diagnostics and effective clinical treatments by providing direct insight into clinically relevant genetic information. Thus far, many CRISPR/Cas systems have been repurposed for diagnostic functions and are revolutionizing the accessibility of robust diagnostic tools due to their high flexibility, sensitivity and specificity. As RNA-guided targeted recognition effectors, Cas9 variants have been utilized for a variety of diagnostic applications, including biosensing assays, imaging assays and target enrichment for next-generation sequencing (NGS), thereby enabling the development of flexible and cost-effective tests. In addition, the ensuing discovery of Cas proteins (Cas12 and Cas13) with collateral cleavage activities has facilitated the development of numerous diagnostic tools for rapid and portable detection, and these tools have great potential for point-of-care settings. However, representative reviews proposed on this topic are mainly confined to classical biosensing applications; thus, a comprehensive and systematic description of this fast-developing field is required. In this review, based on the detection principle, we provide a detailed classification and comprehensive discussion of recent works that harness these CRISPR-based diagnostic tools from a new perspective. Furthermore, current challenges and future perspectives of CRISPR-based diagnostics are outlined.},
}
@article {pmid32729496,
year = {2020},
author = {Wang, L and Shen, X and Wang, T and Chen, P and Qi, N and Yin, BC and Ye, BC},
title = {A lateral flow strip combined with Cas9 nickase-triggered amplification reaction for dual food-borne pathogen detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {165},
number = {},
pages = {112364},
doi = {10.1016/j.bios.2020.112364},
pmid = {32729496},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA Primers ; *Deoxyribonuclease I ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; },
abstract = {Nucleic acid-based detection methods are accurate and rapid, which are widely-used in food-borne pathogen detection. However, traditional nucleic acid-based detection methods usually rely on special instruments, weakening their practicality for on-site tests in resource-limited locations. In this work, we developed a convenient and affordable method for food-borne pathogen detection based on a lateral flow strip combined with Cas9 nickase-triggered isothermal DNA amplification, which allows instrument-free and dual target detection. The genomic DNAs of two most common foodborne pathogens, Salmonella typhimurium and Escherichia coli, were simultaneously amplified in a one-pot reaction using specific sgRNAs and primers. The amplicons of genomic DNAs were double-labelled by digoxin/biotin and FITC/biotin tags, respectively, and directly visualized on a simple lateral flow strip. Our method exhibited a high specificity and sensitivity with a detection limit of 100 copies for genomic DNAs and 100 CFU/mL for bacteria. We believe that this method has potential to provide a convenient and low-cost point-of-care test for pathogen detection in the food quality surveillance.},
}
@article {pmid32729326,
year = {2021},
author = {Zhang, QS and Tiyaboonchai, A and Nygaard, S and Baradar, K and Major, A and Balaji, N and Grompe, M},
title = {Induced Liver Regeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1.},
journal = {Human gene therapy},
volume = {32},
number = {5-6},
pages = {294-301},
pmid = {32729326},
issn = {1557-7422},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Hepatocytes ; Liver Regeneration ; Mice ; *Tyrosinemias/genetics/therapy ; },
abstract = {The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.},
}
@article {pmid32729074,
year = {2021},
author = {Lerner, T and Kluesner, M and Tasakis, RN and Moriarity, BS and Papavasiliou, FN and Pecori, R},
title = {C-to-U RNA Editing: From Computational Detection to Experimental Validation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2181},
number = {},
pages = {51-67},
doi = {10.1007/978-1-0716-0787-9_4},
pmid = {32729074},
issn = {1940-6029},
mesh = {APOBEC-1 Deaminase/antagonists &amp; inhibitors/*genetics ; Animals ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Cytidine/chemistry/*genetics ; High-Throughput Nucleotide Sequencing ; Macrophages/cytology/*metabolism ; Mice ; RAW 264.7 Cells ; RNA Editing/*genetics ; RNA, Messenger/genetics ; Uridine/chemistry/*genetics ; },
abstract = {The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell types, with respect to which transcripts are edited and the level to which they are edited. When considering these potential sources of error and variability, an RNA-seq comparison between wild-type APOBEC1 sample and a matched control with an APOBEC1 knockout is a reliable method for the discrimination of true sites edited by APOBEC1. Here we present a detailed description of a method for studying APOBEC1 RNA editing, specifically in the murine macrophage cell line RAW 264.7. Our method covers the production of an APOBEC1 knockout cell line using the CRISPR/Cas9 system, through to experimental validation and quantification of editing sites (where we discuss a recently published algorithm (termed MultiEditR) which allows for the detection and quantification of RNA editing from Sanger sequencing). Importantly, this same protocol can be adapted to any RNA modification detectable by RNA-seq analysis for which the responsible protein is known.},
}
@article {pmid32728216,
year = {2020},
author = {Banik, SM and Pedram, K and Wisnovsky, S and Ahn, G and Riley, NM and Bertozzi, CR},
title = {Lysosome-targeting chimaeras for degradation of extracellular proteins.},
journal = {Nature},
volume = {584},
number = {7820},
pages = {291-297},
pmid = {32728216},
issn = {1476-4687},
support = {R01 CA227942/CA/NCI NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; T32 GM120007/GM/NIGMS NIH HHS/United States ; K00CA21245403/NH/NIH HHS/United States ; K00 CA212454/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antibodies/chemistry/metabolism ; Antigens, CD/metabolism ; Apolipoprotein E4/metabolism ; B7-H1 Antigen/metabolism ; CRISPR-Cas Systems ; Cell Line ; ErbB Receptors/metabolism ; Extracellular Space/*metabolism ; Female ; Glycopeptides/chemical synthesis/metabolism ; Humans ; Ligands ; Lysosomes/*metabolism ; Membrane Proteins/chemistry/*metabolism ; Mice ; Protein Domains ; Protein Transport ; *Proteolysis ; Receptor, IGF Type 2/metabolism ; Receptors, Transferrin/metabolism ; Recombinant Fusion Proteins/chemical synthesis/chemistry/*metabolism ; Solubility ; Substrate Specificity ; },
abstract = {The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)[1,2] and others (for example, dTAGs[3], Trim-Away[4], chaperone-mediated autophagy targeting[5] and SNIPERs[6]) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes[7]-are key agents in cancer, ageing-related diseases and autoimmune disorders[8], and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.},
}
@article {pmid32728076,
year = {2020},
author = {Pavani, G and Laurent, M and Fabiano, A and Cantelli, E and Sakkal, A and Corre, G and Lenting, PJ and Concordet, JP and Toueille, M and Miccio, A and Amendola, M},
title = {Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3778},
pmid = {32728076},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Engineering/*methods ; Cell Line ; Female ; *Gene Editing ; Gene Expression Regulation ; Hematopoietic Stem Cell Transplantation/*methods ; Hematopoietic Stem Cells/*metabolism ; Hemophilia A/therapy ; Humans ; Metabolic Diseases/therapy ; Mice ; Promoter Regions, Genetic/genetics ; Transplantation, Autologous/methods ; Transplantation, Heterologous ; alpha-Globins/genetics/metabolism ; },
abstract = {Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.},
}
@article {pmid32728052,
year = {2020},
author = {Gussow, AB and Park, AE and Borges, AL and Shmakov, SA and Makarova, KS and Wolf, YI and Bondy-Denomy, J and Koonin, EV},
title = {Machine-learning approach expands the repertoire of anti-CRISPR protein families.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3784},
pmid = {32728052},
issn = {2041-1723},
mesh = {Archaea/genetics/virology ; Bacteria/genetics/virology ; Bacteriophages/*genetics ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/genetics ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Datasets as Topic ; Gene Editing/methods ; Host-Parasite Interactions/genetics ; *Machine Learning ; Sequence Analysis, Protein/*methods ; Sequence Homology, Amino Acid ; Viral Proteins/*genetics ; },
abstract = {The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.},
}
@article {pmid32727858,
year = {2020},
author = {Repizo, GD and Espariz, M and Seravalle, JL and Díaz Miloslavich, JI and Steimbrüch, BA and Shuman, HA and Viale, AM},
title = {Acinetobacter baumannii NCIMB8209: a Rare Environmental Strain Displaying Extensive Insertion Sequence-Mediated Genome Remodeling Resulting in the Loss of Exposed Cell Structures and Defensive Mechanisms.},
journal = {mSphere},
volume = {5},
number = {4},
pages = {},
pmid = {32727858},
issn = {2379-5042},
support = {R21 AI115203/AI/NIAID NIH HHS/United States ; },
mesh = {Acinetobacter baumannii/drug effects/*genetics ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Multiple, Bacterial/*genetics ; Environmental Microbiology ; Genes, Bacterial/*genetics ; Genetic Variation ; *Genomic Islands ; Genomics ; Phylogeny ; Plants/microbiology ; Virulence Factors/genetics ; Whole Genome Sequencing ; },
abstract = {Acinetobacter baumannii represents nowadays an important nosocomial pathogen of poorly defined reservoirs outside the clinical setting. Here, we conducted whole-genome sequencing analysis of the Acinetobacter sp. NCIMB8209 collection strain, isolated in 1943 from the aerobic degradation (retting) of desert guayule shrubs. Strain NCIMB8209 contained a 3.75-Mb chromosome and a plasmid of 134 kb. Phylogenetic analysis based on core genes indicated NCIMB8209 affiliation to A. baumannii, a result supported by the identification of a chromosomal blaOXA-51-like gene. Seven genomic islands lacking antimicrobial resistance determinants, 5 regions encompassing phage-related genes, and notably, 93 insertion sequences (IS) were found in this genome. NCIMB8209 harbors most genes linked to persistence and virulence described in contemporary A. baumannii clinical strains, but many of the genes encoding components of surface structures are interrupted by IS. Moreover, defense genetic islands against biological aggressors such as type 6 secretion systems or CRISPR-cas are absent from this genome. These findings correlate with a low capacity of NCIMB8209 to form biofilm and pellicle, low motility on semisolid medium, and low virulence toward Galleria mellonella and Caenorhabditis elegans Searching for catabolic genes and concomitant metabolic assays revealed the ability of NCIMB8209 to grow on a wide range of substances produced by plants, including aromatic acids and defense compounds against external aggressors. All the above features strongly suggest that NCIMB8209 has evolved specific adaptive features to a particular environmental niche. Moreover, they also revealed that the remarkable genetic plasticity identified in contemporary A. baumannii clinical strains represents an intrinsic characteristic of the species.IMPORTANCEAcinetobacter baumannii is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) opportunistic pathogen, with poorly defined natural habitats/reservoirs outside the clinical setting. A. baumannii arose from the Acinetobacter calcoaceticus-A. baumannii complex as the result of a population bottleneck, followed by a recent population expansion from a few clinically relevant clones endowed with an arsenal of resistance and virulence genes. Still, the identification of virulence traits and the evolutionary paths leading to a pathogenic lifestyle has remained elusive, and thus, the study of nonclinical ("environmental") A. baumannii isolates is necessary. We conducted here comparative genomic and virulence studies on A. baumannii NCMBI8209 isolated in 1943 from the microbiota responsible for the decomposition of guayule, and therefore well differentiated both temporally and epidemiologically from the multidrug-resistant strains that are predominant nowadays. Our work provides insights on the adaptive strategies used by A. baumannii to escape from host defenses and may help the adoption of measures aimed to limit its further dissemination.},
}
@article {pmid32727031,
year = {2020},
author = {Liang, M and Sui, T and Liu, Z and Chen, M and Liu, H and Shan, H and Lai, L and Li, Z},
title = {AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects.},
journal = {Cells},
volume = {9},
number = {8},
pages = {},
pmid = {32727031},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Transfection ; },
abstract = {The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly "shut off" nuclease activity of both CBE and ABE systems.},
}
@article {pmid32726977,
year = {2020},
author = {Chokhachi Baradaran, P and Kozovska, Z and Furdova, A and Smolkova, B},
title = {Targeting Epigenetic Modifications in Uveal Melanoma.},
journal = {International journal of molecular sciences},
volume = {21},
number = {15},
pages = {},
pmid = {32726977},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; *DNA Methylation ; *DNA, Neoplasm/genetics/metabolism ; *Epigenesis, Genetic ; *Gene Expression Regulation, Neoplastic ; *Genetic Therapy ; Histones/genetics/metabolism ; Humans ; *Melanoma/genetics/metabolism/therapy ; Neoplasm Proteins/genetics/metabolism ; Protein Processing, Post-Translational ; *Uveal Neoplasms/genetics/metabolism/therapy ; },
abstract = {Uveal melanoma (UM), the most common intraocular malignancy in adults, is a rare subset of melanoma. Despite effective primary therapy, around 50% of patients will develop the metastatic disease. Several clinical trials have been evaluated for patients with advanced UM, though outcomes remain dismal due to the lack of efficient therapies. Epigenetic dysregulation consisting of aberrant DNA methylation, histone modifications, and small non-coding RNA expression, silencing tumor suppressor genes, or activating oncogenes, have been shown to play a significant role in UM initiation and progression. Given that there is no evidence any approach improves results so far, adopting combination therapies, incorporating a new generation of epigenetic drugs targeting these alterations, may pave the way for novel promising therapeutic options. Furthermore, the fusion of effector enzymes with nuclease-deficient Cas9 (dCas9) in clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system equips a potent tool for locus-specific erasure or establishment of DNA methylation as well as histone modifications and, therefore, transcriptional regulation of specific genes. Both, CRISPR-dCas9 potential for driver epigenetic alterations discovery, and possibilities for their targeting in UM are highlighted in this review.},
}
@article {pmid32726578,
year = {2020},
author = {Hickey, KL and Dickson, K and Cogan, JZ and Replogle, JM and Schoof, M and D'Orazio, KN and Sinha, NK and Hussmann, JA and Jost, M and Frost, A and Green, R and Weissman, JS and Kostova, KK},
title = {GIGYF2 and 4EHP Inhibit Translation Initiation of Defective Messenger RNAs to Assist Ribosome-Associated Quality Control.},
journal = {Molecular cell},
volume = {79},
number = {6},
pages = {950-962.e6},
pmid = {32726578},
issn = {1097-4164},
support = {R37 GM059425/GM/NIGMS NIH HHS/United States ; T32 GM007445/GM/NIGMS NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 AG041826/AG/NIA NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; DP5 OD028147/OD/NIH HHS/United States ; K99 GM130964/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carrier Proteins/*genetics ; Eukaryotic Initiation Factor-4E/*genetics ; Humans ; Mice ; *Peptide Chain Initiation, Translational ; Protein Biosynthesis/genetics ; Protein Processing, Post-Translational/genetics ; Quality Control ; RNA, Messenger/genetics ; Ribosomes/*genetics ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Ribosome-associated quality control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to degradation of faulty mRNA and partially synthesized polypeptides. Here we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific inhibition of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.},
}
@article {pmid32725389,
year = {2020},
author = {Sung, CK and Yim, H},
title = {CRISPR-mediated promoter de/methylation technologies for gene regulation.},
journal = {Archives of pharmacal research},
volume = {43},
number = {7},
pages = {705-713},
pmid = {32725389},
issn = {1976-3786},
support = {SC2 GM122686/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Methylation ; Gene Expression Regulation/genetics ; Humans ; Promoter Regions, Genetic/*genetics ; },
abstract = {DNA methylation on cytosines of CpG dinucleotides is well established as a basis of epigenetic regulation in mammalian cells. Since aberrant regulation of DNA methylation in promoters of tumor suppressor genes or proto-oncogenes may contribute to the initiation and progression of various types of human cancer, sequence-specific methylation and demethylation technologies could have great clinical benefit. The CRISPR-Cas9 protein with a guide RNA can target DNA sequences regardless of the methylation status of the target site, making this system superb for precise methylation editing and gene regulation. Targeted methylation-editing technologies employing the dCas9 fusion proteins have been shown to be highly effective in gene regulation without altering the DNA sequence. In this review, we discuss epigenetic alterations in tumorigenesis as well as various dCas9 fusion technologies and their usages in site-specific methylation editing and gene regulation.},
}
@article {pmid32725371,
year = {2021},
author = {Thiesler, H and Beimdiek, J and Hildebrandt, H},
title = {Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {78},
number = {4},
pages = {1637-1653},
pmid = {32725371},
issn = {1420-9071},
mesh = {Animals ; Antigens, CD/genetics ; Antigens, Differentiation, B-Lymphocyte/genetics ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Feedback, Physiological/drug effects ; Golgi Apparatus/drug effects/genetics ; Humans ; Inflammation/*genetics/immunology/pathology ; Lipopolysaccharides/toxicity ; Macrophage Activation/drug effects/immunology ; Macrophages/drug effects/immunology/metabolism ; Mice ; Microglia/immunology/*metabolism/pathology ; Phagocytosis/drug effects ; Sialic Acid Binding Immunoglobulin-like Lectins/*genetics/immunology ; Sialic Acids/*genetics/immunology ; },
abstract = {Polysialic acid (polySia) emerges as a novel regulator of microglia activity. We recently identified polysialylated proteins in the Golgi compartment of murine microglia that are released in response to inflammatory stimulation. Since exogenously added polySia is able to attenuate the inflammatory response, we proposed that the release of polysialylated proteins constitutes a mechanism for negative feedback regulation of microglia activation. Here, we demonstrate that translocation of polySia from the Golgi to the cell surface can be induced by calcium depletion of the Golgi compartment and that polysialylated proteins are continuously released for at least 24 h after the onset of inflammatory stimulation. The latter was unexpected, because polySia signals detected by immunocytochemistry are rapidly depleted. However, it indicates that the amount of released polySia is much higher than anticipated based on immunostaining. This may be crucial for microglial responses during traumatic brain injury (TBI), as we detected polySia signals in activated microglia around a stab wound in the adult mouse brain. In BV2 microglia, the putative polySia receptor Siglec-E is internalized during lipopolysaccharide (LPS)-induced activation and in response to polySia exposure, indicating interaction. Correspondingly, CRISPR/Cas9-mediated Siglec-E knockout prevents inhibition of pro inflammatory activation by exogenously added polySia and leads to a strong increase of the LPS response. A comparable increase of LPS-induced activation has been observed in microglia with abolished polySia synthesis. Together, these results indicate that the release of the microglia-intrinsic polySia pool, as implicated in TBI, inhibits the inflammatory response by acting as a trans-activating ligand of Siglec-E.},
}
@article {pmid32724143,
year = {2020},
author = {Udompaisarn, S and Toopaang, W and Sae-Ueng, U and Srisuksam, C and Wichienchote, N and Wasuwan, R and Nahar, NAS and Tanticharoen, M and Amnuaykanjanasin, A},
title = {The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12630},
pmid = {32724143},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; Beauveria/*cytology/*enzymology/isolation &amp; purification/pathogenicity ; CRISPR-Cas Systems/genetics ; Cell Wall/*enzymology/ultrastructure ; DNA End-Joining Repair/genetics ; Fluorescence ; Fungal Proteins/*metabolism ; Gene Editing ; Genetic Complementation Test ; Genetic Loci ; Insecta/microbiology ; Microbial Viability ; Mutagenesis/genetics ; Mutation/genetics ; Phagocytosis ; Polyketide Synthases/*metabolism ; Spores, Fungal/physiology/ultrastructure ; },
abstract = {Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.},
}
@article {pmid32724069,
year = {2020},
author = {Jeong, SY and Lyu, J and Kim, JA and Oh, IH},
title = {Ryk modulates the niche activity of mesenchymal stromal cells by fine-tuning canonical Wnt signaling.},
journal = {Experimental &amp; molecular medicine},
volume = {52},
number = {7},
pages = {1140-1151},
pmid = {32724069},
issn = {2092-6413},
support = {2017M3A9B3061947//National Research Foundation of Korea (NRF)/International ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Colony-Forming Units Assay ; Hematopoiesis ; Homozygote ; Ligands ; Mesenchymal Stem Cells/*metabolism ; Mice, Inbred C57BL ; Protein Domains ; Receptor Protein-Tyrosine Kinases/chemistry/*metabolism ; *Wnt Signaling Pathway ; },
abstract = {The importance of modulating the intensity of Wnt signaling has been highlighted in various biological models, but their mechanisms remain unclear. In this study, we found that Ryk-an atypical Wnt receptor with a pseudokinase domain-has a Wnt-modulating effect in bone marrow stromal cells to control hematopoiesis-supporting activities. We first found that Ryk is predominantly expressed in the mesenchymal stromal cells (MSCs) of the bone marrow (BM) compared with hematopoietic cells. Downregulation of Ryk in MSCs decreased their clonogenic activity and ability to support self-renewing expansion of primitive hematopoietic progenitors (HPCs) in response to canonical Wnt ligands. In contrast, under high concentrations of Wnt, Ryk exerted suppressive effects on the transactivation of target genes and HPC-supporting effects in MSCs, thus fine-tuning the signaling intensity of Wnt in BM stromal cells. This ability of Ryk to modulate the HPC-supporting niche activity of MSCs was abrogated by induction of deletion mutants of Ryk lacking the intracellular domain or extracellular domain, indicating that the pseudokinase-containing intracellular domain mediates the Wnt-modulating effects in response to extracellular Wnt ligands. These findings indicate that the ability of the BM microenvironment to respond to extracellular signals and support hematopoiesis may be fine-tuned by Ryk via modulation of Wnt signaling intensity to coordinate hematopoietic activity.},
}
@article {pmid32724054,
year = {2020},
author = {Jin, YH and Liao, B and Migaud, H and Davie, A},
title = {Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12600},
pmid = {32724054},
issn = {2045-2322},
mesh = {Animals ; Aquaculture ; Argonaute Proteins/*genetics ; *CRISPR-Cas Systems ; Fertilization in Vitro ; *Founder Effect ; Gene Knockdown Techniques ; Microinjections ; *Mutation ; Tilapia/*genetics/physiology ; },
abstract = {The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0.},
}
@article {pmid32723866,
year = {2020},
author = {Stachler, AE and Wörtz, J and Alkhnbashi, OS and Turgeman-Grott, I and Smith, R and Allers, T and Backofen, R and Gophna, U and Marchfelder, A},
title = {Adaptation induced by self-targeting in a type I-B CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {39},
pages = {13502-13515},
pmid = {32723866},
issn = {1083-351X},
mesh = {Adaptation, Physiological/*genetics ; CRISPR-Cas Systems/*genetics ; DNA, Archaeal/genetics ; Genome, Archaeal/genetics ; Haloferax volcanii/cytology/*genetics ; High-Throughput Nucleotide Sequencing ; },
abstract = {Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas-mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.},
}
@article {pmid32723863,
year = {2020},
author = {Van Eck, J},
title = {Applying gene editing to tailor precise genetic modifications in plants.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {38},
pages = {13267-13276},
pmid = {32723863},
issn = {1083-351X},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Plant ; Plants, Genetically Modified/*genetics ; },
abstract = {The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering, and trait modification for crop improvement. Development of genome editing methodologies began ∼20 years ago, first with meganucleases and followed by zinc finger nucleases, transcriptional activator-like effector nucleases and, most recently, clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species, including important food crops, such as maize, wheat, rice, and potatoes. This review summarizes the various approaches, including the most recent designs being used to make modifications from as small as a single-base-pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock out or modulate through activation and repression. Also discussed are prerequisites necessary for CRISPR/Cas-mediated editing as well as the current challenges.},
}
@article {pmid32721120,
year = {2020},
author = {Hoellerbauer, P and Kufeld, M and Arora, S and Wu, HJ and Feldman, HM and Paddison, PJ},
title = {A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures.},
journal = {Cancer reports (Hoboken, N.J.)},
volume = {3},
number = {5},
pages = {e1269},
pmid = {32721120},
issn = {2573-8348},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; P30 DK056465/DK/NIDDK NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; R01 CA190957/CA/NCI NIH HHS/United States ; T32 CA080416/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Glioblastoma/*genetics/pathology ; Humans ; Neoplastic Stem Cells/*pathology ; Primary Cell Culture/*methods ; Proof of Concept Study ; RNA-Seq ; },
abstract = {BACKGROUND: CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.<br><br>AIMS: To create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient-derived glioblastoma stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments.<br><br>METHODS AND RESULTS: We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl 3'phosphorothioate-modified sgRNAs and purified Cas9 protein. Insertion-deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3&#x2009;days, without the need to create clonal lines for simple loss-of-function experiments. Using Western blotting, we observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. Furthermore, by using RNA-seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in glioblastoma, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.<br><br>CONCLUSION: Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.},
}
@article {pmid32719542,
year = {2020},
author = {Cai, EP and Ishikawa, Y and Zhang, W and Leite, NC and Li, J and Hou, S and Kiaf, B and Hollister-Lock, J and Yilmaz, NK and Schiffer, CA and Melton, DA and Kissler, S and Yi, P},
title = {Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes.},
journal = {Nature metabolism},
volume = {2},
number = {9},
pages = {934-945},
pmid = {32719542},
issn = {2522-5812},
support = {P30 DK036836/DK/NIDDK NIH HHS/United States ; R01 DK120445/DK/NIDDK NIH HHS/United States ; T32 DK007260/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity/drug effects ; *CRISPR-Cas Systems ; Diabetes Mellitus, Type 1/*drug therapy/immunology/pathology ; Endoplasmic Reticulum Stress ; Enzyme Inhibitors/pharmacology ; Female ; *Genome-Wide Association Study ; Induced Pluripotent Stem Cells/immunology ; Insulin-Secreting Cells/*drug effects/immunology/pathology ; Islets of Langerhans Transplantation ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Knockout ; Monoamine Oxidase/*drug effects ; Mutation ; Pargyline/pharmacology ; },
abstract = {Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.},
}
@article {pmid32719478,
year = {2021},
author = {Umkehrer, C and Holstein, F and Formenti, L and Jude, J and Froussios, K and Neumann, T and Cronin, SM and Haas, L and Lipp, JJ and Burkard, TR and Fellner, M and Wiesner, T and Zuber, J and Obenauf, AC},
title = {Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.},
journal = {Nature biotechnology},
volume = {39},
number = {2},
pages = {174-178},
pmid = {32719478},
issn = {1546-1696},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; *Cell Separation ; Clone Cells/*metabolism ; Female ; *Genes, Reporter ; Humans ; Melanoma/pathology ; Mice, Inbred C57BL ; Mitogen-Activated Protein Kinase Kinases/antagonists &amp; inhibitors ; Protein Kinase Inhibitors/pharmacology ; raf Kinases/antagonists &amp; inhibitors ; },
abstract = {We developed a functional lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise clonal tracing of millions of cells with the ability to retrospectively isolate founding clones alive before and during selection, allowing functional experiments. Using CaTCH, we captured rare clones representing as little as 0.001% of a population and investigated the emergence of resistance to targeted melanoma therapy in vivo.},
}
@article {pmid32718240,
year = {2020},
author = {Rocha, LFM and Braga, LAM and Mota, FB},
title = {Gene Editing for Treatment and Prevention of Human Diseases: A Global Survey of Gene Editing-Related Researchers.},
journal = {Human gene therapy},
volume = {31},
number = {15-16},
pages = {852-862},
doi = {10.1089/hum.2020.136},
pmid = {32718240},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Research ; *Genetic Therapy ; *Genome, Human ; Humans ; Research Personnel/psychology/*statistics &amp; numerical data ; Surveys and Questionnaires ; },
abstract = {In the next decades, gene editing technologies are expected to be used in the treatment and prevention of human diseases. Yet, the future uses of gene editing in medicine are still unknown, including its applicability and effectiveness to the treatment and prevention of infectious diseases, cancer, and monogenic and polygenic hereditary diseases. This study aims to address this gap by analyzing the views of over 1,000 gene editing-related researchers from all over the world. Some of our survey results show that, in the next 10 years, DNA double-strand breaks are expected to be the main method for gene editing, and CRISPR-Cas systems to be the mainstream programmable nuclease. In the same period, gene editing is expected to have more applicability and effectiveness to treat and prevent infectious diseases and cancer. Off-targeting mutations, reaching therapeutic levels of editing efficiency, difficulties in targeting specific tissues in vivo, and regulatory and ethical challenges are among the most relevant factors that might hamper the use of gene editing in humans. In conclusion, our results suggest that gene editing might become a reality to the treatment and prevention of a variety of human diseases in the coming 10 years. If the future confirms these researchers' expectations, gene editing could change the way medicine, health systems, and public health deal with the treatment and prevention of human diseases.},
}
@article {pmid32717311,
year = {2020},
author = {Sabzehzari, M and Zeinali, M and Naghavi, MR},
title = {CRISPR-based metabolic editing: Next-generation metabolic engineering in plants.},
journal = {Gene},
volume = {759},
number = {},
pages = {144993},
doi = {10.1016/j.gene.2020.144993},
pmid = {32717311},
issn = {1879-0038},
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; Metabolic Engineering/*methods ; Plant Breeding/*methods ; },
abstract = {Plants generate many secondary metabolites, so called phyto-metabolites, which can be used as toxins, dyes, drugs, and insecticides in bio-warfare plus bio-terrorism, industry, medicine, and agriculture, respectively. To 2013, the first generation metabolic engineering approaches like miRNA-based manipulation were widely adopted by researchers in biosciences. However, the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) genome editing system revolutionized metabolic engineering due to its unique features so that scientists could manipulate the biosynthetic pathways of phyto-metabolites through approaches like miRNA-mediated CRISPR-Cas9. According to the increasing importance of the genome editing in plant sciences, we discussed the current findings on CRISPR-based manipulation of phyto-metabolites in plants, especially medicinal ones, and suggested the ideas to phyto-metabolic editing.},
}
@article {pmid32717029,
year = {2020},
author = {Basar, R and Daher, M and Uprety, N and Gokdemir, E and Alsuliman, A and Ensley, E and Ozcan, G and Mendt, M and Hernandez Sanabria, M and Kerbauy, LN and Nunez Cortes, AK and Li, L and Banerjee, PP and Muniz-Feliciano, L and Acharya, S and Fowlkes, NW and Lu, J and Li, S and Mielke, S and Kaplan, M and Nandivada, V and Bdaiwi, M and Kontoyiannis, AD and Li, Y and Liu, E and Ang, S and Marin, D and Brunetti, L and Gundry, MC and Turk, R and Schubert, MS and Rettig, GR and McNeill, MS and Kurgan, G and Behlke, MA and Champlin, R and Shpall, EJ and Rezvani, K},
title = {Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells.},
journal = {Blood advances},
volume = {4},
number = {14},
pages = {3357-3367},
pmid = {32717029},
issn = {2473-9537},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA061508/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Receptors, Glucocorticoid/genetics ; T-Lymphocytes ; },
abstract = {Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.},
}
@article {pmid32715554,
year = {2020},
author = {Chaverra-Rodriguez, D and Dalla Benetta, E and Heu, CC and Rasgon, JL and Ferree, PM and Akbari, OS},
title = {Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein.},
journal = {Insect molecular biology},
volume = {29},
number = {6},
pages = {569-577},
doi = {10.1111/imb.12663},
pmid = {32715554},
issn = {1365-2583},
support = {R21AI111175//NIH/NIAID/International ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Female ; *Gene Editing ; Germ Cells/*metabolism ; Insect Proteins/*genetics ; Male ; *Mutagenesis ; Ovary ; Ribonucleoproteins/*genetics ; Wasps/*genetics/growth &amp; development ; },
abstract = {CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used the Drosophila melanogaster-derived peptide 'P2C' fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of the cinnabar gene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR-applied genetic manipulation in this and other rising model organisms.},
}
@article {pmid32713893,
year = {2020},
author = {Wake, Y and Kaneko, T},
title = {Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation.},
journal = {The Journal of reproduction and development},
volume = {66},
number = {5},
pages = {469-473},
pmid = {32713893},
issn = {1348-4400},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/*methods ; Embryo Transfer ; Female ; Fluorescent Dyes/pharmacology ; Gene Editing/*methods ; Genome ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Microinjections ; Microscopy, Fluorescence ; RNA, Guide ; },
abstract = {Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.},
}
@article {pmid32712926,
year = {2021},
author = {Tuck, AC and Bühler, M},
title = {Long Non-coding RNA Depletion Using Self-Cleaving Ribozymes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2167},
number = {},
pages = {287-301},
doi = {10.1007/978-1-0716-0716-9_16},
pmid = {32712926},
issn = {1940-6029},
support = {WT103977/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Stem Cells/enzymology/*metabolism ; Gene Editing/*methods ; Mice ; Nucleic Acid Conformation ; RNA, Catalytic/*genetics/metabolism ; RNA, Long Noncoding/*genetics/metabolism ; RNA, Viral/*genetics ; Recombination, Genetic ; },
abstract = {Some long non-coding RNA (lncRNA) genes encode a functional RNA product, whereas others act as DNA elements or via the act of transcription . We describe here a ribozyme-based approach to deplete an endogenous lncRNA in mouse embryonic stem cells, with minimal disruption of its gene. This enables the role of the lncRNA product to be tested.},
}
@article {pmid32712922,
year = {2021},
author = {Berkhout, B and Gao, Z and Herrera-Carrillo, E},
title = {Design and Evaluation of Guide RNA Transcripts with a 3'-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2167},
number = {},
pages = {205-224},
pmid = {32712922},
issn = {1940-6029},
support = {R01 AI145045/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; Blotting, Northern ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/*metabolism ; Endonucleases/genetics/metabolism ; Enzyme Assays/methods ; Flow Cytometry ; Gene Editing/*methods ; Gene Knockout Techniques ; Gene Silencing ; Genes, Reporter ; Genetic Vectors ; HEK293 Cells ; HeLa Cells ; Hepatitis Delta Virus/*genetics/*metabolism ; Humans ; INDEL Mutation ; Luciferases ; RNA, Catalytic/genetics/*metabolism ; RNA, Guide/*genetics/metabolism ; Receptors, CCR5/genetics ; },
abstract = {The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5' and 3'-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5' terminus, but the Pol III enzyme generates small RNAs with 3' U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.},
}
@article {pmid32712502,
year = {2020},
author = {Molina, R and Sofos, N and Montoya, G},
title = {Structural basis of CRISPR-Cas Type III prokaryotic defence systems.},
journal = {Current opinion in structural biology},
volume = {65},
number = {},
pages = {119-129},
doi = {10.1016/j.sbi.2020.06.010},
pmid = {32712502},
issn = {1879-033X},
mesh = {*Archaea/genetics ; *Bacteria/genetics ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Evolution, Molecular ; Plasmids ; },
abstract = {CRISPR loci and CRISPR-associated (Cas) genes encode an adaptive immune system that protects many bacterial and almost all archaea against invasive genetic elements from bacteriophages and plasmids. Several classes of CRISPR systems have been characterized, of which the type III CRISPR systems exhibit the most unique functions. Members of type III cleave both RNA and DNA not only through their corresponding effector complexes but also by CRISPR-Cas associated proteins activated by second messengers produced by those effector complexes. Furthermore, the recent discovery of second messenger degrading proteins called ring nucleases adds an extra regulatory layer to fine-tune these immunity systems. Here, we review the defense mechanisms that govern type III CRISPR interference immunity systems focusing on the structural information available.},
}
@article {pmid32711817,
year = {2020},
author = {Hohmann, SS and Ilieva, M and Michel, TM},
title = {In vitro models for ASD-patient-derived iPSCs and cerebral organoids.},
journal = {Progress in molecular biology and translational science},
volume = {173},
number = {},
pages = {355-375},
doi = {10.1016/bs.pmbts.2020.04.019},
pmid = {32711817},
issn = {1878-0814},
mesh = {Autism Spectrum Disorder/*pathology ; Brain/growth &amp; development/*pathology ; CRISPR-Cas Systems/genetics ; Humans ; Induced Pluripotent Stem Cells/*pathology ; *Models, Biological ; Organoids/*pathology ; },
abstract = {Autism spectrum disorder (ASD) is a set of pervasive neurodevelopmental disorders. The causation is multigenic in most cases, which makes it difficult to model the condition in vitro. Advances in pluripotent stem cell technology has made it possible to generate in vitro models of human brain development. Induced pluripotent stem cells (iPSCs) can be generated from somatic cells and have the ability to differentiate to all of the body's cells. This chapter aims to give an overview of the iPSC technology for generating neural cells and cerebral organoids as models for neurodevelopment and how these models are utilized in the study of ASD. The combination of iPSC technology and the genetic modification tool CRISPR/Cas9 is described, and current limitations and future perspectives of iPSC technology is discussed.},
}
@article {pmid32711280,
year = {2021},
author = {Liu, H and Wang, J and Zeng, H and Liu, X and Jiang, W and Wang, Y and Ouyang, W and Tang, X},
title = {RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification.},
journal = {Food chemistry},
volume = {334},
number = {},
pages = {127608},
doi = {10.1016/j.foodchem.2020.127608},
pmid = {32711280},
issn = {1873-7072},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Endodeoxyribonucleases/genetics ; Fluorescence ; Food Analysis/*methods ; Food Contamination/*analysis ; Food Microbiology/*methods ; Food Safety ; Meat ; Nucleic Acid Amplification Techniques/*methods ; Plants, Genetically Modified/genetics ; RNA, Guide ; Recombinases/genetics ; Sensitivity and Specificity ; },
abstract = {Food analysis to ensure food safety and quality are relevant to all countries. This study aimed to develop a detection technique by combining recombinase polymerase amplification with CRISPR-Cas12a for food safety (termed RPA-Cas12a-FS). Our data showed that this novel method could be detected via fluorescence intensity for the molecular identification of foodborne pathogenic bacteria, genetically modified crops, and meat adulteration. After optimization, the sensitivity and stability of RPA-Cas12a-FS was further enhanced. The RPA-Cas12a-FS system could specifically detect target gene levels as low as 10 copies in 45 min at 37 °C. The RPA-Cas12a-FS system was sensitive both using standard samples in the lab and using samples from the field, which indicated that this detection method was practical. In conclusion, a simple, rapid, and highly sensitive detection method based on CRISPR-Cas12a was developed for molecular identification in the food safety field without requiring technical expertise or ancillary equipment.},
}
@article {pmid32711250,
year = {2020},
author = {Niu, G and Jin, Z and Zhang, C and He, D and Gao, X and Zou, C and Zhang, W and Ding, J and Das, BC and Severinov, K and Hitzeroth, II and Debata, PR and Ma, X and Tian, X and Gao, Q and Wu, J and You, Z and Tian, R and Cui, Z and Fan, W and Xie, W and Huang, Z and Cao, C and Xu, W and Xie, H and Xu, H and Tang, X and Wang, Y and Yu, Z and Han, H and Tan, S and Chen, S and Hu, Z},
title = {An effective vaginal gel to deliver CRISPR/Cas9 system encapsulated in poly (β-amino ester) nanoparticles for vaginal gene therapy.},
journal = {EBioMedicine},
volume = {58},
number = {},
pages = {102897},
pmid = {32711250},
issn = {2352-3964},
mesh = {Administration, Intravaginal ; Animals ; Bentonite/chemistry ; CRISPR-Cas Systems ; Cells, Cultured ; Cervix Uteri/chemistry/*cytology ; Endogenous Retroviruses/drug effects/*genetics ; Female ; Gene Dosage/*drug effects ; Gene Editing ; Genetic Therapy ; Mice ; Models, Animal ; Nanoparticles ; Plasmids/administration &amp; dosage/genetics ; Polymers/*chemistry ; RNA, Guide/*administration &amp; dosage ; Silicates/chemistry ; Swine ; Vaginal Creams, Foams, and Jellies ; },
abstract = {BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery.<br><br>METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (&#x3b2;-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT).<br><br>FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity.<br><br>INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract.<br><br>FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.},
}
@article {pmid32711027,
year = {2020},
author = {Xu, Q and Zhang, Z and Zhao, L and Qin, Y and Cai, H and Geng, Z and Zhu, X and Zhang, W and Zhang, Y and Tan, J and Wang, J and Zhou, J},
title = {Tropism-facilitated delivery of CRISPR/Cas9 system with chimeric antigen receptor-extracellular vesicles against B-cell malignancies.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {326},
number = {},
pages = {455-467},
doi = {10.1016/j.jconrel.2020.07.033},
pmid = {32711027},
issn = {1873-4995},
mesh = {B-Lymphocytes ; CRISPR-Cas Systems ; *Extracellular Vesicles ; Gene Editing ; Humans ; *Neoplasms/genetics ; *Receptors, Chimeric Antigen/genetics ; Tropism ; },
abstract = {The CRISPR/Cas9 system is an efficient genome-editing system that has been successfully applied in the field of gene therapy. However, clinical applications of the CRISPR/Cas9 system are limited by the delivery method and safety concerns. Extracellular Vesicles (EVs) can be released from almost every type of cell, and they act as shuttles to convey molecules between cells. Here, we used EVs derived from epithelial cells as a biosafety delivery platform for the CRISPR/Cas9 system and modified the EVs with a chimeric-antigen receptor (CAR) to give them selective tropism to tumors. Compared to normal EVs, CAR-EVs accumulated in cancer tumors rapidly and released the CRISPR/Cas9 system targeting the MYC oncogene efficiently, both in vitro and in vivo. Taken together, the combination of EV and CAR was confirmed to be a novel strategy facilitating the use of natural gene therapy platforms in cancer treatment in this proof-of-concept research.},
}
@article {pmid32710848,
year = {2020},
author = {Dabelsteen, S and Pallesen, EMH and Marinova, IN and Nielsen, MI and Adamopoulou, M and Rømer, TB and Levann, A and Andersen, MM and Ye, Z and Thein, D and Bennett, EP and Büll, C and Moons, SJ and Boltje, T and Clausen, H and Vakhrushev, SY and Bagdonaite, I and Wandall, HH},
title = {Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.},
journal = {Developmental cell},
volume = {54},
number = {5},
pages = {669-684.e7},
pmid = {32710848},
issn = {1878-1551},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Epithelium/*physiology ; Gene Library ; Glycoproteins/genetics ; Glycosylation ; Humans ; Polysaccharides/*genetics ; Skin/metabolism/pathology ; },
abstract = {The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.},
}
@article {pmid32710421,
year = {2020},
author = {Chen, H and Gregor, T},
title = {Using RNA Tags for Multicolor Live Imaging of Chromatin Loci and Transcription in Drosophila Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2166},
number = {},
pages = {373-384},
pmid = {32710421},
issn = {1940-6029},
support = {R01 GM097275/GM/NIGMS NIH HHS/United States ; U01 DA047730/DA/NIDA NIH HHS/United States ; U01 DK127429/DK/NIDDK NIH HHS/United States ; U01 EB021239/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Chromatin/*genetics ; Color ; Drosophila Proteins/genetics ; Drosophila melanogaster/*embryology/*genetics ; Female ; Fluorescent Dyes/metabolism ; Gene Editing/methods ; Gene Expression Regulation, Developmental ; Gene Knock-In Techniques ; *Genetic Loci ; Green Fluorescent Proteins/*genetics/metabolism ; Male ; Microscopy, Confocal ; RNA/*genetics ; Transcription, Genetic/*genetics ; },
abstract = {Elucidating the biological implications of higher order chromatin architectures in animal development requires simultaneous, quantitative measurements of chromatin dynamics and transcriptional activity in living specimen. Here we describe a multicolor labeling and live imaging approach in embryos of the fruit fly Drosophila melanogaster. The method allows simultaneous measurement of movements of specific loci and their transcriptional activity for developmental genes, enabling new approaches to probe the interaction between 3D chromatin architecture and regulation of gene expression in development.},
}
@article {pmid32710420,
year = {2020},
author = {Wu, X and Ying, Y and Mao, S and Krueger, CJ and Chen, AK},
title = {Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2166},
number = {},
pages = {357-372},
doi = {10.1007/978-1-0716-0712-1_21},
pmid = {32710420},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Centromere/genetics ; Chromatin/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Fluorescent Dyes/metabolism ; *Genetic Loci ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Oligonucleotide Probes/genetics ; RNA, Guide/*genetics ; Telomere/genetics ; Transfection ; },
abstract = {The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein 9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the sgRNA with molecular beacons, a class of small molecule dye-based, fluorogenic oligonucleotide probes, and demonstrated that the resulting system, termed CRISPR/MB, could be more sensitive and quantitative than conventional approaches employing FP reporters in detecting single telomere loci. In this chapter, we describe detailed protocols for the synthesis of CRISPR/MB, as well as its applications for imaging single telomere and centromere loci in live mammalian cells.},
}
@article {pmid32710419,
year = {2020},
author = {Khosravi, S and Dreissig, S and Schindele, P and Wolter, F and Rutten, T and Puchta, H and Houben, A},
title = {Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific Guide RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2166},
number = {},
pages = {343-356},
doi = {10.1007/978-1-0716-0712-1_20},
pmid = {32710419},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Chromatin/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genetic Loci ; Green Fluorescent Proteins/genetics ; Microscopy, Confocal/methods ; Plant Cells/*metabolism ; Plant Leaves/*cytology ; RNA, Guide/*genetics ; Staphylococcus aureus/genetics ; Streptococcus pyogenes/genetics ; Telomere/*genetics/metabolism ; Tobacco/*cytology ; },
abstract = {Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.},
}
@article {pmid32710418,
year = {2020},
author = {Schindele, P and Wolter, F and Puchta, H},
title = {CRISPR Guide RNA Design Guidelines for Efficient Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2166},
number = {},
pages = {331-342},
doi = {10.1007/978-1-0716-0712-1_19},
pmid = {32710418},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drosophila/genetics ; Gene Editing/*methods ; Genome ; HEK293 Cells ; Humans ; Nucleic Acid Conformation ; Nucleotides/chemistry ; RNA, Guide/chemistry/*genetics ; Streptococcus pyogenes/enzymology ; },
abstract = {The simple applicability and facile target programming of the CRISPR/Cas9-system abolish the major boundaries of previous genome editing tools, making it the tool of choice for generating site-specific genome alterations. Its versatility and efficacy have been demonstrated in various organisms; however, accurately predicting guide RNA efficiencies remains an organism-independent challenge. Thus, designing optimal guide RNAs is essential to maximize the experimental outcome. Here, we summarize the current knowledge for guide RNA design and highlight discrepancies between different experimental systems.},
}
@article {pmid32709966,
year = {2020},
author = {Lester, PJ and Bulgarella, M and Baty, JW and Dearden, PK and Guhlin, J and Kean, JM},
title = {The potential for a CRISPR gene drive to eradicate or suppress globally invasive social wasps.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12398},
pmid = {32709966},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Drive Technology ; Internationality ; *Introduced Species ; Pest Control, Biological/*methods ; Population Dynamics ; Wasps/*genetics ; },
abstract = {CRISPR gene drives have potential for widespread and cost-efficient pest control, but are highly controversial. We examined a potential gene drive targeting spermatogenesis to control the invasive common wasp (Vespula vulgaris) in New Zealand. Vespula wasps are haplodiploid. Their life cycle makes gene drive production challenging, as nests are initiated by single fertilized queens in spring followed by several cohorts of sterile female workers and the production of reproductives in autumn. We show that different spermatogenesis genes have different levels of variation between introduced and native ranges, enabling a potential 'precision drive' that could target the reduced genetic diversity and genotypes within the invaded range. In vitro testing showed guide-RNA target specificity and efficacy that was dependent on the gene target within Vespula, but no cross-reactivity in other Hymenoptera. Mathematical modelling incorporating the genetic and life history traits of Vespula wasps identified characteristics for a male sterility drive to achieve population control. There was a trade-off between drive infiltration and impact: a drive causing complete male sterility would not spread, while partial sterility could be effective in limiting population size if the homing rate is high. Our results indicate that gene drives may offer viable suppression for wasps and other haplodiploid pests.},
}
@article {pmid32709883,
year = {2020},
author = {Aubert, L and Nandagopal, N and Steinhart, Z and Lavoie, G and Nourreddine, S and Berman, J and Saba-El-Leil, MK and Papadopoli, D and Lin, S and Hart, T and Macleod, G and Topisirovic, I and Gaboury, L and Fahrni, CJ and Schramek, D and Meloche, S and Angers, S and Roux, PP},
title = {Copper bioavailability is a KRAS-specific vulnerability in colorectal cancer.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3701},
pmid = {32709883},
issn = {2041-1723},
support = {R01 GM067169/GM/NIGMS NIH HHS/United States ; R35 GM136404/GM/NIGMS NIH HHS/United States ; MOP-142374//CIHR/Canada ; PJT-152995//CIHR/Canada ; },
mesh = {Animals ; Biological Availability ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms/genetics/*metabolism/pathology ; Copper/*metabolism ; Copper-Transporting ATPases/metabolism ; Female ; Humans ; Intestinal Mucosa/pathology ; Mice ; Mice, Knockout ; Mice, Nude ; Mice, SCID ; Mutation ; Proto-Oncogene Proteins p21(ras)/*genetics/*metabolism ; },
abstract = {Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.},
}
@article {pmid32708074,
year = {2020},
author = {Cambria, E and Arlt, MJE and Wandel, S and Krupkova, O and Hitzl, W and Passini, FS and Hausmann, ON and Snedeker, JG and Ferguson, SJ and Wuertz-Kozak, K},
title = {TRPV4 Inhibition and CRISPR-Cas9 Knockout Reduce Inflammation Induced by Hyperphysiological Stretching in Human Annulus Fibrosus Cells.},
journal = {Cells},
volume = {9},
number = {7},
pages = {},
pmid = {32708074},
issn = {2073-4409},
mesh = {Adolescent ; Adult ; Aged ; Annulus Fibrosus/*physiology ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Dinoprostone/metabolism ; Female ; Gene Expression Regulation ; *Gene Knockout Techniques ; Humans ; Inflammation Mediators/metabolism ; Interleukin-8/genetics/metabolism ; Male ; Middle Aged ; Phosphorylation ; *Stress, Mechanical ; TRPV Cation Channels/*antagonists &amp; inhibitors/metabolism ; p38 Mitogen-Activated Protein Kinases/metabolism ; },
abstract = {Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.},
}
@article {pmid32707302,
year = {2020},
author = {Oo, ZM and Adlat, S and Sah, RK and Myint, MZZ and Hayel, F and Chen, Y and Htoo, H and Bah, FB and Bahadar, N and Chan, MK and Zhang, L and Feng, X and Zheng, Y},
title = {Brain transcriptome study through CRISPR/Cas9 mediated mouse Dip2c gene knock-out.},
journal = {Gene},
volume = {758},
number = {},
pages = {144975},
doi = {10.1016/j.gene.2020.144975},
pmid = {32707302},
issn = {1879-0038},
mesh = {Animals ; Brain/cytology/growth &amp; development/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Deletion ; Gene Editing/methods ; Gene Expression Regulation/*genetics ; Gene Knockout Techniques ; Intracellular Signaling Peptides and Proteins/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mice, Knockout ; Neoplasm Proteins/*genetics ; RNA, Guide/genetics ; Transcriptome/*genetics ; },
abstract = {Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2C[Δ160kb]), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2c[Δ2bp]) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2c[Δ2bp]. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including 'memory', 'neuropeptide signaling pathway', and 'response to amphetamine' while KEGG analysis shows that 'neuroactive ligand-receptor interaction pathway' is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.},
}
@article {pmid32707148,
year = {2020},
author = {Huang, D and Miller, M and Ashok, B and Jain, S and Peppas, NA},
title = {CRISPR/Cas systems to overcome challenges in developing the next generation of T cells for cancer therapy.},
journal = {Advanced drug delivery reviews},
volume = {158},
number = {},
pages = {17-35},
pmid = {32707148},
issn = {1872-8294},
support = {R01 EB022025/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*physiology ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; Neoplasms/*drug therapy ; Receptors, Antigen, T-Cell/immunology ; Receptors, Chimeric Antigen/*immunology ; T-Lymphocytes/immunology ; },
abstract = {Genetically engineered immune cells with chimeric antigen receptors (CAR) or modified T cell receptors (TCR) have demonstrated their potential as a potent class of new cancer therapeutic strategy. Despite the clinical success of autologous CD19 CAR T cells in hematological malignancies, allogeneic T cells exhibit many advantages over their autologous counterparts and have recently gathered widespread attention due to the emergence of multiplex genome editing techniques, particularly CRISPR/Cas systems. Furthermore, genetically engineered T cells face a host of major challenges in solid tumors that are not as significant for blood cancers such as T cell targeted delivery, target specificity, proliferation, persistence, and the immunosuppressive tumor microenvironment. We take this opportunity to analyze recent strategies to develop allogeneic T cells, specifically in consideration of CRISPR/Cas and its delivery systems for multiplex gene editing. Additionally, we discuss the current methods used to delivery CRISPR/Cas systems for immunotherapeutic applications, and the challenges to continued development of novel delivery systems. We also provide a comprehensive analysis of the major challenges that genetically engineered T cells face in solid tumors along with the most recent strategies to overcome these barriers, with an emphasis on CRISPR-based approaches. We illustrate the synergistic prospects for how the combination of synthetic biology and immune-oncology could pave the way for designing the next generation of precision cancer therapy.},
}
@article {pmid32706513,
year = {2020},
author = {Hong, JW and Jeong, CY and Yu, JH and Kim, SB and Kang, SK and Kim, SW and Kim, NS and Kim, KY and Park, JW},
title = {Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system.},
journal = {Biotechnology progress},
volume = {36},
number = {6},
pages = {e3054},
doi = {10.1002/btpr.3054},
pmid = {32706513},
issn = {1520-6033},
support = {PJ01270901//Rural Development Administration/International ; },
mesh = {Animals ; Bombyx/*genetics ; CRISPR-Cas Systems/genetics ; DNA Shuffling/*methods ; Gene Editing/*methods ; Kynurenine 3-Monooxygenase/*genetics ; },
abstract = {Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.},
}
@article {pmid32705648,
year = {2020},
author = {Kashfi, H and Jinks, N and Nateri, AS},
title = {Generating and Utilizing Murine Cas9-Expressing Intestinal Organoids for Large-Scale Knockout Genetic Screening.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2171},
number = {},
pages = {257-269},
doi = {10.1007/978-1-0716-0747-3_17},
pmid = {32705648},
issn = {1940-6029},
support = {NC/P001793/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Cell Proliferation/genetics/physiology ; Gene Editing ; Lentivirus/genetics ; Mice ; Mice, Transgenic ; Organoids/*cytology/*metabolism ; RNA, Guide/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; },
abstract = {Organoid culture faithfully reproduces the in vivo characteristics of the intestinal/colon epithelium and elucidates molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumorigenesis. CRISPR-Cas9 based editing technology has provided promising opportunities for targeted loss-of-function mutations at chosen sites in the genome of eukaryotes. Herein, we demonstrate a CRISPR/Cas9-mediated mutagenesis-based screening method using murine intestinal organoids by investigating the phenotypical morphology of Cas9-expressing murine intestinal organoids. Murine intestinal crypts can be isolated and seeded into Matrigel and grown into stable organoid lines. Organoids subsequently transduced and selected to generate Cas9 expressing organoids. These organoids can be further transduced with the second lentiviruses expressing guide RNA (gRNA) (s) and screened for 8-10 days using bright-field and fluorescent microscopy to determine possible morphological or phenotypical abnormalities. Via phenotypical screening analysis, the candidate knockouts can be selected based on differential abnormal growth pattern vs their untransduced or lenti-GFP transduced controls. Further assessment of these knockout organoids can be done via phalloidin and propidium iodide (PI) staining, proliferation assay and qRT-PCR and also biochemical analysis. This CRISPR/Cas9 organoid mutagenesis-based screening method provides a reliable and rapid approach for investigating large numbers of genes with unknown/poorly identified biological functions. Knockout intestinal organoids can be associated with the key biological function of the gene(s) in development, homeostasis, disease progression, tumorigenesis, and drug screening, thereby reducing and potentially replacing animal models.},
}
@article {pmid32705644,
year = {2020},
author = {Rajendra, C and Wald, T and Barber, K and Spence, JR and Fattahi, F and Klein, OD},
title = {Generation of Knockout Gene-Edited Human Intestinal Organoids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2171},
number = {},
pages = {215-230},
pmid = {32705644},
issn = {1940-6029},
support = {T32 DK007762/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cells, Cultured ; Embryonic Stem Cells/*cytology/*metabolism ; Gene Editing ; Humans ; Intestinal Mucosa/*cytology ; Organoids/*cytology/*metabolism ; Transfection ; },
abstract = {We discuss a methodology to generate and study knockout gene-edited human intestinal organoids. We describe the generation of knockout human embryonic stem cell lines that we then differentiate into mature human intestinal organoid tissue in Matrigel using several growth factors. We also discuss a pair of assays that can be used to study the integrity of the intestinal epithelial barrier of the human intestinal organoids under inflammatory stress conditions.},
}
@article {pmid32705536,
year = {2020},
author = {Park, CW and Bae, JS and Ryu, KY},
title = {Simultaneous Disruption of Both Polyubiquitin Genes Affects Proteasome Function and Decreases Cellular Proliferation.},
journal = {Cell biochemistry and biophysics},
volume = {78},
number = {3},
pages = {321-329},
doi = {10.1007/s12013-020-00933-2},
pmid = {32705536},
issn = {1559-0283},
mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Phosphorylation ; Polyubiquitin/*chemistry ; Proteasome Endopeptidase Complex/*chemistry ; Transfection ; Ubiquitin/*chemistry ; Ubiquitin C/chemistry/metabolism ; },
abstract = {The ubiquitin (Ub) proteasome system is important for maintaining protein homeostasis and has various roles in cell signaling, proliferation, and cell cycle regulation. In mammals, Ub is encoded by two monoubiquitin and two polyubiquitin genes. Although reduced levels of Ub due to the disruption of one polyubiquitin gene are known to decrease cell proliferation, the effect of disrupting both polyubiquitin genes remains elusive. Polyubiquitin gene Ubc knockout mice are embryonically lethal and polyubiquitin gene Ubb knockout mice are infertile. Thus, it is difficult to study the effects of double knockouts (DKOs). In the present study, the CRISPR/Cas9 system was used to simultaneously knockout both polyubiquitin genes, UBB and UBC, in HEK293T and HeLa cells. In DKO cells, growth decreased significantly compared to the control cells. We observed reduced proteasome function and reduced levels of free Ub in DKO cells. However, the levels of purified proteasome were not different between control and DKO cells, although the mRNA levels of proteasomal subunits were significantly increased in latter. We propose that the reduction of Ub levels, by disruption of both polyubiquitin genes, resulted in an altered proteasomal status, leading to the reduced proteasome activity, and decreased cellular proliferation.},
}
@article {pmid32705255,
year = {2020},
author = {Shao, Z and Wang, K and Zhang, S and Yuan, J and Liao, X and Wu, C and Zou, Y and Ha, Y and Shen, Z and Guo, J and Jie, W},
title = {Ingenuity pathway analysis of differentially expressed genes involved in signaling pathways and molecular networks in RhoE gene‑edited cardiomyocytes.},
journal = {International journal of molecular medicine},
volume = {46},
number = {3},
pages = {1225-1238},
pmid = {32705255},
issn = {1791-244X},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Gene Expression Profiling ; *Gene Regulatory Networks ; Humans ; Myocytes, Cardiac/*metabolism ; Rats ; *Signal Transduction ; rho GTP-Binding Proteins/*genetics ; },
abstract = {RhoE/Rnd3 is an atypical member of the Rho superfamily of proteins, However, the global biological function profile of this protein remains unsolved. In the present study, a RhoE‑knockout H9C2 cardiomyocyte cell line was established using CRISPR/Cas9 technology, following which differentially expressed genes (DEGs) between the knockout and wild‑type cell lines were screened using whole genome expression gene chips. A total of 829 DEGs, including 417 upregulated and 412 downregulated, were identified using the threshold of fold changes ≥1.2 and P<0.05. Using the ingenuity pathways analysis system with a threshold of ‑Log (P‑value)>2, 67 canonical pathways were found to be enriched. Many of the detected signaling pathways, including that of oncostatin M signaling, were found to be associated with the inflammatory response. Subsequent disease and function analysis indicated that apart from cardiovascular disease and development function, RhoE may also be involved in other diseases and function, including organismal survival, cancer, organismal injury and abnormalities, cell‑to‑cell signaling and interaction, and molecular transport. In addition, 885 upstream regulators were enriched, including 59 molecules that were predicated to be strongly activated (Z‑score >2) and 60 molecules that were predicated to be significantly inhibited (Z‑scores <‑2). In particular, 33 regulatory effects and 25 networks were revealed to be associated with the DEGs. Among them, the most significant regulatory effects were 'adhesion of endothelial cells' and 'recruitment of myeloid cells' and the top network was 'neurological disease', 'hereditary disorder, organismal injury and abnormalities'. In conclusion, the present study successfully edited the RhoE gene in H9C2 cells using CRISPR/Cas9 technology and subsequently analyzed the enriched DEGs along with their associated canonical signaling pathways, diseases and functions classification, upstream regulatory molecules, regulatory effects and interaction networks. The results of the present study should facilitate the discovery of the global biological and functional properties of RhoE and provide new insights into role of RhoE in human diseases, especially those in the cardiovascular system.},
}
@article {pmid32703438,
year = {2020},
author = {Shirai, Y and Daimon, T},
title = {Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum.},
journal = {Biochemical and biophysical research communications},
volume = {529},
number = {2},
pages = {372-378},
doi = {10.1016/j.bbrc.2020.05.214},
pmid = {32703438},
issn = {1090-2104},
mesh = {Animals ; *Eye Color ; Female ; Gene Editing ; Genes, Insect ; Genetic Loci ; Male ; Mutation ; Tribolium/*genetics ; },
abstract = {Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection.},
}
@article {pmid32703430,
year = {2020},
author = {Gonzalez-Salinas, F and Rojo, R and Martinez-Amador, C and Herrera-Gamboa, J and Trevino, V},
title = {Transcriptomic and cellular analyses of CRISPR/Cas9-mediated edition of FASN show inhibition of aggressive characteristics in breast cancer cells.},
journal = {Biochemical and biophysical research communications},
volume = {529},
number = {2},
pages = {321-327},
doi = {10.1016/j.bbrc.2020.05.172},
pmid = {32703430},
issn = {1090-2104},
mesh = {Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Cell Movement ; Cell Proliferation ; Fatty Acid Synthase, Type I/*genetics ; Female ; Humans ; MCF-7 Cells ; Mutation ; *Transcriptome ; },
abstract = {Several genes are significantly mutated in breast cancer but only a small percentage of mutations are well-known to contribute to cancer development. FASN is involved in de novo lipogenesis and the regulation of ERα signaling. However, the effect of genetic mutations affecting FASN in breast cancer has not thoroughly studied. Therefore, we used the CRISPR/Cas9 system to edit the FASN locus in MCF-7 cells and evaluated its biological effect. We obtained four clones carrying mutations and frameshifts in the acyl-transferase domain of FASN. We found that clones had reduced proliferation, migration, viability, and showed alterations in cell cycle profiles. RNA-Seq analysis demonstrates that a lack of fully functional FASN may have a more significant role in proliferation-related genes than in lipid metabolism. We conclude that functional knockouts in FASN contributes to decrease the proliferation and migration of breast cancer cells contrary to point mutations in breast cancer patients.},
}
@article {pmid32703416,
year = {2020},
author = {Antonson, P and Apolinário, LM and Shamekh, MM and Humire, P and Poutanen, M and Ohlsson, C and Nalvarte, I and Gustafsson, J&#xc5;},
title = {Generation of an all-exon Esr2 deleted mouse line: Effects on fertility.},
journal = {Biochemical and biophysical research communications},
volume = {529},
number = {2},
pages = {231-237},
doi = {10.1016/j.bbrc.2020.06.063},
pmid = {32703416},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Estrogen Receptor beta/*genetics ; Exons ; Female ; *Fertility ; Gene Deletion ; Male ; Mice ; Ovary/physiology ; Ovulation ; },
abstract = {Estrogen receptor beta (ERβ), encoded by the Esr2 gene, is one of two nuclear receptors that mediate the functions of the steroid hormone estradiol. The binding of estradiol to the receptor results in enhanced transcription of many genes that have estrogen response elements in promoter or enhancer regions. Several genetically modified mouse lines with mutations or deletions of exons in the Esr2 gene have been developed and results from analysis of these are not completely consistent, especially regarding ERβ's role in fertility. To address these controversies, we have used the CRISPR/Cas9 genome editing system to make a deletion of the entire Esr2 gene in the mouse genome and determined the effect of this mutation on fertility. We show that female Esr2 deleted mice, Esr2[ΔE1-10], are subfertile at young age, with fewer litters and smaller litter size, and that they become infertile/have severely reduced fertility at around six months of age, while the male Esr2[ΔE1-10] mice are fertile. Ovaries from Esr2[ΔE1-10] mice are smaller than those from wild-type littermates and the morphology of the ovary displays very few corpora lutea, indicating a defect in ovulation. We also show that the estradiol levels are reduced at diestrus, the phase in the estrous cycle when levels are expected to start to increase before ovulation. Our results verify that ERβ has an important function in female reproduction, likely as a regulator of serum estradiol levels, and that its loss does not affect male reproductive function.},
}
@article {pmid32702356,
year = {2020},
author = {Gao, Y and Zhang, X and Zhang, X and Yuan, J and Xiang, J and Li, F},
title = {CRISPR/Cas9-mediated mutation reveals Pax6 is essential for development of the compound eye in Decapoda Exopalaemon carinicauda.},
journal = {Developmental biology},
volume = {465},
number = {2},
pages = {157-167},
doi = {10.1016/j.ydbio.2020.07.001},
pmid = {32702356},
issn = {1095-564X},
mesh = {Animals ; *Arthropod Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Compound Eye, Arthropod/*embryology ; *Decapoda/embryology/genetics ; Gene Editing ; *Mutation ; *PAX6 Transcription Factor/genetics/metabolism ; },
abstract = {The compound eye in crustaceans is a main eye type in the animal kingdom, knowledge about the mechanism to determine the development of compound eye is very limited. Paired box protein 6 (Pax6) is generally regarded as a master regulator for eye development. In the present study, a genome-based analysis of the Pax6 gene in the ridge tail white prawn Exopalaemon carinicauda was performed and two members of Pax6 homologs, named Ec-Eyeless (EcEy) and Ec-Twin of eyeless (EcToy) were identified. To understand the function of these two homologs of Pax6 gene in the prawn, the CRISPR/Cas9 genome editing technique was applied to generate EcEy and EcToy knock-out (KO) prawns and their phenotypes were analyzed. The surviving EcEy-KO embryos and larvae exhibited severe abnormal eye morphology, suggesting that EcEy is necessary for the compound eye development in prawn, while no mutant phenotype was found in EcToy-KO individuals. These findings highlighted the conservative role of Pax6 gene in the compound eye formation, and the functional differentiation between EcEy and EcToy gene may reveal a novel regulating mechanism of Pax6 on the compound eye development in the decapods. These data will provide important information for understanding the regulation mechanism for crustacean compound eye development.},
}
@article {pmid32701961,
year = {2020},
author = {Douglas, C and Turner, JMA},
title = {Advances and challenges in genetic technologies to produce single-sex litters.},
journal = {PLoS genetics},
volume = {16},
number = {7},
pages = {e1008898},
pmid = {32701961},
issn = {1553-7404},
support = {FC001193/MRC_/Medical Research Council/United Kingdom ; FC001193/CRUK_/Cancer Research UK/United Kingdom ; FC001193/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/methods ; Male ; Reproduction/*genetics ; *Sex Determination Analysis ; Sex Determination Processes/*genetics ; },
abstract = {There is currently a requirement for single-sex litters for many applications, including agriculture, pest control, and reducing animal culling in line with the 3Rs principles: Reduction, Replacement, and Refinement. The advent of CRISPR/Cas9 genome editing presents a new opportunity with which to potentially generate all-female or all-male litters. We review some of the historical nongenetic strategies employed to generate single-sex litters and investigate how genetic and genome editing techniques are currently being used to produce all-male or all-female progeny. Lastly, we speculate on future technologies for generating single-sex litters and the possible associated challenges.},
}
@article {pmid32700830,
year = {2020},
author = {Ostrominski, JW and Yada, RC and Sato, N and Klein, M and Blinova, K and Patel, D and Valadez, R and Palisoc, M and Pittaluga, S and Peng, KW and San, H and Lin, Y and Basuli, F and Zhang, X and Swenson, RE and Haigney, M and Choyke, PL and Zou, J and Boehm, M and Hong, SG and Dunbar, CE},
title = {CRISPR/Cas9-mediated introduction of the sodium/iodide symporter gene enables noninvasive in vivo tracking of induced pluripotent stem cell-derived cardiomyocytes.},
journal = {Stem cells translational medicine},
volume = {9},
number = {10},
pages = {1203-1217},
pmid = {32700830},
issn = {2157-6580},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Disease Models, Animal ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mice ; Myocytes, Cardiac/*metabolism ; Symporters/*genetics ; Transfection ; },
abstract = {Techniques that enable longitudinal tracking of cell fate after myocardial delivery are imperative for optimizing the efficacy of cell-based cardiac therapies. However, these approaches have been underutilized in preclinical models and clinical trials, and there is considerable demand for site-specific strategies achieving long-term expression of reporter genes compatible with safe noninvasive imaging. In this study, the rhesus sodium/iodide symporter (NIS) gene was incorporated into rhesus macaque induced pluripotent stem cells (RhiPSCs) via CRISPR/Cas9. Cardiomyocytes derived from NIS-RhiPSCs (NIS-RhiPSC-CMs) exhibited overall similar morphological and electrophysiological characteristics compared to parental control RhiPSC-CMs at baseline and with exposure to physiological levels of sodium iodide. Mice were injected intramyocardially with 2 million NIS-RhiPSC-CMs immediately following myocardial infarction, and serial positron emission tomography/computed tomography was performed with [18] F-tetrafluoroborate to monitor transplanted cells in vivo. NIS-RhiPSC-CMs could be detected until study conclusion at 8 to 10 weeks postinjection. This NIS-based molecular imaging platform, with optimal safety and sensitivity characteristics, is primed for translation into large-animal preclinical models and clinical trials.},
}
@article {pmid32700574,
year = {2021},
author = {Odongoo, B and Ohara, H and Ngarashi, D and Kaneko, T and Kunihiro, Y and Mashimo, T and Nabika, T},
title = {Pathophysiological significance of Stim1 mutation in sympathetic response to stress and cardiovascular phenotypes in SHRSP/Izm: In vivo evaluation by creation of a novel gene knock-in rat using CRISPR/Cas9.},
journal = {Clinical and experimental hypertension (New York, N.Y. : 1993)},
volume = {43},
number = {1},
pages = {34-41},
doi = {10.1080/10641963.2020.1797085},
pmid = {32700574},
issn = {1525-6006},
mesh = {Animals ; Astrocytes/*metabolism ; Blood Pressure ; CRISPR-Cas Systems ; Calcium-Binding Proteins/metabolism ; Cardiovascular System/*physiopathology ; Gene Knock-In Techniques ; Heart Rate ; Male ; Membrane Proteins/metabolism ; Mutation ; Norepinephrine/urine ; Phenotype ; Rats ; Rats, Inbred SHR ; Stress, Physiological/*genetics/physiology ; Stromal Interaction Molecule 1/*genetics/*metabolism ; Sympathetic Nervous System/*physiopathology ; },
abstract = {Genetic approach using rat congenic lines between SHRSP/Izm and WKY/Izm identified stromal interaction molecule 1 (Stim1), an essential component of store-operated Ca[2+] entry (SOCE), as a promising candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP. Since SHRSP has a nonsense mutation in Stim1 resulting in the expression of a truncated form of STIM1 that caused reduction of SOCE activity in primary cultured cerebral astrocytes, we created SHRSP/Izm knocked-in with the wild-type Stim1 (KI SHRSP) by the CRISPR/Cas9 method to investigate whether the functional recovery of STIM1 would mitigate sympatho-excitation to stress in vivo in SHRSP. No potential off-target nucleotide substitutions/deletions/insertions were found in KI SHRSP. Western blotting and fluorescent Ca[2+] imaging of astrocytes confirmed wild-type STIM1 expression and restored SOCE activity in astrocytes from KI SHRSP, respectively. Blood pressure (BP) measured by the tail-cuff method at 12, 16, and 20 weeks of age did not significantly differ between SHRSP and KI SHRSP, while the heart rate of KI SHRSP at 16 and 20 weeks of age was significantly lower than that of age-matched SHRSP. Unexpectedly, the sympathetic response to stress (evaluated with urinary excretion of norepinephrine under cold stress and BP elevation under cold/restraint stress) did not significantly differ between SHRSP and KI SHRSP. The present results indicated that the functional deficit of STIM1 was not a genetic determinant of the exaggerated sympathetic response to stress in SHRSP and that it would be necessary to explore other candidates within the congenic fragment on chromosome 1.},
}
@article {pmid32699298,
year = {2020},
author = {Sharon, DM and Nesdoly, S and Yang, HJ and Gélinas, JF and Xia, Y and Ansorge, S and Kamen, AA},
title = {A pooled genome-wide screening strategy to identify and rank influenza host restriction factors in cell-based vaccine production platforms.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12166},
pmid = {32699298},
issn = {2045-2322},
support = {CIHR-CPG-151958//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Survival ; Gene Editing ; Gene Ontology ; Genes, Reporter ; Genetic Vectors/genetics/metabolism ; *Genome, Viral ; HEK293 Cells ; Humans ; Influenza A Virus, H1N1 Subtype/*genetics/isolation &amp; purification/physiology ; Influenza Vaccines/genetics/immunology/*metabolism ; Influenza, Human/pathology/prevention &amp; control/virology ; RNA, Guide/metabolism ; Virus Replication ; },
abstract = {Cell-derived influenza vaccines provide better protection and a host of other advantages compared to the egg-derived vaccines that currently dominate the market, but their widespread use is hampered by a lack of high yield, low cost production platforms. Identification and knockout of innate immune and metabolic restriction factors within relevant host cell lines used to grow the virus could offer a means to substantially increase vaccine yield. In this paper, we describe and validate a novel genome-wide pooled CRISPR/Cas9 screening strategy that incorporates a reporter virus and a FACS selection step to identify and rank restriction factors in a given vaccine production cell line. Using the HEK-293SF cell line and A/PuertoRico/8/1934 H1N1 influenza as a model, we identify 64 putative influenza restriction factors to direct the creation of high yield knockout cell lines. In addition, gene ontology and protein complex enrichment analysis of this list of putative restriction factors offers broader insights into the primary host cell determinants of viral yield in cell-based vaccine production systems. Overall, this work will advance efforts to address the public health burden posed by influenza.},
}
@article {pmid32699244,
year = {2020},
author = {Koepke, L and Winter, B and Grenzner, A and Regensburger, K and Engelhart, S and van der Merwe, JA and Krebs, S and Blum, H and Kirchhoff, F and Sparrer, KMJ},
title = {An improved method for high-throughput quantification of autophagy in mammalian cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12241},
pmid = {32699244},
issn = {2045-2322},
support = {794803/MCCC_/Marie Curie/United Kingdom ; },
mesh = {Animals ; Autophagy/*immunology ; CRISPR-Cas Systems/immunology ; Cell Line ; Cell Line, Tumor ; HEK293 Cells ; HeLa Cells ; High-Throughput Screening Assays/*methods ; Humans ; Infections/immunology ; Jurkat Cells ; Mammals/*immunology ; T-Lymphocytes/immunology ; THP-1 Cells ; },
abstract = {Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.},
}
@article {pmid32699230,
year = {2020},
author = {Sakano, D and Inoue, A and Enomoto, T and Imasaka, M and Okada, S and Yokota, M and Koike, M and Araki, K and Kume, S},
title = {Insulin2[Q104del] (Kuma) mutant mice develop diabetes with dominant inheritance.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12187},
pmid = {32699230},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Endoplasmic Reticulum Stress/genetics ; Female ; Gene Editing ; Glucose Tolerance Test ; Hyperglycemia/drug therapy/genetics/*pathology ; Insulin/*genetics/therapeutic use ; Insulin-Secreting Cells/cytology/metabolism ; Islets of Langerhans/physiology/ultrastructure ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Microscopy, Electron ; Oligodeoxyribonucleotides/metabolism ; Organ Size ; },
abstract = {Insulin gene mutations have been identified to cause monogenic diabetes, and most of which developed permanent neonatal diabetes at young ages before 6 months of age in humans. To establish an animal model of permanent diabetes, we performed genome editing using the CRISPR/Cas9 system. We generated a novel Kuma mutant mice with p.Q104del in the Insulin2 (Ins2) gene in a BRJ background that exhibits a severe immune deficiency. Kuma mutant mice are non-obese and developed hyperglycemia from 3 weeks after birth in both males and females, which are inherited in a dominant mode. Kuma mutant mice displayed reduced insulin protein levels from 3-weeks-old, which seem to be caused by the low stability of the mutant insulin protein. Kuma mutant showed a reduction in islet size and islet mass. Electron microscopic analysis revealed a marked decrease in the number and size of insulin granules in the beta-cells of the mutant mice. Hyperglycemia of the mutant can be rescued by insulin administration. Our results present a novel insulin mutation that causes permanent early-onset diabetes, which provides a model useful for islet transplantation studies.},
}
@article {pmid32698485,
year = {2020},
author = {Key, J and Harter, PN and Sen, NE and Gradhand, E and Auburger, G and Gispert, S},
title = {Mid-Gestation lethality of Atxn2l-Ablated Mice.},
journal = {International journal of molecular sciences},
volume = {21},
number = {14},
pages = {},
pmid = {32698485},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Embryo Loss/*genetics/pathology ; Female ; *Gene Deletion ; Gene Expression Regulation, Developmental ; Humans ; Mice/*embryology/genetics ; Pregnancy ; },
abstract = {Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.},
}
@article {pmid32698333,
year = {2020},
author = {Zhang, B},
title = {CRISPR/Cas9: A Robust Genome-Editing Tool with Versatile Functions and Endless Application.},
journal = {International journal of molecular sciences},
volume = {21},
number = {14},
pages = {},
pmid = {32698333},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Genome, Plant ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {Since a potential genome editing tool was first recognized in 2012 [...].},
}
@article {pmid32697075,
year = {2020},
author = {Wei, T and Cheng, Q and Farbiak, L and Anderson, DG and Langer, R and Siegwart, DJ},
title = {Delivery of Tissue-Targeted Scalpels: Opportunities and Challenges for In Vivo CRISPR/Cas-Based Genome Editing.},
journal = {ACS nano},
volume = {14},
number = {8},
pages = {9243-9262},
pmid = {32697075},
issn = {1936-086X},
support = {R01 EB000244/EB/NIBIB NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; R37 EB000244/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Transfer Techniques ; Genetic Therapy ; },
abstract = {CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward understanding gene modification methods in vitro have led to substantial improvements in ex vivo genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly in vivo. In this Perspective, we focus on progress toward in vivo delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues via local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for in vivo gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for in vivo genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for in vivo delivery of CRISPR/Cas9 systems to guide the development of future therapies.},
}
@article {pmid32696754,
year = {2020},
author = {Wen, D and Huang, R and Xie, J and Wen, H and Lin, S},
title = {[Construction of ACT-1 human undifferentiated thyroid cancer cell line with knockout of axis inhibition protein 1 (AXIN1) gene using CRISPR/Cas9].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {36},
number = {5},
pages = {419-424},
pmid = {32696754},
issn = {1007-8738},
mesh = {Adaptor Proteins, Signal Transducing ; Axin Protein ; *CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; Humans ; RNA, Guide/genetics ; *Thyroid Neoplasms/genetics ; },
abstract = {Objective To construct the axis inhibition protein 1 (AXIN1) gene-knockout ACT-1 human undifferentiated thyroid cancer single clone cell line. Methods Molecular cloning technology and clustered regularly interspaced short palindromic repeats/Cas9 nuclease (CRISPR/Cas9) were used to construct AXIN1 gene-knockout single clone cell lines. Real-time quantitative PCR and Western blotting were used to detect AXIN1 mRNA and protein levels of ACT-1 cells, respectively. Results T7 detection results showed two effective single guide RNAs (sgRNAs) Cr3 and Cr5 were successfully constructed; enzyme digestion identification and sequencing showed AXIN1-targeted sgRNA viral vectors carrying green fluorescent protein (GFP) were successfully constructed. We successfully obtained 4 monoclonal ACT-1 undifferentiated thyroid cancer cell lines. AXIN1 mRNA and protein levels in the gene-knockout group were significantly reduced. Conclusion The ACT-1 undifferentiated thyroid cancer cell line with AXIN1 gene knockout has been successfully constructed using CRISPR/Cas9.},
}
@article {pmid32696736,
year = {2020},
author = {Xu, F and Su, C and Wu, T and Chen, H and Zhang, P and Liu, Y and Lan, Y and Li, J},
title = {[CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells inhibits their phagocytosis to Klebsiella pneumoniae].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {36},
number = {6},
pages = {481-486},
pmid = {32696736},
issn = {1007-8738},
mesh = {Animals ; *CRISPR-Cas Systems ; *Klebsiella pneumoniae/genetics ; Mice ; Phagocytosis ; RAW 264.7 Cells ; RNA, Guide ; Receptors, G-Protein-Coupled ; },
abstract = {Objective To construct cell line RAW264.7 with stable knockout of GPR43 gene using CRISPR/Cas9 system, and explore the role and mechanism of GPR43 gene in Klebsiella pneumoniae infection. Methods Three pairs of small-guide RNA (sgRNA) targeting the GPR43 gene were designed and inserted into plasmid pLenticrisprV2. The recombinant plasmid pLenticrisprV2 containing sgRNA was packaged using a lentivirus packaging system. RAW264.7 cells were transfected with viruses, and monoclonal cells were screened using puromycin. The genomic DNA was extracted from the amplified monoclonal cells. The GPR43 gene-related sequences were sequenced and compared with the wild-type GPR43 gene to confirm the cell line with successful knockout (GPR43[-/-] RAW264.7 cells). The expression of GPR43 protein was detected by Western blotting. After GPR43[-/-] RAW264.7 cells were transfected with Klebsiella pneumoniae, the changes in the expression of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in the cells were detected using real-time quantitative PCR. Additionally, the phagocytic capacity of RAW264.7 cells after GPR43 knockout was observed. Results Western blotting confirmed that GPR43 protein was not expressed in the selected monoclonal cells, and DNA sequencing showed that 34 bases were missing at the insertion site of sgRNA, which proved that GPR43 gene was successfully knocked out. After GPR43[-/-] RAW264.7 cells were transfected with Klebsiella pneumoniae, the levels of IL-1β, IL-6 and TNF-α expression in the cells were all lower than those in the control group, and the phagocytic capacity of GPR43[-/-] RAW264.7 cells to Klebsiella pneumoniae decreased. Conclusion CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells can inhibit their phagocytosis for Klebsiella pneumoniae and production of inflammatory cytokines.},
}
@article {pmid32696132,
year = {2020},
author = {Tang, R and Xu, Z},
title = {Gene therapy: a double-edged sword with great powers.},
journal = {Molecular and cellular biochemistry},
volume = {474},
number = {1-2},
pages = {73-81},
doi = {10.1007/s11010-020-03834-3},
pmid = {32696132},
issn = {1573-4919},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Transfer Techniques/*standards ; *Genetic Predisposition to Disease ; Genetic Therapy/methods/*trends ; Humans ; Immunotherapy, Adoptive/*methods ; },
abstract = {Gene therapy is the treatment of a disease through transferring genetic material into cells of the patients. In the recent several years, gene therapy has experienced rapid progress and achieved huge success. Over two dozens of gene therapies have been approved for clinical use by the drug regulatory agencies from different countries. However, concerns about its efficacy and safety have accompanied gene therapy since its birth. In the present manuscript, we first introduce various strategies employed in gene therapy, which includes ex vivo gene delivery v.s. in vivo gene delivery; gene addition v.s. genome editing; inherited disease v.s. acquired disease; and somatic gene therapy v.s. germline gene therapy. Then we discuss the clinical outcomes of some approved gene therapies. We finish our discussion with the safety issues related to gene therapy. We will see that with the technology improvement, somatic gene therapy has been proved to be efficient and safe enough for clinical practice. However, germline gene therapy has important efficiency and safety issues at present, and should not be put into clinical practice before these issues are solved.},
}
@article {pmid32696070,
year = {2021},
author = {Ciesiolka, A and Stroynowska-Czerwinska, A and Joachimiak, P and Ciolak, A and Kozlowska, E and Michalak, M and Dabrowska, M and Olejniczak, M and Raczynska, KD and Zielinska, D and Wozna-Wysocka, M and Krzyzosiak, WJ and Fiszer, A},
title = {Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {78},
number = {4},
pages = {1577-1596},
pmid = {32696070},
issn = {1420-9071},
mesh = {Alleles ; Argonaute Proteins/*genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Humans ; Huntingtin Protein/*genetics ; Huntington Disease/genetics/pathology/*therapy ; MicroRNAs/chemical synthesis/*genetics/pharmacology ; Mutation/genetics ; Open Reading Frames/genetics ; Peptides/genetics ; Protein Biosynthesis/drug effects ; RNA Interference ; Trinucleotide Repeat Expansion/drug effects/genetics ; },
abstract = {Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.},
}
@article {pmid32695773,
year = {2020},
author = {Zhang, J and Zhang, D and Zhu, J and Liu, H and Liang, S and Luo, Y},
title = {Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {726},
pmid = {32695773},
issn = {2296-4185},
abstract = {Streptomyces strains produce a great number of valuable natural products. With the development of genome sequencing, a vast number of biosynthetic gene clusters with high potential for use in the discovery of valuable clinical drugs have been revealed. Therefore, emerging needs for tools to manipulate these biosynthetic pathways are presented. Although the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) system has exhibited great capabilities for gene editing in multiple Streptomyces strains, it has failed to work in some newly discovered strains and some important industrial strains. Additionally, the protospacer adjacent motif (PAM) recognition scope of this system sometimes limits its applications for generating precise site mutations and insertions. Here, we developed three efficient CRISPR-FnCas12a systems for multiplex genome editing in several Streptomyces strains. Each system exhibited advantages for different applications. The CRISPR-FnCas12a1 system was efficiently applied in the industrial strain Streptomyces hygroscopicus, in which SpCas9 does not work well. The CRISPR-FnCas12a2 system was used to delete large fragments ranging from 21.4 to 128 kb. Additionally, the CRISPR-FnCas12a3 system employing the engineered FnCas12a mutant EP16, which recognizes a broad spectrum of PAM sequences, was used to precisely perform site mutations and insertions. The CRISPR-FnCas12a3 system addressed the limitation of TTN PAM recognition in Streptomyces strains with high GC contents. In summary, all the CRISPR-FnCas12a systems developed in this study are powerful tools for precise and multiplex genome editing in Streptomyces strains.},
}
@article {pmid32695770,
year = {2020},
author = {Ding, W and Zhang, Y and Shi, S},
title = {Development and Application of CRISPR/Cas in Microbial Biotechnology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {711},
pmid = {32695770},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has been rapidly developed as versatile genomic engineering tools with high efficiency, accuracy and flexibility, and has revolutionized traditional methods for applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas system for genome engineering are discussed, including the Cas protein, the guide RNA and the donor DNA. Following an overview of various CRISPR/Cas tools for genome engineering, including gene activation, gene interference, orthogonal CRISPR systems and precise single base editing, we highlighted the application of CRISPR/Cas toolbox for multiplexed engineering and high throughput screening. We then summarize recent applications of CRISPR/Cas systems in metabolic engineering toward production of chemicals and natural compounds, and end with perspectives of future advancements.},
}
@article {pmid32695284,
year = {2020},
author = {Dara, M and Talebzadeh, M},
title = {CRISPR/Cas as a Potential Diagnosis Technique for COVID-19.},
journal = {Avicenna journal of medical biotechnology},
volume = {12},
number = {3},
pages = {201-202},
pmid = {32695284},
issn = {2008-2835},
}
@article {pmid32694731,
year = {2020},
author = {Aregger, M and Lawson, KA and Billmann, M and Costanzo, M and Tong, AHY and Chan, K and Rahman, M and Brown, KR and Ross, C and Usaj, M and Nedyalkova, L and Sizova, O and Habsid, A and Pawling, J and Lin, ZY and Abdouni, H and Wong, CJ and Weiss, A and Mero, P and Dennis, JW and Gingras, AC and Myers, CL and Andrews, BJ and Boone, C and Moffat, J},
title = {Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.},
journal = {Nature metabolism},
volume = {2},
number = {6},
pages = {499-513},
pmid = {32694731},
issn = {2522-5812},
support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; MOP-142375//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Chromosome Mapping ; Fatty Acid Synthase, Type I/genetics/metabolism ; Fatty Acids/*biosynthesis ; Humans ; Lipid Metabolism/*genetics ; Lipogenesis/genetics ; Membrane Proteins/*genetics/*metabolism ; Nerve Tissue Proteins/genetics/metabolism ; Signal Transduction ; Starvation/genetics/metabolism ; Sterol Regulatory Element Binding Protein 2/genetics/metabolism ; },
abstract = {The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.},
}
@article {pmid32694729,
year = {2020},
author = {Tellez, K and Hang, Y and Gu, X and Chang, CA and Stein, RW and Kim, SK},
title = {In vivo studies of glucagon secretion by human islets transplanted in mice.},
journal = {Nature metabolism},
volume = {2},
number = {6},
pages = {547-557},
pmid = {32694729},
issn = {2522-5812},
support = {R01 DK090570/DK/NIDDK NIH HHS/United States ; R01 DK107507/DK/NIDDK NIH HHS/United States ; R01 DK108817/DK/NIDDK NIH HHS/United States ; UC4 DK098085/DK/NIDDK NIH HHS/United States ; P30 DK020593/DK/NIDDK NIH HHS/United States ; U01 DK120447/DK/NIDDK NIH HHS/United States ; P30 DK116074/DK/NIDDK NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; UC4 DK097610/DK/NIDDK NIH HHS/United States ; U24 DK059637/DK/NIDDK NIH HHS/United States ; R24 DK106755/DK/NIDDK NIH HHS/United States ; R01 DK050203/DK/NIDDK NIH HHS/United States ; },
mesh = {Adult ; Animals ; CRISPR-Cas Systems ; Diabetes Mellitus, Type 2/metabolism ; Female ; Glucagon/genetics/*metabolism ; Glucagon-Secreting Cells/metabolism ; Glucose Tolerance Test ; Humans ; Islets of Langerhans/*metabolism ; *Islets of Langerhans Transplantation ; Liver/metabolism ; Liver Glycogen/metabolism ; Male ; Mice ; Mice, Knockout ; Middle Aged ; },
abstract = {Little is known about regulated glucagon secretion by human islet α-cells compared to insulin secretion from β-cells, despite conclusive evidence of dysfunction in both cell types in diabetes mellitus. Distinct insulins in humans and mice permit in vivo studies of human β-cell regulation after human islet transplantation in immunocompromised mice, whereas identical glucagon sequences prevent analogous in vivo measures of glucagon output from human α-cells. Here, we use CRISPR-Cas9 editing to remove glucagon codons 2-29 in immunocompromised NSG mice, preserving the production of other proglucagon-derived hormones. Glucagon knockout NSG (GKO-NSG) mice have metabolic, liver and pancreatic phenotypes associated with glucagon-signalling deficits that revert after transplantation of human islets from non-diabetic donors. Glucagon hypersecretion by transplanted islets from donors with type 2 diabetes revealed islet-intrinsic defects. We suggest that GKO-NSG mice provide an unprecedented resource to investigate human α-cell regulation in vivo.},
}
@article {pmid32694532,
year = {2020},
author = {Voordeckers, K and Colding, C and Grasso, L and Pardo, B and Hoes, L and Kominek, J and Gielens, K and Dekoster, K and Gordon, J and Van der Zande, E and Bircham, P and Swings, T and Michiels, J and Van Loo, P and Nuyts, S and Pasero, P and Lisby, M and Verstrepen, KJ},
title = {Ethanol exposure increases mutation rate through error-prone polymerases.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3664},
pmid = {32694532},
issn = {2041-1723},
support = {FC001202/CRUK_/Cancer Research UK/United Kingdom ; FC001202/WT_/Wellcome Trust/United Kingdom ; FC001202/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/metabolism ; DNA Replication/*drug effects ; DNA, Fungal/genetics ; DNA-Directed DNA Polymerase/*metabolism ; Ethanol/*toxicity ; Mutagenesis ; Mutagenicity Tests ; *Mutation Rate ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Saccharomyces cerevisiae/drug effects/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.},
}
@article {pmid32694513,
year = {2020},
author = {Mulholland, CB and Traube, FR and Ugur, E and Parsa, E and Eckl, EM and Schönung, M and Modic, M and Bartoschek, MD and Stolz, P and Ryan, J and Carell, T and Leonhardt, H and Bultmann, S},
title = {Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12066},
pmid = {32694513},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Differentiation ; Chromatography, High Pressure Liquid ; Cytosine/*metabolism ; DNA Methylation ; DNA-Binding Proteins/*genetics/metabolism ; Dioxygenases ; Embryonic Stem Cells/*cytology/*metabolism ; Epigenesis, Genetic ; Mice ; Mice, Knockout ; *Oxidation-Reduction ; Proteome ; Proteomics ; Proto-Oncogene Proteins/*genetics/metabolism ; Tandem Mass Spectrometry ; },
abstract = {Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.},
}
@article {pmid32694114,
year = {2020},
author = {Li, X and Shi, W and Geng, LZ and Xu, JP},
title = {[Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {6},
pages = {556-564},
doi = {10.16288/j.yczz.20-017},
pmid = {32694114},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant/genetics ; Plants ; Ribonucleoproteins/genetics ; },
abstract = {The CRISPR/Cas system is the most popular genome editing technology in recent years and has been widely used in crop improvement. Compared with introducing the CRISPR/Cas system into plant cells with DNA constructs, introducing CRISPR/Cas ribonucleoprotein (RNP) to perform genome editing excels in rapid action, low off-target rates and is free of DNA insertions in editing plants. However, efficient delivery of CRISPR/Cas RNP into plant cells and achieving high editing frequency are still very challenging, which limits the extensive implementation of CRISPR/Cas RNP-mediated genome editing in plants. In this review, we summarize the progress of protein and RNP delivery methods in plant cells, and provide new perspectives of further development and future applications of the CRISPR/Cas RNP technology in plant genome editing.},
}
@article {pmid32694110,
year = {2020},
author = {Qin, RY and Wei, PC},
title = {[Prime editing creates a novel dimension of plant precise genome editing].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {6},
pages = {519-523},
doi = {10.16288/j.yczz.20-125},
pmid = {32694110},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Gene Editing ; Genome, Plant/genetics ; *Oryza/genetics ; },
abstract = {The precise genome editing has not been well established in plants, largely because of the limited frequency of homology recombination and the delivery barrier of donor templates. Recently, Dr. Caixia Gao's group from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, developed a series of plant prime editors (PPEs), which mediats the prime editing in the genomes of rice and wheat. The PPE systems are able to generate all 12 kinds of programmable base substitutions, as well desired multiplex nucleotide substitutions and small deletions or insertions without DNA double-strand breaks, thus providing versatile tools for precise plant genome editing. Herein, we introduce the structure and the editing capacity of the PPEs. The attemp on efficiency enhancements of PPEs and other PPEs are also discussed, which may provide a reference for appropriate application of PPEs in plants and also for continuous optimization of the editing tools.},
}
@article {pmid32694105,
year = {2020},
author = {Chen, YN and Lu, J},
title = {[Application of CRISPR/Cas9 mediated gene editing in trees].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {7},
pages = {657-668},
doi = {10.16288/j.yczz.20-092},
pmid = {32694105},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plants/genetics ; *Trees/genetics ; },
abstract = {The CRISPR/Cas9 system, which can induce precise modifications at a target gene, has been recognized as the most promising gene editing technology, and has played an important role in precision crop breeding. It also provides a new strategy for fundamental researches and molecular breeding of forest trees. Recently, CRISPR/Cas9-mediated gene editing has been applied more extensively in tree genetic studies. It has not only succeeded in developing new drought- resistance or disease-resistant cultivars, but also shows a great potential in regulating lignin biosynthesis and shortening the breeding cycle of forest trees. In this review, we summarize the application and advance of CRISPR/Cas9 in gene function identification and genetic improvement of forest plants. We also discuss the related problems and future perspectives. This review aims to provide a useful reference for tree functional genomics and genetic engineering breeding.},
}
@article {pmid32694104,
year = {2020},
author = {Li, GL and Yang, SX and Wu, ZF and Zhang, XW},
title = {[Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {7},
pages = {641-656},
doi = {10.16288/j.yczz.20-056},
pmid = {32694104},
issn = {0253-9772},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Knock-In Techniques ; Humans ; Recombinational DNA Repair ; },
abstract = {Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.},
}
@article {pmid32694014,
year = {2020},
author = {Li, J and Wang, Z and He, G and Ma, L and Deng, XW},
title = {CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {5},
pages = {263-272},
doi = {10.1016/j.jgg.2020.05.004},
pmid = {32694014},
issn = {1673-8527},
mesh = {Bread ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Mutagenesis/genetics ; Mutation/genetics ; Oryza/genetics ; Pollen/*genetics/growth &amp; development ; Promoter Regions, Genetic/genetics ; RNA Polymerase III/*genetics ; RNA, Guide ; Seeds/genetics/growth &amp; development ; Triticum/*genetics/growth &amp; development ; },
abstract = {Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops. High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat. The emerging effective genome editing tool, CRISPR/Cas9 system, makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles. To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat, we compared four different RNA polymerase (Pol) III promoters (TaU3p, TaU6p, OsU3p, and OsU6p) and three types of sgRNA scaffold in the protoplast system. We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective. The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles, whose orthologs, OsNP1 in rice and ZmIPE1 in maize, encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility. Triple homozygous mutations in TaNP1 genes result in complete male sterility. We further demonstrated that any one wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility. Taken together, this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.},
}
@article {pmid32692933,
year = {2020},
author = {Wambach, JA and Yang, P and Wegner, DJ and Heins, HB and Luke, C and Li, F and White, FV and Cole, FS},
title = {Functional Genomics of ABCA3 Variants.},
journal = {American journal of respiratory cell and molecular biology},
volume = {63},
number = {4},
pages = {436-443},
pmid = {32692933},
issn = {1535-4989},
support = {R01 HL149853/HL/NHLBI NIH HHS/United States ; R21 HL120760/HL/NHLBI NIH HHS/United States ; R33 HL120760/HL/NHLBI NIH HHS/United States ; },
mesh = {A549 Cells ; ATP-Binding Cassette Transporters/*genetics ; Adenosine Triphosphatases/genetics ; Alveolar Epithelial Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA, Complementary/genetics ; Fluorescent Antibody Technique/methods ; Gene Editing/methods ; Genome/*genetics ; Genomics/methods ; Humans ; Lung/metabolism ; Lung Diseases, Interstitial/genetics ; Mutation, Missense/*genetics ; },
abstract = {Rare or private, biallelic variants in the ABCA3 (ATP-binding cassette transporter A3) gene are the most common monogenic cause of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 disease-associated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPase-mediated phospholipid transport (type II). Therapies remain limited and nonspecific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus in A549 cells with CRISPR/Cas9 genome editing. Next, to generate a parent cell line (A549/ABCA3[-/-]) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS cassette, FACS, and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural vesicle phenotype) of two individual ABCA3 mutants (type I mutant, p.L101P; type II mutant, p.E292V) in A549/ABCA3[-/-] cells and in both A549 cells and primary, human alveolar type II cells that transiently express each cDNA after adenoviral-mediated transduction. We also confirmed pharmacologic rescue of ABCA3 variant-encoded mistrafficking and vesicle diameter in A549/ABCA3[-/-] cells that express p.G1421R (type I mutant). A549/ABCA3[-/-] cells provide a scalable, genetically versatile, physiologically relevant functional genomics platform for discovery of variant-specific mechanisms that disrupt ABCA3 function and for screening of potential ABCA3 pharmacologic correctors.},
}
@article {pmid32691935,
year = {2020},
author = {Ai, Y and Wu, S and Zou, C and Wei, H},
title = {LINC00941 promotes oral squamous cell carcinoma progression via activating CAPRIN2 and canonical WNT/β-catenin signaling pathway.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {18},
pages = {10512-10524},
pmid = {32691935},
issn = {1582-4934},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinoma, Squamous Cell/genetics/*pathology ; Cell Division ; Cells, Cultured ; DNA, Neoplasm/genetics/ultrastructure ; Disease Progression ; E1A-Associated p300 Protein/physiology ; Gene Expression Regulation, Neoplastic ; Genes, Reporter ; Histone Code ; Keratinocytes ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mouth Neoplasms/genetics/*pathology ; Neoplasm Proteins/biosynthesis/genetics/*physiology ; Neoplasm Transplantation ; Promoter Regions, Genetic/genetics ; RNA, Guide/administration &amp; dosage/genetics ; RNA, Long Noncoding/genetics/*metabolism ; RNA, Neoplasm/biosynthesis/genetics/*physiology ; RNA-Binding Proteins/biosynthesis/genetics/*physiology ; Recombinant Proteins/metabolism ; Tumor Stem Cell Assay ; Up-Regulation ; Wnt Signaling Pathway/genetics/*physiology ; },
abstract = {Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in many cancer developments. Previous studies showed that lncRNA LINC00941 was aberrantly expressed in oral squamous cell carcinoma (OSCC). However, its role in OSCC development remains elusive. In this study, we demonstrated that in OSCC cells, EP300 activates LINC00941 transcription through up-regulating its promoter H3K27ac modification. Up-regulated LINC00941 in turn activates CAPRIN2 expression by looping to CAPRIN2 promoter. Functional assays suggest that both LINC00941 and CAPRIN2 play pivotal roles in promoting OSCC cell proliferation and colony formation. In vivo assay further confirmed the role of LINC00941 in promoting OSCC cell tumour formation. Lastly, we showed that the role of LINC00941 and CAPRIN2 in OSCC progression was mediated through activating the canonical WNT/β-catenin signaling pathway. Thus, LINC00941/CAPRIN2/ WNT/β-catenin signaling pathway provides new therapeutic targets for OSCC treatment.},
}
@article {pmid32690971,
year = {2021},
author = {Kurt, IC and Zhou, R and Iyer, S and Garcia, SP and Miller, BR and Langner, LM and Grünewald, J and Joung, JK},
title = {CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells.},
journal = {Nature biotechnology},
volume = {39},
number = {1},
pages = {41-46},
pmid = {32690971},
issn = {1546-1696},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cytidine Deaminase/metabolism ; Cytosine/*metabolism ; DNA/genetics/metabolism ; Gene Editing/*methods ; Guanine/metabolism ; HEK293 Cells ; Humans ; },
abstract = {CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications[1-4] but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities[5,6]. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.},
}
@article {pmid32690970,
year = {2021},
author = {Zhao, D and Li, J and Li, S and Xin, X and Hu, M and Price, MA and Rosser, SJ and Bi, C and Zhang, X},
title = {Glycosylase base editors enable C-to-A and C-to-G base changes.},
journal = {Nature biotechnology},
volume = {39},
number = {1},
pages = {35-40},
pmid = {32690970},
issn = {1546-1696},
mesh = {APOBEC-1 Deaminase/genetics/metabolism ; Adenine/metabolism ; Animals ; Base Pairing/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cytidine Deaminase ; Cytosine/*metabolism ; *DNA Glycosylases ; DNA Repair/genetics ; Deoxyribonuclease I/genetics/metabolism ; Escherichia coli/genetics ; Gene Editing/*methods ; Guanine/metabolism ; Rats ; Uracil-DNA Glycosidase ; },
abstract = {Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.},
}
@article {pmid32690674,
year = {2020},
author = {Bowman, EK and Deaner, M and Cheng, JF and Evans, R and Oberortner, E and Yoshikuni, Y and Alper, HS},
title = {Bidirectional titration of yeast gene expression using a pooled CRISPR guide RNA approach.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {31},
pages = {18424-18430},
pmid = {32690674},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Fungal ; Gene Library ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Fungal/*genetics/metabolism ; RNA, Guide/*genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; },
abstract = {Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae's metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.},
}
@article {pmid32687866,
year = {2020},
author = {Southern, SJ and Oyston, PCF},
title = {Genome editing of Francisella tularensis using (CRISPR-Cas9).},
journal = {Journal of microbiological methods},
volume = {176},
number = {},
pages = {106004},
doi = {10.1016/j.mimet.2020.106004},
pmid = {32687866},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems ; Francisella tularensis/*genetics ; Gene Editing/*methods ; *Genome, Bacterial ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system is a powerful tool for gene editing in eukaryotic genomes but is still being developed for editing bacterial genomes. Here we describe the construction of an all-in-one vector for generating potentially scarless deletion mutants in Francisella tularensis LVS using a CRISPR-Cas9-based system.},
}
@article {pmid32687855,
year = {2020},
author = {Mehravar, M and Roshandel, E and Salimi, M and Chegeni, R and Gholizadeh, M and Mohammadi, MH and Hajifathali, A},
title = {Utilization of CRISPR/Cas9 gene editing in cellular therapies for lymphoid malignancies.},
journal = {Immunology letters},
volume = {226},
number = {},
pages = {71-82},
doi = {10.1016/j.imlet.2020.07.003},
pmid = {32687855},
issn = {1879-0542},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; Humans ; Immunotherapy/*methods ; Leukemia, Lymphoid/*therapy ; Lymphocytes/*immunology ; },
abstract = {The ability to change the genetic information of immune cells is a powerful tool for basic and clinical settings. CRISPR/Cas9 gene editing technology by providing an efficient approach has accelerated immune cell therapy of cancers. Lymphoid cancers comprise a wide array of disease including lymphoma, multiple myeloma and lymphocytic leukemia. Here, we review therapeutic applications of the CRISPR/Cas9 technology for immune cell therapy of common lymphoid malignancies. We describe current and future therapeutic application of CRISPR/Cas9 technology with the focus on the production and applications of engineered hematopoietic and immune cells against lymphoid malignancies. Furthermore, we provide an overview of the possible challenges and optimization of CRISPR/Cas9 system for ex- and in vivo applications within recent years.},
}
@article {pmid32687187,
year = {2020},
author = {Kim, H and Lee, WJ and Oh, Y and Kang, SH and Hur, JK and Lee, H and Song, W and Lim, KS and Park, YH and Song, BS and Jin, YB and Jun, BH and Jung, C and Lee, DS and Kim, SU and Lee, SH},
title = {Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide.},
journal = {Nucleic acids research},
volume = {48},
number = {15},
pages = {8601-8616},
pmid = {32687187},
issn = {1362-4962},
mesh = {Bacterial Proteins/*genetics ; Base Pair Mismatch/genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; DNA Cleavage ; Endodeoxyribonucleases/*genetics ; Gene Editing ; Humans ; Models, Molecular ; Mutation/genetics ; Nucleic Acid Conformation ; RNA/genetics ; RNA, Circular/genetics ; RNA, Guide/*genetics ; },
abstract = {The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.},
}
@article {pmid32687165,
year = {2020},
author = {Geisinger, JM and Stearns, T},
title = {CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells.},
journal = {Nucleic acids research},
volume = {48},
number = {16},
pages = {9067-9081},
pmid = {32687165},
issn = {1362-4962},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; R35 GM130286/GM/NIGMS NIH HHS/United States ; F32 GM122214/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Cell Cycle Checkpoints/genetics ; Cell Line ; DNA Damage/genetics ; Gene Editing/methods ; Humans ; Retinoblastoma Binding Proteins/*genetics ; Tumor Suppressor Protein p53/*genetics ; Tumor Suppressor p53-Binding Protein 1/*genetics ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53+/+ cells. Using dCas9, we determined this arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells compared to TP53-/- cells and DD-Cas9, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing recovery in primary and TP53+/+ cell lines.},
}
@article {pmid32686823,
year = {2020},
author = {Okada, K and Takase, R and Hamaoka, Y and Honda, A and Ikeda, A and Hokazono, Y and Maeda, Y and Hayasaka, O and Kotani, T and Komatsu, M and Shiozaki, K},
title = {Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes.},
journal = {The Biochemical journal},
volume = {477},
number = {15},
pages = {2841-2857},
doi = {10.1042/BCJ20200348},
pmid = {32686823},
issn = {1470-8728},
mesh = {Animals ; Animals, Genetically Modified ; Body Weight/genetics ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Nonmammalian ; Female ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Glycoproteins/genetics/metabolism ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Male ; Mucolipidoses/etiology/genetics ; N-Acetylneuraminic Acid/metabolism ; Neuraminidase/*genetics/*metabolism ; Osteogenesis/genetics ; Phenotype ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both α2-3 and α2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Furthermore, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild-type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa, lamp1a, and tfeb were up-regulated in adult Neu1-KO muscle as compared with WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.},
}
@article {pmid32685983,
year = {2020},
author = {Li, S and Jia, Z and Wang, K and Du, L and Li, H and Lin, Z and Ye, X},
title = {Screening and functional characterization of candidate resistance genes to powdery mildew from Dasypyrum villosum#4 in a wheat line Pm97033.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {11},
pages = {3067-3083},
doi = {10.1007/s00122-020-03655-4},
pmid = {32685983},
issn = {1432-2242},
mesh = {Ascomycota/*pathogenicity ; CRISPR-Cas Systems ; Disease Resistance/*genetics ; *Genes, Plant ; Leucine-Rich Repeat Proteins ; Lipoxygenase/genetics ; Plant Diseases/*genetics/microbiology ; Plant Immunity ; Plants, Genetically Modified/microbiology ; Poaceae/*genetics ; Proteins/genetics ; Transcriptome ; Translocation, Genetic ; Triticum/genetics ; },
abstract = {Three genes designated DvLox, Pm21#4, and Pm21#4-H identified in a wheat-Dasypyrum villosum#4 T6V#4S·6DL translocation line Pm97033 conferred wheat for powdery mildew resistance. Powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt) is one of the most devastating diseases in wheat. To date, only a few genes conferring resistance to wheat PM are cloned. Dasypyrum villosum is a wild relative of wheat, which provides Pm21 conferring wheat immunity to PM. In this study, we obtained many differentially expressed genes (DEGs) from a wheat-D. villosum#4 T6V#4S·6DL translocation line Pm97033 using RNA-sequencing. Among them, 7 DEGs associated with pathogen resistance were up-regulated in front of Bgt infection. Virus-induced gene silencing and transformation assays demonstrated that two of them, DvLox and Pm21#4 encoding a lipoxygenase and a encoding coiled-coil/nucleotide-binding site/leucine-rich repeat resistance protein, conferred wheat PM resistance. The transgenic wheat plants expressing DvLox enhanced PM resistance, and the transgenic wheat plants expressing Pm21#4 showed PM immunity. The Pm21#4-silenced Pm97033 plants by the cluster regularly interspaced short palindromic repeats-associated endonuclease (CRISPR/Cas9) system were susceptible to PM. Thus, Pm21#4 is a key gene contributing PM immune resistance in Pm97033. Constitutively expression of Pm21#4-H, which is silenced in Pm97033 and D. villosum#4, endowed a PM-susceptible wheat variety Fielder with PM immune resistance.},
}
@article {pmid32685932,
year = {2020},
author = {Tran, NT and Trombke, J and Rajewsky, K and Chu, VT},
title = {Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100028},
pmid = {32685932},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Knock-In Techniques/*methods ; Hematopoietic Stem Cells/*cytology ; *Homologous Recombination ; Mice ; Stem Cells/*cytology ; },
abstract = {Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis. For complete details on the use and execution of this protocol, please refer to Tran et al. (2019).},
}
@article {pmid32684419,
year = {2020},
author = {Biswas, S and Tian, J and Li, R and Chen, X and Luo, Z and Chen, M and Zhao, X and Zhang, D and Persson, S and Yuan, Z and Shi, J},
title = {Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {5},
pages = {273-280},
doi = {10.1016/j.jgg.2020.04.004},
pmid = {32684419},
issn = {1673-8527},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; *DNA Shuffling ; Homozygote ; INDEL Mutation/genetics ; Mutant Proteins/genetics ; Oryza/*genetics ; Plant Proteins/*genetics ; Plants, Genetically Modified ; },
abstract = {Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T2 to T4 generations, of which the selection of T1 and T2 was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T2 plants. All indel mutants passed stably to T3 or T4 without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.},
}
@article {pmid32682455,
year = {2020},
author = {Yoon, AR and Jung, BK and Choi, E and Chung, E and Hong, J and Kim, JS and Koo, T and Yun, CO},
title = {CRISPR-Cas12a with an oAd Induces Precise and Cancer-Specific Genomic Reprogramming of EGFR and Efficient Tumor Regression.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {10},
pages = {2286-2296},
pmid = {32682455},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; ErbB Receptors/*genetics ; *Gene Editing ; Genetic Vectors/*genetics ; Humans ; Neoplasms/genetics/therapy ; *Oncolytic Virotherapy ; Oncolytic Viruses/*genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas12a represents a class 2/type V CRISPR RNA-guided endonuclease, holding promise as a precise genome-editing tool in vitro and in vivo. For efficient delivery of the CRISPR-Cas system into cancer, oncolytic adenovirus (oAd) has been recognized as a promising alternative vehicle to conventional cancer therapy, owing to its cancer specificity; however, to our knowledge, it has not been used for genome editing. In this study, we show that CRISPR-Cas12a mediated by oAd disrupts the oncogenic signaling pathway with excellent cancer specificity. The intratumoral delivery of a single oAd co-expressing a Cas12a and a CRISPR RNA (crRNA) targeting the epidermal growth factor receptor (EGFR) gene (oAd/Cas12a/crEGFR) induces efficient and precise editing of the targeted EGFR gene in a cancer-specific manner, without detectable off-target nuclease activity. Importantly, oAd/Cas12a/crEGFR elicits a potent antitumor effect via robust induction of apoptosis and inhibition of tumor cell proliferation, ultimately leading to complete tumor regression in a subset of treated mice. Collectively, in this study we show precise genomic reprogramming via a single oAd vector-mediated CRISPR-Cas system and the feasibility of such system as an alternative cancer therapy.},
}
@article {pmid32682409,
year = {2020},
author = {Zhang, F and Liu, R and Zhang, H and Liu, C and Liu, C and Lu, Y},
title = {Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells.},
journal = {BMC cancer},
volume = {20},
number = {1},
pages = {673},
pmid = {32682409},
issn = {1471-2407},
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Brain Neoplasms/drug therapy/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Doxorubicin/pharmacology/therapeutic use ; Drug Resistance, Neoplasm/genetics ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Glioblastoma/drug therapy/*genetics/pathology ; Humans ; Mice ; Neoplasm Grading ; Neoplastic Stem Cells/*pathology ; Oncogenes/genetics ; RNA-Binding Proteins/*genetics ; Temozolomide/pharmacology/therapeutic use ; Up-Regulation ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Glioblastoma is devastating cancer with a high frequency of occurrence and poor survival rate and it is urgent to discover novel glioblastoma-specific antigens for the therapy. Cancer-germline genes are known to be related to the formation and progression of several cancer types by promoting tumor transformation. Dazl is one such germline gene and is up-regulated in a few germ cell cancers. In this study, we analyzed the expression of Dazl in human glioblastoma tissues and cells, and investigated its significance in proliferation, migration, invasion and chemoresistance of the glioblastoma cell lines.<br><br>METHODS: We evaluated the expression of Dazl in different pathologic grades of glioblastoma tissues by immunohistochemistry. We assessed the expression of Dazl in glioblastoma cells and normal human astrocytes (NHA) cells by western blotting and RT-qPCR. Then we generated Dazl knockout glioblastoma cell lines using the CRISPR/Cas9 gene-editing technology to explore the cellular function of Dazl. We detected the proliferation and germline traits via CCK-8 assays and alkaline phosphatase staining, respectively. Boyden chamber assays were performed to measure glioblastoma cell migration and invasion. Crystal violet staining was used to determine the number of viable cells after the treatment of Doxorubicin and Temozolomide. Finally, we used subcutaneous xenograft studies to measure the growth of tumors in vivo.<br><br>RESULTS: We found that Dazl was upregulated in glioblastoma tissues and glioblastoma cell lines. Dazl knockdown glioblastoma cells showed decreased cellular proliferation, migration, invasion, and resistance in vitro, and inhibited the initiation of glioblastoma in vivo. The glioblastoma cell lines A172, U251, and LN229 were found to express stem cell markers CD133, Oct4, Nanog, and Sox2. The expression of these markers was downregulated in Dazl-deficient cells.<br><br>CONCLUSIONS: Our results indicated that Dazl contributes to the tumorigenicity of glioblastoma via reducing cell stemness. Therefore, cancer-germline genes might represent a new paradigm of glioblastoma-initiating cells in the treatment of malignant tumors.},
}
@article {pmid32681501,
year = {2020},
author = {Stojic, L},
title = {Tuning the Expression of Long Noncoding RNA Loci with CRISPR Interference.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2161},
number = {},
pages = {1-16},
doi = {10.1007/978-1-0716-0680-3_1},
pmid = {32681501},
issn = {1940-6029},
support = {C14303/A17197/CRUK_/Cancer Research UK/United Kingdom ; A24455/CRUK_/Cancer Research UK/United Kingdom ; A20412/CRUK_/Cancer Research UK/United Kingdom ; WT202878/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HeLa Cells ; Humans ; *RNA Interference ; RNA, Guide/genetics/metabolism ; RNA, Long Noncoding/*genetics/metabolism ; },
abstract = {Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression networks. Over 50,000 lncRNA loci have been annotated in the human genome, but only a subset has been involved in regulation of key cellular processes, organismal development, and diseases. Hence, the functional role for the majority of the lncRNA genes remains unknown. With the recent developments of different CRISPR/Cas9 technologies, the function of lncRNAs can now be examined. CRISPR interference (CRISPRi) is one of these methods that can be used to inhibit the expression of any genomic locus including lncRNAs. This system utilizes catalytically inactive (d)Cas9 fused to KRAB repression domain and single guide RNA against targeted genomic locus. Since CRISPRi has negligible off-target effects and does not involve changes in the underlying genomic DNA sequence, it represents a valuable addition to the existing armamentarium used to investigate lncRNA biology.},
}
@article {pmid32681448,
year = {2021},
author = {Han, B and Zhang, Y and Bi, X and Zhou, Y and Krueger, CJ and Hu, X and Zhu, Z and Tong, X and Zhang, B},
title = {Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators.},
journal = {Protein &amp; cell},
volume = {12},
number = {1},
pages = {39-56},
pmid = {32681448},
issn = {1674-8018},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; *DNA End-Joining Repair ; DNA, Circular/genetics/metabolism ; Embryo, Nonmammalian ; Gene Editing/*methods ; *Gene Knock-In Techniques ; *Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; *Genotyping Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Integrases/genetics/metabolism ; Luminescent Proteins/genetics/metabolism ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/embryology/*genetics/metabolism ; },
abstract = {Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.},
}
@article {pmid32681048,
year = {2020},
author = {Kang, SH and Lee, WJ and An, JH and Lee, JH and Kim, YH and Kim, H and Oh, Y and Park, YH and Jin, YB and Jun, BH and Hur, JK and Kim, SU and Lee, SH},
title = {Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3596},
pmid = {32681048},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; Gene Editing ; Humans ; Mutation ; RNA, Guide/genetics ; },
abstract = {CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.},
}
@article {pmid32681021,
year = {2020},
author = {Liu, MS and Gong, S and Yu, HH and Jung, K and Johnson, KA and Taylor, DW},
title = {Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3576},
pmid = {32681021},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; DNA, Bacterial/chemistry/genetics/*metabolism ; Kinetics ; Streptococcus pyogenes/chemistry/*enzymology/genetics/metabolism ; },
abstract = {CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.},
}
@article {pmid32680590,
year = {2021},
author = {Wang, Y and Liu, Y and Zheng, P and Sun, J and Wang, M},
title = {Microbial Base Editing: A Powerful Emerging Technology for Microbial Genome Engineering.},
journal = {Trends in biotechnology},
volume = {39},
number = {2},
pages = {165-180},
doi = {10.1016/j.tibtech.2020.06.010},
pmid = {32680590},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/trends ; *Genome, Microbial/genetics ; Homologous Recombination ; Technology/trends ; },
abstract = {Genome engineering is crucial for answering fundamental questions about, and exploring practical applications of, microorganisms. Various microbial genome-engineering tools, including CRISPR/Cas-enhanced homologous recombination (HR), have been developed, with ever-improving simplicity, efficiency, and applicability. Recently, a powerful emerging technology based on CRISPR/Cas-nucleobase deaminase fusions, known as base editing, opened new avenues for microbial genome engineering. Base editing enables nucleotide transition without inducing lethal double-stranded (ds)DNA cleavage, adding foreign donor DNA, or depending on inefficient HR. Here, we review ongoing efforts to develop and apply base editing to engineer industrially and clinically relevant microorganisms. We also summarize bioinformatics tools that would greatly facilitate guide (g)RNA design and sequencing data analysis and discuss the future challenges and prospects associated with this technology.},
}
@article {pmid32679613,
year = {2020},
author = {Zhu, XX and Zhan, QM and Wei, YY and Yan, AF and Feng, J and Liu, L and Lu, SS and Tang, DS},
title = {CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {55},
number = {10},
pages = {1314-1327},
doi = {10.1111/rda.13775},
pmid = {32679613},
issn = {1439-0531},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Gene Knockout Techniques/veterinary ; Male ; Muscle Fibers, Skeletal/physiology ; Myostatin/*genetics ; Nuclear Transfer Techniques/veterinary ; Pork Meat ; Swine ; Swine, Miniature/*genetics/growth &amp; development ; },
abstract = {CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.},
}
@article {pmid32678895,
year = {2020},
author = {Blouin, JM and Ged, C and Lalanne, M and Lamrissi-Garcia, I and Morice-Picard, F and Costet, P and Daher, R and Moreau-Gaudry, F and Bedel, A and Puy, H and Gouya, L and Karim, Z and Richard, E},
title = {Iron chelation rescues hemolytic anemia and skin photosensitivity in congenital erythropoietic porphyria.},
journal = {Blood},
volume = {136},
number = {21},
pages = {2457-2468},
doi = {10.1182/blood.2020006037},
pmid = {32678895},
issn = {1528-0020},
mesh = {5-Aminolevulinate Synthetase/antagonists &amp; inhibitors/biosynthesis/genetics ; Adult ; Anemia, Hemolytic/*drug therapy/etiology ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Deferiprone/*therapeutic use ; Disease Models, Animal ; Erythroid Cells/drug effects/metabolism ; Female ; Gene Knock-In Techniques ; Humans ; Iron/metabolism ; Iron Chelating Agents/*therapeutic use ; Iron Overload/*drug therapy/etiology ; Leukemia, Erythroblastic, Acute/pathology ; Mice ; Peripheral Blood Stem Cells/drug effects/metabolism ; Photosensitivity Disorders/*drug therapy/etiology ; Porphyria, Acute Intermittent/metabolism ; Porphyria, Erythropoietic/complications/*drug therapy ; Porphyrins/biosynthesis ; RNA Interference ; RNA, Small Interfering/pharmacology ; },
abstract = {Congenital erythropoietic porphyria (CEP) is an inborn error of heme synthesis resulting from uroporphyrinogen III synthase (UROS) deficiency and the accumulation of nonphysiological porphyrin isomer I metabolites. Clinical features are heterogeneous among patients with CEP but usually combine skin photosensitivity and chronic hemolytic anemia, the severity of which is related to porphyrin overload. Therapeutic options include symptomatic strategies only and are unsatisfactory. One promising approach to treating CEP is to reduce the erythroid production of porphyrins through substrate reduction therapy by inhibiting 5-aminolevulinate synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthetic pathway. We efficiently reduced porphyrin accumulation after RNA interference-mediated downregulation of ALAS2 in human erythroid cellular models of CEP disease. Taking advantage of the physiological iron-dependent posttranscriptional regulation of ALAS2, we evaluated whether iron chelation with deferiprone could decrease ALAS2 expression and subsequent porphyrin production in vitro and in vivo in a CEP murine model. Treatment with deferiprone of UROS-deficient erythroid cell lines and peripheral blood CD34+-derived erythroid cultures from a patient with CEP inhibited iron-dependent protein ALAS2 and iron-responsive element-binding protein 2 expression and reduced porphyrin production. Furthermore, porphyrin accumulation progressively decreased in red blood cells and urine, and skin photosensitivity in CEP mice treated with deferiprone (1 or 3 mg/mL in drinking water) for 26 weeks was reversed. Hemolysis and iron overload improved upon iron chelation with full correction of anemia in CEP mice treated at the highest dose of deferiprone. Our findings highlight, in both mouse and human models, the therapeutic potential of iron restriction to modulate the phenotype in CEP.},
}
@article {pmid32678143,
year = {2020},
author = {von der Heyde, B and Emmanouilidou, A and Mazzaferro, E and Vicenzi, S and Höijer, I and Klingström, T and Jumaa, S and Dethlefsen, O and Snieder, H and de Geus, E and Ameur, A and Ingelsson, E and Allalou, A and Brooke, HL and den Hoed, M},
title = {Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11831},
pmid = {32678143},
issn = {2045-2322},
support = {R01 DK106236/DK/NIDDK NIH HHS/United States ; R01 DK107786/DK/NIDDK NIH HHS/United States ; U01 DK105554/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Bradycardia/diagnostic imaging/*genetics/metabolism/physiopathology ; CRISPR-Cas Systems ; Cardiovascular Agents/pharmacology ; Embryo, Nonmammalian ; Genes, Reporter ; Genome-Wide Association Study ; Green Fluorescent Proteins/genetics/metabolism ; Heart Rate/drug effects/*physiology ; Humans ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists &amp; inhibitors/*genetics/metabolism ; Ivabradine/pharmacology ; Meta-Analysis as Topic ; Myocardial Contraction/drug effects/*physiology ; Myocytes, Smooth Muscle/cytology/drug effects/metabolism ; Optical Imaging/methods ; Pleckstrin Homology Domains/genetics ; RGS Proteins/*genetics/metabolism ; Zebrafish ; },
abstract = {A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine-an open channel blocker of HCNs-for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).},
}
@article {pmid32677892,
year = {2020},
author = {Barakate, A and Keir, E and Oakey, H and Halpin, C},
title = {Stimulation of homologous recombination in plants expressing heterologous recombinases.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {336},
pmid = {32677892},
issn = {1471-2229},
support = {BB/F020872/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Gene Expression ; *Gene Targeting ; *Homologous Recombination ; Homozygote ; Meiosis/genetics ; Mutation ; Pollen/enzymology/genetics ; Recombinases/*genetics ; Tobacco/enzymology/*genetics ; Transgenes ; },
abstract = {BACKGROUND: Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants.<br><br>RESULTS: We expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive &#x3b2;-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility.<br><br>CONCLUSION: Expression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants.},
}
@article {pmid32677821,
year = {2020},
author = {Yuan, X and Yang, C and He, Q and Chen, J and Yu, D and Li, J and Zhai, S and Qin, Z and Du, K and Chu, Z and Qin, P},
title = {Current and Perspective Diagnostic Techniques for COVID-19.},
journal = {ACS infectious diseases},
volume = {6},
number = {8},
pages = {1998-2016},
pmid = {32677821},
issn = {2373-8227},
mesh = {Betacoronavirus/*chemistry/*genetics/isolation &amp; purification ; COVID-19 ; CRISPR-Cas Systems ; Coronavirus Infections/*diagnosis/virology ; Cytopathogenic Effect, Viral ; Fluorescence Resonance Energy Transfer/methods ; Humans ; Immunochemistry/methods ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; Pneumonia, Viral/*diagnosis/virology ; RNA, Viral/genetics ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Spectrum Analysis, Raman/methods ; Tomography, X-Ray Computed/methods ; Whole Genome Sequencing/methods ; },
abstract = {Since late December 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-negative rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathology and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomography will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.},
}
@article {pmid32675376,
year = {2020},
author = {Pausch, P and Al-Shayeb, B and Bisom-Rapp, E and Tsuchida, CA and Li, Z and Cress, BF and Knott, GJ and Jacobsen, SE and Banfield, JF and Doudna, JA},
title = {CRISPR-CasΦ from huge phages is a hypercompact genome editor.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6501},
pages = {333-337},
pmid = {32675376},
issn = {1095-9203},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
abstract = {CRISPR-Cas systems are found widely in prokaryotes, where they provide adaptive immunity against virus infection and plasmid transformation. We describe a minimal functional CRISPR-Cas system, comprising a single ~70-kilodalton protein, CasΦ, and a CRISPR array, encoded exclusively in the genomes of huge bacteriophages. CasΦ uses a single active site for both CRISPR RNA (crRNA) processing and crRNA-guided DNA cutting to target foreign nucleic acids. This hypercompact system is active in vitro and in human and plant cells with expanded target recognition capabilities relative to other CRISPR-Cas proteins. Useful for genome editing and DNA detection but with a molecular weight half that of Cas9 and Cas12a genome-editing enzymes, CasΦ offers advantages for cellular delivery that expand the genome editing toolbox.},
}
@article {pmid32674762,
year = {2020},
author = {Peng, L and Zhou, J and Yin, L and Man, S and Ma, L},
title = {Integration of logic gates to CRISPR/Cas12a system for rapid and sensitive detection of pathogenic bacterial genes.},
journal = {Analytica chimica acta},
volume = {1125},
number = {},
pages = {162-168},
doi = {10.1016/j.aca.2020.05.017},
pmid = {32674762},
issn = {1873-4324},
mesh = {Animals ; Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clostridiales/enzymology ; *Computers, Molecular ; DNA, Single-Stranded/chemistry ; Endodeoxyribonucleases/chemistry ; Fluorescent Dyes/chemistry ; Food Contamination/analysis ; Humans ; Limit of Detection ; *Logic ; Luminescent Measurements ; Milk/microbiology ; Staphylococcus aureus/chemistry/*isolation &amp; purification ; },
abstract = {For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by using CRISPR-Cas12a system. These logic gates utilised the intrinsic advantages of programmability, sequence specificity and high base resolution of CRISPR-Cas12a system. Among them, the AND gate owned the potentials as a built-in biosensor that responded rapidly to external pathogenic bacteria such as Staphylococcus aureus with high sensitivity and specificity. We applied the CRISPR-Cas12a based bacterial detection after a target-amplification using PCR. The total sample-to-answer time was appropriately 2.0 h, the limit of detection (LOD) was 10[3] CFU/mL, and the dynamic range was 10[3]-10[7] CFU/mL. Also, the sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. Our results not only validated the possibility of using CRISPR-Cas systems for constructing bio-computing devices but also provided a prototype of biosensor for rapid and intelligent pathogenic bacteria detection.},
}
@article {pmid32674730,
year = {2020},
author = {Iqubal, A and Iqubal, MK and Khan, A and Ali, J and Baboota, S and Haque, SE},
title = {Gene Therapy, A Novel Therapeutic Tool for Neurological Disorders: Current Progress, Challenges and Future Prospective.},
journal = {Current gene therapy},
volume = {20},
number = {3},
pages = {184-194},
doi = {10.2174/1566523220999200716111502},
pmid = {32674730},
issn = {1875-5631},
mesh = {Alzheimer Disease/genetics/therapy ; Amyotrophic Lateral Sclerosis/genetics/therapy ; Brain/pathology ; CRISPR-Cas Systems/*genetics ; Gene Transfer Techniques/*trends ; *Genetic Therapy ; Humans ; Huntington Disease/genetics/therapy ; Lysosomal Storage Diseases/genetics/therapy ; Nervous System Diseases/genetics/pathology/*therapy ; Parkinson Disease/genetics/therapy ; },
abstract = {Neurological disorders are one of the major threat for health care system as they put enormous socioeconomic burden. All aged populations are susceptible to one or other neurological problems with symptoms of neuroinflammation, neurodegeneration and cognitive dysfunction. At present, available pharmacotherapeutics are insufficient to treat these diseased conditions and in most cases, they provide only palliative effect. It was also found that the molecular etiology of neurological disorders is directly linked with the alteration in genetic makeup, which can be inherited or triggered by the injury, environmental toxins and by some existing disease. Therefore, to take care of this situation, gene therapy has emerged as an advanced modality that claims to permanently cure the disease by deletion, silencing or edition of faulty genes and by insertion of healthier genes. In this modality, vectors (viral and non-viral) are used to deliver targeted gene into a specific region of the brain via various routes. At present, gene therapy has shown positive outcomes in complex neurological disorders, such as Parkinson's disease, Alzheimer's disease, Huntington disease, Multiple sclerosis, Amyotrophic lateral sclerosis and in lysosomal storage disease. However, there are some limitations such as immunogenic reactions non-specificity of viral vectors and a lack of effective biomarkers to understand the efficacy of therapy. Considerable progress has been made to improve vector design, gene selection and targeted delivery. This review article deals with the current status of gene therapy in neurological disorders along with its clinical relevance, challenges and future prospective.},
}
@article {pmid32673314,
year = {2020},
author = {Mosbach, V and Viterbo, D and Descorps-Declère, S and Poggi, L and Vaysse-Zinkhöfer, W and Richard, GF},
title = {Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions.},
journal = {PLoS genetics},
volume = {16},
number = {7},
pages = {e1008924},
pmid = {32673314},
issn = {1553-7404},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Chromosome Deletion ; Chromosomes, Fungal/genetics ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA Ligase ATP/genetics ; DNA Repair/genetics ; DNA-Binding Proteins/genetics ; Endonucleases/genetics ; Gene Conversion/genetics ; Genome, Human/genetics ; Humans ; Myotonic Dystrophy/*genetics/pathology ; Rad52 DNA Repair and Recombination Protein/genetics ; *Recombination, Genetic ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics ; Trinucleotide Repeat Expansion/genetics ; Trinucleotide Repeats/*genetics ; },
abstract = {Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.},
}
@article {pmid32672881,
year = {2020},
author = {Sexton, JT and Tabor, JJ},
title = {Multiplexing cell-cell communication.},
journal = {Molecular systems biology},
volume = {16},
number = {7},
pages = {e9618},
pmid = {32672881},
issn = {1744-4292},
mesh = {*CRISPR-Cas Systems ; Cell Communication/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/metabolism ; Homoserine/genetics/metabolism ; Metabolic Engineering/*methods ; Promoter Regions, Genetic ; Quorum Sensing/*genetics ; RNA, Guide ; Recombinant Proteins ; Small Molecule Libraries ; Synthetic Biology/*methods ; },
abstract = {The engineering of advanced multicellular behaviors, such as the programmed growth of biofilms or tissues, requires cells to communicate multiple aspects of physiological information. Unfortunately, few cell-cell communication systems have been developed for synthetic biology. Here, we engineer a genetically encoded channel selector device that enables a single communication system to transmit two separate intercellular conversations. Our design comprises multiplexer and demultiplexer sub-circuits constructed from a total of 12 CRISPRi-based transcriptional logic gates, an acyl homoserine lactone-based communication module, and three inducible promoters that enable small molecule control over the conversations. Experimentally parameterized mathematical models of the sub-components predict the steady state and dynamical performance of the full system. Multiplexed cell-cell communication has applications in synthetic development, metabolic engineering, and other areas requiring the coordination of multiple pathways among a community of cells.},
}
@article {pmid32672817,
year = {2020},
author = {Tian, J and Yang, G and Gu, Y and Sun, X and Lu, Y and Jiang, W},
title = {Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces.},
journal = {Nucleic acids research},
volume = {48},
number = {14},
pages = {8188-8202},
pmid = {32672817},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Industrial Microbiology/*methods ; *Quorum Sensing ; Sirolimus/metabolism ; Streptomyces/*genetics/metabolism ; },
abstract = {Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growth, indicating that it can achieve a trade-off between essential pathways and product synthesis. Collectively, this study provides a convenient and effective strategy for strain improvement and shows potential for application in other industrial microorganisms.},
}
@article {pmid32671994,
year = {2020},
author = {Prattapong, P and Ngernsombat, C and Aimjongjun, S and Janvilisri, T},
title = {CRISPR/Cas9-mediated double knockout of SRPK1 and SRPK2 in a nasopharyngeal carcinoma cell line.},
journal = {Cancer reports (Hoboken, N.J.)},
volume = {3},
number = {2},
pages = {e1224},
pmid = {32671994},
issn = {2573-8348},
mesh = {Alternative Splicing ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Gene Knockout Techniques/*methods ; Humans ; Nasopharyngeal Carcinoma/*genetics ; Nasopharyngeal Neoplasms/*genetics ; Protein Serine-Threonine Kinases/*genetics/physiology ; },
abstract = {BACKGROUND: Serine-arginine protein kinase (SRPK) is a regulator of alternative splicing events via phosphorylation of splicing factor proteins. Oncogenic roles of SRPK1 and SRPK2 have been reported in various types of cancer. To date, only SRPK1/2 specific inhibitors and small interfering RNA (siRNA) have been used for halting their function momentarily; however, there is no attempt to generate SRPK1/2 stable knockout cancer cells as a tool to investigate their roles in tumorigenesis.<br><br>AIM: Our objective is therefore to establish a nasopharyngeal carcinoma (NPC) cell line with stable SRPK1 or SRPK2 knockout and SRPK1/2 double knockout as a model to investigate their potential roles in NPC.<br><br>METHODS AND RESULTS: CNE1 was selected as a representative of NPC cell lines to create single and double knockout of SRPK1/2 proteins. SRPK1/2 KO plasmid with cas9, green fluorescent protein (GFP), and gRNA expression was cotransfected with SRPK1/2 homology-directed repair (HDR) plasmid containing puromycin resistance, red fluorescent protein (RFP), and 5' and 3' arm sequence for homologous recombination to CNE1 cells. The transfected CNE1 cells with GFP and RFP expression were sorted through fluorescence-activated cell sorting for further treatment with puromycin containing medium. This step generated stable single knockout of SRPK1 and SRPK2. The SRPK2 knockout NPC cells were used as a precursor for double knockout generation via transfection with Cre plasmid for excision of inserted material to generate puromycin-sensitive SRPK2 knockout clone. The puromycin-sensitive SRPK2 knockout cells were transfected with SRPK1 KO/HDR plasmid and treated with puromycin-containing medium. The puromycin-resistant cells of SRPK1/2 stable double knockout were expanded, and the corresponding protein expression was confirmed by western immunoblotting analysis.<br><br>CONCLUSION: Single and double knockout of SRPK1/2 were established using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated 9 (Cas9) system in an NPC cell line as a model for investigation of their splicing mechanism in NPC.},
}
@article {pmid32671729,
year = {2021},
author = {Liu, Z and Liao, Z and Chen, Y and Zhou, L and Huangting, W and Xiao, H},
title = {Research on CRISPR/system in major cancers and its potential in cancer treatments.},
journal = {Clinical &amp; translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico},
volume = {23},
number = {3},
pages = {425-433},
pmid = {32671729},
issn = {1699-3055},
mesh = {Bacterial Proteins ; CRISPR-Associated Protein 9 ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colorectal Neoplasms/genetics/therapy ; Endodeoxyribonucleases ; Female ; Gene Editing/*methods ; Gene Knockout Techniques ; Genetic Therapy ; Humans ; Immunotherapy/methods ; Liver Neoplasms/genetics/therapy ; Lung Neoplasms/genetics/therapy ; Lymphoma/genetics/therapy ; Male ; Mutation ; Neoplasms/*genetics/*therapy ; Prostatic Neoplasms/genetics/therapy ; Research ; Uterine Cervical Neoplasms/genetics/therapy/virology ; },
abstract = {Cancer is a serious public health problem in the world and the prevention and control of cancer has become one of the health strategies of governments around the world. According to the data of the International Agency for Research on Cancer (IARC), about 8 million people die of cancer every year in the world. With the continuous progress of medical technology, there are many methods to treat cancer at present. However, many treatment methods have achieved different therapeutic effects, some of them have obvious toxic and side effects. Therefore, it is necessary to study simpler and more effective new therapies for alleviating pain and prolonging lifetime of patients. In this view, we focus on the application progress of CRISPR system in some major cancers and its potential in cancer treatments.},
}
@article {pmid32669716,
year = {2020},
author = {Rodriguez-Fraticelli, AE and Weinreb, C and Wang, SW and Migueles, RP and Jankovic, M and Usart, M and Klein, AM and Lowell, S and Camargo, FD},
title = {Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.},
journal = {Nature},
volume = {583},
number = {7817},
pages = {585-589},
pmid = {32669716},
issn = {1476-4687},
support = {K99 HL146983/HL/NHLBI NIH HHS/United States ; R01 HL141402/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P01 HL131477/HL/NHLBI NIH HHS/United States ; //Wellcome Trust/United Kingdom ; R33 CA212697/CA/NCI NIH HHS/United States ; P01HL13147/NH/NIH HHS/United States ; HL128850-01A1/NH/NIH HHS/United States ; MR/K017047/1/MRC_/Medical Research Council/United Kingdom ; R01 HL128850/HL/NHLBI NIH HHS/United States ; WT103789AIA/WT_/Wellcome Trust/United Kingdom ; R33CA212697-01/NH/NIH HHS/United States ; },
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; CRISPR-Cas Systems ; *Cell Lineage ; Cell Self Renewal ; Female ; *Hematopoiesis ; Hematopoietic Stem Cells/*cytology/*metabolism ; Mice ; *Single-Cell Analysis ; },
abstract = {Bone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)[1,2]. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined[3-11]. Recent advances in single-cell RNA sequencing have revealed transcriptional differences among HSCs, providing a possible explanation for their functional heterogeneity[12-17]. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Analysis of differential gene expression between clones with distinct behaviour revealed an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor TCF15 to be required and sufficient to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programmes associated with functional stem cell heterogeneity and identifies a mechanism for the maintenance of the self-renewing HSC state.},
}
@article {pmid32669702,
year = {2020},
author = {},
title = {Mitochondrial genome editing: another win for curiosity-driven research.},
journal = {Nature},
volume = {583},
number = {7816},
pages = {332},
doi = {10.1038/d41586-020-02094-x},
pmid = {32669702},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; DNA, Mitochondrial ; *Gene Editing ; *Genome, Mitochondrial ; },
}
@article {pmid32669560,
year = {2020},
author = {Salvà-Serra, F and Jaén-Luchoro, D and Jakobsson, HE and Gonzales-Siles, L and Karlsson, R and Busquets, A and Gomila, M and Bennasar-Figueras, A and Russell, JE and Fazal, MA and Alexander, S and Moore, ERB},
title = {Complete genome sequences of Streptococcus pyogenes type strain reveal 100%-match between PacBio-solo and Illumina-Oxford Nanopore hybrid assemblies.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11656},
pmid = {32669560},
issn = {2045-2322},
support = {/WT_/Wellcome Trust/United Kingdom ; 101503/Z/13/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Bacterial Typing Techniques ; Base Sequence ; CRISPR-Cas Systems ; *Chromosome Mapping ; DNA, Bacterial/*genetics/metabolism ; Genome, Bacterial ; *High-Throughput Nucleotide Sequencing ; Nanopores ; Prophages/genetics ; Sequence Analysis, DNA ; Streptococcus pyogenes/classification/*genetics/virology ; Virulence Factors/*genetics/metabolism ; Whole Genome Sequencing/*methods ; },
abstract = {We present the first complete, closed genome sequences of Streptococcus pyogenes strains NCTC 8198[T] and CCUG 4207[T], the type strain of the type species of the genus Streptococcus and an important human pathogen that causes a wide range of infectious diseases. S. pyogenes NCTC 8198[T] and CCUG 4207[T] are derived from deposit of the same strain at two different culture collections. NCTC 8198[T] was sequenced, using a PacBio platform; the genome sequence was assembled de novo, using HGAP. CCUG 4207[T] was sequenced and a de novo hybrid assembly was generated, using SPAdes, combining Illumina and Oxford Nanopore sequence reads. Both strategies yielded closed genome sequences of 1,914,862 bp, identical in length and sequence identity. Combining short-read Illumina and long-read Oxford Nanopore sequence data circumvented the expected error rate of the nanopore sequencing technology, producing a genome sequence indistinguishable to the one determined with PacBio. Sequence analyses revealed five prophage regions, a CRISPR-Cas system, numerous virulence factors and no relevant antibiotic resistance genes. These two complete genome sequences of the type strain of S. pyogenes will effectively serve as valuable taxonomic and genomic references for infectious disease diagnostics, as well as references for future studies and applications within the genus Streptococcus.},
}
@article {pmid32668582,
year = {2020},
author = {Qu, M and Essemine, J and Li, M and Chang, S and Chang, T and Chen, GY and Zhu, XG},
title = {Genome-Wide Association Study Unravels LRK1 as a Dark Respiration Regulator in Rice (Oryza sativa L.).},
journal = {International journal of molecular sciences},
volume = {21},
number = {14},
pages = {},
pmid = {32668582},
issn = {1422-0067},
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems ; Carbon Cycle ; Carbon Dioxide/metabolism ; Cell Respiration ; Chlorophyll/metabolism ; Circadian Rhythm/genetics/physiology ; Darkness ; Gene Expression Regulation, Plant/radiation effects ; Gene-Environment Interaction ; *Genes, Plant ; Genome-Wide Association Study ; Greenhouse Effect ; Haplotypes/genetics ; Hot Temperature ; Oryza/genetics/growth &amp; development/*metabolism/radiation effects ; Photosynthesis ; Plant Leaves/*metabolism/radiation effects ; Plant Proteins/*genetics/physiology ; Polymorphism, Single Nucleotide ; Protein Kinases/*genetics/physiology ; Sequence Alignment ; },
abstract = {Respiration is a major plant physiological process that generates adenosine triphosphate (ATP) to support the various pathways involved in the plant growth and development. After decades of focused research on basic mechanisms of respiration, the processes and major proteins involved in respiration are well elucidated. However, much less is known about the natural variation of respiration. Here we conducted a survey on the natural variation of leaf dark respiration (Rd) in a global rice minicore diversity panel and applied a genome-wide association study (GWAS) in rice (Oryza sativa L.) to determine candidate loci associated with Rd. This rice minicore diversity panel consists of 206 accessions, which were grown under both growth room (GR) and field conditions. We found that Rd shows high single-nucleotide polymorphism (SNP) heritability under GR and it is significantly affected by genotype-environment interactions. Rd also exhibits strong positive correlation to the leaf thickness and chlorophyll content. GWAS results of Rd collected under GR and field show an overlapped genomic region in the chromosome 3 (Chr.3), which contains a lead SNP (3m29440628). There are 12 candidate genes within this region; among them, three genes show significantly higher expression levels in accessions with high Rd. Particularly, we observed that the LRK1 gene, annotated as leucine rich repeat receptor kinase, was up-regulated four times. We further found that a single significantly associated SNPs at the promoter region of LRK1, was strongly correlated with the mean annual temperature of the regions from where minicore accessions were collected. A rice lrk1 mutant shows only ~37% Rd of that of WT and retarded growth following exposure to 35 °C for 30 days, but only 24% reduction in growth was recorded under normal temperature (25 °C). This study demonstrates a substantial natural variation of Rd in rice and that the LRK1 gene can regulate leaf dark respiratory fluxes, especially under high temperature.},
}
@article {pmid32668236,
year = {2020},
author = {Zhu, X and Zhang, H and Mendell, JT},
title = {Ribosome Recycling by ABCE1 Links Lysosomal Function and Iron Homeostasis to 3' UTR-Directed Regulation and Nonsense-Mediated Decay.},
journal = {Cell reports},
volume = {32},
number = {2},
pages = {107895},
pmid = {32668236},
issn = {2211-1247},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; P30 CA142543/CA/NCI NIH HHS/United States ; P50 CA196516/CA/NCI NIH HHS/United States ; R35 CA197311/CA/NCI NIH HHS/United States ; },
mesh = {3' Untranslated Regions/*genetics ; ATP-Binding Cassette Transporters/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Codon, Terminator/genetics ; Female ; *Homeostasis ; Humans ; Iron/*metabolism ; Iron-Sulfur Proteins/metabolism ; Lysosomes/*metabolism ; Male ; Nonsense Mediated mRNA Decay/*genetics ; Oxidative Stress ; Ribosomes/*metabolism ; Transcription, Genetic ; Vacuolar Proton-Translocating ATPases/metabolism ; },
abstract = {Nonsense-mediated decay (NMD) is a pathway that degrades mRNAs containing premature termination codons. Here we describe a genome-wide screen for NMD factors that uncovers an unexpected mechanism that broadly governs 3' untranslated region (UTR)-directed regulation. The screen reveals that NMD requires lysosomal acidification, which allows transferrin-mediated iron uptake, which, in turn, is necessary for iron-sulfur (Fe-S) cluster biogenesis. This pathway maintains the activity of the Fe-S cluster-containing ribosome recycling factor ABCE1, whose impaired function results in movement of ribosomes into 3' UTRs, where they displace exon junction complexes, abrogating NMD. Importantly, these effects extend beyond NMD substrates, with ABCE1 activity required to maintain the accessibility of 3' UTRs to diverse regulators, including microRNAs and RNA binding proteins. Because of the sensitivity of the Fe-S cluster of ABCE1 to iron availability and reactive oxygen species, these findings reveal an unanticipated vulnerability of 3' UTR-directed regulation to lysosomal dysfunction, iron deficiency, and oxidative stress.},
}
@article {pmid32668203,
year = {2020},
author = {Simpson, LM and Macartney, TJ and Nardin, A and Fulcher, LJ and Röth, S and Testa, A and Maniaci, C and Ciulli, A and Ganley, IG and Sapkota, GP},
title = {Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System.},
journal = {Cell chemical biology},
volume = {27},
number = {9},
pages = {1164-1180.e5},
pmid = {32668203},
issn = {2451-9448},
support = {MC_UU_00018/6/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Affinity Labels ; Autophagy-Related Protein-1 Homolog/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics/metabolism ; Cell Line ; Gene Knock-In Techniques ; Green Fluorescent Proteins/genetics/immunology/metabolism ; Humans ; Microtubule-Associated Proteins/genetics/metabolism ; Peptides/chemistry/metabolism ; *Proteolysis ; Recombinant Fusion Proteins/genetics/metabolism ; Single-Chain Antibodies/chemistry/genetics/*metabolism ; Ubiquitination ; ras Proteins/metabolism ; },
abstract = {The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.},
}
@article {pmid32668202,
year = {2020},
author = {Röth, S and Macartney, TJ and Konopacka, A and Chan, KH and Zhou, H and Queisser, MA and Sapkota, GP},
title = {Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System.},
journal = {Cell chemical biology},
volume = {27},
number = {9},
pages = {1151-1163.e6},
pmid = {32668202},
issn = {2451-9448},
support = {MC_UU_00018/6/MRC_/Medical Research Council/United Kingdom ; MC_UU_12016/3/MRC_/Medical Research Council/United Kingdom ; },
mesh = {A549 Cells ; Affinity Labels ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation ; Gene Knock-In Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Microscopy, Fluorescence ; Peptides/chemistry/metabolism ; Protein Isoforms/chemistry/genetics/metabolism ; *Proteolysis ; Proto-Oncogene Proteins p21(ras)/chemistry/genetics/*metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called "AdPROM." Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549[GFPKRAS]) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549[GFPKRAS] led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.},
}
@article {pmid32666500,
year = {2021},
author = {Xiao, J and Xiong, Y and Yang, LT and Wang, JQ and Zhou, ZM and Dong, LW and Shi, XJ and Zhao, X and Luo, J and Song, BL},
title = {POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation.},
journal = {Protein &amp; cell},
volume = {12},
number = {4},
pages = {279-296},
pmid = {32666500},
issn = {1674-8018},
mesh = {CRISPR-Cas Systems ; HeLa Cells ; Humans ; Lipoproteins/biosynthesis/genetics ; Lysosomes/genetics/metabolism ; Membrane Proteins/genetics/*metabolism ; *Signal Transduction ; Sterol Regulatory Element Binding Proteins/genetics/*metabolism ; },
abstract = {Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.},
}
@article {pmid32665647,
year = {2020},
author = {Kim, SG},
title = {The way to true plant genome editing.},
journal = {Nature plants},
volume = {6},
number = {7},
pages = {736-737},
pmid = {32665647},
issn = {2055-0278},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; *RNA Viruses ; RNA, Guide ; },
}
@article {pmid32665635,
year = {2020},
author = {Arroyo Hornero, R and Georgiadis, C and Hua, P and Trzupek, D and He, LZ and Qasim, W and Todd, JA and Ferreira, RC and Wood, KJ and Issa, F and Hester, J},
title = {CD70 expression determines the therapeutic efficacy of expanded human regulatory T cells.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {375},
pmid = {32665635},
issn = {2399-3642},
support = {211122/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; RP-2014-05-007/DH_/Department of Health/United Kingdom ; 107212/A/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CD27 Ligand/genetics/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Female ; Flow Cytometry ; Forkhead Transcription Factors/metabolism ; Gene Editing ; Gene Knockout Techniques ; Humans ; Mice, Inbred BALB C ; Sequence Analysis, RNA ; T-Lymphocytes, Regulatory/*immunology/metabolism/transplantation ; Transcriptome ; Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolism ; },
abstract = {Regulatory T cells (Tregs) are critical mediators of immune homeostasis. The co-stimulatory molecule CD27 is a marker of highly suppressive Tregs, although the role of the CD27-CD70 receptor-ligand interaction in Tregs is not clear. Here we show that after prolonged in vitro stimulation, a significant proportion of human Tregs gain stable CD70 expression while losing CD27. The expression of CD70 in expanded Tregs is associated with a profound loss of regulatory function and an unusual ability to provide CD70-directed co-stimulation to TCR-activated conventional T cells. Genetic deletion of CD70 or its blockade prevents Tregs from delivering this co-stimulatory signal, thus maintaining their regulatory activity. High resolution targeted single-cell RNA sequencing of human peripheral blood confirms the presence of CD27[-]CD70[+] Treg cells. These findings have important implications for Treg-based clinical studies where cells are expanded over extended periods in order to achieve sufficient treatment doses.},
}
@article {pmid32665590,
year = {2020},
author = {Bandaru, S and Tsuji, MH and Shimizu, Y and Usami, K and Lee, S and Takei, NK and Yoshitome, K and Nishimura, Y and Otsuki, T and Ito, T},
title = {Structure-based design of gRNA for Cas13.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11610},
pmid = {32665590},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Endonucleases/*genetics/ultrastructure ; Humans ; Neoplasm Proteins/chemistry/genetics ; *Nucleic Acid Conformation ; Proto-Oncogene Proteins/chemistry/genetics ; RNA/genetics/ultrastructure ; RNA, Double-Stranded/genetics/ultrastructure ; RNA, Guide/genetics/*ultrastructure ; Repressor Proteins/chemistry/genetics ; },
abstract = {Cas13 endonuclease activity depends on the RNA local secondary structure with strong preference for single-stranded (SS) regions. Hence, it becomes indispensable to identify the SS regions for effective Cas13 mediated RNA knockdown. We herein present rational gRNA design by integrating experimental structure-seq data and predicted structural models. Utilizing structure-seq data for XIST transcript, we observed that gRNAs targeting the SS regions significantly induce transcript knockdown and cleavage than those targeting double-stranded (DS) regions. Further, we identified the "central seed region" in the gRNA that upon targeting the SS regions efficiently facilitates Cas13 mediated cleavage. In our following pursuits, we considered the scenario wherein experimental structure-seq data is not available, hence we used SS18-SSX2 fusion transcript indicated in synovial sarcomas and computationally predicted its structure. We observed that gRNAs targeting the SS regions predicted from the structure, efficiently induced necrosis compared to gRNAs that target the DS regions. In conclusion, for the effective RNA knockdown, the Cas13 mediated targeting strategy presented herein emphasizes the designing of gRNAs specifically targeting SS regions by utilizing structural information. Further, this strategy, in turn, can be anticipated to narrow the search space for gRNA design (by exclusively targeting SS regions) especially when lncRNAs are the targets.},
}
@article {pmid32664340,
year = {2020},
author = {Matilla, L and Arrieta, V and Jover, E and Garcia-Peña, A and Martinez-Martinez, E and Sadaba, R and Alvarez, V and Navarro, A and Fernandez-Celis, A and Gainza, A and Santamaria, E and Fernandez-Irigoyen, J and Rossignol, P and Zannad, F and Lopez-Andres, N},
title = {Soluble St2 Induces Cardiac Fibroblast Activation and Collagen Synthesis via Neuropilin-1.},
journal = {Cells},
volume = {9},
number = {7},
pages = {},
pmid = {32664340},
issn = {2073-4409},
mesh = {Animals ; Blotting, Western ; CRISPR-Cas Systems ; Collagen/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts/*metabolism ; Male ; Myocardium/*cytology/*metabolism ; NF-kappa B/metabolism ; Neuropilin-1/*metabolism ; Proteomics/methods ; Rats ; Rats, Wistar ; Real-Time Polymerase Chain Reaction ; Receptors, Interleukin-1/*metabolism ; },
abstract = {Circulating levels of soluble interleukin 1 receptor-like 1 (sST2) are increased in heart failure and associated with poor outcome, likely because of the activation of inflammation and fibrosis. We investigated the pathogenic role of sST2 as an inductor of cardiac fibroblasts activation and collagen synthesis. The effects of sST2 on human cardiac fibroblasts was assessed using proteomics and immunodetection approaches to evidence the upregulation of neuropilin-1 (NRP-1), a regulator of the profibrotic transforming growth factor (TGF)-β1. In parallel, sST2 increased fibroblast activation, collagen and fibrosis mediators. Pharmacological inhibition of nuclear factor-kappa B (NF-κB) restored NRP-1 levels and blocked profibrotic effects induced by sST2. In NRP-1 knockdown cells, sST2 failed to induce fibroblast activation and collagen synthesis. Exogenous NRP-1 enhanced cardiac fibroblast activation and collagen synthesis via NF-κB. In a pressure overload rat model, sST2 was elevated in association with cardiac fibrosis and was positively correlated with NRP-1 expression. Our study shows that sST2 induces human cardiac fibroblasts activation, as well as the synthesis of collagen and profibrotic molecules. These effects are mediated by NRP-1. The blockade of NF-κB restored NRP-1 expression, improving the profibrotic status induced by sST2. These results show a new pathogenic role for sST2 and its mediator, NRP-1, as cardiac fibroblast activators contributing to cardiac fibrosis.},
}
@article {pmid32664329,
year = {2020},
author = {Carusillo, A and Mussolino, C},
title = {DNA Damage: From Threat to Treatment.},
journal = {Cells},
volume = {9},
number = {7},
pages = {},
pmid = {32664329},
issn = {2073-4409},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Damage/genetics/*physiology ; DNA Repair/genetics/physiology ; Gene Editing ; Humans ; },
abstract = {DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.},
}
@article {pmid32663371,
year = {2021},
author = {Azeez, A and Busov, V},
title = {CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility.},
journal = {Plant biotechnology journal},
volume = {19},
number = {1},
pages = {23-25},
pmid = {32663371},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Infertility ; *Populus ; },
}
@article {pmid32663294,
year = {2020},
author = {Banerjee, D and Tateishi-Karimata, H and Ohyama, T and Ghosh, S and Endoh, T and Takahashi, S and Sugimoto, N},
title = {Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12042-12054},
pmid = {32663294},
issn = {1362-4962},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cations ; DNA/*chemistry/metabolism ; Magnesium/chemistry ; Nucleic Acid Hybridization ; Oligonucleotides, Antisense/chemical synthesis/*chemistry ; Potassium Chloride/*chemistry ; RNA/*chemistry/metabolism ; Regression Analysis ; Sodium/chemistry ; Sodium Chloride/*chemistry ; Thermodynamics ; },
abstract = {The stability of Watson-Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR-Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.},
}
@article {pmid32661438,
year = {2021},
author = {DeWeirdt, PC and Sanson, KR and Sangree, AK and Hegde, M and Hanna, RE and Feeley, MN and Griffith, AL and Teng, T and Borys, SM and Strand, C and Joung, JK and Kleinstiver, BP and Pan, X and Huang, A and Doench, JG},
title = {Optimization of AsCas12a for combinatorial genetic screens in human cells.},
journal = {Nature biotechnology},
volume = {39},
number = {1},
pages = {94-104},
pmid = {32661438},
issn = {1546-1696},
support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; U19 AI133524/AI/NIAID NIH HHS/United States ; },
mesh = {Acidaminococcus/genetics ; Apoptosis/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9 ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Gene Library ; HEK293 Cells ; Humans ; *RNA, Guide/genetics/metabolism ; },
abstract = {Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.},
}
@article {pmid32661245,
year = {2020},
author = {Gier, RA and Budinich, KA and Evitt, NH and Cao, Z and Freilich, ES and Chen, Q and Qi, J and Lan, Y and Kohli, RM and Shi, J},
title = {High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3455},
pmid = {32661245},
issn = {2041-1723},
support = {R01 GM118501/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Cell Proliferation ; Endodeoxyribonucleases/*genetics ; Epigenesis, Genetic ; *Gene Editing ; *Gene Expression Regulation, Leukemic ; Gene Library ; Genetic Engineering ; Genome, Human ; HEK293 Cells ; Humans ; K562 Cells ; Leukemia/*genetics ; Mice ; NIH 3T3 Cells ; Protein Domains ; RNA, Guide/genetics ; },
abstract = {CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&amp;Jmjd6, Kat6a&amp;Jmjd6, and Brpf1&amp;Jmjd6 in leukemia cells.},
}
@article {pmid32661163,
year = {2020},
author = {Aryan, A and Anderson, MAE and Biedler, JK and Qi, Y and Overcash, JM and Naumenko, AN and Sharakhova, MV and Mao, C and Adelman, ZN and Tu, Z},
title = {Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {30},
pages = {17702-17709},
pmid = {32661163},
issn = {1091-6490},
support = {R01 AI123338/AI/NIAID NIH HHS/United States ; R21 AI121853/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/*genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Female ; *Flight, Animal ; *Genes, Insect ; *Genetic Association Studies ; Genetic Loci ; Genotype ; Inheritance Patterns ; Male ; Membrane Proteins/*genetics ; Penetrance ; Phenotype ; Promoter Regions, Genetic ; Sex Determination Processes/*genetics ; },
abstract = {A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegyptiNix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.},
}
@article {pmid32659298,
year = {2020},
author = {Beneke, T and Gluenz, E},
title = {Bar-seq strategies for the LeishGEdit toolbox.},
journal = {Molecular and biochemical parasitology},
volume = {239},
number = {},
pages = {111295},
doi = {10.1016/j.molbiopara.2020.111295},
pmid = {32659298},
issn = {1872-9428},
support = {15/16_MSD_83633/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; *DNA Barcoding, Taxonomic ; DNA Primers ; Databases, Nucleic Acid ; *Gene Editing ; Genome, Protozoan ; Kinetoplastida/*genetics ; Leishmania/genetics ; Trypanosoma/genetics ; },
abstract = {The number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we previously developed a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligo sequences are now accessible through our website www.leishgedit.net allowing researchers to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.},
}
@article {pmid32659256,
year = {2020},
author = {Rosenblum, D and Gutkin, A and Dammes, N and Peer, D},
title = {Progress and challenges towards CRISPR/Cas clinical translation.},
journal = {Advanced drug delivery reviews},
volume = {154-155},
number = {},
pages = {176-186},
doi = {10.1016/j.addr.2020.07.004},
pmid = {32659256},
issn = {1872-8294},
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; },
abstract = {CRISPR/Cas systems (clustered regularly interspaced short palindromic repeats) have emerged as powerful tools to manipulate the genome for both research and therapeutic purposes. However, the clinical use of this system is hindered by multiple challenges, such as the rate of off-target effects, editing efficiency, the efficacy of HDR, immunogenicity, as well as development of efficient and safe delivery vehicles that can carry these compounds. Tremendous efforts are being conducted to overcome these challenges, including the discovery and engineering of more precise and efficacious Cas nucleases. Moreover, in recent years multiple viral and non-viral delivery approaches have been explored for in vivo delivery of CRISPR components. Here, we summarize the available CRISPR/Cas toolbox for genome editing as well as the recently developed in vivo delivery vehicles for CRISPR/Cas system. Furthermore, we discuss the remaining challenges for successful clinical translation of this system and highlight the current clinical applications.},
}
@article {pmid32658961,
year = {2020},
author = {Domènech, EB and Andrés, R and López-Iniesta, MJ and Mirra, S and García-Arroyo, R and Milla, S and Sava, F and Andilla, J and Loza-Álvarez, P and de la Villa, P and Gonzàlez-Duarte, R and Marfany, G},
title = {A New Cerkl Mouse Model Generated by CRISPR-Cas9 Shows Progressive Retinal Degeneration and Altered Morphological and Electrophysiological Phenotype.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {61},
number = {8},
pages = {14},
pmid = {32658961},
issn = {1552-5783},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA/*genetics ; DNA Mutational Analysis ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; *Mutation ; Phenotype ; Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism ; Retinal Cone Photoreceptor Cells/*metabolism/pathology ; Retinal Degeneration/*genetics/metabolism/pathology ; Retinal Pigment Epithelium/*metabolism/pathology ; },
abstract = {PURPOSE: Close to 100 genes cause retinitis pigmentosa, a Mendelian rare disease that affects 1 out of 4000 people worldwide. Mutations in the ceramide kinase-like gene (CERKL) are a prevalent cause of autosomal recessive cause retinitis pigmentosa and cone-rod dystrophy, but the functional role of this gene in the retina has yet to be fully determined. We aimed to generate a mouse model that resembles the phenotypic traits of patients carrying CERKL mutations to undertake functional studies and assay therapeutic approaches.<br><br>METHODS: The Cerkl locus has been deleted (around 97 kb of genomic DNA) by gene editing using the CRISPR-Cas9 D10A nickase. Because the deletion of the Cerkl locus is lethal in mice in homozygosis, a double heterozygote mouse model with less than 10% residual Cerkl expression has been generated. The phenotypic alterations of the retina of this new model have been characterized at the morphological and electrophysiological levels.<br><br>RESULTS: This CerklKD/KO model shows retinal degeneration, with a decreased number of cones and progressive photoreceptor loss, poorly stacked photoreceptor outer segment membranes, defective retinal pigment epithelium phagocytosis, and altered electrophysiological recordings in aged retinas.<br><br>CONCLUSIONS: To our knowledge, this is the first Cerkl mouse model to mimic many of the phenotypic traits, including the slow but progressive retinal degeneration, shown by human patients carrying CERKL mutations. This useful model will provide unprecedented insights into the retinal molecular pathways altered in these patients and will contribute to the design of effective treatments.},
}
@article {pmid32658470,
year = {2020},
author = {George, JT and Azhar, M and Aich, M and Sinha, D and Ambi, UB and Maiti, S and Chakraborty, D and Srivatsan, SG},
title = {Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {32},
pages = {13954-13965},
pmid = {32658470},
issn = {1520-5126},
support = {IA/S/16/1/502360/WTDBT_/DBT-Wellcome Trust India Alliance/India ; },
mesh = {CRISPR-Cas Systems/*genetics ; Click Chemistry ; Gene Editing ; Models, Molecular ; RNA/chemistry/*genetics ; RNA Nucleotidyltransferases/chemistry/*genetics/metabolism ; RNA, Guide/genetics ; },
abstract = {Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.},
}
@article {pmid32657546,
year = {2020},
author = {Ho, HI and Fang, JR and Cheung, J and Wang, HH},
title = {Programmable CRISPR-Cas transcriptional activation in bacteria.},
journal = {Molecular systems biology},
volume = {16},
number = {7},
pages = {e9427},
pmid = {32657546},
issn = {1744-4292},
support = {U01 GM110714/GM/NIGMS NIH HHS/United States ; DP5 OD009172/OD/NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Bacteria/drug effects/genetics/metabolism ; CRISPR-Cas Systems/*drug effects ; Clustered Regularly Interspaced Short Palindromic Repeats/drug effects ; Directed Molecular Evolution ; Escherichia coli/*drug effects/genetics/*metabolism ; Gene Expression Regulation, Bacterial/*drug effects/genetics ; Gene Library ; Genes, Reporter/drug effects/genetics ; Promoter Regions, Genetic ; Protein Engineering/*methods ; RNA, Guide/genetics ; Sequence Alignment ; Software ; Transcription Factors/chemistry/genetics/*metabolism ; Transcription, Genetic/*drug effects ; Viral Proteins/chemistry/genetics/*metabolism ; },
abstract = {Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by more than 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.},
}
@article {pmid32657224,
year = {2020},
author = {Kadooka, C and Yamaguchi, M and Okutsu, K and Yoshizaki, Y and Takamine, K and Katayama, T and Maruyama, JI and Tamaki, H and Futagami, T},
title = {A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {84},
number = {10},
pages = {2179-2183},
doi = {10.1080/09168451.2020.1792761},
pmid = {32657224},
issn = {1347-6947},
mesh = {Aspergillus/*genetics ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques/*methods ; Mutation ; },
abstract = {We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.},
}
@article {pmid32654970,
year = {2021},
author = {Salick, MR and Lubeck, E and Riesselman, A and Kaykas, A},
title = {The future of cerebral organoids in drug discovery.},
journal = {Seminars in cell &amp; developmental biology},
volume = {111},
number = {},
pages = {67-73},
doi = {10.1016/j.semcdb.2020.05.024},
pmid = {32654970},
issn = {1096-3634},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cerebral Cortex/drug effects/metabolism/pathology ; Drug Discovery/*methods ; Drugs, Investigational/chemistry/*pharmacology ; Humans ; Induced Pluripotent Stem Cells/cytology/drug effects/metabolism ; Machine Learning ; *Models, Biological ; Mutation ; Nerve Tissue Proteins/genetics/metabolism ; Neurodegenerative Diseases/*drug therapy/genetics/metabolism/pathology ; Neurons/cytology/drug effects/metabolism ; Neuroprotective Agents/chemistry/*pharmacology ; Organoids/*drug effects/metabolism/pathology ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods ; },
abstract = {Until the discovery of human embryonic stem cells and human induced pluripotent stem cells, biotechnology companies were severely limited in the number of human tissues that they could model in large-scale in vitro studies. Until this point, companies have been limited to immortalized cancer lines or a small number of primary cell types that could be extracted and expanded. Nowadays, protocols continue to be developed in the stem cell field, enabling researchers to model an ever-growing library of cell types in controlled, large-scale screens. One differentiation method in particular- cerebral organoids- shows substantial potential in the field of neuroscience and developmental neurobiology. Cerebral organoid technology is still in an early phase of development, and there are several challenges that are currently being addressed by academic and industrial researchers alike. Here we briefly describe some of the early adopters of cerebral organoids, several of the challenges that they are likely facing, and various technologies that are currently being implemented to overcome them.},
}
@article {pmid32654206,
year = {2020},
author = {Schrauben, M and Dempster, E and Lunnon, K},
title = {Applying gene-editing technology to elucidate the functional consequence of genetic and epigenetic variation in Alzheimer's disease.},
journal = {Brain pathology (Zurich, Switzerland)},
volume = {30},
number = {5},
pages = {992-1004},
pmid = {32654206},
issn = {1750-3639},
mesh = {Alzheimer Disease/*genetics/metabolism/*pathology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cognitive Dysfunction ; Epigenesis, Genetic/genetics ; Epigenomics/methods ; Gene Editing/*methods ; Genetic Predisposition to Disease/genetics ; Genomics/methods ; Humans ; Mutation/genetics ; Neurodegenerative Diseases/genetics ; Technology ; },
abstract = {Recent studies have highlighted a potential role of genetic and epigenetic variation in the development of Alzheimer's disease. Application of the CRISPR-Cas genome-editing platform has enabled investigation of the functional impact that Alzheimer's disease-associated gene mutations have on gene expression. Moreover, recent advances in the technology have led to the generation of CRISPR-Cas-based tools that allow for high-throughput interrogation of different risk variants to elucidate the interplay between genomic regulatory features, epigenetic modifications, and chromatin structure. In this review, we examine the various iterations of the CRISPR-Cas system and their potential application for exploring the complex interactions and disruptions in gene regulatory circuits that contribute to Alzheimer's disease.},
}
@article {pmid32653777,
year = {2020},
author = {Fillol-Salom, A and Miguel-Romero, L and Marina, A and Chen, J and Penadés, JR},
title = {Beyond the CRISPR-Cas safeguard: PICI-encoded innate immune systems protect bacteria from bacteriophage predation.},
journal = {Current opinion in microbiology},
volume = {56},
number = {},
pages = {52-58},
doi = {10.1016/j.mib.2020.06.002},
pmid = {32653777},
issn = {1879-0364},
support = {MR/S00940X/1/MRC_/Medical Research Council/United Kingdom ; MR/S00940X/1/MRC_/Medical Research Council/United Kingdom ; MR/M003876/1/MRC_/Medical Research Council/United Kingdom ; BB/N002873/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S003835/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 201531/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Bacteriophages/genetics/*physiology ; CRISPR-Cas Systems ; *Genomic Islands ; Gram-Negative Bacteria/genetics/*immunology/*virology ; Gram-Positive Bacteria/genetics/*immunology/*virology ; Host-Pathogen Interactions ; },
abstract = {Phage satellites are genetic elements that depend on helper phages for induction, packaging and transfer. To promote their lifestyles, they have evolved elegant and sophisticated strategies to inhibit phage reproduction, which will be reviewed here. We will principally focus on the convergent interference mechanisms used by phage-inducible chromosomal islands (PICIs), which are a family of satellite phages present in both Gram-positive and Gram-negative bacteria. While some PICI elements have been extensively studied for their roles in virulence and antibiotic resistance, recent studies have highlighted their relevance in controlling phage ecology and diversity. In many cases, these interference mechanisms are complemented by additional strategies that promote the preferential PICI packaging and dissemination of these elements in nature. Since the PICI-encoded mechanisms target conserved phage mechanisms, we propose here that the PICIs form part of the initial innate immune system that phages must overcome to infect their bacterial host.},
}
@article {pmid32653558,
year = {2020},
author = {Wei, H and Wang, M and Lv, N and Yang, H and Zhao, M and Huang, B and Li, R},
title = {Increased repetitive self-grooming occurs in Pax2 mutant mice generated using CRISPR/Cas9.},
journal = {Behavioural brain research},
volume = {393},
number = {},
pages = {112803},
doi = {10.1016/j.bbr.2020.112803},
pmid = {32653558},
issn = {1872-7549},
mesh = {Animals ; Anxiety/physiopathology ; Autism Spectrum Disorder/*physiopathology/*psychology ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Grooming/*physiology ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation ; PAX2 Transcription Factor/genetics/*physiology ; },
abstract = {Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction and communication, and repetitive or restricted interest and behaviors. However, the specific pathogenesis of ASD is still unclear. It has been widely accepted that genetic and environmental risk factors are associated with the pathogenesis of ASD. Paired box2 (Pax2) gene encodes a transcription factor that plays an important role in the development of the central nervous system of humans and mice. In this study, we constructed Pax2 heterozygous gene knockout (Pax2[+/-]) mice using CRISPR/Cas9 technology and performed several autistic-like behavioral assays, including self-grooming test, sociability approach, the elevated plus maze test and Y maze test. Results showed increased repetitive self-grooming and possible abnormal anxiety-like behavior occur in Pax2[+/-] mice. Furthermore, no changes were observed in the abilities of sociability and working memory in Pax2[+/-] mice compared to wild-type C57BL/6 J mice. Finally, we speculated that possible mechanism of abnormal autistic-like behaviors due to the deletion of Pax2 gene.},
}
@article {pmid32652159,
year = {2020},
author = {Chang, J and Chen, X and Zhang, T and Wang, R and Wang, A and Lan, X and Zhou, Y and Ma, S and Xia, Q},
title = {The novel insight into the outcomes of CRISPR/Cas9 editing intra- and inter-species.},
journal = {International journal of biological macromolecules},
volume = {163},
number = {},
pages = {711-717},
doi = {10.1016/j.ijbiomac.2020.07.039},
pmid = {32652159},
issn = {1879-0003},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Genetic Vectors ; High-Throughput Nucleotide Sequencing ; Humans ; INDEL Mutation ; Mice ; Mutation ; RNA, Guide ; Species Specificity ; Zebrafish ; },
abstract = {The CRISPR/Cas (clustered regularly interspaced short palindromic repeat technology/CRISPR-associated protein) is a widely used and powerful research tool in biosciences and a promising therapeutic agent for treating genetic diseases. Mutations induced by Cas9 are generally considered stochastic and unpredictable, thus hindering its applications where precise genetic alternations are required. Here, through deep sequencing and analysis of genome editing outcomes of multiple sites in four distinct species, we found that Cas9-induced mutations are coincident in mutation types but are significantly different in indel patterns among species. In human and mouse cells, indels were almost evenly distributed at both ends of the cleavage sites. However, the indels mainly appeared at the upstream of cleavage sites in Bombyx mori, while they predominantly occurred downstream of the cleavage sites in the zebrafish Danio rerio. We also found that within a species, indel patterns are sequence dependent, wherein deletions between two adjacent micro-homology sequences were the most frequently observed mutations in the repair spectrum. These results suggested the species differences in DNA repair processes during Cas9-induced gene editing, and the important role of sequence structure at the target site in predicting the gene editing outcome.},
}
@article {pmid32651924,
year = {2020},
author = {Mathony, J and Hoffmann, MD and Niopek, D},
title = {Optogenetics and CRISPR: A New Relationship Built to Last.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2173},
number = {},
pages = {261-281},
doi = {10.1007/978-1-0716-0755-8_18},
pmid = {32651924},
issn = {1940-6029},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Humans ; Optogenetics/*methods ; },
abstract = {Since the breakthrough discoveries that CRISPR-Cas9 nucleases can be easily programmed and employed to induce targeted double-strand breaks in mammalian cells, the gene editing field has grown exponentially. Today, CRISPR technologies based on engineered class II CRISPR effectors facilitate targeted modification of genes and RNA transcripts. Moreover, catalytically impaired CRISPR-Cas variants can be employed as programmable DNA binding domains and used to recruit effector proteins, such as transcriptional regulators, epigenetic modifiers or base-modifying enzymes, to selected genomic loci. The juxtaposition of CRISPR and optogenetics enables spatiotemporally confined and highly dynamic genome perturbations in living cells and animals and holds unprecedented potential for biology and biomedicine.Here, we provide an overview of the state-of-the-art methods for light-control of CRISPR effectors. We will detail the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions. Finally, we will discuss limitations of current optogenetic CRISPR tools and point out routes for future innovation in this emerging field.},
}
@article {pmid32651653,
year = {2020},
author = {Wandrer, F and Frangež, &#x17d; and Liebig, S and John, K and Vondran, F and Wedemeyer, H and Veltmann, C and Pfeffer, TJ and Shibolet, O and Schulze-Osthoff, K and Simon, HU and Bantel, H},
title = {Autophagy alleviates amiodarone-induced hepatotoxicity.},
journal = {Archives of toxicology},
volume = {94},
number = {10},
pages = {3527-3539},
pmid = {32651653},
issn = {1432-0738},
mesh = {Aged ; Amiodarone/*adverse effects ; Anti-Arrhythmia Agents/adverse effects ; Apoptosis/drug effects ; *Autophagy ; CRISPR-Cas Systems ; Cell Survival ; Cells, Cultured ; *Chemical and Drug Induced Liver Injury ; Chloroquine/pharmacology ; Endoplasmic Reticulum Stress/drug effects ; Female ; Gene Knockout Techniques ; Hep G2 Cells ; Hepatocytes/*drug effects ; Humans ; Keratin-18/blood ; Lipid Metabolism/*drug effects ; Male ; },
abstract = {Amiodarone is a widely used antiarrhythmic drug that can cause the development of steatohepatitis as well as liver fibrosis and cirrhosis. The molecular mechanisms of amiodarone-mediated liver injury remain largely unknown. We therefore analyzed amiodarone-mediated hepatocellular injury in patients with chronic heart failure, in primary hepatocytes and HepG2 cells. We found that amiodarone-treated patients with chronic heart failure revealed significantly higher serum levels of caspase-cleaved keratin-18, an apoptosis biomarker, compared to healthy individuals or patients not receiving amiodarone. Furthermore, amiodarone treatment of hepatocytes resulted in apoptosis associated with lipid accumulation and ER-stress induction. Liver cell steatosis was accompanied by enhanced de novo lipogenesis which, after reaching peak levels, declined together with decreased activation of ER stress. The decline of amiodarone-mediated lipotoxicity was associated with protective autophagy induction. In contrast, in hepatocytes treated with the autophagy inhibitor chloroquine as well as in autophagy gene (ATG5 or ATG7)-deficient hepatocytes, amiodarone-triggered toxicity was increased. In conclusion, we demonstrate that amiodarone induces lipid accumulation associated with ER stress and apoptosis in hepatocytes, which is mirrored by increased keratin-18 fragment serum levels in amiodarone-treated patients. Autophagy reduces amiodarone-mediated lipotoxicity and could provide a therapeutic strategy for protection from drug-induced liver injury.},
}
@article {pmid32651565,
year = {2020},
author = {Tong, Y and Whitford, CM and Blin, K and Jørgensen, TS and Weber, T and Lee, SY},
title = {CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes.},
journal = {Nature protocols},
volume = {15},
number = {8},
pages = {2470-2502},
pmid = {32651565},
issn = {1750-2799},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Plasmids/genetics ; Streptomyces/*genetics ; },
abstract = {Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.},
}
@article {pmid32651459,
year = {2020},
author = {Jang, HK and Song, B and Hwang, GH and Bae, S},
title = {Current trends in gene recovery mediated by the CRISPR-Cas system.},
journal = {Experimental &amp; molecular medicine},
volume = {52},
number = {7},
pages = {1016-1027},
pmid = {32651459},
issn = {2092-6413},
support = {2018M3A9H3022412//National Research Foundation (NRF)/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA Cleavage ; DNA Repair/genetics ; Gene Editing ; *Genes ; },
abstract = {The CRISPR-Cas system has undoubtedly revolutionized the genome editing field, enabling targeted gene disruption, regulation, and recovery in a guide RNA-specific manner. In this review, we focus on currently available gene recovery strategies that use CRISPR nucleases, particularly for the treatment of genetic disorders. Through the action of DNA repair mechanisms, CRISPR-mediated DNA cleavage at a genomic target can shift the reading frame to correct abnormal frameshifts, whereas DNA cleavage at two sites, which can induce large deletions or inversions, can correct structural abnormalities in DNA. Homology-mediated or homology-independent gene recovery strategies that require donor DNAs have been developed and widely applied to precisely correct mutated sequences in genes of interest. In contrast to the DNA cleavage-mediated gene correction methods listed above, base-editing tools enable base conversion in the absence of donor DNAs. In addition, CRISPR-associated transposases have been harnessed to generate a targeted knockin, and prime editors have been developed to edit tens of nucleotides in cells. Here, we introduce currently developed gene recovery strategies and discuss the pros and cons of each.},
}
@article {pmid32651405,
year = {2020},
author = {Gans, I and Hartig, EI and Zhu, S and Tilden, AR and Hutchins, LN and Maki, NJ and Graber, JH and Coffman, JA},
title = {Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11415},
pmid = {32651405},
issn = {2045-2322},
support = {P20 GM103423/GM/NIGMS NIH HHS/United States ; P20 GM104318/GM/NIGMS NIH HHS/United States ; R03 HD099468/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; *Frameshift Mutation ; Gene Deletion ; Gene Expression Regulation ; Homozygote ; Humans ; Hydrocortisone/metabolism ; Inflammation ; Kruppel-Like Transcription Factors/*metabolism ; Larva ; Mutation ; RNA-Seq ; Receptors, Glucocorticoid/*metabolism ; Receptors, Mineralocorticoid/metabolism ; Signal Transduction ; *Transcriptome ; Up-Regulation ; Zebrafish/genetics ; Zebrafish Proteins/*metabolism ; },
abstract = {The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR[369-], which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR[369-] larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.},
}
@article {pmid32650483,
year = {2020},
author = {Delos Santos, K and Kwon, E and Moon, NS},
title = {PRPS-Associated Disorders and the Drosophila Model of Arts Syndrome.},
journal = {International journal of molecular sciences},
volume = {21},
number = {14},
pages = {},
pmid = {32650483},
issn = {1422-0067},
support = {162337/CAPMC/CIHR/Canada ; },
mesh = {Animals ; Ataxia/*genetics ; CRISPR-Cas Systems/genetics ; Deaf-Blind Disorders/*genetics ; Disease Models, Animal ; Drosophila melanogaster/*genetics ; Gene Editing/methods ; Genetic Diseases, X-Linked/*genetics ; Humans ; Mutation/genetics ; Ribose-Phosphate Pyrophosphokinase/*genetics ; },
abstract = {While a plethora of genetic techniques have been developed over the past century, modifying specific sequences of the fruit fly genome has been a difficult, if not impossible task. clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 truly redefined molecular genetics and provided new tools to model human diseases in Drosophila melanogaster. This is particularly true for genes whose protein sequences are highly conserved. Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism whose missense mutations are found in several neurological disorders, including Arts syndrome. In addition, PRPS is deregulated in cancer, particularly those that become resistant to cancer therapy. Notably, Drosophila PRPS shares about 90% protein sequence identity with its human orthologs, making it an ideal gene to study via CRISPR/Cas9. In this review, we will summarize recent findings on PRPS mutations in human diseases including cancer and on the molecular mechanisms by which PRPS activity is regulated. We will also discuss potential applications of Drosophila CRISPR/Cas9 to model PRPS-dependent disorders and other metabolic diseases that are associated with nucleotide metabolism.},
}
@article {pmid32649862,
year = {2020},
author = {Olivieri, M and Cho, T and Álvarez-Quilón, A and Li, K and Schellenberg, MJ and Zimmermann, M and Hustedt, N and Rossi, SE and Adam, S and Melo, H and Heijink, AM and Sastre-Moreno, G and Moatti, N and Szilard, RK and McEwan, A and Ling, AK and Serrano-Benitez, A and Ubhi, T and Feng, S and Pawling, J and Delgado-Sainz, I and Ferguson, MW and Dennis, JW and Brown, GW and Cortés-Ledesma, F and Williams, RS and Martin, A and Xu, D and Durocher, D},
title = {A Genetic Map of the Response to DNA Damage in Human Cells.},
journal = {Cell},
volume = {182},
number = {2},
pages = {481-496.e21},
pmid = {32649862},
issn = {1097-4172},
support = {ZIA ES102765/ImNIH/Intramural NIH HHS/United States ; FDN143343//CIHR/Canada ; FRN 123518//CIHR/Canada ; PJT-156330//CIHR/Canada ; },
mesh = {Aminoquinolines/pharmacology ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cytochrome-B(5) Reductase/genetics/metabolism ; *DNA Damage/drug effects ; DNA Helicases/genetics/metabolism ; DNA Repair ; DNA Topoisomerases, Type II/genetics/metabolism ; Gene Regulatory Networks/*physiology ; Humans ; Mice ; Picolinic Acids/pharmacology ; RNA, Guide/metabolism ; Tumor Suppressor Protein p53/deficiency/genetics ; },
abstract = {The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.},
}
@article {pmid32649860,
year = {2020},
author = {Lin, FL and Wang, PY and Chuang, YF and Wang, JH and Wong, VHY and Bui, BV and Liu, GS},
title = {Gene Therapy Intervention in Neovascular Eye Disease: A Recent Update.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {10},
pages = {2120-2138},
pmid = {32649860},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Clinical Trials as Topic ; Disease Management ; Disease Susceptibility ; Eye Diseases/etiology/*pathology/*therapy ; Gene Editing ; *Genetic Therapy/methods ; Humans ; Neovascularization, Pathologic/genetics/*therapy ; Platelet-Derived Growth Factor/genetics/metabolism ; Treatment Outcome ; Vascular Endothelial Growth Factor A/genetics/metabolism ; },
abstract = {Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.},
}
@article {pmid32649732,
year = {2020},
author = {de Oliveira, VC and Gomes Mariano Junior, C and Belizário, JE and Krieger, JE and Fernandes Bressan, F and Roballo, KCS and Fantinato-Neto, P and Meirelles, FV and Chiaratti, MR and Concordet, JP and Ambrósio, CE},
title = {Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.},
journal = {PloS one},
volume = {15},
number = {7},
pages = {e0235856},
pmid = {32649732},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle/*genetics ; Cells, Cultured ; DNA Replication ; DNA, Mitochondrial/genetics ; DNA-Binding Proteins/*genetics ; Fibroblasts/metabolism ; Gene Dosage ; *Gene Editing ; Mitochondria/genetics ; Mitochondrial Proteins/*genetics ; Transcription Factors/*genetics ; },
abstract = {Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.},
}
@article {pmid32649026,
year = {2020},
author = {Kolay, S and Diamond, MI},
title = {Alzheimer's disease risk modifier genes do not affect tau aggregate uptake, seeding or maintenance in cell models.},
journal = {FEBS open bio},
volume = {10},
number = {9},
pages = {1912-1920},
pmid = {32649026},
issn = {2211-5463},
mesh = {Alzheimer Disease/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Genes, Modifier/genetics ; Humans ; *Models, Biological ; Protein Aggregates ; tau Proteins/*metabolism ; },
abstract = {Alzheimer's disease (AD) afflicts millions of people worldwide and is caused by accumulated amyloid beta and tau pathology. Progression of tau pathology in AD may utilize prion mechanisms of propagation in which pathological tau aggregates released from one cell are taken up by neighboring or connected cells and act as templates for their own replication, a process termed 'seeding'. We have used HEK293T cells to model various aspects of pathological tau propagation, including uptake of tau aggregates, induced seeding by exogenous aggregates, seeding caused by Lipofectamine-mediated delivery to the cell interior, and stable maintenance of aggregates in dividing cells. The factors that regulate these processes are not well understood, and we hypothesized that AD risk modifier genes might play a role. We identified 22 genes strongly linked to AD via meta-analysis of genome-wide association study (GWAS). We used CRISPR/Cas9 to individually knock out each gene in HEK293T cells and verified disruption using genomic sequencing. We then tested the effect of gene knockout in tau aggregate uptake, naked and Lipofectamine-mediated seeding, and aggregate maintenance in these cultured cell lines. GWAS gene knockouts had no effect in these models of tau pathology. With obvious caveats due to the model systems used, these results imply that the 22 AD risk modifier genes are unlikely to directly modulate tau uptake, seeding, or aggregate maintenance in a cell-autonomous fashion.},
}
@article {pmid32648491,
year = {2020},
author = {Walsh, JD and Boivin, O and Barr, MM},
title = {What about the males? the C. elegans sexually dimorphic nervous system and a CRISPR-based tool to study males in a hermaphroditic species.},
journal = {Journal of neurogenetics},
volume = {34},
number = {3-4},
pages = {323-334},
pmid = {32648491},
issn = {1563-5260},
support = {K12 GM093854/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R01 DK059418/DK/NIDDK NIH HHS/United States ; R01 DK116606/DK/NIDDK NIH HHS/United States ; },
mesh = {Animal Structures/growth &amp; development/innervation/ultrastructure ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*anatomy &amp; histology/genetics/physiology ; Caenorhabditis elegans Proteins/*genetics/physiology ; Cell Cycle Proteins/*genetics ; Cilia/chemistry ; DNA-Binding Proteins/physiology ; Disease Models, Animal ; Extracellular Vesicles/physiology ; Hermaphroditic Organisms/*physiology/ultrastructure ; Humans ; Interneurons/physiology ; Male ; Nervous System/*anatomy &amp; histology/growth &amp; development ; Neuronal Plasticity ; Neurons/classification/physiology/ultrastructure ; Neurotransmitter Agents/physiology ; Nondisjunction, Genetic ; Polycystic Kidney, Autosomal Dominant/genetics ; *Sex Characteristics ; Sexual Behavior, Animal/physiology ; TRPP Cation Channels/genetics ; Transcription Factors/physiology ; },
abstract = {Sexual dimorphism is a device that supports genetic diversity while providing selective pressure against speciation. This phenomenon is at the core of sexually reproducing organisms. Caenorhabditis elegans provides a unique experimental system where males exist in a primarily hermaphroditic species. Early works of John Sulston, Robert Horvitz, and John White provided a complete map of the hermaphrodite nervous system, and recently the male nervous system was added. This addition completely realized the vision of C. elegans pioneer Sydney Brenner: a model organism with an entirely mapped nervous system. With this 'connectome' of information available, great strides have been made toward understanding concepts such as how a sex-shared nervous system (in hermaphrodites and males) can give rise to sex-specific functions, how neural plasticity plays a role in developing a dimorphic nervous system, and how a shared nervous system receives and processes external cues in a sexually-dimorphic manner to generate sex-specific behaviors. In C. elegans, the intricacies of male-mating behavior have been crucial for studying the function and circuitry of the male-specific nervous system and used as a model for studying human autosomal dominant polycystic kidney disease (ADPKD). With the emergence of CRISPR, a seemingly limitless tool for generating genomic mutations with pinpoint precision, the C. elegans model system will continue to be a useful instrument for pioneering research in the fields of behavior, reproductive biology, and neurogenetics.},
}
@article {pmid32647313,
year = {2021},
author = {Sekar, D and Lakshmanan, G and M, B},
title = {Implications of CRISPR/Cas9 system in Hypertension and its related diseases.},
journal = {Journal of human hypertension},
volume = {35},
number = {7},
pages = {642-644},
pmid = {32647313},
issn = {1476-5527},
mesh = {*CRISPR-Cas Systems ; Humans ; *Hypertension/genetics ; },
}
@article {pmid32647138,
year = {2020},
author = {Chen, Y and Liu, J and Zhi, S and Zheng, Q and Ma, W and Huang, J and Liu, Y and Liu, D and Liang, P and Songyang, Z},
title = {Author Correction: Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3522},
doi = {10.1038/s41467-020-17379-y},
pmid = {32647138},
issn = {2041-1723},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid32647121,
year = {2020},
author = {Moses, C and Hodgetts, SI and Nugent, F and Ben-Ary, G and Park, KK and Blancafort, P and Harvey, AR},
title = {Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11393},
pmid = {32647121},
issn = {2045-2322},
support = {R01CA170370/NH/NIH HHS/United States ; R01DA036906/NH/NIH HHS/United States ; },
mesh = {5' Untranslated Regions/genetics ; Animals ; Axons/physiology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Nerve Regeneration/*genetics ; Neuronal Outgrowth/genetics ; Optic Nerve/physiology ; Optic Nerve Injuries/therapy ; PTEN Phosphohydrolase/*genetics ; Promoter Regions, Genetic/genetics ; RNA Interference ; RNA, Guide/genetics ; RNA, Small Interfering/metabolism ; Rats ; Repressor Proteins/genetics ; Spinal Cord Injuries/therapy ; Transcription, Genetic ; Transduction, Genetic/methods ; },
abstract = {After damage to the adult mammalian central nervous system (CNS), surviving neurons have limited capacity to regenerate and restore functional connectivity. Conditional genetic deletion of PTEN results in robust CNS axon regrowth, while PTEN repression with short hairpin RNA (shRNA) improves regeneration but to a lesser extent, likely due to suboptimal PTEN mRNA knockdown using this approach. Here we employed the CRISPR/dCas9 system to repress PTEN transcription in neural cells. We targeted the PTEN proximal promoter and 5' untranslated region with dCas9 fused to the repressor protein Krüppel-associated box (KRAB). dCas9-KRAB delivered in a lentiviral vector with one CRISPR guide RNA (gRNA) achieved potent and specific PTEN repression in human cell line models and neural cells derived from human iPSCs, and induced histone (H)3 methylation and deacetylation at the PTEN promoter. The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript, a construct previously used in CNS injury models. The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells, and enhanced neurite outgrowth after nerve growth factor stimulation. PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.},
}
@article {pmid32646995,
year = {2020},
author = {Guo, CJ},
title = {Modulating microbiome metabolites in vivo.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6500},
pages = {153},
doi = {10.1126/science.abc5620},
pmid = {32646995},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; Clostridium/*genetics/*metabolism ; Gastrointestinal Microbiome/*genetics ; *Host Microbial Interactions ; Humans ; },
}
@article {pmid32646486,
year = {2020},
author = {Hosur, V and Low, BE and Li, D and Stafford, GA and Kohar, V and Shultz, LD and Wiles, MV},
title = {Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {168},
pmid = {32646486},
issn = {1474-760X},
support = {R21 OD027052/OD/NIH HHS/United States ; P30 CA034196/CA/NCI NIH HHS/United States ; P30CA034196/CA/NCI NIH HHS/United States ; R24 OD026440/OD/NIH HHS/United States ; R21 OD027052/CD/ODCDC CDC HHS/United States ; ODO26440//NIH Office of the Director/International ; AI132963//National Institute of Allergy and Infectious Diseases/International ; },
mesh = {Adaptation, Biological ; Animals ; CRISPR-Cas Systems ; *Exons ; Female ; *Gene Expression ; Gene Targeting/*methods ; Male ; Membrane Proteins/*genetics ; Mice ; Mice, Knockout ; Mutation ; *Phenotype ; Pregnancy ; },
abstract = {BACKGROUND: Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies-definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9.<br><br>RESULTS: We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes.<br><br>CONCLUSIONS: Exon skipping is a widespread phenomenon occurring across the genome. These findings have significant implications for the application of genome editing in both basic research and clinical practice.},
}
@article {pmid32645367,
year = {2020},
author = {Harrington, LB and Ma, E and Chen, JS and Witte, IP and Gertz, D and Paez-Espino, D and Al-Shayeb, B and Kyrpides, NC and Burstein, D and Banfield, JF and Doudna, JA},
title = {A scoutRNA Is Required for Some Type V CRISPR-Cas Systems.},
journal = {Molecular cell},
volume = {79},
number = {3},
pages = {416-424.e5},
pmid = {32645367},
issn = {1097-4164},
support = {U01AI142817-02/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; R01 AI092531/AI/NIAID NIH HHS/United States ; 1817593//National Science Foundation/International ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteria/classification/*genetics/immunology/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial/chemistry/genetics/metabolism ; Endodeoxyribonucleases/*genetics/metabolism ; Escherichia coli/genetics/immunology/metabolism ; Genome, Bacterial/*immunology ; Nucleic Acid Conformation ; Phylogeny ; RNA, Bacterial/chemistry/*genetics/metabolism ; RNA, Guide/genetics/metabolism ; RNA, Small Untranslated/chemistry/*genetics/metabolism ; Sequence Alignment ; Sequence Homology, Nucleic Acid ; },
abstract = {CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.},
}
@article {pmid32645350,
year = {2020},
author = {Johnson, WE},
title = {Not Your Typical Anti-CRISPR.},
journal = {Cell host &amp; microbe},
volume = {28},
number = {1},
pages = {1-2},
doi = {10.1016/j.chom.2020.06.016},
pmid = {32645350},
issn = {1934-6069},
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Listeria ; Lysogeny ; },
abstract = {In this issue of Cell Host &amp; Microbe, two papers by Osuna et al. describe the characterization of AcrIIA1, an anti-CRISPR protein distributed widely among Listeria phages. AcrIIA1 functions as an anti-CRISPR and as a dynamic repressor of acr loci, suggesting it may play an important role in lysogeny.},
}
@article {pmid32645003,
year = {2020},
author = {Morton, DJ and Jalloh, B and Kim, L and Kremsky, I and Nair, RJ and Nguyen, KB and Rounds, JC and Sterrett, MC and Brown, B and Le, T and Karkare, MC and McGaughey, KD and Sheng, S and Leung, SW and Fasken, MB and Moberg, KH and Corbett, AH},
title = {A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons.},
journal = {PLoS genetics},
volume = {16},
number = {7},
pages = {e1008901},
pmid = {32645003},
issn = {1553-7404},
support = {R01 GM130147/GM/NIGMS NIH HHS/United States ; T32 GM008367/GM/NIGMS NIH HHS/United States ; F31 NS103595/NS/NINDS NIH HHS/United States ; R01 MH107305/MH/NIMH NIH HHS/United States ; F31 GM134649/GM/NIGMS NIH HHS/United States ; F31 HD088043/HD/NICHD NIH HHS/United States ; F32 GM125350/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Substitution/genetics ; Animals ; CRISPR-Cas Systems/genetics ; Cerebellar Diseases/*genetics/pathology ; Cerebellum/metabolism/pathology ; Cytoskeletal Proteins/*genetics ; Disease Models, Animal ; Drosophila melanogaster/*genetics ; Exosome Multienzyme Ribonuclease Complex/*genetics ; Exosomes/genetics ; Humans ; Mutation/genetics ; Nerve Tissue Proteins/*genetics ; Neurons/*metabolism/pathology ; RNA/genetics ; RNA-Binding Proteins/*genetics ; },
abstract = {The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.},
}
@article {pmid32995532,
year = {2019},
author = {Collias, D and Marshall, R and Collins, SP and Beisel, CL and Noireaux, V},
title = {An educational module to explore CRISPR technologies with a cell-free transcription-translation system.},
journal = {Synthetic biology (Oxford, England)},
volume = {4},
number = {1},
pages = {ysz005},
pmid = {32995532},
issn = {2397-7000},
abstract = {Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.},
}
@article {pmid32528662,
year = {2020},
author = {Baker, C and Hayden, MS},
title = {Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease.},
journal = {F1000Research},
volume = {9},
number = {},
pages = {281},
pmid = {32528662},
issn = {2046-1402},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Dermatology ; *Gene Editing ; Genetic Therapy ; Humans ; Skin Diseases/genetics/*therapy ; },
abstract = {The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research. We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.},
}
@article {pmid32644271,
year = {2020},
author = {Nesbit, KT and Hamdoun, A},
title = {Embryo, larval, and juvenile staging of Lytechinus pictus from fertilization through sexual maturation.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {249},
number = {11},
pages = {1334-1346},
pmid = {32644271},
issn = {1097-0177},
support = {R01 ES027921/ES/NIEHS NIH HHS/United States ; 1840844//National Science Foundation/International ; ES030318/NH/NIH HHS/United States ; ES027921/NH/NIH HHS/United States ; R01 ES030318/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; Embryo, Nonmammalian/*embryology ; *Embryonic Development ; Female ; Larva/growth &amp; development ; Lytechinus/*embryology ; Male ; *Sexual Maturation ; },
abstract = {BACKGROUND: Sea urchin embryos have been used for more than a century in the study of fertilization and early development. However, several of the species used, such as Strongylocentrotus purpuratus, have long generation times making them suboptimal for transgenerational studies.<br><br>RESULTS: Here, we present an overview of the development of a rapidly developing echinoderm species, Lytechinus pictus, from fertilization through sexual maturation. When grown at room temperature (20&#xb0;C) embryos complete the first cell cycle in 90&#x2009;minutes, followed by subsequent cleavages every 45&#x2009;minutes, leading to hatching at 9 hours postfertilization (hpf). The swimming embryos gastrulate from 12 to 36 hpf and produce the cells which subsequently give rise to the larval skeleton and immunocytes. Larvae begin to feed at 2&#x2009;days and metamorphose by 3&#x2009;weeks. Juveniles reach sexual maturity at 4 to 6 months of age, depending on individual growth rate.<br><br>CONCLUSIONS: This staging scheme lays a foundation for future studies in L. pictus, which share many of the attractive features of other urchins but have the key advantage of rapid development to sexual maturation. This is significant for multigenerational and genetic studies newly enabled by CRISPR-CAS mediated gene editing.},
}
@article {pmid32644042,
year = {2020},
author = {Zhang, W and Chen, Y and Yang, J and Zhang, J and Yu, J and Wang, M and Zhao, X and Wei, K and Wan, X and Xu, X and Jiang, Y and Chen, J and Gao, S and Mao, Z},
title = {A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32644042},
issn = {2050-084X},
support = {2018YFC2000100//Chinese National Program on Key Basic Research Project/International ; 19JC1415300//the key project of the Science and Technology of Shanghai Municipality/International ; 19QA1409600//the Shanghai Rising-Star Program/International ; 2017ZZ02015//the Shanghai Municipal Medical and Health Discipline Construction Projects/International ; 2018QNRC001//the Young Elite Scientist Sponsorship Program by CAST/International ; 2017YFA010330//Chinese National Program on Key Basic Research Project/International ; 2016YFA0100400//Chinese National Program on Key Basic Research Project/International ; 31871438//the National Science Foundation of China/International ; 81972457//the National Science Foundation of China/International ; 31721003//the National Science Foundation of China/International ; 31871446//the National Science Foundation of China/International ; 19XD1403000//the Fundamental Research Funds for the Central Universities, Program of Shanghai Academic Research Leader/International ; 19SG18//Shuguang Program" of Shanghai Education Development Foundation and Shanghai Municipal Education Commission"/International ; 2017YFA0103300//Chinese National Program on Key Basic Research Project/International ; 31871438//National Science Foundation of China/International ; 81972457//National Science Foundation of China/International ; 31721003//National Science Foundation of China/International ; 31871446//National Science Foundation of China/International ; 19XD1403000//Program of Shanghai Academic Research Leader/International ; 19SG18//"Shuguang Program" of Shanghai Education Development Foundation and Shanghai Municipal Education Commission/International ; 19JC1415300//The key project of the Science and Technology of Shanghai Municipality/International ; 19QA1409600//Shanghai Rising-Star Program/International ; 2017ZZ02015//Shanghai Municipal Medical and Health Discipline Construction Projects/International ; 2018QNRC001//The Young Elite Scientist Sponsorship Program by CAST/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chromones/*analysis ; *Gene Editing ; HEK293 Cells ; *High-Throughput Screening Assays ; Humans ; Mice ; },
abstract = {Directly modulating the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) - two independent pathways for repairing DNA double-strand breaks (DSBs) - has the potential to improve the efficiency of gene targeting by CRISPR/Cas9. Here, we have developed a rapid and easy-to-score screening approach for identifying small molecules that affect the choice between the two DSB repair pathways. Using this tool, we identified a small molecule, farrerol, that promotes HR but does not affect NHEJ. Further mechanistic studies indicate that farrerol functions through stimulating the recruitment of RAD51 to DSB sites. Importantly, we demonstrated that farrerol effectively promotes precise targeted integration in human cells, mouse cells and mouse embryos at multiple genomic loci. In addition, treating cells with farrerol did not have any obvious negative effect on genomic stability. Moreover, farrerol significantly improved the knock-in efficiency in blastocysts, and the subsequently generated knock-in mice retained the capacity for germline transmission.},
}
@article {pmid32643563,
year = {2020},
author = {Wang, X and Shang, X and Huang, X},
title = {Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods.},
journal = {Emerging microbes &amp; infections},
volume = {9},
number = {1},
pages = {1682-1691},
pmid = {32643563},
issn = {2222-1751},
mesh = {CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; Communicable Diseases/*diagnosis ; Humans ; Molecular Diagnostic Techniques/economics/*methods ; RNA, Guide/pharmacology ; Sensitivity and Specificity ; },
abstract = {Ideal methods for detecting pathogens should be sensitive, specific, rapid, cost-effective and instrument-free. Conventional nucleic acid pathogen detection strategies, mostly PCR-based techniques, have various limitations, such as expensive equipment, reagents and skilled performance. Recently, CRISPR/Cas-based methods have burst onto the scene, with the potential to power the pathogen detection field. Here we introduce these unique methods and discuss its hurdles and promises.},
}
@article {pmid32643177,
year = {2020},
author = {Newman, A and Starrs, L and Burgio, G},
title = {Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage: Techniques for Detecting, Predicting, and Mitigating the On- and off-target Effects of Cas9 Editing.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {42},
number = {9},
pages = {e2000047},
doi = {10.1002/bies.202000047},
pmid = {32643177},
issn = {1521-1878},
support = {APP1143008//National Health and Medical Research Council of Australia/International ; DP180101494//National Health and Medical Research Council of Australia/International ; 81772214//National Natural Science Foundation of China/International ; //National Collaborative Research Infrastructure Strategy (NCRIS)/International ; //Phenomics Australian/International ; //Australian Government Research Training Program Scholarship/International ; DP180101494//Australian Research Council/International ; APP1143008//National Health and Medical Research Council/International ; //National Collaborative Research Infrastructure/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Genomics ; Humans ; RNA, Guide/genetics ; },
abstract = {Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.},
}
@article {pmid32642978,
year = {2020},
author = {Afzal, S and Sirohi, P and Singh, NK},
title = {A review of CRISPR associated genome engineering: application, advances and future prospects of genome targeting tool for crop improvement.},
journal = {Biotechnology letters},
volume = {42},
number = {9},
pages = {1611-1632},
doi = {10.1007/s10529-020-02950-w},
pmid = {32642978},
issn = {1573-6776},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural ; Epigenomics ; *Gene Editing ; *Plants, Genetically Modified ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
abstract = {The Cas9 nuclease initiates double-stranded breaks at the target position in DNA, which are repaired by the intracellular restoration pathways to eliminate or insert pieces of DNA. CRISPR-Cas9 is proficient and cost-effective since cutting is guided by a piece of RNA instead of protein. Emphasis on this technology, in contrast with two recognized genome editing platforms (i.e., ZFNs and TALENs), is provided. This review evaluates the benefits of chemically synthesized gRNAs as well as the integration of chemical amendments to improve gene editing efficiencies. CRISPR is an indispensable means in biological investigations and is now as well transforming varied fields of biotechnology and agriculture. Recent advancement in targetable epigenomic-editing tools allows researchers to dispense direct functional and transcriptional significance to locus-explicit chromatin adjustments encompassing gene regulation and editing. An account of diverse sgRNA design tools is provided, principally on their target competence prediction model, off-target recognition algorithm, and generation of instructive annotations. The modern systems that have been utilized to deliver CRISPR-Cas9 in vivo and in vitro for crop improvement viz. nutritional enhancement, production of drought-tolerant and disease-resistant plants, are also highlighted. The conclusion is focused on upcoming directions, biosafety concerns, and expansive prospects of CRISPR technologies.},
}
@article {pmid32642859,
year = {2020},
author = {Wolabu, TW and Park, JJ and Chen, M and Cong, L and Ge, Y and Jiang, Q and Debnath, S and Li, G and Wen, J and Wang, Z},
title = {Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.},
journal = {Planta},
volume = {252},
number = {2},
pages = {15},
pmid = {32642859},
issn = {1432-2048},
mesh = {Agrobacterium/genetics ; Alleles ; Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Homozygote ; Medicago truncatula/*genetics ; Mutation ; Phenotype ; Promoter Regions, Genetic/genetics ; },
abstract = {An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.},
}
@article {pmid32642121,
year = {2020},
author = {Lui, NS and Benson, J and He, H and Imielski, BR and Kunder, CA and Liou, DZ and Backhus, LM and Berry, MF and Shrager, JB},
title = {Sub-solid lung adenocarcinoma in Asian versus Caucasian patients: different biology but similar outcomes.},
journal = {Journal of thoracic disease},
volume = {12},
number = {5},
pages = {2161-2171},
pmid = {32642121},
issn = {2072-1439},
abstract = {BACKGROUND: Asian and Caucasian patients with lung cancer have been compared in several database studies, with conflicting findings regarding survival. However, these studies did not include proportion of ground-glass opacity or mutational status in their analyses. Asian patients commonly develop sub-solid lung adenocarcinomas that harbor EGFR mutations, which have a better prognosis. We hypothesized that among patients undergoing surgery for sub-solid lung adenocarcinomas, Asian patients have better survival compared to Caucasian patients.<br><br>METHODS: We identified Asian and Caucasian patients who underwent surgical resection for a sub-solid lung adenocarcinoma from 2002 to 2015 at our institution. Sub-solid was defined as &#x2265;10% ground-glass opacity on preoperative CT scan or &#x2265;10% lepidic component on surgical pathology. Time-to-event multivariable analysis was performed to determine which characteristics were associated with recurrence and survival.<br><br>RESULTS: Two hundred twenty-four patients were included with median follow up 48 months. Asian patients were more likely to be never smokers (76.3% vs. 29.0%, P<0.01) and have an EGFR mutation (69.4% vs. 25.6% of those tested, P<0.01), while Caucasian patients were more likely to have a KRAS mutation (23.5% vs. 4.9% of those tested, P<0.01). There was a trend towards Asian patients having a higher proportion of ground-glass opacity (38.8% vs. 30.5%, P=0.11). Time-to-event multivariable analysis showed that higher proportion of ground-glass opacity was significantly associated with better recurrence-free survival (HR 0.76 per 20% increase, P=0.02). However, mutational status and race did not have a significant impact on recurrence-free or overall survival.<br><br>CONCLUSIONS: Asian and Caucasian patients with sub-solid lung adenocarcinoma have different tumor biology, but recurrence-free and overall survival after surgical resection is similar.},
}
@article {pmid32641982,
year = {2020},
author = {Sun, X and Zhang, R and Chen, H and Du, X and Chen, S and Huang, J and Liu, M and Xu, M and Luo, F and Jin, M and Su, N and Qi, H and Yang, J and Tan, Q and Zhang, D and Ni, Z and Liang, S and Zhang, B and Chen, D and Zhang, X and Luo, L and Chen, L and Xie, Y},
title = {Fgfr3 mutation disrupts chondrogenesis and bone ossification in zebrafish model mimicking CATSHL syndrome partially via enhanced Wnt/β-catenin signaling.},
journal = {Theranostics},
volume = {10},
number = {16},
pages = {7111-7130},
pmid = {32641982},
issn = {1838-7640},
mesh = {Animals ; Animals, Genetically Modified ; Bone Diseases, Developmental/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Proliferation/genetics ; Chondrocytes/*pathology ; Chondrogenesis/*genetics ; Disease Models, Animal ; Embryo, Nonmammalian ; Gene Knockout Techniques ; Hand Deformities, Congenital/*genetics/pathology ; Hearing Loss/*genetics/pathology ; Hedgehog Proteins/metabolism ; Humans ; Mutation ; Receptor, Fibroblast Growth Factor, Type 3/*genetics ; Skull/*embryology ; Wnt Signaling Pathway/genetics ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {CATSHL syndrome, characterized by camptodactyly, tall stature and hearing loss, is caused by loss-of-function mutations of fibroblast growth factor receptors 3 (FGFR3) gene. Most manifestations of patients with CATSHL syndrome start to develop in the embryonic stage, such as skeletal overgrowth, craniofacial abnormalities, however, the pathogenesis of these phenotypes especially the early maldevelopment remains incompletely understood. Furthermore, there are no effective therapeutic targets for this skeleton dysplasia. Methods: We generated fgfr3 knockout zebrafish by CRISPR/Cas9 technology to study the developmental mechanisms and therapeutic targets of CATSHL syndrome. Several zebrafish transgenic lines labeling osteoblasts and chondrocytes, and live Alizarin red staining were used to analyze the dynamical skeleton development in fgfr3 mutants. Western blotting, whole mount in situ hybridization, Edu labeling based cell proliferation assay and Wnt/β-catenin signaling antagonist were used to explore the potential mechanisms and therapeutic targets. Results: We found that fgfr3 mutant zebrafish, staring from early development stage, showed craniofacial bone malformation with microcephaly and delayed closure of cranial sutures, chondroma-like lesion and abnormal development of auditory sensory organs, partially resembling the clinical manifestations of patients with CATSHL syndrome. Further studies showed that fgfr3 regulates the patterning and shaping of pharyngeal arches and the timely ossification of craniofacial skeleton. The abnormal development of pharyngeal arch cartilage is related to the augmented hypertrophy and disordered arrangement of chondrocytes, while decreased proliferation, differentiation and mineralization of osteoblasts may be involved in the delayed maturation of skull bones. Furthermore, we revealed that deficiency of fgfr3 leads to enhanced IHH signaling and up-regulated canonical Wnt/β-catenin signaling, and pharmacological inhibition of Wnt/β-catenin could partially alleviate the phenotypes of fgfr3 mutants. Conclusions: Our study further reveals some novel phenotypes and underlying developmental mechanism of CATSHL syndrome, which deepens our understanding of the pathogenesis of CATSHL and the role of fgfr3 in skeleton development. Our findings provide evidence that modulation of Wnt/β-catenin activity could be a potential therapy for CATSHL syndrome and related skeleton diseases.},
}
@article {pmid32641861,
year = {2020},
author = {Kampmann, M},
title = {CRISPR-based functional genomics for neurological disease.},
journal = {Nature reviews. Neurology},
volume = {16},
number = {9},
pages = {465-480},
pmid = {32641861},
issn = {1759-4766},
support = {DP2 GM119139/GM/NIGMS NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; U01 MH115747/MH/NIMH NIH HHS/United States ; R56 AG057528/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods/trends ; Genome-Wide Association Study/methods/*trends ; Genomics/methods/*trends ; Humans ; Induced Pluripotent Stem Cells/physiology ; Nervous System Diseases/*diagnosis/*genetics ; },
abstract = {Neurodegenerative, neurodevelopmental and neuropsychiatric disorders are among the greatest public health challenges, as many lack disease-modifying treatments. A major reason for the absence of effective therapies is our limited understanding of the causative molecular and cellular mechanisms. Genome-wide association studies are providing a growing catalogue of disease-associated genetic variants, and the next challenge is to elucidate how these variants cause disease and to translate this understanding into therapies. This Review describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases. The bacterial CRISPR system can be used in experimental disease models to edit genomes and to control gene expression levels through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These genetic perturbations can be implemented in massively parallel genetic screens to evaluate the functional consequences for human cells. CRISPR screens are particularly powerful in combination with induced pluripotent stem cell technology, which enables the derivation of differentiated cell types, such as neurons and glia, and brain organoids from cells obtained from patients. Modelling of disease-associated changes in gene expression via CRISPRi and CRISPRa can pinpoint causal changes. In addition, genetic modifier screens can be used to elucidate disease mechanisms and causal determinants of cell type-selective vulnerability and to identify therapeutic targets.},
}
@article {pmid32641851,
year = {2020},
author = {},
title = {Focus on CRISPR tools and therapies.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {765},
doi = {10.1038/s41587-020-0611-3},
pmid = {32641851},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Humans ; },
}
@article {pmid32641850,
year = {2020},
author = {},
title = {The clinical cutting edge.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {767},
doi = {10.1038/s41587-020-0612-2},
pmid = {32641850},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/trends ; Genetic Therapy/*trends ; Genome, Human/genetics ; Humans ; Leber Congenital Amaurosis/*therapy ; },
}
@article {pmid32641847,
year = {2020},
author = {},
title = {A COVID-19 model.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {773},
doi = {10.1038/s41587-020-0606-0},
pmid = {32641847},
issn = {1546-1696},
mesh = {Angiotensin-Converting Enzyme 2 ; Animals ; COVID-19 ; *CRISPR-Cas Systems ; Coronavirus Infections/genetics/pathology ; *Disease Models, Animal ; Gene Knock-In Techniques ; Humans ; Mice ; Pandemics ; Peptidyl-Dipeptidase A/*genetics ; Pneumonia, Viral/genetics/pathology ; },
}
@article {pmid32641792,
year = {2020},
author = {Ledford, H},
title = {Scientists make precise gene edits to mitochondrial DNA for first time.},
journal = {Nature},
volume = {583},
number = {7816},
pages = {343},
pmid = {32641792},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Cytidine Deaminase ; DNA, Mitochondrial ; *Gene Editing ; },
}
@article {pmid32640832,
year = {2020},
author = {Sessions, KJ and Chen, YY and Hodge, CA and Hudson, TR and Eszterhas, SK and Hayden, MS and Howell, AL},
title = {Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates.},
journal = {AIDS research and human retroviruses},
volume = {36},
number = {10},
pages = {862-874},
pmid = {32640832},
issn = {1931-8405},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *HIV Infections/genetics ; *HIV-1/genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.},
}
@article {pmid32640650,
year = {2020},
author = {Melfi, R and Cancemi, P and Chiavetta, R and Barra, V and Lentini, L and Di Leonardo, A},
title = {Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons.},
journal = {International journal of molecular sciences},
volume = {21},
number = {13},
pages = {},
pmid = {32640650},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Codon, Nonsense/*genetics ; Cystic Fibrosis/*genetics/metabolism/pathology ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/metabolism ; Gene Editing/*methods ; Humans ; *Mutation ; RNA, Messenger/*genetics ; },
abstract = {Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool "REPAIRv2" (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFP[opal] and CFTR[W1282X], respectively.},
}
@article {pmid32640256,
year = {2020},
author = {Loo, CS and Gatchalian, J and Liang, Y and Leblanc, M and Xie, M and Ho, J and Venkatraghavan, B and Hargreaves, DC and Zheng, Y},
title = {A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function.},
journal = {Immunity},
volume = {53},
number = {1},
pages = {143-157.e8},
pmid = {32640256},
issn = {1097-4180},
support = {S10 OD023689/OD/NIH HHS/United States ; R01 AI107027/AI/NIAID NIH HHS/United States ; F32 GM128377/GM/NIGMS NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; R01 AI151123/AI/NIAID NIH HHS/United States ; T32 CA009370/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity/immunology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/genetics ; HEK293 Cells ; Humans ; Mice, Inbred C57BL ; Mice, Knockout ; Neoplasms/*immunology ; Nucleosomes/immunology ; RNA, Guide/genetics ; T-Lymphocytes, Regulatory/cytology/immunology/*metabolism ; Transcription Factors/genetics/*metabolism ; },
abstract = {Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.},
}
@article {pmid32640102,
year = {2021},
author = {Cheng, H and Hao, M and Ding, B and Mei, D and Wang, W and Wang, H and Zhou, R and Liu, J and Li, C and Hu, Q},
title = {Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.},
journal = {Plant biotechnology journal},
volume = {19},
number = {1},
pages = {87-97},
pmid = {32640102},
issn = {1467-7652},
mesh = {*Brassica napus/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Herbicide Resistance ; },
abstract = {CRISPR/Cas-base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A-PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base-editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base-edited plants of ALS conferred high herbicide resistance, while base-edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T0 to T1 generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off-target sites, large-scale SNP variations were determined through whole-genome sequencing between some base-edited and wild-type plants. These results revealed that A3A-PBE base-editing system could effectively convert C to T substitution with high-editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.},
}
@article {pmid32636858,
year = {2020},
author = {Nguyen, V and Riley, S and Nagel, S and Fisk, I and Searle, IR},
title = {Common Vetch: A Drought Tolerant, High Protein Neglected Leguminous Crop With Potential as a Sustainable Food Source.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {818},
pmid = {32636858},
issn = {1664-462X},
abstract = {Global demand for protein is predicted to increase by 50% by 2050. To meet the increasing demand whilst ensuring sustainability, protein sources that generate low-greenhouse gas emissions are required, and protein-rich legume seeds have the potential to make a significant contribution. Legumes like common vetch (Vicia sativa) that grow in marginal cropping zones and are drought tolerant and resilient to changeable annual weather patterns, will be in high demand as the climate changes. In common vetch, the inability to eliminate the γ-glutamyl-β-cyano-alanine (GBCA) toxin present in the seed has hindered its utility as a human and animal food for many decades, leaving this highly resilient species an "orphan" legume. However, the availability of the vetch genome and transcriptome data together with the application of CRISPR-Cas genome editing technologies lay the foundations to eliminate the GBCA toxin constraint. In the near future, we anticipate that a zero-toxin vetch variety will become a significant contributor to global protein demand.},
}
@article {pmid32636489,
year = {2020},
author = {Weber, J and Braun, CJ and Saur, D and Rad, R},
title = {In vivo functional screening for systems-level integrative cancer genomics.},
journal = {Nature reviews. Cancer},
volume = {20},
number = {10},
pages = {573-593},
pmid = {32636489},
issn = {1474-1768},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Transformation, Neoplastic/genetics/metabolism ; Cell Transformation, Viral ; DNA Transposable Elements ; Early Detection of Cancer ; *Genetic Association Studies/methods ; *Genetic Predisposition to Disease ; Genetic Testing/methods ; *Genomics/methods ; Humans ; Mutagenesis/drug effects/radiation effects ; Neoplasms/*diagnosis/*genetics/therapy ; Translational Research, Biomedical ; },
abstract = {With the genetic portraits of all major human malignancies now available, we next face the challenge of characterizing the function of mutated genes, their downstream targets, interactions and molecular networks. Moreover, poorly understood at the functional level are also non-mutated but dysregulated genomes, epigenomes or transcriptomes. Breakthroughs in manipulative mouse genetics offer new opportunities to probe the interplay of molecules, cells and systemic signals underlying disease pathogenesis in higher organisms. Herein, we review functional screening strategies in mice using genetic perturbation and chemical mutagenesis. We outline the spectrum of genetic tools that exist, such as transposons, CRISPR and RNAi and describe discoveries emerging from their use. Genome-wide or targeted screens are being used to uncover genomic and regulatory landscapes in oncogenesis, metastasis or drug resistance. Versatile screening systems support experimentation in diverse genetic and spatio-temporal settings to integrate molecular, cellular or environmental context-dependencies. We also review the combination of in vivo screening and barcoding strategies to study genetic interactions and quantitative cancer dynamics during tumour evolution. These scalable functional genomics approaches are transforming our ability to interrogate complex biological systems.},
}
@article {pmid32636482,
year = {2020},
author = {Pi, M and Xu, F and Ye, R and Nishimoto, SK and Kesterson, RA and Williams, RW and Lu, L and Quarles, LD},
title = {Humanized GPRC6A[KGKY] is a gain-of-function polymorphism in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11143},
pmid = {32636482},
issn = {2045-2322},
support = {P30CA13148/NH/NIH HHS/United States ; P30 DK074038/DK/NIDDK NIH HHS/United States ; P30 DK056336/DK/NIDDK NIH HHS/United States ; P30 AR048311/AR/NIAMS NIH HHS/United States ; R01 AR037308/AR/NIAMS NIH HHS/United States ; R01 DK120567/DK/NIDDK NIH HHS/United States ; P60 DK079626/DK/NIDDK NIH HHS/United States ; R01 DK120567/NH/NIH HHS/United States ; },
mesh = {Animals ; Blood Glucose/analysis ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Energy Metabolism/*genetics ; Fibroblast Growth Factors/blood ; Gene Editing/methods ; Gene Knock-In Techniques/methods ; Glucose Tolerance Test ; Humans ; Insulin/blood ; Male ; Mice ; Mice, Transgenic ; Polymorphism, Genetic/*genetics ; Real-Time Polymerase Chain Reaction ; Receptors, G-Protein-Coupled/*genetics/physiology ; },
abstract = {GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-[KGKY-knockin] mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-[KGKY-knockin] mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-[KGKY-knockin] mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-[KGKY] variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.},
}
@article {pmid32636400,
year = {2020},
author = {Bae, HS and Jin, YK and Ham, S and Kim, HK and Shin, H and Cho, GB and Lee, KJ and Lee, H and Kim, KM and Koo, OJ and Jang, G and Lee, JM and Lee, JY},
title = {CRISRP/Cas9-mediated knockout of Mct8 reveals a functional involvement of Mct8 in testis and sperm development in a rat.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11148},
pmid = {32636400},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knockdown Techniques/methods ; Male ; Monocarboxylic Acid Transporters/genetics/metabolism/*physiology ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis/genetics/physiology ; Spermatozoa/*growth &amp; development/physiology ; Testis/*growth &amp; development/metabolism ; Thyroid Gland/metabolism/physiology ; },
abstract = {Thyroid hormone (TH) has long been believed to play a minor role in male reproduction. However, evidences from experimental model of thyrotoxicosis or hypothyroidism suggests its role in spermatogenesis. Cellular action of TH requires membrane transport via specific transporters such as monocarboxylate transporter 8 (MCT8). SLC16A2 (encodes for MCT8) inactivating mutation in humans can lead to Allan-Herndon Dudley-syndrome, a X-linked psychomotor and growth retardation. These patients present cryptorchidism which suggests a role of MCT8 during spermatogenesis. In this study, we found that Mct8 is highly expressed during early postnatal development and decreases its expression in the adulthood of testis of wild-type male rats. Histological analysis revealed that spermatogonia largely lacks MCT8 expression while spermatocytes and maturing spermatids highly express MCT8. To further understand the role of Mct8 during spermatogenesis, we generated Slc16a2 (encodes MCT8) knockout rats using CRISPR/Cas9. Serum THs (T3 and T4) level were significantly altered in Slc16a2 knockout rats when compared to wild-type littermates during early to late postnatal development. Unlike Slc16a2 knockout mice, Slc16a2 knockout rats showed growth delay during early to late postnatal development. In adult Slc16a2 knockout rats, we observed reduced sperm motility and viability. Collectively, our data unveil a functional involvement of MCT8 in spermatogenesis, underscoring the importance of TH signaling and action during spermatogenesis.},
}
@article {pmid32635161,
year = {2020},
author = {Meshalkina, DA and Glushchenko, AS and Kysil, EV and Mizgirev, IV and Frolov, A},
title = {SpCas9- and LbCas12a-Mediated DNA Editing Produce Different Gene Knockout Outcomes in Zebrafish Embryos.},
journal = {Genes},
volume = {11},
number = {7},
pages = {},
pmid = {32635161},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism/standards ; CRISPR-Cas Systems ; Gene Editing/*methods/standards ; Gene Knockout Techniques/*methods/standards ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.},
}
@article {pmid32633466,
year = {2020},
author = {Ying, ZM and Wang, F and Chu, X and Yu, RQ and Jiang, JH},
title = {Activatable CRISPR Transcriptional Circuits Generate Functional RNA for mRNA Sensing and Silencing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {42},
pages = {18599-18604},
doi = {10.1002/anie.202004751},
pmid = {32633466},
issn = {1521-3773},
mesh = {Apoptosis ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Gene Silencing ; HeLa Cells ; Humans ; Microscopy, Confocal ; Nucleic Acid Hybridization ; Plasmids/genetics/metabolism ; RNA, Guide/*metabolism ; RNA, Messenger/analysis/*metabolism ; RNA, Small Interfering/metabolism ; Survivin/genetics ; Transcriptional Activation ; },
abstract = {CRISPR-dCas9 systems that are precisely activated by cell-specific information facilitate the development of smart sensors or therapeutic strategies. We report the development of an activatable dCas9 transcriptional circuit that enables sensing and silencing of mRNA in living cells using hybridization-mediated structure switching for gRNA activation. The gRNA is designed with the spacer sequence blocked by a hairpin structure, and mRNA hybridization induces gRNA structure switching and activates the transcription of reporter RNA. An mRNA sensor developed using a light-up RNA reporter shows high sensitivity and fast-response imaging of survivin mRNA in cells under drug treatments and different cell lines. Furthermore, a feedback circuit is engineered by incorporating a small hairpin RNA in the reporter RNA, demonstrating a smart strategy for dynamic sensing and silencing of survivin with induced tumor cell apoptosis. This circuit illustrates a broadly applicable platform for the development of cell-specific sensing and therapeutic strategies.},
}
@article {pmid32633292,
year = {2020},
author = {Gong, S and Chen, Y and Pan, W and Li, N and Tang, B},
title = {An in vitro site-specific cleavage assay of CRISPR-Cas9 using a personal glucose meter.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {62},
pages = {8850-8853},
doi = {10.1039/d0cc03505j},
pmid = {32633292},
issn = {1364-548X},
mesh = {Base Sequence ; Blood Glucose Self-Monitoring/economics/*methods ; CRISPR-Cas Systems/*genetics ; Cost-Benefit Analysis ; },
abstract = {A sensitive and cost-effective assay based on a personal glucose meter was developed to detect the in vitro site-specific cleavage efficiency of CRISPR-Cas9.},
}
@article {pmid32632863,
year = {2020},
author = {Fernandes, LGV and Nascimento, ALTO},
title = {Specific Gene Silencing in Leptospira biflexa by RNA-Guided Catalytically Inactive Cas9 (dCas9).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2134},
number = {},
pages = {109-122},
doi = {10.1007/978-1-0716-0459-5_10},
pmid = {32632863},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques/*methods ; Gene Silencing/*physiology ; Leptospira/*genetics ; Plasmids/genetics ; RNA, Catalytic/*genetics ; RNA, Guide/*genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Easy, practical, and affordable gene silencing techniques are constantly progressing, and genetic tools such as TALEs, RNAi, and CRISPR/Cas9 have emerged as new techniques for understanding the basic biology and virulence mechanisms of pathogenic organisms, including bacteria. Here, we describe one-step targeted gene silencing in Leptospira biflexa by using plasmids expressing catalytically inactive Streptococcus pyogenes Cas9 (dCas9) and a single-guide RNA (sgRNA) capable of pairing to the coding strand of a desired gene.},
}
@article {pmid32632303,
year = {2020},
author = {Song, M and Kim, HK and Lee, S and Kim, Y and Seo, SY and Park, J and Choi, JW and Jang, H and Shin, JH and Min, S and Quan, Z and Kim, JH and Kang, HC and Yoon, S and Kim, HH},
title = {Sequence-specific prediction of the efficiencies of adenine and cytosine base editors.},
journal = {Nature biotechnology},
volume = {38},
number = {9},
pages = {1037-1043},
pmid = {32632303},
issn = {1546-1696},
mesh = {*Adenine ; Aminohydrolases/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; *Cytosine ; Cytosine Deaminase/metabolism ; Gene Editing/*methods ; Genetic Engineering ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Point Mutation ; RNA, Guide/genetics ; Targeted Gene Repair/*methods ; },
abstract = {Base editors, including adenine base editors (ABEs)[1] and cytosine base editors (CBEs)[2,3], are widely used to induce point mutations. However, determining whether a specific nucleotide in its genomic context can be edited requires time-consuming experiments. Furthermore, when the editable window contains multiple target nucleotides, various genotypic products can be generated. To develop computational tools to predict base-editing efficiency and outcome product frequencies, we first evaluated the efficiencies of an ABE and a CBE and the outcome product frequencies at 13,504 and 14,157 target sequences, respectively, in human cells. We found that there were only modest asymmetric correlations between the activities of the base editors and Cas9 at the same targets. Using deep-learning-based computational modeling, we built tools to predict the efficiencies and outcome frequencies of ABE- and CBE-directed editing at any target sequence, with Pearson correlations ranging from 0.50 to 0.95. These tools and results will facilitate modeling and therapeutic correction of genetic diseases by base editing.},
}
@article {pmid32631952,
year = {2020},
author = {Toyoshima, Y and Nakamura, K and Tokita, R and Teramoto, N and Sugihara, H and Kato, H and Yamanouchi, K and Minami, S},
title = {Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {33},
pages = {11914-11927},
pmid = {32631952},
issn = {1083-351X},
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; Female ; Gene Knockdown Techniques ; Glucose/metabolism ; Glucose Tolerance Test ; Insulin/*metabolism ; Insulin Receptor Substrate Proteins/genetics/*metabolism ; Male ; Rats/genetics/*growth &amp; development/metabolism ; },
abstract = {Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired β-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.},
}
@article {pmid32631881,
year = {2020},
author = {Barrangou, R and Sontheimer, EJ},
title = {Shutting down RNA-targeting CRISPR.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6499},
pages = {31-32},
doi = {10.1126/science.abc8243},
pmid = {32631881},
issn = {1095-9203},
support = {R01 GM125797/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA ; },
}
@article {pmid32631827,
year = {2020},
author = {Hao, M and He, Y and Zhang, H and Liao, XP and Liu, YH and Sun, J and Du, H and Kreiswirth, BN and Chen, L},
title = {CRISPR-Cas9-Mediated Carbapenemase Gene and Plasmid Curing in Carbapenem-Resistant Enterobacteriaceae.},
journal = {Antimicrobial agents and chemotherapy},
volume = {64},
number = {9},
pages = {},
pmid = {32631827},
issn = {1098-6596},
support = {R01 AI090155/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Carbapenem-Resistant Enterobacteriaceae/genetics/metabolism ; Enterobacter ; *Enterobacteriaceae Infections ; Microbial Sensitivity Tests ; Plasmids/genetics ; beta-Lactamases/genetics/metabolism ; },
abstract = {Combating plasmid-mediated carbapenem resistance is essential to control and prevent the dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Here, we conducted a proof-of-concept study to demonstrate that CRISPR-Cas9-mediated resistance gene and plasmid curing can effectively resensitize CRE to carbapenems. A novel CRISPR-Cas9-mediated plasmid-curing system (pCasCure) was developed and electrotransferred into various clinical CRE isolates. The results showed that pCasCure can effectively cure blaKPC, blaNDM, and blaOXA-48 in various Enterobacteriaceae species of Klebsiella pneumoniae, Escherichia coli, Enterobacter hormaechei, Enterobacter xiangfangensis, and Serratia marcescens clinical isolates, with a >94% curing efficiency. In addition, we also demonstrated that pCasCure can efficiently eliminate several epidemic carbapenem-resistant plasmids, including the blaKPC-harboring IncFIIK-pKpQIL and IncN pKp58_N plasmids, the blaOXA-48-harboring pOXA-48-like plasmid, and the blaNDM-harboring IncX3 plasmid, by targeting their replication and partitioning (parA in pKpQIL) genes. However, curing the blaOXA-48 gene failed to eliminate its corresponding pOXA-48-like plasmid in clinical K. pneumoniae isolate 49210, while further next-generation sequencing revealed that it was due to IS1R-mediated recombination outside the CRISPR-Cas9 cleavage site resulting in blaOXA-48 truncation and, therefore, escaped plasmid curing. Nevertheless, the curing of carbapenemase genes or plasmids, including the truncation of blaOXA-48 in 49210, successfully restore their susceptibility to carbapenems, with a >8-fold reduction of MIC values in all tested isolates. Taken together, our study confirmed the concept of using CRISPR-Cas9-mediated carbapenemase gene and plasmid curing to resensitize CRE to carbapenems. Further work is needed to integrate pCasCure in an optimal delivery system to make it applicable for clinical intervention.},
}
@article {pmid32630835,
year = {2020},
author = {Naeem, M and Majeed, S and Hoque, MZ and Ahmad, I},
title = {Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing.},
journal = {Cells},
volume = {9},
number = {7},
pages = {},
pmid = {32630835},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; DNA Repair ; Gene Editing/*methods/standards ; Humans ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine.},
}
@article {pmid32630695,
year = {2020},
author = {Nawaz, G and Usman, B and Peng, H and Zhao, N and Yuan, R and Liu, Y and Li, R},
title = {Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line.},
journal = {Genes},
volume = {11},
number = {7},
pages = {},
pmid = {32630695},
issn = {2073-4425},
mesh = {Ascomycota/pathogenicity ; CRISPR-Cas Systems ; *Disease Resistance ; *Mutation ; Oryza/*genetics/immunology/microbiology ; Plant Proteins/chemistry/genetics/metabolism ; Proline-Rich Protein Domains ; Proteome/*genetics/metabolism ; },
abstract = {Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.},
}
@article {pmid32630625,
year = {2020},
author = {Almughem, FA and Aldossary, AM and Tawfik, EA and Alomary, MN and Alharbi, WS and Alshahrani, MY and Alshehri, AA},
title = {Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies.},
journal = {Pharmaceutics},
volume = {12},
number = {7},
pages = {},
pmid = {32630625},
issn = {1999-4923},
abstract = {Cystic Fibrosis (CF), an autosomal recessive genetic disease, is caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation reduces the release of chloride ions (Cl[-]) in epithelial tissues, and hyperactivates the epithelial sodium channels (ENaC) which aid in the absorption of sodium ions (Na[+]). Consequently, the mucus becomes dehydrated and thickened, making it a suitable medium for microbial growth. CF causes several chronic lung complications like thickened mucus, bacterial infection and inflammation, progressive loss of lung function, and ultimately, death. Until recently, the standard of clinical care in CF treatment had focused on preventing and treating the disease complications. In this review, we have summarized the current knowledge on CF pathogenesis and provided an outlook on the current therapeutic approaches relevant to CF (i.e., CFTR modulators and ENaC inhibitors). The enormous potential in targeting bacterial biofilms using antibiofilm peptides, and the innovative therapeutic strategies in using the CRISPR/Cas approach as a gene-editing tool to repair the CFTR mutation have been reviewed. Finally, we have discussed the wide range of drug delivery systems available, particularly non-viral vectors, and the optimal properties of nanocarriers which are essential for successful drug delivery to the lungs.},
}
@article {pmid32630614,
year = {2020},
author = {Mirgayazova, R and Khadiullina, R and Chasov, V and Mingaleeva, R and Miftakhova, R and Rizvanov, A and Bulatov, E},
title = {Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option?.},
journal = {Genes},
volume = {11},
number = {6},
pages = {},
pmid = {32630614},
issn = {2073-4425},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Repair/genetics ; *Gene Editing ; Gene Expression Regulation/genetics ; Genome/genetics ; Humans ; Mutation/genetics ; Neoplasms/genetics/*therapy ; Tumor Suppressor Protein p53/*genetics/therapeutic use ; },
abstract = {The TP53 gene encodes the transcription factor and oncosuppressor p53 protein that regulates a multitude of intracellular metabolic pathways involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence. In many cases, alterations (e.g., mutations of the TP53 gene) negatively affect these pathways resulting in tumor development. Recent advances in genome manipulation technologies, CRISPR/Cas9, in particular, brought us closer to therapeutic gene editing for the treatment of cancer and hereditary diseases. Genome-editing therapies for blood disorders, blindness, and cancer are currently being evaluated in clinical trials. Eventually CRISPR/Cas9 technology is expected to target TP53 as the most mutated gene in all types of cancers. A majority of TP53 mutations are missense which brings immense opportunities for the CRISPR/Cas9 system that has been successfully used for correcting single nucleotides in various models, both in vitro and in vivo. In this review, we highlight the recent clinical applications of CRISPR/Cas9 technology for therapeutic genome editing and discuss its perspectives for editing TP53 and regulating transcription of p53 pathway genes.},
}
@article {pmid32628723,
year = {2020},
author = {Wilson, SK and Heckendorn, J and Martorelli Di Genova, B and Koch, LL and Rooney, PJ and Morrissette, N and Lebrun, M and Knoll, LJ},
title = {A Toxoplasma gondii patatin-like phospholipase contributes to host cell invasion.},
journal = {PLoS pathogens},
volume = {16},
number = {7},
pages = {e1008650},
pmid = {32628723},
issn = {1553-7374},
support = {R01 AI144016/AI/NIAID NIH HHS/United States ; T32 AI007414/AI/NIAID NIH HHS/United States ; F32 AI065023/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; Phospholipases/genetics/metabolism ; Protozoan Proteins/genetics/*metabolism ; Toxoplasma/genetics/*pathogenicity ; Toxoplasmosis/enzymology/*metabolism ; Virulence ; Virulence Factors/*metabolism ; },
abstract = {Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.},
}
@article {pmid32628328,
year = {2020},
author = {Dettmer, R and Naujok, O},
title = {Design and Derivation of Multi-Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n-Mediated Homology-Directed Repair.},
journal = {Current protocols in stem cell biology},
volume = {54},
number = {1},
pages = {e116},
doi = {10.1002/cpsc.116},
pmid = {32628328},
issn = {1938-8969},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line ; Clone Cells ; Electroporation ; *Genes, Reporter ; Genotyping Techniques ; Humans ; Pluripotent Stem Cells/cytology/drug effects/*metabolism ; *Recombinational DNA Repair/drug effects ; Transfection ; },
abstract = {During the past decade, RNA-guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) has become a powerful tool for gene editing of human pluripotent stem cells (PSCs). Using paired CRISPR/Cas9 nickases (CRISPR/Cas9n) it is furthermore possible to reduce off-target effects that may typically occur with traditional CRISPR/Cas9 systems while maintaining high on-target efficiencies. With this technology and a well-designed homology-directed repair vector (HDR), we are now able to integrate transgenes into specific gene loci of PSCs in an allele conserving way. In this protocol we describe CRISPR/Cas9n design and homology directed repair vector design, transfection of human pluripotent stem cells and selection and expansion of generated cell clones. © 2020 The Authors. Basic Protocol 1: Repair template design and CRISPR/Cas9n construction Basic Protocol 2: Transfection of human pluripotent stem cells by electroporation Basic Protocol 3: Genotyping of generated cell clones.},
}
@article {pmid32627965,
year = {2020},
author = {Gonçalves, E and Segura-Cabrera, A and Pacini, C and Picco, G and Behan, FM and Jaaks, P and Coker, EA and van der Meer, D and Barthorpe, A and Lightfoot, H and Mironenko, T and Beck, A and Richardson, L and Yang, W and Lleshi, E and Hall, J and Tolley, C and Hall, C and Mali, I and Thomas, F and Morris, J and Leach, AR and Lynch, JT and Sidders, B and Crafter, C and Iorio, F and Fawell, S and Garnett, MJ},
title = {Drug mechanism-of-action discovery through the integration of pharmacological and CRISPR screens.},
journal = {Molecular systems biology},
volume = {16},
number = {7},
pages = {e9405},
pmid = {32627965},
issn = {1744-4292},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Antineoplastic Agents/*pharmacology/toxicity ; Biomarkers/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Development/*methods ; Drug Screening Assays, Antitumor/*methods ; Gene Knockout Techniques ; Gene Regulatory Networks/*drug effects/genetics ; Genetic Fitness/*drug effects/genetics ; Genomics ; Humans ; Linear Models ; Membrane Proteins/genetics/metabolism ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists &amp; inhibitors ; Pharmaceutical Preparations/metabolism ; Protein Interaction Maps/*drug effects ; Software ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development.},
}
@article {pmid32623905,
year = {2020},
author = {Hanses, U and Kleinsorge, M and Roos, L and Yigit, G and Li, Y and Barbarics, B and El-Battrawy, I and Lan, H and Tiburcy, M and Hindmarsh, R and Lenz, C and Salinas, G and Diecke, S and Müller, C and Adham, I and Altmüller, J and Nürnberg, P and Paul, T and Zimmermann, WH and Hasenfuss, G and Wollnik, B and Cyganek, L},
title = {Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome-Associated Cardiomyopathy.},
journal = {Circulation},
volume = {142},
number = {11},
pages = {1059-1076},
doi = {10.1161/CIRCULATIONAHA.119.044794},
pmid = {32623905},
issn = {1524-4539},
mesh = {*CRISPR-Cas Systems ; *Cardiomyopathies/genetics/metabolism/therapy ; *Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Introns ; *Models, Cardiovascular ; *Mutation ; Myocytes, Cardiac/*metabolism ; *Noonan Syndrome/genetics/metabolism/therapy ; *Transcription Factors/genetics/metabolism ; },
abstract = {BACKGROUND: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS-mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking.<br><br>METHODS: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 (LZTR1). We generated induced pluripotent stem cell-derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level.<br><br>RESULTS: Patients' induced pluripotent stem cell-derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS-mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype.<br><br>CONCLUSIONS: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell-derived cardiomyocytes from NS patients carrying biallelic variants in LZTR1 and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.},
}
@article {pmid32623622,
year = {2020},
author = {Wang, G and Wang, C and Lu, G and Wang, W and Mao, G and Habben, JE and Song, C and Wang, J and Chen, J and Gao, Y and Liu, J and Greene, TW},
title = {Knockouts of a late flowering gene via CRISPR-Cas9 confer early maturity in rice at multiple field locations.},
journal = {Plant molecular biology},
volume = {104},
number = {1-2},
pages = {137-150},
doi = {10.1007/s11103-020-01031-w},
pmid = {32623622},
issn = {1573-5028},
mesh = {Alleles ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats ; Flowers/*genetics/metabolism ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Oryza/*genetics/metabolism ; Phenotype ; Plant Development/genetics ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified/genetics ; Reproduction ; Sequence Alignment ; },
abstract = {OsGhd7 gene was discovered by screening our rice activation tagging population. CRISPR-Cas9 created knockouts of OsGhd7 conferred early flowering and early maturity in rice varieties across multiple geographical locations in China. Our research shows that OsGhd7 is a good target for breeding early maturity rice varieties, and an excellent example of the advantages of applying the CRISPR-Cas9 technology for trait improvement. Flowering time (heading date) is an important trait for crop cultivation and yield. In this study, we discovered a late flowering gene OsGhd7 by screening our rice activation tagging population, and demonstrated that overexpression of OsGhd7 delayed flowering time in rice, and the delay in flowering time depended on the transgene expression level. OsGhd7 is a functional allele of the Ghd7 gene family; knockouts of OsGhd7 generated by CRISPR-Cas9 significantly accelerated flowering time and the earliness of the flowering time depended on field location. The homozygous OsGhd7 knockout lines showed approximately 8, 10, and 20 days earlier flowering than controls at three different locations in China (Changsha City, Sanya City, and Beijing City, respectively) that varied from 18.25° N to 39.90° N. Furthermore, knockouts of OsGhd7 also showed an early flowering phenotype in different rice varieties, indicating OsGhd7 can be used as a common target gene for using the CRISPR technology to modulate rice flowering time. The importance of OsGhd7 and CRISPR technology for breeding early maturity rice varieties are discussed.},
}
@article {pmid32621766,
year = {2020},
author = {Garcia-Peterson, LM and Ndiaye, MA and Chhabra, G and Singh, CK and Guzmán-Pérez, G and Iczkowski, KA and Ahmad, N},
title = {CRISPR/Cas9-mediated Knockout of SIRT6 Imparts Remarkable Antiproliferative Response in Human Melanoma Cells in vitro and in vivo.},
journal = {Photochemistry and photobiology},
volume = {96},
number = {6},
pages = {1314-1320},
pmid = {32621766},
issn = {1751-1097},
support = {1I01BX004221/VA/VA/United States ; P30 AR066524/AR/NIAMS NIH HHS/United States ; P30AR066524/AR/NIAMS NIH HHS/United States ; IK6 BX003780/BX/BLRD VA/United States ; I01CX001441/VA/VA/United States ; I01 CX001441/CX/CSRD VA/United States ; I01 BX004221/BX/BLRD VA/United States ; R01 CA176748/CA/NCI NIH HHS/United States ; IK6BX003780/VA/VA/United States ; R01 AR059130/AR/NIAMS NIH HHS/United States ; R01CA176748/NH/NIH HHS/United States ; R01AR059130/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Proliferation/*genetics ; Female ; Gene Knockdown Techniques ; Humans ; In Vitro Techniques ; Male ; Melanoma/genetics/*pathology ; Mice ; Mice, Nude ; Sirtuins/*genetics ; Skin Neoplasms/genetics/*pathology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Melanoma is one of the most aggressive, potentially fatal forms of skin cancer and has been shown to be associated with solar ultraviolet radiation-dependent initiation and progression. Despite remarkable recent advances with targeted and immune therapeutics, lasting and recurrence-free survival remain significant concerns. Therefore, additional novel mechanism-based approaches are needed for effective melanoma management. The sirtuin SIRT6 appears to have a pro-proliferative function in melanocytic cells. In this study, we determined the effects of genetic manipulation of SIRT6 in human melanoma cells, in vitro and in vivo. Our data demonstrated that CRISPR/Cas9-mediated knockout (KO) of SIRT6 in A375 melanoma cells resulted in a significant (1) decrease in growth, viability and clonogenic survival and (2) induction of G1-phase cell cycle arrest. Further, employing a RT[2] Profiler PCR array containing 84 key transformation and tumorigenesis genes, we found that SIRT6 KO resulted in modulation of genes involved in angiogenesis, apoptosis, cellular senescence, epithelial-to-mesenchymal transition, hypoxia signaling and telomere maintenance. Finally, we found significantly decreased tumorigenicity of SIRT6 KO A375 cells in athymic nude mice. Our data provide strong evidence that SIRT6 promotes melanoma cell survival, both in vitro and in vivo, and could be exploited as a target for melanoma management.},
}
@article {pmid32621079,
year = {2020},
author = {Chang, AX and Chen, B and Yang, AG and Hu, RS and Feng, QF and Chen, M and Yang, XN and Luo, CG and Li, YY and Wang, YY},
title = {The trichome-specific acetolactate synthase NtALS1 gene, is involved in acylsugar biosynthesis in tobacco (Nicotiana tabacum L.).},
journal = {Planta},
volume = {252},
number = {1},
pages = {13},
doi = {10.1007/s00425-020-03418-x},
pmid = {32621079},
issn = {1432-2048},
mesh = {Acetolactate Synthase/*genetics/metabolism ; Arabidopsis Proteins/genetics ; CRISPR-Cas Systems ; Chloroplasts/enzymology ; Diploidy ; Gene Expression Regulation, Plant ; Phylogeny ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Sugars/*metabolism ; Tobacco/genetics/*metabolism ; Trichomes/*enzymology/genetics ; },
abstract = {MAIN CONCLUSION: NtALS1 is specifically expressed in glandular trichomes, and can improve the content of acylsugars in tobacco.<br><br>ABTRACT: The glandular trichomes of many species in the Solanaceae family play an important role in plant defense. These epidermal outgrowths exhibit specialized secondary metabolism, including the production of structurally diverse acylsugars that function in defense against insects and have substantial developmental potential for commercial uses. However, our current understanding of genes involved in acyl chain biosynthesis of acylsugars remains poor in tobacco. In this study, we identified three acetolactate synthase (ALS) genes in tobacco through homology-based gene prediction using Arabidopsis ALS. Quantitative real-time PCR (qRT-PCR) and tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two ntals1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two ntals1 mutants were significantly lower than those in the wild type. Through phylogenetic tree analysis, we also identified NtALS1 orthologs that may be involved in acylsugar biosynthesis in other Solanaceae species. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco.},
}
@article {pmid32620105,
year = {2020},
author = {Liu, L and Zhao, D and Ye, L and Zhan, T and Xiong, B and Hu, M and Bi, C and Zhang, X},
title = {A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3).},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {136},
pmid = {32620105},
issn = {1475-2859},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/virology ; Genome, Viral ; Industrial Microbiology ; Microorganisms, Genetically-Modified/virology ; },
abstract = {Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21's defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21's to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.},
}
@article {pmid32620100,
year = {2020},
author = {Zeng, X and Luo, Y and Vu, NTQ and Shen, S and Xia, K and Zhang, M},
title = {CRISPR/Cas9-mediated mutation of OsSWEET14 in rice cv. Zhonghua11 confers resistance to Xanthomonas oryzae pv. oryzae without yield penalty.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {313},
pmid = {32620100},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems ; Disease Resistance/genetics ; Monosaccharide Transport Proteins/*genetics/immunology/metabolism ; Mutagenesis ; Oryza/*genetics/growth &amp; development ; Plant Diseases/genetics/*microbiology ; Transcriptome ; *Xanthomonas ; },
abstract = {BACKGROUND: Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment.<br><br>RESULTS: In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield.<br><br>CONCLUSIONS: Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.},
}
@article {pmid32619853,
year = {2020},
author = {Gjaltema, RAF and Rots, MG},
title = {Advances of epigenetic editing.},
journal = {Current opinion in chemical biology},
volume = {57},
number = {},
pages = {75-81},
doi = {10.1016/j.cbpa.2020.04.020},
pmid = {32619853},
issn = {1879-0402},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Epigenomics/methods ; Gene Editing/*methods ; Genome ; Histone Code ; Humans ; },
abstract = {Epigenetic editing refers to the locus-specific targeting of epigenetic enzymes to rewrite the local epigenetic landscape of an endogenous genomic site, often with the aim of transcriptional reprogramming. Implementing clustered regularly interspaced short palindromic repeat-dCas9 greatly accelerated the advancement of epigenetic editing, yielding preclinical therapeutic successes using a variety of epigenetic enzymes. Here, we review the current applications of these epigenetic editing tools in mammals and shed light on biochemical improvements that facilitate versatile applications.},
}
@article {pmid32619491,
year = {2020},
author = {Kaserman, JE and Hurley, K and Dodge, M and Villacorta-Martin, C and Vedaie, M and Jean, JC and Liberti, DC and James, MF and Higgins, MI and Lee, NJ and Washko, GR and San Jose Estepar, R and Teckman, J and Kotton, DN and Wilson, AA},
title = {A Highly Phenotyped Open Access Repository of Alpha-1 Antitrypsin Deficiency Pluripotent Stem Cells.},
journal = {Stem cell reports},
volume = {15},
number = {1},
pages = {242-255},
pmid = {32619491},
issn = {2213-6711},
support = {U01 TR001810/TR/NCATS NIH HHS/United States ; R24 HL123828/HL/NHLBI NIH HHS/United States ; T32 HL007035/HL/NHLBI NIH HHS/United States ; R01 DK117940/DK/NIDDK NIH HHS/United States ; R01 DK101501/DK/NIDDK NIH HHS/United States ; RC2 HL101535/HL/NHLBI NIH HHS/United States ; S10 OD021587/OD/NIH HHS/United States ; },
mesh = {*Access to Information ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage ; *Databases as Topic ; Endoderm/pathology ; Female ; Gene Editing ; Genetic Loci ; Genotype ; Hepatocytes/pathology ; Humans ; Induced Pluripotent Stem Cells/*pathology ; Lung/diagnostic imaging/pathology ; Male ; Middle Aged ; Mutation/genetics ; Phenotype ; Transcriptome/genetics ; alpha 1-Antitrypsin/genetics ; alpha 1-Antitrypsin Deficiency/diagnostic imaging/genetics/*pathology ; },
abstract = {Individuals with the genetic disorder alpha-1 antitrypsin deficiency (AATD) are at risk of developing lung and liver disease. Patient induced pluripotent stem cells (iPSCs) have been found to model features of AATD pathogenesis but only a handful of AATD patient iPSC lines have been published. To capture the significant phenotypic diversity of the patient population, we describe here the establishment and characterization of a curated repository of AATD iPSCs with associated disease-relevant clinical data. To highlight the utility of the repository, we selected a subset of iPSC lines for functional characterization. Selected lines were differentiated to generate both hepatic and lung cell lineages and analyzed by RNA sequencing. In addition, two iPSC lines were targeted using CRISPR/Cas9 editing to accomplish scarless repair. Repository iPSCs are available to investigators for studies of disease pathogenesis and therapeutic discovery.},
}
@article {pmid32619467,
year = {2020},
author = {Clement, K and Hsu, JY and Canver, MC and Joung, JK and Pinello, L},
title = {Technologies and Computational Analysis Strategies for CRISPR Applications.},
journal = {Molecular cell},
volume = {79},
number = {1},
pages = {11-29},
pmid = {32619467},
issn = {1097-4164},
support = {R00 HG008399/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; R35 HG010717/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Computational Biology/*methods ; *Epigenomics ; *Gene Editing ; *Genome, Human ; Humans ; },
abstract = {The CRISPR-Cas system offers a programmable platform for eukaryotic genome and epigenome editing. The ability to perform targeted genetic and epigenetic perturbations enables researchers to perform a variety of tasks, ranging from investigating questions in basic biology to potentially developing novel therapeutics for the treatment of disease. While CRISPR systems have been engineered to target DNA and RNA with increased precision, efficiency, and flexibility, assays to identify off-target editing are becoming more comprehensive and sensitive. Furthermore, techniques to perform high-throughput genome and epigenome editing can be paired with a variety of readouts and are uncovering important cellular functions and mechanisms. These technological advances drive and are driven by accompanying computational approaches. Here, we briefly present available CRISPR technologies and review key computational advances and considerations for various CRISPR applications. In particular, we focus on the analysis of on- and off-target editing and CRISPR pooled screen data.},
}
@article {pmid32619241,
year = {2020},
author = {Indikova, I and Indik, S},
title = {Highly efficient 'hit-and-run' genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts.},
journal = {Nucleic acids research},
volume = {48},
number = {14},
pages = {8178-8187},
pmid = {32619241},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Vectors/genetics ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Nanoparticles/chemistry ; *Response Elements ; THP-1 Cells ; Transduction, Genetic/methods ; },
abstract = {The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.},
}
@article {pmid32618081,
year = {2020},
author = {Guan, X and Zhang, H and Qin, H and Chen, C and Hu, Z and Tan, J and Zeng, L},
title = {CRISPR/Cas9-mediated whole genomic wide knockout screening identifies mitochondrial ribosomal proteins involving in oxygen-glucose deprivation/reperfusion resistance.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {16},
pages = {9313-9322},
pmid = {32618081},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; Glucose/*deficiency ; Humans ; Mitochondrial Proteins/*antagonists &amp; inhibitors/genetics ; Neuroblastoma/genetics/metabolism/*pathology ; Oxidative Stress ; Oxygen/*metabolism ; Reperfusion Injury/*physiopathology ; Ribosomal Proteins/*antagonists &amp; inhibitors/genetics ; Tumor Cells, Cultured ; },
abstract = {Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia-reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK-N-BE(2) cells to oxygen-glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia-reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine-type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR-induced apoptosis. We also demonstrated that OGDR down-regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome-scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia-reperfusion damage.},
}
@article {pmid32617796,
year = {2021},
author = {Yoshimi, K and Oka, Y and Miyasaka, Y and Kotani, Y and Yasumura, M and Uno, Y and Hattori, K and Tanigawa, A and Sato, M and Oya, M and Nakamura, K and Matsushita, N and Kobayashi, K and Mashimo, T},
title = {Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats.},
journal = {Human genetics},
volume = {140},
number = {2},
pages = {277-287},
pmid = {32617796},
issn = {1432-1203},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA End-Joining Repair/*genetics ; Exons/genetics ; Female ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Genome/genetics ; Introns/genetics ; Mice ; Mice, Inbred C57BL ; Mutation/genetics ; Plasmids/*genetics ; Rats ; Rats, Long-Evans ; Rats, Wistar ; Recombinational DNA Repair/*genetics ; },
abstract = {CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.},
}
@article {pmid32617133,
year = {2020},
author = {Godoy, PRDV and Pour Khavari, A and Rizzo, M and Sakamoto-Hojo, ET and Haghdoost, S},
title = {Targeting NRF2, Regulator of Antioxidant System, to Sensitize Glioblastoma Neurosphere Cells to Radiation-Induced Oxidative Stress.},
journal = {Oxidative medicine and cellular longevity},
volume = {2020},
number = {},
pages = {2534643},
pmid = {32617133},
issn = {1942-0994},
mesh = {Antioxidants/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/radiation effects ; Dose-Response Relationship, Radiation ; *Gamma Rays ; Glioblastoma/*pathology ; Humans ; Models, Biological ; NF-E2-Related Factor 2/*metabolism ; Neoplasm Proteins/metabolism ; Neoplastic Stem Cells/metabolism/pathology ; Nerve Tissue Proteins/metabolism ; Oxidative Stress/*radiation effects ; RNA-Binding Proteins/metabolism ; Radiation Tolerance/radiation effects ; Spheroids, Cellular/*pathology/radiation effects ; },
abstract = {The presence of glioma stem cells (GSCs), which are enriched in neurospheres, may be connected to the radioresistance of glioblastoma (GBM) due to their enhanced antioxidant defense and elevated DNA repair capacity. The aim was to evaluate the responses to different radiation qualities and to reduce radioresistance of U87MG cells, a GBM cell line. U87MG cells were cultured in a 3D model and irradiated with low (24 mGy/h) and high (0.39 Gy/min) dose rates of low LET gamma and high LET carbon ions (1-2 Gy/min). Thereafter, expression of proteins related to oxidative stress response, extracellular 8-oxo-dG, and neurospheres were determined. LD50 for carbon ions was significantly lower compared to LD50 of high and low dose rate gamma radiation. A significantly higher level of 8-oxo-dG was detected in the media of cells exposed to a low dose rate as compared to a high dose rate of gamma or carbon ions. A downregulation of oxidative stress proteins was also observed (NRF2, hMTH1, and SOD1). The NRF2 gene was knocked down by CRISPR/Cas9 in neurosphere cells, resulting in less self-renewal, more differentiated cells, and less proliferation capacity after irradiation with low and high dose rate gamma rays. Overall, U87MG glioma neurospheres presented differential responses to distinct radiation qualities and NRF2 plays an important role in cellular sensitivity to radiation.},
}
@article {pmid32616820,
year = {2020},
author = {Hagag, IT and Wight, DJ and Bartsch, D and Sid, H and Jordan, I and Bertzbach, LD and Schusser, B and Kaufer, BB},
title = {Abrogation of Marek's disease virus replication using CRISPR/Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10919},
pmid = {32616820},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Chick Embryo ; Chickens ; Ducks ; Genes, Viral ; HEK293 Cells ; Humans ; Mardivirus/*drug effects/genetics/physiology ; Marek Disease/prevention &amp; control ; Marek Disease Vaccines ; Mutation ; Proof of Concept Study ; RNA, Guide/genetics/*pharmacology ; Specific Pathogen-Free Organisms ; Virus Replication/*drug effects/genetics ; },
abstract = {Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.},
}
@article {pmid32616331,
year = {2020},
author = {Yang, L and Chen, J},
title = {A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing.},
journal = {Trends in biochemical sciences},
volume = {45},
number = {10},
pages = {874-888},
doi = {10.1016/j.tibs.2020.06.003},
pmid = {32616331},
issn = {0968-0004},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Mutation ; },
abstract = {Two major moieties in genome editing are required for precise genetic changes: the locator moiety for target binding and the effector moiety for genetic engineering. By taking advantage of CRISPR/Cas, which consists of different modules for independent target binding and cleavage, a spectrum of precise and versatile genome editing technologies have been developed for broad applications in biomedical research, biotechnology, and therapeutics. Here, we briefly summarize the progress of genome editing systems from a view of both locator and effector moieties and highlight the advance of newly reported CRISPR-conjugated base editing and prime editing systems. We also underscore distinct mechanisms of off-target effects in CRISPR-conjugated systems and further discuss possible strategies to reduce off-target mutations in the future.},
}
@article {pmid32615922,
year = {2020},
author = {Duru, IC and Andreevskaya, M and Laine, P and Rode, TM and Ylinen, A and Løvdal, T and Bar, N and Crauwels, P and Riedel, CU and Bucur, FI and Nicolau, AI and Auvinen, P},
title = {Genomic characterization of the most barotolerant Listeria monocytogenes RO15 strain compared to reference strains used to evaluate food high pressure processing.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {455},
pmid = {32615922},
issn = {1471-2164},
mesh = {CRISPR-Cas Systems ; DNA Methylation ; *Food Preservation ; *Genome, Bacterial ; Genomics ; Listeria monocytogenes/*genetics ; Microbial Viability ; Pressure ; RNA-Seq ; Reference Standards ; },
abstract = {BACKGROUND: High pressure processing (HPP; i.e. 100-600 MPa pressure depending on product) is a non-thermal preservation technique adopted by the food industry to decrease significantly foodborne pathogens, including Listeria monocytogenes, from food. However, susceptibility towards pressure differs among diverse strains of L. monocytogenes and it is unclear if this is due to their intrinsic characteristics related to genomic content. Here, we tested the barotolerance of 10 different L. monocytogenes strains, from food and food processing environments and widely used reference strains including clinical isolate, to pressure treatments with 400 and 600 MPa. Genome sequencing and genome comparison of the tested L. monocytogenes strains were performed to investigate the relation between genomic profile and pressure tolerance.<br><br>RESULTS: None of the tested strains were tolerant to 600&#x2009;MPa. A reduction of more than 5 log10 was observed for all strains after 1&#x2009;min 600&#x2009;MPa pressure treatment. L. monocytogenes strain RO15 showed no significant reduction in viable cell counts after 400&#x2009;MPa for 1&#x2009;min and was therefore defined as barotolerant. Genome analysis of so far unsequenced L. monocytogenes strain RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 allowed us to compare the gene content of all strains tested. This revealed that the three most pressure tolerant strains had more than one CRISPR system with self-targeting spacers. Furthermore, several anti-CRISPR genes were detected in these strains. Pan-genome analysis showed that 10 prophage genes were significantly associated with the three most barotolerant strains.<br><br>CONCLUSIONS: L. monocytogenes strain RO15 was the most pressure tolerant among the selected strains. Genome comparison suggests that there might be a relationship between prophages and pressure tolerance in L. monocytogenes.},
}
@article {pmid32615907,
year = {2020},
author = {Chen, P and Visokay, S and Abrams, JM},
title = {Drosophila GFAT1 and GFAT2 enzymes encode obligate developmental functions.},
journal = {Fly},
volume = {14},
number = {1-4},
pages = {3-9},
pmid = {32615907},
issn = {1933-6942},
support = {R01 CA222579/CA/NCI NIH HHS/United States ; R01 GM072124/GM/NIGMS NIH HHS/United States ; R01 GM115682/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Dietary Supplements ; Drosophila Proteins/genetics/*metabolism ; Gene Expression Regulation, Developmental/*physiology ; Gene Expression Regulation, Enzymologic ; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics/*metabolism ; Larva ; Mutation ; Survival ; },
abstract = {Glutamine: fructose-6-phosphate amidotransferase (GFAT) enzymes catalyse the first committed step of the hexosamine biosynthesis pathway (HBP) using glutamine and fructose-6-phosphate to form glucosamine-6-phosphate (GlcN6P). Numerous species (e.g. mouse, rat, zebrafish, chicken) including humans and Drosophila encode two broadly expressed copies of this enzyme but whether these perform redundant, partially overlapping or distinct functions is not known. To address this question, we produced single gene null mutations in the fly counterparts of gfat1 and gfat2. Deletions for either enzyme were fully lethal and homozygotes lacking either GFAT1 or GFAT2 died at or prior to the first instar larval stage. Therefore, when genetically eliminated, neither isoform was able to compensate for the other. Importantly, dietary supplementation with D-glucosamine-6-phosphate rescued GFAT2 deficiency and restored viability to gfat2[-/-] mutants. In contrast, glucosamine-6-phosphate did not rescue gfat1[-/-] animals.},
}
@article {pmid32615024,
year = {2020},
author = {Otte, K and Kühne, NM and Furrer, AD and Baena Lozada, LP and Lutz, VT and Schilling, T and Hertel, R},
title = {A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages.},
journal = {Letters in applied microbiology},
volume = {71},
number = {6},
pages = {588-595},
doi = {10.1111/lam.13349},
pmid = {32615024},
issn = {1472-765X},
mesh = {Bacillus Phages/*genetics/physiology ; Bacillus subtilis/*virology ; *CRISPR-Cas Systems ; Genetic Engineering ; Mutagenesis ; RNA, Guide/genetics/metabolism ; },
abstract = {Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.},
}
@article {pmid32614871,
year = {2020},
author = {Kirchner, S and Reuter, S and Westphal, A and Mrowka, R},
title = {Decipher the complexity of cis-regulatory regions by a modified Cas9.},
journal = {PloS one},
volume = {15},
number = {7},
pages = {e0235530},
pmid = {32614871},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genes, Reporter ; HEK293 Cells ; Humans ; Luciferases, Firefly/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Regulatory Sequences, Nucleic Acid/*genetics ; Renin/genetics ; },
abstract = {BACKGROUND: Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context.<br><br>METHODS: With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models.<br><br>RESULTS: SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data.<br><br>CONCLUSION: By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.},
}
@article {pmid32613263,
year = {2020},
author = {Ma, J and Hancock, WG and Nifong, JM and Kernodle, SP and Lewis, RS},
title = {Identification and editing of a hybrid lethality gene expands the range of interspecific hybridization potential in Nicotiana.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {10},
pages = {2915-2925},
pmid = {32613263},
issn = {1432-2242},
mesh = {CRISPR-Cas Systems ; Crosses, Genetic ; DNA Transposable Elements ; Gene Editing ; *Genes, Lethal ; *Genes, Plant ; *Hybridization, Genetic ; Tobacco/*genetics ; },
abstract = {Identification and inactivation of hybrid lethality genes can be used to expand the available gene pool for improvement of a cultivated crop species. Hybrid lethality is one genetic mechanism that contributes to reproductive isolation in plants and serves as a barrier to use of diverse germplasm for improvement of cultivated species. A classic example is the seedling lethality exhibited by progeny from the Nicotiana tabacum × N. africana interspecific cross. In order to increase the body of knowledge on mechanisms of hybrid lethality in plants, and to potentially develop tools to circumvent them, we utilized a transposon tagging strategy to identify a candidate gene involved in the control of this reaction. N. tabacum gene Nt6549g30 was identified to code for a class of coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) proteins, the largest class of plant defense proteins. Gene editing, along with other experiments, was used to verify that Nt6549g30 is the gene at the N. tabacum Hybrid Lethality 1 (NtHL1) locus controlling the hybrid lethality reaction in crosses with N. africana. Gene editing of Nt6549g30 was also used to reverse interspecific seedling lethality in crosses between N. tabacum and eight of nine additional tested species from section Suaveolentes. Results further implicate the role of disease resistance-like genes in the evolution of plant species and demonstrate the possibility of expanding the gene pool for a crop species through gene editing.},
}
@article {pmid32611756,
year = {2020},
author = {Busse, DC and Habgood-Coote, D and Clare, S and Brandt, C and Bassano, I and Kaforou, M and Herberg, J and Levin, M and Eléouët, JF and Kellam, P and Tregoning, JS},
title = {Interferon-Induced Protein 44 and Interferon-Induced Protein 44-Like Restrict Replication of Respiratory Syncytial Virus.},
journal = {Journal of virology},
volume = {94},
number = {18},
pages = {},
pmid = {32611756},
issn = {1098-5514},
support = {/WT_/Wellcome Trust/United Kingdom ; 206508/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 109056/Z/15/A/WT_/Wellcome Trust/United Kingdom ; },
mesh = {A549 Cells ; Animals ; Antigens/genetics/*immunology ; Biological Assay ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytoskeletal Proteins/deficiency/genetics/*immunology ; Disease Models, Animal ; Epithelial Cells ; Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; Host-Pathogen Interactions/genetics/*immunology ; Humans ; Immunity, Innate ; Infant ; Mice ; Mice, Knockout ; Respiratory Syncytial Virus Infections/genetics/*immunology/virology ; Respiratory Syncytial Virus, Human/genetics/*immunology ; Signal Transduction ; Tumor Suppressor Proteins/deficiency/genetics/*immunology ; Virus Replication ; },
abstract = {Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.},
}
@article {pmid32611519,
year = {2020},
author = {Zhao, X and Jin, C and Dong, T and Sun, Z and Zheng, X and Feng, B and Cheng, Z and Li, X and Tao, Y and Wu, H},
title = {Characterization of promoters for adeno-associated virus mediated efficient Cas9 activation in adult Cas9 knock-in murine cochleae.},
journal = {Hearing research},
volume = {394},
number = {},
pages = {107999},
doi = {10.1016/j.heares.2020.107999},
pmid = {32611519},
issn = {1878-5891},
mesh = {Animals ; CRISPR-Cas Systems ; *Cochlea ; Cytomegalovirus Infections ; *Dependovirus/genetics ; Genetic Vectors ; Hearing Loss ; Mice ; Promoter Regions, Genetic ; },
abstract = {CRISPR/Cas9 gene editing enables the treatment of hearing loss in congenitally deaf neonatal mice via both viral and non-viral delivery. While adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene replacement in the inner ear, application of the AAV-mediated CRISPR/Cas9 gene-editing approach for this purpose is yet to be documented. Based on our previous findings, we focused on the effects of several AAVs delivered via canalostomy injection in adult mice. Among the AAVs examined, AAV8 showed the greatest efficiency and specificity in transducing inner hair cells (IHC). The ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knock-in (Cas9 KI) mice was further evaluated. We compared the effects of six different promoters (CMV, CAG, hSyn, CaMKIIa, GFAP, and ALB) of AAV8 delivered to the inner ear of adult Cas9 KI mice. Our findings showed that three AAV groups (CMV, CAG and hSyn promoters) infected the inner ear efficiently with different tropisms. Notably, AAVs with CMV, CAG, and hSyn promoters infected diverse cell types in mature murine cochleae, including IHCs. In particular, AAV8-hSyn showed high affinity to IHCs and spiral ganglion neurons (SGN). Neither the AAV8 virus itself (except AAV8-CAG) nor the surgical procedures used caused damage to HCs or impaired normal hearing. Our findings indicated that injection of AAV-Cre into mature inner ear efficiently induces Cas9 activation to achieve safe and efficient gene editing and different constituent promoters confer diverse infection patterns in cochlea, expanding the repertoire of gene-editing tools for regulating gene expression in target cells of the inner ear as part of the collective effort to rescue genetic hearing loss and develop effective gene therapy techniques.},
}
@article {pmid32610343,
year = {2020},
author = {Elitt, MS and Barbar, L and Shick, HE and Powers, BE and Maeno-Hikichi, Y and Madhavan, M and Allan, KC and Nawash, BS and Gevorgyan, AS and Hung, S and Nevin, ZS and Olsen, HE and Hitomi, M and Schlatzer, DM and Zhao, HT and Swayze, A and LePage, DF and Jiang, W and Conlon, RA and Rigo, F and Tesar, PJ},
title = {Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease.},
journal = {Nature},
volume = {585},
number = {7825},
pages = {397-403},
pmid = {32610343},
issn = {1476-4687},
support = {S10 OD024981/OD/NIH HHS/United States ; TL1 TR002549/TR/NCATS NIH HHS/United States ; S10 OD026882/OD/NIH HHS/United States ; S10 OD016164/OD/NIH HHS/United States ; P30 CA043703/CA/NCI NIH HHS/United States ; T32 NS077888/NS/NINDS NIH HHS/United States ; R01 NS093357/NS/NINDS NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; F30 HD084167/HD/NICHD NIH HHS/United States ; F30 HD096784/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Gene Editing ; Hypoxia/metabolism ; Male ; Mice ; Mice, Mutant Strains ; Motor Activity/genetics ; Myelin Proteolipid Protein/*deficiency/genetics/metabolism ; Myelin Sheath/metabolism ; Oligodendroglia/metabolism ; Oligonucleotides, Antisense/administration &amp; dosage/genetics ; Pelizaeus-Merzbacher Disease/*genetics/metabolism/*therapy ; Point Mutation ; Respiratory Function Tests ; Survival Analysis ; },
abstract = {Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD)[1,2]. Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD[1,3-5]. Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1[jp]) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.},
}
@article {pmid32610012,
year = {2020},
author = {Xia, PF and Casini, I and Schulz, S and Klask, CM and Angenent, LT and Molitor, B},
title = {Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination.},
journal = {ACS synthetic biology},
volume = {9},
number = {8},
pages = {2162-2171},
doi = {10.1021/acssynbio.0c00226},
pmid = {32610012},
issn = {2161-5063},
mesh = {Acetates/metabolism ; Acetyl Coenzyme A/genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Carbon Cycle ; Clostridium/genetics/*metabolism ; Codon, Terminator ; Deamination ; Gene Editing/*methods ; Genome, Bacterial ; },
abstract = {Acetogenic bacteria are rising in popularity as chassis microbes for biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome and the potential for off-target events. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux toward improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general.},
}
@article {pmid32609820,
year = {2020},
author = {Kuo, J and Yuan, R and Sánchez, C and Paulsson, J and Silver, PA},
title = {Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {48},
number = {14},
pages = {8165-8177},
pmid = {32609820},
issn = {1362-4962},
support = {HR0011-16-2-0049//Defense Advanced Research Projects Agency/International ; F32 GM125108/GM/NIGMS NIH HHS/United States ; 1615487//National Science Foundation/International ; 1562497//National Science Foundation/International ; 1517372//National Science Foundation/International ; },
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/metabolism ; Escherichia coli ; Microfluidics/instrumentation/*methods ; *Periodicity ; RNA, Guide/genetics/*metabolism ; Single-Cell Analysis/instrumentation/methods ; },
abstract = {In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ h at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally independent behavior, as the RNAs control each others' transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.},
}
@article {pmid32609721,
year = {2020},
author = {Damasceno, JD and Reis-Cunha, J and Crouch, K and Beraldi, D and Lapsley, C and Tosi, LRO and Bartholomeu, D and McCulloch, R},
title = {Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.},
journal = {PLoS genetics},
volume = {16},
number = {7},
pages = {e1008828},
pmid = {32609721},
issn = {1553-7404},
support = {MR/S019472/1/MRC_/Medical Research Council/United Kingdom ; G0401553/MRC_/Medical Research Council/United Kingdom ; BB/R017166/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N016165/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 104111/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA Damage/genetics ; DNA Repair/genetics ; DNA Replication/genetics ; Gene Knockout Techniques ; Genome/genetics ; Homologous Recombination/*genetics ; Humans ; Leishmania major/*genetics/pathogenicity ; Leishmaniasis, Cutaneous/*genetics/parasitology ; Rad51 Recombinase/*genetics ; },
abstract = {Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.},
}
@article {pmid32609329,
year = {2020},
author = {Delgado-Benito, V and Berruezo-Llacuna, M and Altwasser, R and Winkler, W and Sundaravinayagam, D and Balasubramanian, S and Caganova, M and Graf, R and Rahjouei, A and Henke, MT and Driesner, M and Keller, L and Prigione, A and Janz, M and Akalin, A and Di Virgilio, M},
title = {PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification.},
journal = {The Journal of experimental medicine},
volume = {217},
number = {10},
pages = {},
pmid = {32609329},
issn = {1540-9538},
mesh = {Animals ; *Antibody Diversity ; B-Lymphocytes/immunology/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Death ; Cell Differentiation ; Cell Line ; Female ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression Regulation ; Genes, Immunoglobulin/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; },
abstract = {The establishment of protective humoral immunity is dependent on the ability of mature B cells to undergo antibody gene diversification while adjusting to the physiological stressors induced by activation with the antigen. Mature B cells diversify their antibody genes by class switch recombination (CSR) and somatic hypermutation (SHM), which are both dependent on efficient induction of activation-induced cytidine deaminase (AID). Here, we identified PDGFA-associated protein 1 (Pdap1) as an essential regulator of cellular homeostasis in mature B cells. Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4) and induction of the ISR transcriptional program, increased cell death, and defective AID expression. As a consequence, loss of Pdap1 reduces germinal center B cell formation and impairs CSR and SHM. Thus, Pdap1 protects mature B cells against chronic ISR activation and ensures efficient antibody diversification by promoting their survival and optimal function.},
}
@article {pmid32609115,
year = {2020},
author = {Sun, HH and He, F and Wang, T and Yin, BC and Ye, BC},
title = {A Cas12a-mediated cascade amplification method for microRNA detection.},
journal = {The Analyst},
volume = {145},
number = {16},
pages = {5547-5552},
doi = {10.1039/d0an00370k},
pmid = {32609115},
issn = {1364-5528},
mesh = {CRISPR-Cas Systems ; DNA ; *DNA, Catalytic ; *MicroRNAs/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Here, we took advantage of Cas12a trans-cleavage activity to develop an enzyme-assisted cascade amplification method for isothermal miRNA detection. A target miRNA-initiated ligation reaction would allow for the production of transcription templates that triggered the transcriptional amplification of RNA strands. These RNA strands were cleaved by the 8-17E DNAzyme to generate crRNAs and recycled RNAs which have the same sequence as the target miRNA. The amplified abundant crRNAs bound to Cas12a and dsDNA activators to form the complex, which trans-cleaved the ssDNA reporters to generate a fluorescence signal for miRNA quantitative analysis. The proposed method exhibits a femtomolar limit of detection and a good specificity in distinguishing the homologous sequences of miRNAs. Its practical application ability was further tested in different cell lines.},
}
@article {pmid32608968,
year = {2020},
author = {Zhao, X and Zeng, L and Mei, Q and Luo, Y},
title = {Allosteric Probe-Initiated Wash-Free Method for Sensitive Extracellular Vesicle Detection through Dual Cycle-Assisted CRISPR-Cas12a.},
journal = {ACS sensors},
volume = {5},
number = {7},
pages = {2239-2246},
doi = {10.1021/acssensors.0c00944},
pmid = {32608968},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Extracellular Vesicles ; RNA ; },
abstract = {Extracellular vesicles (EVs) are emerging as promising biomarkers for cancer diagnosis and therapy. Recognizing low-abundance EVs from clinical samples in an easy-to-operate way is highly desired but remains a challenge. Herein, we established an allosteric probe-initiated dual cycle amplification-assisted CRISPR-Cas12a (AID-Cas) platform for sensitive detection of EVs in a wash-free way. In AID-Cas, the allosteric probe can specifically recognize and bind with target EVs and thus initiate the following dual-cycle amplification. Subsequently, the amplified products were transcribed to generate numerous single-stranded RNAs, which could work as crRNA to trigger the trans-cleavage of CRISPR-Cas12a. Consequently, the proposed approach achieved a good linear response to extracted EVs in a concentration range from 10[2] to 10[6] particles/μL. Because of its high sensitivity, together with its wash-free convenience, the proposed strategy could have promising clinical potentials for early diagnosis of cancers.},
}
@article {pmid32606841,
year = {2020},
author = {Wang, G and Song, G and Xu, Y},
title = {Association of CRISPR/Cas System with the Drug Resistance in Klebsiella pneumoniae.},
journal = {Infection and drug resistance},
volume = {13},
number = {},
pages = {1929-1935},
pmid = {32606841},
issn = {1178-6973},
abstract = {BACKGROUND: Klebsiella pneumoniae is a common opportunistic pathogen and its production of extended-spectrum β-lactamases (ESBL) and carbapenemases leads to drug resistance. Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (Cas) are widespread in the genome of many bacteria and are a defense mechanism against foreign invaders such as plasmids and viruses.<br><br>PURPOSE: To investigate the prevalence of the CRISPR/Cas system in wild type strains of K. pneumoniae in the hospital and its association with drug resistance.<br><br>MATERIALS AND METHODS: A total of 136 strains were collected and characterized their susceptibility to antimicrobial agents. The prevalence of CRISPR/Cas system was detected by PCR and DNA sequencing was analyzed by CRISPRFinder. The statistical analysis of the results was performed by SPSS.<br><br>RESULTS: We found that 50/136 (37%) isolates produced ESBL and 30/136 (22%) isolates were resistant to carbapenems. These isolates were liable to be multidrug resistant against &#x3b2;-lactams, quinolones, and aminoglycosides. Among the carbapenem-resistant isolates, blaKPC was the main drug resistance-associated gene and different types of ESBL and AmpC genes were present. Resistance to &#x3b2;-lactams, quinolones, aminoglycosides, tetracyclines, and &#x3b2;-lactams/enzyme inhibitor were higher in absence of the CRISPR/Cas system. Eighteen spacers within the CRISPR arrays matched with the genomes of plasmids or phages, some of which carried drug resistance genes.<br><br>CONCLUSION: ESBL-producing and carbapenem-resistant K. pneumoniae are more likely to develop multidrug resistance and show an inverse correlation between drug resistance and CRISPR/Cas system. Absence of CRISPR/Cas modules allow for the acquisition of external drug resistance genes.},
}
@article {pmid32606465,
year = {2020},
author = {Ledford, H},
title = {CRISPR gene editing in human embryos wreaks chromosomal mayhem.},
journal = {Nature},
volume = {583},
number = {7814},
pages = {17-18},
pmid = {32606465},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; Chromosomes, Human/*genetics ; *Embryo Research ; *Gene Editing ; Genetic Diseases, Inborn/genetics/therapy ; Germ-Line Mutation/genetics ; Humans ; Mutation ; Octamer Transcription Factor-3/genetics ; Patient Safety ; Preprints as Topic ; Recombination, Genetic ; *Sequence Deletion ; },
}
@article {pmid32604937,
year = {2020},
author = {Ryczek, N and Hryhorowicz, M and Lipiński, D and Zeyland, J and Słomski, R},
title = {Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation.},
journal = {Genes},
volume = {11},
number = {6},
pages = {},
pmid = {32604937},
issn = {2073-4425},
mesh = {Animals ; Animals, Genetically Modified/genetics ; Asialoglycoprotein Receptor/genetics ; CRISPR-Cas Systems/*genetics ; Galactosyltransferases/genetics ; Gene Knockout Techniques ; Genetic Vectors/*genetics ; Heterografts ; Humans ; Mixed Function Oxygenases/genetics ; Mutation/genetics ; Swine/*genetics ; *Transplantation, Heterologous ; von Willebrand Factor/genetics ; },
abstract = {The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (Sus scrofa domestica) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine GGTA1, CMAH, β4GalNT2, vWF, ASGR1 genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies.},
}
@article {pmid32604025,
year = {2020},
author = {Tripathi, L and Ntui, VO and Tripathi, JN},
title = {CRISPR/Cas9-based genome editing of banana for disease resistance.},
journal = {Current opinion in plant biology},
volume = {56},
number = {},
pages = {118-126},
doi = {10.1016/j.pbi.2020.05.003},
pmid = {32604025},
issn = {1879-0356},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Resistance/genetics ; *Gene Editing ; Genome, Plant/genetics ; Humans ; *Musa/genetics ; },
abstract = {Banana production is severely constrained by many pathogens and pests, particularly where a number of them are co-existing. The use of disease-resistant banana varieties is one of the most effective ways to mitigate the negative impacts of pathogens on banana production. Recent advances in new breeding techniques have the potential to accelerate breeding of banana for disease resistance. The CRISPR/Cas9 based genome editing has emerged as the most powerful tool for crop improvement due to its capability of creating precise alterations in plant genome and trait stacking through multiplexing. Recently, the robust CRISPR/Cas9-based genome editing of banana has been established, which can be applied for developing disease-resistant varieties. This article presents a synopsis of recent advancements and perspectives on the application of genome editing for generating disease-resistant banana varieties. It also summarizes the current status of regulatory requirements for the release of genome-edited crop varieties among different countries.},
}
@article {pmid32603815,
year = {2021},
author = {Yu, W and Wu, Z},
title = {Ocular delivery of CRISPR/Cas genome editing components for treatment of eye diseases.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {181-195},
doi = {10.1016/j.addr.2020.06.011},
pmid = {32603815},
issn = {1872-8294},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Administration Routes ; Eye Diseases/*genetics/*therapy ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; },
abstract = {A variety of inherited or multifactorial ocular diseases call for novel treatment paradigms. The newly developed genome editing technology, CRISPR, has shown great promise in treating these diseases, but delivery of the CRISPR/Cas components to target ocular tissues and cells requires appropriate use of vectors and routes of administration to ensure safety, efficacy and specificity. Although adeno-associated viral (AAV) vectors are thus far the most commonly used tool for ocular gene delivery, sustained expression of CRISPR/Cas components may cause immune reactions and an increased risk of off-target editing. In this review, we summarize the ocular administration routes and discuss the advantages and disadvantages of viral and non-viral vectors for delivery of CRISPR/Cas components to the eye. We review the existing studies of CRISPR/Cas genome editing for ocular diseases and discuss the major challenges of the technology in ocular applications. We also discuss the most recently developed CRISPR tools such as base editing and prime editing which may be used for future ocular applications.},
}
@article {pmid32603681,
year = {2021},
author = {Zhang, Y and Karakikes, I},
title = {Translating genomic insights into cardiovascular medicine: Opportunities and challenges of CRISPR-Cas9.},
journal = {Trends in cardiovascular medicine},
volume = {31},
number = {6},
pages = {341-348},
pmid = {32603681},
issn = {1873-2615},
support = {R00 HL104002/HL/NHLBI NIH HHS/United States ; R01 HL139679/HL/NHLBI NIH HHS/United States ; R01 HL150414/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cardiovascular Diseases/*genetics/metabolism/physiopathology/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Loci ; Genetic Predisposition to Disease ; Genetic Therapy ; Genome-Wide Association Study ; *Genomics ; Humans ; Phenotype ; },
abstract = {The growing appreciation of human genetics and genomics in cardiovascular disease (CVD) accompanied by the technological breakthroughs in genome editing, particularly the CRISPR-Cas9 technologies, has presented an unprecedented opportunity to explore the application of genome editing in cardiovascular medicine. The ever-growing genome editing toolbox includes an assortment of CRISPR-Cas systems with increasing efficiency, precision, flexibility, and targeting capacity. Over the past decade, the advent of large-scale genotyping technologies and genome-wide association studies (GWAS) has provided numerous genotype-phenotype associations for diseases with complex traits. Notably, a growing number of loss-of-function mutations have been associated with favorable CVD risk-factor profiles that may confer protection. Combining the newly gained insights of human genetics with recent breakthrough technologies, such as the CRISPR-Cas9 technologies, holds great promise in elucidating novel disease mechanisms and transforming genes into medicines. Nonetheless, translating genetic insights into novel therapeuties remains challenging. Applications of "in body" genome editing for CVD treatment and engineering cardioprotection remain mostly theoretical. Here we highlight the recent advances of the CRISPR-based genome editing toolbox and discuss the potential and challenges of CRISPR-based technologies for translating GWAS findings into genomic medicines.},
}
@article {pmid32603414,
year = {2020},
author = {Naeimi Kararoudi, M and Nagai, Y and Elmas, E and de Souza Fernandes Pereira, M and Ali, SA and Imus, PH and Wethington, D and Borrello, IM and Lee, DA and Ghiaur, G},
title = {CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity.},
journal = {Blood},
volume = {136},
number = {21},
pages = {2416-2427},
pmid = {32603414},
issn = {1528-0020},
support = {K08 HL127269/HL/NHLBI NIH HHS/United States ; R03 HL145226/HL/NHLBI NIH HHS/United States ; },
mesh = {ADP-ribosyl Cyclase 1/*deficiency/genetics ; Adoptive Transfer ; Animals ; Antibodies, Monoclonal/*pharmacology ; Antibody-Dependent Cell Cytotoxicity ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytotoxicity, Immunologic/*drug effects ; Humans ; Immunotherapy ; Killer Cells, Natural/drug effects/*immunology/transplantation ; Male ; Membrane Glycoproteins/*deficiency/genetics ; Mice ; Mice, Inbred NOD ; Multiple Myeloma/*pathology ; NAD/metabolism ; Oxidative Phosphorylation ; Specific Pathogen-Free Organisms ; Tretinoin/pharmacology ; Whole Genome Sequencing ; },
abstract = {Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.},
}
@article {pmid32602833,
year = {2020},
author = {Gong, Y and Bi, Y and Li, Z and Li, Y and Yao, Y and Long, Q and Pu, T and Chen, C and Liu, T and Dong, S and Cun, W},
title = {High-efficiency nonhomologous insertion of a foreign gene into the herpes simplex virus genome.},
journal = {The Journal of general virology},
volume = {101},
number = {9},
pages = {982-996},
pmid = {32602833},
issn = {1465-2099},
mesh = {Animals ; CRISPR-Cas Systems ; Chlorocebus aethiops ; DNA End-Joining Repair ; *Gene Knock-In Techniques ; *Genome, Viral ; HEK293 Cells ; Herpesvirus 1, Human/*genetics ; Humans ; *Mutagenesis, Insertional ; Plasmids ; Vero Cells ; },
abstract = {Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45 % after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70-100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.},
}
@article {pmid32601467,
year = {2020},
author = {Laidlaw, BJ and Duan, L and Xu, Y and Vazquez, SE and Cyster, JG},
title = {The transcription factor Hhex cooperates with the corepressor Tle3 to promote memory B cell development.},
journal = {Nature immunology},
volume = {21},
number = {9},
pages = {1082-1093},
pmid = {32601467},
issn = {1529-2916},
support = {T32 AI007019/AI/NIAID NIH HHS/United States ; DRG-2265-16/HHMI/Howard Hughes Medical Institute/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; R01 AI045073/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AI040098/AI/NIAID NIH HHS/United States ; T32 AI007334/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocyte Subsets/*immunology ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Cell Differentiation ; Co-Repressor Proteins/genetics/*metabolism ; Female ; Gene Expression Regulation ; Germinal Center/*immunology ; Homeodomain Proteins/genetics/*metabolism ; Immunologic Memory ; Lymphocyte Activation ; Male ; Mice ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-6/genetics/metabolism ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcription Factors/genetics/*metabolism ; },
abstract = {Memory B cells (MBCs) are essential for long-lived humoral immunity. However, the transcription factors involved in MBC differentiation are poorly defined. Here, using single-cell RNA sequencing analysis, we identified a population of germinal center (GC) B cells in the process of differentiating into MBCs. Using an inducible CRISPR-Cas9 screening approach, we identified the hematopoietically expressed homeobox protein Hhex as a transcription factor regulating MBC differentiation. The corepressor Tle3 was also identified in the screen and was found to interact with Hhex to promote MBC development. Bcl-6 directly repressed Hhex in GC B cells. Reciprocally, Hhex-deficient MBCs exhibited increased Bcl6 expression and reduced expression of the Bcl-6 target gene Bcl2. Overexpression of Bcl-2 was able to rescue MBC differentiation in Hhex-deficient cells. We also identified Ski as an Hhex-induced transcription factor involved in MBC differentiation. These findings establish an important role for Hhex-Tle3 in regulating the transcriptional circuitry governing MBC differentiation.},
}
@article {pmid32601435,
year = {2020},
author = {van Haasteren, J and Li, J and Scheideler, OJ and Murthy, N and Schaffer, DV},
title = {The delivery challenge: fulfilling the promise of therapeutic genome editing.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {845-855},
pmid = {32601435},
issn = {1546-1696},
support = {UG3 NS115599/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Genetic Diseases, Inborn/genetics/*therapy ; *Genetic Therapy ; Genetic Vectors/genetics/therapeutic use ; Humans ; Nanoparticles/therapeutic use ; Protein Engineering ; },
abstract = {Genome editing has the potential to treat an extensive range of incurable monogenic and complex diseases. In particular, advances in sequence-specific nuclease technologies have dramatically accelerated the development of therapeutic genome editing strategies that are based on either the knockout of disease-causing genes or the repair of endogenous mutated genes. These technologies are progressing into human clinical trials. However, challenges remain before the therapeutic potential of genome editing can be fully realized. Delivery technologies that have serendipitously been developed over the past couple decades in the protein and nucleic acid delivery fields have been crucial to genome editing success to date, including adeno-associated viral and lentiviral vectors for gene therapy and lipid nanoparticle and other non-viral vectors for nucleic acid and protein delivery. However, the efficiency and tissue targeting capabilities of these vehicles must be further improved. In addition, the genome editing enzymes themselves need to be optimized, and challenges regarding their editing efficiency, specificity and immunogenicity must be addressed. Emerging protein engineering and synthetic chemistry approaches can offer solutions and enable the development of safe and efficacious clinical genome editing.},
}
@article {pmid32601432,
year = {2020},
author = {Wang, S and Zong, Y and Lin, Q and Zhang, H and Chai, Z and Zhang, D and Chen, K and Qiu, JL and Gao, C},
title = {Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.},
journal = {Nature biotechnology},
volume = {38},
number = {12},
pages = {1460-1465},
pmid = {32601432},
issn = {1546-1696},
mesh = {CRISPR-Associated Protein 9/*metabolism ; Cytidine Deaminase/*metabolism ; Deamination ; Gene Editing ; Humans ; Oryza/*genetics/microbiology ; Plant Diseases/microbiology ; Proteins/*metabolism ; Protoplasts/metabolism ; Regeneration ; *Sequence Deletion ; Triticum/*genetics ; },
abstract = {Short insertions and deletions can be produced in plant genomes using CRISPR-Cas editors, but reliable production of larger deletions in specific target sites has proven difficult to achieve. We report the development of a series of APOBEC-Cas9 fusion-induced deletion systems (AFIDs) that combine Cas9 with human APOBEC3A (A3A), uracil DNA-glucosidase and apurinic or apyrimidinic site lyase. In rice and wheat, AFID-3 generated deletions from 5'-deaminated C bases to the Cas9-cleavage site. Approximately one-third of deletions produced using AFID-3 in rice and wheat protoplasts (30.2%) and regenerated plants (34.8%) were predictable. We show that eAFID-3, in which the A3A in AFID-3 is replaced with truncated APOBEC3B (A3Bctd), produced more uniform deletions from the preferred TC motif to the double-strand break. AFIDs could be applied to study regulatory regions and protein domains to improve crop plants.},
}
@article {pmid32601426,
year = {2020},
author = {Ochoa-Fernandez, R and Abel, NB and Wieland, FG and Schlegel, J and Koch, LA and Miller, JB and Engesser, R and Giuriani, G and Brandl, SM and Timmer, J and Weber, W and Ott, T and Simon, R and Zurbriggen, MD},
title = {Optogenetic control of gene expression in plants in the presence of ambient white light.},
journal = {Nature methods},
volume = {17},
number = {7},
pages = {717-725},
pmid = {32601426},
issn = {1548-7105},
mesh = {Arabidopsis/genetics/immunology ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Plant ; *Light ; Models, Theoretical ; *Optogenetics ; Plants, Genetically Modified ; },
abstract = {Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.},
}
@article {pmid32601419,
year = {2020},
author = {Ma, X and Zhang, X and Liu, H and Li, Z},
title = {Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9.},
journal = {Nature plants},
volume = {6},
number = {7},
pages = {773-779},
pmid = {32601419},
issn = {2055-0278},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plants/*genetics ; Tobacco/genetics ; },
abstract = {Genome-editing technologies using CRISPR-Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR-Cas reagent delivery methods may be associated with unanticipated genome changes or damage[1,2], with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis[3]. Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR-Cas9 ribonucleoproteins into protoplasts[4] or in vitro fertilized zygotes[5]. However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR-Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency[6-9]. Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR-Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR-Cas9 gene editing in plants through virus infection.},
}
@article {pmid32601418,
year = {2020},
author = {Dinesh-Kumar, SP and Voytas, DF},
title = {Editing through infection.},
journal = {Nature plants},
volume = {6},
number = {7},
pages = {738-739},
pmid = {32601418},
issn = {2055-0278},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Infections ; RNA Editing ; },
}
@article {pmid32601291,
year = {2020},
author = {Kamble, PG and Hetty, S and Vranic, M and Almby, K and Castillejo-López, C and Abalo, XM and Pereira, MJ and Eriksson, JW},
title = {Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10565},
pmid = {32601291},
issn = {2045-2322},
mesh = {Adipocytes/*metabolism ; Adipogenesis/genetics ; Adipose Tissue/metabolism ; Adult ; Aged ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; Middle Aged ; PPAR gamma/genetics ; Proof of Concept Study ; Tacrolimus Binding Proteins/genetics ; },
abstract = {CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology.},
}
@article {pmid32599531,
year = {2020},
author = {Csörgő, B and Nyerges, A and Pál, C},
title = {Targeted mutagenesis of multiple chromosomal regions in microbes.},
journal = {Current opinion in microbiology},
volume = {57},
number = {},
pages = {22-30},
pmid = {32599531},
issn = {1879-0364},
mesh = {Bacteria/*genetics ; CRISPR-Cas Systems ; Chromosomes, Bacterial/*genetics ; Genetic Engineering/*methods ; *Mutagenesis ; Plasmids/genetics ; },
abstract = {Directed evolution allows the effective engineering of proteins, biosynthetic pathways, and cellular functions. Traditional plasmid-based methods generally subject one or occasionally multiple genes-of-interest to mutagenesis, require time-consuming manual interventions, and the genes that are subjected to mutagenesis are outside of their native genomic context. Other methods mutagenize the whole genome unselectively which may distort the outcome. Recent recombineering- and CRISPR-based technologies radically change this field by allowing exceedingly high mutation rates at multiple, predefined loci in their native genomic context. In this review, we focus on recent technologies that potentially allow accelerated tunable mutagenesis at multiple genomic loci in the native genomic context of these target sequences. These technologies will be compared by four main criteria, including the scale of mutagenesis, portability to multiple microbial species, off-target mutagenesis, and cost-effectiveness. Finally, we discuss how these technical advances open new avenues in basic research and biotechnology.},
}
@article {pmid32599515,
year = {2020},
author = {Fontana, J and Sparkman-Yager, D and Zalatan, JG and Carothers, JM},
title = {Challenges and opportunities with CRISPR activation in bacteria for data-driven metabolic engineering.},
journal = {Current opinion in biotechnology},
volume = {64},
number = {},
pages = {190-198},
doi = {10.1016/j.copbio.2020.04.005},
pmid = {32599515},
issn = {1879-0429},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Metabolic Engineering ; RNA, Guide ; },
abstract = {Creating CRISPR gene activation (CRISPRa) technologies in industrially promising bacteria could be transformative for accelerating data-driven metabolic engineering and strain design. CRISPRa has been widely used in eukaryotes, but applications in bacterial systems have remained limited. Recent work shows that multiple features of bacterial promoters impose stringent requirements on CRISPRa-mediated gene activation. However, by systematically defining rules for effective bacterial CRISPRa sites and developing new approaches for encoding complex functions in engineered guide RNAs, there are now clear routes to generalize synthetic gene regulation in bacteria. When combined with multi-omics data collection and machine learning, the full development of bacterial CRISPRa will dramatically improve the ability to rapidly engineer bacteria for bioproduction through accelerated design-build-test-learn cycles.},
}
@article {pmid32598833,
year = {2021},
author = {Lima, NS and Martínez, AG},
title = {Biotechnological challenges: The scope of genome editing.},
journal = {JBRA assisted reproduction},
volume = {25},
number = {1},
pages = {150-154},
pmid = {32598833},
issn = {1518-0557},
mesh = {Animals ; Bacteria ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; *Physicians ; Plants/genetics ; },
abstract = {CRISPR/Cas9 can be considered as the biotechnological breakthrough of the century. Genome editing technologies have developed in a vertiginous way. While the genome editing of species, including animals, plants and bacteria has become a commonly used method, the application of CRISPR-Cas9 in human embryos has led to debates and interdisciplinary discussions. This brings multiple challenges for both scientists and those who must regulate the use of these techniques.},
}
@article {pmid32597917,
year = {2020},
author = {Chen, J and Jiang, F and Huang, CW and Lin, L},
title = {Rapid genotypic antibiotic susceptibility test using CRISPR-Cas12a for urinary tract infection.},
journal = {The Analyst},
volume = {145},
number = {15},
pages = {5226-5231},
doi = {10.1039/d0an00947d},
pmid = {32597917},
issn = {1364-5528},
mesh = {Anti-Bacterial Agents/pharmacology ; Bacteria/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Urinary Tract Infections/diagnosis/drug therapy ; },
abstract = {The current clinical protocol to conduct a bacterial antibiotic susceptibility test (AST) requires at least 18 hours, and cannot be accomplished during a single visit for patients. Here, a new method based on the technique of CRISPR-Cas12a is utilized to accomplish a bacterial genotypic AST within one hour with good accuracy. Two amplification approaches are employed and compared: (1) enriching the bacterial concentration by culturing in growth media; and (2) amplifying target DNA from raw samples by recombinase polymerase amplification (RPA). The results show that CRISPR combined with RPA can rapidly and accurately provide a bacterial genotypic AST of urine samples with urinary tract infections for precise antibiotic treatment. As such, this technology could open a new class of rapid bacterial genotypic AST for various infectious diseases.},
}
@article {pmid32597846,
year = {2020},
author = {Wilson, AM and Wingfield, BD},
title = {CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {160},
pages = {},
doi = {10.3791/61367},
pmid = {32597846},
issn = {1940-087X},
mesh = {Animals ; Ascomycota/*chemistry ; CRISPR-Cas Systems/*genetics ; Drosophila melanogaster ; Gene Editing/*methods ; Plants/*chemistry ; },
abstract = {The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.},
}
@article {pmid32597755,
year = {2020},
author = {Athukoralage, JS and McQuarrie, S and Grüschow, S and Graham, S and Gloster, TM and White, MF},
title = {Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32597755},
issn = {2050-084X},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 204821/Z/16/Z//Wellcome Trust Institutional Strategic Support Fund/International ; BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Archaeoglobus fulgidus/*enzymology/genetics ; CRISPR-Associated Proteins/metabolism ; Catalysis ; Catalytic Domain ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/metabolism ; Escherichia coli/metabolism ; Kinetics ; Methanosarcina ; Models, Molecular ; Oligonucleotides/chemistry ; Protein Multimerization ; RNA/metabolism ; Ribonucleases/genetics/*metabolism ; Second Messenger Systems ; Signal Transduction ; },
abstract = {Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.},
}
@article {pmid32597486,
year = {2020},
author = {Babbs, A and Chatzopoulou, M and Edwards, B and Squire, SE and Wilkinson, IVL and Wynne, GM and Russell, AJ and Davies, KE},
title = {From diagnosis to therapy in Duchenne muscular dystrophy.},
journal = {Biochemical Society transactions},
volume = {48},
number = {3},
pages = {813-821},
pmid = {32597486},
issn = {1470-8752},
support = {MR/N010698/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Codon, Terminator ; Dystrophin/genetics ; Exons ; Genetic Therapy/methods ; Humans ; Male ; Muscular Dystrophy, Duchenne/*diagnosis/genetics/*therapy ; },
abstract = {Genetic approaches for the diagnosis and treatment of inherited muscle diseases have advanced rapidly in recent years. Many of the advances have occurred in the treatment of Duchenne muscular dystrophy (DMD), a muscle wasting disease where affected boys are typically wheelchair bound by age 12 years and generally die in their twenties from respiratory failure or cardiomyopathy. Dystrophin is a 421 kD protein which links F-actin to the extracellular matrix via the dystrophin-associated protein complex (DAPC) at the muscle membrane. In the absence of dystrophin, the DAPC is lost, making the muscle membrane more susceptible to contraction-induced injury. The identification of the gene causing DMD in 1986 resulted in improved diagnosis of the disease and the identification of hotspots for mutation. There is currently no effective treatment. However, there are several promising genetic therapeutic approaches at the preclinical stage or in clinical trials including read-through of stop codons, exon skipping, delivery of dystrophin minigenes and the modulation of expression of the dystrophin related protein, utrophin. In spite of significant progress, the problem of targeting all muscles, including diaphragm and heart at sufficiently high levels, remains a challenge. Any therapy also needs to consider the immune response and some treatments are mutation specific and therefore limited to a subgroup of patients. This short review provides a summary of the current status of DMD therapy with a particular focus on those genetic strategies that have been taken to the clinic.},
}
@article {pmid32597009,
year = {2020},
author = {Castro Machado, F and Bittencourt-Cunha, P and Malvezzi, AM and Arico, M and Radio, S and Smircich, P and Zoltner, M and Field, MC and Schenkman, S},
title = {EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.},
journal = {Cellular microbiology},
volume = {22},
number = {11},
pages = {e13243},
doi = {10.1111/cmi.13243},
pmid = {32597009},
issn = {1462-5822},
support = {203134/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Chagas Disease/*parasitology ; Eukaryotic Initiation Factor-2/genetics/*metabolism ; Gene Expression Regulation ; Humans ; Life Cycle Stages ; Mutation ; Parasitemia ; Phosphorylation ; Protein Biosynthesis ; Proteome/metabolism ; Protozoan Proteins/analysis/biosynthesis/*metabolism ; Trypanosoma cruzi/growth &amp; development/*metabolism/pathogenicity ; Virulence ; },
abstract = {Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNA[met] to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2α[T169A] or with an endogenous CRISPR/Cas9-generated eIF2α[T169A] mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2α[T169A] mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.},
}
@article {pmid32596446,
year = {2020},
author = {Saha, C and Mohanraju, P and Stubbs, A and Dugar, G and Hoogstrate, Y and Kremers, GJ and van Cappellen, WA and Horst-Kreft, D and Laffeber, C and Lebbink, JHG and Bruens, S and Gaskin, D and Beerens, D and Klunder, M and Joosten, R and Demmers, JAA and van Gent, D and Mouton, JW and van der Spek, PJ and van der Oost, J and van Baarlen, P and Louwen, R},
title = {Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA.},
journal = {Science advances},
volume = {6},
number = {25},
pages = {eaaz4849},
pmid = {32596446},
issn = {2375-2548},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; *Campylobacter jejuni/genetics/metabolism ; DNA/genetics ; Gene Editing ; Humans ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.},
}
@article {pmid32595062,
year = {2020},
author = {Chong, ZS and Wright, GJ and Sharma, S},
title = {Investigating Cellular Recognition Using CRISPR/Cas9 Genetic Screening.},
journal = {Trends in cell biology},
volume = {30},
number = {8},
pages = {619-627},
doi = {10.1016/j.tcb.2020.05.005},
pmid = {32595062},
issn = {1879-3088},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Membrane/metabolism ; *Genetic Testing ; Humans ; Models, Biological ; Signal Transduction ; },
abstract = {Neighbouring cells can recognise and communicate with each other by direct binding between cell surface receptor and ligand pairs. Examples of cellular recognition events include pathogen entry into a host cell, sperm-egg fusion, and self/nonself discrimination by the immune system. Despite growing appreciation of cell surface recognition molecules as potential therapeutic targets, identifying key factors contributing to cellular recognition remains technically challenging to perform on a genome-wide scale. Recently, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) knockout or activation (CRISPR-KO/CRISPRa) screens have been applied to identify the molecular determinants of cellular recognition. In this review, we discuss how CRISPR-KO/CRISPRa screening has contributed to our understanding of cellular recognition processes, and how it can be applied to investigate these important interactions in a range of biological contexts.},
}
@article {pmid32592086,
year = {2020},
author = {Fan, R and Chai, Z and Xing, S and Chen, K and Qiu, F and Chai, T and Qiu, JL and Zhang, Z and Zhang, H and Gao, C},
title = {Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand.},
journal = {Science China. Life sciences},
volume = {63},
number = {11},
pages = {1619-1630},
doi = {10.1007/s11427-020-1722-0},
pmid = {32592086},
issn = {1869-1889},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; DNA/chemistry/*metabolism ; DNA Breaks, Double-Stranded ; Deoxyribonuclease I/genetics/*metabolism ; Gene Editing ; Mutation ; RNA, Guide/*chemistry ; Streptococcus pyogenes/enzymology ; },
abstract = {The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.},
}
@article {pmid32592028,
year = {2020},
author = {Varela, C and Bartel, C and Onetto, C and Borneman, A},
title = {Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {16},
pages = {7105-7115},
doi = {10.1007/s00253-020-10750-5},
pmid = {32592028},
issn = {1432-0614},
mesh = {Alleles ; Biotechnology/*methods ; Brettanomyces/drug effects/*genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Deletion ; *Genome, Fungal ; Sulfites/pharmacology ; Transformation, Genetic ; },
abstract = {The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis. Key points • The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis. • Homozygous diploid deletions are possible with step-wise transformations. • Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.},
}
@article {pmid32591530,
year = {2020},
author = {Wei, T and Cheng, Q and Min, YL and Olson, EN and Siegwart, DJ},
title = {Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3232},
pmid = {32591530},
issn = {2041-1723},
support = {R01 HL130253/HL/NHLBI NIH HHS/United States ; P30 CA142543/CA/NCI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cations ; DNA, Neoplasm/isolation &amp; purification ; Dystrophin/genetics ; *Gene Editing ; HeLa Cells ; Humans ; Lipids/chemistry ; Mice, Inbred C57BL ; Nanoparticles/*chemistry ; Organ Specificity/*genetics ; Ribonucleoproteins/*metabolism ; },
abstract = {CRISPR-Cas9 has emerged as a powerful technology that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) to target and edit DNA. However, many therapeutic targets cannot currently be accessed due to the lack of carriers that can deliver RNPs systemically. Here, we report a generalizable methodology that allows engineering of modified lipid nanoparticles to efficiently deliver RNPs into cells and edit tissues including muscle, brain, liver, and lungs. Intravenous injection facilitated tissue-specific, multiplexed editing of six genes in mouse lungs. High carrier potency was leveraged to create organ-specific cancer models in livers and lungs of mice though facile knockout of multiple genes. The developed carriers were also able to deliver RNPs to restore dystrophin expression in DMD mice and significantly decrease serum PCSK9 level in C57BL/6 mice. Application of this generalizable strategy will facilitate broad nanoparticle development for a variety of disease targets amenable to protein delivery and precise gene correction approaches.},
}
@article {pmid32591003,
year = {2020},
author = {De Masi, C and Spitalieri, P and Murdocca, M and Novelli, G and Sangiuolo, F},
title = {Application of CRISPR/Cas9 to human-induced pluripotent stem cells: from gene editing to drug discovery.},
journal = {Human genomics},
volume = {14},
number = {1},
pages = {25},
pmid = {32591003},
issn = {1479-7364},
mesh = {Animals ; *CRISPR-Cas Systems ; Cellular Reprogramming ; *Drug Discovery ; *Gene Editing ; *Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism/transplantation ; Virus Diseases/genetics/*therapy ; },
abstract = {Human-induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 gene editing system represent two instruments of basic and translational research, which both allow to acquire deep insight about the molecular bases of many diseases but also to develop pharmacological research.This review is focused to draw up the latest technique of gene editing applied on hiPSCs, exploiting some of the genetic manipulation directed to the discovery of innovative therapeutic strategies. There are many expediencies provided by the use of hiPSCs, which can represent a disease model clinically relevant and predictive, with a great potential if associated to CRISPR/Cas9 technology, a gene editing tool powered by ease and precision never seen before.Here, we describe the possible applications of CRISPR/Cas9 to hiPSCs: from drug development to drug screening and from gene therapy to the induction of the immunological response to specific virus infection, such as HIV and SARS-Cov-2.},
}
@article {pmid32589945,
year = {2020},
author = {Lee, H and Beilhartz, GL and Kucharska, I and Raman, S and Cui, H and Lam, MHY and Liang, H and Rubinstein, JL and Schramek, D and Julien, JP and Melnyk, RA and Taipale, M},
title = {Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.},
journal = {Cell},
volume = {182},
number = {2},
pages = {345-356.e16},
pmid = {32589945},
issn = {1097-4172},
support = {RN292258 &#x2013; 366017//CIHR/Canada ; },
mesh = {Animals ; Bacterial Toxins/chemistry/*metabolism/toxicity ; Binding Sites ; CRISPR-Cas Systems/genetics ; Cell Line ; Clostridium sordellii/*metabolism ; Cryoelectron Microscopy ; Edema/pathology/prevention &amp; control ; Female ; Humans ; Lung/drug effects/pathology ; Mice ; Mice, Inbred C57BL ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins/biosynthesis/isolation &amp; purification/therapeutic use ; Semaphorins/chemistry/genetics/*metabolism ; },
abstract = {Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.},
}
@article {pmid32589698,
year = {2020},
author = {Veldman, J and Visser, L and Huberts-Kregel, M and Muller, N and Hepkema, B and van den Berg, A and Diepstra, A},
title = {Rosetting T cells in Hodgkin lymphoma are activated by immunological synapse components HLA class II and CD58.},
journal = {Blood},
volume = {136},
number = {21},
pages = {2437-2441},
pmid = {32589698},
issn = {1528-0020},
mesh = {CD2 Antigens/antagonists &amp; inhibitors ; CD4-Positive T-Lymphocytes/*immunology ; CD58 Antigens/biosynthesis/genetics/*immunology ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line, Tumor ; Coculture Techniques ; Gene Knockout Techniques ; Histocompatibility Antigens Class II/*immunology ; Hodgkin Disease/*immunology ; Humans ; Immunological Synapses/*immunology ; Lymphocyte Activation/*immunology ; Protein Interaction Mapping ; Receptors, Antigen, T-Cell/*immunology ; *Rosette Formation ; },
abstract = {A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II-matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.},
}
@article {pmid32588959,
year = {2020},
author = {Niehs, SP and Kumpfmüller, J and Dose, B and Little, RF and Ishida, K and Flórez, LV and Kaltenpoth, M and Hertweck, C},
title = {Insect-Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non-Canonical Polyketide Chain Termination.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {51},
pages = {23122-23126},
pmid = {32588959},
issn = {1521-3773},
mesh = {4-Butyrolactone/*analogs &amp; derivatives/biosynthesis/chemistry/pharmacology ; Animals ; Antifungal Agents/chemistry/metabolism/*pharmacology ; Burkholderia/*chemistry/genetics/metabolism ; Coleoptera ; Hypocreales/*drug effects ; Microbial Sensitivity Tests ; Polyketides/chemistry/metabolism/*pharmacology ; },
abstract = {Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously-overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol-derived C3 building block. The key role of an A-factor synthase (AfsA)-like offloading domain was corroborated by CRISPR-Cas-mediated gene editing, which facilitated precise excision within a PKS domain.},
}
@article {pmid32588039,
year = {2020},
author = {Sun, J and Lu, Y and Nozawa, K and Xu, Z and Morohoshi, A and Castaneda, JM and Noda, T and Miyata, H and Abbasi, F and Shawki, HH and Takahashi, S and Devlin, DJ and Yu, Z and Matzuk, RM and Garcia, TX and Matzuk, MM and Ikawa, M},
title = {CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†.},
journal = {Biology of reproduction},
volume = {103},
number = {2},
pages = {183-194},
pmid = {32588039},
issn = {1529-7268},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD095341/HD/NICHD NIH HHS/United States ; T32 GM120011/GM/NIGMS NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; T32 GM088129/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Epididymis/*metabolism ; Gene Editing ; Male ; Mice ; Phylogeny ; Reproduction/*genetics ; Sperm Motility/genetics ; Spermatogenesis/genetics ; Testis/*metabolism ; },
abstract = {Developing a safe and effective male contraceptive remains a challenge in the field of medical science. Molecules that selectively target the male reproductive tract and whose targets are indispensable for male reproductive function serve among the best candidates for a novel non-hormonal male contraceptive method. To determine the function of these genes in vivo, mutant mice carrying disrupted testis- or epididymis-enriched genes were generated by zygote microinjection or electroporation of the CRISPR/Cas9 components. Male fecundity was determined by consecutively pairing knockout males with wild-type females and comparing the fecundity of wild-type controls. Phenotypic analyses of testis appearance and weight, testis and epididymis histology, and sperm movement were further carried out to examine any potential spermatogenic or sperm maturation defect in mutant males. In this study, we uncovered 13 testis- or epididymis-enriched evolutionarily conserved genes that are individually dispensable for male fertility in mice. Owing to their dispensable nature, it is not feasible to use these targets for the development of a male contraceptive.},
}
@article {pmid32587597,
year = {2020},
author = {Brandt, KM and Gunn, H and Moretti, N and Zemetra, RS},
title = {A Streamlined Protocol for Wheat (Triticum aestivum) Protoplast Isolation and Transformation With CRISPR-Cas Ribonucleoprotein Complexes.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {769},
pmid = {32587597},
issn = {1664-462X},
abstract = {The genetic engineering method CRISPR has been touted as an efficient, inexpensive, easily used, and targeted genetic modification technology that is widely suggested as having the potential to solve many of the problems facing agriculture now and in the future. Like all new technologies, however, it is not without challenges. One of the most difficult challenges to anticipate and detect is gene targets that are inaccessible due to the chromatin state at their specific location. There is currently no way to predict this during the process of designing a sgRNA target, and the only way to detect this issue before spending time and resources on full transformations is to test the cleavage ability of the sgRNA in vivo. In wheat, this is possible using protoplast isolation and PEG transformation with Cas9 ribonucleoprotein complexes. Therefore, we have developed a streamlined protocol for testing the accessibility of sgRNA targets in wheat. The first steps involve digesting wheat leaf tissue in an enzymatic solution and then isolating viable protoplasts using filters and a sucrose gradient. The protoplasts are then transformed using Cas9 ribonucleoprotein complexes via PEG-mediated transformation. DNA is isolated from the CRISPR-Cas-edited protoplasts and PCR is performed to amplify the gene target region. The PCR product is then used to assess the editing efficiency of the chosen sgRNA using Sanger sequencing. This simplified protocol for the isolation and transformation of wheat protoplast cells using Cas9 ribonucleoprotein complexes streamlines CRISPR transformation projects by allowing for a fast and easy test of sgRNA accessibility in vivo.},
}
@article {pmid32587369,
year = {2020},
author = {Li, H and Hu, Z and Jiang, H and Pu, J and Selli, I and Qiu, J and Zhang, B and Feng, J},
title = {TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10343},
pmid = {32587369},
issn = {2045-2322},
support = {R01 NS102148/NS/NINDS NIH HHS/United States ; },
mesh = {5-Methylcytosine/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; DNA Methylation/physiology ; Epigenesis, Genetic ; Frameshift Mutation ; Gene Expression Regulation, Developmental ; Human Embryonic Stem Cells/*physiology ; Humans ; Mixed Function Oxygenases/*deficiency/genetics ; Neural Plate/*growth &amp; development ; Neurogenesis/*genetics ; Neurons/physiology ; PAX6 Transcription Factor/*genetics ; Promoter Regions, Genetic/genetics ; Proto-Oncogene Proteins/*deficiency/genetics ; SOXB1 Transcription Factors/genetics ; Teratoma/genetics/pathology ; },
abstract = {The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN[+]SOX1[+] neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.},
}
@article {pmid32587263,
year = {2020},
author = {Huang, JY and Kan, SH and Sandfeld, EK and Dalton, ND and Rangel, AD and Chan, Y and Davis-Turak, J and Neumann, J and Wang, RY},
title = {CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10321},
pmid = {32587263},
issn = {2045-2322},
support = {T32 GM008243/GM/NIGMS NIH HHS/United States ; },
mesh = {Age of Onset ; Animals ; CRISPR-Cas Systems/genetics ; Cardiomyopathy, Hypertrophic/*genetics/pathology ; Disease Models, Animal ; Female ; Gene Knock-In Techniques ; Glycogen/metabolism ; Glycogen Storage Disease Type II/complications/*genetics ; Humans ; Infant ; Male ; Mice ; Mice, Transgenic ; Muscle Weakness/*genetics/pathology/physiopathology ; Muscle, Skeletal/pathology/physiopathology ; Myocardium/pathology ; RNA, Guide/genetics ; alpha-Glucosidases/*genetics/metabolism ; },
abstract = {Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaa[c.1826] locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaa[c.1826dupA] murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.},
}
@article {pmid32587261,
year = {2020},
author = {Christidi, E and Huang, H and Shafaattalab, S and Maillet, A and Lin, E and Huang, K and Laksman, Z and Davis, MK and Tibbits, GF and Brunham, LR},
title = {Variation in RARG increases susceptibility to doxorubicin-induced cardiotoxicity in patient specific induced pluripotent stem cell-derived cardiomyocytes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10363},
pmid = {32587261},
issn = {2045-2322},
mesh = {Adult ; Aged ; Aged, 80 and over ; Antibiotics, Antineoplastic/adverse effects ; CRISPR-Cas Systems ; Cardiotoxicity/etiology/metabolism/*pathology ; Case-Control Studies ; Doxorubicin/*adverse effects ; Female ; Follow-Up Studies ; Humans ; Induced Pluripotent Stem Cells/drug effects/*pathology ; Male ; Middle Aged ; *Mutation ; Myocytes, Cardiac/drug effects/*pathology ; Neoplasms/*drug therapy/pathology ; Receptors, Retinoic Acid/antagonists &amp; inhibitors/*genetics/metabolism ; Tumor Cells, Cultured ; },
abstract = {Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DNA breaks, reactive oxygen species production, and cell death. Conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in DIC.},
}
@article {pmid32587248,
year = {2020},
author = {van der Wel, T and Hilhorst, R and den Dulk, H and van den Hooven, T and Prins, NM and Wijnakker, JAPM and Florea, BI and Lenselink, EB and van Westen, GJP and Ruijtenbeek, R and Overkleeft, HS and Kaptein, A and Barf, T and van der Stelt, M},
title = {Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3216},
pmid = {32587248},
issn = {2041-1723},
mesh = {ATP-Binding Cassette Transporters/chemistry ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Fluorescence Resonance Energy Transfer/*methods ; *Fluorescent Dyes/chemistry/metabolism ; Gene Editing ; Humans ; Macrophages/metabolism ; Mutation ; Neutrophils ; Phagocytosis ; Protein-Tyrosine Kinases/genetics/metabolism ; *Proto-Oncogene Proteins c-fes/chemistry/genetics/metabolism ; Signal Transduction ; Syk Kinase/metabolism ; },
abstract = {Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.},
}
@article {pmid32586150,
year = {2020},
author = {Carlaw, TM and Zhang, LH and Ross, CJD},
title = {CRISPR/Cas9 Editing: Sparking Discussion on Safety in Light of the Need for New Therapeutics.},
journal = {Human gene therapy},
volume = {31},
number = {15-16},
pages = {794-807},
doi = {10.1089/hum.2020.111},
pmid = {32586150},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy/*methods ; *Genome, Human ; Humans ; *Mutation ; },
abstract = {Recent advances in genome sequencing have greatly improved our ability to understand and identify the causes of genetic diseases. However, there remains an urgent need for innovative, safe, and effective treatments for these diseases. CRISPR-based genome editing systems have become important and powerful tools in the laboratory, and efforts are underway to translate these into patient therapies. Therapeutic base editing is one form of genome engineering that has gained much interest because of its simplicity, specificity, and effectiveness. Base editors are a fusion of a partially deactivated Cas9 enzyme with nickase function, together with a base-modifying enzyme. They are capable of precisely targeting and repairing a pathogenic mutation to restore the normal function of a gene, ideally without disturbing the rest of the genome. In the past year, research has identified new safety concerns of base editors and sparked new innovations to improve their safety. In this review, we provide an overview of the recent advances in the safety and effectiveness of therapeutic base editors and prime editing.},
}
@article {pmid32585766,
year = {2020},
author = {Thomsen, EA and Rovsing, AB and Anderson, MV and Due, H and Huang, J and Luo, Y and Dybkaer, K and Mikkelsen, JG},
title = {Identification of BLNK and BTK as mediators of rituximab-induced programmed cell death by CRISPR screens in GCB-subtype diffuse large B-cell lymphoma.},
journal = {Molecular oncology},
volume = {14},
number = {9},
pages = {1978-1997},
pmid = {32585766},
issn = {1878-0261},
mesh = {Adaptor Proteins, Signal Transducing/genetics/metabolism ; Agammaglobulinaemia Tyrosine Kinase/*genetics/metabolism ; Alleles ; Antigens, CD20/metabolism ; *Apoptosis/drug effects/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Germinal Center/drug effects/*pathology ; HEK293 Cells ; Humans ; Lymphoma, Large B-Cell, Diffuse/*drug therapy/*genetics ; Mutation/genetics ; Rituximab/pharmacology/*therapeutic use ; Serum/metabolism ; },
abstract = {Diffuse large B-cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R-CHOP, which consists of a cancer drug combination supplemented with the humanized CD20-targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome-wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells. Along with the CD20-encoding MS4A1 gene, we identify genes related to B-cell receptor (BCR) signaling as mediators of the intracellular signaling response to rituximab. More specifically, the B-cell linker protein (BLNK) and Bruton's tyrosine kinase (BTK) genes stand out as pivotal genes in facilitating direct rituximab-induced apoptosis through mechanisms that occur alongside complement-dependent cytotoxicity (CDC). Our findings demonstrate that rituximab triggers BCR signaling in a BLNK- and BTK-dependent manner and support the existing notion that intertwined CD20 and BCR signaling pathways in germinal center B-cell-like-subtype DLBCL lead to programmed cell death.},
}
@article {pmid32585623,
year = {2020},
author = {Yin, L and Zhao, F and Sun, H and Wang, Z and Huang, Y and Zhu, W and Xu, F and Mei, S and Liu, X and Zhang, D and Wei, L and Cen, S and Hu, S and Liang, C and Guo, F},
title = {CRISPR-Cas13a Inhibits HIV-1 Infection.},
journal = {Molecular therapy. Nucleic acids},
volume = {21},
number = {},
pages = {147-155},
pmid = {32585623},
issn = {2162-2531},
abstract = {CRISPR-Cas provides bacteria and archaea with immunity against invading phages and foreign plasmid DNA and has been successfully adapted for gene editing in a variety of species. The class 2 type VI CRISPR-Cas effector Cas13a targets and cleaves RNA, providing protection against RNA phages. Here we report the repurposing of CRISPR-Cas13a to inhibit human immunodeficiency virus type 1 (HIV-1) infection through targeting HIV-1 RNA and diminishing viral gene expression. We observed strong inhibition of HIV-1 infection by CRISPR-Cas13a in human cells. We showed that CRISPR-Cas13a not only diminishes the level of newly synthesized viral RNA, either from the transfected plasmid DNA or from the viral DNA, which is integrated into cellular DNA, but it also targets and destroys the viral RNA that enters cells within viral capsid, leading to strong inhibition of HIV-1 infection. Together, our results suggest that CRISPR-Cas13a provides a potential novel tool to treat viral diseases in humans.},
}
@article {pmid32584552,
year = {2020},
author = {Zhang, JJ and Moore, BS},
title = {Site-Directed Mutagenesis of Large Biosynthetic Gene Clusters via Oligonucleotide Recombineering and CRISPR/Cas9 Targeting.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1917-1922},
pmid = {32584552},
issn = {2161-5063},
support = {F31 AI129299/AI/NIAID NIH HHS/United States ; R01 GM085770/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *Multigene Family ; Mutagenesis, Site-Directed/*methods ; Oligonucleotides/*genetics ; Peptide Synthases/genetics ; Plasmids/genetics ; Point Mutation ; Polyketide Synthases/genetics ; *Recombination, Genetic ; Saccharomyces cerevisiae/genetics ; },
abstract = {Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to the large number and size of some genes involved in specialized metabolism, notably those encoding modular polyketide synthase and nonribosomal peptide synthetase megaproteins, it remains difficult to introduce precise genetic mutations to probe domain activity or alter chemical product formation. Here, we report the development and validation of a robust method combining oligonucleotide recombineering and CRISPR/Cas9 targeting for rapid site-directed mutagenesis of cloned pathways, which can be directly transferred to a heterologous host for expression. We rapidly generated 12 point mutations and identified several important determinants of successful mutagenesis, including the protospacer/PAM sequence and presence of regions of local homology. Our approach may be broadly applicable for researchers interested in probing natural product biosynthesis or performing pathway engineering.},
}
@article {pmid32584551,
year = {2020},
author = {Yu, J and Cho, E and Choi, YG and Jeong, YK and Na, Y and Kim, JS and Cho, SR and Woo, JS and Bae, S},
title = {Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1591-1598},
doi = {10.1021/acssynbio.0c00090},
pmid = {32584551},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Cell Adhesion Molecules, Neuronal/analysis/genetics/*metabolism ; Cell Line, Tumor ; Cell Survival/drug effects ; Chromatography, High Pressure Liquid ; Extracellular Matrix Proteins/analysis/genetics/*metabolism ; Gene Editing/*methods ; Humans ; Microscopy, Fluorescence ; Nerve Tissue Proteins/analysis/genetics/*metabolism ; Plasmids/genetics/metabolism ; RNA, Guide/metabolism ; Recombinant Proteins/analysis/biosynthesis/isolation &amp; purification/pharmacology ; Reelin Protein ; Serine Endopeptidases/analysis/genetics/*metabolism ; Tandem Mass Spectrometry ; },
abstract = {The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.},
}
@article {pmid32584145,
year = {2020},
author = {Buchman, AB and Brogan, DJ and Sun, R and Yang, T and Hsu, PD and Akbari, OS},
title = {Programmable RNA Targeting Using CasRx in Flies.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {164-176},
pmid = {32584145},
issn = {2573-1602},
support = {DP2 AI152071/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/*genetics ; Drosophila Proteins/*genetics ; Drosophila melanogaster/*genetics ; Gene Editing/*methods ; Phenotype ; RNA/*genetics ; RNA, Guide/genetics ; Ribonucleases/genetics ; },
abstract = {CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.},
}
@article {pmid32584144,
year = {2020},
author = {Khlghatyan, J and Beaulieu, JM},
title = {CRISPR-Cas9-Mediated Intersectional Knockout of Glycogen Synthase Kinase 3β in D2 Receptor-Expressing Medial Prefrontal Cortex Neurons Reveals Contributions to Emotional Regulation.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {198-210},
pmid = {32584144},
issn = {2573-1602},
support = {MOP-136916//CIHR/Canada ; },
mesh = {Affect ; Animals ; Brain/metabolism ; *CRISPR-Cas Systems ; Emotional Regulation/*physiology ; Emotions ; Glycogen Synthase Kinase 3 beta/*genetics/metabolism ; Male ; Mice ; Mice, Knockout ; Mutation ; Neurons/*metabolism ; Prefrontal Cortex/*metabolism ; Receptors, Dopamine D2/*genetics/*metabolism ; Signal Transduction ; },
abstract = {Glycogen synthase kinase 3β (GSK3β) activity is regulated by dopamine D2 receptor signaling and can be inhibited by psychoactive drugs in a D2 receptor-dependent manner. However, GSK3β is ubiquitously expressed in the brain, and D2 receptor-expressing cells are distributed as a mosaic in multiple cortical regions. This complicates the interrogation of GSK3β functions in cortical D2 cells in a circuit-defined manner using conventional animal models. We used a CRISPR-Cas9-mediated intersectional approach to achieve targeted deletion of GSK3β in D2-expressing neurons of the adult medial prefrontal cortex (mPFC). Isolation and analysis of ribosome-associated RNA specifically from mPFC D2 neurons lacking GSK3β demonstrated large-scale translatome alterations. Deletion of GSK3β in mPFC D2 neurons revealed its contribution to anxiety-related, cognitive, and social behaviors. Our results underscore the viability of an intersectional knockout approach to study functions of a ubiquitous gene in a network-defined fashion while uncovering the contribution of GSK3β expressed in mPFC D2 neurons in the regulation of behavioral dimensions related to mood and emotions. This advances our understanding of GSK3β action at a brain circuit level and can potentially lead to the development of circuit selective therapeutics.},
}
@article {pmid32584143,
year = {2020},
author = {Bothmer, A and Gareau, KW and Abdulkerim, HS and Buquicchio, F and Cohen, L and Viswanathan, R and Zuris, JA and Marco, E and Fernandez, CA and Myer, VE and Cotta-Ramusino, C},
title = {Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells.},
journal = {The CRISPR journal},
volume = {3},
number = {3},
pages = {177-187},
doi = {10.1089/crispr.2019.0074},
pmid = {32584143},
issn = {2573-1602},
mesh = {CD4-Positive T-Lymphocytes ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Human ; Humans ; In Situ Hybridization, Fluorescence ; Multifactorial Inheritance ; RNA, Guide ; Streptococcus pyogenes ; *T-Lymphocytes ; *Translocation, Genetic ; },
abstract = {Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.},
}
@article {pmid32583968,
year = {2020},
author = {Zhao, J and Li, B and Ma, J and Jin, W and Ma, X},
title = {Photoactivatable RNA N[6] -Methyladenosine Editing with CRISPR-Cas13.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {16},
number = {30},
pages = {e1907301},
doi = {10.1002/smll.201907301},
pmid = {32583968},
issn = {1613-6829},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Optogenetics ; *RNA/genetics ; },
abstract = {RNA has important and diverse biological roles, but the molecular methods to manipulate it spatiotemporally are limited. Here, an engineered photoactivatable RNA N[6] -methyladenosine (m[6] A) editing system with CRISPR-Cas13 is designed to direct specific m[6] A editing. Light-inducible heterodimerizing proteins CIBN and CRY2PHR are fused to catalytically inactive PguCas13 (dCas13) and m[6] A effectors, respectively. This system, referred to as PAMEC, enables the spatiotemporal control of m[6] A editing in response to blue light. Further optimization of this system to create a highly efficient version, known as PAMEC[R] , allows the manipulation of multiple genes robustly and simultaneously. When coupled with an upconversion nanoparticle film, the optogenetic operation window is extended from the visible range to tissue-penetrable near-infrared wavelengths, which offers an appealing avenue to remotely control RNA editing. These results show that PAMEC is a promising optogenetic platform for flexible and efficient targeting of RNA, with broad applicability for epitranscriptome engineering, imaging, and future therapeutic development.},
}
@article {pmid32583364,
year = {2020},
author = {Liu, Z and Liao, Z and Chen, Y and Han, L and Yin, Q and Xiao, H},
title = {Application of Various Delivery Methods for CRISPR/dCas9.},
journal = {Molecular biotechnology},
volume = {62},
number = {8},
pages = {355-363},
doi = {10.1007/s12033-020-00258-8},
pmid = {32583364},
issn = {1559-0305},
mesh = {Animals ; *CRISPR-Cas Systems ; Drug Carriers ; *Drug Delivery Systems ; Electroporation ; *Gene Editing ; Genetic Vectors ; Humans ; Mice ; },
abstract = {As gene-editing technology has become more and more popular in the life sciences, CRISPR has brought good news to scientific researchers because of its efficiency, convenience, and wide application. Its wide application has also promoted the development of basic scientific research, agriculture, basic medicine, and clinical treatment. However, how the CRISPR/dCas9 system is effectively delivered to the target organs or cells is still unknown. This paper briefly introduces the CRISPR/dCas9 system and then lists some common delivery methods and their characteristics.},
}
@article {pmid32583135,
year = {2020},
author = {Hashemi, A},
title = {CRISPR-Cas9/CRISPRi tools for cell factory construction in E. coli.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {36},
number = {7},
pages = {96},
doi = {10.1007/s11274-020-02872-9},
pmid = {32583135},
issn = {1573-0972},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Escherichia coli/*genetics/*metabolism ; Gene Editing/*methods ; Metabolic Engineering ; },
abstract = {The innovative CRISPR-Cas based genome editing technology provides some functionality and advantages such as the high efficiency and specificity as well as ease of handling. Both aspects of the CRISPR-Cas9 system including genetic engineering and gene regulation are advantageously applicable to the construction of microbial cell factories. As one of the most extensively used cell factories, E. coli has been engineered to produce various high value-added chemical compounds such as pharmaceuticals, biochemicals, and biofuels. Therefore, to improve the production of valuable metabolites, many investigations have been performed by focusing on CRISPR-Cas- based metabolic engineering of this host. In the current review, the biology underlying CRISPR-Cas9 system was briefly explained and then the applications of CRISPR-Cas9/CRISPRi tools were considered for cell factory construction in E. coli.},
}
@article {pmid32581653,
year = {2020},
author = {Watanabe, N and Kidokoro, M and Tanaka, M and Inoue, S and Tsuji, T and Akatuska, H and Okada, C and Iida, Y and Okada, Y and Suzuki, Y and Sato, T and Yahata, T and Hirayama, N and Nakagawa, Y and Inokuchi, S},
title = {Podoplanin is indispensable for cell motility and platelet-induced epithelial-to-mesenchymal transition-related gene expression in esophagus squamous carcinoma TE11A cells.},
journal = {Cancer cell international},
volume = {20},
number = {},
pages = {263},
pmid = {32581653},
issn = {1475-2867},
abstract = {BACKGROUND: The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood.<br><br>METHODS: Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed.<br><br>RESULTS: PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-&#x3b2;-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-&#x3b2;-induced EMT induction.<br><br>CONCLUSIONS: These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.},
}
@article {pmid32581101,
year = {2020},
author = {Hatterschide, J and Brantly, AC and Grace, M and Munger, K and White, EA},
title = {A Conserved Amino Acid in the C Terminus of Human Papillomavirus E7 Mediates Binding to PTPN14 and Repression of Epithelial Differentiation.},
journal = {Journal of virology},
volume = {94},
number = {17},
pages = {},
pmid = {32581101},
issn = {1098-5514},
support = {R01 AI148431/AI/NIAID NIH HHS/United States ; R01 CA066980/CA/NCI NIH HHS/United States ; T32 AI007324/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acids/*metabolism ; CRISPR-Cas Systems ; Calmodulin-Binding Proteins/metabolism ; Cell Differentiation ; Cell Line ; Gene Knockout Techniques ; Human papillomavirus 16 ; Humans ; Keratinocytes/metabolism/virology ; Mutation ; Papillomaviridae/*metabolism ; Papillomavirus E7 Proteins/genetics/*metabolism ; Papillomavirus Infections/*virology ; Protein Binding ; Protein Tyrosine Phosphatases, Non-Receptor/genetics/*metabolism ; Sequence Alignment ; Transcriptome ; Ubiquitin-Protein Ligases/metabolism ; },
abstract = {The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPVs bind to the host cellular nonreceptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Here, we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates the interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes, and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now, we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the life span of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte life span extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity.IMPORTANCE The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds the putative tumor suppressor PTPN14 and targets it for degradation using the ubiquitin ligase UBR4. PTPN14 binds to a C-terminal arginine highly conserved in diverse HPV E7. Our previous efforts to understand how PTPN14 degradation contributes to the carcinogenic activity of high-risk HPV E7 used variants of E7 unable to bind to UBR4. Now, by directly manipulating E7 binding to PTPN14 and using a PTPN14 knockout rescue experiment, we demonstrate that the degradation of PTPN14 is required for high-risk HPV18 E7 to extend keratinocyte life span. Our data show that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated, and the HPV life cycle depends upon keratinocyte differentiation. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for HPV-mediated carcinogenesis and the HPV life cycle.},
}
@article {pmid32581075,
year = {2020},
author = {Mosterd, C and Moineau, S},
title = {Characterization of a Type II-A CRISPR-Cas System in Streptococcus mutans.},
journal = {mSphere},
volume = {5},
number = {3},
pages = {},
pmid = {32581075},
issn = {2379-5042},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Plasmids/genetics ; Streptococcus Phages/genetics/metabolism ; Streptococcus mutans/*genetics/virology ; },
abstract = {Streptococcus mutans and its virulent phages are important members of the human oral microbiota. S. mutans is also the primary causal agent of dental caries. To survive in this ecological niche, S. mutans must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of S. mutans strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5' end of the CRISPR locus and ectopically. In comparisons of the cas genes of P42S to those of other strains of S. mutans, cas1, cas2, and csn2 appear to be highly conserved within the species. However, more diversity was observed with cas9 While the nuclease domains of S. mutans Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain S. mutans UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species.IMPORTANCE CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, Streptococcus mutans strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.},
}
@article {pmid32580834,
year = {2020},
author = {Shin, J and Oh, JW},
title = {Development of CRISPR/Cas9 system for targeted DNA modifications and recent improvements in modification efficiency and specificity.},
journal = {BMB reports},
volume = {53},
number = {7},
pages = {341-348},
pmid = {32580834},
issn = {1976-670X},
mesh = {CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; Endonucleases/genetics ; Gene Editing/methods ; Genetic Engineering/history/*methods ; Genetic Therapy/methods ; History, 20th Century ; History, 21st Century ; },
abstract = {The targeted nuclease clustered, regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) system has recently emerged as a prominent gene manipulation method. Because of its ease in programming targeted DNA/protein binding through RNA in a vast range of organisms, this prokaryotic defense system is a versatile tool with many applications in the research field as well as high potential in agricultural and clinical improvements. This review will present a brief history that led to its discovery and adaptation. We also present some of its restrictions, and modifications that have been performed to overcome such restrictions, focusing specifically on the most common CRISPR/Cas9 mediated non-homologous end joint repair. [BMB Reports 2020; 53(7): 341-348].},
}
@article {pmid32580383,
year = {2020},
author = {Hao, W and Wang, L and Li, S},
title = {FKBP5 Regulates RIG-I-Mediated NF-κB Activation and Influenza A Virus Infection.},
journal = {Viruses},
volume = {12},
number = {6},
pages = {},
pmid = {32580383},
issn = {1999-4915},
support = {R15 AI126360/AI/NIAID NIH HHS/United States ; R01 AI141399/AI/NIAID NIH HHS/United States ; R21 AI137750/AI/NIAID NIH HHS/United States ; },
mesh = {A549 Cells ; CRISPR-Cas Systems/genetics ; Cell Line ; DEAD Box Protein 58/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Immunity, Innate/immunology ; Influenza A virus/*immunology ; Influenza, Human/*immunology/pathology ; NF-KappaB Inhibitor alpha/*metabolism ; NF-kappa B/metabolism ; Receptors, Immunologic ; Tacrolimus Binding Proteins/genetics/*metabolism ; },
abstract = {Influenza A virus (IAV) is a highly transmissible respiratory pathogen and is a constant threat to global health with considerable economic and social impact. Influenza viral RNA is sensed by host pattern recognition receptors (PRRs), such as the Toll-like receptor 7 (TLR7) and retinoic acid-inducible gene I (RIG-I). The activation of these PRRs instigates the interferon regulatory factor (IRF) and nuclear factor kappa B (NF-κB) signaling pathways that induce the expression of interferon-stimulated genes (ISGs) and inflammatory genes. FK506-binding protein 5 (FKBP5) has been implied in the IκBα kinase (IKK) complex. However, the role of FKBP5 in the RIG-I signaling and IAV infection is not well elucidated. Here, we demonstrate that the knockout of FKBP5 increases IAV infection. Furthermore, FKBP5 binds IKKα, which is critical for RIG-I-induced innate immune responses and ISG expression. Taken together, FKBP5 is a novel anti-influenza host factor that restricts IAV infection by the activation of RIG-I-mediated NF-κB signaling.},
}
@article {pmid32580029,
year = {2020},
author = {Sorial, AK and Hofer, IMJ and Tselepi, M and Cheung, K and Parker, E and Deehan, DJ and Rice, SJ and Loughlin, J},
title = {Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC.},
journal = {Osteoarthritis and cartilage},
volume = {28},
number = {11},
pages = {1448-1458},
pmid = {32580029},
issn = {1522-9653},
support = {MR/P020941/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adipose Tissue/*metabolism ; Adult ; Aged ; Aged, 80 and over ; Arthroplasty, Replacement ; CRISPR-Cas Systems ; Cartilage, Articular/*metabolism ; Cell Line ; Chondrocytes/*metabolism ; CpG Islands ; DNA Methylation ; Epigenesis, Genetic ; Female ; Gene Expression ; Gene Knockdown Techniques ; Genetic Predisposition to Disease ; Glycosaminoglycans/biosynthesis ; Humans ; Male ; Mesenchymal Stem Cells/*metabolism ; Middle Aged ; Osteoarthritis, Hip/blood/*genetics/metabolism/surgery ; Osteoarthritis, Knee/blood/*genetics/metabolism/surgery ; Plectin/blood/*genetics/metabolism ; Quantitative Trait Loci ; Sequence Analysis, RNA ; Synovial Membrane/*metabolism ; Wnt Signaling Pathway/genetics ; },
abstract = {OBJECTIVE: In cartilage, the osteoarthritis (OA) associated single nucleotide polymorphism (SNP) rs11780978 correlates with differential expression of PLEC, and with differential methylation of PLEC CpG dinucleotides, forming eQTLs and mQTLs respectively. This implies that methylation links chondrocyte genotype and phenotype, thus driving the functional effect of this genetic risk signal. PLEC encodes plectin, a cytoskeletal protein that enables tissues to respond to mechanical forces. We sought to assess whether these PLEC functional effects were cartilage specific.<br><br>METHOD: Cartilage, fat pad, synovium and peripheral blood were collected from patients undergoing arthroplasty. PLEC CpGs were analysed for mQTLs and allelic expression imbalance (AEI) was performed to test for eQTLs. Plectin was knocked down in a mesenchymal stem cell (MSC) line using CRISPR/Cas9 and cells phenotyped by RNA-sequencing.<br><br>RESULTS: mQTLs were discovered in fat pad, synovium and blood. Their effects were however stronger in the joint tissues and of comparable effect between these tissues. We observed AEI in synovium in the same direction as for cartilage and correlations between methylation and PLEC expression. Knocking-down plectin impacted on pathways reported to have a role in OA, including Wnt signalling, glycosaminoglycan biosynthesis and immune regulation.<br><br>CONCLUSIONS: Synovium is also a target of the rs11780978 OA association functionally operating on PLEC. In fat pad, mQTLs were identified but these did not correlate with PLEC expression, suggesting the functional effect is not joint-wide. Our study highlights interplay between genetic risk, DNA methylation and gene expression in OA, and reveals clear differences between tissues from the same diseased joint.},
}
@article {pmid32579898,
year = {2020},
author = {Vergara-Mendoza, M and Gomez-Quiroz, LE and Miranda-Labra, RU and Fuentes-Romero, LL and Romero-Rodríguez, DP and González-Ruiz, J and Hernández-Rizo, S and Viveros-Rogel, M},
title = {Regulation of Cas9 by viral proteins Tat and Rev for HIV-1 inactivation.},
journal = {Antiviral research},
volume = {180},
number = {},
pages = {104856},
doi = {10.1016/j.antiviral.2020.104856},
pmid = {32579898},
issn = {1872-9096},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Viral ; HEK293 Cells ; HIV-1/genetics ; Humans ; RNA, Guide/genetics ; *Virus Inactivation ; Virus Replication/genetics ; rev Gene Products, Human Immunodeficiency Virus/*genetics ; tat Gene Products, Human Immunodeficiency Virus/*genetics ; },
abstract = {While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells. In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.},
}
@article {pmid32579048,
year = {2020},
author = {Shariati, K and Pappalardo, Z and Chopra, DG and Yiv, N and Sheen, R and Ku, G},
title = {Selective monitoring of insulin secretion after CRISPR interference in intact pancreatic islets despite submaximal infection.},
journal = {Islets},
volume = {12},
number = {3},
pages = {59-69},
pmid = {32579048},
issn = {1938-2022},
support = {K08 DK087945/DK/NIDDK NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; R01 DK107650/DK/NIDDK NIH HHS/United States ; R01 DK118337/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Bodily Secretions ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Knockdown Techniques ; Insulin/*metabolism ; Islets of Langerhans/*metabolism ; Mice ; RNA Interference ; },
abstract = {Virus-mediated gene knockdown in intact pancreatic islets is technically challenging due to poor infection of the center of the islet. Because the cells that do not have knockdown have normal insulin secretion, measuring changes in insulin secretion after gene knockdown is challenging. We describe a method to monitor insulin secretion from only the beta cells with knockdown of a gene of interest in intact islets using a single lentivirus containing a guide RNA, a luciferase insulin secretion reporter and a dCas9-KRAB cassette. This method allows rapid and inexpensive monitoring of insulin secretion from only those beta cells with knockdown, circumventing the problem of incomplete islet infection.},
}
@article {pmid32577410,
year = {2020},
author = {Kanitchinda, S and Srisala, J and Suebsing, R and Prachumwat, A and Chaijarasphong, T},
title = {CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei.},
journal = {Biotechnology reports (Amsterdam, Netherlands)},
volume = {27},
number = {},
pages = {e00485},
pmid = {32577410},
issn = {2215-017X},
abstract = {Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.},
}
@article {pmid32576837,
year = {2020},
author = {Lamsfus-Calle, A and Daniel-Moreno, A and Antony, JS and Epting, T and Heumos, L and Baskaran, P and Admard, J and Casadei, N and Latifi, N and Siegmund, DM and Kormann, MSD and Handgretinger, R and Mezger, M},
title = {Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34[+] HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10133},
pmid = {32576837},
issn = {2045-2322},
mesh = {Anemia, Sickle Cell/*genetics/therapy ; Antigens, CD34 ; *CRISPR-Cas Systems ; Cells, Cultured ; Fetal Hemoglobin/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Humans ; Kruppel-Like Transcription Factors/*genetics ; Molecular Targeted Therapy ; Mutation ; Repressor Proteins/*genetics ; gamma-Globins/*genetics ; },
abstract = {β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.},
}
@article {pmid32576161,
year = {2020},
author = {Gratacap, RL and Regan, T and Dehler, CE and Martin, SAM and Boudinot, P and Collet, B and Houston, RD},
title = {Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {35},
pmid = {32576161},
issn = {1472-6750},
support = {BB/R008612/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S004343/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/20002172/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/30002275/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002070/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R008973/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Survival ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Disease Resistance/genetics ; Gene Editing/*methods ; Genome ; Lentivirus/*genetics ; Salmonidae/*genetics ; },
abstract = {BACKGROUND: Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques.<br><br>RESULTS: In the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated.<br><br>CONCLUSIONS: The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.},
}
@article {pmid32575125,
year = {2020},
author = {Zeng, Z and Han, N and Liu, C and Buerte, B and Zhou, C and Chen, J and Wang, M and Zhang, Y and Tang, Y and Zhu, M and Wang, J and Yang, Y and Bian, H},
title = {Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing.},
journal = {Annals of botany},
volume = {126},
number = {5},
pages = {929-942},
pmid = {32575125},
issn = {1095-8290},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Hordeum/genetics ; Humans ; Tocopherols ; *Tocotrienols ; Vitamin E ; },
abstract = {BACKGROUND AND AIMS: Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley.<br><br>METHODS: Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants.<br><br>KEY RESULTS: Mutagenesis efficiency among T0 regenerated plantlets was 50-65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of &#x3b3;- and &#x3b4;-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production.<br><br>CONCLUSIONS: Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.},
}
@article {pmid32574856,
year = {2020},
author = {Barone, P and Wu, E and Lenderts, B and Anand, A and Gordon-Kamm, W and Svitashev, S and Kumar, S},
title = {Efficient Gene Targeting in Maize Using Inducible CRISPR-Cas9 and Marker-free Donor Template.},
journal = {Molecular plant},
volume = {13},
number = {8},
pages = {1219-1227},
doi = {10.1016/j.molp.2020.06.008},
pmid = {32574856},
issn = {1752-9867},
mesh = {*CRISPR-Cas Systems ; Gene Targeting/*methods ; Genetic Markers ; Kanamycin Kinase/genetics ; Mutagenesis, Insertional ; Zea mays/*genetics ; },
abstract = {CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here, we report high-frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate being observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports in which the original chimeric GT plants required extensive progeny screening in the next generation to identify non-chimeric GT individuals, our method provides non-chimeric heritable GT in one generation.},
}
@article {pmid32573804,
year = {2020},
author = {Petkova, MV and Stantzou, A and Morin, A and Petrova, O and Morales-Gonzalez, S and Seifert, F and Bellec-Dyevre, J and Manoliu, T and Goyenvalle, A and Garcia, L and Richard, I and Laplace-Builhé, C and Schuelke, M and Amthor, H},
title = {Live-imaging of revertant and therapeutically restored dystrophin in the Dmd[EGFP-mdx] mouse model for Duchenne muscular dystrophy.},
journal = {Neuropathology and applied neurobiology},
volume = {46},
number = {6},
pages = {602-614},
doi = {10.1111/nan.12639},
pmid = {32573804},
issn = {1365-2990},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Dystrophin/*genetics ; Exons ; Gene Editing ; Genetic Therapy ; Humans ; Mice ; Mice, Inbred mdx ; Mice, Transgenic ; Muscular Dystrophy, Duchenne/*genetics/therapy ; Retina/metabolism ; Sarcolemma/metabolism/ultrastructure ; },
abstract = {BACKGROUND: Dmd[mdx] , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies.<br><br>METHODS: We generated a Dmd[EGFP-mdx] reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision.<br><br>RESULTS: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation.<br><br>CONCLUSION: Intravital imaging of Dmd[EGFP-mdx] muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.},
}
@article {pmid32573700,
year = {2020},
author = {Prokoph, N and Probst, NA and Lee, LC and Monahan, JM and Matthews, JD and Liang, HC and Bahnsen, K and Montes-Mojarro, IA and Karaca-Atabay, E and Sharma, GG and Malik, V and Larose, H and Forde, SD and Ducray, SP and Lobello, C and Wang, Q and Luan, SL and Pospíšilová, &#x160; and Gambacorti-Passerini, C and Burke, GAA and Pervez, S and Attarbaschi, A and Janíková, A and Pacquement, H and Landman-Parker, J and Lambilliotte, A and Schleiermacher, G and Klapper, W and Jauch, R and Woessmann, W and Vassal, G and Kenner, L and Merkel, O and Mologni, L and Chiarle, R and Brugières, L and Geoerger, B and Barbieri, I and Turner, SD},
title = {IL10RA modulates crizotinib sensitivity in NPM1-ALK+ anaplastic large cell lymphoma.},
journal = {Blood},
volume = {136},
number = {14},
pages = {1657-1669},
pmid = {32573700},
issn = {1528-0020},
support = {R01 CA196703/CA/NCI NIH HHS/United States ; RG86786//Cancer Research UK/United Kingdom ; },
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line ; Crizotinib/*pharmacology/therapeutic use ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/*genetics ; Gene Editing ; Gene Expression ; Humans ; Immunohistochemistry ; Interleukin-10 Receptor alpha Subunit/*genetics/metabolism ; Lymphoma, Large-Cell, Anaplastic/drug therapy/*genetics/metabolism/pathology ; Models, Biological ; Nucleophosmin ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Protein-Tyrosine Kinases/*genetics/metabolism ; STAT3 Transcription Factor/metabolism ; Signal Transduction/drug effects ; },
abstract = {Anaplastic large cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive anaplastic lymphoma kinase (ALK) fusion protein. ALK inhibitors, such as crizotinib, provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by nucleophosmin 1 (NPM1)-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54% to 90% in clinical trials; however, a subset of patients progressed within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) activation and knockout screens in ALCL cell lines, combined with RNA sequencing data derived from ALK inhibitor-relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of interleukin 10 receptor subunit alpha (IL10RA). Elevated IL10RA expression rewires the STAT3 signaling pathway, bypassing otherwise critical phosphorylation by NPM1-ALK. IL-10RA expression does not correlate with response to standard chemotherapy in pediatric patients, suggesting that a combination of crizotinib and chemotherapy could prevent ALK inhibitor resistance-specific relapse.},
}
@article {pmid32573207,
year = {2020},
author = {Feng, W and Newbigging, AM and Le, C and Pang, B and Peng, H and Cao, Y and Wu, J and Abbas, G and Song, J and Wang, DB and Cui, M and Tao, J and Tyrrell, DL and Zhang, XE and Zhang, H and Le, XC},
title = {Molecular Diagnosis of COVID-19: Challenges and Research Needs.},
journal = {Analytical chemistry},
volume = {92},
number = {15},
pages = {10196-10209},
pmid = {32573207},
issn = {1520-6882},
mesh = {Betacoronavirus/chemistry/*isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques ; Coronavirus Infections/*diagnosis ; False Negative Reactions ; High-Throughput Nucleotide Sequencing ; Humans ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; Pandemics ; Pneumonia, Viral/*diagnosis ; Point-of-Care Testing ; RNA, Viral/*analysis ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2 ; Specimen Handling/methods ; Viral Load ; Viral Proteins/analysis ; Waste Water/analysis ; },
abstract = {Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.},
}
@article {pmid32572269,
year = {2020},
author = {Anzalone, AV and Koblan, LW and Liu, DR},
title = {Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {824-844},
pmid = {32572269},
issn = {1546-1696},
support = {U01 AI142756/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Endonucleases/genetics ; *Gene Editing/methods ; Genome/*genetics ; Humans ; Recombinases/genetics ; Transposases/genetics ; },
abstract = {The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.},
}
@article {pmid32572256,
year = {2020},
author = {Gretarsson, KH and Hackett, JA},
title = {Dppa2 and Dppa4 counteract de novo methylation to establish a permissive epigenome for development.},
journal = {Nature structural &amp; molecular biology},
volume = {27},
number = {8},
pages = {706-716},
pmid = {32572256},
issn = {1545-9985},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *DNA Methylation ; Epigenome ; Gene Expression Regulation, Developmental ; Long Interspersed Nucleotide Elements ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Nuclear Proteins/*genetics/metabolism ; Single-Cell Analysis ; Transcription Factors/*genetics/metabolism ; },
abstract = {Early mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation dynamics, we coupled a single-cell ratiometric DNA methylation reporter with unbiased CRISPR screening in murine embryonic stem cells (ESCs). We identify key genes and regulatory pathways that drive global DNA hypomethylation, and characterize roles for Cop1 and Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their absence, developmental genes and evolutionarily young LINE1 elements, which are specifically bound by DPPA2, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, lineage-associated genes and LINE1 acquire a repressive epigenetic memory, which renders them incompetent for activation during future lineage specification. Dppa2/4 thereby sculpt the pluripotent epigenome by facilitating H3K4me3 and bivalency to counteract de novo methylation, a function co-opted by evolutionarily young LINE1 to evade epigenetic decommissioning.},
}
@article {pmid32572238,
year = {2020},
author = {Choi, J and Diao, H and Faliti, CE and Truong, J and Rossi, M and Bélanger, S and Yu, B and Goldrath, AW and Pipkin, ME and Crotty, S},
title = {Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.},
journal = {Nature immunology},
volume = {21},
number = {7},
pages = {777-789},
pmid = {32572238},
issn = {1529-2916},
support = {P30 CA023100/CA/NCI NIH HHS/United States ; R01 AI072543/AI/NIAID NIH HHS/United States ; S10 RR027366/RR/NCRR NIH HHS/United States ; U19 AI109976/AI/NIAID NIH HHS/United States ; },
mesh = {Adoptive Transfer ; Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics/immunology ; Cell Line ; Cell Movement/genetics/immunology ; Chromatin Immunoprecipitation Sequencing ; Cytokines/immunology/metabolism ; Female ; Gene Expression Regulation/*immunology ; Gene Regulatory Networks ; Germinal Center/cytology/*immunology ; Humans ; Male ; Mice ; Mutation ; Promoter Regions, Genetic/genetics ; Proto-Oncogene Proteins c-bcl-6/genetics/*metabolism ; RNA-Seq ; Repressor Proteins/*genetics/metabolism ; Signal Transduction/genetics/immunology ; T-Lymphocytes, Helper-Inducer/*immunology/metabolism ; },
abstract = {T follicular helper (TFH) cells are a distinct type of CD4[+] T cells that are essential for most antibody and B lymphocyte responses. TFH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl-6 controls TFH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits.},
}
@article {pmid32571941,
year = {2020},
author = {Ravindran, S},
title = {QnAs with Zachary B. Lippman.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {26},
pages = {14624-14625},
pmid = {32571941},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Epistasis, Genetic ; *Gene Editing ; *Genetics ; Genome, Plant/*genetics ; Humans ; Lycopersicon esculentum/genetics ; },
}
@article {pmid32570971,
year = {2020},
author = {Sánchez-Hernández, S and Aguilar-González, A and Guijarro-Albaladejo, B and Maldonado-Pérez, N and Ramos-Hernández, I and Cortijo-Gutiérrez, M and Sánchez Martín, RM and Benabdellah, K and Martin, F},
title = {Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System.},
journal = {Cells},
volume = {9},
number = {6},
pages = {},
pmid = {32570971},
issn = {2073-4409},
mesh = {*CRISPR-Cas Systems ; DNA, Recombinant/genetics ; Gene Editing/*methods ; Gene Targeting/adverse effects/methods ; Genes, Reporter ; Genetic Engineering ; Genetic Therapy/adverse effects/methods ; Genetic Vectors ; *Homologous Recombination ; Humans ; K562 Cells ; Lentivirus/genetics ; *Models, Genetic ; Wiskott-Aldrich Syndrome Protein/genetics ; },
abstract = {In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.},
}
@article {pmid32570896,
year = {2020},
author = {Pourcel, C and Midoux, C and Vergnaud, G and Latino, L},
title = {The Basis for Natural Multiresistance to Phage in Pseudomonas aeruginosa.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {9},
number = {6},
pages = {},
pmid = {32570896},
issn = {2079-6382},
abstract = {Pseudomonas aeruginosa is responsible for long-term infections and is particularly resistant to treatments when hiding inside the extracellular matrix or biofilms. Phage therapy might represent an alternative to antibiotic treatment, but up to 10% of clinical strains appear to resist multiple phages. We investigated the characteristics of P. aeruginosa clinical strains naturally resistant to phages and compared them to highly susceptible strains. The phage-resistant strains were defective in lipopolysaccharide (LPS) biosynthesis, were nonmotile and displayed an important degree of autolysis, releasing phages and pyocins. Complete genome sequencing of three resistant strains showed the existence of a large accessory genome made of multiple insertion elements, genomic islands, pyocins and prophages, including two phages performing lateral transduction. Mutations were found in genes responsible for the synthesis of LPS and/or type IV pilus, the major receptors for most phages. CRISPR-Cas systems appeared to be absent or inactive in phage-resistant strains, confirming that they do not play a role in the resistance to lytic phages but control the insertion of exogenous sequences. We show that, despite their apparent weakness, the multiphage-resistant strains described in this study displayed selective advantages through the possession of various functions, including weapons to eliminate other strains of the same or closely related species.},
}
@article {pmid32570107,
year = {2020},
author = {Sasnauskas, G and Siksnys, V},
title = {CRISPR adaptation from a structural perspective.},
journal = {Current opinion in structural biology},
volume = {65},
number = {},
pages = {17-25},
doi = {10.1016/j.sbi.2020.05.015},
pmid = {32570107},
issn = {1879-033X},
mesh = {*Bacterial Proteins/metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; DNA Viruses/*metabolism ; Escherichia coli/*genetics/virology ; Host Microbial Interactions ; Protein Binding ; *Viruses/genetics ; },
abstract = {Bacterial CRISPR-Cas systems provide adaptive immunity against viruses and other mobile genome elements. During the adaptation step cells become immunized by insertion of short fragments of foreign DNA, termed spacers, into the genomic region called a CRISPR array. Selection, processing and insertion of new spacers is an elaborate and precisely orchestrated reaction, which relies on the Cas1-Cas2 integrase complex and accessory proteins that vary among different types of CRISPR-Cas systems. This review focuses on CRISPR adaptation from the structural perspective, with the spotlight on adaptation proteins employed by type I and type II CRISPR-Cas systems.},
}
@article {pmid32569867,
year = {2020},
author = {Yang, C and Fu, Y and Huang, C and Hu, D and Zhou, K and Hao, Y and Chu, B and Yang, Y and Qian, Z},
title = {Chlorin e6 and CRISPR-Cas9 dual-loading system with deep penetration for a synergistic tumoral photodynamic-immunotherapy.},
journal = {Biomaterials},
volume = {255},
number = {},
pages = {120194},
doi = {10.1016/j.biomaterials.2020.120194},
pmid = {32569867},
issn = {1878-5905},
mesh = {Animals ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chlorophyllides ; Immunotherapy ; *Nanoparticles ; *Photochemotherapy ; *Porphyrins ; },
abstract = {Photodynamic therapy (PDT) is a relatively safe and clinically promising treatment to combat primary tumors, especially epidermal carcinoma, while has negligible effects on distant metastasis. Therefore, this work reports a multifunctional nanosystem (HPR@CCP) exerting a combined photodynamic and immunotherapy to amplify the therapeutic effect on primary tumors and distant metastasis. Specifically, this nanosystem was obtained by electrostatic adsorption of a negatively charged hyaluronic acid "shell" with a positively charged "core" consisting of the CRISPR-Cas9 system targeting the Ptpn2 gene (Cas9-Ptpn2) and a modified mitochondria-targeting chlorin e6 (TPP-PEI-Ce6). Cell experiments demonstrated that the HPR@CCP nanoparticles possessed very high transfection efficiency on B16F10 cells, and TPP-PEI-Ce6 in the nanoparticles resulted in a significant PDT efficacy due to the efficient singlet oxygen generation in mitochondria under laser-irradiation. The accumulation of the nanoparticles in the tumor by active and passive tumor-targeting in vivo led to the disruption of the Ptpn2 gene by the Cas9-Ptpn2 plasmids in the nanocarriers, thus sensitizing tumors to immunotherapy by the increase of the IFN-γ and TNF-α signaling and the promotion of the proliferation of CD8[+] T cells. In addition, Hyaluronidase was administered in advance to destroy the hyaluronic acid in the condensed extracellular matrix and to remove the hyaluronic acid "shell" from the nanosystem, subsequently leading to an enhanced penetration of oxygen and therapeutic agents. Fortunately, the primary and distant tumors in the experimental animals were remarkably inhibited after the combination of PDT-immunotherapy, thus, this easy-to-built nanomedicine could be used as a potential combination therapy against tumors.},
}
@article {pmid32569517,
year = {2020},
author = {Zhang, W and Mitchell, LA and Bader, JS and Boeke, JD},
title = {Synthetic Genomes.},
journal = {Annual review of biochemistry},
volume = {89},
number = {},
pages = {77-101},
doi = {10.1146/annurev-biochem-013118-110704},
pmid = {32569517},
issn = {1545-4509},
support = {RM1 HG009491/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA/chemistry/*genetics/metabolism ; Escherichia coli/genetics/metabolism ; Gene Editing/*methods ; *Gene Transfer Techniques ; *Genes, Synthetic ; Genetic Engineering/*methods ; *Genome ; Humans ; Oligonucleotides/chemical synthesis/metabolism ; Plasmids/chemistry/metabolism ; Poliovirus/genetics/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Spheroplasts/genetics/metabolism ; },
abstract = {DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.},
}
@article {pmid32568829,
year = {2020},
author = {Zhang, L and Wu, F and Zhao, J},
title = {Transmembrane protein 45A regulates the proliferation, migration, and invasion of glioma cells through nuclear factor kappa-B.},
journal = {Anti-cancer drugs},
volume = {31},
number = {9},
pages = {900-907},
doi = {10.1097/CAD.0000000000000890},
pmid = {32568829},
issn = {1473-5741},
mesh = {Aged ; Brain Neoplasms/*metabolism/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/physiology ; Female ; Gene Knockout Techniques ; Glioma/genetics/*metabolism/*pathology ; Humans ; Male ; Membrane Proteins/biosynthesis/genetics/*metabolism ; Middle Aged ; NF-kappa B/*metabolism ; Prognosis ; },
abstract = {Gliomas are the most common and aggressive type of primary brain cancer in adults. The expression of transmembrane protein 45A (TMEM45A) in glioma patients and glioma cell lines was analyzed by quantitative real-time PCR. The influence of TMEM45A on the survival of glioma patients was also explored in this study. To verify the interaction between TMEM45A and key genes, correlation analysis of expression levels and the siRNA knock down method were performed. TMEM45A was upregulated in glioma tissues, and its overexpression was strongly correlated with the poor survival of glioma patients. Experiments using the overexpression and knock down of TMEM45A were carried out to demonstrate its correlation with enhanced proliferation, migration, and invasion in glioma cells. Nuclear factor kappa-B (NFκB) expression was shown to be a downstream factor of TMEM45A in glioma cells. In conclusion, TMEM45A is an oncogenic gene in glioma. The proliferation, migration, and invasion of gliomas could be effectively impeded by inhibition of TMEM45A, and the cancer-promoting effect of TMEM45A on gliomas was involved with the NFκB pathway.},
}
@article {pmid32568240,
year = {2020},
author = {Sharma, S and Wright, GJ},
title = {Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {160},
pages = {},
doi = {10.3791/60803},
pmid = {32568240},
issn = {1940-087X},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Genetic Testing/*methods ; Genome/*genetics ; Humans ; Receptors, Cell Surface/*metabolism ; Transfection ; },
abstract = {Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.},
}
@article {pmid32567999,
year = {2020},
author = {Gianni, D and Farrow, S},
title = {Functional Genomics for Target Identification.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {6},
pages = {531-534},
doi = {10.1177/2472555220927692},
pmid = {32567999},
issn = {2472-5560},
mesh = {CRISPR-Cas Systems/*genetics ; Genomics/*trends ; Humans ; RNA Interference ; RNA, Small Interfering/*genetics ; },
}
@article {pmid32567268,
year = {2020},
author = {Ma, B and Shen, W and Wang, X and Li, Z and Xu, K},
title = {[Gene edited animal models applied in human disease research].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {5},
pages = {849-860},
doi = {10.13345/j.cjb.190395},
pmid = {32567268},
issn = {1872-2075},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Disease Models, Animal ; *Gene Editing ; Humans ; },
abstract = {Recently, with the development and the continuous improvement of various CRISPR systems represented by CRISPR/Cas9, gene editing technology has been gradually improved, and widely applied to the preparation of animal models of human diseases. The gene edited animal models provide important materials for the study of pathogenesis, pathological process, prevention and treatment of human diseases. At present, the gene edited animal models used in human disease research include mainly the rodent models represented by mice and rats, and large animal models represented by pigs. Among them, rodents differ greatly from humans in all aspects of their bodies and have short life span as well, which cannot provide effective evaluation and long-term tracking for the research and treatment of human diseases. On the other hand, pig is closer to human in physiology, anatomy, nutrition and genetics, which provides an important animal model in the field of organ transplantation and human disease research. In this paper, the application of the gene edited animal models was summarized in the researches of 5 human diseases such as neurodegenerative diseases, familial hypertrophic cardiomyopathy, cancer, immunodeficiency diseases and metabolic diseases. We hope this paper will provide a reference for the research of human diseases and the preparation of relative animal models.},
}
@article {pmid32567265,
year = {2020},
author = {Yang, J and Ma, X and Wang, X and Zhang, Z and Wang, S and Qin, H and Mao, S and Lu, F},
title = {[Advances in gene editing of Corynebacterium glutamate].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {5},
pages = {820-828},
doi = {10.13345/j.cjb.190403},
pmid = {32567265},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Corynebacterium glutamicum/genetics ; *Gene Editing/trends ; Glutamic Acid ; *Industrial Microbiology/trends ; },
abstract = {Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.},
}
@article {pmid32567100,
year = {2020},
author = {Leandro, J and Dodatko, T and DeVita, RJ and Chen, H and Stauffer, B and Yu, C and Houten, SM},
title = {Deletion of 2-aminoadipic semialdehyde synthase limits metabolite accumulation in cell and mouse models for glutaric aciduria type 1.},
journal = {Journal of inherited metabolic disease},
volume = {43},
number = {6},
pages = {1154-1164},
doi = {10.1002/jimd.12276},
pmid = {32567100},
issn = {1573-2665},
mesh = {2-Aminoadipic Acid/*analogs &amp; derivatives/genetics/metabolism ; Amino Acid Metabolism, Inborn Errors/genetics/*metabolism/therapy ; Animals ; Brain/*metabolism/pathology ; Brain Diseases, Metabolic/genetics/*metabolism/therapy ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Glutarates/*metabolism ; Glutaryl-CoA Dehydrogenase/*deficiency/genetics/metabolism ; HEK293 Cells ; Humans ; Liver/*metabolism/pathology ; Male ; Mice ; Mice, Knockout ; },
abstract = {Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by acute encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. We investigated the efficacy of substrate reduction through inhibition of 2-aminoadipic semialdehyde synthase (AASS), an enzyme upstream of the defective glutaryl-CoA dehydrogenase (GCDH), in a cell line and mouse model of GA1. We show that loss of AASS function in GCDH-deficient HEK-293 cells leads to an approximately fivefold reduction in the established GA1 clinical biomarker glutarylcarnitine. In the GA1 mouse model, deletion of Aass leads to a 4.3-, 3.8-, and 3.2-fold decrease in the glutaric acid levels in urine, brain, and liver, respectively. Parallel decreases were observed in urine and brain 3-hydroxyglutaric acid levels, and plasma, urine, and brain glutarylcarnitine levels. These in vivo data demonstrate that the saccharopine pathway is the main source of glutaric acid production in the brain and periphery of a mouse model for GA1, and support the notion that pharmacological inhibition of AASS may represent an attractive strategy to treat GA1.},
}
@article {pmid32567018,
year = {2022},
author = {Liao, JQ and Zhou, G and Zhou, Y},
title = {Generation of Monoclonal iPSC Lines with Stable Cas9 Expression and High Cas9 Activity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2454},
number = {},
pages = {575-588},
pmid = {32567018},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; Gene Editing/methods ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {CRISPR/Cas9-mediated gene editing has been rapidly and widely applied in many organisms for delicate genetic manipulation, including human-induced pluripotent stem cells (iPSCs). Gene editing in human iPSCs is promising for genetics and biomedical research due to that gene-edited iPSC still possesses the potential to be differentiated into any cell lineages. In many cases, the generation of Cas9 expressing cell lines is a prerequisite toward performing successful editing of multiplex genes of interest. Here, we describe a simple, effective method to generate stable Cas9 expressing human iPSCs with high Cas9 activity. In this method, stable Cas9 expressing monoclonal human iPSC lines were generated through lentiviral transduction of Cas9 cassette, followed by blasticidin selection and subcloning with low seeding density. After colonies isolation and expansion, a BFP-GFP reporter assay was applied to validate the Cas9 activities of multiple monoclonal lines by flow cytometry (FACS). These Cas9 expressing human iPSCs generated by our method are single cell-derived monoclonal lines with homogenous population and Cas9 activity of up to 99%.},
}
@article {pmid32566443,
year = {2020},
author = {Yarra, R and Cao, H and Jin, L and Mengdi, Y and Zhou, L},
title = {CRISPR/Cas mediated base editing: a practical approach for genome editing in oil palm.},
journal = {3 Biotech},
volume = {10},
number = {7},
pages = {306},
pmid = {32566443},
issn = {2190-572X},
abstract = {The improvement of the yield and quality of oil palm via precise genome editing has been indispensable goal for oil palm breeders. Genome editing via the CRISPR/Cas9 (CRISPR-associated protein 9) system, ZFN (zinc finger nucleases) and TALEN (transcription activator-like effector nucleases) has flourished as an efficient technology for precise target modifications in the genomes of various crops. Among the genome editing technologies, base editing approach has emerged as novel technology that could generate single base changes i.e. irreversible conversion of one target base in to other in a programmable manner. A base editor (adenine or cytosine) is a fusion of catalytically inactive CRISPR-Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations. However, till date no such genetic modifications have ever been developed in oil palm via base editing technology. Precise genome editing via base editing approach can be a challenging task in oil palm due to its complex genome as well as difficulties in tissue culture and genetic transformation methods. However, availability of whole genome sequencing data in oil palm provides a platform for developing the base editing technology. Here, we briefly review the potential application and future implications of base editing technology for the genetic improvement of oil palm.},
}
@article {pmid32565723,
year = {2020},
author = {Sun, H and Ding, JM and Zheng, HH and Lv, KJ and Hu, YF and Luo, YH and Wu, X and Pei, WJ and Wang, LZ and Wu, MC and Zhang, Y and Gao, JL},
title = {The Effects of Sidt2 on the Inflammatory Pathway in Mouse Mesangial Cells.},
journal = {Mediators of inflammation},
volume = {2020},
number = {},
pages = {3560793},
pmid = {32565723},
issn = {1466-1861},
mesh = {Animals ; CRISPR-Cas Systems ; Cytokines/metabolism ; *Gene Expression Profiling ; Gene Expression Regulation ; Glomerular Filtration Rate ; I-kappa B Kinase/metabolism ; Inflammation/*metabolism ; Lentivirus/genetics ; Lipopolysaccharides/metabolism ; Lysosomes/metabolism ; MAP Kinase Signaling System ; Mesangial Cells/*metabolism ; Mice ; Mice, Knockout ; NF-KappaB Inhibitor alpha/metabolism ; Nucleotide Transport Proteins/*metabolism ; Signal Transduction ; Transcription Factor RelA/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {In patients with chronic kidney disease, the abnormal activation of inflammatory pathways is usually an important factor leading to renal fibrosis and further deterioration of renal function. Finding effective intervention targets of the inflammatory signaling pathway is an important way to treat chronic kidney disease. As a newly discovered lysosomal membrane protein, the correlation between SID1 transmembrane family member 2 (Sidt2) and the inflammatory signaling pathway has not been reported. The aim of this study was to investigate the effect of Sidt2 on inflammation by inhibiting the expression of the Sidt2 gene in a mouse mesangial cell line mediated by a lentiviral CRISPR/Cas9 vector. Hematoxylin and eosin staining and microscopy found that the mesangial cells lost their normal morphology after inhibiting the expression of Sidt2, showing that the cell body became smaller, the edge between the cells was unclear, and part of the nucleus was pyknotic and fragmented, appearing blue-black. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2 [-/-] group were higher than those of the Sidt2 [+/+] group. p-Jak2 and IL6 increased in the Jak/Stat pathway, and p-ERK and p-P38 increased in the MAPK pathway. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2 [+/+]+LPS group were significantly higher than those in the Sidt2 [+/+] group. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2 [-/-]+LPS group were higher than those in the Sidt2 [-/-] group. The expressions of p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2 [-/-]+LPS group were higher than those in the Sidt2 [+/+]+LPS group. In the Jak/Stat pathway, the protein expressions of p-Jak2 and IL6 in the Sidt2 [+/+]+LPS group were higher than those in the Sidt2 [+/+] group. The expressions of p-Jak2 and IL6 in the Sidt2 [-/-]+LPS group were higher than those in the Sidt2 [-/-] group. The expressions of p-Jak2 and IL6 in the Sidt2 [-/-]+LPS group were higher than those in the Sidt2 [+/+]+LPS group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the MAPK pathway in the Sidt2 [+/+]+LPS group were higher than those in the Sidt2 [+/+] group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2 [-/-]+LPS group were higher than those in the Sidt2 [-/-] group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2 [-/-]+LPS group were higher than those in the Sidt2 [+/+]+LPS group. These data suggested that deletion of the Sidt2 gene changed the three inflammatory signal pathways, eventually leading to the damage of glomerular mesangial cells in mice.},
}
@article {pmid32564746,
year = {2021},
author = {Khatibi, S and Sahebkar, A and Aghaee-Bakhtiari, SH},
title = {CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.},
journal = {Current pharmaceutical biotechnology},
volume = {22},
number = {4},
pages = {468-479},
doi = {10.2174/1389201021666200621161610},
pmid = {32564746},
issn = {1873-4316},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy/*methods ; Humans ; },
abstract = {Gene therapy has been a long lasting goal for scientists, and there are many optimal methods and tools to correct disease-causing mutations in humans. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been progressively adopted for the assessment a treatment of human diseases, including thalassemia, Parkinson's disease, cystic fibrosis, glaucoma, Huntington's disease, and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS). CRISPR sequences belong to the bacterial immune system, which includes the nuclease Cas enzyme and an RNA sequence. The RNA sequence is unique and pathogen-specific, and identifies and binds to the DNA of invasive viruses, allowing the nuclease Cas enzyme to cut the identified DNA and destroy the invasive viruses. This feature provides the possibility to edit mutations in the DNA sequence of live cells by replacing a specific targeted RNA sequence with the RNA sequence in the CRISPR system. Previous studies have reported the improvement steps in confrontation with human diseases caused by single-nucleotide mutations using this system. In this review, we first introduce CRISPR and its functions and then elaborate on the use of CRISPR in the treatment of human diseases.},
}
@article {pmid32564655,
year = {2020},
author = {Selkova, P and Vasileva, A and Pobegalov, G and Musharova, O and Arseniev, A and Kazalov, M and Zyubko, T and Shcheglova, N and Artamonova, T and Khodorkovskii, M and Severinov, K and Fedorova, I},
title = {Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA.},
journal = {RNA biology},
volume = {17},
number = {10},
pages = {1472-1479},
pmid = {32564655},
issn = {1555-8584},
mesh = {Base Sequence ; Binding Sites ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; Gene Editing ; Models, Molecular ; Nucleic Acid Conformation ; *RNA Cleavage ; RNA, Guide/*genetics/metabolism ; Recombinant Proteins ; },
abstract = {Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5'-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17-19 downstream of PAM in the non-target DNA strand, and after the 22[nd] position of target strand, producing 3-5 nucleotide-long 5'-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6-8nt 5'-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.},
}
@article {pmid32564646,
year = {2020},
author = {Timashev, LA and De Lange, T},
title = {Characterization of t-loop formation by TRF2.},
journal = {Nucleus (Austin, Tex.)},
volume = {11},
number = {1},
pages = {164-177},
pmid = {32564646},
issn = {1949-1042},
support = {27302C0028/ES/NIEHS NIH HHS/United States ; R35 CA210036/CA/NCI NIH HHS/United States ; S10 RR031855/RR/NCRR NIH HHS/United States ; R56 AG016642/AG/NIA NIH HHS/United States ; R01 AG016642/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Mice ; Mice, Knockout ; Proteomics ; Rad51 Recombinase/genetics/metabolism ; Telomeric Repeat Binding Protein 2/genetics/*metabolism ; },
abstract = {T-loops are thought to hide telomeres from DNA damage signaling and DSB repair pathways. T-loop formation requires the shelterin component TRF2, which represses ATM signaling and NHEJ. Here we establish that TRF2 alone, in the absence of other shelterin proteins can form t-loops. Mouse and human cells contain two isoforms of TRF2, one of which is uncharacterized. We show that both isoforms protect telomeres and form t-loops. The isoforms are not cell cycle regulated and t-loops are present in G1, S, and G2. Using the DNA wrapping deficient TRF2 Topless mutant, we confirm its inability to form t-loops and repress ATM. However, since the mutant is also defective in repression of NHEJ and telomeric localization, the role of topological changes in telomere protection remains unclear. Finally, we show that Rad51 does not affect t-loop frequencies or telomere protection. Therefore, alternative models for how TRF2 forms t-loops should be explored.},
}
@article {pmid32563468,
year = {2020},
author = {Sun, M and Li, H and Li, Y and Xiang, H and Liu, Y and He, Y and Qi, M and Li, T},
title = {Tomato YABBY2b controls plant height through regulating indole-3-acetic acid-amido synthetase (GH3.8) expression.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {297},
number = {},
pages = {110530},
doi = {10.1016/j.plantsci.2020.110530},
pmid = {32563468},
issn = {1873-2259},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Flowers/growth &amp; development ; Fruit/growth &amp; development ; Gene Editing ; Gene Expression Profiling ; Indoleacetic Acids/metabolism ; Ligases/metabolism/*physiology ; Lycopersicon esculentum/enzymology/genetics/*growth &amp; development ; Plant Growth Regulators/metabolism ; Plant Proteins/metabolism/*physiology ; Polymerase Chain Reaction ; Transcription Factors/metabolism/*physiology ; Two-Hybrid System Techniques ; },
abstract = {Dwarfing is a desirable agronomic trait in cultivation management. Dwarf plants are lodging-resistant, compact, and perform well under high-density planting. The use of dwarf genetic resources is one approach to improve crop yield. YABBY2b in tomato (Solanum lycopersicum) encodes a transcription factor that regulates plant height. In this study, we created YABBY2b knockout mutant lines, and the resulting yabby2b plants exhibited reduced height and smaller flowers and fruits. The RNA-seq analysis showed that 17 genes responding to gibberellin and auxin were differentially expressed. We hypothesized that indole-3-acetic acid-amido synthetase GH3.8 (GH3.8) played a crucial role in the resulting yabby2b dwarf phenotype. Further analysis showed that YABBY2b suppresses GH3.8 gene expression by directly binding to its promoter, and that this contributes to auxin-mediated repression of GH3.8. Moreover, the silencing of GH3.8 led to increased plant height. Combined, our data suggest that YABBY2b may positively regulate plant height in tomato by inhibiting the expression of growth suppressor GH3.8.},
}
@article {pmid32563463,
year = {2020},
author = {Zhang, C and Wang, J and Wang, X and Li, C and Ye, Z and Zhang, J},
title = {UF, a WOX gene, regulates a novel phenotype of un-fused flower in tomato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {297},
number = {},
pages = {110523},
doi = {10.1016/j.plantsci.2020.110523},
pmid = {32563463},
issn = {1873-2259},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Plant/genetics ; Flowers/anatomy &amp; histology/genetics/*growth &amp; development ; Gene Editing ; Genes, Plant/genetics/*physiology ; Lycopersicon esculentum/anatomy &amp; histology/*genetics/growth &amp; development ; Microscopy, Electron, Scanning ; Phenotype ; Phylogeny ; Plant Leaves/anatomy &amp; histology/genetics/growth &amp; development ; Real-Time Polymerase Chain Reaction ; Transcription Factors/genetics/physiology ; },
abstract = {Flower formation is a basic condition for fruit set in all flowering plants. The normal stamen of tomato flower fused together to form a yellow cylinder surrounding the carpels. In this study, we identified an un-fused flower (uf) tomato mutant that is defective in petal, carpal and stamen fusion and lateral outgrowth. After RNA-seq-based BSA (BSR), the candidate region location was identified in the long arm of chromosome 3. Using map-based cloning with InDel and CAPS markers, the UF candidate gene was mapped in a 104 kb region. In this region, a WOX (WUSCHEL-related homeobox) transcription factor SlWOX1 was considered as a candidate of UF as there is a 72bp deletion in its second exon in uf mutant. The mutations of SlWOX1 generated by CRISPR/CAS9 approach under wild-type background reproduced the phenotypes of uf mutant, indicating that the SlWOX1 gene is indeed UF. Interestingly, expression analysis of organ lateral polarity determinant genes showed that abaxial genes (SlYABBY5 and SlARF4) and adaxial genes (AS and HD-ZIPIII) were significantly down-regulated in the uf mutant, which is different to that in Arabidopsis and petunia. In conclusion, this work revealed a novel function of SlWOX1 in the regulation of flower development in tomato.},
}
@article {pmid32563459,
year = {2020},
author = {Wang, S and Li, Q and Zhao, L and Fu, S and Qin, L and Wei, Y and Fu, YB and Wang, H},
title = {Arabidopsis UBC22, an E2 able to catalyze lysine-11 specific ubiquitin linkage formation, has multiple functions in plant growth and immunity.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {297},
number = {},
pages = {110520},
doi = {10.1016/j.plantsci.2020.110520},
pmid = {32563459},
issn = {1873-2259},
mesh = {Arabidopsis/*enzymology/genetics/growth &amp; development/immunology ; Arabidopsis Proteins/genetics/*physiology ; Botrytis ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Gene Editing ; Gene Expression Profiling ; Gene Knockout Techniques ; Plant Diseases/immunology/microbiology ; RNA, Plant/genetics ; Real-Time Polymerase Chain Reaction ; Ubiquitin-Conjugating Enzymes/genetics/*physiology ; Ubiquitination/genetics ; },
abstract = {Protein ubiquitination is critical for various biological processes in eukaryotes. A ubiquitin (Ub) chain can be linked through one of the seven lysine (K) residues or the N-terminus methionine of the Ub, and the Ub-conjugating enzymes called E2s play a critical role in determining the linkage specificity of Ub chains. Further, while K48-linked polyubiquitin chain is important for protein degradation, much less is known about the functions of other types of polyubiquitin chains in plants. We showed previously that UBC22 is unique in its ability to catalyze K11-dependent Ub dimer formation in vitro and ubc22 knockout mutants had defects in megasporogenesis. In this study, further analyses of the Arabidopsis ubc22 mutants revealed four subtypes of plants based on the phenotypic changes in vegetative growth. These four subtypes appeared consistently in the plants of three independent ubc22 mutants. Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in different subtypes of mutant plants. In one subtype, the mutant plants had increased expression of genes related to plant defenses and showed enhanced resistance to a necrotrophic plant pathogen. These results suggest multiple functions of UBC22 during plant development and stress response.},
}
@article {pmid32563448,
year = {2020},
author = {Navarro-Serna, S and Vilarino, M and Park, I and Gadea, J and Ross, PJ},
title = {Livestock Gene Editing by One-step Embryo Manipulation.},
journal = {Journal of equine veterinary science},
volume = {89},
number = {},
pages = {103025},
doi = {10.1016/j.jevs.2020.103025},
pmid = {32563448},
issn = {0737-0806},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/veterinary ; *Livestock ; },
abstract = {The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.},
}
@article {pmid32561905,
year = {2020},
author = {Park, S and Shimada, K and Fujihara, Y and Xu, Z and Shimada, K and Larasati, T and Pratiwi, P and Matzuk, RM and Devlin, DJ and Yu, Z and Garcia, TX and Matzuk, MM and Ikawa, M},
title = {CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity.},
journal = {Biology of reproduction},
volume = {103},
number = {2},
pages = {195-204},
pmid = {32561905},
issn = {1529-7268},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD095341/HD/NICHD NIH HHS/United States ; T32 GM120011/GM/NIGMS NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; T32 GM088129/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Fertility/*genetics ; Gene Editing ; Male ; Mice ; Mice, Knockout ; Spermatogenesis/genetics ; Testis/*metabolism ; },
abstract = {As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.},
}
@article {pmid32561814,
year = {2020},
author = {Søndergaard, JN and Geng, K and Sommerauer, C and Atanasoai, I and Yin, X and Kutter, C},
title = {Successful delivery of large-size CRISPR/Cas9 vectors in hard-to-transfect human cells using small plasmids.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {319},
pmid = {32561814},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; Humans ; Plasmids/*genetics ; Transfection/*methods ; },
abstract = {With the rise of new powerful genome engineering technologies, such as CRISPR/Cas9, cell models can be engineered effectively to accelerate basic and disease research. The most critical step in this procedure is the efficient delivery of foreign nucleic acids into cells by cellular transfection. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9-19 kb), they result in low transfection efficiency and cell viability, and thus subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. At its core, the technique is based on adding exogenous small plasmids of a defined size to the transfection mixture.},
}
@article {pmid32561775,
year = {2020},
author = {Hashikawa, Y and Hayashi, R and Tajima, M and Okubo, T and Azuma, S and Kuwamura, M and Takai, N and Osada, Y and Kunihiro, Y and Mashimo, T and Nishida, K},
title = {Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {9957},
pmid = {32561775},
issn = {2045-2322},
mesh = {Animals ; B-Lymphocytes/immunology ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/immunology ; Female ; Gene Knockout Techniques/methods ; Interleukin Receptor Common gamma Subunit/genetics/immunology ; Rabbits ; Skin/immunology ; T-Lymphocytes/immunology ; Thymus Gland/immunology ; X-Linked Combined Immunodeficiency Diseases/*genetics/immunology ; },
abstract = {Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.},
}
@article {pmid32561716,
year = {2020},
author = {Chen, Y and Liu, J and Zhi, S and Zheng, Q and Ma, W and Huang, J and Liu, Y and Liu, D and Liang, P and Songyang, Z},
title = {Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3136},
pmid = {32561716},
issn = {2041-1723},
mesh = {Bacterial Proteins/genetics/isolation &amp; purification/*metabolism ; CRISPR-Associated Proteins/genetics/isolation &amp; purification/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Pseudomonas aeruginosa/genetics ; Recombinant Proteins/genetics/isolation &amp; purification/metabolism ; Transcriptional Activation ; Transfection ; },
abstract = {Class 2 CRISPR-Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR-Cas constitutes ~60% of all the CRISPR-Cas systems. However, only type I-B and I-E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I-F system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type I-F Cas proteins, we activate gene transcription in human cells. In most cases, type I-F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type I-F system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type I-F CRISPR-Cas in human cells.},
}
@article {pmid32560695,
year = {2020},
author = {Santillán Martínez, MI and Bracuto, V and Koseoglou, E and Appiano, M and Jacobsen, E and Visser, RGF and Wolters, AA and Bai, Y},
title = {CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {284},
pmid = {32560695},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems ; Disease Resistance/genetics ; Glucosyltransferases/*genetics/metabolism ; Lycopersicon esculentum/enzymology/*growth &amp; development/microbiology ; Mutagenesis ; Plant Diseases/*genetics/microbiology ; Plant Proteins/*genetics/metabolism ; },
abstract = {BACKGROUND: The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On).<br><br>RESULTS: To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants.<br><br>CONCLUSION: CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.},
}
@article {pmid32559432,
year = {2020},
author = {Kaeuferle, T and Deisenberger, L and Jablonowski, L and Stief, TA and Blaeschke, F and Willier, S and Feuchtinger, T},
title = {CRISPR-Cas9-Mediated Glucocorticoid Resistance in Virus-Specific T Cells for Adoptive T Cell Therapy Posttransplantation.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {9},
pages = {1965-1973},
pmid = {32559432},
issn = {1525-0024},
mesh = {Adoptive Transfer/*methods ; *CRISPR-Cas Systems ; Cell Engineering/*methods ; Cell Proliferation/genetics ; Cells, Cultured ; Cyclosporine/pharmacology ; Cytokines/metabolism ; Drug Resistance/*genetics ; Gene Knockout Techniques/methods ; Glucocorticoids/*therapeutic use ; Hematopoietic Stem Cell Transplantation/*adverse effects ; Humans ; Lymphocyte Activation/immunology ; Receptors, Glucocorticoid/deficiency/genetics ; T-Lymphocytes/drug effects/*immunology/metabolism ; Viral Matrix Proteins/immunology ; Virus Diseases/*etiology/immunology/*therapy ; },
abstract = {Immunosuppression posttransplantation exposes patients to an increased risk for refractory viral infections as an important cause of morbidity and mortality. Protective T cell immunity can be restored by adoptive T cell transfer, but ongoing immunosuppression limits efficacy of T cell responses. In order to deliver protection against viral pathogens and allow at the same time necessary steroid therapy, we generated glucocorticoid-resistant T cells by CRISPR-Cas9-mediated knockout of the glucocorticoid receptor in primary human virus-specific T cell products. Characterization of the T cell product revealed high efficiency of glucocorticoid receptor knockout and high purity of virus-specific T cells. This tandem T cell engineering preserved protective T cell functionality, such as cytotoxicity, CD107a degranulation, proliferative capacity, and cytokine release patterns. Virus-specific T cells with glucocorticoid receptor knockout were resistant to the suppressive effect of dexamethasone treatment on lymphocyte proliferation and cytokine secretion (tumor necrosis factor alpha [TNF-α], interleukin-4 [IL-4], IL-6, and sFas). Additionally, glucocorticoid receptor knockout cells remained sensitive to cyclosporine A treatment, thereby providing a rescue approach for patients in case of safety issues. This novel approach provides a therapeutic option for the treatment of patients with viral infections after transplantation who are receiving glucocorticoid therapy.},
}
@article {pmid32559405,
year = {2020},
author = {Lopatina, A and Tal, N and Sorek, R},
title = {Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy.},
journal = {Annual review of virology},
volume = {7},
number = {1},
pages = {371-384},
doi = {10.1146/annurev-virology-011620-040628},
pmid = {32559405},
issn = {2327-0578},
mesh = {Bacteria/genetics/*immunology/*virology ; Bacterial Infections/*immunology/prevention &amp; control/virology ; Bacteriophages/immunology/*pathogenicity ; CRISPR-Cas Systems ; Humans ; Toxin-Antitoxin Systems ; },
abstract = {Facing frequent phage challenges, bacteria have evolved numerous mechanisms to resist phage infection. A commonly used phage resistance strategy is abortive infection (Abi), in which the infected cell commits suicide before the phage can complete its replication cycle. Abi prevents the phage epidemic from spreading to nearby cells, thus protecting the bacterial colony. The Abi strategy is manifested by a plethora of mechanistically diverse defense systems that are abundant in bacterial genomes. In turn, phages have developed equally diverse mechanisms to overcome bacterial Abi. This review summarizes the current knowledge on bacterial defense via cell suicide. It describes the principles of Abi, details how these principles are implemented in a variety of natural defense systems, and discusses phage counter-defense mechanisms.},
}
@article {pmid32559396,
year = {2021},
author = {Mosterd, C and Rousseau, GM and Moineau, S},
title = {A short overview of the CRISPR-Cas adaptation stage.},
journal = {Canadian journal of microbiology},
volume = {67},
number = {1},
pages = {1-12},
doi = {10.1139/cjm-2020-0212},
pmid = {32559396},
issn = {1480-3275},
mesh = {Adaptation, Physiological/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; Gram-Positive Bacteria/genetics ; Streptococcus thermophilus/*genetics ; },
abstract = {CRISPR research began over 30 years ago with the incidental discovery of an unusual nucleotide arrangement in the Escherichia coli genome. It took 20 years to find the main function of CRISPR-Cas systems as an adaptive defence mechanism against invading nucleic acids, and our knowledge of their biology has steadily increased ever since. In parallel, the number of applications derived from CRISPR-Cas systems has risen spectacularly. The CRISPR-based genome editing tool is arguably the most exciting application in both basic and applied research. Lately, CRISPR-Cas research has partially shifted to the least understood aspect of its biology: the ability of CRISPR-Cas systems to acquire new immunities during the so-called adaptation step. To date, the most efficient natural system to readily acquire new spacers is the type II-A system of the gram-positive dairy bacterium Streptococcus thermophilus. The discovery of additional systems able to acquire new spacers will hopefully draw more attention to this step of CRISPR-Cas biology. This review focuses on the breakthroughs that have helped to unravel the adaptation phase and on questions that remain to be answered.},
}
@article {pmid32558098,
year = {2020},
author = {Finger-Bou, M and Orsi, E and van der Oost, J and Staals, RHJ},
title = {CRISPR with a Happy Ending: Non-Templated DNA Repair for Prokaryotic Genome Engineering.},
journal = {Biotechnology journal},
volume = {15},
number = {7},
pages = {e1900404},
doi = {10.1002/biot.201900404},
pmid = {32558098},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; *Genetic Engineering ; Genome, Bacterial/*genetics ; },
abstract = {The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR-Cas and other nucleases mediate double-strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template-independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non-homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.},
}
@article {pmid32556263,
year = {2020},
author = {Roy, D and Huguet, KT and Grenier, F and Burrus, V},
title = {IncC conjugative plasmids and SXT/R391 elements repair double-strand breaks caused by CRISPR-Cas during conjugation.},
journal = {Nucleic acids research},
volume = {48},
number = {16},
pages = {8815-8827},
pmid = {32556263},
issn = {1362-4962},
support = {PJT-153071//CIHR/Canada ; },
mesh = {Bacteriophage lambda/genetics ; *CRISPR-Cas Systems ; *Conjugation, Genetic ; DNA Restriction-Modification Enzymes/*genetics ; Escherichia coli/*genetics ; Gene Transfer, Horizontal ; Operon ; Plasmids/*genetics ; Vibrio cholerae/*genetics ; },
abstract = {Bacteria have evolved defence mechanisms against bacteriophages. Restriction-modification systems provide innate immunity by degrading invading DNAs that lack proper methylation. CRISPR-Cas systems provide adaptive immunity by sampling the genome of past invaders and cutting the DNA of closely related DNA molecules. These barriers also restrict horizontal gene transfer mediated by conjugative plasmids. IncC conjugative plasmids are important contributors to the global dissemination of multidrug resistance among pathogenic bacteria infecting animals and humans. Here, we show that IncC conjugative plasmids are highly resilient to host defence systems during entry into a new host by conjugation. Using a TnSeq strategy, we uncover a conserved operon containing five genes (vcrx089-vcrx093) that confer a novel host defence evasion (hde) phenotype. We show that vcrx089-vcrx090 promote resistance against type I restriction-modification, whereas vcrx091-vcxr093 promote CRISPR-Cas evasion by repairing double-strand DNA breaks via recombination between short sequence repeats. vcrx091, vcrx092 and vcrx093 encode a single-strand binding protein, and a single-strand annealing recombinase and double-strand exonuclease related to Redβ and λExo of bacteriophage λ, respectively. Homologous genes of the integrative and conjugative element R391 also provide CRISPR-Cas evasion. Hence, the conserved hde operon considerably broadens the host range of large families of mobile elements spreading multidrug resistance.},
}
@article {pmid32556171,
year = {2020},
author = {Wu, F and Shim, J and Gong, T and Tan, C},
title = {Orthogonal tuning of gene expression noise using CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {48},
number = {13},
pages = {7606},
doi = {10.1093/nar/gkaa537},
pmid = {32556171},
issn = {1362-4962},
support = {R21 EB025938/EB/NIBIB NIH HHS/United States ; },
}
@article {pmid32556168,
year = {2020},
author = {Padilha, VA and Alkhnbashi, OS and Shah, SA and de Carvalho, ACPLF and Backofen, R},
title = {CRISPRcasIdentifier: Machine learning for accurate identification and classification of CRISPR-Cas systems.},
journal = {GigaScience},
volume = {9},
number = {6},
pages = {},
pmid = {32556168},
issn = {2047-217X},
mesh = {Algorithms ; Archaea/genetics ; Bacteria/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; Genome, Archaeal ; Genome, Bacterial ; Genomics/*methods ; Machine Learning ; Workflow ; },
abstract = {BACKGROUND: CRISPR-Cas genes are extraordinarily diverse and evolve rapidly when compared to other prokaryotic genes. With the rapid increase in newly sequenced archaeal and bacterial genomes, manual identification of CRISPR-Cas systems is no longer viable. Thus, an automated approach is required for advancing our understanding of the evolution and diversity of these systems and for finding new candidates for genome engineering in eukaryotic models.<br><br>RESULTS: We introduce CRISPRcasIdentifier, a new machine learning-based tool that combines regression and classification models for the prediction of potentially missing proteins in instances of CRISPR-Cas systems and the prediction of their respective subtypes. In contrast to other available tools, CRISPRcasIdentifier can both detect cas genes and extract potential association rules that reveal functional modules for CRISPR-Cas systems. In our experimental benchmark on the most recently published and comprehensive CRISPR-Cas system dataset, CRISPRcasIdentifier was compared with recent and state-of-the-art tools. According to the experimental results, CRISPRcasIdentifier presented the best Cas protein identification and subtype classification performance.<br><br>CONCLUSIONS: Overall, our tool greatly extends the classification of CRISPR cassettes and, for the first time, predicts missing Cas proteins and association rules between Cas proteins. Additionally, we investigated the properties of CRISPR subtypes. The proposed tool relies not only on the knowledge of manual CRISPR annotation but also on models trained using machine learning.},
}
@article {pmid32555532,
year = {2020},
author = {Ultee, E and van der Aart, LT and Zhang, L and van Dissel, D and Diebolder, CA and van Wezel, GP and Claessen, D and Briegel, A},
title = {Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {314},
pmid = {32555532},
issn = {2399-3642},
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Wall/*chemistry/metabolism ; Cryoelectron Microscopy ; Gene Expression Regulation, Bacterial ; Peptidoglycan/chemistry/metabolism ; Polysaccharides/metabolism ; Streptomyces coelicolor/*cytology/genetics/growth &amp; development ; Teichoic Acids/chemistry/*metabolism ; Tomography/methods ; },
abstract = {The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall of Streptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down of tagO expression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that the Streptomyces cell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.},
}
@article {pmid32555510,
year = {2020},
author = {Geng, BC and Choi, KH and Wang, SZ and Chen, P and Pan, XD and Dong, NG and Ko, JK and Zhu, H},
title = {A simple, quick, and efficient CRISPR/Cas9 genome editing method for human induced pluripotent stem cells.},
journal = {Acta pharmacologica Sinica},
volume = {41},
number = {11},
pages = {1427-1432},
pmid = {32555510},
issn = {1745-7254},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Plasmids ; Ubiquitin Thiolesterase/genetics ; },
abstract = {Induced pluripotent stem cells (iPSCs) have become an essential research platform to study different human diseases once being discovered by Dr. Shinya Yamanaka in 2006. Another breakthrough in biomedical research is the application of CRISPR/Cas9 system for genome editing in mammalian cells. Although numerous studies have been done to develop methods for gene editing in iPSCs, the current approaches suffer from several limitations, including time and labor consuming, low editing efficiency, and potential off-target effects. In the current study, we report an electroporation-mediated plasmid CRISPR/Cas9 delivery approach for genome editing in iPSCs. With this approach, an edited iPSC cell line could be obtained within 2 weeks. In addition, the transit introducing of CRISPR/Cas9 machinery could minimize genomic integration of Cas9 gene, which avoided potential long-term side effects of Cas9 enzyme. We showed that CRISPR/Cas9-mediated genomic editing did not affect pluripotency and differentiation ability of iPSCs. With the quickly evolving of both iPSC and CRISPR/Cas9-mediated genome editing research fields, we believe that our method can significantly facilitate the application of genome editing in iPSCs research.},
}
@article {pmid32555504,
year = {2020},
author = {Carlisle, SM and Trainor, PJ and Hong, KU and Doll, MA and Hein, DW},
title = {CRISPR/Cas9 knockout of human arylamine N-acetyltransferase 1 in MDA-MB-231 breast cancer cells suggests a role in cellular metabolism.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {9804},
pmid = {32555504},
issn = {2045-2322},
support = {R25 CA134283/CA/NCI NIH HHS/United States ; T32 ES011564/ES/NIEHS NIH HHS/United States ; },
mesh = {Acetylation ; Analysis of Variance ; Arylamine N-Acetyltransferase/genetics/*metabolism ; Breast Neoplasms/*enzymology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatography, Liquid ; Female ; Gene Knockout Techniques ; Humans ; Isoenzymes/genetics/*metabolism ; Metabolic Networks and Pathways/genetics ; Metabolome/genetics ; Substrate Specificity ; Tandem Mass Spectrometry ; },
abstract = {Human arylamine N-acetyltransferase 1 (NAT1), present in all tissues, is classically described as a phase-II xenobiotic metabolizing enzyme but can also catalyze the hydrolysis of acetyl-Coenzyme A (acetyl-CoA) in the absence of an arylamine substrate using folate as a cofactor. NAT1 activity varies inter-individually and has been shown to be overexpressed in estrogen receptor-positive (ER+) breast cancers. NAT1 has also been implicated in breast cancer progression however the exact role of NAT1 remains unknown. The objective of this study was to evaluate the effect of varying levels of NAT1 N-acetylation activity in MDA-MB-231 breast cancer cells on global cellular metabolism and to probe for unknown endogenous NAT1 substrates. Global, untargeted metabolomics was conducted via ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on MDA-MB-231 breast cancer cell lines constructed with siRNA and CRISPR/Cas9 technologies to vary only in NAT1 N-acetylation activity. Many metabolites were differentially abundant in NAT1-modified cell lines compared to the Scrambled parental cell line. N-acetylasparagine and N-acetylputrescine abundances were strongly positively correlated (r = 0.986 and r = 0.944, respectively) with NAT1 N-acetylation activity whereas saccharopine abundance was strongly inversely correlated (r = -0.876). Two of the most striking observations were a reduction in de novo pyrimidine biosynthesis and defective β-oxidation of fatty acids in the absence of NAT1. We have shown that NAT1 expression differentially affects cellular metabolism dependent on the level of expression. Our results support the hypothesis that NAT1 is not just a xenobiotic metabolizing enzyme and may have a role in endogenous cellular metabolism.},
}
@article {pmid32555443,
year = {2021},
author = {Li, FF and Liang, YL and Han, XS and Guan, YN and Chen, J and Wu, P and Zhao, XX and Jing, Q},
title = {ADP receptor P2y12 prevents excessive primitive hematopoiesis in zebrafish by inhibiting Gata1.},
journal = {Acta pharmacologica Sinica},
volume = {42},
number = {3},
pages = {414-421},
pmid = {32555443},
issn = {1745-7254},
mesh = {Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cell Differentiation/physiology ; Embryo, Nonmammalian/physiology ; Endothelium, Vascular/physiology ; Erythrocytes/physiology ; Female ; GATA1 Transcription Factor/*antagonists &amp; inhibitors/metabolism ; Gene Expression Regulation, Developmental/physiology ; Gene Knockout Techniques ; Hematopoiesis/genetics/*physiology ; Male ; Mutation ; Receptors, Purinergic P2Y12/genetics/*physiology ; Up-Regulation/physiology ; Zebrafish ; Zebrafish Proteins/*antagonists &amp; inhibitors/metabolism ; },
abstract = {In the past two decades, purinergic signaling has emerged as a key regulator of hematopoiesis in physiological and pathological conditions. ADP receptor P2y12 is a crucial component of this signaling, but whether it is involved in primitive hematopoiesis remains unknown. To elucidate the function of P2y12 and provide new insights for drug development, we established a zebrafish P2y12 mutant by CRISPR/Cas 9-based genetic modification system, and investigated whether P2y12 acted as an important regulator for primitive hematopoiesis. By using mass spectrometry (MS) combined with RNA sequencing, we showed that absence of P2y12 induced excessive erythropoiesis, evidenced by significantly increased expression of mature erythrocytes marker α-globin (Hbae1 and Hbae3), β-globin (Hbbe1 and Hbbe3). Expression pattern analysis showed that P2y12 was mainly expressed in red blood cells and endothelial cells of early zebrafish embryos. Further studies revealed that primitive erythroid progenitor marker Gata1 was markedly up-regulated. Remarkably, inhibition of Gata1 by injection of Gata1 morpholino could rescue the erythroid abnormality in P2y12 mutants. The present study demonstrates the essential role of purinergic signaling in differentiation of proerythrocytes during primitive hematopoiesis, and provides potential targets for treatment of blood-related disease and drug development.},
}
@article {pmid32553350,
year = {2020},
author = {Huang, Z and Tian, D and Liu, Y and Lin, Z and Lyon, CJ and Lai, W and Fusco, D and Drouin, A and Yin, X and Hu, T and Ning, B},
title = {Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {164},
number = {},
pages = {112316},
pmid = {32553350},
issn = {1873-4235},
support = {U54 GM104940/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; Betacoronavirus/genetics/*isolation &amp; purification ; Biosensing Techniques/methods/statistics &amp; numerical data ; COVID-19 ; *CRISPR-Cas Systems ; Coronavirus Infections/*diagnosis/virology ; Fluorescent Dyes ; Genes, Viral ; High-Throughput Nucleotide Sequencing/methods/statistics &amp; numerical data ; Humans ; Nucleic Acid Amplification Techniques/methods/statistics &amp; numerical data ; Pandemics ; Pneumonia, Viral/*diagnosis/virology ; Predictive Value of Tests ; RNA, Viral/analysis/genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods/statistics &amp; numerical data ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.},
}
@article {pmid32552324,
year = {2020},
author = {Assimos, DG},
title = {Re: CRISPR/Cas9 Engineering of Albino Cystinuria Type A Mice.},
journal = {The Journal of urology},
volume = {204},
number = {3},
pages = {613-614},
doi = {10.1097/JU.0000000000001173.03},
pmid = {32552324},
issn = {1527-3792},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Cystinuria ; Mice ; },
}
@article {pmid32551581,
year = {2020},
author = {Oh, EJ and Liu, R and Liang, L and Freed, EF and Eckert, CA and Gill, RT},
title = {Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform.},
journal = {ACS synthetic biology},
volume = {9},
number = {8},
pages = {2197-2202},
doi = {10.1021/acssynbio.0c00159},
pmid = {32551581},
issn = {2161-5063},
mesh = {Antigen-Antibody Reactions ; CRISPR-Cas Systems/genetics ; Flow Cytometry ; Gene Editing/methods ; Hydrogen-Ion Concentration ; Mutagenesis, Site-Directed ; Protein Engineering ; Protein Stability ; Saccharomyces cerevisiae/genetics/*metabolism ; Single-Chain Antibodies/chemistry/genetics/*metabolism ; },
abstract = {Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.},
}
@article {pmid32551562,
year = {2020},
author = {Yu, S and Price, MA and Wang, Y and Liu, Y and Guo, Y and Ni, X and Rosser, SJ and Bi, C and Wang, M},
title = {CRISPR-dCas9 Mediated Cytosine Deaminase Base Editing in Bacillus subtilis.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1781-1789},
doi = {10.1021/acssynbio.0c00151},
pmid = {32551562},
issn = {2161-5063},
support = {BB/M00029X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacillus subtilis/*enzymology/*genetics ; Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytidine Deaminase/*genetics ; Cytosine Deaminase/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Genetic Loci ; Genome, Bacterial ; Plasmids/genetics/metabolism ; Point Mutation ; Streptococcus pyogenes/enzymology ; },
abstract = {Base editing technology based on clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in Bacillus subtilis utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from S. pyogenes) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in B. subtilis has a 5 nt editing window, which is similar to previously reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in B. subtilis. Finally, we applied multiplex genome editing to generate a B. subtilis 168 mutant strain with eight inactive extracellular protease genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in B. subtilis and industrial applications in the future.},
}
@article {pmid32550896,
year = {2020},
author = {Huang, X and Chen, Z and Liu, Y},
title = {RNAi-mediated control of CRISPR functions.},
journal = {Theranostics},
volume = {10},
number = {15},
pages = {6661-6673},
pmid = {32550896},
issn = {1838-7640},
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Enoxacin/*pharmacology ; Gene Editing/*methods ; Humans ; Mice ; Mice, Inbred C57BL ; MicroRNAs/*genetics/metabolism ; Models, Animal ; RNA Interference/*drug effects ; RNA, Guide/*genetics/metabolism ; },
abstract = {CRISPR-Cas9 has become a versatile tool for genome editing and regulation, and strategies to effectively control its activity have attracted much attention. RNAi, also a gene-regulating tool, is used as another mechanism by which eukaryotes resist the invasion of foreign genetic material. Methods: In this study, we analyzed the quantitative inhibition of the CRISPR system by using artificial miRNAs (amiRNAs) combined with the RNAi enhancer enoxacin to improve the targeting specificity of the CRISPR system. Furthermore, we examined the feasibility of improving the efficiency of gene editing and regulation by blocking the effects of natural intracellular miRNAs on sgRNAs. Results: amiRNAs targeting the sgRNA were used to control its expression, and the small molecule drug denoxacin was utilized to enhance this effect, especially in the presence of Cas9. amiRNA/enoxacin inhibited CRISPR-mediated gene editing and regulation both in vitro and in vivo and could tune sgRNA-targeting specificity. Furthermore, CRISPR efficiency was increased by blocking the effects of endogenous miRNAs. Conclusion: Our study provides an efficient molecular switch for conditional regulation of CRISPR activities in mammalian cells and also presents potentially useful approaches for solving current key issues of off-target effects and low targeting efficiency.},
}
@article {pmid32549675,
year = {2020},
author = {Santosh Kumar, VV and Verma, RK and Yadav, SK and Yadav, P and Watts, A and Rao, MV and Chinnusamy, V},
title = {CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {26},
number = {6},
pages = {1099-1110},
pmid = {32549675},
issn = {0971-5894},
abstract = {Development of abiotic stress tolerant rice cultivars is necessary for sustainable rice production under the scenario of global climate change, dwindling fresh water resources and increase in salt affected areas. Several genes from rice have been functionally validated by using EMS mutants and transgenics. Often, many of these desirable alleles are not available indica rice which is mainly cultivated, and where available, introgression of these alleles into elite cultivars is a time and labour intensive process, in addition to the potential introgression of non-desirable genes due to linkage. CRISPR-Cas technology helps development of elite cultivars with desirable alleles by precision gene editing. Hence, this study was carried out to create mutant alleles of drought and salt tolerance (DST) gene by using CRISPR-Cas9 gene editing in indica rice cv. MTU1010. We used two different gRNAs to target regions of DST protein that might be involved in protein-protein interaction and successfully generated different mutant alleles of DST gene. We selected homozygous dst mutant with 366 bp deletion between the two gRNAs for phenotypic analysis. This 366 bp deletion led to the deletion of amino acid residues from 184 to 305 in frame, and hence the mutant was named as dst [∆184-305] . The dst [∆184-305] mutation induced by CRISPR-Cas9 method in DST gene in indica rice cv. MTU1010 phenocopied EMS-induced dst (N69D) mutation reported earlier in japonica cultivar. The dst [∆184-305] mutant produced leaves with broader width and reduced stomatal density, and thus enhanced leaf water retention under dehydration stress. Our study showed that the reduction in stomatal density in loss of function mutants of dst is, at least, in part due to downregulation of stomatal developmental genes SPCH1, MUTE and ICE1. The Cas9-free dst [∆184-305] mutant exhibited moderate level tolerance to osmotic stress and high level of salt stress in seedling stage. Thus, dst mutant alleles generated in this study will be useful for improving drought and salt tolerance and grain yield in indica rice cultivars.},
}
@article {pmid32549410,
year = {2020},
author = {Sarrou, E and Richmond, L and Carmody, RJ and Gibson, B and Keeshan, K},
title = {CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis.},
journal = {International journal of molecular sciences},
volume = {21},
number = {12},
pages = {},
pmid = {32549410},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Chromosome Breakpoints ; Disease Models, Animal ; Gene Editing/*methods ; HEK293 Cells ; Hematopoietic Stem Cells/*cytology/metabolism ; Histone-Lysine N-Methyltransferase/*genetics ; Humans ; Leukemia/*genetics ; Mice ; Models, Biological ; Myeloid-Lymphoid Leukemia Protein/*genetics ; NIH 3T3 Cells ; Nuclear Proteins/*genetics ; Proto-Oncogene Proteins c-kit/*genetics ; },
abstract = {Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.},
}
@article {pmid32548827,
year = {2020},
author = {Shvets, E and Mendoza-Topaz, C},
title = {Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2169},
number = {},
pages = {149-166},
doi = {10.1007/978-1-0716-0732-9_14},
pmid = {32548827},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Carrier Proteins/*genetics/metabolism ; Caveolae/metabolism ; Caveolin 1/*genetics/metabolism ; Cloning, Molecular/methods ; Electroporation/methods ; Flow Cytometry ; Fluorescent Antibody Technique/methods ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Green Fluorescent Proteins/genetics ; Membrane Proteins/genetics/*metabolism ; Mice ; NIH 3T3 Cells ; Plasmids/genetics ; RNA-Binding Proteins/genetics/*metabolism ; },
abstract = {Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells. In both instances, the modifications were achieved by using Cas9 delivery on plasmid DNA by electroporation and by utilizing FACS cell sorting for selection or enrichment of edited population of cells. We also provide a list with tested gRNA sequences to successfully produce KI and KO of other caveolar components.},
}
@article {pmid32546815,
year = {2020},
author = {Noguchi, H and Miyagi-Shiohira, C and Nakashima, Y and Kinjo, T and Saitoh, I and Watanabe, M},
title = {Mutations in the C1 element of the insulin promoter lead to diabetic phenotypes in homozygous mice.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {309},
pmid = {32546815},
issn = {2399-3642},
mesh = {Animals ; CRISPR-Cas Systems ; Diabetes Mellitus, Experimental/*genetics/pathology ; Female ; Genetic Vectors ; Insulin/*genetics/metabolism ; Insulin-Secreting Cells ; Male ; Mice, Inbred C57BL ; *Mutation ; Phenotype ; *Promoter Regions, Genetic ; },
abstract = {Genome editing technologies such as CRISPR-Cas9 are widely used to establish causal associations between mutations and phenotypes. However, CRISPR-Cas9 is rarely used to analyze promoter regions. The insulin promoter region (approximately 1,000 bp) directs β cell-specific expression of insulin, which in vitro studies show is regulated by ubiquitous, as well as pancreatic, β cell-specific transcription factors. However, we are unaware of any confirmatory in vivo studies. Here, we used CRISPR-Cas9 technology to generate mice with mutations in the promoter regions of the insulin I (Ins1) and II (Ins2) genes. We generated 4 homozygous diabetic mice with 2 distinct mutations in the highly conserved C1 elements in each of the Ins1 and Ins2 promoters (3 deletions and 1 replacement in total). Remarkably, all mice with homozygous or heterozygous mutations in other loci were not diabetic. Thus, the C1 element in mice is required for Ins transcription in vivo.},
}
@article {pmid32546686,
year = {2020},
author = {Zafar, H and Lin, C and Bar-Joseph, Z},
title = {Single-cell lineage tracing by integrating CRISPR-Cas9 mutations with transcriptomic data.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3055},
pmid = {32546686},
issn = {2041-1723},
mesh = {*Algorithms ; Animals ; Brain/cytology ; *CRISPR-Cas Systems ; Caenorhabditis elegans/embryology/genetics ; Cell Differentiation/genetics ; Cell Lineage/*genetics ; Data Interpretation, Statistical ; Embryo, Nonmammalian/cytology ; Gene Expression Profiling/methods/*statistics &amp; numerical data ; Likelihood Functions ; Mutation ; Single-Cell Analysis/*methods/statistics &amp; numerical data ; Zebrafish/genetics ; },
abstract = {Recent studies combine two novel technologies, single-cell RNA-sequencing and CRISPR-Cas9 barcode editing for elucidating developmental lineages at the whole organism level. While these studies provided several insights, they face several computational challenges. First, lineages are reconstructed based on noisy and often saturated random mutation data. Additionally, due to the randomness of the mutations, lineages from multiple experiments cannot be combined to reconstruct a species-invariant lineage tree. To address these issues we developed a statistical method, LinTIMaT, which reconstructs cell lineages using a maximum-likelihood framework by integrating mutation and expression data. Our analysis shows that expression data helps resolve the ambiguities arising in when lineages are inferred based on mutations alone, while also enabling the integration of different individual lineages for the reconstruction of an invariant lineage tree. LinTIMaT lineages have better cell type coherence, improve the functional significance of gene sets and provide new insights on progenitors and differentiation pathways.},
}
@article {pmid32546280,
year = {2020},
author = {Li, C and Zong, Y and Jin, S and Zhu, H and Lin, D and Li, S and Qiu, JL and Wang, Y and Gao, C},
title = {SWISS: multiplexed orthogonal genome editing in plants with a Cas9 nickase and engineered CRISPR RNA scaffolds.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {141},
pmid = {32546280},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Plant ; Oryza ; },
abstract = {We describe here a CRISPR simultaneous and wide-editing induced by a single system (SWISS), in which RNA aptamers engineered in crRNA scaffold recruit their cognate binding proteins fused with cytidine deaminase and adenosine deaminase to Cas9 nickase target sites to generate multiplexed base editing. By using paired sgRNAs, SWISS can produce insertions/deletions in addition to base editing. Rice mutants are generated using the SWISS system with efficiencies of cytosine conversion of 25.5%, adenine conversion of 16.4%, indels of 52.7%, and simultaneous triple mutations of 7.3%. The SWISS system provides a powerful tool for multi-functional genome editing in plants.},
}
@article {pmid32546241,
year = {2020},
author = {Zhou, X and Chen, N and Xu, H and Zhou, X and Wang, J and Fang, X and Zhang, Y and Li, Y and Yang, J and Wang, X},
title = {Regulation of Hippo-YAP signaling by insulin-like growth factor-1 receptor in the tumorigenesis of diffuse large B-cell lymphoma.},
journal = {Journal of hematology &amp; oncology},
volume = {13},
number = {1},
pages = {77},
pmid = {32546241},
issn = {1756-8722},
mesh = {Adaptor Proteins, Signal Transducing/antagonists &amp; inhibitors/deficiency/*physiology ; Aged ; Animals ; Apoptosis/drug effects ; B-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/drug effects ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Hippo Signaling Pathway ; Humans ; Insulin-Like Growth Factor I/pharmacology ; Lymphoma, Large B-Cell, Diffuse/etiology/*metabolism/pathology ; Male ; Mice ; Mice, SCID ; Middle Aged ; Neoplasm Proteins/antagonists &amp; inhibitors/*physiology ; Podophyllotoxin/analogs &amp; derivatives/pharmacology ; Protein Serine-Threonine Kinases/*physiology ; Pseudolymphoma/metabolism ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Random Allocation ; Receptor, IGF Type 1/antagonists &amp; inhibitors/biosynthesis/genetics/*physiology ; Signal Transduction/*physiology ; Transcription Factors/antagonists &amp; inhibitors/deficiency/*physiology ; Tyrphostins/pharmacology/therapeutic use ; Verteporfin/pharmacology ; Xenograft Model Antitumor Assays ; YAP-Signaling Proteins ; },
abstract = {BACKGROUND: Hippo-Yes-associated protein (YAP) signaling is a key regulator of organ size and tumorigenesis, yet the underlying molecular mechanism is still poorly understood. At present, the significance of the Hippo-YAP pathway in diffuse large B-cell lymphoma (DLBCL) is ill-defined.<br><br>METHODS: The expression of YAP in DLBCL was determined in public database and clinical specimens. The effects of YAP knockdown, CRISPR/Cas9-mediated YAP deletion, and YAP inhibitor treatment on cell proliferation and the cell cycle were evaluated both in vitro and in vivo. RNA sequencing was conducted to detect dysregulated RNAs in YAP-knockout DLBCL cells. The regulatory effects of insulin-like growth factor-1 receptor (IGF-1R) on Hippo-YAP signaling were explored by targeted inhibition and rescue experiments.<br><br>RESULTS: High expression of YAP was significantly correlated with disease progression and poor prognosis. Knockdown of YAP expression suppressed cell proliferation and induced cell cycle arrest in DLBCL cells. Verteporfin (VP), a benzoporphyrin derivative, exerted an anti-tumor effect by regulating the expression of YAP and the downstream target genes, CTGF and CYR61. In vitro and in vivo studies revealed that deletion of YAP expression with a CRISPR/Cas9 genome editing system significantly restrained tumor growth. Moreover, downregulation of IGF-1R expression led to a remarkable decrease in YAP expression. In contrast, exposure to IGF-1 promoted YAP expression and reversed the inhibition of YAP expression induced by IGF-1R inhibitors.<br><br>CONCLUSIONS: Our study highlights the critical role of YAP in the pathogenesis of DLBCL and uncovers the regulatory effect of IGF-1R on Hippo-YAP signaling, suggesting a novel therapeutic strategy for DLBCL.},
}
@article {pmid32544548,
year = {2020},
author = {Ali, HS and Boshra, MS and El Meteini, MS and Shafei, AE and Matboli, M},
title = {lncRNA- RP11-156p1.3, novel diagnostic and therapeutic targeting via CRISPR/Cas9 editing in hepatocellular carcinoma.},
journal = {Genomics},
volume = {112},
number = {5},
pages = {3306-3314},
doi = {10.1016/j.ygeno.2020.06.020},
pmid = {32544548},
issn = {1089-8646},
mesh = {Biomarkers, Tumor/blood/genetics/metabolism ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/diagnosis/*genetics/metabolism/therapy ; Female ; Gene Editing ; Hep G2 Cells ; Humans ; Liver/metabolism ; Liver Neoplasms/diagnosis/genetics/therapy ; Male ; Membrane Proteins/blood/genetics/metabolism ; MicroRNAs/blood/metabolism ; Middle Aged ; RNA, Long Noncoding/blood/*metabolism ; RNA, Messenger/blood/metabolism ; },
abstract = {We aim to characterize the expression of RNA panel in HCC. We assessed the expression of HCC-associated mRNA, miRNA and lncRNA network by real time PCR in sera and tissue samples. In a proof-of-principle approach, CRISPR cas9 mediated knock out for lncRNA- RP11-156p1.3 was performed in HEPG2 cell line to validate the role of the chosen RNA in HCC pathogenesis. The differential expression of RFTN1 mRNA, lncRNA- RP11-156p1.3 and miRNA-4764-5p was statistically different among the studied groups. After CRISPR cas9 mediated knockout of lncRNA- RP11-156p1.3 in HEPG2 cells, there was significant decrease in cell count and viability with reversal of the expression of the chosen RNAs. The chosen RNAs play a significant role in HCC pathogenesis and may be potential diagnostic and therapeutic targets.},
}
@article {pmid32544441,
year = {2020},
author = {Weissman, JL and Stoltzfus, A and Westra, ER and Johnson, PLF},
title = {Avoidance of Self during CRISPR Immunization.},
journal = {Trends in microbiology},
volume = {28},
number = {7},
pages = {543-553},
doi = {10.1016/j.tim.2020.02.005},
pmid = {32544441},
issn = {1878-4380},
mesh = {Bacteria/genetics/*virology ; Bacterial Physiological Phenomena/*immunology ; Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Immunity, Innate/physiology ; },
abstract = {The battle between microbes and their viruses is ancient and ongoing. Clustered regularly interspaced short palindromic repeat (CRISPR) immunity, the first and, to date, only form of adaptive immunity found in prokaryotes, represents a flexible mechanism to recall past infections while also adapting to a changing pathogenic environment. Critical to the role of CRISPR as an adaptive immune mechanism is its capacity for self versus non-self recognition when acquiring novel immune memories. Yet, CRISPR systems vary widely in both how and to what degree they can distinguish foreign from self-derived genetic material. We document known and hypothesized mechanisms that bias the acquisition of immune memory towards non-self targets. We demonstrate that diversity is the rule, with many widespread but no universal mechanisms for self versus non-self recognition.},
}
@article {pmid32544402,
year = {2020},
author = {Boender, AJ and Young, LJ},
title = {Oxytocin, vasopressin and social behavior in the age of genome editing: A comparative perspective.},
journal = {Hormones and behavior},
volume = {124},
number = {},
pages = {104780},
pmid = {32544402},
issn = {1095-6867},
support = {P50 MH100023/MH/NIMH NIH HHS/United States ; P51 OD011132/OD/NIH HHS/United States ; R01 MH112788/MH/NIMH NIH HHS/United States ; R21 MH114151/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Biobehavioral Sciences/history/trends ; Brain/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing/history/trends ; History, 21st Century ; Mice ; Oxytocin/*physiology ; Receptors, Oxytocin/genetics ; Receptors, Vasopressin/genetics ; *Social Behavior ; Vasopressins/*physiology ; },
abstract = {Behavioral neuroendocrinology has a rich history of using diverse model organisms to elucidate general principles and evolution of hormone-brain-behavior relationships. The oxytocin and vasopressin systems have been studied in many species, revealing their role in regulating social behaviors. Oxytocin and vasopressin receptors show remarkable species and individual differences in distribution in the brain that have been linked to diversity in social behaviors. New technologies allow for unprecedented interrogation of the genes and neural circuitry regulating behaviors, but these approaches often require transgenic models and are most often used in mice. Here we discuss seminal findings relating the oxytocin and vasopressin systems to social behavior with a focus on non-traditional animal models. We then evaluate the potential of using CRISPR/Cas9 genome editing to examine the roles of genes and enable circuit dissection, manipulation and activity monitoring of the oxytocin and vasopressin systems. We believe that it is essential to incorporate these genetic and circuit level techniques in comparative behavioral neuroendocrinology research to ensure that our field remains innovative and attractive for the next generation of investigators and funding agencies.},
}
@article {pmid32544385,
year = {2020},
author = {Lowey, B and Whiteley, AT and Keszei, AFA and Morehouse, BR and Mathews, IT and Antine, SP and Cabrera, VJ and Kashin, D and Niemann, P and Jain, M and Schwede, F and Mekalanos, JJ and Shao, S and Lee, ASY and Kranzusch, PJ},
title = {CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.},
journal = {Cell},
volume = {182},
number = {1},
pages = {38-49.e17},
pmid = {32544385},
issn = {1097-4172},
support = {R01 AI026289/AI/NIAID NIH HHS/United States ; R01 ES027595/ES/NIEHS NIH HHS/United States ; F31 CA236405/CA/NCI NIH HHS/United States ; T32 CA207021/CA/NCI NIH HHS/United States ; R37 AI018045/AI/NIAID NIH HHS/United States ; P42 ES010337/ES/NIEHS NIH HHS/United States ; R01 AI018045/AI/NIAID NIH HHS/United States ; F32 GM133063/GM/NIGMS NIH HHS/United States ; S10 OD020025/OD/NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Bacteria/*virology ; Bacterial Proteins/chemistry/metabolism ; Bacteriophages/*metabolism ; *CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; *Immunity ; Ligands ; Mutagenesis/genetics ; Nucleotidyltransferases/metabolism ; Oligonucleotides/*metabolism ; Protein Binding ; Second Messenger Systems ; *Signal Transduction ; },
abstract = {cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.},
}
@article {pmid32543746,
year = {2020},
author = {McCann, JV and Bischoff, SR and Zhang, Y and Cowley, DO and Sanchez-Gonzalez, V and Daaboul, GD and Dudley, AC},
title = {Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {7},
pages = {e23369},
pmid = {32543746},
issn = {1526-968X},
support = {F31 CA213793/CA/NCI NIH HHS/United States ; RO1-CA177874/CA/NCI NIH HHS/United States ; R01 CA177875/CA/NCI NIH HHS/United States ; P30 CA044579/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Endothelium, Vascular/drug effects/metabolism ; Extracellular Vesicles/*metabolism ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Integrases/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Tetraspanin 30/*genetics/metabolism ; },
abstract = {Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFP[loxP/stop/loxP] knock-in cassette that enables the specific labeling of circulating CD63[+] vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-Cre[ERT2] mice to generate CD63[emGFP+] vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP[+] vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63[+] EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.},
}
@article {pmid32542850,
year = {2021},
author = {Homma, Y and Hiragi, S and Fukuda, M},
title = {Rab family of small GTPases: an updated view on their regulation and functions.},
journal = {The FEBS journal},
volume = {288},
number = {1},
pages = {36-55},
pmid = {32542850},
issn = {1742-4658},
mesh = {Animals ; Biological Transport ; Eukaryotic Cells/cytology/metabolism ; GTPase-Activating Proteins/classification/*genetics/metabolism ; Guanine Nucleotide Exchange Factors/classification/*genetics/metabolism ; Guanosine Diphosphate/metabolism ; Guanosine Triphosphate/metabolism ; Humans ; Organelles/chemistry/*metabolism ; Phosphorylation ; Phylogeny ; *Protein Processing, Post-Translational ; Saccharomyces cerevisiae/genetics/metabolism ; Terminology as Topic ; Transport Vesicles/chemistry/*metabolism ; rab GTP-Binding Proteins/classification/*genetics/metabolism ; },
abstract = {The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP-loaded) state and an inactive (GDP-loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post-translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector-recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up-to-date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.},
}
@article {pmid32542601,
year = {2020},
author = {Moore, T and Chao-Shern, C and DeDionisio, L and Christie, KA and Nesbit, MA},
title = {Gene Editing for Corneal Stromal Regeneration.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2145},
number = {},
pages = {59-75},
pmid = {32542601},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Cornea/cytology/growth &amp; development ; Corneal Stroma/*cytology/growth &amp; development ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; RNA, Guide/genetics ; Regeneration/*genetics ; },
abstract = {CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.},
}
@article {pmid32542389,
year = {2020},
author = {Dhoonmoon, A and Schleicher, EM and Clements, KE and Nicolae, CM and Moldovan, GL},
title = {Genome-wide CRISPR synthetic lethality screen identifies a role for the ADP-ribosyltransferase PARP14 in DNA replication dynamics controlled by ATR.},
journal = {Nucleic acids research},
volume = {48},
number = {13},
pages = {7252-7264},
pmid = {32542389},
issn = {1362-4962},
support = {F31 CA243301/CA/NCI NIH HHS/United States ; R01 ES026184/ES/NIEHS NIH HHS/United States ; R01 GM134681/GM/NIGMS NIH HHS/United States ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Checkpoint Kinase 1/genetics/metabolism ; *DNA Replication ; Humans ; Poly(ADP-ribose) Polymerases/genetics/*metabolism ; *Synthetic Lethal Mutations ; },
abstract = {The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.},
}
@article {pmid32541958,
year = {2020},
author = {Lazzarotto, CR and Malinin, NL and Li, Y and Zhang, R and Yang, Y and Lee, G and Cowley, E and He, Y and Lan, X and Jividen, K and Katta, V and Kolmakova, NG and Petersen, CT and Qi, Q and Strelcov, E and Maragh, S and Krenciute, G and Ma, J and Cheng, Y and Tsai, SQ},
title = {CHANGE-seq reveals genetic and epigenetic effects on CRISPR-Cas9 genome-wide activity.},
journal = {Nature biotechnology},
volume = {38},
number = {11},
pages = {1317-1327},
pmid = {32541958},
issn = {1546-1696},
support = {U01 EB029373/EB/NIBIB NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; U01 HL145793/HL/NHLBI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; U01 AI157189/AI/NIAID NIH HHS/United States ; 2017093/DDCF_/Doris Duke Charitable Foundation/United States ; R35 GM133614/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chromatin/genetics ; *Epigenesis, Genetic ; Gene Editing ; Genetic Variation ; Genome, Human ; *High-Throughput Nucleotide Sequencing ; Humans ; Machine Learning ; },
abstract = {Current methods can illuminate the genome-wide activity of CRISPR-Cas9 nucleases, but are not easily scalable to the throughput needed to fully understand the principles that govern Cas9 specificity. Here we describe 'circularization for high-throughput analysis of nuclease genome-wide effects by sequencing' (CHANGE-seq), a scalable, automatable tagmentation-based method for measuring the genome-wide activity of Cas9 in vitro. We applied CHANGE-seq to 110 single guide RNA targets across 13 therapeutically relevant loci in human primary T cells and identified 201,934 off-target sites, enabling the training of a machine learning model to predict off-target activity. Comparing matched genome-wide off-target, chromatin modification and accessibility, and transcriptional data, we found that cellular off-target activity was two to four times more likely to occur near active promoters, enhancers and transcribed regions. Finally, CHANGE-seq analysis of six targets across eight individual genomes revealed that human single-nucleotide variation had significant effects on activity at ~15.2% of off-target sites analyzed. CHANGE-seq is a simplified, sensitive and scalable approach to understanding the specificity of genome editors.},
}
@article {pmid32541946,
year = {2020},
author = {Costafreda, MI and Abbasi, A and Lu, H and Kaplan, G},
title = {Exosome mimicry by a HAVCR1-NPC1 pathway of endosomal fusion mediates hepatitis A virus infection.},
journal = {Nature microbiology},
volume = {5},
number = {9},
pages = {1096-1106},
pmid = {32541946},
issn = {2058-5276},
support = {FD999999/ImFDA/Intramural FDA HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carrier Proteins/metabolism ; Cell Line ; Ebolavirus ; Endocytosis ; Endosomes/*metabolism/virology ; Exosomes/*metabolism/virology ; Gene Knockout Techniques ; HEK293 Cells ; Hepatitis A/immunology/*metabolism/virology ; Hepatitis A Virus Cellular Receptor 1/genetics/*metabolism ; Hepatitis A virus/*metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Membrane Glycoproteins ; Niemann-Pick C1 Protein ; Transcriptome ; Viral Fusion Proteins/metabolism ; Virion/metabolism ; Virus Internalization ; },
abstract = {Cell-to-cell communication by exosomes controls normal and pathogenic processes[1,2]. Viruses can spread in exosomes and thereby avoid immune recognition[3]. While biogenesis, binding and uptake of exosomes are well characterized[4,5], delivery of exosome cargo into the cytoplasm is poorly understood[3]. We report that the phosphatidylserine receptor HAVCR1 (refs. [6,7]) and the cholesterol transporter NPC1 (ref. [8]) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome[9], are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway, which Ebola virus exploits to infect cells[9], mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.},
}
@article {pmid32541929,
year = {2021},
author = {Lee, EJ and Han, JC and Park, DY and Cho, J and Kee, C},
title = {Effect of connective tissue growth factor gene editing using adeno-associated virus-mediated CRISPR-Cas9 on rabbit glaucoma filtering surgery outcomes.},
journal = {Gene therapy},
volume = {28},
number = {5},
pages = {277-286},
pmid = {32541929},
issn = {1476-5462},
mesh = {Animals ; CRISPR-Cas Systems ; Connective Tissue Growth Factor/genetics ; Dependovirus/genetics ; *Filtering Surgery ; Gene Editing ; *Glaucoma/genetics/surgery ; Rabbits ; },
abstract = {Suppressing excessive wound healing responses is critical to ensure surgical success in glaucoma filtration surgery (GFS). Currently used adjunctive materials can lead to side effects due to the nonselectivity in cell inhibition and may require repeated applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system may become a compelling opportunity in glaucoma surgery due to its high selectivity and permanent effect. Connective tissue growth factor (CTGF) is one of the most potent stimulators of tissue fibrosis in the eye. Therefore, we tested the effect of CTGF suppression using the CRISPR-Cas9 system on GFS fibrosis. We used an adeno-associated virus (AAV)-CRISPR-Cas9 system and confirmed successful CTGF suppression was achieved in fibroblasts in vitro through western blot analysis and deep sequencing. In the in vivo intereye-comparison rabbit GFS model, CRISPR-CTGF-treated eyes showed significantly better survival of the surgery site, less subconjunctival fibrosis, limited collagen deposition, and reduced cellularity than untreated eyes. Our results suggest a new possibility of CRISPR-Cas9-mediated CTGF suppression to improve human GFS outcomes.},
}
@article {pmid32541832,
year = {2020},
author = {Masuda, T and Amann, L and Sankowski, R and Staszewski, O and Lenz, M and D Errico, P and Snaidero, N and Costa Jordão, MJ and Böttcher, C and Kierdorf, K and Jung, S and Priller, J and Misgeld, T and Vlachos, A and Meyer-Luehmann, M and Knobeloch, KP and Prinz, M},
title = {Novel Hexb-based tools for studying microglia in the CNS.},
journal = {Nature immunology},
volume = {21},
number = {7},
pages = {802-815},
pmid = {32541832},
issn = {1529-2916},
support = {MC_PC_16031/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Brain/cytology/immunology/*pathology ; CRISPR-Cas Systems/genetics ; Encephalomyelitis, Autoimmune, Experimental/immunology/*pathology ; Facial Nerve Injuries/immunology/*pathology ; Gene Knock-In Techniques ; Genes, Reporter/genetics ; Genetic Loci/genetics ; Humans ; Intravital Microscopy ; Luminescent Agents/chemistry ; Luminescent Proteins/chemistry/genetics ; Macrophages/immunology/metabolism ; Mice ; Microglia/immunology/*metabolism ; NIH 3T3 Cells ; RNA-Seq ; Single-Cell Analysis ; Transfection ; beta-Hexosaminidase beta Chain/genetics/*metabolism ; },
abstract = {Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated Hexb[tdTomato] mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.},
}
@article {pmid32541822,
year = {2020},
author = {Cui, J and Techakriengkrai, N and Nedumpun, T and Suradhat, S},
title = {Abrogation of PRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {9617},
pmid = {32541822},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Cell Line ; Flow Cytometry ; *Gene Editing/methods ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Porcine respiratory and reproductive syndrome virus/*metabolism/pathogenicity ; RNA, Messenger/metabolism ; RNA, Viral/*metabolism ; Swine ; },
abstract = {CRISPR/Cas9 enables dsDNA viral genome engineering. However, the lack of RNA targeting activities limits the ability of CRISPR/Cas9 to combat RNA viruses. The recently identified class II type VI CRISPR/Cas effectors (Cas13) are RNA-targeting CRISPR enzymes that enable RNA cleavage in mammalian and plant cells. We sought to knockdown the viral RNA of porcine reproductive and respiratory syndrome virus (PRRSV) directly by exploiting the CRISPR/Cas13b system. Effective mRNA cleavage by CRISPR/Cas13b-mediated CRISPR RNA (crRNA) targeting the ORF5 and ORF7 genes of PRRSV was observed. To address the need for uniform delivery of the Cas13b protein and crRNAs, an all-in-one system expressing Cas13b and duplexed crRNA cassettes was developed. Delivery of a single vector carrying double crRNAs enabled the simultaneous knockdown of two PRRSV genes. Transgenic MARC-145 cells stably expressing the Cas13b effector and crRNA mediated by lentiviral-based transduction showed a robust ability to splice the PRRSV genomic RNA and subgenomic RNAs; viral infection was almost completely abrogated by the combination of double crRNAs simultaneously targeting the ORF5 and ORF7 genes. Our study indicated that the CRISPR/Cas13b system can effectively knockdown the PRRSV genome in vitro and can potentially be used as a potent therapeutic antiviral strategy.},
}
@article {pmid32541689,
year = {2020},
author = {Niu, A and McDougal, A and Ning, B and Safa, F and Luk, A and Mushatt, DM and Nachabe, A and Zwezdaryk, KJ and Robinson, J and Peterson, T and Socola, F and Safah, H and Hu, T and Saba, NS},
title = {COVID-19 in allogeneic stem cell transplant: high false-negative probability and role of CRISPR and convalescent plasma.},
journal = {Bone marrow transplantation},
volume = {55},
number = {12},
pages = {2354-2356},
pmid = {32541689},
issn = {1476-5365},
support = {U54 GM104940/GM/NIGMS NIH HHS/United States ; },
mesh = {Aged ; Allografts ; COVID-19/*diagnosis/*etiology/therapy ; COVID-19 Nucleic Acid Testing/*methods ; COVID-19 Serological Testing ; CRISPR-Cas Systems ; False Negative Reactions ; Female ; Hematopoietic Stem Cell Transplantation/*adverse effects ; Humans ; Immunization, Passive ; Immunocompromised Host ; Leukemia, B-Cell/complications/immunology/therapy ; Leukemia, Myeloid, Acute/complications/immunology/therapy ; Male ; Middle Aged ; Pandemics ; SARS-CoV-2 ; },
}
@article {pmid32541681,
year = {2020},
author = {Croci, S and Carriero, ML and Capitani, K and Daga, S and Donati, F and Papa, FT and Frullanti, E and Lopergolo, D and Lamacchia, V and Tita, R and Giliberti, A and Benetti, E and Niccheri, F and Furini, S and Lo Rizzo, C and Conticello, SG and Renieri, A and Meloni, I},
title = {AAV-mediated FOXG1 gene editing in human Rett primary cells.},
journal = {European journal of human genetics : EJHG},
volume = {28},
number = {10},
pages = {1446-1458},
pmid = {32541681},
issn = {1476-5438},
mesh = {Adult ; *CRISPR-Cas Systems ; Cell Transdifferentiation ; Cells, Cultured ; Cellular Reprogramming Techniques/methods ; Child, Preschool ; Dependovirus/genetics ; Female ; Fibroblasts/cytology/metabolism ; Forkhead Transcription Factors/*genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Male ; Nerve Tissue Proteins/*genetics/metabolism ; Neurons/cytology/metabolism ; Rett Syndrome/*genetics/pathology/therapy ; },
abstract = {Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry[+]/EGFP[+] transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20-35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.},
}
@article {pmid32541005,
year = {2020},
author = {Goodson, NB and Kaufman, MA and Park, KU and Brzezinski, JA},
title = {Simultaneous deletion of Prdm1 and Vsx2 enhancers in the retina alters photoreceptor and bipolar cell fate specification, yet differs from deleting both genes.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {13},
pages = {},
doi = {10.1242/dev.190272},
pmid = {32541005},
issn = {1477-9129},
support = {R01 EY024272/EY/NEI NIH HHS/United States ; T32 NS099042/NS/NINDS NIH HHS/United States ; TL1 TR002533/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Cell Differentiation/genetics/physiology ; Homeodomain Proteins/genetics/*metabolism ; Mice ; Otx Transcription Factors/genetics/metabolism ; Photoreceptor Cells/*metabolism ; Positive Regulatory Domain I-Binding Factor 1/genetics/*metabolism ; Retina/cytology/*metabolism ; Retinal Bipolar Cells/cytology/metabolism ; Transcription Factors/genetics/*metabolism ; },
abstract = {The transcription factor OTX2 is required for photoreceptor and bipolar cell formation in the retina. It directly activates the transcription factors Prdm1 and Vsx2 through cell type-specific enhancers. PRDM1 and VSX2 work in opposition, such that PRDM1 promotes photoreceptor fate and VSX2 bipolar cell fate. To determine how OTX2+ cell fates are regulated in mice, we deleted Prdm1 and Vsx2 or their cell type-specific enhancers simultaneously using a CRISPR/Cas9 in vivo retina electroporation strategy. Double gene or enhancer targeting effectively removed PRDM1 and VSX2 protein expression. However, double enhancer targeting favored bipolar fate outcomes, whereas double gene targeting favored photoreceptor fate. Both conditions generated excess amacrine cells. Combined, these fate changes suggest that photoreceptors are a default fate outcome in OTX2+ cells and that VSX2 must be present in a narrow temporal window to drive bipolar cell formation. Prdm1 and Vsx2 also appear to redundantly restrict the competence of OTX2+ cells, preventing amacrine cell formation. By taking a combinatorial deletion approach of both coding sequences and enhancers, our work provides new insights into the complex regulatory mechanisms that control cell fate choice.},
}
@article {pmid32541004,
year = {2020},
author = {Al Tanoury, Z and Rao, J and Tassy, O and Gobert, B and Gapon, S and Garnier, JM and Wagner, E and Hick, A and Hall, A and Gussoni, E and Pourquié, O},
title = {Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {12},
pages = {},
pmid = {32541004},
issn = {1477-9129},
support = {R01 AR074526/AR/NIAMS NIH HHS/United States ; U54 HD090255/HD/NICHD NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Lineage ; Cell Self Renewal ; Cells, Cultured ; Genes, Reporter ; Genetic Loci ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Muscle Fibers, Skeletal/cytology/metabolism ; Myogenin/genetics ; PAX7 Transcription Factor/genetics/*metabolism ; RNA, Guide/metabolism ; Satellite Cells, Skeletal Muscle/cytology/*metabolism ; },
abstract = {Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7[+] myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7[+] myogenic precursors. We show that the PAX7[+] cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.},
}
@article {pmid32540968,
year = {2020},
author = {Abdisalaam, S and Bhattacharya, S and Mukherjee, S and Sinha, D and Srinivasan, K and Zhu, M and Akbay, EA and Sadek, HA and Shay, JW and Asaithamby, A},
title = {Dysfunctional telomeres trigger cellular senescence mediated by cyclic GMP-AMP synthase.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {32},
pages = {11144-11160},
pmid = {32540968},
issn = {1083-351X},
support = {R01 HL115275/HL/NHLBI NIH HHS/United States ; P30 CA142543/CA/NCI NIH HHS/United States ; C06 RR030414/RR/NCRR NIH HHS/United States ; P50 CA070907/CA/NCI NIH HHS/United States ; R01 AG053341/AG/NIA NIH HHS/United States ; },
mesh = {Cell Cycle ; Cellular Senescence/*genetics ; DNA Breaks, Double-Stranded ; Humans ; Ligases/*metabolism ; Nucleotides, Cyclic/*metabolism ; Signal Transduction ; *Telomere ; },
abstract = {Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double strand breaks specifically in the telomeres, ChIP, telomere immunofluorescence, fluorescence in situ hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.},
}
@article {pmid32539998,
year = {2020},
author = {Miyamoto, T and Takada, R and Tobimatsu, Y and Suzuki, S and Yamamura, M and Osakabe, K and Osakabe, Y and Sakamoto, M and Umezawa, T},
title = {Double knockout of OsWRKY36 and OsWRKY102 boosts lignification with altering culm morphology of rice.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {296},
number = {},
pages = {110466},
doi = {10.1016/j.plantsci.2020.110466},
pmid = {32539998},
issn = {1873-2259},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Gene Editing ; Gene Knockout Techniques ; Lignin/*metabolism ; Magnetic Resonance Spectroscopy ; Oryza/*anatomy &amp; histology/genetics/metabolism ; Phylogeny ; Plant Proteins/metabolism/*physiology ; Plant Stems/*anatomy &amp; histology/genetics/metabolism ; Plants, Genetically Modified ; Transcription Factors/metabolism/*physiology ; },
abstract = {Breeding to enrich lignin, a major component of lignocelluloses, in plants contributes to enhanced applications of lignocellulosic biomass into solid biofuels and valuable aromatic chemicals. To collect information on enhancing lignin deposition in grass species, important lignocellulose feedstocks, we generated rice (Oryza sativa) transgenic lines deficient in OsWRKY36 and OsWRKY102, which encode putative transcriptional repressors for secondary cell wall formation. We used CRISPR/Cas9-mediated targeted mutagenesis and closely characterized their altered cell walls using chemical and nuclear magnetic resonance (NMR) methods. Both OsWRKY36 and OsWRKY102 mutations significantly increased lignin content by up to 28 % and 32 %, respectively. Additionally, OsWRKY36/OsWRKY102-double-mutant lines displayed lignin enrichment of cell walls (by up to 41 %) with substantially altered culm morphology over the single-mutant lines as well as the wild-type controls. Our chemical and NMR analyses showed that relative abundances of guaiacyl and p-coumarate units were slightly higher and lower, respectively, in the WRKY mutant lignins compared with those in the wild-type lignins. Our results provide evidence that both OsWRKY36 and OsWRKY102 are associated with repression of rice lignification.},
}
@article {pmid32539804,
year = {2020},
author = {Tuminauskaite, D and Norkunaite, D and Fiodorovaite, M and Tumas, S and Songailiene, I and Tamulaitiene, G and Sinkunas, T},
title = {DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {65},
pmid = {32539804},
issn = {1741-7007},
support = {S-MIP-17-47//Lietuvos Mokslo Taryba/International ; },
mesh = {Aggregatibacter actinomycetemcomitans/*genetics/metabolism ; *CRISPR-Cas Systems ; *Genes, Bacterial ; R-Loop Structures/*genetics ; },
abstract = {BACKGROUND: CRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems.<br><br>RESULTS: Here we examined the mechanism of DNA interference provided by the type I-F1 system from Aggregatibacter actinomycetemcomitans D7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32&#x2009;nt but also with shorter or longer (14-176&#x2009;nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at -1 or -2,-1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage.<br><br>CONCLUSIONS: We provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop.},
}
@article {pmid32536469,
year = {2020},
author = {Lacey, SF and Fraietta, JA},
title = {First Trial of CRISPR-Edited T cells in Lung Cancer.},
journal = {Trends in molecular medicine},
volume = {26},
number = {8},
pages = {713-715},
doi = {10.1016/j.molmed.2020.06.001},
pmid = {32536469},
issn = {1471-499X},
mesh = {CRISPR-Cas Systems ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Clustered Regularly Interspaced Short Palindromic Repeats ; Feasibility Studies ; Gene Editing ; Humans ; *Lung Neoplasms/genetics/therapy ; T-Lymphocytes ; },
abstract = {The clinical application of CRISPR-Cas9 gene editing has been eagerly awaited since the first description of the technique in 2013. Lu and colleagues now describe the treatment of 12 patients with nonsmall-cell lung cancer (NSCLC) with PD-1 gene-edited bulk autologous T cells, with results supporting both the feasibility and safety of gene editing in cell therapy.},
}
@article {pmid32535164,
year = {2020},
author = {Agarwal, N},
title = {Construction of a novel CRISPRi-based tool for silencing of multiple genes in Mycobacterium tuberculosis.},
journal = {Plasmid},
volume = {110},
number = {},
pages = {102515},
doi = {10.1016/j.plasmid.2020.102515},
pmid = {32535164},
issn = {1095-9890},
mesh = {Bacterial Proteins/*antagonists &amp; inhibitors/*genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Gene Expression Regulation, Bacterial ; *Gene Silencing ; Mycobacterium tuberculosis/*genetics/growth &amp; development ; Plasmids/*genetics ; },
abstract = {Due to lipid-rich cell wall, slow growth and pathogenic nature, it is difficult to manipulate Mycobacterium tuberculosis (Mtb) genome by conventional tools. Recently we have introduced a novel CRISPRi approach for repression of genes in mycobacteria. Although the existing CRISPRi plasmid is proven useful for silencing individual targets, disruption of multiple ORFs remains challenging in mycobacteria. Herein, we report construction of the guide sequence expressing plasmid, pGrna to facilitate cloning and expression of multiple guide sequence cassettes targeting a versatile set of Mtb genes from a single plasmid. Using the modified plasmid, pGrna2, it was shown that expression of all the 10 extracellular sigma factor-encoding genes together with sigB and sigF can be efficiently repressed in Mtb expressing dCas9. In vitro growth analysis indicates that simultaneous knockdown of these non-essential transcriptional regulators is lethal for growth. Importantly, the Δ12sig strain exhibits sensitivity to transcriptional inhibitor rifampicin and oxidative stress diamide, further implying involvement of these genes in controlling bacterial stress response. To the best of my knowledge, this is the first report wherein 12 genes have been efficiently silenced together in a single recombinant strain of Mtb. The modified pGrna2 plasmid offers a powerful tool to decipher the functioning of genes that are redundant or regulate a particular metabolic pathway and can be useful in identification of novel anti-tuberculosis drug targets.},
}
@article {pmid32534122,
year = {2020},
author = {Li, G and Zhang, X and Wang, H and Liu, D and Li, Z and Wu, Z and Yang, H},
title = {Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.},
journal = {The international journal of biochemistry &amp; cell biology},
volume = {125},
number = {},
pages = {105790},
doi = {10.1016/j.biocel.2020.105790},
pmid = {32534122},
issn = {1878-5875},
mesh = {Acetylation ; Animals ; Benzofurans/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; Cells, Cultured ; Chromatin Immunoprecipitation ; DNA End-Joining Repair/*drug effects ; DNA Repair/drug effects ; Gene Editing/*methods ; HEK293 Cells ; Histone Deacetylase Inhibitors/*pharmacology ; Humans ; Hydroxamic Acids/*pharmacology ; Ku Autoantigen/metabolism ; Poly (ADP-Ribose) Polymerase-1/metabolism ; Rad51 Recombinase/genetics/metabolism ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Recombinational DNA Repair/*drug effects ; Swine ; },
abstract = {Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.},
}
@article {pmid32534063,
year = {2020},
author = {Fourier, N and Zolty, M and Azriel, A and Tedesco, D and Levi, BZ},
title = {MafK Mediates Chromatin Remodeling to Silence IRF8 Expression in Non-immune Cells in a Cell Type-SpecificManner.},
journal = {Journal of molecular biology},
volume = {432},
number = {16},
pages = {4544-4560},
doi = {10.1016/j.jmb.2020.06.005},
pmid = {32534063},
issn = {1089-8638},
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Chromatin/*metabolism ; Chromatin Assembly and Disassembly ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Interferon Regulatory Factors/chemistry/*genetics/metabolism ; MafK Transcription Factor/*genetics/*metabolism ; Mice ; NIH 3T3 Cells ; Organ Specificity ; RAW 264.7 Cells ; RNA, Small Interfering/pharmacology ; },
abstract = {The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors, and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific transcription factor and its expression is augmented by interferon-γ. Therefore, it serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. Ahigh-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-Seq analysis revealed three MafK binding regions (-25 kb, -20 kb, and IRF8 6th intron) within the IRF8 locus. These MafK binding sites are sufficient to repress a reporter gene when cloned in genome-integrated lentiviral reporter constructs in only expression-restrictive cells. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding sites in mediating repressed chromatin assembly. Finally, a more thorough genomic analysis was performed, using CRISPR-Cas9 to delete MafK-int6 binding region in IRF8 expression-restrictive cells. Deleted clones exhibited an accessible chromatin conformation within the IRF8 locus that was accompanied by a significant increase in basal expression of IRF8 that was further induced by interferon-γ. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression in a celltype-specific manner.},
}
@article {pmid32533362,
year = {2020},
author = {Li, Y and Yang, L and Yang, J and Shi, J and Chai, P and Ge, S and Wang, Y and Fan, X and Jia, R},
title = {A novel variant in GPAA1, encoding a GPI transamidase complex protein, causes inherited vascular anomalies with various phenotypes.},
journal = {Human genetics},
volume = {139},
number = {12},
pages = {1499-1511},
doi = {10.1007/s00439-020-02192-w},
pmid = {32533362},
issn = {1432-1203},
mesh = {Acyltransferases/genetics ; Adult ; Animals ; CRISPR-Cas Systems/genetics ; Cell Movement/genetics ; Cell Proliferation/genetics ; Endothelial Cells/metabolism/pathology ; Exons/genetics ; Exophthalmos/*genetics/pathology ; Female ; Humans ; Male ; Membrane Glycoproteins/*genetics ; Middle Aged ; Multiprotein Complexes/*genetics ; Pedigree ; Phenotype ; Vascular Malformations/*genetics/pathology ; Whole Exome Sequencing ; Zebrafish/genetics ; },
abstract = {Vascular anomalies (VAs), comprising wide subtypes of tumors and malformations, are often caused by variants in multiple tyrosine kinase (TK) receptor signaling pathways including TIE2, PIK3CA and GNAQ/11. Yet, a portion of individuals with clinical features of VA do not have variants in these genes, suggesting that there are undiscovered pathogenic factors underlying these patients and possibly with overlapping phenotypes. Here, we identified one rare non-synonymous variant (c.968A > G) in the seventh exon of GPAA1 (Glycosylphosphatidylinositol Anchor Attachment Protein 1), shared by the four affected members of a large pedigree with multiple types of VA using whole-exome sequencing. GPAA1 encodes a glycosylphosphatidylinositol (GPI) transamidase complex protein. This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum (ER). We showed such variant led to scarce expression of GPAA1 protein in vascular endothelium and induced a localization change from ER membrane to cytoplasm and nucleus. In addition, expressing wild-type GPAA1 in endothelial cells had an effect to inhibit cell proliferation and migration, while expressing variant GPAA1 led to overgrowth and overmigration, indicating a loss of the quiescent status. Finally, a gpaa1-deficient zebrafish model displayed several types of developmental defects as well as vascular dysplasia, demonstrating that GPAA1 is involved in angiogenesis and vascular remodeling. Altogether, our results indicate that the rare coding variant in GPAA1 (c.968A > G) is causally related to familial forms of VAs.},
}
@article {pmid32532987,
year = {2020},
author = {Du, M and Jillette, N and Zhu, JJ and Li, S and Cheng, AW},
title = {CRISPR artificial splicing factors.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2973},
pmid = {32532987},
issn = {2041-1723},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {*Alternative Splicing ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Line, Tumor ; Exons/*genetics ; Fibroblasts/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Muscular Atrophy, Spinal/genetics/metabolism/pathology ; RNA/*genetics/metabolism ; RNA Isoforms/genetics/metabolism ; RNA Splicing Factors/*genetics/metabolism ; Survival of Motor Neuron 2 Protein/genetics/metabolism ; },
abstract = {Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.},
}
@article {pmid32532970,
year = {2020},
author = {Wetzel, L and Blanchard, S and Rama, S and Beier, V and Kaufmann, A and Wollert, T},
title = {TECPR1 promotes aggrephagy by direct recruitment of LC3C autophagosomes to lysosomes.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2993},
pmid = {32532970},
issn = {2041-1723},
mesh = {Autophagosomes/*metabolism/ultrastructure ; Autophagy/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Endosomes/metabolism ; HeLa Cells ; Humans ; Lysosomes/*metabolism/ultrastructure ; Membrane Proteins/chemistry/genetics/*metabolism ; Microscopy, Confocal ; Microscopy, Immunoelectron ; Microtubule-Associated Proteins/chemistry/genetics/*metabolism ; Neural Stem Cells/cytology/*metabolism ; Neurodegenerative Diseases/metabolism ; Protein Aggregates ; Protein Aggregation, Pathological ; Protein Binding ; Protein Transport ; Proteolysis ; RNA Interference ; },
abstract = {The accumulation of protein aggregates is involved in the onset of many neurodegenerative diseases. Aggrephagy is a selective type of autophagy that counteracts neurodegeneration by degrading such aggregates. In this study, we found that LC3C cooperates with lysosomal TECPR1 to promote the degradation of disease-related protein aggregates in neural stem cells. The N-terminal WD-repeat domain of TECPR1 selectively binds LC3C which decorates matured autophagosomes. The interaction of LC3C and TECPR1 promotes the recruitment of autophagosomes to lysosomes for degradation. Augmented expression of TECPR1 in neural stem cells reduces the number of protein aggregates by promoting their autophagic clearance, whereas knockdown of LC3C inhibits aggrephagy. The PH domain of TECPR1 selectively interacts with PtdIns(4)P to target TECPR1 to PtdIns(4)P containing lysosomes. Exchanging the PH against a tandem-FYVE domain targets TECPR1 ectopically to endosomes. This leads to an accumulation of LC3C autophagosomes at endosomes and prevents their delivery to lysosomes.},
}
@article {pmid32532247,
year = {2020},
author = {Hooton, S and D'Angelantonio, D and Hu, Y and Connerton, PL and Aprea, G and Connerton, IF},
title = {Campylobacter bacteriophage DA10: an excised temperate bacteriophage targeted by CRISPR-cas.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {400},
pmid = {32532247},
issn = {1471-2164},
support = {[grant number BB/P02355X/1]//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacteriophages/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; Campylobacter/immunology/*virology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Open Reading Frames ; Prophages/genetics ; Sequence Homology ; },
abstract = {BACKGROUND: Lytic bacteriophages that infect Campylobacter spp. have been utilized to develop therapeutic/decontamination techniques. However, the association of Campylobacter spp. and bacteriophages has been the focus of several strands of research aimed at understanding the complex relationships that have developed between predators and prey over evolutionary time. The activities of endogenous temperate bacteriophages have been used to evaluate genomic rearrangements and differential protein expression in host cells, and mechanisms of resistance to bacteriophage infection in campylobacters such as phase variation and CRISPR-mediated immunity.<br><br>RESULTS: Temperate bacteriophage DA10 represents a novel excised and infective virus capable of replication in a restricted set of C. jejuni and C. coli hosts. Whole genome sequencing reveals that DA10 (35,379&#x2009;bp) forms part of a novel group of temperate bacteriophages that have limited distribution among database host genome sequences. Analysis of potential host genomes reveals a robust response against DA10 and DA10-like bacteriophages is driven by CRISPR-mediated immunity with 75% of DA10 ORFs represented as ~&#x2009;30&#x2009;bp spacer sequences in numerous Campylobacter Type II-C CRISPR arrays. Several DA10-like homologues have been identified in a small sub-set of C. jejuni and C. coli genome sequences (ranging from near complete integrated prophage sequences to fragments recognisable in the sequence read archive).<br><br>CONCLUSIONS: A complete intact DA10-like prophage in C. jejuni CJ677CC520 provides evidence that the associations between host and DA10-like bacteriophages are long-standing in evolutionary timescales. Extensive nucleotide substitution and loss can be observed in the integrated DA10-like prophage of CJ677CC520 compared to other relatives as observed through pairwise genome comparisons. Examining factors that have limited the population expansion of the prophage, while others appear to have thrived and prospered (Mu-like, CJIE-like, and lytic Campylobacter bacteriophages) will assist in identifying the underlying evolutionary processes in the natural environment.},
}
@article {pmid32532133,
year = {2020},
author = {Park, H and Shin, J and Choi, H and Cho, B and Kim, J},
title = {Valproic Acid Significantly Improves CRISPR/Cas9-Mediated Gene Editing.},
journal = {Cells},
volume = {9},
number = {6},
pages = {},
pmid = {32532133},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/*drug effects ; Gene Editing/*methods ; Humans ; Mice ; Mice, Transgenic ; Valproic Acid/pharmacology/*therapeutic use ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has emerged as a powerful technology, with the potential to generate transgenic animals. Particularly, efficient and precise genetic editing with CRISPR/Cas9 offers immense prospects in various biotechnological applications. Here, we report that the histone deacetylase inhibitor valproic acid (VPA) significantly increases the efficiency of CRISPR/Cas9-mediated gene editing in mouse embryonic stem cells and embryos. This effect may be caused through globally enhanced chromatin accessibility, as indicate by histone hyperacetylation. Taken together, our results suggest that VPA can be used to increase the efficacy of CRISPR/Cas9 in generating transgenic systems.},
}
@article {pmid32531864,
year = {2020},
author = {Chow, SH and Deo, P and Yeung, ATY and Kostoulias, XP and Jeon, Y and Gao, ML and Seidi, A and Olivier, FAB and Sridhar, S and Nethercott, C and Cameron, D and Robertson, AAB and Robert, R and Mackay, CR and Traven, A and Jin, ZB and Hale, C and Dougan, G and Peleg, AY and Naderer, T},
title = {Targeting NLRP3 and Staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages.},
journal = {Journal of leukocyte biology},
volume = {108},
number = {3},
pages = {967-981},
doi = {10.1002/JLB.4MA0420-497R},
pmid = {32531864},
issn = {1938-3673},
mesh = {Animals ; Bacterial Proteins/*antagonists &amp; inhibitors ; Bacterial Toxins/*antagonists &amp; inhibitors ; CD11b Antigen/immunology ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Exotoxins/*antagonists &amp; inhibitors/deficiency ; Gene Knock-In Techniques ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Interleukin-1beta/metabolism ; Leukocidins/*antagonists &amp; inhibitors ; Leukocyte Common Antigens/physiology ; Lung/immunology/microbiology ; Macrophages/cytology/*drug effects/immunology ; Mice ; Mice, Inbred C57BL ; Monocytes/cytology ; NLR Family, Pyrin Domain-Containing 3 Protein/*antagonists &amp; inhibitors ; Peptide Fragments/immunology ; Pneumonia, Staphylococcal/immunology ; Protein Subunits ; Receptor, Anaphylatoxin C5a/deficiency/*drug effects/genetics/physiology ; Recombinant Proteins/metabolism ; *Staphylococcus aureus/physiology ; },
abstract = {Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1β secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1β levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1β secretion after PVL exposure and controls S. aureus lung burdens.},
}
@article {pmid32531863,
year = {2020},
author = {Li, Q and Wu, G and Zhao, Y and Wang, B and Zhao, B and Kong, D and Wei, H and Chen, C and Wang, H},
title = {CRISPR/Cas9-mediated knockout and overexpression studies reveal a role of maize phytochrome C in regulating flowering time and plant height.},
journal = {Plant biotechnology journal},
volume = {18},
number = {12},
pages = {2520-2532},
pmid = {32531863},
issn = {1467-7652},
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant/genetics ; *Phytochrome/genetics/metabolism ; Phytochrome B/metabolism ; Zea mays/genetics/metabolism ; },
abstract = {Maize is a major staple crop widely used for food, feedstocks and industrial products. Shade-avoidance syndrome (SAS), which is triggered when plants sense competition of light from neighbouring vegetation, is detrimental for maize yield production under high-density planting conditions. Previous studies have shown that the red and far-red photoreceptor phytochromes are responsible for perceiving the shading signals and triggering SAS in Arabidopsis; however, their roles in maize are less clear. In this study, we examined the expression patterns of ZmPHYC1 and ZmPHYC2 and found that ZmPHYC1, but not ZmPHYC2, is highly expressed in leaves and is regulated by the circadian clock. Both ZmPHYC1 and ZmPHYC2 proteins are localized to both the nucleus and cytoplasm under light conditions and both of them can interact with themselves or with ZmPHYBs. Heterologous expression of ZmPHYCs can complement the Arabidopsis phyC-2 mutant under constant red light conditions and confer an attenuated SAS in Arabidopsis in response to shading. Double knockout mutants of ZmPHYC1 and ZmPHYC2 created using the CRISPR/Cas9 technology display a moderate early-flowering phenotype under long-day conditions, whereas ZmPHYC2 overexpression plants exhibit a moderately reduced plant height and ear height. Together, these results provided new insight into the function of ZmPHYCs and guidance for breeding high-density tolerant maize cultivars.},
}
@article {pmid32531470,
year = {2020},
author = {Bhagwan, JR and Mosqueira, D and Chairez-Cantu, K and Mannhardt, I and Bodbin, SE and Bakar, M and Smith, JGW and Denning, C},
title = {Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics.},
journal = {Journal of molecular and cellular cardiology},
volume = {145},
number = {},
pages = {43-53},
pmid = {32531470},
issn = {1095-8584},
support = {SP/15/9/31605/BHF_/British Heart Foundation/United Kingdom ; RM/13/30157/BHF_/British Heart Foundation/United Kingdom ; MR/M017354/1/MRC_/Medical Research Council/United Kingdom ; NC/C013105/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; PG/14/59/31000/BHF_/British Heart Foundation/United Kingdom ; NC/C013202/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; RG/14/1/30588/BHF_/British Heart Foundation/United Kingdom ; P47352/CRM/BHF_/British Heart Foundation/United Kingdom ; MR/L012618/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Actins/genetics/metabolism ; Arrhythmias, Cardiac/complications/genetics/physiopathology ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Calcium/metabolism ; Cardiac Myosins/genetics/metabolism ; Cardiomyopathy, Hypertrophic/genetics/*pathology/physiopathology/*therapy ; Cell Line ; Cell Respiration ; Gene Expression Regulation ; Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/metabolism ; *Models, Cardiovascular ; Mutation/genetics ; Myocardial Contraction ; Myocytes, Cardiac/metabolism ; Myosin Heavy Chains/genetics/metabolism ; Optogenetics ; Phenotype ; Tissue Engineering ; },
abstract = {BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics.<br><br>METHODS: We directly compared the p.&#x3b2;-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines.<br><br>RESULTS: Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.&#x3b2;-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs.<br><br>CONCLUSIONS: Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a 'one-size fits all' treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy.},
}
@article {pmid32531194,
year = {2020},
author = {VanderWall, KB and Huang, KC and Pan, Y and Lavekar, SS and Fligor, CM and Allsop, AR and Lentsch, KA and Dang, P and Zhang, C and Tseng, HC and Cummins, TR and Meyer, JS},
title = {Retinal Ganglion Cells With a Glaucoma OPTN(E50K) Mutation Exhibit Neurodegenerative Phenotypes when Derived from Three-Dimensional Retinal Organoids.},
journal = {Stem cell reports},
volume = {15},
number = {1},
pages = {52-66},
pmid = {32531194},
issn = {2213-6711},
support = {R01 EY024984/EY/NEI NIH HHS/United States ; R21 EY031120/EY/NEI NIH HHS/United States ; UL1 TR002529/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Apoptosis ; Autophagy ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/*genetics ; Cell Differentiation/genetics ; Disease Models, Animal ; Down-Regulation/genetics ; Gene Editing ; Glaucoma/*genetics ; Humans ; Membrane Transport Proteins/*genetics ; Microtubule-Associated Proteins/metabolism ; Mutation/*genetics ; Nerve Degeneration/*pathology ; Organoids/*pathology ; Phenotype ; Retinal Ganglion Cells/*pathology ; Sequence Analysis, RNA ; },
abstract = {Retinal ganglion cells (RGCs) serve as the connection between the eye and the brain, with this connection disrupted in glaucoma. Numerous cellular mechanisms have been associated with glaucomatous neurodegeneration, and useful cellular models of glaucoma allow for the precise analysis of degenerative phenotypes. Human pluripotent stem cells (hPSCs) serve as powerful tools for studying human disease, particularly cellular mechanisms underlying neurodegeneration. Thus, efforts focused upon hPSCs with an E50K mutation in the Optineurin (OPTN) gene, a leading cause of inherited forms of glaucoma. CRISPR/Cas9 gene editing introduced the OPTN(E50K) mutation into existing lines of hPSCs, as well as generating isogenic controls from patient-derived lines. RGCs differentiated from OPTN(E50K) hPSCs exhibited numerous neurodegenerative deficits, including neurite retraction, autophagy dysfunction, apoptosis, and increased excitability. These results demonstrate the utility of OPTN(E50K) RGCs as an in vitro model of neurodegeneration, with the opportunity to develop novel therapeutic approaches for glaucoma.},
}
@article {pmid32531193,
year = {2020},
author = {Hunkler, HJ and Hoepfner, J and Huang, CK and Chatterjee, S and Jara-Avaca, M and Gruh, I and Bolesani, E and Zweigerdt, R and Thum, T and Bär, C},
title = {The Long Non-coding RNA Cyrano Is Dispensable for Pluripotency of Murine and Human Pluripotent Stem Cells.},
journal = {Stem cell reports},
volume = {15},
number = {1},
pages = {13-21},
pmid = {32531193},
issn = {2213-6711},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Self Renewal/genetics ; Gene Silencing ; Human Embryonic Stem Cells/metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mice, Knockout ; RNA, Long Noncoding/genetics/*metabolism ; Transcriptome/genetics ; },
abstract = {Pluripotency is tightly regulated and is crucial for stem cells and their implementation for regenerative medicine. Non-coding RNAs, especially long non-coding RNAs (lncRNAs) emerged as orchestrators of versatile (patho)-physiological processes on the transcriptional and post-transcriptional level. Cyrano, a well-conserved lncRNA, is highly expressed in stem cells suggesting an important role in pluripotency, which we aimed to investigate in loss-off-function (LOF) experiments. Cyrano was described previously to be essential for the maintenance of mouse embryonic stem cell (ESC) pluripotency. In contrast, using different genetic models, we here found Cyrano to be dispensable in murine and human iPSCs and in human ESCs. RNA sequencing revealed only a moderate influence of Cyrano on the global transcriptome. In line, Cyrano-depleted iPSCs retained the potential to differentiate into the three germ layers. In conclusion, different methods were applied for LOF studies to rule out potential off-target effects. These approaches revealed that Cyrano does not impact pluripotency.},
}
@article {pmid32531165,
year = {2020},
author = {Liu, W and An, C and Shu, X and Meng, X and Yao, Y and Zhang, J and Chen, F and Xiang, H and Yang, S and Gao, X and Gao, SS},
title = {A Dual-Plasmid CRISPR/Cas System for Mycotoxin Elimination in Polykaryotic Industrial Fungi.},
journal = {ACS synthetic biology},
volume = {9},
number = {8},
pages = {2087-2095},
doi = {10.1021/acssynbio.0c00178},
pmid = {32531165},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Chromatography, High Pressure Liquid ; Citrinin/analysis/*biosynthesis ; Fungal Proteins/genetics/metabolism ; Gene Editing/*methods ; Monascus/genetics/*metabolism ; Multigene Family ; Mutagenesis ; Plasmids/genetics/*metabolism ; },
abstract = {Mycotoxin contamination causes disease and death in both humans and animals. Monascus Red, produced by Monascus purpureus, is used as a food colorant. However, its application is limited by contamination of the nephrotoxin citrinin, which is also produced by the fungus. Suppressing citrinin production by genetic engineering is difficult in a polykaryotic fungus such as M. purpureus. Hence, we developed a CRISPR/Cas system to delete large genomic fragments in polykaryotic fungi. Protoplast preparation and regeneration were optimized, and a dual-plasmid CRISPR/Cas system was designed to enable the deletion of the 15-kb citrinin biosynthetic gene cluster in M. purpureus industrial strain KL-001. The obtained homokaryotic mutants were stable, and citrinin was unambiguously eliminated. Moreover, the Monascus Red pigment production was increased by 2-5%. Our approach provides a powerful solution to solve this long-standing problem in the food industry and enables engineering of polykaryotic fungi for mycotoxin eliminations.},
}
@article {pmid32531157,
year = {2020},
author = {Horie, F and Endo, K and Ito, K},
title = {Artificial Protein-Responsive Riboswitches Upregulate Non-AUG Translation Initiation in Yeast.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1623-1631},
doi = {10.1021/acssynbio.0c00206},
pmid = {32531157},
issn = {2161-5063},
mesh = {Aptamers, Nucleotide/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Codon, Initiator ; Peptide Chain Initiation, Translational ; Plasmids/genetics/metabolism ; RNA, Guide/metabolism ; Ribosomes/metabolism ; *Riboswitch ; Saccharomyces cerevisiae/*genetics ; Transcriptional Activation ; Up-Regulation ; },
abstract = {Artificial control of gene expression is one of the core technologies for engineering biological systems. Riboswitches are cis-acting elements on mRNA that regulate gene expression in a ligand-dependent manner often seen in prokaryotes, but rarely in eukaryotes. Because of the poor variety of such elements available in eukaryotic systems, the number of artificially engineered eukaryotic riboswitches, especially of the upregulation type, is still limited. Here, we developed a design principle for upregulation-type riboswitches that utilize non-AUG initiation induced by ribosomal stalling in a ligand-dependent manner in Saccharomyces cerevisiae. Our design principle simply required the proper positioning of a near-cognate start codon relative to the RNA aptamer. Intriguingly, the CUG codon was the most preferable for non-AUG ON switches in terms of output level and switch performance. This work establishes novel choices for artificial genetic control in eukaryotes with versatile potential for industrial and biomedical applications as well as basic research.},
}
@article {pmid32530374,
year = {2020},
author = {Parveen, S and Akhtar, N and Ghauri, MA and Akhtar, K},
title = {Conventional genetic manipulation of desulfurizing bacteria and prospects of using CRISPR-Cas systems for enhanced desulfurization activity.},
journal = {Critical reviews in microbiology},
volume = {46},
number = {3},
pages = {300-320},
doi = {10.1080/1040841X.2020.1772195},
pmid = {32530374},
issn = {1549-7828},
mesh = {*Biotransformation ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Industrial Microbiology ; Operon ; Sulfur Compounds/metabolism ; Sulfur-Reducing Bacteria/*genetics ; },
abstract = {Highly active and stable biocatalysts are the prerequisite for industrial scale application of the biodesulfurization process. Scientists are making efforts for increasing the desulfurizing activity of native strains by employing various genetic engineering approaches. Nevertheless, the achieved desulfurization rate is lower than the industrial requirements. Thus, there is a dire need to use efficient genetic tools for precise genome editing of desulfurizing bacteria for enhanced efficiency. In comparison to the previously used genetic engineering tools the newly developed CRISPR-Cas is a more efficient and simple genetic tool that has been successfully applied for targeted genome modification of eukaryotes as well as prokaryotes. In this paper, we have reviewed the approaches, previously used to enhance the biodesulfurization rates of the sulfur metabolizing microorganisms and have discussed the potential of CRISPR-Cas systems in engineering desulfurizing biocatalysts. We have also proposed a model to construct competent desulfurizing recombinants involving use of CRISPR-Cas technology. The model can be used to over-express the dsz genes under a constitutive promoter in a suitable heterologous host, to get a steady expression of desulfurization pathway. This may serve as an inducement to develop better performing desulfurizing recombinant strains using CRISPR-Cas systems, which can be helpful in increasing the rate of biodesulfurization in future.},
}
@article {pmid32529426,
year = {2020},
author = {Lee, HK and Macgregor, S and Goring, DR},
title = {A Toolkit for Teasing Apart the Early Stages of Pollen-Stigma Interactions in Arabidopsis thaliana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2160},
number = {},
pages = {13-28},
doi = {10.1007/978-1-0716-0672-8_2},
pmid = {32529426},
issn = {1940-6029},
mesh = {Arabidopsis ; CRISPR-Cas Systems ; Gene Editing/*methods ; Mutation ; Ovule/genetics/*physiology ; Plant Breeding/*methods ; *Plant Infertility ; Pollen/genetics/*physiology ; Self-Incompatibility in Flowering Plants ; },
abstract = {In hermaphroditic flowering plants, the female pistil serves as the main gatekeeper of mate acceptance as several mechanisms are present to prevent fertilization by unsuitable pollen. The characteristic Brassicaceae dry stigma at the top of pistil represents the first layer that requires pollen recognition to elicit appropriate physiological responses from the pistil. Successful pollen-stigma interactions then lead to pollen hydration, pollen germination, and pollen tube entry into the stigmatic surface. To assess these early stages in detail, our lab has used three experimental procedures to quantitatively and qualitatively characterize the outcome of compatible pollen-stigma interactions that would ultimately lead to the successful fertilization. These assays are also useful for assessing self-incompatible pollinations and mutations that affect these pathways. The model organism, Arabidopsis thaliana, offers an excellent platform for these investigations as loss-of-function or gain-of-function mutants can be easily generated using CRISPR/Cas9 technology, existing T-DNA insertion mutant collections, and heterologous expression constructs, respectively. Here, we provide a detailed description of the methods for these inexpensive assays that can be reliably used to assess pollen-stigma interactions and used to identify new players regulating these processes.},
}
@article {pmid32528975,
year = {2020},
author = {Müller-Esparza, H and Osorio-Valeriano, M and Steube, N and Thanbichler, M and Randau, L},
title = {Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {98},
pmid = {32528975},
issn = {2296-889X},
abstract = {CRISPR-Cas systems employ ribonucleoprotein complexes to identify nucleic acid targets with complementarity to bound CRISPR RNAs. Analyses of the high diversification of these effector complexes suggest that they can exhibit a wide spectrum of target requirements and binding affinities. Therefore, streamlined analysis techniques to study the interactions between nucleic acids and proteins are necessary to facilitate the characterization and comparison of CRISPR-Cas effector activities. Bio-layer Interferometry (BLI) is a technique that measures the interference pattern of white light that is reflected from a layer of biomolecules immobilized on the surface of a sensor tip (bio-layers) in real time and in solution. As streptavidin-coated sensors and biotinylated oligonucleotides are commercially available, this method enables straightforward measurements of the interaction of CRISPR-Cas complexes with different targets in a qualitative and quantitative fashion. Here, we present a general method to carry out binding assays with the Type I-Fv complex from Shewanella putrefaciens and the Type I-F complex from Shewanella baltica as model effectors. We report target specificities, dissociation constants and interactions with the Anti-CRISPR protein AcrF7 to highlight possible applications of this technique.},
}
@article {pmid32528479,
year = {2020},
author = {Huang, RS and Shih, HA and Lai, MC and Chang, YJ and Lin, S},
title = {Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {1008},
pmid = {32528479},
issn = {1664-3224},
mesh = {*Antibody-Dependent Cell Cytotoxicity ; Antigens, Differentiation, T-Lymphocyte/genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Survival ; *Clustered Regularly Interspaced Short Palindromic Repeats ; GPI-Linked Proteins/genetics/metabolism ; Gene Expression Regulation ; *Gene Targeting ; HEK293 Cells ; HeLa Cells ; Humans ; Killer Cells, Natural/*immunology/metabolism ; Neoplasms/immunology/metabolism/pathology/*therapy ; RNA, Guide/genetics/metabolism ; Receptors, IgG/genetics/metabolism ; },
abstract = {Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.},
}
@article {pmid32528454,
year = {2020},
author = {Wang, G and Liu, Q and Pei, Z and Wang, L and Tian, P and Liu, Z and Zhao, J and Zhang, H and Chen, W},
title = {The Diversity of the CRISPR-Cas System and Prophages Present in the Genome Reveals the Co-evolution of Bifidobacterium pseudocatenulatum and Phages.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1088},
pmid = {32528454},
issn = {1664-302X},
abstract = {Diverse CRISPR-Cas systems constitute an indispensable part of the bacterial adaptive immune system against viral infections. However, to escape from this immune system, bacteriophages have also evolved corresponding anti-defense measures. We investigated the diversity of CRISPR-Cas systems and the presence of prophages in the genomes of 66 Bifidobacterium pseudocatenulatum strains. Our findings revealed a high occurrence of complete CRISPR-Cas systems (62%, 41/66) in the B. pseudocatenulatum genomes. Subtypes I-C, I-U and II-A, were found to be widespread in this species. No significant association was found between the number of bacterial CRISPR spacers and its host's age. This study on prophages within B. pseudocatenulatum genomes revealed that prophage genes related to distinct functional modules became degraded at different levels, indicating that these prophages were not likely to enter lytic cycle spontaneously. Further, the evolutionary analysis of prophages in this study revealed that they might be derived from different phage ancestors. Notably, self-targeting phenomenon within B. pseudocatenulatum and Anti-CRISPR (Acr) coding genes in prophages was observed. Overall, our results indicate that the competition between B. pseudocatenulatum and phages is a major driving factor for the genomic diversity of both partners.},
}
@article {pmid32528131,
year = {2020},
author = {Awah, CU and Chen, L and Bansal, M and Mahajan, A and Winter, J and Lad, M and Warnke, L and Gonzalez-Buendia, E and Park, C and Zhang, D and Feldstein, E and Yu, D and Zannikou, M and Balyasnikova, IV and Martuscello, R and Konerman, S and Győrffy, B and Burdett, KB and Scholtens, DM and Stupp, R and Ahmed, A and Hsu, P and Sonabend, AM},
title = {Ribosomal protein S11 influences glioma response to TOP2 poisons.},
journal = {Oncogene},
volume = {39},
number = {27},
pages = {5068-5081},
pmid = {32528131},
issn = {1476-5594},
support = {DP5 OD021356/OD/NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; P30 CA060553/CA/NCI NIH HHS/United States ; P50 CA221747/CA/NCI NIH HHS/United States ; },
mesh = {Apoptotic Protease-Activating Factor 1/metabolism ; Brain Neoplasms/*drug therapy/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Breaks, Double-Stranded/drug effects ; DNA Repair/drug effects ; DNA Topoisomerases, Type II/metabolism ; Doxorubicin/pharmacology ; Etoposide/*pharmacology ; Gene Knockout Techniques ; Glioblastoma/*drug therapy/genetics ; Humans ; Ribosomal Proteins/*metabolism ; Topoisomerase II Inhibitors/*pharmacology ; },
abstract = {Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas, the most malignant of all primary brain tumors in adults, is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, and 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of proapoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers.},
}
@article {pmid32527834,
year = {2020},
author = {Liu, Y and Zou, RS and He, S and Nihongaki, Y and Li, X and Razavi, S and Wu, B and Ha, T},
title = {Very fast CRISPR on demand.},
journal = {Science (New York, N.Y.)},
volume = {368},
number = {6496},
pages = {1265-1269},
pmid = {32527834},
issn = {1095-9203},
support = {R35 GM122569/GM/NIGMS NIH HHS/United States ; T32 GM136577/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA Cleavage/*radiation effects ; DNA Repair/*genetics ; Gene Editing/*methods ; HEK293 Cells ; Histones/metabolism ; Humans ; Light ; MRE11 Homologue Protein/genetics ; Optical Imaging/methods ; Phosphorylation ; Single-Cell Analysis/*methods ; },
abstract = {CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.},
}
@article {pmid32527526,
year = {2020},
author = {Yang, Y and Liu, G and Chen, X and Liu, M and Zhan, C and Liu, X and Bai, Z},
title = {High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris.},
journal = {Enzyme and microbial technology},
volume = {138},
number = {},
pages = {109556},
doi = {10.1016/j.enzmictec.2020.109556},
pmid = {32527526},
issn = {1879-0909},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Genome, Fungal/genetics ; Nucleotide Motifs ; Plasmids/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/chemistry/*genetics ; Saccharomycetales/*genetics/growth &amp; development ; },
abstract = {Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.},
}
@article {pmid32527434,
year = {2020},
author = {Douris, V and Denecke, S and Van Leeuwen, T and Bass, C and Nauen, R and Vontas, J},
title = {Using CRISPR/Cas9 genome modification to understand the genetic basis of insecticide resistance: Drosophila and beyond.},
journal = {Pesticide biochemistry and physiology},
volume = {167},
number = {},
pages = {104595},
doi = {10.1016/j.pestbp.2020.104595},
pmid = {32527434},
issn = {1095-9939},
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila ; Drosophila melanogaster ; *Insecticide Resistance ; *Insecticides ; },
abstract = {Chemical insecticides are a major tool for the control of many of the world's most damaging arthropod pests. However, their intensive application is often associated with the emergence of resistance, sometimes with serious implications for sustainable pest control. To mitigate failure of insecticide-based control tools, the mechanisms by which insects have evolved resistance must be elucidated. This includes both identification and functional characterization of putative resistance genes and/or mutations. Research on this topic has been greatly facilitated by using powerful genetic model insects like Drosophila melanogaster, and more recently by advances in genome modification technology, notably CRISPR/Cas9. Here, we present the advances that have been made through the application of genome modification technology in insecticide resistance research. The majority of the work conducted in the field to date has made use of genetic tools and resources available in D. melanogaster. This has greatly enhanced our understanding of resistance mechanisms, especially those mediated by insensitivity of the pesticide target-site. We discuss this progress for a series of different insecticide targets, but also report a number of unsuccessful or inconclusive attempts that highlight some inherent limitations of using Drosophila to characterize resistance mechanisms identified in arthropod pests. We also discuss an experimental framework that may circumvent current limitations while retaining the genetic versatility and robustness that Drosophila has to offer. Finally, we describe examples of direct CRISPR/Cas9 use in non-model pest species, an approach that will likely find much wider application in the near future.},
}
@article {pmid32526160,
year = {2020},
author = {Alix, E and Godlee, C and Cerny, O and Blundell, S and Tocci, R and Matthews, S and Liu, M and Pruneda, JN and Swatek, KN and Komander, D and Sleap, T and Holden, DW},
title = {The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules.},
journal = {Cell host &amp; microbe},
volume = {28},
number = {1},
pages = {54-68.e7},
pmid = {32526160},
issn = {1934-6069},
support = {209411/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 724804/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Antigen Presentation ; Bacterial Proteins/*physiology ; CRISPR-Cas Systems ; Cell Line ; Dendritic Cells/immunology/microbiology ; Female ; Histocompatibility Antigens Class II/*metabolism ; Host-Pathogen Interactions ; Humans ; Lymphocyte Activation ; Membrane Proteins/*physiology ; Mice ; Mice, Inbred C57BL ; Mutation ; Protein Binding ; Salmonella Infections/immunology/microbiology ; Salmonella typhimurium/*physiology ; T-Lymphocytopenia, Idiopathic CD4-Positive/immunology/microbiology ; Ubiquitin-Protein Ligases/*physiology ; *Ubiquitination ; Virulence ; },
abstract = {The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4[+] T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.},
}
@article {pmid32526136,
year = {2020},
author = {Roux, I and Woodcraft, C and Hu, J and Wolters, R and Gilchrist, CLM and Chooi, YH},
title = {CRISPR-Mediated Activation of Biosynthetic Gene Clusters for Bioactive Molecule Discovery in Filamentous Fungi.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1843-1854},
doi = {10.1021/acssynbio.0c00197},
pmid = {32526136},
issn = {2161-5063},
mesh = {Aspergillus nidulans/*genetics ; Bacterial Proteins/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Culture Media ; Drug Discovery/*methods ; Endodeoxyribonucleases/genetics ; Firmicutes/enzymology ; *Genes, Fungal ; *Multigene Family ; Peptide Synthases/genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Streptococcus pyogenes/enzymology ; Temperature ; Transcription, Genetic/genetics ; *Transcriptional Activation ; },
abstract = {Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGCs could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multigene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12a[D156R]-VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.},
}
@article {pmid32525845,
year = {2020},
author = {Xiao, S and Shimura, D and Baum, R and Hernandez, DM and Agvanian, S and Nagaoka, Y and Katsumata, M and Lampe, PD and Kleber, AG and Hong, T and Shaw, RM},
title = {Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias.},
journal = {The Journal of clinical investigation},
volume = {130},
number = {9},
pages = {4858-4870},
pmid = {32525845},
issn = {1558-8238},
support = {R01 HL133286/HL/NHLBI NIH HHS/United States ; R01 HL136463/HL/NHLBI NIH HHS/United States ; P30 DK017047/DK/NIDDK NIH HHS/United States ; R01 HL138577/HL/NHLBI NIH HHS/United States ; R01 HL152691/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Arrhythmias, Cardiac/genetics/*metabolism/pathology ; CRISPR-Cas Systems ; Connexin 43/genetics/*metabolism ; Gap Junctions/genetics/*metabolism/pathology ; Heart Ventricles/*metabolism/pathology ; Mice ; Mice, Mutant Strains ; Protein Transport ; *Proteolysis ; },
abstract = {Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.},
}
@article {pmid32525760,
year = {2021},
author = {Breunig, CT and Köferle, A and Neuner, AM and Wiesbeck, MF and Baumann, V and Stricker, SH},
title = {CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing.},
journal = {Physiological reviews},
volume = {101},
number = {1},
pages = {177-211},
doi = {10.1152/physrev.00034.2019},
pmid = {32525760},
issn = {1522-1210},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Physiological Phenomena/*physiology ; *Epigenesis, Genetic ; Epigenomics ; *Gene Editing ; Genetic Engineering/*methods ; Humans ; Physiology/*methods ; Transcription, Genetic ; },
abstract = {Given the large amount of genome-wide data that have been collected during the last decades, a good understanding of how and why cells change during development, homeostasis, and disease might be expected. Unfortunately, the opposite is true; triggers that cause cellular state changes remain elusive, and the underlying molecular mechanisms are poorly understood. Although genes with the potential to influence cell states are known, the historic dependency on methods that manipulate gene expression outside the endogenous chromatin context has prevented us from understanding how cells organize, interpret, and protect cellular programs. Fortunately, recent methodological innovations are now providing options to answer these outstanding questions, by allowing to target and manipulate individual genomic and epigenomic loci. In particular, three experimental approaches are now feasible due to DNA targeting tools, namely, activation and/or repression of master transcription factors in their endogenous chromatin context; targeting transcription factors to endogenous, alternative, or inaccessible sites; and finally, functional manipulation of the chromatin context. In this article, we discuss the molecular basis of DNA targeting tools and review the potential of these new technologies before we summarize how these have already been used for the manipulation of cellular states and hypothesize about future applications.},
}
@article {pmid32525258,
year = {2020},
author = {Rondon-Galeano, M and Skoczylas, R and Bower, NI and Simons, C and Gordon, E and Francois, M and Koltowska, K and Hogan, BM},
title = {MAFB modulates the maturation of lymphatic vascular networks in mice.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {249},
number = {10},
pages = {1201-1216},
doi = {10.1002/dvdy.209},
pmid = {32525258},
issn = {1097-0177},
support = {1083811//National Health and Medical Research Council/International ; 1107755//National Health and Medical Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Crosses, Genetic ; Genome ; Genotype ; In Situ Hybridization ; Lymphangiogenesis/*physiology ; Lymphatic Vessels/*metabolism ; MafB Transcription Factor/*physiology ; Mice ; Mice, Knockout ; Mutation ; RNA, Messenger/metabolism ; Signal Transduction ; Time Factors ; },
abstract = {BACKGROUND: Lymphatic vessels play key roles in tissue fluid homeostasis, immune cell trafficking and in diverse disease settings. Lymphangiogenesis requires lymphatic endothelial cell (LEC) differentiation, proliferation, migration, and co-ordinated network formation, yet the transcriptional regulators underpinning these processes remain to be fully understood. The transcription factor MAFB was recently identified as essential for lymphangiogenesis in zebrafish and in cultured human LECs. MAFB is activated in response to VEGFC-VEGFR3 signaling and acts as a downstream effector. However, it remains unclear if the role of MAFB in lymphatic development is conserved in the mammalian embryo.<br><br>RESULTS: We generated a Mafb loss-of-function mouse using CRISPR/Cas9 gene editing. Mafb mutant mice presented with perinatal lethality associated with cyanosis. We identify a role for MAFB in modifying lymphatic network morphogenesis in the developing dermis, as well as developing and postnatal diaphragm. Furthermore, mutant vessels displayed excessive smooth muscle cell coverage, suggestive of a defect in the maturation of lymphatic networks.<br><br>CONCLUSIONS: This work confirms a conserved role for MAFB in murine lymphatics that is subtle and modulatory and may suggest redundancy in MAF family transcription factors during lymphangiogenesis.},
}
@article {pmid32524868,
year = {2020},
author = {Ercu, M and Markó, L and Schächterle, C and Tsvetkov, D and Cui, Y and Maghsodi, S and Bartolomaeus, TUP and Maass, PG and Zühlke, K and Gregersen, N and Hübner, N and Hodge, R and Mühl, A and Pohl, B and Illas, RM and Geelhaar, A and Walter, S and Napieczynska, H and Schelenz, S and Taube, M and Heuser, A and Anistan, YM and Qadri, F and Todiras, M and Plehm, R and Popova, E and Langanki, R and Eichhorst, J and Lehmann, M and Wiesner, B and Russwurm, M and Forslund, SK and Kamer, I and Müller, DN and Gollasch, M and Aydin, A and Bähring, S and Bader, M and Luft, FC and Klussmann, E},
title = {Phosphodiesterase 3A and Arterial Hypertension.},
journal = {Circulation},
volume = {142},
number = {2},
pages = {133-149},
doi = {10.1161/CIRCULATIONAHA.119.043061},
pmid = {32524868},
issn = {1524-4539},
mesh = {Alleles ; Amino Acid Substitution ; Animals ; Animals, Genetically Modified ; Arterial Pressure ; Biomarkers/blood/urine ; Brachydactyly/diagnosis/genetics ; CRISPR-Cas Systems ; Cyclic Nucleotide Phosphodiesterases, Type 3/*genetics/metabolism ; DNA Mutational Analysis ; Disease Models, Animal ; Enzyme Activation ; Gene Targeting ; *Genetic Association Studies/methods ; *Genetic Predisposition to Disease ; Genotype ; Hypertension/*genetics ; Immunohistochemistry ; Isoenzymes ; Male ; *Mutation ; Pedigree ; Phenotype ; Radiography ; Rats ; Renin-Angiotensin System/genetics ; },
abstract = {BACKGROUND: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (PDE3A); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking.<br><br>METHODS: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways.<br><br>RESULTS: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3&#x3b8; adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function.<br><br>CONCLUSIONS: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.},
}
@article {pmid32524554,
year = {2020},
author = {Popiel, E and Derry, WB},
title = {Generation and Analysis of CCM Phenotypes in C. elegans.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2152},
number = {},
pages = {191-205},
doi = {10.1007/978-1-0716-0640-7_15},
pmid = {32524554},
issn = {1940-6029},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/genetics/metabolism ; Gene Editing ; Genetic Predisposition to Disease ; Hemangioma, Cavernous, Central Nervous System/diagnosis/*etiology/*metabolism ; Mutagenesis ; Mutation ; *Phenotype ; Ribonucleoproteins/metabolism ; },
abstract = {This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes kri-1 and ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.},
}
@article {pmid32524552,
year = {2020},
author = {Schwefel, K and Spiegler, S and Much, CD and Felbor, U and Rath, M},
title = {CRISPR/Cas9-mediated Generation of Human Endothelial Cell Knockout Models of CCM Disease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2152},
number = {},
pages = {169-177},
doi = {10.1007/978-1-0716-0640-7_13},
pmid = {32524552},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Endothelial Cells/*metabolism ; *Gene Editing ; *Gene Knockout Techniques ; Genetic Association Studies ; Genetic Predisposition to Disease ; Hemangioma, Cavernous, Central Nervous System/diagnosis/*genetics/metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; },
abstract = {The CRISPR/Cas9 system is a versatile tool that enables targeted genome editing in various cell types, including hard-to-transfect endothelial cells. The required crRNA, tracrRNA, and Cas9 protein have mostly been introduced into endothelial cells by viral transduction or plasmid transfection so far. We here describe an effective lipofection-based delivery of pre-complexed crRNA:tracrRNA:Cas9 ribonucleoproteins into human umbilical vein endothelial cells (HUVEC) and immortalized HUVEC (CI-huVEC). Complete inactivation of either CCM1, CCM2, or CCM3 in endothelial cells mimics the situation in cavernous lesions of CCM patients and thus represents a suitable model for future studies.},
}
@article {pmid32524473,
year = {2020},
author = {Lovell, L and Bramley, J and Buchser, W},
title = {Planning and Analysis of Axon Degeneration Screening Experiments.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2143},
number = {},
pages = {63-82},
doi = {10.1007/978-1-0716-0585-1_6},
pmid = {32524473},
issn = {1940-6029},
mesh = {Animals ; Axons/*physiology ; Axotomy ; CRISPR-Cas Systems ; Combinatorial Chemistry Techniques ; Computer Simulation ; Drug Evaluation, Preclinical/methods ; Finches/embryology ; High-Throughput Screening Assays/instrumentation/*methods ; Image Processing, Computer-Assisted ; Nerve Degeneration/*physiopathology ; Phenotype ; Quality Control ; RNA Interference ; Retinal Ganglion Cells/cytology ; Sensitivity and Specificity ; Support Vector Machine ; },
abstract = {A network of intersecting molecular pathways interacts to initiate and execute axon destruction. Maximum protection against axon degeneration likely requires more than manipulation of a single target. Here, we describe the process of designing a high-throughput arrayed screening assay for the identification of key factors responsible for axon destruction and/or protection. First, we go over some existing screens in the literature, then discuss the planning, tracking, analysis, and statistics around such a screening experiment. Prioritization of perturbations may allow laboratories to cost-effectively explore the process of screening. We also present the pairing of a combinatorial drug screen with a machine learning algorithm, predicting how to best modulate neurodegenerative and neuroprotective components.},
}
@article {pmid32523110,
year = {2020},
author = {Kiga, K and Tan, XE and Ibarra-Chávez, R and Watanabe, S and Aiba, Y and Sato'o, Y and Li, FY and Sasahara, T and Cui, B and Kawauchi, M and Boonsiri, T and Thitiananpakorn, K and Taki, Y and Azam, AH and Suzuki, M and Penadés, JR and Cui, L},
title = {Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2934},
pmid = {32523110},
issn = {2041-1723},
support = {MC_PC_17160/MRC_/Medical Research Council/United Kingdom ; MC_PC_18048/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Anti-Infective Agents/*pharmacology ; CRISPR-Cas Systems/*genetics ; Escherichia coli/drug effects/genetics ; Methicillin-Resistant Staphylococcus aureus/drug effects ; Microbial Sensitivity Tests ; Staphylococcus aureus/drug effects/genetics ; },
abstract = {The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.},
}
@article {pmid32522930,
year = {2020},
author = {Yamakawa-Kobayashi, K and Ohhara, Y and Kawashima, T and Ohishi, Y and Kayashima, Y},
title = {Loss of CNDP causes a shorter lifespan and higher sensitivity to oxidative stress in Drosophila melanogaster.},
journal = {Biomedical research (Tokyo, Japan)},
volume = {41},
number = {3},
pages = {131-138},
doi = {10.2220/biomedres.41.131},
pmid = {32522930},
issn = {1880-313X},
mesh = {Animals ; Animals, Genetically Modified ; Antioxidants/metabolism ; Base Sequence ; CRISPR-Cas Systems ; Carnosine/metabolism ; Dipeptidases/deficiency/*genetics ; Drosophila Proteins/deficiency/*genetics ; Drosophila melanogaster/drug effects/enzymology/*genetics/growth &amp; development ; Gene Editing ; Gene Expression ; Glutathione/metabolism ; Hydrogen Peroxide/pharmacology ; Longevity/drug effects/*genetics ; Male ; *Mutation ; Oxidative Stress ; Paraquat/pharmacology ; Reactive Oxygen Species/*metabolism ; },
abstract = {Increasing oxidative stress seems to be the result of an imbalance between free radical production and antioxidant defenses. During the course of aging, oxidative stress causes tissue/cellular damage, which is implicated in numerous age-related diseases. Carnosinase (CN or CNDP) is dipeptidase, which is associated with carnosine and/or glutathione (GSH) metabolism, those are the most abundant naturally occurring endogenous dipeptide and tripeptides with antioxidant and free radical scavenger properties. In the present study, we generated Drosophila cndp (dcndp) mutant flies using the CRISPR/Cas9 system to study the roles of dcndp in vivo. We demonstrate that dcndp mutant flies exhibit shorter lifespan and increased sensitivity to paraquat or hydrogen peroxide (H2O2) induced oxidative stress. These results suggest that dcndp maintains homeostatic conditions, protecting cells and tissues against the harmful effects of oxidative stress in the course of aging.},
}
@article {pmid32522888,
year = {2020},
author = {Bae, B and Gruner, HN and Lynch, M and Feng, T and So, K and Oliver, D and Mastick, GS and Yan, W and Pieraut, S and Miura, P},
title = {Elimination of Calm1 long 3'-UTR mRNA isoform by CRISPR-Cas9 gene editing impairs dorsal root ganglion development and hippocampal neuron activation in mice.},
journal = {RNA (New York, N.Y.)},
volume = {26},
number = {10},
pages = {1414-1430},
pmid = {32522888},
issn = {1469-9001},
support = {P30 GM110767/GM/NIGMS NIH HHS/United States ; R35 GM138319/GM/NIGMS NIH HHS/United States ; R01 NS114219/NS/NINDS NIH HHS/United States ; R01 EY025205/EY/NEI NIH HHS/United States ; P20 GM103650/GM/NIGMS NIH HHS/United States ; P20 GM103554/GM/NIGMS NIH HHS/United States ; R56 NS114219/NS/NINDS NIH HHS/United States ; },
mesh = {3' Untranslated Regions/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Calmodulin/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Ganglia, Spinal/*physiology ; Gene Editing/methods ; Hippocampus/*physiology ; Mice ; Mice, Inbred C57BL ; Neurons/*physiology ; Polyadenylation/genetics ; Pregnancy ; RNA Isoforms/*genetics ; RNA, Messenger/*genetics ; },
abstract = {The majority of mouse and human genes are subject to alternative cleavage and polyadenylation (APA), which most often leads to the expression of two or more alternative length 3' untranslated region (3'-UTR) mRNA isoforms. In neural tissues, there is enhanced expression of APA isoforms with longer 3'-UTRs on a global scale, but the physiological relevance of these alternative 3'-UTR isoforms is poorly understood. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that generates short (Calm1-S) and long (Calm1-L) 3'-UTR mRNA isoforms via APA. We found Calm1-L expression to be largely restricted to neural tissues in mice including the dorsal root ganglion (DRG) and hippocampus, whereas Calm1-S was more broadly expressed. smFISH revealed that both Calm1-S and Calm1-L were subcellularly localized to neural processes of primary hippocampal neurons. In contrast, cultured DRG showed restriction of Calm1-L to soma. To investigate the in vivo functions of Calm1-L, we implemented a CRISPR-Cas9 gene editing strategy to delete a small region encompassing the Calm1 distal poly(A) site. This eliminated Calm1-L expression while maintaining expression of Calm1-S Mice lacking Calm1-L (Calm1[ΔL/ΔL]) exhibited disorganized DRG migration in embryos, and reduced experience-induced neuronal activation in the adult hippocampus. These data indicate that Calm1-L plays functional roles in the central and peripheral nervous systems.},
}
@article {pmid32522852,
year = {2020},
author = {Thamamongood, T and Aebischer, A and Wagner, V and Chang, MW and Elling, R and Benner, C and García-Sastre, A and Kochs, G and Beer, M and Schwemmle, M},
title = {A Genome-Wide CRISPR-Cas9 Screen Reveals the Requirement of Host Cell Sulfation for Schmallenberg Virus Infection.},
journal = {Journal of virology},
volume = {94},
number = {17},
pages = {},
pmid = {32522852},
issn = {1098-5514},
support = {U19 AI135972/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bunyaviridae ; Bunyaviridae Infections/*genetics/*virology ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats ; Europe ; Gene Knockout Techniques ; HEK293 Cells ; Heparitin Sulfate/metabolism ; Humans ; Livestock ; Membrane Glycoproteins/genetics ; Orthobunyavirus/*genetics/pathogenicity/*physiology ; Rift Valley fever virus ; Sulfate Transporters/metabolism ; Sulfotransferases/metabolism ; Vero Cells ; Virus Attachment ; },
abstract = {Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses.IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.},
}
@article {pmid32522779,
year = {2020},
author = {Liu, Y and Pan, C and Ye, L and Si, Y and Bi, C and Hua, X and Yu, Y and Zhu, L and Wang, H},
title = {Nonclassical Biofilms Induced by DNA Breaks in Klebsiella pneumoniae.},
journal = {mSphere},
volume = {5},
number = {3},
pages = {},
pmid = {32522779},
issn = {2379-5042},
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Biofilms/*classification/drug effects/*growth &amp; development ; CRISPR-Cas Systems ; *DNA Breaks ; Gene Expression Regulation, Bacterial ; Humans ; Klebsiella Infections/microbiology ; Klebsiella pneumoniae/drug effects/*genetics/*physiology ; Liver/microbiology ; Mice ; },
abstract = {Biofilms usually form when the density of bacteria increases during the middle to late periods of growth in culture, commonly induced by quorum-sensing systems. Biofilms attach to the surfaces of either living or nonliving objects and protect bacteria against antibiotics and a host's immune system. Here, a novel type of biofilm (the "R-biofilm") is reported. These biofilms were formed by clinically isolated Klebsiella pneumoniae strains following double-stranded-DNA breaks (DSBs), while undamaged bacteria did not form classic biofilms even in the later stages of growth. R-biofilms had a fixed ring-like or discoid shape with good ductility and could protect many living bacterial cells within. We show that extracellular proteins and DNAs released, probably by dead bacteria, were the core structural materials of R-biofilms. We anticipate that novel signaling pathways besides the bacterial SOS response are involved in R-biofilm formation. The observations in this study suggest a limitation to the use of the currently popular Cas9-mediated bactericidal tools to eliminate certain bacteria because the resulting DSBs may lead to the formation of these protective R-biofilms.IMPORTANCE Many pathogenic bacteria can form biofilm matrices that consist of complex molecules such as polysaccharides, proteins, and DNA. These biofilms help the bacteria to infect and colonize a host. Such biofilms may attach and develop on the surfaces of indwelling medical devices or other supportive environments. This study found that following double-strand breaks in their DNA, Klebsiella pneumoniae cells can form a novel type of biofilm with ring-like or discoid morphology. This biofilm structure, named the "R-biofilm," helps protect the bacteria against adverse conditions such as exposure to ethanol, hydrogen peroxide, and UV radiation.},
}
@article {pmid32521708,
year = {2020},
author = {McBeath, E and Parker-Thornburg, J and Fujii, Y and Aryal, N and Smith, C and Hofmann, MC and Abe, JI and Fujiwara, K},
title = {Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice.},
journal = {Genes},
volume = {11},
number = {6},
pages = {},
pmid = {32521708},
issn = {2073-4425},
support = {R01HL13019/NH/NIH HHS/United States ; R01 HD081244/HD/NICHD NIH HHS/United States ; R01HD081244/NH/NIH HHS/United States ; R01HL149303/NH/NIH HHS/United States ; R03 HD101650/HD/NICHD NIH HHS/United States ; P30CA016672/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R50 CA211121/CA/NCI NIH HHS/United States ; R01HL134740/NH/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Blastocyst/metabolism ; CRISPR-Cas Systems/*genetics ; *Genetic Engineering ; Male ; Mice ; Mice, Knockout ; Mutation/genetics ; RNA, Guide/*genetics ; },
abstract = {Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.},
}
@article {pmid32521498,
year = {2020},
author = {Dodsworth, BT and Hatje, K and Meyer, CA and Flynn, R and Cowley, SA},
title = {Rates of homology directed repair of CRISPR-Cas9 induced double strand breaks are lower in naïve compared to primed human pluripotent stem cells.},
journal = {Stem cell research},
volume = {46},
number = {},
pages = {101852},
pmid = {32521498},
issn = {1876-7753},
support = {MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Gene Editing ; Humans ; *Pluripotent Stem Cells ; Recombinational DNA Repair ; },
abstract = {Gene editing in human pluripotent stem cells (hPSC) is a powerful tool for understanding biology, for drug discovery and gene therapy. Naïve hPSC have been suggested to be superior for gene editing compared to conventional 'primed' hPSC. Using droplet digital PCR, we uncover the kinetics of Cas9-induced double strand break repair in conventional hPSC. Cut but unrepaired alleles reach their maximum after 12-24 h. Homology directed repair plateaus after 24 h, whereas repair by non-homologous end joining continues until 48 h after Cas9 introduction. Using this method, we demonstrate that the rate of homology directed repair to resolve Cas9-induced double strand breaks is 40% lower in naïve hPSC compared to conventional hPSC, correlating with, and feasibly explained by, a higher number of cells in G1 phase of the cell cycle in naïve hPSC. Therefore, naïve hPSC are less efficient for CRISPR/Cas9-mediated homology directed repair.},
}
@article {pmid32521225,
year = {2020},
author = {Dirac-Svejstrup, AB and Walker, J and Faull, P and Encheva, V and Akimov, V and Puglia, M and Perkins, D and Kümper, S and Hunjan, SS and Blagoev, B and Snijders, AP and Powell, DJ and Svejstrup, JQ},
title = {DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.},
journal = {Molecular cell},
volume = {79},
number = {2},
pages = {332-341.e7},
pmid = {32521225},
issn = {1097-4164},
support = {/WT_/Wellcome Trust/United Kingdom ; FC001166/CRUK_/Cancer Research UK/United Kingdom ; FC001166/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Aspartic Acid Proteases/genetics/*metabolism ; Binding Sites ; CRISPR-Cas Systems ; Cell Line ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Nuclear Respiratory Factor 1/*metabolism ; Protein Biosynthesis ; Proteolysis ; Ubiquitin/*metabolism ; },
abstract = {The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.},
}
@article {pmid32519675,
year = {2020},
author = {Cofsky, JC and Karandur, D and Huang, CJ and Witte, IP and Kuriyan, J and Doudna, JA},
title = {CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32519675},
issn = {2050-084X},
support = {T32 GM008295/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; MCB-1817593//National Science Foundation/International ; },
mesh = {*Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; DNA/genetics/metabolism ; *DNA Breaks, Double-Stranded ; *Endodeoxyribonucleases/genetics/metabolism ; Escherichia coli/genetics ; Gene Editing/*methods ; R-Loop Structures/*genetics ; RNA, Guide/genetics/metabolism ; },
abstract = {Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3' side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.},
}
@article {pmid32519310,
year = {2020},
author = {Hirata, M and Wittayarat, M and Namula, Z and Le, QA and Lin, Q and Nguyen, NT and Takebayashi, K and Sato, Y and Tanihara, F and Otoi, T},
title = {Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation.},
journal = {Molecular biology reports},
volume = {47},
number = {7},
pages = {5073-5079},
doi = {10.1007/s11033-020-05576-3},
pmid = {32519310},
issn = {1573-4978},
mesh = {Animals ; Blastocyst/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Electroporation/*methods/veterinary ; Female ; Fertilization in Vitro/methods/veterinary ; Galactosyltransferases/genetics ; Gene Targeting/*methods/veterinary ; Ghrelin/genetics ; Homeodomain Proteins/genetics ; Mixed Function Oxygenases/genetics ; RNA, Guide/genetics ; Swine/*genetics/physiology ; Trans-Activators/genetics ; Zygote/metabolism ; },
abstract = {The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.},
}
@article {pmid32518401,
year = {2020},
author = {Wessels, HH and Méndez-Mancilla, A and Guo, X and Legut, M and Daniloski, Z and Sanjana, NE},
title = {Massively parallel Cas13 screens reveal principles for guide RNA design.},
journal = {Nature biotechnology},
volume = {38},
number = {6},
pages = {722-727},
pmid = {32518401},
issn = {1546-1696},
support = {DP2 HG010099/HG/NHGRI NIH HHS/United States ; R00 HG008171/HG/NHGRI NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Computational Biology/*methods ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; HEK293 Cells ; Humans ; RNA, Guide/*genetics ; Sequence Analysis, RNA ; },
abstract = {Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs (gRNAs), we conducted massively parallel screens targeting messenger RNAs (mRNAs) of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15-21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability, testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.},
}
@article {pmid32518237,
year = {2020},
author = {Teng, C and Zhang, H and Hammond, R and Huang, K and Meyers, BC and Walbot, V},
title = {Dicer-like 5 deficiency confers temperature-sensitive male sterility in maize.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2912},
pmid = {32518237},
issn = {2041-1723},
mesh = {Alleles ; CRISPR-Cas Systems ; *DNA Methylation ; Gene Editing ; *Gene Expression Regulation, Plant ; Mutagenesis ; Mutation ; Plant Infertility/*genetics ; Plant Proteins/genetics/*physiology ; RNA, Plant/genetics ; RNA, Small Interfering/metabolism ; Temperature ; Zea mays/*genetics ; },
abstract = {Small RNAs play important roles during plant development by regulating transcript levels of target mRNAs, maintaining genome integrity, and reinforcing DNA methylation. Dicer-like 5 (Dcl5) is proposed to be responsible for precise slicing in many monocots to generate diverse 24-nt phased, secondary small interfering RNAs (phasiRNAs), which are exceptionally abundant in meiotic anthers of diverse flowering plants. The importance and functions of these phasiRNAs remain unclear. Here, we characterized several mutants of dcl5, including alleles generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and a transposon-disrupted allele. We report that dcl5 mutants have few or no 24-nt phasiRNAs, develop short anthers with defective tapetal cells, and exhibit temperature-sensitive male fertility. We propose that DCL5 and 24-nt phasiRNAs are critical for fertility under growth regimes for optimal yield.},
}
@article {pmid32518161,
year = {2020},
author = {Hu, L and Li, H and Chi, Z and He, J},
title = {Loss of the RNA-binding protein Rbm15 disrupts liver maturation in zebrafish.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {33},
pages = {11466-11472},
pmid = {32518161},
issn = {1083-351X},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; *Gene Deletion ; *Gene Expression Regulation, Developmental ; Hepatocytes/cytology/metabolism ; Liver/cytology/*embryology ; Zebrafish/*embryology/genetics ; },
abstract = {Liver organogenesis begins with hepatic precursors in the foregut endoderm, followed by hepatoblast specification, differentiation, outgrowth, and maturation for the formation of functional hepatocytes. Although several signaling pathways and critical factors that regulate liver specification, differentiation, and proliferation have been identified, little is known about how liver maturation is regulated. Here, we used a screen for mutations affecting liver development in zebrafish and identified a cq96 mutant that exhibits a specific defect in liver maturation. Results from positional cloning revealed that cq96 encodes an RNA-binding protein, Rbm15, which is an evolutionarily conserved Spen family protein and known to play a crucial role in RNA m6A modification, nuclear export, and alternative splicing. However, a function of Rbm15 in embryonic liver development has not been reported. We found that Rbm15 is specifically expressed in the liver after its differentiation. CRISPR/Cas9-mediated loss of rbm15 repressed hepatic maturation, but did not affect hepatoblast specification, differentiation, and hepatocyte proliferation and apoptosis. Additional experiments disclosed that the mTOR complex 1 (mTORC1) pathway is highly activated in rbm15-deficient hepatocytes. Moreover, rapamycin treatment partially restored normal hepatic gene expression as well as the nuclear location of the transcription factor Hnf4a. Taken together, these results reveal an unexpected role of Rbm15 in liver maturation.},
}
@article {pmid32517731,
year = {2020},
author = {Vitiello, M and Evangelista, M and Zhang, Y and Salmena, L and Pandolfi, PP and Poliseno, L},
title = {PTENP1 is a ceRNA for PTEN: it's CRISPR clear.},
journal = {Journal of hematology &amp; oncology},
volume = {13},
number = {1},
pages = {73},
pmid = {32517731},
issn = {1756-8722},
support = {R35 CA197529/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma/pathology ; Binding, Competitive ; *CRISPR-Cas Systems ; Cell Division ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Male ; MicroRNAs/genetics/metabolism ; Neoplasm Proteins/biosynthesis/*genetics ; PTEN Phosphohydrolase/biosynthesis/*genetics ; Prostatic Neoplasms/pathology ; Proto-Oncogene Proteins c-akt/biosynthesis/genetics ; Pseudogenes/*genetics ; RNA Interference ; RNA, Guide/genetics ; RNA, Messenger/biosynthesis/genetics ; RNA, Neoplasm/*genetics/metabolism ; RNA, Small Interfering/genetics/pharmacology ; Sequence Homology, Nucleic Acid ; },
abstract = {Here we apply state-of-the-art CRISPR technologies to study the impact that PTENP1 pseudogene transcript has on the expression levels of its parental gene PTEN, and hence on the output of AKT signaling in cancer. Our data expand the repertoire of approaches that can be used to dissect competing endogenous RNA (ceRNA)-based interactions, while providing further experimental evidence in support of the very first one that we discovered.},
}
@article {pmid32517191,
year = {2020},
author = {Zhang, J and Jin, M and Yang, Y and Liu, L and Yang, Y and Gómez, I and Bravo, A and Soberón, M and Xiao, Y and Liu, K},
title = {The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda.},
journal = {Toxins},
volume = {12},
number = {6},
pages = {},
pmid = {32517191},
issn = {2072-6651},
support = {2017YFD0200400//The&#xa0;National&#xa0;Key&#xa0;R&amp;D Program of China/International ; },
mesh = {Animals ; Bacillus thuringiensis Toxins/*pharmacology ; CRISPR-Cas Systems ; Cadherins/genetics/*metabolism ; Cell Line ; Dose-Response Relationship, Drug ; Endotoxins/*pharmacology ; Hemolysin Proteins/*pharmacology ; Insect Proteins/genetics/*metabolism ; *Insecticide Resistance ; Larva/drug effects/metabolism ; Lethal Dose 50 ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/genetics/metabolism ; Mutation ; Spodoptera/*drug effects/embryology/genetics/metabolism ; },
abstract = {It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.},
}
@article {pmid32516948,
year = {2020},
author = {Ansari, WA and Chandanshive, SU and Bhatt, V and Nadaf, AB and Vats, S and Katara, JL and Sonah, H and Deshmukh, R},
title = {Genome Editing in Cereals: Approaches, Applications and Challenges.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
pmid = {32516948},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Edible Grain/*genetics ; *Gene Editing ; *Genome, Plant ; *Genomics/methods ; Plants, Genetically Modified ; Stress, Physiological ; Transformation, Genetic ; },
abstract = {Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.},
}
@article {pmid32516591,
year = {2020},
author = {Gurusamy, D and Henning, AN and Yamamoto, TN and Yu, Z and Zacharakis, N and Krishna, S and Kishton, RJ and Vodnala, SK and Eidizadeh, A and Jia, L and Kariya, CM and Black, MA and Eil, R and Palmer, DC and Pan, JH and Sukumar, M and Patel, SJ and Restifo, NP},
title = {Multi-phenotype CRISPR-Cas9 Screen Identifies p38 Kinase as a Target for Adoptive Immunotherapies.},
journal = {Cancer cell},
volume = {37},
number = {6},
pages = {818-833.e9},
doi = {10.1016/j.ccell.2020.05.004},
pmid = {32516591},
issn = {1878-3686},
mesh = {Animals ; Breast Neoplasms/immunology/pathology/*therapy ; *CRISPR-Cas Systems ; Cell Differentiation ; Female ; Genetic Engineering ; Immunotherapy, Adoptive/*methods ; Male ; Melanoma, Experimental/immunology/pathology/*therapy ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; *Phenotype ; Receptors, Antigen, T-Cell/physiology ; T-Lymphocytes/immunology/*transplantation ; p38 Mitogen-Activated Protein Kinases/*antagonists &amp; inhibitors/genetics ; },
abstract = {T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.},
}
@article {pmid32516586,
year = {2020},
author = {Chan, JD and Beavis, PA and Darcy, PK},
title = {p38 Kinase: A Key Target for Driving Potent T Cells for Adoptive Immunotherapy.},
journal = {Cancer cell},
volume = {37},
number = {6},
pages = {756-758},
doi = {10.1016/j.ccell.2020.05.012},
pmid = {32516586},
issn = {1878-3686},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Immunotherapy, Adoptive ; Phenotype ; *T-Lymphocytes ; },
abstract = {In this issue of Cancer Cell, Gurusamy et al. use a CRISPR-Cas9 screening approach to demonstrate that deletion of p38 increases multiple phenotypic qualities of effective anti-tumor T cells. Preconditioning T cells with a p38 inhibitor enhances anti-tumor efficacy of adoptive immunotherapy.},
}
@article {pmid32515907,
year = {2020},
author = {Toda, E and Okamoto, T},
title = {CRISPR/Cas9-Based Genome Editing Using Rice Zygotes.},
journal = {Current protocols in plant biology},
volume = {5},
number = {2},
pages = {e20111},
doi = {10.1002/cppb.20111},
pmid = {32515907},
issn = {2379-8068},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Oryza/*genetics ; Zygote ; },
abstract = {Genome-editing technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Its application has been hindered in many cases, however, due to technical hurdles, such as the low rate of macromolecule delivery into plant cells and tissues or difficulties in plant transformation and regeneration. Here, a protocol for CRISPR/Cas9-based genome editing using rice zygotes is described. The genome-editing system is constructed via polyethylene glycol/calcium-mediated transfection with CRISPR/Cas9 components in rice zygotes, which are produced by in vitro fertilization of isolated rice gametes. Plasmid DNA harboring a CRISPR/Cas9 expression cassette or preassembled Cas9 protein-guide RNA ribonucleoproteins is transfected into zygotes, resulting in the regeneration of plants with a high frequency of the targeted mutation, which is either mono-allelic or bi-allelic, in the range of about 4% to 64%. Application of the present method has the potential to advance the molecular breeding of other crop species as well as rice. © 2020 Wiley Periodicals LLC.},
}
@article {pmid32515053,
year = {2020},
author = {Schwefel, K and Spiegler, S and Kirchmaier, BC and Dellweg, PKE and Much, CD and Pané-Farré, J and Strom, TM and Riedel, K and Felbor, U and Rath, M},
title = {Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {7},
pages = {9018-9033},
doi = {10.1096/fj.201902888R},
pmid = {32515053},
issn = {1530-6860},
mesh = {Apoptosis Regulatory Proteins/*antagonists &amp; inhibitors/genetics ; CRISPR-Cas Systems ; Carrier Proteins/*antagonists &amp; inhibitors/genetics ; Cells, Cultured ; Endothelium, Vascular/*cytology/metabolism/pathology ; Fibronectins/genetics/*metabolism ; Humans ; KRIT1 Protein/*antagonists &amp; inhibitors/genetics ; Membrane Proteins/*antagonists &amp; inhibitors/genetics ; Phenotype ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors/genetics ; },
abstract = {Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3[-/-] ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.},
}
@article {pmid32514112,
year = {2020},
author = {Michlits, G and Jude, J and Hinterndorfer, M and de Almeida, M and Vainorius, G and Hubmann, M and Neumann, T and Schleiffer, A and Burkard, TR and Fellner, M and Gijsbertsen, M and Traunbauer, A and Zuber, J and Elling, U},
title = {Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.},
journal = {Nature methods},
volume = {17},
number = {7},
pages = {708-716},
pmid = {32514112},
issn = {1548-7105},
mesh = {*Alleles ; Animals ; Benchmarking ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Datasets as Topic ; Humans ; Mice ; Mutation ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.},
}
@article {pmid32514099,
year = {2020},
author = {Villanueva, MT},
title = {Base editing rescues hearing in mice.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {7},
pages = {443},
pmid = {32514099},
issn = {1474-1784},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing ; Mice ; },
}
@article {pmid32514036,
year = {2020},
author = {Tsuchimatsu, T and Kakui, H and Yamazaki, M and Marona, C and Tsutsui, H and Hedhly, A and Meng, D and Sato, Y and Städler, T and Grossniklaus, U and Kanaoka, MM and Lenhard, M and Nordborg, M and Shimizu, KK},
title = {Adaptive reduction of male gamete number in the selfing plant Arabidopsis thaliana.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2885},
pmid = {32514036},
issn = {2041-1723},
mesh = {Adaptation, Physiological/*genetics ; Arabidopsis/*genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems/genetics ; Evolution, Molecular ; Genes, Plant/*genetics ; Mutation ; Plants, Genetically Modified ; Pollen/cytology/*genetics/metabolism ; Reproduction/genetics ; Sequence Homology, Nucleic Acid ; },
abstract = {The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.},
}
@article {pmid32513871,
year = {2020},
author = {Van Orden, MJ and Newsom, S and Rajan, R},
title = {CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {32},
pages = {10956-10968},
pmid = {32513871},
issn = {1083-351X},
support = {P20 GM103640/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; DNA/metabolism ; *Phylogeny ; Protein Binding ; },
abstract = {CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader-repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3' end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader-repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1-Cas2 varied, with G1 stringently requiring a 5-nucleotide 3' overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein-DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1-Cas2 toolset for inserting small DNAs into diverse DNA targets.},
}
@article {pmid32513688,
year = {2020},
author = {Saha, LK and Wakasugi, M and Akter, S and Prasad, R and Wilson, SH and Shimizu, N and Sasanuma, H and Huang, SN and Agama, K and Pommier, Y and Matsunaga, T and Hirota, K and Iwai, S and Nakazawa, Y and Ogi, T and Takeda, S},
title = {Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {25},
pages = {14412-14420},
pmid = {32513688},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems/genetics ; DNA Breaks, Single-Stranded/*radiation effects ; DNA Polymerase beta/genetics/metabolism ; *DNA Repair ; DNA Topoisomerases, Type I/*metabolism ; Fibroblasts ; Gene Knockout Techniques ; Humans ; MCF-7 Cells ; Primary Cell Culture ; Skin/cytology/pathology/radiation effects ; Ultraviolet Rays/*adverse effects ; X-ray Repair Cross Complementing Protein 1/genetics/metabolism ; Xeroderma Pigmentosum/etiology/pathology ; Xeroderma Pigmentosum Group A Protein/genetics/metabolism ; },
abstract = {Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.},
}
@article {pmid32513585,
year = {2020},
author = {Marzec, M and Hensel, G},
title = {Prime Editing: Game Changer for Modifying Plant Genomes.},
journal = {Trends in plant science},
volume = {25},
number = {8},
pages = {722-724},
doi = {10.1016/j.tplants.2020.05.008},
pmid = {32513585},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Genome, Plant/genetics ; Humans ; Mutation ; },
abstract = {Prime editing, developed by Anzalone et al., brings genome editing to a new level, because this approach allows introduction of all mutation types, including insertions, deletions, and all putative 12 types of base-to-base conversions. Previously tested in human cells, this technique has been adapted for use in plants by Lin et al.},
}
@article {pmid32512864,
year = {2020},
author = {Cui, J and Morgan, D and Cheng, DH and Foo, SL and Yap, GLR and Ampomah, PB and Arora, S and Sachaphibulkij, K and Periaswamy, B and Fairhurst, AM and De Sessions, PF and Lim, LHK},
title = {RNA-Sequencing-Based Transcriptomic Analysis Reveals a Role for Annexin-A1 in Classical and Influenza A Virus-Induced Autophagy.},
journal = {Cells},
volume = {9},
number = {6},
pages = {},
pmid = {32512864},
issn = {2073-4409},
mesh = {A549 Cells ; Animals ; Annexin A1/genetics/*metabolism ; Autophagosomes/metabolism ; Autophagy/*genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism/pathology ; *Gene Expression Profiling ; Gene Expression Regulation ; Gene Ontology ; Humans ; Influenza A virus/*physiology ; Lung/pathology ; Mice, Inbred BALB C ; Mutation/genetics ; RNA, Messenger/genetics/metabolism ; *Sequence Analysis, RNA ; TOR Serine-Threonine Kinases/metabolism ; },
abstract = {Influenza viruses have been shown to use autophagy for their survival. However, the proteins and mechanisms involved in the autophagic process triggered by the influenza virus are unclear. Annexin-A1 (ANXA1) is an immunomodulatory protein involved in the regulation of the immune response and Influenza A virus (IAV) replication. In this study, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated protein 9) deletion of ANXA1, combined with the next-generation sequencing, we systematically analyzed the critical role of ANXA1 in IAV infection as well as the detailed processes governing IAV infection, such as macroautophagy. A number of differentially expressed genes were uniquely expressed in influenza A virus-infected A549 parental cells and A549 ∆ANXA1 cells, which were enriched in the immune system and infection-related pathways. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the role of ANXA1 in autophagy. To validate this, the effect of mechanistic target of rapamycin (mTOR) inhibitors, starvation and influenza infection on autophagy was determined, and our results demonstrate that ANXA1 enhances autophagy induced by conventional autophagy inducers and influenza virus. These results will help us to understand the underlying mechanisms of IAV infection and provide a potential therapeutic target for restricting influenza viral replication and infection.},
}
@article {pmid32512313,
year = {2020},
author = {Pyott, DE and Fei, Y and Molnar, A},
title = {Potential for gene editing in antiviral resistance.},
journal = {Current opinion in virology},
volume = {42},
number = {},
pages = {47-52},
doi = {10.1016/j.coviro.2020.04.005},
pmid = {32512313},
issn = {1879-6265},
mesh = {Disease Resistance ; *Gene Editing ; Genome, Plant ; Plant Diseases/genetics/immunology/*virology ; Plant Proteins/genetics/immunology ; Plant Viruses/genetics/*physiology ; Plants/*genetics/immunology/virology ; },
abstract = {The discovery of CRISPR/Cas systems and their subsequent application in genome modifications and in gene expression control have fundamentally changed both basic and applied research. They have already been employed to generate novel virus resistance traits either by modifying host factors in the plant genome or by directly inducing targeted virus degradation. Here we summarise the latest developments in this field and discuss the potential applications and concerns around this technology.},
}
@article {pmid32511611,
year = {2020},
author = {Nemudryi, A and Nemudraia, A and Wiegand, T and Surya, K and Buyukyoruk, M and Vanderwood, KK and Wilkinson, R and Wiedenheft, B},
title = {Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater.},
journal = {medRxiv : the preprint server for health sciences},
volume = {},
number = {},
pages = {},
pmid = {32511611},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
abstract = {SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 52-day time course. We show that changes in SARS-CoV-2 RNA concentrations correlate with local COVID-19 epidemiological data (R2=0.9), though detection in wastewater trails symptom onset dates by 5-8 days. We determine a near complete (98.5%) SARS-CoV-2 genome sequence from the wastewater and use phylogenic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for high-resolution genotyping of the predominant strains circulating in a community.},
}
@article {pmid32510745,
year = {2021},
author = {Xu, Z and Li, Y and Li, M and Xiang, H and Yan, A},
title = {Harnessing the type I CRISPR-Cas systems for genome editing in prokaryotes.},
journal = {Environmental microbiology},
volume = {23},
number = {2},
pages = {542-558},
doi = {10.1111/1462-2920.15116},
pmid = {32510745},
issn = {1462-2920},
mesh = {Bacteria/enzymology/*genetics ; CRISPR-Cas Systems ; DNA, Bacterial/genetics ; *Gene Editing ; *Genome, Bacterial ; },
abstract = {Genetic analysis is crucial to the understanding, exploitation, and control of microorganisms. The advent of CRISPR-Cas-based genome-editing techniques, particularly those mediated by the single-effector (Cas9 and Cas12a) class 2 CRISPR-Cas systems, has revolutionized the genetics in model eukaryotic organisms. However, their applications in prokaryotes are rather limited, largely owing to the exceptional diversity of DNA homeostasis in microorganisms and severe cytotoxicity of overexpressing these nuclease proteins in certain genotypes. Remarkably, CRISPR-Cas systems belonging to different classes and types are continuously identified in prokaryotic genomes and serve as a deep reservoir for expansion of the CRISPR-based genetic toolkits. ~90% of the CRISPR-Cas systems identified so far belong to the class 1 system which hinges on multi-protein effector complexes for DNA interference. Harnessing these widespread native CRISPR-Cas systems for 'built-in' genome editing represents an emerging and powerful genetic tool in prokaryotes, especially in the genetically recalcitrant non-model species and strains. In this progress review, we introduce the general workflow of this emerging editing platform and summarize its establishment in a growing number of prokaryotes by harnessing the most widespread, diverse type I CRISPR-Cas systems present in their genomes. We also discuss the various factors affecting the success and efficiency of this editing platform and the corresponding solutions.},
}
@article {pmid32510617,
year = {2021},
author = {Zittersteijn, HA and Gonçalves, MAFV and Hoeben, RC},
title = {A primer to gene therapy: Progress, prospects, and problems.},
journal = {Journal of inherited metabolic disease},
volume = {44},
number = {1},
pages = {54-71},
pmid = {32510617},
issn = {1573-2665},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/methods/*trends ; Gene Transfer Techniques ; Genetic Diseases, Inborn/therapy ; Genetic Therapy/methods/*trends ; Genetic Vectors ; Humans ; Viruses/genetics ; },
abstract = {Genetic therapies based on gene addition have witnessed a variety of clinical successes and the first therapeutic products have been approved for clinical use. Moreover, innovative gene editing techniques are starting to offer new opportunities in which the mutations that underlie genetic diseases can be directly corrected in afflicted somatic cells. The toolboxes underpinning these DNA modifying technologies are expanding with great pace. Concerning the ongoing efforts for their implementation, viral vector-based gene delivery systems have acquired center-stage, providing new hopes for patients with inherited and acquired disorders. Specifically, the application of genetic therapies using viral vectors for the treatment of inborn metabolic disorders is growing and clinical applications are starting to appear. While the field has matured from the technology perspective and has yielded efficacious products, it is the perception of many stakeholders that from the regulatory side further developments are urgently needed. In this review, we summarize the features of state-of-the-art viral vector systems and the corresponding gene-centered therapies they seek to deliver. Moreover, a brief summary is also given on emerging gene editing approaches built on CRISPR-Cas9 nucleases and, more recently, nickases, including base editors and prime editors. Finally, we will point at some regulatory aspects that may deserve further attention for translating these technological developments into actual advanced therapy medicinal products (ATMPs).},
}
@article {pmid32510613,
year = {2020},
author = {Sobh, A and Loguinov, A and Zhou, J and Jenkitkasemwong, S and Zeidan, R and El Ahmadie, N and Tagmount, A and Knutson, M and Fraenkel, PG and Vulpe, CD},
title = {Genetic screens reveal CCDC115 as a modulator of erythroid iron and heme trafficking.},
journal = {American journal of hematology},
volume = {95},
number = {9},
pages = {1085-1098},
doi = {10.1002/ajh.25899},
pmid = {32510613},
issn = {1096-8652},
support = {R01 GM083198/NH/NIH HHS/United States ; R01 DK085250/NH/NIH HHS/United States ; },
mesh = {Biological Transport, Active ; CRISPR-Cas Systems ; Erythroid Cells/cytology/*metabolism ; Genetic Testing ; HEK293 Cells ; Heme/genetics/*metabolism ; Humans ; Iron/*metabolism ; K562 Cells ; *Nerve Tissue Proteins/genetics/metabolism ; },
abstract = {Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.},
}
@article {pmid32508782,
year = {2020},
author = {Castillo, JA and Secaira-Morocho, H and Maldonado, S and Sarmiento, KN},
title = {Diversity and Evolutionary Dynamics of Antiphage Defense Systems in Ralstonia solanacearum Species Complex.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {961},
pmid = {32508782},
issn = {1664-302X},
abstract = {Over the years, many researchers have reported a great diversity of bacteriophages infecting members of the Ralstonia solanacearum species complex (RSSC). This diversity has driven bacterial evolution by leading the emergence and maintenance of bacterial defense systems to combat phage infection. In this work, we present an in silico study of the arsenal of defense systems that RSSC harbors and their evolutionary history. For this purpose, we used a combination of genomic, phylogenetic and associative methods. We found that in addition to the CRISPR-Cas system already reported, there are eight other antiphage defense systems including the well-known Restriction-Modification and Toxin-Antitoxin systems. Furthermore, we found a tenth defense system, which is dedicated to reducing the incidence of plasmid transformation in bacteria. We undertook an analysis of the gene gain and loss patterns of the defense systems in 15 genomes of RSSC. Results indicate that the dynamics are inclined toward the gain of defense genes as opposed to the rest of the genes that were preferably lost throughout evolution. This was confirmed by evidence on independent gene acquisition that has occurred by profuse horizontal transfer. The mutation and recombination rates were calculated as a proxy of evolutionary rates. Again, genes encoding the defense systems follow different rates of evolution respect to the rest of the genes. These results lead us to conclude that the evolution of RSSC defense systems is highly dynamic and responds to a different evolutionary regime than the rest of the genes in the genomes of RSSC.},
}
@article {pmid32507978,
year = {2020},
author = {Xiao, J and Deng, J and Zhang, Q and Ma, P and Lv, L and Zhang, Y and Li, C and Zhang, Y},
title = {Targeting human cytomegalovirus IE genes by CRISPR/Cas9 nuclease effectively inhibits viral replication and reactivation.},
journal = {Archives of virology},
volume = {165},
number = {8},
pages = {1827-1835},
doi = {10.1007/s00705-020-04687-3},
pmid = {32507978},
issn = {1432-8798},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytomegalovirus/*genetics ; Cytomegalovirus Infections/virology ; DNA, Viral/genetics ; Endonucleases/*genetics ; Fibroblasts/virology ; Gene Editing/methods ; Gene Expression/genetics ; Genes, Viral/*genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; THP-1 Cells ; Virus Replication/*genetics ; },
abstract = {Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.},
}
@article {pmid32506752,
year = {2021},
author = {Humbert, O and Samuelson, C and Kiem, HP},
title = {CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside.},
journal = {British journal of haematology},
volume = {192},
number = {1},
pages = {33-49},
pmid = {32506752},
issn = {1365-2141},
support = {R01 HL136135/HL/NHLBI NIH HHS/United States ; R01 HL147324/HL/NHLBI NIH HHS/United States ; UM1 AI126623/AI/NIAID NIH HHS/United States ; U19 AI096111/AI/NIAID NIH HHS/United States ; R01 AI135953/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematologic Diseases/genetics/*therapy ; Humans ; },
abstract = {Genome editing therapies represent a significant advancement in next-generation, precision medicine for the management of haematological diseases, and CRISPR/Cas9 has to date been the most successful implementation platform. From discovery in bacteria and archaea over three decades ago, through intensive basic research and pre-clinical development phases involving the modification of therapeutically relevant cell types, CRISPR/Cas9 genome editing is now being investigated in ongoing clinic trials. Despite the widespread enthusiasm brought by this new technology, significant challenges remain before genome editing can be routinely recommended and implemented in the clinic. These include risks of genotoxicity resulting from off-target DNA cleavage or chromosomal rearrangement, and suboptimal efficacy of homology-directed repair editing strategies, which thus limit therapeutic options. Practical hurdles such as high costs and inaccessibility to patients outside specialised centres must also be addressed. Future improvements in this rapidly developing field should circumvent current limitations with novel editing platforms and with the simplification of clinical protocols using in vivo delivery of editing reagents.},
}
@article {pmid32506497,
year = {2020},
author = {Xiang, L and Qi, F and Jiang, L and Tan, J and Deng, C and Wei, Z and Jin, S and Huang, G},
title = {CRISPR-dCas9-mediated knockdown of prtR, an essential gene in Pseudomonas aeruginosa.},
journal = {Letters in applied microbiology},
volume = {71},
number = {4},
pages = {386-393},
doi = {10.1111/lam.13337},
pmid = {32506497},
issn = {1472-765X},
mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques/*methods ; Genes, Essential ; Promoter Regions, Genetic ; Pseudomonas aeruginosa/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; },
abstract = {Pseudomonas aeruginosa is a widely distributed non-fermentative Gram-negative opportunistic pathogen that is often responsible for nosocomial infections. Gene interference is a potentially valuable tool for investigating essential genes in P. aeruginosa. To establish a gene interference platform in P. aeruginosa, CRISPR system was used with an inactive Cas9 protein. The CRISPR-dCas9 system was cloned into pHERD20T, a shuttle vector with arabinose inducible promoter, and was further modified to target a regulatory gene prtR that is essential for the viability of P. aeruginosa. Cells expressing the prtR-targeting CRISPR interference (CRISPRi) showed growth defect in an arabinose dose-dependent manner. A high-throughput RNA sequencing analysis of bacterial cells with or without the CRISPRi-mediated prtR inhibition indicated that prtRis a global regulator affecting multiple biological processes. In conclusion, the CRISPR-dCas9-based gene knockdown system has been successfully implemented in P. aeruginosa and demonstrated to be an effective tool in the investigation of essential or difficult-to-inactivate genes in this species.},
}
@article {pmid32506418,
year = {2021},
author = {Ramezankhani, R and Minaei, N and Haddadi, M and Torabi, S and Hesaraki, M and Mirzaei, H and Vosough, M and Verfaillie, CM},
title = {Gene editing technology for improving life quality: A dream coming true?.},
journal = {Clinical genetics},
volume = {99},
number = {1},
pages = {67-83},
doi = {10.1111/cge.13794},
pmid = {32506418},
issn = {1399-0004},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Diseases, Inborn/*genetics/therapy ; Humans ; Quality of Life ; },
abstract = {The fact that monogenic diseases are related to mutations in one specific gene, make gene correction one of the promising strategies in the future to treat genetic diseases or alleviate their symptoms. From this perspective, and along with recent advances in technology, genome editing tools have gained momentum and developed fast. In fact, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) are regarded as novel technologies which are able to correct a number of genetic aberrations in vitro and in vivo. The number of ongoing clinical trials employing these tools has been increased showing the encouraging outcomes of these tools. However, there are still some major challenges with respect to the safety profile and directed delivery of them. In this paper, we provided updated information regarding the history, nature, methods of delivery, and application of the above-mentioned gene editing tools along with the meganucleases (an older similar tool) based on published in vitro and in vivo studies and introduced clinical trials which employed these technologies.},
}
@article {pmid32504339,
year = {2021},
author = {Liu, Q and Jiao, X and Meng, X and Wang, C and Xu, C and Tian, Z and Xie, C and Li, G and Li, J and Yu, H and Wang, K},
title = {FED: a web tool for foreign element detection of genome-edited organism.},
journal = {Science China. Life sciences},
volume = {64},
number = {1},
pages = {167-170},
pmid = {32504339},
issn = {1869-1889},
mesh = {Animals ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Gene Editing/*methods ; Genome/*genetics ; Humans ; *Internet ; Reproducibility of Results ; },
}
@article {pmid32504260,
year = {2020},
author = {Liu, X and Wu, D and Shan, T and Xu, S and Qin, R and Li, H and Negm, M and Wu, D and Li, J},
title = {The trihelix transcription factor OsGTγ-2 is involved adaption to salt stress in rice.},
journal = {Plant molecular biology},
volume = {103},
number = {4-5},
pages = {545-560},
doi = {10.1007/s11103-020-01010-1},
pmid = {32504260},
issn = {1573-5028},
mesh = {Abscisic Acid/metabolism ; Acclimatization/genetics/*physiology ; Adaptation, Physiological ; CRISPR-Cas Systems ; Cation Transport Proteins/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation, Plant ; Oryza/genetics/growth &amp; development/*physiology ; Plant Development ; Plant Proteins/genetics/metabolism ; Plant Roots/metabolism ; Salinity ; Salt Stress/*physiology ; Salt Tolerance/*genetics ; Seedlings/genetics ; Seeds/metabolism ; Sodium/metabolism ; Sodium-Hydrogen Exchangers/metabolism ; Stress, Physiological/genetics ; Symporters/metabolism ; Transcription Factors/genetics/*metabolism ; },
abstract = {OsGTγ-2, a trihelix transcription factor, is a positive regulator of rice responses to salt stress by regulating the expression of ion transporters. Salinity stress seriously restricts rice growth and yield. Trihelix transcription factors (GT factors) specifically bind to GT elements and play a diverse role in plant morphological development and responses to abiotic stresses. In our previous study, we found that the GT-1 element (GAAAAA) is a key element in the salinity-induced OsRAV2 promoter. Here, we identified a rice OsGTγ family member, OsGTγ-2, which directly interacted with the GT-1 element in the OsRAV2 promoter. OsGTγ-2 specifically targeted the nucleus, was mainly expressed in roots, sheathes, stems and seeds, and was induced by salinity, osmotic and oxidative stresses and abscisic acid (ABA). The seed germination rate, seedling growth and survival rate under salinity stress was improved in OsGTγ-2 overexpressing lines (PZmUbi::OsGTγ-2). In contrast, CRISPR/Cas9-mediated OsGTγ-2 knockout lines (osgtγ-2) showed salt-hypersensitive phenotypes. In response to salt stress, different Na[+] and K[+] acclamation patterns were observed in PZmUbi::OsGTγ-2 lines and osgtγ-2 plants were observed. The molecular mechanism of OsGTγ-2 in rice salt adaptation was also investigated. Several major genes responsible for ion transporting, such as the OsHKT2; 1, OsHKT1; 3 and OsNHX1 were transcriptionally regulated by OsGTγ-2. A subsequent yeast one-hybrid assay and EMSA indicated that OsGTγ-2 directly interacted with the promoters of OsHKT2; 1, OsNHX1 and OsHKT1; 3. Taken together, these results suggest that OsGTγ-2 is an important positive regulator involved in rice responses to salt stress and suggest a potential role for OsGTγ-2 in regulating salinity adaptation in rice.},
}
@article {pmid32504093,
year = {2020},
author = {Ababneh, NA and Scaber, J and Flynn, R and Douglas, A and Barbagallo, P and Candalija, A and Turner, MR and Sims, D and Dafinca, R and Cowley, SA and Talbot, K},
title = {Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair.},
journal = {Human molecular genetics},
volume = {29},
number = {13},
pages = {2200-2217},
pmid = {32504093},
issn = {1460-2083},
support = {MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; CL-2015-26-001/DH_/Department of Health/United Kingdom ; MR/L002167/1/MRC_/Medical Research Council/United Kingdom ; TURNER/OCT18/989-797/MNDA_/Motor Neurone Disease Association/United Kingdom ; MR/P007023/1/MRC_/Medical Research Council/United Kingdom ; WTISSF121302/WT_/Wellcome Trust/United Kingdom ; J-0901/PUK_/Parkinson's UK/United Kingdom ; 090532/Z/09/Z/WT_/Wellcome Trust/United Kingdom ; TALBOT-MUTIHAC/APR15/832-791/MNDA_/Motor Neurone Disease Association/United Kingdom ; MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; TALBOT/JULY13/820-791/MNDA_/Motor Neurone Disease Association/United Kingdom ; SCABER/JULY13/945-795/MNDA_/Motor Neurone Disease Association/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; TALBOT/OCT16/889-792/MNDA_/Motor Neurone Disease Association/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics/pathology ; C9orf72 Protein/*genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; DNA Repeat Expansion/genetics ; Female ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Motor Neurons/*metabolism/pathology ; Phenotype ; Recombinational DNA Repair/genetics ; },
abstract = {The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.},
}
@article {pmid32504042,
year = {2020},
author = {Stangl, C and de Blank, S and Renkens, I and Westera, L and Verbeek, T and Valle-Inclan, JE and González, RC and Henssen, AG and van Roosmalen, MJ and Stam, RW and Voest, EE and Kloosterman, WP and van Haaften, G and Monroe, GR},
title = {Partner independent fusion gene detection by multiplexed CRISPR-Cas9 enrichment and long read nanopore sequencing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2861},
pmid = {32504042},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *Gene Fusion ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; *Nanopore Sequencing ; Neoplasms/*diagnosis/genetics ; Sequence Analysis, DNA ; },
abstract = {Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location - to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection.},
}
@article {pmid32503625,
year = {2020},
author = {Seabra, CM and Aneichyk, T and Erdin, S and Tai, DJC and De Esch, CEF and Razaz, P and An, Y and Manavalan, P and Ragavendran, A and Stortchevoi, A and Abad, C and Young, JI and Maciel, P and Talkowski, ME and Gusella, JF},
title = {Transcriptional consequences of MBD5 disruption in mouse brain and CRISPR-derived neurons.},
journal = {Molecular autism},
volume = {11},
number = {1},
pages = {45},
pmid = {32503625},
issn = {2040-2392},
support = {P01 GM061354/GM/NIGMS NIH HHS/United States ; R01 NS093200/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Autism Spectrum Disorder/etiology/metabolism ; Brain/*metabolism ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; Disease Models, Animal ; Gene Expression Regulation, Developmental ; Gene Targeting ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Methyl-CpG-Binding Protein 2/*genetics ; Mice ; Mice, Transgenic ; *Mutation ; Neurons/cytology/*metabolism ; *Transcription, Genetic ; },
abstract = {BACKGROUND: MBD5, encoding the methyl-CpG-binding domain 5 protein, has been proposed as a necessary and sufficient driver of the 2q23.1 microdeletion syndrome. De novo missense and protein-truncating variants from exome sequencing studies have directly implicated MBD5 in the etiology of autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDDs). However, little is known concerning the specific function(s) of MBD5.<br><br>METHODS: To gain insight into the complex interactions associated with alteration of MBD5 in individuals with ASD and related NDDs, we explored the transcriptional landscape of MBD5 haploinsufficiency across multiple mouse brain regions of a heterozygous hypomorphic Mbd5[+/GT] mouse model, and compared these results to CRISPR-mediated mutations of MBD5 in human iPSC-derived neuronal models.<br><br>RESULTS: Gene expression analyses across three brain regions from Mbd5[+/GT] mice showed subtle transcriptional changes, with cortex displaying the most widespread changes following Mbd5 reduction, indicating context-dependent effects. Comparison with MBD5 reduction in human neuronal cells reinforced the context-dependence of gene expression changes due to MBD5 deficiency. Gene co-expression network analyses revealed gene clusters that were associated with reduced MBD5 expression and enriched for terms related to ciliary function.<br><br>LIMITATIONS: These analyses included a limited number of mouse brain regions and neuronal models, and the effects of the gene knockdown are subtle. As such, these results will not reflect the full extent of MBD5 disruption across human brain regions during early neurodevelopment in ASD, or capture the diverse spectrum of cell-type-specific changes associated with MBD5 alterations.<br><br>CONCLUSIONS: Our study points to modest and context-dependent transcriptional consequences of Mbd5 disruption in the brain. It also suggests a possible link between MBD5 and perturbations in ciliary function, which is an established pathogenic mechanism in developmental disorders and syndromes.},
}
@article {pmid32503371,
year = {2020},
author = {Wiegand, T and Karambelkar, S and Bondy-Denomy, J and Wiedenheft, B},
title = {Structures and Strategies of Anti-CRISPR-Mediated Immune Suppression.},
journal = {Annual review of microbiology},
volume = {74},
number = {},
pages = {21-37},
pmid = {32503371},
issn = {1545-3251},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Associated Protein 9/genetics/*immunology ; CRISPR-Cas Systems/*genetics/*immunology/physiology ; Evolution, Molecular ; Protein Binding ; Viral Proteins/*genetics/immunology ; },
abstract = {More than 50 protein families have been identified that inhibit CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-mediated adaptive immune systems. Here, we analyze the available anti-CRISPR (Acr) structures and describe common themes and unique mechanisms of stoichiometric and enzymatic suppressors of CRISPR-Cas. Stoichiometric inhibitors often function as molecular decoys of protein-binding partners or nucleic acid targets, while enzymatic suppressors covalently modify Cas ribonucleoprotein complexes or degrade immune signaling molecules. We review mechanistic insights that have been revealed by structures of Acrs, discuss some of the trade-offs associated with each of these strategies, and highlight how Acrs are regulated and deployed in the race to overcome adaptive immunity.},
}
@article {pmid32502342,
year = {2020},
author = {Piñero-Lambea, C and Garcia-Ramallo, E and Martinez, S and Delgado, J and Serrano, L and Lluch-Senar, M},
title = {Mycoplasma pneumoniae Genome Editing Based on Oligo Recombineering and Cas9-Mediated Counterselection.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1693-1704},
pmid = {32502342},
issn = {2161-5063},
mesh = {Bacillus subtilis/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; *Genome, Bacterial ; Mycoplasma pneumoniae/*genetics ; Plasmids/genetics/metabolism ; Point Mutation ; },
abstract = {Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10[-2], outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains.},
}
@article {pmid32501846,
year = {2020},
author = {Ye, L and Wang, J and Teque, F and Xie, F and Tan, Y and Kan, YW and Levy, JA},
title = {Generation of HIV-1-infected patients' gene-edited induced pluripotent stem cells using feeder-free culture conditions.},
journal = {AIDS (London, England)},
volume = {34},
number = {8},
pages = {1127-1139},
doi = {10.1097/QAD.0000000000002535},
pmid = {32501846},
issn = {1473-5571},
support = {P01 DK088760/DK/NIDDK NIH HHS/United States ; },
mesh = {Cell Differentiation/genetics/physiology ; Family Characteristics ; Gene Editing ; Genetic Vectors/genetics/*metabolism ; HIV Infections/diagnosis ; HIV-1/*genetics ; Humans ; Induced Pluripotent Stem Cells/*physiology ; Leukocytes, Mononuclear/*cytology/metabolism ; },
abstract = {OBJECTIVES: The discovery of induced pluripotent stem cells (iPSC) has brought promise to regenerative medicine as it breaks the ethical barrier of using embryonic stem cells. Such cell culture-derived patient-specific autologous stem cells are needed for transplantation. Here we report deriving HIV-1-infected patients' iPSC lines under transgene-free methods and under feeder-free and xeno-free culture conditions to meet the requirement for clinical application.<br><br>METHODS AND RESULTS: We have reprogrammed patients' peripheral blood mononuclear cells with EBNA1/OriP episomal vectors, or a defective and persistent Sendai virus vector (SeVdp) to ensure a nonintegrating iPSC generation. Both single picked and pooled iPSC lines demonstrated high pluripotency and were able to differentiate into various lineage cells in vivo. The established cell lines could be modified by genetic editing using the TALENs or CRISPR/Cas 9 technology to have a bi-allelic CCR5&#x394;32 mutations seamlessly. All generated iPSC lines and modified cell lines had no evidence of HIV integration and maintained normal karyotype after expansion.<br><br>CONCLUSIONS: This study provides a reproducible simple procedure for generating therapeutic grade iPSCs from HIV-infected patients and for engineering these cells to possess a naturally occurring genotype for resistance to HIV-1 infection when differentiated into immune cells.},
}
@article {pmid32501770,
year = {2020},
author = {de Puig, H and Bosch, I and Collins, JJ and Gehrke, L},
title = {Point-of-Care Devices to Detect Zika and Other Emerging Viruses.},
journal = {Annual review of biomedical engineering},
volume = {22},
number = {},
pages = {371-386},
doi = {10.1146/annurev-bioeng-060418-052240},
pmid = {32501770},
issn = {1545-4274},
support = {R21 AI100190/AI/NIAID NIH HHS/United States ; R33 AI100190/AI/NIAID NIH HHS/United States ; U19 AI131135/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Computational Biology ; DNA/analysis ; Humans ; Immunoassay/*methods ; Metal Nanoparticles/chemistry ; Mice ; Nanoparticles/chemistry ; Nanotechnology/methods ; Nucleic Acids/chemistry ; *Point-of-Care Systems ; Sensitivity and Specificity ; Synthetic Biology/*methods ; *Zika Virus ; Zika Virus Infection/*diagnosis ; },
abstract = {Rapid diagnostic tests (point-of-care devices) are critical components of informed patient care and public health monitoring (surveillance applications). We propose that among the many rapid diagnostics platforms that have been tested or are in development, lateral flow immunoassays and synthetic biology-based diagnostics (including CRISPR-based diagnostics) represent the best overall options given their ease of use, scalability for manufacturing, sensitivity, and specificity. This review describes the identification of lateral flow immunoassay monoclonal antibody pairs that detect and distinguish between closely related pathogens and that are used in combination with functionalized multicolored nanoparticles and computational methods to deconvolute data. We also highlight the promise of synthetic biology-based diagnostic tests, which use synthetic genetic circuits that activate upon recognition of a pathogen-associated nucleic acid sequence, and discuss how the combined or parallel use of lateral flow immunoassays and synthetic biology tools may represent the future of scalable rapid diagnostics.},
}
@article {pmid32501639,
year = {2020},
author = {Char, SN and Lee, H and Yang, B},
title = {Use of CRISPR/Cas9 for Targeted Mutagenesis in Sorghum.},
journal = {Current protocols in plant biology},
volume = {5},
number = {2},
pages = {e20112},
doi = {10.1002/cppb.20112},
pmid = {32501639},
issn = {2379-8068},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Mutagenesis ; Sorghum/*genetics ; },
abstract = {Sorghum (Sorghum bicolor) fulfills the demand for bioenergy resources and also provides substantial diet calories to the world's population. Therefore, many biological studies use sorghum as a research model for improvement of the domesticated food and bioenergy crops. Furthermore, leveraging genome editing systems in a plethora of grass plant species has been extensively studied with no exception in sorghum. However, a protocol that details the genome editing strategies using CRISPR/Cas9 and that combines an efficient tissue culture and transformation platform in sorghum based on Agrobacterium-mediated DNA transfer has yet to be reported. This protocol outlines the steps and workflow from design of sorghum CRISPR target sites using BTx623 as a reference genome, construction of sorghum CRISPR/Cas9 plasmids, tissue culture, to Agrobacterium-mediated transformation followed by genotyping of CRISPR/Cas9 induced mutants. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 expression vector to analysis of CRISPR-edited plants Basic Protocol 2: Stable transformation of sorghum Support Protocol: Management of sorghum plants in a greenhouse.},
}
@article {pmid32500600,
year = {2020},
author = {Hamaker, NK and Lee, KH},
title = {A Site-Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9-Mediated Genome Editing Strategies in CHO Cells.},
journal = {Biotechnology journal},
volume = {15},
number = {8},
pages = {e2000057},
doi = {10.1002/biot.202000057},
pmid = {32500600},
issn = {1860-7314},
support = {70NANB17H002/GM/NIGMS NIH HHS/United States ; 70NANB17H002/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Biotechnology/methods/trends ; CHO Cells ; *CRISPR-Cas Systems/genetics ; Cricetinae ; Cricetulus ; *Gene Editing ; Genes, Reporter ; },
abstract = {Targeted gene knockout and site-specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (site-specific integration and genome alteration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual-fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology.},
}
@article {pmid32500359,
year = {2020},
author = {Benayas, B and Sastre, I and López-Martín, S and Oo, A and Kim, B and Bullido, MJ and Aldudo, J and Yáñez-Mó, M},
title = {Tetraspanin CD81 regulates HSV-1 infection.},
journal = {Medical microbiology and immunology},
volume = {209},
number = {4},
pages = {489-498},
pmid = {32500359},
issn = {1432-1831},
support = {AI136581/NH/NIH HHS/United States ; R01 AI136581/AI/NIAID NIH HHS/United States ; R01 AI150451/AI/NIAID NIH HHS/United States ; AI150451/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Knockout Techniques ; Herpesviridae Infections/*virology ; Herpesvirus 1, Human/pathogenicity/*physiology ; Humans ; SAM Domain and HD Domain-Containing Protein 1/metabolism ; Tetraspanin 28/*genetics/*metabolism ; Viral Envelope Proteins/metabolism ; Virion/metabolism ; Virus Internalization ; Virus Replication ; },
abstract = {Different members of the tetraspanin superfamily have been described to regulate different virus infectious cycles at several stages: viral entry, viral replication or virion exit or infectivity. In addition, tetraspanin CD81 regulates HIV reverse transcription through its association with the dNTP hydrolase SAMHD1. Here we aimed at analysing the role of CD81 in Herpes simplex virus 1 infectivity using a neuroblastoma cell model. For this purpose, we generated a CD81 KO cell line using the CRISPR/Cas9 technology. Despite being CD81 a plasma membrane protein, CD81 KO cells showed no defects in viral entry nor in the expression of early protein markers. In contrast, glycoprotein B and C, which require viral DNA replication for their expression, were significantly reduced in CD81 KO infected cells. Indeed, HSV-1 DNA replication and the formation of new infectious particles were severely compromised in CD81 KO cells. We could not detect significant changes in SAMHD1 total expression levels, but a relocalization into endosomal structures was observed in CD81 KO cells. In summary, CD81 KO cells showed impaired viral DNA replication and produced greatly diminished viral titers.},
}
@article {pmid32499641,
year = {2020},
author = {Cortez, JT and Montauti, E and Shifrut, E and Gatchalian, J and Zhang, Y and Shaked, O and Xu, Y and Roth, TL and Simeonov, DR and Zhang, Y and Chen, S and Li, Z and Woo, JM and Ho, J and Vogel, IA and Prator, GY and Zhang, B and Lee, Y and Sun, Z and Ifergan, I and Van Gool, F and Hargreaves, DC and Bluestone, JA and Marson, A and Fang, D},
title = {CRISPR screen in regulatory T cells reveals modulators of Foxp3.},
journal = {Nature},
volume = {582},
number = {7812},
pages = {416-420},
pmid = {32499641},
issn = {1476-4687},
support = {K99 CA184043/CA/NCI NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; R35 GM128943/GM/NIGMS NIH HHS/United States ; T32 CA009370/CA/NCI NIH HHS/United States ; S10 OD021822/OD/NIH HHS/United States ; R01 AI151123/AI/NIAID NIH HHS/United States ; T32 AI125222/AI/NIAID NIH HHS/United States ; T32 GM008568/GM/NIGMS NIH HHS/United States ; T32 AI007334/AI/NIAID NIH HHS/United States ; R01 AI079056/AI/NIAID NIH HHS/United States ; R01 AI108634/AI/NIAID NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; R00 CA184043/CA/NCI NIH HHS/United States ; R56 AI079056/AI/NIAID NIH HHS/United States ; F32 GM128377/GM/NIGMS NIH HHS/United States ; R01 CA232347/CA/NCI NIH HHS/United States ; F31 CA220801/CA/NCI NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity/immunology ; *CRISPR-Cas Systems ; Cells, Cultured ; Forkhead Transcription Factors/biosynthesis/*metabolism ; Gene Editing ; Gene Expression Regulation ; Humans ; Immunotherapy ; Male ; Mice ; Neoplasms/genetics/immunology/pathology/prevention &amp; control ; Protein Stability ; Reproducibility of Results ; T-Lymphocytes, Regulatory/cytology/immunology/*metabolism ; Ubiquitin Thiolesterase/deficiency/metabolism ; Ubiquitin-Protein Ligases/deficiency/metabolism ; },
abstract = {Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity[1]. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties[2], can promote autoimmunity and/or facilitate more effective tumour immunity[3,4]. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.},
}
@article {pmid32499620,
year = {2020},
author = {Tang, L},
title = {PAM-less is more.},
journal = {Nature methods},
volume = {17},
number = {6},
pages = {559},
pmid = {32499620},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Macrophages ; },
}
@article {pmid32499527,
year = {2020},
author = {Ka, D and Oh, H and Park, E and Kim, JH and Bae, E},
title = {Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD[+] degradation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2816},
pmid = {32499527},
issn = {2041-1723},
mesh = {Bacillus cereus/metabolism ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Cloning, Molecular ; Crystallography, X-Ray ; DNA Mutational Analysis ; Escherichia coli/metabolism/*virology ; Hydrolases/metabolism ; Kinetics ; Mutation ; NAD/*metabolism ; Protein Domains ; Protein Structure, Secondary ; Toll-Like Receptors/metabolism ; },
abstract = {The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD[+] cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD[+] cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD[+] degradation.},
}
@article {pmid32499444,
year = {2020},
author = {Qian, J and Lu, ZX and Mancuso, CP and Jhuang, HY and Del Carmen Barajas-Ornelas, R and Boswell, SA and Ramírez-Guadiana, FH and Jones, V and Sonti, A and Sedlack, K and Artzi, L and Jung, G and Arammash, M and Pettit, ME and Melfi, M and Lyon, L and Owen, SV and Baym, M and Khalil, AS and Silver, PA and Rudner, DZ and Springer, M},
title = {Barcoded microbial system for high-resolution object provenance.},
journal = {Science (New York, N.Y.)},
volume = {368},
number = {6495},
pages = {1135-1140},
doi = {10.1126/science.aba5584},
pmid = {32499444},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; DNA Barcoding, Taxonomic/*methods ; DNA, Bacterial/genetics/*isolation &amp; purification ; DNA, Fungal/genetics/*isolation &amp; purification ; *Environmental Microbiology ; Microbiota/*genetics ; RNA, Guide ; Spores/*genetics ; },
abstract = {Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.},
}
@article {pmid32499410,
year = {2020},
author = {Rice, PA and Craig, NL and Dyda, F},
title = {Comment on "RNA-guided DNA insertion with CRISPR-associated transposases".},
journal = {Science (New York, N.Y.)},
volume = {368},
number = {6495},
pages = {},
doi = {10.1126/science.abb2022},
pmid = {32499410},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements ; RNA ; *Transposases ; },
abstract = {Strecker et al (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.},
}
@article {pmid32499234,
year = {2020},
author = {Perkons, NR and Johnson, O and Pilla, G and Profka, E and Mercadante, M and Ackerman, D and Gade, TPF},
title = {Functional Genetic Screening Enables Theranostic Molecular Imaging in Cancer.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {26},
number = {17},
pages = {4581-4589},
pmid = {32499234},
issn = {1557-3265},
support = {DP5 OD021391/OD/NIH HHS/United States ; F30 CA232388/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carbon-13 Magnetic Resonance Spectroscopy/*methods ; Carcinoma, Hepatocellular/chemically induced/*diagnosis/genetics/pathology ; Datasets as Topic ; Diethylnitrosamine/administration &amp; dosage/toxicity ; Early Detection of Cancer/methods ; Humans ; L-Lactate Dehydrogenase/antagonists &amp; inhibitors/genetics/metabolism ; Lactic Acid/metabolism ; Liver/diagnostic imaging/pathology ; Liver Neoplasms/chemically induced/*diagnosis/genetics/pathology ; Liver Neoplasms, Experimental/chemically induced/*diagnosis/genetics/pathology ; Male ; Molecular Imaging/*methods ; Molecular Probes/administration &amp; dosage/pharmacokinetics ; Precision Medicine/methods ; Proof of Concept Study ; Pyruvic Acid/metabolism ; Rats ; },
abstract = {PURPOSE: Targeted therapies for cancer have accelerated the need for functional imaging strategies that inform therapeutic efficacy. This study assesses the potential of functional genetic screening to integrate therapeutic target identification with imaging probe selection through a proof-of-principle characterization of a therapy-probe pair using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopic imaging (MRSI).<br><br>EXPERIMENTAL DESIGN: CRISPR-negative selection screens from a public dataset were used to identify the relative dependence of 625 cancer cell lines on 18,333 genes. Follow-up screening was performed in hepatocellular carcinoma with a focused CRISPR library targeting imaging-related genes. Hyperpolarized [1-[13]C]-pyruvate was injected before and after lactate dehydrogenase inhibitor (LDHi) administration in male Wistar rats with autochthonous hepatocellular carcinoma. MRSI evaluated intratumoral pyruvate metabolism, while T2-weighted segmentations quantified tumor growth.<br><br>RESULTS: Genetic screening data identified differential metabolic vulnerabilities in 17 unique cancer types that could be imaged with existing probes. Among these, hepatocellular carcinoma required lactate dehydrogenase (LDH) for growth more than the 29 other cancer types in this database. LDH inhibition led to a decrease in lactate generation (P < 0.001) and precipitated dose-dependent growth inhibition (P < 0.01 overall, P < 0.05 for dose dependence). Intratumoral alanine production after inhibition predicted the degree of growth reduction (P < 0.001).<br><br>CONCLUSIONS: These findings demonstrate that DNP-MRSI of LDH activity using hyperpolarized [1-[13]C]-pyruvate is a theranostic strategy for hepatocellular carcinoma, enabling quantification of intratumoral LDHi pharmacodynamics and therapeutic efficacy prediction. This work lays the foundation for a novel theranostic platform wherein functional genetic screening informs imaging probe selection to quantify therapeutic efficacy on a cancer-by-cancer basis.},
}
@article {pmid32499184,
year = {2021},
author = {Oliver, JD and Duscher, D and Hu, MS},
title = {Engineering a Future with VCA: Applying Genetic Circuits to Engineer Tissues for Vascularized Composite Allotransplantation.},
journal = {Journal of plastic, reconstructive &amp; aesthetic surgery : JPRAS},
volume = {74},
number = {1},
pages = {223-243},
doi = {10.1016/j.bjps.2020.05.056},
pmid = {32499184},
issn = {1878-0539},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Immunity/genetics ; *Immunosuppression Therapy ; *Vascularized Composite Allotransplantation ; },
}
@article {pmid32499102,
year = {2020},
author = {Peng, X and Mayo-Muñoz, D and Bhoobalan-Chitty, Y and Martínez-Álvarez, L},
title = {Anti-CRISPR Proteins in Archaea.},
journal = {Trends in microbiology},
volume = {28},
number = {11},
pages = {913-921},
doi = {10.1016/j.tim.2020.05.007},
pmid = {32499102},
issn = {1878-4380},
mesh = {Archaea/genetics/*immunology/virology ; Archaeal Proteins/genetics/*immunology ; Archaeal Viruses/genetics/*physiology ; *CRISPR-Cas Systems ; Rudiviridae/genetics/*physiology ; },
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas immune systems. To date, Acrs inhibiting types I, II, III, V, and VI CRISPR-Cas systems have been characterized. While most known Acrs are derived from bacterial phages and prophages, very few have been characterized in the domain Archaea, despite the nearly ubiquitous presence of CRISPR-Cas in archaeal cells. Here we summarize the discovery and characterization of the archaeal Acrs with the representatives encoded by a model archaeal virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2). AcrID1 inhibits subtype I-D CRISPR-Cas immunity through direct interaction with the large subunit Cas10d of the effector complex, and AcrIIIB1 inhibits subtype III-B CRISPR-Cas immunity through a mechanism interfering with middle/late gene targeting. Future development of efficient screening methods will be key to uncovering the diversity of archaeal Acrs.},
}
@article {pmid32497618,
year = {2020},
author = {Galani, V and Papagiannitsis, CC and Petinaki, E},
title = {First description of ST409 OXA-23-producing Acinetobacter baumannii, carrying a CST8 CRISPR/Cas system, in Central Greece.},
journal = {Journal of global antimicrobial resistance},
volume = {22},
number = {},
pages = {137-138},
doi = {10.1016/j.jgar.2020.05.008},
pmid = {32497618},
issn = {2213-7173},
mesh = {*Acinetobacter Infections ; *Acinetobacter baumannii/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Greece ; Humans ; beta-Lactamases/genetics/metabolism ; },
}
@article {pmid32497313,
year = {2020},
author = {Nateghi Rostami, M},
title = {CRISPR/Cas9 gene drive technology to control transmission of vector-borne parasitic infections.},
journal = {Parasite immunology},
volume = {42},
number = {9},
pages = {e12762},
doi = {10.1111/pim.12762},
pmid = {32497313},
issn = {1365-3024},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Drive Technology ; Humans ; *Insect Vectors ; Malaria/*prevention &amp; control/transmission ; },
abstract = {Gene drive is the process of copying of an endonuclease-containing cassette that leads to increased frequency of inheritance of the desired traits in a targeted population. CRISPR/Cas9 technology is advancing genetic manipulation of insects in the field of gene drive experiments. The CRISPR/Cas9 drive could be engineered for genetic manipulation of parasites and/or vectors for disease control. A number of promising CRISPR/Cas9-based gene drive strategies that interfere with parasite development or impairs the reproductive capability of the insect vector have been proposed in the laboratory for blocking transmission of malaria and leishmaniasis. Still several technical and ethical challenges remain to be addressed, and none appear insuperable in this field.},
}
@article {pmid32496535,
year = {2020},
author = {Mullally, G and van Aelst, K and Naqvi, MM and Diffin, FM and Karvelis, T and Gasiunas, G and Siksnys, V and Szczelkun, MD},
title = {5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.},
journal = {Nucleic acids research},
volume = {48},
number = {12},
pages = {6811-6823},
pmid = {32496535},
issn = {1362-4962},
support = {BB/L01386X1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 788405/ERC_/European Research Council/International ; BB/L000873/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L000873/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S001239/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems/*genetics ; *DNA Cleavage ; Gene Editing ; Nucleic Acid Conformation ; R-Loop Structures/*genetics ; RNA, Guide/*genetics/ultrastructure ; Ribonucleoproteins/genetics/ultrastructure ; Single Molecule Imaging ; Streptococcus pyogenes/genetics ; },
abstract = {A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.},
}
@article {pmid32496191,
year = {2020},
author = {Booth, DS and King, N},
title = {Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32496191},
issn = {2050-084X},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Choanoflagellata/*genetics/*growth &amp; development ; Gene Editing ; Genome, Protozoan ; Lectins, C-Type/genetics ; Protozoan Proteins/genetics ; Reverse Genetics/*methods ; },
abstract = {In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.},
}
@article {pmid32496007,
year = {2020},
author = {Zhang, W and Kataoka, M and Yen Doan, H and Wu, FT and Haga, K and Takeda, N and Muramatsu, M and Li, TC},
title = {Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.},
journal = {Transboundary and emerging diseases},
volume = {67},
number = {6},
pages = {2849-2859},
doi = {10.1111/tbed.13655},
pmid = {32496007},
issn = {1865-1682},
mesh = {Animals ; Antibodies, Viral/blood ; Blotting, Western/veterinary ; CRISPR-Cas Systems ; Cell Line ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Feces/virology ; High-Throughput Nucleotide Sequencing/veterinary ; Immunoglobulin G/blood ; Microscopy, Electron/veterinary ; Orthoreovirus, Mammalian/genetics/immunology/*isolation &amp; purification ; Phylogeny ; Picornaviridae/*pathogenicity ; Picornaviridae Infections/*veterinary/virology ; RNA, Viral/genetics ; Reoviridae Infections/*veterinary/virology ; Swine ; Swine Diseases/*virology ; },
abstract = {Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.},
}
@article {pmid32494996,
year = {2020},
author = {Wei, YY and Zhan, QM and Zhu, XX and Yan, AF and Feng, J and Liu, L and Li, JH and Tang, DS},
title = {Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method.},
journal = {Biotechnology letters},
volume = {42},
number = {11},
pages = {2091-2109},
pmid = {32494996},
issn = {1573-6776},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Electromagnetic Phenomena ; Gene Editing/*methods ; Insulin-Like Growth Factor II/*genetics ; Mutation ; Myostatin/*genetics ; Selective Breeding ; Single-Cell Analysis ; Swine ; Transfection/*methods ; },
abstract = {OBJECTIVES: Guangdong Small-ear Spotted (GDSS) pigs are a pig breed native to China that possesses unfortunate disadvantages, such as slow growth rate, low lean-meat percentage, and reduced feed utilization. In contrast to traditional genetic breeding methods with long cycle time and high cost, CRISPR/Cas9-mediated gene editing for the modification of the pig genome can quickly improve production traits, and therefore this technique exhibits important potential in the genetic improvement and resource development of GDSS pigs. In the present study, we aimed to establish an efficient CRISPR/Cas9-mediated gene-editing system for GDSS pig cells by optimizing the electrotransfection parameters, and to realize efficient CRISPR/Cas9-mediated gene editing of GDSS pig cells.<br><br>RESULTS: After optimization of electrotransfection parameters for the transfection of GDSS pig cells, we demonstrated that a voltage of 150&#xa0;V and a single pulse with a pulse duration of 20&#xa0;ms were the optimal electrotransfection parameters for gene editing in these cells. In addition, our study generated GDSS pig single-cell colonies with biallelic mutations in the myostatin (MSTN) gene and insulin-like growth factor 2 (IGF2) intron-3 locus, which play an important role in pig muscle growth and muscle development. The single-cell colonies showed no foreign gene integration or off-target effects, and maintained normal cell morphology and viability. These gene-edited, single-cell colonies can in the future be used as donor cells to generate MSTN- and IGF2-edited GDSS pigs using somatic cell nuclear transfer (SCNT).<br><br>CONCLUSIONS: This study establishes the foundation for genetic improvement and resource development of GDSS pigs using CRISPR/Cas9-mediated gene editing combined with SCNT.},
}
@article {pmid32494703,
year = {2020},
author = {Wang, F and Hao, Y and Li, Q and Li, J and Zhang, H and Zhang, X and Wang, L and Bustamante, C and Fan, C},
title = {Programming PAM antennae for efficient CRISPR-Cas9 DNA editing.},
journal = {Science advances},
volume = {6},
number = {19},
pages = {eaay9948},
pmid = {32494703},
issn = {2375-2548},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/genetics ; Endonucleases/metabolism ; Gene Editing/methods ; RNA, Guide/genetics ; },
abstract = {Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami-based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.},
}
@article {pmid32494588,
year = {2020},
author = {Ling, X and Xie, B and Gao, X and Chang, L and Zheng, W and Chen, H and Huang, Y and Tan, L and Li, M and Liu, T},
title = {Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates.},
journal = {Science advances},
volume = {6},
number = {15},
pages = {eaaz0051},
pmid = {32494588},
issn = {2375-2548},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/genetics/metabolism ; *Gene Editing/methods ; Mice ; Oligonucleotides/genetics ; Recombinational DNA Repair ; },
abstract = {Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9-mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9-based genome-editing systems.},
}
@article {pmid32494076,
year = {2020},
author = {Tsanni, A},
title = {Bolstering Africa's coronavirus detection efforts.},
journal = {Nature},
volume = {582},
number = {7810},
pages = {140},
doi = {10.1038/d41586-020-01607-y},
pmid = {32494076},
issn = {1476-4687},
mesh = {Africa/epidemiology ; Betacoronavirus/*genetics/*isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems/genetics ; *Clinical Laboratory Techniques ; Coronavirus Infections/*diagnosis/epidemiology/*virology ; Genome, Viral/genetics ; Genomics ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis/epidemiology/*virology ; Polymerase Chain Reaction ; SARS-CoV-2 ; Time Factors ; },
}
@article {pmid32493719,
year = {2020},
author = {Reding, K and Pick, L},
title = {High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus.},
journal = {Genetics},
volume = {215},
number = {4},
pages = {1027-1037},
pmid = {32493719},
issn = {1943-2631},
support = {R01 GM113230/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Eye Color/*genetics ; Female ; *Gene Editing ; Hemiptera/genetics/*physiology ; Insect Proteins/*genetics/metabolism ; Male ; *Mutagenesis ; },
abstract = {In this manuscript, we report that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is highly efficient in the hemipteran Oncopeltus fasciatus The white gene is well characterized in Drosophila where mutation causes loss of eye pigmentation; white is a reliable marker for transgenesis and other genetic manipulations. Accordingly, white has been targeted in a number of nonmodel insects to establish tools for genetic studies. Here, we generated mutations in the Of-white (Of-w) locus using CRISPR/Cas9. We found that Of-w is required for pigmentation throughout the body of Oncopeltus, not just the ommatidia. High rates of somatic mosaicism were observed in the injected generation, reflecting biallelic mutations, and a high rate of germline mutation was evidenced by the large proportion of heterozygous G1s. However, Of-w mutations are homozygous lethal; G2 homozygotes lacked pigment dispersion throughout the body and did not hatch, precluding the establishment of a stable mutant line. Embryonic and parental RNA interference (RNAi) were subsequently performed to rule out off-target mutations producing the observed phenotype and to evaluate the efficacy of RNAi in ablating gene function compared to a loss-of-function mutation. RNAi knockdowns phenocopied Of-w homozygotes, with an unusual accumulation of orange granules observed in unhatched embryos. This is, to our knowledge, the first CRISPR/Cas9-targeted mutation generated in Oncopeltus While we were unable to establish white as a useful visible marker for Oncopeltus, these findings are instructive for the selection of visible markers in nonmodel species and reveal an unusual role for an ortholog of a classic Drosophila gene.},
}
@article {pmid32493587,
year = {2020},
author = {Uygun, ZO and Yeniay, L and Gi Rgi N Sağın, F},
title = {CRISPR-dCas9 powered impedimetric biosensor for label-free detection of circulating tumor DNAs.},
journal = {Analytica chimica acta},
volume = {1121},
number = {},
pages = {35-41},
doi = {10.1016/j.aca.2020.04.009},
pmid = {32493587},
issn = {1873-4324},
mesh = {Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; Circulating Tumor DNA/*blood ; Class I Phosphatidylinositol 3-Kinases/genetics ; Dielectric Spectroscopy ; Electrodes ; Graphite/chemistry ; Humans ; Limit of Detection ; RNA, Guide/metabolism ; Reproducibility of Results ; },
abstract = {Label-free biosensors which can be integrated into lab-on-a-chip platforms have the advantage of using small volumes for rapid and inexpensive measurements contrary to label-based technologies which are often more costly and time-consuming. In this study, graphene oxide screen printed electrodes (GPHOXE) were modified by deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor for label-free detection of circulating tumor DNAs (ctDNA). This was achieved by detection of a tumor related mutation (PIK3CA exon 9 mutation) via sequence-specific recognition followed by electrochemical impedance spectroscopy (EIS) analysis. The biosensor showed high specificity as there was no impedance signal for other ctDNA sequences, even the single nucleotide mismatch. dCas9-sgRNA modified biosensor demonstrated linear detection limits between 2 and 20 nM for 120 bp ctDNA's in 40 s. The calibration curve showed good linearity, LOD was calculated as 0.65 nM and LOQ was calculated as 1.92 nM. Selectivity and repeatability studies were carried out in real blood samples and the recovery was higher than 96%. In conclusion, dCas9-sgRNA was effectively immobilized and optimized on GPHOXE as the selective biorecognition receptor of this ultrafast impedimetric biosensor. The CRISPR-dCas9 powered impedimetric system showed good selectivity, high repeatability and good recovery properties. This is the first literature to report the use of CRISPR/Cas technology as a label-free tool that can be used in an impedimetric system for detection of ctDNA's.},
}
@article {pmid32493511,
year = {2020},
author = {Song, L and Xie, K},
title = {Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing.},
journal = {Microbiome},
volume = {8},
number = {1},
pages = {80},
pmid = {32493511},
issn = {2049-2618},
support = {31622047//National Natural Science Foundation of China/International ; 2018ZX08010-05B//National Transgenic Science and Technology Program/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; Genetic Engineering ; *High-Throughput Nucleotide Sequencing/methods ; *Microbiota/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {BACKGROUND: High-throughput sequencing of bacterial 16S rRNA gene (16S-seq) is a useful and common method for studying bacterial community structures. However, contamination of the 16S rRNA genes from the mitochondrion and plastid hinders the sensitive bacterial 16S-seq in plant microbiota profiling, especially for some plant species such as rice. To date, efficiently mitigating such host contamination without a bias is challenging in 16S rRNA gene-based amplicon sequencing.<br><br>RESULTS: We developed Cas-16S-seq method to reduce abundant host contamination for plant microbiota profiling. This method utilizes the Cas9 nuclease and specific guide RNA (gRNA) to cut 16S rRNA targets during library construction, thereby removing host contamination in 16S-seq. We used rice as an example to validate the feasibility and effectiveness of Cas-16S-seq. We established a bioinformatics pipeline to design gRNAs that specifically target rice 16S rRNA genes without bacterial 16S rRNA off-targets. We compared the effectiveness of Cas-16S-seq with that of the commonly used 16S-seq method for artificially mixed 16S rRNA gene communities, paddy soil, rice root, and phyllosphere samples. The results showed that Cas-16S-seq substantially reduces the fraction of rice 16S rRNA gene sequences from 63.2 to 2.9% in root samples and from 99.4 to 11.6% in phyllosphere samples on average. Consequently, Cas-16S-seq detected more bacterial species than the 16S-seq in plant samples. Importantly, when analyzing soil samples, Cas-16S-seq and 16S-seq showed almost identical bacterial communities, suggesting that Cas-16S-seq with host-specific gRNAs that we designed has no off-target in rice microbiota profiling.<br><br>CONCLUSION: Our Cas-16S-seq can efficiently remove abundant host contamination without a bias for 16S rRNA gene-based amplicon sequencing, thereby enabling deeper bacterial community profiling with a low cost and high flexibility. Thus, we anticipate that this method would be a useful tool for plant microbiomics. Video Abstract.},
}
@article {pmid32493474,
year = {2020},
author = {Shaw, S and Knüsel, S and Hoenner, S and Roditi, I},
title = {A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei.},
journal = {BMC research notes},
volume = {13},
number = {1},
pages = {268},
pmid = {32493474},
issn = {1756-0500},
mesh = {*CRISPR-Cas Systems ; DNA-Directed RNA Polymerases/genetics ; *Gene Editing ; Genome, Protozoan/*genetics ; RNA, Guide/genetics ; Trypanosoma brucei brucei/*genetics ; Viral Proteins/genetics ; },
abstract = {OBJECTIVE: Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line.<br><br>RESULTS: We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.},
}
@article {pmid32493396,
year = {2020},
author = {de Boer, CG and Ray, JP and Hacohen, N and Regev, A},
title = {MAUDE: inferring expression changes in sorting-based CRISPR screens.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {134},
pmid = {32493396},
issn = {1474-760X},
support = {K99-HG009920-01/HG/NHGRI NIH HHS/United States ; 5 F32 AI129249/NH/NIH HHS/United States ; RM1HG006193/NH/NIH HHS/United States ; R01HG008131-01/NH/NIH HHS/United States ; //CIHR/Canada ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression ; *Genetic Techniques ; Models, Genetic ; *RNA, Guide ; *Software ; },
abstract = {Improved methods are needed to model CRISPR screen data for interrogation of genetic elements that alter reporter gene expression readout. We create MAUDE (Mean Alterations Using Discrete Expression) for quantifying the impact of guide RNAs on a target gene's expression in a pooled, sorting-based expression screen. MAUDE quantifies guide-level effects by modeling the distribution of cells across sorting expression bins. It then combines guides to estimate the statistical significance and effect size of targeted genetic elements. We demonstrate that MAUDE outperforms previous approaches and provide experimental design guidelines to best leverage MAUDE, which is available on https://github.com/Carldeboer/MAUDE.},
}
@article {pmid32492427,
year = {2020},
author = {Knupp, A and Mishra, S and Martinez, R and Braggin, JE and Szabo, M and Kinoshita, C and Hailey, DW and Small, SA and Jayadev, S and Young, JE},
title = {Depletion of the AD Risk Gene SORL1 Selectively Impairs Neuronal Endosomal Traffic Independent of Amyloidogenic APP Processing.},
journal = {Cell reports},
volume = {31},
number = {9},
pages = {107719},
pmid = {32492427},
issn = {2211-1247},
support = {P30 AG066462/AG/NIA NIH HHS/United States ; R01 AG062148/AG/NIA NIH HHS/United States ; T32 AG052354/AG/NIA NIH HHS/United States ; },
mesh = {Alzheimer Disease/metabolism/pathology ; Amyloid Precursor Protein Secretases/antagonists &amp; inhibitors/metabolism ; Amyloid beta-Peptides/metabolism ; Amyloid beta-Protein Precursor/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Endosomes/*metabolism ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; LDL-Receptor Related Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Membrane Transport Proteins/genetics/*metabolism ; Neurons/cytology/metabolism ; Protein Transport ; RNA Interference ; RNA, Guide/metabolism ; RNA, Small Interfering/metabolism ; },
abstract = {SORL1/SORLA is a sorting receptor involved in retromer-related endosomal traffic and an Alzheimer's disease (AD) risk gene. Using CRISPR-Cas9, we deplete SORL1 in hiPSCs to ask if loss of SORL1 contributes to AD pathogenesis by endosome dysfunction. SORL1-deficient hiPSC neurons show early endosome enlargement, a hallmark cytopathology of AD. There is no effect of SORL1 depletion on endosome size in hiPSC microglia, suggesting a selective effect on neuronal endosomal trafficking. We validate defects in neuronal endosomal traffic by showing altered localization of amyloid precursor protein (APP) in early endosomes, a site of APP cleavage by the β-secretase (BACE). Inhibition of BACE does not rescue endosome enlargement in SORL1-deficient neurons, suggesting that this phenotype is independent of amyloidogenic APP processing. Our data, together with recent findings, underscore how sporadic AD pathways regulating endosomal trafficking and autosomal-dominant AD pathways regulating APP cleavage independently converge on the defining cytopathology of AD.},
}
@article {pmid32492426,
year = {2020},
author = {Romera-Branchat, M and Severing, E and Pocard, C and Ohr, H and Vincent, C and Née, G and Martinez-Gallegos, R and Jang, S and Andrés, F and Madrigal, P and Coupland, G},
title = {Functional Divergence of the Arabidopsis Florigen-Interacting bZIP Transcription Factors FD and FDP.},
journal = {Cell reports},
volume = {31},
number = {9},
pages = {107717},
pmid = {32492426},
issn = {2211-1247},
mesh = {Abscisic Acid/pharmacology ; Arabidopsis/genetics/*metabolism ; Arabidopsis Proteins/classification/genetics/*metabolism ; Basic-Leucine Zipper Transcription Factors/classification/genetics/*metabolism ; CCAAT-Enhancer-Binding Protein-beta/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Florigen/*metabolism ; Flowers/genetics/metabolism ; Gene Editing ; Gene Expression Regulation, Plant/drug effects ; Genotype ; Homeodomain Proteins/genetics/metabolism ; MADS Domain Proteins/genetics/metabolism ; Mutagenesis ; Phylogeny ; Plants, Genetically Modified/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Flowering of many plant species depends on interactions between basic leucine zipper (bZIP) transcription factors and systemically transported florigen proteins. Members of the genus Arabidopsis contain two of these bZIPs, FD and FDP, which we show have largely complementary expression patterns in shoot apices before and during flowering. CRISPR-Cas9-induced null mutants for FDP flower slightly earlier than wild-type, whereas fd mutants are late flowering. Identical G-box sequences are enriched at FD and FDP binding sites, but only FD binds to genes involved in flowering and only fd alters their transcription. However, both proteins bind to genes involved in responses to the phytohormone abscisic acid (ABA), which controls developmental and stress responses. Many of these genes are differentially expressed in both fd and fdp mutant seedlings, which also show reduced ABA sensitivity. Thus, florigen-interacting bZIPs have distinct functions in flowering dependent on their expression patterns and, at earlier stages in development, play common roles in phytohormone signaling.},
}
@article {pmid32492006,
year = {2020},
author = {Pi, Y and He, KZ and Zhang, WQ and Dong, ZQ and Jiang, FG and Jiang, KJ and Guo, S},
title = {[Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish].},
journal = {Molekuliarnaia biologiia},
volume = {54},
number = {3},
pages = {435-444},
doi = {10.31857/S0026898420030131},
pmid = {32492006},
issn = {0026-8984},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques ; Genes, Reporter ; *Homologous Recombination ; *Zebrafish/genetics ; },
abstract = {Homology-directed (HD) genome modification offers an opportunity to precisely modify the genome. Despite reported successful cases, for many loci, precise genome editing remains challenging and inefficient in vivo. Here we report an effort to precisely knock-in a GFP reporter into gad locus mediated by CRISPR/Cas9 system in the zebrafish Danio rerio. PCR artifact was detected in testing for homologous recombination (HR), but was mitigated by optimizing PCR condition and decreasing the injected targeting plasmid concentration. Under this optimized condition, time course analysis revealed a decline of the HR-positive embryos at embryogenesis progressed. GFP signals also diminished at later developmental stages. The GFP signals were consistent with PCR detection, both of which suggested the loss of targeted insertion events at later stages. Such loss of insertion might be one underlying reason for the inability to obtain germ-line transgenic lines with GFP knocked into the gad locus. Our results suggest that the low HR efficiency associated with CRISPR-mediated knock-in is in part due to loss of insertion after targeted integration into the gad locus.},
}
@article {pmid32491259,
year = {2021},
author = {Kücükköse, C and Taskin, AA and Marada, A and Brummer, T and Dennerlein, S and Vögtle, FN},
title = {Functional coupling of presequence processing and degradation in human mitochondria.},
journal = {The FEBS journal},
volume = {288},
number = {2},
pages = {600-613},
doi = {10.1111/febs.15358},
pmid = {32491259},
issn = {1742-4658},
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems ; Cell Fractionation ; Cell Proliferation ; *Feedback, Physiological ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Metalloendopeptidases/*genetics/metabolism ; Mitochondria/*genetics/metabolism/pathology ; Mitochondrial Proteins/deficiency/*genetics ; Mutation ; Oligopeptides/genetics/metabolism ; Oxidative Phosphorylation ; Proteolysis ; Serine Endopeptidases/deficiency/*genetics ; Stress, Physiological/genetics ; },
abstract = {The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PreP[R183Q] patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.},
}
@article {pmid32490205,
year = {2020},
author = {Sun, W and Wang, J and Hu, Q and Zhou, X and Khademhosseini, A and Gu, Z},
title = {CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation.},
journal = {Science advances},
volume = {6},
number = {21},
pages = {eaba2983},
pmid = {32490205},
issn = {2375-2548},
support = {R01 GM126571/GM/NIGMS NIH HHS/United States ; R01 HL140951/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cholesterol ; DNA/genetics/metabolism ; Gene Editing ; Polymers/metabolism ; *Proprotein Convertase 9/genetics/metabolism ; RNA/genetics ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)-based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).},
}
@article {pmid32490186,
year = {2020},
author = {Moroz-Omori, EV and Satyapertiwi, D and Ramel, MC and Høgset, H and Sunyovszki, IK and Liu, Z and Wojciechowski, JP and Zhang, Y and Grigsby, CL and Brito, L and Bugeon, L and Dallman, MJ and Stevens, MM},
title = {Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation.},
journal = {ACS central science},
volume = {6},
number = {5},
pages = {695-703},
pmid = {32490186},
issn = {2374-7943},
support = {MR/R015651/1/MRC_/Medical Research Council/United Kingdom ; RE/18/4/34215/BHF_/British Heart Foundation/United Kingdom ; },
abstract = {The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.},
}
@article {pmid32488146,
year = {2020},
author = {Schwinn, MK and Steffen, LS and Zimmerman, K and Wood, KV and Machleidt, T},
title = {A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {8953},
pmid = {32488146},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Luminescent Measurements/*methods ; Luminescent Proteins/*analysis ; Plasmids ; Proteins/*analysis ; },
abstract = {The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.},
}
@article {pmid32488144,
year = {2020},
author = {Eskandari-Shahraki, M and Prud'homme, B and Bergeron, F and Manjunath, P},
title = {Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {8982},
pmid = {32488144},
issn = {2045-2322},
support = {MOP-130274//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/International ; },
mesh = {Animals ; Animals, Newborn ; Body Weight/genetics ; *CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Male ; Mice, Knockout/*genetics ; Seminal Vesicle Secretory Proteins/*genetics/*physiology ; Sperm Motility/*genetics ; },
abstract = {The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.},
}
@article {pmid32488111,
year = {2020},
author = {Russell, R and Carnese, PP and Hennings, TG and Walker, EM and Russ, HA and Liu, JS and Giacometti, S and Stein, R and Hebrok, M},
title = {Loss of the transcription factor MAFB limits β-cell derivation from human PSCs.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2742},
pmid = {32488111},
issn = {2041-1723},
support = {P30 DK063720/DK/NIDDK NIH HHS/United States ; R01 DK090570/DK/NIDDK NIH HHS/United States ; R01 DK105831/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Female ; Gene Editing ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Glucagon/metabolism ; Glucagon-Secreting Cells/metabolism ; Humans ; Insulin/metabolism ; Insulin-Secreting Cells/*metabolism ; Islets of Langerhans/metabolism ; MafB Transcription Factor/*genetics/*metabolism ; Male ; Mice ; Pancreatic Stellate Cells/*metabolism ; Stem Cells ; Transcriptome ; },
abstract = {Next generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human β-cells postnatally, while its expression is restricted to embryonic and neo-natal β-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to β-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.},
}
@article {pmid32488086,
year = {2020},
author = {Santos-Moreno, J and Tasiudi, E and Stelling, J and Schaerli, Y},
title = {Multistable and dynamic CRISPRi-based synthetic circuits.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2746},
pmid = {32488086},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/metabolism ; Escherichia coli Proteins/biosynthesis/genetics ; *Gene Expression ; Gene Regulatory Networks/*genetics ; Genetic Engineering ; },
abstract = {Gene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable synthetic circuit construction. Here we use CRISPRi to build a synthetic oscillator ("CRISPRlator"), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allow us to achieve robust circuit behaviors in Escherichia coli populations. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.},
}
@article {pmid32487737,
year = {2020},
author = {Rao, C and Malaguti, M and Mason, JO and Lowell, S},
title = {The transcription factor E2A drives neural differentiation in pluripotent cells.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {12},
pages = {},
pmid = {32487737},
issn = {1477-9129},
support = {/WT_/Wellcome Trust/United Kingdom ; WT103789AIA/WT_/Wellcome Trust/United Kingdom ; MR/K501293/1/MRC_/Medical Research Council/United Kingdom ; 108906/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {3' Untranslated Regions ; Animals ; Basic Helix-Loop-Helix Transcription Factors/deficiency/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Lineage ; Cell Self Renewal ; Dimerization ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Neurons/cytology/*metabolism ; Octamer Transcription Factor-3/deficiency/genetics/metabolism ; RNA, Guide/metabolism ; SOXB1 Transcription Factors/deficiency/genetics/metabolism ; Transcriptome ; Up-Regulation ; },
abstract = {The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.},
}
@article {pmid32487497,
year = {2020},
author = {Todokoro, T and Bando, H and Kotaka, A and Tsutsumi, H and Hata, Y and Ishida, H},
title = {Identification of a novel pyrithiamine resistance marker gene thiI for genome co-editing in Aspergillus oryzae.},
journal = {Journal of bioscience and bioengineering},
volume = {130},
number = {3},
pages = {227-232},
doi = {10.1016/j.jbiosc.2020.04.013},
pmid = {32487497},
issn = {1347-4421},
mesh = {Aspergillus oryzae/*drug effects/*genetics ; CRISPR-Cas Systems/genetics ; Drug Resistance, Fungal/*genetics ; *Gene Editing ; Genes, Fungal/*genetics ; Genetic Markers/*genetics ; Pyrithiamine/*pharmacology ; },
abstract = {Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.},
}
@article {pmid32487115,
year = {2020},
author = {Wu, X and Ma, W and Mei, C and Chen, X and Yao, Y and Liu, Y and Qin, X and Yuan, Y},
title = {Description of CRISPR/Cas9 development and its prospect in hepatocellular carcinoma treatment.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {39},
number = {1},
pages = {97},
pmid = {32487115},
issn = {1756-9966},
mesh = {Animals ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/pathology/*therapy ; *Gene Editing ; *Genetic Therapy ; Humans ; Liver Neoplasms/genetics/pathology/*therapy ; Neoplasm Proteins/antagonists &amp; inhibitors/*genetics ; },
abstract = {Hepatocellular carcinoma (HCC) is one of the most common malignancies today. Patients suffer from HCC since its high malignancy and limited treatment means. With the development of genetic research, new therapeutic strategy comes up in the way of gene editing. Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) was discovered as an immune sequence in bacteria and archaea. After artificial transformation and follow-up research, it is widely used as a gene editing tool. In this review, the development of CRISPR/Cas9 is summarized in retrospect. Through the evaluation of novel research in HCC, it is concluded that CRISPR/Cas9 would promote cancer research and provide a new tool for genetic treatment in prospect.},
}
@article {pmid32487025,
year = {2020},
author = {Muhammad Rafid, AH and Toufikuzzaman, M and Rahman, MS and Rahman, MS},
title = {CRISPRpred(SEQ): a sequence-based method for sgRNA on target activity prediction using traditional machine learning.},
journal = {BMC bioinformatics},
volume = {21},
number = {1},
pages = {223},
pmid = {32487025},
issn = {1471-2105},
mesh = {Algorithms ; Area Under Curve ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Databases, Genetic ; Deep Learning ; Gene Editing/*methods ; HEK293 Cells ; Humans ; *Machine Learning ; RNA, Guide/*genetics ; ROC Curve ; Reproducibility of Results ; },
abstract = {BACKGROUND: The latest works on CRISPR genome editing tools mainly employs deep learning techniques. However, deep learning models lack explainability and they are harder to reproduce. We were motivated to build an accurate genome editing tool using sequence-based features and traditional machine learning that can compete with deep learning models.<br><br>RESULTS: In this paper, we present CRISPRpred(SEQ), a method for sgRNA on-target activity prediction that leverages only traditional machine learning techniques and hand-crafted features extracted from sgRNA sequences. We compare the results of CRISPRpred(SEQ) with that of DeepCRISPR, the current state-of-the-art, which uses a deep learning pipeline. Despite using only traditional machine learning methods, we have been able to beat DeepCRISPR for the three out of four cell lines in the benchmark dataset convincingly (2.174%, 6.905% and 8.119% improvement for the three cell lines).<br><br>CONCLUSION: CRISPRpred(SEQ) has been able to convincingly beat DeepCRISPR in 3 out of 4 cell lines. We believe that by exploring further, one can design better features only using the sgRNA sequences and can come up with a better method leveraging only traditional machine learning algorithms that can fully beat the deep learning models.},
}
@article {pmid32486929,
year = {2021},
author = {Jiang, M and Ye, Y and Li, J},
title = {Core Hairpin Structure of SpCas9 sgRNA Functions in a Sequence- and Spatial Conformation-Dependent Manner.},
journal = {SLAS technology},
volume = {26},
number = {1},
pages = {92-102},
doi = {10.1177/2472630320922813},
pmid = {32486929},
issn = {2472-6311},
mesh = {*CRISPR-Cas Systems ; DNA ; Gene Editing ; *RNA, Guide/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a widely used genome-editing tool with great clinical potential. However, its application is limited because of low editing efficiency of some target sequences and off-target effects. As this system contains only the Cas9 protein and a single-guide RNA (sgRNA; engineered from crRNA and tracrRNA), the structure and function of these components should be studied in detail to address the current clinical needs. Consequently, we investigated the structural and sequence features of the core hairpin (the first stem loop of sgRNA) of SpCas9 sgRNA. We showed that the core hairpin structure of sgRNA is essential for SpCas9/sgRNA-mediated DNA cleavage and that the internal loop structure in the core hairpin plays a vital role in target DNA cleavage. We observed that the root stem structure within the core hairpin preferentially forms Watson-Crick base pairs and should be of a specific length to maintain an appropriate spatial conformation for Cas9 binding. However, the length of the leaf stem structure of the core hairpin is flexible, having a variable nucleotide composition. Furthermore, extension of the leaf stem structure enhances the DNA cleavage activity of the Cas9/sgRNA complex, and this could be used to enhance the efficiency of gene editing. These observations provide insight into the sgRNA/Cas9 interaction, indicating that sgRNA modification could be a strategy for improved DNA editing efficiency, and optimized sgRNA can be further used for genome-wide functional screening and clinical application.},
}
@article {pmid32486913,
year = {2020},
author = {Curti, LA and Pereyra-Bonnet, F and Repizo, GD and Fay, JV and Salvatierra, K and Blariza, MJ and Ibañez-Alegre, D and Rinflerch, AR and Miretti, M and Gimenez, CA},
title = {CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease.},
journal = {Emerging microbes &amp; infections},
volume = {9},
number = {1},
pages = {1140-1148},
pmid = {32486913},
issn = {2222-1751},
mesh = {Bacterial Proteins/*genetics/*pharmacology ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; Dengue/virology ; Dengue Virus/genetics ; Drug Resistance, Bacterial/*genetics ; Endodeoxyribonucleases/*genetics ; Fluorescent Dyes ; Gene Editing/*methods ; Hantaan virus/genetics ; Humans ; Molecular Diagnostic Techniques ; RNA Viruses/*genetics/pathogenicity ; RNA, Viral/genetics ; Zika Virus/genetics ; beta-Lactamases/*pharmacology ; },
abstract = {CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.},
}
@article {pmid32486292,
year = {2020},
author = {Lee, J and Kim, DH and Lee, K},
title = {Current Approaches and Applications in Avian Genome Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
pmid = {32486292},
issn = {1422-0067},
mesh = {Adenoviridae/genetics ; Agriculture ; Animal Husbandry/trends ; Animals ; Animals, Genetically Modified ; Biotechnology/*methods ; CRISPR-Cas Systems ; Chickens/*genetics ; Gene Editing/*methods ; *Genome ; Germ Cells/cytology ; Models, Animal ; Oocytes/cytology ; Ovulation ; Poultry ; Quail ; },
abstract = {Advances in genome-editing technologies and sequencing of animal genomes enable researchers to generate genome-edited (GE) livestock as valuable animal models that benefit biological researches and biomedical and agricultural industries. As birds are an important species in biology and agriculture, their genome editing has gained significant interest and is mainly performed by using a primordial germ cell (PGC)-mediated method because pronuclear injection is not practical in the avian species. In this method, PGCs can be isolated, cultured, genetically edited in vitro, and injected into a recipient embryo to produce GE offspring. Recently, a couple of GE quail have been generated by using the newly developed adenovirus-mediated method. Without technically required in vitro procedures of the PGC-mediated method, direct injection of adenovirus into the avian blastoderm in the freshly laid eggs resulted in the production of germ-line chimera and GE offspring. As more approaches are available in avian genome editing, avian research in various fields will progress rapidly. In this review, we describe the development of avian genome editing and scientific and industrial applications of GE avian species.},
}
@article {pmid32486234,
year = {2020},
author = {Yip, BH},
title = {Recent Advances in CRISPR/Cas9 Delivery Strategies.},
journal = {Biomolecules},
volume = {10},
number = {6},
pages = {},
pmid = {32486234},
issn = {2218-273X},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; *Gene Transfer Techniques ; Humans ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.},
}
@article {pmid32486152,
year = {2020},
author = {Maule, G and Arosio, D and Cereseto, A},
title = {Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
pmid = {32486152},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Cystic Fibrosis/*therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; Disease Models, Animal ; *Gene Editing ; Genetic Therapy/*methods/*trends ; Genome, Human ; Humans ; Mice ; Mutation ; Oligonucleotides, Antisense ; Phenotype ; },
abstract = {Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies.},
}
@article {pmid32486059,
year = {2020},
author = {Laanto, E and Mäkelä, K and Hoikkala, V and Ravantti, JJ and Sundberg, LR},
title = {Adapting a Phage to Combat Phage Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {9},
number = {6},
pages = {},
pmid = {32486059},
issn = {2079-6382},
abstract = {Phage therapy is becoming a widely recognized alternative for fighting pathogenic bacteria due to increasing antibiotic resistance problems. However, one of the common concerns related to the use of phages is the evolution of bacterial resistance against the phages, putatively disabling the treatment. Experimental adaptation of the phage (phage training) to infect a resistant host has been used to combat this problem. Yet, there is very little information on the trade-offs of phage infectivity and host range. Here we co-cultured a myophage FCV-1 with its host, the fish pathogen Flavobacterium columnare, in lake water and monitored the interaction for a one-month period. Phage resistance was detected within one day of co-culture in the majority of the bacterial isolates (16 out of the 18 co-evolved clones). The primary phage resistance mechanism suggests defense via surface modifications, as the phage numbers rose in the first two days of the experiment and remained stable thereafter. However, one bacterial isolate had acquired a spacer in its CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas locus, indicating that also CRISPR-Cas defense was employed in the phage-host interactions. After a week of co-culture, a phage isolate was obtained that was able to infect 18 out of the 32 otherwise resistant clones isolated during the experiment. Phage genome sequencing revealed several mutations in two open reading frames (ORFs) likely to be involved in the regained infectivity of the evolved phage. Their location in the genome suggests that they encode tail genes. Characterization of this evolved phage, however, showed a direct cost for the ability to infect several otherwise resistant clones-adsorption was significantly lower than in the ancestral phage. This work describes a method for adapting the phage to overcome phage resistance in a fish pathogenic system.},
}
@article {pmid32485960,
year = {2020},
author = {Eom, YW and Akter, R and Li, W and Lee, S and Hwang, S and Kim, J and Cho, MY},
title = {M1 Macrophages Promote TRAIL Expression in Adipose Tissue-Derived Stem Cells, Which Suppresses Colitis-Associated Colon Cancer by Increasing Apoptosis of CD133[+] Cancer Stem Cells and Decreasing M2 Macrophage Population.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
pmid = {32485960},
issn = {1422-0067},
mesh = {AC133 Antigen/metabolism ; Adipose Tissue/cytology ; Adult ; Animals ; *Apoptosis ; Azoxymethane ; CRISPR-Cas Systems ; Cell Line, Tumor ; Colitis/*complications/metabolism ; Colitis-Associated Neoplasms/complications/*metabolism ; Colon/pathology ; Dextran Sulfate ; Humans ; Macrophages/*metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Neoplastic Stem Cells/cytology ; Stem Cells/*metabolism ; TNF-Related Apoptosis-Inducing Ligand/*metabolism ; },
abstract = {We have previously reported that adipose tissue-derived stem cells (ASCs) cultured at high cell density can induce cancer cell death through the expression of type I interferons and tumor necrosis factor (TNF)-related apoptosis-inducing ligands (TRAIL). Here, we investigated whether TRAIL-expressing ASCs induced by M1 macrophages can alleviate colitis-associated cancer in an azoxymethane (AOM)/dextran sodium sulfate (DSS) animal model. M1 macrophages significantly increased the TRAIL expression in ASCs, which induced the apoptosis of LoVo cells in a TRAIL-dependent manner. However, CD133[knockout] LoVo cells, generated using the CRISPR-Cas9 gene-editing system, were resistant to TRAIL. In the AOM/DSS-induced colitis-associated cancer model, the intraperitoneal transplantation of TRAIL-expressing ASCs significantly suppressed colon cancer development. Moreover, immunohistochemical staining revealed a low CD133 expression in tumors from the AOM/DSS + ASCs group when compared with tumors from the untreated group. Additionally, the ASC treatment selectively reduced the number of M2 macrophages in tumoral (45.7 ± 4.2) and non-tumoral mucosa (30.3 ± 1.5) in AOM/DSS + ASCs-treated animals relative to those in the untreated group (tumor 71.7 ± 11.2, non-tumor 94.3 ± 12.5; p < 0.001). Thus, TRAIL-expressing ASCs are promising agents for anti-tumor therapy, particularly to alleviate colon cancer by inducing the apoptosis of CD133[+] cancer stem cells and decreasing the M2 macrophage population.},
}
@article {pmid32485269,
year = {2020},
author = {Sabbir, MG and Taylor, CG and Zahradka, P},
title = {Hypomorphic CAMKK2 in EA.hy926 endothelial cells causes abnormal transferrin trafficking, iron homeostasis and glucose metabolism.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {10},
pages = {118763},
doi = {10.1016/j.bbamcr.2020.118763},
pmid = {32485269},
issn = {1879-2596},
mesh = {Adult ; Alternative Splicing/genetics ; Antigens, CD/metabolism ; Aorta/metabolism ; CRISPR-Cas Systems/genetics ; Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics/*metabolism ; Cell Nucleus/metabolism ; Cell Respiration ; Endoplasmic Reticulum/metabolism ; Endothelial Cells/*metabolism/ultrastructure ; Endothelium, Vascular/metabolism ; Exons/genetics ; Glucose/*metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism ; Glycolysis ; HEK293 Cells ; *Homeostasis ; Humans ; Iron/*metabolism ; Mitochondria/metabolism ; Monocytes/metabolism ; Organ Specificity ; Protein Transport ; RNA, Messenger/genetics/metabolism ; Receptors, Transferrin/metabolism ; Transferrin/*metabolism ; Voltage-Dependent Anion Channel 1/metabolism ; },
abstract = {We recently reported that loss of calcium/calmodulin-dependent protein kinase kinase-2 (CAMKK2), a serine/threonine kinase activated by intracellular calcium, in mice leads to tissue-specific aberrant turnover of transferrin (TF), a receptor-mediated iron-transporter that supplies iron to tissues. Iron dyshomeostasis is associated with the pathogenesis of several diseases, making TF transport relevant to health. In this study, we used hemizygous CAMKK2 hypomorphic human endothelial cell (EA.hy926) clones to demonstrate that cells with reduced CAMKK2 exhibit increased TF uptake and transcytosis, and decreased intracellular trafficking to subcellular organelles compared to wild-type. The abnormal TF trafficking in CAMKK2 hypomorphic cells correlated with a reduction in intracellular iron content and defective glucose metabolism including glycolysis and mitochondrial respiration. CAMKK2 deficiency also caused reduction in GAPDH and VDAC1 protein level which correlated to defective bioenergetics function. These findings have identified a novel mechanistic link between abnormal calcium signaling, iron dyshomeostasis and metabolic dysfunction involving CAMKK2.},
}
@article {pmid32485215,
year = {2020},
author = {Zou, MM and Wang, Q and Chu, LN and Vasseur, L and Zhai, YL and Qin, YD and He, WY and Yang, G and Zhou, YY and Peng, L and You, MS},
title = {CRISPR/Cas9-induced vitellogenin knockout lead to incomplete embryonic development in Plutella xylostella.},
journal = {Insect biochemistry and molecular biology},
volume = {123},
number = {},
pages = {103406},
doi = {10.1016/j.ibmb.2020.103406},
pmid = {32485215},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Embryonic Development/genetics ; Gene Knockout Techniques ; Insect Proteins/genetics/metabolism ; Larva/metabolism ; *Moths/embryology/genetics ; Oogenesis/genetics ; Oviposition ; Pest Control/methods ; Transcriptome ; *Vitellogenins/genetics/metabolism ; },
abstract = {Vitellogenin (Vg) is important for insect egg maturation and embryo development. In the present study, we characterized the molecular structure and expression profile of Vg gene, and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops, using CRISPR/Cas9 system. The P. xylostella Vg (PxVg) included all conserved domains and motifs that were commonly found in most insect Vgs except for the polyserine tract. PxVg gene was highly expressed in female pupae and adults. PxVg protein was detected in eggs and female adults. PxVg was mainly expressed in the fat body and its protein was detected in most tissues, except in the midgut. CRISPR/Cas9-induced PxVg knockout successfully constructed a homozygous mutant strain with a 5-base pair nucleotide deletion. No PxVg protein was found in the mutant individuals and in their ovaries. There were no significant differences between wild (WT) and mutant (Mut-5) types of P. xylostella in terms of ovariole length and the number of fully developed oocytes in newly emerged females. No significant difference was observed in the number of eggs laid within two days, but there was a lower egg hatchability (84% for WT vs. 47% for Mut-5). This is the first study presenting the functions of Vg in ovary development, egg maturation, oviposition and embryonic development of P. xylostella. Our results suggest that the reproductive functions of Vg may be species-specific in insects. It is possible that Vg may not be the major egg yolk protein precursor in P. xylostella. Other "functional Vgs" closely involved in the yolk formation and oogenesis would need to be further explored in P. xylostella.},
}
@article {pmid32485207,
year = {2021},
author = {Ely, A and Singh, P and Smith, TS and Arbuthnot, P},
title = {In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {134-146},
doi = {10.1016/j.addr.2020.05.010},
pmid = {32485207},
issn = {1872-8294},
mesh = {Antiviral Agents/therapeutic use ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dose-Response Relationship, Drug ; Gene Editing ; Genetic Therapy/*methods ; Genetic Vectors/administration &amp; dosage ; Hepatitis B Vaccines/administration &amp; dosage ; Hepatitis B, Chronic/drug therapy/*genetics/prevention &amp; control/*therapy ; Nanoparticles/chemistry ; RNA, Messenger/*administration &amp; dosage ; Ribonucleases/administration &amp; dosage ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases/metabolism ; },
abstract = {Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. The ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.},
}
@article {pmid32485164,
year = {2020},
author = {Sun, SH and Chen, Q and Gu, HJ and Yang, G and Wang, YX and Huang, XY and Liu, SS and Zhang, NN and Li, XF and Xiong, R and Guo, Y and Deng, YQ and Huang, WJ and Liu, Q and Liu, QM and Shen, YL and Zhou, Y and Yang, X and Zhao, TY and Fan, CF and Zhou, YS and Qin, CF and Wang, YC},
title = {A Mouse Model of SARS-CoV-2 Infection and Pathogenesis.},
journal = {Cell host &amp; microbe},
volume = {28},
number = {1},
pages = {124-133.e4},
pmid = {32485164},
issn = {1934-6069},
mesh = {Aging ; Angiotensin-Converting Enzyme 2 ; Animals ; Betacoronavirus/*physiology ; Brain/virology ; COVID-19 ; CRISPR-Cas Systems ; *Coronavirus Infections/pathology/virology ; Cytokines/blood ; *Disease Models, Animal ; Gene Knock-In Techniques ; Lung/pathology/virology ; Lung Diseases, Interstitial/pathology ; *Mice, Inbred C57BL ; Nose/virology ; *Pandemics ; Peptidyl-Dipeptidase A/genetics/metabolism ; *Pneumonia, Viral/pathology/virology ; RNA, Viral/analysis ; SARS-CoV-2 ; Stomach/virology ; Trachea/virology ; Viral Load ; Virus Replication ; },
abstract = {Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.},
}
@article {pmid32485106,
year = {2020},
author = {Liu, Y and Chen, J and Crisante, D and Jaramillo Lopez, JM and Mahadevan, R},
title = {Dynamic Cell Programming with Quorum Sensing-Controlled CRISPRi Circuit.},
journal = {ACS synthetic biology},
volume = {9},
number = {6},
pages = {1284-1291},
doi = {10.1021/acssynbio.0c00148},
pmid = {32485106},
issn = {2161-5063},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/*genetics ; DNA Replication/drug effects ; Escherichia coli/drug effects/genetics/metabolism ; Gene Editing ; Green Fluorescent Proteins/genetics/metabolism ; Quorum Sensing/*genetics ; RNA, Guide/metabolism ; Synthetic Biology/*methods ; },
abstract = {Synthetic biology is enabling rapid advances in the areas of biomanufacturing and live therapeutics. Dynamic circuits that can be used to regulate cellular resources and microbial community behavior represent a defining focus of synthetic biology, and have attracted tremendous interest. However, the existing dynamic circuits are mostly gene editing-dependent or cell lysis-based, which limits their broad and convenient application, and in some cases, such lysis-based circuits can suffer from genetic instability due to evolution. There is limited research in quorum sensing-assisted CRISPRi, which can function in a gene editing-independent manner. Here, we constructed a series of quorum sensing controlled CRISPRi systems (Q-CRISPRi), which can dynamically program bacteria by using customized sgRNA without introducing cell lysis. We successfully applied Q-CRISPRi circuits to dynamically program gene expression, population density, phenotype, physical property, and community composition of microbial consortia. The strategies reported here represent methods for dynamic cell programming and could be effective in programming industrially and medically important microorganisms to offer better control of their metabolism and behavior.},
}
@article {pmid32485105,
year = {2020},
author = {Tou, CJ and Schaffer, DV and Dueber, JE},
title = {Targeted Diversification in the S. cerevisiae Genome with CRISPR-Guided DNA Polymerase I.},
journal = {ACS synthetic biology},
volume = {9},
number = {7},
pages = {1911-1916},
doi = {10.1021/acssynbio.0c00149},
pmid = {32485105},
issn = {2161-5063},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Chromosomes, Fungal/genetics ; DNA Polymerase I/*genetics ; DNA Replication/genetics ; Deoxyribonuclease I/genetics ; Escherichia coli/genetics ; Gene Editing/methods ; Genetic Loci ; *Genome, Fungal ; Mutagenesis ; Nucleotides/genetics ; Point Mutation ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/*genetics ; },
abstract = {New technologies to target nucleotide diversification in vivo are promising enabling strategies to perform directed evolution for engineering applications and forward genetics for addressing biological questions. Recently, we reported EvolvR-a system that employs CRISPR-guided Cas9 nickases fused to nick-translating, error-prone DNA polymerases to diversify targeted genomic loci-in E. coli. As CRISPR-Cas9 has shown activity across diverse cell types, EvolvR has the potential to be ported into other organisms, including eukaryotes, if nick-translating polymerases can be active across species. Here, we implement and characterize EvolvR's function in Saccharomyces cerevisiae, representing a key first step to enable EvolvR-mediated mutagenesis in eukaryotes. This advance will be useful for mutagenesis of user-defined loci in the yeast chromosomes for both engineering and basic research applications, and it furthermore provides a platform to develop the EvolvR technology for performance in higher eukaryotes.},
}
@article {pmid32485068,
year = {2020},
author = {Zeng, D and Liu, T and Ma, X and Wang, B and Zheng, Z and Zhang, Y and Xie, X and Yang, B and Zhao, Z and Zhu, Q and Liu, YG},
title = {Quantitative regulation of Waxy expression by CRISPR/Cas9-based promoter and 5'UTR-intron editing improves grain quality in rice.},
journal = {Plant biotechnology journal},
volume = {18},
number = {12},
pages = {2385-2387},
pmid = {32485068},
issn = {1467-7652},
mesh = {5' Untranslated Regions/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Introns ; *Oryza/genetics ; Waxes ; },
}
@article {pmid32483723,
year = {2020},
author = {Wang, X and Lv, S and Liu, T and Wei, J and Qu, S and Lu, Y and Zhang, J and Oo, S and Zhang, B and Pan, X and Liu, H},
title = {CRISPR/Cas9 genome editing shows the important role of AZC_2928 gene in nitrogen-fixing bacteria of plants.},
journal = {Functional &amp; integrative genomics},
volume = {20},
number = {5},
pages = {657-668},
doi = {10.1007/s10142-020-00739-8},
pmid = {32483723},
issn = {1438-7948},
mesh = {Azorhizobium caulinodans/*genetics/growth &amp; development/physiology ; Bacterial Proteins/chemistry/genetics/*physiology ; Biofilms ; *CRISPR-Cas Systems ; Chemotaxis ; *Gene Editing ; Gene Knockout Techniques ; Genes, Bacterial ; Nitrogen Fixation ; Plant Root Nodulation ; Sequence Analysis, Protein ; Sesbania/microbiology/physiology ; },
abstract = {AZC_2928 gene (GenBank accession no. BAF88926.1) of Azorhizobium caulinodans ORS571 has sequence homology to 2,3-aminomutases. However, its function is unknown. In this study, we are for the first time to knock out the gene completely in A. caulinodans ORS571 using the current advanced genome editing tool, CRISPR/Cas9. Our results show that the editing efficiency is 34% and AZC_2928 plays an extremely important role in regulating the formation of chemotaxis and biofilm. CRISPR/Cas9 knockout of AZC_2928 (△AZC_2928) significantly enhanced chemotaxis and biofilm formation. Both chemotaxis and biofilm formation play an important role in nitrogen-fixing bacteria and their interaction with their host plants. Interestingly, AZC_2928 did not affect the motility of A. caulinodans ORS571 and the nodulation formation in their natural host plant, Sesbania rostrata. Due to rhizobia needing to form bacteroids for symbiotic nitrogen fixation in mature nodules, AZC_2928 might have a direct influence on nitrogen fixation efficiency rather than the number of nodulations.},
}
@article {pmid32483603,
year = {2020},
author = {Stenger, D and Stief, TA and Kaeuferle, T and Willier, S and Rataj, F and Schober, K and Vick, B and Lotfi, R and Wagner, B and Grünewald, TGP and Kobold, S and Busch, DH and Jeremias, I and Blaeschke, F and Feuchtinger, T},
title = {Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR.},
journal = {Blood},
volume = {136},
number = {12},
pages = {1407-1418},
pmid = {32483603},
issn = {1528-0020},
support = {681524/ERC_/European Research Council/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; Immunotherapy, Adoptive/*methods ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology/pathology/*therapy ; Receptors, Antigen, T-Cell/genetics/*immunology ; Transduction, Genetic ; Tumor Cells, Cultured ; },
abstract = {Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.},
}
@article {pmid32483454,
year = {2020},
author = {Liu, H and Li, H and Liang, Y and Du, X and Yang, C and Yang, L and Xie, J and Zhao, R and Tong, Y and Qiu, S and Song, H},
title = {Phage-delivered sensitisation with subsequent antibiotic treatment reveals sustained effect against antimicrobial resistant bacteria.},
journal = {Theranostics},
volume = {10},
number = {14},
pages = {6310-6321},
pmid = {32483454},
issn = {1838-7640},
mesh = {Animals ; Anti-Bacterial Agents/*administration &amp; dosage ; Bacteriophages/*physiology ; CRISPR-Cas Systems ; Disease Models, Animal ; Drug Resistance, Bacterial ; Escherichia coli/*drug effects/genetics/virology ; Escherichia coli Infections/*drug therapy/microbiology ; Female ; Lysogeny ; Mice ; Mice, Inbred BALB C ; },
abstract = {Temperate phages integrated with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems have been gaining attention as potential strategies for combating bacteria resistant to antimicrobials. To further advance this technology, phage recombination procedure should be improved, and the bactericidal effect should be examined in detail and compared with conventional lytic phage strategy. The possibility of the emergence of mutational resistance, a phenomenon commonly observed with lytic phage therapy, should be illustrated. Methods: Here, we developed a novel one-step cloning method to fulfil the recombination of CRISPR/Cas9 system within the genome of a new isolated lysogenic Escherichia coli phage. Then, we proposed and developed a phage-delivered resistance eradication with subsequent antibiotic treatment (PRESA) strategy. The removal efficiency and antimicrobial effect of the plasmids were analysed. Long-term antimicrobial effect was evaluated by continued OD600 monitoring for 240 hours to illustrate the potential mutational resistance, compared with the lytic phage strategy. The treatment effect of PRESA was evaluated in vivo by determining bacterial loads in the skin and intestine of infected mice, in contrast with lytic phage therapy. Genome sequencing was performed to identify mutations in bacterial cells treated with phage strategies. Results: Phage-delivered CRISPR targeting efficiently eradicated and blocked the transfer of the antibiotic resistance plasmid. PRESA decreased the bacterial load by over 6- and 5-logs in vitro and in vivo, respectively. Importantly, while lytic phages induced mutational phage resistance at 24 h in vitro and 48 hours in vivo, PRESA demonstrated a constant effect and revealed no resistant mutants. Genes involved in DNA mismatch repair were upregulated in cells undergoing Cas9-based plasmid cleavage, which may reduce the development of mutations. Conclusion: The PRESA strategy for eradicating resistant bacteria showed high bactericidal efficacy and a sustained inhibition effect against resistant bacteria. By restoring the efficacy of low-cost antibiotics, PRESA could be developed as an efficient and economical therapy for infections of antibiotic resistant bacteria.},
}
@article {pmid32483441,
year = {2020},
author = {Liao, TT and Lin, CC and Jiang, JK and Yang, SH and Teng, HW and Yang, MH},
title = {Harnessing stemness and PD-L1 expression by AT-rich interaction domain-containing protein 3B in colorectal cancer.},
journal = {Theranostics},
volume = {10},
number = {14},
pages = {6095-6112},
pmid = {32483441},
issn = {1838-7640},
mesh = {Animals ; B7-H1 Antigen/immunology/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Colorectal Neoplasms/immunology/metabolism/*pathology ; Computational Biology/methods ; DNA-Binding Proteins/*metabolism ; Databases, Genetic ; Epigenesis, Genetic ; Female ; Gene Knockout Techniques ; Humans ; Immunotherapy ; Jumonji Domain-Containing Histone Demethylases/*metabolism ; Mice ; Mice, Nude ; Neoplastic Stem Cells/immunology/metabolism/*pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Background and Aims: Cancer stem cells (CSCs) have been shown to be responsible for the tumor initiation, metastasis, and therapeutic resistance of colorectal cancer (CRC). Recent studies have also indicated the importance of CSCs in escaping immune surveillance. However, the coordinated epigenetic control of the stem cell signature and the key molecule(s) involved in immunosurveillance of colorectal CSCs (CRCSCs) are unclear. Here, we investigated the role of a histone modifier, AT-rich interaction domain-containing protein 3B (ARID3B), in CRC. Methods: CRC patient-derived xenografts (PDXs) with knockout of ARID3B induced by CRISPR/Cas9 in vivo were used. Molecular/cellular biology assays were performed. Clinical data obtained from The Cancer Genome Atlas, as well as from our cohort (Taipei Veterans General Hospital), were analyzed. Results: ARID3B was crucial for the growth of CRC, and ARID3B promoted the stem-like features of CRC. Mechanistically, ARID3B activated Notch target genes, intestinal stem cell (ISC) genes, and programmed death-ligand 1 (PD-L1) through the recruitment of lysine-specific demethylase 4C (KDM4C) to modulate the chromatin configuration for transcriptional activation. Clinical sample analyses showed that the coexpression of ARID3B and the Notch target HES1 correlated with a worse outcome and that ARID3B and PD-L1 were highly expressed in the consensus molecular subtype 4 of CRC. Pharmacological inhibition of KDM4 activity reversed the ARID3B-induced signature. Conclusion: We reveal a noncanonical Notch pathway for activating Notch target genes, ISC genes, and PD-L1 in CRC. This finding explains the immune escape of CRCSCs and indicates a potential group that may benefit from immune checkpoint inhibitors. Epigenetic drugs for reversing stem-like features of CRC should also be investigated.},
}
@article {pmid32483365,
year = {2020},
author = {Sakata, RC and Ishiguro, S and Mori, H and Tanaka, M and Tatsuno, K and Ueda, H and Yamamoto, S and Seki, M and Masuyama, N and Nishida, K and Nishimasu, H and Arakawa, K and Kondo, A and Nureki, O and Tomita, M and Aburatani, H and Yachie, N},
title = {Base editors for simultaneous introduction of C-to-T and A-to-G mutations.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {865-869},
pmid = {32483365},
issn = {1546-1696},
mesh = {Adenine/metabolism ; Adenosine Deaminase/genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cytosine/metabolism ; DNA/*genetics ; Deoxyribonuclease I/genetics ; *Gene Editing ; Genome, Human/genetics ; Glycoproteins/genetics ; Guanine/metabolism ; HEK293 Cells ; Humans ; Mutation/genetics ; Nuclear Proteins/genetics ; RNA/genetics ; },
abstract = {We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.},
}
@article {pmid32483364,
year = {2020},
author = {Grünewald, J and Zhou, R and Lareau, CA and Garcia, SP and Iyer, S and Miller, BR and Langner, LM and Hsu, JY and Aryee, MJ and Joung, JK},
title = {A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {861-864},
pmid = {32483364},
issn = {1546-1696},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Adenine/chemistry ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cytosine/chemistry ; Cytosine Deaminase/*genetics ; *Gene Editing ; HEK293 Cells ; Humans ; Mutation/genetics ; RNA/genetics ; },
abstract = {Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR-Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors.},
}
@article {pmid32483363,
year = {2020},
author = {Zhang, X and Zhu, B and Chen, L and Xie, L and Yu, W and Wang, Y and Li, L and Yin, S and Yang, L and Hu, H and Han, H and Li, Y and Wang, L and Chen, G and Ma, X and Geng, H and Huang, W and Pang, X and Yang, Z and Wu, Y and Siwko, S and Kurita, R and Nakamura, Y and Yang, L and Liu, M and Li, D},
title = {Dual base editor catalyzes both cytosine and adenine base conversions in human cells.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {856-860},
pmid = {32483363},
issn = {1546-1696},
mesh = {*Adenine ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; *Cytosine ; Deoxyribonuclease I/genetics ; Gene Editing/*methods ; Humans ; RNA/genetics ; },
abstract = {Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&amp;C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&amp;C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.},
}
@article {pmid32483187,
year = {2020},
author = {Hirschi, M and Lu, WT and Santiago-Frangos, A and Wilkinson, R and Golden, SM and Davidson, AR and Lander, GC and Wiedenheft, B},
title = {AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2730},
pmid = {32483187},
issn = {2041-1723},
support = {DP2 EB020402/EB/NIBIB NIH HHS/United States ; S10 OD021634/OD/NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Bacteria/genetics/*metabolism/virology ; Bacterial Proteins/chemistry/genetics/metabolism ; Bacteriophages/genetics/*metabolism ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/chemistry/genetics/*metabolism ; Models, Molecular ; Multiprotein Complexes/chemistry/metabolism/ultrastructure ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation ; Proteus penneri/genetics/metabolism/virology ; RNA, Guide/chemistry/genetics/*metabolism ; Sequence Homology, Amino Acid ; Viral Proteins/chemistry/genetics/*metabolism ; },
abstract = {Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.},
}
@article {pmid32483117,
year = {2020},
author = {Rose, JC and Popp, NA and Richardson, CD and Stephany, JJ and Mathieu, J and Wei, CT and Corn, JE and Maly, DJ and Fowler, DM},
title = {Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2697},
pmid = {32483117},
issn = {2041-1723},
support = {R01 GM109110/GM/NIGMS NIH HHS/United States ; R01 GM086858/GM/NIGMS NIH HHS/United States ; F30 CA189793/CA/NCI NIH HHS/United States ; T32 GM007266/GM/NIGMS NIH HHS/United States ; P30 DK017047/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Binding Sites/genetics ; Biocatalysis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; DNA Repair ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mice ; Models, Genetic ; *Mutation ; RNA, Guide/*genetics/metabolism ; },
abstract = {CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.},
}
@article {pmid32479629,
year = {2020},
author = {Becker, M and Noll-Puchta, H and Amend, D and Nolte, F and Fuchs, C and Jeremias, I and Braun, CJ},
title = {CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.},
journal = {Nucleic acids research},
volume = {48},
number = {13},
pages = {e78},
pmid = {32479629},
issn = {1362-4962},
support = {681524/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Cloning, Molecular ; *Gene Library ; Genome ; Humans ; Mice ; Oligonucleotides/genetics ; RNA, Guide/*genetics ; },
abstract = {The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.},
}
@article {pmid32479612,
year = {2020},
author = {Wu, F and Shim, J and Gong, T and Tan, C},
title = {Orthogonal tuning of gene expression noise using CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {48},
number = {13},
pages = {e76},
pmid = {32479612},
issn = {1362-4962},
support = {R21 EB025938/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cloning, Molecular/*methods ; Escherichia coli/*genetics ; Gene Expression/*genetics ; Gene Expression Regulation, Bacterial ; Genes, Reporter/genetics ; RNA, Guide/*genetics ; },
abstract = {The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.},
}
@article {pmid32479262,
year = {2020},
author = {Wolf, G and de Iaco, A and Sun, MA and Bruno, M and Tinkham, M and Hoang, D and Mitra, A and Ralls, S and Trono, D and Macfarlan, TS},
title = {KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32479262},
issn = {2050-084X},
support = {1ZIAHD008933//Eunice Kennedy Shriver National Institute of Child Health and Human Development/International ; 310030_152879/SNSF_/Swiss National Science Foundation/Switzerland ; 310030B_173337/SNSF_/Swiss National Science Foundation/Switzerland ; No. 268721/ERC_/European Research Council/International ; No 694658/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; *DNA/chemistry/genetics/metabolism ; DNA Transposable Elements/*genetics ; Evolution, Molecular ; Gene Editing ; Mice ; Mice, Knockout ; Mutation/genetics ; *Repressor Proteins/chemistry/genetics/metabolism ; Retroelements/*genetics ; Zinc Fingers/*genetics ; },
abstract = {The Krüppel-associated box zinc finger protein (KRAB-ZFP) family diversified in mammals. The majority of human KRAB-ZFPs bind transposable elements (TEs), however, since most TEs are inactive in humans it is unclear whether KRAB-ZFPs emerged to suppress TEs. We demonstrate that many recently emerged murine KRAB-ZFPs also bind to TEs, including the active ETn, IAP, and L1 families. Using a CRISPR/Cas9-based engineering approach, we genetically deleted five large clusters of KRAB-ZFPs and demonstrate that target TEs are de-repressed, unleashing TE-encoded enhancers. Homozygous knockout mice lacking one of two KRAB-ZFP gene clusters on chromosome 2 and chromosome 4 were nonetheless viable. In pedigrees of chromosome 4 cluster KRAB-ZFP mutants, we identified numerous novel ETn insertions with a modest increase in mutants. Our data strongly support the current model that recent waves of retrotransposon activity drove the expansion of KRAB-ZFP genes in mice and that many KRAB-ZFPs play a redundant role restricting TE activity.},
}
@article {pmid32478678,
year = {2020},
author = {Chemello, F and Bassel-Duby, R and Olson, EN},
title = {Correction of muscular dystrophies by CRISPR gene editing.},
journal = {The Journal of clinical investigation},
volume = {130},
number = {6},
pages = {2766-2776},
pmid = {32478678},
issn = {1558-8238},
support = {R01 HL138426/HL/NHLBI NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Dystrophin/biosynthesis/genetics ; *Gene Editing ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; *Mutation ; },
abstract = {Muscular dystrophies are debilitating disorders that result in progressive weakness and degeneration of skeletal muscle. Although the genetic mutations and clinical abnormalities of a variety of neuromuscular diseases are well known, no curative therapies have been developed to date. The advent of genome editing technology provides new opportunities to correct the underlying mutations responsible for many monogenic neuromuscular diseases. For example, Duchenne muscular dystrophy, which is caused by mutations in the dystrophin gene, has been successfully corrected in mice, dogs, and human cells through CRISPR/Cas9 editing. In this Review, we focus on the potential for, and challenges of, correcting muscular dystrophies by editing disease-causing mutations at the genomic level. Ideally, because muscle tissues are extremely long-lived, CRISPR technology could offer a one-time treatment for muscular dystrophies by correcting the culprit genomic mutations and enabling normal expression of the repaired gene.},
}
@article {pmid32478492,
year = {2020},
author = {Tan, YY and Du, H and Wu, X and Liu, YH and Jiang, M and Song, SY and Wu, L and Shu, QY},
title = {Gene editing: an instrument for practical application of gene biology to plant breeding.},
journal = {Journal of Zhejiang University. Science. B},
volume = {21},
number = {6},
pages = {460-473},
pmid = {32478492},
issn = {1862-1783},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; Gene Expression Regulation, Plant ; *Plant Breeding ; Plants, Genetically Modified ; RNA Processing, Post-Transcriptional ; Transcription, Genetic ; },
abstract = {Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.},
}
@article {pmid32478351,
year = {2020},
author = {Hsieh, K and Zhao, G and Wang, TH},
title = {Applying biosensor development concepts to improve preamplification-free CRISPR/Cas12a-Dx.},
journal = {The Analyst},
volume = {145},
number = {14},
pages = {4880-4888},
pmid = {32478351},
issn = {1364-5528},
support = {R01 AI138978/AI/NIAID NIH HHS/United States ; R01 AI137272/AI/NIAID NIH HHS/United States ; R01 AI117032/AI/NIAID NIH HHS/United States ; S10 OD021567/OD/NIH HHS/United States ; U01 CA214165/CA/NCI NIH HHS/United States ; UG3 CA211457/CA/NCI NIH HHS/United States ; },
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded ; Limit of Detection ; },
abstract = {Development of CRISPR/Cas-based in vitro diagnostic devices, or CRISPR/Cas-Dx, has become an intensely researched area. Among the different classes of CRISPR/Cas-Dx, the class based on the Cas12a enzyme (i.e., CRISPR/Cas12a-Dx or simply Cas12a-Dx), is predominantly employed for detecting DNA targets. Current research in Cas12a-Dx has focused on appending Cas12a-Dx to preamplification techniques or coupling Cas12a-Dx to different detection modalities, which has inevitably overshadowed the detection performance of Cas12a-Dx and overlooked its intrinsic detection capability without preamplification. We recognize that Cas12a-Dx, which relies on DNA-activated Cas12a to cleave single-stranded DNA, shares significant similarity with other nuclease-based DNA biosensors, whose performances can be influenced by parameters ranging from the reaction buffer to the reaction temperature. We are thus inspired to probe the limits of preamplification-free Cas12a-Dx by exploring and systematically evaluating several potential parameters that may impact its detection sensitivity and time. Using a previously reported fluorescence-based Cas12a-Dx as the test bed, we have identified that the Cas12a enzyme, the reaction buffer, the substrate label, the substrate concentration, and the reaction temperature can be optimized to significantly improve the signal-to-background ratio and the reaction rate of Cas12a-Dx. Based on these findings, we have improved the limit of detection (LOD) of the Cas12a-Dx to 100 fM, while reduced the time-to-positive to <46 min, representing the most sensitive LOD without preamplification and the fastest time-to-positive for this LOD to date. More broadly, our work provides a roadmap for further advancing Cas12a-Dx and perhaps other classes of CRISPR/Cas-Dx.},
}
@article {pmid32476557,
year = {2020},
author = {O'Shea, P and Wildenhain, J and Leveridge, M and Revankar, C and Yang, JP and Bradley, J and Firth, M and Pilling, J and Piper, D and Chesnut, J and Isherwood, B},
title = {A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {6},
pages = {618-633},
doi = {10.1177/2472555220926160},
pmid = {32476557},
issn = {2472-5560},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Library ; Genes, Reporter/genetics ; Genome, Human/genetics ; High-Throughput Screening Assays/methods ; Humans ; NF-kappa B/*genetics ; Phosphotransferases/classification/genetics ; RNA, Guide/genetics ; Signal Transduction/genetics ; Tumor Necrosis Factor-alpha/*genetics ; beta-Lactamases/genetics ; },
abstract = {CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.},
}
@article {pmid32474999,
year = {2020},
author = {Pan, Y and Sun, Y and Wang, Y and Zhang, Z},
title = {Barcode sequence could be a good target for developing a species-specific anti-parasite agent based on CRISPR-Cas9.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {7},
pages = {9393-9404},
doi = {10.1096/fj.202000118RR},
pmid = {32474999},
issn = {1530-6860},
mesh = {Animals ; *CRISPR-Cas Systems ; Ciliophora/*genetics/growth &amp; development/metabolism ; Ciliophora Infections/parasitology/*veterinary ; Fish Diseases/*parasitology ; *Gene Editing ; Protozoan Proteins/*genetics ; RNA, Guide/*genetics ; },
abstract = {Parasitic infections are a severe issue in many regions of the world. We assume that if a chemical can destroy a DNA barcode sequence, then this chemical could be developed as a species-specific parasiticidal agent. To test this hypothesis, we designed sgRNAs that target the sequences of both a DNA barcode (ITS-2) and a control (5.8S rDNA) in Cryptocaryon irritans. In in vivo tests, we found that exposure to Cas9 mRNA mixed with sgRNAs was able to significantly reduce the hatching rate of tomont and the survival rate of theront. Quantitative Real-time PCR demonstrated that the DNAs of tomont and theront exposed to sgRNAs and Cas9 mRNA were significantly disrupted, no matter whether they were exposed to a single sgRNA or a mixture of two sgRNAs. DNA sequencing also suggested the test group that was exposed to a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments and the test group exposed to two sgRNAs combined with Cas9-induced deletion of large pieces. The findings and principles provided by this study contribute to the development of novel nucleic acid therapeutic drugs for cryptocaryoniasis and other parasitic diseases and provide insight into the development of species-specific parasiticidal agents.},
}
@article {pmid32474864,
year = {2020},
author = {Butterfield, KT and McGrath, PS and Han, CM and Kogut, I and Bilousova, G},
title = {Generation of an Induced Pluripotent Stem Cell Line with the Constitutive EGFP Reporter.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2155},
number = {},
pages = {11-21},
pmid = {32474864},
issn = {1940-6029},
support = {P30 AR057212/AR/NIAMS NIH HHS/United States ; R21 AR074642/AR/NIAMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; *Gene Expression ; Gene Knock-In Techniques ; Gene Targeting ; *Genes, Reporter ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Green Fluorescent Proteins/*genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; },
abstract = {The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.},
}
@article {pmid32474853,
year = {2020},
author = {Xie, P and Mo, JL and Liu, JH and Li, X and Tan, LM and Zhang, W and Zhou, HH and Liu, ZQ},
title = {Pharmacogenomics of 5-fluorouracil in colorectal cancer: review and update.},
journal = {Cellular oncology (Dordrecht)},
volume = {43},
number = {6},
pages = {989-1001},
pmid = {32474853},
issn = {2211-3436},
mesh = {Animals ; Autophagy/genetics ; Colorectal Neoplasms/*drug therapy/*genetics/pathology ; Drug Resistance, Neoplasm/genetics ; Fluorouracil/*therapeutic use ; Humans ; *Pharmacogenetics ; Signal Transduction/genetics ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is a disease with high morbidity and mortality rates. 5-fluorouracil (5-FU) is the first-line recommended drug for chemotherapy in patients with CRC, and it has a good effect on a variety of other solid tumors as well. Unfortunately, however, due to the emergence of drug resistance the effectiveness of treatment may be greatly reduced. In the past decade, major progress has been made in the field of 5-FU drug resistance in terms of molecular mechanisms, pre-clinical (animal) models and clinical trials.<br><br>CONCLUSIONS: In this article we systematically review and update current knowledge on 5-FU pharmacogenomics related to drug uptake and activation, the expression and activity of target enzymes (DPD, TS and MTHFR) and key signaling pathways in CRC. Furthermore, a summary of drug combination strategies aimed at targeting specific genes and/or pathways to reverse 5-FU resistance is provided. Based on this, we suggest that causal relationships between genes, pathways and drug sensitivity should be systematically considered from a multidimensional perspective. In the design of research methods, emerging technologies such as CRISPR-Cas, TALENS and patient-derived xenograft models should be applied as far as possible to improve the accuracy of clinically relevant results.},
}
@article {pmid32473055,
year = {2020},
author = {Merker, L and Schindele, P and Huang, TK and Wolter, F and Puchta, H},
title = {Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a.},
journal = {Plant biotechnology journal},
volume = {18},
number = {12},
pages = {2382-2384},
pmid = {32473055},
issn = {1467-7652},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Targeting ; Temperature ; },
}
@article {pmid32472422,
year = {2020},
author = {Safari, I and Baradaran-Rafii, A and Issazadeh-Navikas, S and Elahi, E},
title = {CHST6 mutations identified in Iranian MCD patients and CHST6 mutations reported worldwide identify targets for gene editing approaches including the CRISPR/Cas system.},
journal = {International ophthalmology},
volume = {40},
number = {9},
pages = {2223-2235},
doi = {10.1007/s10792-020-01401-9},
pmid = {32472422},
issn = {1573-2630},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; *Corneal Dystrophies, Hereditary/genetics ; DNA Mutational Analysis ; *Gene Editing ; Humans ; Iran ; Mutation ; Sulfotransferases ; },
abstract = {PURPOSE: To identify CHST6 mutations in Iranians macular corneal dystrophy (MCD) patients and also to assess distribution of amino acids in the encoded protein that are affected by CHST6 mutations reported hitherto in various populations in order to predict gene regions that may be appropriate targets for gene editing approaches including the CRISPR/Cas system. The analysis will also reveal biologically and functionally important regions of the protein.<br><br>METHODS: Mutation screening of CHST6 by sequencing was performed on 21 Iranian MCD-affected probands. Previously reported MCD causing CHST6 mutations were identified by searches in NCBI.<br><br>RESULTS: Nineteen CHST6 mutations were found among the 21 Iranian patients, most of which were missense mutations and six of which were novel. Totally, 189 mutations among 375 MCD patients have been found worldwide, and 134 of these are missense mutations. The distribution of 88 amino acids affected by missense mutations along the length of the encoded protein was not random, and four regions of possible mutation clustering were noted. 25% of patients harbored mutations in a DNA region consisting of only 36 nucleotides.<br><br>CONCLUSION: Similar to most populations, CHST6 mutations among Iranians are very heterogeneous as indicated by finding 19 different mutations among 21 MCD patients. Nevertheless, identification of four potential mutation clusters identifies regions that are most suitable for gene therapy targeting by the CRISPR/Cas approach. Additionally, the mutation clusters identify regions with potential structural and/or functional importance. Consistent with this, the amino acids in these regions are well conserved among various membrane-bound sulfotransferases.},
}
@article {pmid32472264,
year = {2020},
author = {Li, Q and Lu, L and Liu, H and Bai, X and Zhou, X and Wu, B and Yuan, M and Yang, L and Xing, Y},
title = {A minor QTL, SG3, encoding an R2R3-MYB protein, negatively controls grain length in rice.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {8},
pages = {2387-2399},
doi = {10.1007/s00122-020-03606-z},
pmid = {32472264},
issn = {1432-2242},
mesh = {Alleles ; CRISPR-Cas Systems ; Cell Nucleus/metabolism ; Chromosome Mapping/*methods ; Codon, Nonsense ; Edible Grain/*genetics ; Exons ; Gene Expression Regulation, Plant/genetics ; Gene Knockout Techniques ; *Genes, Plant ; INDEL Mutation ; Oryza/*genetics/growth &amp; development ; Phylogeny ; Plant Breeding/*methods ; Plants, Genetically Modified ; Polymorphism, Single Nucleotide ; Quantitative Trait Loci ; Seeds/*genetics/growth &amp; development ; Transcription Factors/*genetics/metabolism ; },
abstract = {SG3, an R2R3 MYB protein coding gene that tightly linked to a major QTLGS3, negatively regulates grain length while dependent on the status ofGS3in rice. It is often very difficult to isolate a minor QTL that is closely linked to a major QTL in rice. In this study, we focused on the isolation of a minor grain length QTL, small grain 3 (SG3), which is closely linked to the major QTL grain size 3 (GS3). The genetic effect of SG3 on grain length was dependent on GS3 status. Its genetic effect was larger in the presence of nonfunctional sg3 than functional SG3. A large number of near-isogenic F2 plants in which GS3 was fixed with nonfunctional alleles were developed to clone SG3. A gene encoding an R2R3 MYB domain transcriptional regulator was identified as the candidate gene for SG3. SG3 overexpression and knockdown plants showed shortened and elongated grains, respectively, which demonstrated that SG3 acts as a negative regulator of grain length. SG3 was preferentially expressed in panicles after flowering, and SG3 acted as a transcription activator. Comparative sequencing analysis identified a 12-bp insertion in the third exon of NYZ that led to a frameshift and resulted in a premature stop codon. The insertion/deletion was associated with grain length in the presence of functional GS3 in the indica subspecies. SG3 and GS3 were frequently in coupling phase in indica rice, making them good targets for the breeding of cultivars with short or long grains. The isolation of the SG3 MYB gene provides new gene resource and contributes to the regulatory network of grain length in rice.},
}
@article {pmid32471984,
year = {2020},
author = {Lukasheva, V and Devost, D and Le Gouill, C and Namkung, Y and Martin, RD and Longpré, JM and Amraei, M and Shinjo, Y and Hogue, M and Lagacé, M and Breton, B and Aoki, J and Tanny, JC and Laporte, SA and Pineyro, G and Inoue, A and Bouvier, M and Hébert, TE},
title = {Signal profiling of the β1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β1AR and β2AR.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {8779},
pmid = {32471984},
issn = {2045-2322},
support = {MOP11215//CIHR/Canada ; FDN148431//CIHR/Canada ; PJT159687//CIHR/Canada ; },
mesh = {Biosensing Techniques/methods ; CRISPR-Cas Systems ; Calcium/metabolism ; GTP-Binding Protein alpha Subunits, G12-G13/*metabolism ; GTP-Binding Protein alpha Subunits, Gs/*metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Ligands ; Phenotype ; Receptors, Adrenergic, beta-1/genetics/*metabolism ; Receptors, Adrenergic, beta-2/genetics/*metabolism ; Signal Transduction ; },
abstract = {A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β1-adrenergic receptor (β1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the β1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.},
}
@article {pmid32471865,
year = {2020},
author = {Das, S and Banerjee, A and Kamran, M and Ejazi, SA and Asad, M and Ali, N and Chakrabarti, S},
title = {A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {29},
pages = {9934-9947},
pmid = {32471865},
issn = {1083-351X},
support = {MR/P027989/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Antiprotozoal Agents/*pharmacology ; CRISPR-Cas Systems ; Cricetinae ; Dinucleoside Phosphates/*pharmacology ; Gene Knockout Techniques ; Heat-Shock Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Leishmania donovani/genetics/*metabolism ; Leishmaniasis, Visceral/*drug therapy/genetics/metabolism ; Macrophages/metabolism/*parasitology ; Mice ; Protozoan Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P[1],P[5]-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.},
}
@article {pmid32471232,
year = {2020},
author = {Yun, Y and Ha, Y},
title = {CRISPR/Cas9-Mediated Gene Correction to Understand ALS.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
pmid = {32471232},
issn = {1422-0067},
mesh = {Amyotrophic Lateral Sclerosis/genetics/*therapy ; Animals ; C9orf72 Protein/genetics ; *CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Humans ; RNA-Binding Protein FUS/genetics ; Superoxide Dismutase-1/genetics ; Targeted Gene Repair/*methods ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the death of motor neurons in the spinal cord and brainstem. ALS has a diverse genetic origin; at least 20 genes have been shown to be related to ALS. Most familial and sporadic cases of ALS are caused by variants of the SOD1, C9orf72, FUS, and TARDBP genes. Genome editing using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9) can provide insights into the underlying genetics and pathophysiology of ALS. By correcting common mutations associated with ALS in animal models and patient-derived induced pluripotent stem cells (iPSCs), CRISPR/Cas9 has been used to verify the effects of ALS-associated mutations and observe phenotype differences between patient-derived and gene-corrected iPSCs. This technology has also been used to create mutations to investigate the pathophysiology of ALS. Here, we review recent studies that have used CRISPR/Cas9 to understand the genetic underpinnings of ALS.},
}
@article {pmid32470556,
year = {2020},
author = {Xu, H and Zhang, X and Cai, Z and Dong, X and Chen, G and Li, Z and Qiu, L and He, L and Liang, B and Liu, X and Liu, J},
title = {An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {22},
number = {8},
pages = {1020-1029},
doi = {10.1016/j.jmoldx.2020.04.212},
pmid = {32470556},
issn = {1943-7811},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Bacterial/genetics ; Data Accuracy ; Endodeoxyribonucleases/*genetics ; Feasibility Studies ; Humans ; Limit of Detection ; Mycobacterium tuberculosis/*genetics ; Nucleic Acid Amplification Techniques/*methods ; RNA Cleavage/*genetics ; RNA, Guide/*genetics ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/*diagnosis/microbiology ; },
abstract = {Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R[2] = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.},
}
@article {pmid32470504,
year = {2020},
author = {Ebrahimi, V and Hashemi, A},
title = {Challenges of in vitro genome editing with CRISPR/Cas9 and possible solutions: A review.},
journal = {Gene},
volume = {753},
number = {},
pages = {144813},
doi = {10.1016/j.gene.2020.144813},
pmid = {32470504},
issn = {1879-0038},
mesh = {Animals ; Bacteria/genetics ; Bacterial Proteins/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods/*trends ; Genome, Bacterial/genetics ; Humans ; Metabolic Engineering/methods/*trends ; },
abstract = {Microbial production of bio-based ingredients often requires metabolically engineered bacterial strains with the edited genome. Genome editing tools are also essential for gene identification and investigating genotype-phenotype connections. Currently, one of the most common tools of genome editing is based on a natural bacterial adaptive immune system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) due to its simple, rapid, and efficient activities. Although successful in some in vitro systems, its application as an approach of metabolic engineering and genome editing is still not so extensive. Here, we discuss existing barriers and challenges of the CRISPR/Cas9 editing tool for in vitro systems. Firstly, we aim to briefly introduce the CRISPR/Cas9 method as an in vitro gene editing tool. Next, we discuss existing obstacles to CRISPR-based editing in bacterial and in vitro model systems and offer guidelines to help achieve editing in an expanded range of in vitro systems.},
}
@article {pmid32470463,
year = {2020},
author = {Strezoska, &#x17d; and Dickerson, SM and Maksimova, E and Chou, E and Gross, MM and Hemphill, K and Hardcastle, T and Perkett, M and Stombaugh, J and Miller, GW and Anderson, EM and Vermeulen, A and Smith, AVB},
title = {CRISPR-mediated transcriptional activation with synthetic guide RNA.},
journal = {Journal of biotechnology},
volume = {319},
number = {},
pages = {25-35},
doi = {10.1016/j.jbiotec.2020.05.005},
pmid = {32470463},
issn = {1873-4863},
mesh = {Animals ; Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mice ; NIH 3T3 Cells ; *RNA, Guide ; Transcriptional Activation/*genetics ; },
abstract = {The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.},
}
@article {pmid32470403,
year = {2020},
author = {Sullenger, BA},
title = {RGEN Editing of RNA and DNA: The Long and Winding Road from Catalytic RNAs to CRISPR to the Clinic.},
journal = {Cell},
volume = {181},
number = {5},
pages = {955-960},
doi = {10.1016/j.cell.2020.04.050},
pmid = {32470403},
issn = {1097-4172},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Endonucleases/metabolism ; Gene Editing/*methods/*trends ; Mutation ; RNA/genetics ; RNA Editing/*genetics/physiology ; RNA, Catalytic/genetics ; RNA, Guide/genetics ; },
abstract = {The first clinical studies utilizing RNA-guided endonucleases (RGENs) to therapeutically edit RNA and DNA in cancer patients were recently published. These groundbreaking technological advances promise to revolutionize genetic therapy and, as I discuss, represent the culmination of decades of innovative work to engineer RGENs for such editing applications.},
}
@article {pmid32470292,
year = {2020},
author = {Shin, S and Kim, SH and Shin, SW and Grav, LM and Pedersen, LE and Lee, JS and Lee, GM},
title = {Comprehensive Analysis of Genomic Safe Harbors as Target Sites for Stable Expression of the Heterologous Gene in HEK293 Cells.},
journal = {ACS synthetic biology},
volume = {9},
number = {6},
pages = {1263-1269},
doi = {10.1021/acssynbio.0c00097},
pmid = {32470292},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Gene Targeting/*methods ; Genes, Reporter ; Genetic Loci ; HEK293 Cells ; Humans ; Plasmids/genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics/metabolism ; RNA, Messenger/metabolism ; RNA, Untranslated/*genetics ; Receptors, CCR5/*genetics ; Simian virus 40/genetics ; Transgenes/*genetics ; },
abstract = {Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human genomic safe harbors (GSHs)-AAVS1, CCR5, and human ROSA26 loci-were evaluated with respect to the transgene expression level and stability in human embryonic kidney (HEK293) cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity. Transgene expression at the AAVS1 locus was sustained without selection for approximately 3 months. Furthermore, the CMV promoter showed the highest expression, followed by the EF1α, SV40, and TK promoters at the AAVS1 locus. Master cell lines were created using CRISPR/Cas9-mediated integration of the landing pad into the AAVS1 locus and were used for faster generation of recombinant cell lines that produce therapeutic proteins with recombinase-mediated cassette exchange.},
}
@article {pmid32469948,
year = {2020},
author = {Lee, MH and Hsu, TL and Lin, JJ and Lin, YJ and Kao, YY and Chang, JJ and Li, WH},
title = {Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0233492},
pmid = {32469948},
issn = {1932-6203},
mesh = {Biotechnology ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Fungal ; Glycoproteins/biosynthesis/chemistry/genetics ; Glycosylation ; Humans ; Kluyveromyces/*genetics/*metabolism ; Mannosidases/genetics/metabolism ; Mannosyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Metabolic Engineering/*methods ; N-Acetylglucosaminyltransferases/genetics/metabolism ; Polysaccharides/*biosynthesis/*chemistry/genetics ; Recombinant Proteins/biosynthesis/chemistry/genetics ; },
abstract = {Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (β-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.},
}
@article {pmid32468716,
year = {2020},
author = {Meyer, M},
title = {The CRISPR babies controversy: Responsibility and regulation in the spotlight.},
journal = {EMBO reports},
volume = {21},
number = {7},
pages = {e50307},
pmid = {32468716},
issn = {1469-3178},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Mammalian ; *Gene Editing ; Germ Cells ; Humans ; },
abstract = {The genome editing of human embryos by He Jianjui and the announcement to do so by Denis Rebrikov should spur the research community into discussing robust and transparent governance for human germline modificition.},
}
@article {pmid32468663,
year = {2020},
author = {Hu, Y and Li, J and Zhu, Y and Li, M and Lin, J and Yang, L and Wang, C and Lu, Z},
title = {Development and characterization of an Otp conditional loss of function allele.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {9},
pages = {e23370},
doi = {10.1002/dvg.23370},
pmid = {32468663},
issn = {1526-968X},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques/*methods ; Homeodomain Proteins/*genetics/metabolism ; Hypothalamus/cytology/metabolism ; *Loss of Function Mutation ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins/*genetics/metabolism ; Neurons/metabolism ; },
abstract = {Orthopedia (Otp) is a homeodomain transcription factor that plays an essential role in the development of hypothalamic neurosecretory systems. Loss of Otp results in the failure of differentiation of key hypothalamic neuroendocrine cell types, and pups die soon after birth. Although the constitutive knockout Otp mouse model (Otp [KO]) has significantly expanded our understanding of Otp's function in vivo, a conditional loss of function Otp allele that enables tissue or cell-type specific ablation of Otp has not been developed. Here, we used CRISPR/Cas9 gene-editing technology to generate a conditional Otp knockout mouse line in which exon 2 of the murine Otp gene is flanked by LoxP sites (Otp [f/f]). Crossing the Otp [f/f] mouse with Agrp-Ires-cre mouse, we demonstrate the requirement for Otp in the continuous differentiation of AgRP neurons after cell fate determination. We also show that the residual AgRP neurons in Agrp-Ires-cre;Otp [f/f] mice project to similar downstream target regions. This newly developed Otp [f/f] mouse can be used to explore the functions of Otp with cell-type or temporal specificity.},
}
@article {pmid32468475,
year = {2020},
author = {Buiani, R},
title = {Gene Editing, Sexual Reproduction, and the Arts: The Present, the Future, and the Imagined.},
journal = {Advances in experimental medicine and biology},
volume = {1195},
number = {},
pages = {177},
doi = {10.1007/978-3-030-32633-3_25},
pmid = {32468475},
issn = {0065-2598},
mesh = {*Art ; CRISPR-Cas Systems ; Gene Editing/*ethics/*trends ; Humans ; Reproductive Techniques, Assisted/*ethics/*trends ; },
abstract = {In recent years, popular culture has been graced with countless news announcing novel developments in genome editing. While many experiments are still in their early stages, genome editing seems very promising. Often betraying a sensationalist and triumphant tone, news coverage focuses on the potentials that these developments will have for the advancement of the human species, i.e., the eradication of disease, the extension of life, the improvement of the body and its appearance, etc. The future looks hopeful and unproblematic according to these accounts. On the opposite end of the spectrum, some may wonder whether these developments pose a potential worsening of the human condition: Are these developments safe? What are the ethical implications? Who will benefit from these developments? Given today's social divisions and cultural conflicts, these voices predict a rather unpromising future and warn against the pursue of innovation at any cost.},
}
@article {pmid32467995,
year = {2020},
author = {Alagoz, M and Kherad, N},
title = {Advance genome editing technologies in the treatment of human diseases: CRISPR therapy (Review).},
journal = {International journal of molecular medicine},
volume = {46},
number = {2},
pages = {521-534},
pmid = {32467995},
issn = {1791-244X},
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Humans ; Neurodegenerative Diseases/genetics ; },
abstract = {Genome editing techniques are considered to be one of the most challenging yet efficient tools for assisting therapeutic approaches. Several studies have focused on the development of novel methods to improve the efficiency of gene editing, as well as minimise their off‑target effects. Clustered regularly interspaced short palindromic repeats (CRISPR)‑associated protein (Cas9) is a tool that has revolutionised genome editing technologies. New applications of CRISPR/Cas9 in a broad range of diseases have demonstrated its efficiency and have been used in ex vivo models of somatic and pluripotent stem cells, as well as in in vivo animal models, and may eventually be used to correct defective genes. The focus of the present review was the recent applications of CRISPR/Cas9 and its contribution to the treatment of challenging human diseases, such as various types of cancer, neurodegenerative diseases and a broad spectrum of other disorders. CRISPR technology is a novel method for disease treatment, enhancing the effectiveness of drugs and improving the development of personalised medicine.},
}
@article {pmid32467331,
year = {2020},
author = {Meeske, AJ and Jia, N and Cassel, AK and Kozlova, A and Liao, J and Wiedmann, M and Patel, DJ and Marraffini, LA},
title = {A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6499},
pages = {54-59},
pmid = {32467331},
issn = {1095-9203},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages/genetics/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; Endonucleases/*metabolism ; Listeria/*virology ; RNA Stability ; RNA, Guide/genetics ; RNA, Viral/metabolism ; Viral Proteins/genetics/*metabolism ; },
abstract = {The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.},
}
@article {pmid32467316,
year = {2020},
author = {van der Lelij, P and Newman, JA and Lieb, S and Jude, J and Katis, V and Hoffmann, T and Hinterndorfer, M and Bader, G and Kraut, N and Pearson, MA and Peters, JM and Zuber, J and Gileadi, O and Petronczki, M},
title = {STAG1 vulnerabilities for exploiting cohesin synthetic lethality in STAG2-deficient cancers.},
journal = {Life science alliance},
volume = {3},
number = {7},
pages = {},
pmid = {32467316},
issn = {2575-1077},
support = {/WT_/Wellcome Trust/United Kingdom ; 106169/ZZ14/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Cycle Proteins/*deficiency/*genetics/metabolism ; Cell Line, Tumor ; Chromosomal Proteins, Non-Histone/*genetics ; Disease Susceptibility ; Gene Silencing ; Gene Targeting ; Genome-Wide Association Study ; Humans ; Models, Molecular ; Neoplasms/*genetics/metabolism/pathology ; Nuclear Proteins/chemistry/*genetics/metabolism ; Protein Binding ; Proteolysis ; Structure-Activity Relationship ; *Synthetic Lethal Mutations ; },
abstract = {The cohesin subunit STAG2 has emerged as a recurrently inactivated tumor suppressor in human cancers. Using candidate approaches, recent studies have revealed a synthetic lethal interaction between STAG2 and its paralog STAG1 To systematically probe genetic vulnerabilities in the absence of STAG2, we have performed genome-wide CRISPR screens in isogenic cell lines and identified STAG1 as the most prominent and selective dependency of STAG2-deficient cells. Using an inducible degron system, we show that chemical genetic degradation of STAG1 protein results in the loss of sister chromatid cohesion and rapid cell death in STAG2-deficient cells, while sparing STAG2-wild-type cells. Biochemical assays and X-ray crystallography identify STAG1 regions that interact with the RAD21 subunit of the cohesin complex. STAG1 mutations that abrogate this interaction selectively compromise the viability of STAG2-deficient cells. Our work highlights the degradation of STAG1 and inhibition of its interaction with RAD21 as promising therapeutic strategies. These findings lay the groundwork for the development of STAG1-directed small molecules to exploit synthetic lethality in STAG2-mutated tumors.},
}
@article {pmid32467232,
year = {2020},
author = {Long, J and Galvan, DL and Mise, K and Kanwar, YS and Li, L and Poungavrin, N and Overbeek, PA and Chang, BH and Danesh, FR},
title = {Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {47},
pages = {15840-15852},
pmid = {32467232},
issn = {1083-351X},
support = {R01 DK060632/DK/NIDDK NIH HHS/United States ; R01 DK060635/DK/NIDDK NIH HHS/United States ; R01 DK078900/DK/NIDDK NIH HHS/United States ; R01 DK091310/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/*metabolism ; Cell Line, Tumor ; *Gene Expression Regulation ; Glucose/genetics/*metabolism ; Histone Deacetylase 1/genetics/metabolism ; Humans ; Mice ; Podocytes/*metabolism ; RNA, Long Noncoding/*biosynthesis/genetics ; Repressor Proteins/genetics/metabolism ; *Transcription, Genetic ; },
abstract = {Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.},
}
@article {pmid32466470,
year = {2020},
author = {Han, JP and Chang, YJ and Song, DW and Choi, BS and Koo, OJ and Yi, SY and Park, TS and Yeom, SC},
title = {High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
pmid = {32466470},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Mice ; Mice, Inbred C57BL ; *Mitosis ; NIH 3T3 Cells ; *Nuclear Localization Signals ; Recombinant Proteins/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.},
}
@article {pmid32466303,
year = {2020},
author = {Bischoff, N and Wimberger, S and Maresca, M and Brakebusch, C},
title = {Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?.},
journal = {Cells},
volume = {9},
number = {5},
pages = {},
pmid = {32466303},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Cycle/genetics ; DNA Damage/genetics ; DNA Repair/genetics ; *Gene Editing ; Humans ; Small Molecule Libraries/*metabolism ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.},
}
@article {pmid32466287,
year = {2020},
author = {Ibragimova, S and Szebenyi, C and Sinka, R and Alzyoud, EI and Homa, M and Vágvölgyi, C and Nagy, G and Papp, T},
title = {CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera.},
journal = {International journal of molecular sciences},
volume = {21},
number = {10},
pages = {},
pmid = {32466287},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Fungal Proteins/genetics ; Mucorales/*genetics ; *Mutagenesis ; Orotidine-5'-Phosphate Decarboxylase/genetics ; },
abstract = {Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5'-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.},
}
@article {pmid32466123,
year = {2020},
author = {Mianné, J and Bourguignon, C and Nguyen Van, C and Fieldès, M and Nasri, A and Assou, S and De Vos, J},
title = {Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology.},
journal = {Cells},
volume = {9},
number = {5},
pages = {},
pmid = {32466123},
issn = {2073-4409},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Knock-In Techniques ; Humans ; INDEL Mutation/genetics ; Pluripotent Stem Cells/*metabolism ; *Transgenes ; },
abstract = {Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).},
}
@article {pmid32464217,
year = {2020},
author = {Zheng, R and Li, Y and Wang, L and Fang, X and Zhang, J and He, L and Yang, L and Li, D and Geng, H},
title = {CRISPR/Cas9-mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1.},
journal = {Kidney international},
volume = {98},
number = {4},
pages = {947-957},
doi = {10.1016/j.kint.2020.04.049},
pmid = {32464217},
issn = {1523-1755},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; *Hyperoxaluria ; *Hyperoxaluria, Primary/genetics/therapy ; Metabolic Networks and Pathways ; Rats ; Transaminases/genetics ; },
abstract = {Primary hyperoxaluria type I is caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT), leading to accumulation of glyoxylate and subsequent production of oxalate and urolithiasis. Here, we generated a novel rat model of primary hyperoxaluria type I that carries a D205N mutation in the partially humanized Agxt gene through the CRISPR/Cas9 system. The AgxtD205N mutant rats showed undetectable alanine glyoxylate aminotransferase protein expression, developed hyperoxaluria at 1 month of age and exhibited severe renal calcium oxalate deposition after ethylene glycol challenge. This suggests our novel model is more relevant to the human disease than existing animal models. To test whether this model could be used for the development of innovative therapeutics, SaCas9 targeting hydroxyacid oxidase 1, responsible for metabolizing glycolate into glyoxylate, was delivered via adeno-associated viral vectors into newborn rats with primary hyperoxaluria type 1. This approach generated nearly 30% indels in the Hao1 gene in the liver, leading to 42% lower urine oxalate levels in the treated group than in the control group and preventing the rats with primary hyperoxaluria type 1 from undergoing severe nephrocalcinosis for at least 12 months. Thus, our results demonstrate that this partially humanized AgxtD205N rat strain is a high-performing model of primary hyperoxaluria type 1 for understanding pathology, and the development of novel therapeutics, such as reprogramming of the metabolic pathway through genome editing.},
}
@article {pmid32464135,
year = {2020},
author = {Kim, Y and Ahmed, S and Al Baki, MA and Kumar, S and Kim, K and Park, Y and Stanley, D},
title = {Deletion mutant of PGE2 receptor using CRISPR-Cas9 exhibits larval immunosuppression and adult infertility in a lepidopteran insect, Spodoptera exigua.},
journal = {Developmental and comparative immunology},
volume = {111},
number = {},
pages = {103743},
doi = {10.1016/j.dci.2020.103743},
pmid = {32464135},
issn = {1879-0089},
mesh = {Animals ; Bacterial Infections/*immunology ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Cell Growth Processes ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Female ; Hemocytes/*physiology ; Immune Tolerance/genetics ; Immunity, Cellular ; Infertility, Female/genetics ; Insect Proteins/*genetics ; Larva ; RNA Interference ; Receptors, Prostaglandin E/*genetics ; Reproduction ; Sequence Deletion/*genetics ; Sf9 Cells ; Signal Transduction ; Spodoptera/*physiology ; },
abstract = {Prostaglandins (PGs) mediate various physiological processes in insects and other invertebrates, but there is very little information on PG receptors. This study identified a PGE2 receptor (SePGE2R) in the lepidopteran insect, Spodoptera exigua, and addressed its functional association with cellular immunity, development, and reproduction. SePGE2R is expressed in most developmental stages and tissues. After SePGR2R expression knock down by RNA interference (RNAi), larval nodule formation (clears bacterial infections from circulating hemolymph) was severely suppressed coupled with reduced F-actin growth in hemocytes. Treating female adults with RNAi prevented nurse cell dumping in follicles and interfered with oocyte development. SePGE2R was heterologously expressed in Sf9 cells, in which the endogenous S. frugiperda PGE2R was knocked down by small interfering RNA. This transiently expressed SePGE2R responded to PGE2, but not other PGs, with dose-dependent up-regulation of intracellular cAMP concentrations. Treating S. exigua larvae with PGE2 led to activation of a trimeric Gαs subunit, protein kinase A (PKA), and Rho family small intracellular G proteins in hemocytes. A deletion mutant of SePGE2R was generated using CRISPR/Cas9 which exhibited severely retarded larval development and adult reproduction. We infer that PGE2R mediates insect immune and reproductive processes via a PKA signal pathway.},
}
@article {pmid32462967,
year = {2020},
author = {Rubbini, D and Cornet, C and Terriente, J and Di Donato, V},
title = {CRISPR Meets Zebrafish: Accelerating the Discovery of New Therapeutic Targets.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {6},
pages = {552-567},
doi = {10.1177/2472555220926920},
pmid = {32462967},
issn = {2472-5560},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Drug Discovery/*trends ; Gene Editing/*trends ; High-Throughput Screening Assays/*trends ; Humans ; Zebrafish/genetics ; },
abstract = {Bringing a new drug to the market costs an average of US$2.6 billion and takes more than 10 years from discovery to regulatory approval. Despite the need to reduce cost and time to increase productivity, pharma companies tend to crowd their efforts in the same indications and drug targets. This results in the commercialization of drugs that share the same mechanism of action (MoA) and, in many cases, equivalent efficacies among them-an outcome that helps neither patients nor the balance sheet of the companies trying to bring therapeutics to the same patient population. Indeed, the discovery of new therapeutic targets, based on a deeper understanding of the disease biology, would likely provide more innovative MoAs and potentially greater drug efficacies. It would also bring better chances for identifying appropriate treatments according to the patient's genetic stratification. Nowadays, we count with an enormous amount of unprocessed information on potential disease targets that could be extracted from omics data obtained from patient samples. In addition, hundreds of pharmacological and genetic screenings have been performed to identify innovative drug targets. Traditionally, rodents have been the animal models of choice to perform functional genomic studies. The high experimental cost, combined with the low throughput provided by those models, however, is a bottleneck for discovering and validating novel genetic disease associations. To overcome these limitations, we propose that zebrafish, in conjunction with the use of CRISPR/Cas9 genome-editing tools, could streamline functional genomic processes to bring biologically relevant knowledge on innovative disease targets in a shorter time frame.},
}
@article {pmid32461556,
year = {2020},
author = {Lõhmussaar, K and Kopper, O and Korving, J and Begthel, H and Vreuls, CPH and van Es, JH and Clevers, H},
title = {Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2660},
pmid = {32461556},
issn = {2041-1723},
mesh = {Animals ; Antineoplastic Agents/pharmacology ; BRCA1 Protein/genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems/*genetics ; Epithelium/pathology ; Fallopian Tubes/pathology ; Female ; Gene Editing/methods ; Mice ; Mutation ; Neurofibromatosis 1/genetics ; Organ Culture Techniques/methods ; *Organoids/drug effects/physiopathology ; Ovarian Neoplasms/drug therapy/*etiology/genetics ; Ovary/pathology ; PTEN Phosphohydrolase/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {High-grade serous ovarian cancer (HG-SOC)-often referred to as a "silent killer"-is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients.},
}
@article {pmid32460957,
year = {2020},
author = {Zhang, Q and Fu, Y and Thakur, C and Bi, Z and Wadgaonkar, P and Qiu, Y and Xu, L and Rice, M and Zhang, W and Almutairy, B and Chen, F},
title = {CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA.},
journal = {Biochemical and biophysical research communications},
volume = {528},
number = {1},
pages = {54-61},
pmid = {32460957},
issn = {1090-2104},
support = {P30 ES020957/ES/NIEHS NIH HHS/United States ; R01 ES028263/ES/NIEHS NIH HHS/United States ; R01 ES028335/ES/NIEHS NIH HHS/United States ; },
mesh = {Alternative Splicing/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Dioxygenases/genetics ; Exons/genetics ; *Gene Editing ; Histone Demethylases/genetics ; Humans ; Nuclear Proteins/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; Sequence Deletion ; },
abstract = {The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.},
}
@article {pmid32459325,
year = {2020},
author = {Wang, J and Dai, W and Li, J and Xie, R and Dunstan, RA and Stubenrauch, C and Zhang, Y and Lithgow, T},
title = {PaCRISPR: a server for predicting and visualizing anti-CRISPR proteins.},
journal = {Nucleic acids research},
volume = {48},
number = {W1},
pages = {W348-W357},
pmid = {32459325},
issn = {1362-4962},
mesh = {*Bacteriophages ; *CRISPR-Cas Systems ; Computer Graphics ; Machine Learning ; Sequence Analysis, Protein ; *Software ; Viral Proteins/*chemistry ; },
abstract = {Anti-CRISPRs are widespread amongst bacteriophage and promote bacteriophage infection by inactivating the bacterial host's CRISPR-Cas defence system. Identifying and characterizing anti-CRISPR proteins opens an avenue to explore and control CRISPR-Cas machineries for the development of new CRISPR-Cas based biotechnological and therapeutic tools. Past studies have identified anti-CRISPRs in several model phage genomes, but a challenge exists to comprehensively screen for anti-CRISPRs accurately and efficiently from genome and metagenome sequence data. Here, we have developed an ensemble learning based predictor, PaCRISPR, to accurately identify anti-CRISPRs from protein datasets derived from genome and metagenome sequencing projects. PaCRISPR employs different types of feature recognition united within an ensemble framework. Extensive cross-validation and independent tests show that PaCRISPR achieves a significantly more accurate performance compared with homology-based baseline predictors and an existing toolkit. The performance of PaCRISPR was further validated in discovering anti-CRISPRs that were not part of the training for PaCRISPR, but which were recently demonstrated to function as anti-CRISPRs for phage infections. Data visualization on anti-CRISPR relationships, highlighting sequence similarity and phylogenetic considerations, is part of the output from the PaCRISPR toolkit, which is freely available at http://pacrispr.erc.monash.edu/.},
}
@article {pmid32458463,
year = {2020},
author = {Tan, SI and Yu, PJ and Ng, IS},
title = {CRISPRi-mediated programming essential gene can as a Direct Enzymatic Performance Evaluation &amp; Determination (DEPEND) system.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {9},
pages = {2842-2851},
doi = {10.1002/bit.27443},
pmid = {32458463},
issn = {1097-0290},
support = {MOST 108-2218-E-006-006//Ministry of Science and Technology, Taiwan/International ; MOST 108-2221-E-006-004-MY3//Ministry of Science and Technology, Taiwan/International ; MOST 108-2621-M-006-015//Ministry of Science and Technology, Taiwan/International ; },
mesh = {5-Aminolevulinate Synthetase/genetics/metabolism ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; Carbonic Anhydrases/genetics/metabolism ; Escherichia coli/genetics ; Genes, Essential/*genetics ; RNA, Guide/genetics ; Recombinant Proteins/*genetics/metabolism ; },
abstract = {Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time-consuming and labor-intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO2 uptake. CRISPRi::CA comprises a whole-cell biosensor to monitor CO2 concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation &amp; Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony-forming units. Furthermore, the enzymatic performance of 5-aminolevulinic acid synthetase (ALAS), which converts glycine and succinate-CoA to release a molecule of CO2 , has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time-saving, and flexible technology for the screening and inspection of high-performance enzymes, which may accelerate protein engineering in the future.},
}
@article {pmid32458346,
year = {2020},
author = {Chen, B and Deng, S and Ge, T and Ye, M and Yu, J and Lin, S and Ma, W and Songyang, Z},
title = {Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in.},
journal = {Protein &amp; cell},
volume = {11},
number = {9},
pages = {641-660},
pmid = {32458346},
issn = {1674-8018},
mesh = {*CRISPR-Cas Systems ; *Cell Tracking ; *Gene Expression Profiling ; *Gene Knock-In Techniques ; HEK293 Cells ; Humans ; *Proteomics ; RNA, Long Noncoding/genetics/*metabolism ; },
abstract = {In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.},
}
@article {pmid32457510,
year = {2020},
author = {Burgess, DJ},
title = {A mouse with history.},
journal = {Nature reviews. Genetics},
volume = {21},
number = {7},
pages = {388},
pmid = {32457510},
issn = {1471-0064},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; *Transcriptome ; },
}
@article {pmid32456805,
year = {2020},
author = {Zhang, D and Zhang, B},
title = {SpRY: Engineered CRISPR/Cas9 Harnesses New Genome-Editing Power.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {8},
pages = {546-548},
doi = {10.1016/j.tig.2020.05.001},
pmid = {32456805},
issn = {0168-9525},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; Streptococcus pyogenes/genetics ; },
abstract = {Due to protospacer adjacent motif (PAM) requirements, CRISPR/Cas9 cannot access many genetic loci. A recent study by Walton et al. structurally engineered Streptococcus pyogenes Cas9 (SpCas9) to near-PAMless SpRY that can target most DNA sequences with high editing efficiency and flexibility. This newly engineered SpRY will potentially expand genome-editing capabilities for basic and applied research.},
}
@article {pmid32456131,
year = {2020},
author = {Scherer, A and Stephens, VR and McGivney, GR and Gutierrez, WR and Laverty, EA and Knepper-Adrian, V and Dodd, RD},
title = {Distinct Tumor Microenvironments Are a Defining Feature of Strain-Specific CRISPR/Cas9-Induced MPNSTs.},
journal = {Genes},
volume = {11},
number = {5},
pages = {},
pmid = {32456131},
issn = {2073-4425},
support = {T32 GM067795/GM/NIGMS NIH HHS/United States ; U54 CA196519/CA/NCI NIH HHS/United States ; R25 GM116686/NH/NIH HHS/United States ; T32 GM067795/NH/NIH HHS/United States ; P30 CA086862/NH/NIH HHS/United States ; T32 GM007337/GM/NIGMS NIH HHS/United States ; T32 CA078586/NH/NIH HHS/United States ; },
mesh = {Animals ; CD4-Positive T-Lymphocytes/metabolism/pathology ; CRISPR-Cas Systems/*genetics ; Carcinogenesis/*genetics ; Disease Models, Animal ; Gene Editing ; Gene Expression Regulation, Neoplastic/genetics ; Genetic Association Studies ; Humans ; Mice ; Mice, Inbred BALB C ; Neurofibrosarcoma/diagnosis/*genetics/pathology ; Tumor Microenvironment/*genetics ; },
abstract = {The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype-phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is observed in C57BL/6, 129X1 and 129/SvJae mice. Indel pattern analysis demonstrates that indel frequency, type and size are similar across all genetic backgrounds. Gene expression and IHC analysis identify multiple strain-dependent differences in CD4+ T cell infiltration and myeloid cell populations, including M2 macrophages and mast cells. These data highlight important strain-specific phenotypes of genomically-matched MPNSTs that have implications for the design of future studies using similar in vivo gene editing approaches.},
}
@article {pmid32455882,
year = {2020},
author = {Sledzinski, P and Nowaczyk, M and Olejniczak, M},
title = {Computational Tools and Resources Supporting CRISPR-Cas Experiments.},
journal = {Cells},
volume = {9},
number = {5},
pages = {},
pmid = {32455882},
issn = {2073-4409},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Computational Biology/*methods ; DNA Repair/genetics ; Gene Editing ; RNA, Guide/genetics ; },
abstract = {The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor.},
}
@article {pmid32453716,
year = {2020},
author = {Latour, BL and Van De Weghe, JC and Rusterholz, TD and Letteboer, SJ and Gomez, A and Shaheen, R and Gesemann, M and Karamzade, A and Asadollahi, M and Barroso-Gil, M and Chitre, M and Grout, ME and van Reeuwijk, J and van Beersum, SE and Miller, CV and Dempsey, JC and Morsy, H and , and Bamshad, MJ and , and Nickerson, DA and Neuhauss, SC and Boldt, K and Ueffing, M and Keramatipour, M and Sayer, JA and Alkuraya, FS and Bachmann-Gagescu, R and Roepman, R and Doherty, D},
title = {Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome.},
journal = {The Journal of clinical investigation},
volume = {130},
number = {8},
pages = {4423-4439},
pmid = {32453716},
issn = {1558-8238},
support = {U54 HG006493/HG/NHGRI NIH HHS/United States ; U54 HD083091/HD/NICHD NIH HHS/United States ; F32 HD095599/HD/NICHD NIH HHS/United States ; P50 HD103524/HD/NICHD NIH HHS/United States ; /DH_/Department of Health/United Kingdom ; K99 HD100554/HD/NICHD NIH HHS/United States ; UM1 HG006493/HG/NHGRI NIH HHS/United States ; R01 NS064077/NS/NINDS NIH HHS/United States ; },
mesh = {*Abnormalities, Multiple/genetics/metabolism ; Acetylation ; Animals ; *Armadillo Domain Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cerebellum/*abnormalities/metabolism ; *Cilia/genetics/metabolism ; Disease Models, Animal ; *Eye Abnormalities/genetics/metabolism ; Humans ; *Kidney Diseases, Cystic/genetics/metabolism ; Peptides/genetics/metabolism ; Retina/*abnormalities/metabolism ; *Zebrafish/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; },
abstract = {Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.},
}
@article {pmid32453626,
year = {2020},
author = {Reimann, V and Ziemann, M and Li, H and Zhu, T and Behler, J and Lu, X and Hess, WR},
title = {Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups.},
journal = {RNA biology},
volume = {17},
number = {10},
pages = {1442-1453},
pmid = {32453626},
issn = {1555-8584},
mesh = {Anabaena/*enzymology/genetics ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/*metabolism ; Enzyme Activation ; Gene Expression Profiling ; Gene Expression Regulation, Bacterial ; RNA Processing, Post-Transcriptional ; RNA, Bacterial/genetics ; RNA, Small Untranslated ; Sequence Deletion ; Substrate Specificity ; Transcription, Genetic ; Transcriptome ; },
abstract = {Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavour other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighbouring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.},
}
@article {pmid32451449,
year = {2020},
author = {Beying, N and Schmidt, C and Pacher, M and Houben, A and Puchta, H},
title = {CRISPR-Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis.},
journal = {Nature plants},
volume = {6},
number = {6},
pages = {638-645},
pmid = {32451449},
issn = {2055-0278},
mesh = {Arabidopsis/enzymology/*genetics ; *CRISPR-Cas Systems ; *Chromosomes, Plant ; Endonucleases/analysis ; Plant Proteins/analysis ; *Translocation, Genetic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits[1-4]. Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus[5], we were able to induce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note, translocation frequency was about five times more efficient in the absence of the classical non-homologous end-joining pathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner, fixing or breaking genetic linkages between traits on different chromosomes. Controlled restructuring of plant genomes has the potential to transform plant breeding.},
}
@article {pmid32450806,
year = {2020},
author = {Tian, Y and Chen, K and Li, X and Zheng, Y and Chen, F},
title = {Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2-2.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {233},
pmid = {32450806},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics/metabolism ; Gene Knockout Techniques ; Oleic Acids/chemistry ; Plant Oils/analysis/*chemistry ; Plant Proteins/*genetics/metabolism ; Seeds/chemistry/metabolism ; Tobacco/chemistry/enzymology/*genetics ; },
abstract = {BACKGROUND: Tobacco seed oil could be used as an appropriate feedstock for biodiesel production. However, the high linoleic acid content of tobacco seed oil makes it susceptible to oxidation. Altering the fatty acid profile by increasing the content of oleic acid could improve the properties of biodiesel produced from tobacco seed oil.<br><br>RESULTS: Four FAD2 genes, NtFAD2-1a, NtFAD2-1b, NtFAD2-2a, and NtFAD2-2b, were identified in allotetraploid tobacco genome. Phylogenetic analysis of protein sequences showed that NtFAD2-1a and NtFAD2-2a originated from N. tomentosiformis, while NtFAD2-1b and NtFAD2-2b from N. sylvestris. Expression analysis revealed that NtFAD2-2a and NtFAD2-2b transcripts were more abundant in developing seeds than in other tissues, while NtFAD2-1a and NtFAD2-1b showed low transcript levels in developing seed. Phylogenic analysis showed that NtFAD2-2a and NtFAD2-2b were seed-type FAD2 genes. Heterologous expression in yeast cells demonstrated that both NtFAD2-2a and NtFAD2-2b protein could introduce a double bond at the &#x394;[12] position of fatty acid chain. The fatty acid profile analysis of tobacco fad2-2 mutant seeds derived from CRISPR-Cas9 edited plants showed dramatic increase of oleic acid content from 11% to over 79%, whereas linoleic acid decreased from 72 to 7%. In addition, the fatty acid composition of leaf was not affected in fad2-2 mutant plants.<br><br>CONCLUSION: Our data showed that knockout of seed-type FAD2 genes in tobacco could significantly increase the oleic acid content in seed oil. This research suggests that CRISPR-Cas9 system offers a rapid and highly efficient method in the tobacco seed lipid engineering programs.},
}
@article {pmid32450802,
year = {2020},
author = {Wada, N and Ueta, R and Osakabe, Y and Osakabe, K},
title = {Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {234},
pmid = {32450802},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.},
}
@article {pmid32450202,
year = {2020},
author = {Dheer, P and Rautela, I and Sharma, V and Dhiman, M and Sharma, A and Sharma, N and Sharma, MD},
title = {Evolution in crop improvement approaches and future prospects of molecular markers to CRISPR/Cas9 system.},
journal = {Gene},
volume = {753},
number = {},
pages = {144795},
doi = {10.1016/j.gene.2020.144795},
pmid = {32450202},
issn = {1879-0038},
mesh = {Agriculture/*methods ; Biomarkers ; Biotechnology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Crops, Agricultural/*genetics ; Disease Resistance/genetics ; Gene Editing/methods ; Gene Targeting/methods ; Genetic Engineering/*methods ; Genome, Plant/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The advent of genetic selection and genome modification method assure about a real novel reformation in biotechnology and genetic engineering. With the extensive capabilities of molecular markers of them being stable, cost-effective and easy to use, they ultimately become a potent tool for variety of applications such a gene targeting, selection, editing, functional genomics; mainly for the improvisation of commercially important crops. Three main benefits of molecular marker in the field of agriculture and crop improvement programmes first, reduction of the duration of breeding programmes, second, they allow creation of new genetic variation and genetic diversity of plants and third most promising benefit is help in production of engineered plant for disease resistance, or resistance from pathogen and herbicides. This review is anticipated to present an outline how the techniques have been evolved from the simple conventional applications of DNA based molecular markers to highly throughput CRISPR technology and geared the crop yield. Techniques like using Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems have revolutionised in the field of genome editing. These have been promptly accepted in both the research and commercial industry. On the whole, the widespread use of molecular markers with their types, their appliance in plant breeding along with the advances in genetic selection and genome editing together being a novel strategy to boost crop yield has been reviewed.},
}
@article {pmid32449788,
year = {2020},
author = {Zhang, F and Cheng, D and Wang, S and Zhu, J},
title = {Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {9},
pages = {2816-2826},
pmid = {32449788},
issn = {1097-0290},
support = {R01 GM071725/GM/NIGMS NIH HHS/United States ; R01GM071725/NH/NIH HHS/United States ; //Spokane County Health Sciences and Service Authority/International ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Homologous Recombination/*genetics ; Humans ; Mice ; Plasmids/genetics ; },
abstract = {Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.},
}
@article {pmid32449724,
year = {2020},
author = {Wang, Y and Smith, AJH and Hay, DC},
title = {Introducing Point Mutations into Human Pluripotent Stem Cells using Seamless Genome Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {159},
pages = {},
doi = {10.3791/61152},
pmid = {32449724},
issn = {1940-087X},
support = {MC_UU_00016/10/MRC_/Medical Research Council/United Kingdom ; MR/K017047/1/MRC_/Medical Research Council/United Kingdom ; MR/L022974/1/MRC_/Medical Research Council/United Kingdom ; TC/16/37/CSO_/Chief Scientist Office/United Kingdom ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Gene Editing/*methods ; Genetic Vectors/genetics ; Genome, Human ; Humans ; Pluripotent Stem Cells/*metabolism ; Point Mutation/*genetics ; },
abstract = {Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4α) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4α locus.},
}
@article {pmid32448919,
year = {2020},
author = {Huang, H and Cui, T and Zhang, L and Yang, Q and Yang, Y and Xie, K and Fan, C and Zhou, Y},
title = {Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {8},
pages = {2401-2411},
doi = {10.1007/s00122-020-03607-y},
pmid = {32448919},
issn = {1432-2242},
mesh = {Alleles ; Brassica napus/*genetics/metabolism ; CRISPR-Cas Systems ; Chromatography, Gas ; Chromatography, Liquid ; Fatty Acid Desaturases/*genetics/metabolism ; Fatty Acids/*metabolism ; Frameshift Mutation ; Gene Editing/*methods ; *Genes, Plant ; Genotype ; Hypocotyl/genetics/metabolism ; Linoleic Acid/analysis ; Mutation ; Oleic Acid/analysis ; Plant Leaves/genetics/growth &amp; development/metabolism ; Plant Proteins/*genetics/metabolism ; Plant Roots/genetics/metabolism ; Plants, Genetically Modified/genetics ; Polyploidy ; RNA-Seq ; Seedlings/genetics/growth &amp; development/metabolism ; Seeds/chemistry/*genetics/growth &amp; development/metabolism ; alpha-Linolenic Acid/analysis ; },
abstract = {Genomic editing with CRISPR/Cas9 system can simultaneously modify multiple copies of theBnaFAD2 gene to develop novel variations in fatty acids profiles in polyploidy rapeseed. Fatty acid composition affects edible and processing quality of vegetable oil and has been one of the primary targets for genetic modification in oilseed crops including rapeseed (Brassica napus). Fatty acid desaturase 2 gene, FAD2, is a key player that affects three major fatty acids, namely oleic, linoleic and linolenic acid, in oilseed plants. Previously, we showed that there are four copies of BnaFAD2 in allotetraploid rapeseed. In this study, we further established spatiotemporal expression pattern of each copy of BnaFAD2 using published RNA-seq data. Genomic editing technology based on CRISPR/Cas9 system was used to mutate all the copies of BnaFAD2 to create novel allelic variations in oleic acid and other fatty acid levels. A number of mutants at two targeting sites were identified, and the phenotypic variation in the mutants was systematically evaluated. The oleic acid content in the seed of the mutants increased significantly with the highest exceeding 80% compared with wild type of 66.43%, while linoleic and linolenic acid contents decreased accordingly. Mutations on BnaFAD2.A5 caused more dramatic changes of fatty acid profile than the mutations on BnaFAD2.C5 alleles that were identified with gene editing technique for the first time. Moreover, combining different mutated alleles of BnaFAD2 can even broaden the variation more dramatically. It was found that effects of different mutation types at BnaFAD2 alleles on oleic levels varied, indicating a possibility to manipulate fatty acid levels by precise mutation at specific region of a gene.},
}
@article {pmid32448328,
year = {2020},
author = {Su, B and Song, D and Zhu, H},
title = {Homology-dependent recombination of large synthetic pathways into E. coli genome via λ-Red and CRISPR/Cas9 dependent selection methodology.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {108},
pmid = {32448328},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems ; *Chromosomes, Bacterial ; Escherichia coli/*genetics ; *Gene Editing ; *Homologous Recombination ; *Metabolic Engineering ; },
abstract = {BACKGROUND: Metabolic engineering frequently needs genomic integration of many heterologous genes for biosynthetic pathway assembly. Despite great progresses in genome editing for the model microorganism Escherichia coli, the integration of large pathway into genome for stabilized chemical production is still challenging compared with small DNA integration.<br><br>RESULTS: We have developed a &#x3bb;-Red assisted homology-dependent recombination for large synthetic pathway integration in E. coli. With this approach, we can integrate as large as 12&#xa0;kb DNA module into the chromosome of E. coli W3110 in a single step. The efficiency of this method can reach 100%, thus markedly improve the integration efficiency and overcome the limitation of the integration size adopted the common method. Furthermore, the limiting step in the methylerythritol 4-phosphate (MEP) pathway and lycopene synthetic pathway were integrated into the W3110 genome using our system. Subsequently, the yields of the final strain were increased 106 and 4.4-fold compared to the initial strain and the reference strain, respectively.<br><br>CONCLUSIONS: In addition to pre-existing method, our system presents an optional strategy for avoiding using plasmids and a valuable tool for large synthetic pathway assembly in E. coli.},
}
@article {pmid32448155,
year = {2020},
author = {Cianfanelli, FR and Cunrath, O and Bumann, D},
title = {Efficient dual-negative selection for bacterial genome editing.},
journal = {BMC microbiology},
volume = {20},
number = {1},
pages = {129},
pmid = {32448155},
issn = {1471-2180},
support = {310030_156818//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (CH)/International ; },
mesh = {CRISPR-Cas Systems ; Conjugation, Genetic ; Drug Resistance, Multiple, Bacterial ; Escherichia coli/*genetics ; Gene Editing/*methods ; Genes, Transgenic, Suicide ; Genome, Bacterial ; Mutation ; Plasmids/*genetics ; Pseudomonas aeruginosa/*genetics ; Salmonella enterica/*genetics ; },
abstract = {BACKGROUND: Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering.<br><br>RESULTS: Here, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in-frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates.<br><br>CONCLUSIONS: Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.},
}
@article {pmid32446977,
year = {2020},
author = {Tyagi, S and Kumar, R and Das, A and Won, SY and Shukla, P},
title = {CRISPR-Cas9 system: A genome-editing tool with endless possibilities.},
journal = {Journal of biotechnology},
volume = {319},
number = {},
pages = {36-53},
doi = {10.1016/j.jbiotec.2020.05.008},
pmid = {32446977},
issn = {1873-4863},
mesh = {Animals ; Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing/ethics/methods/standards ; Humans ; Plants, Genetically Modified ; },
abstract = {The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.},
}
@article {pmid32446740,
year = {2020},
author = {Mi, X and Li, Z and Yan, J and Li, Y and Zheng, J and Zhuang, Z and Yang, W and Gong, L and Shi, J},
title = {Activation of HIF-1 signaling ameliorates liver steatosis in zebrafish atp7b deficiency (Wilson's disease) models.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1866},
number = {10},
pages = {165842},
doi = {10.1016/j.bbadis.2020.165842},
pmid = {32446740},
issn = {1879-260X},
mesh = {Animals ; CRISPR-Cas Systems ; Copper/metabolism/toxicity ; Copper-Transporting ATPases/*genetics ; Disease Models, Animal ; Fatty Liver/genetics/*metabolism/pathology ; Female ; Gene Knockout Techniques ; Hep G2 Cells ; Hepatocytes/metabolism ; Hepatolenticular Degeneration/*genetics/*metabolism ; Humans ; Hypoxia-Inducible Factor 1/*metabolism ; Liver/*metabolism/pathology ; Male ; Mutation ; Signal Transduction/*physiology ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {Wilson's disease is an autosomal recessive disease characterized by excess copper accumulated in the liver and brain. It is caused by mutations in the copper transporter gene ATP7B. However, based on the poor understanding of the transcriptional program involved in the pathogenesis of Wilson's disease and the lack of more safe and efficient therapies, the identification of novel pathways and the establishment of complementary model systems of Wilson's disease are urgently needed. Herein, we generated two zebrafish atp7b-mutant lines using the CRISPR/Cas9 editing system, and the mutants developed hepatic and behavioral deficits similar to those observed in humans with Wilson's disease. Interestingly, we found that atp7b-deficient zebrafish embryos developed liver steatosis under low-dose Cu exposure, and behavioral deficits appeared under high-dose Cu exposure. Analyses of publicly available transcriptomic data from ATP7B-knockout HepG2 cells demonstrated that the HIF-1 signaling pathway is downregulated in ATP7B-knockout HepG2 cells compared with wildtype cells following Cu exposure. The HIF-1 signaling pathway was also downregulated in our atp7b-deficient zebrafish mutants following Cu exposure. Furthermore, we demonstrate that activation of the HIF-1 signaling pathway with the chemical compound FG-4592 or DMOG ameliorates liver steatosis and reduces accumulated Cu levels in zebrafish atp7b deficiency models. These findings introduce a novel prospect that modulation of the HIF-1 signaling pathway should be explored as a novel strategy to reduce copper toxicity in Wilson's disease patients.},
}
@article {pmid32446218,
year = {2020},
author = {Lilianty, J and Nur Patria, Y and Stanley, EG and Elefanty, AG and Bateman, JF and Lamandé, SR},
title = {Generation of a heterozygous COL2A1 (p.R989C) spondyloepiphyseal dysplasia congenita mutation iPSC line, MCRIi001-B, using CRISPR/Cas9 gene editing.},
journal = {Stem cell research},
volume = {45},
number = {},
pages = {101843},
doi = {10.1016/j.scr.2020.101843},
pmid = {32446218},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Collagen Type II/genetics ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells ; Mutation/genetics ; Osteochondrodysplasias/congenital ; },
abstract = {To produce an in vitro model of the human chondrodysplasia, spondyloepiphyseal dysplasia congenita, we used CRISPR/Cas9 gene editing to generate a heterozygous patient COL2A1 mutation in an established control human iPSC line. The gene-edited heterozygous COL2A1 p.R989C line had a normal karyotype, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. When differentiated into cartilage this cell line and the parental isogenic control may be used to explore disease mechanisms and evaluate therapeutic approaches.},
}
@article {pmid32445790,
year = {2020},
author = {Yamagata, T and Raveau, M and Kobayashi, K and Miyamoto, H and Tatsukawa, T and Ogiwara, I and Itohara, S and Hensch, TK and Yamakawa, K},
title = {CRISPR/dCas9-based Scn1a gene activation in inhibitory neurons ameliorates epileptic and behavioral phenotypes of Dravet syndrome model mice.},
journal = {Neurobiology of disease},
volume = {141},
number = {},
pages = {104954},
doi = {10.1016/j.nbd.2020.104954},
pmid = {32445790},
issn = {1095-953X},
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Disease Models, Animal ; Epilepsies, Myoclonic/*genetics/*physiopathology/prevention &amp; control ; Epilepsy/*genetics/*physiopathology/prevention &amp; control ; Female ; GABAergic Neurons/physiology ; Genetic Therapy/methods ; HEK293 Cells ; Humans ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; NAV1.1 Voltage-Gated Sodium Channel/*genetics/*physiology ; Neurons/*physiology ; Phenotype ; },
abstract = {Dravet syndrome is a severe infantile-onset epileptic encephalopathy which begins with febrile seizures and is caused by heterozygous loss-of-function mutations of the voltage-gated sodium channel gene SCN1A. We designed a CRISPR-based gene therapy for Scn1a-haplodeficient mice using multiple guide RNAs (gRNAs) in the promoter regions together with the nuclease-deficient Cas9 fused to transcription activators (dCas9-VPR) to trigger the transcription of SCN1A or Scn1a in vitro. We tested the effect of this strategy in vivo using an adeno-associated virus (AAV) mediated system targeting inhibitory neurons and investigating febrile seizures and behavioral parameters. In both the human and mouse genes multiple guide RNAs (gRNAs) in the upstream, rather than downstream, promoter region showed high and synergistic activities to increase the transcription of SCN1A or Scn1a in cultured cells. Intravenous injections of AAV particles containing the optimal combination of 4 gRNAs into transgenic mice with Scn1a-haplodeficiency and inhibitory neuron-specific expression of dCas9-VPR at four weeks of age increased Nav1.1 expression in parvalbumin-positive GABAergic neurons, ameliorated their febrile seizures and improved their behavioral impairments. Although the usage of transgenic mice and rather modest improvements in seizures and abnormal behaviors hamper direct clinical application, our results indicate that the upregulation of Scn1a expression in the inhibitory neurons can significantly improve the phenotypes, even when applied after the juvenile stages. Our findings also suggest that the decrease in Nav1.1 is directly involved in the symptoms seen in adults with Dravet syndrome and open a way to improve this condition.},
}
@article {pmid32444599,
year = {2020},
author = {Yang, H and Yang, S and Jing, L and Huang, L and Chen, L and Zhao, X and Yang, W and Pan, Y and Yin, P and Qin, ZS and Tang, B and Li, S and Li, XJ},
title = {Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2582},
pmid = {32444599},
issn = {2041-1723},
support = {R56 AG019206/AG/NIA NIH HHS/United States ; R01 NS095181/NS/NINDS NIH HHS/United States ; R01 NS095279/NS/NINDS NIH HHS/United States ; R01 NS101701/NS/NINDS NIH HHS/United States ; R01 NS036232/NS/NINDS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/metabolism ; Age Factors ; Animals ; CRISPR-Cas Systems ; Cell Nucleus/metabolism ; Corpus Striatum/metabolism/pathology ; Exons ; Female ; Gene Knock-In Techniques ; Huntingtin Protein/*genetics/*metabolism ; Huntington Disease/*etiology/genetics ; Male ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Mutation ; Phenotype ; },
abstract = {Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.},
}
@article {pmid32443745,
year = {2020},
author = {Korablev, A and Lukyanchikova, V and Serova, I and Battulin, N},
title = {On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes.},
journal = {International journal of molecular sciences},
volume = {21},
number = {10},
pages = {},
pmid = {32443745},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; Gene Knock-In Techniques/*adverse effects/methods ; Male ; Mice ; Mice, Inbred C57BL ; Recombinational DNA Repair ; Zygote/*metabolism ; },
abstract = {Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.},
}
@article {pmid32442502,
year = {2020},
author = {Shilton, AK and Marraffini, LA},
title = {Shoot the Messenger! A New Phage Weapon to Neutralize the Type III CRISPR Immune Response.},
journal = {Molecular cell},
volume = {78},
number = {4},
pages = {568-569},
doi = {10.1016/j.molcel.2020.04.011},
pmid = {32442502},
issn = {1097-4164},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine Nucleotides ; *Bacteriophages ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oligoribonucleotides ; },
abstract = {Athukoralage et al. (2020) identify a new anti-CRISPR (Acr) that degrades cA4, a cyclic oligo-adenylate second messenger produced during the type III CRISPR immune response. This provides an effective way by which invaders can bypass downstream CRISPR effectors that rely on this signaling molecule.},
}
@article {pmid32442413,
year = {2020},
author = {Kerek, EM and Cromwell, CR and Hubbard, BP},
title = {CRISPR Lights up In Situ Protein Evolution.},
journal = {Cell chemical biology},
volume = {27},
number = {5},
pages = {475-478},
doi = {10.1016/j.chembiol.2020.05.005},
pmid = {32442413},
issn = {2451-9448},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Organelles ; },
abstract = {In this issue of Cell Chemical Biology, Erdogan et al. (2020) describe a new CRISPR/Cas9-based strategy for performing directed evolution of mammalian proteins in situ. Using this technique to select functional mRuby3 variants within lysosomes, they identify mCRISPRed, a fluorescent protein that displays robust stability and activity at low pH.},
}
@article {pmid32442401,
year = {2020},
author = {Haley, B and Roudnicky, F},
title = {Functional Genomics for Cancer Drug Target Discovery.},
journal = {Cancer cell},
volume = {38},
number = {1},
pages = {31-43},
doi = {10.1016/j.ccell.2020.04.006},
pmid = {32442401},
issn = {1878-3686},
mesh = {Animals ; Antineoplastic Agents/*therapeutic use ; CRISPR-Cas Systems/genetics ; Drug Discovery/*methods ; Gene Editing/methods ; Genetic Testing/*methods ; Genome, Human/*genetics ; Genomics/*methods ; Humans ; Neoplasms/diagnosis/genetics/*therapy ; },
abstract = {Functional genomics describes a field of biology that uses a range of approaches for assessing gene function with high-throughput molecular, genetic, and cellular technologies. The near limitless potential for applying these concepts to study the activities of all genetic loci has completely upended how today's cancer biologists tackle drug target discovery. We provide an overview of contemporary functional genomics platforms, highlighting areas of distinction and complementarity across technologies, so as to aid in the development or interpretation of cancer-focused screening efforts.},
}
@article {pmid32442184,
year = {2020},
author = {Dong, H and Wang, D and Bai, Z and Yuan, Y and Yang, W and Zhang, Y and Ni, H and Jiang, L},
title = {Generation of imidazolinone herbicide resistant trait in Arabidopsis.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0233503},
pmid = {32442184},
issn = {1932-6203},
mesh = {Acetolactate Synthase/genetics ; Amino Acid Substitution ; Arabidopsis/*drug effects/*genetics ; Arabidopsis Proteins/genetics ; Base Sequence ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Gene Editing ; Genes, Plant ; Herbicide Resistance/*genetics ; Herbicides/pharmacology ; Imidazolines/pharmacology ; Mutagenesis, Site-Directed ; Plant Breeding ; Plants, Genetically Modified/drug effects/genetics ; Selection, Genetic ; Weed Control ; },
abstract = {Recently-emerged base editing technologies could create single base mutations at precise genomic positions without generation DNA double strand breaks. Herbicide resistant mutations have been successfully introduced to different plant species, including Arabidopsis, watermelon, wheat, potato and tomato via C to T (or G to A on the complementary strand) base editors (CBE) at the P197 position of endogenous acetolactate synthase (ALS) genes. Additionally, G to A conversion to another conserved amino acid S653 on ALS gene could confer tolerance to imidazolinone herbicides. However, no such mutation was successfully generated via CBE, likely due to the target C base is outside of the classic base editing window. Since CBE driven by egg cell (EC) specific promoter would re-edit the wild type alleles in egg cells and early embryos, we hypothesized the diversity of base editing outcomes could be largely increased at later generations to allow selection of desired herbicide resistant mutants. To test this hypothesis, we aimed to introduce C to T conversion to the complement strand of S653 codon at ALS gene, hosting a C at the 10th position within the 20-nt spacer sequence outside of the classic base editing window. While we did not detect base-edited T1 plants, efficient and diverse base edits emerged at later generations. Herbicide resistant mutants with different editing outcomes were recovered when T3 and T4 seeds were subject to herbicide selection. As expected, most herbicide resistant plants contained S653N mutation as a result of G10 to A10. Our results showed that CBE could create imidazolinone herbicide resistant trait in Arabidopsis and be potentially applied to crops to facilitate weed control.},
}
@article {pmid32441252,
year = {2020},
author = {Bowden, AR and Morales-Juarez, DA and Sczaniecka-Clift, M and Agudo, MM and Lukashchuk, N and Thomas, JC and Jackson, SP},
title = {Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32441252},
issn = {2050-084X},
support = {C6/A11224/CRUK_/Cancer Research UK/United Kingdom ; C6946/A24843/CRUK_/Cancer Research UK/United Kingdom ; WT203144//Wellcome/International ; 205253/Z/16/Z//Wellcome/International ; C6/A18796/CRUK_/Cancer Research UK/United Kingdom ; 206388/Z/17/Z//Wellcome/International ; /WT_/Wellcome Trust/United Kingdom ; 304302648//Consejo Nacional de Ciencia y Tecnolog&#xed;a/International ; },
mesh = {Apoptosis ; *CRISPR-Cas Systems ; Cell Line ; DNA Damage ; Epithelial Cells ; Gene Deletion ; Gene Editing ; Gene Expression Regulation ; Humans ; Tumor Suppressor Protein p53/genetics/*metabolism ; },
abstract = {CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.},
}
@article {pmid32439761,
year = {2020},
author = {Brandt, ZJ and Echert, AE and Bostrom, JR and North, PN and Link, BA},
title = {Core Hippo pathway components act as a brake on Yap and Taz in the development and maintenance of the biliary network.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {12},
pages = {},
pmid = {32439761},
issn = {1477-9129},
support = {R01 EY029267/EY/NEI NIH HHS/United States ; },
mesh = {Acyltransferases ; Animals ; Animals, Genetically Modified/growth &amp; development/metabolism ; Biliary Tract/anatomy &amp; histology/growth &amp; development/*metabolism ; CRISPR-Cas Systems/genetics ; Carboxylic Ester Hydrolases/metabolism ; Gallbladder/anatomy &amp; histology/growth &amp; development/metabolism ; Larva/growth &amp; development/metabolism ; Liver/anatomy &amp; histology/metabolism ; Phenotype ; Protein Serine-Threonine Kinases/deficiency/genetics/*metabolism ; Serine-Threonine Kinase 3 ; Signal Transduction ; Trans-Activators/genetics/*metabolism ; Transcription Factors/genetics/*metabolism ; YAP-Signaling Proteins ; Zebrafish/growth &amp; development/metabolism ; Zebrafish Proteins/deficiency/genetics/*metabolism ; },
abstract = {The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1[-/-] hepatocytes is able to overcome these initial deficits and allows sav1[-/-] fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1[-/-] rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.},
}
@article {pmid32439759,
year = {2020},
author = {Mereu, L and Morf, MK and Spiri, S and Gutierrez, P and Escobar-Restrepo, JM and Daube, M and Walser, M and Hajnal, A},
title = {Polarized epidermal growth factor secretion ensures robust vulval cell fate specification in Caenorhabditis elegans.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {11},
pages = {},
pmid = {32439759},
issn = {1477-9129},
mesh = {Animals ; Animals, Genetically Modified/growth &amp; development/metabolism ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/growth &amp; development/*metabolism ; Caenorhabditis elegans Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Epidermal Growth Factor/antagonists &amp; inhibitors/genetics/*metabolism ; ErbB Receptors/metabolism ; Female ; Gene Editing ; Larva/metabolism ; Mitogen-Activated Protein Kinases/metabolism ; Mutagenesis ; Netrins/genetics/metabolism ; RNA Interference ; RNA-Dependent RNA Polymerase/genetics/metabolism ; Signal Transduction ; Stem Cells/cytology/metabolism ; Vulva/cytology/growth &amp; development/*metabolism ; ras GTPase-Activating Proteins/antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC.},
}
@article {pmid32439756,
year = {2020},
author = {Kesavan, G and Machate, A and Hans, S and Brand, M},
title = {Cell-fate plasticity, adhesion and cell sorting complementarily establish a sharp midbrain-hindbrain boundary.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {11},
pages = {},
doi = {10.1242/dev.186882},
pmid = {32439756},
issn = {1477-9129},
mesh = {Animals ; Animals, Genetically Modified/growth &amp; development/metabolism ; CRISPR-Cas Systems/genetics ; Cadherins/genetics/metabolism ; Cell Adhesion/*physiology ; Cell Lineage ; Embryo, Nonmammalian/metabolism ; Ephrins/antagonists &amp; inhibitors/genetics/metabolism ; Gastrulation ; Gene Editing ; Mesencephalon/*metabolism/pathology ; Microscopy, Atomic Force ; Microscopy, Fluorescence ; Morpholinos/metabolism ; Otx Transcription Factors/genetics/metabolism ; Rhombencephalon/*metabolism/pathology ; Signal Transduction ; Time-Lapse Imaging ; Zebrafish/growth &amp; development/*metabolism ; Zebrafish Proteins/genetics/metabolism ; },
abstract = {The formation and maintenance of sharp boundaries between groups of cells play a vital role during embryonic development as they serve to compartmentalize cells with similar fates. Some of these boundaries also act as organizers, with the ability to induce specific cell fates and morphogenesis in the surrounding cells. The midbrain-hindbrain boundary (MHB) is such an organizer: it acts as a lineage restriction boundary to prevent the intermingling of cells with different developmental fates. However, the mechanisms underlying the lineage restriction process remain unclear. Here, using novel fluorescent knock-in reporters, live imaging, Cre/lox-mediated lineage tracing, atomic force microscopy-based cell adhesion assays and mutant analysis, we analyze the process of lineage restriction at the MHB and provide mechanistic details. Specifically, we show that lineage restriction occurs by the end of gastrulation, and that the subsequent formation of sharp gene expression boundaries in the developing MHB occur through complementary mechanisms, i.e. cell-fate plasticity and cell sorting. Furthermore, we show that cell sorting at the MHB involves differential adhesion among midbrain and hindbrain cells that is mediated by N-cadherin and Eph-ephrin signaling.},
}
@article {pmid32439162,
year = {2020},
author = {Kaneko, T and Tanaka, S},
title = {Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts.},
journal = {Biochemical and biophysical research communications},
volume = {527},
number = {4},
pages = {1039-1042},
doi = {10.1016/j.bbrc.2020.05.034},
pmid = {32439162},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cumulus Cells/*cytology ; Electroporation/*methods ; Embryo, Mammalian/metabolism ; Female ; Gene Editing/*methods ; Male ; Mice, Inbred ICR ; Oviducts/*cytology ; },
abstract = {Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00-17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00-11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5-7) compared with the control without operation (11).},
}
@article {pmid32439069,
year = {2020},
author = {Hahn, E and Hiemenz, M},
title = {Therapeutic Gene Editing with CRISPR: A Laboratory Medicine Perspective.},
journal = {Clinics in laboratory medicine},
volume = {40},
number = {2},
pages = {205-219},
doi = {10.1016/j.cll.2020.02.008},
pmid = {32439069},
issn = {1557-9832},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genetic Therapy ; Humans ; Immunotherapy, Adoptive ; Sequence Analysis, DNA ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
abstract = {Therapeutic gene editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system offers significant improvements in specificity and programmability compared with previous methods. CRISPR editing strategies can be used ex vivo and in vivo with many theoretic disease applications. Off-target effects of CRISPR-mediated gene editing are an important outcome to be aware of, minimize, and detect. The current methods of regulatory approval for personalized therapies are complex and may be proved inefficient as these therapies are implemented more widely. The role of pathologists and laboratory medicine practitioners is vital to the clinical implementation of therapeutic gene editing.},
}
@article {pmid32439068,
year = {2020},
author = {Koulisis, N and Nagiel, A},
title = {Precision Therapy for Inherited Retinal Disease: At the Forefront of Genomic Medicine.},
journal = {Clinics in laboratory medicine},
volume = {40},
number = {2},
pages = {189-204},
doi = {10.1016/j.cll.2020.02.007},
pmid = {32439068},
issn = {1557-9832},
mesh = {CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Gene Editing ; *Genetic Therapy ; *Genomics ; High-Throughput Nucleotide Sequencing ; Molecular Diagnostic Techniques ; *Precision Medicine ; *Retinal Diseases/diagnosis/genetics/therapy ; Sequence Analysis, DNA ; },
abstract = {Inherited retinal diseases (IRDs) represent a diverse array of conditions characterized by dysfunction or loss of 1 or more retinal cell types. Next-generation sequencing has enabled rapid and relatively inexpensive genotyping, with more than 250 genes identified as responsible for IRDs. This expansion in molecular diagnostic accuracy, in combination with the retina's relative accessibility and immune privilege, has fostered the development of precision therapies to treat these myriad conditions. Novel techniques are being used in early trials. Precision molecular therapies for IRDs hold great promise as diagnostic and treatment strategies continue to expand.},
}
@article {pmid32438486,
year = {2020},
author = {Wang, Q and Alariqi, M and Wang, F and Li, B and Ding, X and Rui, H and Li, Y and Xu, Z and Qin, L and Sun, L and Li, J and Zou, J and Lindsey, K and Zhang, X and Jin, S},
title = {The application of a heat-inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants.},
journal = {Plant biotechnology journal},
volume = {18},
number = {12},
pages = {2436-2443},
pmid = {32438486},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; Gossypium ; Hot Temperature ; Humans ; Plants, Genetically Modified/genetics ; Tetraploidy ; },
abstract = {The CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low-frequency off-target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium-mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off-target mutation was detected by whole-genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.},
}
@article {pmid32437010,
year = {2020},
author = {Zhang, J and Hong, W and Guo, L and Wang, Y and Wang, Y},
title = {Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {9},
pages = {2911-2917},
doi = {10.1002/bit.27435},
pmid = {32437010},
issn = {1097-0290},
support = {2018-67021-27715//National Institute of Food and Agriculture/International ; ALA014-1017025//USDA-NIFA/International ; //Ocean University of China-Auburn University (OUC-AU) Grants/International ; //Auburn University Intramural Grants Program (IGP)/International ; //Alabama Agricultural Experiment Station/International ; },
mesh = {Batch Cell Culture Techniques ; Butyrates/metabolism ; CRISPR-Cas Systems/*genetics ; Clostridium tyrobutyricum/*genetics/metabolism ; Fermentation ; Gene Editing/*methods ; Plasmids/*genetics ; Transformation, Bacterial/*genetics ; },
abstract = {Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 10[3] CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.},
}
@article {pmid32434907,
year = {2020},
author = {Kapolka, NJ and Taghon, GJ and Rowe, JB and Morgan, WM and Enten, JF and Lambert, NA and Isom, DG},
title = {DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {23},
pages = {13117-13126},
pmid = {32434907},
issn = {1091-6490},
support = {R01 GM130142/GM/NIGMS NIH HHS/United States ; R03 TR002908/TR/NCATS NIH HHS/United States ; R35 GM119518/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cost-Benefit Analysis ; HEK293 Cells ; High-Throughput Screening Assays/economics/*methods ; Humans ; Ligands ; Receptors, G-Protein-Coupled/agonists/antagonists &amp; inhibitors/genetics/*metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Signal Transduction/drug effects ; },
abstract = {More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs.},
}
@article {pmid32433962,
year = {2020},
author = {Abe, T and Inoue, KI and Furuta, Y and Kiyonari, H},
title = {Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes.},
journal = {Cell reports},
volume = {31},
number = {7},
pages = {107653},
doi = {10.1016/j.celrep.2020.107653},
pmid = {32433962},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; Mice ; Mice, Knockout ; S Phase/*genetics ; Zygote/*metabolism ; },
abstract = {In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.},
}
@article {pmid32433960,
year = {2020},
author = {Riggan, L and Hildreth, AD and Rolot, M and Wong, YY and Satyadi, W and Sun, R and Huerta, C and O'Sullivan, TE},
title = {CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells.},
journal = {Cell reports},
volume = {31},
number = {7},
pages = {107651},
pmid = {32433960},
issn = {2211-1247},
support = {R01 AI145997/AI/NIAID NIH HHS/United States ; T32 AI007323/AI/NIAID NIH HHS/United States ; P30 DK063491/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Immunity, Innate/*genetics ; Mice ; Ribonucleoproteins/*metabolism ; },
abstract = {CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity, the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system.},
}
@article {pmid32433763,
year = {2020},
author = {Roberts, A and Barrangou, R},
title = {Applications of CRISPR-Cas systems in lactic acid bacteria.},
journal = {FEMS microbiology reviews},
volume = {44},
number = {5},
pages = {523-537},
doi = {10.1093/femsre/fuaa016},
pmid = {32433763},
issn = {1574-6976},
mesh = {*CRISPR-Cas Systems ; Industrial Microbiology/*trends ; Lactobacillales/*genetics ; Microbiota/genetics ; },
abstract = {As a phenotypically and phylogenetically diverse group, lactic acid bacteria are found in a variety of natural environments and occupy important roles in medicine, biotechnology, food and agriculture. The widespread use of lactic acid bacteria across these industries fuels the need for new and functionally diverse strains that may be utilized as starter cultures or probiotics. Originally characterized in lactic acid bacteria, CRISPR-Cas systems and derived molecular machines can be used natively or exogenously to engineer new strains with enhanced functional attributes. Research on CRISPR-Cas biology and its applications has exploded over the past decade with studies spanning from the initial characterization of CRISPR-Cas immunity in Streptococcus thermophilus to the use of CRISPR-Cas for clinical gene therapies. Here, we discuss CRISPR-Cas classification, overview CRISPR biology and mechanism of action, and discuss current and future applications in lactic acid bacteria, opening new avenues for their industrial exploitation and manipulation of microbiomes.},
}
@article {pmid32433547,
year = {2020},
author = {Richter, MF and Zhao, KT and Eton, E and Lapinaite, A and Newby, GA and Thuronyi, BW and Wilson, C and Koblan, LW and Zeng, J and Bauer, DE and Doudna, JA and Liu, DR},
title = {Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {883-891},
pmid = {32433547},
issn = {1546-1696},
support = {R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; T32 GM095450/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; },
mesh = {Adenine/*metabolism ; Adenosine Deaminase/genetics ; Bacteriophages/genetics ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; Gene Editing ; HEK293 Cells ; Humans ; Mutagenesis/genetics ; Mutation/genetics ; RNA/*genetics ; },
abstract = {Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.},
}
@article {pmid32431295,
year = {2020},
author = {Liu, SY and Yi, GQ and Tang, ZL and Chen, B},
title = {Progress on genome-wide CRISPR/Cas9 screening for functional genes and regulatory elements.},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {5},
pages = {435-443},
doi = {10.16288/j.yczz.19-390},
pmid = {32431295},
issn = {0253-9772},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {The CRISPR/Cas9 system is a powerful tool which has been extensively used for genome editing in the past few years. Nuclease-dead Cas9 (CRISPR/dCas9), a Cas9 protein mutant without splicing ability, along with loss-of- function (LOF), gain-of-function (GOF), or non-coding genes scanning approaches can reveal genome-scale functional determinants. CRISPR/Cas9 has been widely adopted to decipher disease mechanisms and pinpoint drug targets in the life science field, and also provide novel insights into animal genetics and breeding. In this review, we summarize the research progress in high-throughput CRISPR/Cas9 screening for revealing the functional genes and regulatory elements in the whole genome. We also highlight the applications of CRISPR/Cas9 system in the animal cells, providing a reference for gene editing and other related research in related fields.},
}
@article {pmid32429836,
year = {2020},
author = {Sriboon, S and Li, H and Guo, C and Senkhamwong, T and Dai, C and Liu, K},
title = {Knock-out of TERMINAL FLOWER 1 genes altered flowering time and plant architecture in Brassica napus.},
journal = {BMC genetics},
volume = {21},
number = {1},
pages = {52},
pmid = {32429836},
issn = {1471-2156},
support = {2016YFD0101007//National Key Research and Development Program of China./International ; },
mesh = {Amino Acid Sequence ; Brassica napus/*genetics/physiology ; CRISPR-Cas Systems ; Flowers/*physiology ; Gene Dosage ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; *Genes, Plant ; Phenotype ; Phylogeny ; Plants, Genetically Modified/physiology ; Sequence Alignment ; },
abstract = {BACKGROUND: TERMINAL FLOWER 1 (TFL1) is a member of phosphatidylethanolamine-binding protein (PEBP) family, which plays an important role in the determination of floral meristem identity and regulates flowering time in higher plants.<br><br>RESULTS: Five BnaTFL1 gene copies were identified in the genome of Brassica napus. The phylogenetic analysis indicated that all five BnaTFL1 gene copies were clustered with their corresponding homologous copies in the ancestral species, B. rapa and B. oleracea. The expression of the BnaTFL1s were confined to flower buds, flowers, seeds, siliques and stem tissues and displayed distinct expression profiles. Knockout mutants of BnaC03.TFL1 generated by CRISPR/Cas9 exhibited early flowering phenotype, while the knockout mutants of the other gene copies had similar flowering time as the wild type. Furthermore, knock-out mutants of individual BnaTFL1 gene copy displayed altered plant architecture. The plant height, branch initiation height, branch number, silique number, number of seeds per silique and number of siliques on the main inflorescence were significantly reduced in the BnaTFL1 mutants.<br><br>CONCLUSIONS: Our results indicated that BnaC03.TFL1 negatively regulates flowering time in B. napus. BnaC03.TFL1 together with the other BnaTFL1 paralogues are essential for controlling the plant architecture.},
}
@article {pmid32428469,
year = {2020},
author = {Yaguchi, S and Yaguchi, J and Suzuki, H and Kinjo, S and Kiyomoto, M and Ikeo, K and Yamamoto, T},
title = {Establishment of homozygous knock-out sea urchins.},
journal = {Current biology : CB},
volume = {30},
number = {10},
pages = {R427-R429},
doi = {10.1016/j.cub.2020.03.057},
pmid = {32428469},
issn = {1879-0445},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; DNA/chemistry/genetics ; Female ; Gene Expression Regulation, Enzymologic ; Gene Knockout Techniques/*methods ; Homozygote ; Male ; Mutation ; Polyketide Synthases/*genetics ; Sea Urchins/*genetics ; },
abstract = {Yaguchi et al. establish a homozygous knock-out sea urchin line by applying the CRISPR-Cas9 system to a new model species, Temnopleurus reevesii, whose breeding cycle takes about half a year.},
}
@article {pmid32428441,
year = {2020},
author = {Gough, V and Gersbach, CA},
title = {Immunity to Cas9 as an Obstacle to Persistent Genome Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {6},
pages = {1389-1391},
pmid = {32428441},
issn = {1525-0024},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}
@article {pmid32428200,
year = {2020},
author = {Wüst, RCI and Houtkooper, RH and Auwerx, J},
title = {Confounding factors from inducible systems for spatiotemporal gene expression regulation.},
journal = {The Journal of cell biology},
volume = {219},
number = {7},
pages = {},
pmid = {32428200},
issn = {1540-8140},
mesh = {Animals ; *Artifacts ; *CRISPR-Cas Systems ; Doxycycline/adverse effects ; Gene Editing/*methods ; Gene Expression Regulation ; *Genome ; Integrases/genetics/metabolism ; Mice ; Mice, Transgenic ; Mitochondria/drug effects/metabolism ; Receptors, Estrogen/genetics/metabolism ; Response Elements/*drug effects ; Tamoxifen/adverse effects ; Tetracycline/adverse effects ; Transfection/methods ; },
abstract = {Spatiotemporally regulated targeted gene manipulation is a common way to study the effect of gene variants on phenotypic traits, but the Cre/loxP and Tet-On/Tet-Off systems can affect whole-organism physiology and function due to off-target effects. We highlight some of these adverse effects, including whole-body endocrinology and disturbances in the gut microbiome and in mitochondrial and metabolic function.},
}
@article {pmid32427850,
year = {2020},
author = {Chen, H and Zeng, Y and Yang, Y and Huang, L and Tang, B and Zhang, H and Hao, F and Liu, W and Li, Y and Liu, Y and Zhang, X and Zhang, R and Zhang, Y and Li, Y and Wang, K and He, H and Wang, Z and Fan, G and Yang, H and Bao, A and Shang, Z and Chen, J and Wang, W and Qiu, Q},
title = {Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2494},
pmid = {32427850},
issn = {2041-1723},
mesh = {Alleles ; CRISPR-Cas Systems ; Chromosomes, Plant/*genetics ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Medicago sativa/*genetics ; Mutation ; Phenotype ; Plant Breeding/methods ; *Tetraploidy ; Transgenes/*genetics ; },
abstract = {Artificially improving traits of cultivated alfalfa (Medicago sativa L.), one of the most important forage crops, is challenging due to the lack of a reference genome and an efficient genome editing protocol, which mainly result from its autotetraploidy and self-incompatibility. Here, we generate an allele-aware chromosome-level genome assembly for the cultivated alfalfa consisting of 32 allelic chromosomes by integrating high-fidelity single-molecule sequencing and Hi-C data. We further establish an efficient CRISPR/Cas9-based genome editing protocol on the basis of this genome assembly and precisely introduce tetra-allelic mutations into null mutants that display obvious phenotype changes. The mutated alleles and phenotypes of null mutants can be stably inherited in generations in a transgene-free manner by cross pollination, which may help in bypassing the debate about transgenic plants. The presented genome and CRISPR/Cas9-based transgene-free genome editing protocol provide key foundations for accelerating research and molecular breeding of this important forage crop.},
}
@article {pmid32426943,
year = {2020},
author = {Budhagatapalli, N and Halbach, T and Hiekel, S and Büchner, H and Müller, AE and Kumlehn, J},
title = {Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.},
journal = {Plant biotechnology journal},
volume = {18},
number = {12},
pages = {2376-2378},
pmid = {32426943},
issn = {1467-7652},
mesh = {Bread ; CRISPR-Cas Systems ; Haploidy ; Mutagenesis ; Mutagenesis, Site-Directed ; Pollination ; *RNA, Guide ; Triticum/genetics ; *Zea mays/genetics ; },
}
@article {pmid32426802,
year = {2021},
author = {Singhal, J and Chikara, S and Horne, D and Awasthi, S and Salgia, R and Singhal, SS},
title = {Targeting RLIP with CRISPR/Cas9 controls tumor growth.},
journal = {Carcinogenesis},
volume = {42},
number = {1},
pages = {48-57},
pmid = {32426802},
issn = {1460-2180},
support = {P30 CA033572/CA/NCI NIH HHS/United States ; },
mesh = {ATP-Binding Cassette Transporters/*genetics/metabolism ; Apoptosis/genetics ; Breast Neoplasms/genetics/*therapy ; CRISPR-Cas Systems/*genetics ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Survival/genetics ; Female ; GTPase-Activating Proteins/*genetics/metabolism ; Gene Editing/methods ; Gene Knockdown Techniques ; Genetic Therapy/*methods ; Humans ; Proof of Concept Study ; RNA, Guide/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the 'guardian of the genome' because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo.},
}
@article {pmid32424520,
year = {2020},
author = {Wang, G and Sukumar, S},
title = {Unpredicted central inversion in a sgRNA flanked by inverted repeats.},
journal = {Molecular biology reports},
volume = {47},
number = {8},
pages = {6375-6378},
doi = {10.1007/s11033-020-05524-1},
pmid = {32424520},
issn = {1573-4978},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Escherichia coli/*genetics ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; Homeodomain Proteins/genetics ; Humans ; *Inverted Repeat Sequences ; RNA, Guide/*genetics ; Transformation, Genetic ; },
abstract = {In genome engineering, sgRNAs define the genomic target to be modified in CRISPR/Cas9 system. Either for single gene editing or genome-wide screens, sgRNAs are cloned into plasmid vectors. During our performance of CRISPR/Cas9 gene knock out, we found that the central part of a sgRNA was inverted after transformation into Escherichia coli. Interestingly, the inverted portion was found to be flanked by inverted repeats, and sealing of nicks inside the plasmid could correct the inversion. This type of sgRNA recombination completely changed its original sequence and should be noted during sgRNA design and performance of CRISPR/Cas9.},
}
@article {pmid32424339,
year = {2020},
author = {Guo, R and Zhang, Y and Teng, M and Jiang, C and Schineller, M and Zhao, B and Doench, JG and O'Reilly, RJ and Cesarman, E and Giulino-Roth, L and Gewurz, BE},
title = {DNA methylation enzymes and PRC1 restrict B-cell Epstein-Barr virus oncoprotein expression.},
journal = {Nature microbiology},
volume = {5},
number = {8},
pages = {1051-1063},
pmid = {32424339},
issn = {2058-5276},
support = {R01 CA047006/CA/NCI NIH HHS/United States ; K08 CA219473/CA/NCI NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; R01 CA228700/CA/NCI NIH HHS/United States ; R01 AI137337/AI/NIAID NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R35 CA047006/CA/NCI NIH HHS/United States ; },
mesh = {Antigens, Viral ; B-Lymphocytes/*virology ; Burkitt Lymphoma ; CCAAT-Enhancer-Binding Proteins ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism/*pharmacology ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism ; DNA (Cytosine-5-)-Methyltransferases ; DNA Methylation/*physiology ; Epstein-Barr Virus Nuclear Antigens ; Gene Expression Regulation, Viral/*drug effects ; Genes, Viral ; Genome, Viral ; Herpesvirus 4, Human/*drug effects/*genetics/metabolism ; Humans ; Oncogene Proteins/genetics/*metabolism ; Ubiquitin-Protein Ligases ; },
abstract = {To accomplish the remarkable task of lifelong infection, the Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programmes to navigate the B-cell compartment and evade immune responses. The transforming programme, consisting of highly immunogenic EBV nuclear antigen (EBNA) and latent membrane proteins (LMPs), is expressed in newly infected B lymphocytes and in post-transplant lymphomas. On memory cell differentiation and in most EBV-associated Burkitt's lymphomas, all but one viral antigen are repressed for immunoevasion. To gain insights into the epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR-Cas9 screen was performed in EBV and Burkitt's lymphoma cells. Here, we show that the ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and its DNA methyltransferase partner DNA methyltransferase I (DNMT1) are critical for the restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency programme switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal centre B cells, the Burkitt's lymphoma cell of origin, providing a molecular link between B-cell state and the EBV latency programme. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.},
}
@article {pmid32424272,
year = {2020},
author = {Zuo, E and Sun, Y and Yuan, T and He, B and Zhou, C and Ying, W and Liu, J and Wei, W and Zeng, R and Li, Y and Yang, H},
title = {A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects.},
journal = {Nature methods},
volume = {17},
number = {6},
pages = {600-604},
pmid = {32424272},
issn = {1548-7105},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Cytosine/*metabolism ; DNA/*genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mutation ; Point Mutation ; RNA/*genetics ; },
abstract = {Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.},
}
@article {pmid32424075,
year = {2020},
author = {Chang, J and Wang, R and Yu, K and Zhang, T and Chen, X and Liu, Y and Shi, R and Wang, X and Xia, Q and Ma, S},
title = {Genome-wide CRISPR screening reveals genes essential for cell viability and resistance to abiotic and biotic stresses in Bombyx mori.},
journal = {Genome research},
volume = {30},
number = {5},
pages = {757-767},
pmid = {32424075},
issn = {1549-5469},
mesh = {Animals ; Bombyx/*genetics/virology ; *CRISPR-Cas Systems ; Cell Line ; Cell Survival/genetics ; *Genes, Essential ; *Genes, Insect ; Genome, Insect ; Host-Pathogen Interactions ; RNA ; Stress, Physiological/genetics ; Temperature ; },
abstract = {High-throughput genetic screens are powerful methods to interrogate gene function on a genome-wide scale and identify genes responsible to certain stresses. Here, we developed a piggyBac strategy to deliver pooled sgRNA libraries stably into cell lines. We used this strategy to conduct a screen based on genome-wide clustered regularly interspaced short palindromic repeat technology (CRISPR)-Cas9 in Bombyx mori cells. We first constructed a single guide RNA (sgRNA) library containing 94,000 sgRNAs, which targeted 16,571 protein-coding genes. We then generated knockout collections in BmE cells using the piggyBac transposon. We identified 1006 genes that are essential for cell viability under normal growth conditions. Of the identified genes, 82.4% (829 genes) were homologous to essential genes in seven animal species. We also identified 838 genes whose loss facilitated cell growth. Next, we performed context-specific positive screens for resistance to biotic or nonbiotic stresses using temperature and baculovirus separately, which identified several key genes and pathways from each screen. Collectively, our results provide a novel and versatile platform for functional annotations of B. mori genomes and deciphering key genes responsible for various conditions. This study also shows the effectiveness, practicality, and convenience of genome-wide CRISPR screens in nonmodel organisms.},
}
@article {pmid32423474,
year = {2020},
author = {Zhang, Y and Held, MA and Showalter, AM},
title = {Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {221},
pmid = {32423474},
issn = {1471-2229},
mesh = {Amino Acid Sequence ; Arabidopsis/enzymology/*genetics ; *CRISPR-Cas Systems ; Galactans/*metabolism ; },
abstract = {BACKGROUND: Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated.<br><br>RESULTS: Three members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knock-out mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage.<br><br>CONCLUSIONS: This research provides genetic evidence that GLCAT14A-C function in the transfer of GlcA to AGPs, which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation.},
}
@article {pmid32422965,
year = {2020},
author = {Morales-Cámara, S and Alexandre-Moreno, S and Bonet-Fernández, JM and Atienzar-Aroca, R and Aroca-Aguilar, JD and Ferre-Fernández, JJ and Méndez, CD and Morales, L and Fernández-Sánchez, L and Cuenca, N and Coca-Prados, M and Martínez-de-la-Casa, JM and Garcia-Feijoo, J and Escribano, J},
title = {Role of GUCA1C in Primary Congenital Glaucoma and in the Retina: Functional Evaluation in Zebrafish.},
journal = {Genes},
volume = {11},
number = {5},
pages = {},
pmid = {32422965},
issn = {2073-4425},
mesh = {Adult ; Amino Acid Sequence ; Animals ; Apoptosis ; Base Sequence ; CRISPR-Cas Systems ; Female ; Gene Editing ; Gene Knockout Techniques ; Glaucoma/congenital/*genetics ; Gliosis/genetics/pathology ; Guanylate Cyclase-Activating Proteins/genetics/*physiology ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Middle Aged ; Pedigree ; Retina/*metabolism/pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment ; Sequence Homology, Amino Acid ; Zebrafish ; Zebrafish Proteins/genetics/*physiology ; },
abstract = {Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.},
}
@article {pmid32422653,
year = {2020},
author = {Hecht, TK and Blank, B and Steger, M and Lopez, V and Beck, G and Ramazanov, B and Mann, M and Tagliabracci, V and von Blume, J},
title = {Fam20C regulates protein secretion by Cab45 phosphorylation.},
journal = {The Journal of cell biology},
volume = {219},
number = {6},
pages = {},
pmid = {32422653},
issn = {1540-8140},
support = {R01 GM134083/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics/*metabolism ; Casein Kinase I/deficiency/genetics/*metabolism ; Cell Line, Tumor ; Extracellular Matrix Proteins/deficiency/genetics/*metabolism ; Gene Knockout Techniques ; Glycoproteins/genetics/*metabolism ; Humans ; Isoantigens/metabolism ; Mutation ; Phosphorylation ; Protein Transport/*genetics/physiology ; RNA, Small Interfering ; Recombinant Proteins ; Seminal Plasma Proteins/metabolism ; trans-Golgi Network/*metabolism ; },
abstract = {The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. Here, we show that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. Our findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN.},
}
@article {pmid32422169,
year = {2020},
author = {Przewrocka, J and Rowan, A and Rosenthal, R and Kanu, N and Swanton, C},
title = {Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting.},
journal = {Annals of oncology : official journal of the European Society for Medical Oncology},
volume = {31},
number = {9},
pages = {1270-1273},
pmid = {32422169},
issn = {1569-8041},
support = {FC001169/MRC_/Medical Research Council/United Kingdom ; FC001169/WT_/Wellcome Trust/United Kingdom ; FC001169/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Chromosomal Instability ; *Gene Targeting ; Humans ; },
}
@article {pmid32421986,
year = {2020},
author = {Hashmat, R and Yousaf, MZ and Rahman, Z and Anjum, KM and Yaqoob, A and Imran, M},
title = {CRISPR-CAS Replacing Antiviral Drugs against HIV: An Update.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {30},
number = {1},
pages = {77-83},
doi = {10.1615/CritRevEukaryotGeneExpr.2020028233},
pmid = {32421986},
issn = {1045-4403},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genetic Therapy ; Genome, Viral/genetics ; HIV Infections/genetics/*therapy/virology ; HIV-1/genetics/pathogenicity ; Humans ; RNA, Guide/genetics ; Rats ; },
abstract = {Various antiretroviral drugs do not kill or cure the human immunodeficiency virus (HIV) but do prevent the replication of the virus. The combination of antiretroviral drugs is known as highly active antiretroviral therapy (HAART). Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attacks on this proviral DNA. The biggest achievement might be the complete elimination of HIV from infected cells. A study revealed that the tail injection, in transgenic mice and rats having HIV-1 genome, of an adenoassociated virus (AAV) vector expressing a short version of the Cas9 endonuclease (saCas9) and the gRNAs resulted in the cleavage of integrated HIV-1 DNA and excision of a DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, kidney, and circulating lymphocytes. HIV-1 has capacity to escape the attack on its genome from most of inhibitors. Thus, to achieve successful antiretroviral treatment, combinations of several antiviral therapies have been applied that are based on two important facts. The first is that multiple drugs lead to synergistic or additive inhibition, and the second is that the combinational therapy increases chances of drug resistance. The success that has been achieved with the help of the genetically engineered tool CRISPR is that dCas9 protein alone can efficiently silence viral gene expression in bacteria with sgRNA. All the reported investigations have indicated that CRISPR/Cas9 can be used as immune machinery into human cells in the form of novel antivirus tools.},
}
@article {pmid32421984,
year = {2020},
author = {Ashfaq, UA and Khalid, H},
title = {CRISPR/CAS9-Mediated Antiviral Activity: A Tool to Combat Viral Infection.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {30},
number = {1},
pages = {45-56},
doi = {10.1615/CritRevEukaryotGeneExpr.2020028207},
pmid = {32421984},
issn = {1045-4403},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genetic Therapy ; Humans ; Virus Diseases/genetics/*therapy/virology ; },
abstract = {Viruses hijack host cellular receptors and functions for replication, thereby posing a complication in identifying therapeutic targets. The CRISPR/Cas 9 platform has revolutionized gene-editing modalities in a wide range of cells and organisms with high potential in therapeutics. Recently, it has been put to work targeting human pathogenic viruses that interrupt receptors and functions with viral replication. This review encompasses major discoveries in CRISPR/Cas as an antiviral strategy. Additionally, challenges that demand consideration prior to its use in the clinic as part of the antiviral armamentarium are briefly addressed.},
}
@article {pmid32421859,
year = {2020},
author = {Li, T and Li, F and Lin, J and Zhang, Y and Zhang, Q and Sun, Y and Chen, X and Xu, M and Wang, X and Li, Q},
title = {Deletion of c16orf45 in zebrafish results in a low fertilization rate and increased thigmotaxis.},
journal = {Developmental psychobiology},
volume = {62},
number = {8},
pages = {1003-1010},
doi = {10.1002/dev.21984},
pmid = {32421859},
issn = {1098-2302},
support = {81771632//National Natural Science Foundation of China/International ; 81271509//National Natural Science Foundation of China/International ; 81601329//National Natural Science Foundation of China/International ; 13JC1403700//Shanghai Municipal Commission of Science and Technology Program/International ; ZXDF089002//Shanghai Municipal Education Commission/International ; },
mesh = {Animals ; Animals, Genetically Modified ; Behavior, Animal/*physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Nonmammalian ; Exoribonucleases/*physiology ; Female ; Fertility/*genetics ; Fertilization/*genetics ; Gene Deletion ; Male ; Neurodevelopmental Disorders/*genetics ; Zebrafish ; Zebrafish Proteins/*physiology ; },
abstract = {c16orf45 is located at 16p13.11, an important locus related to neurodevelopmental diseases. Clinical studies have demonstrated that c16orf45 is associated with various neurodevelopmental diseases. To further elucidate the effect of c16orf45 on neural development, we constructed a zebrafish model with a stably inherited c16orf45 deletion via CRISPR/Cas9 technology. We found that deletion of c16orf45 significantly reduced the zebrafish fertilization rate, and both females and males showed reduced fertility. Meanwhile, the homozygous c16orf45 knockout zebrafish showed a developmental delay at 24 hr postfertilization (hpf). However, morphological changes were not apparent after 2 days postfertilization (dpf). Notably, the results of behavioral experiments revealed increased thigmotaxis in c16orf45[-][/][-] zebrafish at 2 months. In conclusion, these findings demonstrate that c16orf45 plays an important role in nervous system and reproductive system.},
}
@article {pmid32421777,
year = {2020},
author = {Garrett, S and Shiimori, M and Watts, EA and Clark, L and Graveley, BR and Terns, MP},
title = {Primed CRISPR DNA uptake in Pyrococcus furiosus.},
journal = {Nucleic acids research},
volume = {48},
number = {11},
pages = {6120-6135},
pmid = {32421777},
issn = {1362-4962},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptation, Physiological/*immunology ; Base Pairing ; Base Sequence ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics/*metabolism ; DNA Helicases/metabolism ; Mutation ; Nucleic Acid Hybridization ; Plasmids/genetics/metabolism ; Pyrococcus furiosus/*genetics/*immunology/metabolism ; RNA/genetics/metabolism ; Ribonucleoproteins/genetics/immunology/metabolism ; },
abstract = {CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.},
}
@article {pmid32421776,
year = {2020},
author = {Russo, M and Sobh, A and Zhang, P and Loguinov, A and Tagmount, A and Vulpe, CD and Liu, B},
title = {Functional Pathway Identification With CRISPR/Cas9 Genome-wide Gene Disruption in Human Dopaminergic Neuronal Cells Following Chronic Treatment With Dieldrin.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {176},
number = {2},
pages = {366-381},
doi = {10.1093/toxsci/kfaa071},
pmid = {32421776},
issn = {1096-0929},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Dieldrin/toxicity ; Dopaminergic Neurons/*drug effects ; Humans ; *Pesticides/toxicity ; },
abstract = {Organochlorine pesticides, once widely used, are extremely persistent and bio-accumulative in the environment. Epidemiological studies have implicated that environmental exposure to organochlorine pesticides including dieldrin is a risk factor for the development of Parkinson's disease. However, the pertinent mechanisms of action remain poorly understood. In this study, we carried out a genome-wide (Brunello library, 19 114 genes, 76 411 sgRNAs) CRISPR/Cas9 screen in human dopaminergic SH-SY5Y neuronal cells exposed to a chronic treatment (30 days) with dieldrin to identify cellular pathways that are functionally related to the chronic cellular toxicity. Our results indicate that dieldrin toxicity was enhanced by gene disruption of specific components of the ubiquitin proteasome system as well as, surprisingly, the protein degradation pathways previously implicated in inherited forms of Parkinson's disease, centered on Parkin. In addition, disruption of regulatory components of the mTOR pathway which integrates cellular responses to both intra- and extracellular signals and is a central regulator for cell metabolism, growth, proliferation, and survival, led to increased sensitivity to dieldrin-induced cellular toxicity. This study is one of the first to apply a genome-wide CRISPR/Cas9-based functional gene disruption screening approach in an adherent neuronal cell line to globally decipher cellular mechanisms that contribute to environmental toxicant-induced neurotoxicity and provides novel insight into the dopaminergic neurotoxicity associated with chronic exposure to dieldrin.},
}
@article {pmid32421209,
year = {2020},
author = {Bouchaut, B and Asveld, L},
title = {Safe-by-Design: Stakeholders' Perceptions and Expectations of How to Deal with Uncertain Risks of Emerging Biotechnologies in the Netherlands.},
journal = {Risk analysis : an official publication of the Society for Risk Analysis},
volume = {40},
number = {8},
pages = {1632-1644},
pmid = {32421209},
issn = {1539-6924},
mesh = {*Biotechnology ; Netherlands ; *Risk ; Safety ; *Uncertainty ; },
abstract = {Advanced gene editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas have increased the pace of developments in the field of industrial biotechnology. Such techniques imply new possibilities when working with living organisms, possibly leading to uncertain risks. In the Netherlands, current policy fails to address these uncertain risks because risk classification is determined process-wise (i.e., genetically modified organism [GMO] and non-GMO), there is a strong focus on quantifiable risks, and the linearity within current governance (science-policy-society) hinders iterative communication between stakeholders, leaving limited room to anticipate uncertainties at an early stage of development. A suggested concept to overcome these shortcomings is the Safe-by-Design (SbD) approach, which, theoretically, allows stakeholders to iteratively incorporate safety measures throughout a technology's development process, creating a dynamic environment for the anticipation of uncertain risks. Although this concept originates from chemical engineering and is already widely applied in nanotechnology, for the field of biotechnology, there is no agreed upon definition yet. To explore the possibilities of SbD for future governance of biotechnology, we should gain insight in how various stakeholders perceive notions of risk, safety, and inherent safety, and what this implies for the applicability of SbD for risk governance concerning industrial biotechnology. Our empirical research reveals three main themes: (1) diverging expectations with regard to safety and risks, and to establish an acceptable level of risk; (2) different applications of SbD and inherent safety, namely, product- and process-wise; and (3) unclarity in allocating responsibilities to stakeholders in the development process of a biotechnology and within society.},
}
@article {pmid32416935,
year = {2020},
author = {Levesque, S and Agudelo, D and Doyon, Y},
title = {Rewired Cas9s with Minimal Sequence Constraints.},
journal = {Trends in pharmacological sciences},
volume = {41},
number = {7},
pages = {429-431},
doi = {10.1016/j.tips.2020.04.009},
pmid = {32416935},
issn = {1873-3735},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {The genome editing toolkit is ever expanding. Although CRISPR-Cas systems can target virtually any gene, single-nucleotide resolution is yet to be achieved. Walton and colleagues engineered nucleases and base editors compatible with every protospacer adjacent motif (PAM) to achieve high-precision targeting. Their findings revealed the striking plasticity of Cas9.},
}
@article {pmid32416111,
year = {2021},
author = {Wan, T and Ping, Y},
title = {Delivery of genome-editing biomacromolecules for treatment of lung genetic disorders.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {196-216},
doi = {10.1016/j.addr.2020.05.002},
pmid = {32416111},
issn = {1872-8294},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Administration Routes ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; Lung Diseases/*genetics/*therapy ; Macromolecular Substances ; },
abstract = {Genome-editing systems based on clustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein (CRISPR/Cas), are emerging as a revolutionary technology for the treatment of various genetic diseases. To date, the delivery of genome-editing biomacromolecules by viral or non-viral vectors have been proposed as new therapeutic options for lung genetic disorders, such as cystic fibrosis (CF) and α-1 antitrypsin deficiency (AATD), and it has been accepted that these delivery vectors can introduce CRISPR/Cas9 machineries into target cells or tissues in vitro, ex vivo and in vivo. However, the efficient local or systemic delivery of CRISPR/Cas9 elements to the lung, enabled by either viral or by non-viral carriers, still remains elusive. Herein, we first introduce lung genetic disorders and their current treatment options, and then summarize CRISPR/Cas9-based strategies for the therapeutic genome editing of these disorders. We further summarize the pros and cons of different routes of administration for lung genetic disorders. In particular, the potentials of aerosol delivery for therapeutic CRISPR/Cas9 biomacromolecules for lung genome editing are discussed and highlighted. Finally, current challenges and future outlooks in this emerging area are briefly discussed.},
}
@article {pmid32416058,
year = {2020},
author = {Christie, KA and Robertson, LJ and Conway, C and Blighe, K and DeDionisio, LA and Chao-Shern, C and Kowalczyk, AM and Marshall, J and Turnbull, D and Nesbit, MA and Moore, CBT},
title = {Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {8},
pages = {1846-1857},
pmid = {32416058},
issn = {1525-0024},
mesh = {*Alleles ; Amino Acid Sequence ; Amino Acid Substitution ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Genes, Dominant ; Genetic Diseases, Inborn/*genetics/*therapy ; *Genetic Therapy ; Genomics/methods ; Haplotypes ; Humans ; *Mutation ; Polymorphism, Single Nucleotide ; Precision Medicine ; RNA, Guide ; Transforming Growth Factor beta1/genetics ; },
abstract = {CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor β-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.},
}
@article {pmid32415890,
year = {2020},
author = {Butt, H and Rao, GS and Sedeek, K and Aman, R and Kamel, R and Mahfouz, M},
title = {Engineering herbicide resistance via prime editing in rice.},
journal = {Plant biotechnology journal},
volume = {18},
number = {12},
pages = {2370-2372},
pmid = {32415890},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Herbicide Resistance ; *Oryza/genetics ; },
}
@article {pmid32415294,
year = {2020},
author = {Zhong, Y and Chen, B and Li, M and Wang, D and Jiao, Y and Qi, X and Wang, M and Liu, Z and Chen, C and Wang, Y and Chen, M and Li, J and Xiao, Z and Cheng, D and Liu, W and Boutilier, K and Liu, C and Chen, S},
title = {A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis.},
journal = {Nature plants},
volume = {6},
number = {5},
pages = {466-472},
pmid = {32415294},
issn = {2055-0278},
mesh = {Arabidopsis/*genetics/physiology ; Arabidopsis Proteins/*genetics/physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Genes, Plant/genetics/physiology ; *Haploidy ; Loss of Function Mutation/genetics ; Membrane Proteins/*genetics/physiology ; Plants, Genetically Modified ; },
abstract = {Doubled haploid technology using inducer lines carrying mutations in ZmPLA1/MTL/NLD and ZmDMP[1-4] has revolutionized traditional maize breeding. ZmPLA1/MTL/NLD is conserved in monocots and has been used to extend the system from maize to other monocots[5-7], but no functional orthologue has been identified in dicots, while ZmDMP-like genes exist in both monocots and dicots[4,8,9]. Here, we report that loss-of-function mutations in the Arabidopsis thaliana ZmDMP-like genes AtDMP8 and AtDMP9 induce maternal haploids, with an average haploid induction rate of 2.1 ± 1.1%. In addition, to facilitate haploid seed identification in dicots, we established an efficient FAST-Red fluorescent marker-based haploid identification system that enables the identification of haploid seeds with >90% accuracy. These results show that mutations in DMP genes also trigger haploid induction in dicots. The conserved expression patterns and amino acid sequences of ZmDMP-like genes in dicots suggest that DMP mutations could be used to develop in vivo haploid induction systems in dicots.},
}
@article {pmid32415280,
year = {2020},
author = {Das, S and Chandrasekaran, AP and Suresh, B and Haq, S and Kang, JH and Lee, SJ and Kim, J and Kim, J and Lee, S and Kim, HH and Kim, KS and Ramakrishna, S},
title = {Genome-scale screening of deubiquitinase subfamily identifies USP3 as a stabilizer of Cdc25A regulating cell cycle in cancer.},
journal = {Cell death and differentiation},
volume = {27},
number = {11},
pages = {3004-3020},
pmid = {32415280},
issn = {1476-5403},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Male ; Mice ; Mice, Nude ; Survival Analysis ; Ubiquitin-Specific Proteases/*genetics/metabolism ; *Ubiquitination ; Uterine Cervical Neoplasms/*metabolism/pathology ; Xenograft Model Antitumor Assays ; cdc25 Phosphatases/*genetics/metabolism ; },
abstract = {Conventional screening methods for deubiquitinating enzymes (DUBs) have important limitations. A loss-of-function study based on the knockout of DUB genes in mammalian cells can provide an excellent model for exploring DUB function. Here, we used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs), a DUB subfamily, and then systematically screened for DUBs that stabilize the Cdc25A oncoprotein. USP3 was identified as a deubiquitinase of Cdc25A. USP3 depletion reduces the Cdc25A protein level, resulting in a significant delay in cell-cycle progression, and reduces the growth of cervical tumor xenografts in nude mice. Clinically, USP3 expression is positively correlated with Cdc25A protein expression and the poorest survival in breast cancer. We envision that our DUB knockout library kit will facilitate genome-scale screening of functional DUBs for target proteins of interest in a wide range of biomedical fields.},
}
@article {pmid32415249,
year = {2020},
author = {Mullard, A},
title = {Gene-editing pipeline takes off.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {6},
pages = {367-372},
doi = {10.1038/d41573-020-00096-y},
pmid = {32415249},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems/*genetics ; Computational Biology/*methods/trends ; *Gene Editing ; Humans ; Medical Oncology/*methods/trends ; },
}
@article {pmid32415248,
year = {2020},
author = {Mullard, A},
title = {Jennifer Doudna.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {6},
pages = {380-381},
doi = {10.1038/d41573-020-00095-z},
pmid = {32415248},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems/genetics ; Drug Discovery/*methods/*trends ; Gene Editing ; *Genetic Research ; Genome-Wide Association Study ; },
}
@article {pmid32415193,
year = {2020},
author = {Bhokisham, N and VanArsdale, E and Stephens, KT and Hauk, P and Payne, GF and Bentley, WE},
title = {A redox-based electrogenetic CRISPR system to connect with and control biological information networks.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2427},
pmid = {32415193},
issn = {2041-1723},
support = {R21 EB024102/EB/NIBIB NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Electrochemistry ; Electrodes ; Escherichia coli/metabolism ; Escherichia coli Proteins/metabolism ; Ferricyanides/chemistry ; Gene Expression Regulation, Bacterial ; Genetic Engineering/*methods ; *Oxidation-Reduction ; Oxidative Stress ; Plasmids/metabolism ; Promoter Regions, Genetic ; Pyocyanine/chemistry ; Quorum Sensing ; Regulon ; Salmonella enterica/metabolism ; Spectrometry, Fluorescence ; },
abstract = {Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.},
}
@article {pmid32413572,
year = {2020},
author = {Liu, D and Zhao, X and Tang, A and Xu, X and Liu, S and Zha, L and Ma, W and Zheng, J and Shi, M},
title = {CRISPR screen in mechanism and target discovery for cancer immunotherapy.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1874},
number = {1},
pages = {188378},
doi = {10.1016/j.bbcan.2020.188378},
pmid = {32413572},
issn = {1879-2561},
mesh = {Animals ; Antineoplastic Agents, Immunological/pharmacology/*therapeutic use ; Biomarkers, Tumor/antagonists &amp; inhibitors/*genetics/immunology ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Drug Discovery/*methods ; Drug Resistance, Neoplasm/drug effects/genetics ; Drug Screening Assays, Antitumor/methods ; Gene Expression Regulation, Neoplastic/drug effects/immunology ; Genomics/methods ; Humans ; Molecular Targeted Therapy/methods ; Neoplasms/*drug therapy/genetics/immunology ; T-Lymphocytes/drug effects/immunology ; Tumor Escape/drug effects/genetics ; },
abstract = {CRISPR/Cas-based genetic perturbation screens have emerged as powerful tools for large-scale identification of new targets for cancer immunotherapy. Various strategies of CRISPR screen have been used for immune-oncology (IO) target discovery. The genomic sequences targeted by CRISPR/Cas system range from coding sequences to non-coding RNA/DNA, including miRNAs, LncRNAs, circRNAs, promoters, and enhancers, which may be potential targets for future pharmacological and therapeutic interventions. Rapid progresses have been witnessed in finding novel targets for enhancing tumor antigen presentation, sensitizing of tumor cells to immune-mediated cytotoxicity, and reinvigorating tumor-specific T cells by using CRISPR technologies. In combination with other strategies, the detailed characteristics of the targets for immunotherapy have been obtained by CRISPR screen. In this review, we present an overview of recent progresses in the development of CRISPR-based screens for IO target identification and discuss the challenges and possible solutions in this rapidly growing field.},
}
@article {pmid32413320,
year = {2020},
author = {Bowling, S and Sritharan, D and Osorio, FG and Nguyen, M and Cheung, P and Rodriguez-Fraticelli, A and Patel, S and Yuan, WC and Fujiwara, Y and Li, BE and Orkin, SH and Hormoz, S and Camargo, FD},
title = {An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.},
journal = {Cell},
volume = {181},
number = {6},
pages = {1410-1422.e27},
pmid = {32413320},
issn = {1097-4172},
support = {F31 CA235893/CA/NCI NIH HHS/United States ; R01 HL128850/HL/NHLBI NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; P01 HL131477/HL/NHLBI NIH HHS/United States ; U54 DK110805/DK/NIDDK NIH HHS/United States ; R01 DK123216/DK/NIDDK NIH HHS/United States ; R00 GM118910/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Lineage/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Flow Cytometry/methods ; Hematopoietic Stem Cells/physiology ; Male ; Mice ; Transcriptome/*genetics ; Transduction, Genetic/methods ; },
abstract = {Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.},
}
@article {pmid32413257,
year = {2020},
author = {Boonekamp, FJ and Dashko, S and Duiker, D and Gehrmann, T and van den Broek, M and den Ridder, M and Pabst, M and Robert, V and Abeel, T and Postma, ED and Daran, JM and Daran-Lapujade, P},
title = {Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences.},
journal = {ACS synthetic biology},
volume = {9},
number = {6},
pages = {1361-1375},
pmid = {32413257},
issn = {2161-5063},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; DNA/*chemistry ; Gene Editing ; Glycolysis/genetics ; RNA/*chemistry ; Research Design ; Saccharomyces cerevisiae/genetics/metabolism ; Synthetic Biology/*methods ; },
abstract = {The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavors, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented, and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The 13 genes encoding proteins involved in the major pathway for sugar utilization (i.e., glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance, and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.},
}
@article {pmid32413049,
year = {2020},
author = {Liu, B and Diaz Arguello, OA and Chen, D and Chen, S and Saber, A and Haisma, HJ},
title = {CRISPR-mediated ablation of overexpressed EGFR in combination with sunitinib significantly suppresses renal cell carcinoma proliferation.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0232985},
pmid = {32413049},
issn = {1932-6203},
mesh = {A549 Cells ; Antineoplastic Agents/therapeutic use ; Apoptosis/drug effects/genetics ; *CRISPR-Cas Systems ; Carcinoma, Renal Cell/genetics/pathology/*therapy ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Cisplatin/pharmacology ; Combined Modality Therapy ; Drug Resistance, Neoplasm/genetics ; ErbB Receptors/antagonists &amp; inhibitors/genetics ; Gene Knockout Techniques/*methods ; Genes, erbB-1 ; HEK293 Cells ; HeLa Cells ; Humans ; Kidney Neoplasms/genetics/pathology/*therapy ; MAP Kinase Signaling System/drug effects/genetics ; Models, Biological ; Protein Kinase Inhibitors/therapeutic use ; Sunitinib/*therapeutic use ; },
abstract = {Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma.},
}
@article {pmid32412410,
year = {2020},
author = {Wierson, WA and Welker, JM and Almeida, MP and Mann, CM and Webster, DA and Torrie, ME and Weiss, TJ and Kambakam, S and Vollbrecht, MK and Lan, M and McKeighan, KC and Levey, J and Ming, Z and Wehmeier, A and Mikelson, CS and Haltom, JA and Kwan, KM and Chien, CB and Balciunas, D and Ekker, SC and Clark, KJ and Webber, BR and Moriarity, BS and Solin, SL and Carlson, DF and Dobbs, DL and McGrail, M and Essner, J},
title = {Efficient targeted integration directed by short homology in zebrafish and mammalian cells.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32412410},
issn = {2050-084X},
support = {GM088424/NH/NIH HHS/United States ; R01 EY025780/EY/NEI NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; GM63904/NH/NIH HHS/United States ; R01 GM063904/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblasts/metabolism ; Gene Expression Regulation ; *Gene Knock-In Techniques ; *Genes, Reporter ; Green Fluorescent Proteins/*genetics/metabolism ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; Sequence Homology, Nucleic Acid ; Sus scrofa ; Transcription Activator-Like Effector Nucleases/*genetics/metabolism ; Zebrafish/*genetics ; },
abstract = {Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.},
}
@article {pmid32411384,
year = {2020},
author = {Lin, J and Feng, M and Zhang, H and She, Q},
title = {Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase.},
journal = {Cell discovery},
volume = {6},
number = {},
pages = {29},
pmid = {32411384},
issn = {2056-5968},
abstract = {Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in Escherichia coli. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3' anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct states of ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3' anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match, as in the noncognate target RNA (NTR), to the 5' tag of crRNA. In addition, further increasing the number of 3' anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.},
}
@article {pmid32409581,
year = {2020},
author = {Collins, MP and Stransky, LA and Forgac, M},
title = {AKT Ser/Thr kinase increases V-ATPase-dependent lysosomal acidification in response to amino acid starvation in mammalian cells.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {28},
pages = {9433-9444},
pmid = {32409581},
issn = {1083-351X},
support = {F31 CA189321/CA/NCI NIH HHS/United States ; R01 GM034478/GM/NIGMS NIH HHS/United States ; R37 GM034478/GM/NIGMS NIH HHS/United States ; },
mesh = {AMP-Activated Protein Kinases/metabolism ; Amino Acids ; Animals ; Cyclic AMP-Dependent Protein Kinases/metabolism ; HEK293 Cells ; Heterocyclic Compounds, 3-Ring/pharmacology ; Humans ; Lysosomes/*enzymology ; Mice ; Mice, Knockout ; Proto-Oncogene Proteins c-akt/antagonists &amp; inhibitors/*metabolism ; Vacuolar Proton-Translocating ATPases/*metabolism ; },
abstract = {The vacuolar H[+]-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by the regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.},
}
@article {pmid32409578,
year = {2020},
author = {Morimoto, K and Suzuki, N and Tanida, I and Kakuta, S and Furuta, Y and Uchiyama, Y and Hanada, K and Suzuki, Y and Yamaji, T},
title = {Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {28},
pages = {9490-9501},
pmid = {32409578},
issn = {1083-351X},
mesh = {Animals ; Galactosyltransferases/genetics/metabolism ; Globosides/genetics/*metabolism ; Glycolipids/genetics/*metabolism ; HeLa Cells ; Humans ; Mice ; Receptors, Cell Surface/genetics/*metabolism ; Shiga Toxin/genetics/*metabolism ; },
abstract = {Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.},
}
@article {pmid32408895,
year = {2020},
author = {Thom, CS and Jobaliya, CD and Lorenz, K and Maguire, JA and Gagne, A and Gadue, P and French, DL and Voight, BF},
title = {Tropomyosin 1 genetically constrains in vitro hematopoiesis.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {52},
pmid = {32408895},
issn = {1741-7007},
support = {R56 DK101478/DK/NIDDK NIH HHS/United States ; R01 HL130698/HL/NHLBI NIH HHS/United States ; T32HD043021//National Institute of Child Health and Human Development/International ; R01 DK101478/DK/NIDDK NIH HHS/United States ; R01DK101478/DK/NIDDK NIH HHS/United States ; T32 HD043021/HD/NICHD NIH HHS/United States ; },
mesh = {Blood Platelets/*metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Hematopoiesis/*genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; In Vitro Techniques ; Tropomyosin/deficiency/*metabolism ; },
abstract = {BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.<br><br>RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.<br><br>CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.},
}
@article {pmid32408568,
year = {2020},
author = {Pan, M and Nethery, MA and Hidalgo-Cantabrana, C and Barrangou, R},
title = {Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium.},
journal = {Microorganisms},
volume = {8},
number = {5},
pages = {},
pmid = {32408568},
issn = {2076-2607},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.},
}
@article {pmid32408486,
year = {2020},
author = {Dai, Y and Hu, G and Dupas, A and Medina, L and Blandels, N and Clemente, HS and Ladouce, N and Badawi, M and Hernandez-Raquet, G and Mounet, F and Grima-Pettenati, J and Cassan-Wang, H},
title = {Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.},
journal = {International journal of molecular sciences},
volume = {21},
number = {10},
pages = {},
pmid = {32408486},
issn = {1422-0067},
mesh = {Aldehyde Oxidoreductases/genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Eucalyptus/*genetics/metabolism ; Gene Editing/*methods ; Genes, Plant/*genetics ; Lignin/biosynthesis/genetics ; Multivariate Analysis ; Mutation ; Plant Roots/*genetics/metabolism ; Plants, Genetically Modified ; Spectroscopy, Fourier Transform Infrared ; Transcription Factors/genetics/metabolism ; Wood/*genetics/metabolism ; },
abstract = {Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.},
}
@article {pmid32408049,
year = {2020},
author = {Ramongolalaina, C},
title = {Dual-luciferase assay and siRNA silencing for nodD1 to study the competitiveness of Bradyrhizobium diazoefficiens USDA110 in soybean nodulation.},
journal = {Microbiological research},
volume = {237},
number = {},
pages = {126488},
doi = {10.1016/j.micres.2020.126488},
pmid = {32408049},
issn = {1618-0623},
mesh = {Bacterial Proteins/*genetics ; Bradyrhizobium/*genetics ; CRISPR-Cas Systems ; Fluorescent Dyes/analysis ; Genes, Bacterial ; Luciferases, Renilla ; Nitrogen Fixation/genetics ; Plant Development ; *Plant Root Nodulation ; Plant Roots/microbiology ; RNA, Small Interfering ; Soil Microbiology ; Soybeans/*microbiology ; Symbiosis ; Transformation, Bacterial ; },
abstract = {The symbiosis of soybean with Bradyrhizobium diazoefficiens USDA110, which always competes with other rhizobia in the field, is of great agronomic and environmental importance. Herein, a dual-luciferase reporter assay was utilized to monitor the dynamics of two dominant bradyrhizobia infecting roots of soybean. More explicitly, luciferase-tagged B. diazoefficiens USDA110 (USDA110-FLuc) and Bradyrhizobium elkanii USDA 94 (USDA94-RLuc) were designed, co-inoculated into soybean seeds, and observed for their colonization in root nodules by bioluminescence imaging. The results showed that USDA110-FLuc initiated infection earlier than USDA94-RLuc, but its occupancy in the nodules decreased as the plant grew. A nodulation test showed that nodD1 mutant USDA110 strains, including CRISPR engineered mutants, were less competitive than wild type. I constructed siRNAs to knockdown nodD1 at different target sites and transformed them into the bacteria. Surprisingly, although siRNAs - with 3' end target sites - were able to repress up to 65% of nodD1 expression, the profiling of total RNAs with a bioanalyzer revealed that 23S/16S-rRNA ratios of siRNA-transformed and wild type USDA110 strains were similar, but lower than that of nodD1 mutant. In short, the current work - while reporting the competitiveness of B. diazoefficiens USDA110 in early occupancy of soybean nodules and the gene nodD1 as a key determinant of this infection - gives an insight on siRNA silencing in microbes, and demonstrates a highly efficient imaging approach that could entail many new avenues for many biological research fields.},
}
@article {pmid32406907,
year = {2020},
author = {Fueller, J and Herbst, K and Meurer, M and Gubicza, K and Kurtulmus, B and Knopf, JD and Kirrmaier, D and Buchmuller, BC and Pereira, G and Lemberg, MK and Knop, M},
title = {CRISPR-Cas12a-assisted PCR tagging of mammalian genes.},
journal = {The Journal of cell biology},
volume = {219},
number = {6},
pages = {},
pmid = {32406907},
issn = {1540-8140},
mesh = {Alleles ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endodeoxyribonucleases/genetics/metabolism ; Gene Targeting/*methods ; Genes, Reporter ; High-Throughput Nucleotide Sequencing ; Homologous Recombination ; Humans ; Luminescent Proteins/genetics/metabolism ; Oligonucleotides/genetics ; Polymerase Chain Reaction/*methods ; RNA, Guide/genetics ; Transfection ; },
abstract = {Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.},
}
@article {pmid32406632,
year = {2020},
author = {Burbery, R},
title = {Embryonic Regulation and Research: What Is the Status of Human Germline Genome Editing in Australia?.},
journal = {Journal of law and medicine},
volume = {27},
number = {3},
pages = {718-740},
pmid = {32406632},
issn = {1320-159X},
mesh = {Australia ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Germ Cells ; Humans ; },
abstract = {This article considers the status of human germline genome editing in Australia. Through an analysis of the current State, Territory and Commonwealth legal and ethical regulatory frameworks, and incorporating the capability approach to health justice proposed by Martha Nussbaum and Amartya Sen, the article argues that heritable alterations using CRISPR/Cas9 technology in a clinical context are inevitable in Australia, so the law needs to respond adequately to these scientific advances. The article concludes that human germline genome editing is currently not, and should not, be lawful in Australia, except for research purposes.},
}
@article {pmid32406490,
year = {2020},
author = {Demirci, S and Leonard, A and Tisdale, JF},
title = {Genome editing strategies for fetal hemoglobin induction in beta-hemoglobinopathies.},
journal = {Human molecular genetics},
volume = {29},
number = {R1},
pages = {R100-R106},
pmid = {32406490},
issn = {1460-2083},
mesh = {*CRISPR-Cas Systems ; Fetal Hemoglobin/*metabolism ; *Gene Editing ; *Genetic Therapy ; Hemoglobinopathies/genetics/*therapy ; Humans ; beta-Globins/antagonists &amp; inhibitors/*genetics ; },
abstract = {Genome editing to correct a defective β-globin gene or induce fetal globin (HbF) for patients with beta-hemoglobinopathies has the potential to be a curative strategy available to all. HbF reactivation has long been an area of intense interest given the HbF inhibition of sickle hemoglobin (HbS) polymerization. Patients with HbS who also have high HbF tend to have less severe or even minimal clinical manifestations. Approaches to genetically engineer high HbF include de novo generation of naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations, editing of transcriptional HbF repressors or their binding sites and/or regulating epigenetic intermediates controlling HbF expression. Recent preclinical and early clinical trial data show encouraging results; however, long-term follow-up is lacking, and the safety and efficacy concerns of genome editing remain.},
}
@article {pmid32405769,
year = {2020},
author = {Biswas, S and Li, R and Hong, J and Zhao, X and Yuan, Z and Zhang, D and Shi, J},
title = {Effective identification of CRISPR/Cas9-induced and naturally occurred mutations in rice using a multiplex ligation-dependent probe amplification-based method.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {8},
pages = {2323-2334},
doi = {10.1007/s00122-020-03600-5},
pmid = {32405769},
issn = {1432-2242},
mesh = {*CRISPR-Cas Systems ; Electrophoresis, Capillary ; Gene Editing/*methods ; Genetic Variation ; Genome, Plant ; INDEL Mutation ; Multiplex Polymerase Chain Reaction/*methods ; *Mutagenesis ; Mutation ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; Sensitivity and Specificity ; },
abstract = {A multiplex ligation-dependent probe amplification (MLPA)-based method was developed and successfully utilized to efficiently detect both CRISPR/Cas9-induced and naturally occurred mutations in rice. The site-specific nuclease-based CRISPR/Cas9 system has emerged as one of the most efficient genome editing tools to modify multiple genomic targets simultaneously in various organisms, including plants for both fundamental and applied researches. Screening for both on-target and off-target mutations in CRISPR/Cas9-generated mutants at the early stages is an indispensable step for functional analysis and subsequent application. Various methods have been developed to detect CRISPR/Cas9-induced mutations in plants. Still, very few have focused on the detection of both on- and off-targets simultaneously, let alone the detection of natural mutations. Here, we report a multiplex capable method that allows to detect CRISPR/Cas9 induced on- and off-target mutations as well as naturally occurred mutation based on a multiplex ligation-dependent probe amplification (MLPA) method. We demonstrated that unlike other methods, the modified target-specific MLPA method can accurately identify any INDELs generated naturally or by the CRISPR/Cas9 system and that it can detect natural variation and zygosity of the CRISPR/Cas9-generated mutants in rice as well. Furthermore, its high sensitivity allowed to define INDELs down to 1 bp and substitutions to a single nucleotide. Therefore, this sensitive, reliable, and cheap method would further accelerate functional analysis and marker-assisted breeding in plants, including rice.},
}
@article {pmid32404985,
year = {2020},
author = {Tsai, CN and Yu, SC and Lee, CW and Pang, JS and Wu, CH and Lin, SE and Chung, YH and Tsai, CL and Hsieh, SY and Yu, MC},
title = {SOX4 activates CXCL12 in hepatocellular carcinoma cells to modulate endothelial cell migration and angiogenesis in vivo.},
journal = {Oncogene},
volume = {39},
number = {24},
pages = {4695-4710},
pmid = {32404985},
issn = {1476-5594},
mesh = {Animals ; CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/blood supply/genetics/metabolism/pathology ; Cell Line, Tumor ; *Cell Movement ; Chemokine CXCL12/genetics/*metabolism ; *Human Umbilical Vein Endothelial Cells/metabolism/pathology ; Humans ; *Liver Neoplasms, Experimental/blood supply/genetics/metabolism/pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; *Neoplasm Proteins/genetics/metabolism ; *Neovascularization, Pathologic/genetics/metabolism/pathology ; SOXC Transcription Factors/genetics/*metabolism ; *Thrombosis/genetics/metabolism/pathology ; Tumor Microenvironment/genetics ; },
abstract = {The overexpression of SOX4 in various kinds of cancer cells was associated with poor prognosis for patients. The role of SOX4 in angiogenesis and tumor microenvironment modulation was recently documented in breast cancer but remains unclear in hepatocellular carcinoma (HCC). In our study, the clinical relevance of SOX4 overexpression in HCC and its role in the tumor microenvironment were investigated. The overexpression of SOX4 (SOX4[high]) in tumor lesions was associated with higher microvessel density (P = 0.012), tumor thrombosis formation (P = 0.012), distant metastasis (P < 0.001), and an independent prognostic factor for disease-free survival in HCC patients (P = 0.048). Endogenous SOX4 knockout in Hep3B cells by the CRISPR/cas9 system reduced the expression of CXCL12, which, in turn, attenuated chemotaxis in human umbilical vein endothelial cells, tube formation in vitro, reduced tumor growth, reticular fiber production, and angiogenesis in vivo in a xenograft mouse model. Treatment with an antagonist targeting CXCR4 (AMD3100), a receptor of CXCL12, inhibited chemotaxis and tube formation in endothelial cells in vitro. The CXCL12 promoter was activated by ectopic expression of a Flag-tagged SOX4 plasmid, endogenous SOX4 knockdown abolished promoter activity of CXCL12 as shown by luciferase assays, and an association with the CXCL12 promoter was identified via chromatin immunoprecipitation in HCC cells. In conclusion, SOX4 modulates the CXCL12 promoter in HCC cells. The secretory CXCL12, in turn, modulates CXCR4 in endothelial cells, reticular fibers to regulate the tumor microenvironment and modulate neovascularization, which might contribute to the distant metastasis of tumors.},
}
@article {pmid32404434,
year = {2020},
author = {Kumar, N and Sharma, S and Kumar, R and Tripathi, BN and Barua, S and Ly, H and Rouse, BT},
title = {Host-Directed Antiviral Therapy.},
journal = {Clinical microbiology reviews},
volume = {33},
number = {3},
pages = {},
pmid = {32404434},
issn = {1098-6618},
mesh = {Animals ; *CRISPR-Cas Systems ; *Drug Development ; Gene Targeting ; Host-Derived Cellular Factors/*antagonists &amp; inhibitors/genetics ; Host-Pathogen Interactions/genetics ; Humans ; Mice ; *RNA, Small Interfering ; Virus Replication/*genetics ; Viruses/genetics ; },
abstract = {Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.},
}
@article {pmid32403926,
year = {2020},
author = {Modell, AE and Siriwardena, SU and Choudhary, A},
title = {A Jumbo Phage Forms a Nucleus-like Compartment to Evade Bacterial Defense Systems.},
journal = {Biochemistry},
volume = {59},
number = {20},
pages = {1869-1870},
doi = {10.1021/acs.biochem.0c00273},
pmid = {32403926},
issn = {1520-4995},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Viral ; Immune System ; },
}
@article {pmid32402659,
year = {2020},
author = {Vangheluwe, N and Swinnen, G and de Koning, R and Meyer, P and Houben, M and Huybrechts, M and Sajeev, N and Rienstra, J and Boer, D},
title = {Give CRISPR a Chance: the GeneSprout Initiative.},
journal = {Trends in plant science},
volume = {25},
number = {7},
pages = {624-627},
doi = {10.1016/j.tplants.2020.04.011},
pmid = {32402659},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Plant Breeding ; Plants/genetics ; },
abstract = {Did you know that a group of early-career researchers launched an initiative enabling open dialog on new plant breeding techniques, such as genome editing? We developed a wide-ranging initiative that aims to facilitate public engagement and provide a platform for young plant scientists to encourage participation in science communication.},
}
@article {pmid32402073,
year = {2020},
author = {Yi, H and Huang, L and Yang, B and Gomez, J and Zhang, H and Yin, Y},
title = {AcrFinder: genome mining anti-CRISPR operons in prokaryotes and their viruses.},
journal = {Nucleic acids research},
volume = {48},
number = {W1},
pages = {W358-W365},
pmid = {32402073},
issn = {1362-4962},
mesh = {Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Databases, Genetic ; Genome, Archaeal ; Genome, Bacterial ; Genomics/methods ; *Operon ; *Software ; Viral Proteins/*genetics ; },
abstract = {Anti-CRISPR (Acr) proteins encoded by (pro)phages/(pro)viruses have a great potential to enable a more controllable genome editing. However, genome mining new Acr proteins is challenging due to the lack of a conserved functional domain and the low sequence similarity among experimentally characterized Acr proteins. We introduce here AcrFinder, a web server (http://bcb.unl.edu/AcrFinder) that combines three well-accepted ideas used by previous experimental studies to pre-screen genomic data for Acr candidates. These ideas include homology search, guilt-by-association (GBA), and CRISPR-Cas self-targeting spacers. Compared to existing bioinformatics tools, AcrFinder has the following unique functions: (i) it is the first online server specifically mining genomes for Acr-Aca operons; (ii) it provides a most comprehensive Acr and Aca (Acr-associated regulator) database (populated by GBA-based Acr and Aca datasets); (iii) it combines homology-based, GBA-based, and self-targeting approaches in one software package; and (iv) it provides a user-friendly web interface to take both nucleotide and protein sequence files as inputs, and output a result page with graphic representation of the genomic contexts of Acr-Aca operons. The leave-one-out cross-validation on experimentally characterized Acr-Aca operons showed that AcrFinder had a 100% recall. AcrFinder will be a valuable web resource to help experimental microbiologists discover new Anti-CRISPRs.},
}
@article {pmid32401787,
year = {2020},
author = {Picariello, T and Hou, Y and Kubo, T and McNeill, NA and Yanagisawa, HA and Oda, T and Witman, GB},
title = {TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0232594},
pmid = {32401787},
issn = {1932-6203},
support = {R35 GM122574/GM/NIGMS NIH HHS/United States ; R37 GM030626/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics ; DNA/genetics ; Gene Editing/*methods ; Mutagenesis, Insertional/*methods ; RNA, Guide/genetics ; },
abstract = {Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.},
}
@article {pmid32401601,
year = {2020},
author = {Eykelenboom, JK and Tanaka, TU},
title = {Zooming in on chromosome dynamics.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {19},
number = {12},
pages = {1422-1432},
pmid = {32401601},
issn = {1551-4005},
support = {/WT_/Wellcome Trust/United Kingdom ; 322682/ERC_/European Research Council/International ; 096535/Z/11/Z/WT_/Wellcome Trust/United Kingdom ; BB/S007768/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Survival ; Chromosomes/*metabolism ; Genetic Loci ; Humans ; },
abstract = {Until recently, our understanding of chromosome organization in higher eukaryotic cells has been based on analyses of large-scale, low-resolution changes in chromosomes structure. More recently, CRISPR-Cas9 technologies have allowed us to "zoom in" and visualize specific chromosome regions in live cells so that we can begin to examine in detail the dynamics of chromosome organization in individual cells. In this review, we discuss traditional methods of chromosome locus visualization and look at how CRISPR-Cas9 gene-targeting methodologies have helped improve their application. We also describe recent developments of the CRISPR-Cas9 technology that enable visualization of specific chromosome regions without the requirement for complex genetic manipulation.},
}
@article {pmid32401488,
year = {2020},
author = {Cencic, R and Naineni, SK and Pugsley, L and Senéchal, P and Sahni, A and Pelletier, J},
title = {CRISPR-Based Screen Links an Inhibitor of Nonsense-Mediated Decay to eIF4A3 Target Engagement.},
journal = {ACS chemical biology},
volume = {15},
number = {6},
pages = {1621-1629},
doi = {10.1021/acschembio.0c00253},
pmid = {32401488},
issn = {1554-8937},
support = {FDN-148366//CIHR/Canada ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; DEAD-box RNA Helicases/genetics/*metabolism ; Eukaryotic Initiation Factor-4A/genetics/*metabolism ; Humans ; Mutation ; *Nonsense Mediated mRNA Decay ; },
abstract = {Eukaryotic initiation factor (eIF) 4A3 is a DEAD-box RNA helicase and a core component of the exon-junction complex (EJC). The EJC marks the location of exon:exon junctions following the removal of introns by splicing and plays a critical role in an mRNA surveillance program known as nonsense-mediated decay (NMD). NMD is often triggered by the presence of a premature termination codon (PTC) upstream of the EJC, leading to degradation of the variant mRNA which prevents synthesis of a potentially harmful, truncated polypeptide. One approach by which to treat rare diseases where the underlying cause is a PTC is thus to prevent NMD, while stimulating readthrough of the PTC. Hence, there is much interest in inhibiting NMD, and recently a set of small molecules, 1,4-diacylpiperazine derivatives, targeting eIF4A3 has been developed and shown to harbor such activity. Herein, we undertake a CRISPR/Cas9-based variomics screen to identify eIF4A3 alleles resistant to said compounds. Our results provide genetic evidence linking compound bioactivity to eIF4A3 engagement.},
}
@article {pmid32401064,
year = {2020},
author = {Xiang, X and Qian, K and Zhang, Z and Lin, F and Xie, Y and Liu, Y and Yang, Z},
title = {CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia.},
journal = {Journal of drug targeting},
volume = {28},
number = {7-8},
pages = {727-731},
pmid = {32401064},
issn = {1029-2330},
mesh = {COVID-19 ; COVID-19 Testing ; *CRISPR-Cas Systems ; Clinical Laboratory Techniques ; Coronavirus Infections/*diagnosis ; Humans ; *Molecular Diagnostic Techniques ; Pandemics ; Pneumonia, Viral/*diagnosis/virology ; },
abstract = {Emerging infectious diseases, the persistent potential for destabilising pandemics, remain a global threat leading to excessive morbidity and mortality. The current outbreak of pneumonia caused by 2019 novel coronavirus (COVID-19) illustrated difficulties in lack of effective drugs for treatment. Accurate and rapid diagnostic tools are essential for early recognition and treatment of infectious diseases, allowing timely implementation of infection control, improved clinical care and other public health measures to stop the spread of the disease. CRISPR-Cas technology speed up the development of infectious disease diagnostics with high rapid and accurate. In this review, we summarise current advance regarding diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13, in the development of fast, accurate and portable diagnostic tests and highlight the potential of CRISPR-Cas13 in COVID-19 Pneumonia and other emerging infectious diseases diagnosis.},
}
@article {pmid32400332,
year = {2020},
author = {Ghaffari, M and Sanadgol, N and Abdollahi, M},
title = {A Systematic Review of Current Progresses in the Nucleic Acid-Based Therapies for Neurodegeneration with Implications for Alzheimer's Disease.},
journal = {Mini reviews in medicinal chemistry},
volume = {20},
number = {15},
pages = {1499-1517},
doi = {10.2174/1389557520666200513122357},
pmid = {32400332},
issn = {1875-5607},
support = {UOZ-GR-9618-5//University of Zabol/International ; },
mesh = {Alzheimer Disease/*drug therapy/pathology ; Amyloid beta-Peptides/immunology/metabolism ; Antagomirs/therapeutic use ; Aptamers, Nucleotide/chemistry/metabolism/therapeutic use ; CRISPR-Cas Systems/genetics ; Humans ; MicroRNAs/antagonists &amp; inhibitors/metabolism ; Nucleic Acids/*therapeutic use ; Oligonucleotides, Antisense/therapeutic use ; RNA, Small Interfering/therapeutic use ; },
abstract = {Recently, manipulation of gene expression and switching genes on or off highlight the potential of nucleic acid-based therapies (NA-BTs). Alzheimer's Disease (AD) is a common devastating neurodegenerative disease (NDs) responsible for 60-80% of all cases of dementia and predicted as a main public health concern among aged populations. The aim of this study was to outline the current research in the field of NA-BTs for the treatment of AD disabilities, including strategies to suppress the memory and learning defects, to promote recovery processes, and to reinforce social relationships in these patients. This review was performed via evaluating PubMed reported studies from January 2010 to November 2019. Also, reference lists were checked to find additional studies. All intermediation or complementarity of animal models, case-control and cohort studies, and controlled trials (CTs) on specific NA-BTs to AD were acceptable, although in vitro studies were excluded due to the considerable diversities and heterogeneities. After removing the duplicates according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) instruction, we merged remaining titles across search databases. There are 48 ongoing studies related to the application of nucleic acids in the treatment and diagnosis of AD where more consideration is given to DNA targeting strategies (18 targets for vectors and aptamers), antisense oligonucleotides (10 targets), micro-RNAs mimics (7 targets), antagomiRs (6 targets), small interferences-RNAs (5 targets), as well as mRNAs (2 targets) respectively. All of these targets are grouped into 4 categories according to their role in molecular pathways where amyloid-β (18 targets), neural survival (11 targets), memory and cognition (8 targets), and tau (3 targets) are more targeted pathways, respectively. With recent successes in the systemic delivery of nucleic acids via intravenous injection; it is worth investing in the production of new-generation medicines. There are still several challenges for NA-BTs including, their delivery to the effective modulators, mass production at low cost, sustaining efficacy and minimizing off-target effects. Regarding miRNA-based therapies, given the obvious involvement of miRNAs in numerous facets of brain disease, and the many sophisticated techniques for delivery to the brain, miRNA-based therapies will make new hope for the treatment of neurological diseases such as AD.},
}
@article {pmid32400327,
year = {2020},
author = {Rösner, J and Wellmeyer, B and Merzendorfer, H},
title = {Tribolium castaneum: A Model for Investigating the Mode of Action of Insecticides and Mechanisms of Resistance.},
journal = {Current pharmaceutical design},
volume = {26},
number = {29},
pages = {3554-3568},
doi = {10.2174/1381612826666200513113140},
pmid = {32400327},
issn = {1873-4286},
mesh = {Animals ; Base Sequence ; *Insecticides/pharmacology ; RNA Interference ; *Tribolium/genetics ; },
abstract = {The red flour beetle, Tribolium castaneum, is a worldwide insect pest of stored products, particularly food grains, and a powerful model organism for developmental, physiological and applied entomological research on coleopteran species. Among coleopterans, T. castaneum has the most fully sequenced and annotated genome and consequently provides the most advanced genetic model of a coleopteran pest. The beetle is also easy to culture and has a short generation time. Research on this beetle is further assisted by the availability of expressed sequence tags and transcriptomic data. Most importantly, it exhibits a very robust response to systemic RNA interference (RNAi), and a database of RNAi phenotypes (iBeetle) is available. Finally, classical transposonbased techniques together with CRISPR/Cas-mediated gene knockout and genome editing allow the creation of transgenic lines. As T. castaneum develops resistance rapidly to many classes of insecticides including organophosphates, methyl carbamates, pyrethroids, neonicotinoids and insect growth regulators such as chitin synthesis inhibitors, it is further a suitable test system for studying resistance mechanisms. In this review, we will summarize recent advances in research focusing on the mode of action of insecticides and mechanisms of resistance identified using T. castaneum as a pest model.},
}
@article {pmid32398353,
year = {2020},
author = {Cotterell, J and Vila-Cejudo, M and Batlle-Morera, L and Sharpe, J},
title = {Endogenous CRISPR/Cas9 arrays for scalable whole-organism lineage tracing.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {9},
pages = {},
doi = {10.1242/dev.184481},
pmid = {32398353},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genetic Engineering ; Mice ; Mutation/genetics ; Phylogeny ; Zebrafish ; },
abstract = {The past decade has seen a renewed appreciation of the central importance of cellular lineages to many questions in biology (especially organogenesis, stem cells and tumor biology). This has been driven in part by a renaissance in genetic clonal-labeling techniques. Recent approaches are based on accelerated mutation of DNA sequences, which can then be sequenced from individual cells to re-create a 'phylogenetic' tree of cell lineage. However, current approaches depend on making transgenic alterations to the genome in question, which limit their application. Here, we introduce a new method that completely avoids the need for prior genetic engineering, by identifying endogenous CRISPR/Cas9 target arrays suitable for lineage analysis. In both mouse and zebrafish, we identify the highest quality compact arrays as judged by equal base composition, 5' G sequence, minimal likelihood of residing in the functional genome, minimal off targets and ease of amplification. We validate multiple high-quality endogenous CRISPR/Cas9 arrays, demonstrating their utility for lineage tracing. Our pragmatically scalable technique thus can produce deep and broad lineages in vivo, while removing the dependence on genetic engineering.},
}
@article {pmid32398038,
year = {2020},
author = {Bánfalvi, Z and Csákvári, E and Villányi, V and Kondrák, M},
title = {Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {25},
pmid = {32398038},
issn = {1472-6750},
support = {K-120641//NKFI/International ; },
mesh = {Agrobacterium/*genetics ; Base Sequence ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial ; Gene Editing/*methods ; Gene Transfer Techniques ; Genes, Plant ; Genome, Plant ; Mutation ; Oxidoreductases/*genetics ; Phenotype ; Plant Leaves ; Plants, Genetically Modified/*genetics ; Sequence Analysis, DNA ; Solanum tuberosum/*genetics ; *Transgenes ; },
abstract = {BACKGROUND: Gene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible.<br><br>RESULTS: In this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right- and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2-10%. Interestingly, while large deletions (>&#x2009;100&#x2009;bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato.<br><br>CONCLUSIONS: Gene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.},
}
@article {pmid32397339,
year = {2020},
author = {Wang, L and Lim, CK and Klotz, MG},
title = {High Synteny and Sequence Identity between Genomes of Nitrosococcus oceani Strains Isolated from Different Oceanic Gyres Reveals Genome Economization and Autochthonous Clonal Evolution.},
journal = {Microorganisms},
volume = {8},
number = {5},
pages = {},
pmid = {32397339},
issn = {2076-2607},
abstract = {The ammonia-oxidizing obligate aerobic chemolithoautotrophic gammaproteobacterium, Nitrosococcus oceani, is omnipresent in the world's oceans and as such important to the global nitrogen cycle. We generated and compared high quality draft genome sequences of N. oceani strains isolated from the Northeast (AFC27) and Southeast (AFC132) Pacific Ocean and the coastal waters near Barbados at the interface between the Caribbean Sea and the North Atlantic Ocean (C-27) with the recently published Draft Genome Sequence of N. oceani Strain NS58 (West Pacific Ocean) and the complete genome sequence of N. oceani C-107, the type strain (ATCC 19707) isolated from the open North Atlantic, with the goal to identify indicators for the evolutionary origin of the species. The genomes of strains C-107, NS58, C-27, and AFC27 were highly conserved in content and synteny, and these four genomes contained one nearly sequence-identical plasmid. The genome of strain AFC132 revealed the presence of genetic inventory unknown from other marine ammonia-oxidizing bacteria such as genes encoding NiFe-hydrogenase and a non-ribosomal peptide synthetase (NRPS)-like siderophore biosynthesis module. Comparative genome analysis in context with the literature suggests that AFC132 represents a metabolically more diverse ancestral lineage to the other strains with C-107 and NS58 potentially being the youngest. The results suggest that the N. oceani species evolved by genome economization characterized by the loss of genes encoding catabolic diversity while acquiring a higher redundancy in inventory dedicated to nitrogen catabolism, both of which could have been facilitated by their rich complements of CRISPR/Cas and Restriction Modification systems.},
}
@article {pmid32397082,
year = {2020},
author = {Zhao, Y and Li, G and Chen, Y and Lu, Y},
title = {Challenges and Advances in Genome Editing Technologies in Streptomyces.},
journal = {Biomolecules},
volume = {10},
number = {5},
pages = {},
pmid = {32397082},
issn = {2218-273X},
support = {31970083 and 31400083//National Natural Science Foundation of China/International ; 2019YFA0905400//the National Key R&amp;D Program/International ; 2018ZX09711001-006-012//the National Mega-project for Innovative Drugs/International ; NY2018015//the Key R&amp;D Plan of Zhenjiang City/International ; NY2017001//the Key R&amp;D Plan of Dantu District/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods/*trends ; Integrases/metabolism ; Recombination, Genetic/genetics ; Streptomyces/*genetics ; },
abstract = {The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.},
}
@article {pmid32396992,
year = {2020},
author = {Baerwald, MR and Goodbla, AM and Nagarajan, RP and Gootenberg, JS and Abudayyeh, OO and Zhang, F and Schreier, AD},
title = {Rapid and accurate species identification for ecological studies and monitoring using CRISPR-based SHERLOCK.},
journal = {Molecular ecology resources},
volume = {20},
number = {4},
pages = {961-970},
pmid = {32396992},
issn = {1755-0998},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; Ecology ; Fishes/*genetics ; San Francisco ; },
abstract = {One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.},
}
@article {pmid32396228,
year = {2020},
author = {Wu, F and Qiao, X and Zhao, Y and Zhang, Z and Gao, Y and Shi, L and Du, H and Wang, L and Zhang, YJ and Zhang, Y and Liu, L and Wang, Q and Kong, D},
title = {Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Cas12b/C2c1.},
journal = {Journal of integrative plant biology},
volume = {62},
number = {11},
pages = {1653-1658},
doi = {10.1111/jipb.12944},
pmid = {32396228},
issn = {1744-7909},
mesh = {Arabidopsis/genetics/*metabolism ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genome, Plant/*genetics ; Mutagenesis ; Mutation/genetics ; },
abstract = {Cas12b/C2c1 is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice. To explore the potential applications of the CRISPR-Cas12b system in the dicot Arabidopsis thaliana, we selected BvCas12b and BhCas12b v4 for analysis. We successfully used both endonucleases to induce mutations, perform multiplex genome editing, and create large deletions at multiple loci. No significant mutations were detected at potential off-target sites. Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPR-Cas12b systems for genome editing in Arabidopsis.},
}
@article {pmid32395634,
year = {2020},
author = {Pirona, AC and Oktriani, R and Boettcher, M and Hoheisel, JD},
title = {Process for an efficient lentiviral cell transduction.},
journal = {Biology methods &amp; protocols},
volume = {5},
number = {1},
pages = {bpaa005},
pmid = {32395634},
issn = {2396-8923},
abstract = {The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation. Here, we present a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures.},
}
@article {pmid32395632,
year = {2020},
author = {Culp, E and Richman, C and Sharanya, D and Jhaveri, N and van den Berg, W and Gupta, BP},
title = {Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system.},
journal = {Biology methods &amp; protocols},
volume = {5},
number = {1},
pages = {bpaa003},
pmid = {32395632},
issn = {2396-8923},
abstract = {The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.},
}
@article {pmid32394708,
year = {2020},
author = {Xie, T and Chen, X and Guo, T and Rong, H and Chen, Z and Sun, Q and Batley, J and Jiang, J and Wang, Y},
title = {Targeted Knockout of BnTT2 Homologues for Yellow-Seeded Brassica napus with Reduced Flavonoids and Improved Fatty Acid Composition.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {20},
pages = {5676-5690},
doi = {10.1021/acs.jafc.0c01126},
pmid = {32394708},
issn = {1520-5118},
mesh = {Brassica napus/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; Color ; Fatty Acids/*chemistry/metabolism ; Flavonoids/*chemistry/metabolism ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified/chemistry/genetics/metabolism ; Seeds/chemistry/genetics/*metabolism ; },
abstract = {Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the BnTT2 mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.},
}
@article {pmid32393904,
year = {2020},
author = {Lin, Q and Zong, Y and Xue, C and Wang, S and Jin, S and Zhu, Z and Wang, Y and Anzalone, AV and Raguram, A and Doman, JL and Liu, DR and Gao, C},
title = {Prime genome editing in rice and wheat.},
journal = {Nature biotechnology},
volume = {38},
number = {5},
pages = {582-585},
pmid = {32393904},
issn = {1546-1696},
support = {U01 AI142756/AI/NIAID NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; Gene Editing/*methods ; Genome, Plant ; Oryza/genetics/*growth &amp; development ; Triticum/genetics/*growth &amp; development ; },
abstract = {Prime editors, which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.},
}
@article {pmid32393889,
year = {2020},
author = {Zhang, X and Chen, L and Zhu, B and Wang, L and Chen, C and Hong, M and Huang, Y and Li, H and Han, H and Cai, B and Yu, W and Yin, S and Yang, L and Yang, Z and Liu, M and Zhang, Y and Mao, Z and Wu, Y and Liu, M and Li, D},
title = {Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain.},
journal = {Nature cell biology},
volume = {22},
number = {6},
pages = {740-750},
pmid = {32393889},
issn = {1476-4679},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Differentiation ; Cytidine/chemistry/*genetics ; DNA-Binding Proteins/genetics/*metabolism ; Embryo, Mammalian/cytology/metabolism ; Female ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; *Mutation ; Protein Domains ; Rad51 Recombinase/genetics/*metabolism ; },
abstract = {Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.},
}
@article {pmid32393885,
year = {2020},
author = {Katti, A and Dow, LE},
title = {Base editing goes into hyperdrive.},
journal = {Nature cell biology},
volume = {22},
number = {6},
pages = {617-618},
pmid = {32393885},
issn = {1476-4679},
support = {F31 CA247351/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cytidine ; *DNA-Binding Proteins ; Gene Editing ; },
}
@article {pmid32393878,
year = {2020},
author = {Xiao, Y and Offringa, R},
title = {PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX.},
journal = {Nature plants},
volume = {6},
number = {5},
pages = {544-555},
pmid = {32393878},
issn = {2055-0278},
mesh = {3-Phosphoinositide-Dependent Protein Kinases/metabolism/*physiology ; Arabidopsis/*growth &amp; development/physiology ; Arabidopsis Proteins/metabolism/*physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Indoleacetic Acids/*metabolism ; Phloem/*growth &amp; development ; Phosphorylation ; Plant Growth Regulators/*metabolism ; },
abstract = {The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a conserved master regulator of AGC kinases in eukaryotic organisms. pdk1 loss of function causes a lethal phenotype in animals and yeasts, but only mild phenotypic defects in Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1-encoding genes, PDK1 and PDK2. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate true loss-of-function pdk1 alleles, which, when combined with pdk2 alleles, showed severe developmental defects including fused cotyledons, a short primary root, dwarf stature and defects in male fertility. We obtained evidence that PDK1 is responsible for AGC1 kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX) activation by phosphorylation during vascular development, and that the PDK1 phospholipid-binding Pleckstrin Homology domain is not required for this process. Our data indicate that PDK1 regulates polar auxin transport by activating AGC1 clade kinases, resulting in PIN phosphorylation.},
}
@article {pmid32393832,
year = {2020},
author = {Wheeler, EC and Vu, AQ and Einstein, JM and DiSalvo, M and Ahmed, N and Van Nostrand, EL and Shishkin, AA and Jin, W and Allbritton, NL and Yeo, GW},
title = {Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors.},
journal = {Nature methods},
volume = {17},
number = {6},
pages = {636-642},
pmid = {32393832},
issn = {1548-7105},
support = {F31 CA206233/CA/NCI NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; P50 GM085764/GM/NIGMS NIH HHS/United States ; F31 CA217173/CA/NCI NIH HHS/United States ; R01 EY024556/EY/NEI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; T32 GM008666/GM/NIGMS NIH HHS/United States ; K99 HG009530/HG/NHGRI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytoplasm/metabolism ; Humans ; Machine Learning ; Microscopy, Confocal/*methods ; Oxidative Stress/*genetics ; Protein Aggregates/genetics ; RNA, Guide/*genetics ; RNA-Binding Proteins/*genetics ; Tissue Array Analysis/*methods ; },
abstract = {Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR-Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein-RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.},
}
@article {pmid32393822,
year = {2020},
author = {Chatterjee, P and Jakimo, N and Lee, J and Amrani, N and Rodríguez, T and Koseki, SRT and Tysinger, E and Qing, R and Hao, S and Sontheimer, EJ and Jacobson, J},
title = {An engineered ScCas9 with broad PAM range and high specificity and activity.},
journal = {Nature biotechnology},
volume = {38},
number = {10},
pages = {1154-1158},
pmid = {32393822},
issn = {1546-1696},
support = {R01 GM115911/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Motifs/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Cleavage ; Gene Editing/methods ; Streptococcus/genetics ; },
abstract = {CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9-Sc[++] and a higher-fidelity mutant HiFi-Sc[++]-that have simultaneously broad 5'-NNG-3' PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc[++] and HiFi-Sc[++] extend the use of CRISPR editing for diverse applications.},
}
@article {pmid32393821,
year = {2020},
author = {Simoni, A and Hammond, AM and Beaghton, AK and Galizi, R and Taxiarchi, C and Kyrou, K and Meacci, D and Gribble, M and Morselli, G and Burt, A and Nolan, T and Crisanti, A},
title = {A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae.},
journal = {Nature biotechnology},
volume = {38},
number = {9},
pages = {1054-1060},
pmid = {32393821},
issn = {1546-1696},
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics/metabolism ; Female ; Gene Drive Technology/*methods ; Insect Proteins/genetics/metabolism ; Malaria/prevention &amp; control/*transmission ; Male ; Mosquito Control ; Mosquito Vectors/*genetics ; Sex Determination Processes/*genetics ; X Chromosome/genetics/metabolism ; },
abstract = {Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.},
}
@article {pmid32393674,
year = {2020},
author = {Wang, Z and Wang, Y and Wang, S and Gorzalski, AJ and McSwiggin, H and Yu, T and Castaneda-Garcia, K and Prince, B and Wang, H and Zheng, H and Yan, W},
title = {Efficient genome editing by CRISPR-Mb3Cas12a in mice.},
journal = {Journal of cell science},
volume = {133},
number = {9},
pages = {},
pmid = {32393674},
issn = {1477-9137},
support = {P30 GM110767/GM/NIGMS NIH HHS/United States ; P50 HD098593/HD/NICHD NIH HHS/United States ; R01 HD085506/HD/NICHD NIH HHS/United States ; R21 HD071736/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Mice ; Moraxella ; RNA ; },
abstract = {As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequence-targeting CRISPR (cr)RNAs of 23 nt spacers are used, >70% of the founders obtained are edited. Moreover, the use of Mb3Cas12a tagged to monomeric streptavidin (mSA) in conjunction with biotinylated DNA donor template leads to high knock-in efficiency in two-cell mouse embryos, with 40% of founders obtained containing the desired knock-in sequences.},
}
@article {pmid32393110,
year = {2020},
author = {Zhou, Y and Tang, Y and Fu, P and Tian, D and Yu, L and Huang, Y and Li, G and Li, M and Wang, Y and Yang, Z and Xu, X and Yin, Z and Zhou, D and Poirel, L and Jiang, X},
title = {The type I-E CRISPR-Cas system influences the acquisition of blaKPC-IncF plasmid in Klebsiella pneumonia.},
journal = {Emerging microbes &amp; infections},
volume = {9},
number = {1},
pages = {1011-1022},
pmid = {32393110},
issn = {2222-1751},
mesh = {Bacterial Proteins/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification ; China/epidemiology ; *Gene Transfer, Horizontal ; Humans ; Klebsiella Infections/epidemiology ; Klebsiella pneumoniae/*genetics/isolation &amp; purification ; Molecular Epidemiology ; Plasmids/isolation &amp; purification ; Pneumonia/epidemiology/microbiology ; beta-Lactamases/genetics/isolation &amp; purification ; },
abstract = {Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and blaKPC-IncF plasmids. CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer. Because of blaKPC-IncF plasmids are favored by CG258 K. pneumoniae, it was of interest to examine the co-distribution of CRISPR and blaKPC-IncF plasmids in such isolates. We collected 459 clinical K. pneumoniae isolates in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that CRISPR-Cas system was significantly scarce in the CG258 lineage and blaKPC-positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in K. pneumoniae could effectively hindered blaKPC-IncF plasmids invasion and existence. Notably, most blaKPC-IncF plasmids were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of blaKPC in K. pneumoniae populations, and the scarcity of CRISPR-Cas system was one of potential factors leading to the propagation of blaKPC-IncF plasmids in CG258 K. pneumoniae.},
}
@article {pmid32392580,
year = {2021},
author = {Niu, X and Deng, K and Liu, L and Yang, K and Hu, X},
title = {A statistical framework for predicting critical regions of p53-dependent enhancers.},
journal = {Briefings in bioinformatics},
volume = {22},
number = {3},
pages = {},
pmid = {32392580},
issn = {1477-4054},
mesh = {CRISPR-Cas Systems ; Datasets as Topic ; *Enhancer Elements, Genetic ; *Genes, p53 ; Humans ; RNA, Guide/genetics ; },
abstract = {P53 is the 'guardian of the genome' and is responsible for regulating cell cycle and apoptosis. The genomic p53 binding regions, where activating transcriptional factors and cofactors like p300 simultaneously bind, are called 'p53-dependent enhancers', which play an important role in tumorigenesis. Current experimental assays generally provide a broad peak of each enhancer element, leaving our knowledge about critical enhancer regions (CERs) limited. Under the inspiration of enhancer dissection by CRISPR-Cas9 screen library on genome-wide p53 binding sites, here we introduce a statistical framework called 'Computational CRISPR Strategy' (CCS), to predict whether a given DNA fragment will be a p53-dependent CER by employing 7-mer as feature extractions along with random forest as the regressor. When training on a p53 CRISPR enhancer dataset, CCS not only accurately fitted the top-ranked enriched single guide RNAs (sgRNAs) but also successfully reproduced two known CERs that were validated by experiments. When applying it to an independent testing dataset on a tilling of a 2K-b genomic region of CRISPR-deCDKN1A-Lib, the trained model shows great generalizability by identifying a CER containing five top-ranked sgRNAs. A feature importance analysis further indicates that top-ranked 7-mers are mapped onto informative TF motifs including POU5F1 and SOX5, which are differentially enriched in p53-dependent CERs and are potential factors to make a general p53 binding site to form a p53-dependent CER, providing the interpretability of the trained model. Our results demonstrate that CCS is an alternative way of the CRISPR experiment to screen the genome for mapping p53-dependent CERs.},
}
@article {pmid32391789,
year = {2020},
author = {Sollberger, G and Streeck, R and Apel, F and Caffrey, BE and Skoultchi, AI and Zychlinsky, A},
title = {Linker histone H1.2 and H1.4 affect the neutrophil lineage determination.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32391789},
issn = {2050-084X},
support = {R01 GM116143/GM/NIGMS NIH HHS/United States ; GM116143/GM/NIGMS NIH HHS/United States ; P2EZP3_148748//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/International ; P300P3_158518//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/International ; },
mesh = {Animals ; Bone Marrow/physiology ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Eosinophils/cytology ; Gene Expression Regulation ; Gene Knockout Techniques ; Hematopoiesis ; Hematopoietic Stem Cells/*cytology ; Histones/*physiology ; Humans ; Mice ; Microscopy, Electron, Transmission ; Neutrophils/*cytology ; Transcription Factors/physiology ; },
abstract = {Neutrophils are important innate immune cells that tackle invading pathogens with different effector mechanisms. They acquire this antimicrobial potential during their maturation in the bone marrow, where they differentiate from hematopoietic stem cells in a process called granulopoiesis. Mature neutrophils are terminally differentiated and short-lived with a high turnover rate. Here, we show a critical role for linker histone H1 on the differentiation and function of neutrophils using a genome-wide CRISPR/Cas9 screen in the human cell line PLB-985. We systematically disrupted expression of somatic H1 subtypes to show that individual H1 subtypes affect PLB-985 maturation in opposite ways. Loss of H1.2 and H1.4 induced an eosinophil-like transcriptional program, thereby negatively regulating the differentiation into the neutrophil lineage. Importantly, H1 subtypes also affect neutrophil differentiation and the eosinophil-directed bias of murine bone marrow stem cells, demonstrating an unexpected subtype-specific role for H1 in granulopoiesis.},
}
@article {pmid32391788,
year = {2020},
author = {Damiano-Guercio, J and Kurzawa, L and Mueller, J and Dimchev, G and Schaks, M and Nemethova, M and Pokrant, T and Brühmann, S and Linkner, J and Blanchoin, L and Sixt, M and Rottner, K and Faix, J},
title = {Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32391788},
issn = {2050-084X},
support = {FA 330/11-1//Deutsche Forschungsgemeinschaft/International ; AAA 741773//H2020 European Research Council/International ; CoG 724373//H2020 European Research Council/International ; RO2414/5-1//Deutsche Forschungsgemeinschaft/International ; GRK2223/1//Technische Universit&#xe4;t Braunschweig/International ; },
mesh = {Actin Capping Proteins/metabolism ; Actin Cytoskeleton/metabolism ; Actin-Related Protein 2-3 Complex/metabolism ; Actins/*metabolism ; Animals ; CRISPR-Cas Systems ; *Cell Adhesion ; Cell Line, Tumor ; *Cell Movement ; DNA-Binding Proteins/*genetics/*metabolism ; Fibroblasts ; Focal Adhesions ; Gene Knockout Techniques ; Integrins/metabolism ; Melanoma, Experimental ; Mice ; NIH 3T3 Cells ; Polymerization ; Pseudopodia/physiology ; Recombinant Proteins/genetics/metabolism ; },
abstract = {Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.},
}
@article {pmid32390420,
year = {2020},
author = {Yin, K and Ding, X and Li, Z and Zhao, H and Cooper, K and Liu, C},
title = {Dynamic Aqueous Multiphase Reaction System for One-Pot CRISPR-Cas12a-Based Ultrasensitive and Quantitative Molecular Diagnosis.},
journal = {Analytical chemistry},
volume = {92},
number = {12},
pages = {8561-8568},
pmid = {32390420},
issn = {1520-6882},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R21 TW010625/TW/FIC NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA, Viral/*genetics ; Endodeoxyribonucleases/*genetics ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; *Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques ; *Real-Time Polymerase Chain Reaction ; Water/chemistry ; },
abstract = {Recently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than 1 h. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pretreatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.},
}
@article {pmid32389642,
year = {2020},
author = {Xiao, L and Ma, L and Wang, Z and Yu, Y and Lye, SJ and Shan, Y and Wei, Y},
title = {Deciphering a distinct regulatory network of TEAD4, CDX2 and GATA3 in humans for trophoblast transition from embryonic stem cells.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {9},
pages = {118736},
doi = {10.1016/j.bbamcr.2020.118736},
pmid = {32389642},
issn = {1879-2596},
mesh = {CDX2 Transcription Factor/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; DNA-Binding Proteins/*genetics/metabolism ; Ectopic Gene Expression ; Embryonic Stem Cells/*cytology/*metabolism ; GATA3 Transcription Factor/*genetics/metabolism ; Gene Expression Regulation, Developmental ; Gene Targeting ; Humans ; Muscle Proteins/*genetics/metabolism ; TEA Domain Transcription Factors ; Transcription Factors/*genetics/metabolism ; Trophoblasts/*cytology/*metabolism ; },
abstract = {The placenta is an essential organ for the fetus, but its regulatory mechanism for formation of functional trophoblast lineage remains elusive in humans. Although widely known in mice, TEAD4 and its downstream targets CDX2 and GATA3 have not been determined in human models. In this work, we used a human model of trophoblast transition from BAP (BMP4, A83-01 and PD173074)-treated human embryonic stem cells (hESCs) and performed multiple gain- and loss-of-function tests of TEAD4, CDX2 or GATA3 to study their roles during this process. Although hESCs with TEAD4 deletion maintain pluripotency, their trophoblast transition potentials are attenuated. This impaired trophoblast transition could be rescued by separately overexpressing TEAD4, CDX2 or GATA3. Furthermore, trophoblast transition from hESCs is also attenuated by knockout of CDX2 but remains unaffected with deletion of GATA3. However, CDX2-overexpressed hESCs maintain pluripotency, whereas overexpression of GATA3 in hESCs leads to spontaneous differentiation including trophoblast lineage. In brief, our findings using a human model of trophoblast transition from BAP-treated hESCs reveal transcription roles of TEAD4, CDX2 and GATA in humans that are different from those in mice. We hope that this evidence can aid in understanding the distinct transcriptional network regulating trophoblast development in humans.},
}
@article {pmid32387190,
year = {2020},
author = {Mukama, O and Nie, C and Habimana, JD and Meng, X and Ting, Y and Songwe, F and Al Farga, A and Mugisha, S and Rwibasira, P and Zhang, Y and Zeng, L},
title = {Synergetic performance of isothermal amplification techniques and lateral flow approach for nucleic acid diagnostics.},
journal = {Analytical biochemistry},
volume = {600},
number = {},
pages = {113762},
doi = {10.1016/j.ab.2020.113762},
pmid = {32387190},
issn = {1096-0309},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques ; Nucleic Acids/*genetics ; },
abstract = {The advancement in developing sensitive, rapid, and specific sensing tools is crucial in diagnostics and biotechnological applications. Although various isothermal amplification approaches exist for the detection and identification of nucleic acids, post-amplicon analysis is still based on traditional methods such as gel electrophoresis, colorimetry, turbidity, which could be non-specific and inconvenient. Thus, this review will first elaborate various isothermal amplification techniques (principle, merits, and demerits) and their potentials when combined with lateral flow approach for point-of-care nucleic acid diagnostics. Different methods for monitoring carryover contamination resulting from amplification product contamination will be discussed. Then, we will present recent advances in diagnostics with both target pre-amplification and CRISPR-Cas systems, which exhibit collateral cleavage of target nucleic acid and a reporter single strand nucleic acid within the vicinity. When the reporter is fluorophore-labeled, it provides a detectable signal by fluorescence or lateral flow biosensors. Lastly, we will discuss how CRISPR-Cas system based diagnostics could be more effective, affordable and portable for on-site detection.},
}
@article {pmid32386748,
year = {2020},
author = {Vukmirovic, D and Seymour, C and Mothersill, C},
title = {Deciphering and simulating models of radiation genotoxicity with CRISPR/Cas9 systems.},
journal = {Mutation research. Reviews in mutation research},
volume = {783},
number = {},
pages = {108298},
doi = {10.1016/j.mrrev.2020.108298},
pmid = {32386748},
issn = {1388-2139},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; DNA/metabolism/*radiation effects ; *DNA Breaks, Double-Stranded ; DNA Damage ; DNA Repair ; DNA, Mitochondrial/metabolism/radiation effects ; Humans ; *Models, Genetic ; Mutagenicity Tests/*methods ; Radiation, Ionizing ; },
abstract = {This short review explores the utility and applications of CRISPR/Cas9 systems in radiobiology. Specifically, in the context of experimentally simulating genotoxic effects of Ionizing Radiation (IR) to determine the contributions from DNA targets and 'Complex Double-Stranded Breaks' (complex DSBs) to the IR response. To elucidate this objective, this review considers applications of CRISPR/Cas9 on nuclear DNA targets to recognize the respective 'nucleocentric' response. The article also highlights contributions from mitochondrial DNA (mtDNA) - an often under-recognized target in radiobiology. This objective requires accurate experimental simulation of IR-like effects and parameters with the CRISPR/Cas9 systems. Therefore, the role of anti-CRISPR proteins in modulating enzyme activity to simulate dose rate - an important factor in radiobiology experiments is an important topic of this review. The applications of auxiliary domains on the Cas9 nuclease to simulate oxidative base damage and multiple stressor experiments are also topics of discussion. Ultimately, incorporation of CRISPR/Cas9 experiments into computational parameters in radiobiology models of IR damage and shortcomings to the technology are discussed as well. Altogether, the simulation of IR parameters and lack of damage to non-DNA targets in the CRISPR/Cas9 system lends this rapidly emerging tool as an effective model of IR induced DNA damage. Therefore, this literature review ultimately considers the relevance of complex DSBs to radiobiology with respect to using the CRISPR/Cas9 system as an effective experimental tool in models of IR induced effects.},
}
@article {pmid32385892,
year = {2021},
author = {Cai, W and Luo, T and Mao, L and Wang, M},
title = {Spatiotemporal Delivery of CRISPR/Cas9 Genome Editing Machinery Using Stimuli-Responsive Vehicles.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {60},
number = {16},
pages = {8596-8606},
doi = {10.1002/anie.202005644},
pmid = {32385892},
issn = {1521-3773},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; Humans ; },
abstract = {Recent innovations in genome editing have enabled the precise manipulation of the genetic information of mammalians, and benefitted the development of next-generation gene therapy. Despite these advances, several barriers to the clinical translation of genome editing remain, including the intracellular delivery of genome editing machinery, and the risk of off-target editing effect. Here, we review the recent advance of spatiotemporal delivery of CRISPR/Cas9 genome editing machinery, which is composed of programmable Cas9 nuclease and a single-guide RNA (sgRNA) using stimuli-responsive nanoparticles. We discuss the specific chemistries that have been used for controlled Cas9/sgRNA delivery and intracellular release in the presence of endogenous or external signals. These methodologies can leverage biological signals found locally within disease cells, or exogenous signals administrated with spatiotemporal control, through which an improved genome editing could be achieved. We also discuss the future in exploiting these approaches for fundamental biomedical applications and therapeutic genome editing.},
}
@article {pmid32385085,
year = {2020},
author = {Sun, D and Mao, X and Fei, M and Chen, Z and Zhu, T and Qiu, J},
title = {Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {14},
pages = {},
pmid = {32385085},
issn = {1098-5336},
mesh = {*CRISPR-Cas Systems ; DNA-Binding Proteins/*genetics/metabolism ; Escherichia coli/*genetics/metabolism ; Escherichia coli Proteins/*genetics/metabolism ; Molecular Chaperones/*genetics/metabolism ; *Transformation, Bacterial ; },
abstract = {Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (P cas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli Although the H-NS paralogue StpA also binds P cas , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of P cas Third, by comparing transcriptional activities of the intact P cas and the P cas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in P cas Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P cas Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coliIMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.},
}
@article {pmid32385078,
year = {2020},
author = {Wasels, F and Chartier, G and Hocq, R and Lopes Ferreira, N},
title = {A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {13},
pages = {},
pmid = {32385078},
issn = {1098-5336},
mesh = {Alcohol Oxidoreductases/*genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Clostridium acetobutylicum/enzymology/*genetics ; Gene Editing ; },
abstract = {Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.},
}
@article {pmid32384610,
year = {2020},
author = {Sürün, D and Schneider, A and Mircetic, J and Neumann, K and Lansing, F and Paszkowski-Rogacz, M and Hänchen, V and Lee-Kirsch, MA and Buchholz, F},
title = {Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors.},
journal = {Genes},
volume = {11},
number = {5},
pages = {},
pmid = {32384610},
issn = {2073-4425},
mesh = {Adenine/*chemistry ; Aminohydrolases ; Autoimmune Diseases of the Nervous System/genetics ; Base Sequence ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line ; Cellular Reprogramming Techniques ; Cytosine/*chemistry ; Embryoid Bodies ; Gene Editing/*methods ; Genes, p53 ; HEK293 Cells ; Humans ; Imidazoles/pharmacology ; Induced Pluripotent Stem Cells/*metabolism ; *Mutagenesis ; *Mutation ; Nervous System Malformations/genetics ; Piperazines/pharmacology ; RNA, Guide/genetics ; RNA, Messenger/*genetics/metabolism ; SAM Domain and HD Domain-Containing Protein 1/genetics ; Transfection ; },
abstract = {In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.},
}
@article {pmid32383796,
year = {2020},
author = {Common, J and Walker-Sünderhauf, D and van Houte, S and Westra, ER},
title = {Diversity in CRISPR-based immunity protects susceptible genotypes by restricting phage spread and evolution.},
journal = {Journal of evolutionary biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jeb.13638},
pmid = {32383796},
issn = {1420-9101},
support = {MR/N013794/1/MRC_/Medical Research Council/United Kingdom ; BB/J014400/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M009122/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.},
}
@article {pmid32383535,
year = {2020},
author = {Das, AS and Basu, A and Kumar, R and Borah, PK and Bakshi, S and Sharma, M and Duary, RK and Ray, PS and Mukhopadhyay, R},
title = {Post-transcriptional regulation of C-C motif chemokine ligand 2 expression by ribosomal protein L22 during LPS-mediated inflammation.},
journal = {The FEBS journal},
volume = {287},
number = {17},
pages = {3794-3813},
doi = {10.1111/febs.15362},
pmid = {32383535},
issn = {1742-4658},
mesh = {5' Untranslated Regions ; Active Transport, Cell Nucleus ; Base Sequence ; CRISPR-Cas Systems ; Cell Movement ; Chemokine CCL2/*biosynthesis/genetics ; Humans ; Inflammation/chemically induced/*genetics/metabolism ; Lipopolysaccharides/*toxicity ; MCF-7 Cells ; Models, Molecular ; Neoplasm Proteins/metabolism ; Protein Conformation ; Protein Interaction Mapping ; *Protein Processing, Post-Translational ; RNA Helicases/metabolism ; RNA Stability ; RNA, Messenger/metabolism ; RNA-Binding Proteins/*physiology ; Ribosomal Proteins/deficiency/*physiology ; Sequence Alignment ; Sequence Homology, Nucleic Acid ; THP-1 Cells ; Trans-Activators/metabolism ; },
abstract = {Monocyte infiltration to the site of pathogenic invasion is critical for inflammatory response and host defence. However, this process demands precise regulation as uncontrolled migration of monocytes to the site delays resolution of inflammation and ultimately promotes chronic inflammation. C-C motif chemokine ligand 2 (CCL2) plays a key role in monocyte migration, and hence, its expression should be tightly regulated. Here, we report a post-transcriptional regulation of CCL2 involving the large ribosomal subunit protein L22 (RPL22) in LPS-activated, differentiated THP-1 cells. Early events following LPS treatment include transcriptional upregulation of RPL22 and its nuclear accumulation. The protein binds to the first 20 nt sequence of the 5'UTR of ccl2 mRNA. Simultaneous nuclear translocation of up-frameshift-1 protein and its interaction with RPL22 results in cytoplasmic degradation of the ccl2 mRNA at a later stage. Removal of RPL22 from cells results in increased expression of CCL2 in response to LPS causing disproportionate migration of monocytes. We propose that post-transcriptional regulation of CCL2 by RPL22 fine-tunes monocyte infiltration during a pathogenic insult and maintains homeostasis of the immune response critical to resolution of inflammation. DATABASES: Microarray data are available in NCBI GEO database (Accession No GSE126525).},
}
@article {pmid32382982,
year = {2020},
author = {Zhang, Q and Yin, K and Liu, G and Li, S and Li, M and Qiu, JL},
title = {Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites.},
journal = {Science China. Life sciences},
volume = {63},
number = {12},
pages = {1918-1927},
pmid = {32382982},
issn = {1869-1889},
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; DNA, Plant/genetics/metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Exodeoxyribonucleases/genetics/*metabolism ; Gene Editing/*methods ; Genome, Plant/genetics ; INDEL Mutation ; Oryza/genetics ; Plants, Genetically Modified ; Recombinant Fusion Proteins/metabolism ; },
abstract = {CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease (T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.},
}
@article {pmid32381591,
year = {2020},
author = {Bálint, &#x160; and Müller, S and Fischer, R and Kessler, BM and Harkiolaki, M and Valitutti, S and Dustin, ML},
title = {Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells.},
journal = {Science (New York, N.Y.)},
volume = {368},
number = {6493},
pages = {897-901},
pmid = {32381591},
issn = {1095-9203},
support = {/WT_/Wellcome Trust/United Kingdom ; 100262/WT_/Wellcome Trust/United Kingdom ; 670930/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems ; *Cytotoxicity, Immunologic ; Exocytosis ; Gene Editing ; Granzymes/*metabolism ; Humans ; K562 Cells ; Multiprotein Complexes/*metabolism ; Perforin/*metabolism ; T-Lymphocytes, Cytotoxic/*metabolism ; Thrombospondin 1/genetics/*metabolism ; Tomography, X-Ray ; },
abstract = {Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.},
}
@article {pmid32380756,
year = {2020},
author = {Murphy, BG and Wolf, T and Vogel, H and Castillo, D and Woolard, K},
title = {An RNA-Directed Gene Editing Strategy for Attenuating the Infectious Potential of Feline Immunodeficiency Virus-Infected Cells: A Proof of Concept.},
journal = {Viruses},
volume = {12},
number = {5},
pages = {},
pmid = {32380756},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems ; Cats ; Feline Acquired Immunodeficiency Syndrome/therapy/*virology ; Gene Editing ; HIV/genetics/physiology ; HIV Infections/therapy/virology ; Humans ; Immunodeficiency Virus, Feline/*genetics/physiology ; RNA, Viral/*genetics ; T-Lymphocytes/virology ; },
abstract = {Modern antiretroviral therapy for immunodeficiency viruses, although remarkably effective in controlling viral transcription, and overt virus-associated morbidity, has failed to absolutely eradicate retroviruses from their infected hosts as a result of proviral integration in long-lived reservoir cells. Immunodeficiency virus-infected patients are therefore consigned to lifelong antiviral therapy as a means to control viremia, viral transmission, and infection-associated morbidity. Unfortunately, lifelong antiviral therapies can be difficult for patients to continuously maintain and may be associated with therapy-specific morbidities. Patient advocates have argued for new methods to achieve retroviral eradication. As a proof-of-concept study, a lentivirus-delivered RNA-directed gene editing strategy was utilized in a series of in vitro experiments in an attempt to attenuate the feline immunodeficiency virus (FIV) proviral load, viral transcription, and production of infectious virions. We found that a feline T lymphocyte cell line (MCH5-4) treated with an FIV-specific clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing tool resulted in a reduction of cell-free viral RNA relative to control cells. Decreased infectious potential was demonstrated in a two-step FIV infection study-naïve MCH5-4 cells infected with cell-free FIV harvested from FIV-infected and CRISPR lentivirus-treated cells had less integrated proviral DNA than control cells. This study represents the initial steps towards the development of an effective method of proviral eradication in an immunodeficiency virus-infected host.},
}
@article {pmid32380730,
year = {2020},
author = {Pavan, E and Ormazabal, M and Peruzzo, P and Vaena, E and Rozenfeld, P and Dardis, A},
title = {CRISPR/Cas9 Editing for Gaucher Disease Modelling.},
journal = {International journal of molecular sciences},
volume = {21},
number = {9},
pages = {},
pmid = {32380730},
issn = {1422-0067},
mesh = {Biomarkers ; *CRISPR-Cas Systems ; Cell Line ; Disease Susceptibility ; Endoplasmic Reticulum Stress ; Endoplasmic Reticulum-Associated Degradation ; Gaucher Disease/*genetics/metabolism/pathology ; *Gene Editing ; Gene Expression ; Glucosylceramidase/genetics ; Humans ; Inflammation Mediators/metabolism ; *Models, Biological ; Monocytes/metabolism ; Mutation ; Unfolded Protein Response ; },
abstract = {Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson's disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.},
}
@article {pmid32380204,
year = {2020},
author = {Wang, Y and Shahi, PK and Xie, R and Zhang, H and Abdeen, AA and Yodsanit, N and Ma, Z and Saha, K and Pattnaik, BR and Gong, S},
title = {A pH-responsive silica-metal-organic framework hybrid nanoparticle for the delivery of hydrophilic drugs, nucleic acids, and CRISPR-Cas9 genome-editing machineries.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {324},
number = {},
pages = {194-203},
pmid = {32380204},
issn = {1873-4995},
support = {R01 HL129785/HL/NHLBI NIH HHS/United States ; UG3 NS111688/NS/NINDS NIH HHS/United States ; R01 EY024995/EY/NEI NIH HHS/United States ; R01 HL143469/HL/NHLBI NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Hydrogen-Ion Concentration ; *Metal-Organic Frameworks ; Mice ; *Nanoparticles ; *Nucleic Acids ; *Pharmaceutical Preparations ; Silicon Dioxide ; },
abstract = {Efficient delivery of hydrophilic drugs, nucleic acids, proteins, and any combination thereof is essential for various biomedical applications. Herein, we report a straightforward, yet versatile approach to efficiently encapsulate and deliver various hydrophilic payloads using a pH-responsive silica-metal-organic framework hybrid nanoparticle (SMOF NP) consisting of both silica and zeolitic imidazole framework (ZIF). This unique SMOF NP offers a high loading content and efficiency, excellent stability, and robust intracellular delivery of a variety of payloads, including hydrophilic small molecule drugs (e.g., doxorubicin hydrochloride), nucleic acids (e.g., DNA and mRNA), and genome-editing machineries (e.g., Cas9-sgRNA ribonucleoprotein (RNP), and RNP together with donor DNA (e.g., RNP + ssODN)). The superior drug delivery/gene transfection/genome-editing efficiencies of the SMOF NP are attributed to its pH-controlled release and endosomal escape capabilities due to the proton sponge effect enabled by the imidazole moieties in the SMOF NPs. Moreover, the surface of the SMOF NP can be easily customized (e.g., PEGylation and ligand conjugation) via various functional groups incorporated into the silica component. RNP-loaded SMOF NPs induced efficient genome editing in vivo in murine retinal pigment epithelium (RPE) tissue via subretinal injection, providing a highly promising nanoplatform for the delivery of a wide range of hydrophilic payloads.},
}
@article {pmid32380039,
year = {2020},
author = {He, C and Jaffar Ali, D and Xu, H and Kumaravel, S and Si, K and Li, Y and Sun, B and Ma, J and Xiao, Z},
title = {Epithelial cell -derived microvesicles: A safe delivery platform of CRISPR/Cas9 conferring synergistic anti-tumor effect with sorafenib.},
journal = {Experimental cell research},
volume = {392},
number = {2},
pages = {112040},
doi = {10.1016/j.yexcr.2020.112040},
pmid = {32380039},
issn = {1090-2422},
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/pathology/*therapy ; Cell Proliferation ; Cell-Derived Microparticles/metabolism/*pathology ; Combined Modality Therapy ; Epithelial Cells/metabolism/*pathology ; Female ; Gene Editing ; Humans ; Liver Neoplasms/genetics/pathology/*therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Sorafenib/*pharmacology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; ras GTPase-Activating Proteins/*antagonists &amp; inhibitors/genetics ; },
abstract = {Safe and efficient intracellular delivery of CRISPR/Cas9 is a key step for effective therapeutic genome editing in a wide range of diseases. This remains challenging due to multiple drawbacks of the currently available vehicles. Here we report that epithelial cell -derived microvesicles (MVs) function as safe and natural carriers for efficient delivery of CRISPR/Cas9 to treat cancer. In our study, compared to epithelial cell -derived MVs, cancer -derived MVs were quickly absorbed intracellularly by recipient cancer cells in vitro and showed selective accumulation in tumors of HepG2 xenografts in vivo, due to their cancer cell tropism dependent targeting. Surprisingly, synergistic anti-tumor effect of sgIQ 1.1 loaded Cas9MVs/HEK293 + sorafenib was better than sgIQ 1.1 + Cas9MVs/HepG2 + sorafenib in vitro. In addition, qPCR results showed that miR-21 and miR-181a expression were upregulated in HepG2 cells treated with cancer cell -derived MVs that might support the cancer progression. Further, treatment of HepG2 xenografts with sgIQ 1.1 loaded Cas9MVs/HEK293 showed enhanced anti-cancer effect than sgIQ 1.1 + Cas9MVs/HepG2. Therefore, we conclude that normal cells -derived MVs can act as better and safe natural delivery systems for cancer therapeutics in the future.},
}
@article {pmid32379967,
year = {2020},
author = {Choi, SY and Woo, HM},
title = {CRISPRi-dCas12a: A dCas12a-Mediated CRISPR Interference for Repression of Multiple Genes and Metabolic Engineering in Cyanobacteria.},
journal = {ACS synthetic biology},
volume = {9},
number = {9},
pages = {2351-2361},
doi = {10.1021/acssynbio.0c00091},
pmid = {32379967},
issn = {2161-5063},
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Chromatography, Gas ; Gene Expression Regulation, Bacterial ; Metabolic Engineering/*methods ; Oligonucleotide Array Sequence Analysis ; Photosynthesis ; Plasmids/genetics/metabolism ; Repressor Proteins/deficiency/*genetics ; Squalene/analysis/chemistry/metabolism ; Synechococcus/chemistry/genetics/*metabolism ; },
abstract = {In cyanobacteria, metabolic engineering using synthetic biology tools is limited to build a biosolar cell factory that converts CO2 to value-added chemicals, as repression of essential genes has not been achieved. In this study, we developed a dCas12a-mediated CRISPR interference system (CRISPRi-dCas12a) in cyanobacteria that effectively blocked the transcriptional initiation by means of a CRISPR-RNA (crRNA) and 19-nt direct repeat, resulting in 53-94% gene repression. The repression of multiple genes in a single crRNA array was also successfully achieved without a loss in repression strength. In addition, as a demonstration of the dCas12a-mediated CRISPRi for metabolic engineering, photosynthetic squalene production was improved by repressing the essential genes of either acnB encoding for aconitase or cpcB2 encoding for phycocyanin β-subunit in Synechococcus elongatus PCC 7942. The ability to regulate gene repression will promote the construction of biosolar cell factories to produce value-added chemicals.},
}
@article {pmid32378871,
year = {2020},
author = {Wang, S and Wei, L and Wang, JQ and Ji, H and Xiong, W and Liu, J and Yin, P and Tian, T and Zhou, X},
title = {Light-Driven Activation of RNA-Guided Nucleic Acid Cleavage.},
journal = {ACS chemical biology},
volume = {15},
number = {6},
pages = {1455-1463},
doi = {10.1021/acschembio.0c00105},
pmid = {32378871},
issn = {1554-8937},
mesh = {Acetylation/radiation effects ; CRISPR-Cas Systems/*radiation effects ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/radiation effects ; Gene Editing/*methods ; Humans ; *Light ; RNA Cleavage/radiation effects ; RNA, Guide/chemistry/*genetics ; },
abstract = {As one of the most favorable stimuli, photoactivation provides an advantageous way to manipulate biological objects. In the current study, we have successfully demonstrated the use of light activation guide RNA (gRNA) strategy for controlling CRISPR systems. By conjugating photolabile protecting groups, the CRISPR functions became minimal, but exposure of acylated gRNAs to 365 nm light triggers the removal of masking groups, leading to the rescue of CRISPR functions. Furthermore, our strategy has been successfully used to control gene editing in human cells. This proof-of-concept study therefore demonstrates the promising potential of our strategy to versatile applications in chemical biology.},
}
@article {pmid32376790,
year = {2021},
author = {Lu, C and Zhang, Y and Qin, Y and Xu, Q and Zhou, R and Cui, Y and Zhu, Y and Zhang, X and Zhang, J and Wei, X and Wang, M and Hang, B and Mao, JH and Snijders, AM and Liu, M and Hu, Z and Shen, H and Zhou, Z and Guo, X and Wu, X and Wang, X and Xia, Y},
title = {Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis.},
journal = {Journal of medical genetics},
volume = {58},
number = {1},
pages = {56-65},
doi = {10.1136/jmedgenet-2019-106598},
pmid = {32376790},
issn = {1468-6244},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Chromosomes, Human, X/*genetics ; Exome/genetics ; Humans ; Male ; Mice ; Mice, Knockout ; Repressor Proteins/*genetics ; Sperm Motility/genetics ; Spermatogenesis/*genetics ; Spermatogonia/metabolism/pathology ; Testis/*growth &amp; development/pathology ; Whole Exome Sequencing ; },
abstract = {BACKGROUND: Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood.<br><br>METHODS: We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system.<br><br>RESULTS: Four low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure.<br><br>CONCLUSION: Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.},
}
@article {pmid32376684,
year = {2020},
author = {Alam, SMD and Tsukamoto, Y and Ogawa, M and Senoo, Y and Ikeda, K and Tashima, Y and Takeuchi, H and Okajima, T},
title = {N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {25},
pages = {8560-8574},
pmid = {32376684},
issn = {1083-351X},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromatography, High Pressure Liquid ; Endoplasmic Reticulum/*metabolism ; Endoplasmic Reticulum Stress/drug effects ; Gene Editing ; Glycopeptides/analysis ; Glycosylation ; Humans ; Mice ; Mutagenesis, Site-Directed ; N-Acetylglucosaminyltransferases/deficiency/genetics/*metabolism ; Receptor, Notch1/genetics/metabolism ; Sequence Alignment ; Tandem Mass Spectrometry ; Tunicamycin/pharmacology ; },
abstract = {Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.},
}
@article {pmid32376630,
year = {2020},
author = {Specht, DA and Xu, Y and Lambert, G},
title = {Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {21},
pages = {11274-11282},
pmid = {32376630},
issn = {1091-6490},
support = {R35 GM133759/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/genetics/*metabolism ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/chemistry/genetics/*metabolism ; Escherichia coli/genetics ; Francisella ; High-Throughput Screening Assays/methods ; RNA Interference ; RNA, Guide ; Thermodynamics ; },
abstract = {The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.},
}
@article {pmid32376152,
year = {2020},
author = {Itoh, M and Kawagoe, S and Tamai, K and Nakagawa, H and Asahina, A and Okano, HJ},
title = {Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using the CRISPR/Cas9 and piggyBac transposon system.},
journal = {Journal of dermatological science},
volume = {98},
number = {3},
pages = {163-172},
doi = {10.1016/j.jdermsci.2020.04.004},
pmid = {32376152},
issn = {1873-569X},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Collagen Type VII/*genetics/metabolism ; DNA Transposable Elements/genetics ; Epidermolysis Bullosa Dystrophica/genetics/*therapy ; Gene Editing/*methods ; Genetic Therapy/methods ; Homologous Recombination ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Keratinocytes/metabolism/*transplantation ; Mutation ; },
abstract = {BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use.<br><br>OBJECTIVE: In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation.<br><br>METHODS: For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression.<br><br>RESULTS: After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their "stemness". When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored.<br><br>CONCLUSIONS: Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.},
}
@article {pmid32376086,
year = {2020},
author = {Zheng, HX and Sun, X and Zhang, XS and Sui, N},
title = {m[6]A Editing: New Tool to Improve Crop Quality?.},
journal = {Trends in plant science},
volume = {25},
number = {9},
pages = {859-867},
doi = {10.1016/j.tplants.2020.04.005},
pmid = {32376086},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Plant Development ; Plants/genetics ; RNA ; },
abstract = {N[6]-methyladenosine (m[6]A) is the most common type of eukaryotic mRNA modification. It plays an important role in regulating plant growth and development and stress resistance. m[6]A modification influences nearly all aspects of RNA metabolism and functionality and has great potential for improving crop quality. However, changing m[6]A modification levels as a whole may have unpredictable effects, making it impossible to accurately predict the effect of specific m[6]A modifications on RNA. In this opinion article, the main challenges and possible solutions for exploring m[6]A modification functions in plant systems are discussed. An m[6]A editing platform that uses new high-throughput methods to identify m[6]A modification at single-base resolution, and genome editing for selective editing of specific m[6]A sites for crop improvement is proposed.},
}
@article {pmid32375580,
year = {2020},
author = {Gee, S and Nelson, N and Bornot, A and Carter, N and Cuomo, ME and Dovedi, SJ and Smith, PD and Gianni, D and Baker, DJ},
title = {Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {6},
pages = {581-590},
doi = {10.1177/2472555220916457},
pmid = {32375580},
issn = {2472-5560},
mesh = {Adenocarcinoma of Lung/*drug therapy/genetics/immunology ; Antibodies, Anti-Idiotypic/immunology/isolation &amp; purification ; B7-H1 Antigen/antagonists &amp; inhibitors/*genetics/immunology ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/*genetics/immunology ; Cell Line, Tumor ; Coculture Techniques ; Drug Screening Assays, Antitumor ; ErbB Receptors/genetics/immunology ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Immune Checkpoint Inhibitors/immunology/isolation &amp; purification/pharmacology ; Muromonab-CD3/immunology/isolation &amp; purification ; Protein Serine-Threonine Kinases/*genetics/immunology ; Proto-Oncogene Proteins/*genetics/immunology ; T-Lymphocytes/immunology/pathology ; },
abstract = {Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.},
}
@article {pmid32375333,
year = {2020},
author = {Tian, R and Pan, Y and Etheridge, THA and Deshmukh, H and Gulick, D and Gibson, G and Bao, G and Lee, CM},
title = {Pitfalls in Single Clone CRISPR-Cas9 Mutagenesis to Fine-map Regulatory Intervals.},
journal = {Genes},
volume = {11},
number = {5},
pages = {},
pmid = {32375333},
issn = {2073-4425},
support = {1R01HG008146-01A/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Causality ; Cell Lineage ; Chromosome Mapping/*methods ; Clone Cells ; Gene Deletion ; Genome-Wide Association Study/*methods ; HL-60 Cells ; Humans ; Leukopoiesis/genetics ; Multifactorial Inheritance/*genetics ; *Mutagenesis ; Neutrophils/cytology ; *Polymorphism, Single Nucleotide ; Quantitative Trait Loci/*genetics ; Quantitative Trait, Heritable ; RNA-Seq ; Reproducibility of Results ; *Scientific Experimental Error ; Sequence Deletion ; Single-Cell Analysis/*methods ; },
abstract = {The majority of genetic variants affecting complex traits map to regulatory regions of genes, and typically lie in credible intervals of 100 or more SNPs. Fine mapping of the causal variant(s) at a locus depends on assays that are able to discriminate the effects of polymorphisms or mutations on gene expression. Here, we evaluated a moderate-throughput CRISPR-Cas9 mutagenesis approach, based on replicated measurement of transcript abundance in single-cell clones, by deleting candidate regulatory SNPs, affecting four genes known to be affected by large-effect expression Quantitative Trait Loci (eQTL) in leukocytes, and using Fluidigm qRT-PCR to monitor gene expression in HL60 pro-myeloid human cells. We concluded that there were multiple constraints that rendered the approach generally infeasible for fine mapping. These included the non-targetability of many regulatory SNPs, clonal variability of single-cell derivatives, and expense. Power calculations based on the measured variance attributable to major sources of experimental error indicated that typical eQTL explaining 10% of the variation in expression of a gene would usually require at least eight biological replicates of each clone. Scanning across credible intervals with this approach is not recommended.},
}
@article {pmid32374858,
year = {2020},
author = {Zhao, Y and Boeke, JD},
title = {CRISPR-Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference.},
journal = {Nucleic acids research},
volume = {48},
number = {10},
pages = {5788-5798},
pmid = {32374858},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Deoxyribonucleases/metabolism ; Francisella/enzymology ; Gene Editing/*methods ; Promoter Regions, Genetic ; RNA/metabolism ; RNA Polymerase II/metabolism ; Ribonucleases/metabolism ; Schizosaccharomyces/genetics ; },
abstract = {The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.},
}
@article {pmid32374791,
year = {2020},
author = {Nadipuram, SM and Thind, AC and Rayatpisheh, S and Wohlschlegel, JA and Bradley, PJ},
title = {Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0232552},
pmid = {32374791},
issn = {1932-6203},
support = {R01 AI123360/AI/NIAID NIH HHS/United States ; T32 AI007323/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Biotinylation ; Brain/metabolism/parasitology ; CRISPR-Cas Systems ; Female ; Gene Knockout Techniques ; Genes, Protozoan ; Humans ; Life Cycle Stages ; Mice ; Mice, Inbred C57BL ; Proteome/metabolism ; Protozoan Proteins/genetics/*metabolism ; Toxoplasma/genetics/growth &amp; development/*physiology ; Toxoplasmosis, Animal/metabolism/parasitology ; Toxoplasmosis, Cerebral/metabolism/parasitology ; Vacuoles/metabolism ; Virulence ; },
abstract = {Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing life-long chronic infection in any mammalian host. During the intracellular life cycle, the parasite secretes an array of proteins into the parasitophorous vacuole (PV) where it resides. Specialized organelles called the dense granules secrete GRA proteins that are known to participate in nutrient acquisition, immune evasion, and host cell-cycle manipulation. Although many GRAs have been discovered which are expressed during the acute infection mediated by tachyzoites, little is known about those that participate in the chronic infection mediated by the bradyzoite form of the parasite. In this study, we sought to uncover novel bradyzoite-upregulated GRA proteins using proximity biotinylation, which we previously used to examine the secreted proteome of the tachyzoites. Using a fusion of the bradyzoite upregulated protein MAG1 to BirA* as bait and a strain with improved switch efficiency, we identified a number of novel GRA proteins which are expressed in bradyzoites. After using the CRISPR/Cas9 system to characterize these proteins by gene knockout, we focused on one of these GRAs (GRA55) and found it was important for the establishment or maintenance of cysts in the mouse brain. These findings highlight new components of the GRA proteome of the tissue-cyst life stage of T. gondii and identify potential targets that are important for maintenance of parasite persistence in vivo.},
}
@article {pmid32374759,
year = {2020},
author = {Bare, DJ and Cherny, VV and DeCoursey, TE and Abukhdeir, AM and Morgan, D},
title = {Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0227522},
pmid = {32374759},
issn = {1932-6203},
support = {R35 GM126902/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Movement/genetics ; Cell Proliferation/*genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Heterografts ; Humans ; Hydrogen Peroxide/pharmacology ; Immunohistochemistry ; Ion Channels/*genetics ; Membrane Proteins/*genetics ; Mice ; NADPH Oxidases/genetics ; RNA, Small Interfering/genetics ; Triple Negative Breast Neoplasms/*genetics/pathology ; },
abstract = {Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.},
}
@article {pmid32373229,
year = {2020},
author = {Fajrial, AK and He, QQ and Wirusanti, NI and Slansky, JE and Ding, X},
title = {A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing.},
journal = {Theranostics},
volume = {10},
number = {12},
pages = {5532-5549},
pmid = {32373229},
issn = {1838-7640},
mesh = {Animals ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Gene Editing/*methods ; Gene Transfer Techniques/*trends ; Genetic Therapy/*methods ; Humans ; Nanotechnology/*methods ; },
abstract = {Gene editing is a versatile technique in biomedicine that promotes fundamental research as well as clinical therapy. The development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing machinery has accelerated the application of gene editing. However, the delivery of CRISPR components often suffers when using conventional transfection methods, such as viral transduction and chemical vectors, due to limited packaging size and inefficiency toward certain cell types. In this review, we discuss physical transfection methods for CRISPR gene editing which can overcome these limitations. We outline different types of physical transfection methods, highlight novel techniques to deliver CRISPR components, and emphasize the role of micro and nanotechnology to improve transfection performance. We present our perspectives on the limitations of current technology and provide insights on the future developments of physical transfection methods.},
}
@article {pmid32372650,
year = {2020},
author = {Hejlesen, R and Füchtbauer, EM},
title = {Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.},
journal = {BioTechniques},
volume = {68},
number = {6},
pages = {345-348},
doi = {10.2144/btn-2019-0104},
pmid = {32372650},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems/*genetics ; Cloning, Molecular/*methods ; DNA/chemistry/*genetics ; DNA Repair/genetics ; Gene Editing/methods ; Humans ; Mutagenesis, Site-Directed/*methods ; Mutation/genetics ; Plasmids/chemistry/genetics ; },
abstract = {We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.},
}
@article {pmid32372640,
year = {2020},
author = {Shu, P and Li, Z and Min, D and Zhang, X and Ai, W and Li, J and Zhou, J and Li, Z and Li, F and Li, X},
title = {CRISPR/Cas9-Mediated SlMYC2 Mutagenesis Adverse to Tomato Plant Growth and MeJA-Induced Fruit Resistance to Botrytis cinerea.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {20},
pages = {5529-5538},
doi = {10.1021/acs.jafc.9b08069},
pmid = {32372640},
issn = {1520-5118},
mesh = {Acetates/*adverse effects/pharmacology ; Botrytis/*physiology ; CRISPR-Cas Systems ; Cyclopentanes/*adverse effects/pharmacology ; Disease Resistance ; Flowers/genetics/growth &amp; development/immunology ; Fruit/drug effects/genetics/immunology/microbiology ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/drug effects/*genetics/immunology/microbiology ; Mutagenesis ; Oxylipins/*adverse effects/pharmacology ; Plant Diseases/immunology/*microbiology ; Plant Growth Regulators/*pharmacology ; Plant Proteins/*genetics/immunology ; Plants, Genetically Modified/genetics/growth &amp; development/*immunology/microbiology ; },
abstract = {Methyl jasmonate (MeJA), a natural phytohormone, played a critical role not only in plant growth but also in plant defense response to biotic and abiotic stresses. MYC2, a basic helix-loop-helix transcription factor, is a master regulator in MeJA signaling pathway. In the present work, slmyc2 mutants were generated by the clustered regularly interspaced short palindromic repeats and associated Cas9 protein (CRISPR/Cas9) system to investigate the role of SlMYC2 in tomato plant growth and fruit disease resistance induced by exogenous MeJA. The results showed that slmyc2 mutants possessed a higher number of flowers and a lower fruit setting rate in comparison with wild-type plants. In addition, the fruit shape of slmyc2 mutant was prolate, while the control fruits were oblate. Knockout of SlMYC2 significantly decreased the activities of disease defensive and antioxidant enzymes, as well as the expression levels of pathogen-related (PR) genes (SlPR-1 and SlPR-STH2) and the key genes related to jasmonic acid (JA) biosynthesis and signaling pathway including allene oxide cyclase (SlAOC), lipoxygenase D (SlLOXD), SlMYC2, and coronatine insensitive 1 (SlCOI1), and consequently aggravated the disease symptoms. By contrast, the disease symptoms were largely reduced in MeJA-treated fruit that possessed higher activities of these enzymes and expression levels of genes. However, the induction effects of MeJA on fruit disease resistance and these enzymes' activities and genes' expressions were significantly attenuated by knockout of SlMYC2. Therefore, the results indicated that SlMYC2 played positive regulatory roles not only in the growth of tomato plants but also in MeJA-induced disease resistance and the antioxidant process in tomato fruits.},
}
@article {pmid32372485,
year = {2020},
author = {Yang, S and Wu, F and Peng, S and Wang, F and Chen, Y and Yuan, Y and Weng, X and Zhou, X},
title = {A m[6] A Sensing Method by Its Impact on the Stability of RNA Double Helix.},
journal = {Chemistry &amp; biodiversity},
volume = {17},
number = {7},
pages = {e2000050},
doi = {10.1002/cbdv.202000050},
pmid = {32372485},
issn = {1612-1880},
mesh = {Adenosine/*analogs &amp; derivatives/analysis/pharmacology ; Fluorescence ; RNA/*drug effects ; RNA Stability/drug effects ; Structure-Activity Relationship ; },
abstract = {N[6] -Methyladenosine (m[6] A) is one of the most important RNA modifications in epigenetics. The development of detection method for m[6] A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m[6] A modification and target RNAs without m[6] A modification. We verified the fact that the presence of m[6] A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m[6] A detection methods in the future.},
}
@article {pmid32372479,
year = {2020},
author = {Hua, K and Jiang, Y and Tao, X and Zhu, JK},
title = {Precision genome engineering in rice using prime editing system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2167-2169},
pmid = {32372479},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Oryza/genetics ; },
}
@article {pmid32371585,
year = {2020},
author = {Milton, CK and Self, AJ and Clarke, PA and Banerji, U and Piccioni, F and Root, DE and Whittaker, SR},
title = {A Genome-scale CRISPR Screen Identifies the ERBB and mTOR Signaling Networks as Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer.},
journal = {Molecular cancer therapeutics},
volume = {19},
number = {7},
pages = {1423-1435},
pmid = {32371585},
issn = {1538-8514},
support = {RIF2016_A16/PANCREATICCANUK_/Pancreatic Cancer UK/United Kingdom ; },
mesh = {Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; ErbB Receptors/antagonists &amp; inhibitors/*genetics ; Gene Expression Regulation, Neoplastic/*drug effects ; Genome, Human ; High-Throughput Screening Assays ; Humans ; Pancreatic Neoplasms/*drug therapy/genetics/pathology ; Phosphatidylinositol 3-Kinases/chemistry ; Phosphoinositide-3 Kinase Inhibitors/*pharmacology ; Phosphorylation ; Small Molecule Libraries/*pharmacology ; TOR Serine-Threonine Kinases/antagonists &amp; inhibitors/*genetics ; Tumor Cells, Cultured ; },
abstract = {KRAS mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for Kras-induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergize with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB family receptor tyrosine kinase signaling and mTOR signaling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB family or mTOR was synergistic with PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signaling axis were associated with decreased survival of patients with pancreatic cancer. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signaling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer.},
}
@article {pmid32371471,
year = {2020},
author = {Bezuidt, OKI and Lebre, PH and Pierneef, R and León-Sobrino, C and Adriaenssens, EM and Cowan, DA and Van de Peer, Y and Makhalanyane, TP},
title = {Phages Actively Challenge Niche Communities in Antarctic Soils.},
journal = {mSystems},
volume = {5},
number = {3},
pages = {},
pmid = {32371471},
issn = {2379-5077},
support = {BBS/E/F/000PR10353/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {By modulating the structure, diversity, and trophic outputs of microbial communities, phages play crucial roles in many biomes. In oligotrophic polar deserts, the effects of katabatic winds, constrained nutrients, and low water availability are known to limit microbial activity. Although phages may substantially govern trophic interactions in cold deserts, relatively little is known regarding the precise ecological mechanisms. Here, we provide the first evidence of widespread antiphage innate immunity in Antarctic environments using metagenomic sequence data from hypolith communities as model systems. In particular, immunity systems such as DISARM and BREX are shown to be dominant systems in these communities. Additionally, we show a direct correlation between the CRISPR-Cas adaptive immunity and the metavirome of hypolith communities, suggesting the existence of dynamic host-phage interactions. In addition to providing the first exploration of immune systems in cold deserts, our results suggest that phages actively challenge niche communities in Antarctic polar deserts. We provide evidence suggesting that the regulatory role played by phages in this system is an important determinant of bacterial host interactions in this environment.IMPORTANCE In Antarctic environments, the combination of both abiotic and biotic stressors results in simple trophic levels dominated by microbiomes. Although the past two decades have revealed substantial insights regarding the diversity and structure of microbiomes, we lack mechanistic insights regarding community interactions and how phages may affect these. By providing the first evidence of widespread antiphage innate immunity, we shed light on phage-host dynamics in Antarctic niche communities. Our analyses reveal several antiphage defense systems, including DISARM and BREX, which appear to dominate in cold desert niche communities. In contrast, our analyses revealed that genes which encode antiphage adaptive immunity were underrepresented in these communities, suggesting lower infection frequencies in cold edaphic environments. We propose that by actively challenging niche communities, phages play crucial roles in the diversification of Antarctic communities.},
}
@article {pmid32371470,
year = {2020},
author = {Li, T and Zhang, Y and Dong, K and Kuo, CJ and Li, C and Zhu, YQ and Qin, J and Li, QT and Chang, YF and Guo, X and Zhu, Y},
title = {Isolation and Characterization of the Novel Phage JD032 and Global Transcriptomic Response during JD032 Infection of Clostridioides difficile Ribotype 078.},
journal = {mSystems},
volume = {5},
number = {3},
pages = {},
pmid = {32371470},
issn = {2379-5077},
abstract = {Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions.IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.},
}
@article {pmid32371392,
year = {2020},
author = {Chen, H and Shi, Z and Guo, J and Chang, KJ and Chen, Q and Yao, CH and Haigis, MC and Shi, Y},
title = {The human mitochondrial 12S rRNA m[4]C methyltransferase METTL15 is required for mitochondrial function.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {25},
pages = {8505-8513},
pmid = {32371392},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/metabolism ; Evolution, Molecular ; Gene Editing ; Genome, Mitochondrial ; Glycolysis ; Humans ; Kinetics ; Methylation ; Methyltransferases/genetics/*metabolism ; Microscopy, Fluorescence ; Mitochondria/genetics/*metabolism ; RNA, Messenger/metabolism ; RNA, Mitochondrial/metabolism ; RNA, Ribosomal/genetics/*metabolism ; Substrate Specificity ; },
abstract = {Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N[4]-methylation (m[4]C) at position 839 (m[4]C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m[4]C839 both in vivo and in vitro We tracked the evolutionary history of RNA m[4]C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m[4]C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m[4]C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.},
}
@article {pmid32371236,
year = {2020},
author = {van Wyk, N and Kroukamp, H and Espinosa, MI and von Wallbrunn, C and Wendland, J and Pretorius, IS},
title = {Blending wine yeast phenotypes with the aid of CRISPR DNA editing technologies.},
journal = {International journal of food microbiology},
volume = {324},
number = {},
pages = {108615},
doi = {10.1016/j.ijfoodmicro.2020.108615},
pmid = {32371236},
issn = {1879-3460},
mesh = {Acetic Acid/analysis/metabolism ; *CRISPR-Cas Systems ; Fermentation ; Gene Editing ; Glycerol/analysis/metabolism ; Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Phenotype ; Proteins/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Wine/analysis/*microbiology ; },
abstract = {In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.},
}
@article {pmid32370494,
year = {2020},
author = {Nouri, R and Jiang, Y and Lian, XL and Guan, W},
title = {Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN).},
journal = {ACS sensors},
volume = {5},
number = {5},
pages = {1273-1280},
doi = {10.1021/acssensors.0c00497},
pmid = {32370494},
issn = {2379-3694},
support = {R21 EB026035/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; *HIV-1/genetics ; *Nanopores ; },
abstract = {Nucleic acid detection methods are crucial for many fields such as pathogen detection and genotyping. Solid-state nanopore sensors represent a promising platform for nucleic acid detection due to its unique single molecule sensitivity and label-free electronic sensing. Here, we demonstrated the use of the glass nanopore for highly sensitive quantification of single-stranded circular DNAs (reporters), which could be degraded under the trans-cleavage activity of the target-specific CRISPR-Cas12a. We developed and optimized the Cas12a assay for HIV-1 analysis. We validated the concept of the solid-state CRISPR-Cas12a-assisted nanopores (SCAN) to specifically detect the HIV-1 DNAs. We showed that the glass nanopore sensor is effective in monitoring the cleavage activity of the target DNA-activated Cas12a. We developed a model to predict the total experimental time needed for making a statistically confident positive/negative call in a qualitative test. The SCAN concept combines the much-needed specificity and sensitivity into a single platform, and we anticipate that the SCAN would provide a compact, rapid, and low-cost method for nucleic acid detection at the point of care.},
}
@article {pmid32369642,
year = {2020},
author = {Tikunov, YM and Roohanitaziani, R and Meijer-Dekens, F and Molthoff, J and Paulo, J and Finkers, R and Capel, I and Carvajal Moreno, F and Maliepaard, C and Nijenhuis-de Vries, M and Labrie, CW and Verkerke, W and van Heusden, AW and van Eeuwijk, F and Visser, RGF and Bovy, AG},
title = {The genetic and functional analysis of flavor in commercial tomato: the FLORAL4 gene underlies a QTL for floral aroma volatiles in tomato fruit.},
journal = {The Plant journal : for cell and molecular biology},
volume = {103},
number = {3},
pages = {1189-1204},
pmid = {32369642},
issn = {1365-313X},
mesh = {Borates/*metabolism/standards ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Plant/genetics ; Food Quality ; Fructose/*analogs &amp; derivatives/metabolism/standards ; Gene Editing ; Genes, Plant/*genetics/physiology ; Lycopersicon esculentum/*genetics/metabolism/standards ; Phenylalanine/metabolism ; Quantitative Trait Loci/*genetics ; Quantitative Trait, Heritable ; Volatile Organic Compounds/metabolism ; },
abstract = {Tomato (Solanum lycopersicum L.) has become a popular model for genetic studies of fruit flavor in the last two decades. In this article we present a study of tomato fruit flavor, including an analysis of the genetic, metabolic and sensorial variation of a collection of contemporary commercial glasshouse tomato cultivars, followed by a validation of the associations found by quantitative trait locus (QTL) analysis of representative biparental segregating populations. This led to the identification of the major sensorial and chemical components determining fruit flavor variation and detection of the underlying QTLs. The high representation of QTL haplotypes in the breeders' germplasm suggests that there is great potential for applying these QTLs in current breeding programs aimed at improving tomato flavor. A QTL on chromosome 4 was found to affect the levels of the phenylalanine-derived volatiles (PHEVs) 2-phenylethanol, phenylacetaldehyde and 1-nitro-2-phenylethane. Fruits of near-isogenic lines contrasting for this locus and in the composition of PHEVs significantly differed in the perception of fruity and rose-hip-like aroma. The PHEV locus was fine mapped, which allowed for the identification of FLORAL4 as a candidate gene for PHEV regulation. Using a gene-editing-based (CRISPR-CAS9) reverse-genetics approach, FLORAL4 was demonstrated to be the key factor in this QTL affecting PHEV accumulation in tomato fruit.},
}
@article {pmid32368102,
year = {2020},
author = {Gholizadeh, P and Köse, &#x15e; and Dao, S and Ganbarov, K and Tanomand, A and Dal, T and Aghazadeh, M and Ghotaslou, R and Ahangarzadeh Rezaee, M and Yousefi, B and Samadi Kafil, H},
title = {How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance.},
journal = {Infection and drug resistance},
volume = {13},
number = {},
pages = {1111-1121},
pmid = {32368102},
issn = {1178-6973},
abstract = {Rapid emergence of antibiotic-resistant bacteria has made it harder for us to combat infectious diseases and to develop new antibiotics. The clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) system, as a bacterial adaptive immune system, is recognized as one of the new strategies for controlling antibiotic-resistant strains. The programmable Cas nuclease of this system used against bacterial genomic sequences could be lethal or could help reduce resistance of bacteria to antibiotics. Therefore, this study aims to review using the CRISPR-Cas system to promote sensitizing bacteria to antibiotics. We envision that CRISPR-Cas approaches may open novel ways for the development of smart antibiotics, which could eliminate multidrug-resistant (MDR) pathogens and differentiate between beneficial and pathogenic microorganisms. These systems can be exploited to quantitatively and selectively eliminate individual bacterial strains based on a sequence-specific manner, creating opportunities in the treatment of MDR infections, the study of microbial consortia, and the control of industrial fermentation.},
}
@article {pmid32367443,
year = {2020},
author = {Ye, S and Enghiad, B and Zhao, H and Takano, E},
title = {Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {47},
number = {4-5},
pages = {413-423},
pmid = {32367443},
issn = {1476-5535},
support = {P01 GM077596/GM/NIGMS NIH HHS/United States ; R01 AI144967/AI/NIAID NIH HHS/United States ; AI144967/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Multigene Family ; Plasmids/genetics ; *Recombination, Genetic ; Streptomyces/*genetics/metabolism ; Streptomyces coelicolor/genetics/metabolism ; },
abstract = {CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.},
}
@article {pmid32367255,
year = {2020},
author = {Wu, YC and Wang, IJ},
title = {Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish.},
journal = {Molecular genetics and genomics : MGG},
volume = {295},
number = {4},
pages = {911-922},
doi = {10.1007/s00438-020-01681-x},
pmid = {32367255},
issn = {1617-4623},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Embryo, Nonmammalian ; Embryonic Development ; Gene Expression Regulation, Enzymologic ; Gene Knockout Techniques ; Germ-Line Mutation ; Green Fluorescent Proteins/genetics ; Heat-Shock Response/*genetics ; Melanins/biosynthesis/genetics ; Monophenol Monooxygenase/*genetics ; Myosin Light Chains/genetics ; Promoter Regions, Genetic/genetics ; RNA/genetics ; Zebrafish/*genetics/growth &amp; development ; Zebrafish Proteins/genetics ; },
abstract = {Tyrosinase (TYR) converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into L-dopaquinone, which can produce melanin pigment. The abrogation of the functional activity of TYR can result in albino skin and eye diseases because of a deficiency in melanin pigment production. In this study, we developed and characterized an inducible knockout TYR platform comprising the heat-inducible heat-shock-promoter-70-driving CRISPR/Cas9 system and a zU6-promoter-driving tyr single guide RNA (sgRNA) system to investigate the temporal expression of TYR genes. To overcome the difficulty of identifying zebrafish germline integrations and facilitate the observation of Cas9 expression, heart-specific cmlc2:enhanced green fluorescent protein (EGFP; used to confirm tyr sgRNA expression) and two selectable markers (P2A-mCherry and internal ribosomal entry site-EGFP) were applied in our system. Heat shock treatment administered to Cas9 transgenic embryos induced mCherry or EGFP fluorescence expression throughout the embryos' bodies, and Cas9 protein was detected 1 h after heat shock treatment. Mutations were created by direct injection and line crossing, which led to mosaic and complete depigmentation phenotypes in approximately 50% and 100% of the embryos, respectively. Using our system, conditional TYR knockout in zebrafish was achieved efficiently and simply.},
}
@article {pmid32367056,
year = {2020},
author = {Goh, KJ and Chen, JH and Rocha, N and Semple, RK},
title = {Human pluripotent stem cell-based models suggest preadipocyte senescence as a possible cause of metabolic complications of Werner and Bloom Syndromes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7490},
pmid = {32367056},
issn = {2045-2322},
support = {//Wellcome Trust/United Kingdom ; MC_UU_00014/5/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adipocytes/*metabolism/pathology ; *Bloom Syndrome/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Cell Line ; *Cellular Senescence ; Gene Deletion ; Human Embryonic Stem Cells/*metabolism/pathology ; Humans ; *Models, Biological ; *Werner Syndrome/genetics/metabolism/pathology ; },
abstract = {Werner Syndrome (WS) and Bloom Syndrome (BS) are disorders of DNA damage repair caused by biallelic disruption of the WRN or BLM DNA helicases respectively. Both are commonly associated with insulin resistant diabetes, usually accompanied by dyslipidemia and fatty liver, as seen in lipodystrophies. In keeping with this, progressive reduction of subcutaneous adipose tissue is commonly observed. To interrogate the underlying cause of adipose tissue dysfunction in these syndromes, CRISPR/Cas9 genome editing was used to generate human pluripotent stem cell (hPSC) lacking either functional WRN or BLM helicase. No deleterious effects were observed in WRN[-/-] or BLM[-/-] embryonic stem cells, however upon their differentiation into adipocyte precursors (AP), premature senescence emerged, impairing later stages of adipogenesis. The resulting adipocytes were also found to be senescent, with increased levels of senescent markers and senescence-associated secretory phenotype (SASP) components. SASP components initiate and reinforce senescence in adjacent cells, which is likely to create a positive feedback loop of cellular senescence within the adipocyte precursor compartment, as demonstrated in normal ageing. Such a scenario could progressively attenuate adipose mass and function, giving rise to "lipodystrophy-like" insulin resistance. Further assessment of pharmacological senolytic strategies are warranted to mitigate this component of Werner and Bloom syndromes.},
}
@article {pmid32366884,
year = {2020},
author = {Pinzon-Arteaga, C and Snyder, MD and Lazzarotto, CR and Moreno, NF and Juras, R and Raudsepp, T and Golding, MC and Varner, DD and Long, CR},
title = {Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7411},
pmid = {32366884},
issn = {2045-2322},
mesh = {Animals ; Apoptosis ; Biotechnology/methods ; *CRISPR-Cas Systems ; Cell Line ; *Exons ; Fibroblasts/*metabolism ; *Gene Editing ; Genetic Engineering/methods ; Glycogen Storage Disease Type IV/*genetics/therapy/veterinary ; Homologous Recombination ; Horses ; Karyotyping ; Phenotype ; *Point Mutation ; RNA, Guide/genetics ; Skin/metabolism ; },
abstract = {Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1[102C>A]). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1[102C>A] mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.},
}
@article {pmid32366131,
year = {2020},
author = {McCarthy, MW},
title = {Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine.},
journal = {Expert review of anti-infective therapy},
volume = {18},
number = {8},
pages = {799-805},
pmid = {32366131},
issn = {1744-8336},
mesh = {Betacoronavirus ; COVID-19 ; *CRISPR-Cas Systems ; Coronavirus Infections/therapy ; Gene Editing/methods ; Genetic Therapy/*methods ; *Hospital Medicine ; Humans ; Pandemics ; Pneumonia, Viral/therapy ; SARS-CoV-2 ; },
abstract = {INTRODUCTION: Clustered regularly interspaced short palindromic repeats (CRISPR) are segments of nucleic acid that play a role in prokaryotic defense and form the basis of a genome editing technology that allows permanent alteration of genetic material. This methodology, known as CRISPR-Cas9, is poised to revolutionize molecular biology, but no literature yet exists on how these advances will affect hospitalists.<br><br>AREAS COVERED: These specialists in inpatient medicine care for a wide variety of hospitalized patients, including those with infectious disease, cancer, cardiovascular disease, autoimmune disease, hematologic disease, and a variety of other conditions that may soon be impacted by advances in gene-modifying technology provided by CRISPR-Cas9. A Literature search was performed using PubMed [1 December 2019-17 April 2020].<br><br>EXPERT OPINION: This paper reviews the remarkable diagnostic and therapeutic potential of the CRISPR-Cas9 platform and concludes with a look at ethical issues and technical hurdles pertaining to the implementation of permanent gene modification in the practice of Hospital Medicine.},
}
@article {pmid32364943,
year = {2020},
author = {Mukama, O and Wu, J and Li, Z and Liang, Q and Yi, Z and Lu, X and Liu, Y and Liu, Y and Hussain, M and Makafe, GG and Liu, J and Xu, N and Zeng, L},
title = {An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids.},
journal = {Biosensors &amp; bioelectronics},
volume = {159},
number = {},
pages = {112143},
doi = {10.1016/j.bios.2020.112143},
pmid = {32364943},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/standards ; *CRISPR-Cas Systems ; DNA, Bacterial ; DNA, Single-Stranded ; Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques ; *Nucleic Acids ; *Point-of-Care Systems ; Sensitivity and Specificity ; },
abstract = {CRISPR/Cas systems have displayed remarkable potential in developing novel biosensing applications for nucleic acid detection owing to the collateral cleavage activity of Cas effector proteins (Cas12, Cas13, etc.). Despite tremendous progress in recent years, the existing CRISPR/Cas based biosensing platforms have several limitations, including reliance on proper amplification methods, expensive fluorescence detection equipment, or lateral flow biosensor (LFB). Herein, we report a simple, inexpensive, and ultrasensitive DNA probe based LFB with CRISPR/Cas and loop-mediated Isothermal Amplification (namely CIA). The concept behind this approach is a non-detectable test line on the LFB when the Cas effector protein collaterally cleaves the cognate target and an ssDNA reporter sequence. The CIA based LFB can detect as low as a single copy cloned Pseudomonas aeruginosa acyltransferase gene, 1 cfu/ml plasmid containing E. coli DH5α pure cultures, as well as clinical samples without DNA extraction/purification or advanced apparatuses. No cross-reactivity with other non-target bacteria was observed. The naked eye result readout was obtained in 15 min of LAMP amplification, 30 min of Cas12 reaction, and 5 min of LFB readout. This platform is robust and of low cost for on-site testing.},
}
@article {pmid32364557,
year = {2020},
author = {Gong, Y and Tian, S and Xuan, Y and Zhang, S},
title = {Lipid and polymer mediated CRISPR/Cas9 gene editing.},
journal = {Journal of materials chemistry. B},
volume = {8},
number = {20},
pages = {4369-4386},
doi = {10.1039/d0tb00207k},
pmid = {32364557},
issn = {2050-7518},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Lipids/*chemistry ; Molecular Structure ; Particle Size ; Polymers/*chemistry ; Surface Properties ; },
abstract = {A clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) system is the most widely used tool for gene editing. Due to the advantages of simple operation, high efficiency and high specificity, it has been used in various fields. In this review, we introduce the latest research in the delivery technology of CRISPR/Cas9 based on lipids and polymers, which are synthesized through chemical methods. Many successful examples and methods are discussed. In addition, we discuss the potential of these delivery systems used for CRISPR/Cas9 in clinical applications in the future.},
}
@article {pmid32363688,
year = {2020},
author = {Wang, X and Yu, B and Jin, Q and Zhang, J and Yan, B and Yang, L and Li, Y and Li, Q and Wang, P and Sun, C and Liu, M and Tian, L and Sun, Y},
title = {Regulation of laryngeal squamous cell cancer progression by the lncRNA RP11-159K7.2/miR-206/DNMT3A axis.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {12},
pages = {6781-6795},
pmid = {32363688},
issn = {1582-4934},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Carcinoma, Squamous Cell/*genetics/*pathology ; Cell Line, Tumor ; Cell Proliferation ; DNA (Cytosine-5-)-Methyltransferases/genetics/*metabolism ; DNA Methylation/genetics ; DNA Methyltransferase 3A ; *Disease Progression ; Feedback, Physiological ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Laryngeal Neoplasms/*genetics/*pathology ; Male ; Mice, Inbred BALB C ; MicroRNAs/genetics/*metabolism ; Middle Aged ; Multivariate Analysis ; Neoplasm Invasiveness ; Prognosis ; RNA, Long Noncoding/genetics/*metabolism ; Up-Regulation/genetics ; },
abstract = {Long non-coding RNAs (lncRNAs), which are longer than 200 nt, have been proved to play a role in promoting or inhibiting cancer progression. The following study investigated the role and underlying mechanisms of lncRNA RP11-159K7.2 in laryngeal squamous cell carcinoma (LSCC) progression. Briefly, in situ hybridization (ISH) and real-time quantitative PCR (RT-qPCR) showed higher expression of RP11-159K7.2 in LSCC tissues and cell lines. Patients with low expression level of RP11-159K7.2 lived longer compared to those with high expression of RP11-159K7.2 (χ[2] = 39.111, ***P < 0.001). Multivariate Cox regression analysis suggested that lncRNA RP11-159K7.2 was an independent prognostic factor for LSCC patients (HR = 2.961, ***P < 0.001). Furthermore, to investigate the potential involvement of RP11-159K7.2 in the development of LSCC, we knocked out the expression of endogenous RP11-159K7.2 in TU-212 cells and AMC-HN-8 cells via CRISPR/Cas9 double vector lentiviral system. RP11-159K7.2 knockout decreased LSCC cell growth and invasion both in vitro and in vivo. Mechanically, we found that RP11-159K7.2 could positively regulate the expression of DNMT3A by sponging miR-206. In addition, a feedback loop was also discovered between DNMT3A and miR-206. To sum up, these findings suggest that lncRNA RP11-159K7.2 could be used as a potential biomarker for prognosis and treatment of LSCC.},
}
@article {pmid32363186,
year = {2020},
author = {Whelan, AI and Gutti, P and Lema, MA},
title = {Gene Editing Regulation and Innovation Economics.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {303},
pmid = {32363186},
issn = {2296-4185},
abstract = {Argentina was the first country that enacted regulatory criteria to assess if organisms resulting from new breeding techniques (NBTs) are to be regarded as genetically modified organisms (GMOs) or not. The country has now accumulated 4 year of experience applying such criteria, reaching a considerable number of cases, composed mostly of gene-edited plants, animals, and microorganisms of agricultural use. This article explores the effects on economic innovation of such regulatory experience. This is done by comparing the cases of products derived from gene editing and other NBTs that have been presented to the regulatory system, against the cases of GMOs that have been deregulated in the country. Albeit preliminary, this analysis suggests that products from gene editing will have different profiles and market release rates compared with the first wave of products from the so called "modern biotechnology." Gene editing products seems to follow a much faster development rate from bench to market. Such development is driven by a more diverse group of developers, and led mostly by small and medium enterprises (SMEs) and public research institutions. In addition, product profiles are also more diversified in terms of traits and organisms. The inferences of these findings for the agricultural and biotechnology sectors, particularly in developing countries, are discussed.},
}
@article {pmid32361465,
year = {2020},
author = {Yao, M and Yang, Q and Lian, M and Su, P and Cui, X and Ren, T and Wang, X and Wu, C and Zheng, Y},
title = {Generation of Dip2a homozygous knockout murine ES cell line IBMSe001-A-1 via CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {45},
number = {},
pages = {101778},
doi = {10.1016/j.scr.2020.101778},
pmid = {32361465},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Mice ; Nuclear Proteins ; Technology ; },
abstract = {DIP2A mutation is associated with abnormal brain development and diseases including dyslexia, autism and Alzheimer's disease. However, the role and the involved mechanisms remain unknown. To study the biological function of DIP2A during mESCs neural differentiation in early neural development, we generated a Dip2a homozygous knockout 46C ESC cell line using CRISPR/Cas9 genome editing technology. The eighth exon of Dip2a gene was replaced with PGK-Puro-P2A-mCherry. This 46C-Dip2a KO cell line offers a useful resource to investigate the molecular mechanisms of DIP2A in the process of cell fate determination, as well as a potential source of building disease mouse model.},
}
@article {pmid32361310,
year = {2020},
author = {Liu, CL and Huang, CY and Chen, HC and Lu, HE and Hsieh, PCH and Lee, JJ},
title = {Generation of a gene corrected human isogenic IBMS-iPSC-014-C from polycystic-kidney-disease induced pluripotent stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {45},
number = {},
pages = {101784},
doi = {10.1016/j.scr.2020.101784},
pmid = {32361310},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Induced Pluripotent Stem Cells ; Kidney ; },
abstract = {We report the engendering an isogenic iPSC line from the IBMS-iPSC-014-05 with homozygous correction of the R803X, Chr4: 88989098C > T in PKD2, using CRISPR/Cas9 technology. The results from the isogenic control, IBMS-iPSC-014-05C, showed that mutation had been corrected, while maintaining normal morphology, pluripotency, and differentiation capacity into three germ layers.},
}
@article {pmid32360576,
year = {2021},
author = {Song, X and Liu, C and Wang, N and Huang, H and He, S and Gong, C and Wei, Y},
title = {Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {158-180},
doi = {10.1016/j.addr.2020.04.010},
pmid = {32360576},
issn = {1872-8294},
mesh = {Animals ; Antineoplastic Agents, Immunological/*administration &amp; dosage ; Apoptosis/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Delivery Systems ; Epigenesis, Genetic/physiology ; Gene Editing ; Genes, Neoplasm/physiology ; Genetic Therapy/*methods ; Genetic Vectors/administration &amp; dosage ; Humans ; Neoplasms/*genetics/*therapy ; Tumor Microenvironment ; },
abstract = {The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are efficient and versatile gene editing tools, which offer enormous potential to treat cancer by editing genome, transcriptome or epigenome of tumor cells and/or immune cells. A large body of works have been done with CRISPR/Cas systems for genetic modification, and 16 clinical trials were conducted to treat cancer by ex vivo or in vivo gene editing approaches. Now, promising preclinical works have begun using CRISPR/Cas systems in vivo. However, efficient and safe delivery of CRISPR/Cas systems in vivo is still a critical challenge for their clinical applications. This article summarizes delivery of CRISPR/Cas systems by physical methods, viral vectors and non-viral vectors for cancer gene therapy and immunotherapy. The prospects for the development of physical methods, viral vectors and non-viral vectors for delivery of CRISPR/Cas systems are reviewed, and promising advances in cancer treatment using CRISPR/Cas systems are discussed.},
}
@article {pmid32360447,
year = {2021},
author = {Wang, S and Li, H and Kou, Z and Ren, F and Jin, Y and Yang, L and Dong, X and Yang, M and Zhao, J and Liu, H and Dong, N and Jia, L and Chen, X and Zhou, Y and Qiu, S and Hao, R and Song, H},
title = {Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system.},
journal = {Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases},
volume = {27},
number = {3},
pages = {443-450},
doi = {10.1016/j.cmi.2020.04.018},
pmid = {32360447},
issn = {1469-0691},
mesh = {Antiviral Agents/*pharmacology ; *CRISPR-Cas Systems ; DNA, Viral/*genetics ; Drug Resistance, Viral/*genetics ; Genotype ; Hepatitis B virus/*drug effects/genetics ; Mutation ; Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; },
abstract = {OBJECTIVES: Undetectable or low-level hepatitis B virus (HBV) DNA and drug resistance mutations in patients may increase the risk of HBV transmission or cause active viral replication and other clinical problems. Here, we established a highly sensitive and practical method for HBV and drug resistance detection using a polymerase chain reaction (PCR) -based CRISPR-Cas13a detection system (referred to as PCR-CRISPR) and evaluated its detection capability using clinical samples.<br><br>METHODS: Specific CRISPR RNAs (crRNAs) are designed for HBV DNA detection and YMDD (tyrosine-methionine-aspartate-aspartate)&#xa0;variant identification. The HBV DNA was detected in 312 serum samples for HBV diagnosis using quantification PCR (qPCR) and PCR-CRISPR. Additionally, 424 serum samples for YMDD testing were detected by qPCR, direct sequencing, and our assay.<br><br>RESULTS: Using PCR-CRISPR, one copy per test of HBV DNA was detected with HBV-1 crRNA in 15&#xa0;min after PCR amplification. Consistent results with qPCR were observed for 302 samples, while the remaining 10 samples with low-level HBV DNA were detectable by PCR-CRISPR and droplet digital PCR but not by qPCR. PCR-CRISPR diagnosed all 412 drug-resistant samples detected by the YMDD detection qPCR kit and direct sequencing, as well as the other 12 drug-resistant samples with low-level HBV DNA undetectable by qPCR and direct sequencing.<br><br>CONCLUSIONS: We developed a novel PCR-CRISPR method for highly sensitive and specific detection of HBV DNA and drug resistance mutations. One copy per test for HBV DNA and YMDD drug resistance mutations could be detected. This method has wide application prospects for the early detection of HBV infection, drug resistance monitoring and treatment guidance.},
}
@article {pmid32359477,
year = {2020},
author = {Soleimany, AP and Bhatia, SN},
title = {Activity-Based Diagnostics: An Emerging Paradigm for Disease Detection and Monitoring.},
journal = {Trends in molecular medicine},
volume = {26},
number = {5},
pages = {450-468},
pmid = {32359477},
issn = {1471-499X},
support = {/HHMI/Howard Hughes Medical Institute/United States ; T32 GM008313/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Biomarkers/metabolism ; Drug Monitoring/*methods ; Humans ; Infections/*diagnosis/metabolism ; Noncommunicable Diseases/*prevention &amp; control ; Precision Medicine/methods ; },
abstract = {Diagnostics to accurately detect disease and monitor therapeutic response are essential for effective clinical management. Bioengineering, chemical biology, molecular biology, and computer science tools are converging to guide the design of diagnostics that leverage enzymatic activity to measure or produce biomarkers of disease. We review recent advances in the development of these 'activity-based diagnostics' (ABDx) and their application in infectious and noncommunicable diseases. We highlight efforts towards both molecular probes that respond to disease-specific catalytic activity to produce a diagnostic readout, as well as diagnostics that use enzymes as an engineered component of their sense-and-respond cascade. These technologies exemplify how integrating techniques from multiple disciplines with preclinical validation has enabled ABDx that may realize the goals of precision medicine.},
}
@article {pmid32358986,
year = {2020},
author = {Pröbsting, M and Schenke, D and Hossain, R and Häder, C and Thurau, T and Wighardt, L and Schuster, A and Zhou, Z and Ye, W and Rietz, S and Leckband, G and Cai, D},
title = {Loss of function of CRT1a (calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape (Brassica napus).},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2328-2344},
pmid = {32358986},
issn = {1467-7652},
mesh = {*Arabidopsis/genetics ; *Brassica napus/genetics ; Calreticulin ; Plant Diseases/genetics ; *Verticillium ; },
abstract = {Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up-regulation of so-called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up-regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild-type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas-induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele-specific sequencing suggests a major effect of mutations in the CRT1a A-genome copy, while the C-genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.},
}
@article {pmid32358068,
year = {2020},
author = {Rowe, JB and Taghon, GJ and Kapolka, NJ and Morgan, WM and Isom, DG},
title = {CRISPR-addressable yeast strains with applications in human G protein-coupled receptor profiling and synthetic biology.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {24},
pages = {8262-8271},
pmid = {32358068},
issn = {1083-351X},
support = {R03 TR002908/TR/NCATS NIH HHS/United States ; R35 GM119518/GM/NIGMS NIH HHS/United States ; },
mesh = {Autocrine Communication ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Dosage ; Genes, Reporter ; Humans ; Metabolic Engineering ; Pheromones/metabolism ; Receptors, G-Protein-Coupled/*metabolism ; Receptors, Mating Factor/metabolism ; Receptors, Somatostatin/metabolism ; Reproducibility of Results ; Saccharomyces cerevisiae/*metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; Signal Transduction ; Somatostatin/analogs &amp; derivatives/pharmacology ; *Synthetic Biology ; },
abstract = {Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.},
}
@article {pmid32358066,
year = {2020},
author = {Melville, DB and Studer, S and Schekman, R},
title = {Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {25},
pages = {8401-8412},
pmid = {32358066},
issn = {1083-351X},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Binding Sites ; CRISPR-Cas Systems/genetics ; Cell Line ; Dimerization ; Gene Editing ; Guanosine Triphosphate/chemistry/metabolism ; Humans ; Molecular Dynamics Simulation ; Monomeric GTP-Binding Proteins/chemistry/deficiency/genetics/*metabolism ; Phylogeny ; Protein Binding ; Protein Conformation, alpha-Helical ; Recombinant Fusion Proteins/biosynthesis/chemistry/isolation &amp; purification ; Sequence Alignment ; Vesicular Transport Proteins/chemistry/*metabolism ; },
abstract = {Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.},
}
@article {pmid32358065,
year = {2020},
author = {Traughber, CA and Opoku, E and Brubaker, G and Major, J and Lu, H and Lorkowski, SW and Neumann, C and Hardaway, A and Chung, YM and Gulshan, K and Sharifi, N and Brown, JM and Smith, JD},
title = {Uptake of high-density lipoprotein by scavenger receptor class B type 1 is associated with prostate cancer proliferation and tumor progression in mice.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {24},
pages = {8252-8261},
pmid = {32358065},
issn = {1083-351X},
support = {R01 DK120679/DK/NIDDK NIH HHS/United States ; R01 HL128268/HL/NHLBI NIH HHS/United States ; R01 HL148158/HL/NHLBI NIH HHS/United States ; R01 HL128628/HL/NHLBI NIH HHS/United States ; P01 HL147823/HL/NHLBI NIH HHS/United States ; R01 CA190289/CA/NCI NIH HHS/United States ; P50 AA024333/AA/NIAAA NIH HHS/United States ; R01 CA172382/CA/NCI NIH HHS/United States ; P30 CA043703/CA/NCI NIH HHS/United States ; R01 CA236780/CA/NCI NIH HHS/United States ; F31 HL134231/HL/NHLBI NIH HHS/United States ; T32 GM088088/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Cell Line, Tumor ; Cell Proliferation ; Cholesterol/metabolism ; *Disease Progression ; Gene Expression Regulation, Neoplastic ; Humans ; Lipoproteins, HDL/*metabolism ; Male ; Mice, Inbred C57BL ; Prostatic Neoplasms/genetics/*metabolism/*pathology ; Scavenger Receptors, Class B/*metabolism ; Up-Regulation/genetics ; },
abstract = {High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however, its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1[+/+]) and SR-B1 KO (SR-B1[-/-]) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1[-/-] prostate cancer cells formed smaller tumors in WT hosts than SR-B1[+/+] cells in the same host model. Increased tumor volume was overall associated with reduced survival. We conclude that knocking out SR-B1 in prostate cancer tumors reduces HDL-associated increases in prostate cancer cell proliferation and disease progression.},
}
@article {pmid32358057,
year = {2020},
author = {Esbin, MN and Whitney, ON and Chong, S and Maurer, A and Darzacq, X and Tjian, R},
title = {Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection.},
journal = {RNA (New York, N.Y.)},
volume = {26},
number = {7},
pages = {771-783},
pmid = {32358057},
issn = {1469-9001},
support = {T32 GM007232/GM/NIGMS NIH HHS/United States ; T32 GM008295/GM/NIGMS NIH HHS/United States ; T32 GM098218/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; COVID-19 Testing ; CRISPR-Cas Systems ; Centers for Disease Control and Prevention, U.S. ; *Clinical Laboratory Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/diagnosis ; Humans ; Nasopharynx/virology ; Nucleic Acid Amplification Techniques/instrumentation/*methods ; Point-of-Care Systems ; RNA, Viral/genetics/isolation &amp; purification ; Real-Time Polymerase Chain Reaction/instrumentation/*methods ; SARS-CoV-2 ; Time Factors ; United States ; Workflow ; },
abstract = {The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.},
}
@article {pmid32357907,
year = {2020},
author = {Perez Kerkvliet, C and Dwyer, AR and Diep, CH and Oakley, RH and Liddle, C and Cidlowski, JA and Lange, CA},
title = {Glucocorticoid receptors are required effectors of TGFβ1-induced p38 MAPK signaling to advanced cancer phenotypes in triple-negative breast cancer.},
journal = {Breast cancer research : BCR},
volume = {22},
number = {1},
pages = {39},
pmid = {32357907},
issn = {1465-542X},
support = {ES090057//National Institute of Health/International ; UL1 TR002494/TR/NCATS NIH HHS/United States ; T32 GM008244/GM/NIGMS NIH HHS/United States ; R01 CA192178/CA/NCI NIH HHS/United States ; T32 CA009138/CA/NCI NIH HHS/United States ; F30 CA228261/CA/NCI NIH HHS/United States ; },
mesh = {Biomarkers, Tumor/*metabolism ; Cell Line, Tumor ; Cell Movement ; Extracellular Matrix Proteins/*metabolism ; Female ; Gene Editing ; Humans ; Neoplasm Staging ; Phosphorylation ; Receptors, Glucocorticoid/*metabolism ; Transcriptome ; Transforming Growth Factor beta/*metabolism ; Triple Negative Breast Neoplasms/metabolism/*pathology ; Tumor Microenvironment ; p38 Mitogen-Activated Protein Kinases/*metabolism ; },
abstract = {BACKGROUND: Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple-negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress.<br><br>METHODS: We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGF&#x3b2;1 in the absence of exogenously added GR ligands. Regulation of selected basal and TGF&#x3b2;1-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures.<br><br>RESULTS: In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGF&#x3b2;1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3&#x3b6; were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feedforward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGF&#x3b2;1 predicts shortened overall survival in breast cancer patients.<br><br>CONCLUSIONS: Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic "sensor" of intrinsic stimuli as well as extrinsic factors rich within the TME (TGF&#x3b2;1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.},
}
@article {pmid32357367,
year = {2020},
author = {Freund, EC and Lock, JY and Oh, J and Maculins, T and Delamarre, L and Bohlen, CJ and Haley, B and Murthy, A},
title = {Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9.},
journal = {The Journal of experimental medicine},
volume = {217},
number = {7},
pages = {},
pmid = {32357367},
issn = {1540-9538},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Dendritic Cells/metabolism ; Gene Deletion ; Gene Editing ; *Gene Knockout Techniques ; *Gene Transfer Techniques ; Genetic Engineering ; Genome ; Humans ; Macrophages/metabolism ; Mice ; Monocytes/metabolism ; Myeloid Cells/*metabolism ; Phagocytosis ; Phenotype ; RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; Viruses/metabolism ; },
abstract = {Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.},
}
@article {pmid32356906,
year = {2020},
author = {Cheng, L and Min, D and He, RL and Cheng, ZH and Liu, DF and Yu, HQ},
title = {Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {8},
pages = {2389-2400},
doi = {10.1002/bit.27368},
pmid = {32356906},
issn = {1097-0290},
support = {21590812//National Natural Science Foundation of China/International ; 21607146//National Natural Science Foundation of China/International ; 51878638//National Natural Science Foundation of China/International ; 51821006//National Natural Science Foundation of China/International ; 2018YFA0901301//National Key Research and Development Program of China/International ; 2018YFC0406303//National Key Research and Development Program of China/International ; },
mesh = {Acetylglucosamine/metabolism ; *Biodegradation, Environmental ; CRISPR-Cas Systems ; Carbon/*metabolism ; Environmental Pollutants/*metabolism ; Gene Editing/*methods ; *Shewanella/genetics/metabolism ; },
abstract = {Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.},
}
@article {pmid32356894,
year = {2020},
author = {Li, J and Chen, Z and Chen, F and Xie, G and Ling, Y and Peng, Y and Lin, Y and Luo, N and Chiang, CM and Wang, H},
title = {Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.},
journal = {Nucleic acids research},
volume = {48},
number = {10},
pages = {5684-5694},
pmid = {32356894},
issn = {1362-4962},
support = {R01 CA251698/CA/NCI NIH HHS/United States ; },
mesh = {5' Untranslated Regions ; Adenosine/analogs &amp; derivatives/metabolism ; AlkB Homolog 5, RNA Demethylase/genetics/*metabolism ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Cell Proliferation ; Demethylation ; HEK293 Cells ; HeLa Cells ; Humans ; Oncogenes ; Prevotella/enzymology ; Protein Engineering ; RNA, Messenger/chemistry/*metabolism ; Recombinant Fusion Proteins/metabolism ; },
abstract = {Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.},
}
@article {pmid32356712,
year = {2020},
author = {Ballouhey, O and Bartoli, M and Levy, N},
title = {[CRISPR-Cas9 for muscle dystrophies].},
journal = {Medecine sciences : M/S},
volume = {36},
number = {4},
pages = {358-366},
doi = {10.1051/medsci/2020081},
pmid = {32356712},
issn = {1958-5381},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; Humans ; Muscular Dystrophies/*genetics/*therapy ; Therapies, Investigational/methods/trends ; },
abstract = {Muscular dystrophies are a group of rare muscular disorders characterized by weakness and progressive degeneration of the muscle. They are diseases of genetic origin caused by the mutation of one or more genes involved in muscle function. Despite significant progress made in the field of biotherapies in recent years, there is as yet no curative treatment available for these diseases. Studies conducted since the discovery of the CRISPR-Cas9 genomic editing tool have nevertheless led to significant and promising advances in the treatment of muscular dystrophies. CRISPR-Cas9 system allows a stable and permanent edition of the genome and should make it possible to avoid long, partially efficient and repetitive treatments. In this review, we will discuss the latest therapeutic advances obtained using the CRISPR-Cas9 system in genetic muscular dystrophies.},
}
@article {pmid32355295,
year = {2020},
author = {Wu, M and Zhang, X and Wei, W and Long, L and An, S and Gao, G},
title = {CRISPR/Cas9 mediated genetic resource for unknown kinase and phosphatase genes in Drosophila.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7383},
pmid = {32355295},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; Drosophila melanogaster ; Humans ; Phosphoric Monoester Hydrolases/*genetics ; Protein Kinases/*genetics ; },
abstract = {Kinases and phosphatases are crucial for cellular processes and animal development. Various sets of resources in Drosophila have contributed significantly to the identification of kinases, phosphatases and their regulators. However, there are still many kinases, phosphatases and associate genes with unknown functions in the Drosophila genome. In this study, we utilized a CRISPR/Cas9 strategy to generate stable mutants for these unknown kinases, phosphatases and associate factors in Drosophila. For all the 156 unknown gene loci, we totally obtained 385 mutant alleles of 105 candidates, with 18 failure due to low efficiency of selected gRNAs and other 33 failure due to few recovered F0, which indicated high probability of lethal genes. From all the 105 mutated genes, we observed 9 whose mutants were lethal and another 4 sterile, most of which with human orthologs referred in OMIM, representing their huge value for human disease research. Here, we deliver these mutants as an open resource for more interesting studies.},
}
@article {pmid32355287,
year = {2020},
author = {Liu, X and Liu, T and Shang, Y and Dai, P and Zhang, W and Lee, BJ and Huang, M and Yang, D and Wu, Q and Liu, LD and Zheng, X and Zhou, BO and Dong, J and Yeap, LS and Hu, J and Xiao, T and Zha, S and Casellas, R and Liu, XS and Meng, FL},
title = {ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells.},
journal = {Cell research},
volume = {30},
number = {9},
pages = {732-744},
pmid = {32355287},
issn = {1748-7838},
support = {P01 CA174653/CA/NCI NIH HHS/United States ; R01 CA158073/CA/NCI NIH HHS/United States ; R01 CA215067/CA/NCI NIH HHS/United States ; R01 CA226852/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA/*metabolism ; DNA Damage/genetics ; DNA End-Joining Repair ; DNA Helicases/*metabolism ; DNA-Binding Proteins/metabolism ; Gene Regulatory Networks ; HEK293 Cells ; Humans ; Immunoglobulin Class Switching ; Immunoglobulin G/metabolism ; Mammals/*genetics ; Mice, Knockout ; Mutation/genetics ; Protein Binding ; V(D)J Recombination/*genetics ; },
abstract = {Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.},
}
@article {pmid32355272,
year = {2020},
author = {Audebert, C and Bonardi, F and Caboche, S and Guyot, K and Touzet, H and Merlin, S and Gantois, N and Creusy, C and Meloni, D and Mouray, A and Viscogliosi, E and Certad, G and Benamrouz-Vanneste, S and Chabé, M},
title = {Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7316},
pmid = {32355272},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Computational Biology ; Cryptosporidiosis/*parasitology ; Cryptosporidium parvum/*genetics/pathogenicity ; Feces ; Female ; Genome ; Genome, Protozoan ; Humans ; Male ; Mice ; Mice, SCID ; Middle Aged ; Oocysts ; Phenotype ; Virulence/*genetics ; Virulence Factors/*genetics ; Young Adult ; },
abstract = {Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.},
}
@article {pmid32355226,
year = {2021},
author = {Ali, G and Tariq, MA and Shahid, K and Ahmad, FJ and Akram, J},
title = {Advances in genome editing: the technology of choice for precise and efficient β-thalassemia treatment.},
journal = {Gene therapy},
volume = {28},
number = {1-2},
pages = {6-15},
pmid = {32355226},
issn = {1476-5462},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; Retrospective Studies ; Technology ; beta-Globins/genetics ; *beta-Thalassemia/genetics/therapy ; },
abstract = {Beta (β)-thalassemia is one of the most significant hemoglobinopathy worldwide. The high prevalence of the β-thalassemia carriers aggravates the disease burden for patients and national economies in the developing world. The survival of β-thalassemia patients solely relies on repeated transfusions, which eventually results into multi-organ damage. The fetal γ-globin genes are ordinarily silenced at birth and replaced by the adult β-globin genes. However, mutations that cause lifelong persistence of fetal γ-globin, ameliorate the debilitating effects of β-globin mutations. Therefore, therapeutically reactivating the fetal γ-globin gene is a prime focus of researchers. CRISPR/Cas9 is the most common approach to correct disease causative mutations or to enhance or disrupt the expression of proteins to mitigate the effects of the disease. CRISPR/cas9 and prime gene editing to correct mutations in hematopoietic stem cells of β-thalassemia patients has been considered a novel therapeutic approach for effective hemoglobin production. However, genome-editing technologies, along with all advantages, have shown some disadvantages due to either random insertions or deletions at the target site of edition or non-specific targeting in genome. Therefore, the focus of this review is to compare pros and cons of these editing technologies and to elaborate the retrospective scope of gene therapy for β-thalassemia patients.},
}
@article {pmid32355159,
year = {2020},
author = {Wienert, B and Nguyen, DN and Guenther, A and Feng, SJ and Locke, MN and Wyman, SK and Shin, J and Kazane, KR and Gregory, GL and Carter, MAM and Wright, F and Conklin, BR and Marson, A and Richardson, CD and Corn, JE},
title = {Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2109},
pmid = {32355159},
issn = {2041-1723},
support = {L40 AI140341/AI/NIAID NIH HHS/United States ; P30 AI027763/AI/NIAID NIH HHS/United States ; DP2 HL141006/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Cycle Proteins/*antagonists &amp; inhibitors/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing ; Genetic Engineering/methods ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Homologous Recombination ; Humans ; K562 Cells ; Phenotype ; Protein Serine-Threonine Kinases/*antagonists &amp; inhibitors/*genetics ; RNA, Guide/metabolism ; *Recombinational DNA Repair ; S Phase ; },
abstract = {Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.},
}
@article {pmid32354746,
year = {2020},
author = {Chen, X and Gao, YQ and Zheng, YY and Wang, W and Wang, P and Liang, J and Zhao, W and Tao, T and Sun, J and Wei, L and Li, Y and Zhou, Y and Gan, Z and Zhang, X and Chen, HQ and Zhu, MS},
title = {The intragenic microRNA miR199A1 in the dynamin 2 gene contributes to the pathology of X-linked centronuclear myopathy.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {26},
pages = {8656-8667},
pmid = {32354746},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Dynamin II/analysis/*genetics ; Female ; Longevity ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/analysis/*genetics ; Muscle Strength ; Muscle, Skeletal/metabolism/pathology ; Myopathies, Structural, Congenital/*genetics/pathology ; },
abstract = {Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1[-/y] disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1[-/-] with Mtm1[-/y] mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.},
}
@article {pmid32354171,
year = {2020},
author = {Ulman, A and Skrzypek, K and Konieczny, P and Mussolino, C and Cathomen, T and Majka, M},
title = {Genome Editing of the SNAI1 Gene in Rhabdomyosarcoma: A Novel Model for Studies of Its Role.},
journal = {Cells},
volume = {9},
number = {5},
pages = {},
pmid = {32354171},
issn = {2073-4409},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; Gene Expression ; HEK293 Cells ; Histone Deacetylase 1/metabolism ; Humans ; Rhabdomyosarcoma/*genetics ; Snail Family Transcription Factors/*genetics/metabolism ; Transcription Activator-Like Effector Nucleases ; },
abstract = {Genome editing (GE) tools and RNA interference technology enable the modulation of gene expression in cancer research. While GE mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALEN) activity can be used to induce gene knockouts, shRNA interacts with the targeted transcript, resulting in gene knockdown. Here, we compare three different methods for SNAI1 knockout or knockdown in rhabdomyosarcoma (RMS) cells. RMS is the most common sarcoma in children and its development has been previously associated with SNAI1 transcription factor activity. To investigate the role of SNAI1 in RMS development, we compared CRISPR/Cas9, TALEN, and shRNA tools to identify the most efficient tool for the modulation of SNAI1 expression with biological effects. Subsequently, the genome sequence, transcript levels, and protein expression of SNAI1 were evaluated. The modulation of SNAI1 using three different approaches affected the morphology of the cells and modulated the expression of myogenic factors and HDAC1. Our study revealed a similar effectiveness of the tested methods. Nevertheless, the low efficiency of the GE tools was a limiting factor in obtaining biallelic gene knockouts. To conclude, we established and characterized three different models of SNAI1 knockout and knockdown that might be used in further studies investigating the role of SNAI1 in RMS.},
}
@article {pmid32354036,
year = {2020},
author = {Horii, T and Kobayashi, R and Kimura, M and Morita, S and Hatada, I},
title = {Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.},
journal = {Cells},
volume = {9},
number = {5},
pages = {},
pmid = {32354036},
issn = {2073-4409},
mesh = {Alleles ; Animals ; Blastomeres/*metabolism ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Cell Fusion/methods ; Cytochalasin B/metabolism/pharmacology ; Electroporation/methods ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Female ; Genetic Engineering/*methods ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Knockout ; Zygote/drug effects/metabolism ; },
abstract = {The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca[2+])-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca[2+]-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca[2+]-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.},
}
@article {pmid32353627,
year = {2020},
author = {Li, J and Li, H and Chen, J and Yan, L and Xia, L},
title = {Toward Precision Genome Editing in Crop Plants.},
journal = {Molecular plant},
volume = {13},
number = {6},
pages = {811-813},
doi = {10.1016/j.molp.2020.04.008},
pmid = {32353627},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/*genetics ; *Gene Editing ; *Genome, Plant ; },
}
@article {pmid32353475,
year = {2020},
author = {Dhanjal, JK and Dammalapati, S and Pal, S and Sundar, D},
title = {Evaluation of off-targets predicted by sgRNA design tools.},
journal = {Genomics},
volume = {112},
number = {5},
pages = {3609-3614},
doi = {10.1016/j.ygeno.2020.04.024},
pmid = {32353475},
issn = {1089-8646},
mesh = {Algorithms ; *CRISPR-Cas Systems ; *Machine Learning ; RNA/*genetics ; RNA Editing ; },
abstract = {The ease of programming CRISPR/Cas9 system for targeting a specific location within the genome has paved way for many clinical and industrial applications. However, its widespread use is still limited owing to its off-target effects. Though this off-target activity has been reported to be dependent on both sgRNA sequence and experimental conditions, a clear understanding of the factors imparting specificity to CRISPR/Cas9 system is important. A machine learning-based computational model has been developed for prediction of off-targets with more likelihood to be cleaved in vivo with an accuracy of 91.49%. The sequence features important for the prediction of positive off-targets were found to be accessibility, mismatches, GC-content and position-specific conservation of nucleotides. The instructions and code to generate the dataset and reproduce the analysis has been made available at http://web.iitd.ac.in/crispcut/off-targets/.},
}
@article {pmid32353388,
year = {2020},
author = {Mitić, D and Radovčić, M and Markulin, D and Ivančić-Baće, I},
title = {StpA represses CRISPR-Cas immunity in H-NS deficient Escherichia coli.},
journal = {Biochimie},
volume = {174},
number = {},
pages = {136-143},
doi = {10.1016/j.biochi.2020.04.020},
pmid = {32353388},
issn = {1638-6183},
mesh = {*CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/*metabolism ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/genetics/*metabolism ; Fimbriae Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Molecular Chaperones/genetics/*metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Functional CRISPR-Cas systems provide many bacteria and most archaea with adaptive immunity against invading DNA elements. CRISPR arrays store DNA fragments of previous infections while products of cas genes provide immunity by integrating new DNA fragments and using this information to recognize and destroy invading DNA. Escherichia coli contains the CRISPR-Cas type I-E system in which foreign DNA targets are recognized by Cascade, a crRNA-guided complex comprising five proteins (CasA, CasB, CasC, CasD, CasE), and degraded by Cas3. In E. coli the CRISPR-Cas type I-E system is repressed by the histone-like nucleoid-structuring protein H-NS. H-NS repression can be relieved either by inactivation of the hns gene or by elevated levels of the H-NS antagonist LeuO, which induces higher transcript levels of cas genes than was observed for Δhns cells. This suggests that derepression in Δhns cells is incomplete and that an additional repressor could be involved in the silencing. One such candidate is the H-NS paralog protein StpA, which has DNA binding preferences similar to those of H-NS. Here we show that overexpression of StpA in Δhns cells containing anti-lambda spacers abolishes resistance to λvir infection and reduces transcription of the casA gene. In cells lacking hns and stpA genes, the transcript levels of the casA gene are higher than Δhns and similar to wt cells overexpressing LeuO. Taken together, these results suggest that Cascade genes in E. coli are repressed by the StpA protein when H-NS is absent.},
}
@article {pmid32353252,
year = {2020},
author = {Abbott, TR and Dhamdhere, G and Liu, Y and Lin, X and Goudy, L and Zeng, L and Chemparathy, A and Chmura, S and Heaton, NS and Debs, R and Pande, T and Endy, D and La Russa, MF and Lewis, DB and Qi, LS},
title = {Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.},
journal = {Cell},
volume = {181},
number = {4},
pages = {865-876.e12},
pmid = {32353252},
issn = {1097-4172},
mesh = {A549 Cells ; Antibiotic Prophylaxis/methods ; Antiviral Agents/*pharmacology ; Base Sequence ; Betacoronavirus/*drug effects/genetics/growth &amp; development ; COVID-19 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computer Simulation ; Conserved Sequence ; Coronavirus/drug effects/genetics/growth &amp; development ; Coronavirus Infections/drug therapy ; Coronavirus Nucleocapsid Proteins ; Coronavirus RNA-Dependent RNA Polymerase ; Epithelial Cells/virology ; Humans ; Influenza A Virus, H1N1 Subtype/*drug effects/genetics/growth &amp; development ; Lung/pathology/virology ; Nucleocapsid Proteins/genetics ; Pandemics ; Phosphoproteins ; Phylogeny ; Pneumonia, Viral/drug therapy ; RNA, Viral/*antagonists &amp; inhibitors ; RNA-Dependent RNA Polymerase/genetics ; SARS-CoV-2 ; Viral Nonstructural Proteins/genetics ; },
abstract = {The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.},
}
@article {pmid32352514,
year = {2020},
author = {Calvo-Villamañán, A and Ng, JW and Planel, R and Ménager, H and Chen, A and Cui, L and Bikard, D},
title = {On-target activity predictions enable improved CRISPR-dCas9 screens in bacteria.},
journal = {Nucleic acids research},
volume = {48},
number = {11},
pages = {e64},
pmid = {32352514},
issn = {1362-4962},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA-Directed RNA Polymerases/antagonists &amp; inhibitors ; Datasets as Topic ; Escherichia coli/*genetics ; *High-Throughput Screening Assays ; Linear Models ; RNA, Guide/*genetics ; Reproducibility of Results ; },
abstract = {The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.},
}
@article {pmid32350961,
year = {2020},
author = {Dang, L and Li, G and Wang, X and Huang, S and Zhang, Y and Miao, Y and Zeng, L and Cui, S and Huang, X},
title = {Comparison of gene disruption induced by cytosine base editing-mediated iSTOP with CRISPR/Cas9-mediated frameshift.},
journal = {Cell proliferation},
volume = {53},
number = {5},
pages = {e12820},
pmid = {32350961},
issn = {1365-2184},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytosine/*metabolism ; Frameshift Mutation/*genetics ; Gene Editing/methods ; Genotype ; HEK293 Cells ; Humans ; Plasmids/genetics ; RNA, Messenger/genetics ; Transfection/methods ; },
abstract = {OBJECTIVES: Recently developed CRISPR-dependent cytosine base editor (CBE), converting four codons (CAA, CAG, CGA and TGG) into stop codons without DNA double-strand breaks (DSB), serves as an efficient gene disruption strategy besides uncontrollable CRISPR-mediated frameshift. However, the detailed difference of gene knockout between the two systems has not been clarified.<br><br>MATERIALS AND METHODS: Here, we selected some sgRNAs with different position background, then HEK293T cells were transfected with CBE/Cas9 plasmids together with sgRNAs. GFP-positive cells were harvested by fluorescence-activated cell sorting (FACS) 48&#xa0;hours after transfection. Genomic DNA was collected for deep sequencing to analyse editing efficiency and genotype. RNA and protein were extracted to analyse gene mRNA level using qPCR analysis and Western blot.<br><br>RESULTS: Here, we compared the gene disruption by CBE-mediated iSTOP with CRISPR/Cas9-mediated frameshift. We found BE-mediated gene knockout yielded fewer genotypes. BE-mediated gene editing precisely achieved silencing of two neighbouring genes, while CRISPR/Cas9 may delete the large fragment between two target sites. All of three stop codons could efficiently disrupt the target genes. It is worth notifying, Cas9-mediated gene knockout showed a more impact on neighbouring genes mRNA level than the BE editor.<br><br>CONCLUSIONS: Our results reveal the differences between the two gene knockout strategies and provide useful information for choosing the appropriate gene disruption strategy.},
}
@article {pmid32350510,
year = {2020},
author = {Landmesser, U and Poller, W and Tsimikas, S and Most, P and Paneni, F and Lüscher, TF},
title = {From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.},
journal = {European heart journal},
volume = {41},
number = {40},
pages = {3884-3899},
doi = {10.1093/eurheartj/ehaa229},
pmid = {32350510},
issn = {1522-9645},
mesh = {Angiopoietin-Like Protein 3 ; Angiopoietin-like Proteins ; Animals ; *Cardiovascular Diseases/genetics/prevention &amp; control ; Gene Silencing ; Humans ; *Hypercholesterolemia ; *Nucleic Acids/therapeutic use ; Proprotein Convertase 9/genetics ; RNA ; },
abstract = {Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.},
}
@article {pmid32350113,
year = {2020},
author = {McDonald, IM and Graves, LM},
title = {Enigmatic MELK: The controversy surrounding its complex role in cancer.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {24},
pages = {8195-8203},
pmid = {32350113},
issn = {1083-351X},
support = {R01 CA199064/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Cell Proliferation ; Humans ; Models, Biological ; Neoplasms/*enzymology/pathology ; Protein Serine-Threonine Kinases/*metabolism ; },
abstract = {The Ser/Thr protein kinase MELK (maternal embryonic leucine zipper kinase) has been considered an attractive therapeutic target for managing cancer since 2005. Studies using expression analysis have indicated that MELK expression is higher in numerous cancer cells and tissues than in their normal, nonneoplastic counterparts. Further, RNAi-mediated MELK depletion impairs proliferation of multiple cancers, including triple-negative breast cancer (TNBC), and these growth defects can be rescued with exogenous WT MELK, but not kinase-dead MELK complementation. Pharmacological MELK inhibition with OTS167 (alternatively called OTSSP167) and NVS-MELK8a, among other small molecules, also impairs cancer cell growth. These collective results led to MELK being classified as essential for cancer proliferation. More recently, in 2017, the proliferation of TNBC and other cancer cell lines was reported to be unaffected by genetic CRISPR/Cas9-mediated MELK deletion, calling into question the essentiality of this kinase in cancer. To date, the requirement of MELK in cancer remains controversial, and mechanisms underlying the disparate growth effects observed with RNAi, pharmacological inhibition, and CRISPR remain unclear. Our objective with this review is to highlight the evidence on both sides of this controversy, to provide commentary on the purported requirement of MELK in cancer, and to emphasize the need for continued elucidation of the functions of MELK.},
}
@article {pmid32349666,
year = {2020},
author = {Brandt, K and Nethery, MA and O'Flaherty, S and Barrangou, R},
title = {Genomic characterization of Lactobacillus fermentum DSM 20052.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {328},
pmid = {32349666},
issn = {1471-2164},
mesh = {CRISPR-Cas Systems/genetics ; Genetic Variation ; *Genome, Bacterial ; Genomic Islands ; Genomics ; Interspersed Repetitive Sequences ; Lactobacillus fermentum/classification/*genetics ; Phylogeny ; },
abstract = {BACKGROUND: Lactobacillus fermentum, a member of the lactic acid bacteria complex, has recently garnered increased attention due to documented antagonistic properties and interest in assessing the probiotic potential of select strains that may provide human health benefits. Here, we genomically characterize L. fermentum using the type strain DSM 20052 as a canonical representative of this species.<br><br>RESULTS: We determined the polished whole genome sequence of this type strain and compared it to 37 available genome sequences within this species. Results reveal genetic diversity across nine clades, with variable content encompassing mobile genetic elements, CRISPR-Cas immune systems and genomic islands, as well as numerous genome rearrangements. Interestingly, we determined a high frequency of occurrence of diverse Type I, II, and III CRISPR-Cas systems in 72% of the genomes, with a high level of strain hypervariability.<br><br>CONCLUSIONS: These findings provide a basis for the genetic characterization of L. fermentum strains of scientific and commercial interest. Furthermore, our study enables genomic-informed selection of strains with specific traits for commercial product formulation, and establishes a framework for the functional characterization of features of interest.},
}
@article {pmid32349563,
year = {2020},
author = {Tromp, TR and Stroes, ESG and Hovingh, GK},
title = {Gene-based therapy in lipid management: the winding road from promise to practice.},
journal = {Expert opinion on investigational drugs},
volume = {29},
number = {5},
pages = {483-493},
doi = {10.1080/13543784.2020.1757070},
pmid = {32349563},
issn = {1744-7658},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiovascular Diseases/etiology/*prevention &amp; control ; Genetic Therapy/*methods ; Heart Disease Risk Factors ; Humans ; Hypercholesterolemia/complications/genetics/*therapy ; Lipid Metabolism/genetics ; Oligonucleotides, Antisense/administration &amp; dosage ; },
abstract = {INTRODUCTION: Cardiovascular disease (CVD) is a leading cause of morbidity and mortality. High plasma low-density lipoprotein cholesterol (LDL-C) levels are a key CVD-risk factor. Triglyceride-rich remnant particles and lipoprotein(a) (Lp[a]) are also causally related to CVD. Consequently, therapeutic strategies for lowering LDL-C and triglyceride levels are widely used in routine clinical practice; however, specific Lp(a) lowering agents are not available. Many patients do not achieve guideline-recommended lipid levels with currently available therapies; hence, novel targets and treatment modalities are eagerly sought.<br><br>AREAS COVERED: We discuss the milestones on the trajectory toward the full application of gene-based therapies in daily clinical practice. We describe the different methods, ranging from antisense oligonucleotides to liver-directed gene therapy and Crispr-cas9 modification to target the pivotal players in lipid metabolism: PCSK9, APOB, ANGPTL3, Lp(a), LDLR, and apoC-III.<br><br>EXPERT OPINION: While acknowledging their different stages of development, gene-based therapies are likely to invoke a paradigm shift in lipid management because they allow us to target previously undruggable targets. Moreover, their low dosing frequency, high target selectivity, and relatively predictable adverse event profile are considered major advantages over current lipid-lowering therapies.},
}
@article {pmid32349249,
year = {2020},
author = {Fuster-García, C and García-Bohórquez, B and Rodríguez-Muñoz, A and Millán, JM and García-García, G},
title = {Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies.},
journal = {Genes},
volume = {11},
number = {5},
pages = {},
pmid = {32349249},
issn = {2073-4425},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Frameshift Mutation/genetics ; Gene Editing/*methods ; *Genetic Engineering ; Genetic Heterogeneity ; Genetic Therapy/trends ; Humans ; Phenotype ; Retinal Dystrophies/*genetics/pathology/therapy ; },
abstract = {Inherited retinal dystrophies are an assorted group of rare diseases that collectively account for the major cause of visual impairment of genetic origin worldwide. Besides clinically, these vision loss disorders present a high genetic and allelic heterogeneity. To date, over 250 genes have been associated to retinal dystrophies with reported causative variants of every nature (nonsense, missense, frameshift, splice-site, large rearrangements, and so forth). Except for a fistful of mutations, most of them are private and affect one or few families, making it a challenge to ratify the newly identified candidate genes or the pathogenicity of dubious variants in disease-associated loci. A recurrent option involves altering the gene in in vitro or in vivo systems to contrast the resulting phenotype and molecular imprint. To validate specific mutations, the process must rely on simulating the precise genetic change, which, until recently, proved to be a difficult endeavor. The rise of the CRISPR/Cas9 technology and its adaptation for genetic engineering now offers a resourceful suite of tools to alleviate the process of functional studies. Here we review the implementation of these RNA-programmable Cas9 nucleases in culture-based and animal models to elucidate the role of novel genes and variants in retinal dystrophies.},
}
@article {pmid32349121,
year = {2020},
author = {Ackerman, CM and Myhrvold, C and Thakku, SG and Freije, CA and Metsky, HC and Yang, DK and Ye, SH and Boehm, CK and Kosoko-Thoroddsen, TF and Kehe, J and Nguyen, TG and Carter, A and Kulesa, A and Barnes, JR and Dugan, VG and Hung, DT and Blainey, PC and Sabeti, PC},
title = {Massively multiplexed nucleic acid detection with Cas13.},
journal = {Nature},
volume = {582},
number = {7811},
pages = {277-282},
pmid = {32349121},
issn = {1476-4687},
support = {F32 CA236425/CA/NCI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Betacoronavirus/genetics/isolation &amp; purification ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*genetics ; Drug Resistance, Viral/genetics ; Genome, Viral/genetics ; HIV/classification/genetics/isolation &amp; purification ; Humans ; Influenza A virus/classification/genetics/isolation &amp; purification ; Microfluidic Analytical Techniques/instrumentation/*methods ; RNA, Guide/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; Virus Diseases/*diagnosis/*virology ; },
abstract = {The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples[1-3] while simultaneously testing for many pathogens[4-6]. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents[7] self-organize in a microwell array[8] to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health[9-11].},
}
@article {pmid32348779,
year = {2020},
author = {Lin, P and Qin, S and Pu, Q and Wang, Z and Wu, Q and Gao, P and Schettler, J and Guo, K and Li, R and Li, G and Huang, C and Wei, Y and Gao, GF and Jiang, J and Wu, M},
title = {CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.},
journal = {Molecular cell},
volume = {78},
number = {5},
pages = {850-861.e5},
pmid = {32348779},
issn = {1097-4164},
support = {R03 AI097532/AI/NIAID NIH HHS/United States ; R01 AI138203/AI/NIAID NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; R01 AI109317/AI/NIAID NIH HHS/United States ; P20 GM103442/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacteria/genetics ; Bacteriophages/genetics ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/genetics ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Leptotrichia/genetics/metabolism ; RNA/genetics ; RNA Editing/genetics/*physiology ; },
abstract = {Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.},
}
@article {pmid32348727,
year = {2020},
author = {Michels, BE and Mosa, MH and Streibl, BI and Zhan, T and Menche, C and Abou-El-Ardat, K and Darvishi, T and Członka, E and Wagner, S and Winter, J and Medyouf, H and Boutros, M and Farin, HF},
title = {Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids.},
journal = {Cell stem cell},
volume = {26},
number = {5},
pages = {782-792.e7},
doi = {10.1016/j.stem.2020.04.003},
pmid = {32348727},
issn = {1875-9777},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Colon ; Genes, Tumor Suppressor ; Humans ; *Organoids ; },
abstract = {Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC[-/-];KRAS[G12D] mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.},
}
@article {pmid32347937,
year = {2020},
author = {Samolygo, A and Athukoralage, JS and Graham, S and White, MF},
title = {Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence.},
journal = {Nucleic acids research},
volume = {48},
number = {11},
pages = {6149-6156},
pmid = {32347937},
issn = {1362-4962},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacteria/*enzymology ; Bacterial Proteins/*metabolism ; Binding Sites ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Models, Molecular ; Protein Domains ; RNA/metabolism ; RNA Stability ; Ribonucleases/*metabolism ; Second Messenger Systems ; },
abstract = {Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.},
}
@article {pmid32347797,
year = {2020},
author = {Ghinia Tegla, MG and Buenaventura, DF and Kim, DY and Thakurdin, C and Gonzalez, KC and Emerson, MM},
title = {OTX2 represses sister cell fate choices in the developing retina to promote photoreceptor specification.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32347797},
issn = {2050-084X},
support = {T34 GM007639/GM/NIGMS NIH HHS/United States ; T34GM007639/GM/NIGMS NIH HHS/United States ; G12 MD007603/MD/NIMHD NIH HHS/United States ; R01EY024982/EY/NEI NIH HHS/United States ; 3G12MD007603/MD/NIMHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Chickens ; Female ; Gene Editing ; Gene Regulatory Networks ; Male ; Mutation ; Otx Transcription Factors/genetics/*physiology ; PAX6 Transcription Factor/analysis ; Photoreceptor Cells, Vertebrate/*physiology ; Retina/*embryology ; Sequence Analysis, RNA ; Single-Cell Analysis ; },
abstract = {During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices.},
}
@article {pmid32347717,
year = {2020},
author = {Lu, M and Tokuyasu, TA},
title = {CRISPR-Cas13-Based RNA-Interacting Protein Detection in Living Cells.},
journal = {Biochemistry},
volume = {59},
number = {19},
pages = {1791-1792},
doi = {10.1021/acs.biochem.0c00281},
pmid = {32347717},
issn = {1520-4995},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/genetics ; },
}
@article {pmid32347065,
year = {2020},
author = {Wang, K and An, W and Liu, Y and Liu, D and Feng, L and Wang, J and Huang, J and Liu, X and Liang, W},
title = {[Disruption of OsRhoGDI2 by CRISPR/Cas9 technology leads to semi-dwarf in rice].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {4},
pages = {707-715},
doi = {10.13345/j.cjb.190333},
pmid = {32347065},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems ; *Genes, Plant/genetics ; *Oryza/genetics/growth &amp; development ; Plants, Genetically Modified ; rho Guanine Nucleotide Dissociation Inhibitor beta/genetics ; },
abstract = {OsRhoGDI2 was isolated as a putative partner of Rho protein family member OsRacD from rice panicles by yeast two-hybrid, but its function remains unknown. In order to identify the function of OsRhoGDI2, OsRhoGDI2 knockout mutants were created by CRISPR/Cas9 technology. The results showed that two different homozygous mutants were obtained in T0 generation, and eight kinds homozygous mutants were identified in T1 generation. Sequence analysis revealed that the base substitution or base deletion occurred near the editing targets of the gene in knockout rice, and it could be expected that the truncated OsRhoGDI2 proteins lacking the RhoGDI conserved domain would be generated. Phenotype analysis showed that the OsRhoGDI2 knockout rice plants were significantly lower than the control plants. Statistical analysis confirmed that the significant decrease of plant height was due to the shortening of the second and third internodes, suggesting that OsRhoGDI2 gene may be related with rice height control.},
}
@article {pmid32346859,
year = {2020},
author = {Zhang, Q and Jiang, B and Du, Z and Chasin, LA},
title = {A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {8},
pages = {2401-2409},
doi = {10.1002/bit.27367},
pmid = {32346859},
issn = {1097-0290},
support = {//Merck &amp; Co., Inc./International ; },
mesh = {Animals ; *Antibodies, Monoclonal/genetics/metabolism ; CHO Cells ; CRISPR-Cas Systems ; Cell Line ; Cricetinae ; Cricetulus ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Purines/metabolism ; Pyrimidines/metabolism ; *Recombinant Proteins/genetics/metabolism ; Transfection ; },
abstract = {Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.},
}
@article {pmid32346757,
year = {2020},
author = {Schaffert, L and Schneiker-Bekel, S and Gierhake, J and Droste, J and Persicke, M and Rosen, W and Pühler, A and Kalinowski, J},
title = {Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {12},
pages = {5395-5408},
pmid = {32346757},
issn = {1432-0614},
mesh = {ATP-Binding Cassette Transporters/*genetics/metabolism ; Acarbose/*metabolism ; Actinoplanes/*genetics/*metabolism ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Carbohydrate Metabolism ; Gene Deletion ; Multigene Family ; Protein Binding ; Proteome/metabolism ; Starch/metabolism ; },
abstract = {Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8-16% higher product titers when grown in maltose-containing medium.},
}
@article {pmid32346083,
year = {2020},
author = {Soliman, SHA and Stark, AE and Gardner, ML and Harshman, SW and Breece, CC and Amari, F and Orlacchio, A and Chen, M and Tessari, A and Martin, JA and Visone, R and Freitas, MA and La Perle, KMD and Palmieri, D and Coppola, V},
title = {Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7138},
pmid = {32346083},
issn = {2045-2322},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cytoskeletal Proteins/genetics/*metabolism ; Immunohistochemistry ; Mice ; Mice, Knockout ; Nuclear Proteins/genetics/*metabolism ; Phosphoproteins/*metabolism ; Protein Binding ; RNA, Messenger/metabolism ; RNA-Binding Proteins/*metabolism ; },
abstract = {The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.},
}
@article {pmid32346064,
year = {2020},
author = {Adamski, V and Hattermann, K and Kubelt, C and Cohrs, G and Lucius, R and Synowitz, M and Sebens, S and Held-Feindt, J},
title = {Entry and exit of chemotherapeutically-promoted cellular dormancy in glioblastoma cells is differentially affected by the chemokines CXCL12, CXCL16, and CX3CL1.},
journal = {Oncogene},
volume = {39},
number = {22},
pages = {4421-4435},
pmid = {32346064},
issn = {1476-5594},
mesh = {Antineoplastic Agents, Alkylating/*pharmacology ; Brain Neoplasms/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chemokine CX3CL1 ; Chemokine CXCL12 ; Chemokine CXCL16 ; Gene Expression Regulation, Neoplastic/*drug effects ; Glioblastoma/*pathology ; Humans ; Primary Cell Culture ; Real-Time Polymerase Chain Reaction ; Temozolomide/*pharmacology ; Transcriptome ; },
abstract = {Glioblastoma multiforme (GBM) is a malignant brain tumor that evades therapy regimens. Since cellular dormancy is one strategy for surviving, and since chemokines determine the environmental conditions in which dormancy occurs, we investigated how chemokines affect temozolomide (TMZ)-promoted cellular dormancy entry and exit in GBM cells. TMZ administration over ten days promoted cellular dormancy entry, whereas discontinuing TMZ for a further 15 days resulted in resumption of proliferation. Co-administration of a chemokine cocktail containing CXCL12, CXCL16, and CX3CL1 resulted in both delayed entry and exit from cellular dormancy. A microarray-based transcriptome analysis in LN229 GBM cells revealed that cellular dormancy entry was characterized by an increased expression of CCL2 and SAA2, while THSD4, FSTL3, and VEGFC were upregulated during dormancy exit. Co-stimulation with the chemokine cocktail reduced upregulation of identified genes. After verifying the appearance of identified genes in human GBM primary cultures and ex vivo samples, we clarified whether each chemokine alone impacts cellular dormancy mechanisms using specific antagonists and selective CRISPR/Cas9 clones. While expression of CCL2 and SAA2 in LN229 cells was altered by the CXCL12-CXCR4-CXCR7 axis, CXCL16 and CX3CL1 contributed to reduced upregulation of THSD4 and, to a weaker extent, of VEGFC. The influence on FSTL3 expression depended on the entire chemokine cocktail. Effects of chemokines on dormancy entry and exit-associated genes were detectable in human GBM primary cells, too, even if in a more complex, cell-specific manner. Thus, chemokines play a significant role in the regulation of TMZ-promoted cellular dormancy in GBMs.},
}
@article {pmid32345641,
year = {2020},
author = {DeWerff, SJ and Bautista, MA and Pauly, M and Zhang, C and Whitaker, RJ},
title = {Killer Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism.},
journal = {mBio},
volume = {11},
number = {2},
pages = {},
pmid = {32345641},
issn = {2150-7511},
mesh = {Archaea/*virology ; *Bacteriophages ; Biological Evolution ; CRISPR-Cas Systems/*immunology ; Evolution, Molecular ; Genome, Viral ; *Host Microbial Interactions ; *Sulfolobus/genetics/virology ; Symbiosis ; },
abstract = {Theory, simulation, and experimental evolution demonstrate that diversified CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of host-virus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immune Sulfolobus islandicus cells and immune-deficient strains that are infected by the chronic virus SSV9. We demonstrate that Sulfolobus islandicus cells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversified CRISPR-Cas immunity.IMPORTANCE Multiple studies, especially those focusing on the role of lytic viruses in key model systems, have shown the importance of viruses in shaping microbial populations. However, it has become increasingly clear that viruses with a long host-virus interaction, such as those with a chronic lifestyle, can be important drivers of evolution and have large impacts on host ecology. In this work, we describe one such interaction with the acidic crenarchaeon Sulfolobus islandicus and its chronic virus Sulfolobus spindle-shaped virus 9. Our work expands the view in which this symbiosis between host and virus evolved, describing a killing phenotype which we hypothesize has evolved in part due to the high prevalence and diversity of CRISPR-Cas immunity seen in natural populations. We explore the implications of this phenotype in population dynamics and host ecology, as well as the implications of mutualism between this virus-host pair.},
}
@article {pmid32345633,
year = {2020},
author = {Prezza, G and Heckel, T and Dietrich, S and Homberger, C and Westermann, AJ and Vogel, J},
title = {Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.},
journal = {RNA (New York, N.Y.)},
volume = {26},
number = {8},
pages = {1069-1078},
pmid = {32345633},
issn = {1469-9001},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/*genetics ; DNA, Complementary/genetics ; Gene Expression Profiling/methods ; Gene Library ; High-Throughput Nucleotide Sequencing/methods ; Nucleic Acid Hybridization/methods ; RNA, Bacterial/*genetics ; RNA, Ribosomal/*genetics ; RNA-Seq/*methods ; Sequence Analysis, RNA/methods ; },
abstract = {A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.},
}
@article {pmid32344896,
year = {2020},
author = {Xu, X and Hulshoff, MS and Tan, X and Zeisberg, M and Zeisberg, EM},
title = {CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications.},
journal = {International journal of molecular sciences},
volume = {21},
number = {9},
pages = {},
pmid = {32344896},
issn = {1422-0067},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Chromatin/ultrastructure ; DNA/metabolism ; Endodeoxyribonucleases/*metabolism ; Endonucleases/metabolism ; Epigenomics/*methods ; Gene Editing/*methods ; Gene Transfer Techniques ; Green Fluorescent Proteins/analysis/genetics ; Optical Imaging ; RNA, Guide/genetics ; Recombinant Fusion Proteins/analysis ; Substrate Specificity ; Telomere/ultrastructure ; Transcription Activator-Like Effector Nucleases/metabolism ; Transcription Activator-Like Effectors/metabolism ; Transcription, Genetic ; Zinc Fingers ; },
abstract = {The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.},
}
@article {pmid32344599,
year = {2020},
author = {Monsur, MB and Shao, G and Lv, Y and Ahmad, S and Wei, X and Hu, P and Tang, S},
title = {Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants.},
journal = {Genes},
volume = {11},
number = {4},
pages = {},
pmid = {32344599},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Engineering ; Plant Proteins/*genetics ; Plants/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach.},
}
@article {pmid32344465,
year = {2020},
author = {Mohammadzadeh, I and Qujeq, D and Yousefi, T and Ferns, GA and Maniati, M and Vaghari-Tabari, M},
title = {CRISPR/Cas9 gene editing: A new therapeutic approach in the treatment of infection and autoimmunity.},
journal = {IUBMB life},
volume = {72},
number = {8},
pages = {1603-1621},
doi = {10.1002/iub.2296},
pmid = {32344465},
issn = {1521-6551},
mesh = {Autoimmune Diseases/*genetics/therapy ; Autoimmunity/*genetics/immunology ; Bacteria/genetics/pathogenicity/virology ; CRISPR-Associated Protein 9/*genetics/therapeutic use ; CRISPR-Cas Systems/*genetics ; Genome, Bacterial/genetics ; Genome, Viral/genetics ; Humans ; RNA/genetics/therapeutic use ; Virus Diseases/genetics/therapy/virology ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) may be viewed as an adaptive bacterial immune system. When a virus infects a bacterium, a fragment of the virus genome is inserted into the CRISPR sequence of the bacterial genome as a memory. When the bacterium becomes infected again with the same virus, an RNA molecule that is a transcript of the memory sequence, directs Cas9, an endonuclease, to the complementary region of the virus genome, and Cas9 disables the virus by a double-strand break. In recent years, studies have shown that by designing synthetic RNA molecules and delivering them along with Cas9 into eukaryotic cells, different regions of the cell's genome can be targeted and manipulated. These findings have drawn much attention to this new technology and it has been shown that CRISPR/Cas9 gene editing can be used to treat some human diseases. These include infectious diseases and autoimmune diseases. In this review article, in addition to a brief overview of the biology of the CRISPR/Cas9 system, we collected the most recent findings on the applications of CRISPR/Cas9 technology for better investigation of the pathogenesis and treatment of viral infections (human immunodeficiency virus infection, hepatitis virus infections, and onco-virus infections), non-viral infections (parasitic, fungal, and bacterial infections), and autoimmune diseases.},
}
@article {pmid32343689,
year = {2020},
author = {Gisler, S and Maia, ARR and Chandrasekaran, G and Kopparam, J and van Lohuizen, M},
title = {A genome-wide enrichment screen identifies NUMA1-loss as a resistance mechanism against mitotic cell-death induced by BMI1 inhibition.},
journal = {PloS one},
volume = {15},
number = {4},
pages = {e0227592},
pmid = {32343689},
issn = {1932-6203},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/drug effects/genetics ; CRISPR-Cas Systems/genetics ; Carcinogenesis/drug effects/*genetics ; Cell Cycle Proteins/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Drug Resistance, Neoplasm/*genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; M Phase Cell Cycle Checkpoints/drug effects ; Mutation ; Neoplasms/drug therapy/*genetics/pathology ; Polycomb Repressive Complex 1/antagonists &amp; inhibitors/genetics/*metabolism ; RNA, Small Interfering/metabolism ; },
abstract = {BMI1 is a core protein of the polycomb repressive complex 1 (PRC1) that is overexpressed in several cancer types, making it a promising target for cancer therapies. However, the underlying mechanisms and interactions associated with BMI1-induced tumorigenesis are often context-dependent and complex. Here, we performed a drug resistance screen on mutagenized human haploid HAP1 cells treated with BMI1 inhibitor PTC-318 to find new genetic and mechanistic features associated with BMI1-dependent cancer cell proliferation. Our screen identified NUMA1-mutations as the most significant inducer of PTC-318 cell death resistance. Independent validations on NUMA1-proficient HAP1 and non-small cell lung cancer cell lines exposed to BMI1 inhibition by PTC-318 or BMI1 knockdown resulted in cell death following mitotic arrest. Interestingly, cells with CRISPR-Cas9 derived NUMA1 knockout also showed a mitotic arrest phenotype following BMI1 inhibition but, contrary to cells with wildtype NUMA1, these cells were resistant to BMI1-dependent cell death. The current study brings new insights to BMI1 inhibition-induced mitotic lethality in cancer cells and presents a previously unknown role of NUMA1 in this process.},
}
@article {pmid32343230,
year = {2020},
author = {Cruz-Becerra, G and Kadonaga, JT},
title = {Enhancement of homology-directed repair with chromatin donor templates in cells.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32343230},
issn = {2050-084X},
support = {R35 GM118060/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; Drosophila ; *Gene Editing/methods ; HEK293 Cells ; Humans ; Recombinational DNA Repair/*genetics ; Transfection/methods ; },
abstract = {A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes.},
}
@article {pmid32342312,
year = {2020},
author = {Silaeva, YY and Kalmykov, VA and Varlamova, EA and Korshunov, EN and Korshunova, DS and Kubekina, MV and Shtil, AA and Roninson, IB and Deykin, AV},
title = {Genome Editing As an Approach to the Study of in Vivo Transcription Reprogramming.},
journal = {Doklady. Biochemistry and biophysics},
volume = {490},
number = {1},
pages = {43-46},
pmid = {32342312},
issn = {1608-3091},
mesh = {Animals ; CRISPR-Cas Systems ; Catalytic Domain ; Cyclin-Dependent Kinase 8/*metabolism ; Exons ; Gene Editing/*methods ; *Genome ; Heterozygote ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Mutation ; Oligonucleotides/genetics ; *RNA, Guide ; *Transcription, Genetic ; },
abstract = {CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'-3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.},
}
@article {pmid32341578,
year = {2020},
author = {Lu, Y and Xue, J and Deng, T and Zhou, X and Yu, K and Deng, L and Huang, M and Yi, X and Liang, M and Wang, Y and Shen, H and Tong, R and Wang, W and Li, L and Song, J and Li, J and Su, X and Ding, Z and Gong, Y and Zhu, J and Wang, Y and Zou, B and Zhang, Y and Li, Y and Zhou, L and Liu, Y and Yu, M and Wang, Y and Zhang, X and Yin, L and Xia, X and Zeng, Y and Zhou, Q and Ying, B and Chen, C and Wei, Y and Li, W and Mok, T},
title = {Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer.},
journal = {Nature medicine},
volume = {26},
number = {5},
pages = {732-740},
pmid = {32341578},
issn = {1546-170X},
mesh = {Adolescent ; Adult ; Aged ; CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/immunology/pathology/*therapy ; Drug Resistance, Neoplasm/immunology ; Feasibility Studies ; Female ; Gene Editing/*methods ; Genetic Therapy/adverse effects/methods ; Humans ; *Immunotherapy, Adoptive/adverse effects/methods ; Lung Neoplasms/immunology/pathology/*therapy ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor/*genetics/metabolism ; T-Lymphocytes/metabolism/*transplantation ; Treatment Outcome ; Young Adult ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of immune checkpoint genes could improve the efficacy of T cell therapy, but the first necessary undertaking is to understand the safety and feasibility. Here, we report results from a first-in-human phase I clinical trial of CRISPR-Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856). Primary endpoints were safety and feasibility, and the secondary endpoint was efficacy. The exploratory objectives included tracking of edited T cells. All prespecified endpoints were met. PD-1-edited T cells were manufactured ex vivo by cotransfection using electroporation of Cas9 and single guide RNA plasmids. A total of 22 patients were enrolled; 17 had sufficient edited T cells for infusion, and 12 were able to receive treatment. All treatment-related adverse events were grade 1/2. Edited T cells were detectable in peripheral blood after infusion. The median progression-free survival was 7.7 weeks (95% confidence interval, 6.9 to 8.5 weeks) and median overall survival was 42.6 weeks (95% confidence interval, 10.3-74.9 weeks). The median mutation frequency of off-target events was 0.05% (range, 0-0.25%) at 18 candidate sites by next generation sequencing. We conclude that clinical application of CRISPR-Cas9 gene-edited T cells is generally safe and feasible. Future trials should use superior gene editing approaches to improve therapeutic efficacy.},
}
@article {pmid32341525,
year = {2020},
author = {Wang, B and Lin, Z and Li, X and Zhao, Y and Zhao, B and Wu, G and Ma, X and Wang, H and Xie, Y and Li, Q and Song, G and Kong, D and Zheng, Z and Wei, H and Shen, R and Wu, H and Chen, C and Meng, Z and Wang, T and Li, Y and Li, X and Chen, Y and Lai, J and Hufford, MB and Ross-Ibarra, J and He, H and Wang, H},
title = {Genome-wide selection and genetic improvement during modern maize breeding.},
journal = {Nature genetics},
volume = {52},
number = {6},
pages = {565-571},
pmid = {32341525},
issn = {1546-1718},
mesh = {CRISPR-Cas Systems ; China ; Genome, Plant ; *Genome-Wide Association Study ; Linkage Disequilibrium ; Phenotype ; Plant Breeding/*methods ; Plant Proteins/genetics ; Plants, Genetically Modified ; Polymorphism, Single Nucleotide ; Quantitative Trait, Heritable ; Reproducibility of Results ; United States ; Zea mays/*genetics/physiology ; },
abstract = {Since the development of single-hybrid maize breeding programs in the first half of the twentieth century[1], maize yields have increased over sevenfold, and much of that increase can be attributed to tolerance of increased planting density[2-4]. To explore the genomic basis underlying the dramatic yield increase in maize, we conducted a comprehensive analysis of the genomic and phenotypic changes associated with modern maize breeding through chronological sampling of 350 elite inbred lines representing multiple eras of germplasm from both China and the United States. We document several convergent phenotypic changes in both countries. Using genome-wide association and selection scan methods, we identify 160 loci underlying adaptive agronomic phenotypes and more than 1,800 genomic regions representing the targets of selection during modern breeding. This work demonstrates the use of the breeding-era approach for identifying breeding signatures and lays the foundation for future genomics-enabled maize breeding.},
}
@article {pmid32341482,
year = {2020},
author = {Burgio, G},
title = {Gene conversion following CRISPR/Cas9 DNA cleavage: an overlooked effect.},
journal = {Gene therapy},
volume = {27},
number = {6},
pages = {245-246},
pmid = {32341482},
issn = {1476-5462},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *DNA Cleavage ; Gene Conversion ; Gene Editing ; RNA, Guide ; },
}
@article {pmid32341348,
year = {2020},
author = {Hopke, A and Scherer, A and Kreuzburg, S and Abers, MS and Zerbe, CS and Dinauer, MC and Mansour, MK and Irimia, D},
title = {Neutrophil swarming delays the growth of clusters of pathogenic fungi.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2031},
pmid = {32341348},
issn = {2041-1723},
support = {R01 GM092804/GM/NIGMS NIH HHS/United States ; R01 AI132638/AI/NIAID NIH HHS/United States ; R01 HL140837/HL/NHLBI NIH HHS/United States ; P41 EB002503/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Candida albicans/*growth &amp; development ; Candidiasis/microbiology ; Cell Line ; Extracellular Traps/metabolism ; Granulocyte Colony-Stimulating Factor/pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology ; Granulomatous Disease, Chronic/microbiology ; Humans ; Image Processing, Computer-Assisted ; Microarray Analysis ; NADPH Oxidases/metabolism ; Neutrophils/*cytology/*microbiology ; Peroxidase/metabolism ; Reactive Oxygen Species/metabolism ; },
abstract = {Neutrophils employ several mechanisms to restrict fungi, including the action of enzymes such as myeloperoxidase (MPO) or NADPH oxidase, and the release of neutrophil extracellular traps (NETs). Moreover, they cooperate, forming "swarms" to attack fungi that are larger than individual neutrophils. Here, we designed an assay for studying how these mechanisms work together and contribute to neutrophil's ability to contain clusters of live Candida. We find that neutrophil swarming over Candida clusters delays germination through the action of MPO and NADPH oxidase, and restricts fungal growth through NET release within the swarm. In comparison with neutrophils from healthy subjects, those from patients with chronic granulomatous disease produce larger swarms against Candida, but their release of NETs is delayed, resulting in impaired control of fungal growth. We also show that granulocyte colony-stimulating factors (GCSF and GM-CSF) enhance swarming and neutrophil ability to restrict fungal growth, even during treatment with chemical inhibitors that disrupt neutrophil function.},
}
@article {pmid32340238,
year = {2020},
author = {Seys, FM and Rowe, P and Bolt, EL and Humphreys, CM and Minton, NP},
title = {A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences.},
journal = {Genes},
volume = {11},
number = {4},
pages = {},
pmid = {32340238},
issn = {2073-4425},
support = {BB/L013940/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/K00283X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L502030/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Clostridium/*genetics/metabolism ; *Gene Editing ; *Gene Knockout Techniques ; *Genetic Complementation Test ; Genetic Engineering/*methods ; Genome, Bacterial ; Reference Standards ; },
abstract = {Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for 'gold standard' complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) 'bookmark' sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of 'watermark' sequences into the complementing genes.},
}
@article {pmid32339904,
year = {2020},
author = {Chen, S and Luo, Z and Ward, C and Ibañez, DP and Liu, H and Zhong, X and Sharma, NK and Qin, B and Fan, W and Wang, D},
title = {Generation of two LRRK2 homozygous knockout human induced pluripotent stem cell lines using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {45},
number = {},
pages = {101804},
doi = {10.1016/j.scr.2020.101804},
pmid = {32339904},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics ; Mutation ; *Parkinson Disease/genetics ; },
abstract = {Mutations in the Leucine rich repeat kinase 2 (LRRK2) gene are found in both familial and sporadic Parkinson's disease (PD), and are also associated with immune-related disorders including Crohn's disease (CD) and leprosy. We have generated two homozygous LRRK2 knockout human induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 in a well-characterized human iPSC clone. The LRRK2 knockout cell lines retained normal morphology, gene expression, and the capacity to differentiate into cell types of the three germ layers. These cell lines are valuable for elucidating the role of LRRK2 in innate immunity and PD.},
}
@article {pmid32339532,
year = {2020},
author = {Leung, AW and Broton, C and Bogacheva, MS and Xiao, AZ and Garcia-Castro, MI and Lou, YR},
title = {RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells.},
journal = {Journal of molecular biology},
volume = {432},
number = {13},
pages = {3956-3964},
doi = {10.1016/j.jmb.2020.04.017},
pmid = {32339532},
issn = {1089-8638},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Humans ; Loss of Function Mutation/genetics ; Mutagenesis/*genetics ; Pluripotent Stem Cells/*cytology ; RNA/*genetics ; RNA, Guide ; Transfection ; },
abstract = {Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.},
}
@article {pmid32339389,
year = {2020},
author = {Huang, L and Li, Q and Zhang, C and Chu, R and Gu, Z and Tan, H and Zhao, D and Fan, X and Liu, Q},
title = {Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2164-2166},
pmid = {32339389},
issn = {1467-7652},
mesh = {Alleles ; Amylose ; CRISPR-Cas Systems/genetics ; *Oryza/genetics/metabolism ; *Starch Synthase/genetics ; },
}
@article {pmid32338600,
year = {2020},
author = {Meinke, S and Goldammer, G and Weber, AI and Tarabykin, V and Neumann, A and Preussner, M and Heyd, F},
title = {Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32338600},
issn = {2050-084X},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics ; Cell Line ; *Gene Expression Regulation, Developmental ; Humans ; *Introns ; Mice ; RNA Splicing ; Repressor Proteins/*genetics ; Serine-Arginine Splicing Factors/*genetics ; Spliceosomes/*genetics ; },
abstract = {Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that Srsf10 expression is controlled through an autoregulated minor intron, tightly correlating Srsf10 with minor spliceosome abundance across different tissues and differentiation stages in mammals. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing Srsf10 autoregulation by Crispr/Cas9-mediated deletion of the autoregulatory exon induces expression of all SR proteins in a human cell line. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing.},
}
@article {pmid32338598,
year = {2020},
author = {Athukoralage, JS and Graham, S and Rouillon, C and Grüschow, S and Czekster, CM and White, MF},
title = {The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32338598},
issn = {2050-084X},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Escherichia coli/enzymology/genetics ; *Host Microbial Interactions ; Nucleotides, Cyclic/*metabolism ; *Signal Transduction ; Sulfolobus solfataricus/genetics/metabolism ; Viruses/*enzymology/*genetics ; },
abstract = {Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an 'off-switch' for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.},
}
@article {pmid32338048,
year = {2020},
author = {Hong, L and Zhang, C and Jiang, Y and Liu, H and Huang, H and Guo, D},
title = {Therapeutic status and the prospect of CRISPR/Cas9 gene editing in multiple myeloma.},
journal = {Future oncology (London, England)},
volume = {16},
number = {16},
pages = {1125-1136},
doi = {10.2217/fon-2019-0822},
pmid = {32338048},
issn = {1744-8301},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Immunotherapy/*methods ; Multiple Myeloma/*genetics/immunology/*therapy ; },
abstract = {In recent years, CRISPR/Cas9, a novel gene-editing technology, has shown considerable potential in the design of novel research methods and future options for treating multiple myeloma (MM). The use of CRISPR/Cas9 promises faster and more accurate identification and validation of target genes. In this review, we summarize the current research status of the application of CRISPR technology in MM, especially in detecting the expression of MM gene, exploring the mechanism of drug action, screening for drug-resistant genes, developing immunotherapy and screening for new drug targets. Given the tremendous progress that has been made, we believe that CRISPR/Cas9 possesses great potential in MM-related clinical practice.},
}
@article {pmid32337966,
year = {2020},
author = {Wang, Y and Ke, Y and Liu, W and Sun, Y and Ding, X},
title = {A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level.},
journal = {ACS sensors},
volume = {5},
number = {5},
pages = {1427-1435},
doi = {10.1021/acssensors.0c00320},
pmid = {32337966},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems ; DNA ; *Escherichia coli O157/genetics ; Polymerase Chain Reaction ; },
abstract = {Bacterial contamination accounts for more than half of food poisoning cases. Conventional methods such as colony-counting and general polymerase chain reaction are time-consuming, instrument-dependent, and sometimes not accurate. Herein, we developed a novel one-pot toolbox with precision and ultra sensitivity (OCTOPUS) platform for foodborne pathogen detection based on the mechanism in which Cas12a nontarget binding unleashes its collateral DNase activity. We demonstrated its application on two widespread foodborne bacteria, namely, E. coli O157:H7 and Streptococcus aureus, using specific crRNA targeting rfbE and nuc gene, respectively. For better sensitivity, recombinase polymerase amplification (RPA) was integrated without product purification. This one-pot detection, that is, RPA reagent, crRNA, and ssDNA-FQ reporter are all in one tube with the subsequent addition of Cas12a enzyme, was able to detect genomic DNA at the attomolar level. It omits an extra cap-opening process to avoid practical inconvenience and possible cross-sample contamination. Moreover, we demonstrated this platform for a real food matrix. A simple water boiling method for genome extraction together with one-pot assay achieved a limit of detection value of 1 CFU/mL in less than 50 min. This OCTOPUS technique integrates bacterial genome extraction, preamplification based on RPA, and Cas12a/crRNA cleavage assay.},
}
@article {pmid32336701,
year = {2020},
author = {Lin, J and Li, Z and Feng, Z and Fang, Z and Chen, J and Chen, W and Liang, W and Chen, Q},
title = {Pseudorabies virus (PRV) strain with defects in gE, gC, and TK genes protects piglets against an emerging PRV variant.},
journal = {The Journal of veterinary medical science},
volume = {82},
number = {6},
pages = {846-855},
pmid = {32336701},
issn = {1347-7439},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Deletion ; Genes, Viral ; HEK293 Cells ; Herpesvirus 1, Suid/*genetics/*immunology ; Humans ; Immunization ; Injections, Intramuscular ; Mice, Inbred BALB C ; Pseudorabies/*prevention &amp; control/virology ; Pseudorabies Vaccines/*administration &amp; dosage/immunology ; Swine ; Swine Diseases/*prevention &amp; control/virology ; Vaccines, Attenuated ; },
abstract = {The prevalence of an emerging variant of the pseudorabies virus (PRV) has been causing serious losses to farmers in China. Moreover, the commercially available PRV vaccine often fails to provide thorough protection. Therefore, in this study, we generated a PRV-∆gC\gE∆TK strain with defects in gC, gE, and TK of PRV. Compared to the parental PRV strain and the single gene deletion strains (PRV-∆gC, PRV-∆gE, and PRV-∆TK), PRV-∆gC\gE∆TK grew slowly, and exhibited fewer and smaller plaques on swine testis (ST) cells. Furthermore, animal experiment results showed that mice that were immunized intramuscularly with PRV-∆gC\gE∆TK, survived throughout the experiment with no observed clinical symptoms, and were completely protected against PRV challenge. Additionally, deletion of the gC, gE, and TK genes significantly alleviated viral damage in the brain. Furthermore, one-day-old weaned piglets immunized intramuscularly with PRV-∆gC\gE∆TK elicited higher levels of gB antibodies against both the emerging PRV variant and the parental PRV, exhibited full protection against challenge with both variants, and showed neutralization capacity against PRV. These data suggest that PRV-∆gC\gE∆TK is a promising vaccine candidate for the control of pseudorabies.},
}
@article {pmid32336015,
year = {2020},
author = {Tan, J and Zhao, Y and Wang, B and Hao, Y and Wang, Y and Li, Y and Luo, W and Zong, W and Li, G and Chen, S and Ma, K and Xie, X and Chen, L and Liu, YG and Zhu, Q},
title = {Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining.},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2161-2163},
pmid = {32336015},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; Gene Editing ; Genomics ; },
}
@article {pmid32335155,
year = {2020},
author = {Yuan, F and Sun, M and Liu, H and Qian, F},
title = {Albumin-conjugated drug is irresistible by single gene mutation of endocytic system: Verification by genome-wide CRISPR-Cas9 loss-of-function screens.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {323},
number = {},
pages = {311-320},
doi = {10.1016/j.jconrel.2020.04.035},
pmid = {32335155},
issn = {1873-4995},
mesh = {Albumins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mutation ; *Pharmaceutical Preparations ; },
abstract = {Albumin-conjugated drugs attain KRAS mutant cancer targeting through KRAS-enhanced macropinocytosis and intensified lysosomal degradation due to reduced neonatal Fc receptor (FcRn) expression. The cytosolic delivery of active payloads relies on endocytosis and subsequent intracellular processing of albumin delivery vehicles, wherein complex regulatory mechanisms and molecular machineries are closely involved. Despite the obvious merit of KRAS targeting, could such an endocytic process involving extra molecular regulators also bring about extra vulnerabilities to albumin-conjugated drugs, particularly, unexpected drug resistance? To assess such risks, here we performed an unbiased drug resistance mechanism comparison in pancreatic cancer, between free triptolide (TP, a potent cytotoxin) and albumin-conjugated TP, using genome-wide CRISPR-Cas9 loss-of-function screens. GTF2H5, a subunit of GTF2H transcription factor complex, was the only hit identified regardless of forms of TP treatment. With drug efficacy tests on GTF2H5 knockout clones, we further concluded that GTF2H5 deficiency conferred drug resistance primarily due to the pharmacological mechanism of action (MoA) of TP. In addition, molecules previously considered to be able to affect endocytosis and intracellular processing were not enriched during the screening with albumin-conjugated TP. With the aid of genome-wide CRISPR-Cas9 loss-of-function screens, we conclude that the pharmacological resistance of the active payload, rather than any potential loss-of-function mutations in endocytic molecular machineries, is the solely crucial drug resistance mechanism of albumin-conjugated drugs.},
}
@article {pmid32335151,
year = {2020},
author = {Trautmann, C and Bock, A and Urbach, A and Hübner, CA and Engmann, O},
title = {Acute vitamin B12 supplementation evokes antidepressant response and alters Ntrk-2.},
journal = {Neuropharmacology},
volume = {171},
number = {},
pages = {108112},
doi = {10.1016/j.neuropharm.2020.108112},
pmid = {32335151},
issn = {1873-7064},
mesh = {Animals ; Antidepressive Agents/*pharmacology ; Behavior, Animal ; CRISPR-Cas Systems ; Cell Line ; DNA Methylation/drug effects ; Depression/*drug therapy/psychology ; Epigenomics ; Female ; Gene Expression/drug effects ; Male ; Membrane Glycoproteins/*antagonists &amp; inhibitors ; Mice ; Mice, Inbred C57BL ; Prefrontal Cortex/drug effects/metabolism ; Protein-Tyrosine Kinases/*antagonists &amp; inhibitors ; Receptor, trkB/drug effects ; Vitamin B 12/*pharmacology ; },
abstract = {Depression is the leading cause of disability worldwide. Although most research into risk factors focuses on stress, dietary factors also have a strong link with depression. For instance, chronic vitamin B12-supplementation may reduce depression risk and helps to reverse the prodepressive effects of early life stress in animal models. However, it is still unclear whether a single acute dose of vitamin B12 is sufficient to induce antidepressant effects on molecular or behavioral levels. Based on pharmacological work and CRISPR-dCas9 epigenome editing in Neuro2A-cells we provide in vitro evidence for a link between vitamin B12, gene expression and DNA methylation of the antidepressant-associated gene Ntrk-2, which codes for the BDNF-receptor TRKB. Using stress-induction protocols in C57Bl/6 J mice combined with behavioral testing and subsequent molecular tissue analysis, we establish in vivo evidence for antidepressant effects of vitamin B12. Acute supplementation with vitamin B12, but not folic acid, selectively altered DNA methylation and gene expression of Ntrk-2 in vitro, albeit DNA methylation and Ntrk-2 gene expression do not correlate in vivo. Importantly, one acute vitamin B12 injection improved multiple behavioral measures in tests for antidepressant action and at the same time reversed the effects of chronic and acute stress on Ntrk-2 levels in vivo, however causality has not been proven at this stage. Taken together, acute vitamin B12 supplementation can reverse stress effects on Ntrk-2 gene expression and improve behaviors that are associated with depression-like behavior in mice. Our findings encourage further investigation of vitamin B12-supplementation as a novel model for antidepressant action.},
}
@article {pmid32334681,
year = {2020},
author = {Chang, W and Liu, W and Shen, H and Chen, S and Liao, P and Liu, Y},
title = {Molecular AND logic gate for multiple single-nucleotide mutations detection based on CRISPR/Cas9n system-trigged signal amplification.},
journal = {Analytica chimica acta},
volume = {1112},
number = {},
pages = {46-53},
doi = {10.1016/j.aca.2020.03.058},
pmid = {32334681},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems/*genetics ; Fluorescence ; Mutation ; *Nucleic Acid Amplification Techniques ; },
abstract = {Precise detection of single-nucleotide mutations (SNMs) is extremely important in various biomedical applications, but the simultaneous detection of multiple SNMs remains a great challenge. Herein, we developed a new method based on CRISPR/Cas9 system for multiple SNMs detection. The CRISPR/Cas9 system transduces the nucleic acid into an intermediate trigger to initiate the isothermal amplification reaction and further form fluorescence signals. According to this strategy, we established nucleic acid bio-computing operations-molecule logic gate in simultaneous distinction of the genetic locus. We demonstrate that the fluorescence signals generated from different input combinations can be used to discriminate the multiple genetic locus, and the molecular logic gate has great potential in single-base mismatch detection. In addition, the successful assay of real samples indicates that the novel strategy could further adapt for the pathogenic monitoring and biomedical research.},
}
@article {pmid32334589,
year = {2020},
author = {Luo, Y and Ge, M and Wang, B and Sun, C and Wang, J and Dong, Y and Xi, JJ},
title = {CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {93},
pmid = {32334589},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Rhodobacter sphaeroides/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled.<br><br>RESULTS: Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones.<br><br>CONCLUSIONS: Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.},
}
@article {pmid32334351,
year = {2020},
author = {Wang, YQ and Li, W and Zhuang, JL and Liu, YD and Shapleigh, JP},
title = {Bacteriophage-mediated extracellular DNA release is important for the structural stability of aerobic granular sludge.},
journal = {The Science of the total environment},
volume = {726},
number = {},
pages = {138392},
doi = {10.1016/j.scitotenv.2020.138392},
pmid = {32334351},
issn = {1879-1026},
mesh = {Bacteria ; *Bacteriophages ; Bioreactors ; DNA ; Metagenome ; *Sewage ; },
abstract = {The aim of this study was to investigate the microbial characteristics and the structural role of exDNA in different size AGSs. Metagenomic results showed that exDNA has a significantly lower GC content, ~46.0%, than the ~65.0% GC of intracellular DNA (inDNA). Taxonomic predictions showed most of the reads from the exDNA that could be taxonomically assigned were from members of the phyla Bacteroidetes (55.0-64.2% of the total exDNA reads). Assigned inDNA reads were mainly from Proteobacteria (50.9-57.8%) or Actinobacteria (18.0-28.0%). Reads mapping showed that exDNA read depths were similar across all predicted open reading frames from assembled genomes that were assigned as Bacteroidetes which is consistent with cell lysis as a source of exDNA. Enrichment of CRISPR-CAS proteins in exDNA reads and CRISPR spacers in Bacteroidetes associated draft genomes suggested that bacteriophage infection may be an important cause of lysis of these cells. A critical role for this exDNA was found using DNase I digestion experiments which showed that the exDNA was vital for the structural stability of relatively small sized AGS but not for the larger sized AGS. The characteristics of exDNA in AGSs revealed in this work provide a new perspective on AGS components and structural stability.},
}
@article {pmid32334080,
year = {2021},
author = {Mizuno-Iijima, S and Ayabe, S and Kato, K and Matoba, S and Ikeda, Y and Dinh, TTH and Le, HT and Suzuki, H and Nakashima, K and Hasegawa, Y and Hamada, Y and Tanimoto, Y and Daitoku, Y and Iki, N and Ishida, M and Ibrahim, EAE and Nakashiba, T and Hamada, M and Murata, K and Miwa, Y and Okada-Iwabu, M and Iwabu, M and Yagami, KI and Ogura, A and Obata, Y and Takahashi, S and Mizuno, S and Yoshiki, A and Sugiyama, F},
title = {Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.},
journal = {Methods (San Diego, Calif.)},
volume = {191},
number = {},
pages = {23-31},
doi = {10.1016/j.ymeth.2020.04.007},
pmid = {32334080},
issn = {1095-9130},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Cycle Proteins ; DNA-Binding Proteins ; *Gene Editing ; Gene Knock-In Techniques ; HEK293 Cells ; Humans ; Mice ; Zygote ; },
abstract = {Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.},
}
@article {pmid32332872,
year = {2020},
author = {Croci, S and Carriero, ML and Capitani, K and Daga, S and Donati, F and Frullanti, E and Lamacchia, V and Tita, R and Giliberti, A and Valentino, F and Benetti, E and Ciabattini, A and Furini, S and Lo Rizzo, C and Pinto, AM and Conticello, SG and Renieri, A and Meloni, I},
title = {High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot.},
journal = {European journal of human genetics : EJHG},
volume = {28},
number = {9},
pages = {1231-1242},
pmid = {32332872},
issn = {1476-5438},
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Cellular Reprogramming ; Fibroblasts/cytology/metabolism ; *Gene Editing ; Genetic Therapy/methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Methyl-CpG-Binding Protein 2/*genetics/metabolism ; *Mutation, Missense ; Neurons/cytology/metabolism ; *Recombinational DNA Repair ; Rett Syndrome/*genetics/therapy ; },
abstract = {Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.},
}
@article {pmid32332735,
year = {2020},
author = {Jiang, T and Henderson, JM and Coote, K and Cheng, Y and Valley, HC and Zhang, XO and Wang, Q and Rhym, LH and Cao, Y and Newby, GA and Bihler, H and Mense, M and Weng, Z and Anderson, DG and McCaffrey, AP and Liu, DR and Xue, W},
title = {Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1979},
pmid = {32332735},
issn = {2041-1723},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine/*chemistry ; Alleles ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Codon ; Codon, Nonsense ; Cystic Fibrosis/pathology ; Gene Editing ; HEK293 Cells ; Humans ; Mice ; Mutation ; Nucleotides ; Phenotype ; Plasmids ; RNA, Guide/*chemistry ; RNA, Messenger/*chemistry ; Transfection ; Uridine/analogs &amp; derivatives/chemistry ; },
abstract = {CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.},
}
@article {pmid32331715,
year = {2020},
author = {Kun, RS and Meng, J and Salazar-Cerezo, S and Mäkelä, MR and de Vries, RP and Garrigues, S},
title = {CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass.},
journal = {Enzyme and microbial technology},
volume = {136},
number = {},
pages = {109508},
doi = {10.1016/j.enzmictec.2020.109508},
pmid = {32331715},
issn = {1879-0909},
mesh = {Aspergillus niger/*genetics ; Biomass ; *CRISPR-Cas Systems ; Carbohydrate Metabolism ; Gene Editing/economics/*methods ; *Genome, Fungal ; Genomics/methods ; Industrial Microbiology ; Plants/*metabolism ; Point Mutation ; Transcription Factors/*genetics ; },
abstract = {The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of d-xylose and l-arabinose from wheat bran, and d-galacturonic acid from sugar beet pulp.},
}
@article {pmid32330873,
year = {2020},
author = {Hewes, AM and Sansbury, BM and Barth, S and Tarcic, G and Kmiec, EB},
title = {gRNA Sequence Heterology Tolerance Catalyzed by CRISPR/Cas in an In Vitro Homology-Directed Repair Reaction.},
journal = {Molecular therapy. Nucleic acids},
volume = {20},
number = {},
pages = {568-579},
pmid = {32330873},
issn = {2162-2531},
abstract = {CRISPR and associated Cas nucleases are genetic engineering tools revolutionizing innovative approaches to cancer and inherited diseases. CRISPR-directed gene editing relies heavily on proper DNA sequence alignment between the guide RNA (gRNA)/CRISPR complex and its genomic target. Accurate hybridization of complementary DNA initiates gene editing in human cells, but inherent gRNA sequence variation that could influence the gene editing reaction has been clearly established among diverse genetic populations. As this technology advances toward clinical implementation, it will be essential to assess what degree of gRNA variation generates unwanted and erroneous CRISPR activity. With the use of a system in which a cell-free extract catalyzes nonhomologous end joining (NHEJ) and homology-directed repair (HDR), it is possible to observe a more representative population of all forms of gene editing outcomes. In this manuscript, we demonstrate CRISPR/Cas complexation at heterologous binding sites that facilitate precise and error-prone HDR. The tolerance of mispairing between the gRNA and target site of the DNA to enable HDR is surprisingly high and greatly influenced by polarity of the donor DNA strand in the reaction. These results suggest that some collateral genomic activity could occur at unintended sites in CRISPR-directed gene editing in human cells.},
}
@article {pmid32330446,
year = {2020},
author = {Kwon, JB and Vankara, A and Ettyreddy, AR and Bohning, JD and Gersbach, CA},
title = {Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-Based Transcriptional Activators.},
journal = {Stem cell reports},
volume = {14},
number = {5},
pages = {755-769},
pmid = {32330446},
issn = {2213-6711},
support = {UH3 TR002142/TR/NCATS NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; U01 HL156348/HL/NHLBI NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; R21 AR072265/AR/NIAMS NIH HHS/United States ; U01 EB028901/EB/NIBIB NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; *Cell Lineage ; Cells, Cultured ; Cellular Reprogramming Techniques/*methods ; Dystrophin/genetics/metabolism ; Epigenesis, Genetic ; Female ; Gene Editing/methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Infant, Newborn ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Muscle Development ; Myoblasts/cytology/*metabolism ; PAX7 Transcription Factor/genetics/*metabolism ; },
abstract = {Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin[+] myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.},
}
@article {pmid32330228,
year = {2020},
author = {Hu, S and Du, J and Chen, N and Jia, R and Zhang, J and Liu, X and Yang, L},
title = {In Vivo CRISPR/Cas9-Mediated Genome Editing Mitigates Photoreceptor Degeneration in a Mouse Model of X-Linked Retinitis Pigmentosa.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {61},
number = {4},
pages = {31},
pmid = {32330228},
issn = {1552-5783},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; China ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Injections, Intraocular ; Male ; Mice ; Mice, Knockout ; Random Allocation ; Retinal Degeneration/genetics/*therapy ; Retinitis Pigmentosa/*genetics/physiopathology/*therapy ; Risk Assessment ; Sensitivity and Specificity ; },
abstract = {PURPOSE: Retinitis pigmentosa GTPase regulator (RPGR)-related X-linked retinitis pigmentosa is associated with one of the most severe phenotypes among inherited retinal disease. The aim of this study was to investigate Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-mediated gene editing therapy in a mouse model of Rpgr.<br><br>METHODS: The Rpgr-/yCas9+/WT male mice were used for this study. At 6 months of age, they received a single subretinal injection of adeno-associated virus vectors carrying sgRNA and donor template separately, and therapeutic effect was examined after 1, 6, and 12 months.<br><br>RESULTS: Rpgr knockout mouse showed slow but progressive age-related retinal degeneration, which emulates the disease occurring in humans. Significant photoreceptor preservation was observed in the treated part of the retina, in sharp contrast to the untreated part of the retina in the same eye after 6 and 12 months. It was surprising that precise modification at the target locus as demonstrated by genomic DNA sequencing in the post-mitotic photoreceptor was observed. Moreover, the therapeutic effect lasts for up to 12 months and no off-target effects were shown.<br><br>CONCLUSIONS: Our study strongly demonstrates that gene editing therapy is a promising therapeutic strategy to treat inherited retinal degeneration.},
}
@article {pmid32329776,
year = {2020},
author = {Jacobsen, T and Ttofali, F and Liao, C and Manchalu, S and Gray, BN and Beisel, CL},
title = {Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs.},
journal = {Nucleic acids research},
volume = {48},
number = {10},
pages = {5624-5638},
pmid = {32329776},
issn = {1362-4962},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/classification/genetics/*metabolism ; CRISPR-Associated Proteins/chemistry/classification/genetics/*metabolism ; DNA Cleavage ; Endodeoxyribonucleases/chemistry/classification/genetics/*metabolism ; HEK293 Cells ; Humans ; Mutation ; Phylogeny ; Prevotella/enzymology ; Protein Biosynthesis ; Protein Domains ; Transcription, Genetic ; },
abstract = {CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.},
}
@article {pmid32329714,
year = {2020},
author = {Hays, SG and Seed, KD},
title = {Dominant Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32329714},
issn = {2050-084X},
support = {R01 AI127652/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems/genetics ; Cholera/*microbiology ; *Hepatitis Delta Virus ; Time Factors ; Vibrio cholerae/*genetics ; },
abstract = {Bacteria, bacteriophages that prey upon them, and mobile genetic elements (MGEs) compete in dynamic environments, evolving strategies to sense the milieu. The first discovered environmental sensing by phages, lysis inhibition, has only been characterized and studied in the limited context of T-even coliphages. Here, we discover lysis inhibition in the etiological agent of the diarrheal disease cholera, Vibrio cholerae, infected by ICP1, a phage ubiquitous in clinical samples. This work identifies the ICP1-encoded holin, teaA, and antiholin, arrA, that mediate lysis inhibition. Further, we show that an MGE, the defensive phage satellite PLE, collapses lysis inhibition. Through lysis inhibition disruption a conserved PLE protein, LidI, is sufficient to limit the phage produced from infection, bottlenecking ICP1. These studies link a novel incarnation of the classic lysis inhibition phenomenon with conserved defensive function of a phage satellite in a disease context, highlighting the importance of lysis timing during infection and parasitization.},
}
@article {pmid32329685,
year = {2020},
author = {Mian-Ling, Z and Yun-Qi, C and Chao-Chun, Z},
title = {Prader-Willi Syndrome: Molecular Mechanism and Epigenetic Therapy.},
journal = {Current gene therapy},
volume = {20},
number = {1},
pages = {36-43},
doi = {10.2174/1566523220666200424085336},
pmid = {32329685},
issn = {1875-5631},
mesh = {CRISPR-Cas Systems/genetics ; Chromosomes, Human, Pair 15/genetics ; Epigenesis, Genetic/*genetics ; Epigenome/genetics ; Genetic Therapy/*trends ; Genomic Imprinting/*genetics ; Humans ; Maternal Inheritance/genetics ; Paternal Inheritance/genetics ; Prader-Willi Syndrome/genetics/pathology/*therapy ; },
abstract = {Prader-Willi syndrome (PWS) is an imprinted neurodevelopmental disease characterized by cognitive impairments, developmental delay, hyperphagia, obesity, and sleep abnormalities. It is caused by a lack of expression of the paternally active genes in the PWS imprinting center on chromosome 15 (15q11.2-q13). Owing to the imprinted gene regulation, the same genes in the maternal chromosome, 15q11-q13, are intact in structure but repressed at the transcriptional level because of the epigenetic mechanism. The specific molecular defect underlying PWS provides an opportunity to explore epigenetic therapy to reactivate the expression of repressed PWS genes inherited from the maternal chromosome. The purpose of this review is to summarize the main advances in the molecular study of PWS and discuss current and future perspectives on the development of CRISPR/Cas9- mediated epigenome editing in the epigenetic therapy of PWS. Twelve studies on the molecular mechanism or epigenetic therapy of PWS were included in the review. Although our understanding of the molecular basis of PWS has changed fundamentally, there has been a little progress in the epigenetic therapy of PWS that targets its underlying genetic defects.},
}
@article {pmid32329213,
year = {2020},
author = {Fatma, Z and Schultz, JC and Zhao, H},
title = {Recent advances in domesticating non-model microorganisms.},
journal = {Biotechnology progress},
volume = {36},
number = {5},
pages = {e3008},
doi = {10.1002/btpr.3008},
pmid = {32329213},
issn = {1520-6033},
support = {SC0018420//U.S. Department of Energy/International ; SC0018260//U.S. Department of Energy/International ; },
mesh = {*Bacteria/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Fungi/genetics/metabolism ; *Metabolic Engineering ; Regulatory Sequences, Nucleic Acid/genetics ; *Synthetic Biology ; },
abstract = {Non-model microorganisms have been increasingly explored as microbial cell factories for production of chemicals, fuels, and materials owing to their unique physiology and metabolic capabilities. However, these microorganisms often lack facile genetic tools for strain development, which hinders their adoption as production hosts. In this review, we describe recent advances in domestication of non-model microorganisms, including bacteria, actinobacteria, cyanobacteria, yeast, and fungi, with a focus on the development of genetic tools. In addition, we highlight some successful applications of non-model microorganisms as microbial cell factories.},
}
@article {pmid32328868,
year = {2020},
author = {Sunitha, S and Rock, CD},
title = {CRISPR/Cas9-mediated targeted mutagenesis of TAS4 and MYBA7 loci in grapevine rootstock 101-14.},
journal = {Transgenic research},
volume = {29},
number = {3},
pages = {355-367},
pmid = {32328868},
issn = {1573-9368},
support = {15-0214-SA//California Department of Food and Agriculture/International ; 17-0514-SA//California Department of Food and Agriculture/International ; },
mesh = {Anthocyanins/*biosynthesis/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; Genome, Plant ; Mutagenesis ; Plant Proteins/*genetics/metabolism ; Plant Roots/genetics/metabolism ; Plants, Genetically Modified/*genetics/metabolism ; Transcription Factors/*genetics/metabolism ; Vitis/*genetics/metabolism ; },
abstract = {Pierce's disease (PD) of grapevine (Vitis vinifera) is caused by the bacterium Xylella fastidiosa and is vectored by xylem sap-sucking insects, whereas Grapevine Red Blotch Virus (GRBV) causes Red Blotch Disease and is transmitted in the laboratory by alfalfa leafhopper Spissistilus festinus. The significance of anthocyanin accumulations in distinct tissues of grapevine by these pathogens is unknown, but vector feeding preferences and olfactory cues from host anthocyanins may be important for these disease etiologies. Phosphate, sugar, and UV light are known to regulate anthocyanin accumulation via miR828 and Trans-Acting Small-interfering locus4 (TAS4), specifically in grape by production of phased TAS4a/b/c small-interfering RNAs that are differentially expressed and target MYBA5/6/7 transcription factor transcripts for post-transcriptional slicing and antisense-mediated silencing. To generate materials that can critically test these genes' functions in PD and GRBV disease symptoms, we produced transgenic grape plants targeting TAS4b and MYBA7 using CRISPR/Cas9 technology. We obtained five MYBA7 lines all with bi-allelic editing events and no off-targets detected at genomic loci with homology to the guide sequence. We obtained two independent edited TAS4b lines; one bi-allelic, the other heterozygous while both had fortuitous evidences of bi-allelic TAS4a off-target editing events at the paralogous locus. No visible anthocyanin accumulation phenotypes were observed in regenerated plants, possibly due to the presence of genetically redundant TAS4c and MYBA5/6 loci or absence of inductive environmental stress conditions. The editing events encompass single base insertions and di/trinucleotide deletions of Vvi-TAS4a/b and Vvi-MYBA7 at expected positions 3 nt upstream from the guideRNA proximal adjacent motifs NGG. We also identified evidences of homologous recombinations of TAS4a with TAS4b at the TAS4a off-target in one of the TAS4b lines, resulting in a chimeric locus with a bi-allelic polymorphism, supporting independent recombination events in transgenic plants associated with apparent high Cas9 activities. The lack of obvious visible pigment phenotypes in edited plants precluded pathogen challenge tests of the role of anthocyanins in host PD and GRBV resistance/tolerance mechanisms. Nonetheless, we demonstrate successful genome-editing of non-coding RNA and MYB transcription factor loci which can serve future characterizations of the functions of TAS4a/b/c and MYBA7 in developmental, physiological, and environmental biotic/abiotic stress response pathways important for value-added nutraceutical synthesis and pathogen responses of winegrape.},
}
@article {pmid32328608,
year = {2020},
author = {Zhang, NN and Wang, CN and Ni, X},
title = {[Construction of transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {72},
number = {2},
pages = {148-156},
pmid = {32328608},
issn = {0371-0874},
mesh = {Adrenal Cortex/*enzymology ; Animals ; CRISPR-Cas Systems ; Cystathionine gamma-Lyase/genetics ; Integrases/genetics/*metabolism ; Mice ; *Mice, Transgenic ; Zona Fasciculata/*enzymology ; },
abstract = {The adrenal gland is an important endocrine organ of human body. CYP11B1 gene was specifically expressed in the zona fasciculata in adrenal cortex. In order to better study the function of genes specifically expressed in the zona fasciculata in adrenal cortex, the mice with Cre recombinase specifically expressed in the zona fasciculata in adrenal cortex were constructed. It was then confirmed that CYP11B1 was specifically expressed in adrenal glands. Then, using CRISPR/Cas9 technique, CYP11B1-2A-GfpCre recombinant vector was constructed and subsequently injected into the fertilized eggs of mice. It was confirmed that the Cre gene was mainly expressed in the zona fasciculata in adrenal cortex of CYP11B1Cre mice by using mTmG and LacZ staining. The CYP11B1Cre mice were then mated with cystathionine γ-lyase (CTH)[f/f] mice, thereby generating CTH[f/f]/CYP11B1Cre mice. It was also confirmed that CTH gene in the zona fasciculata in adrenal cortex was specifically knocked out in these mice. These results suggest that transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex were constructed successfully. This animal model can be a powerful tool for the study of the function of genes expressed in the zona fasciculata in adrenal cortex.},
}
@article {pmid32327470,
year = {2020},
author = {Opsomer, R and Contino, S and Perrin, F and Gualdani, R and Tasiaux, B and Doyen, P and Vergouts, M and Vrancx, C and Doshina, A and Pierrot, N and Octave, JN and Gailly, P and Stanga, S and Kienlen-Campard, P},
title = {Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release.},
journal = {eNeuro},
volume = {7},
number = {3},
pages = {},
pmid = {32327470},
issn = {2373-2822},
mesh = {*Alzheimer Disease/genetics ; Amyloid beta-Peptides ; *Amyloid beta-Protein Precursor/genetics ; Animals ; Basic Helix-Loop-Helix Transcription Factors ; Humans ; Mice ; Synaptic Transmission ; Transcription Factors ; gamma-Aminobutyric Acid ; },
abstract = {The amyloid precursor protein (APP) has been extensively studied as the precursor of the β-amyloid (Aβ) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP-/- neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP-/- mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.},
}
@article {pmid32327447,
year = {2020},
author = {Lee, HJ and Kim, HJ and Lee, SJ},
title = {CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs.},
journal = {Genome research},
volume = {30},
number = {5},
pages = {768-775},
pmid = {32327447},
issn = {1549-5469},
mesh = {Base Pair Mismatch ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Gene Editing/*methods ; Genome, Bacterial ; Mutagenesis ; Mutation ; Plasmids/genetics ; RNA/chemistry ; },
abstract = {Genome editing has been revolutionized by the CRISPR-Cas9 system. CRISPR-Cas9 is composed of single-molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a protospacer adjacent motif (PAM) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR-Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR-Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced two- to four-base mutations in galK in Escherichia coli with high editing efficiencies (81%-86%). However, single-point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed one- or two-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli A single-point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 36%-95% of negatively selected E. coli cells using single-base mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single-base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.},
}
@article {pmid32327358,
year = {2020},
author = {Gao, Y and Dai, X and Aoi, Y and Takebayashi, Y and Yang, L and Guo, X and Zeng, Q and Yu, H and Kasahara, H and Zhao, Y},
title = {Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {3},
pages = {157-165},
pmid = {32327358},
issn = {1673-8527},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {Amidohydrolases/*genetics/isolation &amp; purification ; Arabidopsis/*genetics/metabolism ; Arabidopsis Proteins/*genetics/isolation &amp; purification ; CRISPR-Cas Systems/genetics ; Chromosomes/genetics ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Genome, Plant/genetics ; Indoleacetic Acids/*metabolism ; Mutation/genetics ; },
abstract = {Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAMHYDROLASE1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.},
}
@article {pmid32326901,
year = {2020},
author = {Hahn, F and Korolev, A and Sanjurjo Loures, L and Nekrasov, V},
title = {A modular cloning toolkit for genome editing in plants.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {179},
pmid = {32326901},
issn = {1471-2229},
support = {BB/P016855/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cloning, Molecular/*methods ; *Gene Editing ; *Genome, Plant ; Protoplasts ; RNA, Guide ; Triticum/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.<br><br>RESULTS: Here we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.<br><br>CONCLUSIONS: We believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.},
}
@article {pmid32326617,
year = {2020},
author = {Mikolajewicz, N and Komarova, SV},
title = {Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.},
journal = {International journal of molecular sciences},
volume = {21},
number = {8},
pages = {},
pmid = {32326617},
issn = {1422-0067},
support = {PJT-165939/CAPMC/CIHR/Canada ; },
mesh = {Adenosine Diphosphate/pharmacology ; Adenosine Triphosphate/pharmacology ; Animals ; Bone Density/genetics ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Line ; Cell Proliferation/drug effects/*genetics ; Cells, Cultured ; Cyclic AMP/metabolism ; Gene Knockout Techniques ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Osteoblasts/*metabolism ; Osteogenesis/drug effects/*genetics ; Phosphorylation ; Purinergic Antagonists/metabolism/*pharmacology ; Receptors, Purinergic P2Y/genetics/*metabolism ; Signal Transduction/drug effects/*genetics ; Uridine Diphosphate Glucose/metabolism/pharmacology ; Uridine Diphosphate Sugars/*metabolism/pharmacology ; },
abstract = {The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca[2+]]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca[2+] signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14[-/-] mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.},
}
@article {pmid32326615,
year = {2020},
author = {Malek, N and Mrówczyńska, E and Michrowska, A and Mazurkiewicz, E and Pavlyk, I and Mazur, AJ},
title = {Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells' Motility and Focal Adhesion Formation.},
journal = {International journal of molecular sciences},
volume = {21},
number = {8},
pages = {},
pmid = {32326615},
issn = {1422-0067},
mesh = {Actins/analysis/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/drug effects/genetics ; Focal Adhesions/drug effects/genetics/*metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Humans ; Lysophospholipids/pharmacology ; Melanoma/genetics/*metabolism ; Neoplasm Invasiveness/genetics ; Protein Isoforms/metabolism ; Signal Transduction/drug effects/genetics ; Stress Fibers/genetics/metabolism ; Tetradecanoylphorbol Acetate/analogs &amp; derivatives/pharmacology ; },
abstract = {Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells' FA formation and motility.},
}
@article {pmid32326099,
year = {2020},
author = {Becskei, A},
title = {Tuning up Transcription Factors for Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {25},
number = {8},
pages = {},
pmid = {32326099},
issn = {1420-3049},
mesh = {Animals ; Biotechnology/methods ; CRISPR-Cas Systems ; Clinical Trials as Topic ; DNA-Binding Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; *Gene Expression Regulation ; Gene Transfer Techniques ; *Genetic Therapy/methods ; Humans ; Protein Binding ; Repressor Proteins/genetics/metabolism ; Transcription Factors/chemistry/*genetics/*metabolism ; *Transcription, Genetic ; },
abstract = {The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, transcription activator-like effector (TALE) and CRISPR-dCas9 revealed common principles behind their efficacy, which can aid the optimization of transcriptional activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor natural and synthetic transcription factors to the needs of specific applications.},
}
@article {pmid32325942,
year = {2020},
author = {Luo, J and Teng, M and Zai, X and Tang, N and Zhang, Y and Mandviwala, A and Reddy, VRAP and Baigent, S and Yao, Y and Nair, V},
title = {Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System.},
journal = {Viruses},
volume = {12},
number = {4},
pages = {},
pmid = {32325942},
issn = {1999-4915},
support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032, BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cells, Cultured ; Chick Embryo ; Cloning, Molecular ; *Gene Editing ; Gene Knockdown Techniques ; Genome, Viral ; Marek Disease/*genetics ; MicroRNAs/*genetics ; *Mutagenesis ; Open Reading Frames ; RNA, Guide ; RNA, Messenger/genetics ; RNA, Viral/*genetics ; Sequence Analysis, DNA ; Virus Replication ; },
abstract = {The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.},
}
@article {pmid32325051,
year = {2020},
author = {Osuna, BA and Karambelkar, S and Mahendra, C and Sarbach, A and Johnson, MC and Kilcher, S and Bondy-Denomy, J},
title = {Critical Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor.},
journal = {Cell host &amp; microbe},
volume = {28},
number = {1},
pages = {23-30.e5},
pmid = {32325051},
issn = {1934-6069},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; T32 GM067547/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/metabolism ; Bacteriophages/*physiology ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation ; Genetic Engineering ; Host-Pathogen Interactions ; Listeria monocytogenes/*virology ; Promoter Regions, Genetic ; Protein Binding ; Repressor Proteins/genetics/*metabolism ; Viral Proteins/genetics/*metabolism ; },
abstract = {Bacteriophages must rapidly deploy anti-CRISPR proteins (Acrs) to inactivate the RNA-guided nucleases that enforce CRISPR-Cas adaptive immunity in their bacterial hosts. Listeria monocytogenes temperate phages encode up to three anti-Cas9 proteins, with acrIIA1 always present. AcrIIA1 binds and inhibits Cas9 with its C-terminal domain; however, the function of its highly conserved N-terminal domain (NTD) is unknown. Here, we report that the AcrIIA1[NTD] is a critical transcriptional repressor of the strong anti-CRISPR promoter. A rapid burst of anti-CRISPR transcription occurs during phage infection and the subsequent negative feedback by AcrIIA1[NTD] is required for optimal phage replication, even in the absence of CRISPR-Cas immunity. In the presence of CRISPR-Cas immunity, full-length AcrIIA1 uses its two-domain architecture to act as a "Cas9 sensor," tuning acr expression according to Cas9 levels. Finally, we identify AcrIIA1[NTD] homologs in other Firmicutes and demonstrate that they have been co-opted by hosts as "anti-anti-CRISPRs," repressing phage anti-CRISPR deployment.},
}
@article {pmid32325050,
year = {2020},
author = {Osuna, BA and Karambelkar, S and Mahendra, C and Christie, KA and Garcia, B and Davidson, AR and Kleinstiver, BP and Kilcher, S and Bondy-Denomy, J},
title = {Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes.},
journal = {Cell host &amp; microbe},
volume = {28},
number = {1},
pages = {31-40.e9},
pmid = {32325050},
issn = {1934-6069},
support = {DP5 OD021344/OD/NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Listeria ; Lysogeny ; },
abstract = {Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.},
}
@article {pmid32324799,
year = {2020},
author = {Glazier, VE and Krysan, DJ},
title = {Genetic interaction analysis comes to the diploid human pathogen Candida albicans.},
journal = {PLoS pathogens},
volume = {16},
number = {4},
pages = {e1008399},
pmid = {32324799},
issn = {1553-7374},
support = {R01 AI133409/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Candida albicans/*genetics/metabolism/*pathogenicity ; Epistasis, Genetic ; Fungal Proteins/*genetics/metabolism ; *Gene Deletion ; *Gene Expression Regulation, Fungal ; *Genome, Fungal ; Humans ; Models, Genetic ; },
}
@article {pmid32323721,
year = {2020},
author = {Hao, J and Li, C and Lin, C and Hao, Y and Yu, X and Xia, Y and Gao, F and Jiang, Z and Wang, D},
title = {Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis.},
journal = {Bioscience reports},
volume = {40},
number = {5},
pages = {},
pmid = {32323721},
issn = {1573-4935},
mesh = {A549 Cells ; Adenine/*metabolism ; *Apoptosis ; CRISPR-Cas Systems ; *Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Methylation ; MicroRNAs/*genetics/metabolism ; *Point Mutation ; *RNA Processing, Post-Transcriptional ; RNA, Guide/*genetics/metabolism ; RNA, Long Noncoding/*genetics/metabolism ; },
abstract = {Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T-A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.},
}
@article {pmid32323394,
year = {2020},
author = {Iida, M and Suzuki, M and Sakane, Y and Nishide, H and Uchiyama, I and Yamamoto, T and Suzuki, KT and Fujii, S},
title = {A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon sequencing.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {25},
number = {7},
pages = {498-509},
doi = {10.1111/gtc.12775},
pmid = {32323394},
issn = {1365-2443},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Female ; Frameshift Mutation ; Gene Editing/*methods ; Gene Library ; Genetic Association Studies ; Genotype ; High-Throughput Nucleotide Sequencing ; INDEL Mutation ; Male ; Phenotype ; Software ; Workflow ; Xenopus/*genetics ; },
abstract = {Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.},
}
@article {pmid32322250,
year = {2020},
author = {Yang, L and Li, W and Ujiroghene, OJ and Yang, Y and Lu, J and Zhang, S and Pang, X and Lv, J},
title = {Occurrence and Diversity of CRISPR Loci in Lactobacillus casei Group.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {624},
pmid = {32322250},
issn = {1664-302X},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system that resists foreign genes through nuclease targeting in bacteria and archaea. In this study, we analyzed 68 strains of Lactobacillus casei group from the NCBI GenBank database, and bioinformatic tools were used to investigate the occurrence and diversity of CRISPR system. The results showed that a total of 30 CRISPR loci were identified from 27 strains. Apart from three strains which contained double loci with distinguishable distributed sites, most strains contained only one CRISPR locus. The analysis of direct repeat (DR) sequences showed that all DR could form stable RNA secondary structures. The CRISPR spacers showed diversity, and their origin and evolution were revealed through the investigation of their spacer sequences. In addition, a large number of CRISPR spacers showed perfect homologies to phage and plasmid sequences. Collectively, our results would contribute to researches of resistance in L. casei group, and also provide a new vision on the diversity and evolution of CRISPR/Cas system.},
}
@article {pmid32322010,
year = {2020},
author = {Baquero, DP and Contursi, P and Piochi, M and Bartolucci, S and Liu, Y and Cvirkaite-Krupovic, V and Prangishvili, D and Krupovic, M},
title = {New virus isolates from Italian hydrothermal environments underscore the biogeographic pattern in archaeal virus communities.},
journal = {The ISME journal},
volume = {14},
number = {7},
pages = {1821-1833},
pmid = {32322010},
issn = {1751-7370},
mesh = {*Archaeal Viruses/genetics ; DNA Viruses/genetics ; Genome, Viral ; Humans ; Italy ; *Rudiviridae/genetics ; *Viruses ; },
abstract = {Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus, Acidianus, and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.},
}
@article {pmid32321973,
year = {2020},
author = {Punetha, M and Chouhan, VS and Sonwane, A and Singh, G and Bag, S and Green, JA and Whitworth, K and Sarkar, M},
title = {Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {6849},
pmid = {32321973},
issn = {2045-2322},
mesh = {Animals ; Buffaloes/genetics/*metabolism ; *CRISPR-Cas Systems ; Corpus Luteum/*metabolism ; Female ; Fibroblast Growth Factor 2/*biosynthesis/genetics ; Gene Editing ; *Gene Expression Regulation ; Gene Knockout Techniques ; Vascular Endothelial Growth Factor A/*biosynthesis/genetics ; },
abstract = {The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation. However, their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The present study was conducted to understand the role of EGR mediated regulation of VEGF A and FGF 2 signaling in buffalo luteal cells. Towards this goal, luteal cells were cultured and treated with VEGF A and FGF 2 and the mRNA expression pattern of EGR family members were documented. The EGR 1 message was found to be up-regulated in luteal cells of buffalo at 72 hours of culture. The functional validation of EGR 1 gene was accomplished by knocking out (KO) of EGR 1 in cultured luteal cells by CRISPR/Cas9 mediated gene editing technology. The EGR 1 KO cells were then cultured and stimulated with VEGF A and FGF 2. It was observed that VEGF A and FGF 2 induced angiogenesis, cell proliferation and steroidogenesis in wild type luteal cells, whereas the response of the growth factors was attenuated in the EGR 1 KO cells. Taken together our study provides evidence convincingly that both VEGF and FGF mediate their biological action through a common intermediate, EGR 1, to regulate corpus luteum function of buffalo.},
}
@article {pmid32321936,
year = {2020},
author = {Guillen, RX and Beckley, JR and Chen, JS and Gould, KL},
title = {CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {6797},
pmid = {32321936},
issn = {2045-2322},
support = {P30 DK058404/DK/NIDDK NIH HHS/United States ; R01 GM112989/GM/NIGMS NIH HHS/United States ; R35 GM131799/GM/NIGMS NIH HHS/United States ; R25 GM062459/GM/NIGMS NIH HHS/United States ; P30 CA068485/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Casein Kinase 1 epsilon/*genetics/metabolism ; Casein Kinase Idelta/*genetics/metabolism ; Cell Nucleus/genetics/metabolism ; Centrosome/metabolism ; Endocytosis/*genetics ; Gene Targeting/*methods ; Guanine Nucleotide Exchange Factors ; HEK293 Cells ; HeLa Cells ; Humans ; Interphase/genetics ; Phosphoproteins/*genetics/metabolism ; Phosphorylation ; Protein Binding ; Substrate Specificity ; },
abstract = {Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis. Here, we have investigated the localization and interactomes of endogenously tagged CK1δ and CK1ε during interphase and mitosis. CK1δ and CK1ε localize to centrosomes throughout the cell cycle, and in interphase cells to the nucleus, and in both a diffuse and punctate pattern in the cytoplasm. Also, for the first time, they were detected at the midbody during cell division. Mass spectrometry analysis identified a total of 181 proteins co-purifying with a Venus multifunctional (VM)-tagged CK1δ and/or CK1ε. GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), a protein required for efficient endocytosis, was consistently one of the most abundant interacting partners. We demonstrate that GAPVD1 is a substrate of CK1δ/ε with up to 38 phosphorylated residues in vitro and in vivo. Wildtype and a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, were able to rescue defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1. Our results indicate that GAPVD1 is an important interacting partner and substrate of CK1δ/ε for endocytosis.},
}
@article {pmid32321775,
year = {2020},
author = {Kagoya, Y and Guo, T and Yeung, B and Saso, K and Anczurowski, M and Wang, CH and Murata, K and Sugata, K and Saijo, H and Matsunaga, Y and Ohashi, Y and Butler, MO and Hirano, N},
title = {Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.},
journal = {Cancer immunology research},
volume = {8},
number = {7},
pages = {926-936},
doi = {10.1158/2326-6066.CIR-18-0508},
pmid = {32321775},
issn = {2326-6074},
mesh = {Allografts ; Animals ; Antigens, CD19/immunology ; CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Histocompatibility Antigens Class I/*chemistry/genetics ; Histocompatibility Antigens Class II/*chemistry/genetics ; Humans ; Immunotherapy, Adoptive/*methods ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasms/immunology/metabolism/*therapy ; Receptors, Antigen, T-Cell/antagonists &amp; inhibitors/genetics/*immunology ; Receptors, Chimeric Antigen/*immunology ; },
abstract = {Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.},
}
@article {pmid32320749,
year = {2020},
author = {Schwaller, J},
title = {Learning from mouse models of MLL fusion gene-driven acute leukemia.},
journal = {Biochimica et biophysica acta. Gene regulatory mechanisms},
volume = {1863},
number = {8},
pages = {194550},
doi = {10.1016/j.bbagrm.2020.194550},
pmid = {32320749},
issn = {1876-4320},
mesh = {Animals ; Bone Marrow Cells ; CRISPR-Cas Systems ; Cell Transformation, Neoplastic/genetics ; Disease Models, Animal ; Hematopoietic Stem Cells/metabolism ; Histone-Lysine N-Methyltransferase ; Homeodomain Proteins/metabolism ; Humans ; Leukemia/*genetics ; Leukemia, Myeloid, Acute/genetics ; Mice ; MicroRNAs ; Myeloid-Lymphoid Leukemia Protein/*genetics/*metabolism ; Nuclear Proteins ; Oncogene Proteins, Fusion/*genetics/*metabolism ; Tumor Microenvironment ; },
abstract = {5-10% of human acute leukemias carry chromosomal translocations involving the mixed lineage leukemia (MLL) gene that result in the expression of chimeric protein fusing MLL to >80 different partners of which AF4, ENL and AF9 are the most prevalent. In contrast to many other leukemia-associated mutations, several MLL-fusions are powerful oncogenes that transform hematopoietic stem cells but also more committed progenitor cells. Here, I review different approaches that were used to express MLL fusions in the murine hematopoietic system which often, but not always, resulted in highly penetrant and transplantable leukemias that closely phenocopied the human disease. Due to its simple and reliable nature, reconstitution of irradiated mice with bone marrow cells retrovirally expressing the MLL-AF9 fusion became the most frequently in vivo model to study the biology of acute myeloid leukemia (AML). I review some of the most influential studies that used this model to dissect critical protein interactions, the impact of epigenetic regulators, microRNAs and microenvironment-dependent signals for MLL fusion-driven leukemia. In addition, I highlight studies that used this model for shRNA- or genome editing-based screens for cellular vulnerabilities that allowed to identify novel therapeutic targets of which some entered clinical trials. Finally, I discuss some inherent characteristics of the widely used mouse model based on retroviral expression of the MLL-AF9 fusion that can limit general conclusions for the biology of AML. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.},
}
@article {pmid32318908,
year = {2020},
author = {Li, Y and Yan, W},
title = {Exploitation of genetic resources based on regulome and gene editing in crops.},
journal = {Science China. Life sciences},
volume = {63},
number = {9},
pages = {1406-1409},
pmid = {32318908},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genome, Plant ; Plants, Genetically Modified/*genetics ; Proteomics ; },
}
@article {pmid32318747,
year = {2020},
author = {Lee, EY and Yu, JY and Paek, AR and Lee, SH and Jang, H and Cho, SY and Kim, JH and Kang, HG and Yun, T and Oh, SE and Park, SY and You, HJ},
title = {Targeting TJP1 attenuates cell-cell aggregation and modulates chemosensitivity against doxorubicin in leiomyosarcoma.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {98},
number = {5},
pages = {761-773},
doi = {10.1007/s00109-020-01909-8},
pmid = {32318747},
issn = {1432-1440},
mesh = {Antibiotics, Antineoplastic/*pharmacology ; CRISPR-Cas Systems ; Cell Communication/*drug effects/*genetics ; Cell Line, Tumor ; Cell Proliferation ; Doxorubicin/*pharmacology ; *Drug Resistance, Neoplasm ; Gene Editing ; Gene Expression Profiling ; Gene Silencing ; Humans ; Janus Kinases/metabolism ; Leiomyosarcoma/drug therapy/*genetics/metabolism/pathology ; NF-kappa B/metabolism ; RNA, Small Interfering/genetics ; STAT Transcription Factors/metabolism ; Signal Transduction ; Transcriptome ; Zonula Occludens-1 Protein/antagonists &amp; inhibitors/*genetics ; },
abstract = {Tight junction protein 1 (TJP1) is a membrane-associated cytosolic protein important for cell-cell communication in intercellular barriers in epithelial and non-epithelial cells. Here, we explored the functional involvement of TJP1 in non-epithelial tumors such as soft tissue sarcoma, especially in leiomyosarcoma (LMS). TJP1 expression in soft tissue sarcoma was analyzed in normal and tumor tissues as well as from public datasets such as the TCGA provisional dataset, in which TJP1 expression was compared with other subtypes such as undifferentiated sarcomas, and myxofibrosarcomas. SK-LMS-1 cell lines with reduced TJP1 expression showed attenuated anchorage-independent colony formation as well as reduced intercellular aggregation on non-coated culture plates compared with control as well as parental SK-LMS-1 cells. Transcriptome profiling following TJP1 knockdown in SK-LMS-1 cells suggested the involvement of several signaling pathways, including NF-κB pathway and growth factor receptor signaling. In addition, TJP1 downregulation induced enhanced response against anti-cancer agents, doxorubicin and gefitinib. Taken together, these results suggest that TJP1 contributes to sarcoma genesis and might be useful therapeutic target. KEY MESSAGES: • TJP1 expression at RNA level higher in tumor than in normal tissues of sarcoma. • Targeting TJP1 attenuates cell-cell aggregation and anchorage-independent growth. • Targeting TJP1 is beneficial in anti-cancer therapy in LMS.},
}
@article {pmid32317636,
year = {2020},
author = {Ulgherait, M and Chen, A and McAllister, SF and Kim, HX and Delventhal, R and Wayne, CR and Garcia, CJ and Recinos, Y and Oliva, M and Canman, JC and Picard, M and Owusu-Ansah, E and Shirasu-Hiza, M},
title = {Circadian regulation of mitochondrial uncoupling and lifespan.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1927},
pmid = {32317636},
issn = {2041-1723},
support = {R01 GM130764/GM/NIGMS NIH HHS/United States ; T32 HL120826/HL/NHLBI NIH HHS/United States ; R01 GM117407/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; F31 GM125363/GM/NIGMS NIH HHS/United States ; R35 GM119793/GM/NIGMS NIH HHS/United States ; R21 DK112074/DK/NIDDK NIH HHS/United States ; R35 GM124717/GM/NIGMS NIH HHS/United States ; R01 AG045842/AG/NIA NIH HHS/United States ; R21 AR077312/AR/NIAMS NIH HHS/United States ; T32 DK007328/DK/NIDDK NIH HHS/United States ; R35 GM127049/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis ; Cell Proliferation ; Circadian Clocks ; *Circadian Rhythm ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/*metabolism ; Homeostasis ; Intestines/pathology ; Longevity ; Male ; Membrane Potential, Mitochondrial ; Membrane Transport Proteins/*metabolism ; Mitochondria/*metabolism ; Mutation ; Oxidative Stress/physiology ; Oxygen Consumption ; Period Circadian Proteins/*metabolism ; Uncoupling Protein 1/metabolism ; },
abstract = {Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.},
}
@article {pmid32317048,
year = {2020},
author = {Strauss, RP and Audsley, KM and Passman, AM and van Vuuren, JH and Finch-Edmondson, ML and Callus, BA and Yeoh, GC},
title = {Loss of ARF/INK4A Promotes Liver Progenitor Cell Transformation Toward Tumorigenicity Supporting Their Role in Hepatocarcinogenesis.},
journal = {Gene expression},
volume = {20},
number = {1},
pages = {39-52},
pmid = {32317048},
issn = {1555-3884},
mesh = {Animals ; Azacitidine/pharmacology ; CRISPR-Cas Systems ; Cell Line, Transformed ; Cell Transformation, Neoplastic/*genetics ; Cyclin-Dependent Kinase Inhibitor p16/biosynthesis/deficiency/*physiology ; DNA Methylation/drug effects ; Epithelial-Mesenchymal Transition ; Gene Deletion ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; Genes, p16 ; Liver/cytology/embryology ; Liver Neoplasms, Experimental/*genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Proteins/biosynthesis/genetics/physiology ; Phenotype ; Snail Family Transcription Factors/biosynthesis/genetics ; Stem Cells/*pathology ; Tumor Stem Cell Assay ; Vimentin/biosynthesis/genetics ; },
abstract = {Liver progenitor cells (LPCs) contribute to liver regeneration during chronic damage and are implicated as cells of origin for liver cancers including hepatocellular carcinoma (HCC). The CDKN2A locus, which encodes the tumor suppressors alternate reading frame protein (ARF) and INK4A, was identified as one of the most frequently altered genes in HCC. This study demonstrates that inactivation of CDKN2A enhances tumorigenic transformation of LPCs. The level of ARF and INK4A expression was determined in a panel of transformed and nontransformed wild-type LPC lines. Moreover, the transforming potential of LPCs with inactivated CDKN2A was shown to be enhanced in LPCs derived from Arf[-/-] and CDKN2A[fl/fl] mice and in wild-type LPCs following CRISPR-Cas9 suppression of CDKN2A. ARF and INK4A abundance is consistently reduced or ablated following LPC transformation. Arf[-/-] and CDKN2A[-/-] LPCs displayed hallmarks of transformation such as anchorage-independent and more rapid growth than control LPC lines with unaltered CDKN2A. Transformation was not immediate, suggesting that the loss of CDKN2A alone is insufficient. Further analysis revealed decreased p21 expression as well as reduced epithelial markers and increased mesenchymal markers, indicative of epithelial-to-mesenchymal transition, following inactivation of the CDKN2A gene were required for tumorigenic transformation. Loss of ARF and INK4A enhances the propensity of LPCs to undergo a tumorigenic transformation. As LPCs represent a cancer stem cell candidate, identifying CDKN2A as a driver of LPC transformation highlights ARF and INK4A as viable prognostic markers and therapeutic targets for HCC.},
}
@article {pmid32316275,
year = {2020},
author = {Nakazawa, T and Natsume, A and Nishimura, F and Morimoto, T and Matsuda, R and Nakamura, M and Yamada, S and Nakagawa, I and Motoyama, Y and Park, YS and Tsujimura, T and Wakabayashi, T and Nakase, H},
title = {Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-Generation CAR-T Cells Targeting EGFRvIII on In Vitro Human Glioblastoma Cell Growth.},
journal = {Cells},
volume = {9},
number = {4},
pages = {},
pmid = {32316275},
issn = {2073-4409},
mesh = {Brain Neoplasms/*genetics/immunology/therapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/genetics ; ErbB Receptors/*immunology ; Exons ; Glioblastoma/*genetics/immunology/therapy ; Humans ; Immunotherapy, Adoptive/*methods ; Programmed Cell Death 1 Receptor/*genetics/metabolism ; Receptors, Chimeric Antigen/*genetics/immunology/metabolism ; T-Lymphocytes/*immunology ; },
abstract = {Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. However, the antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells in GBM are limited. The inhibitory effect is induced by the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on GBM cells. In the present study, PD-1-disrupted EvCAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing GBM cells without altering the T-cell phenotype and the expression of other checkpoint receptors. In the future, the in vivo antitumor effect of this vector should be evaluated in order to determine if it could be applied clinically for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM.},
}
@article {pmid32316223,
year = {2020},
author = {Syding, LA and Nickl, P and Kasparek, P and Sedlacek, R},
title = {CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review.},
journal = {Cells},
volume = {9},
number = {4},
pages = {},
pmid = {32316223},
issn = {2073-4409},
mesh = {Angelman Syndrome/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA Methylation ; Diabetes Mellitus/genetics/*metabolism ; Epigenesis, Genetic ; Epigenome/drug effects/*genetics ; Gene Editing/*methods ; Genomic Imprinting/genetics ; Humans ; Infant, Newborn, Diseases/genetics/*metabolism ; Prader-Willi Syndrome/genetics/*metabolism ; Rare Diseases/genetics/metabolism ; Silver-Russell Syndrome/genetics/*metabolism ; },
abstract = {Imprinting diseases (IDs) are rare congenital disorders caused by aberrant dosages of imprinted genes. Rare IDs are comprised by a group of several distinct disorders that share a great deal of homology in terms of genetic etiologies and symptoms. Disruption of genetic or epigenetic mechanisms can cause issues with regulating the expression of imprinted genes, thus leading to disease. Genetic mutations affect the imprinted genes, duplications, deletions, and uniparental disomy (UPD) are reoccurring phenomena causing imprinting diseases. Epigenetic alterations on methylation marks in imprinting control centers (ICRs) also alters the expression patterns and the majority of patients with rare IDs carries intact but either silenced or overexpressed imprinted genes. Canonical CRISPR/Cas9 editing relying on double-stranded DNA break repair has little to offer in terms of therapeutics for rare IDs. Instead CRISPR/Cas9 can be used in a more sophisticated way by targeting the epigenome. Catalytically dead Cas9 (dCas9) tethered with effector enzymes such as DNA de- and methyltransferases and histone code editors in addition to systems such as CRISPRa and CRISPRi have been shown to have high epigenome editing efficiency in eukaryotic cells. This new era of CRISPR epigenome editors could arguably be a game-changer for curing and treating rare IDs by refined activation and silencing of disturbed imprinted gene expression. This review describes major CRISPR-based epigenome editors and points out their potential use in research and therapy of rare imprinting diseases.},
}
@article {pmid32315755,
year = {2021},
author = {Rahman, MM and Tollefsbol, TO},
title = {Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects.},
journal = {Methods (San Diego, Calif.)},
volume = {187},
number = {},
pages = {77-91},
pmid = {32315755},
issn = {1095-9130},
support = {R01 CA178441/CA/NCI NIH HHS/United States ; R01 CA204346/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Carcinogenesis/genetics ; Cell Line, Tumor ; Clinical Trials, Phase I as Topic ; Disease Models, Animal ; *Epigenesis, Genetic ; Gene Editing/*methods ; Gene Expression Regulation, Neoplastic ; Genetic Therapy/*methods ; Humans ; Mice ; Mice, Transgenic ; Neoplasms/genetics/*therapy ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Cancer therapeutics is an ever-evolving field due to incessant demands for effective and precise treatment options. Over the last few decades, cancer treatment strategies have shifted somewhat from surgery to targeted precision medicine. CRISPR-dCas9 is an emerging version of precision cancer therapy that has been adapted from the prokaryotic CRISPR-Cas system. Once ligated to epigenetic effectors (EE), CRISPR-dCas9 can function as an epigenetic editing tool and CRISPR-dCas9-EE complexes could be exploited to alter cancerous epigenetic features associated with different cancer hallmarks. In this article, we discuss the rationale of epigenetic editing as a therapeutic strategy against cancer. We also outline how sgRNA-dCas9 was derived from the CRISPR-Cas system. In addition, the current status of sgRNA-dCas9 use (in vivo and in vitro) in cancer is updated with a molecular illustration of CRISPR-dCas9-mediated epigenetic and transcriptional modulation. As sgRNA-dCas9 is still at the developmental phase, challenges are inherent to its use. We evaluate major challenges in targeting cancer with sgRNA-dCas9 such as off-target effects, lack of sgRNA designing rubrics, target site selection dilemmas and deficient sgRNA-dCas9 delivery systems. Finally, we appraise the sgRNA-dCas9 as a prospective cancer therapeutic by summarizing ongoing improvements of sgRNA-dCas9 methodology.},
}
@article {pmid32315232,
year = {2020},
author = {Shola, DTN and Yang, C and Kewaldar, VS and Kar, P and Bustos, V},
title = {New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {109-122},
pmid = {32315232},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; DNA, Single-Stranded/genetics ; Gene Editing/*methods ; Humans ; Plasmids/genetics ; },
abstract = {CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR-Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5 kbase, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps. Hence, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein.},
}
@article {pmid32315231,
year = {2020},
author = {Joberty, G and Fälth-Savitski, M and Paulmann, M and Bösche, M and Doce, C and Cheng, AT and Drewes, G and Grandi, P},
title = {A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {123-134},
pmid = {32315231},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Gene Silencing/physiology ; Hep G2 Cells ; Humans ; INDEL Mutation/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.},
}
@article {pmid32315230,
year = {2020},
author = {Davies, K and Jínek, M},
title = {The CRISPR-RNA World: An Interview with Martin Jínek.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {68-72},
doi = {10.1089/crispr.2020.29091.mji},
pmid = {32315230},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/*methods/*trends ; History, 20th Century ; History, 21st Century ; Humans ; RNA ; },
}
@article {pmid32315229,
year = {2020},
author = {Davies, K and Barrangou, R},
title = {COVID-19 and the CRISPR Community Response.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {66-67},
doi = {10.1089/crispr.2020.29092.rba},
pmid = {32315229},
issn = {2573-1602},
mesh = {Betacoronavirus ; Biomedical Research/trends ; COVID-19 ; COVID-19 Testing ; *CRISPR-Cas Systems ; Clinical Laboratory Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/*diagnosis/drug therapy/*therapy ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis/*therapy ; SARS-CoV-2 ; },
}
@article {pmid32315227,
year = {2020},
author = {Liu, Z and Schiel, JA and Maksimova, E and Strezoska, &#x17d; and Zhao, G and Anderson, EM and Wu, Y and Warren, J and Bartels, A and van Brabant Smith, A and Lowe, CE and Forbes, KP},
title = {ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {97-108},
pmid = {32315227},
issn = {2573-1602},
mesh = {Animals ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Endodeoxyribonucleases/genetics/*metabolism ; Endonucleases/genetics ; Eubacterium/*enzymology/genetics/metabolism ; Gene Editing/*methods ; Genome/genetics ; HCT116 Cells ; Humans ; Mice ; RNA, Guide/genetics ; Rats ; },
abstract = {MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.},
}
@article {pmid32315225,
year = {2020},
author = {Heu, CC and McCullough, FM and Luan, J and Rasgon, JL},
title = {CRISPR-Cas9-Based Genome Editing in the Silverleaf Whitefly (Bemisia tabaci).},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {89-96},
pmid = {32315225},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Editing/*methods ; Germ Cells/physiology ; Germ-Line Mutation/genetics ; Hemiptera/*genetics ; Ovary/metabolism ; Plant Viruses/genetics ; },
abstract = {Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject and which have high mortality post injection. We developed a CRISPR-Cas9 gene editing protocol based on injection of vitellogenic adult females rather than embryos ("ReMOT Control"). We identified an ovary-targeting peptide ligand ("BtKV") that, when fused to Cas9 and injected into adult females, transduced the ribonucleoprotein complex to the germline, resulting in efficient, heritable editing of the offspring genome. In contrast to embryo injection, adult injection is easy and does not require specialized equipment. Development of easy-to-use gene editing protocols for B. tabaci will allow researchers to apply the power of reverse genetic approaches to this species and will lead to novel control methods for this devastating pest insect.},
}
@article {pmid32315224,
year = {2020},
author = {Arguedas-Ramírez, G},
title = {Ethics and Global Governance of Human Germline Genome Editing: The Problem of Techno-Scientific Colonialist Paternalism.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {83-88},
doi = {10.1089/crispr.2019.0045},
pmid = {32315224},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Genome, Human ; Germ Cells ; Germ-Line Mutation/*ethics/genetics ; Government ; Humans ; Paternalism/ethics ; },
abstract = {I want to enrich the debate about the ethics and governance of human germline editing (HGE) by emphasizing an underappreciated, yet important, set of concerns regarding exclusionary practices, norms, and efforts that impede a broader discussion about the subject. The possibility for establishing a binding, global, regulatory framework is influenced by economic and geopolitical factors as well as historical processes and sociopolitical problems, such as anti-scientific social movements and the politicization of science. Likewise, it is influenced by different understanding, epistemic resources, and goals between the CRISPR/genome editing community and the rest of society. In this Perspective, I explain the concept of "techno-scientific colonialist paternalism" and why it negatively affects our discussion around HGE. I also discuss the pitfalls of scientific self-regulation, and finally, I advocate that the implementation of HGE should cease to allow time and care for a thoughtful global discussion to emerge.},
}
@article {pmid32315223,
year = {2020},
author = {Soni, S},
title = {CRISPR and the Law: A South African Perspective.},
journal = {The CRISPR journal},
volume = {3},
number = {2},
pages = {81-82},
doi = {10.1089/crispr.2020.29089.sso},
pmid = {32315223},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; History, 20th Century ; History, 21st Century ; Humans ; },
}
@article {pmid32315156,
year = {2020},
author = {Taharabaru, T and Yokoyama, R and Higashi, T and Mohammed, AFA and Inoue, M and Maeda, Y and Niidome, T and Onodera, R and Motoyama, K},
title = {Genome Editing in a Wide Area of the Brain Using Dendrimer-Based Ternary Polyplexes of Cas9 Ribonucleoprotein.},
journal = {ACS applied materials &amp; interfaces},
volume = {12},
number = {19},
pages = {21386-21397},
doi = {10.1021/acsami.9b21667},
pmid = {32315156},
issn = {1944-8252},
mesh = {Animals ; Brain/*metabolism ; CRISPR-Associated Protein 9/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Dendrimers/*chemistry ; Drug Carriers/*chemistry ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; Mice, Inbred BALB C ; Ribonucleoproteins/*pharmacology ; },
abstract = {A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-β-cyclodextrin conjugate (GUG-β-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-β-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-β-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-β-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.},
}
@article {pmid32315033,
year = {2020},
author = {Smith, CJ and Castanon, O and Said, K and Volf, V and Khoshakhlagh, P and Hornick, A and Ferreira, R and Wu, CT and Güell, M and Garg, S and Ng, AHM and Myllykallio, H and Church, GM},
title = {Enabling large-scale genome editing at repetitive elements by reducing DNA nicking.},
journal = {Nucleic acids research},
volume = {48},
number = {9},
pages = {5183-5195},
pmid = {32315033},
issn = {1362-4962},
support = {RM1 HG008525/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins ; CRISPR-Cas Systems ; Cell Survival ; Endodeoxyribonucleases ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Mutation ; RNA ; *Retroelements ; },
abstract = {To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.},
}
@article {pmid32314277,
year = {2020},
author = {Sun, BZ},
title = {Review of Altered inheritance: CRISPR and the ethics of human genome editing by Françoise Baylis.},
journal = {Monash bioethics review},
volume = {38},
number = {1},
pages = {91-93},
doi = {10.1007/s40592-020-00106-0},
pmid = {32314277},
issn = {1836-6716},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Databases, Genetic ; *Gene Editing ; Genome, Human ; Humans ; },
}
@article {pmid32314149,
year = {2020},
author = {Yue, X and Xia, T and Wang, S and Dong, H and Li, Y},
title = {Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9-RecA system.},
journal = {Biotechnology letters},
volume = {42},
number = {9},
pages = {1699-1706},
doi = {10.1007/s10529-020-02893-2},
pmid = {32314149},
issn = {1573-6776},
mesh = {Actinobacteria/*genetics ; Actinomyces/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Mutation/genetics ; Rec A Recombinases/*genetics ; },
abstract = {OBJECTIVE: To develop an inducible CRISPR/Cas9-Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively.<br><br>RESULTS: Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9-RecA system was confirmed to produce more A40926 with an approximate yield of 200&#xa0;mg&#xa0;L[-1] than that of around 150&#xa0;mg&#xa0;L[-1] produced by the wild-type strain.<br><br>CONCLUSIONS: This inducible CRISPR/Cas9-RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.},
}
@article {pmid32314039,
year = {2020},
author = {Kleinschnitz, K and Vießmann, N and Jordan, M and Heidmann, SK},
title = {Condensin I is required for faithful meiosis in Drosophila males.},
journal = {Chromosoma},
volume = {129},
number = {2},
pages = {141-160},
pmid = {32314039},
issn = {1432-0886},
mesh = {Adenosine Triphosphatases/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line ; Chromosome Segregation ; DNA-Binding Proteins/genetics/*metabolism ; Drosophila/*genetics/*metabolism ; Drosophila Proteins/genetics/metabolism ; Fertility/genetics ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression ; Genes, Reporter ; Genetic Engineering ; Male ; Meiosis/*physiology ; Multiprotein Complexes/genetics/*metabolism ; Nondisjunction, Genetic ; Protein Binding ; Protein Interaction Domains and Motifs ; Proteolysis ; RNA Interference ; },
abstract = {The heteropentameric condensin complexes play vital roles in the formation and faithful segregation of mitotic chromosomes in eukaryotes. While the different contributions of the two common condensin complexes, condensin I and condensin II, to chromosome morphology and behavior in mitosis have been thoroughly investigated, much less is known about the specific roles of the two complexes during meiotic divisions. In Drosophila melanogaster, faithful mitotic divisions depend on functional condensin I, but not on condensin II. However, meiotic divisions in Drosophila males require functional condensin II subunits. The role of condensin I during male meiosis in Drosophila has been unresolved. Here, we show that condensin I-specific subunits localize to meiotic chromatin in both meiosis I and II during Drosophila spermatogenesis. Live cell imaging reveals defects during meiotic divisions after RNAi-mediated knockdown of condensin I-specific mRNAs. This phenotype correlates with reduced male fertility and an increase in nondisjunction events both in meiosis I and meiosis II. Consistently, a reduction in male fertility was also observed after proteasome-mediated degradation of the condensin I subunit Barren. Taken together, our results demonstrate an essential role of condensin I during male meiosis in Drosophila melanogaster.},
}
@article {pmid32313931,
year = {2020},
author = {Kopp, ND and Nygaard, KR and Liu, Y and McCullough, KB and Maloney, SE and Gabel, HW and Dougherty, JD},
title = {Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA binding and long-term behavioral consequences.},
journal = {Human molecular genetics},
volume = {29},
number = {9},
pages = {1498-1519},
pmid = {32313931},
issn = {1460-2083},
support = {R01 MH107515/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Brain/metabolism/pathology ; CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems/genetics ; DNA-Binding Proteins/genetics ; Disease Models, Animal ; Embryonic Development/genetics ; Gene Editing ; Gene Expression Regulation, Developmental/genetics ; Humans ; Mice ; Muscle Proteins/*genetics ; Trans-Activators/*genetics ; Transcription Factors, TFII/*genetics ; Transcription, Genetic/genetics ; Williams Syndrome/*genetics/pathology ; },
abstract = {Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.},
}
@article {pmid32313254,
year = {2020},
author = {Wienert, B and Wyman, SK and Yeh, CD and Conklin, BR and Corn, JE},
title = {CRISPR off-target detection with DISCOVER-seq.},
journal = {Nature protocols},
volume = {15},
number = {5},
pages = {1775-1799},
pmid = {32313254},
issn = {1750-2799},
support = {R01 EY028249/EY/NEI NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; DNA Repair ; Gene Editing/*methods ; Humans ; K562 Cells ; Mice ; Sequence Analysis, DNA ; },
abstract = {DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) is a broadly applicable approach for unbiased CRISPR-Cas off-target identification in cells and tissues. It leverages the recruitment of DNA repair factors to double-strand breaks (DSBs) after genome editing with CRISPR nucleases. Here, we describe a detailed experimental protocol and analysis pipeline with which to perform DISCOVER-seq. The principle of this method is to track the precise recruitment of MRE11 to DSBs by chromatin immunoprecipitation followed by next-generation sequencing. A customized open-source bioinformatics pipeline, BLENDER (blunt end finder), then identifies off-target sequences genome wide. DISCOVER-seq is capable of finding and measuring off-targets in primary cells and in situ. The two main advantages of DISCOVER-seq are (i) low false-positive rates because DNA repair enzyme binding is required for genome edits to occur and (ii) its applicability to a wide variety of systems, including patient-derived cells and animal models. The whole protocol, including the analysis, can be completed within 2 weeks.},
}
@article {pmid32313011,
year = {2020},
author = {Després, PC and Dubé, AK and Seki, M and Yachie, N and Landry, CR},
title = {Perturbing proteomes at single residue resolution using base editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1871},
pmid = {32313011},
issn = {2041-1723},
support = {364920//CIHR/Canada ; 384483//CIHR/Canada ; 387697//CIHR/Canada ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genes, Essential ; Genome ; Genome, Fungal ; High-Throughput Nucleotide Sequencing ; *High-Throughput Screening Assays ; Molecular Sequence Data ; Mutagenesis ; Mutation ; Proteome/*genetics ; RNA, Guide/genetics ; Yeasts/*genetics ; },
abstract = {Base editors derived from CRISPR-Cas9 systems and DNA editing enzymes offer an unprecedented opportunity for the precise modification of genes, but have yet to be used at a genome-scale throughput. Here, we test the ability of the Target-AID base editor to systematically modify genes genome-wide by targeting yeast essential genes. We mutate around 17,000 individual sites in parallel across more than 1500 genes. We identify over 700 sites at which mutations have a significant impact on fitness. Using previously determined and preferred Target-AID mutational outcomes, we find that gRNAs with significant effects on fitness are enriched in variants predicted to be deleterious based on residue conservation and predicted protein destabilization. We identify key features influencing effective gRNAs in the context of base editing. Our results show that base editing is a powerful tool to identify key amino acid residues at the scale of proteomes.},
}
@article {pmid32312838,
year = {2020},
author = {Ng, WA and Ma, A and Chen, M and Reed, BH},
title = {A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {6},
pages = {1893-1901},
pmid = {32312838},
issn = {2160-1836},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Expression ; Mutation ; Phenotype ; },
abstract = {We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.},
}
@article {pmid32312593,
year = {2020},
author = {Borrero-de Acuña, JM and Poblete-Castro, I},
title = {Expanding the Reach of Recombineering to Environmental Bacteria.},
journal = {Trends in biotechnology},
volume = {38},
number = {7},
pages = {684-685},
doi = {10.1016/j.tibtech.2020.03.014},
pmid = {32312593},
issn = {1879-3096},
mesh = {Biotechnology/trends ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; *Genetic Engineering ; Metagenomics/trends ; Mutation/genetics ; Pseudomonas putida/*genetics/metabolism ; Recombination, Genetic/*genetics ; },
abstract = {Broadening the application of recombineering technologies in biotechnologically important bacteria poses significant challenges. Aparicio et al. present a vital breakthrough for efficient single-stranded recombineering by utilizing a thermoinducible system in Pseudomonas putida. This offers a simple genome-editing tool towards creating superior biocatalysts for the synthesis of chemicals and for bioremediation endeavors.},
}
@article {pmid32310310,
year = {2020},
author = {Liu, Y and Wang, B},
title = {A Novel Eukaryote-Like CRISPR Activation Tool in Bacteria: Features and Capabilities.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {42},
number = {6},
pages = {e1900252},
doi = {10.1002/bies.201900252},
pmid = {32310310},
issn = {1521-1878},
support = {MR/S018875/1//UKRI Future Leaders Fellowship/International ; RPG-2015-445//Leverhulme Trust/International ; BB/N007212/1//UK BBSRC/International ; MR/S018875/1/MRC_/Medical Research Council/United Kingdom ; 202078/Z/16/Z//Wellcome Trust Seed Award in Science/International ; },
mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Eukaryota/genetics ; Transcriptional Activation ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) activation (CRISPRa) in bacteria is an attractive method for programmable gene activation. Recently, a eukaryote-like, σ[54] -dependent CRISPRa system has been reported. It exhibits high dynamic ranges and permits flexible target site selection. Here, an overview of the existing strategies of CRISPRa in bacteria is presented, and the characteristics and design principles of the CRISPRa system are introduced. Possible scenarios for applying the eukaryote-like CRISPRa system is discussed with corresponding suggestions for performance optimization and future functional expansion. The authors envision the new eukaryote-like CRISPRa system enabling novel designs in multiplexed gene regulation and promoting research in the σ[54] -dependent gene regulatory networks among a variety of biotechnology relevant or disease-associated bacterial species.},
}
@article {pmid32310226,
year = {2020},
author = {Nishizono, H and Darwish, M and Uosaki, H and Masuyama, N and Seki, M and Abe, H and Yachie, N and Yasuda, R},
title = {Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {158},
pages = {},
doi = {10.3791/60808},
pmid = {32310226},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Cas Systems ; Cryopreservation ; Electroporation ; *Embryo, Mammalian ; Fertilization in Vitro ; Genetic Engineering/*methods ; Humans ; Mice ; Mice, Transgenic ; },
abstract = {The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.},
}
@article {pmid32308928,
year = {2020},
author = {Menon, AV and Sohn, JI and Nam, JW},
title = {CGD: Comprehensive guide designer for CRISPR-Cas systems.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {814-820},
pmid = {32308928},
issn = {2001-0370},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems, including dead Cas9 (dCas9), Cas9, and Cas12a, have revolutionized genome engineering in mammalian somatic cells. Although computational tools that assess the target sites of CRISPR-Cas systems are inevitably important for designing efficient guide RNAs (gRNAs), they exhibit generalization issues in selecting features and do not provide optimal results in a comprehensive manner. Here, we introduce a Comprehensive Guide Designer (CGD) for four different CRISPR systems, which utilizes the machine learning algorithm, Elastic Net Logistic Regression (ENLOR), to autonomously generalize the models. CGD contains specific models trained with public datasets generated by CRISPRi, CRISPRa, CRISPR-Cas9, and CRISPR-Cas12a (designated as CGDi, CGDa, CGD9, and CGD12a, respectively) in an unbiased manner. The trained CGD models were benchmarked to other regression-based machine learning models, such as ElasticNet Linear Regression (ENLR), Random Forest and Boruta (RFB), and Extreme Gradient Boosting (Xgboost) with inbuilt feature selection. Evaluation with independent test datasets showed that CGD models outperformed the pre-existing methods in predicting the efficacy of gRNAs. All CGD source codes and datasets are available at GitHub (https://github.com/vipinmenon1989/CGD), and the CGD webserver can be accessed at http://big.hanyang.ac.kr:2195/CGD.},
}
@article {pmid32308773,
year = {2020},
author = {Gao, Q and Ouyang, W and Kang, B and Han, X and Xiong, Y and Ding, R and Li, Y and Wang, F and Huang, L and Chen, L and Wang, D and Dong, X and Zhang, Z and Li, Y and Ze, B and Hou, Y and Yang, H and Ma, Y and Gu, Y and Chao, CC},
title = {Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression.},
journal = {Theranostics},
volume = {10},
number = {11},
pages = {5137-5153},
pmid = {32308773},
issn = {1838-7640},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Gene Editing/*methods ; Humans ; Male ; Mice ; *Mutation ; Neoplasms/*genetics/pathology/*therapy ; Proto-Oncogene Proteins p21(ras)/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Rationale: KRAS is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. Methods: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the KRAS G12S mutant allele or repress its transcription, respectively, with the goal of treating KRAS-driven cancers. Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. Conclusion: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic KRAS mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.},
}
@article {pmid32307772,
year = {2020},
author = {Chiu, YW and Hori, Y and Ebinuma, I and Sato, H and Hara, N and Ikeuchi, T and Tomita, T},
title = {Identification of calcium and integrin-binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9-based screening system.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {6},
pages = {7661-7674},
doi = {10.1096/fj.201902966RR},
pmid = {32307772},
issn = {1530-6860},
mesh = {Alzheimer Disease/metabolism ; Amyloid Precursor Protein Secretases/metabolism ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/metabolism ; Animals ; Brain/metabolism ; CRISPR-Cas Systems/*physiology ; Calcium-Binding Proteins/*metabolism ; Carrier Proteins/metabolism ; Cell Line ; Cell Line, Tumor ; Cell Membrane/metabolism ; HEK293 Cells ; Humans ; Mice ; Neurons/metabolism ; Protein Binding/physiology ; Protein Transport/physiology ; Synapsins/metabolism ; Up-Regulation/physiology ; },
abstract = {The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.},
}
@article {pmid32307552,
year = {2020},
author = {Harel, T and Griffin, JN and Arbogast, T and Monroe, TO and Palombo, F and Martinelli, M and Seri, M and Pippucci, T and Elpeleg, O and Katsanis, N},
title = {Loss of function mutations in CCDC32 cause a congenital syndrome characterized by craniofacial, cardiac and neurodevelopmental anomalies.},
journal = {Human molecular genetics},
volume = {29},
number = {9},
pages = {1489-1497},
pmid = {32307552},
issn = {1460-2083},
support = {R01 DK072301/DK/NIDDK NIH HHS/United States ; R01 GM121317/GM/NIGMS NIH HHS/United States ; R01 HD042601/HD/NICHD NIH HHS/United States ; T32 DK108738/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cilia/genetics/pathology ; Ciliopathies/complications/*genetics/pathology ; Congenital Abnormalities/*genetics/pathology ; Craniofacial Abnormalities/complications/genetics/pathology ; Exome/genetics ; Female ; Heart Defects, Congenital/complications/*genetics/pathology ; Homozygote ; Humans ; Loss of Function Mutation/genetics ; Male ; Neurodevelopmental Disorders/complications/*genetics/pathology ; Pedigree ; Phenotype ; Whole Exome Sequencing ; Zebrafish/genetics ; },
abstract = {Despite the wide use of genomics to investigate the molecular basis of rare congenital malformations, a significant fraction of patients remains bereft of diagnosis. As part of our continuous effort to recruit and perform genomic and functional studies on such cohorts, we investigated the genetic and mechanistic cause of disease in two independent consanguineous families affected by overlapping craniofacial, cardiac, laterality and neurodevelopmental anomalies. Using whole exome sequencing, we identified homozygous frameshift CCDC32 variants in three affected individuals. Functional analysis in a zebrafish model revealed that ccdc32 depletion recapitulates the human phenotypes. Because some of the patient phenotypes overlap defects common to ciliopathies, we asked if loss of CCDC32 might contribute to the dysfunction of this organelle. Consistent with this hypothesis, we show that ccdc32 is required for normal cilia formation in zebrafish embryos and mammalian cell culture, arguing that ciliary defects are at least partially involved in the pathomechanism of this disorder.},
}
@article {pmid32303549,
year = {2020},
author = {Philippe, C and Levesque, S and Dion, MB and Tremblay, DM and Horvath, P and Lüth, N and Cambillau, C and Franz, C and Neve, H and Fremaux, C and Heller, KJ and Moineau, S},
title = {Novel Genus of Phages Infecting Streptococcus thermophilus: Genomic and Morphological Characterization.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {13},
pages = {},
pmid = {32303549},
issn = {1098-5336},
mesh = {Microscopy, Electron, Transmission ; Sequence Analysis, DNA ; Siphoviridae/*classification/genetics/ultrastructure ; Streptococcus Phages/*classification/genetics/ultrastructure ; Streptococcus thermophilus/*virology ; },
abstract = {Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.},
}
@article {pmid32302779,
year = {2020},
author = {Peng, R and Zhang, R and Zhang, J and Tan, P and Han, Y and Zhang, K and Lin, G and Xie, J and Li, J},
title = {Continual Improvement of the Reliability of EML4-ALK Rearrangement Detection in Non-Small-Cell Lung Cancer: A Long-Term Comparison of ALK Detection in China.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {22},
number = {7},
pages = {876-884},
doi = {10.1016/j.jmoldx.2020.03.007},
pmid = {32302779},
issn = {1943-7811},
mesh = {Anaplastic Lymphoma Kinase/antagonists &amp; inhibitors/*genetics ; Animals ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*genetics ; Cell Line, Tumor ; China/epidemiology ; Female ; *Gene Rearrangement ; HEK293 Cells ; Humans ; In Situ Hybridization, Fluorescence/methods ; Laboratory Proficiency Testing/*methods ; Lung Neoplasms/*genetics ; Mice ; Mice, Nude ; Oncogene Proteins, Fusion/*genetics ; Precision Medicine/methods ; *Quality Improvement ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Tumor Burden/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.},
}
@article {pmid32302591,
year = {2020},
author = {Roth, TL and Li, PJ and Blaeschke, F and Nies, JF and Apathy, R and Mowery, C and Yu, R and Nguyen, MLT and Lee, Y and Truong, A and Hiatt, J and Wu, D and Nguyen, DN and Goodman, D and Bluestone, JA and Ye, CJ and Roybal, K and Shifrut, E and Marson, A},
title = {Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.},
journal = {Cell},
volume = {181},
number = {3},
pages = {728-744.e21},
pmid = {32302591},
issn = {1097-4172},
support = {F30 DK120213/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; L30 AI113413/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; S10 OD021822/OD/NIH HHS/United States ; P30 AR070155/AR/NIAMS NIH HHS/United States ; R01 DK119979/DK/NIDDK NIH HHS/United States ; P50 AI150476/AI/NIAID NIH HHS/United States ; DP2 DA042423/DA/NIDA NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; T32 AI007334/AI/NIAID NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Blood Cells ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Knock-In Techniques/*methods ; Genetic Engineering/*methods ; Humans ; Immunotherapy/*methods ; Mice ; Mice, Inbred NOD ; Mice, SCID ; RNA, Guide/genetics ; Single-Cell Analysis/methods ; T-Lymphocytes ; Transcriptome/genetics ; },
abstract = {Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.},
}
@article {pmid32302524,
year = {2020},
author = {Tian, S and Liu, Y and Wu, H and Liu, H and Zeng, J and Choi, MY and Chen, H and Gerhard, R and Dong, M},
title = {Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.},
journal = {Cell host &amp; microbe},
volume = {27},
number = {5},
pages = {782-792.e7},
pmid = {32302524},
issn = {1934-6069},
support = {R21 NS106159/NS/NINDS NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 HL146134/HL/NHLBI NIH HHS/United States ; R01 HL093242/HL/NHLBI NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R21 CA235533/CA/NCI NIH HHS/United States ; R01 HL137229/HL/NHLBI NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 HL118676/HL/NHLBI NIH HHS/United States ; R01 HL130845/HL/NHLBI NIH HHS/United States ; },
mesh = {A549 Cells ; Animals ; Bacterial Proteins ; Bacterial Toxins/*metabolism ; Binding Sites ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clostridium sordellii/*genetics/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Female ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Lung Neoplasms ; Male ; Mice ; Protein Binding ; Semaphorins/*genetics/*isolation &amp; purification/metabolism ; Virulence Factors/metabolism ; },
abstract = {The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.},
}
@article {pmid32302231,
year = {2020},
author = {Philippidis, A},
title = {One Small Dose, One Giant Leap for CRISPR Gene Editing.},
journal = {Human gene therapy},
volume = {31},
number = {7-8},
pages = {402-404},
doi = {10.1089/hum.2020.29119.bfs},
pmid = {32302231},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid32300998,
year = {2020},
author = {Wu, C and Chen, Y and Qiu, Y and Niu, X and Zhu, N and Chen, J and Yao, H and Wang, W and Ma, Y},
title = {A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.},
journal = {Biotechnology letters},
volume = {42},
number = {7},
pages = {1203-1210},
doi = {10.1007/s10529-020-02887-0},
pmid = {32300998},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Fungal/*genetics ; Hypocreales/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; Transcription, Genetic/genetics ; },
abstract = {OBJECTIVE: To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription.<br><br>RESULTS: Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500&#xa0;bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events.<br><br>CONCLUSIONS: We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.},
}
@article {pmid32300337,
year = {2020},
author = {Xu, A and Wang, D and Ding, Y and Zheng, Y and Wang, B and Wei, Q and Wang, S and Yang, L and Ma, LZ},
title = {Integrated Comparative Genomic Analysis and Phenotypic Profiling of Pseudomonas aeruginosa Isolates From Crude Oil.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {519},
pmid = {32300337},
issn = {1664-302X},
abstract = {Pseudomonas aeruginosa is an environmental microorganism that can thrive in diverse ecological niches including plants, animals, water, soil, and crude oil. It also one of the microorganism widely used in tertiary recovery of crude oil and bioremediation. However, the genomic information regarding the mechanisms of survival and adapation of this bacterium in crude oil is still limited. In this study, three Pseudomonads strains (named as IMP66, IMP67, and IMP68) isolated from crude oil were taken for whole-genome sequencing by using a hybridized PacBio and Illumina approach. The phylogeny analysis showed that the three strains were all P. aeruginosa species and clustered in clade 1, the group with PAO1 as a representitive. Subsequent comparative genomic analysis revealed a high degree of individual genomic plasticity, with a probable alkane degradation genomic island, one type I-F CRISPR-Cas system and several prophages integrated into their genomes. Nine genes encoding alkane hydroxylases (AHs) homologs were found in each strain, which might enable these strains to degrade alkane in crude oil. P. aeruginosa can produce rhamnolipids (RLs) biosurfactant to emulsify oil, which enables their survival in crude oil enviroments. Our previous report showed that IMP67 and IMP68 were high RLs producers, while IMP66 produced little RLs. Genomic analysis suggested that their RLs yield was not likely due to differences at genetic level. We then further analyzed the quorum sensing (QS) signal molecules that regulate RLs synthesis. IMP67 and IMP68 produced more N-acyl-homoserine lactones (AHLs) signal molecules than that of PAO1 and IMP66, which could explain their high RLs yield. This study provides evidence for adaptation of P. aeruginosa in crude oil and proposes the potential application of IMP67 and IMP68 in microbial-enhanced oil recovery and bioremediation.},
}
@article {pmid32300245,
year = {2020},
author = {Broughton, JP and Deng, X and Yu, G and Fasching, CL and Servellita, V and Singh, J and Miao, X and Streithorst, JA and Granados, A and Sotomayor-Gonzalez, A and Zorn, K and Gopez, A and Hsu, E and Gu, W and Miller, S and Pan, CY and Guevara, H and Wadford, DA and Chen, JS and Chiu, CY},
title = {CRISPR-Cas12-based detection of SARS-CoV-2.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {870-874},
pmid = {32300245},
issn = {1546-1696},
support = {R21 AI129455/AI/NIAID NIH HHS/United States ; R33 AI120977/AI/NIAID NIH HHS/United States ; R33-AI129455//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/International ; R01-HL105704//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/International ; R01 HL105704/HL/NHLBI NIH HHS/United States ; },
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; COVID-19 Vaccines ; *CRISPR-Cas Systems ; *Clinical Laboratory Techniques ; Coronavirus Infections/diagnosis/virology ; Humans ; Nucleic Acid Amplification Techniques/*methods ; Pandemics ; Pneumonia, Viral/diagnosis/virology ; RNA, Guide/genetics ; SARS-CoV-2 ; Time Factors ; },
abstract = {An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.},
}
@article {pmid32300176,
year = {2020},
author = {Huang, M and Feng, X and Su, D and Wang, G and Wang, C and Tang, M and Paulucci-Holthauzen, A and Hart, T and Chen, J},
title = {Genome-wide CRISPR screen uncovers a synergistic effect of combining Haspin and Aurora kinase B inhibition.},
journal = {Oncogene},
volume = {39},
number = {21},
pages = {4312-4322},
pmid = {32300176},
issn = {1476-5594},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA210929/CA/NCI NIH HHS/United States ; },
mesh = {A549 Cells ; *Aurora Kinase B/antagonists &amp; inhibitors/genetics/metabolism ; *CRISPR-Cas Systems ; Genome-Wide Association Study ; HCT116 Cells ; Humans ; *Intracellular Signaling Peptides and Proteins/antagonists &amp; inhibitors/genetics/metabolism ; *Neoplasm Proteins/antagonists &amp; inhibitors/genetics/metabolism ; *Neoplasms/drug therapy/enzymology/genetics ; Piperazines/*pharmacology ; Protein Kinase Inhibitors/*pharmacokinetics ; *Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Aurora kinases are a family of serine/threonine kinases vital for cell division. Because of the overexpression of Aurora kinases in a broad range of cancers and their important roles in mitosis, inhibitors targeting Aurora kinases have attracted attention in cancer therapy. VX-680 is an effective pan-Aurora kinase inhibitor; however, its clinical efficacy was not satisfying. In this study, we performed CRISPR/Cas9 screens to identify genes whose depletion shows synthetic lethality with VX-680. The top hit from these screens was GSG2 (also known as Haspin), a serine/threonine kinase that phosphorylates histone H3 at Thr-3 during mitosis. Moreover, both Haspin knockout and Haspin inhibitor-treated HCT116 cells were hypersensitive to VX-680. Furthermore, we showed that the synthetic lethal interaction between Haspin depletion and VX-680 was mediated by the inhibition of Haspin with Aurora kinase B (AURKB), but not with Aurora kinase A (AURKA). Strikingly, combined inhibition of Haspin and AURKB had a better efficacy than single-agent treatment in both head and neck squamous cell carcinoma and non-small cell lung cancer. Taken together, our findings have uncovered a synthetic lethal interaction between AURKB and Haspin, which provides a strong rationale for this combination therapy for cancer patients.},
}
@article {pmid32300046,
year = {2020},
author = {Wang, Q and Zhuang, P and Huang, H and Li, L and Liu, L and Webber, HC and Dalal, R and Siew, L and Fligor, CM and Chang, KC and Nahmou, M and Kreymerman, A and Sun, Y and Meyer, JS and Goldberg, JL and Hu, Y},
title = {Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {40},
number = {20},
pages = {3896-3914},
pmid = {32300046},
issn = {1529-2401},
support = {T32 EY027816/EY/NEI NIH HHS/United States ; P30 EY026877/EY/NEI NIH HHS/United States ; R01 EY023295/EY/NEI NIH HHS/United States ; R01 EY024984/EY/NEI NIH HHS/United States ; R01 EY026766/EY/NEI NIH HHS/United States ; U24 EY029903/EY/NEI NIH HHS/United States ; U01 EY027261/EY/NEI NIH HHS/United States ; K08 EY022058/EY/NEI NIH HHS/United States ; R01 EY024932/EY/NEI NIH HHS/United States ; R21 EY031063/EY/NEI NIH HHS/United States ; R01 EY025295/EY/NEI NIH HHS/United States ; R01 EY028106/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Dependovirus/genetics ; Female ; Gene Deletion ; Gene Expression Regulation/*genetics ; Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells ; Male ; Mice ; Mice, Inbred C57BL ; Optic Nerve/pathology ; Optic Nerve Diseases/genetics/therapy ; RNA Editing/*genetics ; Retinal Ganglion Cells/*metabolism/pathology ; Transgenes/genetics ; gamma-Synuclein/*genetics ; },
abstract = {Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.},
}
@article {pmid32299216,
year = {2020},
author = {Huang, L and Tian, H and Luo, J and Song, N and Wu, J},
title = {CRISPR/Cas9 Based Knockout of miR-145 Affects Intracellular Fatty Acid Metabolism by Targeting INSIG1 in Goat Mammary Epithelial Cells.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {18},
pages = {5138-5146},
doi = {10.1021/acs.jafc.0c00845},
pmid = {32299216},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; Epithelial Cells/*metabolism ; Fatty Acids/*metabolism ; Female ; Gene Knockout Techniques ; Goats/*genetics/metabolism ; Intracellular Signaling Peptides and Proteins/*genetics/metabolism ; Mammary Glands, Animal/*metabolism ; MicroRNAs/*genetics/metabolism ; },
abstract = {MiR-145 modulates fatty acid metabolism by regulating the expression of fatty acid metabolism-related genes in goat mammary epithelial cells. Previous studies using RNAi methods have clarified the function of miR-145 in lipogenesis. However, there are limiting factors such as short-term and inconsistent inhibition efficiency in RNAi method. On the basis of previous miR-145 functional studies, this study aims to knock out miR-145 and validate the function using CRISPR/Cas9 technology. We successfully obtained the single cell clone which had single nucleotide deletion around the Drosha processing site. The expression of miR-145 was significantly decreased, and the mRNA and protein expression of target gene INSIG1 were both increased by RT-qPCR and Western blot. The expression of fatty acid metabolism-associated gene (DGAT1, AGPAT6, TIP47, ADFP, CD36, ACSL1, ATGL, ACOX, CPT1A, FADS2, ELOVL5, PPARA, SCD1, FASN, and ACACA) were decreased. The contents of triacylglycerol and cholesterol were significantly inhibited. The percentage of C17:0 and C18:0 saturated fatty acid increased. Taken together, these data suggested that knockout of miR-145 could inhibit TAG and cholesterol contents and affect fatty acid composition through regulating the expression of fatty acid metabolism-related genes. These findings provide a sufficient theoretical basis for improving goat milk quality by miR-145.},
}
@article {pmid32299104,
year = {2020},
author = {Khan, DH and Mullokandov, M and Wu, Y and Voisin, V and Gronda, M and Hurren, R and Wang, X and MacLean, N and Jeyaraju, DV and Jitkova, Y and Xu, GW and Laister, R and Seneviratne, A and Blatman, ZM and Ketela, T and Bader, GD and Marhon, SA and De Carvalho, DD and Minden, MD and Gross, A and Schimmer, AD},
title = {Mitochondrial carrier homolog 2 is necessary for AML survival.},
journal = {Blood},
volume = {136},
number = {1},
pages = {81-92},
doi = {10.1182/blood.2019000106},
pmid = {32299104},
issn = {1528-0020},
support = {/CAPMC/CIHR/Canada ; },
mesh = {Acetylation ; Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Fetal Blood/cytology ; Gene Expression Regulation, Leukemic/genetics ; Gene Knockdown Techniques ; Histones/metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mitochondria/*metabolism ; Mitochondrial Membrane Transport Proteins/*physiology ; Myeloid-Lymphoid Leukemia Protein/physiology ; Neoplasm Proteins/*physiology ; Oncogene Proteins, Fusion/physiology ; Protein Processing, Post-Translational ; Pyruvic Acid/metabolism ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; },
abstract = {Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.},
}
@article {pmid32299090,
year = {2020},
author = {Huang, P and Peslak, SA and Lan, X and Khandros, E and Yano, JA and Sharma, M and Keller, CA and Giardine, B and Qin, K and Abdulmalik, O and Hardison, RC and Shi, J and Blobel, GA},
title = {The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression.},
journal = {Blood},
volume = {135},
number = {24},
pages = {2121-2132},
pmid = {32299090},
issn = {1528-0020},
support = {R56 DK065806/DK/NIDDK NIH HHS/United States ; R01 HL119479/HL/NHLBI NIH HHS/United States ; R01 DK054937/DK/NIDDK NIH HHS/United States ; R24 DK106766/DK/NIDDK NIH HHS/United States ; U54 HG006998/HG/NHGRI NIH HHS/United States ; },
mesh = {Activating Transcription Factor 4/*genetics ; Anemia, Sickle Cell/blood/genetics/therapy ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Enhancer Elements, Genetic ; Erythroblasts/metabolism ; Fetal Hemoglobin/*genetics ; Gene Expression Regulation ; Gene Silencing ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Protein Serine-Threonine Kinases/deficiency/genetics ; Repressor Proteins/*genetics ; Species Specificity ; beta-Thalassemia/blood/genetics/therapy ; eIF-2 Kinase/*genetics ; gamma-Globins/biosynthesis/genetics ; },
abstract = {Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and β-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.},
}
@article {pmid32298665,
year = {2020},
author = {Chen, Y and Jiang, H and Wang, T and He, D and Tian, R and Cui, Z and Tian, X and Gao, Q and Ma, X and Yang, J and Wu, J and Tan, S and Xu, H and Tang, X and Wang, Y and Yu, Z and Han, H and Das, BC and Severinov, K and Hitzeroth, II and Debata, PR and Xu, W and Fan, W and Jin, Z and Cao, C and Yu, M and Xie, W and Huang, Z and Hu, Z and You, Z},
title = {In vitro and in vivo growth inhibition of human cervical cancer cells via human papillomavirus E6/E7 mRNAs' cleavage by CRISPR/Cas13a system.},
journal = {Antiviral research},
volume = {178},
number = {},
pages = {104794},
doi = {10.1016/j.antiviral.2020.104794},
pmid = {32298665},
issn = {1872-9096},
mesh = {Animals ; Apoptosis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; DNA Breaks, Double-Stranded ; DNA-Binding Proteins/*genetics ; Down-Regulation ; Female ; Genetic Therapy ; HeLa Cells ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins, Viral/*genetics ; Papillomavirus E7 Proteins/*genetics ; Papillomavirus Infections/virology ; RNA, Messenger/genetics/*metabolism ; RNA, Viral/genetics/metabolism ; Repressor Proteins/*genetics ; Retinoblastoma Binding Proteins/genetics ; Tumor Suppressor Protein p53/genetics ; Ubiquitin-Protein Ligases/genetics ; Up-Regulation ; Uterine Cervical Neoplasms/*pathology/virology ; Xenograft Model Antitumor Assays ; },
abstract = {Sustained infection of high-risk human papillomavirus (HR-HPVs), especially HPV16 and HPV18, is a major cause of cervical cancer. E6 and E7 oncoproteins, encoded by the HPV genome, are critical for transformation and maintenance of malignant phenotypes of cervical cancer. Here, we used an emerging programmable clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13a system to cleave HPV 16/18 E6/E7 messenger RNAs (mRNAs). The results showed that customized CRISPR/Cas13a system effectively and specifically knocked down HPV 16/18 E6/E7 mRNAs, inducing growth inhibition and apoptosis in HPV16-positive SiHa and HPV18-positive HeLa Cell lines, but not in HPV-negative C33A cell line. Simultaneously, we detected downregulation of E6/E7 oncoproteins and upregulation of tumor suppressor P53 and RB proteins. In addition, we used subcutaneous xenograft tumor growth assays to find that the weight and volume of tumors in the SiHa-16E6CR1 group knocked down by the CRISPR/Cas13a system were significantly lower than those in the SiHa-VECTOR group lacking crRNA. Our study demonstrated that targeting HPV E6/E7 mRNAs by the CRISPR/Cas13a system may be a candidate therapeutic strategy for HPV-related cervical cancer.},
}
@article {pmid32298599,
year = {2020},
author = {Zou, P and Duan, L and Zhang, S and Bai, X and Liu, Z and Jin, F and Sun, H and Xu, W and Chen, R},
title = {Target Specificity of the CRISPR-Cas9 System in Arabidopsis thaliana, Oryza sativa, and Glycine max Genomes.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {27},
number = {10},
pages = {1544-1552},
doi = {10.1089/cmb.2019.0453},
pmid = {32298599},
issn = {1557-8666},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; Computational Biology ; Gene Editing/methods ; Gene Targeting/adverse effects/methods ; Genome, Plant ; Oryza/*genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Soybeans/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), a class of immune-associated sequences in bacteria, have been developed as a powerful tool for editing eukaryotic genomes in diverse cells and organisms in recent years. The CRISPR-Cas9 system can recognize upstream 20 nucleotides (guide sequence) adjacent to the protospacer-adjacent motif site and trigger double-stranded DNA cleavage as well as DNA repair mechanisms, which eventually result in knockout, knockin, or site-specific mutagenesis. However, off-target effect caused by guide sequence misrecognition is the major drawback and restricts its widespread application. In this study, global analysis of specificities of all guide sequences in Arabidopsis thaliana, Oryza sativa (rice), and Glycine max (soybean) were performed. As a result, a simple pipeline and three genome-wide databases were established and shared for the scientific society. For each target site of CRISPR-Cas9, specificity score and off-target number were calculated and evaluated. The mean values of off-target numbers for A. thaliana, rice, and soybean were determined as 27.5, 57.3, and 174.7, respectively. Comparative analysis among these plants suggested that the frequency of off-target effects was correlated to genome size, chromosomal locus, gene density, and guanine-cytosine (GC) content. Our results contributed to the better understanding of CRISPR-Cas9 system in plants and would help to minimize the off-target effect during its applications in the future.},
}
@article {pmid32298586,
year = {2020},
author = {Choudhury, A and Fankhauser, RG and Freed, EF and Oh, EJ and Morgenthaler, AB and Bassalo, MC and Copley, SD and Kaar, JL and Gill, RT},
title = {Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {9},
number = {5},
pages = {1083-1099},
doi = {10.1021/acssynbio.9b00440},
pmid = {32298586},
issn = {2161-5063},
support = {R01 GM124365/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Escherichia coli/*genetics/metabolism ; Galactokinase/genetics ; Gene Editing/*methods ; Mutation ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; },
abstract = {In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.},
}
@article {pmid32298553,
year = {2020},
author = {Leonova, EI and Gainetdinov, RR},
title = {CRISPR/Cas9 Technology in Translational Biomedicine.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {54},
number = {3},
pages = {354-370},
doi = {10.33594/000000224},
pmid = {32298553},
issn = {1421-9778},
support = {N 19-75-30008//Russian Science Foundation/Russia ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics/physiology ; Caspase 9/chemistry/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Gene Expression Regulation/*genetics ; Genetic Engineering/methods ; Genetic Therapy/*methods ; Homologous Recombination/genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - RNA-guided Cas9 endonuclease system has provided a fast and efficient method for precise genome editing in diverse mammalian species, including humans. The CRISPR/Cas9 technology allows generation of modifications into site-specific locations of the selected genes in one major step by carrying deletions, insertions or DNA donor-directed precise sequence modifications. Cas9 forms a nucleoprotein complex with a sequence-specific guide RNA to create double-stranded breaks in complementary DNA target. Further, double-stranded break repair machinery leads to the intended gene modifications. The CRISPR/Cas9 system is widely used technique for genome modification, editing and other biotechnology applications, such as functional annotation, a system for visualization of specific genomic loci and transcriptional control of genes. CRISPR/Cas9-mediated manipulation of the laboratory animal genomes has contributed to the understanding of gene functions and has become a popular approach for modeling human disorders. Furthermore, the growing application of CRISPR-Cas9 system to human genes emerges as an extremely powerful technology for the molecular characterization and treatment of human disease. In this review we present the essential principles of CRISPR/Cas9 technology and the recent advances in its use in translational biomedicine.},
}
@article {pmid32297291,
year = {2020},
author = {Ghorbani Faal, P and Farsi, M and Seifi, A and Mirshamsi Kakhki, A},
title = {Virus-induced CRISPR-Cas9 system improved resistance against tomato yellow leaf curl virus.},
journal = {Molecular biology reports},
volume = {47},
number = {5},
pages = {3369-3376},
doi = {10.1007/s11033-020-05409-3},
pmid = {32297291},
issn = {1573-4978},
mesh = {Begomovirus/*genetics/pathogenicity ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Lycopersicon esculentum/*genetics ; Plant Diseases/genetics ; Plant Viruses/genetics ; Plants, Genetically Modified/genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential off-target effects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overexpressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infiltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a significant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efficiency of using an inducible promoter in CRISPR-Cas9 constructs.},
}
@article {pmid32296572,
year = {2020},
author = {Zhang, J and You, Y},
title = {CRISPR-Cas13a system: a novel approach to precision oncology.},
journal = {Cancer biology &amp; medicine},
volume = {17},
number = {1},
pages = {6-8},
pmid = {32296572},
issn = {2095-3941},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; Medical Oncology/*methods/trends ; Neoplasms/genetics/*therapy ; Precision Medicine/*methods/trends ; },
}
@article {pmid32296011,
year = {2020},
author = {Li, H and Yang, Y and Hong, W and Huang, M and Wu, M and Zhao, X},
title = {Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.},
journal = {Signal transduction and targeted therapy},
volume = {5},
number = {1},
pages = {1},
pmid = {32296011},
issn = {2059-3635},
support = {No. 81602492//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*trends ; Genetic Diseases, Inborn/*genetics/therapy ; Genome, Human/genetics ; Humans ; Molecular Targeted Therapy/*trends ; },
abstract = {Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.},
}
@article {pmid32295056,
year = {2020},
author = {Kobayashi, Y and Aoshima, T and Ito, R and Shinmura, R and Ohtsuka, M and Akasaka, E and Sato, M and Takabayashi, S},
title = {Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain.},
journal = {Cells},
volume = {9},
number = {4},
pages = {},
pmid = {32295056},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Electroporation/methods ; Female ; Gene Editing/*methods ; Male ; Mice ; Mice, Inbred C57BL ; Oviducts/cytology/*metabolism ; Pregnancy ; },
abstract = {Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350-400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare's serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.},
}
@article {pmid32295026,
year = {2020},
author = {Anwar, A and Kim, JK},
title = {Transgenic Breeding Approaches for Improving Abiotic Stress Tolerance: Recent Progress and Future Perspectives.},
journal = {International journal of molecular sciences},
volume = {21},
number = {8},
pages = {},
pmid = {32295026},
issn = {1422-0067},
mesh = {*Adaptation, Biological ; Agriculture ; CRISPR-Cas Systems ; Crops, Agricultural/*genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Genetic Engineering ; MicroRNAs ; *Plant Breeding ; *Plants, Genetically Modified ; Quantitative Trait Loci ; *Stress, Physiological/genetics ; },
abstract = {The recent rapid climate changes and increasing global population have led to an increased incidence of abiotic stress and decreased crop productivity. Environmental stresses, such as temperature, drought, nutrient deficiency, salinity, and heavy metal stresses, are major challenges for agriculture, and they lead to a significant reduction in crop growth and productivity. Abiotic stress is a very complex phenomenon, involving a variety of physiological and biochemical changes in plant cells. Plants exposed to abiotic stress exhibit enhanced levels of ROS (reactive oxygen species), which are highly reactive and toxic and affect the biosynthesis of chlorophyll, photosynthetic capacity, and carbohydrate, protein, lipid, and antioxidant enzyme activities. Transgenic breeding offers a suitable alternative to conventional breeding to achieve plant genetic improvements. Over the last two decades, genetic engineering/transgenic breeding techniques demonstrated remarkable developments in manipulations of the genes for the induction of desired characteristics into transgenic plants. Transgenic approaches provide us with access to identify the candidate genes, miRNAs, and transcription factors (TFs) that are involved in specific plant processes, thus enabling an integrated knowledge of the molecular and physiological mechanisms influencing the plant tolerance and productivity. The accuracy and precision of this phenomenon assures great success in the future of plant improvements. Hence, transgenic breeding has proven to be a promising tool for abiotic stress improvement in crops. This review focuses on the potential and successful applications, recent progress, and future perspectives of transgenic breeding for improving abiotic stress tolerance and productivity in plants.},
}
@article {pmid32294528,
year = {2020},
author = {Tao, X and Xu, T and Kempher, ML and Liu, J and Zhou, J},
title = {Precise promoter integration improves cellulose bioconversion and thermotolerance in Clostridium cellulolyticum.},
journal = {Metabolic engineering},
volume = {60},
number = {},
pages = {110-118},
doi = {10.1016/j.ymben.2020.03.013},
pmid = {32294528},
issn = {1096-7184},
mesh = {Bioengineering ; Biomass ; CRISPR-Cas Systems ; Cellulose/*metabolism ; Clostridium cellulolyticum/*genetics/metabolism/ultrastructure ; DNA Transposable Elements ; DNA, Bacterial/genetics ; Ethanol/metabolism ; Fermentation ; Lactic Acid/metabolism ; Microarray Analysis ; Multigene Family/genetics ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; Thermotolerance ; },
abstract = {Lignocellulose has been used for production of sustainable biofuels and value-added chemicals. However, the low-efficiency bioconversion of lignocellulose greatly contributes to a high production cost. Here, we employed CRISPR-Cas9 editing to improve cellulose degradation efficiency by editing a regulatory element of the cip-cel gene cluster in Clostridium cellulolyticum. Insertion of a synthetic promoter (P4) and an endogenous promoter (P2) in the mspI-deficient parental strain (Δ2866) created chromosomal integrants, P4-2866 and P2-2866, respectively. Both engineered strains increased the transcript abundance of downstream polycistronic genes and enhanced in vitro cellulolytic activities of isolated cellulosomes. A high cellulose load of 20 g/L suppressed cellulose degradation in the parental strain in the first 150 h fermentation; whereas P4-2866 and P2-2866 hydrolyzed 29% and 53% of the cellulose, respectively. Both engineered strains also demonstrated a greater growth rate and a higher cell biomass yield. Interestingly, the Δ2866 parental strain demonstrated better thermotolerance than the wildtype strain, and promoter insertion further enhanced thermotolerance. Similar improvements in cell growth and cellulose degradation were reproduced by promoter insertion in the wildtype strain and a lactate production-defective mutant (LM). P2 insertion in LM increased ethanol titer by 65%. Together, the editing of regulatory elements of catabolic gene clusters provides new perspectives on improving cellulose bioconversion in microbes.},
}
@article {pmid32294405,
year = {2020},
author = {Ostendorf, T and Zillinger, T and Andryka, K and Schlee-Guimaraes, TM and Schmitz, S and Marx, S and Bayrak, K and Linke, R and Salgert, S and Wegner, J and Grasser, T and Bauersachs, S and Soltesz, L and Hübner, MP and Nastaly, M and Coch, C and Kettwig, M and Roehl, I and Henneke, M and Hoerauf, A and Barchet, W and Gärtner, J and Schlee, M and Hartmann, G and Bartok, E},
title = {Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.},
journal = {Immunity},
volume = {52},
number = {4},
pages = {591-605.e6},
doi = {10.1016/j.immuni.2020.03.009},
pmid = {32294405},
issn = {1097-4180},
mesh = {CRISPR-Cas Systems ; Cell Line ; Endoribonucleases/immunology/*metabolism ; Erythrocytes/immunology/parasitology ; Escherichia coli/chemistry/immunology ; Gene Editing/methods ; Humans ; Listeria monocytogenes/chemistry/immunology ; Monocytes/*immunology/microbiology/parasitology ; Neutrophils/*immunology/microbiology/parasitology ; Plasmodium falciparum/chemistry/immunology ; Primary Cell Culture ; RNA Stability ; RNA, Bacterial/immunology/*metabolism ; RNA, Protozoan/immunology/*metabolism ; Serratia marcescens/chemistry/immunology ; Staphylococcus aureus/chemistry/immunology ; Streptococcus/chemistry/immunology ; THP-1 Cells ; Toll-Like Receptor 8/immunology/*metabolism ; },
abstract = {Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2[-/-] or RNASET2[-/-] but not RNASE2[-/-]RNASET2[-/-] cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.},
}
@article {pmid32294089,
year = {2020},
author = {Fajardo-Ortiz, D and Shattuck, A and Hornbostel, S},
title = {Mapping the coevolution, leadership and financing of research on viral vectors, RNAi, CRISPR/Cas9 and other genomic editing technologies.},
journal = {PloS one},
volume = {15},
number = {4},
pages = {e0227593},
pmid = {32294089},
issn = {1932-6203},
mesh = {Biomedical Research/economics/methods/organization &amp; administration/*trends ; Biomedical Technology/economics/methods/organization &amp; administration/*trends ; CRISPR-Cas Systems ; China ; Financing, Organized/economics/methods/*trends ; Gene Editing/economics/methods/*trends ; Genetic Vectors ; Inventions/economics/*trends ; Leadership ; Politics ; RNA Interference ; United States ; Viruses/genetics ; },
abstract = {Genomic editing technologies are developing rapidly, promising significant developments for biomedicine, agriculture and other fields. In the present investigation, we analyzed and compared the process of innovation for six genomic technologies: viral vectors, RNAi, TALENs, meganucleases, ZFNs and CRISPR/Cas including the profile of the main research institutions and their funders, to understand how innovation evolved and what institutions influenced research trajectories. A Web of Science search of papers on viral vectors RNAi, CRISPR/Cas, TALENs, ZFNs and meganucleases was used to build a citation network of 16,746 papers. An analysis of network clustering combined with text mining was performed. For viral vectors, a long-term process of incremental innovation was identified, which was largely publicly funded in the United States and the European Union. The trajectory of RNAi research included clusters related to the study of RNAi as a biological phenomenon and its use in functional genomics, biomedicine and pest control. A British philanthropic organization and a US pharmaceutical company played a key role in the development of basic RNAi research and clinical application respectively, in addition to government and academic institutions. In the case of CRISPR/Cas research, basic science discoveries led to the technical improvements, and these two in turn provided the information required for the development of biomedical, agricultural, livestock and industrial applications. The trajectory of CRISPR/Cas research exhibits a geopolitical division of the investigation efforts between the US, as the main producer and funder of basic research and technical improvements, and Chinese research institutions increasingly leading applied research. Our results reflect a change in the model for financing science, with reduced public financing for basic science and applied research on publicly funded technological developments in the US, and the emergence of China as a scientific superpower, with implications for the development of applications of genomic technologies.},
}
@article {pmid32293244,
year = {2020},
author = {Silveira, MC and Rocha-de-Souza, CM and Albano, RM and de Oliveira Santos, IC and Carvalho-Assef, APD},
title = {Exploring the success of Brazilian endemic clone Pseudomonas aeruginosa ST277 and its association with the CRISPR-Cas system type I-C.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {255},
pmid = {32293244},
issn = {1471-2164},
mesh = {Bacterial Proteins/*genetics ; Brazil/epidemiology ; CRISPR-Cas Systems ; Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Microbial/genetics ; Genome, Bacterial ; Genomic Islands ; Humans ; Phylogeny ; Polymorphism, Single Nucleotide ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/*genetics/pathogenicity ; beta-Lactamases/*genetics ; },
abstract = {BACKGROUND: The Brazilian endemic clone Pseudomonas aeruginosa ST277 carries important antibiotic resistance determinants, highlighting the gene coding for SPM-1 carbapenemase. However, the resistance and persistence of this clone is apparently restricted to the Brazilian territory. To understand the differences between Brazilian strains from those isolated in other countries, we performed a phylogenetic analysis of 47 P. aeruginosa ST277 genomes as well as analyzed the virulence and resistance gene profiles. Furthermore, we evaluated the distribution of genomic islands and assessed in detail the characteristics of the CRISPR-Cas immunity system in these isolates.<br><br>RESULTS: The Brazilian genomes presented a typical set of resistance and virulence determinants, genomic islands and a high frequency of the CRISPR-Cas system type I-C. Even though the ST277 genomes are closely related, the phylogenetic analysis showed that the Brazilian strains share a great number of exclusively SNPs when compared to other ST277 genomes. We also observed a standard CRISPR spacers content for P. aeruginosa ST277, confirming a strong link between sequence type and spacer acquisition. Most CRISPR spacer targets were phage sequences.<br><br>CONCLUSIONS: Based on our findings, P. aeruginosa ST277 strains circulating in Brazil characteristically acquired In163 and PAGI-25, which can distinguish them from strains that do not accumulate resistance mechanisms and can be found on the Asian, European and North American continents. The distinctive genetic elements accumulated in Brazilian samples can contribute to the resistance, pathogenicity and transmission success that characterize the ST277 in this country.},
}
@article {pmid32292777,
year = {2020},
author = {Cen, YK and Lin, JG and Wang, YL and Wang, JY and Liu, ZQ and Zheng, YG},
title = {The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {232},
pmid = {32292777},
issn = {2296-4185},
abstract = {In recent years, there has been a noticeable increase in research interests on the Fusarium species, which includes prevalent plant pathogens and human pathogens, common microbial food contaminants and industrial microbes. Taken the advantage of gibberellin synthesis, Fusarium fujikuroi succeed in being a prevalent plant pathogen. At the meanwhile, F. fujikuroi was utilized for industrial production of gibberellins, a group of extensively applied phytohormone. F. fujikuroi has been known for its outstanding performance in gibberellin production for almost 100 years. Research activities relate to this species has lasted for a very long period. The slow development in biological investigation of F. fujikuroi is largely due to the lack of efficient research technologies and molecular tools. During the past decade, technologies to analyze the molecular basis of host-pathogen interactions and metabolic regulations have been developed rapidly, especially on the aspects of genetic manipulation. At the meanwhile, the industrial fermentation technologies kept sustained development. In this article, we reviewed the currently available research tools/methods for F. fujikuroi research, focusing on the topics about genetic engineering and gibberellin production.},
}
@article {pmid32292501,
year = {2020},
author = {Wu, SS and Li, QC and Yin, CQ and Xue, W and Song, CQ},
title = {Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.},
journal = {Theranostics},
volume = {10},
number = {10},
pages = {4374-4382},
pmid = {32292501},
issn = {1838-7640},
support = {DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Genetic Diseases, Inborn/*therapy ; *Genetic Therapy ; Humans ; },
abstract = {CRISPR/Cas genome editing is a simple, cost effective, and highly specific technique for introducing genetic variations. In mammalian cells, CRISPR/Cas can facilitate non-homologous end joining, homology- directed repair, and single-base exchanges. Cas9/Cas12a nuclease, dCas9 transcriptional regulators, base editors, PRIME editors and RNA editing tools are widely used in basic research. Currently, a variety of CRISPR/Cas-based therapeutics are being investigated in clinical trials. Among many new findings that have advanced the field, we highlight a few recent advances that are relevant to CRISPR/Cas-based gene therapies for monogenic human genetic diseases.},
}
@article {pmid32291290,
year = {2020},
author = {Li, R and Vavrik, C and Danna, CH},
title = {Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {6},
pages = {2033-2042},
pmid = {32291290},
issn = {2160-1836},
mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.},
}
@article {pmid32290857,
year = {2020},
author = {Jones, MG and Khodaverdian, A and Quinn, JJ and Chan, MM and Hussmann, JA and Wang, R and Xu, C and Weissman, JS and Yosef, N},
title = {Inference of single-cell phylogenies from lineage tracing data using Cassiopeia.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {92},
pmid = {32290857},
issn = {1474-760X},
support = {U19 AI090023/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F32 GM125247/GM/NIGMS NIH HHS/United States ; T32 HG000047/HG/NHGRI NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; T32 GM067547/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems ; *Cell Lineage ; Humans ; Mutation ; *Phylogeny ; *Single-Cell Analysis ; },
abstract = {The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together, these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.},
}
@article {pmid32290427,
year = {2020},
author = {Wang, X and Xu, Y and Huang, J and Jin, W and Yang, Y and Wu, Y},
title = {CRISPR-Mediated Knockout of the ABCC2 Gene in Ostrinia furnacalis Confers High-Level Resistance to the Bacillus thuringiensis Cry1Fa Toxin.},
journal = {Toxins},
volume = {12},
number = {4},
pages = {},
pmid = {32290427},
issn = {2072-6651},
support = {2019ZX08012004-005//National Major Science and Technology Projects of China/International ; KYT201803//Fundamental Research Funds for the Central Universities/International ; },
mesh = {Animals ; Bacillus thuringiensis/genetics/*metabolism ; Bacillus thuringiensis Toxins/genetics/*metabolism ; CRISPR-Cas Systems ; Endotoxins/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Gene Knockout Techniques ; Hemolysin Proteins/genetics/*metabolism ; Insect Proteins/deficiency/*genetics ; Insecticide Resistance/genetics ; Moths/*genetics/metabolism ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/deficiency/*genetics ; *Pest Control, Biological ; Plants, Genetically Modified/genetics/metabolism/*parasitology ; Zea mays/genetics/metabolism/*parasitology ; },
abstract = {The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalisABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.},
}
@article {pmid32289863,
year = {2020},
author = {Hearn, JI and Green, TN and Chopra, M and Nursalim, YNS and Ladvanszky, L and Knowlton, N and Blenkiron, C and Poulsen, RC and Singleton, DC and Bohlander, SK and Kalev-Zylinska, ML},
title = {N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation.},
journal = {Thrombosis and haemostasis},
volume = {120},
number = {4},
pages = {671-686},
pmid = {32289863},
issn = {2567-689X},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems ; Calcium/metabolism ; Calcium Signaling ; Carcinogenesis ; Cell Differentiation ; Cell Line, Tumor ; Endoplasmic Reticulum Stress/genetics ; Erythrocytes/*physiology ; Homeostasis ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Megakaryocytes/*physiology ; Receptors, N-Methyl-D-Aspartate/genetics/*metabolism ; Thrombopoiesis ; },
abstract = {The release of calcium ions (Ca[2+]) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca[2+] influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 [-/-] cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca[2+] levels were higher in Meg-01-GRIN1 [-/-] cells, but ER Ca[2+] release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca[2+]. Microarray analysis revealed that Meg-01-GRIN1 [-/-] cells had deregulated expression of transcripts involved in Ca[2+] metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca[2+] regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca[2+] homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.},
}
@article {pmid32289369,
year = {2020},
author = {Xu, L and Dai, Q and Shi, Z and Liu, X and Gao, L and Wang, Z and Zhu, X and Li, Z},
title = {Accurate MRSA identification through dual-functional aptamer and CRISPR-Cas12a assisted rolling circle amplification.},
journal = {Journal of microbiological methods},
volume = {173},
number = {},
pages = {105917},
doi = {10.1016/j.mimet.2020.105917},
pmid = {32289369},
issn = {1872-8359},
mesh = {Aptamers, Nucleotide ; Bacteriological Techniques/*methods ; Biosensing Techniques/methods ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fluorescent Dyes ; Methicillin-Resistant Staphylococcus aureus/*genetics/*isolation &amp; purification ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Infectious diseases have become one of the most threatening global challenge with high morbidity and mortality, bringing great difficulties to clinical diagnosis and treatment. New strategy for high-specific and sensitive bacteria detection are urgently needed in facing the crisis of worldwide antibiotic resistance. Herein, a novel method through the integration of dual aptamer technology and CRISPR-Cas12a assisted rolling circle amplification (RCA) was present to obtain both accurate identification and high-sensitive detection of Methicillin-Resistant Staphylococcus Aureus (MRSA). The specificity inherited from the dual functionalized aptamers initiated bioconjugation to specifically recognize the protein targets on the surface of bacteria. Besides the target activity, the functionalized aptamer could also convert the protein recognition to nucleic acids signals. Through the integration of attached RCA and CRISPR-Cas12a assisted trans-cleavage, dual amplification of the nucleic acid signal was obtained. Based on this, we have extended the application of CRISPR-Cas12a from the nucleic acid detection to bacteria detection. As a result, the proposed method was demonstrated to be with significantly improved sensitivity towards MRSA detection. We believe that the novel integrated strategy would diversify the existing pool of bacterial detection and inspire the development of promising drug candidates in the future.},
}
@article {pmid32289196,
year = {2020},
author = {Yu, Y and Wang, X and Sun, H and Liang, Q and Wang, W and Zhang, C and Bian, X and Cao, Q and Li, Q and Xie, Y and Ma, D and Li, Z and Sun, J},
title = {Improving CRISPR-Cas-mediated RNA targeting and gene editing using SPLCV replicon-based expression vectors in Nicotiana benthamiana.},
journal = {Plant biotechnology journal},
volume = {18},
number = {10},
pages = {1993-1995},
pmid = {32289196},
issn = {1467-7652},
}
@article {pmid32286628,
year = {2020},
author = {Eitzinger, S and Asif, A and Watters, KE and Iavarone, AT and Knott, GJ and Doudna, JA and Minhas, FUAA},
title = {Machine learning predicts new anti-CRISPR proteins.},
journal = {Nucleic acids research},
volume = {48},
number = {9},
pages = {4698-4708},
pmid = {32286628},
issn = {1362-4962},
support = {S10 OD020062/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacterial Proteins/chemistry/*genetics ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; *Machine Learning ; Prophages/genetics ; Proteome ; Sequence Analysis, Protein ; Streptococcus/enzymology/genetics ; },
abstract = {The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.},
}
@article {pmid32284612,
year = {2020},
author = {Zeng, J and Wu, Y and Ren, C and Bonanno, J and Shen, AH and Shea, D and Gehrke, JM and Clement, K and Luk, K and Yao, Q and Kim, R and Wolfe, SA and Manis, JP and Pinello, L and Joung, JK and Bauer, DE},
title = {Therapeutic base editing of human hematopoietic stem cells.},
journal = {Nature medicine},
volume = {26},
number = {4},
pages = {535-541},
pmid = {32284612},
issn = {1546-170X},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*pathology/therapy ; Animals ; Antigens, CD34/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Feasibility Studies ; Female ; *Gene Editing/methods ; Gene Targeting/methods ; Genetic Therapy/*methods ; Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells/*metabolism/pathology ; Heterografts ; Humans ; Mice ; Mice, Inbred NOD ; Mice, Transgenic ; Primary Cell Culture ; Repressor Proteins/*genetics/metabolism ; beta-Thalassemia/pathology/therapy ; gamma-Globins/*genetics/metabolism ; },
abstract = {Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34[+] hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.},
}
@article {pmid32284602,
year = {2020},
author = {Mathony, J and Harteveld, Z and Schmelas, C and Upmeier Zu Belzen, J and Aschenbrenner, S and Sun, W and Hoffmann, MD and Stengl, C and Scheck, A and Georgeon, S and Rosset, S and Wang, Y and Grimm, D and Eils, R and Correia, BE and Niopek, D},
title = {Computational design of anti-CRISPR proteins with improved inhibition potency.},
journal = {Nature chemical biology},
volume = {16},
number = {7},
pages = {725-730},
pmid = {32284602},
issn = {1552-4469},
mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Human ; HEK293 Cells ; Hepatocytes/cytology/metabolism ; Humans ; MicroRNAs/*genetics/metabolism ; Models, Molecular ; Mutagenesis, Insertional ; Neisseria meningitidis/enzymology/genetics ; Plasmids/chemistry/metabolism ; Protein Domains ; Protein Engineering/*methods ; Protein Structure, Secondary ; RNA, Guide/genetics/metabolism ; Staphylococcus aureus/enzymology/genetics ; },
abstract = {Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.},
}
@article {pmid32284587,
year = {2020},
author = {Hanna, RE and Doench, JG},
title = {Design and analysis of CRISPR-Cas experiments.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {813-823},
pmid = {32284587},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Engineering/trends ; Genome/genetics ; Humans ; RNA, Guide/*genetics ; *Software ; },
abstract = {A large and ever-expanding set of CRISPR-Cas systems now enables the rapid and flexible manipulation of genomes in both targeted and large-scale experiments. Numerous software tools and analytical methods have been developed for the design and analysis of CRISPR-Cas experiments, including resources to design optimal guide RNAs for various modes of manipulation and to analyze the results of such experiments. A major recent focus has been the development of comprehensive tools for use on data from large-scale CRISPR-based genetic screens. As this field continues to progress, a clear ongoing challenge is not only to innovate, but to actively maintain and improve existing tools so that researchers across disciplines can rely on a stable set of excellent computational resources for CRISPR-Cas experiments.},
}
@article {pmid32284586,
year = {2020},
author = {Gaudelli, NM and Lam, DK and Rees, HA and Solá-Esteves, NM and Barrera, LA and Born, DA and Edwards, A and Gehrke, JM and Lee, SJ and Liquori, AJ and Murray, R and Packer, MS and Rinaldi, C and Slaymaker, IM and Yen, J and Young, LE and Ciaramella, G},
title = {Directed evolution of adenine base editors with increased activity and therapeutic application.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {892-900},
pmid = {32284586},
issn = {1546-1696},
mesh = {Adenine/*metabolism ; Adenosine Deaminase ; CRISPR-Cas Systems/*genetics ; Cytosine/*metabolism ; DNA/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Mutation/genetics ; RNA, Guide/*genetics ; },
abstract = {The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34[+] cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.},
}
@article {pmid32284553,
year = {2020},
author = {Kaminski, MM and Alcantar, MA and Lape, IT and Greensmith, R and Huske, AC and Valeri, JA and Marty, FM and Klämbt, V and Azzi, J and Akalin, E and Riella, LV and Collins, JJ},
title = {A CRISPR-based assay for the detection of opportunistic infections post-transplantation and for the monitoring of transplant rejection.},
journal = {Nature biomedical engineering},
volume = {4},
number = {6},
pages = {601-609},
pmid = {32284553},
issn = {2157-846X},
mesh = {Biomarkers/blood/urine ; *CRISPR-Cas Systems ; Chemokine CXCL9/blood/urine ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytomegalovirus/genetics/isolation &amp; purification ; Cytomegalovirus Infections/diagnosis ; DNA, Viral/blood/genetics/urine ; Graft Rejection/*virology ; Humans ; Kidney ; Kidney Diseases/virology ; Kidney Transplantation/adverse effects ; Male ; Middle Aged ; Opportunistic Infections/*diagnosis ; Pathology, Molecular/*methods ; Point-of-Care Testing ; Polyomavirus/genetics/isolation &amp; purification ; Polyomavirus Infections/diagnosis ; Postoperative Complications/diagnosis ; RNA, Messenger ; Tumor Virus Infections/diagnosis ; },
abstract = {In organ transplantation, infection and rejection are major causes of graft loss. They are linked by the net state of immunosuppression. To diagnose and treat these conditions earlier, and to improve long-term patient outcomes, refined strategies for the monitoring of patients after graft transplantation are needed. Here, we show that a fast and inexpensive assay based on CRISPR-Cas13 accurately detects BK polyomavirus DNA and cytomegalovirus DNA from patient-derived blood and urine samples, as well as CXCL9 messenger RNA (a marker of graft rejection) at elevated levels in urine samples from patients experiencing acute kidney transplant rejection. The assay, which we adapted for lateral-flow readout, enables-via simple visualization-the post-transplantation monitoring of common opportunistic viral infections and of graft rejection, and should facilitate point-of-care post-transplantation monitoring.},
}
@article {pmid32284347,
year = {2020},
author = {Douglas, P and Ye, R and Radhamani, S and Cobban, A and Jenkins, NP and Bartlett, E and Roveredo, J and Kettenbach, AN and Lees-Miller, SP},
title = {Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin.},
journal = {Molecular and cellular biology},
volume = {40},
number = {13},
pages = {},
pmid = {32284347},
issn = {1098-5549},
support = {P01 CA092584/CA/NCI NIH HHS/United States ; R35 GM119455/GM/NIGMS NIH HHS/United States ; },
mesh = {A549 Cells ; Anaphase-Promoting Complex-Cyclosome/*antagonists &amp; inhibitors/metabolism ; Animals ; Antineoplastic Agents/*pharmacology ; Aurora Kinase A/metabolism ; CRISPR-Cas Systems ; Carbamates/pharmacology ; Cell Cycle Proteins/metabolism ; Contractile Proteins/*genetics/metabolism ; DNA-Activated Protein Kinase/*genetics/metabolism ; Diamines/pharmacology ; Down-Regulation/drug effects ; Enzyme Inhibitors/*pharmacology ; HeLa Cells ; Humans ; Nocodazole/*pharmacology ; Phosphorylation/drug effects ; Protein Serine-Threonine Kinases/metabolism ; Proto-Oncogene Proteins/metabolism ; Up-Regulation/drug effects ; Xenopus ; },
abstract = {The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.},
}
@article {pmid32284320,
year = {2020},
author = {Ratner, HK and Weiss, DS},
title = {Francisella novicida CRISPR-Cas Systems Can Functionally Complement Each Other in DNA Defense while Providing Target Flexibility.},
journal = {Journal of bacteriology},
volume = {202},
number = {12},
pages = {},
pmid = {32284320},
issn = {1098-5530},
support = {R01 AI110701/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; DNA, Bacterial/genetics/metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Female ; Francisella/*enzymology/genetics/pathogenicity ; Gene Expression Regulation, Bacterial ; Gram-Negative Bacterial Infections/microbiology ; Humans ; Mice ; Mice, Inbred C57BL ; Plasmids/genetics/metabolism ; Virulence ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that facilitate protection of bacteria and archaea against infection by external mobile genetic elements. The model pathogen Francisella novicida encodes a CRISPR-Cas12a (FnoCas12a) system and a CRISPR-Cas9 (FnoCas9) system, the latter of which has an additional and noncanonical function in bacterial virulence. Here, we investigated and compared the functional roles of the FnoCas12a and FnoCas9 systems in transformation inhibition and bacterial virulence. Unlike FnoCas9, FnoCas12a was not required for F. novicida virulence. However, both systems were highly effective at plasmid restriction and acted independently of each other. We further identified a critical protospacer-adjacent motif (PAM) necessary for transformation inhibition by FnoCas12a, demonstrating a greater flexibility for target identification by FnoCas12a than previously appreciated and a specificity that is distinct from that of FnoCas9. The effectors of the two systems exhibited different patterns of expression at the mRNA level, suggesting that they may confer distinct benefits to the bacterium in diverse environments. These data suggest that due to the differences between the two CRISPR-Cas systems, together they may provide F. novicida with a more comprehensive defense against foreign nucleic acids. Finally, we demonstrated that the FnoCas12a and FnoCas9 machineries could be simultaneously engineered to restrict the same nonnative target, thereby expanding the toolset for prokaryotic genome manipulation.IMPORTANCE CRISPR-Cas9 and CRISPR-Cas12a systems have been widely commandeered for genome engineering. However, they originate in prokaryotes, where they function as adaptive immune systems. The details of this activity and relationship between these systems within native host organisms have been minimally explored. The human pathogen Francisella novicida contains both of these systems, with the Cas9 system also exhibiting a second activity, modulating virulence through transcriptional regulation. We compared and contrasted the ability of these two systems to control virulence and restrict DNA within their native host bacterium, highlighting differences and similarities in these two functions. Collectively, our results indicate that these two distinct and reprogrammable endogenous systems provide F. novicida with a more comprehensive defense against mobile genetic elements.},
}
@article {pmid32282899,
year = {2020},
author = {Gao, Z and Fan, M and Das, AT and Herrera-Carrillo, E and Berkhout, B},
title = {Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA.},
journal = {Nucleic acids research},
volume = {48},
number = {10},
pages = {5527-5539},
pmid = {32282899},
issn = {1362-4962},
support = {R01 AI145045/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Cell Line ; DNA, Viral/chemistry ; Endodeoxyribonucleases/*metabolism ; Gene Editing ; Genome, Viral ; HEK293 Cells ; HIV/*genetics ; Humans ; Mutation ; RNA/chemistry ; T-Lymphocytes/virology ; },
abstract = {The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.},
}
@article {pmid32281426,
year = {2020},
author = {Aghamiri, S and Talaei, S and Roshanzamiri, S and Zandsalimi, F and Fazeli, E and Aliyu, M and Kheiry Avarvand, O and Ebrahimi, Z and Keshavarz-Fathi, M and Ghanbarian, H},
title = {Delivery of genome editing tools: A promising strategy for HPV-related cervical malignancy therapy.},
journal = {Expert opinion on drug delivery},
volume = {17},
number = {6},
pages = {753-766},
doi = {10.1080/17425247.2020.1747429},
pmid = {32281426},
issn = {1744-7593},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/genetics ; Female ; *Gene Editing ; Humans ; Papillomavirus Infections/*complications/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Uterine Cervical Neoplasms/genetics/*virology ; },
abstract = {INTRODUCTION: Persistent high-risk human papillomavirus infection is the main cause of various types of cancer especially cervical cancer. The E6 and E7 oncoproteins of HPV play critical roles in promoting carcinogenesis and cancer cell growth. As a result, E6 and E7 oncogenes are considered as promising therapeutic targets for cervical cancer. Recently, the development of genome-editing technologies including transcription activator-like effector nucleases (TALEN), meganucleases (MNs), zinc finger nucleases (ZFN), and more importantly clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) has sparked a revolution in the cervical cancer-targeted therapy. However, due to immunogenicity, off-target effect, renal clearance, guide RNA (gRNA) nuclease degradation, and difficult direct transportation into the cytoplasm and nucleus, the safe and effective delivery is considered as the Achilles' heel of this robust strategy.<br><br>AREAS COVERED: In this review, we discuss cutting-edge available strategies for in vivo delivery of genome-editing technologies for HPV-induced cervical cancer therapy. Moreover, the combination of genome-editing tools and other therapies has been fully discussed.<br><br>EXPERT OPINION: The combination of nanoparticle-based delivery systems and genome-editing tools is a promising powerful strategy for cervical cancer therapy. The most significant limitations of this strategy that need to be focused on are low efficiency and off-target events.},
}
@article {pmid32279281,
year = {2020},
author = {Zhang, Y and Zhang, Q and Chen, QJ},
title = {Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems.},
journal = {Science China. Life sciences},
volume = {63},
number = {10},
pages = {1491-1498},
doi = {10.1007/s11427-020-1685-9},
pmid = {32279281},
issn = {1869-1889},
mesh = {Agrobacterium/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Vectors/*genetics ; Genome, Bacterial/genetics ; Genome, Plant/*genetics ; Plant Development/genetics ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics/growth &amp; development ; Plasmids/genetics ; },
abstract = {Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants. Agrobacterium-mediated genetic transformation (AMGT) is the preferred method of CRISPR/Cas reagent delivery, and researchers have recently made great improvements to this process. In this article, we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents. We give an overview of the development of AMGT vectors including binary vector, superbinary vector, dual binary vector, and ternary vector systems. We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains. We focus in particular on the ternary vector system and the resources we developed. In summary, it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems, which are often integrated with morphogenic regulators. The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.},
}
@article {pmid32278427,
year = {2020},
author = {Zhang, M and Liu, C and Shi, Y and Wu, J and Wu, J and Chen, H},
title = {Selective endpoint visualized detection of Vibrio parahaemolyticus with CRISPR/Cas12a assisted PCR using thermal cycler for on-site application.},
journal = {Talanta},
volume = {214},
number = {},
pages = {120818},
doi = {10.1016/j.talanta.2020.120818},
pmid = {32278427},
issn = {1873-3573},
mesh = {Animals ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Electrochemical Techniques ; Food Contamination/analysis ; Particle Size ; Penaeidae/*microbiology ; *Polymerase Chain Reaction ; RNA, Bacterial/*genetics ; Surface Properties ; *Temperature ; Vibrio parahaemolyticus/genetics/*isolation &amp; purification ; },
abstract = {Vibrio parahaemolyticus is a major cause of seafood-associated food poisoning. It is of great significance to develop an accurate, simple and cost-effective method to identify infected seafood, especially for on-site application. Polymerase chain reaction (PCR) remains the golden standard for nucleic acid detection. But traditional methods heavily reply on sophisticated instrument and specialized operators, which limits the application for on-site detections. Here we developed a novel, specific and visualized detection method for PCR based on CRISPR/Cas12a system. On a low-cost thermal cycler, amplification reaction can be conducted easily. The CRISPR/Cas12a system was specifically designed to evaluate amplicons, eliminating false positive results. Besides the negative samples remained colorless, the positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye using a homemade UV device. The presented detection method was verified by detecting shrimp samples. The limit of detection is 1.02 × 10[2] copies/μL. This presented method provided a new strategy for specific endpoint detection of PCR and advanced its application in field for food safety assurance.},
}
@article {pmid32278382,
year = {2020},
author = {Ou, L and Przybilla, MJ and Ahlat, O and Kim, S and Overn, P and Jarnes, J and O'Sullivan, MG and Whitley, CB},
title = {A Highly Efficacious PS Gene Editing System Corrects Metabolic and Neurological Complications of Mucopolysaccharidosis Type I.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {6},
pages = {1442-1454},
pmid = {32278382},
issn = {1525-0024},
mesh = {Animals ; Brain/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/genetics ; Disease Models, Animal ; Enzyme Activation ; Gene Dosage ; Gene Editing/*methods ; *Gene Expression ; Gene Order ; Gene Transfer Techniques ; *Genetic Therapy/adverse effects/methods ; Genetic Vectors/genetics ; Humans ; Iduronidase/*genetics ; Liver/metabolism/pathology ; Mice ; Mucopolysaccharidosis I/*genetics/metabolism/*therapy ; RNA, Guide ; *Transgenes ; Treatment Outcome ; },
abstract = {Our previous study delivered zinc finger nucleases to treat mice with mucopolysaccharidosis type I (MPS I), resulting in a phase I/II clinical trial (ClinicalTrials.gov: NCT02702115). However, in the clinical trial, the efficacy needs to be improved due to the low transgene expression level. To this end, we designed a proprietary system (PS) gene editing approach with CRISPR to insert a promoterless α-l-iduronidase (IDUA) cDNA sequence into the albumin locus of hepatocytes. In this study, adeno-associated virus 8 (AAV8) vectors delivering the PS gene editing system were injected into neonatal and adult MPS I mice. IDUA enzyme activity in the brain significantly increased, while storage levels were normalized. Neurobehavioral tests showed that treated mice had better memory and learning ability. Also, histological analysis showed efficacy reflected by the absence of foam cells in the liver and vacuolation in neuronal cells. No vector-associated toxicity or increased tumorigenesis risk was observed. Moreover, no off-target effects were detected through the unbiased genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) analysis. In summary, these results showed the safety and efficacy of the PS in treating MPS I and paved the way for clinical studies. Additionally, as a therapeutic platform, the PS has the potential to treat other lysosomal diseases.},
}
@article {pmid32278307,
year = {2020},
author = {Yang, YC and Chen, YH and Kao, JH and Ching, C and Liu, IJ and Wang, CC and Tsai, CH and Wu, FY and Liu, CJ and Chen, PJ and Chen, DS and Yang, HC},
title = {Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing.},
journal = {Molecular therapy. Nucleic acids},
volume = {20},
number = {},
pages = {480-490},
pmid = {32278307},
issn = {2162-2531},
abstract = {Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated "base editors" (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.},
}
@article {pmid32277852,
year = {2020},
author = {Przanowska, RK and Sobierajska, E and Su, Z and Jensen, K and Przanowski, P and Nagdas, S and Kashatus, JA and Kashatus, DF and Bhatnagar, S and Lukens, JR and Dutta, A},
title = {miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {6},
pages = {7687-7702},
pmid = {32277852},
issn = {1530-6860},
support = {T32 GM007267/GM/NIGMS NIH HHS/United States ; R01 CA200755/CA/NCI NIH HHS/United States ; R01 AR067712/AR/NIAMS NIH HHS/United States ; 18PRE33990261/AHA_/American Heart Association-American Stroke Association/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Cell Proliferation/genetics ; HEK293 Cells ; Humans ; Mice ; Mice, Knockout ; MicroRNAs/*genetics ; Mitochondria/*genetics ; Muscle Development/*genetics ; Muscle, Skeletal/*physiology ; Muscular Diseases/genetics ; Myoblasts, Skeletal/*physiology ; },
abstract = {miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.},
}
@article {pmid32277804,
year = {2020},
author = {Nakayama, T and Grainger, RM and Cha, SW},
title = {Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {6},
pages = {e23366},
doi = {10.1002/dvg.23366},
pmid = {32277804},
issn = {1526-968X},
support = {EY022954/NH/NIH HHS/United States ; K01 DK101618/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry/*genetics ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Larva/metabolism ; Melanocytes/metabolism ; Membrane Transport Proteins/*genetics/metabolism ; Phosphorothioate Oligonucleotides/chemistry/genetics ; *Recombinational DNA Repair ; Skin Pigmentation ; Xenopus laevis ; Zygote/metabolism ; },
abstract = {We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).},
}
@article {pmid32277725,
year = {2020},
author = {Deng, X and Iwagawa, T and Fukushima, M and Watanabe, S},
title = {Characterization of human-induced pluripotent stem cells carrying homozygous RB1 gene deletion.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {25},
number = {7},
pages = {510-517},
doi = {10.1111/gtc.12771},
pmid = {32277725},
issn = {1365-2443},
mesh = {CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Cell Proliferation/*genetics ; Gene Deletion ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Nanog Homeobox Protein/metabolism ; Octamer Transcription Factor-3/metabolism ; Organoids/*metabolism ; Phenotype ; Retina/growth &amp; development/*metabolism ; Retinal Neoplasms/genetics/metabolism ; Retinoblastoma/genetics/metabolism ; Retinoblastoma Binding Proteins/*genetics/metabolism ; Stage-Specific Embryonic Antigens/metabolism ; Ubiquitin-Protein Ligases/*genetics/metabolism ; },
abstract = {Retinoblastoma is an infant cancer that results from loss of RB1 expression in both alleles. The RB1 gene was the first reported cancer suppressor gene; however, the mechanism by which RB1 loss causes cancer in the retina has not yet been clarified. Human-induced pluripotent stem cells (iPSCs) provide an ideal tool for mechanistic research regarding retinoblastoma. However, because RB1 is a tumor suppressor, loss of both alleles of RB1 in human iPS cells may affect the phenotype of the cells. To examine this possibility, we established human iPSCs with deletions in both alleles of RB1 by CRISPR/Cas9 technique to characterize the associated phenotype. We first examined the expression of RB1 transcripts by RT-qPCR, and RB1 transcripts were expressed in immature hiPSCs and then the expression levels of RB1 transcripts consistently increased during retinal organoid differentiation in human iPSCs. Expression levels of immature markers including SSEA4, OCT3/4 and NANOG were indistinguishable between control iPSCs and RB1 knockout iPSCs. Proliferative activity was also unaffected by homozygous RB1 deletion. Taken together, we showed that homozygous deletion of RB1 did not affect the maturation and proliferation statuses of human iPSCs.},
}
@article {pmid32277708,
year = {2020},
author = {Chang, Y and Hellwarth, PB and Randolph, LN and Sun, Y and Xing, Y and Zhu, W and Lian, XL and Bao, X},
title = {Fluorescent indicators for continuous and lineage-specific reporting of cell-cycle phases in human pluripotent stem cells.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {7},
pages = {2177-2186},
pmid = {32277708},
issn = {1097-0290},
support = {R01 HL142627/HL/NHLBI NIH HHS/United States ; R56 HL142627/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cell Cycle ; Cell Line ; Gene Editing ; Genes, Reporter ; Humans ; Microscopy, Fluorescence ; Pluripotent Stem Cells/*cytology/metabolism ; Ubiquitination ; },
abstract = {Proper cell-cycle progression is essential for the self-renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination-based cell-cycle indicator (FUCCI) has allowed the dual-color visualization of the G1 and S/G2 /M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage-specific FUCCI reporters have not yet been developed to analyze complex tissue-specific cell-cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell-cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell-cycle-dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac-specific TNNT2-FUCCI reporter for lineage-specific cell-cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell-cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration.},
}
@article {pmid32277572,
year = {2020},
author = {Li, C and Liu, H and Hu, YC and Lan, Y and Jiang, R},
title = {Generation and characterization of Six2 conditional mice.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {7},
pages = {e23365},
pmid = {32277572},
issn = {1526-968X},
support = {R01 DE027046/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Craniofacial Abnormalities/*genetics/pathology ; Female ; Gene Editing/methods ; Gene Targeting/methods ; Homeodomain Proteins/*genetics/metabolism ; Integrases/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Phenotype ; Transcription Factors/*genetics/metabolism ; Wnt1 Protein/genetics/metabolism ; },
abstract = {Heterozygous deletion of Six2, which encodes a member of sine oculis homeobox family transcription factors, has recently been associated with the frontonasal dysplasia syndrome FND4. Previous studies showed that Six2 is expressed in multiple tissues during craniofacial development in mice, including embryonic head mesoderm, postmigratory frontonasal neural crest cells, and epithelial and mesenchymal cells of the developing palate and nasal structures. Whereas Six2 [-/-] mice exhibited cranial base defects but did not recapitulate frontonasal phenotypes of FND4 patients, Six1 [-/-] Six2 [-/-] double mutant mice showed severe craniofacial defects including midline facial clefting. The complex phenotypes of FND4 patients and of Six1 [-/-] Six2 [-/-] mutant mice indicate that Six2 plays crucial roles in distinct cell types at multiple stages of craniofacial morphogenesis. Here we report generation of mice carrying insertions of a pair of loxP sites flanking exon-1 of the Six2 gene (Six2 [f] allele) using CRISPR/Cas9-mediated genome editing. We show that the Six2 [f] allele functions normally and is effectively inactivated by Cre-mediated recombination in vivo. Furthermore, we show that Six2 [f/f] ;Wnt1-Cre mice recapitulated cranial base defects but not neonatal lethality of Six2 [-/-] mice. These results indicate that Six2 [f/f] mice enable systematic investigation of cell type- and stage-specific Six2 function in development and disease.},
}
@article {pmid32277462,
year = {2020},
author = {Bhandawat, A and Sharma, V and Rishi, V and K Roy, J},
title = {Biolistic Delivery of Programmable Nuclease (CRISPR/Cas9) in Bread Wheat.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2124},
number = {},
pages = {309-329},
doi = {10.1007/978-1-0716-0356-7_17},
pmid = {32277462},
issn = {1940-6029},
mesh = {Biolistics/*methods ; Bread ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Endonucleases/*metabolism ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Mutation/genetics ; Protoplasts/metabolism ; RNA, Guide/genetics ; Reproducibility of Results ; Ribonucleoproteins/metabolism ; Transcription, Genetic ; Triticum/embryology/*genetics ; },
abstract = {The discovery of site-specific programmable nucleases has led to a major breakthrough in the area of genome editing. In the past few years, CRISPR/Cas system has been utilized for genome editing of a large number of crops including cereals like wheat, rice, maize, and barley. In terms of consumption, wheat is second only to rice as the most important crop of the world. In the present chapter, we describe biolistic delivery method of ribonucleoprotein (RNP) complexes of programmable nuclease (CRISPR/Cas9) for targeted genome editing and selection-free screening of transformants in wheat. The method not only overcomes the problem of random integration into the genome but also reduces the off-targets. Besides the step-by-step protocol, plausible challenges and ways to overcome them are also discussed. By using the described method of biolistic delivery of CRISPR/Cas9 in plant systems, genome-edited plants can be identified within 11 weeks.},
}
@article {pmid32277460,
year = {2020},
author = {Imani, J and Kogel, KH},
title = {Plant Transformation Techniques: Agrobacterium- and Microparticle-Mediated Gene Transfer in Cereal Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2124},
number = {},
pages = {281-294},
doi = {10.1007/978-1-0716-0356-7_15},
pmid = {32277460},
issn = {1940-6029},
mesh = {Agrobacterium/*genetics ; Agrobacterium tumefaciens/genetics ; Biolistics ; DNA, Plant/genetics ; Edible Grain/*genetics ; *Gene Transfer Techniques ; Genetic Vectors/metabolism ; Glucuronidase/metabolism ; Hordeum/embryology/*genetics ; Plants, Genetically Modified ; Sterilization ; *Transformation, Genetic ; Triticum/embryology/*genetics ; },
abstract = {Biotechnological methods for targeted gene transfers into plants are key for successful breeding in the twenty-first century and thus essential for the survival of humanity. Two decades ago, genetic transformation of crop plants was not routine, and it was all but impossible with important cereals such as barley and wheat. The recent focus on crop plant genomics-yet based on the Arabidopsis toolbox-boosted the research for more efficient plant transformation protocols, thereby considerably widened the number of genetically tractable crops. Moreover, modern genome editing methods such as the CRISPR/Cas technique are game changers in plant breeding, though heavily dependent on technical optimization of plant transformation. Basically, there are two successful ways of introducing DNA into plant cells: one is making use of a living DNA vector, namely, microbes such as the soil bacterium Agrobacterium tumefaciens that infects plants and naturally transfers and subsequently integrates DNA into the plant genome. The other method uses a direct physical transfer of DNA by means of microinjection, microprojectile bombardment, or polymers such as polyethylene glycol. Both ways subsequently require sophisticated strategies for selecting and multiplying the transformed cells under tissue culture conditions to develop into a fully functional plant with the new desirable characteristics. Here we discuss practical and theoretical aspects of cereal crop plant transformation by Agrobacterium-mediated transformation and microparticle bombardment. Using immature embryos as explants, the efficiency of cereal transformation is compelling, reaching today up to 80% transformation efficiency.},
}
@article {pmid32276963,
year = {2020},
author = {Bian, T and Tagmount, A and Vulpe, C and Vijendra, KC and Xing, C},
title = {CXL146, a Novel 4H-Chromene Derivative, Targets GRP78 to Selectively Eliminate Multidrug-Resistant Cancer Cells.},
journal = {Molecular pharmacology},
volume = {97},
number = {6},
pages = {402-408},
pmid = {32276963},
issn = {1521-0111},
support = {R01 CA163864/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; Benzopyrans/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Resistance, Multiple/drug effects ; Drug Resistance, Neoplasm/*drug effects ; Drug Screening Assays, Antitumor ; Endoplasmic Reticulum Chaperone BiP ; HL-60 Cells ; Heat-Shock Proteins/drug effects/*metabolism ; Humans ; Mitoxantrone/pharmacology ; },
abstract = {The 78-kDa glucose-regulated protein (GRP78), an endoplasmic reticulum (ER) chaperone, is a master regulator of the ER stress. A number of studies revealed that high levels of GRP78 protein in cancer cells confer multidrug resistance (MDR) to therapeutic treatment. Therefore, drug candidate that reduces GRP78 may represent a novel approach to eliminate MDR cancer cells. Our earlier studies showed that a set of 4H-chromene derivatives induced selective cytotoxicity in MDR cancer cells. In the present study, we elucidated its selective mechanism in four MDR cancer cell lines with one lead candidate (CXL146). Cytotoxicity results confirmed the selective cytotoxicity of CXL146 toward the MDR cancer cell lines. We noted significant overexpression of GRP78 in all four MDR cell lines compared with the parental cell lines. Unexpectedly, CXL146 treatment rapidly and dose-dependently reduced GRP78 protein in MDR cancer cell lines. Using human leukemia (HL) 60/mitoxantrone (MX) 2 cell line as the model, we demonstrated that CXL146 treatment activated the unfolded protein response (UPR); as evidenced by the activation of inositol-requiring enzyme 1α, protein kinase R-like ER kinase, and activating transcription factor 6. CXL146-induced UPR activation led to a series of downstream events, including extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation, which contributed to CXL146-induced apoptosis. Targeted reduction in GRP78 resulted in reduced sensitivity of HL60/MX2 toward CXL146. Long-term sublethal CXL146 exposure also led to reduction in GRP78 in HL60/MX2. These data collectively support GRP78 as the target of CXL146 in MDR treatment. Interestingly, HL60/MX2 upon long-term sublethal CXL146 exposure regained sensitivity to mitoxantrone treatment. Therefore, further exploration of CXL146 as a novel therapy in treating MDR cancer cells is warranted. SIGNIFICANCE STATEMENT: Multidrug resistance is one major challenge to cancer treatment. This study provides evidence that cancer cells overexpress 78-kDa glucose-regulated protein (GRP78) as a mechanism to acquire resistance to standard cancer therapies. A chromene-based small molecule, CXL146, selectively eliminates cancer cells with GRP78 overexpression via activating unfolded protein response-mediated apoptosis. Further characterization indicates that CXL146 and standard therapies complementarily target different populations of cancer cells, supporting the potential of CXL146 to overcome multidrug resistance in cancer treatment.},
}
@article {pmid32276588,
year = {2020},
author = {Faulkner, J and Jiang, P and Farris, D and Walker, R and Dai, Z},
title = {CRISPR/CAS9-mediated knockout of Abi1 inhibits p185[Bcr-Abl]-induced leukemogenesis and signal transduction to ERK and PI3K/Akt pathways.},
journal = {Journal of hematology &amp; oncology},
volume = {13},
number = {1},
pages = {34},
pmid = {32276588},
issn = {1756-8722},
support = {R15 CA191476/CA/NCI NIH HHS/United States ; R21 CA187303/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Animals ; CRISPR-Cas Systems ; Carcinogenesis/*genetics/metabolism ; Cell Line, Tumor ; Cytoskeletal Proteins/*genetics ; Female ; Fusion Proteins, bcr-abl/*genetics ; Leukemia/*genetics/metabolism ; MAP Kinase Signaling System ; Mice, Inbred BALB C ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Proto-Oncogene Proteins c-bcr/*genetics ; *Signal Transduction ; },
abstract = {BACKGROUND: Abl interactor 1 (Abi1) is a downstream target of Abl tyrosine kinases and a component of the WAVE regulatory complex (WRC) that plays an important role in regulating actin cytoskeleton remodeling and membrane receptor signaling. While studies using short hairpin RNA (shRNA) have suggested that Abi1 plays a critical role in Bcr-Abl-induced leukemogenesis, the mechanism involved is not clear.<br><br>METHODS: In this study, we knocked out Abi1 expression in p185[Bcr-Abl]-transformed hematopoietic cells using CRISPR/Cas9-mediated gene editing technology. The effects of Abi1 deficiency on actin cytoskeleton remodeling, the Bcr-Abl signaling, IL-3 independent growth, and SDF-induced chemotaxis in these cells were examined by various in vitro assays. The leukemogenic activity of these cells was evaluated by a syngeneic mouse transplantation model.<br><br>RESULTS: We show here that Abi1 deficiency reduced the IL3-independent growth and SDF-1&#x3b1;-mediated chemotaxis in p185[Bcr-Abl]-transformed hematopoietic cells and inhibited Bcr-Abl-induced abnormal actin remodeling. Depletion of Abi1 also impaired the Bcr-Abl signaling to the ERK and PI3 kinase/Akt pathways. Remarkably, the p185[Bcr-Abl]-transformed cells with Abi1 deficiency lost their ability to develop leukemia in syngeneic mice. Even though these cells developed drug tolerance in vitro after prolonged selection with imatinib as their parental cells, the imatinib-tolerant cells remain incapable of leukemogenesis in vivo.<br><br>CONCLUSIONS: Together, this study highlights an essential role of Abi1 in Bcr-Abl-induced leukemogenesis and provides a model system for dissecting the Abi1 signaling in Bcr-Abl-positive leukemia.},
}
@article {pmid32276445,
year = {2020},
author = {Ahmar, S and Gill, RA and Jung, KH and Faheem, A and Qasim, MU and Mubeen, M and Zhou, W},
title = {Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook.},
journal = {International journal of molecular sciences},
volume = {21},
number = {7},
pages = {},
pmid = {32276445},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Plant ; Plant Breeding/*methods ; Plants/genetics ; Plants, Genetically Modified ; },
abstract = {In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.},
}
@article {pmid32276422,
year = {2020},
author = {Xu, K and Han, CX and Zhou, H and Ding, JM and Xu, Z and Yang, LY and He, C and Akinyemi, F and Zheng, YM and Qin, C and Luo, HX and Meng, H},
title = {Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle.},
journal = {International journal of molecular sciences},
volume = {21},
number = {7},
pages = {},
pmid = {32276422},
issn = {1422-0067},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Chickens ; Gene Editing ; *Gene Expression Profiling ; *Gene Knockout Techniques ; Myostatin/*genetics ; },
abstract = {Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.},
}
@article {pmid32276275,
year = {2020},
author = {Svaton, M and Skvarova Kramarzova, K and Kanderova, V and Mancikova, A and Smisek, P and Jesina, P and Krijt, J and Stiburkova, B and Dobrovolny, R and Sokolova, J and Bakardjieva-Mihaylova, V and Vodickova, E and Rackova, M and Stuchly, J and Kalina, T and Stary, J and Trka, J and Fronkova, E and Kozich, V},
title = {A homozygous deletion in the SLC19A1 gene as a cause of folate-dependent recurrent megaloblastic anemia.},
journal = {Blood},
volume = {135},
number = {26},
pages = {2427-2431},
pmid = {32276275},
issn = {1528-0020},
mesh = {Adolescent ; Anemia, Megaloblastic/drug therapy/*genetics ; CRISPR-Cas Systems ; Cells, Cultured ; Clone Cells ; Folic Acid/*therapeutic use ; Frameshift Mutation ; Gene Knockout Techniques ; Homozygote ; Humans ; Hyperhomocysteinemia/drug therapy/*genetics ; K562 Cells ; Male ; Recurrence ; Sequence Deletion ; Sodium-Hydrogen Exchanger 1/*deficiency/genetics ; Vitamin B 12/therapeutic use ; Whole Exome Sequencing ; },
}
@article {pmid32276113,
year = {2020},
author = {Zhu, GH and Chereddy, SCRR and Howell, JL and Palli, SR},
title = {Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation.},
journal = {Insect biochemistry and molecular biology},
volume = {122},
number = {},
pages = {103373},
doi = {10.1016/j.ibmb.2020.103373},
pmid = {32276113},
issn = {1879-0240},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/*instrumentation ; Larva/genetics/growth &amp; development/metabolism ; RNA, Guide/*genetics ; Spodoptera/*genetics/growth &amp; development/metabolism ; },
abstract = {The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.},
}
@article {pmid32275950,
year = {2021},
author = {Niu, D and Ma, X and Yuan, T and Niu, Y and Xu, Y and Sun, Z and Ping, Y and Li, W and Zhang, J and Wang, T and Church, GM},
title = {Porcine genome engineering for xenotransplantation.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {229-245},
doi = {10.1016/j.addr.2020.04.001},
pmid = {32275950},
issn = {1872-8294},
mesh = {Animals ; CRISPR-Cas Systems ; Endogenous Retroviruses ; Genetic Engineering/*methods ; Graft Rejection/physiopathology/prevention &amp; control ; Homologous Recombination ; Humans ; Immunosuppressive Agents/therapeutic use ; Inflammation/physiopathology/prevention &amp; control ; Recombinant Fusion Proteins ; Swine ; Swine Diseases/prevention &amp; control ; Transplantation, Heterologous/*methods ; },
abstract = {The extreme shortage of human donor organs for treatment of patients with end-stage organ failures is well known. Xenotransplantation, which might provide unlimited organ supply, is a most promising strategy to solve this problem. Domestic pigs are regarded as ideal organ-source animals owing to similarity in anatomy, physiology and organ size to humans as well as high reproductive capacity and low maintenance cost. However, several barriers, which include immune rejection, inflammation and coagulative dysfunctions, as well as the cross-species transmission risk of porcine endogenous retrovirus, blocked the pig-to-human xenotransplantation. With the rapid development of genome engineering technologies and the potent immunosuppressive medications in recent years, these barriers could be eliminated through genetic modification of pig genome together with the administration of effective immunosuppressants. A number of candidate genes involved in the regulation of immune response, inflammation and coagulation have been explored to optimize porcine xenograft survival in non-human primate recipients. PERV inactivation in pigs has also been accomplished to firmly address the safety issue in pig-to-human xenotransplantation. Many encouraging preclinical milestones have been achieved with some organs surviving for years. Therefore, the clinical trials of some promising organs, such as islet, kidney and heart, are aimed to be launched in the near future.},
}
@article {pmid32275780,
year = {2020},
author = {Nieves-Cordones, M and Lara, A and Silva, M and Amo, J and Rodriguez-Sepulveda, P and Rivero, RM and Martínez, V and Botella, MA and Rubio, F},
title = {Root high-affinity K[+] and Cs[+] uptake and plant fertility in tomato plants are dependent on the activity of the high-affinity K[+] transporter SlHAK5.},
journal = {Plant, cell &amp; environment},
volume = {43},
number = {7},
pages = {1707-1721},
doi = {10.1111/pce.13769},
pmid = {32275780},
issn = {1365-3040},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cesium/*metabolism ; Flowers/metabolism ; Fruit/growth &amp; development ; Gene Editing ; Lycopersicon esculentum/*metabolism/physiology ; Plant Proteins/metabolism/*physiology ; Plant Roots/*metabolism ; Plants, Genetically Modified ; Pollen Tube/growth &amp; development ; Potassium/*metabolism ; Potassium Channels/metabolism/*physiology ; Reproduction ; Seeds/growth &amp; development ; },
abstract = {Root K[+] acquisition is a key process for plant growth and development, extensively studied in the model plant Arabidopsis thaliana. Because important differences may exist among species, translational research supported by specific studies is needed in crops such as tomato. Here we present a reverse genetics study to demonstrate the role of the SlHAK5 K[+] transporter in tomato K[+] nutrition, Cs[+] accumulation and its fertility. slhak5 KO lines, generated by CRISPR-Cas edition, were characterized in growth experiments, Rb[+] and Cs[+] uptake tests and root cells K[+] -induced plasma membrane depolarizations. Pollen viability and its K[+] accumulation capacity were estimated by using the K[+] -sensitive dye Ion Potassium Green 4. SlHAK5 is the major system for high-affinity root K[+] uptake required for plant growth at low K[+] , even in the presence of salinity. It also constitutes a pathway for Cs[+] entry in tomato plants with a strong impact on fruit Cs[+] accumulation. SlHAK5 also contributes to pollen K[+] uptake and viability and its absence produces almost seedless fruits. Knowledge gained into SlHAK5 can serve as a model for other crops with fleshy fruits and it can help to generate tools to develop low Cs[+] or seedless fruits crops.},
}
@article {pmid32275651,
year = {2020},
author = {Willems, J and de Jong, APH and Scheefhals, N and Mertens, E and Catsburg, LAE and Poorthuis, RB and de Winter, F and Verhaagen, J and Meye, FJ and MacGillavry, HD},
title = {ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons.},
journal = {PLoS biology},
volume = {18},
number = {4},
pages = {e3000665},
pmid = {32275651},
issn = {1545-7885},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Dependovirus/genetics ; Epitopes/*genetics ; Female ; Gene Editing/*methods ; Gene Knock-In Techniques ; Genes, Reporter ; Genetic Vectors ; Genome ; Green Fluorescent Proteins/genetics/metabolism ; Male ; Mice, Transgenic ; Microscopy, Fluorescence ; Molecular Imaging/methods ; Neurons/*immunology/physiology ; Organ Culture Techniques ; Proteins/*genetics/immunology/metabolism ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate/genetics/metabolism ; Spatio-Temporal Analysis ; },
abstract = {The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.},
}
@article {pmid32275383,
year = {2020},
author = {Li, S and Liu, Q and Zhong, Z and Deng, Z and Sun, Y},
title = {Exploration of Hygromycin B Biosynthesis Utilizing CRISPR-Cas9-Associated Base Editing.},
journal = {ACS chemical biology},
volume = {15},
number = {6},
pages = {1417-1423},
doi = {10.1021/acschembio.0c00071},
pmid = {32275383},
issn = {1554-8937},
mesh = {Bacterial Proteins/genetics/metabolism ; *Biosynthetic Pathways ; CRISPR-Cas Systems ; Gene Editing/methods ; Glycosyltransferases/genetics/metabolism ; Hygromycin B/*metabolism ; Streptomyces/enzymology/genetics/*metabolism ; Substrate Specificity ; },
abstract = {Hygromycin B is an aminoglycoside antibiotic widely used in industry and biological research. However, most of its biosynthetic pathway has not been completely identified due to the immense difficulty in genetic manipulation of the producing strain. To address this problem, we developed an efficient system that combines clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-associated base editing and site-specific recombination instead of conventional double-crossover-based homologous recombination. This strategy was successfully applied to the in vivo inactivation of five candidate genes involved in the biosynthesis of hygromycin B by generating stop codons or mutating conserved residues within the encoding region. The results revealed that HygJ, HygL, and HygD are responsible for successive dehydrogenation, transamination, and transglycosylation of nucleoside diphosphate (NDP)-heptose. Notably, HygY acts as an unusual radical S-adenosylmethionine (SAM)-dependent epimerase for hydroxyl carbons, and HygM serves as a versatile methyltransferase in multiple parallel metabolic networks. Based on in vivo and in vitro evidence, the biosynthetic pathway for hygromycin B is proposed.},
}
@article {pmid32274568,
year = {2020},
author = {Bonet-Fernández, JM and Aroca-Aguilar, JD and Corton, M and Ramírez, AI and Alexandre-Moreno, S and García-Antón, MT and Salazar, JJ and Ferre-Fernández, JJ and Atienzar-Aroca, R and Villaverde, C and Iancu, I and Tamayo, A and Méndez-Hernández, CD and Morales-Fernández, L and Rojas, B and Ayuso, C and Coca-Prados, M and Martinez-de-la-Casa, JM and García-Feijoo, J and Escribano, J},
title = {CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.},
journal = {Human genetics},
volume = {139},
number = {10},
pages = {1209-1231},
doi = {10.1007/s00439-020-02164-0},
pmid = {32274568},
issn = {1432-1203},
mesh = {Adult ; Animals ; Anterior Chamber/metabolism/pathology/surgery ; CRISPR-Cas Systems ; Case-Control Studies ; Complement C3/deficiency/*genetics ; Embryo, Nonmammalian ; Extracellular Matrix/*metabolism/pathology ; Eye Abnormalities/*genetics/metabolism/pathology/surgery ; Female ; Gene Editing ; Gene Expression ; Genes, Recessive ; Glaucoma/*genetics/metabolism/pathology/surgery ; High-Throughput Nucleotide Sequencing ; Humans ; *Loss of Function Mutation ; Male ; Middle Aged ; Pedigree ; Trabecular Meshwork/metabolism/pathology/surgery ; Trabeculectomy ; Trypsin Inhibitor, Kazal Pancreatic/deficiency/*genetics ; Zebrafish ; alpha-Macroglobulins/deficiency/*genetics ; },
abstract = {Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.},
}
@article {pmid32274563,
year = {2020},
author = {Brito, LF and Schultenkämper, K and Passaglia, LMP and Wendisch, VF},
title = {CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {11},
pages = {5095-5106},
pmid = {32274563},
issn = {1432-0614},
mesh = {Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; Chromosomes ; *Gene Expression ; L-Iditol 2-Dehydrogenase/metabolism ; Paenibacillus/enzymology/*genetics ; Promoter Regions, Genetic ; RNA, Guide ; Sorbitol/metabolism ; },
abstract = {Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species.Key points• CRISPRi toward gene repression was applied for the first time in Paenibacillus.• CRISPRi of spo0A and yaaT depleted spores and increased biofilms in SBR5.• CRISPRi-based ydjJ repression decreased specific activity of sorbitol dehydrogenase.},
}
@article {pmid32273496,
year = {2020},
author = {Jiang, Y and Wei, J and Cui, H and Liu, C and Zhi, Y and Jiang, Z and Li, Z and Li, S and Yang, Z and Wang, X and Qian, P and Zhang, C and Zhong, C and Su, XZ and Yuan, J},
title = {An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1764},
pmid = {32273496},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Culicidae/*metabolism/parasitology ; Cyclic GMP/metabolism ; Cyclic GMP-Dependent Protein Kinases/metabolism ; Gametogenesis/*drug effects ; Gene Editing/methods ; Intracellular Membranes/*metabolism ; Malaria/parasitology ; Mosquito Vectors/metabolism/parasitology ; Plasmodium/genetics/metabolism/physiology ; Protozoan Proteins/genetics/*metabolism ; Xanthurenates/metabolism/*pharmacology ; },
abstract = {Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca[2+] mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission.},
}
@article {pmid32273345,
year = {2020},
author = {Ling, L and Mulaka, M and Munro, J and Dass, S and Mather, MW and Riscoe, MK and Llinás, M and Zhou, J and Ke, H},
title = {Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {21},
pages = {7235-7248},
pmid = {32273345},
issn = {1083-351X},
support = {R01 AI028398/AI/NIAID NIH HHS/United States ; I01 BX003312/BX/BLRD VA/United States ; R56 AI100569/AI/NIAID NIH HHS/United States ; R01 AI100569/AI/NIAID NIH HHS/United States ; K22 AI127702/AI/NIAID NIH HHS/United States ; },
mesh = {Antimalarials/*pharmacology ; Humans ; Malaria, Falciparum/drug therapy/genetics/*metabolism ; Mitochondria/genetics/*metabolism ; Mitochondrial Proteins/genetics/metabolism ; Mitochondrial Ribosomes/*metabolism ; Plasmodium falciparum/genetics/*metabolism ; Protozoan Proteins/genetics/metabolism ; Ribosomal Proteins/genetics/metabolism ; },
abstract = {The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼27 small, fragmented rRNA genes having lengths of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum is essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of the mitoribosome in sustaining the metabolic status of the parasite mitochondrion remains unclear. The small ribosomal subunit in P. falciparum has 14 annotated mitoribosomal proteins, and employing a CRISPR/Cas9-based conditional knockdown tool, here we verified the location and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, and PfmtRPS18). Using immuno-EM, we provide evidence that the P. falciparum mitoribosome is closely associated with the mitochondrial inner membrane. Upon knockdown of the mitoribosome, parasites became hypersensitive to inhibitors targeting mitochondrial Complex III (bc1), dihydroorotate dehydrogenase (DHOD), and the F1F0-ATP synthase complex. Furthermore, the mitoribosome knockdown blocked the pyrimidine biosynthesis pathway and reduced the cellular pool of pyrimidine nucleotides. These results suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capacity of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors that target mitochondrial functions.},
}
@article {pmid32272843,
year = {2020},
author = {Wang, G and Tian, W and Liu, X and Ren, W and Liu, C},
title = {New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting.},
journal = {Analytical chemistry},
volume = {92},
number = {9},
pages = {6702-6708},
doi = {10.1021/acs.analchem.0c00680},
pmid = {32272843},
issn = {1520-6882},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Endodeoxyribonucleases/*genetics ; HEK293 Cells ; Humans ; MicroRNAs/*genetics ; Time Factors ; Transcription, Genetic/*genetics ; },
abstract = {Current CRISPR-Cas-based nucleic acid sensing methods relying on the preassembled Cas-crRNA complexes are generally limited to the detection of protospacer-adjacent motif (PAM)-containing sequences, and nonspecific backgrounds are inevitable. Herein, we propose a new CRISPR-derived microRNA sensing mechanism based on rolling circle transcription (RCT)-unleashed self-recruiting of crRNA by Cas12a (Cas12a-SCR). In Cas12a-SCR, target microRNA can specifically trigger RCT to produce a long single-strand RNA with numerous pre-crRNA repeats, which can be trimmed and recruited by Cas12a actively. This new target-initiated, real-time producing, trimming, and self-assembling manner of Cas12a-crRNA remarkably suppresses the nonspecific background and relieves the stringent requirement of PAM site in the target sequence. Thus, the universality of the Cas12a-SCR toward different nucleic acid sequences is greatly expanded.},
}
@article {pmid32272060,
year = {2020},
author = {Zhou, H and Su, J and Hu, X and Zhou, C and Li, H and Chen, Z and Xiao, Q and Wang, B and Wu, W and Sun, Y and Zhou, Y and Tang, C and Liu, F and Wang, L and Feng, C and Liu, M and Li, S and Zhang, Y and Xu, H and Yao, H and Shi, L and Yang, H},
title = {Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice.},
journal = {Cell},
volume = {181},
number = {3},
pages = {590-603.e16},
doi = {10.1016/j.cell.2020.03.024},
pmid = {32272060},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Cell Differentiation/physiology ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Dopamine/metabolism ; Gene Expression Regulation/genetics ; Heterogeneous-Nuclear Ribonucleoproteins/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Nervous System Diseases/metabolism ; Neurogenesis/*physiology ; Neuroglia/*metabolism ; Neurons/metabolism ; Parkinson Disease/metabolism ; Polypyrimidine Tract-Binding Protein/genetics/metabolism ; Retinal Ganglion Cells/*metabolism/physiology ; },
abstract = {Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.},
}
@article {pmid32271968,
year = {2020},
author = {Zhang, D and Hussain, A and Manghwar, H and Xie, K and Xie, S and Zhao, S and Larkin, RM and Qing, P and Jin, S and Ding, F},
title = {Genome editing with the CRISPR-Cas system: an art, ethics and global regulatory perspective.},
journal = {Plant biotechnology journal},
volume = {18},
number = {8},
pages = {1651-1669},
pmid = {32271968},
issn = {1467-7652},
mesh = {Agriculture ; Animals ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Plants/genetics ; },
abstract = {Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR-Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR-Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR-Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre-clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR-Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR-Cas-modified organisms/products.},
}
@article {pmid32271547,
year = {2020},
author = {Cooper, RM and Hasty, J},
title = {One-Day Construction of Multiplex Arrays to Harness Natural CRISPR-Cas Systems.},
journal = {ACS synthetic biology},
volume = {9},
number = {5},
pages = {1129-1137},
pmid = {32271547},
issn = {2161-5063},
support = {R01 GM069811/GM/NIGMS NIH HHS/United States ; },
mesh = {Acinetobacter/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; DNA Primers/metabolism ; Gene Editing/*methods ; Plasmids/genetics/metabolism ; },
abstract = {CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable-we assembled 9-spacer arrays with 1 day's worth of work. We used this method to harness the endogenous CRISPR-Cas system of the highly competent bacterium Acinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense versus DNA acquisition in naturally competent A. baylyi.},
}
@article {pmid32270472,
year = {2020},
author = {Huang, LH and Liu, QJ and Sun, XW and Li, XJ and Liu, M and Jia, SR and Xie, YY and Zhong, C},
title = {Tailoring bacterial cellulose structure through CRISPR interference-mediated downregulation of galU in Komagataeibacter xylinus CGMCC 2955.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {7},
pages = {2165-2176},
doi = {10.1002/bit.27351},
pmid = {32270472},
issn = {1097-0290},
support = {21978219//National Natural Science Foundation of China/International ; 21576212//National Natural Science Foundation of China/International ; 19JCZDJC34700//Natural Science Foundation of Tianjin City/International ; 19PTSYJC00060//Natural Science Foundation of Tianjin City/International ; },
mesh = {Bacterial Proteins/*genetics ; CRISPR-Cas Systems ; Cellulose/chemistry/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Down-Regulation ; Gluconacetobacter xylinus/*genetics ; Transcriptome ; UTP-Glucose-1-Phosphate Uridylyltransferase/*genetics ; },
abstract = {Diverse applications of bacterial cellulose (BC) have different requirements in terms of its structural characteristics. culturing Komagataeibacter xylinus CGMCC 2955, BC structure changes with alterations in oxygen tension. Here, the K. xylinus CGMCC 2955 transcriptome was analyzed under different oxygen tensions. Transcriptome and genome analysis indicated that BC structure is related to the rate of BC synthesis and cell growth, and galU is an essential gene that controls the carbon metabolic flux between the BC synthesis pathway and the pentose phosphate (PP) pathway. The CRISPR interference (CRISPRi) system was utilized in K. xylinus CGMCC 2955 to control the expression levels of galU. By overexpressing galU and interfering with different sites of galU sequences using CRISPRi, we obtained strains with varying expression levels of galU (3.20-3014.84%). By testing the characteristics of BC, we found that the porosity of BC (range: 62.99-90.66%) was negative with galU expression levels. However, the crystallinity of BC (range: 56.25-85.99%) was positive with galU expression levels; galU expression levels in engineered strains were lower than those in the control strains. Herein, we propose a new method for regulating the structure of BC to provide a theoretical basis for its application in different fields.},
}
@article {pmid32270227,
year = {2020},
author = {Kobaisi, F and Fayyad, N and Sulpice, E and Badran, B and Fayyad-Kazan, H and Rachidi, W and Gidrol, X},
title = {High-throughput synthetic rescue for exhaustive characterization of suppressor mutations in human genes.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {21},
pages = {4209-4222},
pmid = {32270227},
issn = {1420-9071},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; *Mutagenesis ; Neoplasms/genetics ; RNA Interference ; *Suppression, Genetic ; },
abstract = {Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment. Here, we first review all known suppressor mutations' mechanisms characterized by genetic screens on model species like yeast or flies. However, human suppressor mutations are scarce, despite some being discovered based on orthologue genes. Because of recent advances in high-throughput screening, developing an inventory of human suppressor mutations for pathological processes seems achievable. In addition, we review several screening methods for suppressor mutations in cultured human cells through knock-out, knock-down or random mutagenesis screens on large scale. We provide examples of studies published over the past years that opened new therapeutic avenues, particularly in oncology.},
}
@article {pmid32270050,
year = {2019},
author = {Evanoff, M and Komor, AC},
title = {Base Editors: Modular Tools for the Introduction of Point Mutations in Living Cells.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {5},
pages = {483-491},
pmid = {32270050},
issn = {2397-8554},
support = {T32 GM007240/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/chemistry/genetics ; DNA, Single-Stranded/chemistry/metabolism ; Endonucleases/chemistry/*genetics ; Gene Editing/*methods ; Genetic Enhancement ; *Point Mutation ; RNA, Guide/chemistry/metabolism ; Recombination, Genetic/genetics ; Synthetic Biology ; Transcription, Genetic ; },
abstract = {Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.},
}
@article {pmid32269948,
year = {2020},
author = {Wang, JH and Tseng, SY and Tung, KC},
title = {Genomic investigation of emerging zoonotic pathogen Shewanella xiamenensis.},
journal = {Ci ji yi xue za zhi = Tzu-chi medical journal},
volume = {32},
number = {2},
pages = {162-166},
pmid = {32269948},
issn = {1016-3190},
abstract = {OBJECTIVE: Shewanella xiamenensis is an emerging zoonotic pathogen commonly found in aquatic ecosystem. Clustered regularly interspaced short palindromic repeats (CRISPR) and (CRISPR)-associated gene systems act as adaptive immune system of prokaryotes. Recently, growing evidence suggested their role in bacterial virulence and resistance. Despite its medical importance, little is known about the genomic characteristics of S. xiamenensis.<br><br>MATERIALS AND METHODS: Strain ZYW6 was isolated from Epinephelus awoara. We sequenced the 16S rRNA gene and blast against the GenBank bacterial database. Antibiotic susceptibility tests and interpretation were performed by automatic VITEK 2 system. We extracted the genomic DNA with QIAGEN Genomic-tip 100/G kit and QIAGEN Genomic DNA Buffer Set. Whole-genome shotgun sequencing was performed using the Illumina MiSeq sequencer. To identify the CRISPR-Cas System in the genome of S. xiamenensis ZYW6, the Integrated Microbial Genomes and Microbiomes and CRISPRFinder were used.<br><br>RESULTS: We characterized the genome of a S. xiamenensis strain. The genome is 4,765,190 bp in length and encodes 4262 open-reading frames. Type I CRISPR-Cas system and serine biosynthesis genes were identified.<br><br>CONCLUSION: Our results demonstrate the genetic structure of CRISPR-Cas system, l-serine synthesis, and oxacillinase in S. xiamenensis. The report of antibiotics resistance genes in the study might indicate a possible reservoir of antimicrobial drug resistance determinants in food animal, resulting in potential infection source. The findings provide insights into the structure and composition of CRISPR-Cas system in S. xiamenensis and foundation for future biological validation.},
}
@article {pmid32268246,
year = {2020},
author = {Seo, HH and Han, HW and Kim, JH},
title = {Generation of a TLR7 heterozygous knockout line (WAe009-A-18) from human embryonic stem cells using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {45},
number = {},
pages = {101735},
doi = {10.1016/j.scr.2020.101735},
pmid = {32268246},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Heterozygote ; *Human Embryonic Stem Cells ; Humans ; Toll-Like Receptor 7/genetics ; },
abstract = {Toll-like receptor 7 (TLR7) is a member of the toll-like receptor (TLR) family that is essential in the innate immune system. In this study, we established a heterozygous TLR7 knockout H9 cell line using CRISPR/Cas9. TLR7 knockout H9 cells maintained their pluripotency and exhibited the ability to differentiate into the three germ layers without any karyotype abnormalities.},
}
@article {pmid32268192,
year = {2020},
author = {Hu, T and Zhou, J and Tong, Y and Su, P and Li, X and Liu, Y and Liu, N and Wu, X and Zhang, Y and Wang, J and Gao, L and Tu, L and Lu, Y and Jiang, Z and Zhou, YJ and Gao, W and Huang, L},
title = {Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast.},
journal = {Metabolic engineering},
volume = {60},
number = {},
pages = {87-96},
doi = {10.1016/j.ymben.2020.03.011},
pmid = {32268192},
issn = {1096-7184},
mesh = {Biosynthetic Pathways ; CRISPR-Cas Systems ; Computer Simulation ; Diterpenes/chemistry/*metabolism ; Fermentation ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways ; Mutant Chimeric Proteins/genetics/*metabolism ; Mutation ; Plasmids ; Saccharomyces cerevisiae/*genetics/*metabolism ; Terpenes/*metabolism ; },
abstract = {Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal compounds. In this study, comprehensive engineering strategies were applied to construct a high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L[-1] geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein modification, which resulted in a final yield of 550.7 mg L[-1] in shake flasks and 3.5 g L[-1] in a 5-L bioreactor. This work offers an efficient and green process for the production of the important intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the biotechnological production of valuable natural diterpenes.},
}
@article {pmid32267478,
year = {2020},
author = {Hampton, T},
title = {With First CRISPR Trials, Gene Editing Moves Toward the Clinic.},
journal = {JAMA},
volume = {323},
number = {16},
pages = {1537-1539},
doi = {10.1001/jama.2020.3438},
pmid = {32267478},
issn = {1538-3598},
mesh = {*Bioethical Issues ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/ethics/*methods ; Humans ; Leber Congenital Amaurosis/genetics/*therapy ; Neoplasms/*therapy ; },
}
@article {pmid32265856,
year = {2020},
author = {Iranzo, J and Faure, G and Wolf, YI and Koonin, EV},
title = {Game-Theoretical Modeling of Interviral Conflicts Mediated by Mini-CRISPR Arrays.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {381},
pmid = {32265856},
issn = {1664-302X},
abstract = {All cellular organisms coevolve with multiple viruses, so that both virus-host and intervirus conflicts are major factors of evolution. Accordingly, hosts evolve multiple, elaborate defense systems and viruses respond by evolving means of antidefense. Although less thoroughly characterized, several dedicated mechanisms of intervirus competition have been described as well. Recently, the genomes of some bacterial and archaeal viruses have been shown to harbor CRISPR mini-arrays that typically contain a single spacer targeting a closely related virus. The involvement of mini-arrays in an intervirus conflict has been experimentally demonstrated for a pair of archaeal viruses. We model the evolution of virus-encoded CRISPR mini-arrays using a game theoretical approach. Analysis of the model reveals multiple equilibria that include mutual targeting, unidirectional targeting, no targeting, cyclic polymorphism, and bistability. The choice between these evolutionary regimes depends on the model parameters including the coinfection frequency, differential productivity of the conflicting viruses, and the fitness cost of mini-arrays. At high coinfection frequencies, the model becomes a version of the Prisoner's dilemma in which defection, i.e., mutual targeting between the competing viruses, is the winning strategy.},
}
@article {pmid32265471,
year = {2020},
author = {Menchaca, A and Dos Santos-Neto, PC and Souza-Neves, M and Cuadro, F and Mulet, AP and Tesson, L and Chenouard, V and Guiffès, A and Heslan, JM and Gantier, M and Anegón, I and Crispo, M},
title = {Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {5995},
pmid = {32265471},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/*methods ; Male ; Membrane Proteins/*genetics ; Microinjections ; Mutation ; Oligodeoxyribonucleotides/*genetics ; Recombinational DNA Repair ; Sheep/embryology/*genetics ; },
abstract = {Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.},
}
@article {pmid32264803,
year = {2020},
author = {Akram, F and Ikram Ul Haq, and Ahmed, Z and Khan, H and Ali, MS},
title = {CRISPR-Cas9, A Promising Therapeutic Tool for Cancer Therapy: A Review.},
journal = {Protein and peptide letters},
volume = {27},
number = {10},
pages = {931-944},
doi = {10.2174/0929866527666200407112432},
pmid = {32264803},
issn = {1875-5305},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; Humans ; *Neoplasms/genetics/therapy ; },
abstract = {Cancer is one of the most leading causes of mortality all over the world and remains a foremost social and economic burden. Mutations in the genome of individuals are taking place more frequently due to the excessive progress of xenobiotics and industrialization in the present world. With the progress in the field of molecular biology, it is possible to alter the genome and to observe the functional changes derived from genetic modulation using gene-editing technologies. Several therapies have been applied for the treatment of malignancy which affect the normal body cells; however, more effort is required to develop vsome latest therapeutic approaches for cancer biology and oncology exploiting these molecular biology advances. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (Cas9) system has emerged as a powerful technology for cancer therapy because of its great accuracy and efficiency. Genome editing technologies have demonstrated a plethora of benefits to the biological sciences. CRISPR- Cas9, a versatile gene editing tool, has become a robust strategy for making alterations to the genome of organisms and a potent weapon in the arsenal of tumor treatment. It has revealed an excellent clinical potential for cancer therapy by discovering novel targets and has provided the researchers with the perception about how tumors respond to drug therapy. Stern efforts are in progress to enhance its efficiency of sequence specific targeting and consequently repressing offtarget effects. CRISPR-Cas9 uses specific proteins to convalesce mutations at genetic level. In CRISPR-Cas9 system, RNA-guided Cas9 endonuclease harnesses gene mutation, DNA deletion or insertion, transcriptional activation or repression, multiplex targeting only by manipulating 20-nucleotide components of RNA. Originally, CRISPR-Cas9 system was used by bacteria for their defense against different bacteriophages, and recently this system is receiving noteworthy appreciation due to its emerging role in the treatment of genetic disorders and carcinogenesis. CRISPR-Cas9 can be employed to promptly engineer oncolytic viruses and immune cells for cancer therapeutic applications. More notably, it has the ability to precisely edit genes not only in model organisms but also in human being that permits its use in therapeutic analysis. It also plays a significant role in the development of complete genomic libraries for cancer patients. In this review, we have highlighted the involvement of CRISPR-Cas9 system in cancer therapy accompanied by its prospective applications in various types of malignancy and cancer biology. In addition, some other conspicuous functions of this unique system have also been discussed beyond genome editing.},
}
@article {pmid32257106,
year = {2020},
author = {Xu, M},
title = {CCR5-Δ32 biology, gene editing, and warnings for the future of CRISPR-Cas9 as a human and humane gene editing tool.},
journal = {Cell &amp; bioscience},
volume = {10},
number = {},
pages = {48},
pmid = {32257106},
issn = {2045-3701},
abstract = {BACKGROUND: Biomedical technologies have not just improved human health but also assisted in the creation of human life. Since the first birth of a healthy baby by in vitro fertilization (IVF) 40 years ago, IVF has been the mainstay treatment for couples struggling with infertility. This technology, in addition to increasingly accessible genetic testing, has made it possible for countless couples to have children. Since CRISPR-Cas9 gene editing was described in 2015, its potential for targeting genetic diseases has been much anticipated. However, the potential of using CRISPR-Cas9 for human germline modification has led to many fears of "designer babies" and widespread concerns for the impact of this technology on human evolution and its implications in Social Darwinism. In addition to these ethical/moral concerns, there remain many unknowns about CRISPR-Cas9 technology and endless unanticipated consequence to gene editing.<br><br>METHODS: In this paper, we analyze the current progresses of CRISPR-Cas9 technology and discuss the theoretical advantages of certain allelic variances in the C-C chemokine receptor 5 gene (CCR5) in the setting of recent ethical/moral concerns regarding&#xa0;gene editing using the CRISPR-Cas9 system.<br><br>RESULTS: These uncertainties have been highlighted recently by the birth of Chinese twins whose C-C chemokine receptor 5 (CCR5) gene had been inactivated via CRISPR-Cas9 to be theoretically protective against HIV infection. CCR5 signaling is critical for the successful infection of human immunodeficiency virus (HIV) and people with homozygous inactivating CCR5-&#x394;32 mutations have been shown to be protected against HIV infection. Those with the CCR5-&#x394;32/&#x394;32 mutation also have greater neuroplasticity, allowing for improved recovery from neurological trauma, and decreased Chagas cardiomyopathy. However, the CCR5-&#x394;32/&#x394;32 mutation has also been associated with earlier clinical manifestations for West Nile infection, ambiguous effects on osteoclast function, and a four-fold increased mortality from influenza infection. These detrimental health impacts, in addition to the confounding factor that these CRISPR babies do not carry this exact CCR5-&#x394;32/&#x394;32 mutation, lead to many questions regarding the children's future health and the moral conundrum of their birth. The creation and birth of these babies was not completed with any scientific, ethical, or governmental oversight, which has spurned the acceleration of talks regarding global regulations for human genetic editing.<br><br>CONCLUSIONS: Although we can try to regulate for ethical, health-related only use of this technology, moral and governmental oversights need to be supplemented by technical regulations. For instance, whole genome sequencing needs to be used to eliminate off-target mutations that could affect the health and safety of infants born to this process. Like Pandora's Box, we cannot pretend to forget CRISPR-Cas9 technology, all we can do is ensure a safe, moral, and equitable used of this technology.},
}
@article {pmid32256462,
year = {2020},
author = {Álvarez, VE and Quiroga, MP and Galán, AV and Vilacoba, E and Quiroga, C and Ramírez, MS and Centrón, D},
title = {Crucial Role of the Accessory Genome in the Evolutionary Trajectory of Acinetobacter baumannii Global Clone 1.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {342},
pmid = {32256462},
issn = {1664-302X},
abstract = {Acinetobacter baumannii is one of the most important nosocomial pathogens able to rapidly develop extensive drug resistance. Here, we study the role of accessory genome in the success of the globally disseminated clone 1 (GC1) with functional and genomic approaches. Comparative genomics was performed with available GC1 genomes (n = 106) against other A. baumannii high-risk and sporadic clones. Genetic traits related to accessory genome were found common and conserved along time as two novel regions of genome plasticity, and a CRISPR-Cas system acquired before clonal diversification located at the same loci as "sedentary" modules. Although identified within hotspot for recombination, other block of accessory genome was also "sedentary" in lineage 1 of GC1 with signs of microevolution as the AbaR0-type genomic island (GI) identified in A144 and in A155 strains which were maintained one month in independent experiments without antimicrobial pressure. The prophage YMC/09/02/B1251_ABA_BP was found to be "mobile" since, although it was shared by all GC1 genomes, it showed high intrinsic microevolution as well as mobility to different insertion sites. Interestingly, a wide variety of Insertion Sequences (IS), probably acquired by the flow of plasmids related to Rep_3 superfamily was found. These IS showed dissimilar genomic location amongst GC1 genomes presumably associated with promptly niche adaptation. On the other hand, a type VI secretion system and three efflux pumps were subjected to deep processes of genomic loss in A. baumannii but not in GC1. As a whole, these findings suggest that preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1. Competition of A144 and A155 versus A118 (ST 404/ND) without antimicrobial pressure suggested a higher ability of GC1 to grow over a clone with sporadic behavior which explains, from an ecological perspective, the global achievement of this successful pandemic clone in the hospital habitat. Together, these data suggest an essential role of still unknown properties of "mobile" and "sedentary" accessory genome that is preserved over time under different antibiotic or stress conditions.},
}
@article {pmid32255179,
year = {2020},
author = {Rabinowitz, R and Abadi, S and Almog, S and Offen, D},
title = {Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing.},
journal = {Nucleic acids research},
volume = {48},
number = {W1},
pages = {W340-W347},
pmid = {32255179},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Variation ; Humans ; *Point Mutation ; *Software ; },
abstract = {Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A deaminase enzyme is fused to a deactivated Cas and enables transition conversions. The diversified repertoire of base editors provides a wide range of base editing possibilities. However, existing base editors cannot induce transversion substitutions and activate only within a specified region relative to the binding site, thus, they cannot precisely correct every point mutation. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a point mutation. When a precise correction is impossible, BE-FF aims to mutate bystander nucleotides in order to induce synonymous corrections that will correct the coding sequence. To measure BE-FF practicality, we analysed a database of human pathogenic point mutations. Out of the transition mutations, 60.9% coding sequences could be corrected. Notably, 19.4% of the feasible corrections were not achieved by precise corrections but only by synonymous corrections. Moreover, 298 cases of transversion-derived pathogenic mutations were detected to be potentially repairable by base editing via synonymous corrections, although base editing is considered impractical for such mutations.},
}
@article {pmid32253539,
year = {2020},
author = {Li, F and Liu, Q and Li, X and Zhang, C and Li, J and Sun, W and Liu, D and Xiao, D and Tian, C},
title = {Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.},
journal = {Biotechnology letters},
volume = {42},
number = {7},
pages = {1181-1191},
doi = {10.1007/s10529-020-02882-5},
pmid = {32253539},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems/*genetics ; Genetic Engineering/*methods ; Glucan 1,4-alpha-Glucosidase/genetics/*metabolism ; Recombinant Fusion Proteins/genetics/*metabolism ; *Sordariales/genetics/metabolism ; Viral Proteins/genetics ; },
abstract = {OBJECTIVE: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass.<br><br>RESULTS: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9&#xa0;&#xd7;His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0-9.0 and thermostable stability at 65&#xa0;&#xb0;C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyR&#x394;alp-1&#x394;res-1&#x394;cre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT.<br><br>CONCLUSIONS: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.},
}
@article {pmid32252832,
year = {2020},
author = {McVeigh, P},
title = {Post-genomic progress in helminth parasitology.},
journal = {Parasitology},
volume = {147},
number = {8},
pages = {835-840},
pmid = {32252832},
issn = {1469-8161},
mesh = {Animals ; Anthelmintics/therapeutic use ; CRISPR-Cas Systems ; Drug Resistance/genetics ; *Genome, Helminth ; Genomics ; Helminthiasis/diagnosis ; *Helminths/drug effects/genetics/metabolism/parasitology ; Humans ; Parasitology/*trends ; Pathology, Molecular ; Proteomics ; RNA Interference ; Transcriptome ; },
abstract = {Helminth parasitology is an important discipline, which poses often unique technical challenges. One challenge is that helminth parasites, particularly those in humans, are often difficult to obtain alive and in sufficient quantities for study; another is the challenge of studying these organisms in vitro - no helminth parasite life cycle has been fully recapitulated outside of a host. Arguably, the key issue retarding progress in helminth parasitology has been a lack of experimental tools and resources, certainly relative to the riches that have driven many parasitologists to adopt free-living model organisms as surrogate systems. In response to these needs, the past 10-12 years have seen the beginnings of helminth parasitology's journey into the 'omics' era, with the release of abundant sequencing resources, and the functional genomics tools with which to test biological hypotheses. To reflect this progress, the 2019 Autumn Symposium of the British Society for Parasitology was held in Queen's University Belfast on the topic of 'post-genomic progress in helminth parasitology'. This issue presents examples of the current state of play in the field, while this editorial summarizes how genomic datasets and functional genomic tools have stimulated impressive recent progress in our understanding of parasite biology.},
}
@article {pmid32252431,
year = {2020},
author = {Yang, Y and Li, N and Qiu, J and Ge, H and Qin, X},
title = {Identification of the Repressive Domain of the Negative Circadian Clock Component CHRONO.},
journal = {International journal of molecular sciences},
volume = {21},
number = {7},
pages = {},
pmid = {32252431},
issn = {1422-0067},
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems ; Cell Nucleus/metabolism ; *Circadian Clocks ; Circadian Rhythm Signaling Peptides and Proteins/chemistry/*genetics/metabolism ; Codon Usage ; Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques ; Humans ; Period Circadian Proteins/chemistry/genetics/metabolism ; Protein Binding ; Protein Conformation ; *Protein Interaction Domains and Motifs ; Protein Transport ; Recombinant Proteins/genetics ; Spectrum Analysis ; Structure-Activity Relationship ; },
abstract = {Circadian rhythm is an endogenous, self-sustainable oscillation that participates in regulating organisms' physiological activities. Key to this oscillation is a negative feedback by the main clock components Periods and Cryptochromes that repress the transcriptional activity of BMAL1/CLOCK (defined in the Abbreviations) complexes. In addition, a novel repressor, CHRONO, has been identified recently, but details of CHRONO's function during repressing the circadian cycle remain unclear. Here we report that a domain of CHRONO mainly composed of α-helixes is critical to repression through the exploitation of protein-protein interactions according to luciferase reporter assays, co-immunoprecipitation, immunofluorescence, genome editing, and structural information analysis via circular dichroism spectroscopy. This repression is fulfilled by interactions between CHRONO and a region on the C-terminus of BMAL1 where Cryptochrome and CBP (defined in the Abbreviations) bind. Our resultsindicate that CHRONO and PER differentially function as BMAL1/CLOCK-dependent repressors. Besides, the N-terminus of CHRONO is important for its nuclear localization. We further develop a repression model of how PER, CRY, and CHRONO proteins associate with BMAL1, respectively.},
}
@article {pmid32252379,
year = {2020},
author = {Kim, TH and Kern, C and Zhou, H},
title = {Knockout of IRF7 Highlights its Modulator Function of Host Response Against Avian Influenza Virus and the Involvement of MAPK and TOR Signaling Pathways in Chicken.},
journal = {Genes},
volume = {11},
number = {4},
pages = {},
pmid = {32252379},
issn = {2073-4425},
mesh = {Animals ; *CRISPR-Cas Systems ; Chick Embryo ; Chickens ; Gene Expression Profiling ; Influenza A virus/*immunology ; Influenza in Birds/*immunology/metabolism/virology ; Interferon Regulatory Factor-7/*antagonists &amp; inhibitors/genetics/metabolism ; Mitogen-Activated Protein Kinases/genetics/*metabolism ; TOR Serine-Threonine Kinases/genetics/*metabolism ; },
abstract = {Interferon regulatory factor 7 (IRF7) is known as the master transcription factor of the type I interferon response in mammalian species along with IRF3. Yet birds only have IRF7, while they are missing IRF3, with a smaller repertoire of immune-related genes, which leads to a distinctive immune response in chickens compared to in mammals. In order to understand the functional role of IRF7 in the regulation of the antiviral response against avian influenza virus in chickens, we generated IRF7[-/-] chicken embryonic fibroblast (DF-1) cell lines and respective controls (IRF7[wt]) by utilizing the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. IRF7 knockout resulted in increased viral titers of low pathogenic avian influenza viruses. Further RNA-sequencing performed on H6N2-infected IRF7[-/-] and IRF7[wt] cell lines revealed that the deletion of IRF7 resulted in the significant down-regulation of antiviral effectors and the differential expression of genes in the MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) signaling pathways. Dynamic gene expression profiling of the host response between the wildtype and IRF7 knockout revealed potential signaling pathways involving AP1 (activator protein 1), NF-κB (nuclear factor kappa B) and inflammatory cytokines that may complement chicken IRF7. Our findings in this study provide novel insights that have not been reported previously, and lay a solid foundation for enhancing our understanding of the host antiviral response against the avian influenza virus in chickens.},
}
@article {pmid32252259,
year = {2020},
author = {Reicher, A and Harris, AL and Prinz, F and Kiesslich, T and Wei, M and Öllinger, R and Rad, R and Pichler, M and Kwong, LN},
title = {Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.},
journal = {International journal of molecular sciences},
volume = {21},
number = {7},
pages = {},
pmid = {32252259},
issn = {1422-0067},
support = {5T32CA217789-03/NH/NIH HHS/United States ; W 1226/FWF_/Austrian Science Fund FWF/Austria ; T32 CA217789/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Chromosome Breakpoints ; *Chromosome Inversion ; *Gene Editing ; *Gene Fusion ; Gene Targeting ; Genetic Loci ; Humans ; RNA, Guide ; RNA-Binding Proteins/*genetics ; Receptor, Fibroblast Growth Factor, Type 2/*genetics ; },
abstract = {Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10-15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2-Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.},
}
@article {pmid32251518,
year = {2020},
author = {Watanabe, Y and Okuya, K and Takada, Y and Kinoshita, M and Yokoi, S and Chisada, S and Kamei, Y and Tatsukawa, H and Yamamoto, N and Abe, H and Hashimoto, H and Hitomi, K},
title = {Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation.},
journal = {Journal of biochemistry},
volume = {168},
number = {3},
pages = {213-222},
doi = {10.1093/jb/mvaa038},
pmid = {32251518},
issn = {1756-2651},
mesh = {Animals ; Animals, Genetically Modified ; Behavior, Animal ; Brain/metabolism ; CRISPR-Cas Systems ; Fish Proteins/*genetics ; GTP-Binding Proteins/*genetics/*metabolism ; Gene Editing ; Humans ; *Movement ; Oryzias/*genetics/*metabolism ; Phenotype ; Protein Glutamine gamma Glutamyltransferase 2 ; Transglutaminases/*genetics/*metabolism ; },
abstract = {Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 using the CRISPR/Cas9 and transcription activator-like effector nucleases systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank-diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that orthologue of human TG2 was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type transglutaminase and the nervous system and the associated behaviour.},
}
@article {pmid32251383,
year = {2020},
author = {Cheng, Q and Wei, T and Farbiak, L and Johnson, LT and Dilliard, SA and Siegwart, DJ},
title = {Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.},
journal = {Nature nanotechnology},
volume = {15},
number = {4},
pages = {313-320},
pmid = {32251383},
issn = {1748-3395},
support = {P30 CA142543/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Drug Delivery Systems ; *Gene Editing ; Mice ; Nanoparticles/*chemistry ; Organ Specificity ; *RNA, Messenger/chemistry/pharmacology ; },
abstract = {CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.},
}
@article {pmid32250930,
year = {2020},
author = {Wu, H and Qian, C and Wu, C and Wang, Z and Wang, D and Ye, Z and Ping, J and Wu, J and Ji, F},
title = {End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {157},
number = {},
pages = {112153},
doi = {10.1016/j.bios.2020.112153},
pmid = {32250930},
issn = {1873-4235},
mesh = {Biosensing Techniques/instrumentation/*methods ; *CRISPR-Cas Systems ; Equipment Design ; Food Safety ; Lectins/genetics ; Molecular Diagnostic Techniques/instrumentation/methods ; Nucleic Acid Amplification Techniques/instrumentation/methods ; Plants, Genetically Modified/*genetics ; Polymerase Chain Reaction/instrumentation/methods ; Soybeans/*genetics ; Zea mays/*genetics ; },
abstract = {A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready[@]). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.},
}
@article {pmid32250339,
year = {2020},
author = {Hatcher, A and Yu, K and Meyer, J and Aiba, I and Deneen, B and Noebels, JL},
title = {Pathogenesis of peritumoral hyperexcitability in an immunocompetent CRISPR-based glioblastoma model.},
journal = {The Journal of clinical investigation},
volume = {130},
number = {5},
pages = {2286-2300},
pmid = {32250339},
issn = {1558-8238},
support = {R01 CA223388/CA/NCI NIH HHS/United States ; U01 CA217842/CA/NCI NIH HHS/United States ; R01 CA217105/CA/NCI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; R01 NS029709/NS/NINDS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 HL092332/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Brain/metabolism/pathology/physiopathology ; *Brain Neoplasms/genetics/metabolism/pathology/physiopathology ; *CRISPR-Cas Systems ; Gene Deletion ; *Glioblastoma/genetics/metabolism/pathology/physiopathology ; Mice ; Mice, Knockout ; *Neoplasms, Experimental/genetics/metabolism/pathology/physiopathology ; *Seizures/genetics/metabolism/pathology/physiopathology ; *Synaptic Transmission ; },
abstract = {Seizures often herald the clinical appearance of gliomas or appear at later stages. Dissecting their precise evolution and cellular pathogenesis in brain malignancies could inform the development of staged therapies for these highly pharmaco-resistant epilepsies. Studies in immunodeficient xenograft models have identified local interneuron loss and excess glial glutamate release as chief contributors to network disinhibition, but how hyperexcitability in the peritumoral microenvironment evolves in an immunocompetent brain is unclear. We generated gliomas in WT mice via in utero deletion of key tumor suppressor genes and serially monitored cortical epileptogenesis during tumor infiltration with in vivo electrophysiology and GCAMP7 calcium imaging, revealing a reproducible progression from hyperexcitability to convulsive seizures. Long before seizures, coincident with loss of inhibitory cells and their protective scaffolding, gain of glial glutamate antiporter xCT expression, and reactive astrocytosis, we detected local Iba1+ microglial inflammation that intensified and later extended far beyond tumor boundaries. Hitherto unrecognized episodes of cortical spreading depolarization that arose frequently from the peritumoral region may provide a mechanism for transient neurological deficits. Early blockade of glial xCT activity inhibited later seizures, and genomic reduction of host brain excitability by deleting MapT suppressed molecular markers of epileptogenesis and seizures. Our studies confirmed xenograft tumor-driven pathobiology and revealed early and late components of tumor-related epileptogenesis in a genetically tractable, immunocompetent mouse model of glioma, allowing the complex dissection of tumor versus host pathogenic seizure mechanisms.},
}
@article {pmid32249828,
year = {2020},
author = {Gonatopoulos-Pournatzis, T and Aregger, M and Brown, KR and Farhangmehr, S and Braunschweig, U and Ward, HN and Ha, KCH and Weiss, A and Billmann, M and Durbic, T and Myers, CL and Blencowe, BJ and Moffat, J},
title = {Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.},
journal = {Nature biotechnology},
volume = {38},
number = {5},
pages = {638-648},
pmid = {32249828},
issn = {1546-1696},
support = {MOP-142375//CIHR/Canada ; },
mesh = {Alternative Splicing ; Animals ; Bacterial Proteins/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Endodeoxyribonucleases/*metabolism ; Gene Editing/*methods ; *Gene Regulatory Networks ; Genetic Fitness ; Humans ; Machine Learning ; Male ; Mice ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism ; },
abstract = {Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.},
}
@article {pmid32248654,
year = {2020},
author = {Urick, ME and Bell, DW},
title = {Proteomic profiling of FBXW7-mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target.},
journal = {Cancer medicine},
volume = {9},
number = {11},
pages = {3863-3874},
pmid = {32248654},
issn = {2045-7634},
support = {Z01 HG200338/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Cystadenocarcinoma, Serous/genetics/metabolism/*pathology ; Endometrial Neoplasms/genetics/metabolism/*pathology ; F-Box-WD Repeat-Containing Protein 7/antagonists &amp; inhibitors/genetics/*metabolism ; Female ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; Humans ; *Mutation ; Proteome/analysis/*metabolism ; Tumor Cells, Cultured ; },
abstract = {BACKGROUND: Despite advancements over the past decade revealing molecular aberrations characteristic of endometrial cancer (EC) subtypes, serous ECs remain difficult to treat and associated with poor outcomes. This is due, in part, to the rarity of these tumors within clinical trials and the inability to directly target the most frequent genomic abnormalities. One of the most commonly somatically mutated genes in serous ECs is the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7).<br><br>METHODS: To identify changes in protein expression associated with FBXW7 mutation, we clustered regularly interspaced short palindromic repeats (CRISPR)-edited ARK4 FBXW7 nonmutant serous EC cells to insert recurrent FBXW7 mutations. We then compared the liquid chromatography tandem mass spectrometry-based proteomic profiles of CRISPR-edited ARK1 and ARK4 serous EC cells to matched parental cells.<br><br>RESULTS: Among distinct total and phosphorylated proteins that were significantly differentially expressed in FBXW7-mutant cell lines compared to matched parental lines, we identified increased PADI2 (peptidyl arginine deiminase 2) expression in all ARK1 and ARK4 CRISPR-edited FBXW7-mutant cell lines. We further confirmed the correlation between FBXW7 mutation and increased PADI2 expression in a third biological background, JHUEM-1 endometrioid EC cells. Finally, we established that PADI2 protein is expressed in primary serous endometrial tumors.<br><br>CONCLUSION: Our findings provide novel insight into proteomic changes associated with FBXW7 mutation in serous ECs and identify PADI2 as a novel potential therapeutic target for these tumors.},
}
@article {pmid32248582,
year = {2020},
author = {Tan, J and Yu, W},
title = {CRISPR as a tool in tumor therapy: A short review.},
journal = {Biotechnology and applied biochemistry},
volume = {67},
number = {6},
pages = {875-879},
doi = {10.1002/bab.1913},
pmid = {32248582},
issn = {1470-8744},
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell Engineering ; *Gene Knock-In Techniques ; *Gene Knockout Techniques ; Humans ; *Neoplasms/genetics/metabolism/therapy ; },
abstract = {Tumors remain a health concern worldwide, and gene therapy is a useful approach for treating tumors. The Clustered regulatory interspaced short palindromic repeat (CRISPR) system, an efficient and robust gene-editing system originating from bacteria, serves as an important tool in gene therapy. In this review, we briefly summarize the usage of CRISPR (including in gene knockout and gene knock-in applications) and the CRISPRa system in tumor therapy, including its delivery approaches and the general fields in which it has been applied. We summarize the application of the CRISPR system in tumor therapy in two aspects: its application in tumor cell modification and its application in drug engineering. This review also summarizes the advantages and challenges of using the CRISPR system in tumor therapy.},
}
@article {pmid32246439,
year = {2020},
author = {Jia, F and Li, X and Zhang, C and Tang, X},
title = {The expanded development and application of CRISPR system for sensitive nucleotide detection.},
journal = {Protein &amp; cell},
volume = {11},
number = {9},
pages = {624-629},
pmid = {32246439},
issn = {1674-8018},
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; RNA, Viral/*genetics ; Viruses/*genetics ; },
}
@article {pmid32245970,
year = {2020},
author = {Yao, L and Shabestary, K and Björk, SM and Asplund-Samuelsson, J and Joensson, HN and Jahn, M and Hudson, EP},
title = {Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1666},
pmid = {32245970},
issn = {2041-1723},
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; Lactic Acid/metabolism ; Peroxiredoxins/*genetics/metabolism ; Photobioreactors/*microbiology ; Synechocystis/*genetics/metabolism ; },
abstract = {Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.},
}
@article {pmid32245807,
year = {2020},
author = {Phelan, JD and Staudt, LM},
title = {CRISPR-based technology to silence the expression of IncRNAs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {15},
pages = {8225-8227},
pmid = {32245807},
issn = {1091-6490},
support = {P01 CA229100/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
}
@article {pmid32245761,
year = {2020},
author = {Meile, S and Sarbach, A and Du, J and Schuppler, M and Saez, C and Loessner, MJ and Kilcher, S},
title = {Engineered Reporter Phages for Rapid Bioluminescence-Based Detection and Differentiation of Viable Listeria Cells.},
journal = {Applied and environmental microbiology},
volume = {86},
number = {11},
pages = {},
pmid = {32245761},
issn = {1098-5336},
mesh = {Bacteriophages/*chemistry ; Listeria/*physiology ; Luciferases/*chemistry ; Luminescent Measurements/*methods ; *Microbial Viability ; },
abstract = {The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for de novo genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable Listeria cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian, and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of L. monocytogenes in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 h faster than culture-dependent approaches.IMPORTANCE Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near online assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection but also enable an early estimation of the potential virulence of Listeria isolates from food production and processing sites.},
}
@article {pmid32245271,
year = {2020},
author = {Vidyanti, AN and Hsieh, JY and Lin, KJ and Fang, YC and Setyopranoto, I and Hu, CJ},
title = {Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion.},
journal = {International journal of molecular sciences},
volume = {21},
number = {6},
pages = {},
pmid = {32245271},
issn = {1422-0067},
mesh = {Amyloid beta-Peptides/metabolism ; Animals ; Behavior Rating Scale ; Brain Ischemia/diagnostic imaging/genetics/*metabolism/physiopathology ; CRISPR-Cas Systems ; Carotid Stenosis ; *Cerebrovascular Circulation ; Chronic Disease ; Dementia, Vascular/physiopathology ; Disease Models, Animal ; Gene Knockout Techniques ; HMGB1 Protein/genetics/*metabolism ; Hippocampus/diagnostic imaging/pathology/physiopathology ; Interleukin-1beta/metabolism ; Interleukin-6/metabolism ; Magnetic Resonance Imaging ; Male ; Mice ; Mice, Inbred C57BL ; Psychomotor Performance ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {The pathophysiology of vascular cognitive impairment (VCI) is associated with chronic cerebral hypoperfusion (CCH). Increased high-mobility group box protein 1 (HMGB1), a nonhistone protein involved in injury and inflammation, has been established in the acute phase of CCH. However, the role of HMGB1 in the chronic phase of CCH remains unclear. We developed a novel animal model of CCH with a modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice. Cerebral blood flow (CBF) reduction, the expression of HMGB1 and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6), and brain pathology were assessed. Furthermore, we evaluated the effect of HMGB1 suppression through bilateral intrahippocampus injection with the CRISPR/Cas9 knockout plasmid. Three months after CCH induction, CBF decreased to 30-50% with significant cognitive decline in BCCAO mice. The 7T-aMRI showed hippocampal atrophy, but amyloid positron imaging tomography showed nonsignificant amyloid-beta accumulation. Increased levels of HMGB1, TNF-α, IL-1β, and IL-6 were observed 3 months after BCCAO. HMGB1 suppression with CRISPR/Cas9 knockout plasmid restored TNF-α, IL-1β, and IL-6 and attenuated hippocampal atrophy and cognitive decline. We believe that HMGB1 plays a pivotal role in CCH-induced VCI pathophysiology and can be a potential therapeutic target of VCI.},
}
@article {pmid32244056,
year = {2020},
author = {Araldi, RP and Khalil, C and Grignet, PH and Teixeira, MR and de Melo, TC and Módolo, DG and Fernandes, LGV and Ruiz, J and de Souza, EB},
title = {Medical applications of clustered regularly interspaced short palindromic repeats (CRISPR/Cas) tool: A comprehensive overview.},
journal = {Gene},
volume = {745},
number = {},
pages = {144636},
doi = {10.1016/j.gene.2020.144636},
pmid = {32244056},
issn = {1879-0038},
mesh = {Adaptive Immunity/genetics ; Animals ; Bacteria/genetics/immunology ; CRISPR-Cas Systems/*genetics/immunology ; Containment of Biohazards ; DNA Breaks, Double-Stranded ; DNA Repair ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/adverse effects/*methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Since the discovery of the double helix and the introduction of genetic engineering, the possibility to develop new strategies to manipulate the genome has fascinated scientists around the world. Currently scientists have the knowledge andabilitytoedit the genomes. Several methodologies of gene editing have been established, all of them working like "scissor", creating double strand breaks at specific spots. The introduction of a new technology, which was adapted from the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas bacterial immune system, has revolutionized the genetic therapy field, as it allows a much more precise editing of gene than the previously described tools and, therefore, to prevent and treat disease in humans. This review aims to revisit the genome editing history that led to the rediscovery of the CRISPR/Cas technology and to explore the technical aspects, applications and perspectives of this fascinating, powerful, precise, simpler and cheaper technology in different fields.},
}
@article {pmid32243905,
year = {2020},
author = {Huang 黄镜梅, JM and Rao 饶聪, C and Wang 王帅, S and He 何林凤, LF and Zhao 赵思琪, SQ and Zhou 周丽琪, LQ and Zhao 赵云霞, YX and Yang 杨凤霞, FX and Gao 高聪芬, CF and Wu 吴顺凡, SF},
title = {Multiple target-site mutations occurring in lepidopterans confer resistance to diamide insecticides.},
journal = {Insect biochemistry and molecular biology},
volume = {121},
number = {},
pages = {103367},
doi = {10.1016/j.ibmb.2020.103367},
pmid = {32243905},
issn = {1879-0240},
mesh = {Amino Acid Sequence ; Animals ; Benzamides/pharmacology ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics/metabolism ; Insect Proteins/chemistry/*genetics/metabolism ; Insecticide Resistance/*genetics ; Insecticides/*pharmacology ; Moths/drug effects/*genetics/metabolism ; *Mutation ; Pyrazoles/pharmacology ; Ryanodine Receptor Calcium Release Channel/chemistry/*genetics/metabolism ; Sequence Alignment ; Sulfones/pharmacology ; ortho-Aminobenzoates/pharmacology ; },
abstract = {Diamide resistant phenotypes have evolved in the field and the resistance has been attributed to target-site mutations in some lepidopteran pests. In this study, we documented the resistance status of Chilo suppressalis to chlorantraniliprole during 2016-2018 in seven provinces of China. To investigate the possible role of target-site mutations as known from lepidopterans, we sequenced respective domains of the RyR gene of C. suppressalis with different levels of diamide resistance. The results revealed that I4758M (corresponding to I4790M in P. xylostella), Y4667D/C (numbered according to C. suppressalis), G4915E (corresponding to G4946E in P. xylostella), and one novel Y4891F (numbered according to C. suppressalis) RyR target-site mutations were present. The contribution of these mutations was further investigated by diamide toxicity bioassays with eight genome modified Drosophila melanogaster lines. The study showed that genome modified flies bearing the Y4667D mutation (corresponding to the Y4667D and I4758M simultaneous mutation in C. suppressalis) exhibited high resistance ratios to chlorantraniliprole (1542.8-fold), cyantraniliprole (487.9-fold) and tetrachlorantraniliprole (290.1-fold). The M4758I and G4915E simultaneous mutations (corresponding to single G4915E mutation in C. suppressalis) showed high resistance ratios to chlorantraniliprole (153.1-fold) and cyantraniliprole (323.5-fold), and relatively low resistance to flubendiamide (28.9-fold) and tetrachlorantraniliprole (25.2-fold). These findings suggest that multiple point mutations in RyR confer diamide resistance of C. suppressalis. The results contribute to a better understanding of insect diamide resistance mechanisms and provide insights on the impact of RyR target-site mutations in insects.},
}
@article {pmid32243904,
year = {2020},
author = {Zhang, R and Zhang, Z and Huang, Y and Qian, A and Tan, A},
title = {A single ortholog of teashirt and tiptop regulates larval pigmentation and adult appendage patterning in Bombyx mori.},
journal = {Insect biochemistry and molecular biology},
volume = {121},
number = {},
pages = {103369},
doi = {10.1016/j.ibmb.2020.103369},
pmid = {32243904},
issn = {1879-0240},
mesh = {Animals ; Bombyx/anatomy &amp; histology/*genetics/growth &amp; development/metabolism ; *CRISPR-Cas Systems ; Extremities/anatomy &amp; histology ; *Gain of Function Mutation ; Insect Proteins/*genetics/metabolism ; Larva/genetics/growth &amp; development/physiology ; *Loss of Function Mutation ; Pigmentation/genetics ; },
abstract = {Two paralogous genes, teashirt (tsh) and tiptop (tio), encode zinc-finger transcription factors and play important roles in insect growth and development. In the fruit fly, Drosophila melanogaster, tsh promotes trunk segmental identities and contributes to the patterning of other tissues during the embryonic stage. During the adult stage, tsh contributes to the specification and patterning of appendages, including the leg, wing and eye. While tio acts redundantly with tsh, flies lacking tio function are viable without deleterious phenotypes. This gene pair is present in the genomes of all Drosophila species but only as a single homologue in several other insect species. In Oncopeltus fasciatus and Tribolium castaneum, tsh/tio has been functionally characterized as specifying the identity of the leg during the adult stage. However, in lepidopteran insects which include large numbers of pests in agriculture and forestry, as well as the major silk producer silkworm Bombyx mori, the biological functions of tsh/tio are still poorly understood. In the current study, we performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B. mori. The results show that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage. RNA-seq analysis and subsequent q-RT-PCR analysis revealed that depletion of tsh/tio significantly elevated the expression of the kynurenine 3-monooxygenase gene, as well as melanin synthase-related genes during the larval stage. Furthermore, ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality. These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage patterning, as well as the novel function of regulating larval pigmentation in B. mori, providing novel insights into how tsh/tio regulates insect growth and development.},
}
@article {pmid32243863,
year = {2020},
author = {Thamm, A and Saunders, TE and Dolan, L},
title = {MpFEW RHIZOIDS1 miRNA-Mediated Lateral Inhibition Controls Rhizoid Cell Patterning in Marchantia polymorpha.},
journal = {Current biology : CB},
volume = {30},
number = {10},
pages = {1905-1915.e4},
doi = {10.1016/j.cub.2020.03.032},
pmid = {32243863},
issn = {1879-0445},
support = {R25 GM067110/GM/NIGMS NIH HHS/United States ; BB/J014427/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Marchantia/genetics/growth &amp; development/*metabolism ; MicroRNAs/genetics/*metabolism ; Models, Biological ; Plant Proteins/genetics/*metabolism ; Plant Roots/cytology/*growth &amp; development/metabolism ; Plants, Genetically Modified ; RNA, Plant/genetics/*metabolism ; },
abstract = {Lateral inhibition patterns differentiated cell types among equivalent cells during development in bacteria, metazoans, and plants. Tip-growing rhizoid cells develop among flat epidermal cells in the epidermis of the early-diverging land plant Marchantia polymorpha. We show that the majority of rhizoid cells develop individually, but some develop in linear, one-dimensional groups (chains) of between 2 and 7 rhizoid cells in wild-type plants. The distribution of rhizoid cells can be accounted for within a simple cellular automata model of lateral inhibition. The model predicted that in the absence of lateral inhibition, two-dimensional rhizoid cell groups (clusters) form. These can be larger than those formed with lateral inhibition. M. polymorpha rhizoid differentiation is positively regulated by the ROOT HAIR DEFECTIVE SIX-LIKE1 (MpRSL1) basic-helix-loop-helix (bHLH) transcription factor, which is directly repressed by the FEW RHIZOIDS1 (MpFRH1) microRNA (miRNA). To test if MpFRH1 miRNA acts during lateral inhibition, we generated loss-of-function (lof) mutants without the MpFRH1 miRNA. Two-dimensional clusters of rhizoids develop in Mpfrh1[lof] mutants as predicted by the model for plants that lack lateral inhibition. Furthermore, two-dimensional clusters of up to 9 rhizoid cells developed in the Mpfrh1[lof] mutants compared to a maximum number of 7 observed in wild-type groups. The higher steady-state levels of MpRSL1 mRNA in Mpfrh1[lof] mutants indicate that MpFRH1-mediated lateral inhibition involves the repression of MpRSL1 activity. Together, the modeling and genetic data indicate that MpFRH1 miRNA mediates lateral inhibition by repressing MpRSL1 during pattern formation in the M. polymorpha epidermis.},
}
@article {pmid32243862,
year = {2020},
author = {Collins, C and Majekodunmi, A and Mitchell, B},
title = {Centriole Number and the Accumulation of Microtubules Modulate the Timing of Apical Insertion during Radial Intercalation.},
journal = {Current biology : CB},
volume = {30},
number = {10},
pages = {1958-1964.e3},
pmid = {32243862},
issn = {1879-0445},
support = {F32 GM119322/GM/NIGMS NIH HHS/United States ; R01 GM113922/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Division/*physiology ; Centrioles/*physiology ; Embryo, Nonmammalian ; Female ; Gene Expression Regulation, Developmental ; Male ; Microtubules/*physiology ; Plasmids ; RNA, Messenger ; Skin/*cytology ; Xenopus laevis ; },
abstract = {Centrioles are microtubule (MT)-based structures that provide important functions during cell migration, cell division, and cell signaling [1]. Modulating centriole number in 3D cell cultures has been shown to influence protrusive behavior [2-5]. Here, we address in vivo the role of centrioles and the accumulation of MTs on the protrusive behavior required during the initiation of radial intercalation. Radial intercalation is an important developmental process whereby cells undergo polarized movements and interdigitate into a more superficial layer [6, 7]. It is commonly employed during metamorphic events, such as the tissue thinning coupled with expansion or during the introduction of different cell types into an epithelium. During radial intercalation, cells emerge from a basal layer by undergoing a process of apical migration, apical insertion, and expansion [8]. In Xenopus skin, multiciliated cells (MCCs), which contain ∼150 centrioles, and ionocytes (ICs), which contain two centrioles, differentiate during the same developmental window, but MCCs complete intercalation prior to ICs. Here, we utilize this difference in timing to create a quantifiable assay for insertion and find that the timing of insertion is modulated by changes in centriole number and the accumulation of acetylated MTs. Additionally, centrioles align between the nucleus and the leading edge creating an axis of migration with apically oriented (+) ends. Using the MT (-) end protein CAMSAP1 fused to the apically positioned Par6 protein, we have artificially reversed the orientation of MTs and find that the accumulation of MTs in either orientation is sufficient to promote apical insertion.},
}
@article {pmid32243835,
year = {2020},
author = {Teboul, L and Herault, Y and Wells, S and Qasim, W and Pavlovic, G},
title = {Variability in Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical Safety.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {6},
pages = {1422-1431},
pmid = {32243835},
issn = {1525-0024},
support = {MC_UP_1502/1/MRC_/Medical Research Council/United Kingdom ; RP-2014-05-007/DH_/Department of Health/United Kingdom ; A410/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animal Experimentation ; Animals ; CRISPR-Cas Systems ; Clinical Studies as Topic ; Gene Editing/*methods/*standards ; Gene Expression ; Gene Transfer Techniques ; Genetic Therapy/adverse effects/methods/standards ; Humans ; Organ Specificity ; Reproducibility of Results ; Risk Assessment ; Translational Research, Biomedical/methods/standards ; },
abstract = {Genome editing tools have already revolutionized biomedical research and are also expected to have an important impact in the clinic. However, their extensive use in research has revealed much unpredictability, both off and on target, in the outcome of their application. We discuss the challenges associated with this unpredictability, both for research and in the clinic. For the former, an extensive validation of the model is essential. For the latter, potential unpredicted activity does not preclude the use of these tools but requires that molecular evidence to underpin the relevant risk:benefit evaluation is available. Safe and successful clinical application will also depend on the mode of delivery and the cellular context.},
}
@article {pmid32243786,
year = {2020},
author = {Wang, D and Zhang, F and Gao, G},
title = {CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors.},
journal = {Cell},
volume = {181},
number = {1},
pages = {136-150},
pmid = {32243786},
issn = {1097-4172},
support = {UG3 HL147367/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 NS076991/NS/NINDS NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; R01 HL097088/HL/NHLBI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/*therapeutic use ; Humans ; },
abstract = {The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.},
}
@article {pmid32242409,
year = {2020},
author = {Bao, Y and Jiang, Y and Xiong, E and Tian, T and Zhang, Z and Lv, J and Li, Y and Zhou, X},
title = {CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage.},
journal = {ACS sensors},
volume = {5},
number = {4},
pages = {1082-1091},
doi = {10.1021/acssensors.0c00034},
pmid = {32242409},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems/*physiology ; Humans ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/*methods ; },
abstract = {Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format.},
}
@article {pmid32242237,
year = {2020},
author = {Xu, H and Qu, C and Gan, L and Sun, K and Tan, J and Liu, X and Jiang, Z and Tian, W and Liu, W and Zhang, S and Yang, Y and Jiang, L and Zhu, X and Zhang, L},
title = {Deletion of the Impg2 gene causes the degeneration of rod and cone cells in mice.},
journal = {Human molecular genetics},
volume = {29},
number = {10},
pages = {1624-1634},
doi = {10.1093/hmg/ddaa062},
pmid = {32242237},
issn = {1460-2083},
mesh = {Animals ; Autophagy/genetics ; CRISPR-Cas Systems/genetics ; Cell Death/genetics ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress/*genetics ; Heat-Shock Proteins/genetics ; Humans ; Mice ; Mice, Knockout ; Protein Disulfide-Isomerases/genetics ; Proteoglycans/*genetics ; Retina/*metabolism/pathology ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Retinal Degeneration/*genetics/pathology ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Rhodopsin/*genetics ; Transcription Factor CHOP/genetics ; },
abstract = {Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.},
}
@article {pmid32242071,
year = {2020},
author = {Jun, S and Lim, H and Chun, H and Lee, JH and Bang, D},
title = {Single-cell analysis of a mutant library generated using CRISPR-guided deaminase in human melanoma cells.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {154},
pmid = {32242071},
issn = {2399-3642},
mesh = {Antineoplastic Agents/pharmacology ; Biomarkers, Tumor/*genetics ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytidine Deaminase/*genetics/metabolism ; Female ; *Gene Editing ; *Gene Library ; HEK293 Cells ; Humans ; Melanoma/drug therapy/*genetics/metabolism/pathology ; *Mutation ; RNA, Guide/genetics ; RNA-Seq ; *Single-Cell Analysis ; Skin Neoplasms/drug therapy/*genetics/metabolism/pathology ; Vemurafenib/pharmacology ; },
abstract = {CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNA-guided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutation-associated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the human melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.},
}
@article {pmid32241913,
year = {2020},
author = {Mekler, V and Kuznedelov, K and Severinov, K},
title = {Quantification of the affinities of CRISPR-Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {19},
pages = {6509-6517},
pmid = {32241913},
issn = {1083-351X},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry ; *CRISPR-Cas Systems ; Francisella/*chemistry ; RNA, Bacterial/*chemistry ; RNA, Guide/*chemistry ; Staphylococcus aureus/*chemistry ; Streptococcus pyogenes/*chemistry ; },
abstract = {The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9-gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9-gRNA, SaCas9-gRNA, and FnCas9-gRNA, respectively) and of three engineered SpCas9-gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a "beacon" assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9-gRNA binding to fluorescently labeled target DNA derivatives called "Cas9 beacons." We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9-gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.},
}
@article {pmid32241803,
year = {2020},
author = {Mona, B and Villarreal, J and Savage, TK and Kollipara, RK and Boisvert, BE and Johnson, JE},
title = {Positive autofeedback regulation of Ptf1a transcription generates the levels of PTF1A required to generate itch circuit neurons.},
journal = {Genes &amp; development},
volume = {34},
number = {9-10},
pages = {621-636},
pmid = {32241803},
issn = {1549-5477},
support = {R01 HD037932/HD/NICHD NIH HHS/United States ; R37 HD091856/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Enhancer Elements, Genetic/genetics ; Feedback, Physiological/*physiology ; Gene Expression Regulation/*physiology ; Mice ; Mutation ; Neurons/cytology/*physiology ; Pruritus/*genetics ; Spinal Cord ; Transcription Factors/*genetics/metabolism ; },
abstract = {Peripheral somatosensory input is modulated in the dorsal spinal cord by a network of excitatory and inhibitory interneurons. PTF1A is a transcription factor essential in dorsal neural tube progenitors for specification of these inhibitory neurons. Thus, mechanisms regulating Ptf1a expression are key for generating neuronal circuits underlying somatosensory behaviors. Mutations targeted to distinct cis-regulatory elements for Ptf1a in mice, tested the in vivo contribution of each element individually and in combination. Mutations in an autoregulatory enhancer resulted in reduced levels of PTF1A, and reduced numbers of specific dorsal spinal cord inhibitory neurons, particularly those expressing Pdyn and Gal Although these mutants survive postnatally, at ∼3-5 wk they elicit a severe scratching phenotype. Behaviorally, the mutants have increased sensitivity to itch, but acute sensitivity to other sensory stimuli such as mechanical or thermal pain is unaffected. We demonstrate a requirement for positive transcriptional autoregulatory feedback to attain the level of the neuronal specification factor PTF1A necessary for generating correctly balanced neuronal circuits.},
}
@article {pmid32241274,
year = {2020},
author = {Liu, E and Zhang, ZZ and Cheng, X and Liu, X and Cheng, L},
title = {SCNrank: spectral clustering for network-based ranking to reveal potential drug targets and its application in pancreatic ductal adenocarcinoma.},
journal = {BMC medical genomics},
volume = {13},
number = {Suppl 5},
pages = {50},
pmid = {32241274},
issn = {1755-8794},
mesh = {*Algorithms ; Antineoplastic Agents/*therapeutic use ; Biomarkers, Tumor/*antagonists &amp; inhibitors/genetics ; Carcinoma, Pancreatic Ductal/drug therapy/genetics/pathology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic/*drug effects ; Gene Regulatory Networks/*drug effects ; Humans ; *Molecular Targeted Therapy ; Pancreatic Neoplasms/*drug therapy/genetics/pathology ; Prognosis ; Protein Interaction Maps ; Survival Rate ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy. Due to its wide heterogeneity, PDAC acts aggressively and responds poorly to most chemotherapies, causing an urgent need for the development of new therapeutic strategies. Cell lines have been used as the foundation for drug development and disease modeling. CRISPR-Cas9 plays a key role in every step-in drug discovery: from target identification and validation to preclinical cancer cell testing. Using cell-line models and CRISPR-Cas9 technology together make drug target prediction feasible. However, there is still a large gap between predicted results and actionable targets in real tumors. Biological network models provide great modus to mimic genetic interactions in real biological systems, which can benefit gene perturbation studies and potential target identification for treating PDAC. Nevertheless, building a network model that takes cell-line data and CRISPR-Cas9 data as input to accurately predict potential targets that will respond well on real tissue remains unsolved.<br><br>METHODS: We developed a novel algorithm 'Spectral Clustering for Network-based target Ranking' (SCNrank) that systematically integrates three types of data: expression profiles from tumor tissue, normal tissue and cell-line PDAC; protein-protein interaction network (PPI); and CRISPR-Cas9 data to prioritize potential drug targets for PDAC. The whole algorithm can be classified into three steps: 1. using STRING PPI network skeleton, SCNrank constructs tissue-specific networks with PDAC tumor and normal pancreas tissues from expression profiles; 2. With the same network skeleton, SCNrank constructs cell-line-specific networks using the cell-line PDAC expression profiles and CRISPR-Cas 9 data from pancreatic cancer cell-lines; 3. SCNrank applies a novel spectral clustering approach to reduce data dimension and generate gene clusters that carry common features from both networks. Finally, SCNrank applies a scoring scheme called 'Target Influence score' (TI), which estimates a given target's influence towards the cluster it belongs to, for scoring and ranking each drug target.<br><br>RESULTS: We applied SCNrank to analyze 263 expression profiles, CRPSPR-Cas9 data from 22 different pancreatic cancer cell-lines and the STRING protein-protein interaction (PPI) network. With SCNrank, we successfully constructed an integrated tissue PDAC network and an integrated cell-line PDAC network, both of which contain 4414 selected genes that are overexpressed in tumor tissue samples. After clustering, 4414 genes are distributed into 198 clusters, which include 367 targets of FDA approved drugs. These drug targets are all scored and ranked by their TI scores, which we defined to measure their influence towards the network. We validated top-ranked targets in three aspects: Firstly, mapping them onto the existing clinical drug targets of PDAC to measure the concordance. Secondly, we performed enrichment analysis to these drug targets and the clusters there are within, to reveal functional associations between clusters and PDAC; Thirdly, we performed survival analysis for the top-ranked targets to connect targets with clinical outcomes. Survival analysis reveals that overexpression of three top-ranked genes, PGK1, HMMR and POLE2, significantly increases the risk of death in PDAC patients.<br><br>CONCLUSION: SCNrank is an unbiased algorithm that systematically integrates multiple types of omics data to do potential drug target selection and ranking. SCNrank shows great capability in predicting drug targets for PDAC. Pancreatic cancer-associated gene candidates predicted by our SCNrank approach have the potential to guide genetics-based anti-pancreatic drug discovery.},
}
@article {pmid32241200,
year = {2020},
author = {Knoedler, JR and Ávila-Mendoza, J and Subramani, A and Denver, RJ},
title = {The Paralogous Krüppel-like Factors 9 and 13 Regulate the Mammalian Cellular Circadian Clock Output Gene Dbp.},
journal = {Journal of biological rhythms},
volume = {35},
number = {3},
pages = {257-274},
pmid = {32241200},
issn = {1552-4531},
support = {R01 NS046690/NS/NINDS NIH HHS/United States ; R21 NS088062/NS/NINDS NIH HHS/United States ; T32 HD079342/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CLOCK Proteins/genetics ; CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics ; Cell Line ; Circadian Clocks/*genetics ; Circadian Rhythm ; DNA-Binding Proteins/*genetics ; Gene Expression Regulation ; Gene Knockout Techniques ; Kruppel-Like Transcription Factors/*genetics ; Male ; Mice ; Mice, Knockout ; Repressor Proteins/*genetics ; Transcription Factors/*genetics ; Transcription, Genetic ; },
abstract = {An intricate transcription-translation feedback loop (TTFL) governs cellular circadian rhythms in mammals. Here, we report that the zinc finger transcription factor Krüppel-like factor 9 (KLF9) is regulated by this TTFL, it associates in chromatin at the core circadian clock and clock-output genes, and it acts to modulate transcription of the clock-output gene Dbp. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with Per1, Per3, Dbp, Tef, Bhlhe40, Bhlhe41, Nr1d1, and Nr1d2. Of the 3514 KLF9 peaks identified in HT22 cells, 1028 contain E-box sequences to which the transcriptional activators CLOCK and BMAL1 may bind, a frequency significantly greater than expected by chance. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene Dbp, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basal Dbp transcription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfected Dbp reporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within the Dbp locus that are in close proximity to E-boxes. Knockout of Klf9 or the paralogous gene Klf13 using CRISPR/Cas9 genome editing in HT22 cells had no effect on Dbp expression, but combined knockout of both genes strongly impaired circadian Dbp mRNA oscillation. Like KLF9, KLF13 also showed association in chromatin with clock- and clock-output genes, and forced expression of KLF13 inhibited the actions of CLOCK+BMAL1 on Dbp transcription. Our results suggest novel and partly overlapping roles for KLF9 and KLF13 in modulating cellular circadian clock output by a mechanism involving direct interaction with the core TTFL.},
}
@article {pmid32239615,
year = {2020},
author = {Grosshans, HK and Fischer, TT and Steinle, JA and Brill, AL and Ehrlich, BE},
title = {Neuronal Calcium Sensor 1 is up-regulated in response to stress to promote cell survival and motility in cancer cells.},
journal = {Molecular oncology},
volume = {14},
number = {6},
pages = {1134-1151},
pmid = {32239615},
issn = {1878-0261},
support = {UL1 TR001863/TR/NCATS NIH HHS/United States ; T32 GM007324/GM/NIGMS NIH HHS/United States ; P01 DK057751/DK/NIDDK NIH HHS/United States ; F31 DK118836/DK/NIDDK NIH HHS/United States ; P30 DK034989/DK/NIDDK NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Cell Line, Tumor ; Cell Movement/drug effects/*genetics ; Cell Survival/drug effects/genetics ; Chelating Agents/pharmacology ; Cytosol/metabolism ; Endoplasmic Reticulum/drug effects/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism ; NF-kappa B/metabolism ; Neoplasms/*genetics/*pathology ; Neuronal Calcium-Sensor Proteins/*genetics/metabolism ; Neuropeptides/*genetics/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Signal Transduction ; Stress, Physiological/drug effects/*genetics ; Tumor Necrosis Factor-alpha/metabolism ; Up-Regulation/drug effects/*genetics ; },
abstract = {Changes in intracellular calcium (Ca[2+]) signaling can modulate cellular machinery required for cancer progression. Neuronal calcium sensor 1 (NCS1) is a ubiquitously expressed Ca[2+] -binding protein that promotes tumor aggressiveness by enhancing cell survival and metastasis. However, the underlying mechanism by which NCS1 contributes to increased tumor aggressiveness has yet to be identified. In this study, we aimed to determine (a) whether NCS1 expression changes in response to external stimuli, (b) the importance of NCS1 for cell survival and migration, and (c) the cellular mechanism(s) through which NSC1 modulates these outcomes. We found that NCS1 abundance increases under conditions of stress, most prominently after stimulation with the pro-inflammatory cytokine tumor necrosis factor α, in a manner dependent on nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). We found that NFκB signaling is activated in human breast cancer tissue, which was accompanied by an increase in NCS1 mRNA expression. Further exploration into the relevance of NCS1 in breast cancer progression showed that knockout of NCS1 (NCS1 KO) caused decreased cell survival and motility, increased baseline intracellular Ca[2+] levels, and decreased inositol 1,4,5-trisphosphate-mediated Ca[2+] responses. Protein kinase B (Akt) activity was decreased in NCS1 KO cells, which could be rescued by buffering intracellular Ca[2+] . Conversely, Akt activity was increased in cells overexpressing NCS1 (NCS1 OE). We therefore conclude that NCS1 acts as cellular stress response protein up-regulated by stress-induced NFκB signaling and that NCS1 influences cell survival and motility through effects on Ca[2+] signaling and Akt pathway activation.},
}
@article {pmid32239370,
year = {2020},
author = {Hou, J and Cao, X and Cheng, Y and Wang, X},
title = {Roles of TP53 gene in the development of resistance to PI3K inhibitor resistances in CRISPR-Cas9-edited lung adenocarcinoma cells.},
journal = {Cell biology and toxicology},
volume = {36},
number = {5},
pages = {481-492},
doi = {10.1007/s10565-020-09523-7},
pmid = {32239370},
issn = {1573-6822},
mesh = {A549 Cells ; Adenocarcinoma of Lung/enzymology/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; DNA, Neoplasm/chemistry ; Drug Resistance, Neoplasm/drug effects/*genetics ; *Gene Editing ; *Genes, p53 ; Humans ; Lung Neoplasms/enzymology/*genetics ; Models, Biological ; Phosphatidylinositol 3-Kinases/genetics/*metabolism ; Protein Isoforms/antagonists &amp; inhibitors/genetics/metabolism ; Protein Kinase Inhibitors/chemistry/*pharmacology ; Small Molecule Libraries/chemistry/pharmacology ; },
abstract = {The mutation rates of tumor suppressor protein p53 gene (TP53) are high in lung adenocarcinoma and promote the development of acquired drug resistance. The present study evaluated the p53-dependent role in lung cancer cell sensitivity to PI3K-specific inhibitors, PI3K-associated inhibitors, PI3K-non-related inhibitors, and protein-based stimuli using designed p53 mutation. We found that the deletion of p53 key regions from amino acid 96 to 393 with the CRISPR-Cas9 altered multi-dimensional structure and sequencing of p53, probably leading the secondary changes in chemical structures and properties of PI3K subunit proteins or in interactions between p53 and PI3K isoform genes. The p53-dependent cell sensitivity varied among target specificities, drug chemical properties, mechanism-specific signal pathways, and drug efficacies, independently upon the size of molecules. The effects of the designed p53 mutation highly depend upon p53-involved molecular mechanisms in the cell. Our results indicate that lung cancer cell resistance to drug can develop with dynamic formations of p53 mutations changing the cell sensitivity. This may explain the real-time occurrence of cancer cell resistance to drug treatment, during which drugs may induce the new mutations of p53. Thus, it is important to dynamically monitor the formation of new mutations during the therapy and discover new drug resistance-specific targets.},
}
@article {pmid32239050,
year = {2020},
author = {Lim, S and Kim, J and Kim, Y and Xu, D and Clark, DS},
title = {CRISPR/Cas-directed programmable assembly of multi-enzyme complexes.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {36},
pages = {4950-4953},
doi = {10.1039/d0cc01174f},
pmid = {32239050},
issn = {1364-548X},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Multienzyme Complexes/*genetics/metabolism ; Streptococcus pyogenes/enzymology ; },
abstract = {We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.},
}
@article {pmid32238925,
year = {2020},
author = {Han, K and Pierce, SE and Li, A and Spees, K and Anderson, GR and Seoane, JA and Lo, YH and Dubreuil, M and Olivas, M and Kamber, RA and Wainberg, M and Kostyrko, K and Kelly, MR and Yousefi, M and Simpkins, SW and Yao, D and Lee, K and Kuo, CJ and Jackson, PK and Sweet-Cordero, A and Kundaje, A and Gentles, AJ and Curtis, C and Winslow, MM and Bassik, MC},
title = {CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.},
journal = {Nature},
volume = {580},
number = {7801},
pages = {136-141},
pmid = {32238925},
issn = {1476-4687},
support = {T32 CA009302/CA/NCI NIH HHS/United States ; DP2 HD084069/HD/NICHD NIH HHS/United States ; K00 CA212433/CA/NCI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; U01 CA217851/CA/NCI NIH HHS/United States ; U01 CA199216/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma/genetics/metabolism/pathology ; Amino Acid Motifs ; Animals ; CRISPR-Cas Systems/*genetics ; Carboxypeptidases/antagonists &amp; inhibitors/deficiency/genetics/metabolism ; Cell Culture Techniques/*methods ; Cell Proliferation/*genetics ; Female ; Genome, Human/*genetics ; Humans ; Lung Neoplasms/*genetics/metabolism/*pathology ; Mice ; Molecular Targeted Therapy ; Mutation ; Phenotype ; Receptor, IGF Type 1/chemistry/metabolism ; Signal Transduction ; Spheroids, Cellular/metabolism/*pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Cancer genomics studies have identified thousands of putative cancer driver genes[1]. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif[2] from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.},
}
@article {pmid32238808,
year = {2020},
author = {Fontana, J and Dong, C and Kiattisewee, C and Chavali, VP and Tickman, BI and Carothers, JM and Zalatan, JG},
title = {Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1618},
pmid = {32238808},
issn = {2041-1723},
mesh = {Bacteria/*genetics ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Escherichia coli Proteins ; *Gene Expression Regulation, Bacterial ; Genes, Bacterial/genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Trans-Activators ; Transcriptional Activation ; },
abstract = {In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2-4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ[70]-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.},
}
@article {pmid32238445,
year = {2020},
author = {Vigouroux, A and Bikard, D},
title = {CRISPR Tools To Control Gene Expression in Bacteria.},
journal = {Microbiology and molecular biology reviews : MMBR},
volume = {84},
number = {2},
pages = {},
pmid = {32238445},
issn = {1098-5557},
mesh = {Bacteria/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression ; Genetic Engineering/*methods ; },
abstract = {CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.},
}
@article {pmid32236513,
year = {2020},
author = {Jackman, M and Marcozzi, C and Barbiero, M and Pardo, M and Yu, L and Tyson, AL and Choudhary, JS and Pines, J},
title = {Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint.},
journal = {The Journal of cell biology},
volume = {219},
number = {6},
pages = {},
pmid = {32236513},
issn = {1540-8140},
support = {/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {CDC2 Protein Kinase/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/drug effects/genetics ; Cell Cycle Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Cyclin B1/genetics/*metabolism ; Exons ; Gene Editing ; Genomic Instability/genetics ; HeLa Cells ; Humans ; Kinetochores/*metabolism ; Mad2 Proteins/genetics/metabolism ; Mass Spectrometry ; Mutation ; Nuclear Envelope/metabolism ; Nuclear Pore/*metabolism ; Nuclear Pore Complex Proteins/metabolism ; Nuclear Proteins/metabolism ; Protein Binding ; Protein Serine-Threonine Kinases/antagonists &amp; inhibitors ; Protein-Tyrosine Kinases/antagonists &amp; inhibitors ; Proto-Oncogene Proteins/metabolism ; Signal Transduction/*genetics/physiology ; Spindle Apparatus/metabolism ; },
abstract = {How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability.},
}
@article {pmid32235908,
year = {2020},
author = {Burgess, DJ},
title = {CRISPR screens beyond Cas9.},
journal = {Nature reviews. Genetics},
volume = {21},
number = {5},
pages = {273},
pmid = {32235908},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Exons ; Gene Editing ; Genomics ; },
}
@article {pmid32234883,
year = {2020},
author = {Sekino, Y and Hagura, T and Han, X and Babasaki, T and Goto, K and Inoue, S and Hayashi, T and Teishima, J and Shigeta, M and Taniyama, D and Kuraoka, K and Sentani, K and Yasui, W and Matsubara, A},
title = {PTEN Is Involved in Sunitinib and Sorafenib Resistance in Renal Cell Carcinoma.},
journal = {Anticancer research},
volume = {40},
number = {4},
pages = {1943-1951},
doi = {10.21873/anticanres.14149},
pmid = {32234883},
issn = {1791-7530},
mesh = {Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; Carcinoma, Renal Cell/*drug therapy/genetics/pathology ; Drug Resistance, Neoplasm/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Kaplan-Meier Estimate ; Male ; PTEN Phosphohydrolase/*genetics ; Progression-Free Survival ; Protein Kinase Inhibitors/pharmacology ; Sorafenib/*pharmacology ; Spheroids, Cellular/drug effects ; Sunitinib/*pharmacology ; },
abstract = {BACKGROUND/AIM: Targeted receptor tyrosine kinase inhibitor (TKI) is a standard treatment in advanced renal cell carcinoma (RCC). However, the role of PTEN in TKI resistance remains poorly understood. We aimed to determine the functional role of PTEN knockout and analyse the predictive significance of PTEN expression for TKI treatment in RCC.<br><br>MATERIALS AND METHODS: We developed PTEN knockout cells in RCC cell lines using the CRISPR-Cas9 system and analysed the effect of PTEN knockout on spheroid formation and resistance to sunitinib and sorafenib.<br><br>RESULTS: PTEN knockout promoted spheroid formation and decreased sunitinib/sorafenib sensitivity in RCC cell lines. PTEN immunohistochemistry in 74 metastatic RCCs treated with sunitinib and sorafenib revealed negative PTEN expression in 23% of samples. Kaplan-Meier analysis showed a significant association of negative PTEN expression with poor progression-free survival in metastatic RCC treated with sunitinib and sorafenib (p=0.024) or sunitinib alone (p=0.009).<br><br>CONCLUSION: PTEN may be a biomarker and therapeutic target in patients with metastatic RCC.},
}
@article {pmid32234808,
year = {2020},
author = {Vanderboor, CMG and Thibeault, PE and Nixon, KCJ and Gros, R and Kramer, J and Ramachandran, R},
title = {Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and β-Arrestin.},
journal = {Molecular pharmacology},
volume = {97},
number = {6},
pages = {365-376},
doi = {10.1124/mol.119.118232},
pmid = {32234808},
issn = {1521-0111},
support = {376560//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Membrane/*metabolism/*pathology ; Cell Shape ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Muscle, Smooth, Vascular/metabolism ; Rats ; Rats, Inbred WKY ; Receptors, G-Protein-Coupled/metabolism ; Receptors, Thrombin/agonists/*metabolism ; Signal Transduction ; beta-Arrestins/*metabolism ; rhoA GTP-Binding Protein/*metabolism ; },
abstract = {Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the β-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and β-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.},
}
@article {pmid32234056,
year = {2020},
author = {Liu, SJ and Malatesta, M and Lien, BV and Saha, P and Thombare, SS and Hong, SJ and Pedraza, L and Koontz, M and Seo, K and Horlbeck, MA and He, D and Birk, HS and Jain, M and Olsen, HE and Akeson, M and Weissman, JS and Monje, M and Gupta, N and Raleigh, DR and Ullian, EM and Lim, DA},
title = {CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {83},
pmid = {32234056},
issn = {1474-760X},
support = {HG010053/NH/NIH HHS/United States ; R21 NS101395/NS/NINDS NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; 1R21 NS101395-01/NH/NIH HHS/United States ; F30 NS092319-01/NH/NIH HHS/United States ; I01 BX000252/BX/BLRD VA/United States ; R01 HG010053/HG/NHGRI NIH HHS/United States ; 1R01NS091544/NH/NIH HHS/United States ; R03AG063157/NH/NIH HHS/United States ; R01 NS091544/NS/NINDS NIH HHS/United States ; T32 HD007470/HD/NICHD NIH HHS/United States ; R03 AG063157/AG/NIA NIH HHS/United States ; P30 EY002162/EY/NEI NIH HHS/United States ; },
mesh = {Adult ; Astrocytes ; Brain ; Brain Neoplasms/genetics/pathology/radiotherapy/*therapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Combined Modality Therapy ; Glioblastoma/genetics/pathology/radiotherapy/*therapy ; Humans ; Oligonucleotides, Antisense ; Organoids ; RNA, Long Noncoding/*antagonists &amp; inhibitors ; Radiation Tolerance ; },
abstract = {BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients.<br><br>RESULTS: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy.<br><br>CONCLUSIONS: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.},
}
@article {pmid32234052,
year = {2020},
author = {Horii, T and Morita, S and Hino, S and Kimura, M and Hino, Y and Kogo, H and Nakao, M and Hatada, I},
title = {Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {77},
pmid = {32234052},
issn = {1474-760X},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Methylation ; *Disease Models, Animal ; Embryonic Stem Cells/metabolism ; *Epigenesis, Genetic ; *Epigenome ; Epigenomics/methods ; Humans ; *Mice ; RNA, Long Noncoding/genetics ; Silver-Russell Syndrome/diagnostic imaging/*genetics ; Zygote/metabolism ; },
abstract = {BACKGROUND: Epigenetic modifications, including DNA methylation, play an important role in gene silencing and genome stability. Consequently, epigenetic dysregulation can cause several diseases, such as cancer, obesity, diabetes, autism, and imprinting disorders.<br><br>RESULTS: We validate three methods for the generation of epigenome-edited mice using the dCas9-SunTag and single-chain variable fragment-TET1 catalytic domain. We generate model mice for Silver-Russell syndrome (SRS), an imprinting disorder, by target-specific DNA demethylation in the H19 differentially methylated region. Like SRS patients, these mice show H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation.<br><br>CONCLUSION: This is the first report of an imprinting disease model animal generated by targeted demethylation of specific loci of the epigenome in fertilized eggs. Epigenome-edited animals are also useful for exploring the causative epimutations in epigenetic diseases.},
}
@article {pmid32233423,
year = {2020},
author = {Smith, CW and Nandu, N and Kachwala, MJ and Chen, YS and Uyar, TB and Yigit, MV},
title = {Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates.},
journal = {Biochemistry},
volume = {59},
number = {15},
pages = {1474-1481},
pmid = {32233423},
issn = {1520-4995},
support = {R15 GM128115/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/*analysis ; CRISPR-Associated Proteins/*analysis ; CRISPR-Cas Systems ; DNA/*chemistry ; Endodeoxyribonucleases/*analysis ; Fluorescent Dyes/*chemistry ; Spectrometry, Fluorescence ; },
abstract = {The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through trans-cleavage upon activation with a specific target DNA. This shredding activity offered the potential for development of ssDNA-templated probes with fluorescent dye (F) and quencher (Q) labels. However, the formulations of double-stranded DNA (dsDNA)-templated fluorescent probes have not been reported possibly due to unknown (or limited) activity of Cas12a against short dsDNAs. The ssDNA probes have been shown to be powerful for diagnostic applications; however, limiting the probe selections to short ssDNAs could be restrictive from an application and probe diversification standpoint. Here, we report a dsDNA substrate (probe-full) for probing Cas12a trans-cleavage activity upon target detection. A diverse set of Cas12a substrates with alternating dsDNA character were designed and studied using fluorescence spectroscopy. We have observed that probe-full without any nick displayed trans-cleavage performance that was better than that of the form that contains a nick. Different experimental conditions of salt concentration, target concentration, and mismatch tolerance were examined to evaluate the probe performance. The activity of Cas12a was programmed for a dsDNA frame copied from a tobacco curly shoot virus (TCSV) or hepatitis B virus (HepBV) genome by using crRNA against TCSV or HepBV, respectively. While on-target activity offered detection of as little as 10 pM dsDNA target, off-target activity was not observed even at 1 nM control DNAs. This study demonstrates that trans-cleavage of Cas12a is not limited to ssDNA substrates, and Cas12a-based diagnostics can be extended to dsDNA substrates.},
}
@article {pmid32233134,
year = {2020},
author = {Chang, YJ and Bae, J and Zhao, Y and Lee, G and Han, J and Lee, YH and Koo, OJ and Seo, S and Choi, YK and Yeom, SC},
title = {In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal.},
journal = {Journal of veterinary science},
volume = {21},
number = {2},
pages = {e26},
pmid = {32233134},
issn = {1976-555X},
support = {NRF-2015R1C1A1A01051949/NRF/National Research Foundation of Korea/Korea ; 2017M3A9B4061409/NRF/National Research Foundation of Korea/Korea ; 2018R1A1A1A05078158/NRF/National Research Foundation of Korea/Korea ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Campylobacter jejuni/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; *Gene Targeting ; Mice ; Mice, Transgenic ; Pancreatic Neoplasms/*microbiology ; Streptococcus pyogenes/*genetics ; },
abstract = {Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.},
}
@article {pmid32232367,
year = {2020},
author = {Suárez, GA and Dugan, KR and Renda, BA and Leonard, SP and Gangavarapu, LS and Barrick, JE},
title = {Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining.},
journal = {Nucleic acids research},
volume = {48},
number = {8},
pages = {4585-4600},
pmid = {32232367},
issn = {1362-4962},
mesh = {Acinetobacter/*genetics/growth &amp; development ; CRISPR-Cas Systems ; Gene Deletion ; Genes, Bacterial ; Genetic Engineering/*methods ; *Genome, Bacterial ; Transformation, Bacterial ; },
abstract = {One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.},
}
@article {pmid32232035,
year = {2020},
author = {Calderón-Franco, D and Lin, Q and van Loosdrecht, MCM and Abbas, B and Weissbrodt, DG},
title = {Anticipating Xenogenic Pollution at the Source: Impact of Sterilizations on DNA Release From Microbial Cultures.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {171},
pmid = {32232035},
issn = {2296-4185},
abstract = {The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121°C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.},
}
@article {pmid32231336,
year = {2020},
author = {Replogle, JM and Norman, TM and Xu, A and Hussmann, JA and Chen, J and Cogan, JZ and Meer, EJ and Terry, JM and Riordan, DP and Srinivas, N and Fiddes, IT and Arthur, JG and Alvarado, LJ and Pfeiffer, KA and Mikkelsen, TS and Weissman, JS and Adamson, B},
title = {Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.},
journal = {Nature biotechnology},
volume = {38},
number = {8},
pages = {954-961},
pmid = {32231336},
issn = {1546-1696},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R00 GM134154/GM/NIGMS NIH HHS/United States ; K99 GM134154/GM/NIGMS NIH HHS/United States ; F31 NS115380/NS/NINDS NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; Gene Targeting ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Nucleic Acid Amplification Techniques/*methods ; RNA, Guide/*genetics ; Single-Cell Analysis ; Transcriptome ; },
abstract = {Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.},
}
@article {pmid32231334,
year = {2020},
author = {Taketani, M and Zhang, J and Zhang, S and Triassi, AJ and Huang, YJ and Griffith, LG and Voigt, CA},
title = {Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron.},
journal = {Nature biotechnology},
volume = {38},
number = {8},
pages = {962-969},
pmid = {32231334},
issn = {1546-1696},
support = {P50 GM098792/GM/NIGMS NIH HHS/United States ; R01 EB021908/EB/NIBIB NIH HHS/United States ; },
mesh = {*Automation ; Bacteroides thetaiotaomicron/*genetics ; CRISPR-Cas Systems ; Gastrointestinal Microbiome ; *Gene Regulatory Networks ; Humans ; RNA, Guide ; Transcription, Genetic ; },
abstract = {Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome[1]. Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron[2-5]. We report implementation of Cello circuit design automation software[6] for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR-dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.},
}
@article {pmid32231122,
year = {2020},
author = {Abbasi, F and Kodani, M and Emori, C and Kiyozumi, D and Mori, M and Fujihara, Y and Ikawa, M},
title = {CRISPR/Cas9-Mediated Genome Editing Reveals Oosp Family Genes are Dispensable for Female Fertility in Mice.},
journal = {Cells},
volume = {9},
number = {4},
pages = {},
pmid = {32231122},
issn = {2073-4409},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/*genetics ; Computer Simulation ; Conserved Sequence ; Female ; Fertility/*physiology ; Gene Deletion ; *Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Mice, Knockout ; Mice, Mutant Strains ; *Multigene Family ; Ovary/metabolism ; Phenotype ; Pregnancy Proteins/chemistry/genetics/*metabolism ; },
abstract = {There are over 200 genes that are predicted to be solely expressed in the oocyte and ovary, and thousands more that have expression patterns in the female reproductive tract. Unfortunately, many of their physiological functions, such as their roles in oogenesis or fertilization, have yet to be elucidated. Previous knockout (KO) mice studies have proven that many of the genes that were once thought to be essential for fertility are dispensable in vivo. Therefore, it is extremely important to confirm the roles of all genes before spending immense time studying them in vitro. To do this, our laboratory analyzes the functions of ovary and oocyte-enriched genes in vivo through generating CRISPR/Cas9 KO mice and examining their fertility. In this study, we have knocked out three Oosp family genes (Oosp1, Oosp2, and Oosp3) that have expression patterns linked to the female reproductive system and found that the triple KO (TKO) mutant mice generated exhibited decreased prolificacy but were not infertile; thus, these genes may potentially be dispensable for fertility. We also generated Cd160 and Egfl6 KO mice and found these genes are individually dispensable for female fertility. KO mice with no phenotypic data are seldom published, but we believe that this information must be shared to prevent unnecessary experimentation by other laboratories.},
}
@article {pmid32230903,
year = {2020},
author = {Peddle, CF and Fry, LE and McClements, ME and MacLaren, RE},
title = {CRISPR Interference-Potential Application in Retinal Disease.},
journal = {International journal of molecular sciences},
volume = {21},
number = {7},
pages = {},
pmid = {32230903},
issn = {1422-0067},
support = {MC_PC_18059/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; Gene Knockdown Techniques ; Gene Silencing ; Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells ; RNA Interference ; RNA, Guide/metabolism ; Retinal Diseases/*genetics/*metabolism/therapy ; Transcription, Genetic ; },
abstract = {The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive 'dead' Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profile as a therapy. Pre-clinical studies have demonstrated the versatility of CRISPR interference for gene silencing both in vivo and in ex vivo modification of iPSCs for transplantation. Applying CRISPR interference techniques for the treatment of autosomal dominant inherited retinal diseases is promising but there are few in vivo studies to date. This review details how CRISPR interference might be used to treat retinal diseases and addresses potential challenges for clinical translation.},
}
@article {pmid32230824,
year = {2020},
author = {Zhang, Q and Zhang, L and Ross, P and Zhao, J and Zhang, H and Chen, W},
title = {Comparative Genomics of Lactobacillus crispatus from the Gut and Vagina Reveals Genetic Diversity and Lifestyle Adaptation.},
journal = {Genes},
volume = {11},
number = {4},
pages = {},
pmid = {32230824},
issn = {2073-4425},
mesh = {Animals ; Chickens ; Feces/*microbiology ; Female ; Gastrointestinal Microbiome/*genetics ; Gastrointestinal Tract/*microbiology ; *Genetic Variation ; Genome, Bacterial ; Genomics ; Humans ; Lactobacillus crispatus/*genetics ; *Life Style ; Phylogeny ; Vagina/*microbiology ; },
abstract = {Lactobacillus crispatus colonizes the human feces, human vagina, and the crops and ceca of chicken. To explore the genetic characteristics and evolutionary relationships of L. crispatus isolated from different niches, we selected 37 strains isolated from the human vagina (n = 17), human feces (n = 11), and chicken feces (n = 9), and used comparative genomics to explore the genetic information of L. crispatus from the feces and vagina. No significant difference was found in the three sources of genomic features such as genome size, GC content, and number of protein coding sequences (CDS). However, in a phylogenetic tree constructed based on core genes, vagina-derived L. crispatus and feces-derived strains were each clustered separately. Therefore, the niche exerted an important impact on the evolution of L. crispatus. According to gene annotation, the L. crispatus derived from the vagina possessed a high abundance of genes related to acid tolerance, redox reactions, pullulanase, and carbohydrate-binding modules (CBMs). These genes helped L. crispatus to better adapt to the acidic environment of the vagina and obtain more nutrients, maintaining its dominance in the vagina in competition with other strains. In feces-derived bacteria, more genes encoding CRISPR/Cas system, glycoside hydrolases (GHs) family, and tetracycline/lincomycin resistance genes were found to adapt to the complex intestinal environment. This study highlights the evolutionary relationship of L. crispatus strains isolated from the vagina and feces, and the adaptation of L. crispatus to the host environment.},
}
@article {pmid32230737,
year = {2020},
author = {Li, J and Fang, K and Rong, Z and Li, X and Ren, X and Ma, H and Chen, H and Li, X and Qian, P},
title = {Comparison of gE/gI- and TK/gE/gI-Gene-Deleted Pseudorabies Virus Vaccines Mediated by CRISPR/Cas9 and Cre/Lox Systems.},
journal = {Viruses},
volume = {12},
number = {4},
pages = {},
pmid = {32230737},
issn = {1999-4915},
mesh = {Amino Acid Sequence ; Animals ; Antibodies, Viral/immunology ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; Gene Targeting ; Genetic Engineering ; Genome, Viral ; HEK293 Cells ; Herpesvirus 1, Suid/*genetics/*immunology/isolation &amp; purification ; *Homologous Recombination ; Humans ; Integrases/*metabolism ; Mice ; Pseudorabies Vaccines/*genetics/*immunology ; Swine ; T-Lymphocyte Subsets/immunology/metabolism ; Virulence ; },
abstract = {Pseudorabies (PR), caused by pseudorabies virus (PRV), is an acute and febrile infectious disease in swine. To eradicate PR, a more efficacious vaccine needs to be developed. Here, the gE/gI- and TK/gE/gI-gene-deleted recombinant PRV (rGXΔgE/gI and rGXΔTK/gE/gI) are constructed through CRISPR/Cas9 and Cre/Lox systems. We found that the rGXΔTK/gE/gI was safer than rGXΔgE/gI in mice. Additionally, the effects of rGXΔgE/gI and rGXΔTK/gE/gI were further evaluated in swine. The rGXΔgE/gI and rGXΔTK/gE/gI significantly increased numbers of IFN-γ-producing CD4+ and CD8+ T-cells in swine, whereas there was no difference between rGXΔgE/gI and rGXΔTK/gE/gI. Moreover, rGXΔgE/gI and rGXΔTK/gE/gI promoted a PRV-specific humoral immune response. The PRV-specific humoral immune response induced by rGXΔgE/gI was consistent with that caused by rGXΔTK/gE/gI. After the challenge, swine vaccinated with rGXΔgE/gI and rGXΔTK/gE/gI showed no clinical signs and viral shedding. However, histopathological detection revealed that rGXΔgE/gI, not rGXΔTK/gE/gI, caused pathological lesions in brain and lung tissues. In summary, these results demonstrate that the TK/gE/gI-gene-deleted recombinant PRV was safer compared with rGXΔgE/gI in swine. The data imply that the TK/gE/gI-gene-deleted recombinant PRV may be a more efficacious vaccine candidate for the prevention of PR.},
}
@article {pmid32229273,
year = {2020},
author = {Wu, QW and Kapfhammer, JP},
title = {The CRISPR-Cas13a system interferes with Purkinje cell dendritic development.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {7},
pages = {118710},
doi = {10.1016/j.bbamcr.2020.118710},
pmid = {32229273},
issn = {1879-2596},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cerebellar Cortex/growth &amp; development/metabolism ; Dendritic Cells/cytology/*metabolism ; Mice ; Primary Cell Culture ; Purkinje Cells/cytology/*metabolism ; },
}
@article {pmid32228671,
year = {2020},
author = {Macdonald, SJ and Highfill, CA},
title = {A naturally-occurring 22-bp coding deletion in Ugt86Dd reduces nicotine resistance in Drosophila melanogaster.},
journal = {BMC research notes},
volume = {13},
number = {1},
pages = {188},
pmid = {32228671},
issn = {1756-0500},
support = {R01 ES029922/ES/NIEHS NIH HHS/United States ; R01 OD010974/OD/NIH HHS/United States ; R01-ES029922/NH/NIH HHS/United States ; R01-OD010974/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; *Drosophila melanogaster/drug effects/genetics ; Drug Resistance/*genetics ; *Gene Deletion ; Gene Editing ; Nicotine/*pharmacology ; Nicotinic Agonists/*pharmacology ; },
abstract = {OBJECTIVE: Segregating genetic variants contribute to the response to toxic, xenobiotic compounds, and identifying these causative sites can help describe the mechanisms underlying metabolism of toxic compounds. In previous work we implicated the detoxification gene Ugt86Dd in the genetic control of larval nicotine resistance in Drosophila melanogaster. Furthermore, we suggested that a naturally-occurring 22-bp deletion that leads to a stop codon in exon 2 of the gene markedly reduces resistance. Here we use homology directed CRISPR/Cas9 gene editing to specifically test this hypothesis.<br><br>RESULTS: We edited chromosome three from an inbred strain named A4 which carries the insertion allele at Ugt86Dd, successfully generated four alleles carrying the 22-bp Ugt86Dd deletion, and substituted edited chromosomes back into the A4 background. The original A4 strain, and an un-edited control strain in the same A4 background, show no significant difference in egg-to-adult or larva-to-adult viability on either control media or nicotine-supplemented media, and only slightly delayed development in nicotine media. However, strains carrying the 22-bp deletion showed reduced viability in nicotine conditions, and significantly longer development. Our data strongly suggest that the naturally-occurring 22-bp insertion/deletion event in Ugt86Dd directly impacts variation in nicotine resistance in D. melanogaster.},
}
@article {pmid32227559,
year = {2020},
author = {Lamas-Toranzo, I and Martínez-Moro, A and O Callaghan, E and Millán-Blanca, G and Sánchez, JM and Lonergan, P and Bermejo-Álvarez, P},
title = {RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.},
journal = {Molecular reproduction and development},
volume = {87},
number = {5},
pages = {542-549},
pmid = {32227559},
issn = {1098-2795},
mesh = {Animals ; Animals, Genetically Modified ; Benzamides/*pharmacology ; CRISPR-Cas Systems/*drug effects/physiology ; Cattle/*embryology ; Cells, Cultured ; DNA Breaks, Double-Stranded/drug effects ; DNA End-Joining Repair/*drug effects ; Embryo Culture Techniques ; Embryo, Mammalian ; Gene Editing/methods/veterinary ; *Gene Knock-In Techniques/methods/veterinary ; Gene Targeting/methods/veterinary ; Sulfonamides/*pharmacology ; },
abstract = {Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.},
}
@article {pmid32226015,
year = {2020},
author = {Hu, Z and Wang, S and Zhang, C and Gao, N and Li, M and Wang, D and Wang, D and Liu, D and Liu, H and Ong, SG and Wang, H and Wang, Y},
title = {A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope.},
journal = {PLoS biology},
volume = {18},
number = {3},
pages = {e3000686},
pmid = {32226015},
issn = {1545-7885},
support = {R00 HL130416/HL/NHLBI NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; DNA/chemistry/genetics/*metabolism ; Dependovirus/genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Nucleotide Motifs ; Protein Engineering ; Staphylococcus/*enzymology/genetics ; Substrate Specificity ; },
abstract = {Compact CRISPR/Cas9 systems that can be packaged into an adeno-associated virus (AAV) hold great promise for gene therapy. Unfortunately, currently available small Cas9 nucleases either display low activity or require a long protospacer adjacent motif (PAM) sequence, limiting their extensive applications. Here, we screened a panel of Cas9 nucleases and identified a small Cas9 ortholog from Staphylococcus auricularis (SauriCas9), which recognizes a simple NNGG PAM, displays high activity for genome editing, and is compact enough to be packaged into an AAV for genome editing. Moreover, the conversion of adenine and cytosine bases can be achieved by fusing SauriCas9 to the cytidine and adenine deaminase. Therefore, SauriCas9 holds great potential for both basic research and clinical applications.},
}
@article {pmid32223888,
year = {2020},
author = {Schelling, MA and Sashital, DG},
title = {An adaptable defense.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32223888},
issn = {2050-084X},
mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The response of bacteria to the threat posed by phages depends on their local environment.},
}
@article {pmid32223887,
year = {2020},
author = {Pyenson, NC and Marraffini, LA},
title = {Co-evolution within structured bacterial communities results in multiple expansion of CRISPR loci and enhanced immunity.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32223887},
issn = {2050-084X},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/*physiology ; *Biological Coevolution ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; DNA, Viral/genetics ; *Microbiota ; RNA, Guide/genetics ; Staphylococcus aureus/*genetics/virology ; },
abstract = {Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria through the insertion of a short sequence from the invader's genome, known as the 'spacer', into the CRISPR locus. Spacers are transcribed into guide RNAs that direct the Cas9 nuclease to its target on the invader. In liquid cultures, most bacteria acquire a single spacer. Multiple spacer integration is a rare event which significance for immunity is poorly understood. Here, we found that when phage infections occur on solid media, a high proportion of the surviving colonies display complex morphologies that contain cells with multiple spacers. This is the result of the viral-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages. Our results reveal the versatility of CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.},
}
@article {pmid32222157,
year = {2020},
author = {Li, J and Wang, L and Hua, X and Tang, H and Chen, R and Yang, T and Das, S and Xiao, J},
title = {CRISPR/Cas9-Mediated miR-29b Editing as a Treatment of Different Types of Muscle Atrophy in Mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {5},
pages = {1359-1372},
pmid = {32222157},
issn = {1525-0024},
support = {R01 HL122547/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiotensin II/adverse effects ; Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Immobilization/adverse effects ; Injections, Intramuscular ; Male ; Mice ; Mice, Inbred C57BL ; MicroRNAs/*administration &amp; dosage/*genetics ; Muscle Denervation/adverse effects ; Muscular Atrophy/chemically induced/pathology/*therapy ; Myoblasts, Skeletal/metabolism ; RNA, Guide/genetics ; RNA, Messenger/genetics ; Signal Transduction/genetics ; Treatment Outcome ; },
abstract = {Muscle atrophy is the loss of skeletal muscle mass and strength in response to diverse catabolic stimuli. At present, no effective treatments except exercise have been shown to reduce muscle atrophy clinically. Here, we report that CRISPR/Cas9-mediated genome editing through local injection into gastrocnemius muscles or tibialis anterior muscle efficiently targets the biogenesis processing sites in pre-miR-29b. In vivo, this CRISPR-based treatment prevented the muscle atrophy induced by angiotensin II (AngII), immobilization, and denervation via activation of the AKT-FOXO3A-mTOR signaling pathway and protected against AngII-induced myocyte apoptosis in mice, leading to significantly increased exercise capacity. Our work establishes CRISPR/Cas9-based gene targeting on miRNA as a potential durable therapy for the treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo.},
}
@article {pmid32221923,
year = {2020},
author = {Zhang, WW and Lypaczewski, P and Matlashewski, G},
title = {Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2116},
number = {},
pages = {199-224},
doi = {10.1007/978-1-0716-0294-2_14},
pmid = {32221923},
issn = {1940-6029},
support = {MOP125996//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular/methods ; Gene Editing/*methods ; Genes, Protozoan/*genetics ; Genetic Vectors/genetics ; Humans ; Leishmania/*genetics/isolation &amp; purification ; Mutation ; Parasitology/methods ; RNA, Guide/genetics ; Transfection/methods ; },
abstract = {CRISPR-Cas9 is an RNA guided endonuclease derived from the bacterium Streptococcus pyogenes. Due to its simplicity, versatility, and high efficiency, it has been widely used for genome editing in a variety of organisms including the protozoan parasite Leishmania, the causative agent of human leishmaniasis. Compared to the traditional homologous recombination gene targeting method, CRISPR-Cas9 has been shown to be a more efficient method to delete or disrupt Leishmania genes, generate point mutations, and add tags to endogenous genes. Notably, the stable CRISPR expression systems were shown to delete multicopy family Leishmania genes and genes present in multiploid chromosomes, identify essential Leishmania genes, and create specific chromosome translocations. In this chapter, we describe detailed procedures on using the stable CRISPR expression system for genome editing in Leishmania. These procedures include CRISPR targeting site selection, gRNA design, cloning single and double gRNA coding sequences into the Leishmania CRISPR vector pLdCN, oligonucleotide donor and drug resistance selection donor design, Leishmania cell transfection, screening, and isolation of CRISPR-edited mutants. As the principles of gene editing are generally similar, many of these procedures could also apply to the transient Leishmania CRISPR systems described by other labs.},
}
@article {pmid32221922,
year = {2020},
author = {Lander, N and Chiurillo, MA and Docampo, R},
title = {CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2116},
number = {},
pages = {177-197},
doi = {10.1007/978-1-0716-0294-2_13},
pmid = {32221922},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Calcium Channels/genetics/metabolism ; Calcium Signaling/*genetics ; Chagas Disease/parasitology ; Energy Metabolism/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genes, Protozoan/*genetics ; Genetic Vectors/genetics ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Life Cycle Stages ; Parasitology/methods ; Phosphoprotein Phosphatases/genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; RNA, Guide/genetics ; Trypanosoma cruzi/*genetics/metabolism ; },
abstract = {Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca[2+] transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca[2+] increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.},
}
@article {pmid32221665,
year = {2020},
author = {Zhi, J and Liu, X and Li, D and Huang, Y and Yan, S and Cao, B and Qiu, Z},
title = {CRISPR/Cas9-mediated SlAN2 mutants reveal various regulatory models of anthocyanin biosynthesis in tomato plant.},
journal = {Plant cell reports},
volume = {39},
number = {6},
pages = {799-809},
doi = {10.1007/s00299-020-02531-1},
pmid = {32221665},
issn = {1432-203X},
mesh = {Anthocyanins/*biosynthesis/*genetics ; *CRISPR-Cas Systems ; Cotyledon/genetics/metabolism ; Fruit/*genetics/*metabolism ; Gene Expression Regulation, Plant ; Hypocotyl/genetics/metabolism ; Lycopersicon esculentum/*genetics/metabolism ; Mutation ; Phenotype ; Plant Development ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Sequence Alignment ; Sequence Analysis, DNA ; Transcription Factors/*genetics ; Transcriptome ; Transformation, Genetic ; },
abstract = {Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar 'Indigo Rose.' Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar 'Indigo Rose' using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. 'Indigo Rose,' while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. 'Indigo Rose.' Our findings further our understanding of anthocyanin production in tomato and other plant species.},
}
@article {pmid32221408,
year = {2020},
author = {Wu, R and Chai, B and Cole, JR and Gunturu, SK and Guo, X and Tian, R and Gu, JD and Zhou, J and Tiedje, JM},
title = {Targeted assemblies of cas1 suggest CRISPR-Cas's response to soil warming.},
journal = {The ISME journal},
volume = {14},
number = {7},
pages = {1651-1662},
pmid = {32221408},
issn = {1751-7370},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome ; Oklahoma ; *Soil ; },
abstract = {There is an increasing interest in the clustered regularly interspaced short palindromic repeats CRISPR-associated protein (CRISPR-Cas) system to reveal potential virus-host dynamics. The universal and most conserved Cas protein, cas1 is an ideal marker to elucidate CRISPR-Cas ecology. We constructed eight Hidden Markov Models (HMMs) and assembled cas1 directly from metagenomes by a targeted-gene assembler, Xander, to improve detection capacity and resolve the diverse CRISPR-Cas systems. The eight HMMs were first validated by recovering all 17 cas1 subtypes from the simulated metagenome generated from 91 prokaryotic genomes across 11 phyla. We challenged the targeted method with 48 metagenomes from a tallgrass prairie in Central Oklahoma recovering 3394 cas1. Among those, 88 were near full length, 5 times more than in de-novo assemblies from the Oklahoma metagenomes. To validate the host assignment by cas1, the targeted-assembled cas1 was mapped to the de-novo assembled contigs. All the phylum assignments of those mapped contigs were assigned independent of CRISPR-Cas genes on the same contigs and consistent with the host taxonomies predicted by the mapped cas1. We then investigated whether 8 years of soil warming altered cas1 prevalence within the communities. A shift in microbial abundances was observed during the year with the biggest temperature differential (mean 4.16 °C above ambient). cas1 prevalence increased and even in the phyla with decreased microbial abundances over the next 3 years, suggesting increasing virus-host interactions in response to soil warming. This targeted method provides an alternative means to effectively mine cas1 from metagenomes and uncover the host communities.},
}
@article {pmid32221291,
year = {2020},
author = {Garcia-Doval, C and Schwede, F and Berk, C and Rostøl, JT and Niewoehner, O and Tejero, O and Hall, J and Marraffini, LA and Jinek, M},
title = {Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1596},
pmid = {32221291},
issn = {2041-1723},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine Nucleotides/*chemistry/*metabolism ; Bacterial Proteins/chemistry/metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; Endonucleases/*chemistry/*metabolism ; Enzyme Activation ; Models, Molecular ; Oligoribonucleotides/*chemistry/*metabolism ; Protein Domains ; RNA Stability ; },
abstract = {Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.},
}
@article {pmid32220383,
year = {2020},
author = {Zuo, W and Depotter, JR and Doehlemann, G},
title = {Cas9HF1 enhanced specificity in Ustilago maydis.},
journal = {Fungal biology},
volume = {124},
number = {3-4},
pages = {228-234},
doi = {10.1016/j.funbio.2020.02.006},
pmid = {32220383},
issn = {1878-6146},
mesh = {Basidiomycota/*genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Fungal ; Sensitivity and Specificity ; Whole Genome Sequencing ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.},
}
@article {pmid32220334,
year = {2020},
author = {Armenta, DA and Dixon, SJ},
title = {Investigating Nonapoptotic Cell Death Using Chemical Biology Approaches.},
journal = {Cell chemical biology},
volume = {27},
number = {4},
pages = {376-386},
pmid = {32220334},
issn = {2451-9448},
support = {R01 GM122923/GM/NIGMS NIH HHS/United States ; R01 NS121319/NS/NINDS NIH HHS/United States ; T32 GM007276/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Transport System y+/antagonists &amp; inhibitors/metabolism ; CRISPR-Cas Systems/genetics ; Coenzyme A Ligases/genetics/metabolism ; *Ferroptosis/drug effects ; Humans ; Iron Regulatory Protein 2/antagonists &amp; inhibitors/genetics/metabolism ; Molecular Probes/chemistry/metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase/antagonists &amp; inhibitors/genetics/metabolism ; RNA, Small Interfering/metabolism ; Small Molecule Libraries/*chemistry/metabolism/pharmacology ; },
abstract = {Nonapoptotic cell death is important for human health and disease. Here, we show how various tools and techniques drawn from the chemical biology field have played a central role in the discovery and characterization of nonapoptotic cell death pathways. Focusing on the example of ferroptosis, we describe how phenotypic screening, chemoproteomics, chemical genetic analysis, and other methods enabled the elucidation of this pathway. Synthetic small-molecule inducers and inhibitors of ferroptosis identified in early studies have now been leveraged to identify an even broader set of compounds that affect ferroptosis and to validate new chemical methods and probes for various ferroptosis-associated processes. A number of limitations associated with specific chemical biology tools or techniques have also emerged and must be carefully considered. Nevertheless, the study of ferroptosis provides a roadmap for how chemical biology methods may be used to discover and characterize nonapoptotic cell death mechanisms.},
}
@article {pmid32219745,
year = {2020},
author = {Lang, L and Teng, Y},
title = {Using Genome-Editing Tools to Develop a Novel In Situ Coincidence Reporter Assay for Screening ATAD3A Transcriptional Inhibitors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2138},
number = {},
pages = {159-166},
pmid = {32219745},
issn = {1940-6029},
support = {R01 DE028351/DE/NIDCR NIH HHS/United States ; R03 DE028387/DE/NIDCR NIH HHS/United States ; },
mesh = {ATPases Associated with Diverse Cellular Activities/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Epigenesis, Genetic/genetics ; Gene Editing/methods ; Gene Expression/genetics ; Genes, Reporter/*genetics ; Genetic Markers/genetics ; Humans ; Membrane Proteins/*genetics ; Mitochondrial Proteins/*genetics ; Promoter Regions, Genetic/genetics ; Transcription, Genetic/*genetics ; Transgenes/genetics ; },
abstract = {Transgene-based reporter gene assays have been used for discovery of inhibitors targeting vital gene transcription. In traditional assays, the reporter gene is commonly fused with a cloned promoter and integrated into a random genomic location. This has been widely applied but significantly dampened by disadvantages, including incomplete cis-acting elements, the influence of foreign epigenetic environments, and generation of false hits that disrupt the luciferase reporter activity. Therefore, there is a need to develop novel strategies for developing in situ reporter assays closely mimicking endogenous gene expression without disrupting its function. By employing the CRISPR-Cas9 system, we developed an effective in situ coincidence reporter system with a selection marker in the endogenous locus of ATAD3A, which provides a means of screening for transcription-targeted lead compounds with high confidence.},
}
@article {pmid32219408,
year = {2020},
author = {George, AF and Jang, KS and Nyegaard, M and Neidleman, J and Spitzer, TL and Xie, G and Chen, JC and Herzig, E and Laustsen, A and Marques de Menezes, EG and Houshdaran, S and Pilcher, CD and Norris, PJ and Jakobsen, MR and Greene, WC and Giudice, LC and Roan, NR},
title = {Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.},
journal = {Human reproduction (Oxford, England)},
volume = {35},
number = {3},
pages = {617-640},
pmid = {32219408},
issn = {1460-2350},
support = {T32 AI007334/AI/NIAID NIH HHS/United States ; R21 AI116252/AI/NIAID NIH HHS/United States ; R01 AI127219/AI/NIAID NIH HHS/United States ; R21 AI122821/AI/NIAID NIH HHS/United States ; P50 HD055764/HD/NICHD NIH HHS/United States ; },
mesh = {Cross-Sectional Studies ; *Decidua/physiology ; Endometriosis ; Endometrium/cytology ; Female ; Fibroblasts/*cytology ; Humans ; Interleukin-11/genetics/*physiology ; Polycystic Ovary Syndrome ; *Semen ; },
abstract = {STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)?<br><br>SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization.<br><br>WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis.<br><br>STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n&#xa0;=&#x2009;9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization.<br><br>eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs.<br><br>SP promoted decidualization both in the absence and presence of steroid hormones (P&#xa0;<&#x2009;0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization.<br><br>LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640.<br><br>This study is limited to in vitro analyses.<br><br>Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation.<br><br>This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.},
}
@article {pmid32218601,
year = {2020},
author = {Huang, JK and Samassekou, K and Alhmadi, HB and VanDerway, DR and Diaz, JD and Seiver, JA and McClenahan, SW and Holt, SM and Wen, L},
title = {Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology.},
journal = {PloS one},
volume = {15},
number = {3},
pages = {e0230915},
pmid = {32218601},
issn = {1932-6203},
mesh = {Alcohol Oxidoreductases/deficiency/*genetics/*metabolism ; Biotransformation ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; *Gene Knockout Techniques ; Mutation ; Nocardia/*enzymology/genetics ; },
abstract = {Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.},
}
@article {pmid32218510,
year = {2020},
author = {Mahendra, C and Christie, KA and Osuna, BA and Pinilla-Redondo, R and Kleinstiver, BP and Bondy-Denomy, J},
title = {Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.},
journal = {Nature microbiology},
volume = {5},
number = {4},
pages = {620-629},
pmid = {32218510},
issn = {2058-5276},
support = {R01GM127489//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/International ; DP5 OD021344/OD/NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; R00-CA218870//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/International ; R01 GM127489/GM/NIGMS NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Conjugation, Genetic ; DNA/antagonists &amp; inhibitors/genetics/metabolism ; Enterococcus/genetics/virology ; *Gene Transfer, Horizontal ; HEK293 Cells ; Humans ; Listeria/genetics/virology ; Plasmids/chemistry/*metabolism ; Protein Binding ; RNA, Guide/*antagonists &amp; inhibitors/genetics/metabolism ; Staphylococcus/genetics/virology ; Streptococcus/genetics/virology ; },
abstract = {CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.},
}
@article {pmid32217751,
year = {2020},
author = {Walton, RT and Christie, KA and Whittaker, MN and Kleinstiver, BP},
title = {Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.},
journal = {Science (New York, N.Y.)},
volume = {368},
number = {6488},
pages = {290-296},
pmid = {32217751},
issn = {1095-9203},
support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting/*methods ; *Genetic Predisposition to Disease ; HEK293 Cells ; Humans ; Mutagenesis ; Protein Domains ; Substrate Specificity ; },
abstract = {Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.},
}
@article {pmid32217638,
year = {2020},
author = {Yan, HHN and Siu, HC and Ho, SL and Yue, SSK and Gao, Y and Tsui, WY and Chan, D and Chan, AS and Wong, JWH and Man, AHY and Lee, BCH and Chan, ASY and Chan, AKW and Hui, HS and Cheung, AKL and Law, WL and Lo, OSH and Yuen, ST and Clevers, H and Leung, SY},
title = {Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles.},
journal = {Gut},
volume = {69},
number = {12},
pages = {2165-2179},
doi = {10.1136/gutjnl-2019-320019},
pmid = {32217638},
issn = {1468-3288},
mesh = {Adenomatous Polyposis Coli Protein/genetics ; Bone Morphogenetic Protein 2/genetics ; Bone Morphogenetic Protein Receptors, Type I/genetics ; CRISPR-Cas Systems ; Cell Adhesion Molecules/genetics ; Colorectal Neoplasms/*genetics ; Gene Expression Profiling ; Gene Fusion ; *Genetic Profile ; Humans ; Models, Genetic ; Mutation ; Organoids/*pathology ; Receptor Protein-Tyrosine Kinases/genetics ; Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics ; Smad4 Protein/genetics ; Thrombospondins/genetics ; Tissue Banks ; Ubiquitin-Protein Ligases/genetics ; Up-Regulation ; Whole Exome Sequencing ; },
abstract = {OBJECTIVE: Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs.<br><br>DESIGN: We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed.<br><br>RESULTS: We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time.<br><br>CONCLUSIONS: These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.},
}
@article {pmid32217510,
year = {2020},
author = {Tang, LC and Gu, F},
title = {Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM.},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {3},
pages = {236-249},
doi = {10.16288/j.yczz.19-297},
pmid = {32217510},
issn = {0253-9772},
mesh = {*Amino Acid Motifs ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; RNA, Guide/*genetics ; },
abstract = {The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.},
}
@article {pmid32216029,
year = {2020},
author = {Karunarathna, NL and Wang, H and Harloff, HJ and Jiang, L and Jung, C},
title = {Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes.},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2251-2266},
pmid = {32216029},
issn = {1467-7652},
mesh = {*Brassica napus/genetics ; Fatty Acids ; Humans ; *Plant Oils ; Polyploidy ; Seeds/genetics ; },
abstract = {Plant-based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a worldwide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA-seq and quantitative PCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigour and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.},
}
@article {pmid32215707,
year = {2020},
author = {Mo, XH and Zhang, H and Wang, TM and Zhang, C and Zhang, C and Xing, XH and Yang, S},
title = {Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {10},
pages = {4515-4532},
doi = {10.1007/s00253-020-10543-w},
pmid = {32215707},
issn = {1432-0614},
mesh = {Biosynthetic Pathways/*genetics ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Carotenoids/*metabolism ; Gene Knockdown Techniques ; Metabolic Networks and Pathways ; Methylobacterium extorquens/*enzymology/*genetics ; Oxidoreductases/*genetics/metabolism ; Phylogeny ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Streptococcus pyogenes/enzymology/genetics ; },
abstract = {The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.},
}
@article {pmid32215703,
year = {2020},
author = {Wu, D and Xie, W and Li, X and Cai, G and Lu, J and Xie, G},
title = {Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {10},
pages = {4435-4444},
doi = {10.1007/s00253-020-10549-4},
pmid = {32215703},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; *Fermentation ; Genome, Fungal ; Membrane Transport Proteins/genetics/metabolism ; Metabolic Engineering ; Oryza/*microbiology ; Recombination, Genetic ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Urethane/*metabolism ; Wine/*analysis ; },
abstract = {Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85[DUR1,2/DUR3]-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85[DUR1,2/DUR3]-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.},
}
@article {pmid32214056,
year = {2020},
author = {Libetti, D and Bernardini, A and Sertic, S and Messina, G and Dolfini, D and Mantovani, R},
title = {The Switch from NF-YAl to NF-YAs Isoform Impairs Myotubes Formation.},
journal = {Cells},
volume = {9},
number = {3},
pages = {},
pmid = {32214056},
issn = {2073-4409},
mesh = {Animals ; Base Sequence ; CCAAT-Binding Factor/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Fusion ; Cell Line ; Clone Cells ; Exons/genetics ; Gene Expression Regulation ; Mice ; Muscle Fibers, Skeletal/cytology/*metabolism ; MyoD Protein/metabolism ; Myogenin/metabolism ; Protein Isoforms/genetics/metabolism ; Transcription Factors/metabolism ; },
abstract = {NF-YA, the regulatory subunit of the trimeric transcription factor (TF) NF-Y, is regulated by alternative splicing (AS) generating two major isoforms, "long" (NF-YAl) and "short" (NF-YAs). Muscle cells express NF-YAl. We ablated exon 3 in mouse C2C12 cells by a four-guide CRISPR/Cas9n strategy, obtaining clones expressing exclusively NF-YAs (C2-YAl-KO). C2-YAl-KO cells grow normally, but are unable to differentiate. Myogenin and-to a lesser extent, MyoD- levels are substantially lower in C2-YAl-KO, before and after differentiation. Expression of the fusogenic Myomaker and Myomixer genes, crucial for the early phases of the process, is not induced. Myomaker and Myomixer promoters are bound by MyoD and Myogenin, and Myogenin overexpression induces their expression in C2-YAl-KO. NF-Y inactivation reduces MyoD and Myogenin, but not directly: the Myogenin promoter is CCAAT-less, and the canonical CCAAT of the MyoD promoter is not bound by NF-Y in vivo. We propose that NF-YAl, but not NF-YAs, maintains muscle commitment by indirectly regulating Myogenin and MyoD expression in C2C12 cells. These experiments are the first genetic evidence that the two NF-YA isoforms have functionally distinct roles.},
}
@article {pmid32213923,
year = {2020},
author = {Lim, KRQ and Nguyen, Q and Dzierlega, K and Huang, Y and Yokota, T},
title = {CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32213923},
issn = {2073-4425},
support = {FDN 143251//CIHR/Canada ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Editing/methods ; Haplorhini ; Murinae ; Muscular Dystrophy, Duchenne/*genetics/pathology ; Rabbits ; Swine ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.},
}
@article {pmid32213627,
year = {2020},
author = {Lek, A and Zhang, Y and Woodman, KG and Huang, S and DeSimone, AM and Cohen, J and Ho, V and Conner, J and Mead, L and Kodani, A and Pakula, A and Sanjana, N and King, OD and Jones, PL and Wagner, KR and Lek, M and Kunkel, LM},
title = {Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy.},
journal = {Science translational medicine},
volume = {12},
number = {536},
pages = {},
pmid = {32213627},
issn = {1946-6242},
support = {P50 HD060848/HD/NICHD NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; DP2 HG010099/HG/NHGRI NIH HHS/United States ; R00 HG008171/HG/NHGRI NIH HHS/United States ; U54 HD090255/HD/NICHD NIH HHS/United States ; UL1 TR001863/TR/NCATS NIH HHS/United States ; R01 AR062587/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Homeodomain Proteins/genetics/metabolism ; Humans ; *Muscular Dystrophy, Facioscapulohumeral/genetics/therapy ; Zebrafish/genetics/metabolism ; },
abstract = {The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.},
}
@article {pmid32213107,
year = {2020},
author = {Qiao, C and Liu, W and Jiang, H and He, M and Yang, Q and Xing, Y},
title = {Integrated analysis of miRNA and mRNA expression profiles in p53-edited PFF cells.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {19},
number = {8},
pages = {949-959},
pmid = {32213107},
issn = {1551-4005},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Movement/genetics ; Cell Proliferation/genetics ; Fetus/*cytology ; Fibroblasts/*metabolism ; Gene Editing/*methods ; Gene Expression Profiling ; Gene Knockdown Techniques ; Gene Regulatory Networks ; High-Throughput Nucleotide Sequencing/methods ; MicroRNAs/*genetics ; RNA, Messenger/*genetics ; Sequence Analysis, RNA ; Signal Transduction/genetics ; Swine/embryology ; *Transcriptome ; Tumor Suppressor Protein p53/*genetics/metabolism ; },
abstract = {p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations.Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.},
}
@article {pmid32212662,
year = {2020},
author = {Thonnekottu, D and Chatterjee, D},
title = {CRISPR-Cas9 Genome Interrogation: A Facilitated Subdiffusive Target Search Strategy.},
journal = {The journal of physical chemistry. B},
volume = {124},
number = {16},
pages = {3271-3282},
doi = {10.1021/acs.jpcb.0c00086},
pmid = {32212662},
issn = {1520-5207},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Engineering ; Genome ; },
abstract = {The functional application of RNA-guided CRISPR-associated Cas9 protein, a bacterial immune system-based protein complex, via which in vivo, highly specific, and well-regulated, gene-editing processes are being monitored at an unprecedented level, has led to remarkable progress in genetic engineering and technology. The complicated in vivo process of genome interrogation followed by gene editing by the Cas9 complex was recently reported by Knight et al. (Science, 2015, 350, 823-826) using an elegant single-particle tracking method, aided by the two-photon fluorescence correlation spectroscopic technique. In contrast to the usually observed fast target-searching and protein-binding events in biophysical systems, an interesting slow genome-interrogation process by the RNA-guided CRISPR-Cas9 system through a crowded chromatin environment of a mammalian cell has been revealed in Knight et al.'s study. Motivated by this experiment, in this paper, we provide a generalized theoretical framework to capture this particular target-searching mechanism of the CRISPR-Cas9 protein complex. We show that an analysis on the basis of 3D subdiffusion under a cylindrical volume, created by several nonspecific off-target interactions from the DNA strands, can capture the essential details of the process. Moreover, on the basis of this model, we quantify the dynamics of this process and estimate the survival probability, first passage time, and the intensity correlation function of the tagged proteins of the experiment. The results from our theoretical predictions are found to be consistent with the experimental observations, and hence, seem to provide a plausible microscopic picture of the process.},
}
@article {pmid32210344,
year = {2020},
author = {Rathore, A and Iketani, S and Wang, P and Jia, M and Sahi, V and Ho, DD},
title = {CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {5350},
pmid = {32210344},
issn = {2045-2322},
support = {R01 AI134328/AI/NIAID NIH HHS/United States ; T32 AI106711/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Polymerase III/genetics ; DNA-Binding Proteins/genetics ; DNA-Directed RNA Polymerases/genetics ; Deubiquitinating Enzyme CYLD/genetics ; Deubiquitinating Enzymes/antagonists &amp; inhibitors/*genetics ; Endopeptidases/genetics ; Enzyme Inhibitors/pharmacology ; Gene Knockout Techniques/*methods ; HIV-1/genetics/*physiology ; Host-Pathogen Interactions/genetics/physiology ; Humans ; Jurkat Cells ; Nucleoproteins/genetics ; Proteasome Endopeptidase Complex/genetics ; RNA-Binding Proteins/genetics ; Transcription Factors/genetics ; Ubiquitin Thiolesterase/genetics ; *Virus Latency/drug effects ; },
abstract = {The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.},
}
@article {pmid32209973,
year = {2020},
author = {Meyer, M and Kuffner, K and Winter, J and Neumann, ID and Wetzel, CH and Jurek, B},
title = {Myocyte Enhancer Factor 2A (MEF2A) Defines Oxytocin-Induced Morphological Effects and Regulates Mitochondrial Function in Neurons.},
journal = {International journal of molecular sciences},
volume = {21},
number = {6},
pages = {},
pmid = {32209973},
issn = {1422-0067},
mesh = {Animals ; Cell Line ; Cell Respiration ; Gene Expression Regulation ; Gene Knockdown Techniques ; MEF2 Transcription Factors/genetics/metabolism ; Mitochondria/drug effects/*genetics/*metabolism ; Neurons/drug effects/*metabolism ; Oxytocin/*metabolism/pharmacology ; Rats ; },
abstract = {The neuropeptide oxytocin (OT) is a well-described modulator of socio-emotional traits, such as anxiety, stress, social behavior, and pair bonding. However, when dysregulated, it is associated with adverse psychiatric traits, such as various aspects of autism spectrum disorder (ASD). In this study, we identify the transcription factor myocyte enhancer factor 2A (MEF2A) as the common link between OT and cellular changes symptomatic for ASD, encompassing neuronal morphology, connectivity, and mitochondrial function. We provide evidence for MEF2A as the decisive factor defining the cellular response to OT: while OT induces neurite retraction in MEF2A expressing neurons, OT causes neurite outgrowth in absence of MEF2A. A CRISPR-Cas-mediated knockout of MEF2A and retransfection of an active version or permanently inactive mutant, respectively, validated our findings. We also identified the phosphatase calcineurin as the main upstream regulator of OT-induced MEF2A signaling. Further, MEF2A signaling dampens mitochondrial functioning in neurons, as MEF2A knockout cells show increased maximal cellular respiration, spare respiratory capacity, and total cellular ATP. In summary, we reveal a central role for OT-induced MEF2A activity as major regulator of cellular morphology as well as neuronal connectivity and mitochondrial functioning, with broad implications for a potential treatment of disorders based on morphological alterations or mitochondrial dysfunction.},
}
@article {pmid32209669,
year = {2020},
author = {Deem, MW},
title = {CRISPR recognizes as many phage types as possible without overwhelming the Cas machinery.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {14},
pages = {7550-7552},
pmid = {32209669},
issn = {1091-6490},
mesh = {Bacteria ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
}
@article {pmid32209486,
year = {2020},
author = {Hunker, AC and Soden, ME and Krayushkina, D and Heymann, G and Awatramani, R and Zweifel, LS},
title = {Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System.},
journal = {Cell reports},
volume = {30},
number = {12},
pages = {4303-4316.e6},
pmid = {32209486},
issn = {2211-1247},
support = {R01 MH104450/MH/NIMH NIH HHS/United States ; P30 DA048736/DA/NIDA NIH HHS/United States ; T32 AA007455/AA/NIAAA NIH HHS/United States ; R01 MH110556/MH/NIMH NIH HHS/United States ; F31 MH116549/MH/NIMH NIH HHS/United States ; T32 GM007270/GM/NIGMS NIH HHS/United States ; R01 DA044315/DA/NIDA NIH HHS/United States ; },
mesh = {Aging/*genetics ; Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Nucleotidyltransferases/metabolism ; Dependovirus/*genetics ; GABA Plasma Membrane Transport Proteins/metabolism ; Genetic Vectors/*metabolism ; Glutamic Acid/metabolism ; Mice, Inbred C57BL ; Mutagenesis/*genetics ; Mutation/genetics ; Nervous System/*metabolism ; Optogenetics ; Phenotype ; },
abstract = {Mice engineered for conditional, cell type-specific gene inactivation have dominated the field of mouse genetics because of the high efficiency of Cre-loxP-mediated recombination. Recent advances in CRISPR/Cas9 technologies have provided alternatives for rapid gene mutagenesis for loss-of-function (LOF) analysis. Whether these strategies can be streamlined for rapid genetic analysis with the efficiencies comparable with those of conventional genetic approaches has yet to be established. We show that a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA (sgRNA) are as efficient as conventional conditional gene knockout and can be adapted for use in either Cre- or Flp-driver mouse lines. The efficacy of this approach is demonstrated for the analysis of GABAergic, glutamatergic, and monoaminergic neurotransmission. Using this strategy, we reveal insight into the role of GABAergic regulation of midbrain GABA-producing neurons in psychomotor activation.},
}
@article {pmid32208677,
year = {2020},
author = {Ramesh, A and Ong, T and Garcia, JA and Adams, J and Wheeldon, I},
title = {Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica.},
journal = {ACS synthetic biology},
volume = {9},
number = {4},
pages = {967-971},
doi = {10.1021/acssynbio.9b00498},
pmid = {32208677},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal/*genetics ; Genome, Fungal/genetics ; RNA, Guide/*genetics ; Yarrowia/*genetics ; },
abstract = {Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.},
}
@article {pmid32208444,
year = {2020},
author = {Buglo, E and Sarmiento, E and Martuscelli, NB and Sant, DW and Danzi, MC and Abrams, AJ and Dallman, JE and Züchner, S},
title = {Genetic compensation in a stable slc25a46 mutant zebrafish: A case for using F0 CRISPR mutagenesis to study phenotypes caused by inherited disease.},
journal = {PloS one},
volume = {15},
number = {3},
pages = {e0230566},
pmid = {32208444},
issn = {1932-6203},
support = {R01 NS072248/NS/NINDS NIH HHS/United States ; R01 NS075764/NS/NINDS NIH HHS/United States ; R21 HD093021/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cerebellar Ataxia/genetics ; Disease Models, Animal ; Female ; Gene Targeting ; Male ; Mitochondrial Membrane Transport Proteins/*genetics ; Mutagenesis ; Mutation ; Optic Atrophy/genetics ; Peripheral Nervous System Diseases/genetics ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {A phenomenon of genetic compensation is commonly observed when an organism with a disease-bearing mutation shows incomplete penetrance of the disease phenotype. Such incomplete phenotypic penetrance, or genetic compensation, is more commonly found in stable knockout models, rather than transient knockdown models. As such, these incidents present a challenge for the disease modeling field, although a deeper understanding of genetic compensation may also hold the key for novel therapeutic interventions. In our study we created a knockout model of slc25a46 gene, which is a recently discovered important player in mitochondrial dynamics, and deleterious mutations in which are known to cause peripheral neuropathy, optic atrophy and cerebellar ataxia. We report a case of genetic compensation in a stable slc25a46 homozygous zebrafish mutant (hereafter referred as "mutant"), in contrast to a penetrant disease phenotype in the first generation (F0) slc25a46 mosaic mutant (hereafter referred as "crispant"), generated with CRISPR/Cas-9 technology. We show that the crispant phenotype is specific and rescuable. By performing mRNA sequencing, we define significant changes in slc25a46 mutant's gene expression profile, which are largely absent in crispants. We find that among the most significantly altered mRNAs, anxa6 gene stands out as a functionally relevant player in mitochondrial dynamics. We also find that our genetic compensation case does not arise from mechanisms driven by mutant mRNA decay. Our study contributes to the growing evidence of the genetic compensation phenomenon and presents novel insights about Slc25a46 function. Furthermore, our study provides the evidence for the efficiency of F0 CRISPR screens for disease candidate genes, which may be used to advance the field of functional genetics.},
}
@article {pmid32207772,
year = {2020},
author = {Auckland, P and Roscioli, E and Coker, HLE and McAinsh, AD},
title = {CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes.},
journal = {The Journal of cell biology},
volume = {219},
number = {5},
pages = {},
pmid = {32207772},
issn = {1540-8140},
support = {208384/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 106151/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromosomal Proteins, Non-Histone/*genetics ; Chromosome Segregation/*genetics ; Chromosomes/genetics ; Dyneins/genetics ; HeLa Cells ; Humans ; *Kinetochores ; Microfilament Proteins/*genetics ; Microtubule-Associated Proteins/*genetics ; Microtubules/genetics ; Mutant Proteins/genetics ; Protein Binding/genetics ; Spindle Apparatus/genetics ; },
abstract = {Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.},
}
@article {pmid32207531,
year = {2020},
author = {Hirakawa, MP and Krishnakumar, R and Timlin, JA and Carney, JP and Butler, KS},
title = {Gene editing and CRISPR in the clinic: current and future perspectives.},
journal = {Bioscience reports},
volume = {40},
number = {4},
pages = {},
pmid = {32207531},
issn = {1573-4935},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Culture Techniques ; Clinical Trials as Topic ; Drug Delivery Systems/methods/trends ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; Genetic Vectors/administration &amp; dosage/genetics ; Humans ; Immunotherapy, Adoptive/*methods/trends ; Mice ; Models, Animal ; Nanoparticles ; Receptors, Chimeric Antigen/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Translational Research, Biomedical/*methods/trends ; Zinc Finger Nucleases/genetics ; },
abstract = {Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.},
}
@article {pmid32206715,
year = {2020},
author = {Sun, H and Fu, S and Cui, S and Yin, X and Sun, X and Qi, X and Cui, K and Wang, J and Ma, L and Liu, FY and Liao, FF and Wang, XH and Yi, M and Wan, Y},
title = {Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain.},
journal = {Science advances},
volume = {6},
number = {12},
pages = {eaay6687},
pmid = {32206715},
issn = {2375-2548},
mesh = {Age Factors ; Animals ; Biomarkers ; Brain/*metabolism ; *CRISPR-Cas Systems ; Dependovirus/genetics ; *Gene Editing ; Gene Knockdown Techniques ; Genetic Loci ; Genetic Vectors/genetics ; Male ; Memory ; Neurons/metabolism ; RNA, Guide ; Rats ; },
abstract = {A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system-based technique, and its combined application with anterograde/retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection- and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down the cbp (CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection- and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection- and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies.},
}
@article {pmid32205365,
year = {2020},
author = {Wang, M and Zhang, G and Zhang, Y and Cui, X and Wang, S and Gao, S and Wang, Y and Liu, Y and Bae, JH and Yang, WH and Qi, LS and Wang, L and Liu, R},
title = {Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through Integrin-AKT Signaling Pathway in Lung Cancer.},
journal = {Molecular cancer research : MCR},
volume = {18},
number = {7},
pages = {943-954},
doi = {10.1158/1541-7786.MCR-19-1033},
pmid = {32205365},
issn = {1557-3125},
support = {P30 CA013148/CA/NCI NIH HHS/United States ; },
mesh = {A549 Cells ; Adenocarcinoma of Lung/genetics/metabolism/*pathology ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; *Down-Regulation ; Epithelial-Mesenchymal Transition ; Female ; Fibrinogen/*genetics/*metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Integrins/*metabolism ; Lung Neoplasms/genetics/metabolism/*pathology ; Male ; Mice ; Neoplasm Metastasis ; Neoplasm Transplantation ; Proto-Oncogene Proteins c-akt/metabolism ; Signal Transduction ; },
abstract = {Fibrinogen is an extracellular matrix protein composed of three polypeptide chains with fibrinogen alpha (FGA), beta (FGB) and gamma (FGG). Although fibrinogen and its related fragments are involved in tumor angiogenesis and metastasis, their functional roles are incompatible. A recent genome-scale screening reveals that loss of FGA affects the acceleration of tumor growth and metastasis of lung cancer, but the mechanism remains elusive. We used CRISPR/Cas9 genome editing to knockout (KO) FGA in human lung adenocarcinoma (LUAD) cell lines A549 and H1299. By colony formation, transwell migration and matrix invasion assays, FGA KO increased cell proliferation, migration, and invasion but decreased the expressions of epithelial-mesenchymal transition marker E-cadherin and cytokeratin 5/8 in A549 and H1299 cells. However, administration of FGA inhibited cell proliferation and migration but induced apoptosis in A549 cells. Of note, FGA KO cells indirectly cocultured by transwells with FGA wild-type cells increased FGA in the culture medium, leading to decreased migration of FGA KO cells. Furthermore, our functional analysis identified a direct interaction of FGA with integrin α5 as well as FGA-integrin signaling that regulated the AKT-mTOR signaling pathway in A549 cells. In addition, we validated that FGA KO increased tumor growth and metastasis through activation of AKT signaling in an A549 xenograft model. IMPLICATIONS: These findings demonstrate that that loss of FGA facilities tumor growth and metastasis through the integrin-AKT signaling pathway in lung cancer.},
}
@article {pmid32203915,
year = {2020},
author = {Fatima, A and Schuster, J and Akram, T and Sobol, M and Hoeber, J and Dahl, N},
title = {Generation of a human Neurochondrin deficient iPSC line KICRi002-A-3 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {44},
number = {},
pages = {101758},
doi = {10.1016/j.scr.2020.101758},
pmid = {32203915},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Induced Pluripotent Stem Cells ; Mice ; Nerve Tissue Proteins ; },
abstract = {The role of Neurochondrin (NCDN) in humans is not well understood. Mice with a conditional Ncdn knock-out show epileptic seizures, depressive-like behaviours and impaired spatial learning. Using CRISPR/Cas9, we generated a Neurochondrin deficient human iPSC line KICRi002-A-3 carrying a homozygous 752 bp deletion / 2 bp insertion in the NCDN gene. The iPSC line maintained a normal 46,XY karyotype, expressed pluripotency markers and exhibited capability to differentiate into the three germ layers in vitro. Off-target editing was excluded and Neurochondrin expression was not detectable. The iPSC line offers a valuable resource to study the role of Neurochondrin during human neurogenesis.},
}
@article {pmid32203679,
year = {2020},
author = {Mo, F and Heslop, HE and Mamonkin, M},
title = {CRISPR-Edited Immune Effectors: The End of the Beginning.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {4},
pages = {995-996},
pmid = {32203679},
issn = {1525-0024},
support = {P50 CA126752/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Neoplasms ; T-Lymphocytes ; },
}
@article {pmid32203462,
year = {2020},
author = {Lin, KH and Rutter, JC and Xie, A and Pardieu, B and Winn, ET and Bello, RD and Forget, A and Itzykson, R and Ahn, YR and Dai, Z and Sobhan, RT and Anderson, GR and Singleton, KR and Decker, AE and Winter, PS and Locasale, JW and Crawford, L and Puissant, A and Wood, KC},
title = {Using antagonistic pleiotropy to design a chemotherapy-induced evolutionary trap to target drug resistance in cancer.},
journal = {Nature genetics},
volume = {52},
number = {4},
pages = {408-417},
pmid = {32203462},
issn = {1546-1718},
support = {F30 CA206348/CA/NCI NIH HHS/United States ; F31 CA195967/CA/NCI NIH HHS/United States ; R01 CA207083/CA/NCI NIH HHS/United States ; T32 GM007171/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptation, Physiological/genetics ; Animals ; Biological Evolution ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Environment ; Genetic Fitness/genetics ; Genetic Pleiotropy/*genetics ; HEK293 Cells ; HL-60 Cells ; Humans ; Mice ; Neoplasms/*genetics ; Nuclear Proteins/genetics ; Phenotype ; Quantitative Trait Loci/genetics ; Transcription Factors/genetics ; },
abstract = {Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.},
}
@article {pmid32203418,
year = {2020},
author = {Zhao, J and Wang, M and Chang, L and Yu, J and Song, A and Liu, C and Huang, W and Zhang, T and Wu, X and Shen, X and Zhu, B and Li, G},
title = {RYBP/YAF2-PRC1 complexes and histone H1-dependent chromatin compaction mediate propagation of H2AK119ub1 during cell division.},
journal = {Nature cell biology},
volume = {22},
number = {4},
pages = {439-452},
pmid = {32203418},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics/metabolism ; Cell Division ; Epigenesis, Genetic ; Feedback, Physiological ; Gene Deletion ; Gene Editing ; HEK293 Cells ; Histones/*genetics/metabolism ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Muscle Proteins/*genetics/metabolism ; Nucleosomes/chemistry/metabolism ; Polycomb Repressive Complex 1/*genetics/metabolism ; Protein Binding ; Protein Isoforms/genetics/metabolism ; *Protein Processing, Post-Translational ; Repressor Proteins/*genetics/metabolism ; Ubiquitination ; },
abstract = {Stable propagation of epigenetic information is important for maintaining cell identity in multicellular organisms. However, it remains largely unknown how mono-ubiquitinated histone H2A on lysine 119 (H2AK119ub1) is established and stably propagated during cell division. In this study, we found that the proteins RYBP and YAF2 each specifically bind H2AK119ub1 to recruit the RYBP-PRC1 or YAF2-PRC1 complex to catalyse the ubiquitination of H2A on neighbouring nucleosomes through a positive-feedback model. Additionally, we demonstrated that histone H1-compacted chromatin enhances the distal propagation of H2AK119ub1, thereby reinforcing the inheritance of H2AK119ub1 during cell division. Moreover, we showed that either disruption of RYBP/YAF2-PRC1 activity or impairment of histone H1-dependent chromatin compaction resulted in a significant defect of the maintenance of H2AK119ub1. Therefore, our results suggest that histone H1-dependent chromatin compaction plays a critical role in the stable propagation of H2AK119ub1 by RYBP/YAF2-PRC1 during cell division.},
}
@article {pmid32203410,
year = {2020},
author = {Borges, AL and Castro, B and Govindarajan, S and Solvik, T and Escalante, V and Bondy-Denomy, J},
title = {Bacterial alginate regulators and phage homologs repress CRISPR-Cas immunity.},
journal = {Nature microbiology},
volume = {5},
number = {5},
pages = {679-687},
pmid = {32203410},
issn = {2058-5276},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Alginates/*metabolism ; Bacteria/genetics/*metabolism/*virology ; Bacterial Proteins/genetics/metabolism ; Bacteriophages/*genetics ; Base Sequence ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics ; Gene Expression Regulation, Bacterial ; Genes, Bacterial/genetics ; Genes, Regulator/genetics ; Immunity ; Pseudomonas Phages/genetics ; Pseudomonas aeruginosa/metabolism ; Transcription Factors ; Transcription, Genetic ; },
abstract = {CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection[1]. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases[2]. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB-AlgB), previously characterized in the regulation of Pseudomonas aeruginosa alginate biosynthesis[3,4], as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR-Cas system. Downstream of KinB-AlgB, activators of alginate production AlgU (a σ[E] orthologue) and AlgR repress CRISPR-Cas activity during planktonic and surface-associated growth[5]. AmrZ, another alginate regulator[6], is triggered to repress CRISPR-Cas immunity upon surface association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme and carry hijacked homologs of AmrZ that repress CRISPR-Cas expression and activity. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.},
}
@article {pmid32203383,
year = {2020},
author = {Marino, ND and Pinilla-Redondo, R and Csörgő, B and Bondy-Denomy, J},
title = {Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies.},
journal = {Nature methods},
volume = {17},
number = {5},
pages = {471-479},
pmid = {32203383},
issn = {1548-7105},
support = {DP5 OD021344/OD/NIH HHS/United States ; F32 GM133127/GM/NIGMS NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Enzyme Inhibitors/*pharmacology ; *Gene Editing ; *Gene Expression Regulation ; Humans ; Protein Engineering ; },
abstract = {Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, a diverse family of prokaryotic adaptive immune systems, have emerged as a biotechnological tool and therapeutic. The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems.},
}
@article {pmid32203294,
year = {2020},
author = {Sheridan, C},
title = {Fast, portable tests come online to curb coronavirus pandemic.},
journal = {Nature biotechnology},
volume = {38},
number = {5},
pages = {515-518},
doi = {10.1038/d41587-020-00010-2},
pmid = {32203294},
issn = {1546-1696},
mesh = {Betacoronavirus/*isolation &amp; purification/pathogenicity ; COVID-19 ; CRISPR-Cas Systems/genetics ; Coronavirus Infections/*diagnosis/epidemiology/prevention &amp; control/virology ; Disease Outbreaks/*prevention &amp; control ; Humans ; Pandemics/*prevention &amp; control ; Pneumonia, Viral/*diagnosis/epidemiology/prevention &amp; control/virology ; Public Health ; SARS-CoV-2 ; },
}
@article {pmid32203198,
year = {2020},
author = {Preece, R and Georgiadis, C and Gkazi, SA and Etuk, A and Christi, A and Qasim, W},
title = {'Mini' U6 Pol III promoter exhibits nucleosome redundancy and supports multiplexed coupling of CRISPR/Cas9 effects.},
journal = {Gene therapy},
volume = {27},
number = {9},
pages = {451-458},
pmid = {32203198},
issn = {1476-5462},
support = {RP-2014-05-007/DH_/Department of Health/United Kingdom ; IS-BRC-1215-20012//Great Ormond Street Hospital for Children (GOSH)/International ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Humans ; *Nucleosomes/genetics ; Promoter Regions, Genetic ; RNA, Guide ; },
abstract = {RNA polymerase III (Pol III) promoters express short non-coding RNAs and have been adopted for expression of microRNA, interference RNA, and CRISPR single guide RNA (sgRNA). Vectors incorporating H1 and U6 Pol III promoters are being applied for therapeutic genome editing, including multiplexed CRISPR/Cas9 effects. We report a nucleosome-depleted, minimal U6 promoter, which when embedded within lentiviral long terminal repeat (LTR) regions, supports high level transcriptional activity. Furthermore, duplex minimal H1 &amp; U6 promoters transcribed dual sgRNAs for simultaneous disruption of T cell receptor (TCR) and human leukocyte antigen (HLA) molecules, supporting efficient generation of 'universal' CAR T cells.},
}
@article {pmid32200959,
year = {2020},
author = {Paul, B and Montoya, G},
title = {CRISPR-Cas12a: Functional overview and applications.},
journal = {Biomedical journal},
volume = {43},
number = {1},
pages = {8-17},
pmid = {32200959},
issn = {2320-2890},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/*genetics/metabolism ; *Gene Editing/methods ; Humans ; RNA/genetics ; },
abstract = {Prokaryotes have developed an adaptive immune system called Clustered regularly interspaced short palindromic repeats (CRISPR) to combat attacks by foreign mobile genetic elements (MGEs) such as plasmids and phages. In the past decade, the widely characterized CRISPR-Cas9 enzyme has been redesigned to trigger a genome editing revolution. Class II type V CRISPR-Cas12a is a new RNA guided endonuclease that has been recently harnessed as an alternative genome editing tool, which is emerging as a powerful molecular scissor to consider in the genome editing application landscape. In this review, we aim to provide a mechanistic insight into the working mechanism of Cas12a, comparing it with Cas9, and eventually provide an overview of its current applications in genome editing and biotechnology applications.},
}
@article {pmid32200874,
year = {2020},
author = {Otten, ABC and Sun, BK},
title = {Research Techniques Made Simple: CRISPR Genetic Screens.},
journal = {The Journal of investigative dermatology},
volume = {140},
number = {4},
pages = {723-728.e1},
pmid = {32200874},
issn = {1523-1747},
support = {2019088/DDCF_/Doris Duke Charitable Foundation/United States ; K08 AR067853/AR/NIAMS NIH HHS/United States ; R03 AR075844/AR/NIAMS NIH HHS/United States ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Testing ; Humans ; Phenotype ; *Research Design ; },
abstract = {CRISPR and Cas proteins, often referred to as CRISPR/Cas, are the components of a bacterial genome editing system that can be used to perturb genes in cells and tissues. A classic application is to use CRISPR/Cas to generate genetic loss-of-function. When performed at large scale and combined with deep sequencing techniques, CRISPR-based perturbations can be performed in a high throughput setting to screen many candidate genomic elements for their roles in a phenotype of interest. Here, we discuss major considerations in the design, execution, and analysis of CRISPR screens. We focus on CRISPR knockout screens but also review adaptations to the CRISPR/Cas system that highlight the versatility of the system to make other types of experimental genetic changes as well. We also discuss examples of CRISPR genetic screens in investigative dermatology and how they may be used to answer key scientific questions in the field.},
}
@article {pmid32200531,
year = {2020},
author = {Chen, B and Niu, Y and Wang, H and Wang, K and Yang, H and Li, W},
title = {Recent advances in CRISPR research.},
journal = {Protein &amp; cell},
volume = {11},
number = {11},
pages = {786-791},
pmid = {32200531},
issn = {1674-8018},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid32200382,
year = {2020},
author = {Potlapalli, BP and Schubert, V and Metje-Sprink, J and Liehr, T and Houben, A},
title = {Application of Tris-HCl Allows the Specific Labeling of Regularly Prepared Chromosomes by CRISPR-FISH.},
journal = {Cytogenetic and genome research},
volume = {160},
number = {3},
pages = {156-165},
doi = {10.1159/000506720},
pmid = {32200382},
issn = {1424-859X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Chromosomes/*genetics ; In Situ Hybridization, Fluorescence/*methods ; Mice ; RNA, Guide/genetics ; Secale/genetics ; Soybeans/*genetics ; Tobacco/genetics ; Triticum/genetics ; Zea mays/genetics ; },
abstract = {Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe the further development of the RNA-guided endonuclease-in situ labeling (RGEN-ISL) method CRISPR-FISH. Using soybean and mouse chromosomes, we demonstrate that the treatment of conventionally fixed chromosomes (in ethanol or methanol:acetic acid) with 40 mM Tris-HCl (pH 9.0) for 30 minutes at 37°C prior to CRISPR-FISH allows the application of this method for the detection of high-copy sequences. Wheat, rye, maize, and Nicotiana benthamiana were used to confirm the applicability of the identified CRISPR-FISH conditions also in other species.},
}
@article {pmid32198888,
year = {2020},
author = {Foster, K and Grüschow, S and Bailey, S and White, MF and Terns, MP},
title = {Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity.},
journal = {Nucleic acids research},
volume = {48},
number = {8},
pages = {4418-4434},
pmid = {32198888},
issn = {1362-4962},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; R01 GM097330/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; F31 GM125365/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaeal Proteins/chemistry/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Catalytic Domain ; Deoxyribonucleases/*metabolism ; Endoribonucleases/chemistry/*metabolism ; Plasmids ; Protein Domains ; Pyrococcus furiosus/*enzymology/genetics/immunology/metabolism ; Ribonucleoproteins/metabolism ; Second Messenger Systems ; },
abstract = {Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.},
}
@article {pmid32198625,
year = {2020},
author = {Han, HA and Pang, JKS and Soh, BS},
title = {Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {98},
number = {5},
pages = {615-632},
pmid = {32198625},
issn = {1432-1440},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Repair ; Disease Management ; *Gene Editing ; Gene Expression Regulation ; Gene Targeting ; Genetic Predisposition to Disease ; *Genetic Therapy/methods ; Humans ; MicroRNAs/genetics ; Models, Animal ; Organ Specificity/genetics ; Promoter Regions, Genetic ; RNA, Guide ; },
abstract = {The rapid advancement of genome editing technologies has opened up new possibilities in the field of medicine. Nuclease-based techniques such as the CRISPR/Cas9 system are now used to target genetically linked disorders that were previously hard-to-treat. The CRISPR/Cas9 gene editing approach wields several advantages over its contemporary editing systems, notably in the ease of component design, implementation and the option of multiplex genome editing. While results from the early phase clinical trials have been encouraging, the small patient population recruited into these trials hinders a conclusive assessment on the safety aspects of the CRISPR/Cas9 therapy. Potential safety concerns include the lack of fidelity in the CRISPR/Cas9 system which may lead to unintended DNA modifications at non-targeted gene loci. This review focuses modifications to the CRISPR/Cas9 components that can mitigate off-target effects in in vitro and preclinical models and its translatability to gene therapy in patient populations.},
}
@article {pmid32198457,
year = {2020},
author = {Josling, GA and Russell, TJ and Venezia, J and Orchard, L and van Biljon, R and Painter, HJ and Llinás, M},
title = {Dissecting the role of PfAP2-G in malaria gametocytogenesis.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1503},
pmid = {32198457},
issn = {2041-1723},
support = {R01 AI125565/AI/NIAID NIH HHS/United States ; T32 GM125592/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Erythrocytes/parasitology ; Gene Expression Regulation ; Genes, Protozoan/genetics ; Malaria/*parasitology/transmission ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/*genetics/*growth &amp; development/metabolism ; Protozoan Proteins/blood/*genetics/*metabolism ; Transcription Factors/metabolism ; },
abstract = {In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. The transcription factor PfAP2-G is a key determinant of sexual commitment that orchestrates this crucial cell fate decision. Here we identify the direct targets of PfAP2-G and demonstrate that it dynamically binds hundreds of sites across the genome. We find that PfAP2-G is a transcriptional activator of early gametocyte genes, and identify differences in PfAP2-G occupancy between gametocytes derived via next-cycle and same-cycle conversion. Our data implicate PfAP2-G not only as a transcriptional activator of gametocyte genes, but also as a potential regulator of genes important for red blood cell invasion. We also find that regulation by PfAP2-G requires interaction with a second transcription factor, PfAP2-I. These results clarify the functional role of PfAP2-G during sexual commitment and early gametocytogenesis.},
}
@article {pmid32198422,
year = {2020},
author = {Hanzawa, N and Hashimoto, K and Yuan, X and Kawahori, K and Tsujimoto, K and Hamaguchi, M and Tanaka, T and Nagaoka, Y and Nishina, H and Morita, S and Hatada, I and Yamada, T and Ogawa, Y},
title = {Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {5181},
pmid = {32198422},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Demethylation ; DNA Methylation ; Epigenesis, Genetic ; Epigenome ; Fibroblast Growth Factors/*genetics/metabolism ; Gene Editing/methods ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; PPAR alpha/genetics/metabolism ; Promoter Regions, Genetic/genetics ; },
abstract = {Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.},
}
@article {pmid32198276,
year = {2020},
author = {Hollywood, JA and Przepiorski, A and D'Souza, RF and Sreebhavan, S and Wolvetang, EJ and Harrison, PT and Davidson, AJ and Holm, TM},
title = {Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis.},
journal = {Journal of the American Society of Nephrology : JASN},
volume = {31},
number = {5},
pages = {962-982},
pmid = {32198276},
issn = {1533-3450},
mesh = {Amino Acid Transport Systems, Neutral/deficiency/genetics ; Animals ; Autophagy/drug effects ; CRISPR-Cas Systems ; Cell Line ; Cysteamine/pharmacology/*therapeutic use ; Cystine/blood ; Cystinosis/*drug therapy ; *Disease Models, Animal ; Drug Evaluation, Preclinical ; Drug Therapy, Combination ; Everolimus/pharmacology/*therapeutic use ; Gene Editing ; Heterografts ; Humans ; Induced Pluripotent Stem Cells/metabolism/*transplantation/ultrastructure ; Lysosomes/drug effects/ultrastructure ; Mice ; Mice, SCID ; Organoids/metabolism/*transplantation ; Phenotype ; TOR Serine-Threonine Kinases/*antagonists &amp; inhibitors ; },
abstract = {BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments.<br><br>METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls.<br><br>RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities.<br><br>CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.},
}
@article {pmid32198222,
year = {2020},
author = {Nicolai, CJ and Wolf, N and Chang, IC and Kirn, G and Marcus, A and Ndubaku, CO and McWhirter, SM and Raulet, DH},
title = {NK cells mediate clearance of CD8[+] T cell-resistant tumors in response to STING agonists.},
journal = {Science immunology},
volume = {5},
number = {45},
pages = {},
pmid = {32198222},
issn = {2470-9468},
support = {F31 CA228381/CA/NCI NIH HHS/United States ; R01 AI113041/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CD8-Positive T-Lymphocytes/*drug effects/*immunology ; CRISPR-Cas Systems/drug effects/immunology ; Interferon Type I/pharmacology ; Killer Cells, Natural/drug effects/*immunology ; Membrane Proteins/*agonists/immunology ; Mice ; Mice, Congenic ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Knockout ; Neoplasms/*drug therapy/*immunology/pathology ; Tumor Cells, Cultured ; },
abstract = {Several immunotherapy approaches that mobilize CD8[+] T cell responses stimulate tumor rejection, and some, such as checkpoint blockade, have been approved for several cancer indications and show impressive increases in patient survival. However, tumors may evade CD8[+] T cell recognition via loss of MHC molecules or because they contain few or no neoantigens. Therefore, approaches are needed to combat CD8[+] T cell-resistant cancers. STING-activating cyclic dinucleotides (CDNs) are a new class of immune-stimulating agents that elicit impressive CD8[+] T cell-mediated tumor rejection in preclinical tumor models and are now being tested in clinical trials. Here, we demonstrate powerful CDN-induced, natural killer (NK) cell-mediated tumor rejection in numerous tumor models, independent of CD8[+] T cells. CDNs enhanced NK cell activation, cytotoxicity, and antitumor effects in part by inducing type I interferon (IFN). IFN acted in part directly on NK cells in vivo and in part indirectly via the induction of IL-15 and IL-15 receptors, which were important for CDN-induced NK activation and tumor control. After in vivo administration of CDNs, dendritic cells (DCs) up-regulated IL-15Rα in an IFN-dependent manner. Mice lacking the type I IFN receptor specifically on DCs had reduced NK cell activation and tumor control. Therapeutics that activate NK cells, such as CDNs, checkpoint inhibitors, NK cell engagers, and cytokines, may represent next-generation approaches to cancer immunotherapy.},
}
@article {pmid32197890,
year = {2020},
author = {Zheng, C and Schneider, JW and Hsieh, J},
title = {Role of RB1 in human embryonic stem cell-derived retinal organoids.},
journal = {Developmental biology},
volume = {462},
number = {2},
pages = {197-207},
pmid = {32197890},
issn = {1095-564X},
support = {R01 NS089770/NS/NINDS NIH HHS/United States ; R01 NS093992/NS/NINDS NIH HHS/United States ; R01 NS113516/NS/NINDS NIH HHS/United States ; R21 AG066496/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Apoptosis/physiology ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Human Embryonic Stem Cells/cytology/*metabolism ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Organoids/cytology ; Pluripotent Stem Cells/cytology ; Retina/*embryology/physiology ; Retinal Ganglion Cells/metabolism ; Retinal Neoplasms/metabolism ; Retinoblastoma/metabolism ; Retinoblastoma Binding Proteins/*metabolism/physiology ; Ubiquitin-Protein Ligases/*metabolism/physiology ; },
abstract = {Three-dimensional (3D) organoid models derived from human pluripotent stem cells provide a platform for studying human development and understanding disease mechanisms. Most studies that examine biallelic inactivation of the cell cycle regulator Retinoblastoma 1 (RB1) and the link to retinoblastoma is in mice, however, less is known regarding the pathophysiological role of RB1 during human retinal development. To study the role of RB1 in early human retinal development and tumor formation, we generated retinal organoids from CRISPR/Cas9-derived RB1-null human embryonic stem cells (hESCs). We showed that RB is abundantly expressed in retinal progenitor cells in retinal organoids and loss of RB1 promotes S-phase entry. Furthermore, loss of RB1 resulted in widespread apoptosis and reduced the number of photoreceptor, ganglion, and bipolar cells. Interestingly, RB1 mutation in retinal organoids did not result in retinoblastoma formation in vitro or in the vitreous body of NOD/SCID immunodeficient mice. Together, our work identifies a crucial function for RB1 in human retinal development and suggests that RB1 deletion alone is not sufficient for tumor development, at least in human retinal organoids.},
}
@article {pmid32197474,
year = {2020},
author = {Binda, CS and Klaver, B and Berkhout, B and Das, AT},
title = {CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.},
journal = {Viruses},
volume = {12},
number = {3},
pages = {},
pmid = {32197474},
issn = {1999-4915},
support = {R01 AI145045/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Targeting ; Genome, Viral ; HIV-1/*physiology ; Humans ; *Margins of Excision ; *Mutation ; Polymerase Chain Reaction ; Proviruses/*genetics ; RNA, Guide/*genetics ; Virus Replication/genetics ; },
abstract = {Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.},
}
@article {pmid32197234,
year = {2020},
author = {Xu, Y and Lu, J and Wu, J and Jiang, R and Guo, C and Tang, Y and Wang, H and Kong, S and Wang, S},
title = {HOXA10 co-factor MEIS1 is required for the decidualization in human endometrial stromal cell.},
journal = {Journal of molecular endocrinology},
volume = {64},
number = {4},
pages = {249-258},
doi = {10.1530/JME-19-0100},
pmid = {32197234},
issn = {1479-6813},
mesh = {CRISPR-Cas Systems/genetics ; Cells, Cultured ; Decidua/metabolism/*physiology ; Embryo Implantation/genetics ; Endometrium/cytology/*physiology ; Female ; Gene Knockout Techniques ; Gene Regulatory Networks/physiology ; HEK293 Cells ; Homeobox A10 Proteins/metabolism ; Humans ; Myeloid Ecotropic Viral Integration Site 1 Protein/antagonists &amp; inhibitors/genetics/metabolism/*physiology ; Protein Binding ; RNA, Small Interfering/pharmacology ; Stromal Cells/physiology ; },
abstract = {Decidualization is a critical process for embryo implantation and pregnancy maintenance in humans. The homeobox gene HOXA10 has been widely studied in endometrial receptivity establishment and decidualization. MEIS1, a three-amino-acid loop extension (TALE) family homeobox gene, has been proven to be a co-factor for HOXA10 in mouse uterus. However, the interaction between MEIS1 and HOXA10 in the human decidual cells remains to be elucidated. siRNA and CRISPR-Cas9 were employed to knockdown and knockout MEIS1 in the cultured human endometrial stromal cells, and it was found that MEIS1 deficiency leads to impaired decidualization. The physical interaction between the MEIS1 and HOXA10 in human endometrial stromal cell was confirmed by immunoprecipitation. Moreover, KAT2B and ETA were proved to be downregulated in the absence of MEIS1, and luciferase reporter and ChIP assays demonstrated that MEIS1-HOXA10 complex binds to the promoters of KAT2B and ETA and regulates their activity. Overexpression of KAT2B and ETA can partially rescue the decidualization defects in MEIS1-knockout HESCs. Taken together, these data suggest that MEIS1 plays an indispensable role in decidualization in human endometrial stromal cells, and MEIS1 interacts with HOXA10 to regulate the downstream genes, such as KAT2B and ETA. These findings will contribute to our understanding about the regulatory network in the process of decidualization in humans.},
}
@article {pmid32197113,
year = {2020},
author = {Petrus-Reurer, S and Winblad, N and Kumar, P and Gorchs, L and Chrobok, M and Wagner, AK and Bartuma, H and Lardner, E and Aronsson, M and Plaza Reyes, &#xc1; and André, H and Alici, E and Kaipe, H and Kvanta, A and Lanner, F},
title = {Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II.},
journal = {Stem cell reports},
volume = {14},
number = {4},
pages = {648-662},
pmid = {32197113},
issn = {2213-6711},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cytotoxicity, Immunologic ; Epithelial Cells/*metabolism ; Heterografts ; Histocompatibility Antigens Class I/*metabolism ; Histocompatibility Antigens Class II/*metabolism ; Human Embryonic Stem Cells/*cytology/metabolism ; Humans ; Immunomodulation ; Nuclear Proteins/metabolism ; Polymorphism, Single Nucleotide/genetics ; Retinal Pigment Epithelium/*cytology ; T-Lymphocytes/metabolism ; Trans-Activators/metabolism ; beta 2-Microglobulin/metabolism ; },
abstract = {Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.},
}
@article {pmid32196554,
year = {2020},
author = {Hardouin, P and Goulet, A},
title = {Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?.},
journal = {Biochemical Society transactions},
volume = {48},
number = {2},
pages = {507-516},
doi = {10.1042/BST20190638},
pmid = {32196554},
issn = {1470-8752},
support = {R01 GM129325/GM/NIGMS NIH HHS/United States ; P41 GM103311/GM/NIGMS NIH HHS/United States ; },
mesh = {Allosteric Site ; Archaea/genetics/virology ; Bacteria/genetics/*virology ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Evolution, Molecular ; Models, Molecular ; Protein Domains ; Structure-Activity Relationship ; },
abstract = {Bacteriophages (phages) and their preys are engaged in an evolutionary arms race driving the co-adaptation of their attack and defense mechanisms. In this context, phages have evolved diverse anti-CRISPR proteins to evade the bacterial CRISPR-Cas immune system, and propagate. Anti-CRISPR proteins do not share much resemblance with each other and with proteins of known function, which raises intriguing questions particularly relating to their modes of action. In recent years, there have been many structure-function studies shedding light on different CRISPR-Cas inhibition strategies. As the anti-CRISPR field of research is rapidly growing, it is opportune to review the current knowledge on these proteins, with particular emphasis on the molecular strategies deployed to inactivate distinct steps of CRISPR-Cas immunity. Anti-CRISPR proteins can be orthosteric or allosteric inhibitors of CRISPR-Cas machineries, as well as enzymes that irreversibly modify CRISPR-Cas components. This repertoire of CRISPR-Cas inhibition mechanisms will likely expand in the future, providing fundamental knowledge on phage-bacteria interactions and offering great perspectives for the development of biotechnological tools to fine-tune CRISPR-Cas-based gene edition.},
}
@article {pmid32195227,
year = {2020},
author = {Zheng, Y and Li, J and Wang, B and Han, J and Hao, Y and Wang, S and Ma, X and Yang, S and Ma, L and Yi, L and Peng, W},
title = {Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {62},
pmid = {32195227},
issn = {2296-4185},
abstract = {Establishment of production platforms through prokaryotic engineering in microbial organisms would be one of the most efficient means for chemicals, protein, and biofuels production. Despite the fact that CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies have readily emerged as powerful and versatile tools for genetic manipulations, their applications are generally limited in prokaryotes, possibly owing to the large size and severe cytotoxicity of the heterogeneous Cas (CRISPR-associated) effector. Nevertheless, the rich natural occurrence of CRISPR-Cas systems in many bacteria and most archaea holds great potential for endogenous CRISPR-based prokaryotic engineering. The endogenous CRISPR-Cas systems, with type I systems that constitute the most abundant and diverse group, would be repurposed as genetic manipulation tools once they are identified and characterized as functional in their native hosts. This article reviews the major progress made in understanding the mechanisms of invading DNA immunity by type I CRISPR-Cas and summarizes the practical applications of endogenous type I CRISPR-based toolkits for prokaryotic engineering.},
}
@article {pmid32195078,
year = {2020},
author = {Manghwar, H and Li, B and Ding, X and Hussain, A and Lindsey, K and Zhang, X and Jin, S},
title = {CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {7},
number = {6},
pages = {1902312},
pmid = {32195078},
issn = {2198-3844},
abstract = {Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator-Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost-effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off-target effects. Efforts are being made to reduce the off-target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off-target effects. Here, the current GE tools, the off-target effects generated by GE technology, types of off-target effects, mechanisms of off-target effects, major concerns, and outcomes of off-target effects in plants and animals are summarized. The methods to detect off-target effects, tools for single-guide RNA (sgRNA) design, evaluation and prediction of off-target effects, and strategies to increase the on-target efficiency and mitigate the off-target impact on intended genome-editing outcomes are summarized.},
}
@article {pmid32194972,
year = {2020},
author = {Li, Q and Sapkota, M and van der Knaap, E},
title = {Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement.},
journal = {Horticulture research},
volume = {7},
number = {},
pages = {36},
pmid = {32194972},
issn = {2662-6810},
abstract = {Directed breeding of horticultural crops is essential for increasing yield, nutritional content, and consumer-valued characteristics such as shape and color of the produce. However, limited genetic diversity restricts the amount of crop improvement that can be achieved through conventional breeding approaches. Natural genetic changes in cis-regulatory regions of genes play important roles in shaping phenotypic diversity by altering their expression. Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement through the introduction of genetic variation in a targeted manner. The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) provides a more refined method for modulating gene expression and creating phenotypic diversity to benefit crop improvement. Here, we focus on the current applications of CRISPR/Cas-mediated cis-engineering in horticultural crops. We describe strategies and limitations for its use in crop improvement, including de novo cis-regulatory element (CRE) discovery, precise genome editing, and transgene-free genome editing. In addition, we discuss the challenges and prospects regarding current technologies and achievements. CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural crops that are better able to adapt to climate change and providing food for an increasing world population.},
}
@article {pmid32194873,
year = {2020},
author = {Han, B and Meng, X and Wu, P and Li, Z and Li, S and Zhang, Y and Zha, C and Ye, Q and Jiang, C and Cai, J and Jiang, T},
title = {ATRX/EZH2 complex epigenetically regulates FADD/PARP1 axis, contributing to TMZ resistance in glioma.},
journal = {Theranostics},
volume = {10},
number = {7},
pages = {3351-3365},
pmid = {32194873},
issn = {1838-7640},
mesh = {Animals ; Antineoplastic Agents, Alkylating/*pharmacology/therapeutic use ; Brain Neoplasms/*drug therapy/genetics/metabolism ; CRISPR-Cas Systems ; DNA Damage ; *DNA Methylation ; DNA Repair ; DNA, Neoplasm/genetics ; DNA-Binding Proteins/physiology ; Dioxygenases ; Drug Resistance, Neoplasm/*genetics ; Enhancer of Zeste Homolog 2 Protein/genetics/*physiology ; Fas-Associated Death Domain Protein/*physiology ; Gene Editing ; Gene Expression Regulation, Neoplastic/*genetics ; Gene Knockout Techniques ; Glioma/*drug therapy/genetics/metabolism ; Histone Code ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Proteins/*physiology ; Poly (ADP-Ribose) Polymerase-1/*physiology ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/physiology ; STAT5 Transcription Factor/physiology ; Temozolomide/*pharmacology/therapeutic use ; Tumor Stem Cell Assay ; Up-Regulation ; X-linked Nuclear Protein/antagonists &amp; inhibitors/genetics/*physiology ; Xenograft Model Antitumor Assays ; },
abstract = {Rationale: Glioma is the most common primary malignant brain tumor in adults. Chemoresistance of temozolomide (TMZ), the first-line chemotherapeutic agent, is a major issue in the management of patients with glioma. Alterations of alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene constitute one of the most prevalent genetic abnormalities in gliomas. Therefore, elucidation of the role of ATRX contributing to TMZ resistance in glioma is urgently needed. Methods: We performed the bioinformatics analysis of gene expression, and DNA methylation profiling, as well as RNA and ChIP-seq data sets. CRISPR-Cas9 gene editing system was used to achieve the ATRX knockout in TMZ resistant cells. In vitro and in vivo experiments were carried out to investigate the role of ATRX contributing to TMZ resistance in glioma. Results: We found that ATRX expression was upregulated via DNA demethylation mediated by STAT5b/TET2 complex and strengthened DNA damage repair by stabilizing PARP1 protein in TMZ resistant cells. ATRX elicited PARP1 stabilization by the down-regulating of FADD expression via the H3K27me3 enrichment, which was dependent on ATRX/EZH2 complex in TMZ resistant cells. Magnetic resonance imaging (MRI) revealed that the PARP inhibitor together with TMZ inhibited glioma growth in ATRX wild type TMZ resistant intracranial xenograft models. Conclusions: The present study further illustrated the novel mechanism of the ATRX/PARP1 axis contributing to TMZ resistance. Our results provided substantial new evidence that PARP inhibitor might be a potential adjuvant agent in overcoming ATRX mediated TMZ resistance in glioma.},
}
@article {pmid32194858,
year = {2020},
author = {Wu, J and Tang, B and Tang, Y},
title = {Allele-specific genome targeting in the development of precision medicine.},
journal = {Theranostics},
volume = {10},
number = {7},
pages = {3118-3137},
pmid = {32194858},
issn = {1838-7640},
mesh = {*Alleles ; CRISPR-Cas Systems ; Computational Biology ; DNA End-Joining Repair ; Drug Design ; Epigenome ; Gene Editing/ethics/*methods ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy ; Genetic Variation ; Genome, Human/*genetics ; Haploinsufficiency ; Histocompatibility ; Humans ; Induced Pluripotent Stem Cells/immunology ; Mutagenesis, Site-Directed ; Neoplasms/genetics/therapy ; Polymorphism, Single Nucleotide ; Precision Medicine/ethics/*methods ; RNA, Guide/genetics ; Recombinational DNA Repair ; },
abstract = {The CRISPR-based genome editing holds immense potential to fix disease-causing mutations, however, must also handle substantial natural genetic variations between individuals. Previous studies have shown that mismatches between the single guide RNA (sgRNA) and genomic DNA may negatively impact sgRNA efficiencies and lead to imprecise specificity prediction. Hence, the genetic variations bring about a great challenge for designing platinum sgRNAs in large human populations. However, they also provide a promising entry for designing allele-specific sgRNAs for the treatment of each individual. The CRISPR system is rather specific, with the potential ability to discriminate between similar alleles, even based on a single nucleotide difference. Genetic variants contribute to the discrimination capabilities, once they generate a novel protospacer adjacent motif (PAM) site or locate in the seed region near an available PAM. Therefore, it can be leveraged to establish allele-specific targeting in numerous dominant human disorders, by selectively ablating the deleterious alleles. So far, allele-specific CRISPR has been increasingly implemented not only in treating dominantly inherited diseases, but also in research areas such as genome imprinting, haploinsufficiency, spatiotemporal loci imaging and immunocompatible manipulations. In this review, we will describe the working principles of allele-specific genome manipulations by virtue of expanding engineering tools of CRISPR. And then we will review new advances in the versatile applications of allele-specific CRISPR targeting in treating human genetic diseases, as well as in a series of other interesting research areas. Lastly, we will discuss their potential therapeutic utilities and considerations in the era of precision medicine.},
}
@article {pmid32194561,
year = {2020},
author = {Brandt, LJB and Barnkob, MB and Michaels, YS and Heiselberg, J and Barington, T},
title = {Emerging Approaches for Regulation and Control of CAR T Cells: A Mini Review.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {326},
pmid = {32194561},
issn = {1664-3224},
mesh = {Antigens, Neoplasm/immunology ; CRISPR-Cas Systems ; Cell Hypoxia ; Cetuximab/pharmacology/therapeutic use ; Cytokine Release Syndrome/etiology/*prevention &amp; control ; Cytokines/biosynthesis ; Genes, Transgenic, Suicide ; Humans ; *Immunotherapy, Adoptive/adverse effects/methods ; Interleukin 1 Receptor Antagonist Protein/biosynthesis ; Lymphocyte Activation ; Protease Inhibitors/pharmacology/therapeutic use ; Protein Binding ; Protein Domains ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; Receptors, Chimeric Antigen/genetics/immunology ; Rituximab/pharmacology/therapeutic use ; T-Cell Antigen Receptor Specificity ; T-Lymphocyte Subsets/drug effects/*immunology/transplantation ; Tetracycline/pharmacology ; Transcription, Genetic/drug effects ; Transfection ; Tumor Microenvironment ; },
abstract = {Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers. However, widespread application of the therapy is currently limited by potentially life-threatening toxicities due to a lack of control of the highly potent transfused cells. Researchers have therefore developed several regulatory mechanisms in order to control CAR T cells in vivo. Clinical adoption of these control systems will depend on several factors, including the need for temporal and spatial control, the immunogenicity of the requisite components as well as whether the system allows reversible control or induces permanent elimination. Here we describe currently available and emerging control methods and review their function, advantages, and limitations.},
}
@article {pmid32194155,
year = {2020},
author = {Jiménez, A and Hoff, B and Revuelta, JL},
title = {Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1.},
journal = {New biotechnology},
volume = {57},
number = {},
pages = {29-33},
doi = {10.1016/j.nbt.2020.02.002},
pmid = {32194155},
issn = {1876-4347},
mesh = {Bacterial Proteins/*genetics ; Clostridiales/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Eremothecium/*genetics ; *Gene Editing ; },
abstract = {CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific 5'-NGG-3' PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5'-TTTN-3') to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system.},
}
@article {pmid32193338,
year = {2020},
author = {Yadav, V and Sun, S and Coelho, MA and Heitman, J},
title = {Centromere scission drives chromosome shuffling and reproductive isolation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {14},
pages = {7917-7928},
pmid = {32193338},
issn = {1091-6490},
support = {R37 AI039115/AI/NIAID NIH HHS/United States ; R56 AI112595/AI/NIAID NIH HHS/United States ; R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI112595/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Centromere/*genetics ; Chromosomal Instability/genetics ; Chromosomes/genetics ; Cryptococcosis/*genetics/microbiology ; Cryptococcus neoformans/*genetics/pathogenicity ; DNA Breaks, Double-Stranded ; Genome, Fungal/genetics ; Genomics ; Humans ; *Reproductive Isolation ; Translocation, Genetic/genetics ; },
abstract = {A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.},
}
@article {pmid32192326,
year = {2020},
author = {Tu, K and Deng, H and Kong, L and Wang, Y and Yang, T and Hu, Q and Hu, M and Yang, C and Zhang, Z},
title = {Reshaping Tumor Immune Microenvironment through Acidity-Responsive Nanoparticles Featured with CRISPR/Cas9-Mediated Programmed Death-Ligand 1 Attenuation and Chemotherapeutics-Induced Immunogenic Cell Death.},
journal = {ACS applied materials &amp; interfaces},
volume = {12},
number = {14},
pages = {16018-16030},
doi = {10.1021/acsami.9b23084},
pmid = {32192326},
issn = {1944-8252},
mesh = {Acids/chemistry ; Animals ; B7-H1 Antigen/genetics/immunology ; CRISPR-Cas Systems/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cyclin-Dependent Kinase 5/genetics ; Humans ; Immunogenic Cell Death/drug effects/genetics ; Melanoma, Experimental/genetics/*immunology/pathology/therapy ; Mice ; Nanoparticles/*chemistry/therapeutic use ; Paclitaxel/chemistry/immunology/*pharmacology ; T-Lymphocytes/drug effects/immunology ; Transfection ; Tumor Microenvironment/drug effects ; },
abstract = {Blocking immune checkpoints with monoclonal antibody has been verified to achieve potential clinical successes for cancer immunotherapy. However, its application has been impeded by the "cold" tumor microenvironment. Here, weak acidity-responsive nanoparticles co-loaded with CRISPR/Cas9 and paclitaxel (PTX) with the ability to convert "cold" tumor into "hot" tumor are reported. The nanoparticles exhibited high cargo packaging capacity, superior transfection efficiency, well biocompatibility, and effective tumor accumulation. The CRISPR/Cas9 encapsulated in nanoparticles could specifically knock out cyclin-dependent kinase 5 gene to significantly attenuate the expression of programmed death-ligand 1 on tumor cells. More importantly, PTX co-delivered in nanoparticles could significantly induce immunogenic cell death, reduce regulatory T lymphocytes, repolarize tumor-associated macrophages, and enhance antitumor immunity. Therefore, the nanoparticles could effectively convert cold tumor into hot tumor, achieve effective tumor growth inhibition, and prolong overall survival from 16 to 36 days. This research provided a referable strategy for the development of combinatorial immunotherapy and chemotherapy.},
}
@article {pmid32191876,
year = {2020},
author = {Watson, CJ and Monstad-Rios, AT and Bhimani, RM and Gistelinck, C and Willaert, A and Coucke, P and Hsu, YH and Kwon, RY},
title = {Phenomics-Based Quantification of CRISPR-Induced Mosaicism in Zebrafish.},
journal = {Cell systems},
volume = {10},
number = {3},
pages = {275-286.e5},
pmid = {32191876},
issn = {2405-4720},
support = {K01 AR066061/AR/NIAMS NIH HHS/United States ; R01 AR072199/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Biological Variation, Population ; Bone Morphogenetic Protein 1/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mosaicism/*embryology/veterinary ; Phenomics/*methods ; Phenotype ; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics ; Zebrafish/genetics ; },
abstract = {Genetic mosaicism can manifest as spatially variable phenotypes that vary from site to site within an organism. Here, we use imaging-based phenomics to quantitate phenotypes at many sites within the axial skeleton of CRISPR-edited G0 zebrafish. Through characterization of loss-of-function cell clusters in the developing skeleton, we identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. We quantitate the phenotypic mosaicism produced by somatic mutations of two genes, plod2 and bmp1a, implicated in human osteogenesis imperfecta. Comparison of somatic, CRISPR-generated G0 mutants to homozygous germline mutants reveals phenotypic convergence, suggesting that CRISPR screens of G0 animals can faithfully recapitulate the biology of inbred disease models. We describe statistical frameworks for phenomic analysis of spatial phenotypic variation present in somatic G0 mutants. In sum, this study defines an approach for decoding spatially variable phenotypes generated during CRISPR-based screens.},
}
@article {pmid32191373,
year = {2020},
author = {Sashidhar, N and Harloff, HJ and Potgieter, L and Jung, C},
title = {Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds.},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2241-2250},
pmid = {32191373},
issn = {1467-7652},
mesh = {*Brassica napus/genetics ; Gene Editing ; Humans ; Phytic Acid ; Seeds/genetics ; Tetraploidy ; },
abstract = {Commercialization of Brassica napus. L (oilseed rape) meal as protein diet is gaining more attention due to its well-balanced amino acid and protein contents. Phytic acid (PA) is a major source of phosphorus in plants but is considered as anti-nutritive for monogastric animals including humans due to its adverse effects on essential mineral absorption. The undigested PA causes eutrophication, which potentially threatens aquatic life. PA accounts to 2-5% in mature seeds of oilseed rape and is synthesized by complex pathways involving multiple enzymes. Breeding polyploids for recessive traits is challenging as gene functions are encoded by several paralogs. Gene redundancy often requires to knock out several gene copies to study their underlying effects. Therefore, we adopted CRISPR-Cas9 mutagenesis to knock out three functional paralogs of BnITPK. We obtained low PA mutants with an increase of free phosphorus in the canola grade spring cultivar Haydn. These mutants could mark an important milestone in rapeseed breeding with an increase in protein value and no adverse effects on oil contents.},
}
@article {pmid32191315,
year = {2020},
author = {Hayashi, S and Watanabe, M and Kobayashi, M and Tohge, T and Hashimoto, T and Shoji, T},
title = {Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco.},
journal = {Plant &amp; cell physiology},
volume = {61},
number = {6},
pages = {1041-1053},
doi = {10.1093/pcp/pcaa036},
pmid = {32191315},
issn = {1471-9053},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Plant/*genetics ; Gene Knockout Techniques ; Metabolic Networks and Pathways/genetics ; Nicotine/*biosynthesis ; Plant Leaves/metabolism ; Plant Proteins/genetics/*metabolism ; Plant Roots/metabolism ; Plants, Genetically Modified ; Tobacco/*genetics ; Transcription Factors/genetics/*metabolism ; },
abstract = {The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.},
}
@article {pmid32191207,
year = {2020},
author = {Güven, A and Kalebic, N and Long, KR and Florio, M and Vaid, S and Brandl, H and Stenzel, D and Huttner, WB},
title = {Extracellular matrix-inducing Sox9 promotes both basal progenitor proliferation and gliogenesis in developing neocortex.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32191207},
issn = {2050-084X},
support = {G0701018/MRC_/Medical Research Council/United Kingdom ; G1100578/MRC_/Medical Research Council/United Kingdom ; MR/N004272/1/MRC_/Medical Research Council/United Kingdom ; 250197/ERC_/European Research Council/International ; MR/R006237/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Differentiation/genetics ; Cell Proliferation ; Extracellular Matrix/*metabolism ; Ferrets ; Gene Expression Regulation ; Gene Knockdown Techniques ; Humans ; Mice ; Neocortex/*physiology ; Neural Stem Cells/cytology/*metabolism ; Neurogenesis/*physiology ; Neuroglia/cytology/*metabolism ; Neurons/cytology/metabolism ; SOX9 Transcription Factor/*genetics/metabolism ; Signal Transduction ; },
abstract = {Neocortex expansion is largely based on the proliferative capacity of basal progenitors (BPs), which is increased by extracellular matrix (ECM) components via integrin signaling. Here we show that the transcription factor Sox9 drives expression of ECM components and that laminin 211 increases BP proliferation in embryonic mouse neocortex. We show that Sox9 is expressed in human and ferret BPs and is required for BP proliferation in embryonic ferret neocortex. Conditional Sox9 expression in the mouse BP lineage, where it normally is not expressed, increases BP proliferation, reduces Tbr2 levels and induces Olig2 expression, indicative of premature gliogenesis. Conditional Sox9 expression also results in cell-non-autonomous stimulation of BP proliferation followed by increased upper-layer neuron production. Our findings demonstrate that Sox9 exerts concerted effects on transcription, BP proliferation, neuron production, and neurogenic vs. gliogenic BP cell fate, suggesting that Sox9 may have contributed to promote neocortical expansion.},
}
@article {pmid32190127,
year = {2020},
author = {Kulkarni, A and Yu, W and Moon, AS and Pandey, A and Hanley, KA and Xu, J},
title = {Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes.},
journal = {Journal of genomics},
volume = {8},
number = {},
pages = {30-36},
pmid = {32190127},
issn = {1839-9940},
support = {P20 GM103451/GM/NIGMS NIH HHS/United States ; SC1 AI112786/AI/NIAID NIH HHS/United States ; },
abstract = {In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.},
}
@article {pmid32189556,
year = {2020},
author = {Ding, M and Tyrchan, C and Bäck, E and Östling, J and Schubert, S and McCrae, C},
title = {Combined siRNA and Small-Molecule Phenotypic Screening Identifies Targets Regulating Rhinovirus Replication in Primary Human Bronchial Epithelial Cells.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {6},
pages = {634-645},
doi = {10.1177/2472555220909726},
pmid = {32189556},
issn = {2472-5560},
mesh = {Bronchi/drug effects/virology ; CRISPR-Cas Systems/genetics ; Epithelial Cells/drug effects/virology ; Gene Regulatory Networks/drug effects ; Humans ; Intramolecular Transferases/*genetics ; Molecular Targeted Therapy ; RNA, Small Interfering/genetics ; Rhinovirus/*drug effects/pathogenicity ; Small Molecule Libraries/pharmacology ; Virus Diseases/*drug therapy/genetics/virology ; Virus Replication/*drug effects ; },
abstract = {Human rhinovirus (RV) is the most common cause of acute upper respiratory tract infections and has recently been shown to play a significant role in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). There is a significant unmet medical need for agents for the prevention and/or treatment of exacerbations triggered by human RV infection. Phenotypic drug discovery programs using different perturbation modalities, for example, siRNA, small-molecule compounds, and CRISPR, hold significant value for identifying novel drug targets. We have previously reported the identification of lanosterol synthase as a novel regulator of RV2 replication through a phenotypic screen of a library of siRNAs against druggable genes in normal human bronchial epithelial (NHBE) cells. Here, we describe a follow-up phenotypic screen of small-molecule compounds that are annotated to be pharmacological regulators of target genes that were identified to significantly affect RV2 replication in the siRNA primary screen of 10,500 druggable genes. Two hundred seventy small-molecule compounds selected for interacting with 122 target gene hits were screened in the primary RV2 assay in NHBE cells by quantifying viral replication via in situ hybridization followed by secondary quantitative PCR-based assays for RV2, RV14, and RV16. The described follow-up phenotypic screening allowed us to identify Fms-related tyrosine kinase 4 (FLT4) as a novel target regulating RV replication. We demonstrate that a combination of siRNA and small-molecule compound screening models is a useful phenotypic drug discovery approach for the identification of novel drug targets.},
}
@article {pmid32188851,
year = {2020},
author = {Schmidt, H and Collier, TC and Hanemaaijer, MJ and Houston, PD and Lee, Y and Lanzaro, GC},
title = {Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1425},
pmid = {32188851},
issn = {2041-1723},
support = {U01 CK000516/CK/NCEZID CDC HHS/United States ; R56 AI130277/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/*genetics ; Alleles ; Animals ; Anopheles/*genetics ; CRISPR-Cas Systems ; Female ; Gene Frequency ; *Genome, Insect ; Insect Proteins/genetics ; Mosquito Vectors/genetics ; },
abstract = {A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.},
}
@article {pmid32188734,
year = {2020},
author = {Xu, S and Zhou, L and Liang, X and Zhou, Y and Chen, H and Yan, S and Wang, Y},
title = {Novel Cell-Virus-Virophage Tripartite Infection Systems Discovered in the Freshwater Lake Dishui Lake in Shanghai, China.},
journal = {Journal of virology},
volume = {94},
number = {11},
pages = {},
pmid = {32188734},
issn = {1098-5514},
mesh = {China ; Chlorophyta/*virology ; DNA, Viral/*genetics ; Lakes ; Metagenomics ; *Phylogeny ; *Virophages/classification/genetics ; *Water Microbiology ; },
abstract = {Virophages are small parasitic double-stranded DNA (dsDNA) viruses of giant dsDNA viruses infecting unicellular eukaryotes. Except for a few isolated virophages characterized by parasitization mechanisms, features of virophages discovered in metagenomic data sets remain largely unknown. Here, the complete genomes of seven virophages (26.6 to 31.5 kbp) and four large DNA viruses (190.4 to 392.5 kbp) that coexist in the freshwater lake Dishui Lake, Shanghai, China, have been identified based on environmental metagenomic investigation. Both genomic and phylogenetic analyses indicate that Dishui Lake virophages (DSLVs) are closely related to each other and to other lake virophages, and Dishui Lake large DNA viruses are affiliated with the micro-green alga-infecting Prasinovirus of the Phycodnaviridae (named Dishui Lake phycodnaviruses [DSLPVs]) and protist (protozoan and alga)-infecting Mimiviridae (named Dishui Lake large alga virus [DSLLAV]). The DSLVs possess more genes with closer homology to that of large alga viruses than to that of giant protozoan viruses. Furthermore, the DSLVs are strongly associated with large green alga viruses, including DSLPV4 and DSLLAV1, based on codon usage as well as oligonucleotide frequency and correlation analyses. Surprisingly, a nonhomologous CRISPR-Cas like system is found in DSLLAV1, which appears to protect DSLLAV1 from the parasitization of DSLV5 and DSLV8. These results suggest that novel cell-virus-virophage (CVv) tripartite infection systems of green algae, large green alga virus (Phycodnaviridae- and Mimiviridae-related), and virophage exist in Dishui Lake, which will contribute to further deep investigations of the evolutionary interaction of virophages and large alga viruses as well as of the essential roles that the CVv plays in the ecology of algae.IMPORTANCE Virophages are small parasitizing viruses of large/giant viruses. To our knowledge, the few isolated virophages all parasitize giant protozoan viruses (Mimiviridae) for propagation and form a tripartite infection system with hosts, here named the cell-virus-virophage (CVv) system. However, the CVv system remains largely unknown in environmental metagenomic data sets. In this study, we systematically investigated the metagenomic data set from the freshwater lake Dishui Lake, Shanghai, China. Consequently, four novel large alga viruses and seven virophages were discovered to coexist in Dishui Lake. Surprisingly, a novel CVv tripartite infection system comprising green algae, large green alga viruses (Phycodnaviridae- and Mimiviridae-related), and virophages was identified based on genetic link, genomic signature, and CRISPR system analyses. Meanwhile, a nonhomologous CRISPR-like system was found in Dishui Lake large alga viruses, which appears to protect the virus host from the infection of Dishui Lake virophages (DSLVs). These findings are critical to give insight into the potential significance of CVv in global evolution and ecology.},
}
@article {pmid32187529,
year = {2020},
author = {Schmid-Burgk, JL and Gao, L and Li, D and Gardner, Z and Strecker, J and Lash, B and Zhang, F},
title = {Highly Parallel Profiling of Cas9 Variant Specificity.},
journal = {Molecular cell},
volume = {78},
number = {4},
pages = {794-800.e8},
pmid = {32187529},
issn = {1097-4164},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *Gene Editing ; *Genetic Variation ; HEK293 Cells ; Humans ; K562 Cells ; RNA, Guide/*genetics ; },
abstract = {Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.},
}
@article {pmid32187363,
year = {2020},
author = {Chen, HM and Marques, JG and Sugino, K and Wei, D and Miyares, RL and Lee, T},
title = {CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila.},
journal = {Nucleic acids research},
volume = {48},
number = {8},
pages = {4344-4356},
pmid = {32187363},
issn = {1362-4962},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {5' Untranslated Regions ; Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Drosophila Proteins/genetics ; *Gene Editing ; Genome, Insect ; INDEL Mutation ; Male ; *Mutagenesis ; Nerve Tissue Proteins/genetics ; Spermatozoa/metabolism ; },
abstract = {The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-transcriptional regulation.},
}
@article {pmid32186918,
year = {2020},
author = {Davidson, AR and Lu, WT and Stanley, SY and Wang, J and Mejdani, M and Trost, CN and Hicks, BT and Lee, J and Sontheimer, EJ},
title = {Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.},
journal = {Annual review of biochemistry},
volume = {89},
number = {},
pages = {309-332},
pmid = {32186918},
issn = {1545-4509},
support = {R01 GM125797/GM/NIGMS NIH HHS/United States ; FDN-15427//CIHR/Canada ; },
mesh = {Archaea/genetics/immunology/virology ; Bacteria/genetics/immunology/virology ; Bacterial Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Biological Coevolution ; CRISPR-Associated Proteins/antagonists &amp; inhibitors/genetics/metabolism ; CRISPR-Cas Systems/*drug effects ; DNA/antagonists &amp; inhibitors/chemistry/genetics/metabolism ; DNA Cleavage/drug effects ; Endodeoxyribonucleases/antagonists &amp; inhibitors/genetics/metabolism ; Gene Editing/*methods ; Humans ; Models, Molecular ; Multigene Family ; Protein Binding ; Protein Multimerization/drug effects ; RNA, Guide/genetics/metabolism ; Small Molecule Libraries/chemistry/metabolism/*pharmacology ; Viral Proteins/chemistry/*genetics/metabolism/pharmacology ; Viruses/*genetics/metabolism/pathogenicity ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.},
}
@article {pmid32185620,
year = {2020},
author = {Chang, Y and Shao, J and Gao, Y and Liu, W and Gao, Z and Hu, Y and Chang, H},
title = {Reporter gene knock-in into Marc-145 cells using CRISPR/Cas9-mediated homologous recombination.},
journal = {Biotechnology letters},
volume = {42},
number = {8},
pages = {1317-1325},
doi = {10.1007/s10529-020-02860-x},
pmid = {32185620},
issn = {1573-6776},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chlorocebus aethiops ; Gene Knock-In Techniques/*methods ; Genes, Reporter/*genetics ; Green Fluorescent Proteins/genetics/metabolism ; Homologous Recombination/*genetics ; Polymerase Chain Reaction ; Recombinant Proteins/genetics/metabolism ; },
abstract = {OBJECTIVES: Marc-145 cells (monkey embryonic kidney epithelial cells) play a critical role in the biotechnology industry as certain virus host cells. To investigate the expression of enhanced green fluorescent protein (eGFP) gene as a foreign gene in Marc-145 cells, which we developed an approach of foreign gene site-specific knock-in into Marc-145 cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and putatively explored appropriate genomic recombination sites in Marc-145 cells.<br><br>RESULTS: Our study demonstrated that the specific homologous recombination (HR) site between the Rac GTPase activating protein 1 (RACGAP1) and the acid-sensing ion channel subunit 1 (ASIC1) genes of the 11th chromosome could be used as the target site of Cas9 for the generation of target gene knock-in into Marc-145 cells, by the insertion of the eGFP cassette into the specific HR site and subsequent expression.<br><br>CONCLUSIONS: Junction PCR, sequencing, Southern blot and fluorescence assay determined eGFP gene-specific knock-in HR site between the RACGAP1 and ASIC1 genes of the 11th chromosome, which was identified by the genomic safe harbours in Marc-145 cells. Our study encouraged a broader range of applications, such as Marc-145 cells development and engineering for virus adaption and yield increase in the vaccine biotechnology industry.},
}
@article {pmid32184230,
year = {2020},
author = {Baral, SS and Lieux, ME and DiMario, PJ},
title = {Nucleolar stress in Drosophila neuroblasts, a model for human ribosomopathies.},
journal = {Biology open},
volume = {9},
number = {4},
pages = {},
pmid = {32184230},
issn = {2046-6390},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Nucleolus/*genetics/*metabolism ; Disease Models, Animal ; Disease Susceptibility ; Drosophila/embryology/*genetics/*metabolism ; Drosophila Proteins/genetics/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockdown Techniques ; Humans ; Larva ; Neural Stem Cells/cytology/*metabolism ; Organogenesis/genetics ; RNA-Binding Proteins/genetics/metabolism ; Ribosomes/metabolism ; *Stress, Physiological ; },
abstract = {Different stem cells or progenitor cells display variable threshold requirements for functional ribosomes. This is particularly true for several human ribosomopathies in which select embryonic neural crest cells or adult bone marrow stem cells, but not others, show lethality due to failures in ribosome biogenesis or function (now known as nucleolar stress). To determine if various Drosophila neuroblasts display differential sensitivities to nucleolar stress, we used CRISPR-Cas9 to disrupt the Nopp140 gene that encodes two splice variant ribosome biogenesis factors (RBFs). Disruption of Nopp140 induced nucleolar stress that arrested larvae in the second instar stage. While the majority of larval neuroblasts arrested development, the mushroom body (MB) neuroblasts continued to proliferate as shown by their maintenance of deadpan, a neuroblast-specific transcription factor, and by their continued EdU incorporation. MB neuroblasts in wild-type larvae appeared to contain more fibrillarin and Nopp140 in their nucleoli as compared to other neuroblasts, indicating that MB neuroblasts stockpile RBFs as they proliferate in late embryogenesis while other neuroblasts normally enter quiescence. A greater abundance of Nopp140 encoded by maternal transcripts in Nopp140-/- MB neuroblasts of 1----2-day-old larvae likely rendered these cells more resilient to nucleolar stress.},
}
@article {pmid32184217,
year = {2020},
author = {Banas, K and Rivera-Torres, N and Bialk, P and Yoo, BC and Kmiec, EB},
title = {Kinetics of Nuclear Uptake and Site-Specific DNA Cleavage during CRISPR-Directed Gene Editing in Solid Tumor Cells.},
journal = {Molecular cancer research : MCR},
volume = {18},
number = {6},
pages = {891-902},
pmid = {32184217},
issn = {1557-3125},
support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; P20 GM109021/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Nucleus/genetics/*metabolism ; *DNA Cleavage ; *Gene Editing ; *Genome, Human ; Humans ; Kinetics ; Lung Neoplasms/*genetics/*pathology ; Tumor Cells, Cultured ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed gene editing is approaching clinical implementation in cancer. Thus, it is imperative to define the molecular framework upon which safe and efficacious therapeutic strategies can be built. Two important reaction parameters include the biological time frame within which the CRISPR/Cas complex enters the nucleus and executes gene editing, and the method of discrimination that the CRISPR/Cas complex utilizes to target tumor cell, but not normal cell, genomes. We are developing CRISPR-directed gene editing for the treatment of non-small cell lung carcinoma focusing on disabling Nuclear Factor Erythroid 2-Related Factor-Like (NRF2), a transcription factor that regulates chemoresistance and whose genetic disruption would enhance chemosensitivity. In this report, we define the time frame of cellular events that surround the initialization of CRISPR-directed gene editing as a function of the nuclear penetration and the execution of NRF2 gene disruption. We also identify a unique protospacer adjacent motif that facilitates site-specific cleavage of the NRF2 gene present only in tumor genomes. IMPLICATIONS: Our results begin to set a scientifically meritorious foundation for the exploitation of CRISPR-directed gene editing as an augmentative therapy for lung cancer and other solid tumors. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/6/891/F1.large.jpg.},
}
@article {pmid32183816,
year = {2020},
author = {Xie, JY and Wei, JX and Lv, LH and Han, QF and Yang, WB and Li, GL and Wang, PX and Wu, SB and Duan, JX and Zhuo, WF and Liu, PQ and Min, J},
title = {Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma.},
journal = {Cell communication and signaling : CCS},
volume = {18},
number = {1},
pages = {46},
pmid = {32183816},
issn = {1478-811X},
mesh = {Angiopoietin-2/*physiology ; Carcinoma, Hepatocellular/*metabolism ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Exosomes/metabolism ; Gene Expression Regulation, Neoplastic ; Human Umbilical Vein Endothelial Cells ; Humans ; Liver Neoplasms/*metabolism ; Neovascularization, Pathologic ; },
abstract = {BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown.<br><br>METHODS: We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay.<br><br>RESULTS: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC.<br><br>CONCLUSIONS: Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy. Video Abstract.},
}
@article {pmid32182692,
year = {2020},
author = {Dabrowska, M and Ciolak, A and Kozlowska, E and Fiszer, A and Olejniczak, M},
title = {Generation of New Isogenic Models of Huntington's Disease Using CRISPR-Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {21},
number = {5},
pages = {},
pmid = {32182692},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; HEK293 Cells ; Humans ; Huntingtin Protein/genetics/metabolism ; Huntington Disease/*genetics ; Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.},
}
@article {pmid32181354,
year = {2020},
author = {Champer, SE and Oh, SY and Liu, C and Wen, Z and Clark, AG and Messer, PW and Champer, J},
title = {Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs.},
journal = {Science advances},
volume = {6},
number = {10},
pages = {eaaz0525},
pmid = {32181354},
issn = {2375-2548},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Anopheles/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila melanogaster/*genetics/metabolism ; Female ; Gene Drive Technology/*methods ; Gene Editing/methods ; Male ; Models, Genetic ; RNA, Guide/*genetics/metabolism ; },
abstract = {The rapid evolution of resistance alleles poses a major obstacle for genetic manipulation of populations with CRISPR homing gene drives. One proposed solution is using multiple guide RNAs (gRNAs), allowing a drive to function even if some resistant target sites are present. Here, we develop a model of homing mechanisms parameterized by experimental studies. Our model incorporates several factors affecting drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology, Cas9 activity saturation, gRNA activity level variance, and incomplete homology-directed repair. We find that homing drives have an optimal number of gRNAs, usually between two and eight, depending on the specific drive type and performance parameters. These results contradict the notion that resistance rates can be reduced to arbitrarily low levels by gRNA multiplexing and highlight the need for combined approaches to counter resistance evolution in CRISPR homing drives.},
}
@article {pmid32180319,
year = {2020},
author = {Shahabipour, F and Oskuee, RK and Dehghani, H and Shokrgozar, MA and Aninwene, GE and Bonakdar, S},
title = {Cell-cell interaction in a coculture system consisting of CRISPR/Cas9 mediated GFP knock-in HUVECs and MG-63 cells in alginate-GelMA based nanocomposites hydrogel as a 3D scaffold.},
journal = {Journal of biomedical materials research. Part A},
volume = {108},
number = {8},
pages = {1596-1606},
doi = {10.1002/jbm.a.36928},
pmid = {32180319},
issn = {1552-4965},
mesh = {Alginates/chemistry ; CRISPR-Cas Systems ; *Cell Communication ; Cell Line ; Coculture Techniques/methods ; Gene Knock-In Techniques/methods ; Green Fluorescent Proteins/analysis/*genetics ; Human Umbilical Vein Endothelial Cells/*cytology/metabolism ; Humans ; Osteoblasts/*cytology/metabolism ; Tissue Scaffolds/chemistry ; },
abstract = {The interaction between osteogenic and angiogenic cells through a coculturing system in biocompatible materials has been considered for successfully engineering vascularized bone tissue equivalents. In this study, we developed a hydrogel-blended scaffold consisted of gelatin methacryloyl (GelMA) and alginate enriched with hydroxyapatite nanoparticles (HAP) to model an in vitro prevascularized bone construct. The hydrogel-based scaffold revealed a higher mechanical stiffness than those of pure (GelMA), alginate, and (GelMA+ HAP) hydrogels. In the present study, we generated a green fluorescent protein (GFP) knock-in umbilical vein endothelial cells (HUVECs) cell line using the CRISPR/Cas9 technology. The GFP was inserted into the human-like ROSA locus of HUVECs genome. HUVECs expressing GFP were cocultured with OB-like cells (MG-63) within three-dimensionally (3D) fabricated hydrogel to investigate the response of cocultured osteoblasts and endothelial cells in a 3D structure. Cell viability under the 3D cocultured gel was higher than the 3D monocultured. Compared to the 3D monocultured condition, the cells were aligned and developed into the vessel-like structures. During 14 days of culture periods, the cells displayed actin protrusions by the formation of spike-like filopodia in the 3D cocultured model. Angiogenic and osteogenic-related genes such as CD31, vWF, and osteocalcin showed higher expression in the cocultured versus the monocultured. These results have collectively indicated that the 3D cocultured hydrogel facilitates interaction among cells, thereby having a greater effect on angiogenic and osteogenic properties in the absence of induction media.},
}
@article {pmid32179706,
year = {2020},
author = {Cabrera-Serrano, M and Coote, DJ and Azmanov, D and Goullee, H and Andersen, E and McLean, C and Davis, M and Ishimura, R and Stark, Z and Vallat, JM and Komatsu, M and Kornberg, A and Ryan, M and Laing, NG and Ravenscroft, G},
title = {A homozygous UBA5 pathogenic variant causes a fatal congenital neuropathy.},
journal = {Journal of medical genetics},
volume = {57},
number = {12},
pages = {835-842},
doi = {10.1136/jmedgenet-2019-106496},
pmid = {32179706},
issn = {1468-6244},
mesh = {Ataxia/genetics/pathology ; CRISPR-Cas Systems/*genetics ; Central Nervous System/metabolism/pathology ; Consanguinity ; Epilepsy/genetics/pathology ; Female ; Gene Expression Regulation/genetics ; Genetic Linkage ; HEK293 Cells ; Homozygote ; Humans ; Infant ; Intellectual Disability/*genetics/pathology ; Male ; Movement Disorders/genetics/pathology ; Mutation/genetics ; Nervous System Malformations/*genetics/pathology ; Pedigree ; Peripheral Nerves/metabolism/pathology ; Proteins/*genetics ; Ubiquitin-Activating Enzymes/*genetics ; },
abstract = {BACKGROUND: UBA5 is the activating enzyme of UFM1 in the ufmylation post-translational modification system. Different neurological phenotypes have been associated with UBA5 pathogenic variants including epilepsy, intellectual disability, movement disorders and ataxia.<br><br>METHODS AND RESULTS: We describe a large multigenerational consanguineous family presenting with a severe congenital neuropathy causing early death in infancy. Whole exome sequencing and linkage analysis identified a novel homozygous UBA5 NM_024818.3 c.31C>T (p.Arg11Trp) mutation. Protein expression assays in mouse tissue showed similar levels of UBA5 in peripheral nerves to the central nervous system. CRISPR-Cas9 edited HEK (human embrionic kidney) cells homozygous for the UBA5 p.Arg11Trp mutation showed reduced levels of UBA5 protein compared with the wild-type. The mutant p.Arg11Trp UBA5 protein shows reduced ability to activate UFM1.<br><br>CONCLUSION: This report expands the phenotypical spectrum of UBA5 mutations to include fatal peripheral neuropathy.},
}
@article {pmid32179649,
year = {2020},
author = {Suzuki, E and Ogawa, N and Takeda, TA and Nishito, Y and Tanaka, YK and Fujiwara, T and Matsunaga, M and Ueda, S and Kubo, N and Tsuji, T and Fukunaka, A and Yamazaki, T and Taylor, KM and Ogra, Y and Kambe, T},
title = {Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {17},
pages = {5669-5684},
pmid = {32179649},
issn = {1083-351X},
mesh = {Alkaline Phosphatase/*metabolism ; Animals ; Avian Proteins/metabolism ; Cation Transport Proteins/*metabolism ; Cell Line ; Chickens ; *Enzyme Activation ; Golgi Apparatus/metabolism ; Humans ; Protein Multimerization ; Zinc/*metabolism ; },
abstract = {Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.},
}
@article {pmid32178548,
year = {2020},
author = {Marchev, AS and Yordanova, ZP and Georgiev, MI},
title = {Green (cell) factories for advanced production of plant secondary metabolites.},
journal = {Critical reviews in biotechnology},
volume = {40},
number = {4},
pages = {443-458},
doi = {10.1080/07388551.2020.1731414},
pmid = {32178548},
issn = {1549-7801},
mesh = {*Bioreactors ; CRISPR-Cas Systems ; Gene Editing ; Metabolic Engineering/*methods ; Plant Cells/*metabolism ; Plants/genetics/metabolism ; *Secondary Metabolism ; },
abstract = {For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant in vitro systems. The unique structural features of plant-derived secondary metabolites, such as their safety profile, multi-target spectrum and "metabolite likeness," have led to the establishment of many plant-derived drugs, comprising approximately a quarter of all drugs approved by the Food and Drug Administration and/or European Medicinal Agency. However, there are still many challenges to overcome to enhance the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant secondary metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this review, we present an integrated overview of the possible avenues for enhancing the biosynthesis of high-value marketable molecules produced by plant in vitro systems. These include metabolic engineering and CRISPR/Cas9 technology for the regulation of plant metabolism through overexpression/repression of single or multiple structural genes or transcriptional factors. The use of NMR-based metabolomics for monitoring metabolite concentrations and additionally as a tool to study the dynamics of plant cell metabolism and nutritional management is discussed here. Different types of bioreactor systems, their modification and optimal process parameters for the lab- or industrial-scale production of plant secondary metabolites are specified.},
}
@article {pmid32176842,
year = {2020},
author = {Bhardwaj, R and Augustynek, BS and Ercan-Herbst, E and Kandasamy, P and Seedorf, M and Peinelt, C and Hediger, MA},
title = {Ca[2+]/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca[2+]-Dependent Inactivation of the Orai1 Channel.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {54},
number = {2},
pages = {252-270},
doi = {10.33594/000000218},
pmid = {32176842},
issn = {1421-9778},
support = {CRSII5_180326, CRSII3_160782//Schweizerischer Nationalfonds (SNF), Sinergia Grant/Switzerland ; },
mesh = {CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Channels/genetics/*metabolism ; Calcium Signaling ; Calmodulin/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Hydrophobic and Hydrophilic Interactions ; Membrane Proteins/metabolism ; Models, Chemical ; Models, Molecular ; Mutation ; Neoplasm Proteins/*chemistry/genetics/metabolism/*physiology ; ORAI1 Protein/chemistry/genetics/*metabolism ; Protein Binding ; Protein Domains ; Stromal Interaction Molecule 1/*chemistry/genetics/metabolism/*physiology ; Up-Regulation ; },
abstract = {BACKGROUND/AIMS: Store-operated Ca[2+] entry (SOCE) through plasma membrane Ca[2+] channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca[2+] store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca[2+]-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca[2+] release-activated Ca[2+] current (ICRAC). A major regulatory mechanism that prevents deleterious Ca[2+] overload is the slow Ca[2+]-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca[2+]/calmodulin (Ca[2+]/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca[2+]/CaM-mediated SCDI of ICRAC is as yet unclear.<br><br>METHODS: The Ca[2+]/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca[2+] influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca[2+]/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology.<br><br>RESULTS: Here we identified residues Leu[374]/Val[375] (H1) and Leu[390]/Phe[391] (H2) within SOAR that serve as hydrophobic anchor sites for Ca[2+]/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca[2+]/CaM binding. Single residue mutations of Phe[391] to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca[2+]/CaM. Hence, the role of H2 residues in Ca[2+]/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca[2+]/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca[2+]/CaM, proving a direct role of STIM1 in coordinating SCDI.<br><br>CONCLUSION: Taken together, we propose a mechanistic model, wherein binding of Ca[2+]/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca[2+]/CaM are functionally coordinated to control ICRAC.},
}
@article {pmid32176542,
year = {2020},
author = {Feng, W and Liu, C and Spinozzi, S and Wang, L and Evans, SM and Chen, J},
title = {Identifying the Cardiac Dyad Proteome In Vivo by a BioID2 Knock-In Strategy.},
journal = {Circulation},
volume = {141},
number = {11},
pages = {940-942},
pmid = {32176542},
issn = {1524-4539},
support = {R01 HL130295/HL/NHLBI NIH HHS/United States ; R01 HL137957/HL/NHLBI NIH HHS/United States ; R01 HL144872/HL/NHLBI NIH HHS/United States ; R01 HL146759/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Biotinylation ; CRISPR-Cas Systems ; Excitation Contraction Coupling/*genetics ; Gene Editing ; Gene Knock-In Techniques/*methods ; Gene Ontology ; Membrane Proteins/genetics/metabolism/*physiology ; Mice ; Muscle Proteins/genetics/metabolism/*physiology ; Myocytes, Cardiac/*physiology ; Oligopeptides ; Protein Interaction Mapping ; *Proteome ; Recombinant Fusion Proteins/metabolism ; Sarcoplasmic Reticulum/metabolism ; Tandem Mass Spectrometry ; },
}
@article {pmid32176433,
year = {2020},
author = {Lima Leite, E and Gautron, A and Deplanche, M and Nicolas, A and Ossemond, J and Nguyen, MT and do Carmo, FLR and Gilot, D and Azevedo, V and Götz, F and Le Loir, Y and Otto, M and Berkova, N},
title = {Involvement of caspase-1 in inflammasomes activation and bacterial clearance in S. aureus-infected osteoblast-like MG-63 cells.},
journal = {Cellular microbiology},
volume = {22},
number = {8},
pages = {e13204},
doi = {10.1111/cmi.13204},
pmid = {32176433},
issn = {1462-5822},
support = {ZIA AI000904/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Caspase 1/*genetics/*immunology ; Cell Line ; Gene Deletion ; Humans ; Inflammasomes/*immunology/metabolism ; Interleukin-1beta/genetics/immunology ; Osteoblasts/*microbiology ; Staphylococcus aureus/*pathogenicity ; THP-1 Cells ; },
abstract = {Staphylococcus aureus, a versatile Gram-positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. We show here that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knock-out CASP1 [-/-] MG-63 cells, which were generated using CRISPR-Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1β production. Furthermore, we found that the lack of caspase-1 in CASP1 [-/-] MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 [-/-] MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance.},
}
@article {pmid32175691,
year = {2020},
author = {Choudhury, A and Fenster, JA and Fankhauser, RG and Kaar, JL and Tenaillon, O and Gill, RT},
title = {CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.},
journal = {Molecular systems biology},
volume = {16},
number = {3},
pages = {e9265},
pmid = {32175691},
issn = {1744-4292},
mesh = {CRISPR-Cas Systems ; DNA-Directed RNA Polymerases/genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/genetics ; *Genes, Essential ; Genetic Engineering/*methods ; *Mutation ; RNA, Guide/pharmacology ; Recombination, Genetic ; },
abstract = {Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.},
}
@article {pmid32175589,
year = {2020},
author = {Zhu, CS and Liu, CY and Qiu, XY and Xie, SS and Li, WY and Zhu, L and Zhu, LY},
title = {Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {7},
pages = {2279-2294},
doi = {10.1002/bit.27334},
pmid = {32175589},
issn = {1097-0290},
support = {31500686//National Natural Science Foundation of China/International ; 31870855//National Natural Science Foundation of China/International ; 2019RS2030//"Huxiang Young Talents Plan" Project of Hunan Province/International ; 2017JJ3358//Hunan Provincial Natural Science Foundation of China/International ; ZK17-03-58//National University of Defense Technology Project/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/analysis/*genetics ; Humans ; Polymorphism, Single Nucleotide ; RNA/analysis/*genetics ; },
abstract = {Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.},
}
@article {pmid32175288,
year = {2020},
author = {Lander, N and Cruz-Bustos, T and Docampo, R},
title = {A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {68},
pmid = {32175288},
issn = {2235-2988},
support = {R21 AI140421/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Down-Regulation ; *Gene Silencing ; Glucosamine/analogs &amp; derivatives/metabolism/pharmacology ; Glucose-6-Phosphate/analogs &amp; derivatives/metabolism ; Inorganic Pyrophosphatase/*genetics/metabolism ; Phosphoproteins/*genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; RNA, Catalytic/metabolism ; *Riboswitch ; Trypanosoma cruzi/enzymology/*genetics/growth &amp; development ; },
abstract = {Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.},
}
@article {pmid32174896,
year = {2020},
author = {Jiang, J and Yang, B and Ross, RP and Stanton, C and Zhao, J and Zhang, H and Chen, W},
title = {Comparative Genomics of Pediococcus pentosaceus Isolated From Different Niches Reveals Genetic Diversity in Carbohydrate Metabolism and Immune System.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {253},
pmid = {32174896},
issn = {1664-302X},
abstract = {Pediococcus pentosaceus isolated from fermented food and the gastrointestinal tracts of humans and animals have been widely identified, and some strains have been reported to reduce inflammation, encephalopathy, obesity and fatty liver in animals. In this study, the genomes of 65 P. pentosaceus strains isolated from human and animal feces and different fermented food were sequenced and comparative genomics analysis was performed on all strains along with nine sequenced representative strains to preliminarily reveal the lifestyle of P. pentosaceus, and investigate the genomic diversity within this species. The results reveal that P. pentosaceus is not host-specific, and shares core genes encoding proteins related to translation, ribosomal structure and biogenesis and signal transduction mechanisms, while its genetic diversity relates mainly to carbohydrate metabolism, and horizontally transferred DNA, especially prophages and bacteriocins encoded on plasmids. Additionally, this is the first report of a type IIA CRISPR/Cas system in P. pentosaceus. This work provides expanded resources of P. pentosaceus genomes, and offers a framework for understanding the biotechnological potential of this species.},
}
@article {pmid32174491,
year = {2020},
author = {Jeong, CB and Kang, HM and Hong, SA and Byeon, E and Lee, JS and Lee, YH and Choi, IY and Bae, S and Lee, JS},
title = {Generation of albino via SLC45a2 gene targeting by CRISPR/Cas9 in the marine medaka Oryzias melastigma.},
journal = {Marine pollution bulletin},
volume = {154},
number = {},
pages = {111038},
doi = {10.1016/j.marpolbul.2020.111038},
pmid = {32174491},
issn = {1879-3363},
mesh = {*Albinism ; Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Targeting ; *Oryzias ; },
abstract = {To produce albinism in the marine medaka Oryzias melastigma, we disrupted the solute carrier family 45 (SLC45a2) gene by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with a single guide RNA (sgRNA). Selected sgRNAs were able to target a SLC45a2 gene as confirmed by genotyping and heteroduplex mobility assay (HMA). Of the survived embryos after injection, 54.2% and 60.0% embryos exhibited albinism phenotype by sgRNA1 and sgRNA2, respectively. Deep sequencing at the on-target sites showed different insertion and deletion (indel) mutation profiles near the DNA cleavage sites, indicating high efficacy of producing SLC45a2 knock-out mutants by this method. Moreover, HMA at the potential off-target sites revealed that off-target activity would be induced at a low rate, or not induced at all. This albino marine medaka will be a good model for marine molecular ecotoxicology in establishment of diverse in vivo endpoints, and the application of this efficient gene targeting method in the marine medaka would be useful tool for mechanistic approaches.},
}
@article {pmid32173581,
year = {2020},
author = {Tonelli, F and Cotti, S and Leoni, L and Besio, R and Gioia, R and Marchese, L and Giorgetti, S and Villani, S and Gistelinck, C and Wagener, R and Kobbe, B and Fiedler, IAK and Larionova, D and Busse, B and Eyre, D and Rossi, A and Witten, PE and Forlino, A},
title = {Crtap and p3h1 knock out zebrafish support defective collagen chaperoning as the cause of their osteogenesis imperfecta phenotype.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {90},
number = {},
pages = {40-60},
doi = {10.1016/j.matbio.2020.03.004},
pmid = {32173581},
issn = {1569-1802},
support = {GGP13098/TI_/Telethon/Italy ; },
mesh = {Animals ; CRISPR-Cas Systems ; Collagen Type I/*metabolism ; Collagen Type II/*metabolism ; Cyclophilins/genetics ; Disease Models, Animal ; Extracellular Matrix Proteins/*genetics ; Gene Knockout Techniques ; Hydroxylation ; Osteogenesis Imperfecta/*genetics/metabolism ; Phenotype ; Prolyl Hydroxylases/chemistry/*genetics ; Zebrafish ; Zebrafish Proteins/chemistry/genetics ; },
abstract = {Prolyl 3-hydroxylation is a rare collagen type I post translational modification in fibrillar collagens. The primary 3Hyp substrate sites in type I collagen are targeted by an endoplasmic reticulum (ER) complex composed by cartilage associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and prolyl cis/trans isomerase B, whose mutations cause recessive forms of osteogenesis imperfecta with impaired levels of α1(I)3Hyp986. The absence of collagen type I 3Hyp in wild type zebrafish provides the unique opportunity to clarify the role of the complex in vertebrate. Zebrafish knock outs for crtap and p3h1 were generated by CRISPR/Cas9. Mutant fish have the typical OI patients' reduced size, body disproportion and altered mineralization. Vertebral body fusions, deformities and fractures are accompanied to reduced size, thickness and bone volume. Intracellularly, collagen type I is overmodified, and partially retained causing enlarged ER cisternae. In the extracellular matrix the abnormal collagen type I assembles in disorganized fibers characterized by altered diameter. The data support the defective chaperone role of the 3-hydroxylation complex as the primary cause of the skeletal phenotype.},
}
@article {pmid32173285,
year = {2020},
author = {Ye, C and Chen, Z and Liu, Z and Wang, F and He, X},
title = {Defining endogenous barcoding sites for CRISPR/Cas9-based cell lineage tracing in zebrafish.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {2},
pages = {85-91},
doi = {10.1016/j.jgg.2019.11.012},
pmid = {32173285},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Lineage/*genetics ; *DNA Barcoding, Taxonomic ; Genome/genetics ; Humans ; Single-Cell Analysis ; Transcriptome/genetics ; Zebrafish/classification/*genetics ; },
abstract = {There is a growing interest in developing experimental methods for tracking the developmental cell lineages of a complex organism. The recently developed CRISPR/Cas9-based barcoding method is, although highly promising, difficult to scale up because it relies on exogenous barcoding sequences that are engineered into the genome. In this study, we characterized 78 high-quality endogenous sites in the zebrafish genome that can be used as CRISPR/Cas9-based barcoding sites. The 78 sites are all highly expressed in most of the cell types according to single-cell RNA sequencing (scRNA-seq) data. Hence, the barcoding information of the 78 endogenous sites is recovered by the available scRNA-seq platforms, enabling simultaneous characterization of cell type and cell lineage information.},
}
@article {pmid32170285,
year = {2020},
author = {Ming, M and Ren, Q and Pan, C and He, Y and Zhang, Y and Liu, S and Zhong, Z and Wang, J and Malzahn, AA and Wu, J and Zheng, X and Zhang, Y and Qi, Y},
title = {CRISPR-Cas12b enables efficient plant genome engineering.},
journal = {Nature plants},
volume = {6},
number = {3},
pages = {202-208},
pmid = {32170285},
issn = {2055-0278},
mesh = {Alicyclobacillus/*genetics ; Bacillus/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome, Plant ; Oryza/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12b is a newly emerged genome engineering system. Here, we compared Cas12b from Alicyclobacillus acidoterrestris (Aac), Alicyclobacillus acidiphilus (Aa), Bacillus thermoamylovorans (Bth) and Bacillus hisashii (Bh) for genome engineering in rice, an important crop. We found AaCas12b was more efficient than AacCas12b and BthCas12b for targeted mutagenesis, which was further demonstrated in multiplexed genome editing. We also engineered the Cas12b systems for targeted transcriptional repression and activation. Our work establishes Cas12b as the third promising CRISPR system, after Cas9 and Cas12a, for plant genome engineering.},
}
@article {pmid32170140,
year = {2020},
author = {Iwasaki, RS and Ozdilek, BA and Garst, AD and Choudhury, A and Batey, RT},
title = {Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1394},
pmid = {32170140},
issn = {2041-1723},
support = {R01 GM073850/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/*genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Nucleic Acid Conformation ; RNA, Guide ; Theophylline ; },
abstract = {CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.},
}
@article {pmid32170079,
year = {2020},
author = {Gee, P and Lung, MSY and Okuzaki, Y and Sasakawa, N and Iguchi, T and Makita, Y and Hozumi, H and Miura, Y and Yang, LF and Iwasaki, M and Wang, XH and Waller, MA and Shirai, N and Abe, YO and Fujita, Y and Watanabe, K and Kagita, A and Iwabuchi, KA and Yasuda, M and Xu, H and Noda, T and Komano, J and Sakurai, H and Inukai, N and Hotta, A},
title = {Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1334},
pmid = {32170079},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cell Survival ; Dimerization ; Exons/*genetics ; Extracellular Vesicles/*metabolism ; Gene Editing ; Genetic Vectors/metabolism ; HEK293 Cells ; HIV Protease/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Ligands ; Luciferases/metabolism ; Nanoparticles/*chemistry ; RNA Splicing/genetics ; RNA, Catalytic/metabolism ; RNA, Guide/*metabolism ; Ribonucleoproteins/metabolism ; Tissue Donors ; tat Gene Products, Human Immunodeficiency Virus/metabolism ; },
abstract = {Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.},
}
@article {pmid32170016,
year = {2020},
author = {Zhang, K and Wang, S and Li, S and Zhu, Y and Pintilie, GD and Mou, TC and Schmid, MF and Huang, Z and Chiu, W},
title = {Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {13},
pages = {7176-7182},
pmid = {32170016},
issn = {1091-6490},
support = {P20 GM103546/GM/NIGMS NIH HHS/United States ; P41 GM103832/GM/NIGMS NIH HHS/United States ; R01 GM079429/GM/NIGMS NIH HHS/United States ; S10 OD021600/OD/NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*metabolism/ultrastructure ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Host-Pathogen Interactions ; Pseudomonas aeruginosa ; Viral Proteins/*metabolism/ultrastructure ; },
abstract = {Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.},
}
@article {pmid32169855,
year = {2020},
author = {Lawrence, CM},
title = {A moonlighting nuclease puts CRISPR in its place.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {11},
pages = {3415-3416},
pmid = {32169855},
issn = {1083-351X},
mesh = {*CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Polymerase III ; Endonucleases/genetics/metabolism ; Escherichia coli/metabolism ; *Escherichia coli Proteins/metabolism ; Exonucleases ; },
abstract = {Integration of spacers into CRISPR loci requires the Cas1/Cas2 integrase complex, frequently in combination with Cas4 exonuclease. However, several CRISPR-Cas systems lack Cas4. Whether Cas4-like activity is dispensable in these systems or provided by an unidentified actor was not known. In this issue of the Journal of Biological Chemistry, Ramachandran et al. show that in subtype I-E systems, Cas4-like activity is supplied by DnaQ-superfamily exonucleases, providing a beautiful example of cellular machinery moonlighting in support of CRISPR-Cas adaptive immunity.},
}
@article {pmid32169537,
year = {2020},
author = {Wan, T and Chen, Y and Pan, Q and Xu, X and Kang, Y and Gao, X and Huang, F and Wu, C and Ping, Y},
title = {Genome editing of mutant KRAS through supramolecular polymer-mediated delivery of Cas9 ribonucleoprotein for colorectal cancer therapy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {322},
number = {},
pages = {236-247},
doi = {10.1016/j.jconrel.2020.03.015},
pmid = {32169537},
issn = {1873-4995},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Colorectal Neoplasms/drug therapy/genetics ; *Gene Editing ; Humans ; Mice ; Polymers ; Proto-Oncogene Proteins p21(ras) ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR (clustered, regularly interspaced, short palindromic repeats)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful genome-editing tool to correct genetic disorders. However, successful intracellular delivery of CRISPR/Cas9, especially in the form of ribonucleoprotein (RNP), remains elusive for clinical translation. Herein, we describe a supramolecular polymer that can mediate efficient controlled delivery of Cas9 RNP in vitro and in vivo. This supramolecular polymer system is prepared by complexing disulfide-bridged biguanidyl adamantine (Ad-SS-GD) with β-cyclodextrin-conjugated low-molecular-weight polyethyleneimime (CP) through supramolecular assembly to generate CP/Ad-SS-GD. Due to multiple, strong hydrogen bonding and salt bridge effects, CP/Ad-SS-GD well interact with Cas9 RNP to form stable nanocomplex CP/Ad-SS-GD/RNP, which can be readily released in the reductive intracellular milieu as a result of the cleavage of disulfide bonds. The supramolecular polymer ensures the efficient intracellular delivery and the release of Cas9 RNP into 293T cells and colorectal cancer (CRC) cells, thus displaying high genome-editing activity in vitro. Importantly, we also found that hyaluronic acid (HA)-decorated CP/Ad-SS-GD/RNP nanocomplexes targeting mutant KRAS effectively inhibit tumor growth as well as metastasis in the tumor-bearing mouse models. Collectively, our findings provide a promising therapeutic strategy against mutant KRAS for the treatment of CRC-activated RAS pathways, offering a new therapeutic genome-editing modality for the colorectal cancer treatment.},
}
@article {pmid32169520,
year = {2020},
author = {Satyam, R and Bhardwaj, T and Jha, NK and Jha, SK and Nand, P},
title = {Toward a chimeric vaccine against multiple isolates of Mycobacteroides - An integrative approach.},
journal = {Life sciences},
volume = {250},
number = {},
pages = {117541},
doi = {10.1016/j.lfs.2020.117541},
pmid = {32169520},
issn = {1879-0631},
mesh = {Alleles ; B-Lymphocytes/immunology ; Bacterial Vaccines/*chemistry ; Bacteriophages ; CRISPR-Cas Systems ; Computational Biology ; Drug Resistance, Bacterial ; Epitopes ; Epitopes, T-Lymphocyte/genetics ; Gastrointestinal Microbiome ; Genome, Bacterial ; Genomics ; Gram-Positive Bacterial Infections/*prevention &amp; control ; Histocompatibility Antigens Class I/metabolism ; Histocompatibility Antigens Class II/metabolism ; Humans ; Immunotherapy ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Mycobacteriaceae/*genetics/pathogenicity ; Proteome ; *Vaccinology ; Virulence ; },
abstract = {AIM: Nontuberculous mycobacterial (NTM) infection such as endophthalmitis, dacryocystitis, and canaliculitis are pervasive across the globe and are currently managed by antibiotics. However, the recent cases of Mycobacteroides developing drug resistance reported along with the improper practice of medicine intrigued us to explore its genomic and proteomic canvas at a global scale and develop a chimeric vaccine against Mycobacteroides.<br><br>MAIN METHODS: We carried out a vivid genomic study on five recently sequenced strains of Mycobacteroides and explored their Pan-core genome/proteome in three different phases. The promiscuous antigenic proteins were identified via a subtractive proteomics approach that qualified for virulence causation, resistance and essentiality factors for this notorious bacterium. An integrated pipeline was developed for the identification of B-Cell, MHC (Major histocompatibility complex) class I and II epitopes.<br><br>KEY FINDINGS: Phase I identified the shreds of evidence of reductive evolution and propensity of the Pan-genome of Mycobacteroides getting closed soon. Phase II and Phase III produced 8 vaccine constructs. Our final vaccine construct, V6 qualified for all tests such as absence for allergenicity, presence of antigenicity, etc. V6 contains &#x3b2;-defensin as an adjuvant, linkers, Lysosomal-associated membrane protein 1 (LAMP1) signal peptide, and PADRE (Pan HLA-DR epitopes) amino acid sequence. Besides, V6 also interacts with a maximum number of MHC molecules and the TLR4/MD2 (Toll-like receptor 4/Myeloid differentiation factor 2) complex confirmed by docking and molecular dynamics simulation studies.<br><br>SIGNIFICANCE: The knowledge harnessed from the current study can help improve the current treatment regimens or in an event of an outbreak and propel further related studies.},
}
@article {pmid32168764,
year = {2020},
author = {Xu, Y and Liu, H and Pan, H and Wang, X and Zhang, Y and Yao, B and Li, N and Lai, L and Li, Z},
title = {CRISPR/Cas9-mediated Disruption of Fibroblast Growth Factor 5 in Rabbits Results in a Systemic Long Hair Phenotype by Prolonging Anagen.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32168764},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Exons/genetics ; Fibroblast Growth Factor 5/*genetics ; Hair/*growth &amp; development ; Hair Follicle/*growth &amp; development/metabolism ; Phenotype ; RNA, Messenger/genetics ; Rabbits ; },
abstract = {Hair growth and morphology are generally regulated by the hair cycle in mammals. Fibroblast Growth Factor 5 (FGF5), which is a hair cycle regulator, has a role in regulating the hair cycle during the transition from the anagen phase to the catagen phase, and a hereditary long hair phenotype has been widely reported when FGF5 is mutated in humans and other species. However, there has been no such report in rabbits. Thus, the first exon of rabbit FGF5 was disrupted by the CRISPR/Cas9 system, and the phenotype of FGF5[-/-] rabbits was characterized while using hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, quantitative PCR, scanning electron microscopy, and western blotting. The results showed a significant and systemic long hair phenotype in the FGF5[-/-] rabbits, which indicated that FGF5 is a negative regulator of hair growth. In addition, a decreased diameter of the fiber and a higher area proportion of hair follicle clusters were determined in FGF5[-/-] rabbits as compared with the WT rabbits. Further investigation verified that prolonging the anagen phase in rabbits, with decreased BMP2/4 pathway signaling and increased VERSICAN pathway signaling, caused the systemic long hair phenotype. Taken together, these results indicate a systemic long hair phenotype by prolonging anagen in FGF5[-/-] rabbits, which could be widely used for Fur production and an ideal model for studying the mechanism of long hair in the future.},
}
@article {pmid32168334,
year = {2020},
author = {Fasulo, B and Meccariello, A and Morgan, M and Borufka, C and Papathanos, PA and Windbichler, N},
title = {A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters.},
journal = {PLoS genetics},
volume = {16},
number = {3},
pages = {e1008647},
pmid = {32168334},
issn = {1553-7404},
support = {BB/P000843/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Drosophila Proteins/genetics ; Drosophila melanogaster/genetics ; Endonucleases/genetics ; Female ; Gene Editing/*methods ; Male ; Models, Animal ; Pest Control, Biological/methods ; Sex Determination Processes/*genetics ; Sex Preselection/*methods ; Sex Ratio ; Spermatogenesis/genetics ; X Chromosome/genetics ; },
abstract = {Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.},
}
@article {pmid32167008,
year = {2020},
author = {Yu, L and Wang, L and Yi, H and Wu, X},
title = {Beneficial effects of LRP6-CRISPR on prevention of alcohol-related liver injury surpassed fecal microbiota transplant in a rat model.},
journal = {Gut microbes},
volume = {11},
number = {4},
pages = {1015-1029},
pmid = {32167008},
issn = {1949-0984},
mesh = {Animals ; Bacteria/classification/genetics/*growth &amp; development/isolation &amp; purification ; *CRISPR-Cas Systems ; Dysbiosis/microbiology ; *Fecal Microbiota Transplantation ; Feces/microbiology ; *Gastrointestinal Microbiome ; Genetic Therapy ; Liver Cirrhosis, Alcoholic/microbiology/*prevention &amp; control ; Liver Diseases, Alcoholic/microbiology/*therapy ; Low Density Lipoprotein Receptor-Related Protein-6/*genetics/metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Symbiosis ; },
abstract = {Alcohol intake can modify gut microbiota composition, increase gut permeability, and promote liver fibrogenesis. LRP6 is a signal transmembrane protein and a co-receptor for the canonical Wnt signaling pathway. This study compared the curative effect of LRP6-CRISPR on alcohol-related liver injury with that of traditional fecal microbiota transplant (FMT) and investigated the alteration of the gut microbiome following the treatment. A rat model of alcohol-related liver injury was established and injected with lentiviral vectors expressing LRP6-CRISPR or administered with fecal filtrate from healthy rats, with healthy rat served as the control. Liver tissues of rats were examined by HE staining, Sirius staining, and Oil red O staining, respectively. The expression of LRP6 and fibrosis biomarkers were tested by PCR. The fecal sample of rats was collected and examined by 16S rRNA sequencing. Our data indicated that LRP6-CRISPR was more efficient in the prevention of alcohol-related liver injury than FMT. Microbiome analysis showed that alcohol-related liver injury related to gut microbiota dysbiosis, while treatment with LRP6-CRISPR or FMT increased gut microflora diversity and improved gut symbiosis. Further, bacteria specific to the disease stages were identified. Genera Romboutsia, Escherichia-Shigella, Pseudomonas, Turicibacter, and Helicobacter were prevalent in the intestine of rats with alcohol-related liver injury, while the domination of Lactobacillus was found in rats treated with LRP6-CRISPR or FMT. Besides, Lactobacillus and genera belonging to family Lachnospiraceae, Bacteroidales S24-7 group, and Ruminococcaceae were enriched in healthy rats. LRP6-CRISPR and FMT have beneficial effects on the prevention of alcohol-related liver injury, and correspondently, both treatments altered the disrupted gut microflora to a healthy one.},
}
@article {pmid32166879,
year = {2020},
author = {Tanihara, F and Hirata, M and Thi Nguyen, N and Anh Le, Q and Hirano, T and Otoi, T},
title = {Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics.},
journal = {Molecular reproduction and development},
volume = {87},
number = {4},
pages = {471-481},
doi = {10.1002/mrd.23335},
pmid = {32166879},
issn = {1098-2795},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Diabetes Mellitus ; Disease Models, Animal ; Embryo Transfer ; Female ; Gene Editing/*methods ; Homeodomain Proteins/*genetics/metabolism ; Male ; *Mosaicism ; Mutation Rate ; Pancreas/metabolism/pathology ; *Phenotype ; Semen/metabolism ; Spermatozoa/metabolism ; Swine/*genetics ; Trans-Activators/*genetics/metabolism ; Zygote/metabolism ; },
abstract = {Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.},
}
@article {pmid32165679,
year = {2020},
author = {Rohiwal, SS and Dvorakova, N and Klima, J and Vaskovicova, M and Senigl, F and Slouf, M and Pavlova, E and Stepanek, P and Babuka, D and Benes, H and Ellederova, Z and Stieger, K},
title = {Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {4619},
pmid = {32165679},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell Survival ; Chemical Phenomena ; Colloids ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; *Gene Transfer Techniques ; Genes, Reporter ; HEK293 Cells ; Humans ; *Magnetite Nanoparticles/chemistry/ultrastructure ; Particle Size ; Plasmids/genetics ; *Polyethyleneimine/chemistry ; Static Electricity ; Transfection/*methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.},
}
@article {pmid32165588,
year = {2020},
author = {Loganathan, SK and Schleicher, K and Malik, A and Quevedo, R and Langille, E and Teng, K and Oh, RH and Rathod, B and Tsai, R and Samavarchi-Tehrani, P and Pugh, TJ and Gingras, AC and Schramek, D},
title = {Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.},
journal = {Science (New York, N.Y.)},
volume = {367},
number = {6483},
pages = {1264-1269},
doi = {10.1126/science.aax0902},
pmid = {32165588},
issn = {1095-9203},
support = {//Canadian Institute of Health Research/International ; },
mesh = {ADAM10 Protein/genetics ; Amyloid Precursor Protein Secretases/genetics ; Animals ; CRISPR-Cas Systems ; Female ; *Genes, Tumor Suppressor ; *Genetic Predisposition to Disease ; Genetic Testing ; HEK293 Cells ; Head and Neck Neoplasms/*genetics ; Humans ; LIM Domain Proteins/genetics ; Male ; Membrane Proteins/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Mutation ; Receptors, Notch/genetics ; Signal Transduction/genetics ; Squamous Cell Carcinoma of Head and Neck/*genetics ; Tumor Suppressor Proteins/*genetics ; },
abstract = {In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.},
}
@article {pmid32165308,
year = {2020},
author = {Rothschild, SC and Ingram, SR and Lu, FI and Thisse, B and Thisse, C and Parkerson, JA and Tombes, RM},
title = {Genetic compensation of γ CaMKII, an evolutionarily conserved gene.},
journal = {Gene},
volume = {742},
number = {},
pages = {144567},
doi = {10.1016/j.gene.2020.144567},
pmid = {32165308},
issn = {1879-0038},
mesh = {Animals ; Animals, Genetically Modified ; Biological Evolution ; CRISPR-Cas Systems/genetics ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/*genetics ; Embryo, Nonmammalian ; *Gene Expression Regulation, Developmental ; Loss of Function Mutation ; Mutagenesis ; Zebrafish/*genetics/growth &amp; development ; Zebrafish Proteins/*genetics ; },
abstract = {CaMKII is a Ca[2+]/CaM-dependent protein kinase encoded by a family of conserved genes found throughout all metazoan species and expressed from fertilization into adulthood. One of these genes, camk2g1, is particularly important during early development as determined by pharmacologic, dominant negative and antisense morpholino approaches in zebrafish. Four other teleost fish species (cavefish, medaka, stickleback, and tilapia), exhibit sequence conservation of camk2g1 and duplication of the same CaMKII genes. A homozygous mutant of camk2g1 was generated in zebrafish using TALEN technology but yielded none of the phenotypic alterations seen using all other approaches and was reproductively viable. However, these camk2g1 mutant embryos showed a 4-fold over-expression of its paralog camk2g2. None of the other camk2 genes showed such transcriptional elevation, in fact, some of these genes were suppressed to 10% of wild type levels. In contrast, G0 camk2g1 CRISPR/Cas9 embryos recapitulated nearly all of the altered phenotypes observed in camk2g1 morphants, including renal, aural and ciliary defects. These findings validate the importance of this gene family during early zebrafish development and provide evidence for gene-specific transcriptional cross-talk consistent with genetic compensation.},
}
@article {pmid32164660,
year = {2020},
author = {Champer, J and Kim, IK and Champer, SE and Clark, AG and Messer, PW},
title = {Performance analysis of novel toxin-antidote CRISPR gene drive systems.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {27},
pmid = {32164660},
issn = {1741-7007},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Antidotes/*pharmacology ; Antitoxins/*pharmacology ; *CRISPR-Cas Systems ; Gene Drive Technology/*methods ; Genes, Essential ; Haploinsufficiency ; Models, Genetic ; },
abstract = {BACKGROUND: CRISPR gene drive systems allow the rapid spread of a genetic construct throughout a population. Such systems promise novel strategies for the management of vector-borne diseases and invasive species by suppressing a target population or modifying it with a desired trait. However, current homing-type drives have two potential shortcomings. First, they can be thwarted by the rapid evolution of resistance. Second, they lack any mechanism for confinement to a specific target population. In this study, we conduct a comprehensive performance assessment of several new types of CRISPR-based gene drive systems employing toxin-antidote (TA) principles, which should be less prone to resistance and allow for the confinement of drives to a target population due to invasion frequency thresholds.<br><br>RESULTS: The underlying principle of the proposed CRISPR toxin-antidote gene drives is to disrupt an essential target gene while also providing rescue by a recoded version of the target as part of the drive allele. Thus, drive alleles tend to remain viable, while wild-type targets are disrupted and often rendered nonviable, thereby increasing the relative frequency of the drive allele. Using individual-based simulations, we show that Toxin-Antidote Recessive Embryo (TARE) drives targeting an haplosufficient but essential gene (lethal when both copies are disrupted) can enable the design of robust, regionally confined population modification strategies with high flexibility in choosing promoters and targets. Toxin-Antidote Dominant Embryo (TADE) drives require a haplolethal target gene and a germline-restricted promoter, but they could permit faster regional population modification and even regionally confined population suppression. Toxin-Antidote Dominant Sperm (TADS) drives can be used for population modification or suppression. These drives are expected to spread rapidly and could employ a variety of promoters, but unlike TARE and TADE, they would not be regionally confined and also require highly specific target genes.<br><br>CONCLUSIONS: Overall, our results suggest that CRISPR-based TA gene drives provide promising candidates for flexible ecological engineering strategies in a variety of organisms.},
}
@article {pmid32164255,
year = {2020},
author = {Lanigan, TM and Kopera, HC and Saunders, TL},
title = {Principles of Genetic Engineering.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32164255},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Targeting/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods/standards/trends ; Humans ; },
abstract = {Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.},
}
@article {pmid32163387,
year = {2020},
author = {Gerashchenkov, GA and Rozhnova, NA and Kuluev, BR and Kiryanova, OY and Gumerova, GR and Knyazev, AV and Vershinina, ZR and Mikhailova, EV and Chemeris, DA and Matniyazov, RT and Baimiev, AK and Gubaidullin, IM and Baimiev, AK and Chemeris, AV},
title = {[Design of Guide RNA for CRISPR/Cas Plant Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {54},
number = {1},
pages = {29-50},
doi = {10.31857/S0026898420010061},
pmid = {32163387},
issn = {0026-8984},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.},
}
@article {pmid32162131,
year = {2020},
author = {Safier, LZ and Zuccaro, MV and Egli, D},
title = {Efficient SNP editing in haploid human pluripotent stem cells.},
journal = {Journal of assisted reproduction and genetics},
volume = {37},
number = {4},
pages = {735-745},
pmid = {32162131},
issn = {1573-7330},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Centromere/genetics ; Embryonic Stem Cells/cytology ; Gene Editing ; Haploidy ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Mutation/genetics ; Organic Anion Transporters, Sodium-Dependent/*genetics ; Parthenogenesis/genetics ; RNA, Guide/*genetics ; Symporters/*genetics ; Whole Exome Sequencing ; },
abstract = {PURPOSE: To correct a potentially damaging mutation in haploid human embryonic stem cells.<br><br>METHODS: Exome sequencing was performed on DNA extracted from parthenogenetically derived embryonic stem cell line (pES12). An SLC10A2 gene mutation, which affects bile acid transport, was chosen as mutation of interest in this proof of concept study to attempt correction in human pluripotent haploid cells. Confirmation of the mutation was verified, and guide RNA and a correction template was designed in preparation of performing CRISPR. Haploid cells underwent serial fluorescence activated cell sorting (FACS) with Hoechst 33342 to create an increasingly haploid (1n) enriched culture. Nucleofection was performed on p. 37 and then cells were sorted for 1n DNA content with +GFP to identify the haploid cells that expressed Cas9 tagged with GFP.<br><br>RESULTS: 104,686 haploid GFP + cells were collected. Cells were cultured, individual colonies picked, and 48 clones were sent for Sanger sequencing. CRIPSR efficiency was 77.1%, with 7/48 (14.6%) clones resulting in a corrected SLC10A2 mutation. Confirmation of persistence of haploid cells was achieved with repeated FACS sorting and centromere quantification. Given the large number of passages and exposure to CRISPR, we also performed analysis of karyotypes and of off-target effects. Cells evaluated were karyotypically normal and there was no evident off target effects.<br><br>CONCLUSIONS: CRISPR/Cas9 can be effectively utilized to edit mutations in haploid human embryonic stem cells. Establishment and maintenance of a haploid cell culture provides a novel way to utilize CRISPR/Cas9 in gene editing, particularly in the study of recessive alleles.},
}
@article {pmid32161259,
year = {2020},
author = {Mahameed, M and Boukeileh, S and Obiedat, A and Darawshi, O and Dipta, P and Rimon, A and McLennan, G and Fassler, R and Reichmann, D and Karni, R and Preisinger, C and Wilhelm, T and Huber, M and Tirosh, B},
title = {Pharmacological induction of selective endoplasmic reticulum retention as a strategy for cancer therapy.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1304},
pmid = {32161259},
issn = {2041-1723},
mesh = {Acetamides/pharmacology/therapeutic use ; Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/*drug therapy/pathology ; Cell Line, Tumor ; Cyclohexylamines/pharmacology/therapeutic use ; Endoplasmic Reticulum/*drug effects/metabolism ; Endoplasmic Reticulum Stress/drug effects ; Gene Knockout Techniques ; Golgi Apparatus/metabolism ; Humans ; Liver Neoplasms/*drug therapy/pathology ; Male ; Membrane Proteins/genetics/metabolism ; Mice ; Molecular Chaperones/genetics/metabolism ; Nelfinavir/pharmacology/therapeutic use ; Xenograft Model Antitumor Assays ; eIF-2 Kinase/genetics/*metabolism ; },
abstract = {The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.},
}
@article {pmid32161257,
year = {2020},
author = {Dubois, ML and Meller, A and Samandi, S and Brunelle, M and Frion, J and Brunet, MA and Toupin, A and Beaudoin, MC and Jacques, JF and Lévesque, D and Scott, MS and Lavigne, P and Roucou, X and Boisvert, FM},
title = {UBB pseudogene 4 encodes functional ubiquitin variants.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1306},
pmid = {32161257},
issn = {2041-1723},
support = {398925//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Division ; Cell Nucleus/metabolism ; Cloning, Molecular ; Datasets as Topic ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Lamin Type A/*metabolism ; Proteomics ; Pseudogenes/*genetics ; RNA-Seq ; Ubiquitin/*genetics/metabolism ; Ubiquitination ; },
abstract = {Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes Ub[KEKS] (Q2K, K33E, Q49K, N60S). The functional consequences of Ub[KEKS] conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that Ub[KEKS] modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.},
}
@article {pmid32161190,
year = {2020},
author = {Martin, E and Minet, N and Boschat, AC and Sanquer, S and Sobrino, S and Lenoir, C and de Villartay, JP and Leite-de-Moraes, M and Picard, C and Soudais, C and Bourne, T and Hambleton, S and Hughes, SM and Wynn, RF and Briggs, TA and , and Patel, S and Lawrence, MG and Fischer, A and Arkwright, PD and Latour, S},
title = {Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation.},
journal = {JCI insight},
volume = {5},
number = {5},
pages = {},
pmid = {32161190},
issn = {2379-3708},
support = {/WT_/Wellcome Trust/United Kingdom ; /CRUK_/Cancer Research UK/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Carbon-Nitrogen Ligases/*genetics ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; *Homozygote ; Humans ; Immunophenotyping ; Jurkat Cells ; Lymphocyte Activation ; Lymphocytes/*immunology ; *Mutation ; },
abstract = {Cytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%-90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma.},
}
@article {pmid32161176,
year = {2020},
author = {Reddy, VRAP and Sadigh, Y and Tang, N and Yao, Y and Nair, V},
title = {Novel Insights into the Roles of Bcl-2 Homolog Nr-13 (vNr-13) Encoded by Herpesvirus of Turkeys in the Virus Replication Cycle, Mitochondrial Networks, and Apoptosis Inhibition.},
journal = {Journal of virology},
volume = {94},
number = {10},
pages = {},
pmid = {32161176},
issn = {1098-5514},
support = {BBS/E/I/00007032/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014262/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/K002465/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007030/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Apoptosis/*physiology ; Avian Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Chickens/metabolism ; Endoplasmic Reticulum/metabolism ; Herpesviridae/*genetics ; Herpesviridae Infections/virology ; Lymphoma, B-Cell/immunology ; Membrane Proteins/genetics/*metabolism ; Mitochondria/*metabolism ; Poultry Diseases/virology ; Sequence Alignment ; Sequence Analysis ; Turkeys/*virology ; Viral Proteins/genetics/*metabolism ; Viral Vaccines/immunology ; Virus Replication/*physiology ; },
abstract = {The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-ΔvNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-ΔvNr-13 showed similar growth kinetics; however, at early time points, HVT-ΔvNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-ΔvNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells.IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek's disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.},
}
@article {pmid32161170,
year = {2020},
author = {Seltzer, J and Moorad, R and Schifano, JM and Landis, JT and Dittmer, DP},
title = {Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma.},
journal = {Journal of virology},
volume = {94},
number = {10},
pages = {},
pmid = {32161170},
issn = {1098-5514},
support = {P01 CA019014/CA/NCI NIH HHS/United States ; R01 CA163217/CA/NCI NIH HHS/United States ; R01 CA239583/CA/NCI NIH HHS/United States ; R01 DE018304/DE/NIDCR NIH HHS/United States ; },
mesh = {B-Lymphocytes ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Herpesvirus 8, Human/*genetics/physiology ; Humans ; Interleukin-1 Receptor-Associated Kinases/*genetics/*metabolism ; Interleukin-10/metabolism ; Interleukin-1beta/metabolism ; Lymphoma, Primary Effusion/*metabolism/*virology ; Myeloid Differentiation Factor 88/*genetics/*metabolism ; NF-kappa B/metabolism ; Sequence Analysis ; Signal Transduction/*genetics ; Transcriptome ; },
abstract = {Kaposi sarcoma-associated herpesvirus (KSHV) is necessary but not sufficient for primary effusion lymphoma (PEL) development. Alterations in cellular signaling pathways are also a characteristic of PEL. Other B cell lymphomas have acquired an oncogenic mutation in the myeloid differentiation primary response 88 (MYD88) gene. The MYD88 L265P mutant results in the activation of interleukin-1 receptor associated kinase (IRAK). To probe IRAK/MYD88 signaling in PEL, we employed CRISPR/Cas9 technology to generate stable deletion clones in BCBL-1Cas9 and BC-1Cas9 cells. To look for off-target effects, we determined the complete exome of the BCBL-1Cas9 and BC-1Cas9 cells. Deletion of either MYD88, IRAK4, or IRAK1 abolished interleukin-1 beta (IL-1β) signaling; however, we were able to grow stable subclones from each population. Transcriptome sequencing (RNA-seq) analysis of IRAK4 knockout cell lines (IRAK4 KOs) showed that the IRAK pathway induced cellular signals constitutively, independent of IL-1β stimulation, which was abrogated by deletion of IRAK4. Transient complementation with IRAK1 increased NF-κB activity in MYD88 KO, IRAK1 KO, and IRAK4 KO cells even in the absence of IL-1β. IL-10, a hallmark of PEL, was dependent on the IRAK pathway, as IRAK4 KOs showed reduced IL-10 levels. We surmise that, unlike B cell receptor (BCR) signaling, MYD88/IRAK signaling is constitutively active in PEL, but that under cell culture conditions, PEL rapidly became independent of this pathway.IMPORTANCE One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.},
}
@article {pmid32161149,
year = {2020},
author = {Peters, JM},
title = {mSphere of Influence: Comprehensive Genetic Analysis.},
journal = {mSphere},
volume = {5},
number = {2},
pages = {},
pmid = {32161149},
issn = {2379-5042},
mesh = {Bacillus subtilis/genetics ; CRISPR-Cas Systems ; *Gene Regulatory Networks ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Jason M. Peters works in the fields of antibiotic resistance and biofuel production. In this mSphere of Influence article, he reflects on how the paper "A global genetic interaction network maps a wiring diagram of cellular function" by Costanzo et al. (Science 353:aaf1420, 2016, https://doi.org/10.1126/science.aaf1420) has impacted his work by highlighting the power of gene networks to uncover new biology.},
}
@article {pmid32161115,
year = {2020},
author = {Murugan, K and Seetharam, AS and Severin, AJ and Sashital, DG},
title = {CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {17},
pages = {5538-5553},
pmid = {32161115},
issn = {1083-351X},
mesh = {Acidaminococcus/*enzymology/genetics/metabolism ; Bacterial Proteins/genetics/*metabolism ; Base Pair Mismatch ; Base Sequence ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/*genetics/metabolism ; DNA Cleavage ; Endodeoxyribonucleases/genetics/*metabolism ; Francisella/*enzymology/genetics/metabolism ; Gene Editing/methods ; Gene Expression ; },
abstract = {Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.},
}
@article {pmid32160917,
year = {2020},
author = {Tang, X and Suo, J and Liang, L and Duan, C and Hu, D and Gu, X and Yu, Y and Liu, X and Cui, S and Suo, X},
title = {Genetic modification of the protozoan Eimeria tenella using the CRISPR/Cas9 system.},
journal = {Veterinary research},
volume = {51},
number = {1},
pages = {41},
pmid = {32160917},
issn = {1297-9716},
mesh = {*CRISPR-Cas Systems ; Eimeria tenella/*genetics ; Gene Editing/*veterinary ; *Genes, Protozoan ; },
abstract = {Eimeria tenella has emerged as valuable model organism for studying the biology and immunology of protozoan parasites with the establishment of the reverse genetic manipulation platform. In this report, we described the application of CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (endonuclease) system for efficient genetic editing in E. tenella, and showed that the CRISPR/Cas9 system mediates site-specific double-strand DNA breaks with a single guide RNA. Using this system, we successfully tagged the endogenous microneme protein 2 (EtMic2) by inserting the red fluorescent protein into the C-terminal of EtMic2. Our results extended the utility of the CRISPR/Cas9-mediated genetic modification system to E. tenella, and opened a new avenue for targeted investigation of gene functions in apicomplexan parasites.},
}
@article {pmid32160575,
year = {2020},
author = {Maki, JA and Cavallin, JE and Lott, KG and Saari, TW and Ankley, GT and Villeneuve, DL},
title = {A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas).},
journal = {Aquatic toxicology (Amsterdam, Netherlands)},
volume = {222},
number = {},
pages = {105464},
pmid = {32160575},
issn = {1879-1514},
support = {EPA999999//Intramural EPA/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cyprinidae/*genetics/growth &amp; development/metabolism ; Embryo, Nonmammalian/*drug effects/enzymology ; *Embryonic Development/drug effects/genetics ; Melanins/genetics ; Monophenol Monooxygenase/genetics ; Mutation ; Phenotype ; Pigmentation/genetics ; Water Pollutants, Chemical/*toxicity ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.},
}
@article {pmid32160520,
year = {2020},
author = {Shinohara, T and Kanatsu-Shinohara, M},
title = {Transgenesis and Genome Editing of Mouse Spermatogonial Stem Cells by Lentivirus Pseudotyped with Sendai Virus F Protein.},
journal = {Stem cell reports},
volume = {14},
number = {3},
pages = {447-461},
pmid = {32160520},
issn = {2213-6711},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Gene Transfer Techniques ; *Genome ; Kinetics ; Lentivirus/*metabolism ; Male ; Mice, Transgenic ; Phenotype ; Sendai virus/*metabolism ; Sertoli Cells/metabolism ; Spermatogenesis/genetics ; Spermatogonia/*cytology ; Stem Cells/*metabolism ; Viral Fusion Proteins/*metabolism ; Virus Integration ; },
abstract = {Spermatogonial stem cells (SSCs) serve as a resource for producing genetically modified animals. However, genetic manipulation of SSCs has met with limited success. Here, we show efficient gene transfer into SSCs via a lentivirus (FV-LV) using a fusion protein (F), a Sendai virus (SV) envelope protein involved in virion/cell membrane fusion. FV-LVs transduced cultured SSCs more efficiently than conventional LVs. Although SSCs infected with SV failed to produce offspring, those transduced with FV-LVs were fertile. In vivo microinjection showed that FV-LVs could penetrate not only the basement membrane of the seminiferous tubules but also the blood-testis barrier, which resulted in successful transduction of both spermatogenic cells and testicular somatic cells. Cultured SSCs transfected with FV-LVs that express drug-inducible CRISPR/Cas9 against Kit or Sycp3 showed impaired spermatogenesis upon transplantation and drug treatment in vivo. Thus, FV-LVs provide an efficient method for functional analysis of genes involved in SSCs and spermatogenesis.},
}
@article {pmid32160465,
year = {2020},
author = {Liow, LT and Go, MK and Chang, MW and Yew, WS},
title = {Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum.},
journal = {ACS synthetic biology},
volume = {9},
number = {4},
pages = {953-961},
doi = {10.1021/acssynbio.0c00064},
pmid = {32160465},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Chromobacterium/genetics/metabolism ; Cyanides/isolation &amp; purification/metabolism ; Drug Resistance, Bacterial/genetics ; Electronic Waste ; Genetic Engineering/*methods ; Gold/isolation &amp; purification/metabolism ; Promoter Regions, Genetic/genetics ; Synthetic Biology/*methods ; },
abstract = {Chromobacterium violaceum has been of interest recently due to its cyanogenic ability and its potential role in environmental sustainability via the biorecovery of gold from electronic waste. However, as with many nonmodel bacteria, there are limited genetic tools to implement the use of this Gram-negative chassis in synthetic biology. We propose a system that involves assaying spontaneous antibiotic resistances and using broad host range vectors to develop episomal vectors for nonmodel Gram-negative bacteria. These developed vectors can subsequently be used to characterize inducible promoters for gene expressions and implementing CRISPRi to inhibit endogenous gene expression for further studies. Here, we developed the first episomal genetic toolkit for C. violaceum consisting of two origins of replication, three antibiotic resistance genes, and four inducible promoter systems. We examined the occurrences of spontaneous resistances of the bacterium to the chosen selection markers to prevent incidences of false positives. We also tested broad host range vectors from four different incompatibility groups and characterized four inducible promoter systems, which potentially can be applied in other Gram-negative nonmodel bacteria. CRISPRi was also implemented to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building in this chassis and other nonmodel bacteria for synthetic biology and biotechnological applications.},
}
@article {pmid32160419,
year = {2020},
author = {Huang, H and Zhong, L and Zhou, J and Hou, Y and Zhang, Z and Xing, X and Sun, J},
title = {Leydig-like cells derived from reprogrammed human foreskin fibroblasts by CRISPR/dCas9 increase the level of serum testosterone in castrated male rats.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {7},
pages = {3971-3981},
pmid = {32160419},
issn = {1582-4934},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Castration ; Cellular Reprogramming/*genetics ; Fibroblasts/cytology/metabolism ; Foreskin/cytology/metabolism ; Humans ; Leydig Cells/cytology/metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis/*genetics ; Testosterone/*blood ; },
abstract = {In the past few years, Leydig cell (LC) transplantation has been regarded as an effective strategy for providing physiological patterns of testosterone in vivo. Recently, we have successfully converted human foreskin fibroblasts (HFFs) into functional Leydig-like cells (iLCs) in vitro by using the CRISPR/dCas9 system, which shows promising potential for seed cells. However, it is not known whether the reprogrammed iLCs can survive or restore serum testosterone levels in vivo. Therefore, in this study, we evaluate whether reprogrammed iLCs can restore the serum testosterone levels of castrated rats when they are transplanted into the fibrous capsule. We first developed the castrated Sprague Dawley rat model through bilateral orchiectomy and subsequently injected extracellular matrix gel containing transplanted cells into the fibrous capsule of castrated rats. Finally, we evaluated dynamic serum levels of testosterone and luteinizing hormone (LH) in castrated rats, the survival of implanted iLCs, and the expression levels of Leydig steroidogenic enzymes by immunofluorescence staining and Western blotting. Our results demonstrated that implanted iLCs could partially restore the serum testosterone level of castrated rats, weakly mimic the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis for a short period, and survive and secrete testosterone, through 6 weeks after transplantation. Therefore, this study may be valuable for treating male hypogonadism in the future.},
}
@article {pmid32160370,
year = {2020},
author = {Zhou, W and Brown, W and Bardhan, A and Delaney, M and Ilk, AS and Rauen, RR and Kahn, SI and Tsang, M and Deiters, A},
title = {Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {23},
pages = {8998-9003},
pmid = {32160370},
issn = {1521-3773},
support = {R21 HD085206/HD/NICHD NIH HHS/United States ; R01GM112728/NH/NIH HHS/United States ; T32GM088119/NH/NIH HHS/United States ; R01 GM112728/GM/NIGMS NIH HHS/United States ; T32 GM088119/GM/NIGMS NIH HHS/United States ; R21HD085206/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing/*methods ; Nucleic Acid Hybridization ; RNA, Guide/*genetics ; Spatio-Temporal Analysis ; Time Factors ; *Zebrafish ; },
abstract = {We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5'-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.},
}
@article {pmid32159950,
year = {2020},
author = {Aman, R and Mahas, A and Mahfouz, M},
title = {Nucleic Acid Detection Using CRISPR/Cas Biosensing Technologies.},
journal = {ACS synthetic biology},
volume = {9},
number = {6},
pages = {1226-1233},
doi = {10.1021/acssynbio.9b00507},
pmid = {32159950},
issn = {2161-5063},
mesh = {Animals ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; DNA/*analysis/metabolism ; Gene Editing ; Humans ; RNA, Guide/metabolism ; Virus Diseases/diagnosis ; },
abstract = {For infectious diseases, rapid and accurate identification of the pathogen is critical for effective management and treatment, but diagnosis remains challenging, particularly in resource-limited areas. Methods that accurately detect pathogen nucleic acids can provide robust, accurate, rapid, and ultrasensitive technologies for point-of-care diagnosis of pathogens, and thus yield information that is invaluable for disease management and treatment. Several technologies, mostly PCR-based, have been employed for pathogen detection; however, these require expensive reagents and equipment, and skilled personnel. CRISPR/Cas systems have been used for genome editing, based on their ability to accurately recognize and cleave specific DNA and RNA sequences. Moreover, following recognition of the target sequence, certain CRISPR/Cas systems including orthologues of Cas13, Cas12a, and Cas14 exhibit collateral nonspecific catalytic activities that can be employed for nucleic acid detection, for example by degradation of a labeled nucleic acid to produce a fluorescent signal. CRISPR/Cas systems are amenable to multiplexing, thereby enabling a single diagnostic test to identify multiple targets down to attomolar (10[-18] mol/L) concentrations of target molecules. Developing devices that couple CRISPR/Cas with lateral flow systems may allow inexpensive, accurate, highly sensitive, in-field deployable diagnostics. These sensors have myriad applications, from human health to agriculture. In this review, we discuss the recent advances in the field of CRISPR-based biosensing technologies and highlight insights of their potential use in a myriad of applications.},
}
@article {pmid32159922,
year = {2020},
author = {Fernández, A and Morín, M and Muñoz-Santos, D and Josa, S and Montero, A and Rubio-Fernández, M and Cantero, M and Fernández, J and Del Hierro, MJ and Castrillo, M and Moreno-Pelayo, M&#xc1; and Montoliu, L},
title = {Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents.},
journal = {Current protocols in mouse biology},
volume = {10},
number = {1},
pages = {e69},
doi = {10.1002/cpmo.69},
pmid = {32159922},
issn = {2161-2617},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genotyping Techniques/instrumentation/*methods ; Indicators and Reagents ; Mice ; Mice, Transgenic ; *Models, Animal ; },
abstract = {The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley &amp; Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.},
}
@article {pmid32159216,
year = {2020},
author = {Warma, A and Ndiaye, K},
title = {Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†.},
journal = {Biology of reproduction},
volume = {102},
number = {6},
pages = {1177-1190},
doi = {10.1093/biolre/ioaa030},
pmid = {32159216},
issn = {1529-7268},
mesh = {Animals ; Antibodies ; CRISPR-Cas Systems ; *Cattle ; Connective Tissue Growth Factor/genetics/metabolism ; Female ; Granulosa Cells ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; Mitogen-Activated Protein Kinase 3/genetics/metabolism ; Protein Serine-Threonine Kinases/genetics/*metabolism ; },
abstract = {Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.},
}
@article {pmid32159213,
year = {2020},
author = {Gao, B and Sabnis, R and Costantini, T and Jinkerson, R and Sun, Q},
title = {A peek in the micro-sized world: a review of design principles, engineering tools, and applications of engineered microbial community.},
journal = {Biochemical Society transactions},
volume = {48},
number = {2},
pages = {399-409},
doi = {10.1042/BST20190172},
pmid = {32159213},
issn = {1470-8752},
mesh = {Animals ; Biodegradation, Environmental ; Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; Gene Transfer Techniques ; Genetic Engineering/*methods ; Humans ; *Microbial Consortia ; *Microbial Interactions ; Synthetic Biology/*methods ; Waste Disposal, Fluid ; },
abstract = {Microbial communities drive diverse processes that impact nearly everything on this planet, from global biogeochemical cycles to human health. Harnessing the power of these microorganisms could provide solutions to many of the challenges that face society. However, naturally occurring microbial communities are not optimized for anthropogenic use. An emerging area of research is focusing on engineering synthetic microbial communities to carry out predefined functions. Microbial community engineers are applying design principles like top-down and bottom-up approaches to create synthetic microbial communities having a myriad of real-life applications in health care, disease prevention, and environmental remediation. Multiple genetic engineering tools and delivery approaches can be used to 'knock-in' new gene functions into microbial communities. A systematic study of the microbial interactions, community assembling principles, and engineering tools are necessary for us to understand the microbial community and to better utilize them. Continued analysis and effort are required to further the current and potential applications of synthetic microbial communities.},
}
@article {pmid32158257,
year = {2020},
author = {Du, T and Jia, X and Dong, X and Ru, X and Li, L and Wang, Y and Liu, J and Feng, G and Wen, T},
title = {Cosmc Disruption-Mediated Aberrant O-glycosylation Suppresses Breast Cancer Cell Growth via Impairment of CD44.},
journal = {Cancer management and research},
volume = {12},
number = {},
pages = {511-522},
pmid = {32158257},
issn = {1179-1322},
abstract = {BACKGROUND: Breast cancer remains the most lethal malignancy in women worldwide. Aberrant O-glycosylation is closely related to many human diseases, including breast carcinoma; however, its precise role in cancer development is insufficiently understood. Cosmc is an endoplasmic reticulum-localized chaperone that regulates the O-glycosylation of proteins. Cosmc dysfunction results in inactive T-synthase and expression of truncated O-glycans such as Tn antigen. Here we investigated the impact of Cosmc disruption-mediated aberrant O-glycosylation on breast cancer cell development through in vitro and in vivo experiments.<br><br>MATERIALS AND METHODS: We deleted the Cosmc gene in two breast cancer cell lines (MCF7, T47D) using the CRISPR/Cas-9 system and then measured the expression levels of Tn antigen. The proliferation of Tn-positive cells was examined by RTCA, colony formation and in vivo experiments. The effects of Cosmc deficiency on glycoprotein CD44 and MAPK pathway were also determined.<br><br>RESULTS: Both in vitro and in vivo studies showed that Cosmc deficiency markedly suppressed breast cancer cell growth compared with the corresponding controls. Mechanistically, Cosmc disruption impaired the protein expression of CD44 and the associated MAPK signaling pathway; the latter plays a crucial role in cell proliferation. Reconstitution of CD44 substantially reversed the observed alterations, confirming that CD44 requires normal O-glycosylation for its proper expression and activation of the related signaling pathway.<br><br>CONCLUSION: This study showed that Cosmc deficiency-mediated aberrant O-glycosylation suppressed breast cancer cell growth, which was likely mediated by the impairment of CD44 expression.},
}
@article {pmid32157873,
year = {2020},
author = {Li, J and Yang, S and Zuo, C and Dai, L and Guo, Y and Xie, G},
title = {Applying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow Concentrations.},
journal = {ACS sensors},
volume = {5},
number = {4},
pages = {970-977},
doi = {10.1021/acssensors.9b02305},
pmid = {32157873},
issn = {2379-3694},
mesh = {Biosensing Techniques/*methods ; CRISPR-Cas Systems/*physiology ; Humans ; },
abstract = {Efficient signal amplification is essential to construct ultrasensitive biosensors for biologically relevant species with abundant concomitant interferences. Here, we apply LbaCas12a as a signal amplifier to develop a versatile CRISPR-Cas12a platform to detect a wide range of analytes in ultralow concentrations. The platform relies on the indiscriminate single-stranded DNase activity of LbaCas12a, which recognizes single-stranded DNA intermediates generated by non-DNA targets down to femtomolar concentrations and subsequently enhances the fluorescence signal output. With the help of functional nucleotides (DNAzyme and aptamer), ultrasensitive bioassays for Pb[2+] and Acinetobacter baumannii have been designed with a limit of detection down to ∼0.053 nM and ∼3 CFU/mL, respectively. It also allows simultaneous detection of four microRNAs (miRNAs) at a picomolar concentration without significant interferences by other counterparts, suggesting the potential of multiplexed miRNA expression profiles analysis in high throughput. Given the versatility and generality of the CRISPR-Cas12a platform, we expect the current work to advance the application of CRISPR-Cas-based platforms in bioanalysis and provide new insights into ultrasensitive biosensor design.},
}
@article {pmid32157792,
year = {2020},
author = {Liu, X and Zhang, Y and Han, Y and Lu, W and Yang, J and Tian, J and Sun, P and Yu, T and Hu, Y and Zhang, H and Huang, P and Liu, P},
title = {Overexpression of GLT1D1 induces immunosuppression through glycosylation of PD-L1 and predicts poor prognosis in B-cell lymphoma.},
journal = {Molecular oncology},
volume = {14},
number = {5},
pages = {1028-1044},
pmid = {32157792},
issn = {1878-0261},
mesh = {Animals ; B7-H1 Antigen/*metabolism ; Biomarkers, Tumor/genetics/metabolism ; CD8-Positive T-Lymphocytes/enzymology/*immunology/*metabolism ; CRISPR-Cas Systems ; Carcinogenesis/*genetics/immunology ; Cell Line, Tumor ; Cell Survival/genetics ; Coculture Techniques ; Databases, Genetic ; Female ; Gene Knockdown Techniques ; Glycosylation ; Humans ; *Immunosuppression Therapy ; Lymphoma, B-Cell/genetics/*immunology/*metabolism/mortality ; Lymphoma, Large B-Cell, Diffuse/genetics/immunology/metabolism/mortality ; Mice ; Mice, Inbred C57BL ; Polysaccharides/metabolism ; Prognosis ; RNA, Small Interfering ; T-Lymphocytes/immunology ; Tunicamycin/pharmacology ; },
abstract = {B-cell non-Hodgkin's lymphoma (NHL) is a class of heterogeneous diseases with variable clinical outcomes. Immunosuppression is particularly common in the subtypes of lymphoma with poor prognosis, but the underlying mechanism remains unclear. Using a RT-PCR array analysis, we have identified that glycosyltransferase 1 domain-containing 1 (GLT1D1), an enzyme that transfers glycosyl groups to proteins, is highly upregulated in the incurable subtype of B-cell NHL and in early relapse diffuse large B-cell lymphoma. Analysis of clinical specimens revealed that GLT1D1 expression was positively correlated with the level of glycosylated programmed cell death-ligand 1 (PD-L1) in B-cell NHL and that high GLT1D1 expression was associated with poor prognosis. Mechanistically, we showed that GLT1D1 transferred N-linked glycans to PD-L1, thus promoting the immunosuppressive function of glycosylated PD-L1. Downregulation of GLT1D1 resulted in a decrease of glycosylated PD-L1 and enhanced cytotoxic T-cell function against lymphoma cells. In vivo, overexpression of GLT1D1 promoted tumor growth by facilitating tumor immune escape through increased levels of PD-L1. Our work has identified GLT1D1 as a predictive biomarker for B-cell NHL. It has also shown that this enzyme enhances PD-L1 stabilization via N-glycosylation, thus promoting immunosuppression and tumor growth. As such, GLT1D1 might be a novel therapeutic target for the treatment of B-NHL.},
}
@article {pmid32157557,
year = {2020},
author = {Guo, J and Zhang, Q and Su, Y and Lu, X and Wang, Y and Yin, M and Hu, W and Wen, W and Lei, QY},
title = {Arginine methylation of ribose-5-phosphate isomerase A senses glucose to promote human colorectal cancer cell survival.},
journal = {Science China. Life sciences},
volume = {63},
number = {9},
pages = {1394-1405},
pmid = {32157557},
issn = {1869-1889},
mesh = {Aldose-Ketose Isomerases/*metabolism ; Amino Acid Sequence ; Animals ; Arginine/*chemistry ; CRISPR-Cas Systems ; Catalytic Domain ; Cell Line, Tumor ; Cell Survival ; Colorectal Neoplasms/*metabolism ; Gene Knockout Techniques ; Glucose/*metabolism ; Humans ; Methylation ; Mice ; Mice, Nude ; NADP/metabolism ; Oxidation-Reduction ; Pentose Phosphate Pathway ; Protein Binding ; Protein-Arginine N-Methyltransferases/metabolism ; Reactive Oxygen Species/metabolism ; Up-Regulation ; },
abstract = {Cancer cells remodel their metabolic network to adapt to variable nutrient availability. Pentose phosphate pathway (PPP) plays protective and biosynthetic roles by oxidizing glucose to generate reducing power and ribose. How cancer cells modulate PPP activity in response to glucose supply remains unclear. Here we show that ribose-5-phosphate isomerase A (RPIA), an enzyme in PPP, directly interacts with co-activator associated arginine methyltransferase 1 (CARM1) and is methylated at arginine 42 (R42). R42 methylation up-regulates the catalytic activity of RPIA. Furthermore, glucose deprivation strengthens the binding of CARM1 with RPIA to induce R42 hypermethylation. Insufficient glucose supply links to RPIA hypermethylation at R42, which increases oxidative PPP flux. RPIA methylation supports ROS clearance by enhancing NADPH production and fuels nucleic acid synthesis by increasing ribose supply. Importantly, RPIA methylation at R42 significantly potentiates colorectal cancer cell survival under glucose starvation. Collectively, RPIA methylation connects glucose availability to nucleotide synthesis and redox homeostasis.},
}
@article {pmid32157225,
year = {2020},
author = {Ledford, H},
title = {CRISPR treatment inserted directly into the body for first time.},
journal = {Nature},
volume = {579},
number = {7798},
pages = {185},
doi = {10.1038/d41586-020-00655-8},
pmid = {32157225},
issn = {1476-4687},
mesh = {Antigens, Neoplasm/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytoskeletal Proteins/genetics ; Gene Editing/*methods/trends ; *Genetic Therapy ; Genetic Vectors/genetics ; Humans ; Leber Congenital Amaurosis/genetics/*therapy ; Mutation ; },
}
@article {pmid32157203,
year = {2020},
author = {Fenner, A},
title = {CRISPR-Cas9 ERβ deletion reveals roles in prostate.},
journal = {Nature reviews. Urology},
volume = {17},
number = {4},
pages = {192-193},
pmid = {32157203},
issn = {1759-4820},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Estrogen Receptor beta ; Male ; Mice ; Phenotype ; Prostate ; },
}
@article {pmid32156810,
year = {2020},
author = {Ma, H and Qian, W and Bambouskova, M and Collins, PL and Porter, SI and Byrum, AK and Zhang, R and Artyomov, M and Oltz, EM and Mosammaparast, N and Miner, JJ and Diamond, MS},
title = {Barrier-to-Autointegration Factor 1 Protects against a Basal cGAS-STING Response.},
journal = {mBio},
volume = {11},
number = {2},
pages = {},
pmid = {32156810},
issn = {2150-7511},
support = {R01 AI127774/AI/NIAID NIH HHS/United States ; R01 CA193318/CA/NCI NIH HHS/United States ; P01 CA092584/CA/NCI NIH HHS/United States ; R01 AI143982/AI/NIAID NIH HHS/United States ; R01 AI145296/AI/NIAID NIH HHS/United States ; R01 CA227001/CA/NCI NIH HHS/United States ; R01 AI134035/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; DNA-Binding Proteins/genetics/*immunology ; Gene Editing ; Gene Expression Regulation ; Homeostasis/immunology ; *Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Interferons/immunology ; Membrane Proteins/*immunology ; Mice ; Microglia/immunology ; Nuclear Proteins/genetics/*immunology ; Nucleotidyltransferases/*immunology ; Signal Transduction ; },
abstract = {Although the pathogen recognition receptor pathways that activate cell-intrinsic antiviral responses are well delineated, less is known about how the host regulates this response to prevent sustained signaling and possible immune-mediated damage. Using a genome-wide CRISPR-Cas9 screening approach to identify host factors that modulate interferon-stimulated gene (ISG) expression, we identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1), a previously described inhibitor of retrovirus integration, as a modulator of basal cell-intrinsic immunity. Ablation of Banf1 by gene editing resulted in chromatin activation near host defense genes with associated increased expression of ISGs, including Oas2, Rsad2 (viperin), Ifit1, and ISG15 The phenotype in Banf1-deficient cells occurred through a cGAS-, STING-, and IRF3-dependent signaling axis, was associated with reduced infection of RNA and DNA viruses, and was reversed in Banf1 complemented cells. Confocal microscopy and biochemical studies revealed that a loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline. Our study identifies an undescribed role for Banf1 in regulating the levels of cytoplasmic DNA and cGAS-dependent ISG homeostasis and suggests possible therapeutic directions for promoting or inhibiting cell-intrinsic innate immune responses.IMPORTANCE Although the interferon (IFN) signaling pathway is a key host mechanism to restrict infection of a diverse range of viral pathogens, its unrestrained activity either at baseline or in the context of an immune response can result in host cell damage and injury. Here, we used a genome-wide CRISPR-Cas9 screen and identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1) as a modulator of basal cell-intrinsic immunity. A loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline, which triggered IFN-stimulated gene expression via a cGAS-STING-IRF3 axis that did not require type I IFN or STAT1 signaling. Our experiments define a regulatory network in which Banf1 limits basal inflammation by preventing self DNA accumulation in the cytosol.},
}
@article {pmid32156803,
year = {2020},
author = {Selle, K and Fletcher, JR and Tuson, H and Schmitt, DS and McMillan, L and Vridhambal, GS and Rivera, AJ and Montgomery, SA and Fortier, LC and Barrangou, R and Theriot, CM and Ousterout, DG},
title = {In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.},
journal = {mBio},
volume = {11},
number = {2},
pages = {},
pmid = {32156803},
issn = {2150-7511},
support = {P30 CA016086/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Bacteriophages/*genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clostridioides difficile/*genetics ; Enterocolitis, Pseudomembranous/microbiology/therapy ; Female ; Genetic Engineering ; Male ; Mice ; Mice, Inbred C57BL ; },
abstract = {Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.IMPORTANCEClostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficilein vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.},
}
@article {pmid32156754,
year = {2020},
author = {Hoshika, S and Sun, X and Kuranaga, E and Umetsu, D},
title = {Reduction of endocytic activity accelerates cell elimination during tissue remodeling of the Drosophila epidermal epithelium.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {7},
pages = {},
doi = {10.1242/dev.179648},
pmid = {32156754},
issn = {1477-9129},
mesh = {Adherens Junctions/genetics/metabolism ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cadherins/genetics/metabolism ; Caspases/genetics/metabolism ; Cell Death/physiology ; *Drosophila/cytology/physiology ; Embryo, Nonmammalian ; Endocytosis/*physiology ; Epidermis/*physiology ; Epithelium/*physiology ; Gene Editing ; Gene Expression Regulation, Developmental ; Metamorphosis, Biological/*physiology ; Myosin Type II/genetics/metabolism ; },
abstract = {Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Cells that are no longer needed are quickly removed without compromising the barrier function of the tissue. During metamorphosis, insects undergo developmentally programmed tissue remodeling. However, the mechanisms that regulate this rapid tissue remodeling are not precisely understood. Here, we show that the temporal dynamics of endocytosis modulate physiological cell properties to prime larval epidermal cells for cell elimination. Endocytic activity gradually reduces as tissue remodeling progresses. This reduced endocytic activity accelerates cell elimination through the regulation of Myosin II subcellular reorganization, junctional E-cadherin levels, and caspase activation. Whereas the increased Myosin II dynamics accelerates cell elimination, E-cadherin plays a protective role against cell elimination. Reduced E-cadherin is involved in the amplification of caspase activation by forming a positive-feedback loop with caspase. These findings reveal the role of endocytosis in preventing cell elimination and in the cell-property switching initiated by the temporal dynamics of endocytic activity to achieve rapid cell elimination during tissue remodeling.},
}
@article {pmid32156733,
year = {2020},
author = {Watters, KE and Shivram, H and Fellmann, C and Lew, RJ and McMahon, B and Doudna, JA},
title = {Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {12},
pages = {6531-6539},
pmid = {32156733},
issn = {1091-6490},
support = {K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Amino Acid Sequence ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Conserved Sequence ; DNA/metabolism ; Enzyme Inhibitors/*metabolism ; Gene Editing ; Genome, Bacterial/genetics ; HEK293 Cells ; Humans ; Inverted Repeat Sequences ; Staphylococcus/chemistry/*genetics ; Staphylococcus aureus/enzymology ; },
abstract = {Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9.},
}
@article {pmid32156541,
year = {2020},
author = {Ge, L and Dong, X and Gong, X and Kang, J and Zhang, Y and Quan, F},
title = {Mutation in myostatin 3'UTR promotes C2C12 myoblast proliferation and differentiation by blocking the translation of MSTN.},
journal = {International journal of biological macromolecules},
volume = {154},
number = {},
pages = {634-643},
doi = {10.1016/j.ijbiomac.2020.03.043},
pmid = {32156541},
issn = {1879-0003},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; MicroRNAs/metabolism ; Mutation ; Myoblasts/*cytology ; Myostatin/*genetics/physiology ; Phenotype ; },
abstract = {The point mutation in myostatin (MSTN) can produce the Texel sheep double muscle phenotype. However, whether other species have the same mode of action as MSTN and whether breeding materials can be obtained through cross-species genetic editing remain unclear. The mutation in the mouse MSTN 3'UTR could create a target site for mmu-miR-1/206, as verified by the dual luciferase reporter system. A C2C12 cell model with the mutation in MSTN 3'UTR was constructed using CRISPR/Cas9 gene editing. Then, the mRNA and protein expression of MSTN was analyzed in the mutant C2C12 cell model. Results revealed that the mutation blocked the translational level of MSTN. By inhibiting mmu-mir-206, low expression of MSTN protein in mutant C2C12 cell can be rescued. Furthermore, the proliferation and differentiation abilities of the mutant C2C12 cell model were tested by RT-PCR, CCK8 analysis, EDU (5-ethynyl-2'-deoxyuridine) proliferation analysis, immunofluorescence analysis, Western blot, and myotube fusion statistics. This study may serve as a reference for elucidating the function and molecular mechanism of MSTN and as a foundation for accurate breeding improvement.},
}
@article {pmid32156257,
year = {2020},
author = {Berry, LK and Thomas, GH and Thorpe, PH},
title = {CATS: Cas9-assisted tag switching. A high-throughput method for exchanging genomic peptide tags in yeast.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {221},
pmid = {32156257},
issn = {1471-2164},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Gene Editing ; Green Fluorescent Proteins/*genetics/metabolism ; High-Throughput Screening Assays ; Peptides/*genetics/metabolism ; Plasmids/genetics ; RNA, Guide/genetics ; Saccharomyces cerevisiae/genetics/*growth &amp; development ; Staining and Labeling ; },
abstract = {BACKGROUND: The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library.<br><br>RESULTS: We present a new recombination-based method, CATS - Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining.<br><br>CONCLUSIONS: The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.},
}
@article {pmid32155151,
year = {2020},
author = {Wang, R and Graham, S and Sun, N and McCarthy, D and Peng, R and Erickson, J and Oconnor, L and Zhu, X and Wurbel, M and Dunstan, R and Westmoreland, S and Chung, N and Ghayur, T and Gu, J},
title = {CRISPR/Cas9-targeting of CD40 in hematopoietic stem cells limits immune activation mediated by anti-CD40.},
journal = {PloS one},
volume = {15},
number = {3},
pages = {e0228221},
pmid = {32155151},
issn = {1932-6203},
mesh = {Animals ; CD40 Antigens/*immunology/metabolism ; CRISPR-Cas Systems/*genetics ; Colitis/immunology/therapy ; Gene Expression Regulation ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism ; Mice ; },
abstract = {Inflammatory bowel diseases (IBD) are complex, multifactorial disorders characterized by chronic relapsing intestinal inflammation. IBD is diagnosed around 1 in 1000 individuals in Western countries with globally increasing incident rates. Association studies have identified hundreds of genes that are linked to IBD and potentially regulate its pathology. The further dissection of the genetic network underlining IBD pathogenesis and pathophysiology is hindered by the limited capacity to functionally characterize each genetic association, including generating knockout animal models for every associated gene. Cutting-edge CRISPR/Cas9-based technology may transform the field of IBD research by efficiently and effectively introducing genetic alterations. In the present study, we used CRISPR/Cas9-based technologies to genetically modify hematopoietic stem cells. Through cell sorting and bone marrow transplantation, we established a system to knock out target gene expression by over 90% in the immune system of reconstituted animals. Using a CD40-mediated colitis model, we further validated our CRISPR/Cas9-based platform for investigating gene function in experimental IBD. In doing so, we developed a model system that delivers genetically modified mice in a manner much faster than conventional methodology, significantly reducing the time from target identification to in vivo target validation and expediting drug development.},
}
@article {pmid32154189,
year = {2020},
author = {Romagnoli, BAA and Holetz, FB and Alves, LR and Goldenberg, S},
title = {RNA Binding Proteins and Gene Expression Regulation in Trypanosoma cruzi.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {56},
pmid = {32154189},
issn = {2235-2988},
mesh = {Gene Expression Regulation ; Protozoan Proteins/genetics/metabolism ; RNA, Messenger/genetics ; RNA-Binding Proteins/genetics/metabolism ; *Trypanosoma cruzi/genetics/metabolism ; },
abstract = {The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi, the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation.},
}
@article {pmid32152696,
year = {2020},
author = {Zhang, M and Liu, Q and Yang, X and Xu, J and Liu, G and Yao, X and Ren, R and Xu, J and Lou, L},
title = {CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum.},
journal = {Plant cell reports},
volume = {39},
number = {5},
pages = {589-595},
pmid = {32152696},
issn = {1432-203X},
mesh = {*CRISPR-Cas Systems ; Citrullus/*genetics/microbiology ; Disease Resistance/*genetics ; *Fusarium ; *Gene Editing/methods ; Loss of Function Mutation ; Mutagenesis ; Peptide Hormones/*genetics ; Plant Diseases/*microbiology ; Plant Growth Regulators/*genetics ; Plants, Genetically Modified ; Seedlings/genetics/microbiology ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9-mediated editing of Clpsk1 enhanced watermelon resistance to Fusarium oxysporum. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be an effective genome-editing tool for crop improvement. Previous studies described that Phytosulfokine (PSK) signalling attenuates plant immune response. In this work, we employed the CRISPR/Cas9 system to knockout Clpsk1 gene, encoding the PSK precursor, to confer enhanced watermelon resistance to Fusarium oxysporum f.sp. niveum (FON). Interactions between PSK and FON were analysed and it was found that transcript of Clpsk1 was significantly induced upon FON infection. Meanwhile, application of exogenous PSK increased the pathogen growth. Then, one sgRNA, which targeted the first exon of Clpsk1, was selected for construction of pRGEB32-CAS9-gRNA-Clpsk1 expression cassette. The construct was then transformed to watermelon through Agrobacterium tumefaciens-mediated transformation method. Six mutant plants were obtained and three types of mutations at the expected position were identified based on Sanger sequencing. Resistance evaluation indicated that Clpsk1 loss-of-function rendered watermelon seedlings more resistant to infection by FON. These results indicate that CRISPR/Cas9-mediated gene modification is an effective approach for watermelon improvement.},
}
@article {pmid32152597,
year = {2020},
author = {Gao, H and Gadlage, MJ and Lafitte, HR and Lenderts, B and Yang, M and Schroder, M and Farrell, J and Snopek, K and Peterson, D and Feigenbutz, L and Jones, S and St Clair, G and Rahe, M and Sanyour-Doyel, N and Peng, C and Wang, L and Young, JK and Beatty, M and Dahlke, B and Hazebroek, J and Greene, TW and Cigan, AM and Chilcoat, ND and Meeley, RB},
title = {Superior field performance of waxy corn engineered using CRISPR-Cas9.},
journal = {Nature biotechnology},
volume = {38},
number = {5},
pages = {579-581},
pmid = {32152597},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; Crop Production ; Gene Editing/methods ; Genetic Introgression ; Plant Proteins/*genetics ; *Quantitative Trait Loci ; Sequence Deletion ; Zea mays/genetics/*growth &amp; development ; },
abstract = {We created waxy corn hybrids by CRISPR-Cas9 editing of a waxy allele in 12 elite inbred maize lines, a process that was more than a year faster than conventional trait introgression using backcrossing and marker-assisted selection. Field trials at 25 locations showed that CRISPR-waxy hybrids were agronomically superior to introgressed hybrids, producing on average 5.5 bushels per acre higher yield.},
}
@article {pmid32152546,
year = {2020},
author = {Girardi, E and César-Razquin, A and Lindinger, S and Papakostas, K and Konecka, J and Hemmerich, J and Kickinger, S and Kartnig, F and Gürtl, B and Klavins, K and Sedlyarov, V and Ingles-Prieto, A and Fiume, G and Koren, A and Lardeau, CH and Kumaran Kandasamy, R and Kubicek, S and Ecker, GF and Superti-Furga, G},
title = {A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.},
journal = {Nature chemical biology},
volume = {16},
number = {4},
pages = {469-478},
pmid = {32152546},
issn = {1552-4469},
support = {P 29250/FWF_/Austrian Science Fund FWF/Austria ; W 1232/FWF_/Austrian Science Fund FWF/Austria ; 695214/ERC_/European Research Council/International ; 772437/ERC_/European Research Council/International ; I 2192/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Amino Acid Transport Systems, Neutral/genetics/metabolism ; Antineoplastic Agents ; Biochemical Phenomena ; Biological Transport/genetics/physiology ; CRISPR-Cas Systems ; Cation Transport Proteins/genetics/metabolism ; Drug Resistance/*genetics/physiology ; Genetic Testing ; Humans ; Monocarboxylic Acid Transporters/genetics/metabolism ; Monosaccharide Transport Proteins/genetics/metabolism ; Protein Transport/physiology ; Solute Carrier Proteins/*metabolism/physiology ; Symporters/genetics/metabolism ; },
abstract = {Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.},
}
@article {pmid32152514,
year = {2020},
author = {Campbell, KJ and Blyth, K},
title = {Somatic base editing to model oncogenic drivers in breast cancer.},
journal = {Lab animal},
volume = {49},
number = {4},
pages = {115-116},
pmid = {32152514},
issn = {1548-4475},
mesh = {Animals ; *CRISPR-Cas Systems ; Carcinogenesis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; },
}
@article {pmid32152313,
year = {2020},
author = {McCarty, NS and Graham, AE and Studená, L and Ledesma-Amaro, R},
title = {Multiplexed CRISPR technologies for gene editing and transcriptional regulation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1281},
pmid = {32152313},
issn = {2041-1723},
support = {BB/R01602X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Expression Regulation ; Humans ; RNA, Guide/genetics ; *Transcription, Genetic ; },
abstract = {Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.},
}
@article {pmid32152282,
year = {2020},
author = {Sybirna, A and Tang, WWC and Pierson Smela, M and Dietmann, S and Gruhn, WH and Brosh, R and Surani, MA},
title = {A critical role of PRDM14 in human primordial germ cell fate revealed by inducible degrons.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1282},
pmid = {32152282},
issn = {2041-1723},
support = {MR/P009948/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; /CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Cell Lineage ; DNA-Binding Proteins/genetics/*metabolism ; Embryonic Stem Cells/cytology/metabolism ; Gene Expression Regulation ; Germ Cells/*cytology/*metabolism ; HEK293 Cells ; Humans ; Indoleacetic Acids/pharmacology ; Mice ; Promoter Regions, Genetic/genetics ; Protein Binding ; *Proteolysis ; RNA-Binding Proteins/genetics/*metabolism ; Transcription Factors/genetics/*metabolism ; Transcriptome/genetics ; },
abstract = {PRDM14 is a crucial regulator of mouse primordial germ cells (mPGCs), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion. We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of hPGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts.},
}
@article {pmid32152089,
year = {2020},
author = {Ates, KM and Wang, T and Moreland, T and Veeranan-Karmegam, R and Ma, M and Jeter, C and Anand, P and Wenzel, W and Kim, HG and Wolfe, LA and Stephen, J and Adams, DR and Markello, T and Tifft, CJ and Settlage, R and Gahl, WA and Gonsalvez, GB and Malicdan, MC and Flanagan-Steet, H and Pan, YA},
title = {Deficiency in the endocytic adaptor proteins PHETA1/2 impairs renal and craniofacial development.},
journal = {Disease models &amp; mechanisms},
volume = {13},
number = {5},
pages = {},
pmid = {32152089},
issn = {1754-8411},
support = {R01 GM086524/GM/NIGMS NIH HHS/United States ; R01 GM100088/GM/NIGMS NIH HHS/United States ; R21 GM119016/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/chemistry/*deficiency/metabolism ; Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Cathepsin K/metabolism ; Cell Differentiation ; Chondrocytes/pathology ; Cilia/pathology ; Collagen Type II/metabolism ; *Endocytosis ; Face/*embryology ; Genes, Dominant ; HeLa Cells ; Humans ; Kidney/*embryology ; Morphogenesis ; Motor Activity ; Mutation/genetics ; Pronephros/pathology ; Skull/*embryology ; Undiagnosed Diseases/diagnostic imaging/genetics ; Vesicular Transport Proteins/chemistry/genetics ; Zebrafish ; Zebrafish Proteins/chemistry/*deficiency/metabolism ; },
abstract = {A critical barrier in the treatment of endosomal and lysosomal diseases is the lack of understanding of the in vivo functions of the putative causative genes. We addressed this by investigating a key pair of endocytic adaptor proteins, PH domain-containing endocytic trafficking adaptor 1 and 2 (PHETA1/2; also known as FAM109A/B, Ses1/2, IPIP27A/B), which interact with the protein product of OCRL, the causative gene for Lowe syndrome. Here, we conducted the first study of PHETA1/2 in vivo, utilizing the zebrafish system. We found that impairment of both zebrafish orthologs, pheta1 and pheta2, disrupted endocytosis and ciliogenesis in renal tissues. In addition, pheta1/2 mutant animals exhibited reduced jaw size and delayed chondrocyte differentiation, indicating a role in craniofacial development. Deficiency of pheta1/2 resulted in dysregulation of cathepsin K, which led to an increased abundance of type II collagen in craniofacial cartilages, a marker of immature cartilage extracellular matrix. Cathepsin K inhibition rescued the craniofacial phenotypes in the pheta1/2 double mutants. The abnormal renal and craniofacial phenotypes in the pheta1/2 mutant animals were consistent with the clinical presentation of a patient with a de novo arginine (R) to cysteine (C) variant (R6C) of PHETA1. Expressing the patient-specific variant in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results provide insights into the in vivo roles of PHETA1/2 and suggest that the R6C variant is contributory to the pathogenesis of disease in the patient.This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid32152070,
year = {2020},
author = {Nüssing, S and House, IG and Kearney, CJ and Chen, AXY and Vervoort, SJ and Beavis, PA and Oliaro, J and Johnstone, RW and Trapani, JA and Parish, IA},
title = {Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {204},
number = {8},
pages = {2308-2315},
doi = {10.4049/jimmunol.1901396},
pmid = {32152070},
issn = {1550-6606},
mesh = {Animals ; CD8-Positive T-Lymphocytes/*immunology ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/immunology ; Electroporation ; Female ; *Gene Editing ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Polymorphism, Single Nucleotide/genetics/immunology ; },
abstract = {CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8[+] T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8[+] T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.},
}
@article {pmid32151954,
year = {2020},
author = {Yoshimatsu, S and Sato, T and Yamamoto, M and Sasaki, E and Nakajima, M and Nakamura, M and Shiozawa, S and Noce, T and Okano, H},
title = {Generation of a male common marmoset embryonic stem cell line DSY127-BV8VT1 carrying double reporters specific for the germ cell linage using the CRISPR-Cas9 and PiggyBac transposase systems.},
journal = {Stem cell research},
volume = {44},
number = {},
pages = {101740},
doi = {10.1016/j.scr.2020.101740},
pmid = {32151954},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Callithrix ; Cell Differentiation ; *Cell Line ; *Embryonic Stem Cells ; Germ Cells ; Male ; *Transposases ; },
abstract = {BLIMP1 (PRDM1) and VASA (DDX4) play pivotal roles in the development of the germ cell linage. Importantly, these genes are specifically expressed in germ cells; BLIMP1 in primordial germ cells (PGCs) to early-stage gonocytes, and VASA in migration-stage PGCs to mature gametes. The high reproductive efficiency of common marmosets (marmosets; Callithrix jacchus) makes them advantageous for use in germ cell research. We herein report the generation of a male marmoset embryonic stem cell (ESC) line harboring BLIMP1 and DDX4 double reporters. This ESC line will be a useful tool for investigating male gametogenesis in non-human primates.},
}
@article {pmid32151271,
year = {2020},
author = {Bodapati, S and Daley, TP and Lin, X and Zou, J and Qi, LS},
title = {A benchmark of algorithms for the analysis of pooled CRISPR screens.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {62},
pmid = {32151271},
issn = {1474-760X},
support = {R21 MD012867/MD/NIMHD NIH HHS/United States ; P30 AG059307/AG/NIA NIH HHS/United States ; },
mesh = {*Algorithms ; *CRISPR-Cas Systems ; Computer Simulation ; RNA, Small Interfering ; Sequence Analysis ; },
abstract = {Genome-wide pooled CRISPR-Cas-mediated knockout, activation, and repression screens are powerful tools for functional genomic investigations. Despite their increasing importance, there is currently little guidance on how to design and analyze CRISPR-pooled screens. Here, we provide a review of the commonly used algorithms in the computational analysis of pooled CRISPR screens. We develop a comprehensive simulation framework to benchmark and compare the performance of these algorithms using both synthetic and real datasets. Our findings inform parameter choices of CRISPR screens and provide guidance to researchers on the design and analysis of pooled CRISPR screens.},
}
@article {pmid32151165,
year = {2020},
author = {Graff, GD and Sherkow, JS},
title = {Models of Technology Transfer for Genome-Editing Technologies.},
journal = {Annual review of genomics and human genetics},
volume = {21},
number = {},
pages = {509-534},
doi = {10.1146/annurev-genom-121119-100145},
pmid = {32151165},
issn = {1545-293X},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Patents as Topic ; *Technology Transfer ; },
abstract = {Many of the fundamental inventions of genome editing, including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR, were first made at universities and patented to encourage commercial development. This gave rise to a diversity of technology transfer models but also conflicts among them. Against a broader historical and policy backdrop of university patenting and special challenges concerning research tools, we review the patent estates of genome editing and the diversity of technology transfer models employed to commercialize them, including deposit in the public domain, open access contracts, material transfer agreements, nonexclusive and exclusive licenses, surrogate licenses, and aggregated licenses. Advantages are found in this diversity, allowing experimentation and competition that we characterize as a federalism model of technology transfer. A notable feature of genome editing has been the rise and success of third-party licensing intermediaries. At the same time, the rapid pace of development of genome-editing technology is likely to erode the importance of patent estates and licensing regimes and may mitigate the effect of overly broad patents, giving rise to new substitutes to effectuate commercialization.},
}
@article {pmid32150860,
year = {2020},
author = {Yasuhara-Bell, J and Arif, M and Busot, GY and Mann, R and Rodoni, B and Stack, JP},
title = {Comparative Genomic Analysis Confirms Five Genetic Populations of the Select Agent, Rathayibacter toxicus.},
journal = {Microorganisms},
volume = {8},
number = {3},
pages = {},
pmid = {32150860},
issn = {2076-2607},
abstract = {Rathayibacter toxicus is a Gram-positive, nematode-vectored bacterium that infects several grass species in the family Poaceae. Unique in its genus, R. toxicus has the smallest genome, possesses a complete CRISPR-Cas system, a vancomycin-resistance cassette, produces tunicamycin, a corynetoxin responsible for livestock deaths in Australia, and is designated a Select Agent in the United States. In-depth, genome-wide analyses performed in this study support the previously designated five genetic populations, with a core genome comprising approximately 80% of the genome for all populations. Results varied as a function of the type of analysis and when using different bioinformatics tools for the same analysis; e.g., some programs failed to identify specific genomic regions that were actually present. The software variance highlights the need to verify bioinformatics results by additional methods; e.g., PCR, mapping genes to genomes, use of multiple algorithms). These analyses suggest the following relationships among populations: RT-IV ↔ RT-I ↔ RT-II ↔ RT-III ↔ RT-V, with RT-IV and RT-V being the most unrelated. This is the most comprehensive analysis of R. toxicus that included populations RT-I and RT-V. Future studies require underrepresented populations and more recent isolates from varied hosts and geographic locations.},
}
@article {pmid32150575,
year = {2020},
author = {Shields, RC and Walker, AR and Maricic, N and Chakraborty, B and Underhill, SAM and Burne, RA},
title = {Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.},
journal = {PLoS pathogens},
volume = {16},
number = {3},
pages = {e1008344},
pmid = {32150575},
issn = {1553-7374},
support = {T90 DE021990/DE/NIDCR NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*physiology ; Gene Expression Regulation, Bacterial/*physiology ; Genome, Bacterial/*physiology ; Genome-Wide Association Study ; RNA, Bacterial/biosynthesis/genetics ; RNA, Guide/biosynthesis/genetics ; *Streptococcus mutans/genetics/metabolism ; },
abstract = {A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.},
}
@article {pmid32150344,
year = {2020},
author = {Johnson, CG and Chen, T and Furey, N and Hemmingsen, MG and Bissig, KD},
title = {Somatic Liver Knockout (SLiK): A Quick and Efficient Way to Generate Liver-Specific Knockout Mice Using Multiplex CRISPR/Cas9 Gene Editing.},
journal = {Current protocols in molecular biology},
volume = {130},
number = {1},
pages = {e117},
pmid = {32150344},
issn = {1934-3647},
support = {R56 DK115461/DK/NIDDK NIH HHS/United States ; T32 GM088129/GM/NIGMS NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; P30 CA014236/CA/NCI NIH HHS/United States ; R01 HL134510/HL/NHLBI NIH HHS/United States ; R01 DK115461/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Hepatocytes/*metabolism ; Liver/*metabolism ; Mice ; Mice, Knockout ; RNA, Guide/genetics ; Transfection/methods ; },
abstract = {Somatic liver knockout (SLiK) is a method developed to rapidly generate a liver-specific knockout of one or several genes. This technique combines the strengths of CRISPR/Cas9 gene editing and hydrodynamic tail-vein injection, a simple in vivo method for transfection of hepatocytes, to harness the powerful selection pressure of tyrosinemic livers to replace host hepatocytes with any desired gene deletion. In this protocol, we will describe sgRNA design and cloning, hydrodynamic tail-vein injection of targeting constructs, and screening and validation methods for efficient in vivo gene editing. © 2020 by John Wiley &amp; Sons, Inc. Support Protocol 1: sgRNA design Support Protocol 2: sgRNA construction: daisy chaining multiple sgRNAs Basic Protocol: Delivery of DNA by hydrodynamic tail-vein injection and liver repopulation of edited hepatocytes Support Protocol 3: Validation of CRISPR/Cas9 cutting in vivo.},
}
@article {pmid32149449,
year = {2020},
author = {Westra, ER},
title = {Interview with Professor Edze Westra, NERC Independent Fellow. University of Exeter.},
journal = {Cellular microbiology},
volume = {22},
number = {6},
pages = {e13202},
doi = {10.1111/cmi.13202},
pmid = {32149449},
issn = {1462-5822},
mesh = {CRISPR-Cas Systems ; *Host-Pathogen Interactions/genetics/physiology ; Humans ; Microbiology ; Research ; },
}
@article {pmid32422031,
year = {2019},
author = {Ren, H and Hua, Z and Meng, J and Molenaar, A and Bi, Y and Cheng, N and Zheng, X},
title = {Generation of Acsl4 Gene Knockout Mouse Model by CRISPR/Cas9-Mediated Genome Engineering.},
journal = {Critical reviews in biomedical engineering},
volume = {47},
number = {5},
pages = {419-426},
doi = {10.1615/CritRevBiomedEng.2019030342},
pmid = {32422031},
issn = {1943-619X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Coenzyme A Ligases/*genetics ; Disease Models, Animal ; Female ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Lipid Metabolism Disorders ; Male ; Mice ; Mice, Knockout/*genetics ; },
abstract = {Acyl-CoA synthetase 4 (Acsl4) is involved in lipid synthesis and fatty acid degradation, and disruption of its function causes lipid metabolism disorder in various species. Herein, we report the generation of Acsl4 knockout (KO) mice using the CRISPR/Cas9 gene editing system to study its effects on lipid deposition. In this report, a large 12kb deletion in the Acsl4 gene was performed by coinjection of Cas9 mRNA and two guide RNAs (sgRNAs) into mouse fertilized oocytes. Six mutant mice carrying target mutations were examined by PCR analysis and direct sequencing. The gene modified mice remained healthy and displayed normal behavior. All the mutant F0 mice were mated with wild mice to produce the F1 generation, and only 1 F1 mutant mouse was obtained.},
}
@article {pmid32421996,
year = {2019},
author = {Ergoren, MC and Idlibi, R},
title = {Therapeutic Genome Editing by CRISPR/Cas9-Mediated Strategy to Cure Genetic Disorders in Humans: Guide for Molecular Surgeons.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {29},
number = {5},
pages = {387-399},
doi = {10.1615/CritRevEukaryotGeneExpr.2019025930},
pmid = {32421996},
issn = {1045-4403},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Engineering ; *Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells ; Surgeons ; },
abstract = {Genetic engineering scientists have shown a wide increase especially on therapeutic promise which has gained considerable awareness. This goes back to a variety of tools including the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This technique has shown an efficient flourish in genome editing and treatment of genetic diseases. Due to its specificity and ease of use it has been widely adopted for a variety of applications involving human diseases, treatment, therapeutics in cancer and biotechnology. Albeit studies have shown that CRISPR/Cas9 technique is more proficient, particular and manipulable than past eras of genetic modulation instruments. Additionally, it can be further enhanced by the application of different developments raising its general productivity in a higher recurrence of genome modifications and decreasing its off-target impacts. Here in this review this technique will be introduced with discussing its relation with the genome engineering and gene editing especially in the therapeutic area. Furthermore, the specificity of the application is explained and the contribution of Induced Human Pluripotent Stem Cells (iPSCs) in gene manipulation and how CRISPR technology expanded its application is discussed. Lastly, we describe the novel effective applications of the CRISPR/Cas9 technology and propose some enhancements that might improve the technique with highlighting the future approach and remarks.},
}
@article {pmid32188556,
year = {2019},
author = {Lee, K and Farrell, K and Uh, K},
title = {Application of genome-editing systems to enhance available pig resources for agriculture and biomedicine.},
journal = {Reproduction, fertility, and development},
volume = {32},
number = {2},
pages = {40-49},
pmid = {32188556},
issn = {1031-3613},
support = {R21 OD027062/OD/NIH HHS/United States ; },
mesh = {Agriculture/methods/*trends ; Animals ; Animals, Genetically Modified ; Biomedical Research/methods/*trends ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods/*veterinary ; Genetic Engineering/methods/trends/*veterinary ; Genetic Enhancement/methods ; Swine/*genetics ; },
abstract = {Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.},
}
@article {pmid32288252,
year = {2018},
author = {Koo, B and Kim, DE and Kweon, J and Jin, CE and Kim, SH and Kim, Y and Shin, Y},
title = {CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases.},
journal = {Sensors and actuators. B, Chemical},
volume = {273},
number = {},
pages = {316-321},
pmid = {32288252},
issn = {0925-4005},
abstract = {Rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9-mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. We addressed the clinical utility of this CRISPR/dCas9-mediated biosensor in tick-borne illnesses including scrub typhus (ST) and severe fever with thrombocytopenia syndrome (SFTS), whose clinical presentations are too similar to be easily differentiated. By using CRISPR/dCas9-mediated biosensor, we achieved single molecule sensitivity for the detection of ST (0.54 aM) and SFTS (0.63 aM); this detection sensitivity is 100 times more sensitive than that of RT-PCR assay. Finally, CRISPR/dCas9-mediated biosensor was able to clearly distinguish between ST and SFTS in serum samples within 20 min. We believe that CRISPR/dCas9-mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications.},
}
@article {pmid32161796,
year = {2018},
author = {Anton, T and Karg, E and Bultmann, S},
title = {Applications of the CRISPR/Cas system beyond gene editing.},
journal = {Biology methods &amp; protocols},
volume = {3},
number = {1},
pages = {bpy002},
pmid = {32161796},
issn = {2396-8923},
abstract = {Since the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) as a tool for gene editing a plethora of locus-specific as well as genome-wide approaches have been developed that allow efficient and reproducible manipulation of genomic sequences. However, the seemingly unbound potential of CRISPR/Cas does not stop with its utilization as a site-directed nuclease. Mutations in its catalytic centers render Cas9 (dCas9) a universal recruitment platform that can be utilized to control transcription, visualize DNA sequences, investigate in situ proteome compositions and manipulate epigenetic modifications at user-defined genomic loci. In this review, we give a comprehensive introduction and overview of the development, improvement and application of recent dCas9-based approaches.},
}
@article {pmid32148769,
year = {2020},
author = {Kumar, A},
title = {Jump around: transposons in and out of the laboratory.},
journal = {F1000Research},
volume = {9},
number = {},
pages = {},
pmid = {32148769},
issn = {2046-1402},
mesh = {DNA Barcoding, Taxonomic ; *DNA Transposable Elements ; Genetic Therapy ; Humans ; *Mutagenesis, Insertional ; },
abstract = {Since Barbara McClintock's groundbreaking discovery of mobile DNA sequences some 70 years ago, transposable elements have come to be recognized as important mutagenic agents impacting genome composition, genome evolution, and human health. Transposable elements are a major constituent of prokaryotic and eukaryotic genomes, and the transposition mechanisms enabling transposon proliferation over evolutionary time remain engaging topics for study, suggesting complex interactions with the host, both antagonistic and mutualistic. The impact of transposition is profound, as over 100 human heritable diseases have been attributed to transposon insertions. Transposition can be highly mutagenic, perturbing genome integrity and gene expression in a wide range of organisms. This mutagenic potential has been exploited in the laboratory, where transposons have long been utilized for phenotypic screening and the generation of defined mutant libraries. More recently, barcoding applications and methods for RNA-directed transposition are being used towards new phenotypic screens and studies relevant for gene therapy. Thus, transposable elements are significant in affecting biology both in vivo and in the laboratory, and this review will survey advances in understanding the biological role of transposons and relevant laboratory applications of these powerful molecular tools.},
}
@article {pmid32147886,
year = {2020},
author = {He, XY and Ren, XH and Peng, Y and Zhang, JP and Ai, SL and Liu, BY and Xu, C and Cheng, SX},
title = {Aptamer/Peptide-Functionalized Genome-Editing System for Effective Immune Restoration through Reversal of PD-L1-Mediated Cancer Immunosuppression.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {32},
number = {17},
pages = {e2000208},
doi = {10.1002/adma.202000208},
pmid = {32147886},
issn = {1521-4095},
mesh = {Animals ; Apoptosis ; Aptamers, Nucleotide/*chemistry ; B7-H1 Antigen/*metabolism ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Gene Editing/*methods ; Humans ; Hyaluronic Acid/chemistry ; Immunosuppression Therapy ; Nanoparticles/chemistry ; Oligodeoxyribonucleotides/chemistry ; Peptides/*chemistry ; Plasmids/chemistry/metabolism ; Programmed Cell Death 1 Receptor/metabolism ; beta Catenin/deficiency/genetics/metabolism ; },
abstract = {Effective reversal of tumor immunosuppression is of critical importance in cancer therapy. A multifunctional delivery vector that can effectively deliver CRISPR-Cas9 plasmid for β-catenin knockout to reverse tumor immunosuppression is constructed. The multi-functionalized delivery vector is decorated with aptamer-conjugated hyaluronic acid and peptide-conjugated hyaluronic acid to combine the tumor cell/nuclear targeting function of AS1411 with the cell penetrating/nuclear translocation function of TAT-NLS. Due to the significantly enhanced plasmid enrichment in malignant cell nuclei, the genome editing system can induce effective β-catenin knockout and suppress Wnt/β-catenin pathway, resulting in notably downregulated proteins involved in tumor progression and immunosuppression. Programmed death-ligand 1 (PD-L1) downregulation in edited tumor cells not only releases the PD-1/PD-L1 brake to improve the cancer killing capability of CD8[+] T cells, but also enhances antitumor immune responses of immune cells. This provides a facile strategy to reverse tumor immunosuppression and to restore immunosurveillance and activate anti-tumor immunity.},
}
@article {pmid32147798,
year = {2020},
author = {Lambert, M and Leijonhufvud, C and Segerberg, F and Melenhorst, JJ and Carlsten, M},
title = {CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2121},
number = {},
pages = {213-239},
doi = {10.1007/978-1-0716-0338-3_18},
pmid = {32147798},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cell Movement/immunology ; Flow Cytometry/*methods ; Gene Editing/history/*methods ; Gene Knockout Techniques/history/*methods ; History, 20th Century ; History, 21st Century ; Humans ; Killer Cells, Natural/*metabolism ; Real-Time Polymerase Chain Reaction/*methods ; Sequence Analysis, DNA/methods ; },
abstract = {Natural killer (NK) cells are cytotoxic lymphocytes of our immune system with the ability to identify and kill certain virally infected and tumor-transformed cells. During the past 15 years, it has become increasingly clear that NK cells are involved in tumor immune surveillance and that they can be utilized to treat cancer patients. However, their ability to induce durable responses in settings of adoptive cell therapy needs to be further improved. One possible approach is to genetically engineer NK cells to augment their cytotoxicity per se, but also their ability to persist in vivo and home to the tumor-bearing tissue. In recent years, investigators have explored the potential of viral transduction and mRNA electroporation to modify NK cells. Although these methods have generated promising data, they are associated with certain limitations. With the increasing advances in the CRISPR/Cas9 technology, investigators have now turned their attention toward using this technology with NK cells as an alternative method. In this book chapter, we introduce NK cells and provide an historical overview of techniques to genetically engineer lymphocytes. Further, we elucidate protocols for inducing double-strand breaks in NK cells via CRISPR/Cas9 together with readouts to address its efficacy and functional outcome. We also discuss the pros and cons of the described readouts. The overall aim of this book chapter is to help introduce the CRISPR/Cas9 technology to the broader audience of NK cell researchers.},
}
@article {pmid32147450,
year = {2021},
author = {Tang, H and Zhao, X and Jiang, X},
title = {Synthetic multi-layer nanoparticles for CRISPR-Cas9 genome editing.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {55-78},
doi = {10.1016/j.addr.2020.03.001},
pmid = {32147450},
issn = {1872-8294},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Chemistry, Pharmaceutical ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Dendrimers/chemistry ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Vectors/*administration &amp; dosage ; Gold/chemistry ; Humans ; Lanthanoid Series Elements/chemistry ; Lipids/chemistry ; Mice ; Nanoparticles/*chemistry ; Polymers/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) has great potential to revolutionize biomedical research and disease therapy. The specific and efficient genome editing strongly depends on high efficiency of delivery of the CRISPR payloads. However, optimization of CRISPR delivery vehicles still remains a major obstacle. Recently, various non-viral vectors have been utilized to deliver CRISPR tools. Many of these vectors have multi-layer structures assembled. In this review, we will introduce the development of CRISPR-Cas9 systems and their general therapeutic applications by summarizing current CRISPR-Cas9 based clinical trials. We will highlight the multi-layer nanoparticles (NPs) that have been developed to deliver CRISPR cargos in vitro and in vivo for various purposes, as well the potential building blocks of multi-layer NPs. We will also discuss the challenges in making the CRISPR tools into viable pharmaceutical products and provide potential solutions on efficiency and biosafety issues.},
}
@article {pmid32146695,
year = {2020},
author = {Capdeville, N and Schindele, P and Puchta, H},
title = {Application of CRISPR/Cas-mediated base editing for directed protein evolution in plants.},
journal = {Science China. Life sciences},
volume = {63},
number = {4},
pages = {613-616},
doi = {10.1007/s11427-020-1655-9},
pmid = {32146695},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Escherichia coli/genetics ; Evolution, Molecular ; Gene Editing/*methods ; Humans ; Mutagenesis/genetics ; Mutation/genetics ; Plant Proteins/*genetics ; Plants/genetics ; Staphylococcus aureus/genetics ; },
}
@article {pmid32144266,
year = {2020},
author = {Wang, Z and Baulcombe, DC},
title = {Transposon age and non-CG methylation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1221},
pmid = {32144266},
issn = {2041-1723},
support = {BB/R018529/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/metabolism ; DNA Methylation/*genetics ; DNA Transposable Elements/*genetics ; Evolution, Molecular ; Gene Silencing ; Lycopersicon esculentum/genetics/growth &amp; development ; Mutation/genetics ; Nucleotides/*genetics ; Phenotype ; Plant Proteins/metabolism ; RNA Polymerase II/metabolism ; Terminal Repeat Sequences/genetics ; },
abstract = {Silencing of transposable elements (TEs) is established by small RNA-directed DNA methylation (RdDM). Maintenance of silencing is then based on a combination of RdDM and RNA-independent mechanisms involving DNA methyltransferase MET1 and chromodomain DNA methyltransferases (CMTs). Involvement of RdDM, according to this model should decrease with TE age but here we show a different pattern in tomato and Arabidopsis. In these species the CMTs silence long terminal repeat (LTR) transposons in the distal chromatin that are younger than those affected by RdDM. To account for these findings we propose that, after establishment of primary RdDM as in the original model, there is an RNA-independent maintenance phase involving CMTs followed by secondary RdDM. This progression of epigenetic silencing in the gene-rich distal chromatin is likely to influence the transcriptome either in cis or in trans depending on whether the mechanisms are RNA-dependent or -independent.},
}
@article {pmid32144253,
year = {2020},
author = {Kulcsár, PI and Tálas, A and Tóth, E and Nyeste, A and Ligeti, Z and Welker, Z and Welker, E},
title = {Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5'G-extended sgRNAs.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1223},
pmid = {32144253},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/chemistry/*genetics ; CRISPR-Cas Systems/*genetics ; Crystallography, X-Ray ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mutagenesis ; Mutation ; Protein Structure, Secondary/genetics ; RNA, Guide/*genetics ; Structure-Activity Relationship ; },
abstract = {Increased fidelity mutants of the SpCas9 nuclease constitute the most promising approach to mitigating its off-target effects. However, these variants are effective only in a restricted target space, and many of them are reported to work less efficiently when applied in clinically relevant, pre-assembled, ribonucleoprotein forms. The low tolerance to 5'-extended, 21G-sgRNAs contributes, to a great extent, to their decreased performance. Here, we report the generation of Blackjack SpCas9 variant that shows increased fidelity yet remain effective with 21G-sgRNAs. Introducing Blackjack mutations into previously reported increased fidelity variants make them effective with 21G-sgRNAs and increases their fidelity. Two "Blackjack" nucleases, eSpCas9-plus and SpCas9-HF1-plus are superior variants of eSpCas9 and SpCas9-HF1, respectively, possessing matching on-target activity and fidelity but retaining activity with 21G-sgRNAs. They facilitate the use of existing pooled sgRNA libraries with higher specificity and show similar activities whether delivered as plasmids or as pre-assembled ribonucleoproteins.},
}
@article {pmid32142798,
year = {2020},
author = {Kocher, T and March, OP and Bischof, J and Liemberger, B and Hainzl, S and Klausegger, A and Hoog, A and Strunk, D and Bauer, JW and Koller, U},
title = {Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa.},
journal = {The Journal of investigative dermatology},
volume = {140},
number = {10},
pages = {1985-1993.e5},
doi = {10.1016/j.jid.2020.02.012},
pmid = {32142798},
issn = {1523-1747},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Collagen Type VII/*genetics ; DNA End-Joining Repair ; Epidermolysis Bullosa Dystrophica/*genetics ; Female ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; Keratinocytes/metabolism ; Mutation ; Ribonucleoproteins/pharmacology ; },
abstract = {End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.},
}
@article {pmid32142753,
year = {2020},
author = {Lin, J and Fan, Y and Lin, X},
title = {Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-Cas9 expression (TRACE) system.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {138},
number = {},
pages = {103364},
pmid = {32142753},
issn = {1096-0937},
support = {R01 AI140719/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cryptococcus neoformans/*genetics ; Electroporation/*methods ; Gene Editing/methods ; },
abstract = {The basidiomycete Cryptococcus neoformans is not only a clinically important pathogen, but also a model organism for studying microbial pathogenesis and eukaryotic biology. One key factor behind its rise as a model organism is its genetic amenability. The widely used methods for transforming the C. neoformans species complex are Agrobacterium-mediated transformation (AMT) for random insertional mutagenesis and biolistic transformation for targeted mutagenesis. Electroporation was introduced to C. neoformans in early 1990s. Although electroporation is economic and yields a large number of transformants, introduced DNA rarely integrates into cryptococcal genome, which limits its use. Biolistic transformation, although costly and inefficient, has been the only method used in targeted mutagenesis in the past two decades. Several modifications, including the use of a donor DNA with split markers, a drug-resistant selection marker, and a recipient strain deficient in non-homologous end joining (NHEJ), have since modestly increased the frequency of genome integration and the rate of homologous replacement of the DNA introduced by electroporation. However, electroporation was not the method of choice for transformation until the recent adoption of CRISPR-Cas9 systems. We have developed a Transient CRISPR-Cas9 coupled with Electroporation System (TRACE), which dramatically facilitates targeted mutagenesis in the Cryptococcus species complex. TRACE combines the high transformation efficiency of electroporation with the high rates of DNA integration due to the transiently expressed CRISPR-Cas9. Here, we briefly discussed the history of electroporation for Cryptococcus transformation and provided detailed procedures for electroporation and the cassettes construction of the TRACE system for various genetic manipulations.},
}
@article {pmid32142727,
year = {2020},
author = {Kovacsics, D and Brózik, A and Tihanyi, B and Matula, Z and Borsy, A and Mészáros, N and Szabó, E and Németh, E and Fóthi, &#xc1; and Zámbó, B and Szüts, D and Várady, G and Orbán, TI and Apáti, &#xc1; and Sarkadi, B},
title = {Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells.},
journal = {Biochemical pharmacology},
volume = {175},
number = {},
pages = {113865},
doi = {10.1016/j.bcp.2020.113865},
pmid = {32142727},
issn = {1873-2968},
mesh = {A549 Cells ; ATP Binding Cassette Transporter, Subfamily G, Member 2/*genetics ; Antineoplastic Agents/pharmacology ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques ; Drug Resistance, Multiple/genetics ; Drug Resistance, Neoplasm/genetics ; ErbB Receptors/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Knockdown Techniques ; Genes, Reporter ; Humans ; Neoplasm Proteins/*genetics ; Plasmids ; },
abstract = {Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design.},
}
@article {pmid32142691,
year = {2020},
author = {Barrangou, R and Sontheimer, EJ},
title = {CRISPR Shields: Fending Off Diverse Cas Nucleases with Nucleus-like Structures.},
journal = {Molecular cell},
volume = {77},
number = {5},
pages = {934-936},
doi = {10.1016/j.molcel.2020.02.015},
pmid = {32142691},
issn = {1097-4164},
support = {R01 GM125797/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; RNA ; },
abstract = {Two recent studies have uncovered a novel means by which bacteriophages thwart host immunity. Mendoza et al. (2020) and Malone et al. (2020) demonstrate that a nucleus-like proteinaceous structure shields phage DNA from CRISPR-associated nucleases encompassing Cascade-Cas3, Cas9, and Cas12.},
}
@article {pmid32142629,
year = {2020},
author = {Erdogan, M and Fabritius, A and Basquin, J and Griesbeck, O},
title = {Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells.},
journal = {Cell chemical biology},
volume = {27},
number = {5},
pages = {610-621.e5},
doi = {10.1016/j.chembiol.2020.02.004},
pmid = {32142629},
issn = {2451-9448},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Luminescent Proteins/*genetics ; Lysosomes/genetics ; Models, Molecular ; },
abstract = {Engineered proteins must be phenotypically selected for function in the appropriate physiological context. Here, we present a versatile approach that allows generating panels of mammalian cells that express diversified heterologous protein libraries in the cytosol or subcellular compartments under stable conditions and in a single-variant-per-cell manner. To this end we adapt CRISPR/Cas9 editing technology to diversify targeted stretches of a protein of interest in situ. We demonstrate the utility of the approach by in situ engineering and intra-lysosome specific selection of an extremely pH-resistant long Stokes shift red fluorescent protein variant. Tailoring properties to specific conditions of cellular sub-compartments or organelles of mammalian cells can be an important asset to optimize various proteins, protein-based tools, and biosensors for distinct functions.},
}
@article {pmid32142605,
year = {2020},
author = {Wrist, A and Sun, W and Summers, RM},
title = {The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research.},
journal = {ACS synthetic biology},
volume = {9},
number = {4},
pages = {682-697},
doi = {10.1021/acssynbio.9b00475},
pmid = {32142605},
issn = {2161-5063},
mesh = {*Aptamers, Nucleotide ; CRISPR-Cas Systems ; *Cell Engineering ; Cell Line ; Humans ; RNA Interference ; *Riboswitch ; Synthetic Biology ; *Theophylline ; },
abstract = {The theophylline aptamer was isolated from an oligonucleotide library in 1994. Since that time, the aptamer has found wide utility, particularly in synthetic biology, cellular engineering, and diagnostic applications. The primary application of the theophylline aptamer is in the construction and characterization of synthetic riboswitches for regulation of gene expression. These riboswitches have been used to control cellular motility, regulate carbon metabolism, construct logic gates, screen for mutant enzymes, and control apoptosis. Other applications of the theophylline aptamer in cellular engineering include regulation of RNA interference and genome editing through CRISPR systems. Here we describe the uses of the theophylline aptamer for cellular engineering over the past 25 years. In so doing, we also highlight important synthetic biology applications to control gene expression in a ligand-dependent manner.},
}
@article {pmid32142408,
year = {2020},
author = {Kovač, A and Miskey, C and Menzel, M and Grueso, E and Gogol-Döring, A and Ivics, Z},
title = {RNA-guided retargeting of Sleeping Beauty transposition in human cells.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32142408},
issn = {2050-084X},
mesh = {CRISPR-Cas Systems ; Chromosomes, Human, X ; Genetic Therapy ; Genome, Human ; HeLa Cells ; Humans ; Hypoxanthine Phosphoribosyltransferase ; Multigene Family ; RNA, Guide/genetics/*metabolism ; Reproducibility of Results ; Retroelements ; Transposases/genetics/*metabolism ; },
abstract = {An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.},
}
@article {pmid32142118,
year = {2020},
author = {Kalwani, P and Rath, D and Ballal, A},
title = {Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.},
journal = {The Biochemical journal},
volume = {477},
number = {5},
pages = {971-983},
doi = {10.1042/BCJ20200026},
pmid = {32142118},
issn = {1470-8728},
mesh = {Anabaena/genetics/*metabolism ; Bacterial Proteins/*chemistry/genetics/*metabolism ; CRISPR-Associated Proteins/*chemistry/genetics/*metabolism ; CRISPR-Cas Systems/*physiology ; Cyanobacteria/chemistry/genetics/metabolism ; Protein Structure, Secondary ; },
abstract = {The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.},
}
@article {pmid32141890,
year = {2020},
author = {Maepa, MB and Jacobs, R and van den Berg, F and Arbuthnot, P},
title = {Recent developments with advancing gene therapy to treat chronic infection with hepatitis B virus.},
journal = {Current opinion in HIV and AIDS},
volume = {15},
number = {3},
pages = {200-207},
doi = {10.1097/COH.0000000000000623},
pmid = {32141890},
issn = {1746-6318},
mesh = {DNA, Viral ; Genetic Therapy ; *HIV Infections ; Hepatitis B virus/genetics ; *Hepatitis B, Chronic/therapy ; Humans ; Virus Replication ; },
abstract = {PURPOSE OF REVIEW: The available vaccine and therapies against hepatitis B virus (HBV) rarely eliminate chronic infection with the virus. High mortality resulting from complicating cirrhosis and hepatocellular carcinoma makes improving anti-HBV therapy an important priority. Recent advances with using gene therapy to counter HBV have potential and are the focus of this review.<br><br>RECENT FINDINGS: The stable replication-competent HBV intermediate comprising covalently closed circular DNA (cccDNA) is the template for expression of all viral genes. Inactivating cccDNA has thus been a focus of research aimed at achieving cure for HBV infection. Many studies have reported profound inhibition of replication of the virus using silencing and editing techniques. Therapeutic gene silencing with synthetic short interfering RNA is now in clinical trials. Ability to mutate and permanently inactivate cccDNA with engineered gene editors, such as those derived from CRISPR/Cas or TALENs, is particularly appealing but has not yet reached clinical evaluation.<br><br>SUMMARY: Gene silencing and gene editing potentially provide the means to cure HBV infection. However, achieving efficient delivery of therapeutic sequences, ensuring their specificity of action and progress with other antiviral strategies are likely to determine utility of gene therapy for chronic HBV infection.},
}
@article {pmid32141701,
year = {2020},
author = {Herrero, AB and Quwaider, D and Corchete, LA and Mateos, MV and García-Sanz, R and Gutiérrez, NC},
title = {FAM46C controls antibody production by the polyadenylation of immunoglobulin mRNAs and inhibits cell migration in multiple myeloma.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {7},
pages = {4171-4182},
pmid = {32141701},
issn = {1582-4934},
mesh = {Antibody Formation/*genetics/immunology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Gene Knockout Techniques ; Humans ; Immunoglobulins/biosynthesis/*immunology ; Multiple Myeloma/*genetics/immunology/pathology ; Nucleotidyltransferases/*genetics/immunology ; Polyadenylation/immunology ; RNA, Messenger/genetics ; Signal Transduction/genetics ; Unfolded Protein Response ; },
abstract = {FAM46C, frequently mutated in multiple myeloma (MM), has recently been shown to encode a non-canonical poly(A) polymerase (ncPAP). However, its target mRNAs and its role in MM pathogenesis remain mostly unknown. Using CRISPR-Cas9 technology and gene expression analysis, we found that the inactivation of FAM46C in MM down-regulates immunoglobulins (Igs) and several mRNAs encoding ER-resident proteins, including some involved in unfolded protein response and others that affect glycosylation. Interestingly, we show that FAM46C expression is induced during plasma cell (PC) differentiation and that Ig mRNAs encoding heavy and light chains are substrates of the ncPAP, as revealed by poly(A) tail-length determination assays. The absence of the ncPAP results in Ig mRNA poly(A) tail-shortening, leading to a reduction in mRNA and protein abundance. On the other hand, loss of FAM46C up-regulates metastasis-associated lncRNA MALAT1 and results in a sharp increase in the migration ability. This phenotype depends mainly on the activation of PI3K/Rac1 signalling, which might have significant therapeutic implications. In conclusion, our results identify Ig mRNAs as targets of FAM46C, reveal an important function of this protein during PC maturation to increase antibody production and suggest that its role as a tumour suppressor might be related to the inhibition of myeloma cell migration.},
}
@article {pmid32140907,
year = {2020},
author = {Gong, Y and Yang, B and Chen, W},
title = {Systematic genome editing of the genes spanning an entire chromosome by CRISPR/Cas9 in a vertebrate-zebrafish (Danio rerio).},
journal = {Science China. Life sciences},
volume = {63},
number = {7},
pages = {1096-1097},
pmid = {32140907},
issn = {1869-1889},
support = {R01 DK109407/DK/NIDDK NIH HHS/United States ; R01 DK117147/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Base Sequence ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Chromosomes/genetics ; Databases, Genetic ; Gene Editing/*methods ; Genome ; Transcriptome/*genetics ; Zebrafish ; Zebrafish Proteins/genetics ; },
}
@article {pmid32140725,
year = {2020},
author = {Zhang, Z and Sun, W and Shi, T and Lu, P and Zhuang, M and Liu, JL},
title = {Capturing RNA-protein interaction via CRUIS.},
journal = {Nucleic acids research},
volume = {48},
number = {9},
pages = {e52},
pmid = {32140725},
issn = {1362-4962},
mesh = {*CRISPR-Associated Proteins ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Immunoprecipitation ; Mass Spectrometry ; RNA/metabolism ; RNA-Binding Proteins/*metabolism ; *Ribonucleases ; },
abstract = {No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA-protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA-protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA-protein interactions in their natural environment, and provides new insights into RNA biology.},
}
@article {pmid32139868,
year = {2020},
author = {Zhou, Y and Lin, L and Wang, H and Zhang, Z and Zhou, J and Jiao, N},
title = {Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {98},
pmid = {32139868},
issn = {2399-3642},
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coumaric Acids/*metabolism ; *Gene Editing ; Lignin/*metabolism ; Polyhydroxyalkanoates/*metabolism ; Pseudomonas putida/genetics/*metabolism ; Vanillic Acid/metabolism ; },
abstract = {Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value. However, its recalcitrance and toxicity poses a challenge for ferulic acid-to-bioproducts bioconversion. Here, we develop a genome editing strategy for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9n-λ-Red system with pyrF as a selection marker, which maintains cell viability and genetic stability, increases mutation efficiency, and simplifies genetic manipulation. Via this method, four functional modules, comprised of nine genes involved in ferulic acid catabolism and polyhydroxyalkanoate biosynthesis, were integrated into the genome, generating the KTc9n20 strain. After metabolic engineering and optimization of C/N ratio, polyhydroxyalkanoate production was increased to ~270 mg/L, coupled with ~20 mM ferulic acid consumption. This study not only establishes a simple and efficient genome editing strategy, but also offers an encouraging example of how to apply this method to improve microbial aromatic compound bioconversion.},
}
@article {pmid32139608,
year = {2020},
author = {Fichtner, AS and Karunakaran, MM and Gu, S and Boughter, CT and Borowska, MT and Starick, L and Nöhren, A and Göbel, TW and Adams, EJ and Herrmann, T},
title = {Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {12},
pages = {6697-6707},
pmid = {32139608},
issn = {1091-6490},
support = {T32 EB009412/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Antibodies, Monoclonal/*immunology ; Butyrophilins/antagonists &amp; inhibitors/genetics/immunology/*metabolism ; CRISPR-Cas Systems ; Camelids, New World ; Female ; HEK293 Cells ; Hemiterpenes/*pharmacology ; Humans ; Lymphocyte Activation/drug effects/*immunology ; Male ; Mice ; Mice, Inbred BALB C ; Organophosphorus Compounds/*pharmacology ; Protein Binding ; Receptors, Antigen, T-Cell, gamma-delta/drug effects/immunology/metabolism ; T-Lymphocyte Subsets/drug effects/*immunology/metabolism ; },
abstract = {Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.},
}
@article {pmid32139597,
year = {2020},
author = {Wiggenhauser, LM and Qi, H and Stoll, SJ and Metzger, L and Bennewitz, K and Poschet, G and Krenning, G and Hillebrands, JL and Hammes, HP and Kroll, J},
title = {Activation of Retinal Angiogenesis in Hyperglycemic pdx1 [-/-] Zebrafish Mutants.},
journal = {Diabetes},
volume = {69},
number = {5},
pages = {1020-1031},
doi = {10.2337/db19-0873},
pmid = {32139597},
issn = {1939-327X},
mesh = {Animals ; Blood Glucose ; CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation/drug effects ; Homeodomain Proteins/genetics/*metabolism ; *Hyperglycemia ; Larva ; *Neovascularization, Pathologic ; Nitric Oxide/metabolism ; Phthalazines/pharmacology ; Protein Kinase Inhibitors/pharmacology ; Pyridines/pharmacology ; Retinal Neovascularization ; Retinal Vessels/*physiology ; Trans-Activators/genetics/*metabolism ; Vascular Endothelial Growth Factor A/metabolism ; Zebrafish ; },
abstract = {Progression from the initial vascular response upon hyperglycemia to a proliferative stage with neovacularizations is the hallmark of proliferative diabetic retinopathy. Here, we report on the novel diabetic pdx1 [-/-] zebrafish mutant as a model for diabetic retinopathy that lacks the transcription factor pdx1 through CRISPR-Cas9-mediated gene knockout leading to disturbed pancreatic development and hyperglycemia. Larval pdx1 [-/-] mutants prominently show vasodilation of blood vessels through increased vascular thickness in the hyaloid network as direct developmental precursor of the adult retinal vasculature in zebrafish. In adult pdx1 [-/-] mutants, impaired glucose homeostasis induces increased hyperbranching and hypersprouting with new vessel formation in the retina and aggravation of the vascular alterations from the larval to the adult stage. Both vascular aspects respond to antiangiogenic and antihyperglycemic pharmacological interventions in the larval stage and are accompanied by alterations in the nitric oxide metabolism. Thus, the pdx1 [-/-] mutant represents a novel model to study mechanisms of hyperglycemia-induced retinopathy wherein extensive proangiogenic alterations in blood vessel morphology and metabolic alterations underlie the vascular phenotype.},
}
@article {pmid32139545,
year = {2020},
author = {Chen, J and Brunner, AD and Cogan, JZ and Nuñez, JK and Fields, AP and Adamson, B and Itzhak, DN and Li, JY and Mann, M and Leonetti, MD and Weissman, JS},
title = {Pervasive functional translation of noncanonical human open reading frames.},
journal = {Science (New York, N.Y.)},
volume = {367},
number = {6482},
pages = {1140-1146},
pmid = {32139545},
issn = {1095-9203},
support = {K99 GM134154/GM/NIGMS NIH HHS/United States ; R00 GM134154/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Humans ; *Open Reading Frames ; Operon ; Peptides/*genetics ; Protein Biosynthesis/*genetics ; *RNA, Messenger/genetics/metabolism ; Ribosomes/metabolism ; Transcriptome ; },
abstract = {Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.},
}
@article {pmid32138651,
year = {2020},
author = {Cui, Y and Liao, X and Peng, S and Tang, T and Huang, C and Yang, C},
title = {OffScan: a universal and fast CRISPR off-target sites detection tool.},
journal = {BMC genomics},
volume = {21},
number = {Suppl 1},
pages = {872},
pmid = {32138651},
issn = {1471-2164},
mesh = {Algorithms ; Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; Endonucleases/metabolism ; Gene Editing/*methods ; Humans ; Mice ; Mutation ; RNA, Guide/*genetics ; Zebrafish/genetics ; },
abstract = {BACKGROUND: The Type II clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) is a powerful genome editing technology, which is more and more popular in gene function analysis. In CRISPR/Cas, RNA guides Cas nuclease to the target site to perform DNA modification.<br><br>RESULTS: The performance of CRISPR/Cas depends on well-designed single guide RNA (sgRNA). However, the off-target effect of sgRNA leads to undesired mutations in genome and limits the use of CRISPR/Cas. Here, we present OffScan, a universal and fast CRISPR off-target detection tool.<br><br>CONCLUSIONS: OffScan is not limited by the number of mismatches and allows custom protospacer-adjacent motif (PAM), which is the target site by Cas protein. Besides, OffScan adopts the FM-index, which efficiently improves query speed and reduce memory consumption.},
}
@article {pmid32138192,
year = {2020},
author = {Bouzroud, S and Gasparini, K and Hu, G and Barbosa, MAM and Rosa, BL and Fahr, M and Bendaou, N and Bouzayen, M and Zsögön, A and Smouni, A and Zouine, M},
title = {Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32138192},
issn = {2073-4425},
mesh = {Abscisic Acid/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant/genetics ; Lycopersicon esculentum/*genetics/growth &amp; development ; Osmotic Pressure/physiology ; Plant Development/genetics ; Plant Leaves/genetics/growth &amp; development ; Plant Proteins/*genetics ; Plants, Genetically Modified/genetics/growth &amp; development ; Salinity ; Salt Stress/*genetics ; Salt Tolerance/*genetics ; },
abstract = {Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.},
}
@article {pmid32132707,
year = {2020},
author = {Guo, X and Aviles, G and Liu, Y and Tian, R and Unger, BA and Lin, YT and Wiita, AP and Xu, K and Correia, MA and Kampmann, M},
title = {Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.},
journal = {Nature},
volume = {579},
number = {7799},
pages = {427-432},
pmid = {32132707},
issn = {1476-4687},
support = {1S10OD010786-01/NH/NIH HHS/United States ; R01 GM044037/GM/NIGMS NIH HHS/United States ; R01 DK026506/DK/NIDDK NIH HHS/United States ; DP2 GM123500/GM/NIGMS NIH HHS/United States ; DK26506/NH/NIH HHS/United States ; OD022552/NH/NIH HHS/United States ; R56 DK026506/DK/NIDDK NIH HHS/United States ; GM44037/NH/NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; GM119139/NH/NIH HHS/United States ; },
mesh = {Activating Transcription Factor 4/biosynthesis/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cytosol/enzymology/*metabolism ; Enzyme Activation ; Eukaryotic Initiation Factor-2/metabolism ; Humans ; Male ; Metalloendopeptidases/*metabolism ; Mitochondria/*metabolism/*pathology ; Mitochondrial Proteins/chemistry/*metabolism ; Molecular Chaperones/metabolism ; Phosphorylation ; Protein Binding ; *Stress, Physiological ; eIF-2 Kinase/*metabolism ; },
abstract = {In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF4[1-3]. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.},
}
@article {pmid32132663,
year = {2020},
author = {Zheng, Z and Zhang, Y and Liu, Z and Dong, Z and Xie, C and Bravo, A and Soberón, M and Mahillon, J and Sun, M and Peng, D},
title = {The CRISPR-Cas systems were selectively inactivated during evolution of Bacillus cereus group for adaptation to diverse environments.},
journal = {The ISME journal},
volume = {14},
number = {6},
pages = {1479-1493},
pmid = {32132663},
issn = {1751-7370},
mesh = {Acclimatization ; Bacillus ; Bacillus cereus/*physiology ; *CRISPR-Cas Systems ; Environment ; Gene Transfer, Horizontal ; },
abstract = {CRISPR-Cas systems are considered as barriers to horizontal gene transfer (HGT). However, the influence of such systems on HGT within species is unclear. Also, little is known about the impact of CRISPR-Cas systems on bacterial evolution at the population level. Here, using Bacillus cereus sensu lato as model, we investigate the interplay between CRISPR-Cas systems and HGT at the population scale. We found that only a small fraction of the strains have CRISPR-Cas systems (13.9% of 1871), and most of such systems are defective based on their gene content analysis. Comparative genomic analysis revealed that the CRISPR-Cas systems are barriers to HGT within this group, since strains harboring active systems contain less mobile genetic elements (MGEs), have lower fraction of unique genes and also display limited environmental distributions than strains without active CRISPR-Cas systems. The introduction of a functional CRISPR-Cas system into a strain lacking the system resulted in reduced adaptability to various stresses and decreased pathogenicity of the transformant strain, indicating that B. cereus group strains could benefit from inactivating such systems. Our work provides a large-scale case to support that the CRISPR-Cas systems are barriers to HGT within species, and that in the B. cereus group the inactivation of CRISPR-Cas systems correlated with acquisition of MGEs that could result in better adaptation to diverse environments.},
}
@article {pmid32132530,
year = {2020},
author = {Dong, OX and Yu, S and Jain, R and Zhang, N and Duong, PQ and Butler, C and Li, Y and Lipzen, A and Martin, JA and Barry, KW and Schmutz, J and Tian, L and Ronald, PC},
title = {Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1178},
pmid = {32132530},
issn = {2041-1723},
mesh = {Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Carotenoids/analysis/*metabolism ; DNA, Plant/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Genome, Plant/genetics ; Oryza/*genetics/metabolism ; Plant Breeding/*methods ; Plants, Genetically Modified ; Seeds/chemistry ; Whole Genome Sequencing ; },
abstract = {Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.},
}
@article {pmid32130724,
year = {2020},
author = {Meng, Y and Liu, Y and Dakou, E and Gutierrez, GJ and Leyns, L},
title = {Polycomb group RING finger protein 5 influences several developmental signaling pathways during the in vitro differentiation of mouse embryonic stem cells.},
journal = {Development, growth &amp; differentiation},
volume = {62},
number = {4},
pages = {232-242},
doi = {10.1111/dgd.12659},
pmid = {32130724},
issn = {1440-169X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Line ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells/*cytology/*metabolism ; Polycomb-Group Proteins/deficiency/genetics/*metabolism ; *Signal Transduction/genetics ; },
abstract = {Polycomb group (PcG) RING finger protein 5 (PCGF5) is a core component of the so-called Polycomb repressive complex 1.5 (PRC1.5), which is involved in epigenetic transcriptional repression. To explore the developmental function of Pcgf5, we generated Pcgf5 knockout (Pcgf5[-/-]) mouse embryonic stem cell (mESC) lines with the help of CRISPR/Cas9 technology. We subjected the Pcgf5[-/-] and wild-type (WT) mESCs to a differentiation protocol toward mesodermal-cardiac cell types as aggregated embryoid bodies (EBs) and we found that knockout of Pcgf5 delayed the generation of the three germ layers, especially the ectoderm. Further, disruption of Pcgf5 impacted the epithelial-mesenchymal transition during EB morphogenesis and differentially affected the gene expression of essential developmental signaling pathways such as Nodal and Wnt. Finally, we also unveiled that loss of Pcgf5 induced the repression of genes involved in the Notch pathway, which may explain the enhancement of cardiomyocyte maturation and the dampening of ectodermal-neural differentiation observed in the Pcgf5[-/-] EBs.},
}
@article {pmid32130564,
year = {2020},
author = {Liu, H and Hou, G and Wang, P and Guo, G and Wang, Y and Yang, N and Rehman, MNU and Li, C and Li, Q and Zheng, J and Zeng, J and Li, S},
title = {A double-locus scarless genome editing system in Escherichia coli.},
journal = {Biotechnology letters},
volume = {42},
number = {8},
pages = {1457-1465},
doi = {10.1007/s10529-020-02856-7},
pmid = {32130564},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; Escherichia coli/*genetics ; Gene Editing/*methods ; Genome, Bacterial/*genetics ; Plasmids/genetics ; Recombination, Genetic/genetics ; },
abstract = {OBJECTIVE: To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette.<br><br>RESULTS: A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and &#x3bb; Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88&#x2009;&#xb1;&#x2009;5.5% (point mutation), 39.7&#x2009;&#xb1;&#x2009;4.3% (deletion/insertion), and 57.8&#x2009;&#xb1;&#x2009;3.4%-58.5&#x2009;&#xb1;&#x2009;4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40&#xa0;bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96&#x2009;&#xb1;&#x2009;3% in 4&#xa0;h) than the help plasmid carrying cas9 and &#x3bb; Red (92&#x2009;&#xb1;&#x2009;2% in 9&#xa0;h).<br><br>CONCLUSIONS: The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.},
}
@article {pmid32130260,
year = {2020},
author = {Tsukumo, Y and Naito, M and Suzuki, T},
title = {Influence of EGFR-activating mutations on sensitivity to tyrosine kinase inhibitors in a KRAS mutant non-small cell lung cancer cell line.},
journal = {PloS one},
volume = {15},
number = {3},
pages = {e0229712},
pmid = {32130260},
issn = {1932-6203},
mesh = {A549 Cells ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/*genetics ; Cell Proliferation/drug effects ; Clone Cells ; ErbB Receptors/genetics ; Gene Dosage ; Gene Editing ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Lung Neoplasms/*genetics ; Mutation/*genetics ; Phosphorylation/drug effects ; Protein Kinase Inhibitors/*pharmacology ; Proto-Oncogene Proteins p21(ras)/*genetics ; },
abstract = {In non-small cell lung cancer (NSCLC), oncogenic driver mutations including those in KRAS and EGFR are typically mutually exclusive. However, recent reports indicate that multiple driver mutations are found in a certain percentage of cancers, and that the therapeutic responses of such cases with co-mutations of driver genes are largely unclear. Here, using CRISPR-Cas9-mediated genome editing, we generated isogenic cell lines harboring one or two copies of an EGFR-activating mutation from the human NSCLC cell line A549, which is known to harbor a homozygous KRAS gene mutation. In comparison with parent cells with KRAS mutation alone, cells with concomitant EGFR mutation exhibited higher sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) but not to conventional anti-cancer drugs. In particular, cells with two copies of EGFR mutation were markedly more sensitive to EGFR-TKIs compared with parent cells. Thus, the presence of concomitant EGFR mutation can affect the TKI response of KRAS-mutated cells, implying that EGFR-TKI may represent an effective treatment option against NSCLC with EGFR/KRAS co-mutation.},
}
@article {pmid32129762,
year = {2020},
author = {Gigante, ED and Taylor, MR and Ivanova, AA and Kahn, RA and Caspary, T},
title = {ARL13B regulates Sonic hedgehog signaling from outside primary cilia.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32129762},
issn = {2050-084X},
support = {R01 NS090029/NS/NINDS NIH HHS/United States ; R35 GM122568/GM/NIGMS NIH HHS/United States ; R35 GM122549/GM/NIGMS NIH HHS/United States ; T32 NS096050/NS/NINDS NIH HHS/United States ; F31 NS106755/NS/NINDS NIH HHS/United States ; },
mesh = {ADP-Ribosylation Factors/*genetics/*metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cilia/*metabolism ; Embryonic Development/genetics ; Gene Editing ; Gene Expression ; Hedgehog Proteins/*metabolism ; Mice ; Mice, Knockout ; Mutation ; Phenotype ; *Signal Transduction ; },
abstract = {ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13B[V358A] protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13b[V358A/V358A] mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13b[V358A/V358A] cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B's role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.},
}
@article {pmid32129700,
year = {2020},
author = {Salant, GM and Tat, KL and Goodrich, JA and Kugel, JF},
title = {miR-206 knockout shows it is critical for myogenesis and directly regulates newly identified target mRNAs.},
journal = {RNA biology},
volume = {17},
number = {7},
pages = {956-965},
pmid = {32129700},
issn = {1555-8584},
support = {R01 GM068414/GM/NIGMS NIH HHS/United States ; T32 GM008759/GM/NIGMS NIH HHS/United States ; R01 AR067821/AR/NIAMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Gene Expression Regulation ; *Gene Knockout Techniques ; High-Throughput Nucleotide Sequencing ; Mice ; MicroRNAs/*genetics ; Muscle Development/*genetics ; Myoblasts/metabolism ; *RNA Interference ; RNA, Messenger/*genetics ; },
abstract = {The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six of these targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.},
}
@article {pmid32129468,
year = {2020},
author = {Wu, Y and Liu, Y and Lv, X and Li, J and Du, G and Liu, L},
title = {CAMERS-B: CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {6},
pages = {1817-1825},
doi = {10.1002/bit.27322},
pmid = {32129468},
issn = {1097-0290},
support = {31600068//National Natural Science Foundation of China/International ; 31622001//National Natural Science Foundation of China/International ; 31671845//National Natural Science Foundation of China/International ; 31930085//National Natural Science Foundation of China/International ; JUSRP51713B//Fundamental Research Funds for the Central Universities/International ; 2018YFA0900300//National Key Research and Development Program of China/International ; 111-2-06//Higher Education Discipline Innovation Project/International ; KYCX18_1802//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/International ; },
mesh = {Bacillus subtilis/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Francisella/*genetics ; Gene Editing/*methods ; Genome, Bacterial ; },
abstract = {The clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been widely used in genome editing and transcriptional regulation. In this study, by engineering the Francisella novicida U112 CRISPR/Cpf1 system, a powerful tool called CRISPR/Cpf1 assisted multiple-genes editing and regulation system for B. subtilis was constructed for engineering Bacillus subtilis, and a synthetic oligos mediated assembly of CRISPR RNA (crRNA) array method was created to build crRNA array. This system can achieve the double genes in-frame knocking out, multiple point mutations (up to six), or single gene insertion at a time with 100% efficiency. In addition, transcriptional regulation systems were also developed using the DNase deactivated Cas protein (dCpf1) and a transcription factor RemA, which can implement repression and activation on multiple-genes concurrently. Finally, as a proof-of-concept demonstration, the synthesis pathways of N-acetylglucosamine and acetoin in B. subtilis were engineered by using this system. Overall, we provide effective tools for genome editing and metabolic engineering of B. subtilis cell factories to produce various biochemicals.},
}
@article {pmid32129035,
year = {2019},
author = {Nicol, D and Eckstein, L},
title = {Gene Editing Clinical Trials Could Slip through Australian Regulatory Cracks.},
journal = {Journal of law and medicine},
volume = {27},
number = {2},
pages = {274-283},
pmid = {32129035},
issn = {1320-159X},
mesh = {Australia ; *CRISPR-Cas Systems ; Clinical Trials as Topic/*legislation &amp; jurisprudence ; Gene Editing/*legislation &amp; jurisprudence ; Humans ; },
abstract = {In this column we explore the regulatory environment within which clinical trials involving new genome editing techniques are undertaken. Ostensibly, there is regulatory congestion in this area, with overlapping obligations through the national scheme for regulating gene technology, the national scheme for regulating the supply of therapeutic goods, and the human research ethics system, predominantly administered at the institutional level. In practice, however, the oversight of gene editing clinical trials is left almost entirely to human research ethics committees. Given the uncertain risks associated with such novel technologies, we conclude that it is opportune to reconsider the rigour of current Australian processes for assessing clinical trials involving gene-editing technology.},
}
@article {pmid32128412,
year = {2020},
author = {Zhang, Y and Li, H and Min, YL and Sanchez-Ortiz, E and Huang, J and Mireault, AA and Shelton, JM and Kim, J and Mammen, PPA and Bassel-Duby, R and Olson, EN},
title = {Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system.},
journal = {Science advances},
volume = {6},
number = {8},
pages = {eaay6812},
pmid = {32128412},
issn = {2375-2548},
support = {P50 HD087351/HD/NICHD NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/*genetics ; Disease Models, Animal ; Dystrophin/*genetics ; Exons ; Gene Dosage ; *Gene Editing ; Gene Expression ; Gene Targeting ; Gene Transfer Techniques ; *Genetic Therapy ; Genetic Vectors/*genetics ; Mice ; Muscle, Skeletal/metabolism ; Muscular Dystrophy, Duchenne/*genetics/therapy ; Mutation ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9-mediated "single-cut" genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9-mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.},
}
@article {pmid32127692,
year = {2020},
author = {van der Oost, J and Mougiakos, I},
title = {First structural insights into CRISPR-Cas-guided DNA transposition.},
journal = {Cell research},
volume = {30},
number = {3},
pages = {193-194},
pmid = {32127692},
issn = {1748-7838},
mesh = {Bacterial Proteins/metabolism ; *CRISPR-Cas Systems ; *DNA Transposable Elements ; DNA, Bacterial ; *Recombination, Genetic ; Transposases/*metabolism ; Vibrio cholerae/*genetics ; },
}
@article {pmid32126327,
year = {2020},
author = {Fatima, A and Schuster, J and Akram, T and González, CM and Sobol, M and Hoeber, J and Dahl, N},
title = {Incontinentia pigmenti: Generation of an IKBKG deficient human iPSC line (KICRi002-A-1) on a 46,XY background using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {44},
number = {},
pages = {101739},
doi = {10.1016/j.scr.2020.101739},
pmid = {32126327},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; *Cell Line ; Female ; Humans ; I-kappa B Kinase/genetics/metabolism ; *Incontinentia Pigmenti/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Male ; Mutation ; },
abstract = {Incontinentia pigmenti (IP) is an X-linked dominant neuroectodermal dysplasia caused by loss-of-function mutations in the IKBKG gene. Using CRISPR/Cas9 technology, we generated an IKBKG knock-out iPSC line (KICRi002-A-1) on a 46,XY background. The iPSC line showed a normal karyotype, expressed pluripotency markers and exhibited capability to differentiate into the three germ layers in vitro. Off-target editing was excluded and no IKBKG mRNA expression could be detected. Our line offers a useful resource to elucidate mechanisms caused by IKBKG deficiency that leads to disrupted male fetal development and for drug screening to improve treatment of female patients with IP.},
}
@article {pmid32126126,
year = {2020},
author = {Lacchini, E and Kiegle, E and Castellani, M and Adam, H and Jouannic, S and Gregis, V and Kater, MM},
title = {CRISPR-mediated accelerated domestication of African rice landraces.},
journal = {PloS one},
volume = {15},
number = {3},
pages = {e0229782},
pmid = {32126126},
issn = {1932-6203},
mesh = {Acclimatization/genetics ; Africa ; CRISPR-Cas Systems/genetics ; DNA, Plant/genetics ; *Domestication ; Edible Grain/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genes, Plant/genetics ; Genetic Loci/genetics ; Oryza/*genetics ; Plant Breeding/*methods ; Plants, Genetically Modified/genetics ; Proof of Concept Study ; Time Factors ; },
abstract = {African Oryza glaberrima and Oryza sativa landraces are considered valuable resources for breeding traits due to their adaptation to local environmental and soil conditions. They often possess superior resistance to endemic pests and tolerance to drought and nutrient deficiencies when compared to the "imported" high production Asian rice varieties. In contrast, "domestication traits" such as seed shattering, lodging, and seed yield are not well established in these African landraces. Therefore, the use of these African varieties for high production agriculture is limited by unpredictable yield and grain quality. We are addressing this shortcoming by developing protocols for genetically transforming African landraces to allow the use of CRISPR-Cas mediated breeding approaches. Here we use as proof of concept the cultivated African landrace Kabre to target selected known "domestication loci" and improve the agronomic potential of Kabre rice. Stable genetic transformation with CRISPR-Cas9-based vectors generated single and simultaneous multiple gene knockouts. Plants with reduced stature to diminish lodging were generated by disrupting the HTD1 gene. Furthermore, three loci shown to control seed size and/or yield (GS3, GW2 and GN1A) were targeted using a multiplex CRISPR-Cas9 construct. This resulted in mutants with significantly improved seed yield. Our study provides an example of how new breeding technologies can accelerate the development of highly productive African landrace rice varieties, an important advancement considering that Africa is a hotspot for worldwide population growth and therefore prone to food shortage.},
}
@article {pmid32126082,
year = {2020},
author = {Quiroga Artigas, G and Lapébie, P and Leclère, L and Bauknecht, P and Uveira, J and Chevalier, S and Jékely, G and Momose, T and Houliston, E},
title = {A G protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia.},
journal = {PLoS biology},
volume = {18},
number = {3},
pages = {e3000614},
pmid = {32126082},
issn = {1545-7885},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cyclic AMP/metabolism ; Female ; Gene Expression ; Hydrozoa/genetics/*physiology ; Male ; Mutation ; Neuropeptides/*metabolism ; Oocytes/*physiology ; Phylogeny ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Receptors, Neuropeptide/genetics/metabolism ; },
abstract = {The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based "deorphanization" of candidate oocyte-expressed G protein-coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.},
}
@article {pmid32125907,
year = {2020},
author = {Abati, E and Bresolin, N and Comi, G and Corti, S},
title = {Silence superoxide dismutase 1 (SOD1): a promising therapeutic target for amyotrophic lateral sclerosis (ALS).},
journal = {Expert opinion on therapeutic targets},
volume = {24},
number = {4},
pages = {295-310},
doi = {10.1080/14728222.2020.1738390},
pmid = {32125907},
issn = {1744-7631},
mesh = {Amyotrophic Lateral Sclerosis/genetics/physiopathology/*therapy ; Animals ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; *Gene Silencing ; Humans ; Molecular Targeted Therapy ; Mutation ; Oligonucleotides, Antisense/administration &amp; dosage ; RNA Interference ; Superoxide Dismutase-1/*genetics ; },
abstract = {Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive and incurable neurodegenerative disorder that targets upper and lower motor neurons and leads to fatal muscle paralysis. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for 15% of familial ALS cases, but several studies have indicated that SOD1 dysfunction may also play a pathogenic role in sporadic ALS. SOD1 induces numerous toxic effects through the pathological misfolding and aggregation of mutant SOD1 species, hence a reduction of the levels of toxic variants appears to be a promising therapeutic strategy for SOD1-related ALS. Several methods are used to modulate gene expression in vivo; these include RNA interference, antisense oligonucleotides (ASOs) and CRISPR/Cas9 technology.Areas covered: This paper examines the current approaches for gene silencing and the progress made in silencing SOD1 in vivo. It progresses to shed light on the key results and pitfalls of these studies and highlights the future challenges and new perspectives for this exciting research field.Expert opinion: Gene silencing strategies targeting SOD1 may represent effective approaches for familial and sporadic ALS-related neurodegeneration; however, the risk of off-target effects must be minimized, and effective and minimally invasive delivery strategies should be fine-tuned.},
}
@article {pmid32124927,
year = {2020},
author = {Xu, M and Weng, Q and Ji, J},
title = {Applications and advances of CRISPR/Cas9 in animal cancer model.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {235-241},
doi = {10.1093/bfgp/elaa002},
pmid = {32124927},
issn = {2041-2657},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Editing ; Humans ; Neoplasms/*genetics ; },
abstract = {The recent developments of clustered regularly interspaced short palindromic repeats(CRISPR)/-associate protein 9 (CRISPR/Cas9) have got scientific interests due to the straightforward, efficient and versatile talents of it. Furthermore, the CRISPR/Cas9 system has democratized access to gene editing in many biological fields, including cancer. Cancer development is a multistep process caused by innate and acquired mutations and leads to the initiation and progression of tumorigenesis. It is obvious that establishing appropriate animal cancer models which can simulate human cancers is crucial for cancer research currently. Since the emergence of CRISPR/Cas9, considerable efforts have been taken by researchers to apply this technology in generating animal cancer models. Although there is still a long way to go we are happy to see the achievements we have made and the promising future we have.},
}
@article {pmid32124178,
year = {2020},
author = {Ding, N and Qin, Q and Wu, X and Miller, R and Zaitlin, D and Li, D and Yang, S},
title = {Antagonistic regulation of axillary bud outgrowth by the BRANCHED genes in tobacco.},
journal = {Plant molecular biology},
volume = {103},
number = {1-2},
pages = {185-196},
pmid = {32124178},
issn = {1573-5028},
mesh = {CRISPR-Cas Systems ; Down-Regulation ; Gene Expression Regulation, Plant ; *Genes, Plant ; Phylogeny ; Plant Development/*genetics ; RNA Interference ; Signal Transduction ; Tobacco/genetics/*growth &amp; development ; Transcriptome ; Up-Regulation ; },
abstract = {As a key integrator of shoot branching, BRANCHED 1 (BRC1) coordinates and is orchestrated by endogenous and environmental signals involved in the regulation of axillary bud outgrowth. In the present study, we characterized the regulatory roles of five BRC gene members in tobacco (Nicotiana tabacum L.) using CRISPR site-directed mutagenesis and overexpression assays. It was shown that lateral branching was negatively regulated by NtBRC1A-1, 1B-1, and 1B-2, but was unexpectedly promoted by NtBRC2A. Suppression of bud growth may be attained by direct binding of NtBRCs to the Tassels Replace Upper Ears 1 (TRU1) genes. It was speculated that NtBRC2A probably confers a dominant negative effect by interfering with the branching-inhibitory BRC1 genes. Our results suggested that highly homologous gene family members may function antagonistically in the same signaling pathway. However, the molecular mechanism underlying NtBRC2A-mediated outgrowth of axillary buds needs to be further addressed. KEY MESSAGE: Axillary bud outgrowth in general is negatively regulated by the BRANCHED gene. Here we show that the BRANCHED genes play opposing regulatory roles in tobacco lateral branching.},
}
@article {pmid32123387,
year = {2020},
author = {Bratovič, M and Fonfara, I and Chylinski, K and Gálvez, EJC and Sullivan, TJ and Boerno, S and Timmermann, B and Boettcher, M and Charpentier, E},
title = {Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches.},
journal = {Nature chemical biology},
volume = {16},
number = {5},
pages = {587-595},
pmid = {32123387},
issn = {1552-4469},
mesh = {Arginine/*chemistry ; CRISPR-Associated Protein 9/*chemistry/*metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; DNA Mismatch Repair ; Escherichia coli/genetics ; HEK293 Cells ; Humans ; MCF-7 Cells ; Protein Conformation ; RNA/metabolism ; },
abstract = {The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.},
}
@article {pmid32123354,
year = {2020},
author = {Rollie, C and Chevallereau, A and Watson, BNJ and Chyou, TY and Fradet, O and McLeod, I and Fineran, PC and Brown, CM and Gandon, S and Westra, ER},
title = {Publisher Correction: Targeting of temperate phages drives loss of type I CRISPR-Cas systems.},
journal = {Nature},
volume = {579},
number = {7799},
pages = {E10},
doi = {10.1038/s41586-020-2089-z},
pmid = {32123354},
issn = {1476-4687},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid32123335,
year = {2020},
author = {Artegiani, B and Hendriks, D and Beumer, J and Kok, R and Zheng, X and Joore, I and Chuva de Sousa Lopes, S and van Zon, J and Tans, S and Clevers, H},
title = {Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing.},
journal = {Nature cell biology},
volume = {22},
number = {3},
pages = {321-331},
pmid = {32123335},
issn = {1476-4679},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knock-In Techniques/*methods ; Hepatocytes/cytology/ultrastructure ; Humans ; Intestines/cytology ; Liver/cytology ; Organoids/*cytology/ultrastructure ; Spindle Apparatus/ultrastructure ; Tumor Suppressor Protein p53/physiology ; },
abstract = {CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.},
}
@article {pmid32123334,
year = {2020},
author = {Yang, Q and Oost, KC and Liberali, P},
title = {Engineering human knock-in organoids.},
journal = {Nature cell biology},
volume = {22},
number = {3},
pages = {261-263},
pmid = {32123334},
issn = {1476-4679},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Organoids ; Tissue Engineering ; },
}
@article {pmid32123105,
year = {2020},
author = {Ivanov, IE and Wright, AV and Cofsky, JC and Aris, KDP and Doudna, JA and Bryant, Z},
title = {Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {11},
pages = {5853-5860},
pmid = {32123105},
issn = {1091-6490},
support = {R01 GM106159/GM/NIGMS NIH HHS/United States ; T32 GM008295/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Base Pairing ; CRISPR-Associated Proteins/*chemistry/metabolism ; *CRISPR-Cas Systems ; DNA/*chemistry ; DNA Cleavage ; Endonucleases/metabolism ; Gene Editing ; Genome ; R-Loop Structures ; RNA/chemistry ; RNA, Guide/metabolism ; },
abstract = {The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.},
}
@article {pmid32122959,
year = {2020},
author = {Macias, VM and McKeand, S and Chaverra-Rodriguez, D and Hughes, GL and Fazekas, A and Pujhari, S and Jasinskiene, N and James, AA and Rasgon, JL},
title = {Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {4},
pages = {1353-1360},
pmid = {32122959},
issn = {2160-1836},
support = {R21 AI111175/AI/NIAID NIH HHS/United States ; R01 AI029746/AI/NIAID NIH HHS/United States ; R01 AI128201/AI/NIAID NIH HHS/United States ; R21 AI138074/AI/NIAID NIH HHS/United States ; R21 AI129507/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Anopheles/genetics ; CRISPR-Cas Systems ; Female ; Gene Editing ; *Malaria ; Mosquito Vectors/genetics ; },
abstract = {Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.},
}
@article {pmid32122911,
year = {2020},
author = {Diaz-de-la-Loza, MD and Loker, R and Mann, RS and Thompson, BJ},
title = {Control of tissue morphogenesis by the HOX gene Ultrabithorax.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {5},
pages = {},
pmid = {32122911},
issn = {1477-9129},
support = {R01 GM054510/GM/NIGMS NIH HHS/United States ; FC001180/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; FC001180/CRUK_/Cancer Research UK/United Kingdom ; R35 GM118336/GM/NIGMS NIH HHS/United States ; 102853/B/13/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; Drosophila melanogaster/*embryology/genetics ; Homeodomain Proteins/*genetics ; Matrix Metalloproteinase Inhibitors/metabolism ; Membrane Proteins/genetics ; Morphogenesis/*genetics ; Serine Endopeptidases/genetics ; Transcription Factors/*genetics ; Wings, Animal/*embryology ; },
abstract = {Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. Here, we show that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. Our results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.},
}
@article {pmid32122593,
year = {2020},
author = {Liu, Y and Xu, Z and Zhang, Y and Yu, M and Wang, S and Gao, Y and Liu, C and Zhang, Y and Gao, L and Qi, X and Cui, H and Pan, Q and Li, K and Wang, X},
title = {Marek's disease virus as a CRISPR/Cas9 delivery system to defend against avian leukosis virus infection in chickens.},
journal = {Veterinary microbiology},
volume = {242},
number = {},
pages = {108589},
doi = {10.1016/j.vetmic.2020.108589},
pmid = {32122593},
issn = {1873-2542},
mesh = {Animals ; Avian Leukosis/*prevention &amp; control/virology ; Avian Leukosis Virus/genetics/pathogenicity ; *CRISPR-Cas Systems ; Chickens/virology ; Fibroblasts/virology ; Gene Editing/methods ; Genetic Vectors ; Genome, Viral ; Herpesvirus 2, Gallid/*genetics ; Marek Disease Vaccines/*genetics ; Poultry Diseases/*prevention &amp; control/virology ; RNA, Guide/genetics ; RNA, Viral/genetics ; Specific Pathogen-Free Organisms ; Virus Integration ; },
abstract = {The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens.},
}
@article {pmid32122549,
year = {2020},
author = {Wang, J and Balan, V and Marincola, F},
title = {CRISPR technology for immuno-oncology applications.},
journal = {Methods in enzymology},
volume = {635},
number = {},
pages = {251-266},
doi = {10.1016/bs.mie.2019.05.018},
pmid = {32122549},
issn = {1557-7988},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; Technology ; },
abstract = {Emergency of the CRISPR technology, a new genome editing tool, revolutionized the biomedical research field in the past 6 years. At the same time, recent advances in cancer immunotherapy reinvigorated our hope to cure most if not all cancer patients with further development of various treatment options. A combination of the CRISPR technology with immuno-oncology research will undoubtedly accelerate the development of new cancer therapies. This review will focus on the CRISPR system and its applications in immune-oncology including identification of immune-oncology gene targets, generation of cancer animal models, and enabling better cell design and manufacture for adoptive cellular therapies.},
}
@article {pmid32122544,
year = {2020},
author = {Bommareddy, PK and Peters, C and Kaufman, HL},
title = {Generation and validation of recombinant herpes simplex type 1 viruses (HSV-1) using CRISPR/Cas9 genetic disruption.},
journal = {Methods in enzymology},
volume = {635},
number = {},
pages = {167-184},
doi = {10.1016/bs.mie.2019.08.011},
pmid = {32122544},
issn = {1557-7988},
mesh = {CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; *Herpes Simplex/therapy ; *Herpesvirus 1, Human/genetics ; Humans ; *Melanoma/genetics/therapy ; *Oncolytic Virotherapy ; },
abstract = {Herpes simplex virus type 1 (HSV-1) is a large DNA virus that has been popular for oncolytic virus development in pre-clinical research and clinical trials. An oncolytic HSV-1 encoding granulocyte-macrophage colony stimulating factor (GM-CSF), designated talimogene laherparepvec (T-VEC) was approved for the treatment of patients with advanced melanoma in 2015. There are numerous advantages of HSV-1 for oncolytic development, including the ease of recombinant engineering, presence of non-essential genes allowing attenuation of pathogenicity and space for foreign transgene expression. In addition, most recombinants retain sensitivity to acyclovir providing an additional safety feature. In this chapter, we will focus on the key methods for the development of oncolytic HSV-1 vectors and some of the commonly utilized laboratory protocols used to characterize and assess the structure and oncolytic activity of recombinant HSV-1 viruses.},
}
@article {pmid32119776,
year = {2020},
author = {Li, W and Chan, C and Zeng, C and Turk, R and Behlke, MA and Cheng, X and Dong, Y},
title = {Rational Design of Small Molecules to Enhance Genome Editing Efficiency by Selectively Targeting Distinct Functional States of CRISPR-Cas12a.},
journal = {Bioconjugate chemistry},
volume = {31},
number = {3},
pages = {542-546},
pmid = {32119776},
issn = {1520-4812},
support = {R01 HL136652/HL/NHLBI NIH HHS/United States ; },
mesh = {Acidaminococcus/enzymology ; CRISPR-Cas Systems/*genetics ; *Drug Design ; Endodeoxyribonucleases/chemistry/*metabolism ; Gene Editing/*methods ; Molecular Dynamics Simulation ; Protein Conformation ; Small Molecule Libraries/*pharmacology ; },
abstract = {CRISPR-Cas12a, a type-V CRISPR-Cas endonuclease, is an effective genome editing platform. To improve the gene editing efficiency of Cas12a, we rationally designed small molecule enhancers through a combined computational approach. First, we used extensive molecular dynamics (MD) simulations to explore the conformational landscape of Cas12a from Acidaminococcus (AsCas12a), revealing distinct conformational states that could be targeted by small molecules to modulate its genome editing function. We then identified 57 compounds that showed different binding behavior and stabilizing effects on these distinct conformational states using molecular docking. After experimental testing 6 of these 57 compounds, compound 1, quinazoline-2,4(1H,3H)-dione, was found particularly promising in enhancing the AsCas12a-mediated genome editing efficiency in human cells. Compound 1 was shown to act like a molecular "glue" at the interface between AsCas12a and crRNA near the 5'-handle region, thus specifically stabilizing the enzyme-crRNA complex. These results provide a new paradigm for future design of small molecules to modulate the genome editing of the CRISPR-Cas systems.},
}
@article {pmid32118581,
year = {2020},
author = {Jo, Y and Kim, SS and Garland, K and Fuentes, I and DiCarlo, LM and Ellis, JL and Fu, X and Booth, SL and Evers, BM and DeBose-Boyd, RA},
title = {Enhanced ER-associated degradation of HMG CoA reductase causes embryonic lethality associated with Ubiad1 deficiency.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32118581},
issn = {2050-084X},
support = {P01 HL020948/HL/NHLBI NIH HHS/United States ; R01 GM134700/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Dimethylallyltranstransferase/*deficiency ; Endoplasmic Reticulum/*metabolism ; Female ; Fetal Death/etiology ; Gene Editing ; Gene Knockout Techniques ; Hydroxymethylglutaryl CoA Reductases/*metabolism ; Male ; Mice/embryology ; Mice, Knockout ; },
abstract = {UbiA prenyltransferase domain-containing protein-1 (UBIAD1) synthesizes the vitamin K subtype menaquinone-4 (MK-4). Previous studies in cultured cells (Schumacher et al., 2015) revealed that UBIAD1 also inhibits endoplasmic reticulum (ER)-associated degradation (ERAD) of ubiquitinated HMG CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway that produces cholesterol and essential nonsterol isoprenoids. Gene knockout studies were previously attempted to explore the function of UBIAD1 in mice; however, homozygous germ-line elimination of the Ubiad1 gene caused embryonic lethality. We now report that homozygous deletion of Ubiad1 is produced in knockin mice expressing ubiquitination/ERAD-resistant HMGCR. Thus, embryonic lethality of Ubiad1 deficiency results from depletion of mevalonate-derived products owing to enhanced ERAD of HMGCR rather than from reduced synthesis of MK-4. These findings provide genetic evidence for the significance of UBIAD1 in regulation of cholesterol synthesis and offer the opportunity in future studies for the discovery of new physiological roles of MK-4.},
}
@article {pmid32118319,
year = {2020},
author = {Zhou, L and Li, R and Zhang, R and Peng, R and Chen, K and Gao, P and Zhang, J and Zhang, R and Li, J},
title = {Utilizing CRISPR/Cas9 technology to prepare lymphoblastoid cell lines harboring genetic mutations for generating quality control materials in genetic testing.},
journal = {Journal of clinical laboratory analysis},
volume = {34},
number = {7},
pages = {e23256},
pmid = {32118319},
issn = {1098-2825},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line ; Electroporation/methods ; Gene Editing/*methods ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing ; Humans ; *Mutation ; Quality Control ; RNA, Guide/genetics ; Transfection ; Whole Genome Sequencing ; alpha-Thalassemia/*genetics ; },
abstract = {BACKGROUND: To meet the requirements of the rapidly progressing genetic testing technologies in clinical laboratories, assuring the quality of genetic tests by utilizing appropriate quality control materials is of paramount importance. The CRISPR/Cas9 technology was used to prepare quality control materials because genome-edited human cell lines are one of the major resources for quality control materials.<br><br>METHODS: In this study, in vitro transcribed sgRNA were transfected into a Cas9-expressing lymphoblastoid cell line (LCL)-by electroporation-to simulate the SEA-type deletion observed in &#x3b1;-thalassemia. The edited positive cell line was screened and identified by polymerase chain reaction (PCR) followed by Sanger sequencing. The whole-genome sequencing was also performed to show evidence of predicted mutation.<br><br>RESULTS: The results showed that electroporation of the in vitro transcribed gRNAs into stable Cas9-expressing LCL was a more efficient gene-editing technique as compared to plasmid-mediated transfection, and that the positive rates could reach up to 35.9%. The predominance of indel sizes relative to the predicted deletion length was clustered between 10 and 0&#xa0;bp. The results of whole-genome sequencing also demonstrated the existence of SEA-type deletion of &#x3b1;-thalassemia.<br><br>CONCLUSIONS: Gene-editing based on Cas9-expressing LCL by electroporation of sgRNA was a more efficient approach to introduce mutations for generating quality control materials for genetic testing. The edited lymphoblastoid cell lines were feasible to serve as quality control materials in genetic testing.},
}
@article {pmid32117254,
year = {2020},
author = {Boettcher, AN and Li, Y and Ahrens, AP and Kiupel, M and Byrne, KA and Loving, CL and Cino-Ozuna, AG and Wiarda, JE and Adur, M and Schultz, B and Swanson, JJ and Snella, EM and Ho, CS and Charley, SE and Kiefer, ZE and Cunnick, JE and Putz, EJ and Dell'Anna, G and Jens, J and Sathe, S and Goldman, F and Westin, ER and Dekkers, JCM and Ross, JW and Tuggle, CK},
title = {Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART[-/-]IL2RG[-/Y] SCID Pigs.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {100},
pmid = {32117254},
issn = {1664-3224},
support = {R24 OD019813/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Antigens, CD34 ; *Bone Marrow Transplantation ; CRISPR-Cas Systems ; Cell Differentiation ; Chimera ; DNA-Binding Proteins/deficiency ; Disease Models, Animal ; Gene Targeting ; Genetic Engineering ; Graft Survival ; Host vs Graft Reaction ; Humans ; Interleukin Receptor Common gamma Subunit/*genetics ; Killer Cells, Natural ; Models, Animal ; Severe Combined Immunodeficiency/*genetics ; Swine ; T-Lymphocytes/metabolism ; Transplantation, Heterologous ; },
abstract = {Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T[-] B[-] NK[-] SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)[-/-] genetic background. We confirmed ART[-/-]IL2RG[-/Y] pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART[-/-]IL2RG[-/Y] male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34[+] selected cord blood cells into the fetal ART[-/-]IL2RG[-/Y] SCID pigs. At birth, human CD45[+] CD3ε[+] cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.},
}
@article {pmid32117179,
year = {2020},
author = {Watanabe, S and Cui, B and Kiga, K and Aiba, Y and Tan, XE and Sato'o, Y and Kawauchi, M and Boonsiri, T and Thitiananpakorn, K and Taki, Y and Li, FY and Azam, AH and Nakada, Y and Sasahara, T and Cui, L},
title = {Corrigendum: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {179},
doi = {10.3389/fmicb.2020.00179},
pmid = {32117179},
issn = {1664-302X},
abstract = {[This corrects the article DOI: 10.3389/fmicb.2019.02838.].},
}
@article {pmid32114677,
year = {2020},
author = {Pryzhkova, MV and Jordan, PW},
title = {Adaptation of Human Testicular Niche Cells for Pluripotent Stem Cell and Testis Development Research.},
journal = {Tissue engineering and regenerative medicine},
volume = {17},
number = {2},
pages = {223-235},
pmid = {32114677},
issn = {2212-5469},
support = {R01 GM117155/GM/NIGMS NIH HHS/United States ; KY Cha Award//American Society for Reproductive Medicine/International ; R01GM117155/GM/NIGMS NIH HHS/United States ; },
mesh = {Biomarkers/metabolism ; CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Cells, Cultured ; Coculture Techniques ; Humans ; Male ; Pluripotent Stem Cells/*cytology/*metabolism ; SOX9 Transcription Factor/metabolism ; Sertoli Cells/cytology/metabolism ; Spermatogonia ; Testis/*cytology/*metabolism ; },
abstract = {BACKGROUND: Human testicular cells are greatly valuable to the research community as tools for studying testicular physiology and the effects of environmental pollutants. Because adult testicular cells have a limited self-organization capacity and life span, we investigated whether human pluripotent stem cells (hPSCs) can be used together with testicular cells to move a step closer toward making an optimal model of the human testis.<br><br>METHODS: We used in vitro culture of donor testicular cells under serum-containing and chemically defined conditions. CRISPR-Cas9 technology was applied to introduce fluorescent transgenes (mCherry2 and EGFP) into hPSCs and testicular cells. hPSC-derived spheroids were co-cultured with human testicular cells in mini-spin bioreactors.<br><br>RESULTS: Traditional cell culture conditions used for maintenance of testicular somatic cells generally contain serum and pose limitations on evaluating the role of active molecules on cell functions. We established that chemically defined culture conditions can be used to maintain testicular cells without the loss of proliferative activity. These cultures demonstrate marker expression which is characteristic of common testicular cell types: Sertoli, Leydig, endothelial, myoid cells, and macrophages. In order to model testicular physiology, it is important to be able to perform live cell microscopy. Thus, we generated fluorescent protein-expressing human testicular cells and hPSCs and demonstrated that these cell types can be successfully co-cultured for prolonged periods of time in a three-dimensional microenvironment.<br><br>CONCLUSION: Our research extends the possible applications of human testis-derived somatic cells and shows that they can be used together with hPSCs for further studies of human male reproductive biology.},
}
@article {pmid32114158,
year = {2020},
author = {Dermauw, W and Jonckheere, W and Riga, M and Livadaras, I and Vontas, J and Van Leeuwen, T},
title = {Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae.},
journal = {Insect biochemistry and molecular biology},
volume = {120},
number = {},
pages = {103347},
doi = {10.1016/j.ibmb.2020.103347},
pmid = {32114158},
issn = {1879-0240},
mesh = {Animals ; Arthropod Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; *Mutagenesis ; Oxidoreductases/*genetics/metabolism ; Tetranychidae/*genetics ; },
abstract = {The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.},
}
@article {pmid32114092,
year = {2020},
author = {Gao, X and Jin, Z and Tan, X and Zhang, C and Zou, C and Zhang, W and Ding, J and Das, BC and Severinov, K and Hitzeroth, II and Debata, PR and He, D and Ma, X and Tian, X and Gao, Q and Wu, J and Tian, R and Cui, Z and Fan, W and Huang, Z and Cao, C and Bao, Y and Tan, S and Hu, Z},
title = {Hyperbranched poly(β-amino ester) based polyplex nanopaticles for delivery of CRISPR/Cas9 system and treatment of HPV infection associated cervical cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {321},
number = {},
pages = {654-668},
doi = {10.1016/j.jconrel.2020.02.045},
pmid = {32114092},
issn = {1873-4995},
mesh = {Animals ; CRISPR-Cas Systems ; Drug Delivery Systems ; Esters ; Female ; Humans ; Mice ; Mice, Nude ; Papillomavirus E7 Proteins/genetics ; *Papillomavirus Infections/therapy ; *Polymers ; *Uterine Cervical Neoplasms/genetics/therapy ; },
abstract = {Persistent high-risk HPV infection is the main factor for cervical cancer. HPV E7 oncogene plays an important role in HPV carcinogenesis. Down-regulation of E7 oncogene expression could induce growth inhibition in HPV-positive cells and thus treats HPV related cervical cancer. Here we developed a non-virus gene vector based on poly(amide-amine)-poly(β-amino ester) hyperbranched copolymer (hPPC) for the delivery of CRISPR/Cas9 system to specifically cleave HPV E7 oncogene in HPV-positive cervical cancer cells. The diameter of polyplex nanoparticles (NPs) formed by hPPCs/linear poly(β-amino ester) (PBAE) and plasmids were approximately 300 nm. These hPPCs/PBAE-green fluorescence protein plasmids polyplex NPs showed high transfection efficiency and low toxicity in cells and mouse organs. By cleaving HPV16 E7 oncogene, reducing the expression of HPV16 E7 protein and increasing intracellular retinoblastoma 1 (RB1) amount, hPPCs/PBAE-CRISPR/Cas9 therapeutic plasmids polyplex NPs, especially highly branched hPPC1-plasmids polyplex NPs, exhibited strong growth inhibition of cervical cancer cells in vitro and xenograft tumors in nude mice. Together, the hPPCs/PBAE polyplex NPs to deliver HPV16 E7 targeted CRISPR/Cas9 system in this study could potentially be applied to treat HPV-related cervical cancer.},
}
@article {pmid32112663,
year = {2020},
author = {Yang, J and Xu, S and Wang, HC},
title = {Heterogeneity of fibroblasts from radicular cyst influenced osteoclastogenesis and bone destruction.},
journal = {Oral diseases},
volume = {26},
number = {5},
pages = {983-997},
doi = {10.1111/odi.13317},
pmid = {32112663},
issn = {1601-0825},
mesh = {Animals ; *Fibroblasts ; *Osteogenesis/genetics ; *Radicular Cyst ; Retrospective Studies ; },
abstract = {AIM: To analyze the heterogeneity of fibroblasts isolated from the fibrous capsules of radicular cysts and explore the effects of fibroblast subsets on bone destruction.<br><br>METHODOLOGY: Radicular cysts were divided into groups according to varying perilesional sclerosis identified by radiograph. Colony-forming units (CFUs) were isolated from the fibrous capsules of cysts, by which Trap&#xa0;+&#xa0;MNCs were induced, and the expression of osteoclastogenesis-related genes was compared among groups by real-time PCR. The variances in gene profiles of CFUs were identified by principal component analysis, and then, CFUs were divided into subsets using cluster analysis. The induction of Trap&#xa0;+&#xa0;MNCs and related gene expression was compared among subsets, and osteoclastogenic induction was blocked by IST-9 or bevacizumab. The fibroblast subsets in cysts were investigated by retrospective immunostaining with IST-9, VEGF-A, and CD34. A fibroblast subset that underwent gene editing by CRISPR/Cas was injected into the site of bone defects in animal models, and the in vivo effects on osteoclastogenesis were investigated.<br><br>RESULTS: The fibroblast CFUs isolated from radicular cysts with perilesional unsclerotized cysts induced more Trap&#xa0;+&#xa0;MNCs than those with perilesional sclerotic cysts (p&#xa0;<&#xa0;.05). Most fibroblast CFUs from unsclerotized cysts belonged to Cluster 2, which induced more Trap&#xa0;+&#xa0;MNCs (p&#xa0;<&#xa0;.05) and highly expressed genes facilitating osteoclastogenesis; these results were different from those of Cluster 1 (p&#xa0;<&#xa0;.05), in which most CFUs were isolated from perilesional sclerotic cysts or controls (p&#xa0;<&#xa0;.05). The high expression of EDA&#xa0;+&#xa0;FN and VEGF-A was investigated in both the fibroblasts of Cluster 2 and the fibrous capsules of unsclerotized cysts (p&#xa0;<&#xa0;.05), and the number of Trap&#xa0;+&#xa0;MNCs induced by Cluster 2 was decreased by treatment with IST-9 and bevacizumab (p&#xa0;<&#xa0;.05). Consistently, EDA exon exclusion significantly decreased the osteoclastogenic induction of fibroblasts from Cluster 2 in vivo (p&#xa0;<&#xa0;.05).<br><br>CONCLUSION: The fibrous capsules of radicular cysts contain heterogeneous fibroblasts that can form subsets exhibiting different effects on osteoclastogenesis. The subset, which depending on the autocrine effects of EDA&#xa0;+&#xa0;FN on VEGF-A, mainly contributes to the osteoclastogenesis and bone destruction of radicular cysts. The regulation of the proportion of subsets is a possible strategy for artificially interfering with osteoclastogenesis.},
}
@article {pmid32112565,
year = {2020},
author = {Freen-van Heeren, JJ and Popović, B and Guislain, A and Wolkers, MC},
title = {Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production.},
journal = {European journal of immunology},
volume = {50},
number = {7},
pages = {949-958},
pmid = {32112565},
issn = {1521-4141},
support = {10132//KWF Kankerbestrijding/International ; 917.14.214//Dutch National Science Foundation/International ; //Oncode Institute/International ; },
mesh = {*AU Rich Elements ; CD8-Positive T-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Humans ; Interferon-gamma/genetics/*immunology ; MART-1 Antigen/genetics/immunology ; Male ; Receptors, Antigen, T-Cell/genetics/immunology ; },
abstract = {Long-lasting CD8[+] T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes.},
}
@article {pmid32112380,
year = {2020},
author = {Jin, Y and Shen, Y and Weintraub, NL and Tang, Y},
title = {Using iRFP Genetic Labeling Technology to Track Tumorogenesis of Transplanted CRISPR/Cas9-Edited iPSC in Skeletal Muscle.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2126},
number = {},
pages = {73-83},
doi = {10.1007/978-1-0716-0364-2_7},
pmid = {32112380},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism/*pathology ; Cell Tracking/methods ; Dystrophin/deficiency/*genetics ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism/transplantation ; Luminescent Proteins/genetics/*metabolism ; Mice ; Molecular Imaging ; Muscle, Skeletal/*cytology/metabolism ; Muscular Dystrophy, Animal/genetics/*physiopathology/therapy ; Spectroscopy, Near-Infrared ; Stem Cell Transplantation/*adverse effects ; },
abstract = {Tumorigenesis and attendant safety risks are significant concerns of induced pluripotent stem cell (iPSC)-based therapies. Thus, it is crucial to evaluate iPSC proliferation, differentiation, and tumor formation after transplantation. Several approaches have been employed for tracking the donor cells, including fluorescent protein and luciferase, but both have limitations. Here, we introduce a protocol using iRFP genetic labeling technology to track tumor formation of iPSCs in skeletal muscle after CRISPR/Cas9 gene editing.},
}
@article {pmid32111843,
year = {2020},
author = {de Jong, OG and Murphy, DE and Mäger, I and Willms, E and Garcia-Guerra, A and Gitz-Francois, JJ and Lefferts, J and Gupta, D and Steenbeek, SC and van Rheenen, J and El Andaloussi, S and Schiffelers, RM and Wood, MJA and Vader, P},
title = {A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1113},
pmid = {32111843},
issn = {2041-1723},
support = {BB/M024393/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Biological Transport ; *CRISPR-Cas Systems ; Cell Communication ; Cell Line ; Endocytosis/genetics ; Extracellular Vesicles/genetics/*metabolism ; Fluorescence ; Genes, Reporter/genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Small Untranslated/genetics/*metabolism ; },
abstract = {Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.},
}
@article {pmid32110989,
year = {2020},
author = {Sato, M and Miyagasako, R and Takabayashi, S and Ohtsuka, M and Hatada, I and Horii, T},
title = {Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice.},
journal = {Cells},
volume = {9},
number = {3},
pages = {},
pmid = {32110989},
issn = {2073-4409},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Dextrans/chemistry ; Exons/genetics ; Female ; Fluorescence ; Gene Editing ; *Gene Transfer Techniques ; INDEL Mutation/genetics ; Introns/genetics ; Methyl-CpG-Binding Protein 2/genetics ; Mice, Inbred C57BL ; Mice, Transgenic ; Nucleic Acids/*metabolism ; Oviducts/*metabolism ; },
abstract = {Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4-1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4-1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called "sequential i-GONAD (si-GONAD)."},
}
@article {pmid32109417,
year = {2020},
author = {Jiang, W and Oikonomou, P and Tavazoie, S},
title = {Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.},
journal = {Cell},
volume = {180},
number = {5},
pages = {1002-1017.e31},
pmid = {32109417},
issn = {1097-4172},
support = {R01 AI077562/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics ; Genome, Bacterial/*genetics ; *Genomic Library ; Humans ; RNA, Bacterial/genetics ; RNA, Guide/genetics ; Staphylococcus aureus/*genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.},
}
@article {pmid32109227,
year = {2020},
author = {Champer, J and Lee, E and Yang, E and Liu, C and Clark, AG and Messer, PW},
title = {A toxin-antidote CRISPR gene drive system for regional population modification.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1082},
pmid = {32109227},
issn = {2041-1723},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *Antidotes ; CRISPR-Cas Systems/genetics ; Drosophila/genetics ; Drosophila Proteins/*genetics ; Female ; Gene Drive Technology/*methods ; Genetics, Population ; Heterozygote ; *Models, Genetic ; Mutation ; Protein Engineering/*methods ; },
abstract = {Engineered gene drives based on a homing mechanism could rapidly spread genetic alterations through a population. However, such drives face a major obstacle in the form of resistance against the drive. In addition, they are expected to be highly invasive. Here, we introduce the Toxin-Antidote Recessive Embryo (TARE) drive. It functions by disrupting a target gene, forming recessive lethal alleles, while rescuing drive-carrying individuals with a recoded version of the target. Modeling shows that such drives will have threshold-dependent invasion dynamics, spreading only when introduced above a fitness-dependent frequency. We demonstrate a TARE drive in Drosophila with 88-95% transmission by female heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. Our results suggest that TARE drives constitute promising candidates for the development of effective, flexible, and regionally confinable drives for population modification.},
}
@article {pmid32108566,
year = {2020},
author = {Tekedar, HC and Blom, J and Kalindamar, S and Nho, S and Karsi, A and Lawrence, ML},
title = {Comparative genomics of the fish pathogens Edwardsiella ictaluri 93-146 and Edwardsiella piscicida C07-087.},
journal = {Microbial genomics},
volume = {6},
number = {2},
pages = {},
pmid = {32108566},
issn = {2057-5858},
mesh = {Animals ; Catfishes/microbiology ; Edwardsiella/*genetics/isolation &amp; purification/metabolism ; Edwardsiella ictaluri/*genetics/isolation &amp; purification/metabolism ; Enterobacteriaceae Infections/microbiology/*veterinary ; Fish Diseases/*microbiology ; *Genome, Bacterial ; Genomics ; Phylogeny ; },
abstract = {Edwardsiella ictaluri and Edwardsiella piscicida are important fish pathogens affecting cultured and wild fish worldwide. To investigate the genome-level differences and similarities between catfish-adapted strains in these two species, the complete E. ictaluri 93-146 and E. piscicida C07-087 genomes were evaluated by applying comparative genomics analysis. All available complete (10) and non-complete (19) genomes from five Edwardsiella species were also included in a systematic analysis. Average nucleotide identity and core-genome phylogenetic tree analyses indicated that the five Edwardsiella species were separated from each other. Pan-/core-genome analyses for the 29 strains from the five species showed that genus Edwardsiella members have 9474 genes in their pan genome, while the core genome consists of 1421 genes. Orthology cluster analysis showed that E. ictaluri and E. piscicida genomes have the greatest number of shared clusters. However, E. ictaluri and E. piscicida also have unique features; for example, the E. ictaluri genome encodes urease enzymes and cytochrome o ubiquinol oxidase subunits, whereas E. piscicida genomes encode tetrathionate reductase operons, capsular polysaccharide synthesis enzymes and vibrioferrin-related genes. Additionally, we report for what is believed to be the first time that E. ictaluri 93-146 and three other E. ictaluri genomes encode a type IV secretion system (T4SS), whereas none of the E. piscicida genomes encode this system. Additionally, the E. piscicida C07-087 genome encodes two different type VI secretion systems. E. ictaluri genomes tend to encode more insertion elements, phage regions and genomic islands than E. piscicida. We speculate that the T4SS could contribute to the increased number of mobilome elements in E. ictaluri compared to E. piscicida. Two of the E. piscicida genomes encode full CRISPR-Cas regions, whereas none of the E. ictaluri genomes encode Cas proteins. Overall, comparison of the E. ictaluri and E. piscicida genomes reveals unique features and provides new insights on pathogenicity that may reflect the host adaptation of the two species.},
}
@article {pmid32106616,
year = {2020},
author = {Morita, S and Horii, T and Kimura, M and Hatada, I},
title = {Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform.},
journal = {International journal of molecular sciences},
volume = {21},
number = {5},
pages = {},
pmid = {32106616},
issn = {1422-0067},
mesh = {A549 Cells ; Basic-Leucine Zipper Transcription Factors/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Genetic Engineering/*methods ; Humans ; Mixed Function Oxygenases/genetics/metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Single-Chain Antibodies/genetics/metabolism ; *Up-Regulation ; },
abstract = {Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9-VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9-SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9-SunTag format synergistically activates gene expression using a single sgRNA.},
}
@article {pmid32105778,
year = {2020},
author = {Huynh, N and Wang, S and King-Jones, K},
title = {Spatial and temporal control of gene manipulation in Drosophila via drug-activated Cas9 nucleases.},
journal = {Insect biochemistry and molecular biology},
volume = {120},
number = {},
pages = {103336},
doi = {10.1016/j.ibmb.2020.103336},
pmid = {32105778},
issn = {1879-0240},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Endonucleases ; *Gene Editing ; *Gene Expression ; },
abstract = {Advances in CRISPR/Cas9 have revolutionized molecular biology and greatly facilitated the ability to manipulate gene function through the creation of precisely engineered mutants. We recently reported a collection of modular gateway-compatible Cas9/gRNA Drosophila lines to interfere with gene expression in a tissue-specific manner, including polytene tissues. However, most current in vivo CRISPR/Cas9 tools cannot temporally control the induction of Cas9 or gRNAs via external stimuli such as RU486. A drug-inducible CRISPR/Cas9 system would allow studying genes at later stages where early lethality is an issue. This would be especially useful when combined with tissue-specific expression of Cas9 or gRNAs, allowing for full spatiotemporal control. Here, we present a RU486-inducible version of Cas9 and also show that a Rapamycin-inducible Cas9, previously used in mammalian cell culture, works in Drosophila as well. Both RU486 and rapamycin-inducible Cas9 work in vivo and in Drosophila cell culture. We also present split Cas9 constructs for rapamycin-dependent gene disruption and activation. These approaches establish drug-inducible and thus temporally controlled CRISPR/Cas9 tools for gene disruption and expression in a living model organism. Our CRISPR/Cas9 vector collection can be easily adapted for any tissue and provides higher fidelity compared to RNAi approaches.},
}
@article {pmid32105327,
year = {2020},
author = {Lujan, H and Romer, E and Salisbury, R and Hussain, S and Sayes, C},
title = {Determining the Biological Mechanisms of Action for Environmental Exposures: Applying CRISPR/Cas9 to Toxicological Assessments.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {175},
number = {1},
pages = {5-18},
doi = {10.1093/toxsci/kfaa028},
pmid = {32105327},
issn = {1096-0929},
mesh = {Animals ; *CRISPR-Cas Systems ; Diffusion of Innovation ; *Environmental Exposure ; Environmental Pollutants/*toxicity ; Gene Expression Regulation ; *Gene Targeting ; Humans ; Risk Assessment ; Signal Transduction ; *Toxicity Tests ; },
abstract = {Toxicology is a constantly evolving field, especially in the area of developing alternatives to animal testing. Toxicological research must evolve and utilize adaptive technologies in an effort to improve public, environmental, and occupational health. The most commonly cited mechanisms of toxic action after exposure to a chemical or particle test substance is oxidative stress. However, because oxidative stress involves a plethora of genes and proteins, the exact mechanism(s) are not commonly defined. Exact mechanisms of toxicity can be revealed using an emerging laboratory technique referred to as CRISPR (clustered regularly interspaced short palindromic repeats). This article reviews the most common CRISPR techniques utilized today and how each may be applied in Toxicological Sciences. Specifically, the CRISPR/CRISPR-associated protein complex is used for single gene knock-outs, whereas CRISPR interference/activation is used for silencing or activating (respectively) ribonucleic acid. Finally, CRISPR libraries are used for knocking-out entire gene pathways. This review highlights the application of CRISPR in toxicology to elucidate the exact mechanism through which toxicants perturb normal cellular functions.},
}
@article {pmid32103777,
year = {2020},
author = {Sharma, R and Liang, Y and Lee, MY and Pidatala, VR and Mortimer, JC and Scheller, HV},
title = {Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.},
journal = {BMC research notes},
volume = {13},
number = {1},
pages = {116},
pmid = {32103777},
issn = {1756-0500},
mesh = {Agrobacterium tumefaciens/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant/genetics ; Genomics/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Plant Leaves/*genetics/metabolism ; Plants, Genetically Modified ; Protoplasts/metabolism ; RNA, Guide/genetics/metabolism ; Reproducibility of Results ; Sorghum/*genetics/metabolism ; Tobacco/cytology/metabolism ; *Transformation, Genetic ; },
abstract = {OBJECTIVES: Sorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable.<br><br>RESULTS: Here, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.},
}
@article {pmid32103062,
year = {2020},
author = {Vermilyea, SC and Babinski, A and Tran, N and To, S and Guthrie, S and Kluss, JH and Schmidt, JK and Wiepz, GJ and Meyer, MG and Murphy, ME and Cookson, MR and Emborg, ME and Golos, TG},
title = {In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson's Disease in Common Marmosets.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {3447},
pmid = {32103062},
issn = {2045-2322},
support = {UL1 TR000427/TR/NCATS NIH HHS/United States ; R24 OD019803/OD/NIH HHS/United States ; UL1 TR002373/TR/NCATS NIH HHS/United States ; P51 OD011106/OD/NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Autophagy ; CRISPR-Cas Systems/*genetics ; Callithrix ; Cell Differentiation ; Disease Models, Animal ; Dopaminergic Neurons/cytology/metabolism ; Embryonic Stem Cells/cytology/metabolism ; Endoplasmic Reticulum Stress ; Gene Editing/*methods ; Induced Pluripotent Stem Cells/cytology/metabolism ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry/*genetics/metabolism ; Mutagenesis, Site-Directed ; Neurites/physiology ; Parkinson Disease/genetics/*pathology ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Up-Regulation ; },
abstract = {Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.},
}
@article {pmid32102844,
year = {2020},
author = {Liu, HJ and Jian, L and Xu, J and Zhang, Q and Zhang, M and Jin, M and Peng, Y and Yan, J and Han, B and Liu, J and Gao, F and Liu, X and Huang, L and Wei, W and Ding, Y and Yang, X and Li, Z and Zhang, M and Sun, J and Bai, M and Song, W and Chen, H and Sun, X and Li, W and Lu, Y and Liu, Y and Zhao, J and Qian, Y and Jackson, D and Fernie, AR and Yan, J},
title = {High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize.},
journal = {The Plant cell},
volume = {32},
number = {5},
pages = {1397-1413},
pmid = {32102844},
issn = {1532-298X},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Repair/genetics ; Gene Editing ; *Genes, Plant ; Mutagenesis/*genetics ; Mutation/genetics ; Plants, Genetically Modified ; Plasmids/genetics ; *Quantitative Trait, Heritable ; RNA, Guide/genetics ; Reproducibility of Results ; Templates, Genetic ; Transformation, Genetic ; Zea mays/*genetics ; },
abstract = {Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.},
}
@article {pmid32102843,
year = {2020},
author = {Salomé, PA},
title = {A Roadmap toward Large-Scale Genome Editing in Crops.},
journal = {The Plant cell},
volume = {32},
number = {5},
pages = {1340-1341},
pmid = {32102843},
issn = {1532-298X},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/genetics ; *Gene Editing ; Mutagenesis ; *Zea mays ; },
}
@article {pmid32102684,
year = {2020},
author = {Cui, YR and Wang, SJ and Chen, J and Li, J and Chen, W and Wang, S and Meng, B and Zhu, W and Zhang, Z and Yang, B and Jiang, B and Yang, G and Ma, P and Liu, J},
title = {Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {51},
pmid = {32102684},
issn = {1474-760X},
mesh = {Allosteric Regulation ; Bacteriophage M13 ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/metabolism ; *CRISPR-Cas Systems ; Capsid Proteins/chemistry ; Gene Editing/methods ; HEK293 Cells ; Humans ; K562 Cells ; Peptide Fragments/chemistry/genetics/*metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.<br><br>RESULTS: We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.<br><br>CONCLUSION: G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.},
}
@article {pmid32102453,
year = {2020},
author = {Lee, S and Shin, Y and Kim, K and Song, Y and Kim, Y and Kang, SW},
title = {Protein Translocation Acquires Substrate Selectivity Through ER Stress-Induced Reassembly of Translocon Auxiliary Components.},
journal = {Cells},
volume = {9},
number = {2},
pages = {},
pmid = {32102453},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics/*metabolism ; Calnexin/metabolism ; Cell Line ; Cricetinae ; Endoplasmic Reticulum/*metabolism ; Endoplasmic Reticulum Stress/*genetics ; Gene Editing ; Humans ; Membrane Glycoproteins/genetics/*metabolism ; Mutant Proteins/chemistry/metabolism ; Oxidation-Reduction ; Prion Proteins/*chemistry/genetics/*metabolism ; Protein Transport ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Receptors, Peptide/genetics/*metabolism ; Substrate Specificity ; },
abstract = {Protein import across the endoplasmic reticulum membrane is physiologically regulated in a substrate-selective manner to ensure the protection of stressed ER from the overload of misfolded proteins. However, it is poorly understood how different types of substrates are accurately distinguished and disqualified during translocational regulation. In this study, we found poorly assembled translocon-associated protein (TRAP) complexes in stressed ER. Immunoaffinity purification identified calnexin in the TRAP complex in which poor assembly inhibited membrane insertion of the prion protein (PrP) in a transmembrane sequence-selective manner, through translocational regulation. This reaction was induced selectively by redox perturbation, rather than calcium depletion, in the ER. The liberation of ERp57 from calnexin appeared to be the reason for the redox sensitivity. Stress-independent disruption of the TRAP complex prevented a pathogenic transmembrane form of PrP (ctmPrP) from accumulating in the ER. This study uncovered a previously unappreciated role for calnexin in assisting the redox-sensitive function of the TRAP complex and provided insights into the ER stress-induced reassembly of translocon auxiliary components as a key mechanism by which protein translocation acquires substrate selectivity.},
}
@article {pmid32102340,
year = {2020},
author = {Xu, C and Tang, L and Liang, Y and Jiao, S and Yu, H and Luo, H},
title = {Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber.},
journal = {Molecules (Basel, Switzerland)},
volume = {25},
number = {4},
pages = {},
pmid = {32102340},
issn = {1420-3049},
mesh = {Aminohydrolases/chemistry/genetics/*metabolism ; Bacterial Proteins/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Chaperonin 60/chemistry/genetics/*metabolism ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli/*enzymology/genetics ; Gene Editing ; Gene Expression ; Genetic Engineering/methods ; *Genome, Bacterial ; Humans ; Kinetics ; Models, Molecular ; Plasmids/chemistry/metabolism ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Stability ; Recombinant Fusion Proteins/chemistry/genetics/metabolism ; Rhodococcus/*enzymology/genetics ; },
abstract = {For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32-47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.},
}
@article {pmid32101536,
year = {2020},
author = {Liu, C and Zhao, J and Lu, W and Dai, Y and Hockings, J and Zhou, Y and Nussinov, R and Eng, C and Cheng, F},
title = {Individualized genetic network analysis reveals new therapeutic vulnerabilities in 6,700 cancer genomes.},
journal = {PLoS computational biology},
volume = {16},
number = {2},
pages = {e1007701},
pmid = {32101536},
issn = {1553-7358},
support = {HHSN261200800001E/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/therapeutic use ; BRCA2 Protein/genetics ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Carcinogenesis ; Cell Line, Tumor ; Computational Biology/*methods ; Exome ; *Gene Regulatory Networks ; Genetic Testing ; Genomics ; Humans ; Inhibitory Concentration 50 ; Models, Theoretical ; Mutation ; Neoplasms/drug therapy/*genetics ; Pharmacogenetics ; Precision Medicine ; Protein Interaction Mapping ; Survival Rate ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Tumor-specific genomic alterations allow systematic identification of genetic interactions that promote tumorigenesis and tumor vulnerabilities, offering novel strategies for development of targeted therapies for individual patients. We develop an Individualized Network-based Co-Mutation (INCM) methodology by inspecting over 2.5 million nonsynonymous somatic mutations derived from 6,789 tumor exomes across 14 cancer types from The Cancer Genome Atlas. Our INCM analysis reveals a higher genetic interaction burden on the significantly mutated genes, experimentally validated cancer genes, chromosome regulatory factors, and DNA damage repair genes, as compared to human pan-cancer essential genes identified by CRISPR-Cas9 screenings on 324 cancer cell lines. We find that genes involved in the cancer type-specific genetic subnetworks identified by INCM are significantly enriched in established cancer pathways, and the INCM-inferred putative genetic interactions are correlated with patient survival. By analyzing drug pharmacogenomics profiles from the Genomics of Drug Sensitivity in Cancer database, we show that the network-predicted putative genetic interactions (e.g., BRCA2-TP53) are significantly correlated with sensitivity/resistance of multiple therapeutic agents. We experimentally validated that afatinib has the strongest cytotoxic activity on BT474 (IC50 = 55.5 nM, BRCA2 and TP53 co-mutant) compared to MCF7 (IC50 = 7.7 μM, both BRCA2 and TP53 wild type) and MDA-MB-231 (IC50 = 7.9 μM, BRCA2 wild type but TP53 mutant). Finally, drug-target network analysis reveals several potential druggable genetic interactions by targeting tumor vulnerabilities. This study offers a powerful network-based methodology for identification of candidate therapeutic pathways that target tumor vulnerabilities and prioritization of potential pharmacogenomics biomarkers for development of personalized cancer medicine.},
}
@article {pmid32101273,
year = {2020},
author = {Zhou, Q and Zhang, Y and Zou, Y and Yin, T and Yang, J},
title = {Human embryo gene editing: God's scalpel or Pandora's box?.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {154-163},
doi = {10.1093/bfgp/elz025},
pmid = {32101273},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genetic Therapy ; Genome, Human/*genetics ; Humans ; },
abstract = {Gene editing refers to the site-specific modification of the genome, which mainly focuses on basic research, model organism construction and treatment and prevention of disease. Since the first application of CRISPR/Cas9 on the human embryo genome in 2015, the controversy over embryo gene editing (abbreviated as EGE in the following text) has never stopped. At present, the main contradictions focus on (1) ideal application prospects and immature technologies; (2) scientific progress and ethical supervision; and (3) definition of reasonable application scope. In fact, whether the EGE is 'God's scalpel' or 'Pandora's box' depends on the maturity of the technology and ethical supervision. This non-systematic review included English articles in NCBI, technical documents from the Human Fertilization and Embryology Authority as well as reports in the media, which performed from 1980 to 2018 with the following search terms: 'gene editing, human embryo, sequence-specific nuclease (SSN) (CRISPR/Cas, TALENT, ZFN), ethical consideration, gene therapy.' Based on the research status of EGE, this paper summarizes the technical defects and ethical controversies, enumerates the optimization measures and looks forward to the application prospect, aimed at providing some suggestions for the development trend. We should regard the research and development of EGE optimistically, improve and innovate the technology boldly and apply its clinical practice carefully.},
}
@article {pmid32101166,
year = {2020},
author = {Kaur, G and Burroughs, AM and Iyer, LM and Aravind, L},
title = {Highly regulated, diversifying NTP-dependent biological conflict systems with implications for the emergence of multicellularity.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32101166},
issn = {2050-084X},
mesh = {ATPases Associated with Diverse Cellular Activities/metabolism ; Archaea/*cytology/genetics/metabolism ; Bacteria/*cytology/genetics/metabolism ; Biological Evolution ; CRISPR-Cas Systems ; Nucleotides/*metabolism ; Prokaryotic Cells ; },
abstract = {Social cellular aggregation or multicellular organization pose increased risk of transmission of infections through the system upon infection of a single cell. The generality of the evolutionary responses to this outside of Metazoa remains unclear. We report the discovery of several thematically unified, remarkable biological conflict systems preponderantly present in multicellular prokaryotes. These combine thresholding mechanisms utilizing NTPase chaperones (the MoxR-vWA couple), GTPases and proteolytic cascades with hypervariable effectors, which vary either by using a reverse transcriptase-dependent diversity-generating system or through a system of acquisition of diverse protein modules, typically in inactive form, from various cellular subsystems. Conciliant lines of evidence indicate their deployment against invasive entities, like viruses, to limit their spread in multicellular/social contexts via physical containment, dominant-negative interactions or apoptosis. These findings argue for both a similar operational 'grammar' and shared protein domains in the sensing and limiting of infections during the multiple emergences of multicellularity.},
}
@article {pmid32100378,
year = {2020},
author = {Kim, GD and Lee, JH and Song, S and Kim, SW and Han, JS and Shin, SP and Park, BC and Park, TS},
title = {Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {4},
pages = {5688-5696},
doi = {10.1096/fj.201903035R},
pmid = {32100378},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/genetics/growth &amp; development/*metabolism ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; Germ Cells/cytology/*metabolism ; Muscle, Skeletal/cytology/*metabolism ; Myostatin/antagonists &amp; inhibitors/*physiology ; Phenotype ; },
abstract = {Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.},
}
@article {pmid32100335,
year = {2020},
author = {Meccariello, A and Tsoumani, KT and Gravina, A and Primo, P and Buonanno, M and Mathiopoulos, KD and Saccone, G},
title = {Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.},
journal = {Archives of insect biochemistry and physiology},
volume = {104},
number = {2},
pages = {e21667},
doi = {10.1002/arch.21667},
pmid = {32100335},
issn = {1520-6327},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Insect Proteins/*genetics ; *Mutagenesis ; Ribonucleoproteins/*genetics ; Tephritidae/*genetics ; },
abstract = {The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.},
}
@article {pmid32098826,
year = {2020},
author = {Georgiou, A and Stewart, A and Cunningham, D and Banerji, U and Whittaker, SR},
title = {Inactivation of NF1 Promotes Resistance to EGFR Inhibition in KRAS/NRAS/BRAF[V600] -Wild-Type Colorectal Cancer.},
journal = {Molecular cancer research : MCR},
volume = {18},
number = {6},
pages = {835-846},
pmid = {32098826},
issn = {1557-3125},
support = {11566/CRUK_/Cancer Research UK/United Kingdom ; RP-2016-07-028/DH_/Department of Health/United Kingdom ; },
mesh = {Apoptosis ; Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Colorectal Neoplasms/*drug therapy/genetics/pathology ; *Drug Resistance, Neoplasm ; ErbB Receptors/antagonists &amp; inhibitors ; GTP Phosphohydrolases/genetics ; *Gene Expression Regulation, Neoplastic ; Humans ; Membrane Proteins/genetics ; *Mutation ; Neurofibromin 1/*antagonists &amp; inhibitors/genetics ; Protein Kinase Inhibitors/*pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; Tumor Cells, Cultured ; },
abstract = {Through the use of an unbiased, genome-scale CRISPR modifier screen, we identified NF1 suppression as a mechanism of resistance to EGFR inhibition in NRAS/KRAS/BRAF[V600] -wild-type colorectal cancer cells. Reduced NF1 expression permitted sustained signaling through the MAPK pathway to promote cell proliferation in the presence of EGFR inhibition. Targeting of MEK in combination with EGFR inhibition leads to synergistic antiproliferative activity. Human KRAS/NRAS/BRAF[V600] -wild-type colorectal cancer cell lines with NF1 mutations displayed reduced NF1 mRNA or protein expression and were resistant to EGFR blockade by gefitinib or cetuximab. Cooccurring loss-of-function mutations in PTEN were associated with resistance to dual EGFR/MEK inhibition but cotreatment with a PI3K inhibitor further suppressed proliferation. Loss of NF1 may be a useful biomarker to identify patients that are less likely to benefit from single-agent anti-EGFR therapy in colorectal cancer and may direct potential combination strategies. IMPLICATIONS: This study suggests that further clinical validation of NF1 status as predictor of response to anti-EGFR targeting antibodies in patients with colorectal cancer with KRAS/NRAS/BRAF[V600] -wild-type tumors is warranted.},
}
@article {pmid32098766,
year = {2020},
author = {Raja, DA and Subramaniam, Y and Aggarwal, A and Gotherwal, V and Babu, A and Tanwar, J and Motiani, RK and Sivasubbu, S and Gokhale, RS and Natarajan, VT},
title = {Histone variant dictates fate biasing of neural crest cells to melanocyte lineage.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {5},
pages = {},
doi = {10.1242/dev.182576},
pmid = {32098766},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line, Tumor ; Cell Lineage ; Embryonic Stem Cells/*cytology ; Gene Regulatory Networks/genetics ; Histones/*genetics ; Melanocytes/*cytology ; Melanoma, Experimental ; Mice ; Microphthalmia-Associated Transcription Factor/*genetics ; Neural Crest/*cytology ; Zebrafish/embryology ; Zebrafish Proteins/*genetics ; },
abstract = {In the neural crest lineage, progressive fate restriction and stem cell assignment are crucial for both development and regeneration. Whereas fate commitment events have distinct transcriptional footprints, fate biasing is often transitory and metastable, and is thought to be moulded by epigenetic programmes. Therefore, the molecular basis of specification is difficult to define. In this study, we established a role for a histone variant, H2a.z.2, in specification of the melanocyte lineage from multipotent neural crest cells. H2a.z.2 silencing reduces the number of melanocyte precursors in developing zebrafish embryos and from mouse embryonic stem cells in vitro We demonstrate that this histone variant occupies nucleosomes in the promoter of the key melanocyte determinant mitf, and enhances its induction. CRISPR/Cas9-based targeted mutagenesis of this gene in zebrafish drastically reduces adult melanocytes, as well as their regeneration. Thereby, our study establishes the role of a histone variant upstream of the core gene regulatory network in the neural crest lineage. This epigenetic mark is a key determinant of cell fate and facilitates gene activation by external instructive signals, thereby establishing melanocyte fate identity.},
}
@article {pmid32098761,
year = {2020},
author = {Sandoval, A and Elahi, H and Ploski, JE},
title = {Genetically Engineering the Nervous System with CRISPR-Cas.},
journal = {eNeuro},
volume = {7},
number = {2},
pages = {},
pmid = {32098761},
issn = {2373-2822},
support = {R21 MH109945/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Neurons ; },
abstract = {The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in next-generation sequencing (NGS) and gene-editing technology have begun to close this gap. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type. This technology has advanced our understanding of complex neurologic diseases by enabling the rapid generation of novel, disease-relevant in vitro and transgenic animal models. In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for genetic interrogation of the normal and diseased nervous system. Additionally, we discuss limitations of the CRISPR editing system and suggest how future modifications to existing platforms may advance our understanding of the brain.},
}
@article {pmid32098759,
year = {2020},
author = {Hafezi, Y and Sruba, SR and Tarrash, SR and Wolfner, MF and Clark, AG},
title = {Dissecting Fertility Functions of Drosophila Y Chromosome Genes with CRISPR.},
journal = {Genetics},
volume = {214},
number = {4},
pages = {977-990},
pmid = {32098759},
issn = {1943-2631},
support = {P40 OD018537/OD/NIH HHS/United States ; R01 GM119125/GM/NIGMS NIH HHS/United States ; R01 HD059060/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chromosomes, Insect/*genetics ; Drosophila Proteins/*genetics/metabolism ; Drosophila melanogaster ; Gene Editing ; Infertility, Male/*genetics ; Male ; Mutation ; Y Chromosome/*genetics ; },
abstract = {Gene-poor, repeat-rich regions of the genome are poorly understood and have been understudied due to technical challenges and the misconception that they are degenerating "junk." Yet multiple lines of evidence indicate these regions may be an important source of variation that could drive adaptation and species divergence, particularly through regulation of fertility. The ∼40 Mb Y chromosome of Drosophila melanogaster contains only 16 known protein-coding genes, and is highly repetitive and entirely heterochromatic. Most of the genes originated from duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting functional constraint. We devised a genetic strategy for recovering and retaining stocks with sterile Y-linked mutations and combined it with CRISPR to create mutants with deletions that disrupt three Y-linked genes. Two genes, PRY and FDY, had no previously identified functions. We found that PRY mutant males are subfertile, but FDY mutant males had no detectable fertility defects. FDY, the newest known gene on the Y chromosome, may have fertility effects that are conditional or too subtle to detect. The third gene, CCY, had been predicted but never formally shown to be required for male fertility. CRISPR targeting and RNA interference of CCY caused male sterility. Surprisingly, however, our CCY mutants were sterile even in the presence of an extra wild-type Y chromosome, suggesting that perturbation of the Y chromosome can lead to dominant sterility. Our approach provides an important step toward understanding the complex functions of the Y chromosome and parsing which functions are accomplished by genes vs. repeat elements.},
}
@article {pmid32098368,
year = {2020},
author = {Lee, J and Kim, DH and Lee, K},
title = {Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide.},
journal = {International journal of molecular sciences},
volume = {21},
number = {4},
pages = {},
pmid = {32098368},
issn = {1422-0067},
mesh = {Amino Acid Sequence ; Animals ; Avian Proteins/*genetics ; Base Sequence ; CRISPR-Cas Systems ; Coturnix/*genetics ; Cysteine/*genetics ; Female ; Gene Knockout Techniques ; Hyperplasia ; Male ; Muscle Development/genetics ; Muscle, Skeletal/*metabolism/pathology ; *Mutation ; Myostatin/*genetics ; Sequence Deletion ; Sequence Homology, Amino Acid ; },
abstract = {Mutation in myostatin (MSTN), a negative regulator of muscle growth in skeletal muscle, resulted in increased muscle mass in mammals and fishes. However, MSTN mutation in avian species has not been reported. The objective of this study was to generate MSTN mutation in quail and investigate the effect of MSTN mutation in avian muscle growth. Recently, a new targeted gene knockout approach for the avian species has been developed using an adenoviral CRISPR/Cas9 system. By injecting the recombinant adenovirus containing CRISPR/Cas9 into the quail blastoderm, potential germline chimeras were generated and offspring with three base-pair deletion in the targeted region of the MSTN gene was identified. This non-frameshift mutation in MSTN resulted in deletion of cysteine 42 in the MSTN propeptide region and homozygous mutant quail showed significantly increased body weight and muscle mass with muscle hyperplasia compared to heterozygous mutant and wild-type quail. In addition, decreased fat pad weight and increased heart weight were observed in MSTN mutant quail in an age- and sex-dependent manner, respectively. Taken together, these data indicate anti-myogenic function of MSTN in the avian species and the importance of cysteine 42 in regulating MSTN function.},
}
@article {pmid32098042,
year = {2020},
author = {Plavec, TV and Berlec, A},
title = {Safety Aspects of Genetically Modified Lactic Acid Bacteria.},
journal = {Microorganisms},
volume = {8},
number = {2},
pages = {},
pmid = {32098042},
issn = {2076-2607},
abstract = {Lactic acid bacteria (LAB) have a long history of use in the food industry. Some species are part of the normal human microbiota and have beneficial properties for human health. Their long-standing use and considerable biotechnological potential have led to the development of various systems for their engineering. Together with novel approaches such as CRISPR-Cas, the established systems for engineering now allow significant improvements to LAB strains. Nevertheless, genetically modified LAB (GM-LAB) still encounter disapproval and are under extensive regulatory requirements. This review presents data on the prospects for LAB to obtain 'generally recognized as safe' (GRAS) status. Genetic modification of LAB is discussed, together with problems that can arise from their engineering, including their dissemination into the environment and the spread of antibiotic resistance markers. Possible solutions that would allow the use of GM-LAB are described, such as biocontainment, alternative selection markers, and use of homologous DNA. The use of GM-LAB as cell factories in closed systems that prevent their environmental release is the least problematic aspect, and this is also discussed.},
}
@article {pmid32097710,
year = {2020},
author = {Chen, S and Yao, Y and Zhang, Y and Fan, G},
title = {CRISPR system: Discovery, development and off-target detection.},
journal = {Cellular signalling},
volume = {70},
number = {},
pages = {109577},
doi = {10.1016/j.cellsig.2020.109577},
pmid = {32097710},
issn = {1873-3913},
mesh = {Archaea/*genetics ; Bacteria/*genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Archaeal ; DNA, Bacterial ; *Gene Editing ; },
abstract = {As a revolutionary gene editing tool based on the adaptive immune defense mechanism of bacteria and archaea against exogenous DNA invasion, CRISPR/Cas system shows many remarkable characteristics over ZFNs and TALENs. However, off-target effect remains as one of the major imperfection of CRISPR/Cas system, hindering its further application in translational research. In this review, we highlight major breakthrough cross the development/application of this powerful toolkit, and summarize feasible methods for detecting potential off-target effects during genetic manipulation. We hope this review will assist scientists for accurate genomic editing in their future research.},
}
@article {pmid32097584,
year = {2020},
author = {Hung, IC and Chen, TM and Lin, JP and Tai, YL and Shen, TL and Lee, SJ},
title = {Wnt5b integrates Fak1a to mediate gastrulation cell movements via Rac1 and Cdc42.},
journal = {Open biology},
volume = {10},
number = {2},
pages = {190273},
pmid = {32097584},
issn = {2046-2441},
mesh = {Actins ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Movement ; Focal Adhesion Kinase 1/*genetics/metabolism ; Gastrulation ; Mice ; Mutation ; Signal Transduction ; Wnt-5a Protein/genetics/*metabolism ; Zebrafish/*embryology/genetics/metabolism ; Zebrafish Proteins/genetics/*metabolism ; cdc42 GTP-Binding Protein/genetics/*metabolism ; rac1 GTP-Binding Protein/genetics/*metabolism ; },
abstract = {Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.},
}
@article {pmid32097570,
year = {2020},
author = {Sebastian, RM and Shoulders, MD},
title = {Chemical Biology Framework to Illuminate Proteostasis.},
journal = {Annual review of biochemistry},
volume = {89},
number = {},
pages = {529-555},
pmid = {32097570},
issn = {1545-4509},
support = {DP2 GM119162/GM/NIGMS NIH HHS/United States ; R01 AR071443/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Expression Regulation ; Half-Life ; Heat-Shock Response/drug effects ; Humans ; Molecular Chaperones/*genetics/metabolism ; *Molecular Probe Techniques ; Protein Aggregates ; Protein Engineering/methods ; Protein Folding/drug effects ; Protein Transport/drug effects ; Proteome/chemistry/*genetics/metabolism ; Proteostasis/drug effects/*genetics ; Proteostasis Deficiencies/*genetics/metabolism/pathology ; Signal Transduction ; Small Molecule Libraries/chemical synthesis/pharmacology ; Unfolded Protein Response/drug effects ; },
abstract = {Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.},
}
@article {pmid32097467,
year = {2020},
author = {Pauli, C and Liu, Y and Rohde, C and Cui, C and Fijalkowska, D and Gerloff, D and Walter, C and Krijgsveld, J and Dugas, M and Edemir, B and Pabst, C and Müller, LP and Zhou, F and Müller-Tidow, C},
title = {Site-specific methylation of 18S ribosomal RNA by SNORD42A is required for acute myeloid leukemia cell proliferation.},
journal = {Blood},
volume = {135},
number = {23},
pages = {2059-2070},
doi = {10.1182/blood.2019004121},
pmid = {32097467},
issn = {1528-0020},
mesh = {CRISPR-Cas Systems ; *Cell Proliferation ; *DNA Methylation ; Humans ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; RNA, Ribosomal, 18S/chemistry/*genetics ; RNA, Small Nucleolar/genetics/*metabolism ; Ribosomal Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Tumor Cells, Cultured ; },
abstract = {Noncoding RNAs, including small nucleolar RNAs (snoRNAs), play important roles in leukemogenesis, but the relevant mechanisms remain incompletely understood. We performed snoRNA-focused CRISPR-Cas9 knockout library screenings that targeted the entire snoRNAnome and corresponding host genes. The C/D box containing SNORD42A was identified as an essential modulator for acute myeloid leukemia (AML) cell survival and proliferation in multiple human leukemia cell lines. In line, SNORD42A was consistently expressed at higher levels in primary AML patient samples than in CD34+ progenitors, monocytes, and granulocytes. Functionally, knockout of SNORD42A reduced colony formation capability and inhibited proliferation. The SNORD42A acts as a C/D box snoRNA and directs 2'-O-methylation at uridine 116 of 18S ribosomal RNA (rRNA). Deletion of SNORD42A decreased 18S-U116 2'-O-methylation, which was associated with a specific decrease in the translation of ribosomal proteins. In line, the cell size of SNORD42A deletion carrying leukemia cells was decreased. Taken together, these findings establish that high-level expression of SNORD42A with concomitant U116 18S rRNA 2'-O-methylation is essential for leukemia cell growth and survival.},
}
@article {pmid32096600,
year = {2020},
author = {Jacinto, FV and Link, W and Ferreira, BI},
title = {CRISPR/Cas9-mediated genome editing: From basic research to translational medicine.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {7},
pages = {3766-3778},
pmid = {32096600},
issn = {1582-4934},
support = {#748585/MCCC_/Marie Curie/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Genetic Therapy/*trends ; Humans ; Models, Animal ; Mutation/genetics ; Translational Research, Biomedical/*trends ; },
abstract = {The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.},
}
@article {pmid32096325,
year = {2020},
author = {Wu, J and Chen, C and Xian, G and Liu, D and Lin, L and Yin, S and Sun, Q and Fang, Y and Zhang, H and Wang, Y},
title = {Engineering herbicide-resistant oilseed rape by CRISPR/Cas9-mediated cytosine base-editing.},
journal = {Plant biotechnology journal},
volume = {18},
number = {9},
pages = {1857-1859},
pmid = {32096325},
issn = {1467-7652},
mesh = {*Brassica napus/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine ; *Herbicides/pharmacology ; Plants, Genetically Modified/genetics ; },
}
@article {pmid32096222,
year = {2020},
author = {Tiraboschi, E and Martina, S and van der Ent, W and Grzyb, K and Gawel, K and Cordero-Maldonado, ML and Poovathingal, SK and Heintz, S and Satheesh, SV and Brattespe, J and Xu, J and Suster, M and Skupin, A and Esguerra, CV},
title = {New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.},
journal = {Epilepsia},
volume = {61},
number = {3},
pages = {549-560},
doi = {10.1111/epi.16456},
pmid = {32096222},
issn = {1528-1167},
support = {//Centre for Molecular Medicine Norway (NCMM) Start up grant/International ; C14/BM/7975668/CaSCAD//National Research Fund of Luxembourg/International ; INTER/DFG/17/11583046 MechEPI (Mechanisms of Epileptogenesis)//National Research Fund of Luxembourg/International ; },
mesh = {Animals ; Anticonvulsants/pharmacology ; Brain/drug effects/metabolism/pathology/*physiopathology ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Diazepam/pharmacology ; Disease Models, Animal ; Electroencephalography ; Epilepsies, Myoclonic/*genetics/metabolism/pathology/physiopathology ; Fenfluramine/pharmacology ; GABAergic Neurons/drug effects/metabolism/pathology ; Gene Expression Profiling ; Gliosis/genetics/pathology ; Locomotion/drug effects ; Mutation, Missense ; NAV1.1 Voltage-Gated Sodium Channel/*genetics/metabolism ; Neuronal Plasticity/drug effects/*genetics ; RNA-Seq ; Real-Time Polymerase Chain Reaction ; Serotonin 5-HT2 Receptor Agonists/pharmacology ; Single-Cell Analysis ; Zebrafish ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {OBJECTIVE: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine.<br><br>METHODS: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled &#x3b3;-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae.<br><br>RESULTS: Homozygous mutant (scn1lab[mut/mut]) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean&#xa0;=&#xa0;7.5 per 20-minute recording; P&#xa0;<&#xa0;.0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1lab[mut/mut] larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P&#xa0;<&#xa0;.001; n&#xa0;=&#xa0;15 mutant, n&#xa0;=&#xa0;16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1lab[mut/mut] larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1lab[mut/mut] larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1lab[mut/mut] larval brains that were rescued by fenfluramine but not diazepam.<br><br>SIGNIFICANCE: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1lab[mut/mut] brain, and present first-time evidence for potential disease modification by fenfluramine.},
}
@article {pmid32096010,
year = {2020},
author = {Townsend, MH and Bennion, K and Robison, RA and O'Neill, KL},
title = {Paving the way towards universal treatment with allogenic T cells.},
journal = {Immunologic research},
volume = {68},
number = {1},
pages = {63-70},
pmid = {32096010},
issn = {1559-0755},
mesh = {Animals ; CRISPR-Cas Systems ; Genetic Engineering ; Graft vs Host Disease/*prevention &amp; control ; Humans ; Immunotherapy, Adoptive/*methods ; Isoantigens/genetics/immunology/*metabolism ; Neoplasms/immunology/*therapy ; Precision Medicine ; T-Lymphocytes/*physiology/transplantation ; Transcription Activator-Like Effector Nucleases/metabolism ; Transplantation, Homologous ; },
abstract = {With several different CAR T cell therapies under advanced phases of clinical trials, and the first FDA-approved CAR treatments in 2017 (Yescarta and Kymriah), CAR T cell therapy has become one of the most promising therapies for the treatment of certain types of cancer. This success has bred an opportunity to optimize the production of CAR T cells for easier patient access. CAR T cell therapy is a rather expensive and personalized process that requires expensive measures to collect cells from patients, engineer those cells, and re-infuse the cells into the patient with adequate quality controls at each phase. With this in mind, significant attempts at creating a "universal" CAR T cell are underway in order to create an "off-the-shelf" product that would reduce the expense and time required for traditional CAR T cell treatment. The primary obstacle facing this endeavor is avoiding graft-versus-host disease that accompanies allogeneic transplants between genetically dissimilar individuals. With the advent of CRISPR and TALEN technology, editing the genome of allogeneic cells has become very possible, and several groups have provided initial data analyzing the effects of CAR T cells that have been edited to avoid host rejection and avoid endogenous TCR alloreactivity. These engineered cells not only have to avoid GVHD but also have to retain their anti-tumor efficacy in vivo. Here, we expand on the recent efforts and strides that have been made in the design and testing of universal allogeneic CAR T cells.},
}
@article {pmid32095891,
year = {2020},
author = {Ding, Y and Han, D and Cui, HL},
title = {Halorussus halophilus sp. nov., A Novel Halophilic Archaeon Isolated from a Marine Solar Saltern.},
journal = {Current microbiology},
volume = {77},
number = {7},
pages = {1321-1327},
doi = {10.1007/s00284-020-01921-8},
pmid = {32095891},
issn = {1432-0991},
mesh = {Base Composition ; China ; DNA, Archaeal/genetics ; Genome, Archaeal/genetics ; Geologic Sediments/microbiology ; *Halobacteriaceae/chemistry/classification/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {The halophilic archaeal strain ZS-3[T] (= CGMCC 1.12866[T] = JCM 30239[T]) was isolated from a sediment sample of Zhoushan marine solar saltern, P. R. China. Phylogenetic analyses based on 16S rRNA, rpoB' genes and the concatenation of 738 protein sequences reveal that strain ZS-3[T] was related to members of the genus Halorussus. The OrthoANI and in silico DDH values between strain ZS-3[T] and the current Halorussus members are much lower than the threshold values proposed as the species boundary (ANI 95-96% and in silico DDH 70%), suggesting that strain ZS-3[T] represents a novel species of Halorussus (Halorussus halophilus sp. nov.). Diverse phenotypic characteristics differentiate strain ZS-3[T] from current Halorussus members. Since the strain expressed diverse hydrolyzing enzyme activity, its complete genome was sequenced. The genome of strain ZS-3[T] was found to be 4,450,731 bp with total GC content of 61.51%, and comprises one chromosome and three plasmids. A total of 4694 protein coding genes, 43 tRNA genes and 6 rRNA genes were predicted. A CRISPR-Cas system was also detected. The genome encodes sixteen putative glycoside hydrolases, nine extracellular proteases, seventeen aminopeptidases, seven carboxypeptidases, one esterase and one nitrite reductase. The exploration of the hydrolase genes may expand our understanding of adapted mechanism of halophilic archaea surviving optimally in hypersaline environments where containing organic matter. Meanwhile, various hydrolyzing enzymes may extend this microorganism for further applications in salt-based fermentation.},
}
@article {pmid32095812,
year = {2020},
author = {NandyMazumdar, M and Yin, S and Paranjapye, A and Kerschner, JL and Swahn, H and Ge, A and Leir, SH and Harris, A},
title = {Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells.},
journal = {Nucleic acids research},
volume = {48},
number = {7},
pages = {3513-3524},
pmid = {32095812},
issn = {1362-4962},
support = {R01 HL094585/HL/NHLBI NIH HHS/United States ; T32 GM008056/GM/NIGMS NIH HHS/United States ; },
mesh = {CCAAT-Enhancer-Binding Protein-beta/metabolism ; CRISPR-Cas Systems ; Caco-2 Cells ; Cell Line ; Chromatin/chemistry ; Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis/*genetics ; *Enhancer Elements, Genetic ; Epithelial Cells/metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Promoter Regions, Genetic ; RNA Polymerase II/metabolism ; Respiratory Mucosa/*metabolism ; Sequence Deletion ; Trans-Activators/metabolism ; },
abstract = {The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanisms controlling its transcription are likely divergent between them. Here, we determine how two extragenic CREs that are prominent in epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44 and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here, we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter, suggesting non-redundant enhancers. The deletions also greatly reduced promoter interactions with the 5' TAD boundary. We show substantial recruitment of RNAPII to the -35 kb element and identify CEBPβ as a key activator of airway expression of CFTR, likely through occupancy at this CRE and the gene promoter.},
}
@article {pmid32095520,
year = {2020},
author = {Wang, L and Yang, Y and Breton, C and Bell, P and Li, M and Zhang, J and Che, Y and Saveliev, A and He, Z and White, J and Latshaw, C and Xu, C and McMenamin, D and Yu, H and Morizono, H and Batshaw, ML and Wilson, JM},
title = {A mutation-independent CRISPR-Cas9-mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency.},
journal = {Science advances},
volume = {6},
number = {7},
pages = {eaax5701},
pmid = {32095520},
issn = {2375-2548},
support = {P01 HD057247/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Repair/genetics ; Dependovirus/genetics ; Dietary Proteins ; Disease Models, Animal ; *Gene Targeting ; Genetic Loci ; *Genetic Therapy ; Genetic Vectors/metabolism ; INDEL Mutation/genetics ; Liver/enzymology/pathology ; Male ; Mice ; Mutation/*genetics ; Ornithine Carbamoyltransferase/genetics/metabolism ; Ornithine Carbamoyltransferase Deficiency Disease/*genetics/*therapy ; Time Factors ; },
abstract = {Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spf[ash] mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.},
}
@article {pmid32095517,
year = {2020},
author = {Skryabin, BV and Kummerfeld, DM and Gubar, L and Seeger, B and Kaiser, H and Stegemann, A and Roth, J and Meuth, SG and Pavenstädt, H and Sherwood, J and Pap, T and Wedlich-Söldner, R and Sunderkötter, C and Schwartz, YB and Brosius, J and Rozhdestvensky, TS},
title = {Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events.},
journal = {Science advances},
volume = {6},
number = {7},
pages = {eaax2941},
pmid = {32095517},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Crosses, Genetic ; DNA/*genetics ; Female ; *Gene Editing ; Gene Targeting ; Genetic Loci ; Genome ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; *Templates, Genetic ; },
abstract = {CRISPR-Cas9-mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.},
}
@article {pmid32094657,
year = {2020},
author = {Liu, Y and Liu, Z and Cao, Z and Wei, W},
title = {Reply to: Fitness effects of CRISPR/Cas9-targeting of long noncoding RNA genes.},
journal = {Nature biotechnology},
volume = {38},
number = {5},
pages = {577-578},
pmid = {32094657},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; RNA, Guide ; *RNA, Long Noncoding ; },
}
@article {pmid32094656,
year = {2020},
author = {Horlbeck, MA and Liu, SJ and Chang, HY and Lim, DA and Weissman, JS},
title = {Fitness effects of CRISPR/Cas9-targeting of long noncoding RNA genes.},
journal = {Nature biotechnology},
volume = {38},
number = {5},
pages = {573-576},
pmid = {32094656},
issn = {1546-1696},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F30 NS092319/NS/NINDS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; R35 CA209919/CA/NCI NIH HHS/United States ; R01 NS091544/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; RNA, Guide ; *RNA, Long Noncoding ; },
}
@article {pmid32094150,
year = {2020},
author = {McCann, JL and Salamango, DJ and Law, EK and Brown, WL and Harris, RS},
title = {MagnEdit-interacting factors that recruit DNA-editing enzymes to single base targets.},
journal = {Life science alliance},
volume = {3},
number = {4},
pages = {},
pmid = {32094150},
issn = {2575-1077},
support = {K99 AI147811/AI/NIAID NIH HHS/United States ; P01 CA234228/CA/NCI NIH HHS/United States ; T90 DE022732/DE/NIDCR NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {APOBEC Deaminases/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytidine Deaminase/*genetics ; Cytosine/chemistry ; DNA/genetics ; Deoxyribonuclease I/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome/genetics ; Heterogeneous-Nuclear Ribonucleoproteins/*genetics/metabolism ; Minor Histocompatibility Antigens/*genetics ; Nuclear Proteins/*genetics/metabolism ; Proof of Concept Study ; Transcription Factors/*genetics/metabolism ; },
abstract = {Although CRISPR/Cas9 technology has created a renaissance in genome engineering, particularly for gene knockout generation, methods to introduce precise single base changes are also highly desirable. The covalent fusion of a DNA-editing enzyme such as APOBEC to a Cas9 nickase complex has heightened hopes for such precision genome engineering. However, current cytosine base editors are prone to undesirable off-target mutations, including, most frequently, target-adjacent mutations. Here, we report a method to "attract" the DNA deaminase, APOBEC3B, to a target cytosine base for specific editing with minimal damage to adjacent cytosine bases. The key to this system is fusing an APOBEC-interacting protein (not APOBEC itself) to Cas9n, which attracts nuclear APOBEC3B transiently to the target site for editing. Several APOBEC3B interactors were tested and one, hnRNPUL1, demonstrated proof-of-concept with successful C-to-T editing of episomal and chromosomal substrates and lower frequencies of target-adjacent events.},
}
@article {pmid32094061,
year = {2020},
author = {Zhang, F and Li, X and He, M and Ye, D and Xiong, F and Amin, G and Zhu, Z and Sun, Y},
title = {Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {47},
number = {1},
pages = {37-47},
doi = {10.1016/j.jgg.2019.12.004},
pmid = {32094061},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques ; Gene Targeting ; Genome/*genetics ; Germ Cells/growth &amp; development ; Humans ; Mutation/genetics ; Phenotype ; RNA, Guide/genetics ; Zebrafish/*genetics/growth &amp; development ; Zygote/*growth &amp; development ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell (PGC) transplantation (PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic (MZ) mutants of tcf7l1a (gene essential for head development), pou5f3 (gene essential for zygotic genome activation) and chd (gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1a and chd, as MZtcf7l1a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.},
}
@article {pmid32093670,
year = {2020},
author = {Michno, JM and Virdi, K and Stec, AO and Liu, J and Wang, X and Xiong, Y and Stupar, RM},
title = {Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {10},
pmid = {32093670},
issn = {1472-6750},
support = {2015-33522-24096//National Institute of Food and Agriculture/International ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; High-Throughput Nucleotide Sequencing ; *Mutation ; Plants, Genetically Modified/growth &amp; development ; Quantitative Trait, Heritable ; Sequence Analysis, DNA ; Soybeans/*genetics/growth &amp; development ; Transgenes ; },
abstract = {BACKGROUND: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results.<br><br>RESULTS: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission.<br><br>CONCLUSIONS: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.},
}
@article {pmid32091258,
year = {2020},
author = {Brokowski, C and Adli, M},
title = {Ethical Considerations in Therapeutic Clinical Trials Involving Novel Human Germline-Editing Technology.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {18-26},
doi = {10.1089/crispr.2019.0051},
pmid = {32091258},
issn = {2573-1602},
mesh = {Beneficence ; CRISPR-Cas Systems/genetics ; Clinical Trials as Topic/*ethics/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/methods ; Germ Cells/metabolism ; Germ-Line Mutation/*genetics ; Humans ; },
abstract = {Much of the international community opposes editing the human germline. Yet, given enough experience to become better acquainted with strengths and limitations, prominent international figures are cautiously optimistic about using CRISPR-like novel technologies for clinical applications. Not only might such applications be morally (ethically) permissible, but clinical trials for therapeutic aims could be necessary. Here, we assess critical dimensions of early-phase trials deploying germline-editing technologies for "bench-to-bedside" translation. While assuming no overarching position favoring or opposing such research, our discussion primarily focuses on normative considerations. First, we evaluate the imperative of conducting trials to produce reliable, reproducible knowledge and advancement, if possible, for human diseases that are incurable and/or whose treatments are deficient. Second, we address complexities in assessing risk and potential-benefit profiles. Third, we review the moral foundations of trial participation through well-established and accepted bioethical principles: autonomy, nonmaleficence, beneficence, and distributive justice. Finally, we raise critical questions about the scope of regulatory authority and investigator and funder accountability for these applications that could have everlasting impacts.},
}
@article {pmid32091257,
year = {2020},
author = {Ranisch, R and Rudolph, T and Cremer, HJ and Knoepffler, N},
title = {Ordo-Responsibility for Germline Gene Editing.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {37-43},
doi = {10.1089/crispr.2019.0040},
pmid = {32091257},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/*legislation &amp; jurisprudence/methods ; Genome, Human/genetics ; Germ Cells/metabolism ; Germ-Line Mutation/genetics ; Government ; Human Rights/*ethics ; Humans ; Morals ; Reproductive Rights/ethics ; },
abstract = {The case of twins born with genes modified by He Jiankui highlights the need for international governance of germline gene editing (GGE). This article proposes a global framework that utilizes "ordo-responsibilities." This is a pragmatic ethical approach open to pluralism and grounded in principles of human dignity and human rights. Ordo-responsibility is pragmatic in (1) accepting generally available values on a global level (e.g., human dignity, human rights) and (2) seeking achievable implementation. Genetic science is practiced globally in ways that transcend borders. As such, its practice must take account of the vast complexity of cultural, ethical, legal, and anthropological convictions. Here, we explain the basic structure of an appropriate rule-finding process, outline a possible pathway toward an international framework, and discuss minimal requirements that are needed in that endeavor. We thereby contribute to the debate on how to govern genome-editing technologies and GGE globally.},
}
@article {pmid32091256,
year = {2020},
author = {Gleim, S and Lubieniechi, S and Smyth, SJ},
title = {CRISPR-Cas9 Application in Canadian Public and Private Plant Breeding.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {44-51},
doi = {10.1089/crispr.2019.0061},
pmid = {32091256},
issn = {2573-1602},
mesh = {Agriculture/methods ; CRISPR-Cas Systems/genetics ; Canada ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Humans ; Plant Breeding/*methods ; Plants/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Plant-breeding technologies have expanded, accelerating breeding research beyond the confines of current regulations. The application of genome editing, such as CRISPR-Cas9, do not neatly fit into existing regulatory frameworks, creating uncertainty as to whether they can be regarded as conventionally developed varieties without further regulation. This research presents the current views of Canadian plant breeders based on a national survey of plant breeders. There is evidence that a review of existing regulations is required, as >60% anticipate the use of genome-editing technologies in the next few years. This paper reviews plant-breeding practices under the context of present plants with novel trait (PNT) regulations and where plant breeders place the use of CRISPR-Cas9 within the suite of available genome-editing options. This paper establishes when and why, or why not, breeders choose to introduce CRISPR-Cas9 into their research over other plant-breeding applications.},
}
@article {pmid32091255,
year = {2020},
author = {Wiegand, T and Wiedenheft, B},
title = {CRISPR Surveillance Turns Transposon Taxi.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {10-12},
pmid = {32091255},
issn = {2573-1602},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Transposable Elements/*genetics ; },
}
@article {pmid32091254,
year = {2020},
author = {Locke, LG},
title = {The Promise of CRISPR for Human Germline Editing and the Perils of "Playing God".},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {27-31},
pmid = {32091254},
issn = {2573-1602},
mesh = {Biotechnology/*ethics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/methods ; Germ Cells/metabolism ; Germ-Line Mutation/*genetics ; Humans ; },
abstract = {In the midst of the media and professional exuberance regarding the potential benefits of CRISPR technology, voices of criticism and caution have also arisen. One of the thorniest such cautions has been the common objection that CRISPR allows bioscientists to "play God," particularly when it comes to potentially editing the human germline. Many in the biotechnology field are unsure how to address this concern. What does it mean, particularly for bioscientists who may not have any rational or rhetorical categories for God? In this article, I explore possible meanings of "playing God" and the arguments for how those meanings might be applied in the utilization of CRISPR technology for human germline editing. I then test the validity of those arguments and explore potential counterarguments. Finally, I discuss how members of the bioscience community might respond to the objection of "playing God" and contribute to that dialogue in ways that could impact the future of CRISPR development and applications.},
}
@article {pmid32091253,
year = {2020},
author = {Niemiec, E and Howard, HC},
title = {Germline Genome Editing Research: What Are Gamete Donors (Not) Informed About in Consent Forms?.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {52-63},
pmid = {32091253},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Compensation and Redress/ethics ; Consent Forms/ethics ; Female ; Gene Editing/*ethics/methods ; Genome, Human/genetics ; Germ Cells/metabolism ; Germ-Line Mutation/genetics ; Humans ; Informed Consent/*ethics ; Male ; Oocyte Donation/ethics ; Oocytes ; Spermatozoa ; Tissue Donors/*ethics ; },
abstract = {The potential for using germline genome editing (GGE) in humans has garnered a lot of attention, both for its scientific possibilities as well as for the ethical, legal, and social challenges it ignites. The ethical debate has focused primarily on the suggestions of using GGE to establish a pregnancy (i.e., to offer it in a clinical setting), which is, to date, illegal in many jurisdictions. The use of GGE in research (where a pregnancy would not be established) has received much less attention, despite the fact that it raises serious ethical and social issues as well. Herein, we report on the analysis of informed consent forms for egg and sperm donation used in a widely publicized study where genome editing was used to correct a disease-causing genetic mutation in human embryos. Importantly, embryos were created using eggs and sperm obtained specifically for these experiments. The analysis indicates deficiencies in how the forms addressed various issues, including limited and potentially misleading information about the sensitive nature of the study, the lack of an explicit mention of genomic sequencing, as well as the poor readability of the forms. Furthermore, the arguably high compensation of U.S.$5,000 for egg donors raises questions about undue inducement to participate in research. Moreover, since the procurement of eggs involves serious health risks, it may be questioned whether research requiring such a procedure should be pursued. If such experiments are continued, donors should be informed about all relevant aspects in order to make informed decisions about participating.},
}
@article {pmid32091252,
year = {2020},
author = {Cornuault, JK and Moineau, S},
title = {A Jumbo Formation in the Viral Game Plan.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {14-17},
doi = {10.1089/crispr.2020.29082.jco},
pmid = {32091252},
issn = {2573-1602},
mesh = {Bacteria/*genetics/immunology ; Bacteriophages/*genetics/immunology/metabolism ; CRISPR-Cas Systems/*genetics/immunology ; },
}
@article {pmid32091251,
year = {2020},
author = {Pruett-Miller, SM},
title = {Cancer Screens: Better Together.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {12-14},
doi = {10.1089/crispr.2020.29084.spm},
pmid = {32091251},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Humans ; Neoplasms/*genetics ; Oncogenes ; },
}
@article {pmid32091250,
year = {2020},
author = {Thaldar, D and Shozi, B},
title = {Procreative Non-Maleficence: A South African Human Rights Perspective on Heritable Human Genome Editing.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {32-36},
pmid = {32091250},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/*legislation &amp; jurisprudence/methods ; Genome, Human/genetics ; Germ Cells/metabolism ; Germ-Line Mutation/genetics ; Humans ; Reproduction/*ethics/genetics ; Reproductive Rights/ethics ; Reproductive Techniques/trends ; South Africa ; },
abstract = {If the safety and efficacy issues relating to heritable genome editing can be resolved, how should liberal democratic societies regulate the use of this technology by prospective parents who wish to effect edits to the genomes of their prospective children? We suggest that recent developments in South African law can be useful in this regard. The country's apex court recently recognized as a legal principle that the scope of possible reproductive decisions that parents may make when using new reproductive technologies excludes decisions that will cause harm to the prospective child-the principle of procreative non-maleficence. We suggest that the principle of procreative non-maleficence provides a mechanism for striking an equitable balance between two competing interests that are given legal recognition in most liberal democracies: the reproductive rights of prospective parents and the state's duty to protect child welfare.},
}
@article {pmid32091248,
year = {2020},
author = {Davies, K and Marraffini, L},
title = {Major Insights into Microbiology: An Interview with Luciano Marraffini.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {5-9},
doi = {10.1089/crispr.2020.29080.lma},
pmid = {32091248},
issn = {2573-1602},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods/*trends ; History, 20th Century ; History, 21st Century ; Humans ; },
}
@article {pmid32091246,
year = {2020},
author = {Soni, S},
title = {Cautious Progress Toward Clinical Application of Human Gene Editing.},
journal = {The CRISPR journal},
volume = {3},
number = {1},
pages = {3-4},
doi = {10.1089/crispr.2020.29083.sso},
pmid = {32091246},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Humans ; },
}
@article {pmid32090878,
year = {2020},
author = {Xu, W and Jin, T and Dai, Y and Liu, CC},
title = {Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems.},
journal = {Biosensors &amp; bioelectronics},
volume = {155},
number = {},
pages = {112100},
doi = {10.1016/j.bios.2020.112100},
pmid = {32090878},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; DNA/*analysis ; *Electrochemical Techniques ; Gene Editing ; Humans ; Limit of Detection ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {Robust developments of personalized medicine for next-generation healthcare highlight the need for sensitive and accurate point-of-care platforms for quantification of disease biomarkers. Broad presentations of clustered regularly interspaced short palindromic repeats (CRISPR) as an accurate gene editing tool also indicate that the high-specificity and programmability of CRISPR system can be utilized for the development of biosensing systems. Herein, we present a CRISPR Cas system enhanced electrochemical DNA (E-DNA) sensor with unprecedented sensitivity and accuracy. The principle of the E-DNA sensor is the target induced conformational change of the surface signaling probe (containing an electrochemical tag), leading to the variation of the electron transfer rate of the electrochemical tag. With the introduction of CRISPR cleavage activity into the E-DNA sensor, a more apparent signal change between with and without the presence of the target can be achieved. We compared the performance of Cas9 and Cas12a enhanced E-DNA sensor and optimized the chemical environment of CRISPR, achieving a femto-molar detection limit without enzymatic amplification. Moreover, we correlated the CRISPR cleavage signal with the original E-DNA signal as a strategy to indicate potential mismatches in the target sequence. Comparing with classic DNA electrochemistry based mutation detection strategy, CRISPR enhanced E-DNA sensor can determine the presence of a single mutation at an unknown concentration condition. Overall, we believe that the CRISPR enhanced E-DNA sensing strategy will be of especially high utility for point-of-care systems owing to the programmability, modularity, high-sensitivity and high-accuracy.},
}
@article {pmid32090297,
year = {2020},
author = {Zhang, XK and Wang, DN and Chen, J and Liu, ZJ and Wei, LJ and Hua, Q},
title = {Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica.},
journal = {Biotechnology letters},
volume = {42},
number = {6},
pages = {945-956},
doi = {10.1007/s10529-020-02844-x},
pmid = {32090297},
issn = {1573-6776},
mesh = {Bioreactors ; CRISPR-Cas Systems/genetics ; Fermentation ; Glucose/metabolism ; Metabolic Engineering/*methods ; Yarrowia/*genetics/metabolism ; beta Carotene/analysis/*metabolism ; },
abstract = {OBJECTIVE: Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient β-carotene-producing Yarrowia lipolytica cell factories for potential industrial application.<br><br>RESULTS: The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of &#x3b2;-carotene in Y. lipolytica XK2. Furthermore, &#x3b2;-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408&#xa0;mg/L &#x3b2;-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2.<br><br>CONCLUSIONS: 4.5&#xa0;g/L &#x3b2;-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.},
}
@article {pmid32088728,
year = {2021},
author = {Zhang, B and Chen, Y and Shi, X and Zhou, M and Bao, L and Hatanpaa, KJ and Patel, T and DeBerardinis, RJ and Wang, Y and Luo, W},
title = {Regulation of branched-chain amino acid metabolism by hypoxia-inducible factor in glioblastoma.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {78},
number = {1},
pages = {195-206},
pmid = {32088728},
issn = {1420-9071},
support = {CA168746/CA/NCI NIH HHS/United States ; R00 CA168746/CA/NCI NIH HHS/United States ; CA222393/CA/NCI NIH HHS/United States ; AG066166/AG/NIA NIH HHS/United States ; R35 GM124693/GM/NIGMS NIH HHS/United States ; R00 NS078049/NS/NINDS NIH HHS/United States ; GM124693/NH/NIH HHS/United States ; R01 CA222393/CA/NCI NIH HHS/United States ; R01 AG066166/AG/NIA NIH HHS/United States ; },
mesh = {Amino Acids, Branched-Chain/*metabolism ; Basic Helix-Loop-Helix Transcription Factors/deficiency/genetics/*metabolism ; Brain Neoplasms/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Glioblastoma/metabolism/pathology ; Glutamic Acid/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/deficiency/genetics/*metabolism ; Large Neutral Amino Acid-Transporter 1/genetics/metabolism ; Protein Binding ; Transaminases/antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Hypoxia-inducible factors (HIFs) mediate metabolic reprogramming in response to hypoxia. However, the role of HIFs in branched-chain amino acid (BCAA) metabolism remains unknown. Here we show that hypoxia upregulates mRNA and protein levels of the BCAA transporter LAT1 and the BCAA metabolic enzyme BCAT1, but not their paralogs LAT2-4 and BCAT2, in human glioblastoma (GBM) cell lines as well as primary GBM cells. Hypoxia-induced LAT1 protein upregulation is mediated by both HIF-1 and HIF-2 in GBM cells. Although both HIF-1α and HIF-2α directly bind to the hypoxia response element at the first intron of the human BCAT1 gene, HIF-1α is exclusively responsible for hypoxia-induced BCAT1 expression in GBM cells. Knockout of HIF-1α and HIF-2α significantly reduces glutamate labeling from BCAAs in GBM cells under hypoxia, which provides functional evidence for HIF-mediated reprogramming of BCAA metabolism. Genetic or pharmacological inhibition of BCAT1 inhibits GBM cell growth under hypoxia. Together, these findings uncover a previously unrecognized HIF-dependent metabolic pathway that increases GBM cell growth under conditions of hypoxic stress.},
}
@article {pmid32088047,
year = {2020},
author = {Mukai, C and Nelson, JL and Cheong, SH and Diel de Amorim, M and Travis, AJ},
title = {Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog.},
journal = {Theriogenology},
volume = {150},
number = {},
pages = {347-352},
doi = {10.1016/j.theriogenology.2020.02.003},
pmid = {32088047},
issn = {1879-3231},
mesh = {Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair/physiology ; Dogs/*physiology ; Embryo Transfer ; Fertilization in Vitro/*veterinary ; Fibroblast Growth Factor 5/genetics/metabolism ; Gene Editing/methods/*veterinary ; Gene Expression Regulation ; Genotype ; In Vitro Oocyte Maturation Techniques/*veterinary ; Keratins, Hair-Specific/genetics/metabolism ; Oocytes/*physiology ; Time Factors ; Zygote/*physiology ; },
abstract = {Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.},
}
@article {pmid32088034,
year = {2020},
author = {Menchaca, A and Dos Santos-Neto, PC and Mulet, AP and Crispo, M},
title = {CRISPR in livestock: From editing to printing.},
journal = {Theriogenology},
volume = {150},
number = {},
pages = {247-254},
pmid = {32088034},
issn = {1879-3231},
mesh = {Animals ; *Animals, Genetically Modified ; Biotechnology ; *CRISPR-Cas Systems ; *Gene Editing ; Livestock/*genetics ; Printing, Three-Dimensional ; },
abstract = {Precise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time- and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.},
}
@article {pmid32087201,
year = {2020},
author = {Devesa-Guerra, I and Morales-Ruiz, T and Pérez-Roldán, J and Parrilla-Doblas, JT and Dorado-León, M and García-Ortiz, MV and Ariza, RR and Roldán-Arjona, T},
title = {DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.},
journal = {Journal of molecular biology},
volume = {432},
number = {7},
pages = {2204-2216},
doi = {10.1016/j.jmb.2020.02.007},
pmid = {32087201},
issn = {1089-8638},
mesh = {5-Methylcytosine/*chemistry ; Arabidopsis/*genetics/metabolism ; Arabidopsis Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Epigenesis, Genetic ; *Gene Editing ; Nuclear Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; Transcriptional Activation ; },
abstract = {Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.},
}
@article {pmid32087195,
year = {2020},
author = {Duncan, S and Delage, S and Chioran, A and Sirbu, O and Brown, TJ and Ringuette, MJ},
title = {The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes.},
journal = {Developmental biology},
volume = {461},
number = {2},
pages = {197-209},
doi = {10.1016/j.ydbio.2020.02.011},
pmid = {32087195},
issn = {1095-564X},
mesh = {Adipocytes/cytology ; Animals ; Animals, Genetically Modified ; Basement Membrane/*metabolism ; Binding Sites ; COP-Coated Vesicles/metabolism ; CRISPR-Cas Systems ; Cell Size ; Collagen Type IV/*metabolism ; Cystine/metabolism ; Drosophila Proteins/*physiology ; Drosophila melanogaster/growth &amp; development/metabolism ; Fat Body/cytology/growth &amp; development ; Genes, Lethal ; Hemocytes/metabolism ; Larva ; Molecular Chaperones/*physiology ; Osteonectin/chemistry/deficiency/genetics/*physiology ; Protein Domains ; Wings, Animal/growth &amp; development ; },
abstract = {The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. We report that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2[nd] instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2[nd] instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Removal of the disulfide bridge in the C-terminal EF-hand2 of SPARC, which is known to enhance Col(IV) binding, did not lead to larval lethality; however, a less intense fat body phenotype was observed. Additionally, both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2[nd] instar.},
}
@article {pmid32086804,
year = {2020},
author = {Shin, SW and Lee, JS},
title = {Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {6},
pages = {1895-1903},
doi = {10.1002/bit.27315},
pmid = {32086804},
issn = {1097-0290},
support = {2018R1C1B6001423//National Research Foundation of Korea/International ; 2019R1A6A1A11051471//National Research Foundation of Korea/International ; },
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cricetulus ; Gene Knock-In Techniques/*methods ; Plasmids/genetics ; RNA, Guide/genetics ; *Transgenes ; },
abstract = {Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development (CLD). CRISPR/Cas9 with the homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requiring a selection process to find a targeted integrant in CHO cells. Here, we explored several parameters that influence the targeting efficiency using a promoter-trap-based single- or double-knock-in (KI) monitoring system. A simple change in the donor template design by the addition of single-guide RNA recognition sequences strongly increased KI efficiency (2.9-36.0 fold), depending on integration sites and cell culture mode, compared to conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled us to obtain populations with ~1-4% of double-KI cells without additional enrichment procedures. Thus, this simple optimized strategy not only allows efficient CRISPR/Cas9-mediated TI in CHO cells but also paves the way for the applicability of multiplexed KIs in one experimental step without the need for sequential and independent CHO-CLD procedures.},
}
@article {pmid32086048,
year = {2020},
author = {Kalds, P and Gao, Y and Zhou, S and Cai, B and Huang, X and Wang, X and Chen, Y},
title = {Redesigning small ruminant genomes with CRISPR toolkit: Overview and perspectives.},
journal = {Theriogenology},
volume = {147},
number = {},
pages = {25-33},
doi = {10.1016/j.theriogenology.2020.02.015},
pmid = {32086048},
issn = {1879-3231},
mesh = {Animals ; *CRISPR-Cas Systems ; *Genome ; Goats/*genetics ; Humans ; Sheep/*genetics ; },
abstract = {Genetic modification is a rapidly developing field in which numerous significant breakthroughs have been achieved. Over the last few decades, genetic modification has evolved from insertional transgenesis to gene targeting and editing and, more recently, to base and prime editing using CRISPR-derived systems. Currently, CRISPR-based genome editing systems are showing great potential for generating gene-edited offspring with defined genetic characteristics. Domestic small ruminants (sheep and goats) have shown great potential as large animal models for genome engineering. Ovine and caprine genomes have been engineered using CRISPR-based systems for numerous purposes. These include generating superior agricultural breeds, expression of therapeutic agents in mammary glands, and developing animal models to be used in the study of human genetic disorders and regenerative medicine. The creation of these models has been facilitated by the continuous emergence and development of genetic modification tools. In this review, we provide an overview on how CRISPR-based systems have been used in the generation of gene-edited small ruminants through the two main pathways (embryonic microinjection and somatic cell nuclear transfer) and highlight the ovine and caprine genes that have been targeted via knockout, knockin, HDR-mediated point mutation, and base editing approaches, as well as the aims of these specific manipulations.},
}
@article {pmid32085798,
year = {2020},
author = {Shaw, L and Williams, RL and Hamill, KJ},
title = {CRISPR-Cas9-mediated labelling of the C-terminus of human laminin β1 leads to secretion inhibition.},
journal = {BMC research notes},
volume = {13},
number = {1},
pages = {90},
pmid = {32085798},
issn = {1756-0500},
support = {BB/L020513/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P0257731/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {A549 Cells ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Humans ; Laminin/*chemistry/*metabolism ; },
abstract = {OBJECTIVES: The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LMβ1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models.<br><br>RESULTS: CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LM&#x3b2;1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LM&#x3b2;1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LM&#x3b2;1 inhibits LM secretion, and is not a viable approach for use in animal models.},
}
@article {pmid32085579,
year = {2020},
author = {Hao, M and Wang, Z and Qiao, H and Yin, P and Qiao, J and Qi, H},
title = {Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli.},
journal = {Cells},
volume = {9},
number = {2},
pages = {},
pmid = {32085579},
issn = {2073-4409},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; DNA Replication/*genetics ; DNA, Circular/biosynthesis/genetics ; DNA, Single-Stranded/*biosynthesis/*genetics ; Escherichia coli/*genetics ; Gene Editing/*methods ; Nucleotides/metabolism ; Plasmids/genetics ; RNA, Guide/metabolism ; },
abstract = {As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.},
}
@article {pmid32084428,
year = {2020},
author = {Deem, TL and Collins, JB and DeVost, MH and Parker, CO and Saroka, SC and Zoldork, RJ and Gutierrez, F and Russell, JM and Lantz, CS},
title = {Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.},
journal = {Immunology letters},
volume = {221},
number = {},
pages = {18-26},
pmid = {32084428},
issn = {1879-0542},
support = {R15 AI094443/AI/NIAID NIH HHS/United States ; R15 AI119797/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Editing ; *Gene Expression ; Gene Order ; Gene Targeting ; *Genes, Reporter ; Genetic Vectors/genetics ; Humans ; Interleukin-3/*biosynthesis/*genetics ; Lymphocyte Activation/genetics/immunology ; Male ; Mice ; *Mice, Transgenic ; T-Lymphocyte Subsets/immunology/metabolism ; *Transgenes ; },
abstract = {Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1[+] CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.},
}
@article {pmid32084388,
year = {2020},
author = {Geurts, MH and de Poel, E and Amatngalim, GD and Oka, R and Meijers, FM and Kruisselbrink, E and van Mourik, P and Berkers, G and de Winter-de Groot, KM and Michel, S and Muilwijk, D and Aalbers, BL and Mullenders, J and Boj, SF and Suen, SWF and Brunsveld, JE and Janssens, HM and Mall, MA and Graeber, SY and van Boxtel, R and van der Ent, CK and Beekman, JM and Clevers, H},
title = {CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank.},
journal = {Cell stem cell},
volume = {26},
number = {4},
pages = {503-510.e7},
doi = {10.1016/j.stem.2020.01.019},
pmid = {32084388},
issn = {1875-9777},
support = {C6307/A29058/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Adenine ; Biological Specimen Banks ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Codon, Nonsense ; *Cystic Fibrosis/genetics ; Gene Editing ; Humans ; Organoids/metabolism ; },
abstract = {Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.},
}
@article {pmid32084371,
year = {2020},
author = {McCarron, A and Cmielewski, P and Reyne, N and McIntyre, C and Finnie, J and Craig, F and Rout-Pitt, N and Delhove, J and Schjenken, JE and Chan, HY and Boog, B and Knight, E and Gilmore, RC and O'Neal, WK and Boucher, RC and Parsons, D and Donnelley, M},
title = {Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.},
journal = {The American journal of pathology},
volume = {190},
number = {5},
pages = {977-993},
doi = {10.1016/j.ajpath.2020.01.009},
pmid = {32084371},
issn = {1525-2191},
mesh = {Animals ; CRISPR-Cas Systems ; *Cystic Fibrosis/genetics/pathology ; Cystic Fibrosis Transmembrane Conductance Regulator/*deficiency/genetics ; *Disease Models, Animal ; Gene Editing/*methods ; Humans ; Mice, Knockout ; Mutation ; Phenotype ; Rats ; Rats, Sprague-Dawley ; },
abstract = {Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.},
}
@article {pmid32081273,
year = {2020},
author = {Li, QF and Zhou, Y and Xiong, M and Ren, XY and Han, L and Wang, JD and Zhang, CQ and Fan, XL and Liu, QQ},
title = {Gibberellin recovers seed germination in rice with impaired brassinosteroid signalling.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {293},
number = {},
pages = {110435},
doi = {10.1016/j.plantsci.2020.110435},
pmid = {32081273},
issn = {1873-2259},
mesh = {Brassinosteroids/*metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Gene Ontology ; Germination/genetics/*physiology ; Gibberellins/*metabolism ; Oryza/genetics/*metabolism ; Plant Proteins/genetics/metabolism ; Protein Interaction Maps ; Proteomics ; RNA, Messenger/metabolism ; Seeds/genetics/growth &amp; development/*metabolism ; Signal Transduction/*physiology ; },
abstract = {Seed germination is essential for ensuring grain yield and quality. Germination rate, uniformity, and post-germination growth all contribute to cultivation. Although the phytohormones gibberellin (GA) and brassinosteroid (BR) are known to regulate germination, the underlying mechanism of their crosstalk in co-regulating rice seed germination remains unclear. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach was employed to identify target proteins responsive to GA during recovery of germination in BR-deficient and BR-insensitive rice. A total of 42 differentially abundant proteins were identified in both BR-deficient and BR-insensitive plants, and most were altered consistently in the two groups. Gene Ontology (GO) analysis revealed enrichment in proteins with binding and catalytic activity. A potential protein-protein interaction network was constructed using STRING analysis, and five Late Embryogenesis Abundant (LEA) family members were markedly down-regulated at both mRNA transcript and protein levels. These LEA genes were specifically expressed in rice seeds, especially during the latter stages of seed development. Mutation of LEA33 affected rice grain size and seed germination, possibly by reducing BR accumulation and enhancing GA biosynthesis. The findings improve our knowledge of the mechanisms by which GA and BR coordinate seed germination.},
}
@article {pmid32081255,
year = {2020},
author = {Yang, C and Huang, Y and Lv, W and Zhang, Y and Bhat, JA and Kong, J and Xing, H and Zhao, J and Zhao, T},
title = {GmNAC8 acts as a positive regulator in soybean drought stress.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {293},
number = {},
pages = {110442},
doi = {10.1016/j.plantsci.2020.110442},
pmid = {32081255},
issn = {1873-2259},
mesh = {Abscisic Acid/metabolism ; Acclimatization/genetics/physiology ; CRISPR-Cas Systems ; *Droughts ; Ethylenes/metabolism ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Genes, Plant/genetics ; Mutagenesis ; Plant Leaves/metabolism ; Plant Proteins/*genetics/*metabolism ; Plants, Genetically Modified ; Salicylic Acid/metabolism ; Soybeans/*genetics/*physiology ; Stress, Physiological/*genetics/*physiology ; Tobacco ; Transcription Factors/metabolism ; Transcriptome ; },
abstract = {NAC proteins represent one of the largest transcription factor (TF) families involved in the regulation of plant development and the response to abiotic stress. In the present study, we elucidated the detailed role of GmNAC8 in the regulation of drought stress tolerance in soybean. The GmNAC8 protein was localized in the nucleus, and expression of the GmNAC8 gene was significantly induced in response to drought, abscisic acid (ABA), ethylene (ETH) and salicylic acid (SA) treatments. Thus, we generated GmNAC8 overexpression (OE1 and OE2) and GmNAC8 knockout (KO1 and KO2) lines to determine the role of GmNAC8 in drought stress tolerance. Our results revealed that, compared with the wild type (WT) plant, GmNAC8 overexpression and GmNAC8 knockout lines exhibited significantly higher and lower drought tolerance, respectively. Furthermore, the SOD activity and proline content were significantly higher in the GmNAC8 overexpression lines and significantly lower in the GmNAC8 knockout lines than in the WT plants under drought stress. In addition, GmNAC8 protein was found to physically interact with the drought-induced protein GmDi19-3 in the nucleus. Moreover, the GmDi19-3 expression pattern showed the same trend as the GmNAC8 gene did under drought and hormone (ABA, ETH and SA) treatments, and GmDi19-3 overexpression lines (GmDi19-3-OE9, GmDi19-3-OE10 and GmDi19-3-OE31) showed enhanced drought tolerance compared to that of the WT plants. Hence, the above results indicated that GmNAC8 acts as a positive regulator of drought tolerance in soybean and inferred that GmNAC8 probably functions by interacting with another positive regulatory protein, GmDi19-3.},
}
@article {pmid32081254,
year = {2020},
author = {Dai, X and Liu, N and Wang, L and Li, J and Zheng, X and Xiang, F and Liu, Z},
title = {MYB94 and MYB96 additively inhibit callus formation via directly repressing LBD29 expression in Arabidopsis thaliana.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {293},
number = {},
pages = {110323},
doi = {10.1016/j.plantsci.2019.110323},
pmid = {32081254},
issn = {1873-2259},
mesh = {Arabidopsis/genetics/growth &amp; development/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; Bony Callus/growth &amp; development ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Indoleacetic Acids/metabolism ; Plant Roots/growth &amp; development/metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Trans-Activators/genetics/*metabolism ; Transcription Factors/*metabolism ; Transcriptome ; },
abstract = {Plant somatic cells can be reprogrammed during in vitro culture. Callus induction is the initial step of a typical plant regeneration system. Recent studies showed that auxin-induced callus formation in multiple organs occurs from the pericycle or pericycle-like cells via a root developmental pathway. However, the molecular control of callus formation is largely unknown. Here, two MYB transcription factors, MYB94 and MYB96, were shown to play negative roles in auxin-induced callus formation in Arabidopsis. MYB94 and MYB96 were expressed in the newly formed callus. myb96, myb94, and myb94 myb96 generated more calli than the WT, with myb94 myb96 producing the most. MYB94 and MYB96 repressed expression of LATERAL ORGAN BOUNDARIES-DOMAIN 29 (LBD29) via directly binding to the gene's promoter. The loss of function of LBD29 partly rescued the callus formation defect of myb94 myb96. Our findings found MYB94 and MYB96 to be important repressors of callus formation and MYB94/96-LBD29 as a new regulatory pathway acting in parallel with ARF7/19-LBDs' pathway to modulate in vitro callus formation.},
}
@article {pmid32080068,
year = {2020},
author = {Frith, KH},
title = {CRISPR-Cas: What Is It and Why Should Nurses Care?.},
journal = {Nursing education perspectives},
volume = {41},
number = {2},
pages = {136-137},
doi = {10.1097/01.NEP.0000000000000644},
pmid = {32080068},
issn = {1536-5026},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Nurses/*psychology ; },
}
@article {pmid32079488,
year = {2020},
author = {Luo, F and Long, K and Li, X and Mai, M and Zhong, Z and Li, S and Li, P and Zhou, S and Zhang, T and Long, X and Jin, L and Li, M},
title = {Deficient of LRRC8A attenuates hypoxia-induced necrosis in 3T3-L1 cells.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {84},
number = {6},
pages = {1139-1145},
doi = {10.1080/09168451.2020.1730689},
pmid = {32079488},
issn = {1347-6947},
mesh = {3T3-L1 Cells ; Animals ; CRISPR-Cas Systems ; Cell Hypoxia/*genetics ; Cell Survival/genetics ; Female ; *Gene Expression ; Gene Knockout Techniques ; Hypoxia/*genetics ; Membrane Proteins/*genetics/*metabolism ; Mice ; Myoblasts, Cardiac/metabolism ; Necrosis/genetics ; Rats ; Respiratory Mucosa/metabolism ; Swine ; Transfection ; },
abstract = {Under acute hypoxia, multiple ion channels on the cell membrane are activated, causing cell swelling and eventually necrosis. LRRC8A is an indispensable protein of the volume-regulated anion channel (VRAC), which participates in swelling and the acceleration of cell necrosis. In this study, we revealed a dynamic change in the expression level of the LRRC8 family during hypoxia in 3T3-L1 cells. The disruption of LRRC8A in 3T3-L1 cells was also associated with a significant anti-necrotic phenotype upon hypoxia accompanied by the reduced expression of necrosis-related genes. In vivo, differential expression of LRRC8 family members was also identified between high-altitude pigs and their low-altitude relatives. Taken these findings together, this study demonstrates the involvement of LRRC8A in hypoxia-induced cell necrosis.},
}
@article {pmid32079396,
year = {2020},
author = {Ray, A and Di Felice, R},
title = {Protein-Mutation-Induced Conformational Changes of the DNA and Nuclease Domain in CRISPR/Cas9 Systems by Molecular Dynamics Simulations.},
journal = {The journal of physical chemistry. B},
volume = {124},
number = {11},
pages = {2168-2179},
doi = {10.1021/acs.jpcb.9b07722},
pmid = {32079396},
issn = {1520-5207},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; DNA Cleavage ; Molecular Dynamics Simulation ; Mutation ; },
abstract = {Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems offer a unique protocol for genome editing in eukaryotic cells. The nuclease activity of Cas9 has been harnessed to perform precise genome editing by creating double-strand breaks. However, the nuclease activity of Cas9 can be triggered when there is imperfect complementarity between the RNA guide sequence and an off-target genomic site, which is a major limitation of the CRISPR technique for practical applications. Hence, understanding the binding mechanisms in CRISPR/Cas9 for predicting ways to increase cleavage specificity is a timely research target. One way to understand and tune the binding strength is to study wild-type and mutant Cas9, in complex with a guide RNA and a target DNA. We have performed classical all-atom MD simulations over a cumulative time scale of 13.5 μs of CRISPR/Cas9 ternary complexes with the wild-type Cas9 from Streptococcus pyogenes and three of its mutants: K855A, H982A, and the combination K855A+H982A, selected from the outcome of experimental work. Our results reveal significant structural impact of the mutations, with implications for specificity. We find that the "unwound" part of the nontarget DNA strand exhibits enhanced flexibility in complexes with Cas9 mutants and tries to move away from the HNH/RuvC interface, where it is otherwise stabilized by electrostatic couplings in the wild-type complex. Our findings refine an electrostatic model by which cleavage specificity can be optimized through protein mutations.},
}
@article {pmid32079278,
year = {2020},
author = {Katsouda, A and Peleli, M and Asimakopoulou, A and Papapetropoulos, A and Beis, D},
title = {Generation and Characterization of a CRISPR/Cas9 -Induced 3-mst Deficient Zebrafish.},
journal = {Biomolecules},
volume = {10},
number = {2},
pages = {},
pmid = {32079278},
issn = {2218-273X},
support = {886//Hellenic Foundation for Research and Innovation/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Hydrogen Sulfide/metabolism ; *Oxidative Stress ; Regeneration ; Sulfurtransferases/*genetics/metabolism ; Zebrafish/embryology/*genetics/physiology ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H2S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced 3-mst mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The 3-mst zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H2S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H2S levels were reduced in 3-mst[-/-] zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following 3-mst deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in 3-mst deficient zebrafish. Overall, we provide evidence for the expression of 3-mst in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.},
}
@article {pmid32078805,
year = {2020},
author = {Komáromy, AM},
title = {CRISPR-Cas9 Disruption of Aquaporin 1: An Alternative to Glaucoma Eye Drop Therapy?.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {3},
pages = {706-708},
pmid = {32078805},
issn = {1525-0024},
support = {R01 EY025752/EY/NEI NIH HHS/United States ; },
mesh = {Aquaporin 1 ; *CRISPR-Cas Systems ; Ciliary Body ; Gene Editing ; Genetic Therapy ; *Glaucoma ; Humans ; Ophthalmic Solutions ; },
}
@article {pmid32078438,
year = {2020},
author = {Napierski, NC and Granger, K and Langlais, PR and Moran, HR and Strom, J and Touma, K and Harris, SP},
title = {A Novel "Cut and Paste" Method for In Situ Replacement of cMyBP-C Reveals a New Role for cMyBP-C in the Regulation of Contractile Oscillations.},
journal = {Circulation research},
volume = {126},
number = {6},
pages = {737-749},
pmid = {32078438},
issn = {1524-4571},
support = {R01 HL080367/HL/NHLBI NIH HHS/United States ; R01 HL140925/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Calcium Signaling ; Carrier Proteins/chemistry/*genetics/metabolism ; Endopeptidases/genetics/metabolism ; Gene Editing/*methods ; Mice ; Mice, Inbred C57BL ; *Myocardial Contraction ; Protein Domains ; Protein Engineering/*methods ; Sarcomeres/*metabolism/physiology ; },
abstract = {RATIONALE: cMyBP-C (cardiac myosin-binding protein-C) is a critical regulator of heart contraction, but the mechanisms by which cMyBP-C affects actin and myosin are only partly understood. A primary obstacle is that cMyBP-C localization on thick filaments may be a key factor defining its interactions, but most in vitro studies cannot duplicate the unique spatial arrangement of cMyBP-C within the sarcomere.<br><br>OBJECTIVE: The goal of this study was to validate a novel hybrid genetic/protein engineering approach for rapid manipulation of cMyBP-C in sarcomeres in situ.<br><br>METHODS AND RESULTS: We designed a novel cut and paste approach for removal and replacement of cMyBP-C N'-terminal domains (C0-C7) in detergent-permeabilized cardiomyocytes from gene-edited Spy-C mice. Spy-C mice express a TEVp (tobacco etch virus protease) cleavage site and a SpyTag (st) between cMyBP-C domains C7 and C8. A cut is achieved using TEVp which cleaves cMyBP-C to create a soluble N'-terminal &#x3b3;C0C7 (endogenous [genetically encoded] N'-terminal domains C0 to C7 of cardiac myosin binding protein-C) fragment and an insoluble C'-terminal SpyTag-C8-C10 fragment that remains associated with thick filaments. Paste of new recombinant (r)C0C7 domains is achieved by a covalent bond formed between SpyCatcher (-sc; encoded at the C'-termini of recombinant proteins) and SpyTag. Results show that loss of &#x3b3;C0C7 reduced myofilament Ca[2+] sensitivity and increased cross-bridge cycling (ktr) at submaximal [Ca[2+]]. Acute loss of &#x3b3;C0C7 also induced auto-oscillatory contractions at submaximal [Ca[2+]]. Ligation of rC0C7 (exogenous [recombinant] N'-terminal domains C0 to C7 of cardiac myosin binding protein-C)-sc returned pCa50 and ktr to control values and abolished oscillations, but phosphorylated (p)-rC0C7-sc did not completely rescue these effects.<br><br>CONCLUSIONS: We describe a robust new approach for acute removal and replacement of cMyBP-C in situ. The method revealed a novel role for cMyBP-C N'-terminal domains to damp sarcomere-driven contractile waves (so-called spontaneous oscillatory contractions). Because phosphorylated (p)-rC0C7-sc was less effective at damping contractile oscillations, results suggest that spontaneous oscillatory contractions may contribute to enhanced contractility in response to inotropic stimuli.},
}
@article {pmid32078250,
year = {2020},
author = {Beckermann, TM and Welch, RC and Williams, FM and Mortlock, DP and Sha, F and Ikizler, TA and Woodard, LE and Wilson, MH},
title = {CRISPR/Cas9 engineering of albino cystinuria Type A mice.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {5},
pages = {e23357},
pmid = {32078250},
issn = {1526-968X},
support = {I01 BX004258/BX/BLRD VA/United States ; IK2 BX002797/BX/BLRD VA/United States ; I01 BX002190/BX/BLRD VA/United States ; P30 CA068485/CA/NCI NIH HHS/United States ; I01 BX004845/BX/BLRD VA/United States ; P30 DK114809/DK/NIDDK NIH HHS/United States ; P30 DK020593/DK/NIDDK NIH HHS/United States ; R01 DK093660/DK/NIDDK NIH HHS/United States ; },
mesh = {Amino Acid Transport Systems, Basic/*genetics ; Amino Acid Transport Systems, Neutral/*genetics ; Animals ; CRISPR-Cas Systems ; Cysteine/urine ; Cystinuria/*genetics/pathology ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; *Mutation ; },
abstract = {Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co-injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off-target editing. No off-target indels were observed for the top 10 predicted off-targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.},
}
@article {pmid32077487,
year = {2020},
author = {Price, MA and Cruz, R and Bryson, J and Escalettes, F and Rosser, SJ},
title = {Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {6},
pages = {1805-1816},
doi = {10.1002/bit.27312},
pmid = {32077487},
issn = {1097-0290},
support = {BB/M00029X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacillus subtilis/*genetics ; Bacterial Proteins/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*genetics ; Eubacterium/*enzymology/genetics ; Gene Editing/*methods ; Point Mutation ; },
abstract = {The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&amp;D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.},
}
@article {pmid32076642,
year = {2020},
author = {Aschenbrenner, S and Kallenberger, SM and Hoffmann, MD and Huck, A and Eils, R and Niopek, D},
title = {Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.},
journal = {Science advances},
volume = {6},
number = {6},
pages = {eaay0187},
pmid = {32076642},
issn = {2375-2548},
mesh = {CRISPR-Associated Protein 9/chemistry/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Enzyme Activation ; *Gene Editing ; *Gene Targeting/methods ; Genes, Reporter ; Genetic Loci ; Humans ; Kinetics ; Models, Theoretical ; *Recombinant Fusion Proteins ; Substrate Specificity ; },
abstract = {The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.},
}
@article {pmid32076262,
year = {2020},
author = {Kim, S and Loeff, L and Colombo, S and Jergic, S and Brouns, SJJ and Joo, C},
title = {Selective loading and processing of prespacers for precise CRISPR adaptation.},
journal = {Nature},
volume = {579},
number = {7797},
pages = {141-145},
pmid = {32076262},
issn = {1476-4687},
mesh = {Base Pairing ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Single-Stranded/*genetics/metabolism ; Exodeoxyribonuclease V/metabolism ; Exonucleases/metabolism ; Fluorescence ; Gene Editing/*methods ; Kinetics ; Recombination, Genetic/genetics ; Time Factors ; },
abstract = {CRISPR-Cas immunity protects prokaryotes against invading genetic elements[1]. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)[2-5]. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear[6,7]. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.},
}
@article {pmid32075916,
year = {2020},
author = {Warner, M and Wu, WF and Montanholi, L and Nalvarte, I and Antonson, P and Gustafsson, JA},
title = {Ventral prostate and mammary gland phenotype in mice with complete deletion of the ERβ gene.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {9},
pages = {4902-4909},
pmid = {32075916},
issn = {1091-6490},
mesh = {Androgens/metabolism ; Animals ; CRISPR-Cas Systems ; Chemokines/metabolism ; DNA-Binding Proteins/metabolism ; Epithelium/metabolism ; Estrogen Receptor alpha/metabolism ; Estrogen Receptor beta/*genetics ; Estrogens/metabolism ; Eye Proteins ; Female ; Hyperplasia/pathology ; Inflammation ; Ki-67 Antigen/metabolism ; Male ; Mammary Glands, Animal/*metabolism ; Mice ; Mice, Knockout ; Nerve Tissue Proteins/metabolism ; *Phenotype ; Prostate/*metabolism/pathology ; RNA, Messenger/metabolism ; Receptors, Androgen/*metabolism ; *Sequence Deletion ; Signal Transduction ; Stromal Cells ; Trans-Activators ; Transcription Factors/metabolism ; Transcriptome ; Tumor Suppressor Proteins/metabolism ; },
abstract = {Disagreements about the phenotype of estrogen receptor β (ERβ) knockout mouse, created by removing the DNA-binding domain of the ERβ gene or interruption of the gene with a neocassette (Oliver Smithies ERβ knockout mice [ERβ[OS-/-]]), prompted us to create an ERβ knockout mouse by deleting the ERβ gene with the use of CRISPR/Cas9 technology. We confirmed that the ERβ gene was eliminated from the mouse genome and that no ERβ mRNA or protein was detectable in tissues of this mouse. Overall the phenotype of the ventral prostate (VP) and mammary gland (MG) in ERβ[crispr-/-] mice was similar to, but more severe than, that in the ERβ[OS-/-]mice. In the VP of 6-mo-old ERβ[crispr-/-] mice there was epithelial hyperplasia, fibroplasia, inflammation, stromal overgrowth, and intraductal cancer-like lesions. This was accompanied by an increase in Ki67 and P63 and loss in DACH1 and PURα, two androgen receptor (AR) repressors. In the MG there was overexpression of estrogen receptor α and progesterone receptor, loss of collagen, increase in proliferation and expression of metalloproteases, and invasive epithelium. Surprisingly, by 18 mo of age, the number of hyperplastic foci was reduced, the ducts of the VP and MG became atrophic, and, in the VP, there was massive immune infiltration and massive desquamation of the luminal epithelial cells. These changes were coincident with reduced levels of androgens in males and estrogens in females. We conclude that ERβ is a tumor suppressor gene in the VP and MG where its loss increases the activity AR and ERα, respectively.},
}
@article {pmid32075512,
year = {2020},
author = {Bor, B and Collins, AJ and Murugkar, PP and Balasubramanian, S and To, TT and Hendrickson, EL and Bedree, JK and Bidlack, FB and Johnston, CD and Shi, W and McLean, JS and He, X and Dewhirst, FE},
title = {Insights Obtained by Culturing Saccharibacteria With Their Bacterial Hosts.},
journal = {Journal of dental research},
volume = {99},
number = {6},
pages = {685-694},
pmid = {32075512},
issn = {1544-0591},
support = {F32 DE025548/DE/NIDCR NIH HHS/United States ; K99 DE027719/DE/NIDCR NIH HHS/United States ; F31 DE026057/DE/NIDCR NIH HHS/United States ; R00 DE027719/DE/NIDCR NIH HHS/United States ; R01 DE023810/DE/NIDCR NIH HHS/United States ; R01 DE026186/DE/NIDCR NIH HHS/United States ; R01 DE024468/DE/NIDCR NIH HHS/United States ; R01 DE027850/DE/NIDCR NIH HHS/United States ; R37 DE016937/DE/NIDCR NIH HHS/United States ; R01 DE020102/DE/NIDCR NIH HHS/United States ; },
mesh = {Actinomyces ; *Bacteria/genetics ; Genome, Bacterial ; Humans ; In Situ Hybridization, Fluorescence ; *Microbiota ; Mouth ; },
abstract = {Oral microbiome research has moved from asking "Who's there?" to "What are they doing?" Understanding what microbes "do" involves multiple approaches, including obtaining genomic information and examining the interspecies interactions. Recently we isolated a human oral Saccharibacteria (TM7) bacterium, HMT-952, strain TM7x, which is an ultrasmall parasite of the oral bacterium Actinomyces odontolyticus. The host-parasite interactions, such as phage-bacterium or Saccharibacteria-host bacterium, are understudied areas with large potential for insight. The Saccharibacteria phylum is a member of Candidate Phyla Radiation, a large lineage previously devoid of cultivated members. However, expanding our understanding of Saccharibacteria-host interactions requires examining multiple phylogenetically distinct Saccharibacteria-host pairs. Here we report the isolation of 3 additional Saccharibacteria species from the human oral cavity in binary coculture with their bacterial hosts. They were obtained by filtering ultrasmall Saccharibacteria cells free of other larger bacteria and inoculating them into cultures of potential host bacteria. The binary cocultures obtained could be stably passaged and studied. Complete closed genomes were obtained and allowed full genome analyses. All have small genomes (<1 Mb) characteristic of parasitic species and dramatically limited de novo synthetic pathway capabilities but include either restriction modification or CRISPR-Cas systems as part of an innate defense against foreign DNA. High levels of gene synteny exist among Saccharibacteria species. Having isolates growing in coculture with their hosts allowed time course studies of growth and parasite-host interactions by phase contrast, fluorescence in situ hybridization, and scanning electron microscopy. The cells of the 4 oral Saccharibacteria species are ultrasmall and could be seen attached to their larger Actinobacteria hosts. Parasite attachment appears to lead to host cell death and lysis. The successful cultivation of Saccharibacteria species has significantly expanded our understanding of these ultrasmall Candidate Phyla Radiation bacteria.},
}
@article {pmid32073884,
year = {2020},
author = {Deng, M and Liu, Z and Chen, B and Cai, Y and Wan, Y and Wang, F},
title = {Locus-Specific Regulation of Xist Expression Using the CRISPR-Cas9-Based System.},
journal = {DNA and cell biology},
volume = {39},
number = {4},
pages = {572-578},
doi = {10.1089/dna.2019.4945},
pmid = {32073884},
issn = {1557-7430},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Catalytic Domain/genetics ; Cell Line ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; DNA Methylation/*genetics ; Decitabine/pharmacology ; Enzyme Inhibitors/pharmacology ; Female ; Fibroblasts ; Gene Expression Regulation/*genetics ; Goats ; RNA, Guide/genetics ; RNA, Long Noncoding/*genetics ; },
abstract = {DNA methylation inhibitor or loss and gain of function of DNA methylation key players were widely used to investigate the regulation of X inactive-specific transcript (Xist) expression by DNA methylation, which results in global change of DNA methylation. Here, we reported a novel method for regulation of Xist using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. First, Xist expression was increased in 5-aza-2'-deoxycytidine-treated female goat fibroblast cells. Second, three single-guide RNAs (sgRNAs) that target the Xist differential methylation region (DMR) were inserted to deactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase Dnmt3a coexpression plasmid. Bisulfite PCR analysis and quantitative real-time PCR revealed that the methylation level of the DMR was significantly increased, while the expression of Xist was downregulated in all three sgRNAs, compared with the mock-transfected cells. Third, the methylation activity at the sites of 37 bp from the protospacer-adjacent motif sequence showed the strong change relative to the mock-transfected cells. Furthermore, genome-wide DNA methylation and expression of the DNA methylation key players were not statistically changed in all three sgRNAs. Therefore, we confirmed that Xist expression was regulated by DNA methylation, and directed DNA methylation of Xist DMR at locus-specific solution decreased Xist expression.},
}
@article {pmid32073434,
year = {2020},
author = {Agache, I and Cojanu, C and Laculiceanu, A and Rogozea, L},
title = {Genetics and epigenetics of allergy.},
journal = {Current opinion in allergy and clinical immunology},
volume = {20},
number = {3},
pages = {223-232},
doi = {10.1097/ACI.0000000000000634},
pmid = {32073434},
issn = {1473-6322},
mesh = {Acetylation ; Animals ; Biomarkers ; CRISPR-Cas Systems/genetics ; DNA Methylation/immunology ; Disease Models, Animal ; Epigenesis, Genetic/*immunology ; Gene Editing/methods ; Gene-Environment Interaction ; *Genetic Predisposition to Disease ; Histones/genetics/immunology ; Humans ; Hypersensitivity/diagnosis/*genetics/immunology/therapy ; Precision Medicine/*methods ; Probiotics/administration &amp; dosage ; },
abstract = {PURPOSE OF REVIEW: Allergic diseases are prototypic examples for gene × environment-wide interactions. This review considers the current evidence for genetic and epigenetic mechanisms in allergic diseases and highlights barriers and facilitators for the implementation of these novel tools both for research and clinical practice.<br><br>RECENT FINDINGS: The value of whole-genome sequencing studies and the use of polygenic risk score analysis in homogeneous well characterized populations are currently being tested. Epigenetic mechanisms are known to play a crucial role in the pathogenesis of allergic disorders, especially through mediating the effects of the environmental factors, well recognized risk modifiers. There is emerging evidence for the immune-modulatory role of probiotics through epigenetic changes. Direct or indirect targeting of epigenetic mechanisms affect expression of the genes favouring the development of allergic diseases and can improve tissue biology. The ability to specifically edit the epigenome, especially using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, holds the promise of enhancing understanding of how epigenetic modifications function and enabling manipulation of cell phenotype for research or therapeutic purposes.<br><br>SUMMARY: Additional research in the role of genetic and epigenetic mechanisms in relation to allergic diseases' endotypes is needed. An international project characterizing the human epigenome in relation to allergic diseases is warranted.},
}
@article {pmid32072789,
year = {2020},
author = {Li, J and Liu, Y and Wang, Y and Yu, P and Zheng, P and Wang, M},
title = {[Optimization of base editing in Corynebacterium glutamicum].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {1},
pages = {143-151},
doi = {10.13345/j.cjb.190192},
pmid = {32072789},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Corynebacterium glutamicum ; Gene Editing ; RNA, Guide ; },
abstract = {In recent years, CRISPR/Cas9-mediated base editing has been developed to a powerful genome editing tool, providing advantages such as without introducing double-stranded DNA break, a donor template and relying on host homologous recombination repair pathway, and has been widely applied in animals, plants, yeast and bacteria. In previous study, our group developed a multiplex automated base editing method (MACBETH) in the important industrial model strain Corynebacterium glutamicum. In this study, to further optimize the method and improve the base editing efficiency in C. glutamicum, we first constructed a green fluorescent protein (GFP) reporter-based detection system. The point mutation in the inactivated GFP protein can be edited to restore the GFP fluorescence. By combining with flow cytometry analysis, the base-editing efficiency can be quickly calculated. Then, the base editor with the target gRNA was constructed, and the editing efficiency with the initial editing condition was (13.11±0.21)%. Based on this result, the editing conditions were optimized and the result indicated that the best medium is CGXII, the best initial OD600 of induction is 0.05, the best induction time is 20 h, and the best IPTG concentration is 0.01 mmol/L. After optimization, the editing efficiency was improved to (30.35±0.75)%, which was 1.3-fold of that in initial condition. Finally, endogenous genomic loci of C. glutamicum were selected to assess if the optimized condition can improve genome editing in other loci. Editing efficiency of different loci in optimized condition were improved to 1.7-2.5 fold of that in original condition, indicating the effectiveness and versatility of the optimized condition. Our research will promote the better application of base editing technology in C. glutamicum.},
}
@article {pmid32072776,
year = {2020},
author = {Ouyang, J and Xue, S and Zhou, Q and Cui, H},
title = {[Research progress and applications of gene editing technology CRISPR/Cas in zebrafish].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {1},
pages = {1-12},
doi = {10.13345/j.cjb.190178},
pmid = {32072776},
issn = {1872-2075},
mesh = {Animals ; Archaea ; *CRISPR-Cas Systems ; *Gene Editing ; Zebrafish ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) are acquired immune system in bacteria and archaea. This system is used in site-directed gene editing. Recently, scientists discovered new CRISPR-associated (Cas) proteins, in which Cas12a-mediated gene editing can significantly reduce the off-target rate. In this article, we review CRISPR/Cas system's discovery of history, composition, classification, and working principle. The latest research progress of the CRISPR/Cas system, and its application in zebrafish are introduced.},
}
@article {pmid32071397,
year = {2020},
author = {Kato, H and Asamitsu, K and Sun, W and Kitajima, S and Yoshizawa-Sugata, N and Okamoto, T and Masai, H and Poellinger, L},
title = {Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.},
journal = {Oncogene},
volume = {39},
number = {16},
pages = {3322-3335},
pmid = {32071397},
issn = {1476-5594},
mesh = {Amino Acid Substitution/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics ; Colorectal Neoplasms/*genetics/pathology ; DNA-Binding Proteins/*genetics ; Gene Expression Regulation, Neoplastic/genetics ; HCT116 Cells ; Histone Demethylases/*genetics ; Histone-Lysine N-Methyltransferase/genetics ; Humans ; Jumonji Domain-Containing Histone Demethylases/genetics ; Mutation/genetics ; Nuclear Proteins/*genetics ; Tetratricopeptide Repeat/*genetics ; Transcription Factors/*genetics ; },
abstract = {The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.},
}
@article {pmid32071273,
year = {2020},
author = {Schulte, LN and Schweinlin, M and Westermann, AJ and Janga, H and Santos, SC and Appenzeller, S and Walles, H and Vogel, J and Metzger, M},
title = {An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection.},
journal = {mBio},
volume = {11},
number = {1},
pages = {},
pmid = {32071273},
issn = {2150-7511},
mesh = {Animals ; CRISPR-Cas Systems ; Caco-2 Cells ; Coculture Techniques/*methods ; Disease Models, Animal ; Epithelial Cells/microbiology ; Epithelium/microbiology ; Gastroenteritis/microbiology ; Gene Expression Regulation, Bacterial ; Host-Pathogen Interactions/*physiology ; Humans ; Intestines/*microbiology ; Killer Cells, Natural ; Mice ; STAT3 Transcription Factor/metabolism ; Salmonella Infections/immunology/*microbiology ; Salmonella typhimurium/genetics/immunology ; Transcriptome ; Type III Secretion Systems ; },
abstract = {A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.},
}
@article {pmid32071241,
year = {2020},
author = {Bradde, S and Nourmohammad, A and Goyal, S and Balasubramanian, V},
title = {The size of the immune repertoire of bacteria.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {10},
pages = {5144-5151},
pmid = {32071241},
issn = {1091-6490},
mesh = {*Adaptive Immunity ; Bacteria/*genetics/*virology ; *Bacteriophages ; CRISPR-Cas Systems/*physiology ; },
abstract = {Some bacteria and archaea possess an immune system, based on the CRISPR-Cas mechanism, that confers adaptive immunity against viruses. In such species, individual prokaryotes maintain cassettes of viral DNA elements called spacers as a memory of past infections. Typically, the cassettes contain several dozen expressed spacers. Given that bacteria can have very large genomes and since having more spacers should confer a better memory, it is puzzling that so little genetic space would be devoted by prokaryotes to their adaptive immune systems. Here, assuming that CRISPR functions as a long-term memory-based defense against a diverse landscape of viral species, we identify a fundamental tradeoff between the amount of immune memory and effectiveness of response to a given threat. This tradeoff implies an optimal size for the prokaryotic immune repertoire in the observational range.},
}
@article {pmid32071193,
year = {2020},
author = {Zirin, J and Hu, Y and Liu, L and Yang-Zhou, D and Colbeth, R and Yan, D and Ewen-Campen, B and Tao, R and Vogt, E and VanNest, S and Cavers, C and Villalta, C and Comjean, A and Sun, J and Wang, X and Jia, Y and Zhu, R and Peng, P and Yu, J and Shen, D and Qiu, Y and Ayisi, L and Ragoowansi, H and Fenton, E and Efrem, S and Parks, A and Saito, K and Kondo, S and Perkins, L and Mohr, SE and Ni, J and Perrimon, N},
title = {Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies.},
journal = {Genetics},
volume = {214},
number = {4},
pages = {755-767},
pmid = {32071193},
issn = {1943-2631},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; R24 RR032668/RR/NCRR NIH HHS/United States ; R24 OD021997/OD/NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; R01 GM084947/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems ; *Databases, Genetic ; Drosophila melanogaster/*genetics ; Gain of Function Mutation ; Genetic Engineering/methods ; Loss of Function Mutation ; },
abstract = {The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.},
}
@article {pmid32071159,
year = {2020},
author = {Brooks, MR and Padilla-Vélez, L and Khan, TA and Qureshi, AA and Pieper, JB and Maddox, CW and Alam, MT},
title = {Prophage-Mediated Disruption of Genetic Competence in Staphylococcus pseudintermedius.},
journal = {mSystems},
volume = {5},
number = {1},
pages = {},
pmid = {32071159},
issn = {2379-5077},
support = {T35 OD011145/OD/NIH HHS/United States ; },
abstract = {Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of soft tissue infections in dogs and occasionally infects humans. Hypervirulent multidrug-resistant (MDR) MRSP clones have emerged globally. The sequence types ST71 and ST68, the major epidemic clones of Europe and North America, respectively, have spread to other regions. The genetic factors underlying the success of these clones have not been investigated thoroughly. Here, we performed a comprehensive genomic analysis of 371 S. pseudintermedius isolates to dissect the differences between major clonal lineages. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, restriction-modification (RM), and CRISPR/Cas systems differs significantly among MRSP clones. The isolates with GyrA+GrlA mutations, conferring fluoroquinolone resistance, carry more of these genes than those without GyrA+GrlA mutations. ST71 and ST68 clones carry lineage-specific prophages with genes that are likely associated with their increased fitness and virulence. We have discovered that a prophage, SpST71A, is inserted within the comGA gene of the late competence operon comG in the ST71 lineage. A functional comG is essential for natural genetic competence, which is one of the major modes of horizontal gene transfer (HGT) in bacteria. The RM and CRISPR/Cas systems, both major genetic barriers to HGT, are also lineage specific. Clones harboring CRISPR/Cas or a prophage-disrupted comG exhibited less genetic diversity and lower rates of recombination than clones lacking these systems. After Listeria monocytogenes, this is the second example of prophage-mediated competence disruption reported in any bacteria. These findings are important for understanding the evolution and clonal expansion of MDR MRSP clones.IMPORTANCE Staphylococcus pseudintermedius is a bacterium responsible for clinically important infections in dogs and can infect humans. In this study, we performed genomic analysis of 371 S. pseudintermedius isolates to understand the evolution of antibiotic resistance and virulence in this organism. The analysis covered significant reported clones, including ST71 and ST68, the major epidemic clones of Europe and North America, respectively. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, and horizontal gene transfer differs among clones. ST71 and ST68 carry prophages with novel virulence and antibiotic resistance genes. Importantly, site-specific integration of a prophage, SpST71A, has led to the disruption of the genetic competence operon comG in ST71 clone. A functional comG is essential for the natural uptake of foreign DNA and thus plays an important role in the evolution of bacteria. This study provides insight into the emergence and evolution of antibiotic resistance and virulence in S. pseudintermedius, which may help in efforts to combat this pathogen.},
}
@article {pmid32071146,
year = {2021},
author = {Basheer, F and Vassiliou, G},
title = {Mouse Models of Myeloid Malignancies.},
journal = {Cold Spring Harbor perspectives in medicine},
volume = {11},
number = {1},
pages = {},
pmid = {32071146},
issn = {2157-1422},
support = {MC_PC_12009/MRC_/Medical Research Council/United Kingdom ; MC_PC_17230/MRC_/Medical Research Council/United Kingdom ; C22324/A23015/CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {Animals ; Bone Marrow Neoplasms/*genetics ; CRISPR-Cas Systems/*genetics ; Clonal Hematopoiesis/genetics ; Disease Models, Animal ; Gene Editing ; Humans ; Leukemia, Myeloid, Acute/genetics ; Mice ; Myelodysplastic Syndromes/genetics ; },
abstract = {Mouse models of human myeloid malignancies support the detailed and focused investigation of selected driver mutations and represent powerful tools in the study of these diseases. Carefully developed murine models can closely recapitulate human myeloid malignancies in vivo, enabling the interrogation of a number of aspects of these diseases including their preclinical course, interactions with the microenvironment, effects of pharmacological agents, and the role of non-cell-autonomous factors, as well as the synergy between co-occurring mutations. Importantly, advances in gene-editing technologies, particularly CRISPR-Cas9, have opened new avenues for the development and study of genetically modified mice and also enable the direct modification of mouse and human hematopoietic cells. In this review we provide a concise overview of some of the important mouse models that have advanced our understanding of myeloid leukemogenesis with an emphasis on models relevant to clonal hematopoiesis, myelodysplastic syndromes, and acute myeloid leukemia with a normal karyotype.},
}
@article {pmid32070859,
year = {2020},
author = {Zuo, Z and Liu, J},
title = {Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage.},
journal = {Current opinion in structural biology},
volume = {62},
number = {},
pages = {166-174},
pmid = {32070859},
issn = {1879-033X},
support = {R15 HL147265/HL/NHLBI NIH HHS/United States ; },
mesh = {Allosteric Regulation ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; DNA/*metabolism ; DNA Cleavage ; Humans ; Protein Engineering/*methods ; RNA, Guide/*metabolism ; },
abstract = {The CRISPR-Cas9 system from Streptococcus pyogenes has been exploited as a programmable RNA-guided DNA-targeting and DNA-editing platform. This evolutionary tool enables diverse genetic manipulations with unprecedented precision and ease. Cas9 is an allosteric enzyme, which is allosterically regulated in conformational activation, target recognition, and DNA cleavage. Here, we outline the underlying allosteric control over the Cas9 complex assembly and targeting specificity. We further review the strategies for mitigating intrinsic Cas9 off-target effects through allosteric modulations and the advances in engineering controllable Cas9 systems that are responsive to external allosteric signals. Future development of highly specific, tunable CRISPR-Cas9 systems through allosteric modulations would greatly benefit applications that require both conditional control and high precision.},
}
@article {pmid32070424,
year = {2020},
author = {Xu, J and Zheng, Z and Du, X and Shi, B and Wang, J and Gao, D and Zhu, Q and Chen, X and Han, J},
title = {A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG.},
journal = {Stem cell research &amp; therapy},
volume = {11},
number = {1},
pages = {67},
pmid = {32070424},
issn = {1757-6512},
mesh = {Activins/*metabolism ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Nanog Homeobox Protein/*metabolism ; Pluripotent Stem Cells/*metabolism ; Signal Transduction ; Swine ; },
abstract = {BACKGROUND: NANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG is a transcription factor highly expressed in pig pre-implantation embryos, indicating that it is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not fully understood in this animal.<br><br>METHODS: In this study, pig NANOG tdTomato knock-in reporter positive PC-iPS cells were established using CRISPR/Cas9. The resulting cell line was treated with several cytokines and their corresponding inhibitors to identify pathways that regulate NANOG expression. The pathways examined were LIF (leukemia inhibitory factor)/IL6 (interleukin 6)-STAT3, FGF (fibroblast growth factor)/ERK, IGF1 (insulin-like growth factor 1)/PIP3 (phosphoinositide 3-kinase)-AKT, Activin A/SMAD, and BMP4 (bone morphogenetic proteins)/SMAD.<br><br>RESULTS: Our experiments showed that the Activin A/SMAD pathway is directly associated with activation of NANOG expression in the pig, as is also the case in mice and humans. Activin A directly regulates the expression of pig NANOG via SMAD2/3; inhibition of this pathway by SB431542 resulted in inhibition of NANOG expression.<br><br>CONCLUSIONS: Our results show that Activin A plays an important regulatory role in NANOG-mediated pluripotency in pig iPS cells. Activin A treatment may be therefore an effective method for de novo derivation of authentic embryonic stem cells (ESCs) from pig pre-implantation embryos.},
}
@article {pmid32070421,
year = {2020},
author = {Dicks, A and Wu, CL and Steward, N and Adkar, SS and Gersbach, CA and Guilak, F},
title = {Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.},
journal = {Stem cell research &amp; therapy},
volume = {11},
number = {1},
pages = {66},
pmid = {32070421},
issn = {1757-6512},
support = {K99 AR075899/AR/NIAMS NIH HHS/United States ; R00 AR075899/AR/NIAMS NIH HHS/United States ; T32 GM007171/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Cells, Cultured ; Chondrocytes/*metabolism ; Chondrogenesis/*genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Prospective Studies ; },
abstract = {BACKGROUND: Articular cartilage shows little or no capacity for intrinsic repair, generating a critical need of regenerative therapies for joint injuries and diseases such as osteoarthritis. Human-induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering and in vitro human disease modeling; however, off-target differentiation remains a challenge during hiPSC chondrogenesis. Therefore, the objective of this study was to identify cell surface markers that define the true chondroprogenitor population and use these markers to purify iPSCs as a means of improving the homogeneity and efficiency of hiPSC chondrogenic differentiation.<br><br>METHODS: We used a CRISPR-Cas9-edited COL2A1-GFP knock-in reporter hiPSC line, coupled with a surface marker screen, to identify a novel chondroprogenitor population. Single-cell RNA sequencing was then used to analyze the distinct clusters within the population. An unpaired t test with Welch's correction or an unpaired Kolmogorov-Smirnov test was performed with significance reported at a 95% confidence interval.<br><br>RESULTS: Chondroprogenitors expressing CD146, CD166, and PDGFR&#x3b2;, but not CD45, made up an average of 16.8% of the total population. Under chondrogenic culture conditions, these triple-positive chondroprogenitor cells demonstrated decreased heterogeneity as measured by single-cell RNA sequencing with fewer clusters (9 clusters in unsorted vs. 6 in sorted populations) closer together. Additionally, there was more robust and homogenous matrix production (unsorted: 1.5&#x2009;ng/ng vs. sorted: 19.9&#x2009;ng/ng sGAG/DNA; p&#x2009;<&#x2009;0.001) with significantly higher chondrogenic gene expression (i.e., SOX9, COL2A1, ACAN; p&#x2009;<&#x2009;0.05).<br><br>CONCLUSIONS: Overall, this study has identified a unique hiPSC-derived subpopulation of chondroprogenitors that are CD146[+]/CD166[+]/PDGFR&#x3b2;[+]/CD45[-] and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.},
}
@article {pmid32069391,
year = {2020},
author = {Polino, AJ and Nasamu, AS and Niles, JC and Goldberg, DE},
title = {Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin V.},
journal = {ACS infectious diseases},
volume = {6},
number = {4},
pages = {738-746},
pmid = {32069391},
issn = {2373-8227},
support = {R01 AI112508/AI/NIAID NIH HHS/United States ; R01 AI138447/AI/NIAID NIH HHS/United States ; P50 GM098792/GM/NIGMS NIH HHS/United States ; },
mesh = {Aspartic Acid Endopeptidases/*antagonists &amp; inhibitors ; CRISPR-Cas Systems ; Erythrocytes/parasitology ; Humans ; Inhibitory Concentration 50 ; Life Cycle Stages/drug effects ; Plasmodium falciparum/*drug effects/*enzymology/growth &amp; development ; Protein Processing, Post-Translational/genetics ; Protozoan Proteins/*antagonists &amp; inhibitors ; },
abstract = {Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This protein export program is essential for parasite survival. Hence export-related proteins could be potential drug targets. One essential enzyme in this pathway is plasmepsin V (PMV), an aspartic protease that processes export-destined proteins in the parasite endoplasmic reticulum (ER) at the Plasmodium export element (PEXEL) motif. Despite long-standing interest in this enzyme, functional studies have been hindered by the inability of previous technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a system for stringent post-transcriptional regulation allowing a tightly controlled, tunable knockdown of PMV. Using this system, we found that PMV must be dramatically depleted to affect parasite growth, suggesting the parasite maintains this enzyme in substantial excess. Surprisingly, depletion of PMV arrested parasite growth immediately after RBC invasion, significantly before the death from exported protein deficit that has previously been described. The data suggest that PMV inhibitors can halt parasite growth at two distinct points in the parasite life cycle. However, overcoming the functional excess of PMV in the parasite may require inhibitor concentrations far beyond the enzyme's IC50.},
}
@article {pmid32067617,
year = {2020},
author = {Taheri, F and Taghizadeh, E and Pour, MJR and Rostami, D and Renani, PG and Rastgar-Moghadam, A and Hayat, SMG},
title = {Limb-girdle Muscular Dystrophy and Therapy: Insights into Cell and Gene-based Approaches.},
journal = {Current gene therapy},
volume = {19},
number = {6},
pages = {386-394},
doi = {10.2174/1566523220666200218113526},
pmid = {32067617},
issn = {1875-5631},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Exons ; Gene Editing ; *Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells ; Mesenchymal Stem Cells ; Mice ; Muscular Dystrophies, Limb-Girdle/*genetics/*therapy ; RNA, Small Interfering ; Transplants ; },
abstract = {The Limb-Girdle Muscular Dystrophies (LGMD) are genetically heterogeneous disorders, responsible for muscle wasting and severe form of dystrophies. Despite the critical developments in the insight and information of pathomechanisms of limb-girdle muscular dystrophy, any definitive treatments do not exist, and current strategies are only based on the improvement of the signs of disorder and to enhance the life quality without resolving an underlying cause. There is a crucial relationship between pharmacological therapy and different consequences; therefore, other treatment strategies will be required. New approaches, such as gene replacement, gene transfer, exon skipping, siRNA knockdown, and anti-myostatin therapy, which can target specific cellular or molecular mechanism of LGMD, could be a promising avenue for the treatment. Recently, genome engineering strategies with a focus on molecular tools such as CRISPR-Cas9 are used to different types of neuromuscular disorders and show the highest potential for clinical translation of these therapies. Thus, recent advancements and challenges in the field will be reviewed in this paper.},
}
@article {pmid32067153,
year = {2020},
author = {Gonzalez, TL and Hancock, M and Sun, S and Gersch, CL and Larios, JM and David, W and Hu, J and Hayes, DF and Wang, S and Rae, JM},
title = {Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer.},
journal = {Breast cancer research and treatment},
volume = {180},
number = {3},
pages = {611-622},
doi = {10.1007/s10549-020-05564-y},
pmid = {32067153},
issn = {1573-7217},
support = {T32ES007062/ES/NIEHS NIH HHS/United States ; },
mesh = {Breast Neoplasms/*drug therapy/genetics/*metabolism/pathology ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Estrogen Antagonists/*pharmacology ; Estrogen Receptor alpha/antagonists &amp; inhibitors/*genetics/metabolism ; Estrogens/*pharmacology ; Female ; Humans ; *Mutation ; Proteolysis ; Tumor Cells, Cultured ; },
abstract = {PURPOSE: Studies have identified several estrogen receptor α (ERα) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens.<br><br>METHODS: ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ER&#x3b1; via the ubiquitin-proteasome pathway.<br><br>RESULTS: Wild-type MCF-7 and ER&#x3b1; LBD mutant cells were treated with ERD-148 (10&#xa0;pM-1&#xa0;&#xb5;M) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1&#xa0;nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1&#xa0;nM ERD-148 for 24&#xa0;h exhibited reduced ER&#x3b1; protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24&#xa0;h of ERD-148 treatment at 10&#xa0;nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the ~&#x2009;IC90 observed in the ER-positive cells.<br><br>CONCLUSION: ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ER&#x3b1; with comparable potency to Fulvestrant with marginal non-specific toxicity.},
}
@article {pmid32066938,
year = {2020},
author = {Gutierrez-Arcelus, M and Baglaenko, Y and Arora, J and Hannes, S and Luo, Y and Amariuta, T and Teslovich, N and Rao, DA and Ermann, J and Jonsson, AH and , and Navarrete, C and Rich, SS and Taylor, KD and Rotter, JI and Gregersen, PK and Esko, T and Brenner, MB and Raychaudhuri, S},
title = {Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci.},
journal = {Nature genetics},
volume = {52},
number = {3},
pages = {247-253},
pmid = {32066938},
issn = {1546-1718},
support = {HHSN268201500003C/HL/NHLBI NIH HHS/United States ; U54 HG003067/HG/NHGRI NIH HHS/United States ; N01HC95163/HL/NHLBI NIH HHS/United States ; UL1 TR001079/TR/NCATS NIH HHS/United States ; N01HC95169/HL/NHLBI NIH HHS/United States ; U19 AI111224/AI/NIAID NIH HHS/United States ; N01HC95168/HL/NHLBI NIH HHS/United States ; P30 DK063491/DK/NIDDK NIH HHS/United States ; HHSN268201800001C/HL/NHLBI NIH HHS/United States ; N01HC95165/HL/NHLBI NIH HHS/United States ; N01HC95159/HL/NHLBI NIH HHS/United States ; UL1 TR000040/TR/NCATS NIH HHS/United States ; UL1 TR001881/TR/NCATS NIH HHS/United States ; HHSN268201000001I/HL/NHLBI NIH HHS/United States ; N01HC95160/HL/NHLBI NIH HHS/United States ; R01 HL120393/HL/NHLBI NIH HHS/United States ; T32 HG002295/HG/NHGRI NIH HHS/United States ; N01HC95164/HL/NHLBI NIH HHS/United States ; U01 GM092691/GM/NIGMS NIH HHS/United States ; N01HC95162/HL/NHLBI NIH HHS/United States ; N01HC95161/HL/NHLBI NIH HHS/United States ; UL1 TR001420/TR/NCATS NIH HHS/United States ; R01 AR063759/AR/NIAMS NIH HHS/United States ; HHSN268201500003I/HL/NHLBI NIH HHS/United States ; U01 HG009379/HG/NHGRI NIH HHS/United States ; N01HC95167/HL/NHLBI NIH HHS/United States ; UH2 AR067677/AR/NIAMS NIH HHS/United States ; R01 HL117626/HL/NHLBI NIH HHS/United States ; N01HC95166/HL/NHLBI NIH HHS/United States ; MR/R013926/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; Autoimmunity/*genetics ; CD4-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cell Line ; Gene Expression Regulation ; Genetic Loci ; *Genetic Variation ; Genotyping Techniques ; HLA Antigens/*genetics/metabolism ; HLA-DQ beta-Chains/*genetics/metabolism ; Humans ; Immunity, Cellular ; Lymphocyte Activation/*genetics ; Promoter Regions, Genetic/*genetics ; T-Lymphocytes, Regulatory ; },
abstract = {Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4[+] T cell regulatory elements[1-3]. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity[4,5]. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4[+] T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR-Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.},
}
@article {pmid32066906,
year = {2020},
author = {Li, X and Qian, X and Wang, B and Xia, Y and Zheng, Y and Du, L and Xu, D and Xing, D and DePinho, RA and Lu, Z},
title = {Programmable base editing of mutated TERT promoter inhibits brain tumour growth.},
journal = {Nature cell biology},
volume = {22},
number = {3},
pages = {282-288},
pmid = {32066906},
issn = {1476-4679},
support = {R01 CA084628/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Brain Neoplasms/*genetics/metabolism/pathology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cellular Senescence ; *Gene Editing ; Glioblastoma/*genetics/metabolism/pathology ; Humans ; Male ; Mice, Nude ; Middle Aged ; Mutation ; Promoter Regions, Genetic ; Telomerase/*genetics/metabolism ; Telomere Shortening ; Transcription Factors/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR interference and programmable base editing have transformed the manipulation of eukaryotic genomes for potential therapeutic applications[1-4]. Here, we exploited CRISPR interference and programmable base editing to determine their potential in editing a TERT gene promoter-activating mutation, which occurs in many diverse cancer types, particularly glioblastoma[5-8]. Correction of the -124C>T TERT promoter mutation to -124C was achieved using a single guide RNA (sgRNA)-guided and catalytically impaired Campylobacter jejuni CRISPR-associated protein 9-fused adenine base editor (CjABE). This modification blocked the binding of members of the E26 transcription factor family to the TERT promoter, reduced TERT transcription and TERT protein expression, and induced cancer-cell senescence and proliferative arrest. Local injection of adeno-associated viruses expressing sgRNA-guided CjABE inhibited the growth of gliomas harbouring TERT-promoter mutations. These preclinical proof-of-concept studies establish the feasibility of gene editing as a therapeutic approach for cancer and validate activated TERT-promoter mutations as a cancer-specific therapeutic target.},
}
@article {pmid32065582,
year = {2020},
author = {George, G and Ninagawa, S and Yagi, H and Saito, T and Ishikawa, T and Sakuma, T and Yamamoto, T and Imami, K and Ishihama, Y and Kato, K and Okada, T and Mori, K},
title = {EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32065582},
issn = {2050-084X},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carrier Proteins/*metabolism ; Catalysis ; *Endoplasmic Reticulum-Associated Degradation ; Gene Editing ; Gene Knockdown Techniques ; Glycoproteins/*metabolism ; HCT116 Cells ; Humans ; Mannose/*metabolism ; Mannosidases/metabolism ; Polymerase Chain Reaction ; alpha-Mannosidase/*metabolism ; },
abstract = {Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.},
}
@article {pmid32065238,
year = {2020},
author = {Bicak, M and Wang, X and Gao, X and Xu, X and Väänänen, RM and Taimen, P and Lilja, H and Pettersson, K and Klein, RJ},
title = {Prostate cancer risk SNP rs10993994 is a trans-eQTL for SNHG11 mediated through MSMB.},
journal = {Human molecular genetics},
volume = {29},
number = {10},
pages = {1581-1591},
pmid = {32065238},
issn = {1460-2083},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA092629/CA/NCI NIH HHS/United States ; S10 OD018522/OD/NIH HHS/United States ; S10 OD026880/OD/NIH HHS/United States ; R01 CA175491/CA/NCI NIH HHS/United States ; R03 CA165082/CA/NCI NIH HHS/United States ; U01 HG007033/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Expression Regulation/genetics ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Genotype ; Humans ; Male ; Polymorphism, Single Nucleotide/genetics ; Prostatic Neoplasms/*genetics/pathology ; Prostatic Secretory Proteins/*genetics ; Quantitative Trait Loci/genetics ; RNA, Long Noncoding/*genetics ; RNA, Untranslated/*genetics ; },
abstract = {How genome-wide association studies-identified single-nucleotide polymorphisms (SNPs) affect remote genes remains unknown. Expression quantitative trait locus (eQTL) association meta-analysis on 496 prostate tumor and 602 normal prostate samples with 117 SNPs revealed novel cis-eQTLs and trans-eQTLs. Mediation testing and colocalization analysis demonstrate that MSMB is a cis-acting mediator for SNHG11 (P < 0.01). Removing rs10993994 in LNCaP cell lines by CRISPR/Cas9 editing shows that the C-allele corresponds with an over 100-fold increase in MSMB expression and 5-fold increase in SNHG11 compared with the T-allele. Colocalization analysis confirmed that the same set of SNPs associated with MSMB expression is associated with SNHG11 expression (posterior probability of shared variants is 66.6% in tumor and 91.4% in benign). These analyses further demonstrate variants driving MSMB expression differ in tumor and normal, suggesting regulatory network rewiring during tumorigenesis.},
}
@article {pmid32065127,
year = {2020},
author = {Sinakevitch, I and Kurtzman, Z and Choi, HG and Ruiz Pardo, DA and Dahan, RA and Klein, N and Bugarija, B and Wendlandt, E and Smith, BH},
title = {Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {155},
pages = {},
doi = {10.3791/59993},
pmid = {32065127},
issn = {1940-087X},
support = {R01 GM113967/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Antibodies/*metabolism ; Bees/*metabolism ; Brain/*metabolism ; CRISPR-Cas Systems/*genetics ; Dieldrin/*metabolism ; Drosophila melanogaster/genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; Rabbits ; Receptors, Metabotropic Glutamate/*metabolism ; },
abstract = {Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.},
}
@article {pmid32064511,
year = {2020},
author = {DiNapoli, SE and Martinez-McFaline, R and Gribbin, CK and Wrighton, PJ and Balgobin, CA and Nelson, I and Leonard, A and Maskin, CR and Shwartz, A and Quenzer, ED and Mailhiot, D and Kao, C and McConnell, SC and de Jong, JLO and Goessling, W and Houvras, Y},
title = {Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.},
journal = {Nucleic acids research},
volume = {48},
number = {7},
pages = {e38},
pmid = {32064511},
issn = {1362-4962},
support = {TL1 TR002386/TR/NCATS NIH HHS/United States ; UL1 TR002384/TR/NCATS NIH HHS/United States ; F31 CA192813/CA/NCI NIH HHS/United States ; F31 CA213997/CA/NCI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; TL1 TR000459/TR/NCATS NIH HHS/United States ; F32 AA025271/AA/NIAAA NIH HHS/United States ; R01 DK090311/DK/NIDDK NIH HHS/United States ; R01 DK105198/DK/NIDDK NIH HHS/United States ; R24 OD017870/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Fluorescent Dyes ; *Gene Editing ; Green Fluorescent Proteins/genetics ; INDEL Mutation ; Indicators and Reagents ; Melanocytes ; Nitroreductases/genetics ; RNA/chemistry ; *Recombinational DNA Repair ; Templates, Genetic ; Zebrafish/embryology/genetics ; },
abstract = {CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.},
}
@article {pmid32063424,
year = {2020},
author = {Vona, B and Doll, J and Hofrichter, MAH and Haaf, T and Varshney, GK},
title = {Small fish, big prospects: using zebrafish to unravel the mechanisms of hereditary hearing loss.},
journal = {Hearing research},
volume = {397},
number = {},
pages = {107906},
pmid = {32063424},
issn = {1878-5891},
support = {P20 GM103636/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Hearing/genetics ; *Hearing Loss/genetics ; Humans ; Zebrafish/genetics ; },
abstract = {Over the past decade, advancements in high-throughput sequencing have greatly enhanced our knowledge of the mutational signatures responsible for hereditary hearing loss. In its present state, the field has a largely uncensored view of protein coding changes in a growing number of genes that have been associated with hereditary hearing loss, and many more that have been proposed as candidate genes. Sequencing data can now be generated using methods that have become widespread and affordable. The greatest hurdles facing the field concern functional validation of uncharacterized genes and rapid application to human diseases, including hearing and balance disorders. To date, over 30 hearing-related disease models exist in zebrafish. New genome editing technologies, including CRISPR/Cas9 will accelerate the functional validation of hearing loss genes and variants in zebrafish. Here, we discuss current progress in the field and recent advances in genome editing approaches.},
}
@article {pmid32061885,
year = {2020},
author = {Vega-Cuesta, P and Ruiz-Gómez, A and Molnar, C and Organista, MF and Resnik-Docampo, M and Falo-Sanjuan, J and López-Varea, A and de Celis, JF},
title = {Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.},
journal = {Developmental biology},
volume = {461},
number = {2},
pages = {172-183},
doi = {10.1016/j.ydbio.2020.02.009},
pmid = {32061885},
issn = {1095-564X},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/*physiology ; Drosophila melanogaster/genetics/*physiology ; ErbB Receptors ; Female ; Gene Editing ; Genetic Association Studies ; Insulin/*physiology ; Longevity/genetics ; Male ; Membrane Proteins/genetics/*physiology ; Phosphatidylinositol 3-Kinases/genetics/metabolism ; Protein Interaction Mapping ; Proto-Oncogene Proteins c-raf/genetics/physiology ; Receptor Protein-Tyrosine Kinases/physiology ; Receptors, Invertebrate Peptide ; Recombinant Fusion Proteins/metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Signal Transduction/physiology ; Wings, Animal/growth &amp; development/ultrastructure ; ras Proteins/genetics/*physiology ; },
abstract = {Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.},
}
@article {pmid32061775,
year = {2020},
author = {Zhang, J and Cui, WW and Du, C and Huang, Y and Pi, X and Guo, W and Wang, J and Huang, W and Chen, D and Li, J and Li, H and Zhang, J and Ma, Y and Mu, H and Zhang, S and Liu, M and Cui, X and Hu, Y},
title = {Knockout of DNase1l1l abrogates lens denucleation process and causes cataract in zebrafish.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1866},
number = {5},
pages = {165724},
doi = {10.1016/j.bbadis.2020.165724},
pmid = {32061775},
issn = {1879-260X},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Cataract/*genetics/pathology ; Cell Nucleus/metabolism ; Deoxyribonucleases/*genetics/metabolism ; Disease Models, Animal ; Embryo, Nonmammalian ; Epithelial Cells/cytology/metabolism ; Female ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Heat Shock Transcription Factors/genetics/*metabolism ; Humans ; Lens, Crystalline/cytology/*embryology/metabolism/pathology ; Male ; Zebrafish ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {Removal of nuclei in lens fiber cells is required for organelle-free zone (OFZ) formation during lens development. Defect in degradation of nuclear DNA leads to cataract formation. DNase2β degrades nuclear DNA of lens fiber cells during lens differentiation in mouse. Hsf4 is the principal heat shock transcription factor in lens and facilitates the lens differentiation. Knockout of Hsf4 in mouse and zebrafish resulted in lens developmental defect that was characterized by retaining of nuclei in lens fiber cells. In previous in vitro studies, we found that Hsf4 promoted DNase2β expression in human and mouse lens epithelial cells. In this study, it was found that, instead of DNase2β, DNase1l1l is uniquely expressed in zebrafish lens and was absent in Hsf4[-/-] zebrafish lens. Using CRISPR-Cas9 technology, a DNase1l1l knockout zebrafish line was constructed, which developed cataract. Deletion of DNase1l1l totally abrogated lens primary and secondary fiber cell denucleation process, whereas had little effect on the clearance of other organelles. The transcriptional regulation of DNase1l1l was dramatically impaired in Hsf4[-/-] zebrafish lens. Rescue of DNase1l1l mRNA into Hsf4[-/-] zebrafish embryos alleviated its defect in lens fiber cell denucleation. Our results in vivo demonstrated that DNase1l1l is the primary DNase responsible for nuclear DNA degradation in lens fiber cells, and Hsf4 can transcriptionally activate DNase1l1l expression in zebrafish.},
}
@article {pmid32061705,
year = {2020},
author = {Lee, J and Bayarsaikhan, D and Bayarsaikhan, G and Kim, JS and Schwarzbach, E and Lee, B},
title = {Recent advances in genome editing of stem cells for drug discovery and therapeutic application.},
journal = {Pharmacology &amp; therapeutics},
volume = {209},
number = {},
pages = {107501},
doi = {10.1016/j.pharmthera.2020.107501},
pmid = {32061705},
issn = {1879-016X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Drug Discovery/methods/*trends ; Gene Editing/methods/*trends ; Genetic Engineering/methods/*trends ; Genetic Therapy/methods/*trends ; Humans ; Stem Cell Transplantation/methods/*trends ; Stem Cells/physiology ; },
abstract = {Genome engineering technologies right from viral vector-mediated to protein-based editing- which include zinc finger nucleases, TALENs, and CRISPR/Cas systems-have been improved significantly. These technologies have facilitated drug discovery and have resulted in the development of potential curative therapies for many intractable diseases. They can efficiently correct genetic errors; however, these technologies have limitations, such as off-target effects and possible safety issues, which need to be considered when employing these techniques in humans. Significant efforts have been made to overcome these limitations and to accelerate the clinical implementation of these technologies. In this review, we focus on the recent technological advancements in genome engineering and their applications in stem cells to enable efficient discovery of drugs and treatment of intractable diseases.},
}
@article {pmid32061647,
year = {2020},
author = {Wang, M and Zhang, S and Shi, Y and Yang, Y and Wu, Y},
title = {Global gene expression changes induced by knockout of a protease gene cluster in Helicoverpa armigera with CRISPR/Cas9.},
journal = {Journal of insect physiology},
volume = {122},
number = {},
pages = {104023},
doi = {10.1016/j.jinsphys.2020.104023},
pmid = {32061647},
issn = {1879-1611},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Expression ; Gene Knockout Techniques/*methods ; Gene Silencing ; Genes, Insect ; Insect Proteins/genetics/metabolism ; Moths/*genetics/metabolism ; Multigene Family ; Serine Proteases/*genetics/metabolism ; Transcriptome ; Trypsin/genetics/metabolism ; },
abstract = {Helicoverpa armigera is one of the most serious agricultural insect pests of global importance. It is highly polyphagous and depends on digestive serine proteases to degrade proteins to peptides and to amino acids. H. armigera has evolved adaptive ability to compensate for the inhibition of plant defensive protease inhibitors (PIs) in its diet by overproduction of digestive enzymes. As far as we know, compensation for deletion of serine protease genes has not yet been studied in any herbivorous insect. In this study, we used CRISPR/Cas9 to knock out a cluster of 18 trypsin-like genes in H. armigera. Compared with the wild type SCD strain, activities of the total proteases, trypsins and chymotrypsins were not significantly changed in the gene cluster knockout strain (Tryp-KO). RNA-seq data showed 1492 upregulated and 461 downregulated DEGs in Try-KO. GO function classification and KEGG pathway analyses revealed these differentially expressed genes were enriched for terms related to binding, catalytic activity, metabolic process and signal transduction. In regard to serine protease genes, 35 were upregulated and 12 downregulated in Tryp-KO strain. Our study indicated that H. armigera can compensate for the deleted protease genes by overexpression of other trypsin and chymotrypsin genes in order to maintain its genetic and metabolic robustness. It also suggests that genetic perturbations created by genome editing tools can induce global gene expression changes.},
}
@article {pmid32061586,
year = {2020},
author = {Yoo, S and Nair, S and Kim, HJ and Kim, Y and Lee, C and Lee, G and Park, JH},
title = {Knock-in mutations of scarecrow, a Drosophila homolog of mammalian Nkx2.1, reveal a novel function required for development of the optic lobe in Drosophila melanogaster.},
journal = {Developmental biology},
volume = {461},
number = {2},
pages = {145-159},
doi = {10.1016/j.ydbio.2020.02.008},
pmid = {32061586},
issn = {1095-564X},
mesh = {Alleles ; Animals ; Brain/growth &amp; development ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/*physiology ; Drosophila melanogaster/embryology/*genetics/growth &amp; development ; Embryo, Nonmammalian ; Exons/genetics ; Gene Editing ; Gene Expression Regulation, Developmental ; Gene Knock-In Techniques ; Genes, Reporter ; Homeodomain Proteins/genetics/*physiology ; Intracellular Signaling Peptides and Proteins/physiology ; Larva ; Membrane Proteins/physiology ; Mutation ; Nerve Tissue Proteins/genetics/metabolism ; Optic Lobe, Nonmammalian/*embryology/growth &amp; development ; Receptors, Notch/physiology ; },
abstract = {scarecrow (scro) gene encodes a Drosophila homolog of mammalian Nkx2.1 that belongs to an evolutionally conserved NK2 family. Nkx2.1 has been well known for its role in the development of hypothalamus, lung, thyroid gland, and brain. However, little is known about biological roles of scro. To understand scro functions, we generated two types of knock-in mutant alleles, substituting part of either exon-2 or exon-3 for EGFP (or Gal4) by employing the CRISPR/Cas9 genome editing tool. Using these mutations, we characterized spatio-temporal expression patterns of the scro gene and its mutant phenotypes. Homozygous knock-in mutants are lethal during embryonic and early larval development. In developing embryos, scro is exclusively expressed in the pharyngeal primordia and numerous neural clusters in the central nervous system (CNS). In postembryonic stages, the most prominent scro expression is detected in the larval and adult optic lobes, suggesting that scro plays a role for the development and/or function of this tissue type. Notch signaling is the earliest factor known to act for the development of the optic lobe. scro mutants lacked mitotic cells and Delta expression in the optic anlagen, and showed altered expression of several proneural and neurogenic genes including Delta and Notch. Furthermore, scro mutants showed grossly deformed neuroepithelial (NE) cells in the developing optic lobe and severely malformed adult optic lobes, the phenotypes of which are shown in Notch or Delta mutants, suggesting scro acting epistatic to the Notch signaling. From these data together, we propose that scro plays an essential role for the development of the optic lobe, possibly acting as a regional specification factor.},
}
@article {pmid32061011,
year = {2020},
author = {Zhang, Y and Ran, Y and Nagy, I and Lenk, I and Qiu, JL and Asp, T and Jensen, CS and Gao, C},
title = {Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {9},
pages = {1854-1856},
pmid = {32061011},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Lolium/genetics ; Mutagenesis/genetics ; },
}
@article {pmid32060484,
year = {2020},
author = {Mäkelä, J and Sherratt, DJ},
title = {Catching an invader.},
journal = {Nature reviews. Microbiology},
volume = {18},
number = {4},
pages = {194},
pmid = {32060484},
issn = {1740-1534},
support = {200782/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; DNA, Viral ; Escherichia coli/metabolism/virology ; Streptococcus pyogenes/metabolism/virology ; },
}
@article {pmid32058903,
year = {2020},
author = {Chang, KS and Kim, J and Park, H and Hong, SJ and Lee, CG and Jin, E},
title = {Enhanced lipid productivity in AGP knockout marine microalga Tetraselmis sp. using a DNA-free CRISPR-Cas9 RNP method.},
journal = {Bioresource technology},
volume = {303},
number = {},
pages = {122932},
doi = {10.1016/j.biortech.2020.122932},
pmid = {32058903},
issn = {1873-2976},
mesh = {CRISPR-Cas Systems ; Glucose-1-Phosphate Adenylyltransferase ; Lipids ; *Microalgae ; Ribonucleoproteins ; },
abstract = {A marine green microalga, Tetraselmis sp., has been studied for the production of biomass and lipids in seawater culture. Since carbohydrate and lipid biosynthesis are competitive metabolic pathways, we attempted to increase lipid synthesis in Tetraselmis by inhibiting carbohydrate synthesis. The main regulatory enzyme in the starch synthesis pathway is ADP-glucose pyrophosphorylase (AGP). AGP loss-of-function mutants were developed using the CRISPR-Cas9 ribonucleoprotein (RNP) delivery system. AGP mutants showed a slight decrease in growth. However, the lipid content in two AGP mutants was significantly enhanced by 2.7 and 3.1 fold (21.1% and 24.1% of DCW), respectively, compared to that in the wild type (7.68% of DCW) under nitrogen starvation. This study is an example of metabolic engineering by genetic editing using the CRISPR-Cas9 RNP method in marine green microalgae. Consequently, starchless Tetraselmis mutants might be considered potential producers of lipids in seawater cultures.},
}
@article {pmid32058568,
year = {2020},
author = {Wu, M and Hu, N and Du, X and Wei, J},
title = {Application of CRISPR/Cas9 technology in sepsis research.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {229-234},
doi = {10.1093/bfgp/elz040},
pmid = {32058568},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Gene Transfer Techniques ; Humans ; Sepsis/*genetics ; },
abstract = {CRISPR/Cas9, as a new genome-editing tool, offers new approaches to understand and treat diseases, which is being rapidly applied in various areas of biomedical research including sepsis field. The type II prokaryotic CRISPR/Cas system uses a single-guide RNA (sgRNA) to target the Cas9 nuclease to a specific genomic sequence, which is introduced into disease models for functional characterization and for testing of therapeutic strategies. This incredibly precise technology can be used for therapeutic research of gene-related diseases and to program any sequence in a target cell. Most importantly, the multifunctional capacity of this technology allows simultaneous editing of several genes. In this review, we focus on the basic principles, advantages and limitations of CRISPR/Cas9 and the use of the CRISPR/Cas9 system as a powerful tool in sepsis research and as a new strategy for the treatment of sepsis.},
}
@article {pmid32057858,
year = {2020},
author = {Sun, Y and Liu, M and Yan, C and Yang, H and Wu, Z and Liu, Y and Su, N and Hou, J and Zhang, J and Yang, F and Zhang, J},
title = {CRISPR/Cas9-mediated deletion of β, β-carotene 9', 10'-oxygenase gene (EcBCO2) from Exopalaemon carinicauda.},
journal = {International journal of biological macromolecules},
volume = {151},
number = {},
pages = {168-177},
doi = {10.1016/j.ijbiomac.2020.02.073},
pmid = {32057858},
issn = {1879-0003},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Computational Biology/methods ; *Gene Deletion ; Gene Expression ; Gene Expression Profiling ; Gene Knockdown Techniques ; Gene Targeting ; Oxygenases/*genetics ; Palaemonidae/*genetics ; Phylogeny ; RNA, Guide ; RNA, Messenger/genetics ; },
abstract = {CRISPR/Cas9 technology is an efficient genome editing tool for producing genetically modified animals. Carotenoids color the world around us and their accumulation in animals could be used to culture colorful new verities in animal breeding. β, β-carotene 9', 10'-oxygenase (BCO2) is an important enzyme during β-carotene metabolism. In this research, one full-length cDNA sequence encoding BCO2 (named EcBCO2) were obtained from Exopalaemon carinicauda. The genomic structure analysis showed that EcBCO2 gene was composed of 9 exons and 8 introns. Then, the CRISPR/Cas9-mediated deletion of EcBCO2 gene was generated by co-microinjection of Cas9 mRNA and EcBCO2 sgRNA into one-cell stage embryos of E. carinicauda. Subsequently, the phenotype of EcBCO2-KO prawns was compared with that of wild-type prawns, which showed that EcBCO2-KO resulted in the color change in the hepatopancreas of prawns. In addition, the EcBCO2-KO prawns had a higher survival rate than wild-type prawns when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. These results indicate that BCO2 gene could be used as a candidate gene in molecular marker-assistant breeding of prawns.},
}
@article {pmid32057312,
year = {2020},
author = {Gough, G and O'Brien, NL and Alic, I and Goh, PA and Yeap, YJ and Groet, J and Nizetic, D and Murray, A},
title = {Modeling Down syndrome in cells: From stem cells to organoids.},
journal = {Progress in brain research},
volume = {251},
number = {},
pages = {55-90},
doi = {10.1016/bs.pbr.2019.10.003},
pmid = {32057312},
issn = {1875-7855},
support = {098330/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Down Syndrome ; Humans ; *Models, Biological ; *Organoids ; *Pluripotent Stem Cells ; },
abstract = {Down Syndrome (DS) is a complex chromosomal disorder, with neurological issues, featuring among the symptoms. Primary neuronal cells and tissues are extremely useful, but limited both in supply and experimental manipulability. To better understand the cellular, molecular and pathological mechanisms involved in DS neurodevelopment and neurodegeneration, a range of different cellular models have been developed over the years including human: mouse hybrid cells, transchromosomic mouse embryonic stem cells (ESCs) and human ESC and induced pluripotent stem cells derived from different sources. All of these model systems have provided useful information in the study of DS. Furthermore, different technologies to genetically modify or correct trisomy of either single genes or the whole chromosome have been developed using these cellular models. New techniques and protocols to allow better modeling of cellular mechanisms and disease processes are being developed and the use of cerebral organoids offers great promise for future research into the neural phenotypes seen in DS.},
}
@article {pmid32057173,
year = {2020},
author = {Miao, J and Liu, X and Li, G and Du, X and Liu, X},
title = {Multiple point mutations in PsORP1 gene conferring different resistance levels to oxathiapiprolin confirmed using CRISPR-Cas9 in Phytophthora sojae.},
journal = {Pest management science},
volume = {76},
number = {7},
pages = {2434-2440},
doi = {10.1002/ps.5784},
pmid = {32057173},
issn = {1526-4998},
mesh = {CRISPR-Cas Systems ; Hydrocarbons, Fluorinated ; *Phytophthora ; Plant Diseases ; *Point Mutation ; Pyrazoles ; Soybeans ; },
abstract = {BACKGROUND: Oxathiapiprolin is among the first commercial oxysterol-binding protein inhibitors (OSBPIs) developed by DuPont Corporation and shows excellent activity against plant-pathogenic oomycetes. Although more than 21 target site mutations have been identified in insensitive oomycetes, only G770V, G839W, and ΔN837 have been verified to confer oxathiapiprolin resistance in Phytophthora capsici or P. sojae. The effect of other mutations on OSBPIs sensitivity requires urgent investigation.<br><br>RESULTS: P. sojae transformants containing 16 mutations of PsORP1 were recovered using the CRISPR-Cas9 system. Transformants containing L733W, S768F, S768Y, N837Y, N837F, P861H, L863W, or I877Y showed high oxathiapiprolin resistance, with resistant factors (RFs) >&#x2009;3000. Point mutations S768K, S768I, G770L, G770P, G770A, &#x394;G818/F819, N837I, and I877F exhibited low resistance, with RFs&#x2009;<&#x2009;80. Phenotype assays revealed that the most highly resistant transformants showed enhanced or similar pathogenicity, oospore production, and cyst gemination. However, most transformants displayed decreased sporangia and zoospore production compared with parental wild-type P6497.<br><br>CONCLUSION: This study demonstrated that L733W, S768F, S768Y, N837Y, N837F, P861H, L863W, and I877Y in PsORP1 confer high oxathiapiprolin resistance in P. sojae.},
}
@article {pmid32056963,
year = {2020},
author = {He, Q and Yu, D and Bao, M and Korensky, G and Chen, J and Shin, M and Kim, J and Park, M and Qin, P and Du, K},
title = {High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system.},
journal = {Biosensors &amp; bioelectronics},
volume = {154},
number = {},
pages = {112068},
doi = {10.1016/j.bios.2020.112068},
pmid = {32056963},
issn = {1873-4235},
mesh = {African Swine Fever/*diagnosis/virology ; African Swine Fever Virus/*isolation &amp; purification/pathogenicity ; Animals ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/chemistry ; Fluorescence ; Point-of-Care Systems ; Swine ; Viral Proteins/chemistry/*isolation &amp; purification ; },
abstract = {Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.},
}
@article {pmid32056365,
year = {2020},
author = {Zuo, Y and Xue, Y and Lu, W and Ma, H and Chen, M and Wu, Y and Yang, Y and Hu, Z},
title = {Functional validation of nicotinic acetylcholine receptor (nAChR) α6 as a target of spinosyns in Spodoptera exigua utilizing the CRISPR/Cas9 system.},
journal = {Pest management science},
volume = {76},
number = {7},
pages = {2415-2422},
doi = {10.1002/ps.5782},
pmid = {32056365},
issn = {1526-4998},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Insecticide Resistance ; Insecticides ; Macrolides ; Receptors, Nicotinic ; *Spodoptera ; },
abstract = {BACKGROUND: The beet armyworm, Spodoptera exigua, is a serious agricultural pest that is primarily controlled using chemical insecticides. Recently, resistance to the insecticide spinosad has been described in S. exigua field populations. To date, there has been no functional evidence proving the involvement of the nicotinic acetylcholine receptor (nAChR) α6 mutation in spinosad resistance in S. exigua.<br><br>RESULTS: In this study, using the CRISPR/Cas9 genome-editing system, a homozygous strain (Se&#x3b1;6-KO) with approximately 1760-bp deletion within Se&#x3b1;6 in S. exigua causing a premature truncation of Se&#x3b1;6 was successfully constructed. Insecticide bioassays showed that Se&#x3b1;6-KO exhibited 373-fold higher resistance to spinosad and 850-fold higher resistance to spinetoram compared to WH-S strain with the same genetic background but showed no significant change in susceptibility to emamectin benzoate and chlorantraniliprole. Genetic analysis revealed that Se&#x3b1;6-KO is inherited as an incompletely recessive trait.<br><br>CONCLUSION: The results clearly demonstrated the functional role of Se&#x3b1;6 in resistance to spinosyn insecticides and provide an example of using genome editing to verify a target premature truncation associated with resistance.},
}
@article {pmid32056283,
year = {2020},
author = {Yang, N and Tian, J and Wang, X and Mei, S and Zou, D and Peng, X and Zhu, Y and Yang, Y and Gong, Y and Ke, J and Zhong, R and Chang, J and Miao, X},
title = {A functional variant in TNXB promoter associates with the risk of esophageal squamous-cell carcinoma.},
journal = {Molecular carcinogenesis},
volume = {59},
number = {4},
pages = {439-446},
doi = {10.1002/mc.23166},
pmid = {32056283},
issn = {1098-2744},
mesh = {CRISPR-Cas Systems ; Carcinoma, Squamous Cell/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Esophageal Neoplasms/*genetics/pathology ; Gene Expression Profiling/methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Genetic Predisposition to Disease/*genetics ; Humans ; *Polymorphism, Single Nucleotide ; Promoter Regions, Genetic/*genetics ; RNA Interference ; Risk Factors ; Tenascin/*genetics ; },
abstract = {Our previous study identified a tag single-nucleotide polymorphism (SNP) rs204900 in TNXB associated with risk of esophageal squamous-cell carcinoma (ESCC) in the Chinese population. However, the functional role of TNXB and causal variants had not been interrogated in that study. In the present study, we explored the effects of TNXB expression in the development of ESCC and searched for functional variants in this gene. We found TNXB was downregulated in ESCC tumors. Using small interfering RNAs and CRISPR-Cas9 methods, we identified that both knockdown and knockout of TNXB significantly promoted ESCC cell growth in vitro, suggesting a tumor suppressor role of this gene in ESCC. Through further fine-mapping analysis, we identified that a noncoding variant in the promoter of TNXB, rs411337, predisposed to ESCC risk (odds ratio = 1.36, 95% confidence interval: 1.22-1.51, P = 9.10 × 10[-9]). These findings revealed the functional mechanism of TNXB in the development of ESCC and may contribute to the prevention and treatment of this disease in the future.},
}
@article {pmid32054870,
year = {2020},
author = {Hirotsune, S and Kiyonari, H and Jin, M and Kumamoto, K and Yoshida, K and Shinohara, M and Watanabe, H and Wynshaw-Boris, A and Matsuzaki, F},
title = {Enhanced homologous recombination by the modulation of targeting vector ends.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2518},
pmid = {32054870},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Breaks, Double-Stranded ; Female ; *Gene Editing/methods ; *Gene Targeting/methods ; Genetic Vectors/genetics ; *Homologous Recombination ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Recombinational DNA Repair ; },
abstract = {The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.},
}
@article {pmid32054864,
year = {2020},
author = {Wang, Y and Maeda, Y and Liu, YS and Takada, Y and Ninomiya, A and Hirata, T and Fujita, M and Murakami, Y and Kinoshita, T},
title = {Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {860},
pmid = {32054864},
issn = {2041-1723},
mesh = {Acyltransferases/deficiency/genetics/metabolism ; CRISPR-Cas Systems ; *Endoplasmic Reticulum-Associated Degradation/genetics ; Galactosyltransferases/deficiency/genetics/metabolism ; Gene Knockout Techniques ; Glycosphingolipids/*biosynthesis/genetics ; Glycosylphosphatidylinositols/*biosynthesis/genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Models, Molecular ; Recombinant Fusion Proteins/chemistry/genetics/metabolism ; },
abstract = {Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.},
}
@article {pmid32053781,
year = {2020},
author = {Croteau, FR and Hynes, AP},
title = {In the Anti-CRISPR Jungle, Only the Weak Thrive?.},
journal = {Cell host &amp; microbe},
volume = {27},
number = {2},
pages = {157-159},
doi = {10.1016/j.chom.2020.01.019},
pmid = {32053781},
issn = {1934-6069},
mesh = {Bacteria ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cooperative Behavior ; },
abstract = {If, as we all know, only the strong survive, why do bacterial viruses (phages) encode weak suppressors of a bacterial immune system? In this issue of Cell Host &amp; Microbe, Chevallereau et al. (2019) expertly demonstrate how, in the context of competition with other phages, weakness can be a strength.},
}
@article {pmid32053779,
year = {2020},
author = {White, CW and Caspar, B and Vanyai, HK and Pfleger, KDG and Hill, SJ},
title = {CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.},
journal = {Cell chemical biology},
volume = {27},
number = {5},
pages = {499-510.e7},
pmid = {32053779},
issn = {2451-9448},
support = {MR/N020081/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*Bioluminescence Resonance Energy Transfer Techniques ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; HEK293 Cells ; Humans ; Ligands ; Luciferases/analysis/genetics/metabolism ; Luminescent Proteins/analysis/genetics/metabolism ; Protein Binding ; Protein Conformation ; Receptors, CXCR/analysis/genetics/*metabolism ; Receptors, CXCR4/analysis/genetics/*metabolism ; beta-Arrestins/analysis/genetics/metabolism ; },
abstract = {G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.},
}
@article {pmid32053639,
year = {2020},
author = {Jin, J and Xu, Y and Huo, L and Ma, L and Scott, AW and Pizzi, MP and Li, Y and Wang, Y and Yao, X and Song, S and Ajani, JA},
title = {An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue.},
journal = {PloS one},
volume = {15},
number = {2},
pages = {e0228910},
pmid = {32053639},
issn = {1932-6203},
support = {R01 CA160433/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R21 CA129906/CA/NCI NIH HHS/United States ; R21 CA127672/CA/NCI NIH HHS/United States ; R01 CA172741/CA/NCI NIH HHS/United States ; R01 CA138671/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fluorescent Dyes ; Gene Editing ; Gene Knockout Techniques/*methods ; Genetic Engineering/*methods ; Genetic Vectors ; Humans ; Lentivirus/genetics ; Plasmids ; RNA, Guide/genetics ; },
abstract = {A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.},
}
@article {pmid32053368,
year = {2020},
author = {Scheidler, CM and Vrabel, M and Schneider, S},
title = {Genetic Code Expansion, Protein Expression, and Protein Functionalization in Bacillus subtilis.},
journal = {ACS synthetic biology},
volume = {9},
number = {3},
pages = {486-493},
doi = {10.1021/acssynbio.9b00458},
pmid = {32053368},
issn = {2161-5063},
mesh = {Bacillus subtilis/*genetics/metabolism ; CRISPR-Cas Systems ; Click Chemistry ; Creatine Kinase, MM Form/metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Bacterial/drug effects ; Genetic Code ; Genetic Vectors ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Isopropyl Thiogalactoside/pharmacology ; Lysine/chemistry ; Norbornanes/chemistry ; Protein Engineering/*methods ; Recombinant Proteins/chemistry/genetics/isolation &amp; purification/*metabolism ; },
abstract = {The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry. We used this strategy to site-specifically introduce norbornene lysine into a single chain antibody and functionalize it with fluorophores for the detection of human target proteins.},
}
@article {pmid32053108,
year = {2020},
author = {Port, F and Strein, C and Stricker, M and Rauscher, B and Heigwer, F and Zhou, J and Beyersdörffer, C and Frei, J and Hess, A and Kern, K and Lange, L and Langner, N and Malamud, R and Pavlović, B and Rädecke, K and Schmitt, L and Voos, L and Valentini, E and Boutros, M},
title = {A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {32053108},
issn = {2050-084X},
support = {DECODE//European Research Council/International ; Decode//European Research Council/International ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Drosophila melanogaster/*genetics ; Gene Editing/*methods ; RNA/genetics ; },
abstract = {Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.},
}
@article {pmid32052827,
year = {2020},
author = {Rossi, CC and Pereira, MF and Giambiagi-deMarval, M},
title = {Underrated Staphylococcus species and their role in antimicrobial resistance spreading.},
journal = {Genetics and molecular biology},
volume = {43},
number = {1 suppl 2},
pages = {e20190065},
pmid = {32052827},
issn = {1415-4757},
abstract = {The increasing threat of antimicrobial resistance has shed light on the interconnection between humans, animals, the environment, and their roles in the exchange and spreading of resistance genes. In this review, we present evidences that show that Staphylococcus species, usually referred to as harmless or opportunistic pathogens, represent a threat to human and animal health for acting as reservoirs of antimicrobial resistance genes. The capacity of genetic exchange between isolates of different sources and species of the Staphylococcus genus is discussed with emphasis on mobile genetic elements, the contribution of biofilm formation, and evidences obtained either experimentally or through genome analyses. We also discuss the involvement of CRISPR-Cas systems in the limitation of horizontal gene transfer and its suitability as a molecular clock to describe the history of genetic exchange between staphylococci.},
}
@article {pmid32052006,
year = {2020},
author = {Cheng, X and Fan, S and Wen, C and Du, X},
title = {CRISPR/Cas9 for cancer treatment: technology, clinical applications and challenges.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {209-214},
doi = {10.1093/bfgp/elaa001},
pmid = {32052006},
issn = {2041-2657},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genetic Therapy/methods ; Humans ; Neoplasms/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is described as RNA mediated adaptive immune system defense, which is naturally found in bacteria and archaea. CRISPR-Cas9 has shown great promise for cancer treatment in cancer immunotherapy, manipulation of cancer genome and epigenome and elimination or inactivation of carcinogenic viral infections. However, many challenges remain to be addressed to increase its efficacy, including off-target effects, editing efficiency, fitness of edited cells, immune response and delivery methods. Here, we explain CRISPR-Cas classification and its general function mechanism for gene editing. Then, we summarize these preclinical CRISPR-Cas9-based therapeutic strategies against cancer. Moreover, the challenges and improvements of CRISPR-Cas9 clinical applications will be discussed.},
}
@article {pmid32051598,
year = {2020},
author = {Doudna, JA},
title = {The promise and challenge of therapeutic genome editing.},
journal = {Nature},
volume = {578},
number = {7794},
pages = {229-236},
pmid = {32051598},
issn = {1476-4687},
support = {/HHMI/Howard Hughes Medical Institute/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*genetics/*therapy ; CRISPR-Cas Systems/genetics ; Gene Editing/ethics/*methods/standards/*trends ; Genome, Human/*genetics ; Germ-Line Mutation/genetics ; Humans ; Muscular Dystrophy, Duchenne/*genetics/*therapy ; Organ Specificity/genetics ; Patient Safety ; beta-Globins/genetics ; },
abstract = {Genome editing, which involves the precise manipulation of cellular DNA sequences to alter cell fates and organism traits, has the potential to both improve our understanding of human genetics and cure genetic disease. Here I discuss the scientific, technical and ethical aspects of using CRISPR (clustered regularly interspaced short palindromic repeats) technology for therapeutic applications in humans, focusing on specific examples that highlight both opportunities and challenges. Genome editing is-or will soon be-in the clinic for several diseases, with more applications under development. The rapid pace of the field demands active efforts to ensure that this breakthrough technology is used responsibly to treat, cure and prevent genetic disease.},
}
@article {pmid32051592,
year = {2020},
author = {Al-Shayeb, B and Sachdeva, R and Chen, LX and Ward, F and Munk, P and Devoto, A and Castelle, CJ and Olm, MR and Bouma-Gregson, K and Amano, Y and He, C and Méheust, R and Brooks, B and Thomas, A and Lavy, A and Matheus-Carnevali, P and Sun, C and Goltsman, DSA and Borton, MA and Sharrar, A and Jaffe, AL and Nelson, TC and Kantor, R and Keren, R and Lane, KR and Farag, IF and Lei, S and Finstad, K and Amundson, R and Anantharaman, K and Zhou, J and Probst, AJ and Power, ME and Tringe, SG and Li, WJ and Wrighton, K and Harrison, S and Morowitz, M and Relman, DA and Doudna, JA and Lehours, AC and Warren, L and Cate, JHD and Santini, JM and Banfield, JF},
title = {Clades of huge phages from across Earth's ecosystems.},
journal = {Nature},
volume = {578},
number = {7795},
pages = {425-431},
pmid = {32051592},
issn = {1476-4687},
support = {R01 AI092531/AI/NIAID NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acyl-tRNA Synthetases/genetics ; Animals ; Bacteria/genetics/*virology ; Bacteriophages/*classification/*genetics/isolation &amp; purification/metabolism ; Biodiversity ; CRISPR-Cas Systems/genetics ; *Earth, Planet ; *Ecosystem ; Evolution, Molecular ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Viral ; Genome, Viral/*genetics ; Host Specificity ; Humans ; Lakes/virology ; Molecular Sequence Annotation ; Oceans and Seas ; *Phylogeny ; Prophages/genetics ; Protein Biosynthesis ; RNA, Transfer/genetics ; Ribosomal Proteins/genetics ; Seawater/virology ; Soil Microbiology ; Transcription, Genetic ; },
abstract = {Bacteriophages typically have small genomes[1] and depend on their bacterial hosts for replication[2]. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.},
}
@article {pmid32051236,
year = {2020},
author = {Spoto, M and Guan, C and Fleming, E and Oh, J},
title = {A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes.},
journal = {mSphere},
volume = {5},
number = {1},
pages = {},
pmid = {32051236},
issn = {2379-5042},
support = {DP2 GM126893/GM/NIGMS NIH HHS/United States ; F30 DE027870/DE/NIDCR NIH HHS/United States ; R01 AR078634/AR/NIAMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*instrumentation/methods ; Genome, Bacterial/*genetics ; Programming Languages ; RNA, Guide/genetics ; *Software ; },
abstract = {The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful-focusing on CRISPRi as a potential application-as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species.IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.},
}
@article {pmid32050613,
year = {2020},
author = {Grigonyte, AM and Harrison, C and MacDonald, PR and Montero-Blay, A and Tridgett, M and Duncan, J and Sagona, AP and Constantinidou, C and Jaramillo, A and Millard, A},
title = {Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7.},
journal = {Viruses},
volume = {12},
number = {2},
pages = {},
pmid = {32050613},
issn = {1999-4915},
support = {BB/M017982/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/L015080/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Bacteriophage T7/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Genetic Markers ; Genome, Viral ; *Mutation ; Viral Tail Proteins/genetics ; },
abstract = {With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.},
}
@article {pmid32050051,
year = {2020},
author = {Hou, C and Yang, Y and Xing, Y and Zhan, C and Liu, G and Liu, X and Liu, C and Zhan, J and Xu, D and Bai, Z},
title = {Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.},
journal = {Yeast (Chichester, England)},
volume = {37},
number = {4},
pages = {305-312},
doi = {10.1002/yea.3462},
pmid = {32050051},
issn = {1097-0061},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Culture Media/chemistry ; Fungal Proteins/*genetics/metabolism ; Gene Editing ; Gene Expression Regulation, Fungal ; Methanol/metabolism ; Pichia/*genetics/metabolism ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide ; Transcription Factors ; },
abstract = {A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.},
}
@article {pmid32048998,
year = {2020},
author = {Tuck, AC and Rankova, A and Arpat, AB and Liechti, LA and Hess, D and Iesmantavicius, V and Castelo-Szekely, V and Gatfield, D and Bühler, M},
title = {Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.},
journal = {Molecular cell},
volume = {77},
number = {6},
pages = {1222-1236.e13},
pmid = {32048998},
issn = {1097-4164},
support = {103977/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Apoptosis Regulatory Proteins/*physiology ; CRISPR-Cas Systems ; DNA-Binding Proteins/*physiology ; Exoribonucleases/*physiology ; Gene Expression Regulation ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Open Reading Frames ; *Protein Biosynthesis ; Proto-Oncogene Proteins/physiology ; RNA Helicases/*physiology ; *RNA Stability ; RNA, Messenger/chemistry/genetics/*metabolism ; Ribosomes/genetics/metabolism ; },
abstract = {RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.},
}
@article {pmid32048826,
year = {2020},
author = {Peng, H and Le, C and Wu, J and Li, XF and Zhang, H and Le, XC},
title = {A Genome-Editing Nanomachine Constructed with a Clustered Regularly Interspaced Short Palindromic Repeats System and Activated by Near-Infrared Illumination.},
journal = {ACS nano},
volume = {14},
number = {3},
pages = {2817-2826},
doi = {10.1021/acsnano.9b05276},
pmid = {32048826},
issn = {1936-086X},
support = {/CAPMC/CIHR/Canada ; },
mesh = {A549 Cells ; Apoptosis/genetics ; CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/genetics ; Cell Survival/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; ErbB Receptors/genetics ; *Gene Editing ; HEK293 Cells ; Homeodomain Proteins/genetics ; Humans ; Infrared Rays ; *Nanomedicine ; Protein Serine-Threonine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; Transcription Factors/genetics ; },
abstract = {The RNA-guided CRISPR/Cas9 system is a powerful genome-editing technology with broad applications. Improving delivery efficiency and controllable activity of the CRISPR/Cas9 system is an area of intense research. We report the design, construction, and application of a CRISPR/Cas9 nanomachine (LACM), activated by a near-infrared (NIR) laser, which enables efficient delivery of single-guide RNA (sgRNA) into living cells and achieves controlled release of the sgRNA for the CRISPR/Cas9 activity. The LACM was constructed using a gold nanorod (AuNR) as a carrier that was decorated with dozens of protector DNAs stably hybridizing with the target binding domain of sgRNA. The DNA assembly on the AuNR protected the sgRNA. Irradiation with a NIR laser generated heat on the AuNR, resulting in controlled release of sgRNA, which guided the CRISPR/Cas9 genome editing. Successful editing of the EGFP and EMX1 genes in A549 and HEK293T cells, as well as knocking down of the PLK1 gene to induce apoptosis of the target cells, highlights the promising potential of the LACM for diverse applications.},
}
@article {pmid32047240,
year = {2020},
author = {Wang, X and Ji, P and Fan, H and Dang, L and Wan, W and Liu, S and Li, Y and Yu, W and Li, X and Ma, X and Ma, X and Zhao, Q and Huang, X and Liao, M},
title = {CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {62},
pmid = {32047240},
issn = {2399-3642},
mesh = {African Swine Fever/*diagnosis/virology ; African Swine Fever Virus/*genetics ; Animals ; *CRISPR-Cas Systems ; DNA, Viral ; Genome, Viral ; *Immunoassay ; Molecular Diagnostic Techniques/methods/standards ; *Reagent Strips ; Reproducibility of Results ; Sensitivity and Specificity ; Swine ; },
abstract = {African swine fever virus (ASFV), the aetiological agent of African swine fever (ASF), causes lethal haemorrhagic fever in domestic pigs with high mortality and morbidity and has devastating consequences on the global swine industry. On-site rapid and sensitive detection of ASFV is key to the timely implementation of control. In this study, we developed a rapid, sensitive and instrument-free ASFV detection method based on CRISPR/Cas12a technology and lateral flow detection (named CRISPR/Cas12a-LFD). The limit of detection of CRISPR/Cas12a-LFD is 20 copies of ASFV genomic DNA per reaction, and the detection process can be completed in an hour. The assay showed no cross-reactivity with other swine DNA viruses, and has 100% agreement with real-time PCR detection of ASFV in 149 clinical samples. Overall, the CRISPR/Cas12a-LFD method provides a novel alternative for the portable, simple, sensitive, and specific detection of ASFV and may contribute to the prevention and control of ASF outbreaks.},
}
@article {pmid32046842,
year = {2020},
author = {Wei, CT and Maly, DJ and Fowler, DM},
title = {Temporal and rheostatic control of genome editing with a chemically-inducible Cas9.},
journal = {Methods in enzymology},
volume = {633},
number = {},
pages = {119-141},
doi = {10.1016/bs.mie.2019.11.001},
pmid = {32046842},
issn = {1557-7988},
support = {R01 GM086858/GM/NIGMS NIH HHS/United States ; R01 GM109110/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA Cleavage ; Endonucleases/genetics ; *Gene Editing ; },
abstract = {Nuclease-mediated DNA cleavage and subsequent repair lie at the heart of genome editing, and the RNA-guided endonuclease Cas9 has emerged as the most widely-used tool for facilitating this process. Extensive biochemical and biophysical efforts have revealed much regarding the structure, mechanism, and cellular properties of Cas9. This has enabled engineering of Cas9 variants with enhanced activity, specificity, and other features. However, we lack a detailed understanding of the kinetics of Cas9-mediated DNA cleavage and repair in vivo. To study in vivo Cas9 cleavage kinetics and activity dose-dependence, we have engineered a chemically-inducible, single-component Cas9, ciCas9. ciCas9 allows for temporal and rheostatic control of Cas9 activity using a small molecule activator, A115. We have also developed a droplet-digital PCR-based assay (DSB-ddPCR) to directly quantify Cas9-mediated double-stranded breaks (DSBs). The methods in this chapter describe the application of ciCas9 and DSB-ddPCR to study the kinetics and dose-dependence of Cas9 editing in vivo.},
}
@article {pmid32046217,
year = {2020},
author = {Burmistrz, M and Krakowski, K and Krawczyk-Balska, A},
title = {RNA-Targeting CRISPR-Cas Systems and Their Applications.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32046217},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR-Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR-Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.},
}
@article {pmid32046096,
year = {2020},
author = {Wu, N and Lu, Q and Wang, P and Zhang, Q and Zhang, J and Qu, J and Wang, N},
title = {Construction and Analysis of GmFAD2-1A and GmFAD2-2A Soybean Fatty Acid Desaturase Mutants Based on CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32046096},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics/metabolism ; Fatty Acid Desaturases/chemistry/*genetics/metabolism ; Gene Editing/methods ; *Mutation ; Oleic Acid/biosynthesis ; Plant Breeding/methods ; Plant Proteins/chemistry/*genetics/metabolism ; Seeds/genetics/metabolism ; Soybeans/*genetics/metabolism ; },
abstract = {The soybean fatty acid desaturase family is composed of seven genes, but the function of each gene has not been reported. Bioinformatics was used to analyse the structure of genes in this family, as well as the correlation between Δ12-fatty acid desaturase II (FAD2) expression and oleic acid content on different days after flowering of soybean. In the present study, CRISPR/Cas9 technology was used to construct single and double mutant knockout vectors of functional genes in the FAD2 family. Analysis of the molecular biology and expression patterns of genes in the FAD2 family, namely, GmFAD2-1A (Glyma.10G278000) and GmFAD2-2A (Glyma.19G147300), showed that they had little homology with other soybean FAD2 genes, and that their function was slightly changed. Sequencing of the target showed that the editing efficiency of the GmFAD2-1A and GmFAD2-2A genes was 95% and 55.56%, respectively, and that the double mutant editing efficiency was 66.67%. The mutations were divided into two main types, as follows: base deletion and insertion. A near-infrared grain analyser determined the following results: In the T2 generation, the oleic acid content increased from 17.10% to 73.50%; the linoleic acid content decreased from 62.91% to 12.23%; the protein content increased from 37.69% to 41.16%; in the T3 generation, the oleic acid content increased from 19.15% to 72.02%; the linoleic acid content decreased from 56.58% to 17.27%. In addition, the protein content increased from 37.52% to 40.58% compared to that of the JN38 control variety.},
}
@article {pmid32045522,
year = {2020},
author = {Wang, X and Xiong, E and Tian, T and Cheng, M and Lin, W and Wang, H and Zhang, G and Sun, J and Zhou, X},
title = {Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral Flow Nucleic Acid Assay.},
journal = {ACS nano},
volume = {14},
number = {2},
pages = {2497-2508},
doi = {10.1021/acsnano.0c00022},
pmid = {32045522},
issn = {1936-086X},
mesh = {African Swine Fever Virus/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Listeria monocytogenes/*genetics ; Nucleic Acids/*genetics ; Real-Time Polymerase Chain Reaction ; Swine ; },
abstract = {The lateral flow assay is one of the most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the false-positive signal and tedious and inefficient hybridization steps. Here, we introduce the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a detection limit of hundreds of copies of genome samples with high specificity within 1 h. We further evaluated the performance of CASLFA in a nonlaboratory environment and successfully confirmed 27 ASFV-infected samples from 110 suspected swine serum samples, with an accuracy of 100% when compared to real-time PCR (RT-PCR) assay. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for gene analysis in resource-poor or nonlaboratory environments.},
}
@article {pmid32045111,
year = {2020},
author = {Batianis, C and Kozaeva, E and Damalas, SG and Martín-Pascual, M and Volke, DC and Nikel, PI and Martins Dos Santos, VAP},
title = {An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida.},
journal = {Microbial biotechnology},
volume = {13},
number = {2},
pages = {368-385},
pmid = {32045111},
issn = {1751-7915},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; *Pseudomonas putida/genetics ; RNA, Guide ; },
abstract = {Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single-plasmid CRISPR-interference (CRISPRi) system expressing a nuclease-deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.},
}
@article {pmid32044725,
year = {2020},
author = {Aquino-Jarquin, G},
title = {Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing.},
journal = {Molecular therapy. Nucleic acids},
volume = {19},
number = {},
pages = {1065-1072},
pmid = {32044725},
issn = {2162-2531},
abstract = {One of the most prevalent forms of post-transcriptional RNA modification is the conversion of adenosine-to-inosine (A-to-I), mediated by adenosine deaminase acting on RNA (ADAR) enzymes. The advent of the CRISPR/Cas systems inspires researchers to work actively in the engineering of programmable RNA-guided machines for basic research and biomedical applications. In this regard, CIRTS (CRISPR-Cas-Inspired RNA Targeting System), RESCUE (RNA Editing for Specific C to U Exchange), RESTORE (Recruiting Endogenous ADAR to Specific Transcripts for Oligonucleotide-mediated RNA Editing), and LEAPER (Leveraging Endogenous ADAR for Programmable Editing of RNA) are innovative RNA base-editing platforms that have recently been engineered to perform programmable base conversions on target RNAs mediated by ADAR enzymes in mammalian cells. Thus, these four currently characterized RNA-editing systems constitute novel molecular tools with compelling programmability, specificity, and efficiency that show us some creative ways to take advantage of the engineered deaminases for precise base editing. Moreover, the advanced engineering of these systems permits editing of full-length transcripts containing disease-causing point mutations without the loss of genomic information, providing an attractive alternative for in vivo research and in the therapeutic setting if the challenges encountered in off-target edits and delivery are appropriately addressed. Here, I present an analytical approach of the current status and rapid progress of the novel ADAR-mediated RNA-editing systems when highlighting the qualities of each new RNA-editing platform and how these RNA-targeting strategies could be used to recruit human ADARs on endogenous transcripts, not only for our understanding of RNA-modification-mediated regulation of gene expression but also for editing clinically relevant mutations in a programmable and straightforward manner.},
}
@article {pmid32044035,
year = {2020},
author = {Gu, L and Yuan, H and Lv, X and Li, G and Cong, R and Li, J and Du, G and Liu, L},
title = {High-yield and plasmid-free biocatalytic production of 5-methylpyrazine-2-carboxylic acid by combinatorial genetic elements engineering and genome engineering of Escherichia coli.},
journal = {Enzyme and microbial technology},
volume = {134},
number = {},
pages = {109488},
doi = {10.1016/j.enzmictec.2019.109488},
pmid = {32044035},
issn = {1879-0909},
mesh = {Biocatalysis ; CRISPR-Cas Systems ; Carboxylic Acids/*metabolism ; Escherichia coli/*genetics ; *Genome, Bacterial ; Metabolic Engineering/*methods ; Oxygenases/metabolism ; Pyrazoles/*metabolism ; Substrate Specificity ; },
abstract = {5-Methylpyrazine-2-carboxylic acid (MPCA) is an important pharmaceutical intermediate and is used in the production of hypoglycemic agents and lipid-lowering drugs. This work aimed to develop a whole-cell biocatalytic process for the efficient synthesis of MPCA from 2, 5-dimethylpyrazine (DMP). Firstly, a whole-cell biocatalyst Escherichia coli strain was constructed by plasmid-based expression of xylene monooxygenase (XMO), benzyl alcohol dehydrogenase (BADH), and benzaldehyde dehydrogenase (BZDH) from Pseudomonas putida ATCC 33015, resulting in MPCA titer of 5.0 g/L. Then, the reaction conditions were optimized and the MPCA titer was further increased to 9.1 g/L. Next, the Ribosome Binding Site (RBS) Calculator v2.0 was used to predict and compare the translation initiation rates of the RBS sequences preceding xylM and xylA genes, encoding the two subunits of XMO. By optimizing the RBS sequences preceding xylA, the MPCA titer was increased to 10.2 g/L and the yield of MPCA on DMP reached 0.665 mol/mol. Finally, to achieve plasmid-free production of MPCA, we integrated the genes encoding for XMO, BADH and BZDH in the genome by using CRISPR/Cas9 and further fine-tuned the copy number ratios of xylM and xylA in the genome, improving the MPCA titer to 15.6 g/L and the yield of MPCA on DMP to 1.0 mol/mol. This work developed a high-yield and plasmid-free biocatalysis process for the environmentally friendly production of MPCA with 100% substrate conversion, and paved the way for the commercial production of MPCA in the future.},
}
@article {pmid32043190,
year = {2020},
author = {Yang, S and Cao, X and Yu, W and Li, S and Zhou, YJ},
title = {Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {7},
pages = {3037-3047},
doi = {10.1007/s00253-020-10405-5},
pmid = {32043190},
issn = {1432-0614},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Gene Expression ; Gene Expression Regulation, Fungal ; Genes, Essential/*genetics ; Genetic Markers/genetics ; Genome, Fungal/*genetics ; Genomics ; Metabolic Engineering ; *Mutagenesis, Site-Directed ; Mutation ; Saccharomyces cerevisiae/*genetics/metabolism ; },
abstract = {Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts. KEY POINTS: • A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%. • The optimized three-module method improves the integration efficiency by more than three times. • This method will facilitate the functional genomic analysis and metabolic flux regulation.},
}
@article {pmid32042170,
year = {2020},
author = {Miller, SM and Wang, T and Randolph, PB and Arbab, M and Shen, MW and Huang, TP and Matuszek, Z and Newby, GA and Rees, HA and Liu, DR},
title = {Continuous evolution of SpCas9 variants compatible with non-G PAMs.},
journal = {Nature biotechnology},
volume = {38},
number = {4},
pages = {471-481},
pmid = {32042170},
issn = {1546-1696},
support = {F32 GM119228/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 EB027793/EB/NIBIB NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/*genetics/*metabolism ; DNA Cleavage ; Directed Molecular Evolution ; Gene Editing ; Genetic Variation ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Mutation ; Nucleotide Motifs ; Streptococcus pyogenes/enzymology/genetics ; Substrate Specificity ; },
abstract = {The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer-sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.},
}
@article {pmid32042152,
year = {2020},
author = {Mita, P and Sun, X and Fenyö, D and Kahler, DJ and Li, D and Agmon, N and Wudzinska, A and Keegan, S and Bader, JS and Yun, C and Boeke, JD},
title = {BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.},
journal = {Nature structural &amp; molecular biology},
volume = {27},
number = {2},
pages = {179-191},
pmid = {32042152},
issn = {1545-9985},
support = {P01 AG051449/AG/NIA NIH HHS/United States ; P50 GM107632/GM/NIGMS NIH HHS/United States ; },
mesh = {BRCA1 Protein/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; DNA Breaks, Double-Stranded ; *DNA Repair ; Fanconi Anemia/genetics/metabolism ; Gene Deletion ; HEK293 Cells ; HeLa Cells ; Homologous Recombination ; Humans ; *Long Interspersed Nucleotide Elements ; Microscopy ; *S Phase ; },
abstract = {Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.},
}
@article {pmid32042051,
year = {2020},
author = {Chillappagari, S and Belapurkar, R and Möller, A and Molenda, N and Kracht, M and Rohrbach, S and Schmitz, ML},
title = {SIAH2-mediated and organ-specific restriction of HO-1 expression by a dual mechanism.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2268},
pmid = {32042051},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Ferroptosis ; Fibroblasts ; Gene Knockdown Techniques ; HEK293 Cells ; Heme Oxygenase-1/genetics/*metabolism ; Humans ; Membrane Proteins/genetics/*metabolism ; Mice ; Mice, Knockout ; NF-E2-Related Factor 2/*metabolism ; Nuclear Proteins/genetics/*metabolism ; Oxygen/metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism ; Primary Cell Culture ; Proteasome Endopeptidase Complex/metabolism ; Protein Domains ; Protein Stability ; Proteolysis ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Ubiquitination ; },
abstract = {The intracellular levels of the cytoprotective enzyme heme oxygenase-1 (HO-1) are tightly controlled. Here, we reveal a novel mechanism preventing the exaggerated expression of HO-1. The analysis of mice with a knock-out in the ubiquitin E3 ligase seven in absentia homolog 2 (SIAH2) showed elevated HO-1 protein levels in specific organs such as heart, kidney and skeletal muscle. Increased HO-1 protein amounts were also seen in human cells deleted for the SIAH2 gene. The higher HO-1 levels are not only due to an increased protein stability but also to elevated expression of the HO-1 encoding HMOX1 gene, which depends on the transcription factor nuclear factor E2-related factor 2 (NRF2), a known SIAH2 target. Dependent on its RING (really interesting new gene) domain, expression of SIAH2 mediates proteasome-dependent degradation of its interaction partner HO-1. Additionally SIAH2-deficient cells are also characterized by reduced expression levels of glutathione peroxidase 4 (GPX4), rendering the knock-out cells more sensitive to ferroptosis.},
}
@article {pmid32042045,
year = {2020},
author = {Wang, Y and Wang, M and Zheng, T and Hou, Y and Zhang, P and Tang, T and Wei, J and Du, Q},
title = {Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2269},
pmid = {32042045},
issn = {2045-2322},
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; Endodeoxyribonucleases/metabolism ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mutation ; Nucleotides/metabolism ; RNA, Guide/*genetics ; },
abstract = {To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a "core" sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of 'super' off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.},
}
@article {pmid32041790,
year = {2020},
author = {Duong, T and Rasmussen, NR and Ballato, E and Mote, FS and Reiner, DJ},
title = {The Rheb-TORC1 signaling axis functions as a developmental checkpoint.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {5},
pages = {},
pmid = {32041790},
issn = {1477-9129},
support = {R01 GM121625/GM/NIGMS NIH HHS/United States ; R21 HD090707/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Autophagy/physiology ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics/*growth &amp; development ; Caenorhabditis elegans Proteins/*genetics/*metabolism ; Life Cycle Stages/genetics ; Longevity/physiology ; Mechanistic Target of Rapamycin Complex 1/genetics/*metabolism ; Phosphotransferases (Alcohol Group Acceptor)/*genetics ; RNA Interference ; RNA, Small Interfering/genetics ; Ras Homolog Enriched in Brain Protein/genetics/*metabolism ; Signal Transduction ; },
abstract = {In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development and autophagy. Our CRISPR-generated, fluorescently tagged endogenous Caenorhabditis elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. LET-363/TOR and DAF-15/Raptor are required for development beyond the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs the decision of the animal to progress through development.},
}
@article {pmid32041517,
year = {2020},
author = {Liu, Q and Cheng, X and Liu, G and Li, B and Liu, X},
title = {Deep learning improves the ability of sgRNA off-target propensity prediction.},
journal = {BMC bioinformatics},
volume = {21},
number = {1},
pages = {51},
pmid = {32041517},
issn = {1471-2105},
mesh = {*CRISPR-Cas Systems ; *Deep Learning ; Gene Editing ; Humans ; RNA/metabolism ; },
abstract = {BACKGROUND: CRISPR/Cas9 system, as the third-generation genome editing technology, has been widely applied in target gene repair and gene expression regulation. Selection of appropriate sgRNA can improve the on-target knockout efficacy of CRISPR/Cas9 system with high sensitivity and specificity. However, when CRISPR/Cas9 system is operating, unexpected cleavage may occur at some sites, known as off-target. Presently, a number of prediction methods have been developed to predict the off-target propensity of sgRNA at specific DNA fragments. Most of them use artificial feature extraction operations and machine learning techniques to obtain off-target scores. With the rapid expansion of off-target data and the rapid development of deep learning theory, the existing prediction methods can no longer satisfy the prediction accuracy at the clinical level.<br><br>RESULTS: Here, we propose a prediction method named CnnCrispr to predict the off-target propensity of sgRNA at specific DNA fragments. CnnCrispr automatically trains the sequence features of sgRNA-DNA pairs with GloVe model, and embeds the trained word vector matrix into the deep learning model including biLSTM and CNN with five hidden layers. We conducted performance verification on the data set provided by DeepCrispr, and found that the auROC and auPRC in the "leave-one-sgRNA-out" cross validation could reach 0.957 and 0.429 respectively (the Pearson value and spearman value could reach 0.495 and 0.151 respectively under the same settings).<br><br>CONCLUSION: Our results show that CnnCrispr has better classification and regression performance than the existing states-of-art models. The code for CnnCrispr can be freely downloaded from https://github.com/LQYoLH/CnnCrispr.},
}
@article {pmid32041354,
year = {2020},
author = {Tromp, AT and Van Gent, M and Jansen, JP and Scheepmaker, LM and Velthuizen, A and De Haas, CJC and Van Kessel, KPM and Bardoel, BW and Boettcher, M and McManus, MT and Van Strijp, JAG and Lebbink, RJ and Haas, PA and Spaan, AN},
title = {Host-Receptor Post-Translational Modifications Refine Staphylococcal Leukocidin Cytotoxicity.},
journal = {Toxins},
volume = {12},
number = {2},
pages = {},
pmid = {32041354},
issn = {2072-6651},
support = {UU 2012-5667//KWF Kankerbestrijding/International ; },
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Survival/genetics ; Drug Resistance, Bacterial/genetics ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; HEK293 Cells ; Host Microbial Interactions/*genetics ; Humans ; Leukocidins/genetics/metabolism/*toxicity ; Phagocytes/microbiology/pathology ; Protein Binding ; *Protein Processing, Post-Translational ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Staphylococcal Infections/microbiology/*pathology ; Staphylococcus aureus/genetics/metabolism/*pathogenicity ; U937 Cells ; },
abstract = {Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies post-translational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus.},
}
@article {pmid32040758,
year = {2020},
author = {Shockey, J},
title = {Gene editing in plants: assessing the variables through a simplified case study.},
journal = {Plant molecular biology},
volume = {103},
number = {1-2},
pages = {75-89},
pmid = {32040758},
issn = {1573-5028},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems ; Cytosine/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Genome, Plant ; Guanosine/metabolism ; Mutation ; },
abstract = {Multiple variables that control the relative levels of successful heritable plant genome editing were addressed using simple case studies in Arabidopsis thaliana. The recent advent of genome editing technologies (especially CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats) has revolutionized various fields of scientific research. The process is much more specific than previous mutagenic processes and allows for targeting of nearly any gene of interest for the creation of loss-of-function mutations and many other types of editing, including gene-replacement and gene activation. However, not all CRISPR construct designs are successful, due to several factors, including differences in the strength and cell- or tissue-type specificity of the regulatory elements used to express the Cas9 (CRISPR Associated protein 9) DNA nuclease and single guide RNA components, and differences in the relative editing efficiency at different target areas within a given gene. Here we compare the levels of editing created in Arabidopsis thaliana by CRISPR constructs containing either different promoters, or altered target sites with varied levels of guanine-cytosine base content. Additionally, nuclease activity at sites targeted by imperfectly matched single guide RNAs was observed, suggesting that while the primary goal of most CRISPR construct designs is to achieve rapid, robust, heritable gene editing, the formation of unintended mutations at other genomic loci must be carefully monitored.},
}
@article {pmid32040336,
year = {2020},
author = {Carballar-Lejarazú, R and Kelsey, A and Pham, TB and Bennett, EP and James, AA},
title = {Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species Anopheles stephensi.},
journal = {BioTechniques},
volume = {68},
number = {4},
pages = {172-179},
pmid = {32040336},
issn = {1940-9818},
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair/genetics ; Gene Editing ; INDEL Mutation/*genetics ; Malaria/transmission ; Mosquito Vectors/*genetics ; Mutagenesis, Insertional/genetics ; Polymerase Chain Reaction/*methods ; },
abstract = {CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi: Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes.},
}
@article {pmid32040335,
year = {2020},
author = {Her, NG and Babic, I and Yenugonda, VM and Kesari, S and Nurmemmedov, E},
title = {Cellular thermal shift analysis for interrogation of CRISPR-assisted proteomic changes.},
journal = {BioTechniques},
volume = {68},
number = {4},
pages = {180-184},
doi = {10.2144/btn-2019-0100},
pmid = {32040335},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing/*methods ; Humans ; Mutagenesis, Insertional/*genetics ; Proteome/analysis/*genetics ; Proteomics/*methods ; },
abstract = {CRISPR-Cas9 has proven to be a versatile tool for the discovery of essential genetic elements involved in various disease states. CRISPR-assisted dense mutagenesis focused on therapeutically challenging protein complexes allows us to systematically perturb protein-coding sequences in situ and correlate them with functional readouts. Such perturbations can mimic targeting by therapeutics and serve as a foundation for the discovery of highly specific modulators. However, translation of such genomics data has been challenging due to the missing link for proteomics under the physiological state of the cell. We present a method based on cellular thermal shift assays to easily interrogate proteomic shifts generated by CRISPR-assisted dense mutagenesis, as well as a case focused on NuRD epigenetic complex.},
}
@article {pmid32040210,
year = {2020},
author = {Zhang, W and Liu, Y and Zhou, X and Zhao, R and Wang, H},
title = {Applications of CRISPR-Cas9 in gynecological cancer research.},
journal = {Clinical genetics},
volume = {97},
number = {6},
pages = {827-834},
doi = {10.1111/cge.13717},
pmid = {32040210},
issn = {1399-0004},
mesh = {CRISPR-Cas Systems/*genetics ; Female ; *Gene Editing ; *Genetic Therapy ; Genital Neoplasms, Female/*genetics/therapy ; Humans ; },
abstract = {Gynecological cancers pose a significant threat to women's health worldwide, with cervical cancer, ovarian cancer, and endometrial cancer having high incidences. Current gynecological cancer treatment methods mainly include surgery, chemotherapy, radiotherapy, and chemoradiotherapy. The CRISPR-Cas9 gene editing technology as a new therapeutic method has shown tremendous effect in the treatment of other cancers, promoting research on its potential therapeutic effect in gynecological cancer. In this article, we reviewed the current research status of CRISPR-Cas9 technology in gynecological cancer, focusing on the importance of studying the mechanism of CRISPR-Cas9 in gynecological cancer treatment, thereby laying a foundation for further research on its clinical application.},
}
@article {pmid32038657,
year = {2020},
author = {Cohen, LB and Lindsay, SA and Xu, Y and Lin, SJH and Wasserman, SA},
title = {The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {9},
pmid = {32038657},
issn = {1664-3224},
support = {R01 GM050545/GM/NIGMS NIH HHS/United States ; T32 GM007240/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Antimicrobial Cationic Peptides/genetics/*metabolism ; CRISPR-Cas Systems ; Candida glabrata/*immunology ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/genetics/*immunology/metabolism/*microbiology ; Enterococcus faecalis/*immunology ; Fusarium/*immunology ; Gene Knockout Techniques ; Host-Pathogen Interactions/genetics/immunology ; Hyphae/immunology ; Immunity, Innate ; Signal Transduction/genetics/immunology ; },
abstract = {Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, Δdaisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.},
}
@article {pmid32038537,
year = {2019},
author = {Wimmer, F and Beisel, CL},
title = {CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {3078},
pmid = {32038537},
issn = {1664-302X},
abstract = {CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.},
}
@article {pmid32037739,
year = {2020},
author = {Wang, J and Zhang, C and Feng, B},
title = {The rapidly advancing Class 2 CRISPR-Cas technologies: A customizable toolbox for molecular manipulations.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {6},
pages = {3256-3270},
pmid = {32037739},
issn = {1582-4934},
mesh = {Archaea/*genetics ; Bacteria/*genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Genome/genetics ; High-Throughput Nucleotide Sequencing/methods ; RNA, Guide/genetics ; },
abstract = {The CRISPR-Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid-guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence-specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high-throughput sequencing, the discoveries and engineering of various innovative CRISPR-Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid-guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non-existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications.},
}
@article {pmid32036747,
year = {2020},
author = {Yao, B and Liang, M and Liu, H and Sui, T and Song, Y and Zhang, Y and Deng, J and Xu, Y and Lai, L and Li, Z},
title = {The minimal promoter (P1) of Xist is non-essential for X chromosome inactivation.},
journal = {RNA biology},
volume = {17},
number = {5},
pages = {623-629},
pmid = {32036747},
issn = {1555-8584},
mesh = {Animals ; Biopsy ; CRISPR-Cas Systems ; DNA Mutational Analysis ; Female ; Gene Knockout Techniques ; *Genes, X-Linked ; Immunohistochemistry ; Mice, Transgenic ; *Promoter Regions, Genetic ; RNA, Long Noncoding/*genetics ; Rabbits ; *X Chromosome Inactivation ; },
abstract = {The previous report shows the minimal promoter (P1) contributes to the Xist RNA activation in cells, while the role of the Xist P1 has not yet been investigated in animal individuals. Here, female Xist P1 knockout rabbits (Xist P1[-/-]) were generated for the studies. The results showed that there is no significant difference in transmission ratio, Xist and X-linked genes expression, and Xist RNA localization between the female wild type (WT) and Xist P1[-/-] rabbits, suggesting that P1 is non-essential for Xist expression and XCI in rabbits. Our study has explored the function of Xist P1 in animal level for the first time, and the results provide new ideas for future studies of XCI mechanisms.},
}
@article {pmid32036448,
year = {2020},
author = {Leonard, F and Curtis, LT and Hamed, AR and Zhang, C and Chau, E and Sieving, D and Godin, B and Frieboes, HB},
title = {Nonlinear response to cancer nanotherapy due to macrophage interactions revealed by mathematical modeling and evaluated in a murine model via CRISPR-modulated macrophage polarization.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {69},
number = {5},
pages = {731-744},
pmid = {32036448},
issn = {1432-0851},
support = {R15CA203605/CA/NCI NIH HHS/United States ; R15 CA203605/CA/NCI NIH HHS/United States ; },
mesh = {Albumin-Bound Paclitaxel/*administration &amp; dosage ; Animals ; Breast Neoplasms/immunology/pathology/*therapy ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics/immunology ; Cell Engineering ; Cell Line, Tumor/transplantation ; Coculture Techniques ; Disease Models, Animal ; Female ; Humans ; Liposomes ; Liver Neoplasms/immunology/secondary/*therapy ; Macrophage Activation/*genetics ; Macrophages/*immunology ; Mice ; *Models, Biological ; Nanoparticles ; Spheroids, Cellular ; Treatment Outcome ; Tumor Microenvironment/drug effects/genetics/immunology ; },
abstract = {Tumor-associated macrophages (TAMs) have been shown to both aid and hinder tumor growth, with patient outcomes potentially hinging on the proportion of M1, pro-inflammatory/growth-inhibiting, to M2, growth-supporting, phenotypes. Strategies to stimulate tumor regression by promoting polarization to M1 are a novel approach that harnesses the immune system to enhance therapeutic outcomes, including chemotherapy. We recently found that nanotherapy with mesoporous particles loaded with albumin-bound paclitaxel (MSV-nab-PTX) promotes macrophage polarization towards M1 in breast cancer liver metastases (BCLM). However, it remains unclear to what extent tumor regression can be maximized based on modulation of the macrophage phenotype, especially for poorly perfused tumors such as BCLM. Here, for the first time, a CRISPR system is employed to permanently modulate macrophage polarization in a controlled in vitro setting. This enables the design of 3D co-culture experiments mimicking the BCLM hypovascularized environment with various ratios of polarized macrophages. We implement a mathematical framework to evaluate nanoparticle-mediated chemotherapy in conjunction with TAM polarization. The response is predicted to be not linearly dependent on the M1:M2 ratio. To investigate this phenomenon, the response is simulated via the model for a variety of M1:M2 ratios. The modeling indicates that polarization to an all-M1 population may be less effective than a combination of both M1 and M2. Experimental results with the CRISPR system confirm this model-driven hypothesis. Altogether, this study indicates that response to nanoparticle-mediated chemotherapy targeting poorly perfused tumors may benefit from a fine-tuned M1:M2 ratio that maintains both phenotypes in the tumor microenvironment during treatment.},
}
@article {pmid32035346,
year = {2020},
author = {Pan, M and Barrangou, R},
title = {Combining omics technologies with CRISPR-based genome editing to study food microbes.},
journal = {Current opinion in biotechnology},
volume = {61},
number = {},
pages = {198-208},
doi = {10.1016/j.copbio.2019.12.027},
pmid = {32035346},
issn = {1879-0429},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Probiotics ; },
abstract = {The implementation of omics technologies such as genomics, proteomics and transcriptomics has revolutionized our understanding of microbiomes, and shed light on the functional attributes and mechanisms of action underlying the ability of probiotics to impact host health and starter cultures to drive food fermentation. Recently, molecular machines from CRISPR-Cas systems have redefined the gene editing toolbox and democritized our ability to alter the genome of food microorganisms. An integrated approach in which CRISPR-based genome editing is informed by omics studies is poised to enable the engineering of microorganisms and the formulation of microbiomes impacting the food supply chain. Here, we highlight the current applications of omics technologies in food microorganisms and CRISPR-based genome editing technologies in bacteria, and discuss how this integrated approach enables effective engineering of food microbes to generate enhanced probiotic strains, develop novel biotherapeutics and alter microbial communities in food matrices.},
}
@article {pmid32035013,
year = {2020},
author = {Lin, MH and Pope, BD and Sasaki, T and Keeley, DP and Sherwood, DR and Miner, JH},
title = {Mammalian hemicentin 1 is assembled into tracks in the extracellular matrix of multiple tissues.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {249},
number = {6},
pages = {775-788},
pmid = {32035013},
issn = {1097-0177},
support = {R01 DK078314/DK/NIDDK NIH HHS/United States ; R35 GM118049/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Basement Membrane/metabolism ; CRISPR-Cas Systems/genetics ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/genetics/*metabolism ; Hair Follicle/cytology/metabolism ; Kidney/metabolism ; Mice ; Mice, Knockout ; Sclera/cytology/metabolism ; },
abstract = {BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals.<br><br>RESULTS: Hmcn1 [-/-] mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency.<br><br>CONCLUSION: That Hmcn1 [-/-] , Hmcn2 [-/-] , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.},
}
@article {pmid32034780,
year = {2020},
author = {Clément, CMH and Deffieu, MS and Dorobantu, CM and Baumert, TF and Ayala-Nunez, NV and Mély, Y and Ronde, P and Gaudin, R},
title = {Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.},
journal = {Biology of the cell},
volume = {112},
number = {5},
pages = {140-151},
pmid = {32034780},
issn = {1768-322X},
mesh = {CRISPR-Cas Systems ; Carcinoma, Hepatocellular/metabolism/virology ; Cell Line, Tumor ; Cell Movement ; Claudin-1/*metabolism ; *Gene Knock-In Techniques ; Hepacivirus/*physiology ; Hepatocytes/*metabolism/virology ; Humans ; Liver Neoplasms/metabolism/virology ; *Virus Internalization ; },
abstract = {BACKGROUND INFORMATION: Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival.<br><br>RESULTS: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1[+/+] cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs.<br><br>CONCLUSIONS: The Huh7.5.1 TagRFP-CLDN1[+/+] edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging.<br><br>SIGNIFICANCE: Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.},
}
@article {pmid32034392,
year = {2020},
author = {Solomon, SM},
title = {Genome editing in animals: why FDA regulation matters.},
journal = {Nature biotechnology},
volume = {38},
number = {2},
pages = {142-143},
pmid = {32034392},
issn = {1546-1696},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; *Gene Editing ; Genome ; Germ Cells ; Plasmids ; },
}
@article {pmid32034293,
year = {2020},
author = {Dai, W and Wu, J and Wang, D and Wang, J},
title = {Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting telomeres.},
journal = {Gene therapy},
volume = {27},
number = {6},
pages = {266-280},
pmid = {32034293},
issn = {1476-5462},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Genes, Neoplasm ; Humans ; Mice ; NF-kappa B/genetics/metabolism ; *Neoplasms/genetics/therapy ; Telomere/metabolism ; },
abstract = {The transcription factor NF-κB is an attractive target for cancer therapy due to its over-activation in all tumours; however, NF-κB inhibitors developed in the past decades rarely became drugs due to undesirable side effects. In this study, we developed a gene therapy strategy named NF-κB-activated gene expression (Nage), which could induce the death of cancer cells in vitro and in vivo by utilising NF-κB over-activity in cancer cells, but had no effects on normal cells. Nage was consisted of an NF-κB-specific promoter formed by fusing an NF-κB decoy sequence with a minimal promoter, which could be bound by the intracellular over-activated NF-κB and thus activated the expression of downstream effector genes in an NF-κB-specific manner. In this study, we first confirmed the cancer cell-specific over-activation of NF-κB and then tested the cancer cell specificity of the Nage vector by expressing the reporter gene ZsGreen in various in vitro cultivated cells. We next demonstrated that the Nage vector could be used to express CRISPR/Cas9 protein only in cancer cells. The Cas9 protein was then guided by a sgRNA targeting telomeric DNA and induced cancer cell death. The Nage vector expressing Cas9/sgRNA could be packaged into adeno-associated virus (AAV) and intravenously injected to inhibit tumour growth in mice without visible side effects and toxicity.},
}
@article {pmid32034052,
year = {2020},
author = {Dickinson, MH and Vosshall, LB and Dow, JAT},
title = {Genome editing in non-model organisms opens new horizons for comparative physiology.},
journal = {The Journal of experimental biology},
volume = {223},
number = {Pt Suppl 1},
pages = {},
doi = {10.1242/jeb.221119},
pmid = {32034052},
issn = {1477-9145},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Physiology, Comparative ; },
}
@article {pmid32034051,
year = {2020},
author = {Matthews, BJ and Vosshall, LB},
title = {How to turn an organism into a model organism in 10 'easy' steps.},
journal = {The Journal of experimental biology},
volume = {223},
number = {Pt Suppl 1},
pages = {},
pmid = {32034051},
issn = {1477-9145},
support = {UL1 TR000043/TR/NCATS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Aedes/genetics ; Animals ; CRISPR-Cas Systems ; *Drosophila melanogaster/genetics ; Gene Editing ; Mice ; Mosquito Vectors ; Saccharomyces cerevisiae/genetics ; },
abstract = {Many of the major biological discoveries of the 20th century were made using just six species: Escherichia coli bacteria, Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, Caenorhabditis elegans nematodes, Drosophila melanogaster flies and Mus musculus mice. Our molecular understanding of the cell division cycle, embryonic development, biological clocks and metabolism were all obtained through genetic analysis using these species. Yet the 'big 6' did not start out as genetic model organisms (hereafter 'model organisms'), so how did they mature into such powerful systems? First, these model organisms are abundant human commensals: they are the bacteria in our gut, the yeast in our beer and bread, the nematodes in our compost pile, the flies in our kitchen and the mice in our walls. Because of this, they are cheaply, easily and rapidly bred in the laboratory and in addition were amenable to genetic analysis. How and why should we add additional species to this roster? We argue that specialist species will reveal new secrets in important areas of biology and that with modern technological innovations like next-generation sequencing and CRISPR-Cas9 genome editing, the time is ripe to move beyond the big 6. In this review, we chart a 10-step path to this goal, using our own experience with the Aedes aegypti mosquito, which we built into a model organism for neurobiology in one decade. Insights into the biology of this deadly disease vector require that we work with the mosquito itself rather than modeling its biology in another species.},
}
@article {pmid32034045,
year = {2020},
author = {Courtier-Orgogozo, V and Martin, A},
title = {The coding loci of evolution and domestication: current knowledge and implications for bio-inspired genome editing.},
journal = {The Journal of experimental biology},
volume = {223},
number = {Pt Suppl 1},
pages = {},
doi = {10.1242/jeb.208934},
pmid = {32034045},
issn = {1477-9145},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Domestication ; *Gene Editing ; Phenotype ; Plant Breeding ; Plants ; },
abstract = {One promising application of CRISPR/Cas9 is to create targeted mutations to introduce traits of interest into domesticated organisms. However, a major current limitation for crop and livestock improvement is to identify the precise genes and genetic changes that must be engineered to obtain traits of interest. Here, we discuss the advantages of bio-inspired genome editing, i.e. the engineered introduction of natural mutations that have already been associated with traits of interest in other lineages (breeds, populations or species). To obtain a landscape view of potential targets for genome editing, we used Gephebase (www.gephebase.org), a manually curated database compiling published data about the genes responsible for evolutionary and domesticated changes across eukaryotes, and examined the >1200 mutations that have been identified in the coding regions of more than 700 genes in animals, plants and yeasts. We observe that our genetic knowledge is relatively important for certain traits, such as xenobiotic resistance, and poor for others. We also note that protein-null alleles, often owing to nonsense and frameshift mutations, represent a large fraction of the known loci of domestication (42% of identified coding mutations), compared with intraspecific (27%) and interspecific evolution (11%). Although this trend may be subject to detection, publication and curation biases, it is consistent with the idea that breeders have selected large-effect mutations underlying adaptive traits in specific settings, but that these mutations and associated phenotypes would not survive the vagaries of changing external and internal environments. Our compilation of the loci of evolution and domestication uncovers interesting options for bio-inspired and transgene-free genome editing.},
}
@article {pmid32034043,
year = {2020},
author = {Martin, A and Wolcott, NS and O'Connell, LA},
title = {Bringing immersive science to undergraduate laboratory courses using CRISPR gene knockouts in frogs and butterflies.},
journal = {The Journal of experimental biology},
volume = {223},
number = {Pt Suppl 1},
pages = {},
doi = {10.1242/jeb.208793},
pmid = {32034043},
issn = {1477-9145},
mesh = {Animals ; *Butterflies ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Knockout Techniques ; Humans ; Laboratories ; Students ; },
abstract = {The use of CRISPR/Cas9 for gene editing offers new opportunities for biology students to perform genuine research exploring the gene-to-phenotype relationship. It is important to introduce the next generation of scientists, health practitioners and other members of society to the technical and ethical aspects of gene editing. Here, we share our experience leading hands-on undergraduate laboratory classes, where students formulate hypotheses regarding the roles of candidate genes involved in development, perform loss-of-function experiments using programmable nucleases and analyze the phenotypic effects of mosaic mutant animals. This is enabled by the use of the amphibian Xenopus laevis and the butterfly Vanessa cardui, two organisms that reliably yield hundreds of large and freshly fertilized eggs in a scalable manner. Frogs and butterflies also present opportunities to teach key biological concepts about gene regulation and development. To complement these practical aspects, we describe learning activities aimed at equipping students with a broad understanding of genome editing techniques, their application in fundamental and translational research, and the bioethical challenges they raise. Overall, our work supports the introduction of CRISPR technology into undergraduate classrooms and, when coupled with classroom undergraduate research experiences, enables hypothesis-driven research by undergraduates.},
}
@article {pmid32034041,
year = {2020},
author = {Raban, RR and Marshall, JM and Akbari, OS},
title = {Progress towards engineering gene drives for population control.},
journal = {The Journal of experimental biology},
volume = {223},
number = {Pt Suppl 1},
pages = {},
pmid = {32034041},
issn = {1477-9145},
support = {R21 AI123937/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Drive Technology ; Gene Editing ; Humans ; *Malaria/prevention &amp; control ; Population Control ; *Zika Virus ; *Zika Virus Infection ; },
abstract = {Vector-borne diseases, such as dengue, Zika and malaria, are a major cause of morbidity and mortality worldwide. These diseases have proven difficult to control and currently available management tools are insufficient to eliminate them in many regions. Gene drives have the potential to revolutionize vector-borne disease control. This suite of technologies has advanced rapidly in recent years as a result of the availability of new, more efficient gene editing technologies. Gene drives can favorably bias the inheritance of a linked disease-refractory gene, which could possibly be exploited (i) to generate a vector population incapable of transmitting disease or (ii) to disrupt an essential gene for viability or fertility, which could eventually eliminate a population. Importantly, gene drives vary in characteristics such as their transmission efficiency, confinability and reversibility, and their potential to develop resistance to the drive mechanism. Here, we discuss recent advancements in the gene drive field, and contrast the benefits and limitations of a variety of technologies, as well as approaches to overcome these limitations. We also discuss the current state of each gene drive technology and the technical considerations that need to be addressed on the pathway to field implementation. While there are still many obstacles to overcome, recent progress has brought us closer than ever before to genetic-based vector modification as a tool to support vector-borne disease elimination efforts worldwide.},
}
@article {pmid32033083,
year = {2020},
author = {Veillet, F and Perrot, L and Guyon-Debast, A and Kermarrec, MP and Chauvin, L and Chauvin, JE and Gallois, JL and Mazier, M and Nogué, F},
title = {Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32033083},
issn = {1422-0067},
mesh = {Amino Acid Substitution/genetics ; Bryopsida/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Lycopersicon esculentum/*genetics ; Plants, Genetically Modified/genetics ; Solanum tuberosum/*genetics ; Streptococcus pyogenes/enzymology ; },
abstract = {Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.},
}
@article {pmid32032606,
year = {2020},
author = {Sterner, ZR and Shewade, LH and Mertz, KM and Sturgeon, SM and Buchholz, DR},
title = {Glucocorticoid receptor is required for survival through metamorphosis in the frog Xenopus tropicalis.},
journal = {General and comparative endocrinology},
volume = {291},
number = {},
pages = {113419},
doi = {10.1016/j.ygcen.2020.113419},
pmid = {32032606},
issn = {1095-6840},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; Breeding ; CRISPR-Cas Systems/genetics ; Corticosterone/metabolism ; Female ; Gene Expression Regulation, Developmental/drug effects ; Gene Knockout Techniques ; Larva/genetics/growth &amp; development ; Male ; *Metamorphosis, Biological/genetics ; Mutation/genetics ; Receptors, Glucocorticoid/chemistry/genetics/*metabolism ; Thyroid Hormones/metabolism ; Xenopus/*growth &amp; development/*metabolism ; },
abstract = {Stress hormones, also known as glucocorticoids, are critical for survival at birth in mammals due at least in part to their importance in lung maturation. However, because air breathing is not always required for amphibian survival and because stress hormones have no known developmental impact except to modulate the developmental actions of thyroid hormone (TH), the requirement for stress hormone signaling during metamorphosis is not well understoodi. Here, we produced a glucocorticoid receptor knockout (GRKO) Xenopus line with a frameshift mutation in the first exon of the glucocorticoid receptor. Induction by exogenous corticosterone (CORT, the frog stress hormone) of the CORT response genes, klf9 (Krüppel-like factor 9, also regulated by TH) and ush1g (Usher's syndrome 1G), was completely abrogated in GRKO tadpoles. Surprisingly, GRKO tadpoles developed faster than wild-type tadpoles until forelimb emergence and then developed more slowly until their death at the climax of metamorphosis. Growth rate was not affected in GRKO tadpoles, but they achieved a smaller maximum size. Gene expression analysis of the TH response genes, thrb (TH receptor beta) and klf9 showed reduced expression in the tail at metamorphic climax consistent with the reduced development rate. These results indicate that glucocorticoid receptor is required for survival through metamorphosis and support dual roles for GR signaling in control of developmental rate.},
}
@article {pmid32032579,
year = {2020},
author = {Kroll, JR and Tsiaxiras, J and van Zon, JS},
title = {Variability in β-catenin pulse dynamics in a stochastic cell fate decision in C. elegans.},
journal = {Developmental biology},
volume = {461},
number = {2},
pages = {110-123},
pmid = {32032579},
issn = {1095-564X},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*cytology/genetics ; Caenorhabditis elegans Proteins/biosynthesis/genetics/*metabolism ; Cell Differentiation ; Cell Fusion ; Cell Lineage ; Cytoskeletal Proteins/metabolism ; Female ; Gene Expression Regulation, Developmental ; Genotype ; Homeodomain Proteins/metabolism ; In Situ Hybridization, Fluorescence ; Integumentary System/anatomy &amp; histology ; Membrane Glycoproteins/biosynthesis/genetics ; Recombinant Fusion Proteins/metabolism ; Single-Cell Analysis ; Stochastic Processes ; Time-Lapse Imaging ; Vulva/cytology ; Wnt Signaling Pathway ; beta Catenin/*metabolism ; },
abstract = {During development, cell fate decisions are often highly stochastic, but with the frequency of the different possible fates tightly controlled. To understand how signaling networks control the cell fate frequency of such random decisions, we studied the stochastic decision of the Caenorhabditis elegans P3.p cell to either fuse to the hypodermis or assume vulva precursor cell fate. Using time-lapse microscopy to measure the single-cell dynamics of two key inhibitors of cell fusion, the Hox gene LIN-39 and Wnt signaling through the β-catenin BAR-1, we uncovered significant variability in the dynamics of LIN-39 and BAR-1 levels. Most strikingly, we observed that BAR-1 accumulated in a single, 1-4 h pulse at the time of the P3.p cell fate decision, with strong variability both in pulse slope and time of pulse onset. We found that the time of BAR-1 pulse onset was delayed relative to the time of cell fusion in mutants with low cell fusion frequency, linking BAR-1 pulse timing to cell fate outcome. Overall, a model emerged where animal-to-animal variability in LIN-39 levels and BAR-1 pulse dynamics biases cell fate by modulating their absolute level at the time cell fusion is induced. Our results highlight that timing of cell signaling dynamics, rather than its average level or amplitude, could play an instructive role in determining cell fate.},
}
@article {pmid32032363,
year = {2020},
author = {Drews, K and Calgi, MP and Harrison, WC and Drews, CM and Costa-Pinheiro, P and Shaw, JJP and Jobe, KA and Han, JD and Fox, TE and White, JM and Kester, M},
title = {Glucosylceramide synthase maintains influenza virus entry and infection.},
journal = {PloS one},
volume = {15},
number = {2},
pages = {e0228735},
pmid = {32032363},
issn = {1932-6203},
support = {R01 AI114776/AI/NIAID NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; T32 GM008715/GM/NIGMS NIH HHS/United States ; },
mesh = {A549 Cells ; CRISPR-Cas Systems/genetics ; Gene Editing ; Glucosyltransferases/deficiency/*genetics ; HEK293 Cells ; Humans ; Influenza A virus/*physiology ; Macrolides/pharmacology ; Virus Internalization/drug effects ; },
abstract = {Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.},
}
@article {pmid32031431,
year = {2020},
author = {Dailey, PJ and Elbeik, T and Holodniy, M},
title = {Companion and complementary diagnostics for infectious diseases.},
journal = {Expert review of molecular diagnostics},
volume = {20},
number = {6},
pages = {619-636},
doi = {10.1080/14737159.2020.1724784},
pmid = {32031431},
issn = {1744-8352},
mesh = {Anti-Bacterial Agents/pharmacology/therapeutic use ; Anti-Infective Agents/pharmacology/*therapeutic use ; Antiviral Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Communicable Diseases/diagnosis/*drug therapy/microbiology/virology ; Device Approval ; Drug Approval ; Drug Development ; Drug Resistance, Microbial/*genetics ; Genotyping Techniques ; Humans ; *Microbial Sensitivity Tests ; *Molecular Diagnostic Techniques ; *Point-of-Care Testing ; Precision Medicine/*methods ; United States ; United States Food and Drug Administration ; },
abstract = {INTRODUCTION: Companion diagnostics (CDx) are important in oncology therapeutic decision-making, but specific regulatory-approved CDx for infectious disease treatment are officially lacking. While not approved as CDx, several ID diagnostics are used as CDx. The diagnostics community, manufacturers, and regulatory agencies have made major efforts to ensure that diagnostics for new antimicrobials are available at or near release of new agents.<br><br>AREAS COVERED: This review highlights the status of Complementary and companion diagnostic (c/CDx) in the infectious disease literature, with a focus on genotypic antimicrobial resistance testing against pathogens as a class of diagnostic tests.<br><br>EXPERT OPINION: CRISPR, sepsis markers, and narrow spectrum antimicrobials, in addition to current and emerging technologies, present opportunities for infectious disease c/CDx. Challenges include slow guideline revision, high costs for regulatory approval, lengthy buy in by agencies, discordant pharmaceutical/diagnostic partnerships, and higher treatment costs. The number of patients and available medications used to treat different infectious diseases is well suited to support competing diagnostic tests. However, newer approaches to treatment (for example, narrow spectrum antibiotics), may be well suited for a small number of patients, i.e. a niche market in support of a CDx. The current emphasis is rapid and point-of-care (POC) diagnostic platforms as well as changes in treatment.},
}
@article {pmid32031369,
year = {2020},
author = {Yuan, C and Tian, T and Sun, J and Hu, M and Wang, X and Xiong, E and Cheng, M and Bao, Y and Lin, W and Jiang, J and Yang, C and Chen, Q and Zhang, H and Wang, H and Wang, X and Deng, X and Liao, X and Liu, Y and Wang, Z and Zhang, G and Zhou, X},
title = {Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a/13a System.},
journal = {Analytical chemistry},
volume = {92},
number = {5},
pages = {4029-4037},
doi = {10.1021/acs.analchem.9b05597},
pmid = {32031369},
issn = {1520-6882},
mesh = {African Swine Fever Virus/genetics ; Animals ; Bacteria/genetics ; CRISPR-Cas Systems/*genetics ; Colorimetry/*methods ; DNA Probes/chemistry ; DNA, Viral/analysis/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; MicroRNAs/analysis/chemistry ; Promoter Regions, Genetic ; RNA, Ribosomal, 16S/*analysis/chemistry ; Swine ; },
abstract = {Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 h. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S rDNA or 16S rRNA. A CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity, thus, demonstrating its use as a robust next-generation gene detection platform.},
}
@article {pmid32030512,
year = {2020},
author = {Nakayama, KI and Ishita, Y and Chihara, T and Okumura, M},
title = {Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus.},
journal = {Development genes and evolution},
volume = {230},
number = {3},
pages = {257-264},
doi = {10.1007/s00427-020-00651-y},
pmid = {32030512},
issn = {1432-041X},
support = {18K14716//Japan Society for the Promotion of Science/International ; 18H05369//Japan Society for the Promotion of Science/International ; JP19gm6310003//Japan Agency for Medical Research and Development/International ; JPMXS05S2900002//Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chemotaxis ; Electrophoresis, Microchip/methods ; Gene Editing/*methods ; Genetic Markers ; Genome, Helminth ; Heterozygote ; Microinjections/methods ; Models, Animal ; Mutation ; Nematoda/*genetics ; Phenotype ; RNA, Guide ; },
abstract = {CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus, CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus. We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus.},
}
@article {pmid32029904,
year = {2020},
author = {Wei, Y and Huai, C and Zhou, C and Gao, Y and Chen, L and Zhou, W and Wei, M and Qin, S},
title = {A methylation functional detection hepatic cell system validates correlation between DNA methylation and drug-induced liver injury.},
journal = {The pharmacogenomics journal},
volume = {20},
number = {5},
pages = {717-723},
pmid = {32029904},
issn = {1473-1150},
mesh = {CRISPR-Cas Systems ; Cell Line ; Chemical and Drug Induced Liver Injury/diagnosis/*genetics/metabolism ; Cytochrome P-450 CYP2D6/*genetics/metabolism ; Cytochrome P-450 CYP2E1/*genetics/metabolism ; *DNA Methylation ; Gene Editing ; Hepatocytes/*drug effects/metabolism/pathology ; Humans ; Pharmacogenetics ; *Pharmacogenomic Variants ; Rifampin/*toxicity ; },
abstract = {Drug-induced liver injury (DILI) is a life-threatening, adverse reaction to certain drugs. The onset and extent of DILI can vary drastically in different patients using identical drugs. Association studies suggested that subtle differences in DNA methylation may help explain the individual differences in DILI. However, there are very few experimental methods to confirm such associations. In this study, we established a novel DNA methylation functional detection system in human hepatocytes, using CRISPR/dCas9 for targeted modification of DNA methylation, and set four parameters to indicate the liver injury by cell model. Using this system, we validated the association of hypermethylation of CYP2D6 and CYP2E1 with rifampin-induced DILI. Our results revealed that, following treatment of HepaRG cells with rifampin, the methylation levels of CYP2D6 and CYP2E1 were inversely proportional to cell viability and glutathione content, and directly proportional to caspase 3/7 activity. We expect that our methylation detection system will serve as a useful tool in validating correlations between DNA methylation and DILI in other in vitro systems. Our results establish a foundation for future investigations to better understand the mechanisms underlying DILI and may aid in advancing personalized DILI medicine.},
}
@article {pmid32029872,
year = {2020},
author = {Kitamoto, K and Taketani, Y and Fujii, W and Inamochi, A and Toyono, T and Miyai, T and Yamagami, S and Kuroda, M and Usui, T and Ouchi, Y},
title = {Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2000},
pmid = {32029872},
issn = {2045-2322},
mesh = {Amino Acid Substitution ; Animals ; Arginine/genetics ; CRISPR-Cas Systems ; Corneal Dystrophies, Hereditary/*genetics/pathology ; Corneal Stroma/pathology/ultrastructure ; Cysteine/genetics ; *Disease Models, Animal ; Extracellular Matrix Proteins/*genetics ; Female ; Heterozygote ; Homozygote ; Humans ; Male ; Mice ; Mice, Transgenic ; Microscopy, Electron, Transmission ; Mutation ; Recombinational DNA Repair ; Transforming Growth Factor beta/*genetics ; },
abstract = {Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.},
}
@article {pmid32029861,
year = {2020},
author = {Sevestre, F and Facon, M and Wattebled, F and Szydlowski, N},
title = {Facilitating gene editing in potato: a Single-Nucleotide Polymorphism (SNP) map of the Solanum tuberosum L. cv. Desiree genome.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2045},
pmid = {32029861},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome, Plant/*genetics ; High-Throughput Nucleotide Sequencing ; Molecular Sequence Annotation ; Mutagenesis ; Plant Proteins/genetics ; Plants, Genetically Modified ; Polymorphism, Single Nucleotide ; RNA, Guide/genetics ; *Reverse Genetics ; Solanum tuberosum/*genetics ; Starch Synthase/genetics ; },
abstract = {Genome editing is a powerful tool for plant functional genomics allowing for multiallelic targeted mutagenesis. The recent development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) systems for gene editing in plants allows for simple, cost-effective introduction of site-specific double-stranded DNA breaks. The nuclear genomes of a homozygous doubled-monoploid potato clone (DM) and a heterozygous diploid clone (RH) have been sequenced in 2011. However, common potato cultivars display a highly heterozygous autotetraploid genome thus complicating target design for tetra-allelic gene editing. Here, we report on the SNP physical map of the widely used Solanum tuberosum L. cv. Desiree and on the position of the diverse indels providing an essential tool for target design in genome editing approaches. We used this tool for designing a specific gRNA and successfully knocking-out a newly discovered starch synthase gene (SS6) in potato. Resequencing data are publicly available at the Sequence Read Archive of the NCBI (accession number: PRJNA507597) and will represent a valuable resource for functional genomic studies of various metabolic pathways, cell and plant physiology as well as high-throughput reverse genetics in potato.},
}
@article {pmid32029769,
year = {2020},
author = {Wessel, GM and Kiyomoto, M and Shen, TL and Yajima, M},
title = {Genetic manipulation of the pigment pathway in a sea urchin reveals distinct lineage commitment prior to metamorphosis in the bilateral to radial body plan transition.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1973},
pmid = {32029769},
issn = {2045-2322},
support = {R01 GM125071/GM/NIGMS NIH HHS/United States ; R01 GM126043/GM/NIGMS NIH HHS/United States ; R01 GM132222/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Biosynthetic Pathways/genetics ; Body Patterning/*physiology ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Embryo, Nonmammalian ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; *Metamorphosis, Biological ; Oxygenases/genetics/metabolism ; Pigmentation/*physiology ; Pigments, Biological/*biosynthesis ; Polyketide Synthases/genetics/metabolism ; Polyketides/metabolism ; Sea Urchins/*growth &amp; development ; Transcription Factors/genetics/metabolism ; },
abstract = {Echinoderms display a vast array of pigmentation and patterning in larval and adult life stages. This coloration is thought to be important for immune defense and camouflage. However, neither the cellular nor molecular mechanism that regulates this complex coloration in the adult is known. Here we knocked out three different genes thought to be involved in the pigmentation pathway(s) of larvae and grew the embryos to adulthood. The genes tested were polyketide synthase (PKS), Flavin-dependent monooxygenase family 3 (FMO3) and glial cells missing (GCM). We found that disabling of the PKS gene at fertilization resulted in albinism throughout all life stages and throughout all cells and tissues of this animal, including the immune cells of the coelomocytes. We also learned that FMO3 is an essential modifier of the polyketide. FMO3 activity is essential for larval pigmentation, but in juveniles and adults, loss of FMO3 activity resulted in the animal becoming pastel purple. Linking the LC-MS analysis of this modified pigment to a naturally purple animal suggested a conserved echinochrome profile yielding a pastel purple. We interpret this result as FMO3 modifies the parent polyketide to contribute to the normal brown/green color of the animal, and that in its absence, other biochemical modifications are revealed, perhaps by other members of the large FMO family in this animal. The FMO modularity revealed here may be important in the evolutionary changes between species and for different immune challenges. We also learned that glial cells missing (GCM), a key transcription factor of the endomesoderm gene regulatory network of embryos in the sea urchin, is required for pigmentation throughout the life stages of this sea urchin, but surprisingly, is not essential for larval development, metamorphosis, or maintenance of adulthood. Mosaic knockout of either PKS or GCM revealed spatial lineage commitment in the transition from bilaterality of the larva to a pentaradial body plan of the adult. The cellular lineages identified by pigment presence or absence (wild-type or knock-out lineages, respectively) followed a strict oral/aboral profile. No circumferential segments were seen and instead we observed 10-fold symmetry in the segments of pigment expression. This suggests that the adult lineage commitments in the five outgrowths of the hydropore in the larva are early, complete, fixed, and each bilaterally symmetric. Overall, these results suggest that pigmentation of this animal is genetically determined and dependent on a population of pigment stem cells that are set-aside in a sub-region of each outgrowth of the pentaradial adult rudiment prior to metamorphosis. This study reveals the complex chemistry of pigment applicable to many organisms, and further, provides an insight into the key transitions from bilateral to pentaradial body plans unique to echinoderms.},
}
@article {pmid32029722,
year = {2020},
author = {DeWeirdt, PC and Sangree, AK and Hanna, RE and Sanson, KR and Hegde, M and Strand, C and Persky, NS and Doench, JG},
title = {Genetic screens in isogenic mammalian cell lines without single cell cloning.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {752},
pmid = {32029722},
issn = {2041-1723},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Line ; Clone Cells ; Gene Knockout Techniques ; Gene Library ; Gene Regulatory Networks ; Genetic Testing/*methods ; Humans ; Multigene Family ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists &amp; inhibitors/deficiency/genetics ; Poly (ADP-Ribose) Polymerase-1/antagonists &amp; inhibitors/deficiency/genetics ; Single-Cell Analysis ; bcl-X Protein/antagonists &amp; inhibitors/deficiency/genetics ; },
abstract = {Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.},
}
@article {pmid32029688,
year = {2020},
author = {Ebright, RY and Lee, S and Wittner, BS and Niederhoffer, KL and Nicholson, BT and Bardia, A and Truesdell, S and Wiley, DF and Wesley, B and Li, S and Mai, A and Aceto, N and Vincent-Jordan, N and Szabolcs, A and Chirn, B and Kreuzer, J and Comaills, V and Kalinich, M and Haas, W and Ting, DT and Toner, M and Vasudevan, S and Haber, DA and Maheswaran, S and Micalizzi, DS},
title = {Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis.},
journal = {Science (New York, N.Y.)},
volume = {367},
number = {6485},
pages = {1468-1473},
pmid = {32029688},
issn = {1095-9203},
support = {F30 CA224588/CA/NCI NIH HHS/United States ; U01 EB012493/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R21 CA220103/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; U01 CA214297/CA/NCI NIH HHS/United States ; K12 CA087723/CA/NCI NIH HHS/United States ; R01 GM100202/GM/NIGMS NIH HHS/United States ; F30 CA232407/CA/NCI NIH HHS/United States ; T32 CA009361/CA/NCI NIH HHS/United States ; R01 CA235412/CA/NCI NIH HHS/United States ; R01 CA129933/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/genetics/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; *Neoplasm Metastasis ; Neoplasm Transplantation ; Neoplastic Cells, Circulating/*pathology ; Ribosomal Proteins/*genetics ; Sequence Analysis, RNA ; },
abstract = {Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.},
}
@article {pmid32029687,
year = {2020},
author = {Stadtmauer, EA and Fraietta, JA and Davis, MM and Cohen, AD and Weber, KL and Lancaster, E and Mangan, PA and Kulikovskaya, I and Gupta, M and Chen, F and Tian, L and Gonzalez, VE and Xu, J and Jung, IY and Melenhorst, JJ and Plesa, G and Shea, J and Matlawski, T and Cervini, A and Gaymon, AL and Desjardins, S and Lamontagne, A and Salas-Mckee, J and Fesnak, A and Siegel, DL and Levine, BL and Jadlowsky, JK and Young, RM and Chew, A and Hwang, WT and Hexner, EO and Carreno, BM and Nobles, CL and Bushman, FD and Parker, KR and Qi, Y and Satpathy, AT and Chang, HY and Zhao, Y and Lacey, SF and June, CH},
title = {CRISPR-engineered T cells in patients with refractory cancer.},
journal = {Science (New York, N.Y.)},
volume = {367},
number = {6481},
pages = {},
doi = {10.1126/science.aba7365},
pmid = {32029687},
issn = {1095-9203},
support = {R01 CA120409/CA/NCI NIH HHS/United States ; U01 AG066100/AG/NIA NIH HHS/United States ; },
mesh = {*Adoptive Transfer ; Aged ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Engineering ; Female ; *Gene Editing ; Humans ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor/genetics ; Receptors, Antigen, T-Cell, alpha-beta/*genetics ; T-Lymphocytes/*immunology/*transplantation ; Transgenes ; },
abstract = {CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.},
}
@article {pmid32029609,
year = {2020},
author = {Couzin-Frankel, J},
title = {CRISPR takes on cancer.},
journal = {Science (New York, N.Y.)},
volume = {367},
number = {6478},
pages = {616},
doi = {10.1126/science.367.6478.616},
pmid = {32029609},
issn = {1095-9203},
mesh = {Antigens, Neoplasm/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockdown Techniques ; Humans ; Immunotherapy, Adoptive/*methods ; Membrane Proteins/genetics ; T-Lymphocytes/*immunology/*transplantation ; },
}
@article {pmid32029474,
year = {2020},
author = {Majumder, S and Kono, M and Lee, YT and Byrnes, C and Li, C and Tuymetova, G and Proia, RL},
title = {A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {13},
pages = {4341-4349},
pmid = {32029474},
issn = {1083-351X},
support = {P30 CA138313/CA/NCI NIH HHS/United States ; P30 GM103339/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/*genetics ; CRISPR-Cas Systems/genetics ; Disease Resistance/*genetics ; Gene Expression Regulation ; Gene Knockout Techniques ; Genome, Human/genetics ; Globosides/*genetics ; HeLa Cells ; Humans ; Lipid Metabolism/genetics ; Lipids/biosynthesis/genetics ; Mice ; Mice, Knockout ; Receptors, Aryl Hydrocarbon/*genetics ; Serine C-Palmitoyltransferase/*genetics ; Shiga Toxin/pharmacology ; Signal Transduction/genetics ; Sphingolipids/*biosynthesis/genetics ; Trihexosylceramides/*genetics ; },
abstract = {Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.},
}
@article {pmid32029273,
year = {2020},
author = {Kurihara, T and Kouyama-Suzuki, E and Satoga, M and Li, X and Badawi, M and Thiha, and Baig, DN and Yanagawa, T and Uemura, T and Mori, T and Tabuchi, K},
title = {DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons.},
journal = {Biochemical and biophysical research communications},
volume = {524},
number = {3},
pages = {621-628},
doi = {10.1016/j.bbrc.2020.01.132},
pmid = {32029273},
issn = {1090-2104},
mesh = {Actins/metabolism ; Animals ; Base Sequence ; Brain/*cytology ; CRISPR-Cas Systems/*genetics ; *DNA End-Joining Repair ; *Gene Knock-In Techniques ; Green Fluorescent Proteins/metabolism ; Mice, Inbred ICR ; Neurons/*metabolism ; Pyramidal Cells/metabolism ; RNA, Guide/metabolism ; Rad51 Recombinase ; },
abstract = {Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the β-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the β-actin donor template DNA, and found that the knock-in efficiency was increased to ∼14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different β-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR).},
}
@article {pmid32029227,
year = {2020},
author = {Barnes, TM and Shah, K and Allison, MB and Steinl, GK and Gordian, D and Sabatini, PV and Tomlinson, AJ and Cheng, W and Jones, JC and Zhu, Q and Faber, C and Myers, MG},
title = {Identification of the leptin receptor sequences crucial for the STAT3-Independent control of metabolism.},
journal = {Molecular metabolism},
volume = {32},
number = {},
pages = {168-175},
pmid = {32029227},
issn = {2212-8778},
support = {R01 DK056731/DK/NIDDK NIH HHS/United States ; F30 DK097861/DK/NIDDK NIH HHS/United States ; P30 DK034933/DK/NIDDK NIH HHS/United States ; P30 DK020572/DK/NIDDK NIH HHS/United States ; T32 DK101357/DK/NIDDK NIH HHS/United States ; R37 DK056731/DK/NIDDK NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Obesity/metabolism ; Receptors, Leptin/*genetics/metabolism ; STAT3 Transcription Factor/genetics/*metabolism ; Signal Transduction ; },
abstract = {BACKGROUND: Leptin acts via its receptor, LepRb, on specialized neurons in the brain to modulate energy balance and glucose homeostasis. LepRb→STAT3 signaling plays a crucial role in leptin action, but LepRb also mediates an additional as-yet-unidentified signal (Signal 2) that is important for leptin action. Signal 2 requires LepRb regions in addition to those required for JAK2 activation but operates independently of STAT3 and LepRb phosphorylation sites.<br><br>METHODS: To identify LepRb sequences that mediate Signal 2, we used CRISPR/Cas9 to generate five novel mouse lines containing COOH-terminal truncation mutants of LepRb. We analyzed the metabolic phenotype and measures of hypothalamic function for these mouse lines.<br><br>RESULTS: We found that deletion of LepRb sequences between residues 921 and 960 dramatically worsens metabolic control and alters hypothalamic function relative to smaller truncations. We also found that deletion of the regions including residues 1013-1053 and 960-1013 each decreased obesity compared to deletions that included additional COOH-terminal residues.<br><br>CONCLUSIONS: LepRb sequences between residues 921 and 960 mediate the STAT3 and LepRb phosphorylation-independent second signal that contributes to the control of energy balance and metabolism by leptin/LepRb. In addition to confirming the inhibitory role of the region (residues 961-1013) containing Tyr985, we also identified the region containing residues 1013-1053 (which contains no Tyr residues) as a second potential mediator of LepRb inhibition. Thus, the intracellular domain of LepRb mediates multiple Tyr-independent signals.},
}
@article {pmid32028669,
year = {2020},
author = {Zhao, W and Wang, Y and Liang, FS},
title = {Chemical and Light Inducible Epigenome Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32028669},
issn = {1422-0067},
support = {R21 HG010673/HG/NHGRI NIH HHS/United States ; R21HG010673/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Methylation ; *Epigenesis, Genetic ; *Epigenome ; Gene Editing/*methods ; Humans ; *Light ; Small Molecule Libraries/*pharmacology ; },
abstract = {The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers a revolutionary tool for mechanistic studies in epigenome regulation as well as the development of novel epigenome therapies. Inducible and reversible epigenome editing provides unique temporal control critical for understanding the dynamics and kinetics of epigenome regulation. This review summarizes the progress in the development of spatiotemporal-specific tools using small molecules or light as inducers to achieve the conditional control of epigenome editing and their applications in epigenetic research.},
}
@article {pmid32027839,
year = {2020},
author = {Kempton, HR and Goudy, LE and Love, KS and Qi, LS},
title = {Multiple Input Sensing and Signal Integration Using a Split Cas12a System.},
journal = {Molecular cell},
volume = {78},
number = {1},
pages = {184-191.e3},
doi = {10.1016/j.molcel.2020.01.016},
pmid = {32027839},
issn = {1097-4164},
mesh = {Breast Neoplasms/genetics/therapy ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; *Cell Engineering ; Cell Line, Tumor ; Dimerization ; Female ; HEK293 Cells ; Humans ; Transcriptional Activation ; },
abstract = {The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.},
}
@article {pmid32027059,
year = {2020},
author = {Ni, XY and Zhou, ZD and Huang, J and Qiao, X},
title = {Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster.},
journal = {Archives of insect biochemistry and physiology},
volume = {104},
number = {1},
pages = {e21662},
doi = {10.1002/arch.21662},
pmid = {32027059},
issn = {1520-6327},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Drosophila melanogaster/*genetics ; Gene Editing/*methods ; Genome, Insect ; },
abstract = {Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off-target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5'-NG-3' PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome-wide off-target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome-editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non-NGG PAM site TGA, xCas9 could produce DNA cleavage and indel-mediated disruption on the target gene. However, for other non-NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.},
}
@article {pmid32026884,
year = {2020},
author = {Santillán-Doherty, P and Grether-González, P and Medina-Arellano, MJ and Chan, S and Tapia-Ibargüengoitia, R and Brena-Sesma, I and Fuente, RC and Linares-Salgado, J and Mendoza-Cárdenas, H and Muñoz-Fernández, L and Schiavon, R},
title = {Considerations on genetic engineering: regarding the birth of twins subjected to gene edition.},
journal = {Gaceta medica de Mexico},
volume = {156},
number = {1},
pages = {53-59},
doi = {10.24875/GMM.19005182},
pmid = {32026884},
issn = {0016-3813},
mesh = {Bioethical Issues ; *CRISPR-Cas Systems ; China ; Conflict of Interest ; Female ; Gene Editing/*ethics ; Genetic Engineering/classification/ethics ; Genome, Human ; HIV Infections/prevention &amp; control ; Humans ; Informed Consent/ethics ; Publishing/ethics ; Receptors, CCR5/*genetics ; Research Design ; Sperm Injections, Intracytoplasmic ; Therapeutic Human Experimentation/ethics ; Twins, Dizygotic ; },
abstract = {In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.},
}
@article {pmid32026742,
year = {2020},
author = {Kabadi, A and McDonnell, E and Frank, CL and Drowley, L},
title = {Applications of Functional Genomics for Drug Discovery.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {8},
pages = {823-842},
doi = {10.1177/2472555220902092},
pmid = {32026742},
issn = {2472-5560},
mesh = {CRISPR-Cas Systems/genetics ; Drug Discovery/*trends ; Epigenome/genetics ; Gene Editing ; *Genetic Predisposition to Disease ; Genome, Human/*genetics ; Genome-Wide Association Study ; Genomics/*trends ; Genotype ; Humans ; Phenotype ; RNA, Small Interfering/genetics ; },
abstract = {Many diseases, such as diabetes, autoimmune diseases, cancer, and neurological disorders, are caused by a dysregulation of a complex interplay of genes. Genome-wide association studies have identified thousands of disease-linked polymorphisms in the human population. However, detailing the causative gene expression or functional changes underlying those associations has been elusive in many cases. Functional genomics is an emerging field of research that aims to deconvolute the link between genotype and phenotype by making use of large -omic data sets and next-generation gene and epigenome editing tools to perturb genes of interest. Here we review how functional genomic tools can be used to better understand the biological interplay between genes, improve disease modeling, and identify novel drug targets. Incorporation of functional genomic capabilities into conventional drug development pipelines is predicted to expedite the development of first-in-class therapeutics.},
}
@article {pmid32024996,
year = {2020},
author = {Carlevaro-Fita, J and Lanzós, A and Feuerbach, L and Hong, C and Mas-Ponte, D and Pedersen, JS and , and Johnson, R and , },
title = {Cancer LncRNA Census reveals evidence for deep functional conservation of long noncoding RNAs in tumorigenesis.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {56},
pmid = {32024996},
issn = {2399-3642},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Cell Transformation, Neoplastic/*genetics ; Databases, Genetic ; *Disease Susceptibility ; Evolution, Molecular ; Genome, Human ; Genomics/methods ; Humans ; Neoplasms/*genetics ; Polymorphism, Single Nucleotide ; *RNA, Long Noncoding ; },
abstract = {Long non-coding RNAs (lncRNAs) are a growing focus of cancer genomics studies, creating the need for a resource of lncRNAs with validated cancer roles. Furthermore, it remains debated whether mutated lncRNAs can drive tumorigenesis, and whether such functions could be conserved during evolution. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we introduce the Cancer LncRNA Census (CLC), a compilation of 122 GENCODE lncRNAs with causal roles in cancer phenotypes. In contrast to existing databases, CLC requires strong functional or genetic evidence. CLC genes are enriched amongst driver genes predicted from somatic mutations, and display characteristic genomic features. Strikingly, CLC genes are enriched for driver mutations from unbiased, genome-wide transposon-mutagenesis screens in mice. We identified 10 tumour-causing mutations in orthologues of 8 lncRNAs, including LINC-PINT and NEAT1, but not MALAT1. Thus CLC represents a dataset of high-confidence cancer lncRNAs. Mutagenesis maps are a novel means for identifying deeply-conserved roles of lncRNAs in tumorigenesis.},
}
@article {pmid32024985,
year = {2020},
author = {Bird, L},
title = {MR1-restricted pan-cancer T cells.},
journal = {Nature reviews. Immunology},
volume = {20},
number = {3},
pages = {141},
pmid = {32024985},
issn = {1474-1741},
mesh = {*CRISPR-Cas Systems ; *Early Detection of Cancer ; Histocompatibility Antigens Class I/genetics ; Minor Histocompatibility Antigens ; },
}
@article {pmid32023921,
year = {2020},
author = {Holmes, AC and Zagnoli-Vieira, G and Caldecott, KW and Semler, BL},
title = {Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation.},
journal = {Viruses},
volume = {12},
number = {2},
pages = {},
pmid = {32023921},
issn = {1999-4915},
support = {MRN/N50189X/1/MRC_/Medical Research Council/United Kingdom ; R21 AI126879/AI/NIAID NIH HHS/United States ; R01 AI026765/AI/NIAID NIH HHS/United States ; T32 AI007163/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; DNA-Binding Proteins/*genetics/metabolism ; Epithelial Cells/virology ; Gene Knockout Techniques ; Humans ; Phosphoric Diester Hydrolases/*genetics/metabolism ; Picornaviridae/*genetics/growth &amp; development ; Picornaviridae Infections/*virology ; Polyribosomes/immunology ; RNA, Double-Stranded/genetics ; RNA, Viral/genetics ; Retina/cytology ; },
abstract = {In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5' phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.},
}
@article {pmid32023459,
year = {2020},
author = {Dubreuil, MM and Morgens, DW and Okumoto, K and Honsho, M and Contrepois, K and Lee-McMullen, B and Traber, GM and Sood, RS and Dixon, SJ and Snyder, MP and Fujiki, Y and Bassik, MC},
title = {Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway.},
journal = {Cell reports},
volume = {30},
number = {5},
pages = {1417-1433.e7},
pmid = {32023459},
issn = {2211-1247},
support = {R01 GM122923/GM/NIGMS NIH HHS/United States ; DP2 HD084069/HD/NICHD NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; T32 GM099608/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Catalase/metabolism ; Cytoprotection ; Cytosol/metabolism ; Genome, Human ; Glucose/metabolism ; Glycolysis ; HeLa Cells ; Humans ; K562 Cells ; *Oxidative Stress ; *Pentose Phosphate Pathway ; Peroxisomes/*metabolism ; Phosphogluconate Dehydrogenase ; Protein Transport ; RNA, Small Interfering/metabolism ; Reactive Oxygen Species/metabolism ; },
abstract = {Reactive oxygen species (ROS) play critical roles in metabolism and disease, yet a comprehensive analysis of the cellular response to oxidative stress is lacking. To systematically identify regulators of oxidative stress, we conducted genome-wide Cas9/CRISPR and shRNA screens. This revealed a detailed picture of diverse pathways that control oxidative stress response, ranging from the TCA cycle and DNA repair machineries to iron transport, trafficking, and metabolism. Paradoxically, disrupting the pentose phosphate pathway (PPP) at the level of phosphogluconate dehydrogenase (PGD) protects cells against ROS. This dramatically alters metabolites in the PPP, consistent with rewiring of upper glycolysis to promote antioxidant production. In addition, disruption of peroxisomal import unexpectedly increases resistance to oxidative stress by altering the localization of catalase. Together, these studies provide insights into the roles of peroxisomal matrix import and the PPP in redox biology and represent a rich resource for understanding the cellular response to oxidative stress.},
}
@article {pmid32023146,
year = {2020},
author = {Zhao, K and Foster, J and Ridgway, ND},
title = {Oxysterol-binding protein-related protein 1 variants have opposing cholesterol transport activities from the endolysosomes.},
journal = {Molecular biology of the cell},
volume = {31},
number = {8},
pages = {793-802},
pmid = {32023146},
issn = {1939-4586},
support = {MOP-15284//CIHR/Canada ; },
mesh = {ATP Binding Cassette Transporter 1/metabolism ; Acetyl-CoA C-Acetyltransferase/metabolism ; Amino Acid Motifs ; Biological Transport ; CRISPR-Cas Systems ; Cell Membrane/metabolism ; Cholesterol/*metabolism ; Endoplasmic Reticulum/metabolism ; Endosomes/*metabolism ; HeLa Cells ; Humans ; Intracellular Membranes/metabolism ; Lysosomes/*metabolism ; Membrane Lipids/metabolism ; Protein Domains ; Protein Isoforms/metabolism ; Receptors, LDL/metabolism ; Receptors, Steroid/deficiency/genetics/*metabolism ; Sequence Deletion ; },
abstract = {OSBPL1 encodes the full-length oxysterol-binding protein-related protein ORP1L, which transports LDL-derived cholesterol at membrane contacts between the late endosomes/lysosomes (LEL) and the endoplasmic reticulum (ER). OSBPL1 also encodes the truncated variant ORP1S that contains only the C-terminal lipid binding domain. HeLa cells in which both variants were knocked out (ORP1-null) were used to determine the functional relationship between ORP1L and ORP1S with respect to cellular cholesterol localization and regulation. ORP1-null cells accumulated cholesterol in LEL and had reduced plasma membrane (PM) cholesterol. PM cholesterol was restored by expression of wild-type ORP1S or a phosphatidylinositol phosphate-binding mutant but not by a sterol-binding mutant. Expression of ORP2, another truncated variant, also restored PM cholesterol in ORP1-null cells. Consistent with a LEL-to-PM cholesterol transport activity, a small fraction of ORP1S was detected on the PM. As a consequence of reduced delivery of cholesterol to the PM in ORP1-null cells, cholesterol was diverted to the ER resulting in normalization of de novo cholesterol synthesis. The deficiency in PM cholesterol also reduced ABCA1-dependent cholesterol efflux and LDL receptor activity in ORP1-null cells. We conclude that ORP1S, which lacks discrete membrane-targeting motifs, transports cholesterol from LEL to the PM.},
}
@article {pmid32020912,
year = {2020},
author = {Nandy, D and Maity, A and Mitra, AK},
title = {Target-specific gene delivery in plant systems and their expression: Insights into recent developments.},
journal = {Journal of biosciences},
volume = {45},
number = {},
pages = {},
pmid = {32020912},
issn = {0973-7138},
mesh = {CRISPR-Cas Systems/*genetics ; Endonucleases ; Gene Editing/*methods ; Gene Targeting ; Genetic Engineering/*methods ; Genetic Therapy ; Homologous Recombination/*genetics ; Nanotubes, Carbon ; Plants/*genetics/*microbiology ; Transcription Activator-Like Effector Nucleases/*genetics ; Zinc Finger Nucleases/*genetics ; },
abstract = {In order to improve crop plants in terms of their yield, drought resistance, pest resistance, nutritional value, etc., modern agriculture has relied upon plant genetic engineering. Since the advent of recombinant DNA technology, several tools have been used for genetic transformations in plants such as Agrobacterium tumefaciens, virus-mediated gene transfer, direct gene transfer systems such as electroporation, particle gun, microinjection and chemical methods. All these traditional methods lack specificity and the transgenes are integrated at random sites in the plant DNA. Recently novel techniques for gene targeting have evolved such as engineered nucleases such as Zinc Finger Nucleases, Transcription Activator like effector nucleases, Clustered regular interspaced short palindromic repeats. Other advances include improvement in tools for delivery of gene editing components which include carrier proteins, and carbon nanotubes. The present review focuses on the latest techniques for target specific gene delivery in plants, their expression and future directions in plant biotechnology.},
}
@article {pmid32020639,
year = {2020},
author = {Chen, N and Li, Y and Huang, N and Yao, J and Luo, WF and Jiang, Q},
title = {The Nrf2 activator MIND4-17 protects retinal ganglion cells from high glucose-induced oxidative injury.},
journal = {Journal of cellular physiology},
volume = {235},
number = {10},
pages = {7204-7213},
doi = {10.1002/jcp.29619},
pmid = {32020639},
issn = {1097-4652},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Diabetic Retinopathy/genetics/metabolism/pathology ; Gene Knockout Techniques ; Glucose/*metabolism/toxicity ; Humans ; Kelch-Like ECH-Associated Protein 1/antagonists &amp; inhibitors/genetics/metabolism ; Mice ; Mice, Inbred BALB C ; NF-E2-Related Factor 2/antagonists &amp; inhibitors/genetics/*metabolism ; Oxidative Stress/drug effects ; Retinal Ganglion Cells/*drug effects/*metabolism/pathology ; Signal Transduction/drug effects ; Thiazoles/*pharmacology ; },
abstract = {Diabetic retinopathy (DR) is a leading cause of acquired blindness among adults. High glucose (HG) induces oxidative injury and apoptosis in retinal ganglion cells (RGCs), serving as a primary pathological mechanism of DR. MIND4-17 activates nuclear-factor-E2-related factor 2 (Nrf2) signaling via modifying one cysteine (C151) residue of Kelch-like ECH-associated protein 1 (Keap1). The current study tested its effect in HG-treated primary murine RGCs. We show that MIND4-17 disrupted Keap1-Nrf2 association, leading to Nrf2 protein stabilization and nuclear translocation, causing subsequent expression of key Nrf2 target genes, including heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. Functional studies showed that MIND4-17 pretreatment significantly inhibited HG-induced cytotoxicity and apoptosis in primary murine RGCs. Reactive oxygen species production and oxidative injury in HG-treated murine RGCs were attenuated by MIND4-17. Nrf2 silencing (by targeted small interfering RNA) or knockout (by CRISPR/Cas9 method) abolished MIND4-17-induced RGC cytoprotection against HG. Additionally, Keap1 knockout or silencing mimicked and abolished MIND4-17-induced activity in RGCs. In vivo, MIND4-17 intravitreal injection activated Nrf2 signaling and attenuated retinal dysfunction by light damage in mice. We conclude that MIND4-17 activates Nrf2 signaling to protect murine RGCs from HG-induced oxidative injury.},
}
@article {pmid32020276,
year = {2020},
author = {Ministro, JH and Oliveira, SS and Oliveira, JG and Cardoso, M and Aires-da-Silva, F and Corte-Real, S and Goncalves, J},
title = {Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {6},
pages = {2501-2512},
doi = {10.1007/s00253-020-10423-3},
pmid = {32020276},
issn = {1432-0614},
mesh = {Antibodies/*genetics/immunology ; Antigens/*immunology ; *CRISPR-Cas Systems ; Endoplasmic Reticulum/*immunology ; HEK293 Cells ; High-Throughput Screening Assays ; Humans ; Jurkat Cells ; *Peptide Library ; Proof of Concept Study ; },
abstract = {Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.},
}
@article {pmid32020049,
year = {2020},
author = {Javidi-Parsijani, P and Lyu, P and Makani, V and Sarhan, WM and Yoo, KW and El-Korashi, L and Atala, A and Lu, B},
title = {CRISPR/Cas9 increases mitotic gene conversion in human cells.},
journal = {Gene therapy},
volume = {27},
number = {6},
pages = {281-296},
pmid = {32020049},
issn = {1476-5462},
mesh = {*CRISPR-Cas Systems ; *Gene Conversion ; Gene Editing ; HEK293 Cells ; Humans ; },
abstract = {Gene conversion is a process of transferring genetic material from one homologous sequence to another. Most reported gene conversions are meiotic although mitotic gene conversion is also described. When using CRISPR/Cas9 to target the human hemoglobin subunit beta (HBB) gene, hemoglobin subunit delta (HBD) gene footprints were observed in HBB gene. However, it is unclear whether these were the results of gene conversion or PCR-mediated sequence shuffling between highly homologous sequences. Here we provide evidence that the HBD footprints in HBB were indeed results of gene conversion. We demonstrated that the CRISPR/Cas9 facilitated unidirectional sequence transfer from the homologous gene without double-strand breaks (DSB) to the one with DSBs, and showed that the rates of HBD footprint in HBB were positively correlated to the HBB insertion and deletion rates. We further showed that when targeting HBD gene, HBB footprints could also be observed in HBD gene. The mitotic gene conversion was observed not only in immortalized HEK293T cells, but also in human primary cells. Our work reveals mitotic gene conversion as an often overlooked effect of CRISPR/Cas9-mediated genome editing.},
}
@article {pmid32020014,
year = {2020},
author = {Xu, D and Cai, Y and Tang, L and Han, X and Gao, F and Cao, H and Qi, F and Kapranov, P},
title = {A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1794},
pmid = {32020014},
issn = {2045-2322},
support = {31671382//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; Humans ; RNA, Long Noncoding/genetics/*metabolism ; },
abstract = {Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs - very long intergenic non-coding (vlinc) RNAs - in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.},
}
@article {pmid32019402,
year = {2020},
author = {Wang, W and Wang, X and Luo, C and Pu, Q and Yin, Q and Xu, L and Peng, X and Ma, S and Xia, Q and Liu, S},
title = {Let-7 microRNA is a critical regulator in controlling the growth and function of silk gland in the silkworm.},
journal = {RNA biology},
volume = {17},
number = {5},
pages = {703-717},
pmid = {32019402},
issn = {1555-8584},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*physiology ; CRISPR-Cas Systems ; Energy Metabolism ; Exocrine Glands/*metabolism/pathology ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Knockdown Techniques ; Gene Targeting ; MicroRNAs/chemistry/*genetics ; Models, Biological ; Nucleic Acid Conformation ; Nutrients/metabolism ; RNA Interference ; Signal Transduction ; Silk/*biosynthesis ; Transcriptome ; Transgenes ; },
abstract = {The silk gland is characterized by high protein synthesis. However, the molecular mechanisms controlling silk gland growth and silk protein synthesis remain undetermined. Here we demonstrated that CRISPR/Cas9-based knockdown of let-7 or the whole cluster promoted endoreduplication and enlargement of the silk gland, accompanied by changing silk yield, whereas transgenic overexpression of let-7 led to atrophy and degeneration of the silk gland. Mechanistically, let-7 controls cell growth in the silk gland through coordinating nutrient metabolism processes and energy signalling pathways. Transgenic overexpression of pyruvate carboxylase, a novel target of let-7, resulted in enlargement of the silk glands, which is consistent with the abnormal phenotype of the let-7 knockdown. Overall, our data reveal a previously unknown miRNA-mediated regulation of silk gland growth and physiology and shed light on involvement of let-7 as a critical stabilizer and booster in carbohydrate metabolism, which may have important implications for understanding of the molecular mechanism and physiological function of specialized organs in other species.},
}
@article {pmid32017998,
year = {2020},
author = {Li, C and Li, YH and Li, Y and Lu, H and Hong, H and Tian, Y and Li, H and Zhao, T and Zhou, X and Liu, J and Zhou, X and Jackson, SA and Liu, B and Qiu, LJ},
title = {A Domestication-Associated Gene GmPRR3b Regulates the Circadian Clock and Flowering Time in Soybean.},
journal = {Molecular plant},
volume = {13},
number = {5},
pages = {745-759},
doi = {10.1016/j.molp.2020.01.014},
pmid = {32017998},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/genetics ; Circadian Clocks/*genetics ; *Domestication ; Evolution, Molecular ; Flowers/*genetics ; *Gene Expression Regulation, Plant ; *Genes, Plant ; Genome-Wide Association Study ; Geography ; Haplotypes/genetics ; Mutation/genetics ; Plant Breeding ; Plant Proteins/*genetics/metabolism ; Quantitative Trait Loci/genetics ; RNA, Messenger/genetics/metabolism ; Soybeans/*genetics ; Subcellular Fractions/metabolism ; Time Factors ; Transcription, Genetic ; },
abstract = {Improved soybean cultivars have been adapted to grow at a wide range of latitudes, enabling expansion of cultivation worldwide. However, the genetic basis of this broad adaptation is still not clear. Here, we report the identification of GmPRR3b as a major flowering time regulatory gene that has been selected during domestication and genetic improvement for geographic expansion. Through a genome-wide association study of a diverse soybean landrace panel consisting of 279 accessions, we identified 16 candidate quantitative loci associated with flowering time and maturity time. The strongest signal resides in the known flowering gene E2, verifying the effectiveness of our approach. We detected strong signals associated with both flowering and maturity time in a genomic region containing GmPRR3b. Haplotype analysis revealed that GmPRR3b[H6] is the major form of GmPRR3b that has been utilized during recent breeding of modern cultivars. mRNA profiling analysis showed that GmPRR3b[H6] displays rhythmic and photoperiod-dependent expression and is preferentially induced under long-day conditions. Overexpression of GmPRR3b[H6] increased main stem node number and yield, while knockout of GmPRR3b[H6] using CRISPR/Cas9 technology delayed growth and the floral transition. GmPRR3b[H6] appears to act as a transcriptional repressor of multiple predicted circadian clock genes, including GmCCA1a, which directly upregulates J/GmELF3a to modulate flowering time. The causal SNP (Chr12:5520945) likely endows GmPRR3b[H6] a moderate but appropriate level of activity, leading to early flowering and vigorous growth traits preferentially selected during broad adaptation of landraces and improvement of cultivars.},
}
@article {pmid32017351,
year = {2020},
author = {Göser, V and Kehl, A and Röder, J and Hensel, M},
title = {Role of the ESCRT-III complex in controlling integrity of the Salmonella-containing vacuole.},
journal = {Cellular microbiology},
volume = {22},
number = {6},
pages = {e13176},
doi = {10.1111/cmi.13176},
pmid = {32017351},
issn = {1462-5822},
mesh = {Adaptor Proteins, Signal Transducing ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Endosomal Sorting Complexes Required for Transport/genetics/*metabolism ; Endosomes ; Epithelial Cells/microbiology ; HeLa Cells ; Humans ; Lysosomes ; Salmonella/*metabolism ; Salmonella enterica/metabolism ; Salmonella typhimurium/metabolism ; Type III Secretion Systems/metabolism ; Vacuoles/*microbiology ; },
abstract = {Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2-encoded type III secretion system (SPI2-T3SS). Fusion events of endosomal compartments with the Salmonella-containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella-modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT-III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT-III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2-T3SS-deficient mutant strain was partially abrogated. ESCRT-dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT-III recruitment in Salmonella-infected host cells.},
}
@article {pmid32017225,
year = {2020},
author = {Cicalese, MP and Aiuti, A},
title = {New perspectives in gene therapy for inherited disorders.},
journal = {Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology},
volume = {31 Suppl 24},
number = {},
pages = {5-7},
doi = {10.1111/pai.13149},
pmid = {32017225},
issn = {1399-3038},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy/*methods/trends ; Genetic Vectors ; Heredodegenerative Disorders, Nervous System/genetics/*therapy ; Humans ; Immune System Diseases/genetics/*therapy ; Zinc Finger Nucleases/genetics/*metabolism ; },
abstract = {Gene therapy has become promising in many fields of medicine, as a single treatment could allow long-lasting and curative benefits. New medicines based on cell gene correction are expected to occur in upcoming years and will be hopefully part of the therapeutic armamentarium for inherited and acquired diseases. Issues related to the costs of these new therapies and access to care for all patients, and procedures and expertise needed to facilitate their application worldwide require to be addressed, together with long-term safety and efficacy monitoring.},
}
@article {pmid32017087,
year = {2020},
author = {Li, S and Wang, Z and Tong, H and Li, S and Yan, Y},
title = {TCP11L2 promotes bovine skeletal muscle-derived satellite cell migration and differentiation via FMNL2.},
journal = {Journal of cellular physiology},
volume = {235},
number = {10},
pages = {7183-7193},
doi = {10.1002/jcp.29617},
pmid = {32017087},
issn = {1097-4652},
mesh = {Actin-Related Protein 2-3 Complex/metabolism ; Animals ; CRISPR-Cas Systems ; Cattle ; Cell Differentiation/genetics/physiology ; Cell Movement/genetics/physiology ; Cells, Cultured ; Formins/*metabolism ; Mice ; Muscle Development/genetics/physiology ; Muscle Proteins/chemistry/genetics/*metabolism ; Satellite Cells, Skeletal Muscle/cytology/*metabolism ; t-Complex Genome Region ; },
abstract = {T-complex 11 like 2 (TCP11L2) is a protein containing a serine-rich region in its N-terminal region. However, the function of TCP11L2 is unclear. Here, we showed that TCP11L2 expression gradually increased during muscle-derived satellite cell (MDSC) differentiation in vitro, reaching a peak on Day 3, which is the migration and fusion stage of MDSCs. Using CRISPR/dCas9 gene-editing technology to elevate or repress the expression of TCP11L2, we also showed that TCP11L2 promoted MDSC differentiation. Moreover, wound-healing assays showed that TCP11L2 promoted the migration of MDSCs during differentiation. Additionally, immunofluorescence analyses showed that TCP11L2 was mainly distributed around the microfilament and microtubules. Furthermore, the expression of TCP11L2 affected the expression of actin-related protein 2/3 (ARP2/3) complex. Co-immunoprecipitation assays and immunofluorescence analysis showed that TCP11L2 interacted with formin-like 2 (FMNL2). This protein promoted migration of bovine MDSCs by affecting the expression of ARP2/3. Finally, the activities of TCP11L2 during MDSC differentiation and migration were blocked when FMNL2 was inhibited. Taken together, our data established that TCP11L2 interacted with FMNL2 to promote MDSC migration and differentiation.},
}
@article {pmid32016561,
year = {2020},
author = {Yue, JJ and Hong, CY and Wei, P and Tsai, YC and Lin, CS},
title = {How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis.},
journal = {Rice (New York, N.Y.)},
volume = {13},
number = {1},
pages = {9},
pmid = {32016561},
issn = {1939-8425},
abstract = {The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.},
}
@article {pmid32016291,
year = {2020},
author = {Hampton, T},
title = {DNA Prime Editing: A New CRISPR-Based Method to Correct Most Disease-Causing Mutations.},
journal = {JAMA},
volume = {323},
number = {5},
pages = {405-406},
doi = {10.1001/jama.2019.21827},
pmid = {32016291},
issn = {1538-3598},
mesh = {*CRISPR-Cas Systems ; *DNA ; Gene Editing/*methods ; Humans ; *Mutation ; },
}
@article {pmid32015438,
year = {2020},
author = {Gan, W and Dai, X and Dai, X and Xie, J and Yin, S and Zhu, J and Wang, C and Liu, Y and Guo, J and Wang, M and Liu, J and Hu, J and Quinton, RJ and Ganem, NJ and Liu, P and Asara, JM and Pandolfi, PP and Yang, Y and He, Z and Gao, G and Wei, W},
title = {LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control.},
journal = {Nature cell biology},
volume = {22},
number = {2},
pages = {246-256},
pmid = {32015438},
issn = {1476-4679},
support = {R01 CA200651/CA/NCI NIH HHS/United States ; P30 DK123704/DK/NIDDK NIH HHS/United States ; R00 CA181342/CA/NCI NIH HHS/United States ; R01 GM094777/GM/NIGMS NIH HHS/United States ; P20 GM130457/GM/NIGMS NIH HHS/United States ; P30 EY012196/EY/NEI NIH HHS/United States ; R00 CA207867/CA/NCI NIH HHS/United States ; R01 CA222571/CA/NCI NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; R01 GM117150/GM/NIGMS NIH HHS/United States ; P01 CA120964/CA/NCI NIH HHS/United States ; R01 CA200573/CA/NCI NIH HHS/United States ; R01 CA177910/CA/NCI NIH HHS/United States ; U54 HD090255/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Colonic Neoplasms/genetics/metabolism/pathology ; Female ; Gene Editing ; *Gene Expression Regulation, Developmental ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Heterografts ; Hippo Signaling Pathway ; Humans ; Liver/abnormalities/metabolism ; MCF-7 Cells ; Mechanistic Target of Rapamycin Complex 1/*genetics/metabolism ; Mice ; Mice, Nude ; Mice, Transgenic ; Myocardium/metabolism/pathology ; Neurofibromin 2/deficiency/genetics ; Organ Size ; Protein Serine-Threonine Kinases/deficiency/*genetics/metabolism ; Ras Homolog Enriched in Brain Protein/*genetics/metabolism ; Regulatory-Associated Protein of mTOR/*genetics/metabolism ; Signal Transduction ; Tumor Suppressor Proteins/deficiency/*genetics ; },
abstract = {The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor[+/+] mice, Raptor[D/D] knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.},
}
@article {pmid32015437,
year = {2020},
author = {Smargon, AA and Shi, YJ and Yeo, GW},
title = {RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering.},
journal = {Nature cell biology},
volume = {22},
number = {2},
pages = {143-150},
pmid = {32015437},
issn = {1476-4679},
support = {U41 HG009889/HG/NHGRI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; U19 MH107367/MH/NIMH NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; R01 EY029166/EY/NEI NIH HHS/United States ; T32 EB009380/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Therapy/methods ; Humans ; *Metagenome ; RNA, Guide/genetics/metabolism ; *Transcriptome ; },
abstract = {Deployment of RNA-guided DNA endonuclease CRISPR-Cas technology has led to radical advances in biology. As the functional diversity of CRISPR-Cas and parallel systems is further explored, RNA manipulation is emerging as a powerful mode of CRISPR-based engineering. In this Perspective, we chart progress in the RNA-targeting CRISPR-Cas (RCas) field and illustrate how continuing evolution in scientific discovery translates into applications for RNA biology and insights into mysteries, obstacles, and alternative technologies that lie ahead.},
}
@article {pmid32015436,
year = {2020},
author = {Barucci, G and Cornes, E and Singh, M and Li, B and Ugolini, M and Samolygo, A and Didier, C and Dingli, F and Loew, D and Quarato, P and Cecere, G},
title = {Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants.},
journal = {Nature cell biology},
volume = {22},
number = {2},
pages = {235-245},
pmid = {32015436},
issn = {1476-4679},
support = {679243/ERC_/European Research Council/International ; P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Argonaute Proteins/deficiency/*genetics/metabolism ; Biological Evolution ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/metabolism ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Fertility/genetics ; Gene Editing ; *Gene Silencing ; Histones/*genetics/metabolism ; Inheritance Patterns ; Mutation ; RNA, Antisense/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/*genetics/metabolism ; Repetitive Sequences, Nucleic Acid ; },
abstract = {PIWI-interacting RNAs (piRNAs) promote fertility in many animals. However, whether this is due to their conserved role in repressing repetitive elements (REs) remains unclear. Here, we show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is not caused by derepression of REs or other piRNA targets but, rather, is mediated by epigenetic silencing of all of the replicative histone genes. In the absence of piRNAs, downstream components of the piRNA pathway relocalize from germ granules and piRNA targets to histone mRNAs to synthesize antisense small RNAs (sRNAs) and induce transgenerational silencing. Removal of the downstream components of the piRNA pathway restores histone mRNA expression and fertility in piRNA mutants, and the inheritance of histone sRNAs in wild-type worms adversely affects their fertility for multiple generations. We conclude that sRNA-mediated silencing of histone genes impairs the fertility of piRNA mutants and may serve to maintain piRNAs across evolution.},
}
@article {pmid32015155,
year = {2020},
author = {Cole, KA},
title = {Targeting ATRX Loss through Inhibition of the Cell-Cycle Checkpoint Mediator WEE1.},
journal = {Cancer research},
volume = {80},
number = {3},
pages = {375-376},
doi = {10.1158/0008-5472.CAN-19-3587},
pmid = {32015155},
issn = {1538-7445},
mesh = {Adult ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Child ; Humans ; *Neoplasms ; Nuclear Proteins ; *Protein-Tyrosine Kinases ; Pyrimidinones ; X-linked Nuclear Protein ; },
abstract = {In this issue of Cancer Research, Liang and colleagues perform a genome-wide CRISPR-Cas9-negative loss-of-function screen and identify WEE1 kinase as a therapeutic vulnerability in cells depleted of the ATRX chromatin remodeler gene. Because ATRX mutations are frequently mutated across a variety of pediatric and adult malignancies, this work may contribute to the preclinical rationale for a precision medicine trial of the WEE1 inhibitor AZD1775 (adavosertib) for patients whose tumors demonstrate ATRX loss.See related article by Liang et al., p. 510.},
}
@article {pmid32013858,
year = {2020},
author = {Zhou, X and Yang, B and Stanton, C and Ross, RP and Zhao, J and Zhang, H and Chen, W},
title = {Comparative analysis of Lactobacillus gasseri from Chinese subjects reveals a new species-level taxa.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {119},
pmid = {32013858},
issn = {1471-2164},
mesh = {Adult ; Asians ; Bacterial Proteins/genetics ; Bacteriocins/genetics ; CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; Feces/microbiology ; Genome, Bacterial/genetics ; Genomics/methods ; Humans ; Infant ; Lactobacillus gasseri/*genetics ; Phylogeny ; Probiotics/metabolism ; RNA, Ribosomal, 16S/genetics ; },
abstract = {BACKGROUND: Lactobacillus gasseri as a probiotic has history of safe consumption is prevalent in infants and adults gut microbiota to maintain gut homeostasis.<br><br>RESULTS: In this study, to explore the genomic diversity and mine potential probiotic characteristics of L. gasseri, 92 strains of L. gasseri were isolated from Chinese human feces and identified based on 16&#x2009;s rDNA sequencing, after draft genomes sequencing, further average nucleotide identity (ANI) value and phylogenetic analysis reclassified them as L. paragasseri (n&#x2009;=&#x2009;79) and L. gasseri (n&#x2009;=&#x2009;13), respectively. Their pan/core-genomes were determined, revealing that L. paragasseri had an open pan-genome. Comparative analysis was carried out to identify genetic features, and the results indicated that 39 strains of L. paragasseri harboured Type II-A CRISPR-Cas system while 12 strains of L. gasseri contained Type I-E and II-A CRISPR-Cas systems. Bacteriocin operons and the number of carbohydrate-active enzymes were significantly different between the two species.<br><br>CONCLUSIONS: This is the first time to study pan/core-genome of L. gasseri and L. paragasseri, and compare their genetic diversity, and all the results provided better understating on genetics of the two species.},
}
@article {pmid32013399,
year = {2020},
author = {Zhang, D and Yan, Y and Que, H and Yang, T and Cheng, X and Ding, S and Zhang, X and Cheng, W},
title = {CRISPR/Cas12a-Mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing.},
journal = {ACS sensors},
volume = {5},
number = {2},
pages = {557-562},
doi = {10.1021/acssensors.9b02461},
pmid = {32013399},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/*chemistry ; Electrochemistry/*methods ; Humans ; Nucleic Acids/*chemistry ; },
abstract = {A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.},
}
@article {pmid32013077,
year = {2020},
author = {Erwood, S and Gu, B},
title = {Embryo-Based Large Fragment Knock-in in Mammals: Why, How and What's Next.},
journal = {Genes},
volume = {11},
number = {2},
pages = {},
pmid = {32013077},
issn = {2073-4425},
support = {FDN-143334//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA Repair ; *Embryo, Mammalian ; Gene Editing ; Gene Knock-In Techniques/*methods ; Mice ; },
abstract = {Endonuclease-mediated genome editing technologies, most notably CRISPR/Cas9, have revolutionized animal genetics by allowing for precise genome editing directly through embryo manipulations. As endonuclease-mediated model generation became commonplace, large fragment knock-in remained one of the most challenging types of genetic modification. Due to their unique value in biological and biomedical research, however, a diverse range of technological innovations have been developed to achieve efficient large fragment knock-in in mammalian animal model generation, with a particular focus on mice. Here, we first discuss some examples that illustrate the importance of large fragment knock-in animal models and then detail a subset of the recent technological advancements that have allowed for efficient large fragment knock-in. Finally, we envision the future development of even larger fragment knock-ins performed in even larger animal models, the next step in expanding the potential of large fragment knock-in in animal models.},
}
@article {pmid32011704,
year = {2019},
author = {Wurihan, W and Huang, Y and Weber, AM and Wu, X and Fan, H},
title = {Nonspecific toxicities of Streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis.},
journal = {Pathogens and disease},
volume = {77},
number = {9},
pages = {},
pmid = {32011704},
issn = {2049-632X},
support = {R21 AI122034/AI/NIAID NIH HHS/United States ; R21 AI140167/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Chlamydia trachomatis/*genetics/metabolism ; RNA, Guide/antagonists &amp; inhibitors/metabolism ; Staphylococcus aureus/*genetics ; Streptococcus pyogenes/*genetics ; },
abstract = {Chlamydiae are common, important pathogens for humans and animals alike. Despite recent advancement in genetics, scientists are still searching for efficient tools to knock out or knock down the expression of chromosomal genes. We attempted to adopt a dCas9-based CRISPR interference (CRISPRi) technology to conditionally knock down gene expression in Chlamydia trachomatis using an anhydrotetracycline (ATC)-inducible expression system. Surprisingly, expression of the commonly used Streptococcus pyogenes dCas9 in C. trachomatis causes strong inhibition in the absence of any guide RNA (gRNA). Staphylococcus aureus dCas9 also shows strong toxicity in the presence of only an empty gRNA scaffold. Toxicity of the S. pyogenes dCas9 is readily observed with as little as 0.2 nM ATC. Growth inhibition by S. aureus dCas9 is evident starting at 1.0 nM ATC. In contrast, C. trachomatis growth was not affected by methionine-tRNA ligase overexpression induced with 10 nM ATC. We conclude that S. pyogenes and S. aureus dCas9 proteins in their current forms have limited utility for chlamydial research and suggest strategies to overcome this problem.},
}
@article {pmid32010936,
year = {2020},
author = {Shiriaeva, A and Fedorov, I and Vyhovskyi, D and Severinov, K},
title = {Detection of CRISPR adaptation.},
journal = {Biochemical Society transactions},
volume = {48},
number = {1},
pages = {257-269},
pmid = {32010936},
issn = {1470-8752},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptation, Physiological ; Adaptive Immunity ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/*genetics ; Polymerase Chain Reaction/methods ; Prokaryotic Cells/immunology ; },
abstract = {Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.},
}
@article {pmid32008319,
year = {2020},
author = {Wittig, S and Songailiene, I and Schmidt, C},
title = {Formation and Stoichiometry of CRISPR-Cascade Complexes with Varying Spacer Lengths Revealed by Native Mass Spectrometry.},
journal = {Journal of the American Society for Mass Spectrometry},
volume = {31},
number = {3},
pages = {538-546},
doi = {10.1021/jasms.9b00011},
pmid = {32008319},
issn = {1879-1123},
mesh = {Amino Acid Sequence ; CRISPR-Associated Proteins/*chemistry ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleotides/chemistry ; Protein Subunits/chemistry ; RNA, Bacterial/*chemistry ; Streptococcus thermophilus/*chemistry ; Tandem Mass Spectrometry ; },
abstract = {The adaptive immune system of bacteria and archaea against viral DNA is based on clustered, regularly interspaced, short palindromic repeats (CRISPRs) which are encoded in the host genome and translated into CRISPR RNAs (crRNAs) containing single spacer sequences complementary to foreign DNA. crRNAs assemble with CRISPR-associated (Cas) proteins forming surveillance complexes that base-pair with viral DNA and mediate its degradation. As specificity of degradation is provided by the crRNA spacer sequence, genetic engineering of the CRISPR system has emerged as a popular molecular tool, for instance, in gene silencing and programmed DNA degradation. Elongating or shortening the crRNA spacer sequence are therefore promising ventures to modify specificity toward the target DNA. However, even though the stoichiometry of wild-type complexes is well established, it is unknown how variations in crRNA spacer length affect their stoichiometry. The CRISPR-associated antiviral defense surveillance complexes of Streptococcus thermophilus (StCascade complexes) contain crRNA and five protein subunits. Using native mass spectrometry, we studied the formation and stoichiometry of StCascade complexes assembled on a set of crRNAs with different spacer lengths. We assigned all relevant complexes and gained insights into the stoichiometry of the complexes as well as their preferred assembly. We found that stable complexes, which incorporate or lose a (Cas7)2(Cse2)1-module, assemble on crRNA varied in length by 12-nucleotide units, while varying crRNA length in six-nucleotide units results in heterogeneous mixtures of complexes. Combining our results from the various variants, we generated an assembly pathway revealing general features of I-E type Cascade complex formation.},
}
@article {pmid32008085,
year = {2020},
author = {Mohammadinejad, R and Biagioni, A and Arunkumar, G and Shapiro, R and Chang, KC and Sedeeq, M and Taiyab, A and Hashemabadi, M and Pardakhty, A and Mandegary, A and Thiery, JP and Aref, AR and Azimi, I},
title = {EMT signaling: potential contribution of CRISPR/Cas gene editing.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {14},
pages = {2701-2722},
doi = {10.1007/s00018-020-03449-3},
pmid = {32008085},
issn = {1420-9071},
mesh = {CRISPR-Cas Systems/*genetics ; Embryonic Development/genetics ; Epithelial-Mesenchymal Transition/*genetics ; Gene Editing/*methods ; Humans ; Organogenesis/genetics ; Signal Transduction/genetics ; },
abstract = {Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.},
}
@article {pmid32008042,
year = {2021},
author = {O'Brien, AR and Burgio, G and Bauer, DC},
title = {Domain-specific introduction to machine learning terminology, pitfalls and opportunities in CRISPR-based gene editing.},
journal = {Briefings in bioinformatics},
volume = {22},
number = {1},
pages = {308-314},
pmid = {32008042},
issn = {1477-4054},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods/standards ; Genomics/methods/standards ; Humans ; *Machine Learning ; },
abstract = {The use of machine learning (ML) has become prevalent in the genome engineering space, with applications ranging from predicting target site efficiency to forecasting the outcome of repair events. However, jargon and ML-specific accuracy measures have made it hard to assess the validity of individual approaches, potentially leading to misinterpretation of ML results. This review aims to close the gap by discussing ML approaches and pitfalls in the context of CRISPR gene-editing applications. Specifically, we address common considerations, such as algorithm choice, as well as problems, such as overestimating accuracy and data interoperability, by providing tangible examples from the genome-engineering domain. Equipping researchers with the knowledge to effectively use ML to better design gene-editing experiments and predict experimental outcomes will help advance the field more rapidly.},
}
@article {pmid32007615,
year = {2020},
author = {Cao, M and Fatma, Z and Song, X and Hsieh, PH and Tran, VG and Lyon, WL and Sayadi, M and Shao, Z and Yoshikuni, Y and Zhao, H},
title = {A genetic toolbox for metabolic engineering of Issatchenkia orientalis.},
journal = {Metabolic engineering},
volume = {59},
number = {},
pages = {87-97},
doi = {10.1016/j.ymben.2020.01.005},
pmid = {32007615},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *DNA, Fungal/genetics/metabolism ; *Metabolic Engineering ; *Pichia/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; },
abstract = {The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.},
}
@article {pmid32006663,
year = {2020},
author = {Kaur, N and Alok, A and Shivani, and Kumar, P and Kaur, N and Awasthi, P and Chaturvedi, S and Pandey, P and Pandey, A and Pandey, AK and Tiwari, S},
title = {CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit.},
journal = {Metabolic engineering},
volume = {59},
number = {},
pages = {76-86},
doi = {10.1016/j.ymben.2020.01.008},
pmid = {32006663},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Fruit/genetics/metabolism ; *Gene Editing ; *Intramolecular Lyases/genetics/metabolism ; *Musa/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; beta Carotene/*biosynthesis/genetics ; },
abstract = {Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.},
}
@article {pmid32006655,
year = {2020},
author = {Zhu, J and Zhou, X and Huang, X and Du, Z},
title = {Bacterial expression, purification, and initial characterization of a full-length Cas13b protein from Porphyromonas gingivalis.},
journal = {Protein expression and purification},
volume = {169},
number = {},
pages = {105588},
pmid = {32006655},
issn = {1096-0279},
support = {R15 GM131366/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/biosynthesis/chemistry/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/biosynthesis/chemistry/metabolism ; Escherichia coli/genetics/metabolism ; Porphyromonas gingivalis/*genetics/metabolism ; Recombinant Proteins ; },
abstract = {The CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (and probably functional) diversities may exist. It is therefore important to study CRISPR-Cas13b cases from different bacterial species. Here we report the expression, purification, and initial characterization of a Cas13b endonuclease that is associated with the 8th putative CRISPR locus from Porphyromonas gingivalis genome (Pgi8Cas13b). The full-length Pgi8Cas13b protein (1119 residues) was successfully expressed in E. Coli cells, and purified by affinity and ion-exchange chromatography methods. The purified protein is biologically active, being able to bind its cognate crRNA with high specificity and affinity. Preparation of biologically active Pgi8Cas13b protein provides the basis for further in vitro biochemical and biophysical studies of the Pgi8Cas13b CRISPR system.},
}
@article {pmid32006629,
year = {2020},
author = {Huang, S and Geng, A},
title = {High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration.},
journal = {Journal of biotechnology},
volume = {310},
number = {},
pages = {13-20},
doi = {10.1016/j.jbiotec.2020.01.014},
pmid = {32006629},
issn = {1873-4863},
mesh = {Butylene Glycols/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome, Fungal ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/biosynthesis/genetics ; },
abstract = {CRISPR Cas9 system is becoming an emerging genome-editing platform and has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. In this study, we developed a novel replicative and integrative CRISPR Cas9 genome-editing platform for large DNA construct in vivo assembly, replication, and high-copy genome integration in Saccharomyces cerevisiae. It harnessed advantages of autonomous replicative sequence in S. cerevisiae, in vivo DNA assembly, CRISPR Cas9, and delta integration. Enhanced green fluorescent protein was used as a marker to confirm large DNA construct in vivo assembly and genome integration. Based on this platform, an efficient 2,3- BDO producing yeast strain was rapidly constructed with up to 25-copy genome integration of 2,3-BDO biosynthesis pathway. Further strain engineering was conducted by multiplex disruption of ADH1, PDC1, PDC5 and MTH1 using a 2μ-based replicative CRISPR Cas9 plasmid containing donor DNAs. As a result, the 2,3-BDO titer was improved by 3.9 folds compared to that obtained by the initially engineered yeast and 50.5 g/L 2,3-BDO was produced by the final engineered yeast strain 36aS5-CFBDO in fed-batch fermentation without strain evolution and process optimization. This study demonstrated that the new replicative and integrative CRISPR Cas9 genome-editing platform was promising in generating an efficient 2,3-BDO-producing S. cerevisiae strain.},
}
@article {pmid32006418,
year = {2020},
author = {Dannenmann, B and Nasri, M and Welte, K and Skokowa, J},
title = {CRISPR/Cas9 Genome Editing of Human-Induced Pluripotent Stem Cells Followed by Granulocytic Differentiation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {471-483},
doi = {10.1007/978-1-0716-0290-4_27},
pmid = {32006418},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Electroporation/methods ; Gene Editing/*methods ; Granulocytes/*cytology/metabolism ; *Hematopoiesis ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Transfection/methods ; },
abstract = {Research on patient-derived induced pluripotent stem cells (iPSCs) could immensely benefit from the implementation of CRISPR/Cas9 genome editing of iPSCs, creating unique opportunities such as the establishment of isogenic iPSC lines for disease modeling or personalized patient-specific drug screenings. Here we describe a stepwise protocol of safe, efficient, and selection-free CRISPR/Cas9-mediated gene correction or knockout in human iPSCs followed by 3D spin-embryoid body (EB)-based hematopoietic/neutrophilic iPSC-differentiation.},
}
@article {pmid32006417,
year = {2020},
author = {Mir, P and Ritter, M and Welte, K and Skokowa, J and Klimiankou, M},
title = {Gene Knockout in Hematopoietic Stem and Progenitor Cells Followed by Granulocytic Differentiation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {455-469},
doi = {10.1007/978-1-0716-0290-4_26},
pmid = {32006417},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Granulocytes/*cytology/metabolism ; Hematopoietic Stem Cells/*cytology/metabolism ; Humans ; *Leukopoiesis ; RNA, Guide/genetics ; },
abstract = {In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.},
}
@article {pmid32006416,
year = {2020},
author = {Bai, B and Myklebust, JH and Wälchli, S},
title = {Gene Editing in B-Lymphoma Cell Lines Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {445-454},
doi = {10.1007/978-1-0716-0290-4_25},
pmid = {32006416},
issn = {1940-6029},
mesh = {B-Lymphocytes/metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/methods ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; Lymphoma, B-Cell/*genetics/therapy ; RNA, Guide/genetics ; Transduction, Genetic/methods ; },
abstract = {Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. Here, we present a strategy for sequential delivery of the two essential components, Cas9 and sgRNA, into B-lymphoid cell lines. Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.},
}
@article {pmid32006415,
year = {2020},
author = {Laoharawee, K and Johnson, MJ and Moriarity, BS},
title = {CRISPR/Cas9-Mediated Genome Engineering of Primary Human B Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {435-444},
doi = {10.1007/978-1-0716-0290-4_24},
pmid = {32006415},
issn = {1940-6029},
mesh = {B-Lymphocytes/cytology/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Humans ; Leukocytes, Mononuclear/cytology/metabolism ; },
abstract = {The CRISPR/Cas9 system allows for site-specific gene editing and genome engineering of primary human cells. Here we describe methods for gene editing and genome engineering of B cells isolated from human peripheral blood mononuclear cells using CRISPR/Cas9. Editing frequencies of up to 90% and integration rates greater than 60% can be achieved with this method.},
}
@article {pmid32006414,
year = {2020},
author = {Zhang, X and Cheng, C and Sun, W and Wang, H},
title = {Engineering T Cells Using CRISPR/Cas9 for Cancer Therapy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {419-433},
doi = {10.1007/978-1-0716-0290-4_23},
pmid = {32006414},
issn = {1940-6029},
mesh = {Adoptive Transfer/*methods ; Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Neoplasms/genetics/immunology/*therapy ; *T-Lymphocytes/immunology/metabolism/transplantation ; },
abstract = {Recent advances in the development of gene editing technologies, especially the CRISPR/Cas 9 system, have substantially enhanced our ability to make precise and efficient changes in the genomes of various cells. In particular, the genetic engineering of T cells holds huge potential to improve the efficacy and safety of T cells-based cancer therapy. Due to its ease of use and high efficiency, CRISPR/Cas9 enables efficient gene knockout, site-specific knock-in, and genome-wide screen in T cells. Here we review the current progress of applying gene editing to T-cell therapy, focusing on the technical aspects of the CRISPR/Cas9 platform. We also discuss the challenges and future prospects.},
}
@article {pmid32006413,
year = {2020},
author = {Wälchli, S and Sioud, M},
title = {Next Generation of Adoptive T Cell Therapy Using CRISPR/Cas9 Technology: Universal or Boosted?.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {407-417},
doi = {10.1007/978-1-0716-0290-4_22},
pmid = {32006413},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/adverse effects/*methods ; T-Lymphocytes/metabolism/transplantation ; },
abstract = {Adoptive T cell therapy (ACT) using either chimeric antigen receptor (CAR)- or T cell receptor (TCR)-engineered lymphocytes has emerged as a promising strategy to treat cancer. However, this therapy is still facing enormous challenges such as poor quality of autologous T cells, T cell exhaustion, and the immune suppressive tumor microenvironments. Additionally, graft-versus-host disease is an issue that must be addressed to allow the use of allogeneic T cells. Strategies to overcome these therapeutic challenges using gene editing technology are now being developed. One strategy is to disrupt TCR and/or MHC expression in healthy donor T cells to generate T cells for universal use. Another strategy is to improve the quality of patient's T cells by eliminating either the expression of selected immune checkpoint receptors or negative regulators of TCR signaling and/or T-cell homeostasis. Here, we review the use of CRISPR-Cas9 platform in T cell engineering with a focus on the development of universal T cells and boosted autologous cells for next-generation ACT.},
}
@article {pmid32006412,
year = {2020},
author = {Prykhozhij, SV and Cordeiro-Santanach, A and Caceres, L and Berman, JN},
title = {Genome Editing in Zebrafish Using High-Fidelity Cas9 Nucleases: Choosing the Right Nuclease for the Task.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {385-405},
doi = {10.1007/978-1-0716-0290-4_21},
pmid = {32006412},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Microinjections ; Mutagenesis, Site-Directed ; RNA, Guide/genetics ; Zebrafish/*genetics ; },
abstract = {Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.},
}
@article {pmid32006411,
year = {2020},
author = {Sahoo, N and Cuello, V and Udawant, S and Litif, C and Mustard, JA and Keniry, M},
title = {CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {365-383},
pmid = {32006411},
issn = {1940-6029},
support = {SC3 GM132053/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Forkhead Box Protein O3/genetics ; Gene Editing/*methods ; Genetic Vectors/genetics ; Humans ; Mutation ; RNA, Guide/genetics ; },
abstract = {CRISPR Cas9 genome editing allows researchers to modify genes in a multitude of ways including to obtain deletions, epitope-tagged loci, and knock-in mutations. Within 6 years of its initial application, CRISPR-Cas9 genome editing has been widely employed, but disadvantages to this method, such as low modification efficiencies and off-target effects, need careful consideration. Obtaining custom donor vectors can also be expensive and time-consuming. This chapter details strategies to overcome barriers to CRISPR-Cas9 genome editing as well as recent developments in employing this technique.},
}
@article {pmid32006410,
year = {2020},
author = {Hiranniramol, K and Chen, Y and Wang, X},
title = {CRISPR/Cas9 Guide RNA Design Rules for Predicting Activity.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {351-364},
doi = {10.1007/978-1-0716-0290-4_19},
pmid = {32006410},
issn = {1940-6029},
support = {R01 DE026471/DE/NIDCR NIH HHS/United States ; R01 GM089784/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Genomics/methods ; Humans ; Models, Genetic ; RNA, Guide/*genetics ; },
abstract = {A critical stage in performing gene editing experiments using the CRISPR/Cas9 system is the design of guide RNA (gRNA). In this chapter, we conduct a review of the current gRNA design rules for maximizing on-target Cas9 activity while minimizing off-target activity. In addition, we present some of the currently available computational tools for gRNA activity prediction and assay design.},
}
@article {pmid32006393,
year = {2020},
author = {Lennox, KA and Behlke, MA},
title = {Chemical Modifications in RNA Interference and CRISPR/Cas Genome Editing Reagents.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {23-55},
doi = {10.1007/978-1-0716-0290-4_2},
pmid = {32006393},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Indicators and Reagents ; Oligonucleotides/chemistry/genetics ; *RNA Interference ; RNA, Guide/chemistry/genetics ; RNA, Small Interfering/chemistry/genetics ; },
abstract = {Chemically modified oligonucleotides (ONs) are routinely used in the laboratory to assess gene function, and clinical advances are rapidly progressing as continual efforts are being made to optimize ON efficacy. Over the years, RNA interference (RNAi) has become one of the main tools used to inhibit RNA expression across a wide variety of species. Efforts have been made to improve the exogenous delivery of the double-stranded RNA components to the endogenous intracellular RNAi machinery to direct efficacious degradation of a user-defined RNA target. More recently, synthetic RNA ONs are being used to mimic the bacterial-derived CRISPR/Cas system to direct specific editing of the mammalian genome. Both of these techniques rely on the use of various chemical modifications to the RNA phosphate backbone or sugar in specific positions throughout the ONs to improve the desired biological outcome. Relevant chemical modifications also include conjugated targeting ligands to assist ON delivery to specific cell types. Chemical modifications are most beneficial for therapeutically relevant ONs, as they serve to enhance target binding, increase drug longevity, facilitate cell-specific targeting, improve internalization into productive intracellular compartments, and mitigate both sequence-specific as well as immune-related off-target effects (OTEs). The knowledge gained from years of optimizing RNAi reagents and characterizing the biochemical and biophysical properties of each chemical modification will hopefully accelerate the CRISPR/Cas technology into the clinic, as well as further expand the use of RNAi to treat currently undruggable diseases. This review discusses the most commonly employed chemical modifications in RNAi reagents and CRISPR/Cas guide RNAs and provides an overview of select publications that have demonstrated success in improving ON efficacy and/or mitigating undesired OTEs.},
}
@article {pmid32006392,
year = {2020},
author = {Sioud, M},
title = {RNA and CRISPR Interferences: Past, Present, and Future Perspectives.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2115},
number = {},
pages = {1-22},
doi = {10.1007/978-1-0716-0290-4_1},
pmid = {32006392},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/methods ; Humans ; RNA/genetics ; *RNA Interference ; RNA, Small Interfering/genetics ; },
abstract = {RNA interference (RNAi), a natural gene silencing process, is a widely used technique in basic research, preclinical studies, and drug development strategies. Although the technique has great potential to generate new human therapies and treat undruggable diseases, the clinical application of RNAi is still challenging primarily because of the delivery problem and potential off-target effects. Over the past two decades, great efforts have been undertaken to develop delivery agents and chemical modifications to overcome these challenges. Such advances in RNA delivery and chemical modifications have benefited researchers who are developing gene-editing therapies based on CRISPR-Cas9, an RNA-guided endonuclease, which is already having a major impact on biology and medicine. Here, I review the discovery of these two interference tools, identify the technical challenges yet to be overcome and provide some perspectives on how these two RNA-based technologies can be harnessed to treat human diseases.},
}
@article {pmid32005981,
year = {2020},
author = {Kouprina, N and Noskov, VN and Larionov, V},
title = {Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast.},
journal = {Nature protocols},
volume = {15},
number = {3},
pages = {734-749},
pmid = {32005981},
issn = {1750-2799},
support = {ZIA BC010413/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cloning, Molecular/*methods ; DNA Replication ; DNA, Fungal/*genetics ; Genetic Vectors ; *Genome, Fungal ; Plasmids ; Replication Origin ; Saccharomyces cerevisiae/*genetics ; Transformation, Genetic ; },
abstract = {Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.},
}
@article {pmid32005950,
year = {2020},
author = {Kang, W and Zhao, X and Sun, Z and Dong, T and Jin, C and Tong, L and Zhu, W and Tao, Y and Wu, H},
title = {Adeno-associated virus vector enables safe and efficient Cas9 activation in neonatal and adult Cas9 knockin murine cochleae.},
journal = {Gene therapy},
volume = {27},
number = {7-8},
pages = {392-405},
pmid = {32005950},
issn = {1476-5462},
mesh = {Animals ; CRISPR-Cas Systems ; *Cochlea ; *Dependovirus/genetics ; *Gene Transfer Techniques ; *Genetic Vectors ; Mice ; },
abstract = {Adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene manipulation in the inner ear. For example, hair cells (HCs) and multiple other cell types can be transduced by the local injection of AAVs into the inner ear. However, application of the AAV-mediated CRISPR/Cas9 gene-editing approach to the inner ear in adult mice has not yet been studied. Based on our previous work, we investigated several AAV serotypes in neonatal and adult mice in parallel, and found that AAV8 had the top efficiency to transduce inner HCs. We then tested the ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knockin mice, and observed significant Cas9 activation in the inner ear of both neonatal and adult animals. Neither the AAV8 virus itself nor the surgical procedures used to deliver it-cochleostomy for neonatal mice and canalostomy for adult mice-caused any damage to HCs or impaired normal hearing. Our studies indicate that the local injection of AAV8-Cre can induce Cas9 activation to perform safe and efficient gene editing in the inner ear, expanding the repertoire of gene-editing tools for regulating gene expression in the inner ear as a part of efforts to rescue genetic hearing loss, initiate regeneration of HCs, or develop gene therapy techniques.},
}
@article {pmid32005926,
year = {2020},
author = {de Almeida Monteiro Melo Ferraz, M and Nagashima, JB and Venzac, B and Le Gac, S and Songsasen, N},
title = {A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1575},
pmid = {32005926},
issn = {2045-2322},
support = {F32 HD090854/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carcinoma in Situ/metabolism/*veterinary ; Dog Diseases/*metabolism ; Dogs ; Female ; Fluorescent Antibody Technique ; Gene Editing ; Lab-On-A-Chip Devices/*veterinary ; Ovarian Neoplasms/metabolism/*veterinary ; Oviducts/*metabolism ; Polymerase Chain Reaction ; },
abstract = {Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC.},
}
@article {pmid32005820,
year = {2020},
author = {Tan, J and Zhang, F and Karcher, D and Bock, R},
title = {Expanding the genome-targeting scope and the site selectivity of high-precision base editors.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {629},
pmid = {32005820},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; Gene Editing/*methods ; *Genome, Fungal ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Base editors (BEs) are RNA-guided CRISPR-Cas-derived genome editing tools that induce single-nucleotide changes. The limitations of current BEs lie in their low precision (especially when multiple target nucleotides of the deaminase are present within the activity window) and their restriction to targets that are in proper distance from the PAM sequence. We have recently developed high-precision cytidine BEs by engineering CDA1 truncations and nCas9 fusions that predominantly edit nucleotide C-18 relative to the PAM sequence NGG. Here, by testing fusions with Cas9 variants that recognize alternative PAMs, we provide a series of high-precision BEs that greatly expand the versatility of base editing. In addition, we obtained BEs that selectively edit C-15 or C-16. We also show that our high-precision BEs can substantially reduce off-target effect. These improved base editing tools will be widely applicable in basic research, biotechnology and gene therapy.},
}
@article {pmid32005668,
year = {2020},
author = {Wu, X and Schnitzler, GR and Gao, GF and Diamond, B and Baker, AR and Kaplan, B and Williamson, K and Westlake, L and Lorrey, S and Lewis, TA and Garvie, CW and Lange, M and Hayat, S and Seidel, H and Doench, J and Cherniack, AD and Kopitz, C and Meyerson, M and Greulich, H},
title = {Mechanistic insights into cancer cell killing through interaction of phosphodiesterase 3A and schlafen family member 12.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {11},
pages = {3431-3446},
pmid = {32005668},
issn = {1083-351X},
support = {R35 CA197568/CA/NCI NIH HHS/United States ; },
mesh = {Base Sequence ; Biomarkers, Tumor/metabolism ; CRISPR-Cas Systems/genetics ; Catalytic Domain ; Cell Death/drug effects ; Cell Line, Tumor ; Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry/*metabolism ; Frameshift Mutation/genetics ; Genome ; Heterozygote ; Humans ; Intracellular Signaling Peptides and Proteins/*metabolism ; Neoplasms/*pathology ; Protein Binding/drug effects ; Pyridazines/pharmacology ; },
abstract = {Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.},
}
@article {pmid32005374,
year = {2020},
author = {Loriato, VAP and Martins, LGC and Euclydes, NC and Reis, PAB and Duarte, CEM and Fontes, EPB},
title = {Engineering resistance against geminiviruses: A review of suppressed natural defenses and the use of RNAi and the CRISPR/Cas system.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {292},
number = {},
pages = {110410},
doi = {10.1016/j.plantsci.2020.110410},
pmid = {32005374},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/immunology ; Disease Resistance/genetics ; Geminiviridae/*physiology ; Genetic Engineering ; *Host-Pathogen Interactions ; Plant Diseases/genetics/*immunology ; Plant Immunity/*genetics ; Plants, Genetically Modified/genetics/immunology ; *RNA Interference ; },
abstract = {The Geminiviridae family is one of the most successful and largest families of plant viruses that infect a large variety of important dicotyledonous and monocotyledonous crops and cause significant yield losses worldwide. This broad spectrum of host range is only possible because geminiviruses have evolved sophisticated strategies to overcome the arsenal of antiviral defenses in such diverse plant species. In addition, geminiviruses evolve rapidly through recombination and pseudo-recombination to naturally create a great diversity of virus species with divergent genome sequences giving the virus an advantage over the host recognition system. Therefore, it is not surprising that efficient molecular strategies to combat geminivirus infection under open field conditions have not been fully addressed. In this review, we present the anti-geminiviral arsenal of plant defenses, the evolved virulence strategies of geminiviruses to overcome these plant defenses and the most recent strategies that have been engineered for transgenic resistance. Although, the in vitro reactivation of suppressed natural defenses as well as the use of RNAi and CRISPR/Cas systems hold the potential for achieving broad-range resistance and/or immunity, potential drawbacks have been associated with each case.},
}
@article {pmid32005150,
year = {2020},
author = {Borys, SM and Younger, ST},
title = {Identification of functional regulatory elements in the human genome using pooled CRISPR screens.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {107},
pmid = {32005150},
issn = {1471-2164},
mesh = {Binding Sites ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Gene Knockout Techniques ; Genome, Human ; Humans ; Neoplasms/*genetics ; Regulatory Elements, Transcriptional ; Tumor Suppressor Protein p53/*chemistry/*genetics/metabolism ; },
abstract = {BACKGROUND: Genome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise < 2% of the sequence space in the human genome leaving a substantial portion of the genome uninterrogated. Noncoding regions of the genome harbor important regulatory elements (e.g. promoters, enhancers, silencers) that influence cellular processes but high-throughput methods for evaluating their essentiality have yet to be established.<br><br>RESULTS: Here, we describe a CRISPR-based screening approach that facilitates the functional profiling of thousands of noncoding regulatory elements in parallel. We selected the tumor suppressor p53 as a model system and designed a pooled CRISPR library targeting thousands of p53 binding sites throughout the genome. Following transduction into dCas9-KRAB-expressing cells we identified several regulatory elements that influence cell proliferation. Moreover, we uncovered multiple elements that are required for the p53-mediated DNA damage response. Surprisingly, many of these elements are located deep within intergenic regions of the genome that have no prior functional annotations.<br><br>CONCLUSIONS: This work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.},
}
@article {pmid32004507,
year = {2020},
author = {Papanicolaou, KN and Ashok, D and Liu, T and Bauer, TM and Sun, J and Li, Z and da Costa, E and D'Orleans, CC and Nathan, S and Lefer, DJ and Murphy, E and Paolocci, N and Foster, DB and O'Rourke, B},
title = {Global knockout of ROMK potassium channel worsens cardiac ischemia-reperfusion injury but cardiomyocyte-specific knockout does not: Implications for the identity of mitoKATP.},
journal = {Journal of molecular and cellular cardiology},
volume = {139},
number = {},
pages = {176-189},
pmid = {32004507},
issn = {1095-8584},
support = {R01 HL134821/HL/NHLBI NIH HHS/United States ; ZIA HL002066/ImNIH/Intramural NIH HHS/United States ; F31 HL134198/HL/NHLBI NIH HHS/United States ; K12 HL141952/HL/NHLBI NIH HHS/United States ; R01 HL136918/HL/NHLBI NIH HHS/United States ; R01 HL137259/HL/NHLBI NIH HHS/United States ; R01 HL092141/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Electrophysiological Phenomena ; Gene Editing ; Gene Knockout Techniques ; Hemodynamics ; Ischemic Preconditioning, Myocardial ; Mice, Knockout ; Mitochondria, Heart/metabolism ; Myocardial Reperfusion Injury/*metabolism/pathology/physiopathology ; Myocardium/metabolism/pathology ; Myocytes, Cardiac/*metabolism/pathology ; Organ Specificity ; Perfusion ; Phenotype ; Potassium Channels/*metabolism ; Potassium Channels, Inwardly Rectifying/*deficiency/metabolism ; },
abstract = {The renal-outer-medullary‑potassium (ROMK) channel, mutated in Bartter's syndrome, regulates ion exchange in kidney, but its extra-renal functions remain unknown. Additionally, ROMK was postulated to be the pore-forming subunit of the mitochondrial ATP-sensitive K[+] channel (mitoKATP), a mediator of cardioprotection. Using global and cardiomyocyte-specific knockout mice (ROMK-GKO and ROMK-CKO respectively), we characterize the effects of ROMK knockout on mitochondrial ion handling, the response to pharmacological KATP channel modulators, and ischemia/reperfusion (I/R) injury. Mitochondria from ROMK-GKO hearts exhibited a lower threshold for Ca[2+]-triggered permeability transition pore (mPTP) opening but normal matrix volume changes during oxidative phosphorylation. Isolated perfused ROMK-GKO hearts exhibited impaired functional recovery and increased infarct size when I/R was preceded by an ischemic preconditioning (IPC) protocol. Because ROMK-GKO mice exhibited severe renal defects and cardiac remodeling, we further characterized ROMK-CKO hearts to avoid confounding systemic effects. Mitochondria from ROMK-CKO hearts had unchanged matrix volume responses during oxidative phosphorylation and still swelled upon addition of a mitoKATP opener, but exhibited a lower threshold for mPTP opening, similar to GKO mitochondria. Nevertheless, I/R induced damage was not exacerbated in ROMK-CKO hearts, either ex vivo or in vivo. Lastly, we examined the response of ROMK-CKO hearts to ex vivo I/R injury with or without IPC and found that IPC still protected these hearts, suggesting that cardiomyocyte ROMK does not participate significantly in the cardioprotective pathway elicited by IPC. Collectively, our findings from these novel strains of mice suggest that cardiomyocyte ROMK is not a central mediator of mitoKATP function, although it can affect mPTP activation threshold.},
}
@article {pmid32004468,
year = {2020},
author = {Verma, R and Mohl, D and Deshaies, RJ},
title = {Harnessing the Power of Proteolysis for Targeted Protein Inactivation.},
journal = {Molecular cell},
volume = {77},
number = {3},
pages = {446-460},
doi = {10.1016/j.molcel.2020.01.010},
pmid = {32004468},
issn = {1097-4164},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genetic Engineering/*methods/*trends ; Humans ; Morpholinos/genetics ; Protein Engineering/*methods/*trends ; Protein Transport ; Proteolysis ; },
abstract = {Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.},
}
@article {pmid32004439,
year = {2020},
author = {Wertz, MH and Mitchem, MR and Pineda, SS and Hachigian, LJ and Lee, H and Lau, V and Powers, A and Kulicke, R and Madan, GK and Colic, M and Therrien, M and Vernon, A and Beja-Glasser, VF and Hegde, M and Gao, F and Kellis, M and Hart, T and Doench, JG and Heiman, M},
title = {Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.},
journal = {Neuron},
volume = {106},
number = {1},
pages = {76-89.e8},
pmid = {32004439},
issn = {1097-4199},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 NS085880/NS/NINDS NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; T32 EB019940/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Cell Survival/*genetics ; Gene Knockdown Techniques ; Gene Library ; Genes, Essential/genetics ; Huntingtin Protein/*genetics ; Huntington Disease/*genetics ; Mice ; Mice, Transgenic ; NM23 Nucleoside Diphosphate Kinases/genetics ; Neostriatum/*metabolism ; Neurons/*metabolism ; Nucleoside Diphosphate Kinase D/genetics ; Protein Aggregates ; RNA Interference ; RNA, Guide ; RNA, Small Interfering ; Receptors, Dopamine D2/genetics ; Sequence Analysis, RNA ; },
abstract = {Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.},
}
@article {pmid32003793,
year = {2020},
author = {Tang, Y and Fu, P and Zhou, Y and Xie, Y and Jin, J and Wang, B and Yu, L and Huang, Y and Li, G and Li, M and Liang, W and Ou, HY and Jiang, X},
title = {Absence of the type I-E CRISPR-Cas system in Klebsiella pneumoniae clonal complex 258 is associated with dissemination of IncF epidemic resistance plasmids in this clonal complex.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {75},
number = {4},
pages = {890-895},
doi = {10.1093/jac/dkz538},
pmid = {32003793},
issn = {1460-2091},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Epidemics ; *Klebsiella pneumoniae/genetics ; Multilocus Sequence Typing ; Plasmids/genetics ; },
abstract = {BACKGROUND: The pandemics caused by MDR Klebsiella pneumoniae are mostly due to the global dissemination of high-risk clonal complex 258 (CC258) and related IncF epidemic plasmids. However, the factors leading to the epidemiological advantages of CC258-IncF linkage remain obscure. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems, providing adaptive immunity against invading DNA, play an important role in the interactions between plasmids and hosts.<br><br>OBJECTIVES: To investigate the relationship between CRISPR-Cas systems and the high-risk linkage CC258-IncF.<br><br>METHODS: CRISPR-Cas loci were detected among 381 collected K. pneumoniae clinical isolates and 207 K. pneumoniae complete genomes available in GenBank. MLST was used to determine the genetic relatedness of these isolates. Nucleotide BLAST was used to search for protospacers on K. pneumoniae plasmids.<br><br>RESULTS: We observed an epidemic correlation between CRISPR-Cas loci, CC258 and IncF plasmids. Interestingly, most type I-E CRISPR-Cas systems identified carried spacers matching the backbone regions of IncF plasmids.<br><br>CONCLUSIONS: Our results suggest that the absence of type I-E CRISPR-Cas systems in K. pneumoniae CC258 is strongly associated with the dissemination of IncF epidemic plasmids, contributing to the global success of the international high-risk linkage CC258-IncF. Our findings provide new information regarding the dissemination and evolution of the high-risk linkage of K. pneumoniae CC258-IncF and pave the way for new strategies to address the problem of antibiotic resistance.},
}
@article {pmid32003123,
year = {2020},
author = {Wang, H and Wang, P and Liang, X and Li, W and Yang, M and Ma, J and Yue, W and Fan, S},
title = {Down-regulation of endothelial protein C receptor promotes preeclampsia by affecting actin polymerization.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {6},
pages = {3370-3383},
pmid = {32003123},
issn = {1582-4934},
mesh = {Actins/*metabolism ; Adult ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Down-Regulation ; Endothelial Protein C Receptor/*biosynthesis/genetics ; Female ; Gene Knockout Techniques ; Humans ; Hypertension/pathology ; Placenta/*metabolism ; Pre-Eclampsia/*pathology ; Pregnancy ; Pregnancy Complications/pathology ; Protein Serine-Threonine Kinases/metabolism ; Proteinuria/pathology ; Trophoblasts/*cytology ; rac1 GTP-Binding Protein/metabolism ; },
abstract = {Preeclampsia is a severe pregnancy-related disease that is found in 3%-5% of pregnancies worldwide and is primarily related to the decreased proliferation and invasion of trophoblast cells and abnormal uterine spiral artery remodelling. However, studies on the pathogenesis of placental trophoblasts are insufficient, and the aetiology of PE remains unclear. Here, we report that endothelial protein C receptor (EPCR), a transmembrane glycoprotein, was down-regulated in placentas from preeclamptic patients. Moreover, lack of EPCR significantly reduced the trophoblast cell proliferation, invasion and tube formation capabilities. Microscale thermophoresis analysis showed that EPCR directly bound to protease-activated receptor 1 (PAR-1), a G protein-coupled receptor. This change resulted in a substantial reduction in active Rac1 and caused excessive actin rearrangement. Our findings reveal a previously unidentified role of EPCR in the regulation of trophoblast proliferation, invasion and tube formation through promotion of actin polymerization, which is required for normal placental development.},
}
@article {pmid32002907,
year = {2020},
author = {Güell, M},
title = {Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2110},
number = {},
pages = {139-149},
doi = {10.1007/978-1-0716-0255-3_10},
pmid = {32002907},
issn = {1940-6029},
mesh = {Algorithms ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Computational Biology/methods ; Endogenous Retroviruses/*genetics ; Gene Dosage ; *Gene Editing ; Gene Expression ; Genes, Reporter ; Genetic Vectors ; Genome-Wide Association Study/*methods ; Heterografts ; Humans ; RNA, Guide ; Swine ; Transplantation, Heterologous/adverse effects ; Virus Activation/genetics ; },
abstract = {The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.},
}
@article {pmid32002906,
year = {2020},
author = {Muñoz-Santos, D and Montoliu, L and Fernández, A},
title = {Generation of Genetically Modified Mice Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2110},
number = {},
pages = {129-138},
doi = {10.1007/978-1-0716-0255-3_9},
pmid = {32002906},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Gene Targeting ; Genetic Engineering/methods ; Genotyping Techniques ; Mice ; *Mice, Transgenic ; Models, Animal ; Oocytes ; RNA, Guide ; },
abstract = {Using CRISPR-based genome-editing techniques, we are able to generate a variety of new mouse models of several types of diseases. These animal models will be instrumental not only for enabling the comprehension of a particular disease and its underlying molecular mechanism but also as unique recipients for testing novel and innovative therapeutic approaches that are being currently explored. This chapter describes detailed step-by-step protocols, reagents, and equipment required for successful generation of genome-edited mice using CRISPR tools.},
}
@article {pmid32002841,
year = {2020},
author = {Kim, YS and Kim, GR and Park, M and Yang, SC and Park, SH and Won, JE and Lee, JH and Shin, HE and Song, H and Kim, HR},
title = {Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor.},
journal = {Tissue engineering and regenerative medicine},
volume = {17},
number = {1},
pages = {45-53},
pmid = {32002841},
issn = {2212-5469},
support = {NRF-2019R1A6A1A03032888//Ministry of Education/International ; HI17C1133//Ministry of Health and Welfare/International ; },
mesh = {Animals ; Base Sequence ; Blastocyst/metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Electroporation/*methods ; Endonucleases ; Gene Editing/*methods ; Gene Targeting ; Leukemia Inhibitory Factor/genetics/*metabolism ; Mice, Knockout ; Microinjections ; RNA, Guide/genetics ; Zygote/*metabolism ; },
abstract = {BACKROUND: CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system.<br><br>METHODS: The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined.<br><br>RESULTS: Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting.<br><br>CONCLUSION: Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.},
}
@article {pmid32002793,
year = {2020},
author = {Huang, H and Zhang, X and Lv, J and Yang, H and Wang, X and Ma, S and Shao, R and Peng, X and Lin, Y and Rong, Z},
title = {Cell-cell contact-induced gene editing/activation in mammalian cells using a synNotch-CRISPR/Cas9 system.},
journal = {Protein &amp; cell},
volume = {11},
number = {4},
pages = {299-303},
pmid = {32002793},
issn = {1674-8018},
mesh = {CRISPR-Cas Systems/*genetics ; *Cell Communication ; *Gene Editing ; HEK293 Cells ; Humans ; Receptors, Notch/*genetics ; },
}
@article {pmid32002787,
year = {2020},
author = {Loi, M and Trazzi, S and Fuchs, C and Galvani, G and Medici, G and Gennaccaro, L and Tassinari, M and Ciani, E},
title = {Increased DNA Damage and Apoptosis in CDKL5-Deficient Neurons.},
journal = {Molecular neurobiology},
volume = {57},
number = {5},
pages = {2244-2262},
pmid = {32002787},
issn = {1559-1182},
mesh = {Animals ; *Apoptosis/drug effects ; Brain-Derived Neurotrophic Factor/pharmacology ; CRISPR-Cas Systems ; Cell Division ; Cell Line, Tumor ; Cells, Cultured ; *DNA Damage ; Epileptic Syndromes/*genetics/pathology ; Gene Editing ; Hippocampus/cytology ; Histones/analysis ; Humans ; Hydrogen Peroxide/pharmacology ; Kainic Acid/pharmacology ; MAP Kinase Signaling System ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons/drug effects/metabolism/*pathology ; Protein Serine-Threonine Kinases/*deficiency ; Proto-Oncogene Proteins c-akt ; Signal Transduction ; Spasms, Infantile/*genetics/pathology ; Tretinoin/pharmacology ; },
abstract = {Mutations in the CDKL5 gene, which encodes a serine/threonine kinase, causes a rare encephalopathy, characterized by early-onset epilepsy and severe intellectual disability, named CDKL5 deficiency disorder (CDD). In vitro and in vivo studies in mouse models of Cdkl5 deficiency have highlighted the role of CDKL5 in brain development and, in particular, in the morphogenesis and synaptic connectivity of hippocampal and cortical neurons. Interestingly, Cdkl5 deficiency in mice increases vulnerability to excitotoxic stress in hippocampal neurons. However, the mechanism by which CDKL5 controls neuronal survival is far from being understood. To investigate further the function of CDKL5 and dissect the molecular mechanisms underlying neuronal survival, we generated a human neuronal model of CDKL5 deficiency, using CRISPR/Cas9-mediated genome editing. We demonstrated that CDKL5 deletion in human neuroblastoma SH-SY5Y cells not only impairs neuronal maturation but also reduces cell proliferation and survival, with alterations in the AKT and ERK signaling pathways and an increase in the proapoptotic BAX protein and in DNA damage-associated biomarkers (i.e., γH2AX, RAD50, and PARP1). Furthermore, CDKL5-deficient cells were hypersensitive to DNA damage-associated stress, accumulated more DNA damage foci (γH2AX positive) and were more prone to cell death than the controls. Importantly, increased kainic acid-induced cell death of hippocampal neurons of Cdkl5 KO mice correlated with an increased γH2AX immunostaining. The results suggest a previously unknown role for CDKL5 in DNA damage response that could underlie the pro-survival function of CDKL5.},
}
@article {pmid32001819,
year = {2020},
author = {Naert, T and Dimitrakopoulou, D and Tulkens, D and Demuynck, S and Carron, M and Noelanders, R and Eeckhout, L and Van Isterdael, G and Deforce, D and Vanhove, C and Van Dorpe, J and Creytens, D and Vleminckx, K},
title = {RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma in Xenopus tropicalis.},
journal = {Oncogene},
volume = {39},
number = {13},
pages = {2692-2706},
pmid = {32001819},
issn = {1476-5594},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Carcinoma, Neuroendocrine/genetics/*pathology ; Carcinoma, Small Cell/genetics/*pathology ; Disease Models, Animal ; Gene Editing ; Glioblastoma/genetics/*pathology ; Humans ; Pancreatic Neoplasms/genetics/*pathology ; Retinoblastoma-Like Protein p107/genetics/*metabolism ; Signal Transduction/genetics ; Xenopus ; Xenopus Proteins/genetics/*metabolism ; },
abstract = {Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small-cell lung cancer (SCLC), choroid plexus tumors, and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional redundancy between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain insight into this in vivo redundancy. We show that while rb1 inactivation is sufficient to induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking tp53 mutant X. tropicalis line, we demonstrate increased malignancy of rb1/rbl1-mutant glioma towards glioblastoma upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, the tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental setup, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1, generating new insights in the functional redundancy within the retinoblastoma protein family in suppressing neuroendocrine pancreatic cancer and glioma/glioblastoma.},
}
@article {pmid32001790,
year = {2020},
author = {Oladimeji, PO and Bakke, J and Wright, WC and Chen, T},
title = {KANSL2 and MBNL3 are regulators of pancreatic ductal adenocarcinoma invasion.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1485},
pmid = {32001790},
issn = {2045-2322},
support = {P30 CA021765/CA/NCI NIH HHS/United States ; R35 GM118041/GM/NIGMS NIH HHS/United States ; P30-CA21765//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; },
mesh = {CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/*genetics/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Histone Acetyltransferases/antagonists &amp; inhibitors/*genetics ; Humans ; Neoplasm Invasiveness/genetics/pathology ; Pancreatic Neoplasms/*genetics/pathology ; RNA, Small Interfering/genetics ; RNA-Binding Proteins/antagonists &amp; inhibitors/*genetics ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer. One major reason for this is that PDAC quickly metastasizes to other organs, thereby making its treatment difficult. The molecular machinery driving PDAC metastasis is still poorly understood. In this study, we applied an unbiased approach using CRISPR screening to identify genes that strongly regulate invasion (based on an in vitro assessment of their metastatic potential) in PANC-1, a PDAC cell line. Through CRISPR screening, we identified MBNL3 and KANSL2 as strong regulators of invasion in PANC-1 cells. We further validated MBNL3 and KANSL2 as regulators of PANC-1 cell invasion by using the doxycycline-inducible shRNA system. We also showed that MBNL3 and KANSL2 do not affect cell proliferation. Through our efforts, we have established a process to identify genes that regulate cell invasion and can be further investigated as potential targets for therapeutic intervention.},
}
@article {pmid32001684,
year = {2020},
author = {Williams, JB and Li, S and Higgs, EF and Cabanov, A and Wang, X and Huang, H and Gajewski, TF},
title = {Tumor heterogeneity and clonal cooperation influence the immune selection of IFN-γ-signaling mutant cancer cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {602},
pmid = {32001684},
issn = {2041-1723},
support = {T32 AI007090/AI/NIAID NIH HHS/United States ; T32 HD007009/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; B7-H1 Antigen/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Clone Cells ; Cytotoxicity, Immunologic ; *Genetic Heterogeneity ; Humans ; Immunomodulation ; Interferon-gamma/*metabolism ; Lymphocytes, Tumor-Infiltrating/immunology ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation/*genetics ; Neoplasms/*genetics/*pathology ; *Signal Transduction ; T-Lymphocytes/immunology ; },
abstract = {PD-1/PD-L1 blockade can promote robust tumor regression yet secondary resistance often occurs as immune selective pressure drives outgrowth of resistant tumor clones. Here using a genome-wide CRISPR screen in B16.SIY melanoma cells, we confirm Ifngr2 and Jak1 as important genes conferring sensitivity to T cell-mediated killing in vitro. However, when implanted into mice, these Ifngr2- and Jak1-deficient tumors paradoxically are better controlled immunologically. This phenotype maps to defective PD-L1 upregulation on mutant tumor cells, which improves anti-tumor efficacy of CD8[+] T cells. To reconcile these observations with clinical reports of anti-PD-1 resistance linked to emergence of IFN-γ signaling mutants, we show that when mixed with wild-type tumor cells, IFN-γ-insensitive tumor cells indeed grow out, which depends upon PD-L1 expression by wild-type cells. Our results illustrate the complexity of functions for IFN-γ in anti-tumor immunity and demonstrate that intratumor heterogeneity and clonal cooperation can contribute to immunotherapy resistance.},
}
@article {pmid32001436,
year = {2020},
author = {Daniel, E and Barlow, HR and Sutton, GI and Gu, X and Htike, Y and Cowdin, MA and Cleaver, O},
title = {Cyp26b1 is an essential regulator of distal airway epithelial differentiation during lung development.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {4},
pages = {},
pmid = {32001436},
issn = {1477-9129},
support = {R01 DK079862/DK/NIDDK NIH HHS/United States ; R01 HL113498/HL/NHLBI NIH HHS/United States ; R24 DK106743/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Endothelial Cells/cytology ; Epithelial Cells/cytology ; Epithelium/*embryology ; Female ; Gene Expression Regulation, Developmental ; Kidney/embryology ; Lung/*embryology ; Mice ; Mice, Inbred C57BL ; Organogenesis/drug effects ; Pregnancy ; Pregnancy, Animal ; Pulmonary Alveoli/*embryology ; Retinoic Acid 4-Hydroxylase/*genetics/*physiology ; Signal Transduction ; Stem Cells/cytology ; Tretinoin/pharmacology ; },
abstract = {Proper organ development depends on coordinated communication between multiple cell types. Retinoic acid (RA) is an autocrine and paracrine signaling molecule essential for the development of most organs, including the lung. Despite extensive work detailing effects of RA deficiency in early lung morphogenesis, little is known about how RA regulates late gestational lung maturation. Here, we investigate the role of the RA catabolizing protein Cyp26b1 in the lung. Cyp26b1 is highly enriched in lung endothelial cells (ECs) throughout development. We find that loss of Cyp26b1 leads to reduction of alveolar type 1 cells, failure of alveolar inflation and early postnatal lethality in mouse. Furthermore, we observe expansion of distal epithelial progenitors, but no appreciable changes in proximal airways, ECs or stromal populations. Exogenous administration of RA during late gestation partially mimics these defects; however, transcriptional analyses comparing Cyp26b1[-/-] with RA-treated lungs reveal overlapping, but distinct, responses. These data suggest that defects observed in Cyp26b1[-/-] lungs are caused by both RA-dependent and RA-independent mechanisms. This work reports crucial cellular crosstalk during lung development involving Cyp26b1-expressing endothelium and identifies a novel RA modulator in lung development.},
}
@article {pmid32001375,
year = {2020},
author = {Espino-Saldaña, AE and Durán-Ríos, K and Olivares-Hernandez, E and Rodríguez-Ortiz, R and Arellano-Carbajal, F and Martínez-Torres, A},
title = {Temporal and spatial expression of zebrafish mctp genes and evaluation of frameshift alleles of mctp2b.},
journal = {Gene},
volume = {738},
number = {},
pages = {144371},
doi = {10.1016/j.gene.2020.144371},
pmid = {32001375},
issn = {1879-0038},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Calcium/metabolism ; Embryonic Development/genetics ; Endoplasmic Reticulum/metabolism ; Frameshift Mutation/genetics ; Gene Expression Regulation, Developmental/genetics ; HEK293 Cells ; Humans ; Membrane Proteins/genetics/*metabolism ; Receptors, Calcium-Sensing/genetics ; Zebrafish/genetics ; Zebrafish Proteins/genetics ; },
abstract = {MCTPs (multiple C2 domain proteins with two transmembrane regions) have been proposed as novel endoplasmic reticulum calcium sensors; however, their function remains largely unknown. Here we report the structure of the four mctp genes from zebrafish (mctp1a, mctp1b, mctp2a and mctp2b), their diversity, expression pattern during embryonic development and in adult tissue and the effect of knocking down the expression of Mctp2b by CRISPR/Cas9. The four mctp genes are expressed from early development and exhibit differential expression patterns but are found mainly in the nervous and muscular systems. Mctp2b tagged with fluorescent proteins and expressed in HEK-293 cells and neurons of the fish spinal cord localized mostly in the endoplasmic reticulum but also in lysosomes and late and recycling endosomes. Knocking down mctp2b expression impaired embryonic development, suggesting that the functional participation of this gene is relevant, at least during the early stages of development.},
}
@article {pmid32001363,
year = {2020},
author = {Kuang, Y and Li, S and Ren, B and Yan, F and Spetz, C and Li, X and Zhou, X and Zhou, H},
title = {Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms.},
journal = {Molecular plant},
volume = {13},
number = {4},
pages = {565-572},
doi = {10.1016/j.molp.2020.01.010},
pmid = {32001363},
issn = {1752-9867},
mesh = {Acetolactate Synthase/*genetics/metabolism ; Adenine ; Agrobacterium/genetics/metabolism ; CRISPR-Cas Systems ; Cytosine ; Directed Molecular Evolution ; Drug Tolerance/*genetics ; Gene Editing ; Genome, Plant/genetics ; Herbicides/*pharmacology ; Oryza/*drug effects/genetics ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Polymorphism, Single Nucleotide ; },
abstract = {Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, derived from two sites that have never been targeted by natural or human selection during rice domestication, were identified, conferring varying levels of tolerance to the herbicide bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.},
}
@article {pmid32000993,
year = {2020},
author = {Lee, K and Uh, K and Farrell, K},
title = {Current progress of genome editing in livestock.},
journal = {Theriogenology},
volume = {150},
number = {},
pages = {229-235},
pmid = {32000993},
issn = {1879-3231},
support = {R21 OD027062/OD/NIH HHS/United States ; },
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Editing/*veterinary ; Genetic Engineering/*veterinary ; Livestock/*genetics ; },
abstract = {Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.},
}
@article {pmid31999448,
year = {2020},
author = {Siemon, T and Wang, Z and Bian, G and Seitz, T and Ye, Z and Lu, Y and Cheng, S and Ding, Y and Huang, Y and Deng, Z and Liu, T and Christmann, M},
title = {Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {6},
pages = {2760-2765},
doi = {10.1021/jacs.9b12940},
pmid = {31999448},
issn = {1520-5126},
mesh = {CRISPR-Cas Systems ; Escherichia coli/genetics ; *Metabolic Engineering ; Plants/*chemistry ; Saccharomyces cerevisiae/genetics ; Sesquiterpenes, Guaiane/chemical synthesis/*chemistry ; },
abstract = {Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.},
}
@article {pmid31998323,
year = {2019},
author = {Laparidou, M and Schlickenrieder, A and Thoma, T and Lengyel, K and Schusser, B},
title = {Blocking of the CXCR4-CXCL12 Interaction Inhibits the Migration of Chicken B Cells Into the Bursa of Fabricius.},
journal = {Frontiers in immunology},
volume = {10},
number = {},
pages = {3057},
pmid = {31998323},
issn = {1664-3224},
mesh = {Animals ; B-Lymphocytes/immunology ; Bursa of Fabricius/*immunology ; CRISPR-Cas Systems/immunology ; Cell Movement/*immunology ; Chemokine CXCL12/*immunology ; Chemotaxis/immunology ; Chick Embryo ; Chickens/*immunology ; Mice ; Receptors, CXCR4/*immunology ; Signal Transduction/immunology ; },
abstract = {B cells have first been described in chickens as antibody producing cells and were named after the Bursa of Fabricius, a unique organ supporting their development. Understanding different factors mediating the early migration of B cells into the bursa of Fabricius is crucial for the study of B cell biology. While CXCL12 (stromal derived factor 1) was found to play an important role in B lymphocyte trafficking in mammals, its role in the chicken is still unknown. Previous studies indicated that chicken CXCL12 and its receptor CXCR4 are simultaneously expressed during bursal development. In this study, we investigated whether the CXCR4/CXCL12 interaction mediates B cell migration in chicken embryo. We used the CRISPR/Cas9 system to induce a CXCR4 knockout in chicken B cells which led to chemotaxis inhibition toward CXCL12. This was confirmed by adoptive cell transfer and inhibition of the CXCR4/CXCL12 interaction by blocking with the small inhibitor AMD3100. In addition, we found that the chicken exhibits similarities to mice when it comes to CXCR4 being dependent on B cell receptor expression. B cells lacking the B cell receptor failed to migrate toward CXCL12 and showed no response upon CXCL12 stimulation. Overall, we demonstrated the significance of CXCR4/CXCL12 in chicken B cell development in vivo and the importance of the B cell receptor in CXCR4 dependent signaling.},
}
@article {pmid31998256,
year = {2019},
author = {Kamruzzaman, M and Iredell, JR},
title = {CRISPR-Cas System in Antibiotic Resistance Plasmids in Klebsiella pneumoniae.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2934},
pmid = {31998256},
issn = {1664-302X},
abstract = {CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is a microbial adaptive immune system involved in defense against different types of mobile genetic elements. CRISPR-Cas systems are usually found in bacterial and archaeal chromosomes but have also been reported in bacteriophage genomes and in a few mega-plasmids. Klebsiella pneumoniae is an important member of the Enterobacteriaceae with which they share a huge pool of antibiotic resistance genes, mostly via plasmids. CRISPR-Cas systems have been identified in K. pneumoniae chromosomes, but relatively little is known of CRISPR-Cas in the plasmids resident in this species. In this study, we searched for CRISPR-Cas system in 699 complete plasmid sequences (>50-kb) and 217 complete chromosomal sequences of K. pneumoniae from GenBank and analyzed the CRISPR-Cas systems and CRISPR spacers found in plasmids and chromosomes. We found a putative CRISPR-Cas system in the 44 plasmids from Klebsiella species and GenBank search also identified the identical system in three plasmids from other Enterobacteriaceae, with CRISPR spacers targeting different plasmid and chromosome sequences. 45 of 47 plasmids with putative type IV CRISPR had IncFIB replicon and 36 of them had an additional IncHI1B replicon. All plasmids except two are very large (>200 kb) and half of them carried multiple antibiotic resistance genes including bla CTX-M , bla NDM , bla OXA . To our knowledge, this is the first report of multi drug resistance plasmids from Enterobacteriaceae with their own CRISPR-Cas system and it is possible that the plasmid type IV CRISPR may depend on the chromosomal type I-E CRISPRs for their competence. Both chromosomal and plasmid CRISPRs target a large variety of plasmids from this species, further suggesting key roles in the epidemiology of large plasmids.},
}
@article {pmid31996360,
year = {2020},
author = {Lund, C and Yellapragada, V and Vuoristo, S and Balboa, D and Trova, S and Allet, C and Eskici, N and Pulli, K and Giacobini, P and Tuuri, T and Raivio, T},
title = {Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells.},
journal = {Disease models &amp; mechanisms},
volume = {13},
number = {3},
pages = {},
pmid = {31996360},
issn = {1754-8411},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Fetus/cytology ; Fibroblast Growth Factor 8/pharmacology ; *Genes, Reporter ; Gonadotropin-Releasing Hormone/*metabolism ; Humans ; Hypogonadism/genetics ; LIM-Homeodomain Proteins/metabolism ; Neurons/drug effects/*metabolism ; Pluripotent Stem Cells/drug effects/*metabolism ; RNA, Messenger/genetics/metabolism ; Transcription Factors/metabolism ; Transcriptome/*genetics ; Up-Regulation/drug effects/genetics ; },
abstract = {Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid31996144,
year = {2020},
author = {Ren, C and Guo, Y and Kong, J and Lecourieux, F and Dai, Z and Li, S and Liang, Z},
title = {Knockout of VvCCD8 gene in grapevine affects shoot branching.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {47},
pmid = {31996144},
issn = {1471-2229},
mesh = {Arabidopsis Proteins/*genetics ; CRISPR-Cas Systems ; Dioxygenases/*genetics ; Gene Editing ; Gene Knockout Techniques ; Genes, Plant ; Plant Shoots/*genetics/growth &amp; development ; Plants, Genetically Modified ; Vitis/*genetics ; },
abstract = {BACKGROUND: Shoot branching is an important trait of plants that allows them to adapt to environment changes. Strigolactones (SLs) are newly identified plant hormones that inhibit shoot branching in plants. The SL biosynthesis genes CCD7 (carotenoid cleavage dioxygenase 7) and CCD8 have been found to regulate branching in several herbaceous plants by taking advantage of their loss-of-function mutants. However, the role for CCD7 and CCD8 in shoot branching control in grapevine is still unknown due to the lack of corresponding mutants.<br><br>RESULTS: Here we employed the CRISPR/Cas9 system to edit the VvCCD7 and VvCCD8 genes in the grape hybrid 41B. The 41B embryogenic cells can easily be transformed and used for regeneration of the corresponding transformed plants. Sequencing analysis revealed that gene editing has been used successfully to target both VvCCD genes in 41B embryogenic cells. After regeneration, six 41B plantlets were identified as transgenic plants carrying the CCD8-sgRNA expression cassette. Among these, four plants showed mutation in the target region and were selected as ccd8 mutants. These ccd8 mutants showed increased shoot branching compared to the corresponding wild-type plants. In addition, no off-target mutation was detected in the tested mutants at predicted off-target sites.<br><br>CONCLUSIONS: Our results underline the key role of VvCCD8 in the control of grapevine shoot branching.},
}
@article {pmid31995698,
year = {2020},
author = {Urnov, FD},
title = {Prime Time for Genome Editing?.},
journal = {The New England journal of medicine},
volume = {382},
number = {5},
pages = {481-484},
doi = {10.1056/NEJMcibr1914271},
pmid = {31995698},
issn = {1533-4406},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/history/*methods ; Genetic Diseases, Inborn/genetics/therapy ; History, 21st Century ; Humans ; Mammals ; Mutation ; },
}
@article {pmid31995666,
year = {2020},
author = {Gong, T and Zeng, J and Tang, B and Zhou, X and Li, Y},
title = {CRISPR-Cas systems in oral microbiome: From immune defense to physiological regulation.},
journal = {Molecular oral microbiology},
volume = {35},
number = {2},
pages = {41-48},
doi = {10.1111/omi.12279},
pmid = {31995666},
issn = {2041-1014},
mesh = {Archaea/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Humans ; *Microbiota/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins (CRISPR-Cas) system, found in bacteria and archaea, provides sequence-based adaptive immunity against mobile genetic elements, including phages and plasmids. The oral cavity contains approximately 700 prokaryote species harboring known CRISPR-Cas systems, including type I, type II, type III, type V, and type VI, and unidentified CRISPR-Cas systems. There is increasing evidence to suggest that different CRISPR-Cas systems in the human oral microbiome can affect bacterial physiology through different mechanisms. Here, we review the canonical and novel functions of the CRISPR-Cas system, including defense against the invasion of foreign mobile elements, biofilm formation, acquisition of resistance genes, DNA repair, regulation of interspecific competition and intraspecific diversification, stress responses, and gene expression regulation. Overall, the mechanisms involved in CRISPR-Cas systems and their effects on bacterial physiology provide new insights into our understanding of the function and application of methods (including gene editing, modulation of CRISPR-Cas by anti-CRISPR, antimicrobials) on the oral microbiome.},
}
@article {pmid31994379,
year = {2020},
author = {Sun, X and Wang, Y and Zhang, L and Liu, S and Zhang, M and Wang, J and Ning, B and Peng, Y and He, J and Hu, Y and Gao, Z},
title = {CRISPR-Cas9 Triggered Two-Step Isothermal Amplification Method for E. coli O157:H7 Detection Based on a Metal-Organic Framework Platform.},
journal = {Analytical chemistry},
volume = {92},
number = {4},
pages = {3032-3041},
doi = {10.1021/acs.analchem.9b04162},
pmid = {31994379},
issn = {1520-6882},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Escherichia coli O157/genetics/*isolation &amp; purification ; Metal-Organic Frameworks/*chemistry ; *Nucleic Acid Amplification Techniques ; },
abstract = {Escherichia coli O157:H7 has been reported as an important pathogenic bacteria causing serious infection and economic loss. However, detection of Escherichia coli O157:H7 needs improvement, given its current complexity and sensitivity. Herein, we attempt to build a fluorescence sensing method to detect Escherichia coli O157:H7 with easy operation and high efficiency. The target virulence gene sequences are recognized and cleaved by the CRISPR-Cas9 system, and trigger strand displacement amplification and rolling circle amplification. After amplification reactions, massive products can hybridize with the probes, the fluorescence of which are quenched based on a metal-organic framework platform, leading to the fluorescence recovery at typical excitation/emission wavelengths of 480/518 nm. This method exhibits high sensitivity with the detection limit at 4.0 × 10[1] CFU mL[-1] and a wide range from 1.3 × 10[2] CFU mL[-1] to 6.5 × 10[4] CFU mL[-1]. Meanwhile, this assay also shows significant specificity and applies to practical samples with high accuracy. Therefore, our method would have great potential application in bacterial detection, food safety monitoring, or clinical diagnostics.},
}
@article {pmid31992616,
year = {2020},
author = {Yan, MY and Li, SS and Ding, XY and Guo, XP and Jin, Q and Sun, YC},
title = {A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis.},
journal = {mBio},
volume = {11},
number = {1},
pages = {},
pmid = {31992616},
issn = {2150-7511},
mesh = {Bacterial Toxins/genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA End-Joining Repair ; *Gene Editing ; *Genome, Bacterial ; Models, Biological ; Mycobacterium smegmatis/genetics ; Mycobacterium tuberculosis/*genetics/metabolism ; Protein Binding ; RNA, Guide ; Rec A Recombinases/metabolism ; },
abstract = {New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coliIMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.},
}
@article {pmid31991936,
year = {2020},
author = {Gao, Q and Li, G and Sun, H and Xu, M and Wang, H and Ji, J and Wang, D and Yuan, C and Zhao, X},
title = {Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {31991936},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Multigene Family ; *Mutagenesis ; *Oryza/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; *Plants, Genetically Modified/genetics/growth &amp; development ; },
abstract = {The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T0 lines for 15 of the 16 targets. The targeted mutations are detected in the T0 lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T0 plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T1 generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T1 generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.},
}
@article {pmid31991730,
year = {2020},
author = {Fry, LE and Peddle, CF and Barnard, AR and McClements, ME and MacLaren, RE},
title = {RNA editing as a therapeutic approach for retinal gene therapy requiring long coding sequences.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {31991730},
issn = {1422-0067},
support = {MC_PC_19049/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adenosine Deaminase/metabolism ; Animals ; CRISPR-Cas Systems ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Targeting ; Genetic Diseases, Inborn/genetics/therapy ; *Genetic Therapy ; Humans ; *RNA Editing ; RNA-Binding Proteins/metabolism ; Retina/*metabolism/pathology ; Retinal Diseases/genetics/therapy ; *Transgenes ; },
abstract = {RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration.},
}
@article {pmid31991288,
year = {2020},
author = {Su, D and Feng, X and Colic, M and Wang, Y and Zhang, C and Wang, C and Tang, M and Hart, T and Chen, J},
title = {CRISPR/CAS9-based DNA damage response screens reveal gene-drug interactions.},
journal = {DNA repair},
volume = {87},
number = {},
pages = {102803},
pmid = {31991288},
issn = {1568-7856},
support = {R35 GM130119/GM/NIGMS NIH HHS/United States ; R01 CA216437/CA/NCI NIH HHS/United States ; R01 CA216911/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA210929/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; DNA Damage/*genetics ; DNA Polymerase III/genetics ; DNA-Binding Proteins/genetics ; Drug Resistance/*genetics ; Gene Library ; Genes, p53/genetics ; HEK293 Cells ; Humans ; Nucleoproteins/genetics ; Poly(ADP-ribose) Polymerase Inhibitors/metabolism ; Small Molecule Libraries/*pharmacology ; },
abstract = {DNA damage response (DDR) is critically important for cell survival, genome maintenance, and its defect has been exploited therapeutically in cancer treatment. Many DDR-targeting agents have been generated and have entered the clinic and/or clinical trials. In order to provide a global and unbiased view of DDR network, we designed a focused CRISPR library targeting 365 DDR genes and performed CRISPR screens on the responses to several DDR inhibitors and DNA-damaging agents in 293A cells. With these screens, we determined responsive pathways enriched under treatment with different types of small-molecule agents. Additionally, we showed that POLE3/4-deficient cells displayed enhanced sensitivity to an ATR inhibitor, a PARP inhibitor, and camptothecin. Moreover, by performing DDR screens in isogenic TP53 wild-type and TP53 knock-out cell lines, our results suggest that the performance of our CRISPR DDR dropout screens is independent of TP53 status. Collectively, our findings indicate that CRISPR DDR screens can be used to identify potential targets of small-molecule drugs and reveal that TP53 status does not affect the outcome of these screens.},
}
@article {pmid31990070,
year = {2020},
author = {Semina, EV and Rubina, KA and Shmakova, AA and Rysenkova, KD and Klimovich, PS and Aleksanrushkina, NA and Sysoeva, VY and Karagyaur, MN and Tkachuk, VA},
title = {Downregulation of uPAR promotes urokinase translocation into the nucleus and epithelial to mesenchymal transition in neuroblastoma.},
journal = {Journal of cellular physiology},
volume = {235},
number = {9},
pages = {6268-6286},
pmid = {31990070},
issn = {1097-4652},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Nucleus/*genetics ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; Humans ; Membrane Proteins/*genetics ; Neuroblastoma/*genetics/pathology ; Receptors, Urokinase Plasminogen Activator/*genetics ; Signal Transduction ; },
abstract = {The urokinase system is involved in a variety of physiological processes, such as fibrinolysis, matrix remodeling, wound healing, and regeneration. Upon binding to its cognate receptor urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator (uPA) catalyzes the conversion of plasminogen to plasmin and the activation of matrix metalloproteases. Apart from this, uPA-uPAR interaction can lead to the activation of transcription factors, mitogen-activated protein kinase signaling pathways and RTK cascades. Elevated expression of uPA and uPAR is markedly associated with cancer progression and metastasis and correlates with a poor prognosis in clinics. Targeting the urokinase system has proved to be effective in experimental models in vitro and in vivo, however, in clinics the inhibition of the uPA/uPAR system has fallen short of expectations, suggesting that the question of the functional relevance of uPA/uPAR system is far from being moot. Recently, using CRISPR/Cas9 technology, we have shown that uPAR knockout decreases the proliferation of neuroblastoma Neuro2a cells in vitro. In the present study we demonstrate that uPAR expression is essential for maintaining the epithelial phenotype in Neuro2a cells and that uPAR silencing promotes epithelial-mesenchymal transition (EMT) and increased cell migration. Accordingly, uPAR knockout results in the downregulation of epithelial markers (E-cadherin, occludin, and claudin-5) and in the increase of mesenchymal markers (N-cadherin, α-smooth muscle actin, and interleukin-6). In search of the molecular mechanism underlying these changes, we identified uPA as a key component. Two key insights emerged as a result of this work: in the absence of uPAR, uPA is translocated into the nucleus where it is presumably involved in the activation of transcription factors (nuclear factor κB and Snail) resulting in EMT. In uPAR-expressing cells, uPAR functions as a uPA "trap" that binds uPA on the cell surface and promotes controlled uPA internalization and degradation in lysosomes.},
}
@article {pmid31989926,
year = {2020},
author = {Sanor, LD and Flowers, GP and Crews, CM},
title = {Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {31989926},
issn = {2050-084X},
support = {Predoctoral Training Fellowship T32GM007499/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Ambystoma mexicanum/embryology/*genetics/*physiology ; Animals ; *CRISPR-Cas Systems ; Catalase/genetics ; Fetuin-B/genetics ; Gene Editing ; Gene Expression Regulation, Developmental ; Genetic Testing/methods ; *Haploidy ; Karyotype ; Methyltransferases/genetics ; Monophenol Monooxygenase/genetics ; Mutation ; Regeneration ; Sequence Analysis ; },
abstract = {Axolotls and other salamanders can regenerate entire limbs after amputation as adults, and much recent effort has sought to identify the molecular programs controlling this process. While targeted mutagenesis approaches like CRISPR/Cas9 now permit gene-level investigation of these mechanisms, genetic screening in the axolotl requires an extensive commitment of time and space. Previously, we quantified CRISPR/Cas9-generated mutations in the limbs of mosaic mutant axolotls before and after regeneration and found that the regenerated limb is a highfidelity replicate of the original limb (Flowers et al. 2017). Here, we circumvent aforementioned genetic screening limitations and present methods for a multiplex CRISPR/Cas9 haploid screen in chimeric axolotls (MuCHaChA), which is a novel platform for haploid genetic screening in animals to identify genes essential for limb regeneration.},
}
@article {pmid31989837,
year = {2020},
author = {Ling, K and Yang, L and Yang, N and Chen, M and Wang, Y and Liang, S and Li, Y and Jiang, L and Yan, P and Liang, Z},
title = {Gene Targeting of HPV18 E6 and E7 Synchronously by Nonviral Transfection of CRISPR/Cas9 System in Cervical Cancer.},
journal = {Human gene therapy},
volume = {31},
number = {5-6},
pages = {297-308},
doi = {10.1089/hum.2019.246},
pmid = {31989837},
issn = {1557-7422},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; DNA-Binding Proteins/*genetics ; Disease Models, Animal ; Female ; Gene Knockout Techniques ; Gene Targeting ; Genetic Therapy/methods ; HeLa Cells ; Humans ; Mice ; Mice, Nude ; Oncogene Proteins, Viral/*genetics ; Papillomavirus Infections/*therapy ; Transfection ; Tumor Suppressor Protein p53/metabolism ; Uterine Cervical Neoplasms/*therapy/virology ; rho GTP-Binding Proteins/metabolism ; },
abstract = {High-risk human papillomavirus (HPV) E6 and E7 genes display vital oncogenic properties in cervical cancer. Eliminating HPV driver gene or loss of function by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a promising treatment for the HPV-associated cancer. Thus, this study designed a CRISPR/Cas9 system to target the E6 and E7 genes at once, to detect whether it have efficacy in vitro and in vivo. Meanwhile, CRISPR/Cas9 system was measured after transfection with liposomes but virus. Cervical cancer lines (HeLa and SiHa) were used in this study. Sanger sequencing confirmed that the single CRISPR/Cas9 vector [termed E6E7-knockout (KO)] containing guide RNAs could targeting both HPV18 E6 and E7 genes in vitro. In addition, double-targeting E6 and E7 increased p53 protein expression significantly while compared with E6 or E7 targeting, respectively. Mice with xenografts were divided into four groups: three doses of experimental groups (20, 40, and 60 μg) and one control group. The E6E7-KO through liposome delivery was injected into tumors. Tumor growth was measured and protein expression was observed through immunohistochemistry. The toxic side effects in vivo were also evaluated. E6E7-KO induced cell apoptosis and inhibited cell proliferation markedly in vitro. E6E7-KO downregulated the messenger RNA and protein expression of E6 and E7, whereas p53 and p21 protein levels were upregulated accordingly. Notably, E6E7-KO delivery by liposome exhibited an effect in vivo. Tumor growth was inhibited in the E6E7-KO groups, which was accompanied by decreased E6/E7 protein expression and increased p53/p21 protein expression, especially the level of p53 protein expression. Therefore, E6E7-KO could have synergy efficient by p53 pathway. Furthermore, local injection with CRISPR/Cas9 by nonviral delivery may be regarded as a potential therapy for cervical cancer in the future.},
}
@article {pmid31989168,
year = {2020},
author = {Creutzburg, SCA and Wu, WY and Mohanraju, P and Swartjes, T and Alkan, F and Gorodkin, J and Staals, RHJ and van der Oost, J},
title = {Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a.},
journal = {Nucleic acids research},
volume = {48},
number = {6},
pages = {3228-3243},
pmid = {31989168},
issn = {1362-4962},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA Repair/*genetics ; Endodeoxyribonucleases/*genetics ; Escherichia coli/genetics ; *Gene Editing ; Luminescent Proteins/genetics ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.},
}
@article {pmid31988296,
year = {2020},
author = {Woznicki, JA and Flood, P and Bustamante-Garrido, M and Stamou, P and Moloney, G and Fanning, A and Zulquernain, SA and McCarthy, J and Shanahan, F and Melgar, S and Nally, K},
title = {Human BCL-G regulates secretion of inflammatory chemokines but is dispensable for induction of apoptosis by IFN-γ and TNF-α in intestinal epithelial cells.},
journal = {Cell death &amp; disease},
volume = {11},
number = {1},
pages = {68},
pmid = {31988296},
issn = {2041-4889},
mesh = {Apoptosis/*drug effects/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chemokine CCL20/metabolism ; Chemokine CCL5/metabolism ; Chemokines/*metabolism ; Colorectal Neoplasms/genetics/metabolism ; Epithelial Cells/drug effects/*metabolism ; Gene Knockout Techniques ; Humans ; Inflammatory Bowel Diseases/genetics/metabolism ; Interferon-gamma/*pharmacology ; Intestinal Mucosa/*metabolism ; NF-kappa B/metabolism ; Organoids/drug effects ; Proto-Oncogene Proteins c-bcl-2/genetics/*metabolism ; STAT1 Transcription Factor/genetics/metabolism ; Transcription Factors/metabolism ; Tumor Necrosis Factor-alpha/*pharmacology ; Up-Regulation ; },
abstract = {Proteins of the BCL-2 family are evolutionarily conserved modulators of apoptosis that function as sensors of cellular integrity. Over the past three decades multiple BCL-2 family members have been identified, many of which are now fully incorporated into regulatory networks governing the mitochondrial apoptotic pathway. For some, however, an exact role in cell death signalling remains unclear. One such 'orphan' BCL-2 family member is BCL-G (or BCL2L14). In this study we analysed gastrointestinal expression of human BCL-G in health and disease states, and investigated its contribution to inflammation-induced tissue damage by exposing intestinal epithelial cells (IEC) to IFN-γ and TNF-α, two pro-inflammatory mediators associated with gut immunopathology. We found that both BCL-G splice variants - BCL-GS (short) and BCL-GL (long) - were highly expressed in healthy gut tissue, and that their mRNA levels decreased in active inflammatory bowel diseases (for BCL-GS) and colorectal cancer (for BCL-GS/L). In vitro studies revealed that IFN-γ and TNF-α synergised to upregulate BCL-GS/L and to trigger apoptosis in colonic epithelial cell lines and primary human colonic organoids. Using RNAi, we showed that synergistic induction of IEC death was STAT1-dependent while optimal expression of BCL-GS/L required STAT1, NF-κB/p65 and SWI/SNF-associated chromatin remodellers BRM and BRG1. To test the direct contribution of BCL-G to the effects of IFN-γ and TNF-α on epithelial cells, we used RNAi- and CRISPR/Cas9-based perturbations in parallel with isoform-specific overexpression of BCL-G, and found that BCL-G was dispensable for Th1 cytokine-induced apoptosis of human IEC. Instead, we discovered that depletion of BCL-G differentially affected secretion of inflammatory chemokines CCL5 and CCL20, thus uncovering a non-apoptotic immunoregulatory function of this BCL-2 family member. Taken together, our data indicate that BCL-G may be involved in shaping immune responses in the human gut in health and disease states through regulation of chemokine secretion rather than intestinal apoptosis.},
}
@article {pmid31988185,
year = {2020},
author = {Bryan, CD and Casey, MA and Pfeiffer, RL and Jones, BW and Kwan, KM},
title = {Optic cup morphogenesis requires neural crest-mediated basement membrane assembly.},
journal = {Development (Cambridge, England)},
volume = {147},
number = {4},
pages = {},
pmid = {31988185},
issn = {1477-9129},
support = {R01 EY015128/EY/NEI NIH HHS/United States ; R01 EY025780/EY/NEI NIH HHS/United States ; R01 EY028927/EY/NEI NIH HHS/United States ; T32 GM007464/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Basement Membrane/*embryology ; CRISPR-Cas Systems ; Calcium-Binding Proteins/physiology ; Cell Movement ; Electrophoresis, Capillary ; Extracellular Matrix/physiology ; Extracellular Matrix Proteins/physiology ; Eye/*embryology ; Forkhead Transcription Factors/physiology ; Gene Expression Regulation, Developmental ; Genotype ; Mesoderm/embryology ; Microscopy, Electron, Transmission ; Morphogenesis ; Mutation ; Neural Crest/cytology/*embryology ; Organogenesis ; Retina/embryology ; Retinal Pigment Epithelium/embryology ; Signal Transduction ; Transcription Factor AP-2/physiology ; Zebrafish ; Zebrafish Proteins/physiology ; },
abstract = {Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.},
}
@article {pmid31988182,
year = {2020},
author = {Zhu, J and Hay, AN and Potter, AA and Richwine, MW and Sproule, T and LeRoith, T and Wilson, J and Hasham, MG and Roopenian, DC and Leeth, CM},
title = {Abrogated AID Function Prolongs Survival and Diminishes Renal Pathology in the BXSB Mouse Model of Systemic Lupus Erythematosus.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {204},
number = {5},
pages = {1091-1100},
pmid = {31988182},
issn = {1550-6606},
support = {K08 DK101735/DK/NIDDK NIH HHS/United States ; R03 AR066787/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocyte Subsets/*immunology/pathology ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/*immunology ; Disease Models, Animal ; Lupus Erythematosus, Systemic/genetics/*immunology/pathology ; Mice ; Mice, Knockout ; },
abstract = {Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex-mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73[+] B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.},
}
@article {pmid31988004,
year = {2020},
author = {Zaynab, M and Sharif, Y and Fatima, M and Afzal, MZ and Aslam, MM and Raza, MF and Anwar, M and Raza, MA and Sajjad, N and Yang, X and Li, S},
title = {CRISPR/Cas9 to generate plant immunity against pathogen.},
journal = {Microbial pathogenesis},
volume = {141},
number = {},
pages = {103996},
doi = {10.1016/j.micpath.2020.103996},
pmid = {31988004},
issn = {1096-1208},
mesh = {Bacterial Infections ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Disease Resistance/*genetics ; Gene Editing/*methods ; Mycoses ; Plant Immunity/genetics ; Plants, Genetically Modified ; Virus Diseases ; },
abstract = {Different types of molecular approaches have been used for improving resistance against pathogens to secure food. Efficient and advanced genome editing tool as paralleled to earlier techniques like Zinc Finger Nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). The approach of CRISPR/Cas9 has updated our abilities of genetic manipulation in many crops. The assembly of purposes that can be achieved through CRISPR/Cas9 and its related products make it a powerful system that will expose novel prospects in the complex domain of plant-pathogen interactions and will help to develop crop resistance against pathogens. CRISPR/Cas9 engineering permits DNA endonuclease guided by an RNA for a range of genome engineering applications across various eukaryotic species and provides an effective platform to create resistance against bacteria, viruses, insects, and fungi. In this review, we discuss CRISPR-Cas9 engineered crop plants resistant to specific pathogens.},
}
@article {pmid31987937,
year = {2020},
author = {Hausmann, C and Vogt, A and Kerscher, M and Ghoreschi, K and Schäfer-Korting, M and Zoschke, C},
title = {Optimizing skin pharmacotherapy for older patients: the future is at hand but are we ready for it?.},
journal = {Drug discovery today},
volume = {25},
number = {5},
pages = {851-861},
doi = {10.1016/j.drudis.2020.01.011},
pmid = {31987937},
issn = {1878-5832},
mesh = {Administration, Cutaneous ; Aged ; Aging/*drug effects ; Animals ; Bioprinting/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Pharmaceutical Preparations/*administration &amp; dosage ; Skin Diseases/*drug therapy/genetics ; },
abstract = {Age-related changes affect both the local pharmacotherapy of skin diseases and the transdermal administration of drugs. The development of aged skin models disregards the highly individual process of aging, facilitating general conclusions for older patients. Nevertheless, 'omics technology, high-content screening, and non-invasive imaging, as well as bioprinting, CRISPR-Cas, and, patients-on-a-chip, can retrieve personalized information for the generation of in vitro models. Herein, we suggest a strategy to optimize pharmacotherapy for older patients. The technology for relevant human cell-based models is at hand and the consideration of patient heterogeneity is required to unlock their full potential.},
}
@article {pmid31987908,
year = {2020},
author = {Kalkan, BM and Kala, EY and Yuce, M and Karadag Alpaslan, M and Kocabas, F},
title = {Development of gene editing strategies for human β-globin (HBB) gene mutations.},
journal = {Gene},
volume = {734},
number = {},
pages = {144398},
doi = {10.1016/j.gene.2020.144398},
pmid = {31987908},
issn = {1879-0038},
mesh = {Anemia, Sickle Cell/*genetics ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA Repair ; Gene Editing/*methods ; Genetic Vectors ; HEK293 Cells ; Humans ; Mutation ; RNA, Guide ; beta-Globins/*genetics ; },
abstract = {Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene.},
}
@article {pmid31987837,
year = {2020},
author = {Kaneko, T and Nakagawa, Y},
title = {Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos.},
journal = {Cryobiology},
volume = {92},
number = {},
pages = {231-234},
doi = {10.1016/j.cryobiol.2020.01.016},
pmid = {31987837},
issn = {1090-2392},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryopreservation/*methods ; Electroporation/*methods ; Embryo, Mammalian/*cytology ; Embryonic Development ; Endonucleases/genetics/metabolism ; Freezing ; Gene Editing/*methods ; Mice ; Mice, Knockout ; Microinjections ; Rats ; Vitrification ; },
abstract = {Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)," has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.},
}
@article {pmid31987174,
year = {2020},
author = {Xiao, G and Liu, S and Liu, H and He, X and Zhang, S and Liang, Z and Guo, H and Ou, M and Zhou, L and Liu, L and Zhang, T and Zhang, G},
title = {CRISPR/Cas12a-based biosensing platform for precise and efficient screening of CRISPR/Cas9-induced biallelic mutants.},
journal = {Talanta},
volume = {210},
number = {},
pages = {120613},
doi = {10.1016/j.talanta.2019.120613},
pmid = {31987174},
issn = {1873-3573},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Humans ; Mutation ; RNA/genetics ; THP-1 Cells ; },
abstract = {CRISPR/Cas9 is a robust tool to manipulate genes in a wide range of species. Although several methods are introduced to identify the CRISPR/Cas9-induced mutations, they are labor-intensive, costly, and not easy to use or were sequence-limited. Moreover, few of them could identify the biallelic mutants that are the desired outcomes of targeted mutagenesis. Recently, a CRISPR/Cas12a-mediated biosensing platform was developed to detect nucleic acids based on the collateral DNA cleavage activity of Cas12a; it was highly sensitive, specific, rapid, and cost-efficient for genotyping, mutation detection, and single nucleotide polymorphism (SNP) identification, thereby deeming it as an innovative method for screening the CRISPR/Cas9-induced biallelic mutants. Thus, the CRISPR/Cas12a-based biosensing platform has been successfully utilized for screening 23 CRISPR/Cas9-induced biallelic mutants in Thp-1 cells, which were also confirmed by direct sequencing and ELISA. The precision and efficiency of CRISPR/Cas12a-based biosensing platform make it a promising tool for screening of CRISPR/Cas9-induced biallelic mutants in the future.},
}
@article {pmid31985873,
year = {2020},
author = {Liu, X and Qin, R and Li, J and Liao, S and Shan, T and Xu, R and Wu, D and Wei, P},
title = {A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice.},
journal = {Plant biotechnology journal},
volume = {18},
number = {9},
pages = {1845-1847},
pmid = {31985873},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Oryza/genetics ; },
}
@article {pmid31984132,
year = {2020},
author = {Ilyas, M and Mir, A and Efthymiou, S and Houlden, H},
title = {The genetics of intellectual disability: advancing technology and gene editing.},
journal = {F1000Research},
volume = {9},
number = {},
pages = {},
pmid = {31984132},
issn = {2046-1402},
support = {MR/J004758/1/MRC_/Medical Research Council/United Kingdom ; G0601943/MRC_/Medical Research Council/United Kingdom ; G0802760/MRC_/Medical Research Council/United Kingdom ; G1001253/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; G108/638/MRC_/Medical Research Council/United Kingdom ; MR/S005021/1/MRC_/Medical Research Council/United Kingdom ; R605/0717/DMT_/The Dunhill Medical Trust/United Kingdom ; MR/S01165X/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; Intellectual Disability/*genetics ; Neurodevelopmental Disorders/*genetics ; },
abstract = {Intellectual disability (ID) is a neurodevelopmental condition affecting 1-3% of the world's population. Genetic factors play a key role causing the congenital limitations in intellectual functioning and adaptive behavior. The heterogeneity of ID makes it more challenging for genetic and clinical diagnosis, but the advent of large-scale genome sequencing projects in a trio approach has proven very effective. However, many variants are still difficult to interpret. A combined approach of next-generation sequencing and functional, electrophysiological, and bioinformatics analysis has identified new ways to understand the causes of ID and help to interpret novel ID-causing genes. This approach offers new targets for ID therapy and increases the efficiency of ID diagnosis. The most recent functional advancements and new gene editing techniques involving the use of CRISPR-Cas9 allow for targeted editing of DNA in in vitro and more effective mammalian and human tissue-derived disease models. The expansion of genomic analysis of ID patients in diverse and ancient populations can reveal rare novel disease-causing genes.},
}
@article {pmid31983425,
year = {2020},
author = {Kim, DH and Kim, EJ and Kim, DH and Park, SW},
title = {Dact2 is involved in the regulation of epithelial-mesenchymal transition.},
journal = {Biochemical and biophysical research communications},
volume = {524},
number = {1},
pages = {190-197},
doi = {10.1016/j.bbrc.2019.12.090},
pmid = {31983425},
issn = {1090-2104},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Carcinogenesis/metabolism/pathology ; *Epithelial-Mesenchymal Transition ; Gastrointestinal Tract/pathology ; Gene Expression Regulation ; Gene Knockout Techniques ; Humans ; Liver/metabolism/pathology ; Pancreas/pathology ; Wound Healing ; Zebrafish/genetics ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Dishevelled-associated antagonist of beta-catenin 2 (Dact2) is involved in the regulation of intracellular signaling pathways during development. It negatively regulates the Nodal signaling pathway, possibly by promoting lysosomal degradation of Nodal receptors such as TGFBR1, and plays an inhibitory role during the re-epithelialization of skin wounds by attenuating transforming growth factor-β signaling. Dact2 is known to act as a functional tumor suppressor in colon cancer; reduced Dact2 can promote liver cancer progression and suppress gastric cancer proliferation, invasion, and metastasis by inhibiting Wnt signaling. Zebrafish is used as a model of cancer biology because it shows similar tumorigenesis and morphogenesis as in humans and gene manipulation in this organism is possible. This study was performed to explore phenotypic changes in Dact2 knockout zebrafish and investigate the function of Dact2. A 10-base pair deletion Dact2 knockout zebrafish was prepared using the CRISPR-Cas9 genome editing system. Dact2 knockout enhanced the expression of the MMP2 and MMP9 genes, which are related to tumor invasion and migration, and the Snail, VEGF, and ZEB genes, which are related to epithelial-mesenchymal transition (EMT). The absence of Dact2 also resulted in hyperplasia of the gastrointestinal epithelium, fibrosis in the pancreas and liver, increased proliferation of the pancreatic and hepatic bile ducts, and invasive proliferation into the pancreas. A wound healing assay confirmed that the absence of Dact2 enhanced EMT, thus accelerating wound healing. This study suggests that a loss of function of Dact2 impacts EMT-related gene regulation and tumor generation in a zebrafish knockout model, which is a useful model for exploring the mechanisms of these processes.},
}
@article {pmid31982142,
year = {2020},
author = {Adashi, EY and Cohen, IG},
title = {Therapeutic Germline Editing: Sense and Sensibility.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {5},
pages = {315-317},
doi = {10.1016/j.tig.2019.12.011},
pmid = {31982142},
issn = {0168-9525},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Genome, Human/*genetics ; Germ-Line Mutation/*genetics ; Humans ; },
abstract = {Safe and effective heritable editing of the human genome is years away from the clinic because of formidable technical, statutory, regulatory, and societal challenges. In particular, we note the fledgling state of the science, the imperatives of editing efficiency, specificity, and uniformity, and the extant legal roadblock.},
}
@article {pmid31981984,
year = {2020},
author = {Do, HS and Park, SW and Im, I and Seo, D and Yoo, HW and Go, H and Kim, YH and Koh, GY and Lee, BH and Han, YM},
title = {Enhanced thrombospondin-1 causes dysfunction of vascular endothelial cells derived from Fabry disease-induced pluripotent stem cells.},
journal = {EBioMedicine},
volume = {52},
number = {},
pages = {102633},
pmid = {31981984},
issn = {2352-3964},
mesh = {Adult ; Alleles ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Endothelial Cells/*metabolism ; Endothelium, Vascular ; Enzyme Activation ; Fabry Disease/diagnosis/*genetics/*metabolism ; Gene Editing ; Gene Expression ; Gene Expression Profiling ; Gene Knockout Techniques ; Humans ; Immunohistochemistry ; Immunophenotyping ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Mice ; Mice, Knockout ; Middle Aged ; Models, Biological ; Mutation ; Oxidative Stress ; Phenotype ; Thrombospondin 1/*genetics/metabolism ; alpha-Galactosidase/genetics/metabolism ; },
abstract = {BACKGROUND: Fabry disease (FD) is a recessive X-linked lysosomal storage disorder caused by α-galactosidase A (GLA) deficiency. Although the mechanism is unclear, GLA deficiency causes an accumulation of globotriaosylceramide (Gb3), leading to vasculopathy.<br><br>METHODS: To explore the relationship between the accumulation of Gb3 and vasculopathy, induced pluripotent stem cells generated from four Fabry patients (FD-iPSCs) were differentiated into vascular endothelial cells (VECs). Genome editing using CRISPR-Cas9 system was carried out to correct the GLA mutation or to delete Thrombospondin-1 (TSP-1). Global transcriptomes were compared between wild-type (WT)- and FD-VECs by RNA-sequencing analysis.<br><br>FINDINGS: Here, we report that overexpression of TSP-1 contributes to the dysfunction of VECs in FD. VECs originating from FD-iPSCs (FD-VECs) showed aberrant angiogenic functionality even upon treatment with recombinant &#x3b1;-galactosidase. Intriguingly, FD-VECs produced more p-SMAD2 and TSP-1 than WT-VECs. We also found elevated TSP-1 in the peritubular capillaries of renal tissues biopsied from FD patients. Inhibition of SMAD2 signaling or knock out of TSP-1 (TSP-1[-/-]) rescues normal vascular functionality in FD-VECs, like in gene-corrected FD-VECs. In addition, the enhanced oxygen consumption rate is reduced in TSP-1[-/-] FD-VECs.<br><br>INTERPRETATION: The overexpression of TSP-1 secondary to Gb3 accumulation is primarily responsible for the observed FD-VEC dysfunction. Our findings implicate dysfunctional VEC angiogenesis in the peritubular capillaries in some of the complications of Fabry disease.<br><br>FUNDING: This study was supported by grant 2018M3A9H1078330 from the National Research Foundation of the Republic of Korea.},
}
@article {pmid31981492,
year = {2020},
author = {Wu, J and Bell, OH and Copland, DA and Young, A and Pooley, JR and Maswood, R and Evans, RS and Khaw, PT and Ali, RR and Dick, AD and Chu, CJ},
title = {Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {3},
pages = {820-829},
pmid = {31981492},
issn = {1525-0024},
mesh = {Animals ; Aquaporin 1/*genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems ; Ciliary Body/*metabolism ; Dependovirus/genetics ; *Gene Editing ; Gene Expression ; Gene Targeting ; *Genetic Therapy/methods ; Genetic Vectors/genetics ; Glaucoma/diagnosis/*genetics/physiopathology/*therapy ; Mice ; Retina/metabolism/pathology ; Transduction, Genetic ; Transgenes ; },
abstract = {Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm[2]) compared with untreated eyes (25 ± 5/mm[2]; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.},
}
@article {pmid31981443,
year = {2020},
author = {Wang, L and Sun, S and Wu, T and Liu, L and Sun, X and Cai, Y and Li, J and Jia, H and Yuan, S and Chen, L and Jiang, B and Wu, C and Hou, W and Han, T},
title = {Natural variation and CRISPR/Cas9-mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean.},
journal = {Plant biotechnology journal},
volume = {18},
number = {9},
pages = {1869-1881},
pmid = {31981443},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; China ; Flowers/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Mutation/genetics ; *Photoperiod ; Plant Proteins/genetics/metabolism ; *Soybeans/genetics/metabolism ; },
abstract = {Flowering time is a critical determinant of the geographic distribution and regional adaptability of soybean (Glycine max) and is strongly regulated by photoperiod and temperature. In this study, quantitative trait locus (QTL) mapping and subsequent candidate gene analysis revealed that GmPRR37, encoding a pseudo-response regulator protein, is responsible for the major QTL qFT12-2, which was identified from a population of 308 recombinant inbred lines (RILs) derived from a cross between a very late-flowering soybean cultivar, 'Zigongdongdou (ZGDD)', and an extremely early-flowering cultivar, 'Heihe27 (HH27)', in multiple environments. Comparative analysis of parental sequencing data confirmed that HH27 contains a non-sense mutation that causes the loss of the CCT domain in the GmPRR37 protein. CRISPR/Cas9-induced Gmprr37-ZGDD mutants in soybean exhibited early flowering under natural long-day (NLD) conditions. Overexpression of GmPRR37 significantly delayed the flowering of transgenic soybean plants compared with wild-type under long photoperiod conditions. In addition, both the knockout and overexpression of GmPRR37 in soybean showed no significant phenotypic alterations in flowering time under short-day (SD) conditions. Furthermore, GmPRR37 down-regulated the expression of the flowering-promoting FT homologues GmFT2a and GmFT5a, and up-regulated flowering-inhibiting FT homologue GmFT1a expression under long-day (LD) conditions. We analysed haplotypes of GmPRR37 among 180 cultivars collected across China and found natural Gmprr37 mutants flower earlier and enable soybean to be cultivated at higher latitudes. This study demonstrates that GmPRR37 controls soybean photoperiodic flowering and provides opportunities to breed optimized cultivars with adaptation to specific regions and farming systems.},
}
@article {pmid31981430,
year = {2020},
author = {Chen, L and Cai, Y and Qu, M and Wang, L and Sun, H and Jiang, B and Wu, T and Liu, L and Sun, S and Wu, C and Yao, W and Yuan, S and Han, T and Hou, W},
title = {Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b, an ortholog of FLOWERING LOCUS T.},
journal = {Plant, cell &amp; environment},
volume = {43},
number = {4},
pages = {934-944},
pmid = {31981430},
issn = {1365-3040},
mesh = {Adaptation, Physiological/genetics ; Arabidopsis Proteins/genetics/physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cloning, Molecular ; Flowers/growth &amp; development ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Genetic Variation/genetics/physiology ; Geography ; Photoperiod ; Plant Proteins/*genetics/physiology ; Soybeans/genetics/growth &amp; development/*physiology ; Transcription Factors/*genetics/physiology ; Transcriptome ; },
abstract = {Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a 'weight' model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.},
}
@article {pmid31980824,
year = {2020},
author = {Szentes, S and Zsibrita, N and Koncz, M and Zsigmond, E and Salamon, P and Pletl, Z and Kiss, A},
title = {I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.},
journal = {Nucleic acids research},
volume = {48},
number = {5},
pages = {e28},
pmid = {31980824},
issn = {1362-4962},
mesh = {Base Sequence ; Binding Sites ; Binding, Competitive ; *Biological Assay ; CRISPR-Cas Systems ; DNA, Bacterial/*genetics/metabolism ; DNA-Directed RNA Polymerases/*genetics/metabolism ; Deoxyribonucleases, Type II Site-Specific/genetics/metabolism ; Escherichia coli/*genetics/metabolism ; Escherichia coli Proteins/genetics ; Lac Repressors/deficiency/genetics ; Plasmids/chemistry/metabolism ; Promoter Regions, Genetic ; Protein Binding ; *Transcription, Genetic ; beta-Galactosidase/genetics/metabolism ; },
abstract = {We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.},
}
@article {pmid31980818,
year = {2020},
author = {Nimkar, S and Anand, B},
title = {Cas3/I-C mediated target DNA recognition and cleavage during CRISPR interference are independent of the composition and architecture of Cascade surveillance complex.},
journal = {Nucleic acids research},
volume = {48},
number = {5},
pages = {2486-2501},
pmid = {31980818},
issn = {1362-4962},
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Associated Proteins/chemistry/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Conserved Sequence ; DNA/*metabolism ; Evolution, Molecular ; Models, Biological ; Protein Domains ; },
abstract = {In type I CRISPR-Cas system, Cas3-a nuclease cum helicase-in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organization of Cascade subunits in type I-C influence the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues-which are unique to type I-C-located in the uncharacterized C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the non-target strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.},
}
@article {pmid31980629,
year = {2020},
author = {Carpenter, MD and Hu, Q and Bond, AM and Lombroso, SI and Czarnecki, KS and Lim, CJ and Song, H and Wimmer, ME and Pierce, RC and Heller, EA},
title = {Nr4a1 suppresses cocaine-induced behavior via epigenetic regulation of homeostatic target genes.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {504},
pmid = {31980629},
issn = {2041-1723},
support = {P01 NS097206/NS/NINDS NIH HHS/United States ; T32 GM008076/GM/NIGMS NIH HHS/United States ; DP1 DA044250/DA/NIDA NIH HHS/United States ; R21 DA039038/DA/NIDA NIH HHS/United States ; R01 DA033641/DA/NIDA NIH HHS/United States ; R01 DA015214/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal/*drug effects ; CRISPR-Cas Systems/genetics ; Cocaine/administration &amp; dosage/*pharmacology ; *Epigenesis, Genetic/drug effects ; Female ; Histones/metabolism ; Homeostasis/*drug effects/genetics ; Male ; Mice, Inbred C57BL ; Neurons/drug effects/metabolism ; Nuclear Receptor Subfamily 4, Group A, Member 1/*metabolism ; Phenylacetates/pharmacology ; Promoter Regions, Genetic/genetics ; Protein Processing, Post-Translational ; Synapsins/metabolism ; },
abstract = {Endogenous homeostatic mechanisms can restore normal neuronal function following cocaine-induced neuroadaptations. Such mechanisms may be exploited to develop novel therapies for cocaine addiction, but a molecular target has not yet been identified. Here we profiled mouse gene expression during early and late cocaine abstinence to identify putative regulators of neural homeostasis. Cocaine activated the transcription factor, Nr4a1, and its target gene, Cartpt, a key molecule involved in dopamine metabolism. Sustained activation of Cartpt at late abstinence was coupled with depletion of the repressive histone modification, H3K27me3, and enrichment of activating marks, H3K27ac and H3K4me3. Using both CRISPR-mediated and small molecule Nr4a1 activation, we demonstrated the direct causal role of Nr4a1 in sustained activation of Cartpt and in attenuation of cocaine-evoked behavior. Our findings provide evidence that targeting abstinence-induced homeostatic gene expression is a potential therapeutic target in cocaine addiction.},
}
@article {pmid31980609,
year = {2020},
author = {Li, K and Liu, Y and Cao, H and Zhang, Y and Gu, Z and Liu, X and Yu, A and Kaphle, P and Dickerson, KE and Ni, M and Xu, J},
title = {Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {485},
pmid = {31980609},
issn = {2041-1723},
support = {K12 HD068369/HD/NICHD NIH HHS/United States ; R01 CA230631/CA/NCI NIH HHS/United States ; R01 DK111430/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; Female ; Gene Editing/*methods ; HEK293 Cells ; Hematopoiesis/genetics ; Humans ; Jurkat Cells ; K562 Cells ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Mice, Transgenic ; Neoplasms/genetics ; RNA, Guide/genetics ; T-Cell Acute Lymphocytic Leukemia Protein 1/genetics ; },
abstract = {Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.},
}
@article {pmid31980606,
year = {2020},
author = {Nishiguchi, KM and Fujita, K and Miya, F and Katayama, S and Nakazawa, T},
title = {Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {482},
pmid = {31980606},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Cyclic Nucleotide Phosphodiesterases, Type 6/deficiency/genetics ; DNA End-Joining Repair ; Dependovirus/*genetics ; Eye Proteins/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Photoreceptor Cells, Vertebrate/*physiology ; Retinal Dystrophies/*genetics/physiopathology/*therapy ; Targeted Gene Repair/methods ; Transducin/deficiency/genetics ; Visual Acuity/genetics/physiology ; },
abstract = {Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.},
}
@article {pmid31980167,
year = {2020},
author = {Jeon, EH and Park, TS and Jang, Y and Hwang, E and Kim, SJ and Song, KD and Weinstein, DA and Lee, YM and Park, BC and Jun, HS},
title = {Glucose-6-phosphate transporter mediates macrophage proliferation and functions by regulating glycolysis and mitochondrial respiration.},
journal = {Biochemical and biophysical research communications},
volume = {524},
number = {1},
pages = {89-95},
doi = {10.1016/j.bbrc.2020.01.043},
pmid = {31980167},
issn = {1090-2104},
mesh = {Animals ; Antiporters/*genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation ; Glucose/metabolism ; Glycogen Storage Disease Type I/*metabolism ; Glycolysis ; Humans ; Macrophages/cytology/*metabolism ; Mice ; Mitochondria/metabolism ; Models, Animal ; Monosaccharide Transport Proteins/*genetics/*metabolism ; Mutation ; Neutrophils/metabolism ; Oxidation-Reduction ; Phenotype ; Phosphorylation ; Swine ; },
abstract = {Glycogen storage disease type Ib (GSD-Ib), caused by a deficiency in glucose-6-phosphate transporter (G6PT), is characterized by disrupted glucose homeostasis, inflammatory bowel disease, neutropenia, and neutrophil dysfunction. The purpose of this study was to investigate the role of G6PT on macrophage functions and metabolism. Peritoneal macrophages of G6pt[-/-] mice were lower in number and their effector functions including migration, superoxide production, and phagocytosis were impaired. To investigate the underlying mechanisms of macrophage dysfunction, the G6PT gene was mutated in porcine alveolar macrophage 3D4/31 cells using the CRISPR/Cas9 technology. The G6PT-deficient macrophages exhibited significant decline in cell growth, bactericidal activity, and antiviral response. These phenotypes are associated with the impaired glycolysis and mitochondrial oxidative phosphorylation. We therefore propose that the G6PT-mediated metabolism is essential for effector functions of macrophage, the immune deficiencies observed in GSD-Ib extend beyond neutropenia and neutrophil dysfunction, and future therapeutic targets aimed both the neutrophils and macrophages may be necessary.},
}
@article {pmid31980032,
year = {2020},
author = {Yang, L and Zhu, Y and Yu, H and Cheng, X and Chen, S and Chu, Y and Huang, H and Zhang, J and Li, W},
title = {scMAGeCK links genotypes with multiple phenotypes in single-cell CRISPR screens.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {19},
pmid = {31980032},
issn = {1474-760X},
support = {R01 HG010753/HG/NHGRI NIH HHS/United States ; },
mesh = {Algorithms ; Animals ; *CRISPR-Cas Systems ; Cell Proliferation ; Cluster Analysis ; Embryonic Stem Cells/metabolism ; Enhancer Elements, Genetic ; Genes, Tumor Suppressor ; *Genotype ; Mice ; Oncogenes ; *Phenotype ; *RNA-Seq ; Single-Cell Analysis/*methods ; },
abstract = {We present scMAGeCK, a computational framework to identify genomic elements associated with multiple expression-based phenotypes in CRISPR/Cas9 functional screening that uses single-cell RNA-seq as readout. scMAGeCK outperforms existing methods, identifies genes and enhancers with known and novel functions in cell proliferation, and enables an unbiased construction of genotype-phenotype network. Single-cell CRISPR screening on mouse embryonic stem cells identifies key genes associated with different pluripotency states. Applying scMAGeCK on multiple datasets, we identify key factors that improve the power of single-cell CRISPR screening. Collectively, scMAGeCK is a novel tool to study genotype-phenotype relationships at a single-cell level.},
}
@article {pmid31980014,
year = {2020},
author = {Sanderson, H and Ortega-Polo, R and Zaheer, R and Goji, N and Amoako, KK and Brown, RS and Majury, A and Liss, SN and McAllister, TA},
title = {Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.},
journal = {BMC microbiology},
volume = {20},
number = {1},
pages = {20},
pmid = {31980014},
issn = {1471-2180},
support = {001//Genomics Research and Development Initiative/International ; },
mesh = {Bacterial Proteins/*genetics ; *Drug Resistance, Multiple, Bacterial ; Enterococcus faecium/*genetics ; Genome Size ; Genomics/*methods ; Interspersed Repetitive Sequences ; Multilocus Sequence Typing ; Phylogeny ; Vancomycin Resistance ; Virulence Factors/genetics ; Waste Water/*microbiology ; Whole Genome Sequencing ; },
abstract = {BACKGROUND: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted.<br><br>RESULTS: VRE isolates, including E. faecalis (n&#x2009;=&#x2009;24), E. faecium (n&#x2009;=&#x2009;11), E. casseliflavus (n&#x2009;=&#x2009;2) and E. gallinarum (n&#x2009;=&#x2009;2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p&#x2009;<&#x2009;0.001) and E. faecalis (p&#x2009;<&#x2009;0.001) and with the number of AMR genes in E. faecium (p&#x2009;=&#x2009;0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers.<br><br>CONCLUSIONS: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.},
}
@article {pmid31979133,
year = {2020},
author = {Nguyen, Q and Lim, KRQ and Yokota, T},
title = {Genome Editing for the Understanding and Treatment of Inherited Cardiomyopathies.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {31979133},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiomyopathies/*genetics/*therapy ; Gene Editing/methods ; Genome/*genetics ; Humans ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {Cardiomyopathies are diseases of heart muscle, a significant percentage of which are genetic in origin. Cardiomyopathies can be classified as dilated, hypertrophic, restrictive, arrhythmogenic right ventricular or left ventricular non-compaction, although mixed morphologies are possible. A subset of neuromuscular disorders, notably Duchenne and Becker muscular dystrophies, are also characterized by cardiomyopathy aside from skeletal myopathy. The global burden of cardiomyopathies is certainly high, necessitating further research and novel therapies. Genome editing tools, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as increasingly important technologies in studying this group of cardiovascular disorders. In this review, we discuss the applications of genome editing in the understanding and treatment of cardiomyopathy. We also describe recent advances in genome editing that may help improve these applications, and some future prospects for genome editing in cardiomyopathy treatment.},
}
@article {pmid31978714,
year = {2020},
author = {Xu, C and Zhou, Z and Liu, C and Kang, X and Zhong, X and Zhang, Q and Xu, Y},
title = {Generation of a DAPK1 knockout first (conditional ready) human embryonic stem cell line (ZSSYe001-A) by CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {43},
number = {},
pages = {101693},
doi = {10.1016/j.scr.2019.101693},
pmid = {31978714},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Death-Associated Protein Kinases/*genetics ; Human Embryonic Stem Cells/*metabolism ; Humans ; Male ; },
abstract = {Death-associated protein kinase 1 (DAPK1) is a Ca[2+]/calmodulin regulated Ser/Thr kinase involved in various cellular processes including cell death, autophagy and inflammation. Its dysregulation has been linked to tumour metastasis, anti-viral responses, Alzheimer's disease and other neurological disorders. To further investigate the role of DAPK1 in these processes, we generated a DAPK1 knockout first (conditional ready) human embryonic stem (hES) cell line in which the endogenous DAPK1 can be easily restored with expression of FLPe. This cell line provides an ideal model to study the role of DAPK1 in human development and various pathologies related to DAPK1 dysregulation in vitro.},
}
@article {pmid31978056,
year = {2020},
author = {Emmer, BT and Lascuna, PJ and Tang, VT and Kotnik, EN and Saunders, TL and Khoriaty, R and Ginsburg, D},
title = {Murine Surf4 is essential for early embryonic development.},
journal = {PloS one},
volume = {15},
number = {1},
pages = {e0227450},
pmid = {31978056},
issn = {1932-6203},
support = {T32 HL007853/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; K08 HL148552/HL/NHLBI NIH HHS/United States ; K08 HL128794/HL/NHLBI NIH HHS/United States ; R01 HL148333/HL/NHLBI NIH HHS/United States ; KL2 TR002241/TR/NCATS NIH HHS/United States ; R35 HL135793/HL/NHLBI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Apolipoproteins B/blood/metabolism ; CRISPR-Cas Systems/genetics ; Cholesterol/blood ; *Embryonic Development ; Gene Editing ; Heterozygote ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Knockout ; Proprotein Convertase 9/blood/metabolism ; },
abstract = {Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4. Heterozygous Surf4+/- mice exhibit grossly normal appearance, behavior, body weight, fecundity, and organ development, with no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol, and a detectable accumulation of intrahepatic apoliprotein B. Homozygous Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. In contrast to the milder murine phenotypes associated with deficiency of known SURF4 cargoes, the embryonic lethality of Surf4-/- mice implies the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.},
}
@article {pmid31977149,
year = {2020},
author = {Bauer, R and Neffgen, N and Grempels, A and Furitsch, M and Mauerer, S and Barbaqadze, S and Haase, G and Kestler, H and Spellerberg, B},
title = {Heterogeneity of Streptococcus anginosus ß-hemolysis in relation to CRISPR/Cas.},
journal = {Molecular oral microbiology},
volume = {35},
number = {2},
pages = {56-65},
doi = {10.1111/omi.12278},
pmid = {31977149},
issn = {2041-1014},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Hemolysis ; Humans ; Phylogeny ; Streptococcus/genetics ; *Streptococcus anginosus/genetics ; },
abstract = {Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß-hemolytic due to the presence of an SLS-like gene cluster. However, the majority of strains do not display ß-hemolysis. To investigate ß-hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß-hemolytic and non-ß-hemolytic strains. All of the ß-hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß-hemolysis do not harbor sag genes. Phylogenetic analysis of the ß-hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß-hemolytic versus non-ß-hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non-ß-hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß-hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self-targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß-hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus.},
}
@article {pmid31975506,
year = {2020},
author = {Yang, B and McJunkin, K},
title = {CRISPR screening strategies for microRNA target identification.},
journal = {The FEBS journal},
volume = {287},
number = {14},
pages = {2914-2922},
doi = {10.1111/febs.15218},
pmid = {31975506},
issn = {1742-4658},
support = {ZIA DK075147/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Computational Biology/*methods ; *Gene Expression Regulation ; Humans ; MicroRNAs/*genetics ; Phenotype ; RNA, Messenger/genetics/*metabolism ; },
abstract = {Identifying microRNA (miRNA) target genes remains a major challenge in understanding the roles miRNAs play in gene regulation. Furthermore, understanding which miRNA-target interactions are the most biologically important is even more difficult. We present CRISPR-based strategies to identify essential miRNA binding sites. First, CRISPR knockout screens can easily be adapted to identify genes whose inactivation suppresses miRNA mutant phenotypes. Second, a custom approach to target individual miRNA binding sites via CRISPR can identify sites whose mutation recapitulates miRNA mutant phenotypes. We emphasize that the latter approach requires a readout of mutational profile (rather than single guide RNA abundance) when applied in a negative selection setting. Overall, the advent of CRISPR technology alongside improving empirical means of miRNA target identification will accelerate our dissection of miRNA gene regulatory networks.},
}
@article {pmid31974923,
year = {2020},
author = {Golchin, A and Shams, F and Karami, F},
title = {Advancing Mesenchymal Stem Cell Therapy with CRISPR/Cas9 for Clinical Trial Studies.},
journal = {Advances in experimental medicine and biology},
volume = {1247},
number = {},
pages = {89-100},
doi = {10.1007/5584_2019_459},
pmid = {31974923},
issn = {0065-2598},
mesh = {*CRISPR-Cas Systems ; *Clinical Trials as Topic ; Gene Editing ; Humans ; Mesenchymal Stem Cell Transplantation/*methods ; Mesenchymal Stem Cells/*metabolism ; Regenerative Medicine ; },
abstract = {Currently, regenerative medicine and cellular-based therapy have been in the center of attention worldwide in advanced medical technology. Mesenchymal stem cell (MSC) as a suitable stem cell source for cell-based therapy has been shown to be safe and effective in multiple clinical trial studies (CTSs) of several diseases. Despite the advantages, MSC needs more investigation to enhance its therapeutic application. The CRISPR/Cas system is a novel technique for editing of genes that is being explored as a means to improve MSCs therapeutic usage. In this study, we review the recent studies that explore CRISPR potency in gene engineering of MSCs, which have great relevance in MSC-based therapies. However, CRISPR/Cas technology make possible specific targeting of loci in target genes, but next-generation MSC-based therapies to achieve extensive clinical application need dedicated efforts.},
}
@article {pmid31974649,
year = {2020},
author = {Larroude, M and Trabelsi, H and Nicaud, JM and Rossignol, T},
title = {A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity.},
journal = {Biotechnology letters},
volume = {42},
number = {5},
pages = {773-785},
pmid = {31974649},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems ; Fungal Proteins/*genetics ; Gene Editing/*methods ; Homologous Recombination ; Mutation ; RNA, Guide/genetics ; Yarrowia/genetics/*growth &amp; development ; },
abstract = {OBJECTIVES: The construction and validation of a set of Yarrowia lipolytica CRISPR/Cas9 vectors containing six different markers that allows virtually any genetic background to be edited, including those of wild-type strains.<br><br>RESULTS: Using the Golden Gate method, we assembled a set of six CRISPR/Cas9 vectors, each containing a different selection marker, to be used for editing the genome of the industrial yeast Y. lipolytica. This vector set is available via Addgene. Any guide RNA (gRNA) sequence can be easily and rapidly introduced in any of these vectors using Golden Gate assembly. We successfully edited six different genes in a variety of genetic backgrounds, including those of wild-type strains, with five of the six vectors. Use of these vectors strongly improved homologous recombination and cassette integration at a specific locus.<br><br>CONCLUSIONS: We have created a versatile and modular set of CRISPR/Cas9 vectors that will allow any Y. lipolytica strain to be rapidly edited; this tool will facilitate experimentation with any prototroph wild-type strains displaying interesting features.},
}
@article {pmid31974435,
year = {2020},
author = {Quijada-Álamo, M and Hernández-Sánchez, M and Alonso-Pérez, V and Rodríguez-Vicente, AE and García-Tuñón, I and Martín-Izquierdo, M and Hernández-Sánchez, JM and Herrero, AB and Bastida, JM and San Segundo, L and Gruber, M and García, JL and Yin, S and Ten Hacken, E and Benito, R and Ordóñez, JL and Wu, CJ and Hernández-Rivas, JM},
title = {CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition.},
journal = {Leukemia},
volume = {34},
number = {6},
pages = {1599-1612},
pmid = {31974435},
issn = {1476-5551},
support = {P01 CA206978/CA/NCI NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; R01 CA216273/CA/NCI NIH HHS/United States ; },
mesh = {Adenine/analogs &amp; derivatives ; Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology ; Ataxia Telangiectasia Mutated Proteins/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosome Deletion ; Chromosomes, Human, Pair 11/genetics ; Drug Synergism ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/*genetics ; Mice ; Mutagenesis, Site-Directed/*methods ; Mutation ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Piperidines ; Poly (ADP-Ribose) Polymerase-1/antagonists &amp; inhibitors ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Proto-Oncogene Proteins c-bcr/antagonists &amp; inhibitors ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Xenograft Model Antitumor Assays ; },
abstract = {The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.},
}
@article {pmid31974430,
year = {2020},
author = {Sakurai, T and Kamiyoshi, A and Kawate, H and Watanabe, S and Sato, M and Shindo, T},
title = {Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1091},
pmid = {31974430},
issn = {2045-2322},
support = {16K15233//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; 19K07477//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; },
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Gene Targeting ; Male ; Mice/*genetics ; Mice, Inbred ICR ; Mice, Transgenic ; *Mosaicism ; },
abstract = {The CRISPR/Cas9 system is widely used to generate gene-edited animals. Here, we developed an efficient system for generating genetically modified mice using maternal Cas9 from Cas9 transgenic mice. Using this system, we achieved lower mosaicism and higher rates of knock-in success, gene-editing, and birth compared to the similar parameters obtained using exogenously administered Cas9 (mRNA/protein) system. Furthermore, we successfully induced simultaneous mutations at multiple loci (a maximum of nine). Our novel gene-editing system based on maternal Cas9 could potentially facilitate the generation of mice with single and multiple gene modifications.},
}
@article {pmid31974191,
year = {2021},
author = {Chen, J and An, B and Yu, B and Peng, X and Yuan, H and Yang, Q and Chen, X and Yu, T and Wang, L and Zhang, X and Wang, H and Zou, X and Pang, D and Ouyang, H and Tang, X},
title = {CRISPR/Cas9-mediated knockin of human factor IX into swine factor IX locus effectively alleviates bleeding in hemophilia B pigs.},
journal = {Haematologica},
volume = {106},
number = {3},
pages = {829-837},
pmid = {31974191},
issn = {1592-8721},
mesh = {Animals ; CRISPR-Cas Systems ; Factor IX/genetics ; *Hemophilia A/genetics ; *Hemophilia B/genetics/therapy ; Hemorrhage/genetics ; Humans ; Swine ; },
abstract = {Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities in the coagulation factor IX gene. Without prophylactic treatment, patients experience frequent spontaneous bleeding episodes. Well-characterized animal models are valuable for determining the pathobiology of the disease and testing novel therapeutic innovations. Here, we generated a porcine model of hemophilia B using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. Moreover, we tested the possibility of hemophilia B therapy by gene insertion. Frequent spontaneous joint bleeding episodes that occurred in hemophilia B pigs allowed a thorough investigation of the pathological process of hemophilic arthropathy. In contrast to the hemophilia B pigs, which showed a severe bleeding tendency and joint damage, the transgenic pigs carrying human coagulation factor IX exhibited a partial improvement of bleeding. In summary, this study not only offers a translational hemophilia B model for exploring the pathological process of hemophilic arthropathy but also provides a possibility for the permanent correction of hemophilia in the future by genome editing in situ.},
}
@article {pmid31973584,
year = {2020},
author = {Zhen, S and Liu, Y and Lu, J and Tuo, X and Yang, X and Chen, H and Chen, W and Li, X},
title = {Human Papillomavirus Oncogene Manipulation Using Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 Delivered by pH-Sensitive Cationic Liposomes.},
journal = {Human gene therapy},
volume = {31},
number = {5-6},
pages = {309-324},
doi = {10.1089/hum.2019.312},
pmid = {31973584},
issn = {1557-7422},
mesh = {Alphapapillomavirus ; Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Female ; Gene Editing ; Genetic Therapy ; Humans ; Hydrogen-Ion Concentration ; Liposomes/*therapeutic use ; Mice ; Mice, Nude ; Nanoparticles/therapeutic use ; Oncogene Proteins, Viral/*genetics ; Papillomavirus E7 Proteins/*genetics ; Papillomavirus Infections/therapy ; Repressor Proteins/*genetics ; Uterine Cervical Neoplasms/*therapy/virology ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology enables targeted gene editing, but cancer gene therapy with this approach requires improvements to enable safe and efficient delivery of CRISPR/Cas9 to tumors. We developed and evaluated a self-assembled liposome to selectively deliver CRISPR/Cas9 to cancer tissues. Our CRISPR/Cas9 system effectively inhibited proliferation of human papillomavirus (HPV) 16-positive cervical cancer cells and induced apoptosis by inactivating the HR-HPV16E6/E7 oncogene. Based on this system, we prepared a long-circulating pH-sensitive cationic nano-liposome complex with a high cell targeting and gene knockout rate. Intratumoral injection of cationic liposomes targeted to splicing HPV16 E6/E7 in nude mice significantly inhibited tumor growth without significant toxicity in vivo. Liposomes that targeted HPV16 E6/E7 splicing were established as a basis for treatment of HPV16-positive cervical cancer drug candidates. Our study demonstrates that this liposome offers an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine-based cancer therapeutics.},
}
@article {pmid31973565,
year = {2020},
author = {Zhou, X},
title = {Empowering chimeric antigen receptor T-cell therapy with CRISPR.},
journal = {BioTechniques},
volume = {68},
number = {4},
pages = {169-171},
doi = {10.2144/btn-2019-0107},
pmid = {31973565},
issn = {1940-9818},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Immunotherapy, Adoptive ; *Receptors, Chimeric Antigen ; },
}
@article {pmid31972154,
year = {2020},
author = {Pandelakis, M and Delgado, E and Ebrahimkhani, MR},
title = {CRISPR-Based Synthetic Transcription Factors In Vivo: The Future of Therapeutic Cellular Programming.},
journal = {Cell systems},
volume = {10},
number = {1},
pages = {1-14},
pmid = {31972154},
issn = {2405-4720},
support = {R01 EB024562/EB/NIBIB NIH HHS/United States ; R01 EB028532/EB/NIBIB NIH HHS/United States ; R01 HL141805/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Genetic Engineering/*methods ; Humans ; Transcription Factors/*metabolism ; },
abstract = {Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR/Cas9-based transcriptional regulators and epigenome editors have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. For this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Better understanding of the design principle controlling gene expression together with technologies that enable spatiotemporal control of transcriptional engineering are fundamental for rational design, improved efficacy, and ultimately safe translation to humans. In this review, we will discuss recent advances and integrative strategies that can help pave the path toward a new class of transcriptional therapeutics.},
}
@article {pmid31971562,
year = {2020},
author = {Hiranniramol, K and Chen, Y and Liu, W and Wang, X},
title = {Generalizable sgRNA design for improved CRISPR/Cas9 editing efficiency.},
journal = {Bioinformatics (Oxford, England)},
volume = {36},
number = {9},
pages = {2684-2689},
pmid = {31971562},
issn = {1367-4811},
support = {R01 DE026471/DE/NIDCR NIH HHS/United States ; R01 GM089784/GM/NIGMS NIH HHS/United States ; R41 GM126682/GM/NIGMS NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *RNA, Guide/genetics ; },
abstract = {MOTIVATION: The development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions.<br><br>RESULTS: We employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools.<br><br>The new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid31970591,
year = {2019},
author = {Knott, MML and Hölting, TLB and Ohmura, S and Kirchner, T and Cidre-Aranaz, F and Grünewald, TGP},
title = {Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies.},
journal = {Cancer metastasis reviews},
volume = {38},
number = {4},
pages = {625-642},
pmid = {31970591},
issn = {1573-7233},
mesh = {Animals ; Bone Neoplasms/drug therapy/genetics/metabolism/*therapy ; CRISPR-Cas Systems ; Child ; Genetic Therapy/*methods ; Humans ; Oncogene Proteins, Fusion/*genetics/*metabolism ; Pediatrics/methods ; Rhabdomyosarcoma, Alveolar/drug therapy/metabolism/therapy ; Sarcoma/drug therapy/genetics/metabolism/*therapy ; Sarcoma, Ewing/genetics/metabolism/*therapy ; },
abstract = {While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the "undruggable" into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments. Graphical abstract Scheme depicting the different targeting strategies of fusion oncogenes in pediatric fusion-driven sarcomas. Fusion oncogenes can be targeted on their DNA level (1), RNA level (2), protein level (3), and by targeting downstream functions and interaction partners (4).},
}
@article {pmid31970368,
year = {2020},
author = {Cui, Y and Tang, Y and Liang, M and Ji, Q and Zeng, Y and Chen, H and Lan, J and Jin, P and Wang, L and Song, G and Lou, J},
title = {Direct observation of the formation of a CRISPR-Cas12a R-loop complex at the single-molecule level.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {14},
pages = {2123-2126},
doi = {10.1039/c9cc08325a},
pmid = {31970368},
issn = {1364-548X},
mesh = {CRISPR-Cas Systems/genetics ; DNA/*chemical synthesis/chemistry/genetics ; Optical Tweezers ; Thermodynamics ; },
abstract = {Here, we develop an optical tweezers-based single-molecule manipulation assay to detect the formation of an R-loop complex in the Cas12a system and characterize its thermodynamic stability. We found that the formation of the R-loop complex induces a two-step unfolding of a DNA hairpin containing the target sequence, the non-target sequence binds loosely to Cas12a and can be easily released from the complex, and the Nuc domain of Cas12a plays key roles in target binding and R-loop formation.},
}
@article {pmid31969710,
year = {2020},
author = {Rollie, C and Chevallereau, A and Watson, BNJ and Chyou, TY and Fradet, O and McLeod, I and Fineran, PC and Brown, CM and Gandon, S and Westra, ER},
title = {Targeting of temperate phages drives loss of type I CRISPR-Cas systems.},
journal = {Nature},
volume = {578},
number = {7793},
pages = {149-153},
pmid = {31969710},
issn = {1476-4687},
support = {714478/ERC_/European Research Council/International ; },
mesh = {Bacteria/*genetics/immunology/virology ; Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation, Viral ; Lysogeny/genetics ; Prophages/genetics ; },
abstract = {On infection of their host, temperate viruses that infect bacteria (bacteriophages; hereafter referred to as phages) enter either a lytic or a lysogenic cycle. The former results in lysis of bacterial cells and phage release (resulting in horizontal transmission), whereas lysogeny is characterized by the integration of the phage into the host genome, and dormancy (resulting in vertical transmission)[1]. Previous co-culture experiments using bacteria and mutants of temperate phages that are locked in the lytic cycle have shown that CRISPR-Cas systems can efficiently eliminate the invading phages[2,3]. Here we show that, when challenged with wild-type temperate phages (which can become lysogenic), type I CRISPR-Cas immune systems cannot eliminate the phages from the bacterial population. Furthermore, our data suggest that, in this context, CRISPR-Cas immune systems are maladaptive to the host, owing to the severe immunopathological effects that are brought about by imperfect matching of spacers to the integrated phage sequences (prophages). These fitness costs drive the loss of CRISPR-Cas from bacterial populations, unless the phage carries anti-CRISPR (acr) genes that suppress the immune system of the host. Using bioinformatics, we show that this imperfect targeting is likely to occur frequently in nature. These findings help to explain the patchy distribution of CRISPR-Cas immune systems within and between bacterial species, and highlight the strong selective benefits of phage-encoded acr genes for both the phage and the host under these circumstances.},
}
@article {pmid31969475,
year = {2020},
author = {Glover, L},
title = {mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics.},
journal = {mSphere},
volume = {5},
number = {1},
pages = {},
pmid = {31969475},
issn = {2379-5042},
mesh = {Antigenic Variation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Repair ; Gene Editing ; Proteomics/*methods ; Recombination, Genetic ; Trypanosoma brucei brucei/genetics ; },
abstract = {Lucy Glover's research focuses on the role of DNA repair and recombination in antigenic variation in the parasite Trypanosoma brucei, the causative agent of both human and animal African trypanosomiasis. In this mSphere of Influence article, she reflects on how "A CRISPR-based approach for proteomic analysis of a single genomic locus" by Z. J. Waldrip, S. D. Byrum, A. J. Storey, J. Gao, et al. (Epigenetics 9:1207-1211, 2014, https://doi.org/10.4161/epi.29919) made an impact on her research by taking the precision of CRISPR-Cas9 and repurposing it to look at single-locus proteomics. By using this technology in trypanosomes, Dr. Glover and her colleagues could study the dynamic accumulation of repair proteins after specific damage and gain insight into how the location of a double-strand break (DSB) dictates repair pathway choice and how this may influence immune evasion in these parasites.},
}
@article {pmid31965382,
year = {2020},
author = {Banakar, R and Schubert, M and Collingwood, M and Vakulskas, C and Eggenberger, AL and Wang, K},
title = {Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene.},
journal = {Rice (New York, N.Y.)},
volume = {13},
number = {1},
pages = {4},
pmid = {31965382},
issn = {1939-8425},
abstract = {BACKGROUND: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site.<br><br>FINDINGS: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1-2&#x2009;bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2&#x2009;bp to 20&#x2009;bp without the loss of the PAM site.<br><br>CONCLUSIONS: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.},
}
@article {pmid31964810,
year = {2020},
author = {Koslová, A and Trefil, P and Mucksová, J and Reinišová, M and Plachý, J and Kalina, J and Kučerová, D and Geryk, J and Krchlíková, V and Lejčková, B and Hejnar, J},
title = {Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {4},
pages = {2108-2112},
pmid = {31964810},
issn = {1091-6490},
mesh = {Animals ; Animals, Genetically Modified/genetics/immunology/virology ; Avian Leukosis/genetics/*immunology/virology ; Avian Leukosis Virus/classification/*genetics/physiology ; Avian Proteins/*genetics/immunology ; CRISPR-Cas Systems ; Chickens ; Disease Resistance ; Female ; Gene Editing ; Male ; Poultry Diseases/genetics/*immunology/virology ; Sodium-Hydrogen Exchanger 1/*genetics/immunology ; },
abstract = {Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na[+]/H[+] exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.},
}
@article {pmid31964350,
year = {2020},
author = {Bai, H and Liu, L and An, K and Lu, X and Harrison, M and Zhao, Y and Yan, R and Lu, Z and Li, S and Lin, S and Liang, F and Qin, W},
title = {CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {67},
pmid = {31964350},
issn = {1471-2164},
mesh = {Amino Acid Substitution ; Animals ; *CRISPR-Cas Systems ; *DNA, Single-Stranded ; *Gene Editing ; Gene Knock-In Techniques ; Gene Targeting ; Genetic Predisposition to Disease ; Humans ; Loss of Function Mutation ; RNA, Guide ; Sequence Analysis, DNA ; Zebrafish/*genetics ; },
abstract = {BACKGROUND: Gene targeting by homology-directed repair (HDR) can precisely edit the genome and is a versatile tool for biomedical research. However, the efficiency of HDR-based modification is still low in many model organisms including zebrafish. Recently, long single-stranded DNA (lssDNA) molecules have been developed as efficient alternative donor templates to mediate HDR for the generation of conditional mouse alleles. Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long single-stranded DNA template to produce more efficient and precise mutations in zebrafish.<br><br>RESULTS: The efficiency of knock-ins was assessed by phenotypic rescue at the tyrosinase (tyr) locus and confirmed by sequencing. zLOST was found to be a successful optimised rescue strategy: using zLOST containing a tyr repair site, we restored pigmentation in at least one melanocyte in close to 98% of albino tyr[25del/25del] embryos, although more than half of the larvae had only a small number of pigmented cells. Sequence analysis showed that there was precise HDR dependent repair of the tyr locus in these rescued pigmented embryos. Furthermore, quantification of zLOST knock-in efficiency at the rps14, nop56 and th loci by next generation sequencing demonstrated that zLOST showed a clear improvement. We utilised the HDR efficiency of zLOST to precisely model specific human disease mutations in zebrafish with ease. Finally, we determined that this method can achieve a germline transmission rate of up to 31.8%.<br><br>CONCLUSIONS: In summary, these results show that zLOST is a useful method of zebrafish genome editing, particularly for generating desired mutations by targeted DNA knock-in through HDR.},
}
@article {pmid31963842,
year = {2020},
author = {Wen, L and Zhao, C and Song, J and Ma, L and Ruan, J and Xia, X and Chen, YE and Zhang, J and Ma, PX and Xu, J},
title = {CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells.},
journal = {International journal of molecular sciences},
volume = {21},
number = {2},
pages = {},
pmid = {31963842},
issn = {1422-0067},
support = {AG055863/NH/NIH HHS/United States ; TR001711/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Female ; Gene Knockout Techniques/*methods ; Haploinsufficiency ; HeLa Cells ; Humans ; INDEL Mutation ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Telomerase/*genetics/*metabolism ; Uterine Cervical Neoplasms/genetics/metabolism/*pathology ; },
abstract = {Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.},
}
@article {pmid31963583,
year = {2020},
author = {Akutsu, SN and Fujita, K and Tomioka, K and Miyamoto, T and Matsuura, S},
title = {Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders.},
journal = {Cells},
volume = {9},
number = {1},
pages = {},
pmid = {31963583},
issn = {2073-4409},
mesh = {Aneuploidy ; CRISPR-Cas Systems ; Cellular Reprogramming Techniques/*methods ; Chromosome Disorders/*genetics/metabolism/*therapy ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Down Syndrome/*genetics/therapy ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Mosaicism ; Sex Chromosomes/*genetics/pathology ; Trisomy/*genetics ; },
abstract = {Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.},
}
@article {pmid31963209,
year = {2020},
author = {Adamson, CS and Nevels, MM},
title = {Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function.},
journal = {Viruses},
volume = {12},
number = {1},
pages = {},
pmid = {31963209},
issn = {1999-4915},
support = {MR/P022146/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Antiviral Agents/therapeutic use ; CRISPR-Cas Systems ; Cytomegalovirus/drug effects/*genetics ; Cytomegalovirus Infections/therapy ; *Gene Expression Regulation, Viral ; Genes, Immediate-Early/*genetics ; Humans ; Immediate-Early Proteins/drug effects/genetics/*metabolism ; RNA Interference ; RNA, Catalytic/drug effects/genetics ; Viral Proteins/drug effects/genetics/metabolism ; Virus Replication/drug effects ; },
abstract = {The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. "Bright and early" events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.},
}
@article {pmid31961902,
year = {2020},
author = {Mata López, S and Balog-Alvarez, C and Vitha, S and Bettis, AK and Canessa, EH and Kornegay, JN and Nghiem, PP},
title = {Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy.},
journal = {PloS one},
volume = {15},
number = {1},
pages = {e0228072},
pmid = {31961902},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dogs ; Dystrophin/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Mutation ; Myoblasts/cytology/*metabolism ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.},
}
@article {pmid31960911,
year = {2020},
author = {Gravesteijn, G and Dauwerse, JG and Overzier, M and Brouwer, G and Hegeman, I and Mulder, AA and Baas, F and Kruit, MC and Terwindt, GM and van Duinen, SG and Jost, CR and Aartsma-Rus, A and Lesnik Oberstein, SAJ and Rutten, JW},
title = {Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients.},
journal = {Human molecular genetics},
volume = {29},
number = {11},
pages = {1853-1863},
pmid = {31960911},
issn = {1460-2083},
mesh = {Adult ; Aged ; Biopsy ; CADASIL/diagnostic imaging/*genetics/metabolism/physiopathology ; CRISPR-Cas Systems/genetics ; Cysteine/*genetics ; Exons/genetics ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Protein Aggregation, Pathological/diagnostic imaging/*genetics/metabolism/pathology ; Receptor, Notch3/*genetics ; Severity of Illness Index ; Skin/chemistry/diagnostic imaging ; White Matter/diagnostic imaging/*metabolism/pathology ; },
abstract = {CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.},
}
@article {pmid31960794,
year = {2020},
author = {Li, M and Yang, T and Kandul, NP and Bui, M and Gamez, S and Raban, R and Bennett, J and Sánchez C, HM and Lanzaro, GC and Schmidt, H and Lee, Y and Marshall, JM and Akbari, OS},
title = {Development of a confinable gene drive system in the human disease vector Aedes aegypti.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {31960794},
issn = {2050-084X},
support = {1U01CK000516/CC/CDC HHS/United States ; },
mesh = {Aedes/*genetics/physiology ; Animals ; Animals, Genetically Modified/genetics/physiology ; CRISPR-Cas Systems/genetics ; Female ; *Gene Drive Technology ; Male ; Mosquito Vectors/*genetics/physiology ; RNA, Guide/genetics ; },
abstract = {Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.},
}
@article {pmid31960370,
year = {2020},
author = {Meadows, SK and Brandsmeier, LA and Newberry, KM and Betti, MJ and Nesmith, AS and Mackiewicz, M and Partridge, EC and Mendenhall, EM and Myers, RM},
title = {Epitope Tagging ChIP-Seq of DNA Binding Proteins Using CETCh-Seq.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2117},
number = {},
pages = {3-34},
doi = {10.1007/978-1-0716-0301-7_1},
pmid = {31960370},
issn = {1940-6029},
support = {U54 HG006998/HG/NHGRI NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems ; Cell Adhesion ; Chromatin Immunoprecipitation Sequencing ; Epitopes/*metabolism ; Gene Editing ; Hep G2 Cells ; Humans ; Protein Binding ; Transcription Factors/*analysis/*genetics ; },
abstract = {Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) has been used to identify transcription factor (TF) binding proteins throughout the genome. Unfortunately, this approach traditionally requires commercially available, ChIP-seq grade antibodies that frequently fail to generate acceptable datasets. To obtain data for the many TFs for which there is no appropriate antibody, we recently developed a new method for performing ChIP-seq by epitope tagging endogenous TFs using CRISPR/Cas9 genome editing technology (CETCh-seq). Here, we describe our general protocol of CETCh-seq for both adherent and nonadherent cell lines using a commercially available FLAG antibody.},
}
@article {pmid31960185,
year = {2020},
author = {Soonsanga, S and Luxananil, P and Promdonkoy, B},
title = {Modulation of Cas9 level for efficient CRISPR-Cas9-mediated chromosomal and plasmid gene deletion in Bacillus thuringiensis.},
journal = {Biotechnology letters},
volume = {42},
number = {4},
pages = {625-632},
doi = {10.1007/s10529-020-02809-0},
pmid = {31960185},
issn = {1573-6776},
mesh = {Bacillus thuringiensis/*genetics ; Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; *Gene Deletion ; Gene Editing/methods ; Plasmids/genetics ; Promoter Regions, Genetic ; Streptococcus pyogenes/metabolism ; },
abstract = {OBJECTIVES: To set up an efficient gene editing system in Bacillus thuringiensis (Bt) using CRISPR-Cas9 by demonstrating deletion of chromosomal and plasmid genes.<br><br>RESULTS: CRISPR-Cas9 from Streptococcus pyogenes was found to function in Bt cells, resulting in DNA cleavage that is lethal to the cells. The system was assessed for its ability to mediate gene editing by knock-out of the protease genes nprA (neutral protease A) and aprA (alkaline protease A). Gene editing was not detected when the Bacillus-derived pBCX was used to carry CRISPR-Cas9 elements and a DNA repair template. When the Cas9 promoter was replaced with the sporulation-specific promoter cyt2A, a Bt &#x2206;nprA clone was obtained, but this plasmid construct did not give reproducible results. Bt &#x2206;nprA &#x2206;aprA and Bt &#x2206;aprA deletion mutants were finally generated when the Lactobacillus plantarum-derived plasmid pLPPR9 was used, likely due to its lower copy number reducing Cas9 toxicity. Only three to four clones each needed to be screened to identify the desired gene-modified mutants. Conversely, efficient editing of the plasmid vip3A gene required the use of pBCX and longer homology sequences for the repair template.<br><br>CONCLUSIONS: Capitalizing on the differential impact of plasmid copy number and homology arm length, we devised distinct yet simple and efficient approaches to chromosomal and plasmid gene deletion for Bt that condense the screening process, minimize screening, and facilitate multiple consecutive gene editing steps.},
}
@article {pmid31960055,
year = {2020},
author = {Cardenas-Diaz, FL and Leavens, KF and Kishore, S and Osorio-Quintero, C and Chen, YJ and Stanger, BZ and Wang, P and French, D and Gadue, P},
title = {A Dual Reporter EndoC-βH1 Human β-Cell Line for Efficient Quantification of Calcium Flux and Insulin Secretion.},
journal = {Endocrinology},
volume = {161},
number = {2},
pages = {},
pmid = {31960055},
issn = {1945-7170},
support = {K12 DK094723/DK/NIDDK NIH HHS/United States ; UC4 DK104196/DK/NIDDK NIH HHS/United States ; R01 DK118155/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Calcium Signaling ; *Cell Line ; Down-Regulation ; Gene Knockout Techniques ; *Genes, Reporter ; Homeodomain Proteins/genetics ; Humans ; *Insulin Secretion ; *Insulin-Secreting Cells ; Trans-Activators/genetics ; },
abstract = {Human in vitro model systems of diabetes are critical to both study disease pathophysiology and offer a platform for drug testing. We have generated a set of tools in the human β-cell line EndoC-βH1 that allows the efficient and inexpensive characterization of β-cell physiology and phenotypes driven by disruption of candidate genes. First, we generated a dual reporter line that expresses a preproinsulin-luciferase fusion protein along with GCaMP6s. This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. Using these tools, we demonstrate that the generation of the reporter human β-cell line was highly efficient and validated that luciferase activity could accurately reflect insulin secretion. Second, we used a lentiviral vector carrying the CRISPR-Cas9 system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPR-Cas9 lentiviral system. As a proof of principle, we disrupt the β-cell master regulator, PDX1, and show that mutant EndoC-βH1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-βH1 cells exhibit decreased expression of the β-cell-specific genes MAFA and NKX6.1 and increased GCG expression. The system presented here provides a platform to quickly and easily test β-cell functionality in wildtype and cells lacking a gene of interest.},
}
@article {pmid31959982,
year = {2020},
author = {Crowther, MD and Dolton, G and Legut, M and Caillaud, ME and Lloyd, A and Attaf, M and Galloway, SAE and Rius, C and Farrell, CP and Szomolay, B and Ager, A and Parker, AL and Fuller, A and Donia, M and McCluskey, J and Rossjohn, J and Svane, IM and Phillips, JD and Sewell, AK},
title = {Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.},
journal = {Nature immunology},
volume = {21},
number = {2},
pages = {178-185},
pmid = {31959982},
issn = {1529-2916},
support = {//Wellcome Trust/United Kingdom ; 100327//Wellcome Trust/United Kingdom ; MR/L008742/1/MRC_/Medical Research Council/United Kingdom ; U54 DK110858/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cytotoxicity, Immunologic/*immunology ; Genome-Wide Association Study ; Histocompatibility Antigens Class I/*immunology ; Humans ; Immunotherapy/methods ; Lymphocyte Activation/immunology ; Mice ; Minor Histocompatibility Antigens/*immunology ; Neoplasms/*immunology ; Receptors, Antigen, T-Cell/*immunology ; T-Lymphocyte Subsets/*immunology ; },
abstract = {Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.},
}
@article {pmid31959981,
year = {2020},
author = {Mori, L and De Libero, G},
title = {'Bohemian Rhapsody' of MR1T cells.},
journal = {Nature immunology},
volume = {21},
number = {2},
pages = {108-110},
pmid = {31959981},
issn = {1529-2916},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Histocompatibility Antigens Class I ; *T-Lymphocytes ; },
}
@article {pmid31959876,
year = {2020},
author = {Yun, Y and Hong, SA and Kim, KK and Baek, D and Lee, D and Londhe, AM and Lee, M and Yu, J and McEachin, ZT and Bassell, GJ and Bowser, R and Hales, CM and Cho, SR and Kim, J and Pae, AN and Cheong, E and Kim, S and Boulis, NM and Bae, S and Ha, Y},
title = {CRISPR-mediated gene correction links the ATP7A M1311V mutations with amyotrophic lateral sclerosis pathogenesis in one individual.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {33},
pmid = {31959876},
issn = {2399-3642},
mesh = {*Amino Acid Substitution ; Amyotrophic Lateral Sclerosis/diagnosis/*etiology/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Copper-Transporting ATPases/*genetics/metabolism ; DNA Mutational Analysis ; *Gene Editing ; Genetic Association Studies ; Genetic Predisposition to Disease ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Male ; *Mutation ; Neurons/metabolism ; RNA, Guide ; Whole Genome Sequencing ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a severe disease causing motor neuron death, but a complete cure has not been developed and related genes have not been defined in more than 80% of cases. Here we compared whole genome sequencing results from a male ALS patient and his healthy parents to identify relevant variants, and chose one variant in the X-linked ATP7A gene, M1311V, as a strong disease-linked candidate after profound examination. Although this variant is not rare in the Ashkenazi Jewish population according to results in the genome aggregation database (gnomAD), CRISPR-mediated gene correction of this mutation in patient-derived and re-differentiated motor neurons drastically rescued neuronal activities and functions. These results suggest that the ATP7A M1311V mutation has a potential responsibility for ALS in this patient and might be a potential therapeutic target, revealed here by a personalized medicine strategy.},
}
@article {pmid31959800,
year = {2020},
author = {Hazelbaker, DZ and Beccard, A and Angelini, G and Mazzucato, P and Messana, A and Lam, D and Eggan, K and Barrett, LE},
title = {A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {635},
pmid = {31959800},
issn = {2045-2322},
mesh = {Basic Helix-Loop-Helix Transcription Factors ; *CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Transposable Elements ; Drosophila Proteins ; *Gene Transfer Techniques ; *Genetic Vectors ; Humans ; *Pluripotent Stem Cells ; *RNA, Guide ; Transgenes ; },
abstract = {CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. Collectively, our results provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.},
}
@article {pmid31959747,
year = {2020},
author = {Huang, T and Liu, Z and Zheng, Y and Feng, T and Gao, Q and Zeng, W},
title = {YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m[6]A/mRNA pathway.},
journal = {Cell death &amp; disease},
volume = {11},
number = {1},
pages = {37},
pmid = {31959747},
issn = {2041-4889},
mesh = {Adenosine/*analogs &amp; derivatives/metabolism ; Animals ; Apoptosis ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Adhesion/genetics ; Cell Cycle ; Cell Line ; Cell Movement ; Cell Proliferation ; Extracellular Matrix/metabolism ; Gene Deletion ; Gene Expression Regulation ; Male ; Matrix Metalloproteinases/*metabolism ; Mice, Knockout ; Phenotype ; RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Spermatogonia/*cytology/metabolism ; },
abstract = {As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m[6]A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m[6]A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m[6]A/mRNA degradation pathway.},
}
@article {pmid31959210,
year = {2020},
author = {Watanabe, S and Oiwa, K and Murata, Y and Komine, O and Sobue, A and Endo, F and Takahashi, E and Yamanaka, K},
title = {ALS-linked TDP-43[M337V] knock-in mice exhibit splicing deregulation without neurodegeneration.},
journal = {Molecular brain},
volume = {13},
number = {1},
pages = {8},
pmid = {31959210},
issn = {1756-6606},
mesh = {Alternative Splicing/genetics/*physiology ; Amyotrophic Lateral Sclerosis/genetics ; Animals ; Base Sequence ; Brain/metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/*genetics/physiology ; Exons/genetics ; Gene Knock-In Techniques ; Humans ; Mice ; *Mutation, Missense ; *Point Mutation ; RNA, Messenger/metabolism ; Spinal Cord/metabolism ; },
abstract = {Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43), a DNA/RNA binding protein, is a pathological signature of amyotrophic lateral sclerosis (ALS). Missense mutations in the TARDBP gene are also found in inherited and sporadic ALS, indicating that dysfunction in TDP-43 is causative for ALS. To model TDP-43-linked ALS in rodents, we generated TDP-43 knock-in mice with inherited ALS patient-derived TDP-43[M337V] mutation. Homozygous TDP-43[M337V] mice developed normally without exhibiting detectable motor dysfunction and neurodegeneration. However, splicing of mRNAs regulated by TDP-43 was deregulated in the spinal cords of TDP-43[M337V] mice. Together with the recently reported TDP-43 knock-in mice with ALS-linked mutations, our finding indicates that ALS patient-derived mutations in the TARDBP gene at a carboxyl-terminal domain of TDP-43 may cause a gain of splicing function by TDP-43, however, were insufficient to induce robust neurodegeneration in mice.},
}
@article {pmid31959180,
year = {2020},
author = {Jo, A and Ringel-Scaia, VM and McDaniel, DK and Thomas, CA and Zhang, R and Riffle, JS and Allen, IC and Davis, RM},
title = {Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR-Cas9 plasmid.},
journal = {Journal of nanobiotechnology},
volume = {18},
number = {1},
pages = {16},
pmid = {31959180},
issn = {1477-3155},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; DNA/chemistry ; Fluorescent Dyes/chemistry ; Gene Transfer Techniques ; Macrophages/metabolism ; Mice ; Nanoparticles/*chemistry ; Organosilicon Compounds/chemistry ; Plasmids ; Polylactic Acid-Polyglycolic Acid Copolymer/*chemistry ; Transfection ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells.<br><br>RESULTS: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160&#xa0;nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6&#xa0;wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24&#xa0;h and corresponded to ~&#x2009;2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24&#xa0;h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells.<br><br>CONCLUSIONS: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.},
}
@article {pmid31958883,
year = {2020},
author = {Liu, K and Sun, B and You, H and Tu, JL and Yu, X and Zhao, P and Xu, JW},
title = {Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system.},
journal = {Microbial biotechnology},
volume = {13},
number = {2},
pages = {386-396},
pmid = {31958883},
issn = {1751-7915},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Deletion ; Gene Editing ; *Reishi/genetics ; },
abstract = {Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin-resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9-mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5'-monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde-3-phosphate dehydrogenase gene intron in the Cas9 plasmid is 14-18 in 10[7] protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA-directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.},
}
@article {pmid31958362,
year = {2020},
author = {Navarro-Hernandez, IC and López-Ortega, O and Acevedo-Ochoa, E and Cervantes-Díaz, R and Romero-Ramírez, S and Sosa-Hernández, VA and Meza-Sánchez, DE and Juárez-Vega, G and Pérez-Martínez, CA and Chávez-Munguía, B and Galván-Hernández, A and Antillón, A and Ortega-Blake, I and Santos-Argumedo, L and Hernández-Hernández, JM and Maravillas-Montero, JL},
title = {Tetraspanin 33 (TSPAN33) regulates endocytosis and migration of human B lymphocytes by affecting the tension of the plasma membrane.},
journal = {The FEBS journal},
volume = {287},
number = {16},
pages = {3449-3471},
doi = {10.1111/febs.15216},
pmid = {31958362},
issn = {1742-4658},
mesh = {B-Lymphocytes/*metabolism/ultrastructure ; CRISPR-Cas Systems ; Cell Adhesion/genetics ; Cell Line, Tumor ; Cell Membrane/*metabolism ; Cell Movement/*genetics ; Endocytosis/*genetics ; Gene Knockdown Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Microscopy, Confocal ; Microscopy, Electron ; Phagocytosis/genetics ; Stress, Mechanical ; Tetraspanins/*genetics/metabolism ; },
abstract = {B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody-secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid-bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin-enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin-induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane.},
}
@article {pmid31956898,
year = {2020},
author = {Tian, B and Minero, GAS and Fock, J and Dufva, M and Hansen, MF},
title = {CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification.},
journal = {Nucleic acids research},
volume = {48},
number = {5},
pages = {e30},
pmid = {31956898},
issn = {1362-4962},
mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/*genetics/metabolism ; Endonucleases/*genetics/metabolism ; Gene Editing/methods ; Nucleic Acid Amplification Techniques/*standards ; RNA, Guide/genetics/metabolism ; Reference Standards ; },
abstract = {False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.},
}
@article {pmid31956094,
year = {2020},
author = {Liu, PF and Wu, Q},
title = {Probing 3D genome by CRISPR/Cas9.},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {1},
pages = {18-31},
doi = {10.16288/j.yczz.19-246},
pmid = {31956094},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome ; *Genomics ; },
abstract = {CRISPR/Cas9 system has significant advantages in gene editing strategy, offering cost-effective and efficient means to modify and edit the genomes of animals, plants, and microorganisms. Three-dimensional (3D) genome is an emerging and interdisciplinary field catapulted by combined technological breakthroughs of chromosome conformation capture with next-generation sequencing and live imaging with super-resolution microscopy. An important aspect of 3D genomics is to model structural variations and label specific genomic fragments to investigate the effects of manipulation of genomic elements on gene expression regulation, cell development and differentiation, and spatial location of chromosomal regions. Therefore, CRISPR/Cas9 system and its derivative technologies of DNA-fragment editing are excellent toolboxes for investigating dynamics and functions of the higher-order chromatin organization and three-dimensional genome structure. In this review, we describe the opportunities and challenges of CRISPR as well as its derivative technologies in 3D genome research, thereby providing some critical references and future research directions in the field.},
}
@article {pmid31955242,
year = {2020},
author = {Li, M and Liu, X and Dai, S and Xiao, H and Qi, S and Li, Y and Zheng, Q and Jie, M and Cheng, CHK and Wang, D},
title = {Regulation of spermatogenesis and reproductive capacity by Igf3 in tilapia.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {23},
pages = {4921-4938},
doi = {10.1007/s00018-019-03439-0},
pmid = {31955242},
issn = {1420-9071},
mesh = {Androgens/pharmacology ; Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Proliferation/drug effects ; Gene Expression Regulation, Developmental/drug effects ; Male ; Mice, Knockout ; Mutation/genetics ; RNA, Messenger/genetics/metabolism ; *Reproduction/drug effects/genetics ; Signal Transduction/drug effects/genetics ; Somatomedins/genetics/*metabolism ; *Spermatogenesis/drug effects/genetics ; Spermatogonia/cytology/drug effects/metabolism ; Testis/drug effects/metabolism ; Tilapia/genetics/*physiology ; },
abstract = {A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3[-/-] testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3[-/-] males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.},
}
@article {pmid31954772,
year = {2020},
author = {Beisel, CL},
title = {Methods for characterizing, applying, and teaching CRISPR-Cas systems.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {1-2},
doi = {10.1016/j.ymeth.2020.01.004},
pmid = {31954772},
issn = {1095-9130},
mesh = {Bacteria/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Molecular Biology/*education/methods ; Teaching ; },
}
@article {pmid31953707,
year = {2020},
author = {Xu, Y and Meng, X and Wang, J and Qin, B and Wang, K and Li, J and Wang, C and Yu, H},
title = {ScCas9 recognizes NNG protospacer adjacent motif in genome editing of rice.},
journal = {Science China. Life sciences},
volume = {63},
number = {3},
pages = {450-452},
doi = {10.1007/s11427-019-1630-2},
pmid = {31953707},
issn = {1869-1889},
mesh = {Amino Acid Sequence ; Bacteria ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing ; Gene Expression Regulation ; Humans ; Mutation ; Oryza/*genetics ; },
}
@article {pmid31953404,
year = {2020},
author = {López Del Amo, V and Bishop, AL and Sánchez C, HM and Bennett, JB and Feng, X and Marshall, JM and Bier, E and Gantz, VM},
title = {A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {352},
pmid = {31953404},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Base Sequence ; CRISPR-Cas Systems ; Diptera ; Ecosystem ; Female ; Gene Drive Technology/*methods ; Gene Editing ; Genes, X-Linked ; Genetics, Population/*methods ; Male ; Models, Theoretical ; RNA, Guide/genetics ; Transgenes ; },
abstract = {CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.},
}
@article {pmid31953347,
year = {2020},
author = {Zhang, L and He, A and Chen, B and Bi, J and Chen, J and Guo, D and Qian, Y and Wang, W and Shi, T and Zhao, Z and Shi, J and An, W and Attenello, F and Lu, W},
title = {A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.},
journal = {Genome research},
volume = {30},
number = {2},
pages = {155-163},
pmid = {31953347},
issn = {1549-5469},
mesh = {Antineoplastic Agents, Alkylating/pharmacology ; Base Sequence ; CRISPR-Cas Systems/genetics ; Calcium-Binding Proteins/*genetics ; Carrier Proteins/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Regulatory Networks/genetics ; Glioma/*drug therapy/genetics/pathology ; Humans ; Neoplasm Proteins/genetics ; RNA, Long Noncoding/*genetics ; Temozolomide/*pharmacology ; Transcription Factors/*genetics ; },
abstract = {Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.},
}
@article {pmid31953191,
year = {2020},
author = {Chen, W and Dong, Y and Saqib, HSA and Vasseur, L and Zhou, W and Zheng, L and Lai, Y and Ma, X and Lin, L and Xu, X and Bai, J and He, W and You, M},
title = {Functions of duplicated glucosinolate sulfatases in the development and host adaptation of Plutella xylostella.},
journal = {Insect biochemistry and molecular biology},
volume = {119},
number = {},
pages = {103316},
doi = {10.1016/j.ibmb.2020.103316},
pmid = {31953191},
issn = {1879-0240},
mesh = {*Adaptation, Biological ; Animals ; CRISPR-Cas Systems ; Female ; Gene Duplication ; *Genes, Insect ; Glucosinolates/metabolism ; Insect Proteins/*genetics/metabolism ; Larva/enzymology/genetics/growth &amp; development ; Male ; Moths/enzymology/*genetics/growth &amp; development ; Mutation ; Sulfatases/*genetics/metabolism ; },
abstract = {Evolutionary adaptations of herbivorous insects are often dictated by the necessity to withstand a corresponding evolutionary innovation in host plant defense. Glucosinolate sulfatase (GSS) enzyme activity is considered a central adaptation strategy in Plutella xylostella against glucosinolates (GS)-myrosinase defense system in the Brassicales. The high functional versatility of sulfatases suggests that they may perform other vital roles in the process of growth and development. Here, we used a CRISPR/Cas9 system to generate stable homozygous single/double mutant lines of gss1 or/and gss2 with no predicted off-target effects, to analyze the functions of the pair of duplicated genes in the development and host adaptation of P. xylostella. The bioassays showed that, when fed on their usual artificial diet, significant reduction in egg hatching rate and final larval survival rate of the single mutant line of gss2 compared with the original strain or mutant lines of gss1, revealing unexpected functions of GSS2 in embryonic and larval development. When larvae of homozygous mutant lines were transferred onto a new food, Arabidopsis thaliana, no induced effect at protein level of GSS1/2 or gene expression level of gss1/gss2 was detected. The absence of GSS1 or GSS2 reduced the survival rate of larvae and prolonged the duration of the larval stage, indicating that both GSS1 and GSS2 played an important role in adaptation to host plants. The versatile functions of duplicated GSSs in this study provide a foundation for further research to understand potential functions of other sulfatase members and support evidence of adaptation in herbivorous insects.},
}
@article {pmid31953182,
year = {2020},
author = {Rojas-Sánchez, U and López-Calleja, AC and Millán-Chiu, BE and Fernández, F and Loske, AM and Gómez-Lim, MA},
title = {Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.},
journal = {Protein expression and purification},
volume = {168},
number = {},
pages = {105570},
doi = {10.1016/j.pep.2020.105570},
pmid = {31953182},
issn = {1096-0279},
mesh = {Aspergillus niger/*genetics/metabolism ; CRISPR-Cas Systems ; Cloning, Molecular ; Erythropoietin/*biosynthesis/genetics ; Fructose-Bisphosphatase/chemistry/genetics ; Gene Expression ; Gene Knockdown Techniques ; *Genes, Fungal ; Genetic Vectors/chemistry/metabolism ; Glycosylation ; Humans ; *Introns ; Plasmids/chemistry/*metabolism ; Promoter Regions, Genetic ; Protein Stability ; Proteolysis ; RNA, Messenger/*genetics/metabolism ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L[-1] by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.},
}
@article {pmid31952017,
year = {2020},
author = {Locatelli, A},
title = {Towards a novel therapy against AIDS.},
journal = {Medical hypotheses},
volume = {137},
number = {},
pages = {109569},
doi = {10.1016/j.mehy.2020.109569},
pmid = {31952017},
issn = {1532-2777},
mesh = {*Acquired Immunodeficiency Syndrome/therapy ; Animals ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Mice ; RNA, Guide ; },
abstract = {AIDS is an infectious disease that kills over a million people per year. Very recently, Dash et al have for the first time reached the functional cure in HIV-infected humanized mice using CRISPR-Cas9 in combination with LASER ART, and this with a success of one third. Here, I use a theoretical approach to design a therapeutic strategy applicable to humans and different from that of Dash et al. The experimental treatment presented here includes the injection of an Env-directed integrase-defective CRISPR gene-editing lentiviral vector able to express quintuplex gRNAs plus the humanized SpCas9 and the puromycin resistance gene linked by T2A, preceded by a plasma/leukapheresis and the injection of an immunosuppressive cocktail, and followed by an in vivo positive selection. My protocol could have a major impact on HIV-infected people in the event of confirmation by a clinical trial, and it is possible that it becomes a reference treatment against AIDS, although, for the moment, it is only at the stage of hypothesis and theory.},
}
@article {pmid31951830,
year = {2020},
author = {Jang, HK and Bae, S},
title = {i-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {2},
pages = {348-349},
pmid = {31951830},
issn = {1525-0024},
mesh = {*Adenine ; CRISPR-Cas Systems ; Codon, Initiator ; *Gene Editing ; Mutation ; },
}
@article {pmid31951545,
year = {2020},
author = {Davis, BJ and O'Connell, MR},
title = {Put on Your Para-spectacles: The Development of Optimized CRISPR-Cas13-Based Approaches to Image RNA Dynamics in Real Time.},
journal = {Molecular cell},
volume = {77},
number = {2},
pages = {207-209},
doi = {10.1016/j.molcel.2019.12.018},
pmid = {31951545},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Eyeglasses ; *RNA ; },
abstract = {Live-cell RNA imaging is a powerful approach to observe the real-time dynamics of RNA metabolism. Two recent papers describe an optimized fluorescence-based CRISPR-Cas13 approach to image colocalized or repeat-containing RNAs in real time, as well as demonstrate simultaneous RNA-DNA labeling by using Cas13 and Cas9 in tandem.},
}
@article {pmid31950837,
year = {2020},
author = {Mohammadinejad, R and Sassan, H and Pardakhty, A and Hashemabadi, M and Ashrafizadeh, M and Dehshahri, A and Mandegary, A},
title = {ZEB1 and ZEB2 gene editing mediated by CRISPR/Cas9 in A549 cell line.},
journal = {Bratislavske lekarske listy},
volume = {121},
number = {1},
pages = {31-36},
doi = {10.4149/BLL_2020_005},
pmid = {31950837},
issn = {0006-9248},
mesh = {A549 Cells ; *CRISPR-Cas Systems ; *Carcinoma, Non-Small-Cell Lung/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; Humans ; *Lung Neoplasms/genetics ; *Zinc Finger E-box Binding Homeobox 2/genetics ; *Zinc Finger E-box-Binding Homeobox 1/genetics ; },
abstract = {OBJECTIVES: One of the best approaches for recognition of protein function is the induction of mutations for a gene knockout. In line with this strategy, gene editing tools allow researchers to induce these mutations. Lung cancer is one of the leading causes of death worldwide. ZEB1 and ZEB2 genes are the candidates for this disease.<br><br>METHODS: The ZEB1 and ZEB2 knockout in the non-small cell lung cancer cell line (A549 cell) was investigated. Purification of recombination plasmids was performed from bacteria and then was transported to the A549 cell line. The deletion of ZEB1 and ZEB2 were examined by PCR.<br><br>RESULTS: The results demonstrated the mutation and deletion in ZEB1 and ZEB2 genes. Based on the findings of this study, A549 cells were transfected with the vectors carrying the sgRNA/Cas9, simultaneously. The DNA fragment demonstrated the presence of indels in target sites as well as provided the potential of CRISPR/Cas9 system.<br><br>CONCLUSION: CRISPR/Cas9 offers a great potential as an efficient technique for editing of ZEB1 and ZEB2 genes in A549 cell line (Tab. 1, Fig. 6, Ref. 44).},
}
@article {pmid31950445,
year = {2020},
author = {Dong, J and Kan, B and Liu, H and Zhan, M and Wang, S and Xu, G and Han, R and Ni, Y},
title = {CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced L-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase.},
journal = {Applied biochemistry and biotechnology},
volume = {191},
number = {3},
pages = {955-967},
doi = {10.1007/s12010-020-03231-y},
pmid = {31950445},
issn = {1559-0291},
mesh = {Arginine/metabolism ; Bioreactors ; *CRISPR-Cas Systems ; Citrulline/metabolism ; Corynebacterium glutamicum/enzymology/*genetics ; Escherichia coli/genetics/metabolism ; Fermentation ; Genome, Bacterial ; Glucose/metabolism ; Glutamate Synthase (NADH)/*metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenases/*metabolism ; Glycolysis ; Industrial Microbiology ; Metabolic Engineering ; NADP/metabolism ; Ornithine/*biosynthesis ; Plasmids/genetics ; Recombinant Proteins/metabolism ; Transcription, Genetic ; },
abstract = {Here, Corynebacterium glutamicum SNK118 was metabolically engineered for L-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of L-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26 g/L with productivity of 1.23 g/L/h (over 72 h) and yield of 0.414 g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of L-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote L-ornithine production by C. glutamicum strains.},
}
@article {pmid31950325,
year = {2020},
author = {Moreira, D and Pereira, AM and Lopes, AL and Coimbra, S},
title = {The best CRISPR/Cas9 versus RNA interference approaches for Arabinogalactan proteins' study.},
journal = {Molecular biology reports},
volume = {47},
number = {3},
pages = {2315-2325},
pmid = {31950325},
issn = {1573-4978},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation ; Gene Silencing ; Gene Targeting ; Humans ; Mucoproteins/*genetics/metabolism ; Plant Proteins/genetics/metabolism ; *RNA Interference ; RNA, Small Interfering/genetics ; Research ; },
abstract = {Arabinogalactan Proteins (AGPs) are hydroxyproline-rich proteins containing a high proportion of carbohydrates, widely spread in the plant kingdom. AGPs have been suggested to play important roles in plant development processes, especially in sexual plant reproduction. Nevertheless, the functions of a large number of these molecules, remains to be discovered. In this review, we discuss two revolutionary genetic techniques that are able to decode the roles of these glycoproteins in an easy and efficient way. The RNA interference is a frequently technique used in plant biology that promotes genes silencing. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR/Cas9), emerged a few years ago as a revolutionary genome-editing technique that has allowed null mutants to be obtained in a wide variety of organisms, including plants. The two techniques have some differences between them and depending on the research objective, these may work as advantage or disadvantage. In the present work, we propose the use of the two techniques to obtain AGP mutants easily and quickly, helping to unravel the role of AGPs, surely a great asset for the future.},
}
@article {pmid31950239,
year = {2020},
author = {Walther, M and Schrahn, S and Krauss, V and Lein, S and Kessler, J and Jenuwein, T and Reuter, G},
title = {Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis.},
journal = {Chromosoma},
volume = {129},
number = {1},
pages = {83-98},
pmid = {31950239},
issn = {1432-0886},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; Blastula/embryology/*metabolism ; CRISPR-Cas Systems ; Centrosome ; Chromatin Assembly and Disassembly ; Cloning, Molecular ; Drosophila/classification/*genetics/*metabolism ; Drosophila Proteins/genetics/metabolism ; Embryonic Development/*genetics ; Female ; Gene Expression Regulation, Developmental ; Genome-Wide Association Study ; Heterochromatin/*genetics/*metabolism ; Histones/*metabolism ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Male ; Mutation ; Phylogeny ; },
abstract = {Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.},
}
@article {pmid31950135,
year = {2020},
author = {Li, C and Mei, H and Hu, Y},
title = {Applications and explorations of CRISPR/Cas9 in CAR T-cell therapy.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {175-182},
pmid = {31950135},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; },
abstract = {Chimeric antigen receptor(CAR) T-cell therapy has shown remarkable effects and promising prospects in patients with refractory or relapsed malignancies, pending further progress in the next-generation CAR T cells with more optimized structure, enhanced efficacy and reduced toxicities. The clustered regulatory interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology holds immense promise for advancing the field owing to its flexibility, simplicity, high efficiency and multiplexing in precise genome editing. Herein, we review the applications and explorations of CRISPR/Cas9 technology in constructing allogenic universal CAR T cells, disrupting inhibitory signaling to enhance potency and exploration of safer and more controllable novel CAR T cells.},
}
@article {pmid31949143,
year = {2020},
author = {Sirvent, S and Vallejo, AF and Davies, J and Clayton, K and Wu, Z and Woo, J and Riddell, J and Chaudhri, VK and Stumpf, P and Nazlamova, LA and Wheway, G and Rose-Zerilli, M and West, J and Pujato, M and Chen, X and Woelk, CH and MacArthur, B and Ardern-Jones, M and Friedmann, PS and Weirauch, MT and Singh, H and Polak, ME},
title = {Genomic programming of IRF4-expressing human Langerhans cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {313},
pmid = {31949143},
issn = {2041-1723},
support = {MC_PC_15078/MRC_/Medical Research Council/United Kingdom ; P30 AR070549/AR/NIAMS NIH HHS/United States ; },
mesh = {Antigen Presentation/genetics ; Basic-Leucine Zipper Transcription Factors/metabolism ; CRISPR-Cas Systems ; Cell Movement ; Cytokines/metabolism ; Gene Editing ; Gene Expression Profiling ; *Genomics ; Histocompatibility Antigens Class I ; Histocompatibility Antigens Class II ; Humans ; Interferon Regulatory Factors/*genetics/*metabolism ; Langerhans Cells/immunology/*metabolism ; NF-kappa B/metabolism ; Proto-Oncogene Proteins/metabolism ; Repressor Proteins/metabolism ; Trans-Activators/metabolism ; Transcription, Genetic ; Transcriptional Activation ; Up-Regulation ; },
abstract = {Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.},
}
@article {pmid31947593,
year = {2020},
author = {Jia, Y and Yang, B and Ross, P and Stanton, C and Zhang, H and Zhao, J and Chen, W},
title = {Comparative Genomics Analysis of Lactobacillus mucosae from Different Niches.},
journal = {Genes},
volume = {11},
number = {1},
pages = {},
pmid = {31947593},
issn = {2073-4425},
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Bacterial ; Humans ; Lactobacillus/*genetics/isolation &amp; purification ; *Operon ; Species Specificity ; },
abstract = {The potential probiotic benefits of Lactobacillus mucosae have received increasing attention. To investigate the genetic diversity of L. mucosae, comparative genomic analyses of 93 strains isolated from different niches (human and animal gut, human vagina, etc.) and eight strains of published genomes were conducted. The results showed that the core genome of L. mucosae mainly encoded translation and transcription, amino acid biosynthesis, sugar metabolism, and defense function while the pan-genomic curve tended to be close. The genetic diversity of L. mucosae mainly reflected in carbohydrate metabolism and immune/competitive-related factors, such as exopolysaccharide (EPS), enterolysin A, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. It was worth noting that this research firstly predicted the complete EPS operon shared among L. mucosae. Additionally, the type IIIA CRISPR-Cas system was discovered in L. mucosae for the first time. This work provided new ideas for the study of this species.},
}
@article {pmid31945616,
year = {2020},
author = {Li, Y and Glass, Z and Huang, M and Chen, ZY and Xu, Q},
title = {Ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications.},
journal = {Biomaterials},
volume = {234},
number = {},
pages = {119711},
pmid = {31945616},
issn = {1878-5905},
support = {R01 DC016875/DC/NIDCD NIH HHS/United States ; R01 EB027170/EB/NIBIB NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Therapy ; Genome ; Humans ; },
abstract = {The recently developed CRISPR/Cas9 technology has revolutionized the genome engineering field. Since 2016, increasing number of studies regarding CRISPR therapeutics have entered clinical trials, most of which are focusing on the ex vivo genome editing. In this review, we highlight the ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications. In these studies, CRISPR/Cas9 tools were used to edit cells in vitro and the successfully edited cells were considered as therapeutics, which can be introduced into patients to treat diseases. Considering a large number of previous reviews have been focused on the CRISPR/Cas9 delivery methods and materials, this review provides a different perspective, by mainly introducing the targeted conditions and design strategies for ex vivo CRISPR/Cas9 therapeutics. Brief descriptions of the history, functionality, and applications of CRISPR/Cas9 systems will be introduced first, followed by the design strategies and most significant results from previous research that used ex vivo CRISPR/Cas9 genome editing for the treatment of conditions or diseases. The last part of this review includes general information about the status of CRISPR/Cas9 therapeutics in clinical trials. We also discuss some of the challenges as well as the opportunities in this research area.},
}
@article {pmid31945142,
year = {2020},
author = {Morio, F and Lombardi, L and Butler, G},
title = {The CRISPR toolbox in medical mycology: State of the art and perspectives.},
journal = {PLoS pathogens},
volume = {16},
number = {1},
pages = {e1008201},
pmid = {31945142},
issn = {1553-7374},
mesh = {Aspergillus fumigatus/genetics ; *CRISPR-Cas Systems ; Cryptococcus neoformans/genetics ; Forecasting ; Gene Editing ; Humans ; Mucorales/genetics ; Mycology/*methods ; },
abstract = {Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR-Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR-Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.},
}
@article {pmid31944664,
year = {2020},
author = {Ou, B and Jiang, B and Jin, D and Yang, Y and Zhang, M and Zhang, D and Zhao, H and Xu, M and Song, H and Wu, W and Chen, M and Lu, T and Huang, J and Seo, H and Garcia, C and Zheng, W and Guo, W and Lu, Y and Jiang, Y and Yang, S and Kaushik, RS and Li, X and Zhang, W and Zhu, G},
title = {Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.},
journal = {ACS synthetic biology},
volume = {9},
number = {2},
pages = {412-426},
doi = {10.1021/acssynbio.9b00430},
pmid = {31944664},
issn = {2161-5063},
mesh = {Adhesins, Escherichia coli/immunology ; Animals ; Antibodies, Bacterial/blood/immunology ; Bacterial Adhesion ; CRISPR-Cas Systems/*genetics ; Cell Line ; *Chromosomes, Bacterial ; Enterotoxigenic Escherichia coli/*genetics/immunology ; Epithelial Cells/cytology/metabolism ; Escherichia coli Proteins/genetics/immunology ; Female ; Immunoglobulin G/immunology/metabolism ; Mice ; Mice, Inbred BALB C ; Multigene Family ; Swine ; },
abstract = {F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4[+] or both F4[+] and F18[+] ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4[+]/F18[+] ETEC infection.},
}
@article {pmid31943709,
year = {2020},
author = {},
title = {Mucopolysaccharidosis Type I Phenotypically Corrected with Edited Hematopoietic Stem Cells: Instead of altering the IDUA gene, a protein was inserted in a repurposable place in the genome known as a "safe harbor locus".},
journal = {American journal of medical genetics. Part A},
volume = {182},
number = {2},
pages = {275-276},
doi = {10.1002/ajmg.a.61223},
pmid = {31943709},
issn = {1552-4833},
mesh = {Animals ; Antigens, CD34/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; Humans ; Iduronidase/*genetics ; Mice ; Mucopolysaccharidosis I/*genetics/pathology/therapy ; Phenotype ; Receptors, CCR5/*genetics ; },
}
@article {pmid31943634,
year = {2020},
author = {Palumbo, CM and Gutierrez-Bujari, JM and O'Geen, H and Segal, DJ and Beal, PA},
title = {Versatile 3' Functionalization of CRISPR Single Guide RNA.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {21},
number = {11},
pages = {1633-1640},
pmid = {31943634},
issn = {1439-7633},
support = {T32 GM113770/GM/NIGMS NIH HHS/United States ; R01 GM080784/GM/NIGMS NIH HHS/United States ; T36 GM008784/GM/NIGMS NIH HHS/United States ; R21 HG010559/HG/NHGRI NIH HHS/United States ; },
mesh = {Binding Sites ; Biotin/*chemistry ; Biotinylation ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cross-Linking Reagents/chemistry ; DNA/*chemistry/metabolism ; DNA Cleavage ; Fluorescent Dyes/chemistry ; Gene Editing/*methods ; Gene Expression ; HEK293 Cells ; Heterogeneous-Nuclear Ribonucleoprotein K/genetics/metabolism ; Humans ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; RNA, Guide/*chemistry/genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; Y-Box-Binding Protein 1/genetics/metabolism ; },
abstract = {Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3'-end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3'-end was broadly tolerated by Streptococcus pyogenes Cas9 in an in vitro DNA cleavage assay. The 3'-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.},
}
@article {pmid31943080,
year = {2020},
author = {Yu, J and Xiang, X and Huang, J and Liang, X and Pan, X and Dong, Z and Petersen, TS and Qu, K and Yang, L and Zhao, X and Li, S and Zheng, T and Xu, Z and Liu, C and Han, P and Xu, F and Yang, H and Liu, X and Zhang, X and Bolund, L and Luo, Y and Lin, L},
title = {Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.},
journal = {Nucleic acids research},
volume = {48},
number = {5},
pages = {e25},
pmid = {31943080},
issn = {1362-4962},
mesh = {Alleles ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Circular/*genetics/metabolism ; Gene Editing/methods ; HEK293 Cells ; Haplotypes ; Hep G2 Cells ; Humans ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; },
abstract = {Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.},
}
@article {pmid31943070,
year = {2020},
author = {Fedorova, I and Arseniev, A and Selkova, P and Pobegalov, G and Goryanin, I and Vasileva, A and Musharova, O and Abramova, M and Kazalov, M and Zyubko, T and Artamonova, T and Artamonova, D and Shmakov, S and Khodorkovskii, M and Severinov, K},
title = {DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system.},
journal = {Nucleic acids research},
volume = {48},
number = {4},
pages = {2026-2034},
pmid = {31943070},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/*chemistry/genetics ; CRISPR-Cas Systems/*genetics ; Clostridium cellulolyticum/*enzymology ; Crystallography, X-Ray ; DNA/*chemistry/genetics ; Gene Editing ; Mutation ; Nucleotide Motifs/genetics ; RNA, Guide/genetics ; Streptococcus pyogenes/enzymology ; Substrate Specificity ; },
abstract = {Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.},
}
@article {pmid31942685,
year = {2020},
author = {Hao, Y and Zong, W and Zeng, D and Han, J and Chen, S and Tang, J and Zhao, Z and Li, X and Ma, K and Xie, X and Zhu, Q and Chen, Y and Zhao, X and Guo, J and Liu, YG},
title = {Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants.},
journal = {Science China. Life sciences},
volume = {63},
number = {6},
pages = {933-935},
pmid = {31942685},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Genetic Vectors ; Genome, Plant ; Plants, Genetically Modified/*genetics ; Promoter Regions, Genetic/*genetics ; RNA, Small Nuclear/*genetics ; },
}
@article {pmid31942067,
year = {2020},
author = {Athukoralage, JS and McMahon, SA and Zhang, C and Grüschow, S and Graham, S and Krupovic, M and Whitaker, RJ and Gloster, TM and White, MF},
title = {An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.},
journal = {Nature},
volume = {577},
number = {7791},
pages = {572-575},
pmid = {31942067},
issn = {1476-4687},
support = {BB/G011400/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R008035/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adenine Nucleotides/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/metabolism ; CRISPR-Cas Systems/*immunology ; DNA, Viral/metabolism ; Endonucleases/chemistry/*metabolism ; Host Microbial Interactions/*immunology ; Models, Molecular ; Nucleotides, Cyclic/chemistry/metabolism ; Oligoribonucleotides/chemistry/metabolism ; Phylogeny ; Signal Transduction ; Sulfolobus/genetics/immunology/metabolism/*virology ; Viral Proteins/chemistry/classification/*metabolism ; Viruses/*enzymology/immunology ; },
abstract = {The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)[1,2] and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)[3-5]. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain[6], sculpting a powerful antiviral response[7-10] that can drive viruses to extinction[7,8]. Cyclic nucleotides are increasingly implicated in host-pathogen interactions[11-13]. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.},
}
@article {pmid31942057,
year = {2020},
author = {Dolgin, E},
title = {The kill-switch for CRISPR that could make gene-editing safer.},
journal = {Nature},
volume = {577},
number = {7790},
pages = {308-310},
pmid = {31942057},
issn = {1476-4687},
mesh = {Animals ; Bacteria/genetics/immunology/virology ; Bacteriophages/immunology/physiology ; Biohazard Release/*prevention &amp; control ; Biosensing Techniques ; Biotechnology/*methods ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/metabolism ; *CRISPR-Cas Systems ; Culicidae/genetics ; Female ; Gene Drive Technology/*methods/standards ; Gene Editing/*methods/*standards ; Humans ; Male ; Optogenetics ; },
}
@article {pmid31942051,
year = {2020},
author = {Hampton, HG and Watson, BNJ and Fineran, PC},
title = {The arms race between bacteria and their phage foes.},
journal = {Nature},
volume = {577},
number = {7790},
pages = {327-336},
pmid = {31942051},
issn = {1476-4687},
mesh = {Adsorption ; Animals ; Bacteria/growth &amp; development/*immunology/*virology ; Bacteriophages/*immunology/metabolism ; CRISPR-Cas Systems/physiology ; Host Microbial Interactions/*immunology ; Humans ; },
abstract = {Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.},
}
@article {pmid31941961,
year = {2020},
author = {Straume, AH and Kjærner-Semb, E and Ove Skaftnesmo, K and Güralp, H and Kleppe, L and Wargelius, A and Edvardsen, RB},
title = {Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {409},
pmid = {31941961},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; Embryo, Mammalian/cytology/metabolism ; Fish Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Gene Editing ; *INDEL Mutation ; Membrane Transport Proteins/chemistry/genetics/*metabolism ; *Recombinational DNA Repair ; Salmo salar/embryology/*genetics ; },
abstract = {Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5'-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.},
}
@article {pmid31941712,
year = {2020},
author = {Chen, X and Chen, Y and Xin, H and Wan, T and Ping, Y},
title = {Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {5},
pages = {2395-2405},
pmid = {31941712},
issn = {1091-6490},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics/radiation effects ; Gene Editing/*methods ; Gold/chemistry ; HEK293 Cells ; Humans ; Infrared Rays ; Nanotubes/*chemistry ; *Optogenetics ; Plasmids/genetics ; Promoter Regions, Genetic ; },
abstract = {We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.},
}
@article {pmid31941083,
year = {2020},
author = {Yang, D and Wang, Z and Ma, J and Fu, Q and Wu, L and Wang, H and Wang, S and Yan, Y and Sun, J},
title = {Glycine Cleavage System and cAMP Receptor Protein Co-Regulate CRISPR/cas3 Expression to Resist Bacteriophage.},
journal = {Viruses},
volume = {12},
number = {1},
pages = {},
pmid = {31941083},
issn = {1999-4915},
mesh = {Amino Acid Oxidoreductases/*genetics ; Bacteriophages/*genetics/pathogenicity ; CRISPR-Associated Proteins/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cyclic AMP Receptor Protein/*genetics ; DNA Helicases/*genetics ; Escherichia coli/genetics/virology ; Escherichia coli Proteins/*genetics ; Gene Expression Regulation, Bacterial ; Multienzyme Complexes/*genetics ; Mutation ; Transferases/*genetics ; },
abstract = {The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N[5], N[10]-methylene tetrahydrofolate (N[5], N[10]-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N[5], N[10]-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.},
}
@article {pmid31939740,
year = {2020},
author = {Mastro, TL and Preza, A and Basu, S and Chattarji, S and Till, SM and Kind, PC and Kennedy, MB},
title = {A sex difference in the response of the rodent postsynaptic density to synGAP haploinsufficiency.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {31939740},
issn = {2050-084X},
support = {MR/P006213/1/MRC_/Medical Research Council/United Kingdom ; R01 MH115456/MH/NIMH NIH HHS/United States ; MH15456/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Female ; GTPase-Activating Proteins/genetics/*metabolism ; *Haploinsufficiency ; Male ; Mice ; Mice, Knockout ; Phosphorylation ; Post-Synaptic Density/*metabolism ; Sex Factors ; },
abstract = {SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of Syngap1 in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young Syngap1[+/-]mice. Here we show that only females and not males show a highly significant correlation between an increase in TARP and a decrease in synGAP in the PSDs of Syngap1[+/-]rodents. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP haploinsufficiency.},
}
@article {pmid31939194,
year = {2020},
author = {Huo, Z and Tu, J and Lee, DF and Zhao, R},
title = {Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2108},
number = {},
pages = {355-369},
doi = {10.1007/978-1-0716-0247-8_29},
pmid = {31939194},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cloning, Molecular ; *Gene Editing ; Gene Order ; *Gene Targeting ; *Genetic Engineering ; Genetic Vectors ; Humans ; *Mutation ; Plasmids ; RNA, Guide ; },
abstract = {CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system has been widely accepted and employed in gene editing of a vast range of cell types. Comparing to zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs), CRISPR shows less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene, but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome-wide screening. Here we will describe the procedure of generating stable cell lines with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.},
}
@article {pmid31939193,
year = {2020},
author = {Hu, O and Provvido, A and Zhu, Y},
title = {Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2108},
number = {},
pages = {345-353},
doi = {10.1007/978-1-0716-0247-8_28},
pmid = {31939193},
issn = {1940-6029},
support = {R15 CA213426/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Expression ; *Gene Knockout Techniques ; Genetic Vectors/genetics ; Humans ; Receptors, Interleukin-17/*genetics ; Single-Cell Analysis ; Transfection ; },
abstract = {CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here we present a methodological approach of establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). Similar approaches can be used for generating mammalian cell lines with gene knockout for other genes of interest.},
}
@article {pmid31937945,
year = {2020},
author = {},
title = {Towards better base editing.},
journal = {Nature biomedical engineering},
volume = {4},
number = {1},
pages = {1},
doi = {10.1038/s41551-020-0515-z},
pmid = {31937945},
issn = {2157-846X},
mesh = {Base Pairing/*genetics ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
}
@article {pmid31937939,
year = {2020},
author = {Kim, HK and Lee, S and Kim, Y and Park, J and Min, S and Choi, JW and Huang, TP and Yoon, S and Liu, DR and Kim, HH},
title = {High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells.},
journal = {Nature biomedical engineering},
volume = {4},
number = {1},
pages = {111-124},
pmid = {31937939},
issn = {2157-846X},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Deep Learning ; Gene Editing/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/physiology ; Streptococcus pyogenes/genetics ; },
abstract = {The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from Streptococcus pyogenes (SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.},
}
@article {pmid31937772,
year = {2020},
author = {Shen, J and Zhou, X and Shan, Y and Yue, H and Huang, R and Hu, J and Xing, D},
title = {Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {267},
pmid = {31937772},
issn = {2041-1723},
mesh = {Allosteric Regulation ; Animals ; Aptamers, Nucleotide ; Bacteria/*isolation &amp; purification ; CRISPR-Cas Systems ; Catalysis ; DNA, Bacterial/genetics ; Fluorescence ; Food Microbiology ; Mice ; Microbiological Techniques/*methods ; Milk/microbiology ; Real-Time Polymerase Chain Reaction ; Salmonella Infections/blood/diagnosis ; Salmonella enteritidis/isolation &amp; purification ; },
abstract = {The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called 'APC-Cas') that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 10[5] CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S. Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens.},
}
@article {pmid31937679,
year = {2020},
author = {Weissman, JL and Johnson, PLF},
title = {Network-Based Prediction of Novel CRISPR-Associated Genes in Metagenomes.},
journal = {mSystems},
volume = {5},
number = {1},
pages = {},
pmid = {31937679},
issn = {2379-5077},
abstract = {A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording "memories" of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection.IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to "remember" past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples ("metagenomes"), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes.},
}
@article {pmid31937638,
year = {2020},
author = {Guzzo, M and Castro, LK and Reisch, CR and Guo, MS and Laub, MT},
title = {A CRISPR Interference System for Efficient and Rapid Gene Knockdown in Caulobacter crescentus.},
journal = {mBio},
volume = {11},
number = {1},
pages = {},
pmid = {31937638},
issn = {2150-7511},
support = {R01 GM082899/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Caulobacter crescentus/*genetics ; DNA-Binding Proteins/genetics ; Down-Regulation ; *Gene Knockdown Techniques ; *Genes, Essential ; Streptococcus/genetics ; Streptococcus thermophilus/genetics ; Transcription Factors/genetics ; },
abstract = {CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knock down gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in CaulobacterIMPORTANCECaulobacter crescentus is a major model organism for understanding cell cycle regulation and cellular asymmetry. The current genetic tools for deleting or silencing the expression of individual genes, particularly those essential for viability, are time-consuming and labor-intensive, which limits global genetic studies. Here, we optimized CRISPR interference (CRISPRi) for use in Caulobacter Using Streptococcus thermophilus CRISPR3 or Streptococcus pasteurianus CRISPR systems, we show that the coexpression of a catalytically dead form of Cas9 (dCas9) with a single guide RNA (sgRNA) containing a seed region that targets the promoter region of a gene of interest efficiently downregulates the expression of the targeted gene. We also demonstrate that multiple sgRNAs can be produced in parallel to enable the facile silencing of multiple genes, opening the door to systematic genetic interaction studies. In sum, our work now provides a rapid, specific, and powerful new tool for silencing gene expression in C. crescentus and possibly other alphaproteobacteria.},
}
@article {pmid31937548,
year = {2020},
author = {Deecker, SR and Ensminger, AW},
title = {Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {3},
pages = {1039-1050},
pmid = {31937548},
issn = {2160-1836},
support = {PHT-148819//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; Genomics ; Legionella pneumophila/*genetics ; Phylogeny ; Plasmids ; },
abstract = {In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system's array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.},
}
@article {pmid31937317,
year = {2020},
author = {Klepsch, V and Pommermayr, M and Humer, D and Brigo, N and Hermann-Kleiter, N and Baier, G},
title = {Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy.},
journal = {Cell communication and signaling : CCS},
volume = {18},
number = {1},
pages = {8},
pmid = {31937317},
issn = {1478-811X},
support = {P 30324/FWF_/Austrian Science Fund FWF/Austria ; P 31383/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; CTLA-4 Antigen/metabolism ; Cells, Cultured ; Gene Deletion ; Immune Checkpoint Inhibitors/pharmacology/*therapeutic use ; Immunity/drug effects ; Mice, Inbred C57BL ; Mutagenesis/genetics ; Neoplasms/*drug therapy/*immunology/pathology ; Programmed Cell Death 1 Receptor/metabolism ; RNA, Guide/metabolism ; Repressor Proteins/deficiency/*metabolism ; Reproducibility of Results ; T-Lymphocytes/drug effects/*immunology ; },
abstract = {BACKGROUND: NR2F6 has been proposed as an alternative cancer immune checkpoint in the effector T cell compartment. However, a realistic assessment of the in vivo therapeutic potential of NR2F6 requires acute depletion.<br><br>METHODS: Employing primary T cells isolated from Cas9-transgenic mice for electroporation of chemically synthesized sgRNA, we established a CRISPR/Cas9-mediated acute knockout protocol of Nr2f6 in primary mouse T cells.<br><br>RESULTS: Analyzing these Nr2f6[CRISPR/Cas9 knockout] T cells, we reproducibly observed a hyper-reactive effector phenotype upon CD3/CD28 stimulation in vitro, highly reminiscent to Nr2f6[-/-] T cells. Importantly, CRISPR/Cas9-mediated Nr2f6 ablation prior to adoptive cell therapy (ACT) of autologous polyclonal T cells into wild-type tumor-bearing recipient mice in combination with PD-L1 or CTLA-4 tumor immune checkpoint blockade significantly delayed MC38 tumor progression and induced superior survival, thus further validating a T cell-inhibitory function of NR2F6 during tumor progression.<br><br>CONCLUSIONS: These findings indicate that Nr2f6[CRISPR/Cas9 knockout] T cells are comparable to germline Nr2f6[-/-] T cells, a result providing an independent confirmation of the immune checkpoint function of lymphatic NR2F6. Taken together, CRISPR/Cas9-mediated acute Nr2f6 gene ablation in primary mouse T cells prior to ACT appeared feasible for potentiating established PD-L1 and CTLA-4 blockade therapies, thereby pioneering NR2F6 inhibition as a sensitizing target for augmented tumor regression. Video abstract.},
}
@article {pmid31936769,
year = {2020},
author = {Cui, L and Wang, X and Huang, D and Zhao, Y and Feng, J and Lu, Q and Pu, Q and Wang, Y and Cheng, G and Wu, M and Dai, M},
title = {CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems.},
journal = {Pathogens (Basel, Switzerland)},
volume = {9},
number = {1},
pages = {},
pmid = {31936769},
issn = {2076-0817},
support = {P20 GM103442/GM/NIGMS NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; },
abstract = {Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria.},
}
@article {pmid31936280,
year = {2020},
author = {Wang, S and Yang, B and Ross, RP and Stanton, C and Zhao, J and Zhang, H and Chen, W},
title = {Comparative Genomics Analysis of Lactobacillus ruminis from Different Niches.},
journal = {Genes},
volume = {11},
number = {1},
pages = {},
pmid = {31936280},
issn = {2073-4425},
mesh = {Adaptation, Biological/genetics ; Animals ; CRISPR-Cas Systems/genetics ; Comparative Genomic Hybridization/methods ; Feces/microbiology ; Genome, Bacterial/genetics ; Genomics/methods ; Humans ; Intestines/microbiology ; Lactobacillus/*genetics/isolation &amp; purification ; Phylogeny ; },
abstract = {Lactobacillus ruminis is a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals. Although a few genomes of L. ruminis were published, most of them were animal derived. To explore the genetic diversity and potential niche-specific adaptation changes of L. ruminis, in the current work, draft genomes of 81 L. ruminis strains isolated from human, bovine, piglet, and other animals were sequenced, and comparative genomic analysis was performed. The genome size and GC content of L. ruminis on average were 2.16 Mb and 43.65%, respectively. Both the origin and the sampling distance of these strains had a great influence on the phylogenetic relationship. For carbohydrate utilization, the human-derived L. ruminis strains had a higher consistency in the utilization of carbon source compared to the animal-derived strains. L. ruminis mainly increased the competitiveness of niches by producing class II bacteriocins. The type of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system presented in L. ruminis was mainly subtype IIA. The diversity of CRISPR/Cas locus depended on the high denaturation of spacer number and sequence, although cas1 protein was relatively conservative. The genetic differences in those newly sequenced L. ruminis strains highlighted the gene gains and losses attributed to niche adaptations.},
}
@article {pmid31935425,
year = {2020},
author = {Zhou, W and Niu, YJ and Nie, ZW and Kim, JY and Xu, YN and Yan, CG and Cui, XS},
title = {Nuclear accumulation of pyruvate dehydrogenase alpha 1 promotes histone acetylation and is essential for zygotic genome activation in porcine embryos.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {4},
pages = {118648},
doi = {10.1016/j.bbamcr.2020.118648},
pmid = {31935425},
issn = {1879-2596},
mesh = {Acetylation ; Animals ; CRISPR-Cas Systems ; Cell Nucleus/*enzymology ; Embryo, Mammalian/enzymology/metabolism ; Embryonic Development/*genetics ; Gene Editing ; Gene Expression ; Genome ; Histones/*metabolism ; Pyruvate Dehydrogenase (Lipoamide)/genetics/*metabolism ; Swine ; Zygote/*enzymology/metabolism ; },
abstract = {Porcine zygotic genome activation (ZGA) occurs along with global epigenetic remodeling at the 4-cell stage. These processes are regulated by histone acetylation, which requires acetyl-coenzyme A (CoA). Pyruvate dehydrogenase complex (PDC) is a crucial enzyme in glucose metabolism that converts pyruvate into acetyl-CoA. In mammalian cells, acetyl-CoA is produced by pyruvate dehydrogenase alpha 1 (PDHA1) translocated into the nucleus in special conditions. To determine whether zygotic PDHA1 plays a critical role in promoting histone acetylation during ZGA, a CRISPR/Cas9 genome editing system using multiple guide RNAs was employed to generate a PDHA1-targeted parthenogenetic embryo model. Results of immunofluorescent staining showed that the nuclear accumulation of PDHA1 during ZGA was significantly inhibited by PDHA1 targeting. Meanwhile, the 4-cell arrest rate significantly increased at 72 h after activation, indicating impeded embryonic development. In addition, nuclear histone acetylation significantly decreased when PDHA1 was targeted, and quantitative PCR showed that expression of several zygotic genes was significantly decreased in the PDHA1-targeting group compared to the control group. Overexpression of PDHA1 recovered the nuclear PDHA1, H3K9Ac and H3K27Ac and EIF1A expression levels. Moreover, the 5-to-8-cell-stage embryo development rate was only partially rescued. In conclusion, expression of zygotic origin PDHA1 contributes to porcine ZGA by maintaining histone acetylation in porcine embryos.},
}
@article {pmid31935373,
year = {2020},
author = {Chen, CC and Li, B and Millman, SE and Chen, C and Li, X and Morris, JP and Mayle, A and Ho, YJ and Loizou, E and Liu, H and Qin, W and Shah, H and Violante, S and Cross, JR and Lowe, SW and Zhang, L},
title = {Vitamin B6 Addiction in Acute Myeloid Leukemia.},
journal = {Cancer cell},
volume = {37},
number = {1},
pages = {71-84.e7},
pmid = {31935373},
issn = {1878-3686},
support = {R01 CA190261/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; U01 HL127522/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; GTP Phosphohydrolases/metabolism ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute/*enzymology ; Membrane Proteins/metabolism ; Mice ; Monomeric GTP-Binding Proteins/metabolism ; Phosphotransferases/genetics/*metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; Polyamines/metabolism ; Pyridoxal Phosphate/*metabolism ; RNA, Small Interfering/metabolism ; Vitamin B 6/*metabolism ; },
abstract = {Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.},
}
@article {pmid31935181,
year = {2020},
author = {Ho, J and Zhao, M and Wojcik, S and Taiaroa, G and Butler, M and Poulter, R},
title = {The application of the CRISPR-Cas9 system in Pseudomonas syringae pv. actinidiae.},
journal = {Journal of medical microbiology},
volume = {69},
number = {3},
pages = {478-486},
doi = {10.1099/jmm.0.001124},
pmid = {31935181},
issn = {1473-5644},
mesh = {Actinidia/microbiology ; CRISPR-Cas Systems/genetics/*physiology ; Escherichia coli/physiology ; Fruit/microbiology ; Gene Deletion ; Gene Knockout Techniques ; Gene Targeting ; Genetic Engineering ; Peptide Synthases/genetics ; Plasmids ; Pseudomonas syringae/genetics/*physiology ; Sequence Analysis, DNA ; Streptococcus pyogenes/physiology ; Whole Genome Sequencing ; },
abstract = {Introduction. Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world.Aim. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species.Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa.Results. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of S. pyogenes, which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site.Conclusion. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.},
}
@article {pmid31933285,
year = {2019},
author = {Hirsch, F and Lemaitre, C and Chneiweiss, H and Montoliu, L},
title = {Genome Editing: Promoting Responsible Research.},
journal = {Pharmaceutical medicine},
volume = {33},
number = {3},
pages = {187-191},
pmid = {31933285},
issn = {1179-1993},
mesh = {Animals ; CRISPR-Cas Systems ; Ethics, Research ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Genome, Human ; Humans ; },
abstract = {For more than 40 years, scientists have been developing tools and technologies for genome modification; however, initial progress was slow and few outside of the molecular biology community took an interest in the field. Everything has dramatically changed with the recent appearance of the so-called precision approaches, and especially with the 'CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) revolution'. With great powers come great responsibilities. CRISPR-derived technologies have been proven efficient, cheap, rather easy and fast, and provided universal access to genome modification techniques beyond the leading research centers and reference laboratories. The popularization of techniques to manipulate the human genome and that of all other living beings consequently raises many essential questions, on the ethical and legal sides, both for the scientific community and the lay public. In order to mitigate excessive hype and concern among citizens, a call for the mobilization of the various stakeholders is now urgent through a global governance of genome editing.},
}
@article {pmid31933198,
year = {2020},
author = {Shang, W and Wang, F and Zhu, Q and Wang, L and Wang, H},
title = {CRISPR/Cas9-Based Genetic Screening to Study T-Cell Function.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2111},
number = {},
pages = {59-70},
doi = {10.1007/978-1-0716-0266-9_5},
pmid = {31933198},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Gene Regulatory Networks ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Jurkat Cells ; Lentivirus/genetics ; Lymphocyte Activation ; RNA, Guide/*pharmacology ; Sequence Analysis, DNA/*methods ; Signal Transduction ; T-Lymphocytes/*immunology ; Transduction, Genetic ; },
abstract = {T-cell-based cancer immunotherapies have emerged as a promising approach for cancer treatment, highlighting the importance of understanding the regulation of T-cell function. However, the molecular mechanisms underlying T-cell activation are not fully understood. The CRISPR/Cas9 system can serve as a robust method to systematically study signaling pathways. In this chapter, we describe details of using the CRISPR screen to identify regulators in TCR signaling, from the sgRNA library construction to genomic DNA sequencing. We also add some notes to further help readers performing the CRISPR screen. This approach can be readily adapted to study the activation of other immune cells, including B cells and dendritic cells.},
}
@article {pmid31932729,
year = {2020},
author = {Jost, M and Santos, DA and Saunders, RA and Horlbeck, MA and Hawkins, JS and Scaria, SM and Norman, TM and Hussmann, JA and Liem, CR and Gross, CA and Weissman, JS},
title = {Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs.},
journal = {Nature biotechnology},
volume = {38},
number = {3},
pages = {355-364},
pmid = {31932729},
issn = {1546-1696},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; U01 CA217882/CA/NCI NIH HHS/United States ; R35 GM118061/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; K99 GM130964/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; F32 GM116331/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; Deep Learning ; Gene Editing ; *Gene Expression ; Genomic Library ; HeLa Cells ; Humans ; K562 Cells ; Phenotype ; RNA, Guide/*genetics ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; },
abstract = {A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.},
}
@article {pmid31932727,
year = {2020},
author = {Li, C and Zhang, R and Meng, X and Chen, S and Zong, Y and Lu, C and Qiu, JL and Chen, YH and Li, J and Gao, C},
title = {Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors.},
journal = {Nature biotechnology},
volume = {38},
number = {7},
pages = {875-882},
pmid = {31932727},
issn = {1546-1696},
mesh = {Adenine/metabolism ; CRISPR-Cas Systems/*genetics ; Cytosine/metabolism ; Deoxyribonuclease I ; *Gene Editing ; Genes, Plant/genetics ; Genome, Plant/*genetics ; Humans ; Mutagenesis/*genetics ; Mutation/genetics ; Oryza/genetics/growth &amp; development ; RNA, Guide ; },
abstract = {Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited[1]. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C > T efficiency up to 61.61% and simultaneous C > T and A > G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing.},
}
@article {pmid31932482,
year = {2020},
author = {Gustafson, EA and Seymour, KA and Sigrist, K and Rooij, DGDE and Freiman, RN},
title = {ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.},
journal = {Molecular and cellular biology},
volume = {40},
number = {7},
pages = {},
pmid = {31932482},
issn = {1098-5549},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Female ; HEK293 Cells ; Humans ; Infertility, Male/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Ovary/metabolism ; Protamines/genetics/metabolism ; Protein Domains/genetics ; Spermatogenesis/*genetics ; TATA-Binding Protein Associated Factors/*genetics ; Testis/metabolism ; Transcription Factor TFIID/*genetics ; Transcription Factors/genetics/*metabolism ; Transcriptional Activation/genetics ; Two-Hybrid System Techniques ; },
abstract = {TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.},
}
@article {pmid31932164,
year = {2020},
author = {Lau, RK and Ye, Q and Birkholz, EA and Berg, KR and Patel, L and Mathews, IT and Watrous, JD and Ego, K and Whiteley, AT and Lowey, B and Mekalanos, JJ and Kranzusch, PJ and Jain, M and Pogliano, J and Corbett, KD},
title = {Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.},
journal = {Molecular cell},
volume = {77},
number = {4},
pages = {723-733.e6},
pmid = {31932164},
issn = {1097-4164},
support = {R01 AI026289/AI/NIAID NIH HHS/United States ; R01 ES027595/ES/NIEHS NIH HHS/United States ; R37 AI018045/AI/NIAID NIH HHS/United States ; F31 CA236405/CA/NCI NIH HHS/United States ; T32 HL007444/HL/NHLBI NIH HHS/United States ; T32 GM007752/GM/NIGMS NIH HHS/United States ; K01 DK116917/DK/NIDDK NIH HHS/United States ; R01 AI018045/AI/NIAID NIH HHS/United States ; S10 OD020025/OD/NIH HHS/United States ; P30 DK063491/DK/NIDDK NIH HHS/United States ; R01 GM129245/GM/NIGMS NIH HHS/United States ; T32 GM127235/GM/NIGMS NIH HHS/United States ; R21 AI148814/AI/NIAID NIH HHS/United States ; },
mesh = {Allosteric Regulation ; Bacterial Proteins/*chemistry/*metabolism ; Bacteriophage lambda/genetics/physiology ; CRISPR-Cas Systems ; DNA Cleavage ; DNA Restriction Enzymes/chemistry ; Deoxyribonuclease I/*chemistry/*metabolism ; Escherichia coli/enzymology/immunology/*virology ; Genome, Viral ; Protein Multimerization ; Second Messenger Systems ; },
abstract = {Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.},
}
@article {pmid31931564,
year = {2020},
author = {Alyami, MZ and Alsaiari, SK and Li, Y and Qutub, SS and Aleisa, FA and Sougrat, R and Merzaban, JS and Khashab, NM},
title = {Cell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {4},
pages = {1715-1720},
doi = {10.1021/jacs.9b11638},
pmid = {31931564},
issn = {1520-5126},
mesh = {Animals ; *Biomimetics ; *CRISPR-Cas Systems ; HeLa Cells ; Heterografts ; Humans ; MCF-7 Cells ; Metal-Organic Frameworks/*chemistry ; Mice ; },
abstract = {Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C[3]-ZIF(cell membrane type). Incubation of C[3]-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C[3]-ZIFMCF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C[3]-ZIFHELA. In vivo testing confirmed the selectivity of C[3]-ZIFMCF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.},
}
@article {pmid31930919,
year = {2020},
author = {Yang, YB and Tang, YD and Hu, Y and Yu, F and Xiong, JY and Sun, MX and Lyu, C and Peng, JM and Tian, ZJ and Cai, XH and An, TQ},
title = {Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.},
journal = {Nano letters},
volume = {20},
number = {2},
pages = {1417-1427},
doi = {10.1021/acs.nanolett.9b05103},
pmid = {31930919},
issn = {1530-6992},
mesh = {CRISPR-Cas Systems/*genetics ; Capsid ; HeLa Cells ; Herpesvirus 1, Suid/*isolation &amp; purification/ultrastructure ; Humans ; Quantum Dots/*chemistry ; Virion/genetics/*isolation &amp; purification ; },
abstract = {Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.},
}
@article {pmid31930530,
year = {2020},
author = {Annunziato, S and Lutz, C and Henneman, L and Bhin, J and Wong, K and Siteur, B and van Gerwen, B and de Korte-Grimmerink, R and Zafra, MP and Schatoff, EM and Drenth, AP and van der Burg, E and Eijkman, T and Mukherjee, S and Boroviak, K and Wessels, LF and van de Ven, M and Huijbers, IJ and Adams, DJ and Dow, LE and Jonkers, J},
title = {In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.},
journal = {The EMBO journal},
volume = {39},
number = {5},
pages = {e102169},
pmid = {31930530},
issn = {1460-2075},
support = {93512009//Netherlands Genomics Initiative (NGI) Zenith/International ; F31 CA224800/CA/NCI NIH HHS/United States ; 85300120//Cancer Systems Biology Center (CSBC)/International ; 184032303//National Roadmap grant for Large-Scale Research Facilities/International ; 319661//ERC Synergy project CombatCancer/International ; VICI 91814643//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/International ; //EC | FP7 | FP7 Ideas: European Research Council (FP7 Ideas)/International ; 024001028//Cancer Genomics Netherlands (CGCNL)/International ; //Oncode Institute/International ; },
mesh = {Animals ; Breast Neoplasms/*genetics ; *CRISPR-Cas Systems ; Disease Models, Animal ; Female ; *Gene Editing ; Male ; Mice ; Mice, Transgenic ; Mutation ; },
abstract = {Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.},
}
@article {pmid31928144,
year = {2020},
author = {Zhao, Q and Li, C and Yu, M and Sun, Y and Wang, J and Ma, L and Sun, X and Lu, B},
title = {HuR stabilizes HTT mRNA via interacting with its exon 11 in a mutant HTT-dependent manner.},
journal = {RNA biology},
volume = {17},
number = {4},
pages = {500-516},
pmid = {31928144},
issn = {1555-8584},
mesh = {3' Untranslated Regions ; Animals ; Binding Sites ; CRISPR-Cas Systems ; Cell Line ; ELAV-Like Protein 1/genetics/*metabolism ; Exons ; Feedback, Physiological ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Huntingtin Protein/*chemistry/*genetics ; Huntington Disease/*genetics/metabolism ; Mice ; Mitogen-Activated Protein Kinase 11/*metabolism ; *Mutation ; RNA Stability ; },
abstract = {Huntington's Disease (HD) is a monogenetic neurodegenerative disorder mainly caused by the cytotoxicity of the mutant HTT protein (mHTT) encoded by the mutant HTT gene. Lowering HTT mRNA has been extensively studied as a potential therapeutic strategy, but how its level is regulated endogenously has been unclear. Here we report that the RNA-binding protein (RBP) HuR interacts with and stabilizes HTT mRNA in an mHTT-dependent manner. In HD cells but not wild-type cells, siRNA knockdown or CRISPR-induced heterozygous knockout of HuR decreased HTT mRNA stability. HuR interacted with HTT mRNA at a conserved site in exon 11 rather than the 3'-UTR region of the mRNA. Interestingly, this interaction was dependent on the presence of mHTT, likely via the activation of MAPK11, which enhanced cytosolic localization of the HuR protein. Thus, mHTT, MAPK11 and HuR may form a positive feedback loop that stabilizes HTT mRNA and enhances mHTT accumulation, which may contribute to HD progression. Our data reveal a novel regulatory mechanism of HTT mRNA via non-canonical binding of HuR.},
}
@article {pmid31927726,
year = {2020},
author = {Li, R and Zeng, W and Ma, M and Wei, Z and Liu, H and Liu, X and Wang, M and Shi, X and Zeng, J and Yang, L and Mo, D and Liu, X and Chen, Y and He, Z},
title = {Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs.},
journal = {Transgenic research},
volume = {29},
number = {1},
pages = {149-163},
pmid = {31927726},
issn = {1573-9368},
support = {2019A1515011134//Natural Science Foundation of Guangdong Province/International ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing ; Male ; *Muscle Development ; Muscle, Skeletal/*cytology ; Myostatin/antagonists &amp; inhibitors/*genetics ; Protein Sorting Signals/*genetics ; Swine ; },
abstract = {Myostatin (MSTN), a member of the transforming growth factor-β superfamily, is a negative regulator of muscle growth and development. Disruption of the MSTN gene in various mammalian species markedly promotes muscle growth. Previous studies have mainly focused on the disruption of the MSTN peptide coding region in pigs but not on the modification of the signal peptide region. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was used to successfully introduce two mutations (PVD20H and GP19del) in the MSTN signal peptide region of the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Both mutations in signal peptide increased the muscle mass without inhibiting the production of mature MSTN peptide in the cells. Histological analysis revealed that the enhanced muscle mass in MSTN[+/PVD20H] pig was mainly due to an increase in the number of muscle fibers. The expression of MSTN in the longissimus dorsi muscle of MSTN[+/PVD20H] and MSTN[KO/PVD20H] pigs was significantly downregulated, whereas that of myogenic regulatory factors, including MyoD, Myogenin, and Myf-5, was significantly upregulated when compared to those in the longissimus dorsi muscle of wild-type pigs. Meanwhile, the mutations also activated the PI3K/Akt pathway. The results of this study indicated that precise editing of the MSTN signal peptide can enhance porcine muscle development without markedly affecting the expression of mature MSTN peptide, which could exert other beneficial biological functions in the edited pigs.},
}
@article {pmid31927725,
year = {2020},
author = {Paschon, V and Correia, FF and Morena, BC and da Silva, VA and Dos Santos, GB and da Silva, MCC and Cristante, AF and Willerth, SM and Perrin, FE and Kihara, AH},
title = {CRISPR, Prime Editing, Optogenetics, and DREADDs: New Therapeutic Approaches Provided by Emerging Technologies in the Treatment of Spinal Cord Injury.},
journal = {Molecular neurobiology},
volume = {57},
number = {4},
pages = {2085-2100},
pmid = {31927725},
issn = {1559-1182},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Designer Drugs/*therapeutic use ; *Gene Editing ; Humans ; *Optogenetics ; Spinal Cord Injuries/*therapy ; Translational Research, Biomedical ; },
abstract = {Spinal cord injury (SCI) causes temporary disabilities or permanent effects including neuropathic pain and spastiscity. The damage often results from mechanical trauma, which in turn triggers the neuroinflammatory process. Neuroinflammation plays essential roles in the structural, biochemical, and cellular changes that take place in the spinal cord after the injury. Indeed, SCI activates many different signaling pathways that coordinate the resulting cellular responses. While neuroinflammation serves as a physiological reaction to harmful stimuli, it is clear that long-lasting inflammatory response leads to aggravation of the neurodegenerative processes, becoming detrimental to recovery post-injury. In this context, we present some possible therapeutic targets in these activated signaling pathways and provide new perspectives for SCI treatment based on recently developed technologies, including clustered regularly interspaced short palindromic repeats (CRISPR)-based methods (including prime editing), optogenetics, and designer receptor exclusively activated by designer drugs (DREADDs). We critically analyze the recent advances in the deployment of these methods focusing on the control of the initial neuroinflammatory response. We then propose alternatives and provide new avenues for SCI treatment based on these emerging technologies.},
}
@article {pmid31927331,
year = {2020},
author = {Weng, Y and Huang, Q and Li, C and Yang, Y and Wang, X and Yu, J and Huang, Y and Liang, XJ},
title = {Improved Nucleic Acid Therapy with Advanced Nanoscale Biotechnology.},
journal = {Molecular therapy. Nucleic acids},
volume = {19},
number = {},
pages = {581-601},
pmid = {31927331},
issn = {2162-2531},
abstract = {Due to a series of systemic and intracellular obstacles in nucleic acid (NA) therapy, including fast degradation in blood, renal clearance, poor cellular uptake, and inefficient endosomal escape, NAs may need delivery methods to transport to the cell nucleus or cytosol to be effective. Advanced nanoscale biotechnology-associated strategies, such as controlling the particle size, charge, drug loading, response to environmental signals, or other physical/chemical properties of delivery carriers, have provided great help for the in vivo and in vitro delivery of NA therapeutics. In this review, we introduce the characteristics of different NA modalities and illustrate how advanced nanoscale biotechnology assists NA therapy. The specific features and challenges of various nanocarriers in clinical and preclinical studies are summarized and discussed. With the help of advanced nanoscale biotechnology, some of the major barriers to the development of NA therapy will eventually be overcome in the near future.},
}
@article {pmid31926765,
year = {2020},
author = {Wang, R and Angenent, GC and Seymour, G and de Maagd, RA},
title = {Revisiting the Role of Master Regulators in Tomato Ripening.},
journal = {Trends in plant science},
volume = {25},
number = {3},
pages = {291-301},
doi = {10.1016/j.tplants.2019.11.005},
pmid = {31926765},
issn = {1878-4372},
mesh = {Fruit ; Gene Expression Regulation, Plant ; *Lycopersicon esculentum ; Phenotype ; Plant Proteins ; },
abstract = {The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are 'master regulators'. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.},
}
@article {pmid31926228,
year = {2020},
author = {Tian, Y and Liu, RR and Xian, WD and Xiong, M and Xiao, M and Li, WJ},
title = {A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency.},
journal = {International journal of biological macromolecules},
volume = {147},
number = {},
pages = {376-384},
doi = {10.1016/j.ijbiomac.2020.01.079},
pmid = {31926228},
issn = {1879-0003},
mesh = {*Alicyclobacillus/enzymology/genetics/isolation &amp; purification ; *Bacterial Proteins/chemistry/genetics ; *Brevibacillus/enzymology/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Endonucleases/genetics ; Hot Springs/*microbiology ; Water Microbiology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (Kcat/Km (s[-][1] M[-][1]) = 8.80 × 10[11]), which is significantly higher than that of AacCas12b (Kcat/Km (s[-][1] M[-][1]) = 7.56 × 10[8]) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.},
}
@article {pmid31925767,
year = {2020},
author = {Chojnacka-Puchta, L and Sawicka, D},
title = {CRISPR/Cas9 gene editing in a chicken model: current approaches and applications.},
journal = {Journal of applied genetics},
volume = {61},
number = {2},
pages = {221-229},
pmid = {31925767},
issn = {2190-3883},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Chickens/*genetics ; Disease Models, Animal ; Egg Hypersensitivity/*genetics/pathology ; *Gene Editing ; Genome/genetics ; Germ Cells/cytology/metabolism ; Humans ; Phenotype ; },
abstract = {Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken bioreactors, production of knock-in/out chickens, low-allergenicity eggs, and disease-resistance models. New strategies, including CRISPR/Cas9, have made gene editing easy and highly efficient in comparison to the well-known process of homologous recombination. The clustered regularly interspaced short palindromic repeats (CRISPR) technique enables us to understand the function of genes and/or to modify the animal phenotype to fit a specific scientific or production target. To facilitate chicken genome engineering applications, we present a concise description of the method and current application of the CRISPR/Cas9 system in chickens. Different strategies for delivering sgRNAs and the Cas9 protein, we also present extensively. Furthermore, we describe a new gesicle technology as a way to deliver Cas9/sgRNA complexes into target cells, and we discuss the advantages and describe basal applications of the CRISPR/Cas9 system in a chicken model.},
}
@article {pmid31925391,
year = {2020},
author = {Jia, N and Xie, W and de la Cruz, MJ and Eng, ET and Patel, DJ},
title = {Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.},
journal = {Cell research},
volume = {30},
number = {2},
pages = {182-184},
pmid = {31925391},
issn = {1748-7838},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; S10 RR029205/RR/NCRR NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/metabolism ; CRISPR-Cas Systems/*genetics ; DNA/*chemistry/*metabolism ; DNA Transposable Elements/*genetics ; Structure-Activity Relationship ; Vibrio cholerae/metabolism ; },
}
@article {pmid31925293,
year = {2020},
author = {Lee, HK and Smith, HE and Liu, C and Willi, M and Hennighausen, L},
title = {Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {19},
pmid = {31925293},
issn = {2399-3642},
mesh = {*Adenine ; Animals ; Base Sequence ; CRISPR-Cas Systems ; *Cytosine ; *Embryo, Mammalian ; Female ; Gene Editing ; Gene Targeting ; Genome ; Male ; Mice ; *Mutation ; Point Mutation ; *RNA Editing ; RNA, Guide ; },
abstract = {Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.},
}
@article {pmid31923454,
year = {2020},
author = {McCloskey, AG and Miskelly, MG and Moore, CBT and Nesbit, MA and Christie, KA and Owolabi, AI and Flatt, PR and McKillop, AM},
title = {CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function.},
journal = {Peptides},
volume = {125},
number = {},
pages = {170251},
doi = {10.1016/j.peptides.2019.170251},
pmid = {31923454},
issn = {1873-5169},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Diabetes Mellitus, Experimental/genetics/*metabolism/physiopathology ; Disease Models, Animal ; Gastric Inhibitory Polypeptide/*metabolism ; Gene Editing/*methods ; Glucagon-Like Peptide 1/*metabolism ; Humans ; Insulin-Secreting Cells/*metabolism ; Male ; Mice ; Receptors, Cannabinoid/chemistry/genetics/*metabolism ; },
abstract = {G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic target. This study aimed to assess the metabolic functionality of GPR55 ligands using CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55 signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10[-7]-10[-4]M) stimulated insulin secretion (p < 0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4) beta cells, with 2-2.7-fold (p < 0.001) increase demonstrated in BRIN-BD11 cells (10[-4]M). The insulinotropic effect of Abn-CBD (42 %), AM251 (30 %) and PEA (53 %) were impaired (p < 0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602 completely abolished (p < 0.001). Gpr55 ablation abolished the release of intracellular Ca[2+] upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-CBD and AM251 (1.7-3-fold; p < 0.01) was greatly diminished (p < 0.001) in Gpr55 null cells. Orally administered Abn-CBD and AM251 (0.1 μmol/kgBW) improved GIP (p < 0.05-p < 0.01), GLP-1 (p < 0.05-p < 0.001), glucose tolerance (p < 0.001) and circulating insulin (p < 0.05-p < 0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p < 0.05) and insulin release (p < 0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects of Abn-CBD (p < 0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1 release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a novel approach for the treatment of type-2-diabetes.},
}
@article {pmid31923323,
year = {2020},
author = {Du, M and Zhou, K and Liu, Y and Deng, L and Zhang, X and Lin, L and Zhou, M and Zhao, W and Wen, C and Xing, J and Li, CB and Li, C},
title = {A biotechnology-based male-sterility system for hybrid seed production in tomato.},
journal = {The Plant journal : for cell and molecular biology},
volume = {102},
number = {5},
pages = {1090-1100},
pmid = {31923323},
issn = {1365-313X},
mesh = {Biotechnology/*methods ; CRISPR-Cas Systems ; Lycopersicon esculentum/metabolism/*physiology ; Plant Infertility/genetics/physiology ; Seeds/metabolism/*physiology ; },
abstract = {Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male-sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology-based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male-sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated mutagenesis of a stamen-specific gene SlSTR1 and devised a transgenic maintainer by transforming male-sterile plants with a fertility-restoration gene linked to a seedling-colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male-sterile plant segregated into 50% non-transgenic male-sterile plants and 50% male-fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male-sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.},
}
@article {pmid31922351,
year = {2020},
author = {Meng, B and Ip, NCY and Abbink, TEM and Kenyon, JC and Lever, AML},
title = {ESCRT-II functions by linking to ESCRT-I in human immunodeficiency virus-1 budding.},
journal = {Cellular microbiology},
volume = {22},
number = {5},
pages = {e13161},
pmid = {31922351},
issn = {1462-5822},
support = {MR/N022939/1/MRC_/Medical Research Council/United Kingdom ; MR/N022939/1//UK Medical Research Council/International ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Endosomal Sorting Complexes Required for Transport/*genetics/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; HIV Infections/*virology ; HIV-1/*metabolism ; Humans ; T-Lymphocytes ; Ubiquitin/metabolism ; Virus Replication ; },
abstract = {Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT-I and -III in HIV budding being firmly established, participation of ESCRT-II in this process has been controversial. EAP45 is a critical component of ESCRT-II. Previously, we utilised a CRISPR-Cas9 EAP45 knockout cell line to assess the involvement of ESCRT-II in HIV replication. We demonstrated that the absence of ESCRT-II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re-introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT-II. EAP45 acts through the YPXL-ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT-II in the late stages of HIV replication and reinforces the notion that ESCRT-II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis.},
}
@article {pmid31922192,
year = {2020},
author = {Hidalgo-Cantabrana, C and Barrangou, R},
title = {Characterization and applications of Type I CRISPR-Cas systems.},
journal = {Biochemical Society transactions},
volume = {48},
number = {1},
pages = {15-23},
doi = {10.1042/BST20190119},
pmid = {31922192},
issn = {1470-8752},
mesh = {Adaptive Immunity ; Animals ; Bacteria/genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genome, Bacterial ; Genome, Human ; Humans ; Plasmids/genetics ; },
abstract = {CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.},
}
@article {pmid31921404,
year = {2020},
author = {Martin, P and Wagh, V and Reis, SA and Erdin, S and Beauchamp, RL and Shaikh, G and Talkowski, M and Thiele, E and Sheridan, SD and Haggarty, SJ and Ramesh, V},
title = {TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling.},
journal = {Molecular autism},
volume = {11},
number = {1},
pages = {2},
pmid = {31921404},
issn = {2040-2392},
support = {R01 NS109540/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Codon, Nonsense ; Eukaryotic Initiation Factor-4E/*metabolism ; Gene Editing ; Germ-Line Mutation ; Humans ; Induced Pluripotent Stem Cells/cytology ; Intracellular Signaling Peptides and Proteins/*metabolism ; Mechanistic Target of Rapamycin Complex 1/antagonists &amp; inhibitors ; Neural Stem Cells/*metabolism ; Neurogenesis ; Phenotype ; Protein Serine-Threonine Kinases/*metabolism ; RNA-Seq ; Signal Transduction ; Sirolimus ; *Tuberous Sclerosis ; Tuberous Sclerosis Complex 1 Protein/genetics ; },
abstract = {BACKGROUND: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin.<br><br>METHODS: Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq.<br><br>RESULTS: Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs.<br><br>CONCLUSION: MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.},
}
@article {pmid31921246,
year = {2019},
author = {Wesseler, J and Politiek, H and Zilberman, D},
title = {The Economics of Regulating New Plant Breeding Technologies - Implications for the Bioeconomy Illustrated by a Survey Among Dutch Plant Breeders.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {1597},
pmid = {31921246},
issn = {1664-462X},
abstract = {New plant breeding technologies (NPBTs) are increasingly used for developing new plants with novel traits. The science tells us that those plants in general are as safe as than those once developed using "conventional" plant breeding methods. The knowledge about the induced changes and properties of the new plants by using NPBTs is more precise. This should lead to the conclusion that plants developed using NPBTs should not be regulated differently than those developed using "conventional" plant breeding methods. This contribution discusses the economics of regulating new plant breeding technologies. We first develop the theoretical model and elaborate on the different regulatory approaches being used and compare their advantages and disadvantages. Then we provide a perspectives on EU regulation around mutagenesis-based New Plant Breeding Techniques (NPBT), formed by new insights from a survey among Dutch plant breeding companies. The survey measures the attitude of breeding companies towards the ruling of the EU Court of Justice that subjected the use of CRISPR-Cas in the development of new plant varieties under the general EU regulations around GMOs. The results show that plant breeders experience a financial barrier because of the ruling, with perceived negative impact on competitiveness and investments in CRISPR-Cas as a result. The degree of negative impact differs however significantly among seed-sectors and company sizes. One of the most striking results was the relative optimism of companies in the sector about more lenient legislation in the next five years, despite the stated negative effects.},
}
@article {pmid31921143,
year = {2019},
author = {Zhuang, X and Veltri, DP and Long, EO},
title = {Genome-Wide CRISPR Screen Reveals Cancer Cell Resistance to NK Cells Induced by NK-Derived IFN-γ.},
journal = {Frontiers in immunology},
volume = {10},
number = {},
pages = {2879},
pmid = {31921143},
issn = {1664-3224},
mesh = {B7 Antigens/genetics/immunology ; CRISPR-Cas Systems ; *Carcinoma, Renal Cell/genetics/immunology/pathology ; Genome-Wide Association Study ; Humans ; *Interferon-gamma/genetics/immunology ; K562 Cells ; Kidney Neoplasms/genetics/immunology/pathology ; Killer Cells, Natural/*immunology/pathology ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/immunology/pathology ; Natural Cytotoxicity Triggering Receptor 3/genetics/immunology ; Receptors, Interferon/genetics/immunology ; *Tumor Escape ; },
abstract = {The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.},
}
@article {pmid31921024,
year = {2019},
author = {Watanabe, S and Cui, B and Kiga, K and Aiba, Y and Tan, XE and Sato'o, Y and Kawauchi, M and Boonsiri, T and Thitiananpakorn, K and Taki, Y and Li, FY and Azam, AH and Nakada, Y and Sasahara, T and Cui, L},
title = {Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2838},
pmid = {31921024},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.},
}
@article {pmid31919672,
year = {2020},
author = {Zhao, Z and Zhang, RA and Fu, GY and Zhang, R and Nie, YF and Sun, C and Wu, M},
title = {The Complete Genome of Emcibacter congregatus ZYL[T], a Marine Bacterium Encoding a CRISPR-Cas 9 Immune System.},
journal = {Current microbiology},
volume = {77},
number = {5},
pages = {762-768},
pmid = {31919672},
issn = {1432-0991},
mesh = {Alphaproteobacteria/*genetics ; Aquatic Organisms/genetics ; Base Composition ; CRISPR-Cas Systems/*genetics ; China ; Gene Editing ; *Genome, Bacterial ; Genomics ; Geologic Sediments/microbiology ; Phylogeny ; Sequence Analysis, DNA ; },
abstract = {Emcibacter congregatus ZYL[T] was isolated from a sediment sample cultured in situ in a coast located in the East China Sea. The genome of E. congregatus ZYL[T] was sequenced and assembled into one single circular chromosome with the size of 4,189,011 bp and G+C content of 52.6%. Genomic annotation showed that E. congregatus ZYL[T] had an intact Type II-C CRISPR-Cas system consists of three cas genes (cas 9, cas 1, and cas 2), 34 direct repeat sequences with the length of 36 bp, and 33 spacers. The predicted Cas 9 protein was smaller than most of existing genome editing tools. This structure might have potential in developing new gene editing system and uncovering the regulatory mechanisms of CRISPR-Cas system. Besides, the comparison between E. congregatus ZYL[T] and its relative species living in neritic environments unraveled some common traits of the defective strategies of these bacteria to face inshore challenges including the motility, multidrug resistance, and universal efflux pumps.},
}
@article {pmid31919495,
year = {2020},
author = {Kukhtar, D and Rubio-Peña, K and Serrat, X and Cerón, J},
title = {Mimicking of splicing-related retinitis pigmentosa mutations in C. elegans allow drug screens and identification of disease modifiers.},
journal = {Human molecular genetics},
volume = {29},
number = {5},
pages = {756-765},
doi = {10.1093/hmg/ddz315},
pmid = {31919495},
issn = {1460-2083},
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans ; Genes, Dominant ; High-Throughput Screening Assays ; Humans ; *Mutation, Missense ; Pharmaceutical Preparations/*administration &amp; dosage ; RNA Interference ; *RNA Splicing ; RNA Splicing Factors/antagonists &amp; inhibitors/*genetics ; RNA-Binding Proteins/antagonists &amp; inhibitors/*genetics ; Retinitis Pigmentosa/drug therapy/genetics/*pathology ; Ribonucleoproteins, Small Nuclear/antagonists &amp; inhibitors/*genetics ; },
abstract = {CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1, whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.},
}
@article {pmid31919444,
year = {2020},
author = {},
title = {When the world needs to know.},
journal = {Nature biotechnology},
volume = {38},
number = {1},
pages = {1},
doi = {10.1038/s41587-019-0396-4},
pmid = {31919444},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; *Ethics, Research ; Fertilization in Vitro ; Gene Editing ; Humans ; Publications/ethics ; },
}
@article {pmid31919360,
year = {2020},
author = {Li, B and Clohisey, SM and Chia, BS and Wang, B and Cui, A and Eisenhaure, T and Schweitzer, LD and Hoover, P and Parkinson, NJ and Nachshon, A and Smith, N and Regan, T and Farr, D and Gutmann, MU and Bukhari, SI and Law, A and Sangesland, M and Gat-Viks, I and Digard, P and Vasudevan, S and Lingwood, D and Dockrell, DH and Doench, JG and Baillie, JK and Hacohen, N},
title = {Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {164},
pmid = {31919360},
issn = {2041-1723},
support = {NIH P50HG006193//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; U19 AI057229/AI/NIAID NIH HHS/United States ; R21 CA220103/CA/NCI NIH HHS/United States ; MR/N02995X/1/MRC_/Medical Research Council/United Kingdom ; R01 GM100202/GM/NIGMS NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; R01 AI137057/AI/NIAID NIH HHS/United States ; 103258/Z/13/Z//Wellcome Trust (Wellcome)/International ; P50 HG006193/HG/NHGRI NIH HHS/United States ; DP2 DA042422/DA/NIDA NIH HHS/United States ; R01 AI124378/AI/NIAID NIH HHS/United States ; //Wellcome Trust/United Kingdom ; },
mesh = {A549 Cells ; Adaptor Proteins, Signal Transducing/genetics ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dibenzothiepins ; Genetic Predisposition to Disease/*genetics ; Genome-Wide Association Study ; Host-Pathogen Interactions/*genetics ; Humans ; Influenza A virus/*pathogenicity ; Influenza, Human/*genetics/*pathology ; Membrane Proteins/genetics ; Methyltransferases/metabolism ; Morpholines ; Nerve Tissue Proteins/genetics ; Oxazines/pharmacology ; Pyridines/pharmacology ; Pyridones ; Thiepins/pharmacology ; Triazines/pharmacology ; Vacuolar Proton-Translocating ATPases/metabolism ; Virus Internalization ; },
abstract = {Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.},
}
@article {pmid31917956,
year = {2020},
author = {Zhang, L and Xu, W and Gao, X and Li, W and Qi, S and Guo, D and Ajayi, OE and Ding, SW and Wu, Q},
title = {lncRNA Sensing of a Viral Suppressor of RNAi Activates Non-canonical Innate Immune Signaling in Drosophila.},
journal = {Cell host &amp; microbe},
volume = {27},
number = {1},
pages = {115-128.e8},
doi = {10.1016/j.chom.2019.12.006},
pmid = {31917956},
issn = {1934-6069},
mesh = {Animals ; Antimicrobial Cationic Peptides/genetics/metabolism ; CRISPR-Cas Systems ; Carrier Proteins/*metabolism ; Dicistroviridae/genetics/*immunology/pathogenicity ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/genetics/*immunology ; Gene Knockdown Techniques ; Immunity, Innate ; RNA Interference/immunology ; *RNA, Long Noncoding/genetics/immunology/metabolism ; Signal Transduction ; Viral Proteins/metabolism ; Virulence Factors/metabolism ; },
abstract = {Antiviral immunity in insects is mediated by the RNA interference (RNAi) pathway. Viruses evade antiviral RNAi by expressing virulence factors known as viral suppressors of RNAi (VSR). Here, we report the identification of VINR, a Drosophila VSR-interacting long non-coding (lnc) RNA that activates non-canonical innate immune signaling upon detection of the dsRNA-binding VSR of Drosophila C virus (DCV). VINR is required for the induction of antimicrobial peptide (AMP) genes but dispensable for antiviral RNAi. VINR functions by preventing the ubiquitin proteasome-dependent degradation of Cactin, a coiled-coil and arginine-serine-rich domain-containing protein that regulates a non-cannonical antimicrobial pathway for AMP induction. CRISPR-Cas9 knockout of VINR in Drosophila cells enhances DCV replication independently of antiviral RNAi, and VINR-knockout adult flies exhibit enhanced disease susceptibility to DCV and bacteria. Our findings reveal a counter counter-defense strategy activated by a lncRNA in response to the viral suppression of the primary antiviral RNAi immunity.},
}
@article {pmid31917244,
year = {2020},
author = {Gui, S and Taning, CNT and Wei, D and Smagghe, G},
title = {First report on CRISPR/Cas9-targeted mutagenesis in the Colorado potato beetle, Leptinotarsa decemlineata.},
journal = {Journal of insect physiology},
volume = {121},
number = {},
pages = {104013},
doi = {10.1016/j.jinsphys.2020.104013},
pmid = {31917244},
issn = {1879-1611},
mesh = {Animals ; *CRISPR-Cas Systems ; Coleoptera/*genetics ; Gene Editing/*methods ; Gene Silencing ; Genome, Insect ; Insect Control ; Larva/growth &amp; development ; *Mutagenesis ; Pest Control ; RNA Interference ; },
abstract = {Leptinotarsa decemlineata (Say), commonly known as the Colorado potato beetle (CPB), is an agricultural important pest for potatoes and other solanaceous plants. The CRISPR/Cas system is an efficient genome editing technology, which could be exploited to study the biology of CPB and possibly also lead to the development of better environmentally friendly pest management strategies. However, the use of CRISPR/Cas9 has been limited to only a few model insects. Here, for the first time, a CRISPR/Cas9 protocol for mutagenesis studies in CPB was developed. A gene with a clear phenotype such as the vestigial gene (vest), known to be involved in wing development in other insect species, was selected as a good indicator for the knockout study. First, vest was functionally characterized in CPB by using RNAi technology for knockdown studies. Once the expected deformed wing phenotypes were observed, a CRISPR/Cas9 work flow was established for mutagenesis in CPB. By co-injecting the Cas9 protein and a vest-guide RNA into 539 CPB eggs of <1 h old, sixty-two successfully developed to adults, among which mutation in the vest loci was confirmed in 5 of the 18 wingless CPBs (29% phenotypic mutation efficiency). The mutation in vest resulted in a clear phenotype in the CPBs, which developed to adulthood with no hindwing and elytron formed. Altogether, this study provides for the first time a useful methodology involving the use of the CRISPR/Cas9 system for mutagenesis studies in one of the most important pest insects.},
}
@article {pmid31916673,
year = {2020},
author = {Wang, M and Xu, Z and Gosavi, G and Ren, B and Cao, Y and Kuang, Y and Zhou, C and Spetz, C and Yan, F and Zhou, X and Zhou, H},
title = {Targeted base editing in rice with CRISPR/ScCas9 system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {8},
pages = {1645-1647},
pmid = {31916673},
issn = {1467-7652},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Oryza/genetics ; },
}
@article {pmid31915817,
year = {2020},
author = {Ahmad, S and Wei, X and Sheng, Z and Hu, P and Tang, S},
title = {CRISPR/Cas9 for development of disease resistance in plants: recent progress, limitations and future prospects.},
journal = {Briefings in functional genomics},
volume = {19},
number = {1},
pages = {26-39},
doi = {10.1093/bfgp/elz041},
pmid = {31915817},
issn = {2041-2657},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/*immunology ; Disease Resistance/*immunology ; Gene Editing/*methods ; Genome, Plant ; Host-Pathogen Interactions/*immunology ; Plant Breeding/*methods ; Plant Diseases/*immunology ; },
abstract = {Several plant pathogens severely affect crop yield and quality, thereby threatening global food security. In order to cope with this challenge, genetic improvement of plant disease resistance is required for sustainable agricultural production, for which conventional breeding is unlikely to do enough. Luckily, genome editing systems that particularly clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has revolutionized crop improvement by enabling robust and precise targeted genome modifications. It paves the way towards new methods for genetic improvement of plant disease resistance and accelerates resistance breeding. In this review, the challenges, limitations and prospects for conventional breeding and the applications of CRISPR/Cas9 system for the development of transgene-free disease-resistant crops are discussed.},
}
@article {pmid31915386,
year = {2020},
author = {Si, X and Zhang, H and Wang, Y and Chen, K and Gao, C},
title = {Manipulating gene translation in plants by CRISPR-Cas9-mediated genome editing of upstream open reading frames.},
journal = {Nature protocols},
volume = {15},
number = {2},
pages = {338-363},
pmid = {31915386},
issn = {1750-2799},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Open Reading Frames/*genetics ; Plants/*genetics ; Protein Biosynthesis/*genetics ; *Transcription, Genetic ; },
abstract = {Gene expression is regulated by multiple processes, and the translation of mRNAs into proteins is an especially critical step. Upstream open reading frames (uORFs) are widespread cis-elements in eukaryotic genes that usually suppress the translation of downstream primary ORFs (pORFs). Here, we describe a protocol for fine-tuning gene translation in plants by editing endogenous uORFs with the CRISPR-Cas9 system. The method we present readily yields transgene-free uorf mutant offspring. We provide detailed protocols for predicting uORFs and testing their effects on downstream pORFs using a dual-luciferase reporter system, designing and constructing single guide RNA (sgRNA)-Cas9 vectors, identifying transgene-free uorf mutants, and finally comparing the mRNA, protein and phenotypic levels of target genes in uorf mutants and controls. Predicting uORFs and confirming their effects in protoplasts takes only 2-3 weeks, and transgene-free mutants with edited target uORFs controlling different levels of pORF translation can be obtained within 4 months. Unlike previous methods, our strategy achieves fine-tuning of gene translation in transgene-free derivatives, which accelerates the analysis of gene function and the improvement of crop traits.},
}
@article {pmid31915280,
year = {2020},
author = {Labeau, A and Simon-Loriere, E and Hafirassou, ML and Bonnet-Madin, L and Tessier, S and Zamborlini, A and Dupré, T and Seta, N and Schwartz, O and Chaix, ML and Delaugerre, C and Amara, A and Meertens, L},
title = {A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection.},
journal = {Journal of virology},
volume = {94},
number = {7},
pages = {},
pmid = {31915280},
issn = {1098-5514},
mesh = {Animals ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; Dengue/*virology ; Dengue Virus/*physiology ; Endoplasmic Reticulum/metabolism ; Fibroblasts/metabolism ; Glycoproteins/metabolism ; Glycosylation ; Glycosylphosphatidylinositols/metabolism ; HEK293 Cells ; Humans ; Mannose/chemistry ; Mannosyltransferases/*metabolism ; Oligosaccharides/chemistry ; RNA, Guide/metabolism ; RNA, Viral/chemistry ; Vero Cells ; Virus Replication ; },
abstract = {Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection.IMPORTANCE Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.},
}
@article {pmid31915253,
year = {2020},
author = {Kim, BH and Zhang, G},
title = {Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {3},
pages = {1029-1037},
pmid = {31915253},
issn = {2160-1836},
support = {R35 GM124913/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chromosomes ; Embryo, Nonmammalian ; Exons ; Gene Knockout Techniques ; Mitochondrial Proteins/genetics ; Mutation ; RNA, Guide ; Transcription Factors/genetics ; Ubiquitin-Protein Ligases/genetics ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F1 fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.},
}
@article {pmid31914687,
year = {2020},
author = {Liu, Z and Shan, H and Chen, S and Chen, M and Song, Y and Lai, L and Li, Z},
title = {Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {1},
pages = {588-596},
doi = {10.1096/fj.201901587R},
pmid = {31914687},
issn = {1530-6860},
mesh = {Adenine/chemistry ; Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cytosine/chemistry ; Embryo, Mammalian/cytology/*metabolism ; Fibroblast Growth Factor 5/genetics ; Gene Editing/*methods ; Homeodomain Proteins/genetics ; Models, Animal ; *Mutation ; Nucleotide Motifs/*genetics ; Rabbits ; Streptococcus pyogenes/genetics ; },
abstract = {Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits. Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases.},
}
@article {pmid31914674,
year = {2020},
author = {Watanabe, H and Higashimoto, K and Miyake, N and Morita, S and Horii, T and Kimura, M and Suzuki, T and Maeda, T and Hidaka, H and Aoki, S and Yatsuki, H and Okamoto, N and Uemura, T and Hatada, I and Matsumoto, N and Soejima, H},
title = {DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {1},
pages = {960-973},
pmid = {31914674},
issn = {1530-6860},
mesh = {CRISPR-Cas Systems ; Child ; Child, Preschool ; *DNA Methylation ; Enhancer Elements, Genetic ; Epigenome ; Female ; Gene Deletion ; Genomic Imprinting ; HEK293 Cells ; Histone-Lysine N-Methyltransferase/*genetics ; Histones/chemistry ; Humans ; Infant ; Infant, Newborn ; Insulin-Like Growth Factor II/*genetics ; Lysine/chemistry ; Male ; Phenotype ; Point Mutation ; Promoter Regions, Genetic ; Sotos Syndrome/*genetics ; },
abstract = {Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.},
}
@article {pmid31914652,
year = {2020},
author = {Roberston, MJ and Raghunathan, S and Potaman, VN and Zhang, F and Stewart, MD and McConnell, BK and Schwartz, RJ},
title = {CRISPR-Cas9-induced IGF1 gene activation as a tool for enhancing muscle differentiation via multiple isoform expression.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {1},
pages = {555-570},
pmid = {31914652},
issn = {1530-6860},
support = {R15 HL124458/HL/NHLBI NIH HHS/United States ; R15 HL141963/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Anti-Inflammatory Agents/pharmacology ; Base Sequence ; *CRISPR-Cas Systems ; *Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dexamethasone/pharmacology ; Humans ; Insulin-Like Growth Factor I/genetics/*metabolism ; Mice ; Muscle, Skeletal/*cytology/drug effects/metabolism ; Muscular Atrophy/chemically induced/metabolism/*pathology ; Myoblasts/*cytology/drug effects/metabolism ; Phosphorylation ; Protein Isoforms ; RNA, Messenger/genetics/metabolism ; Sequence Homology ; Signal Transduction ; *Transcriptional Activation ; },
abstract = {Muscle wasting, or muscle atrophy, can occur with age, injury, and disease; it affects the quality of life and complicates treatment. Insulin-like growth factor 1 (IGF1) is a key positive regulator of muscle mass. The IGF1/Igf1 gene encodes multiple protein isoforms that differ in tissue expression, potency, and function, particularly in cellular proliferation and differentiation, as well as in systemic versus localized signaling. Genome engineering is a novel strategy for increasing gene expression and has the potential to recapitulate the diverse biology seen in IGF1 signaling through the overexpression of multiple IGF1 isoforms. Using a CRISPR-Cas9 gene activation approach, we showed that the expression of multiple IGF1 or Igf1 mRNA variants can be increased in human and mouse skeletal muscle myoblast cell lines using a single-guide RNA (sgRNA). We found increased IGF1 protein levels in the cell culture media and increased cellular phosphorylation of AKT1, the main effector of IGF1 signaling. We also showed that the expression of Class 1 or Class 2 mRNA variants can be selectively increased by changing the sgRNA target location. The expression of multiple IGF1 or Igf1 mRNA transcript variants in human and mouse skeletal muscle myoblasts promoted myotube differentiation and prevented dexamethasone-induced atrophy in myotubes in vitro. Our findings suggest that this novel approach for enhancing IGF1 signaling has potential therapeutic applications for treating skeletal muscle atrophy.},
}
@article {pmid31914418,
year = {2020},
author = {Ramachandran, A and Summerville, L and Learn, BA and DeBell, L and Bailey, S},
title = {Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {11},
pages = {3403-3414},
pmid = {31914418},
issn = {1083-351X},
support = {R01 GM097330/GM/NIGMS NIH HHS/United States ; T32 CA009110/CA/NCI NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Proteins/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Polymerase III/metabolism ; Escherichia coli/*enzymology/*genetics ; Escherichia coli Proteins/metabolism ; Exonucleases/*metabolism ; Nucleotide Motifs/genetics ; Sequence Analysis, DNA ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3' overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3'-5' exonucleases DnaQ and ExoT can trim long 3' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3'-5' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.},
}
@article {pmid31914394,
year = {2020},
author = {Kondo, S and Takahashi, T and Yamagata, N and Imanishi, Y and Katow, H and Hiramatsu, S and Lynn, K and Abe, A and Kumaraswamy, A and Tanimoto, H},
title = {Neurochemical Organization of the Drosophila Brain Visualized by Endogenously Tagged Neurotransmitter Receptors.},
journal = {Cell reports},
volume = {30},
number = {1},
pages = {284-297.e5},
doi = {10.1016/j.celrep.2019.12.018},
pmid = {31914394},
issn = {2211-1247},
mesh = {Animals ; Brain/*diagnostic imaging ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/genetics/*metabolism ; Ethanol/adverse effects ; Gene Expression Regulation ; Genes, Reporter ; Receptors, Dopamine/metabolism ; Receptors, Neurotransmitter/*metabolism ; },
abstract = {Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. Here we describe a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, we profile the expression of 75 neurotransmitter receptors in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, we convert T2A-GAL4 into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. Our resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain.},
}
@article {pmid31914134,
year = {2020},
author = {Yamazaki, H and Shirakawa, K and Matsumoto, T and Kazuma, Y and Matsui, H and Horisawa, Y and Stanford, E and Sarca, AD and Shirakawa, R and Shindo, K and Takaori-Kondo, A},
title = {APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.},
journal = {PloS one},
volume = {15},
number = {1},
pages = {e0223463},
pmid = {31914134},
issn = {1932-6203},
mesh = {Antibodies, Monoclonal, Humanized/pharmacology ; Bortezomib/pharmacology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Nucleus/genetics ; Cytidine Deaminase/*genetics ; DNA Damage/*genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Green Fluorescent Proteins/genetics ; Humans ; Lenalidomide/pharmacology ; Minor Histocompatibility Antigens/*genetics ; Multiple Myeloma/drug therapy/*genetics/pathology/radiotherapy ; Mutation/genetics ; Phosphorylcholine/analogs &amp; derivatives/pharmacology ; Polymethacrylic Acids/pharmacology ; RNA, Small Interfering/genetics ; Radiation ; Signal Transduction/drug effects ; },
abstract = {Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.},
}
@article {pmid31913359,
year = {2020},
author = {Wang, B and Xu, W and Yang, H},
title = {Structural basis of a Tn7-like transposase recruitment and DNA loading to CRISPR-Cas surveillance complex.},
journal = {Cell research},
volume = {30},
number = {2},
pages = {185-187},
pmid = {31913359},
issn = {1748-7838},
mesh = {Bacterial Proteins/chemistry/metabolism ; CRISPR-Cas Systems/*genetics ; DNA/*metabolism ; Models, Molecular ; Transposases/*chemistry/metabolism ; Vibrio cholerae/metabolism ; },
}
@article {pmid31913120,
year = {2020},
author = {Hickman, AB and Kailasan, S and Genzor, P and Haase, AD and Dyda, F},
title = {Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {31913120},
issn = {2050-084X},
support = {S10 RR028976/RR/NCRR NIH HHS/United States ; },
mesh = {Archaeal Proteins/*chemistry/genetics/metabolism ; CRISPR-Associated Proteins/chemistry/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/*genetics/metabolism ; DNA Transposable Elements ; DNA, Archaeal/chemistry/genetics/metabolism ; DNA, Intergenic ; Methanosarcina/*enzymology/genetics ; Models, Molecular ; Nucleic Acid Conformation ; Protein Conformation ; Protein Multimerization ; Transposases/*chemistry/genetics/*metabolism ; },
abstract = {Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.},
}
@article {pmid31912450,
year = {2020},
author = {Farooq, R and Hussain, K and Tariq, M and Farooq, A and Mustafa, M},
title = {CRISPR/Cas9: targeted genome editing for the treatment of hereditary hearing loss.},
journal = {Journal of applied genetics},
volume = {61},
number = {1},
pages = {51-65},
pmid = {31912450},
issn = {2190-3883},
mesh = {Animals ; Biomarkers ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing ; Gene Expression Regulation ; Gene Transfer Techniques ; Genetic Diseases, Inborn/*genetics/*therapy ; Genetic Predisposition to Disease ; Genetic Therapy ; Genetic Vectors/genetics ; Hair Cells, Auditory/physiology ; Hearing Loss/*genetics/*therapy ; Humans ; Mice ; Protein Binding ; RNA Interference ; Regeneration ; Ribonucleoproteins/metabolism ; Transfection/methods ; Treatment Outcome ; },
abstract = {Hereditary hearing loss (HHL) is a neurosensory disorder that affects every 1/500 newborns worldwide and nearly 1/3 people over the age of 65. Congenital deafness is inherited as monogenetic or polygenic disorder. The delicacy, tissue heterogeneity, deep location of the inner ear down the brainstem, and minute quantity of cells present in cochlea are the major challenges for current therapeutic approaches to cure deafness. Targeted genome editing is considered a suitable approach to treat HHL since it can target defective molecular components of auditory transduction to restore normal cochlear function. With the advent of CRISPR/Cas9 technique, targeted genome editing and biomedical research have been revolutionized. The robustness and simplicity of this technology lie in its design and delivery methods. It can directly deliver a complex of Cas9 endonuclease and single guide RNA (sgRNA) into zygote using either vector-mediated stable transfection or transient delivery of ribonucleoproteins complexes. This strategy induces DNA double strand breaks (DSBs) at target site followed by endogenous DNA repairing mechanisms of the cell. CRISPR/Cas9 has been successfully used in model animals to edit hearing genes like calcium and integrin-binding protein 2, myosin VIIA, Xin-actin binding repeat containing 2, leucine-zipper and sterile-alpha motif kinase Zak, epiphycan, transmembrane channel-like protein 1, and cadherin 23. This review discusses the utility of lipid-mediated transient delivery of Cas9/sgRNA complexes, an efficient way to restore hearing in humans, suffering from HHL. Notwithstanding, challenges like PAM requirement, HDR efficiency, off-target activity, and optimized delivery systems need to be addressed.},
}
@article {pmid31911736,
year = {2019},
author = {Eissenberg, JC},
title = {The Brave New World of Gene Editing and Molecular Medicine.},
journal = {Missouri medicine},
volume = {116},
number = {6},
pages = {497-502},
pmid = {31911736},
issn = {0026-6620},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics ; Genetic Therapy/*ethics ; Humans ; Molecular Medicine/trends ; },
abstract = {Gene therapy has long been a promise of molecular biology. So far, that promise has largely been unrealized. The advent of gene editing using technology adapted from bacteria may finally usher in the era of gene therapy.},
}
@article {pmid31911695,
year = {2020},
author = {Ledford, H},
title = {Quest to use CRISPR against disease gains ground.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {156},
pmid = {31911695},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *HIV Infections ; Humans ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Stem Cells ; },
}
@article {pmid31911671,
year = {2020},
author = {Wang, L and Li, L and Ma, Y and Hu, H and Li, Q and Yang, Y and Liu, W and Yin, S and Li, W and Fu, B and Kurita, R and Nakamura, Y and Liu, M and Lai, Y and Li, D},
title = {Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies.},
journal = {Cell research},
volume = {30},
number = {3},
pages = {276-278},
pmid = {31911671},
issn = {1748-7838},
support = {81670470//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Hematopoietic Stem Cells ; *Hemoglobinopathies/genetics/therapy ; Humans ; Promoter Regions, Genetic ; *gamma-Globins/genetics/metabolism ; },
}
@article {pmid31911609,
year = {2020},
author = {Munck, C and Sheth, RU and Freedberg, DE and Wang, HH},
title = {Recording mobile DNA in the gut microbiota using an Escherichia coli CRISPR-Cas spacer acquisition platform.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {95},
pmid = {31911609},
issn = {2041-1723},
support = {R01 AI132403/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Computational Biology ; DNA, Bacterial/*genetics/metabolism ; Escherichia coli/*genetics/metabolism ; Feces/microbiology ; *Gastrointestinal Microbiome ; Gene Transfer, Horizontal ; Humans ; *Interspersed Repetitive Sequences ; Plasmids/genetics/metabolism ; },
abstract = {The flow of genetic material between bacteria is central to the adaptation and evolution of bacterial genomes. However, our knowledge about DNA transfer within complex microbiomes is lacking, with most studies of horizontal gene transfer (HGT) relying on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers. Here, we utilize the CRISPR-Cas spacer acquisition process to detect DNA acquisition events from complex microbiota in real-time and at nucleotide resolution. In this system, an E. coli recording strain is exposed to a microbial sample and spacers are acquired from transferred plasmids and permanently stored in genomic CRISPR arrays. Sequencing and analysis of acquired spacers enables identification of the transferred plasmids. This approach allowed us to identify individual mobile elements without relying on phenotypic markers or post-transfer replication. We found that HGT into the recording strain in human clinical fecal samples can be extensive and is driven by different plasmid types, with the IncX type being the most actively transferred.},
}
@article {pmid31911131,
year = {2019},
author = {Li, J and Hong, S and Chen, W and Zuo, E and Yang, H},
title = {Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {11},
pages = {513-521},
doi = {10.1016/j.jgg.2019.11.002},
pmid = {31911131},
issn = {1673-8527},
mesh = {CRISPR-Associated Protein 9/genetics ; Chromatin Immunoprecipitation ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing ; Humans ; RNA, Guide/genetics ; Whole Genome Sequencing/methods ; },
abstract = {CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors. Currently, poor efficiency and off-target problems have impeded the application of CRISPR systems. The on-target efficiency has been improved in several advanced versions of CRISPR systems, whereas the off-target detection still remains a key challenge. Here, we outline the different versions of CRISPR systems and off-target detection strategies, discuss the merits and limitations of off-target detection methods, and provide potential implications for further gene editing research.},
}
@article {pmid31910234,
year = {2020},
author = {Alcaraz, E and Vilardell, J and Borgo, C and Sarró, E and Plana, M and Marin, O and Pinna, LA and Bayascas, JR and Meseguer, A and Salvi, M and Itarte, E and Ruzzene, M},
title = {Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells.},
journal = {PloS one},
volume = {15},
number = {1},
pages = {e0227340},
pmid = {31910234},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Casein Kinase II/*genetics ; Cell Line ; Chromones/pharmacology ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Enzymologic/drug effects ; Gene Knockout Techniques ; Glycogen Synthase Kinase 3 beta/*genetics ; Humans ; Kidney/drug effects/metabolism ; MAP Kinase Signaling System/drug effects ; Morpholines/pharmacology ; Naphthyridines/pharmacology ; Oncogene Protein v-akt/*genetics ; Phenazines ; Phosphatidylinositol 3-Kinases/genetics ; Phosphorylation/drug effects ; Protein Isoforms ; Ribosomal Protein S6 Kinases, 90-kDa/genetics ; Signal Transduction/drug effects ; Snail Family Transcription Factors/*genetics ; },
abstract = {The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells.},
}
@article {pmid31910089,
year = {2020},
author = {Tatineni, S and Stewart, LR and Sanfaçon, H and Wang, X and Navas-Castillo, J and Hajimorad, MR},
title = {Fundamental Aspects of Plant Viruses-An Overview on Focus Issue Articles.},
journal = {Phytopathology},
volume = {110},
number = {1},
pages = {6-9},
doi = {10.1094/PHYTO-10-19-0404-FI},
pmid = {31910089},
issn = {0031-949X},
mesh = {Plant Diseases/virology ; *Plant Pathology ; *Plant Viruses/physiology ; },
abstract = {Given the importance of and rapid research progress in plant virology in recent years, this Focus Issue broadly emphasizes advances in fundamental aspects of virus infection cycles and epidemiology. This Focus Issue comprises three review articles and 18 research articles. The research articles cover broad research areas on the identification of novel viruses, the development of detection methods, reverse genetics systems and functional genomics for plant viruses, vector and seed transmission studies, viral population studies, virus-virus interactions and their effect on vector transmission, and management strategies of viral diseases. The three review articles discuss recent developments in application of prokaryotic clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) technology for plant virus resistance, mixed viral infections and their role in disease synergism and cross-protection, and viral transmission by whiteflies. The following briefly summarizes the articles appearing in this Focus Issue.},
}
@article {pmid31909863,
year = {2020},
author = {Yagoubat, A and Crobu, L and Berry, L and Kuk, N and Lefebvre, M and Sarrazin, A and Bastien, P and Sterkers, Y},
title = {Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region preservation.},
journal = {Cellular microbiology},
volume = {22},
number = {5},
pages = {e13159},
doi = {10.1111/cmi.13159},
pmid = {31909863},
issn = {1462-5822},
mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Gene Knockout Techniques/*methods ; Integrases ; Leishmania/*genetics ; Proto-Oncogene Proteins c-crk/genetics ; Recombination, Genetic ; Transfection ; },
abstract = {Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.},
}
@article {pmid31907834,
year = {2019},
author = {Xia, Y and Huang, G and Zhu, Y},
title = {Sustainable plant disease control: biotic information flow and behavior manipulation.},
journal = {Science China. Life sciences},
volume = {62},
number = {12},
pages = {1710-1713},
doi = {10.1007/s11427-019-1599-y},
pmid = {31907834},
issn = {1869-1889},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Gene Silencing ; Genome ; Insecta/drug effects ; Insecticides/metabolism ; Pest Control, Biological ; Plant Development ; Plant Diseases/*genetics/microbiology/*prevention &amp; control ; Plant Proteins/*genetics/*metabolism ; RNA/metabolism ; },
}
@article {pmid31907374,
year = {2020},
author = {Lebar, T and Lainšček, D and Merljak, E and Aupič, J and Jerala, R},
title = {A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells.},
journal = {Nature chemical biology},
volume = {16},
number = {5},
pages = {513-519},
pmid = {31907374},
issn = {1552-4469},
support = {787115/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Genetic Engineering/*methods ; HEK293 Cells ; Humans ; Mammals/genetics ; Mice ; NIH 3T3 Cells ; Peptides/*genetics/*metabolism ; Recombinant Proteins/genetics/metabolism ; Transcription, Genetic ; },
abstract = {Protein interactions guide most cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR-dCas9-based transcriptional activators and to amplify the response of light and small molecule-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.},
}
@article {pmid31907146,
year = {2019},
author = {Chen, G and Cheng, D and Chen, B},
title = {[Development of CRISPR technology and its application in bone and cartilage tissue engineering].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {12},
pages = {1515-1520},
pmid = {31907146},
issn = {1673-4254},
mesh = {CRISPR-Cas Systems ; Cartilage ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide ; *Tissue Engineering ; },
abstract = {The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.},
}
@article {pmid31906943,
year = {2020},
author = {Yeo, WL and Heng, E and Tan, LL and Lim, YW and Ching, KC and Tsai, DJ and Jhang, YW and Lauderdale, TL and Shia, KS and Zhao, H and Ang, EL and Zhang, MM and Lim, YH and Wong, FT},
title = {Biosynthetic engineering of the antifungal, anti-MRSA auroramycin.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {3},
pmid = {31906943},
issn = {1475-2859},
mesh = {Anti-Bacterial Agents/*biosynthesis/chemistry/pharmacology ; Antifungal Agents/*chemistry/pharmacology ; Biosynthetic Pathways/*genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Metabolic Engineering/*methods ; Methicillin-Resistant Staphylococcus aureus/drug effects ; Microbial Sensitivity Tests ; Polyenes/chemistry ; Streptomyces/*genetics/metabolism ; },
abstract = {Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.},
}
@article {pmid31905010,
year = {2020},
author = {Zhang, K and Su, L and Wu, J},
title = {Recent Advances in Recombinant Protein Production by Bacillus subtilis.},
journal = {Annual review of food science and technology},
volume = {11},
number = {},
pages = {295-318},
doi = {10.1146/annurev-food-032519-051750},
pmid = {31905010},
issn = {1941-1421},
mesh = {Bacillus subtilis/*metabolism ; CRISPR-Cas Systems ; Gene Editing ; Proteomics ; Recombinant Proteins/biosynthesis ; },
abstract = {Bacillus subtilis has become a widely used microbial cell factory for the production of recombinant proteins, especially those associated with foods and food processing. Recent advances in genetic manipulation and proteomic analysis have been used to greatly improve protein production in B. subtilis. This review begins with a discussion of genome-editing technologies and application of the CRISPR-Cas9 system to B. subtilis. A summary of the characteristics of crucial legacy strains is followed by suggestions regarding the choice of origin strain for genetic manipulation. Finally, the review analyzes the genes and operons of B. subtilis that are important for the production of secretory proteins and provides suggestions and examples of how they can be altered to improve protein production. This review is intended to promote the engineering of this valuable microbial cell factory for better recombinant protein production.},
}
@article {pmid31904924,
year = {2020},
author = {Gamboa, L and Phung, EV and Li, H and Meyers, JP and Hart, AC and Miller, IC and Kwong, GA},
title = {Heat-Triggered Remote Control of CRISPR-dCas9 for Tunable Transcriptional Modulation.},
journal = {ACS chemical biology},
volume = {15},
number = {2},
pages = {533-542},
pmid = {31904924},
issn = {1554-8937},
support = {DP2 HD091793/HD/NICHD NIH HHS/United States ; T32 EB006343/EB/NIBIB NIH HHS/United States ; UL1 TR000454/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Chemokine CCL21/metabolism ; Genes, Switch ; Granzymes/metabolism ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; HSP70 Heat-Shock Proteins/genetics ; *Heating ; Herpes Simplex Virus Protein Vmw65/genetics ; Humans ; Kruppel-Like Transcription Factors/genetics ; Mice, Nude ; Protein Domains ; Recombinant Fusion Proteins/genetics ; Repressor Proteins/genetics ; Simplexvirus/chemistry ; Transcription, Genetic/physiology ; Transcriptional Activation/*physiology ; },
abstract = {CRISPR-associated proteins (Cas) are enabling powerful new approaches to control mammalian cell functions, yet the lack of spatially defined, noninvasive modalities limits their use as biological tools. Here, we integrate thermal gene switches with dCas9 complexes to confer remote control of gene activation and suppression with short pulses of heat. Using a thermal switch constructed from the heat shock protein A6 (HSPA6) locus, we show that a single heat pulse 3-5 °C above basal temperature is sufficient to trigger expression of dCas9 complexes. We demonstrate that dCas9 fused to the transcriptional activator VP64 is functional after heat activation, and, depending on the number of heat pulses, drives transcription of endogenous genes GzmB and CCL21 to levels equivalent to that achieved by a constitutive viral promoter. Across a range of input temperatures, we find that downstream protein expression of GzmB closely correlates with transcript levels (R[2] = 0.99). Using dCas9 fused with the transcriptional suppressor KRAB, we show that longitudinal suppression of the reporter d2GFP depends on key thermal input parameters including pulse magnitude, number of pulses, and dose fractionation. In living mice, we extend our study using photothermal heating to spatially target implanted cells to suppress d2GFP in vivo. Our study establishes a noninvasive and targeted approach to harness Cas-based proteins for modulation of gene expression to complement current methods for remote control of cell function.},
}
@article {pmid31904830,
year = {2020},
author = {Heo, J and Larner, JM and Brautigan, DL},
title = {Protein kinase CK2 phosphorylation of SAPS3 subunit increases PP6 phosphatase activity with Aurora A kinase.},
journal = {The Biochemical journal},
volume = {477},
number = {2},
pages = {431-444},
doi = {10.1042/BCJ20190740},
pmid = {31904830},
issn = {1470-8728},
support = {P30 CA044579/CA/NCI NIH HHS/United States ; },
mesh = {Alanine/genetics ; Amino Acid Substitution/genetics ; Aurora Kinase A/chemistry/*genetics ; CRISPR-Cas Systems/genetics ; Casein Kinase II/*chemistry/genetics ; Catalytic Domain/genetics ; Enzyme Inhibitors/pharmacology ; HeLa Cells ; Humans ; Phosphoprotein Phosphatases/antagonists &amp; inhibitors/chemistry/*genetics ; Phosphorylation/genetics ; Protein Binding/drug effects ; RNA, Small Interfering/genetics ; Substrate Specificity/drug effects ; },
abstract = {Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.},
}
@article {pmid31904282,
year = {2020},
author = {Isobe, K and Raghuram, V and Krishnan, L and Chou, CL and Yang, CR and Knepper, MA},
title = {CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.},
journal = {American journal of physiology. Renal physiology},
volume = {318},
number = {3},
pages = {F600-F616},
pmid = {31904282},
issn = {1522-1466},
support = {ZIA HL001285/ImNIH/Intramural NIH HHS/United States ; ZIA HL006129/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; Aquaporin 2/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Gene Deletion ; Gene Expression Regulation ; Mice ; Mutation ; Myosin-Light-Chain Kinase/genetics/*metabolism ; Protein Transport ; Proteomics/*methods ; },
abstract = {Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.},
}
@article {pmid31903905,
year = {2019},
author = {Beretta, M and Mouquet, H},
title = {[CRISPR-Cas9 editing of HIV-1 neutralizing human B cells].},
journal = {Medecine sciences : M/S},
volume = {35},
number = {12},
pages = {993-996},
doi = {10.1051/medsci/2019196},
pmid = {31903905},
issn = {1958-5381},
mesh = {Animals ; Antibodies, Neutralizing/*genetics/metabolism ; B-Lymphocytes/*immunology/*metabolism ; Bioengineering/methods/trends ; CRISPR-Cas Systems/*physiology ; Gene Editing/*methods ; HIV Infections/immunology/therapy ; HIV-1/*immunology ; Humans ; },
}
@article {pmid31903793,
year = {2020},
author = {Zlobin, NE and Lebedeva, MV and Taranov, VV},
title = {CRISPR/Cas9 genome editing through in planta transformation.},
journal = {Critical reviews in biotechnology},
volume = {40},
number = {2},
pages = {153-168},
doi = {10.1080/07388551.2019.1709795},
pmid = {31903793},
issn = {1549-7801},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant/*genetics ; Plants/*genetics ; Plants, Genetically Modified ; Transformation, Genetic ; },
abstract = {In this review, the application of CRISPR/Cas9 plant genome editing using alternative transformation methods is discussed. Genome editing by the CRISPR/Cas9 system is usually implemented via the generation of transgenic plants carrying Cas9 and sgRNA genes in the genome. Transgenic plants are usually developed by in vitro regeneration from single transformed cells, which requires using different in vitro culture-based methods. Despite their common application, these methods have some disadvantages and limitations. Thus, some methods of plant transformation that do not depend on in vitro regeneration have been developed. These methods are known as "in planta" transformation. The main focus of this review is the so-called floral dip in planta transformation method, although other approaches are also described. The main features of in planta transformation in the context of CRISPR/Cas9 genome editing are discussed. Furthermore, multiple ways to increase the effectiveness of this approach and to broaden its use in different plant species are considered.},
}
@article {pmid31903140,
year = {2020},
author = {Zhang, Q and Thakur, C and Fu, Y and Bi, Z and Wadgaonkar, P and Xu, L and Liu, Z and Liu, W and Wang, J and Kidder, BL and Chen, F},
title = {Mdig promotes oncogenic gene expression through antagonizing repressive histone methylation markers.},
journal = {Theranostics},
volume = {10},
number = {2},
pages = {602-614},
pmid = {31903140},
issn = {1838-7640},
mesh = {CRISPR-Cas Systems/genetics ; Cells, Cultured ; *DNA Methylation ; Dioxygenases/genetics/*metabolism ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques/methods ; Histone Demethylases/genetics/*metabolism ; Histones/*chemistry/genetics ; Humans ; Lung/metabolism/pathology ; Lung Neoplasms/genetics/metabolism/*pathology ; Nuclear Proteins/genetics/*metabolism ; *Oncogenes ; Promoter Regions, Genetic ; },
abstract = {The mineral dust-induced gene (mdig) is overexpressed in a number of human cancers, suggesting critical roles of this gene played on the pathogenesis of cancers. Unlike several other JmjC-domain containing proteins that exhibit histone demethylase activity, it remains enigmatic whether mdig is involved in the demethylation processes of the histone proteins. Methods: To provide direct evidence suggesting contribution of mdig to the demethylation of histone proteins, we recently examined the histone methylation profiles in human bronchial epithelial cells as well as two cancer cell lines with mdig knockout through CRISPR-Cas9 gene editing. Results: Global histone methylation analysis revealed a pronounced increase of the repressive histone trimethylation in three different cell types with mdig depletion, including trimethylation of lysines 9 and 27 on histone H3 (H3K9me3, H3K27me3) and trimethylation of lysine 20 of histone H4 (H4K20me3). Importantly, data from both ChIP-seq and RNA-seq suggested that genetic disruption of mdig enriches repressive histone trimethylation and inhibits expression of target genes in the oncogenic pathways of cell growth, stemness of the cells, tissue fibrosis, and cell motility. Conclusion: Taken together, our study provides the first insight into the molecular effects of mdig as an antagonist for repressive histone methylation markers and suggests that targeting mdig may represent a new area to explore in cancer therapy.},
}
@article {pmid31902585,
year = {2019},
author = {Liang, P and Wen, J and Huang, J},
title = {Off-target effects of cytidine base editor and adenine base editor: What can we do?.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {11},
pages = {509-512},
doi = {10.1016/j.jgg.2019.09.004},
pmid = {31902585},
issn = {1673-8527},
mesh = {Adenine/metabolism ; CRISPR-Cas Systems/genetics/physiology ; Cytidine/*metabolism ; Cytidine Deaminase/genetics/metabolism ; Gene Editing ; Guanine/metabolism ; Humans ; },
}
@article {pmid31902584,
year = {2019},
author = {Wu, J and Yin, H},
title = {Engineering guide RNA to reduce the off-target effects of CRISPR.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {11},
pages = {523-529},
doi = {10.1016/j.jgg.2019.11.003},
pmid = {31902584},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; RNA, Guide/*genetics ; },
abstract = {As versatile and robust genome editing tools, clustered regularly interspaced short palindromic repeats (CRISPR) technologies have been broadly used in basic research, biotechnology, and therapeutic development. Off-target mutagenesis by CRISPR systems has been demonstrated, and various methods have been developed to markedly increase their specificity. In this review, we highlight the efforts of producing and modifying guide RNA (gRNA) to minimize off-target activities, including sequence and structure design, tuning expression and chemical modification. The modalities of gRNA engineering can be applied across CRISPR systems. In conjunction with CRISPR protein effectors, the engineered gRNA enables efficient and precise genome editing.},
}
@article {pmid31902487,
year = {2020},
author = {Yin, H and Li, Z and Zhang, J and Huang, J and Kang, H and Tian, J and Qu, L},
title = {Construction of a US7/US8/UL23/US3-deleted recombinant pseudorabies virus and evaluation of its pathogenicity in dogs.},
journal = {Veterinary microbiology},
volume = {240},
number = {},
pages = {108543},
doi = {10.1016/j.vetmic.2019.108543},
pmid = {31902487},
issn = {1873-2542},
mesh = {Animals ; Antibodies, Viral/blood ; CRISPR-Cas Systems ; Dogs ; *Gene Deletion ; Herpesvirus 1, Suid/*genetics/growth &amp; development/*pathogenicity ; Pseudorabies/immunology/*virology ; Rabies Vaccines/administration &amp; dosage/genetics/*immunology ; Vaccination ; Vaccines, Attenuated/administration &amp; dosage/immunology ; Vaccines, Synthetic/administration &amp; dosage/immunology ; Viral Envelope Proteins/genetics ; Viral Proteins/*genetics ; Virulence ; },
abstract = {Since 2011, to control the spread of pseudorabies (PR), US7/US8/UL23-deleted recombinant PRV (rPRV) vaccines based on current variants have been developed. The vaccines can provide effective immune protection to pigs, but fur-bearing animals, such as dogs, foxes, and minks, are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs. It is suspected that the attenuated PRV vaccine strain is not safe for these fur-bearing animals. To confirm this, we construct a US7/US8/UL23-deleted and a US7/US8/UL23/US3-deleted rPRV based on PRV GL isolated from fox using the CRISPR/Cas9 method. Growth kinetics in vitro and pathogenicity in dogs were compared between the wild type and both rPRVs. The results showed that the growth kinetics of wild-type PRV and US7/US8/UL23-deleted rPRV were faster than those of US7/US8/UL23/US3-deleted recombinant PRV from 24 h to 48 h post infection. Moreover, PRV GL- and rPRV[delUS7/US8/UL23]-infected cells formed cell-cell fusion, but the rPRV[delUS7/US8/UL23/US3]-infected cells did not. Dogs challenged with wild-type PRV or US7/US8/UL23-deleted rPRV showed obvious nervous symptoms, and all the dogs died, but the group challenged with the US7/US8/UL23/US3-deleted rPRV did not show any nervous symptoms, and all the dogs survived for the duration of the experiment. Tissue viral load analyses also showed that the virulence of the US7/US8/UL23/US3-deleted rPRV was significantly reduced in dogs. This study provides evidence that the US7/US8/UL23-deleted rPRV variant still exhibits high virulence for dogs and also highlights the role of the US3 gene in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine.},
}
@article {pmid31902276,
year = {2020},
author = {Cheresiz, SV and Volgin, AD and Kokorina Evsyukova, A and Bashirzade, AAO and Demin, KA and de Abreu, MS and Amstislavskaya, TG and Kalueff, AV},
title = {Understanding neurobehavioral genetics of zebrafish.},
journal = {Journal of neurogenetics},
volume = {34},
number = {2},
pages = {203-215},
doi = {10.1080/01677063.2019.1698565},
pmid = {31902276},
issn = {1563-5260},
mesh = {Animals ; Animals, Genetically Modified ; Behavior, Animal ; CRISPR-Cas Systems ; Cell Lineage ; Central Nervous System Diseases/genetics ; Gene Editing/methods ; Gene Targeting/methods ; Genetics, Behavioral/*methods ; Models, Animal ; Neurosciences/*methods ; Quantitative Trait Loci ; RNA, Bacterial ; RNA, Small Interfering/genetics ; Reverse Genetics/methods ; Species Specificity ; Zebrafish/classification/*genetics ; Zebrafish Proteins/biosynthesis/genetics ; },
abstract = {Due to its fully sequenced genome, high genetic homology to humans, external fertilization, fast development, transparency of embryos, low cost and active reproduction, the zebrafish (Danio rerio) has become a novel promising model organism in biomedicine. Zebrafish are a useful tool in genetic and neuroscience research, including linking various genetic mutations to brain mechanisms using forward and reverse genetics. These approaches have produced novel models of rare genetic CNS disorders and common brain illnesses, such as addiction, aggression, anxiety and depression. Genetically modified zebrafish also foster neuroanatomical studies, manipulating neural circuits and linking them to different behaviors. Here, we discuss recent advances in neurogenetics of zebrafish, and evaluate their unique strengths, inherent limitations and the rapidly growing potential for elucidating the conserved roles of genes in neuropsychiatric disorders.},
}
@article {pmid31901956,
year = {2020},
author = {Levi, O and Arava, Y},
title = {Expanding the CRISPR/Cas9 Toolbox for Gene Engineering in S. cerevisiae.},
journal = {Current microbiology},
volume = {77},
number = {3},
pages = {468-478},
pmid = {31901956},
issn = {1432-0991},
mesh = {*CRISPR-Cas Systems ; Genetic Engineering/*methods ; Genetic Vectors ; *Genome, Fungal ; *Homologous Recombination ; Plasmids/*genetics ; RNA, Guide/genetics ; Saccharomyces cerevisiae/*genetics ; },
abstract = {The yeast S. cerevisiae serves as a model organism for many decades. Numerous molecular tools have been developed throughout the years to engineer its genome. Specifically, homologous recombination protocols allowed gene deletion, replacement and tagging of almost every S. cerevisiae gene, thus enabling mechanistic understanding of various cellular processes. Recently, CRISPR/Cas9-based approaches have been adapted to the yeast system, simplifying the protocols to manipulate this organism. In CRISPR/Cas9 systems, guide-RNA directs a site-specific double-strand DNA cleavage by the Cas9 nuclease. The directed cleavage enhances homologous recombination events, thereby facilitating changes to desired genomic loci. The use of a single vector to express both guide-RNA and Cas9 enzyme may simplify genomic manipulations and was used to introduce double-strand breaks at artificial sites (Anand et al. in Nature 544(7650):377-380, 2017. https://doi.org/10.1038/nature22046) or within selection markers (Ryan et al. in Cold Spring Harbor Protoc, 2014. https://doi.org/10.1101/pdb.prot086827). Here, we generalize this approach to demonstrate its utility in modifying natural genomic loci. We devise vectors to perform common genetic manipulations in S. cerevisiae, including gene deletion, single-base mutations, introduction of site-specific polymorphism and tag insertion. Notably, a vector that efficiently cleaves within GFP was generated, allowing replacing a GFP tag with other sequences. This vector may be of utility for replacing any gene tagged with GFP by a sequence of choice. Importantly, we demonstrate the efficiency of chemically synthesized 80-mer homologous DNA as a substrate for recombination, alleviating the need for PCR steps in the procedure. In all presented applications, high efficiency of the expected gene alteration and no other change in the genomic loci were obtained. Overall, this work expands the repertoire of single-plasmid CRISPR/cas9 approaches and provides a facile alternative to manipulate the yeast genome.},
}
@article {pmid31901860,
year = {2020},
author = {Rubio, A},
title = {Integration of CRISPR-Cas9, shRNA with other genomic data provides reliable predicions of gene essentiality.},
journal = {EBioMedicine},
volume = {51},
number = {},
pages = {102577},
pmid = {31901860},
issn = {2352-3964},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Damage/genetics ; DNA Repair/genetics ; *Genes, Essential ; *Genome ; Humans ; RNA, Small Interfering/*metabolism ; },
}
@article {pmid31901692,
year = {2020},
author = {Ryu, JY and Won, EJ and Lee, HAR and Kim, JH and Hui, E and Kim, HP and Yoon, TJ},
title = {Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy.},
journal = {Biomaterials},
volume = {232},
number = {},
pages = {119736},
doi = {10.1016/j.biomaterials.2019.119736},
pmid = {31901692},
issn = {1878-5905},
mesh = {Alopecia/genetics/therapy ; Animals ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {Compared to a plasmid, viral, and other delivery systems, direct Cas9/sgRNA protein delivery has several advantages such as low off-targeting effects and non-integration, but it still has limitations due to low transfer efficiency. As such, the CRISPR/Cas9 system is being developed in combination with nano-carrier technology to enhance delivery efficiency and biocompatibility. We designed a microbubble-nanoliposomal particle as a Cas9/sgRNA riboprotein complex carrier, which effectively facilitates local delivery to a specific site when agitated by ultrasound activation. In practice, we successfully transferred the protein constructs into dermal papilla cells in the hair follicle of androgenic alopecia animals by microbubble cavitation induced sonoporation of our particle. The delivered Cas9/sgRNA recognized and edited specifically the target gene with high efficiency in vitro and in vivo, thus recovering hair growth. We demonstrated the topical application of ultrasound-activated nanoparticles for androgenic alopecia therapy through the suppression of SRD5A2 protein production by CRISPR-based genomic editing.},
}
@article {pmid31901636,
year = {2020},
author = {Broeders, M and Herrero-Hernandez, P and Ernst, MPT and van der Ploeg, AT and Pijnappel, WWMP},
title = {Sharpening the Molecular Scissors: Advances in Gene-Editing Technology.},
journal = {iScience},
volume = {23},
number = {1},
pages = {100789},
pmid = {31901636},
issn = {2589-0042},
abstract = {The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate gene expression by targeting transcription factors or epigenetic machineries to DNA; or to target and modify RNA. Endogenous repair mechanisms are used to make the modifications required in DNA; they include non-homologous end joining, homology-directed repair, homology-independent targeted integration, microhomology-mediated end joining, base-excision repair, and mismatch repair. Off-target effects can be monitored using in silico prediction and sequencing and minimized using Cas proteins with higher accuracy, such as high-fidelity Cas9, enhanced-specificity Cas9, and hyperaccurate Cas9. Alternatives to Cas9 have been identified, including Cpf1, Cas12a, Cas12b, and smaller Cas9 orthologs such as CjCas9. Delivery of gene-editing components is performed ex vivo using standard techniques or in vivo using AAV, lipid nanoparticles, or cell-penetrating peptides. Clinical development of gene-editing technology is progressing in several fields, including immunotherapy in cancer treatment, antiviral therapy for HIV infection, and treatment of genetic disorders such as β-thalassemia, sickle cell disease, lysosomal storage disorders, and retinal dystrophy. Here we review these technological advances and the challenges to their clinical implementation.},
}
@article {pmid31901522,
year = {2020},
author = {Chevallereau, A and Meaden, S and Fradet, O and Landsberger, M and Maestri, A and Biswas, A and Gandon, S and van Houte, S and Westra, ER},
title = {Exploitation of the Cooperative Behaviors of Anti-CRISPR Phages.},
journal = {Cell host &amp; microbe},
volume = {27},
number = {2},
pages = {189-198.e6},
pmid = {31901522},
issn = {1934-6069},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Bacteria/genetics ; Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Directed Molecular Evolution ; Host Microbial Interactions ; Viral Proteins/genetics/metabolism ; },
abstract = {Bacteriophages encoding anti-CRISPR proteins (Acrs) must cooperate to overcome phage resistance mediated by the bacterial immune system CRISPR-Cas, where the first phage blocks CRISPR-Cas immunity in order to allow a second Acr phage to successfully replicate. However, in nature, bacteria are frequently not pre-immunized, and phage populations are often not clonal, exhibiting variations in Acr presence and strength. We explored how interactions between Acr phages and initially sensitive bacteria evolve, both in the presence and absence of competing phages lacking Acrs. We find that Acr phages benefit "Acr-negative" phages by limiting the evolution of CRISPR-based resistance and helping Acr-negative phages to replicate on resistant host sub-populations. These benefits depend on the strength of CRISPR-Cas inhibitors and result in strong Acrs providing smaller fitness advantages than weaker ones when Acr phages compete with Acr-negative phages. These results indicate that different Acr types shape the evolutionary dynamics and social interactions of phage populations in natural communities.},
}
@article {pmid31900489,
year = {2020},
author = {Wang, J and Xiang, X and Bolund, L and Zhang, X and Cheng, L and Luo, Y},
title = {GNL-Scorer: a generalized model for predicting CRISPR on-target activity by machine learning and featurization.},
journal = {Journal of molecular cell biology},
volume = {12},
number = {11},
pages = {909-911},
pmid = {31900489},
issn = {1759-4685},
mesh = {Bayes Theorem ; CRISPR-Cas Systems/*genetics ; Humans ; *Machine Learning ; *Models, Genetic ; RNA, Guide/genetics ; Regression Analysis ; *Software ; },
}
@article {pmid31900423,
year = {2020},
author = {Maggio, I and Zittersteijn, HA and Wang, Q and Liu, J and Janssen, JM and Ojeda, IT and van der Maarel, SM and Lankester, AC and Hoeben, RC and Gonçalves, MAFV},
title = {Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components.},
journal = {Gene therapy},
volume = {27},
number = {5},
pages = {209-225},
pmid = {31900423},
issn = {1476-5462},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Genetic Vectors/genetics ; RNA, Guide/genetics ; },
abstract = {Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.},
}
@article {pmid31900392,
year = {2020},
author = {Wang, SR and Wu, LY and Huang, HY and Xiong, W and Liu, J and Wei, L and Yin, P and Tian, T and Zhou, X},
title = {Conditional control of RNA-guided nucleic acid cleavage and gene editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {91},
pmid = {31900392},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Endonucleases/metabolism ; *Gene Editing ; HeLa Cells ; Humans ; RNA/genetics/*metabolism ; RNA, Guide/genetics/*metabolism ; },
abstract = {Prokaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.},
}
@article {pmid31900288,
year = {2020},
author = {Agudelo, D and Carter, S and Velimirovic, M and Duringer, A and Rivest, JF and Levesque, S and Loehr, J and Mouchiroud, M and Cyr, D and Waters, PJ and Laplante, M and Moineau, S and Goulet, A and Doyon, Y},
title = {Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.},
journal = {Genome research},
volume = {30},
number = {1},
pages = {107-117},
pmid = {31900288},
issn = {1549-5469},
support = {P41 GM103311/GM/NIGMS NIH HHS/United States ; R01 GM129325/GM/NIGMS NIH HHS/United States ; //CIHR/Canada ; },
mesh = {Animals ; CRISPR-Associated Protein 9/chemistry/*metabolism ; *CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; *Gene Editing ; Humans ; Mammals ; Mice ; Mice, Knockout ; Streptococcus thermophilus/*enzymology/*genetics ; Structure-Activity Relationship ; Substrate Specificity ; },
abstract = {Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.},
}
@article {pmid31900175,
year = {2020},
author = {Xu, H and Fang, T and Omran, RP and Whiteway, M and Jiang, L},
title = {RNA sequencing reveals an additional Crz1-binding motif in promoters of its target genes in the human fungal pathogen Candida albicans.},
journal = {Cell communication and signaling : CCS},
volume = {18},
number = {1},
pages = {1},
pmid = {31900175},
issn = {1478-811X},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Candida albicans/*genetics/*pathogenicity ; Fungal Proteins/*genetics/metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Fungal ; *Genes, Fungal ; Humans ; Models, Biological ; Mutation/genetics ; Nucleotide Motifs/*genetics ; Phenotype ; *Promoter Regions, Genetic ; Protein Binding ; *Sequence Analysis, RNA ; Transcriptome/genetics ; },
abstract = {BACKGROUND: The calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in Candida albicans, but it has not been experimentally demonstrated.<br><br>METHODS: An inactivation mutant for CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2&#x2009;M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively.<br><br>RESULTS: RNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5'-GGAGGC(G/A)C(T/A)G-3'], which is different from the putative CaCrz1-binding motif [5'-G(C/T)GGT-3'] identified in the previous study, but similar to Saccharomyces cerevisiae ScCrz1-binding motif [5'-GNGGC(G/T)CA-3']. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression.<br><br>CONCLUSIONS: CaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5'-TGAGGGACTG-3' site in its promoter. Video abstract.},
}
@article {pmid31899623,
year = {2020},
author = {Kopniczky, MB and Canavan, C and McClymont, DW and Crone, MA and Suckling, L and Goetzmann, B and Siciliano, V and MacDonald, JT and Jensen, K and Freemont, PS},
title = {Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology.},
journal = {ACS synthetic biology},
volume = {9},
number = {1},
pages = {144-156},
doi = {10.1021/acssynbio.9b00437},
pmid = {31899623},
issn = {2161-5063},
support = {BB/M025632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Binding Sites ; CRISPR-Associated Protein 9/chemistry/genetics ; CRISPR-Cas Systems ; Cell Engineering/*methods ; Cell-Free System ; Escherichia coli/genetics ; Gene Regulatory Networks ; Genetic Engineering/*methods ; HeLa Cells ; Humans ; Internal Ribosome Entry Sites/genetics ; Plasmids/genetics ; Promoter Regions, Genetic ; Protein Biosynthesis/*genetics ; Reproducibility of Results ; Synthetic Biology/methods ; Transcription, Genetic/genetics ; },
abstract = {The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.},
}
@article {pmid31898344,
year = {2020},
author = {Wang, H and Zhu, N and Ye, X and Wang, L and Wang, B and Shan, W and Lai, X and Tan, Y and Fu, S and Xiao, H and Huang, H},
title = {PTPN21-CDSlong isoform inhibits the response of acute lymphoblastic leukemia cells to NK-mediated lysis via the KIR/HLA-I axis.},
journal = {Journal of cellular biochemistry},
volume = {121},
number = {5-6},
pages = {3298-3312},
doi = {10.1002/jcb.29601},
pmid = {31898344},
issn = {1097-4644},
mesh = {CRISPR-Cas Systems ; Cell Death ; Cell Line, Tumor ; Cytotoxicity, Immunologic/immunology ; Gene Editing ; *Gene Expression Regulation, Leukemic ; Histocompatibility Antigens Class I/immunology ; Humans ; Killer Cells, Natural/immunology/metabolism ; Molecular Chaperones/metabolism ; Phosphoric Monoester Hydrolases/metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*metabolism ; Protein Isoforms ; Protein Tyrosine Phosphatases, Non-Receptor/*chemistry/*metabolism ; RNA-Seq ; },
abstract = {Protein tyrosine phosphatase non-receptor type 21 (PTPN21) is a member of the non-receptor tyrosine phosphatase family. We have found that PTPN21 is mutated in relapsed Philadelphia chromosome-negative acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation. PTPN21 consists of three types of isoforms according to the length of the protein encoded. However, the roles of different isoforms in leukemic cells have not been elucidated. In the study, PTPN21 isoform constitution in five ALL cell lines were identified by transcriptome polymerase chain reaction combined with Sanger sequencing, and the relationship between PTPN21 isoforms and sensitivity to natural killer (NK) cells mediated killing in ALL cell lines were further assessed by knock-out of different isoforms of PTPN21 using CRISPR-Cas9 technique. Subsequently, we explored the functional mechanisms through RNA sequencing and confirmatory testing. The results showed that there was no significant change when all PTPN21 isoforms were knocked out in ALL cells, but the sensitivity of NALM6 cells with PTPN21-CDSlong knock-out (NALM6-PTPN21lk) to NK-mediated killing was significantly increased. Whole transcriptome sequencing and further validation testing showed that human leukocyte antigen class I (HLA-I) molecules were significantly decreased, accompanied by a significantly downregulated expression of antigen presenting-related chaperones in NALM6-PTPN21lk cells. Our results uncovered a previously unknown mechanism that PTPN21-CDSlong and CDSshort isoforms may play opposite roles in NK-mediated killing in ALL cells, and showed that the endogenous PTPN21-CDSlong isoform inhibited ALL cells to NK cell-mediated lysis by regulating the KIR-HLA-I axis.},
}
@article {pmid31897558,
year = {2020},
author = {Zhao, X and Zhang, W and Qiu, X and Mei, Q and Luo, Y and Fu, W},
title = {Rapid and sensitive exosome detection with CRISPR/Cas12a.},
journal = {Analytical and bioanalytical chemistry},
volume = {412},
number = {3},
pages = {601-609},
doi = {10.1007/s00216-019-02211-4},
pmid = {31897558},
issn = {1618-2650},
mesh = {A549 Cells ; Aptamers, Nucleotide/*chemistry ; Biosensing Techniques/*methods ; CRISPR-Cas Systems ; Exosomes/*chemistry/pathology ; Humans ; Lung Neoplasms/pathology ; Nucleic Acid Amplification Techniques/methods ; Tetraspanin 30/*analysis ; },
abstract = {Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 10[3]-6 × 10[7] particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.},
}
@article {pmid31896785,
year = {2020},
author = {Broniewski, JM and Meaden, S and Paterson, S and Buckling, A and Westra, ER},
title = {The effect of phage genetic diversity on bacterial resistance evolution.},
journal = {The ISME journal},
volume = {14},
number = {3},
pages = {828-836},
pmid = {31896785},
issn = {1751-7370},
support = {714478/ERC_/European Research Council/International ; BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Archaea/genetics ; Bacteria/genetics/*immunology/*virology ; Bacteriophages/classification/*genetics/physiology ; Biological Evolution ; CRISPR-Cas Systems ; Genetic Variation ; Pseudomonas aeruginosa/genetics/immunology/virology ; },
abstract = {CRISPR-Cas adaptive immune systems are found in bacteria and archaea and provide defence against phage by inserting phage-derived sequences into CRISPR loci on the host genome to provide sequence specific immunological memory against re-infection. Under laboratory conditions the bacterium Pseudomonas aeruginosa readily evolves the high levels of CRISPR-based immunity against clonal populations of its phage DMS3vir, which in turn causes rapid extinction of the phage. However, in nature phage populations are likely to be more genetically diverse, which could theoretically impact the frequency at which CRISPR-based immunity evolves which in turn can alter phage persistence over time. Here we experimentally test these ideas and found that a smaller proportion of infected bacterial populations evolved CRISPR-based immunity against more genetically diverse phage populations, with the majority of the population evolving a sm preventing phage adsorption and providing generalised defence against a broader range of phage genotypes. However, those cells that do evolve CRISPR-based immunity in response to infection with more genetically diverse phage acquire greater numbers of CRISPR memory sequences in order to resist a wider range of phage genotypes. Despite differences in bacterial resistance evolution, the rates of phage extinction were similar in the context of clonal and diverse phage infections suggesting selection for CRISPR-based immunity or sm-based resistance plays a relatively minor role in the ecological dynamics in this study. Collectively, these data help to understand the drivers of CRISPR-based immunity and their consequences for bacteria-phage coexistence, and, more broadly, when generalised defences will be favoured over more specific defences.},
}
@article {pmid31895004,
year = {2020},
author = {Bennett, HW and Gustavsson, AK and Bayas, CA and Petrov, PN and Mooney, N and Moerner, WE and Jackson, PK},
title = {Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.},
journal = {Molecular biology of the cell},
volume = {31},
number = {7},
pages = {619-639},
pmid = {31895004},
issn = {1939-4586},
support = {R35 GM118067/GM/NIGMS NIH HHS/United States ; R01 GM121565/GM/NIGMS NIH HHS/United States ; R01 GM114276/GM/NIGMS NIH HHS/United States ; K99 GM134187/GM/NIGMS NIH HHS/United States ; T32 AG047126/AG/NIA NIH HHS/United States ; },
mesh = {Biomarkers/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cilia/*metabolism ; Green Fluorescent Proteins/metabolism ; Humans ; *Imaging, Three-Dimensional ; Kinesins/metabolism ; *Microscopy ; Models, Biological ; Mutation/genetics ; NIMA-Related Kinases/metabolism ; Nuclear Proteins/metabolism ; Protein Transport ; *Single Molecule Imaging ; Transcription Factors/*metabolism ; },
abstract = {Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.},
}
@article {pmid31894108,
year = {2019},
author = {Chen, Z and Song, Z and Yang, J and Huang, J and Jiang, H},
title = {Sp7/osterix positively regulates dlx2b and bglap to affect tooth development and bone mineralization in zebrafish larvae.},
journal = {Journal of biosciences},
volume = {44},
number = {6},
pages = {},
pmid = {31894108},
issn = {0973-7138},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calcification, Physiologic/genetics ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/genetics ; Humans ; Larva/genetics/growth &amp; development ; Osteocalcin/genetics ; Osteogenesis/*genetics ; Sp7 Transcription Factor/*genetics ; Tooth/growth &amp; development ; Transcription Factors/genetics ; Zebrafish/*genetics/growth &amp; development ; Zebrafish Proteins/*genetics ; },
abstract = {Osterix (or Sp7) is an important transcription factor that promotes osteoblast differentiation by modulating the expression of a range of target genes. Although many studies have focused on Osterix/Sp7 regulatory mechanisms, the detailed functions have not been fully elucidated. Toward this end, in this study, we used CRISPR/Cas9 technology to knock out the zebrafish sp7 gene, and then analyzed its phenotype and biological function. Two knockout sp7 mutant lines were successfully obtained. The bone mineralization level was significantly reduced in the zebrafish sp7-/- homozygote, resulting in abnormal tooth development in the larvae. Quantitative real-time polymerase chain reaction showed that loss of sp7 led to down-regulated expression of the dlx2b and bglap genes related to tooth development and bone mineralization, respectively. Moreover, cell transfection experiments demonstrated that Sp7 directly regulates the expression of dlx2b and bglap through Sp7-binding sites on the promoter regions of these two genes. Overall, this study provides new insight into the role of Sp7 in bone mineralization and tooth development.},
}
@article {pmid31893521,
year = {2020},
author = {Chen, M and Luo, D},
title = {A CRISPR Path to Cutting-Edge Materials.},
journal = {The New England journal of medicine},
volume = {382},
number = {1},
pages = {85-88},
doi = {10.1056/NEJMcibr1911506},
pmid = {31893521},
issn = {1533-4406},
mesh = {*Bacterial Proteins ; *Biosensing Techniques ; *CRISPR-Associated Protein 9 ; *CRISPR-Associated Proteins ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Cleavage ; *Endodeoxyribonucleases ; Gene Editing/*methods ; Humans ; Hydrogels ; },
}
@article {pmid31893458,
year = {2020},
author = {Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2107},
number = {},
pages = {349-364},
doi = {10.1007/978-1-0716-0235-5_19},
pmid = {31893458},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Cotyledon/genetics/*growth &amp; development ; Gene Editing/*methods ; Mutation ; Plant Breeding ; Soybeans/genetics/*growth &amp; development ; Tissue Culture Techniques ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.},
}
@article {pmid31892993,
year = {2020},
author = {Yamaguchi, H and Suzuki, S and Osana, Y and Kawachi, M},
title = {Genomic Characteristics of the Toxic Bloom-Forming Cyanobacterium Microcystis aeruginosa NIES-102.},
journal = {Journal of genomics},
volume = {8},
number = {},
pages = {1-6},
pmid = {31892993},
issn = {1839-9940},
abstract = {Microcystis aeruginosa, a bloom-forming cyanobacterium distributed mainly in freshwater environments, can be divided into at least 12 groups (A-K and X) based on multi-locus phylogenetic analyses. In this study, we characterized the genome of microcystin-producing M. aeruginosa NIES-102, assigned to group A, isolated from Lake Kasumigaura, Japan. The complete genome sequence of M. aeruginosa NIES-102 comprised a 5.87-Mbp circular chromosome containing 5,330 coding sequences. The genome was the largest among all sequenced genomes for the species. In a comparison with the genome of M. aeruginosa NIES-843, which belongs to the same group, the microcystin biosynthetic gene cluster and CRISPR-Cas locus were highly similar. A synteny analysis revealed small-scale rearrangements between the two genomes. Genes encoding transposases were more abundant in these two genomes than in other Microcystis genomes. Our results improve our understanding of structural genomic changes and adaptation to a changing environment in the species.},
}
@article {pmid31891587,
year = {2019},
author = {Foronda, M and Tarumoto, Y and Schatoff, EM and Leach, BI and Diaz, BJ and Zimmerman, J and Goswami, S and Shusterman, M and Vakoc, CR and Dow, LE},
title = {Tankyrase inhibition sensitizes cells to CDK4 blockade.},
journal = {PloS one},
volume = {14},
number = {12},
pages = {e0226645},
pmid = {31891587},
issn = {1932-6203},
support = {F31 CA224800/CA/NCI NIH HHS/United States ; K22 CA181280/CA/NCI NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; R01 CA195787/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cellular Senescence ; Colorectal Neoplasms/*enzymology/therapy ; Cyclin-Dependent Kinase 4/*genetics ; Cyclin-Dependent Kinase 6/genetics ; Drug Resistance, Neoplasm/*genetics ; G1 Phase Cell Cycle Checkpoints ; Humans ; Mutation ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use ; Tankyrases/*antagonists &amp; inhibitors ; Wnt Signaling Pathway/drug effects ; },
abstract = {Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.},
}
@article {pmid31891494,
year = {2020},
author = {Fan, J and Liu, Y and Liu, L and Huang, Y and Li, X and Huang, W},
title = {A Multifunction Lipid-Based CRISPR-Cas13a Genetic Circuit Delivery System for Bladder Cancer Gene Therapy.},
journal = {ACS synthetic biology},
volume = {9},
number = {2},
pages = {343-355},
doi = {10.1021/acssynbio.9b00349},
pmid = {31891494},
issn = {2161-5063},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Editing ; *Genetic Therapy ; Humans ; Infrared Rays ; Liposomes/chemistry/*metabolism ; Mice ; Mice, Nude ; Photothermal Therapy ; Plasmids/genetics/metabolism ; Transfection ; Urinary Bladder Neoplasms/pathology/therapy ; Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism ; },
abstract = {The treatment of bladder cancer has recently shown minimal progress. Gene therapy mediated by CRISPR provides a new option for bladder cancer treatment. In this study, we developed a versatile liposome system to deliver the CRISPR-Cas13a gene circuits into bladder cancer cells. After in vitro studies and intravesical perfusion studies in mice, this system showed five advantages: (1) CRISPR-Cas13a, a transcriptional targeting and cleavage tool for gene expression editing, did not affect the stability of the cell genome; (2) the prepared liposome systems were targeted to hVEGFR2, which is always highly expressed in bladder cancer cells; (3) the CRISPR-Cas13a sequence was driven by an artificial tumor specific promoter to achieve further targeting; (4) a near-infrared photosensitizer released using near-infrared light was introduced to control the delivery system; and (5) the plasmids were constructed with three crRNA tandem sequences to achieve multiple targeting and wider therapeutic results. This tumor cell targeting lipid delivery system with near-infrared laser-controlled ability provided a versatile strategy for CRISPR-Cas13a based gene therapy of bladder cancer.},
}
@article {pmid31890588,
year = {2020},
author = {Walker, JE and Lanahan, AA and Zheng, T and Toruno, C and Lynd, LR and Cameron, JC and Olson, DG and Eckert, CA},
title = {Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.},
journal = {Metabolic engineering communications},
volume = {10},
number = {},
pages = {e00116},
pmid = {31890588},
issn = {2214-0301},
abstract = {The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.},
}
@article {pmid31890142,
year = {2020},
author = {Liu, G and Zhang, Y and Zhang, T},
title = {Computational approaches for effective CRISPR guide RNA design and evaluation.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {35-44},
pmid = {31890142},
issn = {2001-0370},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/ CRISPR-associated (Cas) system has emerged as the main technology for gene editing. Successful editing by CRISPR requires an appropriate Cas protein and guide RNA. However, low cleavage efficiency and off-target effects hamper the development and application of CRISPR/Cas systems. To predict cleavage efficiency and specificity, numerous computational approaches have been developed for scoring guide RNAs. Most scores are empirical or trained by experimental datasets, and scores are implemented using various computational methods. Herein, we discuss these approaches, focusing mainly on the features or computational methods they utilise. Furthermore, we summarise these tools and give some suggestions for their usage. We also recommend three versatile web-based tools with user-friendly interfaces and preferable functions. The review provides a comprehensive and up-to-date overview of computational approaches for guide RNA design that could help users to select the optimal tools for their research.},
}
@article {pmid31890021,
year = {2019},
author = {Liu, Q and Zhang, Y and Li, F and Li, J and Sun, W and Tian, C},
title = {Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila.},
journal = {Biotechnology for biofuels},
volume = {12},
number = {},
pages = {293},
pmid = {31890021},
issn = {1754-6834},
abstract = {BACKGROUND: Thermophilic filamentous fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR-Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes.<br><br>RESULTS: In this study, we developed an Acidaminococcus sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in M. thermophila. These CRISPR-Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR-Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in M. thermophila via three transformation rounds, using two selectable markers neo and bar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR-Cas9 system and found the two technologies were complementary. Together we called them CRISPR-Cas-assisted marker recycling technology (Camr technology).<br><br>CONCLUSIONS: Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR-Cas12a system or the established CRISPR-Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.},
}
@article {pmid31889638,
year = {2019},
author = {Mao, S and Liu, Y and Huang, S and Huang, X and Chi, T},
title = {Site-directed RNA editing (SDRE): Off-target effects and their countermeasures.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {11},
pages = {531-535},
doi = {10.1016/j.jgg.2019.11.005},
pmid = {31889638},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; RNA Editing/*genetics ; RNA, Guide/*genetics ; },
abstract = {Site-directed RNA editing (SDRE) is invaluable to basic research and clinical applications and has emerged as a new frontier in genome editing. The past few years have witnessed a surge of interest in SDRE, with SDRE tools emerging at a breathtaking pace. However, off-target effects of SDRE remain a tough problem, which constitutes a major hurdle to their clinical applications. Here we discuss the diverse strategies for combating off-target editing, drawing lessons from the published studies as well as our ongoing research. Overall, SDRE is still at its infancy, with significant challenges and exciting opportunities ahead.},
}
@article {pmid31889332,
year = {2020},
author = {Borowicz, P and Chan, H and Medina, D and Gumpelmair, S and Kjelstrup, H and Spurkland, A},
title = {A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9.},
journal = {Scandinavian journal of immunology},
volume = {91},
number = {4},
pages = {e12862},
doi = {10.1111/sji.12862},
pmid = {31889332},
issn = {1365-3083},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Humans ; Jurkat Cells ; Mutation ; *T-Lymphocytes ; *Workflow ; },
abstract = {CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.},
}
@article {pmid31888773,
year = {2019},
author = {Gomez-Giro, G and Arias-Fuenzalida, J and Jarazo, J and Zeuschner, D and Ali, M and Possemis, N and Bolognin, S and Halder, R and Jäger, C and Kuper, WFE and van Hasselt, PM and Zaehres, H and Del Sol, A and van der Putten, H and Schöler, HR and Schwamborn, JC},
title = {Synapse alterations precede neuronal damage and storage pathology in a human cerebral organoid model of CLN3-juvenile neuronal ceroid lipofuscinosis.},
journal = {Acta neuropathologica communications},
volume = {7},
number = {1},
pages = {222},
pmid = {31888773},
issn = {2051-5960},
support = {FNR/PoC16/11559169//Fonds National de la Recherche Luxembourg/International ; Aides &#xe0; la Formation-Recherche//Fonds National de la Recherche Luxembourg/International ; },
mesh = {CRISPR-Cas Systems ; Cerebral Cortex/*growth &amp; development/*pathology ; Endothelial Cells/pathology ; Humans ; Induced Pluripotent Stem Cells/physiology ; Lysosomes/pathology ; Membrane Glycoproteins/*genetics ; Molecular Chaperones/*genetics ; Mutation ; Neuronal Ceroid-Lipofuscinoses/*genetics/*pathology ; Neurons/*pathology ; Organoids ; Synapses/*pathology ; },
abstract = {The juvenile form of neuronal ceroid Lipofuscinosis (JNCL) is the most common form within this group of rare lysosomal storage disorders, causing pediatric neurodegeneration. The genetic disorder, which is caused by recessive mutations affecting the CLN3 gene, features progressive vision loss, cognitive and motor decline and other psychiatric conditions, seizure episodes, leading to premature death. Animal models have traditionally aid the understanding of the disease mechanisms and pathology and are very relevant for biomarker research and therapeutic testing. Nevertheless, there is a need for establishing reliable and predictive human cellular models to study the disease. Since patient material, particularly from children, is scarce and difficult to obtain, we generated an engineered a CLN3-mutant isogenic human induced pluripotent stem cell (hiPSC) line carrying the c.1054C → T pathologic variant, using state of the art CRISPR/Cas9 technology. To prove the suitability of the isogenic pair to model JNCL, we screened for disease-specific phenotypes in non-neuronal two-dimensional cell culture models as well as in cerebral brain organoids. Our data demonstrates that the sole introduction of the pathogenic variant gives rise to classical hallmarks of JNCL in vitro. Additionally, we discovered an alteration of the splicing caused by this particular mutation. Next, we derived cerebral organoids and used them as a neurodevelopmental model to study the particular effects of the CLN3[Q352X] mutation during brain formation in the disease context. About half of the mutation -carrying cerebral organoids completely failed to develop normally. The other half, which escaped this severe defect were used for the analysis of more subtle alterations. In these escapers, whole-transcriptome analysis demonstrated early disease signatures, affecting pathways related to development, corticogenesis and synapses. Complementary metabolomics analysis confirmed decreased levels of cerebral tissue metabolites, some particularly relevant for synapse formation and neurotransmission, such as gamma-amino butyric acid (GABA). Our data suggests that a mutation in CLN3 severely affects brain development. Furthermore, before disease onset, disease -associated neurodevelopmental changes, particular concerning synapse formation and function, occur.},
}
@article {pmid31888216,
year = {2019},
author = {Yoshimura, T and Nakamura, K and Li, C and Fujisawa, M and Shiina, T and Imamura, M and Li, T and Mukaida, N and Matsukawa, A},
title = {Cancer Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Is Dispensable for the Progression of 4T1 Murine Breast Cancer.},
journal = {International journal of molecular sciences},
volume = {20},
number = {24},
pages = {},
pmid = {31888216},
issn = {1422-0067},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Clone Cells ; *Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics/*metabolism ; Granulocytes/metabolism ; Macrophages/metabolism ; Mammary Neoplasms, Animal/genetics/*metabolism/*pathology ; Mice, Inbred BALB C ; Neoplasm Proteins/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Splenomegaly/pathology ; },
abstract = {We previously reported that 4T1 murine breast cancer cells produce GM-CSF that up-regulates macrophage expression of several cancer promoting genes, including Mcp-1/Ccl2, Ccl17 and Rankl, suggesting a critical role of cancer cell-derived GM-CSF in cancer progression. Here, we attempted to define whether 4T1 cell-derived GM-CSF contributes to the expression of these genes by 4T1tumors, and their subsequent progression. Intraperitoneal injection of anti-GM-CSF neutralizing antibody did not decrease the expression of Mcp-1, Ccl17 or Rankl mRNA by 4T1 tumors. To further examine the role of cancer cell-derived GM-CSF, we generated GM-CSF-deficient 4T1 cells by using the Crisper-Cas9 system. As previously demonstrated, 4T1 cells are a mixture of cells and cloning of cells by itself significantly reduced tumor growth and lung metastasis. By contrast, GM-CSF-deficiency did not affect tumor growth, lung metastasis or the expression of these chemokine and cytokine genes in tumor tissues. By in-situ hybridization, the expression of Mcp-1 mRNA was detected in both F4/80-expressing and non-expressing cells in tumors of GM-CSF-deficient cells. These results indicate that cancer cell-derived GM-CSF is dispensable for the tuning of the 4T1 tumor microenvironment and the production of MCP-1, CCL17 or RANKL in the 4T1 tumor microenvironment is likely regulated by redundant mechanisms.},
}
@article {pmid31885264,
year = {2020},
author = {East, KW and Newton, JC and Morzan, UN and Narkhede, YB and Acharya, A and Skeens, E and Jogl, G and Batista, VS and Palermo, G and Lisi, GP},
title = {Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {3},
pages = {1348-1358},
pmid = {31885264},
issn = {1520-5126},
support = {P20 GM109035/GM/NIGMS NIH HHS/United States ; R01 EY027440/EY/NEI NIH HHS/United States ; },
mesh = {Allosteric Regulation ; *CRISPR-Cas Systems ; Deoxyribonucleases/metabolism ; *Molecular Dynamics Simulation ; Nuclear Magnetic Resonance, Biomolecular/*methods ; },
abstract = {CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.},
}
@article {pmid31885201,
year = {2019},
author = {Serçin, &#xd6; and Reither, S and Roidos, P and Ballin, N and Palikyras, S and Baginska, A and Rein, K and Llamazares, M and Halavatyi, A and Winter, H and Muley, T and Jurkowska, RZ and Abdollahi, A and Zenke, FT and Neumann, B and Mardin, BR},
title = {A solid-phase transfection platform for arrayed CRISPR screens.},
journal = {Molecular systems biology},
volume = {15},
number = {12},
pages = {e8983},
pmid = {31885201},
issn = {1744-4292},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*instrumentation ; Genetic Predisposition to Disease ; High-Throughput Screening Assays ; Humans ; Neoplasms/*genetics ; Phenotype ; RNA, Guide/*pharmacology ; Transfection ; },
abstract = {Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high-throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.},
}
@article {pmid31885035,
year = {2019},
author = {Hasan, A and Yeom, HS and Ryu, J and Bode, HB and Kim, Y},
title = {Phenylethylamides derived from bacterial secondary metabolites specifically inhibit an insect serotonin receptor.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {20358},
pmid = {31885035},
issn = {2045-2322},
mesh = {Amino Acid Sequence ; Animals ; Bacteria/classification/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression ; Hemocytes/metabolism ; Immunity, Cellular ; Insect Proteins/*antagonists &amp; inhibitors ; Larva ; Phagocytosis/genetics/immunology ; Phenethylamines/chemistry/*pharmacology ; Phylogeny ; RNA Interference ; Receptors, Serotonin/genetics/*metabolism ; Secondary Metabolism ; Serotonin Antagonists/chemistry/*pharmacology ; },
abstract = {Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Furthermore, knockout mutant of Se-5HTR by CRISPR-Cas9 led to significant reduction of phagocytotic activity of S. exigua hemocytes. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives.},
}
@article {pmid31884594,
year = {2019},
author = {Pendse, ND and Lamas, V and Pawlyk, BS and Maeder, ML and Chen, ZY and Pierce, EA and Liu, Q},
title = {In Vivo Assessment of Potential Therapeutic Approaches for USH2A-Associated Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1185},
number = {},
pages = {91-96},
doi = {10.1007/978-3-030-27378-1_15},
pmid = {31884594},
issn = {0065-2598},
mesh = {Animals ; CRISPR-Cas Systems ; Exons ; Extracellular Matrix Proteins/*genetics ; Humans ; Membrane Proteins/genetics ; Mice ; Mice, Knockout ; Mutation ; Usher Syndromes/genetics/*therapy ; },
abstract = {Mutations in USH2A gene account for most cases of Usher syndrome type II (USH2), characterized by a combination of congenital hearing loss and progressive vision loss. In particular, approximately 30% of USH2A patients harbor a single base pair deletion, c.2299delG, in exon 13 that creates a frameshift and premature stop codon, leading to a nonfunctional USH2A protein. The USH2A protein, also known as usherin, is an extremely large transmembrane protein (5202 aa), which limits the use of conventional AAV-mediated gene therapy; thus development of alternative approaches is required for the treatment of USH2A patients. As usherin contains multiple repetitive domains, we hypothesize that removal of one or more of those domains encoded by mutant exon(s) in the USH2A gene may reconstitute the reading frame and restore the production of a shortened yet adequately functional protein. In this study, we deleted the exon 12 of mouse Ush2a gene (corresponding to exon 13 of human USH2A) using CRISPR/Cas9-based exon-skipping approach and revealed that a shortened form of Ush2a that lacks exon 12 (Ush2a-∆Ex12) is produced and localized correctly in the cochlea. When the Ush2a-∆Ex12 allele is expressed on an Ush2a null background, the Ush2a-∆Ex12 protein can successfully restore the impaired hair cell structure and the auditory function in the Ush2a[-/-] mice. These results demonstrate that CRISPR/Cas9-based exon-skipping strategy holds a great therapeutic potential for the treatment of USH2A patients.},
}
@article {pmid31884373,
year = {2020},
author = {Nur Patria, Y and Lilianty, J and Elefanty, AG and Stanley, EG and Labonne, T and Bateman, JF and Lamandé, SR},
title = {Generation of a SOX9-tdTomato reporter human iPSC line, MCRIi001-A-2, using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {42},
number = {},
pages = {101689},
doi = {10.1016/j.scr.2019.101689},
pmid = {31884373},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Middle Aged ; SOX9 Transcription Factor/*genetics ; Transfection ; },
abstract = {To develop an iPSC SOX9 reporter line for monitoring differentiation into SOX9 expressing cells such as chondrocytes, cranial neural crest and Sertoli cells, we used gene editing to introduce sequences encoding the tdTomato fluorescent protein into the SOX9 locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. Endogenous SOX9 expression was undisturbed and the tdTomato fluorescent reporter mirrored SOX9 mRNA expression. This iPSC line will be useful for assessing iPSC differentiation into SOX9-expressing cells and enrichment by cell sorting.},
}
@article {pmid31884054,
year = {2020},
author = {van Leeuwe, TM and Gerritsen, A and Arentshorst, M and Punt, PJ and Ram, AFJ},
title = {Rab GDP-dissociation inhibitor gdiA is an essential gene required for cell wall chitin deposition in Aspergillus niger.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {136},
number = {},
pages = {103319},
doi = {10.1016/j.fgb.2019.103319},
pmid = {31884054},
issn = {1096-0937},
mesh = {Aspergillus niger/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Chitin/*metabolism ; Fungal Proteins/*genetics ; Gene Deletion ; Gene Editing ; Gene Expression Regulation, Fungal ; *Genes, Essential ; Genome, Fungal ; Genotyping Techniques ; Guanine Nucleotide Dissociation Inhibitors/*genetics ; Polymorphism, Single Nucleotide ; RNA Splicing/genetics ; rab GTP-Binding Proteins/genetics/metabolism ; },
abstract = {The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5' donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.},
}
@article {pmid31883579,
year = {2020},
author = {Wu, H and He, JS and Zhang, F and Ping, J and Wu, J},
title = {Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: Rapid, specific and sensitive.},
journal = {Analytica chimica acta},
volume = {1096},
number = {},
pages = {130-137},
doi = {10.1016/j.aca.2019.10.042},
pmid = {31883579},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; Colorimetry ; DNA Primers/genetics ; Nucleic Acid Amplification Techniques ; Plants, Genetically Modified/*genetics ; *Promoter Regions, Genetic ; RNA, Guide/genetics ; Soybeans/*genetics ; Zea mays/*genetics ; },
abstract = {An ultrafast and convenient method for visually detecting CaMV35S promoter amplicon (amplified products) was established by using CRISPR/Cas12a system coupled with a designed reaction vessel. Genetically modified (GM) soybean (Roundup Ready®) powders containing CaMV35S promoter were employed as detection targets, which were amplified by loop-mediated isothermal amplification (LAMP). The CRISPR/Cas12a system directly mixed with amplified products at 37 °C for 5 min and detection results could be clearly identified by the naked eye under UV light (254 nm). A designed reaction vessel was employed to make operation easier and could effectively prevent contamination at the source. The CRISPR/Cas12a detection system was optimized in our study and the concentration of magnesium ions was proved to be important for the work of CRISPR/Cas12a system. The optimized concentration range of magnesium ions was between 10 mM and 12 mM. Besides, the activated Bst DNA polymerase also had little effects on CRISPR/Cas12a system. The developed method could significantly distinguish the specific and non-specific amplification. And as low as 0.05% transgenic contents in soybean powders could be detected. It would have the potential to be complementary to instrument-based ultrahigh sensitive method and provide a new solution for on-site rapid detection.},
}
@article {pmid31883145,
year = {2020},
author = {Xiong, Q and Xie, C and Zhang, Z and Liu, L and Powell, JT and Shen, Q and Lin, C},
title = {DNA Origami Post-Processing by CRISPR-Cas12a.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {59},
number = {10},
pages = {3956-3960},
pmid = {31883145},
issn = {1521-3773},
support = {DP2 GM114830/GM/NIGMS NIH HHS/United States ; R01 GM132114/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/*metabolism ; CRISPR-Associated Proteins/chemistry/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/*metabolism ; Endodeoxyribonucleases/chemistry/*metabolism ; },
abstract = {Customizable nanostructures built through the DNA-origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting-edge tools for DNA-origami design and preparation, it remains challenging to separate structural components of an architecture built from-thus held together by-a continuous scaffold strand, which in turn limits the modularity and function of the DNA-origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA-origami structures. We target single-stranded (ss) regions of DNA-origami structures and remove them with CRISPR-Cas12a, a hyper-active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post-processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a-like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.},
}
@article {pmid31883094,
year = {2020},
author = {Leist, SR and Cockrell, AS},
title = {Genetically Engineering a Susceptible Mouse Model for MERS-CoV-Induced Acute Respiratory Distress Syndrome.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2099},
number = {},
pages = {137-159},
pmid = {31883094},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Coronavirus Infections/pathology/*virology ; Dipeptidyl Peptidase 4/*genetics/metabolism ; *Disease Models, Animal ; Disease Susceptibility ; Female ; Genetic Engineering ; Humans ; Lung/virology ; Male ; Mice/*genetics ; Mice, Inbred C57BL ; Middle East Respiratory Syndrome Coronavirus/*physiology ; Respiratory Distress Syndrome/pathology/*virology ; },
abstract = {Since 2012, monthly cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. The ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with MERS-CoV in human infections. The inability of MERS-CoV to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. Identification of human dipeptidyl peptidase IV (hDPP4) as the receptor for MERS-CoV infection opened the door for genetic engineering of mice. Precise molecular engineering of mouse DPP4 (mDPP4) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology maintained inherent expression profiles, and limited MERS-CoV susceptibility to tissues that naturally express mDPP4, notably the lower respiratory tract wherein MERS-CoV elicits severe pulmonary pathology. Here, we describe the generation of the 288-330[+/+] MERS-CoV mouse model in which mice were made susceptible to MERS-CoV by modifying two amino acids on mDPP4 (A288 and T330), and the use of adaptive evolution to generate novel MERS-CoV isolates that cause fatal respiratory disease. The 288-330[+/+] mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for MERS-CoV. The chapter starts with a historical perspective on the emergence of MERS-CoV and animal models evaluated for MERS-CoV pathogenesis, and then outlines the development of the 288-330[+/+] mouse model, assays for assessing a MERS-CoV pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice.},
}
@article {pmid31882978,
year = {2019},
author = {Hara, S and Terao, M and Muramatsu, A and Takada, S},
title = {Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {20202},
pmid = {31882978},
issn = {2045-2322},
mesh = {*Alleles ; Animals ; *CRISPR-Cas Systems ; DNA Methylation ; Founder Effect ; Mice ; *Mosaicism ; *Mutation ; },
abstract = {Generation of mutant imprinting control region (ICR) mice using genome editing is an important approach for elucidating ICR functions. IG-DMR is an ICR in the Dlk1-Dio3 imprinted domain that contains functional regions-in both parental alleles-that are essential for embryonic development. One drawback of this approach is that embryonic lethality can occur from aberrant expression of the imprinted genes if IG-DMR gets mutated in either the paternal or maternal allele. To overcome this problem, we generated mosaic mice that contained cells with modified IG-DMR alleles and wild-type cells using the 2CC method that allowed for microinjection of the CRISPR/Cas9 constructs into a blastomere of 2-cell embryos. This method improved the birth rate of the founder pups relative to that obtained using the standard protocol. We also successfully produced mosaic mice in which the tandem repeat array sequence in the IG-DMR had been replaced by homology directed repair. Additionally, paternal transmission of the replaced allele caused aberrant expression of the imprinted genes due to hypomethylation of the IG-DMR, indicating that the replaced allele recapitulated our deletion model. Our results indicate that this method is useful for the generation of mutant mice in which a genomic locus essential for normal development has been genetically edited.},
}
@article {pmid31882637,
year = {2019},
author = {Banakar, R and Eggenberger, AL and Lee, K and Wright, DA and Murugan, K and Zarecor, S and Lawrence-Dill, CJ and Sashital, DG and Wang, K},
title = {High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {19902},
pmid = {31882637},
issn = {2045-2322},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; DNA Fragmentation ; Oryza/*genetics ; Plasmids/*genetics ; RNA, Plant/*genetics ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.},
}
@article {pmid31882627,
year = {2019},
author = {Ferrara, M and Haidukowski, M and Logrieco, AF and Leslie, JF and Mulè, G},
title = {A CRISPR-Cas9 System for Genome Editing of Fusarium proliferatum.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {19836},
pmid = {31882627},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Fumonisins/*metabolism ; Fungal Proteins/genetics/metabolism ; Fusarium/*genetics/metabolism ; Gene Deletion ; Gene Editing/*methods ; Mutation ; Polyketide Synthases/genetics/metabolism ; Reproducibility of Results ; },
abstract = {Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR-Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.},
}
@article {pmid31882576,
year = {2019},
author = {Heffel, MG and Finnigan, GC},
title = {Mathematical modeling of self-contained CRISPR gene drive reversal systems.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {20050},
pmid = {31882576},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Drive Technology ; Humans ; Models, Theoretical ; },
abstract = {There is a critical need for further research into methods to control biological populations. Numerous challenges to agriculture, ecological systems, and human health could be mitigated by the targeted reduction and management of key species (e.g. pests, parasites, and vectors for pathogens). The discovery and adaptation of the CRISPR/Cas editing platform co-opted from bacteria has provided a mechanism for a means to alter an entire population. A CRISPR-based gene drive system can allow for the forced propagation of a genetic element that bypasses Mendelian inheritance which can be used to bias sex determination, install exogenous information, or remove endogenous DNA within an entire species. Laboratory studies have demonstrated the potency by which gene drives can operate within insects and other organisms. However, continued research and eventual application face serious opposition regarding issues of policy, biosafety, effectiveness, and reversal. Previous mathematical work has suggested the use of modified gene drive designs that are limited in spread such as daisy chain or underdominance drives. However, no system has yet been proposed that allows for an inducible reversal mechanism without requiring the introduction of additional individuals. Here, we study gene drive effectiveness, fitness, and inducible drive systems that could respond to external stimuli expanding from a previous frequency-based population model. We find that programmed modification during gene drive propagation could serve as a potent safeguard to either slow or completely reverse drive systems and allow for a return to the original wild-type population.},
}
@article {pmid31882406,
year = {2020},
author = {Kandul, NP and Liu, J and Buchman, A and Gantz, VM and Bier, E and Akbari, OS},
title = {Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {2},
pages = {827-837},
pmid = {31882406},
issn = {2160-1836},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Drive Technology ; Gene Editing ; *Gene Knockout Techniques ; Gene Order ; *Gene Targeting/methods ; Genetic Vectors/genetics ; Genotyping Techniques ; Models, Genetic ; Mutation ; RNA, Guide ; Zygote/metabolism ; },
abstract = {Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.},
}
@article {pmid31882399,
year = {2020},
author = {Ianiri, G and Fang, YF and Dahlmann, TA and Clancey, SA and Janbon, G and Kück, U and Heitman, J},
title = {Mating-Type-Specific Ribosomal Proteins Control Aspects of Sexual Reproduction in Cryptococcus neoformans.},
journal = {Genetics},
volume = {214},
number = {3},
pages = {635-649},
pmid = {31882399},
issn = {1943-2631},
support = {R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; R37 AI039115/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Cryptococcus neoformans/*genetics/growth &amp; development ; Fungal Proteins/genetics ; Genes, Mating Type, Fungal/*genetics ; Haploidy ; Phenotype ; Reproduction/*genetics ; Ribosomal Proteins/*genetics ; },
abstract = {The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39 Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.},
}
@article {pmid31881433,
year = {2020},
author = {Mushtaq, M and Mukhtar, S and Sakina, A and Dar, AA and Bhat, R and Deshmukh, R and Molla, K and Kundoo, AA and Dar, MS},
title = {Tweaking genome-editing approaches for virus interference in crop plants.},
journal = {Plant physiology and biochemistry : PPB},
volume = {147},
number = {},
pages = {242-250},
doi = {10.1016/j.plaphy.2019.12.022},
pmid = {31881433},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems ; *Crops, Agricultural/virology ; *Gene Editing/methods ; *Genome, Plant ; *Plant Viruses/physiology ; Reproducibility of Results ; },
abstract = {Plant viruses infect various economically important crops and cause a serious threat to agriculture. As of now, conventional strategies employed are inadequate to circumvent the proliferation of rapidly evolving plant viruses. In this regard, recent advancement in genome-editing approach looks promising to produce plants resistant to DNA/RNA virus infections. Clustered regularly interspaced palindromic repeats (CRISPR) system has been emerged as a promising genome-editing tool that has received special interest because of its ease, competence and reproducibility. Recent studies have demonstrated that CRISPR/Cas9 system has great potential to confer plant immunity by either directly targeting or cleaving the viral genome in both RNA and DNA viruses. Similarly, the approach can be used for targeting the host susceptibility genes more particularly in case of RNA viruses. In the present review, different approaches and strategies being used to improve plant resistance against devastating viruses are discussed in view of recent advances in CRISPR systems. This review also describes the major pitfalls of CRISPR/Cas9 system that utilizes highly efficient and novel platforms to engineer interference to single and multiple plant RNA viruses.},
}
@article {pmid31880137,
year = {2019},
author = {Chen, X and Chen, L and Li, D},
title = {[Research progress of gene therapy in clinical application].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {12},
pages = {2295-2307},
doi = {10.13345/j.cjb.190363},
pmid = {31880137},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; *Genetic Therapy ; Humans ; },
abstract = {In the 1960s, scientists first raised the idea of curing genetic diseases using gene therapy. This new conceptual strategy aimed to achieve a much longer therapeutic effect by introducing exogenous genetic materials into the patients. After more than five decades of ups and downs, gene therapy has been brought into a new era by those milestone breakthroughs in the 21st century. Here we reviewed and summarized the history and breakthroughs of gene therapy, including some critical clinical trials, approved drugs, and emerging gene editing techniques. We believe that with their unique advantages over traditional therapies, more gene therapies will become practical approaches to genetic diseases and benefit the entire human race.},
}
@article {pmid31879968,
year = {2020},
author = {Carotenuto, P and Hedayat, S and Fassan, M and Cardinale, V and Lampis, A and Guzzardo, V and Vicentini, C and Scarpa, A and Cascione, L and Costantini, D and Carpino, G and Alvaro, D and Ghidini, M and Trevisani, F and Te Poele, R and Salati, M and Ventura, S and Vlachogiannis, G and Hahne, JC and Boulter, L and Forbes, SJ and Guest, RV and Cillo, U and Said-Huntingford, I and Begum, R and Smyth, E and Michalarea, V and Cunningham, D and Rimassa, L and Santoro, A and Roncalli, M and Kirkin, V and Clarke, P and Workman, P and Valeri, N and Braconi, C},
title = {Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.},
journal = {Hepatology (Baltimore, Md.)},
volume = {72},
number = {3},
pages = {982-996},
pmid = {31879968},
issn = {1527-3350},
support = {BRC to ICR/Marsden/DH_/Department of Health/United Kingdom ; CDF TO NV/CRUK_/Cancer Research UK/United Kingdom ; CRUK FUNDED CANCER THERAPEUTICS UNIT AT ICR/CRUK_/Cancer Research UK/United Kingdom ; PW is a CRUK Life Fellow/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Antineoplastic Agents/pharmacology ; *Biliary Tract Neoplasms/drug therapy/metabolism/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; *Cholangiocarcinoma/drug therapy/metabolism/pathology ; Cisplatin/*pharmacology ; Deoxycytidine/*analogs &amp; derivatives/pharmacology ; Drug Discovery ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; High-Throughput Screening Assays/methods ; Humans ; *MicroRNAs/antagonists &amp; inhibitors/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND AND AIMS: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy.<br><br>APPROACH AND RESULTS: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment.<br><br>CONCLUSIONS: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.},
}
@article {pmid31879772,
year = {2020},
author = {Pinilla-Redondo, R and Mayo-Muñoz, D and Russel, J and Garrett, RA and Randau, L and Sørensen, SJ and Shah, SA},
title = {Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids.},
journal = {Nucleic acids research},
volume = {48},
number = {4},
pages = {2000-2012},
pmid = {31879772},
issn = {1362-4962},
mesh = {Archaea/genetics ; Bacteria/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Evolution, Molecular ; Plasmids/*genetics ; },
abstract = {CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR-Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.},
}
@article {pmid31878068,
year = {2019},
author = {Chen, SJ and Wang, SC and Chen, YC},
title = {Antiviral Agents as Therapeutic Strategies Against Cytomegalovirus Infections.},
journal = {Viruses},
volume = {12},
number = {1},
pages = {},
pmid = {31878068},
issn = {1999-4915},
mesh = {Antiviral Agents/*therapeutic use ; CRISPR-Cas Systems ; Cytomegalovirus/*drug effects/physiology ; Cytomegalovirus Infections/complications/*drug therapy/therapy ; *Drug Resistance, Viral ; Humans ; Immunotherapy, Adoptive ; Viral Vaccines ; Virus Latency/*drug effects ; },
abstract = {Cytomegalovirus (CMV) is a threat to human health in the world, particularly for immunologically weak patients. CMV may cause opportunistic infections, congenital infections and central nervous system infections. CMV infections are difficult to treat due to their specific life cycles, mutation, and latency characteristic. Despite recent advances, current drugs used for treating active CMV infections are limited in their efficacy, and the eradication of latent infections is impossible. Current antiviral agents which target the UL54 DNA polymerase are restricted because of nephrotoxicity and viral resistance. CMV also cannot be prevented or eliminated with a vaccine. Fortunately, letermovir which targets the human CMV (HCMV) terminase complex has been recently approved to treat CMV infections in humans. The growing point is developing antiviral agents against both lytically and latently infected cells. The nucleic acid-based therapeutic approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) are being explored to remove acute and/or latent CMV infections. HCMV vaccine is being developed for prophylaxis. Additionally, adoptive T cell therapy (ACT) has been experimentally used to combate drug-resistant and recurrent CMV in patients after cell and/or organ transplantation. Developing antiviral agents is promising in this area to obtain fruitful outcomes and to have a great impact on humans for the therapy of CMV infections.},
}
@article {pmid31877894,
year = {2019},
author = {Bae, T and Kim, H and Kim, JH and Kim, YJ and Lee, SH and Ham, BJ and Hur, JK},
title = {Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences.},
journal = {Molecules (Basel, Switzerland)},
volume = {25},
number = {1},
pages = {},
pmid = {31877894},
issn = {1420-3049},
mesh = {CRISPR-Cas Systems ; DNA, Complementary/genetics ; DNA, Intergenic/genetics ; ErbB Receptors/genetics ; Gene Editing/*methods ; Humans ; *Point Mutation ; },
abstract = {In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.},
}
@article {pmid31877124,
year = {2019},
author = {Garrett, AM and Bosch, PJ and Steffen, DM and Fuller, LC and Marcucci, CG and Koch, AA and Bais, P and Weiner, JA and Burgess, RW},
title = {CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4.},
journal = {PLoS genetics},
volume = {15},
number = {12},
pages = {e1008554},
pmid = {31877124},
issn = {1553-7404},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 NS055272/NS/NINDS NIH HHS/United States ; R21 NS090030/NS/NINDS NIH HHS/United States ; R56 NS055272/NS/NINDS NIH HHS/United States ; },
mesh = {*Alternative Splicing ; Animals ; CRISPR-Cas Systems ; Cadherin Related Proteins ; Cadherins/*genetics/metabolism ; Embryonic Development ; Exons ; Female ; Humans ; INDEL Mutation ; Male ; Mice ; Multigene Family ; *Mutation ; Neurons/cytology/*metabolism ; Protein Isoforms/genetics/metabolism ; Sequence Deletion ; Whole Genome Sequencing ; },
abstract = {The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.},
}
@article {pmid31876477,
year = {2019},
author = {Gabel, I and Moreno, J},
title = {Genome Editing, Ethics, and Politics.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1105-1110},
pmid = {31876477},
issn = {2376-6980},
support = {T32 HG009496/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Embryo Research/ethics/legislation &amp; jurisprudence ; Ethics, Medical ; Gene Editing/ethics/*legislation &amp; jurisprudence ; Genetic Therapy/ethics/legislation &amp; jurisprudence ; Genome, Human ; Humans ; *Politics ; United States ; },
abstract = {For the better part of a dozen years and over 3 US presidential terms, heated debates about the ethics of cloning and embryonic stem cell research helped to define the American political landscape. Current lack of public controversy about regulation of human genome editing does not signal that ethical issues about engineering human embryos have been settled. Rather, while genome editing raises old ethical questions about the value of human life, eugenics, and the weight of unintended consequences, it also came into being in a political landscape that vastly differs from the early aughts when bioethics was last a major topic of political controversy.},
}
@article {pmid31876473,
year = {2019},
author = {McConnell, SC},
title = {An Exclusive Interview With CRISPR.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1079-1088},
doi = {10.1001/amajethics.2019.1079},
pmid = {31876473},
issn = {2376-6980},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics/methods ; Genetic Therapy/adverse effects/*ethics/methods ; Genome, Human/genetics ; Humans ; },
abstract = {This article chronicles a didactic encounter between an ethics-minded physician-scientist and a personified genome editing technology called clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins, commonly abbreviated as CRISPR/Cas, or simply CRISPR. The interview considers clinically and ethically relevant questions about this technology related to patient safety, therapeutic efficacy, equitable access, and global governance of humanity's genetic legacy.},
}
@article {pmid31876471,
year = {2019},
author = {Blasimme, A},
title = {Why Include the Public in Genome Editing Governance Deliberation?.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1065-1070},
doi = {10.1001/amajethics.2019.1065},
pmid = {31876471},
issn = {2376-6980},
mesh = {CRISPR-Cas Systems ; *Community Participation ; Gene Editing/ethics/*legislation &amp; jurisprudence ; Genetic Therapy/ethics/legislation &amp; jurisprudence ; Genome, Human/genetics ; Government Regulation ; Humans ; },
abstract = {With the birth of genetically engineered twins in November 2018, international debate about human genome editing governance has moved from an emphasis on mutual engagement among multiple stakeholders to a self-regulatory model enacted through high-level expert groups with little or no public input. This article reconstructs this paradigm shift and suggests that inclusive public deliberation should still have a role in public decision making about genome editing.},
}
@article {pmid31876467,
year = {2019},
author = {Schweikart, SJ},
title = {What Is Prudent Governance of Human Genome Editing?.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1042-1048},
doi = {10.1001/amajethics.2019.1042},
pmid = {31876467},
issn = {2376-6980},
mesh = {*CRISPR-Cas Systems ; Congresses as Topic ; Gene Editing/ethics/*legislation &amp; jurisprudence ; Genome, Human/genetics ; Humans ; Models, Theoretical ; },
abstract = {CRISPR technology has made questions about how best to regulate human genome editing immediately relevant. A sound and ethical governance structure for human genome editing is necessary, as the consequences of this new technology are far-reaching and profound. Because there are currently many risks associated with genome editing technology, the extent of which are unknown, regulatory prudence is ideal. When considering how best to create a prudent governance scheme, we can look to 2 guiding examples: the Asilomar conference of 1975 and the German Ethics Council guidelines for human germline intervention. Both models offer a path towards prudent regulation in the face of unknown and significant risks.},
}
@article {pmid31876466,
year = {2019},
author = {Thompson, C},
title = {How Should "CRISPRed" Babies Be Monitored Over Their Life Course to Promote Health Equity?.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1036-1041},
doi = {10.1001/amajethics.2019.1036},
pmid = {31876466},
issn = {2376-6980},
support = {209829/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Health Equity/*ethics ; Humans ; },
abstract = {Gene-edited babies who might be born in the future should be monitored over the course of their life. These patients' physical, mental, and social health monitoring should be coordinated by clinicians in ways that anonymize patients' data for privacy protection but also allow for national and international aggregate evaluations. Transnational monitoring efforts should focus on safety and efficacy, social and disability justice, what constitutes the standard of care, and how best to promote both access to care and social and genomic research and innovation. In addition, effective and binding mechanisms for stopping or limiting uses of gene editing technology should be developed.},
}
@article {pmid31876465,
year = {2019},
author = {Lehmann, LS},
title = {Using the 4-S Framework to Guide Conversations With Patients About CRISPR.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1029-1035},
doi = {10.1001/amajethics.2019.1029},
pmid = {31876465},
issn = {2376-6980},
mesh = {*CRISPR-Cas Systems ; Counseling/ethics ; Gene Editing/*ethics ; Genetic Diseases, Inborn/therapy ; Genetic Therapy/adverse effects/*ethics ; Humans ; Patient Education as Topic/*ethics/methods ; Physician-Patient Relations/*ethics ; Risk Factors ; },
abstract = {As patients with genetic diseases seek to have healthy biologically connected children, they will undoubtedly turn to trusted health care professionals for guidance. "Doctor, should I enter a clinical trial to edit my embryos?" is likely to become a query posed by patients with genetic illnesses. Physicians need both empathic communication skills and a framework for responding to this question. Applying the 4-S framework to gene editing can guide clinicians' responses to patients' CRISPR queries by facilitating discussion of (1) safety, (2) significance of harm to be averted, (3) impact on succeeding generations, and (4) social consequences.},
}
@article {pmid31876277,
year = {2019},
author = {Lomov, NA and Viushkov, VS and Petrenko, AP and Syrkina, MS and Rubtsov, MA},
title = {[Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {6},
pages = {982-997},
doi = {10.1134/S0026898419060119},
pmid = {31876277},
issn = {0026-8984},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods/*standards ; },
abstract = {The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii) analyzing clones for presence of the desired mutation. This review presents the methods used to assess the efficiency of the CRISPR/Cas system and to confirm mutation in the target locus and discusses their advantages and disadvantages. It aims to provide information that could help researchers to choose a technique most appropriate for their specific tasks and available resources.},
}
@article {pmid31875674,
year = {2020},
author = {Wang, R and Zhao, X and Chen, X and Qiu, X and Qing, G and Zhang, H and Zhang, L and Hu, X and He, Z and Zhong, D and Wang, Y and Luo, Y},
title = {Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9 Cleavage (RACE) for Highly Specific Detection of Multiple Extracellular Vesicle MicroRNAs.},
journal = {Analytical chemistry},
volume = {92},
number = {2},
pages = {2176-2185},
doi = {10.1021/acs.analchem.9b04814},
pmid = {31875674},
issn = {1520-6882},
mesh = {A549 Cells ; CRISPR-Cas Systems/*genetics ; Extracellular Vesicles/*genetics ; Humans ; MicroRNAs/blood/*genetics ; *Nucleic Acid Amplification Techniques ; *Reverse Transcriptase Polymerase Chain Reaction ; Temperature ; Tumor Cells, Cultured ; },
abstract = {Multiplexed detection of extracellular vesicle (EV)-derived microRNAs (miRNAs) plays a critical role in facilitating disease diagnosis and prognosis evaluation. Herein, we developed a highly specific nucleic acid detection platform for simultaneous quantification of several EV-derived miRNAs in constant temperature by integrating the advantages of a clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases (CRISPR/Cas) system and rolling circular amplification (RCA) techniques. Particularly, the proposed approach demonstrated single-base resolution attributed to the dual-specific recognition from both padlock probe-mediated ligation and protospacer adjacent motif (PAM)-triggered cleavage. The high consistency between the proposed approach RCA-assisted CRISPR/Cas9 cleavage (RACE) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) in detecting EV-derived miRNAs' abundance from both cultured cancer cells and clinical lung cancer patients validated its robustness, revealing its potentials in the screening, diagnosis, and prognosis of various diseases. In summary, RACE is a powerful tool for multiplexed, specific detection of nucleic acids in point-of-care diagnostics and field-deployable analysis.},
}
@article {pmid31875554,
year = {2019},
author = {Shinoda, K and Maman, Y and Canela, A and Schatz, DG and Livak, F and Nussenzweig, A},
title = {Intra-Vκ Cluster Recombination Shapes the Ig Kappa Locus Repertoire.},
journal = {Cell reports},
volume = {29},
number = {13},
pages = {4471-4481.e6},
pmid = {31875554},
issn = {2211-1247},
support = {R01 AI032524/AI/NIAID NIH HHS/United States ; Z01 BC010283/ImNIH/Intramural NIH HHS/United States ; Z01 BC010959/ImNIH/Intramural NIH HHS/United States ; ZIC BC011428/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/cytology/immunology/*metabolism ; CRISPR-Cas Systems ; Clone Cells ; DNA/*genetics/immunology ; DNA Breaks, Double-Stranded ; DNA Ligase ATP/deficiency/genetics/immunology ; Endonucleases/deficiency/genetics/immunology ; Female ; Gene Editing ; Homeodomain Proteins/genetics/immunology ; Immunoglobulin Variable Region/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Multigene Family ; Nuclear Proteins/deficiency/genetics/immunology ; Spleen/cytology/immunology ; V(D)J Recombination/*immunology ; },
abstract = {During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.},
}
@article {pmid31875277,
year = {2020},
author = {Peng, Y and Yang, T and Tang, X and Chen, F and Wang, S},
title = {Construction of an Inducible CRISPR/Cas9 System for CXCR4 Gene and Demonstration of its Effects on MKN-45 Cells.},
journal = {Cell biochemistry and biophysics},
volume = {78},
number = {1},
pages = {23-30},
doi = {10.1007/s12013-019-00898-x},
pmid = {31875277},
issn = {1559-0283},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Movement/drug effects ; Chemokine CXCL12/pharmacology ; Doxycycline/pharmacology ; Exons ; Gene Editing ; Gene Expression/drug effects ; Humans ; Lentivirus/genetics ; Plasmids/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Receptors, CXCR4/*genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system is an effective tool for gene editing. However, this conventional expression system cannot control the timing of gene editing and does not utilize resistance screening markers. Therefore, carrying out CRISPR/Cas9 experiments is extremely inconvenient. Our aim is to develop an inducible lentiviral vector-based gene-editing system for C-X-C chemokine receptor 4 (CXCR4) by CRISPR/Cas9, and to demonstrate its function in MKN-45 cell. The DNA fragments of Blasticidin and T2A-GFP were produced using the lenti-Cas9-BLAST and PX458 plasmids as templates. The PCR products were harvested and cloned into the lenti-guide-puro plasmid to yield the lenti-guide-BLAST-GFP plasmid. Three double-stranded guide RNA (gRNA) sequences targeting the exon 2 of CXCR4 gene were designed online (http://crispr.mit.edu), synthesized, and recombined into the lenti-guide-BLAST-GFP plasmid, to yield the lenti-guide-BLAST-GFP-gRNA plasmid. The pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA plasmids were packaged with lentiviral vectors, which were then transfected into MKN-45 cells, to identify the CXCR4 gene-editing effects using the T7 endonuclease 1 (T7E1) and Western blot assays. The lenti-guide-BLAST-GFP and lenti-guide-BLAST-GFP-gRNA plasmids were successfully constructed and packaged, to yield lentiviral particles. Transfection of the pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA viral vectors could decrease the expression of CXCR4 protein, and lead to gene editing in MKN-45 cells. The efficiencies of gRNA-1, gRNA-2, and gRNA-3 were 45.6%, 53.6%, and 56.7%, respectively. Furthermore, the chemotactic efficiency of the dual viral vector-infected MKN-45 cells was significantly decreased in response to SDF-1. The numbers of migratory cells in the lower chamber of the transwell system were 30.0 ± 0.23, 29.7 ± 1.55, 28.2 ± 1.11 and 36.1 ± 2.00 cells per field (400×) for gRNA-1, gRNA-2, gRNA-3 and the control, respectively (P < 0.05). We constructed an inducible CXCR4 gene-editing, dual-vector CRISPR/Cas9 system, which could induce CXCR4 gene editing in MKN-45 cells in a doxycycline-dependent manner and thus reduce the migration of MKN-45 cells.},
}
@article {pmid31874862,
year = {2020},
author = {Katayama, S and Sato, K and Nakazawa, T},
title = {In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining.},
journal = {Life science alliance},
volume = {3},
number = {1},
pages = {},
pmid = {31874862},
issn = {2575-1077},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Carnitine O-Acetyltransferase/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; DNA End-Joining Repair/*genetics ; Dependovirus/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Genetic Vectors ; Green Fluorescent Proteins/*genetics ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NIH 3T3 Cells ; Retinal Ganglion Cells/metabolism ; Staining and Labeling/*methods ; Staphylococcus aureus/enzymology ; Transfection ; },
abstract = {Gene knockout is important for understanding gene function and genetic disorders. The CRISPR/Cas9 system has great potential to achieve this purpose. However, we cannot distinguish visually whether a gene is knocked out and in how many cells it is knocked out among a population of cells. Here, we developed a new system that enables the labelling of knockout cells with fluorescent protein through microhomology-mediated end joining-based knock-in. Using a combination with recombinant adeno-associated virus, we delivered our system into the retina, where the expression of Staphylococcus aureus Cas9 was driven by a retina ganglion cell (RGC)-specific promoter, and knocked out carnitine acetyltransferase (CAT). We evaluated RGCs and revealed that CAT is required for RGC survival. Furthermore, we applied our system to Keap1 and confirmed that Keap1 is not expressed in fluorescently labelled cells. Our system provides a promising framework for cell type-specific genome editing and fluorescent labelling of gene knockout based on knock-in.},
}
@article {pmid31874693,
year = {2020},
author = {Lin, WR and Ng, IS},
title = {Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation.},
journal = {Enzyme and microbial technology},
volume = {133},
number = {},
pages = {109458},
doi = {10.1016/j.enzmictec.2019.109458},
pmid = {31874693},
issn = {1879-0909},
mesh = {Agrobacterium tumefaciens/genetics ; Biofuels ; Biomass ; *CRISPR-Cas Systems ; Chlorella vulgaris/*genetics/*metabolism ; Fatty Acid Desaturases/genetics ; Green Fluorescent Proteins/genetics ; *Lipid Metabolism ; Lipids/*analysis ; Microalgae/genetics/metabolism ; Plasmids/genetics ; },
abstract = {Microalgae biorefinery is an alternative, sustainable and promising trend to solve the problem of fossil oil depletion and carbon dioxide emission. However, considering the innate limitation of cell growth and oil content in microalgae, to accelerate metabolic balance by CRISPR/Cas9 system is attractive. At first, plasmid based from Agrobacterium tumefaciens and a fragment of mGFP was transformed into Chlorella sorokiniana and Chlorella vulgaris FSP-E by electroporation, respectively. Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM), and an increase of fluorescent was observed by 67% compared with that in wild type, which proved the Agrobacterium-mediated plasmid is suitable for gene insertion in Chlorella species. Consequently, plasmid with similar structure as mentioned previously containing fragment of Cas9 with sgRNA designed on omega-3 fatty acid desaturase (fad3) gene was constructed and showed a higher accumulation of lipid content by 46% (w/w) in C. vulgaris FSP-E. This is first-time to use CRISPR/Cas9 based technology for gene manipulation in Chlorella.},
}
@article {pmid31874686,
year = {2020},
author = {Salazar-Cerezo, S and Kun, RS and de Vries, RP and Garrigues, S},
title = {CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer.},
journal = {Enzyme and microbial technology},
volume = {133},
number = {},
pages = {109463},
doi = {10.1016/j.enzmictec.2019.109463},
pmid = {31874686},
issn = {1879-0909},
mesh = {*CRISPR-Cas Systems ; Enzymes/*biosynthesis ; *Gene Editing ; Genome, Fungal ; Industrial Microbiology/methods ; Penicillium/*enzymology/*genetics ; Phenotype ; },
abstract = {Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.},
}
@article {pmid31874627,
year = {2019},
author = {Bradford, J and Perrin, D},
title = {Improving CRISPR guide design with consensus approaches.},
journal = {BMC genomics},
volume = {20},
number = {Suppl 9},
pages = {931},
pmid = {31874627},
issn = {1471-2164},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Software ; },
abstract = {BACKGROUND: CRISPR-based systems are playing an important role in modern genome engineering. A large number of computational methods have been developed to assist in the identification of suitable guides. However, there is only limited overlap between the guides that each tool identifies. This can motivate further development, but also raises the question of whether it is possible to combine existing tools to improve guide design.<br><br>RESULTS: We considered nine leading guide design tools, and their output when tested using two sets of guides for which experimental validation data is available. We found that consensus approaches were able to outperform individual tools. The best performance (with a precision of up to 0.912) was obtained when combining four of the tools and accepting all guides selected by at least three of them.<br><br>CONCLUSIONS: These results can be used to improve CRISPR-based studies, but also to guide further tool development. However, they only provide a short-term solution as the time and computational resources required to run four tools may be impractical in certain applications.},
}
@article {pmid31874152,
year = {2020},
author = {Bekbulat, F and Schmitt, D and Feldmann, A and Huesmann, H and Eimer, S and Juretschke, T and Beli, P and Behl, C and Kern, A},
title = {RAB18 Loss Interferes With Lipid Droplet Catabolism and Provokes Autophagy Network Adaptations.},
journal = {Journal of molecular biology},
volume = {432},
number = {4},
pages = {1216-1234},
doi = {10.1016/j.jmb.2019.12.031},
pmid = {31874152},
issn = {1089-8638},
mesh = {Autophagosomes/metabolism ; Autophagy/physiology ; CRISPR-Cas Systems/genetics/physiology ; HeLa Cells ; Humans ; Immunoblotting ; Immunohistochemistry ; Lipid Droplets/*chemistry ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; rab GTP-Binding Proteins/genetics/*metabolism ; rab3 GTP-Binding Proteins/genetics/metabolism ; },
abstract = {Autophagy is dependent on appropriate lipid supply for autophagosome formation. The regulation of lipid acquisition and the autophagy network response to lipid-limiting conditions are mostly elusive. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet catabolism, causing an impaired fatty acid release. The resulting reduced lipid-droplet-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network. These adjustments include increased expression and phosphorylation of ATG2B as well as augmented formation of the ATG12-ATG5 conjugate. Moreover, ATG9A shows an enhanced phosphorylation at amino acid residues tyrosine 8 and serine 14, resulting in an increased ATG9A trafficking. Via pharmacological inhibition of Y8 phosphorylation, we demonstrate that this ATG9A modification is important to maintain basal autophagy under RAB18 knockout conditions. However, while the network adaptations are sufficient to maintain basal autophagic activity, they are incapable of ensuring autophagy induction upon starvation, which is characterized by an enhanced lipid demand. Thus, here, we define the molecular role of RAB18 in connecting lipid droplets and autophagy, emphasize the significance of lipid droplets as lipid sources for the degradative pathway, and uncover a remarkable autophagy network plasticity, including phosphorylation-dependent ATG9A activation, to compensate reduced lipid availability in order to rescue basal autophagic activity.},
}
@article {pmid31873747,
year = {2020},
author = {Ota, A and Ishihara, T and Ishihara, N},
title = {Mitochondrial nucleoid morphology and respiratory function are altered in Drp1-deficient HeLa cells.},
journal = {Journal of biochemistry},
volume = {167},
number = {3},
pages = {287-294},
doi = {10.1093/jb/mvz112},
pmid = {31873747},
issn = {1756-2651},
mesh = {CRISPR-Cas Systems ; Cell Respiration/genetics ; DNA, Mitochondrial/genetics/*metabolism ; Dynamins/genetics/*metabolism ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Microscopy, Fluorescence ; Mitochondria/genetics/*metabolism/ultrastructure ; Mitochondrial Dynamics/genetics/physiology ; RNA, Small Interfering ; },
abstract = {Mitochondria are dynamic organelles that frequently divide and fuse with each other. The dynamin-related GTPase protein Drp1 has a key role in mitochondrial fission. To analyse the physiological roles of Drp1 in cultured human cells, we analysed Drp1-deficient HeLa cells established by genome editing using CRISPR/Cas9. Under fluorescent microscopy, not only mitochondria were elongated but their DNA (mtDNA) nucleoids were extremely enlarged in bulb-like mitochondrial structures ('mito-bulbs') in the Drp1-deficient HeLa cells. We further found that respiratory activity, as measured by oxygen consumption rates, was severely repressed in Drp1-deficient HeLa cells and that this was reversible by the co-repression of mitochondrial fusion factors. Although mtDNA copy number was not affected, several respiratory subunits were repressed in Drp1-deficient HeLa cells. These results suggest that mitochondrial fission is required for the maintenance of active respiratory activity and the morphology of mtDNA nucleoids in human cells.},
}
@article {pmid31873222,
year = {2020},
author = {Yu, Y and Guo, Y and Tian, Q and Lan, Y and Yeh, H and Zhang, M and Tasan, I and Jain, S and Zhao, H},
title = {An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.},
journal = {Nature chemical biology},
volume = {16},
number = {4},
pages = {387-390},
pmid = {31873222},
issn = {1552-4469},
support = {U54 DK107965/DK/NIDDK NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; },
mesh = {5' Flanking Region/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Gene Knock-In Techniques/*methods ; Genome/genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Sequence Homology, Nucleic Acid ; },
abstract = {Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.},
}
@article {pmid31873217,
year = {2020},
author = {Kwon, CT and Heo, J and Lemmon, ZH and Capua, Y and Hutton, SF and Van Eck, J and Park, SJ and Lippman, ZB},
title = {Rapid customization of Solanaceae fruit crops for urban agriculture.},
journal = {Nature biotechnology},
volume = {38},
number = {2},
pages = {182-188},
pmid = {31873217},
issn = {1546-1696},
mesh = {Agriculture/*methods ; Base Sequence ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/*physiology ; Fruit/*physiology ; Gene Editing ; Inflorescence/physiology ; Mutation/genetics ; Phylogeny ; Plant Shoots/physiology ; Solanaceae/*physiology ; },
abstract = {Cultivation of crops in urban environments might reduce the environmental impact of food production[1-4]. However, lack of available land in cities and a need for rapid crop cycling, to yield quickly and continuously, mean that so far only lettuce and related 'leafy green' vegetables are cultivated in urban farms[5]. New fruit varieties with architectures and yields suitable for urban farming have proven difficult to breed[1,5]. We identified a regulator of tomato stem length (SlER) and devised a trait-stacking strategy to combine mutations for condensed shoots, rapid flowering (SP5G) and precocious growth termination (SP). Application of our strategy using one-step CRISPR-Cas9 genome editing restructured vine-like tomato plants into compact, early yielding plants suitable for urban agriculture. Field data confirmed that yields were maintained, and we demonstrated cultivation in indoor farming systems. Targeting the same stem length regulator alone in groundcherry, another Solanaceae plant, also enabled engineering to a compact stature. Our approach can expand the repertoire of crops for urban agriculture.},
}
@article {pmid31872379,
year = {2020},
author = {Zhao, Y and Tian, J and Zheng, G and Chen, J and Sun, C and Yang, Z and Zimin, AA and Jiang, W and Deng, Z and Wang, Z and Lu, Y},
title = {Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces.},
journal = {Science China. Life sciences},
volume = {63},
number = {7},
pages = {1053-1062},
doi = {10.1007/s11427-019-1559-y},
pmid = {31872379},
issn = {1869-1889},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cytidine Deaminase/*genetics ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; Genome, Bacterial/genetics ; Guanosine/metabolism ; Immunosuppressive Agents/metabolism ; Point Mutation/genetics ; Promoter Regions, Genetic ; Recombinant Proteins/*genetics ; Sirolimus/metabolism ; Streptomyces/*genetics ; },
abstract = {CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination (HR) ability. Here, a deaminase-assisted base editor dCas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9 (dCas9), the cytidine deaminase from Petromyzon marinus (PmCDA1), the uracil DNA glycosylase inhibitor (UGI) and the protein degradation tag (LVA tag). Using dCas9-CDA-ULstr, we achieved single-, double- and triple-point mutations (cytosine-to-thymine substitutions) at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes (with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the dCas9-CDA-ULstr-based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.},
}
@article {pmid31872378,
year = {2020},
author = {Li, J and Li, HY and Gu, SY and Zi, HX and Jiang, L and Du, JL},
title = {One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting.},
journal = {Science China. Life sciences},
volume = {63},
number = {1},
pages = {59-67},
doi = {10.1007/s11427-019-1607-9},
pmid = {31872378},
issn = {1869-1889},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Expression ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques/methods ; Gene Targeting/*methods ; Genetic Engineering ; Genotype ; Green Fluorescent Proteins/genetics ; Homologous Recombination/*genetics ; Introns/*genetics ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2[zKOIS] which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2[zCKOIS] was switched to a non-functional CKO allele hey2[zCKOIS-inv]associated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.},
}
@article {pmid31872309,
year = {2020},
author = {Wang, C and Wang, G and Gao, Y and Lu, G and Habben, JE and Mao, G and Chen, G and Wang, J and Yang, F and Zhao, X and Zhang, J and Mo, H and Qu, P and Liu, J and Greene, TW},
title = {A cytokinin-activation enzyme-like gene improves grain yield under various field conditions in rice.},
journal = {Plant molecular biology},
volume = {102},
number = {4-5},
pages = {373-388},
pmid = {31872309},
issn = {1573-5028},
mesh = {CRISPR-Cas Systems ; Cytokinins/*metabolism ; Droughts ; Edible Grain/enzymology/*genetics ; Gene Editing ; Gene Expression Regulation, Plant ; Nitrogen ; Oryza/enzymology/*genetics ; Plant Proteins/genetics/*metabolism ; Plant Roots/physiology ; Protein Domains ; Seeds/physiology ; Stress, Physiological ; },
abstract = {CRISPR-edited variants at the 3'-end of OsLOGL5's coding sequence (CDS), significantly increased rice grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Cytokinins impact numerous aspects of plant growth and development. This study reports that constitutive ectopic overexpression of a rice cytokinin-activation enzyme-like gene, OsLOGL5, significantly reduced primary root growth, tiller number, and yield. Conversely, mutations at the 3'-end of OsLOGL5 CDS resulted in normal rice plant morphology but with increased grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Six gene edited variants (Edit A to F) were created and tested in the field. Edit-B and Edit-F plants increased, but Edit-D and Edit-E plants decreased, the panicle number per plant. All OsLOGL5-edited plants significantly increased seed setting rate, total grain numbers, full-filled grain numbers per panicle, and thousand seed weight under drought conditions, suggesting that OsLOGL5 is likely involved in the regulation of both seed development and grain filling processes. Our results indicate that the C-terminal end of OsLOGL5 protein plays an important role in regulating rice yield improvement under different abiotic stress conditions, and OsLOGL5 is important for rice yield enhancement and stability.},
}
@article {pmid31872209,
year = {2020},
author = {Swartjes, T and Staals, RHJ and van der Oost, J},
title = {Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a.},
journal = {Biochemical Society transactions},
volume = {48},
number = {1},
pages = {207-219},
pmid = {31872209},
issn = {1470-8752},
mesh = {Adaptive Immunity ; Bacterial Proteins/chemistry/*genetics ; CRISPR-Associated Protein 9/chemistry/*genetics ; CRISPR-Associated Proteins/chemistry/*genetics ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA Cleavage ; DNA-Binding Proteins ; Deoxyribonucleases/chemistry/*genetics ; Endodeoxyribonucleases/chemistry/*genetics ; Gene Editing/methods ; Humans ; RNA, Guide/*genetics ; },
abstract = {Discovered as an adaptive immune system of prokaryotes, CRISPR-Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges.},
}
@article {pmid31871154,
year = {2020},
author = {Ng, SR and Rideout, WM and Akama-Garren, EH and Bhutkar, A and Mercer, KL and Schenkel, JM and Bronson, RT and Jacks, T},
title = {CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {1},
pages = {513-521},
pmid = {31871154},
issn = {1091-6490},
support = {T32 GM007753/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation/genetics ; Disease Models, Animal ; Disease Progression ; Feasibility Studies ; Gene Editing/*methods ; *Gene Expression Regulation, Neoplastic ; *Genes, Tumor Suppressor ; Humans ; Loss of Function Mutation ; Lung/pathology ; Lung Neoplasms/*genetics/pathology ; Mice ; Mice, Transgenic ; Neoplasm Staging ; Retinoblastoma-Like Protein p107/genetics ; Retinoblastoma-Like Protein p130/genetics ; Small Cell Lung Carcinoma/*genetics/pathology ; Tumor Burden/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.},
}
@article {pmid31871091,
year = {2020},
author = {Li, Q and Zhao, P and Li, L and Zhao, H and Shi, L and Tian, P},
title = {Engineering a CRISPR Interference System To Repress a Class 1 Integron in Escherichia coli.},
journal = {Antimicrobial agents and chemotherapy},
volume = {64},
number = {3},
pages = {},
pmid = {31871091},
issn = {1098-6596},
mesh = {Anti-Bacterial Agents/*pharmacology ; Base Sequence ; *CRISPR-Cas Systems ; Conjugation, Genetic ; Drug Resistance, Multiple, Bacterial/genetics ; Escherichia coli/*drug effects/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; Gene Transfer, Horizontal ; Genetic Engineering/*methods ; Integrases/genetics/metabolism ; *Integrons ; Plasmids/chemistry/metabolism ; RNA, Guide/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfamethoxazole/pharmacology ; Trimethoprim/pharmacology ; },
abstract = {Microbial multidrug resistance (MDR) poses a huge threat to human health. Bacterial acquisition of MDR relies primarily on class 1 integron-involved horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). To date, no strategies other than the use of antibiotics can efficiently cope with MDR. Here, we report that an engineered CRISPR interference (CRISPRi) system can markedly reduce MDR by blocking a class 1 integron in Escherichia coli Using CRISPRi to block plasmid R388 class 1 integron, E. coli recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs (sgRNAs) R3 and R6 led to 8- and 32-fold decreases in half-maximal inhibitory concentrations (IC50) for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR (RT-qPCR) revealed that the strain employing sgRNA R6 exhibited 97% and 84% decreases in the transcriptional levels of the dfrB2 cassette and sul1, two typical ARGs, respectively. RT-qPCR analysis also demonstrated that the strain recruiting sgRNA R3 showed a 96% decrease in the transcriptional level of intI1, and a conjugation assay revealed a 1,000-fold decrease in HGT rates of ARGs. Overall, the sgRNA R3 targeting the 31 bp downstream of the Pc promoter on the intI1 nontemplate strand outperformed other sgRNAs in reducing integron activity. Furthermore, this CRISPRi system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPRi system to reduce the class 1 integron in E. coli This study provides valuable insights for future development of CRISPRi-based antimicrobial agents and cellular therapy to suppress MDR.},
}
@article {pmid31869524,
year = {2020},
author = {Bosch, JA and Knight, S and Kanca, O and Zirin, J and Yang-Zhou, D and Hu, Y and Rodiger, J and Amador, G and Bellen, HJ and Perrimon, N and Mohr, SE},
title = {Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.},
journal = {Current protocols in molecular biology},
volume = {130},
number = {1},
pages = {e112},
pmid = {31869524},
issn = {1934-3647},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; R01 GM067858/GM/NIGMS NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; R24 OD019847/OD/NIH HHS/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/genetics ; Drosophila/*cytology/*genetics ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; *Genes, Insect ; Green Fluorescent Proteins/*genetics ; *Open Reading Frames ; Plasmids/genetics ; RNA, Guide/genetics ; Transfection ; },
abstract = {The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley &amp; Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.},
}
@article {pmid31868891,
year = {2020},
author = {Salanga, MC and Brun, NR and Francolini, RD and Stegeman, JJ and Goldstone, JV},
title = {CRISPR-Cas9-Mutated Pregnane X Receptor (pxr) Retains Pregnenolone-induced Expression of cyp3a65 in Zebrafish (Danio rerio) Larvae.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {174},
number = {1},
pages = {51-62},
pmid = {31868891},
issn = {1096-0929},
support = {P42 ES007381/ES/NIEHS NIH HHS/United States ; R21 HD073805/HD/NICHD NIH HHS/United States ; U24 HG003345/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Aryl Hydrocarbon Hydroxylases/*biosynthesis/genetics ; *CRISPR-Cas Systems ; Cytochrome P-450 Enzyme Inducers/*toxicity ; Enzyme Induction ; *Gene Targeting ; Ligands ; Mutation ; Oxidoreductases, N-Demethylating/*biosynthesis/genetics ; Pregnane X Receptor/*agonists/genetics/metabolism ; Pregnenolone/*toxicity ; Zebrafish/embryology/genetics/*metabolism ; Zebrafish Proteins/*biosynthesis/genetics ; },
abstract = {Pregnane X receptor (PXR; NR1I2) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure, including induction of CYP3A transcription, in many vertebrate species. PXR is activated by a wide range of ligands that differ across species, making functional studies on its role in the chemical defensome most relevant when approached in a species-specific manner. Knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Morpholino knockdown of Pxr in zebrafish indicated a similar requirement. Here, we report on the generation of 2 zebrafish lines each carrying a heritable deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise, larvae homozygous for either of the pxr mutant alleles retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone. Thus, zebrafish carrying pxr alleles with deletions in either the DNA binding or the ligand-binding domains did not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Alternative possibilities are that Pxr is not required for the induction of selected genes, or that truncated yet functional mutant Pxr is sufficient for the downstream transcriptional effects. It is crucial that we develop a better understanding for the role of Pxr in this important biomedical test species. This study highlights the potential for compensatory mechanisms to avoid deleterious effects arising from gene mutations.},
}
@article {pmid31867292,
year = {2019},
author = {Cao, XZ and Wang, JL and Elsheikha, HM and Li, TT and Sun, LX and Liang, QL and Zhang, ZW and Lin, RQ},
title = {Characterization of the Role of Amylo-Alpha-1,6-Glucosidase Protein in the Infectivity of Toxoplasma gondii.},
journal = {Frontiers in cellular and infection microbiology},
volume = {9},
number = {},
pages = {418},
pmid = {31867292},
issn = {2235-2988},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Knockdown Techniques ; Glycogen Debranching Enzyme System/genetics/*metabolism ; Mice ; Mutation ; Protozoan Proteins/genetics/*metabolism ; Toxoplasma/genetics/*metabolism/ultrastructure ; Toxoplasmosis/mortality/*parasitology/pathology ; Virulence ; },
abstract = {In this study, we characterized the role of amylo-alpha-1,6-glucosidase (Aa16GL) in the biology and infectivity of Toxoplasma gondii, using Aa16GL-deficient parasites of type I RH and type II Prugniaud (Pru) strains. The subcellular localization of Aa16GL protein was characterized by tagging a 3 × HA to the 3' end of the Aa16GL gene endogenous locus. Immunostaining of the expressed Aa16GL protein revealed that it is located in several small cytoplasmic puncta. Functional characterization of ΔAa16GL mutants using plaque assay, egress assay and intracellular replication assay showed that parasites lacking Aa16GL exhibit a slight reduction in the growth rate, but remained virulent to mice. Although PruΔAa16GL tachyzoites retained the ability to differentiate into bradyzoites in vitro, they exhibited slight reduction in their ability to form cysts in mice. These findings reveal new properties of Aa16GL and suggest that while it does not have a substantial role in mediating T. gondii infectivity, this protein can influence the formation of parasite cysts in mice.},
}
@article {pmid31865439,
year = {2020},
author = {Wang, PA and Xiao, H and Zhong, JJ},
title = {CRISPR-Cas9 assisted functional gene editing in the mushroom Ganoderma lucidum.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {4},
pages = {1661-1671},
doi = {10.1007/s00253-019-10298-z},
pmid = {31865439},
issn = {1432-0614},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/genetics ; Fermentation ; Gene Editing/*methods ; Industrial Microbiology ; Reishi/*genetics ; },
abstract = {The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.},
}
@article {pmid31863838,
year = {2020},
author = {Liu, F and Selin, C and Zou, Z and Dilantha Fernando, WG},
title = {LmCBP1, a secreted chitin-binding protein, is required for the pathogenicity of Leptosphaeria maculans on Brassica napus.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {136},
number = {},
pages = {103320},
doi = {10.1016/j.fgb.2019.103320},
pmid = {31863838},
issn = {1096-0937},
mesh = {Brassica napus/*microbiology ; CRISPR-Cas Systems ; Carrier Proteins/*genetics/metabolism ; Chitin/metabolism ; DNA, Fungal ; Fungal Proteins/*genetics/metabolism ; Gene Knockout Techniques ; Host-Pathogen Interactions ; Hydrogen Peroxide/pharmacology ; Leptosphaeria/*genetics/metabolism/*pathogenicity ; Phylogeny ; Plant Diseases/*microbiology ; Reactive Oxygen Species/metabolism ; Virulence/genetics ; },
abstract = {Leptosphaeria maculans is the causal agent of blackleg disease on Brassica napus. Determining the underlying functions of genes required for pathogenesis is essential for understanding the infection process. A chitin-binding protein (LmCBP1) was discovered as a pathogenicity factor for the infection of B. napus by L. maculans through gene knockout using the CRISPR-Cas9 system. Chitin-binding activity was demonstrated through a chitin-protein binding assay. A secreted signal peptide was detected using a yeast secreted-signal peptide trap assay. An increased expression level during the infection stage was also observed, suggesting that LmCBP1 is a secreted protein. The knockout mutants showed decreased infection on B. napus, with reduced pathogenicity on ten cultivars with/without diverse R genes. The mutants were more sensitive to H2O2 compared to wild type L. maculans isolate JN3. This study provides evidence of the virulence of a novel chitin-binding protein LmCBP1 on B. napus through mutants created via the CRISPR-Cas9 system.},
}
@article {pmid31863586,
year = {2020},
author = {Cuculis, L and Zhao, C and Abil, Z and Zhao, H and Shukla, D and Schroeder, CM},
title = {Divalent cations promote TALE DNA-binding specificity.},
journal = {Nucleic acids research},
volume = {48},
number = {3},
pages = {1406-1422},
pmid = {31863586},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; Calcium/*chemistry ; Cations, Divalent/*chemistry ; DNA/*chemistry ; DNA-Binding Proteins/chemistry ; Gene Editing ; Magnesium/*chemistry ; Potassium/chemistry ; Protein Binding ; Sodium/chemistry ; Solutions/chemistry ; Transcription Activator-Like Effector Nucleases/*chemistry/metabolism ; },
abstract = {Recent advances in gene editing have been enabled by programmable nucleases such as transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. However, several open questions remain regarding the molecular machinery in these systems, including fundamental search and binding behavior as well as role of off-target binding and specificity. In order to achieve efficient and specific cleavage at target sites, a high degree of target site discrimination must be demonstrated for gene editing applications. In this work, we studied the binding affinity and specificity for a series of TALE proteins under a variety of solution conditions using in vitro fluorescence methods and molecular dynamics (MD) simulations. Remarkably, we identified that TALEs demonstrate high sequence specificity only upon addition of small amounts of certain divalent cations (Mg2+, Ca2+). However, under purely monovalent salt conditions (K+, Na+), TALEs bind to specific and non-specific DNA with nearly equal affinity. Divalent cations preferentially bind to DNA over monovalent cations, which attenuates non-specific interactions between TALEs and DNA and further stabilizes specific interactions. Overall, these results uncover new mechanistic insights into the binding action of TALEs and further provide potential avenues for engineering and application of TALE- or TALEN-based systems for genome editing and regulation.},
}
@article {pmid31862961,
year = {2019},
author = {Dempster, JM and Pacini, C and Pantel, S and Behan, FM and Green, T and Krill-Burger, J and Beaver, CM and Younger, ST and Zhivich, V and Najgebauer, H and Allen, F and Gonçalves, E and Shepherd, R and Doench, JG and Yusa, K and Vazquez, F and Parts, L and Boehm, JS and Golub, TR and Hahn, WC and Root, DE and Garnett, MJ and Tsherniak, A and Iorio, F},
title = {Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5817},
pmid = {31862961},
issn = {2041-1723},
support = {206194/WT_/Wellcome Trust/United Kingdom ; U01 CA176058/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/pharmacology/therapeutic use ; Biomarkers, Tumor/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Datasets as Topic ; Drug Screening Assays, Antitumor/*methods ; Gene Expression Profiling ; Genes, Essential/drug effects/genetics ; Genomics/*methods ; Humans ; Molecular Targeted Therapy/methods ; Neoplasms/drug therapy/*genetics ; Oncogenes/drug effects/genetics ; Precision Medicine/methods ; Reproducibility of Results ; Small Molecule Libraries/pharmacology ; },
abstract = {Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.},
}
@article {pmid31862502,
year = {2020},
author = {Xie, Z and Jiao, H and Xiao, H and Jiang, Y and Liu, Z and Qi, C and Zhao, D and Jiao, S and Yu, T and Tang, X and Pang, D and Ouyang, H},
title = {Generation of pRSAD2 gene knock-in pig via CRISPR/Cas9 technology.},
journal = {Antiviral research},
volume = {174},
number = {},
pages = {104696},
doi = {10.1016/j.antiviral.2019.104696},
pmid = {31862502},
issn = {1872-9096},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line ; Classical Swine Fever/metabolism ; Fibroblasts/virology ; Gene Expression ; *Gene Knock-In Techniques ; Genes, Reporter ; Herpesvirus 1, Suid/metabolism ; Iron-Sulfur Proteins/*genetics ; Swine ; *Transgenes ; Virus Replication/genetics ; },
abstract = {A wide range of endemic and epidemic viruses, including classic swine fever virus (CSFV), pseudorabies virus (PRV) and others, are among the most economically important pathogens in pigs and have severely affected the national economy, human health and animal welfare and productivity. The RSAD2 exhibits antiviral activity against various DNA and RNA viruses. In this study, we successfully accomplished site-specific insertion of the porcine RSAD2 gene (pRSAD2) at the porcine ROSA26 (pROSA26) locus, generating pRSAD2 gene knock-in (pRSAD2-KI) PK-15 cells and porcine foetal fibroblasts (PFFs) via CRISPR/Cas9 technology. Gene expression analysis confirmed that pRSAD2-KI cells stably and efficiently overexpressed the pRSAD2 gene. Furthermore, viral challenge studies in vitro indicated that site-specific integration of the pRSAD2 gene not only effectively reduced CSFV infection but also PRV infection. More importantly, we ultimately successfully produced a pRSAD2-KI pig that constitutively overexpressed the pRSAD2, viral challenge results indicated that fibroblasts isolated from the pRSAD2-KI pig reduced CSFV infection. Taken together, these results suggest that CRISPR/Cas9-mediated knock-in strategy can be used for producing pRSAD2-KI pigs.},
}
@article {pmid31862124,
year = {2020},
author = {Li, B and Niu, Y and Ji, W and Dong, Y},
title = {Strategies for the CRISPR-Based Therapeutics.},
journal = {Trends in pharmacological sciences},
volume = {41},
number = {1},
pages = {55-65},
doi = {10.1016/j.tips.2019.11.006},
pmid = {31862124},
issn = {1873-3735},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Humans ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing technology is an emerging RNA-guided nuclease system initially identified from the microbial adaptive immune systems. In recent years, the CRISPR system has been reprogrammed to target specific regions of the eukaryotic genome and has become a powerful tool for genetic engineering. Researchers have explored many approaches to improve the genome editing activity of the CRISPR-Cas system and deliver its components both ex vivo and in vivo. Moreover, these strategies have been applied to genome editing in preclinical research and clinical trials. In this review, we focus on representative strategies for regulation and delivery of the CRISPR-Cas system, and outline current therapeutic applications in their clinical translation.},
}
@article {pmid31861790,
year = {2019},
author = {Hassa, J and Wibberg, D and Maus, I and Pühler, A and Schlüter, A},
title = {Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter.},
journal = {Microorganisms},
volume = {8},
number = {1},
pages = {},
pmid = {31861790},
issn = {2076-2607},
abstract = {In the thermophilic biogas-producing microbial community, the genus Methanothermobacter was previously described to be frequently abundant. The aim of this study was to establish and analyze the genome sequence of the archaeal strain Methanothermobacter wolfeii SIV6 originating from a thermophilic industrial-scale biogas fermenter and compare it to related reference genomes. The circular chromosome has a size of 1,686,891 bases, featuring a GC content of 48.89%. Comparative analyses considering three completely sequenced Methanothermobacter strains revealed a core genome of 1494 coding sequences and 16 strain specific genes for M. wolfeii SIV6, which include glycosyltransferases and CRISPR/cas associated genes. Moreover, M. wolfeii SIV6 harbors all genes for the hydrogenotrophic methanogenesis pathway and genome-centered metatranscriptomics indicates the high metabolic activity of this strain, with 25.18% of all transcripts per million (TPM) belong to the hydrogenotrophic methanogenesis pathway and 18.02% of these TPM exclusively belonging to the mcr operon. This operon encodes the different subunits of the enzyme methyl-coenzyme M reductase (EC: 2.8.4.1), which catalyzes the final and rate-limiting step during methanogenesis. Finally, fragment recruitment of metagenomic reads from the thermophilic biogas fermenter on the SIV6 genome showed that the strain is abundant (1.2%) within the indigenous microbial community. Detailed analysis of the archaeal isolate M. wolfeii SIV6 indicates its role and function within the microbial community of the thermophilic biogas fermenter, towards a better understanding of the biogas production process and a microbial-based management of this complex process.},
}
@article {pmid31860398,
year = {2019},
author = {Flotte, TR and Gao, G},
title = {Prime Editing: A Novel Cas9-Reverse Transcriptase Fusion May Revolutionize Genome Editing.},
journal = {Human gene therapy},
volume = {30},
number = {12},
pages = {1445-1446},
doi = {10.1089/hum.2019.29098.trf},
pmid = {31860398},
issn = {1557-7422},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 DK098252/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; DNA/biosynthesis/*genetics ; Gene Editing/*methods ; Genetic Therapy/*trends ; Humans ; RNA-Directed DNA Polymerase/genetics ; Recombinational DNA Repair/*genetics ; },
}
@article {pmid31860354,
year = {2019},
author = {Vergnaud, G},
title = {And Then There Were Three: Discovering the Role of CRISPRs.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {359-361},
doi = {10.1089/crispr.2019.29073.gve},
pmid = {31860354},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*history/*methods ; Genome, Bacterial ; History, 20th Century ; History, 21st Century ; Humans ; Yersinia pestis/genetics ; },
}
@article {pmid31860353,
year = {2019},
author = {Hough, SH and Ajetunmobi, A},
title = {A CRISPR Moratorium Isn't Enough: We Need a Boycott.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {343-345},
doi = {10.1089/crispr.2019.0041},
pmid = {31860353},
issn = {2573-1602},
support = {206388/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disclosure/ethics ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Humans ; Information Dissemination/ethics ; Peer Review/ethics/legislation &amp; jurisprudence ; },
}
@article {pmid31860352,
year = {2019},
author = {Ulloa-Navas, MJ and García-Tárraga, P and García-Verdugo, JM and Herranz-Pérez, V},
title = {Immunogold Labeling to Detect Streptococcus pyogenes Cas9 in Cell Culture and Tissues by Electron Microscopy.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {395-405},
doi = {10.1089/crispr.2019.0032},
pmid = {31860352},
issn = {2573-1602},
mesh = {Animals ; Brain ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/immunology ; DNA ; Gene Editing/*methods ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Microscopy, Electron/methods ; Microscopy, Immunoelectron/methods ; RNA, Guide ; Streptococcus pyogenes/*genetics ; },
abstract = {The CRISPR-Cas9 system is a powerful and yet precise DNA-editing tool in rapid development. By combining immunogold labeling and electron microscopy with the novel CRISPR-Cas9 system, we propose a new method to gain insight into the biology of this tool. In this study, we analyzed different Cas9-induced systems such as HEK293T cell line, murine oligodendrocyte progenitor cells, brain and liver to detect Cas9 expression by immunoelectron microscopy. Our results show that while Cas9 expression could be found in the nuclei and nucleopores of transfected HEK293T cells, in transfected oligodendrocyte precursor cells, Cas9 was found in cytoplasmic vesicles. In Cas9 constitutively expressing oligodendrocyte precursors, the enzyme was located in the cytoplasm of nondividing cells. Finally, while in the liver Cas9 was detected in different cell types, in the brain we found no specifically labeled cells. In conclusion, immunoelectron microscopy opens a new spectrum of opportunities to study the CRISPR-Cas9 system in a more precise manner.},
}
@article {pmid31860351,
year = {2019},
author = {Davies, K and Church, GM},
title = {Radical Technology Meets Radical Application: An Interview with George Church.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {346-351},
doi = {10.1089/crispr.2019.29074.gch},
pmid = {31860351},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods/*trends ; Humans ; Technology/methods/trends ; },
}
@article {pmid31860350,
year = {2019},
author = {Mikkelsen, RB and Frederiksen, HRS and Gjerris, M and Holst, B and Hyttel, P and Luo, Y and Freude, K and Sandøe, P},
title = {Genetic Protection Modifications: Moving Beyond the Binary Distinction Between Therapy and Enhancement for Human Genome Editing.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {362-369},
doi = {10.1089/crispr.2019.0024},
pmid = {31860350},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*ethics/methods ; Genetic Predisposition to Disease/genetics/prevention &amp; control ; Genetic Therapy/ethics/methods ; Genome, Human ; Humans ; Primary Prevention/*ethics/*methods ; RNA, Guide/genetics ; },
abstract = {The current debate and policy surrounding the use of genome editing in humans typically relies on a binary distinction between therapy and human enhancement. Here, we argue that this dichotomy fails to take into account perhaps the most significant potential uses of CRISPR-Cas9 genome editing in humans. We argue that genetic treatment of sporadic Alzheimer's disease, breast and ovarian cancer predisposing BRCA1/2 mutations, and the introduction of human immunodeficiency virus resistance in humans should be considered within a new category of genetic protection treatments. We suggest that if this category is not introduced, life-altering research might be unnecessarily limited by current or future policy. Otherwise ad hoc decisions might be made, which introduce a risk of unforeseen moral costs, and might overlook or fail to address some important opportunities.},
}
@article {pmid31860348,
year = {2019},
author = {Barrangou, R},
title = {Foresight is 2020: Ten Bold Predictions for the New CRISPR Year.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {341-342},
doi = {10.1089/crispr.2019.29075.rba},
pmid = {31860348},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/*methods ; Humans ; },
}
@article {pmid31860346,
year = {2019},
author = {LeMieux, J},
title = {CRISPR Jumps in New Directions.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {354-356},
doi = {10.1089/crispr.2019.29077.jlm},
pmid = {31860346},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Transposable Elements/genetics ; Genetic Engineering/*methods ; Humans ; Integrases/genetics ; },
}
@article {pmid31859532,
year = {2020},
author = {Martin, J and Free, T},
title = {A look back at 2019 in BioTechniques.},
journal = {BioTechniques},
volume = {68},
number = {1},
pages = {2-3},
doi = {10.2144/btn-2019-0164},
pmid = {31859532},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems ; Humans ; *Polymerase Chain Reaction ; *Serial Publications ; Social Media ; Urine Specimen Collection/*methods ; },
}
@article {pmid31859038,
year = {2020},
author = {Ali, Z and Mahfouz, MM and Mansoor, S},
title = {CRISPR-TSKO: A Tool for Tissue-Specific Genome Editing in Plants.},
journal = {Trends in plant science},
volume = {25},
number = {2},
pages = {123-126},
doi = {10.1016/j.tplants.2019.12.002},
pmid = {31859038},
issn = {1878-4372},
mesh = {*Arabidopsis ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Mutagenesis ; },
abstract = {Functional genomics is at the core of studying the exact function of genes. However, homozygous knockouts of essential and pleiotropic effectors (almost 10% of the genome) are not always possible, thus, functions of these genes remain obscured. The tissue-specific genome editing tool (CRISPR-TSKO) recently described by Decaestecker et al., can characterize these indispensable genes and has wide applications in plants.},
}
@article {pmid31858701,
year = {2020},
author = {Ye, S and Chen, G and Kohnen, MV and Wang, W and Cai, C and Ding, W and Wu, C and Gu, L and Zheng, Y and Ma, X and Lin, C and Zhu, Q},
title = {Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro).},
journal = {Plant biotechnology journal},
volume = {18},
number = {7},
pages = {1501-1503},
pmid = {31858701},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant/genetics ; },
}
@article {pmid31858277,
year = {2019},
author = {Van Vu, T and Sung, YW and Kim, J and Doan, DTH and Tran, MT and Kim, JY},
title = {Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants.},
journal = {Rice (New York, N.Y.)},
volume = {12},
number = {1},
pages = {95},
pmid = {31858277},
issn = {1939-8425},
abstract = {Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.},
}
@article {pmid31857715,
year = {2020},
author = {Makarova, KS and Wolf, YI and Iranzo, J and Shmakov, SA and Alkhnbashi, OS and Brouns, SJJ and Charpentier, E and Cheng, D and Haft, DH and Horvath, P and Moineau, S and Mojica, FJM and Scott, D and Shah, SA and Siksnys, V and Terns, MP and Venclovas, &#x10c; and White, MF and Yakunin, AF and Yan, W and Zhang, F and Garrett, RA and Backofen, R and van der Oost, J and Barrangou, R and Koonin, EV},
title = {Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.},
journal = {Nature reviews. Microbiology},
volume = {18},
number = {2},
pages = {67-83},
pmid = {31857715},
issn = {1740-1534},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaea/*genetics ; Bacteria/*genetics ; CRISPR-Cas Systems/*genetics/physiology ; *Evolution, Molecular ; Gene Expression Regulation, Archaeal/*physiology ; Gene Expression Regulation, Bacterial/*physiology ; },
abstract = {The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.},
}
@article {pmid31857575,
year = {2019},
author = {Lian, J and Schultz, C and Cao, M and HamediRad, M and Zhao, H},
title = {Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5794},
pmid = {31857575},
issn = {2041-1723},
mesh = {Biotechnology/methods ; CRISPR-Cas Systems/*genetics ; Chromosome Mapping/*methods ; Gene Editing/methods ; Gene Expression Regulation, Fungal ; Genome, Fungal/*genetics ; Genomic Library ; Genomics/*methods ; Genotype ; High-Throughput Nucleotide Sequencing ; High-Throughput Screening Assays/*methods ; Phenotype ; Saccharomyces cerevisiae/genetics ; },
abstract = {Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype-phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.},
}
@article {pmid31857428,
year = {2020},
author = {Benati, D and Patrizi, C and Recchia, A},
title = {Gene editing prospects for treating inherited retinal diseases.},
journal = {Journal of medical genetics},
volume = {57},
number = {7},
pages = {437-444},
doi = {10.1136/jmedgenet-2019-106473},
pmid = {31857428},
issn = {1468-6244},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Gene Transfer Techniques/*trends ; Humans ; Induced Pluripotent Stem Cells/transplantation ; Macular Degeneration/genetics/*therapy ; Optic Atrophy, Hereditary, Leber/genetics/therapy ; Retina/pathology/transplantation ; Retinal Diseases/genetics/*therapy ; Retinitis Pigmentosa/genetics/therapy ; },
abstract = {Retinal diseases (RD) include inherited retinal dystrophy (IRD), for example, retinitis pigmentosa and Leber's congenital amaurosis, or multifactorial forms, for example, age-related macular degeneration (AMD). IRDs are clinically and genetically heterogeneous in nature. To date, more than 200 genes are known to cause IRDs, which perturb the development, function and survival of rod and cone photoreceptors or retinal pigment epithelial cells. Conversely, AMD, the most common cause of blindness in the developed world, is an acquired disease of the macula characterised by progressive visual impairment. To date, available therapeutic approaches for RD include nutritional supplements, neurotrophic factors, antiangiogenic drugs for wet AMD and gene augmentation/interference strategy for IRDs. However, these therapies do not aim at correcting the genetic defect and result in inefficient and expensive treatments. The genome editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, represents an attractive strategy to tackle IRDs without available cure. Indeed, CRISPR/Cas system can permanently and precisely replace or remove genetic mutations causative of a disease, representing a molecular tool to cure a genetic disorder. In this review, we will introduce the mechanism of CRISPR/Cas system, presenting an updated panel of Cas variants and delivery systems, then we will focus on applications of CRISPR/Cas genome editing in the retina, and, as emerging treatment options, in patient-derived induced pluripotent stem cells followed by transplantation of retinal progenitor cells into the eye.},
}
@article {pmid31856197,
year = {2019},
author = {Huhn, SC and Ou, Y and Kumar, A and Liu, R and Du, Z},
title = {High throughput, efficacious gene editing &amp; genome surveillance in Chinese hamster ovary cells.},
journal = {PloS one},
volume = {14},
number = {12},
pages = {e0218653},
pmid = {31856197},
issn = {1932-6203},
mesh = {Animals ; CHO Cells/*metabolism/*physiology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cricetinae/genetics ; Cricetulus ; Gene Editing/methods ; Genetic Engineering/*methods ; Genome/genetics ; High-Throughput Screening Assays/methods ; },
abstract = {Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Furthermore, we validate sequence decomposition as a cost effective, rapid, and accurate method for assessing mutants and eliminating non-clonal CHO populations using only capillary sequencing.},
}
@article {pmid31855178,
year = {2019},
author = {Palit, SA and Vis, D and Stelloo, S and Lieftink, C and Prekovic, S and Bekers, E and Hofland, I and Šuštić, T and Wolters, L and Beijersbergen, R and Bergman, AM and Győrffy, B and Wessels, LF and Zwart, W and van der Heijden, MS},
title = {TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31855178},
issn = {2050-084X},
support = {NKI2014-7080//KWF Kankerbestrijding/International ; Alpe d'HuZes Bas Mulder Award//KWF Kankerbestrijding/International ; VIDI grant//NWO/International ; },
mesh = {Androgen Receptor Antagonists/pharmacology ; Benzamides ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Co-Repressor Proteins/*genetics ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; HEK293 Cells ; Hepatocyte Nuclear Factor 3-alpha/*genetics ; Humans ; Male ; Nitriles ; Phenylthiohydantoin/analogs &amp; derivatives/pharmacology ; Prostate/metabolism ; Prostatic Neoplasms/drug therapy/*genetics/pathology ; Receptors, Androgen/*genetics ; Receptors, Glucocorticoid/genetics ; Transcriptional Activation/drug effects ; },
abstract = {Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor TLE3 conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon TLE3 loss share AR as the top transcriptional regulator, and TLE3 loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a TLE3-negative background. This was consistent with binding of TLE3 and AR at the GR locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized TLE3[KO] cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.},
}
@article {pmid31853989,
year = {2020},
author = {Maier, LK and Marchfelder, A and Randau, L},
title = {Meeting Report: German Genetics Society-Genome Editing with CRISPR.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {42},
number = {2},
pages = {e1900223},
doi = {10.1002/bies.201900223},
pmid = {31853989},
issn = {1521-1878},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genetic Engineering/methods ; Humans ; },
}
@article {pmid31853092,
year = {2019},
author = {},
title = {The scientific events that shaped the decade.},
journal = {Nature},
volume = {576},
number = {7787},
pages = {337-338},
pmid = {31853092},
issn = {1476-4687},
mesh = {*Artificial Intelligence ; Bias ; Big Data ; *CRISPR-Cas Systems ; *Climate Change/statistics &amp; numerical data ; Conservation of Energy Resources ; *Environmental Policy ; Female ; *Gene Editing ; Genomics ; Gravitation ; Humans ; Infant, Newborn ; *Microbiota ; *Physics ; Social Change ; },
}
@article {pmid31853090,
year = {2019},
author = {Castelvecchi, D and Cyranoski, D and Gibney, E and Ledford, H and Maxmen, A and Morello, L and Stoye, E and Subbaraman, N and Tollefson, J and Witze, A},
title = {The science news events that shaped 2019.},
journal = {Nature},
volume = {576},
number = {7787},
pages = {350-353},
doi = {10.1038/d41586-019-03838-0},
pmid = {31853090},
issn = {1476-4687},
mesh = {*Agriculture/trends ; Animals ; Artificial Intelligence ; *Astronomy ; Bullying ; CRISPR-Cas Systems ; *Embryo Research ; *Environmental Policy/legislation &amp; jurisprudence/trends ; European Union/organization &amp; administration ; Extinction, Biological ; *Gene Editing/ethics ; *Global Warming/legislation &amp; jurisprudence/prevention &amp; control ; HIV Infections/therapy/virology ; Hemorrhagic Fever, Ebola/drug therapy/prevention &amp; control ; Humans ; Induced Pluripotent Stem Cells/transplantation ; Leadership ; Mars ; Measles/epidemiology ; Meteoroids ; Minor Planets ; Moon ; Politics ; Sexual Harassment ; *Space Flight ; Stem Cell Research ; Strikes, Employee ; United States ; },
}
@article {pmid31853065,
year = {2020},
author = {Halpin-Healy, TS and Klompe, SE and Sternberg, SH and Fernández, IS},
title = {Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {271-274},
pmid = {31853065},
issn = {1476-4687},
support = {GM103310/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *DNA Transposable Elements ; DNA, Bacterial/*chemistry/genetics ; Models, Molecular ; Nucleic Acid Conformation ; Protein Multimerization ; Protein Structure, Quaternary ; RNA, Bacterial/chemistry ; Vibrio cholerae/*chemistry/genetics ; },
abstract = {Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements[1-3]. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3[4,5], but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons[6,7]. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.},
}
@article {pmid31853027,
year = {2020},
author = {Neff, EP},
title = {CRISPR takes genetic screens forward.},
journal = {Lab animal},
volume = {49},
number = {1},
pages = {13-16},
pmid = {31853027},
issn = {1548-4475},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Humans ; Immunotherapy ; *Neoplasms ; Protein Tyrosine Phosphatase, Non-Receptor Type 2 ; },
}
@article {pmid31852823,
year = {2020},
author = {Wu, Q and Xu, F and Liu, L and Char, SN and Ding, Y and Je, BI and Schmelz, E and Yang, B and Jackson, D},
title = {The maize heterotrimeric G protein β subunit controls shoot meristem development and immune responses.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {3},
pages = {1799-1805},
pmid = {31852823},
issn = {1091-6490},
mesh = {Arabidopsis/genetics/metabolism ; Autoimmunity/physiology ; CRISPR-Cas Systems ; GTP-Binding Protein beta Subunits/chemistry/genetics/*metabolism ; Gene Knockout Techniques ; Meristem/cytology/*growth &amp; development/immunology ; Phenotype ; Plant Immunity/*physiology ; Plant Shoots/cytology/*growth &amp; development/immunology ; Plants, Genetically Modified/genetics/metabolism ; Signal Transduction ; Transcriptome ; Zea mays/*metabolism ; },
abstract = {Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein β subunit gene (Gβ) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.},
}
@article {pmid31852806,
year = {2019},
author = {Nixon, SL and Daly, RA and Borton, MA and Solden, LM and Welch, SA and Cole, DR and Mouser, PJ and Wilkins, MJ and Wrighton, KC},
title = {Genome-Resolved Metagenomics Extends the Environmental Distribution of the Verrucomicrobia Phylum to the Deep Terrestrial Subsurface.},
journal = {mSphere},
volume = {4},
number = {6},
pages = {},
pmid = {31852806},
issn = {2379-5042},
mesh = {Computational Biology ; Genes, Bacterial ; *Genome, Bacterial ; Genomics ; Metabolic Networks and Pathways/genetics ; Metagenomics/*methods ; *Soil Microbiology ; Verrucomicrobia/*classification/*genetics ; },
abstract = {Bacteria of the phylum Verrucomicrobia are prevalent and are particularly common in soil and freshwater environments. Their cosmopolitan distribution and reported capacity for polysaccharide degradation suggests members of Verrucomicrobia are important contributors to carbon cycling across Earth's ecosystems. Despite their prevalence, the Verrucomicrobia are underrepresented in isolate collections and genome databases; consequently, their ecophysiological roles may not be fully realized. Here, we expand genomic sampling of the Verrucomicrobia phylum by describing a novel genus, "Candidatus Marcellius," belonging to the order Opitutales "Ca. Marcellius" was recovered from a shale-derived produced fluid metagenome collected 313 days after hydraulic fracturing, the deepest environment from which a member of the Verrucomicrobia has been recovered to date. We uncover genomic attributes that may explain the capacity of this organism to inhabit a shale gas well, including the potential for utilization of organic polymers common in hydraulic fracturing fluids, nitrogen fixation, adaptation to high salinities, and adaptive immunity via CRISPR-Cas. To illuminate the phylogenetic and environmental distribution of these metabolic and adaptive traits across the Verrucomicrobia phylum, we performed a comparative genomic analysis of 31 publicly available, nearly complete Verrucomicrobia genomes. Our genomic findings extend the environmental distribution of the Verrucomicrobia 2.3 kilometers into the terrestrial subsurface. Moreover, we reveal traits widely encoded across members of the Verrucomicrobia, including the capacity to degrade hemicellulose and to adapt to physical and biological environmental perturbations, thereby contributing to the expansive habitat range reported for this phylum.IMPORTANCE The Verrucomicrobia phylum of bacteria is widespread in many different ecosystems; however, its role in microbial communities remains poorly understood. Verrucomicrobia are often low-abundance community members, yet previous research suggests they play a major role in organic carbon degradation. While Verrucomicrobia remain poorly represented in culture collections, numerous genomes have been reconstructed from metagenomic data sets in recent years. The study of genomes from across the phylum allows for an extensive assessment of their potential ecosystem roles. The significance of this work is (i) the recovery of a novel genus of Verrucomicrobia from 2.3 km in the subsurface with the ability to withstand the extreme conditions that characterize this environment, and (ii) the most extensive assessment of ecophysiological traits encoded by Verrucomicrobia genomes to date. We show that members of this phylum are specialist organic polymer degraders that can withstand a wider range of environmental conditions than previously thought.},
}
@article {pmid31852755,
year = {2020},
author = {Zimmer, AM and Shir-Mohammadi, K and Kwong, RWM and Perry, SF},
title = {Reassessing the contribution of the Na[+]/H[+] exchanger Nhe3b to Na[+] uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing.},
journal = {The Journal of experimental biology},
volume = {223},
number = {Pt 2},
pages = {},
doi = {10.1242/jeb.215111},
pmid = {31852755},
issn = {1477-9145},
mesh = {Animals ; *CRISPR-Cas Systems ; Zebrafish/*genetics/metabolism ; },
abstract = {Freshwater fishes absorb Na[+] from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na[+] uptake is coordinated by (1) Na[+]/H[+] exchanger 3b (Nhe3b) and (2) H[+]-ATPase-powered electrogenic uptake in H[+]-ATPase-rich (HR) cells and by (3) Na[+]-Cl[-]-cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na[+] uptake by larval zebrafish under 'normal' (800 µmol l[-1]) and 'low' (10 µmol l[-1]) Na[+] conditions. We hypothesized that Na[+] uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na[+] where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na[+] uptake was sustained in nhe3b KO larvae under both Na[+] conditions, which led to the exploration of whether compensatory regulation of H[+]-ATPase or Ncc was responsible for maintaining Na[+] uptake in nhe3b KO larvae. mRNA expression of the genes encoding H[+]-ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H[+]-ATPase, which significantly reduced H[+] flux by HR cells, did not reduce Na[+] uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na[+]-Cl[-]-cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H[+]-ATPase morpholino and chloride-free conditions did not reduce Na[+] uptake under normal or low Na[+] These findings highlight the flexibility of the Na[+] uptake system and demonstrate that Nhe3b is expendable to Na[+] uptake in zebrafish and that our understanding of Na[+] uptake mechanisms in this species is incomplete.},
}
@article {pmid31852637,
year = {2019},
author = {Lei, T and Xiao, B and He, Y and Qu, J and Sun, Z and Li, L},
title = {[Development and applications of CRISPR/Cas9 library screening technology in cancer research].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {11},
pages = {1381-1386},
pmid = {31852637},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems ; Early Detection of Cancer ; Gene Knockout Techniques ; Gene Library ; Mice ; },
abstract = {The CRISPR/Cas9 technology has developed rapidly in recent years with fast, simple and accurate editing functions to allow gene knockout, knock in, activation and interference. It has become a powerful genetic screening tool and been widely used in various models including cell lines, mice and zebrafish. The application of CRISPR system in constructing genome library for high-throughput screening is the main strategy for target gene research of diseases, especially neoplasms. Here we summarize the rationales and recent development of CRISPR/Cas9 library screening technology, the strategies for improving the off-target effects, the basic workflow of library screening and the application of this technology in tumor research.},
}
@article {pmid31852439,
year = {2019},
author = {Cheng, Q and Dong, L and Su, T and Li, T and Gan, Z and Nan, H and Lu, S and Fang, C and Kong, L and Li, H and Hou, Z and Kou, K and Tang, Y and Lin, X and Zhao, X and Chen, L and Liu, B and Kong, F},
title = {CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {562},
pmid = {31852439},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genes, Plant ; *Mutagenesis ; Plants, Genetically Modified ; Soybeans/genetics/*growth &amp; development ; },
abstract = {BACKGROUND: Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants.<br><br>RESULTS: Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies.<br><br>CONCLUSIONS: Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35&#x2009;days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.},
}
@article {pmid31851676,
year = {2019},
author = {Katzmeier, F and Aufinger, L and Dupin, A and Quintero, J and Lenz, M and Bauer, L and Klumpe, S and Sherpa, D and Dürr, B and Honemann, M and Styazhkin, I and Simmel, FC and Heymann, M},
title = {A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a.},
journal = {PloS one},
volume = {14},
number = {12},
pages = {e0220091},
pmid = {31851676},
issn = {1932-6203},
mesh = {Bacterial Proteins/*genetics ; Biosensing Techniques/*instrumentation ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; *Fluorescence ; Green Fluorescent Proteins ; In Vitro Techniques ; Limit of Detection ; RNA, Bacterial/*analysis/*genetics ; Transcription, Genetic ; },
abstract = {Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.},
}
@article {pmid31850661,
year = {2020},
author = {Hussain, Q and Shi, J and Scheben, A and Zhan, J and Wang, X and Liu, G and Yan, G and King, GJ and Edwards, D and Wang, H},
title = {Genetic and signalling pathways of dry fruit size: targets for genome editing-based crop improvement.},
journal = {Plant biotechnology journal},
volume = {18},
number = {5},
pages = {1124-1140},
pmid = {31850661},
issn = {1467-7652},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Domestication ; Flowers ; *Fruit/genetics ; *Gene Editing ; },
abstract = {Fruit is seed-bearing structures specific to angiosperm that form from the gynoecium after flowering. Fruit size is an important fitness character for plant evolution and an agronomical trait for crop domestication/improvement. Despite the functional and economic importance of fruit size, the underlying genes and mechanisms are poorly understood, especially for dry fruit types. Improving our understanding of the genomic basis for fruit size opens the potential to apply gene-editing technology such as CRISPR/Cas to modulate fruit size in a range of species. This review examines the genes involved in the regulation of fruit size and identifies their genetic/signalling pathways, including the phytohormones, transcription and elongation factors, ubiquitin-proteasome and microRNA pathways, G-protein and receptor kinases signalling, arabinogalactan and RNA-binding proteins. Interestingly, different plant taxa have conserved functions for various fruit size regulators, suggesting that common genome edits across species may have similar outcomes. Many fruit size regulators identified to date are pleiotropic and affect other organs such as seeds, flowers and leaves, indicating a coordinated regulation. The relationships between fruit size and fruit number/seed number per fruit/seed size, as well as future research questions, are also discussed.},
}
@article {pmid31849223,
year = {2020},
author = {Chen, Q and Tian, T and Xiong, E and Wang, P and Zhou, X},
title = {CRISPR/Cas13a Signal Amplification Linked Immunosorbent Assay for Femtomolar Protein Detection.},
journal = {Analytical chemistry},
volume = {92},
number = {1},
pages = {573-577},
doi = {10.1021/acs.analchem.9b04403},
pmid = {31849223},
issn = {1520-6882},
mesh = {Biomarkers, Tumor/*analysis ; CRISPR-Cas Systems/*genetics ; *Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-6/*analysis ; Vascular Endothelial Growth Factor A/*analysis ; },
abstract = {The enzyme-linked immunosorbent assay (ELISA) is a basic technique used in analytical and clinical investigations. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious diseases. Herein we show a mechanism utilizing the CRISPR/Cas13a-based signal export amplification strategy, which double-amplifies the output signal by T7 RNA polymerase transcription and CRISPR/Cas13a collateral cleavage activity. This process is termed the CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA). The proposed method was validated by detecting an inflammatory factor, human interleukin-6 (human IL-6), and a tumor marker, human vascular endothelial growth factor (human VEGF), which achieved limit of detection (LOD) values of 45.81 fg/mL (2.29 fM) and 32.27 fg/mL (0.81 fM), respectively, demonstrating that CLISA is at least 10[2]-fold more sensitive than conventional ELISA.},
}
@article {pmid31848930,
year = {2020},
author = {Ye, C and Zhang, X and Chen, X and Cao, Q and Zhang, X and Zhou, Y and Li, W and Hong, L and Xie, H and Liu, X and Cao, H and Wang, YJ and Kang, B},
title = {Multiple novel hepatocellular carcinoma signature genes are commonly controlled by the master pluripotency factor OCT4.},
journal = {Cellular oncology (Dordrecht)},
volume = {43},
number = {2},
pages = {279-295},
pmid = {31848930},
issn = {2211-3436},
mesh = {ATPases Associated with Diverse Cellular Activities/genetics ; Adult ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/complications/*genetics ; Cell Cycle Proteins/genetics ; Cell Line, Tumor ; Female ; Gene Expression Profiling/*methods ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Hepatitis B/complications/virology ; Hepatitis B virus/physiology ; Humans ; Kaplan-Meier Estimate ; Liver Neoplasms/complications/*genetics ; Male ; Middle Aged ; Octamer Transcription Factor-3/*genetics ; Thymidine Kinase/genetics ; },
abstract = {BACKGROUND: Worldwide, hepatocellular carcinoma (HCC) is a common solid tumor with a poor prognosis. HCC is often due to hepatitis B virus (HBV) infection. As yet, efficacious HCC treatment regimens for late-stage HCC patients are lacking. Therefore, the identification of more specific and sensitive biomarkers for its early diagnosis and treatment remains an urgent need.<br><br>METHODS: Total RNAs from paired HBV-derived HCC tumors and adjacent peritumor tissues (APTs) were subjected to RNA sequencing (RNA-seq), and differentially expressed genes (DEGs) between HCC tumors and APTs were selected and verified.<br><br>RESULTS: We identified 166 DEGs and found that eight top-ranked and verified DEGs (TK1, CTTN, CEP72, TRIP13, FTH1, FLAD1, CHRM2, AMBP) all contained putative OCT4 binding motifs in their promoter regions. TK1, TRIP13 and OCT4 were found to exhibit concurrent higher expression levels in HCC tumors than in APTs. The mRNA levels of TK1, TRIP13 and OCT4 in a cohort of 384 HCC samples from the TCGA database were all found to be negatively correlated with patient overall survival, relapse-free survival and progression-free survival, underscoring the HCC biomarker status of TK1 and TRIP13 on one hand, and implicating their association with OCT4 on the other hand. Furthermore, OCT4 proteins were found to bind to the promoters of both genes in vitro and in vivo. Knocking out OCT4 in HCC-derived cell lines reduced the expression of TK1 and TRIP13 and significantly decreased their tumorigenicity.<br><br>CONCLUSIONS: Using RNA-seq, we identified several novel HCC signature genes that may serve as biomarkers for its diagnosis and prognosis. Their common transcriptional regulation by OCT4 suggests key roles in the development of HCC, and indicates that OCT4 may serve as a potential therapeutic target.},
}
@article {pmid31848638,
year = {2020},
author = {Li, P and Zhang, L and Li, Z and Xu, C and Du, X and Wu, S},
title = {Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {19},
pages = {3875-3884},
pmid = {31848638},
issn = {1420-9071},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Endodeoxyribonucleases/*genetics ; Gene Editing/*methods ; Genetic Loci ; HEK293 Cells ; Humans ; Plasmids/genetics/metabolism ; RNA, Guide/metabolism ; },
abstract = {Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.},
}
@article {pmid31848331,
year = {2019},
author = {Ujihara, Y and Kanagawa, M and Mohri, S and Takatsu, S and Kobayashi, K and Toda, T and Naruse, K and Katanosaka, Y},
title = {Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5754},
pmid = {31848331},
issn = {2041-1723},
mesh = {Adult ; Age Factors ; Animals ; Animals, Newborn ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Disease Models, Animal ; Dystroglycans/metabolism ; Female ; Gene Knockout Techniques ; Glycosylation ; HEK293 Cells ; Heart Failure/*etiology/pathology ; Heart Ventricles/cytology/pathology ; Humans ; Male ; Membrane Proteins/genetics ; Mice ; Mice, Knockout ; Muscle, Skeletal/cytology/*pathology ; Muscular Dystrophies/*complications/genetics/pathology ; Myocardial Contraction/genetics ; Myocytes, Cardiac/cytology/*pathology ; Primary Cell Culture ; Sarcolemma/pathology ; Transferases/*genetics/metabolism ; },
abstract = {Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.},
}
@article {pmid31848330,
year = {2019},
author = {Woronik, A and Tunström, K and Perry, MW and Neethiraj, R and Stefanescu, C and Celorio-Mancera, MP and Brattström, O and Hill, J and Lehmann, P and Käkelä, R and Wheat, CW},
title = {A transposable element insertion is associated with an alternative life history strategy.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5757},
pmid = {31848330},
issn = {2041-1723},
support = {R00 EY027016/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Butterflies/*physiology ; CRISPR-Cas Systems/genetics ; Color ; DNA Transposable Elements/*genetics ; Female ; Gene Editing/methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; *Genetic Loci ; Homeodomain Proteins/*genetics/metabolism ; *Life History Traits ; Male ; Pigmentation/genetics ; Pigments, Biological/biosynthesis ; Reproduction/genetics ; Sex Factors ; Whole Genome Sequencing ; Wings, Animal/metabolism/ultrastructure ; },
abstract = {Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: either yellow/orange or white. Here we map the locus associated with this ALHS in Colias crocea to a transposable element insertion located downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find white-specific expression of BarH-1 suppresses the formation of pigment granules in wing scales and gives rise to white wing color. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. Together, these findings characterize a mechanism for a female-limited ALHS.},
}
@article {pmid31848272,
year = {2019},
author = {Van Goethem, MW and Swenson, TL and Trubl, G and Roux, S and Northen, TR},
title = {Characteristics of Wetting-Induced Bacteriophage Blooms in Biological Soil Crust.},
journal = {mBio},
volume = {10},
number = {6},
pages = {},
pmid = {31848272},
issn = {2150-7511},
mesh = {Bacillus/physiology/virology ; Bacterial Proteins/chemistry/genetics/metabolism ; *Bacteriophages ; Computational Biology/methods ; *Desert Climate ; Ecosystem ; Firmicutes/genetics/metabolism/virology ; Gene Expression Profiling ; Host-Pathogen Interactions ; Metagenome ; Metagenomics/methods ; *Photosynthesis ; Phylogeny ; *Soil Microbiology ; Structure-Activity Relationship ; },
abstract = {Biological soil crusts (biocrusts) are photosynthetic "hot spots" in deserts and cover ∼12% of the Earth's terrestrial surface, and yet they face an uncertain future given expected shifts in rainfall events. Laboratory wetting of biocrust communities is known to cause a bloom of Firmicutes which rapidly become dominant community members within 2 days after emerging from a sporulated state. We hypothesized that their bacteriophages (phages) would respond to such a dramatic increase in their host's abundance. In our experiment, wetting caused Firmicutes to bloom and triggered a significant depletion of cyanobacterial diversity. We used genome-resolved metagenomics to link phage to their hosts and found that the bloom of the genus Bacillus correlated with a dramatic increase in the number of Caudovirales phages targeting these diverse spore-formers (r = 0.762). After 2 days, we observed dramatic reductions in the relative abundances of Bacillus, while the number of Bacillus phages continued to increase, suggestive of a predator-prey relationship. We found predicted auxiliary metabolic genes (AMGs) associated with sporulation in several Caudovirales genomes, suggesting that phages may influence and even benefit from sporulation dynamics in biocrusts. Prophage elements and CRISPR-Cas repeats in Firmicutes metagenome-assembled genomes (MAGs) provide evidence of recent infection events by phages, which were corroborated by mapping viral contigs to their host MAGs. Combined, these findings suggest that the blooming Firmicutes become primary targets for biocrust Caudovirales phages, consistent with the classical "kill-the-winner" hypothesis.IMPORTANCE This work forms part of an overarching research theme studying the effects of a changing climate on biological soil crust (biocrust) in the Southwestern United States. To our knowledge, this study was the first to characterize bacteriophages in biocrust and offers a view into the ecology of phages in response to a laboratory wetting experiment. The phages identified here represent lineages of Caudovirales, and we found that the dynamics of their interactions with their Firmicutes hosts explain the collapse of a bacterial bloom that was induced by wetting. Moreover, we show that phages carried host-altering metabolic genes and found evidence of proviral infection and CRISPR-Cas repeats within host genomes. Our results suggest that phages exert controls on population density by lysing dominant bacterial hosts and that they further impact biocrust by acquiring host genes for sporulation. Future research should explore how dominant these phages are in other biocrust communities and quantify how much the control and lysis of blooming populations contributes to nutrient cycling in biocrusts.},
}
@article {pmid31846777,
year = {2020},
author = {Sun, Y and Yan, C and Liu, M and Liu, Y and Wang, W and Cheng, W and Yang, F and Zhang, J},
title = {CRISPR/Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda.},
journal = {Fish &amp; shellfish immunology},
volume = {97},
number = {},
pages = {421-431},
doi = {10.1016/j.fsi.2019.12.037},
pmid = {31846777},
issn = {1095-9947},
mesh = {Aeromonas hydrophila/pathogenicity ; Animals ; Arthropod Proteins/*genetics ; *CRISPR-Cas Systems ; Carotenoids/chemistry ; Gene Deletion ; Gene Knockout Techniques/*methods ; Immunity, Innate ; Mutagenesis ; Oxygenases/*genetics ; Palaemonidae/*enzymology/*genetics/microbiology ; Vibrio parahaemolyticus/pathogenicity ; },
abstract = {During the immune defense reaction of invertebrate, a plenty of reactive oxygen species (ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids could function as scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda. EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed. Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 26 EcNinaB-X1-KO prawns were generated and the mutant rate reached 6.5% after embryo injection. The EcNinaB-X1-KO prawns had significant lower mortality than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis.},
}
@article {pmid31845410,
year = {2020},
author = {Tsoumani, KT and Meccariello, A and Mathiopoulos, KD and Papathanos, PA},
title = {Developing CRISPR-based sex-ratio distorters for the genetic control of fruit fly pests: A how to manual.},
journal = {Archives of insect biochemistry and physiology},
volume = {103},
number = {3},
pages = {e21652},
doi = {10.1002/arch.21652},
pmid = {31845410},
issn = {1520-6327},
mesh = {Animals ; *CRISPR-Cas Systems ; Diptera/*genetics ; Female ; Gene Editing ; Insect Control/*methods ; Male ; Pest Control, Biological/*methods ; *Sex Ratio ; },
abstract = {Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.},
}
@article {pmid31844895,
year = {2020},
author = {Ureña-Bailén, G and Lamsfus-Calle, A and Daniel-Moreno, A and Raju, J and Schlegel, P and Seitz, C and Atar, D and Antony, JS and Handgretinger, R and Mezger, M},
title = {CRISPR/Cas9 technology: towards a new generation of improved CAR-T cells for anticancer therapies.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {191-200},
doi = {10.1093/bfgp/elz039},
pmid = {31844895},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Immunotherapy/*methods ; },
abstract = {Chimeric antigen receptor (CAR)-modified T cells have raised among other immunotherapies for cancer treatment, being implemented against B-cell malignancies. Despite the promising outcomes of this innovative technology, CAR-T cells are not exempt from limitations that must yet to be overcome in order to provide reliable and more efficient treatments against other types of cancer. The purpose of this review is to shed light on the field of CAR-T cell gene editing for therapy universalization and further enhancement of antitumor function. Several studies have proven that the disruption of certain key genes is essential to boost immunosuppressive resistance, prevention of fratricide, and clinical safety. Due to its unparalleled simplicity, feasibility to edit multiple gene targets simultaneously, and affordability, CRISPR/CRISPR-associated protein 9 system has been proposed in different clinical trials for such CAR-T cell improvement. The combination of such powerful technologies is expected to provide a new generation of CAR-T cell-based immunotherapies for clinical application.},
}
@article {pmid31844142,
year = {2019},
author = {Masoudi, M and Seki, M and Yazdanparast, R and Yachie, N and Aburatani, H},
title = {A genome-scale CRISPR/Cas9 knockout screening reveals SH3D21 as a sensitizer for gemcitabine.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {19188},
pmid = {31844142},
issn = {2045-2322},
mesh = {Antimetabolites, Antineoplastic/pharmacology ; Apoptosis/drug effects/genetics ; CRISPR-Cas Systems/drug effects/*genetics ; Cell Cycle Checkpoints/drug effects/genetics ; Cell Line ; Cell Line, Tumor ; DNA Replication/drug effects/genetics ; Deoxycytidine/*analogs &amp; derivatives/pharmacology ; Drug Resistance, Neoplasm/drug effects/genetics ; HEK293 Cells ; Humans ; Pancreatic Neoplasms/*drug therapy/*genetics ; },
abstract = {Gemcitabine, 2',2'-difluoro-2'-deoxycytidine, is used as a pro-drug in treatment of variety of solid tumour cancers including pancreatic cancer. After intake, gemcitabine is transferred to the cells by the membrane nucleoside transporter proteins. Once inside the cells, it is converted to gemcitabine triphosphate followed by incorporation into DNA chains where it causes inhibition of DNA replication and thereby cell cycle arrest and apoptosis. Currently gemcitabine is the standard drug for treatment of pancreatic cancer and despite its widespread use its effect is moderate. In this study, we performed a genome-scale CRISPR/Cas9 knockout screening on pancreatic cancer cell line Panc1 to explore the genes that are important for gemcitabine efficacy. We found SH3D21 as a novel gemcitabine sensitizer implying it may act as a therapeutic target for improvement of gemcitabine efficacy in treatment of pancreatic cancer.},
}
@article {pmid31844114,
year = {2019},
author = {Maffei, M and Morelli, C and Graham, E and Patriarca, S and Donzelli, L and Doleschall, B and de Castro Reis, F and Nocchi, L and Chadick, CH and Reymond, L and Corrêa, IR and Johnsson, K and Hackett, JA and Heppenstall, PA},
title = {A ligand-based system for receptor-specific delivery of proteins.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {19214},
pmid = {31844114},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dependovirus/genetics ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Therapy/methods ; Keratinocytes/metabolism ; Ligands ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Proteins/*genetics/*metabolism ; Skin/metabolism ; },
abstract = {Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.},
}
@article {pmid31844051,
year = {2019},
author = {Valderrama, JA and Kulkarni, SS and Nizet, V and Bier, E},
title = {A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5726},
pmid = {31844051},
issn = {2041-1723},
support = {R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Biomedical Technology/methods ; Biotechnology/methods ; CRISPR-Cas Systems/genetics ; Colony Count, Microbial ; DNA Copy Number Variations/*genetics ; Drug Resistance, Bacterial/*genetics ; Escherichia coli/drug effects/genetics ; Gene Drive Technology/*methods ; Genes, Bacterial/*genetics ; Genetic Loci/*genetics ; Genetic Vectors/genetics ; Microbial Sensitivity Tests ; Plasmids/genetics ; RNA, Guide/genetics ; Transformation, Bacterial ; },
abstract = {Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.},
}
@article {pmid31843958,
year = {2020},
author = {Zhao, Y and Sun, X and Yang, X and Zhang, B and Li, S and Han, P and Zhang, B and Wang, X and Li, S and Chang, Y and Wei, W},
title = {Tolerogenic Dendritic Cells Generated by BAFF Silencing Ameliorate Collagen-Induced Arthritis by Modulating the Th17/Regulatory T Cell Balance.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {204},
number = {3},
pages = {518-530},
doi = {10.4049/jimmunol.1900552},
pmid = {31843958},
issn = {1550-6606},
mesh = {Animals ; Arthritis, Experimental/*immunology ; B-Cell Activating Factor/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Dendritic Cells/*immunology ; Forkhead Transcription Factors/genetics/metabolism ; Immune Tolerance ; Immunomodulation ; Male ; Mice ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics/metabolism ; RNA, Small Interfering/genetics ; T-Lymphocytes, Regulatory/*immunology ; Th17 Cells/*immunology ; },
abstract = {Tolerogenic dendritic cells (tolDCs) have received much attention because of their capacity to restore immune homeostasis. RNA interference techniques have been used in several studies to generate tolDCs by inactivating certain molecules that regulate DC maturation and immunologic function. BAFF is a key B cell survival factor that is not only essential for B cell function but also T cell costimulation, and DCs are the major source of BAFF. In this study, we determined whether BAFF gene silencing in mature DCs could lead to a tolerogenic phenotype as well as the potential therapeutic effect of BAFF-silenced DCs on collagen-induced arthritis (CIA) in mice. Meanwhile, CRISPR/Cas9-mediated BAFF[-/-] DC2.4 cells were generated to verify the role of BAFF in DC maturation and functionality. BAFF-silenced DCs and BAFF[-/-] DC2.4 cells exhibited an immature phenotype and functional state. Further, the transplantation of BAFF-silenced DCs significantly alleviated CIA severity in mice, which correlated with a reduction in Th17 populations and increased regulatory T cells. In vitro, BAFF-silenced DCs promoted Foxp3 mRNA and IL-10 expression but inhibited ROR-γt mRNA and IL-17A expression in CD4[+] T cells. Together, BAFF-silenced DCs can alleviate CIA, partly by inducing Foxp3[+] regulatory T cells and suppressing Th17 subsets. Collectively, BAFF plays an important role in interactions between DCs and T cells, which might be a promising genetic target to generate tolDCs for autoimmune arthritis treatment.},
}
@article {pmid31843888,
year = {2020},
author = {Liu, X and Xiao, W and Zhang, Y and Wiley, SE and Zuo, T and Zheng, Y and Chen, N and Chen, L and Wang, X and Zheng, Y and Huang, L and Lin, S and Murphy, AN and Dixon, JE and Xu, P and Guo, X},
title = {Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {1},
pages = {328-336},
pmid = {31843888},
issn = {1091-6490},
support = {R01 DK018024/DK/NIDDK NIH HHS/United States ; R01 GM074830/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Enzyme Assays ; Gene Knock-In Techniques ; Humans ; Membrane Proteins/genetics/*metabolism ; Mice ; Mice, Knockout ; Mice, Transgenic ; Mitochondria/metabolism ; Nuclear Proteins/genetics/*metabolism ; Oxidative Stress ; Phosphoprotein Phosphatases/genetics/*metabolism ; Phosphorylation/physiology ; Proteasome Endopeptidase Complex/genetics/*metabolism ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Protein Subunits/genetics/*metabolism ; RNA, Small Interfering/metabolism ; Serine/metabolism ; Trans-Activators/genetics/metabolism ; },
abstract = {The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.},
}
@article {pmid31843544,
year = {2020},
author = {Navabpour, S and Kwapis, JL and Jarome, TJ},
title = {A neuroscientist's guide to transgenic mice and other genetic tools.},
journal = {Neuroscience and biobehavioral reviews},
volume = {108},
number = {},
pages = {732-748},
pmid = {31843544},
issn = {1873-7528},
support = {K99 AG056596/AG/NIA NIH HHS/United States ; R00 AG056596/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Genetic Techniques ; *Genetic Vectors ; Mice ; *Mice, Transgenic ; Neurosciences/*methods ; },
abstract = {The past decade has produced an explosion in the number and variety of genetic tools available to neuroscientists, resulting in an unprecedented ability to precisely manipulate the genome and epigenome in behaving animals. However, no single resource exists that describes all of the tools available to neuroscientists. Here, we review the genetic, transgenic, and viral techniques that are currently available to probe the complex relationship between genes and cognition. Topics covered include types of traditional transgenic mouse models (knockout, knock-in, reporter lines), inducible systems (Cre-loxP, Tet-On, Tet-Off) and cell- and circuit-specific systems (TetTag, TRAP, DIO-DREADD). Additionally, we provide details on virus-mediated and siRNA/shRNA approaches, as well as a comprehensive discussion of the myriad manipulations that can be made using the CRISPR-Cas9 system, including single base pair editing and spatially- and temporally-regulated gene-specific transcriptional control. Collectively, this review will serve as a guide to assist neuroscientists in identifying and choosing the appropriate genetic tools available to study the complex relationship between the brain and behavior.},
}
@article {pmid31843453,
year = {2020},
author = {Wang, X and Liu, Z and Li, G and Dang, L and Huang, S and He, L and Ma, Y and Li, C and Liu, M and Yang, G and Huang, X and Zhou, F and Ma, X},
title = {Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {2},
pages = {431-440},
pmid = {31843453},
issn = {1525-0024},
mesh = {Adenine/*metabolism ; CRISPR-Cas Systems ; *Codon, Initiator ; *Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; *Gene Silencing ; Genes, Reporter ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; *Mutation ; },
abstract = {Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.},
}
@article {pmid31842424,
year = {2019},
author = {Volkova, EI and Andreyenkova, NG and Andreyenkov, OV and Sidorenko, DS and Zhimulev, IF and Demakov, SA},
title = {Structural and Functional Dissection of the 5' Region of the Notch Gene in Drosophila melanogaster.},
journal = {Genes},
volume = {10},
number = {12},
pages = {},
pmid = {31842424},
issn = {2073-4425},
mesh = {5' Untranslated Regions/genetics ; Animals ; CRISPR-Cas Systems ; Chromosome Structures/genetics ; Drosophila Proteins/*genetics/*metabolism ; Drosophila melanogaster/genetics ; Gene Expression Regulation/genetics ; Homologous Recombination/genetics ; Polytene Chromosomes/genetics ; Receptors, Notch/*genetics/*metabolism ; Structure-Activity Relationship ; },
abstract = {Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5'-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5' nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5'untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5'-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.},
}
@article {pmid31841972,
year = {2020},
author = {Kim, YK and Yu, JH and Min, SH and Park, SW},
title = {Generation of a GLA knock-out human-induced pluripotent stem cell line, KSBCi002-A-1, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {42},
number = {},
pages = {101676},
doi = {10.1016/j.scr.2019.101676},
pmid = {31841972},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Fabry Disease/*genetics/pathology ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mutation ; alpha-Galactosidase/*metabolism ; },
abstract = {Fabry disease is an X-linked inherited disease caused by a mutation in the galactosidase alpha (GLA) gene. Here, we generated a GLA knock-out cell line (GLA-KO hiPSCs) from normal human-induced pluripotent stem cells (hFSiPS1) using the CRISPR-Cas9 genome-editing tool. The GLA-KO hiPSCs maintained normal morphology, karyotypes, expression of stemness markers, and trilineage differentiation potential. Furthermore, the GLA-KO hiPSCs exhibited dissipation of GLA activity and abnormal Globotriaosylceramide (Gb3) accumulation. Our GLA-KO hiPSC line represents a valuable tool for studying the mechanisms involved in Fabry disease and the development of novel therapeutic alternatives to treat this rare condition.},
}
@article {pmid31841614,
year = {2020},
author = {Zhao, C and Devlin, AC and Chouhan, AK and Selvaraj, BT and Stavrou, M and Burr, K and Brivio, V and He, X and Mehta, AR and Story, D and Shaw, CE and Dando, O and Hardingham, GE and Miles, GB and Chandran, S},
title = {Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology.},
journal = {Glia},
volume = {68},
number = {5},
pages = {1046-1064},
pmid = {31841614},
issn = {1098-1136},
support = {MR/T000708/1/MRC_/Medical Research Council/United Kingdom ; SHAW/APR15/970-797/MNDA_/Motor Neurone Disease Association/United Kingdom ; MR/R001162/1/MRC_/Medical Research Council/United Kingdom ; MEHTA/JUL17/948-795/MNDA_/Motor Neurone Disease Association/United Kingdom ; MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; MILES/OCT14/878-792/MNDA_/Motor Neurone Disease Association/United Kingdom ; G0300329/MRC_/Medical Research Council/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; SHAW/NOV14/985-797/MNDA_/Motor Neurone Disease Association/United Kingdom ; },
mesh = {Action Potentials/physiology ; Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology ; Astrocytes/*metabolism/pathology ; C9orf72 Protein/*genetics/metabolism ; Coculture Techniques ; Humans ; Induced Pluripotent Stem Cells/*metabolism/pathology ; Motor Neurons/*metabolism/pathology ; Mutation ; },
abstract = {Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na[+] and K[+] currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.},
}
@article {pmid31840103,
year = {2019},
author = {Sansbury, BM and Hewes, AM and Kmiec, EB},
title = {Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {458},
pmid = {31840103},
issn = {2399-3642},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; *DNA Repair ; *Gene Editing ; Gene Targeting ; *Genetic Variation ; Humans ; },
abstract = {As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.},
}
@article {pmid31840076,
year = {2019},
author = {Rui, Y and Wilson, DR and Choi, J and Varanasi, M and Sanders, K and Karlsson, J and Lim, M and Green, JJ},
title = {Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing.},
journal = {Science advances},
volume = {5},
number = {12},
pages = {eaay3255},
pmid = {31840076},
issn = {2375-2548},
support = {T32 GM007057/GM/NIGMS NIH HHS/United States ; R01 CA228133/CA/NCI NIH HHS/United States ; R01 EB022148/EB/NIBIB NIH HHS/United States ; S10 OD016374/OD/NIH HHS/United States ; P30 EY001765/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cytosol/chemistry ; Gene Editing ; *Gene Transfer Techniques ; Glioma/genetics/pathology/*therapy ; Humans ; Mice ; Nanoparticles/chemistry ; Polymers/chemistry/*pharmacology ; Ribonucleoproteins/genetics ; },
abstract = {Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.},
}
@article {pmid31837184,
year = {2020},
author = {Miyamoto, S and Aoto, K and Hiraide, T and Nakashima, M and Takabayashi, S and Saitsu, H},
title = {Nanopore sequencing reveals a structural alteration of mirror-image duplicated genes in a genome-editing mouse line.},
journal = {Congenital anomalies},
volume = {60},
number = {4},
pages = {120-125},
doi = {10.1111/cga.12364},
pmid = {31837184},
issn = {1741-4520},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Duplication/*genetics ; *Gene Editing ; Humans ; Mice ; *Nanopore Sequencing ; },
abstract = {CRISPR-Cas9 technology has been used in various studies; however, it has also been found to introduce unexpected structural alternations. In this study, we used nanopore sequencing to characterize an unexpected structural alteration of mirror-image duplicated genes in a mouse line, in which we aimed to delete a part of the duplicated genes using genome editing. We removed low-molecular-weight DNA fragments and increased the input, which led to improved sequence performance. With 14.9 Gb input for whole-genome analysis, we detected a complex structural alteration involving inversion and deletion, which appears to be difficult to characterize with short-read sequencers. Therefore, our study clearly showed the utility of nanopore sequencing for characterizing unexpected complex structural alterations caused by genome editing.},
}
@article {pmid31836902,
year = {2020},
author = {N'Tumba-Byn, T and Yamada, M and Seandel, M},
title = {Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.},
journal = {Biology of reproduction},
volume = {102},
number = {4},
pages = {950-962},
pmid = {31836902},
issn = {1529-7268},
support = {DP2 HD080352/HD/NICHD NIH HHS/United States ; },
mesh = {Adult Stem Cells/cytology/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation/*physiology ; Cells, Cultured ; Male ; Mice ; Mice, Knockout ; Receptor, EphB2/genetics/*metabolism ; Signal Transduction/physiology ; Spermatogenesis/physiology ; Spermatogonia/cytology/*metabolism ; },
abstract = {Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.},
}
@article {pmid31836812,
year = {2019},
author = {Hu, X and Cui, Y and Dong, G and Feng, A and Wang, D and Zhao, C and Zhang, Y and Hu, J and Zeng, D and Guo, L and Qian, Q},
title = {Using CRISPR-Cas9 to generate semi-dwarf rice lines in elite landraces.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {19096},
pmid = {31836812},
issn = {2045-2322},
mesh = {Agriculture ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Genes, Plant ; Germination ; Mutation/genetics ; Oryza/*genetics/growth &amp; development ; Phenotype ; Plant Roots/growth &amp; development ; Plants, Genetically Modified ; },
abstract = {Genetic erosion refers to the loss of genetic variation in a crop. In China, only a few original landraces of rice (Oryza sativa) were used in breeding and these became the primary genetic background of modern varieties. Expanding the genetic diversity among Chinese rice varieties and cultivating high-yielding and high-quality varieties with resistance to different biotic and abiotic stresses is critical. Here, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9(Cas9) genome editing system to edit Semi-Dwarf1 (SD1) in the elite landraces Kasalath and TeTePu (TTP), which contain many desired agronomic traits such as tolerance to low phosphorous and broad-spectrum resistance to several diseases and insects. Mutations of SD1 confer shorter plant height for better resistance to lodging. Field trials demonstrated that the yield of the new Kasalath and TTP mutant lines was better than that of the wild type under modern cultivation and that the lines maintained the same desirable agronomic characteristics as their wild-type progenitors. Our results showed that breeding using available landraces in combination with genomic data of different landraces and gene-editing techniques is an effective way to relieve genetic erosion in modern rice varieties.},
}
@article {pmid31836654,
year = {2020},
author = {Bhatta, A and Atianand, M and Jiang, Z and Crabtree, J and Blin, J and Fitzgerald, KA},
title = {A Mitochondrial Micropeptide Is Required for Activation of the Nlrp3 Inflammasome.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {204},
number = {2},
pages = {428-437},
pmid = {31836654},
issn = {1550-6606},
support = {R01 AI083713/AI/NIAID NIH HHS/United States ; T32 AI095213/AI/NIAID NIH HHS/United States ; R37 AI067497/AI/NIAID NIH HHS/United States ; R01 AI067497/AI/NIAID NIH HHS/United States ; R56 AI067497/AI/NIAID NIH HHS/United States ; P01 AI083215/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Cytosol/*metabolism ; Humans ; Immunity, Innate/genetics ; Inflammasomes/*metabolism ; Lipopolysaccharides/immunology ; Macrophages/*physiology ; Mice ; Mitochondria/genetics/*metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/*metabolism ; Peptide Fragments/genetics/*metabolism ; RNA, Long Noncoding/*genetics ; RNA, Small Interfering/genetics ; },
abstract = {Functional peptides encoded by short open reading frames are emerging as important mediators of fundamental biological processes. In this study, we identified a micropeptide produced from a putative long noncoding RNA (lncRNAs) that is important in controlling innate immunity. By studying lncRNAs in mice macrophages, we identified lncRNA 1810058I24Rik, which was downregulated in both human and murine myeloid cells exposed to LPS as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed that this transcript was localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with [35]S-labeled methionine resulted in translation of a 47 aa micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene mitochondrial micropeptide-47 (Mm47). Crispr-Cas9-mediated deletion of Mm47, as well as small interfering RNA studies in mice primary macrophages, showed that the transcriptional response downstream of TLR4 was intact in cells lacking Mm47. In contrast, Mm47-deficient or knockdown cells were compromised for Nlrp3 inflammasome responses. Activation of Nlrc4 or Aim2 inflammasomes were intact in cells lacking Mm47. This study therefore identifies, to our knowledge, a novel mitochondrial micropeptide Mm47 that is required for the activation of the Nlrp3 inflammasome. This work further highlights the functional activity of short open reading frame-encoded peptides and underscores their importance in innate immunity.},
}
@article {pmid31836139,
year = {2020},
author = {An, Y and Park, KH and Lee, M and Kim, TJ and Woo, EJ},
title = {Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system.},
journal = {Biochemical and biophysical research communications},
volume = {523},
number = {1},
pages = {112-116},
doi = {10.1016/j.bbrc.2019.12.046},
pmid = {31836139},
issn = {1090-2104},
mesh = {Apoproteins/chemistry/genetics ; CRISPR-Associated Proteins/*chemistry/genetics ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; Models, Molecular ; Protein Subunits/chemistry/genetics ; },
abstract = {The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the β-hairpin motif (β5-β6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the β-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.},
}
@article {pmid31835565,
year = {2019},
author = {Dorn, A and Puchta, H},
title = {DNA Helicases as Safekeepers of Genome Stability in Plants.},
journal = {Genes},
volume = {10},
number = {12},
pages = {},
pmid = {31835565},
issn = {2073-4425},
mesh = {DNA/metabolism ; DNA Helicases/genetics/*metabolism ; DNA Repair/physiology ; Genome, Plant/*genetics/physiology ; Genomic Instability/genetics/*physiology ; Meiosis/genetics ; Plants/enzymology ; RecQ Helicases/metabolism/physiology ; Recombinant Proteins/genetics/metabolism ; },
abstract = {Genetic information of all organisms is coded in double-stranded DNA. DNA helicases are essential for unwinding this double strand when it comes to replication, repair or transcription of genetic information. In this review, we will focus on what is known about a variety of DNA helicases that are required to ensure genome stability in plants. Due to their sessile lifestyle, plants are especially exposed to harmful environmental factors. Moreover, many crop plants have large and highly repetitive genomes, making them absolutely dependent on the correct interplay of DNA helicases for safeguarding their stability. Although basic features of a number of these enzymes are conserved between plants and other eukaryotes, a more detailed analysis shows surprising peculiarities, partly also between different plant species. This is additionally of high relevance for plant breeding as a number of these helicases are also involved in crossover control during meiosis and influence the outcome of different approaches of CRISPR/Cas based plant genome engineering. Thus, gaining knowledge about plant helicases, their interplay, as well as the manipulation of their pathways, possesses the potential for improving agriculture. In the long run, this might even help us cope with the increasing obstacles of climate change threatening food security in completely new ways.},
}
@article {pmid31835025,
year = {2019},
author = {Yang, L and Yang, B and Chen, J},
title = {One Prime for All Editing.},
journal = {Cell},
volume = {179},
number = {7},
pages = {1448-1450},
doi = {10.1016/j.cell.2019.11.030},
pmid = {31835025},
issn = {1097-4172},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; },
abstract = {Many targeted base transversions, insertions, and deletions remain challenging due to the lack of precise and efficient genome editing technologies. Recently, Anzalone et al. reported a versatile approach to achieve all types of genome edits, shedding new light on correcting most genetic variants associated with diseases.},
}
@article {pmid31834846,
year = {2020},
author = {Liao, W and Liu, Y and Chen, C and Li, J and Du, F and Long, D and Zhang, W},
title = {Distribution of CRISPR-Cas Systems in Clinical Carbapenem-Resistant Klebsiella pneumoniae Strains in a Chinese Tertiary Hospital and Its Potential Relationship with Virulence.},
journal = {Microbial drug resistance (Larchmont, N.Y.)},
volume = {26},
number = {6},
pages = {630-636},
doi = {10.1089/mdr.2019.0276},
pmid = {31834846},
issn = {1931-8448},
mesh = {Adult ; Aged ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/biosynthesis/genetics ; *CRISPR-Cas Systems ; Carbapenem-Resistant Enterobacteriaceae/drug effects/*genetics/*pathogenicity ; China ; DNA, Bacterial ; Female ; Genes, Bacterial ; Humans ; Klebsiella pneumoniae/drug effects/*genetics/*pathogenicity ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Multilocus Sequence Typing ; Tertiary Care Centers ; Virulence/genetics ; beta-Lactamases/biosynthesis/genetics ; },
abstract = {Aim: In this study, we aimed to characterize the CRISPR-Cas systems in clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates and to investigate the potential association of CRISPR-Cas systems with bacterial virulence. Methods: A total of 168 CRKP strains were collected from inpatients in a teaching hospital in Jiangxi Province. Five common carbapenemase genes, subtype genes of the CRISPR-Cas system, and 13 virulence genes were amplified by PCR using specific primers. The potential virulence of all the clinical CRKP strains was tested in a Galleria mellonella infection model. Results: PCR analysis of five common carbapenemase genes revealed the frequency of carbapenemase gene KPC-2 was the highest in the CRISPR-negative strains, compared to CRISPR type I-E* strains or CRISPR type I-E strains (p < 0.01). Isolates having the subtype I-E* CRISPR-Cas system tended to have more virulence genes such as magA, kfu, wcaG, and allS, compared to CRISPR-negative isolates and type I-E CRISPR-Cas isolates (p < 0.01). The average survival time of the larvae infected with the isolates having the subtype I-E* CRISPR-Cas system was significantly shorter than the other two group isolates (p < 0.05). Conclusion: The CRKP strains, which had the subtype I-E CRISPR-Cas system or the subtype I-E* CRISPR-Cas system, showed reduced acquisition of carbapenemase genes compared to CRISPR-negative isolates. Importantly, we first found that a small portion of "CR-hvKP" strains were selected from the CRKP clones, which had the type I-E* CRISPR-Cas systems.},
}
@article {pmid31834612,
year = {2020},
author = {Oh-Hashi, K and Kohno, H and Kandeel, M and Hirata, Y},
title = {Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches.},
journal = {Molecular and cellular biochemistry},
volume = {465},
number = {1-2},
pages = {53-64},
pmid = {31834612},
issn = {1573-4919},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cell Survival/drug effects/genetics ; Cytotoxins/*pharmacology ; Endoplasmic Reticulum Stress/drug effects/genetics ; Endoribonucleases/*deficiency ; *Gene Deletion ; Gene Expression Regulation/*drug effects ; Mice ; *Microtubule-Associated Proteins ; Protein Serine-Threonine Kinases/*deficiency ; },
abstract = {IRE1 is the most conserved endoplasmic reticulum (ER)-resident stress sensor. Its activation not only splices XBP1 but also participates in a variety of cell signaling. We elucidated the role of IRE1α in Neuro2a cells by establishing IRE1α-deficient cells and applying four IRE1 inhibitors. IRE1α deficiency prevented almost all spliced XBP1 (sXBP1) protein expression by treatment with thapsigargin (Tg) and tunicamycin (Tm); these phenomena paralleled the values measured by our two Nanoluciferase-based IRE1 assays. However, cell viability and protein expression of other ER stress-responsive factors in the IRE1α-deficient cells were comparable to those in the parental wild-type cells with or without Tm treatment. Next, we elucidated the IRE1 inhibitory actions and cytotoxicity of four compounds: STF083010, KIRA6, 4μ8C, and toyocamycin. KIRA6 attenuated IRE1 activity in a dose-dependent manner, but it showed severe cytotoxicity even in the IRE1α-deficient cells at a low concentration. The IRE1α-deficient cells were slightly resistant to KIRA6 at 0.1 μM in both the presence and absence of ER stress; however, resistance was not observed at 0.02 μM. Treatment with only KIRA6 at 0.1 μM for 12 h remarkably induced LC3 II, an autophagic marker, in both parental and IRE1α-deficient cells. Co-treatment with KIRA6 and Tm induced LC3 II, cleaved caspase-9, and cleaved caspase-3; however, IRE1α-deficiency did not abolish the expression of these two cleaved caspases. On the other hand, KIRA6 prohibited Tm-induced ATF4 induction in an IRE1-independent manner; however, co-treatment with KIRA6 and Tm also induced LC3 II and two cleaved caspases in the ATF4-deficient Neuro2a cells. Thus, we demonstrate that IRE1α deficiency has little impact on cell viability and expression of ER stress-responsive factors in Neuro2a cells, and the pharmacological actions of KIRA6 include IRE1-independent ways.},
}
@article {pmid31831810,
year = {2019},
author = {Nakamura, M and Okamura, Y and Iwai, H},
title = {Plasmid-based and -free methods using CRISPR/Cas9 system for replacement of targeted genes in Colletotrichum sansevieriae.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18947},
pmid = {31831810},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Colletotrichum/*genetics ; *Gene Targeting ; *Genes, Fungal ; Plasmids/*genetics ; },
abstract = {The CRISPR-Cas9 system has a potential for wide application in organisms that particularly present low homologous integration rates. In this study, we developed three different methods using this system to replace a gene through homology-directed repair in the plant pathogenic fungus Colletotrichum sansevieriae, which has a low recombination frequency. The gene encoding scytalone dehydratase was used as the target so that mutants can be readily distinguished owning to a lack of melanin biosynthesis. First, we performed a plasmid-based method using plasmids containing a Cas9 expression cassette and/or a single-guide RNA (sgRNA) under the control of the endogenous U6 snRNA promoter, and 67 out of 69 (97.1%) transformants exhibited a melanin-deficient phenotype with high efficiency. Second, we performed a transformation using a Cas9 protein/sgRNA complex and obtained 23 out of 28 (82.1%) transformants. Lastly, we developed a hybrid system combining a Cas9 protein and donor DNA-sgRNA expression plasmid, which yielded 75 out of 84 (89.2%) transformants. This system was also applicable to four other genes at different loci of the fungus. This is the first study to establish a CRISPR/Cas9 gene replacement system in Colletotrichum spp. and it presents a potential application for a broad range of use in other species of the genus.},
}
@article {pmid31831639,
year = {2019},
author = {Guo, CJ and Allen, BM and Hiam, KJ and Dodd, D and Van Treuren, W and Higginbottom, S and Nagashima, K and Fischer, CR and Sonnenburg, JL and Spitzer, MH and Fischbach, MA},
title = {Depletion of microbiome-derived molecules in the host using Clostridium genetics.},
journal = {Science (New York, N.Y.)},
volume = {366},
number = {6471},
pages = {},
pmid = {31831639},
issn = {1095-9203},
support = {R01 DK101674/DK/NIDDK NIH HHS/United States ; K08 DK110335/DK/NIDDK NIH HHS/United States ; R01 DK085025/DK/NIDDK NIH HHS/United States ; DP2 HD101401/HD/NICHD NIH HHS/United States ; DP5 OD023056/OD/NIH HHS/United States ; DP1 DK113598/DK/NIDDK NIH HHS/United States ; T32 GM008568/GM/NIGMS NIH HHS/United States ; S10 OD018040/OD/NIH HHS/United States ; R01 DK110174/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Clostridium/*genetics/*metabolism ; Gastrointestinal Microbiome/*genetics ; Gene Deletion ; Gene Editing/*methods ; *Host Microbial Interactions ; Metabolic Networks and Pathways/*genetics ; Mice ; Mice, Inbred Strains ; },
abstract = {The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.},
}
@article {pmid31830738,
year = {2020},
author = {Mc Carlie, S and Boucher, CE and Bragg, RR},
title = {Molecular basis of bacterial disinfectant resistance.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {48},
number = {},
pages = {100672},
doi = {10.1016/j.drup.2019.100672},
pmid = {31830738},
issn = {1532-2084},
mesh = {Animals ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Bacteria/*drug effects/*genetics ; Bacterial Infections/*drug therapy/*genetics ; CRISPR-Cas Systems/genetics ; Disinfectants/*pharmacology/*therapeutic use ; Drug Resistance, Multiple, Bacterial/*genetics ; Humans ; },
abstract = {Antibiotic resistance could accelerate humanity towards an already fast-approaching post-antibiotic era, where disinfectants and effective biosecurity measures will be critically important to control microbial diseases. Disinfectant resistance has the potential to change our way of life from compromising food security to threatening our medical health systems. Resistance to antimicrobial agents occurs through either intrinsic or acquired resistance mechanisms. Acquired resistance occurs through the efficient transfer of mobile genetic elements, which can carry single, or multiple resistance determinants. Drug resistance genes may form part of integrons, transposons and insertions sequences which are capable of intracellular transfer onto plasmids or gene cassettes. Thereafter, resistance plasmids and gene cassettes mobilize by self-transmission between bacteria, increasing the prevalence of drug resistance determinants in a bacterial population. An accumulation of drug resistance genes through these mechanisms gives rise to multidrug resistant (MDR) bacteria. The study of this mobility is integral to safeguard current antibiotics, disinfectants and other antimicrobials. Literature evidence, however, indicates that knowledge regarding disinfectant resistance is severly limited. Genome engineering such as the CRISPR-Cas system, has identified disinfectant resistance genes, and reversed resistance altogether in certain prokaryotes. Demonstrating that these techniques could prove invaluable in the combat against disinfectant resistance by uncovering the secrets of MDR bacteria.},
}
@article {pmid31830647,
year = {2020},
author = {Trionfini, P and Ciampi, O and Romano, E and Benigni, A and Tomasoni, S},
title = {Generation of two isogenic knockout PKD2 iPS cell lines, IRFMNi003-A-1 and IRFMNi003-A-2, using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {42},
number = {},
pages = {101667},
doi = {10.1016/j.scr.2019.101667},
pmid = {31830647},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mutation ; TRPP Cation Channels/*genetics ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can lead to kidney failure resulting in end-stage renal disease. ADPKD is mainly caused by mutations in either the PKD1 and PKD2 genes, encoding for polycystin-1 and polycystin-2, respectively. In order to clarify the disease mechanisms, here we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines in which the PKD2 gene was deleted using CRISPR/Cas9 technology. The PKD2[-/-] iPSCs expressed the main pluripotency markers, were able to differentiate into the three germ layers and had a normal karyotype.},
}
@article {pmid31830033,
year = {2019},
author = {Dunipace, L and Ákos, Z and Stathopoulos, A},
title = {Coacting enhancers can have complementary functions within gene regulatory networks and promote canalization.},
journal = {PLoS genetics},
volume = {15},
number = {12},
pages = {e1008525},
pmid = {31830033},
issn = {1553-7404},
support = {R35 GM118146/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Body Patterning ; CRISPR-Cas Systems ; Drosophila/genetics/*growth &amp; development ; Drosophila Proteins/genetics ; Enhancer Elements, Genetic ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Developmental ; *Gene Regulatory Networks ; Phenotype ; Repressor Proteins/genetics ; Sequence Deletion ; },
abstract = {Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers, brk5' and brk3', that drive similar but not identical expression of the gene brinker (brk) in early Drosophila embryos. Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5' deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3' deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.},
}
@article {pmid31829959,
year = {2019},
author = {Becú-Villalobos, D},
title = {[CRISPR-CAS9 in medicine, the saga continues].},
journal = {Medicina},
volume = {79},
number = {6},
pages = {522-523},
pmid = {31829959},
issn = {1669-9106},
mesh = {CRISPR-Cas Systems/*genetics ; Embryo, Mammalian ; Gene Editing ; Humans ; Mutation/genetics ; },
}
@article {pmid31829621,
year = {2020},
author = {Knoot, CJ and Biswas, S and Pakrasi, HB},
title = {Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973.},
journal = {ACS synthetic biology},
volume = {9},
number = {1},
pages = {132-143},
doi = {10.1021/acssynbio.9b00417},
pmid = {31829621},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; Base Sequence ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; Gene Knockdown Techniques ; Isopropyl Thiogalactoside/pharmacology ; Lac Operon ; Lac Repressors/genetics ; Luminescent Proteins/genetics ; Microorganisms, Genetically-Modified ; Photosynthesis/*genetics ; Photosystem I Protein Complex/genetics ; Plasmids/genetics ; RNA, Guide/genetics ; Synechococcus/*genetics/*growth &amp; development ; },
abstract = {Cyanobacteria are photoautotrophic prokaryotes that serve as key model organisms to study basic photosynthetic processes and are potential carbon-negative production chassis for commodity and high-value chemicals. The development of new synthetic biology tools and improvement of current ones is a requisite for furthering these organisms as models and production vehicles. CRISPR interference (CRISPRi) allows for targeted gene repression using a DNase-dead Cas nuclease ("dCas"). Here, we describe a titratable dCas12a (dCpf1) CRISPRi system and apply it to repress key photosynthetic processes in the fast-growing cyanobacterium Synechococcus sp. UTEX 2973 (S2973). The system relies on a lac repressor system that retains tight regulation in the absence of inducer (0-10% repression) while maintaining the capability for >90% repression of high-abundance gene targets. We determined that dCas12a is less toxic than dCas9. We tested the efficacy of the system toward eYFP and three native targets in S2973: the phycobilisome antenna, glycogen synthesis, and photosystem I (PSI), an essential part of the photosynthetic electron transport chain in oxygenic photoautotrophs. PSI was knocked down indirectly by repressing the protein factor BtpA involved in stabilizing core PSI proteins. We could reduce cellular PSI titer by 87% under photoautotrophic conditions, and we characterized these cells to gain insights into the response of the strain to the low PSI content. The ability to tightly regulate and time the (de)repression of essential genes in trans will allow for the study of photosynthetic processes that are not accessible using knockout mutants.},
}
@article {pmid31829168,
year = {2019},
author = {Liu, W and Meridew, JA and Aravamudhan, A and Ligresti, G and Tschumperlin, DJ and Tan, Q},
title = {Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression.},
journal = {Respiratory research},
volume = {20},
number = {1},
pages = {281},
pmid = {31829168},
issn = {1465-993X},
support = {R01 HL092961/HL/NHLBI NIH HHS/United States ; R01 HL142596/HL/NHLBI NIH HHS/United States ; },
mesh = {Adipogenesis ; CCAAT-Enhancer-Binding Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Case-Control Studies ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblasts/drug effects/*metabolism/pathology ; Fibrosis ; *Gene Editing ; Gene Expression Regulation ; Humans ; Idiopathic Pulmonary Fibrosis/genetics/*metabolism/pathology ; Lung/drug effects/*metabolism/pathology ; Phenotype ; RNA Interference ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology ; },
abstract = {BACKGROUND: Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated.<br><br>METHODS: Lung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-&#x3b2;1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression.<br><br>RESULTS: We found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and &#x3b1;SMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states.<br><br>CONCLUSIONS: CEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.},
}
@article {pmid31829086,
year = {2020},
author = {Liao, KC and Chuo, V and Fagg, WS and Bradrick, SS and Pompon, J and Garcia-Blanco, MA},
title = {The RNA binding protein Quaking represses host interferon response by downregulating MAVS.},
journal = {RNA biology},
volume = {17},
number = {3},
pages = {366-380},
pmid = {31829086},
issn = {1555-8584},
mesh = {A549 Cells ; Adaptor Proteins, Signal Transducing/genetics/*metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation ; Host-Pathogen Interactions/*physiology ; Humans ; Interferon Regulatory Factor-3/genetics/metabolism ; Interferon Type I/genetics/*metabolism ; Phosphorylation ; Poly I-C/genetics/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Respirovirus Infections/metabolism ; Sendai virus/pathogenicity ; },
abstract = {Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNβ transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNβ activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNβ induction. Consistently, ectopic expression of RIG-I activates stronger IFNβ induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.},
}
@article {pmid31828084,
year = {2019},
author = {Wu, J and Tang, Y and Zhang, CL},
title = {Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9.},
journal = {BioMed research international},
volume = {2019},
number = {},
pages = {1039623},
pmid = {31828084},
issn = {2314-6141},
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Expression/genetics ; HEK293 Cells ; Humans ; Huntingtin Protein/*genetics ; Huntington Disease/genetics ; Nerve Degeneration/genetics ; Protein Biosynthesis/genetics ; RNA/*genetics ; Sequence Alignment ; },
abstract = {Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.},
}
@article {pmid31827283,
year = {2019},
author = {Wei, J and Long, L and Zheng, W and Dhungana, Y and Lim, SA and Guy, C and Wang, Y and Wang, YD and Qian, C and Xu, B and Kc, A and Saravia, J and Huang, H and Yu, J and Doench, JG and Geiger, TL and Chi, H},
title = {Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.},
journal = {Nature},
volume = {576},
number = {7787},
pages = {471-476},
pmid = {31827283},
issn = {1476-4687},
support = {AI140761/NH/NIH HHS/United States ; R01 AI131703/AI/NIAID NIH HHS/United States ; CA221290/NH/NIH HHS/United States ; AI150241/NH/NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; AI131703/NH/NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; AI105887/NH/NIH HHS/United States ; R01 CA176624/CA/NCI NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; R01 AI105887/AI/NIAID NIH HHS/United States ; AI150514/NH/NIH HHS/United States ; R01 CA221290/CA/NCI NIH HHS/United States ; R01 GM134382/GM/NIGMS NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/deficiency/metabolism ; CD8-Positive T-Lymphocytes/cytology/*immunology ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Female ; Gene Deletion ; Humans ; Immunotherapy, Adoptive/*methods ; Leukemia/genetics/*immunology/metabolism/*therapy ; Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; Melanoma/genetics/*immunology/metabolism/*therapy ; Mice ; Mitochondria/metabolism ; *Molecular Targeted Therapy ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics/metabolism ; Reproducibility of Results ; Ribonucleases/deficiency/genetics/immunology/*metabolism ; Suppressor of Cytokine Signaling 1 Protein/genetics/metabolism ; Tumor Microenvironment/immunology ; },
abstract = {Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells[1]. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8[+] T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8[+] T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8[+] T cells. By contrast, the targeting of additional signalling factors-including PTPN2 and SOCS1-improves the therapeutic efficacy of REGNASE-1-deficient CD8[+] T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.},
}
@article {pmid31827259,
year = {2020},
author = {Schuh, RS and Gonzalez, EA and Tavares, AMV and Seolin, BG and Elias, LS and Vera, LNP and Kubaski, F and Poletto, E and Giugliani, R and Teixeira, HF and Matte, U and Baldo, G},
title = {Neonatal nonviral gene editing with the CRISPR/Cas9 system improves some cardiovascular, respiratory, and bone disease features of the mucopolysaccharidosis I phenotype in mice.},
journal = {Gene therapy},
volume = {27},
number = {1-2},
pages = {74-84},
pmid = {31827259},
issn = {1476-5462},
mesh = {Animals ; Animals, Newborn ; Bone Diseases/genetics ; CRISPR-Cas Systems/genetics ; Cardiovascular System/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Female ; Gene Editing/methods ; Genetic Therapy/methods ; Glycosaminoglycans/metabolism ; Iduronidase/*genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mucopolysaccharidosis I/genetics/physiopathology/*therapy ; Phenotype ; RNA, Untranslated/*genetics/metabolism ; },
abstract = {Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS I patients may die in the first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua gene aiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in various tissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach "hard-to-treat" tissues to ensure better systemic and neurological effects.},
}
@article {pmid31827197,
year = {2019},
author = {Li, B and Ren, N and Yang, L and Liu, J and Huang, Q},
title = {A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18877},
pmid = {31827197},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genome ; HEK293 Cells ; Humans ; RNA, Guide/*genetics ; *Real-Time Polymerase Chain Reaction ; },
abstract = {CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3' end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.},
}
@article {pmid31826245,
year = {2020},
author = {Azouzi, S and Mikdar, M and Hermand, P and Gautier, EF and Salnot, V and Willemetz, A and Nicolas, G and Vrignaud, C and Raneri, A and Mayeux, P and Bole-Feysot, C and Nitschké, P and Cartron, JP and Colin, Y and Hermine, O and Jedlitschky, G and Cloutier, M and Constanzo-Yanez, J and Ethier, C and Robitaille, N and St-Louis, M and Le Van Kim, C and Peyrard, T},
title = {Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation.},
journal = {Blood},
volume = {135},
number = {6},
pages = {441-448},
pmid = {31826245},
issn = {1528-0020},
mesh = {Blood Group Antigens/*genetics ; Blood Platelets/cytology/metabolism ; CRISPR-Cas Systems ; Erythroid Cells/cytology/metabolism ; Gene Deletion ; Humans ; Multidrug Resistance-Associated Proteins/*genetics ; Phenotype ; *Platelet Aggregation ; },
abstract = {The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.},
}
@article {pmid31825816,
year = {2020},
author = {Benetó, N and Cozar, M and Gort, L and Pacheco, L and Vilageliu, L and Grinberg, D and Canals, I},
title = {Generation of two NAGLU-mutated homozygous cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo B syndrome.},
journal = {Stem cell research},
volume = {42},
number = {},
pages = {101668},
doi = {10.1016/j.scr.2019.101668},
pmid = {31825816},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mucopolysaccharidosis III/*genetics/pathology ; },
}
@article {pmid31825677,
year = {2020},
author = {Zhao, H and Li, Z and Zhu, Y and Hao, B},
title = {A linear-amplification VDJ-seq technique for quantification of immunoglobulin and T cell receptor diversity.},
journal = {Genome},
volume = {63},
number = {3},
pages = {145-153},
doi = {10.1139/gen-2019-0096},
pmid = {31825677},
issn = {1480-3321},
mesh = {Animals ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/*methods ; Immunoglobulins/*genetics ; Mice ; Receptors, Antigen, T-Cell/*genetics ; Sequence Analysis, DNA ; V(D)J Recombination ; },
abstract = {The V(D)J recombination is essential for generating a highly diverse repertoire of antigen receptors expressed on T and B lymphocytes. Here, we developed a linear-amplification VDJ-seq technique for quantifying V(D)J recombination of antigen receptor genes. This technique takes advantage of linear amplification using in vitro transcription and reverse transcription to avoid bias generated by the PCR amplification of low copy number of target DNA. The unrearranged alleles are removed by in vitro cleavage with the CRISPR-Cas9 system. The linear-amplification VDJ-seq assay was applied in quantification of the Vκ-Jκ recombination of the mouse Igκ gene with Jκ capture primers. The Jκ genes were detected in 95.86% of clean reads with more than half containing the Vκ gene, indicating high specificity of capturing and amplification. We also applied this approach to quantify the usage of Jα within the Trav12 gene family of the Tcra gene.},
}
@article {pmid31825599,
year = {2020},
author = {Dalvie, NC and Leal, J and Whittaker, CA and Yang, Y and Brady, JR and Love, KR and Love, JC},
title = {Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii.},
journal = {ACS synthetic biology},
volume = {9},
number = {1},
pages = {26-35},
pmid = {31825599},
issn = {2161-5063},
support = {P30 CA014051/CA/NCI NIH HHS/United States ; T32 GM008334/GM/NIGMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; Base Sequence ; Biological Products ; *CRISPR-Cas Systems ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; *Genome, Fungal ; Glycerol Kinase/genetics ; Glycosylation ; Phenotype ; Promoter Regions, Genetic ; Protein Processing, Post-Translational ; RNA Polymerase III/genetics ; RNA, Guide/*genetics ; Saccharomycetales/*genetics ; Transcriptome ; },
abstract = {There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.},
}
@article {pmid31824468,
year = {2019},
author = {Ipoutcha, T and Tsarmpopoulos, I and Talenton, V and Gaspin, C and Moisan, A and Walker, CA and Brownlie, J and Blanchard, A and Thebault, P and Sirand-Pugnet, P},
title = {Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria (Mollicutes).},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2701},
pmid = {31824468},
issn = {1664-302X},
abstract = {CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5' region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.},
}
@article {pmid31823826,
year = {2019},
author = {Vasquez-Rifo, A and Veksler-Lublinsky, I and Cheng, Z and Ausubel, FM and Ambros, V},
title = {The Pseudomonas aeruginosa accessory genome elements influence virulence towards Caenorhabditis elegans.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {270},
pmid = {31823826},
issn = {1474-760X},
support = {R01GM088365/NH/NIH HHS/United States ; R01GM034028/NH/NIH HHS/United States ; R01AI085581/NH/NIH HHS/United States ; P30DK040561/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*microbiology ; Genome, Bacterial ; Host-Pathogen Interactions/*genetics ; Interspersed Repetitive Sequences ; Pseudomonas aeruginosa/*genetics/*pathogenicity ; },
abstract = {BACKGROUND: Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans to provide evolutionary insights into host-pathogen relationships.<br><br>RESULTS: Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identify accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we find a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence while the presence of CRISPR-Cas associates with higher virulence.<br><br>CONCLUSIONS: Our analysis demonstrates substantial strain variation in P. aeruginosa virulence, mediated by specific accessory genome elements that promote increased or decreased virulence. We exemplify that viral-like accessory genome elements that decrease virulence can be restricted by bacterial CRISPR-Cas immune defense systems, and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in virulence maintenance.},
}
@article {pmid31823200,
year = {2020},
author = {Nazipova, NN and Shabalina, SA},
title = {Understanding off-target effects through hybridization kinetics and thermodynamics.},
journal = {Cell biology and toxicology},
volume = {36},
number = {1},
pages = {11-15},
pmid = {31823200},
issn = {1573-6822},
mesh = {Artifacts ; Base Pair Mismatch/genetics/physiology ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods/trends ; Humans ; Kinetics ; Nucleic Acid Hybridization/*methods ; Thermodynamics ; },
}
@article {pmid31822210,
year = {2019},
author = {Espinosa-Medina, I and Garcia-Marques, J and Cepko, C and Lee, T},
title = {High-throughput dense reconstruction of cell lineages.},
journal = {Open biology},
volume = {9},
number = {12},
pages = {190229},
pmid = {31822210},
issn = {2046-2441},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Lineage/*genetics ; *Cell Tracking/methods ; Computational Biology ; DNA Barcoding, Taxonomic ; *Gene Expression Profiling/methods ; *High-Throughput Nucleotide Sequencing/methods ; Humans ; *Molecular Imaging/methods ; Mutation ; Phylogeny ; Single-Cell Analysis/methods ; },
abstract = {The first meeting exclusively dedicated to the 'High-throughput dense reconstruction of cell lineages' took place at Janelia Research Campus (Howard Hughes Medical Institute) from 14 to 18 April 2019. Organized by Tzumin Lee, Connie Cepko, Jorge Garcia-Marques and Isabel Espinosa-Medina, this meeting echoed the recent eruption of new tools that allow the reconstruction of lineages based on the phylogenetic analysis of DNA mutations induced during development. Combined with single-cell RNA sequencing, these tools promise to solve the lineage of complex model organisms at single-cell resolution. Here, we compile the conference consensus on the technological and computational challenges emerging from the use of the new strategies, as well as potential solutions.},
}
@article {pmid31821678,
year = {2020},
author = {Bi, H and Fei, Q and Li, R and Liu, B and Xia, R and Char, SN and Meyers, BC and Yang, B},
title = {Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production.},
journal = {Plant biotechnology journal},
volume = {18},
number = {7},
pages = {1526-1536},
pmid = {31821678},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *MicroRNAs/genetics ; *Transcription Activator-Like Effector Nucleases ; },
abstract = {MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator-like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild-type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9-mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double-guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single-guide RNA. The difference between the phenotypic severity of miR160a mutants in Col-0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.},
}
@article {pmid31821359,
year = {2019},
author = {Matson, AW and Hosny, N and Swanson, ZA and Hering, BJ and Burlak, C},
title = {Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.},
journal = {PloS one},
volume = {14},
number = {12},
pages = {e0226107},
pmid = {31821359},
issn = {1932-6203},
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems/*genetics ; DNA/genetics/*metabolism ; DNA Cleavage ; Galactosyltransferases/deficiency/*genetics ; Gene Editing/*methods ; RNA, Guide/chemistry/*metabolism ; Ribonucleoproteins/metabolism ; Swine ; },
abstract = {The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.},
}
@article {pmid31821343,
year = {2019},
author = {Elso, CM and Scott, NA and Mariana, L and Masterman, EI and Sutherland, APR and Thomas, HE and Mannering, SI},
title = {Replacing murine insulin 1 with human insulin protects NOD mice from diabetes.},
journal = {PloS one},
volume = {14},
number = {12},
pages = {e0225021},
pmid = {31821343},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems ; Diabetes Mellitus, Type 1/*genetics/metabolism ; Disease Models, Animal ; Humans ; Insulin/*genetics/metabolism ; Insulin-Secreting Cells/*metabolism ; Islets of Langerhans/metabolism ; Mice ; Mice, Inbred NOD ; Pancreas/metabolism ; },
abstract = {Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.},
}
@article {pmid31819946,
year = {2020},
author = {Pei, WD and Zhang, Y and Yin, TL and Yu, Y},
title = {Epigenome editing by CRISPR/Cas9 in clinical settings: possibilities and challenges.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {215-228},
doi = {10.1093/bfgp/elz035},
pmid = {31819946},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenome/genetics ; Gene Editing/methods ; Humans ; },
abstract = {Epigenome editing is a promising approach for both basic research and clinical application. With the convergence of techniques from different fields, regulating gene expression artificially becomes possible. From a clinical point of view, targeted epigenome editing by CRISPR/Cas9 of disease-related genes offers novel therapeutic avenues for many diseases. In this review, we summarize the EpiEffectors used in epigenome editing by CRISPR/Cas9, current applications of epigenome editing and progress made in this field. Moreover, application challenges such as off-target effects, inefficient delivery, stability and immunogenicity are discussed. In conclusion, epigenome editing by CRISPR/Cas9 has broad prospects in the clinic, and future work will promote the application of this technology.},
}
@article {pmid31819262,
year = {2020},
author = {Mendoza, SD and Nieweglowska, ES and Govindarajan, S and Leon, LM and Berry, JD and Tiwari, A and Chaikeeratisak, V and Pogliano, J and Agard, DA and Bondy-Denomy, J},
title = {A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {244-248},
pmid = {31819262},
issn = {1476-4687},
support = {T32 GM007810/GM/NIGMS NIH HHS/United States ; T32 AI060537/AI/NIAID NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; DP5 OD021344/OD/NIH HHS/United States ; R01 GM104556/GM/NIGMS NIH HHS/United States ; R01 GM129245/GM/NIGMS NIH HHS/United States ; R35 GM118099/GM/NIGMS NIH HHS/United States ; GM104556//Natiional Institutes of Health/International ; },
mesh = {CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; DNA, Viral/chemistry ; Genome, Viral ; Pseudomonas Phages/chemistry/*genetics ; Pseudomonas aeruginosa/*immunology/*virology ; Viral Proteins/*chemistry ; },
abstract = {All viruses require strategies to inhibit or evade the immune pathways of cells that they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems, to replicate efficiently[1]. Here we show that jumbo phage ΦKZ segregates its DNA from immunity nucleases of its host, Pseudomonas aeruginosa, by constructing a proteinaceous nucleus-like compartment. ΦKZ is resistant to many immunity mechanisms that target DNA in vivo, including two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI. Cas proteins and restriction enzymes are unable to access the phage DNA throughout the infection, but engineering the relocalization of EcoRI inside the compartment enables targeting of the phage and protection of host cells. Moreover, ΦKZ is sensitive to Cas13a-a CRISPR-Cas enzyme that targets RNA-probably owing to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.},
}
@article {pmid31819217,
year = {2020},
author = {Malone, LM and Warring, SL and Jackson, SA and Warnecke, C and Gardner, PP and Gumy, LF and Fineran, PC},
title = {A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.},
journal = {Nature microbiology},
volume = {5},
number = {1},
pages = {48-55},
pmid = {31819217},
issn = {2058-5276},
mesh = {Bacteriophages/genetics/*physiology/*ultrastructure ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Viral/genetics/metabolism ; Genome, Viral/genetics ; Immune Evasion ; RNA, Viral/genetics/metabolism ; Serratia/genetics/virology ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages[1]. However, DNA modification[2,3], the production of anti-CRISPR proteins[4,5] and potentially other strategies enable phages to evade CRISPR-Cas. Here, we discovered a Serratia jumbo phage that evades type I CRISPR-Cas systems, but is sensitive to type III immunity. Jumbo phage infection resulted in a nucleus-like structure enclosed by a proteinaceous phage shell-a phenomenon only reported recently for distantly related Pseudomonas phages[6,7]. All three native CRISPR-Cas complexes in Serratia-type I-E, I-F and III-A-were spatially excluded from the phage nucleus and phage DNA was not targeted. However, the type III-A system still arrested jumbo phage infection by targeting phage RNA in the cytoplasm in a process requiring Cas7, Cas10 and an accessory nuclease. Type III, but not type I, systems frequently targeted nucleus-forming jumbo phages that were identified in global viral sequence datasets. The ability to recognize jumbo phage RNA and elicit immunity probably contributes to the presence of both RNA- and DNA-targeting CRISPR-Cas systems in many bacteria[1,8]. Together, our results support the model that jumbo phage nucleus-like compartments serve as a barrier to DNA-targeting, but not RNA-targeting, defences, and that this phenomenon is widespread among jumbo phages.},
}
@article {pmid31819203,
year = {2020},
author = {Duan, W and Guo, M and Yi, L and Liu, Y and Li, Z and Ma, Y and Zhang, G and Liu, Y and Bu, H and Song, X and Li, C},
title = {The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model.},
journal = {Gene therapy},
volume = {27},
number = {3-4},
pages = {157-169},
pmid = {31819203},
issn = {1476-5462},
mesh = {Amyotrophic Lateral Sclerosis/genetics/*therapy ; Animals ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; Gene Editing/*methods ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Male ; Mice ; Muscle, Skeletal/metabolism/pathology ; Mutation, Missense ; RNA, Guide/genetics/metabolism ; Superoxide Dismutase-1/*genetics/metabolism ; },
abstract = {The superoxide dismutase 1 (SOD1) mutation is one of the most notable causes of amyotrophic lateral sclerosis (ALS), and modifying the mutant SOD1 gene is the best approach for the treatment of patients with ALS linked to the mutations in this gene. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9)/sgRNA delivered by the adeno-associated virus (AAV) system is a powerful tool for genome editing in the central nervous system (CNS). Here, we tested the capacity of the AAV-SaCas9-sgRNA system to modify mutant SOD1 in SOD1G93A transgenic mice and found that AAV9-SaCas9-sgRNA5 deleted the SOD1 gene, improved the lifespan of SOD1G93A mice by 54.6%, and notably ameliorated the performance of ALS transgenic mice. An immunochemical analysis showed that the expression of mutant SOD1 was very weak in motor neurons expressing SaCas9-sgRNA5. Consequently, the area showing muscle atrophy was more notably restored in the group treated with SaCas9-sgRNA5 compared with the group treated with SaCas9-sgLacZ. In addition, deep sequencing did not show the indel mutation in the gene highly matched to sgRNA5. Hence, AAV9-SaCas9-sgRNA-based gene editing is a feasible potential treatment for patients with ALS linked to SOD1 mutations.},
}
@article {pmid31819086,
year = {2019},
author = {Goldstein, JM and Valido, A and Lewandowski, JP and Walker, RG and Mills, MJ and Messemer, KA and Besseling, P and Lee, KH and Wattrus, SJ and Cho, M and Lee, RT and Wagers, AJ},
title = {Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18613},
pmid = {31819086},
issn = {2045-2322},
support = {F32 AG050395/AG/NIA NIH HHS/United States ; R01 AG057428/AG/NIA NIH HHS/United States ; UL1 TR002541/TR/NCATS NIH HHS/United States ; T32 DK007529/DK/NIDDK NIH HHS/United States ; R01 AG047131/AG/NIA NIH HHS/United States ; R01 AG048917/AG/NIA NIH HHS/United States ; T32 HL007208/HL/NHLBI NIH HHS/United States ; P30 DK036836/DK/NIDDK NIH HHS/United States ; },
mesh = {*Alleles ; Animals ; Bone Morphogenetic Proteins/*genetics ; *CRISPR-Cas Systems ; Female ; *Gene Deletion ; *Gene Editing ; Genes, Reporter ; Genetic Engineering ; Genome ; Glutamic Acid/metabolism ; Green Fluorescent Proteins/metabolism ; Growth Differentiation Factors/*genetics ; Hematopoietic Stem Cells/metabolism ; Homozygote ; Ligands ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Myeloid Cells/metabolism ; Phenotype ; Protein Domains ; Tryptophan/metabolism ; },
abstract = {Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11[WE] allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.},
}
@article {pmid31818875,
year = {2020},
author = {Li, G and Zhang, X and Wang, H and Mo, J and Zhong, C and Shi, J and Zhou, R and Li, Z and Yang, H and Wu, Z and Liu, D},
title = {CRISPR/Cas9-Mediated Integration of Large Transgene into Pig CEP112 Locus.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {2},
pages = {467-473},
pmid = {31818875},
issn = {2160-1836},
mesh = {6-Phytase/genetics ; Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems ; Cells, Cultured ; Female ; Gene Knock-In Techniques ; Genetic Loci ; Glycoside Hydrolases/genetics ; Male ; Pregnancy ; Salivary Glands/*enzymology ; Swine/*genetics ; Transgenes ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating tool that can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knockout, the gene knock-in (KI) efficiency mediated by homology-directed repair remains low, especially for large fragment integration. In this study, we established an efficient method for the CRISPR/Cas9-mediated integration of large transgene cassette, which carries salivary gland-expressed multiple digestion enzymes (≈ 20 kbp) in CEP112 locus in pig fetal fibroblasts (PFFs). Our results showed that using an optimal homology donor with a short and a long arm yielded the best CRISPR/Cas9-mediated KI efficiency in CEP112 locus, and the targeting efficiency in CEP112 locus was higher than in ROSA26 locus. The CEP112 KI cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that KI pig (705) successfully expressed three microbial enzymes (β-glucanase, xylanase, and phytase) in salivary gland. This finding suggested that the CEP112 locus supports exogenous gene expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus in pigs by using our optimal homology arm system and established a modified pig model for foreign digestion enzyme expression in the saliva.},
}
@article {pmid31818368,
year = {2019},
author = {Bastin-Héline, L and de Fouchier, A and Cao, S and Koutroumpa, F and Caballero-Vidal, G and Robakiewicz, S and Monsempes, C and François, MC and Ribeyre, T and Maria, A and Chertemps, T and de Cian, A and Walker, WB and Wang, G and Jacquin-Joly, E and Montagné, N},
title = {A novel lineage of candidate pheromone receptors for sex communication in moths.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31818368},
issn = {2050-084X},
support = {ANR-16-CE02-0003-01//Agence Nationale de la Recherche/International ; ANR-16-CE21-0002-01//Agence Nationale de la Recherche/International ; 31725023//National Natural Science Foundation of China/International ; 31621064//National Natural Science Foundation of China/International ; },
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Drosophila/genetics/metabolism ; Female ; Gene Expression Regulation ; Gene Knockout Techniques ; Insect Proteins/genetics/*metabolism ; Lepidoptera/genetics/metabolism ; Male ; Moths/genetics/*metabolism ; Receptors, Odorant ; Receptors, Pheromone/classification/genetics/*metabolism ; Sex Attractants/*metabolism ; Spodoptera/genetics/metabolism ; Transcriptome ; Xenopus/genetics/metabolism ; },
abstract = {Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication.},
}
@article {pmid31817656,
year = {2019},
author = {Otterbein, H and Lehnert, H and Ungefroren, H},
title = {Negative Control of Cell Migration by Rac1b in Highly Metastatic Pancreatic Cancer Cells Is Mediated by Sequential Induction of Nonactivated Smad3 and Biglycan.},
journal = {Cancers},
volume = {11},
number = {12},
pages = {},
pmid = {31817656},
issn = {2072-6694},
abstract = {Expression of the small GTPase, Ras-related C3 botulinum toxin substrate 1B (RAC1B), a RAC1-related member of the Rho GTPase family, in tumor tissues of pancreatic ductal adenocarcinoma (PDAC) has been shown previously to correlate positively with patient survival, but the underlying mechanism(s) and the target genes involved have remained elusive. Screening of a panel of established PDAC-derived cell lines by immunoblotting indicated that both RAC1B and Mothers against decapentaplegic homolog 3 (SMAD3) were more abundantly expressed in poorly metastatic and well-differentiated lines as opposed to highly metastatic, poorly differentiated ones. Both siRNA-mediated RAC1B knockdown in the transforming growth factor (TGF)-β-sensitive PDAC-derived cell lines, Panc1 and PaCa3, or CRISPR/Cas-mediated knockout of exon 3b of RAC1 in Panc1 cells resulted in a dramatic decrease in the expression of SMAD3. Unexpectedly, the knockdown of SMAD3 reproduced the promigratory activity of a RAC1B knockdown in Panc1 and PaCa3, but not in TGF-β-resistant BxPC3 and Capan1 cells, while forced expression of SMAD3 alone was able to mimic the antimigratory effect of ectopic RAC1B overexpression in Panc1 cells. Moreover, overexpression of SMAD3 was able to rescue Panc1 cells from the RAC1B knockdown-induced increase in cell migration, while knockdown of SMAD3 prevented the RAC1B overexpression-induced decrease in cell migration. Using pharmacological and dominant-negative inhibition of SMAD3 C-terminal phosphorylation, we further show that the migration-inhibiting effect of SMAD3 is independent of its activation by TGF-β. Finally, we provide evidence that the antimigratory program of RAC1B-SMAD3 in Panc1 cells is executed through upregulation of the migration and TGF-β inhibitor, biglycan (BGN). Together, our data suggest that a RAC1B-SMAD3-BGN axis negatively controls cell migration and that SMAD3 can induce antimigratory genes, i.e., BGN independent of its role as a signal transducer for TGF-β. Therefore, targeting this novel pathway for activation is a potential therapeutic strategy in highly metastatic PDAC to interfere with invasion and metastasis.},
}
@article {pmid31817535,
year = {2019},
author = {Kim, HK and Ham, KA and Lee, SW and Choi, HS and Kim, HS and Kim, HK and Shin, HS and Seo, KY and Cho, Y and Nam, KT and Kim, IB and Joe, YA},
title = {Biallelic Deletion of Pxdn in Mice Leads to Anophthalmia and Severe Eye Malformation.},
journal = {International journal of molecular sciences},
volume = {20},
number = {24},
pages = {},
pmid = {31817535},
issn = {1422-0067},
mesh = {Animals ; *Anophthalmos/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Collagen Type IV/genetics/metabolism ; Eye/*growth &amp; development/pathology ; *Gene Deletion ; Mice ; Mice, Knockout ; Peroxidases/*deficiency/metabolism ; Vision, Ocular/genetics ; },
abstract = {Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5' upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.},
}
@article {pmid31817229,
year = {2019},
author = {Zinn, R and Otterbein, H and Lehnert, H and Ungefroren, H},
title = {RAC1B: A Guardian of the Epithelial Phenotype and Protector Against Epithelial-Mesenchymal Transition.},
journal = {Cells},
volume = {8},
number = {12},
pages = {},
pmid = {31817229},
issn = {2073-4409},
mesh = {Antigens, CD/genetics/metabolism ; Cadherins/genetics/metabolism ; Carcinoma/*metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial Cells/drug effects/metabolism/physiology ; *Epithelial-Mesenchymal Transition ; Humans ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Pancreatic Neoplasms/*metabolism ; Phenotype ; Snail Family Transcription Factors/genetics/metabolism ; Transforming Growth Factor beta/pharmacology ; Vimentin/genetics/metabolism ; rac1 GTP-Binding Protein/genetics/*metabolism ; },
abstract = {The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown to potently inhibit transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition (EMT) in pancreatic and breast epithelial cells, but the underlying mechanism has remained obscure. Using a panel of pancreatic ductal adenocarcinoma (PDAC)-derived cell lines of different differentiation stages, we show that RAC1B is more abundantly expressed in well differentiated as opposed to poorly differentiated cells. Interestingly, RNA interference-mediated knockdown of RAC1B decreased expression of the epithelial marker protein E-cadherin, encoded by CDH1, and enhanced its TGF-β1-induced downregulation, whereas ectopic overexpression of RAC1B upregulated CDH1 expression and largely prevented its TGF-β1-induced silencing of CDH1. Conversely, knockdown of RAC1B, or deletion of the RAC1B-specific exon 3b by CRISPR/Cas-mediated genomic editing, enhanced basal and TGF-β1-induced upregulation of mesenchymal markers like Vimentin, and EMT-associated transcription factors such as SNAIL and SLUG. Moreover, we demonstrate that knockout of RAC1B enhanced the cells' migratory activity and derepressed TGF-β1-induced activation of the mitogen-activated protein kinase ERK2. Pharmacological inhibition of ERK1/2 activation in RAC1B-depleted cells rescued cells from the RAC1B knockdown-induced enhancement of cell migration, TGF-β1-induced downregulation of CDH1, and upregulation of SNAI1. We conclude that RAC1B promotes epithelial gene expression and suppresses mesenchymal gene expression by interfering with TGF-β1-induced MEK-ERK signaling, thereby protecting cells from undergoing EMT and EMT-associated responses like acquisition of cell motility.},
}
@article {pmid31816041,
year = {2020},
author = {Shohayeb, B and Ho, U and Yeap, YY and Parton, RG and Millard, SS and Xu, Z and Piper, M and Ng, DCH},
title = {The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.},
journal = {Human molecular genetics},
volume = {29},
number = {2},
pages = {248-263},
doi = {10.1093/hmg/ddz281},
pmid = {31816041},
issn = {1460-2083},
mesh = {Animals ; Anophthalmos/embryology/genetics/metabolism ; Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cells, Cultured ; Cilia/genetics/*metabolism/pathology ; Ciliopathies/embryology/*genetics/metabolism/pathology ; Dwarfism/embryology/genetics/metabolism ; Ependymoglial Cells/cytology/metabolism/pathology ; Fibroblasts/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Microcephaly/embryology/*genetics/metabolism ; Microtubule-Associated Proteins/genetics/*metabolism ; Mutation, Missense ; Neocortex/embryology/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neurogenesis/genetics ; Neuroglia/cytology/metabolism ; Neurons/metabolism ; Tumor Suppressor Proteins/genetics/*metabolism ; },
abstract = {WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.},
}
@article {pmid31813827,
year = {2019},
author = {Hildebrandt, MR and Reuter, MS and Wei, W and Tayebi, N and Liu, J and Sharmin, S and Mulder, J and Lesperance, LS and Brauer, PM and Mok, RSF and Kinnear, C and Piekna, A and Romm, A and Howe, J and Pasceri, P and Meng, G and Rozycki, M and Rodrigues, DC and Martinez, EC and Szego, MJ and Zúñiga-Pflücker, JC and Anderson, MK and Prescott, SA and Rosenblum, ND and Kamath, BM and Mital, S and Scherer, SW and Ellis, J},
title = {Precision Health Resource of Control iPSC Lines for Versatile Multilineage Differentiation.},
journal = {Stem cell reports},
volume = {13},
number = {6},
pages = {1126-1141},
pmid = {31813827},
issn = {2213-6711},
support = {P01 AI102853/AI/NIAID NIH HHS/United States ; THC-135232//CIHR/Canada ; SOP-155609//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; *Cell Differentiation ; *Cell Lineage ; Cell Self Renewal ; Cell Separation ; Ectoderm/cytology/metabolism ; Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; Myocytes, Cardiac/cytology/metabolism ; Neurons/cytology/metabolism ; Organoids ; Phenotype ; T-Lymphocytes/metabolism ; Whole Genome Sequencing ; },
abstract = {Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings.},
}
@article {pmid31811268,
year = {2019},
author = {Kortleve, D and Hammerl, D and Debets, R},
title = {Orthotopic editing of T-cell receptors.},
journal = {Nature biomedical engineering},
volume = {3},
number = {12},
pages = {949-950},
doi = {10.1038/s41551-019-0490-4},
pmid = {31811268},
issn = {2157-846X},
mesh = {Antigens, Neoplasm ; CRISPR-Cas Systems ; Cell Engineering ; *Gene Editing ; Gene Knockout Techniques ; Gene Rearrangement, T-Lymphocyte ; Humans ; Membrane Proteins ; Orthopedic Procedures ; Receptors, Antigen, T-Cell/*genetics ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; },
}
@article {pmid31811188,
year = {2019},
author = {Santos, JM and Oudinet, C and Schöne, L and Dauba, A and Khamlichi, AA},
title = {Essential role of the initial activation signal in isotype selection upon deletion of a transcriptionally committed promoter.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18543},
pmid = {31811188},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Immunoglobulin Class Switching/*genetics ; Immunoglobulin Heavy Chains/*genetics ; Immunoglobulin Isotypes/*genetics ; Mice ; Promoter Regions, Genetic/genetics ; *Recombination, Genetic ; Sequence Deletion ; },
abstract = {Class switch recombination (CSR), which targets exclusively the constant region of the immunoglobulin heavy chain (IgH) locus, plays an important role in humoral immunity by generating different antibody effector functions. The IgH constant locus contains multiple genes controlled by isotype (I) promoters induced by extracellular signals that activate specific I promoters, leading to B cell commitment. However, it is unknown whether after initial commitment to one promoter, non-responsive I promoters are irreversibly silent or if they can be activated after exposure to their specific inducers. Here, we studied the murine cell line CH12, which is committed to produce IgA in response to TGF-β. We show that, although other promoters than Iα are transcriptionally inactive, they are not irreversibly silent. Following deletion of the committed Iα promoter by CRISPR/Cas9, other I promoters display a complex transcriptional pattern largely dependent on the initial committing signal.},
}
@article {pmid31811138,
year = {2019},
author = {Morisaka, H and Yoshimi, K and Okuzaki, Y and Gee, P and Kunihiro, Y and Sonpho, E and Xu, H and Sasakawa, N and Naito, Y and Nakada, S and Yamamoto, T and Sano, S and Hotta, A and Takeda, J and Mashimo, T},
title = {CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5302},
pmid = {31811138},
issn = {2041-1723},
mesh = {CRISPR-Associated Proteins/classification/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; DNA Helicases/metabolism ; Exons ; Gene Editing/*methods ; Gene Expression Regulation/genetics ; Gene Knockout Techniques/methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Muscular Dystrophy, Duchenne/genetics ; Sequence Deletion ; },
abstract = {Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5'-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.},
}
@article {pmid31810603,
year = {2020},
author = {Xia, P and Liu, P and Fu, Q and Liu, C and Luo, Q and Zhang, X and Cheng, L and Qin, T and Zhang, H},
title = {Long noncoding RNA EPIC1 interacts with YAP1 to regulate the cell cycle and promote the growth of pancreatic cancer cells.},
journal = {Biochemical and biophysical research communications},
volume = {522},
number = {4},
pages = {978-985},
doi = {10.1016/j.bbrc.2019.11.167},
pmid = {31810603},
issn = {1090-2104},
mesh = {Adaptor Proteins, Signal Transducing/*metabolism ; Apoptosis/genetics ; Cell Cycle/*genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Pancreatic Neoplasms/*genetics/*pathology ; RNA, Long Noncoding/genetics/*metabolism ; RNA, Small Interfering/metabolism ; Transcription Factors/*metabolism ; Up-Regulation/genetics ; YAP-Signaling Proteins ; },
abstract = {Pancreatic cancer (PC) is a fatal disease; most patients are asymptomatic before the disease enters the advanced stage, but molecular mechanisms of early PC that can be exploited for diagnosis are not clear. Long noncoding RNAs (lncRNAs) play key roles in the progression of PC. In this study, we found that the expression of the lncRNA EPIC1 (Lnc-EPIC1) is high in PC and closely related to tumor size, TNM staging and lymph node metastasis status. Silencing Lnc-EPIC1 by siRNA targeting could significantly inhibit the cell growth and colony formation ability of PC cells and induced G1/S cell cycle arrest and apoptosis in PC cells. Lnc-EPIC1-specific siRNAs could downregulate the expression of cyclins and CDKs, such as CDC20, CDK4 and Cyclin A1. Knocking out YAP1 with the CRISPR/Cas-9 gene editing method recapitulated the effects of the Lnc-EPIC1-specific siRNAs on cell growth, colony formation ability and apoptosis in PC cells. In addition, the Lnc-EPIC1-specific siRNAs did not further inhibit cell growth or promote apoptosis in YAP1-knockout (YAP1-KO) cells. RNA immunoprecipitation (RIP) results showed that there was a direct interaction between Lnc-EPIC1 and YAP1. An Lnc-EPIC1-overexpressing lentiviral vector promoted the growth of PC cells. The results show that Lnc-EPIC1 interacts with YAP1 to promote the progression of PC.},
}
@article {pmid31810530,
year = {2019},
author = {Wang, J and Bai, P and Li, Q and Lin, Y and Huo, D and Ke, F and Zhang, Q and Li, T and Zhao, J},
title = {Interaction between cyanophage MaMV-DC and eight Microcystis strains, revealed by genetic defense systems.},
journal = {Harmful algae},
volume = {85},
number = {},
pages = {101699},
doi = {10.1016/j.hal.2019.101699},
pmid = {31810530},
issn = {1878-1470},
mesh = {*Cyanobacteria ; *Microcystis ; },
abstract = {Cyanophage MaMV-DC is a member of Myoviridae that was reported to specifically infect and lyse Microcystis aeruginosa FACHB-524 among 21 selected cyanobacterial strains. We reidentified the infection specificity of MaMV-DC among seven other Microcystis strains of different species. In our experiments, MaMV-DC infected three Microcystis strains but did not form plaque in Microcystis lawns. This indicated that MaMV-DC is at least a genus- rather than strain-specific virus. Cyanophage MaMV-DC genes were transcribed in M. aeruginosa FACHB-524, M. flos-aquae TF09, M. aeruginosa TA09 and M. wesenbergii DW09, and the growth of these Microcystis strains was inhibited by the addition of MaMV-DC. The predicted defense of eight Microcystis strains by CRISPR-Cas systems has shown mixed consistency with the infection experiment results, suggesting other defense or anti-defense systems play roles during infection process. Restriction-modification (RM) system analysis revealed an abundance of four types of RM proteins that may play roles in defense against cyanophages.},
}
@article {pmid31809194,
year = {2019},
author = {Crowley, VM and Catching, A and Taylor, HN and Borges, AL and Metcalf, J and Bondy-Denomy, J and Jackson, RN},
title = {A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {434-440},
pmid = {31809194},
issn = {2573-1602},
support = {T32 AI060537/AI/NIAID NIH HHS/United States ; T32 GM008284/GM/NIGMS NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; DP5 OD021344/OD/NIH HHS/United States ; },
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plasmids/genetics ; Pseudomonas aeruginosa/*genetics ; RNA Interference/physiology ; RNA, Guide/*genetics ; },
abstract = {Bacteria and archaea use CRISPR-Cas adaptive immune systems to destroy complementary nucleic acids using RNAs derived from CRISPR loci. Here, we provide the first functional evidence for type IV CRISPR-Cas, demonstrating that the system from Pseudomonas aeruginosa strain PA83 mediates RNA-guided interference against a plasmid in vivo, both clearing the plasmid and inhibiting its uptake. This interference depends on the putative NTP-dependent helicase activity of Csf4/DinG.},
}
@article {pmid31805916,
year = {2019},
author = {Ahmed, HMM and Hildebrand, L and Wimmer, EA},
title = {Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {85},
pmid = {31805916},
issn = {1472-6750},
mesh = {Animals ; Animals, Genetically Modified/*genetics/physiology ; CRISPR-Cas Systems ; Drosophila/*genetics/physiology ; Drosophila Proteins/genetics/metabolism ; Female ; Gene Editing ; Male ; Plant Diseases/*parasitology ; Spermatozoa/*cytology/metabolism ; Transgenes ; },
abstract = {BACKGROUND: The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.<br><br>RESULTS: To improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_&#x3b2;2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.<br><br>CONCLUSION: The deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the &#x3b2;2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.},
}
@article {pmid31805259,
year = {2019},
author = {Wilson, KM and Muralidharan, B and Isaacs, AM},
title = {Relax, Don't RAN Translate It.},
journal = {Neuron},
volume = {104},
number = {5},
pages = {827-829},
doi = {10.1016/j.neuron.2019.11.014},
pmid = {31805259},
issn = {1097-4199},
support = {ISAACS/APR15/834-791/MNDA_/Motor Neurone Disease Association/United Kingdom ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics ; C9orf72 Protein ; CRISPR-Cas Systems ; DEAD-box RNA Helicases ; DNA Repeat Expansion ; Frontotemporal Dementia/*genetics ; Humans ; RNA Helicases ; },
abstract = {The (GGGGCC)n repeat expansion in C9orf72, which is the most common cause of frontotemporal dementia and amyotrophic lateral sclerosis, is translated through repeat-associated non-AUG (RAN) translation. In this issue of Neuron, Cheng et al. (2019) report that the helicase DDX3X, which unwinds (or relaxes) RNA, suppresses RAN translation and toxicity.},
}
@article {pmid31804951,
year = {2020},
author = {Cai, P and Otten, ABC and Cheng, B and Ishii, MA and Zhang, W and Huang, B and Qu, K and Sun, BK},
title = {A genome-wide long noncoding RNA CRISPRi screen identifies PRANCR as a novel regulator of epidermal homeostasis.},
journal = {Genome research},
volume = {30},
number = {1},
pages = {22-34},
pmid = {31804951},
issn = {1549-5469},
support = {2019088/DDCF/Doris Duke Charitable Foundation/United States ; K08 AR067853/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Biomarkers ; CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epidermis/*metabolism ; Filaggrin Proteins ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Knockdown Techniques ; *Genome-Wide Association Study/methods ; *Homeostasis ; Humans ; Keratinocytes/metabolism ; MAP Kinase Signaling System ; Mice ; Organogenesis/genetics ; Promoter Regions, Genetic ; *RNA Interference ; RNA, Long Noncoding/*genetics ; Transcriptome ; },
abstract = {Genome-wide association studies indicate that many disease susceptibility regions reside in non-protein-coding regions of the genome. Long noncoding RNAs (lncRNAs) are a major component of the noncoding genome, but their biological impacts are not fully understood. Here, we performed a CRISPR interference (CRISPRi) screen on 2263 epidermis-expressed lncRNAs and identified nine novel candidate lncRNAs regulating keratinocyte proliferation. We further characterized a top hit from the screen, progenitor renewal associated non-coding RNA (PRANCR), using RNA interference-mediated knockdown and phenotypic analysis in organotypic human tissue. PRANCR regulates keratinocyte proliferation, cell cycle progression, and clonogenicity. PRANCR-deficient epidermis displayed impaired stratification with reduced expression of differentiation genes that are altered in human skin diseases, including keratins 1 and 10, filaggrin, and loricrin. Transcriptome analysis showed that PRANCR controls the expression of 1136 genes, with strong enrichment for late cell cycle genes containing a CHR promoter element. In addition, PRANCR depletion led to increased levels of both total and nuclear CDKN1A (also known as p21), which is known to govern both keratinocyte proliferation and differentiation. Collectively, these data show that PRANCR is a novel lncRNA regulating epidermal homeostasis and identify other lncRNA candidates that may have roles in this process as well.},
}
@article {pmid31803983,
year = {2020},
author = {Ortega, MS and Kelleher, AM and O'Neil, E and Benne, J and Cecil, R and Spencer, TE},
title = {NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo.},
journal = {Molecular reproduction and development},
volume = {87},
number = {1},
pages = {152-160},
pmid = {31803983},
issn = {1098-2795},
support = {R01 HD072898/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Cell Lineage/*genetics ; Embryo, Mammalian/*metabolism ; Exons ; Female ; Fertilization in Vitro/methods ; GATA6 Transcription Factor/metabolism ; Gene Expression Regulation, Developmental ; Genotype ; Germ Layers/*metabolism ; Nanog Homeobox Protein/genetics/*metabolism ; RNA, Guide ; Zygote/metabolism ; },
abstract = {During preimplantation development, the embryo undergoes two consecutive lineages specifications. The first cell fate decision determines which cells give rise to the trophectoderm (TE) and the inner cell mass (ICM). Subsequently, the ICM differentiates into hypoblast and epiblast, the latter giving rise to the embryo proper. The transcription factors that govern these cell fate decisions have been extensively studied in the mouse, but are still poorly understood in other mammalian species. In the present study, the role of NANOG in the formation of the epiblast and maintenance of pluripotency in the bovine embryo was investigated. Using a CRISPR-Cas9 approach, guide RNAs were designed to target exon 2, resulting in a functional deletion of bovine NANOG at the zygote stage. Disruption of NANOG resulted in the embryos that form a blastocoel and an ICM composed of hypoblast cells. Furthermore, NANOG-null embryos showed lower expression of epiblast cell markers SOX2 and HA2AFZ, and hypoblast marker GATA6; without affecting the expression of TE markers CDX2 and KRT8. Results indicate that NANOG, has no apparent role in segregation or maintenance of the TE, but it is required to derive and maintain the pluripotent epiblast and during the second lineage commitment in the bovine embryo.},
}
@article {pmid31802333,
year = {2020},
author = {Li, X and Liu, Q and Sun, W and He, Q and Tian, C},
title = {Improving cellulases production by Myceliophthora thermophila through disruption of protease genes.},
journal = {Biotechnology letters},
volume = {42},
number = {2},
pages = {219-229},
doi = {10.1007/s10529-019-02777-0},
pmid = {31802333},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems ; Cellulases/*metabolism ; Fungal Proteins/genetics/metabolism ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Fungal ; Metabolic Engineering ; Mutation ; Peptide Hydrolases/*genetics/metabolism ; Proteolysis ; Sordariales/*enzymology/genetics ; },
abstract = {OBJECTIVE: To identify main protease genes for the proteolytic degradation of cellulases in M. thermophila and generate a lower-proteases fungal host that can be used for further metabolic engineering to increase cellulase production and heterologous protein expression.<br><br>RESULTS: Systematic transcriptomic analysis were conducted on the expression of proteases genes in M. thermophila genome and five highly expressed genes encoding extracellular proteases were selected for mutation analyses. A series of single- and multi-gene mutants of these five selected genes was constructed using the CRISPR-Cas9 technique. Compared with WT, the &#x394;Mtalp1 and the quintuple mutant showed significantly lower protease activity (decreased 52.7% and 58.4%, respectively) and at least double enhanced cellulase production.<br><br>CONCLUSIONS: The results indicated that Mtalp1 is a critical protease gene in cellulase degradation in M. thermophila and disruption of protease genes showed significantly decreased protease activity and obviously enhanced cellulase production in the fermentation broth of &#x394;Mtalp1 and the quintuple mutant.},
}
@article {pmid31801211,
year = {2019},
author = {Brezgin, S and Kostyusheva, A and Kostyushev, D and Chulanov, V},
title = {Dead Cas Systems: Types, Principles, and Applications.},
journal = {International journal of molecular sciences},
volume = {20},
number = {23},
pages = {},
pmid = {31801211},
issn = {1422-0067},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Chromatin/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Communicable Diseases/genetics/metabolism/pathology/*therapy ; DNA Methylation ; Epigenesis, Genetic ; Gene Editing/*methods ; Genetic Diseases, Inborn/genetics/metabolism/pathology/*therapy ; Genome, Human ; Histones/genetics/metabolism ; Humans ; Inflammation/genetics/metabolism/pathology/*therapy ; Neoplasms/genetics/metabolism/pathology/*therapy ; RNA, Guide/genetics/metabolism ; },
abstract = {The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.},
}
@article {pmid31800222,
year = {2020},
author = {Kim, H and Ji, CH and Je, HW and Kim, JP and Kang, HS},
title = {mpCRISTAR: Multiple Plasmid Approach for CRISPR/Cas9 and TAR-Mediated Multiplexed Refactoring of Natural Product Biosynthetic Gene Clusters.},
journal = {ACS synthetic biology},
volume = {9},
number = {1},
pages = {175-180},
doi = {10.1021/acssynbio.9b00382},
pmid = {31800222},
issn = {2161-5063},
mesh = {Base Sequence ; Biological Products/*metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/*methods ; Genotype ; *Multigene Family ; Plasmids/*genetics ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae/*genetics ; Transcription, Genetic/*genetics ; },
abstract = {Multiplexed refactoring provides a tool for rapid transcriptional optimization of biosynthetic gene clusters (BGCs) through simultaneous replacement of multiple native promoters with synthetic counterparts. Here, we present the mpCRISTAR, a multiple plasmid-based CRISPR/Cas9 and TAR (transformation-associated recombination), that enables a rapid and highly efficient, multiplexed refactoring of natural product BGCs in yeast. A series of CRISPR plasmids with different auxotrophic markers that could be stably maintained in yeast cells were constructed to express multiple gRNAs simultaneously. We demonstrated the multiplexing capacity of mpCRISTAR using the actinorhodin biosynthetic gene cluster as a model cluster. mpCRISTAR1, in which each CRISPR plasmid expresses one gRNA, allows for simultaneous replacement of up to four promoter sites with nearly 100% efficiency. By expressing two gRNAs from one CRISPR plasmid, termed mpCRISTAR2, we simultaneously replaced a total of six and eight promoter sites with 68% and 32% efficiency, respectively. The mpCRISTAR could be performed iteratively using two different auxotrophic markers, allowing for refactoring of any type of BGC regardless of their operon complexities. The mpCRISTAR platform we report here would become a useful tool for the discovery of new natural products from transcriptionally silent biosynthetic gene clusters present in microbial genomes.},
}
@article {pmid31800219,
year = {2020},
author = {Xiong, Y and Zhang, J and Yang, Z and Mou, Q and Ma, Y and Xiong, Y and Lu, Y},
title = {Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {1},
pages = {207-213},
pmid = {31800219},
issn = {1520-5126},
support = {R01 GM124316/GM/NIGMS NIH HHS/United States ; R21 MH110975/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/*chemistry ; Humans ; *Point-of-Care Systems ; },
abstract = {Beyond its extraordinary genome editing ability, the CRISPR-Cas systems have opened a new era of biosensing applications due to its high base resolution and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic-acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic-acid targets, we herein report CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detection. The sensors are based on the Cas12a-dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA), and its single-stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA molecules that can lock a DNA activator for Cas12a-crRNA, preventing the ssDNA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by commercially available portable fluorimeters. Using this method, ATP and Na[+] have been detected quantitatively under ambient temperature (25 °C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system significantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanalytical and biomedical applications.},
}
@article {pmid31799604,
year = {2020},
author = {Chen, X and Tasca, F and Wang, Q and Liu, J and Janssen, JM and Brescia, MD and Bellin, M and Szuhai, K and Kenrick, J and Frock, RL and Gonçalves, MAFV},
title = {Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking.},
journal = {Nucleic acids research},
volume = {48},
number = {2},
pages = {974-995},
pmid = {31799604},
issn = {1362-4962},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; DNA/chemistry/*genetics ; DNA Breaks, Double-Stranded ; DNA Breaks, Single-Stranded ; Deoxyribonuclease I/*chemistry/genetics ; Endonucleases/chemistry ; Gene Editing/*methods ; Gene Targeting/methods ; Genome/genetics ; Humans ; Mutation/genetics ; RNA, Guide/chemistry/genetics ; },
abstract = {Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.},
}
@article {pmid31799598,
year = {2020},
author = {Liu, B and Chen, S and Rose, A and Chen, D and Cao, F and Zwinderman, M and Kiemel, D and Aïssi, M and Dekker, FJ and Haisma, HJ},
title = {Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing.},
journal = {Nucleic acids research},
volume = {48},
number = {2},
pages = {517-532},
pmid = {31799598},
issn = {1362-4962},
mesh = {Acetylation/drug effects ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Chromatin/genetics ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Histone Deacetylase 1/antagonists &amp; inhibitors/*genetics ; Histone Deacetylase 2/antagonists &amp; inhibitors/*genetics ; Histone Deacetylase Inhibitors/chemistry/pharmacology ; Histones/chemistry/genetics ; Humans ; },
abstract = {Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.},
}
@article {pmid31798958,
year = {2019},
author = {Buyukyoruk, M and Wiedenheft, B},
title = {Type I-F CRISPR-Cas provides protection from DNA, but not RNA phages.},
journal = {Cell discovery},
volume = {5},
number = {},
pages = {54},
pmid = {31798958},
issn = {2056-5968},
support = {R01 GM110270/GM/NIGMS NIH HHS/United States ; },
}
@article {pmid31798579,
year = {2019},
author = {Pesch, T and Bonati, L and Kelton, W and Parola, C and Ehling, RA and Csepregi, L and Kitamura, D and Reddy, ST},
title = {Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells.},
journal = {Frontiers in immunology},
volume = {10},
number = {},
pages = {2630},
pmid = {31798579},
issn = {1664-3224},
mesh = {Animals ; *B-Lymphocytes ; CRISPR-Cas Systems ; Gene Editing/*methods ; Mice ; Protein Engineering/*methods ; Receptors, Antigen, B-Cell/*genetics/immunology ; Receptors, Artificial/*genetics/immunology ; },
abstract = {Immune cell therapies based on the integration of synthetic antigen receptors comprise a powerful strategy for the treatment of diverse diseases, most notably T cells engineered to express chimeric antigen receptors (CAR) for targeted cancer therapy. In addition to T lymphocytes, B lymphocytes may also represent valuable immune cells that can be engineered for therapeutic purposes such as protein replacement therapy or recombinant antibody production. In this article, we report a promising concept for the molecular design, optimization, and genomic integration of a novel class of synthetic antigen receptors, chimeric B cell receptors (CBCR). We initially optimized CBCR expression and detection by modifying the extracellular surface tag, the transmembrane regions and intracellular signaling domains. For this purpose, we stably integrated a series of CBCR variants using CRISPR-Cas9 into immortalized B cell hybridomas. Subsequently, we developed a reliable and consistent pipeline to precisely introduce cassettes of several kb size into the genome of primary murine B cells also using CRISPR-Cas9 induced HDR. Finally, we were able to show the robust surface expression and antigen recognition of a synthetic CBCR in primary B cells. We anticipate CBCRs and our approach for engineering primary B cells will be a valuable tool for the advancement of future B cell- based immune cell therapies.},
}
@article {pmid31797930,
year = {2019},
author = {Liu, RM and Liang, LL and Freed, E and Chang, H and Oh, E and Liu, ZY and Garst, A and Eckert, CA and Gill, RT},
title = {Synthetic chimeric nucleases function for efficient genome editing.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5524},
pmid = {31797930},
issn = {2041-1723},
mesh = {Bacteria/genetics ; Biotechnology/methods ; *CRISPR-Cas Systems ; Endonucleases/genetics/*metabolism ; Gene Editing/*methods ; Gene Library ; HEK293 Cells ; Humans ; Mutation ; Recombinant Fusion Proteins/genetics/*metabolism ; Reproducibility of Results ; Yeasts/genetics ; },
abstract = {CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.},
}
@article {pmid31797925,
year = {2019},
author = {Luo, X and He, Y and Zhang, C and He, X and Yan, L and Li, M and Hu, T and Hu, Y and Jiang, J and Meng, X and Ji, W and Zhao, X and Zheng, P and Xu, S and Su, B},
title = {Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5525},
pmid = {31797925},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Macaca mulatta/*genetics ; *Mutation ; Whole Genome Sequencing ; },
abstract = {CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.},
}
@article {pmid31797922,
year = {2019},
author = {Watson, BNJ and Vercoe, RB and Salmond, GPC and Westra, ER and Staals, RHJ and Fineran, PC},
title = {Type I-F CRISPR-Cas resistance against virulent phages results in abortive infection and provides population-level immunity.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5526},
pmid = {31797922},
issn = {2041-1723},
support = {BB/K001833/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N008081/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacteria/genetics/*immunology/virology ; Bacterial Infections/immunology/microbiology/virology ; Bacteriophages/genetics/*immunology/physiology ; CRISPR-Cas Systems/genetics/*immunology ; Microbial Viability/genetics/immunology ; Pectobacterium/genetics/*immunology/virology ; Virus Replication/genetics/immunology ; },
abstract = {Type I CRISPR-Cas systems are abundant and widespread adaptive immune systems in bacteria and can greatly enhance bacterial survival in the face of phage infection. Upon phage infection, some CRISPR-Cas immune responses result in bacterial dormancy or slowed growth, which suggests the outcomes for infected cells may vary between systems. Here we demonstrate that type I CRISPR immunity of Pectobacterium atrosepticum leads to suppression of two unrelated virulent phages, ɸTE and ɸM1. Immunity results in an abortive infection response, where infected cells do not survive, but viral propagation is severely decreased, resulting in population protection due to the reduced phage epidemic. Our findings challenge the view of CRISPR-Cas as a system that protects the individual cell and supports growing evidence of abortive infection by some types of CRISPR-Cas systems.},
}
@article {pmid31797562,
year = {2020},
author = {Chen, F and Alphonse, M and Liu, Q},
title = {Strategies for nonviral nanoparticle-based delivery of CRISPR/Cas9 therapeutics.},
journal = {Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology},
volume = {12},
number = {3},
pages = {e1609},
doi = {10.1002/wnan.1609},
pmid = {31797562},
issn = {1939-0041},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing ; *Gene Transfer Techniques ; Humans ; Nanoparticles/*chemistry ; },
abstract = {CRISPR-based genome editing technology has become an important potential therapeutic tool for various diseases. A vital challenge is to reach a safe, efficient, and clinically suitable delivery of a CRISPR-associated protein and a single-guide RNA. A possible translational approach to applying CRISPR-based technology is the use of viral vectors such as adeno-associated virus. However, such vectors give long-term exposure in vivo that may increase potential off-target effects as well as the risk of immunogenicity. Therefore, limitations to clinical applications are addressed using nonviral delivery systems such as nanoparticle-based delivery strategies. Today, the nanoparticle-based delivery approach is becoming more and more attractive in gene therapeutics because of its specific targeting, scale-up efficiency, efficacy of customization, minor stimulation of immune response, and minimal exposure to nucleases. In this review, we will present the most recent advances in developing innovations and potential advantages of the nanoparticle delivery system in CRISPR genome editing. We will also propose potential strategies of CRISPR-based technology for therapeutic and industrial applications. Our review will differ in focus from previous reviews and advance the literature on the subject by (a) focusing on the challenges of the CRISPR/Cas9 delivery system; (b) focusing on the application of nanoparticle-based delivery of CRISPR components (Cas9 and sgRNA), such as lipids and polymeric vectors; (c) discussing the potential nanoparticle-based delivery approaches for CRISPR/Cas9 application. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.},
}
@article {pmid31794887,
year = {2019},
author = {Liu, Z and Yao, M and Yao, H and Hu, G and Qin, B},
title = {Generation of Rybp homozygous knockout murine ES cell line GIBHe001-A-1 by using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101638},
doi = {10.1016/j.scr.2019.101638},
pmid = {31794887},
issn = {1876-7753},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line/*cytology ; Homozygote ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells/*metabolism ; Repressor Proteins/*genetics ; Reproducibility of Results ; },
abstract = {RYBP (Ring1 and YY1 Binding Protein) is critical for pluripotency and differentiation of embryonic stem cells (ESCs). RYBP depletion disturbs both neural and myocardial differentiation of ESCs. Moreover, low level of RYBP is correlated with diseases such as glioblastoma. To study the biological function of RYBP in neural differentiation of ESCs, here we generated Rybp homozygous knockout murine ESC line based on Sox1-GFP reporter using CRISPR/Cas9 genome editing technology. The last two exons of Rybp gene in which contain 115 amino acids have been replaced with PGK-Pruo by homologous recombination.},
}
@article {pmid31794381,
year = {2019},
author = {Bendriem, RM and Singh, S and Aleem, AA and Antonetti, DA and Ross, ME},
title = {Tight junction protein occludin regulates progenitor Self-Renewal and survival in developing cortex.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31794381},
issn = {2050-084X},
support = {Training Fellowship (T32HD060600)/NH/NIH HHS/United States ; R01 NS105477/NS/NINDS NIH HHS/United States ; P01 HD067244/HD/NICHD NIH HHS/United States ; P30 DK020572/DK/NIDDK NIH HHS/United States ; P01HD067244/NH/NIH HHS/United States ; R01NS105477/NH/NIH HHS/United States ; },
mesh = {Aneuploidy ; Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Centrosome/metabolism ; Cerebral Cortex/*growth &amp; development/*metabolism/pathology ; Disease Models, Animal ; Gene Editing ; Humans ; Mice ; Mice, Knockout ; Microcephaly/genetics/pathology ; Microtubules/metabolism ; Mutagenesis ; Mutation ; Neurogenesis/genetics/physiology ; Occludin/genetics/*metabolism ; Spindle Apparatus/metabolism ; Tight Junctions/genetics/*metabolism ; },
abstract = {Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.},
}
@article {pmid31793351,
year = {2020},
author = {Safari, F and Sharifi, M and Farajnia, S and Akbari, B and Karimi Baba Ahmadi, M and Negahdaripour, M and Ghasemi, Y},
title = {The interaction of phages and bacteria: the co-evolutionary arms race.},
journal = {Critical reviews in biotechnology},
volume = {40},
number = {2},
pages = {119-137},
doi = {10.1080/07388551.2019.1674774},
pmid = {31793351},
issn = {1549-7801},
mesh = {Bacteria/*immunology/virology ; Bacteriophages/*physiology ; *Biological Evolution ; CRISPR-Cas Systems ; *Host-Pathogen Interactions ; },
abstract = {Since the dawn of life, bacteria and phages are locked in a constant battle and both are perpetually changing their tactics to overcome each other. Bacteria use various strategies to overcome the invading phages, including adsorption inhibition, restriction-modification (R/E) systems, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, abortive infection (Abi), etc. To counteract, phages employ intelligent tactics for the nullification of bacterial defense systems, such as accessing host receptors, evading R/E systems, and anti-CRISPR proteins. Intense knowledge about the details of these defense pathways is the basis for their broad utilities in various fields of research from microbiology to biotechnology. Hence, in this review, we discuss some strategies used by bacteria to inhibit phage infections as well as phage tactics to circumvent bacterial defense systems. In addition, the application of these strategies will be described as a lesson learned from bacteria and phage combats. The ecological factors that affect the evolution of bacterial immune systems is the other issue represented in this review.},
}
@article {pmid31793074,
year = {2020},
author = {Hanawalt, P and Sweasy, J},
title = {Mechanistic understanding of cellular responses to genomic stress.},
journal = {Environmental and molecular mutagenesis},
volume = {61},
number = {1},
pages = {25-33},
doi = {10.1002/em.22349},
pmid = {31793074},
issn = {1098-2280},
mesh = {Animals ; CRISPR-Cas Systems ; Cellular Microenvironment ; Chromatin/genetics ; DNA/genetics ; *DNA Damage ; *DNA Repair ; Epigenesis, Genetic ; Gene Editing ; *Genomic Instability ; Genomics ; Humans ; Mutagenesis ; Mutation ; SOS Response, Genetics ; },
abstract = {Within the past half century we have learned of multiple pathways for repairing damaged DNA, based upon the intrinsic redundancy of information in its complementary double strands. Mechanistic details of these pathways have provided insights into environmental and endogenous threats to genomic stability. Studies on bacterial responses to ultraviolet light led to the discovery of excision repair, as well as the inducible SOS response to DNA damage. Similar responses in eukaryotes promote upregulation of error-prone translesion DNA polymerases. Recent advances in this burgeoning field include duplex DNA sequencing to provide strikingly accurate profiling of mutational signatures, analyses of gene expression patterns in single cells, CRISPR/Cas9 to generate changes at precise genomic positions, novel roles for RNA in gene expression and DNA repair, phase-separated aqueous environments for specialized cellular transactions, and DNA lesions as epigenetic signals for gene expression. The Environmental Mutagenesis and Genomics Society (EMGS), through the broad range of expertise in its membership, stands at the crossroad of basic understanding of mechanisms for genomic maintenance and the field of genetic toxicology, with the need for regulation of exposures to toxic substances. Our future challenges include devising strategies and technologies to identify individuals who are susceptible to specific genomic stresses, along with basic research on the underlying mechanisms of cellular stress responses that promote disease-causing mutations. As the science moves forward it should also be a responsibility for the EMGS to expand its outreach programs for the enlightenment and benefit of all humans and the biosphere. Environ. Mol. Mutagen. 61:25-33, 2020. © 2019 Wiley Periodicals, Inc.},
}
@article {pmid31792795,
year = {2020},
author = {Khosravi, S and Ishii, T and Dreissig, S and Houben, A},
title = {Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells.},
journal = {Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology},
volume = {28},
number = {1},
pages = {7-17},
pmid = {31792795},
issn = {1573-6849},
mesh = {*CRISPR-Cas Systems ; DNA-Binding Proteins/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Genes, Reporter ; Genetic Engineering/methods ; *Genome, Plant ; *Genomics/methods ; Molecular Imaging ; *Plant Cells/metabolism ; Promoter Regions, Genetic ; Trans-Activators/metabolism ; Zinc Fingers ; },
abstract = {The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science.},
}
@article {pmid31792780,
year = {2020},
author = {Yang, J and Li, J and Wang, J and Sheng, G and Wang, M and Zhao, H and Yang, Y and Wang, Y},
title = {Crystal structure of Cas1 in complex with branched DNA.},
journal = {Science China. Life sciences},
volume = {63},
number = {4},
pages = {516-528},
doi = {10.1007/s11427-019-9827-x},
pmid = {31792780},
issn = {1869-1889},
mesh = {Amino Acid Sequence ; Binding Sites ; CRISPR-Associated Proteins/*chemistry ; CRISPR-Cas Systems ; Crystallization ; DNA/*chemistry/genetics ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Multimerization ; Thermus thermophilus/chemistry/genetics ; },
abstract = {Cas1 is a key component of the CRISPR adaptation complex, which captures and integrates foreign DNA into the CRISPR array, resulting in the generation of new spacers. We have determined crystal structures of Thermus thermophilus Cas1 involved in new spacer acquisition both in complex with branched DNA and in the free state. Cas1 forms an asymmetric dimer without DNA. Conversely, two asymmetrical dimers bound to two branched DNAs result in the formation of a DNA-mediated tetramer, dimer of structurally asymmetrical dimers, in which the two subunits markedly present different conformations. In the DNA binding complex, the N-terminal domain adopts different orientations with respect to the C-terminal domain in the two monomers that form the dimer. Substrate binding triggers a conformational change in the loop 164-177 segment. This loop is also involved in the 3' fork arm and 5' fork arm strand recognition in monomer A and B, respectively. This study provides important insights into the molecular mechanism of new spacer adaptation.},
}
@article {pmid31792738,
year = {2020},
author = {Zheng, K and Jiang, FF and Su, L and Wang, X and Chen, YX and Chen, HC and Liu, ZF},
title = {Highly Efficient Base Editing in Viral Genome Based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein.},
journal = {Virologica Sinica},
volume = {35},
number = {2},
pages = {191-199},
pmid = {31792738},
issn = {1995-820X},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cell Line ; Chromosomes, Artificial, Bacterial/*genetics ; Computational Biology ; Cytidine/genetics ; Cytidine Deaminase/*genetics ; Escherichia coli/genetics ; Gene Editing ; *Genome, Viral ; Mutagenesis ; Plasmids/genetics ; Uridine/genetics ; },
abstract = {Viruses evolve rapidly and continuously threaten animal health and economy, posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine. We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase. We cloned pseudorabies virus genome into bacterial artificial chromosome, and used CRISPR-guided cytidine deaminase to directly convert cytidine (C) to uridine (U) to induce premature stop mutagenesis in viral genes. The editing efficiencies were 100%. Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus (PRV) genomes. Notably, in our study viral genome exists as a plasmid in E. coli, suggesting that this method is virus species-independent. This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.},
}
@article {pmid31792413,
year = {2019},
author = {Platt, RJ},
title = {CRISPR tool modifies genes precisely by copying RNA into the genome.},
journal = {Nature},
volume = {576},
number = {7785},
pages = {48-49},
pmid = {31792413},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; Genome ; RNA ; },
}
@article {pmid31792375,
year = {2019},
author = {},
title = {CRISPR-based technologies for cell biology.},
journal = {Nature cell biology},
volume = {21},
number = {12},
pages = {1463},
doi = {10.1038/s41556-019-0434-y},
pmid = {31792375},
issn = {1476-4679},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Biology ; Gene Editing/methods ; Humans ; },
}
@article {pmid31792362,
year = {2019},
author = {Leinonen, JT and Chen, YC and Pennonen, J and Lehtonen, L and Junna, N and Tukiainen, T and Panula, P and Widén, E},
title = {LIN28B affects gene expression at the hypothalamic-pituitary axis and serum testosterone levels.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18060},
pmid = {31792362},
issn = {2045-2322},
support = {MC_PC_12028/MRC_/Medical Research Council/United Kingdom ; MC_PC_17228/MRC_/Medical Research Council/United Kingdom ; MC_QA137853/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Computational Biology ; Datasets as Topic ; Estrogen Receptor alpha/metabolism ; Female ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Hypothalamo-Hypophyseal System/*metabolism ; Hypothalamus/metabolism ; Male ; Models, Animal ; Pituitary Gland/metabolism ; Polymorphism, Single Nucleotide ; Pro-Opiomelanocortin/*metabolism ; RNA-Binding Proteins/*genetics/*metabolism ; RNA-Seq ; Sexual Maturation/genetics ; Testosterone/*blood/metabolism ; Zebrafish ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Genome-wide association studies (GWAS) have recurrently associated sequence variation nearby LIN28B with pubertal timing, growth and disease. However, the biology linking LIN28B with these traits is still poorly understood. With our study, we sought to elucidate the mechanisms behind the LIN28B associations, with a special focus on studying LIN28B function at the hypothalamic-pituitary (HP) axis that is ultimately responsible for pubertal onset. Using CRISPR-Cas9 technology, we first generated lin28b knockout (KO) zebrafish. Compared to controls, the lin28b KO fish showed both accelerated growth tempo, reduced adult size and increased expression of mitochondrial genes during larval development. Importantly, data from the knockout zebrafish models and adult humans imply that LIN28B expression has potential to affect gene expression in the HP axis. Specifically, our results suggest that LIN28B expression correlates positively with the expression of ESR1 in the hypothalamus and POMC in the pituitary. Moreover, we show how the pubertal timing advancing allele (T) for rs7759938 at the LIN28B locus associates with higher testosterone levels in the UK Biobank data. Overall, we provide novel evidence that LIN28B contributes to the regulation of sex hormone pathways, which might help explain why the gene associates with several distinct traits.},
}
@article {pmid31792341,
year = {2020},
author = {Xing, H and Meng, LH},
title = {CRISPR-cas9: a powerful tool towards precision medicine in cancer treatment.},
journal = {Acta pharmacologica Sinica},
volume = {41},
number = {5},
pages = {583-587},
pmid = {31792341},
issn = {1745-7254},
mesh = {Animals ; Antineoplastic Agents/therapeutic use ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; Neoplasms/*drug therapy/*genetics ; Precision Medicine/*methods ; },
abstract = {Cancer is a highly heterogeneous disease in term of molecular signature even though it is originated from the same tissue type. Cancer heterogeneity may occur during its development or treatment, which is the main cause resulting in drug resistance and recurrence. Precision medicine refers to matching the right medicine to the right patients based on their molecular signatures. Therefore, a thorough understanding of the mechanism of tumorigenesis and drug resistance is essential to precision medicine. CRISPR-cas9 system is a powerful tool for gene editing and CRISPR-based high-throughput screening has been widely applied especially in searching for tumor-driven or synergistic lethal genes aiming to overcome drug resistance. In this review, we describe the progress of CRISPR-cas9-based unbiased screening in precision medicine including identification of new drug targets, biomarkers and elucidation of mechanisms leading to drug resistance. The existing challenges as well as the future directions are also discussed.},
}
@article {pmid31792295,
year = {2019},
author = {Palasin, K and Uechi, T and Yoshihama, M and Srisowanna, N and Choijookhuu, N and Hishikawa, Y and Kenmochi, N and Chotigeat, W},
title = {Abnormal development of zebrafish after knockout and knockdown of ribosomal protein L10a.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18130},
pmid = {31792295},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; DEAD-box RNA Helicases/genetics ; Embryo, Nonmammalian/*abnormalities ; Erythropoiesis/genetics ; GATA1 Transcription Factor/genetics ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Germ Cells/physiology ; Hemoglobins/*genetics ; Oligonucleotides, Antisense ; RNA-Binding Proteins/*genetics/metabolism ; Tumor Suppressor Protein p53/genetics ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {In this study, to investigate the secondary function of Rpl10a in zebrafish development, morpholino antisense oligonucleotides (MOs) were used to knock down the zebrafish ribosomal protein L10a (rpl10a). At 25 hpf (hours post-fertilization), embryos injected with the rpl10a MO showed an abnormal morphology, including short bodies, curved tails, and small yolk sac extensions. We observed pigment reductions, edema, larger yolk sacs, smaller eyes and smaller yolk sac extensions at 50 hpf. In addition, reductions in the expression of primordial germ cell (PGC) marker genes (nanos1 and vasa) were observed in rpl10a knockdown embryos. A rescue experiment using a rpl10a mRNA co-injection showed the recovery of the morphology and red blood cell production similar to wild-type. Moreover, the CRISPR-Cas9 system was used to edit the sequence of rpl10a exon 5, resulting in a homozygous 5-bp deletion in the zebrafish genome. The mutant embryos displayed a morphology similar to that of the knockdown animals. Furthermore, the loss of rpl10a function led to reduced expression of gata1, hbae3, and hbbe1 (erythroid synthesis) and increased tp53 expression. Overall, the results suggested that Rpl10a deficiency caused delays in embryonic development, as well as apoptosis and anemia, in zebrafish.},
}
@article {pmid31792288,
year = {2019},
author = {Neiman, G and Scarafía, MA and La Greca, A and Santín Velazque, NL and Garate, X and Waisman, A and Möbbs, AM and Kasai-Brunswick, TH and Mesquita, F and Martire-Greco, D and Moro, LN and Luzzani, C and Bastos Carvalho, A and Sevlever, GE and Campos de Carvalho, A and Guberman, AS and Miriuka, SG},
title = {Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18077},
pmid = {31792288},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Down-Regulation ; Gene Expression Regulation, Developmental ; HEK293 Cells ; Human Embryonic Stem Cells/*cytology/metabolism ; Humans ; Integrin alpha5/*genetics ; Myocytes, Cardiac/*cytology/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Stem Cell Niche ; },
abstract = {The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.},
}
@article {pmid31792264,
year = {2019},
author = {Munawar, U and Roth, M and Barrio, S and Wajant, H and Siegmund, D and Bargou, RC and Kortüm, KM and Stühmer, T},
title = {Assessment of TP53 lesions for p53 system functionality and drug resistance in multiple myeloma using an isogenic cell line model.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18062},
pmid = {31792264},
issn = {2045-2322},
mesh = {Alleles ; Antineoplastic Agents, Alkylating/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Heterozygote ; Homozygote ; Humans ; Melphalan/pharmacology/therapeutic use ; Multiple Myeloma/drug therapy/*genetics/pathology ; Point Mutation ; Signal Transduction/genetics ; Tumor Suppressor Protein p53/*genetics/metabolism ; },
abstract = {Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.},
}
@article {pmid31792240,
year = {2019},
author = {Sajwan, S and Mannervik, M},
title = {Gene activation by dCas9-CBP and the SAM system differ in target preference.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18104},
pmid = {31792240},
issn = {2045-2322},
mesh = {Acetylation ; Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Chromatin/genetics ; Drosophila/cytology/embryology/*genetics ; Drosophila Proteins/*genetics/metabolism ; Embryo, Nonmammalian ; Female ; Gene Expression Regulation ; Histone Acetyltransferases/genetics/metabolism ; Histones/genetics/metabolism ; Promoter Regions, Genetic ; Protein Domains ; Recombinant Proteins/genetics ; Transcription, Genetic ; p300-CBP Transcription Factors/*genetics/metabolism ; },
abstract = {Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas9 (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. To date, the most efficient dCas9-based activator is the Synergistic Activation Mediator (SAM) system whereby transcription activation domains are directly fused to dCas9 as well as tethered through MS2 loops engineered into the gRNA. Here, we show that dCas9 fused to the catalytic domain of the histone acetyltransferase CBP is a more potent activator than the SAM system at some loci, but less efficient at other locations in Drosophila cells. Our results suggest that different rate-limiting steps in the transcription cycle are affected by dCas9-CBP and the SAM system, and that comparing these activators may be useful for mechanistic studies of transcription as well as for increasing the number of hits in genome-wide overexpression screens.},
}
@article {pmid31791730,
year = {2020},
author = {Lee, SH and Park, YH and Jin, YB and Kim, SU and Hur, JK},
title = {CRISPR Diagnosis and Therapeutics with Single Base Pair Precision.},
journal = {Trends in molecular medicine},
volume = {26},
number = {3},
pages = {337-350},
doi = {10.1016/j.molmed.2019.09.008},
pmid = {31791730},
issn = {1471-499X},
mesh = {Animals ; Base Pairing/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genome/genetics ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats, or CRISPR, has been widely accepted as a versatile genome editing tool with significant potential for medical application. Reliable allele specificity is one of the most critical elements for successful application of this technology to develop high-precision therapeutics and diagnostics. CRISPR-based genome editing tools achieve high-fidelity distinction of single-base differences in target genomic loci by structural identification of CRISPR-associated (Cas) proteins and sequences of the guide RNAs. In this review, we describe the structural features of ribonucleoprotein complex formation by CRISPR proteins and guide RNAs that eventually recognize target DNA sequences. This structural understanding provides the basis for the recent applications of enhanced single-base precision genome editing technologies for effective distinction of specific alleles.},
}
@article {pmid31791724,
year = {2020},
author = {Yang, Q and Zhong, X and Li, Q and Lan, J and Tang, H and Qi, P and Ma, J and Wang, J and Chen, G and Pu, Z and Li, W and Lan, X and Deng, M and Harwood, W and Li, Z and Wei, Y and Zheng, Y and Jiang, Q},
title = {Mutation of the d-hordein gene by RNA-guided Cas9 targeted editing reducing the grain size and changing grain compositions in barley.},
journal = {Food chemistry},
volume = {311},
number = {},
pages = {125892},
doi = {10.1016/j.foodchem.2019.125892},
pmid = {31791724},
issn = {1873-7072},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Glutens/*genetics/metabolism ; Hordeum/genetics/growth &amp; development/*metabolism ; Mutation ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/metabolism ; Prolamins/metabolism ; RNA, Guide/genetics/*metabolism ; Seeds/genetics/metabolism ; Starch/metabolism ; beta-Glucans/metabolism ; },
abstract = {In this study, we successfully knock-out the d-hordein component of barley storage protein using RNA-guided Cas9. Mutation frequencies of 25% and 14% at two different target sites were obtained. Homozygous mutant plants that were T-DNA free were identified in the T1 generation. Barley grains without d-hordein proteins from T2 seeds showed a significantly reduced grain size compared to the parent plant and control non-edited line. The protein matrix surrounding the starch granules was increased, whereas the starch granules themselves were decreased in size in the mutant plants compared to controls. The main effect of a lack of d-hordein was a considerable decrease in the prolamines and an increase in the glutenins. The changes of other grain composition included the increased starch content, amylose content, and β-glucan content. The roles of d-hordein mutation on barley grain size and grain composition remain to be studied.},
}
@article {pmid31791381,
year = {2019},
author = {Mahas, A and Aman, R and Mahfouz, M},
title = {CRISPR-Cas13d mediates robust RNA virus interference in plants.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {263},
pmid = {31791381},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Host-Pathogen Interactions ; *Plant Immunity ; Plant Viruses/*immunology ; RNA Viruses/*immunology ; Tobacco ; },
abstract = {BACKGROUND: CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana.<br><br>RESULTS: Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta.<br><br>CONCLUSIONS: Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.},
}
@article {pmid31790583,
year = {2019},
author = {Krumbach, K and Sonntag, CK and Eggeling, L and Marienhagen, J},
title = {CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of Corynebacterium glutamicum.},
journal = {ACS synthetic biology},
volume = {8},
number = {12},
pages = {2726-2734},
pmid = {31790583},
issn = {2161-5063},
mesh = {Amino Acid Substitution/*genetics ; Bacterial Proteins/*genetics ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Corynebacterium glutamicum/*genetics ; DNA/genetics ; *Gene Editing ; Genetic Vectors/metabolism ; Glutamic Acid/metabolism ; Oligonucleotides/metabolism ; RNA/genetics ; },
abstract = {Against the background of a growing demand for the implementation of environmentally friendly production processes, microorganisms are engineered for the large-scale biosynthesis of chemicals, fuels, or food and feed additives from sustainable resources. Since strain development is expensive and time-consuming, continuous improvement of molecular tools for the genetic modification of the microbial production hosts is absolutely vital. Recently, the CRISPR/Cas12a technology for the engineering of Corynebacterium glutamicum as an important platform organism for industrial amino acid production has been introduced. Here, this system was advanced by designing an easy-to-construct crRNA delivery vector using simple oligonucleotides. In combination with a C. glutamicum strain engineered for the chromosomal expression of the β-galactosidase-encoding lacZ gene, this new plasmid was used to investigate CRISPR/Cas12a targeting and editing at various positions relative to the PAM site. Finally, we used this system to perform codon saturation mutagenesis at critical positions in the mechanosensitive channel MscCG to identify new gain-of-function mutations for increased l-glutamate export. The mutations obtained can be explained by particular demands of the channel on its immediate lipid environment to allow l-glutamate efflux.},
}
@article {pmid31789594,
year = {2019},
author = {Oh, HS and Neuhausser, WM and Eggan, P and Angelova, M and Kirchner, R and Eggan, KC and Knipe, DM},
title = {Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31789594},
issn = {2050-084X},
support = {Q-FASTR Award//Harvard Medical School/International ; P01 AI098681/AI/NIAID NIH HHS/United States ; R21 AI135423/AI/NIAID NIH HHS/United States ; P01 AI098681/NH/NIH HHS/United States ; R21 AI135423/NH/NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Repair/genetics ; DNA, Viral/genetics ; *Gene Editing ; Gene Expression Regulation, Viral ; *Genes, Viral ; Herpesviridae/*genetics ; Herpesvirus 1, Human/genetics ; Humans ; Models, Genetic ; Mutagenesis/genetics ; RNA, Guide/genetics ; Virus Replication/genetics ; },
abstract = {Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.},
}
@article {pmid31789494,
year = {2019},
author = {Echave, MC and Domingues, RMA and Gómez-Florit, M and Pedraz, JL and Reis, RL and Orive, G and Gomes, ME},
title = {Biphasic Hydrogels Integrating Mineralized and Anisotropic Features for Interfacial Tissue Engineering.},
journal = {ACS applied materials &amp; interfaces},
volume = {11},
number = {51},
pages = {47771-47784},
doi = {10.1021/acsami.9b17826},
pmid = {31789494},
issn = {1944-8252},
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Cellulose/chemistry ; Gelatin/chemistry ; Gene Editing/*methods ; Humans ; Hydrogels/chemistry ; Microscopy ; Nanoparticles/chemistry ; Swine ; Tendons/cytology ; Tissue Engineering/*methods ; Transglutaminases/metabolism ; },
abstract = {The innate graded structural and compositional profile of musculoskeletal tissue interfaces is disrupted and replaced by fibrotic tissue in the context of disease and degeneration. Tissue engineering strategies focused on the restoration of the transitional complexity found in those junctions present special relevance for regenerative medicine. Herein, we developed a gelatin-based multiphasic hydrogel system, where sections with distinct composition and microstructure were integrated in a single unit. In each phase, hydroxyapatite particles or cellulose nanocrystals (CNC) were incorporated into an enzymatically cross-linked gelatin network to mimic bone or tendon tissue, respectively. Stiffer hydrogels were produced with the incorporation of mineralized particles, and magnetic alignment of CNC resulted in anisotropic structure formation. The evaluation of the biological commitment with human adipose-derived stem cells toward the tendon-to-bone interface revealed an aligned cell growth and higher synthesis and deposition of tenascin in the anisotropic phase, while the activity of the secreted alkaline phosphatase and the expression of osteopontin were induced in the mineralized phase. These results highlight the potential versatility offered by gelatin-transglutaminase enzyme tandem for the development of strategies that mimic the graded, composite, and complex intersections of the connective tissues.},
}
@article {pmid31789463,
year = {2020},
author = {Pizon, V and Gaudin, N and Poteau, M and Cifuentes-Diaz, C and Demdou, R and Heyer, V and Reina San Martin, B and Azimzadeh, J},
title = {hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning.},
journal = {Biology of the cell},
volume = {112},
number = {1},
pages = {22-37},
doi = {10.1111/boc.201900038},
pmid = {31789463},
issn = {1768-322X},
mesh = {Animals ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*metabolism ; Cell Line ; *Centrioles/metabolism/ultrastructure ; *Centrosome/metabolism/ultrastructure ; Cilia/ultrastructure ; Cytoskeletal Proteins/metabolism ; Cytoskeleton/ultrastructure ; Humans ; Microscopy, Electron ; Microtubules/metabolism/ultrastructure ; RNA, Small Interfering ; Tubulin/*metabolism ; },
abstract = {BACKGROUND: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes.<br><br>RESULTS: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein.<br><br>CONCLUSIONS AND SIGNIFICANCE: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.},
}
@article {pmid31788711,
year = {2020},
author = {Wang, Q and Xie, F and Tong, Y and Habisch, R and Yang, B and Zhang, L and Müller, R and Fu, C},
title = {Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {1},
pages = {225-239},
doi = {10.1007/s00253-019-10223-4},
pmid = {31788711},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; Chromogenic Compounds ; Gene Editing/*methods ; *Genes, Reporter ; *Genome, Bacterial ; High-Throughput Screening Assays ; Multigene Family ; Plasmids/genetics ; Streptomyces coelicolor/*genetics ; },
abstract = {Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.},
}
@article {pmid31788683,
year = {2020},
author = {Chen, M and Xu, J and Zhou, Y and Zhang, S and Zhu, D},
title = {CRISPR-Cas9 genome editing for cancer immunotherapy: opportunities and challenges.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {183-190},
doi = {10.1093/bfgp/elz027},
pmid = {31788683},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Immunotherapy/*methods ; Neoplasms/*genetics ; },
abstract = {Cancer immunotherapy, consisting of antibodies, adoptive T-cell transfer, vaccines and cytokines, is a novel strategy for fighting cancer by artificially stimulating the immune system. It has developed rapidly in recent years, and its efficacy in hematological malignancies and solid tumors has been remarkable. It is regarded as one of the most promising methods for cancer therapy. The current trend in immunotherapy research seeks to improve its efficacy and to ensure the safety of cancer immunotherapy through the use of gene editing technologies. As it is an efficient and simple technology, the CRISPR-Cas9 system is highly anticipated to dramatically strengthen cancer immunotherapy. Intensive research on the CRISPR-Cas9 system has provided increasing confidence to clinicians that this system can be put into clinical use in the near future. This paper reviews the application and challenges of CRISPR-Cas9 in this field, based on various strategies including adaptive cell therapy and antibody therapy, and also highlights the function of CRISPR/Cas9 in the screening of new cancer targets.},
}
@article {pmid31786675,
year = {2020},
author = {Dong, L and Lin, X and Yu, D and Huang, L and Wang, B and Pan, L},
title = {High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {47},
number = {1},
pages = {133-144},
pmid = {31786675},
issn = {1476-5535},
mesh = {Aspergillus niger/genetics/*metabolism ; CRISPR-Cas Systems ; Enzyme Stability ; Ethanol/*metabolism ; Fermentation ; Hot Temperature ; Sordariales/genetics/*metabolism ; Trehalase/genetics/*metabolism ; },
abstract = {Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.},
}
@article {pmid31785780,
year = {2020},
author = {Timmermans, ML and Ross, AC},
title = {Unearthing Hidden Chemical Potential from Discarded Actinobacterial Libraries.},
journal = {Trends in biotechnology},
volume = {38},
number = {1},
pages = {7-9},
doi = {10.1016/j.tibtech.2019.11.003},
pmid = {31785780},
issn = {1879-3096},
mesh = {*Actinobacteria/genetics/metabolism ; *Anti-Bacterial Agents/chemistry/metabolism ; CRISPR-Cas Systems ; *Gene Library ; *Genetic Engineering ; Mutation/genetics ; },
abstract = {The redundancy of natural product biosynthesis in microbes poses a practical challenge for discovering new antimicrobial compounds from bacteria. The recent application of clustered regularly interspaced short palindromic repeats (CRISPR) technology by Culp et al. to inactivate the production of abundant antibiotics generates a metabolic clean slate for the detection and/or isolation of new and less plentiful antibiotics activated in mutant strains.},
}
@article {pmid31784756,
year = {2020},
author = {Nagaki, K and Yamaji, N},
title = {Decrosslinking enables visualization of RNA-guided endonuclease-in situ labeling signals for DNA sequences in plant tissues.},
journal = {Journal of experimental botany},
volume = {71},
number = {6},
pages = {1792-1800},
pmid = {31784756},
issn = {1460-2431},
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA ; *Endonucleases/genetics/metabolism ; Plants/*genetics ; RNA ; *RNA, Guide ; },
abstract = {Information about the positioning of individual loci in the nucleus and the status of epigenetic modifications at these loci in each cell contained in plant tissue increases our understanding of how cells in a tissue coordinate gene expression. To obtain such information, a less damaging method of visualizing DNA in tissue that can be used with immunohistochemistry is required. Recently, a less damaging DNA visualization method using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/associated caspase 9) system, named RNA-guided endonuclease-in situ labeling (RGEN-ISL), was reported. This system made it possible to visualize a target DNA locus in a nucleus fixed on a glass slide with a set of simple operations, but it could not be applied to cells in plant tissues. In this work, we have developed a modified RGEN-ISL method with decrosslinking that made it possible to simultaneously detect the DNA loci and immunohistochemistry signals, including histone modification, in various types of plant tissues and species.},
}
@article {pmid31784531,
year = {2019},
author = {Chylinski, K and Hubmann, M and Hanna, RE and Yanchus, C and Michlits, G and Uijttewaal, ECH and Doench, J and Schramek, D and Elling, U},
title = {CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5454},
pmid = {31784531},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Engineering ; Homologous Recombination ; Integrases ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; Mutagenesis ; RNA Polymerase III ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.},
}
@article {pmid31784416,
year = {2020},
author = {Dudek, AM and Zabaleta, N and Zinn, E and Pillay, S and Zengel, J and Porter, C and Franceschini, JS and Estelien, R and Carette, JE and Zhou, GL and Vandenberghe, LH},
title = {GPR108 Is a Highly Conserved AAV Entry Factor.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {2},
pages = {367-381},
pmid = {31784416},
issn = {1525-0024},
support = {R01 AI130123/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Motifs ; Animals ; CRISPR-Cas Systems ; Capsid Proteins/chemistry/genetics ; *Conserved Sequence ; Dependovirus/classification/*genetics ; Evolution, Molecular ; Gene Transfer Techniques ; Genetic Engineering ; Genetic Therapy ; Genetic Vectors/*genetics ; Genome, Viral ; Golgi Apparatus/metabolism ; Humans ; Phylogeny ; Protein Interaction Domains and Motifs ; },
abstract = {Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.},
}
@article {pmid31784086,
year = {2020},
author = {Bruns, AN and Lo, SH},
title = {Tensin regulates pharyngeal pumping in Caenorhabditis elegans.},
journal = {Biochemical and biophysical research communications},
volume = {522},
number = {3},
pages = {599-603},
pmid = {31784086},
issn = {1090-2104},
support = {R01 HL139473/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/chemistry/genetics/*physiology ; Caenorhabditis elegans Proteins/*metabolism ; Defecation ; Humans ; Pharynx/physiology ; Protein Domains ; Sequence Homology, Amino Acid ; Tensins/*metabolism ; },
abstract = {Tensin is a focal adhesion molecule that is known to regulate cell adhesion, migration, and proliferation. Although there are four tensin homologs (TNS1, TNS2, TNS3, and CTEN/TNS4) in mammals, only one tensin gene is found in Caenorhabditis elegans. Sequence analysis suggests that Caenorhabditis elegans tensin is slightly closer aligned with human TNS1 than with other human tensins. To establish the role of TNS1 in Caenorhabditis elegans, we have generated TNS1 knockout (KO) worms by CRISPR-Cas9 and homologous recombination directed repair approaches. Lack of TNS1 does not appear to affect the development or gross morphology of the worms. Nonetheless, defecation cycles are significantly longer in TNS1 KO worms. In addition, their pharyngeal pumping rate is markedly faster, which is likely due to a shorter pump duration in the KO worms. These findings indicate that TNS1 is not required for the development and survival of Caenorhabditis elegans but point to a critical role in modulating defecation and pharyngeal pumping rates.},
}
@article {pmid31782599,
year = {2020},
author = {Li, J and Qin, R and Zhang, Y and Xu, S and Liu, X and Yang, J and Zhang, X and Wei, P},
title = {Optimizing plant adenine base editor systems by modifying the transgene selection system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {7},
pages = {1495-1497},
pmid = {31782599},
issn = {1467-7652},
mesh = {*Adenine ; CRISPR-Cas Systems ; *Gene Editing ; Plants ; Transgenes ; },
}
@article {pmid31782306,
year = {2020},
author = {Wang, M and Chen, K and Wu, Q and Peng, R and Zhang, R and Li, J},
title = {RCasFISH: CRISPR/dCas9-Mediated in Situ Imaging of mRNA Transcripts in Fixed Cells and Tissues.},
journal = {Analytical chemistry},
volume = {92},
number = {3},
pages = {2468-2475},
doi = {10.1021/acs.analchem.9b03797},
pmid = {31782306},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/*genetics ; Humans ; *In Situ Hybridization, Fluorescence ; Lasers ; Microscopy, Confocal ; RNA, Messenger/*genetics ; Real-Time Polymerase Chain Reaction ; Tissue Fixation ; Tumor Cells, Cultured ; },
abstract = {Effective characterization and imaging of endogenous RNA transcripts have important value in the diagnosis, treatment, and prognosis of diseases. Traditional qRT-PCR as a liquid-based RNA detection method might lead to false-negative results due to the admixture of too many nontarget cells. Also, many in situ RNA imaging methods were hindered by long turnaround time and insufficient signals. Here, we describe and evaluate a CRISPR/dCas9-MS2-based RNA fluorescence in situ hybridization assay (RCasFISH) for in situ amplified imaging and quantification of RNA transcripts in fixed cells as well as formalin-fixed, paraffin-embedded (FFPE) tissue sections at a single-molecular level in individual cells. Compared to single molecular FISH (smFISH), RCasFISH yields brighter dot signals and a better signal-to-noise ratio (SNR) with lower costs and less than 1.5 h of hybridization. In addition, by using human epidermal growth factor receptor 2 (HER2) as a model, we quantified individual HER2 mRNA molecules in clinical breast cancer FFPE tissue sections and demonstrated its potential to resolve FISH-equivocal cases. Therefore, RCasFISH may provide a new approach for gene expression studies in basic research and hold the potential for molecular diagnostic applications.},
}
@article {pmid31782080,
year = {2019},
author = {Ren, M and Zafar, MM and Mo, H and Yang, Z and Li, F},
title = {Fighting against fall armyworm by using multiple genes pyramiding and silencing (MGPS) technology.},
journal = {Science China. Life sciences},
volume = {62},
number = {12},
pages = {1703-1706},
doi = {10.1007/s11427-019-1586-7},
pmid = {31782080},
issn = {1869-1889},
mesh = {Animals ; Bacterial Toxins/*genetics/pharmacology ; CRISPR-Cas Systems/genetics ; Drug Resistance ; Gastrointestinal Absorption ; Gene Silencing ; Gene Transfer Techniques ; Insecticides/*pharmacology ; Moths/*classification/drug effects/*genetics ; Pest Control, Biological/*methods ; Phospholipids/chemistry ; Protein Precursors/*genetics/pharmacology ; RNA/chemistry/metabolism ; },
}
@article {pmid31782056,
year = {2019},
author = {Pahan, K},
title = {A Broad Application of CRISPR Cas9 in Infectious, Inflammatory and Neurodegenerative Diseases.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {4},
pages = {534-536},
pmid = {31782056},
issn = {1557-1904},
support = {I01 BX003033/BX/BLRD VA/United States ; R01 AG050431/AG/NIA NIH HHS/United States ; I01 BX005002/BX/BLRD VA/United States ; R21 NS108025/NS/NINDS NIH HHS/United States ; IK6 BX004982/BX/BLRD VA/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/immunology ; CRISPR-Cas Systems/*genetics/immunology ; Gene Editing/*methods/trends ; HIV Infections/*genetics/immunology/therapy ; Humans ; Inflammation/*genetics/immunology/therapy ; Neurodegenerative Diseases/*genetics/immunology/therapy ; },
abstract = {Being the most important immune-responsive cell type of the CNS, microglia always glorify the so-called crossroad of Neurology, Immunology and Pharmacology. As microglial activation is a hallmark of different neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), HIV-associated neurocognitive disorders (HAND), Amyotrophic lateral sclerosis (ALS), etc., selective targeting of microglial cell signaling may be a valid option to control these neurodegenerative disorders with lesser side effects. This is particularly important as no effective therapies are available against these diseases and available neuroimmune modulators are known to target multiple cell types in a non-cell-specific manner. How we can achieve such specificity? A newly-developed cutting-edge molecular biology tool is rocking biomedical research in recent years so much so that it has already come under major lawsuits between the University of California Berkeley and the MIT-Harvard Broad Institute regarding its ownership rights, probably halting the Nobel committee to announce the most coveted prize to its owners. It is none other than Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In nutshell, the Cas9 enzyme has been paired with the bacterial immune system, CRISPR, to ultimately turn CRISPR/Cas9 as an effective genome editor. Therefore, this special issue has been devoted to highlight some of the recent discoveries on CRISPR/Cas9 in neurodegenerative disorders and explain these discoveries in the light of neuroimmune pharmacology.},
}
@article {pmid31781808,
year = {2020},
author = {Hu, T and Cui, Y and Qu, X},
title = {Characterization and comparison of CRISPR Loci in Streptococcus thermophilus.},
journal = {Archives of microbiology},
volume = {202},
number = {4},
pages = {695-710},
doi = {10.1007/s00203-019-01780-3},
pmid = {31781808},
issn = {1432-072X},
mesh = {Bacteriophages/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/*genetics ; Plasmids/genetics ; Sequence Analysis, DNA ; Streptococcus thermophilus/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) consists of a series of regular repeat-spacer sequences. It can not only act as a natural immune system in most prokaryotes, but also be utilized as the tool of newly developed genome modification and evolutionary researches. Streptococcus thermophilus is an important model organism for the study and application of CRISPR systems. In present study, the occurrence and diversity of CRISPR-Cas systems in the genomes of S. thermophilus were investigated including 4 new sequenced strains CS5, CS9, CS18, CS20, and other 23 strains downloaded from NCBI website. 66 CRISPR/Cas systems were identified among these 27 strains and could divided into four subsystems according to the arrangement of Cas proteins, notably I-E, II-A, II-C and III-A. Overall, 26 type II-C systems, 18 type II-A systems, 13 type III-A systems, 9 type I-E systems were identified. It was mentioned that CS20 contained two type II-C systems which had not been identified in the other 26 S. thermophilus strains. Overall, 1,080 spacers were analyzed and blasted. Sequence identity searches of spacers implied that most spacers derived from partial sequences of exogenous DNA, including various bacteriophages and plasmids. Of note, a large number of novel spacers were found in this study, indicating the unique phage environment they have undergone, especially CS20 strain. In addition, the analysis of the cas1 and cas9 genes revealed the genetic relationship among CRISPR-Cas system in these strains. Furthermore, the analysis of CRISPR spacers also indicated protospacer adjacent motif (PAM) sequences. Summary of PAM sequences could lay the foundations for the application of S. thermophilus CRISPR-Cas system. Our results suggested CS5 and CS18 can be used as model strains in the research of CRISPR-Cas system, and CS20 might have greater application potential in gene editing.},
}
@article {pmid31779484,
year = {2020},
author = {Zhao, H and Li, Y and He, L and Pu, W and Yu, W and Li, Y and Wu, YT and Xu, C and Wei, Y and Ding, Q and Song, BL and Huang, H and Zhou, B},
title = {In Vivo AAV-CRISPR/Cas9-Mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia.},
journal = {Circulation},
volume = {141},
number = {1},
pages = {67-79},
doi = {10.1161/CIRCULATIONAHA.119.042476},
pmid = {31779484},
issn = {1524-4539},
mesh = {Animals ; *Atherosclerosis/genetics/metabolism/pathology/therapy ; *CRISPR-Cas Systems ; *Dependovirus ; *Gene Editing ; *Hyperlipoproteinemia Type II/genetics/metabolism/pathology/therapy ; Mice ; Mice, Transgenic ; Mutation, Missense ; Receptors, LDL/genetics/metabolism ; },
abstract = {BACKGROUND: Mutations in low-density lipoprotein (LDL) receptor (LDLR) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease.<br><br>METHODS: The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, Ldlr[E208X], based on a relevant familial hypercholesterolemia-related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into Ldlr[E208X] mice.<br><br>RESULTS: We found that homogeneous Ldlr[E208X] mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration.<br><br>CONCLUSIONS: Our work shows that in vivo AAV-CRISPR/Cas9-mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.},
}
@article {pmid31778538,
year = {2019},
author = {Msheik, H and El Hayek, S and Bari, MF and Azar, J and Abou-Kheir, W and Kobeissy, F and Vatish, M and Daoud, G},
title = {Transcriptomic profiling of trophoblast fusion using BeWo and JEG-3 cell lines.},
journal = {Molecular human reproduction},
volume = {25},
number = {12},
pages = {811-824},
doi = {10.1093/molehr/gaz061},
pmid = {31778538},
issn = {1460-2407},
support = {MR/J003360/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Communication/*physiology ; Cell Differentiation/physiology ; Cell Fusion ; Cell Line, Tumor ; Colforsin/pharmacology ; Female ; Gene Expression Regulation, Developmental/*genetics ; Humans ; Placenta/cytology/*metabolism ; Pregnancy ; Protein Serine-Threonine Kinases/*genetics ; Trophoblasts/*metabolism ; },
abstract = {In human placenta, alteration in trophoblast differentiation has a major impact on placental maintenance and integrity. However, little is known about the mechanisms that control cytotrophoblast fusion. The BeWo cell line is used to study placental function, since it forms syncytium and secretes hormones after treatment with cAMP or forskolin. In contrast, the JEG-3 cell line fails to undergo substantial fusion. Therefore, BeWo and JEG-3 cells were used to identify a set of genes responsible for trophoblast fusion. Cells were treated with forskolin for 48 h to induce fusion. RNA was extracted, hybridised to Affymetrix HuGene ST1.0 arrays and analysed using system biology. Trophoblast differentiation was evaluated by real-time PCR and immunocytochemistry analysis. Moreover, some of the identified genes were validated by real-time PCR and their functional capacity was demonstrated by western blot using phospho-specific antibodies and CRISPR/cas9 knockdown experiments. Our results identified a list of 32 altered genes in fused BeWo cells compared to JEG-3 cells after forskolin treatment. Among these genes, four were validated by RT-PCR, including salt-inducible kinase 1 (SIK1) gene which is specifically upregulated in BeWo cells upon fusion and activated after 2 min with forskolin. Moreover, silencing of SIK1 completely abolished the fusion. Finally, SIK1 was shown to be at the center of many biological and functional processes, suggesting that it might play a role in trophoblast differentiation. In conclusion, this study identified new target genes implicated in trophoblast fusion. More studies are required to investigate the role of these genes in some placental pathology.},
}
@article {pmid31776930,
year = {2020},
author = {Zhang, J and Späth, SS and Katz, SG},
title = {Genome-Wide CRISPRi/a Screening in an In Vitro Coculture Assay of Human Immune Cells with Tumor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2097},
number = {},
pages = {231-252},
pmid = {31776930},
issn = {1940-6029},
support = {R01 HL131793/HL/NHLBI NIH HHS/United States ; R21 AI121993/AI/NIAID NIH HHS/United States ; R21 CA198561/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Coculture Techniques/*methods ; *Genetic Testing ; *Genome ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lentivirus/metabolism ; Leukocytes/*immunology ; Neoplasms/*pathology ; Oligonucleotides/metabolism ; Plasmids/metabolism ; RNA, Guide/metabolism ; Transformation, Genetic ; Virion/metabolism ; },
abstract = {Cell-based immunotherapy has achieved preclinical success in certain types of cancer patients, with a few approved cell-based products for clinical use. These achievements revitalized the field of cell engineering/ immunotherapy and brought attention to the opportunities that cell-based immunotherapeutics can offer to patients. On the other hand, obvious indications emphasize the need for a better understanding of the biological mechanisms involved in the immune response. This knowledge may not only ameliorate safety and efficacy, but also determine the possibilities and limitations in use of immune cell engineering for cancer treatment, and facilitate developing novel immunotherapeutic strategies. Recently developed technology based on CRISPR-dCas9 has an immense potential to systematically uncover genetic mechanisms by identifying subsets of essential genes involved in interactions of cancer cells with the immune system. This chapter will present a reliable and reproducible general protocol for the application of genome-wide sgRNA gene-editing tools in the recently established two-cell type co-culture, consisting of immune cells as effectors and cancer cells as targets, utilizing CRISPRi/a-dCas9-based technology.},
}
@article {pmid31776929,
year = {2020},
author = {Bailis, W},
title = {CRISPR/Cas9 Gene Targeting in Primary Mouse Bone Marrow-Derived Macrophages.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2097},
number = {},
pages = {223-230},
doi = {10.1007/978-1-0716-0203-4_14},
pmid = {31776929},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Targeting/*methods ; HEK293 Cells ; Humans ; Macrophages/*metabolism ; Mice ; Retroviridae/metabolism ; Transduction, Genetic ; },
abstract = {CRISPR-Cas9 technology allows for rapid, targeted genome editing at nearly any loci with limited off-target effects. Here, we describe a method for using retroviral transduction to deliver single-guide RNA to primary bone marrow-derived macrophages. This protocol allows for high-throughput reverse genetics assays in primary immune cells and is also compatible with retroviral systems for transgene expression.},
}
@article {pmid31776874,
year = {2020},
author = {Xiao, G and Zhang, S and Liang, Z and Li, G and Fang, M and Liu, Y and Zhang, J and Ou, M and He, X and Zhang, T and Zeng, C and Liu, L and Zhang, G},
title = {Identification of Mycobacterium abscessus species and subspecies using the Cas12a/sgRNA-based nucleic acid detection platform.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {39},
number = {3},
pages = {551-558},
pmid = {31776874},
issn = {1435-4373},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; DNA, Bacterial ; Endodeoxyribonucleases/*genetics ; Genes, Bacterial ; Humans ; Molecular Typing/*methods ; Mycobacterium Infections, Nontuberculous/*diagnosis/*microbiology ; Mycobacterium abscessus/*classification/*genetics ; Phylogeny ; Polymerase Chain Reaction ; *RNA, Guide ; Sequence Analysis, DNA ; Workflow ; },
abstract = {The rapidly growing mycobacterium Mycobacterium abscessus is a clinically important organism causing pulmonary and skin diseases. The M. abscessus complex is comprised of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Here, we aimed to develop a Cas12a/sgRNA-based nucleic acid detection platform to identify M. abscessus species and subspecies. By designing specific sgRNA probes targeting rpoB and erm(41), we demonstrated that M. abscessus could be differentiated from other major mycobacterial species and identified at the subspecies level. Using this platform, a total of 38 clinical M. abscessus isolates were identified, 18 as M. abscessus subsp. abscessus and 20 as M. abscessus subsp. massiliense. We concluded that the Cas12a/sgRNA-based nucleic acid detection platform provides an easy-to-use, quick, and cost-effective approach for identification of M. abscessus species and subspecies.},
}
@article {pmid31776399,
year = {2019},
author = {Johansen, IE and Liu, Y and Jørgensen, B and Bennett, EP and Andreasson, E and Nielsen, KL and Blennow, A and Petersen, BL},
title = {High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17715},
pmid = {31776399},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Plant Breeding/*methods ; Solanum tuberosum/*genetics ; Tetraploidy ; },
abstract = {CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.},
}
@article {pmid31776341,
year = {2019},
author = {Shimokawa-Chiba, N and Müller, C and Fujiwara, K and Beckert, B and Ito, K and Wilson, DN and Chiba, S},
title = {Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5397},
pmid = {31776341},
issn = {2041-1723},
mesh = {Bacillus subtilis/*genetics/metabolism ; Bacterial Proteins/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; Escherichia coli Proteins/genetics ; Gene Expression Regulation, Bacterial ; Microorganisms, Genetically-Modified ; Peptide Termination Factors/chemistry/genetics/metabolism ; Protein Biosynthesis ; Protein Conformation ; RNA, Transfer, Amino Acyl/metabolism ; RNA-Binding Proteins/genetics ; Ribosomes/genetics/*metabolism ; },
abstract = {Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.},
}
@article {pmid31775745,
year = {2019},
author = {Karponi, G and Kritas, SK and Papadopoulou, G and Akrioti, EK and Papanikolaou, E and Petridou, E},
title = {Development of a CRISPR/Cas9 system against ruminant animal brucellosis.},
journal = {BMC veterinary research},
volume = {15},
number = {1},
pages = {422},
pmid = {31775745},
issn = {1746-6148},
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; Brucella melitensis/genetics ; Brucellosis/*therapy ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cells, Cultured ; DNA-Directed RNA Polymerases ; Gene Editing/veterinary ; Genetic Therapy/veterinary ; Macrophages/microbiology ; Sheep ; Sheep Diseases/microbiology/*therapy ; },
abstract = {BACKGROUND: Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells.<br><br>RESULTS: We infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella's RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system.<br><br>CONCLUSIONS: Pending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.},
}
@article {pmid31775607,
year = {2019},
author = {Nethery, MA and Henriksen, ED and Daughtry, KV and Johanningsmeier, SD and Barrangou, R},
title = {Comparative genomics of eight Lactobacillus buchneri strains isolated from food spoilage.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {902},
pmid = {31775607},
issn = {1471-2164},
mesh = {Computational Biology/methods ; Evolution, Molecular ; Fermentation ; *Food Microbiology ; *Genome, Bacterial ; *Genomics/methods ; Lactobacillus/classification/*genetics/*isolation &amp; purification ; Molecular Sequence Annotation ; Phylogeny ; Whole Genome Sequencing ; },
abstract = {BACKGROUND: Lactobacillus buchneri is a lactic acid bacterium frequently associated with food bioprocessing and fermentation and has been found to be either beneficial or detrimental to industrial food processes depending on the application. The ability to metabolize lactic acid into acetic acid and 1,2-propandiol makes L. buchneri invaluable to the ensiling process, however, this metabolic activity leads to spoilage in other applications, and is especially damaging to the cucumber fermentation industry. This study aims to augment our genomic understanding of L. buchneri in order to make better use of the species in a wide range of applicable industrial settings.<br><br>RESULTS: Whole-genome sequencing (WGS) was performed on seven phenotypically diverse strains isolated from spoiled, fermented cucumber and the ATCC type strain for L. buchneri, ATCC 4005. Here, we present our findings from the comparison of eight newly-sequenced and assembled genomes against two publicly available closed reference genomes, L. buchneri CD034 and NRRL B-30929. Overall, we see ~&#x2009;50% of all coding sequences are conserved across these ten strains. When these coding sequences are clustered by functional description, the strains appear to be enriched in mobile genetic elements, namely transposons. All isolates harbor at least one CRISPR-Cas system, and many contain putative prophage regions, some of which are targeted by the host's own DNA-encoded spacer sequences.<br><br>CONCLUSIONS: Our findings provide new insights into the genomics of L. buchneri through whole genome sequencing and subsequent characterization of genomic features, building a platform for future studies and identifying elements for potential strain manipulation or engineering.},
}
@article {pmid31775372,
year = {2019},
author = {Nakamura, S and Watanabe, S and Ando, N and Ishihara, M and Sato, M},
title = {Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice.},
journal = {International journal of molecular sciences},
volume = {20},
number = {23},
pages = {},
pmid = {31775372},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/*administration &amp; dosage ; Female ; Fetus/*metabolism ; *Gene Editing ; *Gene Targeting ; Genetic Engineering ; *Genetic Therapy ; Mice ; Placenta/*metabolism ; Pregnancy ; },
abstract = {Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.},
}
@article {pmid31775087,
year = {2019},
author = {Xie, L and Huang, J and Li, X and Dai, L and Lin, X and Zhang, J and Luo, J and Zhang, W},
title = {Generation of a homozygous HDAC6 knockout human embryonic stem cell line by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101610},
doi = {10.1016/j.scr.2019.101610},
pmid = {31775087},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Female ; *Gene Editing ; Histone Deacetylase 6/*antagonists &amp; inhibitors/genetics/metabolism ; *Homozygote ; Human Embryonic Stem Cells/*cytology/metabolism ; Humans ; Karyotype ; },
abstract = {Histone deacetylase 6 (HDAC6) is a unique cytoplasmic enzyme in the HDAC family. The HDAC6 has been shown to play important roles in several biological processes. Meanwhile, it is also an attractive therapeutic target for a variety of diseases. However, the mechanism of HDAC6 function is not fully understood yet, and it is still lacking highly specific targeted drugs. Here, we generated a homozygous HDAC6 knockout human embryonic stem cell (hESC) line, WAe009-A-21 by the CRISPR/Cas9-based gene editing method. The WAe009-A-21 cell line does not express HDAC6 protein, while maintaining normal 46, XX karyotype, pluripotency, and trilineage differentiation potential.},
}
@article {pmid31774847,
year = {2019},
author = {Shridhar, PB and Worley, JN and Gao, X and Yang, X and Noll, LW and Shi, X and Bai, J and Meng, J and Nagaraja, TG},
title = {Analysis of virulence potential of Escherichia coli O145 isolated from cattle feces and hide samples based on whole genome sequencing.},
journal = {PloS one},
volume = {14},
number = {11},
pages = {e0225057},
pmid = {31774847},
issn = {1932-6203},
support = {U01 FD001418/FD/FDA HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cattle ; Drug Resistance, Bacterial/genetics ; Escherichia coli/*genetics/isolation &amp; purification/*pathogenicity ; Feces/*microbiology ; Genome Size ; Genome, Bacterial ; Humans ; Phylogeny ; Plasmids/genetics ; Prophages/genetics ; Virulence/genetics ; *Whole Genome Sequencing ; },
abstract = {Escherichia coli O145 serogroup is one of the big six non-O157 Shiga toxin producing E. coli (STEC) that causes foodborne illnesses in the United States and other countries. Cattle are a major reservoir of STEC, which harbor them in their hindgut and shed in the feces. Cattle feces is the main source of hide and subsequent carcass contaminations during harvest leading to foodborne illnesses in humans. The objective of our study was to determine the virulence potential of STEC O145 strains isolated from cattle feces and hide samples. A total of 71 STEC O145 strains isolated from cattle feces (n = 16), hide (n = 53), and human clinical samples (n = 2) were used in the study. The strains were subjected to whole genome sequencing using Illumina MiSeq platform. The average draft genome size of the fecal, hide, and human clinical strains were 5.41, 5.28, and 5.29 Mb, respectively. The average number of genes associated with mobile genetic elements was 260, 238, and 259, in cattle fecal, hide, and human clinical strains, respectively. All strains belonged to O145:H28 serotype and carried eae subtype γ. Shiga toxin 1a was the most common Shiga toxin gene subtype among the strains, followed by stx2a and stx2c. The strains also carried genes encoding type III secretory system proteins, nle, and plasmid-encoded virulence genes. Phylogenetic analysis revealed clustering of cattle fecal strains separately from hide strains, and the human clinical strains were more closely related to the hide strains. All the strains belonged to sequence type (ST)-32. The virulence gene profile of STEC O145 strains isolated from cattle sources was similar to that of human clinical strains and were phylogenetically closely related to human clinical strains. The genetic analysis suggests the potential of cattle STEC O145 strains to cause human illnesses. Inclusion of more strains from cattle and their environment in the analysis will help in further elucidation of the genetic diversity and virulence potential of cattle O145 strains.},
}
@article {pmid31773990,
year = {2020},
author = {Klatt, D and Cheng, E and Hoffmann, D and Santilli, G and Thrasher, AJ and Brendel, C and Schambach, A},
title = {Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells.},
journal = {Human gene therapy},
volume = {31},
number = {3-4},
pages = {199-210},
pmid = {31773990},
issn = {1557-7422},
support = {104807/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; },
mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Gene Editing ; Gene Expression ; *Gene Silencing ; Gene Targeting ; *Genetic Loci ; Genetic Vectors ; Granulomatous Disease, Chronic/genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Myeloid Cells/cytology/*metabolism ; NADPH Oxidases/deficiency/genetics/metabolism ; Organ Specificity/genetics ; *Promoter Regions, Genetic ; *Transgenes ; },
abstract = {Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral long terminal repeat (LTR)-driven vectors, which semirandomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSCs) and can impede transgene expression during differentiation. In this study, we aimed to correct p47[phox] deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47[phox] transgene into the AAVS1 locus of human induced PSCs (iPSCs) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes, and myeloid-related protein 8 [MRP8]). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoters achieved therapeutically relevant levels of p47[phox] expression and nicotinamide adenine dinucleotide phosphate oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells.},
}
@article {pmid31773942,
year = {2019},
author = {Li, S and Song, Z and Liu, C and Chen, XL and Han, H},
title = {Biomimetic Mineralization-Based CRISPR/Cas9 Ribonucleoprotein Nanoparticles for Gene Editing.},
journal = {ACS applied materials &amp; interfaces},
volume = {11},
number = {51},
pages = {47762-47770},
doi = {10.1021/acsami.9b17598},
pmid = {31773942},
issn = {1944-8252},
mesh = {Biomimetics/methods ; Biotechnology/methods ; CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {Delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex has provided an alternative strategy for the regulation of CRISPR functions, offering a transient and DNA-free means for genomic editing. Chemical methods of delivering the RNPs via nanocomplexes have the potential to address these delivery problems for efficiency, safety, and packaging capacity. Here, we developed a biomimetic mineralization-mediated strategy for efficient DNA-free genome editing by using CRISPR/Cas9 RNPs. We found that the RNPs have the ability to form the biomimetic mineralized RNP nanoparticles (Bm-RNP NPs) quickly in situ and can be effectively delivered into the fungal protoplast cells. Biomimetic mineralization can maintain the natural function of Cas9 protein and protect the sgRNA activity. At the same time, the encapsulated RNPs can be effectively released into the cytoplasm, and the Sytalone dehydratase (SDH) gene can be edited in a targeted manner. Except for phenotypic defects, molecular detections indicated that the delivery of Bm-RNP NPs achieved 20% genomic editing for Magnaporthe oryzae compared to RNPs alone. Moreover, the Bm-RNP NP-mediated editing of the SDH gene significantly affects the appressorium-mediated penetration and invasive growth in M. oryzae. Our system has the advantages of being cheap, fast, and effective, without the traditional transformation process, suggesting the potential application of this DNA-free gene-editing strategy in different organisms.},
}
@article {pmid31772156,
year = {2019},
author = {Steinmann, S and Kunze, P and Hampel, C and Eckstein, M and Bertram Bramsen, J and Muenzner, JK and Carlé, B and Ndreshkjana, B and Kemenes, S and Gasparini, P and Friedrich, O and Andersen, C and Geppert, C and Wang, S and Eyupoglu, I and Bäuerle, T and Hartmann, A and Schneider-Stock, R},
title = {DAPK1 loss triggers tumor invasion in colorectal tumor cells.},
journal = {Cell death &amp; disease},
volume = {10},
number = {12},
pages = {895},
pmid = {31772156},
issn = {2041-4889},
mesh = {Animals ; Antigens, Neoplasm/metabolism ; CRISPR-Cas Systems/genetics ; Cell Adhesion Molecules/metabolism ; Cell Proliferation ; Chick Embryo ; Chorioallantoic Membrane/metabolism ; Clone Cells ; Colorectal Neoplasms/*enzymology/genetics/*pathology ; Death-Associated Protein Kinases/*deficiency/metabolism ; Extracellular Matrix/metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; MAP Kinase Signaling System ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Rats, Wistar ; Reproducibility of Results ; Spheroids, Cellular/metabolism/pathology ; Tumor Burden ; },
abstract = {Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.},
}
@article {pmid31771992,
year = {2019},
author = {Emmert, AS and Iwasawa, E and Shula, C and Schultz, P and Lindquist, D and Dunn, RS and Fugate, EM and Hu, YC and Mangano, FT and Goto, J},
title = {Impaired neural differentiation and glymphatic CSF flow in the Ccdc39 rat model of neonatal hydrocephalus: genetic interaction with L1cam.},
journal = {Disease models &amp; mechanisms},
volume = {12},
number = {11},
pages = {},
pmid = {31771992},
issn = {1754-8411},
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; Cell Death ; Cell Differentiation ; Cerebrospinal Fluid/*physiology ; Cytoskeletal Proteins/genetics ; *Disease Models, Animal ; Glymphatic System/*physiology ; Hydrocephalus/*etiology ; *Mutation ; Neural Cell Adhesion Molecule L1/*genetics ; Neurons/cytology ; Rats ; Rats, Sprague-Dawley ; },
abstract = {Neonatal hydrocephalus affects about one child per 1000 births and is a major congenital brain abnormality. We previously discovered a gene mutation within the coiled-coil domain-containing 39 (Ccdc39) gene, which causes the progressive hydrocephalus (prh) phenotype in mice due to lack of ependymal-cilia-mediated cerebrospinal fluid (CSF) flow. In this study, we used CRISPR/Cas9 to introduce the Ccdc39 gene mutation into rats, which are more suitable for imaging and surgical experiments. The Ccdc39[prh/prh] mutants exhibited mild ventriculomegaly at postnatal day (P)5 that progressed into severe hydrocephalus by P11 (P<0.001). After P11, macrophage and neutrophil invasion along with subarachnoid hemorrhage were observed in mutant brains showing reduced neurofilament density, hypomyelination and increased cell death signals compared with wild-type brains. Significantly more macrophages entered the brain parenchyma at P5 before hemorrhaging was noted and increased expression of a pro-inflammatory factor (monocyte chemoattractant protein-1) was found in the cortical neural and endothelial cells in the mutant brains at P11. Glymphatic-mediated CSF circulation was progressively impaired along the middle cerebral artery from P11 as mutants developed severe hydrocephalus (P<0.001). In addition, Ccdc39[prh/prh] mutants with L1 cell adhesion molecule (L1cam) gene mutation, which causes X-linked human congenital hydrocephalus, showed an accelerated early hydrocephalus phenotype (P<0.05-0.01). Our findings in Ccdc39[prh/prh] mutant rats demonstrate a possible causal role of neuroinflammation in neonatal hydrocephalus development, which involves impaired cortical development and glymphatic CSF flow. Improved understanding of inflammatory responses and the glymphatic system in neonatal hydrocephalus could lead to new therapeutic strategies for this condition.This article has an associated First Person interview with the joint first authors of the paper.},
}
@article {pmid31771289,
year = {2019},
author = {Santos, R and Amaral, O},
title = {Advances in Sphingolipidoses: CRISPR-Cas9 Editing as an Option for Modelling and Therapy.},
journal = {International journal of molecular sciences},
volume = {20},
number = {23},
pages = {},
pmid = {31771289},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Enzyme Replacement Therapy ; Gaucher Disease/genetics/therapy ; Gene Editing/*methods ; Humans ; Sphingolipidoses/genetics/*therapy ; beta-Glucosidase/genetics ; },
abstract = {Sphingolipidoses are inherited genetic diseases characterized by the accumulation of glycosphingolipids. Sphingolipidoses (SP), which usually involve the loss of sphingolipid hydrolase function, are of lysosomal origin, and represent an important group of rare diseases among lysosomal storage disorders. Initial treatments consisted of enzyme replacement therapy, but, in recent decades, various therapeutic approaches have been developed. However, these commonly used treatments for SP fail to be fully effective and do not penetrate the blood-brain barrier. New approaches, such as genome editing, have great potential for both the treatment and study of sphingolipidoses. Here, we review the most recent advances in the treatment and modelling of SP through the application of CRISPR-Cas9 genome editing. CRISPR-Cas9 is currently the most widely used method for genome editing. This technique is versatile; it can be used for altering the regulation of genes involved in sphingolipid degradation and synthesis pathways, interrogating gene function, generating knock out models, or knocking in mutations. CRISPR-Cas9 genome editing is being used as an approach to disease treatment, but more frequently it is utilized to create models of disease. New CRISPR-Cas9-based tools of gene editing with diminished off-targeting effects are evolving and seem to be more promising for the correction of individual mutations. Emerging Prime results and CRISPR-Cas9 difficulties are also discussed.},
}
@article {pmid31769791,
year = {2020},
author = {Sun, J and Wang, J and Zheng, D and Hu, X},
title = {Advances in therapeutic application of CRISPR-Cas9.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {164-174},
doi = {10.1093/bfgp/elz031},
pmid = {31769791},
issn = {2041-2657},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Genetic Therapy ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.},
}
@article {pmid31769589,
year = {2020},
author = {Zhang, P and Du, H and Wang, J and Pu, Y and Yang, C and Yan, R and Yang, H and Cheng, H and Yu, D},
title = {Multiplex CRISPR/Cas9-mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus.},
journal = {Plant biotechnology journal},
volume = {18},
number = {6},
pages = {1384-1395},
pmid = {31769589},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Isoflavones ; Metabolic Engineering ; *Mosaic Viruses ; Plants, Genetically Modified/genetics ; Soybeans/genetics ; },
abstract = {Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant-environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone-3-hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9-mediated multiplex gene-editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII-1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T0 transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T0 triple-mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T3 generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one-third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T2 triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.},
}
@article {pmid31768773,
year = {2020},
author = {Li, ZH and Meng, H and Ma, B and Tao, X and Liu, M and Wang, FQ and Wei, DZ},
title = {Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {47},
number = {1},
pages = {83-96},
pmid = {31768773},
issn = {1476-5535},
mesh = {CRISPR-Cas Systems ; Genome, Fungal ; Plasmids/genetics ; RNA, Fungal/genetics ; RNA, Guide/genetics ; Saccharomyces cerevisiae/*genetics ; },
abstract = {A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.},
}
@article {pmid31768302,
year = {2019},
author = {Cabrera-Contreras, R and Santamaría, RI and Bustos, P and Martínez-Flores, I and Meléndez-Herrada, E and Morelos-Ramírez, R and Barbosa-Amezcua, M and González-Covarrubias, V and Silva-Herzog, E and Soberón, X and González, V},
title = {Genomic diversity of prevalent Staphylococcus epidermidis multidrug-resistant strains isolated from a Children's Hospital in México City in an eight-years survey.},
journal = {PeerJ},
volume = {7},
number = {},
pages = {e8068},
pmid = {31768302},
issn = {2167-8359},
abstract = {Staphylococcus epidermidis is a human commensal and pathogen worldwide distributed. In this work, we surveyed for multi-resistant S. epidermidis strains in eight years at a children's health-care unit in México City. Multidrug-resistant S. epidermidis were present in all years of the study, including resistance to methicillin, beta-lactams, fluoroquinolones, and macrolides. To understand the genetic basis of antibiotic resistance and its association with virulence and gene exchange, we sequenced the genomes of 17 S. epidermidis isolates. Whole-genome nucleotide identities between all the pairs of S. epidermidis strains were about 97% to 99%. We inferred a clonal structure and eight Multilocus Sequence Types (MLSTs) in the S. epidermidis sequenced collection. The profile of virulence includes genes involved in biofilm formation and phenol-soluble modulins (PSMs). Half of the S. epidermidis analyzed lacked the ica operon for biofilm formation. Likely, they are commensal S. epidermidis strains but multi-antibiotic resistant. Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer. Rates of recombination between S. epidermidis strains were more prevalent than the mutation rate and affected the whole genome. Therefore, the multidrug resistance, independently of the pathogenic traits, might explain the persistence of specific highly adapted S. epidermidis clonal lineages in nosocomial settings.},
}
@article {pmid31768221,
year = {2019},
author = {Valenti, MT and Serena, M and Carbonare, LD and Zipeto, D},
title = {CRISPR/Cas system: An emerging technology in stem cell research.},
journal = {World journal of stem cells},
volume = {11},
number = {11},
pages = {937-956},
pmid = {31768221},
issn = {1948-0210},
abstract = {The identification of new and even more precise technologies for modifying and manipulating the genome has been a challenge since the discovery of the DNA double helix. The ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations, eradicating diseases, engineering cells and organisms to achieve new and different functions and obtaining transgenic animals as models for studying specific diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has recently revolutionized genome engineering. The application of this new technology to stem cell research allows disease models to be developed to explore new therapeutic tools. The possibility of translating new systems of molecular knowledge to clinical research is particularly appealing for addressing degenerative diseases. In this review, we describe several applications of CRISPR/Cas9 to stem cells related to degenerative diseases. In addition, we address the challenges and future perspectives regarding the use of CRISPR/Cas9 as an important technology in the medical sciences.},
}
@article {pmid31768018,
year = {2020},
author = {Schneider, E and Pochert, N and Ruess, C and MacPhee, L and Escano, L and Miller, C and Krowiorz, K and Delsing Malmberg, E and Heravi-Moussavi, A and Lorzadeh, A and Ashouri, A and Grasedieck, S and Sperb, N and Kumar Kopparapu, P and Iben, S and Staffas, A and Xiang, P and Rösler, R and Kanduri, M and Larsson, E and Fogelstrand, L and Döhner, H and Döhner, K and Wiese, S and Hirst, M and Keith Humphries, R and Palmqvist, L and Kuchenbauer, F and Rouhi, A},
title = {MicroRNA-708 is a novel regulator of the Hoxa9 program in myeloid cells.},
journal = {Leukemia},
volume = {34},
number = {5},
pages = {1253-1265},
pmid = {31768018},
issn = {1476-5551},
support = {//CIHR/Canada ; },
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Female ; *Gene Expression Regulation, Leukemic ; Hematopoiesis ; Homeodomain Proteins/genetics/*metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; Mice ; Mice, Inbred C57BL ; MicroRNAs/antagonists &amp; inhibitors/*genetics/metabolism ; Myeloid Cells/metabolism/*pathology ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics/*metabolism ; Tumor Cells, Cultured ; },
abstract = {MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.},
}
@article {pmid31767972,
year = {2020},
author = {Zhan, S and Fang, G and Cai, M and Kou, Z and Xu, J and Cao, Y and Bai, L and Zhang, Y and Jiang, Y and Luo, X and Xu, J and Xu, X and Zheng, L and Yu, Z and Yang, H and Zhang, Z and Wang, S and Tomberlin, JK and Zhang, J and Huang, Y},
title = {Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler.},
journal = {Cell research},
volume = {30},
number = {1},
pages = {50-60},
pmid = {31767972},
issn = {1748-7838},
mesh = {Animals ; CRISPR-Cas Systems ; Diptera/*genetics/growth &amp; development/microbiology ; Gene Editing ; Genes, Insect ; *Genome, Insect ; Intestines ; Larva/microbiology ; Microbiota ; Recycling ; Transcriptome ; Waste Products ; },
abstract = {The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is renowned for its bioconversion of organic waste into a sustainable source of animal feed. We report a high-quality genome of 1.1 Gb and a consensus set of 16,770 gene models for this beneficial species. Compared to those of other dipteran species, the BSF genome has undergone a substantial expansion in functional modules related to septic adaptation, including immune system factors, olfactory receptors, and cytochrome P450s. We further profiled midgut transcriptomes and associated microbiomes of BSF larvae fed with representative types of organic waste. We find that the pathways related to digestive system and fighting infection are commonly enriched and that Firmicutes bacteria dominate the microbial community in BSF across all diets. To extend its potential practical applications, we further developed an efficient CRISPR/Cas9-based gene editing approach and implemented this to yield flightless and enhanced feeding capacity phenotypes, both of which could expand BSF production capabilities. Our study provides valuable genomic and technical resources for optimizing BSF lines for industrialization.},
}
@article {pmid31767948,
year = {2019},
author = {Zhao, W and Cheng, L and Quek, C and Bellingham, SA and Hill, AF},
title = {Novel miR-29b target regulation patterns are revealed in two different cell lines.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17449},
pmid = {31767948},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Transformation, Neoplastic ; Cellular Senescence ; Clone Cells ; Gene Editing ; *Gene Expression Regulation ; Gene Knockdown Techniques ; Gene Ontology ; HeLa Cells/*metabolism ; Humans ; Mice ; MicroRNAs/biosynthesis/*genetics ; Models, Molecular ; NIH 3T3 Cells/*metabolism ; Nucleic Acid Conformation ; RNA, Guide/genetics ; Sequence Alignment ; Signal Transduction ; Transcriptome ; },
abstract = {MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs "react" and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells.},
}
@article {pmid31767844,
year = {2019},
author = {McGrath, E and Shin, H and Zhang, L and Phue, JN and Wu, WW and Shen, RF and Jang, YY and Revollo, J and Ye, Z},
title = {Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5353},
pmid = {31767844},
issn = {2041-1723},
support = {R03 HD091264/HD/NICHD NIH HHS/United States ; R01 EB023812/EB/NIBIB NIH HHS/United States ; },
mesh = {APOBEC Deaminases/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/*metabolism ; Cytosine/*metabolism ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Genome, Human/genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mutation ; Plant Cells/metabolism ; Reproducibility of Results ; Whole Genome Sequencing/*methods ; },
abstract = {DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.},
}
@article {pmid31767635,
year = {2020},
author = {Kueh, AJ and Eccles, S and Tang, L and Garnham, AL and May, RE and Herold, MJ and Smyth, GK and Voss, AK and Thomas, T},
title = {HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells.},
journal = {Molecular and cellular biology},
volume = {40},
number = {4},
pages = {},
pmid = {31767635},
issn = {1098-5549},
mesh = {Acetylation ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/*genetics ; DNA Replication/*genetics ; Gene Deletion ; HEK293 Cells ; HeLa Cells ; Histone Acetyltransferases/genetics/*metabolism ; Histones/*metabolism ; Humans ; MCF-7 Cells ; Protein Processing, Post-Translational ; RNA Interference ; RNA, Guide/genetics ; RNA, Small Interfering/genetics ; },
abstract = {HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.},
}
@article {pmid31767004,
year = {2019},
author = {Mougiakos, I and Orsi, E and Ghiffary, MR and Post, W and de Maria, A and Adiego-Perez, B and Kengen, SWM and Weusthuis, RA and van der Oost, J},
title = {Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {204},
pmid = {31767004},
issn = {1475-2859},
mesh = {Bacterial Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Bacterial ; Homologous Recombination ; Hydroxybutyrates/metabolism ; Metabolic Engineering/*methods ; Polyesters/metabolism ; Rhodobacter sphaeroides/*genetics/metabolism ; },
abstract = {BACKGROUND: Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides.<br><br>RESULTS: Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides &#x394;phbB strains under the same conditions is only slightly affected.<br><br>CONCLUSIONS: In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.},
}
@article {pmid31765708,
year = {2020},
author = {Huang, PW and Yang, Q and Zhu, YL and Zhou, J and Sun, K and Mei, YZ and Dai, CC},
title = {The construction of CRISPR-Cas9 system for endophytic Phomopsis liquidambaris and its PmkkA-deficient mutant revealing the effect on rice.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {136},
number = {},
pages = {103301},
doi = {10.1016/j.fgb.2019.103301},
pmid = {31765708},
issn = {1096-0937},
mesh = {Ascomycota/*enzymology/*genetics ; *CRISPR-Cas Systems ; Cell Wall/metabolism ; Endophytes ; Fungal Proteins/genetics ; Gene Knockout Techniques ; Genes, Fungal ; *Host Microbial Interactions ; Ku Autoantigen/genetics ; Mitogen-Activated Protein Kinase Kinases/*genetics ; Mutation ; Oryza/*growth &amp; development/*microbiology ; Reactive Oxygen Species/metabolism ; Signal Transduction/genetics ; },
abstract = {The endophytic fungus Phomopsis liquidambaris efficiently promotes the nitrogen metabolism and growth of host plants such as rice and peanut. However, a lack of genetic tools limits further research regarding the mechanisms of interaction between P. liquidambaris and its host plants. Herein, a CRISPR/Cas9 system for targeted gene disruption in this strain was first constructed and optimized. The knock-out efficiency increased to over 60% when the ku70 or ku80 gene (involved in nonhomologous end-joining, NHEJ) was disrupted. Furthermore, the CRISPR/Cas9 system was applied to disrupt the PmkkA gene, encoding a mitogen-activated protein kinase kinase (MAPKK) in the cell-wall integrity (CWI) MAPK pathway of the strain. The ΔPmkkA mutant strain induced higher reactive oxygen species (ROS) production, chitinase activity and glucanase activity in rice seedlings than wild-type P. liquidambaris (WT), resulting in growth inhibition and strong resistance on rice. These results suggested that the PmkkA gene is crucial during the interaction with rice and may play a role in inhibiting the immune system of host plants. The CRISPR-Cas9 system will be of great use for the study of the interaction between P. liquidambaris and its host plants.},
}
@article {pmid31765060,
year = {2020},
author = {Rodríguez Cruz, PM and Cossins, J and Cheung, J and Maxwell, S and Jayawant, S and Herbst, R and Waithe, D and Kornev, AP and Palace, J and Beeson, D},
title = {Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.},
journal = {Human mutation},
volume = {41},
number = {3},
pages = {619-631},
pmid = {31765060},
issn = {1098-1004},
support = {MR/S005382/1/MRC_/Medical Research Council/United Kingdom ; P 31199/FWF_/Austrian Science Fund FWF/Austria ; MR/M006824/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adrenergic beta-2 Receptor Agonists/pharmacology/therapeutic use ; Alleles ; Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cell Line ; DNA Mutational Analysis ; Female ; Gene Targeting ; Humans ; Mice ; Models, Molecular ; Molecular Conformation ; Muscle Proteins/metabolism ; *Mutation ; Myasthenic Syndromes, Congenital/*diagnosis/drug therapy/*genetics/metabolism ; Pedigree ; Phosphorylation ; Receptor Protein-Tyrosine Kinases/chemistry/*genetics/metabolism ; Receptors, Cholinergic/chemistry/*genetics/metabolism ; Structure-Activity Relationship ; Synapses/*genetics/metabolism ; },
abstract = {MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with β2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.},
}
@article {pmid31764961,
year = {2020},
author = {Cancellieri, S and Canver, MC and Bombieri, N and Giugno, R and Pinello, L},
title = {CRISPRitz: rapid, high-throughput and variant-aware in silico off-target site identification for CRISPR genome editing.},
journal = {Bioinformatics (Oxford, England)},
volume = {36},
number = {7},
pages = {2001-2008},
pmid = {31764961},
issn = {1367-4811},
support = {R00 HG008399/HG/NHGRI NIH HHS/United States ; R35 HG010717/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; RNA, Guide ; Software ; },
abstract = {MOTIVATION: Clustered regularly interspaced short palindromic repeats (CRISPR) technologies allow for facile genomic modification in a site-specific manner. A key step in this process is the in silico design of single guide RNAs to efficiently and specifically target a site of interest. To this end, it is necessary to enumerate all potential off-target sites within a given genome that could be inadvertently altered by nuclease-mediated cleavage. Currently available software for this task is limited by computational efficiency, variant support or annotation, and assessment of the functional impact of potential off-target effects.<br><br>RESULTS: To overcome these limitations, we have developed CRISPRitz, a suite of software tools to support the design and analysis of CRISPR/CRISPR-associated (Cas) experiments. Using efficient data structures combined with parallel computation, we offer a rapid, reliable, and exhaustive search mechanism to enumerate a comprehensive list of putative off-target sites. As proof-of-principle, we performed a head-to-head comparison with other available tools on several datasets. This analysis highlighted the unique features and superior computational performance of CRISPRitz including support for genomic searching with DNA/RNA bulges and mismatches of arbitrary size as specified by the user as well as consideration of genetic variants (variant-aware). In addition, graphical reports are offered for coding and non-coding regions that annotate the potential impact of putative off-target sites that lie within regions of functional genomic annotation (e.g. insulator and chromatin accessible sites from the ENCyclopedia Of DNA Elements [ENCODE] project).<br><br>The software is freely available at: https://github.com/pinellolab/CRISPRitzhttps://github.com/InfOmics/CRISPRitz.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid31763806,
year = {2019},
author = {Tang, Q and Liu, J and Jiang, Y and Zhang, M and Mao, L and Wang, M},
title = {Cell-Selective Messenger RNA Delivery and CRISPR/Cas9 Genome Editing by Modulating the Interface of Phenylboronic Acid-Derived Lipid Nanoparticles and Cellular Surface Sialic Acid.},
journal = {ACS applied materials &amp; interfaces},
volume = {11},
number = {50},
pages = {46585-46590},
doi = {10.1021/acsami.9b17749},
pmid = {31763806},
issn = {1944-8252},
mesh = {Boronic Acids/chemistry ; CRISPR-Cas Systems/drug effects/genetics ; Gene Editing/methods ; *Gene Transfer Techniques ; Genetic Therapy ; HeLa Cells ; Humans ; Lipids/chemistry/*pharmacology ; N-Acetylneuraminic Acid/chemistry/metabolism ; Nanoparticles/*chemistry ; RNA, Messenger/chemistry/*pharmacology ; Transfection/methods ; },
abstract = {Messenger RNA (mRNA) represents an emerging class of nucleic acid therapeutics for genome editing and genetic disease treatment. Delivering exogenous mRNA selectively to cells, however, remains a main challenge to broaden the biomedical application of mRNA and develop targeted gene therapy. Herein, we report cell-selective mRNA delivery and CRISPR/Cas9 genome editing by modulating the interface of phenylboronic acid (PBA) derived lipid nanoparticles (NPs) and cellular surface sialic acid (SA). We design a cationic lipid featuring a PBA group, PBA-BADP, to self-assemble with mRNA into nanoparticles via electrostatic interactions. Importantly, these nanoparticles present free PBA groups on their surface, showing an enhanced cellular uptake by SA-overexpressing cancer cells via the interfacial PBA/SA interaction. It is shown that PBA-BADP/mRNA NPs transfection results in 300 times higher luciferase reporter gene expression in cancer cells than that in noncancer cells. Moreover, we demonstrate that the delivery of tumor suppressor p53 mRNA using PBA-BADP selectively prohibits cancer cell growth, while PBA-BADP/Cas9 mRNA NPs delivery knocks out gene expression of HeLa cancer cells in a much higher efficiency than noncancer cells. We believe these findings could further extend the modulation of PBA and cellular SA interface to advance mRNA delivery and genome editing for new gene therapy.},
}
@article {pmid31763795,
year = {2019},
author = {Laughery, MF and Wyrick, JJ},
title = {Simple CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae.},
journal = {Current protocols in molecular biology},
volume = {129},
number = {1},
pages = {e110},
pmid = {31763795},
issn = {1934-3647},
support = {R21 ES029302/ES/NIEHS NIH HHS/United States ; R21 ES029655/ES/NIEHS NIH HHS/United States ; R21 ES027937/ES/NIEHS NIH HHS/United States ; R01 ES028698/ES/NIEHS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Vectors/genetics ; Plasmids/*genetics ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/*genetics ; },
abstract = {CRISPR-Cas9 has emerged as a powerful method for editing the genome in a wide variety of species, since it can generate a specific DNA break when targeted by the Cas9-bound guide RNA. In yeast, Cas9-targeted DNA breaks are used to promote homologous recombination with a mutagenic template DNA, in order to rapidly generate genome edits (e.g., DNA substitutions, insertions, or deletions) encoded in the template DNA. Since repeated Cas9-induced DNA breaks select against unedited cells, Cas9 can be used to generate marker-free genome edits. Here, we describe a simple protocol for constructing Cas9-expressing plasmids containing a user-designed guide RNA, as well as protocols for using these plasmids for efficient genome editing in yeast. © 2019 by John Wiley &amp; Sons, Inc. Basic Protocol 1: Constructing the guide RNA expression vector Basic Protocol 2: Preparing double-stranded oligonucleotide repair template Alternate Protocol 1: Preparing a single-stranded oligonucleotide repair template Basic Protocol 3: Induce genome editing by co-transformation of yeast Basic Protocol 4: Screening for edited cells Basic Protocol 5: Removing sgRNA/CAS9 expression vector Alternate Protocol 2: Removing pML107-derived sgRNA/CAS9 expression vector.},
}
@article {pmid31763794,
year = {2019},
author = {Kim, HM and Colaiácovo, MP},
title = {CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans.},
journal = {Current protocols in molecular biology},
volume = {129},
number = {1},
pages = {e106},
pmid = {31763794},
issn = {1934-3647},
support = {R01 GM105853/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Genetic Engineering/*methods ; RNA, Guide/genetics ; },
abstract = {The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is being used successfully for efficient and targeted genome editing in various organisms, including the nematode Caenorhabditis elegans. Recent studies have developed a variety of CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: nonhomologous end joining and homologous recombination. Here, we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning (canonical marker-free and cassette selection methods), as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the germline. © 2019 by John Wiley &amp; Sons, Inc. Basic Protocol 1: Guide RNA preparation Alternate Protocol 1: sgRNA cloning using fusion PCR Basic Protocol 2: Preparation of a repair template for homologous recombination Alternate Protocol 2: Preparation of repair template donors for the cassette selection method Basic Protocol 3: Injecting animals Basic Protocol 4: Screening transgenic worms with marker-free method Alternate Protocol 3: Screening transgenic worms with cassette selection method.},
}
@article {pmid31763777,
year = {2019},
author = {Viswanatha, R and Brathwaite, R and Hu, Y and Li, Z and Rodiger, J and Merckaert, P and Chung, V and Mohr, SE and Perrimon, N},
title = {Pooled CRISPR Screens in Drosophila Cells.},
journal = {Current protocols in molecular biology},
volume = {129},
number = {1},
pages = {e111},
pmid = {31763777},
issn = {1934-3647},
support = {P41 GM132087/GM/NIGMS NIH HHS/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; T32 GM007748/GM/NIGMS NIH HHS/United States ; R01 GM084947/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytotoxins/metabolism ; Drosophila melanogaster/cytology ; Gene Library ; High-Throughput Screening Assays/*methods ; RNA, Guide/*genetics ; },
abstract = {High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley &amp; Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.},
}
@article {pmid31763447,
year = {2019},
author = {Zhang, Q and Wen, F and Zhang, S and Jin, J and Bi, L and Lu, Y and Li, M and Xi, XG and Huang, X and Shen, B and Sun, B},
title = {The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation.},
journal = {Science advances},
volume = {5},
number = {11},
pages = {eaaw9807},
pmid = {31763447},
issn = {2375-2548},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/chemistry/genetics/*metabolism ; Gene Editing/methods ; Models, Genetic ; Nucleic Acid Conformation ; Nucleotide Motifs/genetics ; Protein Binding ; RNA, Guide/chemistry/genetics/*metabolism ; Sequence Analysis, DNA/methods ; },
abstract = {Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.},
}
@article {pmid31762718,
year = {2019},
author = {El-Kenawy, A and Benarba, B and Neves, AF and de Araujo, TG and Tan, BL and Gouri, A},
title = {Gene surgery: Potential applications for human diseases.},
journal = {EXCLI journal},
volume = {18},
number = {},
pages = {908-930},
pmid = {31762718},
issn = {1611-2156},
abstract = {Gene therapy became in last decade a new emerging therapeutic era showing promising results against different diseases such as cancer, cardiovascular diseases, diabetes, and neurological disorders. Recently, the genome editing technique for eukaryotic cells called CRISPR-Cas (Clustered Regulatory Interspaced Short Palindromic Repeats) has enriched the field of gene surgery with enhanced applications. In the present review, we summarized the different applications of gene surgery for treating human diseases such as cancer, diabetes, nervous, and cardiovascular diseases, besides the molecular mechanisms involved in these important effects. Several studies support the important therapeutic applications of gene surgery in a large number of health disorders and diseases including β-thalassemia, cancer, immunodeficiencies, diabetes, and neurological disorders. In diabetes, gene surgery was shown to be effective in type 1 diabetes by triggering different signaling pathways. Furthermore, gene surgery, especially that using CRISPR-Cas possessed important application on diagnosis, screening and treatment of several cancers such as lung, liver, pancreatic and colorectal cancer. Nevertheless, gene surgery still presents some limitations such as the design difficulties and costs regarding ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases) use, off-target effects, low transfection efficiency, in vivo delivery-safety and ethical issues.},
}
@article {pmid31761839,
year = {2020},
author = {Yamashita, S and Kogasaka, Y and Hiradate, Y and Tanemura, K and Sendai, Y},
title = {Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation.},
journal = {The Journal of reproduction and development},
volume = {66},
number = {1},
pages = {41-48},
pmid = {31761839},
issn = {1348-4400},
mesh = {Animals ; Blastocyst/*metabolism ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation ; Embryonic Development/*physiology ; Exodeoxyribonucleases/*genetics ; Gene Editing/*methods ; *Mosaicism ; Mutation ; Phosphoproteins/*genetics ; Swine ; Zygote/*metabolism ; },
abstract = {Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.},
}
@article {pmid31761532,
year = {2019},
author = {Morton, AR and Dogan-Artun, N and Faber, ZJ and MacLeod, G and Bartels, CF and Piazza, MS and Allan, KC and Mack, SC and Wang, X and Gimple, RC and Wu, Q and Rubin, BP and Shetty, S and Angers, S and Dirks, PB and Sallari, RC and Lupien, M and Rich, JN and Scacheri, PC},
title = {Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.},
journal = {Cell},
volume = {179},
number = {6},
pages = {1330-1341.e13},
pmid = {31761532},
issn = {1097-4172},
support = {R01 CA169117/CA/NCI NIH HHS/United States ; R01 CA204279/CA/NCI NIH HHS/United States ; TL1 TR002549/TR/NCATS NIH HHS/United States ; R01 CA160356/CA/NCI NIH HHS/United States ; TL1 TR000441/TR/NCATS NIH HHS/United States ; R01 DA043980/DA/NIDA NIH HHS/United States ; R01 CA143237/CA/NCI NIH HHS/United States ; R01 NS087913/NS/NINDS NIH HHS/United States ; R01 NS103434/NS/NINDS NIH HHS/United States ; R01 CA193677/CA/NCI NIH HHS/United States ; R01 CA171652/CA/NCI NIH HHS/United States ; R01 NS089272/NS/NINDS NIH HHS/United States ; F30 CA236313/CA/NCI NIH HHS/United States ; R35 CA197718/CA/NCI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; R01 CA154130/CA/NCI NIH HHS/United States ; },
mesh = {Acetylation ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Chromatin/metabolism ; Chromosomes, Human/*genetics ; DNA, Neoplasm/genetics ; *Enhancer Elements, Genetic ; ErbB Receptors/genetics/metabolism ; *Gene Amplification ; Genes, Neoplasm ; Genetic Loci ; Glioblastoma/genetics/pathology ; Histones/metabolism ; Humans ; Neuroglia/metabolism ; *Oncogenes ; },
abstract = {Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.},
}
@article {pmid31761088,
year = {2019},
author = {Zhang, CC},
title = {Cas9-induced immune response: A potential caution for human genome editing.},
journal = {Biochemical and biophysical research communications},
volume = {520},
number = {4},
pages = {706-707},
doi = {10.1016/j.bbrc.2019.10.025},
pmid = {31761088},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Vectors ; Genome, Human ; Humans ; Monocytes ; STAT6 Transcription Factor ; },
}
@article {pmid31761069,
year = {2019},
author = {Citovsky, V},
title = {My BBRC: From learning biochemistry to editorial decisions.},
journal = {Biochemical and biophysical research communications},
volume = {520},
number = {4},
pages = {672-673},
pmid = {31761069},
issn = {1090-2104},
support = {R01 GM050224/GM/NIGMS NIH HHS/United States ; },
mesh = {*Biochemistry ; Biological Science Disciplines ; CRISPR-Cas Systems/genetics ; *Editorial Policies ; History, 20th Century ; History, 21st Century ; *Learning ; Periodicals as Topic/*history ; Research ; },
}
@article {pmid31760434,
year = {2020},
author = {Liu, H and Wang, K and Jia, Z and Gong, Q and Lin, Z and Du, L and Pei, X and Ye, X},
title = {Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system.},
journal = {Journal of experimental botany},
volume = {71},
number = {4},
pages = {1337-1349},
pmid = {31760434},
issn = {1460-2431},
mesh = {*Agrobacterium/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Haploidy ; Triticum/genetics ; },
abstract = {The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.},
}
@article {pmid31759123,
year = {2021},
author = {Xu, CF and Chen, GJ and Luo, YL and Zhang, Y and Zhao, G and Lu, ZD and Czarna, A and Gu, Z and Wang, J},
title = {Rational designs of in vivo CRISPR-Cas delivery systems.},
journal = {Advanced drug delivery reviews},
volume = {168},
number = {},
pages = {3-29},
doi = {10.1016/j.addr.2019.11.005},
pmid = {31759123},
issn = {1872-8294},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drug Delivery Systems ; Epigenome/genetics ; Exosomes/metabolism ; Gene Editing/*methods ; Gene Regulatory Networks/physiology ; Genetic Vectors/metabolism ; Lipids/chemistry ; Nanoparticles/chemistry ; RNA/metabolism ; Transcription, Genetic/physiology ; },
abstract = {The CRISPR-Cas system initiated a revolution in genome editing when it was, for the first time, demonstrated success in the mammalian cells. Today, scientists are able to readily edit genomes, regulate gene transcription, engineer posttranscriptional events, and image nucleic acids using CRISPR-Cas-based tools. However, to efficiently transport CRISPR-Cas into target tissues/cells remains challenging due to many extra- and intra-cellular barriers, therefore largely limiting the applications of CRISPR-based therapeutics in vivo. In this review, we summarize the features of plasmid-, RNA- and ribonucleoprotein (RNP)-based CRISPR-Cas therapeutics. Then, we survey the current in vivo delivery systems. We specify the requirements for efficient in vivo delivery in clinical settings, and highlight both efficiency and safety for different CRISPR-Cas tools.},
}
@article {pmid31758450,
year = {2020},
author = {Jacot, D and Soldati-Favre, D},
title = {CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2071},
number = {},
pages = {125-141},
doi = {10.1007/978-1-4939-9857-9_7},
pmid = {31758450},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryptosporidium/genetics/pathogenicity ; Gene Knockout Techniques ; Genome, Protozoan/genetics ; Mutagenesis, Site-Directed ; Protein Stability ; Temperature ; Toxoplasma/genetics/*pathogenicity ; },
abstract = {The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.},
}
@article {pmid31757757,
year = {2019},
author = {Yang, LZ and Wang, Y and Li, SQ and Yao, RW and Luan, PF and Wu, H and Carmichael, GG and Chen, LL},
title = {Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.},
journal = {Molecular cell},
volume = {76},
number = {6},
pages = {981-997.e7},
doi = {10.1016/j.molcel.2019.10.024},
pmid = {31757757},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Fluorescent Dyes/chemistry ; Humans ; Molecular Imaging/*methods ; Mucin-4 ; Protein Engineering/methods ; RNA/*physiology ; RNA, Guide/genetics ; RNA, Long Noncoding ; Ribonucleases/genetics/metabolism ; Single Molecule Imaging/*methods ; Staining and Labeling/methods ; },
abstract = {Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.},
}
@article {pmid31757214,
year = {2019},
author = {Hong, W and Guo, F and Yang, M and Xu, D and Zhuang, Z and Niu, B and Bai, Q and Li, X},
title = {Hydroxysteroid sulfotransferase 2B1 affects gastric epithelial function and carcinogenesis induced by a carcinogenic agent.},
journal = {Lipids in health and disease},
volume = {18},
number = {1},
pages = {203},
pmid = {31757214},
issn = {1476-511X},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Carcinogenesis/drug effects/*genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation ; Cholesterol/analogs &amp; derivatives/metabolism ; Gastric Mucosa/drug effects/enzymology/pathology ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Humans ; Hydroxycholesterols/metabolism ; Methylcholanthrene/administration &amp; dosage ; Mice ; Mice, Knockout ; Phosphatidylinositol 3-Kinases/*genetics/metabolism ; Proto-Oncogene Proteins c-akt/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Signal Transduction ; Stomach Neoplasms/chemically induced/enzymology/*genetics/mortality ; Sulfotransferases/antagonists &amp; inhibitors/deficiency/*genetics ; Survival Analysis ; },
abstract = {BACKGROUND: A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear.<br><br>METHODS: A mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1[-/-]) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system.<br><br>RESULTS: The gastric tumor incidence was higher in the SULT2B1[-/-] mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction.<br><br>CONCLUSIONS: The abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.},
}
@article {pmid31756888,
year = {2019},
author = {Arizala, D and Arif, M},
title = {Genome-Wide Analyses Revealed Remarkable Heterogeneity in Pathogenicity Determinants, Antimicrobial Compounds, and CRISPR-Cas Systems of Complex Phytopathogenic Genus Pectobacterium.},
journal = {Pathogens (Basel, Switzerland)},
volume = {8},
number = {4},
pages = {},
pmid = {31756888},
issn = {2076-0817},
abstract = {The Pectobacterium genus comprises pectolytic enterobacteria defined as the causal agents of soft rot, blackleg, and aerial stem rot diseases of potato and economically important crops. In this study, we undertook extensive genome-wide comparative analyses of twelve species that conform the Pectobacterium genus. Bioinformatics approaches outlined a low nucleotide identity of P. parmentieri and P. wasabiae with other species, while P. carotovorum subsp. odoriferum was shown to harbor numerous pseudogenes, which suggests low coding capacity and genomic degradation. The genome atlases allowed for distinguishing distinct DNA structures and highlighted suspicious high transcription zones. The analyses unveiled a noteworthy heterogeneity in the pathogenicity determinants. Specifically, phytotoxins, polysaccharides, iron uptake systems, and the type secretion systems III-V were observed in just some species. Likewise, a comparison of gene clusters encoding antimicrobial compounds put in evidence for high conservation of carotovoricin, whereas a few species possessed the phenazine, carbapenem, and carocins. Moreover, three clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems: I-E, I-F, and III-A were identified. Surrounding some CRISPR-Cas regions, different toxin and antitoxin systems were found, which suggests bacterial suicide in the case of an immune system failure. Multiple whole-genome alignments shed light on to the presence of a novel cellobiose phosphotransferase system (PTS) exclusive to P. parmenteri, and an unreported T5SS conserved in almost all species. Several regions that were associated with virulence, microbe antagonism, and adaptive immune systems were predicted within genomic islands, which underscored the essential role that horizontal gene transfer has imparted in the dynamic evolution and speciation of Pectobacterium species. Overall, the results decipher the different strategies that each species has developed to infect their hosts, outcompete for food resources, and defend against bacteriophages. Our investigation provides novel genetic insights that will assist in understanding the pathogenic lifestyle of Pectobacterium, a genus that jeopardizes the agriculture sustainability of important crops worldwide.},
}
@article {pmid31756390,
year = {2020},
author = {Holding, AN and Cook, HV and Markowetz, F},
title = {Data generation and network reconstruction strategies for single cell transcriptomic profiles of CRISPR-mediated gene perturbations.},
journal = {Biochimica et biophysica acta. Gene regulatory mechanisms},
volume = {1863},
number = {6},
pages = {194441},
doi = {10.1016/j.bbagrm.2019.194441},
pmid = {31756390},
issn = {1876-4320},
support = {BB/R006563/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Cycle ; Gene Regulatory Networks ; RNA-Seq/*methods ; Single-Cell Analysis/*methods ; },
abstract = {Recent advances in single-cell RNA-sequencing (scRNA-seq) in combination with CRISPR/Cas9 technologies have enabled the development of methods for large-scale perturbation studies with transcriptional readouts. These methods are highly scalable and have the potential to provide a wealth of information on the biological networks that underlie cellular response. Here we discuss how to overcome several key challenges to generate and analyse data for the confident reconstruction of models of the underlying cellular network. Some challenges are generic, and apply to analysing any single-cell transcriptomic data, while others are specific to combined single-cell CRISPR/Cas9 data, in particular barcode swapping, knockdown efficiency, multiplicity of infection and potential confounding factors. We also provide a curated collection of published data sets to aid the development of analysis strategies. Finally, we discuss several network reconstruction approaches, including co-expression networks and Bayesian networks, as well as their limitations, and highlight the potential of Nested Effects Models for network reconstruction from scRNA-seq data. This article is part of a Special Issue entitled: Transcriptional Profiles and Regulatory Gene Networks edited by Dr. Dr. Federico Manuel Giorgi and Dr. Shaun Mahony.},
}
@article {pmid31756263,
year = {2020},
author = {Wang, X and Ma, Y and Wang, F and Yang, Y and Wu, S and Wu, Y},
title = {Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella.},
journal = {Pest management science},
volume = {76},
number = {5},
pages = {1618-1625},
doi = {10.1002/ps.5689},
pmid = {31756263},
issn = {1526-4998},
mesh = {Animals ; *CRISPR-Cas Systems ; Insecticide Resistance ; Insecticides ; Moths ; Receptors, Nicotinic ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach.<br><br>RESULTS: Here, we successfully constructed a homozygous strain (Px&#x3b1;6-KO) with a 2-nt deletion mutation of nAChR&#x3b1;6 by CRISPR/Cas9 coupled with non-homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3-TM4 loop region. When compared to the background strain IPP-S, the knockout strain Px&#x3b1;6-KO exhibited 229- and 1462-fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3-fold) effects on the toxicities of imidacloprid, abamectin, &#x3b2;-cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2-nt deletion mutation of nAChR&#x3b1;6 in the Px&#x3b1;6-KO strain.<br><br>CONCLUSION: In vivo functional investigation demonstrates the causality of the Px&#x3b1;6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Px&#x3b1;6-KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. &#xa9; 2019 Society of Chemical Industry.},
}
@article {pmid31756187,
year = {2019},
author = {Cobb, LH and Park, J and Swanson, EA and Beard, MC and McCabe, EM and Rourke, AS and Seo, KS and Olivier, AK and Priddy, LB},
title = {CRISPR-Cas9 modified bacteriophage for treatment of Staphylococcus aureus induced osteomyelitis and soft tissue infection.},
journal = {PloS one},
volume = {14},
number = {11},
pages = {e0220421},
pmid = {31756187},
issn = {1932-6203},
support = {P20 GM103646/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; *Bacteriophages/genetics ; Biofilms ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Fosfomycin/pharmacology ; Gene Editing ; Longitudinal Studies ; Osteomyelitis/microbiology/pathology/*therapy ; Rats ; Rats, Sprague-Dawley ; Soft Tissue Infections/microbiology/pathology/*therapy ; Staphylococcal Infections/pathology/*therapy ; *Staphylococcus aureus ; Vancomycin/pharmacology ; },
abstract = {Osteomyelitis, or bone infection, is often induced by antibiotic resistant Staphylococcus aureus strains of bacteria. Although debridement and long-term administration of antibiotics are the gold standard for osteomyelitis treatment, the increase in prevalence of antibiotic resistant bacterial strains limits the ability of clinicians to effectively treat infection. Bacteriophages (phages), viruses that in a lytic state can effectively kill bacteria, have gained recent attention for their high specificity, abundance in nature, and minimal risk of host toxicity. Previously, we have shown that CRISPR-Cas9 genomic editing techniques could be utilized to expand temperate bacteriophage host range and enhance bactericidal activity through modification of the tail fiber protein. In a dermal infection study, these CRISPR-Cas9 phages reduced bacterial load relative to unmodified phage. Thus we hypothesized this temperate bacteriophage, equipped with the CRISPR-Cas9 bactericidal machinery, would be effective at mitigating infection from a biofilm forming S. aureus strain in vitro and in vivo. In vitro, qualitative fluorescent imaging demonstrated superiority of phage to conventional vancomycin and fosfomycin antibiotics against S. aureus biofilm. Quantitative antibiofilm effects increased over time, at least partially, for all fosfomycin, phage, and fosfomycin-phage (dual) therapeutics delivered via alginate hydrogel. We developed an in vivo rat model of osteomyelitis and soft tissue infection that was reproducible and challenging and enabled longitudinal monitoring of infection progression. Using this model, phage (with and without fosfomycin) delivered via alginate hydrogel were successful in reducing soft tissue infection but not bone infection, based on bacteriological, histological, and scanning electron microscopy analyses. Notably, the efficacy of phage at mitigating soft tissue infection was equal to that of high dose fosfomycin. Future research may utilize this model as a platform for evaluation of therapeutic type and dose, and alternate delivery vehicles for osteomyelitis mitigation.},
}
@article {pmid31754342,
year = {2019},
author = {Chen, B and Gu, P and Jia, J and Liu, W and Liu, Y and Liu, W and Xu, T and Lin, X and Lin, T and Liu, Y and Chen, H and Xu, M and Yuan, J and Zhang, J and Zhang, Y and Xiao, D and Gu, W},
title = {Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection.},
journal = {International journal of biological sciences},
volume = {15},
number = {12},
pages = {2719-2732},
pmid = {31754342},
issn = {1449-2288},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cytoplasm ; Estrus Synchronization/*physiology ; Female ; Gene Editing/*methods ; Male ; Microinjections ; Mutation ; Superovulation ; Swine ; Swine, Miniature/*genetics ; },
abstract = {The Tibet minipig is a rare highland pig breed worldwide and has many applications in biomedical and agricultural research. However, Tibet minipigs are not like domesticated pigs in that their ovulation number is low, which is unfavourable for the collection of zygotes. Partly for this reason, few studies have reported the successful generation of genetically modified Tibet minipigs by zygote injection. To address this issue, we described an efficient way to generate gene-edited Tibet minipigs, the major elements of which include the utilization of synchronized oestrus instead of superovulation to obtain zygotes, optimization of the preparation strategy, and co-injection of clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs) into the cytoplasm of zygotes. We successfully obtained allelic TYR gene knockout (TYR[-/-]) Tibet minipigs with a typical albino phenotype (i.e., red-coloured eyes with light pink-tinted irises and no pigmentation in the skin and hair) as well as TYR[-/-]IL2RG[-/-] and TYR[-/-]RAG1[-/-] Tibet minipigs with typical phenotypes of albinism and immunodeficiency, which was characterized by thymic atrophy and abnormal immunocyte proportions. The overall gene editing efficiency was 75% for the TYR single gene knockout, while for TYR-IL2RG and TYR-RAG1 dual gene editing, the values were 25% and 75%, respectively. No detectable off-target mutations were observed. By intercrossing F0 generation minipigs, targeted genetic mutations can also be transmitted to gene-edited minipigs' offspring through germ line transmission. This study is a valuable exploration for the efficient generation of gene-edited Tibet minipigs with medical research value in the future.},
}
@article {pmid31754336,
year = {2019},
author = {Zhao, C and Wang, Y and Nie, X and Han, X and Liu, H and Li, G and Yang, G and Ruan, J and Ma, Y and Li, X and Cheng, H and Zhao, S and Fang, Y and Xie, S},
title = {Evaluation of the effects of sequence length and microsatellite instability on single-guide RNA activity and specificity.},
journal = {International journal of biological sciences},
volume = {15},
number = {12},
pages = {2641-2653},
pmid = {31754336},
issn = {1449-2288},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Gene Editing ; HEK293 Cells ; Humans ; *Microsatellite Instability ; Plasmids ; RNA, Guide/*metabolism ; Sequence Analysis, RNA ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is effective for genome editing and now widely used in life science research. However, the key factors determining its editing efficiency and off-target cleavage activity for single-guide RNA (sgRNA) are poorly documented. Here, we systematically evaluated the effects of sgRNA length on genome editing efficiency and specificity. Results showed that sgRNA 5'-end lengths can alter genome editing activity. Although the number of predicted off-target sites significantly increased after sgRNA length truncation, sgRNAs with different lengths were highly specific. Because only a few predicted off-targets had detectable cleavage activity as determined by Target capture sequencing (TargetSeq). Interestingly, > 20% of the predicted off-targets contained microsatellites for selected sgRNAs targeting the dystrophin gene, which can produce genomic instability and interfere with accurate assessment of off-target cleavage activity. We found that sgRNA activity and specificity can be sensitively detected by TargetSeq in combination with in silico prediction. Checking whether the on- and off-targets contain microsatellites is necessary to improve the accuracy of analyzing the efficiency of genome editing. Our research provides new features and novel strategies for the accurate assessment of CRISPR sgRNA activity and specificity.},
}
@article {pmid31754267,
year = {2020},
author = {Daga, S and Donati, F and Capitani, K and Croci, S and Tita, R and Giliberti, A and Valentino, F and Benetti, E and Fallerini, C and Niccheri, F and Baldassarri, M and Mencarelli, MA and Frullanti, E and Furini, S and Conticello, SG and Renieri, A and Pinto, AM},
title = {New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells.},
journal = {European journal of human genetics : EJHG},
volume = {28},
number = {4},
pages = {480-490},
pmid = {31754267},
issn = {1476-5438},
mesh = {Adult ; Autoantigens/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Lineage ; Cells, Cultured ; Collagen Type IV/*genetics/metabolism ; Female ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Mutation ; Nephritis, Hereditary/*genetics/pathology/therapy ; Podocytes/cytology/*metabolism ; },
abstract = {Alport syndrome (AS) is an inherited genetic disorder characterized by range of alterations from glomerular basement membrane abnormalities up to end-stage renal disease. Pathogenic variants in the collagen α3, α4, and α5 encoding genes are causative both of the autosomal dominant and of the X-linked forms of AS. Podocytes are the only renal cells that are able to produce the COL(IV)a3-a4a5 heterotrimer. We have previously demonstrated how it is possible to isolate podocyte-lineage cells from urine of patients, providing an easily accessible cellular model closer to the podocytes' physiological conditions. Taking advantage of disease-relevant cell lines, we employed a two-plasmid approach in order to achieve a beneficial and stable variant-specific correction using CRISPR/Cas9 genome editing. One plasmid carries a Donor DNA and a reporter system mCherry/GFP to track the activity of Cas9 in cells. The other plasmid carries a self-cleaving SpCas9 and the variant-specific sgRNA. We have analyzed two stable podocyte-lineage cell lines, harboring a variant in the X-linked COL4A5 (p.(Gly624Asp)) and in the autosomal COL4A3 gene (p.(Gly856Glu)). We have achieved reversion of variants greater than 40% with undesired insertions/deletions lower than 15%. Overall, we have demonstrated a new gene therapy approach directly on patients' cells, key players of Alport pathogenesis, and we have reverted COL4 causative variants towards the wild type state. These results, in combination with preclinical models, could open new frontiers in the management and the treatment of the disorder.},
}
@article {pmid31754144,
year = {2019},
author = {MacLeod, RS and Cawley, KM and Gubrij, I and Nookaew, I and Onal, M and O'Brien, CA},
title = {Effective CRISPR interference of an endogenous gene via a single transgene in mice.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17312},
pmid = {31754144},
issn = {2045-2322},
support = {P20 GM125503/GM/NIGMS NIH HHS/United States ; R01 AR049794/AR/NIAMS NIH HHS/United States ; P20 GM121293/GM/NIGMS NIH HHS/United States ; R25 GM083247/GM/NIGMS NIH HHS/United States ; I01 BX000294/BX/BLRD VA/United States ; M01 RR000080/RR/NCRR NIH HHS/United States ; T32 GM106999/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Mice ; Mice, Transgenic ; RANK Ligand/genetics ; *RNA Interference ; RNA, Guide/genetics ; *Transcription, Genetic ; Transduction, Genetic ; Transgenes/genetics ; },
abstract = {Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcription start-site of target genes. However, there has been limited exploration of CRISPRi in mice. We tested the effectiveness of a single CRISPRi transgene broadly expressing a single guide RNA and a catalytically dead Cas9 fused to the KRAB repressor domain to suppress a well-characterized target gene, Tnfsf11. The phenotype of CRISPRi transgenic mice was compared to mice with germline deletion of Tnfsf11, which are osteopetrotic and do not form lymph nodes. High transgene expression mimicked gene deletion, with failure of lymph node development and classic signs of osteopetrosis such as high bone mass and failure of tooth eruption. Mice with low transgene expression were normal and mice with medium expression displayed an intermediate phenotype. Transgene expression in tissues from these mice correlated inversely with Tnfsf11 mRNA levels. These results demonstrate that a single CRISPRi transgene can effectively suppress a target gene in mice and suggest that this approach may be useful for cell type-specific loss-of-function studies.},
}
@article {pmid31754021,
year = {2019},
author = {Erwood, S and Brewer, RA and Bily, TMI and Maino, E and Zhou, L and Cohn, RD and Ivakine, EA},
title = {Modeling Niemann-Pick disease type C in a human haploid cell line allows for patient variant characterization and clinical interpretation.},
journal = {Genome research},
volume = {29},
number = {12},
pages = {2010-2019},
pmid = {31754021},
issn = {1549-5469},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Genome, Human ; *Haploidy ; Humans ; *Models, Genetic ; Niemann-Pick Disease, Type C/*genetics ; *Whole Genome Sequencing ; },
abstract = {The accurate clinical interpretation of human sequence variation is foundational to personalized medicine. This remains a pressing challenge, however, as genome sequencing becomes routine and new functionally undefined variants rapidly accumulate. Here, we describe a platform for the rapid generation, characterization, and interpretation of genomic variants in haploid cells focusing on Niemann-Pick disease type C (NPC) as an example. NPC is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of unesterified cholesterol and glycolipids. In 95% of cases, NPC is caused by mutations in the NPC1 gene, for which more than 200 unique disease-causing variants have been reported to date. Furthermore, the majority of patients with NPC are compound heterozygotes that often carry at least one private mutation, presenting a challenge for the characterization and classification of individual variants. Here, we have developed the first haploid cell model of NPC. This haploid cell model recapitulates the primary biochemical and molecular phenotypes typically found in patient-derived fibroblasts, illustrating its utility in modeling NPC. Additionally, we show the power of CRISPR/Cas9-mediated base editing in quickly and efficiently generating haploid cell models of individual patient variants in NPC. These models provide a platform for understanding the disease mechanisms underlying individual NPC1 variants while allowing for definitive clinical variant interpretation for NPC.},
}
@article {pmid31753920,
year = {2020},
author = {Bhargava, R and Lopezcolorado, FW and Tsai, LJ and Stark, JM},
title = {The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {1},
pages = {125-137},
pmid = {31753920},
issn = {1083-351X},
support = {P30 CA033572/CA/NCI NIH HHS/United States ; R01 CA197506/CA/NCI NIH HHS/United States ; },
mesh = {1-Acylglycerophosphocholine O-Acyltransferase/genetics/metabolism ; A549 Cells ; CD4 Antigens/genetics/metabolism ; *Chromosome Deletion ; Chromosome Inversion ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA Repair Enzymes/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics ; HEK293 Cells ; Humans ; Promoter Regions, Genetic ; },
abstract = {Clastogen exposure can result in chromosomal rearrangements, including large deletions and inversions that are associated with cancer development. To examine such rearrangements in human cells, here we developed a reporter assay based on endogenous genes on chromosome 12. Using the RNA-guided nuclease Cas9, we induced two DNA double-strand breaks, one each in the GAPDH and CD4 genes, that caused a deletion rearrangement leading to CD4 expression from the GAPDH promoter. We observed that this GAPDH-CD4 deletion rearrangement activates CD4+ cells that can be readily detected by flow cytometry. Similarly, double-strand breaks in the LPCAT3 and CD4 genes induced an LPCAT3-CD4 inversion rearrangement resulting in CD4 expression. Studying the GAPDH-CD4 deletion rearrangement in multiple cell lines, we found that the canonical non-homologous end joining (C-NHEJ) factor XLF promotes these rearrangements. Junction analysis uncovered that the relative contribution of C-NHEJ appears lower in U2OS than in HEK293 and A549 cells. Furthermore, an ATM kinase inhibitor increased C-NHEJ-mediated rearrangements only in U2OS cells. We also found that an XLF residue that is critical for an interaction with the C-NHEJ factor X-ray repair cross-complementing 4 (XRCC4), and XRCC4 itself are each important for promoting both this deletion rearrangement and end joining without insertion/deletion mutations. In summary, a reporter assay based on endogenous genes on chromosome 12 reveals that XLF-dependent C-NHEJ promotes deletion rearrangements in human cells and that cell type-specific differences in the contribution of C-NHEJ and ATM kinase inhibition influence these rearrangements.},
}
@article {pmid31753848,
year = {2020},
author = {Majumder, P and Lee, JT and Rahmberg, AR and Kumar, G and Mi, T and Scharer, CD and Boss, JM},
title = {A super enhancer controls expression and chromatin architecture within the MHC class II locus.},
journal = {The Journal of experimental medicine},
volume = {217},
number = {2},
pages = {},
pmid = {31753848},
issn = {1540-9538},
support = {R01 GM047310/GM/NIGMS NIH HHS/United States ; R01 NS092122/NS/NINDS NIH HHS/United States ; },
mesh = {B-Lymphocytes ; Binding Sites/genetics ; Blood Donors ; Burkitt Lymphoma/pathology ; CCCTC-Binding Factor/metabolism ; CD4-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin/*metabolism ; Chromatin Assembly and Disassembly/*genetics ; Gene Deletion ; Genes, MHC Class II/*genetics ; Genetic Loci ; HLA-DQ alpha-Chains/*genetics/metabolism ; HLA-DRB1 Chains/*genetics/metabolism ; Haplotypes ; Humans ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic/genetics ; },
abstract = {Super enhancers (SEs) play critical roles in cell type-specific gene regulation. The mechanisms by which such elements work are largely unknown. Two SEs termed DR/DQ-SE and XL9-SE are situated within the human MHC class II locus between the HLA-DRB1 and HLA-DQA1 genes and are highly enriched for disease-causing SNPs. To test the function of these elements, we used CRISPR/Cas9 to generate a series of mutants that deleted the SE. Deletion of DR/DQ-SE resulted in reduced expression of HLA-DRB1 and HLA-DQA1 genes. The SEs were found to interact with each other and the promoters of HLA-DRB1 and HLA-DQA1. DR/DQ-SE also interacted with neighboring CTCF binding sites. Importantly, deletion of DR/DQ-SE reduced the local chromatin interactions, implying that it functions as the organizer for the local three-dimensional architecture. These data provide direct mechanisms by which an MHC-II SE contributes to expression of the locus and suggest how variation in these SEs may contribute to human disease and altered immunity.},
}
@article {pmid31752697,
year = {2019},
author = {Xu, W and Song, W and Yang, Y and Wu, Y and Lv, X and Yuan, S and Liu, Y and Yang, J},
title = {Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {511},
pmid = {31752697},
issn = {1471-2229},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Deoxyribonuclease I/genetics/*metabolism ; *Gene Editing ; Nucleotides/genetics ; Oryza/*genetics ; Plant Proteins/genetics/metabolism ; RNA, Guide/*genetics ; RNA, Transfer/genetics ; },
abstract = {BACKGROUND: Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits.<br><br>RESULTS: We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture.<br><br>CONCLUSIONS: We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.},
}
@article {pmid31752385,
year = {2019},
author = {Doyle, TB and Muntean, BS and Ejendal, KF and Hayes, MP and Soto-Velasquez, M and Martemyanov, KA and Dessauer, CW and Hu, CD and Watts, VJ},
title = {Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D1 and D2 Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening.},
journal = {Cells},
volume = {8},
number = {11},
pages = {},
pmid = {31752385},
issn = {2073-4409},
support = {R21 MH096927/MH/NIMH NIH HHS/United States ; K02 DA026405/DA/NIDA NIH HHS/United States ; R01 DA036596/DA/NIDA NIH HHS/United States ; R21 MH101673/MH/NIMH NIH HHS/United States ; R33 MH101673/MH/NIMH NIH HHS/United States ; },
mesh = {Adenylyl Cyclases/*metabolism ; Animals ; CRISPR-Cas Systems ; Carrier Proteins/metabolism ; Cyclic AMP/metabolism ; Dopamine/metabolism ; Drug Discovery ; HEK293 Cells ; Humans ; Mice ; Models, Biological ; Neurons/drug effects/*metabolism ; Protein Binding ; *Signal Transduction/drug effects ; },
abstract = {Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein-NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D1- and D2-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms.},
}
@article {pmid31751606,
year = {2020},
author = {Hensel, G},
title = {Genetic transformation of Triticeae cereals - Summary of almost three-decade's development.},
journal = {Biotechnology advances},
volume = {40},
number = {},
pages = {107484},
doi = {10.1016/j.biotechadv.2019.107484},
pmid = {31751606},
issn = {1873-1899},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; *Edible Grain ; Genetic Engineering ; Plants, Genetically Modified ; Poaceae ; Transformation, Genetic ; *Triticum ; },
abstract = {Triticeae cereals are among the most important crop plants grown worldwide and being used for animal feed, food and beverages. Although breeding efforts evolved over the last ten thousand years our today's crop plants, biotechnological methods would help to speed up the process and incorporate traits impossible by conventional breeding. The main research topics were related to cover the future demand on our agricultural practices to supply sufficient food for a growing world population. Target traits are resistances against viral and fungal diseases, improvement of water and nitrogen use efficiency, to tackle plant architecture, both below and aboveground and to develop varieties that could grow on dry or salty locations. Other applications are considering accumulation of useful compounds or decreasing allergenicity. This review will summarize methods to generate the material including a section how genome engineering using gRNA/Cas (CRISPR/Cas) technology could further improve the methodology and will give an overview about recent and future applications.},
}
@article {pmid31751596,
year = {2020},
author = {Banan, M},
title = {Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells.},
journal = {Journal of biotechnology},
volume = {308},
number = {},
pages = {1-9},
doi = {10.1016/j.jbiotec.2019.11.010},
pmid = {31751596},
issn = {1873-4863},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Gene Knock-In Techniques/*methods ; Humans ; Mammals/*genetics ; Mice ; },
abstract = {Since its inception, the CRISPR/Cas9 technology has been widely utilized for the targeted insertion of donor DNAs into mammalian genomes. A shortcoming with the earlier knock-in (KI) approaches, however, has been the low efficiency of targeted integrations-especially in primary cells and mouse embryos. Since, a variety of novel strategies have been developed towards improving the KI efficiencies in select target cells. In this review, the current applications of CRISPR/Cas9-mediated KIs in mammalian cells are described. Furthermore, the recent strategies which have been developed in order to augment the CRISPR/Cas9-mediated KI efficiencies are summarized.},
}
@article {pmid31749445,
year = {2019},
author = {Harding, HP and Ordonez, A and Allen, F and Parts, L and Inglis, AJ and Williams, RL and Ron, D},
title = {The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31749445},
issn = {2050-084X},
support = {MC_U105184308/MRC_/Medical Research Council/United Kingdom ; C14801/A21211/CRUK_/Cancer Research UK/United Kingdom ; Wellcome 100140//Wellcome/International ; Wellcome 200848/Z/16/Z//Wellcome/International ; },
mesh = {Amino Acids/*metabolism ; Animals ; CHO Cells ; CRISPR-Cas Systems ; Cricetulus ; Endoplasmic Reticulum/metabolism ; Gene Expression Regulation, Enzymologic ; HeLa Cells ; Humans ; Kinetics ; Ligands ; Mice ; Models, Molecular ; Mutagenesis ; Phosphorylation ; Protein Binding ; Protein Conformation ; Protein Serine-Threonine Kinases/chemistry/genetics/*metabolism ; Protein Unfolding ; RNA, Transfer/metabolism ; Ribosomes/chemistry/*metabolism ; Signal Transduction ; Starvation/*metabolism ; Transcriptome ; eIF-2 Kinase/genetics/metabolism ; },
abstract = {The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.},
}
@article {pmid31748735,
year = {2019},
author = {},
title = {Human germline editing needs one message.},
journal = {Nature},
volume = {575},
number = {7783},
pages = {415-416},
doi = {10.1038/d41586-019-03525-0},
pmid = {31748735},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; DNA ; *Gene Editing ; Germ Cells ; Humans ; Tissue Donors ; },
}
@article {pmid31748711,
year = {2020},
author = {Botkin, JR},
title = {The case for banning heritable genome editing.},
journal = {Genetics in medicine : official journal of the American College of Medical Genetics},
volume = {22},
number = {3},
pages = {487-489},
pmid = {31748711},
issn = {1530-0366},
mesh = {CRISPR-Cas Systems/*genetics ; *Ethicists ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Genome, Human/*genetics ; Humans ; },
}
@article {pmid31748650,
year = {2019},
author = {Huang, J and Chen, M and Xu, ES and Luo, L and Ma, Y and Huang, W and Floyd, W and Klann, TS and Kim, SY and Gersbach, CA and Cardona, DM and Kirsch, DG},
title = {Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous Kras[G12D].},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17220},
pmid = {31748650},
issn = {2045-2322},
support = {T32 GM007171/GM/NIGMS NIH HHS/United States ; T32GM007171//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/International ; R35CA197616//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/International ; F30CA206424//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Proliferation ; Cell Transformation, Neoplastic/genetics/metabolism/*pathology ; Cells, Cultured ; Embryo, Mammalian/cytology/metabolism ; Fibroblasts/cytology/metabolism ; Gene Expression Regulation, Neoplastic ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; *Mutation ; Neoplasm Proteins/*antagonists &amp; inhibitors/genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; Sarcoma, Experimental/genetics/pathology/*prevention &amp; control ; Signal Transduction ; },
abstract = {Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in Kras[G12D] immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with Kras[G12D] to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with Kras[G12D] to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.},
}
@article {pmid31748579,
year = {2019},
author = {Lee, JY and Dominguez, AA and Nam, S and Stowers, RS and Qi, LS and Chaudhuri, O},
title = {Identification of cell context-dependent YAP-associated proteins reveals β1 and β4 integrin mediate YAP translocation independently of cell spreading.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17188},
pmid = {31748579},
issn = {2045-2322},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; R37 CA214136/CA/NCI NIH HHS/United States ; S10 RR027425/RR/NCRR NIH HHS/United States ; },
mesh = {Actin Cytoskeleton/*metabolism ; Actins/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cells, Cultured ; Extracellular Matrix/*metabolism ; Humans ; Integrin beta1/chemistry/genetics/*metabolism ; Integrin beta4/chemistry/genetics/*metabolism ; *Mechanotransduction, Cellular ; Phosphorylation ; Protein Processing, Post-Translational ; Protein Transport ; Signal Transduction ; Transcription Factors/genetics/*metabolism ; },
abstract = {Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with β1 integrin under activating conditions, and β4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, β4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.},
}
@article {pmid31748409,
year = {2019},
author = {Yoganand, KN and Muralidharan, M and Nimkar, S and Anand, B},
title = {Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {52},
pages = {20039-20053},
pmid = {31748409},
issn = {1083-351X},
mesh = {Binding Sites ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/chemistry/metabolism ; Electrophoretic Mobility Shift Assay ; Endodeoxyribonucleases/chemistry/genetics/metabolism ; Endonucleases/chemistry/genetics/metabolism ; Escherichia coli/*metabolism ; Escherichia coli Proteins/chemistry/genetics/metabolism ; Exonucleases/metabolism ; Protein Binding ; Protein Structure, Quaternary ; },
abstract = {Prokaryotes deploy CRISPR-Cas-based RNA-guided adaptive immunity to fend off mobile genetic elements such as phages and plasmids. During CRISPR adaptation, which is the first stage of CRISPR immunity, the Cas1-2 integrase complex captures invader-derived prespacer DNA and specifically integrates it at the leader-repeat junction as spacers. For this integration, several variants of CRISPR-Cas systems use Cas4 as an indispensable nuclease for selectively processing the protospacer adjacent motif (PAM) containing prespacers to a defined length. Surprisingly, however, a few CRISPR-Cas systems, such as type I-E, are bereft of Cas4. Despite the absence of Cas4, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. Here, using in vivo and in vitro integration assays, deep sequencing, and exonuclease footprinting, we show that Cas1-2/I-E-via the type I-E-specific extended C-terminal tail of Cas1-displays intrinsic affinity for PAM containing prespacers of variable length in Escherichia coli Although Cas1-2/I-E does not prune the prespacers, its binding protects the prespacer boundaries from exonuclease action. This ensures the pruning of exposed ends by exonucleases to aptly sized substrates for integration into the CRISPR locus. In summary, our work reveals that in a few CRISPR-Cas variants, such as type I-E, the specificity of PAM selection resides with Cas1-2, whereas the prespacer processing is co-opted by cellular non-Cas exonucleases, thereby offsetting the need for Cas4.},
}
@article {pmid31747398,
year = {2019},
author = {Mackow, NA and Shen, J and Adnan, M and Khan, AS and Fries, BC and Diago-Navarro, E},
title = {CRISPR-Cas influences the acquisition of antibiotic resistance in Klebsiella pneumoniae.},
journal = {PloS one},
volume = {14},
number = {11},
pages = {e0225131},
pmid = {31747398},
issn = {1932-6203},
support = {I01 BX003741/BX/BLRD VA/United States ; R21 AI114259/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/toxicity ; Bacterial Proteins/genetics ; CRISPR-Cas Systems ; Carbapenems/toxicity ; *Drug Resistance, Bacterial ; Klebsiella pneumoniae/drug effects/*genetics/pathogenicity ; beta-Lactamases/genetics ; },
abstract = {In the US Carbapenem resistance in Klebsiella pneumoniae (Kp) is primarily attributed to the presence of the genes blaKPC-2 and blaKPC-3, which are transmitted via plasmids. Carbapenem-resistant Kp (CR-Kp) infections are associated with hospital outbreaks. They are difficult to treat, and associated with high mortality rates prompting studies of how resistance is obtained. In this study, we determined the presence of CRISPR-Cas in 304 clinical Kp strains. The CRISPR-Cas system has been found to prevent the spread of plasmids and bacteriophages, and therefore limits the horizontal gene transfer mediated by these mobile genetic elements. Here, we hypothesized that only those Kp strains that lack CRISPR-Cas can acquire CR plasmids, while those strains that have CRISPR-Cas are protected from gaining these plasmids and thus maintain sensitivity to antimicrobials. Our results show that CRISPR-Cas is absent in most clinical Kp strains including the clinically important ST258 clone. ST258 strains that continue to be sensitive to carbapenems also lack CRISPR-Cas. Interestingly, CRISPR-Cas positive strains, all non-ST258, exhibit lower resistance rates to antimicrobials than CRISPR-Cas negative strains. Importantly, we demonstrate that the presence of CRISPR-Cas appears to inhibit the acquisition of blaKPC plasmids in 7 Kp strains. Furthermore, we show that strains that are unable to acquire blaKPC plasmids contain CRISPR spacer sequences highly identical to those found in previously published multidrug-resistance-containing plasmids. Lastly, to our knowledge this is the first paper demonstrating that resistance to blaKPC plasmid invasion in a CRISPR-containing Kp strain can be reversed by deleting the CRISPR-cas cassette.},
}
@article {pmid31746634,
year = {2019},
author = {Ishii, T and Beriain, IM},
title = {Safety of Germline Genome Editing for Genetically Related "Future" Children as Perceived by Parents.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {370-375},
pmid = {31746634},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Child ; Child, Preschool ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fertilization in Vitro/*ethics/methods ; Gene Editing/*ethics/trends ; Genetic Therapy/*ethics/methods ; Germ Cells ; Humans ; Parents ; },
abstract = {The social acceptability of germline genome editing (GGE) depends on its perceived safety, as well as respect for reproductive autonomy. However, it is doubtful that prospective parents sufficiently understand the risks of GGE. In the future, the use of GGE in specific situations seems plausible, as it offers couples potential means to safeguard genetically related future children from a serious disease and overcome infertility due to a gene mutation. Should GGE fail, however, some couples may be obliged to abort affected fetuses, or give birth to adversely affected children, which would be a tragedy. Some children might develop diseases later in life due to overlooked off-target mutations. Compounding this, some parents are unlikely to inform their offspring about the details of conception, hampering necessary follow-up. Prospective parents, scientists and policy makers should carefully discuss the safety implications of GGE for genetically related future children.},
}
@article {pmid31746519,
year = {2020},
author = {O'Neil, EV and Brooks, K and Burns, GW and Ortega, MS and Denicol, AC and Aguiar, LH and Pedroza, GH and Benne, J and Spencer, TE},
title = {Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep.},
journal = {Molecular reproduction and development},
volume = {87},
number = {1},
pages = {142-151},
doi = {10.1002/mrd.23300},
pmid = {31746519},
issn = {1098-2795},
mesh = {Animals ; Blastocyst/*metabolism ; CRISPR-Cas Systems ; Cyclooxygenase 1/metabolism ; Cyclooxygenase 2/genetics/*metabolism ; Embryo Transfer/methods ; Embryonic Development/*genetics ; Endometrium/metabolism ; Female ; Fertilization in Vitro/methods ; Gene Editing ; Gene Expression ; Interferon Type I/biosynthesis ; PPAR gamma/metabolism ; Pregnancy ; Pregnancy Proteins/biosynthesis ; Pregnancy, Animal/*metabolism ; Prostaglandins/biosynthesis ; Sheep/*embryology ; Signal Transduction/genetics ; },
abstract = {Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus-derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.},
}
@article {pmid31746426,
year = {2020},
author = {Liu, Q and Liu, H and Li, L and Dong, X and Ru, X and Fan, X and Wen, T and Liu, J},
title = {ATAD2 predicts poor outcomes in patients with ovarian cancer and is a marker of proliferation.},
journal = {International journal of oncology},
volume = {56},
number = {1},
pages = {219-231},
pmid = {31746426},
issn = {1791-2423},
mesh = {ATPases Associated with Diverse Cellular Activities/antagonists &amp; inhibitors/genetics/*metabolism ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; *Cell Proliferation ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Ovarian Neoplasms/metabolism/*mortality/pathology ; Prognosis ; Survival Rate ; Tumor Cells, Cultured ; },
abstract = {The oncogene ATPase family AAA domain‑ containing protein 2 (ATAD2) has been demonstrated to promote malignancy in a number of different types of tumor; however, its expression and role in ovarian cancer (OC) remain unknown. In the present study, it was demonstrated that ATAD2 acts as both a marker and a driver of cell proliferation in OC. Immunohistochemistry (IHC) and bioinformatics analyses were used to evaluate ATAD2 expression in OC, and multi‑omics integrated analyses were used to dissect which factor resulted in its upregulation. Multiplex IHC assay was used to reveal the specific expression of ATAD2 in proliferating OC cells. CRISPR‑Cas9‑mediated gene editing was performed to investigate the effect of ATAD2 deletion on OC proliferation. The results demonstrated that ATAD2 is elevated in primary OC tissues compared with the adjacent normal tissue and metastases from the stomach. Genetic copy number amplification is a primary cause resulting in upregulation of ATAD2, and this was most frequently observed in OC. High ATAD2 expression was associated with advanced progression and predicted an unfavorable prognosis. ATAD2 could be used to identify cases of OC with a high proliferation signature and could label proliferating cells in OC. CRISPR‑Cas9‑mediated ATAD2 deletion resulted in a significant decrease in both cell proliferation and colony formation ability. Mechanistically, ATAD2‑knockdown resulted in deactivation of the mitogen‑activated protein kinase (MAPK) pathways, particularly the JNK‑MAPK pathway, resulting in suppression of proliferation. Collectively, the data from the present study demonstrated that the ATD2 gene was frequently amplified and protein expression levels were upregulated in OC. Therefore, ATAD2 may serve as an attractive diagnostic and prognostic OC marker, which may be used to identify patients with primary OC, whom are most likely to benefit from ATAD2 gene‑targeted proliferation intervention therapies.},
}
@article {pmid31745933,
year = {2020},
author = {Swinnen, G and Jacobs, T and Pauwels, L and Goossens, A},
title = {CRISPR-Cas-Mediated Gene Knockout in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2083},
number = {},
pages = {321-341},
doi = {10.1007/978-1-4939-9952-1_25},
pmid = {31745933},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing ; *Gene Knockout Techniques ; Gene Targeting ; Genetic Vectors/genetics ; Lycopersicon esculentum/*genetics ; Plants, Genetically Modified ; RNA, Guide ; Workflow ; },
abstract = {Loss-of-function mutants are crucial for plant functional genomics studies. With the advent of CRISPR-Cas genome editing, generating null alleles for one or multiple specific gene(s) has become feasible for many plant species including tomato (Solanum lycopersicum). An easily programmable RNA-guided Cas endonuclease efficiently creates DNA double-strand breaks (DSBs) at targeted genomic sites that can be repaired by nonhomologous end joining (NHEJ) typically leading to small insertions or deletions that can produce null mutations. Here, we describe how to utilize CRISPR-Cas genome editing to obtain stable tomato gene knockout lines.},
}
@article {pmid31745578,
year = {2020},
author = {Sun, S and Wang, X and Wang, K and Cui, X},
title = {Dissection of complex traits of tomato in the post-genome era.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {5},
pages = {1763-1776},
doi = {10.1007/s00122-019-03478-y},
pmid = {31745578},
issn = {1432-2242},
mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; *Gene Editing ; *Genome, Plant ; Lycopersicon esculentum/*genetics/*growth &amp; development ; *Multifactorial Inheritance ; Plant Breeding/*methods ; *Quantitative Trait Loci ; },
abstract = {We present the main advances of dissection of complex traits in tomato by omics, the genes identified to control complex traits and the application of CRISPR/Cas9 in tomato breeding. Complex traits are believed to be under the control of multiple genes, each with different effects and interaction with environmental factors. Advance development of sequencing and molecular technologies has enabled the recognition of the genomic structure of most organisms and the identification of a nearly limitless number of markers that have made it to accelerate the speed of QTL identification and gene cloning. Meanwhile, multiomics have been used to identify the genetic variations among different tomato species, determine the expression profiles of genes in different tissues and at distinct developmental stages, and detect metabolites in different pathways and processes. The combination of these data facilitates to reveal mechanism underlying complex traits. Moreover, mutants generated by mutagens and genome editing provide relatively rich genetic variation for deciphering the complex traits and exploiting them in tomato breeding. In this article, we present the main advances of complex trait dissection in tomato by omics since the release of the tomato genome sequence in 2012. We provide further insight into some tomato complex traits because of the causal genetic variations discovered so far and explore the utilization of CRISPR/Cas9 for the modification of tomato complex traits.},
}
@article {pmid31745574,
year = {2020},
author = {Zhan, Y and Xu, Y and Zheng, P and He, M and Sun, S and Wang, D and Cai, D and Ma, X and Chen, S},
title = {Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {1},
pages = {391-403},
doi = {10.1007/s00253-019-10230-5},
pmid = {31745574},
issn = {1432-0614},
mesh = {Bacillus licheniformis/enzymology/*genetics ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Carboxy-Lyases/genetics ; *Gene Silencing ; Leucine Dehydrogenase/genetics ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways ; Valine/analysis/metabolism ; },
abstract = {Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.},
}
@article {pmid31745563,
year = {2020},
author = {Agmon, N and Temple, J and Tang, Z and Schraink, T and Baron, M and Chen, J and Mita, P and Martin, JA and Tu, BP and Yanai, I and Fenyö, D and Boeke, JD},
title = {Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast.},
journal = {Nucleic acids research},
volume = {48},
number = {1},
pages = {486-499},
pmid = {31745563},
issn = {1362-4962},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R24 DK082840/DK/NIDDK NIH HHS/United States ; },
mesh = {Adenine/*biosynthesis ; Amino Acid Sequence ; Biosynthetic Pathways/*genetics ; CRISPR-Cas Systems ; Carboxy-Lyases/*genetics/metabolism ; Chromosomes, Artificial, Human/*chemistry/metabolism ; Genetic Complementation Test ; Genetic Engineering/methods ; Humans ; Isoenzymes/genetics/metabolism ; Peptide Synthases/*genetics/metabolism ; Phylogeny ; Plasmids/chemistry/metabolism ; Saccharomyces cerevisiae/classification/*genetics/metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; },
abstract = {Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.},
}
@article {pmid31745554,
year = {2020},
author = {Bernheim, A and Bikard, D and Touchon, M and Rocha, EPC},
title = {Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements.},
journal = {Nucleic acids research},
volume = {48},
number = {2},
pages = {748-760},
pmid = {31745554},
issn = {1362-4962},
mesh = {Adaptive Immunity/*genetics ; Bacteriophages/genetics ; CRISPR-Cas Systems/*genetics/immunology ; Genome, Bacterial/genetics/*immunology ; Genomics ; Interspersed Repetitive Sequences/*genetics/immunology ; Plasmids/genetics ; Prokaryotic Cells/immunology ; Prophages/genetics ; },
abstract = {Prokaryotes use CRISPR-Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative and many positive epistatic interactions between Cas subtypes. The latter often result in complex genetic organizations, where a locus has a single adaptation module and diverse interference mechanisms that might provide more effective immunity. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. Many chromosomal CRISPRs lack a neighboring Cas system and they often have repeats compatible with the Cas systems encoded in trans. Phages and 25 000 prophages were almost devoid of CRISPR-Cas systems, whereas 3% of plasmids had CRISPR-Cas systems or isolated CRISPRs. The latter were often compatible with the chromosomal cas clusters, suggesting that plasmids can co-opt the latter. These results highlight the importance of interactions between CRISPRs and cas present in multiple copies and in distinct genomic locations in the function and evolution of bacterial immunity.},
}
@article {pmid31745156,
year = {2019},
author = {Hooghvorst, I and López-Cristoffanini, C and Nogués, S},
title = {Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17077},
pmid = {31745156},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cucurbitaceae/*genetics/growth &amp; development/metabolism ; *Gene Editing ; Genome, Plant ; *Mutation ; Oxidoreductases/*antagonists &amp; inhibitors/genetics/metabolism ; Plant Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Plants, Genetically Modified/*genetics/growth &amp; development/metabolism ; },
abstract = {CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42-45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.},
}
@article {pmid31745112,
year = {2019},
author = {Sullivan, NT and Dampier, W and Chung, CH and Allen, AG and Atkins, A and Pirrone, V and Homan, G and Passic, S and Williams, J and Zhong, W and Kercher, K and Desimone, M and Li, L and C Antell, G and Mell, JC and Ehrlich, GD and Szep, Z and Jacobson, JM and Nonnemacher, MR and Wigdahl, B},
title = {Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17088},
pmid = {31745112},
issn = {2045-2322},
support = {T32 MH079785/MH/NIMH NIH HHS/United States ; P30 MH092177/MH/NIMH NIH HHS/United States ; R01 MH110360/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cohort Studies ; Computational Biology ; Female ; *Gene Editing ; Genome, Viral ; HIV Infections/*genetics/virology ; HIV Long Terminal Repeat/genetics ; HIV-1/*genetics/*growth &amp; development/metabolism ; Humans ; Male ; Middle Aged ; Proviruses/*genetics ; Quasispecies/*genetics ; RNA, Guide/*genetics ; },
abstract = {The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.},
}
@article {pmid31745080,
year = {2019},
author = {Webber, BR and Lonetree, CL and Kluesner, MG and Johnson, MJ and Pomeroy, EJ and Diers, MD and Lahr, WS and Draper, GM and Slipek, NJ and Smeester, BA and Lovendahl, KN and McElroy, AN and Gordon, WR and Osborn, MJ and Moriarity, BS},
title = {Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5222},
pmid = {31745080},
issn = {2041-1723},
support = {P30 CA077598/CA/NCI NIH HHS/United States ; R03 AI144840/AI/NIAID NIH HHS/United States ; R35 GM119483/GM/NIGMS NIH HHS/United States ; T32 AI007313/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Engineering/*methods ; Cells, Cultured ; *DNA Breaks, Double-Stranded ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Immunotherapy, Adoptive/methods ; Reproducibility of Results ; T-Lymphocytes/cytology/*metabolism ; },
abstract = {The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.},
}
@article {pmid31744829,
year = {2020},
author = {Li, F and Huang, Q and Luster, TA and Hu, H and Zhang, H and Ng, WL and Khodadadi-Jamayran, A and Wang, W and Chen, T and Deng, J and Ranieri, M and Fang, Z and Pyon, V and Dowling, CM and Bagdatlioglu, E and Almonte, C and Labbe, K and Silver, H and Rabin, AR and Jani, K and Tsirigos, A and Papagiannakopoulos, T and Hammerman, PS and Velcheti, V and Freeman, GJ and Qi, J and Miller, G and Wong, KK},
title = {In Vivo Epigenetic CRISPR Screen Identifies Asf1a as an Immunotherapeutic Target in Kras-Mutant Lung Adenocarcinoma.},
journal = {Cancer discovery},
volume = {10},
number = {2},
pages = {270-287},
pmid = {31744829},
issn = {2159-8290},
support = {R01 CA216188/CA/NCI NIH HHS/United States ; P50 CA101942/CA/NCI NIH HHS/United States ; R01 CA219670/CA/NCI NIH HHS/United States ; U01 CA233084/CA/NCI NIH HHS/United States ; R01 CA166480/CA/NCI NIH HHS/United States ; R01 CA222218/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma of Lung/*drug therapy/genetics/immunology/pathology ; Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics/*metabolism ; Cell Differentiation/drug effects/genetics ; Cell Line, Tumor ; Disease Models, Animal ; Drug Resistance, Neoplasm/*genetics ; Epigenesis, Genetic/immunology ; Gene Expression Regulation, Neoplastic/immunology ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Immune Checkpoint Inhibitors/*pharmacology/therapeutic use ; Lung/pathology ; Lung Neoplasms/*drug therapy/genetics/immunology/pathology ; Macrophages/drug effects/immunology/metabolism ; Male ; Mice ; Molecular Chaperones/genetics/*metabolism ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/immunology ; Proto-Oncogene Proteins p21(ras)/genetics ; RNA, Guide/genetics ; RNA, Small Interfering/metabolism ; RNA-Seq ; Tumor Microenvironment/drug effects/immunology ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras [G12D]/Trp53 [-/-] LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.},
}
@article {pmid31743839,
year = {2019},
author = {Si, L and Yang, R and Liu, J and Dong, Y and Zhang, H and Xu, X},
title = {Generation of two ERF gene knockout human embryonic stem cell lines using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101644},
doi = {10.1016/j.scr.2019.101644},
pmid = {31743839},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line/*cytology ; *Gene Knockout Techniques ; Human Embryonic Stem Cells/*cytology/*metabolism ; Humans ; Male ; Repressor Proteins/*genetics ; Reproducibility of Results ; },
abstract = {Human ERF gene is a transcription factor involved in development, trophoblast differentiation, apoptosis, and cancer progress. To further understand the exact roles of ERF in these processes, here we report that establishment of two ERF knockout human embryonic stem cell (hESC) lines by CRISPR/Cas9 mediated gene targeting. These cell lines exhibited classical hESC morphology and normal karyotype, and highly expressed pluripotent markers, and had differentiation potential in vitro. These cell lines provide good materials to understand the roles of ERF in development, trophoblast differentiation and craniosynostosis for further studies.},
}
@article {pmid31743770,
year = {2020},
author = {Liu, X and Zhang, P and Zhang, X and Li, X and Bai, Y and Ao, Y and Hexig, B and Guo, X and Liu, D},
title = {Fgf21 knockout mice generated using CRISPR/Cas9 reveal genetic alterations that may affect hair growth.},
journal = {Gene},
volume = {733},
number = {},
pages = {144242},
doi = {10.1016/j.gene.2019.144242},
pmid = {31743770},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Female ; Fibroblast Growth Factors/*genetics/metabolism ; Genetic Engineering/methods ; Hair/*metabolism ; Hair Follicle/*growth &amp; development/metabolism ; MAP Kinase Signaling System/physiology ; Male ; Mice ; Mice, Knockout ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Signal Transduction/physiology ; },
abstract = {OBJECTIVE: To investigate fibroblast growth factor 21 (Fgf21) alterations that may affect hair growth and the underlying molecular mechanisms by constructing Fgf21 global knockout (KO) mice using microinjection-mediated CRISPR/Cas9.<br><br>RESULTS: Following genomic DNA sequencing, we identified 18 mice carrying Ffg21 mutations among the total 63 offspring mice obtained by injecting 340 embryos, which yielded a mutation rate of 28.6 percent. Of these 18 mice, three had both alleles knocked out and 15 were monoallelic KO mice. Compared with the wild-type (WT) mice, the phenotypic analysis showed that the litter size of Fgf21 KO mice significantly reduced (p&#xa0;<&#xa0;0.05), but physiological indexes of the birth weight, gender rate, body weight (0-8&#xa0;week) and body weight of adult male and female were no significant difference (p&#xa0;>&#xa0;0.05). Compared to WT mice, physiological anatomy indicated that the morphological characters of vital organs in Fgf21 KO mice were normal. Depilation experiments demonstrated that compared to the WT mice, the hair regrowth speed was reduced in the Fgf21 KO mice. The number of hair shafts in these mice considerably decreased, as indicated by the tissue sample analyses. Real-time quantitative PCR showed that Erk and Akt expression in the KO mice was significantly decreased (P&#xa0;<&#xa0;0.05), whereas western blotting demonstrated that the expression of Erk and Akt proteins and their phosphorylation levels in KO mice decreased at different rates (P&#xa0;<&#xa0;0.05).<br><br>CONCLUSIONS: Fgf21 was shown to affect hair follicle development and growth cycle, which may be associated with Pi3k/Akt and Mapk/Erk signaling pathways.},
}
@article {pmid31742594,
year = {2019},
author = {Pettingill, P and Weir, GA and Wei, T and Wu, Y and Flower, G and Lalic, T and Handel, A and Duggal, G and Chintawar, S and Cheung, J and Arunasalam, K and Couper, E and Haupt, LM and Griffiths, LR and Bassett, A and Cowley, SA and Cader, MZ},
title = {A causal role for TRESK loss of function in migraine mechanisms.},
journal = {Brain : a journal of neurology},
volume = {142},
number = {12},
pages = {3852-3867},
pmid = {31742594},
issn = {1460-2156},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; J-0901/PUK_/Parkinson's UK/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Humans ; Induced Pluripotent Stem Cells/metabolism ; *Loss of Function Mutation ; Mice ; Migraine Disorders/chemically induced/*genetics/metabolism ; Nitroglycerin ; Nociception/*physiology ; Nociceptors/*metabolism ; Pain Measurement ; Patch-Clamp Techniques ; Potassium Channels/*genetics/metabolism ; },
abstract = {The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.},
}
@article {pmid31742435,
year = {2019},
author = {Wierson, WA and Simone, BW and WareJoncas, Z and Mann, C and Welker, JM and Kar, B and Emch, MJ and Friedberg, I and Gendron, WAC and Barry, MA and Clark, KJ and Dobbs, DL and McGrail, MA and Ekker, SC and Essner, JJ},
title = {Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {417-433},
pmid = {31742435},
issn = {2573-1602},
support = {R01 GM063904/GM/NIGMS NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; R56 GM063904/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/chemistry ; Gene Editing/*methods ; Gene Targeting/methods ; Genetic Engineering/methods ; Genome/genetics ; Humans ; RNA/chemistry ; RNA, Guide/chemistry ; Zebrafish/genetics ; },
abstract = {CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.},
}
@article {pmid31742433,
year = {2019},
author = {Chen, SP and Wang, HH},
title = {An Engineered Cas-Transposon System for Programmable and Site-Directed DNA Transpositions.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {376-394},
pmid = {31742433},
issn = {2573-1602},
support = {F30 DK111145/DK/NIDDK NIH HHS/United States ; T32 GM007367/GM/NIGMS NIH HHS/United States ; DP5 OD009172/OD/NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics/physiology ; DNA Transposable Elements/*genetics/physiology ; Endonucleases/genetics ; Escherichia coli/genetics ; Gene Editing/methods ; Mutagenesis, Insertional/*methods ; Recombination, Genetic/genetics/physiology ; Transposases/genetics/*metabolism ; },
abstract = {Efficient site-directed insertion of heterologous DNA into a genome remains an outstanding challenge. Recombinases that can integrate kilobase-sized DNA constructs are difficult to reprogram to user-defined loci, while genomic insertion using CRISPR-Cas methods relies on inefficient host DNA repair machinery. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a Himar1 transposase fused to a catalytically dead dCas9 nuclease to mediate programmable, site-directed transposition. Using cell-free in vitro assays, we demonstrated that the Himar-dCas9 fusion protein increased the frequency of transposon insertion at a single targeted TA dinucleotide by >300-fold compared to a random transposase, and that site-directed transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We also showed that Himar-dCas9 mediates directed transposition into plasmids in Escherichia coli. This work highlights CasTn as a new modality for host-independent, programmable, site-directed DNA insertions.},
}
@article {pmid31742432,
year = {2019},
author = {Rayner, E and Durin, MA and Thomas, R and Moralli, D and O'Cathail, SM and Tomlinson, I and Green, CM and Lewis, A},
title = {CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {406-416},
pmid = {31742432},
issn = {2573-1602},
support = {MR/P000738/1/MRC_/Medical Research Council/United Kingdom ; MR/P000738/2/MRC_/Medical Research Council/United Kingdom ; 090532/Z/09/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromosomal Instability/*genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytogenetic Analysis/*methods ; Gene Editing/*methods ; Gene Rearrangement/genetics ; Humans ; Mutagenesis/genetics ; Mutation ; Neoplasms/genetics ; RNA, Guide/genetics ; Sequence Deletion/genetics ; },
abstract = {CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small mutations, large deletions of target loci, or large-scale chromosomal rearrangements. A common application of CRISPR-Cas9 is as a tool for creating isogenic cell-line models to study the effects of precise mutations, or variants, on disease traits. Here, we describe the effect of standard CRISPR-Cas9 mutagenesis protocols on well characterized cancer cell lines. We demonstrate that commonly used methods for detecting correctly mutated clones fail to uncover large-scale rearrangements. We show that simple cytogenetic methods can be used to identify clones carrying chromosomal abnormalities and large mutations at target loci. These methods are quick and cost-efficient, and we suggest that such controls should be performed prior to publication of studies based on novel CRISPR-Cas9 mutated cancer cell lines.},
}
@article {pmid31742431,
year = {2019},
author = {Hollister, BM and Gatter, MC and Abdallah, KE and Armsby, AJ and Buscetta, AJ and Byeon, YJJ and Cooper, KE and Desine, S and Persaud, A and Ormond, KE and Bonham, VL},
title = {Perspectives of Sickle Cell Disease Stakeholders on Heritable Genome Editing.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {441-449},
pmid = {31742431},
issn = {2573-1602},
support = {ZIA HG200394/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Adult ; Anemia, Sickle Cell/*genetics/*therapy ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Focus Groups ; Gene Editing/*ethics/methods ; Genetic Therapy/ethics/methods ; Humans ; Male ; Middle Aged ; Stakeholder Participation/psychology ; },
abstract = {Advances in CRISPR technology and the announcement of the first gene-edited babies have sparked a global dialogue about the future of heritable genome editing (HGE). There has been an international call for public input to inform a substantive debate about benefits and risks of HGE. This study investigates the views of the sickle cell disease (SCD) community. We utilized a mixed-methods approach to examine SCD stakeholders' views in the United States. We found SCD stakeholders hold a nuanced view of HGE. Assuming the technology is shown to be safe and effective, they are just as supportive of HGE as genetics professionals, but more supportive than the general public. However, they are also concerned about the potential implications of HGE, despite this support. As discourse surrounding HGE advances, it is crucial to engage disease communities and other key stakeholders whose lives could be altered by these interventions.},
}
@article {pmid31741433,
year = {2019},
author = {Zeng, H and Castillo-Cabrera, J and Manser, M and Lu, B and Yang, Z and Strande, V and Begue, D and Zamponi, R and Qiu, S and Sigoillot, F and Wang, Q and Lindeman, A and Reece-Hoyes, JS and Russ, C and Bonenfant, D and Jiang, X and Wang, Y and Cong, F},
title = {Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31741433},
issn = {2050-084X},
mesh = {A549 Cells ; Adaptor Proteins, Signal Transducing/metabolism ; Animals ; *CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cullin Proteins ; ErbB Receptors/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Guanine Nucleotide Exchange Factors/genetics ; HEK293 Cells ; Humans ; Methionyl Aminopeptidases/metabolism ; Mice ; Mice, Nude ; Receptors, Lysophosphatidic Acid/metabolism ; Signal Transduction ; Transcription Factors/metabolism ; Transcriptome ; Ubiquitin-Protein Ligases/genetics ; YAP-Signaling Proteins ; rhoA GTP-Binding Protein/metabolism ; },
abstract = {EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.},
}
@article {pmid31740840,
year = {2019},
author = {Giesselmann, P and Brändl, B and Raimondeau, E and Bowen, R and Rohrandt, C and Tandon, R and Kretzmer, H and Assum, G and Galonska, C and Siebert, R and Ammerpohl, O and Heron, A and Schneider, SA and Ladewig, J and Koch, P and Schuldt, BM and Graham, JE and Meissner, A and Müller, FJ},
title = {Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing.},
journal = {Nature biotechnology},
volume = {37},
number = {12},
pages = {1478-1481},
pmid = {31740840},
issn = {1546-1696},
mesh = {Algorithms ; Amyotrophic Lateral Sclerosis/genetics ; C9orf72 Protein/genetics ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; DNA Methylation/*genetics ; Genomics/*methods ; Humans ; Microsatellite Repeats/*genetics ; Nanopore Sequencing/*methods ; Nanopores ; },
abstract = {Expansions of short tandem repeats are genetic variants that have been implicated in several neuropsychiatric and other disorders, but their assessment remains challenging with current polymerase-based methods[1-4]. Here we introduce a CRISPR-Cas-based enrichment strategy for nanopore sequencing combined with an algorithm for raw signal analysis. Our method, termed STRique for short tandem repeat identification, quantification and evaluation, integrates conventional sequence mapping of nanopore reads with raw signal alignment for the localization of repeat boundaries and a hidden Markov model-based repeat counting mechanism. We demonstrate the precise quantification of repeat numbers in conjunction with the determination of CpG methylation states in the repeat expansion and in adjacent regions at the single-molecule level without amplification. Our method enables the study of previously inaccessible genomic regions and their epigenetic marks.},
}
@article {pmid31740839,
year = {2019},
author = {Cameron, P and Coons, MM and Klompe, SE and Lied, AM and Smith, SC and Vidal, B and Donohoue, PD and Rotstein, T and Kohrs, BW and Nyer, DB and Kennedy, R and Banh, LM and Williams, C and Toh, MS and Irby, MJ and Edwards, LS and Lin, CH and Owen, ALG and Künne, T and van der Oost, J and Brouns, SJJ and Slorach, EM and Fuller, CK and Gradia, S and Kanner, SB and May, AP and Sternberg, SH},
title = {Harnessing type I CRISPR-Cas systems for genome engineering in human cells.},
journal = {Nature biotechnology},
volume = {37},
number = {12},
pages = {1471-1477},
pmid = {31740839},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; Escherichia coli ; Gene Editing/*methods ; Genome/genetics ; HEK293 Cells ; Humans ; Models, Genetic ; },
abstract = {Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea[1,2]. Target interference relies on a multi-subunit, RNA-guided complex called Cascade[3,4], which recruits a trans-acting helicase-nuclease, Cas3, for target degradation[5-7]. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain[8-11] and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.},
}
@article {pmid31740817,
year = {2020},
author = {Camsund, D and Lawson, MJ and Larsson, J and Jones, D and Zikrin, S and Fange, D and Elf, J},
title = {Time-resolved imaging-based CRISPRi screening.},
journal = {Nature methods},
volume = {17},
number = {1},
pages = {86-92},
pmid = {31740817},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Cell Cycle ; DNA Replication ; Escherichia coli/genetics/growth &amp; development/*metabolism ; Escherichia coli Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Gene Library ; Genotype ; Image Processing, Computer-Assisted/*methods ; Metabolic Engineering/*methods ; Microfluidic Analytical Techniques/*methods ; Microscopy/*methods ; Phenotype ; },
abstract = {Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.},
}
@article {pmid31740807,
year = {2020},
author = {Zhang, ZJ and Pedicord, VA and Peng, T and Hang, HC},
title = {Site-specific acylation of a bacterial virulence regulator attenuates infection.},
journal = {Nature chemical biology},
volume = {16},
number = {1},
pages = {95-103},
pmid = {31740807},
issn = {1552-4469},
support = {R01 AT007671/AT/NCCIH NIH HHS/United States ; R01 GM087544/GM/NIGMS NIH HHS/United States ; R01AT007671//U.S. Department of Health &amp; Human Services | NIH | National Center for Complementary and Integrative Health (NCCIH)/International ; R01GM087544//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/International ; },
mesh = {Animals ; Arginine/chemistry ; Bacterial Proteins/metabolism ; Binding Sites ; Butyrates/chemistry ; CRISPR-Cas Systems ; Fatty Acids/*metabolism ; *Gene Expression Regulation, Bacterial ; Genome, Bacterial ; *Genomic Islands ; Genomics ; Lysine/chemistry ; Mice ; Mice, Inbred C57BL ; Mutagenesis ; Mutation ; Proteomics/methods ; Salmonella typhimurium/*metabolism/pathogenicity ; Trans-Activators/metabolism ; *Virulence ; Virulence Factors/metabolism ; },
abstract = {Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we found that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR-Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells, as well as dissemination in vivo. Moreover, a multiple-site HilA lysine acylation mutant strain of S. Typhimurium was resistant to butyrate inhibition ex vivo and microbiota attenuation in vivo. Our results suggest that prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.},
}
@article {pmid31740621,
year = {2019},
author = {van der Horst, SEM and Cravo, J and Woollard, A and Teapal, J and van den Heuvel, S},
title = {C. elegans Runx/CBFβ suppresses POP-1 TCF to convert asymmetric to proliferative division of stem cell-like seam cells.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {22},
pages = {},
pmid = {31740621},
issn = {1477-9129},
support = {P40 OD010440/OD/NIH HHS/United States ; BB/G018448/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Alleles ; Animals ; Asymmetric Cell Division ; CRISPR-Cas Systems ; Caenorhabditis elegans/*cytology ; Caenorhabditis elegans Proteins/*metabolism ; Cell Differentiation ; Cell Division ; Cell Lineage ; Cell Proliferation ; Core Binding Factor beta Subunit/*metabolism ; DNA-Binding Proteins/*metabolism ; Down-Regulation ; Gene Expression Regulation, Developmental ; Green Fluorescent Proteins/metabolism ; High Mobility Group Proteins/*metabolism ; Male ; RNA Interference ; Repressor Proteins/metabolism ; Stem Cells/*cytology ; Transcription Factors/*metabolism ; Wnt Signaling Pathway ; },
abstract = {A correct balance between proliferative and asymmetric cell divisions underlies normal development, stem cell maintenance and tissue homeostasis. What determines whether cells undergo symmetric or asymmetric cell division is poorly understood. To gain insight into the mechanisms involved, we studied the stem cell-like seam cells in the Caenorhabditis elegans epidermis. Seam cells go through a reproducible pattern of asymmetric divisions, instructed by divergent canonical Wnt/β-catenin signaling, and symmetric divisions that increase the seam cell number. Using time-lapse fluorescence microscopy we observed that symmetric cell divisions maintain asymmetric localization of Wnt/β-catenin pathway components. Our observations, based on lineage-specific knockout and GFP-tagging of endogenous pop-1, support the model that POP-1[TCF] induces differentiation at a high nuclear level, whereas low nuclear POP-1 promotes seam cell self-renewal. Before symmetric division, the transcriptional regulator RNT-1[Runx] and cofactor BRO-1[CBFβ] temporarily bypass Wnt/β-catenin asymmetry by downregulating pop-1 expression. Thereby, RNT-1/BRO-1 appears to render POP-1 below the level required for its repressor function, which converts differentiation into self-renewal. Thus, we found that conserved Runx/CBFβ-type stem cell regulators switch asymmetric to proliferative cell division by opposing TCF-related transcriptional repression.},
}
@article {pmid31740585,
year = {2020},
author = {},
title = {Prime Editing Promises Greater Precision, Safety.},
journal = {Cancer discovery},
volume = {10},
number = {1},
pages = {OF1},
doi = {10.1158/2159-8290.CD-ND2019-014},
pmid = {31740585},
issn = {2159-8290},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Engineering ; *Genetic Therapy ; *Genome, Human ; Humans ; *Precision Medicine ; RNA, Guide ; },
abstract = {Cancer researchers are heralding the arrival of prime editing, an ultra-precise twist on CRISPR genome engineering, as a powerful new tool for disease modeling, target validation, and clinical applications.},
}
@article {pmid31740584,
year = {2019},
author = {Wang, S and Crisman, L and Miller, J and Datta, I and Gulbranson, DR and Tian, Y and Yin, Q and Yu, H and Shen, J},
title = {Inducible Exoc7/Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {52},
pages = {19988-19996},
pmid = {31740584},
issn = {1083-351X},
support = {R01 DK095367/DK/NIDDK NIH HHS/United States ; T32 GM008759/GM/NIGMS NIH HHS/United States ; R35 GM126960/GM/NIGMS NIH HHS/United States ; R01 DK124431/DK/NIDDK NIH HHS/United States ; R00 AI108793/AI/NIAID NIH HHS/United States ; T32 GM142607/GM/NIGMS NIH HHS/United States ; RF1 AG061829/AG/NIA NIH HHS/United States ; R56 DK095367/DK/NIDDK NIH HHS/United States ; K99 AI108793/AI/NIAID NIH HHS/United States ; },
mesh = {Adipocytes/cytology/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Exocytosis/*drug effects ; Gene Editing ; Glucose Transporter Type 4/*metabolism ; HeLa Cells ; Humans ; Insulin/*pharmacology ; Mice ; RNA, Guide/*metabolism ; Signal Transduction ; Vesicular Transport Proteins/deficiency/*genetics/metabolism ; },
abstract = {Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.},
}
@article {pmid31739790,
year = {2019},
author = {Alexander, J and Findlay, GM and Kircher, M and Shendure, J},
title = {Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement.},
journal = {BMC biology},
volume = {17},
number = {1},
pages = {90},
pmid = {31739790},
issn = {1741-7007},
support = {F30 CA213728/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DP1 HG007811/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Ctenophora/*genetics ; Epigenome/genetics ; Gene Editing/*methods ; Genome/*genetics ; },
abstract = {BACKGROUND: Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ.<br><br>RESULTS: In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event.<br><br>CONCLUSIONS: This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.},
}
@article {pmid31739488,
year = {2019},
author = {Lentsch, E and Li, L and Pfeffer, S and Ekici, AB and Taher, L and Pilarsky, C and Grützmann, R},
title = {CRISPR/Cas9-Mediated Knock-Out of Kras[G12D] Mutated Pancreatic Cancer Cell Lines.},
journal = {International journal of molecular sciences},
volume = {20},
number = {22},
pages = {},
pmid = {31739488},
issn = {1422-0067},
mesh = {Alleles ; Amino Acid Substitution ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Expression Profiling ; *Gene Knockout Techniques ; Gene Targeting ; Humans ; *Mutation ; Pancreatic Neoplasms/*genetics ; Proto-Oncogene Mas ; Proto-Oncogene Proteins p21(ras)/*genetics ; Signal Transduction ; },
abstract = {In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the Kras[G12D] protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the Kras[G12D] mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.},
}
@article {pmid31738765,
year = {2019},
author = {Lutz, S and Brion, C and Kliebhan, M and Albert, FW},
title = {DNA variants affecting the expression of numerous genes in trans have diverse mechanisms of action and evolutionary histories.},
journal = {PLoS genetics},
volume = {15},
number = {11},
pages = {e1008375},
pmid = {31738765},
issn = {1553-7404},
support = {R35 GM124676/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA-Binding Proteins/*genetics ; *Evolution, Molecular ; Fatty Acids/genetics/metabolism ; Gene Expression Regulation, Fungal/genetics ; Green Fluorescent Proteins/genetics ; Lipid Metabolism/genetics ; Monosaccharide Transport Proteins/genetics ; Mutation, Missense/genetics ; Phenotype ; Regulatory Sequences, Nucleic Acid/*genetics ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/*genetics ; Stearoyl-CoA Desaturase/*genetics ; Transcription Factors/*genetics ; },
abstract = {DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression.},
}
@article {pmid31736401,
year = {2019},
author = {Towers, CG and Thorburn, A},
title = {Circumventing autophagy inhibition.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {18},
number = {24},
pages = {3421-3431},
pmid = {31736401},
issn = {1551-4005},
support = {R01 CA150925/CA/NCI NIH HHS/United States ; R01 CA190170/CA/NCI NIH HHS/United States ; T32 CA190216/CA/NCI NIH HHS/United States ; },
mesh = {Autophagy/*genetics ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cell Lineage/genetics ; Clonal Evolution/genetics ; Humans ; NF-E2-Related Factor 2/*genetics ; Neoplasms/*genetics/pathology/therapy ; Signal Transduction/genetics ; },
abstract = {Autophagy is cellular recycling process that plays a complex role in cancer. Pre-clinical studies indicating a pro-tumorigenic role of autophagy have led to the launch of dozens of clinical trials combining autophagy inhibition with other standard of care therapies in different tumor types. A recent publication utilized a novel, acute, CRISPR/Cas9 assay to identify cancer cell lines that are exquisitely sensitive to loss of core autophagy genes within the first 7 days. However, weeks later, rare populations of originally autophagy dependent cells were found that could circumvent autophagy inhibition. Analysis of these rare clones revealed that in the process of circumventing loss of autophagy, the cells upregulated NRF2 signaling to maintain protein homeostasis and consequently become more sensitive to proteasome inhibition as well as knock down of NRF2. This review highlights recent publications regarding the role of autophagy in cancer and potential mechanisms cancer cells may be able to commandeer to circumvent autophagy inhibition. We hope to make significant clinical advances by understanding if and when cancer cells will become resistant to autophagy inhibition, and pre-clinical studies may be able to provide insight into the best combinatorial therapies to prevent tumor relapse while on autophagy inhibitors.},
}
@article {pmid31735642,
year = {2020},
author = {Vink, JNA and Martens, KJA and Vlot, M and McKenzie, RE and Almendros, C and Estrada Bonilla, B and Brocken, DJW and Hohlbein, J and Brouns, SJJ},
title = {Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism.},
journal = {Molecular cell},
volume = {77},
number = {1},
pages = {39-50.e10},
doi = {10.1016/j.molcel.2019.10.021},
pmid = {31735642},
issn = {1097-4164},
support = {639707/ERC_/European Research Council/International ; },
mesh = {Bacteria/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; DNA Replication/genetics ; Gene Dosage/genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.},
}
@article {pmid31734927,
year = {2020},
author = {Nguyen, TH and Mai, HTT and Moukouanga, D and Lebrun, M and Bellafiore, S and Champion, A},
title = {CRISPR/Cas9-Mediated Gene Editing of the Jasmonate Biosynthesis OsAOC Gene in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2085},
number = {},
pages = {199-209},
doi = {10.1007/978-1-0716-0142-6_15},
pmid = {31734927},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cyclopentanes/*metabolism ; *Gene Editing ; Intramolecular Oxidoreductases/*genetics/*metabolism ; Mutagenesis ; Oryza/*genetics/*metabolism ; Oxylipins/*metabolism ; Phenotype ; Plasmids/genetics ; RNA, Guide ; },
abstract = {The function of Jasmonate (JA) is well documented in different plant physiological processes as well as in the interactions with their environment. Mutants impaired in JA production and/or signaling are useful materials to study the function of this phytohormone. Genes involved in the JA biosynthesis pathway in rice have been described, but few mutants deficient in JA production and signaling have been identified. Moreover, these mutants are mostly generated through random mutagenesis approaches, such as irradiation, EMS treatment, or T-DNA insertion, and potentially harbor undesired mutations that could affect other biological processes. The CRISPR/Cas9 system is a precise and efficient genome editing tool that creates DNA modification at specific loci and limit undesired mutations.In this chapter, we describe a procedure to generate new JA-deficient mutant using CRISPR/Cas9 system in rice. The Allene Oxide Cyclase (OsAOC) gene is targeted since it is a single copy gene in the JA biosynthesis pathway in rice. The widely used variety Oryza sativa japonica Kitaake has been chosen due to its short life cycle and its ease of genetic transformation. This protocol describes the selection of the 20-nt target sequence, construction of the binary vector, and strategy for selecting the T-DNA-free mutant.},
}
@article {pmid31734746,
year = {2020},
author = {Haeussler, M},
title = {CRISPR off-targets: a question of context.},
journal = {Cell biology and toxicology},
volume = {36},
number = {1},
pages = {5-9},
pmid = {31734746},
issn = {1573-6822},
support = {U41 HG002371/HG/NHGRI NIH HHS/United States ; },
mesh = {Artifacts ; Base Pair Mismatch/genetics/physiology ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/*methods/trends ; Humans ; },
}
@article {pmid31734133,
year = {2019},
author = {Yang, T and Deng, L and Zhao, W and Zhang, R and Jiang, H and Ye, Z and Li, CB and Li, C},
title = {Rapid breeding of pink-fruited tomato hybrids using the CRISPR/Cas9 system.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {10},
pages = {505-508},
doi = {10.1016/j.jgg.2019.10.002},
pmid = {31734133},
issn = {1673-8527},
mesh = {Breeding ; CRISPR-Cas Systems/genetics ; Fruit/genetics/*metabolism/*physiology ; Lycopersicon esculentum/genetics/*metabolism/*physiology ; },
}
@article {pmid31733872,
year = {2020},
author = {Xiong, ZQ and Wei, YY and Kong, LH and Song, X and Yi, HX and Ai, LZ},
title = {Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.},
journal = {Journal of dairy science},
volume = {103},
number = {1},
pages = {161-165},
doi = {10.3168/jds.2019-17346},
pmid = {31733872},
issn = {1525-3198},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Targeting ; Lactobacillales/enzymology/*genetics ; Lactococcus lactis/enzymology/*genetics ; Nisin/metabolism ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; },
abstract = {Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.},
}
@article {pmid31733438,
year = {2019},
author = {Überbacher, C and Obergasteiger, J and Volta, M and Venezia, S and Müller, S and Pesce, I and Pizzi, S and Lamonaca, G and Picard, A and Cattelan, G and Malpeli, G and Zoli, M and Beccano-Kelly, D and Flynn, R and Wade-Martins, R and Pramstaller, PP and Hicks, AA and Cowley, SA and Corti, C},
title = {Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101656},
pmid = {31733438},
issn = {1876-7753},
support = {MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; WTISSF121302/WT_/Wellcome Trust/United Kingdom ; J-0901/PUK_/Parkinson's UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Dopaminergic Neurons/cytology/*metabolism ; *Gene Editing ; *Gene Knock-In Techniques ; Green Fluorescent Proteins/*biosynthesis/genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Microscopy, Fluorescence ; *Polymerase Chain Reaction ; *Transgenes ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.},
}
@article {pmid31731968,
year = {2020},
author = {Lee, SS and Park, J and Heo, YB and Woo, HM},
title = {Case study of xylose conversion to glycolate in Corynebacterium glutamicum: Current limitation and future perspective of the CRISPR-Cas systems.},
journal = {Enzyme and microbial technology},
volume = {132},
number = {},
pages = {109395},
doi = {10.1016/j.enzmictec.2019.109395},
pmid = {31731968},
issn = {1879-0909},
mesh = {*CRISPR-Cas Systems ; Case-Control Studies ; Corynebacterium glutamicum/genetics/*metabolism ; *Gene Editing ; Glycolates/*metabolism ; Metabolic Engineering/*methods ; RNA, Guide ; Xylose/*metabolism ; },
abstract = {RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance the production of value-added chemicals in Corynebacterium glutamicum as an industrial host. Here, we described the biotransformation of xylose to glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic pathway involving heterologous D-tagatose 3-epimerase and L-fuculose kinase/aldolase reactions was introduced in C. glutamicum, resulting in 9.9 ± 0.01 g/L glycolate from 20 g/L xylose at a yield of 0.51 g/g (equal to 1.0 mol/mol). Additional glyoxylate reduction pathway developed by CRISPR-Cas12a recombineering has been introduced and attempted to increase the maximum theoretical molar yield of 2.0 (mol/mol). Due to the limitation of the CRISPR-Cas12a recombineering with TTTV PAM sites, advanced CRISPR-Cas systems were suggested for the next-round metabolic engineering for improving the glycolate yield to overcome the current genome-editing tool for metabolic engineering in C. glutamicum.},
}
@article {pmid31731183,
year = {2019},
author = {Benetó, N and Cozar, M and García-Morant, M and Creus-Bachiller, E and Vilageliu, L and Grinberg, D and Canals, I},
title = {Generation of two compound heterozygous HGSNAT-mutated lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo C syndrome.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101616},
doi = {10.1016/j.scr.2019.101616},
pmid = {31731183},
issn = {1876-7753},
mesh = {Acetyltransferases/*genetics ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Male ; *Models, Biological ; Mucopolysaccharidosis III/*pathology ; Mutation/*genetics ; Reproducibility of Results ; },
}
@article {pmid31730647,
year = {2019},
author = {Snedeker, J and Gibbons, WJ and Paulding, DF and Abdelhamed, Z and Prows, DR and Stottmann, RW},
title = {Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.},
journal = {PLoS genetics},
volume = {15},
number = {11},
pages = {e1008467},
pmid = {31730647},
issn = {1553-7404},
support = {R01 GM112744/GM/NIGMS NIH HHS/United States ; R35 GM131875/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Cilia/genetics ; Embryo, Mammalian ; Embryonic Development/*genetics ; Genotype ; Humans ; Mice ; Mice, Inbred C57BL ; Microcephaly/*genetics/physiopathology ; Prosencephalon/growth &amp; development/metabolism ; Quantitative Trait Loci/*genetics ; Receptors, G-Protein-Coupled/*genetics ; Spina Bifida Cystica/genetics/physiopathology ; Synthetic Lethal Mutations/genetics ; },
abstract = {The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.},
}
@article {pmid31730408,
year = {2020},
author = {Schoger, E and Carroll, KJ and Iyer, LM and McAnally, JR and Tan, W and Liu, N and Noack, C and Shomroni, O and Salinas, G and Groß, J and Herzog, N and Doroudgar, S and Bassel-Duby, R and Zimmermann, WH and Zelarayán, LC},
title = {CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart.},
journal = {Circulation research},
volume = {126},
number = {1},
pages = {6-24},
doi = {10.1161/CIRCRESAHA.118.314522},
pmid = {31730408},
issn = {1524-4571},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Dependovirus/genetics ; Fibroblasts/metabolism ; *Gene Expression Regulation/genetics ; Genes, Synthetic ; Genetic Vectors/genetics ; Heart/growth &amp; development ; Kruppel-Like Transcription Factors/biosynthesis/genetics ; MEF2 Transcription Factors/biosynthesis/genetics ; Mice ; Mice, Transgenic ; Myocardium/*metabolism ; Myocytes, Cardiac/metabolism ; Myosin Heavy Chains/genetics ; Promoter Regions, Genetic ; Protein Domains ; RNA Polymerase III/genetics ; RNA, Guide/genetics ; *Transcriptional Activation ; },
abstract = {RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far.<br><br>OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription.<br><br>METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects.<br><br>CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.},
}
@article {pmid31730297,
year = {2020},
author = {Liu, Q and Zhang, H and Huang, X},
title = {Anti-CRISPR proteins targeting the CRISPR-Cas system enrich the toolkit for genetic engineering.},
journal = {The FEBS journal},
volume = {287},
number = {4},
pages = {626-644},
doi = {10.1111/febs.15139},
pmid = {31730297},
issn = {1742-4658},
mesh = {Archaea/*genetics/immunology/virology ; Bacteria/*genetics/immunology/virology ; Bacteriophages/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Evolution, Molecular ; Gene Editing/*methods ; Genetic Engineering/*methods ; Humans ; RNA, Guide/genetics/immunology ; Repressor Proteins/*genetics/metabolism ; Viral Proteins/*genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune defense systems, which are widely distributed in bacteria and Archaea, can provide sequence-specific protection against foreign DNA or RNA in some cases. However, the evolution of defense systems in bacterial hosts did not lead to the elimination of phages, and some phages carry anti-CRISPR genes that encode products that bind to the components mediating the defense mechanism and thus antagonize CRISPR-Cas immune systems of bacteria. Given the extensive application of CRISPR-Cas9 technologies in gene editing, in this review, we focus on the anti-CRISPR proteins (Acrs) that inhibit CRISPR-Cas systems for gene editing. We describe the discovery of Acrs in immune systems involving type I, II, and V CRISPR-Cas immunity, discuss the potential function of Acrs in inactivating type II and V CRISPR-Cas systems for gene editing and gene modulation, and provide an outlook on the development of important biotechnology tools for genetic engineering using Acrs.},
}
@article {pmid31730252,
year = {2020},
author = {Li, S and Zhang, Y and Xia, L and Qi, Y},
title = {CRISPR-Cas12a enables efficient biallelic gene targeting in rice.},
journal = {Plant biotechnology journal},
volume = {18},
number = {6},
pages = {1351-1353},
pmid = {31730252},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene Targeting ; *Oryza/genetics ; },
}
@article {pmid31729871,
year = {2019},
author = {Liu, J and Wu, T and Lu, X and Wu, X and Liu, S and Zhao, S and Xu, X and Ding, B},
title = {A Self-Assembled Platform Based on Branched DNA for sgRNA/Cas9/Antisense Delivery.},
journal = {Journal of the American Chemical Society},
volume = {141},
number = {48},
pages = {19032-19037},
doi = {10.1021/jacs.9b09043},
pmid = {31729871},
issn = {1520-5126},
mesh = {Animals ; Breast Neoplasms/genetics/*therapy ; *CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; DNA/*chemistry ; Female ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; MCF-7 Cells ; Mice ; Mice, Inbred BALB C ; Nanostructures/chemistry ; Protein Serine-Threonine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; RNA, Antisense/*administration &amp; dosage/genetics/therapeutic use ; RNA, Guide/*administration &amp; dosage/genetics/therapeutic use ; },
abstract = {Precisely assembled DNA nanostructures are promising candidates for the delivery of biomolecule-based therapeutics. Herein, we introduce a facile strategy for the construction of a branched DNA-based nanoplatform for codelivery of gene editing (sgRNA/Cas9, targeting DNA in the nucleus) and gene silencing (antisense, targeting mRNA in the cytoplasm) components for synergistic tumor therapy in vitro and in vivo. In our design, the branched DNA structure can efficiently load a sgRNA/Cas9/antisense complex targeting a tumor-associated gene, PLK1, through DNA self-assembly. With the incorporation of an active targeting aptamer and an endosomal escape peptide by host-guest interaction, the biocompatible DNA nanoplatform demonstrates efficient inhibition of tumor growth without apparent systemic toxicity. This multifunctional DNA nanocarrier provides a new strategy for the development of gene therapeutics.},
}
@article {pmid31729437,
year = {2019},
author = {Datsomor, AK and Olsen, RE and Zic, N and Madaro, A and Bones, AM and Edvardsen, RB and Wargelius, A and Winge, P},
title = {CRISPR/Cas9-mediated editing of Δ5 and Δ6 desaturases impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar L.).},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16888},
pmid = {31729437},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Docosahexaenoic Acids/biosynthesis ; Fatty Acid Desaturases/*genetics ; Fatty Acids, Omega-3/biosynthesis ; Fatty Acids, Unsaturated/*biosynthesis ; Gene Editing/*methods ; Lipogenesis/*genetics ; Metabolic Engineering/methods/veterinary ; Mutagenesis/physiology ; Mutation ; *Salmo salar/genetics/growth &amp; development/metabolism ; },
abstract = {The in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Δ6abc/5[Mt] with mutations in Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2, and Δ6bc[Mt] with mutations in Δ6fads2-b and Δ6fads2-c. F0 fish displaying high degree of gene editing (50-100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Δ6abc/5[Mt] showed reduced 22:6n-3 levels and accumulated Δ6-desaturation substrates (18:2n-6, 18:3n-3) and Δ5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Δ6bc[Mt] showed no effect on Δ6-desaturation compared to WT, suggesting Δ6 Fads2-a as having the predominant Δ6-desaturation activity in salmon, at least in the tissues analyzed. Both Δ6abc/5[Mt] and Δ6bc[Mt] demonstrated significant accumulation of Δ8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Δ6abc/5[Mt] demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Δ8-desaturation activities for salmon Δ6 Fads2 enzymes.},
}
@article {pmid31729390,
year = {2019},
author = {Medvedeva, S and Liu, Y and Koonin, EV and Severinov, K and Prangishvili, D and Krupovic, M},
title = {Virus-borne mini-CRISPR arrays are involved in interviral conflicts.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5204},
pmid = {31729390},
issn = {2041-1723},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaea/virology ; Archaeal Viruses/classification/*genetics/isolation &amp; purification ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Evolution, Molecular ; Genome, Viral ; Phylogeny ; },
abstract = {CRISPR-Cas immunity is at the forefront of antivirus defense in bacteria and archaea and specifically targets viruses carrying protospacers matching the spacers catalogued in the CRISPR arrays. Here, we perform deep sequencing of the CRISPRome-all spacers contained in a microbiome-associated with hyperthermophilic archaea of the order Sulfolobales recovered directly from an environmental sample and from enrichment cultures established in the laboratory. The 25 million CRISPR spacers sequenced from a single sampling site dwarf the diversity of spacers from all available Sulfolobales isolates and display complex temporal dynamics. Comparison of closely related virus strains shows that CRISPR targeting drives virus genome evolution. Furthermore, we show that some archaeal viruses carry mini-CRISPR arrays with 1-2 spacers and preceded by leader sequences but devoid of cas genes. Closely related viruses present in the same population carry spacers against each other. Targeting by these virus-borne spacers represents a distinct mechanism of heterotypic superinfection exclusion and appears to promote archaeal virus speciation.},
}
@article {pmid31728703,
year = {2020},
author = {Liu, W and Rudis, MR and Cheplick, MH and Millwood, RJ and Yang, JP and Ondzighi-Assoume, CA and Montgomery, GA and Burris, KP and Mazarei, M and Chesnut, JD and Stewart, CN},
title = {Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.},
journal = {Plant cell reports},
volume = {39},
number = {2},
pages = {245-257},
pmid = {31728703},
issn = {1432-203X},
mesh = {Agrobacterium ; Biolistics/methods ; *CRISPR-Cas Systems ; Cell Line ; DNA ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genome, Plant ; Mutagenesis ; Plant Cells/*metabolism ; Plants, Genetically Modified ; Protoplasts ; RNA, Guide/*metabolism ; Ribonucleoproteins/*genetics/*metabolism ; Tobacco/genetics ; },
abstract = {A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.},
}
@article {pmid31727983,
year = {2019},
author = {Chin, CL and Goh, JB and Srinivasan, H and Liu, KI and Gowher, A and Shanmugam, R and Lim, HL and Choo, M and Tang, WQ and Tan, AH and Nguyen-Khuong, T and Tan, MH and Ng, SK},
title = {A human expression system based on HEK293 for the stable production of recombinant erythropoietin.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16768},
pmid = {31727983},
issn = {2045-2322},
mesh = {Batch Cell Culture Techniques/*methods ; CRISPR-Cas Systems ; Erythropoietin/*genetics/*metabolism ; Gene Expression ; Gene Knockout Techniques ; Genetic Vectors/genetics ; Glutamate-Ammonia Ligase/*genetics ; Glycosylation ; HEK293 Cells ; Humans ; Models, Biological ; Protein Engineering/*methods ; Recombinant Proteins/metabolism ; },
abstract = {Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.},
}
@article {pmid31727959,
year = {2019},
author = {Chen, H and Shi, M and Gilam, A and Zheng, Q and Zhang, Y and Afrikanova, I and Li, J and Gluzman, Z and Jiang, R and Kong, LJ and Chen-Tsai, RY},
title = {Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16838},
pmid = {31727959},
issn = {2045-2322},
mesh = {Albumins/genetics ; Animals ; CRISPR-Cas Systems ; Dependovirus/*genetics ; Disease Models, Animal ; Factor VIII/chemistry/*genetics ; Gene Editing/*methods ; Genetic Therapy ; Genetic Vectors/administration &amp; dosage ; Hemophilia A/genetics/*therapy ; Humans ; Mice ; Mice, Inbred C57BL ; Protein Domains ; Treatment Outcome ; },
abstract = {Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.},
}
@article {pmid31727877,
year = {2019},
author = {Xue, J and Zhong, S and Sun, BM and Sun, QF and Hu, LY and Pan, SJ},
title = {Lnc-THOR silencing inhibits human glioma cell survival by activating MAGEA6-AMPK signaling.},
journal = {Cell death &amp; disease},
volume = {10},
number = {11},
pages = {866},
pmid = {31727877},
issn = {2041-4889},
mesh = {AMP-Activated Protein Kinase Kinases ; Animals ; Antigens, Neoplasm/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Cell Survival/genetics ; Gene Expression Regulation, Neoplastic ; Glioma/*genetics/pathology ; Humans ; Mice ; Neoplasm Proteins/*genetics ; Protein Kinases/*genetics ; RNA, Long Noncoding/*genetics ; RNA, Small Interfering ; RNA-Binding Proteins/genetics ; Signal Transduction/genetics ; },
abstract = {Long non-coding RNA THOR (Lnc-THOR) binds to IGF2BP1, essential for its function. We here show that Lnc-THOR is expressed in human glioma tissues and cells. Its expression is extremely low or even undetected in normal brain tissues, as well as in human neuronal cells and astrocytes. We show that Lnc-THOR directly binds to IGF2BP1 in established and primary human glioma cells. shRNA-mediated Lnc-THOR knockdown or CRISPR/Cas9-induced Lnc-THOR knockout potently inhibited cell survival and proliferation, while provoking glioma cell apoptosis. Contrarily, forced overexpression of Lnc-THOR promoted glioma cell growth and migration. Importantly, Lnc-THOR shRNA or knockout activated MAGEA6-AMPK signaling in glioma cells. AMPK inactivation, by AMPKα1 shRNA, knockout, or dominant-negative mutation (T172A), attenuated Lnc-THOR shRNA-induced A172 glioma cell apoptosis. Moreover, CRISPR/Cas9-induced IGF2BP1 knockout activated MAGEA6-AMPK signaling as well, causing A172 glioma cell apoptosis. Significantly, Lnc-THOR shRNA was ineffective in IGF2BP1 KO A172 cells. In vivo, Lnc-THOR silencing or knockout potently inhibited subcutaneous A172 xenograft tumor growth in mice. MAGEA6 downregulation and AMPK activation were detected in Lnc-THOR-silenced/-KO A172 tumor tissues. Taken together, Lnc-THOR depletion inhibits human glioma cell survival possibly by activating MAGEA6-AMPK signaling.},
}
@article {pmid31727847,
year = {2019},
author = {Fei, T and Li, W and Peng, J and Xiao, T and Chen, CH and Wu, A and Huang, J and Zang, C and Liu, XS and Brown, M},
title = {Deciphering essential cistromes using genome-wide CRISPR screens.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {50},
pages = {25186-25195},
pmid = {31727847},
issn = {1091-6490},
support = {R01 HG008728/HG/NHGRI NIH HHS/United States ; },
mesh = {Binding Sites ; Breast Neoplasms/genetics/metabolism ; CCCTC-Binding Factor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Genome, Human ; Hepatocyte Nuclear Factor 3-alpha/genetics/*metabolism ; Humans ; Male ; Promoter Regions, Genetic ; Prostatic Neoplasms/genetics/metabolism ; *Regulatory Elements, Transcriptional ; },
abstract = {Although millions of transcription factor binding sites, or cistromes, have been identified across the human genome, defining which of these sites is functional in a given condition remains challenging. Using CRISPR/Cas9 knockout screens and gene essentiality or fitness as the readout, we systematically investigated the essentiality of over 10,000 FOXA1 and CTCF binding sites in breast and prostate cancer cells. We found that essential FOXA1 binding sites act as enhancers to orchestrate the expression of nearby essential genes through the binding of lineage-specific transcription factors. In contrast, CRISPR screens of the CTCF cistrome revealed 2 classes of essential binding sites. The first class of essential CTCF binding sites act like FOXA1 sites as enhancers to regulate the expression of nearby essential genes, while a second class of essential CTCF binding sites was identified at topologically associated domain (TAD) boundaries and display distinct characteristics. Using regression methods trained on our screening data and public epigenetic profiles, we developed a model to predict essential cis-elements with high accuracy. The model for FOXA1 essentiality correctly predicts noncoding variants associated with cancer risk and progression. Taken together, CRISPR screens of cis-regulatory elements can define the essential cistrome of a given factor and can inform the development of predictive models of cistrome function.},
}
@article {pmid31727803,
year = {2019},
author = {Doudna, J},
title = {CRISPR's unwanted anniversary.},
journal = {Science (New York, N.Y.)},
volume = {366},
number = {6467},
pages = {777},
doi = {10.1126/science.aba1751},
pmid = {31727803},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human ; Humans ; },
}
@article {pmid31727736,
year = {2019},
author = {McNamar, R and Abu-Adas, Z and Rothblum, K and Knutson, BA and Rothblum, LI},
title = {Conditional depletion of the RNA polymerase I subunit PAF53 reveals that it is essential for mitosis and enables identification of functional domains.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {52},
pages = {19907-19922},
pmid = {31727736},
issn = {1083-351X},
support = {R01 GM069841/GM/NIGMS NIH HHS/United States ; R01 HL077814/HL/NHLBI NIH HHS/United States ; R03 DE027785/DE/NIDCR NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; DNA, Ribosomal/metabolism ; Dimerization ; Helix-Turn-Helix Motifs ; Indoleacetic Acids/metabolism ; Mice ; *Mitosis ; Protein Subunits/chemistry/genetics/metabolism ; RNA Polymerase I/chemistry/genetics/*metabolism ; RNA, Guide/metabolism ; S Phase Cell Cycle Checkpoints ; Saccharomyces cerevisiae/growth &amp; development/*metabolism ; Saccharomyces cerevisiae Proteins/chemistry/metabolism ; Transcription, Genetic ; },
abstract = {Our knowledge of the mechanism of rDNA transcription has benefited from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory (Nogi, Y., Vu, L., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7026-7030 and Nogi, Y., Yano, R., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3962-3966) developed a system in yeast to identify genes essential for ribosome biogenesis. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast. Thus, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an auxin-inducible degron that enables combining a "genetics-like"approach with biochemistry to study mammalian rDNA transcription. We now show that the mammalian orthologue of yeast RPA49, PAF53, is required for rDNA transcription and mitotic growth. We have studied the domains of the protein required for activity. We have found that the C-terminal, DNA-binding domain (tandem-winged helix), the heterodimerization, and the linker domain were essential. Analysis of the linker identified a putative helix-turn-helix (HTH) DNA-binding domain. This HTH constitutes a second DNA-binding domain within PAF53. The HTH of the yeast and mammalian orthologues is essential for function. In summary, we show that an auxin-dependent degron system can be used to rapidly deplete nucleolar proteins in mammalian cells, that PAF53 is necessary for rDNA transcription and cell growth, and that all three PAF53 domains are necessary for its function.},
}
@article {pmid31727474,
year = {2019},
author = {Manghwar, H and Lindsey, K and Zhang, X and Jin, S},
title = {CRISPR/Cas System: Recent Advances and Future Prospects for Genome Editing.},
journal = {Trends in plant science},
volume = {24},
number = {12},
pages = {1102-1125},
doi = {10.1016/j.tplants.2019.09.006},
pmid = {31727474},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural ; *Gene Editing ; },
abstract = {Genome editing (GE) has revolutionized biological research through the new ability to precisely edit the genomes of living organisms. In recent years, various GE tools have been explored for editing simple and complex genomes. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has widely been used in GE due to its high efficiency, ease of use, and accuracy. It can be used to add desirable and remove undesirable alleles simultaneously in a single event. Here, we discuss various applications of CRISPR/Cas9 in a range of important crops, compare it with other GE tools, and review its mechanism, limitations, and future possibilities. Various newly emerging CRISPR/Cas systems, including base editing (BE), xCas9, and Cas12a (Cpf1), are also considered.},
}
@article {pmid31727372,
year = {2020},
author = {Zhang, X and Lin, Y and Wu, Q and Wang, Y and Chen, GQ},
title = {Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering.},
journal = {Trends in biotechnology},
volume = {38},
number = {7},
pages = {689-700},
doi = {10.1016/j.tibtech.2019.10.006},
pmid = {31727372},
issn = {1879-3096},
mesh = {Bacteria/genetics/metabolism ; Binding Sites/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/trends ; *Metabolic Engineering ; Polyhydroxyalkanoates/*biosynthesis/genetics ; Promoter Regions, Genetic/genetics ; Ribosomes/genetics ; *Synthetic Biology ; },
abstract = {Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biology and DNA-editing approaches have been adopted to engineer cells for more efficient PHA production. Recent advances in synthetic biology applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphology engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biology methods and genome-editing tools contribute to controllable PHA molecular weights and compositions, enhanced PHA accumulation, and easy downstream processing.},
}
@article {pmid31727255,
year = {2019},
author = {Liao, C and Slotkowski, RA and Beisel, CL},
title = {CRATES: A one-step assembly method for Class 2 CRISPR arrays.},
journal = {Methods in enzymology},
volume = {629},
number = {},
pages = {493-511},
doi = {10.1016/bs.mie.2019.04.011},
pmid = {31727255},
issn = {1557-7988},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Cleavage ; Escherichia coli/genetics ; Gene Editing/*methods ; Plasmids/*genetics ; },
abstract = {CRISPR-Cas systems naturally rely on CRISPR arrays to achieve immunity against multiple foreign invaders, where these arrays are also being utilized for multiplexed targeting as part of CRISPR technologies. However, CRISPR arrays have proven difficult to synthesize or assemble to-date due to the repetitive DNA repeats in each array. To overcome this barrier, we recently reported a cloning method we term CRATES (CRISPR Assembly through Trimmed Ends of Spacers) for the single-step, efficient generation of large Class 2 CRISPR arrays. CRATES generates CRISPR arrays through assembly of multiple repeat-spacer subunits using defined junction sequences within the trimmed portion of the CRISPR spacers. These arrays can be utilized by single-effector nucleases associated with Class 2 CRISPR-Cas systems, such as Cas9, Cas12a/Cpf1, or Cas13a/C2c2. Here, we describe in detail the steps for generating arrays utilized by Cas9 and Cas12a as well as composite arrays co-utilized by both nucleases. We also generate a representative three-spacer array and demonstrate multiplexed DNA cleavage through plasmid-clearance assays in Escherichia coli. This method is expected to simplify the study of natural CRISPR arrays and facilitate multiplexed targeting with programmable nucleases from Class 2 Cas nucleases across the myriad applications of CRISPR technologies.},
}
@article {pmid31727171,
year = {2019},
author = {Zhang, S and Zhang, F and Chen, Q and Wan, C and Xiong, J and Xu, J},
title = {CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {38},
number = {1},
pages = {467},
pmid = {31727171},
issn = {1756-9966},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Movement/physiology ; Cell Proliferation/physiology ; Female ; Hep G2 Cells ; Heterografts ; Histone-Lysine N-Methyltransferase/genetics/*metabolism ; Histones/genetics/*metabolism ; Humans ; Liver Neoplasms/genetics/*metabolism/pathology ; Male ; Methylation ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/genetics/*metabolism ; Signal Transduction ; Wnt Proteins/genetics/*metabolism ; },
abstract = {BACKGROUND: The NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/β-catenin signaling pathway.<br><br>METHODS: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/&#x3b2;-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo.<br><br>RESULTS: NSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/&#x3b2;-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice.<br><br>CONCLUSION: In conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.},
}
@article {pmid31727131,
year = {2019},
author = {Choi, BD and Yu, X and Castano, AP and Darr, H and Henderson, DB and Bouffard, AA and Larson, RC and Scarfò, I and Bailey, SR and Gerhard, GM and Frigault, MJ and Leick, MB and Schmidts, A and Sagert, JG and Curry, WT and Carter, BS and Maus, MV},
title = {CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma.},
journal = {Journal for immunotherapy of cancer},
volume = {7},
number = {1},
pages = {304},
pmid = {31727131},
issn = {2051-1426},
support = {K12 CA087723/CA/NCI NIH HHS/United States ; T32 CA071345/CA/NCI NIH HHS/United States ; R25 NS065743/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Brain Neoplasms/immunology/*therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; *ErbB Receptors ; Glioblastoma/immunology/*therapy ; Humans ; *Immunotherapy, Adoptive ; Mice ; Programmed Cell Death 1 Receptor/*genetics ; T-Lymphocytes/immunology ; Xenograft Model Antitumor Assays ; },
abstract = {Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.},
}
@article {pmid31727120,
year = {2019},
author = {So, RWL and Chung, SW and Lau, HHC and Watts, JJ and Gaudette, E and Al-Azzawi, ZAM and Bishay, J and Lin, LT and Joung, J and Wang, X and Schmitt-Ulms, G},
title = {Application of CRISPR genetic screens to investigate neurological diseases.},
journal = {Molecular neurodegeneration},
volume = {14},
number = {1},
pages = {41},
pmid = {31727120},
issn = {1750-1326},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Disease Models, Animal ; *Gene Editing ; Genetic Testing/methods ; Humans ; Neurodegenerative Diseases/*genetics ; },
abstract = {The adoption of CRISPR-Cas9 technology for functional genetic screens has been a transformative advance. Due to its modular nature, this technology can be customized to address a myriad of questions. To date, pooled, genome-scale studies have uncovered genes responsible for survival, proliferation, drug resistance, viral susceptibility, and many other functions. The technology has even been applied to the functional interrogation of the non-coding genome. However, applications of this technology to neurological diseases remain scarce. This shortfall motivated the assembly of a review that will hopefully help researchers moving in this direction find their footing. The emphasis here will be on design considerations and concepts underlying this methodology. We will highlight groundbreaking studies in the CRISPR-Cas9 functional genetics field and discuss strengths and limitations of this technology for neurological disease applications. Finally, we will provide practical guidance on navigating the many choices that need to be made when implementing a CRISPR-Cas9 functional genetic screen for the study of neurological diseases.},
}
@article {pmid31726660,
year = {2019},
author = {Zaman, QU and Chu, W and Hao, M and Shi, Y and Sun, M and Sang, SF and Mei, D and Cheng, H and Liu, J and Li, C and Hu, Q},
title = {CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.},
journal = {Biomolecules},
volume = {9},
number = {11},
pages = {},
pmid = {31726660},
issn = {2218-273X},
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/*genetics ; Brassica napus/*genetics/growth &amp; development ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/*genetics ; Fruit/*genetics/growth &amp; development ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Genome, Plant/genetics ; Mutation/genetics ; },
abstract = {Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum-valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.},
}
@article {pmid31726388,
year = {2019},
author = {Lindel, F and Dodt, CR and Weidner, N and Noll, M and Bergemann, F and Behrendt, R and Fischer, S and Dietrich, J and Cartellieri, M and Hamann, MV and Lindemann, D},
title = {TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components.},
journal = {Molecular therapy. Nucleic acids},
volume = {18},
number = {},
pages = {708-726},
pmid = {31726388},
issn = {2162-2531},
abstract = {The adaptation of CRISPR/Cas technology for use in mammals has revolutionized genome engineering. In particular with regard to clinical application, efficient expression of Cas9 within a narrow time frame is highly desirable to minimize the accumulation of off-target editing. We developed an effective, aptamer-independent retroviral delivery system for Cas9 mRNAs that takes advantage of a unique foamy virus (FV) capability: the efficient encapsidation and transfer of non-viral RNAs. This enabled us to create a FV vector toolbox for efficient, transient delivery (TraFo) of CRISPR/Cas9 components into different target tissues. Co-delivery of Cas9 mRNA by TraFo-Cas9 vectors in combination with retroviral, integration-deficient single guide RNA (sgRNA) expression enhanced efficacy and specificity of gene-inactivation compared with CRISPR/Cas9 lentiviral vector systems. Furthermore, separate TraFo-Cas9 delivery allowed the optional inclusion of a repair matrix for efficient gene correction or tagging as well as the addition of fluorescent negative selection markers for easy identification of off-target editing or incorrect repair events. Thus, the TraFo CRISPR toolbox represents an interesting alternative technology for gene inactivation and gene editing.},
}
@article {pmid31726224,
year = {2020},
author = {Creutzburg, SCA and Swartjes, T and van der Oost, J},
title = {Medium-throughput in vitro detection of DNA cleavage by CRISPR-Cas12a.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {27-31},
doi = {10.1016/j.ymeth.2019.11.005},
pmid = {31726224},
issn = {1095-9130},
mesh = {Bacterial Proteins/*metabolism ; Biosensing Techniques/instrumentation/*methods ; Biotin/chemistry/metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/*isolation &amp; purification/metabolism ; DNA Cleavage ; Endodeoxyribonucleases/*metabolism ; Fluorescent Dyes/*chemistry ; Francisella/enzymology ; RNA, Guide/genetics ; Streptavidin/metabolism ; },
abstract = {Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate.},
}
@article {pmid31726032,
year = {2019},
author = {Uribe, RV and van der Helm, E and Misiakou, MA and Lee, SW and Kol, S and Sommer, MOA},
title = {Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.},
journal = {Cell host &amp; microbe},
volume = {26},
number = {5},
pages = {702},
doi = {10.1016/j.chom.2019.09.005},
pmid = {31726032},
issn = {1934-6069},
mesh = {*Bacteria ; *CRISPR-Cas Systems ; Metagenomics ; },
}
@article {pmid31724704,
year = {2019},
author = {Pflueger, C and Swain, T and Lister, R},
title = {Harnessing targeted DNA methylation and demethylation using dCas9.},
journal = {Essays in biochemistry},
volume = {63},
number = {6},
pages = {813-825},
doi = {10.1042/EBC20190029},
pmid = {31724704},
issn = {1744-1358},
mesh = {Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; DNA/*metabolism ; *DNA Demethylation ; *DNA Methylation ; Epigenomics/methods ; Gene Editing/methods ; Humans ; Streptococcus pyogenes/enzymology ; },
abstract = {DNA methylation is an essential DNA modification that plays a crucial role in genome regulation during differentiation and development, and is disrupted in a range of disease states. The recent development of CRISPR/catalytically dead CRISPR/Cas9 (dCas9)-based targeted DNA methylation editing tools has enabled new insights into the roles and functional relevance of this modification, including its importance at regulatory regions and the role of aberrant methylation in various diseases. However, while these tools are advancing our ability to understand and manipulate this regulatory layer of the genome, they still possess a variety of limitations in efficacy, implementation, and targeting specificity. Effective targeted DNA methylation editing will continue to advance our fundamental understanding of the role of this modification in different genomic and cellular contexts, and further improvements may enable more accurate disease modeling and possible future treatments. In this review, we discuss strategies, considerations, and future directions for targeted DNA methylation editing.},
}
@article {pmid31724187,
year = {2020},
author = {Cazzaniga, S and Kim, M and Bellamoli, F and Jeong, J and Lee, S and Perozeni, F and Pompa, A and Jin, E and Ballottari, M},
title = {Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii.},
journal = {Plant, cell &amp; environment},
volume = {43},
number = {2},
pages = {496-509},
pmid = {31724187},
issn = {1365-3040},
support = {679814/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems ; Carrier Proteins/metabolism ; Chlamydomonas reinhardtii/*genetics/*metabolism ; Chlorophyll/analysis ; Gene Editing ; Gene Expression Regulation, Plant ; Light-Harvesting Protein Complexes/genetics/*metabolism ; Mutation ; Photosynthesis/physiology ; Photosystem II Protein Complex/*genetics/*metabolism ; },
abstract = {Photosystems must balance between light harvesting to fuel the photosynthetic process for CO2 fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment-binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR-Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching.},
}
@article {pmid31723604,
year = {2019},
author = {Kim, HK and Kim, Y and Lee, S and Min, S and Bae, JY and Choi, JW and Park, J and Jung, D and Yoon, S and Kim, HH},
title = {SpCas9 activity prediction by DeepSpCas9, a deep learning-based model with high generalization performance.},
journal = {Science advances},
volume = {5},
number = {11},
pages = {eaax9249},
pmid = {31723604},
issn = {2375-2548},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; *Deep Learning ; Gene Editing/methods ; Humans ; Internet ; Mutation ; RNA, Guide/genetics/*metabolism ; Reproducibility of Results ; },
abstract = {We evaluated SpCas9 activities at 12,832 target sequences using a high-throughput approach based on a human cell library containing single-guide RNA-encoding and target sequence pairs. Deep learning-based training on this large dataset of SpCas9-induced indel frequencies led to the development of a SpCas9 activity-predicting model named DeepSpCas9. When tested against independently generated datasets (our own and those published by other groups), DeepSpCas9 showed high generalization performance. DeepSpCas9 is available at http://deepcrispr.info/DeepSpCas9.},
}
@article {pmid31723075,
year = {2020},
author = {Zetsche, B and Abudayyeh, OO and Gootenberg, JS and Scott, DA and Zhang, F},
title = {A Survey of Genome Editing Activity for 16 Cas12a Orthologs.},
journal = {The Keio journal of medicine},
volume = {69},
number = {3},
pages = {59-65},
pmid = {31723075},
issn = {1880-1293},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; F31 NS059160/NS/NINDS NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; Base Pairing ; Base Sequence ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/*genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; INDEL Mutation ; Isoenzymes/genetics/metabolism ; Moraxella/enzymology/genetics ; Nucleic Acid Conformation ; Plasmids/chemistry/metabolism ; RNA, Guide/chemistry/*genetics/metabolism ; Sequence Alignment ; },
abstract = {The class 2 CRISPR-Cas endonuclease Cas12a (previously known as Cpf1) offers several advantages over Cas9, including the ability to process its own array and the requirement for just a single RNA guide. These attributes make Cas12a promising for many genome engineering applications. To further expand the suite of Cas12a tools available, we tested 16 Cas12a orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cas12a), exhibited robust indel formation. We also showed that Mb3Cas12a displays some tolerance for a shortened PAM (TTN versus the canonical Cas12a PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.},
}
@article {pmid31723061,
year = {2019},
author = {Tsai, IC and Adams, KA and Tzeng, JA and Shennib, O and Tan, PL and Katsanis, N},
title = {Genome-wide suppressor screen identifies USP35/USP38 as therapeutic candidates for ciliopathies.},
journal = {JCI insight},
volume = {4},
number = {22},
pages = {},
pmid = {31723061},
issn = {2379-3708},
support = {P50 MH094268/MH/NIMH NIH HHS/United States ; R01 GM121317/GM/NIGMS NIH HHS/United States ; R01 DK072301/DK/NIDDK NIH HHS/United States ; P50 DK096415/DK/NIDDK NIH HHS/United States ; F32 DK094578/DK/NIDDK NIH HHS/United States ; R01 HD042601/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Bardet-Biedl Syndrome/drug therapy/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Cilia/genetics ; Ciliopathies/*genetics ; Endopeptidases/*genetics ; Genetic Techniques ; Humans ; Microtubule-Associated Proteins/genetics ; Phenotype ; Retinal Degeneration/genetics ; Ubiquitin-Specific Proteases/*genetics ; Wnt Signaling Pathway/genetics ; Zebrafish ; Zebrafish Proteins/genetics ; },
abstract = {The ciliopathies are a group of phenotypically overlapping disorders caused by structural or functional defects in the primary cilium. Although disruption of numerous signaling pathways and cellular trafficking events have been implicated in ciliary pathology, treatment options for affected individuals remain limited. Here, we performed a genome-wide RNAi (RNA interference) screen to identify genetic suppressors of BBS4, one of the genes mutated in Bardet-Biedl syndrome (BBS). We discovered 10 genes that, when silenced, ameliorate BBS4-dependent pathology. One of these encodes USP35, a negative regulator of the ubiquitin proteasome system, suggesting that inhibition of a deubiquitinase, and subsequent facilitation of the clearance of signaling components, might ameliorate BBS-relevant phenotypes. Testing of this hypothesis in transient and stable zebrafish genetic models showed this posit to be true; suppression or ablation of usp35 ameliorated hallmark ciliopathy defects including impaired convergent extension (CE), renal tubule convolution, and retinal degeneration with concomitant clearance of effectors such as β-catenin and rhodopsin. Together, our findings reinforce a direct link between proteasome-dependent degradation and ciliopathies and suggest that augmentation of this system might offer a rational path to novel therapeutic modalities.},
}
@article {pmid31723010,
year = {2020},
author = {Xiao, G and He, X and Zhang, S and Liu, Y and Liang, Z and Liu, H and Zhang, J and Ou, M and Cai, S and Lai, W and Zhang, T and Ren, L and Zhang, G},
title = {Cas12a/Guide RNA-Based Platform for Rapid and Accurate Identification of Major Mycobacterium Species.},
journal = {Journal of clinical microbiology},
volume = {58},
number = {2},
pages = {},
pmid = {31723010},
issn = {1098-660X},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; Endodeoxyribonucleases/*genetics ; Humans ; Mycobacterium/*classification/isolation &amp; purification ; Mycobacterium Infections, Nontuberculous/microbiology ; Mycobacterium avium Complex/genetics/isolation &amp; purification ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Nontuberculous Mycobacteria/genetics/isolation &amp; purification ; *RNA Probes ; RNA, Guide/*genetics ; RNA, Ribosomal, 16S/genetics ; Sensitivity and Specificity ; Tuberculosis/*diagnosis/microbiology ; },
abstract = {Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species (Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium fortuitum) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species.},
}
@article {pmid31722988,
year = {2019},
author = {Anderson, W and Thorpe, J and Long, SA and Rawlings, DJ},
title = {Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {203},
number = {12},
pages = {3166-3178},
pmid = {31722988},
issn = {1550-6606},
support = {DP3 DK111802/DK/NIDDK NIH HHS/United States ; TL1 TR002318/TR/NCATS NIH HHS/United States ; },
mesh = {Alleles ; Autoimmunity/*genetics ; Blood Donors ; CD4-Positive T-Lymphocytes/*metabolism ; CRISPR-Cas Systems/*immunology ; Cells, Cultured ; Gene Editing ; Genome-Wide Association Study ; Humans ; Interleukin-2/metabolism ; Phenotype ; Polymorphism, Single Nucleotide ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics/metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22/genetics ; Receptors, Antigen, T-Cell/metabolism ; Signal Transduction/genetics ; Suppressor of Cytokine Signaling 3 Protein/metabolism ; ZAP-70 Protein-Tyrosine Kinase/genetics ; },
abstract = {Risk of autoimmunity is associated with multiple genetic variants. Genome-wide association studies have linked single-nucleotide polymorphisms in the phosphatases PTPN22 (rs2476601) and PTPN2 (rs1893217) to increased risk for multiple autoimmune diseases. Previous mouse studies of loss of function or risk variants in these genes revealed hyperactive T cell responses, whereas studies of human lymphocytes revealed contrasting phenotypes. To better understand this dichotomy, we established a robust gene editing platform to rapidly address the consequences of loss of function of candidate genes in primary human CD4[+] T cells. Using CRISPR/Cas9, we obtained efficient gene disruption (>80%) of target genes encoding proteins involved in Ag and cytokine receptor signaling pathways including PTPN22 and PTPN2 Loss-of-function data in all genes studied correlated with previous data from mouse models. Further analyses of PTPN2 gene-disrupted T cells demonstrated dynamic effects, by which hyperactive IL-2R signaling promoted compensatory transcriptional events, eventually resulting in T cells that were hyporesponsive to IL-2. These results imply that altered phosphatase activity promotes evolving phenotypes based on Ag experience and/or other programming signals. This approach enables the discovery of molecular mechanisms modulating risk of autoimmunity that have been difficult to parse in traditional mouse models or cross-sectional human studies.},
}
@article {pmid31722958,
year = {2020},
author = {Wei, P and Xue, W and Zhao, Y and Ning, G and Wang, J},
title = {CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.},
journal = {Genome research},
volume = {30},
number = {1},
pages = {95-106},
pmid = {31722958},
issn = {1549-5469},
support = {P40 OD010949/OD/NIH HHS/United States ; },
mesh = {*5' Untranslated Regions ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila/*genetics ; Drosophila Proteins/genetics ; *Evolution, Molecular ; *Gene Library ; Humans ; Transcription Factors/genetics ; },
abstract = {Construction of a genome-wide transgenic UAS-cDNA/ORF library in Drosophila based on the binary GAL4/UAS system has been severely hampered by technical difficulties, although genome-wide cDNA or ORF resources of Drosophila, human, and mouse have been publicly available for more than a decade. Here, we developed a new method named CRISPR-based modular assembly (CRISPRmass) for the high-throughput construction of a genome-wide UAS-cDNA/ORF library from publicly available cDNA/ORF resources. Through cleavage of shared vector sequences of cDNA/ORF plasmids by CRISPR/Cas9 and subsequent insertion of UAS modules by Gibson assembly, the procedure of construction of such a library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. Using CRISPRmass, we generated 5551 UAS-cDNA/ORF constructs covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection, and among them, 5518 were generated within 3 mo by three people. Our results show that CRISPRmass allows modulization, simplicity, efficiency, and adaptability in the generation of a genome-wide UAS-cDNA/ORF plasmid library by using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various genome-wide libraries in general and is an alternative to Gateway technology in high-throughput plasmid library editing. Furthermore, the more than 5500 UAS-cDNA/ORF plasmids of Drosophila genes serve as a powerful resource for gain-of-function (GOF) screening in cultured cells and for generation of a transgenic UAS-cDNA/ORF library in Drosophila.},
}
@article {pmid31722267,
year = {2020},
author = {Kumbha, R and Hosny, N and Matson, A and Steinhoff, M and Hering, BJ and Burlak, C},
title = {Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method.},
journal = {Research in veterinary science},
volume = {128},
number = {},
pages = {59-68},
doi = {10.1016/j.rvsc.2019.10.021},
pmid = {31722267},
issn = {1532-2661},
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Galactosyltransferases/deficiency/*metabolism ; Gene Knockout Techniques ; Nuclear Transfer Techniques/*veterinary ; Oocytes/physiology ; *Sus scrofa ; Transplantation, Heterologous/*veterinary ; },
abstract = {Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6%) and blastocyst rates (39 ± 4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1%). The blastocyst rate (39 ± 1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76%) were comparable to those obtained with wild-type embryos (41.1 ± 0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol.},
}
@article {pmid31722192,
year = {2019},
author = {Garcia, B and Lee, J and Edraki, A and Hidalgo-Reyes, Y and Erwood, S and Mir, A and Trost, CN and Seroussi, U and Stanley, SY and Cohn, RD and Claycomb, JM and Sontheimer, EJ and Maxwell, KL and Davidson, AR},
title = {Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.},
journal = {Cell reports},
volume = {29},
number = {7},
pages = {1739-1746.e5},
pmid = {31722192},
issn = {2211-1247},
support = {R01 GM125797/GM/NIGMS NIH HHS/United States ; //Canadian Institutes for Health/International ; },
mesh = {*CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry ; *CRISPR-Cas Systems ; Enzyme Inhibitors/*chemistry ; *Gene Editing ; HEK293 Cells ; Humans ; },
abstract = {CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.},
}
@article {pmid31720743,
year = {2020},
author = {Song, Y and Sui, T and Zhang, Y and Wang, Y and Chen, M and Deng, J and Chai, Z and Lai, L and Li, Z},
title = {Genetic deletion of a short fragment of glucokinase in rabbit by CRISPR/Cas9 leading to hyperglycemia and other typical features seen in MODY-2.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {16},
pages = {3265-3277},
doi = {10.1007/s00018-019-03354-4},
pmid = {31720743},
issn = {1420-9071},
mesh = {Animals ; Blood Glucose/genetics ; CRISPR-Cas Systems/*genetics ; Diabetes Mellitus, Type 2/*genetics ; Gene Deletion ; Glucokinase/*genetics ; Humans ; Hyperglycemia/*genetics ; Mice ; Mice, Knockout ; Mutation/genetics ; Phenotype ; Rabbits ; },
abstract = {Glucokinase (GCK) is a key enzyme in glucose sensing and glycemic regulation. In humans, mutations in the GCK gene cause maturity-onset diabetes of the young 2 (MODY-2), a disease that is characterized by an early-onset and persistent hyperglycemia. It is known that Gck knockout (KO) is lethal in mice with Gck KO mice dying within 2 weeks after birth. Therefore, Gck KO mice are not suitable for preclinical study and have limited suitability to study the pathophysiological role of glucokinase in vivo. Here, we report the generation of a novel rabbit with a non-frameshift mutation of GCK gene (GCK-NFS) by cytoplasm microinjection of Cas9 mRNA and gRNA. These GCK-NFS rabbits showed typical features of MODY-2 including hyperglycemia and glucose intolerance with similar survival rate and weight compared to wild-type (WT) rabbits. The diabetic phenotype including pancreatic and renal dysfunction was also found in the F1-generation rabbits, indicating that the genetic modification is germline transmissible. Treatment of GCK-NFS rabbit with glimepiride successfully reduced the fasting blood glucose drastically and improved its islet function. In conclusion, this novel GCK mutant rabbit generated with the CRISPR/Cas9 system mimics most, if not all, histopathological and functional defects seen in MODY-2 patients such as hyperglycemia and will be a valuable rabbit model for preclinical studies and drug screening for diabetes as well as for studying the pathophysiological role of glucokinase.},
}
@article {pmid31719150,
year = {2019},
author = {Contreras-Baeza, Y and Sandoval, PY and Alarcón, R and Galaz, A and Cortés-Molina, F and Alegría, K and Baeza-Lehnert, F and Arce-Molina, R and Guequén, A and Flores, CA and San Martín, A and Barros, LF},
title = {Monocarboxylate transporter 4 (MCT4) is a high affinity transporter capable of exporting lactate in high-lactate microenvironments.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {52},
pages = {20135-20147},
pmid = {31719150},
issn = {1083-351X},
mesh = {Biological Transport/drug effects ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Diclofenac/pharmacology ; Fluoresceins/chemistry ; Gene Editing ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Kinetics ; Lactic Acid/*metabolism ; Macrophages/cytology/metabolism ; Monocarboxylic Acid Transporters/antagonists &amp; inhibitors/genetics/*metabolism ; Muscle Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Protein Isoforms/antagonists &amp; inhibitors/genetics/metabolism ; Pyruvic Acid/metabolism ; },
abstract = {Monocarboxylate transporter 4 (MCT4) is an H[+]-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mm, the reported Km of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective Km values for lactate uptake were 1.7, 1.2, and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a Km for pyruvate of 4.2 mm, as opposed to >150 mm reported previously. Parallel assays with the pH-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous Km estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related transporters MCT1 and MCT2, endows cells with the ability to export lactate in high-lactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate.},
}
@article {pmid31714905,
year = {2019},
author = {Anderson, ME and Mavica, J and Shackleford, L and Flis, I and Fochler, S and Basu, S and Alphey, L},
title = {CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say.},
journal = {PloS one},
volume = {14},
number = {11},
pages = {e0224857},
pmid = {31714905},
issn = {1932-6203},
support = {BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 200171/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Aedes/genetics/virology ; Amino Acid Sequence ; Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Culex/*genetics/*virology ; Embryo, Nonmammalian/metabolism ; *Gene Editing ; INDEL Mutation/genetics ; Microinjections ; Mosquito Vectors/*genetics/*virology ; West Nile virus/*physiology ; },
abstract = {Culex quinquefasciatus Say is an opportunistic blood feeder with a wide geographic distribution which is also a major vector for a range of diseases of both animals and humans. CRISPR/Cas technologies have been applied to a wide variety of organisms for both applied and basic research purposes. CRISPR/Cas methods open new possibilities for genetic research in non-model organisms of public health importance. In this work we have adapted microinjection techniques commonly used in other mosquito species to Culex quinquefasciatus, and have shown these to be effective at generating homozygous knock-out mutations of a target gene in one generation. This is the first description of the kmo gene and mutant phenotype in this species.},
}
@article {pmid31713669,
year = {2019},
author = {Yu, Z and Lv, H and Wu, Y and Wei, T and Yang, S and Ju, D and Chen, S},
title = {Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {23-24},
pages = {9593-9606},
doi = {10.1007/s00253-019-10192-8},
pmid = {31713669},
issn = {1432-0614},
mesh = {Acetyl-CoA Carboxylase/genetics/metabolism ; Acyl Coenzyme A/metabolism ; Acyl-CoA Dehydrogenases/genetics/metabolism ; Anti-Bacterial Agents/metabolism/pharmacology ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Crotonates/metabolism ; Gene Expression ; Immunosuppressive Agents/metabolism ; Metabolic Engineering/*methods ; Mutagenesis ; Streptomyces/*genetics/*metabolism ; Tacrolimus/*analogs &amp; derivatives/metabolism ; Ultraviolet Rays ; },
abstract = {FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.},
}
@article {pmid31713617,
year = {2020},
author = {Lee, JS and Lee, JY and Song, DW and Bae, HS and Doo, HM and Yu, HS and Lee, KJ and Kim, HK and Hwang, H and Kwak, G and Kim, D and Kim, S and Hong, YB and Lee, JM and Choi, BO},
title = {Targeted PMP22 TATA-box editing by CRISPR/Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice.},
journal = {Nucleic acids research},
volume = {48},
number = {1},
pages = {130-140},
pmid = {31713617},
issn = {1362-4962},
support = {R01 NS094388/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Axons ; CRISPR-Cas Systems ; Charcot-Marie-Tooth Disease/genetics/metabolism/pathology/*therapy ; Chromosome Duplication ; Chromosomes, Human, Pair 17 ; Disease Models, Animal ; Gene Editing/methods ; Humans ; Injections ; Mice ; Molecular Targeted Therapy/*methods ; Myelin Proteins/*genetics/metabolism ; Myelin Sheath/*metabolism/pathology ; Primary Cell Culture ; Promoter Regions, Genetic ; Schwann Cells/*metabolism/pathology ; Sciatic Nerve/metabolism/pathology ; *TATA Box ; },
abstract = {Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.},
}
@article {pmid31712774,
year = {2020},
author = {Chiarella, AM and Butler, KV and Gryder, BE and Lu, D and Wang, TA and Yu, X and Pomella, S and Khan, J and Jin, J and Hathaway, NA},
title = {Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery.},
journal = {Nature biotechnology},
volume = {38},
number = {1},
pages = {50-55},
pmid = {31712774},
issn = {1546-1696},
support = {R01 GM118653/GM/NIGMS NIH HHS/United States ; R01 GM122749/GM/NIGMS NIH HHS/United States ; R01 HD088626/HD/NICHD NIH HHS/United States ; R21 HD077197/HD/NICHD NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/metabolism ; Chromatin/*metabolism ; Epigenesis, Genetic ; *Gene Expression Regulation ; Genome, Human ; HEK293 Cells ; Humans ; RNA, Guide/metabolism ; Time Factors ; Transcription Factors/metabolism ; },
abstract = {Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.},
}
@article {pmid31712408,
year = {2019},
author = {Lewis, JJ and Geltman, RC and Pollak, PC and Rondem, KE and Van Belleghem, SM and Hubisz, MJ and Munn, PR and Zhang, L and Benson, C and Mazo-Vargas, A and Danko, CG and Counterman, BA and Papa, R and Reed, RD},
title = {Parallel evolution of ancient, pleiotropic enhancers underlies butterfly wing pattern mimicry.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {48},
pages = {24174-24183},
pmid = {31712408},
issn = {1091-6490},
mesh = {Adaptation, Physiological/genetics ; Animals ; Butterflies/*physiology ; CRISPR-Cas Systems ; Chimera ; *Enhancer Elements, Genetic ; Evolution, Molecular ; *Genetic Pleiotropy ; Genome, Insect ; Genome-Wide Association Study ; Insect Proteins/genetics ; Phylogeny ; Pigmentation/genetics/*physiology ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid ; Wings, Animal/*physiology ; },
abstract = {Color pattern mimicry in Heliconius butterflies is a classic case study of complex trait adaptation via selection on a few large effect genes. Association studies have linked color pattern variation to a handful of noncoding regions, yet the presumptive cis-regulatory elements (CREs) that control color patterning remain unknown. Here we combine chromatin assays, DNA sequence associations, and genome editing to functionally characterize 5 cis-regulatory elements of the color pattern gene optix We were surprised to find that the cis-regulatory architecture of optix is characterized by pleiotropy and regulatory fragility, where deletion of individual cis-regulatory elements has broad effects on both color pattern and wing vein development. Remarkably, we found orthologous cis-regulatory elements associate with wing pattern convergence of distantly related comimics, suggesting that parallel coevolution of ancestral elements facilitated pattern mimicry. Our results support a model of color pattern evolution in Heliconius where changes to ancient, multifunctional cis-regulatory elements underlie adaptive radiation.},
}
@article {pmid31712307,
year = {2020},
author = {Wu, CH and Adachi, H and De la Concepcion, JC and Castells-Graells, R and Nekrasov, V and Kamoun, S},
title = {NRC4 Gene Cluster Is Not Essential for Bacterial Flagellin-Triggered Immunity.},
journal = {Plant physiology},
volume = {182},
number = {1},
pages = {455-459},
pmid = {31712307},
issn = {1532-2548},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Flagellin/genetics/*metabolism ; Immunity, Innate/genetics/physiology ; Lycopersicon esculentum/genetics/metabolism/microbiology ; Multigene Family/*genetics ; Mutation/genetics ; Plant Immunity/genetics/physiology ; Tobacco/genetics/metabolism/microbiology ; },
abstract = {CRISPR/Cas9-mediated mutation of NRC2, NRC3, and NRC4 genes did not affect bacterial flagellin-triggered immunity.},
}
@article {pmid31712093,
year = {2020},
author = {Peng, Q and Fang, L and Ding, Z and Wang, D and Peng, G and Xiao, S},
title = {Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.},
journal = {Journal of virological methods},
volume = {276},
number = {},
pages = {113772},
pmid = {31712093},
issn = {1879-0984},
mesh = {Animals ; *CRISPR-Cas Systems ; Coronavirus Infections/prevention &amp; control/*virology ; Genome, Viral ; Porcine epidemic diarrhea virus/*genetics ; Recombination, Genetic ; Swine ; Swine Diseases/*virology ; Viral Vaccines/genetics ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.},
}
@article {pmid31711886,
year = {2020},
author = {Gupta, SK and Dixit, S and Dangi, SK and Kaur, G and Mashooq, M and Karthik, K and Sarkar, M and Mahajan, S and Nagaleekar, VK},
title = {Marker-less deletion of cctA gene of Clostridium chauvoei.},
journal = {Anaerobe},
volume = {61},
number = {},
pages = {102116},
doi = {10.1016/j.anaerobe.2019.102116},
pmid = {31711886},
issn = {1095-8274},
mesh = {Animal Diseases/*microbiology/prevention &amp; control ; Animals ; Animals, Domestic ; Anti-Bacterial Agents/pharmacology ; Antibiotic Prophylaxis ; Bacterial Proteins/*genetics ; CRISPR-Cas Systems ; Clostridium Infections/*veterinary ; Clostridium chauvoei/drug effects/*genetics ; *Gene Deletion ; Gene Editing ; Hemolysin Proteins/*genetics ; Hemolysis ; Mutation ; },
abstract = {Clostridium chauvoei causes blackleg disease in domestic animals, especially cattle and sheep. The pathogen produces several toxins including CctA - a hemolysin and protective antigen. Molecular pathogenesis of the disease is poorly understood, possibly due to lack of genetic manipulation tools for C. chauvoei. In the present study, we report the marker-less deletion of cctA gene using the CRISPR-Cas9 system. The C. chauvoei cctA deletion mutant had negligible hemolytic and significantly reduced cytotoxic activities. To the best of our knowledge, this is the first report of genetic manipulation of C. chauvoei. The method we used in this study can be applied for genetic manipulation of C. chauvoei to better understand the pathogenesis and genetics of the pathogen.},
}
@article {pmid31711197,
year = {2020},
author = {Herrera-Carrillo, E and Gao, Z and Berkhout, B},
title = {CRISPR therapy towards an HIV cure.},
journal = {Briefings in functional genomics},
volume = {19},
number = {3},
pages = {201-208},
pmid = {31711197},
issn = {2041-2657},
support = {R01 AI145045/AI/NIAID NIH HHS/United States ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; HIV Infections/*genetics ; Humans ; Lentivirus/genetics ; },
abstract = {Tools based on RNA interference (RNAi) and the recently developed clustered regularly short palindromic repeats (CRISPR) system enable the selective modification of gene expression, which also makes them attractive therapeutic reagents for combating HIV infection and other infectious diseases. Several parallels can be drawn between the RNAi and CRISPR-Cas9 platforms. An ideal RNAi or CRISPR-Cas9 therapeutic strategy for treating infectious or genetic diseases should exhibit potency, high specificity and safety. However, therapeutic applications of RNAi and CRISPR-Cas9 have been challenged by several major limitations, some of which can be overcome by optimal design of the therapy or the design of improved reagents. In this review, we will discuss some advantages and limitations of anti-HIV strategies based on RNAi and CRISPR-Cas9 with a focus on the efficiency, specificity, off-target effects and delivery methods.},
}
@article {pmid31710422,
year = {2019},
author = {Paix, A and Rasoloson, D and Folkmann, A and Seydoux, G},
title = {Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double-Stranded DNA Donors.},
journal = {Current protocols in molecular biology},
volume = {129},
number = {1},
pages = {e102},
pmid = {31710422},
issn = {1934-3647},
support = {F32 GM117814/GM/NIGMS NIH HHS/United States ; R24 OD023041/OD/NIH HHS/United States ; R37 HD037047/HD/NICHD NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded/*genetics ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; Humans ; Proteins/genetics ; },
abstract = {Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology-directed repair of DNA breaks induced by the Cas9 RNA-guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30-40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9-crRNA-tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors.},
}
@article {pmid31710047,
year = {2019},
author = {Constantinou, SJ and Nguyen, L and Kirschbaum, F and Salazar, VL and Gallant, JR},
title = {Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {152},
pages = {},
doi = {10.3791/60253},
pmid = {31710047},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Electric Fish/embryology/*genetics/growth &amp; development ; *Gene Editing ; *Gene Expression Regulation ; Genome ; Genomics/*methods ; Mutagenesis ; Phenotype ; Sodium Channels/*chemistry/*genetics ; },
abstract = {Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.},
}
@article {pmid31709747,
year = {2019},
author = {},
title = {New Gene Editing Research Hopes to Eliminate Genetically Related Male Infertility.},
journal = {American journal of medical genetics. Part A},
volume = {179},
number = {12},
pages = {2325-2326},
doi = {10.1002/ajmg.a.61374},
pmid = {31709747},
issn = {1552-4833},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; Humans ; Infertility, Male/*genetics/*therapy ; Male ; Research ; },
}
@article {pmid31709036,
year = {2019},
author = {Tang, L and Yang, F and He, X and Xie, H and Liu, X and Fu, J and Xi, H and Lu, X and Liu, C and Song, Z and Qu, J and Zhao, J and Gu, F},
title = {Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.},
journal = {Cell regeneration (London, England)},
volume = {8},
number = {2},
pages = {44-50},
pmid = {31709036},
issn = {2045-9769},
abstract = {Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.},
}
@article {pmid31708910,
year = {2019},
author = {Li, Y and Peng, N},
title = {Endogenous CRISPR-Cas System-Based Genome Editing and Antimicrobials: Review and Prospects.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2471},
pmid = {31708910},
issn = {1664-302X},
abstract = {CRISPR-Cas systems adapt "memories" via spacers from viruses and plasmids to develop adaptive immunity against mobile genetic elements. Mature CRISPR RNAs guide CRISPR-associated nucleases to site-specifically cleave target DNA or RNA, providing an efficient genome engineering tool for organisms of all three kingdoms. Cas9, Cas12, and Cas13 are single proteins with multiple domains that are the most widely used CRISPR nucleases of the Class 2 system. However, these CRISPR endonucleases are large in size, leading to difficulty for manipulation and toxicity for cells. Most archaeal genomes and half of the bacterial genomes encode different types of CRISPR-Cas systems. Therefore, developing endogenous CRISPR-Cas systems-based genome editing will simplify manipulations and increase editing efficiency in prokaryotic cells. Here, we review the current applications and discuss the prospects of using endogenous CRISPR nucleases for genome engineering and CRISPR-based antimicrobials.},
}
@article {pmid31708478,
year = {2019},
author = {Matjusaitis, M and Wagstaff, LJ and Martella, A and Baranowski, B and Blin, C and Gogolok, S and Williams, A and Pollard, SM},
title = {Reprogramming of Fibroblasts to Oligodendrocyte Progenitor-like Cells Using CRISPR/Cas9-Based Synthetic Transcription Factors.},
journal = {Stem cell reports},
volume = {13},
number = {6},
pages = {1053-1067},
pmid = {31708478},
issn = {2213-6711},
support = {MR/T015594/1/MRC_/Medical Research Council/United Kingdom ; MR/P016022/1/MRC_/Medical Research Council/United Kingdom ; MR/S035915/1/MRC_/Medical Research Council/United Kingdom ; BB/M018040/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/K017047/1/MRC_/Medical Research Council/United Kingdom ; A17368/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Animals ; Biomarkers ; *CRISPR-Cas Systems ; Cellular Reprogramming/*genetics ; Fibroblasts/*cytology/*metabolism ; Gene Editing ; Gene Expression ; Mice ; Oligodendrocyte Precursor Cells/*cytology/*metabolism ; Oligodendroglia/cytology/metabolism ; RNA, Guide ; Transcription Factors/*genetics/metabolism ; Transcriptional Activation ; },
abstract = {Cell lineage reprogramming via transgene overexpression of key master regulatory transcription factors has been well documented. However, the poor efficiency and lack of fidelity of this approach is problematic. Synthetic transcription factors (sTFs)-built from the repurposed CRISPR/Cas9 system-can activate endogenous target genes to direct differentiation or trigger lineage reprogramming. Here we explored whether sTFs could be used to steer mouse neural stem cells and mouse embryonic fibroblasts toward the oligodendrocyte lineage. We developed a non-viral modular expression system to enable stable multiplex delivery of pools of sTFs capable of transcriptional activation of three key oligodendrocyte lineage master regulatory genes (Sox10, Olig2, and Nkx6-2). Delivery of these sTFs could enhance neural stem cell differentiation and initiated mouse embryonic fibroblast direct reprograming toward oligodendrocyte progenitor-like cells. Our findings demonstrate the value of sTFs as tools for activating endogenous genes and directing mammalian cell-type identity.},
}
@article {pmid31708433,
year = {2019},
author = {Hoshijima, K and Jurynec, MJ and Klatt Shaw, D and Jacobi, AM and Behlke, MA and Grunwald, DJ},
title = {Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish.},
journal = {Developmental cell},
volume = {51},
number = {5},
pages = {645-657.e4},
pmid = {31708433},
issn = {1878-1551},
support = {P30 CA042014/CA/NCI NIH HHS/United States ; R01 HD081950/HD/NICHD NIH HHS/United States ; T32 GM007464/GM/NIGMS NIH HHS/United States ; T32 HD007491/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; *Gene Deletion ; Gene Editing/*methods ; Loss of Function Mutation ; Zebrafish/*genetics ; },
abstract = {Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.},
}
@article {pmid31707443,
year = {2019},
author = {Dong, Z and Long, J and Huang, L and Hu, Z and Chen, P and Hu, N and Zheng, N and Huang, X and Lu, C and Pan, M},
title = {Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {23-24},
pages = {9583-9592},
doi = {10.1007/s00253-019-10135-3},
pmid = {31707443},
issn = {1432-0614},
mesh = {Animals ; Animals, Genetically Modified/genetics/microbiology/psychology ; Bombyx/*genetics/*microbiology ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Fungal Proteins/genetics ; *Gene Editing ; Gene Expression ; HSP70 Heat-Shock Proteins/*genetics ; Insect Proteins/*genetics ; Larva/genetics/microbiology ; Nosema/genetics/*physiology ; Promoter Regions, Genetic ; Survival Rate ; },
abstract = {The microsporidian Nosema bombycis is an obligate intracellular parasitic fungus that causes devastating disease in sericulture. To date, no efficient biotechnological method to inhibit the proliferation of microspores has been established. Here, we developed a powerful genetic engineering technique involving microsporidia-inducible genome editing in transgenic silkworm that confers resistance to N. bombycis. This system includes an HSP70 promoter-induced expression of the Cas9 protein line and a target BmATAD3A gene line. The double-positive HSP70-Cas9(+)×sgATAD3A(+) lines were obtained by hybridization and activation of the CRISPR/Cas9 system under the condition of microsporidia infection, although it is silenced in uninfected individuals. Genome editing analysis showed that the system could efficiently edit the BmATAD3A gene and induce large deletions. It is notable that the HSP70-induced system could effectively improve the survival rate of transgenic silkworm after microsporidia infection and inhibit the expression of key microsporidia genes. Moreover, no significant developmental differences between the transgenic silkworms infected with microsporidia and normal individuals were observed. In this study, we effectively inhibited microsporidia proliferation in transgenic individuals through disruptive techniques, thereby providing a method for microsporidia treatment and prevention, paving the way for economically advantageous insect breeding.},
}
@article {pmid31707298,
year = {2020},
author = {Zoppo, M and Di Luca, M and Franco, M and Rizzato, C and Lupetti, A and Stringaro, A and De Bernardis, F and Schaudinn, C and Barrasa, MI and Bottai, D and Vyas, VK and Tavanti, A},
title = {CpALS4770 and CpALS4780 contribution to the virulence of Candida parapsilosis.},
journal = {Microbiological research},
volume = {231},
number = {},
pages = {126351},
doi = {10.1016/j.micres.2019.126351},
pmid = {31707298},
issn = {1618-0623},
mesh = {Animals ; Biofilms/growth &amp; development ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Candida parapsilosis/genetics/pathogenicity ; Candidiasis ; Cell Adhesion/*genetics ; Cell Culture Techniques ; Female ; Fungal Proteins/genetics ; Gene Silencing ; Genes, Fungal ; Humans ; Mice ; Mucous Membrane/microbiology ; Virulence/*genetics ; },
abstract = {The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.},
}
@article {pmid31706693,
year = {2020},
author = {Antonacci, A and Scognamiglio, V},
title = {Biotechnological Advances in the Design of Algae-Based Biosensors.},
journal = {Trends in biotechnology},
volume = {38},
number = {3},
pages = {334-347},
doi = {10.1016/j.tibtech.2019.10.005},
pmid = {31706693},
issn = {1879-3096},
mesh = {Atrazine/toxicity ; Biosensing Techniques/*instrumentation/*methods ; Biotechnology/instrumentation/methods ; CRISPR-Cas Systems ; Chlorophyta/*physiology ; Environmental Monitoring ; Equipment Design ; Gene Editing ; Lab-On-A-Chip Devices ; Microfluidic Analytical Techniques/*instrumentation ; Nanostructures/chemistry ; Photosynthesis ; Synechocystis/genetics ; },
abstract = {In addition to their use in biomass production and bioremediation, algae have been extensively exploited in biosensing applications. Algae-based biosensors have demonstrated potential for sensitive, sustainable, and multiplexed detection of analytes of agroenvironmental and security interest. Their advantages include the availability of different algal bioreceptors including whole cells and their photosynthetic subcomponents, their potential to be integrated into dual transduction miniaturized devices, and the opportunity for continuous environmental monitoring. Despite obstacles including limited stability and selectivity, algae-based biosensing is a realistic prospect that has some recent effective applications. Strategic exploitation of cutting-edge technologies including materials science, nanotechnology, microfluidics, and genome editing will help to achieve the full potential of algae-based sensors.},
}
@article {pmid31706628,
year = {2019},
author = {Liu, XS and Jaenisch, R},
title = {Editing the Epigenome to Tackle Brain Disorders.},
journal = {Trends in neurosciences},
volume = {42},
number = {12},
pages = {861-870},
doi = {10.1016/j.tins.2019.10.003},
pmid = {31706628},
issn = {1878-108X},
support = {R01 MH104610/MH/NIMH NIH HHS/United States ; R01 NS088538/NS/NINDS NIH HHS/United States ; R01 GM123511/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Brain Diseases/*genetics ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Epigenomics/*methods ; Gene Editing/*methods ; Humans ; },
abstract = {Genetic studies of epigenetic modifiers such as DNA methyltransferases and histone acetyltransferases have revealed a critical role for epigenetic regulation during brain development and function. Alteration of epigenetic modifications have been documented in a variety of brain disorders, including neurodevelopmental, psychiatric, and neurodegenerative diseases. Development of epigenome editing tools enables a functional dissection of the link between altered epigenetic changes and disease outcomes. Here, we review the development of epigenome editing tools, summarize proof of concept applications focusing on brain disease-associated genes, and discuss the promising application and challenges of epigenome editing to tackle brain disorders.},
}
@article {pmid31704085,
year = {2020},
author = {Pomeroy, EJ and Hunzeker, JT and Kluesner, MG and Lahr, WS and Smeester, BA and Crosby, MR and Lonetree, CL and Yamamoto, K and Bendzick, L and Miller, JS and Geller, MA and Walcheck, B and Felices, M and Webber, BR and Starr, TK and Moriarity, BS},
title = {A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {1},
pages = {52-63},
pmid = {31704085},
issn = {1525-0024},
support = {R01 CA203348/CA/NCI NIH HHS/United States ; R21 CA216652/CA/NCI NIH HHS/United States ; T32 AI007313/AI/NIAID NIH HHS/United States ; U54 CA232561/CA/NCI NIH HHS/United States ; },
mesh = {ADAM17 Protein/genetics ; Adoptive Transfer/*methods ; Animals ; CRISPR-Cas Systems ; Cell Engineering/*methods ; Cytotoxicity, Immunologic/genetics ; Dependovirus ; Female ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Healthy Volunteers ; Humans ; K562 Cells ; Killer Cells, Natural/*immunology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Ovarian Neoplasms/pathology/*therapy ; Parvovirinae/genetics ; Programmed Cell Death 1 Receptor/genetics ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.},
}
@article {pmid31703769,
year = {2019},
author = {Gentner, B and Naldini, L},
title = {In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease.},
journal = {Cell stem cell},
volume = {25},
number = {5},
pages = {592-593},
doi = {10.1016/j.stem.2019.10.004},
pmid = {31703769},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; DNA Breaks ; *Fanconi Anemia ; Genetic Therapy ; Genetic Vectors ; *Hematopoietic Stem Cell Transplantation ; Humans ; Lentivirus/genetics ; Mutation ; },
abstract = {Two recent papers (one by Román-Rodríguez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance.},
}
@article {pmid31703569,
year = {2019},
author = {Zheng, G and Zhu, Q and Dong, J and Lin, X and Zhu, C},
title = {Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {74},
pmid = {31703569},
issn = {1472-6750},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Gene Editing ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mutation/genetics ; Tumor Suppressor Protein p53/genetics/*metabolism ; },
abstract = {BACKGROUND: Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes.<br><br>RESULTS: Here, we generated TRP53-R172P mutant mice (C57BL/6&#x2009;J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.<br><br>CONCLUSIONS: A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.},
}
@article {pmid31702097,
year = {2020},
author = {Niu, Q and Wu, S and Li, Y and Yang, X and Liu, P and Xu, Y and Lang, Z},
title = {Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.},
journal = {Journal of integrative plant biology},
volume = {62},
number = {4},
pages = {398-402},
doi = {10.1111/jipb.12886},
pmid = {31702097},
issn = {1744-7909},
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genome, Plant ; Lycopersicon esculentum/*genetics ; Protoplasts/metabolism ; },
abstract = {The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.},
}
@article {pmid31701460,
year = {2020},
author = {Perretta-Tejedor, N and Freke, G and Seda, M and Long, DA and Jenkins, D},
title = {Generating Mutant Renal Cell Lines Using CRISPR Technologies.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2067},
number = {},
pages = {323-340},
pmid = {31701460},
issn = {1940-6029},
support = {MR/P018629/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Separation/methods ; Cloning, Molecular/methods ; Flow Cytometry/methods ; Gene Editing/*methods ; HEK293 Cells ; Humans ; INDEL Mutation ; RNA, Guide ; },
abstract = {Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles ("indels") at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.},
}
@article {pmid31701407,
year = {2019},
author = {Hong, W and Huang, M and Wei, Y and Wei, X},
title = {A new and promising application of gene editing: CRISPR-controlled smart materials for tissue engineering, bioelectronics, and diagnostics.},
journal = {Science China. Life sciences},
volume = {62},
number = {11},
pages = {1547-1549},
doi = {10.1007/s11427-019-1576-0},
pmid = {31701407},
issn = {1869-1889},
mesh = {Base Sequence ; Biocatalysis ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Diagnostic Techniques and Procedures ; Electronics ; Gene Editing/*methods ; Gene Expression ; Smart Materials/*metabolism ; Tissue Engineering ; Transfection ; },
}
@article {pmid31700063,
year = {2019},
author = {Wang, X and Williams, D and Müller, I and Lemieux, M and Dukart, R and Maia, IBL and Wang, H and Woerman, AL and Schmitt-Ulms, G},
title = {Tau interactome analyses in CRISPR-Cas9 engineered neuronal cells reveal ATPase-dependent binding of wild-type but not P301L Tau to non-muscle myosins.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16238},
pmid = {31700063},
issn = {2045-2322},
support = {137651//CIHR/Canada ; },
mesh = {Adenosine Triphosphatases/*metabolism ; Astrocytes/cytology ; CRISPR-Cas Systems/*genetics ; *Cell Engineering ; Coculture Techniques ; Humans ; *Mutation ; Myosins/metabolism ; Neurons/cytology/*metabolism ; Protein Binding ; *Protein Interaction Mapping ; tau Proteins/genetics/*metabolism ; },
abstract = {Protein interactions of Tau are of interest in efforts to decipher pathogenesis in Alzheimer's disease, a subset of frontotemporal dementias, and other tauopathies. We CRISPR-Cas9 edited two human cell lines to generate broadly adaptable models for neurodegeneration research. We applied the system to inducibly express balanced levels of 3-repeat and 4-repeat wild-type or P301L mutant Tau. Following 12-h induction, quantitative mass spectrometry revealed the Parkinson's disease-causing protein DJ-1 and non-muscle myosins as Tau interactors whose binding to Tau was profoundly influenced by the presence or absence of the P301L mutation. The presence of wild-type Tau stabilized non-muscle myosins at higher steady-state levels. Strikingly, in human differentiated co-cultures of neuronal and glial cells, the preferential interaction of non-muscle myosins to wild-type Tau depended on myosin ATPase activity. Consistently, transgenic P301L Tau mice exhibited reduced phosphorylation of regulatory myosin light chains known to activate this ATPase. The direct link of Tau to non-muscle myosins corroborates independently proposed roles of Tau in maintaining dendritic spines and mitochondrial fission biology, two subcellular niches affected early in tauopathies.},
}
@article {pmid31699974,
year = {2019},
author = {Contadini, C and Monteonofrio, L and Virdia, I and Prodosmo, A and Valente, D and Chessa, L and Musio, A and Fava, LL and Rinaldo, C and Di Rocco, G and Soddu, S},
title = {p53 mitotic centrosome localization preserves centrosome integrity and works as sensor for the mitotic surveillance pathway.},
journal = {Cell death &amp; disease},
volume = {10},
number = {11},
pages = {850},
pmid = {31699974},
issn = {2041-4889},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Centrosome/*metabolism ; Chromosomes, Human ; Humans ; Mice ; *Mitosis ; Myeloid Cell Leukemia Sequence 1 Protein/genetics/*metabolism ; Tumor Suppressor Protein p53/antagonists &amp; inhibitors/genetics/*metabolism ; Tumor Suppressor p53-Binding Protein 1/genetics/*metabolism ; },
abstract = {Centrosomal p53 has been described for three decades but its role is still unclear. We previously reported that, in proliferating human cells, p53 transiently moves to centrosomes at each mitosis. Such p53 mitotic centrosome localization (p53-MCL) occurs independently from DNA damage but requires ATM-mediated p53Ser15 phosphorylation (p53Ser15[P]) on discrete cytoplasmic p53 foci that, through MT dynamics, move to centrosomes during the mitotic spindle formation. Here, we show that inhibition of p53-MCL, obtained by p53 depletion or selective impairment of p53 centrosomal localization, induces centrosome fragmentation in human nontransformed cells. In contrast, tumor cells or mouse cells tolerate p53 depletion, as expected, and p53-MCL inhibition. Such tumor- and species-specific behavior of centrosomal p53 resembles that of the recently identified sensor of centrosome-loss, whose activation triggers the mitotic surveillance pathway in human nontransformed cells but not in tumor cells or mouse cells. The mitotic surveillance pathway prevents the growth of human cells with increased chance of making mitotic errors and accumulating numeral chromosome defects. Thus, we evaluated whether p53-MCL could work as a centrosome-loss sensor and contribute to the activation of the mitotic surveillance pathway. We provide evidence that centrosome-loss triggered by PLK4 inhibition makes p53 orphan of its mitotic dock and promotes accumulation of discrete p53Ser15[P] foci. These p53 foci are required for the recruitment of 53BP1, a key effector of the mitotic surveillance pathway. Consistently, cells from patients with constitutive impairment of p53-MCL, such as ATM- and PCNT-mutant carriers, accumulate numeral chromosome defects. These findings indicate that, in nontransformed human cells, centrosomal p53 contributes to safeguard genome integrity by working as sensor for the mitotic surveillance pathway.},
}
@article {pmid31699823,
year = {2020},
author = {Xiong, Z and Xia, P and Zhu, X and Geng, J and Wang, S and Ye, B and Qin, X and Qu, Y and He, L and Fan, D and Du, Y and Tian, Y and Fan, Z},
title = {Glutamylation of deubiquitinase BAP1 controls self-renewal of hematopoietic stem cells and hematopoiesis.},
journal = {The Journal of experimental medicine},
volume = {217},
number = {2},
pages = {},
pmid = {31699823},
issn = {1540-9538},
mesh = {Animals ; Bone Marrow Transplantation ; CRISPR-Cas Systems ; Carrier Proteins/metabolism ; Cell Self Renewal/*genetics ; Cells, Cultured ; Female ; Glutamic Acid/*metabolism ; Granzymes/metabolism ; HEK293 Cells ; Hematopoiesis/*genetics ; Hematopoietic Stem Cells/*metabolism ; Homeodomain Proteins/genetics/metabolism ; Humans ; Mice ; Mice, Knockout ; Peptide Synthases/metabolism ; Transcription Factors/genetics/metabolism ; Tumor Suppressor Proteins/genetics/*metabolism ; Ubiquitin Thiolesterase/genetics/*metabolism ; Ubiquitination/*genetics ; },
abstract = {All hematopoietic lineages are derived from a limited pool of hematopoietic stem cells (HSCs). Although the mechanisms underlying HSC self-renewal have been extensively studied, little is known about the role of protein glutamylation and deglutamylation in hematopoiesis. Here, we show that carboxypeptidase CCP3 is most highly expressed in BM cells among CCP members. CCP3 deficiency impairs HSC self-renewal and hematopoiesis. Deubiquitinase BAP1 is a substrate for CCP3 in HSCs. BAP1 is glutamylated at Glu651 by TTLL5 and TTLL7, and BAP1-E651A mutation abrogates BAP1 glutamylation. BAP1 glutamylation accelerates its ubiquitination to trigger its degradation. CCP3 can remove glutamylation of BAP1 to promote its stability, which enhances Hoxa1 expression, leading to HSC self-renewal. Bap1E651A mice produce higher numbers of LT-HSCs and peripheral blood cells. Moreover, TTLL5 and TTLL7 deficiencies sustain BAP1 stability to promote HSC self-renewal and hematopoiesis. Therefore, glutamylation and deglutamylation of BAP1 modulate HSC self-renewal and hematopoiesis.},
}
@article {pmid31699116,
year = {2019},
author = {Chen, H and Zhu, C and Zhu, M and Xiong, J and Ma, H and Zhuo, M and Li, S},
title = {High production of valencene in Saccharomyces cerevisiae through metabolic engineering.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {195},
pmid = {31699116},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems/genetics ; Metabolic Engineering/*methods ; Organisms, Genetically Modified/*metabolism ; Plasmids/genetics ; *Saccharomyces cerevisiae/genetics/metabolism ; Sesquiterpenes/*metabolism ; },
abstract = {BACKGROUND: The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive.<br><br>RESULTS: Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3&#xa0;mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield.<br><br>CONCLUSIONS: This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.},
}
@article {pmid31698466,
year = {2019},
author = {Métais, JY and Doerfler, PA and Mayuranathan, T and Bauer, DE and Fowler, SC and Hsieh, MM and Katta, V and Keriwala, S and Lazzarotto, CR and Luk, K and Neel, MD and Perry, SS and Peters, ST and Porter, SN and Ryu, BY and Sharma, A and Shea, D and Tisdale, JF and Uchida, N and Wolfe, SA and Woodard, KJ and Wu, Y and Yao, Y and Zeng, J and Pruett-Miller, S and Tsai, SQ and Weiss, MJ},
title = {Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.},
journal = {Blood advances},
volume = {3},
number = {21},
pages = {3379-3392},
pmid = {31698466},
issn = {2473-9537},
support = {U01 EB029373/EB/NIBIB NIH HHS/United States ; U01 HL145793/HL/NHLBI NIH HHS/United States ; 2017093/DDCF_/Doris Duke Charitable Foundation/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Disease Models, Animal ; Erythropoiesis/genetics ; Fetal Hemoglobin/*genetics ; *Gene Editing ; Gene Expression Regulation ; Gene Targeting ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; Hemoglobinopathies/genetics ; Heterografts ; Humans ; Immunophenotyping ; Mice ; Models, Biological ; Mutation ; Promoter Regions, Genetic ; RNA, Guide ; Sequence Deletion ; gamma-Globins/*genetics ; },
abstract = {Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and β-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.},
}
@article {pmid31697407,
year = {2020},
author = {Chan, B},
title = {Improving the justice-based argument for conducting human gene editing research to cure sickle cell disease.},
journal = {Bioethics},
volume = {34},
number = {2},
pages = {200-202},
doi = {10.1111/bioe.12690},
pmid = {31697407},
issn = {1467-8519},
mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Social Justice ; },
abstract = {In a recent article, Marilyn Baffoe-Bonnie offers three arguments that conducting CRISPR/Cas9 biotechnology research to cure sickle cell disease (SCD) would help address historical and current injustices in SCD research and care. I will grant that the first argument is sound, but show that the second and third arguments suffer from roughly the same defect, which is that they really argue for something else rather than for conducting CRISPR/Cas9 research to cure SCD. I conclude that a better justice-based argument would use only Baffoe-Bonnie's first argument.},
}
@article {pmid31696609,
year = {2020},
author = {Zeng, D and Li, X and Huang, J and Li, Y and Cai, S and Yu, W and Li, Y and Huang, Y and Xie, X and Gong, Q and Tan, J and Zheng, Z and Guo, M and Liu, YG and Zhu, Q},
title = {Engineered Cas9 variant tools expand targeting scope of genome and base editing in rice.},
journal = {Plant biotechnology journal},
volume = {18},
number = {6},
pages = {1348-1350},
pmid = {31696609},
issn = {1467-7652},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Oryza/genetics ; },
}
@article {pmid31696435,
year = {2020},
author = {Qin, XH and Wang, HX and Ma, L and Shen, J and Liu, QH and Xue, L},
title = {Knockout of the Placenta Specific 8 Gene Affects the Proliferation and Migration of Human Embryonic Kidney 293T Cell.},
journal = {Cell biochemistry and biophysics},
volume = {78},
number = {1},
pages = {55-64},
doi = {10.1007/s12013-019-00893-2},
pmid = {31696435},
issn = {1559-0283},
mesh = {CRISPR-Cas Systems/genetics ; Cadherins/metabolism ; Cell Cycle Proteins/metabolism ; Cell Movement ; *Cell Proliferation ; Cyclin A/agonists/metabolism ; Cyclin B1/antagonists &amp; inhibitors/metabolism ; G2 Phase Cell Cycle Checkpoints ; Gene Editing ; HEK293 Cells ; Humans ; M Phase Cell Cycle Checkpoints ; Proteins/*genetics/metabolism ; },
abstract = {Candidate oncogene placenta specific 8 (PLAC8) has been identified to participate in different cellular process and human diseases. However, the effects of PLAC8 on cell proliferation and migration in human kidney cancer (KC) remained unclear. In current study, physiological effects of PLAC8 in immortalized human embryonic kidney cell line (HEK293T) were investigated in vitro. Two PLAC8 knockout (KO) cell lines were established via CRISPR/Cas9-mediated methods combined with fluorescence activated single cell sorting. To classify the characteristic of PLAC8 during cell proliferation and migration in HEK293T, cellular proliferative activity was analyzed by cell counting and colony formation assay. Cell cycle distribution was analyzed by flow cytometry. Cellular motile activity was analyzed by wound-healing and migration assay. Further underlying molecular mechanism was explored via western blot. With the KO cell lines, it was found that PLAC8 KO could decrease cell proliferation. Moreover, the inhibitory effects of PLAC8 KO on cell proliferation were associated with a G2/M arrest in cell cycle progression concomitant with a remarkable inhibition of Cyclin B1 and elevation of Cyclin A. The alteration of cell cycle proteins and E-cadherin might further associate with the enhancement of cell motility. Our study revealed a novel role for PLAC8 in cell proliferation and migration of HEK293T cells, which might shed light on further study of PLAC8 on human KC.},
}
@article {pmid31696232,
year = {2019},
author = {Davies, B},
title = {The technical risks of human gene editing.},
journal = {Human reproduction (Oxford, England)},
volume = {34},
number = {11},
pages = {2104-2111},
pmid = {31696232},
issn = {1460-2350},
support = {203141/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alleles ; CRISPR-Cas Systems ; Evolution, Molecular ; Female ; Gene Deletion ; Gene Editing/*ethics ; Gene Rearrangement ; Genetic Therapy/*adverse effects ; Genotype ; Humans ; Infant, Newborn ; *Mosaicism ; *Mutagenesis ; *Mutation ; Reproductive Medicine/ethics ; Risk ; },
abstract = {A recent report from Dr He Jiankui concerning the birth of twin girls harbouring mutations engineered by CRISPR/Cas nucleases has been met with international condemnation. Beside the serious ethical concerns, there are known technical risks associated with CRISPR/Cas gene editing which further raise questions about how these events could have been allowed to occur. Numerous studies have reported unexpected genomic mutation and mosaicism following the use of CRISPR/Cas nucleases, and it is currently unclear how prevalent these disadvantageous events are and how robust and sensitive the strategies to detect these unwanted events may be. Although Dr Jiankui's study appears to have involved certain checks to ascertain these risks, the decision to implant the manipulated embryos, given these unknowns, must nonetheless be considered reckless. Here I review the technical concerns surrounding genome editing and consider the available data from Dr Jiankui in this context. Although the data remains unpublished, preventing a thorough assessment of what was performed, it seems clear that the rationale behind the undertaking was seriously flawed; the procedures involved substantial technical risks which, when added to the serious ethical concerns, fully justify the widespread criticism that the events have received.},
}
@article {pmid31695185,
year = {2019},
author = {Gorelik, A and Bartual, SG and Borodkin, VS and Varghese, J and Ferenbach, AT and van Aalten, DMF},
title = {Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {11},
pages = {1071-1077},
pmid = {31695185},
issn = {1545-9985},
support = {105310/WT_/Wellcome Trust/United Kingdom ; 110061/WT_/Wellcome Trust/United Kingdom ; 200208/WT_/Wellcome Trust/United Kingdom ; 200208/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Acetylglucosamine/analogs &amp; derivatives/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Glycosylation ; HEK293 Cells ; Humans ; Mice ; Models, Molecular ; N-Acetylglucosaminyltransferases/chemistry/*metabolism ; Protein Processing, Post-Translational ; Substrate Specificity ; beta-N-Acetylhexosaminidases/chemistry/genetics/*metabolism ; },
abstract = {Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.},
}
@article {pmid31695072,
year = {2019},
author = {Scott, T and Urak, R and Soemardy, C and Morris, KV},
title = {Improved Cas9 activity by specific modifications of the tracrRNA.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16104},
pmid = {31695072},
issn = {2045-2322},
support = {P01 AI099783/AI/NIAID NIH HHS/United States ; R01 AI111139/AI/NIAID NIH HHS/United States ; R01 MH113407/MH/NIMH NIH HHS/United States ; },
mesh = {CD4-Positive T-Lymphocytes/metabolism/virology ; CRISPR-Associated Protein 9/chemistry/*metabolism ; Gene Editing/instrumentation/*methods ; HIV Infections/genetics/metabolism/virology ; HIV-1/genetics/physiology ; Humans ; INDEL Mutation ; RNA, Guide/*genetics/metabolism ; Receptors, CCR5/genetics/metabolism ; Ribonucleoproteins/genetics/*metabolism ; Streptococcus pyogenes/enzymology ; },
abstract = {CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.},
}
@article {pmid31695056,
year = {2019},
author = {Zhou, S and da Silva, SD and Siegel, PM and Philip, A},
title = {CD109 acts as a gatekeeper of the epithelial trait by suppressing epithelial to mesenchymal transition in squamous cell carcinoma cells in vitro.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16317},
pmid = {31695056},
issn = {2045-2322},
support = {PJT148916//CIHR/Canada ; ISO-110833//CIHR/Canada ; },
mesh = {Adult ; Aged ; Aged, 80 and over ; Antigens, CD/*genetics/*metabolism ; CRISPR-Cas Systems ; Carcinoma, Squamous Cell/genetics/metabolism/*pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cell Survival ; Epithelial-Mesenchymal Transition ; Female ; GPI-Linked Proteins/genetics/metabolism ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Mouth Neoplasms/genetics/metabolism/*pathology ; Neoplasm Grading ; Neoplasm Proteins/*genetics/*metabolism ; Oligonucleotide Array Sequence Analysis ; Signal Transduction ; Transforming Growth Factor beta/metabolism ; },
abstract = {There is increasing evidence that the expression of CD109, a GPI-anchored cell surface protein is dysregulated in squamous cell carcinoma (SCC). However, the functional role of CD109 in SCC progression is poorly understood. In current study, we demonstrate that CD109 is a critical regulator of epithelial phenotype in SSC cells. CD109 levels inversely correlate with TGF-β signaling, EMT, migration, and invasion in cultured SCC cells. CRISPR/Cas9-mediated knockout CD109 (CD109 KO) in SCC cells represses epithelial traits and promotes the mesenchymal phenotype, as evidenced by elevated expression of mesenchymal proteins and markers of epithelial to mesenchymal transition. Treatment with recombinant CD109 protein causes CD109 KO cells to regain their epithelial traits. CD109 loss results in pronounced alterations of gene expression as detected by microarray analysis and in dysregulation of 15 important signalling pathways as shown by KEGG pathway cluster analysis. Validation using 52 human oral SCC tumor samples show that CD109 levels inversely correlate with tumor grade and the activation state of one such pathway, the TGF-β signaling pathway. Taken together, our findings highlight a novel role for CD109 as a gatekeeper of the epithelial phenotype by regulating TGF-β pathway in SCC cells.},
}
@article {pmid31694954,
year = {2020},
author = {Henderson, LJ and Reoma, LB and Kovacs, JA and Nath, A},
title = {Advances toward Curing HIV-1 Infection in Tissue Reservoirs.},
journal = {Journal of virology},
volume = {94},
number = {3},
pages = {},
pmid = {31694954},
issn = {1098-5514},
mesh = {Brain ; CRISPR-Cas Systems ; *Disease Reservoirs ; Gene Editing/methods ; Genetic Therapy/methods ; HIV Infections/immunology/*therapy/virology ; HIV-1/genetics/immunology/*physiology ; Humans ; Virus Latency ; },
abstract = {A disease of more than 39.6 million people worldwide, HIV-1 infection has no curative therapy. To date, one man has achieved a sterile cure, with millions more hoping to avoid the potential pitfalls of lifelong antiretroviral therapy and other HIV-related disorders, including neurocognitive decline. Recent developments in immunotherapies and gene therapies provide renewed hope in advancing efforts toward a sterilizing or functional cure. On the horizon is research concentrated in multiple separate but potentially complementary domains: vaccine research, viral transcript editing, T-cell effector response targeting including checkpoint inhibitors, and gene editing. Here, we review the concept of targeting the HIV-1 tissue reservoirs, with an emphasis on the central nervous system, and describe relevant new work in functional cure research and strategies for HIV-1 eradication.},
}
@article {pmid31694550,
year = {2019},
author = {Cram, D and Kulkarni, M and Buchwaldt, M and Rajagopalan, N and Bhowmik, P and Rozwadowski, K and Parkin, IAP and Sharpe, AG and Kagale, S},
title = {WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {474},
pmid = {31694550},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Databases, Genetic ; Gene Editing/*methods ; Gene Targeting ; Genome, Plant ; Internet ; *RNA, Guide ; Triticum/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement.<br><br>DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR.<br><br>CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .},
}
@article {pmid31693906,
year = {2019},
author = {Xu, Z and Li, M and Li, Y and Cao, H and Miao, L and Xu, Z and Higuchi, Y and Yamasaki, S and Nishino, K and Woo, PCY and Xiang, H and Yan, A},
title = {Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa.},
journal = {Cell reports},
volume = {29},
number = {6},
pages = {1707-1717.e3},
doi = {10.1016/j.celrep.2019.10.006},
pmid = {31693906},
issn = {2211-1247},
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacterial Outer Membrane Proteins/*genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Membrane Permeability/drug effects ; DNA Gyrase/genetics/metabolism ; Dipeptides/pharmacology ; Drug Resistance, Multiple, Bacterial/*genetics ; Drug Synergism ; Gene Editing/*methods ; Membrane Transport Proteins/*genetics/metabolism ; Microbial Sensitivity Tests ; Mutation ; Pseudomonas aeruginosa/drug effects/*genetics ; Repressor Proteins/genetics/metabolism ; Up-Regulation ; },
abstract = {Despite being fundamentally important and having direct therapeutic implications, the functional genomics of the clinical isolates of multidrug-resistant (MDR) pathogens is often impeded by the lack of genome-editing tools. Here, we report the establishment of a highly efficient, in situ genome-editing technique applicable in clinical and environmental isolates of the prototypic MDR pathogen P. aeruginosa by harnessing the endogenous type I-F CRISPR-Cas systems. Using this approach, we generate various reverse mutations in an epidemic MDR genotype, PA154197, and identify underlying resistance mechanisms that involve the extensive synergy among three different resistance determinants. Screening a series of "ancestor" mutant lines uncovers the remarkable sensitivity of the MDR line PA154197 to a class of small, cationic peptidomimetics, which sensitize PA154197 cells to antibiotics by perturbing outer-membrane permeability. These studies provide a framework for molecular genetics and anti-resistance drug discovery for clinically isolated MDR pathogens.},
}
@article {pmid31693467,
year = {2019},
author = {Valetdinova, KR and Ovechkina, VS and Zakian, SM},
title = {Methods for Correction of the Single-Nucleotide Substitution c.840C&gt;T in Exon 7 of the SMN2 Gene.},
journal = {Biochemistry. Biokhimiia},
volume = {84},
number = {9},
pages = {1074-1084},
doi = {10.1134/S0006297919090104},
pmid = {31693467},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems/genetics ; Cells, Cultured ; Exons/*genetics ; Humans ; Polymorphism, Single Nucleotide/*genetics ; Survival of Motor Neuron 2 Protein/genetics ; },
abstract = {The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.},
}
@article {pmid31693260,
year = {2020},
author = {Li, M and Li, T and Liu, N and Raban, RR and Wang, X and Akbari, OS},
title = {Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats-associated protein 9.},
journal = {Insect molecular biology},
volume = {29},
number = {2},
pages = {214-220},
doi = {10.1111/imb.12626},
pmid = {31693260},
issn = {1365-2583},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Culex/*genetics/growth &amp; development ; Gene Editing/*methods ; *Germ-Line Mutation ; Insect Proteins/*genetics/metabolism ; Mosquito Vectors/*genetics/growth &amp; development ; Mutagenesis ; },
abstract = {Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats-associated protein 9 (CRISPR-Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR-Cas9-targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.},
}
@article {pmid31693214,
year = {2020},
author = {Khanzadi, MN and Khan, AA},
title = {CRISPR/Cas9: Nature's gift to prokaryotes and an auspicious tool in genome editing.},
journal = {Journal of basic microbiology},
volume = {60},
number = {2},
pages = {91-102},
doi = {10.1002/jobm.201900420},
pmid = {31693214},
issn = {1521-4028},
mesh = {Bacteria/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Molecular Biology/*methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of DNA direct repeats found in many prokaryotic genomes. It was discovered in bacteria as their (adaptive) immune system against invading viruses. Cas9 is an endonuclease enzyme linked with the CRISPR system in bacteria. Bacteria use the Cas9 enzyme to chop viral DNA sequences by unwinding it and then finding the complementary base pairs to the guide RNA. CRISPR/Cas9 is a modern and powerful molecular biology approach that is widely used in genome engineering (to activate/repress gene expression). It can be used in vivo to cause targeted genome modifications with better efficiency as compared to meganucleases, zinc-finger nucleases and transcription activator-like effector nucleases. CRISPR/Cas9 is a simple, reliable, and rapid method for causing gene alterations that open new horizons of gene editing in a variety of living organisms, including humans, for the treatment of several diseases. In this short review, we explored the basic mechanisms underlying its working principles along with some of its current applications in a number of diverse fields.},
}
@article {pmid31693213,
year = {2019},
author = {Trimidal, SG and Benjamin, R and Bae, JE and Han, MV and Kong, E and Singer, A and Williams, TS and Yang, B and Schiller, MR},
title = {Can Designer Indels Be Tailored by Gene Editing?: Can Indels Be Customized?.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {41},
number = {12},
pages = {e1900126},
pmid = {31693213},
issn = {1521-1878},
support = {P20 GM121325/GM/NIGMS NIH HHS/United States ; R21 AI116411/AI/NIAID NIH HHS/United States ; R56 AI109156/AI/NIAID NIH HHS/United States ; R56 AI109156/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Humans ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases/metabolism ; },
abstract = {Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical- or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w.},
}
@article {pmid31692906,
year = {2019},
author = {Jo, DH and Song, DW and Cho, CS and Kim, UG and Lee, KJ and Lee, K and Park, SW and Kim, D and Kim, JH and Kim, JS and Kim, S and Kim, JH and Lee, JM},
title = {CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.},
journal = {Science advances},
volume = {5},
number = {10},
pages = {eaax1210},
pmid = {31692906},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dependovirus/metabolism ; Disease Models, Animal ; *Gene Editing ; Genome ; Leber Congenital Amaurosis/*genetics/*therapy ; Male ; Mice ; Mice, Inbred C57BL ; Mutation/genetics ; Phenotype ; Recombinational DNA Repair ; Retina/pathology/physiopathology ; cis-trans-Isomerases/*genetics/metabolism ; },
abstract = {Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.},
}
@article {pmid31691023,
year = {2020},
author = {Barman, A and Deb, B and Chakraborty, S},
title = {A glance at genome editing with CRISPR-Cas9 technology.},
journal = {Current genetics},
volume = {66},
number = {3},
pages = {447-462},
doi = {10.1007/s00294-019-01040-3},
pmid = {31691023},
issn = {1432-0983},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human ; Humans ; },
abstract = {In recent years, CRISPR-Cas9 technology is widely acknowledged for having major applications in the field of biotechnology for editing genome of any organism to treat a variety of complex diseases and for other purposes. The acronym 'CRISPR-Cas' stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated genes. This genetic organization exists in prokaryotic organisms and aids in the development of adaptive immunity since a protein called Cas9 nuclease cleaves specific target nucleic acid sequences from foreign invaders and destroys them. This mode of action has gained interest of the researchers to understand the insights of CRISPR-Cas9 technology. Here, we review that CRISPR-Cas organization is restricted to two classes and possesses different protein effectors. We also review the architecture of CRISPR loci, mechanism involved in genome editing by CRISPR-Cas9 technology and pathways of repairing double-strand breaks (DSBs) generated during the process of genome editing. This review also presents the strategies to increase the Cas9 specificity and reduce off-target activity to achieve accurate genome editing. Further, this review provides information on CRISPR tools used for genome editing, databases that are required for storing data on loci, strategies for delivering CRISPR-Cas9 to cells under study and applications of CRISPR-Cas9 to various fields. Safety measures are implemented on this technology to avoid misuse or ethical issues. We also discuss about the future aspects and potential applications of CRISPR-Cas9 technology required mainly for the treatment of dreadful diseases, crop improvement as well as genetic improvement in human.},
}
@article {pmid31690888,
year = {2019},
author = {Baron, CS and van Oudenaarden, A},
title = {Unravelling cellular relationships during development and regeneration using genetic lineage tracing.},
journal = {Nature reviews. Molecular cell biology},
volume = {20},
number = {12},
pages = {753-765},
doi = {10.1038/s41580-019-0186-3},
pmid = {31690888},
issn = {1471-0080},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Differentiation/*physiology ; Cell Lineage/*physiology ; Embryonic Development/*physiology ; Gene Expression Regulation, Developmental/*physiology ; High-Throughput Nucleotide Sequencing ; Humans ; Regeneration/*physiology ; },
abstract = {Tracking the progeny of single cells is necessary for building lineage trees that recapitulate processes such as embryonic development and stem cell differentiation. In classical lineage tracing experiments, cells are fluorescently labelled to allow identification by microscopy of a limited number of cell clones. To track a larger number of clones in complex tissues, fluorescent proteins are now replaced by heritable DNA barcodes that are read using next-generation sequencing. In prospective lineage tracing, unique DNA barcodes are introduced into single cells through genetic manipulation (using, for example, Cre-mediated recombination or CRISPR-Cas9-mediated editing) and tracked over time. Alternatively, in retrospective lineage tracing, naturally occurring somatic mutations can be used as endogenous DNA barcodes. Finally, single-cell mRNA-sequencing datasets that capture different cell states within a developmental or differentiation trajectory can be used to recapitulate lineages. In this Review, we discuss methods for prospective or retrospective lineage tracing and demonstrate how trajectory reconstruction algorithms can be applied to single-cell mRNA-sequencing datasets to infer developmental or differentiation tracks. We discuss how these approaches are used to understand cell fate during embryogenesis, cell differentiation and tissue regeneration.},
}
@article {pmid31690864,
year = {2019},
author = {Niemiec, E and Howard, HC},
title = {Include egg donors in CRISPR gene-editing debate.},
journal = {Nature},
volume = {575},
number = {7781},
pages = {51},
doi = {10.1038/d41586-019-03388-5},
pmid = {31690864},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Mutation ; },
}
@article {pmid31690654,
year = {2020},
author = {Zhang, Z and Zhang, X and Lin, Z and Wang, J and Liu, H and Zhou, L and Zhong, S and Li, Y and Zhu, C and Lai, J and Li, X and Yu, J and Lin, Z},
title = {A Large Transposon Insertion in the stiff1 Promoter Increases Stalk Strength in Maize.},
journal = {The Plant cell},
volume = {32},
number = {1},
pages = {152-165},
pmid = {31690654},
issn = {1532-298X},
mesh = {Alleles ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Chromosome Mapping ; DNA Transposable Elements/*genetics ; Genes, Plant ; Lignin/metabolism ; Phenotype ; Plant Proteins/genetics/metabolism ; *Promoter Regions, Genetic ; Quantitative Trait Loci ; Sequence Analysis ; Transformation, Genetic ; Zea mays/*genetics ; },
abstract = {Stalk lodging, which is generally determined by stalk strength, results in considerable yield loss and has become a primary threat to maize (Zea mays) yield under high-density planting. However, the molecular genetic basis of maize stalk strength remains unclear, and improvement methods remain inefficient. Here, we combined map-based cloning and association mapping and identified the gene stiff1 underlying a major quantitative trait locus for stalk strength in maize. A 27.2-kb transposable element insertion was present in the promoter of the stiff1 gene, which encodes an F-box domain protein. This transposable element insertion repressed the transcription of stiff1, leading to the increased cellulose and lignin contents in the cell wall and consequently greater stalk strength. Furthermore, a precisely edited allele of stiff1 generated through the CRISPR/Cas9 system resulted in plants with a stronger stalk than the unedited control. Nucleotide diversity analysis revealed that the promoter of stiff1 was under strong selection in the maize stiff-stalk group. Our cloning of stiff1 reveals a case in which a transposable element played an important role in maize improvement. The identification of stiff1 and our edited stiff1 allele pave the way for efficient improvement of maize stalk strength.},
}
@article {pmid31690629,
year = {2020},
author = {Lauer, V and Grampp, S and Platt, J and Lafleur, V and Lombardi, O and Choudhry, H and Kranz, F and Hartmann, A and Wullich, B and Yamamoto, A and Coleman, ML and Ratcliffe, PJ and Mole, DR and Schödel, J},
title = {Hypoxia drives glucose transporter 3 expression through hypoxia-inducible transcription factor (HIF)-mediated induction of the long noncoding RNA NICI.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {13},
pages = {4065-4078},
pmid = {31690629},
issn = {1083-351X},
support = {078333/Z/05/Z/WT_/Wellcome Trust/United Kingdom ; MR/N021053/1/MRC_/Medical Research Council/United Kingdom ; FC001501/CRUK_/Cancer Research UK/United Kingdom ; RG/11/1/28684/BHF_/British Heart Foundation/United Kingdom ; WT091857MA/WT_/Wellcome Trust/United Kingdom ; FC001501/WT_/Wellcome Trust/United Kingdom ; FC001501/MRC_/Medical Research Council/United Kingdom ; A416016/CRUK_/Cancer Research UK/United Kingdom ; 088182/Z/09/Z/WT_/Wellcome Trust/United Kingdom ; RP-2015-06-004/DH_/Department of Health/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Carcinoma, Renal Cell/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; DNA-Binding Proteins/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; Glucose Transporter Type 3/*genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/genetics ; RNA, Long Noncoding/*genetics ; Transcriptional Activation/genetics ; Tumor Hypoxia/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/*genetics ; },
abstract = {Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.},
}
@article {pmid31689989,
year = {2019},
author = {Vats, S and Kumawat, S and Kumar, V and Patil, GB and Joshi, T and Sonah, H and Sharma, TR and Deshmukh, R},
title = {Genome Editing in Plants: Exploration of Technological Advancements and Challenges.},
journal = {Cells},
volume = {8},
number = {11},
pages = {},
pmid = {31689989},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Humans ; Plants/*genetics ; RNA, Guide/genetics ; },
abstract = {Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs.},
}
@article {pmid31688941,
year = {2020},
author = {Abdelmoneim, A and Clark, CL and Mukai, M},
title = {Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {173},
number = {2},
pages = {336-346},
pmid = {31688941},
issn = {1096-0929},
support = {K08 ES025260/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Benzhydryl Compounds/pharmacology ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/drug effects/metabolism ; Endocrine Disruptors/pharmacology ; Environmental Monitoring/methods ; Estradiol/pharmacology ; Estrogens/*metabolism/*pharmacology ; Gene Expression Regulation/drug effects ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/genetics/*metabolism ; Larva/metabolism ; Liver/metabolism ; Molecular Sequence Data ; Phenols/pharmacology ; RNA, Messenger/metabolism ; Vitellogenins/genetics/metabolism ; Zebrafish/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; },
abstract = {An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. Time- and cost-effective biological tools to detect and screen these compounds with potential high-throughput capabilities are in ever-growing demand. We generated a knock-in zebrafish transgenic line with enhanced green fluorescent protein (EGFP) driven by the regulatory region upstream of vitellogenin 1 (vtg1), a well-studied biomarker for estrogenic exposure, using CRISPR/Cas9 technology. Exposure to 17β-estradiol (E2: 0-625 nM) starting at 4-h post-fertilization in dechorionated embryos resulted in the significant induction of hepatic EGFP with ≥5 nM E2 as early as 3-days post-fertilization. Concentration- and time-dependent increase in the percentage of hepatic EGFP-positive larvae and extent of fluorescence expression, categorized into 3 expression levels, were observed with E2 exposure. A strong correlation between the levels of EGFP mRNA, vtg1 mRNA, and EGFP fluorescence levels were detected. Image analysis of the area and intensity of hepatic EGFP fluorescence resulted in high-fidelity quantitative measures that could be used in automated screening applications. In addition, exposure to bisphenol A (0-30 μM) resulted in quantitative responses showing promise for the use of this transgenic line to assess estrogenic activity of endocrine-disrupting chemicals. These results demonstrate that this novel knock-in zebrafish reporter allows for distinct screening of in vivo estrogenic effects, endpoints of which can be used for laboratory testing of samples for estimation of possible human and environmental risks.},
}
@article {pmid31687377,
year = {2019},
author = {Kotagama, OW and Jayasinghe, CD and Abeysinghe, T},
title = {Era of Genomic Medicine: A Narrative Review on CRISPR Technology as a Potential Therapeutic Tool for Human Diseases.},
journal = {BioMed research international},
volume = {2019},
number = {},
pages = {1369682},
pmid = {31687377},
issn = {2314-6141},
mesh = {Animals ; Biotechnology/methods ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genome/*genetics ; Genomics/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) provides acquired immunity in microorganisms against exogenous DNA that may hinder the survival of the organism. Pioneering work by Doudna and Charpentier in 2012 resulted in the creation of the CRISPR/Cas9 genome editing tool on the basis of this concept. The aim of this was to create a rapid, efficient, and versatile genome-editing tool to facilitate genetic manipulation. The mechanism relies on two components: the RNA guide which acts as a sentinel and a Cas protein complex which functions as a highly precise molecular knife. The guide RNA can be modified to match a DNA sequence of interest in the cell and accordingly be used to rectify mutations that may otherwise cause disease. Within a few years following the development of the CRISPR/Cas9 tool, its usage has become ubiquitous. Its influence extends into many fields of biological sciences from biotechnology and biochemistry to molecular biology and biomedical sciences. The following review aims at shedding some light on to the applications of the CRISPR/Cas9 tool in the field of biomedical sciences, particularly gene therapy. An insight with relation to a few of the many diseases that are being tackled with the aid of the CRISPR/Cas9 mechanism and the trends, successes, and challenges of this application as a gene therapy are discussed in this review.},
}
@article {pmid31686530,
year = {2020},
author = {Solar Fernandez, V and Cipolletti, M and Ascenzi, P and Marino, M and Fiocchetti, M},
title = {Neuroglobin As Key Mediator in the 17β-Estradiol-Induced Antioxidant Cell Response to Oxidative Stress.},
journal = {Antioxidants &amp; redox signaling},
volume = {32},
number = {4},
pages = {217-227},
doi = {10.1089/ars.2019.7870},
pmid = {31686530},
issn = {1557-7716},
mesh = {Antioxidants/*pharmacology ; Breast Neoplasms/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Estradiol/*pharmacology ; Female ; Humans ; MCF-7 Cells ; NF-E2-Related Factor 2/*metabolism ; Neuroglobin/*genetics/metabolism ; Oxidative Stress/drug effects ; Reactive Oxygen Species/metabolism ; },
abstract = {Aims: Nuclear factor (erythroid-derived 2)-like-2 factor (NRF-2) is a transcription factor well known to provide an advantage for cancer growth and survival regulating the cellular redox pathway. In breast cancer cells, we recently identified the monomeric heme-globin neuroglobin (NGB) as part of a new mechanism induced by the steroid hormone 17β-estradiol (E2) against oxidative stress. While there is mounting evidence suggesting a critical role of NGB as a sensor of oxidative stress, scarce information is available about its involvement in NRF-2 pathway activation in breast cancer cells. Results: Although NGB is not involved in the rapid E2-induced NRF-2 stability, E2 loses the capacity to regulate the expression of NRF-2-dependent genes in NGB-depleted MCF-7 cells. These data strongly sustain a role of NGB as a compensatory protein in the E2-activated intracellular pathway devoted to the increase of cancer cells tolerance to reactive oxygen species (ROS) generation in stressing conditions acting as key regulator of NRF-2 pathway activity in a time-dependent manner. Innovation: In this study, we identified a new role of NGB in the cell response to oxidative stress. Conclusion: Altogether, reported results open new insights on the NGB effect in regulating intracellular pathways related to cell adaptive response to stress and, as consequence, to cell survival, beyond its direct effect as ROS scavenger, opening new prospective in cancer therapeutic intervention.},
}
@article {pmid31686319,
year = {2019},
author = {Li, G and Liu, YG and Chen, Y},
title = {Genome-editing technologies: the gap between application and policy.},
journal = {Science China. Life sciences},
volume = {62},
number = {11},
pages = {1534-1538},
doi = {10.1007/s11427-019-1566-1},
pmid = {31686319},
issn = {1869-1889},
mesh = {Animals ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Gene Knockdown Techniques ; Genetic Therapy/methods ; Humans ; Patient Safety ; Social Control, Formal ; },
}
@article {pmid31685954,
year = {2019},
author = {Zlotorynski, E},
title = {CRISPR-Cas in its prime.},
journal = {Nature reviews. Molecular cell biology},
volume = {20},
number = {12},
pages = {718-719},
doi = {10.1038/s41580-019-0194-3},
pmid = {31685954},
issn = {1471-0080},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; },
}
@article {pmid31685917,
year = {2019},
author = {Noirot-Gros, MF and Forrester, S and Malato, G and Larsen, PE and Noirot, P},
title = {CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {15954},
pmid = {31685917},
issn = {2045-2322},
mesh = {Biofilms/*growth &amp; development ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytokinesis/genetics ; Gene Editing ; *Gene Expression Regulation, Bacterial ; Gene Silencing ; Pseudomonas fluorescens/*genetics/*growth &amp; development/metabolism ; },
abstract = {Bacterial biofilm formation involves signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Gene inactivation is a powerful approach for functional studies but it is often labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in diverse strain isolates of P. fluorescens, SBW25, WH6 and Pf0-1. We found that CRISPRi is applicable to study complex phenotypes such as cell morphology, motility and biofilm formation over extended periods of time. In SBW25, CRISPRi-mediated silencing of genes encoding the GacA/S two-component system and regulatory proteins associated with the cylic di-GMP signaling messenger produced swarming and biofilm phenotypes similar to those obtained after gene inactivation. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 operon. We conclude that CRISPRi is a reliable and scalable approach to investigate gene networks in the diverse P. fluorescens group.},
}
@article {pmid31685795,
year = {2019},
author = {Moon, SB and Kim, DY and Ko, JH and Kim, YS},
title = {Recent advances in the CRISPR genome editing tool set.},
journal = {Experimental &amp; molecular medicine},
volume = {51},
number = {11},
pages = {1-11},
pmid = {31685795},
issn = {2092-6413},
support = {NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/International ; NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/International ; NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/International ; NRF-2016M3A9B6903343//National Research Foundation of Korea (NRF)/International ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/International ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/International ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/International ; CAP-15-03-KRIBB//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Genetic Engineering/methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Genome editing took a dramatic turn with the development of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system. The CRISPR-Cas system is functionally divided into classes 1 and 2 according to the composition of the effector genes. Class 2 consists of a single effector nuclease, and routine practice of genome editing has been achieved by the development of the Class 2 CRISPR-Cas system, which includes the type II, V, and VI CRISPR-Cas systems. Types II and V can be used for DNA editing, while type VI is employed for RNA editing. CRISPR techniques induce both qualitative and quantitative alterations in gene expression via the double-stranded breakage (DSB) repair pathway, base editing, transposase-dependent DNA integration, and gene regulation using the CRISPR-dCas or type VI CRISPR system. Despite significant technical improvements, technical challenges should be further addressed, including insufficient indel and HDR efficiency, off-target activity, the large size of Cas, PAM restrictions, and immune responses. If sophisticatedly refined, CRISPR technology will harness the process of DNA rewriting, which has potential applications in therapeutics, diagnostics, and biotechnology.},
}
@article {pmid31685631,
year = {2019},
author = {Coale, TH and Moosburner, M and Horák, A and Oborník, M and Barbeau, KA and Allen, AE},
title = {Reduction-dependent siderophore assimilation in a model pennate diatom.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {47},
pages = {23609-23617},
pmid = {31685631},
issn = {1091-6490},
mesh = {Bacterial Outer Membrane Proteins/*metabolism ; Biological Availability ; Biological Transport ; CRISPR-Cas Systems ; Climate Change ; Diatoms/genetics/growth &amp; development/*metabolism ; FMN Reductase/genetics/*metabolism ; Gallium/metabolism ; Gene Knockout Techniques ; Iron/*metabolism ; Membrane Transport Proteins/genetics/*metabolism ; Microbiota ; Oxidation-Reduction ; Phylogeny ; Receptors, Cell Surface/*metabolism ; Recombinant Fusion Proteins/metabolism ; Seawater/chemistry ; Siderophores/*metabolism ; Species Specificity ; },
abstract = {Iron uptake by diatoms is a biochemical process with global biogeochemical implications. In large regions of the surface ocean diatoms are both responsible for the majority of primary production and frequently experiencing iron limitation of growth. The strategies used by these phytoplankton to extract iron from seawater constrain carbon flux into higher trophic levels and sequestration into sediments. In this study we use reverse genetic techniques to target putative iron-acquisition genes in the model pennate diatom Phaeodactylum tricornutum We describe components of a reduction-dependent siderophore acquisition pathway that relies on a bacterial-derived receptor protein and provides a viable alternative to inorganic iron uptake under certain conditions. This form of iron uptake entails a close association between diatoms and siderophore-producing organisms during low-iron conditions. Homologs of these proteins are found distributed across diatom lineages, suggesting the significance of siderophore utilization by diatoms in the marine environment. Evaluation of specific proteins enables us to confirm independent iron-acquisition pathways in diatoms and characterize their preferred substrates. These findings refine our mechanistic understanding of the multiple iron-uptake systems used by diatoms and help us better predict the influence of iron speciation on taxa-specific iron bioavailability.},
}
@article {pmid31685617,
year = {2019},
author = {Xu, M and Kiselar, J and Whited, TL and Hernandez-Sanchez, W and Taylor, DJ},
title = {POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {47},
pages = {23527-23533},
pmid = {31685617},
issn = {1091-6490},
support = {P30 EB009998/EB/NIBIB NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; R01 GM133841/GM/NIGMS NIH HHS/United States ; R01 CA240993/CA/NCI NIH HHS/United States ; R01 GM126218/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Substitution ; CRISPR-Cas Systems ; DNA, Single-Stranded/*metabolism ; HCT116 Cells ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics/metabolism ; *Mutation, Missense ; *Point Mutation ; *Polymorphism, Single Nucleotide ; Protein Binding ; Recombinant Proteins/metabolism ; Shelterin Complex ; Telomere/*metabolism ; Telomere Homeostasis/*physiology ; Telomere-Binding Proteins/genetics/*physiology ; },
abstract = {Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein-protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.},
}
@article {pmid31685615,
year = {2019},
author = {Sagai, T and Amano, T and Maeno, A and Ajima, R and Shiroishi, T},
title = {SHH signaling mediated by a prechordal and brain enhancer controls forebrain organization.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {47},
pages = {23636-23642},
pmid = {31685615},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; Eye Proteins/physiology ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Genes, Reporter ; Hedgehog Proteins/biosynthesis/genetics/*physiology ; Holoprosencephaly/genetics ; Homeodomain Proteins/physiology ; Hypothalamus/abnormalities/embryology/metabolism ; Lac Operon ; Mesencephalon/embryology/metabolism ; Mesoderm/metabolism ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins/biosynthesis/deficiency/genetics/*physiology ; Prosencephalon/abnormalities/*embryology/metabolism ; Signal Transduction ; Transgenes ; },
abstract = {Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.},
}
@article {pmid31685521,
year = {2020},
author = {Bosch, JA and Colbeth, R and Zirin, J and Perrimon, N},
title = {Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of Universal Donor Plasmids.},
journal = {Genetics},
volume = {214},
number = {1},
pages = {75-89},
pmid = {31685521},
issn = {1943-2631},
support = {R24 OD019847/OD/NIH HHS/United States ; P41 GM132087/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM067761/GM/NIGMS NIH HHS/United States ; R01 GM084947/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; Drosophila melanogaster/embryology/*genetics ; Embryo, Nonmammalian ; Gene Knock-In Techniques/*methods ; Genome ; Male ; Mutagenesis, Insertional ; Plasmids/genetics ; },
abstract = {Targeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint [clustered regularly interspaced short palindromic repeat (CRISPR)-assisted insertion tagging] homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and nonhomologous end joining to insert "universal" donor plasmids into the genome. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-red fluorescent protein into seven genes in the fly germ line, producing heritable loss-of-function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, insertions/deletions always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Therefore, homology-independent insertion in Drosophila is a fast and simple alternative to HDR that will enable researchers to dissect gene function.},
}
@article {pmid31684940,
year = {2019},
author = {Arndell, T and Sharma, N and Langridge, P and Baumann, U and Watson-Haigh, NS and Whitford, R},
title = {gRNA validation for wheat genome editing with the CRISPR-Cas9 system.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {71},
pmid = {31684940},
issn = {1472-6750},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Protoplasts/metabolism ; RNA, Guide/*genetics ; Triticum/*genetics ; },
abstract = {BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.<br><br>RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (&#x2265;20&#x2009;bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.<br><br>CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.},
}
@article {pmid31684863,
year = {2019},
author = {Dell'Aglio, E and Dalvit, I and Loubéry, S and Fitzpatrick, TB},
title = {Clarification of the dispensability of PDX1.2 for Arabidopsis viability using CRISPR/Cas9.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {464},
pmid = {31684863},
issn = {1471-2229},
mesh = {Arabidopsis/*physiology ; Arabidopsis Proteins/*genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Gene Expression Regulation, Plant/*genetics ; Hot Temperature ; Loss of Function Mutation/*physiology ; *Phenotype ; },
abstract = {BACKGROUND: PDX1.2 has recently been shown to be a regulator of vitamin B6 biosynthesis in plants and is implicated in biotic and abiotic stress resistance. PDX1.2 expression is strongly and rapidly induced by heat stress. Interestingly, PDX1.2 is restricted to eudicota, wherein it behaves as a non-catalytic pseudoenzyme and is suggested to provide an adaptive advantage to this clade. A first report on an Arabidopsis insertion mutant claims that PDX1.2 is indispensable for viability, being essential for embryogenesis. However, a later study using an independent insertion allele suggests that knockout mutants of pdx1.2 are viable. Therefore, the essentiality of PDX1.2 for Arabidopsis viability is a matter of debate. Given the important implications of PDX1.2 in stress responses, it is imperative to clarify if it is essential for plant viability.<br><br>RESULTS: We have studied the previously reported insertion alleles of PDX1.2, one of which is claimed to be essential for embryogenesis (pdx1.2-1), whereas the other is viable (pdx1.2-2). Our study shows that pdx1.2-1 carries multiple T-DNA insertions, but the T-DNA insertion in PDX1.2 is not responsible for the loss of embryogenesis. By contrast, the pdx1.2-2 allele is an overexpressor of PDX1.2 under standard growth conditions and not a null allele as previously reported. Nonetheless, upregulation of PDX1.2 expression under heat stress is impaired in this mutant line. In wild type Arabidopsis, studies of PDX1.2-YFP fusion proteins show that the protein is enhanced under heat stress conditions. To clarify if PDX1.2 is essential for Arabidopsis viability, we generated several independent mutant lines using the CRISPR-Cas9 gene editing technology. All of these lines are viable and behave similar to wild type under standard growth conditions. Reciprocal crosses of a subset of the CRISPR lines with pdx1.2-1 recovers viability of the latter line and demonstrates that knocking out the functionality of PDX1.2 does not impair embryogenesis.<br><br>CONCLUSIONS: Gene editing reveals that PDX1.2 is dispensable for Arabidopsis viability and resolves conflicting reports in the literature on its function.},
}
@article {pmid31683605,
year = {2019},
author = {Kurilovich, E and Shiriaeva, A and Metlitskaya, A and Morozova, N and Ivancic-Bace, I and Severinov, K and Savitskaya, E},
title = {Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System.},
journal = {Genes},
volume = {10},
number = {11},
pages = {},
pmid = {31683605},
issn = {2073-4425},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {*Adaptation, Physiological ; *CRISPR-Cas Systems ; *DNA Repair ; Escherichia coli/*genetics ; Escherichia coli Proteins/genetics/metabolism ; Exodeoxyribonuclease V/genetics/metabolism ; Exodeoxyribonucleases/genetics/metabolism ; Exonucleases/genetics/metabolism ; Genome, Bacterial ; *Genomic Instability ; },
abstract = {Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.},
}
@article {pmid31683589,
year = {2019},
author = {Kostyusheva, A and Brezgin, S and Bayurova, E and Gordeychuk, I and Isaguliants, M and Goptar, I and Urusov, F and Nikiforova, A and Volchkova, E and Kostyushev, D and Chulanov, V},
title = {ATM and ATR Expression Potentiates HBV Replication and Contributes to Reactivation of HBV Infection upon DNA Damage.},
journal = {Viruses},
volume = {11},
number = {11},
pages = {},
pmid = {31683589},
issn = {1999-4915},
mesh = {Ataxia Telangiectasia Mutated Proteins/drug effects/genetics/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; DNA Damage/drug effects ; DNA Repair/drug effects ; Doxorubicin/pharmacology ; Hepatitis B/virology ; Hepatitis B virus/*growth &amp; development ; Hepatitis B, Chronic/*virology ; Humans ; RNA Interference ; RNA, Small Interfering/pharmacology ; Signal Transduction ; Virus Replication/drug effects/genetics ; },
abstract = {Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.},
}
@article {pmid31683099,
year = {2019},
author = {Lee, Y and Choi, HY and Kwon, A and Park, H and Park, MH and Kim, JW and Kim, MJ and Kim, YO and Kwak, S and Koo, SK},
title = {Generation of a PDX1-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-3, using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101632},
doi = {10.1016/j.scr.2019.101632},
pmid = {31683099},
issn = {1876-7753},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line/*cytology ; *Genes, Reporter ; Green Fluorescent Proteins/*genetics ; Homeodomain Proteins/*genetics ; Humans ; Male ; Trans-Activators/*genetics ; },
abstract = {PDX1 plays a crucial role in the development and maintenance of β-cells and directly regulates pancreatic β-cell-specific transcription factors by binding to the insulin gene. Here, we introduced an EGFP reporter into the C-terminus of PDX1 in KSCBi005-A human induced pluripotent stem cells through homologous recombination using CRISPR/Cas9 nuclease. The cells had a normal karyotype, expressed several pluripotency markers, and maintained their differentiation potential. KSCBi005-A-3 cells can be used to monitor PDX1 expression in live cells during β-cell differentiation; the cell line has been registered at the National Stem Cell Bank, Korea National Institute of Health.},
}
@article {pmid31682806,
year = {2020},
author = {Sidik, H and Ang, CJ and Pouladi, MA},
title = {Huntingtin confers fitness but is not embryonically essential in zebrafish development.},
journal = {Developmental biology},
volume = {458},
number = {1},
pages = {98-105},
doi = {10.1016/j.ydbio.2019.10.037},
pmid = {31682806},
issn = {1095-564X},
mesh = {Amino Acid Sequence ; Animals ; Body Size ; CRISPR-Cas Systems ; Conserved Sequence ; Embryo, Nonmammalian/drug effects/embryology ; Gene Editing ; Gene Knockout Techniques ; Genetic Association Studies ; Genetic Fitness ; Humans ; Huntingtin Protein/chemistry ; *Models, Animal ; Morpholinos/pharmacology ; Nerve Tissue Proteins/deficiency/genetics/*physiology ; Neurulation/genetics ; Sequence Alignment ; Sequence Homology, Amino Acid ; Zebrafish/embryology/*genetics/growth &amp; development ; Zebrafish Proteins/deficiency/genetics/*physiology ; },
abstract = {Attempts to constitutively knockout HTT in rodents resulted in embryonic lethality, curtailing efforts to study HTT function later in development. Here we show that HTT is dispensable for early zebrafish development, contrasting published zebrafish morpholino experiment results. Homozygous HTT knockouts were embryonically viable and appeared developmentally normal through juvenile stages. Comparison of adult fish revealed significant reduction in body size and fitness in knockouts compared to hemizygotes and wildtype fish, indicating an important role for wildtype HTT in postnatal development. Our zebrafish model provides an opportunity to understand the function of wildtype HTT later in development.},
}
@article {pmid31682364,
year = {2019},
author = {Stasi, A and Rodrigues, IP},
title = {Dealing with Patent Fragmentation in Genetics: Can Patent Pools Facilitate the Development of CRISPR Gene-Editing Technology?.},
journal = {Journal of law and medicine},
volume = {26},
number = {4},
pages = {866-873},
pmid = {31682364},
issn = {1320-159X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; },
abstract = {The discovery of CRISPR systems has been one of the most exciting developments in the field of genetics in the past decade. The recent proliferation of intellectual property rights for CRISPR genome editing technology carries the risk of potential bottlenecks for further basic biological research and development of commercial products. To make CRISPR-based technology widely available, the reliance by the industry on efficient methods of collective management of intellectual property rights through patent pools seems inevitable. A packager of patent pools could be used as a mechanism to facilitate transactions in the market for technology and allow interested parties to deal with a single entity. This article argues that, while a global licensing platform could be effectively achieved in non-therapeutic applications of genome editing, it is questionable whether patent pooling would provide the ideal balance of incentive and reward for CRISPR genome editing technologies for human gene therapy.},
}
@article {pmid31681950,
year = {2020},
author = {Bergadà-Pijuan, J and Pulido-Quetglas, C and Vancura, A and Johnson, R},
title = {CASPR, an analysis pipeline for single and paired guide RNA CRISPR screens, reveals optimal target selection for long non-coding RNAs.},
journal = {Bioinformatics (Oxford, England)},
volume = {36},
number = {6},
pages = {1673-1680},
doi = {10.1093/bioinformatics/btz811},
pmid = {31681950},
issn = {1367-4811},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA, Guide ; *RNA, Long Noncoding ; Software ; },
abstract = {MOTIVATION: CRISPR-Cas9 loss-of-function (LOF) pooled screening promises to identify which long non-coding RNAs (lncRNAs), amongst the many thousands to have been annotated so far, are capable of mediating cellular functions. The two principal LOF perturbations, CRISPR-inhibition and CRISPR-deletion, employ one and two guide RNAs, respectively. However, no software solution has the versatility to identify hits across both modalities, and the optimal design parameters for such screens remain poorly understood.<br><br>RESULTS: Here, we present CRISPR Analysis for Single and Paired RNA-guides (CASPR), a user-friendly, end-to-end screen analysis tool. CASPR is compatible with both CRISPRi and CRISPR-del screens, and balances sensitivity and specificity by generating consensus predictions from multiple algorithms. Benchmarking on ground-truth sets of cancer-associated lncRNAs demonstrates CASPR's improved sensitivity with respect to existing methods. Applying CASPR to published screens, we identify two parameters that predict lncRNA hits: expression and annotation quality of the transcription start site. Thus, CASPR is a versatile and complete solution for lncRNA CRISPR screen analysis, and reveals principles for including lncRNAs in screening libraries.<br><br>https://judithbergada.github.io/CASPR/.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid31680172,
year = {2019},
author = {Cali, CP and Park, DS and Lee, EB},
title = {Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.},
journal = {Nucleic acids research},
volume = {47},
number = {22},
pages = {11609-11622},
pmid = {31680172},
issn = {1362-4962},
support = {R01 NS095793/NS/NINDS NIH HHS/United States ; },
mesh = {Amyloid beta-Protein Precursor/*genetics ; Amyotrophic Lateral Sclerosis/*genetics ; C9orf72 Protein/*genetics ; CRISPR-Cas Systems ; Cell Line ; DNA Breaks, Double-Stranded ; DNA Damage/genetics ; DNA Methylation/*genetics ; DNA Repair/genetics ; DNA-Cytosine Methylases/*metabolism ; Frontotemporal Dementia/*genetics ; Gene Knockout Techniques ; Genome, Human/genetics ; HEK293 Cells ; Histones/metabolism ; Humans ; RNA, Guide ; },
abstract = {DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.},
}
@article {pmid31679813,
year = {2019},
author = {Ophinni, Y and Palatini, U and Hayashi, Y and Parrish, NF},
title = {piRNA-Guided CRISPR-like Immunity in Eukaryotes.},
journal = {Trends in immunology},
volume = {40},
number = {11},
pages = {998-1010},
doi = {10.1016/j.it.2019.09.003},
pmid = {31679813},
issn = {1471-4981},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Transposable Elements/*genetics ; Drosophila melanogaster ; Endogenous Retroviruses/*genetics ; Epigenesis, Genetic ; Eukaryota ; Gene Transfer, Horizontal ; Germ Cells/*physiology ; Humans ; Immunity/*genetics ; RNA, Small Interfering/*genetics ; },
abstract = {Eukaryotic genomes contain virus-derived sequences called endogenous virus elements (EVEs). The majority of EVEs are related to retroviruses, which integrate into the host genome in order to replicate. Some retroviral EVEs encode a function; for example, some produce proteins that block infection by related viruses. EVEs derived from nonretroviral viruses - also recently found in many eukaryotic genomes - are more enigmatic. Here, we summarize the evidence that EVEs can act as templates to generate Piwi-interacting RNAs (piRNAs), whose canonical function is sequence-specific silencing of transposable elements (TEs) to maintain genomic integrity. We argue that EVEs may thus enable heritable, sequence-specific antiviral immune memory in eukaryotes - analogous to CRISPR-Cas immunity in prokaryotes.},
}
@article {pmid31678357,
year = {2020},
author = {Yang, L and Li, Y and Wu, Y and Sun, S and Song, Q and Wei, J and Sun, L and Li, M and Wang, D and Zhou, L},
title = {Rln3a is a prerequisite for spermatogenesis and fertility in male fish.},
journal = {The Journal of steroid biochemistry and molecular biology},
volume = {197},
number = {},
pages = {105517},
doi = {10.1016/j.jsbmb.2019.105517},
pmid = {31678357},
issn = {1879-1220},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Cichlids/genetics/*growth &amp; development/metabolism ; *Fertility ; Male ; Mutation ; Phylogeny ; Relaxin/antagonists &amp; inhibitors/genetics/*metabolism ; Sequence Homology ; Sperm Motility ; *Spermatogenesis ; Testis/*growth &amp; development/metabolism ; },
abstract = {The essential roles of Relaxin3 (RLN3) in energy homeostasis had been well investigated, while the mechanisms of RLN3 regulating reproduction remain to be elusive in mammals. Although two rln3 paralogues have been characterized in several teleosts, their functions still remain largely unknown. In this study, two paralogous rln3 genes, represented as rln3a and rln3b, were identified from the testis of Nile tilapia (Oreochromis niloticus). Rln3a was dominantly expressed in testis, while the most abundant rln3b expression was in brain. In situ hybridization demonstrated that rln3a is abundantly expressed in the Leydig cells of the testis. To understand the role of Rln3 in the testicular development, homologous null-rln3a gene mutant line was constructed by CRISPR/Cas9 technology. Morphological observation demonstrated that null mutation of rln3a gene caused testicular hypertrophy and a significant increase of GSI. However, a significant decrease of spermatogenic cells at different phases, i.e. spermatogonia, spermatocytes, spermatids and sperms was found. Silencing of rln3a gene repressed the expression of key genes in germ cell and Leydig cell. Deficiency of Rln3a led to the significant decrease of 11-KT production, which stimulated the up-regulation of both FSH and LH production in the pituitary via a negative feedback manner possibly. Mutation of rln3a in XY fish led to the hypogonadism with sperm deformation, significant decrease of fertility, and sperm motility, revealing as the high mortality of the offspring obtained by crossing the wild type female and rln3a[-/-] XY fish. Interestingly, recombinant human RLN3 injection significantly enhanced the sperm motility in rln3a[-/-] XY fish. Moreover, hCG treatment stimulated the expression of steroidogenic enzyme genes and 11-KT production, which were repressed by rln3a mutation in XY fish. Taken together, this study, for the first time by using a gene knockout model, proved that Rln3a is an indispensable mediator for androgen production in testis via HPG axis, and plays an essential role in spermatogenesis, sperm motility and male fertility in fish.},
}
@article {pmid31677525,
year = {2019},
author = {Lorthongpanich, C and Jiamvoraphong, N and Supakun, P and Damkham, N and Terbto, P and Waeteekul, S and U-Pratya, Y and Laowtammathron, C and Issaragrisil, S},
title = {Generation of a WWTR1 mutation induced pluripotent stem cell line, MUSIi012-A-1, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101634},
doi = {10.1016/j.scr.2019.101634},
pmid = {31677525},
issn = {1876-7753},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line/*cytology ; Female ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Mutation/*genetics ; Reproducibility of Results ; Trans-Activators/*genetics ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; },
abstract = {WWTR1 or TAZ (WWTR1/TAZ) is a transcriptional coactivator that acts as a downstream regulatory target in the Hippo signaling pathway, which plays a pivotal role in regulating cell proliferation and anti-apoptosis. It has been shown in other cell types that WWTR1/TAZ plays a redundant role to its homolog YAP1. Using CRISPR/Cas9 gene editing, we established the WWTR1/TAZ-KO cell line, which features homozygous deletion of WWTR1 gene from human iPSCs. The established WWTR1/YAZ-KO cell line maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and normal karyotype.},
}
@article {pmid31677524,
year = {2019},
author = {Angsutararux, P and Luanpitpong, S and Chingsuwanrote, P and Supraditaporn, K and Waeteekul, S and Terbto, P and Lorthongpanich, C and Laowtammathron, C and U-Pratya, Y and Issaragrisil, S},
title = {Generation of human induced pluripotent stem cell line carrying SCN5AC2204&gt;T Brugada mutation (MUSli009-A-1) introduced by CRISPR/Cas9-mediated genome editing.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101618},
doi = {10.1016/j.scr.2019.101618},
pmid = {31677524},
issn = {1876-7753},
mesh = {Base Sequence ; Brugada Syndrome/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line/*pathology ; *Gene Editing ; Humans ; Induced Pluripotent Stem Cells/*pathology ; Male ; Mutation/*genetics ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) derived from dermal fibroblasts having wild type (WT) SCN5A were engineered by CRISPR/Cas9-mediated genome editing to harbor a specific point mutation (C2204>T) in SCN5A, which results in a substitution of the WT alanine by valine at codon 735 (A735V). The established MUSli009-A-1 hiPSC line has a homozygous C2204>T mutation on exon 14 of SCN5A that was confirmed by DNA sequencing analysis. The cells exhibited normal karyotype, expressed pluripotent markers and retained its capability to differentiate into three germ layers. The cardiomyocytes derived from this line would be a useful model for investigating cardiac channelopathy.},
}
@article {pmid31677059,
year = {2020},
author = {Ghogare, R and Williamson-Benavides, B and Ramírez-Torres, F and Dhingra, A},
title = {CRISPR-associated nucleases: the Dawn of a new age of efficient crop improvement.},
journal = {Transgenic research},
volume = {29},
number = {1},
pages = {1-35},
pmid = {31677059},
issn = {1573-9368},
support = {WPN00011//National Institute of Food and Agriculture/International ; },
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Endonucleases/*metabolism ; *Gene Editing ; Genetic Engineering/*methods/trends ; *Genome, Plant ; Plants, Genetically Modified/*genetics ; },
abstract = {The world stands at a new threshold today. As a planet, we face various challenges, and the key one is how to continue to produce enough food, feed, fiber, and fuel to support the burgeoning population. In the past, plant breeding and the ability to genetically engineer crops contributed to increasing food production. However, both approaches rely on random mixing or integration of genes, and the process can be unpredictable and time-consuming. Given the challenge of limited availability of natural resources and changing environmental conditions, the need to rapidly and precisely improve crops has become urgent. The discovery of CRISPR-associated endonucleases offers a precise yet versatile platform for rapid crop improvement. This review summarizes a brief history of the discovery of CRISPR-associated nucleases and their application in genome editing of various plant species. Also provided is an overview of several new endonucleases reported recently, which can be utilized for editing of specific genes in plants through various forms of DNA sequence alteration. Genome editing, with its ever-expanding toolset, increased efficiency, and its potential integration with the emerging synthetic biology approaches hold promise for efficient crop improvement to meet the challenge of supporting the needs of future generations.},
}
@article {pmid31676843,
year = {2020},
author = {Zhen, S and Li, X},
title = {Liposomal delivery of CRISPR/Cas9.},
journal = {Cancer gene therapy},
volume = {27},
number = {7-8},
pages = {515-527},
pmid = {31676843},
issn = {1476-5500},
mesh = {*CRISPR-Cas Systems ; Drug Carriers ; *Drug Delivery Systems ; Gene Editing/*methods ; Gene Silencing ; Humans ; *Liposomes ; Neoplasms/*therapy ; },
abstract = {Liposomes are one of the most widely investigated carriers for CRISPR/Cas9 delivery. The surface properties of liposomal carriers, including the surface charge, PEGylation, and ligand modification can significantly affect the gene silencing efficiency. Three barriers of systemic CRISPR/Cas9 delivery (long blood circulation, efficient tumor penetration, and efficient cellular uptake/endosomal escape) are analyzed on liposomal carriers with different surface charges, PEGylations, and ligand modifications. Cationic formulations dominate CRISPR/Cas9 delivery and neutral formulations also have good performance while anionic formulations are generally not proper for CRISPR/Cas9 delivery. The PEG dilemma (prolonged blood circulation vs. reduced cellular uptake/endosomal escape) and the side effect of repeated PEGylated formulation (accelerated blood clearance) were discussed. Effects of ligand modification on cationic and neutral formulations were analyzed. Finally, we summarized the achievements in liposomal CRISPR/Cas9 delivery, outlined existing problems, and provided some future perspectives. Liposomes are one of the most widely investigated carriers for CRISPR/Cas9 delivery. The surface properties of liposomal carriers, including the surface charge, PEGylation, and ligand modification can significantly affect the gene silencing efficiency. Three barriers of systemic siRNA delivery (long blood circulation, efficient tumor penetration, and efficient cellular uptake/endosomal escape) are analyzed on liposomal carriers with different surface charges, PEGylations, and ligand modifications. Cationic formulations dominate CRISPR/Cas9 delivery and neutral formulations also have good performance while anionic formulations are generally not proper for CRISPR/Cas9 delivery. The PEG dilemma (prolonged blood circulation vs. reduced cellular uptake/endosomal escape) and the side effect of repeated PEGylated formulation (accelerated blood clearance) were discussed. Effects of ligand modification on cationic and neutral formulations were analyzed. Finally, we summarized the achievements in liposomal CRISPR/Cas9 delivery, outlined existing problems, and provided some future perspectives.},
}
@article {pmid31676749,
year = {2019},
author = {Zhang, X and Feng, J and Chen, S and Yang, H and Dong, Z},
title = {Synergized regulation of NK cell education by NKG2A and specific Ly49 family members.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {5010},
pmid = {31676749},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Histocompatibility Antigens Class I/genetics/immunology/metabolism ; Killer Cells, Natural/*immunology/metabolism ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; *Multigene Family ; NK Cell Lectin-Like Receptor Subfamily A/genetics/*immunology/metabolism ; NK Cell Lectin-Like Receptor Subfamily C/genetics/*immunology/metabolism ; },
abstract = {Mice lacking MHC class-I (MHC-I) display severe defects in natural killer (NK) cell functional maturation, a process designated as "education". Whether self-MHC-I specific Ly49 family receptors and NKG2A, which are closely linked within the NK gene complex (NKC) locus, are essential for NK cell education is still unclear. Here we show, using CRISPR/Cas9-mediated gene deletion, that mice lacking all members of the Ly49 family exhibit a moderate defect in NK cell activity, while mice lacking only two inhibitory Ly49 members, Ly49C and Ly49I, have comparable phenotypes. Furthermore, the deficiency of NKG2A, which recognizes non-classical MHC-Ib molecules, mildly impairs NK cell function. Notably, the combined deletion of NKG2A and the Ly49 family severely compromises the ability of NK cells to mediate "missing-self" and "induced-self" recognition. Therefore, our data provide genetic evidence supporting that NKG2A and the inhibitory members of Ly49 family receptors synergize to regulate NK cell education.},
}
@article {pmid31676591,
year = {2019},
author = {Goossens, R and van den Boogaard, ML and Lemmers, RJLF and Balog, J and van der Vliet, PJ and Willemsen, IM and Schouten, J and Maggio, I and van der Stoep, N and Hoeben, RC and Tapscott, SJ and Geijsen, N and Gonçalves, MAFV and Sacconi, S and Tawil, R and van der Maarel, SM},
title = {Intronic SMCHD1 variants in FSHD: testing the potential for CRISPR-Cas9 genome editing.},
journal = {Journal of medical genetics},
volume = {56},
number = {12},
pages = {828-837},
doi = {10.1136/jmedgenet-2019-106402},
pmid = {31676591},
issn = {1468-6244},
mesh = {Adult ; Aged ; Alleles ; CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Chromatin Assembly and Disassembly/genetics ; Chromosomal Proteins, Non-Histone/*genetics ; Chromosomes, Human, Pair 4/genetics ; DNA Methylation/genetics ; Female ; Gene Editing/methods ; Gene Expression/genetics ; Genetic Predisposition to Disease ; Homeodomain Proteins/*genetics ; Humans ; Male ; Middle Aged ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Facioscapulohumeral/*genetics/physiopathology/therapy ; Mutation/genetics ; },
abstract = {BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1.<br><br>METHODS: We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts.<br><br>RESULTS: We identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4.<br><br>CONCLUSIONS: The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.},
}
@article {pmid31673055,
year = {2019},
author = {Ferreira da Silva, J and Salic, S and Wiedner, M and Datlinger, P and Essletzbichler, P and Hanzl, A and Superti-Furga, G and Bock, C and Winter, G and Loizou, JI},
title = {Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {15751},
pmid = {31673055},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Gene Editing/*methods ; Gene Knockout Techniques ; HSP90 Heat-Shock Proteins/genetics ; Humans ; RNA, Guide/metabolism ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.},
}
@article {pmid31672987,
year = {2019},
author = {Liu, Q and Yu, T and Li, X and Chen, Y and Campbell, K and Nielsen, J and Chen, Y},
title = {Rewiring carbon metabolism in yeast for high level production of aromatic chemicals.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4976},
pmid = {31672987},
issn = {2041-1723},
mesh = {Aldehyde-Lyases/genetics/metabolism ; Amino Acids, Aromatic/*biosynthesis/metabolism ; CRISPR-Cas Systems ; Carbon/*metabolism ; Coumaric Acids ; Glycolysis ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/*genetics ; Propionates/*metabolism ; Saccharomyces cerevisiae/*metabolism ; Saccharomyces cerevisiae Proteins/*metabolism ; Sugar Phosphates/metabolism ; Synthetic Biology ; },
abstract = {The production of bioactive plant compounds using microbial hosts is considered a safe, cost-competitive and scalable approach to their production. However, microbial production of some compounds like aromatic amino acid (AAA)-derived chemicals, remains an outstanding metabolic engineering challenge. Here we present the construction of a Saccharomyces cerevisiae platform strain able to produce high levels of p-coumaric acid, an AAA-derived precursor for many commercially valuable chemicals. This is achieved through engineering the AAA biosynthesis pathway, introducing a phosphoketalose-based pathway to divert glycolytic flux towards erythrose 4-phosphate formation, and optimizing carbon distribution between glycolysis and the AAA biosynthesis pathway by replacing the promoters of several important genes at key nodes between these two pathways. This results in a maximum p-coumaric acid titer of 12.5 g L[-1] and a maximum yield on glucose of 154.9 mg g[-1].},
}
@article {pmid31672965,
year = {2019},
author = {Jillette, N and Du, M and Zhu, JJ and Cardoz, P and Cheng, AW},
title = {Split selectable markers.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4968},
pmid = {31672965},
issn = {2041-1723},
support = {R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cinnamates ; Drug Resistance, Bacterial/*genetics ; Gene Editing ; *Gene Transfer Techniques ; Genetic Engineering/*methods ; Genetic Vectors ; HEK293 Cells ; HeLa Cells ; Humans ; Hygromycin B/analogs &amp; derivatives ; Induced Pluripotent Stem Cells ; *Inteins ; Lentivirus ; Luminescent Proteins/*genetics ; Neomycin ; Nucleosides ; *Protein Splicing ; Puromycin ; Trans-Splicing ; Transgenes/genetics ; },
abstract = {Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.},
}
@article {pmid31672877,
year = {2019},
author = {Cohen, J},
title = {Parents weigh promise and risks of germline editing.},
journal = {Science (New York, N.Y.)},
volume = {366},
number = {6465},
pages = {564-565},
doi = {10.1126/science.366.6465.564},
pmid = {31672877},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Connexin 26 ; Connexins/genetics ; Deafness/genetics/*therapy ; *Gene Editing ; *Genetic Therapy ; Germ Cells ; Humans ; Mutation ; *Parents ; },
}
@article {pmid31672582,
year = {2020},
author = {Yang, TC and Chang, CY and Yarmishyn, AA and Mao, YS and Yang, YP and Wang, ML and Hsu, CC and Yang, HY and Hwang, DK and Chen, SJ and Tsai, ML and Lai, YH and Tzeng, Y and Chang, CC and Chiou, SH},
title = {Carboxylated nanodiamond-mediated CRISPR-Cas9 delivery of human retinoschisis mutation into human iPSCs and mouse retina.},
journal = {Acta biomaterialia},
volume = {101},
number = {},
pages = {484-494},
doi = {10.1016/j.actbio.2019.10.037},
pmid = {31672582},
issn = {1878-7568},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Eye Proteins/genetics ; Gene Editing ; *Gene Transfer Techniques ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Mice, Inbred C57BL ; Mutation/*genetics ; Nanodiamonds/*chemistry ; Photoreceptor Cells, Vertebrate/pathology ; Retina/*metabolism ; Retinoschisis/*genetics ; },
abstract = {Nanodiamonds (NDs) are considered to be relatively safe carbon nanomaterials used for the transmission of DNA, proteins and drugs. The feasibility of utilizing the NDs to deliver CRISPR-Cas9 system for gene editing has not been clearly studied. Therefore, in this study, we aimed to use NDs as the carriers of CRISPR-Cas9 components designed to introduce the mutation in RS1 gene associated with X-linked retinoschisis (XLRS). ND particles with a diameter of 3 nm were functionalized by carboxylation of the surface and covalently conjugated with fluorescent mCherry protein. Two linear DNA constructs were attached to the conjugated mCherry: one encoded Cas9 endonuclease and GFP reporter, another encoded sgRNA and contained insert of HDR template designed to introduce RS1 c.625C>T mutation. Such nanoparticles were successfully delivered and internalized by human iPSCs and mouse retinas, the efficiency of internalization was significantly improved by mixing with BSA. The delivery of ND particles led to introduction of RS1 c.625C>T mutation in both human iPSCs and mouse retinas. Rs1 gene editing in mouse retinas resulted in several pathological features typical for XLRS, such as aberrant photoreceptor structure. To conclude, our ND-based CRISPR-Cas9 delivery system can be utilized as a tool for creating in vitro and in vivo disease models of XLRS. STATEMENT OF SIGNIFICANCE: X-linked retinoschisis (XLRS) is a prevalent hereditary retinal disease, which is caused by mutations in RS1 gene, whose product is important for structural organization of the retina. The recent development of genome editing techniques such as CRISPR-Cas9 significantly improved the prospects for better understanding the pathology and development of treatment for this disease. Firstly, gene editing can allow development of appropriate in vitro and in vivo disease models; secondly, CRISPR-Cas9 can be applied for gene therapy by removing the disease-causative mutation in vivo. The major prerequisite for these approaches is to develop safe and efficient CRISPR-Cas9 delivery system. In this study, we tested specifically modified nanodiamonds for such a delivery system. We were able to introduce Rs1 mutation into the mouse retina and, importantly, observed several XLRS-specific effects.},
}
@article {pmid31672284,
year = {2020},
author = {Chung, CH and Allen, AG and Sullivan, NT and Atkins, A and Nonnemacher, MR and Wigdahl, B and Dampier, W},
title = {Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {1},
pages = {19-28},
pmid = {31672284},
issn = {1525-0024},
support = {P30 MH092177/MH/NIMH NIH HHS/United States ; R01 MH110360/MH/NIMH NIH HHS/United States ; T32 MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {Base Sequence/genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/*methods ; DNA/*genetics ; Databases, Genetic ; Deoxyribonuclease I/genetics ; Gene Editing/*methods ; Genome, Human ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; RNA, Guide/genetics ; RNA-Seq ; Transcription, Genetic ; Transcriptome ; },
abstract = {Defining the variables that impact the specificity of CRISPR/Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targeted loci has also been hypothesized to be an important factor. In this study, functional data from two genome-wide assays, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors that modulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency.},
}
@article {pmid31671275,
year = {2020},
author = {Attwood, KM and Salsman, J and Chung, D and Mathavarajah, S and Van Iderstine, C and Dellaire, G},
title = {PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination.},
journal = {Biochemistry and cell biology = Biochimie et biologie cellulaire},
volume = {98},
number = {3},
pages = {314-326},
doi = {10.1139/bcb-2019-0115},
pmid = {31671275},
issn = {1208-6002},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; DNA Breaks, Double-Stranded ; *Homologous Recombination ; Humans ; In Situ Hybridization, Fluorescence ; Microscopy, Fluorescence ; Promyelocytic Leukemia Protein/*chemistry ; Protein Isoforms ; *Recombinational DNA Repair ; },
abstract = {Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.},
}
@article {pmid31671185,
year = {2019},
author = {Swarts, DC},
title = {Making the cut(s): how Cas12a cleaves target and non-target DNA.},
journal = {Biochemical Society transactions},
volume = {47},
number = {5},
pages = {1499-1510},
doi = {10.1042/BST20190564},
pmid = {31671185},
issn = {1470-8752},
mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; Humans ; RNA, Guide/metabolism ; },
abstract = {CRISPR-Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a-RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR-Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.},
}
@article {pmid31670340,
year = {2019},
author = {Jain, PK and Lo, JH and Rananaware, S and Downing, M and Panda, A and Tai, M and Raghavan, S and Fleming, HE and Bhatia, SN},
title = {Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes.},
journal = {Nanoscale},
volume = {11},
number = {44},
pages = {21317-21323},
pmid = {31670340},
issn = {2040-3372},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; U54 CA151884/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Transfer Techniques ; HeLa Cells ; Humans ; },
abstract = {There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.},
}
@article {pmid31669737,
year = {2020},
author = {Islam, S and Chuensirikulchai, K and Khummuang, S and Keratibumrungpong, T and Kongtawelert, P and Kasinrerk, W and Hatano, S and Nagamachi, A and Honda, H and Watanabe, H},
title = {Accumulation of versican facilitates wound healing: Implication of its initial ADAMTS-cleavage site.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {87},
number = {},
pages = {77-93},
doi = {10.1016/j.matbio.2019.10.006},
pmid = {31669737},
issn = {1569-1802},
mesh = {ADAMTS Proteins/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Extracellular Matrix/metabolism ; Gene Knock-In Techniques ; Hemorrhage/*genetics ; Male ; Mice ; Signal Transduction ; Syndactyly/*genetics ; Transforming Growth Factor beta/metabolism ; Versicans/*chemistry/*genetics/metabolism ; *Wound Healing ; },
abstract = {Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix, and is expressed at high levels in tissues during development and remodeling in pathological conditions. Its core protein is cleaved at a region close to the N-terminal end of CSβ domain by several members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, i.e., ADAMTS-1, 4, 5, 9, 15, and 20. Here, using a CRISPR/Cas9 system, we generated knock-in mice (V1R), which express an ADAMTS cleavage-resistant versican. Some V1R homozygote mice, termed R/R, exhibit syndactyly and organ hemorrhage. In wound healing experiments, R/R wound shows accumulation of versican and activated TGFβ-signaling in the early stage, leading to faster healing than wild type wound. Immunostaining for Ki67, CD31, smooth muscle α-actin, periostin demonstrates higher levels of overall cell proliferation and an increased number of endothelial cells and myofibroblasts. Immunostaining for CD11b and qRT-PCR for macrophage markers revealed increased levels of inflammatory cell infiltration, especially those of M1 macrophages. Cultured R/R dermal fibroblasts revealed increased deposition of versican, type I and III collagens, and hyaluronan, and upregulation of Smad2/3 signaling. Taken together, these results demonstrate that the cleavage site determines versican turnover and that versican plays a central role in the provisional matrix during the wound repair.},
}
@article {pmid31668930,
year = {2019},
author = {Sun, W and Yang, J and Cheng, Z and Amrani, N and Liu, C and Wang, K and Ibraheim, R and Edraki, A and Huang, X and Wang, M and Wang, J and Liu, L and Sheng, G and Yang, Y and Lou, J and Sontheimer, EJ and Wang, Y},
title = {Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States.},
journal = {Molecular cell},
volume = {76},
number = {6},
pages = {938-952.e5},
pmid = {31668930},
issn = {1097-4164},
support = {F31 DK120333/DK/NIDDK NIH HHS/United States ; F31 NS110328/NS/NINDS NIH HHS/United States ; R01 GM125797/GM/NIGMS NIH HHS/United States ; UG3 TR002668/TR/NCATS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/*metabolism ; Binding Sites ; CRISPR-Associated Protein 9/genetics/*metabolism/ultrastructure ; *CRISPR-Cas Systems ; Catalysis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/*metabolism/ultrastructure ; Escherichia coli/enzymology/genetics ; Neisseria meningitidis/*enzymology/genetics ; Protein Binding ; Protein Domains ; RNA, Guide/genetics/metabolism ; Structure-Activity Relationship ; Viral Proteins/genetics/*metabolism/ultrastructure ; },
abstract = {High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNA-bound state. In the HNH catalytic state of Nme1Cas9, the active site is seen poised at the scissile phosphodiester linkage of the target strand, providing a high-resolution view of the active conformation. The HNH active conformation activates the RuvC domain. Our structures explain how Nme1Cas9 and Nme2Cas9 read distinct PAM sequences and how AcrIIC3 inhibits Nme1Cas9 activity. These structures provide insights into Cas9 domain rearrangements, guide-target engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.},
}
@article {pmid31667881,
year = {2020},
author = {Godehardt, AW and Fischer, N and Rauch, P and Gulich, B and Boller, K and Church, GM and Tönjes, RR},
title = {Characterization of porcine endogenous retrovirus particles released by the CRISPR/Cas9 inactivated cell line PK15 clone 15.},
journal = {Xenotransplantation},
volume = {27},
number = {2},
pages = {e12563},
doi = {10.1111/xen.12563},
pmid = {31667881},
issn = {1399-3089},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Cell Line ; Endogenous Retroviruses/*pathogenicity ; Humans ; Proviruses/*pathogenicity ; Swine ; Swine, Miniature/*virology ; Transplantation, Heterologous/adverse effects ; },
abstract = {The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.},
}
@article {pmid31666940,
year = {2019},
author = {Yang, G and Huang, X},
title = {Methods and applications of CRISPR/Cas system for genome editing in stem cells.},
journal = {Cell regeneration (London, England)},
volume = {8},
number = {2},
pages = {33-41},
pmid = {31666940},
issn = {2045-9769},
abstract = {Genome editing technology holds great promise for genome manipulation and gene therapy. While widespread utilization, genome editing has been used to unravel the roles of specific genes in differentiation and pluripotency of stem cells, and reinforce the stem cell-based applications. In this review, we summarize the advances of genome editing technology, as well as the derivative technologies from CRISPR/Cas system, which show tremendous potential in various fields. We also highlight the key findings in the studies of stem cells and regeneration by genome editing technology.},
}
@article {pmid31666694,
year = {2019},
author = {Flavahan, WA and Drier, Y and Johnstone, SE and Hemming, ML and Tarjan, DR and Hegazi, E and Shareef, SJ and Javed, NM and Raut, CP and Eschle, BK and Gokhale, PC and Hornick, JL and Sicinska, ET and Demetri, GD and Bernstein, BE},
title = {Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs.},
journal = {Nature},
volume = {575},
number = {7781},
pages = {229-233},
pmid = {31666694},
issn = {1476-4687},
support = {DP1 CA216873/CA/NCI NIH HHS/United States ; F32 CA203153/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; *Chromosome Aberrations ; DNA Methylation ; Enhancer Elements, Genetic/genetics ; Epigenesis, Genetic ; Fibroblast Growth Factor 4/genetics ; Gastrointestinal Stromal Tumors/enzymology/*genetics/*pathology ; Humans ; Mice ; Mutation ; Oncogenes/*genetics ; Proto-Oncogene Proteins c-kit/antagonists &amp; inhibitors ; Receptors, Fibroblast Growth Factor/antagonists &amp; inhibitors ; Succinate Dehydrogenase/*deficiency/genetics ; },
abstract = {Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood[1-3]. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation[4,5]. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.},
}
@article {pmid31666509,
year = {2019},
author = {Man, JCK and Mohan, RA and Boogaard, MVD and Hilvering, CRE and Jenkins, C and Wakker, V and Bianchi, V and Laat, W and Barnett, P and Boukens, BJ and Christoffels, VM},
title = {An enhancer cluster controls gene activity and topology of the SCN5A-SCN10A locus in vivo.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4943},
pmid = {31666509},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Chromatin ; DNA, Intergenic/genetics ; Enhancer Elements, Genetic/genetics ; Gene Editing ; Gene Expression Regulation ; Heart/*embryology ; Heart Conduction System/*metabolism ; Mice ; Myocardium/*metabolism ; NAV1.5 Voltage-Gated Sodium Channel/*genetics/metabolism ; NAV1.8 Voltage-Gated Sodium Channel/*genetics/metabolism ; Nucleic Acid Conformation ; Regulatory Elements, Transcriptional ; },
abstract = {Mutations and variations in and around SCN5A, encoding the major cardiac sodium channel, influence impulse conduction and are associated with a broad spectrum of arrhythmia disorders. Here, we identify an evolutionary conserved regulatory cluster with super enhancer characteristics downstream of SCN5A, which drives localized cardiac expression and contains conduction velocity-associated variants. We use genome editing to create a series of deletions in the mouse genome and show that the enhancer cluster controls the conformation of a >0.5 Mb genomic region harboring multiple interacting gene promoters and enhancers. We find that this cluster and its individual components are selectively required for cardiac Scn5a expression, normal cardiac conduction and normal embryonic development. Our studies reveal physiological roles of an enhancer cluster in the SCN5A-SCN10A locus, show that it controls the chromatin architecture of the locus and Scn5a expression, and suggest that genetic variants affecting its activity may influence cardiac function.},
}
@article {pmid31666400,
year = {2019},
author = {Sharon, D and Cathelin, S and Mirali, S and Di Trani, JM and Yanofsky, DJ and Keon, KA and Rubinstein, JL and Schimmer, AD and Ketela, T and Chan, SM},
title = {Inhibition of mitochondrial translation overcomes venetoclax resistance in AML through activation of the integrated stress response.},
journal = {Science translational medicine},
volume = {11},
number = {516},
pages = {},
doi = {10.1126/scitranslmed.aax2863},
pmid = {31666400},
issn = {1946-6242},
support = {PJT-159521//CIHR/Canada ; PJT-162186//CIHR/Canada ; },
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; Bridged Bicyclo Compounds, Heterocyclic/*pharmacology ; CRISPR-Cas Systems/genetics ; Cell Death/drug effects ; Cell Line, Tumor ; Cell Respiration/drug effects ; *Drug Resistance, Neoplasm/drug effects ; Electron Transport Complex I/metabolism ; Glycolysis/drug effects ; Humans ; Leukemia, Myeloid, Acute/*pathology ; Mice, Inbred NOD ; Mice, SCID ; Mitochondria/drug effects/*metabolism ; Oxazolidinones/pharmacology ; *Protein Biosynthesis/drug effects ; Proto-Oncogene Proteins c-bcl-2/metabolism ; *Stress, Physiological/drug effects ; Sulfonamides/*pharmacology ; Tetrazoles/pharmacology ; },
abstract = {Venetoclax is a specific B cell lymphoma 2 (BCL-2) inhibitor with promising activity against acute myeloid leukemia (AML), but its clinical efficacy as a single agent or in combination with hypomethylating agents (HMAs), such as azacitidine, is hampered by intrinsic and acquired resistance. Here, we performed a genome-wide CRISPR knockout screen and found that inactivation of genes involved in mitochondrial translation restored sensitivity to venetoclax in resistant AML cells. Pharmacologic inhibition of mitochondrial protein synthesis with antibiotics that target the ribosome, including tedizolid and doxycycline, effectively overcame venetoclax resistance. Mechanistic studies showed that both tedizolid and venetoclax suppressed mitochondrial respiration, with the latter demonstrating inhibitory activity against complex I [nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase] of the electron transport chain (ETC). The drugs cooperated to activate a heightened integrated stress response (ISR), which, in turn, suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. Combination treatment with tedizolid and venetoclax was superior to either agent alone in reducing leukemic burden in mice engrafted with treatment-resistant human AML. The addition of tedizolid to azacitidine and venetoclax further enhanced the killing of resistant AML cells in vitro and in vivo. Our findings demonstrate that inhibition of mitochondrial translation is an effective approach to overcoming venetoclax resistance and provide a rationale for combining tedizolid, azacitidine, and venetoclax as a triplet therapy for AML.},
}
@article {pmid31665741,
year = {2019},
author = {Martino, J and Brunette, GJ and Barroso-González, J and Moiseeva, TN and Smith, CM and Bakkenist, CJ and O'Sullivan, RJ and Bernstein, KA},
title = {The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination.},
journal = {Nucleic acids research},
volume = {47},
number = {19},
pages = {10151-10165},
pmid = {31665741},
issn = {1362-4962},
support = {R01 ES024872/ES/NIEHS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Calcium-Binding Proteins/*genetics ; DNA Damage/genetics ; DNA Repair/genetics ; DNA Replication/genetics ; DNA-Binding Proteins/*genetics ; Genomic Instability/genetics ; Homologous Recombination/*genetics ; Humans ; Multiprotein Complexes/genetics ; Nuclear Proteins/*genetics ; Rad51 Recombinase/genetics ; Rec A Recombinases/*genetics ; Sister Chromatid Exchange/genetics ; Transcription Factors/*genetics ; },
abstract = {RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.},
}
@article {pmid31665284,
year = {2019},
author = {Deaner, M and Alper, HS},
title = {Enhanced scale and scope of genome engineering and regulation using CRISPR/Cas in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {19},
number = {7},
pages = {},
doi = {10.1093/femsyr/foz076},
pmid = {31665284},
issn = {1567-1364},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Expression Regulation, Fungal ; Genetic Engineering/*methods ; *Genome, Fungal ; Metabolic Engineering ; RNA, Guide/genetics ; Saccharomyces cerevisiae/*genetics ; Synthetic Biology ; },
abstract = {Although only 6 years old, the CRISPR system has blossomed into a tool for rapid, on-demand genome engineering and gene regulation in Saccharomyces cerevisiae. In this minireview, we discuss fundamental CRISPR technologies, tools to improve the efficiency and capabilities of gene targeting, and cutting-edge techniques to explore gene editing and transcriptional regulation at genome scale using pooled approaches. The focus is on applications to metabolic engineering with topics including development of techniques to edit the genome in multiplex, tools to enable large numbers of genetic modifications using pooled single-guide RNA libraries and efforts to enable programmable transcriptional regulation using endonuclease-null Cas enzymes.},
}
@article {pmid31664995,
year = {2019},
author = {Cook, NL and Pjanic, M and Emmerich, AG and Rao, AS and Hetty, S and Knowles, JW and Quertermous, T and Castillejo-López, C and Ingelsson, E},
title = {CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation.},
journal = {BMC endocrine disorders},
volume = {19},
number = {1},
pages = {115},
pmid = {31664995},
issn = {1472-6823},
support = {R01 DK120565/DK/NIDDK NIH HHS/United States ; R01 DK106236-01A1/NH/NIH HHS/United States ; R01DK107437/NH/NIH HHS/United States ; R01 DK106236/DK/NIDDK NIH HHS/United States ; P30 DK116074/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Expression Profiling ; Glucose/*metabolism ; Hep G2 Cells ; Hepatocytes/cytology/*metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/*antagonists &amp; inhibitors/genetics/*metabolism ; Lipid Droplets/*metabolism ; *Lipogenesis ; Membrane Proteins/*antagonists &amp; inhibitors/genetics/*metabolism ; Phosphorylation ; Signal Transduction ; },
abstract = {BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.<br><br>METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.<br><br>RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.<br><br>CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.},
}
@article {pmid31664646,
year = {2020},
author = {Morishige, S and Mizuno, S and Ozawa, H and Nakamura, T and Mazahery, A and Nomura, K and Seki, R and Mouri, F and Osaki, K and Yamamura, K and Okamura, T and Nagafuji, K},
title = {CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs.},
journal = {International journal of hematology},
volume = {111},
number = {2},
pages = {225-233},
pmid = {31664646},
issn = {1865-3774},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Therapy/*methods ; Hemophilia B/*genetics/*therapy ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is an efficient genome-editing tool that holds potential for gene therapy. Here, we report an application of this system for gene repair in hemophilia B (HB) using induced pluripotent stem cells (iPSCs). We prepared targeting plasmids with homology arms containing corrected sequences to repair an in-frame deletion in exon 2 of the factor IX (F9) gene and transfected patient-derived iPSCs with the Cas9 nuclease and a guide RNA expression vector. To validate the expression of corrected F9, we attempted to induce the differentiation of iPSCs toward hepatocyte-like cells (HLCs) in vitro. We successfully repaired a disease-causing mutation in HB in patient-derived iPSCs. The transcription product of corrected F9 was confirmed in HLCs differentiated from gene-corrected iPSCs. Although further research should be undertaken to obtain completely functional hepatocytes with secretion of coagulation factor IX, our study provides a proof-of-principle for HB gene therapy using the CRISPR/Cas9 system.},
}
@article {pmid31663848,
year = {2019},
author = {Li, W and Zhang, Y and Han, B and Li, L and Li, M and Lu, X and Chen, C and Lu, M and Zhang, Y and Jia, X and Zhu, Z and Tong, X and Zhang, B},
title = {One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31663848},
issn = {2050-084X},
support = {2018YFA0801000//National Key Research and Development Program of China/International ; 2016YFA0100500//National Key Research and Development Program of China/International ; 2015CB942803//National Key Basic Research Program of China/International ; 31671500//National Natural Science Foundation of China/International ; 31871458//National Natural Science Foundation of China/International ; 81371264//National Natural Science Foundation of China/International ; Qidong-SLS Innovation Fund//Peking University/International ; },
mesh = {*Alleles ; Animals ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genotype ; Staining and Labeling/*methods ; Transcription Factors/genetics ; Zebrafish/*genetics ; Zebrafish Proteins/genetics ; },
abstract = {CRISPR/Cas systems are widely used to knock out genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no 'two-in-one' KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.},
}
@article {pmid31663334,
year = {2019},
author = {Ruiz, E and Talenton, V and Dubrana, MP and Guesdon, G and Lluch-Senar, M and Salin, F and Sirand-Pugnet, P and Arfi, Y and Lartigue, C},
title = {CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System.},
journal = {ACS synthetic biology},
volume = {8},
number = {11},
pages = {2547-2557},
doi = {10.1021/acssynbio.9b00224},
pmid = {31663334},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Cloning, Molecular/*methods ; DNA Cleavage ; DNA, Bacterial/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Loci ; Genome, Bacterial ; Mycoplasma pneumoniae/genetics ; Plasmids/genetics ; RNA, Guide/metabolism ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.},
}
@article {pmid31662324,
year = {2019},
author = {Chen, G and Park, D and Magis, AT and Behera, M and Ramalingam, SS and Owonikoko, TK and Sica, GL and Ye, K and Zhang, C and Chen, Z and Curran, WJ and Deng, X},
title = {Mcl-1 Interacts with Akt to Promote Lung Cancer Progression.},
journal = {Cancer research},
volume = {79},
number = {24},
pages = {6126-6138},
pmid = {31662324},
issn = {1538-7445},
support = {R01 CA200905/CA/NCI NIH HHS/United States ; P50 CA217691/CA/NCI NIH HHS/United States ; P30 CA138292/CA/NCI NIH HHS/United States ; R01 CA193828/CA/NCI NIH HHS/United States ; R01 CA136534/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/drug therapy/mortality/*pathology ; Cell Line, Tumor ; Disease Progression ; Follow-Up Studies ; Gene Knockout Techniques ; Humans ; Kaplan-Meier Estimate ; Lung/pathology ; Lung Neoplasms/drug therapy/mortality/*pathology ; Male ; Mice ; Molecular Docking Simulation ; Myeloid Cell Leukemia Sequence 1 Protein/genetics/*metabolism ; Prognosis ; Protein Binding/drug effects ; Protein Domains/drug effects ; Proto-Oncogene Proteins c-akt/antagonists &amp; inhibitors/*metabolism ; Signal Transduction/drug effects ; Tissue Array Analysis ; Xenograft Model Antitumor Assays ; },
abstract = {Mcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non-small cell lung cancer. Using the Mcl-1-binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer. SIGNIFICANCE: These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.},
}
@article {pmid31661801,
year = {2019},
author = {Yarra, R and Jin, L and Zhao, Z and Cao, H},
title = {Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview.},
journal = {International journal of molecular sciences},
volume = {20},
number = {21},
pages = {},
pmid = {31661801},
issn = {1422-0067},
mesh = {Agrobacterium tumefaciens/genetics/metabolism ; Arecaceae/embryology/*genetics/*growth &amp; development ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Microinjections/methods ; Palm Oil/economics ; Plant Somatic Embryogenesis Techniques/methods ; Polyethylene Glycols/chemistry/pharmacology ; Protoplasts/cytology/drug effects ; Tissue Culture Techniques ; *Transformation, Genetic ; },
abstract = {Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.},
}
@article {pmid31661548,
year = {2019},
author = {Tawarayama, H and Feng, Q and Murayama, N and Suzuki, N and Nakazawa, T},
title = {Cyclin-Dependent Kinase Inhibitor 2b Mediates Excitotoxicity-Induced Death of Retinal Ganglion Cells.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {60},
number = {13},
pages = {4479-4488},
doi = {10.1167/iovs.19-27396},
pmid = {31661548},
issn = {1552-5783},
mesh = {Animals ; Blotting, Western ; CRISPR-Cas Systems ; Cell Death ; Cyclin-Dependent Kinase Inhibitor p15/*physiology ; Cyclin-Dependent Kinase Inhibitor p16/physiology ; Dependovirus ; Excitatory Amino Acid Agonists/*toxicity ; Immunochemistry ; In Situ Hybridization ; Intravitreal Injections ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; N-Methylaspartate/*toxicity ; Parvovirinae/genetics ; Retinal Ganglion Cells/*drug effects/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; },
abstract = {PURPOSE: Glutamate excitotoxicity seems to contribute to retinal ganglion cell (RGC) death in various eye diseases, but the underlying molecular mechanisms are not fully understood. We studied the roles of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, known as cellular stress-related senescence markers, in N-methyl-d-aspartate (NMDA)-induced RGC death.<br><br>METHODS: Gene expression was analyzed using quantitative reverse transcription (qRT)-PCR, in situ hybridization, and immunochemistry. Cdkn2a and Cdkn2b gain- and loss-of-function experiments were performed using the adeno-associated virus type 2 (AAV2)-mediated gene delivery system. AAV2-CRISPR-Cas9-mediated knockout of Cdkn2a or Cdkn2b was validated using cultured cells by T7 endonuclease I assay and Western blot analysis. The effects of altered expression of Cdkn2a and Cdkn2b on NMDA-induced RGC death were evaluated by quantification of RNA binding protein with multiple splicing (Rbpms)-immunoreactive RGCs.<br><br>RESULTS: Intravitreal NMDA injection resulted in upregulation of Cdkn2a and Cdkn2b expression in RGCs of the mouse retina. AAV2-mediated overexpression of Cdkn2b led to increased expression of Cdkn2a in RGCs, but not vice versa. Overexpression of Cdkn2b, but not Cdkn2a, resulted in a further reduction in RGC viability in NMDA-injected retinas. However, excessive levels of Cdkn2a or Cdkn2b had no effect on RGC viability in healthy mice. AAV2-CRISPR-Cas9-mediated knockout of either Cdkn2a or Cdkn2b attenuated NMDA-induced RGC death.<br><br>CONCLUSIONS: Cdkn2a and Cdkn2b have pivotal roles in the regulation of excitotoxic RGC degeneration under NMDA-induced pathologic conditions. Our findings imply that Cdkn2a and Cdkn2b are novel therapeutic targets for ocular diseases displaying excitotoxicity-induced neuronal degeneration.},
}
@article {pmid31659913,
year = {2019},
author = {Zoppo, M and Luca, MD and Villarreal, SN and Poma, N and Barrasa, MI and Bottai, D and Vyas, VK and Tavanti, A},
title = {A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis.},
journal = {Future microbiology},
volume = {14},
number = {},
pages = {1383-1396},
doi = {10.2217/fmb-2019-0168},
pmid = {31659913},
issn = {1746-0921},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Candida parapsilosis/*genetics/growth &amp; development ; Candidiasis/microbiology ; Cell Adhesion ; Cells, Cultured ; Epithelial Cells/microbiology ; Gene Editing/*methods ; *Genes, Fungal ; Humans ; Hyphae/growth &amp; development ; Mouth/cytology ; Multigene Family ; RNA, Guide/genetics ; },
abstract = {Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials &amp; methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.},
}
@article {pmid31659338,
year = {2019},
author = {Eom, JS and Luo, D and Atienza-Grande, G and Yang, J and Ji, C and Thi Luu, V and Huguet-Tapia, JC and Char, SN and Liu, B and Nguyen, H and Schmidt, SM and Szurek, B and Vera Cruz, C and White, FF and Oliva, R and Yang, B and Frommer, WB},
title = {Diagnostic kit for rice blight resistance.},
journal = {Nature biotechnology},
volume = {37},
number = {11},
pages = {1372-1379},
pmid = {31659338},
issn = {1546-1696},
mesh = {Bacterial Proteins/genetics ; Binding Sites ; CRISPR-Cas Systems ; Databases, Genetic ; *Disease Resistance ; Gene Editing ; Gene Expression Regulation, Plant ; Membrane Transport Proteins/chemistry/*genetics/metabolism ; Mutation ; Oryza/genetics/*growth &amp; development/microbiology ; Plant Proteins/genetics ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; Transcription Activator-Like Effectors/*metabolism ; Xanthomonas/metabolism/*pathogenicity ; },
abstract = {Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT-PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR-Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant 'mega' rice lines that will empower farmers to plant lines that are most likely to resist rice blight.},
}
@article {pmid31659337,
year = {2019},
author = {Oliva, R and Ji, C and Atienza-Grande, G and Huguet-Tapia, JC and Perez-Quintero, A and Li, T and Eom, JS and Li, C and Nguyen, H and Liu, B and Auguy, F and Sciallano, C and Luu, VT and Dossa, GS and Cunnac, S and Schmidt, SM and Slamet-Loedin, IH and Vera Cruz, C and Szurek, B and Frommer, WB and White, FF and Yang, B},
title = {Broad-spectrum resistance to bacterial blight in rice using genome editing.},
journal = {Nature biotechnology},
volume = {37},
number = {11},
pages = {1344-1350},
pmid = {31659337},
issn = {1546-1696},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems ; *Disease Resistance ; Gene Editing ; Gene Expression Regulation, Plant ; Membrane Transport Proteins/*genetics ; Mutation ; Oryza/genetics/*growth &amp; development/microbiology ; Plant Proteins/genetics ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; Transcription Activator-Like Effectors/*genetics ; Xanthomonas/genetics/*pathogenicity ; },
abstract = {Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR-Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.},
}
@article {pmid31659326,
year = {2019},
author = {Smits, AH and Ziebell, F and Joberty, G and Zinn, N and Mueller, WF and Clauder-Münster, S and Eberhard, D and Fälth Savitski, M and Grandi, P and Jakob, P and Michon, AM and Sun, H and Tessmer, K and Bürckstümmer, T and Bantscheff, M and Steinmetz, LM and Drewes, G and Huber, W},
title = {Biological plasticity rescues target activity in CRISPR knock outs.},
journal = {Nature methods},
volume = {16},
number = {11},
pages = {1087-1093},
pmid = {31659326},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/genetics ; DNA (Cytosine-5-)-Methyltransferase 1/genetics ; Exons ; *Gene Knockout Techniques ; Humans ; Mutation ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics ; Transcription Factors/genetics ; },
abstract = {Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.},
}
@article {pmid31659303,
year = {2020},
author = {Burgess, DJ},
title = {CRISPR screens come into sight.},
journal = {Nature reviews. Genetics},
volume = {21},
number = {1},
pages = {1},
pmid = {31659303},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; RNA, Guide ; },
}
@article {pmid31659151,
year = {2019},
author = {Zou, X and Ouyang, H and Yu, T and Chen, X and Pang, D and Tang, X and Chen, C},
title = {Preparation of a new type 2 diabetic miniature pig model via the CRISPR/Cas9 system.},
journal = {Cell death &amp; disease},
volume = {10},
number = {11},
pages = {823},
pmid = {31659151},
issn = {2041-4889},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Diabetes Mellitus, Type 2/*genetics/pathology ; Disease Models, Animal ; Humans ; Insulin-Secreting Cells/metabolism/pathology ; Islet Amyloid Polypeptide/*genetics ; Islets of Langerhans/metabolism/pathology ; Lipid Metabolism/*genetics ; Swine ; Swine, Miniature/genetics ; },
abstract = {Diabetes has become one of the major noninfectious diseases that seriously endanger public health. The formation of islet amyloid polypeptide (IAPP) affects the normal physiological functions of the body, such as glucose metabolism and lipid metabolism. The mature human IAPP protein (hIAPP) has a strong tendency to misfold and is considered to be one of the major causes of amyloid changes in islets. Deposition of hIAPP is considered to be one of the leading causes of type 2 diabetes mellitus (T2DM). Miniature pigs are experimental animal models that are well suited for research on gene function and human diabetes. In our study, we obtained IAPP gene-humanized miniature pigs via the CRISPR/Cas9 system and somatic cell nuclear transfer (SCNT) technology. The hIAPP pigs can be used to further study the pathogenesis and related complications of T2DM and to lay a solid foundation for the prevention and treatment of T2DM.},
}
@article {pmid31658850,
year = {2020},
author = {le Sage, C and Lawo, S and Cross, BCS},
title = {CRISPR: A Screener's Guide.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {25},
number = {3},
pages = {233-240},
pmid = {31658850},
issn = {2472-5560},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; },
abstract = {The discovery of CRISPR-Cas9 systems has fueled a rapid expansion of gene editing adoption and has impacted pharmaceutical and biotechnology research substantially. Here, gene editing is used at an industrial scale to identify and validate new biological targets for precision medicines, with functional genomic screening having an increasingly important role. Functional genomic strategies provide a crucial link between observed biological phenomena and the genes that influence and drive those phenomena. Although such studies are not new, the use of CRISPR-Cas9 systems in this arena is providing more robust datasets for target identification and validation. CRISPR-based screening approaches are also useful later in the drug development pipeline for understanding drug resistance and sensitivity ahead of entering clinical trials. This review examines the developing landscape for CRISPR screening technologies within the pharmaceutical industry and explores the next steps for this constantly evolving screening platform.},
}
@article {pmid31658261,
year = {2019},
author = {Liu, Q and He, D and Xie, L},
title = {Prediction of off-target specificity and cell-specific fitness of CRISPR-Cas System using attention boosted deep learning and network-based gene feature.},
journal = {PLoS computational biology},
volume = {15},
number = {10},
pages = {e1007480},
pmid = {31658261},
issn = {1553-7358},
support = {R01 AG057555/AG/NIA NIH HHS/United States ; R01 GM122845/GM/NIGMS NIH HHS/United States ; R01 LM011986/LM/NLM NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Databases, Genetic ; Deep Learning ; Forecasting/*methods ; Gene Editing/methods ; Gene Regulatory Networks/genetics ; Genome/genetics ; Humans ; RNA Editing/genetics ; RNA, Guide/*genetics ; Substrate Specificity/*genetics ; },
abstract = {CRISPR-Cas is a powerful genome editing technology and has a great potential for in vivo gene therapy. Successful translational application of CRISPR-Cas to biomedicine still faces many safety concerns, including off-target side effect, cell fitness problem after CRISPR-Cas treatment, and on-target genome editing side effect in undesired tissues. To solve these issues, it is needed to design sgRNA with high cell-specific efficacy and specificity. Existing single-guide RNA (sgRNA) design tools mainly depend on a sgRNA sequence and the local information of the targeted genome, thus are not sufficient to account for the difference in the cellular response of the same gene in different cell types. To incorporate cell-specific information into the sgRNA design, we develop novel interpretable machine learning models, which integrate features learned from advanced transformer-based deep neural network with cell-specific gene property derived from biological network and gene expression profile, for the prediction of CRISPR-Cas9 and CRISPR-Cas12a efficacy and specificity. In benchmark studies, our models significantly outperform state-of-the-art algorithms. Furthermore, we find that the network-based gene property is critical for the prediction of cell-specific post-treatment cellular response. Our results suggest that the design of efficient and safe CRISPR-Cas needs to consider cell-specific information of genes. Our findings may bolster developing more accurate predictive models of CRISPR-Cas across a broad spectrum of biological conditions as well as provide new insight into developing efficient and safe CRISPR-based gene therapy.},
}
@article {pmid31657874,
year = {2020},
author = {Bae, SJ and Park, BG and Kim, BG and Hahn, JS},
title = {Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System.},
journal = {Biotechnology journal},
volume = {15},
number = {1},
pages = {e1900238},
doi = {10.1002/biot.201900238},
pmid = {31657874},
issn = {1860-7314},
mesh = {CRISPR-Cas Systems/*genetics ; *Cytidine Deaminase/genetics/metabolism ; Gene Editing/*methods ; Genome, Bacterial/*genetics ; Recombinant Fusion Proteins/genetics/metabolism ; Uracil-DNA Glycosidase/genetics ; Yarrowia/*genetics ; },
abstract = {The oleaginous yeast Yarrowia lipolytica has a tendency to use the non-homologous end joining repair (NHEJ) over the homology directed recombination as double-strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target-AID (activation-induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target-specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target-AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.},
}
@article {pmid31654436,
year = {2020},
author = {Yang, JW and Jiang, JH and Wang, HC and Li, CY},
title = {The extra domain A of fibronectin facilitates osteoclastogenesis in radicular cysts through vascular endothelial growth factor.},
journal = {International endodontic journal},
volume = {53},
number = {4},
pages = {478-491},
doi = {10.1111/iej.13241},
pmid = {31654436},
issn = {1365-2591},
mesh = {*Fibronectins ; Humans ; Osteogenesis ; *Radicular Cyst ; Retrospective Studies ; Vascular Endothelial Growth Factor A ; },
abstract = {AIM: To analyse the effects of the alternatively spliced fibronectin (FN) gene and its isoforms on osteoclastogenesis in radicular cysts.<br><br>METHODOLOGY: Specimens of radicular cysts were collected surgically from 22 patients whose radiolucent periapical areas were measured on digital panoramic radiographs before surgery. The associations between the radiolucent areas and FN isoforms, vascular endothelial growth factor (VEGF) expression or micro-vessel density, as well as the relationships amongst them, were analysed by immunohistochemical staining using the antibodies IST-9, BC-1, P1F11, VEGF and CD34. Fibroblasts isolated from those specimens were used to induce Trap&#xa0;+&#xa0;MNCs, and the effects of induction were assessed by blocking FN containing extra domain A (EDA&#xa0;+&#xa0;FN), COX-2 or VEGF in vitro. The effects of EDA exon knockout using CRISPR/Cas system were also assessed. Quantitative PCR was used to analyse relative expression of FN isoforms and osteoclastogenic genes. Data were analysed using linear regression, Spearman's rank correlation analysis, chi-square test and Student's t-test; P&#xa0;<&#xa0;0.05 was considered significant.<br><br>RESULTS: Micro-vessel density and EDA&#xa0;+&#xa0;FN staining were positively associated with the size of radiolucent periapical areas (mm[2] ; P&#xa0;<&#xa0;0.05), consistent with a positive association between Trap&#xa0;+&#xa0;MNCs and VEGF expression in fibroblasts (P&#xa0;<&#xa0;0.05). Blocking the interaction between EDA&#xa0;+&#xa0;FN and fibroblasts inhibited Trap&#xa0;+&#xa0;MNC formation. In addition, EDA exon knockout decreased VEGF expression and inhibited Trap&#xa0;+&#xa0;MNC formation to the extent of blocking VEGF by bevacizumab, but osteoclastogenic induction was restored by recombinant VEGF. Using retrospective clinicopathological data, VEGF staining was shown to be positively associated with EDA&#xa0;+&#xa0;FN staining, micro-vessel density and the size of radiolucent areas (P&#xa0;<&#xa0;0.05).<br><br>CONCLUSION: In fibrous capsules of radicular cysts, the alternatively spliced isoform EDA&#xa0;+&#xa0;FN generated by fibroblasts stimulated VEGF expression via an autocrine effect and then facilitated osteoclastogenesis. Both blockage of VEGF and EDA exon knockout could be used to inhibit bone destruction.},
}
@article {pmid31654427,
year = {2020},
author = {Watanabe, M and Toyomura, T and Wake, H and Liu, K and Teshigawara, K and Takahashi, H and Nishibori, M and Mori, S},
title = {Differential contribution of possible pattern-recognition receptors to advanced glycation end product-induced cellular responses in macrophage-like RAW264.7 cells.},
journal = {Biotechnology and applied biochemistry},
volume = {67},
number = {2},
pages = {265-272},
doi = {10.1002/bab.1843},
pmid = {31654427},
issn = {1470-8744},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Glycation End Products, Advanced/genetics/*metabolism ; Macrophages/*metabolism ; Mice ; RAW 264.7 Cells ; Receptor for Advanced Glycation End Products/genetics/*metabolism ; Toll-Like Receptors/genetics/metabolism ; },
abstract = {Advanced glycation end products (AGEs) are considered to be related to the pathogenesis of some inflammatory diseases. AGEs were reported to stimulate the receptor for AGEs (RAGE), which causes inflammatory reactions. However, recently, toll-like receptors (TLRs), in addition to RAGE, have been reported to be related to AGE-mediated cellular responses, and it remains unclear which receptor is responsible for AGE recognition. To reveal the role of pattern-recognition receptors, including TLRs and/or RAGE, in AGE-mediated cellular responses, we generated macrophage-like RAW264.7 knockout (KO) cells lacking these receptors by genome editing using the CRISPR/Cas9 system and assessed AGE-stimulated changes in these cells. Comparison of the established clones suggested that RAGE partially affects the expression of TLRs. In the KO clone lacking TLR4 and TLR2, AGE-stimulated tumor necrosis factor alpha (TNF-α) expression and phosphorylation of IκBα, p38, and extracellular signal-regulated kinase (ERK) were significantly attenuated, suggesting that AGE-mediated responses are largely dependent on TLRs. On the other hand, on comparison of the AGE-stimulated responses between the KO clone lacking TLR4 and TLR2, and the clone lacking TLR4, TLR2, and RAGE, RAGE played little role in AGE-stimulated TNF-α transcription and ERK phosphorylation. Taken together, this study suggested that AGE-stimulated inflammatory responses occur mainly through TLRs rather than RAGE.},
}
@article {pmid31654413,
year = {2020},
author = {Zhang, L and Zhang, H and Liu, Y and Zhou, J and Shen, W and Liu, L and Li, Q and Chen, X},
title = {A CRISPR-Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {2},
pages = {531-542},
doi = {10.1002/bit.27207},
pmid = {31654413},
issn = {1097-0290},
support = {BK20171138//Natural Science Foundation of Jiangsu Province/International ; 111-2-06//111 Project/International ; none//Top-notch Academic Programs Project of Jiangsu Higher Education Institutions/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; Candida tropicalis/*genetics/metabolism ; DNA, Fungal/genetics ; Gene Editing/*methods ; Genome, Fungal/*genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.},
}
@article {pmid31654081,
year = {2020},
author = {Wang, K and Gong, Q and Ye, X},
title = {Recent developments and applications of genetic transformation and genome editing technologies in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {133},
number = {5},
pages = {1603-1622},
doi = {10.1007/s00122-019-03464-4},
pmid = {31654081},
issn = {1432-2242},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation, Plant ; *Genetic Engineering ; *Genome, Plant ; Plant Proteins/genetics/*metabolism ; *Transformation, Genetic ; Triticum/genetics/*growth &amp; development/metabolism ; },
abstract = {Wheat (Triticum aestivum) is a staple crop across the world and plays a remarkable role in food supplying security. Over the past few decades, basic and applied research on wheat has lagged behind other cereal crops due to the complex and polyploid genome and difficulties in genetic transformation. A breakthrough called as PureWheat was made in the genetic transformation of wheat in 2014 in Asia, leading to a noticeable progress of wheat genome editing. Due to this great achievement, it is predicated that wheat biotechnology revolution is arriving. Genome editing technologies using zinc finger nucleases, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats-associated endonucleases (CRISR/Cas) are becoming powerful tools for crop modification which can help biologists and biotechnologists better understand the processes of mutagenesis and genomic alteration. Among the three genome editing systems, CRISR/Cas has high specificity and activity, and therefore it is widely used in genetic engineering. Generally, the genome editing technologies depend on an efficient genetic transformation system. In this paper, we summarize recent progresses and applications on genetic transformation and genome editing in wheat. We also examine the future aspects of genetic transformation and genome editing. We believe that the technologies for wheat efficient genetic engineering and functional studies will become routine with the emergence of high-quality genomic sequences.},
}
@article {pmid31653950,
year = {2019},
author = {Hanasaki, M and Masumoto, H},
title = {CRISPR/Transposon gene integration (CRITGI) can manage gene expression in a retrotransposon-dependent manner.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {15300},
pmid = {31653950},
issn = {2045-2322},
support = {15K14461//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; },
mesh = {Binding Sites/genetics ; *CRISPR-Cas Systems ; Chromatin/genetics/metabolism ; *Gene Expression Regulation, Fungal ; Genes, Fungal/genetics ; Histones/metabolism ; Lysine/metabolism ; Methylation ; Models, Genetic ; Mutagenesis, Insertional/genetics ; Promoter Regions, Genetic/genetics ; Retroelements/*genetics ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; Transcription, Genetic ; Transcriptional Activation ; },
abstract = {The fine-tuning of gene expression contributes to both basic science and applications. Here, we develop a novel gene expression technology termed CRITGI (CRISPR/Transposon gene integration). CRITGI uses CRISPR/Cas9 to integrate multiple copies of the plasmid pTy1 into Ty1 loci, budding yeast retrotransposons. The pTy1 plasmid harbors a Ty1 consensus sequence for integration, a gene of interest with its own promoter and a selection marker gene. Interestingly, the expression of the pTy1 gene in Ty1 loci could be induced in synthetic complete amino acid depletion medium, which could activate the selection marker gene on pTy1. The induction or repression of the gene on pTy1 depended on Ty1 transcription. Activation of the selection marker gene on pTy1 triggered Ty1 transcription, which led to induction of the gene on pTy1. The gene on pTy1 was not transcribed with Ty1 mRNA; the transcription required its own promoter. Furthermore, the trimethylation of histone H3 on lysine 4, a landmark of transcriptionally active chromatin, accumulated at the 5' end of the gene on pTy1 following selection marker gene activation. Thus, CRITGI is a unique gene regulation system to induce the genes on pTy1 in amino acid depletion medium and utilizes Ty1 transcription to create a chromatin environment favorable for the transcription of the genes on pTy1.},
}
@article {pmid31653696,
year = {2019},
author = {Andreeva, A and Bekkhozhin, Z and Omertassova, N and Baizhumanov, T and Yeltay, G and Akhmetali, M and Toibazar, D and Utepbergenov, D},
title = {The apparent deglycase activity of DJ-1 results from the conversion of free methylglyoxal present in fast equilibrium with hemithioacetals and hemiaminals.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {49},
pages = {18863-18872},
pmid = {31653696},
issn = {1083-351X},
mesh = {Humans ; Parkinson Disease/*metabolism ; Protein Deglycase DJ-1/*metabolism ; Pyruvaldehyde/metabolism ; Serum Albumin, Bovine/metabolism ; },
abstract = {Loss-of-function mutations in the gene encoding human protein DJ-1 cause early onset of Parkinson's disease, suggesting that DJ-1 protects dopaminergic neurons. The molecular mechanisms underlying this neuroprotection are unclear; however, DJ-1 has been suggested to be a GSH-independent glyoxalase that detoxifies methylglyoxal (MGO) by converting it into lactate. It has also been suggested that DJ-1 serves as a deglycase that catalyzes hydrolysis of hemithioacetals and hemiaminals formed by reactions of MGO with the thiol and amino groups of proteins. In this report, we demonstrate that the equilibrium constant of reaction of MGO with thiols is ∼500 m[-1] at 37 °C and that the half-life of the resulting hemithioacetal is only 12 s. These thermodynamic parameters would dictate that a significant fraction of free MGO will be present in a fast equilibrium with hemithioacetals in solution. We found that removal of free MGO by DJ-1's glyoxalase activity forces immediate spontaneous decomposition of hemithioacetals due to the shift in equilibrium position. This spontaneous decomposition of hemithioacetals could be mistaken for deglycase activity of DJ-1. Furthermore, we demonstrate that higher initial concentrations of hemithioacetals are associated with lower rates of DJ-1-mediated conversion of MGO, ruling out the possibility that hemithioacetals are DJ-1 substrates. Experiments with CRISPR/Cas-generated DJ-1-knockout HEK293 cells revealed that DJ-1 does not protect against acute MGO toxicity or carboxymethylation of lysine residues in cells. Combined, our results suggest that DJ-1 does not possess protein deglycase activity.},
}
@article {pmid31653644,
year = {2020},
author = {De Rosa, L and Latella, MC and Secone Seconetti, A and Cattelani, C and Bauer, JW and Bondanza, S and De Luca, M},
title = {Toward Combined Cell and Gene Therapy for Genodermatoses.},
journal = {Cold Spring Harbor perspectives in biology},
volume = {12},
number = {5},
pages = {},
pmid = {31653644},
issn = {1943-0264},
mesh = {Animals ; CRISPR-Cas Systems ; Epidermis/metabolism ; Epidermolysis Bullosa/therapy ; Epidermolysis Bullosa Dystrophica/therapy ; Epidermolysis Bullosa, Junctional/therapy ; Genes, Dominant ; Genes, Recessive ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; Lentivirus/genetics ; Netherton Syndrome/therapy ; Retroviridae/genetics ; Simplexvirus ; Skin/metabolism ; Skin Diseases/*genetics/*therapy ; Stem Cells/cytology ; },
abstract = {To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.},
}
@article {pmid31652763,
year = {2019},
author = {Sun, L and , and Ke, F and Nie, Z and Wang, P and Xu, J},
title = {Citrus Genetic Engineering for Disease Resistance: Past, Present and Future.},
journal = {International journal of molecular sciences},
volume = {20},
number = {21},
pages = {},
pmid = {31652763},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Citrus/*genetics/microbiology/virology ; *Disease Resistance ; Gene Editing/methods ; Genetic Engineering/*methods ; Plant Breeding/*methods ; },
abstract = {Worldwide, citrus is one of the most important fruit crops and is grown in more than 130 countries, predominantly in tropical and subtropical areas. The healthy progress of the citrus industry has been seriously affected by biotic and abiotic stresses. Several diseases, such as canker and huanglongbing, etc., rigorously affect citrus plant growth, fruit quality, and yield. Genetic engineering technologies, such as genetic transformation and genome editing, represent successful and attractive approaches for developing disease-resistant crops. These genetic engineering technologies have been widely used to develop citrus disease-resistant varieties against canker, huanglongbing, and many other fungal and viral diseases. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based systems have made genome editing an indispensable genetic manipulation tool that has been applied to many crops, including citrus. The improved CRISPR systems, such as CRISPR/CRISPR-associated protein (Cas)9 and CRISPR/Cpf1 systems, can provide a promising new corridor for generating citrus varieties that are resistant to different pathogens. The advances in biotechnological tools and the complete genome sequence of several citrus species will undoubtedly improve the breeding for citrus disease resistance with a much greater degree of precision. Here, we attempt to summarize the recent successful progress that has been achieved in the effective application of genetic engineering and genome editing technologies to obtain citrus disease-resistant (bacterial, fungal, and virus) crops. Furthermore, we also discuss the opportunities and challenges of genetic engineering and genome editing technologies for citrus disease resistance.},
}
@article {pmid31651233,
year = {2019},
author = {Wang, L and Zhang, J},
title = {Prediction of sgRNA on-target activity in bacteria by deep learning.},
journal = {BMC bioinformatics},
volume = {20},
number = {1},
pages = {517},
pmid = {31651233},
issn = {1471-2105},
mesh = {Bacteria/*genetics ; CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing ; *Neural Networks, Computer ; RNA, Guide/*genetics ; Sequence Deletion ; },
abstract = {BACKGROUND: One of the main challenges for the CRISPR-Cas9 system is selecting optimal single-guide RNAs (sgRNAs). Recently, deep learning has enhanced sgRNA prediction in eukaryotes. However, the prokaryotic chromatin structure is different from eukaryotes, so models trained on eukaryotes may not apply to prokaryotes.<br><br>RESULTS: We designed and implemented a convolutional neural network to predict sgRNA activity in Escherichia coli. The network was trained and tested on the recently-released sgRNA activity dataset. Our convolutional neural network achieved excellent performance, yielding average Spearman correlation coefficients of 0.5817, 0.7105, and 0.3602, respectively for Cas9, eSpCas9 and Cas9 with a recA coding region deletion. We confirmed that the sgRNA prediction models trained on prokaryotes do not apply to eukaryotes and vice versa. We adopted perturbation-based approaches to analyze distinct biological patterns between prokaryotic and eukaryotic editing. Then, we improved the predictive performance of the prokaryotic Cas9 system by transfer learning. Finally, we determined that potential off-target scores accumulated on a genome-wide scale affect on-target activity, which could slightly improve on-target predictive performance.<br><br>CONCLUSIONS: We developed convolutional neural networks to predict sgRNA activity for wild type and mutant Cas9 in prokaryotes. Our results show that the prediction accuracy of our method is improved over state-of-the-art models.},
}
@article {pmid31649350,
year = {2019},
author = {Opar, A},
title = {CRISPR-edited babies arrived, and regulators are still racing to catch up.},
journal = {Nature medicine},
volume = {25},
number = {11},
pages = {1634-1636},
doi = {10.1038/s41591-019-0641-x},
pmid = {31649350},
issn = {1546-170X},
mesh = {CRISPR-Cas Systems/*genetics ; Cloning, Organism/ethics ; Gene Editing/*ethics ; Humans ; Infant ; },
}
@article {pmid31649251,
year = {2019},
author = {Grajcarek, J and Monlong, J and Nishinaka-Arai, Y and Nakamura, M and Nagai, M and Matsuo, S and Lougheed, D and Sakurai, H and Saito, MK and Bourque, G and Woltjen, K},
title = {Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4856},
pmid = {31649251},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA End-Joining Repair/*genetics ; Gain of Function Mutation ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Loss of Function Mutation ; Sequence Deletion/*genetics ; },
abstract = {The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.},
}
@article {pmid31649173,
year = {2019},
author = {Cohen, J},
title = {Prime editing promises to be a cut above CRISPR.},
journal = {Science (New York, N.Y.)},
volume = {366},
number = {6464},
pages = {406},
doi = {10.1126/science.366.6464.406},
pmid = {31649173},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering/*methods ; RNA, Guide/genetics ; },
}
@article {pmid31648792,
year = {2019},
author = {Li, Y and De la Paz, JA and Jiang, X and Liu, R and Pokkulandra, AP and Bleris, L and Morcos, F},
title = {Coevolutionary Couplings Unravel PAM-Proximal Constraints of CRISPR-SpCas9.},
journal = {Biophysical journal},
volume = {117},
number = {9},
pages = {1684-1691},
pmid = {31648792},
issn = {1542-0086},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Evolution, Molecular ; Fluorescence ; Genes, Reporter ; HEK293 Cells ; Humans ; Nucleotide Motifs/*genetics ; Reproducibility of Results ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system, an immune system analog found in prokaryotes, allows a single-guide RNA to direct a CRISPR-associated protein (Cas) with combined helicase and nuclease activity to DNA. The presence of a specific protospacer adjacent motif (PAM) next to the DNA target site plays a crucial role in determining both efficacy and specificity of gene editing. Herein, we introduce a coevolutionary framework to computationally unveil nonobvious molecular interactions in CRISPR systems and experimentally probe their functional role. Specifically, we use direct coupling analysis, a statistical inference framework used to infer direct coevolutionary couplings, in the context of protein/nucleic acid interactions. Applied to Streptococcus pyogenes Cas9, a Hamiltonian metric obtained from coevolutionary relationships reveals, to our knowledge, novel PAM-proximal nucleotide preferences at the seventh position of S. pyogenes Cas9 PAM (5'-NGRNNNT-3'), which was experimentally confirmed by in vitro and functional assays in human cells. We show that coevolved and conserved interactions point to specific clues toward rationally engineering new generations of Cas9 systems and may eventually help decipher the diversity of this family of proteins.},
}
@article {pmid31647102,
year = {2019},
author = {Zheng, Y and Han, J and Wang, B and Hu, X and Li, R and Shen, W and Ma, X and Ma, L and Yi, L and Yang, S and Peng, W},
title = {Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering.},
journal = {Nucleic acids research},
volume = {47},
number = {21},
pages = {11461-11475},
pmid = {31647102},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Cloning, Molecular/methods ; Gene Deletion ; Gene Editing/*methods ; Genome, Bacterial/genetics ; Metabolic Engineering/*methods ; Organisms, Genetically Modified ; Plasmids/genetics/metabolism ; Zymomonas/*genetics/metabolism ; },
abstract = {Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction-modification (R-M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R-M system. This study might shed light on exploiting and improving CRISPR-Cas systems in other microorganisms for genome editing and metabolic engineering practices.},
}
@article {pmid31647030,
year = {2019},
author = {Wang, Y and Gao, R and Wu, J and Xiong, YC and Wei, J and Zhang, S and Yang, B and Chen, J and Yang, L},
title = {Comparison of cytosine base editors and development of the BEable-GPS database for targeting pathogenic SNVs.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {218},
pmid = {31647030},
issn = {1474-760X},
mesh = {*APOBEC Deaminases ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; Genomics/*methods ; Humans ; *Point Mutation ; },
abstract = {A variety of base editors have been developed to achieve C-to-T editing in different genomic contexts. Here, we compare a panel of five base editors on their C-to-T editing efficiencies and product purity at commonly editable sites, including some human pathogenic C-to-T mutations. We further profile the accessibilities of 20 base editors to all possible pathogenic mutations in silico. Finally, we build the BEable-GPS (Base Editable prediction of Global Pathogenic SNVs) database for users to select proper base editors to model or correct disease-related mutations. The in vivo comparison and in silico profiling catalog the availability of base editors and their broad applications in biomedical studies.},
}
@article {pmid31646186,
year = {2019},
author = {Young, JK and Gasior, SL and Jones, S and Wang, L and Navarro, P and Vickroy, B and Barrangou, R},
title = {The repurposing of type I-E CRISPR-Cascade for gene activation in plants.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {383},
pmid = {31646186},
issn = {2399-3642},
mesh = {Biolistics ; *CRISPR-Cas Systems ; Chromosomes, Plant/genetics ; DNA, Plant/genetics ; Genes, Plant ; Genetic Engineering/*methods ; Plasmids/genetics ; Streptococcus thermophilus/genetics ; Transcriptional Activation ; Zea mays/embryology/*genetics ; },
abstract = {CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 and Cas12a, however, other CRISPR-Cas systems may provide unique opportunities for genome engineering. Indeed, the multi-subunit composition of class 1 systems offers to expand the number of domains and functionalities that may be recruited to a genomic target. Here we report DNA targeting in Zea mays using a class 1 type I-E CRISPR-Cas system from S. thermophilus. First, we engineer its Cascade complex to modulate gene expression by tethering a plant transcriptional activation domain to 3 different subunits. Next, using an immunofluorescent assay, we confirm Cascade cellular complex formation and observe enhanced gene activation when multiple subunits tagged with the transcriptional activator are combined. Finally, we examine Cascade mediated gene activation at chromosomal DNA targets by reprogramming Zea mays cells to change color.},
}
@article {pmid31645729,
year = {2019},
author = {Alseth, EO and Pursey, E and Luján, AM and McLeod, I and Rollie, C and Westra, ER},
title = {Bacterial biodiversity drives the evolution of CRISPR-based phage resistance.},
journal = {Nature},
volume = {574},
number = {7779},
pages = {549-552},
pmid = {31645729},
issn = {1476-4687},
support = {714478/ERC_/European Research Council/International ; },
mesh = {Bacteriophages/*genetics/*immunology/pathogenicity ; *Biodiversity ; CRISPR-Cas Systems/*genetics/immunology ; *Evolution, Molecular ; Pseudomonas aeruginosa/genetics/*immunology/pathogenicity/*virology ; Receptors, Virus/metabolism ; },
abstract = {About half of all bacteria carry genes for CRISPR-Cas adaptive immune systems[1], which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome[2]. Whereas CRISPR loci evolve rapidly in natural environments[3,4], bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions[5,6]. Here we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments[7,8]. Specifically, by using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with the mutation of phage receptors, and therefore tips the balance in favour of the evolution of CRISPR-based resistance. We also demonstrate that this has important knock-on effects for the virulence of P. aeruginosa, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.},
}
@article {pmid31644801,
year = {2020},
author = {Zafar, SA and Zaidi, SS and Gaba, Y and Singla-Pareek, SL and Dhankher, OP and Li, X and Mansoor, S and Pareek, A},
title = {Engineering abiotic stress tolerance via CRISPR/ Cas-mediated genome editing.},
journal = {Journal of experimental botany},
volume = {71},
number = {2},
pages = {470-479},
doi = {10.1093/jxb/erz476},
pmid = {31644801},
issn = {1460-2431},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/*physiology ; Gene Editing ; *Plant Breeding ; Plants, Genetically Modified/genetics/*physiology ; *Stress, Physiological ; },
abstract = {Abiotic stresses, including drought, salinity, temperature, and heavy metals, pose a major challenge for crop production and cause substantial yield reduction worldwide. Breeding tolerant cultivars against these abiotic stresses is the most sustainable and eco-friendly approach to cope with this challenge. Advances in genome editing technologies provide new opportunities for crop improvement by employing precision genome engineering for targeted crop traits. However, the selection of the candidate genes is critical for the success of achieving the desired traits. Broadly speaking, these genes could fall into two major categories, structural and regulatory genes. Structural genes encode proteins that provide stress tolerance directly, whereas regulatory genes act indirectly by controlling the expression of other genes involved in different cellular processes. Additionally, cis-regulatory sequences are also vital for achieving stress tolerance. We propose targeting of these regulatory and/or structural genes along with the cis-regulatory sequences via the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system as a robust, efficient, and practical approach for developing crop varieties resilient to climate change. We also discuss the possibility of creating novel quantitative trait loci for abiotic stress tolerance via the CRISPR/Cas-mediated targeting of promoters. It is hoped that these genome editing tools will not only make a significant contribution towards raising novel plant types having tolerance to multiple abiotic stresses but will also aid in public acceptance of these products in years to come. This article is an attempt to critically evaluate the suitability of available tools and the target genes for obtaining plants with improved tolerance to abiotic stresses.},
}
@article {pmid31644604,
year = {2019},
author = {Roy, A and Zhai, Y and Ortiz, J and Neff, M and Mandal, B and Mukherjee, SK and Pappu, HR},
title = {Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development.},
journal = {PloS one},
volume = {14},
number = {10},
pages = {e0223765},
pmid = {31644604},
issn = {1932-6203},
mesh = {Begomovirus/*genetics ; CRISPR-Cas Systems ; Disease Resistance/*genetics ; *Gene Editing ; *Genome, Viral ; Plant Diseases/*prevention &amp; control/*virology ; Tobacco/genetics/*virology ; Viral Load ; },
abstract = {Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.},
}
@article {pmid31642890,
year = {2019},
author = {Apura, P and Domingues, S and Viegas, SC and Arraiano, CM},
title = {Reprogramming bacteria with RNA regulators.},
journal = {Biochemical Society transactions},
volume = {47},
number = {5},
pages = {1279-1289},
doi = {10.1042/BST20190173},
pmid = {31642890},
issn = {1470-8752},
mesh = {5' Untranslated Regions ; *Bacterial Physiological Phenomena ; Base Pairing ; CRISPR-Cas Systems ; RNA, Bacterial/genetics ; Riboswitch ; *Synthetic Biology ; },
abstract = {The revolution of genomics and growth of systems biology urged the creation of synthetic biology, an engineering discipline aiming at recreating and reprogramming cellular functions for industrial needs. There has been a huge effort in synthetic biology to develop versatile and programmable genetic regulators that would enable the precise control of gene expression. Synthetic RNA components have emerged as a solution, offering a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their compactness, structure and way of action, several types of RNA devices that act on DNA, RNA and protein have been characterized and applied in synthetic biology. RNA-based approaches are more 'economical' for the cell, since they are generally not translated. These RNA-based strategies act on a much shorter time scale than transcription-based ones and can be more efficient than protein-based mechanisms. In this review, we explore these RNA components as building blocks in the RNA synthetic biology field, first by explaining their natural mode of action and secondly discussing how these RNA components have been exploited to rewire bacterial regulatory circuitry.},
}
@article {pmid31642391,
year = {2020},
author = {D'Costa, S and Rich, MJ and Diekman, BO},
title = {Engineered Cartilage from Human Chondrocytes with Homozygous Knockout of Cell Cycle Inhibitor p21.},
journal = {Tissue engineering. Part A},
volume = {26},
number = {7-8},
pages = {441-449},
doi = {10.1089/ten.TEA.2019.0214},
pmid = {31642391},
issn = {1937-335X},
mesh = {Blotting, Western ; Chondrocytes/*cytology/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cyclin-Dependent Kinase Inhibitor p21/genetics/*metabolism ; Gene Editing ; Glycosaminoglycans/metabolism ; Humans ; Immunohistochemistry ; Polymerase Chain Reaction ; Tissue Engineering/*methods ; },
abstract = {Osteoarthritis (OA) is a highly prevalent disease with limited treatment options. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of biallelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell-derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Furthermore, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis. Impact Statement This work provides two important advances to the field of tissue engineering. One is the demonstration that engineered cartilage tissue can be produced from genetically defined populations of primary human chondrocytes. While CRISPR/Cas-9 genome editing has been extensively used in cell lines that divide indefinitely, this work extends the technique to an engineered tissue model system to support investigation of genetic changes that affect cartilage production. A second contribution is the finding that chondrocytes with p21 knockout synthesized more cartilage matrix tissue than unedited controls. This supports the continued investigation of p21 as a potential barrier to effective cartilage regeneration.},
}
@article {pmid31641867,
year = {2019},
author = {Sinha, R and Sharma, B and Dangi, AK and Shukla, P},
title = {Recent metabolomics and gene editing approaches for synthesis of microbial secondary metabolites for drug discovery and development.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {35},
number = {11},
pages = {166},
pmid = {31641867},
issn = {1573-0972},
mesh = {Alkaloids/metabolism ; Antimitotic Agents/metabolism ; Biological Products ; CRISPR-Cas Systems ; Databases, Factual ; Drug Development/*methods ; Drug Discovery/*methods ; Gene Deletion ; Gene Editing/*methods ; Genetic Engineering/methods ; Metabolomics/*methods ; *Microbiological Phenomena ; Multivariate Analysis ; Mutagenesis, Insertional ; Pigments, Biological/metabolism ; *Secondary Metabolism ; Toxins, Biological/metabolism ; },
abstract = {Microbial secondary metabolites (SMs) have been identified as an important natural source of drugs for several metabolic and neurological diseases. Along with biomedical applications, SMs are also used in the food and biochemical industries. SMs include natural products such as pigments, alkaloids, toxins, antimicrobials obtained from cultured microorganisms, while other non-cultivable microorganisms have also acted as a rich source of SMs. But, the isolation of SMs from these sources is a very tedious task. Metabolomics provides complete identification and structural information about the entire cellular metabolome under specific conditions using highly sophisticated instrumentation. Further, gene editing techniques such as cloning and gene refactoring, including advanced CRISPR-Cas, can be used for engineering microbes that have the potential to produce natural SMs that were not produced in native microbial strain. The present review describes integrated metabolomics and gene editing approaches for the synthesis of novel microbial SMs and their potential application towards drug discovery and development.},
}
@article {pmid31641267,
year = {2019},
author = {Ledford, H},
title = {Super-precise new CRISPR tool could tackle a plethora of genetic diseases.},
journal = {Nature},
volume = {574},
number = {7779},
pages = {464-465},
pmid = {31641267},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; },
}
@article {pmid31641261,
year = {2019},
author = {Cyranoski, D},
title = {Russian 'CRISPR-baby' scientist has started editing genes in human eggs with goal of altering deaf gene.},
journal = {Nature},
volume = {574},
number = {7779},
pages = {465-466},
doi = {10.1038/d41586-019-03018-0},
pmid = {31641261},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; Connexin 26 ; Connexins/genetics ; Deafness/*genetics/*prevention &amp; control ; Female ; *Gene Editing ; Genetic Enhancement/ethics ; Goals ; Humans ; Mosaicism ; *Mutation ; Ovum/*metabolism ; Patient Safety/legislation &amp; jurisprudence ; Receptors, CCR5/genetics ; Russia ; Twins/genetics ; },
}
@article {pmid31640958,
year = {2019},
author = {Guo, R and Tian, Y and Jin, X and Chen, H and Wang, G and Huang, X and Li, B and Li, Z and Yang, J},
title = {[Generation of a novel HBeAg transgenic mice using CRISPR/Cas9 technique].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {9},
pages = {1017-1022},
pmid = {31640958},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Genetic Vectors ; Hepatitis B e Antigens/*genetics ; Hepatitis B virus ; Mice ; Mice, Inbred C57BL ; *Mice, Transgenic ; },
abstract = {OBJECTIVE: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique.<br><br>METHODS: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method.<br><br>RESULTS: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice.<br><br>CONCLUSIONS: We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.},
}
@article {pmid31639337,
year = {2020},
author = {Gibboney, S and Orvis, J and Kim, K and Johnson, CJ and Martinez-Feduchi, P and Lowe, EK and Sharma, S and Stolfi, A},
title = {Effector gene expression underlying neuron subtype-specific traits in the Motor Ganglion of Ciona.},
journal = {Developmental biology},
volume = {458},
number = {1},
pages = {52-63},
pmid = {31639337},
issn = {1095-564X},
support = {K99 HD084814/HD/NICHD NIH HHS/United States ; R00 HD084814/HD/NICHD NIH HHS/United States ; U54 HG006332/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Axon Guidance/physiology ; CRISPR-Cas Systems ; Calcium-Binding Proteins/biosynthesis/genetics/physiology ; Central Nervous System/cytology ; Centrosome/physiology ; Ciona intestinalis/cytology/embryology/*genetics/growth &amp; development ; Connectome ; Embryo, Nonmammalian ; Ganglia, Invertebrate/*cytology/growth &amp; development ; Gene Editing ; *Gene Expression Regulation, Developmental ; Interneurons/physiology/ultrastructure ; Larva ; Microtubules/physiology ; Motor Neurons/physiology ; Nerve Tissue Proteins/biosynthesis/genetics/physiology ; Netrin-1/biosynthesis/genetics/physiology ; Neurogenesis ; Neurons/*classification/physiology/ultrastructure ; Repressor Proteins/biosynthesis/genetics/physiology ; Transcriptome ; },
abstract = {The central nervous system of the Ciona larva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog of Ciona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential "effectors" of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular guidance cues part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline. Consistent with our predictions, CRISPR/Cas9-mediated, tissue-specific elimination of two such candidate effectors, Efcab6-related and Netrin1, impaired ddN polarized axon outgrowth across the midline.},
}
@article {pmid31639285,
year = {2019},
author = {Klein, SD and Nguyen, DC and Bhakta, V and Wong, D and Chang, VY and Davidson, TB and Martinez-Agosto, JA},
title = {Mutations in the sonic hedgehog pathway cause macrocephaly-associated conditions due to crosstalk to the PI3K/AKT/mTOR pathway.},
journal = {American journal of medical genetics. Part A},
volume = {179},
number = {12},
pages = {2517-2531},
pmid = {31639285},
issn = {1552-4833},
support = {P50 HD055784/HD/NICHD NIH HHS/United States ; T32 GM008042/GM/NIGMS NIH HHS/United States ; #6-FY12-324//March of Dimes Foundation/International ; K08 HL138305/HL/NHLBI NIH HHS/United States ; P50 CA211015/CA/NCI NIH HHS/United States ; UL1 TR000124/TR/NCATS NIH HHS/United States ; 9172/AS/Autism Speaks/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Child, Preschool ; Female ; Gene Deletion ; Haploinsufficiency ; Hedgehog Proteins/*metabolism ; Humans ; Infant ; Male ; Megalencephaly/diagnosis/*genetics/*metabolism ; Models, Biological ; *Mutation ; Neural Stem Cells ; Phosphatidylinositol 3-Kinases/*metabolism ; Proto-Oncogene Proteins c-akt/*metabolism ; *Signal Transduction ; TOR Serine-Threonine Kinases/*metabolism ; },
abstract = {The hedgehog (Hh) pathway is highly conserved and required for embryonic patterning and determination. Mutations in the Hh pathway are observed in sporadic tumors as well as under syndromic conditions. Common to these syndromes are the findings of polydactyly/syndactyly and brain overgrowth. The latter is also a finding most commonly observed in the cases of mutations in the PI3K/AKT/mTOR pathway. We have identified novel Hh pathway mutations and structural copy number variations in individuals with somatic overgrowth, macrocephaly, dysmorphic facial features, and developmental delay, which phenotypically closely resemble patients with phosphatase and tensin homolog (PTEN) mutations. We hypothesized that brain overgrowth and phenotypic overlap with syndromic overgrowth syndromes in these cases may be due to crosstalk between the Hh and PI3K/AKT/mTOR pathways. To test this, we modeled disease-associated variants by generating PTCH1 and Suppressor of Fused (SUFU) heterozygote cell lines using the CRISPR/Cas9 system. These cells demonstrate activation of PI3K signaling and increased phosphorylation of its downstream target p4EBP1 as well as a distinct cellular phenotype. To further investigate the mechanism underlying this crosstalk, we treated human neural stem cells with sonic hedgehog (SHH) ligand and performed transcriptional analysis of components of the mTOR pathway. These studies identified decreased expression of a set of mTOR negative regulators, leading to its activation. We conclude that there is a significant crosstalk between the SHH and PI3K/AKT/mTOR. We propose that this crosstalk is responsible for why mutations in PTCH1 and SUFU lead to macrocephaly phenotypes similar to those observed in PTEN hamartoma and other overgrowth syndromes associated with mutations in PI3K/AKT/mTOR pathway genes.},
}
@article {pmid31637258,
year = {2019},
author = {Zhang, J and Niu, H and Zhao, ZJ and Fu, X and Wang, Y and Zhang, X and Zhang, F and Zeng, L},
title = {CRISPR/Cas9 Knockout of Bak Mediates Bax Translocation to Mitochondria in response to TNFα/CHX-induced Apoptosis.},
journal = {BioMed research international},
volume = {2019},
number = {},
pages = {9071297},
pmid = {31637258},
issn = {2314-6141},
mesh = {Apoptosis/drug effects/*genetics ; CRISPR-Cas Systems/genetics ; Caspases/*genetics ; Cell Line, Tumor ; Cycloheximide/pharmacology ; Cytosol/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Mitochondria/drug effects/genetics ; Proto-Oncogene Proteins c-bcl-2/genetics ; RNA, Small Interfering/genetics ; Tumor Necrosis Factor-alpha/genetics/pharmacology ; bcl-2 Homologous Antagonist-Killer Protein/*genetics ; bcl-2-Associated X Protein/*genetics ; },
abstract = {TNFα/CHX-induced apoptosis is dependent on caspase-8 activation and regulated by Bcl-2. However, the specific participants and precise mechanisms underlying this apoptotic pathway are poorly understood. The proapoptotic proteins Bak and Bax-members of the Bcl-2 family-are essential for the functioning of the mitochondrial apoptotic pathway. In this study, we used the CRISPR/Cas9 system to knockout Bak in the human SH-SY5Y cell line and determined the effects of this knockout on TNFα/CHX-induced apoptosis. Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and then pro-apoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHX-induced PARP cleavage and caspase activation were blocked in BAK-/- cells or using specific siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using specific siRNA. Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak significantly decreased this translocation. These results indicate that TNFα/CHX-induced apoptosis does not occur in Bak-/- cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent.},
}
@article {pmid31636200,
year = {2019},
author = {Zhang, M and Hill, MC and Kadow, ZA and Suh, JH and Tucker, NR and Hall, AW and Tran, TT and Swinton, PS and Leach, JP and Margulies, KB and Ellinor, PT and Li, N and Martin, JF},
title = {Long-range Pitx2c enhancer-promoter interactions prevent predisposition to atrial fibrillation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {45},
pages = {22692-22698},
pmid = {31636200},
issn = {1091-6490},
support = {R01 HL118761/HL/NHLBI NIH HHS/United States ; U54 HD083092/HD/NICHD NIH HHS/United States ; U54 HG006348/HG/NHGRI NIH HHS/United States ; F30 HL145908/HL/NHLBI NIH HHS/United States ; F31 HL136065/HL/NHLBI NIH HHS/United States ; K01 HL140187/HL/NHLBI NIH HHS/United States ; R01 HL127717/HL/NHLBI NIH HHS/United States ; R01 HL136389/HL/NHLBI NIH HHS/United States ; R01 HL130804/HL/NHLBI NIH HHS/United States ; R01 DE023177/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Atrial Fibrillation/*genetics ; CRISPR-Cas Systems ; Chromosome Mapping ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Homeodomain Proteins/*genetics ; Mice ; Mice, Knockout ; *Promoter Regions, Genetic ; Transcription Factors/*genetics ; },
abstract = {Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to PITX2 Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2 AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2.},
}
@article {pmid31636095,
year = {2019},
author = {Kang, Y and Chu, C and Wang, F and Niu, Y},
title = {CRISPR/Cas9-mediated genome editing in nonhuman primates.},
journal = {Disease models &amp; mechanisms},
volume = {12},
number = {10},
pages = {},
pmid = {31636095},
issn = {1754-8411},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Mosaicism ; Mutation/genetics ; Primates/*genetics ; },
abstract = {Owing to their high similarity to humans, non-human primates (NHPs) provide an exceedingly suitable model for the study of human disease. In this Review, we summarize the history of transgenic NHP models and the progress of CRISPR/Cas9-mediated genome editing in NHPs, from the first proof-of-principle green fluorescent protein-expressing monkeys to sophisticated NHP models of human neurodegenerative disease that accurately phenocopy several complex disease features. We discuss not only the breakthroughs and advantages, but also the potential shortcomings of the application of the CRISPR/Cas9 system to NHPs that have emerged from the expanded understanding of this technology in recent years. Although off-target and mosaic mutations are the main concerns in CRISPR/Cas9-mediated NHP modeling, recent progress in genome editing techniques make it likely that these technical limitations will be overcome soon, bringing excellent prospects to human disease studies.},
}
@article {pmid31636040,
year = {2020},
author = {Morton, LT and Reijmers, RM and Wouters, AK and Kweekel, C and Remst, DFG and Pothast, CR and Falkenburg, JHF and Heemskerk, MHM},
title = {Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {1},
pages = {64-74},
pmid = {31636040},
issn = {1525-0024},
mesh = {Adoptive Transfer/adverse effects/*methods ; Animals ; Antigens, Neoplasm/immunology ; CD8-Positive T-Lymphocytes ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Genes, T-Cell Receptor ; Genetic Therapy/adverse effects/*methods ; Healthy Volunteers ; Humans ; K562 Cells ; Male ; Mice ; Mice, Inbred NOD ; Multiple Myeloma/pathology/*therapy ; Receptors, Antigen, T-Cell, alpha-beta/*genetics/immunology ; Transduction, Genetic ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Generation of an optimal T cell therapeutic expressing high frequencies of transgenic T cell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRαβ reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRαβ was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating T cell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 T cells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited T cell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRαβ chains can be incorporated in current T cell production protocols and is preferential to ensure an improved and safe clinical therapeutic.},
}
@article {pmid31636039,
year = {2020},
author = {Peng, WX and Koirala, P and Zhang, W and Ni, C and Wang, Z and Yang, L and Mo, YY},
title = {lncRNA RMST Enhances DNMT3 Expression through Interaction with HuR.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {1},
pages = {9-18},
pmid = {31636039},
issn = {1525-0024},
support = {R01 CA154989/CA/NCI NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; CRISPR-Cas Systems ; DNA (Cytosine-5-)-Methyltransferases/chemistry/genetics/*metabolism ; DNA Methylation/genetics ; ELAV-Like Protein 1/chemistry/*metabolism ; Enzyme Stability/genetics ; Gene Knockout Techniques ; Genes, Reporter ; HCT116 Cells ; HEK293 Cells ; Humans ; MCF-7 Cells ; RNA, Guide/genetics ; RNA, Long Noncoding/genetics/*metabolism ; Transfection ; Up-Regulation/*genetics ; },
abstract = {Large bodies of studies have shown that the CRISPR/Cas9-based library screening is a very powerful tool for the identification of gene functions. However, most of these studies have focused on protein-coding genes, and, furthermore, very few studies have used gene reporters for screening. In the present study, we generated DNA methyltransferase 3B (DNMT3B) reporter and screened a CRISPR/Cas9 synergistic activation mediator (SAM) library against a focused group of lncRNAs. With this screening approach, we identified Rhabdomyosarcoma 2-Associated Transcript (RMST) as a positive regulator for DNMT3B. This was confirmed by activation of the endogenous RMST by SAM or ectopic expression of RMST. Moreover, RMST knockout (KO) suppresses DNMT3, while rescue with RMST in the KO cells restores the DNMT3 level. Finally, RMST KO suppresses global DNA methylation, leading to the upregulation of methylation-regulated genes. Mechanistically, RMST promotes the interaction between the RNA-binding protein HuR and DNMT3B 3' UTR, increasing the DNMT3B stability. Together, these results not only provide the feasibility of a reporter system for CRISPR library screening but also demonstrate the previously uncharacterized factor RMST as an important player in the modulation of DNA methylation.},
}
@article {pmid31634585,
year = {2020},
author = {Xu, W and Yang, Y and Liu, Y and Kang, G and Wang, F and Li, L and Lv, X and Zhao, S and Yuan, S and Song, J and Wu, Y and Feng, F and He, X and Zhang, C and Song, W and Zhao, J and Yang, J},
title = {Discriminated sgRNAs-Based SurroGate System Greatly Enhances the Screening Efficiency of Plant Base-Edited Cells.},
journal = {Molecular plant},
volume = {13},
number = {1},
pages = {169-180},
doi = {10.1016/j.molp.2019.10.007},
pmid = {31634585},
issn = {1752-9867},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Order ; Genetic Vectors/*genetics ; *Genome, Plant ; Genotype ; Mutation ; Oryza/genetics ; Plant Cells ; RNA, Guide/chemistry/*genetics ; RNA, Plant/chemistry/genetics ; Whole Genome Sequencing ; },
abstract = {The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.},
}
@article {pmid31634458,
year = {2020},
author = {Alisjahbana, A and Mohammad, I and Gao, Y and Evren, E and Ringqvist, E and Willinger, T},
title = {Human macrophages and innate lymphoid cells: Tissue-resident innate immunity in humanized mice.},
journal = {Biochemical pharmacology},
volume = {174},
number = {},
pages = {113672},
doi = {10.1016/j.bcp.2019.113672},
pmid = {31634458},
issn = {1873-2968},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Communicable Diseases/genetics/immunology ; Disease Models, Animal ; Gene Editing ; Humans ; *Immunity, Innate/genetics ; Lymphocytes/*immunology ; Lymphoid Tissue/cytology/*immunology ; Macrophages/*immunology ; Mice ; *Models, Animal ; Neoplasms/genetics/immunology ; Species Specificity ; },
abstract = {Macrophages and innate lymphoid cells (ILCs) are tissue-resident cells that play important roles in organ homeostasis and tissue immunity. Their intricate relationship with the organs they reside in allows them to quickly respond to perturbations of organ homeostasis and environmental challenges, such as infection and tissue injury. Macrophages and ILCs have been extensively studied in mice, yet important species-specific differences exist regarding innate immunity between humans and mice. Complementary to ex-vivo studies with human cells, humanized mice (i.e. mice with a human immune system) offer the opportunity to study human macrophages and ILCs in vivo within their surrounding tissue microenvironments. In this review, we will discuss how humanized mice have helped gain new knowledge about the basic biology of these cells, as well as their function in infectious and malignant conditions. Furthermore, we will highlight active areas of investigation related to human macrophages and ILCs, such as their cellular heterogeneity, ontogeny, tissue residency, and plasticity. In the near future, we expect more fundamental discoveries in these areas through the combined use of improved humanized mouse models together with state-of-the-art technologies, such as single-cell RNA-sequencing and CRISPR/Cas9 genome editing.},
}
@article {pmid31634414,
year = {2019},
author = {Bao, X and Adil, MM and Muckom, R and Zimmermann, JA and Tran, A and Suhy, N and Xu, Y and Sampayo, RG and Clark, DS and Schaffer, DV},
title = {Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {12},
pages = {1556-1566},
doi = {10.1002/stem.3096},
pmid = {31634414},
issn = {1549-4918},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Cell Lineage/*genetics ; Gene Editing/methods ; Gene Expression Regulation/*genetics ; Gene Knock-In Techniques ; Gene Targeting ; Genes, Reporter/*genetics ; Humans ; Pluripotent Stem Cells/*cytology ; Transcription Factors/metabolism ; WT1 Proteins/*genetics ; },
abstract = {Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556-1566.},
}
@article {pmid31634348,
year = {2019},
author = {Serrat, X and Kukhtar, D and Cornes, E and Esteve-Codina, A and Benlloch, H and Cecere, G and Cerón, J},
title = {CRISPR editing of sftb-1/SF3B1 in Caenorhabditis elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing.},
journal = {PLoS genetics},
volume = {15},
number = {10},
pages = {e1008464},
pmid = {31634348},
issn = {1553-7404},
mesh = {Alternative Splicing/drug effects/genetics ; Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/*genetics ; Disease Models, Animal ; High-Throughput Screening Assays/methods ; Homozygote ; Humans ; Mutation, Missense ; Neoplasms/drug therapy/*genetics ; Protein Domains/genetics ; RNA Interference ; RNA Splicing Factors/*genetics ; Ribonucleoprotein, U2 Small Nuclear/*genetics ; Spliceosomes/drug effects ; Synthetic Lethal Mutations ; },
abstract = {SF3B1 is the most frequently mutated splicing factor in cancer. Mutations in SF3B1 likely confer clonal advantages to cancer cells but they may also confer vulnerabilities that can be therapeutically targeted. SF3B1 cancer mutations can be maintained in homozygosis in C. elegans, allowing synthetic lethal screens with a homogeneous population of animals. These mutations cause alternative splicing (AS) defects in C. elegans, as it occurs in SF3B1-mutated human cells. In a screen, we identified RNAi of U2 snRNP components that cause synthetic lethality with sftb-1/SF3B1 mutations. We also detected synthetic interactions between sftb-1 mutants and cancer-related mutations in uaf-2/U2AF1 or rsp-4/SRSF2, demonstrating that this model can identify interactions between mutations that are mutually exclusive in human tumors. Finally, we have edited an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B and herboxidiene. Thus, we have established a multicellular model for SF3B1 mutations amenable for high-throughput genetic and chemical screens.},
}
@article {pmid31634055,
year = {2019},
author = {Hunt, CR and Pandita, TK},
title = {"What's Past is Prologue": Pre-Existing Epigenetic Transcriptional Marks May Also Influence DNA Repair Pathway Choice.},
journal = {Radiation research},
volume = {192},
number = {6},
pages = {577-578},
doi = {10.1667/RR15541.1},
pmid = {31634055},
issn = {1938-5404},
mesh = {CRISPR-Cas Systems ; Chromatin/chemistry ; Chromosomes/genetics ; *DNA Repair ; *Epigenesis, Genetic ; *Homologous Recombination ; Humans ; Transcription, Genetic ; },
}
@article {pmid31633690,
year = {2019},
author = {Sano, S and Wang, Y and Evans, MA and Yura, Y and Sano, M and Ogawa, H and Horitani, K and Doviak, H and Walsh, K},
title = {Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {152},
pages = {},
pmid = {31633690},
issn = {1940-087X},
support = {R01 AG073249/AG/NIA NIH HHS/United States ; R01 HL138014/HL/NHLBI NIH HHS/United States ; R01 HL139819/HL/NHLBI NIH HHS/United States ; R01 HL141256/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Bone Marrow/radiation effects ; Bone Marrow Cells/cytology ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Lineage ; Disease Models, Animal ; *Gene Editing ; Hematopoietic Stem Cells/*metabolism ; Lentivirus/*genetics ; Male ; Mice, Inbred C57BL ; Polyethyleneimine/chemistry ; RNA, Guide/genetics ; },
abstract = {Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.},
}
@article {pmid31633477,
year = {2019},
author = {Liu, W and Yang, C and Liu, Y and Jiang, G},
title = {CRISPR/Cas9 System and its Research Progress in Gene Therapy.},
journal = {Anti-cancer agents in medicinal chemistry},
volume = {19},
number = {16},
pages = {1912-1919},
doi = {10.2174/1871520619666191014103711},
pmid = {31633477},
issn = {1875-5992},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Mutation ; Skin Diseases/genetics/*therapy ; },
abstract = {Genome editing refers to changing the genome sequence of an organism by knockout, insertion, and site mutation, resulting in changes in the genetic information of the organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein-9 nuclease (Cas9) system is a genome editing technique developed by the acquired immune system in the microbes, such as bacteria and archaebacteria, which targets and edits genome sequences according to the principle of complementary base pairing. This technique can be used to edit endogenous genomic DNA sequences in organisms accurately and has been widely used in fields, such as biotechnology, cancer gene therapy, and dermatology. In this review, we summarize the history, structure, mechanism, and application of CRISPR/Cas9 in gene therapy and dermatological diseases.},
}
@article {pmid31633062,
year = {2019},
author = {Ikeda, A and Fujii, W and Sugiura, K and Naito, K},
title = {High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {371},
pmid = {31633062},
issn = {2399-3642},
mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/physiology ; Female ; Gene Editing/*methods ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation ; Polymorphism, Single Nucleotide ; *Zygote/metabolism ; },
abstract = {CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.},
}
@article {pmid31631510,
year = {2019},
author = {Xiong, Z and Xie, Y and Yang, Y and Xue, Y and Wang, D and Lin, S and Chen, D and Lu, D and He, L and Song, B and Yang, Y and Sun, X},
title = {Efficient gene correction of an aberrant splice site in β-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.},
journal = {Journal of cellular and molecular medicine},
volume = {23},
number = {12},
pages = {8046-8057},
pmid = {31631510},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Hematopoiesis ; Humans ; Induced Pluripotent Stem Cells/chemistry/*metabolism ; Mutation ; Oligodeoxyribonucleotides/*genetics ; RNA Cleavage/genetics ; RNA Splice Sites ; RNA Splicing/*genetics ; Whole Exome Sequencing ; beta-Globins/*genetics/metabolism ; beta-Thalassemia/*genetics/metabolism ; },
abstract = {β-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.},
}
@article {pmid31631325,
year = {2020},
author = {Duroy, PO and Bosshard, S and Schmid-Siegert, E and Neuenschwander, S and Arib, G and Lemercier, P and Masternak, J and Roesch, L and Buron, F and Girod, PA and Xenarios, I and Mermod, N},
title = {Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {2},
pages = {466-485},
pmid = {31631325},
issn = {1097-0290},
support = {17196.1 PFLS-LS//Swiss Government Commission for Technology and Innovation/International ; //Universit&#xe9; de Lausanne/International ; //Selexis SA/International ; },
mesh = {Animals ; CHO Cells/*virology ; CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Drug Contamination/prevention &amp; control ; *Endogenous Retroviruses/genetics/metabolism ; Gene Editing ; Genome, Viral/genetics ; Loss of Function Mutation/genetics ; Mutagenesis, Site-Directed/*methods ; *RNA, Viral/genetics/metabolism ; Virion/*genetics ; },
abstract = {The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type-C endogenous retrovirus (ERV) sequences in their genome and to release retroviral-like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type-C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type-C ERV sequences clustering into three functionally conserved groups. Transcripts from one type-C ERV group were full-length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral-like particles, suggesting that this ERV group may produce functional viruses. CRISPR-Cas9 genome editing was used to disrupt the gag gene of the expressed type-C ERV group. Comparison of CRISPR-derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA-loaded viral particles. Clones bearing a Gag loss-of-function mutation in this ERV showed a reduction of RNA-containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.},
}
@article {pmid31631317,
year = {2020},
author = {Karottki, KJC and Hefzi, H and Xiong, K and Shamie, I and Hansen, AH and Li, S and Pedersen, LE and Li, S and Lee, JS and Lee, GM and Kildegaard, HF and Lewis, NE},
title = {Awakening dormant glycosyltransferases in CHO cells with CRISPRa.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {2},
pages = {593-598},
pmid = {31631317},
issn = {1097-0290},
support = {R35 GM119850/GM/NIGMS NIH HHS/United States ; NNF16OC0021638//Novo Nordisk Foundation/International ; NNF10CC1016517//Novo Nordisk Foundation/International ; },
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/*genetics ; Cricetinae ; Cricetulus ; Gene Editing/*methods ; Glycosylation ; Glycosyltransferases/*genetics ; Phenotype ; Polysaccharides/analysis/chemistry ; },
abstract = {Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.},
}
@article {pmid31630543,
year = {2019},
author = {Arkhipov, SN and Potter, DL and Geurts, AM and Pavlov, TS},
title = {Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD.},
journal = {American journal of physiology. Renal physiology},
volume = {317},
number = {6},
pages = {F1649-F1655},
pmid = {31630543},
issn = {1522-1466},
support = {P30 DK090868/DK/NIDDK NIH HHS/United States ; R00 DK105160/DK/NIDDK NIH HHS/United States ; R00 HL116603/HL/NHLBI NIH HHS/United States ; },
mesh = {Adenosine Triphosphate/urine ; Animals ; CRISPR-Cas Systems ; Connexins/biosynthesis/genetics ; Cysts/genetics/*pathology ; Epithelial Sodium Channels/metabolism ; Female ; Gene Knockout Techniques ; Kidney Diseases/genetics/*pathology ; Mutagenesis, Insertional ; Nerve Tissue Proteins/biosynthesis/genetics ; Polycystic Kidney, Autosomal Recessive/genetics/*metabolism ; Pregnancy ; Rats ; Receptors, Purinergic P2X7/genetics/*metabolism ; Sodium/metabolism ; },
abstract = {The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK.P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK.P2rx7[+] littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.},
}
@article {pmid31630114,
year = {2020},
author = {Benson, DL},
title = {Of Molecules and Mechanisms.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {40},
number = {1},
pages = {81-88},
pmid = {31630114},
issn = {1529-2401},
support = {R01 MH103455/MH/NIMH NIH HHS/United States ; R01 MH104491/MH/NIMH NIH HHS/United States ; R01 NS107512/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Exocytosis ; Gene Expression Regulation ; Gene Transfer Techniques/history ; Genes, Reporter ; History, 20th Century ; History, 21st Century ; Humans ; In Situ Hybridization/history/methods ; Microscopy/history/methods ; Molecular Biology/*history/methods ; Nerve Tissue Proteins/genetics/physiology ; Neurosciences/*history ; PDZ Domains ; Polymerase Chain Reaction/history ; Protein Engineering/history ; RNA/genetics ; Recombinant Proteins ; Sequence Analysis, DNA/history/methods ; },
abstract = {Without question, molecular biology drives modern neuroscience. The past 50 years has been nothing short of revolutionary as key findings have moved the field from correlation toward causation. Most obvious are the discoveries and strategies that have been used to build tools for visualizing circuits, measuring activity, and regulating behavior. Less flashy, but arguably as important are the myriad investigations uncovering the actions of single molecules, macromolecular structures, and integrated machines that serve as the basis for constructing cellular and signaling pathways identified in wide-scale gene or RNA studies and for feeding data into informational networks used in systems biology. This review follows the pathways that were opened in neuroscience by major discoveries and set the stage for the next 50 years.},
}
@article {pmid31628330,
year = {2019},
author = {Wagenblast, E and Azkanaz, M and Smith, SA and Shakib, L and McLeod, JL and Krivdova, G and Araújo, J and Shultz, LD and Gan, OI and Dick, JE and Lechman, ER},
title = {Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4730},
pmid = {31628330},
issn = {2041-1723},
support = {R01 AI132963/AI/NIAID NIH HHS/United States ; U01 DK104218/DK/NIDDK NIH HHS/United States ; UC4 DK104218/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Cell Proliferation/*genetics ; Electroporation/methods ; Female ; GATA1 Transcription Factor/genetics/metabolism ; Gene Editing/*methods ; Hematopoietic Stem Cells/cytology/*metabolism ; Humans ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Transplantation, Heterologous ; },
abstract = {In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.},
}
@article {pmid31627701,
year = {2019},
author = {Zatloukalová, P and Krejčíř, R and Valík, D and Vojtěšek, B},
title = {CRISPR-Cas9 as a Tool in Cancer Therapy.},
journal = {Klinicka onkologie : casopis Ceske a Slovenske onkologicke spolecnosti},
volume = {32},
number = {Supplementum 3},
pages = {13-18},
doi = {10.14735/amko20193S},
pmid = {31627701},
issn = {1802-5307},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; *Molecular Targeted Therapy ; Neoplasms/genetics/immunology/*therapy ; Prognosis ; T-Lymphocytes/*immunology ; },
abstract = {BACKGROUND: Genome editing using CRISPR-Cas9 has become one of the basic methods of biological research over a short period of time. This recently discovered system of adaptive immunity of bacteria has been adapted to the needs of science and has become a valuable tool for DNA manipulation. Its simplicity and reliability have contributed to widespread use of the method. Genome editing refers to targeted modifications of genomic DNA with single base pair accuracy. CRISPR-Cas9 differs significantly from previous technologies in the simplicity of directing the enzyme to the target sequence. In the field of cancer research, CRISPR-Cas9 has enabled the development of a number of models for the study of carcinogenesis and drug testing. From a therapeutic point of view, CRISPR-Cas9 has been applied in the field of immunotherapy, especially in ex vivo genetic modifications of the T-cells of patients.<br><br>AIM: Currently, several clinical trials are trying to verify the therapeutic potential of CRISPR-Cas9. Based on these studies, we have summarised the strategies used in the preparation of therapeutic tools useful in cancer therapy.<br><br>CONCLUSION: CRISPR-Cas9 appears to be crucial in basic research, particularly in the study of the function of individual genes involved in carcinogenesis. However, it will still be necessary to optimise the efficacy, safety and specificity of CRISPR-Cas9 before it is used in clinical practice.},
}
@article {pmid31627415,
year = {2019},
author = {Kleinert, RDV and Montoya-Diaz, E and Khera, T and Welsch, K and Tegtmeyer, B and Hoehl, S and Ciesek, S and Brown, RJP},
title = {Yellow Fever: Integrating Current Knowledge with Technological Innovations to Identify Strategies for Controlling a Re-Emerging Virus.},
journal = {Viruses},
volume = {11},
number = {10},
pages = {},
pmid = {31627415},
issn = {1999-4915},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Outbreaks/prevention &amp; control ; *Host-Pathogen Interactions ; Humans ; Mosquito Vectors/genetics/*virology ; Neglected Diseases/prevention &amp; control/virology ; Primates/*virology ; Viral Tropism ; Yellow Fever/immunology/*prevention &amp; control/transmission ; Yellow Fever Vaccine/immunology ; Yellow fever virus/immunology/*pathogenicity ; },
abstract = {Yellow fever virus (YFV) represents a re-emerging zoonotic pathogen, transmitted by mosquito vectors to humans from primate reservoirs. Sporadic outbreaks of YFV occur in endemic tropical regions, causing a viral hemorrhagic fever (VHF) associated with high mortality rates. Despite a highly effective vaccine, no antiviral treatments currently exist. Therefore, YFV represents a neglected tropical disease and is chronically understudied, with many aspects of YFV biology incompletely defined including host range, host-virus interactions and correlates of host immunity and pathogenicity. In this article, we review the current state of YFV research, focusing on the viral lifecycle, host responses to infection, species tropism and the success and associated limitations of the YFV-17D vaccine. In addition, we highlight the current lack of available treatments and use publicly available sequence and structural data to assess global patterns of YFV sequence diversity and identify potential drug targets. Finally, we discuss how technological advances, including real-time epidemiological monitoring of outbreaks using next-generation sequencing and CRISPR/Cas9 modification of vector species, could be utilized in future battles against this re-emerging pathogen which continues to cause devastating disease.},
}
@article {pmid31626775,
year = {2019},
author = {Feldman, D and Singh, A and Schmid-Burgk, JL and Carlson, RJ and Mezger, A and Garrity, AJ and Zhang, F and Blainey, PC},
title = {Optical Pooled Screens in Human Cells.},
journal = {Cell},
volume = {179},
number = {3},
pages = {787-799.e17},
pmid = {31626775},
issn = {1097-4172},
support = {R01 HG009283/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; P50 HG006193/HG/NHGRI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/genetics/metabolism ; *Genetic Testing ; *Genomics ; Humans ; Mediator Complex/genetics ; NF-kappa B/*genetics ; RNA, Guide/genetics ; Transcription Factor RelA/*genetics ; },
abstract = {Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.},
}
@article {pmid31626589,
year = {2019},
author = {Mangas, EL and Rubio, A and Álvarez-Marín, R and Labrador-Herrera, G and Pachón, J and Pachón-Ibáñez, ME and Divina, F and Pérez-Pulido, AJ},
title = {Pangenome of Acinetobacter baumannii uncovers two groups of genomes, one of them with genes involved in CRISPR/Cas defence systems associated with the absence of plasmids and exclusive genes for biofilm formation.},
journal = {Microbial genomics},
volume = {5},
number = {11},
pages = {},
pmid = {31626589},
issn = {2057-5858},
mesh = {Acinetobacter baumannii/*genetics ; Bacteria/genetics ; Bacterial Proteins/genetics ; Biofilms ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial/genetics ; Genomics ; Phylogeny ; Plasmids/*genetics ; },
abstract = {Acinetobacter baumannii is an opportunistic bacterium that causes hospital-acquired infections with a high mortality and morbidity, since there are strains resistant to virtually any kind of antibiotic. The chase to find novel strategies to fight against this microbe can be favoured by knowledge of the complete catalogue of genes of the species, and their relationship with the specific characteristics of different isolates. In this work, we performed a genomics analysis of almost 2500 strains. Two different groups of genomes were found based on the number of shared genes. One of these groups rarely has plasmids, and bears clustered regularly interspaced short palindromic repeat (CRISPR) sequences, in addition to CRISPR-associated genes (cas genes) or restriction-modification system genes. This fact strongly supports the lack of plasmids. Furthermore, the scarce plasmids in this group also bear CRISPR sequences, and specifically contain genes involved in prokaryotic toxin-antitoxin systems that could either act as the still little known CRISPR type IV system or be the precursors of other novel CRISPR/Cas systems. In addition, a limited set of strains present a new cas9-like gene, which may complement the other cas genes in inhibiting the entrance of new plasmids into the bacteria. Finally, this group has exclusive genes involved in biofilm formation, which would connect CRISPR systems to the biogenesis of these bacterial resistance structures.},
}
@article {pmid31625560,
year = {2020},
author = {Alankarage, D and Szot, JO and Pachter, N and Slavotinek, A and Selleri, L and Shieh, JT and Winlaw, D and Giannoulatou, E and Chapman, G and Dunwoodie, SL},
title = {Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.},
journal = {Human molecular genetics},
volume = {29},
number = {7},
pages = {1068-1082},
pmid = {31625560},
issn = {1460-2083},
support = {R01 DE024745/DE/NIDCR NIH HHS/United States ; },
mesh = {Adult ; Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Exome/genetics ; Female ; Heart Defects, Congenital/*genetics/pathology ; Heterozygote ; Humans ; Infant ; Male ; Mice ; Mutation, Missense/genetics ; Pedigree ; Phenotype ; Pre-B-Cell Leukemia Transcription Factor 1/*genetics ; Truncus Arteriosus, Persistent/*genetics/pathology ; Whole Exome Sequencing ; },
abstract = {Pre-B cell leukemia factor 1 (PBX1) is an essential developmental transcription factor, mutations in which have recently been associated with CAKUTHED syndrome, characterized by multiple congenital defects including congenital heart disease (CHD). During analysis of a whole-exome-sequenced cohort of heterogeneous CHD patients, we identified a de novo missense variant, PBX1:c.551G>C p.R184P, in a patient with tetralogy of Fallot with absent pulmonary valve and extra-cardiac phenotypes. Functional analysis of this variant by creating a CRISPR-Cas9 gene-edited mouse model revealed multiple congenital anomalies. Congenital heart defects (persistent truncus arteriosus and ventricular septal defect), hypoplastic lungs, hypoplastic/ectopic kidneys, aplastic adrenal glands and spleen, as well as atretic trachea and palate defects were observed in the homozygous mutant embryos at multiple stages of development. We also observed developmental anomalies in a proportion of heterozygous embryos, suggestive of a dominant mode of inheritance. Analysis of gene expression and protein levels revealed that although Pbx1 transcripts are higher in homozygotes, amounts of PBX1 protein are significantly decreased. Here, we have presented the first functional model of a missense PBX1 variant and provided strong evidence that p.R184P is disease-causal. Our findings also expand the phenotypic spectrum associated with pathogenic PBX1 variants in both humans and mice.},
}
@article {pmid31625446,
year = {2019},
author = {Román, E and Prieto, D and Alonso-Monge, R and Pla, J},
title = {New insights of CRISPR technology in human pathogenic fungi.},
journal = {Future microbiology},
volume = {14},
number = {},
pages = {1243-1255},
doi = {10.2217/fmb-2019-0183},
pmid = {31625446},
issn = {1746-0921},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Fungi/*genetics/pathogenicity ; Gene Editing ; *Genetic Engineering ; *Genome, Fungal ; Humans ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems have emerged as a powerful tool for genome manipulation. Class 2 type II CRISPR/CAS9 is so far the most studied system and has been implemented in many biological systems such as mammalian cells, plants, fungi and bacteria. Fungi are important causes of human diseases worldwide. Genetic manipulation of pathogenic fungi is critical to develop new therapeutic approaches and novel antifungals. We will review here the progress done with CRISPR/CAS9 systems in human pathogenic fungi, with emphasis in Candida albicans and the main modifications that have improved their usefulness in biological research. We finally discuss possible future outcomes and applications to the developed in a near future.},
}
@article {pmid31625429,
year = {2020},
author = {Garcia-Robledo, JE and Barrera, MC and Tobón, GJ},
title = {CRISPR/Cas: from adaptive immune system in prokaryotes to therapeutic weapon against immune-related diseases.},
journal = {International reviews of immunology},
volume = {39},
number = {1},
pages = {11-20},
doi = {10.1080/08830185.2019.1677645},
pmid = {31625429},
issn = {1563-5244},
mesh = {Adaptive Immunity/genetics/immunology ; Archaea/genetics/*immunology ; Autoimmune Diseases/genetics/microbiology/*therapy ; Bacteria/genetics/*immunology ; *CRISPR-Cas Systems ; Eukaryota/*immunology/metabolism ; Gene Editing/methods ; Humans ; Immune System/immunology/metabolism ; Neoplasms/genetics/microbiology/*therapy ; },
abstract = {CRISPR/Cas evolved as an adaptive immune system in bacteria and archaea to inactivate foreign viral and plasmid DNA. However, the capacities of various CRISPR/Cas systems for precise genome editing based on sequence homology also allow their use as tools for genomic and epigenomic modification in eukaryotes. Indeed, these genetic characteristics have proven useful for disease modeling and testing the specific functions of target genes under pathological conditions. Moreover, recent studies provide compelling evidence that CRISPR/Cas systems could be useful therapeutic tools against human diseases, including cancer, monogenic disorders, and autoimmune disorders.HighlightsCRISPR/Cas evolved as an adaptive immune system in bacteria and archaea.CRISPR/Cas systems are nowadays used as tools for genomic modification.CRISPR/Cas systems could be useful therapeutic tools against human disease, including autoimmune conditions.},
}
@article {pmid31624870,
year = {2019},
author = {Almeida, S and Krishnan, G and Rushe, M and Gu, Y and Kankel, MW and Gao, FB},
title = {Production of poly(GA) in C9ORF72 patient motor neurons derived from induced pluripotent stem cells.},
journal = {Acta neuropathologica},
volume = {138},
number = {6},
pages = {1099-1101},
pmid = {31624870},
issn = {1432-0533},
support = {R21 NS112766/NS/NINDS NIH HHS/United States ; R37 NS057553/NS/NINDS NIH HHS/United States ; RF1 NS101986/NS/NINDS NIH HHS/United States ; R01 NS093097/NS/NINDS NIH HHS/United States ; },
mesh = {Brain/metabolism ; C9orf72 Protein/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; DNA-Binding Proteins/metabolism ; HSP70 Heat-Shock Proteins/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Introns ; Motor Neurons/*metabolism ; Neurogenesis ; Proteins/*metabolism ; RNA, Messenger/metabolism ; Sequence Deletion ; },
}
@article {pmid31624845,
year = {2020},
author = {Pourcel, C and Touchon, M and Villeriot, N and Vernadet, JP and Couvin, D and Toffano-Nioche, C and Vergnaud, G},
title = {CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers.},
journal = {Nucleic acids research},
volume = {48},
number = {D1},
pages = {D535-D544},
pmid = {31624845},
issn = {1362-4962},
mesh = {Archaea/classification/enzymology/genetics ; Bacteria/classification/enzymology/genetics ; CRISPR-Associated Proteins/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Databases, Genetic ; *Genome, Archaeal ; *Genome, Bacterial ; Phylogeny ; *Software ; },
abstract = {In Archaea and Bacteria, the arrays called CRISPRs for 'clustered regularly interspaced short palindromic repeats' and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR-Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR-Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.},
}
@article {pmid31624354,
year = {2019},
author = {Bauer, DE},
title = {Production of foetal globin in adult monkeys.},
journal = {Nature biomedical engineering},
volume = {3},
number = {11},
pages = {857-859},
doi = {10.1038/s41551-019-0475-3},
pmid = {31624354},
issn = {2157-846X},
mesh = {Anemia, Sickle Cell/genetics ; Animals ; Bone Marrow Transplantation ; CRISPR-Cas Systems ; Gene Editing/*methods ; Globins/*biosynthesis/*genetics ; Haplorhini ; Hemoglobins/genetics ; Macaca mulatta ; Mutation ; *Stem Cell Transplantation ; Stem Cells ; },
}
@article {pmid31624292,
year = {2019},
author = {Lamas-Toranzo, I and Galiano-Cogolludo, B and Cornudella-Ardiaca, F and Cobos-Figueroa, J and Ousinde, O and Bermejo-Álvarez, P},
title = {Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14900},
pmid = {31624292},
issn = {2045-2322},
mesh = {Animals ; Blastocyst ; CRISPR-Cas Systems/*genetics ; Cattle ; DNA Replication ; Female ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genotyping Techniques ; INDEL Mutation ; Microinjections/methods ; Models, Animal ; *Mosaicism ; Oocytes ; RNA, Guide/administration &amp; dosage/genetics ; Ribonucleoproteins/administration &amp; dosage/genetics ; Zygote ; },
abstract = {Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10-30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.},
}
@article {pmid31624056,
year = {2019},
author = {Zhang, K and Liu, W and Liu, XF and Chen, YS and Liu, XH and He, ZY},
title = {[Generation of cell strains containing point mutations in HPRT1 by CRISPR/Cas9].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {10},
pages = {939-949},
doi = {10.16288/j.yczz.19-108},
pmid = {31624056},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; HEK293 Cells ; HeLa Cells ; Humans ; Hypoxanthine Phosphoribosyltransferase/*genetics ; Lesch-Nyhan Syndrome/genetics ; *Point Mutation ; },
abstract = {Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.},
}
@article {pmid31623959,
year = {2019},
author = {Winter, J and Perez-Pinera, P},
title = {Directed Evolution of CRISPR-Cas9 Base Editors.},
journal = {Trends in biotechnology},
volume = {37},
number = {11},
pages = {1151-1153},
doi = {10.1016/j.tibtech.2019.09.005},
pmid = {31623959},
issn = {1879-3096},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Evolution, Molecular ; Gene Editing/methods ; },
abstract = {A recent publication by Thuronyi et al. described a directed evolution system called phage-assisted continuous evolution (PACE) that was used to generate improved variants of CRISPR-Cas9 base editors. These evolved base editors overcome some of the inherent limitations of the technology such as sequence context preferences, restricted editing windows, and large construct sizes.},
}
@article {pmid31622977,
year = {2019},
author = {Dal-Pra, S and Hodgkinson, CP and Dzau, VJ},
title = {Induced cardiomyocyte maturation: Cardiac transcription factors are necessary but not sufficient.},
journal = {PloS one},
volume = {14},
number = {10},
pages = {e0223842},
pmid = {31622977},
issn = {1932-6203},
support = {R01 HL131814/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Transdifferentiation ; Cells, Cultured ; Cellular Reprogramming ; Fibroblasts/cytology/metabolism ; GATA4 Transcription Factor/antagonists &amp; inhibitors/*genetics/metabolism ; Gene Editing ; MEF2 Transcription Factors/antagonists &amp; inhibitors/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Myocytes, Cardiac/cytology/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; T-Box Domain Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; },
abstract = {The process by which fibroblasts are directly reprogrammed into cardiomyocytes involves two stages; initiation and maturation. Initiation represents the initial expression of factors that induce fibroblasts to transdifferentiate into cardiomyocytes. Following initiation, the cell undergoes a period of maturation before becoming a mature cardiomyocyte. We wanted to understand the role of cardiac development transcription factors in the maturation process. We directly reprogram fibroblasts into cardiomyocytes by a combination of miRNAs (miR combo). The ability of miR combo to induce cardiomyocyte-specific genes in fibroblasts was lost following the knockdown of the cardiac transcription factors Gata4, Mef2C, Tbx5 and Hand2 (GMTH). To further clarify the role of GMTH in miR combo reprogramming we utilized a modified CRISPR-Cas9 approach to activate endogenous GMTH genes. Importantly, both miR combo and the modified CRISPR-Cas9 approach induced comparable levels of GMTH expression. While miR combo was able to reprogram fibroblasts into cardiomyocyte-like cells, the modified CRISPR-Cas9 approach could not. Indeed, we found that cardiomyocyte maturation only occurred with very high levels of GMT expression. Taken together, our data indicates that while endogenous cardiac transcription factors are insufficient to reprogram fibroblasts into mature cardiomyocytes, endogenous cardiac transcription factors are necessary for expression of maturation genes.},
}
@article {pmid31622672,
year = {2020},
author = {Chen, C and Liu, J and Duan, C and Pan, Y and Liu, G},
title = {Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {134},
number = {},
pages = {103279},
doi = {10.1016/j.fgb.2019.103279},
pmid = {31622672},
issn = {1096-0937},
mesh = {Acremonium/*genetics ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Cephalosporins/biosynthesis ; Chimera/*genetics ; DNA, Fungal/*genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Gene Knockout Techniques ; *Genes, Fungal ; Genetic Loci ; Plasmids/genetics ; Promoter Regions, Genetic/*genetics ; RNA, Transfer/genetics ; },
abstract = {Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.},
}
@article {pmid31622618,
year = {2020},
author = {Kawasaki, K and Fujii, M and Sugimoto, S and Ishikawa, K and Matano, M and Ohta, Y and Toshimitsu, K and Takahashi, S and Hosoe, N and Sekine, S and Kanai, T and Sato, T},
title = {Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma.},
journal = {Gastroenterology},
volume = {158},
number = {3},
pages = {638-651.e8},
doi = {10.1053/j.gastro.2019.10.009},
pmid = {31622618},
issn = {1528-0012},
mesh = {Adenoma/*genetics/pathology ; Animals ; CRISPR-Cas Systems ; Colonic Neoplasms/*genetics/pathology ; Eukaryotic Initiation Factor-3/genetics ; Gene Fusion ; Genetic Engineering ; Humans ; Intercellular Signaling Peptides and Proteins/*genetics ; Male ; Mice ; Models, Biological ; Neoplasm Transplantation ; Organoids/*pathology ; Proto-Oncogene Proteins B-raf/genetics ; Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics ; Receptors, G-Protein-Coupled/genetics ; Thrombospondins/*genetics ; Tumor Suppressor Protein p53/genetics ; Wnt Signaling Pathway ; },
abstract = {BACKGROUND &amp; AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids.<br><br>METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5[+] cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells.<br><br>RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAF[V600E] and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAF[V600E] and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells.<br><br>CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.},
}
@article {pmid31622451,
year = {2019},
author = {Iriki, H and Kawata, T and Muramoto, T},
title = {Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.},
journal = {PloS one},
volume = {14},
number = {10},
pages = {e0224128},
pmid = {31622451},
issn = {1932-6203},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Deoxyribonuclease I/genetics/metabolism ; Dictyostelium/*genetics ; Gene Editing/methods ; Point Mutation ; RNA, Guide/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10-30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum.},
}
@article {pmid31621390,
year = {2019},
author = {Zhang, Y and Chi, X and Feng, L and Wu, X and Qi, X},
title = {Improvement of multiplex semi-nested PCR system for screening of rare mutations by high-throughput sequencing.},
journal = {BioTechniques},
volume = {67},
number = {6},
pages = {294-298},
doi = {10.2144/btn-2019-0001},
pmid = {31621390},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genes, Plant/genetics ; Genome, Plant/genetics ; High-Throughput Nucleotide Sequencing/*methods ; Mutation/*genetics ; Oryza/genetics ; Plants, Genetically Modified/genetics ; Polymerase Chain Reaction/*methods ; },
abstract = {The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced rice population. However, this strategy has the potential for further optimization. Here, we describe an improved multiplex semi-nested PCR target-enrichment strategy with simplified processing procedures, reduced false-positive rates and increased mutation detection frequency (1 mutation/73 Kb).},
}
@article {pmid31620779,
year = {2020},
author = {Zhang, Y and Zhang, Z and Zhang, H and Zhao, Y and Zhang, Z and Xiao, J},
title = {PADS Arsenal: a database of prokaryotic defense systems related genes.},
journal = {Nucleic acids research},
volume = {48},
number = {D1},
pages = {D590-D598},
pmid = {31620779},
issn = {1362-4962},
mesh = {Archaea/*genetics/virology ; Archaeal Viruses/pathogenicity ; Bacteria/*genetics/virology ; Bacteriophages/pathogenicity ; CRISPR-Cas Systems ; DNA Restriction-Modification Enzymes ; *Databases, Genetic ; *Host-Pathogen Interactions ; *Software ; },
abstract = {Defense systems are vital weapons for prokaryotes to resist heterologous DNA and survive from the constant invasion of viruses, and they are widely used in biochemistry investigation and antimicrobial drug research. So far, numerous types of defense systems have been discovered, but there is no comprehensive defense systems database to organize prokaryotic defense gene datasets. To fill this gap, we unveil the prokaryotic antiviral defense system (PADS) Arsenal (https://bigd.big.ac.cn/padsarsenal), a public database dedicated to gathering, storing, analyzing and visualizing prokaryotic defense gene datasets. The initial version of PADS Arsenal integrates 18 distinctive categories of defense system with the annotation of 6 600 264 genes retrieved from 63,701 genomes across 33 390 species of archaea and bacteria. PADS Arsenal provides various ways to retrieve defense systems related genes information and visualize them with multifarious function modes. Moreover, an online analysis pipeline is integrated into PADS Arsenal to facilitate annotation and evolutionary analysis of defense genes. PADS Arsenal can also visualize the dynamic variation information of defense genes from pan-genome analysis. Overall, PADS Arsenal is a state-of-the-art open comprehensive resource to accelerate the research of prokaryotic defense systems.},
}
@article {pmid31619674,
year = {2019},
author = {Yahia-Cherbal, H and Rybczynska, M and Lovecchio, D and Stephen, T and Lescale, C and Placek, K and Larghero, J and Rogge, L and Bianchi, E},
title = {NFAT primes the human RORC locus for RORγt expression in CD4[+] T cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4698},
pmid = {31619674},
issn = {2041-1723},
mesh = {CD4-Positive T-Lymphocytes/cytology/metabolism ; CRISPR-Cas Systems ; Cell Lineage ; Flow Cytometry ; *Gene Expression Regulation ; HEK293 Cells ; Histone Code ; Humans ; Jurkat Cells ; NFATC Transcription Factors/*metabolism ; Nuclear Receptor Subfamily 1, Group F, Member 3/*genetics ; Regulatory Elements, Transcriptional/*genetics ; Th17 Cells/cytology/*metabolism ; Thymocytes/cytology/*metabolism ; *Transcriptional Activation ; p300-CBP Transcription Factors/metabolism ; },
abstract = {T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4[+] T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4[+] T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation.},
}
@article {pmid31618682,
year = {2019},
author = {Zhao, Y and Huang, G and Zhang, W},
title = {Mutations in NlInR1 affect normal growth and lifespan in the brown planthopper Nilaparvata lugens.},
journal = {Insect biochemistry and molecular biology},
volume = {115},
number = {},
pages = {103246},
doi = {10.1016/j.ibmb.2019.103246},
pmid = {31618682},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Haploinsufficiency ; Hemiptera/genetics/*growth &amp; development ; Longevity/genetics ; Male ; Receptor, Insulin/*genetics ; },
abstract = {The brown planthopper (BPH) Nilaparvata lugens contains two insulin receptor homologues, designated NlInR1 and NlInR2. NlInR1 is strikingly homologous to the typical InR in insects and vertebrates, containing a ligand-activated intracellular tyrosine kinase catalytic domain. Herein, we report an optimized CRISPR/Cas9 system to induce mutations in the NlInR1 locus in BPH, consisting of a Cas9 plasmid that is specifically expressed in the germline via the Nlvasa promoter and versatile sgRNA expression plasmids under the control of the U6 promoter. We systematically evaluated the efficiency of injection mix compositions and demonstrated an appropriate combination of Cas9/sgRNA to target essential genes. Furthermore, we showed that homozygous mutants for the NlInR1 gene are early embryonic lethal, whereas heterozygous mutants grow more slowly, exhibit a severe reduction in body weight and wing size and live longer than the wild type. Interestingly, the severity of the mutant phenotype was different when targeting distinct important domains of the NlInR1 locus. The severity of the mutant phenotype is similar to that of insulin/insulin-like growth factor (IGF) signaling pathway deficiencies in vertebrates, suggesting a conserved function of NlInR1 in the regulation of development and longevity. Global expression profiling suggests that NlInR1 regulates many cellular processes in BPH, including insulin resistance, phototransduction, metabolism, endocytosis, longevity, biosynthesis and protein processing. Our results also pave the way for understanding the precise molecular mechanism of insulin signaling in wing polyphenism in insects.},
}
@article {pmid31618513,
year = {2020},
author = {Zhao, Y and Yang, X and Zhou, G and Zhang, T},
title = {Engineering plant virus resistance: from RNA silencing to genome editing strategies.},
journal = {Plant biotechnology journal},
volume = {18},
number = {2},
pages = {328-336},
pmid = {31618513},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; *Crops, Agricultural/genetics/virology ; *Disease Resistance/genetics ; *Gene Editing ; Genome, Plant/genetics ; Plant Viruses ; RNA Interference ; },
abstract = {Viral diseases severely affect crop yield and quality, thereby threatening global food security. Genetic improvement of plant virus resistance is essential for sustainable agriculture. In the last decades, several modern technologies were applied in plant antiviral engineering. Here we summarized breakthroughs of the two major antiviral strategies, RNA silencing and genome editing. RNA silencing strategy has been used in antiviral breeding for more than thirty years, and many crops engineered to stably express small RNAs targeting various viruses have been approved for commercial release. Genome editing technology has emerged in the past decade, especially CRISPR/Cas, which provides new methods for genetic improvement of plant virus resistance and accelerates resistance breeding. Finally, we discuss the potential of these technologies for breeding crops, and the challenges and solutions they may face in the future.},
}
@article {pmid31617178,
year = {2020},
author = {Velusamy, T and Gowripalan, A and Tscharke, DC},
title = {CRISPR/Cas9-Based Genome Editing of HSV.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2060},
number = {},
pages = {169-183},
doi = {10.1007/978-1-4939-9814-2_9},
pmid = {31617178},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Genome, Viral ; Herpesvirus 1, Human/*genetics ; Humans ; },
abstract = {The CRISPR/Cas9 gene editing system is a robust and versatile technology that has revolutionized our capacity for genome engineering and is applicable in a wide range of organisms, including large dsDNA viruses. Here we provide an efficient methodology that can be used both for marker-based and for marker-free CRISPR/Cas9-mediated editing of the HSV-1 genome. In our method, Cas9, guide RNAs and a homology-directed repair template are provided to cells by cotransection of plasmids, followed by introduction of the HSV genome by infection. This method offers a great deal of flexibility, facilitating editing of the HSV genome that spans the range from individual nucleotide changes to large deletions and insertions.},
}
@article {pmid31616946,
year = {2019},
author = {Yang, X and Liu, D and Tschaplinski, TJ and Tuskan, GA},
title = {Comparative genomics can provide new insights into the evolutionary mechanisms and gene function in CAM plants.},
journal = {Journal of experimental botany},
volume = {70},
number = {22},
pages = {6539-6547},
pmid = {31616946},
issn = {1460-2431},
mesh = {Carboxylic Acids/*metabolism ; *Evolution, Molecular ; *Genes, Plant ; *Genomics ; Plants/*genetics ; Research ; },
abstract = {Crassulacean acid metabolism (CAM) photosynthesis is an important biological innovation enabling plant adaptation to hot and dry environments. CAM plants feature high water-use efficiency, with potential for sustainable crop production under water-limited conditions. A deep understanding of CAM-related gene function and molecular evolution of CAM plants is critical for exploiting the potential of engineering CAM into C3 crops to enhance crop production on semi-arid or marginal agricultural lands. With the newly emerging genomics resources for multiple CAM species, progress has been made in comparative genomics studies on the molecular basis and subsequently on the evolution of CAM. Here, recent advances in CAM comparative genomics research in constitutive and facultative CAM plants are reviewed, with a focus on the analyses of DNA/protein sequences and gene expression to provide new insights into the path and driving force of CAM evolution and to identify candidate genes involved in CAM-related biological processes. Potential applications of new computational and experimental technologies (e.g. CRISPR/Cas-mediated genome-editing technology) to the comparative and evolutionary genomics research on CAM plants are offered.},
}
@article {pmid31616930,
year = {2020},
author = {Pfeiffer, CA and Meyer, AE and Brooks, KE and Chen, PR and Milano-Foster, J and Spate, LD and Benne, JA and Cecil, RF and Samuel, MS and Ciernia, LA and Spinka, CM and Smith, MF and Wells, KD and Spencer, TE and Prather, RS and Geisert, RD},
title = {Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†.},
journal = {Biology of reproduction},
volume = {102},
number = {2},
pages = {475-488},
pmid = {31616930},
issn = {1529-7268},
support = {U42 OD011140/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Blastocyst/*metabolism ; CRISPR-Cas Systems ; Cyclooxygenase 2/genetics/*metabolism ; Dinoprost/metabolism ; Dinoprostone/metabolism ; Embryo Implantation/*physiology ; Embryonic Development/*physiology ; Endometrium/*metabolism ; Female ; Gene Expression Regulation, Developmental ; Nuclear Transfer Techniques ; Pregnancy ; Swine ; },
abstract = {Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.},
}
@article {pmid31616455,
year = {2019},
author = {Ren, Q and Zhong, Z and Wang, Y and You, Q and Li, Q and Yuan, M and He, Y and Qi, C and Tang, X and Zheng, X and Zhang, T and Qi, Y and Zhang, Y},
title = {Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {1173},
pmid = {31616455},
issn = {1664-462X},
abstract = {CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an Arabidopsis enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9-93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.},
}
@article {pmid31616098,
year = {2019},
author = {Callaway, E},
title = {Geneticists retract study suggesting first CRISPR babies might die early.},
journal = {Nature},
volume = {574},
number = {7778},
pages = {307},
pmid = {31616098},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; Databases, Factual ; Female ; *Gene Editing/ethics ; Germ-Line Mutation/genetics ; HIV Infections/genetics/prevention &amp; control ; Heterozygote ; Humans ; Iceland ; *Patient Safety ; Receptors, CCR5/genetics ; Reproducibility of Results ; *Research Design ; *Research Personnel ; *Retraction of Publication as Topic ; Social Media ; Twins/*genetics ; United Kingdom ; },
}
@article {pmid31615983,
year = {2019},
author = {Wei, L and Lee, D and Law, CT and Zhang, MS and Shen, J and Chin, DW and Zhang, A and Tsang, FH and Wong, CL and Ng, IO and Wong, CC and Wong, CM},
title = {Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4681},
pmid = {31615983},
issn = {2041-1723},
mesh = {Antineoplastic Agents/*therapeutic use ; Apoptosis ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*drug therapy/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Humans ; Liver Neoplasms/*drug therapy/genetics ; Phenylurea Compounds/therapeutic use ; Phosphoglycerate Dehydrogenase/antagonists &amp; inhibitors/*genetics ; Pyridines/therapeutic use ; Quinolines/therapeutic use ; Reactive Oxygen Species/metabolism ; Sorafenib/*therapeutic use ; },
abstract = {Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common. By using genome-wide CRISPR/Cas9 library screening, we identify phosphoglycerate dehydrogenase (PHGDH), the first committed enzyme in the serine synthesis pathway (SSP), as a critical driver for Sorafenib resistance. Sorafenib treatment activates SSP by inducing PHGDH expression. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that inactivation of PHGDH paralyzes the SSP and reduce the production of αKG, serine, and NADPH. Concomitantly, inactivation of PHGDH elevates ROS level and induces HCC apoptosis upon Sorafenib treatment. More strikingly, treatment of PHGDH inhibitor NCT-503 works synergistically with Sorafenib to abolish HCC growth in vivo. Similar findings are also obtained in other FDA-approved tyrosine kinase inhibitors (TKIs), including Regorafenib or Lenvatinib. In summary, our results demonstrate that targeting PHGDH is an effective approach to overcome TKI drug resistance in HCC.},
}
@article {pmid31615875,
year = {2019},
author = {Achuthankutty, D and Thakur, RS and Haahr, P and Hoffmann, S and Drainas, AP and Bizard, AH and Weischenfeldt, J and Hickson, ID and Mailand, N},
title = {Regulation of ETAA1-mediated ATR activation couples DNA replication fidelity and genome stability.},
journal = {The Journal of cell biology},
volume = {218},
number = {12},
pages = {3943-3953},
pmid = {31615875},
issn = {1540-8140},
mesh = {Antigens, Surface/*metabolism ; Ataxia Telangiectasia Mutated Proteins/*metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Chromosome Aberrations ; DNA Damage ; *DNA Replication ; *Gene Expression Regulation, Neoplastic ; Genome, Human ; *Genomic Instability ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Humans ; Mitosis ; Nuclear Proteins/metabolism ; Phosphorylation ; Protein Binding ; Signal Transduction ; },
abstract = {The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9-based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle- and replication stress-dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.},
}
@article {pmid31614382,
year = {2019},
author = {Zhou, Y and Yang, H and Shi, J and Zhang, M and Yang, S and Wang, N and Sun, R and Wang, Z and Fei, J},
title = {Fate Tracing of Isl1+Cells in Adult Mouse Hearts under Physiological and Exercise Conditions.},
journal = {International journal of sports medicine},
volume = {40},
number = {14},
pages = {921-930},
doi = {10.1055/a-0961-1458},
pmid = {31614382},
issn = {1439-3964},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Proliferation/physiology ; Chromosome Mapping ; Heart/embryology/growth &amp; development/*physiology ; LIM-Homeodomain Proteins/*genetics ; Male ; Mice, Inbred C57BL ; Myocardium/*cytology ; Myocytes, Cardiac/*cytology ; *Physical Conditioning, Animal ; Stem Cells/*physiology ; Transcription Factors/*genetics ; },
abstract = {Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1[+]cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1[+]cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1[+]cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1[+]cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.},
}
@article {pmid31613873,
year = {2019},
author = {You, ST and Jhou, YT and Kao, CF and Leu, JY},
title = {Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000433},
pmid = {31613873},
issn = {1545-7885},
mesh = {CRISPR-Cas Systems ; Directed Molecular Evolution ; Ethyl Methanesulfonate/pharmacology ; Gene Editing ; *Gene Expression Regulation, Fungal ; Genes, Reporter ; *Genetic Heterogeneity ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Methylation ; Mutation ; *Protein Processing, Post-Translational ; Protein-Arginine N-Methyltransferases/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; Saccharomyces cerevisiae/drug effects/*genetics/growth &amp; development/metabolism ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; },
abstract = {Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and we also show that two of the Hmt1 methylation targets can suppress noise. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.},
}
@article {pmid31613223,
year = {2019},
author = {Schlichting, M and Díaz, MM and Xin, J and Rosbash, M},
title = {Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31613223},
issn = {2050-084X},
support = {T32 MH019929/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/deficiency/genetics ; Brain/cytology/*metabolism/radiation effects ; CRISPR-Cas Systems ; Cell Communication ; Cell Lineage/genetics ; Circadian Clocks/drug effects/*genetics ; Circadian Rhythm/drug effects/*genetics ; Darkness ; Drosophila Proteins/deficiency/genetics ; Drosophila melanogaster/*genetics/metabolism/radiation effects ; Feedback, Physiological ; Gene Editing ; *Gene Expression Regulation ; Light Signal Transduction/*genetics ; Nerve Net/metabolism/radiation effects ; Neurons/cytology/*metabolism/radiation effects ; Neuropeptides/deficiency/genetics ; Period Circadian Proteins/deficiency/genetics ; Transcription Factors/deficiency/genetics ; },
abstract = {Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, we assayed behavior as well as molecular rhythms within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. We also generated CRISPR-mediated neuron-specific circadian clock knockouts. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.},
}
@article {pmid31613218,
year = {2019},
author = {Delventhal, R and O'Connor, RM and Pantalia, MM and Ulgherait, M and Kim, HX and Basturk, MK and Canman, JC and Shirasu-Hiza, M},
title = {Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31613218},
issn = {2050-084X},
support = {T32 HL120826/HL/NHLBI NIH HHS/United States ; R01GM117407/NH/NIH HHS/United States ; T32 GM007367/GM/NIGMS NIH HHS/United States ; 5T32DK007328/NH/NIH HHS/United States ; R35GM127049/NH/NIH HHS/United States ; R01GM105775/NH/NIH HHS/United States ; R01 GM105775/GM/NIGMS NIH HHS/United States ; 2T32GM007367-42/NH/NIH HHS/United States ; R01 AG045842/AG/NIA NIH HHS/United States ; R01 GM117407/GM/NIGMS NIH HHS/United States ; 5T32HL120826/NH/NIH HHS/United States ; T32 GM007088/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; T32 DK007328/DK/NIDDK NIH HHS/United States ; R35 GM127049/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Brain/cytology/metabolism/radiation effects ; CRISPR-Cas Systems ; Cell Communication ; Cell Lineage/genetics ; Circadian Clocks/drug effects/*genetics ; Circadian Rhythm/drug effects/*genetics ; Darkness ; Drosophila Proteins/deficiency/*genetics ; Drosophila melanogaster/*genetics/metabolism/radiation effects ; Feedback, Physiological ; Gene Editing ; Gene Expression Regulation ; Light Signal Transduction/genetics ; Locomotion/genetics/radiation effects ; Nerve Net/metabolism/radiation effects ; Neurons/cytology/*metabolism/radiation effects ; Neuropeptides/deficiency/*genetics ; Period Circadian Proteins/deficiency/*genetics ; Transcription Factors/deficiency/genetics ; },
abstract = {In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf[+] neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf[+] neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf[+] neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.},
}
@article {pmid31612854,
year = {2019},
author = {Laflamme, C and McKeever, PM and Kumar, R and Schwartz, J and Kolahdouzan, M and Chen, CX and You, Z and Benaliouad, F and Gileadi, O and McBride, HM and Durcan, TM and Edwards, AM and Healy, LM and Robertson, J and McPherson, PS},
title = {Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31612854},
issn = {2050-084X},
support = {Hudson Translational Grant//ALS Society of Canada/International ; ALS RAP//ALS Society of Canada/International ; ALS RAP//ALS Therapy Alliance/International ; ALS RAP/MNDA_/Motor Neurone Disease Association/United Kingdom ; },
mesh = {Amyotrophic Lateral Sclerosis/*diagnosis/genetics/immunology/metabolism ; Animals ; Antibodies, Monoclonal/*chemistry/classification/immunology ; Biomarkers/metabolism ; C9orf72 Protein/*genetics/immunology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Frontotemporal Dementia/*diagnosis/genetics/immunology/metabolism ; Gene Editing ; Gene Expression ; HEK293 Cells ; Humans ; Immunohistochemistry/*standards ; Lysosomes/genetics/metabolism/ultrastructure ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Osteoblasts/metabolism/ultrastructure ; Phagosomes/genetics/metabolism/ultrastructure ; RAW 264.7 Cells ; },
abstract = {Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.},
}
@article {pmid31611891,
year = {2019},
author = {Durut, N and Mittelsten Scheid, O},
title = {The Role of Noncoding RNAs in Double-Strand Break Repair.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {1155},
pmid = {31611891},
issn = {1664-462X},
support = {M 2410/FWF_/Austrian Science Fund FWF/Austria ; },
abstract = {Genome stability is constantly threatened by DNA lesions generated by different environmental factors as well as endogenous processes. If not properly and timely repaired, damaged DNA can lead to mutations or chromosomal rearrangements, well-known reasons for genetic diseases or cancer in mammals, or growth abnormalities and/or sterility in plants. To prevent deleterious consequences of DNA damage, a sophisticated system termed DNA damage response (DDR) detects DNA lesions and initiates DNA repair processes. In addition to many well-studied canonical proteins involved in this process, noncoding RNA (ncRNA) molecules have recently been discovered as important regulators of the DDR pathway, extending the broad functional repertoire of ncRNAs to the maintenance of genome stability. These ncRNAs are mainly connected with double-strand breaks (DSBs), the most dangerous type of DNA lesions. The possibility to intentionally generate site-specific DSBs in the genome with endonucleases constitutes a powerful tool to study, in vivo, how DSBs are processed and how ncRNAs participate in this crucial event. In this review, we will summarize studies reporting the different roles of ncRNAs in DSB repair and discuss how genome editing approaches, especially CRISPR/Cas systems, can assist DNA repair studies. We will summarize knowledge concerning the functional significance of ncRNAs in DNA repair and their contribution to genome stability and integrity, with a focus on plants.},
}
@article {pmid31611562,
year = {2019},
author = {Campbell, BW and Hoyle, JW and Bucciarelli, B and Stec, AO and Samac, DA and Parrott, WA and Stupar, RM},
title = {Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14757},
pmid = {31611562},
issn = {2045-2322},
mesh = {Alleles ; *CRISPR-Cas Systems ; Chromosomes, Plant/genetics ; Gene Editing ; Genes, Plant ; Plant Breeding ; Plants, Genetically Modified/genetics/growth &amp; development ; Soybeans/*genetics/growth &amp; development ; Trichomes/*genetics/growth &amp; development ; },
abstract = {Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.},
}
@article {pmid31611308,
year = {2019},
author = {Graham, MK and Kim, J and Da, J and Brosnan-Cashman, JA and Rizzo, A and Baena Del Valle, JA and Chia, L and Rubenstein, M and Davis, C and Zheng, Q and Cope, L and Considine, M and Haffner, MC and De Marzo, AM and Meeker, AK and Heaphy, CM},
title = {Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells.},
journal = {Molecular cancer research : MCR},
volume = {17},
number = {12},
pages = {2480-2491},
pmid = {31611308},
issn = {1557-3125},
support = {F32 CA213742/CA/NCI NIH HHS/United States ; P30 CA006973/CA/NCI NIH HHS/United States ; R01 CA172380/CA/NCI NIH HHS/United States ; T32 CA009110/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromatin Assembly and Disassembly/genetics ; Chromosomal Instability/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; Humans ; Male ; Mutation ; Prostate/metabolism/pathology ; Prostatic Neoplasms/*genetics/pathology ; RNA/*genetics ; Telomerase/*genetics ; Telomere/genetics ; Telomere Homeostasis/*genetics ; X-linked Nuclear Protein/*genetics ; },
abstract = {A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRX[KO] cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRX[KO] cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRX[KO] TERC[mut] cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. IMPLICATIONS: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.},
}
@article {pmid31610539,
year = {2019},
author = {Němečková, A and Wäsch, C and Schubert, V and Ishii, T and Hřibová, E and Houben, A},
title = {CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication.},
journal = {Cytogenetic and genome research},
volume = {159},
number = {1},
pages = {48-53},
doi = {10.1159/000502600},
pmid = {31610539},
issn = {1424-859X},
mesh = {Amaryllidaceae/*genetics ; Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; Chromatin/metabolism ; Chromosomes/genetics ; DNA Replication/*genetics ; DNA, Plant/genetics ; Endonucleases/genetics ; In Situ Hybridization, Fluorescence/methods ; RNA, Guide/genetics ; Telomere/genetics ; Zea mays/*genetics ; },
abstract = {Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.},
}
@article {pmid31610242,
year = {2020},
author = {Liang, Y and Jiao, S and Wang, M and Yu, H and Shen, Z},
title = {A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.},
journal = {Metabolic engineering},
volume = {57},
number = {},
pages = {13-22},
doi = {10.1016/j.ymben.2019.10.003},
pmid = {31610242},
issn = {1096-7184},
mesh = {Acrylamide/*metabolism ; *Biocatalysis ; *CRISPR-Cas Systems ; *Gene Editing ; *Metabolic Engineering ; *Rhodococcus/genetics/metabolism ; },
abstract = {Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.},
}
@article {pmid31609975,
year = {2019},
author = {Zhang, W and Chen, Z and Zhang, D and Zhao, B and Liu, L and Xie, Z and Yao, Y and Zheng, P},
title = {KHDC3L mutation causes recurrent pregnancy loss by inducing genomic instability of human early embryonic cells.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000468},
pmid = {31609975},
issn = {1545-7885},
mesh = {Abortion, Habitual/*genetics/metabolism/pathology ; Animals ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; DNA Damage ; Embryo, Mammalian ; Female ; Gene Editing ; Gene Expression Regulation ; *Genomic Instability ; Human Embryonic Stem Cells/*metabolism/pathology ; Humans ; Karyotype ; Mice ; Mice, SCID ; Micronuclei, Chromosome-Defective ; Poly (ADP-Ribose) Polymerase-1/*genetics/metabolism ; Pregnancy ; Proteins/*genetics/metabolism ; *Recombinational DNA Repair ; Signal Transduction ; },
abstract = {Recurrent pregnancy loss (RPL) is an important complication in reproductive health. About 50% of RPL cases are unexplained, and understanding the genetic basis is essential for its diagnosis and prognosis. Herein, we report causal KH domain containing 3 like (KHDC3L) mutations in RPL. KHDC3L is expressed in human epiblast cells and ensures their genome stability and viability. Mechanistically, KHDC3L binds to poly(ADP-ribose) polymerase 1 (PARP1) to stimulate its activity. In response to DNA damage, KHDC3L also localizes to DNA damage sites and facilitates homologous recombination (HR)-mediated DNA repair. KHDC3L dysfunction causes PARP1 inhibition and HR repair deficiency, which is synthetically lethal. Notably, we identified two critical residues, Thr145 and Thr156, whose phosphorylation by Ataxia-telangiectasia mutated (ATM) is essential for KHDC3L's functions. Importantly, two deletions of KHDC3L (p.E150_V160del and p.E150_V172del) were detected in female RPL patients, both of which harbor a common loss of Thr156 and are impaired in PARP1 activation and HR repair. In summary, our study reveals both KHDC3L as a new RPL risk gene and its critical function in DNA damage repair pathways.},
}
@article {pmid31609348,
year = {2019},
author = {Cardenas-Diaz, FL and Maguire, JA and Gadue, P and French, DL},
title = {Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {151},
pages = {},
doi = {10.3791/60085},
pmid = {31609348},
issn = {1940-087X},
support = {U01 HL099656/HL/NHLBI NIH HHS/United States ; U01 HL134696/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Base Pairing ; Base Sequence ; CRISPR-Cas Systems/*genetics ; *Genomics ; Humans ; INDEL Mutation/genetics ; Mice ; Mutation/genetics ; Oligodeoxyribonucleotides/metabolism ; Phenotype ; Pluripotent Stem Cells/*metabolism ; RNA, Guide/genetics ; Recombination, Genetic/genetics ; },
abstract = {Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.},
}
@article {pmid31607684,
year = {2019},
author = {Lee, DW and Lee, SK and Rahman, MM and Kim, YJ and Zhang, D and Jeon, JS},
title = {The Role of Rice Vacuolar Invertase2 in Seed Size Control.},
journal = {Molecules and cells},
volume = {42},
number = {10},
pages = {711-720},
pmid = {31607684},
issn = {0219-1032},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Metabolome ; Mutation/genetics ; Organ Size ; Oryza/*enzymology ; Photosynthesis ; Plants, Genetically Modified ; Seeds/*anatomy &amp; histology/*enzymology ; Starch/metabolism ; Sucrose/metabolism ; Vacuoles/*enzymology ; beta-Fructofuranosidase/*metabolism ; },
abstract = {Sink strength optimizes sucrose import, which is fundamental to support developing seed grains and increase crop yields, including those of rice (Oryza sativa). In this regard, little is known about the function of vacuolar invertase (VIN) in controlling sink strength and thereby seed size. Here, in rice we analyzed mutants of two VINs, OsVIN1 and OsVIN2, to examine their role during seed development. In a phenotypic analysis of the T-DNA insertion mutants, only the OsVIN2 mutant osvin2-1 exhibited reduced seed size and grain weight. Scanning electron microscopy analysis revealed that the small seed grains of osvin2-1 can be attributed to a reduction in spikelet size. A significant decrease in VIN activity and hexose level in the osvin2-1 spikelets interfered with spikelet growth. In addition, significant reduction in starch and increase in sucrose, which are characteristic features of reduced turnover and flux of sucrose due to impaired sink strength, were evident in the pre-storage stage of osvin2-1 developing grains. In situ hybridization analysis found that expression of OsVIN2 was predominant in the endocarp of developing grains. A genetically complemented line with a native genomic clone of OsVIN2 rescued reduced VIN activity and seed size. Two additional mutants, osvin2-2 and osvin2-3 generated by the CRISPR/Cas9 method, exhibited phenotypes similar to those of osvin2-1 in spikelet and seed size, VIN activity, and sugar metabolites. These results clearly demonstrate an important role of OsVIN2 as sink strength modulator that is critical for the maintenance of sucrose flux into developing seed grains.},
}
@article {pmid31607545,
year = {2019},
author = {Freije, CA and Myhrvold, C and Boehm, CK and Lin, AE and Welch, NL and Carter, A and Metsky, HC and Luo, CY and Abudayyeh, OO and Gootenberg, JS and Yozwiak, NL and Zhang, F and Sabeti, PC},
title = {Programmable Inhibition and Detection of RNA Viruses Using Cas13.},
journal = {Molecular cell},
volume = {76},
number = {5},
pages = {826-837.e11},
pmid = {31607545},
issn = {1097-4164},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; U19 AI110818/AI/NIAID NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; },
mesh = {A549 Cells ; Animals ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dogs ; Escherichia coli/enzymology/genetics ; Gene Targeting/*methods ; HEK293 Cells ; Humans ; Madin Darby Canine Kidney Cells ; *RNA Stability ; RNA Viruses/*enzymology/genetics ; RNA, Viral/genetics/*metabolism ; Vero Cells ; },
abstract = {The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.},
}
@article {pmid31607505,
year = {2019},
author = {Geng, Y and Yan, H and Li, P and Ren, G and Guo, X and Yin, P and Zhang, L and Qian, Z and Zhao, Z and Sun, YC},
title = {A highly efficient in vivo plasmid editing tool based on CRISPR-Cas12a and phage λ Red recombineering.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {9},
pages = {455-458},
pmid = {31607505},
issn = {1673-8527},
mesh = {Bacteriophage lambda/*genetics ; CRISPR-Cas Systems/*genetics ; Chromosomes, Artificial/genetics ; Chromosomes, Artificial, Bacterial/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Plasmids/*genetics ; },
}
@article {pmid31607332,
year = {2019},
author = {Sun, Y and Liu, D and Shi, M and Zheng, JN},
title = {[ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {27},
number = {5},
pages = {1682-1690},
doi = {10.19746/j.cnki.issn.1009-2137.2019.05.050},
pmid = {31607332},
issn = {1009-2137},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; RNA, Guide ; Receptors, Adrenergic, beta-2/*genetics ; T-Lymphocytes ; },
abstract = {OBJECTIVE: To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy.<br><br>METHODS: Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1&#x3b1;-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of &#x3b2;2 adrenergic receptor (&#x3b2;2-AR).<br><br>RESULTS: The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were &#x3b2;2-AR positive, but the proportion of &#x3b2;2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%.<br><br>CONCLUSION: A method, which is simple and operable, for knocking out &#x3b2;2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of &#x3b2;2-AR in human T cells.},
}
@article {pmid31606929,
year = {2020},
author = {Schindele, P and Puchta, H},
title = {Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing.},
journal = {Plant biotechnology journal},
volume = {18},
number = {5},
pages = {1118-1120},
pmid = {31606929},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/metabolism ; *Gene Editing ; RNA, Guide ; Temperature ; },
}
@article {pmid31606790,
year = {2019},
author = {Girardin, L and Calvez, V and Débarre, F},
title = {Catch Me If You Can: A Spatial Model for a Brake-Driven Gene Drive Reversal.},
journal = {Bulletin of mathematical biology},
volume = {81},
number = {12},
pages = {5054-5088},
doi = {10.1007/s11538-019-00668-z},
pmid = {31606790},
issn = {1522-9602},
mesh = {Animals ; Computer Simulation ; Food Chain ; Gene Drive Technology/adverse effects/*methods/statistics &amp; numerical data ; Genetic Fitness ; Genetics, Population ; Mathematical Concepts ; *Models, Genetic ; Predatory Behavior ; Spatio-Temporal Analysis ; },
abstract = {Population management using artificial gene drives (alleles biasing inheritance, increasing their own transmission to offspring) is becoming a realistic possibility with the development of CRISPR-Cas genetic engineering. A gene drive may, however, have to be stopped. "Antidotes" (brakes) have been suggested, but have been so far only studied in well-mixed populations. Here, we consider a reaction-diffusion system modeling the release of a gene drive (of fitness [Formula: see text]) and a brake (fitness [Formula: see text], [Formula: see text]) in a wild-type population (fitness 1). We prove that whenever the drive fitness is at most 1/2 while the brake fitness is close to 1, coextinction of the brake and the drive occurs in the long run. On the contrary, if the drive fitness is greater than 1/2, then coextinction is impossible: the drive and the brake keep spreading spatially, leaving in the invasion wake a complicated spatiotemporally heterogeneous genetic pattern. Based on numerical experiments, we argue in favor of a global coextinction conjecture provided the drive fitness is at most 1/2, irrespective of the brake fitness. The proof relies upon the study of a related predator-prey system with strong Allee effect on the prey. Our results indicate that some drives may be unstoppable and that if gene drives are ever deployed in nature, threshold drives, that only spread if introduced in high enough frequencies, should be preferred.},
}
@article {pmid31606751,
year = {2019},
author = {Li, XF and Zhou, YW and Cai, PF and Fu, WC and Wang, JH and Chen, JY and Yang, QN},
title = {CRISPR/Cas9 facilitates genomic editing for large-scale functional studies in pluripotent stem cell cultures.},
journal = {Human genetics},
volume = {138},
number = {11-12},
pages = {1217-1225},
pmid = {31606751},
issn = {1432-1203},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Knockout Techniques ; Genomics/*methods ; Humans ; Pluripotent Stem Cells/cytology/*physiology ; },
abstract = {Pluripotent stem cell (PSC) cultures form an integral part of biomedical and medical research due to their capacity to rapidly proliferate and differentiate into hundreds of highly specialized cell types. This makes them a highly useful tool in exploring human physiology and disease. Genomic editing of PSC cultures is an essential method of attaining answers to basic physiological functions, developing in vitro models of human disease, and exploring potential therapeutic strategies and the identification of drug targets. Achieving reliable and efficient genomic editing is an important aspect of using large-scale PSC cultures. The CRISPR/Cas9 genomic editing tool has facilitated highly efficient gene knockout, gene correction, or gene modifications through the design and use of single-guide RNAs which are delivered to the target DNA via Cas9. CRISPR/Cas9 modification of PSCs has furthered the understanding of basic physiology and has been utilized to develop in vitro disease models, to test therapeutic strategies, and to facilitate regenerative or tissue repair approaches. In this review, we discuss the benefits of the CRISPR/Cas9 system in large-scale PSC cultures.},
}
@article {pmid31604991,
year = {2019},
author = {Gul, N and Karlsson, J and Tängemo, C and Linsefors, S and Tuyizere, S and Perkins, R and Ala, C and Zou, Z and Larsson, E and Bergö, MO and Lindahl, P},
title = {The MTH1 inhibitor TH588 is a microtubule-modulating agent that eliminates cancer cells by activating the mitotic surveillance pathway.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14667},
pmid = {31604991},
issn = {2045-2322},
mesh = {Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Carcinoma, Large Cell/*drug therapy/genetics/pathology ; Cell Cycle/drug effects ; Cell Line, Tumor ; DNA Repair Enzymes/antagonists &amp; inhibitors/*genetics ; G1 Phase/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Microtubules/drug effects ; Mitosis/drug effects ; Phosphoric Monoester Hydrolases/antagonists &amp; inhibitors/*genetics ; Pyrimidines/*pharmacology ; Spindle Apparatus/drug effects ; Tubulin Modulators/pharmacology ; Tumor Suppressor Protein p53/genetics ; Ubiquitin Thiolesterase/*genetics ; },
abstract = {The mut-T homolog-1 (MTH1) inhibitor TH588 has shown promise in preclinical cancer studies but its targeting specificity has been questioned. Alternative mechanisms for the anti-cancer effects of TH588 have been suggested but the question remains unresolved. Here, we performed an unbiased CRISPR screen on human lung cancer cells to identify potential mechanisms behind the cytotoxic effect of TH588. The screen identified pathways and complexes involved in mitotic spindle regulation. Using immunofluorescence and live cell imaging, we showed that TH588 rapidly reduced microtubule plus-end mobility, disrupted mitotic spindles, and prolonged mitosis in a concentration-dependent but MTH1-independent manner. These effects activated a USP28-p53 pathway - the mitotic surveillance pathway - that blocked cell cycle reentry after prolonged mitosis; USP28 acted upstream of p53 to arrest TH588-treated cells in the G1-phase of the cell cycle. We conclude that TH588 is a microtubule-modulating agent that activates the mitotic surveillance pathway and thus prevents cancer cells from re-entering the cell cycle.},
}
@article {pmid31604975,
year = {2019},
author = {Iwata, S and Nakadai, H and Fukushi, D and Jose, M and Nagahara, M and Iwamoto, T},
title = {Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14713},
pmid = {31604975},
issn = {2045-2322},
mesh = {Alleles ; Animals ; CRISPR-Associated Protein 9/administration &amp; dosage ; CRISPR-Cas Systems ; Chromosome Inversion/*genetics ; Chromosomes/*genetics ; Electroporation/*methods ; Female ; Genetic Engineering/*methods ; Genome ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Microinjections ; RNA, Guide/administration &amp; dosage ; *Zygote ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.},
}
@article {pmid31604602,
year = {2019},
author = {Fuchsbauer, O and Swuec, P and Zimberger, C and Amigues, B and Levesque, S and Agudelo, D and Duringer, A and Chaves-Sanjuan, A and Spinelli, S and Rousseau, GM and Velimirovic, M and Bolognesi, M and Roussel, A and Cambillau, C and Moineau, S and Doyon, Y and Goulet, A},
title = {Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.},
journal = {Molecular cell},
volume = {76},
number = {6},
pages = {922-937.e7},
doi = {10.1016/j.molcel.2019.09.012},
pmid = {31604602},
issn = {1097-4164},
mesh = {Allosteric Regulation ; Bacteriophages/genetics/*metabolism ; Binding Sites ; CRISPR-Associated Protein 9/genetics/*metabolism/ultrastructure ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/*metabolism/ultrastructure ; Escherichia coli/enzymology/genetics ; Humans ; K562 Cells ; Kinetics ; Mutation ; Protein Binding ; Protein Conformation ; Streptococcus thermophilus/*enzymology/genetics ; Structure-Activity Relationship ; Viral Proteins/genetics/*metabolism/ultrastructure ; },
abstract = {In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.},
}
@article {pmid31604525,
year = {2019},
author = {Shababi, M and Smith, CE and Kacher, M and Alrawi, Z and Villalón, E and Davis, D and Bryda, EC and Lorson, CL},
title = {Development of a novel severe mouse model of spinal muscular atrophy with respiratory distress type 1: FVB-nmd.},
journal = {Biochemical and biophysical research communications},
volume = {520},
number = {2},
pages = {341-346},
pmid = {31604525},
issn = {1090-2104},
support = {R21 NS093175/NS/NINDS NIH HHS/United States ; R21 NS109762/NS/NINDS NIH HHS/United States ; T32 GM008396/GM/NIGMS NIH HHS/United States ; U42 OD010918/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/*genetics/metabolism ; Disease Models, Animal ; Female ; Male ; Mice, Inbred Strains ; Muscle, Skeletal/*pathology ; Muscular Atrophy, Spinal/*etiology ; Neuromuscular Junction/pathology ; Respiratory Distress Syndrome, Newborn/*etiology ; Spinal Cord/metabolism/pathology ; Transcription Factors/*genetics/metabolism ; },
abstract = {Spinal Muscular Atrophy with Respiratory Distress type 1 (SMARD1) is an autosomal recessive disease that develops early during infancy. The gene responsible for disease development is immunoglobulin helicase μ-binding protein 2 (IGHMBP2). IGHMBP2 is a ubiquitously expressed gene but its mutation results in the loss of alpha-motor neurons and subsequent muscle atrophy initially of distal muscles. The current SMARD1 mouse model arose from a spontaneous mutation originally referred to as neuromuscular degeneration (nmd). The nmd mice have the C57BL/6 genetic background and contain an A to G mutation in intron 4 of the endogenous Ighmbp2 gene. This mutation causes aberrant splicing, resulting in only 20-25% of full-length functional protein. Several congenital conditions including hydrocephalus are common in the C57BL/6 background, consistent with our previous observations when developing a gene therapy approach for SMARD1. Additionally, a modifier allele exists on chromosome 13 in nmd mice that can partially suppress the phenotype, resulting in some animals that have extended life spans (up to 200 days). To eliminate the intrinsic complications of the C57BL/6 background and the variation in survival due to the genetic modifier effect, we created a new SMARD1 mouse model that contains the same intron 4 mutation in Ighmbp2 as nmd mice but is now on a FVB congenic background. FVB-nmd are consistently more severe than the original nmd mice with respect to survival, weigh and motor function. The relatively short life span (18-21 days) of FVB-nmd mice allows us to monitor therapeutic efficacy for a variety of novel therapeutics in a timely manner without the complication of the small percentage of longer-lived animals that were observed in our colony of nmd mice.},
}
@article {pmid31603896,
year = {2019},
author = {Chen, W and Zhang, H and Zhang, Y and Wang, Y and Gan, J and Ji, Q},
title = {Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000496},
pmid = {31603896},
issn = {1545-7885},
mesh = {Amino Acid Substitution ; CRISPR-Associated Protein 9/chemistry/*genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; DNA/chemistry/*genetics/metabolism ; Escherichia coli/genetics/metabolism ; Gene Editing ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; Isoenzymes/chemistry/genetics/metabolism ; Klebsiella pneumoniae/genetics/metabolism ; Models, Molecular ; Mutagenesis, Site-Directed/methods ; Mutation ; Nucleotide Motifs ; Protein Binding ; Protein Engineering/methods ; RNA, Guide/chemistry/*genetics/metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Streptococcus pyogenes/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.},
}
@article {pmid31603849,
year = {2019},
author = {Ortez, C and Natera de Benito, D and Carrera García, L and Expósito, J and Nolasco, G and Nascimento, A},
title = {[Advances in the treatment of Duchenne muscular dystrophy].},
journal = {Medicina},
volume = {79 Suppl 3},
number = {},
pages = {77-81},
pmid = {31603849},
issn = {1669-9106},
mesh = {Animals ; CRISPR-Cas Systems ; Dystrophin/genetics ; Genetic Therapy/*methods ; Genotype ; Humans ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne/genetics/*therapy ; Phenotype ; },
abstract = {Duchenne muscular dystrophy is a genetically determined disease, linked to the X chromosome, c haracterized clinically by producing progressive muscle weakness, with an incidence of 1 per 3500-6000 males born. It is caused by the mutation of the DMD gene, which encodes dystrophin, a sub-sarcolemmal protein essential for structural muscle stability. The genetic defects in the DMD gene are divided into: deletions (65%) duplications (5.10%) and point mutations (10-15%). At present there is no curative treatment, the only drug that has been shown to modify the natural history of the disease (independently of the genetic mutation) are corticosteroids, currently indicated in early stages of the disease. In relation to clinical trials, in the last ten years, has experienced great advances in the field of therapeutic options, divided into two major therapeutic targets: 1) the area of gene therapies and 2) trying to reverse or block the pathophysiological processes of the disease, such as inflammation, fibrosis, muscle regeneration, etc. It is likely that an effective treatment for Duchenne muscular dystrophy requires combinations of therapies that address both the primary defect and its secondary pathophysiological consequences.},
}
@article {pmid31603250,
year = {2020},
author = {Soleimani, F and Babaei, E and H Feizi, MA and Fathi, F},
title = {CRISPR-Cas9-mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes.},
journal = {Journal of cellular physiology},
volume = {235},
number = {4},
pages = {3994-4000},
doi = {10.1002/jcp.29295},
pmid = {31603250},
issn = {1097-4652},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; DNA-Activated Protein Kinase/*genetics ; DNA-Binding Proteins/*genetics ; Gene Expression Regulation, Developmental/genetics ; Gene Knockout Techniques ; Mesoderm/growth &amp; development ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; Pluripotent Stem Cells/*metabolism ; },
abstract = {Prkdc encodes for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) playing a key role in nonhomologous end joining pathway during DNA double-strand break repair and also influencing the homologous recombination (HR) repair system by phosphorylation of proteins involved in HR. In addition, Prkdc has other critical functions in biological processes, such as transcriptional regulation, telomere stability, apoptosis, and metabolism. DNA-PKcs upregulates during in vitro differentiation of mouse embryonic stem cells (mESCs). To address the potential role of Prkdc in mESCs pluripotency and in vitro differentiation into ectoderm, mesoderm, and endoderm germ layers under normal physiological conditions, a bi-allelic Prkdc-knockout cell line was generated in the present study by employing CRISPR/Cas9 system, and subsequently, its potential role in stemness and development was studied. The results of the study showed that the expression of pluripotency-associated genes, including Nanog and Sox-2 were overexpressed in the bi-allelic Prkdc-knockout cell line. Also, bi-allelic Prkdc-knockout cell line was shown to have typical mESCs cell morphology, cell cycle distribution, and alkaline phosphatase activity. Furthermore, the results of the study revealed that the expression of several germ layer markers is modulated in Prkdc-knockout lines. In conclusion, the findings of our study demonstrated the role of Prkdc during differentiation and development of ESCs.},
}
@article {pmid31602604,
year = {2019},
author = {Walter, JM and Schubert, MG and Kung, SH and Hawkins, K and Platt, DM and Hernday, AD and Mahatdejkul-Meadows, T and Szeto, W and Chandran, SS and Newman, JD and Horwitz, AA},
title = {Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2049},
number = {},
pages = {39-72},
pmid = {31602604},
issn = {1940-6029},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genetic Engineering/methods ; RNA, Guide/metabolism ; Saccharomyces cerevisiae/*genetics ; Synthetic Biology/methods ; },
abstract = {CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.},
}
@article {pmid31601883,
year = {2019},
author = {Zhao, Y and Tyrishkin, K and Sjaarda, C and Khanal, P and Stafford, J and Rauh, M and Liu, X and Babak, T and Yang, X},
title = {A one-step tRNA-CRISPR system for genome-wide genetic interaction mapping in mammalian cells.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14499},
pmid = {31601883},
issn = {2045-2322},
support = {MOP148629//CIHR/Canada ; },
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/*genetics/pathology ; Cell Survival/genetics ; Chromosome Mapping ; Epistasis, Genetic/genetics ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein/*genetics ; RNA, Transfer/genetics ; Trans-Activators/*genetics ; Transcription Factors/*genetics ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; YAP-Signaling Proteins ; },
abstract = {Mapping genetic interactions in mammalian cells is limited due to technical obstacles. Here we describe a method called TCGI (tRNA-CRISPR for genetic interactions) to generate a high-efficient, barcode-free and scalable pairwise CRISPR libraries in mammalian cells for identifying genetic interactions. We have generated a genome- wide library to identify genes genetically interacting with TAZ in cell viability regulation. Validation of candidate synergistic genes reveals the screening accuracy of 85% and TAZ-MCL1 is characterized as combinational drug targets for non-small cell lung cancer treatments. TCGI has dramatically improved the current methods for mapping genetic interactions and screening drug targets for combinational therapies.},
}
@article {pmid31601834,
year = {2019},
author = {Portugaliza, HP and Llorà-Batlle, O and Rosanas-Urgell, A and Cortés, A},
title = {Reporter lines based on the gexp02 promoter enable early quantification of sexual conversion rates in the malaria parasite Plasmodium falciparum.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14595},
pmid = {31601834},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Erythrocytes/parasitology ; Fluorescent Dyes ; Genes, Reporter ; *Genetic Techniques ; Humans ; Plasmodium falciparum/*genetics ; *Promoter Regions, Genetic ; Protozoan Proteins/genetics ; Transgenes ; },
abstract = {Transmission of malaria parasites from humans to mosquito vectors requires that some asexual parasites differentiate into sexual forms termed gametocytes. The balance between proliferation in the same host and conversion into transmission forms can be altered by the conditions of the environment. The ability to accurately measure the rate of sexual conversion under different conditions is essential for research addressing the mechanisms underlying sexual conversion, and to assess the impact of environmental factors. Here we describe new Plasmodium falciparum transgenic lines with genome-integrated constructs in which a fluorescent reporter is expressed under the control of the promoter of the gexp02 gene. Using these parasite lines, we developed a sexual conversion assay that shortens considerably the time needed for an accurate determination of sexual conversion rates, and dispenses the need to add chemicals to inhibit parasite replication. Furthermore, we demonstrate that gexp02 is expressed specifically in sexual parasites, with expression starting as early as the sexual ring stage, which makes it a candidate marker for circulating sexual rings in epidemiological studies.},
}
@article {pmid31601800,
year = {2019},
author = {Shiriaeva, AA and Savitskaya, E and Datsenko, KA and Vvedenskaya, IO and Fedorova, I and Morozova, N and Metlitskaya, A and Sabantsev, A and Nickels, BE and Severinov, K and Semenova, E},
title = {Detection of spacer precursors formed in vivo during primed CRISPR adaptation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4603},
pmid = {31601800},
issn = {2041-1723},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; R35 GM118059/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Escherichia coli/*genetics/growth &amp; development ; Gene Expression Regulation, Bacterial ; High-Throughput Nucleotide Sequencing/*methods ; Microorganisms, Genetically-Modified ; Transgenes ; },
abstract = {Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.},
}
@article {pmid31601799,
year = {2019},
author = {Kuppers, DA and Arora, S and Lim, Y and Lim, AR and Carter, LM and Corrin, PD and Plaisier, CL and Basom, R and Delrow, JJ and Wang, S and Hansen He, H and Torok-Storb, B and Hsieh, AC and Paddison, PJ},
title = {N[6]-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4596},
pmid = {31601799},
issn = {2041-1723},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; U01 HL099993/HL/NHLBI NIH HHS/United States ; R01 DK119270/DK/NIDDK NIH HHS/United States ; S10 OD020069/OD/NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; R37 CA230617/CA/NCI NIH HHS/United States ; },
mesh = {Adenosine/*analogs &amp; derivatives/genetics ; Antigens, CD34/genetics/metabolism ; Bone Marrow Cells/physiology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Cell Line, Tumor ; Erythropoiesis/*genetics ; Gene Expression Regulation ; Histones/genetics/metabolism ; Humans ; Kruppel-Like Transcription Factors/genetics/metabolism ; Leukemia, Erythroblastic, Acute/genetics ; Methyltransferases/genetics ; Promoter Regions, Genetic ; *Protein Biosynthesis ; RNA Splicing Factors/genetics ; RNA, Messenger/genetics/metabolism ; Regulon ; },
abstract = {Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we utilize a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits are genes coding for the N[6]-methyladenosine (m[6]A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We demonstrate that m[6]A MTase activity promotes erythroid gene expression programs through selective translation of ~300 m[6]A marked mRNAs, including those coding for SETD histone methyltransferases, ribosomal components, and polyA RNA binding proteins. Remarkably, loss of m[6]A marks results in dramatic loss of H3K4me3 marks across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m[6]A MTase subunit and a subset of their mRNAs targets are required for human erythroid specification in primary bone-marrow derived progenitors. Thus, m[6]A mRNA marks promote the translation of a network of genes required for human erythropoiesis.},
}
@article {pmid31601489,
year = {2020},
author = {Kelkar, A and Zhu, Y and Groth, T and Stolfa, G and Stablewski, AB and Singhi, N and Nemeth, M and Neelamegham, S},
title = {Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {1},
pages = {29-41},
pmid = {31601489},
issn = {1525-0024},
support = {P30 CA016056/CA/NCI NIH HHS/United States ; R01 HL103411/HL/NHLBI NIH HHS/United States ; R21 GM126537/GM/NIGMS NIH HHS/United States ; R21 GM133195/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*drug effects/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Doxycycline/*pharmacology ; Enzyme Activation/drug effects ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome, Human ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lentivirus/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.},
}
@article {pmid31600776,
year = {2020},
author = {Strøm, TB and Bjune, K and Leren, TP},
title = {Bone morphogenetic protein 1 cleaves the linker region between ligand-binding repeats 4 and 5 of the LDL receptor and makes the LDL receptor non-functional.},
journal = {Human molecular genetics},
volume = {29},
number = {8},
pages = {1229-1238},
doi = {10.1093/hmg/ddz238},
pmid = {31600776},
issn = {1460-2083},
mesh = {Animals ; Bone Morphogenetic Protein 1/antagonists &amp; inhibitors/*genetics ; CHO Cells ; CRISPR-Cas Systems/genetics ; Cholesterol/*genetics ; Cholesterol, LDL/antagonists &amp; inhibitors/blood/*genetics ; Cricetulus ; Endocytosis/genetics ; Enzyme Inhibitors/pharmacology ; Furin/*genetics ; Gene Expression Regulation/drug effects ; Humans ; Hydrazines/pharmacology ; Ligands ; Lipoproteins, LDL/genetics ; Proprotein Convertases/genetics ; Proteolysis/drug effects ; RNA, Small Interfering/genetics ; Receptors, LDL/antagonists &amp; inhibitors/*genetics ; Thiourea/analogs &amp; derivatives/pharmacology ; },
abstract = {The cell-surface low-density lipoprotein receptor (LDLR) internalizes low-density lipoprotein (LDL) by receptor-mediated endocytosis and plays a key role in the regulation of plasma cholesterol levels. The ligand-binding domain of the LDLR contains seven ligand-binding repeats of approximately 40 residues each. Between ligand-binding repeats 4 and 5, there is a 10-residue linker region that is subject to enzymatic cleavage. The cleaved LDLR is unable to bind LDL. In this study, we have screened a series of enzyme inhibitors in order to identify the enzyme that cleaves the linker region. These studies have identified bone morphogenetic protein 1 (BMP1) as being the cleavage enzyme. This conclusion is based upon the use of the specific BMP1 inhibitor UK 383367, silencing of the BMP1 gene by the use of siRNA or CRISPR/Cas9 technology and overexpression of wild-type BMP1 or the loss-of-function mutant E214A-BMP1. We have also shown that the propeptide of BMP1 has to be cleaved at RSRR120↓ by furin-like proprotein convertases for BMP1 to have an activity towards the LDLR. Targeting BMP1 could represent a novel strategy to increase the number of functioning LDLRs in order to lower plasma LDL cholesterol levels. However, a concern by using BMP1 inhibitors as cholesterol-lowering drugs could be the risk of side effects based on the important role of BMP1 in collagen assembly.},
}
@article {pmid31600234,
year = {2019},
author = {Chen, S and Soehnlen, M and Blom, J and Terrapon, N and Henrissat, B and Walker, ED},
title = {Comparative genomic analyses reveal diverse virulence factors and antimicrobial resistance mechanisms in clinical Elizabethkingia meningoseptica strains.},
journal = {PloS one},
volume = {14},
number = {10},
pages = {e0222648},
pmid = {31600234},
issn = {1932-6203},
support = {R37 AI021884/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/therapeutic use ; Biofilms/growth &amp; development ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Comparative Genomic Hybridization ; DNA-Binding Proteins/genetics ; Drug Resistance, Bacterial/*genetics ; Flavobacteriaceae/*genetics/pathogenicity ; Flavobacteriaceae Infections/drug therapy/epidemiology/*genetics/microbiology ; Genome, Bacterial/*genetics ; Genomics/methods ; Humans ; Phylogeny ; Transcription Factors/genetics ; Virulence Factors/genetics ; },
abstract = {Three human clinical isolates of bacteria (designated strains Em1, Em2 and Em3) had high average nucleotide identity (ANI) to Elizabethkingia meningoseptica. Their genome sizes (3.89, 4.04 and 4.04 Mb) were comparable to those of other Elizabethkingia species and strains, and exhibited open pan-genome characteristics, with two strains being nearly identical and the third divergent. These strains were susceptible only to trimethoprim/sulfamethoxazole and ciprofloxacin amongst 16 antibiotics in minimum inhibitory tests. The resistome exhibited a high diversity of resistance genes, including 5 different lactamase- and 18 efflux protein- encoding genes. Forty-four genes encoding virulence factors were conserved among the strains. Sialic acid transporters and curli synthesis genes were well conserved in E. meningoseptica but absent in E. anophelis and E. miricola. E. meningoseptica carried several genes contributing to biofilm formation. 58 glycoside hydrolases (GH) and 25 putative polysaccharide utilization loci (PULs) were found. The strains carried numerous genes encoding two-component system proteins (56), transcription factor proteins (187~191), and DNA-binding proteins (6~7). Several prophages and CRISPR/Cas elements were uniquely present in the genomes.},
}
@article {pmid31600188,
year = {2019},
author = {Graziano, BR and Town, JP and Sitarska, E and Nagy, TL and Fošnarič, M and Penič, S and Iglič, A and Kralj-Iglič, V and Gov, NS and Diz-Muñoz, A and Weiner, OD},
title = {Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000457},
pmid = {31600188},
issn = {1545-7885},
support = {P30 CA082103/CA/NCI NIH HHS/United States ; R35 GM118167/GM/NIGMS NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; },
mesh = {Actin-Related Protein 2-3 Complex/*genetics/metabolism ; Actins/*genetics/metabolism ; Biomechanical Phenomena ; CRISPR-Cas Systems ; Cell Adhesion/drug effects ; Cell Membrane/drug effects/metabolism/ultrastructure ; Cell Polarity/drug effects/*genetics ; Chemotactic Factors/pharmacology ; Chemotaxis/drug effects/*genetics ; Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; HL-60 Cells ; Humans ; Microscopy, Atomic Force ; N-Formylmethionine Leucyl-Phenylalanine/pharmacology ; Pseudopodia/drug effects/metabolism/ultrastructure ; Signal Transduction ; Surface Properties ; Wiskott-Aldrich Syndrome Protein Family/deficiency/*genetics ; },
abstract = {Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.},
}
@article {pmid31599948,
year = {2019},
author = {Lee, SM and Carlson, AH and Onal, M and Benkusky, NA and Meyer, MB and Pike, JW},
title = {A Control Region Near the Fibroblast Growth Factor 23 Gene Mediates Response to Phosphate, 1,25(OH)2D3, and LPS In Vivo.},
journal = {Endocrinology},
volume = {160},
number = {12},
pages = {2877-2891},
pmid = {31599948},
issn = {1945-7170},
support = {R01 AR064424/AR/NIAMS NIH HHS/United States ; R01 DK118174/DK/NIDDK NIH HHS/United States ; T32 GM008688/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bone and Bones/metabolism ; CRISPR-Cas Systems ; Calcitriol ; Enhancer Elements, Genetic ; Female ; Fibroblast Growth Factor-23 ; Fibroblast Growth Factors/genetics/*metabolism ; *Gene Expression Regulation ; Lipopolysaccharides ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Phosphates/blood ; Promoter Regions, Genetic ; },
abstract = {Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the control of phosphate (P) homeostasis and vitamin D metabolism. Despite advances, however, molecular details of this gene's regulation remain uncertain. In this report, we created mouse strains in which four epigenetically marked FGF23 regulatory regions were individually deleted from the mouse genome using CRISPR/Cas9 gene-editing technology, and the consequences of these mutations were then assessed on Fgf23 expression and regulation in vivo. An initial analysis confirmed that bone expression of Fgf23 and circulating intact FGF23 (iFGF23) were strongly influenced by both chronic dietary P treatment and acute injection of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, further analysis revealed that bone Fgf23 expression and iFGF23 could be rapidly upregulated by dietary P within 3 and 6 hours, respectively; this acute upregulation was lost in the FGF23-PKO mouse containing an Fgf23 proximal enhancer deletion but not in the additional enhancer-deleted mice. Of note, prolonged dietary P treatment over several days led to normalization of FGF23 levels in the FGF23-PKO mouse, suggesting added complexity associated with P regulation of FGF23. Treatment with 1,25(OH)2D3 also revealed a similar loss of Fgf23 induction and blood iFGF23 levels in this mouse. Finally, normal lipopolysaccharide (LPS) induction of Fgf23 expression was also compromised in the FGF23-PKO mouse, a result that, together with our previous report, indicates that the action of LPS on Fgf23 expression is mediated by both proximal and distal Fgf23 enhancers. These in vivo data provide key functional insight into the genomic enhancers through which Fgf23 expression is mediated.},
}
@article {pmid31599687,
year = {2019},
author = {Halpern, J and O'Hara, SE and Doxzen, KW and Witkowsky, LB and Owen, AL},
title = {Societal and Ethical Impacts of Germline Genome Editing: How Can We Secure Human Rights?.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {293-298},
pmid = {31599687},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Gene Editing/*ethics ; Germ Cells ; Human Rights/*ethics ; Humans ; Morals ; Social Values ; },
abstract = {Genome editing has opened up the possibility of heritable alteration of the human germline. The potential of this powerful tool has spurred a call for establishing robust regulatory frameworks to outline permissible uses of genome editing and to map a rational and ethical course. In response, major national scientific bodies and international organizations have convened and released comprehensive reports outlining recommendations for ethical regulatory frameworks. Significantly, these include an emphasis on public participation and the development of principles to guide future applications of genome editing. While essential, public input and principles are not sufficient to ensure ethical uses of this technology. We propose an approach that relies not only on agreed-upon principles and a democratic process but requires a Human Rights Impact Assessment to evaluate the potential burdens that such biomedical interventions may place on human rights.},
}
@article {pmid31599686,
year = {2019},
author = {Wilson, RC and Carroll, D},
title = {The Daunting Economics of Therapeutic Genome Editing.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {280-284},
doi = {10.1089/crispr.2019.0052},
pmid = {31599686},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Gene Editing/*economics/ethics ; Genetic Engineering/*economics/ethics ; Genetic Therapy/economics ; Genome ; Genome, Human/genetics ; Genomics/economics/ethics ; Germ Cells/metabolism/physiology ; Humans ; },
abstract = {There is no shortage of enthusiasm for the clinical potential of CRISPR-based genome editing: many life-changing cures appear to be just around the corner. However, as mature genetic therapies reach the market, it seems that million-dollar price tags are the new normal. Several factors contribute to the extreme pricing of next-generation medicines, including the need to recoup development costs, the undeniable value of these powerful therapies, and the inherent technical challenges of manufacture and delivery. CRISPR technology has been hailed as a great leveler and a democratizing force in biomedicine. But for this principle to hold true in clinical contexts, therapeutic genome editing must avoid several pitfalls that could substantially limit access to its transformative potential, especially in the developing world.},
}
@article {pmid31599683,
year = {2019},
author = {So, D},
title = {The Use and Misuse of Brave New World in the CRISPR Debate.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {316-323},
doi = {10.1089/crispr.2019.0046},
pmid = {31599683},
issn = {2573-1602},
support = {146265//CIHR/Canada ; },
mesh = {Attitude to Health ; CRISPR-Cas Systems ; *Communication ; Ethicists ; Eugenics/trends ; Genetic Engineering/*ethics/*psychology ; Germ Cells ; Humans ; Literature, Modern ; Technology ; Writing ; },
abstract = {When writing about CRISPR and similar technologies, many bioethicists use science-fiction references to help readers picture the ramifications of germline gene editing. By a large margin, the most frequently referenced novel in this debate is Aldous Huxley's 1932 dystopia Brave New World. Despite its iconic status and effectiveness at communicating specific ethical issues, Brave New World provides relatively poor examples of interventions such as gene therapy or enhancement. In addition, the eugenic tropes that Huxley promotes in much of his work make Brave New World an uncomfortable choice for authors who oppose the use of CRISPR for illiberal purposes. Ethicists should consider bringing a wider variety of fiction references into the discourse on genome editing, especially stories that can complement Brave New World with insights about the ethical issues left undeveloped in Huxley's novel.},
}
@article {pmid31599680,
year = {2019},
author = {Macintosh, KL},
title = {Heritable Genome Editing and the Downsides of a Global Moratorium.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {272-279},
doi = {10.1089/crispr.2019.0016},
pmid = {31599680},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Genetic Engineering/*ethics/*legislation &amp; jurisprudence ; Genome ; Genome, Human/genetics ; Genomics/ethics/legislation &amp; jurisprudence ; Germ Cells/metabolism/physiology ; Government ; Humans ; },
abstract = {In 2018, Dr. He Jiankui reported that he had edited human embryos and transferred them to a woman, causing her to give birth to twin girls with modified genomes. An international group of scientists and ethicists responded by proposing a global moratorium on heritable genome editing (HGE). In this article, I oppose this proposal on several grounds. A global moratorium might encourage participating nations to ban HGE or postpone access to it indefinitely. It might also deter or delay basic research that could lead to safe and effective HGE. Lastly, a global moratorium might induce participating nations to adopt or maintain laws and regulations that stigmatize children born with modified genomes. As an alternative, I argue that nations should regulate HGE for safety and efficacy only and without distinguishing between therapeutic and enhancing modifications.},
}
@article {pmid31599679,
year = {2019},
author = {Knoppers, BM and Kleiderman, E},
title = {Heritable Genome Editing: Who Speaks for "Future" Children?.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {285-292},
doi = {10.1089/crispr.2019.0019},
pmid = {31599679},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Child ; Child, Preschool ; Gene Editing/*ethics ; Genetic Engineering/*ethics ; Genetic Therapy/*ethics/methods ; Genome/genetics ; Genome, Human/genetics ; Germ Cells/metabolism/physiology ; Humans ; Infant ; Infant, Newborn ; },
abstract = {Approximately 80% of rare and often incurable and serious conditions affect newborns and children, and roughly half of all rare diseases are considered to have an onset in childhood. Somatic gene therapies are already in clinical trials for spinal muscular atrophy, beta thalassemia, and macular degeneration. If proven to be safe and effective, could heritable genome editing be seen as a form of preventive personalized medicine and as fostering the right to health of the child? The latest calls for global moratoria on clinical applications of heritable genome editing are troubling in that they may create an illusion of control over rogue science and stifle the necessary international debate surrounding an ethically responsible translational path forward. Children are people with distinct rights and interests. An arbitrary moratorium neither fosters their best interests or health nor respects their right to benefit from the advancements of science.},
}
@article {pmid31599678,
year = {2019},
author = {Sherkow, JS},
title = {Controlling CRISPR Through Law: Legal Regimes as Precautionary Principles.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {299-303},
doi = {10.1089/crispr.2019.0029},
pmid = {31599678},
issn = {2573-1602},
mesh = {Animals ; Bioethics/trends ; CRISPR-Cas Systems ; Decision Making/ethics ; Ethical Review/*legislation &amp; jurisprudence ; Genetic Engineering/*ethics/*legislation &amp; jurisprudence ; Government Regulation ; Humans ; Morals ; Politics ; },
abstract = {Since its advent in 2012, CRISPR has spawned a cottage industry of bioethics literature. One principal criticism of the technology is its virtually instant widespread adoption prior to deliberative bodies conducting a meaningful ethical review of its harms and benefits-a violation, to some, of bioethics' "precautionary principle." This view poorly considers, however, the role that the law can play-and does, in fact, play-in policing the introduction of ethically problematic uses of the technology. This Perspective recounts these legal regimes, including regulatory agencies and premarket approval, tort law and deterrence, patents and ethical licenses, funding agencies and review boards, as well as local politics. Identifying these legal regimes and connecting them to the precautionary principle should be instructive for bioethicists and policy makers who wish to conduct ethical reviews of new applications of CRISPR prior to their introduction.},
}
@article {pmid31599677,
year = {2019},
author = {Davies, K and Knoppers, BM},
title = {From Poetry to Policy: An Interview with Bartha Maria Knoppers.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {249-252},
doi = {10.1089/crispr.2019.29071.bkn},
pmid = {31599677},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Genetic Engineering/ethics ; Genetics/*ethics ; History, 20th Century ; History, 21st Century ; Human Genetics/*ethics ; Humans ; },
}
@article {pmid31599676,
year = {2019},
author = {Barrangou, R},
title = {Thinking About CRISPR: The Ethics of Human Genome Editing.},
journal = {The CRISPR journal},
volume = {2},
number = {5},
pages = {247-248},
doi = {10.1089/crispr.2019.29072.rba},
pmid = {31599676},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics ; Genome, Human/genetics ; Humans ; },
}
@article {pmid31597782,
year = {2019},
author = {Passos, V and Zillinger, T and Casartelli, N and Wachs, AS and Xu, S and Malassa, A and Steppich, K and Schilling, H and Franz, S and Todt, D and Steinmann, E and Sutter, K and Dittmer, U and Bohne, J and Schwartz, O and Barchet, W and Goffinet, C},
title = {Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor.},
journal = {Journal of virology},
volume = {93},
number = {24},
pages = {},
pmid = {31597782},
issn = {1098-5514},
mesh = {Anti-HIV Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Line ; Cell Membrane/drug effects/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Genotype ; HEK293 Cells ; HIV Infections/virology ; HIV-1/*drug effects ; Host-Pathogen Interactions/physiology ; Humans ; Interferon-alpha ; Membrane Proteins/genetics/*metabolism/*pharmacology ; Nitriles ; Pyrazoles/pharmacology ; Pyrimidines ; T-Lymphocytes/virology ; Virion/metabolism ; nef Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; },
abstract = {When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.},
}
@article {pmid31597658,
year = {2019},
author = {Ilbay, O and Ambros, V},
title = {Regulation of nuclear-cytoplasmic partitioning by the lin-28-lin-46 pathway reinforces microRNA repression of HBL-1 to confer robust cell-fate progression in C. elegans.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {21},
pages = {},
pmid = {31597658},
issn = {1477-9129},
support = {R01 GM034028/GM/NIGMS NIH HHS/United States ; R35 GM131741/GM/NIGMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; Adaptor Proteins, Signal Transducing/*metabolism ; Alleles ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/*metabolism ; Cell Differentiation ; Cell Lineage ; Cell Nucleus/*metabolism ; Cytoplasm/*metabolism ; DNA-Binding Proteins/*metabolism ; Disease Progression ; Down-Regulation ; Gene Deletion ; *Gene Expression Regulation, Developmental ; MicroRNAs/*metabolism ; Phenotype ; RNA-Binding Proteins/metabolism ; Repressor Proteins/*metabolism ; Transcription Factors/*metabolism ; },
abstract = {MicroRNAs target complementary mRNAs for degradation or translational repression, reducing or preventing protein synthesis. In Caenorhabditis elegans, the transcription factor HBL-1 (Hunchback-like 1) promotes early larval (L2)-stage cell fates, and the let-7 family microRNAs temporally downregulate HBL-1 to enable the L2-to-L3 cell-fate progression. In parallel to let-7-family microRNAs, the conserved RNA-binding protein LIN-28 and its downstream gene lin-46 also act upstream of HBL-1 in regulating the L2-to-L3 cell-fate progression. The molecular function of LIN-46, and how the lin-28-lin-46 pathway regulates HBL-1, are not understood. Here, we report that the regulation of HBL-1 by the lin-28-lin-46 pathway is independent of the let-7/lin-4 microRNA complementary sites (LCSs) in the hbl-1 3'UTR, and involves stage-specific post-translational regulation of HBL-1 nuclear accumulation. We find that LIN-46 is necessary and sufficient to prevent nuclear accumulation of HBL-1. Our results illuminate that robust progression from L2 to L3 cell fates depends on the combination of two distinct modes of HBL-1 downregulation: decreased synthesis of HBL-1 via let-7-family microRNA activity, and decreased nuclear accumulation of HBL-1 via action of the lin-28-lin-46 pathway.},
}
@article {pmid31597252,
year = {2019},
author = {Reuven, N and Adler, J and Broennimann, K and Myers, N and Shaul, Y},
title = {Recruitment of DNA Repair MRN Complex by Intrinsically Disordered Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated Genome Editing.},
journal = {Biomolecules},
volume = {9},
number = {10},
pages = {},
pmid = {31597252},
issn = {2218-273X},
mesh = {Acid Anhydride Hydrolases/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism ; DNA Repair ; DNA Repair Enzymes/*metabolism ; DNA-Binding Proteins/metabolism ; Deoxyribonucleases/*chemistry/genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Herpesvirus 1, Human/genetics/*metabolism ; Humans ; MRE11 Homologue Protein/metabolism ; Nuclear Proteins/metabolism ; Protein Domains ; Recombinant Fusion Proteins/metabolism ; Viral Proteins/*chemistry/genetics/metabolism ; },
abstract = {CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing.},
}
@article {pmid31597119,
year = {2019},
author = {Raposo, VL},
title = {CRISPR-Cas9 and the Promise of a Better Future.},
journal = {European journal of health law},
volume = {26},
number = {4},
pages = {308-329},
doi = {10.1163/15718093-12264438},
pmid = {31597119},
issn = {1571-8093},
mesh = {Biomedical Research/ethics/legislation &amp; jurisprudence ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Europe ; Forecasting ; Gene Editing/ethics/*legislation &amp; jurisprudence ; Genome, Human ; Government Regulation ; Humans ; },
abstract = {Due to its simplicity, low cost and accuracy, CRISPR-Cas9 has become a promising new technique in the field of gene editing. However, despite its virtues, it is not yet immune to scientific hazards and ethical legal concerns. These concerns have been used to justify opposition to genetic manipulation, and have led to some regulations to ban or impose a moratorium based on the precautionary principle. In Europe, regulation mostly comes from the European Union and the Council of Europe, both very cautious towards gene editing. In this article, two arguments on the future legal framework of CRISPR-Cas9 are made. The first is that continued research will contribute to more scientific accuracy; thus, the precautionary principle should promote regulated research to achieve this aim. The second is that most of the legal and ethical concerns surrounding CRISPR-Cas9 are based on unfounded prejudice emanating from a mystical understanding of the human genome.},
}
@article {pmid31596609,
year = {2020},
author = {Koh, KD and Siddiqui, S and Cheng, D and Bonser, LR and Sun, DI and Zlock, LT and Finkbeiner, WE and Woodruff, PG and Erle, DJ},
title = {Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.},
journal = {American journal of respiratory cell and molecular biology},
volume = {62},
number = {3},
pages = {373-381},
pmid = {31596609},
issn = {1535-4989},
support = {P01 HL107202/HL/NHLBI NIH HHS/United States ; K24 HL137013/HL/NHLBI NIH HHS/United States ; U19 AI077439/AI/NIAID NIH HHS/United States ; R35 HL145235/HL/NHLBI NIH HHS/United States ; P30 DK072517/DK/NIDDK NIH HHS/United States ; },
mesh = {Bronchi/cytology ; CRISPR-Cas Systems ; Cells, Cultured ; Down-Regulation ; Epithelial Cells/*drug effects/metabolism ; Gene Expression Regulation ; Gene Targeting/*methods ; Goblet Cells/metabolism ; Humans ; Interleukin-13/*physiology ; Metaplasia ; Mucin 5AC/biosynthesis/genetics ; Mucociliary Clearance/*physiology ; Primary Cell Culture ; Proto-Oncogene Proteins c-ets/deficiency/genetics/*physiology ; RNA, Guide/genetics ; Ribonucleoproteins/administration &amp; dosage/*genetics ; Transcriptome ; },
abstract = {Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.},
}
@article {pmid31596028,
year = {2020},
author = {Ajami, M and Atashi, A and Kaviani, S and Kiani, J and Soleimani, M},
title = {Generation of an in vitro model of β-thalassemia using the CRISPR/Cas9 genome editing system.},
journal = {Journal of cellular biochemistry},
volume = {121},
number = {2},
pages = {1420-1430},
doi = {10.1002/jcb.29377},
pmid = {31596028},
issn = {1097-4644},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Differentiation ; Erythroid Cells/*cytology/metabolism ; *Gene Editing ; Genetic Therapy ; In Vitro Techniques ; Mice ; Mouse Embryonic Stem Cells/*cytology/metabolism ; beta-Globins/antagonists &amp; inhibitors/*genetics ; beta-Thalassemia/*genetics/pathology/therapy ; },
abstract = {β-Thalassemia is a common monogenic disease characterized by defective β-globin chains synthesis. In vitro β-thalassemia-related research on increasing β-like globin genes or identification of factors reducing the severity of the disease, has been performed on mouse erythroleukaemia or K562 cell lines. The aim of this study was the production of an in vitro model of β-thalassemia using the highly efficient CRISPR-Cas9 system. Embryonic stem (ES) cells were nucleofected with guide RNA (gRNA)-Cas9 expression vectors. Molecular testing was done on extracted DNA to assess Hbb-b1 mutation. Analysis of transcription factors and hemoglobin genes were evaluated using quantitative reverse transcription-polymerase chain reaction following erythroid differentiation of ES cells. Sequencing data confirmed Hbb-b1 knockout alleles. Significant expression of erythroid transcription factors was observed in wild-type, Hbb-b1[+/-] and Hbb-b1[-/-] groups (P < .001). Compared with the wild-type group, the absolute number of Hbb-b1 mRNA in Hbb-b1[+/-] group significantly decreased from 6.44 × 10[6] to 3.23 × 10[6] copy number (P < .01), whereas in Hbb-b1[-/-] group had zero expression. The CRISPR/Cas9-mediated Hbb-b1 knockout in ES cells provides accessibility to an in vitro thalassemia model following erythroid differentiation. Considering the need for in vitro and mouse models to investigate the molecular basis of β-thalassemia which also enables testing of therapeutic approaches, this method can be utilized to produce a mouse model of β-thalassemia intermedia (Hbbth1/th1).},
}
@article {pmid31595153,
year = {2019},
author = {Li, P and Meng, Y and Wang, Y and Li, J and Lam, M and Wang, L and Di, LJ},
title = {Nuclear localization of Desmoplakin and its involvement in telomere maintenance.},
journal = {International journal of biological sciences},
volume = {15},
number = {11},
pages = {2350-2362},
pmid = {31595153},
issn = {1449-2288},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; DNA Damage ; Desmoplakins/chemistry/metabolism/*physiology ; HEK293 Cells ; Humans ; Telomerase/metabolism ; Telomere/metabolism ; *Telomere Homeostasis ; Telomere Shortening ; Telomere-Binding Proteins/chemistry/metabolism/*physiology ; },
abstract = {The interaction between genomic DNA and protein fundamentally determines the activity and the function of DNA elements. Capturing the protein complex and identifying the proteins associated with a specific DNA locus is difficult. Herein, we employed CRISPR, the well-known gene-targeting tool in combination with the proximity-dependent labeling tool BioID to capture a specific genome locus associated proteins and to uncover the novel functions of these proteins. By applying this research tool on telomeres, we identified DSP, out of many others, as a convincing telomere binding protein validated by both biochemical and cell-biological approaches. We also provide evidence to demonstrate that the C-terminal domain of DSP is required for its binding to telomere after translocating to the nucleus mediated by NLS sequence of DSP. In addition, we found that the telomere binding of DSP is telomere length dependent as hTERT inhibition or knockdown caused a decrease of telomere length and diminished DSP binding to the telomere. Knockdown of TRF2 also negatively influenced DSP binding to the telomere. Functionally, loss of DSP resulted in the shortened telomere DNA and induced the DNA damage response and cell apoptosis. In conclusion, our studies identified DSP as a novel potential telomere binding protein and highlighted its role in protecting against telomere DNA damage and resultant cell apoptosis.},
}
@article {pmid31594694,
year = {2020},
author = {Shi, M and Kawabe, Y and Ito, A and Kamihira, M},
title = {Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration.},
journal = {Journal of bioscience and bioengineering},
volume = {129},
number = {3},
pages = {363-370},
doi = {10.1016/j.jbiosc.2019.09.011},
pmid = {31594694},
issn = {1347-4421},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Chickens/genetics ; Gene Editing ; Gene Expression ; Genetic Loci ; Ovalbumin/*genetics/metabolism ; RNA, Guide/genetics ; },
abstract = {It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.},
}
@article {pmid31594078,
year = {2019},
author = {Wang, J and Chen, R and Lu, FM},
title = {[Exosome-mediated CRISPR/Cas9 system targets to cut the intercellular transmission function of hepatitis B virus genome].},
journal = {Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology},
volume = {27},
number = {8},
pages = {610-614},
doi = {10.3760/cma.j.issn.1007-3418.2019.08.005},
pmid = {31594078},
issn = {1007-3418},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Exosomes ; Genome, Viral ; Hepatitis B virus/*genetics ; Humans ; Transfection ; },
abstract = {Objective: To determine whether single-stranded guided RNA (gRNA) and Cas9 protein exist in the exosome secreted by cells transfected with CRISPR/Cas9 expression plasmid. Furthermore, to explore whether CRISPR/Cas9 system can edit target genes of peripheral cells through the intercellular transmission of exosomes. Methods: (1) The CRISPR/Cas9 expression plasmid was transfected into HuH7 cells. The supernatant was collected and the exosomes were concentrated and purified by differential centrifugation. The morphology and particle size of exosomes were determined by electronic microscopy and Malvern laser scatter granulometry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the levels of gRNA and Cas9 protein. (2) HuH7 cells transfected with pBB4.5 1.2×HBV and HBV specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, HBV DNA was extracted and sequenced by PCR. Results: There were not only full-length gRNA and Cas9 protein in the exosomes of Huh7 cells transfected with CRISPR / Cas9 expression plasmid. In addition, gene-editing functions were delivered between the cells to achieve the destruction of HBV genome of surrounding cells. Conclusion: The CRISPR-Cas9 gene-editing system has the potential to deliver exosomes between cells via carrying functional gRNA and Cas9 proteins. This phenomenon hints that we are in the process of using the CRISPR/Cas9 system for gene therapy. Therefore, it is necessary to consider the potential effects of exosomes-carried gRNA and Cas9 proteins on the surrounding cells of the distal tubules.},
}
@article {pmid31593471,
year = {2019},
author = {Liu, Q and Cai, J and Zheng, Y and Tan, Y and Wang, Y and Zhang, Z and Zheng, C and Zhao, Y and Liu, C and An, Y and Jiang, C and Shi, L and Kang, C and Liu, Y},
title = {NanoRNP Overcomes Tumor Heterogeneity in Cancer Treatment.},
journal = {Nano letters},
volume = {19},
number = {11},
pages = {7662-7672},
doi = {10.1021/acs.nanolett.9b02501},
pmid = {31593471},
issn = {1530-6992},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Core Binding Factor Alpha 2 Subunit/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Mice ; Mice, Nude ; Nanomedicine/methods ; Neoplasms/genetics/pathology/*therapy ; STAT3 Transcription Factor/genetics ; },
abstract = {Tumor heterogeneity has been one of the most important factors leading to the failure of conventional cancer therapies due to the accumulation of genetically distinct tumor-cell subpopulations during the tumor development process. Due to the diversity of genetic mutations during tumor growth, combining the use of multiple drugs has only achieved limited success in combating heterogeneous tumors. Herein, we report a novel antitumor strategy that effectively addresses tumor heterogeneity by using a CRISPR/Cas9-based nanoRNP carrying a combination of sgRNAs. Such nanoRNP is synthesized from Cas9 ribonucleoprotein, any combinations of required sgRNAs, and a rationally designed responsive polymer that endows nanoRNP with high circulating stability, enhanced tumor accumulation, and the efficient gene editing in targeted tumor cells eventually. By carrying a combination of sgRNAs that targets STAT3 and RUNX1, the nanoRNP exhibited efficient gene expression disruptions on a heterogeneous tumor model with two subsets of cells whose proliferations were sensitive to the reduced expression of STAT3 and RUNX1, respectively, leading to the effective growth inhibition of the heterogeneous tumor. Considering the close relationship between tumor heterogeneity and cancer progression, resistance to therapy, and recurrences, nanoRNP provides a feasible strategy to overcome tumor heterogeneity in the development of more advanced cancer therapy against malignant tumors.},
}
@article {pmid31591751,
year = {2020},
author = {Fischer, K and Rieblinger, B and Hein, R and Sfriso, R and Zuber, J and Fischer, A and Klinger, B and Liang, W and Flisikowski, K and Kurome, M and Zakhartchenko, V and Kessler, B and Wolf, E and Rieben, R and Schwinzer, R and Kind, A and Schnieke, A},
title = {Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.},
journal = {Xenotransplantation},
volume = {27},
number = {1},
pages = {e12560},
doi = {10.1111/xen.12560},
pmid = {31591751},
issn = {1399-3089},
mesh = {Animals ; Antibodies, Heterophile/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Complement System Proteins/metabolism ; Galactosyltransferases/*genetics ; Graft Rejection/*immunology ; HLA Antigens/immunology ; Heterografts/immunology ; Histocompatibility Antigens Class I ; Humans ; *Kidney Transplantation ; Mixed Function Oxygenases/*genetics ; N-Acetylgalactosaminyltransferases/*genetics ; Swine ; Transplantation, Heterologous ; },
abstract = {BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation.<br><br>METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro.<br><br>RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect.<br><br>CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.},
}
@article {pmid31591524,
year = {2019},
author = {Wilson, EA and Anderson, KS},
title = {Weakly immunogenic CRISPR therapies.},
journal = {Nature biomedical engineering},
volume = {3},
number = {10},
pages = {761-762},
doi = {10.1038/s41551-019-0463-7},
pmid = {31591524},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Associated Protein 9/*immunology ; CRISPR-Cas Systems/genetics/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; Cross Reactions/immunology ; Disease Models, Animal ; Gene Editing ; Genetic Therapy/*adverse effects/methods ; Genetic Vectors ; Humans ; Immunity ; Mice ; },
}
@article {pmid31590683,
year = {2019},
author = {Yang, H and Cui, W and Wang, L},
title = {Epigenetic synthetic lethality approaches in cancer therapy.},
journal = {Clinical epigenetics},
volume = {11},
number = {1},
pages = {136},
pmid = {31590683},
issn = {1868-7083},
mesh = {CRISPR-Cas Systems ; Epigenesis, Genetic ; Humans ; Neoplasms/*genetics/therapy ; RNA, Small Interfering/pharmacology/therapeutic use ; *Synthetic Lethal Mutations ; },
abstract = {The onset and development of malignant tumors are closely related to epigenetic modifications, and this has become a research hotspot. In recent years, a variety of epigenetic regulators have been discovered, and corresponding small molecule inhibitors have been developed, but their efficacy in solid tumors is generally poor. With the introduction of the first synthetic lethal drug (the PARP inhibitor olaparib in ovarian cancer with BRCA1 mutation), research into synthetic lethality has also become a hotspot. High-throughput screening with CRISPR-Cas9 and shRNA technology has revealed a large number of synthetic lethal pairs involving epigenetic-related synthetic lethal genes, such as those encoding SWI/SNF complex subunits, PRC2 complex subunits, SETD2, KMT2C, and MLL fusion proteins. In this review, we focus on epigenetic-related synthetic lethal mechanisms, including synthetic lethality between epigenetic mutations and epigenetic inhibitors, epigenetic mutations and non-epigenetic inhibitors, and oncogene mutations and epigenetic inhibitors.},
}
@article {pmid31589905,
year = {2020},
author = {Jin, Y and Liu, M and Sa, R and Fu, H and Cheng, L and Chen, L},
title = {Mouse models of thyroid cancer: Bridging pathogenesis and novel therapeutics.},
journal = {Cancer letters},
volume = {469},
number = {},
pages = {35-53},
doi = {10.1016/j.canlet.2019.09.017},
pmid = {31589905},
issn = {1872-7980},
mesh = {Animals ; Animals, Genetically Modified ; Antineoplastic Agents/*pharmacology/therapeutic use ; Biomarkers, Tumor/analysis/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Disease Progression ; Gene Expression Regulation ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Incidence ; Mice ; Signal Transduction/drug effects/genetics ; Thyroid Gland/drug effects/pathology ; Thyroid Neoplasms/*drug therapy/epidemiology/etiology/pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Due to a global increase in the incidence of thyroid cancer, numerous novel mouse models were established to reveal thyroid cancer pathogenesis and test promising therapeutic strategies, necessitating a comprehensive review of translational medicine that covers (i) the role of mouse models in the research of thyroid cancer pathogenesis, and (ii) preclinical testing of potential anti-thyroid cancer therapeutics. The present review article aims to: (i) describe the current approaches for mouse modeling of thyroid cancer, (ii) provide insight into the biology and genetics of thyroid cancers, and (iii) offer guidance on the use of mouse models for testing potential therapeutics in preclinical settings. Based on research with mouse models of thyroid cancer pathogenesis involving the RTK, RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, SRC, and JAK-STAT signaling pathways, inhibitors of VEGFR, MEK, mTOR, SRC, and STAT3 have been developed as anti-thyroid cancer drugs for "bench-to-bedside" translation. In the future, mouse models of thyroid cancer will be designed to be ''humanized" and "patient-like," offering opportunities to: (i) investigate the pathogenesis of thyroid cancer through target screening based on the CRISPR/Cas system, (ii) test drugs based on new mouse models, and (iii) explore the underlying mechanisms based on multi-omics.},
}
@article {pmid31589887,
year = {2020},
author = {Md Abdullah, AB and Lee, DW and Jung, J and Kim, Y},
title = {Deletion mutant of sPLA2 using CRISPR/Cas9 exhibits immunosuppression, developmental retardation, and failure of oocyte development in legume pod borer, Maruca vitrata.},
journal = {Developmental and comparative immunology},
volume = {103},
number = {},
pages = {103500},
doi = {10.1016/j.dci.2019.103500},
pmid = {31589887},
issn = {1879-0089},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Genes, Insect/physiology ; Immune Tolerance/physiology ; Insect Proteins/*physiology ; Oogenesis/physiology ; Phospholipases A2/*physiology ; Phylogeny ; Spodoptera/*physiology ; },
abstract = {Phospholipase A2 (PLA2) catalyzes release of free fatty acids linked to phospholipids at sn-2 position. Some of these released free fatty acids are used to synthesize eicosanoids that mediate various physiological processes in insects. Although a large number of PLA2s form a superfamily consisting of at least 16 groups, few PLA2s have been identified and characterized in insects. Furthermore, physiological functions of insect PLA2s remain unclear. Clustered regularly interspaced short parlindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been a useful research tool to validate gene function. This study identified and characterized a secretory PLA2 (sPLA2) from legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), and validated its physiological functions using CRISPR/Cas9. An open reading frame of M. vitrata sPLA2 (Mv-sPLA2) encoding 192 amino acids contained signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Mv-sPLA2 was related to other Group III sPLA2s. Mv-sPLA2 was expressed in both larval and adult stages. It was inducible by immune challenge. RNA interference (RNAi) of Mv-sPLA2 significantly suppressed cellular immunity and impaired larval development. Furthermore, RNAi treatment in female adults prevented oocyte development. These physiological alterations were also observed in a mutant line of M. vitrata with Mv-sPLA2 deleted by using CRISPR/Cas9. Mv-sPLA2 was not detected in the mutant line from western blot analysis. Addition of an eicosanoid, PGE2, significantly rescued oocyte development of females of the mutant line. These results suggest that Mv-sPLA2 plays crucial role in immune, developmental, and reproductive processes of M. vitrata.},
}
@article {pmid31589320,
year = {2020},
author = {Dekkers, JF and Whittle, JR and Vaillant, F and Chen, HR and Dawson, C and Liu, K and Geurts, MH and Herold, MJ and Clevers, H and Lindeman, GJ and Visvader, JE},
title = {Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids.},
journal = {Journal of the National Cancer Institute},
volume = {112},
number = {5},
pages = {540-544},
pmid = {31589320},
issn = {1460-2105},
mesh = {Animals ; Breast/cytology/*physiology ; Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/methods ; Gene Knockout Techniques ; Genes, p53 ; Heterografts ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Organoids ; PTEN Phosphohydrolase/genetics ; Retinoblastoma Binding Proteins/genetics ; Tissue Engineering/methods ; Tumor Suppressor Protein p53/genetics ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.},
}
@article {pmid31588686,
year = {2019},
author = {Thach, TT and Bae, DH and Kim, NH and Kang, ES and Lee, BS and Han, K and Kwak, M and Choi, H and Nam, J and Bae, T and Suh, M and Hur, JK and Kim, YH},
title = {Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {15},
number = {46},
pages = {e1903172},
doi = {10.1002/smll.201903172},
pmid = {31588686},
issn = {1613-6829},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; HEK293 Cells ; Humans ; Hydrodynamics ; Lipopeptides/*metabolism ; Liposomes ; Nanoparticles/*chemistry/ultrastructure ; Ribonucleoproteins/*genetics ; },
abstract = {A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.},
}
@article {pmid31588283,
year = {2019},
author = {Greisch, JF and Tamara, S and Scheltema, RA and Maxwell, HWR and Fagerlund, RD and Fineran, PC and Tetter, S and Hilvert, D and Heck, AJR},
title = {Expanding the mass range for UVPD-based native top-down mass spectrometry.},
journal = {Chemical science},
volume = {10},
number = {30},
pages = {7163-7171},
pmid = {31588283},
issn = {2041-6520},
abstract = {Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles.},
}
@article {pmid31587919,
year = {2019},
author = {Cheng, W and Wang, S and Zhang, Z and Morgens, DW and Hayes, LR and Lee, S and Portz, B and Xie, Y and Nguyen, BV and Haney, MS and Yan, S and Dong, D and Coyne, AN and Yang, J and Xian, F and Cleveland, DW and Qiu, Z and Rothstein, JD and Shorter, J and Gao, FB and Bassik, MC and Sun, S},
title = {CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation.},
journal = {Neuron},
volume = {104},
number = {5},
pages = {885-898.e8},
pmid = {31587919},
issn = {1097-4199},
support = {RF1 NS101986/NS/NINDS NIH HHS/United States ; R01 NS107347/NS/NINDS NIH HHS/United States ; R35 GM124824/GM/NIGMS NIH HHS/United States ; K08 NS104273/NS/NINDS NIH HHS/United States ; R37 NS057553/NS/NINDS NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; R01 NS101986/NS/NINDS NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*metabolism ; Animals ; C9orf72 Protein/*biosynthesis ; CRISPR-Cas Systems ; DEAD-box RNA Helicases/*metabolism ; Drosophila ; Frontotemporal Dementia/*metabolism ; Humans ; Protein Biosynthesis/physiology ; Repetitive Sequences, Nucleic Acid ; },
abstract = {Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells. We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.},
}
@article {pmid31587567,
year = {2019},
author = {Zaunbrecher, RJ and Abel, AN and Beussman, K and Leonard, A and von Frieling-Salewsky, M and Fields, PA and Pabon, L and Reinecke, H and Yang, X and Macadangdang, J and Kim, DH and Linke, WA and Sniadecki, NJ and Regnier, M and Murry, CE},
title = {Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.},
journal = {Circulation},
volume = {140},
number = {20},
pages = {1647-1660},
pmid = {31587567},
issn = {1524-4539},
support = {P01 HL094374/HL/NHLBI NIH HHS/United States ; R01 HL128362/HL/NHLBI NIH HHS/United States ; T32 HL007312/HL/NHLBI NIH HHS/United States ; T32 EB001650/EB/NIBIB NIH HHS/United States ; R01 HD048895/HD/NICHD NIH HHS/United States ; U54 DK107979/DK/NIDDK NIH HHS/United States ; R01 HL111197/HL/NHLBI NIH HHS/United States ; F32 HL126332/HL/NHLBI NIH HHS/United States ; R01 HL084642/HL/NHLBI NIH HHS/United States ; R01 HL135143/HL/NHLBI NIH HHS/United States ; R01 HL146436/HL/NHLBI NIH HHS/United States ; R24 HD000836/HD/NICHD NIH HHS/United States ; P01 GM081619/GM/NIGMS NIH HHS/United States ; R01 HL128368/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Calcium Signaling ; Cells, Cultured ; Connectin/genetics/*metabolism ; Fetal Heart/metabolism ; Gene Editing ; Genotype ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Mutation ; *Myocardial Contraction/genetics ; Myocytes, Cardiac/*metabolism ; Phenotype ; Sarcomeres/*metabolism ; },
abstract = {BACKGROUND: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (TTN) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function.<br><br>METHODS: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z[-/-]) and A-band (TTN-A[-/-]) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements.<br><br>RESULTS: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z[-/-]-CMs had sarcomeres and visibly contracted, whereas TTN-A[-/-]-CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z[-/-]-CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z[-/-]-CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin.<br><br>CONCLUSIONS: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.},
}
@article {pmid31586114,
year = {2019},
author = {Zhao, X and Wei, W and Pan, H and Nie, J and Chen, D and Zhang, P and Chen, F and Fu, Q and Zuo, E and Lu, Y and Zhang, M},
title = {Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14315},
pmid = {31586114},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo, Mammalian ; Embryonic Development ; Green Fluorescent Proteins/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic/*embryology/genetics ; *Sex Determination Analysis ; *Y Chromosome ; },
abstract = {Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.},
}
@article {pmid31586095,
year = {2019},
author = {Amoasii, L and Li, H and Zhang, Y and Min, YL and Sanchez-Ortiz, E and Shelton, JM and Long, C and Mireault, AA and Bhattacharyya, S and McAnally, JR and Bassel-Duby, R and Olson, EN},
title = {In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4537},
pmid = {31586095},
issn = {2041-1723},
support = {R01 HL138426/HL/NHLBI NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; Exons/genetics ; Gene Editing/methods ; Genes, Reporter/genetics ; Genetic Therapy/*methods ; Genetic Vectors/chemistry/genetics ; Humans ; Intravital Microscopy/*methods ; Luciferases/chemistry/genetics ; Luminescent Measurements ; Male ; Mice ; Mice, Transgenic ; Muscle, Skeletal/*diagnostic imaging/pathology ; Muscular Dystrophy, Duchenne/diagnostic imaging/genetics/*therapy ; Mutation ; Treatment Outcome ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.},
}
@article {pmid31586074,
year = {2019},
author = {Caeser, R and Di Re, M and Krupka, JA and Gao, J and Lara-Chica, M and Dias, JML and Cooke, SL and Fenner, R and Usheva, Z and Runge, HFP and Beer, PA and Eldaly, H and Pak, HK and Park, CS and Vassiliou, GS and Huntly, BJP and Mupo, A and Bashford-Rogers, RJM and Hodson, DJ},
title = {Genetic modification of primary human B cells to model high-grade lymphoma.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4543},
pmid = {31586074},
issn = {2041-1723},
support = {MC_PC_12009/MRC_/Medical Research Council/United Kingdom ; MR/R009708/1/MRC_/Medical Research Council/United Kingdom ; MR/M008584/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; B-Lymphocytes/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/genetics ; Coculture Techniques/methods ; Genetic Vectors/genetics ; Germinal Center/cytology ; High-Throughput Screening Assays ; Humans ; Lymphoma, Large B-Cell, Diffuse/*genetics/pathology ; Mice ; Neoplasm Grading ; Primary Cell Culture/*methods ; Proto-Oncogene Proteins c-bcl-2/genetics ; Proto-Oncogene Proteins c-bcl-6/genetics ; Proto-Oncogene Proteins c-myc/genetics ; Retroviridae/genetics ; Transduction, Genetic ; Xenograft Model Antitumor Assays ; },
abstract = {Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.},
}
@article {pmid31586052,
year = {2019},
author = {He, W and Zhang, L and Villarreal, OD and Fu, R and Bedford, E and Dou, J and Patel, AY and Bedford, MT and Shi, X and Chen, T and Bartholomew, B and Xu, H},
title = {De novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4541},
pmid = {31586052},
issn = {2041-1723},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; },
mesh = {Algorithms ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Computational Biology/*methods ; Datasets as Topic ; Gene Knockout Techniques ; High-Throughput Screening Assays ; Humans ; Models, Genetic ; Protein Domains/*genetics ; Protein Processing, Post-Translational/genetics ; RNA, Guide/genetics ; SMARCB1 Protein/genetics ; Software ; },
abstract = {High-throughput CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. Here, to facilitate de novo identification of essential protein domains from such screens, we propose ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refer to the protein regions associated with a strong sgRNA dropout effect in the screens. Applied to a published CRISPR tiling screen dataset, ProTiler identifies 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlap with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also reveals unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which is validated experimentally. Surprisingly, the CKHS regions are negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR-Cas9 mediated amino acids loss.},
}
@article {pmid31586047,
year = {2019},
author = {Proto, WR and Siegel, SV and Dankwa, S and Liu, W and Kemp, A and Marsden, S and Zenonos, ZA and Unwin, S and Sharp, PM and Wright, GJ and Hahn, BH and Duraisingh, MT and Rayner, JC},
title = {Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4512},
pmid = {31586047},
issn = {2041-1723},
support = {R37 AI050529/AI/NIAID NIH HHS/United States ; T32 AI007532/AI/NIAID NIH HHS/United States ; 206194/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; P30 AI045008/AI/NIAID NIH HHS/United States ; R01 AI120810/AI/NIAID NIH HHS/United States ; R01 AI091595/AI/NIAID NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Engineering ; Erythrocytes/metabolism/*parasitology ; Evolution, Molecular ; Frameshift Mutation ; Gene Editing ; HEK293 Cells ; Host Specificity/*genetics ; Humans ; Loss of Function Mutation ; Malaria, Falciparum/*parasitology ; Pan troglodytes/parasitology ; Plasmodium falciparum/*genetics/isolation &amp; purification/pathogenicity ; Protozoan Proteins/*genetics ; Sialic Acids/metabolism ; },
abstract = {Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum.},
}
@article {pmid31585845,
year = {2019},
author = {Nussenzweig, PM and McGinn, J and Marraffini, LA},
title = {Cas9 Cleavage of Viral Genomes Primes the Acquisition of New Immunological Memories.},
journal = {Cell host &amp; microbe},
volume = {26},
number = {4},
pages = {515-526.e6},
pmid = {31585845},
issn = {1934-6069},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Intergenic/*genetics ; DNA, Viral/*metabolism ; RNA, Guide/*genetics ; Staphylococcus aureus/*genetics/immunology/virology ; Streptococcus thermophilus/*genetics/immunology/virology ; },
abstract = {Type II CRISPR-Cas systems defend prokaryotes from bacteriophage infection through the acquisition of short viral DNA sequences known as spacers, which are transcribed into short RNA guides to specify the targets of the Cas9 nuclease. To counter the potentially devastating propagation of escaper phages with mutations in the target sequences, the host population acquires many different spacers. Whether and how pre-existing spacers in type II systems affect the acquisition of new ones is unknown. Here, we demonstrate that previously acquired spacers promote additional spacer acquisition from the vicinity of the target DNA site cleaved by Cas9. Therefore, CRISPR immune cells acquire additional spacers at the same time as they destroy the infecting virus. This anticipates the rise of escapers or related viruses that could escape targeting by the first spacer acquired. Our results thus reveal Cas9's role in the generation of immunological memories.},
}
@article {pmid31584620,
year = {2020},
author = {Vermersch, E and Jouve, C and Hulot, JS},
title = {CRISPR/Cas9 gene-editing strategies in cardiovascular cells.},
journal = {Cardiovascular research},
volume = {116},
number = {5},
pages = {894-907},
doi = {10.1093/cvr/cvz250},
pmid = {31584620},
issn = {1755-3245},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cardiovascular Diseases/genetics/metabolism/pathology/*therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Repair ; Embryonic Stem Cells/metabolism ; Epigenesis, Genetic ; *Gene Editing ; Gene Expression Regulation ; Genetic Predisposition to Disease ; *Genetic Therapy/adverse effects ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mutation ; Myocytes, Cardiac/*metabolism/pathology ; Phenotype ; RNA, Guide/genetics/metabolism ; },
abstract = {Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR-Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.},
}
@article {pmid31584167,
year = {2020},
author = {Calvo-Villamañán, A and Bernheim, A and Bikard, D},
title = {Methods for the Analysis and Characterization of Defense Mechanisms Against Horizontal Gene Transfer: CRISPR Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2075},
number = {},
pages = {235-249},
doi = {10.1007/978-1-4939-9877-7_17},
pmid = {31584167},
issn = {1940-6029},
mesh = {Bacteriophages/physiology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Defense Mechanisms ; *Gene Transfer, Horizontal ; Host-Pathogen Interactions ; Plasmids/genetics ; },
abstract = {CRISPR-Cas systems provide RNA-guided adaptive immunity to the majority of archaea and many bacteria. They are able to capture pieces of invading genetic elements in the form of novel spacers in an array of repeats. These elements can then be used as a memory to destroy incoming DNA through the action of RNA-guided nucleases. This chapter describes general procedures to determine the ability of CRISPR-Cas systems to capture novel sequences and to use them to block phages and horizontal gene transfer. All protocols are performed in Staphylococcus aureus using Type II-A CRISPR-Cas systems. Nonetheless, the protocols provided can be adapted to work with other bacteria and other types of CRISPR-Cas systems.},
}
@article {pmid31583880,
year = {2019},
author = {Kong, T and Backes, N and Kalwa, U and Legner, C and Phillips, GJ and Pandey, S},
title = {Adhesive Tape Microfluidics with an Autofocusing Module That Incorporates CRISPR Interference: Applications to Long-Term Bacterial Antibiotic Studies.},
journal = {ACS sensors},
volume = {4},
number = {10},
pages = {2638-2645},
doi = {10.1021/acssensors.9b01031},
pmid = {31583880},
issn = {2379-3694},
mesh = {Adhesives/chemistry ; Amoxicillin/*pharmacology ; Ampicillin/*pharmacology ; Anti-Bacterial Agents/*pharmacology ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Culture Techniques/instrumentation/methods ; Escherichia coli/drug effects/genetics ; Gene Expression ; Lab-On-A-Chip Devices ; Microfluidics/instrumentation/*methods ; Sepharose/chemistry ; },
abstract = {The ability to study bacteria at the single cell level has advanced our insights into microbial physiology and genetics in ways not attainable by studying large populations using more traditional culturing methods. To improve methods to characterize bacteria at the cellular level, we developed a new microfluidic platform that enables cells to be exposed to metabolites in a gradient of concentrations. By designing low-cost, three-dimensional devices with adhesive tapes and tailoring them for bacterial imaging, we avoided the complexities of silicon and polymeric microfabrication. The incorporation of an agarose membrane as the resting substrate, along with a temperature-controlled environmental chamber, allows the culturing of bacterial cells for over 10 h under stable growth or inhibition conditions. Incorporation of an autofocusing module helped the uninterrupted, high-resolution observation of bacteria at the single-cell and at low density population levels. We used the microfluidic platform to record morphological changes in Escherichia coli during ampicillin exposure and to quantify the minimum inhibitory concentration of the antibiotic. We further demonstrated the potential of finely-tuned, incremental gene regulation in a concentration gradient utilizing CRISPR interference (CRISPRi). These low-cost engineering tools, when implemented in combination with genetic approaches such as CRISPRi, should prove useful to uncover new genetic determinants of antibiotic susceptibility and evaluate the long-term effectiveness of antibiotics in bacterial cultures.},
}
@article {pmid31583668,
year = {2020},
author = {Pfalz, M and Gonzalo, A and Christophorou, N and Blary, A and Berard, A and Bessoltane, N and Montes, E and Jaffrelo, L and Poncet, C and Le Paslier, MC and Nesi, N and Charif, D and Jenczewski, E},
title = {Identifying and Isolating Meiotic Mutants in a Polyploid Brassica Crop.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2061},
number = {},
pages = {303-318},
doi = {10.1007/978-1-4939-9818-0_21},
pmid = {31583668},
issn = {1940-6029},
mesh = {Brassica/*genetics ; CRISPR-Cas Systems ; Gene Editing ; Genome, Plant ; Genotype ; *Miosis ; *Mutation ; *Polyploidy ; Recombination, Genetic ; Sequence Analysis, DNA ; Transformation, Genetic ; },
abstract = {This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.},
}
@article {pmid31583448,
year = {2019},
author = {Liu, W and Tang, D and Wang, H and Lian, J and Huang, L and Xu, Z},
title = {Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {21-22},
pages = {8911-8922},
doi = {10.1007/s00253-019-10118-4},
pmid = {31583448},
issn = {1432-0614},
mesh = {Bacterial Proteins/*genetics/metabolism ; Bioreactors/microbiology ; CRISPR-Associated Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Corynebacterium glutamicum/*genetics/*metabolism ; Cysteine/*biosynthesis ; Endodeoxyribonucleases/*genetics/metabolism ; Gene Deletion ; Gene Editing/*methods ; Genome, Bacterial/genetics ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; RNA, Guide/genetics ; Serine/*biosynthesis ; },
abstract = {Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16 bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.},
}
@article {pmid31582646,
year = {2019},
author = {Iida, A and Wang, Z and Sehara-Fujisawa, A},
title = {Disruption of integrin α4 in zebrafish leads to cephalic hemorrhage during development.},
journal = {Genes &amp; genetic systems},
volume = {94},
number = {4},
pages = {177-179},
doi = {10.1266/ggs.19-00033},
pmid = {31582646},
issn = {1880-5779},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Communication/genetics ; Embryonic Development/*genetics ; Gene Editing ; Gene Expression Regulation, Developmental/genetics ; Genome/genetics ; Head/growth &amp; development/pathology ; Hemorrhage/physiopathology ; Heterozygote ; Integrin alpha4/*genetics ; Morphogenesis/*genetics ; Phenotype ; Zebrafish/*genetics/growth &amp; development ; },
abstract = {Integrins, transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix interactions, are heterodimers that consist of an α- and β-subunit. The integrin α4 gene (itgα4) is expressed in various type of cells and tissues. Its biochemical functions and physiological roles have been revealed using cultured cell assays. In contrast, the primary effect caused by itgα4 deletion on vertebrate development is poorly understood, because knockout mice exhibit multiple defects that can lead to embryonic lethality in the uterus. Zebrafish are a convenient vertebrate model to investigate morphogenesis during embryogenesis, because of their external fertilization and subsequent development outside the female's body. Here, we generated a zebrafish mutant line named itgα4 [ko108] using the CRISPR/Cas9 genome editing system; the mutant genome harbored an approximately 2.0-kb deletion in the itgα4 locus. A truncated transcript was detected in itgα4 (+/-) or (-/-) fish but not in (+/+) fish. The mutant transcript was hypothesized to encode a truncated Itgα4 protein due to a premature stop codon. itgα4 (-/-) embryos obtained from the mating of heterozygous parents exhibited no apparent phenotype during development at 24 hours post-fertilization (hpf). However, approximately half of them exhibited cephalic hemorrhage at 48 hpf. The incidence ratio was significantly higher than that in (+/+) or (+/-) embryos. Embryonic hemorrhage has also been reported previously in Itgα4 knockout mice. In contrast, embryonic lethality with the other defects reported in the knockout mice was not observed in our zebrafish model. Therefore, the mutant line itgα4 [ko108] should be a useful model to investigate a physiological function for Itgα4 in the blood circulation system.},
}
@article {pmid31582566,
year = {2019},
author = {Theodorou, I and Courtin, P and Palussière, S and Kulakauskas, S and Bidnenko, E and Péchoux, C and Fenaille, F and Penno, C and Mahony, J and van Sinderen, D and Chapot-Chartier, MP},
title = {A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {46},
pages = {17612-17625},
pmid = {31582566},
issn = {1083-351X},
mesh = {Bacterial Proteins/genetics/metabolism ; Biosynthetic Pathways ; Cell Wall/chemistry/genetics/*metabolism ; Deoxy Sugars/analysis/genetics/metabolism ; Glycosyltransferases/genetics/metabolism ; Lactococcus lactis/chemistry/genetics/*metabolism ; Mannans/analysis/genetics/metabolism ; Multigene Family ; Polysaccharides, Bacterial/analysis/genetics/*metabolism ; },
abstract = {In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.},
}
@article {pmid31582414,
year = {2019},
author = {Singh, P and Patel, RK and Palmer, N and Grenier, JK and Paduch, D and Kaldis, P and Grimson, A and Schimenti, JC},
title = {CDK2 kinase activity is a regulator of male germ cell fate.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {21},
pages = {},
pmid = {31582414},
issn = {1477-9129},
support = {P50 HD076210/HD/NICHD NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Apoptosis ; CRISPR-Cas Systems ; *Cell Differentiation ; *Cell Lineage ; Cell Proliferation ; Cluster Analysis ; Crosses, Genetic ; Cyclin-Dependent Kinase 2/*metabolism ; Germ Cells/cytology/*enzymology ; Heterozygote ; Homeostasis ; Male ; Mass Spectrometry ; Meiosis ; Mice ; Mutagenesis, Site-Directed ; Phenotype ; Phosphorylation ; RNA, Small Cytoplasmic/metabolism ; Seminiferous Tubules/metabolism ; Spermatogenesis ; Spermatogonia/*cytology/metabolism ; Testis/metabolism ; Transcriptome ; },
abstract = {The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter balancing SSC renewal versus terminal differentiation. Here, we report that precise regulation of the cell cycle is crucial for this balance. Whereas cyclin-dependent kinase 2 (Cdk2) is not necessary for mouse viability or gametogenesis stages prior to meiotic prophase I, mice bearing a deregulated allele (Cdk2[Y15S]) are severely deficient in spermatogonial differentiation. This allele disrupts an inhibitory phosphorylation site (Tyr15) for the kinase WEE1. Remarkably, Cdk2[Y15S/Y15S] mice possess abnormal clusters of mitotically active SSC-like cells, but these are eventually removed by apoptosis after failing to differentiate properly. Analyses of lineage markers, germ cell proliferation over time, and single cell RNA-seq data revealed delayed and defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that Cdk2[Y15S] is a gain-of-function allele causing elevated kinase activity, which underlies these differentiation defects. Our results demonstrate that precise regulation of CDK2 kinase activity in male germ cell development is crucial for the gonocyte-to-spermatogonia transition and long-term spermatogenic homeostasis.},
}
@article {pmid31582413,
year = {2019},
author = {Mahamud, MR and Geng, X and Ho, YC and Cha, B and Kim, Y and Ma, J and Chen, L and Myers, G and Camper, S and Mustacich, D and Witte, M and Choi, D and Hong, YK and Chen, H and Varshney, G and Engel, JD and Wang, S and Kim, TH and Lim, KC and Srinivasan, RS},
title = {GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {21},
pages = {},
pmid = {31582413},
issn = {1477-9129},
support = {R01 HL131652/HL/NHLBI NIH HHS/United States ; P30 GM114731/GM/NIGMS NIH HHS/United States ; R01 HL118676/HL/NHLBI NIH HHS/United States ; P20 GM103636/GM/NIGMS NIH HHS/United States ; P20 GM103441/GM/NIGMS NIH HHS/United States ; R01 HL133216/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiopoietin-2/metabolism ; Animals ; CRISPR-Cas Systems ; Calcium-Binding Proteins/metabolism ; Cell Differentiation ; Cell Line ; Claudin-5/metabolism ; EGF Family of Proteins/metabolism ; Endothelial Cells/*metabolism ; Endothelium, Vascular/metabolism ; Female ; GATA2 Transcription Factor/*metabolism ; Gene Deletion ; Humans ; Lymphatic Vessels/cytology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/*metabolism ; *Mutation ; RNA-Seq ; },
abstract = {Mutations in the transcription factor GATA2 cause lymphedema. GATA2 is necessary for the development of lymphatic valves and lymphovenous valves, and for the patterning of lymphatic vessels. Here, we report that GATA2 is not necessary for valvular endothelial cell (VEC) differentiation. Instead, GATA2 is required for VEC maintenance and morphogenesis. GATA2 is also necessary for the expression of the cell junction molecules VE-cadherin and claudin 5 in lymphatic vessels. We identified miR-126 as a target of GATA2, and miR-126[-/-] embryos recapitulate the phenotypes of mice lacking GATA2. Primary human lymphatic endothelial cells (HLECs) lacking GATA2 (HLEC[ΔGATA2]) have altered expression of claudin 5 and VE-cadherin, and blocking miR-126 activity in HLECs phenocopies these changes in expression. Importantly, overexpression of miR-126 in HLEC[ΔGATA2] significantly rescues the cell junction defects. Thus, our work defines a new mechanism of GATA2 activity and uncovers miR-126 as a novel regulator of mammalian lymphatic vascular development.},
}
@article {pmid31582397,
year = {2019},
author = {Pantier, R and Tatar, T and Colby, D and Chambers, I},
title = {Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naïve pluripotency marker.},
journal = {Life science alliance},
volume = {2},
number = {5},
pages = {},
pmid = {31582397},
issn = {2575-1077},
support = {MR/K017047/1/MRC_/Medical Research Council/United Kingdom ; MR/L018497/1/MRC_/Medical Research Council/United Kingdom ; MR/T003162/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Biomarkers/metabolism ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Differentiation ; Cell Line ; Chromatin/metabolism ; DNA-Binding Proteins/chemistry/genetics/*metabolism ; Dioxygenases/chemistry/genetics/*metabolism ; Embryonic Stem Cells/*cytology/metabolism ; Enhancer Elements, Genetic ; Epitopes/*analysis ; Gene Expression ; Mice ; Nanog Homeobox Protein/*metabolism ; Proto-Oncogene Proteins/chemistry/genetics/*metabolism ; },
abstract = {Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2 [Flag-IRES-EGFP] reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.},
}
@article {pmid31581942,
year = {2019},
author = {Shen, W and Zhang, J and Geng, B and Qiu, M and Hu, M and Yang, Q and Bao, W and Xiao, Y and Zheng, Y and Peng, W and Zhang, G and Ma, L and Yang, S},
title = {Establishment and application of a CRISPR-Cas12a assisted genome-editing system in Zymomonas mobilis.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {162},
pmid = {31581942},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/metabolism ; Francisella/enzymology ; Gene Editing/*methods ; *Genome, Bacterial ; Plasmids/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Zymomonas/*genetics/metabolism ; },
abstract = {BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking.<br><br>RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed.<br><br>CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.},
}
@article {pmid31580420,
year = {2019},
author = {Silva, FJ and Ferreira, LC and Campos, VP and Cruz-Magalhães, V and Barros, AF and Andrade, JP and Roberts, DP and de Souza, JT},
title = {Complete Genome Sequence of the Biocontrol Agent Bacillus velezensis UFLA258 and Its Comparison with Related Species: Diversity within the Commons.},
journal = {Genome biology and evolution},
volume = {11},
number = {10},
pages = {2818-2823},
pmid = {31580420},
issn = {1759-6653},
mesh = {Bacillus/classification/*genetics ; *Biological Control Agents ; *Genome, Bacterial ; Genomics ; Phylogeny ; },
abstract = {In this study, the full genome sequence of Bacillus velezensis strain UFLA258, a biological control agent of plant pathogens was obtained, assembled, and annotated. With a comparative genomics approach, in silico analyses of all complete genomes of B. velezensis and closely related species available in the database were performed. The genome of B. velezensis UFLA258 consisted of a single circular chromosome of 3.95 Mb in length, with a mean GC content of 46.69%. It contained 3,949 genes encoding proteins and 27 RNA genes. Analyses based on Average Nucleotide Identity and Digital DNA-DNA Hybridization and a phylogeny with complete sequences of the rpoB gene confirmed that 19 strains deposited in the database as Bacillus amyloliquefaciens were in fact B. velezensis. In total, 115 genomes were analyzed and taxonomically classified as follows: 105 were B. velezensis, 9 were B. amyloliquefaciens, and 1 was Bacillus siamensis. Although these species are phylogenetically close, the combined analyses of several genomic characteristics, such as the presence of biosynthetic genes encoding secondary metabolites, CRISPr/Cas arrays, Average Nucleotide Identity and Digital DNA-DNA Hybridization, and other information on the strains, including isolation source, allowed their unequivocal classification. This genomic analysis expands our knowledge about the closely related species, B. velezensis, B. amyloliquefaciens, and B. siamensis, with emphasis on their taxonomical status.},
}
@article {pmid31578590,
year = {2019},
author = {Fan, Y and Li, J and Wei, W and Fang, H and Duan, Y and Li, N and Zhang, Y and Yu, J and Wang, J},
title = {Ku80 gene knockdown by the CRISPR/Cas9 technique affects the biological functions of human thyroid carcinoma cells.},
journal = {Oncology reports},
volume = {42},
number = {6},
pages = {2486-2498},
pmid = {31578590},
issn = {1791-2431},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Case-Control Studies ; Cell Line, Tumor ; Cell Proliferation/genetics ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Hashimoto Disease/genetics/pathology ; Humans ; Ku Autoantigen/genetics/*metabolism ; RNA, Messenger/metabolism ; Thyroid Cancer, Papillary/*genetics/pathology ; Thyroid Gland/pathology ; Thyroid Neoplasms/*genetics/pathology ; },
abstract = {In the present study, to evaluate the role of Ku80 in thyroid carcinoma (TC), 86 thyroid tissue samples from patients with a spectrum of thyroid disorders were examined for protein levels of Ku80, nuclear factor‑κB (NF‑κB) and RET/TC by immunohistochemistry. Furthermore, in TC cells, Ku80 mRNA was detected by reverse transcription‑quantitative PCR analysis and silenced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 (Cas9) technique to assess its role. An antibody array was used to identify Ku80‑related regulatory genes. The protein levels of Ku80 in the TC tissues were significantly higher than those in non‑neoplastic adjacent tissue samples (P<0.01). The activation of NF‑kB and expression of RET/TC in the TC group were significantly increased (P<0.05) and were correlated with the protein expression of Ku80 (P<0.05). In papillary TC cells, the mRNA levels of Ku80 were high; Ku80 knockdown resulted in reductions in proliferation, invasion and colony formation, increased apoptosis, and reduced levels of proteins involved in MAPK signaling, cell proliferation and apoptosis. The high expression of Ku80 in TC was found to be associated with the expression of RET/TC and activation of NF‑κB, and Ku80 knockdown decreased the malignancy of TC cells.},
}
@article {pmid31578415,
year = {2019},
author = {Khan, AO and White, CW and Pike, JA and Yule, J and Slater, A and Hill, SJ and Poulter, NS and Thomas, SG and Morgan, NV},
title = {Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14219},
pmid = {31578415},
issn = {2045-2322},
support = {PG/16/103/32650//British Heart Foundation (BHF)/International ; PG/15/114/31945//British Heart Foundation (BHF)/International ; PG/13/36/30275//British Heart Foundation (BHF)/International ; FS/15/18/31317//British Heart Foundation (BHF)/International ; MR/N020081/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Clone Cells ; Codon/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Humans ; Ligands ; Luminescent Proteins/analysis/*genetics ; Mutagenesis, Insertional ; Receptors, CXCR4/biosynthesis/genetics ; Single Molecule Imaging/*methods ; Tubulin/biosynthesis/genetics ; },
abstract = {The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.},
}
@article {pmid31578377,
year = {2019},
author = {Pristyazhnyuk, IE and Minina, J and Korablev, A and Serova, I and Fishman, V and Gridina, M and Rozhdestvensky, TS and Gubar, L and Skryabin, BV and Serov, OL},
title = {Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14161},
pmid = {31578377},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Adhesion Molecules, Neuronal/*genetics ; Cells, Cultured ; Chromosomes/genetics ; *Gene Deletion ; *Gene Duplication ; Germ Cells/metabolism ; Homozygote ; Mice ; Mice, Inbred C57BL ; },
abstract = {In a previous study using one-step CRISPR/Cas9 genome editing in mouse zygotes, we created five founders carrying a 1,137 kb deletion and two founders carrying the same deletion, plus a 2,274 kb duplication involving the Cntn6 gene (encoding contactin-6). Using these mice, the present study had the following aims: (i) to establish stage of origin of these rearrangements; (ii) to determine the fate of the deleted DNA fragments; and (iii) to estimate the scale of unpredicted DNA changes accompanying the rearrangements. The present study demonstrated that all targeted deletions and duplications occurred at the one-cell stage and more often in one pronucleus only. FISH analysis revealed that there were no traces of the deleted DNA fragments either within chromosome 6 or on other chromosomes. These data were consistent with the Southern blot analysis showing that chromosomes with deletion often had close to expected sizes of removed DNA fragments. High-throughput DNA sequencing of two homozygotes for duplication demonstrated that there were no unexpected significant or scale DNA changes either at the gRNA and joint sites or other genome sites. Thus, our data suggested that CRISPR/Cas9 technology could generate megabase-sized deletions and duplications in mouse gametes at a reasonably specific level.},
}
@article {pmid31578319,
year = {2019},
author = {Wang, L and Yu, X and Li, M and Sun, G and Zou, L and Li, T and Hou, L and Guo, Y and Shen, D and Qu, D and Cheng, X and Chen, L},
title = {Filamentation initiated by Cas2 and its association with the acquisition process in cells.},
journal = {International journal of oral science},
volume = {11},
number = {3},
pages = {29},
pmid = {31578319},
issn = {2049-3169},
mesh = {Adaptation, Biological/genetics ; Adaptive Immunity ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems/*genetics/*immunology/physiology ; DNA, Bacterial/*genetics ; Endonucleases ; Escherichia coli/genetics/*immunology/metabolism ; Escherichia coli Proteins ; Humans ; Immunity/genetics ; *Oral Health ; },
abstract = {Cas1-and-Cas2-mediated new spacer acquisition is an essential process for bacterial adaptive immunity. The process is critical for the ecology of the oral microflora and oral health. Although molecular mechanisms for spacer acquisition are known, it has never been established if this process is associated with the morphological changes of bacteria. In this study, we demonstrated a novel Cas2-induced filamentation phenotype in E. coli that was regulated by co-expression of the Cas1 protein. A 30 amino acid motif at the carboxyl terminus of Cas2 is necessary for this function. By imaging analysis, we provided evidence to argue that Cas-induced filamentation is a step coupled with new spacer acquisition during which filaments are characterised by polyploidy with asymmetric cell division. This work may open new opportunities to investigate the adaptive immune response and microbial balance for oral health.},
}
@article {pmid31578281,
year = {2019},
author = {Chen, JJ and Nathaniel, DL and Raghavan, P and Nelson, M and Tian, R and Tse, E and Hong, JY and See, SK and Mok, SA and Hein, MY and Southworth, DR and Grinberg, LT and Gestwicki, JE and Leonetti, MD and Kampmann, M},
title = {Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {50},
pages = {18952-18966},
pmid = {31578281},
issn = {1083-351X},
support = {R01 NS059690/NS/NINDS NIH HHS/United States ; P01 AG019724/AG/NIA NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; T32 GM008568/GM/NIGMS NIH HHS/United States ; T32 GM064337/GM/NIGMS NIH HHS/United States ; R56 AG057528/AG/NIA NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; K24 AG053435/AG/NIA NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; S10 OD021741/OD/NIH HHS/United States ; P50 AG023501/AG/NIA NIH HHS/United States ; },
mesh = {Cells, Cultured ; Endosomal Sorting Complexes Required for Transport/*metabolism ; HEK293 Cells ; Humans ; Lysosomes/*metabolism ; Protein Aggregates ; tau Proteins/*metabolism ; },
abstract = {Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.},
}
@article {pmid31576029,
year = {2019},
author = {Maxmen, A},
title = {CRISPR might be the banana's only hope against a deadly fungus.},
journal = {Nature},
volume = {574},
number = {7776},
pages = {15},
pmid = {31576029},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fungi ; *Fusarium ; Gene Editing ; *Musa ; },
}
@article {pmid31576005,
year = {2020},
author = {Meyer, T and Jahn, N and Lindner, S and Röhner, L and Dolnik, A and Weber, D and Scheffold, A and Köpff, S and Paschka, P and Gaidzik, VI and Heckl, D and Wiese, S and Ebert, BL and Döhner, H and Bullinger, L and Döhner, K and Krönke, J},
title = {Functional characterization of BRCC3 mutations in acute myeloid leukemia with t(8;21)(q22;q22.1).},
journal = {Leukemia},
volume = {34},
number = {2},
pages = {404-415},
pmid = {31576005},
issn = {1476-5551},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation/drug effects/genetics ; Cytokines/genetics ; DNA Damage/drug effects/genetics ; Deubiquitinating Enzymes/*genetics ; Doxorubicin/pharmacology ; Granulocyte Colony-Stimulating Factor/genetics ; HEK293 Cells ; Humans ; Inflammasomes/genetics ; Leukemia, Myeloid, Acute/drug therapy/*genetics ; Mice ; Mutation/*genetics ; },
abstract = {BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t(8;21)(q22;q22.1) AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t(8;21)(q22;q22.1) positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t(8;21)(q22;q22.1). Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients.},
}
@article {pmid31574188,
year = {2019},
author = {Matre, PR and Mu, X and Wu, J and Danila, D and Hall, MA and Kolonin, MG and Darabi, R and Huard, J},
title = {CRISPR/Cas9-Based Dystrophin Restoration Reveals a Novel Role for Dystrophin in Bioenergetics and Stress Resistance of Muscle Progenitors.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {12},
pages = {1615-1628},
pmid = {31574188},
issn = {1549-4918},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Polarity/physiology ; Cell Proliferation/genetics ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; Endoplasmic Reticulum Stress/genetics ; Energy Metabolism/genetics ; Epigenesis, Genetic ; Gene Editing ; Genetic Therapy/*methods ; Mice ; Mice, Inbred mdx ; Muscle Fibers, Skeletal/metabolism/*pathology ; Muscular Dystrophy, Duchenne/genetics/pathology/*therapy ; Oxidative Stress/genetics ; Satellite Cells, Skeletal Muscle/*pathology ; Stem Cells/physiology ; },
abstract = {Although the lack of dystrophin expression in muscle myofibers is the central cause of Duchenne muscular dystrophy (DMD), accumulating evidence suggests that DMD may also be a stem cell disease. Recent studies have revealed dystrophin expression in satellite cells and demonstrated that dystrophin deficiency is directly related to abnormalities in satellite cell polarity, asymmetric division, and epigenetic regulation, thus contributing to the manifestation of the DMD phenotype. Although metabolic and mitochondrial dysfunctions have also been associated with the DMD pathophysiology profile, interestingly, the role of dystrophin with respect to stem cells dysfunction has not been elucidated. In the past few years, editing of the gene that encodes dystrophin has emerged as a promising therapeutic approach for DMD, although the effects of dystrophin restoration in stem cells have not been addressed. Herein, we describe our use of a clustered regularly interspaced short palindromic repeats/Cas9-based system to correct the dystrophin mutation in dystrophic (mdx) muscle progenitor cells (MPCs) and show that the expression of dystrophin significantly improved cellular properties of the mdx MPCs in vitro. Our findings reveal that dystrophin-restored mdx MPCs demonstrated improvements in cell proliferation, differentiation, bioenergetics, and resistance to oxidative and endoplasmic reticulum stress. Furthermore, our in vivo studies demonstrated improved transplantation efficiency of the corrected MPCs in the muscles of mdx mice. Our results indicate that changes in cellular energetics and stress resistance via dystrophin restoration enhance muscle progenitor cell function, further validating that dystrophin plays a role in stem cell function and demonstrating the potential for new therapeutic approaches for DMD. Stem Cells 2019;37:1615-1628.},
}
@article {pmid31573887,
year = {2020},
author = {Espino-Saldaña, AE and Rodríguez-Ortiz, R and Pereida-Jaramillo, E and Martínez-Torres, A},
title = {Modeling Neuronal Diseases in Zebrafish in the Era of CRISPR.},
journal = {Current neuropharmacology},
volume = {18},
number = {2},
pages = {136-152},
pmid = {31573887},
issn = {1875-6190},
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Genome ; *Nervous System Diseases ; Zebrafish/*genetics ; },
abstract = {BACKGROUND: Danio rerio is a powerful experimental model for studies in genetics and development. Recently, CRISPR technology has been applied in this species to mimic various human diseases, including those affecting the nervous system. Zebrafish offer multiple experimental advantages: external embryogenesis, rapid development, transparent embryos, short life cycle, and basic neurobiological processes shared with humans. This animal model, together with the CRISPR system, emerging imaging technologies, and novel behavioral approaches, lay the basis for a prominent future in neuropathology and will undoubtedly accelerate our understanding of brain function and its disorders.<br><br>OBJECTIVE: Gather relevant findings from studies that have used CRISPR technologies in zebrafish to explore basic neuronal function and model human diseases.<br><br>METHODS: We systematically reviewed the most recent literature about CRISPR technology applications for understanding brain function and neurological disorders in D. rerio. We highlighted the key role of CRISPR in driving forward our understanding of particular topics in neuroscience.<br><br>RESULTS: We show specific advances in neurobiology when the CRISPR system has been applied in zebrafish and describe how CRISPR is accelerating our understanding of brain organization.<br><br>CONCLUSION: Today, CRISPR is the preferred method to modify genomes of practically any living organism. Despite the rapid development of CRISPR technologies to generate disease models in zebrafish, more efforts are needed to efficiently combine different disciplines to find the etiology and treatments for many brain diseases.},
}
@article {pmid31572350,
year = {2019},
author = {Toro, N and Mestre, MR and Martínez-Abarca, F and González-Delgado, A},
title = {Recruitment of Reverse Transcriptase-Cas1 Fusion Proteins by Type VI-A CRISPR-Cas Systems.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2160},
pmid = {31572350},
issn = {1664-302X},
abstract = {Type VI CRISPR-Cas systems contain a single effector nuclease (Cas13) that exclusively targets single-stranded RNA. It remains unknown how these systems acquire spacers. It has been suggested that type VI systems with adaptation modules can acquire spacers from RNA bacteriophages, but sequence similarities suggest that spacers may provide immunity to DNA phages. We searched databases for Cas13 proteins with linked RTs. We identified two different type VI-A systems with adaptation modules including an RT-Cas1 fusion and Cas2 proteins. Phylogenetic reconstruction analyses revealed that these adaptation modules were recruited by different effector Cas13a proteins, possibly from RT-associated type III-D systems within the bacterial classes Alphaproteobacteria and Clostridia. These type VI-A systems are predicted to acquire spacers from RNA molecules, paving the way for future studies investigating their role in bacterial adaptive immunity and biotechnological applications.},
}
@article {pmid31571409,
year = {2020},
author = {Eschstruth, A and Schneider-Maunoury, S and Giudicelli, F},
title = {Creation of zebrafish knock-in reporter lines in the nefma gene by Cas9-mediated homologous recombination.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {58},
number = {1},
pages = {e23340},
doi = {10.1002/dvg.23340},
pmid = {31571409},
issn = {1526-968X},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Gene Targeting/methods ; Genetic Engineering/*methods ; Genome/genetics ; Homologous Recombination/genetics ; Intermediate Filaments/genetics ; RNA, Guide/genetics ; Zebrafish/genetics ; },
abstract = {CRISPR/Cas9-based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)-induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock-ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9-targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock-in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.},
}
@article {pmid31571107,
year = {2019},
author = {Batır, MB and Şahin, E and Çam, FS},
title = {Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3.},
journal = {Molecular biology reports},
volume = {46},
number = {6},
pages = {6471-6484},
pmid = {31571107},
issn = {1573-4978},
mesh = {Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; *DNA Repair ; DNA, Single-Stranded/metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; *Mutation ; Oligodeoxyribonucleotides/metabolism ; PC-3 Cells ; Prostatic Neoplasms/*genetics ; RNA, Guide/metabolism ; Sequence Analysis, DNA ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Prostate cancer is a common health problem among men worldwide and most of these prostate cancer cases are related to a dysfunctional mutant Tumor Protein p53 (TP53) gene. However, the CRISPR/Cas9 system can be used for repairing of a dysfunctional mutant TP53 gene in combination with donor single-stranded oligodeoxynucleotide (ssODN) via cells' own homology-directed repair (HDR) mechanism. In this study, we aimed to evaluate the CRISPR/Cas9 repairing efficiency on TP53 414delC (p.K139fs*31) null mutation, located in the TP53 gene, of human prostate cancer cell line PC-3 in combination with ssODNs. According to the next-generation sequencing results, TP53 414delC mutation was repaired with an efficiency of 19.95% and 26.0% at the TP53 414delC position with ssODN1 and ssODN2 accompanied by sgRNA2 guided CRISPR/Cas9, respectively. Besides, qPCR and immunofluorescence analysis showed that PC-3 cells, the TP53 414delC mutation of which were repaired, expressed wild type p53 again. Also, significantly increased number of apoptotic cells, driven by the repaired TP53 gene were detected compared to the control cells by flow cytometry analysis. As a result, sgRNA2 guided CRISPR/Cas9 system accompanied by ssODN was shown to effectively repair the TP53 414delC gene region and inhibit the cell proliferation of PC-3 cells. Therefore, the effects of the TP53 414delC mutation repairment in PC-3 cells will be investigated in the in vivo models for tumor clearance analysis in the near future.},
}
@article {pmid31571024,
year = {2019},
author = {Yang, X and Zhou, H and Zhou, X},
title = {Rock paper scissors: CRISPR/Cas9-mediated interference with geminiviruses in plants.},
journal = {Science China. Life sciences},
volume = {62},
number = {10},
pages = {1389-1391},
doi = {10.1007/s11427-019-9825-4},
pmid = {31571024},
issn = {1869-1889},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics/physiology ; Disease Resistance/genetics ; Geminiviridae/*metabolism ; Gene Editing/methods ; Gene Expression Regulation/physiology ; Genome, Viral ; Plant Diseases/*prevention &amp; control ; Plants, Genetically Modified/*genetics/metabolism ; },
}
@article {pmid31570833,
year = {2019},
author = {Levin-Konigsberg, R and Montaño-Rendón, F and Keren-Kaplan, T and Li, R and Ego, B and Mylvaganam, S and DiCiccio, JE and Trimble, WS and Bassik, MC and Bonifacino, JS and Fairn, GD and Grinstein, S},
title = {Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.},
journal = {Nature cell biology},
volume = {21},
number = {10},
pages = {1234-1247},
pmid = {31570833},
issn = {1476-4679},
support = {Z01 HD001607/ImNIH/Intramural NIH HHS/United States ; },
mesh = {ADP-Ribosylation Factors/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Endoplasmic Reticulum/*metabolism/ultrastructure ; Gene Editing ; Gene Expression Regulation ; Humans ; Mice ; Monocytes/*metabolism/ultrastructure ; Phagocytosis ; Phagosomes/*metabolism/ultrastructure ; Phosphatidylinositol Phosphates/*metabolism ; Primary Cell Culture ; Proteolysis ; RAW 264.7 Cells ; RNA, Small Interfering/genetics/metabolism ; Receptors, Steroid/antagonists &amp; inhibitors/*genetics/metabolism ; *Signal Transduction ; Vesicular Transport Proteins/genetics/metabolism ; rab GTP-Binding Proteins/genetics/metabolism ; rab7 GTP-Binding Proteins ; },
abstract = {Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.},
}
@article {pmid31570788,
year = {2020},
author = {Nicolai, S and Mahen, R and Raschellà, G and Marini, A and Pieraccioli, M and Malewicz, M and Venkitaraman, AR and Melino, G},
title = {ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining.},
journal = {Oncogene},
volume = {39},
number = {4},
pages = {754-766},
pmid = {31570788},
issn = {1476-5594},
support = {MC_U132670600/MRC_/Medical Research Council/United Kingdom ; MC_UU_00025/2/MRC_/Medical Research Council/United Kingdom ; MC_UU_12022/1/MRC_/Medical Research Council/United Kingdom ; 100090/12/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA-Binding Proteins/metabolism ; Databases, Genetic ; Humans ; Neoplasms/*genetics/metabolism/*pathology ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Prognosis ; Repressor Proteins/genetics/*metabolism ; Survival Rate ; },
abstract = {Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients' stratification and for the development of personalised therapeutic strategies.},
}
@article {pmid31570734,
year = {2019},
author = {Prolo, LM and Li, A and Owen, SF and Parker, JJ and Foshay, K and Nitta, RT and Morgens, DW and Bolin, S and Wilson, CM and Vega L, JCM and Luo, EJ and Nwagbo, G and Waziri, A and Li, G and Reimer, RJ and Bassik, MC and Grant, GA},
title = {Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14020},
pmid = {31570734},
issn = {2045-2322},
support = {UL1 TR001085/TR/NCATS NIH HHS/United States ; R25 NS065741/NS/NINDS NIH HHS/United States ; 5K08NA075144-05//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/International ; P30 CA124435/CA/NCI NIH HHS/United States ; UL1 TR003142/TR/NCATS NIH HHS/United States ; },
mesh = {Brain Neoplasms/genetics/*pathology ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Glioblastoma/genetics/*pathology ; Humans ; Intracellular Signaling Peptides and Proteins/*metabolism ; Neoplasm Invasiveness/genetics ; Protein Serine-Threonine Kinases/*metabolism ; },
abstract = {Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.},
}
@article {pmid31570731,
year = {2019},
author = {Hanlon, KS and Kleinstiver, BP and Garcia, SP and Zaborowski, MP and Volak, A and Spirig, SE and Muller, A and Sousa, AA and Tsai, SQ and Bengtsson, NE and Lööv, C and Ingelsson, M and Chamberlain, JS and Corey, DP and Aryee, MJ and Joung, JK and Breakefield, XO and Maguire, CA and György, B},
title = {High levels of AAV vector integration into CRISPR-induced DNA breaks.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4439},
pmid = {31570731},
issn = {2041-1723},
support = {P50 AR065139/AR/NIAMS NIH HHS/United States ; R00 CA218870/CA/NCI NIH HHS/United States ; R01 AR044533/AR/NIAMS NIH HHS/United States ; R01 DC017117/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; Bacteriophage lambda/genetics ; Brain ; *CRISPR-Cas Systems ; Cell Line ; Chromosome Mapping ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cochlea ; *DNA Breaks ; Dependovirus/*genetics ; Endonucleases ; Gene Editing/*methods ; Gene Targeting/methods ; Genetic Therapy/methods ; *Genetic Vectors ; Genome ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Muscles ; Neurons/virology ; Targeted Gene Repair/methods ; Treatment Outcome ; Virus Integration/*genetics ; },
abstract = {Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.},
}
@article {pmid31570515,
year = {2019},
author = {Ginley-Hidinger, M and Carleton, JB and Rodriguez, AC and Berrett, KC and Gertz, J},
title = {Sufficiency analysis of estrogen responsive enhancers using synthetic activators.},
journal = {Life science alliance},
volume = {2},
number = {5},
pages = {},
pmid = {31570515},
issn = {2575-1077},
support = {R01 HG008974/HG/NHGRI NIH HHS/United States ; T32 GM007464/GM/NIGMS NIH HHS/United States ; P30 CA042014/CA/NCI NIH HHS/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems ; Cell Line, Tumor ; Enhancer Elements, Genetic/*drug effects ; Estrogen Receptor alpha/*metabolism ; Estrogens/metabolism ; Gene Expression Regulation/drug effects ; Histones/metabolism ; Humans ; Promoter Regions, Genetic/drug effects ; Recombinant Fusion Proteins/*pharmacology ; Transcriptional Activation/*drug effects ; },
abstract = {Multiple regulatory regions bound by the same transcription factor have been shown to simultaneously control a single gene's expression. However, it remains unclear how these regulatory regions combine to regulate transcription. Here, we test the sufficiency of promoter-distal estrogen receptor α-binding sites (ERBSs) for activating gene expression by recruiting synthetic activators in the absence of estrogens. Targeting either dCas9-VP16(10x) or dCas9-p300(core) to ERBS induces H3K27ac and activates nearby expression in a manner similar to an estrogen induction, with dCas9-VP16(10x) acting as a stronger activator. The sufficiency of individual ERBSs is highly correlated with their necessity, indicating an inherent activation potential that is associated with the binding of RNA polymerase II and several transcription factors. By targeting ERBS combinations, we found that ERBSs work independently to control gene expression when bound by synthetic activators. The sufficiency results contrast necessity assays that show synergy between these ERBSs, suggesting that synergy occurs between ERBSs in terms of activator recruitment, whereas directly recruiting activators leads to independent effects on gene expression.},
}
@article {pmid31570165,
year = {2019},
author = {Xue, S and Xu, H and Sun, Z and Shen, H and Chen, S and Ouyang, J and Zhou, Q and Hu, X and Cui, H},
title = {Depletion of TRDMT1 affects 5-methylcytosine modification of mRNA and inhibits HEK293 cell proliferation and migration.},
journal = {Biochemical and biophysical research communications},
volume = {520},
number = {1},
pages = {60-66},
doi = {10.1016/j.bbrc.2019.09.098},
pmid = {31570165},
issn = {1090-2104},
mesh = {5-Methylcytosine/*metabolism ; CRISPR-Cas Systems ; Carcinogenesis ; *Cell Movement ; *Cell Proliferation ; Computational Biology ; DNA (Cytosine-5-)-Methyltransferases/*genetics/*metabolism ; DNA Methylation ; Gene Expression Profiling ; HEK293 Cells ; Humans ; Phenotype ; RNA, Messenger/*metabolism ; RNA-Seq ; Signal Transduction ; },
abstract = {Human TRDMT1 is a transfer RNA (tRNA) methyltransferase for cytosine-5 methylation and has been suggested to be involved in the regulation of numerous developmental processes. However, little is known about the molecular mechanisms or their biological significance. In this study, we investigated the effects of CRISPR-based TRDMT1 knockdown on phenotypes, mRNA m5C modifications and gene expression changes in HEK293 cells. We found that knockdown of TRDMT1 significantly inhibited cell proliferation and migration but had no effect on clonogenic potential. The inhibitory effects could be attenuated by re-expression of TRDMT1 in HEK293 cells. RNA sequencing (RNA-Seq) and RNA bisulfite sequencing (RNA-BisSeq) were performed in TRDMT1 knockdown and wild-type HEK293 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the differentially expressed genes were associated with the cell cycle, RNA transport, and RNA degradation and were enriched in cancer and Notch signaling pathways. We also found that TRDMT1 knockdown could change mRNA methylation levels. For the first time, these findings clarify the role of TRDMT1 in regulating mRNA methylation and inhibiting the proliferation and migration of HEK293 cells. These results provide new insights into a new function of TRDMT1 and elucidate the molecular mechanisms of aberrant RNA m5C during tumorigenesis.},
}
@article {pmid31569754,
year = {2019},
author = {Amoako, DG and Somboro, AM and Abia, ALK and Allam, M and Ismail, A and Bester, LA and Essack, SY},
title = {Genome Mining and Comparative Pathogenomic Analysis of An Endemic Methicillin-Resistant Staphylococcus Aureus (MRSA) Clone, ST612-CC8-t1257-SCCmec_IVd(2B), Isolated in South Africa.},
journal = {Pathogens (Basel, Switzerland)},
volume = {8},
number = {4},
pages = {},
pmid = {31569754},
issn = {2076-0817},
support = {204517/WHO_/World Health Organization/International ; },
abstract = {This study undertook genome mining and comparative genomics to gain genetic insights into the dominance of the methicillin-resistant Staphylococcus aureus (MRSA) endemic clone ST612-CC8-t1257-SCCmec_IVd(2B), obtained from the poultry food chain in South Africa. Functional annotation of the genome revealed a vast array of similar central metabolic, cellular and biochemical networks within the endemic clone crucial for its survival in the microbial community. In-silico analysis of the clone revealed the possession of uniform defense systems, restriction-modification system (type I and IV), accessory gene regulator (type I), arginine catabolic mobile element (type II), and type 1 clustered, regularly interspaced, short palindromic repeat (CRISPR)Cas array (N = 7 ± 1), which offer protection against exogenous attacks. The estimated pathogenic potential predicted a higher probability (average Pscore ≈ 0.927) of the clone being pathogenic to its host. The clone carried a battery of putative virulence determinants whose expression are critical for establishing infection. However, there was a slight difference in their possession of adherence factors (biofilm operon system) and toxins (hemolysins and enterotoxins). Further analysis revealed a conserved environmental tolerance and persistence mechanisms related to stress (oxidative and osmotic), heat shock, sporulation, bacteriocins, and detoxification, which enable it to withstand lethal threats and contribute to its success in diverse ecological niches. Phylogenomic analysis with close sister lineages revealed that the clone was closely related to the MRSA isolate SHV713 from Australia. The results of this bioinformatic analysis provide valuable insights into the biology of this endemic clone.},
}
@article {pmid31569579,
year = {2019},
author = {Gale, GAR and Schiavon Osorio, AA and Mills, LA and Wang, B and Lea-Smith, DJ and McCormick, AJ},
title = {Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.},
journal = {Microorganisms},
volume = {7},
number = {10},
pages = {},
pmid = {31569579},
issn = {2076-2607},
support = {MR/S018875/1/MRC_/Medical Research Council/United Kingdom ; },
abstract = {Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.},
}
@article {pmid31568601,
year = {2019},
author = {Dai, Y and Somoza, RA and Wang, L and Welter, JF and Li, Y and Caplan, AI and Liu, CC},
title = {Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {48},
pages = {17399-17405},
pmid = {31568601},
issn = {1521-3773},
support = {P41 EB021911/EB/NIBIB NIH HHS/United States ; R01 DK113185/DK/NIDDK NIH HHS/United States ; },
mesh = {Acidaminococcus/genetics ; Aptamers, Nucleotide/*chemistry ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; DNA Cleavage ; DNA, Viral/*chemistry ; Electrochemical Techniques ; Electrodes ; Human papillomavirus 16/*genetics ; Humans ; Immobilized Nucleic Acids/*chemistry ; Limit of Detection ; Mesenchymal Stem Cells ; Parvovirus/*genetics ; Sensitivity and Specificity ; Surface Properties ; Transforming Growth Factor beta1/analysis/metabolism ; },
abstract = {An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF-β1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.},
}
@article {pmid31568337,
year = {2019},
author = {Roh, DS and Liao, EC},
title = {Reply: CRISPR Craft: DNA Editing the Reconstructive Ladder.},
journal = {Plastic and reconstructive surgery},
volume = {144},
number = {4},
pages = {715e-716e},
doi = {10.1097/PRS.0000000000006052},
pmid = {31568337},
issn = {1529-4242},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Gene Editing ; *Reconstructive Surgical Procedures ; },
}
@article {pmid31568336,
year = {2019},
author = {DeCoster, RC and Vasconez, HC and Butterfield, TA},
title = {CRISPR Craft: DNA Editing the Reconstructive Ladder.},
journal = {Plastic and reconstructive surgery},
volume = {144},
number = {4},
pages = {714e-715e},
pmid = {31568336},
issn = {1529-4242},
support = {T32 CA160003/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Gene Editing ; *Reconstructive Surgical Procedures ; },
}
@article {pmid31566854,
year = {2019},
author = {Lyu, Y and He, S and Li, J and Jiang, Y and Sun, H and Miao, Y and Pu, K},
title = {A Photolabile Semiconducting Polymer Nanotransducer for Near-Infrared Regulation of CRISPR/Cas9 Gene Editing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {50},
pages = {18197-18201},
doi = {10.1002/anie.201909264},
pmid = {31566854},
issn = {1521-3773},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Fluorescence ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Vectors ; HeLa Cells ; Humans ; Mice, Nude ; Nanostructures/*chemistry ; Photochemical Processes ; Plasmids/genetics ; Polyethylene Glycols/chemistry ; Polyethyleneimine/chemistry ; Polymers/*chemistry ; Proof of Concept Study ; Singlet Oxygen ; },
abstract = {Noninvasive regulation of CRISPR/Cas9 gene editing is conducive to understanding of gene function and development of gene therapy; however, it remains challenging. Herein, a photolabile semiconducting polymer nanotransducer (pSPN) is synthesized to act as the gene vector to deliver CRISPR/Cas9 plasmids into cells and also as the photoregulator to remotely activate gene editing. pSPN comprises a [1] O2 -generating backbone grafted with polyethylenimine brushes through [1] O2 -cleavable linkers. NIR photoirradiation spontaneously triggers the cleavage of gene vectors from pSPN, resulting in the release of CRISPR/Cas9 plasmids and subsequently initiating gene editing. This system affords 15- and 1.8-fold enhancement in repaired gene expression relative to the nonirradiated controls in living cells and mice, respectively. As this approach does not require any specific modifications on biomolecular components, pSPN represents the first generic nanotransducer for in vivo regulation of CRISPR/Cas9 gene editing.},
}
@article {pmid31566614,
year = {2019},
author = {Jin, Y and Shen, Y and Su, X and Weintraub, N and Tang, Y},
title = {CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {151},
pages = {},
pmid = {31566614},
issn = {1940-087X},
support = {R01 AR070029/AR/NIAMS NIH HHS/United States ; R01 HL086555/HL/NHLBI NIH HHS/United States ; R01 HL126949/HL/NHLBI NIH HHS/United States ; R01 HL134354/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dystrophin/*genetics ; Gene Editing/*methods ; Gene Expression Regulation/*genetics ; Induced Pluripotent Stem Cells/*cytology ; Male ; Mice ; Muscles/*cytology ; Mutation ; },
abstract = {Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmd[mdx] mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.},
}
@article {pmid31566583,
year = {2019},
author = {Du, Q and Huynh, LK and Coskun, F and Molina, E and King, MA and Raj, P and Khan, S and Dozmorov, I and Seroogy, CM and Wysocki, CA and Padron, GT and Yates, TR and Markert, ML and de la Morena, MT and van Oers, NS},
title = {FOXN1 compound heterozygous mutations cause selective thymic hypoplasia in humans.},
journal = {The Journal of clinical investigation},
volume = {129},
number = {11},
pages = {4724-4738},
pmid = {31566583},
issn = {1558-8238},
support = {R01 AI114523/AI/NIAID NIH HHS/United States ; R21 AI144140/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Female ; *Forkhead Transcription Factors/genetics/immunology ; *Heterozygote ; Humans ; Male ; Mice ; Mice, Nude ; *Mutation ; Protein Domains ; *Severe Combined Immunodeficiency/genetics/immunology/pathology ; *Thymus Gland/immunology/pathology ; },
abstract = {We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with T-/loB+NK+ SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a T-B+NK+ SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5-amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1.},
}
@article {pmid31566294,
year = {2019},
author = {Kim, J and Lana, B and Torelli, S and Ryan, D and Catapano, F and Ala, P and Luft, C and Stevens, E and Konstantinidis, E and Louzada, S and Fu, B and Paredes-Redondo, A and Chan, AE and Yang, F and Stemple, DL and Liu, P and Ketteler, R and Selwood, DL and Muntoni, F and Lin, YY},
title = {A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.},
journal = {EMBO reports},
volume = {20},
number = {11},
pages = {e47967},
pmid = {31566294},
issn = {1469-3178},
support = {2012-305121//EC|Seventh Framework Programme (FP7)/International ; RG130417//Royal Society/International ; SG/14-15/14//Newlife - The Charity for Disabled Children/International ; 098051//Wellcome Trust/United Kingdom ; 115582//EC|Seventh Framework Programme (FP7)/International ; 92-963//UK Research and Innovation|Medical Research Council (MRC)/International ; MC_U12266B//UK Research and Innovation|Medical Research Council (MRC)/International ; MC_EX_G0800785/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cells, Cultured ; *Drug Evaluation, Preclinical/methods ; Dystroglycans/genetics/*metabolism ; Gene Editing ; Gene Targeting ; Genetic Loci ; Glycosylation/drug effects ; High-Throughput Nucleotide Sequencing ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Molecular Imaging ; Muscular Dystrophies/drug therapy/etiology/metabolism ; Mutation ; N-Acetylglucosaminyltransferases/genetics/metabolism ; Neural Stem Cells/metabolism ; Neurons/metabolism ; Pentosyltransferases/genetics/metabolism ; },
abstract = {Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.},
}
@article {pmid31566126,
year = {2019},
author = {Zhou, LY and Qin, Z and Zhu, YH and He, ZY and Xu, T},
title = {Current RNA-based Therapeutics in Clinical Trials.},
journal = {Current gene therapy},
volume = {19},
number = {3},
pages = {172-196},
doi = {10.2174/1566523219666190719100526},
pmid = {31566126},
issn = {1875-5631},
mesh = {Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; *Clinical Trials as Topic ; Genetic Diseases, Inborn/*genetics/*therapy ; *Genetic Therapy ; Humans ; MicroRNAs/genetics ; Oligoribonucleotides, Antisense/genetics ; RNA, Catalytic ; RNA, Small Interfering/genetics ; },
abstract = {Long-term research on various types of RNAs has led to further understanding of diverse mechanisms, which eventually resulted in the rapid development of RNA-based therapeutics as powerful tools in clinical disease treatment. Some of the developing RNA drugs obey the antisense mechanisms including antisense oligonucleotides, small interfering RNAs, microRNAs, small activating RNAs, and ribozymes. These types of RNAs could be utilized to inhibit/activate gene expression or change splicing to provide functional proteins. In the meantime, some others based on different mechanisms like modified messenger RNAs could replace the dysfunctional endogenous genes to manage some genetic diseases, and aptamers with special three-dimensional structures could bind to specific targets in a high-affinity manner. In addition, the recent most popular CRISPR-Cas technology, consisting of a crucial single guide RNA, could edit DNA directly to generate therapeutic effects. The desired results from recent clinical trials indicated the great potential of RNA-based drugs in the treatment of various diseases, but further studies on improving delivery materials and RNA modifications are required for the novel RNA-based drugs to translate to the clinic. This review focused on the advances and clinical studies of current RNA-based therapeutics, analyzed their challenges and prospects.},
}
@article {pmid31564454,
year = {2019},
author = {Bhoobalan-Chitty, Y and Johansen, TB and Di Cianni, N and Peng, X},
title = {Inhibition of Type III CRISPR-Cas Immunity by an Archaeal Virus-Encoded Anti-CRISPR Protein.},
journal = {Cell},
volume = {179},
number = {2},
pages = {448-458.e11},
doi = {10.1016/j.cell.2019.09.003},
pmid = {31564454},
issn = {1097-4172},
mesh = {CRISPR-Associated Proteins/*physiology ; *CRISPR-Cas Systems ; *Host-Pathogen Interactions ; Ribonucleases/metabolism ; Rudiviridae/*metabolism ; Sulfolobus/*virology ; },
abstract = {Bacteria and archaea possess a striking diversity of CRISPR-Cas systems divided into six types, posing a significant barrier to viral infection. As part of the virus-host arms race, viruses encode protein inhibitors of type I, II, and V CRISPR-Cas systems, but whether there are natural inhibitors of the other, mechanistically distinct CRISPR-Cas types is unknown. Here, we present the discovery of a type III CRISPR-Cas inhibitor, AcrIIIB1, encoded by the Sulfolobus virus SIRV2. AcrIIIB1 exclusively inhibits CRISPR-Cas subtype III-B immunity mediated by the RNase activity of the accessory protein Csx1. AcrIIIB1 does not appear to bind Csx1 but, rather, interacts with two distinct subtype III-B effector complexes-Cmr-α and Cmr-γ-which, in response to protospacer transcript binding, are known to synthesize cyclic oligoadenylates (cOAs) that activate the Csx1 "collateral" RNase. Taken together, we infer that AcrIIIB1 inhibits type III-B CRISPR-Cas immunity by interfering with a Csx1 RNase-related process.},
}
@article {pmid31564075,
year = {2019},
author = {Vo, KTX and Lee, SK and Halane, MK and Song, MY and Hoang, TV and Kim, CY and Park, SY and Jeon, J and Kim, ST and Sohn, KH and Jeon, JS},
title = {Pi5 and Pii Paired NLRs Are Functionally Exchangeable and Confer Similar Disease Resistance Specificity.},
journal = {Molecules and cells},
volume = {42},
number = {9},
pages = {637-645},
pmid = {31564075},
issn = {0219-1032},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; DNA, Complementary/genetics ; Disease Resistance/*genetics ; Gene Expression Regulation, Plant ; *Genes, Plant ; Magnaporthe/*physiology ; Mutation/genetics ; NLR Proteins/*metabolism ; Oryza/genetics/*microbiology ; Plant Diseases/*microbiology ; Plants, Genetically Modified ; },
abstract = {Effector-triggered immunity (ETI) is an effective layer of plant defense initiated upon recognition of avirulence (Avr) effectors from pathogens by cognate plant disease resistance (R) proteins. In rice, a large number of R genes have been characterized from various cultivars and have greatly contributed to breeding programs to improve resistance against the rice blast pathogen Magnaporthe oryzae. The extreme diversity of R gene repertoires is thought to be a result of co-evolutionary history between rice and its pathogens including M. oryzae. Here we show that Pii is an allele of Pi5 by DNA sequence characterization and complementation analysis. Pii-1 and Pii-2 cDNAs were cloned by reverse transcription polymerase chain reaction from the Pii -carrying cultivar Fujisaka5 . The complementation test in susceptible rice cultivar Dongjin demonstrated that the rice blast resistance mediated by Pii , similar to Pi5 , requires the presence of two nucleotide-binding leucine-rich repeat genes, Pii-1 and Pii-2 . Consistent with our hypothesis that Pi5 and Pii are functionally indistinguishable, the replacement of Pii-1 by Pi5-1 and Pii-2 by Pi5-2 , respectively, does not change the level of disease resistance to M. oryzae carrying AVR-Pii. Surprisingly, Exo70F3, required for Pii-mediated resistance, is dispensable for Pi5-mediated resistance. Based on our results, despite similarities observed between Pi5 and Pii, we hypothesize that Pi5 and Pii pairs require partially distinct mechanisms to function.},
}
@article {pmid31562521,
year = {2019},
author = {Liu, D and Chen, M and Mendoza, B and Cheng, H and Hu, R and Li, L and Trinh, CT and Tuskan, GA and Yang, X},
title = {CRISPR/Cas9-mediated targeted mutagenesis for functional genomics research of crassulacean acid metabolism plants.},
journal = {Journal of experimental botany},
volume = {70},
number = {22},
pages = {6621-6629},
pmid = {31562521},
issn = {1460-2431},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Carboxylic Acids/*metabolism ; Databases, Genetic ; *Genomics ; Mutagenesis/*genetics ; Mutation/genetics ; Plants/*genetics ; RNA, Guide/genetics ; *Research ; },
abstract = {Crassulacean acid metabolism (CAM) is an important photosynthetic pathway in diverse lineages of plants featuring high water-use efficiency and drought tolerance. A big challenge facing the CAM research community is to understand the function of the annotated genes in CAM plant genomes. Recently, a new genome editing technology using CRISPR/Cas9 has become a more precise and powerful tool than traditional approaches for functional genomics research in C3 and C4 plants. In this study, we explore the potential of CRISPR/Cas9 to characterize the function of CAM-related genes in the model CAM species Kalanchoë fedtschenkoi. We demonstrate that CRISPR/Cas9 is effective in creating biallelic indel mutagenesis to reveal previously unknown roles of blue light receptor phototropin 2 (KfePHOT2) in the CAM pathway. Knocking out KfePHOT2 reduced stomatal conductance and CO2 fixation in late afternoon and increased stomatal conductance and CO2 fixation during the night, indicating that blue light signaling plays an important role in the CAM pathway. Lastly, we provide a genome-wide guide RNA database targeting 45 183 protein-coding transcripts annotated in the K. fedtschenkoi genome.},
}
@article {pmid31562218,
year = {2019},
author = {Lockhart, J},
title = {Introducing CRISPR-TSKO: A Breakthrough in Precision Gene Editing.},
journal = {The Plant cell},
volume = {31},
number = {12},
pages = {2831-2832},
pmid = {31562218},
issn = {1532-298X},
mesh = {*Arabidopsis ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Mutagenesis ; },
}
@article {pmid31562216,
year = {2019},
author = {Decaestecker, W and Buono, RA and Pfeiffer, ML and Vangheluwe, N and Jourquin, J and Karimi, M and Van Isterdael, G and Beeckman, T and Nowack, MK and Jacobs, TB},
title = {CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis.},
journal = {The Plant cell},
volume = {31},
number = {12},
pages = {2868-2887},
pmid = {31562216},
issn = {1532-298X},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cyclin-Dependent Kinases/genetics/metabolism ; Gene Knockout Techniques/*methods ; Genetic Vectors ; *Mutagenesis ; Organ Specificity/genetics ; Phenotype ; Plant Root Cap/genetics ; Plant Roots/genetics ; Plant Stomata/genetics ; Promoter Regions, Genetic ; },
abstract = {Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.},
}
@article {pmid31560967,
year = {2019},
author = {Zhu, GH and Zheng, MY and Sun, JB and Khuhro, SA and Yan, Q and Huang, Y and Syed, Z and Dong, SL},
title = {CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura.},
journal = {Insect biochemistry and molecular biology},
volume = {115},
number = {},
pages = {103244},
doi = {10.1016/j.ibmb.2019.103244},
pmid = {31560967},
issn = {1879-0240},
mesh = {*Animal Communication ; Animals ; Arthropod Antennae/*physiology ; Base Sequence ; CRISPR-Cas Systems ; Carrier Proteins/*physiology ; Female ; Insect Proteins/*physiology ; Male ; Mutation ; *Olfactory Perception ; Sex Attractants ; Spodoptera/*physiology ; },
abstract = {Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.},
}
@article {pmid31560858,
year = {2019},
author = {Kenawi, M and Rouger, E and Island, ML and Leroyer, P and Robin, F and Rémy, S and Tesson, L and Anegon, I and Nay, K and Derbré, F and Brissot, P and Ropert, M and Cavey, T and Loréal, O},
title = {Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {12},
pages = {13492-13502},
doi = {10.1096/fj.201901106R},
pmid = {31560858},
issn = {1530-6860},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Ceruloplasmin/antagonists &amp; inhibitors/*deficiency/genetics ; *Disease Models, Animal ; Female ; Iron/analysis/*metabolism ; Iron Metabolism Disorders/*complications/genetics/pathology ; Iron Overload/etiology/*pathology ; Liver/metabolism/pathology ; Macrophages/metabolism/*pathology ; Male ; Mutation ; Neurodegenerative Diseases/*complications/genetics/pathology ; Rats ; Rats, Sprague-Dawley ; Sequence Homology ; Spleen/metabolism/pathology ; },
abstract = {Hereditary aceruloplasminemia (HA), related to mutations in the ceruloplasmin (Cp) gene, leads to iron accumulation. Ceruloplasmin ferroxidase activity being considered essential for macrophage iron release, macrophage iron overload is expected, but it is not found in hepatic and splenic macrophages in humans. Our objective was to get a better understanding of the mechanisms leading to iron excess in HA. A clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) knockout of the Cp gene was performed on Sprague-Dawley rats. We evaluated the iron status in plasma, the expression of iron metabolism genes, and the status of other metals whose interactions with iron are increasingly recognized. In Cp[-/-] rats, plasma ceruloplasmin and ferroxidase activity were absent, together with decreased iron concentration and transferrin saturation. Similarly as in humans, the hepatocytes were iron overloaded conversely to hepatic and splenic macrophages. Despite a relative hepcidin deficiency in Cp[-/-] rats and the loss of ferroxidase activity, potentially expected to limit the interaction of iron with transferrin, no increase of plasma non-transferrin-bound iron level was found. Copper was decreased in the spleen, whereas manganese was increased in the plasma. These data suggest that the reported role of ceruloplasmin cannot fully explain the iron hepatosplenic phenotype in HA, encouraging the search for additional mechanisms.-Kenawi, M., Rouger, E., Island, M.-L., Leroyer, P., Robin, F., Remy, S., Tesson, L., Anegon, I., Nay, K., Derbré, F., Brissot, P., Ropert, M., Cavey, T., Loréal, O. Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.},
}
@article {pmid31560841,
year = {2019},
author = {Saeidian, AH and Vahidnezhad, H and Youssefian, L and Sotudeh, S and Sargazi, M and Zeinali, S and Uitto, J},
title = {Hypotrichosis with juvenile macular dystrophy: Combination of whole-genome sequencing and genome-wide homozygosity mapping identifies a large deletion in CDH3 initially undetected by whole-exome sequencing-A lesson from next-generation sequencing.},
journal = {Molecular genetics &amp; genomic medicine},
volume = {7},
number = {11},
pages = {e975},
pmid = {31560841},
issn = {2324-9269},
mesh = {Adolescent ; Cadherins/*genetics ; Chromosome Mapping ; DNA Mutational Analysis/methods ; Female ; High-Throughput Nucleotide Sequencing/*methods ; Homozygote ; Humans ; Hypotrichosis/*congenital/genetics/pathology ; Macular Degeneration/*genetics/*pathology ; Male ; Pedigree ; *Sequence Deletion ; Whole Exome Sequencing/*methods ; Whole Genome Sequencing/*methods ; },
abstract = {BACKGROUND: Hypotrichosis with juvenile macular dystrophy (HJMD) is an autosomal recessive disorder characterized by abnormal growth of scalp hair and juvenile macular degeneration leading to blindness. We have explored the genetic basis of HJMD in a large consanguineous family with 12 affected patients, 1-76 years of age, with characteristic phenotypes.<br><br>METHODS: We first applied genome-wide homozygosity mapping to 10 affected individuals for linkage analysis to identify the genomic region of the defective gene. All affected individuals shared a 7.2&#xa0;Mb region of homozygosity on chromosome 16q21-22.3, which harbored 298 genes, including CDH3, previously associated with HJMD. However, whole-exome sequencing (WES) failed to identify the causative mutation in CDH3.<br><br>RESULTS: Further investigation revealed a missense variant in a gene&#xa0;closely linked to CDH3 (1.4&#xa0;Mb distance: FHOD1: c.1306A>G, p.Arg436Gly). This variant was homozygous in all affected individuals and heterozygous in 18 out of 19 obligate carriers. While this variant was found by bioinformatics predictions to be likely pathogenic, a knock-in mouse for this variant, made by the CRISPR/Cas, showed no disease phenotype. However, using whole-genome sequencing (WGS), we were able to identify a novel Alu recombination-mediated deletion in CDH3:c.del161-811_246&#xa0;+&#xa0;1,044.<br><br>CONCLUSION: WGS was able to identify a deep intronic deletion mutation, not detected by WES.},
}
@article {pmid31560209,
year = {2019},
author = {Park, J and Sung, K and Bak, SY and Koh, HR and Kim, SK},
title = {Positive Identification of DNA Cleavage by CRISPR-Cas9 Using Pyrene Excimer Fluorescence to Detect a Subnanometer Structural Change.},
journal = {The journal of physical chemistry letters},
volume = {10},
number = {20},
pages = {6208-6212},
doi = {10.1021/acs.jpclett.9b01913},
pmid = {31560209},
issn = {1948-7185},
mesh = {Base Pair Mismatch ; CRISPR-Associated Protein 9/*chemistry/genetics ; CRISPR-Cas Systems ; Catalysis ; DNA/*chemistry ; DNA Cleavage ; Fluorescence ; Fluorescent Dyes/*chemistry ; Pyrenes/*chemistry ; RNA, Guide/chemistry/genetics ; },
abstract = {The Cas9 nuclease binds and cleaves DNA through its large-scale structural rearrangements. However, its unique property of not releasing the cleaved DNA has forbidden spectroscopic detection of the cleavage event. Here, we employ a novel fluorescence probe based on pyrene excimer emission to detect a minute structural change not detectable by other methods and demonstrate its applicability to spectroscopic tracking of the Cas9 nuclease activity in time. We show that the intensity of excimer emission depends sensitively on a subtle change in the structural environment of the target nucleic acid, which enables discrimination between cleaved and uncleaved nucleic acids within the DNA/Cas9/gRNA ternary complex. Kinetic parameters were obtained from the temporal evolution of the excimer emission, which revealed that DNA binding is hardly affected by PAM-distal mismatches, whereas the rate of cleavage by Cas9 decreases dramatically even with a 1-bp mismatch. Spectroscopic studies using the pyrene-based probe should be promising for biomolecular systems affected by subnm structural changes.},
}
@article {pmid31559936,
year = {2019},
author = {López-Gutiérrez, B and Cova, M and Izquierdo, L},
title = {A Plasmodium falciparum C-mannosyltransferase is dispensable for parasite asexual blood stage development.},
journal = {Parasitology},
volume = {146},
number = {14},
pages = {1767-1772},
pmid = {31559936},
issn = {1469-8161},
mesh = {Blood/parasitology ; CRISPR-Cas Systems ; Glycosylation ; *Life Cycle Stages ; Loss of Function Mutation ; Mannosyltransferases/genetics/*physiology ; Plasmodium falciparum/*enzymology/genetics/*physiology ; Protozoan Proteins/genetics/*physiology ; Reproduction, Asexual ; Salivary Glands/parasitology ; Thrombospondins/genetics/physiology ; },
abstract = {C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (PfDPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of PfDPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a PfDPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. PfDPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that PfDPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions.},
}
@article {pmid31559572,
year = {2019},
author = {Lu, Y and Shou, J and Jia, Z and Wu, Y and Li, J and Guo, Y and Wu, Q},
title = {Genetic evidence for asymmetric blocking of higher-order chromatin structure by CTCF/cohesin.},
journal = {Protein &amp; cell},
volume = {10},
number = {12},
pages = {914-920},
pmid = {31559572},
issn = {1674-8018},
mesh = {Animals ; CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems ; Cadherins/*genetics ; Cell Cycle Proteins/*genetics ; Cell Line, Tumor ; Chromatin/*genetics ; Chromosomal Proteins, Non-Histone/*genetics ; Humans ; },
}
@article {pmid31558681,
year = {2019},
author = {Wang, R and Zhang, H and Du, J and Xu, J},
title = {Heat resilience in embryonic zebrafish revealed using an in vivo stress granule reporter.},
journal = {Journal of cell science},
volume = {132},
number = {20},
pages = {},
pmid = {31558681},
issn = {1477-9137},
mesh = {Animals ; Animals, Genetically Modified/embryology/genetics ; CRISPR-Cas Systems ; *Cytoplasmic Granules/genetics/metabolism ; *Heat-Shock Response ; Mesencephalon/*embryology ; Neurons/*metabolism ; *RNA Helicases/genetics/metabolism ; *Zebrafish/embryology/genetics ; },
abstract = {Although the regulation of stress granules has become an intensely studied topic, current investigations of stress granule assembly, disassembly and dynamics are mainly performed in cultured cells. Here, we report the establishment of a stress granule reporter to facilitate the real-time study of stress granules in vivo Using CRISPR/Cas9, we fused a green fluorescence protein (GFP) to endogenous G3BP1 in zebrafish. The GFP-G3BP1 reporter faithfully and robustly responded to heat stress in zebrafish embryos and larvae. The induction of stress granules varied by brain regions under the same stress condition, with the midbrain cells showing the highest efficiency and dynamics. Furthermore, pre-conditioning using lower heat stress significantly limited stress granule formation during subsequent higher heat stress. More interestingly, stress granule formation was much more robust in zebrafish embryos than in larvae and coincided with significantly elevated levels of phosphorylated eIF2α and enhanced heat resilience. Therefore, these findings have generated new insights into stress response in zebrafish during early development and demonstrated that the GFP-G3BP1 knock-in zebrafish could be a valuable tool for the investigation of stress granule biology.This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid31558579,
year = {2019},
author = {Zhang, Q and Zhang, Y and Lu, MH and Chai, YP and Jiang, YY and Zhou, Y and Wang, XC and Chen, QJ},
title = {A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize.},
journal = {Plant physiology},
volume = {181},
number = {4},
pages = {1441-1448},
pmid = {31558579},
issn = {1532-2548},
mesh = {Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems/*genetics ; *Genes, Plant ; Genetic Vectors/*genetics ; Morphogenesis/*genetics ; Transformation, Genetic ; Zea mays/*genetics/*growth &amp; development ; },
abstract = {The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.},
}
@article {pmid31558318,
year = {2019},
author = {Di, YH and Sun, XJ and Hu, Z and Jiang, QY and Song, GH and Zhang, B and Zhao, SS and Zhang, H},
title = {Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean.},
journal = {Biochemical and biophysical research communications},
volume = {519},
number = {4},
pages = {819-823},
doi = {10.1016/j.bbrc.2019.09.074},
pmid = {31558318},
issn = {1090-2104},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Promoter Regions, Genetic/*genetics ; Soybeans/*genetics/metabolism ; },
abstract = {Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.},
}
@article {pmid31558095,
year = {2021},
author = {Tanihara, F and Hirata, M and Nguyen, NT and Le, QA and Wittayarat, M and Fahrudin, M and Hirano, T and Otoi, T},
title = {Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes.},
journal = {Animal biotechnology},
volume = {32},
number = {2},
pages = {147-154},
doi = {10.1080/10495398.2019.1668801},
pmid = {31558095},
issn = {1532-2378},
mesh = {Animals ; Animals, Genetically Modified ; Antigens, CD/*genetics ; Antigens, Differentiation, Myelomonocytic/*genetics ; *CRISPR-Cas Systems ; Electroporation/*veterinary ; Embryo Culture Techniques ; Embryo Transfer ; Female ; Fertilization in Vitro ; Gene Deletion ; Pregnancy ; RNA, Guide ; Receptors, Cell Surface/*genetics ; Swine/*genetics ; },
abstract = {CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.},
}
@article {pmid31557657,
year = {2020},
author = {Schindele, A and Dorn, A and Puchta, H},
title = {CRISPR/Cas brings plant biology and breeding into the fast lane.},
journal = {Current opinion in biotechnology},
volume = {61},
number = {},
pages = {7-14},
doi = {10.1016/j.copbio.2019.08.006},
pmid = {31557657},
issn = {1879-0429},
mesh = {Breeding ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Homologous Recombination ; Plant Breeding ; Plants/genetics ; },
abstract = {CRISPR/Cas is in the process of inducing the biggest transformation of plant breeding since the green revolution. Whereas initial efforts focused mainly on changing single traits by error prone non-homologous end joining, the last two years saw a tremendous technical progress achieving more complex genetic, epigenetic and transcriptional changes. The efficiencies of inducing directed changes by homologous recombination have been improved significantly and strategies to break genetic linkages by inducing chromosomal rearrangements have been developed. Cas13 systems have been applied to degrade viral and mRNA in plants. Most importantly, a historical breakthrough was accomplished: By introducing multiple genomic changes simultaneously, domestication of wild species in a single generation has been demonstrated, speeding up breeding dramatically.},
}
@article {pmid31557222,
year = {2019},
author = {Lee, JE and Neumann, M and Duro, DI and Schmid, M},
title = {CRISPR-based tools for targeted transcriptional and epigenetic regulation in plants.},
journal = {PloS one},
volume = {14},
number = {9},
pages = {e0222778},
pmid = {31557222},
issn = {1932-6203},
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/*genetics ; Feasibility Studies ; *Gene Expression Regulation, Plant ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; Histone Code/genetics ; Plants, Genetically Modified/genetics ; Promoter Regions, Genetic/genetics ; *Transcriptional Activation ; },
abstract = {Programmable gene regulators that can modulate the activity of selected targets in trans are a useful tool for probing and manipulating gene function. CRISPR technology provides a convenient method for gene targeting that can also be adapted for multiplexing and other modifications to enable strong regulation by a range of different effectors. We generated a vector toolbox for CRISPR/dCas9-based targeted gene regulation in plants, modified with the previously described MS2 system to amplify the strength of regulation, and using Golden Gate-based cloning to enable rapid vector assembly with a high degree of flexibility in the choice of promoters, effectors and targets. We tested the system using the floral regulator FLOWERING LOCUS T (FT) as a target and a range of different effector domains including the transcriptional activator VP64, the H3K27 acetyltransferase p300 and the H3K9 methyltransferase KRYPTONITE. When transformed into Arabidopsis thaliana, several of the constructs caused altered flowering time phenotypes that were associated with changes in FT expression and/or epigenetic status, thus demonstrating the effectiveness of the system. The MS2-CRISPR/dCas9 system can be used to modulate transcriptional activity and epigenetic status of specific target genes in plants, and provides a versatile tool that can easily be used with different targets and types of regulation for a range of applications.},
}
@article {pmid31556952,
year = {2020},
author = {Milne, N and Tramontin, LRR and Borodina, I},
title = {A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica.},
journal = {FEMS yeast research},
volume = {20},
number = {2},
pages = {},
pmid = {31556952},
issn = {1567-1364},
mesh = {*CRISPR-Cas Systems ; Education, Graduate/methods ; Gene Editing/*methods ; Humans ; Industrial Microbiology ; Metabolic Engineering/*methods ; Saccharomyces cerevisiae/*genetics/metabolism ; Teaching ; Yarrowia/*genetics/metabolism ; },
abstract = {We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at M.Sc. and Ph.D. students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two-week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.},
}
@article {pmid31556487,
year = {2019},
author = {Dandage, R and Landry, CR},
title = {Paralog dependency indirectly affects the robustness of human cells.},
journal = {Molecular systems biology},
volume = {15},
number = {9},
pages = {e8871},
pmid = {31556487},
issn = {1744-4292},
support = {RN348479-387697//Canadian Institute of Health Research (CIHR)/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Cell Line, Tumor ; Computational Biology ; Gene Dosage/*genetics ; Gene Duplication/*genetics ; Genetic Fitness/genetics ; Humans ; Loss of Function Mutation/genetics ; Protein Interaction Maps/*genetics ; },
abstract = {The protective redundancy of paralogous genes partly relies on the fact that they carry their functions independently. However, a significant fraction of paralogous proteins may form functionally dependent pairs, for instance, through heteromerization. As a consequence, one could expect these heteromeric paralogs to be less protective against deleterious mutations. To test this hypothesis, we examined the robustness landscape of gene loss-of-function by CRISPR-Cas9 in more than 450 human cell lines. This landscape shows regions of greater deleteriousness to gene inactivation as a function of key paralog properties. Heteromeric paralogs are more likely to occupy such regions owing to their high expression and large number of protein-protein interaction partners. Further investigation revealed that heteromers may also be under stricter dosage balance, which may also contribute to the higher deleteriousness upon gene inactivation. Finally, we suggest that physical dependency may contribute to the deleteriousness upon loss-of-function as revealed by the correlation between the strength of interactions between paralogs and their higher deleteriousness upon loss of function.},
}
@article {pmid31555818,
year = {2019},
author = {Tsai, PH and Chien, Y and Wang, ML and Hsu, CH and Laurent, B and Chou, SJ and Chang, WC and Chien, CS and Li, HY and Lee, HC and Huo, TI and Hung, JH and Chen, CH and Chiou, SH},
title = {Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network.},
journal = {Nucleic acids research},
volume = {47},
number = {19},
pages = {10115-10133},
pmid = {31555818},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Lineage/genetics ; Cell Self Renewal/genetics ; Cellular Reprogramming/genetics ; DNA-Binding Proteins/*genetics ; *Enhancer Elements, Genetic/genetics ; Gene Expression Regulation, Developmental/genetics ; Humans ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; Mutation/genetics ; Nanog Homeobox Protein/genetics ; Octamer Transcription Factor-3/*genetics ; Pluripotent Stem Cells/metabolism ; SOXB1 Transcription Factors/genetics ; Transcription Factors/*genetics ; Transfection ; },
abstract = {Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.},
}
@article {pmid31555746,
year = {2019},
author = {Duan, J and Sanders, AR and Gejman, PV},
title = {From Schizophrenia Genetics to Disease Biology: Harnessing New Concepts and Technologies.},
journal = {Journal of psychiatry and brain science},
volume = {4},
number = {},
pages = {},
pmid = {31555746},
issn = {2398-385X},
support = {R01 MH106575/MH/NIMH NIH HHS/United States ; R01 MH116281/MH/NIMH NIH HHS/United States ; R21 MH102685/MH/NIMH NIH HHS/United States ; },
abstract = {Schizophrenia (SZ) is a severe mental disorder afflicting around 1% of the population. It is highly heritable but with complex genetics. Recent research has unraveled a plethora of risk loci for SZ. Accordingly, our conceptual understanding of SZ genetics has been rapidly evolving, from oligogenic models towards polygenic or even omnigenic models. A pressing challenge to the field, however, is the translation of the many genetic findings of SZ into disease biology insights leading to more effective treatments. Bridging this gap requires the integration of genetic findings and functional genomics using appropriate cellular models. Harnessing new technologies, such as the development of human induced pluripotent stem cells (hiPSC) and the CRISPR/Cas-based genome/epigenome editing approach are expected to change our understanding of SZ disease biology to a fundamentally higher level. Here, we discuss some new developments.},
}
@article {pmid31555228,
year = {2019},
author = {Zeng, H and Li, C and He, W and Zhang, J and Chen, M and Lei, T and Wu, H and Ling, N and Cai, S and Wang, J and Ding, Y and Wu, Q},
title = {Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter dublinensis Genotyping Based on CRISPR Locus Diversity.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1989},
pmid = {31555228},
issn = {1664-302X},
abstract = {Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter, however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii, C. malonaticus, and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.},
}
@article {pmid31554834,
year = {2019},
author = {Labrie, SJ and Mosterd, C and Loignon, S and Dupuis, M&#xc8; and Desjardins, P and Rousseau, GM and Tremblay, DM and Romero, DA and Horvath, P and Fremaux, C and Moineau, S},
title = {A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {13816},
pmid = {31554834},
issn = {2045-2322},
mesh = {Aminopeptidases/chemistry/*genetics ; Bacterial Proteins/chemistry/genetics ; Catalytic Domain ; Food Microbiology ; *Mutation ; Streptococcus Phages/genetics/*physiology ; Streptococcus thermophilus/enzymology/genetics/*virology ; Virus Replication ; Whole Genome Sequencing ; },
abstract = {Streptococcus thermophilus is a lactic acid bacterium widely used by the dairy industry for the manufacture of yogurt and specialty cheeses. It is also a Gram-positive bacterial model to study phage-host interactions. CRISPR-Cas systems are one of the most prevalent phage resistance mechanisms in S. thermophilus. Little information is available about other host factors involved in phage replication in this food-grade streptococcal species. We used the model strain S. thermophilus SMQ-301 and its virulent phage DT1, harboring the anti-CRISPR protein AcrIIA6, to show that a host gene coding for a methionine aminopeptidase (metAP) is necessary for phage DT1 to complete its lytic cycle. A single mutation in metAP provides S. thermophilus SMQ-301 with strong resistance against phage DT1. The mutation impedes a late step of the lytic cycle since phage adsorption, DNA replication, and protein expression were not affected. When the mutated strain was complemented with the wild-type version of the gene, the phage sensitivity phenotype was restored. When this mutation was introduced into other S. thermophilus strains it provided resistance against cos-type (Sfi21dt1virus genus) phages but replication of pac-type (Sfi11virus genus) phages was not affected. The mutation in the gene coding for the MetAP induces amino acid change in a catalytic domain conserved across many bacterial species. Introducing the same mutation in Streptococcus mutans also provided a phage resistance phenotype, suggesting the wide-ranging importance of the host methionine aminopeptidase in phage replication.},
}
@article {pmid31553918,
year = {2019},
author = {Tran, NT and Sommermann, T and Graf, R and Trombke, J and Pempe, J and Petsch, K and Kühn, R and Rajewsky, K and Chu, VT},
title = {Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells.},
journal = {Cell reports},
volume = {28},
number = {13},
pages = {3510-3522.e5},
pmid = {31553918},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Gene Knock-In Techniques/*methods ; Hematopoietic Stem Cells/*metabolism ; Mice ; Stem Cells/*metabolism ; },
abstract = {Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis.},
}
@article {pmid31553828,
year = {2020},
author = {Peng, F and Zhang, W and Zeng, W and Zhu, JK and Miki, D},
title = {Gene targeting in Arabidopsis via an all-in-one strategy that uses a translational enhancer to aid Cas9 expression.},
journal = {Plant biotechnology journal},
volume = {18},
number = {4},
pages = {892-894},
pmid = {31553828},
issn = {1467-7652},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Gene Targeting ; },
}
@article {pmid31553827,
year = {2020},
author = {Tong, CG and Wu, FH and Yuan, YH and Chen, YR and Lin, CS},
title = {High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes.},
journal = {Plant biotechnology journal},
volume = {18},
number = {4},
pages = {889-891},
pmid = {31553827},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Orchidaceae/*genetics ; },
}
@article {pmid31553754,
year = {2019},
author = {Babaei, M and Liu, Y and Wuerzberger-Davis, SM and McCaslin, EZ and DiRusso, CJ and Yeo, AT and Kagermazova, L and Miyamoto, S and Gilmore, TD},
title = {CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein.},
journal = {PloS one},
volume = {14},
number = {9},
pages = {e0222588},
pmid = {31553754},
issn = {1932-6203},
support = {R01 CA077474/CA/NCI NIH HHS/United States ; R01 GM117350/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; HEK293 Cells ; Humans ; I-kappa B Kinase/*genetics ; NF-kappa B/metabolism ; Regulatory Elements, Transcriptional/*genetics ; Signal Transduction ; },
abstract = {The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.},
}
@article {pmid31552919,
year = {2019},
author = {Lin, B and An, Y and Meng, L and Zhang, H and Song, J and Zhu, Z and Liu, W and Song, Y and Yang, C},
title = {Control of CRISPR-Cas9 with small molecule-activated allosteric aptamer regulating sgRNAs.},
journal = {Chemical communications (Cambridge, England)},
volume = {55},
number = {81},
pages = {12223-12226},
doi = {10.1039/c9cc05531b},
pmid = {31552919},
issn = {1364-548X},
mesh = {Allosteric Regulation ; Aptamers, Nucleotide/*chemistry ; CRISPR-Associated Protein 9/*chemistry ; CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; RNA/*chemistry ; Theophylline/*chemistry ; },
abstract = {The CRISPR-Cas9 system enables facile and efficient genome engineering in living cells and organisms. Small molecule control of CRISPR-Cas9 would allow precise temporal control in applications of the technology. In this work, we developed s[combining low line]mall m[combining low line]olecule-activated allosteric a[combining low line]ptamer r[combining low line]egulat[combining low line]ing (SMART)-sgRNAs as general strategies to control programmable genome editing. With SMART-sgRNAs, temporal control of the CRISPR-Cas9 system against different targets can be achieved, which should facilitate its application in various fields, such as biomedical genome editing, drug screening and chromosome imaging.},
}
@article {pmid31552670,
year = {2020},
author = {Tsapara, G and Andermatt, I and Stoeckli, ET},
title = {Gene Silencing in Chicken Brain Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2047},
number = {},
pages = {439-456},
doi = {10.1007/978-1-4939-9732-9_25},
pmid = {31552670},
issn = {1940-6029},
mesh = {Animals ; Brain/*embryology/metabolism ; CRISPR-Cas Systems ; Chick Embryo ; DNA/*administration &amp; dosage ; Electroporation ; Gene Expression Regulation, Developmental ; Gene Silencing ; RNA Interference ; },
abstract = {Despite the development of brain organoids and neural cultures derived from iPSCs (induced pluripotent stem cells), brain development can only be studied in an animal. The mouse is the most commonly used vertebrate model for the analysis of gene function because of the well-established genetic tools that are available for loss-of-function studies. However, studies of gene function during development can be problematic in mammals. Many genes are active during different stages of development. Absence of gene function during early development may cause aberrant neurogenesis or even embryonic lethality and thus prevent analysis of later stages of development. To avoid these problems, precise temporal control of gene silencing is required.In contrast to mammals, oviparous animals are accessible for experimental manipulations during embryonic development. The combination of accessibility and RNAi- or Crispr/Cas9-based gene silencing makes the chicken embryo a powerful model for developmental studies. Depending on the time window during which gene silencing is attempted, chicken embryos can be used in ovo or ex ovo in a domed dish for easier access during later stages of development. Both techniques allow for precise temporal control of gene silencing during embryonic development.},
}
@article {pmid31552656,
year = {2020},
author = {Farnworth, MS and Eckermann, KN and Ahmed, HMM and Mühlen, DS and He, B and Bucher, G},
title = {The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2047},
number = {},
pages = {191-217},
doi = {10.1007/978-1-4939-9732-9_11},
pmid = {31552656},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; Brain/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Gene Editing/*methods ; Neurons/cytology/metabolism ; Tribolium/genetics/*growth &amp; development ; },
abstract = {With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.},
}
@article {pmid31552655,
year = {2020},
author = {Fritsch, C and Sprecher, SG},
title = {CRISPR/Cas9 Genome Editing to Study Nervous System Development in Drosophila.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2047},
number = {},
pages = {161-189},
doi = {10.1007/978-1-4939-9732-9_10},
pmid = {31552655},
issn = {1940-6029},
mesh = {Alleles ; Animals ; Brain/*cytology/enzymology/*metabolism ; CRISPR-Cas Systems/genetics/physiology ; Drosophila ; Drosophila melanogaster/cytology/enzymology/metabolism ; Gene Editing/*methods ; Models, Genetic ; Plasmids/genetics ; Polymerase Chain Reaction ; },
abstract = {Continuous implementation of new techniques allowing increasingly precise genetic manipulations makes the fruit fly Drosophila melanogaster an impacting model to study the nervous system. While transgenic approaches have been heavily used to investigate how the brain develops, genome editing has been notoriously hard in the fruit fly. The advent of versatile CRISPR/Cas9-based genome editing techniques allow the generation of engineered loci using homologous repair to replace the endogenous genome sequence with a designed template of interest. We here provide a protocol to generate an FRT/FLP-based conditional GFP or HA-flagged gene knockout.},
}
@article {pmid31551987,
year = {2019},
author = {Milicevic, O and Repac, J and Bozic, B and Djordjevic, M and Djordjevic, M},
title = {A Simple Criterion for Inferring CRISPR Array Direction.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2054},
pmid = {31551987},
issn = {1664-302X},
abstract = {Inferring transcriptional direction (orientation) of the CRISPR array is essential for many applications, including systematically investigating non-canonical CRISPR/Cas functions. The standard method, CRISPRDirection (embedded within CRISPRCasFinder), fails to predict the orientation (ND predictions) for ∼37% of the classified CRISPR arrays (>2200 loci); this goes up to >70% for the II-B subtype where non-canonical functions were first experimentally discovered. Alternatively, Potential Orientation (also embedded within CRISPRCasFinder), has a much smaller frequency of ND predictions but might have significantly lower accuracy. We propose a novel simple criterion, where the CRISPR array direction is assigned according to the direction of its associated cas genes (Cas Orientation). We systematically assess the performance of the three methods (Cas Orientation, CRISPRDirection, and Potential Orientation) across all CRISPR/Cas subtypes, by a mutual crosscheck of their predictions, and by comparing them to the experimental dataset. Interestingly, CRISPRDirection agrees much better with Cas Orientation than with Potential Orientation, despite CRISPRDirection and Potential Orientation being mutually related - Potential Orientation corresponding to one of six (heterogeneous) predictors employed by CRISPRDirection - and being unrelated to Cas Orientation. We find that Cas Orientation has much higher accuracy compared to Potential Orientation and comparable accuracy to CRISPRDirection - while accurately assigning an orientation to ∼95% of the CRISPR arrays that are non-determined by CRISPRDirection. Cas Orientation is, at the same time, simple to employ, requiring only (routine for prokaryotes) the prediction of the associated protein coding gene direction.},
}
@article {pmid31551363,
year = {2020},
author = {Liang, J and Zhao, H and Diplas, BH and Liu, S and Liu, J and Wang, D and Lu, Y and Zhu, Q and Wu, J and Wang, W and Yan, H and Zeng, YX and Wang, X and Jiao, Y},
title = {Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition.},
journal = {Cancer research},
volume = {80},
number = {3},
pages = {510-523},
doi = {10.1158/0008-5472.CAN-18-3374},
pmid = {31551363},
issn = {1538-7445},
mesh = {CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*genetics ; Cell Cycle Proteins/genetics ; Humans ; Liver Neoplasms/*genetics ; Nuclear Proteins/genetics ; Protein-Tyrosine Kinases ; Pyrimidinones ; X-linked Nuclear Protein ; },
abstract = {The tumor suppressor gene ATRX is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for ATRX-mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines in vitro, as well as xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage-induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring ATRX mutations, indicating that the synthetic lethal relationship between WEE1 and ATRX could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in ATRX. SIGNIFICANCE: ATRX-mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in ATRX-deficient cancers.See related commentary by Cole, p. 375.},
}
@article {pmid31551131,
year = {2019},
author = {Ajay, J and Vandna, R},
title = {CRISPR-Cas systems ushered in an era of facile DNA-free genome editing.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {1-3},
doi = {10.1016/j.semcdb.2019.09.007},
pmid = {31551131},
issn = {1096-3634},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *DNA/genetics ; *Gene Editing ; Humans ; },
}
@article {pmid31550477,
year = {2019},
author = {Dimitriu, T and Ashby, B and Westra, ER},
title = {Transposition: A CRISPR Way to Get Around.},
journal = {Current biology : CB},
volume = {29},
number = {18},
pages = {R886-R889},
doi = {10.1016/j.cub.2019.08.010},
pmid = {31550477},
issn = {1879-0445},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements ; RNA ; *Transposases ; },
abstract = {CRISPR-Cas systems provide sequence-specific immunity against selfish genetic elements in prokaryotes. Now, two studies show that transposon-encoded variants can guide sequence-specific transposition. These findings have important practical implications but also raise questions of why and how this strategy would benefit transposons.},
}
@article {pmid31549767,
year = {2020},
author = {Nagler, A and Vredevoogd, DW and Alon, M and Cheng, PF and Trabish, S and Kalaora, S and Arafeh, R and Goldin, V and Levesque, MP and Peeper, DS and Samuels, Y},
title = {A genome-wide CRISPR screen identifies FBXO42 involvement in resistance toward MEK inhibition in NRAS-mutant melanoma.},
journal = {Pigment cell &amp; melanoma research},
volume = {33},
number = {2},
pages = {334-344},
pmid = {31549767},
issn = {1755-148X},
support = {CoG- 770854/ERC_/European Research Council/International ; 712977/ERC_/European Research Council/International ; },
mesh = {Base Sequence ; Biomarkers, Tumor/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; F-Box Proteins/*genetics/*metabolism ; GTP Phosphohydrolases/*genetics ; Genetic Testing ; *Genome, Human ; Humans ; MAP Kinase Kinase Kinases/metabolism ; MAP Kinase Signaling System/drug effects/genetics ; Melanoma/drug therapy/*genetics ; Membrane Proteins/*genetics ; Mitogen-Activated Protein Kinase Kinases/antagonists &amp; inhibitors ; Models, Biological ; Mutation/genetics ; Protein Binding/drug effects ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Pyridones/pharmacology/therapeutic use ; Pyrimidinones/pharmacology/therapeutic use ; Skin Neoplasms/drug therapy/*genetics ; },
abstract = {NRAS mutations are the most common alterations among RAS isoforms in cutaneous melanoma, with patients harboring these aggressive tumors having a poor prognosis and low survival rate. The main line of treatment for these patients is MAPK pathway-targeted therapies, such as MEK inhibitors, but, unfortunately, the response to these inhibitors is variable due to tumor resistance. Identifying genetic modifiers involved in resistance toward MEK-targeted therapy may assist in the development of new therapeutic strategies, enhancing treatment response and patient survival. Our whole-genome CRISPR-Cas9 knockout screen identified the target Kelch domain-containing F-Box protein 42 (FBXO42) as a factor involved in NRAS-mutant melanoma-acquired resistance to the MEK1/2 inhibitor trametinib. We further show that FBXO42, an E3 ubiquitin ligase, is involved in the TAK1 signaling pathway, possibly prompting an increase in active P38. In addition, we demonstrate that combining trametinib with the TAK1 inhibitor, takinib, is a far more efficient treatment than trametinib alone in NRAS-mutant melanoma cells. Our findings thus show a new pathway involved in NRAS-mutant melanoma resistance and provide new opportunities for novel therapeutic options.},
}
@article {pmid31549678,
year = {2019},
author = {Zong, Y and Gao, CX},
title = {[Progress on base editing systems].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {9},
pages = {777-800},
doi = {10.16288/j.yczz.19-205},
pmid = {31549678},
issn = {0253-9772},
mesh = {Adenine ; Aminohydrolases ; Animals ; Biotechnology/trends ; *CRISPR-Cas Systems ; Cytosine ; Cytosine Deaminase ; *DNA Breaks, Double-Stranded ; *Gene Editing ; Plants ; },
abstract = {Base editing is a newly developed precise genome editing technique based on the CRISPR/Cas system. According to different base modification enzymes, the current base editing systems can be divided into cytosine base editors (CBE) and adenine base editors (ABE). They use cytosine deaminases or artificially evolved adenine deaminases to perform single-base editing, and achieve C to T (G to A) or A to G (T to C) substitutions, respectively. Due to high efficiency, independence of DNA double-strand breaks, and no need for donor DNA, base editing systems have been successfully applied in diverse species including animals, plants and other organisms since the first report in 2016. Therefore, base editing systems will have a high prospect of providing important support for gene therapy and crop genetic improvement in the future. In this review, we describe the development and current applications of base editing systems for basic research and biotechnology, highlight the challenges, and discuss the directions for future research in this important field. The information presented may facilitate interested researchers to grasp the principles of base editing, to use relevant base editing tools in their own studies, or to innovate new versions of base editing in the future.},
}
@article {pmid31548729,
year = {2019},
author = {Pickar-Oliver, A and Black, JB and Lewis, MM and Mutchnick, KJ and Klann, TS and Gilcrest, KA and Sitton, MJ and Nelson, CE and Barrera, A and Bartelt, LC and Reddy, TE and Beisel, CL and Barrangou, R and Gersbach, CA},
title = {Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells.},
journal = {Nature biotechnology},
volume = {37},
number = {12},
pages = {1493-1501},
pmid = {31548729},
issn = {1546-1696},
support = {T32 GM008555/GM/NIGMS NIH HHS/United States ; DP2 OD008586/OD/NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; R35 GM119561/GM/NIGMS NIH HHS/United States ; F31 NS105419/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics ; Genetic Engineering/*methods ; HEK293 Cells ; Humans ; Listeria monocytogenes/genetics ; RNA, Guide/genetics ; Transcription, Genetic/*genetics ; },
abstract = {Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.},
}
@article {pmid31548728,
year = {2019},
author = {Ye, L and Park, JJ and Dong, MB and Yang, Q and Chow, RD and Peng, L and Du, Y and Guo, J and Dai, X and Wang, G and Errami, Y and Chen, S},
title = {In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma.},
journal = {Nature biotechnology},
volume = {37},
number = {11},
pages = {1302-1313},
pmid = {31548728},
issn = {1546-1696},
support = {U54 CA209992/CA/NCI NIH HHS/United States ; RF1 DA048811/DA/NIDA NIH HHS/United States ; S10 OD018521/OD/NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD/genetics ; CD8-Positive T-Lymphocytes/metabolism/*transplantation ; CRISPR-Cas Systems ; Cell Line, Tumor ; Dependovirus/genetics ; Female ; Gene Editing/*methods ; Glioblastoma/genetics/immunology/*therapy ; Humans ; Immunotherapy, Adoptive ; Male ; Membrane Proteins/*genetics ; Mice ; N-Acetylglucosaminyltransferases/genetics ; Neoplasm Proteins/genetics ; Protein Disulfide-Isomerases/genetics ; RNA, Guide/genetics ; Receptors, Cell Surface/genetics ; Transposases/*genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8[+] T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8[+] T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.},
}
@article {pmid31548722,
year = {2019},
author = {Schrode, N and Ho, SM and Yamamuro, K and Dobbyn, A and Huckins, L and Matos, MR and Cheng, E and Deans, PJM and Flaherty, E and Barretto, N and Topol, A and Alganem, K and Abadali, S and Gregory, J and Hoelzli, E and Phatnani, H and Singh, V and Girish, D and Aronow, B and Mccullumsmith, R and Hoffman, GE and Stahl, EA and Morishita, H and Sklar, P and Brennand, KJ},
title = {Synergistic effects of common schizophrenia risk variants.},
journal = {Nature genetics},
volume = {51},
number = {10},
pages = {1475-1485},
pmid = {31548722},
issn = {1546-1718},
support = {R01 MH109897/MH/NIMH NIH HHS/United States ; S10 OD018522/OD/NIH HHS/United States ; R21 MH107916/MH/NIMH NIH HHS/United States ; R01 MH106056/MH/NIMH NIH HHS/United States ; R01 MH107487/MH/NIMH NIH HHS/United States ; R56 MH101454/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chloride Channels/antagonists &amp; inhibitors/genetics/metabolism ; Female ; Furin/antagonists &amp; inhibitors/genetics/metabolism ; Gene Editing ; *Gene Expression Regulation ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Male ; Monomeric Clathrin Assembly Proteins/antagonists &amp; inhibitors/genetics/metabolism ; *Polymorphism, Single Nucleotide ; *Quantitative Trait Loci ; SNARE Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Schizophrenia/*genetics/*pathology ; },
abstract = {The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.},
}
@article {pmid31548639,
year = {2019},
author = {Kellner, MJ and Koob, JG and Gootenberg, JS and Abudayyeh, OO and Zhang, F},
title = {SHERLOCK: nucleic acid detection with CRISPR nucleases.},
journal = {Nature protocols},
volume = {14},
number = {10},
pages = {2986-3012},
pmid = {31548639},
issn = {1750-2799},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; F31 NS059160/NS/NINDS NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; F30 CA210382/CA/NCI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Primers ; Endonucleases/*genetics/isolation &amp; purification/metabolism ; Humans ; Leptotrichia/genetics ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acids/*analysis/genetics ; Protein Engineering/methods ; RNA, Guide ; Recombinant Proteins/genetics/isolation &amp; purification/metabolism ; Ribonucleases/genetics/isolation &amp; purification/metabolism ; Workflow ; Zika Virus/genetics ; Zika Virus Infection/blood/urine ; },
abstract = {Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.},
}
@article {pmid31548614,
year = {2020},
author = {Ko, T and Sharma, R and Li, S},
title = {Genome-wide screening identifies novel genes implicated in cellular sensitivity to BRAF[V600E] expression.},
journal = {Oncogene},
volume = {39},
number = {4},
pages = {723-738},
pmid = {31548614},
issn = {1476-5594},
mesh = {3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/genetics/*metabolism ; Apoptosis ; CRISPR-Cas Systems ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Fibroblasts/metabolism ; *Genome, Human ; Humans ; MAP Kinase Signaling System ; Melanocytes/metabolism ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; *Mutation ; Neoplasms/genetics/metabolism/*pathology ; Proto-Oncogene Proteins B-raf/*genetics/*metabolism ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Signal Transduction ; },
abstract = {The V600E mutation of BRAF (BRAF[V600E]), which constitutively activates the ERK/MAPK signaling pathway, is frequently found in melanoma and other cancers. Like most other oncogenes, BRAF[V600E] causes oncogenic stress to normal cells, leading to growth arrest (senescence) or apoptosis. Through genome-wide screening, we identified genes implicated in sensitivity of human skin melanocytes and fibroblasts to BRAF[V600E] overexpression. Among the identified genes shared by the two cell types are proto-oncogenes ERK2, a component of the ERK/MAPK pathway, and VAV1, a guanine nucleotide exchange factor for Rho family GTPases that also activates the ERK/MAPK pathway. CDKN1A, which has been known to promote senescence of fibroblasts but not melanocytes, is implicated in sensitivity of the fibroblasts but not the melanocytes to BRAF[V600E] overexpression. Disruptions of GPR4, a pH-sensing G-protein coupled receptor, and DBT, a subunit of the branched chain α-keto acid dehydrogenase that is required for the second and rate-limiting step of branched amino acid catabolism and implicated in maple syrup urine disease, are the most highly selected in the melanocytes upon BRAF[V600E] overexpression. Disruption of DBT severely attenuates ERK/MAPK signaling, p53 activation, and apoptosis in melanocytes, at least in part due to accumulation of branched chain α-keto acids. The expression level of BRAF positively correlates with that of DBT in all cancer types and with that of GPR4 in most cancer types. Overexpression of DBT kills all four melanoma cell lines tested regardless of the presence of BRAF[V600E] mutation. Our findings shed new lights on regulations of oncogenic stress signaling and may be informative for development of novel cancer treatment strategies.},
}
@article {pmid31548591,
year = {2019},
author = {Mair, B and Aldridge, PM and Atwal, RS and Philpott, D and Zhang, M and Masud, SN and Labib, M and Tong, AHY and Sargent, EH and Angers, S and Moffat, J and Kelley, SO},
title = {High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.},
journal = {Nature biomedical engineering},
volume = {3},
number = {10},
pages = {796-805},
doi = {10.1038/s41551-019-0454-8},
pmid = {31548591},
issn = {2157-846X},
support = {//CIHR/Canada ; },
mesh = {CD47 Antigen/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Flow Cytometry ; Gene Editing ; *Genome ; High-Throughput Screening Assays/*methods ; Humans ; Immunomagnetic Separation/*methods ; Immunotherapy ; Lab-On-A-Chip Devices ; Neoplasms/therapy ; *Phenotype ; },
abstract = {Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 10[8] cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.},
}
@article {pmid31548381,
year = {2019},
author = {Tong, Y and Whitford, CM and Robertsen, HL and Blin, K and Jørgensen, TS and Klitgaard, AK and Gren, T and Jiang, X and Weber, T and Lee, SY},
title = {Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {41},
pages = {20366-20375},
pmid = {31548381},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; DNA, Bacterial/genetics ; *Gene Editing ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Genome-Wide Association Study ; Plasmids ; Streptomyces coelicolor/*genetics ; },
abstract = {Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.},
}
@article {pmid31548319,
year = {2019},
author = {Velayutham, TS and Kumar, S and Zhang, X and Kose, N and Walker, DH and Winslow, G and Crowe, JE and McBride, JW},
title = {Ehrlichia chaffeensis Outer Membrane Protein 1-Specific Human Antibody-Mediated Immunity Is Defined by Intracellular TRIM21-Dependent Innate Immune Activation and Extracellular Neutralization.},
journal = {Infection and immunity},
volume = {87},
number = {12},
pages = {},
pmid = {31548319},
issn = {1098-5522},
mesh = {Adenine/analogs &amp; derivatives/pharmacology ; Antibodies, Monoclonal/immunology ; Antibodies, Neutralizing/immunology ; Antigens, Bacterial/genetics/*immunology ; Autophagy/immunology ; Bacterial Adhesion/genetics ; Bacterial Outer Membrane Proteins/*genetics/*immunology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Ehrlichia chaffeensis/genetics/*immunology ; Gene Knockout Techniques ; Humans ; Immunity, Humoral/immunology ; NF-kappa B/genetics ; Ribonucleoproteins/*genetics ; THP-1 Cells ; },
abstract = {Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.},
}
@article {pmid31548010,
year = {2019},
author = {Wang, Y and Wang, Z and Chen, Y and Hua, X and Yu, Y and Ji, Q},
title = {A Highly Efficient CRISPR-Cas9-Based Genome Engineering Platform in Acinetobacter baumannii to Understand the H2O2-Sensing Mechanism of OxyR.},
journal = {Cell chemical biology},
volume = {26},
number = {12},
pages = {1732-1742.e5},
doi = {10.1016/j.chembiol.2019.09.003},
pmid = {31548010},
issn = {2451-9448},
mesh = {Acinetobacter baumannii/drug effects/*genetics ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Drug Resistance, Bacterial/genetics ; Gene Editing/*methods ; Hydrogen Peroxide/*analysis ; Microbial Sensitivity Tests ; Point Mutation ; Repressor Proteins/genetics/*metabolism ; },
abstract = {The rapid emergence of extensively drug-resistant A. baumannii has posed a major threat to global public health, emphasizing the desperate need for novel therapeutic strategies. We report the development of a highly efficient genome-engineering platform in A. baumannii by coupling a Cas9 nuclease-mediated genome cleavage system with the RecAb recombination system. We applied the CRISPR-Cas9/RecAb system to dissect the oxidative stress-sensing mechanism of OxyR by performing alanine scanning mutagenesis of 13 residues residing in the H2O2-sensing pocket, pinpointing new vital factors for H2O2 sensing. Moreover, we developed a cytidine base-editing system, enabling programmed C to T conversions. Exploiting this powerful technique, we systematically investigated the drug-resistant mechanisms in a clinically isolated multidrug-resistant A. baumannii strain by generating premature stop codons in the possible resistance genes, unveiling distinct roles of these genes in drug resistance. The development of these genome-engineering methods will facilitate new therapeutic-means development in A. baumannii and related organisms.},
}
@article {pmid31547486,
year = {2019},
author = {Wang, H and Wu, Y and Zhang, Y and Yang, J and Fan, W and Zhang, H and Zhao, S and Yuan, L and Zhang, P},
title = {CRISPR/Cas9-Based Mutagenesis of Starch Biosynthetic Genes in Sweet Potato (Ipomoea Batatas) for the Improvement of Starch Quality.},
journal = {International journal of molecular sciences},
volume = {20},
number = {19},
pages = {},
pmid = {31547486},
issn = {1422-0067},
mesh = {Arabidopsis/genetics ; *CRISPR-Cas Systems ; *Genes, Plant ; *Ipomoea batatas/genetics/metabolism ; *Mutagenesis ; *Plants, Genetically Modified/genetics/metabolism ; Promoter Regions, Genetic ; *Starch/biosynthesis/genetics ; },
abstract = {CRISPR/Cas9-mediated genome editing is a powerful technology that has been used for the genetic modification of a number of crop species. In order to evaluate the efficacy of CRISPR/Cas9 technology in the root crop, sweet potato (Ipomoea batatas), two starch biosynthetic pathway genes, IbGBSSI (encoding granule-bound starch synthase I), and IbSBEII (encoding starch branching enzyme II), were targeted in the starch-type cultivar Xushu22 and carotenoid-rich cultivar Taizhong6. I. batatas was transformed using a binary vector, in which the Cas9 gene is driven by the Arabidopsis AtUBQ promoter and the guide RNA is controlled by the Arabidopsis AtU6 promoter. A total of 72 Xushu22 and 35 Taizhong6 transgenic lines were generated and analyzed for mutations. The mutation efficiency was 62-92% with multi-allelic mutations in both cultivars. Most of the mutations were nucleotide substitutions that lead to amino acid changes and, less frequently, stop codons. In addition, short nucleotide insertions or deletions were also found in both IbGBSSI and IbSBEII. Furthermore, a 2658 bp deletion was found in one IbSBEII transgenic line. The total starch contents were not significantly changed in IbGBSSI- and IbSBEII-knockout transgenic lines compared to the wild-type control. However, in the allopolyploid sweet potato, the IbGBSSI-knockout reduced, while the IbSBEII-knockout increased, the amylose percentage. Our results demonstrate that CRISPR/Cas9 technology is an effective tool for the improvement of starch qualities in sweet potato and breeding of polyploid root crops.},
}
@article {pmid31546915,
year = {2019},
author = {Scrascia, M and D'Addabbo, P and Roberto, R and Porcelli, F and Oliva, M and Calia, C and Dionisi, AM and Pazzani, C},
title = {Characterization of CRISPR-Cas Systems in Serratia marcescens Isolated from Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae).},
journal = {Microorganisms},
volume = {7},
number = {9},
pages = {},
pmid = {31546915},
issn = {2076-2607},
abstract = {The CRISPR-Cas adaptive immune system has been attracting increasing scientific interest for biological functions and biotechnological applications. Data on the Serratia marcescens system are scarce. Here, we report a comprehensive characterisation of CRISPR-Cas systems identified in S. marcescens strains isolated as secondary symbionts of Rhynchophorus ferrugineus, also known as Red Palm Weevil (RPW), one of the most invasive pests of major cultivated palms. Whole genome sequencing was performed on four strains (S1, S5, S8, and S13), which were isolated from the reproductive apparatus of RPWs. Subtypes I-F and I-E were harboured by S5 and S8, respectively. No CRISPR-Cas system was detected in S1 or S13. Two CRISPR arrays (4 and 51 spacers) were detected in S5 and three arrays (11, 31, and 30 spacers) were detected in S8. The CRISPR-Cas systems were located in the genomic region spanning from ybhR to phnP, as if this were the only region where CRISPR-Cas loci were acquired. This was confirmed by analyzing the S. marcescens complete genomes available in the NCBI database. This region defines a genomic hotspot for horizontally acquired genes and/or CRISPR-Cas systems. This study also supplies the first identification of subtype I-E in S. marcescens.},
}
@article {pmid31545795,
year = {2019},
author = {Cheng, Y and Zhang, N and Hussain, S and Ahmed, S and Yang, W and Wang, S},
title = {Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis.},
journal = {PloS one},
volume = {14},
number = {9},
pages = {e0218583},
pmid = {31545795},
issn = {1932-6203},
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/*genetics ; *CRISPR-Cas Systems ; DNA Mutational Analysis ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Loci ; Genotype ; *Mutation ; Phenotype ; Plants, Genetically Modified ; *Transgenes ; },
abstract = {The CRISPR/Cas9 genome editing technique has been widely used to generate transgene-free mutants in different plant species. Several different methods including fluorescence marker-assisted visual screen of transgene-free mutants and programmed self-elimination of CRISPR/Cas9 construct have been used to increase the efficiency of genome edited transgene-free mutant isolation, but the overall time length required to obtain transgene-free mutants has remained unchanged in these methods. We report here a method for fast generation and easy identification of transgene-free mutants in Arabidopsis. By generating and using a single FT expression cassette-containing CRISPR/Cas9 construct, we targeted two sites of the AITR1 gene. We obtained many early bolting plants in T1 generation, and found that about two thirds of these plants have detectable mutations. We then analyzed T2 generations of two representative lines of genome edited early bolting T1 plants, and identified plants without early bolting phenotype, i.e., transgene-free plants, for both lines. Further more, aitr1 homozygous mutants were successful obtained for both lines from these transgene-free plants. Taken together, these results suggest that the method described here enables fast generation, and at the mean time, easy identification of transgene-free mutants in plants.},
}
@article {pmid31545544,
year = {2020},
author = {Zhang, C and Xu, W and Wang, F and Kang, G and Yuan, S and Lv, X and Li, L and Liu, Y and Yang, J},
title = {Expanding the base editing scope to GA and relaxed NG PAM sites by improved xCas9 system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {4},
pages = {884-886},
pmid = {31545544},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Plants/*genetics ; },
}
@article {pmid31545321,
year = {2019},
author = {Chan, K and Tong, AHY and Brown, KR and Mero, P and Moffat, J},
title = {Pooled CRISPR-Based Genetic Screens in Mammalian Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {151},
pages = {},
doi = {10.3791/59780},
pmid = {31545321},
issn = {1940-087X},
support = {MOP-142375//CIHR/Canada ; PJT-148802//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Genetic Testing/*methods ; Humans ; },
abstract = {Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.},
}
@article {pmid31543470,
year = {2019},
author = {Klatt, D and Cheng, E and Philipp, F and Selich, A and Dahlke, J and Schmidt, RE and Schott, JW and Büning, H and Hoffmann, D and Thrasher, AJ and Schambach, A},
title = {Targeted Repair of p47-CGD in iPSCs by CRISPR/Cas9: Functional Correction without Cleavage in the Highly Homologous Pseudogenes.},
journal = {Stem cell reports},
volume = {13},
number = {4},
pages = {590-598},
pmid = {31543470},
issn = {2213-6711},
support = {104807/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Enzyme Activation ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Loci ; Granulocytes/immunology/metabolism ; Granulomatous Disease, Chronic/*genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Introns ; Macrophages/immunology/metabolism/microbiology ; NADPH Oxidases/*genetics/metabolism ; Phagocytosis/immunology ; Pseudogenes/genetics ; Sequence Homology ; },
abstract = {Mutations in the NADPH oxidase, which is crucial for the respiratory burst in phagocytes, result in chronic granulomatous disease (CGD). The only curative treatment option for CGD patients, who suffer from severe infections, is allogeneic bone marrow transplantation. Over 90% of patients with mutations in the p47[phox] subunit of the oxidase complex carry the deletion c.75_76delGT (ΔGT). This frequent mutation most likely originates via gene conversion from one of the two pseudogenes NCF1B or NCF1C, which are highly homologous to NCF1 (encodes p47[phox]) but carry the ΔGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-ΔGT iPSCs for disease modeling. To avoid unpredictable chromosomal rearrangements by CRISPR/Cas9-mediated cleavage in the pseudogenes, we developed a gene-correction approach to specifically target NCF1 but leave the pseudogenes intact. Functional assays revealed restored NADPH oxidase activity and killing of bacteria in corrected phagocytes as well as the specificity of this approach.},
}
@article {pmid31543367,
year = {2019},
author = {Román-Rodríguez, FJ and Ugalde, L and Álvarez, L and Díez, B and Ramírez, MJ and Risueño, C and Cortón, M and Bogliolo, M and Bernal, S and March, F and Ayuso, C and Hanenberg, H and Sevilla, J and Rodríguez-Perales, S and Torres-Ruiz, R and Surrallés, J and Bueren, JA and Río, P},
title = {NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia.},
journal = {Cell stem cell},
volume = {25},
number = {5},
pages = {607-621.e7},
doi = {10.1016/j.stem.2019.08.016},
pmid = {31543367},
issn = {1875-9777},
mesh = {Alleles ; Animals ; Antigens, CD34/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Proliferation/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; Fanconi Anemia/genetics/metabolism/*therapy ; Fanconi Anemia Complementation Group A Protein/*genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy/*methods ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Hematopoietic Stem Cells/cytology/metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; INDEL Mutation ; Mice ; Mice, Inbred NOD ; Mice, Nude ; },
abstract = {Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34[+] cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.},
}
@article {pmid31541694,
year = {2020},
author = {Xu, X and Wang, Y and Bi, H and Xu, J and Liu, Z and Niu, C and He, L and James, AA and Li, K and Huang, Y},
title = {Mutation of the seminal protease gene, serine protease 2, results in male sterility in diverse lepidopterans.},
journal = {Insect biochemistry and molecular biology},
volume = {116},
number = {},
pages = {103243},
doi = {10.1016/j.ibmb.2019.103243},
pmid = {31541694},
issn = {1879-0240},
mesh = {Animals ; Bombyx/genetics/physiology ; CRISPR-Cas Systems ; Infertility, Male/genetics ; Insect Proteins/*genetics/metabolism ; Male ; Moths/genetics/*physiology ; Mutation ; Reproduction/genetics ; Serine Proteases/*genetics/metabolism ; },
abstract = {Sterile insect technology (SIT) is an environmentally friendly method for pest control. As part of our efforts to develop a strategy that results in engineered male-sterile strains with minimum effects on viability and mating competition, we used CRISPR/Cas9 technology to disrupt Ser2, which encodes a seminal fluid protein, in the model lepidopteran insect, Bombyx mori, and an important agricultural pest, Plutella xylostella. Disruption of Ser2 resulted in dominant heritable male sterility. Wild-type females mated with Ser2-deficient males laid eggs normally, but the eggs did not hatch. We detected no differences in other reproductive behaviors in the mutant males. These results support the conclusion that Ser2 gene is necessary for male reproductive success in diverse lepidopterans. Targeting Ser2 gene has the potential to form the basis for a new strategy for pest control.},
}
@article {pmid31541593,
year = {2020},
author = {Chang, Y and Deng, Y and Li, T and Wang, J and Wang, T and Tan, F and Li, X and Tian, K},
title = {Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a.},
journal = {Transboundary and emerging diseases},
volume = {67},
number = {2},
pages = {564-571},
doi = {10.1111/tbed.13368},
pmid = {31541593},
issn = {1865-1682},
mesh = {Animals ; *CRISPR-Cas Systems ; Fluorescence ; Limit of Detection ; Porcine Reproductive and Respiratory Syndrome/*diagnosis/prevention &amp; control/virology ; Porcine respiratory and reproductive syndrome virus/genetics/*isolation &amp; purification ; RNA, Viral/genetics ; Recombinases/metabolism ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/prevention &amp; control/virology ; Viral Matrix Proteins/*genetics ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) has varied constantly and circulated in the pig industry worldwide. The prevention and control of porcine reproductive and respiratory syndrome (PRRS) is complicated. A visual, sensitive and specific diagnostic method is advantageous to the control of PRRS. The collateral cleavage activity of LwCas13a is activated to degrade non-targeted RNA, when crRNA of LwCas13a bond to target RNA. The enhanced Cas13a detection is the combination of collateral cleavage activity of LwCas13a and recombinase polymerase amplification (RPA). In this study, the enhanced Cas13a detection for PRRSV was established. The novel method was an isothermal detection at 37°C, and the detection can be used for real-time analysis or visual readout. The detection limit of the enhanced Cas13a detection was 172 copies/μl, and there were no cross-reactions with porcine circovirus 2, porcine parvovirus, classical swine fever virus and pseudorabies virus. The enhanced Cas13a detection can work well in clinical samples. In summary, a visual, sensitive and specific nucleic acid detection method based on CRISPR-Cas13a was developed for PRRSV.},
}
@article {pmid31541447,
year = {2020},
author = {Ferrie, AMR and Bhowmik, P and Rajagopalan, N and Kagale, S},
title = {CRISPR/Cas9-Mediated Targeted Mutagenesis in Wheat Doubled Haploids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2072},
number = {},
pages = {183-198},
doi = {10.1007/978-1-4939-9865-4_15},
pmid = {31541447},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Genome, Plant ; Genotype ; *Haploidy ; *Mutagenesis ; Plant Development/genetics ; Plants, Genetically Modified ; RNA, Guide ; Triticum/*genetics ; },
abstract = {CRISPR/Cas9-based genome editing technology has the potential to revolutionize agriculture, but many plant species and/or genotypes are recalcitrant to conventional transformation methods. Additionally, the long generation time of crop plants poses a significant obstacle to effective application of gene editing technology, as it takes a long time to produce modified homozygous genotypes. The haploid single-celled microspores are an attractive target for gene editing experiments, as they enable generation of homozygous doubled haploid mutants in one generation. Here, we describe optimized methods for genome editing of haploid wheat microspores and production of doubled haploid plants by microspore culture.},
}
@article {pmid31541446,
year = {2020},
author = {Bilichak, A and Gaudet, D and Laurie, J},
title = {Emerging Genome Engineering Tools in Crop Research and Breeding.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2072},
number = {},
pages = {165-181},
doi = {10.1007/978-1-4939-9865-4_14},
pmid = {31541446},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Epigenomics/methods ; Gene Editing ; Gene Expression Regulation, Plant ; *Genetic Engineering/methods ; *Genome, Plant ; *Genomics/methods ; *Plant Breeding/methods ; Plant Development/genetics ; Plants, Genetically Modified ; Quantitative Trait, Heritable ; Transcription Activator-Like Effector Nucleases ; },
abstract = {Recent advances in genome engineering are revolutionizing crop research and plant breeding. The ability to make specific modifications to a plant's genetic material creates opportunities for rapid development of elite cultivars with desired traits. The plant genome can be altered in several ways, including targeted introduction of nucleotide changes, deleting DNA segments, introducing exogenous DNA fragments and epigenetic modifications. Targeted changes are mediated by sequence specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR associated protein) systems. Recent advances in engineering chimeric Cas nucleases fused to base editing enzymes permit for even greater precision in base editing and control over gene expression. In addition to gene editing technologies, improvement in delivery systems of exogenous DNA into plant cells have increased the rate of successful gene editing events. Regeneration of fertile plants containing the desired edits remains challenging; however, manipulation of embryogenesis-related genes such as BABY BOOM (BBM) has been shown to facilitate regeneration through tissue culture, often a major hurdle in recalcitrant cultivars. Epigenome reprogramming for improved crop performance is another possibility for future breeders, with recent studies on MutS HOMOLOG 1 (MSH1) demonstrating epigenetic-dependent hybrid vigor in several crops. While these technologies offer plant breeders new tools in creating high yielding, better adapted crop varieties, constantly evolving government policy regarding the cultivation of plants containing transgenes may impede the widespread adoption of some of these techniques. This chapter summarizes advances in genome editing tools and discusses the future of these techniques for crop improvement.},
}
@article {pmid31541261,
year = {2019},
author = {Chutrakul, C and Panchanawaporn, S and Jeennor, S and Anantayanon, J and Vorapreeda, T and Vichai, V and Laoteng, K},
title = {Functional Characterization of Novel U6 RNA Polymerase III Promoters: Their Implication for CRISPR-Cas9-Mediated Gene Editing in Aspergillus oryzae.},
journal = {Current microbiology},
volume = {76},
number = {12},
pages = {1443-1451},
pmid = {31541261},
issn = {1432-0991},
mesh = {Aspergillus oryzae/enzymology/*genetics ; *CRISPR-Cas Systems ; Fungal Proteins/*genetics/metabolism ; *Gene Editing ; Gene Expression Regulation, Fungal ; *Promoter Regions, Genetic ; RNA Polymerase III/*genetics/metabolism ; RNA, Fungal/*genetics/metabolism ; RNA, Guide/*genetics/metabolism ; },
abstract = {U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.},
}
@article {pmid31541109,
year = {2019},
author = {Molina, R and Stella, S and Feng, M and Sofos, N and Jauniskis, V and Pozdnyakova, I and López-Méndez, B and She, Q and Montoya, G},
title = {Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4302},
pmid = {31541109},
issn = {2041-1723},
mesh = {Adenine Nucleotides/*chemistry ; Allosteric Site ; Bacterial Proteins/chemistry ; Binding Sites ; CRISPR-Associated Proteins/*chemistry/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; Endoribonucleases/*chemistry ; Membrane Proteins/chemistry ; Models, Molecular ; Oligoribonucleotides/*chemistry ; Protein Binding ; Protein Domains ; *RNA Stability ; RNA-Binding Proteins/*chemistry/genetics ; Ribonucleases/metabolism ; Second Messenger Systems ; Sulfolobus/*chemistry/genetics ; },
abstract = {Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.},
}
@article {pmid31541098,
year = {2019},
author = {Taghbalout, A and Du, M and Jillette, N and Rosikiewicz, W and Rath, A and Heinen, CD and Li, S and Cheng, AW},
title = {Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4296},
pmid = {31541098},
issn = {2041-1723},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 CA115783/CA/NCI NIH HHS/United States ; R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {5-Methylcytosine/metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Demethylation ; DNA Glycosylases/metabolism ; DNA Methylation ; *DNA Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Mixed Function Oxygenases/genetics ; Oxidation-Reduction ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide/*metabolism ; Sequence Analysis, RNA ; },
abstract = {Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.},
}
@article {pmid31540988,
year = {2019},
author = {Baker, PL and Orf, GS and Kevershan, K and Pyne, ME and Bicer, T and Redding, KE},
title = {Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {23},
pages = {},
pmid = {31540988},
issn = {1098-5336},
mesh = {*CRISPR-Cas Systems ; Clostridiales/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genes, Bacterial ; Photochemical Processes ; },
abstract = {In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome.IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.},
}
@article {pmid31539770,
year = {2019},
author = {Mettelman, RC and O'Brien, A and Whittaker, GR and Baker, SC},
title = {Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus.},
journal = {Virology},
volume = {537},
number = {},
pages = {226-236},
pmid = {31539770},
issn = {1096-0341},
support = {R01 AI085089/AI/NIAID NIH HHS/United States ; T32 AI007508/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Cats ; Cell Line ; Coronavirus, Feline/*growth &amp; development/immunology ; Gene Editing ; Receptor, Interferon alpha-beta/*deficiency ; Receptors, Virus/*biosynthesis/genetics ; Recombinant Proteins/biosynthesis/genetics ; Serine Endopeptidases/*biosynthesis/genetics ; Virus Cultivation/*methods ; Virus Replication ; },
abstract = {Feline coronavirus infection can progress to a fatal infectious peritonitis, which is a widespread feline disease without an effective vaccine. Generating feline cells with reduced ability to respond to interferon (IFN) is an essential step facilitating isolation of new candidate vaccine strains. Here, we describe the use of Crispr/Cas technology to disrupt type I IFN signaling in two feline cell lines, AK-D and Fcwf-4 CU, and evaluate the replication kinetics of a serotype I feline infectious peritonitis virus (FIPV) within these cells. We report that polyclonal cell populations and a clonal isolate, termed Fcwf-4 IRN, exhibited significantly diminished IFN-responsiveness and allowed FIPV replication kinetics comparable to parental cells. Furthermore, we demonstrate that replication of FIPV is enhanced by ectopic expression of a host serine protease, TMPRSS2, in these cells. We discuss the potential of these cells for isolating new clinical strains and for propagating candidate vaccine strains of FIPV.},
}
@article {pmid31538676,
year = {2019},
author = {Chen, W and Yang, F and Xu, X and Kumar, U and He, W and You, M},
title = {Genetic control of Plutella xylostella in omics era.},
journal = {Archives of insect biochemistry and physiology},
volume = {102},
number = {3},
pages = {e21621},
doi = {10.1002/arch.21621},
pmid = {31538676},
issn = {1520-6327},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Female ; Insect Control/*methods ; *Insecticide Resistance ; Moths/*genetics ; Plants, Genetically Modified ; },
abstract = {Diamondback moth, Plutella xylostella (L.), is a specialist pest on cruciferous crops of economic importance. The large-scale use of chemical insecticides for the control of this insect pest has caused a number of challenges to agro-ecosystems. With the advent of the omics era, genetic pest management strategies are becoming increasingly feasible and show a powerful potential for pest control. Here, we review strategies for using transgenic plants and sterile insect techniques for genetic pest management and introduce the major advances in the control of P. xylostella using a female-specific RIDL (release of insects carrying a dominant lethal gene) strategy. Further, the advantages of gene drive developed in combination with sex determination and CRISPR/Cas9 systems are addressed, and the corresponding prospects and implementation issues are discussed. It is predictable that under the policy and regulation of professional committees, the genetic pest control strategy, especially for gene drive, will open a new avenue to sustainable pest management not only for P. xylostella but also for other insect pests.},
}
@article {pmid31538358,
year = {2020},
author = {Yadav, M and Shukla, P},
title = {Efficient engineered probiotics using synthetic biology approaches: A review.},
journal = {Biotechnology and applied biochemistry},
volume = {67},
number = {1},
pages = {22-29},
doi = {10.1002/bab.1822},
pmid = {31538358},
issn = {1470-8744},
mesh = {Biotechnology ; Dietary Supplements ; *Metabolic Engineering ; Probiotics/*metabolism ; Synthetic Biology ; },
abstract = {The uses of probiotics-based food supplements are getting emphasis due to their power to ensure better health conditions. Probiotics have diverse and significant applications in the health sector, so probiotic strains require an understanding of the genome level organizations. Probiotics elucidate various functional parameters that control their metabolic functions. In this review, we have compiled aspects of synthetic biology, which are used for the optimization of metabolic processes in probiotics for their use as a supplement in allopathic medicines. Synthetic biology approaches provide information about diverse biosynthetic pathways and also facilitate the novel metabolic engineering approaches for probiotics strain improvement. We have discussed the synthetic biology approaches for producing engineered probiotics via genetic circuits, expression systems, and genome editing tools like CRISPR-Cas and PEVLAB. This review also enlightens future challenges in the development of engineered probiotics.},
}
@article {pmid31537810,
year = {2019},
author = {Wang, D and Zhang, C and Wang, B and Li, B and Wang, Q and Liu, D and Wang, H and Zhou, Y and Shi, L and Lan, F and Wang, Y},
title = {Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4284},
pmid = {31537810},
issn = {2041-1723},
mesh = {Animals ; Base Sequence/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Deep Learning ; Endonucleases/metabolism ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; INDEL Mutation/genetics ; Internet ; Mice ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; },
abstract = {Highly specific Cas9 nucleases derived from SpCas9 are valuable tools for genome editing, but their wide applications are hampered by a lack of knowledge governing guide RNA (gRNA) activity. Here, we perform a genome-scale screen to measure gRNA activity for two highly specific SpCas9 variants (eSpCas9(1.1) and SpCas9-HF1) and wild-type SpCas9 (WT-SpCas9) in human cells, and obtain indel rates of over 50,000 gRNAs for each nuclease, covering ~20,000 genes. We evaluate the contribution of 1,031 features to gRNA activity and develope models for activity prediction. Our data reveals that a combination of RNN with important biological features outperforms other models for activity prediction. We further demonstrate that our model outperforms other popular gRNA design tools. Finally, we develop an online design tool DeepHF for the three Cas9 nucleases. The database, as well as the designer tool, is freely accessible via a web server, http://www.DeepHF.com/ .},
}
@article {pmid31537809,
year = {2019},
author = {Feng, W and Simpson, DA and Carvajal-Garcia, J and Price, BA and Kumar, RJ and Mose, LE and Wood, RD and Rashid, N and Purvis, JE and Parker, JS and Ramsden, DA and Gupta, GP},
title = {Genetic determinants of cellular addiction to DNA polymerase theta.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4286},
pmid = {31537809},
issn = {2041-1723},
support = {R01 CA222092/CA/NCI NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; T32 GM008719/GM/NIGMS NIH HHS/United States ; R01 GM138834/GM/NIGMS NIH HHS/United States ; F31 CA260794/CA/NCI NIH HHS/United States ; P30 ES010126/ES/NIEHS NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; DP2 HD091800/HD/NICHD NIH HHS/United States ; T32 CA071341/CA/NCI NIH HHS/United States ; },
mesh = {Aminoquinolines/toxicity ; Animals ; Breast Neoplasms/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; DNA-Directed DNA Polymerase/*genetics/metabolism ; HEK293 Cells ; Humans ; Mice ; Mitomycin/toxicity ; Picolinic Acids/toxicity ; },
abstract = {Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.},
}
@article {pmid31537693,
year = {2020},
author = {Tan, YT and Ye, L and Xie, F and Wang, J and Müschen, M and Chen, SJ and Kan, YW and Liu, H},
title = {CRISPR/Cas9-mediated gene deletion efficiently retards the progression of Philadelphia-positive acute lymphoblastic leukemia in a p210 BCR-ABL1[T315I] mutation mouse model.},
journal = {Haematologica},
volume = {105},
number = {5},
pages = {e232-e236},
pmid = {31537693},
issn = {1592-8721},
support = {P01 DK088760/DK/NIDDK NIH HHS/United States ; R35 CA197628/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Fusion Proteins, bcr-abl/genetics/metabolism ; Gene Deletion ; Mice ; Mutation ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics ; },
}
@article {pmid31537455,
year = {2019},
author = {Jubair, L and Fallaha, S and McMillan, NAJ},
title = {Systemic Delivery of CRISPR/Cas9 Targeting HPV Oncogenes Is Effective at Eliminating Established Tumors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {12},
pages = {2091-2099},
pmid = {31537455},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics ; *Drug Delivery Systems ; Female ; Gene Editing ; *Genetic Therapy ; Humans ; Liposomes/administration &amp; dosage ; Mice ; Oncogene Proteins, Viral/antagonists &amp; inhibitors/*genetics ; Papillomaviridae/*genetics ; Papillomavirus Infections/*complications/virology ; Repressor Proteins/antagonists &amp; inhibitors/genetics ; Tumor Cells, Cultured ; Uterine Cervical Neoplasms/genetics/*therapy/virology ; },
abstract = {The recent advancements in CRISPR/Cas9 engineering have resulted in the development of more targeted and potentially safer gene therapies. The challenge in the cancer setting is knowing the driver oncogenes responsible, and the translation of these therapies is hindered by effective and safe delivery methods to target organs with minimal systemic toxicities, on-target specificity of gene editing, and demonstrated lack of long-term adverse events. Using a model system based on cervical cancer, which is driven by the ongoing expression of the human papillomavirus E6 and E7 proteins, we show that CRISPR/Cas9 delivered systemically in vivo using PEGylated liposomes results in tumor elimination and complete survival in treated animals. We compared treatment and editing efficiency of two Cas9 variants, wild-type (WT) Cas9 and the highly specific FokI-dCas9, and showed that the latter was not effective. We also explored high-fidelity repair but found that repair was inefficient, occurring in 6%-8% of cells, whereas non-homologous end joining (NHEJ) was highly efficient, occurring in ∼80% of the cells. Finally, we explored the post gene-editing events in tumors and showed that cell death is induced by apoptosis. Overall, our work demonstrates that in vivo CRISPR/Cas editing treatment of preexisting tumors is completely effective despite the large payloads.},
}
@article {pmid31536613,
year = {2019},
author = {Rock, J},
title = {Tuberculosis drug discovery in the CRISPR era.},
journal = {PLoS pathogens},
volume = {15},
number = {9},
pages = {e1007975},
pmid = {31536613},
issn = {1553-7374},
support = {DP2 AI144850/AI/NIAID NIH HHS/United States ; },
mesh = {Antitubercular Agents/therapeutic use ; Bacterial Proteins/genetics ; CRISPR-Cas Systems ; Drug Delivery Systems ; Drug Discovery/*methods ; Genomics/methods ; Mycobacterium tuberculosis/genetics ; Tuberculosis/*drug therapy/microbiology ; },
abstract = {Stewart Cole and colleagues determined the complete genome sequence of Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), in 1998 [1]. This was a landmark achievement that heralded a new age in TB drug discovery. With the genome sequence in hand, drug discoverers suddenly had thousands of new potential targets to explore. But the excitement has since faded [2]. It is unquestioned that genomics has transformed our understanding of the biology of this pathogen. However, the expectation that the Mtb genome sequence would rapidly lead to new therapeutic interventions remains unfulfilled [3]. One of the (many) reasons for this unrealized potential is that our tools to systematically interrogate the Mtb genome and its drug targets-so-called functional genomics-have been limited. In this Pearl, I argue that the recent development of robust CRISPR-based genetics in Mtb [4] overcomes many prior limitations and holds the potential to close the gap between genomics and TB drug discovery.},
}
@article {pmid31536480,
year = {2019},
author = {Bocharnikov, AV and Keegan, J and Wacleche, VS and Cao, Y and Fonseka, CY and Wang, G and Muise, ES and Zhang, KX and Arazi, A and Keras, G and Li, ZJ and Qu, Y and Gurish, MF and Petri, M and Buyon, JP and Putterman, C and Wofsy, D and James, JA and Guthridge, JM and Diamond, B and Anolik, JH and Mackey, MF and Alves, SE and Nigrovic, PA and Costenbader, KH and Brenner, MB and Lederer, JA and Rao, DA and , },
title = {PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.},
journal = {JCI insight},
volume = {4},
number = {20},
pages = {},
pmid = {31536480},
issn = {2379-3708},
support = {K08 AR072791/AR/NIAMS NIH HHS/United States ; UH2 AR067679/AR/NIAMS NIH HHS/United States ; R01 AR065538/AR/NIAMS NIH HHS/United States ; P30 AR070253/AR/NIAMS NIH HHS/United States ; },
mesh = {Adult ; Aged ; B-Lymphocytes/*immunology ; CD11c Antigen/metabolism ; CRISPR-Cas Systems/genetics ; Case-Control Studies ; Cell Communication/drug effects/genetics/immunology ; Cell Culture Techniques ; Cell Separation ; Cells, Cultured ; Coculture Techniques ; Female ; Flow Cytometry ; Gene Knockout Techniques ; Humans ; Interleukins/antagonists &amp; inhibitors/*metabolism ; Lupus Erythematosus, Systemic/blood/*immunology ; Lymphocyte Activation/drug effects/genetics ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor/metabolism ; Proto-Oncogene Proteins c-maf/genetics/*metabolism ; RNA-Seq ; Receptors, CXCR5/metabolism ; T-Lymphocytes, Helper-Inducer/*immunology/metabolism ; },
abstract = {Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.},
}
@article {pmid31535751,
year = {2020},
author = {Kim, TJ and Sung, JH and Shin, JC and Kim, DY},
title = {CRISPR/Cas-mediated Fubp1 silencing disrupts circadian oscillation of Per1 protein via downregulating Syncrip expression.},
journal = {Cell biology international},
volume = {44},
number = {2},
pages = {424-432},
doi = {10.1002/cbin.11242},
pmid = {31535751},
issn = {1095-8355},
mesh = {Animals ; *CRISPR-Cas Systems ; *Circadian Rhythm ; DNA-Binding Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Down-Regulation ; *Gene Expression Regulation ; *Gene Silencing ; Heterogeneous-Nuclear Ribonucleoproteins/*antagonists &amp; inhibitors/genetics/metabolism ; Mice ; NIH 3T3 Cells ; Period Circadian Proteins/genetics/*metabolism ; RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Most living organisms have physiological and behavioral circadian rhythms controlled by molecular clocks. In mammals, several core clock genes show self-perpetuating oscillation profiles of their messenger RNAs (mRNAs) and proteins through an auto-regulatory transcription-translation feedback loop (TTFL). As a critical component in the molecular clock system, Period 1 (Per1) contributes to the maintenance of circadian rhythm duration predominantly in peripheral clocks. Alterations in Per1 expression and oscillating patterns lead to the development of cancers as well as circadian rhythm abnormalities. In this study, we demonstrate that the phasic profile of Per1 protein was clearly disrupted in CRISPR/Cas-mediated Fubp1-deficient cells. Although Fubp1 does not show rhythmic expression, Fubp1 upregulates the mRNA and protein level of Syncrip, the main post-transcriptional regulator of Per1 protein oscillation. In addition to the diverse physiological functions of Fubp1, including cell-cycle regulation and cellular metabolic control, our results suggest new roles for Fubp1 in the molecular clock system.},
}
@article {pmid31535025,
year = {2019},
author = {Oleinik, N and Kim, J and Roth, BM and Selvam, SP and Gooz, M and Johnson, RH and Lemasters, JJ and Ogretmen, B},
title = {Mitochondrial protein import is regulated by p17/PERMIT to mediate lipid metabolism and cellular stress.},
journal = {Science advances},
volume = {5},
number = {9},
pages = {eaax1978},
pmid = {31535025},
issn = {2375-2548},
support = {R01 CA088932/CA/NCI NIH HHS/United States ; R01 CA214461/CA/NCI NIH HHS/United States ; R01 CA173687/CA/NCI NIH HHS/United States ; R01 DE016572/DE/NIDCR NIH HHS/United States ; P01 CA203628/CA/NCI NIH HHS/United States ; C06 RR015455/RR/NCRR NIH HHS/United States ; P30 CA138313/CA/NCI NIH HHS/United States ; P30 GM103339/GM/NIGMS NIH HHS/United States ; S10 OD018113/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Ceramides/metabolism ; Endoplasmic Reticulum/*metabolism/pathology ; Humans ; Lipid Metabolism ; Membrane Proteins/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism/*pathology ; Mitochondrial Membranes/*metabolism ; Mitochondrial Proteins/antagonists &amp; inhibitors/*physiology ; *Mitophagy ; Protein Transport ; Sphingosine N-Acyltransferase/*physiology ; *Stress, Physiological ; },
abstract = {How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT[-/-] mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.},
}
@article {pmid31534221,
year = {2019},
author = {Osei-Owusu, P and Charlton, TM and Kim, HK and Missiakas, D and Schneewind, O},
title = {FPR1 is the plague receptor on host immune cells.},
journal = {Nature},
volume = {574},
number = {7776},
pages = {57-62},
pmid = {31534221},
issn = {1476-4687},
support = {R01 AI042797/AI/NIAID NIH HHS/United States ; U19 AI107792/AI/NIAID NIH HHS/United States ; U54 AI057153/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Antigens, Bacterial/metabolism ; Bacterial Adhesion ; CRISPR-Cas Systems ; Chemotaxis/immunology ; Disease Models, Animal ; Female ; HEK293 Cells ; Humans ; Macrophages/cytology/immunology/*metabolism/microbiology ; Male ; Mice ; Mice, Inbred C57BL ; Neutrophils/cytology/immunology/*metabolism/microbiology ; Plague/immunology/*microbiology/prevention &amp; control ; Polymorphism, Single Nucleotide/genetics ; Pore Forming Cytotoxic Proteins/metabolism ; Receptors, Formyl Peptide/antagonists &amp; inhibitors/deficiency/genetics/*metabolism ; Type III Secretion Systems/drug effects ; U937 Cells ; Yersinia pestis/chemistry/immunology/*metabolism/pathogenicity ; },
abstract = {The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1[R190W] as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms.},
}
@article {pmid31534175,
year = {2019},
author = {Bernkopf, DB and Brückner, M and Hadjihannas, MV and Behrens, J},
title = {An aggregon in conductin/axin2 regulates Wnt/β-catenin signaling and holds potential for cancer therapy.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4251},
pmid = {31534175},
issn = {2041-1723},
mesh = {Axin Protein/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms/genetics/*pathology ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Wnt Proteins/metabolism ; Wnt Signaling Pathway/*genetics/physiology ; beta Catenin/*metabolism ; },
abstract = {The paralogous scaffold proteins axin and conductin/axin2 are key factors in the negative regulation of the Wnt pathway transcription factor β-catenin, thereby representing interesting targets for signaling regulation. Polymerization of axin proteins is essential for their activity in suppressing Wnt/β-catenin signaling. Notably, conductin shows less polymerization and lower activity than axin. By domain swapping between axin and conductin we here identify an aggregation site in the conductin RGS domain which prevents conductin polymerization. Induction of conductin polymerization by point mutations of this aggregon results in enhanced inhibition of Wnt/β-catenin signaling. Importantly, we identify a short peptide which induces conductin polymerization via masking the aggregon, thereby enhancing β-catenin degradation, inhibiting β-catenin-dependent transcription and repressing growth of colorectal cancer cells. Our study reveals a mechanism for regulating signaling pathways via the polymerization status of scaffold proteins and suggests a strategy for targeted colorectal cancer therapy.},
}
@article {pmid31534142,
year = {2019},
author = {Tarjan, DR and Flavahan, WA and Bernstein, BE},
title = {Epigenome editing strategies for the functional annotation of CTCF insulators.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4258},
pmid = {31534142},
issn = {2041-1723},
support = {DP1 CA216873/CA/NCI NIH HHS/United States ; },
mesh = {Brain Neoplasms/genetics/pathology ; CCCTC-Binding Factor/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics/pathology ; Cell Line ; DNA (Cytosine-5-)-Methyltransferases/*genetics ; DNA Methylation ; DNA Methyltransferase 3A ; Epigenesis, Genetic/genetics ; Gene Editing/*methods ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Promoter Regions, Genetic/genetics ; Recombinant Fusion Proteins/*genetics ; Repressor Proteins/metabolism ; },
abstract = {The human genome is folded into regulatory units termed 'topologically-associated domains' (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.},
}
@article {pmid31533522,
year = {2020},
author = {Wang, J and Zhang, X and Cheng, L and Luo, Y},
title = {An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools.},
journal = {RNA biology},
volume = {17},
number = {1},
pages = {13-22},
pmid = {31533522},
issn = {1555-8584},
mesh = {Algorithms ; *CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing ; Gene Targeting ; Humans ; Machine Learning ; *RNA, Guide ; Sensitivity and Specificity ; },
abstract = {The CRISPR-Cas9 system has become the most promising and versatile tool for genetic manipulation applications. Albeit the technology has been broadly adopted by both academic and pharmaceutic societies, the activity (on-target) and specificity (off-target) of CRISPR-Cas9 are decisive factors for any application of the technology. Several in silico gRNA activity and specificity predicting models and web tools have been developed, making it much more convenient and precise for conducting CRISPR gene editing studies. In this review, we present an overview and comparative analysis of machine and deep learning (MDL)-based algorithms, which are believed to be the most effective and reliable methods for the prediction of CRISPR gRNA on- and off-target activities. As an increasing number of sequence features and characteristics are discovered and are incorporated into the MDL models, the prediction outcome is getting closer to experimental observations. We also introduced the basic principle of CRISPR activity and specificity and summarized the challenges they faced, aiming to facilitate the CRISPR communities to develop more accurate models for applying.},
}
@article {pmid31533452,
year = {2019},
author = {Lambert, JP and Luongo, TS and Tomar, D and Jadiya, P and Gao, E and Zhang, X and Lucchese, AM and Kolmetzky, DW and Shah, NS and Elrod, JW},
title = {MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Stress.},
journal = {Circulation},
volume = {140},
number = {21},
pages = {1720-1733},
pmid = {31533452},
issn = {1524-4539},
support = {P01 HL147841/HL/NHLBI NIH HHS/United States ; P01 HL134608/HL/NHLBI NIH HHS/United States ; R01 HL142271/HL/NHLBI NIH HHS/United States ; R01 HL123966/HL/NHLBI NIH HHS/United States ; R01 HL136954/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Channels/deficiency/genetics/*metabolism ; *Calcium Signaling ; Calcium-Binding Proteins/genetics/metabolism ; Cation Transport Proteins/genetics/metabolism ; Disease Models, Animal ; Energy Metabolism ; Female ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Male ; Membrane Proteins/deficiency/genetics/*metabolism ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria, Heart/*metabolism/pathology ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Mitochondrial Proteins/deficiency/genetics/*metabolism ; Myocardial Contraction ; Myocardial Reperfusion Injury/genetics/*metabolism/pathology/physiopathology ; Myocytes, Cardiac/*metabolism/pathology ; Ventricular Function, Left ; },
abstract = {BACKGROUND: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca[2+] (mCa[2+]) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology.<br><br>METHODS: We created a stable MCUB knockout cell line (MCUB[-/-]) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca[2+] exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex.<br><br>RESULTS: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing mCa[2+] uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa[2+] overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa[2+] uptake impair mitochondrial energetics and contractile function.<br><br>CONCLUSIONS: We detail a new regulatory mechanism to modulate mtCU function and mCa[2+] uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa[2+] uptake and cellular physiology.},
}
@article {pmid31533038,
year = {2019},
author = {Songailiene, I and Rutkauskas, M and Sinkunas, T and Manakova, E and Wittig, S and Schmidt, C and Siksnys, V and Seidel, R},
title = {Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length.},
journal = {Cell reports},
volume = {28},
number = {12},
pages = {3157-3166.e4},
pmid = {31533038},
issn = {2211-1247},
mesh = {CRISPR-Associated Proteins/*chemistry/genetics ; *CRISPR-Cas Systems ; Escherichia coli/*chemistry/genetics ; Escherichia coli Proteins/*chemistry/genetics ; Protein Structure, Secondary ; RNA, Bacterial/*chemistry/genetics ; },
abstract = {The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems.},
}
@article {pmid31532637,
year = {2019},
author = {Li, L and Li, S and Wu, N and Wu, J and Wang, G and Zhao, G and Wang, J},
title = {HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation.},
journal = {ACS synthetic biology},
volume = {8},
number = {10},
pages = {2228-2237},
doi = {10.1021/acssynbio.9b00209},
pmid = {31532637},
issn = {2161-5063},
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA Methylation/*genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; Escherichia coli Proteins/genetics ; Humans ; Nucleic Acid Amplification Techniques/*methods ; Nucleic Acids/*genetics ; Pathology, Molecular/methods ; Polymorphism, Single Nucleotide/genetics ; RNA/genetics ; RNA, Guide/genetics ; },
abstract = {The next-generation CRISPR-based molecular diagnostics has the merits of rapidness, accuracy, and portability. We discovered the Cas12a trans-cleavage activity against collateral single-stranded DNA (ssDNA) and employed the activity to develop a rapid nucleic acid detection system, namely HOLMES (one-hour low-cost multipurpose highly efficient system). Here, with the employment of thermophilic CRISPR-Cas12b, we create HOLMESv2 for four different applications: (1) specifically discriminating single nucleotide polymorphism (SNP); (2) simply detecting virus RNA, human cell mRNA and circular RNA; (3) conveniently quantitating target nucleic acids with a one-step system combined with LAMP amplification in a constant temperature, thus avoiding cross-contamination; (4) accurately quantitating target DNA methylation degree with the combination of Cas12b detection and bisulfite treatment. These results highlight the potential of HOLMESv2 as a promising platform for both molecular diagnostics and epigenetics applications.},
}
@article {pmid31532633,
year = {2019},
author = {Lin, Y and Zou, X and Zheng, Y and Cai, Y and Dai, J},
title = {Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy.},
journal = {ACS synthetic biology},
volume = {8},
number = {10},
pages = {2203-2211},
doi = {10.1021/acssynbio.8b00505},
pmid = {31532633},
issn = {2161-5063},
support = {BB/P02114X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromosomes, Fungal/*genetics ; Genome, Fungal/genetics ; Genomics/methods ; Phenotype ; Saccharomyces cerevisiae/*genetics ; Synthetic Biology/methods ; },
abstract = {Recent advances in DNA synthesis technology have made it possible to rewrite the entire genome of an organism. The major hurdles in this process are efficiently identifying and fixing the defect-inducing sequences (or "bugs") during rewriting. Here, we describe a high-throughput, semiquantitative phenotype assay for evaluating the fitness of synthetic yeast and identifying potential bugs. Growth curves were measured under a carefully chosen set of testing conditions. Statistical analysis revealed strains with subtle defects relative to the wild type, which were targeted for debugging. The effectiveness of the assay was demonstrated by phenotypic profiling of all intermediate synthetic strains of the synthetic yeast chromosome XII. Subsequently, the assay was applied during the process of constructing another synthetic chromosome. Furthermore, we designed an efficient chromosome assembly strategy that integrates iterative megachunk construction with CRISPR/Cas9-mediated assembly of synthetic segments. Together, the semiquantitative assay and refined assembly strategy could greatly facilitate synthetic genomics projects by improving efficiency during both debugging and construction.},
}
@article {pmid31532388,
year = {2019},
author = {Jordan, B},
title = {[Enhancement can do harm].},
journal = {Medecine sciences : M/S},
volume = {35},
number = {8-9},
pages = {709-711},
doi = {10.1051/medsci/2019136},
pmid = {31532388},
issn = {1958-5381},
mesh = {Adult ; Aged ; CRISPR-Cas Systems ; China ; Embryo Research/*ethics ; Gene Editing/*ethics ; Gene Silencing/physiology ; Genetic Enhancement/*ethics ; Genome-Wide Association Study ; HIV Infections/genetics/prevention &amp; control ; HIV-1 ; Humans ; Longevity/*genetics ; Middle Aged ; Receptors, CCR5/genetics ; },
abstract = {Inactivation of the CCR5 gene by CRISPR editing in human embryos, as recently attempted in China, was touted as a positive change for the babies involved since it was expected to impart resistance to HIV infection. However, it turns out that the absence of CCR5 is not neutral but actually decreases fitness, as shown by survival analysis of population data in the UK biobank. This underlines the pitfalls of genetic enhancement, and emphasizes that any germline modification must be preceded by in-depth studies to exclude unforeseen negative effects. ‡.},
}
@article {pmid31531437,
year = {2019},
author = {Ding, F and Huang, X and Gao, X and Xie, M and Pan, G and Li, Q and Song, J and Zhu, X and Zhang, C},
title = {A non-cationic nucleic acid nanogel for the delivery of the CRISPR/Cas9 gene editing tool.},
journal = {Nanoscale},
volume = {11},
number = {37},
pages = {17211-17215},
doi = {10.1039/c9nr05233j},
pmid = {31531437},
issn = {2040-3372},
mesh = {*CRISPR-Cas Systems ; DNA/*chemistry ; *Gene Editing ; *Gene Transfer Techniques ; HeLa Cells ; Humans ; Nanogels/*chemistry ; Polyesters/chemistry/pharmacology ; },
abstract = {Herein, we report a non-cationic DNA-crosslinked nanogel for intracellular delivery of a Cas9 and single guide RNA (Cas9/sgRNA) complex. A DNA-grafted polycaprolactone brush (DNA-g-PCL) is first loaded with the Cas9/sgRNA complex and then crosslinked by DNA linkers via nucleic acid hybridization to form a nanosized hydrogel, in which the gene editing tools are embedded and protected inside. With compact architecture, the Cas9/sgRNA complex-containing nanogel exhibited excellent physiological stability against nuclease digestion and enhanced cellular uptake efficiency, making the delivery system a promising tool for target genome editing.},
}
@article {pmid31531340,
year = {2019},
author = {Sanches-da-Silva, GN and Medeiros, LFS and Lima, FM},
title = {The Potential Use of the CRISPR-Cas System for HIV-1 Gene Therapy.},
journal = {International journal of genomics},
volume = {2019},
number = {},
pages = {8458263},
pmid = {31531340},
issn = {2314-4378},
abstract = {The HIV-1 virus (human immunodeficiency virus) affects 36.9 million people worldwide, with approximately 900000 deaths in 2017. The virus carrier can develop severe immunodeficiency since CD4[+] T lymphocytes are the main target, leading to acquired immunodeficiency syndrome (AIDS). Despite advances in pharmacological treatment, it is still difficult to eliminate latent reservoirs, becoming one of the main obstacles for viral eradication. The CRISPR- (clustered regularly interspaced short palindromic repeat-) Cas system is a genome-editing method which uses a guide RNA, a complementary sequence to the interested site, recruiting a nuclease that can break the viral or the host cell genetic material. From this double-stranded break, cellular repair mechanisms are activated being able to generate deletions, insertions, or substitutions, in order to inactivate specific gene loci, leading to loss of function. The objective of this minireview is to synthesize the current knowledge on the application of CRISPR-Cas-based gene therapy for HIV-1. The strategies encompass all steps of the viral infection cycle, from inhibition of cell invasion, through viral replication and integration inhibition, to excision of the latent provirus. Off-target effects and ethical implications were also discussed to evaluate the safety of the approach and viability of its application in humans, respectively. Although preclinical and clinical tests are still needed, the recent results establish an exciting possibility of applying this technology for prophylaxis and treatment of HIV-1.},
}
@article {pmid31530632,
year = {2019},
author = {Lorente, E and Redondo-Antón, J and Martín-Esteban, A and Guasp, P and Barnea, E and Lauzurica, P and Admon, A and López de Castro, JA},
title = {Substantial Influence of ERAP2 on the HLA-B*40:02 Peptidome: Implications for HLA-B*27-Negative Ankylosing Spondylitis.},
journal = {Molecular &amp; cellular proteomics : MCP},
volume = {18},
number = {11},
pages = {2298-2309},
pmid = {31530632},
issn = {1535-9484},
mesh = {Aminopeptidases/antagonists &amp; inhibitors/genetics/*metabolism ; CRISPR-Cas Systems ; HLA-B Antigens/*metabolism ; HLA-B27 Antigen/*metabolism ; Humans ; Peptide Fragments/*metabolism ; Protein Binding ; Proteome/*analysis ; Spondylitis, Ankylosing/metabolism/*pathology ; },
abstract = {HLA-B*40:02 is one of a few major histocompatibility complex class I (MHC-I) molecules associated with ankylosing spondylitis (AS) independently of HLA-B*27. The endoplasmic reticulum aminopeptidase 2 (ERAP2), an enzyme that process MHC-I ligands and preferentially trims N-terminal basic residues, is also a risk factor for this disease. Like HLA-B*27 and other AS-associated MHC-I molecules, HLA-B*40:02 binds a relatively high percentage of peptides with ERAP2-susceptible residues. In this study, the effects of ERAP2 depletion on the HLA-B*40:02 peptidome were analyzed. ERAP2 protein expression was knocked out by CRISPR in the transfectant cell line C1R-B*40:02, and the differences between the peptidomes from the wild-type and ERAP2-KO cells were determined by label-free quantitative comparisons. The qualitative changes dependent on ERAP2 affected about 5% of the peptidome, but quantitative changes in peptide amounts were much more substantial, reflecting a significant influence of this enzyme on the generation/destruction balance of HLA-B*40:02 ligands. As in HLA-B*27, a major effect was on the frequencies of N-terminal residues. In this position, basic and small residues were increased, and aliphatic/aromatic ones decreased in the ERAP2 knockout. Other peptide positions were also affected. Because most of the non-B*27 MHC-I molecules associated with AS risk bind a relatively high percentage of peptides with N-terminal basic residues, we hypothesize that the non-epistatic association of ERAP2 with AS might be related to the processing of peptides with these residues, thus affecting the peptidomes of AS-associated MHC-I molecules.},
}
@article {pmid31529512,
year = {2020},
author = {Sommer, F and Torraca, V and Kamel, SM and Lombardi, A and Meijer, AH},
title = {Frontline Science: Antagonism between regular and atypical Cxcr3 receptors regulates macrophage migration during infection and injury in zebrafish.},
journal = {Journal of leukocyte biology},
volume = {107},
number = {2},
pages = {185-203},
pmid = {31529512},
issn = {1938-3673},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Movement ; Macrophages/cytology/microbiology/*physiology ; Mutation ; Mycobacterium Infections, Nontuberculous/metabolism/*microbiology/pathology ; Mycobacterium marinum/*physiology ; Protein Conformation ; Receptors, CXCR3/antagonists &amp; inhibitors/classification/genetics/*metabolism ; Zebrafish/microbiology/*physiology ; Zebrafish Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {The CXCR3-CXCL11 chemokine-signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging function, while being unable to signal in the classic G-protein-dependent manner. Two copies of the CXCR3 gene in zebrafish, cxcr3.2 and cxcr3.3, are expressed on macrophages and share a highly conserved ligand-binding site. However, Cxcr3.3 has structural characteristics of ACKRs indicative of a ligand-scavenging role. In contrast, we previously showed that Cxcr3.2 is an active CXCR3 receptor because it is required for macrophage motility and recruitment to sites of mycobacterial infection. In this study, we generated a cxcr3.3 CRISPR-mutant to functionally dissect the antagonistic interplay among the cxcr3 paralogs in the immune response. We observed that cxcr3.3 mutants are more susceptible to mycobacterial infection, whereas cxcr3.2 mutants are more resistant. Furthermore, macrophages in the cxcr3.3 mutant are more motile, show higher activation status, and are recruited more efficiently to sites of infection or injury. Our results suggest that Cxcr3.3 is an ACKR that regulates the activity of Cxcr3.2 by scavenging common ligands and that silencing the scavenging function of Cxcr3.3 results in an exacerbated Cxcr3.2 signaling. In human, splice variants of CXCR3 have antagonistic functions and CXCR3 ligands also interact with ACKRs. Therefore, in zebrafish, an analogous regulatory mechanism appears to have evolved after the cxcr3 gene duplication event, through diversification of conventional and atypical receptor variants.},
}
@article {pmid31527793,
year = {2019},
author = {Diep, J and Ooi, YS and Wilkinson, AW and Peters, CE and Foy, E and Johnson, JR and Zengel, J and Ding, S and Weng, KF and Laufman, O and Jang, G and Xu, J and Young, T and Verschueren, E and Kobluk, KJ and Elias, JE and Sarnow, P and Greenberg, HB and Hüttenhain, R and Nagamine, CM and Andino, R and Krogan, NJ and Gozani, O and Carette, JE},
title = {Enterovirus pathogenesis requires the host methyltransferase SETD3.},
journal = {Nature microbiology},
volume = {4},
number = {12},
pages = {2523-2537},
pmid = {31527793},
issn = {2058-5276},
support = {DP2 AI104557/AI/NIAID NIH HHS/United States ; K99 AI135031/AI/NIAID NIH HHS/United States ; R01 AI021362/AI/NIAID NIH HHS/United States ; P50 GM081879/GM/NIGMS NIH HHS/United States ; R01 GM079641/GM/NIGMS NIH HHS/United States ; R01 AI040085/AI/NIAID NIH HHS/United States ; R01 AI140186/AI/NIAID NIH HHS/United States ; R56 AI021362/AI/NIAID NIH HHS/United States ; P50 AI150476/AI/NIAID NIH HHS/United States ; P01 AI091575/AI/NIAID NIH HHS/United States ; R01 AI141970/AI/NIAID NIH HHS/United States ; U19 AI109662/AI/NIAID NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; R01 AI130123/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Central Nervous System Viral Diseases/virology ; Disease Models, Animal ; Encephalitis, Viral ; Enterovirus/genetics/*metabolism/*pathogenicity ; Enterovirus Infections/virology ; Histone Methyltransferases/genetics/*metabolism ; Methyltransferases/*metabolism ; Mice ; Myelitis/virology ; Neuromuscular Diseases/virology ; Proteolysis ; Viral Proteins ; Virus Replication ; },
abstract = {Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.},
}
@article {pmid31527759,
year = {2019},
author = {Whinn, KS and Kaur, G and Lewis, JS and Schauer, GD and Mueller, SH and Jergic, S and Maynard, H and Gan, ZY and Naganbabu, M and Bruchez, MP and O'Donnell, ME and Dixon, NE and van Oijen, AM and Ghodke, H},
title = {Nuclease dead Cas9 is a programmable roadblock for DNA replication.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {13292},
pmid = {31527759},
issn = {2045-2322},
support = {R01 GM114075/GM/NIGMS NIH HHS/United States ; K99 GM126143/GM/NIGMS NIH HHS/United States ; R01 GM115809/GM/NIGMS NIH HHS/United States ; R01 EB017268/EB/NIBIB NIH HHS/United States ; T32 CA009673/CA/NCI NIH HHS/United States ; R00 GM126143/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; DNA Replication/*genetics ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics ; RNA, Guide/*genetics ; Streptococcus pyogenes/enzymology ; },
abstract = {Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein-DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9-guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.},
}
@article {pmid31527314,
year = {2019},
author = {Rovira Gonzalez, YI and Moyer, AL and LeTexier, NJ and Bratti, AD and Feng, S and Sun, C and Liu, T and Mula, J and Jha, P and Iyer, SR and Lovering, RM and O'Rourke, B and Noh, HL and Suk, S and Kim, JK and Essien Umanah, GK and Wagner, KR},
title = {Mss51 deletion enhances muscle metabolism and glucose homeostasis in mice.},
journal = {JCI insight},
volume = {4},
number = {20},
pages = {},
pmid = {31527314},
issn = {2379-3708},
support = {R01 HL134821/HL/NHLBI NIH HHS/United States ; T32 AR007592/AR/NIAMS NIH HHS/United States ; U24 DK093000/DK/NIDDK NIH HHS/United States ; R01 HL137259/HL/NHLBI NIH HHS/United States ; U2C DK093000/DK/NIDDK NIH HHS/United States ; T32 GM008752/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Diabetes Mellitus, Type 2/etiology/genetics/*metabolism ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Fatty Acids/metabolism ; Female ; Glucose/*metabolism ; Humans ; Insulin ; Insulin Resistance/genetics ; Male ; Mice ; Mice, Knockout ; Mitochondria/metabolism ; Mitochondrial Proteins/*deficiency/genetics ; Muscle Fibers, Skeletal/cytology/*metabolism ; Obesity/etiology/genetics/*metabolism ; Oxidation-Reduction ; Oxidative Phosphorylation ; Oxygen Consumption ; Transcription Factors/*deficiency/genetics ; Weight Gain ; Zinc Fingers ; },
abstract = {Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, β-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid β-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid β-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.},
}
@article {pmid31527312,
year = {2019},
author = {Merola, J and Reschke, M and Pierce, RW and Qin, L and Spindler, S and Baltazar, T and Manes, TD and Lopez-Giraldez, F and Li, G and Bracaglia, LG and Xie, C and Kirkiles-Smith, N and Saltzman, WM and Tietjen, GT and Tellides, G and Pober, JS},
title = {Progenitor-derived human endothelial cells evade alloimmunity by CRISPR/Cas9-mediated complete ablation of MHC expression.},
journal = {JCI insight},
volume = {4},
number = {20},
pages = {},
pmid = {31527312},
issn = {2379-3708},
support = {UL1 TR001863/TR/NCATS NIH HHS/United States ; UL1 TR000142/TR/NCATS NIH HHS/United States ; F30 AI138473/AI/NIAID NIH HHS/United States ; K12 HD047349/HD/NICHD NIH HHS/United States ; K08 HL136898/HL/NHLBI NIH HHS/United States ; S10 OD018521/OD/NIH HHS/United States ; T32 DK007276/DK/NIDDK NIH HHS/United States ; R01 HL085416/HL/NHLBI NIH HHS/United States ; },
mesh = {Allografts/blood supply/*immunology/supply &amp; distribution ; Animals ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cells, Cultured ; Disease Models, Animal ; Endothelial Cells/*immunology/metabolism ; Endothelial Progenitor Cells ; Female ; Fetal Blood/cytology ; Gene Knockout Techniques ; Graft Rejection/blood/immunology/*prevention &amp; control ; Healthy Volunteers ; Humans ; Isoantibodies/immunology ; Killer Cells, Natural/immunology ; Lymphocyte Activation/genetics ; Mice ; Microvessels/cytology/immunology/transplantation ; Nuclear Proteins/*genetics/immunology ; Organ Transplantation/adverse effects/methods ; Primary Cell Culture ; Tissue Engineering/*methods ; Trans-Activators/*genetics/immunology ; beta 2-Microglobulin/*genetics/immunology ; },
abstract = {Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of β2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.},
}
@article {pmid31527264,
year = {2019},
author = {Binger, KJ and Neukam, M and Tattikota, SG and Qadri, F and Puchkov, D and Willmes, DM and Wurmsee, S and Geisberger, S and Dechend, R and Raile, K and Kurth, T and Nguyen, G and Poy, MN and Solimena, M and Muller, DN and Birkenfeld, AL},
title = {Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic β cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {40},
pages = {19983-19988},
pmid = {31527264},
issn = {1091-6490},
mesh = {Animals ; Autophagy ; CRISPR-Cas Systems ; Cytosol/metabolism ; Female ; *Gene Deletion ; Gene Silencing ; Insulin/*metabolism ; Insulin-Secreting Cells/*metabolism ; Insulinoma/metabolism ; Lysosomes/metabolism ; Male ; Mice ; Phenotype ; Promoter Regions, Genetic ; Proton-Translocating ATPases/*genetics ; RNA, Small Interfering/metabolism ; Rats ; Receptors, Cell Surface/*genetics/metabolism ; Receptors, Estrogen/metabolism ; Vacuolar Proton-Translocating ATPases/metabolism ; Vacuoles/metabolism ; },
abstract = {Pancreatic β cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair β cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse β cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining β cell function and diabetes prevention.},
}
@article {pmid31527030,
year = {2019},
author = {Rodrigues, M and McBride, SW and Hullahalli, K and Palmer, KL and Duerkop, BA},
title = {Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.},
journal = {Antimicrobial agents and chemotherapy},
volume = {63},
number = {11},
pages = {},
pmid = {31527030},
issn = {1098-6596},
support = {K01 DK102436/DK/NIDDK NIH HHS/United States ; R01 AI116610/AI/NIAID NIH HHS/United States ; R01 AI141479/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Anti-Bacterial Agents/*pharmacology ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Drug Resistance, Multiple, Bacterial/*genetics ; Enterococcaceae/drug effects/*genetics ; Enterococcus faecalis/drug effects/genetics ; Gene Editing/*methods ; Genes, Bacterial/genetics ; Gram-Positive Bacterial Infections/drug therapy/microbiology ; Mice ; },
abstract = {The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.},
}
@article {pmid31526944,
year = {2019},
author = {Han, HJ and Han, HW and Seo, HH and Kim, JH},
title = {Generation of a KSCBi005-A-5(TLR8KO-A10) homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {40},
number = {},
pages = {101561},
doi = {10.1016/j.scr.2019.101561},
pmid = {31526944},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems ; Cell Line/cytology/*metabolism ; Cellular Reprogramming ; Gene Editing ; Gene Knockout Techniques ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Male ; Middle Aged ; Toll-Like Receptor 8/*genetics/metabolism ; },
abstract = {The Toll like Receptor (TLR) family plays an essential role in pathogen recognition and innate immunity activation. TLR8, an endosomal receptor, can recognize single-stranded RNA viruses, such as influenza virus, Sendai virus, Coxsackie B virus, HIV, and HCV. TLR8 binding to the viral RNA recruits MyD88 and leads to activation of the transcription factor NF-kB and antiviral response. We generated biallelic mutants of the TLR8 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). The TLR8 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.},
}
@article {pmid31526854,
year = {2019},
author = {Ley, D and Pereira, S and Pedersen, LE and Arnsdorf, J and Hefzi, H and Davy, AM and Ha, TK and Wulff, T and Kildegaard, HF and Andersen, MR},
title = {Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion.},
journal = {Metabolic engineering},
volume = {56},
number = {},
pages = {120-129},
doi = {10.1016/j.ymben.2019.09.005},
pmid = {31526854},
issn = {1096-7184},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; *Cellular Reprogramming Techniques ; Cricetulus ; *Gene Editing ; Metabolic Networks and Pathways/*genetics ; },
abstract = {Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells. We aimed to reduce secretion of growth inhibiting metabolic by-products derived from amino acid catabolism including lactate and ammonium. To achieve this, we engineered nine genes in seven different amino acid catabolic pathways using the CRISPR-Cas9 genome editing system. For identification of target genes, we used a metabolic network reconstruction of amino acid catabolism to follow transcriptional changes in response to antibody production, which revealed candidate genes for disruption. We found that disruption of single amino acid catabolic genes reduced specific lactate and ammonium secretion while specific growth rate and integral of viable cell density were increased in many cases. Of particular interest were Hpd and Gad2 disruptions, which show unchanged AA uptake rates, while having growth rates increased up to 19%, and integral of viable cell density as much as 50% higher, and up to 26% decrease in specific ammonium production and to a lesser extent (up to 22%) decrease in lactate production. This study demonstrates the broad potential of engineering amino acid catabolism in CHO cells to achieve improved phenotypes for bioprocessing.},
}
@article {pmid31526571,
year = {2020},
author = {Molla, KA and Yang, Y},
title = {Predicting CRISPR/Cas9-Induced Mutations for Precise Genome Editing.},
journal = {Trends in biotechnology},
volume = {38},
number = {2},
pages = {136-141},
doi = {10.1016/j.tibtech.2019.08.002},
pmid = {31526571},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA Repair/genetics ; Gene Editing/*methods ; *Machine Learning ; Models, Genetic ; *Mutation ; },
abstract = {SpCas9 creates blunt end cuts in the genome and generates random and unpredictable mutations through error-prone repair systems. However, a growing body of recent evidence points instead to Cas9-induced staggered end generation, nonrandomness of mutations, and the predictability of editing outcomes using machine learning models.},
}
@article {pmid31526035,
year = {2019},
author = {Lake, F},
title = {Putting the spotlight on: CRISPR.},
journal = {BioTechniques},
volume = {67},
number = {4},
pages = {150-152},
doi = {10.2144/btn-2019-0099},
pmid = {31526035},
issn = {1940-9818},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila melanogaster/genetics ; Gene Editing/*methods ; Humans ; Protein Engineering ; Receptors, CCR5/genetics ; Research Personnel ; Surveys and Questionnaires ; },
abstract = {Rounding up the results from our recent spotlight on CRISPR - are we still just at the beginning for our CRISPR journey?},
}
@article {pmid31526005,
year = {2019},
author = {Zhao, R and Liu, Y and Zhang, H and Chai, C and Wang, J and Jiang, W and Gu, Y},
title = {CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation.},
journal = {ACS synthetic biology},
volume = {8},
number = {10},
pages = {2270-2279},
doi = {10.1021/acssynbio.9b00033},
pmid = {31526005},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Carbon Cycle/*genetics ; Clostridium/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Fermentation/*genetics ; Gene Deletion ; Gene Editing/methods ; Genome, Bacterial/genetics ; Metabolic Engineering/methods ; },
abstract = {Uncovering the full potential of gas-fermenting Clostridia, attractive autotrophic bacteria capable of using synthesis gases (CO-CO2-H2) to produce a range of chemicals and fuels, for industrial applications relies on having efficient molecular tools for genetic modifications. Although the CRISPR-Cas9-mediated genome editing system has been developed in Clostridia, its use is limited owing to low GC content (approx. 30%) in these anaerobes. Therefore, the effector protein Cas12a, which recognizes T-rich instead of G-rich protospacer-adjacent motifs (PAMs), has evident advantages over Cas9 in CRISPR genome editing in Clostridia. Here, we developed the CRISPR-Cas12a system for efficient gene deletion and regulation in the gas-fermenting Clostridium ljungdahlii species. On the basis of screening for the most suitable Cas12a and significantly improved electrotransformation efficiency that bypassed poor repair efficiency of the Cas12a-caused DNA double-strand break (DSB) in C. ljungdahlii, efficient deletion (80-100%) of four genes (pyrE, pta, adhE1, and ctf) was achieved by using the CRISPR-FnCas12a system. Furthermore, a DNase-deactivated FnCas12a (ddCas12a) was adopted to construct a CRISPRi system to downregulate targeted genes, reaching over 80% repression for most of the chosen binding sites. This CRISPRi system was also used in a butyric acid-producing C. ljungdahlii strain to redirect carbon flux, leading to 20-40% reductions in ethanol titer that were accompanied by increased butyric acid titer. These results demonstrate the high efficiency of the CRISPR-FnCas12a system for genome engineering in C. ljungdahlii, which effectively expands the existing CRISPR-Cas toolbox in gas-fermenting Clostridium species and may play important roles in genetic manipulations where CRISPR-Cas9 is incompetent.},
}
@article {pmid31525995,
year = {2019},
author = {Cui, Y and Dong, H and Ma, Y and Zhang, D},
title = {Strategies for Applying Nonhomologous End Joining-Mediated Genome Editing in Prokaryotes.},
journal = {ACS synthetic biology},
volume = {8},
number = {10},
pages = {2194-2202},
doi = {10.1021/acssynbio.9b00179},
pmid = {31525995},
issn = {2161-5063},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Damage/*genetics ; DNA End-Joining Repair/*genetics ; Gene Editing/methods ; Genome/*genetics ; Humans ; Prokaryotic Cells/*physiology ; },
abstract = {The emergence of genome editing technology based on the CRISPR/Cas system enabled revolutionary progress in genetic engineering. Double-strand breaks (DSBs), which can be induced by the CRISPR/Cas9 system, cause serious DNA damage that can be repaired by a homologous recombination (HR) system or the nonhomologous end joining (NHEJ) pathway. However, many bacterial species have a very weak HR system. Thus, the NHEJ pathway can be used in prokaryotes. Starting with a brief introduction of the mechanism of the NHEJ pathway, this review focuses on current research and details of applications of NHEJ in eukaryotes, which forms the theoretical basis for the application of the NHEJ system in prokaryotes.},
}
@article {pmid31524877,
year = {2019},
author = {Cheng, CK and Wong, THY and Yung, YL and Chan, NCN and Ng, MHL},
title = {Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {151},
pages = {},
doi = {10.3791/60130},
pmid = {31524877},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Core Binding Factor Alpha 2 Subunit/genetics/*metabolism ; Gene Editing/methods ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Leukemia, Myeloid, Acute/*metabolism ; Mice ; RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; },
abstract = {The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.},
}
@article {pmid31523835,
year = {2019},
author = {Sun, J and Nagel, R and Zaal, EA and Ugalde, AP and Han, R and Proost, N and Song, JY and Pataskar, A and Burylo, A and Fu, H and Poelarends, GJ and van de Ven, M and van Tellingen, O and Berkers, CR and Agami, R},
title = {SLC1A3 contributes to L-asparaginase resistance in solid tumors.},
journal = {The EMBO journal},
volume = {38},
number = {21},
pages = {e102147},
pmid = {31523835},
issn = {1460-2075},
support = {201503250056//China Scholarship Council/International ; KWF 0315/2016//Dutch Cancer Society/International ; ERC-PoC 665317/ERC_/European Research Council/International ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; Asparaginase/*pharmacology ; CRISPR-Cas Systems ; Cell Proliferation ; Drug Resistance, Neoplasm/*genetics ; Excitatory Amino Acid Transporter 1/antagonists &amp; inhibitors/genetics/*metabolism ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasms/*drug therapy/enzymology/pathology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.},
}
@article {pmid31523770,
year = {2020},
author = {Chen, W and Ji, Q},
title = {Genetic Manipulation of MRSA Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2069},
number = {},
pages = {113-124},
doi = {10.1007/978-1-4939-9849-4_9},
pmid = {31523770},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Methicillin-Resistant Staphylococcus aureus/*genetics ; *Mutagenesis ; Plasmids/*genetics ; *Point Mutation ; },
abstract = {The clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 system has emerged as an efficient genome engineering method attributed to its high efficiency and versatility. By generating a lethal double-strand DNA break in the target genome, the CRISPR/Cas9 system is capable of selecting the separated crossover events occurring in the traditional genetic manipulation methods in one step, therefore enabling rapid and efficient genome editing in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). By engineering the fusion of a cytidine deaminase APOBEC1 and a Cas9 nickase, a base editor was further developed as a highly efficient gene inactivation and point mutation tool in S. aureus. Here we describe a detailed protocol for CRISPR/Cas9-based genome editing in S. aureus, including genome modification and base editing. This protocol outlines in detail the design of primers, the construction and transformation of editing plasmids, as well as the verification of sequence-specific CRISPR/Cas9-mediated mutagenesis in S. aureus.},
}
@article {pmid31523769,
year = {2020},
author = {Austin, CM and Bose, JL},
title = {Genetic Manipulations of Staphylococcal Chromosomal DNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2069},
number = {},
pages = {103-111},
doi = {10.1007/978-1-4939-9849-4_8},
pmid = {31523769},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Bacterial/*genetics ; *Gene Editing ; *Point Mutation ; Staphylococcus aureus/*genetics ; },
abstract = {Performing genetic manipulation is often key to understanding bacterial gene function. In this chapter, we present the method of allelic exchange using temperature-sensitive plasmids to generate mutations in Staphylococcus, including single-nucleotide mutations, insertions, and gene deletions. In addition, this chapter summarizes other key genetic technologies used for the manipulation of S. aureus, including the CRISPR/Cas9 system and complementation.},
}
@article {pmid31523199,
year = {2019},
author = {Wang, H and Shen, L and Chen, J and Liu, X and Tan, T and Hu, Y and Bai, X and Li, Y and Tian, K and Li, N and Hu, X},
title = {Deletion of CD163 Exon 7 Confers Resistance to Highly Pathogenic Porcine Reproductive and Respiratory Viruses on Pigs.},
journal = {International journal of biological sciences},
volume = {15},
number = {9},
pages = {1993-2005},
pmid = {31523199},
issn = {1449-2288},
mesh = {Animals ; Antigens, CD/*genetics ; Antigens, Differentiation, Myelomonocytic/*genetics ; CRISPR-Cas Systems/genetics ; Exons/*genetics ; Nuclear Transfer Techniques ; Porcine Reproductive and Respiratory Syndrome/*genetics/*prevention &amp; control ; Porcine respiratory and reproductive syndrome virus/*pathogenicity ; Receptors, Cell Surface/*genetics ; Swine ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is a severe infectious disease in the swine industry. PRRSV infection is mediated by porcine CD163 (pCD163). Scavenger receptor cysteine-rich domain 5 coded by exon 7 of pCD163 is essential for PRRSV infection. In this study, we generated CD163 exon 7 deleted (CD163E7D) pigs using CRISPR/Cas9 mediated homologous recombination and somatic cell nuclear transfer (SCNT). The deletion of exon 7 had no adverse effects on CD163-associated functions. Pigs were further challenged with a highly pathogenic PRRSV (HP-PRRSV) strain. The CD163E7D pigs exhibited mild clinical symptoms and had decreased viral loads in blood. All CD163E7D pigs survived the viral challenge, while all the WT pigs displayed severe symptoms, and 2 out of 6 WT pigs died during the challenge. Our results demonstrated that CD163 exon 7 deletion confers resistance to HP-PRRSV infection without impairing the biological functions of CD163.},
}
@article {pmid31522960,
year = {2019},
author = {Bukhari, H and Müller, T},
title = {Endogenous Fluorescence Tagging by CRISPR.},
journal = {Trends in cell biology},
volume = {29},
number = {11},
pages = {912-928},
doi = {10.1016/j.tcb.2019.08.004},
pmid = {31522960},
issn = {1879-3088},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Caenorhabditis elegans/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; DNA End-Joining Repair/genetics ; Fluorescence ; Gene Editing ; Gene Knock-In Techniques/*methods ; Human Embryonic Stem Cells/cytology ; Humans ; Leishmania donovani/genetics ; Luminescent Proteins/*genetics ; Recombinational DNA Repair/genetics ; Transcription Activator-Like Effectors/*metabolism ; Zinc Finger Nucleases/*metabolism ; },
abstract = {Fluorescent proteins have revolutionized biomedical research as they are easy to use for protein tagging, cope without fixation or permeabilization, and thus, enable live cell imaging in various models. Current methods allow easy and quick integration of fluorescent markers to endogenous genes of interest. In this review, we introduce the three central methods, zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and CRISPR, that have been widely used to manipulate cells or organisms. Focusing on CRISPR technology, we give an overview on homology-directed repair (HDR)-, microhomology-mediated end joining (MMEJ)-, and nonhomologous end joining (NHEJ)-based strategies for the knock-in of markers, figure out recent developments of the technique for highly efficient knock-in, and demonstrate pros and cons. We highlight the unique aspects of fluorescent protein knock-ins and pinpoint specific improvements and perspectives, like the combination of editing with stem cell derived organoid development.},
}
@article {pmid31522438,
year = {2019},
author = {Hosseini Rouzbahani, N and Kaviani, S and Vasei, M and Soleimani, M and Azadmanesh, K and Nicknam, MH},
title = {Generation of CCR5-ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immune-mediated Diseases.},
journal = {Iranian journal of allergy, asthma, and immunology},
volume = {18},
number = {3},
pages = {310-319},
doi = {10.18502/ijaai.v18i3.1124},
pmid = {31522438},
issn = {1735-5249},
mesh = {Flow Cytometry/methods ; Gene Editing ; Gene Expression Regulation ; Genes, Reporter ; Genetic Loci ; Genetic Vectors/genetics ; Humans ; Immune System Diseases/*etiology/metabolism/*therapy ; Induced Pluripotent Stem Cells/*metabolism ; RNA, Guide ; Receptors, CCR5/*deficiency/genetics/metabolism ; *Stem Cell Transplantation/methods ; },
abstract = {C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the "Loss of a primer binding site" technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The "Loss of a primer binding site" technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.},
}
@article {pmid31520072,
year = {2019},
author = {Shmakov, SA and Faure, G and Makarova, KS and Wolf, YI and Severinov, KV and Koonin, EV},
title = {Systematic prediction of functionally linked genes in bacterial and archaeal genomes.},
journal = {Nature protocols},
volume = {14},
number = {10},
pages = {3013-3031},
pmid = {31520072},
issn = {1750-2799},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; *Genes, Archaeal ; *Genes, Bacterial ; Genome, Archaeal ; Genome, Bacterial ; Genomics/*methods ; Molecular Sequence Annotation/methods ; Open Reading Frames ; Operon ; },
abstract = {Functionally linked genes in bacterial and archaeal genomes are often organized into operons. However, the composition and architecture of operons are highly variable and frequently differ even among closely related genomes. Therefore, to efficiently extract reliable functional predictions for uncharacterized genes from comparative analyses of the rapidly growing genomic databases, dedicated computational approaches are required. We developed a protocol to systematically and automatically identify genes that are likely to be functionally associated with a 'bait' gene or locus by using relevance metrics. Given a set of bait loci and a genomic database defined by the user, this protocol compares the genomic neighborhoods of the baits to identify genes that are likely to be functionally linked to the baits by calculating the abundance of a given gene within and outside the bait neighborhoods and the distance to the bait. We exemplify the performance of the protocol with three test cases, namely, genes linked to CRISPR-Cas systems using the 'CRISPRicity' metric, genes associated with archaeal proviruses and genes linked to Argonaute genes in halobacteria. The protocol can be run by users with basic computational skills. The computational cost depends on the sizes of the genomic dataset and the list of reference loci and can vary from one CPU-hour to hundreds of hours on a supercomputer.},
}
@article {pmid31519773,
year = {2019},
author = {Pan, H and Yu, W and Zhang, M},
title = {Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi's sarcoma-associated herpesvirus ORF52.},
journal = {Bioscience reports},
volume = {39},
number = {10},
pages = {},
pmid = {31519773},
issn = {1573-4935},
mesh = {Animals ; Bacterial Proteins/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; DNA End-Joining Repair/genetics ; DNA Ligase ATP/genetics/metabolism ; Gene Editing/methods ; Gene Expression Regulation, Viral ; Gene Knockdown Techniques ; Herpesvirus 8, Human/*genetics ; Mice ; Models, Genetic ; Recombinational DNA Repair/*genetics ; Ruminococcus/*genetics ; Tubulin/genetics/metabolism ; Viral Proteins/*genetics ; },
abstract = {Precise genome editing with directed base insertion or targeted point mutations can be achieved by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance in clinical disease therapy. However, HDR efficiency, compared with non-homologous end-joining (NHEJ), is inherently low. To enhance HDR, enabling the insertion of precise genetic modifications, we compared two strategies during surrogate reporter assays in mouse N2A cells: the suppression of DNA ligase IV, a key molecule in NHEJ, using the CasRx (Ruminococcus flavefaciens Cas13d) system, and co-expression of Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52 proteins. We found that suppression of DNA ligase IV promotes HDR efficiency by 1.4-fold. When co-expressed with the Cas9 system, ORF52 improved HDR efficiency by up to 2.1-fold. In addition, we used ORF52 co-expression to modify the ACTB and Tubb3 genes of mouse N2A and E14 cells, which further increased HDR efficiency by approximately two- to four-fold. In conclusion, our data suggest that ORF52 co-expression is effective for enhancing CRISPR/Cas9-mediated HDR, which may be useful for future studies involving precise genome editing.},
}
@article {pmid31519756,
year = {2019},
author = {Hoffman, HK and Fernandez, MV and Groves, NS and Freed, EO and van Engelenburg, SB},
title = {Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {44},
pages = {16266-16281},
pmid = {31519756},
issn = {1083-351X},
support = {R01 AI138625/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cytokinesis/physiology ; DNA-Binding Proteins/genetics/*metabolism ; Endosomal Sorting Complexes Required for Transport/genetics/*metabolism ; Genomics/methods ; HIV-1/metabolism ; HeLa Cells ; Humans ; Jurkat Cells ; Protein Transport ; Transcription Factors/genetics/*metabolism ; Virion/metabolism ; Virus Release ; },
abstract = {The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retroviruses, such as HIV-1, to release virions from infected cells. The crucial roles of the ESCRTs in cellular physiology and viral disease make it imperative to understand the membrane scission mechanism. Current methodological limitations, namely artifacts caused by overexpression of ESCRT subunits, obstruct our understanding of the spatiotemporal organization of the endogenous human ESCRT machinery. Here, we used CRISPR/Cas9-mediated knock-in to tag the critical ESCRT-I component tumor susceptibility 101 (Tsg101) with GFP at its native locus in two widely used human cell types, HeLa epithelial cells and Jurkat T cells. We validated this approach by assessing the function of these knock-in cell lines in cytokinesis, receptor degradation, and virus budding. Using this probe, we measured the incorporation of endogenous Tsg101 in released HIV-1 particles, supporting the notion that the ESCRT machinery initiates virus abscission by scaffolding early-acting ESCRT-I within the head of the budding virus. We anticipate that these validated cell lines will be a valuable tool for interrogating dynamics of the native human ESCRT machinery.},
}
@article {pmid31519690,
year = {2019},
author = {Robertson, FL and Marqués-Torrejón, MA and Morrison, GM and Pollard, SM},
title = {Experimental models and tools to tackle glioblastoma.},
journal = {Disease models &amp; mechanisms},
volume = {12},
number = {9},
pages = {},
pmid = {31519690},
issn = {1754-8411},
support = {A19778/CRUK_/Cancer Research UK/United Kingdom ; A21992/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Animals ; Brain Neoplasms/genetics/pathology ; Genetic Engineering ; Glioblastoma/genetics/*pathology ; Humans ; *Models, Theoretical ; Neoplasm Transplantation ; Neoplastic Stem Cells/pathology ; },
abstract = {Glioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite increasing knowledge of the genetic and epigenetic changes that underlie tumour initiation and growth, the prognosis for GBM patients remains dismal. Genome analysis has failed to lead to success in the clinic. Fresh approaches are needed that can stimulate new discoveries across all levels: cell-intrinsic mechanisms (transcriptional/epigenetic and metabolic), cell-cell signalling, niche and microenvironment, systemic signals, immune regulation, and tissue-level physical forces. GBMs are inherently extremely challenging: tumour detection occurs too late, and cells infiltrate widely, hiding in quiescent states behind the blood-brain barrier. The complexity of the brain tissue also provides varied and complex microenvironments that direct cancer cell fates. Phenotypic heterogeneity is therefore superimposed onto pervasive genetic heterogeneity. Despite this bleak outlook, there are reasons for optimism. A myriad of complementary, and increasingly sophisticated, experimental approaches can now be used across the research pipeline, from simple reductionist models devised to delineate molecular and cellular mechanisms, to complex animal models required for preclinical testing of new therapeutic approaches. No single model can cover the breadth of unresolved questions. This Review therefore aims to guide investigators in choosing the right model for their question. We also discuss the recent convergence of two key technologies: human stem cell and cancer stem cell culture, as well as CRISPR/Cas tools for precise genome manipulations. New functional genetic approaches in tailored models will likely fuel new discoveries, new target identification and new therapeutic strategies to tackle GBM.},
}
@article {pmid31519332,
year = {2020},
author = {Dorman, CJ and Ní Bhriain, N},
title = {CRISPR-Cas, DNA Supercoiling, and Nucleoid-Associated Proteins.},
journal = {Trends in microbiology},
volume = {28},
number = {1},
pages = {19-27},
doi = {10.1016/j.tim.2019.08.004},
pmid = {31519332},
issn = {1878-4380},
mesh = {Bacteria/*genetics ; Bacterial Physiological Phenomena ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA ; Gene Expression Regulation, Bacterial ; Gene Transfer, Horizontal ; Immunity ; Integration Host Factors ; Interspersed Repetitive Sequences ; Promoter Regions, Genetic ; Transcription Factors ; Transcriptome ; },
abstract = {In this opinion article we highlight links between the H-NS nucleoid-associated protein, variable DNA topology, the regulation of CRISPR-cas locus expression, CRISPR-Cas activity, and the recruitment of novel genetic information by the CRISPR array. We propose that the requirement that the invading mobile genetic element be negatively supercoiled limits effective CRISPR action to a window in the bacterial growth cycle when DNA topology is optimal, and that this same window is used for the efficient integration of new spacer sequences at the CRISPR array. H-NS silences CRISPR promoters, and we propose that antagonists of H-NS, such as the LeuO transcription factor, provide a basis for a stochastic genetic switch that acts at random in each cell in the bacterial population. In addition, we wish to propose a mechanism by which mobile genetic elements can suppress CRISPR-cas transcription using H-NS homologues. Although the individual components of this network are known, we propose a new model in which they are integrated and linked to the physiological state of the bacterium. The model provides a basis for cell-to-cell variation in the expression and performance of CRISPR systems in bacterial populations.},
}
@article {pmid31516887,
year = {2019},
author = {Bozorg Qomi, S and Asghari, A and Mojarrad, M},
title = {An Overview of the CRISPR-Based Genomic- and Epigenome-Editing System: Function, Applications, and Challenges.},
journal = {Advanced biomedical research},
volume = {8},
number = {},
pages = {49},
pmid = {31516887},
issn = {2277-9175},
abstract = {Developing a new strategy for an efficient targeted genome editing has always been a great perspective in biology. Although different approaches have been suggested in the last three decades, each one is confronting with limitations. CRISPR-Cas complex is a bacterial-derived system which made a breakthrough in the area of genome editing. This paper presents a brief history of CRISPR genome editing and discusses thoroughly how it works in bacteria and mammalians. At the end, some applications and challenges of this growing research area are also reviewed. In addition to moving the boundaries of genetics, CRISPR-Cas can also provide the ground for fundamental advances in other fields of biological sciences.},
}
@article {pmid31515362,
year = {2019},
author = {Kaiser, J},
title = {CRISPR reveals some cancer drugs hit unexpected targets.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6458},
pages = {1065},
doi = {10.1126/science.365.6458.1065},
pmid = {31515362},
issn = {1095-9203},
mesh = {Antineoplastic Agents/*pharmacology ; *CRISPR-Cas Systems ; Cyclin-Dependent Kinases/antagonists &amp; inhibitors ; *Drug Development ; Gene Silencing ; Humans ; Molecular Targeted Therapy ; Neoplasms/*drug therapy ; Quinolones/pharmacology ; },
}
@article {pmid31513841,
year = {2019},
author = {Hsu, MN and Chang, YH and Truong, VA and Lai, PL and Nguyen, TKN and Hu, YC},
title = {CRISPR technologies for stem cell engineering and regenerative medicine.},
journal = {Biotechnology advances},
volume = {37},
number = {8},
pages = {107447},
doi = {10.1016/j.biotechadv.2019.107447},
pmid = {31513841},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Regenerative Medicine ; Stem Cells ; },
abstract = {CRISPR/Cas9 system exploits the concerted action of Cas9 nuclease and programmable single guide RNA (sgRNA), and has been widely used for genome editing. The Cas9 nuclease activity can be abolished by mutation to yield the catalytically deactivated Cas9 (dCas9). Coupling with the customizable sgRNA for targeting, dCas9 can be fused with transcription repressors to inhibit specific gene expression (CRISPR interference, CRISPRi) or fused with transcription activators to activate the expression of gene of interest (CRISPR activation, CRISPRa). Here we introduce the principles and recent advances of these CRISPR technologies, their delivery vectors and review their applications in stem cell engineering and regenerative medicine. In particular, we focus on in vitro stem cell fate manipulation and in vivo applications such as prevention of retinal and muscular degeneration, neural regeneration, bone regeneration, cartilage tissue engineering, as well as treatment of diseases in blood, skin and liver. Finally, the challenges to translate CRISPR to regenerative medicine and future perspectives are discussed and proposed.},
}
@article {pmid31513626,
year = {2019},
author = {Wu, YW and Yang, SH and Hwangbo, M and Chu, KH},
title = {Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system.},
journal = {PloS one},
volume = {14},
number = {9},
pages = {e0222143},
pmid = {31513626},
issn = {1932-6203},
mesh = {Aeromonadaceae/classification/*growth &amp; development/metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Evolution, Molecular ; Gene Expression Regulation, Bacterial ; Glycerol/metabolism ; Hydroxybutyrates/*metabolism ; Multigene Family ; Phylogeny ; Polyesters/*metabolism ; Salinity ; Stress, Physiological ; Up-Regulation ; Whole Genome Sequencing/*methods ; },
abstract = {Polyhydroxybutyrate (PHB) is biodegradable and renewable and thus considered as a promising alternative to petroleum-based plastics. However, PHB production is costly due to expensive carbon sources for culturing PHB-accumulating microorganisms under sterile conditions. We discovered a hyper PHB-accumulating denitrifying bacterium, Zobellella denitrificans ZD1 (referred as strain ZD1 hereafter) capable of using non-sterile crude glycerol (a waste from biodiesel production) and nitrate to produce high PHB yield under saline conditions. Nevertheless, the underlying genetic mechanisms of PHB production in strain ZD1 have not been elucidated. In this study, we discovered a complete pathway of glycerol conversion to PHB, a novel PHB synthesis gene cluster, a salt-tolerant gene cluster, denitrifying genes, and an assimilatory nitrate reduction gene cluster in the ZD1 genome. Interestingly, the novel PHB synthesis gene cluster was found to be conserved among marine Gammaproteobacteria. Higher levels of PHB accumulation were linked to higher expression levels of the PHB synthesis gene cluster in ZD1 grown with glycerol and nitrate under saline conditions. Additionally, a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas type-I-E antiviral system was found in the ZD1 genome along with a long spacer list, in which most of the spacers belong to either double-stranded DNA viruses or unknown phages. The results of the genome analysis revealed strain ZD1 used the novel PHB gene cluster to produce PHB from non-sterile crude glycerol under saline conditions.},
}
@article {pmid31512216,
year = {2020},
author = {Parker-Thornburg, J},
title = {Breeding Strategies for Genetically Modified Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2066},
number = {},
pages = {163-169},
doi = {10.1007/978-1-4939-9837-1_14},
pmid = {31512216},
issn = {1940-6029},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R50 CA211121/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breeding/*methods ; CRISPR-Cas Systems/*genetics ; *Embryonic Stem Cells ; Germ Cells/growth &amp; development ; Mice ; Mice, Transgenic/*genetics ; Mutation/genetics ; RNA, Guide/genetics ; Zygote/growth &amp; development ; },
abstract = {Genetically modified mice can be generated using a variety of methods. Depending on the method used, the breeding strategy must be modified not only for the initial founding generation of mouse, but to establish individual mouse lines as well. Transgenic founder mice are each unique and must be outcrossed to initially establish the line. Mice that have been targeted using embryonic stem cells will need to be tested for germ line transmission of the modified gene prior to establishing a line. Mice that have been targeted using CRISPR/Cas9 gene-editing endonucleases will often be mosaic, and thus, also require testing for germ line transmission of the mutation of interest.},
}
@article {pmid31512214,
year = {2020},
author = {Aryal, NK and Parker-Thornburg, J},
title = {Genotyping Genetically Modified (GM) Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2066},
number = {},
pages = {133-148},
doi = {10.1007/978-1-4939-9837-1_12},
pmid = {31512214},
issn = {1940-6029},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R50 CA211121/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Stem Cells/metabolism ; Gene Knockout Techniques/*methods ; Genotype ; Mice ; *Mice, Transgenic ; Mutation/genetics ; },
abstract = {Prior to generating a new mouse model, it is important to plan the method that will be used to detect which of the mice generated have the mutation(s) desired. Nearly, all types of mutations may be detected using PCR. However, the choice of primers will differ depending upon the method used to generate the model. Transgenic mice should be genotyped across a unique junction fragment. Targeted ES cells used to generate knock-out or knock-in mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Targeting in ES cells can also be detected using a genomic Southern blot. Mice targeted using CRISPR/Cas9 should have the region of interest amplified using PCR, and then be assessed for size changes (for large changes in sequence) by Surveyor Assay (for gene knock-out and point mutations) and/or sequenced to verify the mutation. Each of these models has a unique requirement for genotyping, and failure to understand the requirements can easily lead to loss of the gene in subsequent generations.},
}
@article {pmid31512210,
year = {2020},
author = {Larson, MA},
title = {Embryo Transfer Surgery.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2066},
number = {},
pages = {101-106},
doi = {10.1007/978-1-4939-9837-1_8},
pmid = {31512210},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryo Implantation/genetics ; Embryo Transfer/*methods ; Fallopian Tubes/surgery ; Female ; Fertilization in Vitro/*methods ; Humans ; Mice ; Microinjections/*methods ; },
abstract = {Embryo transfer surgery is an essential step in the process of generating gene-modified mice, regardless of whether the embryos were modified by DNA, RNA CRISPR components, or embryonic stem cells, or whether they were microinjected or electroporated. Transfer is also a necessary step for assisted reproductive techniques such as rederivation and reanimation by in vitro fertilization. The manipulated embryos must be returned to the reproductive tract of a pseudopregnant recipient female mouse, wherein the transplanted embryos develop to term. Embryos may be transferred to either the oviduct or uterine horn, depending upon the developmental status of the embryos and the stage of the recipient. This chapter will describe the process of transferring embryos surgically to a recipient female.},
}
@article {pmid31512209,
year = {2020},
author = {Gu, B and Gertsenstein, M and Posfai, E},
title = {Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2066},
number = {},
pages = {89-100},
doi = {10.1007/978-1-4939-9837-1_7},
pmid = {31512209},
issn = {1940-6029},
support = {FDN-143334//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryo, Mammalian ; Embryonic Development/*genetics ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Genome/*genetics ; Homologous Recombination/genetics ; Mice ; Microinjections/*methods ; },
abstract = {Large fragment knock-in mouse models such as reporters and conditional mutant mice are important models for biological research. Here we describe 2-cell (2C)-homologous recombination (HR)-CRISPR, a highly efficient method to generate large fragment knock-in mouse models by CRISPR-based genome engineering. Using this method, knock-in founders can be generated routinely in a time frame of about two months with high germline transmission efficiency. 2C-HR-CRISPR will significantly promote the advancement of basic and translational research using genetic mouse models.},
}
@article {pmid31512207,
year = {2020},
author = {Gertsenstein, M and Mianné, J and Teboul, L and Nutter, LMJ},
title = {Targeted Mutations in the Mouse via Embryonic Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2066},
number = {},
pages = {59-82},
doi = {10.1007/978-1-4939-9837-1_5},
pmid = {31512207},
issn = {1940-6029},
support = {MC_UP_1502/3/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Embryonic Stem Cells ; Gene Editing/*methods ; Gene Targeting/*methods ; Homologous Recombination/genetics ; Mice ; Mutation/genetics ; RNA, Guide/genetics ; },
abstract = {Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.},
}
@article {pmid31512206,
year = {2020},
author = {Larson, MA and Gibson, KA and Vivian, JL},
title = {In Vivo Validation of CRISPR Reagents in Preimplantation Mouse Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2066},
number = {},
pages = {47-57},
doi = {10.1007/978-1-4939-9837-1_4},
pmid = {31512206},
issn = {1940-6029},
support = {P30 CA013148/CA/NCI NIH HHS/United States ; P30 HD002528/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *Blastocyst ; CRISPR-Cas Systems/*genetics ; Embryonic Development/*genetics ; Gene Editing/*methods ; Mice ; Zygote/growth &amp; development ; },
abstract = {The CRISPR/Cas9 system has enjoyed enormous success and has now become the standard method of generating gene-modified mouse models. The tools for predicting the activity of CRISPR reagents in the mouse embryo are currently limited and not particularly accurate in predicting if a given reagent will be active. Given the time and cost of generating genetically modified mice, it is highly desirable to use CRISPR reagents that are known to be active in the mouse embryo. In this chapter, we provide a detailed procedure for empirically testing the activity of CRISPR reagents via electroporation into cultured preimplantation mouse embryos. This platform has proven to be rapid, efficient, and applicable to a variety of mouse strains, and can be used for assessing on- and off-target activity through a variety of molecular assays.},
}
@article {pmid31512166,
year = {2019},
author = {Bellizzi, A and Ahye, N and Jalagadugula, G and Wollebo, HS},
title = {A Broad Application of CRISPR Cas9 in Infectious Diseases of Central Nervous System.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {4},
pages = {578-594},
pmid = {31512166},
issn = {1557-1904},
support = {R21 NS096413/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Central Nervous System Diseases/*genetics/metabolism/*therapy ; Gene Editing/methods/*trends ; Genetic Therapy/methods/*trends ; Humans ; },
abstract = {Virus-induced diseases or neurological complications are huge socio-economic burden to human health globally. The complexity of viral-mediated CNS pathology is exacerbated by reemergence of new pathogenic neurotropic viruses of high public relevance. Although the central nervous system is considered as an immune privileged organ and is mainly protected by barrier system, there are a vast majority of neurotropic viruses capable of gaining access and cause diseases. Despite continued growth of the patient population and a number of treatment strategies, there is no successful viral specific therapy available for viral induced CNS diseases. Therefore, there is an urgent need for a clear alternative treatment strategy that can effectively target neurotropic viruses of DNA or RNA genome. To address this need, rapidly growing gene editing technology based on CRISPR/Cas9, provides unprecedented control over viral genome editing and will be an effective, highly specific and versatile tool for targeting CNS viral infection. In this review, we discuss the application of this system to control CNS viral infection and associated neurological disorders and future prospects. Graphical Abstract CRISPR/Cas9 technology as agent control over CNS viral infection.},
}
@article {pmid31511426,
year = {2019},
author = {Lin, A and Giuliano, CJ and Palladino, A and John, KM and Abramowicz, C and Yuan, ML and Sausville, EL and Lukow, DA and Liu, L and Chait, AR and Galluzzo, ZC and Tucker, C and Sheltzer, JM},
title = {Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials.},
journal = {Science translational medicine},
volume = {11},
number = {509},
pages = {},
pmid = {31511426},
issn = {1946-6242},
support = {DP5 OD021385/OD/NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*toxicity ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Clinical Trials as Topic ; Clone Cells ; Cyclin-Dependent Kinases/antagonists &amp; inhibitors/metabolism ; Drug Resistance, Neoplasm/drug effects ; Gene Knockout Techniques ; Genome, Human ; Humans ; Molecular Targeted Therapy ; Quinolones/pharmacology ; RNA Interference/drug effects ; Up-Regulation/drug effects ; },
abstract = {Ninety-seven percent of drug-indication pairs that are tested in clinical trials in oncology never advance to receive U.S. Food and Drug Administration approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well understood. Using CRISPR-Cas9 mutagenesis, we investigated a set of cancer drugs and drug targets in various stages of clinical testing. We show that-contrary to previous reports obtained predominantly with RNA interference and small-molecule inhibitors-the proteins ostensibly targeted by these drugs are nonessential for cancer cell proliferation. Moreover, the efficacy of each drug that we tested was unaffected by the loss of its putative target, indicating that these compounds kill cells via off-target effects. By applying a genetic target-deconvolution strategy, we found that the mischaracterized anticancer agent OTS964 is actually a potent inhibitor of the cyclin-dependent kinase CDK11 and that multiple cancer types are addicted to CDK11 expression. We suggest that stringent genetic validation of the mechanism of action of cancer drugs in the preclinical setting may decrease the number of therapies tested in human patients that fail to provide any clinical benefit.},
}
@article {pmid31511384,
year = {2019},
author = {Zhu, S and Wan, W and Zhang, Y and Shang, W and Pan, X and Zhang, LK and Xiao, G},
title = {Comprehensive Interactome Analysis Reveals that STT3B Is Required for N-Glycosylation of Lassa Virus Glycoprotein.},
journal = {Journal of virology},
volume = {93},
number = {23},
pages = {},
pmid = {31511384},
issn = {1098-5514},
mesh = {CRISPR-Cas Systems ; Cation Transport Proteins ; Cell Line ; Gene Knockout Techniques ; Glycoproteins/*metabolism ; Glycosylation ; HEK293 Cells ; HeLa Cells ; Hexosyltransferases/genetics/*metabolism ; Humans ; Lassa Fever/*metabolism ; Lassa virus/genetics/*metabolism/pathogenicity ; Membrane Proteins/genetics/*metabolism ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins ; Oxidoreductases/metabolism ; Protein Isoforms ; Receptors, Cell Surface ; Tumor Suppressor Proteins/genetics ; Virus Internalization ; },
abstract = {Lassa virus (LASV) is the causative agent of a fatal hemorrhagic fever in humans. The glycoprotein (GP) of LASV mediates viral entry into host cells, and correct processing and modification of GP by host factors is a prerequisite for virus replication. Here, using an affinity purification-coupled mass spectrometry (AP-MS) strategy, 591 host proteins were identified as interactors of LASV GP. Gene ontology analysis was performed to functionally annotate these proteins, and the oligosaccharyltransferase (OST) complex was highly enriched. Functional studies conducted by using CRISPR-Cas9-mediated knockouts showed that STT3A and STT3B, the two catalytically active isoforms of the OST complex, are essential for the propagation of the recombinant arenavirus rLCMV/LASV glycoprotein precursor, mainly via affecting virus infectivity. Knockout of STT3B, but not STT3A, caused hypoglycosylation of LASV GP, indicating a preferential requirement of LASV for the STT3B-OST isoform. Furthermore, double knockout of magnesium transporter 1 (MAGT1) and tumor suppressor candidate 3 (TUSC3), two specific subunits of STT3B-OST, also caused hypoglycosylation of LASV GP and affected virus propagation. Site-directed mutagenesis analysis revealed that the oxidoreductase CXXC active-site motif of MAGT1 or TUSC3 is essential for the glycosylation of LASV GP. NGI-1, a small-molecule OST inhibitor, can effectively reduce virus infectivity without affecting cell viability. The STT3B-dependent N-glycosylation of GP is conserved among other arenaviruses, including both the Old World and New World groups. Our study provided a systematic view of LASV GP-host interactions and revealed the preferential requirement of STT3B for LASV GP N-glycosylation.IMPORTANCE Glycoproteins play vital roles in the arenavirus life cycle by facilitating virus entry and participating in the virus budding process. N-glycosylation of GPs is responsible for their proper functioning; however, little is known about the host factors on which the virus depends for this process. In this study, a comprehensive LASV GP interactome was characterized, and further study revealed that STT3B-dependent N-glycosylation was preferentially required by arenavirus GPs and critical for virus infectivity. The two specific thioredoxin subunits of STT3B-OST MAGT1 and TUSC3 were found to be essential for the N-glycosylation of viral GP. NGI-1, a small-molecule inhibitor of OST, also showed a robust inhibitory effect on arenavirus. Our study provides new insights into LASV GP-host interactions and extends the potential targets for the development of novel therapeutics against Lassa fever in the future.},
}
@article {pmid31509984,
year = {2019},
author = {Jin, DY},
title = {Special Issue: Applications of CRISPR Technology in Virology 2018.},
journal = {Viruses},
volume = {11},
number = {9},
pages = {},
pmid = {31509984},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Genome, Viral ; Host Microbial Interactions/*genetics ; Humans ; Virology/methods ; },
abstract = {Precision genome engineering by CRISPR is a game-changing technology that originates from the study of virus-host interaction and promises to revolutionize virology and antiviral therapy [...].},
}
@article {pmid31509794,
year = {2019},
author = {Verdikt, R and Darcis, G and Ait-Ammar, A and Van Lint, C},
title = {Applications of CRISPR/Cas9 tools in deciphering the mechanisms of HIV-1 persistence.},
journal = {Current opinion in virology},
volume = {38},
number = {},
pages = {63-69},
doi = {10.1016/j.coviro.2019.07.004},
pmid = {31509794},
issn = {1879-6265},
mesh = {*CRISPR-Cas Systems ; Disease Reservoirs ; Gene Editing ; HIV Infections/drug therapy/metabolism/*virology ; HIV-1/*physiology ; Humans ; Proteasome Endopeptidase Complex/metabolism ; Virus Integration ; Virus Latency ; *Virus Replication ; },
abstract = {HIV-1 infection can be controlled but not cured by combination antiretroviral therapy. Indeed, the virus persists in treated individuals in viral reservoirs, the best described of which consisting in latently infected central memory CD4[+] T cells. However, other cell types in other body compartments than in the peripheral blood contribute to HIV-1 persistence. Addressing the molecular mechanisms of HIV-1 persistence and their cell-specific and tissue-specific variations is thus crucial to develop HIV-1 curative strategies. CRISPR/Cas9 editing technologies have revolutionized genetic engineering by their high specificity and their versatility. Multiple applications now allow to investigate the molecular mechanisms of HIV-1 persistence. Here, we review recent advances in CRISPR-based technologies in deciphering HIV-1 gene expression regulation during persistence.},
}
@article {pmid31509742,
year = {2019},
author = {Freeman, AJ and Vervoort, SJ and Ramsbottom, KM and Kelly, MJ and Michie, J and Pijpers, L and Johnstone, RW and Kearney, CJ and Oliaro, J},
title = {Natural Killer Cells Suppress T Cell-Associated Tumor Immune Evasion.},
journal = {Cell reports},
volume = {28},
number = {11},
pages = {2784-2794.e5},
doi = {10.1016/j.celrep.2019.08.017},
pmid = {31509742},
issn = {2211-1247},
mesh = {Animals ; Antigen Presentation/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Coculture Techniques ; Cytotoxicity, Immunologic ; Female ; Gene Ontology ; Histocompatibility Antigens Class I/*metabolism ; Humans ; Immunotherapy ; Interferon-gamma/genetics/*metabolism ; Janus Kinase 1/genetics/metabolism ; Killer Cells, Natural/*immunology ; Male ; Melanoma/genetics/*immunology/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Perforin/genetics/metabolism ; T-Lymphocytes/*immunology ; Transplantation, Heterologous ; Tumor Escape/*genetics/immunology ; },
abstract = {Despite the clinical success of cancer immunotherapies, the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here, we conducted a series of unbiased, genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-γ (IFN-γ) signaling protect tumor cells from NK cell killing. Indeed, Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-γ-driven transcriptional events that regulate MHC I expression. Importantly, tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together, we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells, unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape.},
}
@article {pmid31509667,
year = {2019},
author = {Xu, L and Wang, J and Liu, Y and Xie, L and Su, B and Mou, D and Wang, L and Liu, T and Wang, X and Zhang, B and Zhao, L and Hu, L and Ning, H and Zhang, Y and Deng, K and Liu, L and Lu, X and Zhang, T and Xu, J and Li, C and Wu, H and Deng, H and Chen, H},
title = {CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia.},
journal = {The New England journal of medicine},
volume = {381},
number = {13},
pages = {1240-1247},
doi = {10.1056/NEJMoa1817426},
pmid = {31509667},
issn = {1533-4406},
support = {2017YFA0103000/US/United States/United States ; D171100000517004/US/United States/United States ; 2017YFA0103000/US/United States/United States ; D171100000517004/US/United States/United States ; },
mesh = {Adult ; Anti-Retroviral Agents/therapeutic use ; Blood Cell Count ; CD4 Lymphocyte Count ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HIV Infections/complications/drug therapy/*therapy ; *HIV-1/genetics ; *Hematopoietic Stem Cell Transplantation ; *Hematopoietic Stem Cells ; Humans ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications ; Receptors, CCR5/*genetics ; Viral Load ; },
abstract = {The safety of CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing-related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.).},
}
@article {pmid31509485,
year = {2019},
author = {Matsuda, T and Oinuma, I},
title = {Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9.},
journal = {Molecular biology of the cell},
volume = {30},
number = {22},
pages = {2838-2855},
pmid = {31509485},
issn = {1939-4586},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disks Large Homolog 4 Protein/metabolism ; Gene Editing/methods ; Mice ; Microscopy, Fluorescence/*methods ; Neurons/*metabolism ; Primary Cell Culture ; Rats ; Rats, Wistar ; Single-Cell Analysis/*methods ; Synaptophysin/metabolism ; },
abstract = {Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.},
}
@article {pmid31508509,
year = {2019},
author = {Fang, P and De Souza, C and Minn, K and Chien, J},
title = {Genome-scale CRISPR knockout screen identifies TIGAR as a modifier of PARP inhibitor sensitivity.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {335},
pmid = {31508509},
issn = {2399-3642},
support = {P30 CA168524/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/pharmacology ; Apoptosis Regulatory Proteins/*genetics ; Biomarkers ; *CRISPR-Cas Systems ; Cellular Senescence ; DNA Damage ; Drug Resistance, Neoplasm/*genetics ; Gene Knockdown Techniques ; Genome-Wide Association Study ; Humans ; Phosphoric Monoester Hydrolases/*genetics ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Signal Transduction ; },
abstract = {Treatment of cancer with poly (ADP-ribose) polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination (HR) pathway. Identification of genetic targets that induce or mimic HR deficiencies will extend the clinical utility of PARP inhibitors. Here we perform a CRISPR/Cas9-based genome-scale loss-of-function screen, using the sensitivity of PARP inhibitor olaparib as a surrogate. We identify C12orf5, encoding TP53 induced glycolysis and apoptosis regulator (TIGAR), as a modifier of PARP inhibitor response. We show that TIGAR is amplified in several cancer types, and higher expression of TIGAR associates with poor overall survival in ovarian cancer. TIGAR knockdown enhances sensitivity to olaparib in cancer cells via downregulation of BRCA1 and the Fanconi anemia pathway and increases senescence of these cells by affecting metabolic pathways and increasing the cytotoxic effects of olaparib. Our results indicate TIGAR should be explored as a therapeutic target for treating cancer and extending the use of PARP inhibitors.},
}
@article {pmid31507572,
year = {2019},
author = {Gao, NJ and Al-Bassam, MM and Poudel, S and Wozniak, JM and Gonzalez, DJ and Olson, J and Zengler, K and Nizet, V and Valderrama, JA},
title = {Functional and Proteomic Analysis of Streptococcus pyogenes Virulence Upon Loss of Its Native Cas9 Nuclease.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1967},
pmid = {31507572},
issn = {1664-302X},
support = {R37 AI052453/AI/NIAID NIH HHS/United States ; R01 AI077780/AI/NIAID NIH HHS/United States ; T32 AR064194/AR/NIAMS NIH HHS/United States ; R01 AI145325/AI/NIAID NIH HHS/United States ; T32 GM007752/GM/NIGMS NIH HHS/United States ; T32 GM008666/GM/NIGMS NIH HHS/United States ; },
abstract = {The public health impact of Streptococcus pyogenes (group A Streptococcus, GAS) as a top 10 cause of infection-related mortality in humans contrasts with its benefit to biotechnology as the main natural source of Cas9 nuclease, the key component of the revolutionary CRISPR-Cas9 gene editing platform. Despite widespread knowledge acquired in the last decade on the molecular mechanisms by which GAS Cas9 achieves precise DNA targeting, the functions of Cas9 in the biology and pathogenesis of its native organism remain unknown. In this study, we generated an isogenic serotype M1 GAS mutant deficient in Cas9 protein and compared its behavior and phenotypes to the wild-type parent strain. Absence of Cas9 was linked to reduced GAS epithelial cell adherence, reduced growth in human whole blood ex vivo, and attenuation of virulence in a murine necrotizing skin infection model. Virulence defects of the GAS Δcas9 strain were explored through quantitative proteomic analysis, revealing a significant reduction in the abundance of key GAS virulence determinants. Similarly, deletion of cas9 affected the expression of several known virulence regulatory proteins, indicating that Cas9 impacts the global architecture of GAS gene regulation.},
}
@article {pmid31506266,
year = {2019},
author = {Yin, Y and Yang, B and Entwistle, S},
title = {Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches.},
journal = {mSystems},
volume = {4},
number = {5},
pages = {},
pmid = {31506266},
issn = {2379-5077},
support = {R15 GM114706/GM/NIGMS NIH HHS/United States ; },
abstract = {Anti-CRISPR (Acr) loci/operons encode Acr proteins and Acr-associated (Aca) proteins. Forty-five Acr families have been experimentally characterized inhibiting seven subtypes of CRISPR-Cas systems. We have developed a bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs by combining three computational approaches: homology, guilt-by-association, and self-targeting spacers. Homology search found thousands of Acr homologs in bacterial and viral genomes, but most are homologous to AcrIIA7 and AcrIIA9. Investigating the gene neighborhood of these Acr homologs revealed that only a small percentage (23.0% in bacteria and 8.2% in viruses) of them have neighboring Aca homologs and thus form Acr-Aca operons. Surprisingly, although a self-targeting spacer is a strong indicator of the presence of Acr genes in a genome, a large percentage of Acr-Aca loci are found in bacterial genomes without self-targeting spacers or even without complete CRISPR-Cas systems. Additionally, for Acr homologs from genomes with self-targeting spacers, homology-based Acr family assignments do not always agree with the self-targeting CRISPR-Cas subtypes. Last, by investigating Acr genomic loci coexisting with self-targeting spacers in the same genomes, five known subtypes (I-C, I-E, I-F, II-A, and II-C) and five new subtypes (I-B, III-A, III-B, IV-A, and V-U4) of Acrs were inferred. Based on these findings, we conclude that the discovery of new anti-CRISPRs should not be restricted to genomes with self-targeting spacers and loci with Acr homologs. The evolutionary arms race of CRISPR-Cas systems and anti-CRISPR systems may have driven the adaptive and rapid gain and loss of these elements in closely related genomes.IMPORTANCE As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators.},
}
@article {pmid31506018,
year = {2019},
author = {Hustedt, N and Álvarez-Quilón, A and McEwan, A and Yuan, JY and Cho, T and Koob, L and Hart, T and Durocher, D},
title = {A consensus set of genetic vulnerabilities to ATR inhibition.},
journal = {Open biology},
volume = {9},
number = {9},
pages = {190156},
pmid = {31506018},
issn = {2046-2441},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; FDN143343//CIHR/Canada ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/antagonists &amp; inhibitors/*genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Gene Silencing ; Gene Targeting ; Genes, Reporter ; Genetic Association Studies ; *Genetic Variation ; Humans ; RNA Interference ; RNA, Guide ; },
abstract = {The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.},
}
@article {pmid31506008,
year = {2020},
author = {de Korte, T and Katili, PA and Mohd Yusof, NAN and van Meer, BJ and Saleem, U and Burton, FL and Smith, GL and Clements, P and Mummery, CL and Eschenhagen, T and Hansen, A and Denning, C},
title = {Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?.},
journal = {Annual review of pharmacology and toxicology},
volume = {60},
number = {},
pages = {529-551},
doi = {10.1146/annurev-pharmtox-010919-023309},
pmid = {31506008},
issn = {1545-4304},
support = {RG/15/6/31436/BHF_/British Heart Foundation/United Kingdom ; NC/C013105/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; PG/14/59/31000/BHF_/British Heart Foundation/United Kingdom ; NC/C013202/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; RG/14/1/30588/BHF_/British Heart Foundation/United Kingdom ; NC/K000225/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; SP/15/9/31605/BHF_/British Heart Foundation/United Kingdom ; P47352/CRM/BHF_/British Heart Foundation/United Kingdom ; MR/L012618/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiotoxicity/*prevention &amp; control ; Drug Development/methods ; Drug Discovery/*methods ; Genetic Engineering ; Humans ; Induced Pluripotent Stem Cells/cytology ; Myocytes, Cardiac/cytology/*drug effects ; Precision Medicine/methods ; },
abstract = {In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.},
}
@article {pmid31505675,
year = {2019},
author = {Milon, N and Chantry-Darmon, C and Satge, C and Fustier, MA and Cauet, S and Moreau, S and Callot, C and Bellec, A and Gabrieli, T and Saïas, L and Boutonnet, A and Ginot, F and Bergès, H and Bancaud, A},
title = {μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome.},
journal = {Nucleic acids research},
volume = {47},
number = {15},
pages = {8050-8060},
pmid = {31505675},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial ; Computational Biology/methods ; DNA, Plant/*genetics/isolation &amp; purification ; Electrophoresis, Gel, Pulsed-Field/methods ; Genome, Plant/*genetics ; Medicago truncatula/*genetics ; Reproducibility of Results ; Sequence Analysis, DNA/*methods ; },
abstract = {Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.},
}
@article {pmid31504832,
year = {2019},
author = {González-Delgado, A and Mestre, MR and Martínez-Abarca, F and Toro, N},
title = {Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus.},
journal = {Nucleic acids research},
volume = {47},
number = {19},
pages = {10202-10211},
pmid = {31504832},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endodeoxyribonucleases/*genetics ; Genome, Bacterial/genetics ; Plasmids/genetics ; RNA/genetics ; RNA-Directed DNA Polymerase ; Vibrio vulnificus/genetics ; },
abstract = {The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5'and 3'ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.},
}
@article {pmid31504824,
year = {2019},
author = {Mao, S and Ying, Y and Wu, X and Krueger, CJ and Chen, AK},
title = {CRISPR/dual-FRET molecular beacon for sensitive live-cell imaging of non-repetitive genomic loci.},
journal = {Nucleic acids research},
volume = {47},
number = {20},
pages = {e131},
pmid = {31504824},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer/*methods ; Fluorescent Dyes/chemistry ; *Genetic Loci ; HeLa Cells ; Humans ; RNA, Guide/chemistry/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic imaging systems predominantly rely on fluorescent protein reporters, which lack the optical properties essential for sensitive dynamic imaging. Here, we modified the CRISPR single-guide RNA (sgRNA) to carry two distinct molecular beacons (MBs) that can undergo fluorescence resonance energy transfer (FRET) and demonstrated that the resulting system, CRISPR/dual-FRET MB, enables dynamic imaging of non-repetitive genomic loci with only three unique sgRNAs.},
}
@article {pmid31503550,
year = {2019},
author = {Nyquist, MD and Corella, A and Mohamad, O and Coleman, I and Kaipainen, A and Kuppers, DA and Lucas, JM and Paddison, PJ and Plymate, SR and Nelson, PS and Mostaghel, EA},
title = {Molecular determinants of response to high-dose androgen therapy in prostate cancer.},
journal = {JCI insight},
volume = {4},
number = {19},
pages = {},
pmid = {31503550},
issn = {2379-3708},
support = {R21 CA230138/CA/NCI NIH HHS/United States ; P30 CA015704/CA/NCI NIH HHS/United States ; P50 CA097186/CA/NCI NIH HHS/United States ; I01 BX003324/BX/BLRD VA/United States ; S10 OD020069/OD/NIH HHS/United States ; I01 BX000585/BX/BLRD VA/United States ; P01 CA163227/CA/NCI NIH HHS/United States ; },
mesh = {Androgens/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic/*drug effects ; Gene Knockout Techniques ; Genes, p53/genetics ; Humans ; Male ; Prostatic Neoplasms/*drug therapy/*genetics ; Retinoblastoma Binding Proteins/genetics ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.},
}
@article {pmid31503414,
year = {2019},
author = {Giuliano, CJ and Lin, A and Girish, V and Sheltzer, JM},
title = {Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.},
journal = {Current protocols in molecular biology},
volume = {128},
number = {1},
pages = {e100},
pmid = {31503414},
issn = {1934-3647},
support = {DP5 OD021385/OD/NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Clone Cells ; Gene Knockout Techniques/*methods ; HEK293 Cells ; Humans ; Mammals ; Plasmids ; RNA, Guide ; Transfection ; },
abstract = {CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.},
}
@article {pmid31503392,
year = {2019},
author = {Manna, PT and Davis, LJ and Robinson, MS},
title = {Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.},
journal = {Traffic (Copenhagen, Denmark)},
volume = {20},
number = {12},
pages = {974-982},
pmid = {31503392},
issn = {1600-0854},
support = {086598/WT_/Wellcome Trust/United Kingdom ; 100140/WT_/Wellcome Trust/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; HeLa Cells ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.},
}
@article {pmid31502713,
year = {2019},
author = {Peters, JE},
title = {Targeted transposition with Tn7 elements: safe sites, mobile plasmids, CRISPR/Cas and beyond.},
journal = {Molecular microbiology},
volume = {112},
number = {6},
pages = {1635-1644},
pmid = {31502713},
issn = {1365-2958},
support = {R01 GM129118/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Chromosomes, Bacterial/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Transposable Elements/*genetics/*physiology ; DNA, Bacterial/genetics ; Genomic Islands/genetics ; Plasmids ; },
abstract = {Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7-like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7-like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7-like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for the new dual activities. This expanded understanding of Tn7-like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7-like elements are discussed.},
}
@article {pmid31502661,
year = {2019},
author = {Ou, XY and Wu, XL and Peng, F and Zeng, YJ and Li, HX and Xu, P and Chen, G and Guo, ZW and Yang, JG and Zong, MH and Lou, WY},
title = {Metabolic engineering of a robust Escherichia coli strain with a dual protection system.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {12},
pages = {3333-3348},
doi = {10.1002/bit.27165},
pmid = {31502661},
issn = {1097-0290},
mesh = {*Bacteriophage T7 ; CRISPR-Cas Systems ; Escherichia coli/*genetics/*growth &amp; development/virology ; *Metabolic Engineering ; Metabolic Networks and Pathways ; Microorganisms, Genetically-Modified/*genetics/*growth &amp; development ; },
abstract = {Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&amp;P) system in combination with a CRISPR/Cas9 system. The N&amp;P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&amp;P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 10[7] plaque-forming units/ml and produce GFP with a yield of up to 30 μg/10[9] colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.},
}
@article {pmid31502535,
year = {2019},
author = {Forsberg, KJ and Bhatt, IV and Schmidtke, DT and Javanmardi, K and Dillard, KE and Stoddard, BL and Finkelstein, IJ and Kaiser, BK and Malik, HS},
title = {Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31502535},
issn = {2050-084X},
support = {F31 GM125201/GM/NIGMS NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; R01 GM124131/GM/NIGMS NIH HHS/United States ; F-1808//Welch Foundation/International ; R01 GM105691/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/isolation &amp; purification/*metabolism ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; Enzyme Inhibitors/isolation &amp; purification/*metabolism ; Feces/microbiology/virology ; Humans ; Metagenomics ; *Microbiota ; Mouth/microbiology/virology ; Viral Proteins/genetics/isolation &amp; purification/*metabolism ; },
abstract = {CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli. The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola. We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes.},
}
@article {pmid31502220,
year = {2020},
author = {Grossi, S and Fenini, G and Hennig, P and Di Filippo, M and Beer, HD},
title = {Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2109},
number = {},
pages = {125-145},
doi = {10.1007/7651_2019_262},
pmid = {31502220},
issn = {1940-6029},
mesh = {3T3-L1 Cells ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Feeder Cells/*cytology ; Gene Editing/*methods ; Gene Expression ; Gene Knockout Techniques/*methods ; Humans ; Keratinocytes/*cytology/metabolism ; Mice ; Primary Cell Culture ; },
abstract = {The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.},
}
@article {pmid31502158,
year = {2020},
author = {Balan, V and Wang, J},
title = {The CRISPR System and Cancer Immunotherapy Biomarkers.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2055},
number = {},
pages = {301-322},
doi = {10.1007/978-1-4939-9773-2_14},
pmid = {31502158},
issn = {1940-6029},
mesh = {Biomarkers, Tumor/*genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Immunotherapy ; Neoplasms/drug therapy/*genetics ; Treatment Outcome ; },
abstract = {Recent advances in cancer immunotherapy have shed new light on the possibility to cure most, if not all, cancer patients with further development of various treatment options. The emergency of a new genome editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR) technology, revolutionized the biomedical research field. We envision application of the CRISPR technology in cancer research, diagnosis, and therapy will markedly speed up the development of new treatment options for cancer patients. The CRISPR system and its applications in biomedical research will be discussed with an emphasis on cancer immunotherapy and biomarker development.},
}
@article {pmid31501558,
year = {2019},
author = {Culp, EJ and Yim, G and Waglechner, N and Wang, W and Pawlowski, AC and Wright, GD},
title = {Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics.},
journal = {Nature biotechnology},
volume = {37},
number = {10},
pages = {1149-1154},
doi = {10.1038/s41587-019-0241-9},
pmid = {31501558},
issn = {1546-1696},
support = {MT-14981//CIHR/Canada ; },
mesh = {Anti-Bacterial Agents/*biosynthesis ; CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Escherichia coli ; Gene Expression Regulation, Bacterial ; Multigene Family ; Mutation ; Streptomyces/genetics/*metabolism ; },
abstract = {Actinobacteria, which are one of the largest bacterial phyla and comprise between 13 and 30% of the soil microbiota, are the main source of antibiotic classes in clinical use[1]. During screens for antimicrobials, as many as 50% of actinomycete strains are discarded because they produce a known antibiotic (Supplementary Fig. 1) (ref. [2]). Despite each strain likely having the capacity to produce many compounds, strains are abandoned because the already characterized antibiotic could interfere with screening for, or purification of, newly discovered compounds[3]. We applied CRISPR-Cas9 genome engineering to knockout genes encoding two of the most frequently rediscovered antibiotics, streptothricin or streptomycin, in 11 actinomycete strains. We report that this simple approach led to production of different antibiotics that were otherwise masked. We were able to rapidly discover rare and previously unknown variants of antibiotics including thiolactomycin, amicetin, phenanthroviridin and 5-chloro-3-formylindole. This strategy could be applied to existing strain collections to realize their biosynthetic potential.},
}
@article {pmid31501532,
year = {2019},
author = {Chen, G and Abdeen, AA and Wang, Y and Shahi, PK and Robertson, S and Xie, R and Suzuki, M and Pattnaik, BR and Saha, K and Gong, S},
title = {A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing.},
journal = {Nature nanotechnology},
volume = {14},
number = {10},
pages = {974-980},
pmid = {31501532},
issn = {1748-3395},
support = {R01 HL129785/HL/NHLBI NIH HHS/United States ; R01 NS091540/NS/NINDS NIH HHS/United States ; UG3 NS111688/NS/NINDS NIH HHS/United States ; R01 EY024995/EY/NEI NIH HHS/United States ; R01 HL143469/HL/NHLBI NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*administration &amp; dosage/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Glutathione/chemistry ; HEK293 Cells ; Humans ; Mice ; Nanocapsules/*chemistry ; Polymers/chemistry ; RNA, Guide/*administration &amp; dosage/genetics ; },
abstract = {Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing[1]. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRISPR components and limits the possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating, called a nanocapsule (NC), around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. The NC is synthesized by in situ polymerization, has a hydrodynamic diameter of 25 nm and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle after local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.},
}
@article {pmid31501243,
year = {2019},
author = {Callies, LK and Tadeo, D and Simper, J and Bugge, TH and Szabo, R},
title = {Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {44},
pages = {15987-15996},
pmid = {31501243},
issn = {1083-351X},
support = {ZIC DE000744/ImNIH/Intramural NIH HHS/United States ; ZIG DE000740/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Embryonic Development/genetics ; Epidermis/growth &amp; development/metabolism ; Female ; Fertility/genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Male ; Mice ; *Multigene Family ; Serine Endopeptidases/*genetics/metabolism ; },
abstract = {Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.},
}
@article {pmid31499408,
year = {2019},
author = {Lee, Y and Choi, HY and Kwon, A and Park, H and Park, M and Kim, YO and Kwak, S and Koo, SK},
title = {Generation of a NESTIN-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-1, using CRISPR/Cas9 nuclease.},
journal = {Stem cell research},
volume = {40},
number = {},
pages = {101554},
doi = {10.1016/j.scr.2019.101554},
pmid = {31499408},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line/cytology/*metabolism ; Genes, Reporter ; Green Fluorescent Proteins/*genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Male ; Nestin/*genetics/metabolism ; },
abstract = {NESTIN, an intermediate filament, is a neuroectodermal marker involved in induced pluripotent stem cell (iPSC) differentiation toward neural lineages. Here, we introduced an EGFP reporter into the C-terminus of NESTIN in KSCBi005-A hiPSCs through homologous recombination using CRISPR/Cas9 nuclease. The successfully edited line was confirmed by sequencing and had a normal karyotype. It expressed EGFP upon induction of neural differentiation and exhibited potential for differentiation into three germ layers. KSCBi005-A-1 cells could be used to monitor the expression of NESTIN in differentiated cell types. This cell line is available at the National Stem Cell Bank, Korea National Institute of Health.},
}
@article {pmid31498320,
year = {2019},
author = {Barry, SK and Nakamura, T and Matsuoka, Y and Straub, C and Horch, HW and Extavour, CG},
title = {Injecting Gryllus bimaculatus Eggs.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {150},
pages = {},
pmid = {31498320},
issn = {1940-087X},
support = {P20 GM103423/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Gryllidae ; Microinjections/*instrumentation/*methods ; Needles ; *Ovum ; },
abstract = {Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.},
}
@article {pmid31497003,
year = {2019},
author = {Spencer, BL and Deng, L and Patras, KA and Burcham, ZM and Sanches, GF and Nagao, PE and Doran, KS},
title = {Cas9 Contributes to Group B Streptococcal Colonization and Disease.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1930},
pmid = {31497003},
issn = {1664-302X},
abstract = {Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains a leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20-30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10-15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, has also been observed to modulate virulence in a number of bacterial pathogens. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic Δcas9 mutants. Compared to parental strains, Δcas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 alters transcription in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and Δcas9 mutant strain. Significantly dysregulated genes included those encoding predicted virulence factors, metabolic factors, two-component systems (TCS), and factors important for cell wall formation. These findings were confirmed by qRT-PCR and suggest that Cas9 may regulate a significant portion of the GBS genome. We studied one of the TCS regulators, CiaR, that was significantly downregulated in the Δcas9 mutant strain. RNA-Seq analysis of the WT and ΔciaR strains demonstrated that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may modulate GBS gene expression through other regulators. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of the non-canonical function of Cas9 in GBS colonization and disease.},
}
@article {pmid31495903,
year = {2019},
author = {Yingjun, X and Yuhuan, X and Yuchang, C and Dongzhi, L and Ding, W and Bing, S and Yi, Y and Dian, L and Yanting, X and Zeyu, X and Nengqing, L and Diyu, C and Xiaofang, S},
title = {CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells.},
journal = {Annals of hematology},
volume = {98},
number = {12},
pages = {2661-2671},
pmid = {31495903},
issn = {1432-0584},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Hemoglobins, Abnormal/genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism/pathology ; *alpha-Thalassemia/genetics/metabolism/therapy ; },
abstract = {Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-α[CS]) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.},
}
@article {pmid31495052,
year = {2020},
author = {Pompili, V and Dalla Costa, L and Piazza, S and Pindo, M and Malnoy, M},
title = {Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {3},
pages = {845-858},
pmid = {31495052},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; DNA, Bacterial ; Disease Resistance/*genetics ; Erwinia amylovora/*pathogenicity ; *Gene Editing ; Gene Knockdown Techniques ; Malus/*genetics/microbiology ; Plant Diseases/*genetics/microbiology ; Plants, Genetically Modified/microbiology ; },
abstract = {The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off-target sites revealed no mutation event. Moreover, our construct contained a heat-shock inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.},
}
@article {pmid31494407,
year = {2019},
author = {Mansourian, S and Fandino, RA and Riabinina, O},
title = {Progress in the use of genetic methods to study insect behavior outside Drosophila.},
journal = {Current opinion in insect science},
volume = {36},
number = {},
pages = {45-56},
doi = {10.1016/j.cois.2019.08.001},
pmid = {31494407},
issn = {2214-5753},
mesh = {Animals ; Appetitive Behavior ; *Behavior, Animal ; CRISPR-Cas Systems ; Insecta/*genetics/*physiology ; Mutagenesis, Site-Directed ; Oviposition ; RNA Interference ; Social Behavior ; },
abstract = {In the span of a decade we have seen a rapid progress in the application of genetic tools and genome editing approaches in 'non-model' insects. It is now possible to target sensory receptor genes and neurons, explore their functional roles and manipulate behavioral responses in these insects. In this review, we focus on the latest examples from Diptera, Lepidoptera and Hymenoptera of how applications of genetic tools advanced our understanding of diverse behavioral phenomena. We further discuss genetic methods that could be applied to study insect behavior in the future.},
}
@article {pmid31494246,
year = {2020},
author = {Vemuri, S and Srivastava, R and Mir, Q and Hashemikhabir, S and Dong, XC and Janga, SC},
title = {SliceIt: A genome-wide resource and visualization tool to design CRISPR/Cas9 screens for editing protein-RNA interaction sites in the human genome.},
journal = {Methods (San Diego, Calif.)},
volume = {178},
number = {},
pages = {104-113},
pmid = {31494246},
issn = {1095-9130},
support = {R01 GM123314/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Human/genetics ; Genomics/*methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Several protein-RNA cross linking protocols have been established in recent years to delineate the molecular interaction of an RNA Binding Protein (RBP) and its target RNAs. However, functional dissection of the role of the RBP binding sites in modulating the post-transcriptional fate of the target RNA remains challenging. CRISPR/Cas9 genome editing system is being commonly employed to perturb both coding and noncoding regions in the genome. With the advancements in genome-scale CRISPR/Cas9 screens, it is now possible to not only perturb specific binding sites but also probe the global impact of protein-RNA interaction sites across cell types. Here, we present SliceIt (http://sliceit.soic.iupui.edu/), a database of in silico sgRNA (single guide RNA) library to facilitate conducting such high throughput screens. SliceIt comprises of ~4.8 million unique sgRNAs with an estimated range of 2-8 sgRNAs designed per RBP binding site, for eCLIP experiments of >100 RBPs in HepG2 and K562 cell lines from the ENCODE project. SliceIt provides a user friendly environment, developed using advanced search engine framework, Elasticsearch. It is available in both table and genome browser views facilitating the easy navigation of RBP binding sites, designed sgRNAs, exon expression levels across 53 human tissues along with prevalence of SNPs and GWAS hits on binding sites. Exon expression profiles enable examination of locus specific changes proximal to the binding sites. Users can also upload custom tracks of various file formats directly onto genome browser, to navigate additional genomic features in the genome and compare with other types of omics profiles. All the binding site-centric information is dynamically accessible via "search by gene", "search by coordinates" and "search by RBP" options and readily available to download. Validation of the sgRNA library in SliceIt was performed by selecting RBP binding sites in Lipt1 gene and designing sgRNAs. Effect of CRISPR/Cas9 perturbations on the selected binding sites in HepG2 cell line, was confirmed based on altered proximal exon expression levels using qPCR, further supporting the utility of the resource to design experiments for perturbing protein-RNA interaction networks. Thus, SliceIt provides a one-stop repertoire of guide RNA library to perturb RBP binding sites, along with several layers of functional information to design both low and high throughput CRISPR/Cas9 screens, for studying the phenotypes and diseases associated with RBP binding sites.},
}
@article {pmid31494210,
year = {2019},
author = {Davies, JA and Ireland, S and Harding, S and Sharman, JL and Southan, C and Dominguez-Monedero, A},
title = {Inverse pharmacology: Approaches and tools for introducing druggability into engineered proteins.},
journal = {Biotechnology advances},
volume = {37},
number = {8},
pages = {107439},
pmid = {31494210},
issn = {1873-1899},
support = {BB/M018040/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins ; CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; Humans ; *Protein Engineering ; },
abstract = {A major feature of twenty-first century medical research is the development of therapeutic strategies that use 'biologics' (large molecules, usually engineered proteins) and living cells instead of, or as well as, the small molecules that were the basis of pharmacology in earlier eras. The high power of these techniques can bring correspondingly high risk, and therefore the need for the potential for external control. One way of exerting control on therapeutic proteins is to make them responsive to small molecules; in a clinical context, these small molecules themselves have to be safe. Conventional pharmacology has resulted in thousands of small molecules licensed for use in humans, and detailed structural data on their binding to their protein targets. In principle, these data can be used to facilitate the engineering of drug-responsive modules, taken from natural proteins, into synthetic proteins. This has been done for some years (for example, Cre-ERT2) but usually in a painstaking manner. Recently, we have developed the bioinformatic tool SynPharm to facilitate the design of drug-responsive proteins. In this review, we outline the history of the field, the design and use of the Synpharm tool, and describe our own experiences in engineering druggability into the Cpf1 effector of CRISPR gene editing.},
}
@article {pmid31494053,
year = {2019},
author = {Li, C and Mishra, AS and Gil, S and Wang, M and Georgakopoulou, A and Papayannopoulou, T and Hawkins, RD and Lieber, A},
title = {Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {12},
pages = {2195-2212},
pmid = {31494053},
issn = {1525-0024},
support = {R01 DK101328/DK/NIDDK NIH HHS/United States ; R01 HL141781/HL/NHLBI NIH HHS/United States ; R21 CA193077/CA/NCI NIH HHS/United States ; R01 HL120888/HL/NHLBI NIH HHS/United States ; },
mesh = {Adenoviridae/*genetics ; Animals ; CRISPR-Cas Systems ; Dependovirus/*genetics ; Female ; Genes, Reporter ; Genetic Therapy ; Genetic Vectors/*genetics ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells/cytology/*metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; *Transduction, Genetic ; Transgenes/*physiology ; *Virus Integration ; gamma-Globins/antagonists &amp; inhibitors/genetics ; },
abstract = {Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O[6]BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.},
}
@article {pmid31493817,
year = {2019},
author = {Li, Y and Xu, W and Jerman, S and Sun, Z},
title = {In vivo analysis of renal epithelial cells in zebrafish.},
journal = {Methods in cell biology},
volume = {154},
number = {},
pages = {163-181},
doi = {10.1016/bs.mcb.2019.04.016},
pmid = {31493817},
issn = {0091-679X},
support = {R01 DK113135/DK/NIDDK NIH HHS/United States ; R01 HL125885/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Polarity ; Cilia/metabolism/ultrastructure ; Embryo, Nonmammalian/anatomy &amp; histology/*cytology/metabolism ; Epithelial Cells/*cytology/metabolism ; Female ; Gene Editing/*methods ; Immunohistochemistry/*methods ; Kidney/*cytology/embryology/metabolism ; Male ; Organogenesis/genetics ; RNA, Guide/genetics/metabolism ; Zebrafish ; Zebrafish Proteins/deficiency/*genetics ; },
abstract = {The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.},
}
@article {pmid31493814,
year = {2019},
author = {Drake, KA and Fessler, AR and Carroll, TJ},
title = {Methods for renal lineage tracing: In vivo and beyond.},
journal = {Methods in cell biology},
volume = {154},
number = {},
pages = {121-143},
doi = {10.1016/bs.mcb.2019.06.002},
pmid = {31493814},
issn = {0091-679X},
support = {R01 DK080004/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage/*genetics ; Cell Tracking/*methods ; Doxycycline/pharmacology ; Epithelial Cells/cytology/metabolism ; Fluorescent Antibody Technique/*methods ; Gene Expression/drug effects ; *Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Homeostasis/genetics ; Integrases/*genetics/metabolism ; Kidney/*cytology/growth &amp; development/metabolism ; Luminescent Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Organogenesis/genetics ; Pluripotent Stem Cells/cytology/metabolism ; beta-Galactosidase/genetics/metabolism ; },
abstract = {Lineage tracing has resulted in fundamental discoveries in kidney development and disease and remains a powerful technique to study mechanisms of organogenesis, homeostasis, and repair/regeneration. Following decades of research on the cellular and molecular regulation of renal organogenesis, the kidney has become one of the most well-characterized organs, resulting in exciting advancements in pluripotent stem cell differentiation, tissue bioengineering, and the potential for developing novel regenerative therapies for kidney disease. Lineage tracing, or the labeling of progeny cells arising from a single cell or group of cells, allows for spatial and temporal analyses of dynamic in vivo and in vitro processes. As lineage tracing techniques expand across disciplines of developmental biology, stem cell biology, and regenerative medicine, careful experimental design and interpretation, along with an understanding of the basic principles and technical limitations, are essential for utilizing genetically complex lineage tracing models to further understand kidney development and disease.},
}
@article {pmid31493565,
year = {2019},
author = {Xu, Z and Xu, X and Gong, Q and Li, Z and Li, Y and Wang, S and Yang, Y and Ma, W and Liu, L and Zhu, B and Zou, L and Chen, G},
title = {Engineering Broad-Spectrum Bacterial Blight Resistance by Simultaneously Disrupting Variable TALE-Binding Elements of Multiple Susceptibility Genes in Rice.},
journal = {Molecular plant},
volume = {12},
number = {11},
pages = {1434-1446},
doi = {10.1016/j.molp.2019.08.006},
pmid = {31493565},
issn = {1752-9867},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Disease Resistance/*genetics ; Gene Editing/*methods ; Genetic Predisposition to Disease ; Mutation ; Oryza/*genetics/immunology/*microbiology ; Plant Diseases/immunology/*microbiology ; Transcription Activator-Like Effectors/*metabolism ; },
abstract = {Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, employs the transcription activator-like effectors (TALEs) to induce the expression of the OsSWEET family of putative sugar transporter genes, which function in conferring disease susceptibility (S) in rice plants. To engineer broad-spectrum bacterial blight resistance, we used CRISPR/Cas9-mediated gene editing to disrupt the TALE-binding elements (EBEs) of two S genes, OsSWEET11 and OsSWEET14, in rice cv. Kitaake, which harbors the recessive resistance allele of Xa25/OsSWEET13. The engineered rice line MS14K exhibited broad-spectrum resistance to most Xoo strains with a few exceptions, suggesting that the compatible strains may contain new TALEs. We identified two PthXo2-like TALEs, Tal5LN18 and Tal7PXO61, as major virulence factors in the compatible Xoo strains LN18 and PXO61, respectively, and found that Xoo encodes at least five types of PthXo2-like effectors. Given that PthXo2/PthXo2.1 target OsSWEET13 for transcriptional activation, the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter, and 10 Xa25-like haplotypes were identified. We found that Tal5LN18 and Tal7PXO61 bind slightly different EBE sequences in the OsSWEET13 promoter to activate its expression. CRISPR/Cas9 technology was then used to generate InDels in the EBE of the OsSWEET13 promoter in MS14K to creat a new germplasm with three edited OsSWEET EBEs and broad-spectrum resistance against all Xoo strains tested. Collectively, our findings illustrate how to disarm TALE-S co-evolved loci to generate broad-spectrum resistance through the loss of effector-triggered susceptibility in plants.},
}
@article {pmid31493470,
year = {2019},
author = {Filippova, J and Matveeva, A and Zhuravlev, E and Stepanov, G},
title = {Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems.},
journal = {Biochimie},
volume = {167},
number = {},
pages = {49-60},
doi = {10.1016/j.biochi.2019.09.003},
pmid = {31493470},
issn = {1638-6183},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Fungi/genetics ; Gene Editing/*methods ; Gene Expression ; Humans ; Plants/genetics ; *RNA, Guide/chemical synthesis/chemistry/genetics ; Transfection ; },
abstract = {Genome-editing technologies, in particular, CRISPR systems, are widely used for targeted regulation of gene expression and obtaining modified human and animal cell lines, plants, fungi, and animals with preassigned features. Despite being well described and easy to perform, the most common methods for construction and delivery of CRISPR/Cas9-containing plasmid systems possess significant disadvantages, mostly associated with effects of the presence of exogenous DNA within the cell. Transfection with active ribonucleoprotein complexes of Cas9 with single-guide RNAs (sgRNAs) represents one of the most promising options because of faster production of sgRNAs, the ability of a researcher to control the amount of sgRNA delivered into the cell, and consequently, fewer off-target mutations. Artificial-RNA synthesis strategies allow for the introduction of various modified components, such as backbone alterations, native structural motifs, and labels for visualization. Modifications of RNA can increase its resistance to hydrolysis, alter the thermodynamic stability of RNA-protein and RNA-DNA complexes, and reduce the immunogenic and cytotoxic effects. This review describes various approaches to improving synthetic guide RNA function through nucleotide modification.},
}
@article {pmid31492928,
year = {2019},
author = {Bradley, CA},
title = {In vivo T cell CRISPR screens reveal immunotherapeutic targets.},
journal = {Nature reviews. Cancer},
volume = {19},
number = {11},
pages = {606},
pmid = {31492928},
issn = {1474-1768},
mesh = {CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome ; Humans ; Immunotherapy ; },
}
@article {pmid31492863,
year = {2019},
author = {Gomez-Ospina, N and Scharenberg, SG and Mostrel, N and Bak, RO and Mantri, S and Quadros, RM and Gurumurthy, CB and Lee, C and Bao, G and Suarez, CJ and Khan, S and Sawamoto, K and Tomatsu, S and Raj, N and Attardi, LD and Aurelian, L and Porteus, MH},
title = {Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4045},
pmid = {31492863},
issn = {2041-1723},
support = {K08 NS102398/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD34/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/methods ; *Genome, Human ; Hematopoietic Stem Cell Transplantation/*methods ; Hematopoietic Stem Cells/*metabolism ; Humans ; Iduronidase/genetics/*metabolism ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Mucopolysaccharidosis I/genetics/pathology/*therapy ; NIH 3T3 Cells ; Phenotype ; Receptors, CCR5/genetics/metabolism ; Transplantation, Heterologous ; },
abstract = {Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.},
}
@article {pmid31492858,
year = {2019},
author = {Tycko, J and Wainberg, M and Marinov, GK and Ursu, O and Hess, GT and Ego, BK and Aradhana, and Li, A and Truong, A and Trevino, AE and Spees, K and Yao, D and Kaplow, IM and Greenside, PG and Morgens, DW and Phanstiel, DH and Snyder, MP and Bintu, L and Greenleaf, WJ and Kundaje, A and Bassik, MC},
title = {Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4063},
pmid = {31492858},
issn = {2041-1723},
support = {RM1 HG007735/HG/NHGRI NIH HHS/United States ; R35 GM128645/GM/NIGMS NIH HHS/United States ; R00 HG008662/HG/NHGRI NIH HHS/United States ; P50 HG007735/HG/NHGRI NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Epigenesis, Genetic/genetics ; Epigenomics/methods ; Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Genome, Human/*genetics ; HEK293 Cells ; Humans ; K562 Cells ; RNA, Guide/*genetics ; Regulatory Elements, Transcriptional/*genetics ; },
abstract = {Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.},
}
@article {pmid31492834,
year = {2019},
author = {Tuladhar, R and Yeu, Y and Tyler Piazza, J and Tan, Z and Rene Clemenceau, J and Wu, X and Barrett, Q and Herbert, J and Mathews, DH and Kim, J and Hyun Hwang, T and Lum, L},
title = {CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4056},
pmid = {31492834},
issn = {2041-1723},
support = {R01 GM076485/GM/NIGMS NIH HHS/United States ; R01 CA168761/CA/NCI NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Codon, Nonsense/genetics ; Frameshift Mutation ; Gene Editing/*methods ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; HeLa Cells ; Humans ; INDEL Mutation ; *Mutagenesis ; RNA Stability ; RNA, Messenger/chemistry/*genetics ; },
abstract = {The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.},
}
@article {pmid31492696,
year = {2019},
author = {Dong, Y and Li, H and Zhao, L and Koopman, P and Zhang, F and Huang, JX},
title = {Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {11},
pages = {3645-3651},
pmid = {31492696},
issn = {2160-1836},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; Gene Targeting ; Genome ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Receptors, Progesterone/genetics ; Whole Genome Sequencing ; },
abstract = {The emergence of the CRISPR-Cas9 system has triggered a technical revolution in mammalian genome editing. Compared to traditional gene-targeting strategies, CRISPR-Cas9 technology offers a more efficient and cost-effective approach for generating genetically modified animal models. However, off-target cleavage in CRISPR-mediated genome editing is a major concern in the analysis of phenotypes as well as the selection of therapeutic targets. Here, we analyzed whole-genome sequencing (WGS) data from two knock-out (KO) mouse strains generated by using the CRISPR-Cas9 system targeting the Mmd and Paqr8 loci. A total of nine individuals were sequenced including two parents, four F1 offspring and three uninjected control mice. Using GATK and bcftools software, we identified two off-target events in the founder mice. The two CRISPR-Cas9-induced off-target events were predictable using Cas-OFFinder and were not passed on to the offspring that we investigated. In addition, our results indicated that the number of CRISPR-Cas9-induced mutations was not statistically distinguishable from the background de novo mutations (DNMs).},
}
@article {pmid31492655,
year = {2019},
author = {Soto-Perez, P and Bisanz, JE and Berry, JD and Lam, KN and Bondy-Denomy, J and Turnbaugh, PJ},
title = {CRISPR-Cas System of a Prevalent Human Gut Bacterium Reveals Hyper-targeting against Phages in a Human Virome Catalog.},
journal = {Cell host &amp; microbe},
volume = {26},
number = {3},
pages = {325-335.e5},
pmid = {31492655},
issn = {1934-6069},
support = {DP5 OD021344/OD/NIH HHS/United States ; R01 HL122593/HL/NHLBI NIH HHS/United States ; },
mesh = {Actinobacteria/virology ; Bacteria/genetics/*virology ; Bacteriophages/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; DNA, Bacterial/analysis ; DNA, Viral/analysis ; Databases, Genetic ; Gastrointestinal Tract/*microbiology ; Genome, Bacterial ; Genome, Viral ; Humans ; Metagenomics ; Microbiota/genetics ; Sequence Analysis, DNA ; },
abstract = {Bacteriophages are abundant within the human gastrointestinal tract, yet their interactions with gut bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we show that the type I-C CRISPR-Cas system in the prevalent gut Actinobacterium Eggerthella lenta is transcribed and sufficient for specific targeting of foreign and chromosomal DNA. Comparative analyses of E. lenta CRISPR-Cas systems across (meta)genomes revealed 2 distinct clades according to cas sequence similarity and spacer content. We assembled a human virome database (HuVirDB), encompassing 1,831 samples enriched for viral DNA, to identify protospacers. This revealed matches for a majority of spacers, a marked increase over other databases, and uncovered "hyper-targeted" phage sequences containing multiple protospacers targeted by several E. lenta strains. Finally, we determined the positional mismatch tolerance of observed spacer-protospacer pairs. This work emphasizes the utility of merging computational and experimental approaches for determining the function and targets of CRISPR-Cas systems.},
}
@article {pmid31491085,
year = {2019},
author = {Hogan, AM and Rahman, ASMZ and Lightly, TJ and Cardona, ST},
title = {A Broad-Host-Range CRISPRi Toolkit for Silencing Gene Expression in Burkholderia.},
journal = {ACS synthetic biology},
volume = {8},
number = {10},
pages = {2372-2384},
doi = {10.1021/acssynbio.9b00232},
pmid = {31491085},
issn = {2161-5063},
support = {/CAPMC/CIHR/Canada ; },
mesh = {Bacterial Proteins/genetics ; Burkholderia/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Expression/*genetics ; Gene Silencing/*physiology ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans, and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne gRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi toolkit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderia's pathogenic arsenal.},
}
@article {pmid31488703,
year = {2019},
author = {Wang, H and Nakamura, M and Abbott, TR and Zhao, D and Luo, K and Yu, C and Nguyen, CM and Lo, A and Daley, TP and La Russa, M and Liu, Y and Qi, LS},
title = {CRISPR-mediated live imaging of genome editing and transcription.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6459},
pages = {1301-1305},
doi = {10.1126/science.aax7852},
pmid = {31488703},
issn = {1095-9203},
support = {U01 EB021240/EB/NIBIB NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; DNA/analysis ; DNA Breaks, Double-Stranded ; *Gene Editing ; Genetic Vectors ; HEK293 Cells ; Humans ; In Situ Hybridization, Fluorescence/*methods ; Microscopy, Fluorescence ; Molecular Imaging ; RNA/analysis ; RNA, Guide/genetics ; T-Lymphocytes ; },
abstract = {We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.},
}
@article {pmid31487454,
year = {2019},
author = {Tao, W and Chen, L and Zhao, C and Wu, J and Yan, D and Deng, Z and Sun, Y},
title = {In Vitro Packaging Mediated One-Step Targeted Cloning of Natural Product Pathway.},
journal = {ACS synthetic biology},
volume = {8},
number = {9},
pages = {1991-1997},
doi = {10.1021/acssynbio.9b00248},
pmid = {31487454},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; Biological Products/chemistry/*metabolism ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular ; Gene Editing ; Multigene Family ; Plasmids/genetics/metabolism ; Repressor Proteins/genetics ; Sisomicin/chemistry/metabolism ; Streptomyces/genetics ; Trans-Activators/genetics ; },
abstract = {Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tü3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tü3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways in conjunction with various gene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may be widely adopted for targeted cloning of the natural product pathways.},
}
@article {pmid31484740,
year = {2019},
author = {Shrivastava, R and Tupperwar, N and Drory-Retwitzer, M and Shapira, M},
title = {Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of Leishmania.},
journal = {mSphere},
volume = {4},
number = {5},
pages = {},
pmid = {31484740},
issn = {2379-5042},
mesh = {*Alleles ; Animals ; *CRISPR-Cas Systems ; *Gene Deletion ; Leishmania mexicana/genetics/*pathogenicity ; Macrophages/parasitology ; Mice ; Mutation ; Protozoan Proteins/*genetics ; RAW 264.7 Cells ; },
abstract = {The genomes of Leishmania and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical features and their biological behavior. We therefore hypothesize that each has a specialized function, not limited to protein synthesis. Of the six paralogs, LeishIF4E-3 has a weak cap-binding activity. It participates in the assembly of granules that store inactive transcripts and ribosomal proteins during nutritional stress that is experienced in the sand fly. We investigated the role of LeishIF4E-3 in Leishmania mexicana promastigotes using the CRISPR-Cas9 system. We deleted one of the two LeishIF4E-3 alleles, generating a heterologous deletion mutant with reduced LeishIF4E-3 expression. The mutant showed a decline in de novo protein synthesis and growth kinetics, altered morphology, and impaired infectivity. The mutant cells were rounded and failed to transform into the nectomonad-like form, in response to purine starvation. Furthermore, the infectivity of macrophage cells by the LeishIF4E-3(+/-) mutant was severely reduced. These phenotypic features were not observed in the addback cells, in which expression of LeishIF4E-3 was restored. The observed phenotypic changes correlated with the profile of transcripts associated with LeishIF4E-3. These were enriched for cytoskeleton- and flagellum-encoding genes, along with genes for RNA binding proteins. Our data illustrate the importance of LeishIF4E-3 in translation and in the parasite virulence.IMPORTANCELeishmania species are the causative agents of a spectrum of diseases. Available drug treatment is toxic and expensive, with drug resistance a growing concern. Leishmania parasites migrate between transmitting sand flies and mammalian hosts, experiencing unfavorable extreme conditions. The parasites therefore developed unique mechanisms for promoting a stage-specific program for gene expression, with translation playing a central role. There are six paralogs of the cap-binding protein eIF4E, which vary in their function, expression profiles, and assemblages. Using the CRISPR-Cas9 system for Leishmania, we deleted one of the two LeishIF4E-3 alleles. Expression of LeishIF4E-3 in the deletion mutant was low, leading to reduction in global translation and growth of the mutant cells. Cell morphology also changed, affecting flagellum growth, cell shape, and infectivity. The importance of this study is in highlighting that LeishIF4E-3 is essential for completion of the parasite life cycle. Our study gives new insight into how parasite virulence is determined.},
}
@article {pmid31484079,
year = {2019},
author = {van den Boogaard, M and van Weerd, JH and Bawazeer, AC and Hooijkaas, IB and van de Werken, HJG and Tessadori, F and de Laat, W and Barnett, P and Bakkers, J and Christoffels, VM},
title = {Identification and Characterization of a Transcribed Distal Enhancer Involved in Cardiac Kcnh2 Regulation.},
journal = {Cell reports},
volume = {28},
number = {10},
pages = {2704-2714.e5},
doi = {10.1016/j.celrep.2019.08.007},
pmid = {31484079},
issn = {2211-1247},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; ERG1 Potassium Channel/*genetics/metabolism ; Enhancer Elements, Genetic/*genetics ; Female ; *Gene Expression Regulation ; Genetic Loci ; Heart Ventricles/metabolism ; Humans ; Myocardium/*metabolism ; Promoter Regions, Genetic/genetics ; Protein Isoforms/genetics/metabolism ; RNA, Untranslated/genetics/metabolism ; Sequence Deletion ; *Transcription, Genetic ; Zebrafish ; },
abstract = {The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying IKr, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.},
}
@article {pmid31484075,
year = {2019},
author = {Zhang, R and Earnest, JT and Kim, AS and Winkler, ES and Desai, P and Adams, LJ and Hu, G and Bullock, C and Gold, B and Cherry, S and Diamond, MS},
title = {Expression of the Mxra8 Receptor Promotes Alphavirus Infection and Pathogenesis in Mice and Drosophila.},
journal = {Cell reports},
volume = {28},
number = {10},
pages = {2647-2658.e5},
pmid = {31484075},
issn = {2211-1247},
support = {R01 AI123348/AI/NIAID NIH HHS/United States ; P30 AR073752/AR/NIAMS NIH HHS/United States ; R01 AI095436/AI/NIAID NIH HHS/United States ; R01 AI143673/AI/NIAID NIH HHS/United States ; T32 AI007163/AI/NIAID NIH HHS/United States ; R01 AI114816/AI/NIAID NIH HHS/United States ; },
mesh = {Alphavirus/drug effects/*physiology ; Animals ; Antibodies, Monoclonal/pharmacology ; Arthritis/pathology/virology ; CRISPR-Cas Systems/genetics ; Chikungunya Fever/metabolism/pathology ; Chikungunya virus/drug effects/genetics ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/drug effects/*virology ; Immunoglobulins/deficiency/*metabolism ; Inflammation/pathology ; Membrane Proteins/deficiency/*metabolism ; Mice, Inbred C57BL ; Mutation/genetics ; },
abstract = {Mxra8 is a recently described receptor for multiple alphaviruses, including Chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), and O'nyong nyong (ONNV) viruses. To determine its role in pathogenesis, we generated mice with mutant Mxra8 alleles: an 8-nucleotide deletion that produces a truncated, soluble form (Mxra8[Δ8/Δ8]) and a 97-nucleotide deletion that abolishes Mxra8 expression (Mxra8[Δ97/Δ97]). Mxra8[Δ8/Δ8] and Mxra8[Δ97/Δ97] fibroblasts show reduced CHIKV infection in culture, and Mxra8[Δ8/Δ8] and Mxra8[Δ97/Δ97] mice have decreased infection of musculoskeletal tissues with CHIKV, MAYV, RRV, or ONNV. Less foot swelling is observed in CHIKV-infected Mxra8 mutant mice, which correlated with fewer infiltrating neutrophils and cytokines. A recombinant E2-D71A CHIKV with diminished binding to Mxra8 is attenuated in vivo in wild-type mice. Ectopic Mxra8 expression is sufficient to enhance CHIKV infection and lethality in transgenic flies. These studies establish a role for Mxra8 in the pathogenesis of multiple alphaviruses and suggest that targeting this protein may mitigate disease in humans.},
}
@article {pmid31483929,
year = {2020},
author = {de Maagd, RA and Loonen, A and Chouaref, J and Pelé, A and Meijer-Dekens, F and Fransz, P and Bai, Y},
title = {CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid.},
journal = {Plant biotechnology journal},
volume = {18},
number = {3},
pages = {805-813},
pmid = {31483929},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Crossing Over, Genetic ; Gene Knockout Techniques ; Hybridization, Genetic ; Lycopersicon esculentum/*genetics ; Meiosis ; Plant Breeding ; RecQ Helicases/*genetics ; },
abstract = {Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9-mutagenesis in an interspecific tomato hybrid to knockout RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared with the wild-type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53-fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8-fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. SIGNIFICANCE STATEMENT: Increasing crossover frequency during meiosis can speed up or simplify crop breeding that relies on meiotic crossovers to introduce favourable alleles controlling important traits from wild relatives into crops. Here we show for the first time that knocking out an inhibitor of crossovers in an interspecific hybrid between tomato and its relative wild species using CRISPR/Cas9-mutagenesis results in increased recombination between the two genomes.},
}
@article {pmid31482512,
year = {2019},
author = {Dumeau, CE and Monfort, A and Kissling, L and Swarts, DC and Jinek, M and Wutz, A},
title = {Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1.},
journal = {Transgenic research},
volume = {28},
number = {5-6},
pages = {525-535},
pmid = {31482512},
issn = {1573-9368},
mesh = {Animals ; Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Electroporation ; Endodeoxyribonucleases/*genetics ; *Gene Deletion ; *Gene Transfer Techniques ; Genome/genetics ; Mice ; Mouse Embryonic Stem Cells/metabolism ; Mutation/genetics ; RNA, Guide/genetics ; Zona Pellucida/metabolism ; Zygote/growth &amp; development ; },
abstract = {CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.},
}
@article {pmid31481729,
year = {2018},
author = {},
title = {How to respond to CRISPR babies.},
journal = {Nature},
volume = {564},
number = {7734},
pages = {5},
doi = {10.1038/d41586-018-07634-0},
pmid = {31481729},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Embryo Research/ethics/legislation &amp; jurisprudence ; *Ethics, Research ; Female ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Genetic Therapy ; Germ-Line Mutation/*ethics ; Humans ; Infant, Newborn ; Internationality ; Leadership ; Mitochondrial Replacement Therapy/legislation &amp; jurisprudence ; *Registries/standards ; Research Personnel/ethics/*legislation &amp; jurisprudence ; Twins/*genetics ; },
}
@article {pmid31481684,
year = {2019},
author = {Kumita, W and Sato, K and Suzuki, Y and Kurotaki, Y and Harada, T and Zhou, Y and Kishi, N and Sato, K and Aiba, A and Sakakibara, Y and Feng, G and Okano, H and Sasaki, E},
title = {Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {12719},
pmid = {31481684},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; *CRISPR-Cas Systems ; Callithrix ; *Embryo, Mammalian ; *Gene Editing ; *Gene Knock-In Techniques ; *Gene Knockout Techniques ; },
abstract = {Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.},
}
@article {pmid31481656,
year = {2019},
author = {Young, J and Dominicus, C and Wagener, J and Butterworth, S and Ye, X and Kelly, G and Ordan, M and Saunders, B and Instrell, R and Howell, M and Stewart, A and Treeck, M},
title = {A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3963},
pmid = {31481656},
issn = {2041-1723},
support = {FC001189/CRUK_/Cancer Research UK/United Kingdom ; FC001189/WT_/Wellcome Trust/United Kingdom ; FC001189/MRC_/Medical Research Council/United Kingdom ; R01 AI123457/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques/methods ; Gene Library ; Genome, Protozoan ; Humans ; Mice, Inbred C57BL ; RNA, Guide ; Toxoplasma/*genetics/pathogenicity ; Toxoplasmosis/genetics/parasitology/pathology ; Virulence Factors/*genetics ; },
abstract = {Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1.},
}
@article {pmid31481541,
year = {2019},
author = {Qu, J and Prasad, NK and Yu, MA and Chen, S and Lyden, A and Herrera, N and Silvis, MR and Crawford, E and Looney, MR and Peters, JM and Rosenberg, OS},
title = {Modulating Pathogenesis with Mobile-CRISPRi.},
journal = {Journal of bacteriology},
volume = {201},
number = {22},
pages = {},
pmid = {31481541},
issn = {1098-5530},
support = {K22 AI137122/AI/NIAID NIH HHS/United States ; R01 AI125445/AI/NIAID NIH HHS/United States ; R01 AI128214/AI/NIAID NIH HHS/United States ; R01 EB024014/EB/NIBIB NIH HHS/United States ; T32 GM064337/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockdown Techniques ; Genes, Bacterial ; Immunoblotting ; Male ; Mice ; Mice, Inbred C57BL ; Pneumonia, Bacterial/metabolism/*microbiology ; Pseudomonas Infections/metabolism/*microbiology ; Pseudomonas aeruginosa/genetics/*pathogenicity ; Reverse Transcriptase Polymerase Chain Reaction ; Type III Secretion Systems/genetics ; },
abstract = {Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.},
}
@article {pmid31481454,
year = {2019},
author = {Weisser, M and Ban, N},
title = {Extensions, Extra Factors, and Extreme Complexity: Ribosomal Structures Provide Insights into Eukaryotic Translation.},
journal = {Cold Spring Harbor perspectives in biology},
volume = {11},
number = {9},
pages = {},
pmid = {31481454},
issn = {1943-0264},
mesh = {Animals ; *CRISPR-Cas Systems ; Eukaryotic Cells/*physiology ; Gene Editing ; Genome, Archaeal ; Genome, Bacterial ; Models, Molecular ; Phenotype ; *Protein Biosynthesis ; Protein Conformation ; RNA Splicing ; RNA, Long Noncoding/genetics ; Ribosomes/*physiology ; Transcription, Genetic ; },
abstract = {Although the basic aspects of protein synthesis are preserved in all kingdoms of life, there are many important structural and functional differences between bacterial and the more complex eukaryotic ribosomes. High-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography structures of eukaryotic ribosomes have revealed the complex architectures of eukaryotic ribosomes and species-specific variations in protein and ribosomal RNA (rRNA) extensions. They also enabled structural studies of a range of eukaryotic ribosomal complexes involved in translation initiation, elongation, and termination, revealing unique mechanistic features of the eukaryotic translation process, especially with respect to the identification and recognition of translation start and stop codons on messenger RNAs (mRNAs). Most recently, structural biology has provided insights into the eukaryotic ribosomal biogenesis pathway by visualizing several of its complex intermediates. This review highlights the past decade's structural work on eukaryotic ribosomes and its implications on our understanding of eukaryotic translation.},
}
@article {pmid31480854,
year = {2019},
author = {van Bodegraven, EJ and van Asperen, JV and Sluijs, JA and van Deursen, CBJ and van Strien, ME and Stassen, OMJA and Robe, PAJ and Hol, EM},
title = {GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {11},
pages = {12941-12959},
doi = {10.1096/fj.201900916R},
pmid = {31480854},
issn = {1530-6860},
mesh = {*Alternative Splicing ; Brain Neoplasms/metabolism/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Dual-Specificity Phosphatases/genetics/*physiology ; Extracellular Matrix/metabolism/*pathology ; Gene Knockdown Techniques ; Glial Fibrillary Acidic Protein/*genetics ; Glioma/metabolism/*pathology ; Humans ; Laminin/metabolism ; MAP Kinase Kinase 4/metabolism ; Mitogen-Activated Protein Kinase Phosphatases/genetics/*physiology ; Phosphorylation ; },
abstract = {Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.},
}
@article {pmid31480315,
year = {2019},
author = {Zhang, S and Zhang, R and Gao, J and Gu, T and Song, G and Li, W and Li, D and Li, Y and Li, G},
title = {Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacterium tumefaciens-Mediated CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {20},
number = {17},
pages = {},
pmid = {31480315},
issn = {1422-0067},
mesh = {Agrobacterium/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Genes, Plant ; Genetic Vectors/metabolism ; Genotype ; Inheritance Patterns/*genetics ; Mutagenesis/*genetics ; Mutation Rate ; RNA, Guide/genetics ; Triticum/*genetics ; },
abstract = {The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.},
}
@article {pmid31479876,
year = {2019},
author = {Malerba, N and Benzoni, P and Squeo, GM and Milanesi, R and Giannetti, F and Sadleir, LG and Poke, G and Augello, B and Croce, AI and Barbuti, A and Merla, G},
title = {Generation of the induced human pluripotent stem cell lines CSSi009-A from a patient with a GNB5 pathogenic variant, and CSSi010-A from a CRISPR/Cas9 engineered GNB5 knock-out human cell line.},
journal = {Stem cell research},
volume = {40},
number = {},
pages = {101547},
doi = {10.1016/j.scr.2019.101547},
pmid = {31479876},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line/cytology/*metabolism ; Cells, Cultured ; Child ; Female ; Fibroblasts/cytology/metabolism ; Frameshift Mutation ; GTP-Binding Protein beta Subunits/*genetics/metabolism ; Gene Editing ; Gene Knockout Techniques ; Genetic Diseases, Inborn/*genetics/metabolism/physiopathology ; Genetic Engineering ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Male ; Middle Aged ; },
abstract = {GNB5 loss-of-function pathogenic variants cause IDDCA, a rare autosomal recessive human genetic disease characterized by infantile onset of intellectual disability, sinus bradycardia, hypotonia, visual abnormalities, and epilepsy. We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a patient with the homozygous c.136delG frameshift variant, and a GNB5 knock-out (KO) line by CRISPR/Cas9 editing. hiPSCs express common pluripotency markers and differentiate into the three germ layers. These lines represent a powerful cellular model to study the molecular basis of GNB5-related disorders as well as offer an in vitro model for drug screening.},
}
@article {pmid31478642,
year = {2019},
author = {Li, Y and Mansour, H and Wang, T and Poojari, S and Li, F},
title = {Naked-Eye Detection of Grapevine Red-Blotch Viral Infection Using a Plasmonic CRISPR Cas12a Assay.},
journal = {Analytical chemistry},
volume = {91},
number = {18},
pages = {11510-11513},
doi = {10.1021/acs.analchem.9b03545},
pmid = {31478642},
issn = {1520-6882},
mesh = {Bacterial Proteins/chemistry ; CRISPR-Associated Proteins/chemistry ; CRISPR-Cas Systems ; Colorimetry/*methods ; DNA, Single-Stranded/chemistry ; DNA, Viral/analysis/chemistry ; Endodeoxyribonucleases/chemistry ; Geminiviridae/genetics/*isolation &amp; purification ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Plant Diseases/*virology ; Polymerase Chain Reaction ; Vitis/*virology ; },
abstract = {Herein, we described a novel plasmonic CRISPR Cas12a assay for the visual, colorimetric detection of grapevine viral infections. Our assay generates rapid and specific colorimetric signals for nucleic acid amplicons by combining the unique target-induced incriminate single-stranded DNase activity of Cas12a with plasmon coupling of DNA functionalized gold nanoparticles. The practical applicability of our plasmonic assay was successfully demonstrated through the detection of emerging red-blotch viral infections in grapevine samples collected from commercial vineyards.},
}
@article {pmid31477929,
year = {2019},
author = {McFaline-Figueroa, JL and Hill, AJ and Qiu, X and Jackson, D and Shendure, J and Trapnell, C},
title = {A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition.},
journal = {Nature genetics},
volume = {51},
number = {9},
pages = {1389-1398},
pmid = {31477929},
issn = {1546-1718},
support = {T32 HL007828/HL/NHLBI NIH HHS/United States ; R01 HL118342/HL/NHLBI NIH HHS/United States ; U54 HL145611/HL/NHLBI NIH HHS/United States ; DP2 HD088158/HD/NICHD NIH HHS/United States ; RC2 DK114777/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Epithelial-Mesenchymal Transition/*genetics ; ErbB Receptors/genetics/metabolism ; *Gene Regulatory Networks ; *Genetic Testing ; Humans ; MAP Kinase Kinase 1/genetics/metabolism ; Mammary Glands, Human/*metabolism/pathology ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; Signal Transduction ; Single-Cell Analysis/*methods ; Small Molecule Libraries/pharmacology ; Transcriptome ; Transforming Growth Factor beta/genetics/metabolism ; },
abstract = {Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-β. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.},
}
@article {pmid31477922,
year = {2019},
author = {Grünewald, J and Zhou, R and Iyer, S and Lareau, CA and Garcia, SP and Aryee, MJ and Joung, JK},
title = {CRISPR DNA base editors with reduced RNA off-target and self-editing activities.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1041-1048},
pmid = {31477922},
issn = {1546-1696},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {APOBEC Deaminases/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Cloning, Molecular ; Flow Cytometry ; Gene Editing/*methods ; Gene Expression Regulation, Enzymologic ; Gene Targeting ; HEK293 Cells ; Humans ; Petromyzon ; Protein Conformation ; RNA ; RNA, Guide/genetics ; Streptococcus pyogenes ; Transcriptome ; },
abstract = {Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations[1-4]. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases[5], and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity[5]. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE[6], human activation-induced cytidine deaminase-based CBE[7], and the Petromyzon marinus cytidine deaminase-based CBE Target-AID[4] induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.},
}
@article {pmid31477858,
year = {2019},
author = {Crunkhorn, S},
title = {Expanding the RNA-editing toolbox.},
journal = {Nature reviews. Drug discovery},
volume = {18},
number = {9},
pages = {667},
doi = {10.1038/d41573-019-00128-2},
pmid = {31477858},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; RNA ; *RNA Editing ; },
}
@article {pmid31477243,
year = {2020},
author = {Butt, H and Zaidi, SS and Hassan, N and Mahfouz, M},
title = {CRISPR-Based Directed Evolution for Crop Improvement.},
journal = {Trends in biotechnology},
volume = {38},
number = {3},
pages = {236-240},
doi = {10.1016/j.tibtech.2019.08.001},
pmid = {31477243},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/drug effects/*genetics ; DNA Repair ; Directed Molecular Evolution/*methods ; Gene Editing ; Herbicide Resistance/genetics ; Plants, Genetically Modified ; },
abstract = {Directed evolution involves generating diverse sequence variants of a gene of interest to produce a desirable trait under selective pressure. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) systems can be programmed to target any genomic locus and perform targeted directed evolution. Here, we discuss the opportunities and challenges of this emerging platform for targeted crop improvement.},
}
@article {pmid31476099,
year = {2020},
author = {Xing, WQ and Ma, SY and Liu, YY and Xia, QY},
title = {CRISPR/dCas9-mediated imaging of endogenous genomic loci in living Bombyx mori cells.},
journal = {Insect science},
volume = {27},
number = {6},
pages = {1360-1364},
doi = {10.1111/1744-7917.12722},
pmid = {31476099},
issn = {1744-7917},
mesh = {Animals ; Bombyx/*genetics ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line ; Fibroins/*genetics ; Genes, Insect ; Genome ; Molecular Imaging/methods ; },
}
@article {pmid31475782,
year = {2020},
author = {Wen, Z and Lu, M and Ledesma-Amaro, R and Li, Q and Jin, M and Yang, S},
title = {TargeTron Technology Applicable in Solventogenic Clostridia: Revisiting 12 Years' Advances.},
journal = {Biotechnology journal},
volume = {15},
number = {1},
pages = {e1900284},
doi = {10.1002/biot.201900284},
pmid = {31475782},
issn = {1860-7314},
mesh = {Acetone/metabolism ; Butanols/metabolism ; CRISPR-Cas Systems ; *Clostridium/genetics/metabolism ; Fermentation/physiology ; *Gene Editing ; Metabolic Engineering/*methods ; Solvents/*metabolism ; },
abstract = {Clostridium has great potential in industrial application and medical research. But low DNA repair capacity and plasmids transformation efficiency severely delay development and application of genetic tools based on homologous recombination (HR). TargeTron is a gene editing technique dependent on the mobility of group II introns, rather than homologous recombination, which makes it very suitable for gene disruption of Clostridium. The application of TargeTron technology in solventogenic Clostridium is academically reported in 2007 and this tool has been introduced in various clostridia as it is easy to operate, time saving, and reliable. TargeTron has made great progress in solventogenic Clostridium in the aspects of acetone-butanol-ethanol (ABE) fermentation pathway modification, important functional genes identification, and xylose metabolic pathway analysis and reconstruction. In the review, 12 years' advances of TargeTron technology applicable in solventogenic Clostridium, including its principle, technical characteristics, application, and efforts to expand its capabilities, or to avoid potential drawbacks, are revisisted. Some other technologies as putative competitors or collaborators are also discussed. It is believed that TargeTron combined with CRISPR/Cas-assisted gene/base editing and gene-expression regulation system will make a better future for clostridial genetic modification.},
}
@article {pmid31474367,
year = {2019},
author = {Stanley, SY and Borges, AL and Chen, KH and Swaney, DL and Krogan, NJ and Bondy-Denomy, J and Davidson, AR},
title = {Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription.},
journal = {Cell},
volume = {178},
number = {6},
pages = {1452-1464.e13},
pmid = {31474367},
issn = {1097-4172},
support = {T32 AI060537/AI/NIAID NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; DP5 OD021344/OD/NIH HHS/United States ; U01 MH115747/MH/NIMH NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/virology ; Promoter Regions, Genetic/genetics ; Pseudomonas aeruginosa/virology ; Transcription Factors/genetics ; Transcription, Genetic ; Viral Proteins/*genetics ; },
abstract = {Phages express anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems that would otherwise destroy their genomes. Most acr genes are located adjacent to anti-CRISPR-associated (aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. The conservation of aca genes has served as a signpost for the identification of acr genes, but the function of the proteins encoded by these genes has not been investigated. Here we reveal that an acr-associated promoter drives high levels of acr transcription immediately after phage DNA injection and that Aca proteins subsequently repress this transcription. Without Aca activity, this strong transcription is lethal to a phage. Our results demonstrate how sufficient levels of Acr proteins accumulate early in the infection process to inhibit existing CRISPR-Cas complexes in the host cell. They also imply that the conserved role of Aca proteins is to mitigate the deleterious effects of strong constitutive transcription from acr promoters.},
}
@article {pmid31472270,
year = {2020},
author = {Ziegler, H and Nellen, W},
title = {CRISPR-Cas experiments for schools and the public.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {86-94},
doi = {10.1016/j.ymeth.2019.08.009},
pmid = {31472270},
issn = {1095-9130},
mesh = {Adolescent ; Adult ; Aged ; CRISPR-Cas Systems/*genetics ; Chromogenic Compounds/metabolism ; Color ; DNA Breaks, Double-Stranded ; Education/*methods ; Escherichia coli/enzymology/genetics ; Escherichia coli Proteins/genetics/metabolism ; Galactosides/metabolism ; Gene Editing/*methods ; Humans ; Indoles/metabolism ; Lac Operon/genetics ; Middle Aged ; Molecular Biology/*education ; Plasmids/genetics ; Schools ; Students ; Young Adult ; beta-Galactosidase/genetics/metabolism ; },
abstract = {The "gene scissors" CRISPR-Cas currently revolutionize the field of molecular biology with an enormous impact on society due to the broad application potentials in biomedicine, biotechnology and agriculture. We have developed simple CRISPR-Cas experiments that can serve to introduce pupils, students and non-scientists alike to the fascinating power of targeted gene editing. The experimental course is divided into two parts. In part 1, we target plasmid borne lacZ to convert blue E. coli to white E. coli. In part 2, we analyse the CRISPR-Cas9 mediated double strand breaks in the lacZ gene by a) colony PCR, b) colony cracking gel or c) restriction digest of the plasmids. Experimental work is embedded in short theoretical lecture parts that provide background of CRISPR-Cas and a step-by-step tutorial for the practical work. Though the experiment is robust, inexpensive and simple it should be noted that guidance by an expert instructor is required. Based on our experience, a full day lab course has a positive influence on the participants' attitude towards research in general. This is true for high school students as well as non-scientists (age groups 16-70 years).},
}
@article {pmid31471561,
year = {2020},
author = {Balligand, T and Achouri, Y and Pecquet, C and Gaudray, G and Colau, D and Hug, E and Rahmani, Y and Stroobant, V and Plo, I and Vainchenker, W and Kralovics, R and Van den Eynde, BJ and Defour, JP and Constantinescu, SN},
title = {Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development.},
journal = {Leukemia},
volume = {34},
number = {2},
pages = {510-521},
pmid = {31471561},
issn = {1476-5551},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calreticulin/*genetics ; Exons/*genetics ; Female ; Frameshift Mutation/genetics ; Heart/*physiology ; Hematopoiesis/genetics ; Homozygote ; Male ; Mice ; Primary Myelofibrosis/genetics ; Receptors, Thrombopoietin/genetics ; Thrombocythemia, Essential/*genetics ; Thrombocytosis/genetics ; },
abstract = {Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.},
}
@article {pmid31471004,
year = {2019},
author = {The Lancet Haematology, },
title = {CRISPR-Cas9 gene editing for patients with haemoglobinopathies.},
journal = {The Lancet. Haematology},
volume = {6},
number = {9},
pages = {e438},
doi = {10.1016/S2352-3026(19)30169-3},
pmid = {31471004},
issn = {2352-3026},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Carrier Proteins/genetics ; Gene Editing ; Hemoglobinopathies/genetics/*pathology ; Humans ; Mice ; Nuclear Proteins/genetics ; Repressor Proteins ; },
}
@article {pmid31470649,
year = {2019},
author = {Han, X and Xiong, Y and Zhao, C and Xie, S and Li, C and Li, X and Liu, X and Li, K and Zhao, S and Ruan, J},
title = {Identification of Glyceraldehyde-3-Phosphate Dehydrogenase Gene as an Alternative Safe Harbor Locus in Pig Genome.},
journal = {Genes},
volume = {10},
number = {9},
pages = {},
pmid = {31470649},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Knock-In Techniques/adverse effects/*methods ; Genome ; Glyceraldehyde-3-Phosphate Dehydrogenases/*genetics ; Swine/*genetics ; },
abstract = {The ectopic overexpression of foreign genes in animal genomes is an important strategy for gain-of-function study and establishment of transgenic animal models. Previous studies showed that two loci (Rosa26 and pH11) were identified as safe harbor locus in pig genomes, which means foreign genes can be integrated into this locus for stable expression. Moreover, integration of a transgene may interfere with the endogenous gene expression of the target locus after the foreign fragments are inserted. Here, we provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination. This strategy has no influence on the expression of the endogenous GAPDH gene. Thus, the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knock-ins. This strategy is promising for agricultural breeding and biomedical model applications.},
}
@article {pmid31470095,
year = {2020},
author = {Dong, L and Yu, D and Lin, X and Wang, B and Pan, L},
title = {Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization.},
journal = {Protein expression and purification},
volume = {165},
number = {},
pages = {105482},
doi = {10.1016/j.pep.2019.105482},
pmid = {31470095},
issn = {1096-0279},
mesh = {Ascomycota/*genetics ; Aspergillus niger/*genetics ; CRISPR-Cas Systems ; Chromatography, Gel ; Enzyme Stability ; Fermentation ; Gene Expression ; Hot Temperature ; Hydrogen-Ion Concentration ; Recombinant Proteins/*chemistry/*genetics ; Sequence Analysis, Protein ; Transfection ; Trehalase/*chemistry/*genetics ; },
abstract = {Trehalase catalyzes the conversion of one molecule of trehalose into two glucose molecules. The trehalase TreM from thermophilic fungus Myceliophthora sepedonium was expressed in Aspergillus niger via traditional homologous recombination with trehalase activity of 406.44 U/mL. The multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of the TreM trehalase in Aspergillus niger, which was up to 1943.06 U/mL with a low-background of secreted proteins, 4.8-fold than the transformant obtained via the traditional method. The highest recombinant trehalase activity of the shake fermentation supernatant achieved 4268.29 U/mL when 1.5% glucose was added. Activity assaying showed that the recombinant TreM possessed a specific activity of 679.09 U/mg after gel filtration chromatography purification. The recombinant TreM displayed optimal activity at pH 5.6 and 60 °C and exhibited prominent stability under the conditions of 45-50 °C and pH 4.0-7.5. The activity of recombinant TreM was strongly enhanced by Co[2+] (1, 5 mM), Cu[2+] (1 mM), Mn[2+] (1, 5 mM) and ATP (5 mM), and was greatly inhibited by Cu[2+] (10 mM), EDTA (10 mM) and SDS (10 mM).},
}
@article {pmid31469969,
year = {2020},
author = {Emmer, BT and Ginsburg, D},
title = {Genome Editing and Hematologic Malignancy.},
journal = {Annual review of medicine},
volume = {71},
number = {},
pages = {71-83},
doi = {10.1146/annurev-med-052318-100741},
pmid = {31469969},
issn = {1545-326X},
support = {K08 HL148552/HL/NHLBI NIH HHS/United States ; R35 HL135793/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematologic Neoplasms/diagnosis/*genetics/mortality/therapy ; Humans ; Male ; Neoplasm Invasiveness/pathology ; Neoplasm Staging ; Prognosis ; Risk Assessment ; Survival Analysis ; Treatment Outcome ; },
abstract = {The modern genomic era has seen remarkable advancement in our understanding of the molecular basis for disease, yet translation of basic discoveries into new disease treatments has arguably lagged behind. Recently, breakthroughs in genome editing technologies have created hope for their potential to directly treat the genetic causes of disease. Like any therapeutic intervention, genome editing should be considered in light of its potential risks and benefits. In this review, we highlight the promise of genome editing therapies, as well as the conceptual and technical barriers to their clinical application, with a special emphasis on hematologic malignancies.},
}
@article {pmid31469541,
year = {2019},
author = {Ju, E and Li, T and Ramos da Silva, S and Gao, SJ},
title = {Gold Nanocluster-Mediated Efficient Delivery of Cas9 Protein through pH-Induced Assembly-Disassembly for Inactivation of Virus Oncogenes.},
journal = {ACS applied materials &amp; interfaces},
volume = {11},
number = {38},
pages = {34717-34724},
pmid = {31469541},
issn = {1944-8252},
support = {R01 CA197153/CA/NCI NIH HHS/United States ; R01 CA096512/CA/NCI NIH HHS/United States ; R01 DE025465/DE/NIDCR NIH HHS/United States ; R01 CA132637/CA/NCI NIH HHS/United States ; R01 CA124332/CA/NCI NIH HHS/United States ; R01 CA213275/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/pharmacology ; Female ; *Genes, Viral ; *Gold/chemistry/pharmacology ; HeLa Cells ; Humans ; Hydrogen-Ion Concentration ; *Metal Nanoparticles/chemistry/therapeutic use ; *Oncogenes ; Tumor Suppressor Protein p53/*metabolism ; *Uterine Cervical Neoplasms/drug therapy/genetics/metabolism/virology ; },
abstract = {The CRISPR/Cas gene editing system has been successfully applied to combating bacteria, cancer, virus, and genetic disorders. While viral vectors have been used for the delivery of the CRISPR/Cas9 system, the time required for insert cloning, and virus packaging and standardization, hinders its efficient use. Additionally, the high molecular weight of the Cas9 endonuclease makes it not easy for packing into the vehicles. Herein we report the self-assembly of gold nanoclusters (AuNCs) with SpCas9 protein (SpCas9-AuNCs) under physiological conditions and the efficient delivery of SpCas9 into the cell nucleus. This assembly process is highly dependent on pH. SpCas9-AuNCs are stable at a higher pH but are disassembled at a lower pH. Significantly, this assembly-disassembly process facilitates the delivery of SpCas9 into cells and the cell nucleus, where the SpCas9 exerts its cleavage function. As a proof-of-concept, the assembled SpCas9-AuNCs nanoparticles are successfully used for efficient knockout of the E6 oncogene, restoring the function of tumor-suppressive protein p53 and inducing apoptosis in cervical cancer cells with little effect on normal human cells. The SpCas9-AuNCs are useful for sgRNA functional validation, sgRNA library screening, and genomic manipulation.},
}
@article {pmid31468487,
year = {2020},
author = {Penewit, K and Salipante, SJ},
title = {Genome Editing in Staphylococcus aureus by Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2050},
number = {},
pages = {127-143},
doi = {10.1007/978-1-4939-9740-4_14},
pmid = {31468487},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Electroporation/*methods ; Gene Editing/*methods ; Oligonucleotides/administration &amp; dosage ; Plasmids/administration &amp; dosage ; Staphylococcus aureus/genetics/*growth &amp; development ; Transfection/methods ; },
abstract = {Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered experimental studies of this organism. We recently developed a system for recombineering and CRISPR/Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing precise genomic modifications into the organism and for performing lethal counterselection of unedited cells. These techniques make it possible to scalably and inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and detail general principles which are broadly applicable across a range of organisms for which equivalent systems have been established.},
}
@article {pmid31468486,
year = {2020},
author = {Takemoto, T},
title = {Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically Modified Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2050},
number = {},
pages = {121-126},
doi = {10.1007/978-1-4939-9740-4_13},
pmid = {31468486},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified/growth &amp; development ; CRISPR-Associated Protein 9/*administration &amp; dosage ; CRISPR-Cas Systems ; Electroporation/*methods ; Female ; Male ; Mice ; RNA, Guide/*administration &amp; dosage ; Zygote/chemistry/*growth &amp; development ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for generation of genetically modified mice. In conventional protocols, Cas9 protein (or mRNA) and sgRNA are introduced into zygotes by microinjection. However, microinjection requires special skill and is too time-consuming to treat zygotes on a large scale. Recently, we have developed a simple electroporation method which generates genetically modified mice with high efficiency. Here, we describe our method GEEP (genome editing by electroporation of Cas9 protein). This method facilitates high-throughput genetic analysis of the mouse. This chapter describes the GEEP method to generate genetically modified mice.},
}
@article {pmid31468482,
year = {2020},
author = {Yu, J},
title = {Electroporation of CRISPR-Cas9 into Malignant B Cells for Loss-of-Function Studies of Target Gene Via Knockout.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2050},
number = {},
pages = {85-90},
doi = {10.1007/978-1-4939-9740-4_9},
pmid = {31468482},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Electroporation/*instrumentation ; Gene Editing ; Gene Knockout Techniques/*instrumentation ; Humans ; Loss of Function Mutation ; Lymphoma, B-Cell/*genetics ; Receptor Tyrosine Kinase-like Orphan Receptors/*genetics ; Transfection/instrumentation ; },
abstract = {CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit genomic DNA for studying biology, pathogenesis, and molecular basis of treatment in malignant B cells. Unfortunately, malignant B cells are extremely difficult to transfect by most traditional methods. In this chapter, we describe the use of the Nucleofector™ Technology-based electroporation system with optimized transfection conditions for generating a malignant B cell model, JEKO-1, with ROR1-gene knockout via CRISPR-Cas9 technology.},
}
@article {pmid31468479,
year = {2020},
author = {Espinosa, HD and Mukherjee, P and Patino, C},
title = {Nanofountain Probe Electroporation for Monoclonal Cell Line Generation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2050},
number = {},
pages = {59-68},
doi = {10.1007/978-1-4939-9740-4_6},
pmid = {31468479},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques ; Clone Cells/chemistry/*cytology ; Electroporation/*instrumentation ; Gene Editing/methods ; HEK293 Cells ; Humans ; Single-Cell Analysis ; Transfection/*instrumentation ; },
abstract = {In the field of genetic engineering, the modification of genes to produce stable cell lines has a variety of applications ranging from the development of novel therapeutics to patient specific treatments. To successfully generate a cell line, the gene of interest must be delivered into the cell and integrated into the genome. The efficiency of cell line generation systems therefore depends on the efficiency of delivery of genetically modifying molecules such as plasmids and CRISPR/CAS9 complexes. In this work, we describe a localized electroporation-based system to generate stable monoclonal cell lines. By employing the nanofountain probe electroporation (NFP-E) system, single cells in patterned cultures are selectively transfected with plasmids, grown, and harvested to obtain stably expressing cell lines. Methods for microcontact printing, cell culture, electroporation, and harvesting are detailed in this chapter.},
}
@article {pmid31468291,
year = {2019},
author = {Yan, F and Wang, W and Zhang, J},
title = {CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR-Cas9.},
journal = {Cell biology and toxicology},
volume = {35},
number = {6},
pages = {489-492},
doi = {10.1007/s10565-019-09489-1},
pmid = {31468291},
issn = {1573-6822},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endodeoxyribonucleases/*metabolism ; Gene Editing/*methods/trends ; Humans ; RNA, Guide/genetics/metabolism ; },
}
@article {pmid31467430,
year = {2020},
author = {Kweon, J and Jang, AH and Shin, HR and See, JE and Lee, W and Lee, JW and Chang, S and Kim, K and Kim, Y},
title = {A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants.},
journal = {Oncogene},
volume = {39},
number = {1},
pages = {30-35},
pmid = {31467430},
issn = {1476-5594},
mesh = {BRCA1 Protein/*genetics ; Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Cytosine/chemistry ; DNA Repair/genetics ; Exons/genetics ; Female ; Gene Editing ; Genetic Testing ; Genomic Instability/genetics ; High-Throughput Screening Assays ; Humans ; Loss of Function Mutation/genetics ; Ovarian Neoplasms/*genetics/pathology ; },
abstract = {Genetic mutations in BRCA1, which is crucial for the process of DNA repair and maintenance of genomic integrity, are known to increase markedly the risk of breast and ovarian cancers. Clinical genetic testing has been used to identify new BRCA1 variants; however, functional assessment and determination of their pathogenicity still poses challenges for clinical management. Here, we describe that CRISPR-mediated cytosine base editor, known as BE3, can be used for the functional analysis of BRCA1 variants. We performed CRISPR-mediated base-editing screening using 745 gRNAs targeting all exons in BRCA1 to identify loss-of-function variants and identified variants whose function has heretofore remained unknown, such as c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A. Our results show that CRISPR-mediated base editor is a powerful tool for the reclassification of variants of uncertain significance (VUSs) in BRCA1.},
}
@article {pmid31467167,
year = {2019},
author = {Peng, R and Li, Z and Xu, Y and He, S and Peng, Q and Wu, LA and Wu, Y and Qi, J and Wang, P and Shi, Y and Gao, GF},
title = {Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {38},
pages = {18928-18936},
pmid = {31467167},
issn = {1091-6490},
mesh = {Bacterial Proteins/*antagonists &amp; inhibitors/chemistry/*metabolism ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/chemistry/*metabolism ; Cryoelectron Microscopy ; DNA/metabolism ; Endodeoxyribonucleases/*antagonists &amp; inhibitors/chemistry/*metabolism ; Enzyme Inhibitors/chemistry/*metabolism ; Gene Editing ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Multimerization ; RNA, Guide/metabolism ; },
abstract = {Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.},
}
@article {pmid31467027,
year = {2019},
author = {Chung, JY and Ain, QU and Song, Y and Yong, SB and Kim, YH},
title = {Targeted delivery of CRISPR interference system against Fabp4 to white adipocytes ameliorates obesity, inflammation, hepatic steatosis, and insulin resistance.},
journal = {Genome research},
volume = {29},
number = {9},
pages = {1442-1452},
pmid = {31467027},
issn = {1549-5469},
mesh = {3T3 Cells ; Adipocytes, White/chemistry/cytology ; Animals ; CRISPR-Cas Systems ; Cytokines/metabolism ; Diabetes Mellitus, Type 2/*drug therapy/genetics ; Disease Models, Animal ; Fatty Acid-Binding Proteins/antagonists &amp; inhibitors/*genetics ; Fatty Liver/*drug therapy/genetics ; Gene Expression Regulation ; Gene Knockdown Techniques ; Insulin Resistance ; Mice ; Molecular Targeted Therapy ; Obesity/*drug therapy/genetics ; RNA, Guide/*administration &amp; dosage/pharmacology ; },
abstract = {Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.},
}
@article {pmid31466822,
year = {2019},
author = {Saito, H and Yokobayashi, Y},
title = {Editorial overview: Mammalian synthetic biology: from devices to multicellular systems.},
journal = {Current opinion in chemical biology},
volume = {52},
number = {},
pages = {A1-A2},
doi = {10.1016/j.cbpa.2019.07.010},
pmid = {31466822},
issn = {1879-0402},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Humans ; *Mammals ; *Synthetic Biology ; },
}
@article {pmid31465870,
year = {2019},
author = {Kim, MS and Kim, KH},
title = {Effect of CRISPR/Cas9-mediated knockout of either Mx1 or ISG15 gene in EPC cells on resistance against VHSV infection.},
journal = {Fish &amp; shellfish immunology},
volume = {93},
number = {},
pages = {1041-1046},
doi = {10.1016/j.fsi.2019.08.058},
pmid = {31465870},
issn = {1095-9947},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems/*immunology ; Cell Line, Tumor ; Cyprinidae/genetics/*immunology ; Disease Resistance/*genetics/immunology ; Fish Diseases/genetics/*immunology ; Fish Proteins/*genetics/*immunology ; *Gene Expression ; Gene Knockout Techniques/veterinary ; Hemorrhagic Septicemia, Viral/genetics/immunology ; Interferon Type I/genetics ; Novirhabdovirus/physiology ; Poly I-C/pharmacology ; Sequence Analysis, DNA/veterinary ; Sequence Analysis, Protein/veterinary ; },
abstract = {Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.},
}
@article {pmid31465786,
year = {2020},
author = {Sen, P and Ghosal, S and Hazra, R and Arega, S and Mohanty, R and Kulkarni, KK and Budhwar, R and Ganguly, N},
title = {Transcriptomic analyses of gene expression by CRISPR knockout of miR-214 in cervical cancer cells.},
journal = {Genomics},
volume = {112},
number = {2},
pages = {1490-1499},
doi = {10.1016/j.ygeno.2019.08.020},
pmid = {31465786},
issn = {1089-8646},
mesh = {Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MicroRNAs/*genetics/metabolism ; *Transcriptome ; Uterine Cervical Neoplasms/*genetics ; },
abstract = {In this study, we investigate the effect of one such micro RNA, miR-214 which is frequently down-regulated in cervical cancer. In this study, we either CRISPR knocked out or overexpressed miR-214 in cervical cancer cells and analyzed the global mRNA expression by Next Generation Sequencing (NGS) It was observed that a total of 108 genes were upregulated and 178 downregulated between the samples, above and below the baseline respectively. Gene Ontology and KEGG pathway analysis reveal distinct biological processes and pathways. Analysis of gene regulatory networks also gave different network patterns in the two samples. We confirmed the RNA sequencing data for 10 genes; IFIF27, SMAD3, COX11, TP53INP1, ABL2, FGF8, TNFAIP3, NRG1, SP3 and MDM4 by Real-time PCR. This is the first report on the effect of miR-214 on global mRNA profile in cervical cancer cells. This study also reports new biomarkers for cervical cancer prognosis.},
}
@article {pmid31465441,
year = {2019},
author = {Fleiss, A and O'Donnell, S and Fournier, T and Lu, W and Agier, N and Delmas, S and Schacherer, J and Fischer, G},
title = {Reshuffling yeast chromosomes with CRISPR/Cas9.},
journal = {PLoS genetics},
volume = {15},
number = {8},
pages = {e1008332},
pmid = {31465441},
issn = {1553-7404},
mesh = {Anion Transport Proteins/genetics ; *CRISPR-Cas Systems ; Chromosomes, Fungal/*genetics ; DNA Shuffling/*methods ; Gene Editing/*methods ; Genome, Fungal/genetics ; Promoter Regions, Genetic/genetics ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics ; Translocation, Genetic ; },
abstract = {Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.},
}
@article {pmid31465436,
year = {2019},
author = {Bradford, J and Perrin, D},
title = {A benchmark of computational CRISPR-Cas9 guide design methods.},
journal = {PLoS computational biology},
volume = {15},
number = {8},
pages = {e1007274},
pmid = {31465436},
issn = {1553-7358},
mesh = {Animals ; Benchmarking/methods/statistics &amp; numerical data ; *CRISPR-Cas Systems ; Computational Biology ; Databases, Nucleic Acid/statistics &amp; numerical data ; Gene Editing/*methods/standards/statistics &amp; numerical data ; Mice ; RNA, Guide/*genetics ; Software ; },
abstract = {The popularity of CRISPR-based gene editing has resulted in an abundance of tools to design CRISPR-Cas9 guides. This is also driven by the fact that designing highly specific and efficient guides is a crucial, but not trivial, task in using CRISPR for gene editing. Here, we thoroughly analyse the performance of 18 design tools. They are evaluated based on runtime performance, compute requirements, and guides generated. To achieve this, we implemented a method for auditing system resources while a given tool executes, and tested each tool on datasets of increasing size, derived from the mouse genome. We found that only five tools had a computational performance that would allow them to analyse an entire genome in a reasonable time, and without exhausting computing resources. There was wide variation in the guides identified, with some tools reporting every possible guide while others filtered for predicted efficiency. Some tools also failed to exclude guides that would target multiple positions in the genome. We also considered two collections with over a thousand guides each, for which experimental data is available. There is a lot of variation in performance between the datasets, but the relative order of the tools is partially conserved. Importantly, the most striking result is a lack of consensus between the tools. Our results show that CRISPR-Cas9 guide design tools need further work in order to achieve rapid whole-genome analysis and that improvements in guide design will likely require combining multiple approaches.},
}
@article {pmid31464370,
year = {2019},
author = {Brown, KR and Mair, B and Soste, M and Moffat, J},
title = {CRISPR screens are feasible in TP53 wild-type cells.},
journal = {Molecular systems biology},
volume = {15},
number = {8},
pages = {e8679},
pmid = {31464370},
issn = {1744-4292},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cell Line ; Cell Line, Transformed ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Epithelial Cells/cytology/*metabolism ; Gene Editing/methods/*standards ; Gene Expression Regulation ; HCT116 Cells ; HeLa Cells ; Humans ; Neuroglia/metabolism/pathology ; Organ Specificity ; Proto-Oncogene Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-mdm2/genetics/metabolism ; Quality Control ; RNA, Guide/*genetics/metabolism ; Retinal Pigment Epithelium/cytology/metabolism ; Retinoblastoma Binding Proteins/genetics/metabolism ; Tumor Suppressor Protein p53/*genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.},
}
@article {pmid31464368,
year = {2019},
author = {Haapaniemi, E and Botla, S and Persson, J and Schmierer, B and Taipale, J},
title = {Reply to "CRISPR screens are feasible in TP53 wild-type cells".},
journal = {Molecular systems biology},
volume = {15},
number = {8},
pages = {e9059},
pmid = {31464368},
issn = {1744-4292},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Damage ; *Gene Editing ; Tumor Suppressor Protein p53 ; },
abstract = {Haapaniemi et al address the issues raised by Brown et al and discuss several differences between the analyses performed by the two groups.},
}
@article {pmid31463914,
year = {2020},
author = {Mead, TJ},
title = {Alizarin Red and Alcian Blue Preparations to Visualize the Skeleton.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2043},
number = {},
pages = {207-212},
doi = {10.1007/978-1-4939-9698-8_17},
pmid = {31463914},
issn = {1940-6029},
mesh = {Alcian Blue/*chemistry ; Animals ; Animals, Newborn ; Anthraquinones/*chemistry ; Body Patterning ; Bone and Bones/chemistry/*embryology ; CRISPR-Cas Systems ; Embryonic Development ; Mice ; },
abstract = {Understanding proteolytic remodeling of extracellular matrix involves the generation of global or conditional knockout mice by homologous recombination in embryonic stem cells or their manipulation through new advanced technologies such as CRISPR-Cas9. These models provide opportunities to understand the roles of ADAMTS genes in skeletogenesis. Whole-mount skeletal preparations are necessary for assessment of the skeletal phenotype. They allow for facile visualization of skeletal patterning, size and shape of skeletal elements, and skeletal structure. This protocol describes the staining of the murine skeleton using Alcian blue to identify cartilage and alizarin red to identify bone.},
}
@article {pmid31463899,
year = {2020},
author = {Peris-Torres, C and Serrano, O and Plaza-Calonge, MDC and Rodríguez-Manzaneque, JC},
title = {Inhibition of ADAMTS1 Expression by Lentiviral CRISPR/Cas9 Gene Editing Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2043},
number = {},
pages = {13-24},
doi = {10.1007/978-1-4939-9698-8_2},
pmid = {31463899},
issn = {1940-6029},
mesh = {ADAMTS1 Protein/*antagonists &amp; inhibitors/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; },
abstract = {The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.},
}
@article {pmid31462973,
year = {2019},
author = {Zhang, J and Chen, L and Zhang, J and Wang, Y},
title = {Drug Inducible CRISPR/Cas Systems.},
journal = {Computational and structural biotechnology journal},
volume = {17},
number = {},
pages = {1171-1177},
pmid = {31462973},
issn = {2001-0370},
abstract = {Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have been employed as a powerful versatile technology for programmable gene editing, transcriptional modulation, epigenetic modulation, and genome labeling, etc. Yet better control of their activity is important to accomplish greater precision and to reduce undesired outcomes such as off-target events. The use of small molecules to control CRISPR/Cas activity represents a promising direction. Here, we provide an updated review on multiple drug inducible CRISPR/Cas systems and discuss their distinct properties. We arbitrarily divided the emerging drug inducible CRISPR/Cas systems into two categories based on whether at transcription or protein level does chemical control occurs. The first category includes Tet-On/Off system and Cre-dependent system. The second category includes chemically induced proximity systems, intein splicing system, 4-Hydroxytamoxifen-Estrogen Receptor based nuclear localization systems, allosterically regulated Cas9 system, and destabilizing domain mediated protein degradation systems. Finally, the advantages and limitations of each system were summarized.},
}
@article {pmid31462429,
year = {2019},
author = {Song, X and Wan, X and Huang, T and Zeng, C and Sastry, N and Wu, B and James, CD and Horbinski, C and Nakano, I and Zhang, W and Hu, B and Cheng, SY},
title = {SRSF3-Regulated RNA Alternative Splicing Promotes Glioblastoma Tumorigenicity by Affecting Multiple Cellular Processes.},
journal = {Cancer research},
volume = {79},
number = {20},
pages = {5288-5301},
pmid = {31462429},
issn = {1538-7445},
support = {R21 CA209345/CA/NCI NIH HHS/United States ; R01 NS095642/NS/NINDS NIH HHS/United States ; R01 NS107071/NS/NINDS NIH HHS/United States ; R01 NS102669/NS/NINDS NIH HHS/United States ; UL1 TR001422/TR/NCATS NIH HHS/United States ; P30 CA060553/CA/NCI NIH HHS/United States ; R01 NS093843/NS/NINDS NIH HHS/United States ; P50 CA221747/CA/NCI NIH HHS/United States ; F31 CA232630/CA/NCI NIH HHS/United States ; },
mesh = {*Alternative Splicing ; Animals ; Brain Neoplasms/*genetics/metabolism/pathology ; CRISPR-Cas Systems ; Cell Division ; Cell Line, Tumor ; Cell Self Renewal ; DNA-Binding Proteins/genetics ; Disease Progression ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Glioblastoma/*genetics/metabolism/pathology ; HEK293 Cells ; Heterografts ; Humans ; Mice ; Mice, Nude ; Microtubule-Associated Proteins/genetics ; Neoplasm Proteins/biosynthesis/genetics/*physiology ; Neoplasm Transplantation ; Phosphorylation ; Prognosis ; Protein Isoforms/physiology ; Protein Processing, Post-Translational ; RNA, Messenger/*biosynthesis/genetics ; Serine-Arginine Splicing Factors/antagonists &amp; inhibitors/genetics/*physiology ; Spindle Apparatus/metabolism ; Transcription Factors/genetics ; },
abstract = {Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9'. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. SIGNIFICANCE: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.},
}
@article {pmid31461650,
year = {2019},
author = {Oberlick, EM and Rees, MG and Seashore-Ludlow, B and Vazquez, F and Nelson, GM and Dharia, NV and Weir, BA and Tsherniak, A and Ghandi, M and Krill-Burger, JM and Meyers, RM and Wang, X and Montgomery, P and Root, DE and Bieber, JM and Radko, S and Cheah, JH and Hon, CS and Shamji, AF and Clemons, PA and Park, PJ and Dyer, MA and Golub, TR and Stegmaier, K and Hahn, WC and Stewart, EA and Schreiber, SL and Roberts, CWM},
title = {Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors.},
journal = {Cell reports},
volume = {28},
number = {9},
pages = {2331-2344.e8},
pmid = {31461650},
issn = {2211-1247},
support = {U01 CA176152/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 CA113794/CA/NCI NIH HHS/United States ; F31 CA183558/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; U01 CA217848/CA/NCI NIH HHS/United States ; R01 CA172152/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; HEK293 Cells ; Humans ; Mice ; Mice, Nude ; Mutation ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/*antagonists &amp; inhibitors/genetics ; Rhabdoid Tumor/drug therapy/*genetics ; Small Molecule Libraries/pharmacology ; },
abstract = {Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.},
}
@article {pmid31461646,
year = {2019},
author = {Rasys, AM and Park, S and Ball, RE and Alcala, AJ and Lauderdale, JD and Menke, DB},
title = {CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes.},
journal = {Cell reports},
volume = {28},
number = {9},
pages = {2288-2292.e3},
pmid = {31461646},
issn = {2211-1247},
support = {T32 GM007103/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; *Gene Transfer Techniques ; Lizards/*genetics/physiology ; Male ; Mutation ; Oocytes/*metabolism ; },
abstract = {CRISPR-Cas9-mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are no reptiles with effective methods for targeted mutagenesis. Here, we demonstrate that the microinjection of immature oocytes within the ovaries of Anolis sagrei females enables the production of CRISPR-Cas9-induced mutations. This method is capable of producing F0 embryos and hatchlings with monoallelic or biallelic mutations. We demonstrate that these mutations can be transmitted through the germline to establish genetically modified strains of lizards. Direct tests of gene function can now be performed in Anolis lizards, an important model for studies of reptile evolution and development.},
}
@article {pmid31461619,
year = {2019},
author = {Shao, N and Han, X and Song, Y and Zhang, P and Qin, L},
title = {CRISPR-Cas12a Coupled with Platinum Nanoreporter for Visual Quantification of SNVs on a Volumetric Bar-Chart Chip.},
journal = {Analytical chemistry},
volume = {91},
number = {19},
pages = {12384-12391},
doi = {10.1021/acs.analchem.9b02925},
pmid = {31461619},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/*genetics ; DNA, Single-Stranded/chemistry ; Feasibility Studies ; Magnets/chemistry ; Metal Nanoparticles/*chemistry ; Microspheres ; Nanotechnology/*instrumentation ; Neoplasms/genetics ; Platinum/*chemistry ; *Polymorphism, Single Nucleotide ; },
abstract = {Methods that can detect and quantify single nucleotide variations (SNVs)/single nucleotide polymorphisms (SNPs) are greatly needed in the bioanalytical measurement of gene mutations and polymorphisms. Herein a visual and instrument-free SNV quantification platform is developed. Platinum nanoparticles tethered to magnetic beads by single-stranded DNAs are designed as quantitative readout reporters for a CRISPR-Cas12a nucleic acid detection system. The integration of platinum nanoreporter and CRISPR-Cas system with a volumetric bar-chart chip realizes the volumetric quantification of nucleic acids. This platform enables quantification of multiple cancer mutations in pure DNA samples and mock cell-free DNA samples in serum, with allelic fractions as low as 0.01%. This platform could have great potential in the quantification of SNVs/SNPs as well as other types of nucleic acid targets at the point of care.},
}
@article {pmid31461190,
year = {2019},
author = {Preisler, L and Ben-Yosef, D and Mayshar, Y},
title = {Adenomatous Polyposis Coli as a Major Regulator of Human Embryonic Stem Cells Self-Renewal.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {12},
pages = {1505-1515},
doi = {10.1002/stem.3084},
pmid = {31461190},
issn = {1549-4918},
mesh = {Adenomatous Polyposis Coli/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Embryonic Development/physiology ; Human Embryonic Stem Cells/*cytology ; Humans ; Pluripotent Stem Cells/*cytology ; Wnt Signaling Pathway/physiology ; beta Catenin/*metabolism ; },
abstract = {Human embryonic stem cells (hESCs) provide an essential tool to investigate early human development, study disease pathogenesis, and examine therapeutic interventions. Adenomatous polyposis coli (APC) is a negative regulator of Wnt/β-catenin signaling, implicated in the majority of sporadic colorectal cancers and in the autosomal dominant inherited syndrome familial adenomatous polyposis (FAP). Studies into the role of Wnt/β-catenin signaling in hESCs arrived at conflicting results, due at least in part to variations in culture conditions and the use of external inhibitors and agonists. Here, we directly targeted APC in hESCs carrying a germline APC mutation, derived from affected blastocysts following preimplantation genetic diagnosis (PGD) for FAP, in order to answer open questions regarding the role of APC in regulating pluripotency and differentiation potential of hESCs. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), we generated second hit APC mutations in FAP-hESCs. Despite high CRISPR/Cas9 targeting efficiency and the successful isolation of many clones, none of the isolated clones carried a loss of function mutation in the wild-type (WT) APC allele. Using a fluorescent β-catenin reporter and analysis of mutated-allele frequencies in the APC locus, we show that APC double mutant hESCs robustly activate Wnt/β-catenin signaling that results in rapid differentiation to endodermal and mesodermal lineages. Here, we provide direct evidence for a strict requirement for constant β-catenin degradation through the APC destruction complex in order to maintain pluripotency, highlighting a fundamental role for APC in self-renewal of hESCs. Stem Cells 2019;37:1505-1515.},
}
@article {pmid31460749,
year = {2019},
author = {Wang, B and Wang, R and Wang, D and Wu, J and Li, J and Wang, J and Liu, H and Wang, Y},
title = {Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {91},
number = {19},
pages = {12156-12161},
doi = {10.1021/acs.analchem.9b01526},
pmid = {31460749},
issn = {1520-6882},
mesh = {Bacterial Proteins/chemistry/genetics ; Buffers ; CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems ; DNA/*analysis ; Electrophoresis, Agar Gel ; Endodeoxyribonucleases/chemistry/genetics ; Mycoplasma/genetics ; Nucleic Acid Amplification Techniques/*methods ; },
abstract = {A rapid and sensitive method is crucial for nucleic acid detection. Recently, RNA-guided CRISPR/Cas12a nuclease-based methods present great promise for nucleic acid detection. In the present methods, however, DNA amplification and subsequent Cas12a cleavage is separated and the whole process takes as long as 2 h. Most importantly, the uncapping operation increases the risk of aerosol contamination. In this study, we propose a CRISPR/Cas12a-based method named "Cas12aVDet" for rapid nucleic acid detection. By integrating recombinase polymerase amplification (RPA) with Cas12a cleavage in a single reaction system, the detection can be accomplished in 30 min and uncapping contamination can be avoided. The detection signal can be observed by the naked eye under blue light. This method could detect DNA at single molecule level and demonstrated 100% accuracy for mycoplasma contamination detection, presenting great potential for a variety of nucleic acid detection applications.},
}
@article {pmid31456163,
year = {2019},
author = {Wang, R and Zhang, JY and Lu, KH and Lu, SS and Zhu, XX},
title = {Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing.},
journal = {In vitro cellular &amp; developmental biology. Animal},
volume = {55},
number = {10},
pages = {784-792},
pmid = {31456163},
issn = {1543-706X},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Cell Line ; Female ; Fibroblasts/*physiology ; Gene Editing ; Gene Knockout Techniques ; Homozygote ; Male ; Mutation ; Receptors, Somatotropin/*genetics ; Swine ; Swine, Miniature/*genetics ; },
abstract = {Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.},
}
@article {pmid31455915,
year = {2019},
author = {Callaway, E},
title = {CRISPR cuts turn gels into biological watchdogs.},
journal = {Nature},
volume = {572},
number = {7771},
pages = {574},
pmid = {31455915},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gels ; Gene Editing ; *Smart Materials ; },
}
@article {pmid31455836,
year = {2020},
author = {Zhen, S and Lu, J and Liu, YH and Chen, W and Li, X},
title = {Synergistic antitumor effect on cervical cancer by rational combination of PD1 blockade and CRISPR-Cas9-mediated HPV knockout.},
journal = {Cancer gene therapy},
volume = {27},
number = {3-4},
pages = {168-178},
pmid = {31455836},
issn = {1476-5500},
mesh = {Animals ; Apoptosis/drug effects/immunology ; B7-H1 Antigen/antagonists &amp; inhibitors/*genetics/immunology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Combined Modality Therapy/methods ; Female ; Gene Knockout Techniques ; Genetic Therapy/*methods ; Genetic Vectors/administration &amp; dosage/genetics ; Human papillomavirus 16/*genetics ; Humans ; Mice ; Oncogene Proteins, Viral/genetics ; Papillomavirus E7 Proteins/genetics ; Papillomavirus Infections/immunology/pathology/*therapy/virology ; Plasmids/genetics ; RNA, Guide/genetics ; Repressor Proteins/genetics ; Tumor Microenvironment/genetics/immunology ; Uterine Cervical Neoplasms/immunology/pathology/*therapy/virology ; },
abstract = {Targeted therapy results in objective responses in cervical cancer. However, the responses are short. In contrast, treatment with immune checkpoint inhibitors results in a lower responses rate, but the responses tend to be more durable. Based on these findings, we hypothesized that HPV16 E6/E7-targeted therapy may synergize with the PD-1 pathway blockade to enhance antitumor activity. To test hypothesis, we described for the first time the effects of the CRISPR/Cas9 that was targeted to the HPV and PD1 in vitro and in vivo. Our data showed that gRNA/cas9 targeted HPV16 E6/E7 induced cervical cancer cell SiHa apoptosis, and suggested that overexpression of PD-L1, induced by HPV16 E6/E7, may be responsible for lymphocyte dysfunction. In established SiHa cell- xenografted humanized SCID mice, Administration of gRNA-PD-1 together with gRNA-HPV16 E6/E7 treatment improved the survival and suppressed the tumor growth obviously. In addition, combination treatment increased the population of dendritic cells, CD8+ and CD4+ T lymphocyte cells. According, it enhanced the expression of Th1-associated immune-stimulating genes while reducing the transcription of regulatory/suppressive immune genes, reshaping tumor microenvironment from an immunosuppressive to a stimulatory state. These results demonstrate potent synergistic effects of combination therapy using HPV16 E6/E7-targeted therapy and immune checkpoint blockade PD1, supporting a direct translation of this combination strategy in clinic for the treatment of cervical cancer.},
}
@article {pmid31455729,
year = {2019},
author = {Fujihara, Y and Noda, T and Kobayashi, K and Oji, A and Kobayashi, S and Matsumura, T and Larasati, T and Oura, S and Kojima-Kita, K and Yu, Z and Matzuk, MM and Ikawa, M},
title = {Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {37},
pages = {18498-18506},
pmid = {31455729},
issn = {1091-6490},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Membrane/metabolism ; Disease Models, Animal ; Fallopian Tubes/physiology ; Female ; Fertility/*genetics ; Humans ; Infertility, Male/*genetics ; Male ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Knockout ; Multigene Family/*genetics ; Mutation ; Phosphoric Diester Hydrolases/genetics/metabolism ; Sperm Motility/*genetics ; Spermatozoa/cytology/physiology ; },
abstract = {CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.},
}
@article {pmid31455654,
year = {2019},
author = {Louradour, I and Ghosh, K and Inbar, E and Sacks, DL},
title = {CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major.},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
pmid = {31455654},
issn = {2150-7511},
mesh = {Alleles ; Amino Acid Sequence ; Animals ; *CRISPR-Cas Systems ; Disease Vectors ; Female ; Gene Editing ; Humans ; Insect Proteins/genetics/*metabolism ; Leishmania major/*physiology ; Male ; Mutagenesis ; Mutation ; Phlebotomus/*genetics/immunology/parasitology/physiology ; Sequence Alignment ; },
abstract = {Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites.IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.},
}
@article {pmid31455158,
year = {2019},
author = {Littler, S and Sloss, O and Geary, B and Pierce, A and Whetton, AD and Taylor, SS},
title = {Oncogenic MYC amplifies mitotic perturbations.},
journal = {Open biology},
volume = {9},
number = {8},
pages = {190136},
pmid = {31455158},
issn = {2046-2441},
support = {MR/M008959/1/MRC_/Medical Research Council/United Kingdom ; C1422/A19842/CRUK_/Cancer Research UK/United Kingdom ; C5759/A25254/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Transformation, Neoplastic/*genetics ; Chromosomal Instability ; Chromosome Segregation ; *Gene Amplification ; Gene Expression Regulation ; Genomic Instability ; Humans ; Mitosis/*genetics ; Mutagenesis ; Proto-Oncogene Proteins c-myc/*genetics ; },
abstract = {The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation, apoptosis and pluripotency. When deregulated, MYC drives genomic instability via several mechanisms including aberrant proliferation, replication stress and ROS production. Deregulated MYC also promotes chromosome instability, but less is known about how MYC influences mitosis. Here, we show that deregulating MYC modulates multiple aspects of mitotic chromosome segregation. Cells overexpressing MYC have altered spindle morphology, take longer to align their chromosomes at metaphase and enter anaphase sooner. When challenged with a variety of anti-mitotic drugs, cells overexpressing MYC display more anomalies, the net effect of which is increased micronuclei, a hallmark of chromosome instability. Proteomic analysis showed that MYC modulates multiple networks predicted to influence mitosis, with the mitotic kinase PLK1 identified as a central hub. In turn, we show that MYC modulates several PLK1-dependent processes, namely mitotic entry, spindle assembly and SAC satisfaction. These observations thus underpin the pervasive nature of oncogenic MYC and provide a mechanistic rationale for MYC's ability to drive chromosome instability.},
}
@article {pmid31452351,
year = {2020},
author = {Bai, M and Yuan, J and Kuang, H and Gong, P and Li, S and Zhang, Z and Liu, B and Sun, J and Yang, M and Yang, L and Wang, D and Song, S and Guan, Y},
title = {Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean.},
journal = {Plant biotechnology journal},
volume = {18},
number = {3},
pages = {721-731},
pmid = {31452351},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Mutagenesis ; Root Nodules, Plant/genetics ; Soybeans/*genetics ; },
abstract = {The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR-Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR-Cas9 as a mutant screening tool. Here, we report a pooled CRISPR-Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR-Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1-1/1-2/1-3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.},
}
@article {pmid31452297,
year = {2020},
author = {Yi, P and Goshima, G},
title = {Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens.},
journal = {Plant biotechnology journal},
volume = {18},
number = {3},
pages = {599-601},
pmid = {31452297},
issn = {1467-7652},
mesh = {Bryopsida/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Oligonucleotides/*genetics ; *Transformation, Genetic ; },
}
@article {pmid31452165,
year = {2020},
author = {Sateriale, A and Pawlowic, M and Vinayak, S and Brooks, C and Striepen, B},
title = {Genetic Manipulation of Cryptosporidium parvum with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2052},
number = {},
pages = {219-228},
doi = {10.1007/978-1-4939-9748-0_13},
pmid = {31452165},
issn = {1940-6029},
support = {203134/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; K99 AI137442/AI/NIAID NIH HHS/United States ; T32 AI060546/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line ; Cryptosporidiosis/parasitology ; Cryptosporidium parvum/*genetics/growth &amp; development/isolation &amp; purification ; Genetic Techniques ; Genetic Vectors ; Humans ; Luciferases/genetics/metabolism ; Mice ; Oocysts/genetics/growth &amp; development/isolation &amp; purification ; Transfection/instrumentation/*methods ; Transgenes ; Workflow ; },
abstract = {Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9-driven homologous repair coupled to in vivo propagation within immunodeficient mice. Recent modifications have simplified the initial protocol significantly. This chapter will guide through procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum.},
}
@article {pmid31452146,
year = {2019},
author = {Sharon, D and Chan, SM},
title = {Application of CRISPR-Cas9 Screening Technologies to Study Mitochondrial Biology in Healthy and Disease States.},
journal = {Advances in experimental medicine and biology},
volume = {1158},
number = {},
pages = {269-277},
doi = {10.1007/978-981-13-8367-0_15},
pmid = {31452146},
issn = {0065-2598},
mesh = {*CRISPR-Cas Systems ; Cell Physiological Phenomena/genetics ; *Mitochondria/genetics/pathology ; *Mitochondrial Diseases/genetics ; Research/trends ; },
abstract = {Mitochondria play a central role in maintaining normal cellular homeostasis as well as contributing to the pathogenesis of numerous disease states. The advent of CRISPR-Cas9 screening technologies has greatly accelerated the study of mitochondrial biology. In this chapter, we review the various CRISPR-Cas9 screening platforms that are currently available and prior studies that leveraged this technology to identify genes involved in mitochondrial biology in both healthy and disease states. In addition, we discuss the challenges associated with current CRISPR-Cas9 platforms and potential solutions to further enhance this promising technology.},
}
@article {pmid31451760,
year = {2019},
author = {Tsui, CK and Barfield, RM and Fischer, CR and Morgens, DW and Li, A and Smith, BAH and Gray, MA and Bertozzi, CR and Rabuka, D and Bassik, MC},
title = {CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity.},
journal = {Nature chemical biology},
volume = {15},
number = {10},
pages = {949-958},
pmid = {31451760},
issn = {1552-4469},
support = {R01 CA227942/CA/NCI NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; F31 GM126688/GM/NIGMS NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; T32 GM120007/GM/NIGMS NIH HHS/United States ; },
mesh = {Ado-Trastuzumab Emtansine ; Antineoplastic Agents, Immunological/pharmacology ; *CRISPR-Cas Systems ; Carrier Proteins ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; *Genome-Wide Association Study ; Humans ; Immunoconjugates/*toxicity ; Lysosomes ; Maytansine/*analogs &amp; derivatives/pharmacology ; N-Acetylneuraminic Acid/*pharmacology ; Trastuzumab/*pharmacology ; },
abstract = {Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.},
}
@article {pmid31451697,
year = {2019},
author = {Liu, Y and Wan, X and Wang, B},
title = {Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3693},
pmid = {31451697},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; BB/N007212/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/S018875/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/*genetics/metabolism ; Escherichia coli Proteins/*genetics ; Gene Expression/genetics ; Gene Expression Regulation/genetics ; Metabolic Networks and Pathways/genetics ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; Protein Engineering/*methods ; RNA Polymerase Sigma 54/*genetics ; RNA, Guide/genetics ; Transcriptional Activation/genetics ; },
abstract = {Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on σ[70] promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ[54]-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ[54]-dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications.},
}
@article {pmid31451668,
year = {2019},
author = {Guo, P and Yang, J and Huang, J and Auguste, DT and Moses, MA},
title = {Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {37},
pages = {18295-18303},
pmid = {31451668},
issn = {1091-6490},
support = {DP2 CA174495/CA/NCI NIH HHS/United States ; R01 CA185530/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Disease Models, Animal ; Drug Carriers/*chemistry ; Drug Delivery Systems/*methods ; Female ; *Gene Editing ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Genetic Therapy/methods ; Genetic Vectors ; Humans ; Intercellular Adhesion Molecule-1/genetics ; Lipids/chemistry ; Lipocalin-2/genetics ; Liposomes/chemistry ; Mice ; Mice, Nude ; Nanoparticles/*chemistry ; Triple Negative Breast Neoplasms/*drug therapy/*genetics ; },
abstract = {Triple-negative breast cancer (TNBC), which has the highest mortality rate of all breast cancer, is in urgent need of a therapeutic that hinders the spread and growth of cancer cells. CRISPR genome editing holds the promise of a potential cure for many genetic diseases, including TNBC; however, its clinical translation is being challenged by the lack of safe and effective nonviral delivery systems for in vivo therapeutic genome editing. Here we report the synthesis and application of a noncationic, deformable, and tumor-targeted nanolipogel system (tNLG) for CRISPR genome editing in TNBC tumors. We have demonstrated that tNLGs mediate a potent CRISPR knockout of Lipocalin 2 (Lcn2), a known breast cancer oncogene, in human TNBC cells in vitro and in vivo. The loss of Lcn2 significantly inhibits the migration and the mesenchymal phenotype of human TNBC cells and subsequently attenuates TNBC aggressiveness. In an orthotopic TNBC model, we have shown that systemically administered tNLGs mediated >81% CRISPR knockout of Lcn2 in TNBC tumor tissues, resulting in significant tumor growth suppression (>77%). Our proof-of-principle results provide experimental evidence that tNLGs can be used as a safe, precise, and effective delivery approach for in vivo CRISPR genome editing in TNBC.},
}
@article {pmid31451649,
year = {2019},
author = {Wang, L and Zhao, L and Zhang, X and Zhang, Q and Jia, Y and Wang, G and Li, S and Tian, D and Li, WH and Yang, S},
title = {Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {37},
pages = {18479-18487},
pmid = {31451649},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Cloning, Molecular ; Disease Resistance/*genetics ; Gene Knockout Techniques ; Gene Regulatory Networks/immunology ; Genome, Plant/genetics ; Magnaporthe/*immunology ; NLR Proteins/*genetics/immunology ; Oryza/*genetics/immunology/microbiology ; Phylogeny ; Plant Breeding ; Plant Diseases/genetics/*immunology/microbiology ; Plant Proteins/*genetics/immunology ; Plants, Genetically Modified/genetics/immunology/microbiology ; Sequence Analysis, DNA ; },
abstract = {Tetep is a rice cultivar known for broad-spectrum resistance to blast, a devastating fungal disease. The molecular basis for its broad-spectrum resistance is still poorly understood. Is it because Tetep has many more NLR genes than other cultivars? Or does Tetep possess multiple major NLR genes that can individually confer broad-spectrum resistance to blast? Moreover, are there many interacting NLR pairs in the Tetep genome? We sequenced its genome, obtained a high-quality assembly, and annotated 455 nucleotide-binding site leucine-rich repeat (NLR) genes. We cloned and tested 219 NLR genes as transgenes in 2 susceptible cultivars using 5 to 12 diversified pathogen strains; in many cases, fewer than 12 strains were successfully cultured for testing. Ninety cloned NLRs showed resistance to 1 or more pathogen strains and each strain was recognized by multiple NLRs. However, few NLRs showed resistance to >6 strains, so multiple NLRs are apparently required for Tetep's broad-spectrum resistance to blast. This was further supported by the pedigree analyses, which suggested a correlation between resistance and the number of Tetep-derived NLRs. In developing a method to identify NLR pairs each of which functions as a unit, we found that >20% of the NLRs in the Tetep and 3 other rice genomes are paired. Finally, we designed an extensive set of molecular markers for rapidly introducing clustered and paired NLRs in the Tetep genome for breeding new resistant cultivars. This study increased our understanding of the genetic basis of broad-spectrum blast resistance in rice.},
}
@article {pmid31451425,
year = {2019},
author = {Yao, J and Zeng, H and Zhang, M and Wei, Q and Wang, Y and Yang, H and Lu, Y and Li, R and Xiong, Q and Zhang, L and Chen, Z and Xing, G and Cao, X and Dai, Y},
title = {OSBPL2-disrupted pigs recapitulate dual features of human hearing loss and hypercholesterolaemia.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {8},
pages = {379-387},
doi = {10.1016/j.jgg.2019.06.006},
pmid = {31451425},
issn = {1673-8527},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo Transfer ; Genetic Association Studies ; Genetic Linkage ; Genetic Loci ; *Genetic Predisposition to Disease ; Genotype ; Hearing Loss/diagnosis/*genetics ; Humans ; Hypercholesterolemia/diagnosis/*genetics ; *Mutation ; Phenotype ; Receptors, Steroid/*genetics ; Swine ; Swine, Miniature ; },
abstract = {Oxysterol binding protein like 2 (OSBPL2), an important regulator in cellular lipid metabolism and transport, was identified as a novel deafness-causal gene in our previous work. To resemble the phenotypic features of OSBPL2 mutation in animal models and elucidate the potential genotype-phenotype associations, the OSBPL2-disrupted Bama miniature (BM) pig model was constructed using CRISPR/Cas9-mediated gene editing, somatic cell nuclear transfer (SCNT) and embryo transplantation approaches, and then subjected to phenotypic characterization of auditory function and serum lipid profiles. The OSBPL2-disrupted pigs displayed progressive hearing loss (HL) with degeneration/apoptosis of cochlea hair cells (HCs) and morphological abnormalities in HC stereocilia, as well as hypercholesterolaemia. High-fat diet (HFD) feeding aggravated the development of HL and led to more severe hypercholesterolaemia. The dual phenotypes of progressive HL and hypercholesterolaemia resembled in OSBPL2-disrupted pigs confirmed the implication of OSBPL2 mutation in nonsydromic hearing loss (NSHL) and contributed to the potential linkage between auditory dysfunction and dyslipidaemia/hypercholesterolaemia.},
}
@article {pmid31451030,
year = {2019},
author = {She, J and Wu, Y and Lou, B and Lodd, E and Klems, A and Schmoehl, F and Yuan, Z and Noble, FL and Kroll, J},
title = {Genetic compensation by epob in pronephros development in epoa mutant zebrafish.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {18},
number = {20},
pages = {2683-2696},
pmid = {31451030},
issn = {1551-4005},
mesh = {Animals ; CRISPR-Cas Systems ; Embryo, Nonmammalian/embryology/metabolism/ultrastructure ; Erythropoietin/genetics/*metabolism ; Gene Expression Regulation, Developmental/*genetics ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Heterozygote ; Homozygote ; Microscopy, Electron ; Morpholinos/genetics ; Organogenesis/*genetics ; Pronephros/abnormalities/*embryology/metabolism ; Recombinant Proteins/genetics ; Zebrafish/embryology/*genetics/metabolism ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified epoa being essential for physiological pronephros development in zebrafish, which is driven by blocking apoptosis in developing kidneys. Yet, zebrafish mutants for epoa have not been described so far. In order to compare a transient knockdown vs. permanent knockout for epoa in zebrafish on pronephros development, we used CRISPR/Cas9 technology to generate epoa knockout zebrafish mutants and we performed structural and functional studies on pronephros development. In contrast to epoa morphants, epoa[-/-] zebrafish mutants showed normal pronephros structure; however, a previously uncharacterized gene in zebrafish, named epob, was identified and upregulated in epoa[-/-] mutants. epob knockdown altered pronephros development, which was further aggravated in epoa[-/-] mutants. Likewise, epoa and epob morphants regulated similar and differential gene signatures related to kidney development in zebrafish. In conclusion, stable loss of epoa during embryonic development can be compensated by epob leading to phenotypical discrepancies in epoa knockdown and knockout zebrafish embryos.},
}
@article {pmid31450868,
year = {2019},
author = {Xu, W and Fu, W and Zhu, P and Li, Z and Wang, C and Wang, C and Zhang, Y and Zhu, S},
title = {Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-wide Sequencing.},
journal = {International journal of molecular sciences},
volume = {20},
number = {17},
pages = {},
pmid = {31450868},
issn = {1422-0067},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Genomics/methods ; *Mutagenesis ; Mutation ; Phenotype ; Plants, Genetically Modified ; Whole Genome Sequencing ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%-97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals.},
}
@article {pmid31450674,
year = {2019},
author = {Brandt, ZJ and North, PN and Link, BA},
title = {Somatic Mutations of lats2 Cause Peripheral Nerve Sheath Tumors in Zebrafish.},
journal = {Cells},
volume = {8},
number = {9},
pages = {},
pmid = {31450674},
issn = {2073-4409},
support = {R01 EY029267/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Proliferation ; Gene Editing ; *Mutation ; Neoplasms, Experimental/genetics/metabolism/*pathology ; Nerve Sheath Neoplasms/genetics/metabolism/*pathology ; Protein Serine-Threonine Kinases/*genetics/metabolism ; Serine-Threonine Kinase 3 ; Signal Transduction ; Zebrafish/genetics/growth &amp; development ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {The cellular signaling pathways underlying peripheral nerve sheath tumor (PNST) formation are poorly understood. Hippo signaling has been recently implicated in the biology of various cancers, and is thought to function downstream of mutations in the known PNST driver, NF2. Utilizing CRISPR-Cas9 gene editing, we targeted the canonical Hippo signaling kinase Lats2. We show that, while germline deletion leads to early lethality, targeted somatic mutations of zebrafish lats2 leads to peripheral nerve sheath tumor formation. These peripheral nerve sheath tumors exhibit high levels of Hippo effectors Yap and Taz, suggesting that dysregulation of these transcriptional co-factors drives PNST formation in this model. These data indicate that somatic lats2 deletion in zebrafish can serve as a powerful experimental platform to probe the mechanisms of PNST formation and progression.},
}
@article {pmid31450191,
year = {2019},
author = {Zeng, R and Sidik, H and Robinson, KS and Zhong, FL and Reversade, B and Pouladi, MA},
title = {Generation of four H1 hESC sublines carrying a hemizygous knock-out/mutant MECP2.},
journal = {Stem cell research},
volume = {40},
number = {},
pages = {101533},
doi = {10.1016/j.scr.2019.101533},
pmid = {31450191},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells/*cytology/metabolism ; Exons ; *Gene Editing ; Humans ; Karyotype ; Methyl-CpG-Binding Protein 2/*genetics ; Rett Syndrome/genetics/pathology ; },
abstract = {Rett syndrome (RTT) is a childhood neurodevelopmental disorder caused by mutations in MECP2. To study the molecular mechanisms underlying RTT, four sublines of H1 hESCs were generated, carrying a hemizygous knockout or mutant allele of MECP2. Exons 3 and 4 of MECP2 were targeted using the CRISPR/Cas9 nuclease system.},
}
@article {pmid31450074,
year = {2019},
author = {Das, AT and Binda, CS and Berkhout, B},
title = {Elimination of infectious HIV DNA by CRISPR-Cas9.},
journal = {Current opinion in virology},
volume = {38},
number = {},
pages = {81-88},
pmid = {31450074},
issn = {1879-6265},
support = {R01 AI145045/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Viral ; *Gene Editing ; Genetic Therapy ; *Genome, Viral ; HIV Infections/therapy/*virology ; HIV-1/*genetics ; Humans ; Mutation ; Proviruses/*genetics ; Virus Replication/genetics ; },
abstract = {Current antiretroviral drugs can efficiently block HIV replication and prevent transmission, but do not target the HIV provirus residing in cells that constitute the viral reservoir. Because drug therapy interruption will cause viral rebound from this reservoir, HIV-infected individuals face lifelong treatment. Therefore, novel therapeutic strategies are being investigated that aim to permanently inactivate the proviral DNA, which may lead to a cure. Multiple studies showed that CRISPR-Cas9 genome editing can be used to attack HIV DNA. Here, we will focus on not only how this endonuclease attack can trigger HIV provirus inactivation, but also how virus escape occurs and this can be prevented.},
}
@article {pmid31449515,
year = {2019},
author = {Craig, M and Kaveh, K and Woosley, A and Brown, AS and Goldman, D and Eton, E and Mehta, RM and Dhawan, A and Arai, K and Rahman, MM and Chen, S and Nowak, MA and Goldman, A},
title = {Cooperative adaptation to therapy (CAT) confers resistance in heterogeneous non-small cell lung cancer.},
journal = {PLoS computational biology},
volume = {15},
number = {8},
pages = {e1007278},
pmid = {31449515},
issn = {1553-7358},
support = {DP2 CA238295/CA/NCI NIH HHS/United States ; DP5 OD019851/OD/NIH HHS/United States ; },
mesh = {Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/*physiopathology ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics/physiology ; Coculture Techniques ; Computational Biology ; Computer Simulation ; DEAD-box RNA Helicases/genetics ; Drug Resistance, Multiple/genetics ; Drug Resistance, Neoplasm/genetics ; Humans ; Lung Neoplasms/*drug therapy/genetics/*physiopathology ; Models, Biological ; Mutation ; Ribonuclease III/genetics ; },
abstract = {Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model's parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy.},
}
@article {pmid31447475,
year = {2020},
author = {Suzuki, Y and Kouzuma, A and Watanabe, K},
title = {CRISPR/Cas9-mediated genome editing of Shewanella oneidensis MR-1 using a broad host-range pBBR1-based plasmid.},
journal = {The Journal of general and applied microbiology},
volume = {66},
number = {1},
pages = {41-45},
doi = {10.2323/jgam.2019.04.007},
pmid = {31447475},
issn = {1349-8037},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Frameshift Mutation ; Gene Editing/*methods ; *Genetic Vectors ; Plasmids/*genetics ; Shewanella/*genetics ; },
abstract = {Here, we developed an all-in-one, broad host-range CRISPR/Cas9 vector system widely applicable to genome editing of proteobacteria. Plasmid pBBR1-Cas9 was constructed by cloning the cas9 gene from Streptococcus pyogenes into the broad host-range plasmid pBBR1MCS-2. We evaluated its applicability for frameshift mutagenesis of Shewanella oneidensis MR-1. Significant cell death was observed when MR-1 cells were transformed with a pBBR1-Cas9 derivative that expressed a single-guide RNA targeting the crp gene. However, cell death was partially prevented when a donor DNA fragment containing a modified crp sequence with a frameshift mutation was introduced using the same vector. All transformants (9 colonies) contained the expected frameshift mutation in their chromosomal crp genes. These results indicate that this vector system efficiently introduced CRISPR/Cas9-mediated double-strand DNA breaks and subsequent homology-directed repair. This work provides a simple and powerful genome-editing tool for proteobacteria that can harbor pBBR1-based plasmids.},
}
@article {pmid31447328,
year = {2019},
author = {Fjodorova, M and Louessard, M and Li, Z and De La Fuente, DC and Dyke, E and Brooks, SP and Perrier, AL and Li, M},
title = {CTIP2-Regulated Reduction in PKA-Dependent DARPP32 Phosphorylation in Human Medium Spiny Neurons: Implications for Huntington Disease.},
journal = {Stem cell reports},
volume = {13},
number = {3},
pages = {448-457},
pmid = {31447328},
issn = {2213-6711},
support = {MR/L010305/1/MRC_/Medical Research Council/United Kingdom ; MR/R022429/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Corpus Striatum/metabolism ; Cyclic AMP-Dependent Protein Kinases/*metabolism ; Dopamine and cAMP-Regulated Phosphoprotein 32/*metabolism ; Gene Editing ; Human Embryonic Stem Cells/cytology ; Humans ; Huntington Disease/metabolism/pathology ; Neurons/cytology/*metabolism ; Oxidative Stress ; Phosphorylation ; Receptors, AMPA/metabolism ; Repressor Proteins/deficiency/genetics/*metabolism ; Signal Transduction ; Transcriptome ; Tumor Suppressor Proteins/deficiency/genetics/*metabolism ; },
abstract = {The mechanisms underlying the selective degeneration of medium spiny neurons (MSNs) in Huntington disease (HD) remain largely unknown. CTIP2, a transcription factor expressed by all MSNs, is implicated in HD pathogenesis because of its interactions with mutant huntingtin. Here, we report a key role for CTIP2 in protein phosphorylation via governing protein kinase A (PKA) signaling in human striatal neurons. Transcriptomic analysis of CTIP2-deficient MSNs implicates CTIP2 target genes at the heart of cAMP-Ca[2+] signal integration in the PKA pathway. These findings are further supported by experimental evidence of a substantial reduction in phosphorylation of DARPP32 and GLUR1, two PKA targets in CTIP2-deficient MSNs. Moreover, we show that CTIP2-dependent dysregulation of protein phosphorylation is shared by HD hPSC-derived MSNs and striatal tissues of two HD mouse models. This study therefore establishes an essential role for CTIP2 in human MSN homeostasis and provides mechanistic and potential therapeutic insight into striatal neurodegeneration.},
}
@article {pmid31446895,
year = {2019},
author = {Gurumurthy, CB and O'Brien, AR and Quadros, RM and Adams, J and Alcaide, P and Ayabe, S and Ballard, J and Batra, SK and Beauchamp, MC and Becker, KA and Bernas, G and Brough, D and Carrillo-Salinas, F and Chan, W and Chen, H and Dawson, R and DeMambro, V and D'Hont, J and Dibb, KM and Eudy, JD and Gan, L and Gao, J and Gonzales, A and Guntur, AR and Guo, H and Harms, DW and Harrington, A and Hentges, KE and Humphreys, N and Imai, S and Ishii, H and Iwama, M and Jonasch, E and Karolak, M and Keavney, B and Khin, NC and Konno, M and Kotani, Y and Kunihiro, Y and Lakshmanan, I and Larochelle, C and Lawrence, CB and Li, L and Lindner, V and Liu, XD and Lopez-Castejon, G and Loudon, A and Lowe, J and Jerome-Majewska, LA and Matsusaka, T and Miura, H and Miyasaka, Y and Morpurgo, B and Motyl, K and Nabeshima, YI and Nakade, K and Nakashiba, T and Nakashima, K and Obata, Y and Ogiwara, S and Ouellet, M and Oxburgh, L and Piltz, S and Pinz, I and Ponnusamy, MP and Ray, D and Redder, RJ and Rosen, CJ and Ross, N and Ruhe, MT and Ryzhova, L and Salvador, AM and Alam, SS and Sedlacek, R and Sharma, K and Smith, C and Staes, K and Starrs, L and Sugiyama, F and Takahashi, S and Tanaka, T and Trafford, AW and Uno, Y and Vanhoutte, L and Vanrockeghem, F and Willis, BJ and Wright, CS and Yamauchi, Y and Yi, X and Yoshimi, K and Zhang, X and Zhang, Y and Ohtsuka, M and Das, S and Garry, DJ and Hochepied, T and Thomas, P and Parker-Thornburg, J and Adamson, AD and Yoshiki, A and Schmouth, JF and Golovko, A and Thompson, WR and Lloyd, KCK and Wood, JA and Cowan, M and Mashimo, T and Mizuno, S and Zhu, H and Kasparek, P and Liaw, L and Miano, JM and Burgio, G},
title = {Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {171},
pmid = {31446895},
issn = {1474-760X},
support = {MR/N029992/1/MRC_/Medical Research Council/United Kingdom ; HL 123658/NH/NIH HHS/United States ; U54 GM115516/GM/NIGMS NIH HHS/United States ; MR/L010240/1/MRC_/Medical Research Council/United Kingdom ; K01 AR067858/AR/NIAMS NIH HHS/United States ; P01 CA217798/NH/NIH HHS/United States ; MR/P023576/2/MRC_/Medical Research Council/United Kingdom ; FS/12/57/29717/BHF_/British Heart Foundation/United Kingdom ; CH/13/2/30154/BHF_/British Heart Foundation/United Kingdom ; 097820/Z11/B/WT_/Wellcome Trust/United Kingdom ; P30CA16672/NH/NIH HHS/United States ; RG/15/12/31616/BHF_/British Heart Foundation/United Kingdom ; UM1OD023221/NH/NIH HHS/United States ; P30CA16672//National Institute of Health/International ; P30 CA016672/CA/NCI NIH HHS/United States ; 105610/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; R01 HL144477/HL/NHLBI NIH HHS/United States ; MR/P023576/1/MRC_/Medical Research Council/United Kingdom ; UM1 OD023221/OD/NIH HHS/United States ; R50 CA211121/CA/NCI NIH HHS/United States ; 104192/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; MOP#142452//CIHR/Canada ; P30 CA036727/CA/NCI NIH HHS/United States ; 107851/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; HL138987/NH/NIH HHS/United States ; P30 GM110768/GM/NIGMS NIH HHS/United States ; FS12/57/29717/BHF_/British Heart Foundation/United Kingdom ; P20GM103471/GM/NIGMS NIH HHS/United States ; MR/P011853/1/MRC_/Medical Research Council/United Kingdom ; R01 HL138987/HL/NHLBI NIH HHS/United States ; FS12-57/BHF_/British Heart Foundation/United Kingdom ; R01 HL123658/HL/NHLBI NIH HHS/United States ; MR/M008908/1/MRC_/Medical Research Council/United Kingdom ; BB/N015584/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; UL1 TR001108/TR/NCATS NIH HHS/United States ; 107849/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; P01 CA217798/CA/NCI NIH HHS/United States ; },
mesh = {*Alleles ; Animals ; Blastocyst/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Factor Analysis, Statistical ; Female ; Male ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Mice, Knockout ; Microinjections ; Regression Analysis ; Reproducibility of Results ; },
abstract = {BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).<br><br>RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.<br><br>CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.},
}
@article {pmid31446470,
year = {2019},
author = {Ren, C and Guo, Y and Gathunga, EK and Duan, W and Li, S and Liang, Z},
title = {Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.},
journal = {Plant cell reports},
volume = {38},
number = {12},
pages = {1541-1549},
pmid = {31446470},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Green Fluorescent Proteins/genetics/metabolism ; Oxidoreductases/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene (PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.},
}
@article {pmid31446002,
year = {2019},
author = {Nakagawa, Y and Kaneko, T},
title = {Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm.},
journal = {Cryobiology},
volume = {90},
number = {},
pages = {71-74},
doi = {10.1016/j.cryobiol.2019.08.004},
pmid = {31446002},
issn = {1090-2392},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryopreservation/methods ; Electroporation/*methods ; Female ; Freeze Drying/methods ; Gene Editing/*methods ; Genome/genetics ; Male ; Oocytes/*growth &amp; development ; Rats ; Rats, Transgenic ; Semen Preservation/*methods ; Sperm Injections, Intracytoplasmic/*methods ; Spermatozoa/physiology ; },
abstract = {Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.},
}
@article {pmid31445521,
year = {2019},
author = {Li, X and Sun, T and Wang, X and Tang, J and Liu, Y},
title = {Restore natural fertility of Kit[w]/Kit[wv] mouse with nonobstructive azoospermia through gene editing on SSCs mediated by CRISPR-Cas9.},
journal = {Stem cell research &amp; therapy},
volume = {10},
number = {1},
pages = {271},
pmid = {31445521},
issn = {1757-6512},
mesh = {Adult Germline Stem Cells/physiology ; Animals ; Azoospermia/*genetics ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Female ; Fertility/*genetics ; Gene Editing/methods ; Humans ; Infertility, Male/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Proto-Oncogene Proteins c-kit/*genetics ; Spermatogenesis/genetics ; Spermatogonia/physiology ; Stem Cells/physiology ; Testis/physiology ; },
abstract = {BACKGROUND: Male infertility is a serious social problem in modern society. Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established.<br><br>METHODS: We choose Kit[w]/Kit[wv] mouse as a research model and try to develop a new treatment strategy and "cure" its infertility. Mutant spermatogonial stem cells (SSCs) were isolated from one single unilateral testis of a 14-day-old Kit[w]/Kit[wv] mouse and propagated in vitro. The C to T point mutation on Kit[wv] site of these SSCs was corrected through CRISPR-Cas9-mediated homology-directed repair (HDR) in vitro. Then, the repaired SSCs were screened out, proliferated, and transplanted into the remaining testis, and complete spermatogenesis was established in the recipient testis.<br><br>RESULTS: Healthy offsprings with wild type Kit gene or Kit[w] mutation were obtained through natural mating 4&#xa0;months after SSC transplantation.<br><br>CONCLUSION: In this study, we established an effective new treatment strategy for NOA caused by germ cell gene defects through a combination of SSC isolation, CRISPR-Cas9-mediated gene editing, and SSC transplantation, which brought hope for these NOA patients to restore their natural fertility.},
}
@article {pmid31445394,
year = {2019},
author = {Han, HJ and Seo, HH and Han, HW and Kim, JH},
title = {Generation of a TLR7 homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {40},
number = {},
pages = {101520},
doi = {10.1016/j.scr.2019.101520},
pmid = {31445394},
issn = {1876-7753},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Embryoid Bodies/cytology/metabolism ; Gene Editing ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism/transplantation ; Karyotype ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Teratoma/pathology ; Toll-Like Receptor 7/*genetics ; },
abstract = {Toll Like Receptor (TLR) family plays an important role in the activation of innate immunity against pathogens. TLR7 mediates the recognition of single-stranded RNA viruses, such as human immunodeficiency virus, hepatitis C virus, and influenza virus in endosomes. Here, we generated a TLR7 homozygous knockout human induced pluripotent cell (hiPSC) line, hiPSC-TLR7KO-A59, using the CRISPR/Cas9 genome editing method. The hiPSC-TLR7KO-A59 line maintains normal morphology, pluripotency, and differentiation capacity into three germ layers.},
}
@article {pmid31444855,
year = {2019},
author = {Ramamoorthy, A and Karnes, JH and Finkel, R and Blanchard, R and Pacanowski, M},
title = {Evolution of Next Generation Therapeutics: Past, Present, and Future of Precision Medicines.},
journal = {Clinical and translational science},
volume = {12},
number = {6},
pages = {560-563},
pmid = {31444855},
issn = {1752-8062},
mesh = {CRISPR-Cas Systems/genetics ; *Forecasting ; Genetic Therapy/history/methods/*trends ; History, 20th Century ; History, 21st Century ; Humans ; Pharmacogenetics/history/*trends ; Precision Medicine/history/methods/*trends ; },
}
@article {pmid31444470,
year = {2019},
author = {Suzuki, K and Yamamoto, M and Hernandez-Benitez, R and Li, Z and Wei, C and Soligalla, RD and Aizawa, E and Hatanaka, F and Kurita, M and Reddy, P and Ocampo, A and Hishida, T and Sakurai, M and Nemeth, AN and Nuñez Delicado, E and Campistol, JM and Magistretti, P and Guillen, P and Rodriguez Esteban, C and Gong, J and Yuan, Y and Gu, Y and Liu, GH and López-Otín, C and Wu, J and Zhang, K and Izpisua Belmonte, JC},
title = {Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction.},
journal = {Cell research},
volume = {29},
number = {10},
pages = {804-819},
pmid = {31444470},
issn = {1748-7838},
support = {DP1 DK113616/DK/NIDDK NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; R01 HL123755/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Repair ; Dependovirus/genetics ; GATA3 Transcription Factor/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Genetic Therapy/methods ; Genetic Vectors/metabolism ; Human Embryonic Stem Cells ; Humans ; Introns ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Neurons/cytology/metabolism ; RNA, Guide/metabolism ; Rats ; Tubulin/genetics ; },
abstract = {In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.},
}
@article {pmid31444400,
year = {2019},
author = {Cazzola, A and Schlegel, C and Jansen, I and Bochtler, T and Jauch, A and Krämer, A},
title = {TP53 deficiency permits chromosome abnormalities and karyotype heterogeneity in acute myeloid leukemia.},
journal = {Leukemia},
volume = {33},
number = {11},
pages = {2619-2627},
pmid = {31444400},
issn = {1476-5551},
mesh = {Aneuploidy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; *Chromosome Aberrations ; DNA Damage ; Gene Deletion ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia, Myeloid, Acute/diagnosis/*genetics ; Mutation ; Prognosis ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Abnormal karyotypes are common in cancer cells and frequently observed in acute myeloid leukemia (AML), in which complex karyotype aberrations are associated with poor prognosis. How exactly abnormal karyotypes arise and are propagated in AML is unclear. TP53 mutations and deletions are frequent in complex karyotype AML, suggesting a role of TP53 alterations in the development of chromosome abnormalities. Here, we generated isogenic TP53-knockout versions of the euploid AML cell line EEB to investigate the impact of TP53 on karyotype stability. We show that chromosome abnormalities spontaneously arise in TP53-deficient cells. Numerical aneuploidy could, to some extent, be propagated in a TP53-proficient setting, indicating that it does not necessarily trigger TP53 activation. In contrast, tolerance to structural chromosome aberrations was almost entirely restricted to TP53-knockout clones, all of which were able to continue proliferation in the presence of damaged DNA. Mechanistically, as a source of chromosome aberrations, limited numerical but not structural chromosomal instability was tolerated by TP53-wildtype cells. In contrast, structural instability was found only in TP53-knockout cells. Together, in myeloid cells TP53 loss allows for the development of complex karyotype aberrations and karyotype heterogeneity by perpetuation of chromosome segregation errors.},
}
@article {pmid31444356,
year = {2019},
author = {Miao, C and Wang, Z and Zhang, L and Yao, J and Hua, K and Liu, X and Shi, H and Zhu, JK},
title = {The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3822},
pmid = {31444356},
issn = {2041-1723},
mesh = {Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Edible Grain/physiology ; Gene Expression Regulation, Plant ; Gibberellins/*metabolism ; MicroRNAs/genetics/*metabolism ; Mutagenesis ; Mutation ; Oryza/*physiology ; Plant Breeding ; Plant Dormancy/*genetics ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {The widespread agricultural problem of pre-harvest sprouting (PHS) could potentially be overcome by improving seed dormancy. Here, we report that miR156, an important grain yield regulator, also controls seed dormancy in rice. We found that mutations in one MIR156 subfamily enhance seed dormancy and suppress PHS with negligible effects on shoot architecture and grain size, whereas mutations in another MIR156 subfamily modify shoot architecture and increase grain size but have minimal effects on seed dormancy. Mechanistically, mir156 mutations enhance seed dormancy by suppressing the gibberellin (GA) pathway through de-represssion of the miR156 target gene Ideal Plant Architecture 1 (IPA1), which directly regulates multiple genes in the GA pathway. These results provide an effective method to suppress PHS without compromising productivity, and will facilitate breeding elite crop varieties with ideal plant architectures.},
}
@article {pmid31444345,
year = {2019},
author = {Satchwell, TJ and Wright, KE and Haydn-Smith, KL and Sánchez-Román Terán, F and Moura, PL and Hawksworth, J and Frayne, J and Toye, AM and Baum, J},
title = {Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3806},
pmid = {31444345},
issn = {2041-1723},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Basigin/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Cyclophilins/genetics/metabolism ; Erythroblasts/physiology ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; *Host-Parasite Interactions ; Humans ; Lentivirus/genetics ; Malaria, Falciparum/*parasitology ; Plasmodium falciparum/metabolism/*pathogenicity ; Protein Domains/genetics ; Protozoan Proteins/metabolism ; Reticulocytes/*parasitology/physiology ; Transduction, Genetic ; },
abstract = {Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.},
}
@article {pmid31444197,
year = {2019},
author = {Zhou, D and Jiang, Z and Pang, Q and Zhu, Y and Wang, Q and Qi, Q},
title = {CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {21},
pages = {},
pmid = {31444197},
issn = {1098-5336},
mesh = {Acetylglucosamine/*biosynthesis ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA, Single-Stranded ; Gene Editing/*methods ; Gene Knockout Techniques ; Genes, Bacterial/genetics ; Genetic Engineering ; Genome, Bacterial ; Lactobacillus plantarum/*genetics ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Recombination, Genetic ; },
abstract = {Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications.IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.},
}
@article {pmid31440766,
year = {2019},
author = {Wallace, E and Howard, L and Liu, M and O'Brien, T and Ward, D and Shen, S and Prendiville, T},
title = {Long QT Syndrome: Genetics and Future Perspective.},
journal = {Pediatric cardiology},
volume = {40},
number = {7},
pages = {1419-1430},
pmid = {31440766},
issn = {1432-1971},
mesh = {Electrocardiography ; Heart Ventricles/physiopathology ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Long QT Syndrome/diagnosis/*genetics/metabolism ; Mutation ; Myocytes, Cardiac/metabolism ; },
abstract = {Long QT syndrome (LQTS) is an inherited primary arrhythmia syndrome that may present with malignant arrhythmia and, rarely, risk of sudden death. The clinical symptoms include palpitations, syncope, and anoxic seizures secondary to ventricular arrhythmia, classically torsade de pointes. This predisposition to malignant arrhythmia is from a cardiac ion channelopathy that results in delayed repolarization of the cardiomyocyte action potential. The QT interval on the surface electrocardiogram is a summation of the individual cellular ventricular action potential durations, and hence is a surrogate marker of the abnormal cellular membrane repolarization. Severely affected phenotypes administered current standard of care therapies may not be fully protected from the occurrence of cardiac arrhythmias. There are 17 different subtypes of LQTS associated with monogenic mutations of 15 autosomal dominant genes. It is now possible to model the various LQTS phenotypes through the generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes. RNA interference can silence or suppress the expression of mutant genes. Thus, RNA interference can be a potential therapeutic intervention that may be employed in LQTS to knock out mutant mRNAs which code for the defective proteins. CRISPR/Cas9 is a genome editing technology that offers great potential in elucidating gene function and a potential therapeutic strategy for monogenic disease. Further studies are required to determine whether CRISPR/Cas9 can be employed as an efficacious and safe rescue of the LQTS phenotype. Current progress has raised opportunities to generate in vitro human cardiomyocyte models for drug screening and to explore gene therapy through genome editing.},
}
@article {pmid31440756,
year = {2019},
author = {Liu, C and Chen, CY and Shang, QH and Liu, J},
title = {[Establishment of Ace2 knockout mouse model with CRISPR/Cas9 gene targeting technology].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {71},
number = {4},
pages = {588-596},
pmid = {31440756},
issn = {0371-0874},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Knockout Techniques ; Gene Targeting ; Male ; Mice ; *Mice, Knockout ; RNA, Guide/*genetics ; },
abstract = {The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2[-/Y] mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2[-/Y] mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.},
}
@article {pmid31440241,
year = {2019},
author = {Guo, C and Wang, M and Zhu, Z and He, S and Liu, H and Liu, X and Shi, X and Tang, T and Yu, P and Zeng, J and Yang, L and Cao, Y and Chen, Y and Liu, X and He, Z},
title = {Highly Efficient Generation of Pigs Harboring a Partial Deletion of the CD163 SRCR5 Domain, Which Are Fully Resistant to Porcine Reproductive and Respiratory Syndrome Virus 2 Infection.},
journal = {Frontiers in immunology},
volume = {10},
number = {},
pages = {1846},
pmid = {31440241},
issn = {1664-3224},
mesh = {Animals ; Antigens, CD/*genetics/immunology ; Antigens, Differentiation, Myelomonocytic/*genetics/immunology ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Disease Resistance/genetics ; Embryo Transfer ; Fibroblasts/cytology ; Gene Editing ; Humans ; Macrophages, Alveolar/immunology ; Porcine Reproductive and Respiratory Syndrome/*genetics/immunology/prevention &amp; control ; Porcine respiratory and reproductive syndrome virus/isolation &amp; purification/*pathogenicity ; Protein Domains ; Receptors, Cell Surface/*genetics/immunology ; Selective Breeding ; Sequence Alignment ; Sequence Deletion ; Sequence Homology, Nucleic Acid ; Species Specificity ; Swine/embryology/*genetics ; Viremia/prevention &amp; control ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2 differ in their recognition of CD163. Substitution of porcine CD163 SRCR5 domain with a human CD163-like SRCR8 confers resistance to PRRSV 1 but not PRRSV 2. The deletion of CD163 SRCR5 has been shown to confer resistance to PRRSV 1 in vivo and both PRRSV 1 and 2 in vitro. However, the anti-PRRSV 2 activity of modifying the CD163 SRCR5 domain has not yet been reported. Here, we describe the highly efficient generation of two pig breeds (Liang Guang Small Spotted and Large White pigs) lacking a short region of CD163 SRCR5, including the ligand-binding pocket. We generated a large number of gene-edited Large White pigs of the F0 generation for use in viral challenge studies. The results of this study show that these pigs are completely resistant to infection by species 2 PRRSV, JXA1, and MY strains. There were no clinical symptoms, pathological abnormalities, viremia, or anti-PRRSV antibodies in the CD163 SRCR5-edited pigs compared to wild-type controls after viral challenge. Porcine alveolar macrophages (PAMs) isolated from CD163 SRCR5-edited Large White pigs also displayed resistance to PRRSV in vitro. In addition, CD163 SRCR5-edited PAMs still exhibited a cytokine response to PRRSV infection, and no significant difference was observed in cytokine expression compared to wild-type PAMs. Taken together, these data suggest that CD163 SRCR5-edited pigs are resistant to PRRSV 2, providing a basis for the establishment of PRRSV-resistant pig lines for commercial application and further investigation of the essential region of SRCR5 involved in virus infection.},
}
@article {pmid31439892,
year = {2019},
author = {Liu, B and Song, J and Han, H and Hu, Z and Chen, N and Cui, J and Matsubara, JA and Zhong, J and Lei, H},
title = {Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells.},
journal = {Laboratory investigation; a journal of technical methods and pathology},
volume = {99},
number = {12},
pages = {1874-1886},
pmid = {31439892},
issn = {1530-0307},
support = {R01 EY012509/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Epiretinal Membrane/metabolism ; *Epithelial-Mesenchymal Transition ; HEK293 Cells ; Humans ; Proto-Oncogene Proteins c-mdm2/antagonists &amp; inhibitors/*metabolism ; Retinal Pigment Epithelium/cytology ; Transforming Growth Factor beta2 ; Vitreoretinopathy, Proliferative/*etiology/metabolism/therapy ; },
abstract = {Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.},
}
@article {pmid31439808,
year = {2019},
author = {Lee, J and Mou, H and Ibraheim, R and Liang, SQ and Liu, P and Xue, W and Sontheimer, EJ},
title = {Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {11},
pages = {1421-1431},
pmid = {31439808},
issn = {1469-9001},
support = {DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 GM125797/GM/NIGMS NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/genetics ; *Gene Editing ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; MicroRNAs/*genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Transgenes ; },
abstract = {CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3'-UTR and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3 Nme construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.},
}
@article {pmid31439663,
year = {2019},
author = {Meyer, MB and Lee, SM and Carlson, AH and Benkusky, NA and Kaufmann, M and Jones, G and Pike, JW},
title = {A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {39},
pages = {14467-14481},
pmid = {31439663},
issn = {1083-351X},
support = {R01 DK072281/DK/NIDDK NIH HHS/United States ; R01 DK117475/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Calcitriol/metabolism ; Chromatin/*metabolism ; Cyclic AMP Response Element-Binding Protein/metabolism ; Female ; Fibroblast Growth Factor-23 ; Fibroblast Growth Factors/metabolism ; Kidney/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Parathyroid Hormone/metabolism ; Receptors, Calcitriol/metabolism ; *Response Elements ; Vitamin D3 24-Hydroxylase/*genetics/metabolism ; },
abstract = {Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 In contrast, Cyp24a1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)2D3 Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element-binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1-deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2-deleted strain did not affect 1,25(OH)2D3 induction of renal Cyp24a1 expression yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.},
}
@article {pmid31439014,
year = {2019},
author = {Colic, M and Wang, G and Zimmermann, M and Mascall, K and McLaughlin, M and Bertolet, L and Lenoir, WF and Moffat, J and Angers, S and Durocher, D and Hart, T},
title = {Identifying chemogenetic interactions from CRISPR screens with drugZ.},
journal = {Genome medicine},
volume = {11},
number = {1},
pages = {52},
pmid = {31439014},
issn = {1756-994X},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; 342551//CIHR/Canada ; 361837//CIHR/Canada ; 365646//CIHR/Canada ; FDN143343//CIHR/Canada ; },
mesh = {*Algorithms ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Discovery/*methods ; Genetic Predisposition to Disease ; Humans ; Mutation ; Pharmacogenetics/*methods ; *Pharmacogenomic Variants ; *Software ; },
abstract = {BACKGROUND: Chemogenetic profiling enables the identification of gene mutations that enhance or suppress the activity of chemical compounds. This knowledge provides insights into drug mechanism of action, genetic vulnerabilities, and resistance mechanisms, all of which may help stratify patient populations and improve drug efficacy. CRISPR-based screening enables sensitive detection of drug-gene interactions directly in human cells, but until recently has primarily been used to screen only for resistance mechanisms.<br><br>RESULTS: We present drugZ, an algorithm for identifying both synergistic and suppressor chemogenetic interactions from CRISPR screens. DrugZ identifies synthetic lethal interactions between PARP inhibitors and both known and novel members of the DNA damage repair pathway, confirms KEAP1 loss as a resistance factor for ERK inhibitors in oncogenic KRAS backgrounds, and defines the genetic context for temozolomide activity.<br><br>CONCLUSIONS: DrugZ is an open-source Python software for the analysis of genome-scale drug modifier screens. The software accurately identifies genetic perturbations that enhance or suppress drug activity. Interestingly, analysis of new and previously published data reveals tumor suppressor genes are drug-agnostic resistance genes in drug modifier screens. The software is available at github.com/hart-lab/drugz .},
}
@article {pmid31436510,
year = {2019},
author = {Sukhdev, M},
title = {CRISPRcon: What Does Rome Look Like?.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {193-195},
doi = {10.1089/crispr.2019.29068.msu},
pmid = {31436510},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Gene Editing/ethics/*legislation &amp; jurisprudence ; Policy Making ; },
}
@article {pmid31436509,
year = {2019},
author = {Davies, K and Bolt, A},
title = {The Director's Cut: An Interview with Adam Bolt.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {188-192},
doi = {10.1089/crispr.2019.29067.abo},
pmid = {31436509},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*history/methods ; History, 21st Century ; *Motion Pictures ; },
}
@article {pmid31436507,
year = {2019},
author = {Barrangou, R},
title = {Bringing CRISPR to the Cinema.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {187},
doi = {10.1089/crispr.2019.29070.rba},
pmid = {31436507},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Motion Pictures ; },
}
@article {pmid31436506,
year = {2019},
author = {Standage-Beier, K and Brookhouser, N and Balachandran, P and Zhang, Q and Brafman, DA and Wang, X},
title = {RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and Integration.},
journal = {The CRISPR journal},
volume = {2},
number = {4},
pages = {209-222},
pmid = {31436506},
issn = {2573-1602},
support = {R01 GM106081/GM/NIGMS NIH HHS/United States ; R01 GM131405/GM/NIGMS NIH HHS/United States ; R21 AG056706/AG/NIA NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Recombinant Fusion Proteins ; Saccharomyces cerevisiae/genetics ; Transposon Resolvases ; },
abstract = {CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP-mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.},
}
@article {pmid31436505,
year = {2019},
author = {Gilmore, MS},
title = {The CRISPR-Antibiotic Resistance Connection.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {199-200},
doi = {10.1089/crispr.2019.29065.msg},
pmid = {31436505},
issn = {2573-1602},
mesh = {Bacteria/genetics ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Microbial/*genetics ; Gene Editing/*history ; History, 21st Century ; },
}
@article {pmid31436504,
year = {2019},
author = {Sun, N and Petiwala, S and Lu, C and Hutti, JE and Hu, M and Hu, M and Domanus, MH and Mitra, D and Addo, SN and Miller, CP and Chung, N},
title = {VHL Synthetic Lethality Signatures Uncovered by Genotype-Specific CRISPR-Cas9 Screens.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {230-245},
doi = {10.1089/crispr.2019.0018},
pmid = {31436504},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Carcinoma, Renal Cell/genetics/metabolism ; Cell Line, Tumor ; DNA Damage ; *DNA Repair ; Gene Editing ; Humans ; Kidney Neoplasms/genetics/metabolism ; Selenocysteine/*biosynthesis ; Sequence Analysis, RNA ; *Signal Transduction ; Von Hippel-Lindau Tumor Suppressor Protein/genetics/*metabolism ; von Hippel-Lindau Disease/genetics/metabolism ; },
abstract = {Genome-wide CRISPR-Cas9 essentiality screening represents a powerful approach to identify genetic vulnerabilities in cancer cells. Here, we applied this technology and designed a strategy to identify target genes that are synthetic lethal (SL) with von Hippel-Lindau (VHL) tumor suppressor gene. Inactivation of VHL has been frequently found in clear cell renal cell carcinoma. Its SL partners serve as potential drug targets for the development of targeted cancer therapies. We performed parallel genome-wide CRISPR screens in two pairs of isogenic clear cell renal cell carcinoma cell lines that differ only in the VHL status. Comparative analyses of screening results not only confirmed a well-known role for mTOR signaling in renal carcinoma, but also identified DNA damage response and selenocysteine biosynthesis pathways as novel SL targets in VHL-inactivated cancer cells. Follow-up studies provided cellular and mechanistic insights into SL interactions of these pathway genes with the VHL gene. Our CRISPR and RNA-seq datasets provide a rich resource for future investigation of the function of the VHL tumor suppressor protein. Our work demonstrates the efficiency of CRISPR-based synthetic lethality screening in human isogenic cell pairs. Similar strategies could be employed to unveil SL partners with other oncogenic drivers.},
}
@article {pmid31436503,
year = {2019},
author = {LeMieux, J},
title = {CRISPR-Accelerated Gene Drives Pump the Brakes.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {196-198},
doi = {10.1089/crispr.2019.29069.jlm},
pmid = {31436503},
issn = {2573-1602},
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Culicidae/*genetics ; *Gene Drive Technology ; Gene Editing ; Mosquito Control/*methods ; },
}
@article {pmid31436399,
year = {2019},
author = {Raposo, VL},
title = {The First Chinese Edited Babies: A Leap of Faith in Science.},
journal = {JBRA assisted reproduction},
volume = {23},
number = {3},
pages = {197-199},
pmid = {31436399},
issn = {1518-0557},
mesh = {CRISPR-Cas Systems/*genetics ; China ; *Cloning, Organism/ethics/methods/trends ; *Embryo Research/ethics ; *Ethics, Research ; Female ; *Gene Editing/ethics/methods/trends ; *Genetic Enhancement/ethics/methods ; Genetic Predisposition to Disease ; HIV Infections/genetics ; Humans ; Infant, Newborn ; Pregnancy ; Receptors, CCR5/genetics ; Reproductive Techniques, Assisted/ethics/trends ; },
}
@article {pmid31434745,
year = {2019},
author = {Zhang, WW and Matlashewski, G},
title = {Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania.},
journal = {mSphere},
volume = {4},
number = {4},
pages = {},
pmid = {31434745},
issn = {2379-5042},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair ; DNA, Protozoan/metabolism ; DNA, Single-Stranded/chemistry ; Gene Editing/methods ; *Genome, Protozoan ; Leishmania/*genetics ; Recombination, Genetic ; Staphylococcus aureus ; },
abstract = {CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in LeishmaniaIMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.},
}
@article {pmid31434578,
year = {2019},
author = {Liu, Q and Shi, X and Song, L and Liu, H and Zhou, X and Wang, Q and Zhang, Y and Cai, M},
title = {CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {144},
pmid = {31434578},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Engineering ; Genetic Vectors ; Pichia/*genetics ; RNA, Guide/metabolism ; Synthetic Biology ; },
abstract = {BACKGROUND: Pichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris.<br><br>RESULTS: A non-homologous-end-joining defective strain (&#x394;ku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100&#xa0;bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris.<br><br>CONCLUSIONS: A CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.},
}
@article {pmid31433690,
year = {2019},
author = {Yuan, F and Zhou, J and Xu, L and Jia, W and Chun, L and Xu, XZS and Liu, J},
title = {GABA receptors differentially regulate life span and health span in C. elegans through distinct downstream mechanisms.},
journal = {American journal of physiology. Cell physiology},
volume = {317},
number = {5},
pages = {C953-C963},
pmid = {31433690},
issn = {1522-1563},
support = {R35 GM126917/GM/NIGMS NIH HHS/United States ; },
mesh = {Aging/*genetics/metabolism ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/physiology ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Longevity/*physiology ; Oxidative Stress/physiology ; Receptors, GABA/*genetics/metabolism ; Signal Transduction/*physiology ; },
abstract = {GABA, a prominent inhibitory neurotransmitter, is best known to regulate neuronal functions in the nervous system. However, much less is known about the role of GABA signaling in other physiological processes. Interestingly, recent work showed that GABA signaling can regulate life span via a metabotropic GABAB receptor in Caenorhabditis elegans. However, the role of other types of GABA receptors in life span has not been clearly defined. It is also unclear whether GABA signaling regulates health span. Here, using C. elegans as a model, we systematically interrogated the role of various GABA receptors in both life span and health span. We find that mutations in four different GABA receptors extend health span by promoting resistance to stress and pathogen infection and that two such receptor mutants also show extended life span. Different GABA receptors engage distinct transcriptional factors to regulate life span and health span, and even the same receptor regulates life span and health span via different transcription factors. Our results uncover a novel, profound role of GABA signaling in aging in C. elegans, which is mediated by different GABA receptors coupled to distinct downstream effectors.},
}
@article {pmid31433621,
year = {2019},
author = {Park, S and Beal, PA},
title = {Off-Target Editing by CRISPR-Guided DNA Base Editors.},
journal = {Biochemistry},
volume = {58},
number = {36},
pages = {3727-3734},
pmid = {31433621},
issn = {1520-4995},
support = {R01 GM061115/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Deaminase/metabolism ; Animals ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Cytidine Deaminase/metabolism ; DNA/genetics/*metabolism ; Gene Editing/*methods ; Humans ; RNA Editing ; RNA, Guide/genetics/metabolism ; },
abstract = {Base editing is a genome editing strategy that induces specific single-nucleotide changes within genomic DNA. Two major DNA base editors, cytosine base editors and adenine base editors, that consist of a Cas9 protein linked to a deaminase enzyme that catalyzes targeted base conversion directed by a single-guide RNA have been developed. This strategy has been used widely for precise genome editing because, unlike CRISPR-Cas nuclease-based genome editing systems, this strategy does not create double-strand DNA breaks that often result in high levels of undesirable indels. However, recent papers have reported that DNA base editors can cause substantial off-target editing in both genomic DNA and RNA. The off-target editing described in these studies is primarily independent of guide RNA and arises from the promiscuous reactivity of the deaminase enzymes used in DNA base editors. In this Perspective, we discuss the development of DNA base editors, the guide RNA-independent off-target activity reported in recent studies, and strategies that improve the selectivity of DNA base editors.},
}
@article {pmid31433273,
year = {2020},
author = {Kalinina, NO and Khromov, A and Love, AJ and Taliansky, ME},
title = {CRISPR Applications in Plant Virology: Virus Resistance and Beyond.},
journal = {Phytopathology},
volume = {110},
number = {1},
pages = {18-28},
doi = {10.1094/PHYTO-07-19-0267-IA},
pmid = {31433273},
issn = {0031-949X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Diseases/virology ; *Plant Pathology ; *Plant Viruses ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) is a prokaryotic adaptive immune system which has been reprogrammed into a precise, simple, and efficient gene targeting technology. This emerging technology is revolutionizing various areas of life sciences, medicine, and biotechnology and has raised significant interest among plant biologists, both in basic science and in plant protection and breeding. In this review, we describe the basic principles of CRISPR/Cas systems, and how they can be deployed to model plants and crops for the control, monitoring, and study of the mechanistic aspects of plant virus infections. We discuss how Cas endonucleases can be used to engineer plant virus resistance by directly targeting viral DNA or RNA, as well as how they can inactivate host susceptibility genes. Additionally, other applications of CRISPR/Cas in plant virology such as virus diagnostics and imaging are reviewed. The review also provides a systemic comparison between CRISPR/Cas technology and RNA interference approaches, the latter of which has also been used for development of virus-resistant plants. Finally, we outline challenges to be solved before CRISPR/Cas can produce virus-resistant crop plants which can be marketed.},
}
@article {pmid31433061,
year = {2019},
author = {Wu, X and Zha, J and Koffas, MAG and Dordick, JS},
title = {Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR-dCas9.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {12},
pages = {3149-3159},
doi = {10.1002/bit.27143},
pmid = {31433061},
issn = {1097-0290},
mesh = {Animals ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/drug effects/genetics ; *Gene Deletion ; *Gene Expression Regulation, Bacterial/drug effects/genetics ; Genes, Bacterial ; Humans ; Lysostaphin/*pharmacology ; *Staphylococcus aureus/genetics/growth &amp; development ; },
abstract = {Bacteriolytic enzymes (cell lytic enzymes) are promising alternatives to antibiotics especially in killing drug-resistant bacteria. However, some bacteria slowly become resistant to various classes of peptidoglycan hydrolases, for reasons not well studied, in the presence of growth-supporting nutrients, which are prevalent at sites of infection. Here, we show that Staphylococcus aureus, a human and animal pathogen, while susceptible to the potent staphylolytic enzyme lysostaphin (Lst) in buffered saline, is highly resistant in the rich medium tryptic soy broth (TSB). Through a series of biochemical analysis, we identified that the resistance was due to prevention of Lst-cell binding mediated by the wall teichoic acids (WTAs) present on the cell surface. Inhibition or deletion of the gene tarO responsible for the first step of WTA biosynthesis greatly reduced S. aureus resistance to Lst in TSB. To overcome the resistance, we took advantage of the gene regulation potential of CRISPR-dCas9 and demonstrated that downregulation of tarO, tarH, and/or tarG gene expression, the latter two encoding enzymes that anchor WTAs in the outer layer of cell wall peptidoglycan, sensitized S. aureus to Lst and enabled eradication of the bacterium in TSB in 24 hr. As a result, we elucidate a key mechanism of Lst resistance in metabolically active S. aureus and provide a potential approach for treating life-threatening or hard-to-treat infections caused by Gram-positive pathogens.},
}
@article {pmid31431634,
year = {2019},
author = {Bloomfield, M and Louie, MC},
title = {Chronic cadmium exposure decreases the dependency of MCF7 breast cancer cells on ERα.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {12135},
pmid = {31431634},
issn = {2045-2322},
support = {R15 ES025917/ES/NIEHS NIH HHS/United States ; },
mesh = {Adenocarcinoma/genetics/*metabolism ; Breast Neoplasms/genetics/*metabolism ; CRISPR-Cas Systems ; Cadmium Chloride/*adverse effects ; Cell Movement/physiology ; Cell Proliferation/physiology ; Estrogen Receptor alpha/genetics/*metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MCF-7 Cells ; Neoplasm Invasiveness/physiopathology ; },
abstract = {Cadmium is an environmental contaminant that can activate estrogen receptor alpha (ERα) and contribute to the development and progression of breast cancer. Our lab previously demonstrated that chronic cadmium exposure alters the expression of several ERα-responsive genes and increases the malignancy of breast cancer cells. Although these studies support cadmium's function as a hormone disrupter, the role of ERα in cadmium-induced breast cancer progression remains unclear. To address this, we modulated the expression of ERα and found that while the loss of ERα significantly impaired cancer cell growth, migration, invasion and anchorage-independent growth in both MCF7 and MCF7-Cd cells, cadmium-exposed cells retained a significant advantage in cell growth, migration, and invasion, and partially circumvented the loss of ERα. ERα knockout in MCF7 and MCF7-Cd cells significantly reduced the expression of classical ERα-regulated genes, while non-classical ERα-regulated genes were less impacted by the loss of ERα in MCF7-Cd cells. This is the first study to show that chronic cadmium exposure, even at low levels, can increase the malignancy of breast cancer cells by decreasing their dependency on ERα and increasing the adaptability of the cancer cells.},
}
@article {pmid31430902,
year = {2019},
author = {Razzaq, A and Saleem, F and Kanwal, M and Mustafa, G and Yousaf, S and Imran Arshad, HM and Hameed, MK and Khan, MS and Joyia, FA},
title = {Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox.},
journal = {International journal of molecular sciences},
volume = {20},
number = {16},
pages = {},
pmid = {31430902},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing/*methods ; *Genome, Plant ; Plant Breeding ; Plants/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.},
}
@article {pmid31429483,
year = {2019},
author = {Wu, Q and Wang, B and Zhou, C and Lin, P and Qin, S and Gao, P and Wang, Z and Xia, Z and Wu, M},
title = {Bacterial Type I CRISPR-Cas systems influence inflammasome activation in mammalian host by promoting autophagy.},
journal = {Immunology},
volume = {158},
number = {3},
pages = {240-251},
pmid = {31429483},
issn = {1365-2567},
support = {GM103442/NH/NIH HHS/United States ; 5R01AI109317-04/NH/NIH HHS/United States ; 1R01AI138203-01/NH/NIH HHS/United States ; P20 GM113123/NH/NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Autophagic Cell Death/*immunology ; CRISPR-Cas Systems/*immunology ; Cell Line ; Female ; *Immune Evasion ; Inflammasomes/*immunology ; Male ; Mice ; Pseudomonas Infections/*immunology/pathology ; *Pseudomonas aeruginosa/immunology/pathogenicity ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR-Cas) systems in prokaryotes function at defending against foreign DNAs, providing adaptive immunity to maintain homeostasis. CRISPR-Cas may also influence immune regulation ability in mammalian cells through alterations of pathogenic extent and nature. Recent research has implied that Type I CRISPR-Cas systems of Pseudomonas aeruginosa strain UCBPP-PA14 impede recognition by Toll-like receptor 4, and decrease pro-inflammatory responses both in vitro and in vivo. However, the molecular mechanism by which CRISPR-Cas systems affect host immunity is largely undemonstrated. Here, we explored whether CRISPR-Cas systems can influence autophagy to alter the activation of inflammasome. Using the wild-type PA14 and total CRISPR-Cas region deletion (∆TCR) mutant strain, we elucidated the role and underlying mechanism of Type I CRISPR-Cas systems in bacterial infection, and showed that CRISPR-Cas systems impacted the release of mitochondrial DNA and induction of autophagy. CRISPR-Cas deficiency led to an increase of mitochondrial DNA release, a decrease in autophagy, an increase of inflammasome activation and, ultimately, an elevation of pro-inflammatory response. Our findings illustrate a new important mechanism by which Type I CRISPR-Cas systems control their virulence potency to evade host defense.},
}
@article {pmid31428784,
year = {2019},
author = {Standage-Beier, K and Tekel, SJ and Brookhouser, N and Schwarz, G and Nguyen, T and Wang, X and Brafman, DA},
title = {A transient reporter for editing enrichment (TREE) in human cells.},
journal = {Nucleic acids research},
volume = {47},
number = {19},
pages = {e120},
pmid = {31428784},
issn = {1362-4962},
support = {R01 GM106081/GM/NIGMS NIH HHS/United States ; R01 GM121698/GM/NIGMS NIH HHS/United States ; R01 GM131405/GM/NIGMS NIH HHS/United States ; R21 AG056706/AG/NIA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/metabolism ; RNA, Guide/*genetics ; Transfection/*methods ; },
abstract = {Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.},
}
@article {pmid31428783,
year = {2019},
author = {Birkholz, N and Fagerlund, RD and Smith, LM and Jackson, SA and Fineran, PC},
title = {The autoregulator Aca2 mediates anti-CRISPR repression.},
journal = {Nucleic acids research},
volume = {47},
number = {18},
pages = {9658-9665},
pmid = {31428783},
issn = {1362-4962},
mesh = {Bacteriophages/genetics ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics ; Operon/genetics ; Pectobacterium carotovorum/*genetics ; Promoter Regions, Genetic/genetics ; Protein Domains/genetics ; Transcription Factors/genetics ; *Transcription, Genetic ; Viral Proteins/*genetics ; },
abstract = {CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.},
}
@article {pmid31428700,
year = {2019},
author = {Johnston, AD and Simões-Pires, CA and Suzuki, M and Greally, JM},
title = {High-efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells using a single-stranded donor oligonucleotide strategy.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {312},
pmid = {31428700},
issn = {2399-3642},
support = {T32 GM007288/GM/NIGMS NIH HHS/United States ; },
mesh = {B-Lymphocytes/*metabolism/*virology ; CRISPR-Cas Systems/genetics ; Cell Line, Transformed ; Child ; Clone Cells ; *Gene Editing ; Gene Rearrangement/genetics ; Genetic Loci ; Herpesvirus 4, Human/*metabolism ; Humans ; Oligonucleotides/*metabolism ; },
abstract = {While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a substantial problem when multiple constructs need to be co-expressed. Here we describe a protocol using a single-stranded donor oligonucleotide strategy for 'scarless' editing in lymphoblastoid cells, yielding 12/60 (20%) of clones with homology-directed recombination, when rates of <5-10% are frequently typical for many other cell types. The protocol does not require the use of lentiviruses or stable transfection, permitting lymphoblastoid cell lines to be used for CRISPR-mediated genomic targeting and screening in population genetic studies.},
}
@article {pmid31427601,
year = {2019},
author = {Lin, P and Pu, Q and Wu, Q and Zhou, C and Wang, B and Schettler, J and Wang, Z and Qin, S and Gao, P and Li, R and Li, G and Cheng, Z and Lan, L and Jiang, J and Wu, M},
title = {High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3728},
pmid = {31427601},
issn = {2041-1723},
support = {P20 GM113123/GM/NIGMS NIH HHS/United States ; R01 AI109317/AI/NIAID NIH HHS/United States ; R01 AI138203/AI/NIAID NIH HHS/United States ; R15 AI101973/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/*genetics ; High-Throughput Screening Assays ; Pseudomonas aeruginosa/*genetics ; RNA, Bacterial/*biosynthesis/genetics ; RNA, Small Untranslated/*genetics ; Rho Factor/*antagonists &amp; inhibitors ; Transcription Termination, Genetic/*physiology ; },
abstract = {Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.},
}
@article {pmid31427598,
year = {2019},
author = {Kang, JG and Park, JS and Ko, JH and Kim, YS},
title = {Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11960},
pmid = {31427598},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Cloning, Molecular ; CpG Islands ; *DNA Methylation ; *Epigenesis, Genetic ; *Gene Editing ; *Gene Expression Regulation ; Gene Knock-In Techniques ; Gene Targeting ; Histones/metabolism ; Mice ; NIH 3T3 Cells ; Octamer Transcription Factor-3/chemistry ; Plasmids/genetics ; *Promoter Regions, Genetic ; RNA, Guide ; },
abstract = {Despite the increased interest in epigenetic research, its progress has been hampered by a lack of satisfactory tools to control epigenetic factors in specific genomic regions. Until now, many attempts to manipulate DNA methylation have been made using drugs but these drugs are not target-specific and have global effects on the whole genome. However, due to new genome editing technologies, potential epigenetic factors can now possibly be regulated in a site-specific manner. Here, we demonstrate the utility of CRISPR/Cas9 to modulate methylation at specific CpG sites and to elicit gene expression. We targeted the murine Oct4 gene which is transcriptionally locked due to hypermethylation at the promoter region in NIH3T3 cells. To induce site-specific demethylation at the Oct4 promoter region and its gene expression, we used the CRISPR/Cas9 knock-in and CRISPR/dCas9-Tet1 systems. Using these two approaches, we induced site-specific demethylation at the Oct4 promoter and confirmed the up-regulation of Oct4 expression. Furthermore, we confirmed that the synergistic effect of DNA demethylation and other epigenetic regulations increased the expression of Oct4 significantly. Based on our research, we suggest that our proven epigenetic editing methods can selectively modulate epigenetic factors such as DNA methylation and have promise for various applications in epigenetics.},
}
@article {pmid31427540,
year = {2019},
author = {Hu, Q and Wolfner, MF},
title = {The Drosophila Trpm channel mediates calcium influx during egg activation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {38},
pages = {18994-19000},
pmid = {31427540},
issn = {1091-6490},
support = {R21 HD088744/HD/NICHD NIH HHS/United States ; S10 OD018516/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Signaling/physiology ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Drosophila melanogaster/embryology/*metabolism ; *Embryonic Development ; Female ; Fertilization/*physiology ; Male ; Oocytes/cytology/*metabolism ; TRPM Cation Channels/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.},
}
@article {pmid31426522,
year = {2019},
author = {Shelake, RM and Pramanik, D and Kim, JY},
title = {Exploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era.},
journal = {Microorganisms},
volume = {7},
number = {8},
pages = {},
pmid = {31426522},
issn = {2076-2607},
abstract = {Plants and microbes are co-evolved and interact with each other in nature. Plant-associated microbes, often referred to as plant microbiota, are an integral part of plant life. Depending on the health effects on hosts, plant-microbe (PM) interactions are either beneficial or harmful. The role of microbiota in plant growth promotion (PGP) and protection against various stresses is well known. Recently, our knowledge of community composition of plant microbiome and significant driving factors have significantly improved. So, the use of plant microbiome is a reliable approach for a next green revolution and to meet the global food demand in sustainable and eco-friendly agriculture. An application of the multifaceted PM interactions needs the use of novel tools to know critical genetic and molecular aspects. Recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing (GE) tools are of great interest to explore PM interactions. A systematic understanding of the PM interactions will enable the application of GE tools to enhance the capacity of microbes or plants for agronomic trait improvement. This review focuses on applying GE techniques in plants or associated microbiota for discovering the fundamentals of the PM interactions, disease resistance, PGP activity, and future implications in agriculture.},
}
@article {pmid31424432,
year = {2019},
author = {Akutsu, Y and Doi, M and Furukawa, K and Takagi, Y},
title = {Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {149},
pages = {},
doi = {10.3791/59962},
pmid = {31424432},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Chagas Disease/genetics/*parasitology ; Fibroblasts/metabolism/parasitology ; Gene Editing ; Gene Knockout Techniques/*methods ; Humans ; Life Cycle Stages/*physiology ; Protozoan Proteins/*antagonists &amp; inhibitors/genetics ; Trypanosoma cruzi/*genetics/*growth &amp; development/metabolism ; },
abstract = {Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.},
}
@article {pmid31423617,
year = {2019},
author = {Maroc, L and Fairhead, C},
title = {A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata.},
journal = {Yeast (Chichester, England)},
volume = {36},
number = {12},
pages = {723-731},
doi = {10.1002/yea.3440},
pmid = {31423617},
issn = {1097-0061},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Candida glabrata/*genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; Gene Editing ; Gene Targeting ; Genome, Fungal/*genetics ; Homologous Recombination ; Microbial Viability ; Plasmids ; Promoter Regions, Genetic ; RNA, Guide/genetics ; *Transcriptional Activation ; },
abstract = {In recent years, the CRISPR-Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR-Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR-Cas9 system, from the cutting and repair steps. This has indeed been developed in S. cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C. glabrata is gal[-] and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C. glabrata, for both described outcomes of CRISPR-Cas9-induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C. glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double-strand break repair.},
}
@article {pmid31422865,
year = {2019},
author = {Tian, R and Gachechiladze, MA and Ludwig, CH and Laurie, MT and Hong, JY and Nathaniel, D and Prabhu, AV and Fernandopulle, MS and Patel, R and Abshari, M and Ward, ME and Kampmann, M},
title = {CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.},
journal = {Neuron},
volume = {104},
number = {2},
pages = {239-255.e12},
pmid = {31422865},
issn = {1097-4199},
support = {R56 AG057528/AG/NIA NIH HHS/United States ; ZIA NS003155-01/ImNIH/Intramural NIH HHS/United States ; F30 AG060722/AG/NIA NIH HHS/United States ; ZIA NS003155-03/ImNIH/Intramural NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Survival ; Gene Knockdown Techniques/*methods ; Humans ; *Induced Pluripotent Stem Cells ; Microscopy, Confocal ; Neurons/cytology/*metabolism ; RNA-Seq ; Single-Cell Analysis ; },
abstract = {CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.},
}
@article {pmid31422862,
year = {2019},
author = {Jiang, C and Lin, X and Zhao, Z},
title = {Applications of CRISPR/Cas9 Technology in the Treatment of Lung Cancer.},
journal = {Trends in molecular medicine},
volume = {25},
number = {11},
pages = {1039-1049},
doi = {10.1016/j.molmed.2019.07.007},
pmid = {31422862},
issn = {1471-499X},
mesh = {Animals ; B7-H1 Antigen/genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Drug Resistance/genetics ; ErbB Receptors/genetics ; Gene Editing/*methods/trends ; Gene Frequency ; Gene Silencing ; Genes, Tumor Suppressor ; Genetic Therapy/methods/trends ; Humans ; *Lung Neoplasms/diagnosis/genetics/therapy ; MicroRNAs/genetics ; Models, Animal ; Molecular Targeted Therapy/trends ; Mutation Rate ; Oncogenes/*genetics ; Precision Medicine/trends ; Programmed Cell Death 1 Receptor/genetics/metabolism ; Proto-Oncogenes/genetics ; T-Lymphocytes/metabolism ; gamma Catenin/genetics ; },
abstract = {Since its emergence, the application of CRISPR-associated nuclease 9 (Cas9) technology in cancer research has accelerated studies to investigate many aspects of treatment approaches for lung cancer, including the identification of target genes, construction of animal tumor models, and identification of drug resistance-related genes. Moreover, CRISPR/Cas9 can be used in gene therapy for lung cancer, specifically involving molecular targeted drugs and inhibitors. This article reviews the current landscape of CRISPR/Cas9 applications for lung cancer treatment as a basis for further studies. Given its promising performance, in-depth and systematic research on the application of CRISPR/Cas9 in lung cancer treatment will be necessary in future studies for its successful implementation in clinical practice.},
}
@article {pmid31422074,
year = {2019},
author = {Gordon, DM and Adeosun, SO and Ngwudike, SI and Anderson, CD and Hall, JE and Hinds, TD and Stec, DE},
title = {CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation.},
journal = {Archives of biochemistry and biophysics},
volume = {672},
number = {},
pages = {108072},
pmid = {31422074},
issn = {1096-0384},
support = {P01 HL051971/HL/NHLBI NIH HHS/United States ; L32 MD009154/MD/NIMHD NIH HHS/United States ; F31 DK084958/DK/NIDDK NIH HHS/United States ; P20 GM104357/GM/NIGMS NIH HHS/United States ; K01 HL125445/HL/NHLBI NIH HHS/United States ; P30 DK020572/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Bilirubin/metabolism ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Deletion ; Gene Knockout Techniques ; Hepatocytes/*metabolism ; Lipid Metabolism/*physiology ; Mice ; Oxidative Stress/*physiology ; Oxidoreductases Acting on CH-CH Group Donors/*genetics ; Reactive Oxygen Species/metabolism ; },
abstract = {Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.},
}
@article {pmid31421397,
year = {2019},
author = {Irion, U and Nüsslein-Volhard, C},
title = {The identification of genes involved in the evolution of color patterns in fish.},
journal = {Current opinion in genetics &amp; development},
volume = {57},
number = {},
pages = {31-38},
pmid = {31421397},
issn = {1879-0380},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cichlids/*genetics/physiology ; *Evolution, Molecular ; Gene Expression Regulation, Developmental/genetics ; Pigmentation/*genetics/physiology ; Transcriptome/*genetics ; Zebrafish/genetics/physiology ; },
abstract = {The genetic basis of morphological variation, both within and between species, provides a major topic in evolutionary biology. Teleost fish produce most elaborate color patterns, and among the more than 20000 species a number have been chosen for more detailed analyses because they are suitable to study particular aspects of color pattern evolution. In several fish species, color variants and pattern variants have been collected, transcriptome analyses have been carried out, and the recent advent of gene editing tools, such as CRISPR/Cas9, has allowed the production of mutants. Covering mostly the literature from the last three years, we discuss the cellular basis of coloration and the identification of loci involved in color pattern differences between sister species in cichlids and Danio species, in which cis-regulatory changes seem to prevail.},
}
@article {pmid31419585,
year = {2019},
author = {Melling, GE and Carollo, E and Conlon, R and Simpson, JC and Carter, DRF},
title = {The Challenges and Possibilities of Extracellular Vesicles as Therapeutic Vehicles.},
journal = {European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V},
volume = {144},
number = {},
pages = {50-56},
doi = {10.1016/j.ejpb.2019.08.009},
pmid = {31419585},
issn = {1873-3441},
mesh = {Animals ; CRISPR-Cas Systems/drug effects ; Drug Delivery Systems/*methods ; Extracellular Vesicles/*metabolism ; Humans ; Mesenchymal Stem Cells/drug effects ; Nucleic Acids/metabolism ; Proteins/metabolism ; RNA Interference/drug effects ; Signal Transduction/drug effects ; },
abstract = {Extracellular vesicles (EVs) are small lipid-enclosed particles that can carry various types of cargo, including proteins, nucleic acids and metabolites. They are known to be released by all cell types and can be taken up by other cells, leading to the transfer of the cargo they carry. As such, they represent an important type of intercellular signalling and a natural mechanism for transferring macromolecules between cells. This ability to transfer cargo could be harnessed to deliver therapeutic molecules. Indeed, a growing body of work has described the attempt by the field to utilise EVs to deliver a range of therapeutics including RNAi, CRISPR/Cas9 and chemotherapeutics, to a specific target tissue. However, there are numerous barriers associated with the use of EVs as therapeutic vehicles, including the challenge of efficiently loading therapeutics into EVs, avoiding clearance of the EVs from circulation, targeting the correct tissue type and the inefficiency of internalisation and functional delivery of the cargo. Despite these difficulties, EVs represent a tremendous therapeutic opportunity, both for the delivery of exogenous cargo, as well as the therapeutic benefit of targeting aberrant EV signalling or treating patients with natural EVs, such as those released by mesenchymal stem cells. This review describes current knowledge on the therapeutic potential of EVs and the challenges faced by the field. Many of these challenges are due to a lack of complete understanding of EV function, but further research in this area should continue to yield new solutions that will lead to the use of EVs in the clinic.},
}
@article {pmid31419111,
year = {2019},
author = {Velázquez, E and Lorenzo, V and Al-Ramahi, Y},
title = {Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery.},
journal = {ACS synthetic biology},
volume = {8},
number = {9},
pages = {2186-2193},
doi = {10.1021/acssynbio.9b00293},
pmid = {31419111},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/genetics ; Gene Editing/*methods ; Introns ; Lac Operon/genetics ; Plasmids/genetics/metabolism ; Retroelements/genetics ; },
abstract = {Group II introns were developed some time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-positive and Gram-negative bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of additional genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counterselection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed that directing CRISPR/Cas9 toward the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counterselection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.},
}
@article {pmid31418136,
year = {2019},
author = {Ji, X and Wang, D and Gao, C},
title = {CRISPR editing-mediated antiviral immunity: a versatile source of resistance to combat plant virus infections.},
journal = {Science China. Life sciences},
volume = {62},
number = {9},
pages = {1246-1249},
doi = {10.1007/s11427-019-9722-2},
pmid = {31418136},
issn = {1869-1889},
mesh = {Antiviral Agents/*metabolism ; CRISPR-Cas Systems/*genetics/*immunology ; DNA, Plant/genetics ; Gene Editing ; Gene Expression ; Genome, Bacterial/genetics ; Infections/metabolism ; Mutation ; Plant Diseases/*genetics/virology ; Plant Viruses/genetics/*metabolism ; Virus Diseases/*prevention &amp; control ; },
}
@article {pmid31418127,
year = {2020},
author = {Mills, EM and Barlow, VL and Luk, LYP and Tsai, YH},
title = {Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications.},
journal = {Cell biology and toxicology},
volume = {36},
number = {1},
pages = {17-29},
pmid = {31418127},
issn = {1573-6822},
support = {BB/P009506/1//Biology and Biological Science Research Council/International ; 200730/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods/trends ; Genetic Therapy/*trends ; Humans ; },
abstract = {Progress in targeted gene editing by programmable endonucleases has paved the way for their use in gene therapy. Particularly, Cas9 is an endonuclease with high activity and flexibility, rendering it an attractive option for therapeutic applications in clinical settings. Many disease-causing mutations could potentially be corrected by this versatile new technology. In addition, recently developed switchable Cas9 variants, whose activity can be controlled by an external stimulus, provide an extra level of spatiotemporal control on gene editing and are particularly desirable for certain applications. Here, we discuss the considerations and difficulties for implementing Cas9 to in vivo gene therapy. We put particular emphasis on how switchable Cas9 variants may resolve some of these barriers and advance gene therapy in the clinical setting.},
}
@article {pmid31418019,
year = {2020},
author = {Ly, S and Strus, E and Naidoo, N},
title = {Genetic disruption of the putative binding site for Homer on DmGluRA reduces sleep in Drosophila.},
journal = {Sleep},
volume = {43},
number = {1},
pages = {},
pmid = {31418019},
issn = {1550-9109},
support = {P01 AG017628/AG/NIA NIH HHS/United States ; },
mesh = {Amino Acid Substitution/genetics ; Animals ; Binding Sites/genetics ; CRISPR-Cas Systems ; Drosophila/genetics/*physiology ; Drosophila Proteins/*genetics ; Gene Editing ; Homer Scaffolding Proteins/*metabolism ; Neuronal Plasticity/*genetics/physiology ; Neurons/metabolism ; Receptors, Metabotropic Glutamate/*genetics ; Signal Transduction/physiology ; Sleep/*genetics/physiology ; Synapses/physiology ; },
abstract = {Homer proteins mediate plasticity and signaling at the postsynaptic density of neurons and are necessary for sleep and synaptic remodeling during sleep. The goal of this study was to investigate the mechanisms of sleep regulation by Homer signaling. Using the Drosophila animal model, we demonstrate that knockdown of Homer specifically in the brain reduces sleep and that Drosophila Homer binds to the sole Drosophila mGluR, known as DmGluRA. This is the first evidence that DmGluRA, which bears greatest homology to group II mammalian metabotropic glutamate receptors (mGluRs), shares functional homology with group I mGluRs which couple to Homer proteins in mammals. As sleep is associated with the physical dissociation of Homer and mGluRs proteins at the synapse, we sought to determine the functional necessity of Homer × DmGluRA interaction in sleep regulation. Using the CRISPR/Cas9 gene editing system, we generated a targeted amino acid replacement of the putative binding site for Homer on DmGluRA to prevent Homer and DmGluRA protein binding. We found that loss of the conserved proline-rich PPXXF sequence on DmGluRA reduces Homer/DmGluRA associations and significantly reduces sleep amount. Thus, we identify a conserved mechanism of synaptic plasticity in Drosophila and demonstrate that the interaction of Homer with DmGluRA is necessary to promote sleep.},
}
@article {pmid31417187,
year = {2019},
author = {Secker, KA and Keppeler, H and Duerr-Stoerzer, S and Schmid, H and Schneidawind, D and Hentrich, T and Schulze-Hentrich, JM and Mankel, B and Fend, F and Schneidawind, C},
title = {Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9.},
journal = {Oncogene},
volume = {38},
number = {46},
pages = {7181-7195},
pmid = {31417187},
issn = {1476-5594},
mesh = {Adenosine/analogs &amp; derivatives/pharmacology ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Drug Synergism ; Gene Editing/methods ; Hematopoietic Stem Cells ; Histone-Lysine N-Methyltransferase/*antagonists &amp; inhibitors ; Humans ; Isoquinolines/pharmacology ; *Leukemia/genetics ; *Models, Biological ; Myeloid-Lymphoid Leukemia Protein/genetics ; Phenylurea Compounds/pharmacology ; Protein-Arginine N-Methyltransferases/*antagonists &amp; inhibitors ; Pyrimidines/pharmacology ; },
abstract = {MLL rearrangements play a crucial role in leukemogenesis and comprise a poor prognosis. Therefore, new treatment strategies are urgently needed. We used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4 or -AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient cells faithfully mimicking the nature of the disease. Thus, it serves as a fundamental basis for pharmacological studies: inhibition of histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is one specific therapeutic approach currently tested in clinical trials. However, success was limited by restricted response warranting further investigation of drug combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines and in MLL mouse models. Here, we used DOT1L and PRMT5 inhibitors in our human MLL-rearranged model demonstrating dose-dependent reduced proliferation, impairment of cell cycle, increasing differentiation, apoptosis, downregulation of target genes and sensitization to chemotherapy. Strikingly, the combination of both compounds led to synergistic anti-tumoral effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias.},
}
@article {pmid31417125,
year = {2019},
author = {Morani, F and Doccini, S and Sirica, R and Paterno, M and Pezzini, F and Ricca, I and Simonati, A and Delledonne, M and Santorelli, FM},
title = {Functional Transcriptome Analysis in ARSACS KO Cell Model Reveals a Role of Sacsin in Autophagy.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11878},
pmid = {31417125},
issn = {2045-2322},
mesh = {Autophagy/*genetics ; CRISPR-Cas Systems ; Cells, Cultured ; Computational Biology/methods ; *Gene Expression Profiling/methods ; Gene Knockout Techniques ; Gene Ontology ; Genetic Association Studies ; *Genetic Predisposition to Disease ; Heat-Shock Proteins/*genetics ; Humans ; Mitochondria/genetics/metabolism ; Muscle Spasticity/diagnosis/*genetics/metabolism ; Oxidative Phosphorylation ; Proteasome Endopeptidase Complex/metabolism ; Spinocerebellar Ataxias/*congenital/diagnosis/genetics/metabolism ; TOR Serine-Threonine Kinases/metabolism ; *Transcriptome ; Ubiquitin/metabolism ; },
abstract = {Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS.},
}
@article {pmid31416932,
year = {2019},
author = {Wang, D and Stoveken, HM and Zucca, S and Dao, M and Orlandi, C and Song, C and Masuho, I and Johnston, C and Opperman, KJ and Giles, AC and Gill, MS and Lundquist, EA and Grill, B and Martemyanov, KA},
title = {Genetic behavioral screen identifies an orphan anti-opioid system.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6459},
pages = {1267-1273},
pmid = {31416932},
issn = {1095-9203},
support = {U42 RR024244/RR/NCRR NIH HHS/United States ; F32 DA048579/DA/NIDA NIH HHS/United States ; R21 DA040406/DA/NIDA NIH HHS/United States ; P20 GM103638/GM/NIGMS NIH HHS/United States ; K02 DA026405/DA/NIDA NIH HHS/United States ; R01 DA036596/DA/NIDA NIH HHS/United States ; F32 DA047771/DA/NIDA NIH HHS/United States ; R01 NS072129/NS/NINDS NIH HHS/United States ; },
mesh = {Analgesia ; Animals ; Animals, Genetically Modified ; *Behavior, Animal ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Chromosome Mapping ; Female ; HEK293 Cells ; Humans ; Male ; Mice ; Mice, Knockout ; Morphine/pharmacology ; Nerve Tissue Proteins/*genetics ; Neurons/drug effects ; Orphan Nuclear Receptors/*genetics ; Receptors, G-Protein-Coupled/*genetics ; Receptors, Opioid, mu/*genetics ; Signal Transduction ; },
abstract = {Opioids target the μ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.},
}
@article {pmid31416668,
year = {2019},
author = {Kwart, D and Gregg, A and Scheckel, C and Murphy, EA and Paquet, D and Duffield, M and Fak, J and Olsen, O and Darnell, RB and Tessier-Lavigne, M},
title = {A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ.},
journal = {Neuron},
volume = {104},
number = {2},
pages = {256-270.e5},
doi = {10.1016/j.neuron.2019.07.010},
pmid = {31416668},
issn = {1097-4199},
mesh = {Alzheimer Disease/*genetics/pathology ; Amyloid Precursor Protein Secretases ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/*genetics ; Aspartic Acid Endopeptidases ; CRISPR-Cas Systems ; Cell Line ; Endocytosis/*genetics ; Endosomes/*metabolism/pathology ; Gene Expression Profiling ; Gene Knock-In Techniques ; Heterozygote ; Homozygote ; Humans ; Induced Pluripotent Stem Cells ; Mutation ; Neurons/*metabolism ; Organelle Size ; Peptide Fragments/*metabolism ; Phenotype ; Presenilin-1/*genetics ; Proteomics ; rab5 GTP-Binding Proteins/metabolism ; },
abstract = {Familial Alzheimer's disease (fAD) results from mutations in the amyloid precursor protein (APP) and presenilin (PSEN1 and PSEN2) genes. Here we leveraged recent advances in induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies to generate a panel of isogenic knockin human iPSC lines carrying APP and/or PSEN1 mutations. Global transcriptomic and translatomic profiling revealed that fAD mutations have overlapping effects on the expression of AD-related and endocytosis-associated genes. Mutant neurons also increased Rab5+ early endosome size. APP and PSEN1 mutations had discordant effects on Aβ production but similar effects on APP β C-terminal fragments (β-CTFs), which accumulate in all mutant neurons. Importantly, endosomal dysfunction correlated with accumulation of β-CTFs, not Aβ, and could be rescued by pharmacological modulation of β-secretase (BACE). These data display the utility of our mutant iPSCs in studying AD-related phenotypes in a non-overexpression human-based system and support mounting evidence that β-CTF may be critical in AD pathogenesis.},
}
@article {pmid31416510,
year = {2019},
author = {Ma, F and Shi, CC and Liang, PP and Li, ST and Gu, X and Xiao, X and Hao, H},
title = {[Construction of a mouse model of cblC type methylmalonic acidemia with W203X mutation based on the CRISPR/Cas9 technology].},
journal = {Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics},
volume = {21},
number = {8},
pages = {824-829},
pmid = {31416510},
issn = {1008-8830},
mesh = {Amino Acid Metabolism, Inborn Errors ; Animals ; *CRISPR-Cas Systems ; Carrier Proteins ; Heterozygote ; Mice ; Mutation ; Oxidoreductases ; },
abstract = {OBJECTIVE: To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology.<br><br>METHODS: At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice.<br><br>RESULTS: The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P<0.001).<br><br>CONCLUSIONS: A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.},
}
@article {pmid31416467,
year = {2019},
author = {Keough, KC and Lyalina, S and Olvera, MP and Whalen, S and Conklin, BR and Pollard, KS},
title = {AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {167},
pmid = {31416467},
issn = {1474-760X},
support = {P01HL089707/NH/NIH HHS/United States ; R01MH109907/NH/NIH HHS/United States ; R01 EY028249/EY/NEI NIH HHS/United States ; 1R01EY028249/NH/NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; U01HL100406/NH/NIH HHS/United States ; R01HL130533/NH/NIH HHS/United States ; UM1HL098179/NH/NIH HHS/United States ; },
mesh = {*Alleles ; Base Sequence ; CRISPR-Associated Proteins/metabolism ; Humans ; Polymorphism, Genetic ; RNA, Guide/*genetics ; *Software ; },
abstract = {The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at https://github.com/keoughkath/AlleleAnalyzer .},
}
@article {pmid31415841,
year = {2019},
author = {Xiao, L and Shan, Y and Ma, L and Dunk, C and Yu, Y and Wei, Y},
title = {Tuning FOXD3 expression dose-dependently balances human embryonic stem cells between pluripotency and meso-endoderm fates.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1866},
number = {12},
pages = {118531},
doi = {10.1016/j.bbamcr.2019.118531},
pmid = {31415841},
issn = {1879-2596},
mesh = {CRISPR-Cas Systems/genetics ; Cell Cycle ; Cell Proliferation ; Cells, Cultured ; Endoderm/*metabolism ; Forkhead Transcription Factors/deficiency/*genetics/metabolism ; HEK293 Cells ; Human Embryonic Stem Cells/*metabolism ; Humans ; Phenotype ; },
abstract = {Forkhead box D3 (FOXD3) is a key transcription factor maintaining pluripotency in mouse embryonic stem cells (ESCs). Yet to date studies on its role in human ESCs are quite limited. In this study, we report that deletion of FOXD3 in human ESCs results in loss of pluripotency and spontaneous differentiation toward meso-endoderm. Ectopic overexpression of FOXD3 in hESCs leads to two different phenotypes: Human ESCs expressing high levels of FOXD3 undergo spontaneous meso-endoderm differentiation, whereas those with lower levels of FOXD3 maintain pluripotency. Next we deleted endogenous FOXD3 in the low ectopic expression model and find that addition of exogenous FOXD3 at a low level could rescue FOXD3-deficiency phenotype in hESCs. In summary, our findings suggest that FOXD3 dose-dependently regulates the balance of human ESCs between pluripotency and meso-endoderm fates, which adds to our understanding of the role of FOXD3 in humans.},
}
@article {pmid31413315,
year = {2019},
author = {Liang, M and Li, Z and Wang, W and Liu, J and Liu, L and Zhu, G and Karthik, L and Wang, M and Wang, KF and Wang, Z and Yu, J and Shuai, Y and Yu, J and Zhang, L and Yang, Z and Li, C and Zhang, Q and Shi, T and Zhou, L and Xie, F and Dai, H and Liu, X and Zhang, J and Liu, G and Zhuo, Y and Zhang, B and Liu, C and Li, S and Xia, X and Tong, Y and Liu, Y and Alterovitz, G and Tan, GY and Zhang, LX},
title = {A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3672},
pmid = {31413315},
issn = {2041-1723},
mesh = {Allosteric Regulation ; Biological Assay ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clostridiales ; *Endodeoxyribonucleases ; Humans ; Limit of Detection ; Nucleotide Motifs ; Parabens ; Synthetic Biology ; *Transcription Factors ; Uric Acid/blood ; },
abstract = {Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p-hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.},
}
@article {pmid31413125,
year = {2019},
author = {BeltCappellino, A and Majerciak, V and Lobanov, A and Lack, J and Cam, M and Zheng, ZM},
title = {CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells.},
journal = {Journal of virology},
volume = {93},
number = {21},
pages = {},
pmid = {31413125},
issn = {1098-5514},
support = {ZIA SC010357/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation, Viral ; Gene Knockout Techniques ; Genome, Viral/*genetics ; Genomic Instability ; Herpesvirus 8, Human/*genetics/physiology ; Humans ; RNA, Guide/genetics ; Sequence Inversion ; Viral Regulatory and Accessory Proteins/*genetics/metabolism ; Virus Activation ; Virus Replication ; },
abstract = {Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ∼70 to 150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here, we report the first complete knockout (KO) of viral ORF57 gene from all ∼100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination with multiple rounds of cell selection and single-cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from the KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single-cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out the ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells, where each cell contains only a few copies of the KSHV genome. The ORF57 KO genome was stable in iSLK/Bac16 cells, and, upon lytic induction, was partially rescued by ectopic ORF57 to express viral lytic gene ORF59 and produce infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and the Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high copy numbers.IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology can be applied to knock out the ORF57 gene from all ∼100 copies of the KSHV genome in primary effusion lymphoma (PEL) cells by coexpressing two guide RNAs (gRNAs) and Cas9 from a single expression vector in combination with single-cell cloning. The gene knockout efficiency in this system was evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology also mediated ORF57 inversion in situ in the targeted site of the KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57 knockout (KO) genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of the virus genome and to understand their contributions to viral genetics and the virus life cycle.},
}
@article {pmid31411686,
year = {2019},
author = {Tang, Z and Chen, S and Chen, A and He, B and Zhou, Y and Chai, G and Guo, F and Huang, J},
title = {CasPDB: an integrated and annotated database for Cas proteins from bacteria and archaea.},
journal = {Database : the journal of biological databases and curation},
volume = {2019},
number = {},
pages = {},
pmid = {31411686},
issn = {1758-0463},
mesh = {Archaea/*genetics ; Archaeal Proteins/*genetics ; Bacteria/*genetics ; Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; *Databases, Protein ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) constitute CRISPR-Cas systems, which are antiphage immune systems present in numerous bacterial and most archaeal species. In recent years, CRISPR-Cas systems have been developed into reliable and powerful genome editing tools. Nevertheless, finding similar or better tools from bacteria or archaea remains crucial. This requires the exploration of different CRISPR systems, identification and characterization new Cas proteins. Archives tailored for Cas proteins are urgently needed and necessitate the prediction and grouping of Cas proteins into an information center with all available experimental evidence. Here, we constructed Cas Protein Data Bank (CasPDB), an integrated and annotated online database for Cas proteins from bacteria and archaea. The CasPDB database contains 287 reviewed Cas proteins, 257 745 putative Cas proteins and 3593 Cas operons from 32 023 bacteria species and 1802 archaea species. The database can be freely browsed and searched. The CasPDB web interface also represents all the 3593 putative Cas operons and its components. Among these operons, 328 are members of the type II CRISPR-Cas system.},
}
@article {pmid31410472,
year = {2019},
author = {Josipović, G and Tadić, V and Klasić, M and Zanki, V and Bečeheli, I and Chung, F and Ghantous, A and Keser, T and Madunić, J and Bošković, M and Lauc, G and Herceg, Z and Vojta, A and Zoldoš, V},
title = {Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system.},
journal = {Nucleic acids research},
volume = {47},
number = {18},
pages = {9637-9657},
pmid = {31410472},
issn = {1362-4962},
mesh = {Acyltransferases/genetics ; CRISPR-Cas Systems/*genetics ; Catalytic Domain/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; DNA Methylation/genetics ; DNA Methyltransferase 3A ; *Epigenesis, Genetic ; Gene Editing/*methods ; Gene Expression Regulation/genetics ; Genome/genetics ; Glycosylation ; Hepatocyte Nuclear Factor 1-alpha/genetics ; Humans ; Mixed Function Oxygenases/genetics ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/genetics ; RNA, Guide/genetics ; *Transcription, Genetic ; },
abstract = {Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.},
}
@article {pmid31409685,
year = {2019},
author = {Hullahalli, K and Rodrigues, M and Nguyen, UT and Palmer, K},
title = {Erratum for Hullahalli et al., "An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition".},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
doi = {10.1128/mBio.01775-19},
pmid = {31409685},
issn = {2150-7511},
}
@article {pmid31409682,
year = {2019},
author = {Gloag, ES and Marshall, CW and Snyder, D and Lewin, GR and Harris, JS and Santos-Lopez, A and Chaney, SB and Whiteley, M and Cooper, VS and Wozniak, DJ},
title = {Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
pmid = {31409682},
issn = {2150-7511},
support = {R01 AI061396/AI/NIAID NIH HHS/United States ; R01 AI134895/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics ; Bacteriophages/genetics ; Biofilms/*growth &amp; development ; Biological Evolution ; CRISPR-Associated Proteins/genetics ; Cyclic GMP/metabolism ; Genetic Fitness ; Genome, Bacterial/genetics ; Mutation ; Phenotype ; Pseudomonas Infections/metabolism/*microbiology ; Pseudomonas aeruginosa/*genetics/growth &amp; development/isolation &amp; purification ; Swine ; Wound Infection/metabolism/*microbiology ; },
abstract = {Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.},
}
@article {pmid31408165,
year = {2019},
author = {Liu, L and Lu, JY and Li, F and Xing, X and Li, T and Yang, X and Shen, X},
title = {IDH1 fine-tunes cap-dependent translation initiation.},
journal = {Journal of molecular cell biology},
volume = {11},
number = {10},
pages = {816-828},
pmid = {31408165},
issn = {1759-4685},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryonic Stem Cells/metabolism ; Isocitrate Dehydrogenase/genetics/*metabolism ; Ketoglutaric Acids/metabolism ; Mice ; Polyribosomes/metabolism ; RNA, Messenger/metabolism ; Ribosomes/metabolism ; },
abstract = {The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Its mutation often leads to aberrant gene expression in cancer. IDH1 was reported to bind thousands of RNA transcripts in a sequence-dependent manner; yet, the functional significance of this RNA-binding activity remains elusive. Here, we report that IDH1 promotes mRNA translation via direct associations with polysome mRNA and translation machinery. Comprehensive proteomic analysis in embryonic stem cells (ESCs) revealed striking enrichment of ribosomal proteins and translation regulators in IDH1-bound protein interactomes. We performed ribosomal profiling and analyzed mRNA transcripts that are associated with actively translating polysomes. Interestingly, knockout of IDH1 in ESCs led to significant downregulation of polysome-bound mRNA in IDH1 targets and subtle upregulation of ribosome densities at the start codon, indicating inefficient translation initiation upon loss of IDH1. Tethering IDH1 to a luciferase mRNA via the MS2-MBP system promotes luciferase translation, independently of the catalytic activity of IDH1. Intriguingly, IDH1 fails to enhance luciferase translation driven by an internal ribosome entry site. Together, these results reveal an unforeseen role of IDH1 in fine-tuning cap-dependent translation via the initiation step.},
}
@article {pmid31407519,
year = {2020},
author = {Li, QV and Rosen, BP and Huangfu, D},
title = {Decoding pluripotency: Genetic screens to interrogate the acquisition, maintenance, and exit of pluripotency.},
journal = {Wiley interdisciplinary reviews. Systems biology and medicine},
volume = {12},
number = {1},
pages = {e1464},
pmid = {31407519},
issn = {1939-005X},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 DK096239/DK/NIDDK NIH HHS/United States ; T32 GM008539/GM/NIGMS NIH HHS/United States ; T32GM008539//NIH T32 Training Grant/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells/cytology/physiology ; *Genetic Techniques ; Humans ; Mice ; *Pluripotent Stem Cells/cytology/physiology ; RNA Interference ; Systems Biology/*methods ; },
abstract = {Pluripotent stem cells have the ability to unlimitedly self-renew and differentiate to any somatic cell lineage. A number of systems biology approaches have been used to define this pluripotent state. Complementary to systems level characterization, genetic screens offer a unique avenue to functionally interrogate the pluripotent state and identify the key players in pluripotency acquisition and maintenance, exit of pluripotency, and lineage differentiation. Here we review how genetic screens have helped us decode pluripotency regulation. We will summarize results from RNA interference (RNAi) based screens, discuss recent advances in CRISPR/Cas-based genetic perturbation methods, and how these advances have made it possible to more comprehensively interrogate pluripotency and differentiation through genetic screens. Such investigations will not only provide a better understanding of this unique developmental state, but may enhance our ability to use pluripotent stem cells as an experimental model to study human development and disease progression. Functional interrogation of pluripotency also provides a valuable roadmap for utilizing genetic perturbation to gain systems level understanding of additional cellular states, from later stages of development to pathological disease states. This article is categorized under: Developmental Biology > Stem Cell Biology and Regeneration Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.},
}
@article {pmid31407391,
year = {2020},
author = {Fisicaro, N and Salvaris, EJ and Philip, GK and Wakefield, MJ and Nottle, MB and Hawthorne, WJ and Cowan, PJ},
title = {FokI-dCas9 mediates high-fidelity genome editing in pigs.},
journal = {Xenotransplantation},
volume = {27},
number = {1},
pages = {e12551},
doi = {10.1111/xen.12551},
pmid = {31407391},
issn = {1399-3089},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; DNA Mutational Analysis ; Deoxyribonucleases, Type II Site-Specific/*genetics ; Feasibility Studies ; Gene Editing/*methods ; Mutation/*genetics ; Sus scrofa ; Transplantation, Heterologous ; Whole Genome Sequencing ; },
abstract = {Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.},
}
@article {pmid31407276,
year = {2019},
author = {Ochiai, H},
title = {Real-Time Observation of Localization and Expression (ROLEX) System for Live Imaging of the Transcriptional Activity and Nuclear Position of a Specific Endogenous Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2038},
number = {},
pages = {35-45},
doi = {10.1007/978-1-4939-9674-2_3},
pmid = {31407276},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Nucleus/genetics/*metabolism ; Gene Expression Regulation ; *Genetic Loci ; Mice ; *Microscopy, Fluorescence ; Molecular Imaging/*methods ; Mouse Embryonic Stem Cells/*metabolism ; RNA, Messenger/genetics/*metabolism ; Single Molecule Imaging/*methods ; Time Factors ; *Transcription, Genetic ; },
abstract = {Long genomic DNA is folded in a cell-type-specific manner and stored in the cell nucleus. The higher-order structure of genomic DNA is thought to be important for DNA transcription, repair, and replication. Recent advancements in live cell imaging techniques that enable the labeling of specific genomic loci and RNA have made it possible to capture the dynamic relationships between higher-order genomic structure and gene expression. We have established the real-time observation of localization and expression (ROLEX) system for live imaging of the transcriptional state and nuclear position of a specific endogenous gene. In this chapter, I will introduce the detailed protocol of ROLEX imaging in mouse embryonic stem cells.},
}
@article {pmid31406383,
year = {2019},
author = {Campa, CC and Weisbach, NR and Santinha, AJ and Incarnato, D and Platt, RJ},
title = {Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts.},
journal = {Nature methods},
volume = {16},
number = {9},
pages = {887-893},
pmid = {31406383},
issn = {1548-7105},
support = {26606//Brain and Behavior Research Foundation (Brain &amp; Behavior Research Foundation)/International ; },
mesh = {Acidaminococcus/enzymology ; *CRISPR-Cas Systems ; Endonucleases/genetics/*metabolism ; *Gene Editing ; *Gene Regulatory Networks ; *Genetic Engineering ; *Genome, Human ; HEK293 Cells ; Humans ; Plasmids/genetics ; RNA, Guide/*genetics ; Transcriptional Activation ; },
abstract = {The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.},
}
@article {pmid31406378,
year = {2019},
author = {Durai, V and Bagadia, P and Granja, JM and Satpathy, AT and Kulkarni, DH and Davidson, JT and Wu, R and Patel, SJ and Iwata, A and Liu, TT and Huang, X and Briseño, CG and Grajales-Reyes, GE and Wöhner, M and Tagoh, H and Kee, BL and Newberry, RD and Busslinger, M and Chang, HY and Murphy, TL and Murphy, KM},
title = {Cryptic activation of an Irf8 enhancer governs cDC1 fate specification.},
journal = {Nature immunology},
volume = {20},
number = {9},
pages = {1161-1173},
pmid = {31406378},
issn = {1529-2916},
support = {R01 DK097317/DK/NIDDK NIH HHS/United States ; K08 CA230188/CA/NCI NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; UL1 TR000448/TR/NCATS NIH HHS/United States ; R01 AI106352/AI/NIAID NIH HHS/United States ; UL1 TR002345/TR/NCATS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AI076427/AI/NIAID NIH HHS/United States ; P30 AR073752/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage ; Dendritic Cells/*cytology/immunology ; Enhancer Elements, Genetic/*genetics ; Gene Expression Regulation ; Interferon Regulatory Factors/genetics/*metabolism ; Macrophages/*cytology/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Monocytes/*cytology/metabolism ; Stem Cells/cytology ; Tumor Cells, Cultured ; },
abstract = {Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.},
}
@article {pmid31406271,
year = {2019},
author = {Ehrenhöfer-Wölfer, K and Puchner, T and Schwarz, C and Rippka, J and Blaha-Ostermann, S and Strobl, U and Hörmann, A and Bader, G and Kornigg, S and Zahn, S and Sommergruber, W and Schweifer, N and Zichner, T and Schlattl, A and Neumüller, RA and Shi, J and Vakoc, CR and Kögl, M and Petronczki, M and Kraut, N and Pearson, MA and Wöhrle, S},
title = {SMARCA2-deficiency confers sensitivity to targeted inhibition of SMARCA4 in esophageal squamous cell carcinoma cell lines.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11661},
pmid = {31406271},
issn = {2045-2322},
support = {P01 CA013106/CA/NCI NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; DNA Helicases/*antagonists &amp; inhibitors/genetics ; Epigenesis, Genetic ; Esophageal Neoplasms/*drug therapy/genetics/pathology ; Esophageal Squamous Cell Carcinoma/*drug therapy/genetics/pathology ; Gene Editing ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Loss of Function Mutation ; Molecular Targeted Therapy/methods ; Nuclear Proteins/*antagonists &amp; inhibitors/genetics ; RNA, Guide/genetics ; RNA, Small Interfering/metabolism ; Synthetic Lethal Mutations ; Transcription Factors/*antagonists &amp; inhibitors/deficiency/*genetics ; },
abstract = {SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers.},
}
@article {pmid31406246,
year = {2019},
author = {Gobbi, G and Donati, B and Do Valle, IF and Reggiani, F and Torricelli, F and Remondini, D and Castellani, G and Ambrosetti, DC and Ciarrocchi, A and Sancisi, V},
title = {The Hippo pathway modulates resistance to BET proteins inhibitors in lung cancer cells.},
journal = {Oncogene},
volume = {38},
number = {42},
pages = {6801-6817},
pmid = {31406246},
issn = {1476-5594},
mesh = {A549 Cells ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*metabolism/pathology ; Cell Nucleus/metabolism ; *Drug Resistance, Neoplasm ; Hippo Signaling Pathway ; Humans ; Lung Neoplasms/*metabolism/pathology ; Neoplasm Proteins/*antagonists &amp; inhibitors/metabolism ; Protein Serine-Threonine Kinases/genetics/*metabolism ; *Signal Transduction ; },
abstract = {Inhibitors of BET proteins (BETi) are anti-cancer drugs that have shown efficacy in pre-clinical settings and are currently in clinical trials for different types of cancer, including non-small cell lung cancer (NSCLC). Currently, no predictive biomarker is available to identify patients that may benefit from this treatment. To uncover the mechanisms of resistance to BETi, we performed a genome-scale CRISPR/Cas9 screening in lung cancer cells. We identified three Hippo pathway genes, LATS2, TAOK1, and NF2, as key determinants for sensitivity to BETi. The knockout of these genes induces resistance to BETi, by promoting TAZ nuclear localization and transcriptional activity. Conversely, TAZ expression promotes resistance to these drugs. We also showed that TAZ, YAP, and their partner TEAD are direct targets of BRD4 and that treatment with BETi downregulates their expression. Noticeably, molecular alterations in one or more of these genes are present in a large fraction of NSCLC patients and TAZ amplification or overexpression correlates with a worse outcome in lung adenocarcinoma. Our data define the central role of Hippo pathway in mediating resistance to BETi and provide a rationale for using BETi to counter-act YAP/TAZ-mediated pro-oncogenic activity.},
}
@article {pmid31405997,
year = {2019},
author = {Nance, J and Frøkjær-Jensen, C},
title = {The Caenorhabditis elegans Transgenic Toolbox.},
journal = {Genetics},
volume = {212},
number = {4},
pages = {959-990},
pmid = {31405997},
issn = {1943-2631},
support = {R35 GM118081/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; *Genetic Techniques ; },
abstract = {The power of any genetic model organism is derived, in part, from the ease with which gene expression can be manipulated. The short generation time and invariant developmental lineage have made Caenorhabditis elegans very useful for understanding, e.g., developmental programs, basic cell biology, neurobiology, and aging. Over the last decade, the C. elegans transgenic toolbox has expanded considerably, with the addition of a variety of methods to control expression and modify genes with unprecedented resolution. Here, we provide a comprehensive overview of transgenic methods in C. elegans, with an emphasis on recent advances in transposon-mediated transgenesis, CRISPR/Cas9 gene editing, conditional gene and protein inactivation, and bipartite systems for temporal and spatial control of expression.},
}
@article {pmid31404948,
year = {2019},
author = {Wu, TM and Huang, JZ and Oung, HM and Hsu, YT and Tsai, YC and Hong, CY},
title = {H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.},
journal = {International journal of molecular sciences},
volume = {20},
number = {16},
pages = {},
pmid = {31404948},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Hydrogen Peroxide/*analysis ; Oryza/*genetics ; Phosphotransferases (Alcohol Group Acceptor)/genetics ; Plant Proteins/genetics ; Plants, Genetically Modified/*genetics ; Transgenes ; },
abstract = {Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3'-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.},
}
@article {pmid31404748,
year = {2019},
author = {Menges, J and Cremanns, M and Steenpass, L},
title = {Generation of two H1 hESC sublines carrying deletions of RB1 exon 1/promoter in heterozygous or compound heterozygous state.},
journal = {Stem cell research},
volume = {39},
number = {},
pages = {101517},
doi = {10.1016/j.scr.2019.101517},
pmid = {31404748},
issn = {1876-7753},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Exons/*genetics ; Fluorescent Antibody Technique ; Heterozygote ; Humans ; Karyotyping ; Male ; Microsatellite Repeats/genetics ; Promoter Regions, Genetic/*genetics ; Retinoblastoma/*genetics ; Retinoblastoma Binding Proteins/genetics ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Biallelic inactivation of the retinoblastoma tumor suppressor gene (RB1) causes formation of retinoblastoma, a retinal eye tumor occurring in early childhood. Using the CRISPR/Cas9 nickase system, exon 1 of RB1 was deleted, including the RB1 promoter. As a result, sublines were generated carrying deletions of RB1 exon 1/promoter on one or both alleles.},
}
@article {pmid31404563,
year = {2019},
author = {Tsukamoto, T and Sakai, E and Nishimae, F and Sakurai, F and Mizuguchi, H},
title = {Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells.},
journal = {Journal of biotechnology},
volume = {304},
number = {},
pages = {1-9},
doi = {10.1016/j.jbiotec.2019.08.004},
pmid = {31404563},
issn = {1873-4863},
mesh = {Adenoviridae/genetics/*physiology ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Gene Editing ; Genetic Vectors/physiology ; HEK293 Cells/cytology/virology ; Humans ; RNA, Guide/*genetics ; Viral Load ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.},
}
@article {pmid31403279,
year = {2019},
author = {Qian, C and Wang, R and Wu, H and Zhang, F and Wu, J and Wang, L},
title = {Uracil-Mediated New Photospacer-Adjacent Motif of Cas12a To Realize Visualized DNA Detection at the Single-Copy Level Free from Contamination.},
journal = {Analytical chemistry},
volume = {91},
number = {17},
pages = {11362-11366},
doi = {10.1021/acs.analchem.9b02554},
pmid = {31403279},
issn = {1520-6882},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; DNA Cleavage ; DNA, Single-Stranded/*analysis ; Endodeoxyribonucleases/*metabolism ; Nucleotide Motifs ; Uracil/*metabolism ; },
abstract = {The CRISPR/Cas12a (cpf1) system was reported to indiscriminately cleave single-stranded DNA after binding with target DNA strands. This usually required the target DNA strands to contain the protospacer-adjacent motif (PAM) sequence of TTTN. Herein, we found Cas12a can also recognize another PAM sequence of UUUN resulting in activation of its ssDNA collateral cleavage effect. To make this finding useful, by combining with LAMP, we first realized CRISPR/Cas12a for directly visualized DNA detection at the single-copy level. By treating with UDG enzyme, we made this system free from residual amplicon contamination, which is a big problem in this field. Thus, an ultrasensitive and anticontaminant DNA detection platform, namely, UDG and LAMP and CRISPR (ULC). This new finding would help us better understand the mechanism of Cas12a and expand its application.},
}
@article {pmid31403180,
year = {2019},
author = {Pazmiño-Ibarra, V and Mengual-Martí, A and Targovnik, AM and Herrero, S},
title = {Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {11},
pages = {2823-2833},
doi = {10.1002/bit.27139},
pmid = {31403180},
issn = {1097-0290},
mesh = {Animals ; *Baculoviridae/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing ; *Genetic Vectors ; *Genome, Viral ; *Pest Control, Biological ; Sf9 Cells ; Spodoptera ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system-associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.},
}
@article {pmid31403072,
year = {2019},
author = {Hanewich-Hollatz, MH and Chen, Z and Hochrein, LM and Huang, J and Pierce, NA},
title = {Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.},
journal = {ACS central science},
volume = {5},
number = {7},
pages = {1241-1249},
pmid = {31403072},
issn = {2374-7943},
abstract = {A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.},
}
@article {pmid31402430,
year = {2020},
author = {Kizner, V and Naujock, M and Fischer, S and Jäger, S and Reich, S and Schlotthauer, I and Zuckschwerdt, K and Geiger, T and Hildebrandt, T and Lawless, N and Macartney, T and Dorner-Ciossek, C and Gillardon, F},
title = {CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells.},
journal = {Molecular neurobiology},
volume = {57},
number = {2},
pages = {616-634},
pmid = {31402430},
issn = {1559-1182},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Cell Proliferation ; Cerebral Cortex/*pathology ; DNA/biosynthesis ; *Gene Knockout Techniques ; *Genetic Predisposition to Disease ; Humans ; Induced Pluripotent Stem Cells/*pathology ; Mental Disorders/*genetics ; Neural Stem Cells/metabolism ; Neurites/metabolism ; Neurons/*pathology ; Nuclear Proteins/deficiency/*genetics ; Receptor, ErbB-2/metabolism ; Risk Factors ; rhoA GTP-Binding Protein/metabolism ; },
abstract = {The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.},
}
@article {pmid31402173,
year = {2019},
author = {El Khattabi, L and Zhao, H and Kalchschmidt, J and Young, N and Jung, S and Van Blerkom, P and Kieffer-Kwon, P and Kieffer-Kwon, KR and Park, S and Wang, X and Krebs, J and Tripathi, S and Sakabe, N and Sobreira, DR and Huang, SC and Rao, SSP and Pruett, N and Chauss, D and Sadler, E and Lopez, A and Nóbrega, MA and Aiden, EL and Asturias, FJ and Casellas, R},
title = {A Pliable Mediator Acts as a Functional Rather Than an Architectural Bridge between Promoters and Enhancers.},
journal = {Cell},
volume = {178},
number = {5},
pages = {1145-1158.e20},
pmid = {31402173},
issn = {1097-4172},
support = {DP2 OD008540/OD/NIH HHS/United States ; R01 GM067167/GM/NIGMS NIH HHS/United States ; UM1 HG009375/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/metabolism ; Cells, Cultured ; Chromosomal Proteins, Non-Histone/metabolism ; Cryoelectron Microscopy ; Enhancer Elements, Genetic ; Gene Editing ; Humans ; Male ; Mediator Complex/chemistry/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/cytology/metabolism ; Promoter Regions, Genetic ; Protein Structure, Quaternary ; RNA Polymerase II/genetics/*metabolism ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; },
abstract = {While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.},
}
@article {pmid31402115,
year = {2019},
author = {Estève, J and Blouin, JM and Lalanne, M and Azzi-Martin, L and Dubus, P and Bidet, A and Harambat, J and Llanas, B and Moranvillier, I and Bedel, A and Moreau-Gaudry, F and Richard, E},
title = {Targeted gene therapy in human-induced pluripotent stem cells from a patient with primary hyperoxaluria type 1 using CRISPR/Cas9 technology.},
journal = {Biochemical and biophysical research communications},
volume = {517},
number = {4},
pages = {677-683},
doi = {10.1016/j.bbrc.2019.07.109},
pmid = {31402115},
issn = {1090-2104},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Genetic Loci ; *Genetic Therapy ; Genetic Vectors/metabolism ; Hepatocytes/cytology ; Humans ; Hyperoxaluria, Primary/*genetics/*therapy ; Induced Pluripotent Stem Cells/*pathology ; Mice ; },
abstract = {Primary hyperoxaluria type 1 (PH1) is an inherited metabolic disorder caused by a deficiency of the peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT), which leads to overproduction of oxalate by the liver and results in urolithiasis, nephrocalcinosis and renal failure. The only curative treatment for PH1 is combined liver and kidney transplantation, which is limited by the lack of suitable organs, significant complications, and the life-long requirement for immunosuppressive agents to maintain organ tolerance. Hepatocyte-like cells (HLCs) generated from CRISPR/Cas9 genome-edited human-induced pluripotent stem cells would offer an attractive unlimited source of autologous gene-corrected liver cells as an alternative to orthotopic liver transplantation (OLT). Here we report the CRISPR/Cas9 nuclease-mediated gene targeting of a single-copy AGXT therapeutic minigene into the safe harbour AAVS1 locus in PH1-induced pluripotent stem cells (PH1-iPSCs) without off-target inserts. We obtained a robust expression of a codon-optimized AGT in HLCs derived from AAVS1 locus-edited PH1-iPSCs. Our study provides the proof of concept that CRISPR/Cas9-mediated integration of an AGXT minigene into the AAVS1 safe harbour locus in patient-specific iPSCs is an efficient strategy to generate functionally corrected hepatocytes, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of PH1.},
}
@article {pmid31401729,
year = {2019},
author = {Hsu, CT and Cheng, YJ and Yuan, YH and Hung, WF and Cheng, QW and Wu, FH and Lee, LY and Gelvin, SB and Lin, CS},
title = {Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.},
journal = {Plant molecular biology},
volume = {101},
number = {4-5},
pages = {355-371},
pmid = {31401729},
issn = {1573-5028},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems ; Francisella/genetics ; Gene Editing/*methods ; Homozygote ; Plant Proteins/genetics ; Tetraploidy ; Tobacco/*genetics ; },
abstract = {Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.},
}
@article {pmid31400605,
year = {2019},
author = {Liu, X and Li, G and Zhou, X and Qiao, Y and Wang, R and Tang, S and Liu, J and Wang, L and Huang, X},
title = {Improving Editing Efficiency for the Sequences with NGH PAM Using xCas9-Derived Base Editors.},
journal = {Molecular therapy. Nucleic acids},
volume = {17},
number = {},
pages = {626-635},
pmid = {31400605},
issn = {2162-2531},
abstract = {The development of CRISPR/Cas9-mediated base editors (BEs) provided a versatile tool for precise genome editing. The recently developed xCas9-derived base editors (xBEs) that recognize the NG PAM substantially expand the targeting scope in the genome, while their editing efficiency needs to be improved. Here, we described an improved version of xBEs by fusing the BPNLS and Gam to the N terminus of xBEs (BPNLS-Gam-xBE3 and BPNLS-xABE), and this version of base editor displayed higher targeting efficiency for the majority of detected sites. By using this improved version of xBEs, we successfully created and corrected pathogenic mutations at genomic sites with the NGN protospacer-adjacent motif in human cells. Lastly, we used BPNLS-Gam-xBE3 to model pathogenic mutations in discarded human tripronuclear (3PN) zygotes, and no obvious off-targets and indels were detected. Taken together, the data in our study offer an efficient tool for precise genome editing and, thus, an enriched base editing toolkit.},
}
@article {pmid31399630,
year = {2019},
author = {Honda, A and Tachibana, R and Hamada, K and Morita, K and Mizuno, N and Morita, K and Asano, M},
title = {Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11571},
pmid = {31399630},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Electroporation/methods/veterinary ; Female ; Fertilization in Vitro/methods/veterinary ; Gene Editing/methods/veterinary ; *Gene Knock-In Techniques/methods/veterinary ; *Gene Knockout Techniques/methods/veterinary ; Male ; Rats/*embryology/genetics/physiology ; Rats, Inbred F344/embryology/genetics/physiology ; Rats, Long-Evans/embryology/genetics/physiology ; Rats, Sprague-Dawley/embryology/genetics/physiology ; Rats, Wistar/embryology/genetics/physiology ; Superovulation ; },
abstract = {Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3-47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.},
}
@article {pmid31399578,
year = {2019},
author = {Cheng, TL and Li, S and Yuan, B and Wang, X and Zhou, W and Qiu, Z},
title = {Expanding C-T base editing toolkit with diversified cytidine deaminases.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3612},
pmid = {31399578},
issn = {2041-1723},
mesh = {Base Pairing ; Base Sequence ; CRISPR-Cas Systems ; Cytidine Deaminase/classification/*genetics/metabolism ; *Cytosine ; Gene Editing/*methods ; HCT116 Cells ; HEK293 Cells ; Humans ; *Thymine ; },
abstract = {Base editing tools for cytosine to thymine (C-T) conversion enable genome manipulation at single base-pair resolution with high efficiency. Available base editors (BEs) for C-T conversion (CBEs) have restricted editing scopes and nonnegligible off-target effects, which limit their applications. Here, by screening diversified lamprey cytidine deaminases, we establish various CBEs with expanded and diversified editing scopes, which could be further refined by various fusing strategies, fusing at either N-terminus or C-terminus of nCas9. Furthermore, off-target analysis reveals that several CBEs display improved fidelity. Our study expands the toolkits for C-T conversion, serves as guidance for appropriate choice and offers a framework for benchmarking future improvement of base editing tools.},
}
@article {pmid31399410,
year = {2019},
author = {Maikova, A and Kreis, V and Boutserin, A and Severinov, K and Soutourina, O},
title = {Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {20},
pages = {},
pmid = {31399410},
issn = {1098-5336},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Clostridioides difficile/genetics ; *Gene Editing ; Genetic Engineering/*methods ; Sequence Deletion ; },
abstract = {The human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defense against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system toward autoimmunity allows efficient genome editing in C. difficile We provide a detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference strain 630Δerm and in epidemic C. difficile strain R20291. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.IMPORTANCEClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhea associated with health care in adults in industrialized countries. The incidence of these infections continues to increase, and this trend is accentuated by the general aging of the population. Many questions about the mechanisms contributing to C. difficile's success inside the host remain unanswered. The set of genetic tools available for this pathogen is limited, and new developments are badly needed. C. difficile has developed efficient defense systems that are directed against foreign DNA and that could contribute to its survival in phage-rich gut communities. We show how one such defense system, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential for the use of the CRISPR-Cas system for the development of new therapeutic strategies against C. difficile infections.},
}
@article {pmid31399023,
year = {2019},
author = {Kruse, T and Ratnadevi, CM and Erikstad, HA and Birkeland, NK},
title = {Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph "Candidatus Methylacidiphilum kamchatkense" strain Kam1 and comparison with its closest relatives.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {642},
pmid = {31399023},
issn = {1471-2164},
mesh = {Amino Acid Sequence ; Bacterial Proteins/chemistry/genetics ; Biomass ; Genome, Bacterial/genetics ; *Genomics ; Phylogeny ; Species Specificity ; Verrucomicrobia/*genetics/metabolism ; },
abstract = {BACKGROUND: The candidate genus "Methylacidiphilum" comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the "Ca. Methylacidiphilum" and the sister genus "Ca. Methylacidimicrobium" represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.<br><br>RESULTS: Here we present the closed genome of "Ca. Methylacidiphilum kamchatkense" strain Kam1 and a comparison with the genomes of its two closest relatives "Ca. Methylacidiphilum fumariolicum" strain SolV and "Ca. Methylacidiphilum infernorum" strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of "Ca. Methylacidiphilum" is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between "Ca. M. kamchatkense" strain Kam1 and strains SolV and V4 is &#x2264;95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.<br><br>CONCLUSIONS: Comparing three closed genomes of "Ca. Methylacidiphilum" spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.},
}
@article {pmid31398008,
year = {2019},
author = {Martella, A and Firth, M and Taylor, BJM and Göppert, A and Cuomo, EM and Roth, RG and Dickson, AJ and Fisher, DI},
title = {Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables Development of a Multiplexed, Orthogonal Gene Activation and Repression System.},
journal = {ACS synthetic biology},
volume = {8},
number = {9},
pages = {1998-2006},
doi = {10.1021/acssynbio.8b00527},
pmid = {31398008},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Cellular Reprogramming ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Regulatory Networks/genetics ; HCT116 Cells ; Humans ; Promoter Regions, Genetic ; RNA, Guide/genetics/metabolism ; Signal Transduction/genetics ; Tissue Engineering ; },
abstract = {The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry for diverse applications, including stem cell reprogramming and differentiation, tissue engineering, and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes while downregulating another subset, thus gaining control of gene regulatory networks, signaling pathways, and cellular processes whose activity depends on the expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.},
}
@article {pmid31397669,
year = {2019},
author = {Knott, GJ and Cress, BF and Liu, JJ and Thornton, BW and Lew, RJ and Al-Shayeb, B and Rosenberg, DJ and Hammel, M and Adler, BA and Lobba, MJ and Xu, M and Arkin, AP and Fellmann, C and Doudna, JA},
title = {Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31397669},
issn = {2050-084X},
support = {DGE 1752814//National Science Foundation/International ; RM1HG009490/NH/NIH HHS/United States ; R00GM118909/GM/NIGMS NIH HHS/United States ; Graduate Research Fellowship,DGE 1752814//National Science Foundation/International ; R00 GM118909/GM/NIGMS NIH HHS/United States ; HR0011-17-2-0043//Defense Advanced Research Projects Agency/International ; P50GM082250/NH/NIH HHS/United States ; MCB-1244557//National Science Foundation/International ; },
mesh = {Acidaminococcus/*enzymology/virology ; Bacteriophages/*growth &amp; development ; CRISPR-Cas Systems/*drug effects ; Clostridiales/*enzymology/virology ; Enzyme Inhibitors/*metabolism ; Evolution, Molecular ; *Host-Parasite Interactions ; Viral Proteins/*metabolism ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.},
}
@article {pmid31395785,
year = {2019},
author = {Boettcher, S and Miller, PG and Sharma, R and McConkey, M and Leventhal, M and Krivtsov, AV and Giacomelli, AO and Wong, W and Kim, J and Chao, S and Kurppa, KJ and Yang, X and Milenkowic, K and Piccioni, F and Root, DE and Rücker, FG and Flamand, Y and Neuberg, D and Lindsley, RC and Jänne, PA and Hahn, WC and Jacks, T and Döhner, H and Armstrong, SA and Ebert, BL},
title = {A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6453},
pages = {599-604},
pmid = {31395785},
issn = {1095-9203},
support = {U01 CA199253/CA/NCI NIH HHS/United States ; P50 CA206963/CA/NCI NIH HHS/United States ; R01 HL082945/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32 HG002295/HG/NHGRI NIH HHS/United States ; R01 GM038627/GM/NIGMS NIH HHS/United States ; P01 CA066996/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; T32 HL007623/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gain of Function Mutation ; Genes, Dominant ; Humans ; K562 Cells ; Leukemia, Myeloid, Acute/*genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; *Mutation, Missense ; *Selection, Genetic ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.},
}
@article {pmid31395745,
year = {2019},
author = {Norman, TM and Horlbeck, MA and Replogle, JM and Ge, AY and Xu, A and Jost, M and Gilbert, LA and Weissman, JS},
title = {Exploring genetic interaction manifolds constructed from rich single-cell phenotypes.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6455},
pages = {786-793},
pmid = {31395745},
issn = {1095-9203},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; U01 CA217882/CA/NCI NIH HHS/United States ; R00 CA204602/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; T32 EB009383/EB/NIBIB NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; F32 GM116331/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; K99 CA204602/CA/NCI NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; },
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems ; Calcium-Binding Proteins/genetics ; Cell Cycle Checkpoints/genetics ; Cell Line, Tumor ; *Epistasis, Genetic ; Erythroid Cells/cytology ; Erythropoiesis/genetics ; Female ; Gene Expression Profiling ; Granulocytes/cytology ; Humans ; Microfilament Proteins/genetics ; Proto-Oncogene Proteins c-cbl/genetics ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/*methods ; },
abstract = {How cellular and organismal complexity emerges from combinatorial expression of genes is a central question in biology. High-content phenotyping approaches such as Perturb-seq (single-cell RNA-sequencing pooled CRISPR screens) present an opportunity for exploring such genetic interactions (GIs) at scale. Here, we present an analytical framework for interpreting high-dimensional landscapes of cell states (manifolds) constructed from transcriptional phenotypes. We applied this approach to Perturb-seq profiling of strong GIs mined from a growth-based, gain-of-function GI map. Exploration of this manifold enabled ordering of regulatory pathways, principled classification of GIs (e.g., identifying suppressors), and mechanistic elucidation of synergistic interactions, including an unexpected synergy between CBL and CNN1 driving erythroid differentiation. Finally, we applied recommender system machine learning to predict interactions, facilitating exploration of vastly larger GI manifolds.},
}
@article {pmid31395429,
year = {2019},
author = {Garcia-Marques, J and Yang, CP and Espinosa-Medina, I and Mok, K and Koyama, M and Lee, T},
title = {Unlimited Genetic Switches for Cell-Type-Specific Manipulation.},
journal = {Neuron},
volume = {104},
number = {2},
pages = {227-238.e7},
doi = {10.1016/j.neuron.2019.07.005},
pmid = {31395429},
issn = {1097-4199},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Repair ; Drosophila ; Gene Targeting/*methods ; Genetic Techniques ; RNA, Guide ; Recombinases/genetics ; Zebrafish ; },
abstract = {Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.},
}
@article {pmid31394891,
year = {2019},
author = {Butt, H and Piatek, A and Li, L and S N Reddy, A and M Mahfouz, M},
title = {Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice.},
journal = {Genes},
volume = {10},
number = {8},
pages = {},
pmid = {31394891},
issn = {2073-4425},
mesh = {Alternative Splicing ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Mutagenesis ; Oryza/*genetics ; Plant Proteins/*genetics/metabolism ; Serine-Arginine Splicing Factors/*genetics/metabolism ; },
abstract = {Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.},
}
@article {pmid31394408,
year = {2019},
author = {Readler, JM and AlKahlout, AS and Sharma, P and Excoffon, KJDA},
title = {Isoform specific editing of the coxsackievirus and adenovirus receptor.},
journal = {Virology},
volume = {536},
number = {},
pages = {20-26},
pmid = {31394408},
issn = {1096-0341},
support = {R01 AI127816/AI/NIAID NIH HHS/United States ; R15 AI090625/AI/NIAID NIH HHS/United States ; },
mesh = {Adenoviruses, Human/*genetics/metabolism ; Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coxsackie and Adenovirus Receptor-Like Membrane Protein/*genetics/metabolism ; DNA, Complementary/genetics/metabolism ; Dogs ; Doxycycline/pharmacology ; Exons ; Gene Editing/*methods ; *Gene Expression Regulation, Viral ; Humans ; Madin Darby Canine Kidney Cells ; Promoter Regions, Genetic/drug effects ; Protein Isoforms/genetics/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAR[Ex7]) exhibits basolateral localization, the eight exon-encoded isoform (CAR[Ex8]) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAR[Ex8] cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAR[Ex8] restored sensitivity to adenovirus. These data reinforce the importance of CAR[Ex8] in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.},
}
@article {pmid31393941,
year = {2019},
author = {Li, W and Cho, MY and Lee, S and Jang, M and Park, J and Park, R},
title = {CRISPR-Cas9 mediated CD133 knockout inhibits colon cancer invasion through reduced epithelial-mesenchymal transition.},
journal = {PloS one},
volume = {14},
number = {8},
pages = {e0220860},
pmid = {31393941},
issn = {1932-6203},
mesh = {AC133 Antigen/deficiency/genetics/*pharmacology ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Colonic Neoplasms/genetics/*pathology/therapy ; Cyclic S-Oxides/pharmacology ; Epithelial-Mesenchymal Transition/*genetics ; Gene Knockout Techniques/methods ; Humans ; Neoplasm Invasiveness/*prevention &amp; control ; Neoplastic Stem Cells ; Tumor Cells, Cultured ; Vimentin/metabolism ; },
abstract = {We previously reported that CD133, as a putative cancer stem cell marker, plays an important role in cell proliferation and invasion in colon cancer. To understand the role of CD133 expression in colon cancer, we evaluated the inhibitory effect of CD133 in colon cancer cells. In this study, we generated CD133knockout colon cancer cells (LoVo) using the CRISPR-Cas9 gene editing system. CD133+ colon cancer cells (LoVo) were infected with the lentiviral vector carrying CD133 gRNA and purified cell by culturing single cell colonies. CD133knockout cells was validated by western blot and flow cytometry analysis. In functional study, we observed a significant reduction in cell proliferation and colony formation in CRISPR-Cas9 mediated CD133 knockout cells in compare with control (P < 0.001). We also found the anticancer effect of stattic was dependent on CD133 expression in colon cancer cells. Although CD133knockout cells could not completely block the tumorigenic property, they showed remarkable inhibitory effects on the ability of cell migration and invasion (P < 0.001). In addition, we examined the epithelial mesenchymal transition (EMT)-related protein expression by western blot. The result clearly showed a loss of vimentin expression in CD133knockout cells. Therefore, CRISPR-Cas9 mediated CD133knockout can be an effective treatment modality for CD133+ colon cancer through reducing the characteristics of cancer stem cells.},
}
@article {pmid31393061,
year = {2019},
author = {Ruicci, KM and Plantinga, P and Pinto, N and Khan, MI and Stecho, W and Dhaliwal, SS and Yoo, J and Fung, K and MacNeil, D and Mymryk, JS and Barrett, JW and Howlett, CJ and Nichols, AC},
title = {Disruption of the RICTOR/mTORC2 complex enhances the response of head and neck squamous cell carcinoma cells to PI3K inhibition.},
journal = {Molecular oncology},
volume = {13},
number = {10},
pages = {2160-2177},
pmid = {31393061},
issn = {1878-0261},
support = {MOP 340674//Canadian Institutes for Health Research (CIHR)/International ; },
mesh = {Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cisplatin/pharmacology ; Erlotinib Hydrochloride/pharmacology ; Head and Neck Neoplasms/*drug therapy/genetics/metabolism ; Humans ; Mechanistic Target of Rapamycin Complex 2/genetics/*metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphoinositide-3 Kinase Inhibitors/*pharmacology ; Rapamycin-Insensitive Companion of mTOR Protein/genetics/*metabolism ; Squamous Cell Carcinoma of Head and Neck/*drug therapy/genetics/metabolism ; },
abstract = {Phosphoinositide 3-kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K-PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K-activated Akt signaling is well established in HNSCC, the significance of mTORC2-driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2-specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.},
}
@article {pmid31392987,
year = {2019},
author = {Grüschow, S and Athukoralage, JS and Graham, S and Hoogeboom, T and White, MF},
title = {Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence.},
journal = {Nucleic acids research},
volume = {47},
number = {17},
pages = {9259-9270},
pmid = {31392987},
issn = {1362-4962},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity/immunology ; Adenine Nucleotides/biosynthesis/*genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/immunology ; Interspersed Repetitive Sequences/genetics/immunology ; Mycobacterium tuberculosis/*genetics/immunology ; Oligoribonucleotides/biosynthesis/*genetics ; Prokaryotic Cells/immunology ; RNA Cleavage/genetics/immunology ; Signal Transduction/genetics/immunology ; },
abstract = {The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.},
}
@article {pmid31392986,
year = {2019},
author = {Riesenberg, S and Chintalapati, M and Macak, D and Kanis, P and Maricic, T and Pääbo, S},
title = {Simultaneous precise editing of multiple genes in human cells.},
journal = {Nucleic acids research},
volume = {47},
number = {19},
pages = {e116},
pmid = {31392986},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; Chromosomes ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA-Activated Protein Kinase/*genetics ; Gene Editing/*methods ; HEK293 Cells ; Humans ; INDEL Mutation/genetics ; Oligonucleotides/genetics ; Recombinational DNA Repair/*genetics ; Sequence Deletion/genetics ; },
abstract = {When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.},
}
@article {pmid31392984,
year = {2019},
author = {Kim, JG and Garrett, S and Wei, Y and Graveley, BR and Terns, MP},
title = {CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {47},
number = {16},
pages = {8632-8648},
pmid = {31392984},
issn = {1362-4962},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Endonucleases/*genetics/immunology/metabolism ; Esterification ; *Gene Editing ; Genetic Loci ; *Genome, Bacterial ; Isoenzymes/genetics/immunology/metabolism ; Mutagenesis, Insertional ; Plasmids/chemistry/metabolism ; Streptococcus thermophilus/*genetics/immunology/metabolism/virology ; Viruses/genetics/metabolism ; },
abstract = {CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.},
}
@article {pmid31392916,
year = {2019},
author = {Guo, G and Wang, K and Hu, SS and Tian, T and Liu, P and Mori, T and Chen, P and Johnson, CH and Qin, X},
title = {Autokinase Activity of Casein Kinase 1 δ/ε Governs the Period of Mammalian Circadian Rhythms.},
journal = {Journal of biological rhythms},
volume = {34},
number = {5},
pages = {482-496},
pmid = {31392916},
issn = {1552-4531},
support = {R01 GM067152/GM/NIGMS NIH HHS/United States ; R01 GM107434/GM/NIGMS NIH HHS/United States ; R37 GM067152/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Casein Kinase II/genetics ; Casein Kinase Idelta/*genetics/*metabolism ; *Circadian Clocks ; *Circadian Rhythm ; Gene Knockout Techniques ; Mice ; NIH 3T3 Cells ; Phosphorylation ; },
abstract = {Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε.},
}
@article {pmid31392820,
year = {2020},
author = {Moradpour, M and Abdulah, SNA},
title = {CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {32-44},
pmid = {31392820},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Endonucleases ; *Gene Editing ; Transcriptional Activation ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) and Cas9-associated protein systems provide a powerful genetic manipulation tool that can drive plant research forward. Nuclease-dead Cas9 (dCas9) is an enzymatically inactive mutant of Cas9 in which its endonuclease activity is non-functional. The applications of CRISPR/dCas9 have expanded and diversified in recent years. Originally, dCas9 was used as a CRISPR/Cas9 re-engineering tool that enables targeted expression of any gene or multiple genes through recruitment of transcriptional effector domains without introducing irreversible DNA-damaging mutations. Subsequent applications have made use of its ability to recruit modifying enzymes and reporter proteins to DNA target sites. In this paper, the most recent progress in the applications of CRISPR/dCas9 in plants, which include gene activation and repression, epigenome editing, modulation of chromatin topology, live-cell chromatin imaging and DNA-free genetic modification, will be reviewed. The associated strategies for exploiting the CRISPR/dCas9 system for crop improvement with a dimer of the future of the CRISPR/dCas9 system in the functional genomics of crops and the development of traits will be briefly discussed.},
}
@article {pmid31392536,
year = {2019},
author = {Permyakova, NV and Sidorchuk, YV and Marenkova, TV and Khozeeva, SA and Kuznetsov, VV and Zagorskaya, AA and Rozov, SM and Deineko, EV},
title = {CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells.},
journal = {Molecular biology reports},
volume = {46},
number = {6},
pages = {5735-5743},
pmid = {31392536},
issn = {1573-4978},
mesh = {*Arabidopsis/cytology/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; Cells, Cultured ; DNA, Plant/genetics ; *Gene Silencing ; Genes, Reporter/genetics ; Green Fluorescent Proteins/*genetics/metabolism ; Mutagenesis, Site-Directed/methods ; Mutation/genetics ; Sequence Analysis, DNA ; },
abstract = {Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell suspension cultures. Here, we describe a successful knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture. We transformed seven transgenic suspension cell lines carrying one to three gfp gene copies with a binary vector containing genes coding for Cas9 and guide RNAs targeting the gfp gene. We detected the site-specific mutations by restriction analysis of a gfp amplicon. DNA sequencing of the PCR products confirmed high diversity of insertion-deletion mutations in the cell lines after the editing. We also analyzed gfp mRNA expression by real-time PCR and observed a decrease in gfp transcription after the target site modification. We can conclude that the CRISPR/Cas9 system can be successfully used for introducing site-specific mutations into the genome of cultured suspension cells of Arabidopsis.},
}
@article {pmid31392501,
year = {2019},
author = {Ai, L and Guo, W and Chen, W and Teng, Y and Bai, L},
title = {The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction.},
journal = {Current microbiology},
volume = {76},
number = {11},
pages = {1313-1319},
pmid = {31392501},
issn = {1432-0991},
mesh = {Artemisinins/*metabolism ; CRISPR-Cas Systems ; Fermentation ; Galactose/*metabolism ; Gene Deletion ; Metabolic Engineering ; Repressor Proteins/*genetics/metabolism ; Saccharomyces cerevisiae/*genetics/growth &amp; development/*metabolism ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; Uracil/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has emerged as the dominating tool for genome engineering, while also changes the speed and efficiency of metabolic engineering in conventional and non-conventional yeasts. Among these CRISPR-Cas systems, CRISPR-Cas9 technology has usually been applied for removing unfavorable target genes. Here, we used CRISPR-Cas9 technology to delete the gal80 gene in uracil-deficient strain and had successfully remolded the engineered Saccharomyces cerevisiae that can produce artemisinic acid without galactose induction. An L9(3[4]) orthogonal test was adopted to investigate the effects of different factors on artemisinic acid production. Fermentation medium III with sucrose as carbon sources, 1% inoculum level, and 84-h culture time were identified as the optimal fermentation conditions. Under this condition, the maximum artemisinic acid production by engineered S. cerevisiae 1211-2 was 740 mg/L in shake-flask cultivation level. This study provided an effective approach to reform metabolic pathway of artemisinic acid-producing strain. The engineered S. cerevisiae 1211-2 may be applied to artemisinic acid production by industrial fermentation in the future.},
}
@article {pmid31392299,
year = {2019},
author = {Zhang, F and Song, G and Tian, Y},
title = {Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems.},
journal = {Animal models and experimental medicine},
volume = {2},
number = {2},
pages = {69-75},
pmid = {31392299},
issn = {2576-2095},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene-editing tools. As the natural inhibitors of CRISPR-Cas systems, anti-CRISPRs (Acrs) can be used as the "off-switch" for CRISPR-Cas systems to limit the off-target effects caused by Cas9. Since the discovery of CRISPR-Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.},
}
@article {pmid31391551,
year = {2019},
author = {Zhong, Z and Sepramaniam, S and Chew, XH and Wood, K and Lee, MA and Madan, B and Virshup, DM},
title = {PORCN inhibition synergizes with PI3K/mTOR inhibition in Wnt-addicted cancers.},
journal = {Oncogene},
volume = {38},
number = {40},
pages = {6662-6677},
pmid = {31391551},
issn = {1476-5594},
mesh = {Acyltransferases/*genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/*metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation ; Glucose/metabolism ; Heterografts ; Humans ; Loss of Function Mutation ; Membrane Proteins/*genetics/metabolism ; Mice ; Pancreatic Neoplasms/*metabolism/pathology ; Phosphatidylinositol 3-Kinases/*genetics/metabolism ; TOR Serine-Threonine Kinases/*genetics/metabolism ; Wnt Proteins/*metabolism ; },
abstract = {Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is aggressive and lethal. Although there is an urgent need for effective therapeutics in treating pancreatic cancer, none of the targeted therapies tested in clinical trials to date significantly improve its outcome. PORCN inhibitors show efficacy in preclinical models of Wnt-addicted cancers, including RNF43-mutant pancreatic cancers and have advanced to clinical trials. In this study, we aimed to develop drug combination strategies to further enhance the therapeutic efficacy of the PORCN inhibitor ETC-159. To identify additional druggable vulnerabilities in Wnt-driven pancreatic cancers, we performed an in vivo CRISPR loss-of-function screen. CTNNB1, KRAS, and MYC were reidentified as key oncogenic drivers. Notably, glucose metabolism pathway genes were important in vivo but less so in vitro. Knockout of multiple genes regulating PI3K/mTOR signaling impacted the growth of Wnt-driven pancreatic cancer cells in vivo. Importantly, multiple PI3K/mTOR pathway inhibitors in combination with ETC-159 synergistically suppressed the growth of multiple Wnt-addicted cancer cell lines in soft agar. Furthermore, the combination of the PORCN inhibitor ETC-159 and the pan-PI3K inhibitor GDC-0941 potently suppressed the in vivo growth of RNF43-mutant pancreatic cancer xenografts. This was largely due to enhanced suppressive effects on both cell proliferation and glucose metabolism. These findings demonstrate that dual PORCN and PI3K/mTOR inhibition is a potential strategy for treating Wnt-driven pancreatic cancers.},
}
@article {pmid31391538,
year = {2019},
author = {Bari, VK and Nassar, JA and Kheredin, SM and Gal-On, A and Ron, M and Britt, A and Steele, D and Yoder, J and Aly, R},
title = {CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11438},
pmid = {31391538},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Carotenoids/metabolism ; Dioxygenases/*genetics/metabolism ; Disease Resistance/*genetics ; Exons/genetics ; Gene Expression Regulation, Plant ; Heterocyclic Compounds, 3-Ring/metabolism ; Lactones/metabolism ; Lycopersicon esculentum/enzymology/genetics/*parasitology ; Mutagenesis ; *Orobanche ; Plant Breeding ; Plant Proteins/*genetics/metabolism ; Plant Roots/metabolism ; *Plant Weeds ; Plants, Genetically Modified ; },
abstract = {Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several [CCD8]Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the [CCD8]Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient [CCD8]Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the [CCD8]Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks.},
}
@article {pmid31391532,
year = {2019},
author = {Martens, KJA and van Beljouw, SPB and van der Els, S and Vink, JNA and Baas, S and Vogelaar, GA and Brouns, SJJ and van Baarlen, P and Kleerebezem, M and Hohlbein, J},
title = {Visualisation of dCas9 target search in vivo using an open-microscopy framework.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3552},
pmid = {31391532},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; Gene Dosage ; *Gene Editing ; Lactococcus lactis/genetics/metabolism ; Luminescent Proteins/genetics/metabolism ; Microscopy/instrumentation/*methods ; Models, Genetic ; Monte Carlo Method ; Nucleotide Motifs/genetics ; Plasmids/*genetics ; Recombinant Fusion Proteins/genetics/metabolism ; Single Molecule Imaging/instrumentation/*methods ; Time Factors ; },
abstract = {CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.},
}
@article {pmid31391493,
year = {2019},
author = {Edwards, HS and Krishnakumar, R and Sinha, A and Bird, SW and Patel, KD and Bartsch, MS},
title = {Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11475},
pmid = {31391493},
issn = {2045-2322},
mesh = {Bacteriophage lambda/genetics ; CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; DNA, Viral/genetics ; Escherichia coli/genetics ; HeLa Cells ; High-Throughput Nucleotide Sequencing/instrumentation/*methods ; Humans ; *Models, Genetic ; Nanopores ; Proof of Concept Study ; Sequence Analysis, DNA/instrumentation/*methods ; },
abstract = {The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-by-molecule real-time selective sequencing or "Read Until". As DNA transits a MinION nanopore, real-time pore current data can be accessed and analyzed to provide active feedback to that pore. Fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the strand, providing a novel means of background depletion and/or target enrichment. In contrast to the previously published pattern-matching Read Until approach, our RUBRIC method is the first example of real-time selective sequencing where on-line basecalling enables alignment against conventional nucleic acid references to provide the basis for sequence/reject decisions. We evaluate RUBRIC performance across a range of optimizable parameters, apply it to mixed human/bacteria and CRISPR/Cas9-cut samples, and present a generalized model for estimating real-time selection performance as a function of sample composition and computing configuration.},
}
@article {pmid31391371,
year = {2019},
author = {Yamauchi, T},
title = {[Exploration of novel therapeutic targets in acute myeloid leukemia via genome-wide CRISPR screening].},
journal = {[Rinsho ketsueki] The Japanese journal of clinical hematology},
volume = {60},
number = {7},
pages = {810-817},
doi = {10.11406/rinketsu.60.810},
pmid = {31391371},
issn = {0485-1439},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Leukemia, Myeloid, Acute/*genetics ; Mice ; *RNA Splicing ; *RNA Stability ; RNA, Small Interfering ; },
abstract = {Acute myeloid leukemia (AML) remains a devasting disease. Progress has been made to define molecular mechanisms underlying disease pathogenesis due, in part, to the near-complete understanding of AML genome. Nonetheless, functional studies are necessary to assess the significance of AML-associated mutations and devise urgently needed therapies. Genome-wide knockout screening, employing CRISPR-Cas9 genome editing, is a powerful tool in functional genomics. In this study, genome-wide CRISPR screening was performed using mouse leukemia cell lines developed in our Center, followed by in vivo screening. Among 20,611 genes, 130 AML essential genes were identified, including clinically actionable candidates. It was shown that mRNA decapping enzyme scavenger (DCPS), an enzyme implicated in mRNA decay pathway, is essential for AML survival. ShRNA-mediated gene knockdown and DCPS inhibitor (RG3039) were employed to validate findings. RG3039 induced cell-cycle arrest and apoptosis in vitro. Furthermore, mass spectrometry analysis revealed an association between DCPS and RNA metabolic pathways, and RNA-Seq showed that RG3039 treatment induced aberrant mRNA splicing in AML cells. Importantly, RG3039 exhibited anti-leukemia effects in PDX models. These findings identify DCPS as a novel therapeutic target for AML, shedding new light on the nuclear RNA metabolic pathway in leukemogenesis.},
}
@article {pmid31391281,
year = {2019},
author = {James, CD and Prabhakar, AT and Otoa, R and Evans, MR and Wang, X and Bristol, ML and Zhang, K and Li, R and Morgan, IM},
title = {SAMHD1 Regulates Human Papillomavirus 16-Induced Cell Proliferation and Viral Replication during Differentiation of Keratinocytes.},
journal = {mSphere},
volume = {4},
number = {4},
pages = {},
pmid = {31391281},
issn = {2379-5042},
support = {K99 AI104828/AI/NIAID NIH HHS/United States ; P30 CA016059/CA/NCI NIH HHS/United States ; R00 AI104828/AI/NIAID NIH HHS/United States ; R01 AI141410/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cell Differentiation ; *Cell Proliferation ; Cells, Cultured ; Gene Deletion ; Gene Expression Regulation ; Host-Pathogen Interactions ; Human papillomavirus 16/*physiology ; Humans ; Keratinocytes/*physiology/virology ; Palatine Tonsil/cytology/virology ; SAM Domain and HD Domain-Containing Protein 1/*genetics ; *Virus Replication ; },
abstract = {Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes.IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.},
}
@article {pmid31391273,
year = {2019},
author = {Madigan, VJ and Tyson, TO and Yuziuk, JA and Pillai, M and Moller-Tank, S and Asokan, A},
title = {A CRISPR Screen Identifies the Cell Polarity Determinant Crumbs 3 as an Adeno-associated Virus Restriction Factor in Hepatocytes.},
journal = {Journal of virology},
volume = {93},
number = {21},
pages = {},
pmid = {31391273},
issn = {1098-5514},
support = {R01 GM127708/GM/NIGMS NIH HHS/United States ; R01 HL089221/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Capsid/metabolism ; Cell Line ; Cell Membrane/metabolism ; Cell Polarity ; Claudins/genetics/metabolism ; Dependovirus/genetics/*physiology ; Gene Expression ; Gene Knockout Techniques ; Hepatocytes/*metabolism/physiology/virology ; Humans ; Membrane Glycoproteins/genetics/*metabolism ; Parvoviridae Infections/metabolism/*virology ; Polysaccharides/metabolism ; Serogroup ; Tight Junctions ; Transduction, Genetic ; Virus Attachment ; },
abstract = {Adeno-associated viruses (AAV) are helper-dependent parvoviruses that have been developed into promising gene therapy vectors. Many studies, including a recent unbiased genomic screen, have identified host factors essential for AAV cell entry, but no genome-wide screens that address inhibitory host factors have been reported. Here, we utilize a novel CRISPR screen to identify AAV restriction factors in a human hepatocyte cell line. The major hit from our gain-of-function screen is the apical polarity determinant Crumbs 3 (Crb3). Knockout (KO) of Crb3 enhances AAV transduction, while overexpression exerts the opposite effect. Further, Crb3 appears to restrict AAV transduction in a serotype- and cell type-specific manner. Particularly, for AAV serotype 9 and a rationally engineered AAV variant, we demonstrate that increased availability of galactosylated glycans on the surfaces of Crb3 KO cells, but not the universal AAV receptor, leads to increased capsid attachment and enhanced transduction. We postulate that Crb3 could serve as a key molecular determinant that restricts the availability of AAV glycan attachment factors on the cell surface by maintaining apical-basal polarity and tight junction integrity.IMPORTANCE Adeno-associated viruses (AAVs) have recently emerged at the forefront as gene therapy vectors; however, our understanding of host factors that influence AAV transduction in different cell types is still evolving. In the present study, we perform a genome-scale CRISPR knockout screen to identify cellular host factors that restrict AAV infection in hepatocyte cultures. We discover that Crumbs 3, which determines cellular polarity, also influences the distribution of certain carbohydrate attachment factors on the cell surface. This in turn affects the ability of virions to bind and enter the cells. This study underscores the importance of cell polarity in AAV transduction and provides a potential molecular basis for the differential infectious mechanism(s) in cell culture versus organ systems.},
}
@article {pmid31391075,
year = {2019},
author = {Pacini, V and Petit, F and Querat, B and Laverriere, JN and Cohen-Tannoudji, J and L'hôte, D},
title = {Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.},
journal = {Epigenetics &amp; chromatin},
volume = {12},
number = {1},
pages = {48},
pmid = {31391075},
issn = {1756-8935},
support = {Allocation de recherche 2018//Soci&#xe9;t&#xe9; Fran&#xe7;aise d'Endocrinologie/International ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Chromatin/metabolism ; Chromatin Assembly and Disassembly ; DNA-Directed RNA Polymerases/metabolism ; Enhancer Elements, Genetic ; Estrogen Receptor alpha/*metabolism ; Gonadotrophs/cytology/metabolism ; Histones/metabolism ; Humans ; Mice ; Pituitary Gland/growth &amp; development/metabolism ; Promoter Regions, Genetic ; Sequence Alignment ; Steroidogenic Factor 1/genetics/*metabolism ; Transcription, Genetic ; },
abstract = {BACKGROUND: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood.<br><br>RESULTS: Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ER&#x3b1;. Lastly, we provide evidence that binding of ER&#x3b1; is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription.<br><br>CONCLUSION: This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ER&#x3b1; in this differentiation process.},
}
@article {pmid31390092,
year = {2020},
author = {Zhao, LN and Mondal, D and Warshel, A},
title = {Exploring alternative catalytic mechanisms of the Cas9 HNH domain.},
journal = {Proteins},
volume = {88},
number = {2},
pages = {260-264},
pmid = {31390092},
issn = {1097-0134},
support = {R01 AI055926/AI/NIAID NIH HHS/United States ; R35 GM122472/GM/NIGMS NIH HHS/United States ; },
mesh = {Biocatalysis ; CRISPR-Associated Protein 9/*chemistry/metabolism ; *CRISPR-Cas Systems ; *Catalytic Domain ; Cryoelectron Microscopy ; DNA/*chemistry/metabolism ; *DNA Cleavage ; Gene Editing ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; *Protein Domains ; RNA, Guide/chemistry/metabolism ; },
abstract = {Understanding the reaction mechanism of CRISPR-associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo- and substrate-bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg[2+] . Trying this mechanism with the cryo-EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg[2+] and assume that the cryo-EM structure by itself is a suitable representation of a catalytic-ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell.},
}
@article {pmid31389773,
year = {2019},
author = {Young, CS and Pyle, AD and Spencer, MJ},
title = {CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.},
journal = {Physiology (Bethesda, Md.)},
volume = {34},
number = {5},
pages = {341-353},
pmid = {31389773},
issn = {1548-9221},
support = {R01 AR064327/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Humans ; Neuromuscular Diseases/*genetics ; },
abstract = {This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.},
}
@article {pmid31389128,
year = {2019},
author = {Kim, Y and Lee, SJ and Yoon, HJ and Kim, NK and Lee, BJ and Suh, JY},
title = {Anti-CRISPR AcrIIC3 discriminates between Cas9 orthologs via targeting the variable surface of the HNH nuclease domain.},
journal = {The FEBS journal},
volume = {286},
number = {23},
pages = {4661-4674},
doi = {10.1111/febs.15037},
pmid = {31389128},
issn = {1742-4658},
support = {500-20170161//Creative-Pioneering Researchers Program through Seoul National University/International ; NRF-2016R1C1B2014609//National Research Foundation/International ; PJ013181//Cooperative Research Program for Agriculture Science &amp; Technology Development, Rural Development Administration/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; Calorimetry ; Campylobacter jejuni/genetics ; Chromatography, Gel ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Geobacillus stearothermophilus/genetics ; Magnetic Resonance Spectroscopy ; Mutation/genetics ; Neisseria meningitidis/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems constitute the adaptive immunity of bacteria and archaea, degrading nucleic acids of invading phages and plasmids. In response, phages employ anti-CRISPR (Acr) proteins as a counterdefense mechanism to neutralize the host immunity. AcrIIC3 directly inhibits target DNA cleavage of type II-C Cas9 of Neisseria meningitidis. Here, we show that AcrIIC3 interacts with the HNH nuclease domain of N. meningitidis Cas9 to inhibit its nuclease activity in an allosteric manner. The crystal structure of the AcrIIC3-HNH complex reveals that AcrIIC3 binds opposite the active site on the HNH nuclease domain. AcrIIC3 employs a unique interface for HNH, allowing it to discriminate between Cas9 orthologs, which contrasts with the broad spectrum of Cas9 inhibition by AcrIIC1. Interface residues of HNH provide key electrostatic and hydrophobic interactions that determine the host specificity of AcrIIC3. Mutations that replace HNH interfaces of N. meningitidis Cas9 with those of Geobacillus stearothermophilus Cas9 or Campylobacter jejuni Cas9 significantly attenuate AcrIIC3 binding, illustrating that the divergent interaction surface confers the host specificity of AcrIIC3. Our study demonstrates that the variable sequences of binding interface can define the target specificity of Acr proteins, suggesting potential applications in Cas9 control for gene editing.},
}
@article {pmid31386652,
year = {2019},
author = {Bittermann, E and Abdelhamed, Z and Liegel, RP and Menke, C and Timms, A and Beier, DR and Stottmann, RW},
title = {Differential requirements of tubulin genes in mammalian forebrain development.},
journal = {PLoS genetics},
volume = {15},
number = {8},
pages = {e1008243},
pmid = {31386652},
issn = {1553-7404},
support = {R01 HD036404/HD/NICHD NIH HHS/United States ; R01 NS085023/NS/NINDS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Mammalian ; Ethylnitrosourea/toxicity ; Female ; Gene Deletion ; Gene Editing ; Gene Expression Regulation, Developmental ; Humans ; Male ; Malformations of Cortical Development/*genetics/mortality/pathology ; Mice ; Mice, Transgenic ; Microtubules/genetics ; Models, Animal ; Mutagenesis/drug effects ; Neurogenesis/*genetics ; Sensorimotor Cortex/abnormalities/*embryology ; Species Specificity ; Tubulin/*genetics/metabolism ; },
abstract = {Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.},
}
@article {pmid31386102,
year = {2020},
author = {Li, T and Wang, S and Luo, F and Wu, FX and Wang, J},
title = {MultiGuideScan: a multi-processing tool for designing CRISPR guide RNA libraries.},
journal = {Bioinformatics (Oxford, England)},
volume = {36},
number = {3},
pages = {920-921},
doi = {10.1093/bioinformatics/btz616},
pmid = {31386102},
issn = {1367-4811},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *RNA, Guide ; Software ; },
abstract = {SUMMARY: The recent advance in genome engineering technologies based on CRISPR/Cas9 system is enabling people to systematically understand genomic functions. A short RNA string (the CRISPR guide RNA) can guide the Cas9 endonuclease to specific locations in complex genomes to cut DNA double-strands. The CRISPR guide RNA is essential for gene editing systems. Recently, the GuideScan software is developed to design CRISPR guide RNA libraries, which can be used for genome editing of coding and non-coding genomic regions effectively. However, GuideScan is a serial program and computationally expensive for designing CRISPR guide RNA libraries from large genomes. Here, we present an efficient guide RNA library designing tool (MultiGuideScan) by implementing multiple processes of GuideScan. MultiGuideScan speeds up the guide RNA library designing about 9-12 times on a 32-process mode comparing to GuideScan. MultiGuideScan makes it possible to design guide RNA libraries from large genomes.<br><br>https://github.com/bioinfomaticsCSU/MultiGuideScan.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid31385888,
year = {2019},
author = {Li, P and Zhang, W and Smith, LJ and Ayares, D and Cooper, DKC and Ekser, B},
title = {The potential role of 3D-bioprinting in xenotransplantation.},
journal = {Current opinion in organ transplantation},
volume = {24},
number = {5},
pages = {547-554},
pmid = {31385888},
issn = {1531-7013},
support = {S10 OD023595/OD/NIH HHS/United States ; U19 AI090959/AI/NIAID NIH HHS/United States ; UL1 TR001108/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Bioprinting/*methods ; CRISPR-Cas Systems ; Genetic Engineering ; Humans ; *Printing, Three-Dimensional ; Swine ; Transplantation, Heterologous/*methods ; },
abstract = {PURPOSE OF REVIEW: To review the impact of a new technology, 3D-bioprinting, in xenotransplantation research.<br><br>RECENT FINDINGS: Genetically engineered pigs, beginning with human (h) CD55-transgenic and Gal-knockout pigs, have improved the outcomes of xenotransplantation research. Today, there are more than 30 different genetically engineered pigs either expressing human gene(s) or lacking pig gene(s). CRIPSR/cas9 technology has facilitated the production of multigene pigs (up to nine genes in a single pig), which lack multiple pig xenoantigens, and express human transgenes, such as hCD46, hCD55, hThrombomodulin, hCD39, etc. Although recent studies in nonhuman primates (NHPs) have demonstrated prolonged survival after life-supporting pig kidney, heart, and islet xenotransplantation, researchers have difficulty determining the best genetic combination to test in NHPs because of a potential greater than 100&#x200a;000 genetic combinations. 3D-bioprinting of genetically engineered pig cells: is superior to 2D in-vitro testing, enables organ-specific testing, helps to understand differences in immunogenicity between organs, and is faster and cheaper than testing in NHPs. Moreover, 3D-bioprinted cells can be continuously perfused in a bioreactor, controlling for all variables, except the studied variable.<br><br>SUMMARY: 3D-bioprinting can help in the study of the impact of specific genes (human or pig) in xenotransplantation in a rapid, inexpensive, and reliable way.},
}
@article {pmid31385197,
year = {2019},
author = {Hajizadeh Dastjerdi, A and Newman, A and Burgio, G},
title = {The Expanding Class 2 CRISPR Toolbox: Diversity, Applicability, and Targeting Drawbacks.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {33},
number = {5},
pages = {503-513},
doi = {10.1007/s40259-019-00369-y},
pmid = {31385197},
issn = {1179-190X},
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Humans ; RNA, Guide ; },
abstract = {The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional diversity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.},
}
@article {pmid31384004,
year = {2019},
author = {Baumgarten, S and Bryant, JM and Sinha, A and Reyser, T and Preiser, PR and Dedon, PC and Scherf, A},
title = {Transcriptome-wide dynamics of extensive m[6]A mRNA methylation during Plasmodium falciparum blood-stage development.},
journal = {Nature microbiology},
volume = {4},
number = {12},
pages = {2246-2259},
pmid = {31384004},
issn = {2058-5276},
support = {670301/ERC_/European Research Council/International ; },
mesh = {Adenosine/*analogs &amp; derivatives/metabolism ; CRISPR-Cas Systems ; Erythrocytes/parasitology ; Gene Expression Regulation ; Gene Knockdown Techniques ; Genes, Protozoan ; Humans ; Life Cycle Stages ; Malaria, Falciparum/parasitology ; Methylation ; Methyltransferases/genetics ; Plasmodium falciparum/enzymology/*genetics/*metabolism ; Protozoan Proteins/genetics ; RNA, Messenger/*metabolism ; *Transcriptome ; },
abstract = {Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N[6]-methyladenosine (m[6]A) over the course of blood-stage development. Using mass spectrometry and m[6]A RNA sequencing, we demonstrate that m[6]A is highly developmentally regulated, exceeding m[6]A levels known in any other eukaryote. We characterize a distinct m[6]A writer complex and show that knockdown of the putative m[6]A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m[6]A. In accordance, we find an inverse correlation between m[6]A methylation and mRNA stability or translational efficiency. We further identify two putative m[6]A-binding YTH proteins that are likely to be involved in the regulation of these processes across the parasite's life cycle. Our data demonstrate unique features of an extensive m[6]A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.},
}
@article {pmid31384002,
year = {2019},
author = {Ooi, YS and Majzoub, K and Flynn, RA and Mata, MA and Diep, J and Li, JK and van Buuren, N and Rumachik, N and Johnson, AG and Puschnik, AS and Marceau, CD and Mlera, L and Grabowski, JM and Kirkegaard, K and Bloom, ME and Sarnow, P and Bertozzi, CR and Carette, JE},
title = {An RNA-centric dissection of host complexes controlling flavivirus infection.},
journal = {Nature microbiology},
volume = {4},
number = {12},
pages = {2369-2382},
pmid = {31384002},
issn = {2058-5276},
support = {R37 AI051622/AI/NIAID NIH HHS/United States ; DP2 AI104557/AI/NIAID NIH HHS/United States ; R37 AI047365/AI/NIAID NIH HHS/United States ; R01 AI051622/AI/NIAID NIH HHS/United States ; R01 AI069000/AI/NIAID NIH HHS/United States ; R01 AI134912/AI/NIAID NIH HHS/United States ; R01 AI141970/AI/NIAID NIH HHS/United States ; U19 AI109662/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carrier Proteins ; Cell Line ; Dengue Virus/genetics ; Endoplasmic Reticulum/genetics/metabolism ; Flavivirus/*genetics/pathogenicity/*physiology ; Flavivirus Infections/*virology ; Gene Knockout Techniques ; Host-Pathogen Interactions/genetics ; Humans ; RNA, Viral/*genetics/metabolism ; RNA-Binding Proteins/genetics ; Virus Replication ; Zika Virus/genetics ; },
abstract = {Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.},
}
@article {pmid31383972,
year = {2019},
author = {Liu, XM and Zhou, J and Mao, Y and Ji, Q and Qian, SB},
title = {Programmable RNA N[6]-methyladenosine editing by CRISPR-Cas9 conjugates.},
journal = {Nature chemical biology},
volume = {15},
number = {9},
pages = {865-871},
pmid = {31383972},
issn = {1552-4469},
support = {R01 GM122814/GM/NIGMS NIH HHS/United States ; R21 CA227917/CA/NCI NIH HHS/United States ; 55108556/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenosine/*analogs &amp; derivatives/chemistry/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Humans ; Methyltransferases/classification/*metabolism ; RNA, Messenger/chemistry/genetics/*metabolism ; },
abstract = {RNA modification in the form of N[6]-methyladenosine (m[6]A) regulates nearly all the post-transcriptional processes. The asymmetric m[6]A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m[6]A modifications. Here we report the development of 'm[6]A editing', a powerful approach that enables m[6]A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m[6]A methyltransferase that can be programmed with a guide RNA. The resultant m[6]A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m[6]A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m[6]A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.},
}
@article {pmid31383899,
year = {2019},
author = {Matsuda, T and Oinuma, I},
title = {Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11309},
pmid = {31383899},
issn = {2045-2322},
mesh = {Animals ; Arrestin/analysis/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/methods ; Gene Editing/*methods ; Gene Knock-In Techniques/methods ; Genetic Loci ; Glutamate-Ammonia Ligase/analysis/genetics ; Mice/*genetics ; Nerve Tissue Proteins/analysis/*genetics ; Nervous System/*metabolism ; Neuroglia/cytology/metabolism ; Retinal Rod Photoreceptor Cells/cytology/metabolism ; Rhodopsin/analysis/genetics ; Synaptophysin/analysis/genetics ; },
abstract = {To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.},
}
@article {pmid31381206,
year = {2019},
author = {Wolter, F and Puchta, H},
title = {In planta gene targeting can be enhanced by the use of CRISPR/Cas12a.},
journal = {The Plant journal : for cell and molecular biology},
volume = {100},
number = {5},
pages = {1083-1094},
doi = {10.1111/tpj.14488},
pmid = {31381206},
issn = {1365-313X},
support = {ERC-2016-AdG_741306 CRISBREED/ERC_/European Research Council/International ; },
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/genetics/*metabolism ; Bacterial Proteins/*genetics/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Associated Proteins/*genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Helicases/genetics/*metabolism ; Endodeoxyribonucleases/*genetics/*metabolism ; Gene Targeting/*methods ; Homologous Recombination ; Mutation ; },
abstract = {The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.},
}
@article {pmid31380838,
year = {2019},
author = {Sterner, RM and Cox, MJ and Sakemura, R and Kenderian, SS},
title = {Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {149},
pages = {},
doi = {10.3791/59629},
pmid = {31380838},
issn = {1940-087X},
support = {K12 CA090628/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Granulocyte-Macrophage Colony-Stimulating Factor/*genetics/physiology ; Humans ; *Immunotherapy, Adoptive ; Neoplasms/*therapy ; Receptors, Chimeric Antigen/*immunology ; T-Lymphocytes/*immunology ; },
abstract = {Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSF[k/o] CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.},
}
@article {pmid31380595,
year = {2019},
author = {Yilmaz, O and Patinote, A and Nguyen, T and Com, E and Pineau, C and Bobe, J},
title = {Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio).},
journal = {Molecular reproduction and development},
volume = {86},
number = {9},
pages = {1168-1188},
doi = {10.1002/mrd.23231},
pmid = {31380595},
issn = {1098-2795},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Knockdown Techniques ; *Vitellogenins/genetics/metabolism ; *Zebrafish/embryology/genetics ; *Zebrafish Proteins/genetics/metabolism ; },
abstract = {Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study utilized clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and type-III Vtgs. A multiple CRISPR approach was employed to knockout (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blot analysis showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, revealing a heretofore-unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2-fold in vtg1-KO females, and egg fertility was approximately halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 hr of incubation and in vtg1-KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.},
}
@article {pmid31379988,
year = {2019},
author = {Acosta Lopez, MJ and Trevisson, E and Canton, M and Vazquez-Fonseca, L and Morbidoni, V and Baschiera, E and Frasson, C and Pelosi, L and Rascalou, B and Desbats, MA and Alcázar-Fabra, M and Ríos, JJ and Sánchez-García, A and Basso, G and Navas, P and Pierrel, F and Brea-Calvo, G and Salviati, L},
title = {Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6.},
journal = {Oxidative medicine and cellular longevity},
volume = {2019},
number = {},
pages = {3904905},
pmid = {31379988},
issn = {1942-0994},
mesh = {Adenosine Triphosphate/*metabolism ; Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; Humans ; Mitochondria/metabolism ; Mutagenesis, Site-Directed ; Protein Isoforms/genetics/metabolism ; Protein Structure, Tertiary ; Reactive Oxygen Species/metabolism ; Sequence Alignment ; Ubiquinone/*analogs &amp; derivatives/biosynthesis/genetics/metabolism ; Vanillic Acid/*pharmacology ; },
abstract = {Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.},
}
@article {pmid31378649,
year = {2019},
author = {Su, X and Chen, W and Cai, Q and Liang, P and Chen, Y and Cong, P and Huang, J},
title = {Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {7},
pages = {335-342},
doi = {10.1016/j.jgg.2019.07.002},
pmid = {31378649},
issn = {1673-8527},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; *Gene Editing ; Genes, X-Linked ; *Genome ; *Nuclear Transfer Techniques ; Oocytes/*metabolism ; RNA, Guide ; RNA, Messenger/genetics ; Sequence Analysis, DNA ; Swine ; },
abstract = {Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds. Generation of genetically edited pigs using somatic cell nuclear transfer (SCNT) or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder (F0). Herein, we developed a method termed germinal vesicle oocyte gene editing (GVGE) to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MⅡ transition. Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential. The MⅡ oocytes edited during in vitro maturation (IVM) could develop into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation. Up to 81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos. In conclusion, GVGE might be a valuable method for the generation of non-mosaic maternal allele edited F0 embryos in a short simple step.},
}
@article {pmid31378590,
year = {2019},
author = {Towers, CG and Fitzwalter, BE and Regan, D and Goodspeed, A and Morgan, MJ and Liu, CW and Gustafson, DL and Thorburn, A},
title = {Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition.},
journal = {Developmental cell},
volume = {50},
number = {6},
pages = {690-703.e6},
pmid = {31378590},
issn = {1878-1551},
support = {T32 CA190216/CA/NCI NIH HHS/United States ; P30 CA046934/CA/NCI NIH HHS/United States ; K01 OD022982/CD/ODCDC CDC HHS/United States ; R01 CA190170/CA/NCI NIH HHS/United States ; K01 OD022982/OD/NIH HHS/United States ; R01 CA150925/CA/NCI NIH HHS/United States ; T32 GM007635/GM/NIGMS NIH HHS/United States ; L30 TR002126/TR/NCATS NIH HHS/United States ; },
mesh = {*Adaptation, Physiological/drug effects ; *Autophagy/drug effects ; Autophagy-Related Protein 7/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Clone Cells ; Gene Knockout Techniques ; Genes, Essential ; Humans ; NF-E2-Related Factor 2/*metabolism ; Neoplasms/*metabolism/*pathology ; Proteasome Endopeptidase Complex/metabolism ; Proteasome Inhibitors/pharmacology ; Ribonucleoproteins/metabolism ; *Signal Transduction/drug effects ; *Up-Regulation/drug effects ; },
abstract = {While autophagy is thought to be an essential process in some cancer cells, it is unknown if or how such cancer cells can circumvent autophagy inhibition. To address this, we developed a CRISPR/Cas9 assay with dynamic live-cell imaging to measure acute effects of knockout (KO) of autophagy genes compared to known essential and non-essential genes. In some cancer cells, autophagy is as essential for cancer cell growth as mRNA transcription or translation or DNA replication. However, even these highly autophagy-dependent cancer cells evolve to circumvent loss of autophagy by upregulating NRF2, which is necessary and sufficient for autophagy-dependent cells to circumvent ATG7 KO and maintain protein homeostasis. Importantly, however, this adaptation increases susceptibly to proteasome inhibitors. These studies identify a common mechanism of acquired resistance to autophagy inhibition and show that selection to avoid tumor cell dependency on autophagy creates new, potentially actionable cancer cell susceptibilities.},
}
@article {pmid31378358,
year = {2019},
author = {Guest, M and Goodchild, JA and Bristow, JA and Flemming, AJ},
title = {RDL A301S alone does not confer high levels of resistance to cyclodiene organochlorine or phenyl pyrazole insecticides in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {158},
number = {},
pages = {32-39},
doi = {10.1016/j.pestbp.2019.04.005},
pmid = {31378358},
issn = {1095-9939},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dieldrin/pharmacology ; Endosulfan/pharmacology ; Insecticide Resistance/genetics ; Insecticides/*pharmacology ; Moths/*drug effects/genetics ; Mutation/genetics ; Pyrazoles/pharmacology ; Receptors, GABA-A/genetics ; },
abstract = {Mutations in the GABA-gated chloride channel are associated with resistance to cyclodiene organochlorine and phenyl pyrazole insecticides. The best characterised of these is A301S, which was initially identified in a Dieldrin resistant strain of Drosophila melanogaster. The orthologous mutation has been found in a variety of different crop pests including the diamond back moth Plutella xylostella. However, the contribution of this mutation to resistance in this species remains unclear. We have used the CRISPR/Cas9 system in order to edit Plutella xylostella PxGABARalpha1 to Serine at the 301 orthologous position (282 in PxGABARalpha1) in an insecticide sensitive strain isolated from Vero Beach (VB) USA. In this edited line, no high level of resistance is conferred to Dieldrin, Endosulfan or Fipronil, rather only a subtle shift in sensitivity which could not confer commercially important resistance. We conclude that the high level of commercial resistance to cyclodiene organochlorine and phenyl pyrazole insecticides observed in some field isolates of Plutella xylostella cannot arise from A282S in PxGABARalpha1 alone.},
}
@article {pmid31377338,
year = {2020},
author = {Depardieu, F and Bikard, D},
title = {Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {61-75},
doi = {10.1016/j.ymeth.2019.07.024},
pmid = {31377338},
issn = {1095-9130},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Genetic Vectors/genetics ; Mutation ; Plasmids/genetics ; RNA, Guide/genetics ; Staphylococcus aureus/genetics ; Transcription, Genetic ; },
abstract = {The catalytic null mutant of the Cas9 endonuclease from the bacterial CRISPR immune system, known as dCas9, can be guided by a small RNA to bind DNA sequences of interest and block gene transcription in a strategy known as CRISPRi. This powerful gene silencing method has already been used in a large number of species and in high throughput screens. Here we provide detailed design rules, methods and novel vectors to perform CRISPRi experiments in S. aureus and in E. coli, using the well characterized dCas9 protein from S. pyogenes. In particular, we describe a vector based on plasmid pC194 which is broadly used in Firmicutes, as well as a vector based on the very broad host-range rolling circle plasmid pLZ12, reported to replicate in both Firmicutes and Proteobacteria. A potential caveat of adapting dCas9 tools to various bacterial species is that dCas9 was shown to be toxic when expressed too strongly. We describe a method to optimize the expression level of dCas9 in order to avoid toxicity while ensuring strong on-target repression activity. We demonstrate this method by optimizing a pLZ12 based vector originally developed for S. aureus so that it can work in E. coli. This article should provide all the resources required to perform CRISPRi experiments in a broad range of bacterial species.},
}
@article {pmid31375684,
year = {2019},
author = {Dammert, MA and Brägelmann, J and Olsen, RR and Böhm, S and Monhasery, N and Whitney, CP and Chalishazar, MD and Tumbrink, HL and Guthrie, MR and Klein, S and Ireland, AS and Ryan, J and Schmitt, A and Marx, A and Ozretić, L and Castiglione, R and Lorenz, C and Jachimowicz, RD and Wolf, E and Thomas, RK and Poirier, JT and Büttner, R and Sen, T and Byers, LA and Reinhardt, HC and Letai, A and Oliver, TG and Sos, ML},
title = {MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3485},
pmid = {31375684},
issn = {2041-1723},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R21 CA216504/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Apoptosis/drug effects/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA Damage/drug effects/genetics ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic/drug effects/*genetics ; HEK293 Cells ; Humans ; Lung Neoplasms/drug therapy/*genetics ; Mice ; Molecular Targeted Therapy/methods ; Proto-Oncogene Proteins c-myc/genetics/*metabolism ; RNA, Small Interfering/metabolism ; Small Cell Lung Carcinoma/drug therapy/*genetics ; },
abstract = {MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients.},
}
@article {pmid31375681,
year = {2019},
author = {Johnston, AD and Simões-Pires, CA and Thompson, TV and Suzuki, M and Greally, JM},
title = {Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3472},
pmid = {31375681},
issn = {2041-1723},
support = {P30 DK020541/DK/NIDDK NIH HHS/United States ; T32 GM007288/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Female ; GTPase-Activating Proteins/*genetics/metabolism ; Gene Editing ; Humans ; Male ; Mutagenesis ; Polymorphism, Genetic ; Promoter Regions, Genetic/*genetics ; Protein Binding/genetics ; Transcription Factor RelA/*metabolism ; Whole Genome Sequencing ; },
abstract = {Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.},
}
@article {pmid31375676,
year = {2019},
author = {Shi, C and Luo, P and Du, YT and Chen, H and Huang, X and Cheng, TH and Luo, A and Li, HJ and Yang, WC and Zhao, P and Sun, MX},
title = {Maternal control of suspensor programmed cell death via gibberellin signaling.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3484},
pmid = {31375676},
issn = {2041-1723},
mesh = {Apoptosis/*physiology ; Arabidopsis/growth &amp; development ; CRISPR-Cas Systems/genetics ; Cell Communication/physiology ; *Gene Expression Regulation, Developmental ; *Gene Expression Regulation, Plant ; Gibberellins/*metabolism ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Seeds/*growth &amp; development ; Signal Transduction/physiology ; Tobacco/genetics/growth &amp; development ; Transcription Factors/genetics/metabolism ; },
abstract = {Plant embryos are generated and develop in a stable and well-protected microenvironment surrounded by maternal tissue, which is vital for embryogenesis. However, the signaling mechanisms responsible for maternal tissue-to-proembryo communication are not well understood. Here, we report a pathway for maternal tissue-to-proembryo communication. We identify a DELLA protein, NtCRF1 (NtCYS regulative factor 1), which regulates suspensor programmed cell death (PCD). NtCRF1 can bind to the promoter of NtCYS and regulate the suspensor PCD-switch module NtCYS-NtCP14 in response to gibberellin (GA). We confirm that GA4, as a primary signal triggering suspensor PCD, is generated in the micropylar endothelium by the transient activation of NtGA3oxs in the maternal tissue. Thus, we propose that GA is a maternal-to-proembryo communication signal that is decoded in the proembryo by a GID1-CRF1-CYS-CP14 signaling cascade. Using this mode of communication, maternal tissue precisely controls the embryonic suspensor PCD and is able to nurse the proembryo in a stage-dependent manner.},
}
@article {pmid31375149,
year = {2019},
author = {Yang, F and Cui, P and Lu, Y and Zhang, X},
title = {Requirement of the transcription factor YB-1 for maintaining the stemness of cancer stem cells and reverting differentiated cancer cells into cancer stem cells.},
journal = {Stem cell research &amp; therapy},
volume = {10},
number = {1},
pages = {233},
pmid = {31375149},
issn = {1757-6512},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints ; *Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Female ; Frizzled Receptors/genetics/metabolism ; Glucagon-Like Peptide 1/genetics/metabolism ; Humans ; Mice ; Mice, Nude ; Neoplastic Stem Cells/cytology/*metabolism ; RNA, Guide/metabolism ; Transcription Factors/deficiency/genetics/*metabolism ; },
abstract = {BACKGROUND: Cancer stem cells always express high levels of stemness-associated transcription factors to maintain their features. However, the regulatory mechanism of the stemness of cancer stem cells mediated by transcription factors has not been extensively explored.<br><br>METHODS: The YB-1 gene in cancer stem cells was knocked out by the CRISPR/Cas9 system. The YB-1 knockout cancer stem cells were transfected with a vector expressing YB-1 to rescue YB-1, and then the cell proliferation, cell cycle, apoptosis, and stemness, as well as tumorigenesis in nude mice, were assessed to examine the effect of YB-1 in cancer stem cells. The target genes of YB-1 were confirmed by CHIP-seq. The totipotency or pluripotency of differentiated cancer stem cells were detected by tumorsphere formation assay and quantitative real-time PCR.<br><br>RESULTS: The deletion of YB-1 gene inhibited the proliferation of breast cancer stem cells and melanoma stem cells, leading to cell cycle arrest and apoptosis, and induced irreversible differentiation of cancer stem cells. The tumorigenicity ability of YB-1-deleted cancer stem cells was significantly reduced in vitro and in vivo. The results of ChIP-seq showed that YB-1 maintained the stemness of cancer stem cells by promoting the expressions of stemness-associated genes (FZD-1, p21, GLP-1, GINS1, and Notch2). Furthermore, simultaneous expressions of YB-1 and the other four (SOX2, POU3F2, OCT-4, and OLIG1) or five (SOX2, SALL2, OCT-4, POU3F2, and Bmi-1) transcription factors in YB-1 knockout cancer stem cells restored the stemness of YB-1 knockout cancer stem cells.<br><br>CONCLUSIONS: Our study indicated that YB-1 was required for maintaining the stemness of cancer stem cells and reverting the differentiated tumor cells into cancer stem cells.},
}
@article {pmid31374142,
year = {2020},
author = {Char, SN and Wei, J and Mu, Q and Li, X and Zhang, ZJ and Yu, J and Yang, B},
title = {An Agrobacterium-delivered CRISPR/Cas9 system for targeted mutagenesis in sorghum.},
journal = {Plant biotechnology journal},
volume = {18},
number = {2},
pages = {319-321},
pmid = {31374142},
issn = {1467-7652},
mesh = {*Agrobacterium/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Mutagenesis ; *Plants, Genetically Modified/genetics ; *Sorghum/genetics ; },
}
@article {pmid31373788,
year = {2019},
author = {Shin, J and Kang, S and Song, Y and Jin, S and Lee, JS and Lee, JK and Kim, DR and Kim, SC and Cho, S and Cho, BK},
title = {Genome Engineering of Eubacterium limosum Using Expanded Genetic Tools and the CRISPR-Cas9 System.},
journal = {ACS synthetic biology},
volume = {8},
number = {9},
pages = {2059-2068},
doi = {10.1021/acssynbio.9b00150},
pmid = {31373788},
issn = {2161-5063},
mesh = {Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Drug Resistance, Bacterial/genetics ; Eubacterium/drug effects/*genetics/growth &amp; development ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; },
abstract = {Eubacterium limosum is one of the important bacteria in C1 feedstock utilization as well as in human gut microbiota. Although E. limosum has recently garnered much attention and investigation on a genome-wide scale, a bottleneck for systematic engineering in E. limosum is the lack of available genetic tools and an efficient genome editing platform. To overcome this limitation, we here report expanded genetic tools and the CRISPR-Cas9 system. We have developed an inducible promoter system that enables implementation of the CRISPR-Cas9 system to precisely manipulate target genes of the Wood-Ljungdahl pathway with 100% efficiency. Furthermore, we exploited the effectiveness of CRISPR interference to reduce the expression of target genes, exhibiting substantial repression of several genes in the Wood-Ljungdahl pathway and fructose-PTS system. These expanded genetic tools and CRISPR-Cas9 system comprise powerful and widely applicable genetic tools to accelerate functional genomic study and genome engineering in E. limosum.},
}
@article {pmid31373227,
year = {2019},
author = {Li, F and Hung, SSC and Mohd Khalid, MKN and Wang, JH and Chrysostomou, V and Wong, VHY and Singh, V and Wing, K and Tu, L and Bender, JA and Pébay, A and King, AE and Cook, AL and Wong, RCB and Bui, BV and Hewitt, AW and Liu, GS},
title = {Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina.},
journal = {Human gene therapy},
volume = {30},
number = {11},
pages = {1349-1360},
doi = {10.1089/hum.2019.021},
pmid = {31373227},
issn = {1557-7422},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Electroretinography ; *Gene Editing ; Gene Transfer Techniques ; HEK293 Cells ; Humans ; Mice, Inbred C57BL ; RNA, Guide/genetics ; Reproducibility of Results ; Retina/*metabolism/physiology ; Tomography, Optical Coherence ; },
abstract = {Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.},
}
@article {pmid31373135,
year = {2020},
author = {Zheng, M and Zhang, L and Tang, M and Liu, J and Liu, H and Yang, H and Fan, S and Terzaghi, W and Wang, H and Hua, W},
title = {Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.).},
journal = {Plant biotechnology journal},
volume = {18},
number = {3},
pages = {644-654},
pmid = {31373135},
issn = {1467-7652},
mesh = {Brassica napus/*genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Knockout Techniques ; Genes, Plant ; Mutagenesis ; Plants, Genetically Modified/growth &amp; development ; },
abstract = {Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%-67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi-allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene-free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off-target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi-dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.},
}
@article {pmid31372919,
year = {2019},
author = {Belykh, ES and Maystrenko, TA and Velegzhaninov, IO},
title = {Recent Trends in Enhancing the Resistance of Cultivated Plants to Heavy Metal Stress by Transgenesis and Transcriptional Programming.},
journal = {Molecular biotechnology},
volume = {61},
number = {10},
pages = {725-741},
pmid = {31372919},
issn = {1559-0305},
mesh = {Biodegradation, Environmental ; CRISPR-Cas Systems ; *Disease Resistance ; Gene Expression Regulation, Plant ; Genetic Engineering ; Homeostasis ; Metals, Heavy/*metabolism/toxicity ; Plant Development ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics/*growth &amp; development/metabolism ; },
abstract = {Normal growth and development of high plants strongly depends on the concentration of microelements, including essential heavy metals, in the substrate. However, an excess of those elements may become harmful. Therefore, micronutrient concentrations in plant tissue should be well-balanced and controlled by homeostatic mechanisms. The advancement of knowledge on the regulation of metal homeostasis in plants is important for phytoremediation of metal-contaminated soil and for micronutrient malnutrition control. Experimental data from loss-of-function and gain-of-function studies, including functional descriptions and classifications have presented new opportunities for multiplex CRISPR/dCas9-driven control of gene expression and have opened up new prospects for the goal-seeking regulation of metal homeostasis in plants. The aim of this review is to help for multiplex transcriptional programming targets search by summarizing and analyzing data on possible ways to handle a plant's ability to maintain metal homeostasis.},
}
@article {pmid31372754,
year = {2019},
author = {Fofanov, MV and Morozova, VV and Kozlova, YN and Tikunov, AY and Babkin, IV and Poletaeva, YE and Ryabchikova, EI and Tikunova, NV},
title = {Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.},
journal = {Archives of virology},
volume = {164},
number = {10},
pages = {2637-2640},
doi = {10.1007/s00705-019-04349-z},
pmid = {31372754},
issn = {1432-8798},
mesh = {Bacteriolysis ; Bacteriophages/*classification/genetics/*isolation &amp; purification ; Enterobacteriaceae/*virology ; Genome, Viral ; Microscopy, Electron, Transmission ; *Phylogeny ; Sequence Analysis, DNA ; Siphoviridae/*classification/genetics/*isolation &amp; purification ; Synteny ; Viral Proteins/genetics ; Virion/ultrastructure ; },
abstract = {A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.},
}
@article {pmid31372706,
year = {2019},
author = {Le, Q and Nguyen, V and Park, S},
title = {Recent advances in the engineering and application of streptavidin-like molecules.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {18},
pages = {7355-7365},
doi = {10.1007/s00253-019-10036-5},
pmid = {31372706},
issn = {1432-0614},
mesh = {Binding Sites ; Biotechnology/*methods ; Biotin/*metabolism ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Kinetics ; Nanotechnology ; Protein Binding ; Protein Engineering/*methods ; Streptavidin/*chemistry ; },
abstract = {Streptavidin (SA), and other related proteins, has been isolated from a wide range of organisms, including bacteria, fungi, frogs, fish, and birds. Although their original function is not well understood, they have found an important place in biotechnology based on their unique ability to bind biotin molecules with high affinity and specificity. The SA-biotin interaction is robust and easy to incorporate into different designs, and as such, it is used when reliable molecule interaction is needed under poorly controlled experimental conditions. There are continued efforts to engineer these proteins to modulate their size, valency, and affinity, since the optimum molecular properties vary depending on individual applications. This review will describe recent developments in streptavidin engineering to meet these requirements, including those that form novel oligomeric states, e.g., a monomer, have fewer functional biotin-binding sites, or bind biotin with reduced affinity. We also examine various reported applications of both natural or engineered SA constructs in cell biology, biochemistry, genetics, synthetic chemistry, cancer therapy, drug delivery, and nanotechnology to illustrate the breadth of modern science that is advanced by the endogenous and engineered SA-biotin interactions.},
}
@article {pmid31372658,
year = {2019},
author = {Gam, JJ and DiAndreth, B and Jones, RD and Huh, J and Weiss, R},
title = {A 'poly-transfection' method for rapid, one-pot characterization and optimization of genetic systems.},
journal = {Nucleic acids research},
volume = {47},
number = {18},
pages = {e106},
pmid = {31372658},
issn = {1362-4962},
support = {P50 GM098792/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Expression/genetics ; High-Throughput Screening Assays/*methods ; Humans ; MicroRNAs/genetics ; Transfection/*methods ; },
abstract = {Biological research is relying on increasingly complex genetic systems and circuits to perform sophisticated operations in living cells. Performing these operations often requires simultaneous delivery of many genes, and optimizing the stoichiometry of these genes can yield drastic improvements in performance. However, sufficiently sampling the large design space of gene expression stoichiometries in mammalian cells using current methods is cumbersome, complex, or expensive. We present a 'poly-transfection' method as a simple yet high-throughput alternative that enables comprehensive evaluation of genetic systems in a single, readily-prepared transfection sample. Each cell in a poly-transfection represents an independent measurement at a distinct gene expression stoichiometry, fully leveraging the single-cell nature of transfection experiments. We first benchmark poly-transfection against co-transfection, showing that titration curves for commonly-used regulators agree between the two methods. We then use poly-transfections to efficiently generate new insights, for example in CRISPRa and synthetic miRNA systems. Finally, we use poly-transfection to rapidly engineer a difficult-to-optimize miRNA-based cell classifier for discriminating cancerous cells. One-pot evaluation enabled by poly-transfection accelerates and simplifies the design of genetic systems, providing a new high-information strategy for interrogating biology.},
}
@article {pmid31372638,
year = {2019},
author = {Korkmaz, G and Manber, Z and Lopes, R and Prekovic, S and Schuurman, K and Kim, Y and Teunissen, H and Flach, K and Wit, E and Galli, GG and Zwart, W and Elkon, R and Agami, R},
title = {A CRISPR-Cas9 screen identifies essential CTCF anchor sites for estrogen receptor-driven breast cancer cell proliferation.},
journal = {Nucleic acids research},
volume = {47},
number = {18},
pages = {9557-9572},
pmid = {31372638},
issn = {1362-4962},
mesh = {Binding Sites/genetics ; Breast Neoplasms/*genetics/pathology ; CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems/genetics ; Cell Proliferation/*genetics ; Chromatin/genetics ; Enhancer Elements, Genetic/genetics ; Estrogen Receptor alpha/*genetics ; Female ; Humans ; MCF-7 Cells ; Protein Binding/genetics ; },
abstract = {Estrogen receptor α (ERα) is an enhancer activating transcription factor, a key driver of breast cancer and a main target for cancer therapy. ERα-mediated gene regulation requires proper chromatin-conformation to facilitate interactions between ERα-bound enhancers and their target promoters. A major determinant of chromatin structure is the CCCTC-binding factor (CTCF), that dimerizes and together with cohesin stabilizes chromatin loops and forms the boundaries of topologically associated domains. However, whether CTCF-binding elements (CBEs) are essential for ERα-driven cell proliferation is unknown. To address this question in a global manner, we implemented a CRISPR-based functional genetic screen targeting CBEs located in the vicinity of ERα-bound enhancers. We identified four functional CBEs and demonstrated the role of one of them in inducing chromatin conformation changes in favor of activation of PREX1, a key ERα target gene in breast cancer. Indeed, high PREX1 expression is a bona-fide marker of ERα-dependency in cell lines, and is associated with good outcome after anti-hormonal treatment. Altogether, our data show that distinct CTCF-mediated chromatin structures are required for ERα- driven breast cancer cell proliferation.},
}
@article {pmid31371821,
year = {2019},
author = {Kwon, JB and Gersbach, CA},
title = {Jumping at the chance for precise DNA integration.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1004-1006},
pmid = {31371821},
issn = {1546-1696},
support = {T32 HD040372/HD/NICHD NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Damage ; *Gene Targeting ; *Genome ; *Transposases ; },
}
@article {pmid31371804,
year = {2019},
author = {de la Roche, J and Angsutararux, P and Kempf, H and Janan, M and Bolesani, E and Thiemann, S and Wojciechowski, D and Coffee, M and Franke, A and Schwanke, K and Leffler, A and Luanpitpong, S and Issaragrisil, S and Fischer, M and Zweigerdt, R},
title = {Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of NaV1.5 sodium channels.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {11173},
pmid = {31371804},
issn = {2045-2322},
mesh = {Adult ; Brugada Syndrome/*genetics/pathology ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*cytology ; *Mutation ; Myocytes, Cardiac/*pathology ; NAV1.5 Voltage-Gated Sodium Channel/*genetics ; Patch-Clamp Techniques ; },
abstract = {Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit NaV1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-NaV1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells' immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27-42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-NaV1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs' relevance for investigating channelopathies in a dish.},
}
@article {pmid31371630,
year = {2019},
author = {Jiang, J and Sun, Y and Xiao, R and Wai, K and Ahmad, MJ and Khan, FA and Zhou, H and Li, Z and Zhang, Y and Zhou, A and Zhang, S},
title = {Porcine antiviral activity is increased by CRISPRa-SAM system.},
journal = {Bioscience reports},
volume = {39},
number = {8},
pages = {},
pmid = {31371630},
issn = {1573-4935},
mesh = {Animals ; *CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Mice ; *Myxovirus Resistance Proteins/genetics/immunology ; *N-Acetylgalactosaminyltransferases/genetics/immunology ; Swine ; Swine Diseases/genetics/*immunology ; *Virus Diseases/genetics/immunology ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat activation-synergistic activation mediator system (CRISPRa-SAM) has been efficiently used to up-regulate the targeted genes in human and mouse. But it is not known whether the CRISPRa-SAM system can be used against porcine disease because its two important transcriptional activation domains (P65 and heat shock transcription factor 1 (HSF1)) are from mouse and human, respectively. Pig is one of the most important meat sources, porcine viral infectious diseases cause massive economic losses to the swine industry and threaten the public health. We aimed to investigate whether the CRISPRa-SAM system could increase porcine antiviral activity by mediating two pig-specific target genes (Mx2 and β1,4 N-acetylgalactosaminyltransferase (B4galnt2)). First, we constructed PK-15 and IPEC-J2 cell lines expressing nuclease-deficient Cas9 (dCas9)-vp64 and MS2-P65-HSF1 stably. Next, in these two cell models, we activated Mx2 and B4galnt2 expression through CRISPRa-SAM system. Antiviral activity to PRV or H9N2 was improved in PK-15 cells where Mx2 or B4galnt2 was activated. Altogether, our results demonstrated the potential of CRISPRa-SAM system as a powerful tool for activating pig genes and improving porcine antiviral activity.},
}
@article {pmid31371515,
year = {2019},
author = {Caro, F and Place, NM and Mekalanos, JJ},
title = {Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {34},
pages = {17013-17022},
pmid = {31371515},
issn = {1091-6490},
support = {R01 AI018045/AI/NIAID NIH HHS/United States ; R37 AI018045/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Carrier Proteins/*genetics/metabolism ; Cell Membrane/*genetics/metabolism ; *Gene Knockdown Techniques ; Humans ; Vibrio cholerae/*genetics/metabolism ; },
abstract = {Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or "MOMV" encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.},
}
@article {pmid31371352,
year = {2019},
author = {Engreitz, J and Abudayyeh, O and Gootenberg, J and Zhang, F},
title = {CRISPR Tools for Systematic Studies of RNA Regulation.},
journal = {Cold Spring Harbor perspectives in biology},
volume = {11},
number = {8},
pages = {},
pmid = {31371352},
issn = {1943-0264},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Catalysis ; DNA/genetics ; Gene Editing ; *Gene Expression Regulation ; Genome ; Genomics ; Humans ; Models, Genetic ; Phenotype ; RNA/*genetics ; RNA Processing, Post-Transcriptional ; RNA, Long Noncoding/genetics ; },
abstract = {RNA molecules perform diverse functions in mammalian cells, including transferring genetic information from DNA to protein and playing diverse regulatory roles through interactions with other cellular components. Here, we discuss how clustered regularly interspaced short palindromic repeat (CRISPR)-based technologies for directed perturbations of DNA and RNA are revealing new insights into RNA regulation. First, we review the fundamentals of CRISPR-Cas enzymes and functional genomics tools that leverage these systems. Second, we explore how these new perturbation technologies are transforming the study of regulation of and by RNA, focusing on the functions of DNA regulatory elements and long noncoding RNAs (lncRNAs). Third, we highlight an emerging class of RNA-targeting CRISPR-Cas enzymes that have the potential to catalyze studies of RNA biology by providing tools to directly perturb or measure RNA modifications and functions. Together, these tools enable systematic studies of RNA function and regulation in mammalian cells.},
}
@article {pmid31370935,
year = {2019},
author = {Hanswillemenke, A and Stafforst, T},
title = {Protocols for the generation of caged guideRNAs for light-triggered RNA-targeting with SNAP-ADARs.},
journal = {Methods in enzymology},
volume = {624},
number = {},
pages = {47-68},
doi = {10.1016/bs.mie.2019.06.004},
pmid = {31370935},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Light ; RNA/genetics ; *RNA Editing ; RNA, Guide/*genetics ; Transfection/methods ; },
abstract = {The SNAP-tag technology offers a convenient way to assemble guideRNA-protein conjugates for transcript-specific RNA editing in vitro, in cell culture and in vivo. In contrast to other methods, including CRISPR/Cas-based, the SNAP-tag is small, well expressed and of human origin. Furthermore, the SNAP-ADAR approach enables the ready inclusion of photo control by caging/decaging of the benzylguanine moiety required for the conjugation reaction with the SNAP-tag. Beyond site-directed RNA editing, the method has high potential for various applications in the field of RNA targeting. However, the generation of the required guideRNAs includes some basic chemistry. Here, we provide step-by-step protocols for (a) conduction of photo controlled RNA editing reaction, (b) the generation of photo activatable guideRNAs, and (c) the synthesis of the caged benzylguanine moiety. With this we hope to foster a broader application of these attractive methods to researchers with less experience in chemistry.},
}
@article {pmid31370789,
year = {2019},
author = {Jouanin, A and Schaart, JG and Boyd, LA and Cockram, J and Leigh, FJ and Bates, R and Wallington, EJ and Visser, RGF and Smulders, MJM},
title = {Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {333},
pmid = {31370789},
issn = {1471-2229},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Electrophoresis, Polyacrylamide Gel ; Gene Editing/*methods ; Genes, Plant/*genetics ; Gliadin/*genetics ; Glutens/*genetics/immunology ; Plant Breeding/methods ; Plants, Genetically Modified ; Sequence Alignment ; Triticum/*genetics ; },
abstract = {BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes.<br><br>RESULTS: In this study, we analysed the genetic diversity of over 600 &#x3b1;- and &#x3b3;-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the &#x3b1;- or &#x3b3;-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by &#x3b3;-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon &#x3b3;-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods.<br><br>CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large &#x3b1;- and &#x3b3;-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.},
}
@article {pmid31369818,
year = {2019},
author = {Tanic, J and Wang, Y and Lee, W and Coelho, NM and Glogauer, M and McCulloch, CA},
title = {Adseverin modulates morphology and invasive function of MCF7 cells.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1865},
number = {10},
pages = {2716-2725},
doi = {10.1016/j.bbadis.2019.07.015},
pmid = {31369818},
issn = {1879-260X},
support = {MOP-142250//CIHR/Canada ; },
mesh = {Actins/metabolism ; Breast Neoplasms/metabolism ; CRISPR-Cas Systems ; Cell Movement ; Collagen/metabolism ; Fibronectins/metabolism ; Gelsolin/genetics/*metabolism ; Humans ; MCF-7 Cells/*metabolism ; Phagocytosis ; },
abstract = {Adseverin (Ads) is a Ca[2+]-dependent actin-capping and severing protein that is highly expressed in gastric, prostate and bladder cancer cells. Currently it is unknown whether Ads contributes to the subcortical actin remodeling associated with the formation of cell extensions and matrix invasion in cancer. We compared cell extension formation and matrix degradation in Ads wildtype and Ads-null MCF7 breast cancer cells generated by CRISPR/Cas9. Compared with wildtype, Ads-null cells plated on fibronectin or collagen exhibited a more circular morphology with shorter cell extensions (37% reduction on fibronectin; p < 0.001). Reconstitution of Ads in Ads-null cells restored the formation of cell extensions (p < 0.05). While cell migration on two-dimensional matrices was unchanged by Ads deletion, the formation of cell extensions across Transwell membranes was reduced (~40% reduction, p < 0.05). When plated on fibrillar collagen, compared with wildtype, Ads-null cells showed reduced expression of MT1-MMP, collagen degradation (p < 0.05) and phagocytosis of collagen-coated beads (25% reduction; p = 0.001). We conclude that Ads is involved in the formation of cell extensions and collagen degradation in MCF7 cells, which may in turn affect matrix invasion and metastasis.},
}
@article {pmid31367975,
year = {2019},
author = {Ramalingam, S and Thangavel, S},
title = {CRISPR-Cas9 Probing of Infectious Diseases and Genetic Disorders.},
journal = {Indian journal of pediatrics},
volume = {86},
number = {12},
pages = {1131-1135},
pmid = {31367975},
issn = {0973-7693},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Communicable Diseases/*genetics ; Gene Editing/methods ; Gene Targeting ; Genetic Diseases, Inborn/*genetics ; Genetic Engineering ; Genetic Therapy ; Humans ; },
abstract = {The ability to precisely change the deoxyribonucleic acid (DNA) bases at specific sites offers tremendous advantages in the field of molecular biology and medical biotechnology. Identification of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), revelation of its role in prokaryotic adaptive immunity and subsequent conversion into genome and epigenome engineering system are the landmark research progresses of the decade. The possibilities of deciphering the molecular mechanisms of the disease, identifying the disease targets, generating the disease models, validating the drug targets, developing resistance to the infection and correcting the genotype have brought off much enthusiasm in the field of infectious diseases and genetic disorders. This review focuses on CRISPR/Cas9's impact in the field of infection and genetic disorders.},
}
@article {pmid31367859,
year = {2019},
author = {Li, H and Li, Z and Xiao, N and Su, X and Zhao, S and Zhang, Y and Cui, K and Liu, Q and Shi, D},
title = {Site-specific integration of rotavirus VP6 gene in rabbit β-casein locus by CRISPR/Cas9 system.},
journal = {In vitro cellular &amp; developmental biology. Animal},
volume = {55},
number = {8},
pages = {586-597},
pmid = {31367859},
issn = {1543-706X},
mesh = {Animals ; Antigens, Viral/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Capsid Proteins/*genetics ; Caseins/*genetics ; *Genetic Loci ; Genetic Vectors/metabolism ; Mutagenesis, Site-Directed ; RNA, Guide/genetics ; Rabbits ; Zygote/metabolism ; },
abstract = {Rotavirus (RV) is the leading cause of viral gastroenteritis in neonates and VP6 protein has been discussed as a potential candidate vaccine. CRISPR/Cas9 was the latest generation of gene editing tools that can mediate the site-specific knock-in of exogenous genes, providing strong support for the expression of recombinant proteins. Here, seeking to design a rotavirus vaccine that would be suitable for both mammary-gland-based production and milk-based administration, rabbit β-casein (CSN2) locus was chosen as the target site to integrate the VP6 gene. The efficiency of inducing mutations in different target sites of rabbit CSN2 locus was analyzed and g4 site seems to be the best one to generate mutations (g4 72.76 ± 0.32% vs g1 30.14 ± 1.93%, g2 38.53 ± 0.75%, g3 52.26 ± 1.16%, P < 0.05). We further compared the knock-in efficiency through cytoplasmic injection of two group mixtures (containing 100 ng/μL Cas9 mRNA or Cas9 protein, 20 ng/μL sgRNA4, and 100 ng/μL donor vector) in rabbit zygotes, though the Cas9 mRNA group induced an HDR efficiency as high as 20.0% ± 2.6% than Cas9 protein group (10.3% ± 3.1%), 37.5% of the knock-in events were partial integration in the target site, when Cas9 protein used in the CRISPR/Cas9 system, all of the positive blastocysts showed completely integrated, results showed that the use of Cas9 protein is better than Cas9 mRNA to integrate the correct exogenous gene into the target site. Moreover, the transgenic rabbit that harbored correct integration of VP6 gene was obtained using Cas9 protein group and was used to produce an experimental milk-based rotavirus vaccine. Our research provides a novel strategy to produce rotavirus subunit vaccine and make a foundation for building broader milk-based vaccine protection against other pathogens.},
}
@article {pmid31367845,
year = {2019},
author = {Pei, Y and Lu, M},
title = {Programmable RNA manipulation in living cells.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {24},
pages = {4861-4867},
pmid = {31367845},
issn = {1420-9071},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Targeting/trends ; Humans ; RNA/*genetics ; RNA, Guide/*genetics ; RNA-Binding Proteins/*genetics ; },
abstract = {RNAs are responsible for mediating genetic information flow within the cell. RNA splicing, modification, trafficking, translation, and stability are all controlled at the transcript level. However, biological tools to study and manipulate them in a programmable fashion are currently limited. In this review, we summarize recent advances regarding available RNA-targeting systems discovered so far, including CRISPR-based technologies-Cas9 and Cas13, and programmable RNA-binding proteins-PUF and PPR. These tools allow transcript-specific manipulation in gene expression.},
}
@article {pmid31366989,
year = {2019},
author = {Li, F and Lu, Z and Wu, W and Qian, N and Wang, F and Chen, T},
title = {Optogenetic gene editing in regional skin.},
journal = {Cell research},
volume = {29},
number = {10},
pages = {862-865},
pmid = {31366989},
issn = {1748-7838},
mesh = {Animals ; Arabidopsis/metabolism ; Arabidopsis Proteins/genetics/metabolism ; Bacterial Proteins/genetics ; Basic Helix-Loop-Helix Transcription Factors/metabolism ; CRISPR-Cas Systems/genetics ; Carrier Proteins/genetics ; Cryptochromes/genetics/metabolism ; Doxycycline/pharmacology ; Gene Editing/*methods ; Gene Expression/drug effects ; Histones/genetics/metabolism ; Light ; Mice ; Skin/*metabolism/pathology ; },
}
@article {pmid31366580,
year = {2019},
author = {Humbert, O and Radtke, S and Samuelson, C and Carrillo, RR and Perez, AM and Reddy, SS and Lux, C and Pattabhi, S and Schefter, LE and Negre, O and Lee, CM and Bao, G and Adair, JE and Peterson, CW and Rawlings, DJ and Scharenberg, AM and Kiem, HP},
title = {Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.},
journal = {Science translational medicine},
volume = {11},
number = {503},
pages = {},
pmid = {31366580},
issn = {1946-6242},
support = {R01 HL136135/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Antigens, CD34/metabolism ; CRISPR-Cas Systems/*genetics ; Fetal Hemoglobin/genetics/*metabolism ; Gene Editing ; Genotype ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*cytology ; Humans ; Macaca mulatta ; Primates ; Thy-1 Antigens/metabolism ; },
abstract = {Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34[+]CD90[+]CD45RA[-], allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.},
}
@article {pmid31366028,
year = {2019},
author = {Hajiahmadi, Z and Movahedi, A and Wei, H and Li, D and Orooji, Y and Ruan, H and Zhuge, Q},
title = {Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants.},
journal = {International journal of molecular sciences},
volume = {20},
number = {15},
pages = {},
pmid = {31366028},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods/standards ; Magnoliopsida/genetics ; Plant Breeding/*methods ; RNA, Guide/genetics ; Targeted Gene Repair/*methods/standards ; },
abstract = {The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.},
}
@article {pmid31365872,
year = {2019},
author = {Jiang, C and Trudeau, SJ and Cheong, TC and Guo, R and Teng, M and Wang, LW and Wang, Z and Pighi, C and Gautier-Courteille, C and Ma, Y and Jiang, S and Wang, C and Zhao, B and Paillard, L and Doench, JG and Chiarle, R and Gewurz, BE},
title = {CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation.},
journal = {Cell reports},
volume = {28},
number = {5},
pages = {1307-1322.e8},
pmid = {31365872},
issn = {2211-1247},
support = {R01 AI123420/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA076292/CA/NCI NIH HHS/United States ; R01 CA196703/CA/NCI NIH HHS/United States ; R01 AI137337/AI/NIAID NIH HHS/United States ; },
mesh = {Alcohol Oxidoreductases/genetics/metabolism ; B-Lymphocytes/cytology/*metabolism ; CD40 Antigens/*biosynthesis/genetics ; CELF1 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; DNA-Binding Proteins/genetics/metabolism ; Dual-Specificity Phosphatases/genetics/metabolism ; F-Box Proteins/genetics/metabolism ; Humans ; *MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase Phosphatases/genetics/metabolism ; Protein-Arginine N-Methyltransferases/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-6/genetics/metabolism ; },
abstract = {CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets.},
}
@article {pmid31365173,
year = {2020},
author = {Mishra, R and Joshi, RK and Zhao, K},
title = {Base editing in crops: current advances, limitations and future implications.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {20-31},
pmid = {31365173},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; },
abstract = {Targeted mutagenesis via genome-editing technologies holds great promise in developing improved crop varieties to meet future demands. Point mutations or single nucleotide polymorphisms often determine important agronomic traits of crops. Genome-editing-based single-base changes could generate elite trait variants in crop plants which help in accelerating crop improvement. Among the genome-editing technologies, base editing has emerged as a novel and efficient genome-editing approach which enables direct and irreversible conversion of one target base into another in a programmable manner. A base editor is a fusion of catalytically inactive CRISPR-Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations in the target region enabling precise editing of genomes. In the present review, we have summarized the development of different base-editing platforms. Then, we have focussed on the current advances and the potential applications of this precise technology in crop improvement. The review also sheds light on the limitations associated with this technology. Finally, the future perspectives of this emerging technology towards crop improvement have been highlighted.},
}
@article {pmid31365085,
year = {2019},
author = {Vergnes, B and Gazanion, E and Mariac, C and Du Manoir, M and Sollelis, L and Lopez-Rubio, JJ and Sterkers, Y and Bañuls, AL},
title = {A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {74},
number = {11},
pages = {3231-3239},
doi = {10.1093/jac/dkz334},
pmid = {31365085},
issn = {1460-2091},
mesh = {*Amino Acid Substitution ; Antimony/*pharmacology ; Antiprotozoal Agents/*pharmacology ; CRISPR-Cas Systems ; Calcium/metabolism ; Drug Resistance/genetics ; Gene Editing ; Leishmania/*drug effects/enzymology/genetics ; Mutation ; Nicotinamidase/*genetics ; Parasitic Sensitivity Tests ; Protozoan Proteins/*genetics ; },
abstract = {BACKGROUND: For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms.<br><br>OBJECTIVES: We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo. Here, we found that parasites lacking the pnc1 gene (&#x394;pnc1) are hypersusceptible to the active form of antimony (SbIII) and used these mutant parasites to better understand antimony's mode of action and the mechanisms leading to resistance.<br><br>METHODS: SbIII-resistant WT and &#x394;pnc1 parasites were selected in vitro by a stepwise selection method. NAD(H)/NADP(H) dosages and quantitative RT-PCR experiments were performed to explain the susceptibility differences observed between strains. WGS and a marker-free CRISPR/Cas9 base-editing approach were used to identify and validate the role of a new resistance mutation.<br><br>RESULTS: NAD+-depleted &#x394;pnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. &#x394;pnc1 parasites could become resistant to SbIII by an unknown mechanism. WGS revealed a unique amino acid substitution (H451Y) in an EF-hand domain of an orphan calcium-dependent kinase, recently named SCAMK. When introduced into a WT reference strain by base editing, the H451Y mutation allowed Leishmania parasites to survive at extreme concentrations of SbIII, potentiating the rapid emergence of resistant parasites.<br><br>CONCLUSIONS: These results establish that Leishmania SCAMK is a new central hub of antimony's mode of action and resistance development, and uncover the importance of drug tolerance mutations in the evolution of parasite drug resistance.},
}
@article {pmid31365053,
year = {2019},
author = {Lee, MCS and Lindner, SE and Lopez-Rubio, JJ and Llinás, M},
title = {Cutting back malaria: CRISPR/Cas9 genome editing of Plasmodium.},
journal = {Briefings in functional genomics},
volume = {18},
number = {5},
pages = {281-289},
pmid = {31365053},
issn = {2041-2657},
support = {/WT_/Wellcome Trust/United Kingdom ; R01 AI125565/AI/NIAID NIH HHS/United States ; R21 AI130692/AI/NIAID NIH HHS/United States ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Expression Regulation ; Plasmodium/*genetics ; RNA, Guide/chemistry/genetics ; },
abstract = {CRISPR/Cas9 approaches are revolutionizing our ability to perform functional genomics across a wide range of organisms, including the Plasmodium parasites that cause malaria. The ability to deliver single point mutations, epitope tags and gene deletions at increased speed and scale is enabling our understanding of the biology of these complex parasites, and pointing to potential new therapeutic targets. In this review, we describe some of the biological and technical considerations for designing CRISPR-based experiments, and discuss potential future developments that broaden the applications for CRISPR/Cas9 interrogation of the malaria parasite genome.},
}
@article {pmid31364194,
year = {2019},
author = {Pickerill, ES and Kurtz, RP and Tharp, A and Guerrero Sanz, P and Begum, M and Bernstein, DA},
title = {Pseudouridine synthase 7 impacts Candida albicans rRNA processing and morphological plasticity.},
journal = {Yeast (Chichester, England)},
volume = {36},
number = {11},
pages = {669-677},
pmid = {31364194},
issn = {1097-0061},
support = {R15 AI130950/AI/NIAID NIH HHS/United States ; 1R15AI130950-01/NH/NIH HHS/United States ; },
mesh = {Animals ; Antifungal Agents/pharmacology ; CRISPR-Cas Systems ; Candida albicans/drug effects/enzymology/*genetics ; Candidiasis/microbiology ; Gene Deletion ; Gene Editing ; Humans ; Hydrophobic and Hydrophilic Interactions ; Intramolecular Transferases/genetics/*metabolism ; Larva/microbiology ; Moths/microbiology ; RNA Processing, Post-Transcriptional ; RNA, Fungal/*genetics ; RNA, Ribosomal/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Substrate Specificity ; },
abstract = {RNA can be modified in over 100 distinct ways, and these modifications are critical for function. Pseudouridine synthases catalyse pseudouridylation, one of the most prevalent RNA modifications. Pseudouridine synthase 7 modifies a variety of substrates in Saccharomyces cerevisiae including tRNA, rRNA, snRNA, and mRNA, but the substrates for other budding yeast Pus7 homologues are not known. We used CRISPR-mediated genome editing to disrupt Candida albicans PUS7 and find absence leads to defects in rRNA processing and a decrease in cell surface hydrophobicity. Furthermore, C. albicans Pus7 absence causes temperature sensitivity, defects in filamentation, altered sensitivity to antifungal drugs, and decreased virulence in a wax moth model. In addition, we find C. albicans Pus7 modifies tRNA residues, but does not modify a number of other S. cerevisiae Pus7 substrates. Our data suggests C. albicans Pus7 is important for fungal vigour and may play distinct biological roles than those ascribed to S. cerevisiae Pus7.},
}
@article {pmid31363223,
year = {2019},
author = {Rusk, N},
title = {Human CRISPR.},
journal = {Nature methods},
volume = {16},
number = {8},
pages = {677},
doi = {10.1038/s41592-019-0526-4},
pmid = {31363223},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *RNA ; },
}
@article {pmid31363192,
year = {2019},
author = {Ledford, H},
title = {CRISPR conundrum: Strict European court ruling leaves food-testing labs without a plan.},
journal = {Nature},
volume = {572},
number = {7767},
pages = {15},
doi = {10.1038/d41586-019-02162-x},
pmid = {31363192},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; DNA Mutational Analysis ; Europe ; European Union ; *Food Analysis ; *Food, Genetically Modified ; Gene Editing/*legislation &amp; jurisprudence ; Mutagenesis/*genetics ; Mutation ; *Uncertainty ; },
}
@article {pmid31363095,
year = {2019},
author = {Lang, JF and Toulmin, SA and Brida, KL and Eisenlohr, LC and Davidson, BL},
title = {Standard screening methods underreport AAV-mediated transduction and gene editing.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3415},
pmid = {31363095},
issn = {2041-1723},
support = {R01 NS076631/NS/NINDS NIH HHS/United States ; R21 HD050538/HD/NICHD NIH HHS/United States ; T32 GM007170/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Dependovirus/*genetics ; Gene Editing/*methods ; Genes, Reporter ; Genetic Vectors/*genetics ; Kidney/virology ; Mice ; Mice, Inbred C57BL ; Spleen/virology ; *Transduction, Genetic ; },
abstract = {Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.},
}
@article {pmid31362937,
year = {2019},
author = {Blanchard, Z and Vahrenkamp, JM and Berrett, KC and Arnesen, S and Gertz, J},
title = {Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer.},
journal = {Genome research},
volume = {29},
number = {9},
pages = {1429-1441},
pmid = {31362937},
issn = {1549-5469},
support = {P30 CA042014/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Endometrial Neoplasms/*genetics/metabolism ; Estrogen Receptor alpha/*genetics/metabolism ; Estrogens/metabolism ; Female ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Humans ; *Mutation ; },
abstract = {Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy-resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.},
}
@article {pmid31362039,
year = {2020},
author = {Diallo, M and Hocq, R and Collas, F and Chartier, G and Wasels, F and Wijaya, HS and Werten, MWT and Wolbert, EJH and Kengen, SWM and van der Oost, J and Ferreira, NL and López-Contreras, AM},
title = {Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {51-60},
doi = {10.1016/j.ymeth.2019.07.022},
pmid = {31362039},
issn = {1095-9130},
mesh = {2-Propanol/metabolism ; Butanols/metabolism ; CRISPR-Cas Systems/*genetics ; Cellulase/genetics/metabolism ; Cellulose/metabolism ; Clostridium beijerinckii/*genetics/metabolism ; Ethanol/metabolism ; Fungal Proteins/genetics/metabolism ; Gene Editing/methods ; Genome, Bacterial/genetics ; Industrial Microbiology/methods ; Metabolic Engineering/*methods ; Mutation ; Plasmids/*genetics ; Spores, Bacterial/genetics/growth &amp; development ; Transformation, Bacterial ; },
abstract = {Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.},
}
@article {pmid31362003,
year = {2019},
author = {Karl, M and Sommer, C and Gabriel, CH and Hecklau, K and Venzke, M and Hennig, AF and Radbruch, A and Selbach, M and Baumgrass, R},
title = {Recruitment of Histone Methyltransferase Ehmt1 to Foxp3 TSDR Counteracts Differentiation of Induced Regulatory T Cells.},
journal = {Journal of molecular biology},
volume = {431},
number = {19},
pages = {3606-3625},
doi = {10.1016/j.jmb.2019.07.031},
pmid = {31362003},
issn = {1089-8638},
mesh = {Animals ; Ascorbic Acid/pharmacology ; Base Sequence ; Cell Differentiation/drug effects/genetics ; DNA Methylation/drug effects/genetics ; Demethylation ; Epigenesis, Genetic/drug effects ; Forkhead Transcription Factors/*metabolism ; Histone-Lysine N-Methyltransferase/*metabolism ; Histones/metabolism ; Kruppel-Like Transcription Factors/metabolism ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Genetic ; Nerve Tissue Proteins/metabolism ; T-Lymphocytes, Regulatory/drug effects/*metabolism ; },
abstract = {Differentiation toward CD4[+] regulatory T (Treg) cells is essentially dependent on an epigenetic program at Treg signature genes, which involves remodeling of the Treg-specific demethylated regions (TSDRs). In particular, the epigenetic status of the conserved non-coding sequence 2 of Foxp3 (Foxp3 TSDR) determines expression stability of the master transcription factor and thus Treg lineage identity. However, the molecular mechanisms controlling the epigenetic remodeling at TSDRs in Treg and conventional T cells are largely unknown. Using a combined approach of DNA pull-down and mass spectrometric analysis, we report a novel regulatory mechanism in which transcription factor Wiz recruits the histone methyltransferase Ehmt1 to Foxp3 TSDR. We show that both Wiz and Ehmt1 are crucial for shaping the region with the repressive histone modification H3K9me2 in conventional T cells. Consistently, knocking out either Ehmt1 or Wiz by CRISPR/Cas resulted in the loss of H3K9me2 and enhanced Foxp3 expression during iTreg differentiation. Moreover, the essential role of the Wiz-Ehmt1 interaction as observed at several TSDRs indicates a global function of Ehmt1 in the Treg differentiation program.},
}
@article {pmid31360116,
year = {2019},
author = {Li, X and Hao, F and Hu, X and Wang, H and Dai, B and Wang, X and Liang, H and Cang, M and Liu, D},
title = {Generation of Tβ4 knock-in Cashmere goat using CRISPR/Cas9.},
journal = {International journal of biological sciences},
volume = {15},
number = {8},
pages = {1743-1754},
pmid = {31360116},
issn = {1449-2288},
mesh = {Animals ; CRISPR-Cas Systems ; Goats ; Hair Follicle/metabolism ; Receptors, CCR5/genetics/metabolism ; Thymosin/genetics/*metabolism ; },
abstract = {The cashmere goat breed is known to provide excellent quality cashmere. Here, we attempted to breed high-yielding cashmere goats by specifically inserting the Tβ4 gene into the goat CCR5 locus and provided an animal model for future research. We successfully obtained Tβ4 knock-in goat without any screening and fluorescent markers using CRISPR/Cas9 technology. A series of experiments were performed to examine physical conditions and characteristics of the Tβ4 knock-in goat. The goat exhibited an increase in cashmere yield by 74.5% without affecting the fineness and quality. Additionally, RNA-seq analysis indicated that Tβ4 may promote hair growth by affecting processes such as vasoconstriction, angiogenesis, and vascular permeability around secondary hair follicles. Together, our study can significantly improve the breeding of cashmere goat and thereby increase economic efficiency.},
}
@article {pmid31359433,
year = {2019},
author = {Liu, D and Awazu, A and Sakuma, T and Yamamoto, T and Sakamoto, N},
title = {Establishment of knockout adult sea urchins by using a CRISPR-Cas9 system.},
journal = {Development, growth &amp; differentiation},
volume = {61},
number = {6},
pages = {378-388},
doi = {10.1111/dgd.12624},
pmid = {31359433},
issn = {1440-169X},
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Sea Urchins/*embryology/*genetics ; },
abstract = {Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.},
}
@article {pmid31359007,
year = {2019},
author = {Leenay, RT and Aghazadeh, A and Hiatt, J and Tse, D and Roth, TL and Apathy, R and Shifrut, E and Hultquist, JF and Krogan, N and Wu, Z and Cirolia, G and Canaj, H and Leonetti, MD and Marson, A and May, AP and Zou, J},
title = {Large dataset enables prediction of repair after CRISPR-Cas9 editing in primary T cells.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1034-1037},
pmid = {31359007},
issn = {1546-1696},
support = {P30 AG059307/AG/NIA NIH HHS/United States ; U19 AI135990/AI/NIAID NIH HHS/United States ; K22 AI136691/AI/NIAID NIH HHS/United States ; R01 HG008164/HG/NHGRI NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; P50 AI150476/AI/NIAID NIH HHS/United States ; DP2 DA042423/DA/NIDA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Gene Expression Regulation ; Genome ; Genomics ; Humans ; Induced Pluripotent Stem Cells/physiology ; RNA, Guide/*genetics ; T-Lymphocytes/*physiology ; },
abstract = {Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.},
}
@article {pmid31358636,
year = {2019},
author = {Davis, TH},
title = {Profile of Rodolphe Barrangou.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {32},
pages = {15754-15756},
pmid = {31358636},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems/genetics ; Food Technology/*history ; History, 20th Century ; History, 21st Century ; },
}
@article {pmid31358538,
year = {2019},
author = {Zhang, Y and Zhou, L and Bandyopadhyay, D and Sharma, K and Allen, AJ and Kmieciak, M and Grant, S},
title = {The Covalent CDK7 Inhibitor THZ1 Potently Induces Apoptosis in Multiple Myeloma Cells In Vitro and In Vivo.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {25},
number = {20},
pages = {6195-6205},
pmid = {31358538},
issn = {1557-3265},
support = {P30 CA016059/CA/NCI NIH HHS/United States ; R01 CA205607/CA/NCI NIH HHS/United States ; UH2 TR001373/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology/therapeutic use ; Apoptosis/*drug effects/genetics ; Bortezomib/pharmacology/therapeutic use ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology/therapeutic use ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics ; Cyclin-Dependent Kinases/*antagonists &amp; inhibitors/genetics ; Down-Regulation/drug effects ; Drug Synergism ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Inhibitory Concentration 50 ; Mice ; Multiple Myeloma/*drug therapy/pathology ; Myeloid Cell Leukemia Sequence 1 Protein/genetics ; Oligopeptides/pharmacology/therapeutic use ; Phenylenediamines/*pharmacology/therapeutic use ; Proto-Oncogene Proteins c-myc/genetics ; Pyrimidines/*pharmacology/therapeutic use ; Sulfonamides/pharmacology/therapeutic use ; Transcription, Genetic/drug effects ; Xenograft Model Antitumor Assays ; bcl-X Protein/genetics ; },
abstract = {PURPOSE: The goal of this study was to characterize the activity of the covalent CDK7 inhibitor THZ1 in multiple myeloma models.<br><br>EXPERIMENTAL DESIGN: Multiple myeloma lines were exposed to varying THZ1 concentrations alone or with carfilzomib or ABT-199, after which apoptosis was monitored by flow cytometry, protein expression by Western blot analysis, mRNA by RT-PCR. Analogous studies were performed in cells ectopically expressing c-MYC, MCL-1, or BCL-XL, or CRISPER-Cas CDK7 sgRNA knockout. Primary multiple myeloma cells were exposed to THZ1 &#xb1; carfilzomib or ABT-199. In vivo effects of THZ1 were examined in a systemic U266 xenograft model.<br><br>RESULTS: THZ1 markedly diminished multiple myeloma cell proliferation and survival despite bortezomib or stromal cell resistance in association with G2-M arrest, inactivation of CTD RNA Pol II, dephosphorylation of CDKs 7 as well as 1, 2, and 9, and MCL-1, BCL-xL, and c-MYC mRNA or protein downregulation. Ectopic MCL-1, c-MYC, or BCL-XL expression significantly protected cells from THZ1 lethality. Both THZ1 and CRISPR-Cas CDK7 knockout sharply diminished multiple myeloma cell proliferation and significantly increased carfilzomib and ABT-199 lethality. Parallel effects and interactions were observed in primary CD138[+] (N = 22) or primitive multiple myeloma cells (CD138[-]/CD19[+]/CD20[+]/CD27[+]; N = 16). THZ1 administration [10 mg/kg i.p. twice daily (BID), 5 days/week] significantly improved survival in a systemic multiple myeloma xenograft model with minimal toxicity and induced similar events observed in vitro, for example, MCL-1 and c-MYC downregulation.<br><br>CONCLUSIONS: THZ1 potently reduces multiple myeloma cell proliferation through transcriptional downregulation of MCL-1, BCL-XL, and c-MYC in vitro and in vivo. It warrants further attention as a therapeutic agent in multiple myeloma.},
}
@article {pmid31358055,
year = {2019},
author = {Caron, J and Pène, V and Tolosa, L and Villaret, M and Luce, E and Fourrier, A and Heslan, JM and Saheb, S and Bruckert, E and Gómez-Lechón, MJ and Nguyen, TH and Rosenberg, AR and Weber, A and Dubart-Kupperschmitt, A},
title = {Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection.},
journal = {Stem cell research &amp; therapy},
volume = {10},
number = {1},
pages = {221},
pmid = {31358055},
issn = {1757-6512},
mesh = {Antiviral Agents/pharmacology/therapeutic use ; Apolipoprotein A-II/genetics ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Cholesterol/metabolism ; *Gene Editing ; Hepacivirus/drug effects/physiology ; Hepatitis C/drug therapy/*pathology/virology ; Hepatocytes/cytology/metabolism ; Humans ; Hyperlipoproteinemia Type II/metabolism/*pathology ; Induced Pluripotent Stem Cells/cytology ; Phenotype ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; Proprotein Convertase 9/genetics/metabolism ; Receptors, LDL/*genetics/metabolism ; Sofosbuvir/pharmacology/therapeutic use ; Sterol Regulatory Element Binding Protein 2/genetics/metabolism ; },
abstract = {BACKGROUND: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed.<br><br>METHODS: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells.<br><br>RESULTS: We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis.<br><br>CONCLUSIONS: Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.},
}
@article {pmid31357652,
year = {2019},
author = {Raaijmakers, RHL and Ripken, L and Ausems, CRM and Wansink, DG},
title = {CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities.},
journal = {International journal of molecular sciences},
volume = {20},
number = {15},
pages = {},
pmid = {31357652},
issn = {1422-0067},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; *Gene Editing ; Gene Targeting ; Genetic Association Studies ; Genetic Loci ; Genetic Predisposition to Disease ; *Genetic Therapy ; Humans ; Myotonic Dystrophy/*genetics/*therapy ; Trinucleotide Repeat Expansion ; Trinucleotide Repeats ; },
abstract = {CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the DMPK/DM1-AS gene pair, the autosomal dominant mutation that causes DM1. Expansion of the repeat results in a complex constellation of toxicity at the DNA level, an altered transcriptome and a disturbed proteome. To restore cellular homeostasis and ameliorate DM1 disease symptoms, CRISPR/Cas approaches were directed at the causative mutation in the DNA and the RNA. Specifically, the triplet repeat has been excised from the genome by several laboratories via dual CRISPR/Cas9 cleavage, while one group prevented transcription of the (CTG)n repeat through homology-directed insertion of a polyadenylation signal in DMPK. Independently, catalytically deficient Cas9 (dCas9) was recruited to the (CTG)n repeat to block progression of RNA polymerase II and a dCas9-RNase fusion was shown to degrade expanded (CUG)n RNA. We compare these promising developments in DM1 with those in other microsatellite instability diseases. Finally, we look at hurdles that must be taken to make CRISPR/Cas-mediated editing a therapeutic reality in patients.},
}
@article {pmid31357516,
year = {2019},
author = {Gasparis, S and Przyborowski, M and Kała, M and Nadolska-Orczyk, A},
title = {Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology.},
journal = {Cells},
volume = {8},
number = {8},
pages = {},
pmid = {31357516},
issn = {2073-4409},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cytokinins/*metabolism ; *Gene Editing ; Gene Expression Regulation ; Gene Expression Regulation, Plant ; Hordeum/*physiology ; Humans ; Mutation ; Oxidoreductases/*genetics/metabolism ; Oxidoreductases Acting on CH-NH Group Donors/*genetics/metabolism ; Plant Proteins/genetics/metabolism ; Plant Roots/*physiology ; Plants, Genetically Modified ; RNA, Guide ; },
abstract = {Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields.},
}
@article {pmid31356596,
year = {2019},
author = {Niu, Y and Liu, Z and Nian, X and Xu, X and Zhang, Y},
title = {miR-210 controls the evening phase of circadian locomotor rhythms through repression of Fasciclin 2.},
journal = {PLoS genetics},
volume = {15},
number = {7},
pages = {e1007655},
pmid = {31356596},
issn = {1553-7404},
support = {P20 GM103650/GM/NIGMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; Animals ; CRISPR-Cas Systems ; Cell Adhesion Molecules, Neuronal/*genetics ; Circadian Rhythm ; Darkness ; Drosophila melanogaster/genetics/*physiology ; Gene Knockout Techniques ; *Locomotion ; MicroRNAs/*genetics ; Mutation ; },
abstract = {Circadian clocks control the timing of animal behavioral and physiological rhythms. Fruit flies anticipate daily environmental changes and exhibit two peaks of locomotor activity around dawn and dusk. microRNAs are small non-coding RNAs that play important roles in post-transcriptional regulation. Here we identify Drosophila miR-210 as a critical regulator of circadian rhythms. Under light-dark conditions, flies lacking miR-210 (miR-210KO) exhibit a dramatic 2 hrs phase advance of evening anticipatory behavior. However, circadian rhythms and molecular pacemaker function are intact in miR-210KO flies under constant darkness. Furthermore, we identify that miR-210 determines the evening phase of activity through repression of the cell adhesion molecule Fasciclin 2 (Fas2). Ablation of the miR-210 binding site within the 3' UTR of Fas2 (Fas2ΔmiR-210) by CRISPR-Cas9 advances the evening phase as in miR-210KO. Indeed, miR-210 genetically interacts with Fas2. Moreover, Fas2 abundance is significantly increased in the optic lobe of miR-210KO. In addition, overexpression of Fas2 in the miR-210 expressing cells recapitulates the phase advance behavior phenotype of miR-210KO. Together, these results reveal a novel mechanism by which miR-210 regulates circadian locomotor behavior.},
}
@article {pmid31355948,
year = {2019},
author = {Wang, L and Rubio, MC and Xin, X and Zhang, B and Fan, Q and Wang, Q and Ning, G and Becana, M and Duanmu, D},
title = {CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation.},
journal = {The New phytologist},
volume = {224},
number = {2},
pages = {818-832},
doi = {10.1111/nph.16077},
pmid = {31355948},
issn = {1469-8137},
mesh = {*CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation, Enzymologic/genetics/physiology ; Gene Expression Regulation, Plant/genetics/*physiology ; Isoenzymes/genetics/metabolism ; Leghemoglobin/*genetics/metabolism ; Lotus/genetics/*metabolism ; Nitrogen Fixation/genetics/*physiology ; Plant Root Nodulation/genetics/physiology ; Superoxide Dismutase ; },
abstract = {Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA-sequencing (RNA-seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly-β-hydroxybutyrate and disruption of mitochondria. RNA-seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb-deficient nodules had c. 30-50-fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post-translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.},
}
@article {pmid31355781,
year = {2019},
author = {Lou, WP and Wang, L and Long, C and Liu, L and Fei, JF},
title = {Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {149},
pages = {},
doi = {10.3791/59850},
pmid = {31355781},
issn = {1940-087X},
mesh = {Ambystoma mexicanum ; Animals ; CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems/genetics ; *Electroporation ; Gene Knockout Techniques/*methods ; Neural Stem Cells/*metabolism ; RNA, Guide/*genetics/metabolism ; Regeneration/genetics ; Spinal Cord/cytology/physiology ; },
abstract = {The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.},
}
@article {pmid31354748,
year = {2019},
author = {Kaul, T and Raman, NM and Eswaran, M and Thangaraj, A and Verma, R and Sony, SK and Sathelly, KM and Kaul, R and Yadava, P and Agrawal, PK},
title = {Data Mining by Pluralistic Approach on CRISPR Gene Editing in Plants.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {801},
pmid = {31354748},
issn = {1664-462X},
abstract = {Genome engineering by site-specific nucleases enables reverse genetics and targeted editing of genomes in an efficacious manner. Contemporary revolutionized progress in targeted-genome engineering technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-related RNA-guided endonucleases facilitate coherent interrogation of crop genome function. Evolved as an innate component of the adaptive immune response in bacterial and archaeal systems, CRISPR/Cas system is now identified as a versatile molecular tool that ensures specific and targeted genome modification in plants. Applications of this genome redaction tool-kit include somatic genome editing, rectification of genetic disorders or gene therapy, treatment of infectious diseases, generation of animal models, and crop improvement. We review the utilization of these synthetic nucleases as precision, targeted-genome editing platforms with the inherent potential to accentuate basic science "strengths and shortcomings" of gene function, complement plant breeding techniques for crop improvement, and charter a knowledge base for effective use of editing technology for ever-increasing agricultural demands. Furthermore, the emerging importance of Cpf1, Cas9 nickase, C2c2, as well as other innovative candidates that may prove more effective in driving novel applications in crops are also discussed. The mined data has been prepared as a library and opened for public use at www.lipre.org.},
}
@article {pmid31354680,
year = {2019},
author = {Jamrozy, D and Misra, R and Xu, Z and Ter-Stepanyan, MM and Kocharyan, KS and Cave, R and Hambardzumyan, AD and Mkrtchyan, HV},
title = {Novel Methicillin-Resistant Staphylococcus aureus CC8 Clone Identified in a Hospital Setting in Armenia.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1592},
pmid = {31354680},
issn = {1664-302X},
abstract = {Whole-genome sequencing (WGS) of methicillin-resistant Staphylococcus aureus (MRSA) has been sparse in low- and middle-income countries, therefore, its population structure is unknown for many regions. We conducted a pilot surveillance of MRSA in the maternity ward of a teaching hospital in Armenia, to characterize the genotypes of circulating MRSA clones. In total, 10 MRSA isolates from a hospital environment (n = 4) and patients (n = 6) were recovered between March and May 2015 and April and May 2016, respectively. WGS analysis showed that the isolates belonged to two clonal complexes (CCs): CC8 (n = 8) and CC30 (n = 2). MRSA CC30 isolates carried staphylococcal cassette chromosome mec (SCCmec) type IVa, whereas MRSA CC8 revealed a type-VT-related SCCmec, which contained a CRISPR/Cas array and showed a high similarity to SCCmec found in coagulase-negative staphylococci. All but one MRSA CC8 isolates carried a plasmid identical to the pSK67 and four also carried a pathogenicity island similar to SaPI5. Phylogenetic analysis showed that the MRSA CC8 isolates formed a monophyletic cluster, which emerged around 1995 and was distinct from representatives of globally-distributed MRSA CC8 lineages. WGS characterization of MRSA in countries with no previous S. aureus genomic surveillance can therefore reveal an unrecognized diversity of MRSA lineages.},
}
@article {pmid31354265,
year = {2019},
author = {Hosseini, ES and Nikkhah, M and Hosseinkhani, S},
title = {Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9.},
journal = {International journal of nanomedicine},
volume = {14},
number = {},
pages = {4353-4366},
pmid = {31354265},
issn = {1178-2013},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cations/chemistry ; Cell Death ; Cholesterol/analogs &amp; derivatives/*chemistry ; Electrophoretic Mobility Shift Assay ; Endosomes/metabolism ; Fatty Acids, Monounsaturated/chemistry ; *Gene Editing ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; Liposomes/chemistry ; Nanoparticles/*chemistry ; Particle Size ; Phosphatidylethanolamines/chemistry ; Plasmids/metabolism ; Polyethylene Glycols/chemistry ; Quaternary Ammonium Compounds/chemistry ; RNA, Guide/metabolism ; Static Electricity ; Transfection/*methods ; },
abstract = {Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.},
}
@article {pmid31352406,
year = {2019},
author = {de San Vicente, KM and Schröder, MS and Lombardi, L and Iracane, E and Butler, G},
title = {Correlating Genotype and Phenotype in the Asexual Yeast Candida orthopsilosis Implicates ZCF29 in Sensitivity to Caffeine.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {9},
pages = {3035-3043},
pmid = {31352406},
issn = {2160-1836},
support = {/WT_/Wellcome Trust/United Kingdom ; 097419/Z/11/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Antifungal Agents/pharmacology ; CRISPR-Cas Systems ; Caffeine/*pharmacology ; Candida parapsilosis/*drug effects/genetics/pathogenicity/*physiology ; Codon, Terminator ; Fungal Proteins/*genetics ; Genotype ; Ketoconazole/pharmacology ; Lepidoptera/microbiology ; Microbial Sensitivity Tests ; Microorganisms, Genetically-Modified ; Nystatin/pharmacology ; Phenotype ; Polymorphism, Single Nucleotide ; Virulence/genetics ; },
abstract = {Candida orthopsilosis is diploid asexual yeast that causes human disease. Most C. orthopsilosis isolates arose from at least four separate hybridizations between related, but not identical, parents. Here, we used population genomics data to correlate genotypic and phenotypic variation in 28 C. orthopsilosis isolates. We used cosine similarity scores to identify 65 variants with potential high-impact (deleterious effects) that correlated with specific phenotypes. Of these, 19 were Single Nucleotide Polymorphisms (SNPs) that changed stop or start codons, or splice sites. One variant resulted in a premature stop codon in both alleles of the gene ZCF29 in C. orthopsilosis isolate 185, which correlated with sensitivity to nystatin and caffeine. We used CRISPR-Cas9 editing to introduce this polymorphism into two resistant C. orthopsilosis isolates. Introducing the stop codon resulted in sensitivity to caffeine and to ketoconazole, but not to nystatin. Our analysis shows that it is possible to associate genomic variants with phenotype in asexual Candida species, but that only a small amount of genomic variation can be easily explored.},
}
@article {pmid31352229,
year = {2019},
author = {Cai, C and Wang, X and Zhao, Y and Yi, C and Jin, Z and Zhang, A and Han, L},
title = {Construction of a mavs-inactivated MDCK cell line for facilitating the propagation of canine distemper virus (CDV).},
journal = {Molecular immunology},
volume = {114},
number = {},
pages = {133-138},
doi = {10.1016/j.molimm.2019.06.013},
pmid = {31352229},
issn = {1872-9142},
mesh = {Animals ; Antigens, CD/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Chlorocebus aethiops ; Distemper/genetics ; Distemper Virus, Canine/*genetics ; Dogs ; Humans ; Madin Darby Canine Kidney Cells ; Vero Cells ; },
abstract = {Canine distemper is a highly contagious disease of wild and domestic carnivores. Obtaining of a suitable cell line for canine distemper virus (CDV) propagation is very important for field CDV isolation and vaccine antigen preparation. However, the cell line currently developed cell lines for CDV propagation are a marmoset lymphoid cell line (B95a), which could cause the virus to potentially infect human cells, and canine SLAM-expressing Vero cells, which may cause the virus to lose virulence. Therefore, a canine cell line constructed for efficient CDV propagation would be attractive. In the present study, a Madin-Darby Canine Kidney Epithelial (MDCK) cell line with mavs (mitochondrial antiviral signaling) inactivation was constructed by CRISPR/Cas9 technology. The interferon-I response induced by poly(I:C), an analogue of viral RNA, was significantly blocked in the constructed cell line, designated MDCK-KOmavs. Moreover, the propagation of a filed CDV strain was approximately 100 times higher in MDCK-KOmavs cells than in wild-type MDCK cells. Therefore, in the present study, a canine cell line facilitating CDV propagation was successfully constructed, and the results suggested that the constructed canine cell line was more efficient than the wild-type cell line for the isolation of field CDVs. In addition, the rapid propagation of CDVs to high titers in the constructed MDCK-KOmavs cell line indicated that this cell line could also be an alternative cell line for the preparation of vaccine antigens.},
}
@article {pmid31350845,
year = {2019},
author = {Yan, W and Clarke, H},
title = {New horizons in reproductive biology: a special issue.},
journal = {Biology of reproduction},
volume = {101},
number = {3},
pages = {513},
doi = {10.1093/biolre/ioz138},
pmid = {31350845},
issn = {1529-7268},
support = {P30 GM110767/GM/NIGMS NIH HHS/United States ; P50 HD098593/HD/NICHD NIH HHS/United States ; R01 HD085506/HD/NICHD NIH HHS/United States ; R01 HD099924/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Biology/*trends ; CRISPR-Cas Systems ; Female ; Gene Editing/methods/trends ; History, 20th Century ; History, 21st Century ; Humans ; Infertility/diagnosis/genetics/therapy ; Inventions/*trends ; Male ; RNA-Seq ; Reproduction/*physiology ; },
}
@article {pmid31350780,
year = {2019},
author = {Pu, X and Liu, L and Li, P and Huo, H and Dong, X and Xie, K and Yang, H and Liu, L},
title = {A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.},
journal = {The Plant journal : for cell and molecular biology},
volume = {100},
number = {4},
pages = {863-872},
doi = {10.1111/tpj.14478},
pmid = {31350780},
issn = {1365-313X},
mesh = {Bryopsida/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant ; Mutation Rate ; },
abstract = {Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co-delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss-of-function mutants of multiple genes from different gene families.},
}
@article {pmid31350363,
year = {2019},
author = {Wu, G and Zhao, Y and Shen, R and Wang, B and Xie, Y and Ma, X and Zheng, Z and Wang, H},
title = {Characterization of Maize Phytochrome-Interacting Factors in Light Signaling and Photomorphogenesis.},
journal = {Plant physiology},
volume = {181},
number = {2},
pages = {789-803},
pmid = {31350363},
issn = {1532-2548},
mesh = {Arabidopsis ; CRISPR-Cas Systems ; Light ; Phenotype ; Phytochrome B/*metabolism ; Zea mays/genetics/*metabolism ; },
abstract = {Increasing planting density has been an effective means of increasing maize (Zea mays ssp. mays) yield per unit of land area over the past few decades. However, high-density planting will cause a reduction in the ratio of red to far-red incident light, which could trigger the shade avoidance syndrome and reduce yield. The molecular mechanisms regulating the shade avoidance syndrome are well established in Arabidopsis (Arabidopsis thaliana) but poorly understood in maize. Here, we conducted an initial functional characterization of the maize Phytochrome-Interacting Factor (PIF) gene family in regulating light signaling and photomorphogenesis. The maize genome contains seven distinct PIF genes, which could be grouped into three subfamilies: ZmPIF3s, ZmPIF4s, and ZmPIF5s Similar to the Arabidopsis PIFs, all ZmPIF proteins are exclusively localized to the nucleus and most of them can form nuclear bodies upon light irradiation. We show that all of the ZmPIF proteins could interact with ZmphyB. Heterologous expression of each ZmPIF member could partially or fully rescue the phenotype of the Arabidopsis pifq mutant, and some of these proteins conferred enhanced shade avoidance syndrome in Arabidopsis. Interestingly, all ZmPIF proteins expressed in Arabidopsis are much more stable than their Arabidopsis counterparts upon exposure to red light. Moreover, the Zmpif3, Zmpif4, and Zmpif5 knockout mutants generated via CRISPR/Cas9 technology all showed severely suppressed mesocotyl elongation in dark-grown seedlings and were less responsive to simulated shade treatment. Taken together, our results reveal both conserved and distinct molecular properties of ZmPIFs in regulating light signaling and photomorphogenesis in maize.},
}
@article {pmid31350328,
year = {2019},
author = {Hollstein, PE and Eichner, LJ and Brun, SN and Kamireddy, A and Svensson, RU and Vera, LI and Ross, DS and Rymoff, TJ and Hutchins, A and Galvez, HM and Williams, AE and Shokhirev, MN and Screaton, RA and Berdeaux, R and Shaw, RJ},
title = {The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive Effects of LKB1 in NSCLC.},
journal = {Cancer discovery},
volume = {9},
number = {11},
pages = {1606-1627},
pmid = {31350328},
issn = {2159-8290},
support = {R01 AR059847/AR/NIAMS NIH HHS/United States ; P01 CA120964/CA/NCI NIH HHS/United States ; T32 CA009370/CA/NCI NIH HHS/United States ; R35 CA220538/CA/NCI NIH HHS/United States ; R01 DK092590/DK/NIDDK NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; F32 CA206400/CA/NCI NIH HHS/United States ; T32 DK007541/DK/NIDDK NIH HHS/United States ; },
mesh = {A549 Cells ; AMP-Activated Protein Kinase Kinases ; AMP-Activated Protein Kinases ; Animals ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/genetics/metabolism/*pathology ; Cell Line, Tumor ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/genetics/metabolism/*pathology ; Mice ; Neoplasm Transplantation ; Protein Kinases/genetics ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Proto-Oncogene Proteins p21(ras)/genetics ; Signal Transduction ; Tumor Burden ; },
abstract = {Mutations in the LKB1 (also known as STK11) tumor suppressor are the third most frequent genetic alteration in non-small cell lung cancer (NSCLC). LKB1 encodes a serine/threonine kinase that directly phosphorylates and activates 14 AMPK family kinases ("AMPKRs"). The function of many of the AMPKRs remains obscure, and which are most critical to the tumor-suppressive function of LKB1 remains unknown. Here, we combine CRISPR and genetic analysis of the AMPKR family in NSCLC cell lines and mouse models, revealing a surprising critical role for the SIK subfamily. Conditional genetic loss of Sik1 revealed increased tumor growth in mouse models of Kras-dependent lung cancer, which was further enhanced by loss of the related kinase Sik3. As most known substrates of the SIKs control transcription, gene-expression analysis was performed, revealing upregulation of AP1 and IL6 signaling in common between LKB1- and SIK1/3-deficient tumors. The SIK substrate CRTC2 was required for this effect, as well as for proliferation benefits from SIK loss. SIGNIFICANCE: The tumor suppressor LKB1/STK11 encodes a serine/threonine kinase frequently inactivated in NSCLC. LKB1 activates 14 downstream kinases in the AMPK family controlling growth and metabolism, although which kinases are critical for LKB1 tumor-suppressor function has remained an enigma. Here we unexpectedly found that two understudied kinases, SIK1 and SIK3, are critical targets in lung cancer.This article is highlighted in the In This Issue feature, p. 1469.},
}
@article {pmid31349852,
year = {2019},
author = {Liu, G and Yin, K and Zhang, Q and Gao, C and Qiu, JL},
title = {Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhances Cas9 editing efficiency in vivo.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {145},
pmid = {31349852},
issn = {1474-760X},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Chromatin/*chemistry ; *Gene Editing ; Oryza/genetics ; RNA/genetics ; Trans-Activators/genetics ; *Transcriptional Activation ; },
abstract = {The CRISPR/Cas9 system is unable to edit all targetable genomic sites with full efficiency in vivo. We show that Cas9-mediated editing is more efficient in open chromatin regions than in closed chromatin regions in rice. A construct (Cas9-TV) formed by fusing a synthetic transcription activation domain to Cas9 edits target sites more efficiently, even in closed chromatin regions. Moreover, combining Cas9-TV with a proximally binding dead sgRNA (dsgRNA) further improves editing efficiency up to several folds. The use of Cas9-TV/dsgRNA thus provides a novel strategy for obtaining efficient genome editing in vivo, especially at nuclease-refractory target sites.},
}
@article {pmid31349743,
year = {2019},
author = {Torres-Perez, R and Garcia-Martin, JA and Montoliu, L and Oliveros, JC and Pazos, F},
title = {WeReview: CRISPR Tools-Live Repository of Computational Tools for Assisting CRISPR/Cas Experiments.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {6},
number = {3},
pages = {},
pmid = {31349743},
issn = {2306-5354},
abstract = {Computational tools are essential in the process of designing a CRISPR/Cas experiment for the targeted modification of an organism's genome. Among other functionalities, these tools facilitate the design of a guide-RNA (gRNA) for a given nuclease that maximizes its binding to the intended genomic site, while avoiding binding to undesired sites with similar sequences in the genome of the organism of interest (off-targets). Due to the popularity of this methodology and the rapid pace at which it evolves and changes, new computational tools show up constantly. This rapid turnover, together with the intrinsic high death-rate of bioinformatics tools, mean that many of the published tools become unavailable at some point. Consequently, the traditional ways to inform the community about the landscape of available tools, i.e., reviews in the scientific literature, are not adequate for this fast-moving field. To overcome these limitations, we have developed "WeReview: CRISPR Tools," a live, on-line, user-updatable repository of computational tools to assist researchers in designing CRISPR/Cas experiments. In its web site users can find an updated comprehensive list of tools and search for those fulfilling their specific needs, as well as proposing modifications to the data associated with the tools or the incorporation of new ones.},
}
@article {pmid31349094,
year = {2019},
author = {Lee, J and Le, QV and Yang, G and Oh, YK},
title = {Cas9-edited immune checkpoint blockade PD-1 DNA polyaptamer hydrogel for cancer immunotherapy.},
journal = {Biomaterials},
volume = {218},
number = {},
pages = {119359},
doi = {10.1016/j.biomaterials.2019.119359},
pmid = {31349094},
issn = {1878-5905},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Humans ; Hydrogels/*chemistry ; Immunotherapy/*methods ; Mice ; Mice, Inbred C57BL ; Microscopy, Electron, Scanning ; Programmed Cell Death 1 Receptor/*genetics ; },
abstract = {Immune checkpoint inhibitors have been widely studied in immunotherapy. Although antibodies have been more widely used to block immune checkpoints, DNA aptamers have unique advantages for this purpose. Here, we designed a DNA polyaptamer hydrogel that can be precisely cut by Cas9/sgRNA for programmed release of an immune checkpoint-blocking DNA aptamer. As a representative immune checkpoint inhibitor, we used a PD-1 DNA aptamer. Rolling-circle amplification was used to generate a hydrogel comprising DNA with PD-1 aptamer and an sgRNA-targeting sequence. When mixed with Cas9/sgRNA, the PD-1 DNA aptamer hydrogel (PAH) lost its gel property and liberated the PD-1 aptamer sequence. The precise Cas9/sgRNA-mediated release of the PD-1 DNA aptamer, which was confirmed by gel electrophoresis, was found to effectively activate the cytokine-secretion function of splenocytes. In vivo, molecular imaging revealed that PD-1 DNA polyaptamer hydrogel co-injected with Cas9/sgRNA (Cas9/PAH) remained at the injection site longer than free aptamer and yielded significantly higher antitumor effects and survival than hydrogel or free aptamer. Moreover, increased immune cell filtration was observed at tumor tissues treated with Cas9/PAH. These results suggest that our Cas9/sgRNA-edited immune checkpoint-blocking aptamer hydrogel has strong potential for anticancer immunotherapy.},
}
@article {pmid31348989,
year = {2019},
author = {Qu, Z and Yimer, TA and Xie, S and Wong, F and Yu, S and Liu, X and Han, S and Ma, J and Lu, Z and Hu, X and Qin, Y and Huang, Y and Lv, Y and Li, J and Tang, Z and Liu, F and Liu, M},
title = {Knocking out lca5 in zebrafish causes cone-rod dystrophy due to impaired outer segment protein trafficking.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1865},
number = {10},
pages = {2694-2705},
doi = {10.1016/j.bbadis.2019.07.009},
pmid = {31348989},
issn = {1879-260X},
mesh = {Animals ; CRISPR-Cas Systems ; Cilia/metabolism ; Cone-Rod Dystrophies/*genetics ; Disease Models, Animal ; Eye Proteins/metabolism ; Gene Knockout Techniques ; Genetic Predisposition to Disease/*genetics ; Humans ; Leber Congenital Amaurosis/*genetics/*metabolism/pathology ; Microtubule-Associated Proteins ; Phenotype ; Protein Transport/*physiology ; Retina/metabolism ; Retinal Cone Photoreceptor Cells/pathology ; Retinal Degeneration/genetics/pathology ; Tumor Suppressor Proteins/genetics/metabolism ; Zebrafish/*genetics ; },
abstract = {Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1-2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7 dpf lca5[-/-] zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5[-/-] zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.},
}
@article {pmid31348878,
year = {2019},
author = {Chavez, M and Qi, LS},
title = {Site-Programmable Transposition: Shifting the Paradigm for CRISPR-Cas Systems.},
journal = {Molecular cell},
volume = {75},
number = {2},
pages = {206-208},
doi = {10.1016/j.molcel.2019.07.004},
pmid = {31348878},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; RNA ; Transposases ; },
abstract = {Discoveries by Klompe et al. (2019) and Strecker et al. (2019) elucidate distinct CRISPR-Cas mechanisms for site-specific programmable transposition in prokaryotic organisms.},
}
@article {pmid31348845,
year = {2019},
author = {Nakanishi, T and Maekawa, A and Tabata, H and Yoshioka, T and Pei, Z and Sato, K and Mori, M and Kato, M and Saito, I},
title = {Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.},
journal = {The journal of gene medicine},
volume = {21},
number = {11},
pages = {e3115},
pmid = {31348845},
issn = {1521-2254},
mesh = {Bacteriophage lambda/genetics ; *CRISPR-Cas Systems ; Cosmids/*genetics ; *Gene Amplification ; *Gene Editing ; *Gene Expression ; Gene Order ; Gene Targeting ; Hepatitis B virus/genetics ; Humans ; *RNA, Guide ; Sequence Deletion ; Transfection ; },
abstract = {BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.<br><br>METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection.<br><br>RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites.<br><br>CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.},
}
@article {pmid31347199,
year = {2019},
author = {Zhang, M and Bi, C and Wang, M and Fu, H and Mu, Z and Zhu, Y and Yan, Z},
title = {Analysis of the structures of confirmed and questionable CRISPR loci in 325 Staphylococcus genomes.},
journal = {Journal of basic microbiology},
volume = {59},
number = {9},
pages = {901-913},
doi = {10.1002/jobm.201900124},
pmid = {31347199},
issn = {1521-4028},
mesh = {CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology ; Databases, Genetic ; Genome, Bacterial/*genetics ; Methicillin Resistance/genetics ; Phylogeny ; Staphylococcus/classification/*genetics ; },
abstract = {The CRISPR-Cas (clustered regular interspaced short palindromic repeats and CRISPR-associated proteins) system is a newly discovered immune defense system in the genome of prokaryotes, which can resist the invasion of foreign genetic elements, such as plasmids or phage. In this study, 154 strains of Staphylococcus published in the CRISPRDatabase and 171 strains included in NCBI were downloaded, the confirmed and questionable CRISPR loci of which were analyzed by bioinformatics methods, including their distribution, characteristics of the structure (including the direct repeats, spacers and cas genes), and the relationship between the presence of CRISPR and the mecA gene. Meanwhile, a comprehensive analysis of orphan CRISPR arrays was performed on this basis. A total of 196 confirmed and 1757 questionable CRISPR loci were found in 325 Staphylococcus genomes. Only 25 strains contained cas genes, which were classified into III-A (48.1%) and II-C (51.9%). The difference between the presence of the cas gene and the carrying rate of mecA was statistically significant, and they were negatively correlated. A total of 137 confirmed and 1755 questionable CRISPR loci were assumed to be false-CRISPR. The present study also analyzed the questionable CRISPR array for the first time while analyzing the confirmed CRISPR array in the Staphylococcal genome and screened the false-CRISPR elements in the orphan CRISPR array.},
}
@article {pmid31346683,
year = {2019},
author = {Krogerus, K and Magalhães, F and Kuivanen, J and Gibson, B},
title = {A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {18},
pages = {7597-7615},
pmid = {31346683},
issn = {1432-0614},
mesh = {Beer/microbiology ; CRISPR-Cas Systems ; Dextrins/metabolism ; Fermentation ; Fungal Proteins/*genetics ; *Promoter Regions, Genetic ; Reproducibility of Results ; Reverse Genetics ; Saccharomyces cerevisiae/*genetics/*metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Sequence Deletion ; Starch/*metabolism ; Trisaccharides/*metabolism ; },
abstract = {Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the 'Beer 2'/'Mosaic Beer' brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer's wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where maltotriose use is desired.},
}
@article {pmid31346235,
year = {2019},
author = {Kim, KM and Rana, A and Park, CY},
title = {Orai1 inhibitor STIM2β regulates myogenesis by controlling SOCE dependent transcriptional factors.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {10794},
pmid = {31346235},
issn = {2045-2322},
mesh = {Animals ; Calcium Signaling ; Cell Differentiation ; Cell Line ; Cyclin D1/metabolism ; Cyclin-Dependent Kinase 4/metabolism ; MEF2 Transcription Factors/genetics/metabolism ; Mice ; *Muscle Development ; Myoblasts/cytology/*metabolism ; NFATC Transcription Factors/genetics/*metabolism ; ORAI1 Protein/metabolism ; RNA Splicing ; Stromal Interaction Molecule 2/genetics/*metabolism ; },
abstract = {Store-operated Ca[2+] entry (SOCE), the fundamental Ca[2+] signaling mechanism in myogenesis, is mediated by stromal interaction molecule (STIM), which senses the depletion of endoplasmic reticulum Ca[2+] stores and induces Ca[2+] influx by activating Orai channels in the plasma membrane. Recently, STIM2β, an eight-residue-inserted splice variant of STIM2, was found to act as an inhibitor of SOCE. Although a previous study demonstrated an increase in STIM2β splicing during in vitro differentiation of skeletal muscle, the underlying mechanism and detailed function of STIM2β in myogenesis remain unclear. In this study, we investigated the function of STIM2β in myogenesis using the C2C12 cell line with RNA interference-mediated knockdown and CRISPR-Cas-mediated knockout approaches. Deletion of STIM2β delayed myogenic differentiation through the MEF2C and NFAT4 pathway in C2C12 cells. Further, loss of STIM2β increased cell proliferation by altering Ca[2+] homeostasis and inhibited cell cycle arrest mediated by the cyclin D1-CDK4 degradation pathway. Thus, this study identified a previously unknown function of STIM2β in myogenesis and improves the understanding of how cells effectively regulate the development process via alternative splicing.},
}
@article {pmid31346137,
year = {2019},
author = {Ren, B and Wang, X and Duan, J and Ma, J},
title = {Rhizobial tRNA-derived small RNAs are signal molecules regulating plant nodulation.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6456},
pages = {919-922},
doi = {10.1126/science.aav8907},
pmid = {31346137},
issn = {1095-9203},
mesh = {Argonaute Proteins/genetics ; Bradyrhizobium/genetics/*physiology ; CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; Host Microbial Interactions/*genetics ; Nitrogen Fixation ; Nucleic Acid Conformation ; Plant Proteins/genetics ; Plant Root Nodulation/*genetics ; Plant Roots/metabolism/microbiology ; RNA Interference ; RNA, Bacterial/chemistry/genetics/*physiology ; RNA, Small Untranslated/chemistry/genetics/*physiology ; RNA, Transfer/chemistry/genetics/*physiology ; Soybeans/genetics/metabolism/*microbiology ; },
abstract = {Rhizobial infection and root nodule formation in legumes require recognition of signal molecules produced by the bacteria and their hosts. Here, we show that rhizobial transfer RNA (tRNA)-derived small RNA fragments (tRFs) are signal molecules that modulate host nodulation. Three families of rhizobial tRFs were confirmed to regulate host genes associated with nodule initiation and development through hijacking the host RNA-interference machinery that involves ARGONAUTE 1. Silencing individual tRFs with the use of short tandem target mimics or by overexpressing their targets represses root hair curling and nodule formation, whereas repressing these targets with artificial microRNAs identical to the respective tRFs or mutating these targets with CRISPR-Cas9 promotes nodulation. Our findings thus uncover a bacterial small RNA-mediated mechanism for prokaryote-eukaryote interaction and may pave the way for enhancing nodulation efficiency in legumes.},
}
@article {pmid31346084,
year = {2019},
author = {Orvedahl, A and McAllaster, MR and Sansone, A and Dunlap, BF and Desai, C and Wang, YT and Balce, DR and Luke, CJ and Lee, S and Orchard, RC and Artyomov, MN and Handley, SA and Doench, JG and Silverman, GA and Virgin, HW},
title = {Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {33},
pages = {16497-16506},
pmid = {31346084},
issn = {1091-6490},
support = {U19 AI109725/AI/NIAID NIH HHS/United States ; R01 DK104946/DK/NIDDK NIH HHS/United States ; T32 AI106688/AI/NIAID NIH HHS/United States ; R00 DK116666/DK/NIDDK NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; U19 AI142784/AI/NIAID NIH HHS/United States ; K99 DK116666/DK/NIDDK NIH HHS/United States ; R01 DK114047/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Autophagy/drug effects/*genetics ; Autophagy-Related Protein 5/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Survival/drug effects/genetics ; Cytoprotection/drug effects ; Genome ; Interferon-gamma/*toxicity ; Mice, Knockout ; Myeloid Cells/drug effects/metabolism/*pathology/ultrastructure ; NF-kappa B/metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; Receptors, Tumor Necrosis Factor, Type I/metabolism ; Signal Transduction/drug effects ; Transcriptome/genetics ; Tumor Necrosis Factor-alpha/*toxicity ; },
abstract = {Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.},
}
@article {pmid31344908,
year = {2019},
author = {Bruegmann, T and Deecke, K and Fladung, M},
title = {Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars.},
journal = {International journal of molecular sciences},
volume = {20},
number = {15},
pages = {},
pmid = {31344908},
issn = {1422-0067},
mesh = {Agrobacterium ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Plant/genetics ; Populus/*genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.},
}
@article {pmid31344492,
year = {2019},
author = {Janakiraman, U and Yu, J and Moutal, A and Chinnasamy, D and Boinon, L and Batchelor, SN and Anandhan, A and Khanna, R and Nelson, MA},
title = {TAF1-gene editing alters the morphology and function of the cerebellum and cerebral cortex.},
journal = {Neurobiology of disease},
volume = {132},
number = {},
pages = {104539},
pmid = {31344492},
issn = {1095-953X},
support = {R01 DA042852/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/*genetics ; Cerebellum/abnormalities/*pathology/physiology ; Cerebral Cortex/abnormalities/*pathology/physiology ; Female ; Gene Editing/*methods ; Histone Acetyltransferases/*genetics ; Injections, Intraventricular ; Locomotion/physiology ; Organ Culture Techniques ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; TATA-Binding Protein Associated Factors/*genetics ; Transcription Factor TFIID/*genetics ; },
abstract = {TAF1/MRSX33 intellectual disability syndrome is an X-linked disorder caused by loss-of-function mutations in the TAF1 gene. How these mutations cause dysmorphology, hypotonia, intellectual and motor defects is unknown. Mouse models which have embryonically targeted TAF1 have failed, possibly due to TAF1 being essential for viability, preferentially expressed in early brain development, and intolerant of mutation. Novel animal models are valuable tools for understanding neuronal pathology. Here, we report the development and characterization of a novel animal model for TAF1 ID syndrome in which the TAF1 gene is deleted in embryonic rats using clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) technology and somatic brain transgenesis mediated by lentiviral transduction. Rat pups, post-natal day 3, were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 vectors. Rats were subjected to a battery of behavioral tests followed by histopathological analyses of brains at post-natal day 14 and day 35. TAF1-edited rats exhibited behavioral deficits at both the neonatal and juvenile stages of development. Deletion of TAF1 lead to a hypoplasia and loss of the Purkinje cells. We also observed a decreased in GFAP positive astrocytes and an increase in Iba1 positive microglia within the granular layer of the cerebellum in TAF1-edited animals. Immunostaining revealed a reduction in the expression of the CaV3.1 T-type calcium channel. Abnormal motor symptoms in TAF1-edited rats were associated with irregular cerebellar output caused by changes in the intrinsic activity of the Purkinje cells due to loss of pre-synaptic CaV3.1. This animal model provides a powerful new tool for studies of neuronal dysfunction in conditions associated with TAF1 abnormalities and should prove useful for developing therapeutic strategies to treat TAF1 ID syndrome.},
}
@article {pmid31344316,
year = {2020},
author = {Shao, X and Wu, S and Dou, T and Zhu, H and Hu, C and Huo, H and He, W and Deng, G and Sheng, O and Bi, F and Gao, H and Dong, T and Li, C and Yang, Q and Yi, G},
title = {Using CRISPR/Cas9 genome editing system to create MaGA20ox2 gene-modified semi-dwarf banana.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {17-19},
pmid = {31344316},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Musa/*genetics/growth &amp; development ; Plants, Genetically Modified ; },
}
@article {pmid31344313,
year = {2020},
author = {Li, C and Li, W and Zhou, Z and Chen, H and Xie, C and Lin, Y},
title = {A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene-free bacterial blight-resistant rice.},
journal = {Plant biotechnology journal},
volume = {18},
number = {2},
pages = {313-315},
pmid = {31344313},
issn = {1467-7652},
mesh = {*Breeding/methods ; CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Gene Editing ; *Oryza/genetics/microbiology ; Plant Diseases/genetics/prevention &amp; control ; *Promoter Regions, Genetic/genetics ; Transgenes/genetics ; },
}
@article {pmid31344144,
year = {2019},
author = {Chi, X and Zheng, Q and Jiang, R and Chen-Tsai, RY and Kong, LJ},
title = {A system for site-specific integration of transgenes in mammalian cells.},
journal = {PloS one},
volume = {14},
number = {7},
pages = {e0219842},
pmid = {31344144},
issn = {1932-6203},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems ; Cricetulus ; Gene Expression ; Gene Knock-In Techniques/*methods ; Genetic Loci ; Green Fluorescent Proteins/*genetics/metabolism ; HEK293 Cells ; Homologous Recombination ; Humans ; Integrases/*metabolism ; Recombinant Proteins/*metabolism ; *Transgenes ; },
abstract = {Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins.},
}
@article {pmid31343404,
year = {2019},
author = {Parker, MM and Hao, Y and Guo, F and Pham, B and Chase, R and Platig, J and Cho, MH and Hersh, CP and Thannickal, VJ and Crapo, J and Washko, G and Randell, SH and Silverman, EK and San José Estépar, R and Zhou, X and Castaldi, PJ},
title = {Identification of an emphysema-associated genetic variant near TGFB2 with regulatory effects in lung fibroblasts.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31343404},
issn = {2050-084X},
support = {R01 HL113264/HL/NHLBI NIH HHS/United States ; R01 HL130512/HL/NHLBI NIH HHS/United States ; U01 HL089897/HL/NHLBI NIH HHS/United States ; R01 HL116931/HL/NHLBI NIH HHS/United States ; R01 HL126596/HL/NHLBI NIH HHS/United States ; U01 HL089856/HL/NHLBI NIH HHS/United States ; R01HL089897/HB/NHLBI NIH HHS/United States ; R01HL113264/HB/NHLBI NIH HHS/United States ; P01HL114501/HB/NHLBI NIH HHS/United States ; R01 HL124233/HL/NHLBI NIH HHS/United States ; R01 HL125583/HL/NHLBI NIH HHS/United States ; P01 HL132825/HL/NHLBI NIH HHS/United States ; P01 HL114501/HL/NHLBI NIH HHS/United States ; P01105339/HB/NHLBI NIH HHS/United States ; R01HL089856/HB/NHLBI NIH HHS/United States ; T32 HL007427/HL/NHLBI NIH HHS/United States ; P30 DK065988/DK/NIDDK NIH HHS/United States ; },
mesh = {Aged ; Aged, 80 and over ; Emphysema/*genetics ; Fibroblasts/*physiology ; *Gene Expression Regulation ; *Genetic Loci ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; Lung/*pathology ; Middle Aged ; Transforming Growth Factor beta2/*biosynthesis/genetics ; United States ; },
abstract = {Murine studies have linked TGF-β signaling to emphysema, and human genome-wide association studies (GWAS) studies of lung function and COPD have identified associated regions near genes in the TGF-β superfamily. However, the functional regulatory mechanisms at these loci have not been identified. We performed the largest GWAS of emphysema patterns to date, identifying 10 GWAS loci including an association peak spanning a 200 kb region downstream from TGFB2. Integrative analysis of publicly available eQTL, DNaseI, and chromatin conformation data identified a putative functional variant, rs1690789, that may regulate TGFB2 expression in human fibroblasts. Using chromatin conformation capture, we confirmed that the region containing rs1690789 contacts the TGFB2 promoter in fibroblasts, and CRISPR/Cas-9 targeted deletion of a ~ 100 bp region containing rs1690789 resulted in decreased TGFB2 expression in primary human lung fibroblasts. These data provide novel mechanistic evidence linking genetic variation affecting the TGF-β pathway to emphysema in humans.},
}
@article {pmid31342740,
year = {2019},
author = {Qiu, M and Glass, Z and Xu, Q},
title = {Nonviral Nanoparticles for CRISPR-Based Genome Editing: Is It Just a Simple Adaption of What Have Been Developed for Nucleic Acid Delivery?.},
journal = {Biomacromolecules},
volume = {20},
number = {9},
pages = {3333-3339},
pmid = {31342740},
issn = {1526-4602},
support = {R01 EB027170/EB/NIBIB NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; *Gene Transfer Techniques ; Genetic Therapy ; Humans ; Nanoparticles/*chemistry/therapeutic use ; Nucleic Acids/*genetics/therapeutic use ; },
abstract = {Genome-editing technologies hold tremendous potential for treating genetic diseases. However, the efficient and safe delivery of genome-editing elements to the location of interest, and the achievement of specific targeted gene correction without off-target side effect remains a big challenge. In this Perspective, we highlight recent developments and discuss the challenges of nonviral nanoparticles for the delivery of genome-editing tools. Finally, we will propose promising strategies to improve the delivery efficacy and advance the clinical translation of gene-editing technology.},
}
@article {pmid31342441,
year = {2019},
author = {Coutinho, MF and Matos, L and Santos, JI and Alves, S},
title = {RNA Therapeutics: How Far Have We Gone?.},
journal = {Advances in experimental medicine and biology},
volume = {1157},
number = {},
pages = {133-177},
doi = {10.1007/978-3-030-19966-1_7},
pmid = {31342441},
issn = {0065-2598},
mesh = {*Genetic Therapy/trends ; Humans ; *Oligonucleotides ; *Oligonucleotides, Antisense ; RNA Splicing ; RNA, Messenger ; RNA, Small Interfering ; },
abstract = {In recent years, the RNA molecule became one of the most promising targets for therapeutic intervention. Currently, a large number of RNA-based therapeutics are being investigated both at the basic research level and in late-stage clinical trials. Some of them are even already approved for treatment. RNA-based approaches can act at pre-mRNA level (by splicing modulation/correction using antisense oligonucleotides or U1snRNA vectors), at mRNA level (inhibiting gene expression by siRNAs and antisense oligonucleotides) or at DNA level (by editing mutated sequences through the use of CRISPR/Cas). Other RNA approaches include the delivery of in vitro transcribed (IVT) mRNA or the use of oligonucleotides aptamers. Here we review these approaches and their translation into clinics trying to give a brief overview also on the difficulties to its application as well as the research that is being done to overcome them.},
}
@article {pmid31341289,
year = {2019},
author = {Gurumurthy, CB and Sato, M and Nakamura, A and Inui, M and Kawano, N and Islam, MA and Ogiwara, S and Takabayashi, S and Matsuyama, M and Nakagawa, S and Miura, H and Ohtsuka, M},
title = {Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.},
journal = {Nature protocols},
volume = {14},
number = {8},
pages = {2452-2482},
pmid = {31341289},
issn = {1750-2799},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Electroporation/*methods ; Female ; Gene Editing/*methods ; Male ; Mice ; Microinjections ; Oviducts/physiology ; Pregnancy ; RNA, Guide/administration &amp; dosage/genetics ; RNA, Messenger/administration &amp; dosage/genetics ; },
abstract = {Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.},
}
@article {pmid31341277,
year = {2019},
author = {Kemaladewi, DU and Bassi, PS and Erwood, S and Al-Basha, D and Gawlik, KI and Lindsay, K and Hyatt, E and Kember, R and Place, KM and Marks, RM and Durbeej, M and Prescott, SA and Ivakine, EA and Cohn, RD},
title = {A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.},
journal = {Nature},
volume = {572},
number = {7767},
pages = {125-130},
pmid = {31341277},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Disease Progression ; Female ; Fibrosis/metabolism/pathology ; Gene Editing ; Genes, Modifier/*genetics ; Genetic Therapy/*methods ; Laminin/*genetics/*metabolism ; Male ; Mice ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophies/*genetics/*therapy ; Mutation ; *Up-Regulation ; },
abstract = {Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease[1]. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy[2j]/dy[2j] mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy[2j]/dy[2j] mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.},
}
@article {pmid31341082,
year = {2019},
author = {Hidalgo-Cantabrana, C and Goh, YJ and Pan, M and Sanozky-Dawes, R and Barrangou, R},
title = {Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {32},
pages = {15774-15783},
pmid = {31341082},
issn = {1091-6490},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Loci ; Genetic Variation ; Lactobacillus crispatus/*genetics/ultrastructure ; Nucleotide Motifs/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.},
}
@article {pmid31341074,
year = {2019},
author = {Price, VJ and McBride, SW and Hullahalli, K and Chatterjee, A and Duerkop, BA and Palmer, KL},
title = {Enterococcus faecalis CRISPR-Cas Is a Robust Barrier to Conjugative Antibiotic Resistance Dissemination in the Murine Intestine.},
journal = {mSphere},
volume = {4},
number = {4},
pages = {},
pmid = {31341074},
issn = {2379-5042},
support = {K01 DK102436/DK/NIDDK NIH HHS/United States ; R01 AI116610/AI/NIAID NIH HHS/United States ; R01 AI141479/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; *Conjugation, Genetic ; *Drug Resistance, Bacterial ; Enterococcus faecalis/drug effects/*genetics ; *Gene Transfer, Horizontal ; Intestines/*microbiology ; Mice ; Mice, Inbred C57BL ; Plasmids/genetics ; },
abstract = {CRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions in in vivo models relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogens in vivo, and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids. Enterococcus faecalis is a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessed E. faecalis CRISPR-Cas anti-PRP activity in the mouse intestine and under different in vitro conditions. We observed striking differences in CRISPR-Cas efficiency in vitro versus in vivo With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, while in vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standard in vitro experiments do not adequately model the in vivo antiplasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparent in vitro antiplasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor-to-recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal (healthy) human microbiota can have significant impacts on in vivo antibiotic resistance dissemination.IMPORTANCE CRISPR-Cas is a type of immune system in bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under different in vitro conditions. We observed a robust effect of CRISPR-Cas on in vivo but not in vitro dissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizer Enterococcus faecalis We conclude that standard in vitro experiments currently do not appropriately model the in vivo conditions where antibiotic resistance dissemination occurs between E. faecalis strains in the intestine. Moreover, our results demonstrate that CRISPR-Cas present in native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.},
}
@article {pmid31340950,
year = {2019},
author = {Paulo, DF and Williamson, ME and Arp, AP and Li, F and Sagel, A and Skoda, SR and Sanchez-Gallego, J and Vasquez, M and Quintero, G and Pérez de León, AA and Belikoff, EJ and Azeredo-Espin, AML and McMillan, WO and Concha, C and Scott, MJ},
title = {Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {9},
pages = {3045-3055},
pmid = {31340950},
issn = {2160-1836},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Diptera/embryology/*genetics ; Embryo, Nonmammalian ; Female ; Insect Proteins/*genetics ; Male ; Mutation ; Pest Control/methods ; RNA, Guide ; Sex Determination Processes ; },
abstract = {Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host's tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the Chominivorax and Lcuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the Chominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.},
}
@article {pmid31340865,
year = {2019},
author = {Wong, JH and Shigemizu, D and Yoshii, Y and Akiyama, S and Tanaka, A and Nakagawa, H and Narumiya, S and Fujimoto, A},
title = {Identification of intermediate-sized deletions and inference of their impact on gene expression in a human population.},
journal = {Genome medicine},
volume = {11},
number = {1},
pages = {44},
pmid = {31340865},
issn = {1756-994X},
mesh = {CRISPR-Cas Systems ; Computational Biology/methods ; *Epistasis, Genetic ; *Gene Expression Regulation ; *Genetics, Population ; Genome, Human ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; Humans ; Molecular Sequence Annotation ; Phylogeny ; Quantitative Trait Loci ; Regulatory Sequences, Nucleic Acid ; Reproducibility of Results ; *Sequence Deletion ; Whole Genome Sequencing ; },
abstract = {BACKGROUND: Next-generation sequencing has allowed for the identification of different genetic variations, which are known to contribute to diseases. Of these, insertions and deletions are the second most abundant type of variations in the genome, but their biological importance or disease association is not well-studied, especially for deletions of intermediate sizes.<br><br>METHODS: We identified intermediate-sized deletions from whole-genome sequencing (WGS) data of Japanese samples (n = 174) with a novel deletion calling method which considered multiple samples. These deletions were used to construct a reference panel for use in imputation. Imputation was then conducted using the reference panel and data from 82 publically available Japanese samples with gene expression data. The accuracy of the deletion calling and imputation was examined with Nanopore long-read sequencing technology. We also conducted an expression quantitative trait loci (eQTL) association analysis using the deletions to infer their functional impacts on genes, before characterizing the deletions causal for gene expression level changes.<br><br>RESULTS: We obtained a set of polymorphic 4378 high-confidence deletions and constructed a reference panel. The deletions were successfully imputed into the Japanese samples with high accuracy (97.3%). The eQTL analysis identified 181 deletions (4.1%) suggested as causal for gene expression level changes. The causal deletion candidates were significantly enriched in promoters, super-enhancers, and transcription elongation chromatin states. Generation of deletions in a cell line with the CRISPR-Cas9 system confirmed that they were indeed causative variants for gene expression change. Furthermore, one of the deletions was observed to affect the gene expression levels of a gene it was not located in.<br><br>CONCLUSIONS: This paper reports an accurate deletion calling method for genotype imputation at the whole genome level and shows the importance of intermediate-sized deletions in the human population.},
}
@article {pmid31340764,
year = {2019},
author = {Kumar, A and Birnbaum, MD and Moorthy, BT and Singh, J and Palovcak, A and Patel, DM and Zhang, F},
title = {Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {609},
pmid = {31340764},
issn = {1471-2164},
support = {R01 GM107333/GM/NIGMS NIH HHS/United States ; R01#GM107333/NH/NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems ; Codon, Initiator ; Cricetulus ; Fibroblasts ; Fluorescence ; *Frameshift Mutation ; *Gene Editing ; Genes, Reporter ; HEK293 Cells ; Humans ; *INDEL Mutation ; Internal Ribosome Entry Sites ; Luminescent Proteins/*genetics ; Mice ; RNA, Guide ; },
abstract = {BACKGROUND: Reporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as "Insertion/deletion-activated frame-shift fluorescence protein". To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency.<br><br>RESULTS: To test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location.<br><br>CONCLUSION: The insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools.},
}
@article {pmid31340642,
year = {2019},
author = {Hu, J and Jiang, M and Liu, R and Lv, Y},
title = {Label-Free CRISPR/Cas9 Assay for Site-Specific Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {91},
number = {16},
pages = {10870-10878},
doi = {10.1021/acs.analchem.9b02641},
pmid = {31340642},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide/*genetics ; },
abstract = {The development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a revolutionary step for genome engineering because it enables modification of target genomes. However, many biological applications with the CRISPR/Cas9 system are impeded by off-target effects and loci-dependent nuclease activity with various sgRNAs. Commonly used label-strategy-based CRISPR/Cas9 assays often suffer from possible disturbances to Cas9 activity and a time-consuming labeling procedure. Herein, we for the first time propose a DNA-templated CuNPs-based label-free CRISPR/Cas9 assay, with a low LOD of 0.13 nM and rapid detection in 35 min after CRISPR/Cas9 cleavage. Additionally, the site specificity of the DNA substrate was demonstrated. Through the proposed label-free strategy, a single-base change at a specific loci could lead to a significant reduction of the Cas9 cleavage effect, while the other common genetic modifications might be accepted by the CRIPR/Cas9 system. Therefore, the proposed label-free Cas9 assay may provide a new paradigm for the a priori in vitro CRISPR/Cas9 assay and exploration for in vivo biological applications.},
}
@article {pmid31337983,
year = {2019},
author = {Yamagishi, A and Susaki, M and Takano, Y and Mizusawa, M and Mishima, M and Iijima, M and Kuroda, S and Okada, T and Nakamura, C},
title = {The Structural Function of Nestin in Cell Body Softening is Correlated with Cancer Cell Metastasis.},
journal = {International journal of biological sciences},
volume = {15},
number = {7},
pages = {1546-1556},
pmid = {31337983},
issn = {1449-2288},
mesh = {Actins/*chemistry ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chemotaxis ; Cytoskeleton/chemistry ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Microscopy, Atomic Force ; Neoplasm Invasiveness ; Neoplasm Metastasis/*pathology ; Nestin/chemistry/*physiology ; Protein Domains ; Sequence Analysis, RNA ; Stress, Mechanical ; Vimentin/*chemistry ; },
abstract = {Intermediate filaments play significant roles in governing cell stiffness and invasive ability. Nestin is a type VI intermediate filament protein that is highly expressed in several high-metastatic cancer cells. Although inhibition of nestin expression was shown to reduce the metastatic capacity of tumor cells, the relationship between this protein and the mechanism of cancer cell metastasis remains unclear. Here, we show that nestin softens the cell body of the highly metastatic mouse breast cancer cell line FP10SC2, thereby enhancing the metastasis capacity. Proximity ligation assay demonstrated increased binding between actin and vimentin in nestin knockout cells. Because nestin copolymerizes with vimentin and nestin has an extremely long tail domain in its C-terminal region, we hypothesized that the tail domain functions as a steric inhibitor of the vimentin-actin interaction and suppresses association of vimentin filaments with the cortical actin cytoskeleton, leading to reduced cell stiffness. To demonstrate this function, we mechanically pulled vimentin filaments in living cells using a nanoneedle modified with vimentin-specific antibodies under manipulation by atomic force microscopy (AFM). The tensile test revealed that mobility of vimentin filaments was increased by nestin expression in FP10SC2 cells.},
}
@article {pmid31337724,
year = {2019},
author = {Lum, KK and Howard, TR and Pan, C and Cristea, IM},
title = {Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA.},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
pmid = {31337724},
issn = {2150-7511},
support = {F31 GM120936/GM/NIGMS NIH HHS/United States ; R01 GM114141/GM/NIGMS NIH HHS/United States ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Cytokines/immunology ; DNA, Viral/genetics/*metabolism ; Fibroblasts/*immunology/virology ; HEK293 Cells ; Herpesvirus 1, Human/*genetics/physiology ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Mutation ; Nuclear Proteins/genetics/*metabolism ; Phosphoproteins/genetics/*metabolism ; Protein Domains ; Protein Multimerization ; Proteomics ; Pyrin/genetics/*metabolism ; Virus Replication ; },
abstract = {The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.},
}
@article {pmid31337711,
year = {2019},
author = {Paakinaho, V and Johnson, TA and Presman, DM and Hager, GL},
title = {Glucocorticoid receptor quaternary structure drives chromatin occupancy and transcriptional outcome.},
journal = {Genome research},
volume = {29},
number = {8},
pages = {1223-1234},
pmid = {31337711},
issn = {1549-5469},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin/chemistry/*ultrastructure ; DNA/genetics/metabolism ; Enhancer Elements, Genetic ; Epithelial Cells/*drug effects/metabolism/pathology ; Female ; Gene Editing/methods ; *Genome ; Glucocorticoids/metabolism/*pharmacology ; High-Throughput Nucleotide Sequencing ; Mammary Glands, Animal/metabolism/pathology ; Mice ; Protein Binding ; Protein Structure, Quaternary ; RNA, Messenger/*genetics/metabolism ; Rats ; Receptors, Glucocorticoid/*chemistry/genetics/metabolism ; Transcriptional Activation ; },
abstract = {Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.},
}
@article {pmid31335325,
year = {2019},
author = {Yu, M and Nguyen, ND and Huang, Y and Lin, D and Fujimoto, TN and Molkentine, JM and Deorukhkar, A and Kang, Y and San Lucas, FA and Fernandes, CJ and Koay, EJ and Gupta, S and Ying, H and Koong, AC and Herman, JM and Fleming, JB and Maitra, A and Taniguchi, CM},
title = {Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer.},
journal = {JCI insight},
volume = {5},
number = {16},
pages = {},
pmid = {31335325},
issn = {2379-3708},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; R01 CA227517/CA/NCI NIH HHS/United States ; R01 CA214793/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/*metabolism ; Disease Models, Animal ; Dynamins/antagonists &amp; inhibitors/genetics ; Enzyme Inhibitors/pharmacology ; GTP Phosphohydrolases/genetics ; Leflunomide/pharmacology ; Mice ; Mice, Knockout ; Mitochondria/*metabolism ; Mitochondrial Dynamics/drug effects/*genetics ; Mitophagy/drug effects/*genetics ; Oxidative Phosphorylation/drug effects ; Pancreatic Neoplasms/*metabolism ; Quinazolinones/pharmacology ; Survival Rate ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.},
}
@article {pmid31332749,
year = {2019},
author = {Khoshnejad, M and Brenner, JS and Parhiz, H and Muzykantov, VR},
title = {CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2033},
number = {},
pages = {81-93},
doi = {10.1007/978-1-4939-9654-4_7},
pmid = {31332749},
issn = {1940-6029},
mesh = {Animals ; Antibodies, Monoclonal/*genetics/immunology ; CRISPR-Cas Systems/genetics/immunology ; Gene Editing/*methods ; Genetic Engineering/*methods ; Humans ; Hybridomas/immunology ; Immunoconjugates/*genetics/immunology ; Mice ; Oligopeptides/genetics/immunology ; },
abstract = {Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.},
}
@article {pmid31332488,
year = {2019},
author = {Song, R and Zhai, Q and Sun, L and Huang, E and Zhang, Y and Zhu, Y and Guo, Q and Tian, Y and Zhao, B and Lu, H},
title = {CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {17},
pages = {6919-6932},
pmid = {31332488},
issn = {1432-0614},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Fungi/*genetics ; *Gene Editing ; Gene Targeting ; Genome, Fungal/*genetics ; Industrial Microbiology ; Mutation ; RNA, Guide/genetics ; Transformation, Genetic ; },
abstract = {Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.},
}
@article {pmid31332481,
year = {2020},
author = {Yuan, L and Liu, Q and Wang, Z and Hou, J and Xu, P},
title = {EI24 tethers endoplasmic reticulum and mitochondria to regulate autophagy flux.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {8},
pages = {1591-1606},
pmid = {31332481},
issn = {1420-9071},
mesh = {Animals ; Apoptosis Regulatory Proteins/genetics/*metabolism ; *Autophagy ; CRISPR-Cas Systems ; Cells, Cultured ; Endoplasmic Reticulum/*metabolism ; Female ; HEK293 Cells ; Humans ; Insulin-Secreting Cells/cytology/*metabolism ; Male ; Mice ; Mitochondria/*metabolism ; Nuclear Proteins/genetics/*metabolism ; Voltage-Dependent Anion Channel 1/metabolism ; },
abstract = {Etoposide-induced protein 2.4 (EI24), located on the endoplasmic reticulum (ER) membrane, has been proposed to be an essential autophagy protein. Specific ablation of EI24 in neuronal and liver tissues causes deficiency of autophagy flux. However, the molecular mechanism of the EI24-mediated autophagy process is still poorly understood. Like neurons and hepatic cells, pancreatic β cells are also secretory cells. Pancreatic β cells contain large amounts of ER and continuously synthesize and secrete insulin to maintain blood glucose homeostasis. Yet, the effect of EI24 on autophagy of pancreatic β cells has not been reported. Here, we show that the autophagy process is inhibited in EI24-deficient primary pancreatic β cells. Further mechanistic studies demonstrate that EI24 is enriched at the ER-mitochondria interface and that the C-terminal domain of EI24 is important for the integrity of the mitochondria-associated membrane (MAM) and autophagy flux. Overexpression of EI24, but not the EI24-ΔC mutant, can rescue MAM integrity and decrease the aggregation of p62 and LC3II in the EI24-deficient group. By mass spectrometry-based proteomics following immunoprecipitation, EI24 was found to interact with voltage-dependent anion channel 1 (VDAC1), inositol 1,4,5-trisphosphate receptor (IP3R), and the outer mitochondrial membrane chaperone GRP75. Knockout of EI24 impairs the interaction of IP3R with VDAC1, indicating that these proteins may form a quaternary complex to regulate MAM integrity and the autophagy process.},
}
@article {pmid31332341,
year = {2019},
author = {Moreno, AM and Palmer, N and Alemán, F and Chen, G and Pla, A and Jiang, N and Leong Chew, W and Law, M and Mali, P},
title = {Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.},
journal = {Nature biomedical engineering},
volume = {3},
number = {10},
pages = {806-816},
pmid = {31332341},
issn = {2157-846X},
support = {CDA 16-150/HX/HSRD VA/United States ; R01 AI106005/AI/NIAID NIH HHS/United States ; R01 AI079031/AI/NIAID NIH HHS/United States ; R01 GM123313/GM/NIGMS NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; R01 HG009285/HG/NHGRI NIH HHS/United States ; },
mesh = {Adaptive Immunity ; Animals ; CRISPR-Cas Systems/*genetics ; *Capsid ; Dependovirus/*genetics ; Gene Editing ; Genetic Therapy/*methods ; Genetic Vectors ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Protein Engineering ; },
abstract = {Protein-based therapeutics can activate the adaptive immune system, leading to the production of neutralizing antibodies and the clearance of the treated cells mediated by cytotoxic T cells. Here, we show that the sequential use of immune-orthogonal orthologues of CRISPR-associated protein 9 (Cas9) and adeno-associated viruses (AAVs) evades adaptive immune responses and enables effective gene editing using repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major histocompatibility complex (MHC) proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors and 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologues-which we validated for three Cas9 orthologues in mice-yet we anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice that were previously immunized against the AAV vector and the Cas9 cargo. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.},
}
@article {pmid31332326,
year = {2019},
author = {Thuronyi, BW and Koblan, LW and Levy, JM and Yeh, WH and Zheng, C and Newby, GA and Wilson, C and Bhaumik, M and Shubina-Oleinik, O and Holt, JR and Liu, DR},
title = {Continuous evolution of base editors with expanded target compatibility and improved activity.},
journal = {Nature biotechnology},
volume = {37},
number = {9},
pages = {1070-1079},
pmid = {31332326},
issn = {1546-1696},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 DC013521/DC/NIDCD NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; T32 GM095450/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; },
mesh = {Adenosine Deaminase/genetics/*metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; *Directed Molecular Evolution ; *Gene Editing ; Gene Expression Regulation, Enzymologic ; Gene Targeting ; Humans ; INDEL Mutation ; Mice ; },
abstract = {Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.},
}
@article {pmid31331664,
year = {2019},
author = {Gratacap, RL and Wargelius, A and Edvardsen, RB and Houston, RD},
title = {Potential of Genome Editing to Improve Aquaculture Breeding and Production.},
journal = {Trends in genetics : TIG},
volume = {35},
number = {9},
pages = {672-684},
doi = {10.1016/j.tig.2019.06.006},
pmid = {31331664},
issn = {0168-9525},
support = {BB/R008612/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S004343/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Animals, Genetically Modified ; Aquaculture/*methods ; Breeding/legislation &amp; jurisprudence/*methods ; CRISPR-Cas Systems ; Disease Resistance ; Fertility ; Fishes/*genetics ; Food Supply ; Gene Editing/legislation &amp; jurisprudence/*methods ; Genetic Introgression ; Public Opinion ; Quantitative Trait Loci ; },
abstract = {Aquaculture is the fastest growing food production sector and is rapidly becoming the primary source of seafood for human diets. Selective breeding programs are enabling genetic improvement of production traits, such as disease resistance, but progress is limited by the heritability of the trait and generation interval of the species. New breeding technologies, such as genome editing using CRISPR/Cas9 have the potential to expedite sustainable genetic improvement in aquaculture. Genome editing can rapidly introduce favorable changes to the genome, such as fixing alleles at existing trait loci, creating de novo alleles, or introducing alleles from other strains or species. The high fecundity and external fertilization of most aquaculture species can facilitate genome editing for research and application at a scale that is not possible in farmed terrestrial animals.},
}
@article {pmid31331444,
year = {2019},
author = {Bilousova, G},
title = {Gene Therapy for Skin Fragility Diseases: The New Generation.},
journal = {The Journal of investigative dermatology},
volume = {139},
number = {8},
pages = {1634-1637},
doi = {10.1016/j.jid.2019.04.001},
pmid = {31331444},
issn = {1523-1747},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Epidermolysis Bullosa Dystrophica ; Genetic Therapy ; Humans ; Transcription Activator-Like Effector Nucleases ; },
abstract = {Ex vivo gene therapy is a promising approach to treat devastating skin fragility diseases. March et al. and Takashima et al. report that programmable nucleases-TALENs and CRISPR/Cas9-can safely and efficiently correct genetic defects in cultured adult skin cells, paving the way for broader clinical applications of gene therapies in dermatology.},
}
@article {pmid31331377,
year = {2019},
author = {Tong, Z and Sathe, A and Ebner, B and Qi, P and Veltkamp, C and Gschwend, JE and Holm, PS and Nawroth, R},
title = {Functional genomics identifies predictive markers and clinically actionable resistance mechanisms to CDK4/6 inhibition in bladder cancer.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {38},
number = {1},
pages = {322},
pmid = {31331377},
issn = {1756-9966},
mesh = {Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chickens ; Chorioallantoic Membrane/drug effects ; Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors/genetics ; Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors/genetics ; Drug Resistance, Neoplasm/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; *Genomics ; Heterografts ; Humans ; Mice ; Piperazines/*pharmacology ; Protein Kinase Inhibitors/*pharmacology ; Pyridines/*pharmacology ; Urinary Bladder Neoplasms/*drug therapy/genetics/pathology ; },
abstract = {BACKGROUND: CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer.<br><br>METHODS: A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois membrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal.<br><br>RESULTS: In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in in vivo xenografts.<br><br>CONCLUSIONS: Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.},
}
@article {pmid31330540,
year = {2019},
author = {Han, D and Hong, Y and Mai, X and Hu, Q and Lu, G and Duan, J and Xu, J and Si, X and Zhang, Y},
title = {Systematical study of the mechanistic factors regulating genome dynamics in vivo by CRISPRsie.},
journal = {Journal of molecular cell biology},
volume = {11},
number = {11},
pages = {1018-1020},
pmid = {31330540},
issn = {1759-4685},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; Gene Targeting ; *Genome ; Humans ; Mice ; RNA, Guide ; },
}
@article {pmid31329953,
year = {2019},
author = {Dedow, LK and Bailey-Serres, J},
title = {Searching for a Match: Structure, Function and Application of Sequence-Specific RNA-Binding Proteins.},
journal = {Plant &amp; cell physiology},
volume = {60},
number = {9},
pages = {1927-1938},
doi = {10.1093/pcp/pcz072},
pmid = {31329953},
issn = {1471-9053},
mesh = {Cell Nucleus/metabolism ; Cytoplasm/metabolism ; Plant Proteins/genetics/metabolism ; Plants/*genetics/metabolism ; Plastids/metabolism ; RNA/genetics/metabolism ; *RNA Editing ; RNA-Binding Proteins/*genetics/metabolism ; },
abstract = {Plants encode over 1800 RNA-binding proteins (RBPs) that modulate a myriad of steps in gene regulation from chromatin organization to translation, yet only a small number of these proteins and their target transcripts have been functionally characterized. Two classes of eukaryotic RBPs, pentatricopeptide repeat (PPR) and pumilio/fem-3 binding factors (PUF), recognize and bind to specific sequential RNA sequences through protein-RNA interactions. These modular proteins possess helical structural units containing key residues with high affinity for specific nucleotides, whose sequential order determines binding to a specific target RNA sequence. PPR proteins are nucleus-encoded, but largely regulate post-transcriptional gene regulation within plastids and mitochondria, including splicing, translation and RNA editing. Plant PUFs are involved in gene regulatory processes within the cell nucleus and cytoplasm. The modular structures of PPRs and PUFs that determine sequence specificity has facilitated identification of their RNA targets and biological functions. The protein-based RNA-targeting of PPRs and PUFs contrasts to the prokaryotic cluster regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that target RNAs in prokaryotes. Together the PPR, PUF and CRISPR-Cas systems provide varied opportunities for RNA-targeted engineering applications.},
}
@article {pmid31329043,
year = {2019},
author = {Divaris, K},
title = {The Era of the Genome and Dental Medicine.},
journal = {Journal of dental research},
volume = {98},
number = {9},
pages = {949-955},
pmid = {31329043},
issn = {1544-0591},
support = {U01 DE025046/DE/NIDCR NIH HHS/United States ; },
mesh = {Craniofacial Abnormalities/*genetics ; *Genome, Human ; Humans ; *Oral Health ; Periodontal Diseases/*genetics ; Tooth Diseases/*genetics ; },
abstract = {Understanding the "code of life" and mapping the human genome have been monumental and era-defining scientific landmarks-analogous to setting foot on the moon. The last century has been characterized by exponential advances in our understanding of the biological and specifically molecular basis of health and disease. The early part of the 20th century was marked by fundamental theoretical and scientific advances in understanding heredity, the identification of the DNA molecule and genes, and the elucidation of the central dogma of biology. The second half was characterized by experimental and increasingly molecular investigations, including clinical and population applications. The completion of the Human Genome Project in 2003 and the continuous technological advances have democratized access to this information and the ability to generate health and disease association data; however, the realization of genomic and precision medicine, to practically improve people's health, has lagged. The oral health domain has made great strides and substantially benefited from the last century of advances in genetics and genomics. Observations regarding a hereditary component of dental caries were reported as early as the 1920s. Subsequent breakthroughs were made in the discovery of genetic causes of rare diseases, such as ectodermal dysplasias, orofacial clefts, and other craniofacial and dental anomalies. More recently, genome-wide investigations have been conducted and reported for several diseases and traits, including periodontal disease, dental caries, tooth agenesis, cancers of the head and neck, orofacial pain, temporomandibular disorders, and craniofacial morphometrics. Gene therapies and gene editing with CRISPR/Cas represent the latest frontier surpassed in the era of genomic medicine. Amid rapid genomics progress, several challenges and opportunities lie ahead. Importantly, systematic efforts supported by implementation science are needed to realize the full potential of genomics, including the improvement of public and practitioner genomics literacy, the promotion of individual and population oral health, and the reduction of disparities.},
}
@article {pmid31328964,
year = {2019},
author = {Xu, L and Liu, Y and Han, R},
title = {BEAT: A Python Program to Quantify Base Editing from Sanger Sequencing.},
journal = {The CRISPR journal},
volume = {2},
number = {4},
pages = {223-229},
pmid = {31328964},
issn = {2573-1602},
support = {R01 AR064241/AR/NIAMS NIH HHS/United States ; R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; HEK293 Cells ; Humans ; Sequence Analysis, DNA/*methods ; *Software ; },
abstract = {Through fusing CRISPR-Cas9 nickases with cytidine or adenine deaminases, a new paradigm-shifting class of genome-editing technology, termed "base editors," has recently been developed. Base editors mediate highly efficient, targeted single-base conversion without introducing double-stranded breaks. Analysis of base editing outcomes typically relies on imprecise enzymatic mismatch cleavage assays, time-consuming single-colony sequencing, or expensive next-generation deep sequencing. To overcome these limitations, several groups have recently developed computer programs to measure base-editing efficiency from fluorescence-based Sanger sequencing data such as Edit deconvolution by inference of traces in R (EditR), TIDER, and ICE. These approaches have greatly simplified the quantitation of base-editing experiments. However, the current Sanger sequencing tools lack the capability of batch analysis and producing high-quality images for publication. Here, we provide a base editing analysis tool (BEAT) written in Python to analyze and quantify the base-editing events from Sanger sequencing data in a batch manner, which can also produce intuitive, publication-ready base-editing images.},
}
@article {pmid31328481,
year = {2019},
author = {Wang, Q and Zeng, W and Zhou, J},
title = {[Effect of gene knockout of L-tyrosine transport system on L-tyrosine production in Escherichia coli].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {7},
pages = {1247-1255},
doi = {10.13345/j.cjb.180533},
pmid = {31328481},
issn = {1872-2075},
mesh = {*Escherichia coli ; Escherichia coli Proteins ; Gene Knockout Techniques ; Metabolic Engineering ; Tyrosine ; },
abstract = {L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.},
}
@article {pmid31328227,
year = {2019},
author = {van Tran, N and Ernst, FGM and Hawley, BR and Zorbas, C and Ulryck, N and Hackert, P and Bohnsack, KE and Bohnsack, MT and Jaffrey, SR and Graille, M and Lafontaine, DLJ},
title = {The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.},
journal = {Nucleic acids research},
volume = {47},
number = {15},
pages = {7719-7733},
pmid = {31328227},
issn = {1362-4962},
mesh = {Adenosine/*chemistry/genetics/metabolism ; Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Crystallography, X-Ray ; Gene Deletion ; *Gene Expression Regulation, Neoplastic ; HCT116 Cells ; Humans ; Methyltransferases/*chemistry/genetics/metabolism ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Protein Stability ; RNA, Guide/genetics/metabolism ; RNA, Messenger/*chemistry/genetics/metabolism ; RNA, Ribosomal, 18S/*chemistry/genetics/metabolism ; Signal Transduction ; Substrate Specificity ; },
abstract = {N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.},
}
@article {pmid31327905,
year = {2019},
author = {Baliga, P and Shekar, M and Venugopal, MN},
title = {Detection and characterization of clustered regularly interspaced short palindromic repeat-associated endoribonuclease gene variants in Vibrio parahaemolyticus isolated from seafoods and environment.},
journal = {Veterinary world},
volume = {12},
number = {5},
pages = {689-695},
pmid = {31327905},
issn = {0972-8988},
abstract = {AIM: In Vibrio parahaemolyticus, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated cas6 endoribonuclease gene has been shown to exhibit sequence diversity and has been subtyped into four major types based on its length and composition. In this study, we aimed to detect and characterize the cas6 gene variants prevalent among V. parahaemolyticus strains isolated from seafoods and environment.<br><br>MATERIALS AND METHODS: Novel primers were designed for each of the cas6 subtypes to validate their identification in V. parahaemolyticus by polymerase chain reaction (PCR). In total, 38 V. parahaemolyticus strains isolated from seafoods and environment were screened for the presence of cas6 gene. Few representative PCR products were sequenced, and their phylogenetic relationship was established to available cas6 gene sequences in GenBank database.<br><br>RESULTS: Of the 38 V. parahaemolyticus isolates screened, only about 40% of strains harbored the cas6 endoribonuclease gene, among which 31.6% and 7.9% of the isolates were positive for the presence of the cas6-a and cas6-d subtypes of the gene, respectively. The subtypes cas6-b and cas6-c were absent in strains studied. Sequence and phylogenetic analysis also established the cas6 sequences in this study to match GenBank sequences for cas6-a and cas6-d subtypes.<br><br>CONCLUSION: In V. parahaemolyticus, the Cas6 endoribonuclease is an associated protein of the CRISPR-cas system. CRISPR-positive strains exhibited genotypic variation for this gene. Primers designed in this study would aid in identifying the cas6 genotype and understanding the role of these genotypes in the CRISPR-cas immune system of the pathogen.},
}
@article {pmid31327526,
year = {2019},
author = {Xu, Y and Zhou, P and Cheng, S and Lu, Q and Nowak, K and Hopp, AK and Li, L and Shi, X and Zhou, Z and Gao, W and Li, D and He, H and Liu, X and Ding, J and Hottiger, MO and Shao, F},
title = {A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.},
journal = {Cell},
volume = {178},
number = {3},
pages = {552-566.e20},
doi = {10.1016/j.cell.2019.06.007},
pmid = {31327526},
issn = {1097-4172},
mesh = {ADP-Ribosylation ; Autophagy-Related Proteins/deficiency/genetics/*metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; HeLa Cells ; Humans ; *Macroautophagy ; Microtubule-Associated Proteins/metabolism ; Protein Binding ; Salmonella/*metabolism/pathogenicity ; Type III Secretion Systems/metabolism ; Vacuolar Proton-Translocating ATPases/genetics/*metabolism ; Virulence Factors/*genetics/metabolism ; },
abstract = {Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.},
}
@article {pmid31326596,
year = {2020},
author = {Alkhnbashi, OS and Meier, T and Mitrofanov, A and Backofen, R and Voß, B},
title = {CRISPR-Cas bioinformatics.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {3-11},
doi = {10.1016/j.ymeth.2019.07.013},
pmid = {31326596},
issn = {1095-9130},
mesh = {Algorithms ; CRISPR-Cas Systems/*genetics ; Computational Biology/*methods/trends ; *Gene Editing ; RNA, Guide/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are essential genetic elements in many archaeal and bacterial genomes, playing a key role in a prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas system has also been engineered to facilitate target gene editing in eukaryotic genomes. Bioinformatics played an essential role in the detection and analysis of CRISPR systems and here we review the bioinformatics-based efforts that pushed the field of CRISPR-Cas research further. We discuss the bioinformatics tools that have been published over the last few years and, finally, present the most popular tools for the design of CRISPR-Cas9 guides.},
}
@article {pmid31326354,
year = {2019},
author = {van Kampen, SJ and van Rooij, E},
title = {CRISPR Craze to Transform Cardiac Biology.},
journal = {Trends in molecular medicine},
volume = {25},
number = {9},
pages = {791-802},
doi = {10.1016/j.molmed.2019.06.008},
pmid = {31326354},
issn = {1471-499X},
mesh = {Animals ; Biomarkers ; *CRISPR-Cas Systems ; Disease Susceptibility ; *Gene Editing ; Genetic Predisposition to Disease ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Myocardium/*metabolism ; Myocytes, Cardiac/*metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized many research areas and has rapidly become the gold standard in genome editing by outrivaling all other available tools. Its unprecedented versatility creates the opportunity to modify any aspect of gene regulation. Even though the cardiac field is starting to appreciate the potential of CRISPR, many applications to study cardiac biology and disease so far have remained untouched. In particular, CRISPR-based strategies that act independent of the homology-directed repair pathway could help circumvent issues of modifying the genome of postmitotic cardiomyocytes, which is currently limiting its utility in the heart. Here, we review current applications and future potential for the use of CRISPR to study cardiac biology and disease.},
}
@article {pmid31326273,
year = {2019},
author = {Jia, N and Jones, R and Yang, G and Ouerfelli, O and Patel, DJ},
title = {CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA&gt;p Formation Terminating RNase Activity.},
journal = {Molecular cell},
volume = {75},
number = {5},
pages = {944-956.e6},
pmid = {31326273},
issn = {1097-4164},
support = {P30 GM124165/GM/NIGMS NIH HHS/United States ; R01 AI141507/AI/NIAID NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; S10 RR029205/RR/NCRR NIH HHS/United States ; },
mesh = {Adenine Nucleotides/*chemistry ; Archaeal Proteins/*chemistry ; Binding Sites ; CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Oligoribonucleotides/*chemistry ; Protein Domains ; Ribonucleases/*chemistry ; Thermococcus/*chemistry ; },
abstract = {Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.},
}
@article {pmid31326272,
year = {2019},
author = {Jia, N and Jones, R and Sukenick, G and Patel, DJ},
title = {Second Messenger cA4 Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path.},
journal = {Molecular cell},
volume = {75},
number = {5},
pages = {933-943.e6},
pmid = {31326272},
issn = {1097-4164},
support = {P30 GM124165/GM/NIGMS NIH HHS/United States ; R01 AI141507/AI/NIAID NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; S10 RR029205/RR/NCRR NIH HHS/United States ; },
mesh = {Adenine Nucleotides/*chemistry/metabolism ; Archaeal Proteins/*chemistry/metabolism ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; Oligoribonucleotides/*chemistry/metabolism ; Ribonucleases/*chemistry/metabolism ; *Second Messenger Systems ; Thermococcus/*chemistry/metabolism/ultrastructure ; },
abstract = {Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cAn) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cAn second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppAn), and products (cAn), to decipher mechanistic aspects of cAn formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppAn intermediates and subsequent cAn formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cAn signaling pathway.},
}
@article {pmid31325654,
year = {2019},
author = {Dowrey, T and Schwager, EE and Duong, J and Merkuri, F and Zarate, YA and Fish, JL},
title = {Satb2 regulates proliferation and nuclear integrity of pre-osteoblasts.},
journal = {Bone},
volume = {127},
number = {},
pages = {488-498},
pmid = {31325654},
issn = {1873-2763},
support = {R15 DE026611/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Cell Differentiation/genetics ; Cell Line ; Cell Nucleus/*metabolism ; Cell Nucleus Shape ; Cell Proliferation ; Gene Expression Regulation ; Matrix Attachment Region Binding Proteins/*genetics/metabolism ; Mice ; Models, Biological ; Mutation/genetics ; Osteoblasts/*cytology/*metabolism ; Osteogenesis/genetics ; Transcription Factors/*genetics/metabolism ; },
abstract = {Special AT-rich sequence binding protein 2 (Satb2) is a matrix attachment region (MAR) binding protein. Satb2 impacts skeletal development by regulating gene transcription required for osteogenic differentiation. Although its role as a high-order transcription factor is well supported, other roles for Satb2 in skeletal development remain unclear. In particular, the impact of dosage sensitivity (heterozygous mutations) and variance on phenotypic severity is still not well understood. To further investigate molecular and cellular mechanisms of Satb2-mediated skeletal defects, we used the CRISPR/Cas9 system to generate Satb2 mutations in MC3T3-E1 cells. Our data suggest that, in addition to its role in differentiation, Satb2 regulates progenitor proliferation. We also find that mutations in Satb2 cause chromatin defects including nuclear blebbing and donut-shaped nuclei. These defects may contribute to a slight increase in apoptosis in mutant cells, but apoptosis is insufficient to explain the proliferation defects. Satb2 expression exhibits population-level variation and is most highly expressed from late G1 to late G2. Based on these data, we hypothesize that Satb2 may regulate proliferation through two separate mechanisms. First, Satb2 may regulate the expression of genes necessary for cell cycle progression in pre-osteoblasts. Second, similar to other MAR-binding proteins, Satb2 may participate in DNA replication. We also hypothesize that variation in the severity or penetrance of Satb2-mediated proliferation defects is due to stochastic variation in Satb2 binding to DNA, which may be buffered in some genetic backgrounds. Further elucidation of the role of Satb2 in proliferation has potential impacts on our understanding of both skeletal defects and cancer.},
}
@article {pmid31325492,
year = {2020},
author = {Behler, J and Hess, WR},
title = {Approaches to study CRISPR RNA biogenesis and the key players involved.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {12-26},
doi = {10.1016/j.ymeth.2019.07.015},
pmid = {31325492},
issn = {1095-9130},
mesh = {Adaptive Immunity/genetics ; Archaea/enzymology/genetics/immunology ; Archaeal Proteins/metabolism ; Bacteria/enzymology/genetics/immunology ; Bacterial Proteins/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endoribonucleases/*metabolism ; RNA Processing, Post-Transcriptional/immunology ; RNA, Archaeal/*biosynthesis ; RNA, Bacterial/*biosynthesis ; RNA, Guide/*biosynthesis ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an inheritable and adaptive immune system against phages and foreign genetic elements in many bacteria and archaea. The three stages of CRISPR-Cas immunity comprise adaptation, CRISPR RNA (crRNA) biogenesis and interference. The maturation of the pre-crRNA into mature crRNAs, short guide RNAs that target invading nucleic acids, is crucial for the functionality of CRISPR-Cas defense systems. Mature crRNAs assemble with Cas proteins into the ribonucleoprotein (RNP) effector complex and guide the Cas nucleases to the cognate foreign DNA or RNA target. Experimental approaches to characterize these crRNAs, the specific steps toward their maturation and the involved factors, include RNA-seq analyses, enzyme assays, methods such as cryo-electron microscopy, the crystallization of proteins, or UV-induced protein-RNA crosslinking coupled to mass spectrometry analysis. Complex and multiple interactions exist between CRISPR-cas-encoded specific riboendonucleases such as Cas6, Cas5d and Csf5, endonucleases with dual functions in maturation and interference such as the enzymes of the Cas12 and Cas13 families, and nucleases belonging to the cell's degradosome such as RNase E, PNPase and RNase J, both in the maturation as well as in interference. The results of these studies have yielded a picture of unprecedented diversity of sequences, enzymes and biochemical mechanisms.},
}
@article {pmid31324766,
year = {2019},
author = {Adeyemo, AA and Zaghloul, NA and Chen, G and Doumatey, AP and Leitch, CC and Hostelley, TL and Nesmith, JE and Zhou, J and Bentley, AR and Shriner, D and Fasanmade, O and Okafor, G and Eghan, B and Agyenim-Boateng, K and Chandrasekharappa, S and Adeleye, J and Balogun, W and Owusu, S and Amoah, A and Acheampong, J and Johnson, T and Oli, J and Adebamowo, C and , and Collins, F and Dunston, G and Rotimi, CN},
title = {ZRANB3 is an African-specific type 2 diabetes locus associated with beta-cell mass and insulin response.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3195},
pmid = {31324766},
issn = {2041-1723},
support = {R25 GM055036/GM/NIGMS NIH HHS/United States ; P30 DK072488/DK/NIDDK NIH HHS/United States ; F31 DK115179/DK/NIDDK NIH HHS/United States ; T32 DK098107/DK/NIDDK NIH HHS/United States ; T37 TW000041/TW/FIC NIH HHS/United States ; R01 DK102001/DK/NIDDK NIH HHS/United States ; ZIA HG200362/HG/NHGRI NIH HHS/United States ; },
mesh = {Africa, Northern ; Animals ; Apoptosis ; Base Sequence ; Blood Glucose ; CRISPR-Cas Systems ; DNA Helicases/*genetics/*metabolism ; Diabetes Mellitus, Type 2/*genetics ; Disease Models, Animal ; Female ; Gene Editing ; Gene Knockout Techniques ; *Genetic Association Studies ; Genetic Predisposition to Disease/*genetics ; Genotype ; Ghana ; Glucose/metabolism ; Homozygote ; Humans ; Insulin/*metabolism ; Insulin-Secreting Cells/*metabolism ; Kenya ; Male ; Mice ; Middle Aged ; Mutation ; Nigeria ; Polymorphism, Single Nucleotide ; RNA, Small Interfering ; Transcription Factor 7-Like 2 Protein/genetics ; Transcriptome ; Zebrafish ; },
abstract = {Genome analysis of diverse human populations has contributed to the identification of novel genomic loci for diseases of major clinical and public health impact. Here, we report a genome-wide analysis of type 2 diabetes (T2D) in sub-Saharan Africans, an understudied ancestral group. We analyze ~18 million autosomal SNPs in 5,231 individuals from Nigeria, Ghana and Kenya. We identify a previously-unreported genome-wide significant locus: ZRANB3 (Zinc Finger RANBP2-Type Containing 3, lead SNP p = 2.831 × 10[-9]). Knockdown or genomic knockout of the zebrafish ortholog results in reduction in pancreatic β-cell number which we demonstrate to be due to increased apoptosis in islets. siRNA transfection of murine Zranb3 in MIN6 β-cells results in impaired insulin secretion in response to high glucose, implicating Zranb3 in β-cell functional response to high glucose conditions. We also show transferability in our study of 32 established T2D loci. Our findings advance understanding of the genetics of T2D in non-European ancestry populations.},
}
@article {pmid31324765,
year = {2019},
author = {Niemi, NM and Wilson, GM and Overmyer, KA and Vögtle, FN and Myketin, L and Lohman, DC and Schueler, KL and Attie, AD and Meisinger, C and Coon, JJ and Pagliarini, DJ},
title = {Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3197},
pmid = {31324765},
issn = {2041-1723},
support = {R01 DK101573/DK/NIDDK NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; R01 DK098672/DK/NIDDK NIH HHS/United States ; T32 GM008349/GM/NIGMS NIH HHS/United States ; P41 GM108538/GM/NIGMS NIH HHS/United States ; T32 DK007665/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cloning, Molecular ; Disease Models, Animal ; Energy Metabolism/genetics/physiology ; Female ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lipidomics ; Male ; Membrane Transport Proteins/metabolism ; Metabolism, Inborn Errors/genetics/pathology ; Metabolomics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/*enzymology/*metabolism/ultrastructure ; Mitochondrial Membranes/metabolism ; Mitochondrial Precursor Protein Import Complex Proteins ; Mitochondrial Proteins/genetics/metabolism ; Mutagenesis, Site-Directed ; Phosphorylation ; Protein Phosphatase 2C/*genetics/*metabolism ; Proteomics ; },
abstract = {Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7[-/-] mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7[-/-] tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.},
}
@article {pmid31324451,
year = {2019},
author = {Mosqueira, D and Smith, JGW and Bhagwan, JR and Denning, C},
title = {Modeling Hypertrophic Cardiomyopathy: Mechanistic Insights and Pharmacological Intervention.},
journal = {Trends in molecular medicine},
volume = {25},
number = {9},
pages = {775-790},
doi = {10.1016/j.molmed.2019.06.005},
pmid = {31324451},
issn = {1471-499X},
support = {NC/C013202/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; NC/K000225/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; NC/C013105/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; SP/15/9/31605/BHF_/British Heart Foundation/United Kingdom ; PG/14/59/31000/BHF_/British Heart Foundation/United Kingdom ; RG/14/1/30588/BHF_/British Heart Foundation/United Kingdom ; MR/M017354/1/MRC_/Medical Research Council/United Kingdom ; NC/S001808/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {Animals ; Biomarkers ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/diagnosis/drug therapy/*etiology/*metabolism ; Disease Models, Animal ; *Disease Susceptibility ; Gene Editing ; Humans ; *Models, Biological ; Molecular Targeted Therapy ; Muscle Contraction ; Myocardium/metabolism/pathology ; Myocytes, Cardiac/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Sarcomeres/genetics/metabolism ; },
abstract = {Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular disease where cardiac dysfunction often associates with mutations in sarcomeric genes. Various models based on tissue explants, isolated cardiomyocytes, skinned myofibrils, and purified actin/myosin preparations have uncovered disease hallmarks, enabling the development of putative therapeutics, with some reaching clinical trials. Newly developed human pluripotent stem cell (hPSC)-based models could be complementary by overcoming some of the inconsistencies of earlier systems, whilst challenging and/or clarifying previous findings. In this article we compare recent progress in unveiling multiple HCM mechanisms in different models, highlighting similarities and discrepancies. We explore how insight is facilitating the design of new HCM therapeutics, including those that regulate metabolism, contraction and heart rhythm, providing a future perspective for treatment of HCM.},
}
@article {pmid31323994,
year = {2019},
author = {Rozov, SM and Permyakova, NV and Deineko, EV},
title = {The Problem of the Low Rates of CRISPR/Cas9-Mediated Knock-ins in Plants: Approaches and Solutions.},
journal = {International journal of molecular sciences},
volume = {20},
number = {13},
pages = {},
pmid = {31323994},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Gene Editing ; Genome, Plant/genetics ; Plants/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The main number of genome editing events in plant objects obtained during the last decade with the help of specific nucleases zinc finger (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas are the microindels causing frameshift and subsequent gene knock-out. The knock-ins of genes or their parts, i.e., the insertion of them into a target genome region, are between one and two orders of magnitude less frequent. First and foremost, this is associated with the specific features of the repair systems of higher eukaryotes and the availability of the donor template in accessible proximity during double-strand break (DSB) repair. This review briefs the main repair pathways in plants according to the aspect of their involvement in genome editing. The main methods for increasing the frequency of knock-ins are summarized both along the homologous recombination pathway and non-homologous end joining, which can be used for plant objects.},
}
@article {pmid31323920,
year = {2019},
author = {Milenkovic, VM and Slim, D and Bader, S and Koch, V and Heinl, ES and Alvarez-Carbonell, D and Nothdurfter, C and Rupprecht, R and Wetzel, CH},
title = {CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells.},
journal = {International journal of molecular sciences},
volume = {20},
number = {13},
pages = {},
pmid = {31323920},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Calcium/metabolism ; Cell Line ; Cells, Cultured ; Humans ; Membrane Potential, Mitochondrial/physiology ; Microglia/*metabolism ; Oxidative Phosphorylation ; Receptors, GABA/deficiency/genetics/*metabolism ; Steroids/metabolism ; },
abstract = {The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca[2+] levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.},
}
@article {pmid31323192,
year = {2019},
author = {Nimura, T and Itoh, T and Hagio, H and Hayashi, T and Di Donato, V and Takeuchi, M and Itoh, T and Inoguchi, F and Sato, Y and Yamamoto, N and Katsuyama, Y and Del Bene, F and Shimizu, T and Hibi, M},
title = {Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.},
journal = {Developmental biology},
volume = {455},
number = {2},
pages = {393-408},
doi = {10.1016/j.ydbio.2019.07.010},
pmid = {31323192},
issn = {1095-564X},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Adhesion Molecules, Neuronal/genetics/*physiology ; Cell Movement ; Cerebellum/cytology/*embryology ; Extracellular Matrix Proteins/genetics/*physiology ; Kinesins/metabolism ; Mutation ; Nerve Tissue Proteins/genetics/*physiology ; Purkinje Cells/cytology ; Reelin Protein ; Serine Endopeptidases/genetics/*physiology ; Signal Transduction ; Zebrafish/anatomy &amp; histology/*embryology ; Zebrafish Proteins/genetics/*physiology ; },
abstract = {The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.},
}
@article {pmid31321991,
year = {2019},
author = {Lake, F},
title = {Putting the spotlight on CRISPR.},
journal = {BioTechniques},
volume = {67},
number = {2},
pages = {41},
doi = {10.2144/btn-2019-0084},
pmid = {31321991},
issn = {1940-9818},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Engineering ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Neoplasms/genetics ; },
}
@article {pmid31321702,
year = {2019},
author = {Chimata, MK and Bharti, G},
title = {Regulation of genome edited technologies in India.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {175-181},
pmid = {31321702},
issn = {1573-9368},
mesh = {Biotechnology/*trends ; CRISPR-Cas Systems/*genetics ; Food Safety ; Gene Editing/*trends ; Humans ; India ; Plants, Genetically Modified/*genetics/growth &amp; development ; },
abstract = {In India, genetically modified organisms and products thereof are regulated under the "Rules for the manufacture, use, import, export and storage of hazardous microorganisms, genetically engineered organisms or cells, 1989" (referred to as Rules, 1989) notified under the Environment (Protection) Act, 1986. These Rules are implemented by the Ministry of Environment, Forest and Climate Change, Department of Biotechnology and State Governments though six competent authorities. The Rules, 1989 are supported by series of guidelines on contained research, biologics, confined field trials, food safety assessment, environmental risk assessment etc. The definition of genetic engineering in the Rules, 1989 implies that new genome engineering technologies including gene editing technologies like CRISPR/Cas9 and gene drives may be covered under the rules. The regulatory authorities if required, may also review the experiences of other countries in dealing with such new and emerging technologies.},
}
@article {pmid31321699,
year = {2019},
author = {Kelly, L},
title = {Clarifying the regulation of genome editing in Australia: situation for food.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {161-164},
doi = {10.1007/s11248-019-00159-w},
pmid = {31321699},
issn = {1573-9368},
mesh = {Australia ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics/growth &amp; development ; Gene Editing/methods/*trends ; Humans ; },
}
@article {pmid31321687,
year = {2019},
author = {Nekrasov, V},
title = {Sequence-specific nucleases as tools for enhancing disease resistance in crops.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {75-80},
pmid = {31321687},
issn = {1573-9368},
support = {BB/P016855/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Disease Resistance/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Loss of Function Mutation/genetics ; Plant Diseases/genetics/microbiology ; Plants, Genetically Modified/genetics/growth &amp; development ; Xanthomonas/*genetics ; },
abstract = {Genome editing technologies, such as CRISPR/Cas, have recently become valuable tools for plant reverse genetics as well as crop improvement, including enhancement of disease resistance. Targeting susceptibility (S) genes by genome editing has proven to be a viable strategy for generating resistance to both bacterial and fungal pathogens in various crops. Examples include generating loss-of-function mutations in promoter elements of the SWEET S genes, which are targeted by transcription activator-like effectors secreted by many phytopathogenic Xanthomonas bacteria, as well as in the conserved MLO locus that confers susceptibility to powdery mildew fungal pathogens in many monocots and dicots. In addition to genome editing applications, CRISPR/Cas systems can be used as means of defending plants against viruses via targeting viral genomic DNA or RNA. Genome editing is therefore a highly promising approach that enables engineering disease resistance to various plant pathogens directly in elite cultivar background in a highly precise manner. Unlike conventional crop breeding, genome editing approaches are not relying on lengthy and laborious crosses/back-crosses involving parental and progeny lines and can significantly shorten the breeding timeline. Taking into account the high potential of genome editing technologies for both basic and applied plant science, the recent decision of the European Court of Justice to define transgene-free genetically edited crops as GMOs is, clearly, a backward step for the EU.},
}
@article {pmid31321686,
year = {2019},
author = {Nogué, F and Vergne, P and Chèvre, AM and Chauvin, JE and Bouchabké-Coussa, O and Déjardin, A and Chevreau, E and Hibrand-Saint Oyant, L and Mazier, M and Barret, P and Guiderdoni, E and Sallaud, C and Foucrier, S and Devaux, P and Rogowsky, PM},
title = {Crop plants with improved culture and quality traits for food, feed and other uses.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {65-73},
pmid = {31321686},
issn = {1573-9368},
mesh = {Agriculture/*trends ; Animals ; Arabidopsis/genetics/growth &amp; development ; Brachypodium/genetics/growth &amp; development ; Bryopsida/genetics/growth &amp; development ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics/growth &amp; development ; Gene Editing/*methods ; Genome, Plant/genetics ; Mutagenesis/genetics ; Phenotype ; },
abstract = {The large French research project GENIUS (2012-2019, https://www6.inra.genius-project_eng/) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.},
}
@article {pmid31321685,
year = {2019},
author = {Park, J and Choe, S},
title = {DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {61-64},
pmid = {31321685},
issn = {1573-9368},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; Gene Editing/*trends ; Genome, Plant/genetics ; Mutation ; Protoplasts/metabolism ; Ribonucleoproteins/*genetics ; },
abstract = {Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of desired traits in crossable relatives or failure to generate the wanted phenotypes by random mutagenesis led to develop innovative breeding methods that are truly cross-species and precise. To this end, we devised novel methods of precise genome engineering that are characterized to use pre-assembled CRISPR/Cas9 ribonucleoprotein (RNP) complex instead of using nucleic ands or Agrobacterium. We found that our methods successfully engineered plant genomes without leaving any foreign DNA footprint in the genomes. To facilitate introduction of RNP into plant nucleus, we first obtained protoplasts after removing the transfection barrier, cell wall. Whole plants were regenerated from the single cell of protoplasts that has been engineered with the RNP. Pending the improved way of protoplast regeneration technology especially in crop plants, our methods should help develop novel traits in crop plants in relatively short time with safe and precise way.},
}
@article {pmid31321683,
year = {2019},
author = {Gao, C},
title = {Precision plant breeding using genome editing technologies.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {53-55},
pmid = {31321683},
issn = {1573-9368},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Plant/*genetics ; Plant Breeding ; Plants/genetics ; },
}
@article {pmid31321681,
year = {2019},
author = {Kearns, P},
title = {An overview of OECD activities related to modern techniques of biotechnology and genome editing : OECD conference on genome editing, June 2018.},
journal = {Transgenic research},
volume = {28},
number = {Suppl 2},
pages = {41-44},
pmid = {31321681},
issn = {1573-9368},
mesh = {Biotechnology/*trends ; CRISPR-Cas Systems/*genetics ; Congresses as Topic ; Gene Editing/*trends ; Humans ; Organisation for Economic Co-Operation and Development/*trends ; },
}
@article {pmid31320637,
year = {2019},
author = {Jönsson, ME and Ludvik Brattås, P and Gustafsson, C and Petri, R and Yudovich, D and Pircs, K and Verschuere, S and Madsen, S and Hansson, J and Larsson, J and Månsson, R and Meissner, A and Jakobsson, J},
title = {Activation of neuronal genes via LINE-1 elements upon global DNA demethylation in human neural progenitors.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3182},
pmid = {31320637},
issn = {2041-1723},
mesh = {Brain/embryology ; CRISPR-Cas Systems/genetics ; Chromatin Assembly and Disassembly/genetics ; CpG Islands/genetics ; DNA (Cytosine-5-)-Methyltransferase 1/*genetics ; *DNA Demethylation ; DNA Methylation/*genetics ; Gene Silencing/physiology ; Humans ; Long Interspersed Nucleotide Elements/*genetics ; Neural Stem Cells/*cytology/metabolism ; Transcriptional Activation/genetics ; },
abstract = {DNA methylation contributes to the maintenance of genomic integrity in somatic cells, in part through the silencing of transposable elements. In this study, we use CRISPR-Cas9 technology to delete DNMT1, the DNA methyltransferase key for DNA methylation maintenance, in human neural progenitor cells (hNPCs). We observe that inactivation of DNMT1 in hNPCs results in viable, proliferating cells despite a global loss of DNA CpG-methylation. DNA demethylation leads to specific transcriptional activation and chromatin remodeling of evolutionarily young, hominoid-specific LINE-1 elements (L1s), while older L1s and other classes of transposable elements remain silent. The activated L1s act as alternative promoters for many protein-coding genes involved in neuronal functions, revealing a hominoid-specific L1-based transcriptional network controlled by DNA methylation that influences neuronal protein-coding genes. Our results provide mechanistic insight into the role of DNA methylation in silencing transposable elements in somatic human cells, as well as further implicating L1s in human brain development and disease.},
}
@article {pmid31320583,
year = {2019},
author = {Fandino, RA and Haverkamp, A and Bisch-Knaden, S and Zhang, J and Bucks, S and Nguyen, TAT and Schröder, K and Werckenthin, A and Rybak, J and Stengl, M and Knaden, M and Hansson, BS and Große-Wilde, E},
title = {Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {31},
pages = {15677-15685},
pmid = {31320583},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Cas Systems ; Feeding Behavior/*physiology ; Female ; Insect Proteins/genetics/*metabolism ; Male ; Manduca/genetics/*metabolism ; Oviposition/*physiology ; Receptors, Odorant/genetics/*metabolism ; },
abstract = {The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.},
}
@article {pmid31318118,
year = {2019},
author = {Zhang, L and Wang, L and Xie, Y and Wang, P and Deng, S and Qin, A and Zhang, J and Yu, X and Zheng, W and Jiang, X},
title = {Triple-Targeting Delivery of CRISPR/Cas9 To Reduce the Risk of Cardiovascular Diseases.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {36},
pages = {12404-12408},
doi = {10.1002/anie.201903618},
pmid = {31318118},
issn = {1521-3773},
support = {2017YFA0205901//Minister of Science and Technology of China/International ; 21535001//National Natural Science Foundation of China/International ; 81730051//National Natural Science Foundation of China/International ; 81700382//National Natural Science Foundation of China/International ; 81673039//National Natural Science Foundation of China/International ; 21761142006//National Natural Science Foundation of China/International ; 81671784//National Natural Science Foundation of China/International ; 21505027//National Natural Science Foundation of China/International ; U1601227//National Natural Science Foundation of China/International ; 121D11KYSB20170026//Chinese Academy of Sciences/International ; },
mesh = {Animals ; Asialoglycoprotein Receptor/metabolism ; *CRISPR-Cas Systems ; Cardiovascular Diseases/*prevention &amp; control ; Cell Nucleus/genetics/*metabolism ; Cholesterol, LDL/*blood ; Galactose/chemistry ; *Gene Editing ; Gold/chemistry ; Liver/metabolism ; Mice ; Mutagenesis ; *Mutation ; Nanocomposites/administration &amp; dosage/chemistry ; *PCSK9 Inhibitors ; Peptide Fragments/metabolism ; Proprotein Convertase 9/genetics/metabolism ; tat Gene Products, Human Immunodeficiency Virus/metabolism ; },
abstract = {A high level of low-density lipoprotein cholesterol (LDL-C) in the blood is a major risk factor for coronary heart disease. Herein, we present a triple-targeting strategy to generate a loss-of-function mutation in Pcsk9, which regulates plasma cholesterol levels, using a nanocarrier-delivered CRISPR/Cas9 system. Nuclear localization signal (NLS)-tagged Cas9 and Pcsk9-targeted single guide RNA (sgPcsk9) were complexed with gold nanoclusters (GNCs) modified with cationic HIV-1-transactivating transcriptor (TAT) peptide and further encapsulated in a galactose-modified lipid layer to target the nanoclusters to the liver. The resulting nanoclusters had an in vitro Pcsk9-editing efficiency of about 60 % and resulting in a decrease in plasma LDL-C in mice of approximately 30%. No off-target mutagenesis was detected in 10 sites with high similarity. This approach may have therapeutic potential for the prevention and treatment of cardiovascular disease without side effects.},
}
@article {pmid31317541,
year = {2020},
author = {Ahmad, N and Rahman, MU and Mukhtar, Z and Zafar, Y and Zhang, B},
title = {A critical look on CRISPR-based genome editing in plants.},
journal = {Journal of cellular physiology},
volume = {235},
number = {2},
pages = {666-682},
doi = {10.1002/jcp.29052},
pmid = {31317541},
issn = {1097-4652},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Crops, Agricultural/*genetics ; Epigenesis, Genetic ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plants ; Plants, Genetically Modified/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.},
}
@article {pmid31317533,
year = {2019},
author = {Wang, Y and Liu, Y and Li, J and Yang, Y and Ni, X and Cheng, H and Huang, T and Guo, Y and Ma, H and Zheng, P and Wang, M and Sun, J and Ma, Y},
title = {Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {11},
pages = {3016-3029},
doi = {10.1002/bit.27121},
pmid = {31317533},
issn = {1097-0290},
mesh = {*Aspartate Kinase/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Corynebacterium glutamicum/enzymology/genetics ; *Gene Editing ; },
abstract = {CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG).},
}
@article {pmid31317418,
year = {2019},
author = {Ermert, AL and Nogué, F and Stahl, F and Gans, T and Hughes, J},
title = {CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2026},
number = {},
pages = {237-263},
pmid = {31317418},
issn = {1940-6029},
mesh = {Bryopsida/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics/physiology ; Phytochrome/*metabolism ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics/physiology ; },
abstract = {Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.},
}
@article {pmid31317359,
year = {2019},
author = {Chen, SJ},
title = {Minimizing off-target effects in CRISPR-Cas9 genome editing.},
journal = {Cell biology and toxicology},
volume = {35},
number = {5},
pages = {399-401},
pmid = {31317359},
issn = {1573-6822},
support = {R01 GM063732/GM/NIGMS NIH HHS/United States ; R01 GM117059/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/methods ; Genetic Engineering/*ethics/*methods ; Humans ; RNA, Guide/genetics ; },
}
@article {pmid31316073,
year = {2019},
author = {Liu, J and Srinivasan, S and Li, CY and Ho, IL and Rose, J and Shaheen, M and Wang, G and Yao, W and Deem, A and Bristow, C and Hart, T and Draetta, G},
title = {Pooled library screening with multiplexed Cpf1 library.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3144},
pmid = {31316073},
issn = {2041-1723},
support = {P01 CA117969/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *Gene Library ; Humans ; RNA, Guide/chemistry ; },
abstract = {Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.},
}
@article {pmid31315906,
year = {2019},
author = {Zhang, S and Wang, Y and Jia, L and Wen, X and Du, Z and Wang, C and Hao, Y and Yu, D and Zhou, L and Chen, N and Chen, J and Chen, H and Zhang, H and Celik, I and Gülsoy, G and Luo, J and Qin, B and Cui, X and Liu, Z and Zhang, S and Esteban, MA and Ay, F and Xu, W and Chen, R and Li, W and Hoffman, AR and Hu, JF and Cui, J},
title = {Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing.},
journal = {Genome research},
volume = {29},
number = {9},
pages = {1521-1532},
pmid = {31315906},
issn = {1549-5469},
support = {I01 BX002905/BX/BLRD VA/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cellular Reprogramming ; Chromatin/*genetics ; Fibroblasts/cytology/metabolism ; Mice ; Octamer Transcription Factor-3/*genetics ; Pluripotent Stem Cells/cytology/metabolism ; Promoter Regions, Genetic ; RNA, Long Noncoding/*genetics ; Regulatory Sequences, Nucleic Acid ; SOXB1 Transcription Factors/*genetics ; Sequence Analysis, RNA/*methods ; },
abstract = {Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.},
}
@article {pmid31315144,
year = {2019},
author = {Afolabi, LO and Adeshakin, AO and Sani, MM and Bi, J and Wan, X},
title = {Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9.},
journal = {Immunology},
volume = {158},
number = {2},
pages = {63-69},
pmid = {31315144},
issn = {1365-2567},
mesh = {Adaptive Immunity ; CRISPR-Associated Protein 9/genetics/immunology ; CRISPR-Cas Systems/*immunology ; Cellular Reprogramming/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/immunology ; Cytotoxicity, Immunologic ; Drug Delivery Systems/*methods ; Gene Editing/methods ; *Gene Transfer Techniques ; Humans ; Immunotherapy/methods ; Killer Cells, Natural/*immunology/metabolism ; Metal Nanoparticles/administration &amp; dosage ; Neoplasms/genetics/immunology/pathology/*therapy ; Plasmids/chemistry/immunology ; RNA, Guide/genetics/immunology ; Receptors, Chimeric Antigen/*genetics/immunology ; Tumor Microenvironment/genetics/immunology ; },
abstract = {Natural killer cells are potent cytotoxic lymphocytes specialized in recognizing and eliminating transformed cells, and in orchestrating adaptive anti-tumour immunity. However, NK cells are usually functionally exhausted in the tumour microenvironment. Strategies such as checkpoint blockades are under investigation to overcome NK cell exhaustion in order to boost anti-tumour immunity. The discovery and development of the CRISPR/Cas9 technology offer a flexible and efficient gene-editing capability in modulating various pathways that mediate NK cell exhaustion, and in arming NK cells with novel chimeric antigen receptors to specifically target tumour cells. Despite the high efficiency in its gene-editing capability, difficulty in the delivery of the CRISPR/Cas9 system remains a major bottleneck for its therapeutic applications, particularly for NK cells. The current review discusses feasible approaches to deliver the CRISPR/Cas9 systems, as well as potential strategies in gene-editing for NK cell immunotherapy for cancers.},
}
@article {pmid31314976,
year = {2019},
author = {Dowdy, SF},
title = {Controlling CRISPR-Cas9 Gene Editing.},
journal = {The New England journal of medicine},
volume = {381},
number = {3},
pages = {289-290},
doi = {10.1056/NEJMcibr1906886},
pmid = {31314976},
issn = {1533-4406},
support = {R21 CA234740/CA/NCI NIH HHS/United States ; R21 EY027507/EY/NEI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Vectors ; Humans ; Oligonucleotides/antagonists &amp; inhibitors ; RNA Interference ; RNA, Guide ; *RNAi Therapeutics ; },
}
@article {pmid31313008,
year = {2019},
author = {Li, D and Zhou, H and Zeng, X},
title = {Battling CRISPR-Cas9 off-target genome editing.},
journal = {Cell biology and toxicology},
volume = {35},
number = {5},
pages = {403-406},
doi = {10.1007/s10565-019-09485-5},
pmid = {31313008},
issn = {1573-6822},
mesh = {CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*ethics/methods ; Genetic Engineering/*ethics/*methods ; Humans ; RNA, Guide/genetics ; },
}
@article {pmid31312880,
year = {2020},
author = {Shao, L and Zhang, Y and Pan, X and Liu, B and Liang, C and Zhang, Y and Wang, Y and Yan, B and Xie, W and Sun, Y and Shen, Z and Yu, XY and Li, Y},
title = {Knockout of beta-2 microglobulin enhances cardiac repair by modulating exosome imprinting and inhibiting stem cell-induced immune rejection.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {5},
pages = {937-952},
pmid = {31312880},
issn = {1420-9071},
mesh = {Animals ; Bcl-2-Like Protein 11/metabolism ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Exosomes/metabolism ; Fibrosis/prevention &amp; control ; Human Umbilical Vein Endothelial Cells/cytology/transplantation ; Humans ; Interferon-gamma/pharmacology ; Mesenchymal Stem Cell Transplantation/*methods ; Mesenchymal Stem Cells/cytology/*immunology ; MicroRNAs/genetics ; Myocardial Infarction/*therapy ; Rats ; beta 2-Microglobulin/*genetics/metabolism ; },
abstract = {BACKGROUND AND AIMS: Allogeneic human umbilical mesenchymal stem cells (alloUMSC) are convenient cell source for stem cell-based therapy. However, immune rejection is a major obstacle for clinical application of alloUMSC for cardiac repair after myocardial infarction (MI). The immune rejection is due to the presence of human leukocyte antigen (HLA) class I molecule which is increased during MI. The aim of this study was to knockout HLA light chain β2-microglobulin (B2M) in UMSC to enhance stem cell engraftment and survival after transplantation.<br><br>METHODS AND RESULTS: We developed an innovative strategy using CRISPR/Cas9 to generate UMSC with B2M deletion (B2M[-]UMSC). AlloUMSC injection induced CD8[+] T cell-mediated immune rejection in immune competent rats, whereas no CD8[+] T cell-mediated killing against B2M[-]UMSC was observed even when the cells were treated with IFN-&#x3b3;. Moreover, we demonstrate that UMSC-derived exosomes can inhibit cardiac fibrosis and restore cardiac function, and exosomes derived from B2M[-]UMSC are more efficient than those derived from UMSC, indicating that the beneficial effect of exosomes can be enhanced by modulating exosome's imprinting. Mechanistically, microRNA sequencing identifies miR-24 as a major component of the exosomes from B2M[-]UMSCs. Bioinformatics analysis identifies Bim as a putative target of miR-24. Loss-of-function studies at the cellular level and gain-of-function approaches in exosomes show that the beneficial effects of B2M[-]UMSCs are mediated by the exosome/miR-24/Bim pathway.<br><br>CONCLUSION: Our findings demonstrate that modulation of exosome's imprinting via B2M knockout is an efficient strategy to prevent the immune rejection of alloUMSCs. This study paved the way to the development of new strategies for tissue repair and regeneration without the need for HLA matching.},
}
@article {pmid31309556,
year = {2020},
author = {Ethiraj, P and Sambandam, Y and Hathaway-Schrader, JD and Haque, A and Novince, CM and Reddy, SV},
title = {RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma.},
journal = {Journal of cellular physiology},
volume = {235},
number = {2},
pages = {1663-1673},
pmid = {31309556},
issn = {1097-4652},
support = {K08 DE025337/DE/NIDCR NIH HHS/United States ; R56 AG052511/AG/NIA NIH HHS/United States ; T32 DE017551/DE/NIDCR NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carcinoma, Squamous Cell/*metabolism ; Caspase 1 ; Cell Death ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Mitochondria/metabolism ; Mouth Neoplasms ; RANK Ligand/genetics/*metabolism ; TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism/*pharmacology ; },
abstract = {Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.},
}
@article {pmid31309518,
year = {2020},
author = {Granata, M and Skarmoutsou, E and Mazzarino, MC and Libra, M and D'Amico, F},
title = {Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2109},
number = {},
pages = {67-74},
doi = {10.1007/7651_2019_251},
pmid = {31309518},
issn = {1940-6029},
mesh = {Biopsy ; COP9 Signalosome Complex/*metabolism ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Line ; Humans ; Immunohistochemistry ; Intracellular Signaling Peptides and Proteins/*metabolism ; Keratinocytes/metabolism ; Peptide Hydrolases/*metabolism ; Psoriasis/metabolism/*pathology ; S100 Calcium Binding Protein A7/genetics/*metabolism ; Tissue Embedding ; Tissue Fixation ; },
abstract = {The application of immunohistological methods provides an invaluable contribution in revealing the protein colocalization, which may reflect the occurrence of molecular interaction processes.This chapter describes comprehensive protocols for detection of S100A7/JAB1 colocalization by immunohistochemistry in archival formalin-fixed and paraffin-embedded skin biopsies from healthy and psoriatic subjects. In addition, we provide a protocol for immunocytochemical detection of S100A7/JAB1 colocalization in S100A7 CRISPR-activated human keratinocyte cell line.},
}
@article {pmid31309374,
year = {2019},
author = {Friedrichs, S and Takasu, Y and Kearns, P and Dagallier, B and Oshima, R and Schofield, J and Moreddu, C},
title = {Meeting report of the OECD conference on "Genome Editing: Applications in Agriculture-Implications for Health, Environment and Regulation".},
journal = {Transgenic research},
volume = {28},
number = {3-4},
pages = {419-463},
pmid = {31309374},
issn = {1573-9368},
mesh = {*Agriculture ; CRISPR-Cas Systems ; Congresses as Topic ; *Environment ; Gene Editing/*legislation &amp; jurisprudence/*methods/standards ; *Global Health ; Humans ; *Organisation for Economic Co-Operation and Development ; Research Report ; },
abstract = {The "OECD Conference on Genome Editing: Applications in Agriculture-Implications for Health, Environment and Regulation" was held on the 28-29 June 2018 at the OECD headquarter and conference centre in Paris, France. It brought together policy makers, academia, innovators and other stakeholders involved in the topic, in order to take stock of the current technical developments and implementations of genome editing, as well as their applications in various areas of agriculture and the implications they give rise to (More information on the "OECD Conference on Genome Editing: Applications in Agriculture-Implications for Health, Environment and Regulation" can be found on the OECD Genome Editing hub: http://www.oecd.org/environment/genome-editing-agriculture/ ; the hub also contains the detailed conference programme, the biographies of all conference speakers, the detailed conference abstracts, and the presentations of the two-day conference). The conference aimed to provide a clearer understanding of the regulatory considerations raised by products of genome editing, pointing towards a coherent policy approach to facilitate innovations involving genome editing.},
}
@article {pmid31308554,
year = {2019},
author = {Choi, GCG and Zhou, P and Yuen, CTL and Chan, BKC and Xu, F and Bao, S and Chu, HY and Thean, D and Tan, K and Wong, KH and Zheng, Z and Wong, ASL},
title = {Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.},
journal = {Nature methods},
volume = {16},
number = {8},
pages = {722-730},
doi = {10.1038/s41592-019-0473-0},
pmid = {31308554},
issn = {1548-7105},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; *Mutagenesis ; *Mutation ; Protein Engineering ; RNA, Guide/*genetics ; *Software ; Streptococcus pyogenes/enzymology ; Substrate Specificity ; },
abstract = {The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.},
}
@article {pmid31308503,
year = {2019},
author = {Zhang, Y and Malzahn, AA and Sretenovic, S and Qi, Y},
title = {The emerging and uncultivated potential of CRISPR technology in plant science.},
journal = {Nature plants},
volume = {5},
number = {8},
pages = {778-794},
doi = {10.1038/s41477-019-0461-5},
pmid = {31308503},
issn = {2055-0278},
mesh = {Botany/*trends ; *CRISPR-Cas Systems ; },
abstract = {The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolutionized life science over the past few years. CRISPR was first discovered as an adaptive immune system in bacteria and archaea, and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing, epigenome editing and transcriptome regulation in plants. However, challenges in plant genome editing need to be fully appreciated and solutions explored. This Review intends to provide an informative summary of the latest developments and breakthroughs of CRISPR technology, with a focus on achievements and potential utility in plant biology. Ultimately, CRISPR will not only facilitate basic research, but also accelerate plant breeding and germplasm development. The application of CRISPR to improve germplasm is particularly important in the context of global climate change as well as in the face of current agricultural, environmental and ecological challenges.},
}
@article {pmid31307968,
year = {2019},
author = {Niu, XR and Yin, SM and Chen, X and Shao, TT and Li, DL},
title = {[Gene editing technology and its recent progress in disease therapy].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {7},
pages = {582-598},
doi = {10.16288/j.yczz.19-102},
pmid = {31307968},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; Disease ; Epigenesis, Genetic ; Gene Editing/*trends ; *Genetic Therapy ; Humans ; },
abstract = {Gene editing is a genetic manipulation technology which utilizes bacterial nucleases to accurately and efficiently modify DNA or RNA. Gene editing has broad applications in basic research, breeding, and drug screening, and it is gaining validity and applicability to the therapy of many diseases especially genetic-based disease. In this review, we summarize the development of gene editing technology, its different strategies and applications in the treatment of disease, and the research of gene editing therapy for genetic diseases (including base editor and epigenetic regulation) in the treatment of disorders and diseases of the blood system, liver, muscle and nervous system. Finally, we discuss the future development prospects of gene editing therapy.},
}
@article {pmid31307375,
year = {2019},
author = {Do, PT and Nguyen, CX and Bui, HT and Tran, LTN and Stacey, G and Gillman, JD and Zhang, ZJ and Stacey, MG},
title = {Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {311},
pmid = {31307375},
issn = {1471-2229},
support = {R01 DA007250/DA/NIDA NIH HHS/United States ; T32 DA007250/DA/NIDA NIH HHS/United States ; },
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics ; Gene Editing/*methods ; *Genes, Plant ; Genetic Markers ; Genetic Vectors ; Genotyping Techniques ; Inheritance Patterns ; Plants, Genetically Modified ; *RNA, Guide ; Soybeans/*genetics ; alpha-Linolenic Acid/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds.<br><br>RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation.<br><br>CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.},
}
@article {pmid31304778,
year = {2019},
author = {Krimsky, S},
title = {Breaking the germline barrier in a moral vacuum.},
journal = {Accountability in research},
volume = {26},
number = {6},
pages = {351-368},
doi = {10.1080/08989621.2019.1644171},
pmid = {31304778},
issn = {1545-5815},
mesh = {*CRISPR-Cas Systems ; China ; Embryo Research/*ethics ; Germ Cells/*cytology ; HIV Infections/prevention &amp; control ; Humans ; *Morals ; },
abstract = {In November 2018 a Chinese scientist claimed to have used CRISPR/Cas 9 technology to genetically modify two human embryos that were then gestated in one adult woman through an IVF pregnancy and brought to term. The twin girls are allegedly the first babies born with their prenatal genomes edited. Using both English language and Chinese supporting documents, this paper discusses the background of this human experiment, the social context of Chinese science, and the alleged ethical transgressions of its principal scientist.},
}
@article {pmid31304552,
year = {2019},
author = {Wood, KA and Rowlands, CF and Qureshi, WMS and Thomas, HB and Buczek, WA and Briggs, TA and Hubbard, SJ and Hentges, KE and Newman, WG and O'Keefe, RT},
title = {Disease modeling of core pre-mRNA splicing factor haploinsufficiency.},
journal = {Human molecular genetics},
volume = {28},
number = {22},
pages = {3704-3723},
pmid = {31304552},
issn = {1460-2083},
support = {BB/N000358/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Proliferation/genetics ; Craniofacial Abnormalities/genetics ; Endoplasmic Reticulum Stress/genetics ; Exons ; Gene Expression/genetics ; Gene Expression Regulation, Developmental/genetics ; HEK293 Cells ; Haploinsufficiency/genetics ; Humans ; Introns ; Mandibulofacial Dysostosis/*genetics ; Mutation ; Peptide Elongation Factors/*genetics/metabolism ; Phenotype ; RNA Precursors/metabolism ; RNA Splicing/genetics ; Ribonucleoprotein, U5 Small Nuclear/*genetics/metabolism ; Sequence Analysis, RNA/methods ; Spliceosomes/genetics ; },
abstract = {The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.},
}
@article {pmid31303969,
year = {2019},
author = {Chen, T and Olsen, I},
title = {Porphyromonas gingivalis and its CRISPR-Cas system.},
journal = {Journal of oral microbiology},
volume = {11},
number = {1},
pages = {1638196},
pmid = {31303969},
issn = {2000-2297},
support = {R37 DE016937/DE/NIDCR NIH HHS/United States ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) are immune systems in prokaryotes present in most Bacteria and Archaea. They provide adaptive immunity against foreign elements such as bacteriophages/viruses, plasmids and transposons. During immunization a small sequence of foreign DNA, a so-called spacer is integrated into the CRISPR locus in the host cell. Spacers are then transcribed into small RNA guides that direct cleavage of foreign DNA by Cas nucleases. Immunization through spacer acquisition is transferred vertically to the progeny. It is possible that this genetic immune system of bacteria participates in modulating the microbiome of 'chronic' periodontitis, in which Porphyromonas gingivalis has been identified as a keystone pathogen causing microbial dysbiosis. An in-depth review of our current knowledge on the CRISPR-Cas systems in P. gingivalis is given in this paper with the attempt to understand how this anaerobic bacterium may protect itself in the periodontal pocket where bacteriophages are abundant and even out-number bacteria.},
}
@article {pmid31303577,
year = {2019},
author = {Povedano, JM and Liou, J and Wei, D and Srivatsav, A and Kim, J and Xie, Y and Nijhawan, D and McFadden, DG},
title = {Engineering Forward Genetics into Cultured Cancer Cells for Chemical Target Identification.},
journal = {Cell chemical biology},
volume = {26},
number = {9},
pages = {1315-1321.e3},
pmid = {31303577},
issn = {2451-9448},
support = {R01 CA217333/CA/NCI NIH HHS/United States ; R37 CA226771/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Mismatch Repair/genetics ; Drug Discovery/*methods ; Genetic Engineering/*methods ; Genetic Testing/*methods ; Humans ; Mice ; Neoplasms/genetics ; },
abstract = {Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.},
}
@article {pmid31302752,
year = {2020},
author = {Yuan, H and Yu, T and Wang, L and Yang, L and Zhang, Y and Liu, H and Li, M and Tang, X and Liu, Z and Li, Z and Lu, C and Chen, X and Pang, D and Ouyang, H},
title = {Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {77},
number = {4},
pages = {719-733},
pmid = {31302752},
issn = {1420-9071},
mesh = {Animals ; Blastocyst/metabolism ; CRISPR-Cas Systems ; Cytidine/*genetics ; Galactosyltransferases/genetics ; Gene Editing/*methods ; Genetic Vectors/genetics ; Mixed Function Oxygenases/genetics ; Mutagenesis ; N-Acetylgalactosaminyltransferases/genetics ; RNA, Guide/genetics ; Swine/embryology/*genetics ; },
abstract = {Cytidine base editors (CBEs) have been demonstrated to be useful for precisely inducing C:G-to-T:A base mutations in various organisms. In this study, we showed that the BE4-Gam system induced the targeted C-to-T base conversion in porcine blastocysts at an efficiency of 66.7-71.4% via the injection of a single sgRNA targeting a xeno-antigen-related gene and BE4-Gam mRNA. Furthermore, the efficiency of simultaneous three gene base conversion via the injection of three targeting sgRNAs and BE4-Gam mRNA into porcine parthenogenetic embryos was 18.1%. We also obtained beta-1,4-N-acetyl-galactosaminyl transferase 2, alpha-1,3-galactosyltransferase, and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient pig by somatic cell nuclear transfer, which exhibited significantly decreased activity. In addition, a new CBE version (termed AncBE4max) was used to edit genes in blastocysts and porcine fibroblasts (PFFs) for the first time. While this new version demonstrated a three genes base-editing rate of 71.4% at the porcine GGTA1, B4galNT2, and CMAH loci, it increased the frequency of bystander edits, which ranged from 17.8 to 71.4%. In this study, we efficiently and precisely mutated bases in porcine blastocysts and PFFs using CBEs and successfully generated C-to-T and C-to-G mutations in pigs. These results suggest that CBEs provide a more simple and efficient method for improving economic traits, reducing the breeding cycle, and increasing disease tolerance in pigs, thus aiding in the development of human disease models.},
}
@article {pmid31301239,
year = {2019},
author = {EauClaire, SF and Webb, CJ},
title = {A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae.},
journal = {Yeast (Chichester, England)},
volume = {36},
number = {10},
pages = {607-615},
doi = {10.1002/yea.3432},
pmid = {31301239},
issn = {1097-0061},
mesh = {*Alleles ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Fungal ; Heterozygote ; Mutation ; Phenotype ; RNA Editing ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.},
}
@article {pmid31300803,
year = {2019},
author = {Shao, Y and Lu, N and Xue, X and Qin, Z},
title = {Creating functional chromosome fusions in yeast with CRISPR-Cas9.},
journal = {Nature protocols},
volume = {14},
number = {8},
pages = {2521-2545},
pmid = {31300803},
issn = {1750-2799},
mesh = {CRISPR-Cas Systems/*genetics ; Chromosomes, Fungal/*genetics ; Gene Editing/*methods ; Saccharomyces cerevisiae/*genetics ; },
abstract = {CRISPR-Cas9-facilitated functional chromosome fusion allows the generation of a series of yeast strains with progressively reduced chromosome numbers that are valuable resources for the study of fundamental concepts in chromosome biology, including replication, recombination and segregation. We created a new yeast strain with a single chromosome by using the protocol for chromosome fusion described herein. To ensure the accuracy of chromosome fusions in yeast, the long redundant repetitive sequences near linear chromosomal ends are deleted, and the fusion orders are correspondingly determined. Possible influence on gene expression is minimized to retain gene functionality. This protocol provides experimentally derived guidelines for the generation of functional chromosome fusions in yeast, especially for the deletion of repetitive sequences, the determination of the fusion order and cleavage sites, and primary evaluation of the functionality of chromosome fusions. Beginning with design, one round of typical chromosome fusion and functional verifications can be accomplished within 18 d.},
}
@article {pmid31300729,
year = {2019},
author = {Schneider-Futschik, EK},
title = {Beyond cystic fibrosis transmembrane conductance regulator therapy: a perspective on gene therapy and small molecule treatment for cystic fibrosis.},
journal = {Gene therapy},
volume = {26},
number = {9},
pages = {354-362},
pmid = {31300729},
issn = {1476-5462},
mesh = {Aminophenols/therapeutic use ; Animals ; CRISPR-Cas Systems ; Cystic Fibrosis/genetics/metabolism/*therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/*metabolism ; Gene Transfer Techniques ; *Genetic Therapy ; Genetic Vectors ; Humans ; Quinolones/therapeutic use ; },
abstract = {Cystic fibrosis (CF) is a life-limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent advent of the FDA-approved CFTR modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled treatment of the majority of patients suffering from CF. Even before the identification of the CFTR gene, gene therapy was put forward as a viable treatment option for this genetic condition. However, initial enthusiasm has been hampered as CFTR gene delivery to the lungs has proven to be more challenging than expected. This review covers the contemporary clinical and scientific knowledge base for small molecule CFTR modulator drug therapy, gene delivery vectors and CRISPR/Cas9 gene editing and highlights the prospect of these technologies for future treatment options.},
}
@article {pmid31300537,
year = {2019},
author = {Takeda, H and Kataoka, S and Nakayama, M and Ali, MAE and Oshima, H and Yamamoto, D and Park, JW and Takegami, Y and An, T and Jenkins, NA and Copeland, NG and Oshima, M},
title = {CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {31},
pages = {15635-15644},
pmid = {31300537},
issn = {1091-6490},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Colorectal Neoplasms/genetics/metabolism/pathology ; *Gene Expression Profiling ; *Gene Knockout Techniques ; Humans ; Mice ; Mice, Inbred NOD ; *Neoplasm Proteins/biosynthesis/genetics ; *Organoids/metabolism/pathology ; },
abstract = {Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.},
}
@article {pmid31300418,
year = {2019},
author = {Patil, A and Manzano, M and Gottwein, E},
title = {Genome-wide CRISPR screens reveal genetic mediators of cereblon modulator toxicity in primary effusion lymphoma.},
journal = {Blood advances},
volume = {3},
number = {14},
pages = {2105-2117},
pmid = {31300418},
issn = {2473-9537},
support = {R21 CA210904/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cullin Proteins/metabolism ; Drug Resistance, Neoplasm/genetics ; Endopeptidases/genetics ; Gene Knockdown Techniques ; Genome-Wide Association Study ; Humans ; Lenalidomide/adverse effects/pharmacology ; Lymphoma, Primary Effusion/drug therapy/*etiology/metabolism/pathology ; Models, Biological ; Thalidomide/analogs &amp; derivatives/pharmacology ; Ubiquitin-Protein Ligases ; },
abstract = {Genome-wide CRISPR/Cas9 screens represent a powerful approach to studying mechanisms of drug action and resistance. Cereblon modulating agents (CMs) have recently emerged as candidates for therapeutic intervention in primary effusion lymphoma (PEL), a highly aggressive cancer caused by Kaposi's sarcoma-associated herpesvirus. CMs bind to cereblon (CRBN), the substrate receptor of the cullin-RING type E3 ubiquitin ligase CRL4[CRBN], and thereby trigger the acquisition and proteasomal degradation of neosubstrates. Downstream mechanisms of CM toxicity are incompletely understood, however. To identify novel CM effectors and mechanisms of CM resistance, we performed positive selection CRISPR screens using 3 CMs with increasing toxicity in PEL: lenalidomide (LEN), pomalidomide (POM), and CC-122. Results identified several novel modulators of the activity of CRL4[CRBN] The number of genes whose inactivation confers resistance decreases with increasing CM efficacy. Only inactivation of CRBN conferred complete resistance to CC-122. Inactivation of the E2 ubiquitin conjugating enzyme UBE2G1 also conferred robust resistance against LEN and POM. Inactivation of additional genes, including the Nedd8-specific protease SENP8, conferred resistance to only LEN. SENP8 inactivation indirectly increased levels of unneddylated CUL4A/B, which limits CRL4[CRBN] activity in a dominant negative manner. Accordingly, sensitivity of SENP8-inactivated cells to LEN is restored by overexpression of CRBN. In sum, our screens identify several novel players in CRL4[CRBN] function and define pathways to CM resistance in PEL. These results provide rationale for increasing CM efficacy on patient relapse from a less-efficient CM. Identified genes could finally be developed as biomarkers to predict CM efficacy in PEL and other cancers.},
}
@article {pmid31299082,
year = {2019},
author = {Lyu, P and Javidi-Parsijani, P and Atala, A and Lu, B},
title = {Delivering Cas9/sgRNA ribonucleoprotein (RNP) by lentiviral capsid-based bionanoparticles for efficient 'hit-and-run' genome editing.},
journal = {Nucleic acids research},
volume = {47},
number = {17},
pages = {e99},
pmid = {31299082},
issn = {1362-4962},
mesh = {Aptamers, Nucleotide/chemistry/genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Capsid/metabolism ; Capsid Proteins/genetics ; Gene Editing/*methods ; *Gene Transfer Techniques ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Nanoparticles/chemistry/metabolism ; Ribonucleoproteins/*genetics ; },
abstract = {Transient expression of the CRISPR/Cas9 machinery will not only reduce risks of mutagenesis from off-target activities, but also decrease possible immune response to Cas9 protein. Building on our recent developing of a system able to package up to 100 copies of Cas9 mRNA in each lentivirus-like particle (LVLP) via the specific interaction between aptamer and aptamer-binding proteins (ABP), here we develop a lentiviral capsid-based bionanoparticle system, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP). We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the functions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specific interactions between ABP and aptamer, and sgRNA and Cas9 protein. These RNP bionanoparticles generated Indels on different targets in different cells with efficiencies similar to or better than our recently described Cas9 mRNA LVLPs. The new system showed fast action and reduced off-target rates, and makes it more convenient and efficient in delivering Cas9 RNPs for transient Cas9 expression and efficient genome editing.},
}
@article {pmid31299065,
year = {2019},
author = {Sowińska, W and Wawro, M and Solecka, A and Kasza, A},
title = {Potential limitations of the Sleeping Beauty transposon use in gene expression studies.},
journal = {Acta biochimica Polonica},
volume = {66},
number = {3},
pages = {263-268},
doi = {10.18388/abp.2019_2839},
pmid = {31299065},
issn = {1734-154X},
mesh = {Aged ; Brain Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA Transposable Elements/genetics ; Gene Editing ; Gene Knockout Techniques ; Glioblastoma/genetics/pathology ; Humans ; Interleukin-6/*genetics ; Male ; Mutagenesis, Insertional/*methods ; RNA, Messenger/genetics ; Ribonucleases/*genetics ; Transcription Factors/*genetics ; Transcriptome/*genetics ; Transfection ; Vascular Endothelial Growth Factor A/*genetics ; },
abstract = {MCPIP2 is the least known member of the MCPIP family of proteins. Recently we have found that it is a new RNase involved in transcript turnover. However, the full spectrum of its cellular targets is still unidentified. To discover transcripts which are regulated by this protein we have employed Sleeping Beauty transposons. This tool allows for rapid generation of a stable transgenic cell line with inducible expression of the desired gene. In this study, we analysed how the Sleeping Beauty system itself influences expression of chosen genes, namely IL-6, Regnase-1 and VEGF. We found that the system alone may influence expression of IL-6. Our results indicate that Sleeping Beauty transposons should be used with caution in studies that are focused on changes in the transcript level.},
}
@article {pmid31298604,
year = {2019},
author = {Guo, T and Zheng, F and Zeng, Z and Yang, Y and Li, Q and She, Q and Han, W},
title = {Cmr3 regulates the suppression on cyclic oligoadenylate synthesis by tag complementarity in a Type III-B CRISPR-Cas system.},
journal = {RNA biology},
volume = {16},
number = {10},
pages = {1513-1520},
pmid = {31298604},
issn = {1555-8584},
mesh = {Adenine Nucleotides/*biosynthesis ; Amino Acid Sequence ; *CRISPR-Cas Systems ; DNA Cleavage ; Oligoribonucleotides/*biosynthesis ; Sulfolobus/genetics/metabolism ; },
abstract = {Type III CRISPR-Cas systems code for a multi-subunit ribonucleoprotein (RNP) complex that mediates DNA cleavage and synthesizes cyclic oligoadenylate (cOA) second messenger to confer anti-viral immunity. Both immune activities are to be activated upon binding to target RNA transcripts by their complementarity to crRNA, and autoimmunity avoidance is determined by extended complementarity between the 5'-repeat tag of crRNA and 3'-flanking sequences of target transcripts (anti-tag). However, as to how the strategy could achieve stringent autoimmunity avoidance remained elusive. In this study, we systematically investigated how the complementarity of the crRNA 5'-tag and anti-tag (i.e., tag complementarity) could affect the interference activities (DNA cleavage activity and cOA synthesis activity) of Cmr-α, a type III-B system in Sulfolobus islandicus Rey15A. The results revealed an increasing suppression on both activities by increasing degrees of tag complementarity and a critical function of the 7[th] nucleotide of crRNA in avoiding autoimmunity. More importantly, mutagenesis of Cmr3α exerts either positive or negative effects on the cOA synthesis activity depending on the degrees of tag complementarity, suggesting that the subunit, coupling with the interaction between crRNA tag and anti-tag, function in facilitating immunity and avoiding autoimmunity in Type III-B systems.},
}
@article {pmid31297692,
year = {2019},
author = {Ouyang, Y},
title = {Understanding and breaking down the reproductive barrier between Asian and African cultivated rice: a new start for hybrid rice breeding.},
journal = {Science China. Life sciences},
volume = {62},
number = {8},
pages = {1114-1116},
doi = {10.1007/s11427-019-9592-6},
pmid = {31297692},
issn = {1869-1889},
mesh = {Africa ; Alleles ; Asia ; CRISPR-Cas Systems/genetics ; Genetic Variation ; Hybridization, Genetic ; Mutation ; Oryza/*genetics/*growth &amp; development ; Phylogeny ; Plant Breeding/*methods ; Reproduction ; },
}
@article {pmid31296872,
year = {2019},
author = {Santos-Pereira, JM and Gallardo-Fuentes, L and Neto, A and Acemel, RD and Tena, JJ},
title = {Pioneer and repressive functions of p63 during zebrafish embryonic ectoderm specification.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3049},
pmid = {31296872},
issn = {2041-1723},
support = {IJCI-2016-29884//Ministerio de Econom&#xed;a y Competitividad (Ministry of Economy and Competitiveness)/International ; BFU2014-58449-JIN//Ministerio de Econom&#xed;a y Competitividad (Ministry of Economy and Competitiveness)/International ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Chromatin/metabolism ; Down-Regulation ; Ectoderm/*embryology/metabolism ; Embryo, Nonmammalian ; Embryonic Development/*genetics ; Enhancer Elements, Genetic/genetics ; Epidermis/embryology/metabolism ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Models, Animal ; Neural Plate/*embryology/metabolism ; Phosphoproteins/genetics/*metabolism ; Protein Binding/genetics ; SOXB1 Transcription Factors/genetics/metabolism ; Trans-Activators/genetics/*metabolism ; Zebrafish/embryology ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.},
}
@article {pmid31295508,
year = {2019},
author = {Santos, KO and Costa-Filho, J and Spagnol, KL and Marins, LF},
title = {Comparing methods of genetic manipulation in Bacillus subtilis for expression of recombinant enzyme: Replicative or integrative (CRISPR-Cas9) plasmid?.},
journal = {Journal of microbiological methods},
volume = {164},
number = {},
pages = {105667},
doi = {10.1016/j.mimet.2019.105667},
pmid = {31295508},
issn = {1872-8359},
mesh = {6-Phytase/genetics ; Bacillus subtilis/*genetics ; Bacterial Proteins/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Replication ; Gene Expression Regulation, Bacterial ; Genes, Bacterial/genetics ; *Genetic Techniques ; *Plasmids ; *Recombinant Proteins ; Transformation, Bacterial ; },
abstract = {The present study evaluated the stability of Bacillus subtilis strains transformed with a replicative or integrative plasmid (via CRISPR-Cas9) to express a recombinant phytase. Both transformation methods did not affect the growth of B. subtilis, but the stability of the construct and the enzymatic activity was reduced in the strain transformed with the replicative plasmid.},
}
@article {pmid31295471,
year = {2019},
author = {Gupta, D and Bhattacharjee, O and Mandal, D and Sen, MK and Dey, D and Dasgupta, A and Kazi, TA and Gupta, R and Sinharoy, S and Acharya, K and Chattopadhyay, D and Ravichandiran, V and Roy, S and Ghosh, D},
title = {CRISPR-Cas9 system: A new-fangled dawn in gene editing.},
journal = {Life sciences},
volume = {232},
number = {},
pages = {116636},
doi = {10.1016/j.lfs.2019.116636},
pmid = {31295471},
issn = {1879-0631},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods/trends ; Genome ; Humans ; Plants/genetics ; },
abstract = {Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.},
}
@article {pmid31293824,
year = {2019},
author = {Ashari, KS and Roslan, NS and Omar, AR and Bejo, MH and Ideris, A and Mat Isa, N},
title = {Genome sequencing and analysis of Salmonella enterica subsp. enterica serovar Stanley UPM 517: Insights on its virulence-associated elements and their potentials as vaccine candidates.},
journal = {PeerJ},
volume = {7},
number = {},
pages = {e6948},
pmid = {31293824},
issn = {2167-8359},
abstract = {Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.},
}
@article {pmid31293420,
year = {2019},
author = {Lima, R and Del Fiol, FS and Balcão, VM},
title = {Prospects for the Use of New Technologies to Combat Multidrug-Resistant Bacteria.},
journal = {Frontiers in pharmacology},
volume = {10},
number = {},
pages = {692},
pmid = {31293420},
issn = {1663-9812},
abstract = {The increasing use of antibiotics is being driven by factors such as the aging of the population, increased occurrence of infections, and greater prevalence of chronic diseases that require antimicrobial treatment. The excessive and unnecessary use of antibiotics in humans has led to the emergence of bacteria resistant to the antibiotics currently available, as well as to the selective development of other microorganisms, hence contributing to the widespread dissemination of resistance genes at the environmental level. Due to this, attempts are being made to develop new techniques to combat resistant bacteria, among them the use of strictly lytic bacteriophage particles, CRISPR-Cas, and nanotechnology. The use of these technologies, alone or in combination, is promising for solving a problem that humanity faces today and that could lead to human extinction: the domination of pathogenic bacteria resistant to artificial drugs. This prospective paper discusses the potential of bacteriophage particles, CRISPR-Cas, and nanotechnology for use in combating human (bacterial) infections.},
}
@article {pmid31292210,
year = {2019},
author = {Sanchez, JC and Ollodart, A and Large, CRL and Clough, C and Alvino, GM and Tsuchiya, M and Crane, M and Kwan, EX and Kaeberlein, M and Dunham, MJ and Raghuraman, MK and Brewer, BJ},
title = {Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae.},
journal = {Genetics},
volume = {213},
number = {1},
pages = {229-249},
pmid = {31292210},
issn = {1943-2631},
support = {P41 GM103533/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R35 GM122497/GM/NIGMS NIH HHS/United States ; P30 AG013280/AG/NIA NIH HHS/United States ; T32 HG000035/HG/NHGRI NIH HHS/United States ; P01 AG001751/AG/NIA NIH HHS/United States ; T32 AG000057/AG/NIA NIH HHS/United States ; R01 AG056359/AG/NIA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Chromosomes, Fungal/genetics ; Consensus Sequence ; DNA, Ribosomal/*genetics ; Gene Editing/*methods ; Genetic Fitness ; Genotype ; Phenotype ; *Replication Origin ; Saccharomyces cerevisiae/*genetics ; },
abstract = {The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, thus the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae, developed independently but similar to one developed by others. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9-mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication have on cellular health. We find that long-term growth of the edited clones results in faster-growing suppressors that have acquired segmental aneusomy of the rDNA-containing region of chromosome XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently, and homogeneously edit the rDNA origin via CRISPR/Cas9.},
}
@article {pmid31291570,
year = {2019},
author = {Steinecke, A and Kurabayashi, N and Hayano, Y and Ishino, Y and Taniguchi, H},
title = {In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.},
journal = {Cell reports},
volume = {28},
number = {2},
pages = {325-331.e4},
pmid = {31291570},
issn = {2211-1247},
support = {R01 MH115917/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Genotype ; Mice ; Neurons/*metabolism ; Transfection ; },
abstract = {CRISPR/Cas-based technologies have revolutionized genetic approaches to addressing a wide range of neurobiological questions. The ability of CRISPR/Cas to introduce mutations into target genes allows us to perform in vivo loss-of-function experiments without generating genetically engineered mice. However, the lack of a reliable method to determine genotypes of individual CRISPR/Cas-transfected cells has made it impossible to unambiguously identify the genetic cause of their phenotypes in vivo. Here, we report a strategy for single-cell genotyping in CRISPR/Cas-transfected neurons that were phenotypically characterized in vivo. We show that re-sectioning of cortical slices and subsequent laser microdissection allow us to isolate individual CRISPR/Cas-transfected neurons. Sequencing of PCR products containing a CRISPR/Cas-targeted genomic region in single reference neurons provided genotypes that completely correspond with those deduced from their target protein expression and phenotypes. Thus, our study establishes a powerful strategy to determine the causality between genotypes and phenotypes in CRISPR/Cas-transfected neurons.},
}
@article {pmid31290648,
year = {2019},
author = {Jin, M and Garreau de Loubresse, N and Kim, Y and Kim, J and Yin, P},
title = {Programmable CRISPR-Cas Repression, Activation, and Computation with Sequence-Independent Targets and Triggers.},
journal = {ACS synthetic biology},
volume = {8},
number = {7},
pages = {1583-1589},
doi = {10.1021/acssynbio.9b00141},
pmid = {31290648},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/methods ; Genomics/methods ; Nucleic Acids/genetics ; RNA/genetics ; RNA, Guide/*genetics ; },
abstract = {The programmability of CRISPR-derived Cas9 as a sequence-specific DNA-targeting protein has made it a powerful tool for genomic manipulation in biological research and translational applications. Cas9 activity can be programmably engineered to respond to nucleic acids, but these efforts have focused primarily on single-input control of Cas9, and until recently, they were limited by sequence dependence between parts of the guide RNA and the sequence to be detected. Here, we not only design and present DNA- and RNA-sensing conditional guide RNA (cgRNA) that have no such sequence constraints, but also demonstrate a complete set of logical computations using these designs on DNA and RNA sequence inputs, including AND, OR, NAND, and NOR. The development of sequence-independent nucleic acid-sensing CRISPR-Cas9 systems with multi-input logic computation capabilities could lead to improved genome engineering and regulation as well as the construction of synthetic circuits with broader functionality.},
}
@article {pmid31289302,
year = {2019},
author = {Sakurai, T and Kamiyoshi, A and Takei, N and Watanabe, S and Sato, M and Shindo, T},
title = {Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {9923},
pmid = {31289302},
issn = {2045-2322},
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; *Mutation ; Polymerase Chain Reaction/*methods ; RNA, Untranslated/antagonists &amp; inhibitors/genetics ; Receptor Activity-Modifying Protein 1/antagonists &amp; inhibitors/genetics ; Receptor Activity-Modifying Protein 3/antagonists &amp; inhibitors/genetics ; Taq Polymerase/genetics/*metabolism ; Thermus/enzymology ; },
abstract = {We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5-2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8-2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).},
}
@article {pmid31289301,
year = {2019},
author = {Eaton, SL and Proudfoot, C and Lillico, SG and Skehel, P and Kline, RA and Hamer, K and Rzechorzek, NM and Clutton, E and Gregson, R and King, T and O'Neill, CA and Cooper, JD and Thompson, G and Whitelaw, CB and Wishart, TM},
title = {CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease).},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {9891},
pmid = {31289301},
issn = {2045-2322},
support = {096409/Z/11/Z/WT_/Wellcome Trust/United Kingdom ; BB/P013732/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/International ; MC_EX_MR/S022023/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; BB/J004316/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/International ; MR/S022023/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Male ; *Mutation ; Neuronal Ceroid-Lipofuscinoses/genetics/metabolism/*pathology ; *Phenotype ; Sheep ; Thiolester Hydrolases/*antagonists &amp; inhibitors/genetics ; },
abstract = {The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.},
}
@article {pmid31288753,
year = {2019},
author = {Lam, TJ and Ye, Y},
title = {Long reads reveal the diversification and dynamics of CRISPR reservoir in microbiomes.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {567},
pmid = {31288753},
issn = {1471-2164},
support = {R01 AI108888/AI/NIAID NIH HHS/United States ; R01 AI143254/AI/NIAID NIH HHS/United States ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Intergenic/genetics ; *Genetic Variation ; Microbiota/*genetics ; },
abstract = {BACKGROUND: Sequencing of microbiomes has accelerated the characterization of the diversity of CRISPR-Cas immune systems. However, the utilization of next generation short read sequences for the characterization of CRISPR-Cas dynamics remains limited due to the repetitive nature of CRISPR arrays. CRISPR arrays are comprised of short spacer segments (derived from invaders' genomes) interspaced between flanking repeat sequences. The repetitive structure of CRISPR arrays poses a computational challenge for the accurate assembly of CRISPR arrays from short reads. In this paper we evaluate the use of long read sequences for the analysis of CRISPR-Cas system dynamics in microbiomes.<br><br>RESULTS: We analyzed a dataset of Illumina's TruSeq Synthetic Long-Reads (SLR) derived from a gut microbiome. We showed that long reads captured CRISPR spacers at a high degree of redundancy, which highlights the spacer conservation of spacer sharing CRISPR variants, enabling the study of CRISPR array dynamics in ways difficult to achieve though short read sequences. We introduce compressed spacer graphs, a visual abstraction of spacer sharing CRISPR arrays, to provide a simplified view of complex organizational structures present within CRISPR array dynamics. Utilizing compressed spacer graphs, several key defining characteristics of CRISPR-Cas system dynamics were observed including spacer acquisition and loss events, conservation of the trailer end spacers, and CRISPR arrays' directionality (transcription orientation). Other result highlights include the observation of intense array contraction and expansion events, and reconstruction of a full-length genome for a potential invader (Faecalibacterium phage) based on identified spacers.<br><br>CONCLUSION: We demonstrate in an in silico system that long reads provide the necessary context for characterizing the organization of CRISPR arrays in a microbiome, and reveal dynamic and evolutionary features of CRISPR-Cas systems in a microbial population.},
}
@article {pmid31287866,
year = {2019},
author = {Shinoda, S and Kitagawa, S and Nakagawa, S and Wei, FY and Tomizawa, K and Araki, K and Araki, M and Suzuki, T and Suzuki, T},
title = {Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs.},
journal = {Nucleic acids research},
volume = {47},
number = {16},
pages = {8734-8745},
pmid = {31287866},
issn = {1362-4962},
mesh = {5-Methylcytosine/*metabolism ; Animals ; CRISPR-Cas Systems ; Fibroblasts/metabolism/pathology ; Gene Editing ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Methylation ; Methyltransferases/deficiency/*genetics ; Mice ; Mice, Knockout ; Mitochondria/*genetics/metabolism ; Nucleic Acid Conformation ; Oxidative Phosphorylation ; Primary Cell Culture ; Protein Transport ; *RNA Processing, Post-Transcriptional ; RNA, Messenger/genetics/metabolism ; RNA, Mitochondrial/*genetics/metabolism ; RNA, Transfer/*genetics/metabolism ; S-Adenosylmethionine/metabolism ; },
abstract = {Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.},
}
@article {pmid31287821,
year = {2019},
author = {Nord, H and Dennhag, N and Tydinger, H and von Hofsten, J},
title = {The zebrafish HGF receptor met controls migration of myogenic progenitor cells in appendicular development.},
journal = {PloS one},
volume = {14},
number = {7},
pages = {e0219259},
pmid = {31287821},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Movement/physiology ; Extremities/growth &amp; development ; Gene Expression Regulation, Developmental/genetics ; Muscle Development/physiology ; Muscle Fibers, Skeletal/metabolism ; Muscle, Skeletal/metabolism ; Muscles/metabolism ; Proto-Oncogene Proteins c-met/*genetics/*metabolism ; Stem Cells/metabolism ; Zebrafish/embryology/genetics ; Zebrafish Proteins/metabolism ; },
abstract = {The hepatocyte growth factor receptor C-met plays an important role in cellular migration, which is crucial for many developmental processes as well as for cancer cell metastasis. C-met has been linked to the development of mammalian appendicular muscle, which are derived from migrating muscle progenitor cells (MMPs) from within the somite. Mammalian limbs are homologous to the teleost pectoral and pelvic fins. In this study we used Crispr/Cas9 to mutate the zebrafish met gene and found that the MMP derived musculature of the paired appendages was severely affected. The mutation resulted in a reduced muscle fibre number, in particular in the pectoral abductor, and in a disturbed pectoral fin function. Other MMP derived muscles, such as the sternohyoid muscle and posterior hypaxial muscle were also affected in met mutants. This indicates that the role of met in MMP function and appendicular myogenesis is conserved within vertebrates.},
}
@article {pmid31287004,
year = {2019},
author = {Lalonde, S and Codina-Fauteux, VA and de Bellefon, SM and Leblanc, F and Beaudoin, M and Simon, MM and Dali, R and Kwan, T and Lo, KS and Pastinen, T and Lettre, G},
title = {Integrative analysis of vascular endothelial cell genomic features identifies AIDA as a coronary artery disease candidate gene.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {133},
pmid = {31287004},
issn = {1474-760X},
support = {MOP #136979//CIHR/Canada ; NTC-154083//CIHR/Canada ; U01CA200147/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Coronary Artery Disease/*genetics ; Endothelial Cells/metabolism ; Epigenomics ; Genome-Wide Association Study ; Humans ; Phospholipid Transfer Proteins/*genetics ; Regulatory Elements, Transcriptional ; Transcriptome ; },
abstract = {BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells.<br><br>RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNF&#x3b1;-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA.<br><br>CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.},
}
@article {pmid31286885,
year = {2019},
author = {Reimer, M and Pulakanti, K and Shi, L and Abel, A and Liang, M and Malarkannan, S and Rao, S},
title = {Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression.},
journal = {BMC developmental biology},
volume = {19},
number = {1},
pages = {16},
pmid = {31286885},
issn = {1471-213X},
support = {R01 CA179363/CA/NCI NIH HHS/United States ; R01 CA204231/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; DNA Methylation/*genetics ; DNA-Binding Proteins/*genetics ; Dioxygenases ; Embryonic Stem Cells/*cytology ; Gene Editing ; Gene Expression Regulation, Developmental/genetics ; Mice ; Mice, Knockout ; Proto-Oncogene Proteins/*genetics ; },
abstract = {BACKGROUND: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments.<br><br>RESULTS: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs.<br><br>CONCLUSIONS: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2[-/-] ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.},
}
@article {pmid31286070,
year = {2019},
author = {Horikoshi, N and Sharma, D and Leonard, F and Pandita, RK and Charaka, VK and Hambarde, S and Horikoshi, NT and Gaur Khaitan, P and Chakraborty, S and Cote, J and Godin, B and Hunt, CR and Pandita, TK},
title = {Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {253},
pmid = {31286070},
issn = {2399-3642},
support = {R01 CA129537/CA/NCI NIH HHS/United States ; R01 GM109768/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosome Structures/chemistry ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; Euchromatin/*chemistry ; HEK293 Cells ; HeLa Cells ; Heterochromatin ; Histones/*chemistry ; *Homologous Recombination ; Humans ; Kinetics ; Protein Processing, Post-Translational ; RNA, Guide/genetics ; RNA, Small Interfering/genetics ; },
abstract = {The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.},
}
@article {pmid31286062,
year = {2019},
author = {Görlich, S and Pawolski, D and Zlotnikov, I and Kröger, N},
title = {Control of biosilica morphology and mechanical performance by the conserved diatom gene Silicanin-1.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {245},
pmid = {31286062},
issn = {2399-3642},
mesh = {CRISPR-Cas Systems ; Cell Wall/*physiology ; Diatoms/genetics/*physiology ; Membrane Proteins/genetics/*physiology ; Microscopy, Atomic Force ; Morphogenesis ; Mutagenesis ; *Mutation ; Phenotype ; Plasmids/genetics ; Promoter Regions, Genetic ; Silicon Dioxide/*chemistry ; },
abstract = {The species-specifically patterned biosilica cell walls of diatoms are paradigms for biological mineral morphogenesis and the evolution of lightweight materials with exceptional mechanical performance. Biosilica formation is a membrane-mediated process that occurs in intracellular compartments, termed silica deposition vesicles (SDVs). Silicanin-1 (Sin1) is a highly conserved protein of the SDV membrane, but its role in biosilica formation has remained elusive. Here we generate Sin1 knockout mutants of the diatom Thalassiosira pseudonana. Although the mutants grow normally, they exhibit reduced biosilica content and morphological aberrations, which drastically compromise the strength and stiffness of their cell walls. These results identify Sin1 as essential for the biogenesis of mechanically robust diatom cell walls, thus providing an explanation for the conservation of this gene throughout the diatom realm. This insight paves the way for genetic engineering of silica architectures with desired structures and mechanical performance.},
}
@article {pmid31285745,
year = {2019},
author = {Liu, M and Han, X and Liu, H and Chen, D and Li, Y and Hu, W},
title = {The effects of CRISPR-Cas9 knockout of the TGF-β1 gene on antler cartilage cells in vitro.},
journal = {Cellular &amp; molecular biology letters},
volume = {24},
number = {},
pages = {44},
pmid = {31285745},
issn = {1689-1392},
mesh = {Animals ; Animals, Genetically Modified ; Antlers ; CRISPR-Cas Systems ; Cartilage/*metabolism/physiology ; Cell Line ; *Cell Proliferation ; Deer/genetics/*metabolism/physiology ; Gene Expression Regulation ; Gene Knockout Techniques ; Male ; *Signal Transduction ; Transforming Growth Factor beta1/genetics/*metabolism/physiology ; },
abstract = {BACKGROUND: Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor β (TGF-β). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-β1 during the rapid growth of sika deer antler.<br><br>METHODS: Three CRISPR-Cas9 knockout vectors targeting the TGF-&#x3b2;1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-&#x3b2;1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-&#x3b2; pathway-related genes were determined using a PCR array.<br><br>RESULTS: Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-&#x3b2;1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-&#x3b2; signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-&#x3b2; pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role.<br><br>CONCLUSIONS: TGF-&#x3b2;1 is a newly identified regulatory factor of rapid growth in sika deer antler.},
}
@article {pmid31285607,
year = {2019},
author = {Zhu, X and Clarke, R and Puppala, AK and Chittori, S and Merk, A and Merrill, BJ and Simonović, M and Subramaniam, S},
title = {Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {8},
pages = {679-685},
pmid = {31285607},
issn = {1545-9985},
support = {R01 GM097042/GM/NIGMS NIH HHS/United States ; R01 HD081534/HD/NICHD NIH HHS/United States ; ZIA BC010826-12/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/chemistry/metabolism/*ultrastructure ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/*metabolism/ultrastructure ; Macromolecular Substances/ultrastructure ; Models, Molecular ; Motion ; Protein Conformation ; Protein Domains ; RNA Editing ; RNA, Guide/metabolism ; Streptococcus pyogenes/enzymology ; },
abstract = {The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg[2+]. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.},
}
@article {pmid31285603,
year = {2019},
author = {Taylor, DW},
title = {The final cut: Cas9 editing.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {8},
pages = {669-670},
doi = {10.1038/s41594-019-0267-1},
pmid = {31285603},
issn = {1545-9985},
mesh = {CRISPR-Associated Protein 9/chemistry/metabolism/*ultrastructure ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/metabolism/ultrastructure ; Gene Editing/methods ; Macromolecular Substances/ultrastructure ; Models, Molecular ; Protein Conformation ; *RNA Editing ; Streptococcus pyogenes/enzymology ; },
}
@article {pmid31285598,
year = {2019},
author = {Urnov, FD},
title = {Hijack of CRISPR defences by selfish genes holds clinical promise.},
journal = {Nature},
volume = {571},
number = {7764},
pages = {180-181},
pmid = {31285598},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Gene Editing ; RNA ; },
}
@article {pmid31285596,
year = {2019},
author = {Böttcher, J and Dilworth, D and Reiser, U and Neumüller, RA and Schleicher, M and Petronczki, M and Zeeb, M and Mischerikow, N and Allali-Hassani, A and Szewczyk, MM and Li, F and Kennedy, S and Vedadi, M and Barsyte-Lovejoy, D and Brown, PJ and Huber, KVM and Rogers, CM and Wells, CI and Fedorov, O and Rumpel, K and Zoephel, A and Mayer, M and Wunberg, T and Böse, D and Zahn, S and Arnhof, H and Berger, H and Reiser, C and Hörmann, A and Krammer, T and Corcokovic, M and Sharps, B and Winkler, S and Häring, D and Cockcroft, XL and Fuchs, JE and Müllauer, B and Weiss-Puxbaum, A and Gerstberger, T and Boehmelt, G and Vakoc, CR and Arrowsmith, CH and Pearson, M and McConnell, DB},
title = {Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.},
journal = {Nature chemical biology},
volume = {15},
number = {8},
pages = {822-829},
doi = {10.1038/s41589-019-0310-x},
pmid = {31285596},
issn = {1552-4469},
mesh = {CRISPR-Cas Systems ; Cell Line ; Cell Proliferation/drug effects ; Cell Survival ; Gene Expression Regulation/drug effects ; Histone-Lysine N-Methyltransferase/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Nuclear Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Protein Domains ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; },
abstract = {Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.},
}
@article {pmid31284938,
year = {2019},
author = {Goulin, EH and Galdeano, DM and Granato, LM and Matsumura, EE and Dalio, RJD and Machado, MA},
title = {RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens.},
journal = {Microbiological research},
volume = {226},
number = {},
pages = {1-9},
doi = {10.1016/j.micres.2019.03.006},
pmid = {31284938},
issn = {1618-0623},
mesh = {Bacteria/genetics/pathogenicity ; CRISPR-Cas Systems ; Citrus/*genetics/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural ; Fungi/genetics/pathogenicity ; Gene Editing/methods ; Genetic Engineering ; Plant Diseases/*genetics/*prevention &amp; control ; *RNA Interference ; Viruses/genetics/pathogenicity ; },
abstract = {Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.},
}
@article {pmid31284391,
year = {2019},
author = {Chen, F and Yang, Y and Li, B and Liu, Z and Khan, F and Zhang, T and Zhou, G and Tu, J and Shen, J and Yi, B and Fu, T and Dai, C and Ma, C},
title = {Functional Analysis of M-Locus Protein Kinase Revealed a Novel Regulatory Mechanism of Self-Incompatibility in Brassica napus L.},
journal = {International journal of molecular sciences},
volume = {20},
number = {13},
pages = {},
pmid = {31284391},
issn = {1422-0067},
mesh = {Amino Acid Sequence ; Base Sequence ; Brassica napus/*enzymology/genetics/*physiology ; CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Expression Regulation, Plant ; Mutation/genetics ; Organ Specificity/genetics ; Phylogeny ; Plants, Genetically Modified ; Pollination ; Protein Isoforms/chemistry/genetics/metabolism ; Protein Kinase C/chemistry/genetics/*metabolism ; RNA Interference ; RNA, Messenger/genetics/metabolism ; Self-Incompatibility in Flowering Plants/*physiology ; },
abstract = {Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.},
}
@article {pmid31283004,
year = {2020},
author = {Dok, R and Bamps, M and Glorieux, M and Zhao, P and Sablina, A and Nuyts, S},
title = {Radiosensitization approaches for HPV-positive and HPV-negative head and neck squamous carcinomas.},
journal = {International journal of cancer},
volume = {146},
number = {4},
pages = {1075-1085},
pmid = {31283004},
issn = {1097-0215},
mesh = {Animals ; Benzimidazoles/administration &amp; dosage ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chemoradiotherapy/methods ; Chromones/administration &amp; dosage ; DNA Damage/drug effects/radiation effects ; DNA Repair/drug effects/radiation effects ; Female ; Head and Neck Neoplasms/pathology/*therapy/virology ; Humans ; Male ; Mice ; Middle Aged ; Morpholines/administration &amp; dosage ; Neoplasm Recurrence, Local/pathology/*therapy/virology ; Papillomaviridae/drug effects/genetics ; Papillomavirus Infections/pathology/*therapy/virology ; Radiation-Sensitizing Agents/*administration &amp; dosage ; Radiotherapy Dosage ; Squamous Cell Carcinoma of Head and Neck/pathology/*therapy/virology ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)-negative and HPV-positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV-positive and HPV-negative HNSCCs by performing a targeted CRISPR-Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV-positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end-joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient-derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.},
}
@article {pmid31282887,
year = {2019},
author = {Ma, L and Jang, L and Chen, J and Song, J and Yang, D and Zhang, J and Chen, YE and Xu, J},
title = {CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {148},
pages = {},
doi = {10.3791/59512},
pmid = {31282887},
issn = {1940-087X},
support = {R01 HL109946/HL/NHLBI NIH HHS/United States ; R01 HL136231/HL/NHLBI NIH HHS/United States ; R01 HL159900/HL/NHLBI NIH HHS/United States ; R21 OD023194/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/*methods ; Endonucleases/genetics ; Gene Editing/*methods ; Humans ; Rabbits ; Ribonucleoproteins/*metabolism ; Transfection ; },
abstract = {Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.},
}
@article {pmid31282064,
year = {2019},
author = {Chen, YY and Lin, Y and Han, PY and Jiang, S and Che, L and He, CY and Lin, YC and Lin, ZN},
title = {HBx combined with AFB1 triggers hepatic steatosis via COX-2-mediated necrosome formation and mitochondrial dynamics disorder.},
journal = {Journal of cellular and molecular medicine},
volume = {23},
number = {9},
pages = {5920-5933},
pmid = {31282064},
issn = {1582-4934},
mesh = {Aflatoxin B1/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cyclooxygenase 2/*metabolism ; Dynamins/metabolism ; Fatty Liver/*pathology ; Hep G2 Cells ; Hepatitis B/*pathology ; Hepatitis B virus ; Hepatocytes/transplantation/virology ; Humans ; Lipid Droplets/metabolism ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism ; Mitochondrial Dynamics/*physiology ; Nuclear Pore Complex Proteins/metabolism ; Protein Kinases/metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; RNA-Binding Proteins/metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; Trans-Activators/*metabolism ; Transplantation, Heterologous ; Viral Regulatory and Accessory Proteins/*metabolism ; },
abstract = {Hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure have been recognized as independent risk factors for the occurrence and exacerbation of hepatic steatosis but their combined impacts and the potential mechanisms remain to be further elucidated. Here, we showed that exposure to AFB1 impaired mitochondrial dynamics and increased intracellular lipid droplets (LDs) in the liver of HBV-transgenic mice in vivo and the hepatitis B virus X protein (HBx)-expressing human hepatocytes both ex vivo and in vitro. HBx combined with AFB1 exposure also up-regulated receptor interaction protein 1 (RIP1), receptor interaction protein 3 (RIP3) and activated mixed lineage kinase domain like protein (MLKL), providing evidence of necrosome formation in the hepatocytes. The shift of the mitochondrial dynamics towards imbalance of fission and fusion was rescued when MLKL was inhibited in the HBx and AFB1 co-treated hepatocytes. Most importantly, based on siRNA or CRISPR/Cas9 system, we found that the combination of HBx and AFB1 exposure increased cyclooxygenase-2 (COX-2) to mediate up-regulation of RIP3 and dynamin-related protein 1 (Drp1), which in turn promoted location of RIP3-MLKL necrosome on mitochondria, subsequently exacerbated steatosis in hepatocytes. Taken together, these findings advance the understanding of mechanism associated with HBx and AFB1-induced hepatic necrosome formation, mitochondrial dysfunction and steatosis and make COX-2 a good candidate for treatment.},
}
@article {pmid31280681,
year = {2020},
author = {Hameed, A and Mehmood, MA and Shahid, M and Fatma, S and Khan, A and Ali, S},
title = {Prospects for potato genome editing to engineer resistance against viruses and cold-induced sweetening.},
journal = {GM crops &amp; food},
volume = {11},
number = {4},
pages = {185-205},
pmid = {31280681},
issn = {2164-5701},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; *Plant Viruses ; Solanum tuberosum/*genetics ; },
abstract = {Crop improvement through transgenic technologies is commonly tagged with GMO (genetically-modified-organisms) where the presence of transgene becomes a big question for the society and the legislation authorities. However, new plant breeding techniques like CRISPR/Cas9 system [clustered regularly interspaced palindromic repeats (CRISPR)-associated 9] can overcome these limitations through transgene-free products. Potato (Solanum tuberosum L.) being a major food crop has the potential to feed the rising world population. Unfortunately, the cultivated potato suffers considerable production losses due to several pre- and post-harvest stresses such as plant viruses (majorly RNA viruses) and cold-induced sweetening (CIS; the conversion of sucrose to glucose and fructose inside cell vacuole). A number of strategies, ranging from crop breeding to genetic engineering, have been employed so far in potato for trait improvement. Recently, new breeding techniques have been utilized to knock-out potato genes/factors like eukaryotic translation initiation factors [elF4E and isoform elF(iso)4E)], that interact with viruses to assist viral infection, and vacuolar invertase, a core enzyme in CIS. In this context, CRISPR technology is predicted to reduce the cost of potato production and is likely to pass through the regulatory process being marker and transgene-free. The current review summarizes the potential application of the CRISPR/Cas9 system for traits improvement in potato. Moreover, the prospects for engineering resistance against potato fungal pathogens and current limitations/challenges are discussed.},
}
@article {pmid31280424,
year = {2019},
author = {Mitsui, R and Yamada, R and Ogino, H},
title = {CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {35},
number = {7},
pages = {111},
pmid = {31280424},
issn = {1573-0972},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Gene Expression Regulation, Fungal ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques/methods ; Metabolic Engineering/*methods ; Saccharomyces cerevisiae/genetics/*metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.},
}
@article {pmid31280136,
year = {2019},
author = {Giri, S and Purushottam, M and Viswanath, B and Muddashetty, RS},
title = {Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {39},
number = {},
pages = {101494},
doi = {10.1016/j.scr.2019.101494},
pmid = {31280136},
issn = {1876-7753},
mesh = {Blotting, Western ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics/physiology ; Cell Line ; Embryoid Bodies/cytology/metabolism ; Exons/genetics ; Fragile X Mental Retardation Protein/*genetics ; Genotype ; Human Embryonic Stem Cells/*cytology/*metabolism ; Humans ; Immunohistochemistry ; Karyotype ; RNA-Binding Proteins/genetics ; },
abstract = {Mutations in FMR1 gene is the cause of Fragile X Syndrome (FXS) leading inherited cause of intellectual disability and autism spectrum disorders. FMR1 gene encodes Fragile X Mental Retardation Protein (FMRP) which is a RNA binding protein and play important role in synaptic plasticity and translational regulation in neurons. We have generated a homozygous FMR1 knockout (FMR1-KO) hESC line using CRISPR/Cas9 based genome editing. It created a homozygous 280 nucleotide deletion at exon1, removing the start codon. This FMR1-KO cell line maintains stem cell like morphology, pluripotency, normal karyotype and ability to in-vitro differentiation.},
}
@article {pmid31280073,
year = {2019},
author = {Ju, A and Lee, SW and Lee, YE and Han, KC and Kim, JC and Shin, SC and Park, HJ and EunKyeong Kim, E and Hong, S and Jang, M},
title = {A carrier-free multiplexed gene editing system applicable for suspension cells.},
journal = {Biomaterials},
volume = {217},
number = {},
pages = {119298},
doi = {10.1016/j.biomaterials.2019.119298},
pmid = {31280073},
issn = {1878-5905},
mesh = {Animals ; B7-H1 Antigen/metabolism ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Clathrin/metabolism ; Cytotoxicity, Immunologic ; Endocytosis ; *Gene Editing ; Hepatitis A Virus Cellular Receptor 2/metabolism ; Humans ; Immunity ; Mice, Inbred C57BL ; Mice, Transgenic ; Programmed Cell Death 1 Ligand 2 Protein/metabolism ; Ribonucleoproteins/metabolism ; Suspensions/*chemistry ; },
abstract = {Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8[+] T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8[+] T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70-90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy.},
}
@article {pmid31279828,
year = {2019},
author = {Kamel, M and El-Sayed, A},
title = {Utilization of herpesviridae as recombinant viral vectors in vaccine development against animal pathogens.},
journal = {Virus research},
volume = {270},
number = {},
pages = {197648},
doi = {10.1016/j.virusres.2019.197648},
pmid = {31279828},
issn = {1872-7492},
mesh = {Animals ; CRISPR-Cas Systems ; *Genetic Vectors ; Genome, Viral ; Herpesviridae/*genetics ; Immunity, Humoral ; Vaccines, DNA/immunology ; Vaccines, Synthetic/genetics ; Viral Vaccines/*genetics/immunology ; },
abstract = {Throughout the past few decades, numerous viral species have been generated as vaccine vectors. Every viral vector has its own distinct characteristics. For example, the family herpesviridae encompasses several viruses that have medical and veterinary importance. Attenuated herpesviruses are developed as vectors to convey heterologous immunogens targeting several serious and crucial pathogens. Some of these vectors have already been licensed for use in the veterinary field. One of their prominent features is their capability to accommodate large amount of foreign DNA, and to stimulate both cell-mediated and humoral immune responses. A better understanding of vector-host interaction builds up a robust foundation for the future development of herpesviruses-based vectors. At the time, many molecular tools are applied to enable the generation of herpesvirus-based recombinant vaccine vectors such as BAC technology, homologous and two-step en passant mutagenesis, codon optimization, and the CRISPR/Cas9 system. This review article highlights the most important techniques applied in constructing recombinant herpesviruses vectors, advantages and disadvantages of each recombinant herpesvirus vector, and the most recent research regarding their use to control major animal diseases.},
}
@article {pmid31279547,
year = {2019},
author = {Selle, K and Andersen, JM and Barrangou, R},
title = {Short communication: Transcriptional response to a large genomic island deletion in the dairy starter culture Streptococcus thermophilus.},
journal = {Journal of dairy science},
volume = {102},
number = {9},
pages = {7800-7806},
doi = {10.3168/jds.2019-16397},
pmid = {31279547},
issn = {1525-3198},
mesh = {Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fermentation ; Gene Deletion ; Gene Expression Regulation, Bacterial/physiology ; *Genomic Islands ; Genomics ; Lactic Acid/metabolism ; Lactobacillus delbrueckii/metabolism ; Milk/*microbiology ; Streptococcus thermophilus/genetics/*metabolism ; Transcriptome ; Yogurt/*microbiology ; },
abstract = {Streptococcus thermophilus is a lactic acid bacterium widely used in the syntrophic fermentation of milk into yogurt and cheese. Streptococcus thermophilus has adapted to ferment milk primarily through reductive genome evolution but also through acquisition of genes conferring proto-cooperation with Lactobacillus bulgaricus and efficient metabolism of milk macronutrients. Genomic analysis of Strep. thermophilus strains suggests that mobile genetic elements have contributed to genomic evolution through horizontal gene transfer and genomic plasticity. We previously used the endogenous type II CRISPR-Cas [clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated sequences (Cas)] system in Strep. thermophilus to isolate derivatives lacking the chromosomal mobile genetic element and expandable island that display decreased fitness under routine culturing conditions. Of note, the Lac operon and Leloir pathway genes were deleted in the largest expendable genomic island (102 kbp), rendering the strain incapable of acidifying milk. However, the removal of other open reading frames in the same island had unclear effects on the fitness and regulatory networks of Strep. thermophilus. To uncover the physiological basis for the observed phenotypic changes and underlying regulatory networks affected by deletion of the 102-kbp genomic island in Strep. thermophilus, we analyzed the transcriptome of the mutant that lacked ∼5% of its genome. In addition to the loss of transcripts encoded by the deleted material, we detected a total of 56 genes that were differentially expressed, primarily encompassing 10 select operons. Several predicted metabolic pathways were affected, including amino acid and purine metabolism, oligopeptide transport, and iron transport. Collectively, these results suggest that deletion of a 102-kb genomic island in Strep. thermophilus influences compensatory transcription of starvation stress response genes and metabolic pathways involved in important niche-related adaptation.},
}
@article {pmid31279087,
year = {2019},
author = {Mion, S and Plener, L and Rémy, B and Daudé, D and Chabrière, &#xc9;},
title = {Lactonase SsoPox modulates CRISPR-Cas expression in gram-negative proteobacteria using AHL-based quorum sensing systems.},
journal = {Research in microbiology},
volume = {170},
number = {6-7},
pages = {296-299},
doi = {10.1016/j.resmic.2019.06.004},
pmid = {31279087},
issn = {1769-7123},
mesh = {Acyl-Butyrolactones/*metabolism ; Bacteriophages/genetics/growth &amp; development ; Biofilms/growth &amp; development ; CRISPR-Cas Systems/*genetics ; Chromobacterium/*genetics/isolation &amp; purification/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial/*genetics ; Humans ; Pseudomonas aeruginosa/*genetics/isolation &amp; purification/metabolism ; Quorum Sensing/*genetics ; },
abstract = {Quorum sensing (QS) is a molecular communication system that bacteria use to harmonize the regulation of genes in a cell density-dependent manner. In proteobacteria, QS is involved, among others, in virulence, biofilm formation or CRISPR-Cas gene regulation. Here, we report for the first time the effect of a QS-interfering enzyme to alter the regulation of CRISPR-Cas systems in model and clinical strains of Pseudomonas aeruginosa, as well as in the marine bacterium Chromobacterium violaceum CV12472. The expression of CRISPR-Cas genes decreased in most cases suggesting that enzymatic disruption of QS is promising for modulating phage-bacteria interactions.},
}
@article {pmid31278987,
year = {2019},
author = {Wang, Y and Chen, X and Liu, Z and Xu, J and Li, X and Bi, H and Andongma, AA and Niu, C and Huang, Y},
title = {Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella.},
journal = {Insect biochemistry and molecular biology},
volume = {112},
number = {},
pages = {103180},
doi = {10.1016/j.ibmb.2019.103180},
pmid = {31278987},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Expression Regulation, Developmental ; Genitalia/abnormalities ; Infertility/genetics ; Male ; Moths/*genetics/growth &amp; development ; Mutation ; Phylogeny ; Sequence Alignment ; Sex Differentiation/*genetics ; },
abstract = {DOUBLESEX (DSX): the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in Pxdsx[M] male mutants and male-specific transcripts were detected in Pxdsx[F] female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.},
}
@article {pmid31278903,
year = {2019},
author = {Chen, N and Hu, Z and Yang, Y and Han, H and Lei, H},
title = {Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells.},
journal = {Experimental eye research},
volume = {186},
number = {},
pages = {107716},
doi = {10.1016/j.exer.2019.107716},
pmid = {31278903},
issn = {1096-0007},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Cell Proliferation/physiology ; Cell Survival/physiology ; Cells, Cultured ; Epithelial Cells/*physiology ; Humans ; Mice ; Molecular Targeted Therapy/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-mdm2/*metabolism ; Retinal Pigment Epithelium/*physiology ; Tumor Suppressor Protein p53/metabolism ; Vitreoretinopathy, Proliferative/physiopathology ; Vitreous Body/*metabolism ; },
abstract = {Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2[SNP309G] contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2[SNP309 T/G]. lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 ± 0.2 fold increase in Mdm2 and a 2.0 ± 0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 ± 0.2 fold), survival against apoptosis (2.2 ± 0.2 fold), migration (10 ± 1.5%) and contraction (112.7 ± 14.1 mm[2]) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 - such as proliferative vitreoretinopathy.},
}
@article {pmid31278272,
year = {2019},
author = {Liu, TY and Liu, JJ and Aditham, AJ and Nogales, E and Doudna, JA},
title = {Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3001},
pmid = {31278272},
issn = {2041-1723},
support = {F32 GM122334/GM/NIGMS NIH HHS/United States ; P01 GM051487/GM/NIGMS NIH HHS/United States ; P01GM051487//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/International ; F32GM122334//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/International ; },
mesh = {Adaptive Immunity/*genetics ; Bacteriophages/immunology ; CRISPR-Cas Systems/*genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/immunology ; DNA, Single-Stranded/genetics/immunology/metabolism ; DNA-Directed RNA Polymerases/metabolism ; Plasmids/immunology ; RNA, Guide/genetics/immunology/metabolism ; Staphylococcus epidermidis/*genetics/immunology ; Thermus thermophilus/*genetics/immunology ; Transcription Elongation, Genetic/*immunology ; },
abstract = {Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.},
}
@article {pmid31277669,
year = {2019},
author = {Shabbir, MAB and Shabbir, MZ and Wu, Q and Mahmood, S and Sajid, A and Maan, MK and Ahmed, S and Naveed, U and Hao, H and Yuan, Z},
title = {CRISPR-cas system: biological function in microbes and its use to treat antimicrobial resistant pathogens.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {18},
number = {1},
pages = {21},
pmid = {31277669},
issn = {1476-0711},
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacteria/*drug effects/enzymology/*genetics ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Gene Editing/*methods ; },
abstract = {The development of antibiotic resistance in bacteria is a major public health threat. Infection rates of resistant pathogens continue to rise against nearly all antimicrobials, which has led to development of different strategies to combat the antimicrobial resistance. In this review, we discuss how the newly popular CRISPR-cas system has been applied to combat antibiotic resistance in both extracellular and intracellular pathogens. We also review a recently developed method in which nano-size CRISPR complex was used without any phage to target the mecA gene. However, there is still challenge to practice these methods in field against emerging antimicrobial resistant pathogens.},
}
@article {pmid31277550,
year = {2019},
author = {Corts, AD and Thomason, LC and Gill, RT and Gralnick, JA},
title = {Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection.},
journal = {ACS synthetic biology},
volume = {8},
number = {8},
pages = {1877-1889},
doi = {10.1021/acssynbio.9b00188},
pmid = {31277550},
issn = {2161-5063},
support = {HHSN261200800001E/CA/NCI NIH HHS/United States ; },
mesh = {Bacteria/enzymology/genetics/metabolism ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; DNA, Single-Stranded/genetics ; Electroporation ; Gene Editing ; Mutation/genetics ; Plasmids/genetics ; Recombinases/genetics/metabolism ; Shewanella/*enzymology/genetics/*metabolism ; },
abstract = {Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≃5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatches, deletions, and small insertions. Surprisingly, we found that use of CRISPR/Cas9 alone allows selection of recombinase-independent S. oneidensis mutations, albeit at lower efficiency and frequency. With synthesized single-stranded DNA as substrates for homologous recombination and Cas9 as a counter-selectable marker, this new system provides a rapid, scalable, versatile, and scarless tool that will accelerate progress in Shewanella genomic engineering.},
}
@article {pmid31277549,
year = {2019},
author = {Zuo, F and Zeng, Z and Hammarström, L and Marcotte, H},
title = {Inducible Plasmid Self-Destruction (IPSD) Assisted Genome Engineering in Lactobacilli and Bifidobacteria.},
journal = {ACS synthetic biology},
volume = {8},
number = {8},
pages = {1723-1729},
doi = {10.1021/acssynbio.9b00114},
pmid = {31277549},
issn = {2161-5063},
mesh = {Bifidobacterium/*genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Engineering/methods ; Genome, Bacterial/genetics ; Lactobacillus/*genetics ; Plasmids/*genetics ; },
abstract = {Genome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria, but still have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called inducible plasmid self-destruction (IPSD) was successfully used to perform gene knockout and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.},
}
@article {pmid31276587,
year = {2019},
author = {Van Haute, L and Lee, SY and McCann, BJ and Powell, CA and Bansal, D and Vasiliauskaitė, L and Garone, C and Shin, S and Kim, JS and Frye, M and Gleeson, JG and Miska, EA and Rhee, HW and Minczuk, M},
title = {NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs.},
journal = {Nucleic acids research},
volume = {47},
number = {16},
pages = {8720-8733},
pmid = {31276587},
issn = {1362-4962},
support = {104640/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; MC_UU_00015/4/MRC_/Medical Research Council/United Kingdom ; C6946/A14492/CRUK_/Cancer Research UK/United Kingdom ; 092096/Z/10/Z/WT_/Wellcome Trust/United Kingdom ; C13474/A18583/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {5-Methylcytosine/*metabolism ; Animals ; CRISPR-Cas Systems ; Eczema/*genetics/metabolism/pathology ; Facies ; Fibroblasts/metabolism/pathology ; Gene Editing ; Gene Knockout Techniques ; Growth Disorders/*genetics/metabolism/pathology ; HEK293 Cells ; Humans ; Intellectual Disability/*genetics/metabolism/pathology ; Methylation ; Methyltransferases/deficiency/*genetics ; Mice ; Mice, Knockout ; Microcephaly/*genetics/metabolism/pathology ; Mitochondria/genetics/metabolism ; Nucleic Acid Conformation ; Oxidative Phosphorylation ; Primary Cell Culture ; Protein Transport ; *RNA Processing, Post-Transcriptional ; RNA, Messenger/genetics/metabolism ; RNA, Mitochondrial/*genetics/metabolism ; RNA, Transfer/*genetics/metabolism ; },
abstract = {Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.},
}
@article {pmid31276437,
year = {2019},
author = {Toro, N and Martínez-Abarca, F and Mestre, MR and González-Delgado, A},
title = {Multiple origins of reverse transcriptases linked to CRISPR-Cas systems.},
journal = {RNA biology},
volume = {16},
number = {10},
pages = {1486-1493},
pmid = {31276437},
issn = {1555-8584},
mesh = {*CRISPR-Cas Systems ; Chromosome Mapping ; Genetic Linkage ; Genetic Variation ; Introns ; Phylogeny ; Prokaryotic Cells/metabolism ; RNA-Directed DNA Polymerase/*genetics/metabolism ; },
abstract = {Prokaryotic genomes harbour a plethora of uncharacterized reverse transcriptases (RTs). RTs phylogenetically related to those encoded by group-II introns have been found associated with type III CRISPR-Cas systems, adjacent or fused at the C-terminus to Cas1. It is thought that these RTs may have a relevant function in the CRISPR immune response mediating spacer acquisition from RNA molecules. The origin and relationships of these RTs and the ways in which the various protein domains evolved remain matters of debate. We carried out a large survey of annotated RTs in databases (198,760 sequences) and constructed a large dataset of unique representative sequences (9,141). The combined phylogenetic reconstruction and identification of the RTs and their various protein domains in the vicinity of CRISPR adaptation and effector modules revealed three different origins for these RTs, consistent with their emergence on multiple occasions: a larger group that have evolved from group-II intron RTs, and two minor lineages that may have arisen more recently from Retron/retron-like sequences and Abi-P2 RTs, the latter associated with type I-C systems. We also identified a particular group of RTs associated with CRISPR-cas loci in clade 12, fused C-terminally to an archaeo-eukaryotic primase (AEP), a protein domain (AE-Prim_S_like) forming a particular family within the AEP proper clade. Together, these data provide new insight into the evolution of CRISPR-Cas/RT systems.},
}
@article {pmid31276295,
year = {2019},
author = {Li, G and Zhou, S and Li, C and Cai, B and Yu, H and Ma, B and Huang, Y and Ding, Y and Liu, Y and Ding, Q and He, C and Zhou, J and Wang, Y and Zhou, G and Li, Y and Yan, Y and Hua, J and Petersen, B and Jiang, Y and Sonstegard, T and Huang, X and Chen, Y and Wang, X},
title = {Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair.},
journal = {The FEBS journal},
volume = {286},
number = {23},
pages = {4675-4692},
doi = {10.1111/febs.14983},
pmid = {31276295},
issn = {1742-4658},
support = {2017NY-072//Local Grants/International ; 2018KJXX-009//Local Grants/International ; NXTS201601//Local Grants/International ; CARS-39//China Agriculture Research System/International ; 31772571//National Natural Science Foundation of China/International ; 31572369//National Natural Science Foundation of China/International ; 31872332//National Natural Science Foundation of China/International ; },
mesh = {Alleles ; Animals ; Base Pairing/genetics ; Blotting, Western ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Codon, Nonsense/*genetics ; Fibroblast Growth Factor 5/genetics ; Fibroblasts/cytology/metabolism ; Gene Editing ; Goats ; Hair/*growth &amp; development/*metabolism ; Male ; Mutation/genetics ; Phenotype ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {The ability to alter single bases without homology directed repair (HDR) of double-strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5, which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor (BE3) plasmid and four different sgRNAs into caprine fibroblasts were ineffective in altering FGF5. In contrast, all five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF5 expression. PCR amplicon and whole-genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single-cell embryos. Our results support further optimization of BEs for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation.},
}
@article {pmid31273196,
year = {2019},
author = {Buchmuller, BC and Herbst, K and Meurer, M and Kirrmaier, D and Sass, E and Levy, ED and Knop, M},
title = {Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2960},
pmid = {31273196},
issn = {2041-1723},
support = {KN498/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/International ; INST 35/1314-1 FUGG//Deutsche Forschungsgemeinschaft (German Research Foundation)/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clone Cells ; Gene Library ; Genetic Engineering ; *Genetic Techniques ; Genome, Fungal ; Green Fluorescent Proteins/metabolism ; Nuclear Proteins/metabolism ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.},
}
@article {pmid31273110,
year = {2019},
author = {Hou, Z and Zhang, Y},
title = {Inserting DNA with CRISPR.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6448},
pages = {25-26},
doi = {10.1126/science.aay2056},
pmid = {31273110},
issn = {1095-9203},
support = {R00 GM117268/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; RNA ; *Transposases ; },
}
@article {pmid31273104,
year = {2019},
author = {Cohen, J},
title = {CRISPR patent fight revived.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6448},
pages = {15-16},
doi = {10.1126/science.365.6448.15b},
pmid = {31273104},
issn = {1095-9203},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Mice ; Patents as Topic/*legislation &amp; jurisprudence ; United States ; },
}
@article {pmid31272828,
year = {2019},
author = {Gao, Y and Hisey, E and Bradshaw, TWA and Erata, E and Brown, WE and Courtland, JL and Uezu, A and Xiang, Y and Diao, Y and Soderling, SH},
title = {Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.},
journal = {Neuron},
volume = {103},
number = {4},
pages = {583-597.e8},
pmid = {31272828},
issn = {1097-4199},
support = {R01 MH103374/MH/NIMH NIH HHS/United States ; R01 NS102456/NS/NINDS NIH HHS/United States ; T32 GM007171/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Brain/cytology/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Dependovirus/genetics ; Gene Editing/methods ; Gene Knock-In Techniques/*methods ; Genetic Vectors/genetics ; Genomics/*methods ; Humans ; Immunochemistry/methods ; Inteins ; Mice ; Mutagenesis, Insertional ; Nerve Tissue Proteins/chemistry/genetics ; Protein Engineering/*methods ; *Protein Processing, Post-Translational ; Proteomics ; RNA, Guide/genetics ; Recombinant Fusion Proteins/genetics ; Sequence Homology, Nucleic Acid ; },
abstract = {Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.},
}
@article {pmid31272708,
year = {2019},
author = {Kim, DY and Moon, SB and Kim, YS},
title = {Improving CRISPR Technology to Sustain Animal Welfare: Response to Bailey.},
journal = {Trends in biotechnology},
volume = {37},
number = {9},
pages = {922-923},
doi = {10.1016/j.tibtech.2019.05.010},
pmid = {31272708},
issn = {1879-3096},
mesh = {*Animal Welfare ; Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Genetic Engineering/*ethics/methods ; },
}
@article {pmid31272394,
year = {2019},
author = {Lyzenga, WJ and Harrington, M and Bekkaoui, D and Wigness, M and Hegedus, DD and Rozwadowski, KL},
title = {CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {292},
pmid = {31272394},
issn = {1471-2229},
mesh = {Base Sequence ; Brassicaceae/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Globulins/*genetics/metabolism ; Plant Proteins/*genetics/metabolism ; Seed Storage Proteins/*genetics/metabolism ; Seeds/genetics ; },
abstract = {BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds.<br><br>RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids.<br><br>CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.},
}
@article {pmid31271858,
year = {2019},
author = {Zhang, Y and Shen, S and Zhao, G and Xu, CF and Zhang, HB and Luo, YL and Cao, ZT and Shi, J and Zhao, ZB and Lian, ZX and Wang, J},
title = {In situ repurposing of dendritic cells with CRISPR/Cas9-based nanomedicine to induce transplant tolerance.},
journal = {Biomaterials},
volume = {217},
number = {},
pages = {119302},
doi = {10.1016/j.biomaterials.2019.119302},
pmid = {31271858},
issn = {1878-5905},
mesh = {Animals ; CD40 Antigens/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Dendritic Cells/*immunology ; Disease Models, Animal ; Endocytosis ; Graft Rejection/immunology ; Graft Survival/immunology ; Lymphocyte Activation/immunology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; *Nanomedicine ; RNA, Guide/metabolism ; Transplantation Tolerance/*immunology ; },
abstract = {Organ transplantation is the only effective method to treat end-stage organ failure. However, it is continuously plagued by immune rejection, which is mostly caused by T cell-mediated reactions. Dendritic cells (DCs) are professional antigen-presenting cells, and blocking the costimulatory signaling molecule CD40 in DCs inhibits T cell activation and induces transplant tolerance. In this study, to relieve graft rejection, Cas9 mRNA (mCas9) and a guide RNA targeting the costimulatory molecule CD40 (gCD40) were prepared and encapsulated into poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLAN), denoted CLANmCas9/gCD40. CLAN effectively delivered mCas9/gCD40 into DCs and disrupted CD40 in DCs at the genomic level both in vitro and in vivo. After intravenous injection into an acute mouse skin transplant model, CLANmCas9/gCD40-mediated CD40 disruption significantly inhibited T cell activation, which reduced graft damage and prolonged graft survival. This work provides a promising strategy for reprogramming DCs with nanoparticles carrying the CRISPR/Cas9 system to abate transplant rejection.},
}
@article {pmid31270316,
year = {2019},
author = {Liao, C and Ttofali, F and Slotkowski, RA and Denny, SR and Cecil, TD and Leenay, RT and Keung, AJ and Beisel, CL},
title = {Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2948},
pmid = {31270316},
issn = {2041-1723},
support = {2017-137//Camille and Henry Dreyfus Foundation (Camille and Henry Dreyfus Foundation, Inc.)/International ; R35 GM119561/GM/NIGMS NIH HHS/United States ; Gift #3926//Agilent Technologies/International ; DP1 DA044359/DA/NIDA NIH HHS/United States ; 1DP1DA044359//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; 1R35GM119561//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; },
mesh = {Base Sequence ; CRISPR-Associated Proteins/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; Endonucleases/metabolism ; *Gene Library ; *Genetic Techniques ; HEK293 Cells ; Humans ; Nucleic Acid Conformation ; Plasmids/metabolism ; RNA/*biosynthesis ; RNA, Messenger/genetics/metabolism ; Saccharomyces cerevisiae/metabolism ; },
abstract = {CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.},
}
@article {pmid31270169,
year = {2019},
author = {Guerra, AJ},
title = {mSphere of Influence: Ushering in the CRISPR Revolution to Toxoplasma Biology.},
journal = {mSphere},
volume = {4},
number = {4},
pages = {},
pmid = {31270169},
issn = {2379-5042},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Silencing ; Point Mutation ; Toxoplasma/*genetics ; },
abstract = {Alfredo J. Guerra works in the field of molecular parasitology and structural biology. In this mSphere of Influence article, he reflects on how "Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9" by Bang Shen et al. (mBio 5:e01114-14, 2014, https://doi.org/10.1128/mBio.01114-14) and "Efficient Genome Engineering of Toxoplasma gondii using CRISPR/CAS9" by Saima M. Sidik et al. (PLoS One 9:e100450, 2014, https://doi.org/10.1371/journal.pone.0100450) made an impact on him by successfully implementing strategies to genetically manipulate T. gondii using CRISPR/CAS9 gene editing technology.},
}
@article {pmid31268597,
year = {2019},
author = {Xu, H and Shi, J and Gao, H and Liu, Y and Yang, Z and Shao, F and Dong, N},
title = {The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin.},
journal = {The EMBO journal},
volume = {38},
number = {13},
pages = {e101996},
pmid = {31268597},
issn = {1460-2075},
support = {81788104//National Natural Science Foundation of China (NSFC)/International ; 81671987//National Natural Science Foundation of China (NSFC)/International ; 2017YFA0505900//Ministry of Science and Technology of the People's Republic of China (MOST)/International ; 2016YFA0501500//Ministry of Science and Technology of the People's Republic of China (MOST)/International ; XDB08020202//CAS | Chinese Academy of Sciences Key Project (CAS Key Project)/International ; },
mesh = {Animals ; Antigens, Bacterial/*adverse effects ; Apoptosis Regulatory Proteins/*chemistry/*metabolism ; Bacterial Toxins/*adverse effects ; CRISPR-Cas Systems ; Caspase 1/metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Inflammasomes/drug effects ; Macrophages/*drug effects/metabolism ; Mice ; Protein Domains ; Proteolysis/drug effects ; RAW 264.7 Cells ; RNA, Small Interfering/pharmacology ; Ubiquitin-Conjugating Enzymes/metabolism ; Ubiquitin-Protein Ligases/*chemistry/*metabolism ; Ubiquitination/drug effects ; },
abstract = {Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.},
}
@article {pmid31268303,
year = {2019},
author = {Hirosawa, M and Fujita, Y and Saito, H},
title = {Cell-Type-Specific CRISPR Activation with MicroRNA-Responsive AcrllA4 Switch.},
journal = {ACS synthetic biology},
volume = {8},
number = {7},
pages = {1575-1582},
doi = {10.1021/acssynbio.9b00073},
pmid = {31268303},
issn = {2161-5063},
mesh = {5' Untranslated Regions/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Gene Editing/methods ; Genome/genetics ; HeLa Cells ; Humans ; MicroRNAs/*genetics ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Anti-CRISPR proteins have the potential to regulate CRISPR-Cas systems in a cell-type-specific manner. To selectively edit the genome in target cells, we controlled the expression of AcrllA4, a Streptococcus pyogenes Cas9 inhibitor, based on endogenous microRNA (miRNA) activity. We designed a miRNA-responsive AcrllA4 switch, which is a synthetic mRNA that contains a completely complementary sequence to an arbitrary miRNA at the 5'-UTR region and encodes AcrllA4. Together with the Cas9- or dCas9-VPR-guide RNA complex, this switch functions as a cell-specific Cas9 or dCas9-VPR activator that induces gene knockout or activation depending on the target miRNA. By sensing intracellular miRNAs, the conditional CRISPR-Cas9 ON system that we report could provide a powerful tool for future therapeutic applications and genome engineering.},
}
@article {pmid31267870,
year = {2019},
author = {Lau, CH and Tin, C},
title = {The Synergy between CRISPR and Chemical Engineering.},
journal = {Current gene therapy},
volume = {19},
number = {3},
pages = {147-171},
doi = {10.2174/1566523219666190701100556},
pmid = {31267870},
issn = {1875-5631},
mesh = {*CRISPR-Cas Systems ; Chemical Engineering/*methods ; *Drug Synergism ; *Gene Editing ; Genetic Engineering/*methods ; *Genetic Therapy ; Genetic Vectors/administration &amp; dosage/*genetics ; Humans ; },
abstract = {Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.},
}
@article {pmid31267640,
year = {2019},
author = {Yosef, I and Edry-Botzer, L and Globus, R and Shlomovitz, I and Munitz, A and Gerlic, M and Qimron, U},
title = {A genetic system for biasing the sex ratio in mice.},
journal = {EMBO reports},
volume = {20},
number = {8},
pages = {e48269},
pmid = {31267640},
issn = {1469-3178},
support = {3-14351//Ministry of Science and Technology, Israel/International ; //The Varda and Boaz Dotan Research Center/International ; 268/14//Israel Science Foundation/International ; 1416/15//Israel Science Foundation/International ; 818/18//Israel Science Foundation/International ; 811322//EC | H2020 | H2020 Priority Excellent Science | H2020 European Research Council (ERC)/International ; 336079//EC | FP7 | FP7 Ideas: European Research Council (FP7 Ideas)/International ; },
mesh = {Animals ; Breeding ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; *Chromosomes, Mammalian ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crosses, Genetic ; Female ; Fertilization ; Gene Editing/*methods ; *Genome ; Male ; Mice ; Oocytes/cytology/metabolism ; RNA, Guide/genetics/metabolism ; *Sex Determination Processes ; *Sex Ratio ; Spermatozoa/cytology/metabolism ; },
abstract = {Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.},
}
@article {pmid31267591,
year = {2019},
author = {Kilic, S and Lezaja, A and Gatti, M and Bianco, E and Michelena, J and Imhof, R and Altmeyer, M},
title = {Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments.},
journal = {The EMBO journal},
volume = {38},
number = {16},
pages = {e101379},
pmid = {31267591},
issn = {1460-2075},
support = {150690//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (SNF)/International ; 179057//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (SNF)/International ; 714326//EC | H2020 | H2020 Priority Excellent Science | H2020 European Research Council (ERC)/International ; 16B078//Novartis Stiftung f&#xfc;r Medizinisch-BiologischeForschung (Novartis Foundation for Medical-Biological Research)/International ; KFS-4406-02-2018//Krebsliga Schweiz (Swiss Cancer League)/International ; },
mesh = {Adaptor Proteins, Signal Transducing/metabolism ; Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; *DNA Repair ; Dogs ; Histones/metabolism ; Humans ; Liquid-Liquid Extraction/*methods ; MCF-7 Cells ; Madin Darby Canine Kidney Cells ; Optogenetics ; Osmotic Pressure ; Stress, Physiological ; Tumor Suppressor Protein p53/*metabolism ; Tumor Suppressor p53-Binding Protein 1/genetics/*metabolism ; },
abstract = {The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions.},
}
@article {pmid31267558,
year = {2019},
author = {Paulitschke, V and Eichhoff, O and Gerner, C and Paulitschke, P and Bileck, A and Mohr, T and Cheng, PF and Leitner, A and Guenova, E and Saulite, I and Freiberger, SN and Irmisch, A and Knapp, B and Zila, N and Chatziisaak, TP and Stephan, J and Mangana, J and Kunstfeld, R and Pehamberger, H and Aebersold, R and Dummer, R and Levesque, MP},
title = {Proteomic identification of a marker signature for MAPKi resistance in melanoma.},
journal = {The EMBO journal},
volume = {38},
number = {15},
pages = {e95874},
pmid = {31267558},
issn = {1460-2075},
support = {1334/M//Promedica Stiftung/International ; //MUW Mobility Grant/International ; //Comprehensive Cancer Center Forschungsf&#xf6;rderung der Initiative Krebsforschung, MedUni Wien/International ; FK-14-032//Forschungskredit of the University of Zurich/International ; //URPP "Translational Cancer Research" Grant/International ; //Louis Widmer Grant/International ; //B&#xfc;rgermeistergrant/International ; //Verein Hautkrebsforschung/International ; PMPDP3_151326/SNSF_/Swiss National Science Foundation/Switzerland ; 31003A_166435/SNSF_/Swiss National Science Foundation/Switzerland ; //Verein f&#xfc;r Hautkrebsforschung/International ; KLS 3151-02-2013//Hochspezialisierte Medizin Schwerpunkt Immunologie (HSM-2-Immunologie) Schweiz and Krebsliga Schweiz/International ; //Prof. Bruno Bloch Foundation/International ; },
mesh = {Adult ; Aged ; Carbamates/pharmacology ; Cell Adhesion ; Cell Line, Tumor ; Disease Progression ; *Drug Resistance, Neoplasm ; Epithelial-Mesenchymal Transition ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Insulin-Like Growth Factor Binding Proteins/blood/*metabolism ; Male ; Melanoma/drug therapy/genetics/*metabolism ; Middle Aged ; Protein Interaction Maps ; Protein Kinase Inhibitors/*pharmacology ; Proteomics/*methods ; RNA-Binding Proteins/*metabolism ; Sequence Analysis, RNA ; Sulfonamides/pharmacology ; Survival Analysis ; Up-Regulation ; Vemurafenib/pharmacology ; },
abstract = {MAPK inhibitors (MAPKi) show outstanding clinical response rates in melanoma patients harbouring BRAF mutations, but resistance is common. The ability of melanoma cells to switch from melanocytic to mesenchymal phenotypes appears to be associated with therapeutic resistance. High-throughput, subcellular proteome analyses and RNAseq on two panels of primary melanoma cells that were either sensitive or resistant to MAPKi revealed that only 15 proteins were sufficient to distinguish between these phenotypes. The two proteins with the highest discriminatory power were PTRF and IGFBP7, which were both highly upregulated in the mesenchymal-resistant cells. Proteomic analysis of CRISPR/Cas-derived PTRF knockouts revealed targets involved in lysosomal activation, endocytosis, pH regulation, EMT, TGFβ signalling and cell migration and adhesion, as well as a significantly reduced invasive index and ability to form spheres in 3D culture. Overexpression of PTRF led to MAPKi resistance, increased cell adhesion and sphere formation. In addition, immunohistochemistry of patient samples showed that PTRF expression levels were a significant biomarker of poor progression-free survival, and IGFBP7 levels in patient sera were shown to be higher after relapse.},
}
@article {pmid31267498,
year = {2019},
author = {Ng, CL and Fidock, DA},
title = {Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2013},
number = {},
pages = {123-140},
pmid = {31267498},
issn = {1940-6029},
support = {R01 AI109023/AI/NIAID NIH HHS/United States ; R01 AI124678/AI/NIAID NIH HHS/United States ; R37 AI050234/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antimalarials/therapeutic use ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Malaria, Falciparum/*drug therapy/*prevention &amp; control ; Plasmodium falciparum/drug effects/*pathogenicity ; },
abstract = {Malaria continues to be a global health burden, threatening over 40% of the world's population. Drug resistance in Plasmodium falciparum, the etiological agent of the majority of human malaria cases, is compromising elimination efforts. New approaches to treating drug-resistant malaria benefit from defining resistance liabilities of known antimalarial agents and compounds in development and defining genetic changes that mediate loss of parasite susceptibility. Here, we present protocols for in vitro selection of drug-resistant parasites and for site-directed gene editing of candidate resistance mediators to test for causality.},
}
@article {pmid31266936,
year = {2019},
author = {Dash, PK and Kaminski, R and Bella, R and Su, H and Mathews, S and Ahooyi, TM and Chen, C and Mancuso, P and Sariyer, R and Ferrante, P and Donadoni, M and Robinson, JA and Sillman, B and Lin, Z and Hilaire, JR and Banoub, M and Elango, M and Gautam, N and Mosley, RL and Poluektova, LY and McMillan, J and Bade, AN and Gorantla, S and Sariyer, IK and Burdo, TH and Young, WB and Amini, S and Gordon, J and Jacobson, JM and Edagwa, B and Khalili, K and Gendelman, HE},
title = {Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2753},
pmid = {31266936},
issn = {2041-1723},
support = {P01 DA028555/DA/NIDA NIH HHS/United States ; P30 MH062261/MH/NIMH NIH HHS/United States ; P30MH092177//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/International ; R01NS036126//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/International ; R24 OD018546/OD/NIH HHS/United States ; R01 MH115860/MH/NIMH NIH HHS/United States ; R01MH110360//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/International ; P01 NS043985/NS/NINDS NIH HHS/United States ; P01DA037830//U.S. Department of Health &amp; Human Services | NIH | National Institute on Drug Abuse (NIDA)/International ; R01DA042706//U.S. Department of Health &amp; Human Services | NIH | National Institute on Drug Abuse (NIDA)/International ; R01NS034239//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/International ; P30 MH092177/MH/NIMH NIH HHS/United States ; R01 AG043540/AG/NIA NIH HHS/United States ; P30MH062261//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/International ; R01 MH110360/MH/NIMH NIH HHS/United States ; R01 NS034239/NS/NINDS NIH HHS/United States ; },
mesh = {Adoptive Transfer ; Animals ; Anti-HIV Agents/*administration &amp; dosage ; *CRISPR-Cas Systems ; Combined Modality Therapy ; DNA, Viral/genetics/immunology ; Gene Editing ; HIV Infections/drug therapy/immunology/*therapy/virology ; HIV-1/*genetics/immunology/physiology ; Humans ; Mice ; Treatment Outcome ; Virus Latency ; },
abstract = {Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.},
}
@article {pmid31266898,
year = {2019},
author = {Sims, J and Copenhaver, GP and Schlögelhofer, P},
title = {Meiotic DNA Repair in the Nucleolus Employs a Nonhomologous End-Joining Mechanism.},
journal = {The Plant cell},
volume = {31},
number = {9},
pages = {2259-2275},
pmid = {31266898},
issn = {1532-298X},
mesh = {Arabidopsis/*genetics/physiology ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins ; *Cell Nucleolus ; Chromatin ; Chromosomal Proteins, Non-Histone ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics/*physiology ; DNA Ligases/metabolism ; DNA Repair/*physiology ; DNA, Plant ; DNA, Ribosomal/genetics ; Genomic Instability ; Homologous Recombination ; Meiosis/*physiology ; Plants, Genetically Modified ; Rad51 Recombinase/genetics/metabolism ; },
abstract = {Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo nonallelic exchanges. During meiosis, DNA double-strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR), which subsequently ensures genetic exchange and chromosome disjunction. In Arabidopsis (Arabidopsis thaliana), we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications and meiotic cohesin and allows only very limited meiosis-specific DSB formation. DNA lesions within the rDNA arrays are repaired in an RAD51-independent but LIG4-dependent manner, establishing that nonhomologous end-joining maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats, we validate our findings and demonstrate that the rDNA constitutes an HR-refractory genome environment.},
}
@article {pmid31266804,
year = {2019},
author = {Talbot, BE and Vandorpe, DH and Stotter, BR and Alper, SL and Schlondorff, JS},
title = {Transmembrane insertases and N-glycosylation critically determine synthesis, trafficking, and activity of the nonselective cation channel TRPC6.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {34},
pages = {12655-12669},
pmid = {31266804},
issn = {1083-351X},
support = {P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 DK115438/DK/NIDDK NIH HHS/United States ; T32 DK007726/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Cell Death/drug effects ; Cell Membrane/drug effects/*metabolism ; Gain of Function Mutation ; Glycosylation/drug effects ; HEK293 Cells ; Humans ; Membrane Proteins/metabolism ; N-Acetylglucosaminyltransferases/metabolism ; Podocytes/drug effects/metabolism ; Protein Binding/drug effects ; RNA, Guide/metabolism ; TRPC6 Cation Channel/*metabolism ; },
abstract = {Transient receptor potential cation channel subfamily C member 6 (TRPC6) is a widely expressed ion channel. Gain-of-function mutations in the human TRPC6 channel cause autosomal-dominant focal segmental glomerulosclerosis, but the molecular components involved in disease development remain unclear. Here, we found that overexpression of gain-of-function TRPC6 channel variants is cytotoxic in cultured cells. Exploiting this phenotype in a genome-wide CRISPR/Cas screen for genes whose inactivation rescues cells from TRPC6-associated cytotoxicity, we identified several proteins essential for TRPC6 protein expression, including the endoplasmic reticulum (ER) membrane protein complex transmembrane insertase. We also identified transmembrane protein 208 (TMEM208), a putative component of a signal recognition particle-independent (SND) ER protein-targeting pathway, as being necessary for expression of TRPC6 and several other ion channels and transporters. TRPC6 expression was also diminished by loss of the previously uncharacterized WD repeat domain 83 opposite strand (WDR83OS), which interacted with both TRPC6 and TMEM208. Additionally enriched among the screen hits were genes involved in N-linked protein glycosylation. Deletion of the mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1), necessary for the generation of complex N-linked glycans, abrogated TRPC6 gain-of-function variant-mediated Ca[2+] influx and extracellular signal-regulated kinase activation in HEK cells, but failed to diminish cytotoxicity in cultured podocytes. However, mutating the two TRPC6 N-glycosylation sites abrogated the cytotoxicity of mutant TRPC6 and reduced its surface expression. These results expand the targets of TMEM208-mediated ER translocation to include multipass transmembrane proteins and suggest that TRPC6 N-glycosylation plays multiple roles in modulating channel trafficking and activity.},
}
@article {pmid31266541,
year = {2019},
author = {Schneider, D and Chua, RL and Molitor, N and Hamdan, FH and Rettenmeier, EM and Prokakis, E and Mishra, VK and Kari, V and Wegwitz, F and Johnsen, SA and Kosinsky, RL},
title = {The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells.},
journal = {Clinical epigenetics},
volume = {11},
number = {1},
pages = {98},
pmid = {31266541},
issn = {1868-7083},
mesh = {Apoptosis ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms/*genetics ; Epigenesis, Genetic ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; *Gene Silencing ; HCT116 Cells ; HT29 Cells ; Histones/metabolism ; Humans ; Ubiquitin-Protein Ligases/*genetics ; Ubiquitination ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro.<br><br>METHODS: We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo&#xae; S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK.<br><br>RESULTS: Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis.<br><br>CONCLUSIONS: Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.},
}
@article {pmid31266059,
year = {2019},
author = {Cross, SH and Mckie, L and Keighren, M and West, K and Thaung, C and Davey, T and Soares, DC and Sanchez-Pulido, L and Jackson, IJ},
title = {Missense Mutations in the Human Nanophthalmos Gene TMEM98 Cause Retinal Defects in the Mouse.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {60},
number = {8},
pages = {2875-2887},
pmid = {31266059},
issn = {1552-5783},
support = {MC_PC_U127561112/MRC_/Medical Research Council/United Kingdom ; MC_UU_00007/15/MRC_/Medical Research Council/United Kingdom ; MC_UU_00007/4/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Axial Length, Eye/pathology ; CRISPR-Cas Systems ; Electroretinography ; Female ; Gene Expression Regulation/physiology ; Humans ; Immunohistochemistry ; Male ; Membrane Proteins/*genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Microphthalmos/*genetics/pathology ; Microscopy, Electron, Transmission ; *Mutation, Missense ; Ophthalmoscopy ; Photoreceptor Cells, Vertebrate/*pathology ; Polymerase Chain Reaction ; Retinal Diseases/*genetics/pathology ; },
abstract = {PURPOSE: We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs.<br><br>METHODS: The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes.<br><br>RESULTS: An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions.<br><br>CONCLUSIONS: Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.},
}
@article {pmid31265461,
year = {2019},
author = {Bai, M and Han, Y and Wu, Y and Liao, J and Li, L and Wang, L and Li, Q and Xing, W and Chen, L and Zou, W and Li, J},
title = {Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development.},
journal = {PLoS biology},
volume = {17},
number = {7},
pages = {e3000350},
pmid = {31265461},
issn = {1545-7885},
mesh = {Animals ; Bone Development/*genetics ; CRISPR-Cas Systems ; Cells, Cultured ; *Gene Expression Regulation, Developmental ; Gene Targeting/*methods ; Genetic Testing/*methods ; Haploidy ; Hematopoietic Cell Growth Factors/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Lectins, C-Type/genetics/metabolism ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells/*metabolism ; Relaxin/genetics/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- mice exhibited small skeletal size only at birth, Irx5-/- mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.},
}
@article {pmid31265056,
year = {2020},
author = {Ratti, V and Nanda, S and Eszterhas, SK and Howell, AL and Wallace, DI},
title = {A mathematical model of HIV dynamics treated with a population of gene-edited haematopoietic progenitor cells exhibiting threshold phenomenon.},
journal = {Mathematical medicine and biology : a journal of the IMA},
volume = {37},
number = {2},
pages = {212-242},
doi = {10.1093/imammb/dqz011},
pmid = {31265056},
issn = {1477-8602},
support = {I01 BX003241/BX/BLRD VA/United States ; },
mesh = {Basic Reproduction Number/statistics &amp; numerical data ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes/immunology/virology ; CRISPR-Cas Systems ; Computational Biology ; Computer Simulation ; Gene Editing/methods ; HIV Infections/immunology/*therapy/virology ; *HIV-1/immunology/pathogenicity ; Hematopoietic Stem Cell Transplantation/methods ; Host Microbial Interactions/genetics/immunology ; Humans ; Mathematical Concepts ; *Models, Biological ; Models, Immunological ; Receptors, CCR5/deficiency/genetics ; },
abstract = {The use of gene-editing technology has the potential to excise the CCR5 gene from haematopoietic progenitor cells, rendering their differentiated CD4-positive (CD4+) T cell descendants HIV resistant. In this manuscript, we describe the development of a mathematical model to mimic the therapeutic potential of gene editing of haematopoietic progenitor cells to produce a class of HIV-resistant CD4+ T cells. We define the requirements for the permanent suppression of viral infection using gene editing as a novel therapeutic approach. We develop non-linear ordinary differential equation models to replicate HIV production in an infected host, incorporating the most appropriate aspects found in the many existing clinical models of HIV infection, and extend this model to include compartments representing HIV-resistant immune cells. Through an analysis of model equilibria and stability and computation of $R_0$ for both treated and untreated infections, we show that the proposed therapy has the potential to suppress HIV infection indefinitely and return CD4+ T cell counts to normal levels. A computational study for this treatment shows the potential for a successful 'functional cure' of HIV. A sensitivity analysis illustrates the consistency of numerical results with theoretical results and highlights the parameters requiring better biological justification. Simulations of varying level production of HIV-resistant CD4+ T cells and varying immune enhancements as the result of these indicate a clear threshold response of the model and a range of treatment parameters resulting in a return to normal CD4+ T cell counts.},
}
@article {pmid31264792,
year = {2019},
author = {Huang, H and Zou, X and Zhong, L and Hou, Y and Zhou, J and Zhang, Z and Xing, X and Sun, J},
title = {CRISPR/dCas9-mediated activation of multiple endogenous target genes directly converts human foreskin fibroblasts into Leydig-like cells.},
journal = {Journal of cellular and molecular medicine},
volume = {23},
number = {9},
pages = {6072-6084},
pmid = {31264792},
issn = {1582-4934},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Cellular Reprogramming/*genetics ; Chorionic Gonadotropin/biosynthesis ; Fibroblasts/cytology ; Foreskin/*cytology/growth &amp; development ; GATA4 Transcription Factor/genetics ; Humans ; Leydig Cells/*metabolism ; Male ; RNA, Guide/*genetics ; Steroidogenic Factor 1/genetics ; Testosterone/biosynthesis/genetics ; Transcription Factors/genetics ; Transcriptional Activation/genetics ; },
abstract = {Recently, Leydig cell (LC) transplantation has been revealed as a promising strategy for treating male hypogonadism; however, the key problem restricting the application of LC transplantation is a severe lack of seed cells. It seems that targeted activation of endogenous genes may provide a potential alternative. Therefore, the aim of this study was to determine whether targeted activation of Nr5a1, Gata4 and Dmrt1 (NGD) via the CRISPR/dCas9 synergistic activation mediator system could convert human foreskin fibroblasts (HFFs) into functional Leydig-like cells. We first constructed the stable Hsd3b-dCas9-MPH-HFF cell line using the Hsd3b-EGFP, dCas9-VP64 and MS2-P65-HSF1 lentiviral vectors and then infected it with single guide RNAs. Next, we evaluated the reprogrammed cells for their reprogramming efficiency, testosterone production characteristics and expression levels of Leydig steroidogenic markers by quantitative real-time polymerase chain reaction or Western blotting. Our results showed that the reprogramming efficiency was close to 10% and that the reprogrammed Leydig-like cells secreted testosterone rapidly and, more importantly, responded effectively to stimulation with human chorionic gonadotropin and expressed Leydig steroidogenic markers. Our findings demonstrate that simultaneous targeted activation of the endogenous NGD genes directly reprograms HFFs into functional Leydig-like cells, providing an innovative technology that may have promising potential for the treatment of male androgen deficiency diseases.},
}
@article {pmid31262785,
year = {2019},
author = {Elaimy, AL and Wang, M and Sheel, A and Brown, CW and Walker, MR and Amante, JJ and Xue, W and Chan, A and Baer, CE and Goel, HL and Mercurio, AM},
title = {Real-time imaging of integrin β4 dynamics using a reporter cell line generated by Crispr/Cas9 genome editing.},
journal = {Journal of cell science},
volume = {132},
number = {15},
pages = {},
pmid = {31262785},
issn = {1477-9137},
support = {R01 CA168464/CA/NCI NIH HHS/United States ; F30 CA232657/CA/NCI NIH HHS/United States ; R50 CA221780/CA/NCI NIH HHS/United States ; F30 CA206271/CA/NCI NIH HHS/United States ; R01 CA203439/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Integrin alpha6/genetics/metabolism ; Integrin beta4/genetics/*metabolism ; Microscopy, Video ; Transcription Factors/genetics/metabolism ; YAP-Signaling Proteins ; },
abstract = {The ability to monitor changes in the expression and localization of integrins is essential for understanding their contribution to development, tissue homeostasis and disease. Here, we pioneered the use of Crispr/Cas9 genome editing to tag an allele of the β4 subunit of the α6β4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin β4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin β4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin β4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin β4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin β4 expression in cells proximal to the wound edge was evident, and a population of integrin β4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin β4 dynamics during invasion and metastasis. Moreover, these integrin β4 reporter cells should facilitate studies on the contribution of this integrin to mammary gland biology and cancer.This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid31262744,
year = {2019},
author = {Savona, MR and Rathmell, JC},
title = {Mitochondrial Homeostasis in AML and Gasping for Response in Resistance to BCL2 Blockade.},
journal = {Cancer discovery},
volume = {9},
number = {7},
pages = {831-833},
pmid = {31262744},
issn = {2159-8290},
support = {R01 CA217987/CA/NCI NIH HHS/United States ; },
mesh = {Bridged Bicyclo Compounds, Heterocyclic ; Homeostasis ; Humans ; *Leukemia, Myeloid, Acute ; Proto-Oncogene Proteins c-bcl-2 ; Sulfonamides ; },
abstract = {Understanding resistance to BCL2 inhibition is a critical scientific and clinical challenge. In this issue of Cancer Discovery, two laboratories use unbiased approaches of large loss-of-function CRISPR/Cas 9 screens to discover targetable liabilities in cell signaling and metabolism to acute myeloid leukemia resistant to BCL2 inhibition.See related article by Chen et al., p. 890.See related article by Nechiporuk et al., p. 910.},
}
@article {pmid31262711,
year = {2019},
author = {Aripaka, K and Gudey, SK and Zang, G and Schmidt, A and Åhrling, SS and Österman, L and Bergh, A and von Hofsten, J and Landström, M},
title = {TRAF6 function as a novel co-regulator of Wnt3a target genes in prostate cancer.},
journal = {EBioMedicine},
volume = {45},
number = {},
pages = {192-207},
pmid = {31262711},
issn = {2352-3964},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Inflammation/*genetics/pathology ; Low Density Lipoprotein Receptor-Related Protein-5/genetics ; Male ; Prostatic Neoplasms/*genetics/pathology ; TNF Receptor-Associated Factor 6/*genetics ; Wnt Signaling Pathway/genetics ; Wnt3A Protein/*genetics ; Zebrafish ; Zebrafish Proteins/*genetics ; beta Catenin/genetics ; },
abstract = {BACKGROUND: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of β-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling.<br><br>METHODS: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling.<br><br>FINDINGS: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of &#x3b2;-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes.<br><br>INTERPRETATION: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression. FUND: KAW 2012.0090, CAN 2017/544, Swedish Medical Research Council (2016-02513), Prostatacancerf&#xf6;rbundet, Konung Gustaf V:s Frimurarestiftelse and Cancerforskningsfonden Norrland. The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.},
}
@article {pmid31262344,
year = {2019},
author = {Teng, F and Guo, L and Cui, T and Wang, XG and Xu, K and Gao, Q and Zhou, Q and Li, W},
title = {CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {132},
pmid = {31262344},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; DNA/*analysis ; Escherichia coli ; *Genetic Techniques ; Genetic Testing/*methods ; Humans ; Sensitivity and Specificity ; },
abstract = {CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However, precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an efficient and highly practical platform for DNA detection.},
}
@article {pmid31262257,
year = {2019},
author = {Scholz, I and Lott, SC and Behler, J and Gärtner, K and Hagemann, M and Hess, WR},
title = {Divergent methylation of CRISPR repeats and cas genes in a subtype I-D CRISPR-Cas-system.},
journal = {BMC microbiology},
volume = {19},
number = {1},
pages = {147},
pmid = {31262257},
issn = {1471-2180},
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA (Cytosine-5-)-Methyltransferases ; *DNA Methylation ; DNA, Bacterial/genetics ; Nucleotide Motifs ; Recombination, Genetic ; Sequence Analysis, DNA ; Synechocystis/*genetics ; },
abstract = {BACKGROUND: The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality.<br><br>RESULTS: The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated ([5m]CG[6m]ATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600&#x2009;bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the [m5]CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the [5m]C methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of [m5]CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GG[m4]CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11&#x2009;kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system.<br><br>CONCLUSIONS: The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high [5m]C methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the [5m]C methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of [m5]CGATCG DNA-methylation marks in conjugation and DNA transformation.},
}
@article {pmid31261719,
year = {2019},
author = {Han, SW and Jung, BK and Park, SH and Ryu, KY},
title = {Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {20},
number = {13},
pages = {},
pmid = {31261719},
issn = {1422-0067},
mesh = {Aptamers, Nucleotide/genetics/metabolism ; *CRISPR-Cas Systems ; Genetic Engineering/*methods ; HEK293 Cells ; Humans ; Recombinant Proteins/genetics/metabolism ; Ubiquitin-Conjugating Enzymes/*genetics/metabolism ; Up-Regulation ; },
abstract = {Ubiquitin is required under both normal and stress conditions. Under stress conditions, upregulation of the polyubiquitin gene UBC is essential to meet the requirement of increased ubiquitin levels to confer stress resistance. However, UBC upregulation is usually observed only under stress conditions and not under normal conditions. Therefore, it has not been possible to upregulate UBC under normal conditions to study the effect of excess ubiquitin on cellular machinery. Recently, the CRISPR/Cas9 system has been widely used in biological research as a useful tool to study gene disruption effects. In this study, using an inducible CRISPR/Cas9 variant, a dCas9-VP64 fusion protein, combined with a single guide RNA (sgRNA) containing MS2 aptamer loops and MS2-p65-HSF1, we developed a system to increase the ubiquitin pool via upregulation of UBC. Although it is challenging to upregulate the expression of a gene that is already expressed at high levels, the significance of our system is that UBC upregulation can be induced in an efficient, reversible manner that is compatible with cellular processes, even under normal conditions. This system can be used to study ubiquitin pool dynamics and it will be a useful tool in identifying the role of ubiquitin under normal and stress conditions.},
}
@article {pmid31260812,
year = {2019},
author = {Hua, K and Zhang, J and Botella, JR and Ma, C and Kong, F and Liu, B and Zhu, JK},
title = {Perspectives on the Application of Genome-Editing Technologies in Crop Breeding.},
journal = {Molecular plant},
volume = {12},
number = {8},
pages = {1047-1059},
doi = {10.1016/j.molp.2019.06.009},
pmid = {31260812},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; Genome, Plant/genetics/physiology ; Plant Breeding/*methods ; Plants, Genetically Modified/genetics/*metabolism/*physiology ; },
abstract = {Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progenies of multiple generations derived from time-consuming genetic crosses. Genome-editing systems, in contrast, provide the means to rapidly modify genomes in a precise and predictable way, making it possible to introduce improvements directly into elite varieties. Here, we describe the range of applications available to agricultural researchers using existing genome-editing tools. In addition to providing examples of genome-editing applications in crop breeding, we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement.},
}
@article {pmid31260171,
year = {2020},
author = {Zou, B and Desmidt, AA and Mittal, R and Yan, D and Richmond, M and Tekin, M and Liu, XZ and Lu, Z},
title = {The Generation of Zebrafish Mariner Model Using the CRISPR/Cas9 System.},
journal = {Anatomical record (Hoboken, N.J. : 2007)},
volume = {303},
number = {3},
pages = {556-562},
doi = {10.1002/ar.24221},
pmid = {31260171},
issn = {1932-8494},
support = {R01DC009645/DC/NIDCD NIH HHS/United States ; R01DC012836/DC/NIDCD NIH HHS/United States ; R21DC009879/DC/NIDCD NIH HHS/United States ; R01DC012115/DC/NIDCD NIH HHS/United States ; R01DC012546/DC/NIDCD NIH HHS/United States ; R01DC005575/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal/physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Deafness/*genetics ; Disease Models, Animal ; Gene Editing/*methods ; Myosins/genetics ; *Phenotype ; Zebrafish/genetics ; Zebrafish Proteins/*genetics ; },
abstract = {Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as a powerful tool for gene function studies and has great potential for gene therapy. Although CRISPR/Cas9 has been widely used in many research fields, only a few successful zebrafish models have been established using this technology in hearing research. In this study, we successfully created zebrafish mariner mutants by targeting the motor head domain of Myo7aa using CRISPR/Cas9. The CRISPR/Cas9-generated mutants showed unbalanced swimming behavior and disorganized sterocilia of inner ear hair cells, which resemble the phenotype of the zebrafish mariner mutants. In addition, we found that CRISPR/Cas9-generated mutants have reduced number of stereociliary bundles of inner ear hair cells and have significant hearing loss. Furthermore, phenotypic analysis was performed on F0 larvae within the first week post fertilization, which dramatically shortens data collection period. Therefore, results of this study showed that CRISPR/Cas9 is a quick and effective method to generate zebrafish mutants as a model for studying human genetic deafness. Anat Rec, 303:556-562, 2020. © 2019 American Association for Anatomy.},
}
@article {pmid31257851,
year = {2019},
author = {Liu, BY and He, XY and Xu, C and Ren, XH and Zhuo, RX and Cheng, SX},
title = {Peptide and Aptamer Decorated Delivery System for Targeting Delivery of Cas9/sgRNA Plasmid To Mediate Antitumor Genome Editing.},
journal = {ACS applied materials &amp; interfaces},
volume = {11},
number = {27},
pages = {23870-23879},
doi = {10.1021/acsami.9b05772},
pmid = {31257851},
issn = {1944-8252},
mesh = {Apoptosis/genetics ; *CRISPR-Cas Systems ; DNA, Neoplasm/genetics/metabolism ; *Gene Editing ; *Gene Transfer Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; *Neoplasms/genetics/metabolism/pathology/therapy ; *Peptides/chemistry/pharmacology ; *Plasmids/chemistry/genetics/pharmacology ; Signal Transduction/genetics ; },
abstract = {A multiple-functionalized targeting delivery system was prepared by self-assembly for efficient delivery of Cas9/sgRNA plasmids to targeted tumor cell nuclei. The Cas9/sgRNA plasmids were compacted by protamine in the presence of calcium ions to form nanosized cores, which were further decorated by peptide and aptamer conjugated alginate derivatives. With the help of the nuclear location signal peptide and AS1411 aptamer with specific affinity for nucleolin in the tumor cell membrane and nuclei, the delivery vector can specifically deliver the plasmid to the nuclei of tumorous cells for knocking out the protein tyrosine kinase 2 (PTK2) gene to down-regulate focal adhesion kinase (FAK). The tumor cell apoptosis induced by genome editing is mitochondrial-dependent. In addition, FAK knockout results in negative regulation on the PI3K/AKT signaling pathway. Meanwhile, favorable modulation on various proteins involved in tumor progression can be realized by genome editing. The enhanced E-cadherin and decreased MMPs, vimentin, and VEGF imply the desirable effects of genome editing on suppression of tumor development. Wound healing and transwell assays confirm that the genome editing system can suppress tumor invasion and metastasis in edited cells efficiently. The investigation provides a facile and effective strategy to fabricate multiple-functionalized delivery vectors for genome editing.},
}
@article {pmid31256988,
year = {2019},
author = {Ratner, HK and Escalera-Maurer, A and Le Rhun, A and Jaggavarapu, S and Wozniak, JE and Crispell, EK and Charpentier, E and Weiss, DS},
title = {Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.},
journal = {Molecular cell},
volume = {75},
number = {3},
pages = {498-510.e5},
pmid = {31256988},
issn = {1097-4164},
support = {P51 OD011132/OD/NIH HHS/United States ; R01 AI110701/AI/NIAID NIH HHS/United States ; U54 AI057157/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA Cleavage ; Francisella/*genetics ; Gene Expression Regulation, Bacterial/genetics ; Lipoproteins/biosynthesis/genetics ; RNA/genetics ; Transcription, Genetic ; Virulence/*genetics ; },
abstract = {In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.},
}
@article {pmid31253785,
year = {2019},
author = {Jayavaradhan, R and Pillis, DM and Goodman, M and Zhang, F and Zhang, Y and Andreassen, PR and Malik, P},
title = {CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2866},
pmid = {31253785},
issn = {2041-1723},
support = {UL1 TR001425/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; Gene Editing/*methods ; Humans ; Mutagenesis, Insertional ; Recombinational DNA Repair/*physiology ; Tumor Suppressor p53-Binding Protein 1/genetics/*metabolism ; },
abstract = {Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.},
}
@article {pmid31253768,
year = {2019},
author = {Wu, Y and Zhang, J and Peng, B and Tian, D and Zhang, D and Li, Y and Feng, X and Liu, J and Li, J and Zhang, T and Liu, X and Lu, J and Chen, B and Wang, S},
title = {Generating viable mice with heritable embryonically lethal mutations using the CRISPR-Cas9 system in two-cell embryos.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2883},
pmid = {31253768},
issn = {2041-1723},
support = {91649124//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo, Mammalian/*physiology ; Embryonic Development ; Female ; Gene Editing/methods ; Genetic Engineering/methods ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Knockout ; Mutation ; RNA, Guide ; RNA, Messenger/genetics/metabolism ; },
abstract = {A substantial number of mouse genes, about 25%, are embryonically lethal when knocked out. Using current genetic tools, such as the CRISPR-Cas9 system, it is difficult-or even impossible-to produce viable mice with heritable embryonically lethal mutations. Here, we establish a one-step method for microinjection of CRISPR reagents into one blastomere of two-cell embryos to generate viable chimeric founder mice with a heritable embryonically lethal mutation, of either Virma or Dpm1. By examining founder mice, we identify a phenotype and role of Virma in regulating kidney metabolism in adult mice. Additionally, we generate knockout mice with a heritable postnatally lethal mutation, of either Slc17a5 or Ctla-4, and study its function in vivo. This one-step method provides a convenient system that rapidly generates knockout mice possessing lethal phenotypes. This allows relatively easy in vivo study of the associated genes' functions.},
}
@article {pmid31253764,
year = {2019},
author = {Xie, J and Ge, W and Li, N and Liu, Q and Chen, F and Yang, X and Huang, X and Ouyang, Z and Zhang, Q and Zhao, Y and Liu, Z and Gou, S and Wu, H and Lai, C and Fan, N and Jin, Q and Shi, H and Liang, Y and Lan, T and Quan, L and Li, X and Wang, K and Lai, L},
title = {Efficient base editing for multiple genes and loci in pigs using base editors.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2852},
pmid = {31253764},
issn = {2041-1723},
support = {81702115//National Natural Science Foundation of China (National Science Foundation of China)/International ; 2017YFA0105103//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/International ; 201704030034//Guangzhou Municipal Science and Technology Project/International ; },
mesh = {APOBEC-1 Deaminase ; Animals ; Base Sequence ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; DNA/genetics ; Embryo Culture Techniques ; Embryo, Mammalian ; *Gene Editing ; Genome ; Nuclear Transfer Techniques/veterinary ; *Point Mutation ; RNA, Guide/metabolism ; RNA, Messenger/genetics/metabolism ; Swine/*genetics ; },
abstract = {Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.},
}
@article {pmid31253581,
year = {2019},
author = {Lo Scrudato, M and Poulard, K and Sourd, C and Tomé, S and Klein, AF and Corre, G and Huguet, A and Furling, D and Gourdon, G and Buj-Bello, A},
title = {Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1372-1388},
pmid = {31253581},
issn = {1525-0024},
mesh = {Alternative Splicing ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Nucleus ; Disease Models, Animal ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Vectors/genetics ; Humans ; Mice ; Mice, Knockout ; Muscle, Skeletal/metabolism/pathology ; Myotonic Dystrophy/genetics/therapy ; Myotonin-Protein Kinase/*genetics ; RNA, Guide ; *RNA, Nuclear ; Transduction, Genetic ; *Trinucleotide Repeat Expansion ; },
abstract = {Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy.},
}
@article {pmid31253397,
year = {2019},
author = {Yazawa, R and Nishida, Y and Aoyama, S and Tanida, I and Miyatsuka, T and Suzuki, L and Himuro, M and Haruna, H and Takubo, N and Shimizu, T and Watada, H},
title = {Establishment of a system for screening autophagic flux regulators using a modified fluorescent reporter and CRISPR/Cas9.},
journal = {Biochemical and biophysical research communications},
volume = {516},
number = {3},
pages = {686-692},
doi = {10.1016/j.bbrc.2019.06.129},
pmid = {31253397},
issn = {1090-2104},
mesh = {Animals ; *Autophagy ; Autophagy-Related Protein 7/genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Embryo, Mammalian/cytology ; Fibroblasts/cytology/metabolism ; Gene Expression Regulation ; Green Fluorescent Proteins/chemistry/genetics/*metabolism ; HEK293 Cells ; Humans ; Luminescent Proteins/chemistry/genetics/*metabolism ; Mice, Knockout ; Microscopy, Fluorescence ; Microtubule-Associated Proteins/chemistry/genetics/*metabolism ; },
abstract = {Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.},
}
@article {pmid31253120,
year = {2019},
author = {Hannafon, BN and Cai, A and Calloway, CL and Xu, YF and Zhang, R and Fung, KM and Ding, WQ},
title = {miR-23b and miR-27b are oncogenic microRNAs in breast cancer: evidence from a CRISPR/Cas9 deletion study.},
journal = {BMC cancer},
volume = {19},
number = {1},
pages = {642},
pmid = {31253120},
issn = {1471-2407},
support = {P20 GM103639/GM/NIGMS NIH HHS/United States ; P30 CA225520/CA/NCI NIH HHS/United States ; U54GM104938/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/diet therapy/*genetics/pathology ; CRISPR-Cas Systems ; Cell Movement ; Cell Proliferation ; Cell Survival/drug effects ; Dietary Supplements ; Female ; Fish Oils/administration &amp; dosage/pharmacology ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MCF-7 Cells ; Mice, Nude ; MicroRNAs/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Altered expression of microRNAs (miRNAs) is known to contribute to cancer progression. miR-23b and miR-27b, encoded within the same miRNA cluster, are reported to have both tumor suppressive and oncogenic activity across human cancers, including breast cancer.<br><br>METHODS: To clarify this dichotomous role in breast cancer, miR-23b and miR-27b were knocked out using CRISPR/Cas9 gene knockout technology, and the role of endogenous miR-23b and miR-27b was examined in a breast cancer model system in vitro and in vivo.<br><br>RESULTS: Characterization of the knockout cells in vitro demonstrated that miR-23b and miR-27b are indeed oncogenic miRNAs in MCF7 breast cancer cells. miR-23b and miR-27b knockout reduced tumor growth in xenograft nude mice fed a standard diet, supporting their oncogenic role in vivo. However, when xenograft mice were provided a fish-oil diet, miR-27b depletion, but not miR-23b depletion, compromised fish-oil-induced suppression of xenograft growth, indicating a context-dependent nature of miR-27b oncogenic activity.<br><br>CONCLUSIONS: Our results demonstrate that miR-23b and miR-27b are primarily oncogenic in MCF7 breast cancer cells and that miR-27b may have tumor suppressive activity under certain circumstances.},
}
@article {pmid31252652,
year = {2019},
author = {Khan, AZ and Bilal, M and Mehmood, S and Sharma, A and Iqbal, HMN},
title = {State-of-the-Art Genetic Modalities to Engineer Cyanobacteria for Sustainable Biosynthesis of Biofuel and Fine-Chemicals to Meet Bio-Economy Challenges.},
journal = {Life (Basel, Switzerland)},
volume = {9},
number = {3},
pages = {},
pmid = {31252652},
issn = {2075-1729},
abstract = {In recent years, metabolic engineering of microorganisms has attained much research interest to produce biofuels and industrially pertinent chemicals. Owing to the relatively fast growth rate, genetic malleability, and carbon neutral production process, cyanobacteria has been recognized as a specialized microorganism with a significant biotechnological perspective. Metabolically engineering cyanobacterial strains have shown great potential for the photosynthetic production of an array of valuable native or non-native chemicals and metabolites with profound agricultural and pharmaceutical significance using CO2 as a building block. In recent years, substantial improvements in developing and introducing novel and efficient genetic tools such as genome-scale modeling, high throughput omics analyses, synthetic/system biology tools, metabolic flux analysis and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (CRISPR/cas) systems have been made for engineering cyanobacterial strains. Use of these tools and technologies has led to a greater understanding of the host metabolism, as well as endogenous and heterologous carbon regulation mechanisms which consequently results in the expansion of maximum productive ability and biochemical diversity. This review summarizes recent advances in engineering cyanobacteria to produce biofuel and industrially relevant fine chemicals of high interest. Moreover, the development and applications of cutting-edge toolboxes such as the CRISPR-cas9 system, synthetic biology, high-throughput "omics", and metabolic flux analysis to engineer cyanobacteria for large-scale cultivation are also discussed.},
}
@article {pmid31252430,
year = {2019},
author = {Kieper, SN and Almendros, C and Brouns, SJJ},
title = {Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems.},
journal = {FEMS microbiology letters},
volume = {366},
number = {11},
pages = {},
pmid = {31252430},
issn = {1574-6968},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics/metabolism ; Protein Sorting Signals/genetics/physiology ; Synechocystis/genetics/metabolism ; },
abstract = {Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1-2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genes cas1 and cas2 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation in Synechocystis sp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array.},
}
@article {pmid31251842,
year = {2019},
author = {Cal, L and Suarez-Bregua, P and Braasch, I and Irion, U and Kelsh, R and Cerdá-Reverter, JM and Rotllant, J},
title = {Loss-of-function mutations in the melanocortin 1 receptor cause disruption of dorso-ventral countershading in teleost fish.},
journal = {Pigment cell &amp; melanoma research},
volume = {32},
number = {6},
pages = {817-828},
doi = {10.1111/pcmr.12806},
pmid = {31251842},
issn = {1755-148X},
mesh = {Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; Body Patterning/*genetics ; CRISPR-Cas Systems/genetics ; Loss of Function Mutation/*genetics ; Melanophores/metabolism ; Models, Biological ; Phenotype ; Pigmentation/*genetics ; Receptor, Melanocortin, Type 1/agonists/chemistry/*genetics ; Zebrafish/*embryology/*genetics ; Zebrafish Proteins/metabolism ; },
abstract = {The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in vertebrate pigmentation. Mutations in this gene cause variations in coat coloration in amniotes. Additionally, in mammals MC1R is the main receptor for agouti-signaling protein (ASIP), making it the critical receptor for the establishment of dorsal-ventral countershading. In fish, Mc1r is also involved in pigmentation, but it has been almost exclusively studied in relation to melanosome dispersion activity and as a putative genetic factor involved in dark/light adaptation. However, its role as the crucial component for the Asip1-dependent control of dorsal-ventral pigmentation remains unexplored. Using CRISPR/Cas9, we created mc1r homozygous knockout zebrafish and found that loss-of-function of mc1r causes a reduction of countershading and a general paling of the animals. We find ectopic development of melanophores and xanthophores, accompanied by a decrease in iridophore numbers in the ventral region of mc1r mutants. We also reveal subtle differences in the role of mc1r in repressing pigment cell development between the skin and scale niches in ventral regions.},
}
@article {pmid31251822,
year = {2020},
author = {Zhang, L and Yang, Y and Chai, L and Bu, H and Yang, Y and Huang, H and Ran, J and Zhu, Y and Li, L and Chen, F and Li, W},
title = {FRK plays an oncogenic role in non-small cell lung cancer by enhancing the stemness phenotype via induction of metabolic reprogramming.},
journal = {International journal of cancer},
volume = {146},
number = {1},
pages = {208-222},
doi = {10.1002/ijc.32530},
pmid = {31251822},
issn = {1097-0215},
mesh = {CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*genetics/pathology ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition/genetics ; Female ; Gene Knockdown Techniques ; Humans ; Lung Neoplasms/*genetics/pathology ; Male ; Middle Aged ; Neoplasm Proteins/genetics/*physiology ; Neoplastic Stem Cells/*pathology ; *Oncogenes ; Prognosis ; Protein-Tyrosine Kinases/genetics/*physiology ; },
abstract = {The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-[13] C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.},
}
@article {pmid31251802,
year = {2019},
author = {Argaud, D and Boulanger, MC and Chignon, A and Mkannez, G and Mathieu, P},
title = {Enhancer-mediated enrichment of interacting JMJD3-DDX21 to ENPP2 locus prevents R-loop formation and promotes transcription.},
journal = {Nucleic acids research},
volume = {47},
number = {16},
pages = {8424-8438},
pmid = {31251802},
issn = {1362-4962},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; DEAD-box RNA Helicases/*genetics/metabolism ; DNA/chemistry/*genetics/metabolism ; Enhancer Elements, Genetic ; Gene Editing/methods ; Gene Expression Regulation ; HEK293 Cells ; Histone Demethylases/genetics/metabolism ; Histones/genetics/metabolism ; Humans ; Inflammation ; Jumonji Domain-Containing Histone Demethylases/*genetics/metabolism ; Lipopolysaccharides/pharmacology ; Models, Biological ; NF-kappa B/genetics/metabolism ; Nucleic Acid Conformation ; Phosphoric Diester Hydrolases/*genetics/metabolism ; Protein Binding ; RNA, Messenger/biosynthesis/chemistry/*genetics ; Signal Transduction ; Transcription Initiation Site ; Transcription, Genetic/*drug effects ; },
abstract = {ENPP2, which encodes for the enzyme autotaxin (ATX), is overexpressed during chronic inflammatory diseases and various cancers. However, the molecular mechanism involved in the ENPP2 transcription remains elusive. Here, in HEK 293T cells, we demonstrated that lipopolysaccharide (LPS) increased the transcription process at ENPP2 locus through a NF-кB pathway and a reduction of H3K27me3 level, a histone repressive mark, by the demethylase UTX. Simultaneously, the H3K27me3 demethylase JMJD3/KDM6B was recruited to the transcription start site (TSS), within the gene body and controlled the expression of ENPP2 in a non-enzymatic manner. Mass spectrometry data revealed a novel interaction for JMJD3 with DDX21, a RNA helicase that unwinds R-loops created by nascent transcript and DNA template. Upon LPS treatment, JMJD3 is necessary for DDX21 recruitment at ENPP2 locus allowing the resolution of aberrant R-loops. CRISPR-Cas9-mediated deletion of a distant-acting enhancer decreased the expression of ENPP2 and lowered the recruitment of JMJD3-DDX21 complex at TSS and its progression through the gene body. Taken together, these findings revealed that enhancer-mediated enrichment of novel JMJD3-DDX21 interaction at ENPP2 locus is necessary for nascent transcript synthesis via the resolution of aberrant R-loops formation in response to inflammatory stimulus.},
}
@article {pmid31250381,
year = {2019},
author = {Chang, YJ and Xu, CL and Cui, X and Bassuk, AG and Mahajan, VB and Tsai, YT and Tsang, SH},
title = {CRISPR Base Editing in Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2045},
number = {},
pages = {337-346},
doi = {10.1007/7651_2019_243},
pmid = {31250381},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Cycle ; Cells, Cultured ; Cytidine Deaminase/genetics/*metabolism ; Cytosine/chemistry/metabolism ; Electroporation ; Gene Editing/instrumentation/*methods ; Genome, Human/drug effects ; High-Throughput Nucleotide Sequencing ; Humans ; Induced Pluripotent Stem Cells/chemistry/cytology/drug effects/*metabolism ; Software ; Transfection/methods ; Uracil/chemistry ; },
abstract = {Induced pluripotent stem cells (iPSCs) have demonstrated tremendous potential in numerous disease modeling and regenerative medicine-based therapies. The development of innovative gene transduction and editing technologies has further augmented the potential of iPSCs. Cas9-cytidine deaminases, for example, have developed as an alternative strategy to integrate single-base mutations (C → T or G → A transitions) at specific genomic loci. In this chapter, we specifically describe CRISPR (clustered regularly interspaced short palindromic repeats) base editing in iPSCs for editing precise locations in the genome. This state-of-the-art approach enables highly efficient and accurate modifications in genes. Thus, this technique not only has the potential to have biotechnology and therapeutic applications but also the ability to reveal underlying mechanisms regarding pathologies caused by specific mutations.},
}
@article {pmid31249419,
year = {2019},
author = {Rusk, N},
title = {CRISPR inhibitors.},
journal = {Nature methods},
volume = {16},
number = {7},
pages = {577},
doi = {10.1038/s41592-019-0490-z},
pmid = {31249419},
issn = {1548-7105},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
}
@article {pmid31249142,
year = {2019},
author = {Hou, L and Wang, Y and Liu, Y and Zhang, N and Shamovsky, I and Nudler, E and Tian, B and Dynlacht, BD},
title = {Paf1C regulates RNA polymerase II progression by modulating elongation rate.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {29},
pages = {14583-14592},
pmid = {31249142},
issn = {1091-6490},
support = {R01 GM126891/GM/NIGMS NIH HHS/United States ; R01 GM127267/GM/NIGMS NIH HHS/United States ; R01 GM084089/GM/NIGMS NIH HHS/United States ; R01 GM122395/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM129069/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carrier Proteins/genetics/*metabolism ; Cell Line ; Gene Knockout Techniques ; Histones/metabolism ; Mice ; Myoblasts ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/*metabolism ; RNA, Small Interfering/metabolism ; *Transcription Elongation, Genetic ; *Transcription Termination, Genetic ; Ubiquitination/genetics ; },
abstract = {Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.},
}
@article {pmid31249002,
year = {2019},
author = {Morris, SA},
title = {The evolving concept of cell identity in the single cell era.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {12},
pages = {},
doi = {10.1242/dev.169748},
pmid = {31249002},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans ; Cell Biology/*trends ; Cell Differentiation ; *Cell Lineage ; Epigenesis, Genetic ; Genetic Variation ; Golgi Apparatus/metabolism ; Humans ; Machine Learning ; Mice ; Models, Biological ; Neurons/metabolism ; Phenotype ; Single-Cell Analysis/*methods ; },
abstract = {Fueled by recent advances in single cell biology, we are moving away from qualitative and undersampled assessments of cell identity, toward building quantitative, high-resolution cell atlases. However, it remains challenging to precisely define cell identity, leading to renewed debate surrounding this concept. Here, I present three pillars that I propose are central to the notion of cell identity: phenotype, lineage and state. I explore emerging technologies that are enabling the systematic and unbiased quantification of these properties, and outline how these efforts will enable the construction of a high-resolution, dynamic landscape of cell identity, potentially revealing its underlying molecular regulation to provide new opportunities for understanding and manipulating cell fate.},
}
@article {pmid31248972,
year = {2020},
author = {Nasri, M and Ritter, M and Mir, P and Dannenmann, B and Aghaallaei, N and Amend, D and Makaryan, V and Xu, Y and Fletcher, B and Bernhard, R and Steiert, I and Hahnel, K and Berger, J and Koch, I and Sailer, B and Hipp, K and Zeidler, C and Klimiankou, M and Bajoghli, B and Dale, DC and Welte, K and Skokowa, J},
title = {CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients.},
journal = {Haematologica},
volume = {105},
number = {3},
pages = {598-609},
pmid = {31248972},
issn = {1592-8721},
support = {R01 HL151629/HL/NHLBI NIH HHS/United States ; R24 AI049393/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Congenital Bone Marrow Failure Syndromes ; *Hematopoietic Stem Cell Transplantation ; Humans ; *Induced Pluripotent Stem Cells ; Mutation ; *Neutropenia/congenital/genetics ; },
abstract = {A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 μg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.},
}
@article {pmid31248872,
year = {2019},
author = {Pipe, SW and Selvaraj, SR},
title = {Gene editing in hemophilia: a "CRISPR" choice?.},
journal = {Blood},
volume = {133},
number = {26},
pages = {2733-2734},
doi = {10.1182/blood.2019001180},
pmid = {31248872},
issn = {1528-0020},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Factor IX/genetics ; *Gene Editing ; Gene Targeting ; Hemophilia A/*genetics ; Hemostasis ; Mice ; Mice, Knockout ; },
}
@article {pmid31248723,
year = {2019},
author = {Segal, DJ},
title = {The Right Tools for the Right Job: CRISPR-pass Could Offer Safe Gene Correction for Many Disorders.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1346-1347},
pmid = {31248723},
issn = {1525-0024},
mesh = {Adenine ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Codon, Nonsense ; Gene Editing ; },
}
@article {pmid31248623,
year = {2019},
author = {Lorenzo, D and Esquerda, M and , },
title = {Map of ethical conflicts of the CRISPR-Cas9 gene edition technique.},
journal = {Medicina clinica},
volume = {153},
number = {9},
pages = {357-359},
doi = {10.1016/j.medcli.2019.03.024},
pmid = {31248623},
issn = {1578-8989},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*ethics ; Humans ; },
}
@article {pmid31248401,
year = {2019},
author = {Wilson, LOW and Hetzel, S and Pockrandt, C and Reinert, K and Bauer, DC},
title = {VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {40},
pmid = {31248401},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Computational Biology/*methods ; Gene Editing/*methods/standards ; Gene Targeting/*methods/standards ; Genomics/*methods/standards ; Internet ; Reproducibility of Results ; *Software ; },
abstract = {BACKGROUND: Natural variations in a genome can drastically alter the CRISPR-Cas9 off-target landscape by creating or removing sites. Despite the resulting potential side-effects from such unaccounted for sites, current off-target detection pipelines are not equipped to include variant information. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT).<br><br>RESULTS: VARSCOT identifies only 0.6% of off-targets to be common between 4 individual genomes and the reference, with an average of 82% of off-targets unique to an individual. VARSCOT is the most sensitive detection method for off-targets, finding 40 to 70% more experimentally verified off-targets compared to other popular software tools and its machine learning model allows for CRISPR-Cas9 concentration aware off-target activity scoring.<br><br>CONCLUSIONS: VARSCOT allows researchers to take genomic variation into account when designing individual or population-wide targeting strategies. VARSCOT is available from https://github.com/BauerLab/VARSCOT .},
}
@article {pmid31247227,
year = {2019},
author = {Neil, K and Allard, N and Jordan, D and Rodrigue, S},
title = {Assembly of large mobilizable genetic cargo by double recombinase operated insertion of DNA (DROID).},
journal = {Plasmid},
volume = {104},
number = {},
pages = {102419},
doi = {10.1016/j.plasmid.2019.102419},
pmid = {31247227},
issn = {1095-9890},
support = {159817//CIHR/Canada ; },
mesh = {CRISPR-Cas Systems ; Conjugation, Genetic ; *DNA Transposable Elements ; *Mutagenesis, Insertional ; Recombinases/*metabolism ; },
abstract = {There is an important need to develop new therapeutic tools to modulate the gene content of microbiomes. A potential strategy for microbiome engineering relies on the delivery of genetic payloads by conjugative plasmids. Yet, the introduction of large DNA molecules in conjugative plasmids can be challenging. Here, we describe the Double Recombinase Operated Insertion of DNA (DROID), an efficient method to assemble large DNA molecules without introducing antibiotic resistance genes or other unwanted sequences in the final construct. We exemplify this method by demonstrating that the Bxb1 integrase and FLP recombinase can be used successively to stably insert a relatively large DNA cargo consisting of a CRISPR-Cas9 system in a conjugative plasmid. We further show that the resulting CRISPR-Cas9 mobilization system was able to cure a multi-copy antibiotic resistance plasmid in a target bacterium. In addition to its utility for DNA payload integration in conjugative plasmids, the DROID method could readily be adapted to a multitude of other applications that require the manipulation of large DNA molecules.},
}
@article {pmid31246337,
year = {2020},
author = {Schulman, AH and Oksman-Caldentey, KM and Teeri, TH},
title = {European Court of Justice delivers no justice to Europe on genome-edited crops.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {8-10},
pmid = {31246337},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Europe ; Gene Editing/*legislation &amp; jurisprudence ; Genome, Plant ; Mutagenesis ; *Plants, Genetically Modified ; },
}
@article {pmid31246169,
year = {2019},
author = {Tracey, WD},
title = {The taste of water.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31246169},
issn = {2050-084X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Culicidae/genetics/*physiology ; *Eggs ; Epithelial Sodium Channels/*genetics ; Female ; Fresh Water/chemistry ; Genome, Insect/genetics ; Neurons/metabolism ; Taste/*genetics/physiology ; },
abstract = {Female mosquitos require a specific ion-channel protein to sense the presence of fresh water in which they can lay their eggs.},
}
@article {pmid31243729,
year = {2019},
author = {Yan, M and Li, J},
title = {The evolving CRISPR technology.},
journal = {Protein &amp; cell},
volume = {10},
number = {11},
pages = {783-786},
pmid = {31243729},
issn = {1674-8018},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; },
}
@article {pmid31243272,
year = {2019},
author = {Thavalingam, A and Cheng, Z and Garcia, B and Huang, X and Shah, M and Sun, W and Wang, M and Harrington, L and Hwang, S and Hidalgo-Reyes, Y and Sontheimer, EJ and Doudna, J and Davidson, AR and Moraes, TF and Wang, Y and Maxwell, KL},
title = {Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2806},
pmid = {31243272},
issn = {2041-1723},
support = {RGPIN-2018-06546//Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (Conseil de Recherches en Sciences Naturelles et en G&#xe9;nie du Canada)/International ; GM125797//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/International ; 2017YFA0504203//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/International ; FDN-15427//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/International ; PJT-152918//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/International ; 31725008//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/International ; R01 GM125797/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*metabolism ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Escherichia coli/metabolism ; Gene Editing ; Gene Expression Regulation, Bacterial ; Neisseria/virology ; Ribonucleoproteins/genetics/*metabolism ; Viral Proteins/metabolism/*pharmacology ; },
abstract = {CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.},
}
@article {pmid31243081,
year = {2019},
author = {Markus, BM and Bell, GW and Lorenzi, HA and Lourido, S},
title = {Optimizing Systems for Cas9 Expression in Toxoplasma gondii.},
journal = {mSphere},
volume = {4},
number = {3},
pages = {},
pmid = {31243081},
issn = {2379-5042},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques ; RNA, Guide/*genetics ; Toxoplasma/*genetics ; },
abstract = {CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression.IMPORTANCEToxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species.},
}
@article {pmid31243078,
year = {2019},
author = {Tran, VG and Cao, M and Fatma, Z and Song, X and Zhao, H},
title = {Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis.},
journal = {mSphere},
volume = {4},
number = {3},
pages = {},
pmid = {31243078},
issn = {2379-5042},
mesh = {*CRISPR-Cas Systems ; *Gene Deletion ; *Gene Editing ; Gene Knockout Techniques ; Plasmids/genetics ; Promoter Regions, Genetic ; Saccharomyces cerevisiae/genetics ; Saccharomycetales/*genetics ; },
abstract = {The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1'-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals.IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.},
}
@article {pmid31243056,
year = {2019},
author = {Ianiri, G and Dagotto, G and Sun, S and Heitman, J},
title = {Advancing Functional Genetics Through Agrobacterium-Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast Malassezia.},
journal = {Genetics},
volume = {212},
number = {4},
pages = {1163-1179},
pmid = {31243056},
issn = {1943-2631},
support = {R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; R37 AI039115/AI/NIAID NIH HHS/United States ; },
mesh = {Agrobacterium/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Fungal/genetics ; Gene Deletion ; Malassezia/*genetics ; *Mutagenesis ; Mutagenesis, Insertional ; Reverse Genetics/*methods ; Transformation, Genetic ; },
abstract = {Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.},
}
@article {pmid31242975,
year = {2019},
author = {Xiao, J and Jia, H and Pan, L and Li, Z and Lv, L and Du, B and Zhang, L and Du, F and Huang, Y and Cao, T and Sun, Q and Wei, R and Xing, A and Zhang, Z},
title = {Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {72},
number = {},
pages = {183-190},
doi = {10.1016/j.meegid.2018.06.033},
pmid = {31242975},
issn = {1567-7257},
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/drug effects/genetics ; *Gene Editing/methods ; *Gene Knockdown Techniques ; Genes, Essential ; Mycobacterium smegmatis/drug effects/*genetics/growth &amp; development ; },
abstract = {Despite technical advances in introducing genomic deletions and modulating gene expression, direct inactivation of essential genes in mycobacteria remains difficult. In this study, we described clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to repress the expression of sepF (MSMEG_4219) based on nuclease-deficient CRISPR-associated protein 9 (Cas9) and small guide RNA (sgRNA) specific to the target sequence in Mycobacterium smegmatis. Using this CRISPRi approach, we achieved the repression of sepF by up to 98% in M. smegmatis without off-target effects. The depleted Msm_sepF strains resulted in growth and morphology changes including elongated, filamentous and branched bacterial cells, but the levels of the interacting partners ftsZ and murG were not modified in M. smegmatis. The sepF gene was proven to be an essential gene in M. smegmatis. This study provided an improved and detailed technical procedure for the application of CRISPRi technology in mycobacteria, and this approach was demonstrated to be a simple and efficient tool for regulating the expression of essential genes in M. smegmatis.},
}
@article {pmid31240772,
year = {2020},
author = {Cai, Y and Wang, L and Chen, L and Wu, T and Liu, L and Sun, S and Wu, C and Yao, W and Jiang, B and Yuan, S and Han, T and Hou, W},
title = {Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {298-309},
pmid = {31240772},
issn = {1467-7652},
mesh = {Adaptation, Biological/genetics ; *CRISPR-Cas Systems ; Flowers/*growth &amp; development ; Mutagenesis ; Photoperiod ; Plant Proteins/genetics ; *Quantitative Trait Loci ; Soybeans/*genetics ; },
abstract = {Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean (Glycine max) to diverse latitudes and farming systems. GmFT2a and GmFT5a have been extensively identified as flowering activators and integrators in soybean. Here, we identified two quantitative trait loci (QTLs) regions harbouring GmFT2a and GmFT5a, respectively, associated with different genetic effects on flowering under different photoperiods. We analysed the flowering time of transgenic plants overexpressing GmFT2a or GmFT5a, ft2a mutants, ft5a mutants and ft2aft5a double mutants under long-day (LD) and short-day (SD) conditions. We confirmed that GmFT2a and GmFT5a are not redundant, they collectively regulate flowering time, and the effect of GmFT2a is more prominent than that of GmFT5a under SD conditions whereas GmFT5a has more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions. The ft2aft5a double mutants showed late flowering by about 31.3 days under SD conditions and produced significantly increased numbers of pods and seeds per plant compared to the wild type. We speculate that these mutants may have enormous yield potential for the tropics. In addition, we examined the sequences of these two loci in 202 soybean accessions and investigated the flowering phenotypes, geographical distributions and maturity groups within major haplotypes. These results will contribute to soybean breeding and regional adaptability.},
}
@article {pmid31239056,
year = {2019},
author = {Wayment-Steele, H and Wu, M and Gotrik, M and Das, R},
title = {Evaluating riboswitch optimality.},
journal = {Methods in enzymology},
volume = {623},
number = {},
pages = {417-450},
pmid = {31239056},
issn = {1557-7988},
support = {R35 GM122579/GM/NIGMS NIH HHS/United States ; },
mesh = {Aptamers, Nucleotide/*chemistry ; Biosensing Techniques/methods ; Ligands ; Nucleic Acid Conformation ; RNA Folding ; *Riboswitch ; Thermodynamics ; },
abstract = {Riboswitches are RNA elements that recognize diverse chemical and biomolecular inputs, and transduce this recognition process to genetic, fluorescent, and other engineered outputs using RNA conformational changes. These systems are pervasive in cellular biology and are a promising biotechnology with applications in genetic regulation and biosensing. Here, we derive a simple expression bounding the activation ratio-the proportion of RNA in the active vs. inactive states-for both ON and OFF riboswitches that operate near thermodynamic equilibrium: 1+[I]/Kd[I], where [I] is the input ligand concentration and Kd[I] is the intrinsic dissociation constant of the aptamer module toward the input ligand. A survey of published studies of natural and synthetic riboswitches confirms that the vast majority of empirically measured activation ratios have remained well below this thermodynamic limit. A few natural and synthetic riboswitches achieve activation ratios close to the limit, and these molecules highlight important principles for achieving high riboswitch performance. For several applications, including "light-up" fluorescent sensors and chemically-controlled CRISPR/Cas complexes, the thermodynamic limit has not yet been achieved, suggesting that current tools are operating at suboptimal efficiencies. Future riboswitch studies will benefit from comparing observed activation ratios to this simple expression for the optimal activation ratio. We present experimental and computational suggestions for how to make these quantitative comparisons and suggest new molecular mechanisms that may allow non-equilibrium riboswitches to surpass the derived limit.},
}
@article {pmid31238873,
year = {2019},
author = {Somerville, V and Lutz, S and Schmid, M and Frei, D and Moser, A and Irmler, S and Frey, JE and Ahrens, CH},
title = {Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system.},
journal = {BMC microbiology},
volume = {19},
number = {1},
pages = {143},
pmid = {31238873},
issn = {1471-2180},
mesh = {Bacteria/classification/*virology ; Bacteriophages/*genetics/*physiology ; Biodiversity ; Cheese/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; Lactobacillus delbrueckii/genetics ; Lactobacillus helveticus/genetics ; *Metagenome ; Metagenomics ; Microbiota/*genetics/*physiology ; Plasmids ; RNA, Ribosomal, 16S/genetics ; Whole Genome Sequencing ; },
abstract = {BACKGROUND: Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii.<br><br>RESULTS: Two NWCs from Swiss Gruy&#xe8;re producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats.<br><br>CONCLUSIONS: Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.},
}
@article {pmid31238294,
year = {2019},
author = {Vijay, D and Akhtar, MK and Hess, WR},
title = {Genetic and metabolic advances in the engineering of cyanobacteria.},
journal = {Current opinion in biotechnology},
volume = {59},
number = {},
pages = {150-156},
doi = {10.1016/j.copbio.2019.05.012},
pmid = {31238294},
issn = {1879-0429},
mesh = {Biofuels ; CRISPR-Cas Systems ; Carbon Dioxide ; *Cyanobacteria ; Genetic Engineering ; Metabolic Engineering ; Photosynthesis ; },
abstract = {Cyanobacteria are a group of photosynthetic microorganisms with high commercial potential. They can utilize sunlight directly to convert carbon dioxide or even nitrogen into a variety of industrially relevant chemicals. However, commercial platforms for the renewable and sustainable production of chemicals have yet to be demonstrated for cyanobacteria. Diverse strategies have therefore been employed in recent years to improve the production yields and efficiency of target chemicals. These include the use of CRISPR/Cas systems for mutant selection, synthetic RNA elements for controlling transcription, metabolic network modelling for understanding pathway fluxes, enzyme engineering, improving growth rates, alleviating product toxicity and microbial consortia. More elaborate strategies for engineering cyanobacteria, however, are still very much required if we are to meet the grand challenge of employing cyanobacteria as photosynthetic workhorses for large-scale industrial applications.},
}
@article {pmid31237724,
year = {2019},
author = {Rath, A and Mishra, A and Ferreira, VD and Hu, C and Omerza, G and Kelly, K and Hesse, A and Reddi, HV and Grady, JP and Heinen, CD},
title = {Functional interrogation of Lynch syndrome-associated MSH2 missense variants via CRISPR-Cas9 gene editing in human embryonic stem cells.},
journal = {Human mutation},
volume = {40},
number = {11},
pages = {2044-2056},
pmid = {31237724},
issn = {1098-1004},
support = {R01 CA115783/CA/NCI NIH HHS/United States ; R01 CA222477/CA/NCI NIH HHS/United States ; CA115783/CA/NCI NIH HHS/United States ; CA222477/CA/NCI NIH HHS/United States ; R21 CA181959/CA/NCI NIH HHS/United States ; 13SCB-UCHC-06//State of Connecticut Regenerative Medicine Fund/International ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Colorectal Neoplasms, Hereditary Nonpolyposis/*diagnosis/*genetics ; DNA Damage ; DNA Repair ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Human Embryonic Stem Cells/*metabolism ; Humans ; Microsatellite Instability ; Models, Molecular ; MutS Homolog 2 Protein/chemistry/*genetics ; *Mutation, Missense ; Protein Conformation ; Signal Transduction ; },
abstract = {Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients.},
}
@article {pmid31237422,
year = {2019},
author = {Li, Z and Wang, F and Li, JF},
title = {Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9-Derived Synthetic Gene Activator.},
journal = {Current protocols in molecular biology},
volume = {127},
number = {1},
pages = {e89},
doi = {10.1002/cpmb.89},
pmid = {31237422},
issn = {1934-3647},
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Gene Targeting/*methods ; Genes, Plant/genetics ; Genes, Synthetic/*genetics ; Oryza/*genetics ; Promoter Regions, Genetic ; Protoplasts/metabolism ; RNA, Guide/genetics ; *Transcriptional Activation ; },
abstract = {Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley &amp; Sons, Inc.},
}
@article {pmid31237055,
year = {2019},
author = {Xu, X and Wan, T and Xin, H and Li, D and Pan, H and Wu, J and Ping, Y},
title = {Delivery of CRISPR/Cas9 for therapeutic genome editing.},
journal = {The journal of gene medicine},
volume = {21},
number = {7},
pages = {e3107},
doi = {10.1002/jgm.3107},
pmid = {31237055},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/*methods/trends ; *Gene Transfer Techniques/adverse effects/trends ; Genetic Vectors ; Humans ; Liposomes/chemistry ; Metal Nanoparticles/chemistry ; Peptides/chemistry/genetics ; Polymers/chemistry ; },
abstract = {The clustered, regularly-interspaced, short palindromic repeat (CRISPR)-associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome-editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome-editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off-target effects. Recently, non-viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non-viral vectors, including polymers, liposomes, cell-penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non-viral vectors are also discussed.},
}
@article {pmid31235868,
year = {2019},
author = {Sand, M and Bredenoord, AL and Jongsma, KR},
title = {After the fact-the case of CRISPR babies.},
journal = {European journal of human genetics : EJHG},
volume = {27},
number = {11},
pages = {1621-1624},
pmid = {31235868},
issn = {1476-5438},
mesh = {CRISPR-Cas Systems/*physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Mammalian ; Embryonic Development/genetics ; Gene Editing/ethics/*methods ; Genetic Therapy ; Humans ; Morals ; },
abstract = {The world has been startled by the irresponsible experiment of He Jiankui, who used CRISPR to genetically modify human embryos. In this viewpoint, we explore the phenomenon of moral luck in medicine and its bearing on the limits of simple judgements of the kind "everything that ends well is well" or "someone broke the rules, and is therefore blameworthy". The risks involved in scientific and medical experiments are often brushed aside, when they turn out well. The clinical application of CRISPR in the human germline is presently too risky to be used without more preclinical research and unacceptable without broader societal support, which justifies the call for a moratorium by the scientific community. However, such policies do not determine how to assess cases, where someone was willing to take such risks beyond all rules, guidelines and regulation and succeeds. The policies including the proposed moratorium are as unanimous about the undesirability of current applications of clinical germline editing as they are about the potential importance of this research. What if this potential is achieved by breaking the rules? The paradox of moral luck impinges on this debate. In our analysis, we rebut simplified judgments and advocate a more balanced view on the relation between moral responsibility and the societal consequences of medicine.},
}
@article {pmid31235524,
year = {2019},
author = {Bowin, CF and Inoue, A and Schulte, G},
title = {WNT-3A-induced β-catenin signaling does not require signaling through heterotrimeric G proteins.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {31},
pages = {11677-11684},
pmid = {31235524},
issn = {1083-351X},
mesh = {Dishevelled Proteins ; Gene Editing ; HEK293 Cells ; Heterotrimeric GTP-Binding Proteins/genetics/*metabolism ; Humans ; Low Density Lipoprotein Receptor-Related Protein-6/metabolism ; Pertussis Toxin/pharmacology ; Phosphorylation ; Recombinant Proteins/biosynthesis/isolation &amp; purification/pharmacology ; TCF Transcription Factors/metabolism ; Wnt Signaling Pathway/*drug effects ; Wnt3A Protein/genetics/metabolism/*pharmacology ; beta Catenin/metabolism ; },
abstract = {The network of Wingless/Int-1 (WNT)-induced signaling pathways includes β-catenin-dependent and -independent pathways. β-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, β-catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL receptor-related protein 5/6 (LRP5/6) receptor complex. FZDs are G protein-coupled receptors, but the question of whether heterotrimeric G proteins are involved in WNT/β-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/β-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of Gs/Golf/Gq/G11/G12/G13/Gz in HEK293 (ΔG7) cells, leaving the expression of pertussis toxin (PTX)-sensitive Gi/o proteins unchanged, to assess whether WNT-3A activates WNT/β-catenin signaling in WT and ΔG7 cells devoid of functional G protein signaling. We monitored WNT-3A-induced activation by detection of phosphorylation of LDL receptor-related protein 6 (LRP6), electrophoretic mobility shift of the phosphoprotein Dishevelled (DVL), β-catenin stabilization and dephosphorylation, and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/β-catenin signaling in ΔG7 cells in both the absence and presence of Gi/o-blocking PTX. Furthermore, cells completely devoid of G protein expression, so called Gα-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/β-catenin signaling. These findings corroborate the concept that heterotrimeric G proteins are not required for this FZD- and DVL-mediated signaling branch. Our observations agree with previous results arguing for FZD conformation-dependent functional selectivity between DVL and heterotrimeric G proteins. In conclusion, WNT/β-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins.},
}
@article {pmid31234534,
year = {2019},
author = {Snider, PL and Snider, E and Simmons, O and Lilly, B and Conway, SJ},
title = {Analysis of Uncharacterized mKiaa1211 Expression during Mouse Development and Cardiovascular Morphogenesis.},
journal = {Journal of cardiovascular development and disease},
volume = {6},
number = {2},
pages = {},
pmid = {31234534},
issn = {2308-3425},
support = {R01 HL135657/HL/NHLBI NIH HHS/United States ; R01 HL148165/HL/NHLBI NIH HHS/United States ; HL135657 and HL148165/NH/NIH HHS/United States ; },
abstract = {Mammalian Kiaa1211 and Kiaa1211-like are a homologous pair of uncharacterized, highly conserved genes cloned from fetal and adult brain cDNA libraries. Herein we map the in utero spatiotemporal expression of mKiaa1211 and mKiaa1211L mRNA and their expression patterns in postnatal testis, skin, gastrointestinal, and adipose progenitor tissues. Significantly, mKiaa1211 is present throughout the early stages of mouse heart development, particularly in the second heart field (SHF) lineage as it differentiates from mesenchymal cells into cardiomyocytes. We also show that mKiaa1211 is expressed within several early neuronal tissues destined to give rise to central, peripheral, and sympathetic nervous system structures. Expression profiling revealed that the paralog mKiaa1211L is not expressed during the normal developmental process and that mKiaa1211 expression was noticeably absent from most adult terminally differentiated tissues. Finally, we confirm that a previously uncharacterized CRISPR/CAS-generated mKiaa1211 mouse mutant allele is hypomorphic.},
}
@article {pmid31234407,
year = {2019},
author = {Balestra, D and Branchini, A},
title = {Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies.},
journal = {International journal of molecular sciences},
volume = {20},
number = {12},
pages = {},
pmid = {31234407},
issn = {1422-0067},
mesh = {Animals ; Blood Coagulation Factors/genetics ; CRISPR-Cas Systems ; Coagulation Protein Disorders/*genetics/*therapy ; DNA/genetics ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; RNA, Messenger/genetics ; },
abstract = {Molecular strategies tailored to promote/correct the expression and/or processing of defective coagulation factors would represent innovative therapeutic approaches beyond standard substitutive therapy. Here, we focus on the molecular mechanisms and determinants underlying innovative approaches acting at DNA, mRNA and protein levels in inherited coagulation factor deficiencies, and in particular on: (i) gene editing approaches, which have permitted intervention at the DNA level through the specific recognition, cleavage, repair/correction or activation of target sequences, even in mutated gene contexts; (ii) the rescue of altered pre-mRNA processing through the engineering of key spliceosome components able to promote correct exon recognition and, in turn, the synthesis and secretion of functional factors, as well as the effects on the splicing of missense changes affecting exonic splicing elements; this section includes antisense oligonucleotide- or siRNA-mediated approaches to down-regulate target genes; (iii) the rescue of protein synthesis/function through the induction of ribosome readthrough targeting nonsense variants or the correction of folding defects caused by amino acid substitutions. Overall, these approaches have shown the ability to rescue the expression and/or function of potentially therapeutic levels of coagulation factors in different disease models, thus supporting further studies in the future aimed at evaluating the clinical translatability of these new strategies.},
}
@article {pmid31232162,
year = {2019},
author = {Taylor, HN and Warner, EE and Armbrust, MJ and Crowley, VM and Olsen, KJ and Jackson, RN},
title = {Structural basis of Type IV CRISPR RNA biogenesis by a Cas6 endoribonuclease.},
journal = {RNA biology},
volume = {16},
number = {10},
pages = {1438-1447},
pmid = {31232162},
issn = {1555-8584},
support = {P41 GM103393/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Associated Proteins/chemistry/*metabolism ; Catalysis ; Catalytic Domain ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Firmicutes/genetics/metabolism ; Gene Order ; Models, Molecular ; Molecular Conformation ; Nucleic Acid Conformation ; RNA/*chemistry/*genetics ; RNA Precursors ; Structure-Activity Relationship ; Substrate Specificity ; *Transcription, Genetic ; },
abstract = {Prokaryotic CRISPR-Cas adaptive immune systems rely on small non-coding RNAs derived from CRISPR loci to recognize and destroy complementary nucleic acids. However, the mechanism of Type IV CRISPR RNA (crRNA) biogenesis is poorly understood. To dissect the mechanism of Type IV CRISPR RNA biogenesis, we determined the x-ray crystal structure of the putative Type IV CRISPR associated endoribonuclease Cas6 from Mahella australiensis (Ma Cas6-IV) and characterized its enzymatic activity with RNA cleavage assays. We show that Ma Cas6-IV specifically cleaves Type IV crRNA repeats at the 3' side of a predicted stem loop, with a metal-independent, single-turnover mechanism that relies on a histidine and a tyrosine located within the putative endonuclease active site. Structure and sequence alignments with Cas6 orthologs reveal that although Ma Cas6-IV shares little sequence homology with other Cas6 proteins, all share common structural features that bind distinct crRNA repeat sequences. This analysis of Type IV crRNA biogenesis provides a structural and biochemical framework for understanding the similarities and differences of crRNA biogenesis across multi-subunit Class 1 CRISPR immune systems.},
}
@article {pmid31231990,
year = {2019},
author = {Yan, Y and Zhu, J and Xie, C and Liu, C},
title = {[Regulatory framework of genome-edited products - a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {6},
pages = {921-930},
doi = {10.13345/j.cjb.180418},
pmid = {31231990},
issn = {1872-2075},
mesh = {CRISPR-Cas Systems ; Endonucleases ; *Gene Editing ; *Genome ; Mutagenesis, Site-Directed ; },
abstract = {Genome editing is a genetic engineering technique that uses site-directed cleavage activity of specific artificial nucleases and endogenous DNA damage repair activity to generate insertions, deletions or substitutions in the targeted genomic loci. As the accuracy and efficiency of genome editing is improving and the operation is simple, the application of genome editing is expanding. This article provides an overview of the three major genome editing technologies and genome editing types, and the regulatory frameworks for genome-edited products were summarized in the United States, the European Union, and other countries. At the same time, based on the Chinese safety management principles and systems for genetically modified organisms (GMOs), the authors proposed a regulatory framework for genome-edited products. Genome-edited products should first be classified according to whether containing exogenous genetic components such as Cas9 editing enzymes or not. They should be regulated as traditional genetically modified organisms if they do. Otherwise, the regulation of genome-edited products depends on targeted modifications.},
}
@article {pmid31231788,
year = {2020},
author = {Jiang, C and Meng, L and Yang, B and Luo, X},
title = {Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment.},
journal = {Clinical genetics},
volume = {97},
number = {1},
pages = {73-88},
doi = {10.1111/cge.13589},
pmid = {31231788},
issn = {1399-0004},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Genetic Therapy/*trends ; Genome, Human/genetics ; Humans ; Mutation/genetics ; Neoplasms/genetics/*therapy ; },
abstract = {In recent years, gene editing, especially that using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, has made great progress in the field of gene function. Rapid development of gene editing techniques has contributed to their significance in the field of medicine. Because the CRISPR/Cas9 gene editing tool is not only powerful but also has features such as strong specificity and high efficiency, it can accurately and rapidly screen the whole genome, facilitating the administration of gene therapy for specific diseases. In the field of tumor research, CRISPR/Cas9 can be used to edit genomes to explore the mechanisms of tumor occurrence, development, and metastasis. In these years, this system has been increasingly applied in tumor treatment research. CRISPR/Cas9 can be used to treat tumors by repairing mutations or knocking out specific genes. To date, numerous preliminary studies have been conducted on tumor treatment in related fields. CRISPR/Cas9 holds great promise for gene-level tumor treatment. Personalized and targeted therapy based on CRISPR/Cas9 will possibly shape the development of tumor therapy in the future. In this study, we review the findings of CRISPR/Cas9 for tumor treatment research to provide references for related future studies on the pathogenesis and clinical treatment of tumors.},
}
@article {pmid31231643,
year = {2019},
author = {Wasmer, M},
title = {Roads Forward for European GMO Policy-Uncertainties in Wake of ECJ Judgment Have to be Mitigated by Regulatory Reform.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {7},
number = {},
pages = {132},
pmid = {31231643},
issn = {2296-4185},
abstract = {This article gives an overview of legal and procedural uncertainties regarding genome edited organisms and possible ways forward for European GMO policy. After a recent judgment by the European Court of Justice (ECJ judgment of 25 July 2018, C-528/16), organisms obtained by techniques of genome editing are GMOs and subject to the same obligations as transgenic organisms. Uncertainties emerge if genome edited organisms cannot be distinguished from organisms bred by conventional techniques, such as crossing or random mutagenesis. In this case, identical organisms can be subject to either GMO law or exempt from regulation because of the use of a technique that cannot be identified. Regulatory agencies might not be able to enforce GMO law for such cases in the long term. As other jurisdictions do not regulate such organisms as GMOs, accidental imports might occur and undermine European GMO regulation. In the near future, the EU Commission as well as European and national regulatory agencies will decide on how to apply the updated interpretation of the law. In order to mitigate current legal and procedural uncertainties, a first step forward lies in updating all guidance documents to specifically address genome editing specifically address genome editing, including a solution for providing a unique identifier. In part, the authorization procedure for GMO release can be tailored to different types of organisms by making use of existing flexibilities in GMO law. However, only an amendment to the regulations that govern the process of authorization for GMO release can substantially lower the burden for innovators. In a second step, any way forward has to aim at amending, supplementing or replacing the European GMO Directive (2001/18/EC). The policy options presented in this article presuppose political readiness for reform. This may not be realistic in the current political situation. However, if the problems of current GMO law are just ignored, European competitiveness and research in green biotechnology will suffer.},
}
@article {pmid31230751,
year = {2019},
author = {Helary, L and Castille, J and Passet, B and Vaiman, A and Beauvallet, C and Jaffrezic, F and Charles, M and Tamzini, M and Baraige, F and Letheule, M and Laubier, J and Moazami-Goudarzi, K and Vilotte, JL and Blanquet, V and Duchesne, A},
title = {DNAJC2 is required for mouse early embryonic development.},
journal = {Biochemical and biophysical research communications},
volume = {516},
number = {1},
pages = {258-263},
doi = {10.1016/j.bbrc.2019.06.009},
pmid = {31230751},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/*genetics ; Embryo Implantation ; Embryo Loss/genetics ; Embryo, Mammalian/*embryology/metabolism ; Embryonic Development ; Female ; Gene Deletion ; *Gene Expression Regulation, Developmental ; Mice/*embryology/genetics ; Molecular Chaperones/*genetics ; Pregnancy ; RNA-Binding Proteins/*genetics ; },
abstract = {DNAJC2 protein, also known as ZRF1 or MPP11, acts both as chaperone and as chromatin regulator. It is involved in stem cell differentiation and its expression is associated with various cancer malignancies. However, the role of Dnajc2 gene during mouse embryogenesis has not been assessed so far. To this aim, we invalidated Dnajc2 gene in FVB/Nj mice using the CrispR/Cas9 approach. We showed that this invalidation leads to the early post-implantation lethality of the nullizygous embryos. Furthermore, using siRNAs against Dnajc2 in mouse 1-cell embryos, we showed that maternal Dnajc2 mRNAs may allow for the early preimplantation development of these embryos. Altogether, these data demonstrate for the first time the requirement of DNAJC2 for early mouse embryogenesis.},
}
@article {pmid31230714,
year = {2019},
author = {Rauch, S and He, E and Srienc, M and Zhou, H and Zhang, Z and Dickinson, BC},
title = {Programmable RNA-Guided RNA Effector Proteins Built from Human Parts.},
journal = {Cell},
volume = {178},
number = {1},
pages = {122-134.e12},
pmid = {31230714},
issn = {1097-4172},
support = {P30 CA014599/CA/NCI NIH HHS/United States ; R35 GM119840/GM/NIGMS NIH HHS/United States ; RM1 HG008935/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Protein Biosynthesis ; Protein Engineering/*methods ; Proteolysis ; RNA/*metabolism ; RNA, Guide/*metabolism ; RNA, Small Interfering ; Transcription Activator-Like Effector Nucleases/genetics/*metabolism ; Transfection ; },
abstract = {Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.},
}
@article {pmid31230441,
year = {2019},
author = {Yoder, KE},
title = {A CRISPR/Cas9 library to map the HIV-1 provirus genetic fitness.},
journal = {Acta virologica},
volume = {63},
number = {2},
pages = {129-138},
pmid = {31230441},
issn = {0001-723X},
support = {R01 AI150496/AI/NIAID NIH HHS/United States ; R21 AI122981/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Genetic Fitness ; Genome, Viral ; *Genomic Library ; *HIV-1/genetics ; Humans ; Proviruses/genetics ; },
abstract = {The integrated proviral genome is the major barrier to a cure for HIV-1 infection. Genome editing technologies, such as CRISPR/Cas9, may disable or remove the HIV-1 provirus by introducing DNA double strand breaks at sequence specific sites in the viral genome. Host DNA repair by the error-prone non-homologous end joining pathway generates mutagenic insertions or deletions at the break. CRISPR/Cas9 editing has been shown to reduce replication competent viral genomes in cell culture, but only a minority of possible genome editing targets have been assayed. Currently there is no map of double strand break genetic fitness for HIV-1 to inform the choice of editing targets. However, CRISPR/Cas9 genome editing makes it possible to target double strand breaks along the length of the provirus to generate a double strand break genetic fitness map. We identified all possible HIV-1 targets with different bacterial species of CRISPR/Cas9. This library of guide RNAs was evaluated for GC content and potential off-target sites in the human genome. Complexity of the library was reduced by eliminating duplicate guide RNA targets in the HIV-1 long terminal repeats and targets in the env gene. Although the HIV-1 genome is AT-rich, the S. pyogenes CRISPR/Cas9 with the proto-spacer adjacent motif NGG offers the most HIV-1 guide RNAs. This library of HIV-1 guide RNAs may be used to generate a double strand break genetic fragility map to be further applied to any genome editing technology designed for the HIV-1 provirus. Keywords: HIV-1; genome editing; CRISPR; genetic fitness; guide RNAs.},
}
@article {pmid31229900,
year = {2019},
author = {Li, C and Wang, Q and Peng, Z and Lin, Y and Liu, H and Yang, X and Li, S and Liu, X and Chen, J},
title = {Generation of FOS gene knockout lines from a human embryonic stem cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {39},
number = {},
pages = {101479},
doi = {10.1016/j.scr.2019.101479},
pmid = {31229900},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Cell Differentiation/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Stem Cells/metabolism ; Endothelial Cells ; Gene Knockout Techniques ; Hematopoietic Stem Cells/metabolism ; Human Embryonic Stem Cells/metabolism ; Humans ; Karyotype ; },
abstract = {FOS is component of the AP-1 complex and has been reported to be involved in many cellular functions, including cell proliferation, differentiation, survival, angiogenesis, hematopoiesis and cancer progress. To further understand the exact role of FOS in these processes, here we created two FOS knockout human embryonic stem cell lines by CRISPR/Cas9 mediated gene targeting. These cell lines retained normal morphology and karyotype, normal expression of pluripotent markers, and differentiation potential both in vivo and in vitro. These cell lines can be used to verify whether the FOS mutated produces any affect on endothelial cells and hematopoietic progenitor cells during the hematopoietic differentiation.},
}
@article {pmid31228551,
year = {2019},
author = {Wang, F and Chang, C and Li, R and Zhang, Z and Jiang, H and Zeng, N and Li, D and Chen, L and Xiao, Y and Chen, W and Wang, Q},
title = {Retinol binding protein 4 mediates MEHP-induced glucometabolic abnormalities in HepG2 cells.},
journal = {Toxicology},
volume = {424},
number = {},
pages = {152236},
doi = {10.1016/j.tox.2019.06.007},
pmid = {31228551},
issn = {1879-3185},
mesh = {Apoptosis/drug effects ; CRISPR-Cas Systems ; Diethylhexyl Phthalate/*analogs &amp; derivatives/toxicity ; Gene Deletion ; Gluconeogenesis/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Homeostasis/drug effects ; Humans ; Insulin Resistance/genetics ; Liver/drug effects/metabolism ; Plasticizers/*toxicity ; Retinol-Binding Proteins, Plasma/genetics/*metabolism ; Signal Transduction/genetics ; },
abstract = {Epidemiological and experimental data have implicated the role of di(2-ethylhexyl) phthalate (DEHP) and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in the pathogenesis of metabolic syndrome, including the impairment of hepatic glucose metabolism. To elucidate the underlying mechanism by which DEHP or MEHP perturbs hepatic glucose homeostasis, we compared the effect of DEHP (0-200 μM) and MEHP (0-200 μM) on glucose metabolism in HepG2 cells. In this study, we found that MEHP can induce more severe impairments in glucose homeostasis than DEHP can; these include increased hepatic gluconeogenesis via receptor substrate-1/protein kinase B/fork-head box protein O1 (IRS-1/AKT/FOXO1)-mediated phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC) up-regulation, as well as decreased hepatic glycogen synthesis via glucokinase (GCK) inhibition and IRS-1/AKT/glycogen synthase kinase-3β (GSK-3β)-mediated glycogen synthase (GYS) inactivation. Additionally, our results demonstrated that retinol binding protein 4 (RBP4), an insulin resistance-inducing factor, plays a critical role in the MEHP-induced disorder of glucose homeostasis and the dysfunction of insulin signaling transduction, whereas the deletion of RBP4 by the clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/Cas9) significantly reversed these toxic effects. Although these should be interpreted with caution in view of limited in vivo evidence, the present study provides the first in vitro evidence for potential involvements of RBP4 in disturbance of glucose homeostasis in the MEHP-treated HepG2 cells.},
}
@article {pmid31228550,
year = {2020},
author = {Gramelspacher, MJ and Hou, Z and Zhang, Y},
title = {Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {32-41},
pmid = {31228550},
issn = {1095-9130},
support = {K99 GM117268/GM/NIGMS NIH HHS/United States ; R00 GM117268/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/metabolism ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/*metabolism ; CRISPR-Cas Systems/*genetics ; Enzyme Assays/instrumentation/*methods ; Neisseria meningitidis/enzymology/genetics/virology ; RNA, Guide/genetics/*metabolism ; Viral Proteins/metabolism ; },
abstract = {The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9's RNA-targeting potential.},
}
@article {pmid31228153,
year = {2019},
author = {Filipiak, WE and Hughes, ED and Gavrilina, GB and LaForest, AK and Saunders, TL},
title = {Next Generation Transgenic Rat Model Production.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2018},
number = {},
pages = {97-114},
doi = {10.1007/978-1-4939-9581-3_4},
pmid = {31228153},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Expression ; Genes, Reporter ; Genetic Engineering/*methods ; Microinjections/veterinary ; Models, Animal ; Rats ; Rats, Transgenic/genetics/*growth &amp; development ; Zygote/*growth &amp; development ; },
abstract = {The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.},
}
@article {pmid31228115,
year = {2019},
author = {Ali, Z and Zaidi, SS and Tashkandi, M and Mahfouz, MM},
title = {A Simplified Method to Engineer CRISPR/Cas9-Mediated Geminivirus Resistance in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2028},
number = {},
pages = {167-183},
doi = {10.1007/978-1-4939-9635-3_10},
pmid = {31228115},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; DNA Mutational Analysis ; Disease Resistance/*genetics/immunology ; *Geminiviridae ; *Gene Editing ; *Genetic Engineering ; Genetic Vectors/genetics ; Plant Diseases/*genetics/immunology/*virology ; Plants, Genetically Modified ; RNA, Guide ; Sequence Analysis, DNA ; Viral Load ; },
abstract = {Throughout the world, geminiviruses cause devastating losses in economically important crops, including tomato, cotton, cassava, potato, chili, and cucumber; however, control mechanisms such as genetic resistance remain expensive and ineffective. CRISPR/Cas9 is an adaptive immunity mechanism used by prokaryotes to defend against invading nucleic acids of phages and plasmids. The CRISPR/Cas9 system has been harnessed for targeted genome editing in a variety of eukaryotic species, and in plants, CRISPR/Cas9 has been used to modify or introduce many traits, including virus resistance. Recently, we demonstrated that the CRISPR/Cas9 system could be used to engineer plant immunity against geminiviruses by directly targeting the viral genome for degradation. In this chapter, we describe a detailed method for engineering CRISPR/Cas9-mediated resistance against geminiviruses. This method may provide broad, durable viral resistance, as it can target conserved regions of the viral genome and can also be customized to emerging viral variants. Moreover, this method can be used in many crop species, as it requires little or no knowledge of the host plant's genome.},
}
@article {pmid31228114,
year = {2019},
author = {Osmani, Z and Jin, S and Mikami, M and Endo, M and Atarashi, H and Fujino, K and Yamada, T and Nakahara, KS},
title = {CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2028},
number = {},
pages = {153-165},
doi = {10.1007/978-1-4939-9635-3_9},
pmid = {31228114},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; DNA Mutational Analysis ; *Gene Editing ; Gene Targeting ; Genetic Vectors/genetics ; Mutagenesis ; Phenotype ; Plant Proteins/*genetics ; *Plants, Genetically Modified ; Solanum tuberosum/*genetics ; Transformation, Genetic ; },
abstract = {A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.},
}
@article {pmid31228045,
year = {2019},
author = {Liu, X and Zheng, G and Wang, G and Jiang, W and Li, L and Lu, Y},
title = {Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor.},
journal = {Science China. Life sciences},
volume = {62},
number = {11},
pages = {1492-1505},
doi = {10.1007/s11427-019-9549-8},
pmid = {31228045},
issn = {1869-1889},
mesh = {Anthraquinones/metabolism ; Anti-Bacterial Agents/biosynthesis/*metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Cyclic GMP/*genetics/metabolism ; DNA-Binding Proteins ; Escherichia coli Proteins/*genetics/metabolism ; Fermentation ; Gene Editing ; Gene Expression Regulation, Bacterial ; Mutation ; Phosphorus-Oxygen Lyases/*genetics/metabolism ; Prodigiosin/analogs &amp; derivatives/metabolism ; Streptomyces coelicolor/*metabolism ; Transfection ; },
abstract = {Cyclic dimeric GMP (c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase (DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.},
}
@article {pmid31227932,
year = {2019},
author = {Luo, Y},
title = {Refining CRISPR-based genome and epigenome editing off-targets.},
journal = {Cell biology and toxicology},
volume = {35},
number = {4},
pages = {281-283},
doi = {10.1007/s10565-019-09482-8},
pmid = {31227932},
issn = {1573-6822},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Methylation/genetics ; Epigenome/genetics ; Gene Editing/*ethics/methods ; Genome/genetics ; Humans ; },
}
@article {pmid31227825,
year = {2019},
author = {Tirronen, A and Hokkanen, K and Vuorio, T and Ylä-Herttuala, S},
title = {Recent advances in novel therapies for lipid disorders.},
journal = {Human molecular genetics},
volume = {28},
number = {R1},
pages = {R49-R54},
doi = {10.1093/hmg/ddz132},
pmid = {31227825},
issn = {1460-2083},
mesh = {Animals ; Clinical Trials as Topic ; Combined Modality Therapy ; Disease Management ; Disease Susceptibility ; Drug Evaluation, Preclinical ; Genetic Therapy/methods ; Genetic Vectors/genetics ; Humans ; Hypolipidemic Agents/pharmacology/therapeutic use ; Lipid Metabolism/drug effects/genetics ; Lipid Metabolism Disorders/etiology/metabolism/*therapy ; Treatment Outcome ; },
abstract = {The prevalence of lipid disorders is alarmingly increasing in the Western world. They are the result of either primary causes, such as unhealthy lifestyle choices or inherited risk factors, or secondary causes like other diseases or medication. Atypical changes in the synthesis, processing and catabolism of lipoprotein particles may lead to severe hypercholesterolemia, hypertriglyceridemia or elevated Lp(a). Although cholesterol-lowering drugs are the most prescribed medications, not all patients achieve guideline recommended cholesterol levels with the current treatment options, emphasising the need for new therapies. Also, some lipid disorders do not have any treatment options but rely only on stringent dietary restriction. Patients with untreated lipid disorders carry a severe risk of cardiovascular disease, diabetes, non-alcoholic fatty liver disease and pancreatitis among others. To achieve better treatment outcome, novel selective gene expression and epigenetic targeting therapies are constantly being developed. Therapeutic innovations employing targeted RNA technology utilise small interfering RNAs, antisense oligonucleotides, long non-coding RNAs and microRNAs to regulate target protein production whereas viral gene therapy provides functional therapeutic genes and CRISPR/Cas technology relies on gene editing and transcriptional regulation. In this review, we will discuss the latest advances in clinical trials for novel lipid-lowering therapies and potential new targets in pre-clinical phase.},
}
@article {pmid31227643,
year = {2019},
author = {},
title = {The people behind the papers - Eduardo Leyva-Díaz and Oliver Hobert.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {13},
pages = {},
doi = {10.1242/dev.180869},
pmid = {31227643},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Caenorhabditis elegans/embryology/*genetics ; Central Nervous System/*embryology ; *Developmental Biology/history/trends ; Gene Editing/history/*methods ; History, 20th Century ; History, 21st Century ; Humans ; *Laboratory Personnel/history ; New York ; Organ Specificity/genetics ; },
abstract = {Transcriptional autoregulation occurs when transcription factors bind their own cis-regulatory sequences, ensuring their own continuous expression along with expression of other targets. During development, continued expression of identity-specifying transcription factors can be achieved by autoregulation, but until now formal evidence for a developmental requirement of autoregulation has been lacking. A new paper in Development provides this proof with the help of CRISPR/Cas9 gene editing in the C. elegans nervous system. We caught up with the paper's two authors: postdoc Eduardo Leyva-Díaz and his supervisor Oliver Hobert, Professor of Biological Sciences and HHMI Investigator at Columbia University, New York, to find out more about the work.},
}
@article {pmid31227640,
year = {2019},
author = {Blackburn, NB and Michael, LF and Meikle, PJ and Peralta, JM and Mosior, M and McAhren, S and Bui, HH and Bellinger, MA and Giles, C and Kumar, S and Leandro, AC and Almeida, M and Weir, JM and Mahaney, MC and Dyer, TD and Almasy, L and VandeBerg, JL and Williams-Blangero, S and Glahn, DC and Duggirala, R and Kowala, M and Blangero, J and Curran, JE},
title = {Rare DEGS1 variant significantly alters de novo ceramide synthesis pathway.},
journal = {Journal of lipid research},
volume = {60},
number = {9},
pages = {1630-1639},
pmid = {31227640},
issn = {1539-7262},
support = {R01 HL113323/HL/NHLBI NIH HHS/United States ; P01 HL045522/HL/NHLBI NIH HHS/United States ; C06 RR020547/RR/NCRR NIH HHS/United States ; R37 MH059490/MH/NIMH NIH HHS/United States ; R01 HL140681/HL/NHLBI NIH HHS/United States ; },
mesh = {Blotting, Western ; CRISPR-Cas Systems/genetics ; Ceramides/*biosynthesis/metabolism ; Fatty Acid Desaturases/*genetics ; Female ; Genotype ; Hep G2 Cells ; Humans ; Male ; Mexican Americans ; },
abstract = {The de novo ceramide synthesis pathway is essential to human biology and health, but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry-specific rare functional variant, L175Q, in delta 4-desaturase, sphingolipid 1 (DEGS1), a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders and spur ongoing research of drug targets along this pathway.},
}
@article {pmid31226710,
year = {2019},
author = {Hampton, HG and Patterson, AG and Chang, JT and Taylor, C and Fineran, PC},
title = {GalK limits type I-F CRISPR-Cas expression in a CRP-dependent manner.},
journal = {FEMS microbiology letters},
volume = {366},
number = {11},
pages = {},
doi = {10.1093/femsle/fnz137},
pmid = {31226710},
issn = {1574-6968},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Galactose/metabolism ; Plasmids/genetics ; },
abstract = {CRISPR-Cas adaptive immune systems protect bacteria from phage predation, and other foreign genetic elements such as plasmids. Significant advances have been made regarding how CRISPR-Cas systems elicit immunity; however, comparatively little is known about their regulation. To study CRISPR-Cas regulation, we describe the construction of suicide lacZ-reporter plasmids with different antibiotic resistance cassettes. Through recombination into the host chromosome, single-copy expression can be achieved, thus preserving natural gene expression and maintaining a reporter expression output that reflects regulation within a normal genomic context. Previous work determined that the galactose metabolism gene galM, decreased the expression of the cas operon in Pectobacterium atrosepticum. We used the new integrative reporters to investigate galK, a gene that is located elsewhere in the genome and is responsible for the conversion of α-D-Galactose to Galactose-1-P during galactose metabolism. Deletion of galK led to elevated cas expression in a CRP-dependent manner but had no effect on CRISPR array expression. These results highlight that the metabolic status of the host cell is linked to the induction of CRISPR-Cas immunity.},
}
@article {pmid31226583,
year = {2019},
author = {Wang, Z and Wang, L and Liu, W and Hu, D and Gao, Y and Ge, Q and Liu, X and Li, L and Wang, Y and Wang, S and Li, C},
title = {Pathogenic mechanism and gene correction for LQTS-causing double mutations in KCNQ1 using a pluripotent stem cell model.},
journal = {Stem cell research},
volume = {38},
number = {},
pages = {101483},
doi = {10.1016/j.scr.2019.101483},
pmid = {31226583},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *KCNQ1 Potassium Channel ; *Long QT Syndrome/genetics/metabolism/pathology/therapy ; *Models, Cardiovascular ; *Mutation ; Myocytes, Cardiac/metabolism/pathology ; },
abstract = {AIMS: To establish a KCNQ1 mutant-specific induced pluripotent stem cell (iPSC) model of a Chinese inherited long QT syndrome (LQTS) patient and to explore the pathogenesis of KCNQ1 mutations.<br><br>METHODS AND RESULTS: (1) Two patient-specific iPSC lines from the proband were obtained. (2) The experiments produced spontaneously beating cardiomyocytes (CMs) from patient iPSCs. Splicing mutation c. 605-2A&#x202f;>&#x202f;G in iPSC-derived cardiomyocytes (iPSC-CMs) resulted in the skipping of exon 4, exons 3-4, or exons 3-6 in KCNQ1 transcription what was observed in the patient's peripheral leukocytes. (3) Action potential duration (APD) at 50% and 90% repolarization (APD50 and APD90) of the patient's iPSC-derived ventricular-like-CMs was significantly longer than that of the control. Moreover, early after depolarization (EAD) and coupled beats were observed only in L1-iPSC-CMs. (4) A c.815G&#x202f;>&#x202f;A corrected iPSC line was obtained by using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system.<br><br>CONCLUSION: (1) Cardiomyocytes with spontaneous pulsation were successfully differentiated from LQTS patient-specific iPSC lines. (2) For KCNQ1 splicing mutations, there is a chance that splicing patterns in peripheral leukocytes are similar to that in patient iPSC-CMs. (3) The truncated KCNQ1 proteins induced by such splicing mutation might cause Iks decrease, which in turn produced APD prolongation and triggered activities. (4) Our data showed that CRISPR-Cas9 system could be used to rescue the LQTS-related mutations.},
}
@article {pmid31226337,
year = {2019},
author = {Dong, H and Zheng, J and Yu, D and Wang, B and Pan, L},
title = {Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes.},
journal = {Journal of microbiological methods},
volume = {163},
number = {},
pages = {105655},
doi = {10.1016/j.mimet.2019.105655},
pmid = {31226337},
issn = {1872-8359},
mesh = {Aspergillus niger/*genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Genetic Engineering/methods ; Glucan 1,4-alpha-Glucosidase/biosynthesis ; alpha-Amylases/biosynthesis ; },
abstract = {Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.},
}
@article {pmid31226203,
year = {2019},
author = {Kitsera, N and Rodriguez-Alvarez, M and Emmert, S and Carell, T and Khobta, A},
title = {Nucleotide excision repair of abasic DNA lesions.},
journal = {Nucleic acids research},
volume = {47},
number = {16},
pages = {8537-8547},
pmid = {31226203},
issn = {1362-4962},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Transformed ; DNA/chemistry/*genetics/metabolism ; DNA Damage ; *DNA Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/*genetics/metabolism ; DNA-Binding Proteins/deficiency/*genetics ; Fibroblasts/cytology/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; Genome, Human ; Humans ; Mutation ; Protein Binding ; Skin/cytology/metabolism ; Transcription, Genetic ; Xeroderma Pigmentosum Group A Protein/*genetics/metabolism ; },
abstract = {Apurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, we demonstrate that nucleotide excision repair pathway (NER) efficiently removes BER-resistant AP lesions and significantly enhances the repair of APE1-sensitive ones. Our results further indicate that core NER components XPA and XPF are equally required and that both global genome (GG-NER) and transcription coupled (TC-NER) subpathways contribute to the repair.},
}
@article {pmid31225941,
year = {2019},
author = {de Waal, E and Tran, T and Abbondanza, D and Dey, A and Peterson, C},
title = {An undergraduate laboratory module that uses the CRISPR/Cas9 system to generate frameshift mutations in yeast.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {47},
number = {5},
pages = {573-580},
doi = {10.1002/bmb.21280},
pmid = {31225941},
issn = {1539-3429},
mesh = {CRISPR-Cas Systems/*genetics ; Frameshift Mutation/*genetics ; Humans ; *Laboratories ; Learning ; Saccharomyces cerevisiae/*genetics ; Students ; Universities ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for gene editing and it has become increasingly important for biology students to understand this emerging technique. Most CRISPR laboratory teaching modules use complex metazoan systems or mammalian cell culture which can be expensive. Here, we present a lab module that engages students in learning the fundamentals of CRISPR/Cas9 methodology using the simple and inexpensive model system, Saccharomyces cerevisiae. Students use CRISPR/Cas9 and nonhomologous end joining to generate frameshift insertion and deletion mutations in the CAN1 gene, which are easily selected for using media plates that have canavanine. DNA sequencing is also performed to determine what type of mutation occurred in gene-edited cells. This easy to implement set of experiments has been run as both a 5-week and a shorter 3-week lab module. Learning assessments demonstrate increased understanding in CRISPR-related concepts as well as increased confidence using molecular techniques. Thus, this CRISPR/Cas9 lab module can be added to an existing Genetics, Microbiology, or Molecular Biology lab course to help undergraduate students learn current gene editing techniques with limited effort and cost. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):573-580, 2019.},
}
@article {pmid31225758,
year = {2019},
author = {Chen, S and Yang, L and Li, W},
title = {CRISPR Screening "Big Data" Informs Novel Therapeutic Solutions.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {152-154},
doi = {10.1089/crispr.2019.29062.sch},
pmid = {31225758},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Gene Editing ; Humans ; *Neoplasms ; },
}
@article {pmid31225756,
year = {2019},
author = {Boggio, A and Knoppers, BM and Almqvist, J and Romano, CPR},
title = {The Human Right to Science and the Regulation of Human Germline Engineering.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {134-142},
doi = {10.1089/crispr.2018.0053},
pmid = {31225756},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*legislation &amp; jurisprudence ; Germ Cells ; *Human Rights ; Humans ; },
abstract = {There is currently no international consensus on how human germline engineering should be regulated. Existing national legislation fails to provide the governance framework necessary to regulate germline engineering in the CRISPR era. This is an obstacle to scientific and clinical advancements and inconsistent with human rights requirements. To move forward, we suggest that the human right to science is an ideal starting point for building consensus, at the national and international levels, on governing principles that promote responsible scientific and technological advancements. Regulatory frameworks must recognize the international nature of modern germline genome engineering research, the need for shared governance rather than tech-locked prohibitions, and the fact that humans are not their germline.},
}
@article {pmid31225755,
year = {2019},
author = {Rees, HA and Gaudelli, NM},
title = {A Dimmer-Switch for SpCas9 Activity.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {146-148},
doi = {10.1089/crispr.2019.29060.hre},
pmid = {31225755},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; },
}
@article {pmid31225754,
year = {2019},
author = {Abudayyeh, OO and Gootenberg, JS and Kellner, MJ and Zhang, F},
title = {Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13.},
journal = {The CRISPR journal},
volume = {2},
number = {3},
pages = {165-171},
pmid = {31225754},
issn = {2573-1602},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F30 CA210382/CA/NCI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Plant/*analysis ; Endodeoxyribonucleases ; *Genes, Plant ; Genetic Testing/*methods ; Soybeans/*genetics ; },
abstract = {Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (∼15 min), quantitative, and portable, and can process crude soybean extracts as input material for minimal nucleic acid sample preparation. This field-ready SHERLOCK platform with color-based lateral flow readout can be applied for detection and quantitation of genes in a range of agricultural applications.},
}
@article {pmid31225753,
year = {2019},
author = {Sontheimer, EJ},
title = {X-Tracting a New CRISPR-Cas Genome-Editing Platform from Metagenomic Data Sets.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {148-150},
doi = {10.1089/crispr.2019.29061.ejs},
pmid = {31225753},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Metagenome ; *RNA ; },
}
@article {pmid31225751,
year = {2019},
author = {Fox, R},
title = {Too Much Compromise in Today's CRISPR Pipelines.},
journal = {The CRISPR journal},
volume = {2},
number = {3},
pages = {143-145},
pmid = {31225751},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Eukaryota ; Gene Editing/*methods ; Humans ; },
abstract = {CRISPR-based editing is a revolutionary tool for genome engineering and discovery. The pace of innovation in this young field is truly astonishing, and yet significant gaps remain in the set of capabilities offered. In particular, scalable (massively parallel and multiplex) editing is only available for a small variety of edit types, placing fundamental limits on the kinds of studies researchers can perform. Additional advancements are needed to realize the full potential of the technology.},
}
@article {pmid31225750,
year = {2019},
author = {Barrangou, R},
title = {Taking CRISPR to New Heights.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {133},
doi = {10.1089/crispr.2019.29064.rba},
pmid = {31225750},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}
@article {pmid31225748,
year = {2019},
author = {Newsham, W},
title = {Now on Course, CRISPR J.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {155-156},
doi = {10.1089/crispr.2019.29057.wne},
pmid = {31225748},
issn = {2573-1602},
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Horses/genetics ; },
}
@article {pmid31225747,
year = {2019},
author = {Huston, NC and Tycko, J and Tillotson, EL and Wilson, CJ and Myer, VE and Jayaram, H and Steinberg, BE},
title = {Identification of Guide-Intrinsic Determinants of Cas9 Specificity.},
journal = {The CRISPR journal},
volume = {2},
number = {3},
pages = {172-185},
pmid = {31225747},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Staphylococcus aureus/*enzymology ; Substrate Specificity ; },
abstract = {Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. In silico predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site based entirely on the sequence of the guide RNA (gRNA) and the target. We describe a library-based biochemical assay that directly reports the cleavage efficiency of a particular Cas9-guide complex by measuring both uncleaved and cleaved target molecules over a wide range of mismatched library members. We applied our assay using libraries of targets to evaluate the specificity of Staphylococcus aureus Cas9 under a variety of experimental conditions. Surprisingly, our data show an unexpectedly high variation in the random gRNA:target DNA mismatch tolerance when cleaving with different gRNAs, indicating guide-intrinsic mismatch permissiveness and challenging the assumption of universal specificity models. We use data generated by our assay to create the first off-target, guide-specific cleavage models. The barcoded libraries of targets approach is rapid, highly modular, and capable of generating protein- and guide-specific models, as well as illuminating the biophysics of Cas9 binding versus cutting. These models may be useful in identifying potential off-targets, and the gRNA-intrinsic nature of mismatch tolerance argues for coupling these specificity models with orthogonal methods for a more complete assessment of gRNA specificity.},
}
@article {pmid31223339,
year = {2019},
author = {Cairns, TC and Feurstein, C and Zheng, X and Zheng, P and Sun, J and Meyer, V},
title = {A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger.},
journal = {Biotechnology for biofuels},
volume = {12},
number = {},
pages = {149},
pmid = {31223339},
issn = {1754-6834},
abstract = {BACKGROUND: Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains.<br><br>RESULTS: In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN.<br><br>CONCLUSION: The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.},
}
@article {pmid31223254,
year = {2019},
author = {Hirsch, F and Iphofen, R and Koporc, Z},
title = {Ethics assessment in research proposals adopting CRISPR technology.},
journal = {Biochemia medica},
volume = {29},
number = {2},
pages = {020202},
pmid = {31223254},
issn = {1846-7482},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics ; Humans ; *Research Design ; },
abstract = {The rapid and exponential growth of genome editing has posed many challenges for bioethics. This article briefly explains the nature of the technique and the particularly rapid development of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology. The international and, specifically, European-level systems for assessing the ethical issues consequent on these developments are outlined and discussed. The challenges posed by cases in China are summarized to raise concerns about how a more shared, universally consistent appraisal of bioethical issues can be promoted.},
}
@article {pmid31222853,
year = {2020},
author = {Xu, J and Kang, BC and Naing, AH and Bae, SJ and Kim, JS and Kim, H and Kim, CK},
title = {CRISPR/Cas9-mediated editing of 1-aminocyclopropane-1-carboxylate oxidase1 enhances Petunia flower longevity.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {287-297},
pmid = {31222853},
issn = {1467-7652},
mesh = {Amino Acid Oxidoreductases/*genetics ; *CRISPR-Cas Systems ; Flowers/*growth &amp; development ; *Gene Editing ; Petunia/enzymology/*genetics ; Plants, Genetically Modified ; },
abstract = {The genes that encode the ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) are thought to be involved in flower senescence. Hence, we investigated whether the transcript levels of PhACO genes (PhACO1, PhACO3 and PhACO4) in Petunia cv. Mirage Rose are associated with ethylene production at different flowering stages. High transcript levels were detected in the late flowering stage and linked to high ethylene levels. PhACO1 was subsequently edited using the CRISPR/Cas9 system, and its role in ethylene production was investigated. PhACO1-edited T0 mutant lines, regardless of mutant type (homozygous or monoallelic), exhibited significantly reduced ethylene production and enhanced flower longevity compared with wild-type. Flower longevity and the reduction in ethylene production were observed to be stronger in homozygous plants than in their monoallelic counterparts. Additionally, the transmission of the edited gene to the T1 (lines 6 and 36) generation was also confirmed, with the results for flower longevity and ethylene production proving to be identical to those of the T0 mutant lines. Overall, this study increases the understanding of the role of PhACO1 in petunia flower longevity and also points to the CRISPR/Cas9 system being a powerful tool in the improvement of floricultural quality.},
}
@article {pmid31222183,
year = {2020},
author = {Nair, J and Nair, A and Veerappan, S and Sen, D},
title = {Translatable gene therapy for lung cancer using Crispr CAS9-an exploratory review.},
journal = {Cancer gene therapy},
volume = {27},
number = {3-4},
pages = {116-124},
pmid = {31222183},
issn = {1476-5500},
mesh = {Animals ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/diagnosis/genetics/*therapy ; Cell Line, Tumor ; Chromosomes, Human, Pair 3/genetics ; Chromosomes, Human, Pair 5/genetics ; Chromosomes, Human, Pair 9/genetics ; Clinical Trials as Topic ; Gene Editing/methods ; Gene Knockout Techniques ; Genetic Therapy/*methods ; Humans ; Lung Neoplasms/diagnosis/genetics/*therapy ; Mice ; Mutation ; Sequence Deletion ; Translational Research, Biomedical/*methods ; Tumor Suppressor Proteins/genetics ; },
abstract = {Gene therapy using CRISPR Cas9 technique is rapidly gaining popularity among the scientific community primarily because of its versatility, cost-effectiveness, and high efficacy. While the laboratory-based experiments and findings making use of CRISPR as a gene editing tool are available in ample amounts, the question arises that how much of these findings are actually translatable into measures helping in combating particular disease conditions. In this review, we highlight the important studies and findings done till now in the perspective of lung cancer with an in-depth analysis of various clinical trials associated with the use of CRISPR Cas9 technology in the field of cancer research.},
}
@article {pmid31222014,
year = {2019},
author = {Bell, CC and Fennell, KA and Chan, YC and Rambow, F and Yeung, MM and Vassiliadis, D and Lara, L and Yeh, P and Martelotto, LG and Rogiers, A and Kremer, BE and Barbash, O and Mohammad, HP and Johanson, TM and Burr, ML and Dhar, A and Karpinich, N and Tian, L and Tyler, DS and MacPherson, L and Shi, J and Pinnawala, N and Yew Fong, C and Papenfuss, AT and Grimmond, SM and Dawson, SJ and Allan, RS and Kruger, RG and Vakoc, CR and Goode, DL and Naik, SH and Gilan, O and Lam, EYN and Marine, JC and Prinjha, RK and Dawson, MA},
title = {Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2723},
pmid = {31222014},
issn = {2041-1723},
support = {1106444//Department of Health | National Health and Medical Research Council (NHMRC)/International ; 55008729//Howard Hughes Medical Institute (HHMI)/International ; 1085015//Department of Health | National Health and Medical Research Council (NHMRC)/International ; 1128984//Department of Health | National Health and Medical Research Council (NHMRC)/International ; R01 CA174793/CA/NCI NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; P01 CA013106/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Bone Marrow/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*drug effects ; Epigenesis, Genetic/drug effects ; Female ; Gene Expression Regulation, Leukemic/*drug effects ; HEK293 Cells ; Humans ; Kaplan-Meier Estimate ; Leukemia, Myeloid, Acute/*drug therapy/genetics/mortality/pathology ; Mice ; Mice, Inbred C57BL ; Sequence Analysis, RNA ; Single-Cell Analysis ; Trans-Activators/*antagonists &amp; inhibitors/genetics/metabolism ; Transcription, Genetic/drug effects ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.},
}
@article {pmid31220585,
year = {2020},
author = {Shen, J and Zhou, J and Xu, Y and Xiu, Z},
title = {Prophages contribute to genome plasticity of Klebsiella pneumoniae and may involve the chromosomal integration of ARGs in CG258.},
journal = {Genomics},
volume = {112},
number = {1},
pages = {998-1010},
doi = {10.1016/j.ygeno.2019.06.016},
pmid = {31220585},
issn = {1089-8646},
mesh = {CRISPR-Cas Systems ; Chromosomes, Bacterial ; Drug Resistance, Bacterial/*genetics ; *Genome, Bacterial ; Klebsiella pneumoniae/*genetics ; Prophages/*genetics ; },
abstract = {Klebsiella pneumoniae is an important multidrug-resistant pathogen carrying prophages. Here we explore the contribution of prophages to bacterial evolution and fitness in silico. This study showed prophages contribute to remarkable genome plasticity of K. pneumoniae. The strains of CG258 possess several conserved prophages including the couple of P2-P4 prophages. CRISPR-Cas system has limited impact on the presence of prophages. The strong MLST-depended distribution of CRISPR-Cas and prophages and the high proportion of strains with self-targeting spacers may be the causes. Four core ARGs (blaSHV, fosA and oqxAB) were detected on almost all the chromosomes, but the acquired ARGs were only found in CG258 and CRISPR-positive strains. The factors influencing the chromosomal integration of ARGs in CG258 and CRISPR-positive strains may be different. In CG258, prophages may involve the chromosomal integration of ARGs. For CRISPR-positive strains, the immunity of CRISPR-Cas systems against invading ARG-bearing mobile genetic elements may accelerate the process.},
}
@article {pmid31220528,
year = {2019},
author = {Rahimi, S and Roushandeh, AM and Ebrahimi, A and Samadani, AA and Kuwahara, Y and Roudkenar, MH},
title = {CRISPR/Cas9-mediated knockout of Lcn2 effectively enhanced CDDP-induced apoptosis and reduced cell migration capacity of PC3 cells.},
journal = {Life sciences},
volume = {231},
number = {},
pages = {116586},
doi = {10.1016/j.lfs.2019.116586},
pmid = {31220528},
issn = {1879-0631},
mesh = {Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor/drug effects ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cisplatin/*pharmacology ; Gene Knockout Techniques/methods ; Humans ; Lipocalin-2/*genetics/*metabolism ; Male ; PC-3 Cells ; Prostatic Neoplasms/*drug therapy/genetics/pathology ; Signal Transduction ; },
abstract = {AIMS: Lipocalin 2 (Lcn2/NGAL) belongs to lipocalin superfamily with diverse functions. The precise function of Lcn2, particularly in cancer development, remains to be elucidated yet. In an attempt to knockout of Lcn2 expression by CRISPR/Cas 9 technology in a highly aggressive and invasive prostate cancer cell line and to evaluate the combination therapy with cisplatin (CDDP), this study was conducted.<br><br>MAIN METHODS: Control CRISPR/Cas9 plasmid and homology-directed repair plasmid or validated human Lcn2 CRISPR/Cas9 KO plasmids were co-transfected into PC3 cells using fugene HD transfection reagent. The stable cells were selected in the presence of puromycin. Correspondingly, knock out of Lcn2 was evaluated by RT-PCR, ELISA, and immunocytochemistry. PC3-Scr (control) and Lcn2-KO (PC3 cells in which lcn2 has been knocked out) were treated with or without cisplatin (CDDP). Cell proliferative ability was measured by WST-1 and colony-formation assays. Apoptosis was evaluated by DAPI staining, in situ cell death detection (TUNEL) assay, and cell death detection ELISA plus methods. The migration capabilities were studied by wound healing/scratch and transwell assays.<br><br>KEY FINDINGS: Lcn2 knock out in a highly aggressive and invasive cancer cell like PC3 decreased cell proliferation and increased the sensitivity of CDDP. Conspicuously, loss of Lcn2 expression effectively enhanced CDDP-induced apoptosis in PC3 cells. Lcn2 knock out by CRISPR/Cas9 technology decreased the cell migration capacity of PC3 cells as well.<br><br>SIGNIFICANCE: Lcn2 not only is a valuable and useful biomarker for diagnosis and prognosis of prostate cancer but also and more importantly is a potential novel emerging therapeutic target.},
}
@article {pmid31220273,
year = {2019},
author = {Wong, WR and Brugman, KI and Maher, S and Oh, JY and Howe, K and Kato, M and Sternberg, PW},
title = {Autism-associated missense genetic variants impact locomotion and neurodevelopment in Caenorhabditis elegans.},
journal = {Human molecular genetics},
volume = {28},
number = {13},
pages = {2271-2281},
pmid = {31220273},
issn = {1460-2083},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Autism Spectrum Disorder/*genetics ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Disease Models, Animal ; Fertility/genetics ; Genetic Association Studies ; Locomotion/genetics ; Mutation, Missense ; Neurodevelopmental Disorders/genetics ; Phenotype ; },
abstract = {Autism spectrum disorder (ASD) involves thousands of alleles in over 850 genes, but the current functional inference tools are not sufficient to predict phenotypic changes. As a result, the causal relationship of most of these genetic variants in the pathogenesis of ASD has not yet been demonstrated and an experimental method prioritizing missense alleles for further intensive analysis is crucial. For this purpose, we have designed a pipeline that uses Caenorhabditis elegans as a genetic model to screen for phenotype-changing missense alleles inferred from human ASD studies. We identified highly conserved human ASD-associated missense variants in their C. elegans orthologs, used a CRISPR/Cas9-mediated homology-directed knock-in strategy to generate missense mutants and analyzed their impact on behaviors and development via several broad-spectrum assays. All tested missense alleles were predicted to perturb protein function, but we found only 70% of them showed detectable phenotypic changes in morphology, locomotion or fecundity. Our findings indicate that certain missense variants in the C. elegans orthologs of human CACNA1D, CHD7, CHD8, CUL3, DLG4, GLRA2, NAA15, PTEN, SYNGAP1 and TPH2 impact neurodevelopment and movement functions, elevating these genes as candidates for future study into ASD. Our approach will help prioritize functionally important missense variants for detailed studies in vertebrate models and human cells.},
}
@article {pmid31219803,
year = {2019},
author = {Liu, X and Wei, L and Dong, Q and Liu, L and Zhang, MQ and Xie, Z and Wang, X},
title = {A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass.},
journal = {Aging},
volume = {11},
number = {12},
pages = {4011-4031},
pmid = {31219803},
issn = {1945-4589},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cellular Senescence/*genetics/*physiology ; Fibroblasts/*physiology ; Gene Expression Regulation/*physiology ; Humans ; Lentivirus ; },
abstract = {Cellular senescence is an important mechanism of autonomous tumor suppression, while its consequence such as the senescence-associated secretory phenotype (SASP) may drive tumorigenesis and age-related diseases. Therefore, controlling the cell fate optimally when encountering senescence stress is helpful for anti-cancer or anti-aging treatments. To identify genes essential for senescence establishment or maintenance, we carried out a CRISPR-based screen with a deliberately designed single-guide RNA (sgRNA) library. The library comprised of about 12,000 kinds of sgRNAs targeting 1378 senescence-associated genes selected by integrating the information of literature mining, protein-protein interaction network, and differential gene expression. We successfully detected a dozen gene deficiencies potentially causing senescence bypass, and their phenotypes were further validated with a high true positive rate. RNA-seq analysis showed distinct transcriptome patterns of these bypass cells. Interestingly, in the bypass cells, the expression of SASP genes was maintained or elevated with CHEK2, HAS1, or MDK deficiency; but neutralized with MTOR, CRISPLD2, or MORF4L1 deficiency. Pathways of some age-related neurodegenerative disorders were also downregulated with MTOR, CRISPLD2, or MORF4L1 deficiency. The results demonstrated that disturbing these genes could lead to distinct cell fates as a consequence of senescence bypass, suggesting that they may play essential roles in cellular senescence.},
}
@article {pmid31219653,
year = {2019},
author = {Ahmadzadeh, V and Farajnia, S and Baghban, R and Rahbarnia, L and Zarredar, H},
title = {CRISPR-Cas system: Toward a more efficient technology for genome editing and beyond.},
journal = {Journal of cellular biochemistry},
volume = {120},
number = {10},
pages = {16379-16392},
doi = {10.1002/jcb.29140},
pmid = {31219653},
issn = {1097-4644},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; },
abstract = {Genome engineering technology is of great interest for biomedical research that enables scientists to make specific manipulation in the DNA sequence. Early methods for introducing double-stranded DNA breaks relies on protein-based systems. These platforms have enabled fascinating advances, but all are costly and time-consuming to engineer, preventing these from gaining high-throughput applications. The CRISPR-Cas9 system, co-opted from bacteria, has generated considerable excitement in gene targeting. In this review, we describe gene targeting techniques with an emphasis on recent strategies to improve the specificities of CRISPR-Cas systems for nuclease and non-nuclease applications.},
}
@article {pmid31219587,
year = {2019},
author = {Grainy, J and Garrett, S and Graveley, BR and P Terns, M},
title = {CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.},
journal = {Nucleic acids research},
volume = {47},
number = {14},
pages = {7518-7531},
pmid = {31219587},
issn = {1362-4962},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaeal Proteins/*genetics/metabolism ; Base Sequence ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/*genetics/metabolism ; High-Throughput Nucleotide Sequencing ; Mutation ; Plasmids/genetics ; Pyrococcus furiosus/*genetics/metabolism ; },
abstract = {Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.},
}
@article {pmid31218907,
year = {2019},
author = {Zha, S and Li, Z and Chen, C and Du, Z and Tay, JC and Wang, S},
title = {Beta-2 microglobulin knockout K562 cell-based artificial antigen presenting cells for ex vivo expansion of T lymphocytes.},
journal = {Immunotherapy},
volume = {11},
number = {11},
pages = {967-982},
doi = {10.2217/imt-2018-0211},
pmid = {31218907},
issn = {1750-7448},
mesh = {Adoptive Transfer ; Antigen-Presenting Cells/cytology/*immunology ; Antigens, CD19/genetics/immunology ; CRISPR-Cas Systems ; *Cell Culture Techniques ; *Cell Proliferation ; *Gene Knockout Techniques ; Humans ; K562 Cells ; T-Lymphocytes, Cytotoxic/cytology/*immunology ; beta 2-Microglobulin/*genetics/immunology ; },
abstract = {Aim: The human K562 leukemia cell line as a scaffold of artificial antigen presenting cells (aAPCs) for ex vivo lymphocyte expansion does not usually express major histocompatibility complex (MHC) molecules. However, when stimulated by supernatants from human T lymphocyte cultures, K562 cells upregulate β-2 microglobulin (B2M) and MHC class I expression, which would induce allo-specific T cells. Methods: We disrupted the B2M locus in K562 cells by CRISPR/Cas9 and achieved MHC class I-negative K562 cells. Results: We further generated K562-based MHC class I-negative aAPC line by zinc-finger nuclease mediated insertion of costimulatory molecules into the AAVS1 locus. This aAPC line could attenuate allogeneic immune responses but support robust antigen-independent and CD19 antigen-specific chimeric antigen receptor-T cell expansion in vitro. Conclusion: B2M-knockout K562 cells provide a new scaffold for aAPC construction and broader application in adoptive immunotherapies.},
}
@article {pmid31218589,
year = {2019},
author = {Dong, Z and Qin, Q and Hu, Z and Chen, P and Huang, L and Zhang, X and Tian, T and Lu, C and Pan, M},
title = {Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms.},
journal = {Virologica Sinica},
volume = {34},
number = {4},
pages = {444-453},
pmid = {31218589},
issn = {1995-820X},
mesh = {Animals ; Bombyx/*virology ; *CRISPR-Cas Systems ; Cell Line ; DNA, Viral ; *Gene Editing ; Gene Expression ; *Genetic Vectors ; Nucleopolyhedroviruses/*genetics/physiology ; *Virus Replication ; },
abstract = {Recently the developed single guide (sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. We screened the immediate-early-1 gene (ie-1), the major envelope glycoprotein gene (gp64), and the late expression factor gene (lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector (PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.},
}
@article {pmid31218106,
year = {2019},
author = {Ouyang, Q and Liu, Y and Tan, J and Li, J and Yang, D and Zeng, F and Huang, W and Kong, Y and Liu, Z and Zhou, H and Liu, Y},
title = {Loss of ZNF587B and SULF1 contributed to cisplatin resistance in ovarian cancer cell lines based on Genome-scale CRISPR/Cas9 screening.},
journal = {American journal of cancer research},
volume = {9},
number = {5},
pages = {988-998},
pmid = {31218106},
issn = {2156-6976},
abstract = {Ovarian cancer is one of the most lethal malignancies of the female reproductive system. Platinum-resistance is the major obstacle in the successful treatment of ovarian cancer. Previous studies largely failed to identify the key genes associated with platinum-resistance by using candidate genes testing, bioinformatic analysis and GWAS method. The aim of the study was to utilize the whole human Genome-scale CRISPR-Cas9 knockout (GeCKO) library to screen for novel genes involved in cisplatin resistance in ovarian cancer cell lines. The GeCKO library targeted 19052 genes with 122417 unique guide sequences. Six candidate genes had been screened out including one previously validated gene SULF1 and five novel genes ZNF587B, TADA1, SEMA4G, POTEC and USP17L20. After validated by CCK-8 and RT-PCR analysis, two genes (ZNF587B and SULF1) were discovered to be involved in cisplatin resistance. ZNF587B may serve as a new biomarker for predicting cisplatin resistance.},
}
@article {pmid31217610,
year = {2019},
author = {Ledford, H},
title = {CRISPR babies: when will the world be ready?.},
journal = {Nature},
volume = {570},
number = {7761},
pages = {293-296},
pmid = {31217610},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clinical Trials as Topic ; Female ; Fertilization in Vitro ; Gene Editing/*standards/*trends ; Humans ; Huntington Disease/diagnosis/genetics/prevention &amp; control ; Infant, Newborn ; Male ; Mice ; Mosaicism ; Patient Safety/*standards ; Preimplantation Diagnosis ; Research Design ; Stakeholder Participation ; Time Factors ; Uncertainty ; },
}
@article {pmid31217350,
year = {2019},
author = {Lodd, E and Wiggenhauser, LM and Morgenstern, J and Fleming, TH and Poschet, G and Büttner, M and Tabler, CT and Wohlfart, DP and Nawroth, PP and Kroll, J},
title = {The combination of loss of glyoxalase1 and obesity results in hyperglycemia.},
journal = {JCI insight},
volume = {4},
number = {12},
pages = {},
pmid = {31217350},
issn = {2379-3708},
mesh = {Animals ; CRISPR-Cas Systems ; Diabetes Mellitus, Experimental ; Diabetes Mellitus, Type 2/genetics ; Diet ; Disease Models, Animal ; Gene Knockout Techniques ; Genetic Predisposition to Disease ; Glucose/metabolism ; Hyperglycemia/*etiology/genetics ; Insulin Resistance ; Lactoylglutathione Lyase/genetics/*physiology ; Liver/metabolism ; Male ; Obesity/*complications ; Pyruvaldehyde/metabolism ; Retina/pathology ; Zebrafish/growth &amp; development ; },
abstract = {The increased formation of methylglyoxal (MG) under hyperglycemia is associated with the development of microvascular complications in patients with diabetes mellitus; however, the effects of elevated MG levels in vivo are poorly understood. In zebrafish, a transient knockdown of glyoxalase 1, the main MG detoxifying system, led to the elevation of endogenous MG levels and blood vessel alterations. To evaluate effects of a permanent knockout of glyoxalase 1 in vivo, glo1-/- zebrafish mutants were generated using CRISPR/Cas9. In addition, a diet-induced-obesity zebrafish model was used to analyze glo1-/- zebrafish under high nutrient intake. Glo1-/- zebrafish survived until adulthood without growth deficit and showed increased tissue MG concentrations. Impaired glucose tolerance developed in adult glo1-/- zebrafish and was indicated by increased postprandial blood glucose levels and postprandial S6 kinase activation. Challenged by an overfeeding period, fasting blood glucose levels in glo1-/- zebrafish were increased which translated into retinal blood vessel alterations. Thus, the data have identified a defective MG detoxification as a metabolic prerequisite and glyoxalase 1 alterations as a genetic susceptibility to the development of type 2 diabetes mellitus under high nutrition intake.},
}
@article {pmid31217244,
year = {2019},
author = {Staller, E and Sheppard, CM and Neasham, PJ and Mistry, B and Peacock, TP and Goldhill, DH and Long, JS and Barclay, WS},
title = {ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells.},
journal = {Journal of virology},
volume = {93},
number = {17},
pages = {},
pmid = {31217244},
issn = {1098-5514},
support = {205100//Wellcome Trust/United Kingdom ; BB/L015129/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/K002465/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 104931/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; //Wellcome Trust/United Kingdom ; BB/R013071/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/chemistry/*genetics/metabolism ; Cell Line ; Disease Models, Animal ; Humans ; Influenza A virus/*physiology ; Influenzavirus B/*physiology ; Mice ; Nerve Tissue Proteins/chemistry/*genetics/metabolism ; Nuclear Proteins/chemistry/*genetics/metabolism ; Point Mutation ; Protein Domains ; RNA-Binding Proteins/chemistry/*genetics/metabolism ; RNA-Dependent RNA Polymerase/*metabolism ; Viral Proteins/metabolism ; Virus Replication ; },
abstract = {ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach, we show that the human acidic nuclear phosphoproteins (ANPs) ANP32A and ANP32B are functionally redundant but essential host factors for mammalian-adapted influenza A virus (IAV) and influenza B virus (IBV) replication in human cells. When both proteins are absent from human cells, influenza polymerases are unable to replicate the viral genome, and infectious virus cannot propagate. Provision of exogenous ANP32A or ANP32B recovers polymerase activity and virus growth. We demonstrate that this redundancy is absent in the murine Anp32 orthologues; murine Anp32A is incapable of recovering IAV polymerase activity, while murine Anp32B can do so. Intriguingly, IBV polymerase is able to use murine Anp32A. We show, using a domain swap and point mutations, that the leucine-rich repeat (LRR) 5 region comprises an important functional domain for mammalian ANP32 proteins. Our approach has identified a pair of essential host factors for influenza virus replication and can be harnessed to inform future interventions.IMPORTANCE Influenza virus is the etiological agent behind some of the most devastating infectious disease pandemics to date, and influenza outbreaks still pose a major threat to public health. Influenza virus polymerase, the molecule that copies the viral RNA genome, hijacks cellular proteins to support its replication. Current anti-influenza drugs are aimed against viral proteins, including the polymerase, but RNA viruses like influenza tend to become resistant to such drugs very rapidly. An alternative strategy is to design therapeutics that target the host proteins that are necessary for virus propagation. Here, we show that the human proteins ANP32A and ANP32B are essential for influenza A and B virus replication, such that in their absence cells become impervious to the virus. We map the proviral activity of ANP32 proteins to one region in particular, which could inform future intervention.},
}
@article {pmid31217031,
year = {2019},
author = {Sen, M and Wang, X and Hamdan, FH and Rapp, J and Eggert, J and Kosinsky, RL and Wegwitz, F and Kutschat, AP and Younesi, FS and Gaedcke, J and Grade, M and Hessmann, E and Papantonis, A and Strӧbel, P and Johnsen, SA},
title = {ARID1A facilitates KRAS signaling-regulated enhancer activity in an AP1-dependent manner in colorectal cancer cells.},
journal = {Clinical epigenetics},
volume = {11},
number = {1},
pages = {92},
pmid = {31217031},
issn = {1868-7083},
mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Colorectal Neoplasms/*genetics/metabolism ; *DNA Methylation ; DNA-Binding Proteins/*genetics/*metabolism ; Enhancer Elements, Genetic ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study ; HCT116 Cells ; HT29 Cells ; Humans ; MAP Kinase Signaling System ; Mutation ; Proto-Oncogene Proteins p21(ras)/*genetics ; Signal Transduction ; Transcription Factor AP-1/*genetics ; Transcription Factors/*genetics/*metabolism ; },
abstract = {BACKGROUND: ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity.<br><br>METHODS: To mimic ARID1A-deficient colorectal cancer, we used CRISPR/Cas9-mediated gene editing to inactivate the ARID1A gene in established colorectal cancer cell lines. We integrated gene expression analyses with genome-wide ARID1A occupancy and epigenomic mapping data to decipher ARID1A-dependent transcriptional regulatory mechanisms.<br><br>RESULTS: Interestingly, we found that CRC cell lines harboring KRAS mutations are critically dependent on ARID1A function. In the absence of ARID1A, proliferation of these cell lines is severely impaired, suggesting an essential role for ARID1A in this context. Mechanistically, we showed that ARID1A acts as a co-factor at enhancers occupied by AP1 transcription factors acting downstream of the MEK/ERK pathway. Consistently, loss of ARID1A led to a disruption of KRAS/AP1-dependent enhancer activity, accompanied by a downregulation of expression of the associated target genes.<br><br>CONCLUSIONS: We identify a previously unknown context-dependent tumor-supporting function of ARID1A in CRC downstream of KRAS signaling. Upon the loss of ARID1A in KRAS-mutated cells, enhancers that are co-occupied by ARID1A and the AP1 transcription factors become inactive, thereby leading to decreased target gene expression. Thus, targeting of the BAF complex in KRAS-mutated CRC may offer a unique, previously unknown, context-dependent therapeutic option in CRC.},
}
@article {pmid31216442,
year = {2019},
author = {Skarnes, WC and Pellegrino, E and McDonough, JA},
title = {Improving homology-directed repair efficiency in human stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {18-28},
doi = {10.1016/j.ymeth.2019.06.016},
pmid = {31216442},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; Cell Culture Techniques/instrumentation/methods ; Cell Line ; Culture Media/chemistry ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Gene Editing/*methods ; HEK293 Cells ; Humans ; *Induced Pluripotent Stem Cells ; Oligonucleotides/genetics ; Polymorphism, Single Nucleotide ; RNA, Guide/genetics ; *Recombinational DNA Repair ; Transfection/methods ; },
abstract = {The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7.},
}
@article {pmid31216436,
year = {2019},
author = {Schwartz, C and Cheng, JF and Evans, R and Schwartz, CA and Wagner, JM and Anglin, S and Beitz, A and Pan, W and Lonardi, S and Blenner, M and Alper, HS and Yoshikuni, Y and Wheeldon, I},
title = {Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica.},
journal = {Metabolic engineering},
volume = {55},
number = {},
pages = {102-110},
doi = {10.1016/j.ymben.2019.06.007},
pmid = {31216436},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Library ; *Genes, Fungal ; Yarrowia/*genetics ; },
abstract = {Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.},
}
@article {pmid31216404,
year = {2019},
author = {Hahn, WC},
title = {A CRISPR Way to Identify Cancer Targets.},
journal = {The New England journal of medicine},
volume = {380},
number = {25},
pages = {2475-2477},
doi = {10.1056/NEJMcibr1905048},
pmid = {31216404},
issn = {1533-4406},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Profiling ; *Gene Silencing ; *Genetic Profile ; Humans ; *Loss of Function Mutation ; Microsatellite Instability ; Neoplasms/*genetics ; Precision Medicine ; Werner Syndrome Helicase/genetics/metabolism ; },
}
@article {pmid31216068,
year = {2019},
author = {Sheng, Z and Wang, X and Ma, Y and Zhang, D and Yang, Y and Zhang, P and Zhu, H and Xu, N and Liang, S},
title = {MS-based strategies for identification of protein SUMOylation modification.},
journal = {Electrophoresis},
volume = {40},
number = {21},
pages = {2877-2887},
pmid = {31216068},
issn = {1522-2683},
support = {17ZD045//Health Commission of Sichuan Province/International ; 31470810//National Natural Science Foundation of China/International ; 2014AA020608//National 863 High Tech Foundation/International ; 31470810//National Natural Sciences Foundation of China/International ; 2017-GH02-00062-HZ//Chengdu Science &amp;Technology Program/International ; R01 AA020608/AA/NIAAA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing ; Humans ; *Mass Spectrometry ; Models, Molecular ; *Peptides/analysis/chemistry/isolation &amp; purification ; Recombinant Fusion Proteins ; *Small Ubiquitin-Related Modifier Proteins ; *Sumoylation ; Ubiquitination ; },
abstract = {Protein SUMOylation modification conjugated with small ubiquitin-like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co-modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.},
}
@article {pmid31215287,
year = {2019},
author = {Serova, OV and Orsa, AN and Chachina, NA and Petrenko, AG and Deyev, IE},
title = {с-Met receptor can be activated by extracellular alkaline medium.},
journal = {Journal of receptor and signal transduction research},
volume = {39},
number = {1},
pages = {67-72},
doi = {10.1080/10799893.2019.1620775},
pmid = {31215287},
issn = {1532-4281},
mesh = {Alkalies/*pharmacology ; CRISPR-Cas Systems ; Carcinoma, Renal Cell/drug therapy/*metabolism ; Extracellular Space ; Gene Expression Regulation, Neoplastic/*drug effects ; HeLa Cells ; Humans ; Indoles/pharmacology ; Kidney Neoplasms/drug therapy/*metabolism ; Phosphorylation ; Piperazines/pharmacology ; Proto-Oncogene Proteins c-met/antagonists &amp; inhibitors/genetics/*metabolism ; Sulfonamides/pharmacology ; Tumor Cells, Cultured ; },
abstract = {Receptor tyrosine kinase (RTK) Met or c-Met is a target of hepatocyte growth factor (HGF) and it plays an important role under normal and pathological conditions. Activation of Met signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion, and metastasis. In this article, we describe the ability of Met to activate upon a mild alkali treatment. To identify potential alkali-regulated proteins, CAKI-1 cells were treated with alkaline media and further tested for protein phosphorylation changes. By anti-phosphotyrosine antibody precipitation and lectin chromatography, we identified Met as a major cytoplasmic membrane protein that responded to pH changes by its phosphorylation. The activation of Met by alkali occurred at pH >8.0 and was dose-dependent. Specificity of the Met response to alkali was confirmed by the treatment with Met kinase inhibitor SU11274 and also by Met receptor knockout using CRISPR/CAS9 genome editing system. Both approaches completely blocked the Met phosphorylation response in CAKI-1 cells. Similar pH-dependent Met activation was observed in the HeLa cell line. Our data suggest existence of ligand-independent mechanism of Met receptor activation.},
}
@article {pmid31213543,
year = {2019},
author = {Panganiban, RA and Park, HR and Sun, M and Shumyatcher, M and Himes, BE and Lu, Q},
title = {Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {27},
pages = {13384-13393},
pmid = {31213543},
issn = {1091-6490},
support = {K99 ES029548/ES/NIEHS NIH HHS/United States ; P30 ES000002/ES/NIEHS NIH HHS/United States ; R01 ES029097/ES/NIEHS NIH HHS/United States ; R01 HL133433/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Apoptosis ; *CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; *Endoplasmic Reticulum Stress ; Fibroblasts ; Gene Knockout Techniques ; Genes, Suppressor ; Genome-Wide Association Study ; HEK293 Cells ; Humans ; Mice ; Signal Transduction ; Transcription Factor CHOP/metabolism ; Unfolded Protein Response ; },
abstract = {Sensing misfolded proteins in the endoplasmic reticulum (ER), cells initiate the ER stress response and, when overwhelmed, undergo apoptosis. However, little is known about how cells prevent excessive ER stress response and cell death to restore homeostasis. Here, we report the identification and characterization of cellular suppressors of ER stress-induced apoptosis. Using a genome-wide CRISPR library, we screen for genes whose inactivation further increases ER stress-induced up-regulation of C/EBP homologous protein 10 (CHOP)-the transcription factor central to ER stress-associated apoptosis. Among the top validated hits are two interacting components of the polycomb repressive complex (L3MBTL2 [L(3)Mbt-Like 2] and MGA [MAX gene associated]), and microRNA-124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP expression and sensitizes cells to apoptosis induced by multiple ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells to repress CHOP induction but dissociates from the promoter in the presence of ER stress, whereas miR-124-3 directly targets the IRE1 branch of the ER stress pathway. Our study reveals distinct mechanisms that suppress ER stress-induced apoptosis and may lead to a better understanding of diseases whose pathogenesis is linked to overactive ER stress response.},
}
@article {pmid31213532,
year = {2019},
author = {Boned Del Río, I and Young, LC and Sari, S and Jones, GG and Ringham-Terry, B and Hartig, N and Rejnowicz, E and Lei, W and Bhamra, A and Surinova, S and Rodriguez-Viciana, P},
title = {SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {27},
pages = {13330-13339},
pmid = {31213532},
issn = {1091-6490},
support = {/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Knockout Techniques ; Humans ; Intracellular Signaling Peptides and Proteins/*metabolism ; *MAP Kinase Signaling System ; Phosphorylation ; Protein Multimerization ; raf Kinases/*chemistry/*metabolism ; ras Proteins/metabolism ; },
abstract = {Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.},
}
@article {pmid31212706,
year = {2019},
author = {Vindry, C and Guillin, O and Mangeot, PE and Ohlmann, T and Chavatte, L},
title = {A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[[Ser]Sec] Gene.},
journal = {Cells},
volume = {8},
number = {6},
pages = {},
pmid = {31212706},
issn = {2073-4409},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Codon, Terminator/*genetics ; Gene Editing ; HEK293 Cells ; HeLa Cells ; Humans ; INDEL Mutation/genetics ; Nucleic Acid Conformation ; RNA, Messenger/genetics/metabolism ; RNA, Transfer, Amino Acid-Specific/chemistry/*genetics ; Ribosomes/*metabolism ; Selenium/metabolism ; Selenocysteine/*metabolism ; Selenoproteins/genetics/metabolism ; Virion/*metabolism ; },
abstract = {The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3' UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[[Ser]Sec]). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[[Ser]Sec] and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[[Ser]Sec] gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[[Ser]Sec] and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.},
}
@article {pmid31211904,
year = {2019},
author = {Lander, N and Chiurillo, MA},
title = {State-of-the-art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids.},
journal = {The Journal of eukaryotic microbiology},
volume = {66},
number = {6},
pages = {981-991},
pmid = {31211904},
issn = {1550-7408},
support = {R21 AI140421/AI/NIAID NIH HHS/United States ; R56 AI107663/AI/NIAID NIH HHS/United States ; R01 AI107663/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Protozoan ; Leishmania/*genetics ; Trypanosoma/*genetics ; },
abstract = {CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state-of-the-art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.},
}
@article {pmid31211694,
year = {2019},
author = {De Caneva, A and Porro, F and Bortolussi, G and Sola, R and Lisjak, M and Barzel, A and Giacca, M and Kay, MA and Vlahoviček, K and Zentilin, L and Muro, AF},
title = {Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.},
journal = {JCI insight},
volume = {5},
number = {15},
pages = {},
pmid = {31211694},
issn = {2379-3708},
support = {R01 HL064274/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; Bilirubin ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Complementary ; Disease Models, Animal ; Female ; Gene Targeting/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Genetic Vectors ; Glucuronosyltransferase/*genetics/metabolism ; HEK293 Cells ; Hepatocytes/metabolism ; Humans ; Liver/*metabolism/pathology ; Male ; Metabolic Diseases/*genetics/metabolism/pathology/*therapy ; Mice ; Mice, Knockout ; NIH 3T3 Cells ; Serum Albumin ; Therapeutic Uses ; },
abstract = {Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.},
}
@article {pmid31209374,
year = {2019},
author = {Simon, AJ and d'Oelsnitz, S and Ellington, AD},
title = {Synthetic evolution.},
journal = {Nature biotechnology},
volume = {37},
number = {7},
pages = {730-743},
pmid = {31209374},
issn = {1546-1696},
mesh = {Animals ; CRISPR-Cas Systems ; DNA/chemical synthesis/*genetics ; *Directed Molecular Evolution ; Genetic Engineering ; Genome ; Mutation ; },
abstract = {The combination of modern biotechnologies such as DNA synthesis, λ red recombineering, CRISPR-based editing and next-generation high-throughput sequencing increasingly enables precise manipulation of genes and genomes. Beyond rational design, these technologies also enable the targeted, and potentially continuous, introduction of multiple mutations. While this might seem to be merely a return to natural selection, the ability to target evolution greatly reduces fitness burdens and focuses mutation and selection on those genes and traits that best contribute to a desired phenotype, ultimately throwing evolution into fast forward.},
}
@article {pmid31209355,
year = {2019},
author = {Novick, R},
title = {Targeted killing of virulent Vibrio cholerae.},
journal = {Nature biomedical engineering},
volume = {3},
number = {7},
pages = {507-508},
doi = {10.1038/s41551-019-0419-y},
pmid = {31209355},
issn = {2157-846X},
mesh = {Bacterial Toxins/genetics/*pharmacology ; CRISPR-Cas Systems ; Drug Carriers/*pharmacology ; Drug Delivery Systems/*methods ; Drug Resistance, Bacterial/drug effects/genetics ; Gastrointestinal Microbiome/drug effects ; Humans ; Inteins/genetics ; Plasmids ; Transcription Factors ; Vibrio cholerae/*drug effects/genetics ; Virulence/drug effects/genetics ; Virulence Factors/genetics ; },
}
@article {pmid31209054,
year = {2019},
author = {Lee, J and Ma, J and Lee, K},
title = {Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {27},
pages = {13288-13292},
pmid = {31209054},
issn = {1091-6490},
mesh = {Adenoviridae/genetics ; Animals ; *Blastoderm ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Chimera/genetics ; Coturnix/embryology/*genetics ; Feathers/anatomy &amp; histology ; Female ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; Male ; },
abstract = {Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.},
}
@article {pmid31209053,
year = {2019},
author = {Darbellay, F and Bochaton, C and Lopez-Delisle, L and Mascrez, B and Tschopp, P and Delpretti, S and Zakany, J and Duboule, D},
title = {The constrained architecture of mammalian Hox gene clusters.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {27},
pages = {13424-13433},
pmid = {31209053},
issn = {1091-6490},
mesh = {Alleles ; Animals ; CCCTC-Binding Factor/metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation/genetics ; Genes, Homeobox/*genetics ; Genetic Loci/genetics ; Homeodomain Proteins/genetics ; Mammals/genetics ; Mice ; Multigene Family/*genetics ; Sequence Inversion ; Transcription Factors/genetics ; Transcription, Genetic/genetics ; },
abstract = {In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.},
}
@article {pmid31208775,
year = {2019},
author = {Kurtz, S and Petersen, B},
title = {Pre-determination of sex in pigs by application of CRISPR/Cas system for genome editing.},
journal = {Theriogenology},
volume = {137},
number = {},
pages = {67-74},
doi = {10.1016/j.theriogenology.2019.05.039},
pmid = {31208775},
issn = {1879-3231},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Gene Editing/*veterinary ; Male ; Sex Determination Analysis/veterinary ; Sex Preselection/*veterinary ; Spermatozoa ; Swine/genetics/*physiology ; },
abstract = {In livestock industries, one sex is usually preferred because of the impact on the production (e.g. milk from cows, eggs from laying hens). Furthermore, in pig production, the male-specific boar taint is a big hurdle for consumer acceptance. Consequently, a shift in the ratio towards the desired sex would be a great benefit. The most widely applied method for pre-determination of the sex is fluorescence-activated sperm sorting, which relies on the different DNA content of the X- and Y-chromosomal sperm. However, the successful practical adaption of this method depends on its ease of use. At present, sperm sexing via fluorescence-activated cell sorting (FACS) has only reached commercial application in cattle. Nevertheless, sperm sexing technology still needs to be improved with respect to efficiency and reliability, to obtain high numbers of sexed sperm and less invasive sperm treatment to avoid damage. New genome editing technologies such as Zinc finger nucleases (ZFN), Transcription-activator like endonucleases (TALENs) and the CRISPR/Cas system have emerged and offer great potential to affect determination of the sex at the genome level. The sex-determining region on the Y chromosome (SRY) serves as a main genetic switch of male gender development. It was previously shown that a knockout of the SRY gene in mice and rabbits displayed suppressed testis development in the fetal gonadal ridges resulting in a female phenotype. These new technologies hold great opportunities to pre-determine sex in pigs. However, further investigations are needed to exploit their full potential for practical application.},
}
@article {pmid31208390,
year = {2019},
author = {Petersen, BL and Möller, SR and Mravec, J and Jørgensen, B and Christensen, M and Liu, Y and Wandall, HH and Bennett, EP and Yang, Z},
title = {Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {36},
pmid = {31208390},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Flow Cytometry ; Fluorescence ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/*metabolism ; Microscopy, Fluorescence ; Mutation ; Plant Leaves/cytology/genetics/*metabolism ; Plants, Genetically Modified ; Protoplasts/cytology/*metabolism ; Tobacco/cytology/genetics/*metabolism ; },
abstract = {BACKGROUND: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.<br><br>RESULTS: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.<br><br>CONCLUSIONS: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.},
}
@article {pmid31208154,
year = {2019},
author = {Kang, Y and Xie, H and Zhao, C},
title = {Ankrd45 Is a Novel Ankyrin Repeat Protein Required for Cell Proliferation.},
journal = {Genes},
volume = {10},
number = {6},
pages = {},
pmid = {31208154},
issn = {2073-4425},
mesh = {Animals ; Ankyrin Repeat/*genetics ; Body Patterning/genetics ; CRISPR-Cas Systems/genetics ; Cell Proliferation/*genetics ; Cilia/genetics ; Embryonic Development/*genetics ; Gene Expression Regulation, Developmental/genetics ; HeLa Cells ; Humans ; Larva/genetics/growth &amp; development ; Mitosis/genetics ; Zebrafish/genetics/growth &amp; development ; Zebrafish Proteins/*genetics ; },
abstract = {Ankyrin repeats, the most common protein-protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin repeat-containing proteins play diverse biological functions. Here, we identified the gene ankrd45, which encodes a novel, two ankyrin repeat-containing protein. Zebrafish ankrd45 displayed a tissue specific expression pattern during early development, with high expression in ciliated tissues, including otic vesicles, Kupffer's vesicles, pronephric ducts, and floor plates. Surprisingly, zebrafish ankrd45 mutants were viable and developed grossly normal cilia. In contrast, mutant larvae developed enlarged livers when induced with liver specific expression of Kras[G12V], one of the common mutations of KRAS that leads to cancer in humans. Further, histological analysis suggested that multiple cysts developed in the mutant liver due to cell apoptosis. Similarly, knockdown of ANKRD45 expression with either siRNA or CRISPR/Cas9 methods induced apoptosis in cultured cells, similar to those in zebrafish ankrd45 mutant livers after induction. Using different cell lines, we show that the distribution of ANKRD45 protein was highly dynamic during mitosis. ANKRD45 is preferably localized to the midbody ring during cytokinesis. Together, our results suggest that Ankrd45 is a novel ankyrin repeat protein with a conserved role during cell proliferation in both zebrafish embryos and mammalian cells.},
}
@article {pmid31207994,
year = {2019},
author = {Yan, R and Wang, Z and Ren, Y and Li, H and Liu, N and Sun, H},
title = {Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven.},
journal = {International journal of molecular sciences},
volume = {20},
number = {12},
pages = {},
pmid = {31207994},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Lilium/*genetics ; Oxidoreductases/genetics/metabolism ; Plant Breeding/*methods ; Plant Proteins/genetics/metabolism ; Transformation, Genetic ; },
abstract = {Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum 'White Heaven', respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino-green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.},
}
@article {pmid31207570,
year = {2019},
author = {Johnston, RK and Seamon, KJ and Saada, EA and Podlevsky, JD and Branda, SS and Timlin, JA and Harper, JC},
title = {Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {141},
number = {},
pages = {111361},
doi = {10.1016/j.bios.2019.111361},
pmid = {31207570},
issn = {1873-4235},
mesh = {Bacterial Proteins/*analysis ; Bacteriophages/*chemistry ; Biosensing Techniques/*methods ; CRISPR-Associated Protein 9/*analysis ; CRISPR-Cas Systems ; Immobilized Proteins/chemistry ; Ligands ; Models, Molecular ; Streptococcus pyogenes/*chemistry ; Viral Proteins/*chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.},
}
@article {pmid31207241,
year = {2019},
author = {Urin, V and Shemesh, M and Schreiber, G},
title = {CRISPR/Cas9-based Knockout Strategy Elucidates Components Essential for Type 1 Interferon Signaling in Human HeLa Cells.},
journal = {Journal of molecular biology},
volume = {431},
number = {17},
pages = {3324-3338},
doi = {10.1016/j.jmb.2019.06.007},
pmid = {31207241},
issn = {1089-8638},
mesh = {Antiviral Agents/pharmacology ; *CRISPR-Cas Systems ; Gene Expression Regulation ; Gene Knockout Techniques/*methods ; HeLa Cells ; Humans ; Interferon Regulatory Factor-1/genetics ; Interferon Type I/*genetics ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; Phosphorylation ; Receptor, Interferon alpha-beta/genetics ; STAT1 Transcription Factor/*genetics ; STAT2 Transcription Factor ; STAT3 Transcription Factor/genetics ; STAT6 Transcription Factor ; Signal Transduction/*genetics ; },
abstract = {Type I interferons (IFNs) have a central role in innate and adaptive immunities, proliferation, and cancer surveillance. How IFN binding to its specific receptor, the IFN α and β receptor (IFNAR), can drive such variety of processes is an open question. Here, to systematically and thoroughly investigate the molecular mechanism of IFN signaling, we used a CRISPR/Cas9-based approach in a human cell line (HeLa) to generate knockouts (KOs) of the genes participating in the type 1 IFN signaling cascade. We show that both IFNAR chains (IFNAR1 and IFNAR2) are absolutely required for any IFN-induced signaling. Deletion of either signal transducer and activator of transcription 1 (STAT1) or STAT2 had only a partial effect on IFN-induced antiviral activity or gene induction. However, the deletion of both genes completely abrogated any IFN-induced activity. So did a double STAT2-IFN regulatory factor 1 (IRF1) KO and, to a large extent, a STAT1 KO together with IRF9 knockdown. KO of any of the STATs had no effect on the phosphorylation of other STATs, indicating that they bound IFNAR independently. STAT3 and STAT6 phosphorylations were fully induced by type 1 IFN in the STAT1-STAT2 KO, but did not promote gene induction. Moreover, STAT3 KO did not affect type 1 IFN-induced gene or protein expression. Type 1 IFN also did not activate p38, AKT, or ERK kinase. We conclude that type 1 IFN-induced activities in HeLa cells are mediated by STAT1/STAT2/IRF9, STAT1/STAT1, or STAT2/IRF9 complexes and do not require alternative pathways.},
}
@article {pmid31206994,
year = {2019},
author = {Schachtsiek, J and Stehle, F},
title = {Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.},
journal = {Plant biotechnology journal},
volume = {17},
number = {12},
pages = {2228-2230},
pmid = {31206994},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Nicotine ; Tobacco/*chemistry/*genetics ; },
}
@article {pmid31206027,
year = {2019},
author = {Liu, C and Wan, T and Wang, H and Zhang, S and Ping, Y and Cheng, Y},
title = {A boronic acid-rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing.},
journal = {Science advances},
volume = {5},
number = {6},
pages = {eaaw8922},
pmid = {31206027},
issn = {2375-2548},
mesh = {Animals ; Boronic Acids/*chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Dendrimers/*chemistry/metabolism ; Drug Delivery Systems/*methods ; Fluorescein-5-isothiocyanate/chemistry ; Fluorescent Dyes/chemistry ; Gene Editing/*methods ; *Gene Transfer Techniques ; Green Fluorescent Proteins/chemistry/genetics ; HeLa Cells ; Hemoglobins/chemistry/genetics ; Humans ; Mice ; Models, Molecular ; NIH 3T3 Cells ; Protein Structure, Secondary ; RNA, Guide/genetics/metabolism ; Staining and Labeling/methods ; beta Catenin/chemistry/genetics ; },
abstract = {Cytosolic protein delivery is of central importance for the development of protein-based biotechnologies and therapeutics; however, efficient intracellular delivery of native proteins remains a challenge. Here, we reported a boronic acid-rich dendrimer with unprecedented efficiency for cytosolic delivery of native proteins. The dendrimer could bind with both negatively and positively charged proteins and efficiently delivered 13 cargo proteins into the cytosol of living cells. All the delivered proteins kept their bioactivities after cytosolic delivery. The dendrimer ensures efficient intracellular delivery of Cas9 protein into various cell lines and showed high efficiency in CRISPR-Cas9 genome editing. The rationally designed boronic acid-rich dendrimer permits the development of an efficient platform with high generality for the delivery of native proteins.},
}
@article {pmid31205318,
year = {2019},
author = {Ciurkot, K and Vonk, B and Gorochowski, TE and Roubos, JA and Verwaal, R},
title = {CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {147},
pages = {},
doi = {10.3791/59350},
pmid = {31205318},
issn = {1940-087X},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Saccharomyces cerevisiae/*pathogenicity ; },
abstract = {High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S. cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.},
}
@article {pmid31205227,
year = {2020},
author = {Anderson, KL and Snyder, KM and Ito, D and Lins, DC and Mills, LJ and Weiskopf, K and Ring, NG and Ring, AM and Shimizu, Y and Mescher, MF and Weissman, IL and Modiano, JF},
title = {Evolutionarily conserved resistance to phagocytosis observed in melanoma cells is insensitive to upregulation of pro-phagocytic signals and to CD47 blockade.},
journal = {Melanoma research},
volume = {30},
number = {2},
pages = {147-158},
pmid = {31205227},
issn = {1473-5636},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; P30 CA077598/CA/NCI NIH HHS/United States ; P01 AI035296/AI/NIAID NIH HHS/United States ; F30 CA195973/CA/NCI NIH HHS/United States ; P01 CA139490/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CD47 Antigen/*metabolism ; Cell Line, Tumor ; Humans ; Melanoma/*genetics ; Mice ; Phagocytosis/*physiology ; Signal Transduction ; Transfection ; Up-Regulation ; },
abstract = {Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.},
}
@article {pmid31204998,
year = {2019},
author = {Roberts, AW and Popov, LM and Mitchell, G and Ching, KL and Licht, DJ and Golovkine, G and Barton, GM and Cox, JS},
title = {Cas9[+] conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31204998},
issn = {2050-084X},
support = {Open Philanthropy Fellow//Life Sciences Research Foundation/International ; U19 AI135990/AI/NIAID NIH HHS/United States ; R01AI072429/NH/NIH HHS/United States ; P01AI063302/NH/NIH HHS/United States ; U19AI135990/NH/NIH HHS/United States ; DP1AI24619/NH/NIH HHS/United States ; DP1 AI124619/AI/NIAID NIH HHS/United States ; P01 AI063302/AI/NIAID NIH HHS/United States ; R01 AI072429/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bone Marrow Cells/immunology/metabolism/microbiology ; *CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Gene Editing/*methods ; Homeodomain Proteins/genetics/immunology/metabolism ; Host-Pathogen Interactions/immunology ; Humans ; Listeria monocytogenes/*immunology/physiology ; Macrophages/*immunology/*metabolism/microbiology ; Mice, Transgenic ; Mycobacterium tuberculosis/*immunology/physiology ; Stem Cells/immunology/metabolism ; },
abstract = {Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.},
}
@article {pmid31204011,
year = {2019},
author = {Tian, J and Chang, J and Gong, J and Lou, J and Fu, M and Li, J and Ke, J and Zhu, Y and Gong, Y and Yang, Y and Zou, D and Peng, X and Yang, N and Mei, S and Wang, X and Zhong, R and Cai, K and Miao, X},
title = {Systematic Functional Interrogation of Genes in GWAS Loci Identified ATF1 as a Key Driver in Colorectal Cancer Modulated by a Promoter-Enhancer Interaction.},
journal = {American journal of human genetics},
volume = {105},
number = {1},
pages = {29-47},
pmid = {31204011},
issn = {1537-6605},
mesh = {Activating Transcription Factor 1/antagonists &amp; inhibitors/genetics/*metabolism ; Animals ; Apoptosis ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Proliferation ; Colorectal Neoplasms/genetics/metabolism/*pathology ; *Enhancer Elements, Genetic ; Female ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease ; *Genome-Wide Association Study ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; *Polymorphism, Single Nucleotide ; *Promoter Regions, Genetic ; Quantitative Trait Loci ; RNA Interference ; Risk Factors ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Genome-wide association studies (GWASs) have identified approximately 100 colorectal cancer (CRC) risk loci. However, the causal genes in these loci have not been systematically interrogated. We conducted a high-throughput RNA-interference functional screen to identify the genes essential for proliferation in the CRC risk loci of Asian populations. We found that ATF1, located in the 12q13.12 region, functions as an oncogene that facilitates cell proliferation; ATF1 has the most significant effect of the identified genes and promotes CRC xenograft growth by affecting cell apoptosis. Next, by integrating a fine-mapping analysis, a two-stage affected-control study consisting of 6,213 affected individuals and 10,388 controls, and multipronged experiments, we elucidated that two risk variants, dbSNP: rs61926301 and dbSNP: rs7959129, that located in the ATF1 promoter and first intron, respectively, facilitate a promoter-enhancer interaction, mediated by the synergy of SP1 and GATA3, to upregulate ATF1 expression, thus synergistically predisposing to CRC risk (OR = 1.77, 95% CI = 1.42-2.21, p = 3.16 × 10[-7]; Pmultiplicative-interaction = 1.20 × 10[-22]; Padditive-interaction = 6.50 × 10[-3]). Finally, we performed RNA-seq and ChIP-seq assays in CRC cells treated with ATF1 overexpression in order to dissect the target programs of ATF1. Results showed that ATF1 activates a subset of genes, including BRAF, NRAS, MYC, BIRC2, DAAM1, MAML2, STAT1, ID1, and NKD2, related to apoptosis, Wnt, TGF-β, and MAPK pathways, and these effects could cooperatively increase the risk of CRC. These findings reveal the clinical potential of ATF1 in CRC development and illuminate a promoter-enhancer interaction module between the ATF1 regulatory elements dbSNP: rs61926301 and dbSNP: rs7959129, and they bring us closer to understanding the molecular drivers of cancer.},
}
@article {pmid31203918,
year = {2019},
author = {Olivaes, J and Bonamino, MH and Markoski, MM},
title = {CRISPR/Cas 9 system for the treatment of dilated cardiomyopathy: A hypothesis related to function of a MAP kinase.},
journal = {Medical hypotheses},
volume = {128},
number = {},
pages = {91-93},
doi = {10.1016/j.mehy.2019.05.013},
pmid = {31203918},
issn = {1532-2777},
mesh = {*CRISPR-Cas Systems ; Cardiomyopathy, Dilated/*genetics/*therapy ; DNA/analysis ; *Gene Editing ; Genome, Human ; Heart Transplantation ; Humans ; *MAP Kinase Signaling System ; Models, Theoretical ; Superoxides/metabolism ; Troponin I/metabolism ; },
abstract = {Dilated cardiomyopathy (DCM) is a disease with high incidence and mortality rates. Its therapies have one primary goal, which is to minimize symptoms and it has only one effective approach to healing, the heart transplantation. As it is widely associated with genetic causes, the use of gene therapies, such as the CRISPR/Cas9 system, is a promising alternative to treat DCM. For this purpose, it is necessary to analyze possible target genes for this approach and what would be the implications of their use. Here, we hypothesized that cardiac troponin I type 3 interacting kinase (TNI3K), involved with superoxide production in DCM patients, besides other factors, could be a good target for the use of gene editing.},
}
@article {pmid31203891,
year = {2019},
author = {Huang, Y and Xuan, H and Yang, C and Guo, N and Wang, H and Zhao, J and Xing, H},
title = {GmHsp90A2 is involved in soybean heat stress as a positive regulator.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {285},
number = {},
pages = {26-33},
doi = {10.1016/j.plantsci.2019.04.016},
pmid = {31203891},
issn = {1873-2259},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Heat-Shock Proteins/*physiology ; Heat-Shock Response ; Malondialdehyde/metabolism ; Plant Proteins/*physiology ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Soybeans/*metabolism/physiology ; Two-Hybrid System Techniques ; },
abstract = {Heat shock protein 90 s (Hsp90s), one of the most conserved and abundant molecular chaperones, is an essential component of the protective stress response. A previous study reported at least 12 genes in the GmHsp90s family in soybean and that GmHsp90A2 overexpression enhanced thermotolerance in Arabidopsis thaliana. Here, we investigate the roles of GmHsp90A2 in soybean by utilizing stable transgenic soybean lines overexpressing GmHsp90A2 and mutant lines generated by the CRISPR/Cas9 system. The results showed that compared with wild-type plants (WT) and empty vector control plants (VC), T3 transgenic soybean plants overexpressing GmHsp90A2 exhibited increased tolerance to heat stress through higher chlorophyll and lower malondialdehyde (MDA) contents in plants. Conversely, reduced chlorophyll and increased MDA contents in T2 homozygous GmHsp90A2-knockout mutants indicated decreased tolerance to heat stress. GmHsp90A2 was found to interact with GmHsp90A1 in yeast two-hybrid assays. Furthermore, subcellular localization analyses revealed that GmHsp90A2 was localized to the cytoplasm and cell membrane; as shown by bimolecular fluorescence complementation (BiFC) assays, GmHsp90A2 interacted with GmHsp90A1 in the nucleus and cytoplasm and cell membrane. Hence, we conclude that GmHsp90A1 is able to bind to GmHsp90A2 to form a complex and that this complex enters the nucleus. In summary, GmHsp90A2 might respond to heat stress and positively regulate thermotolerance by interacting with GmHsp90A1.},
}
@article {pmid31203365,
year = {2019},
author = {Wang, P and He, D and Li, B and Guo, Y and Wang, W and Luo, X and Zhao, X and Wang, X},
title = {Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {74},
number = {9},
pages = {2559-2565},
doi = {10.1093/jac/dkz246},
pmid = {31203365},
issn = {1460-2091},
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Conjugation, Genetic ; DNA Transposable Elements/genetics ; Escherichia coli/drug effects/*genetics ; Escherichia coli Infections/*microbiology ; Humans ; Plasmids/*genetics ; RNA, Guide/genetics ; Recombination, Genetic ; },
abstract = {OBJECTIVES: To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates.<br><br>METHODS: Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing.<br><br>RESULTS: In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing.<br><br>CONCLUSIONS: CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.},
}
@article {pmid31202125,
year = {2019},
author = {Yang, J and Wang, X},
title = {Role of long non-coding RNAs in lymphoma: A systematic review and clinical perspectives.},
journal = {Critical reviews in oncology/hematology},
volume = {141},
number = {},
pages = {13-22},
doi = {10.1016/j.critrevonc.2019.05.007},
pmid = {31202125},
issn = {1879-0461},
mesh = {Biomarkers, Pharmacological ; Biomarkers, Tumor/genetics ; Cell Transformation, Neoplastic/genetics ; Epigenesis, Genetic/physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphoma/diagnosis/*genetics/therapy ; Molecular Targeted Therapy/methods ; RNA Interference/physiology ; RNA, Long Noncoding/genetics/*physiology ; },
abstract = {Long non-coding RNAs (lncRNAs), are over 200 nucleotides in length, and they rarely act as templates for protein synthesis. Mounting studies have shown that lncRNAs play a crucial regulatory role in various processes that sustain life, such as epigenetic regulation, cell cycle control, splicing, and post-transcriptional regulation. LncRNAs were aberrantly expressed in most hematological malignancies including lymphoma, participating in tumor suppression or promoting oncogenesis and modulating key genes in different pathways. The specific expression patterns of lncRNAs in lymphoma make them good candidates to be used as diagnostic biomarkers or as therapeutic targets. LncRNAs can be targeted by multiple approaches including nucleic acid therapeutics, CRISPR/Cas genome editing techniques, small molecule inhibitors, and gene therapy. Efforts are made to develop therapeutic strategies aimed at targeting lncRNAs, but there are still some avenues to be covered before they can be applied to the clinical treatment of lymphoma.},
}
@article {pmid31201850,
year = {2019},
author = {Artyukhova, MA and Tyurina, YY and Chu, CT and Zharikova, TM and Bayır, H and Kagan, VE and Timashev, PS},
title = {Interrogating Parkinson's disease associated redox targets: Potential application of CRISPR editing.},
journal = {Free radical biology &amp; medicine},
volume = {144},
number = {},
pages = {279-292},
pmid = {31201850},
issn = {1873-4596},
support = {R01 CA165065/CA/NCI NIH HHS/United States ; U19 AI068021/AI/NIAID NIH HHS/United States ; R01 NS076511/NS/NINDS NIH HHS/United States ; R01 NS061817/NS/NINDS NIH HHS/United States ; P01 HL114453/HL/NHLBI NIH HHS/United States ; R01 GM113908/GM/NIGMS NIH HHS/United States ; R01 AG026389/AG/NIA NIH HHS/United States ; R01 NS101628/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cardiolipins ; Dopaminergic Neurons/metabolism/pathology ; Ferroptosis/genetics ; Gene Editing/*methods ; Humans ; Iron/*metabolism ; Lipid Peroxidation ; Mitochondria/*metabolism/pathology ; Mitophagy ; Mutation ; Oxidation-Reduction ; Parkinson Disease/genetics/metabolism/pathology/*therapy ; Reactive Oxygen Species/metabolism ; Substantia Nigra/metabolism/pathology ; alpha-Synuclein/*genetics/metabolism ; },
abstract = {Loss of dopaminergic neurons in the substantia nigra is one of the pathogenic hallmarks of Parkinson's disease, yet the underlying molecular mechanisms remain enigmatic. While aberrant redox metabolism strongly associated with iron dysregulation and accumulation of dysfunctional mitochondria is considered as one of the major contributors to neurodegeneration and death of dopaminergic cells, the specific anomalies in the molecular machinery and pathways leading to the PD development and progression have not been identified. The high efficiency and relative simplicity of a new genome editing tool, CRISPR/Cas9, make its applications attractive for deciphering molecular changes driving PD-related impairments of redox metabolism and lipid peroxidation in relation to mishandling of iron, aggregation and oligomerization of alpha-synuclein and mitochondrial injury as well as in mechanisms of mitophagy and programs of regulated cell death (apoptosis and ferroptosis). These insights into the mechanisms of PD pathology may be used for the identification of new targets for therapeutic interventions and innovative approaches to genome editing, including CRISPR/Cas9.},
}
@article {pmid31201419,
year = {2019},
author = {Lu, Y and Oura, S and Matsumura, T and Oji, A and Sakurai, N and Fujihara, Y and Shimada, K and Miyata, H and Tobita, T and Noda, T and Castaneda, JM and Kiyozumi, D and Zhang, Q and Larasati, T and Young, SAM and Kodani, M and Huddleston, CA and Robertson, MJ and Coarfa, C and Isotani, A and Aitken, RJ and Okabe, M and Matzuk, MM and Garcia, TX and Ikawa, M},
title = {CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†.},
journal = {Biology of reproduction},
volume = {101},
number = {2},
pages = {501-511},
pmid = {31201419},
issn = {1529-7268},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; R01 HD095341/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Fertility/*genetics ; *Gene Editing ; Gene Expression Regulation/*physiology ; Humans ; Infertility, Male/genetics ; Male ; Mice ; Mice, Knockout ; Testis/*metabolism ; Transcriptome ; },
abstract = {More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.},
}
@article {pmid31201381,
year = {2019},
author = {Memczak, S and Shao, Y and Izpisua Belmonte, JC},
title = {Towards precise, safe genome editing.},
journal = {Cell research},
volume = {29},
number = {9},
pages = {687-689},
pmid = {31201381},
issn = {1748-7838},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; RNA Editing ; Transcriptome ; },
}
@article {pmid31200335,
year = {2019},
author = {Ishiguro, S and Mori, H and Yachie, N},
title = {DNA event recorders send past information of cells to the time of observation.},
journal = {Current opinion in chemical biology},
volume = {52},
number = {},
pages = {54-62},
doi = {10.1016/j.cbpa.2019.05.009},
pmid = {31200335},
issn = {1879-0402},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Communication ; *Cells ; DNA/*genetics ; Humans ; Recombination, Genetic ; Transcriptome ; },
abstract = {While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system. One possible direction to overcome this issue is the development of a cell-embedded 'event recorder' system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past event information stored in 'heritable polymers' of the same cell. This concept has been demonstrated by many synthetic cellular circuits that monitor and transmit a certain set of environmental and intracellular signals into DNA, and have now been further accelerated by recent CRISPR-related technologies. Notably, the discovery of the RT-Cas1-Cas2 system, which acquires sequences of cellular transcripts into a specific host genomic region, has enabled recording of a broader range of molecular profile histories in the DNA tapes of cells, to understand the dynamics of complex biological processes that cannot be addressed by current technologies.},
}
@article {pmid31199673,
year = {2019},
author = {Lu, FI and Wang, YT and Wang, YS and Wu, CY and Li, CC},
title = {Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {9},
pages = {9959-9973},
doi = {10.1096/fj.201900342RR},
pmid = {31199673},
issn = {1530-6860},
mesh = {ADP-Ribosylation Factor 1/antagonists &amp; inhibitors/physiology ; Animals ; CRISPR-Cas Systems ; Cell Movement ; Embryo, Nonmammalian/blood supply ; Embryonic Development ; Endothelial Cells/cytology/metabolism ; Gene Knockdown Techniques ; Genes, Reporter ; Guanine Nucleotide Exchange Factors/antagonists &amp; inhibitors/genetics/*physiology ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Physiologic/genetics/*physiology ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Vascular Endothelial Growth Factor A/*biosynthesis/genetics ; Zebrafish/embryology/genetics ; Zebrafish Proteins/genetics/physiology ; },
abstract = {VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.},
}
@article {pmid31199589,
year = {2019},
author = {Gao, Q and Dong, X and Xu, Q and Zhu, L and Wang, F and Hou, Y and Chao, CC},
title = {Therapeutic potential of CRISPR/Cas9 gene editing in engineered T-cell therapy.},
journal = {Cancer medicine},
volume = {8},
number = {9},
pages = {4254-4264},
pmid = {31199589},
issn = {2045-7634},
mesh = {Animals ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Gene Editing/*methods ; Humans ; Immunotherapy ; Immunotherapy, Adoptive ; Neoplasms/immunology/*therapy ; Receptors, Antigen, T-Cell/*genetics/metabolism ; Receptors, Chimeric Antigen/*genetics/metabolism ; T-Lymphocytes/immunology/*transplantation ; Tumor Microenvironment ; },
abstract = {Cancer patients have been treated with various types of therapies, including conventional strategies like chemo-, radio-, and targeted therapy, as well as immunotherapy like checkpoint inhibitors, vaccine and cell therapy etc. Among the therapeutic alternatives, T-cell therapy like CAR-T (Chimeric Antigen Receptor Engineered T cell) and TCR-T (T Cell Receptor Engineered T cell), has emerged as the most promising therapeutics due to its impressive clinical efficacy. However, there are many challenges and obstacles, such as immunosuppressive tumor microenvironment, manufacturing complexity, and poor infiltration of engrafted cells, etc still, need to be overcome for further treatment with different forms of cancer. Recently, the antitumor activities of CAR-T and TCR-T cells have shown great improvement with the utilization of CRISPR/Cas9 gene editing technology. Thus, the genome editing system could be a powerful genetic tool to use for manipulating T cells and enhancing the efficacy of cell immunotherapy. This review focuses on pros and cons of various gene delivery methods, challenges, and safety issues of CRISPR/Cas9 gene editing application in T-cell-based immunotherapy.},
}
@article {pmid31198998,
year = {2019},
author = {Ohtsuka, M and Sato, M},
title = {i-GONAD: A method for generating genome-edited animals without ex vivo handling of embryos.},
journal = {Development, growth &amp; differentiation},
volume = {61},
number = {5},
pages = {306-315},
doi = {10.1111/dgd.12620},
pmid = {31198998},
issn = {1440-169X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Embryo, Mammalian/metabolism ; Gene Editing/*methods ; Genome/*genetics ; Zygote/metabolism ; },
abstract = {The recent development of genome editing technologies has enabled the creation of genome-edited animals, with alterations at the desired target locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for this purpose because it is simpler and more efficient than other genome editing technologies. The conventional methods for creation of genome-edited animals involve ex vivo handling of embryos (zygotes) for microinjection or in vitro electroporation. However, this process is laborious and time-consuming, and relatively large numbers of animals are used. Furthermore, these methods require specialized skills for handling embryos. In 2015, we reported a novel method for the creation of genome-edited animals without ex vivo handling of embryos. The technology known as Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) involved intraoviductal instillation of genome editing components into a pregnant female and subsequent in vivo electroporation of an entire oviduct. The genome editing components present in the oviductal lumen are transferred to preimplantation embryos in situ for introducing insertion or deletion (indel) mutations at the desired loci. This technology was further improved by optimizing several parameters to develop improved GONAD (i-GONAD) for the efficient generation of mutant or knock-in animals. In this review, we discuss the historical background, potential applications, advantages, and future challenges of GONAD/i-GONAD technology.},
}
@article {pmid31198958,
year = {2019},
author = {Yamamoto, A and Ishida, T and Yoshimura, M and Kimura, Y and Sawa, S},
title = {Developing Heritable Mutations in Arabidopsis thaliana Using a Modified CRISPR/Cas9 Toolkit Comprising PAM-Altered Cas9 Variants and gRNAs.},
journal = {Plant &amp; cell physiology},
volume = {60},
number = {10},
pages = {2255-2262},
doi = {10.1093/pcp/pcz118},
pmid = {31198958},
issn = {1471-9053},
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing ; Mutagenesis ; Mutation ; RNA, Guide/*genetics ; Species Specificity ; Streptococcus pyogenes/*enzymology/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.},
}
@article {pmid31197230,
year = {2019},
author = {Rybicki, EP},
title = {CRISPR-Cas9 strikes out in cassava.},
journal = {Nature biotechnology},
volume = {37},
number = {7},
pages = {727-728},
pmid = {31197230},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems ; *Geminiviridae ; Gene Editing ; Genetic Engineering/methods ; Genetic Predisposition to Disease ; Host-Pathogen Interactions/genetics ; Manihot/genetics ; Plant Diseases/*genetics/*virology ; Plants, Genetically Modified/*virology ; },
}
@article {pmid31197154,
year = {2019},
author = {Naseri, G and Behrend, J and Rieper, L and Mueller-Roeber, B},
title = {COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2615},
pmid = {31197154},
issn = {2041-1723},
support = {031A172//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/International ; 031B0223//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/International ; },
mesh = {Arabidopsis Proteins/genetics/metabolism ; Biosensing Techniques/methods ; Biosynthetic Pathways/*genetics ; CRISPR-Cas Systems/genetics ; Cloning, Molecular/methods ; Flavanones/biosynthesis ; Homologous Recombination/genetics ; Metabolic Engineering/*methods ; Norisoprenoids/biosynthesis ; Promoter Regions, Genetic/genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; Synthetic Biology/methods ; Transcription Factors/genetics/*metabolism ; beta Carotene/biosynthesis ; },
abstract = {Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.},
}
@article {pmid31197149,
year = {2019},
author = {Hojo, MA and Masuda, K and Hojo, H and Nagahata, Y and Yasuda, K and Ohara, D and Takeuchi, Y and Hirota, K and Suzuki, Y and Kawamoto, H and Kawaoka, S},
title = {Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2603},
pmid = {31197149},
issn = {2041-1723},
support = {15H01478//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; 18K15409//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; 18H04810: S.K//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; na//Uehara Memorial Foundation/International ; na//Japan Foundation for Applied Enzymology/International ; JPMJER1303//MEXT | Japan Science and Technology Agency (JST)/International ; },
mesh = {Animals ; Apoptosis/genetics/immunology ; Autoimmunity/*genetics/immunology ; Bcl-2-Like Protein 11/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Enhancer Elements, Genetic/genetics/*immunology ; Female ; Gene Expression Regulation/immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; Receptors, Antigen, T-Cell/immunology/*metabolism ; Signal Transduction/genetics/immunology ; Thymocytes/*physiology ; Thymus Gland/cytology/immunology ; },
abstract = {During thymic negative selection, autoreactive thymocytes carrying T cell receptor (TCR) with overtly strong affinity to self-MHC/self-peptide are removed by Bim-dependent apoptosis, but how Bim is specifically regulated to link TCR activation and apoptosis induction is unclear. Here we identify a murine T cell-specific genomic enhancer E[BAB (Bub1-Acoxl-Bim)], whose deletion leads to accumulation of thymocytes expressing high affinity TCRs. Consistently, E[BAB] knockout mice have defective negative selection and fail to delete autoreactive thymocytes in various settings, with this defect accompanied by reduced Bim expression and apoptosis induction. By contrast, E[BAB] is dispensable for maintaining peripheral T cell homeostasis via Bim-dependent pathways. Our data thus implicate E[BAB] as an important, developmental stage-specific regulator of Bim expression and apoptosis induction to enforce thymic negative selection and suppress autoimmunity. Our study unravels a part of genomic enhancer codes that underlie complex and context-dependent gene regulation in TCR signaling.},
}
@article {pmid31197108,
year = {2019},
author = {Miltner, AM and Mercado-Ayon, Y and Cheema, SK and Zhang, P and Zawadzki, RJ and La Torre, A},
title = {A Novel Reporter Mouse Uncovers Endogenous Brn3b Expression.},
journal = {International journal of molecular sciences},
volume = {20},
number = {12},
pages = {},
pmid = {31197108},
issn = {1422-0067},
support = {R25 GM116690/GM/NIGMS NIH HHS/United States ; R01 EY026942/EY/NEI NIH HHS/United States ; R01EY026942/NH/NIH HHS/United States ; P30 EY012576/EY/NEI NIH HHS/United States ; P30 EY012576/NH/NIH HHS/United States ; T32 EY015387/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Genes, Reporter ; Homeodomain Proteins/*genetics/metabolism ; Luminescent Proteins/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins/genetics/metabolism ; Retina/diagnostic imaging/*metabolism ; Transcription Factor Brn-3B/*genetics/metabolism ; Visual Pathways/diagnostic imaging/metabolism ; },
abstract = {Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.},
}
@article {pmid31196871,
year = {2019},
author = {Brunner, E and Yagi, R and Debrunner, M and Beck-Schneider, D and Burger, A and Escher, E and Mosimann, C and Hausmann, G and Basler, K},
title = {CRISPR-induced double-strand breaks trigger recombination between homologous chromosome arms.},
journal = {Life science alliance},
volume = {2},
number = {3},
pages = {},
pmid = {31196871},
issn = {2575-1077},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; *Chromosomes ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Breaks, Double-Stranded ; Drosophila/genetics ; Gene Editing ; Gene Expression ; Gene Targeting ; Genes, Reporter ; *Homologous Recombination ; Nucleic Acid Conformation ; Phenotype ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.},
}
@article {pmid31195957,
year = {2019},
author = {Luo, J and Chen, W and Xue, L and Tang, B},
title = {Prediction of activity and specificity of CRISPR-Cpf1 using convolutional deep learning neural networks.},
journal = {BMC bioinformatics},
volume = {20},
number = {1},
pages = {332},
pmid = {31195957},
issn = {1471-2105},
mesh = {Algorithms ; Base Sequence ; CRISPR-Cas Systems/*genetics ; *Deep Learning ; Endonucleases/*metabolism ; *Neural Networks, Computer ; RNA, Guide/genetics ; },
abstract = {BACKGROUND: CRISPR-Cpf1 has recently been reported as another RNA-guided endonuclease of class 2 CRISPR-Cas system, which expands the molecular biology toolkit for genome editing. However, most of the online tools and applications to date have been developed primarily for the Cas9. There are a limited number of tools available for the Cpf1.<br><br>RESULTS: We present DeepCpf1, a deep convolution neural networks (CNN) approach to predict Cpf1 guide RNAs on-target activity and off-target effects using their matched and mismatched DNA sequences. Trained on published data sets, DeepCpf1 is superior to other machine learning algorithms and reliably predicts the most efficient and less off-target effects guide RNAs for a given gene. Combined with a permutation importance analysis, the key features of guide RNA sequences are identified, which determine the activity and specificity of genome editing.<br><br>CONCLUSIONS: DeepCpf1 can significantly improve the accuracy of Cpf1-based genome editing and facilitates the generation of optimized guide RNAs libraries.},
}
@article {pmid31195838,
year = {2020},
author = {Pena, SA and Iyengar, R and Eshraghi, RS and Bencie, N and Mittal, J and Aljohani, A and Mittal, R and Eshraghi, AA},
title = {Gene therapy for neurological disorders: challenges and recent advancements.},
journal = {Journal of drug targeting},
volume = {28},
number = {2},
pages = {111-128},
doi = {10.1080/1061186X.2019.1630415},
pmid = {31195838},
issn = {1029-2330},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; Nanoparticles ; Nervous System Diseases/genetics/physiopathology/*therapy ; Polymers/chemistry ; },
abstract = {Major advancements in targeted gene therapy have opened up avenues for the treatment of major neurological disorders through a range of versatile modalities varying from expression of exogenous to suppression of endogenous genes. Recent technological innovations for improved gene sequence delivery have focussed on highly specific viral vector designs, plasmid transfection, nanoparticles, polymer-mediated gene delivery, engineered microRNA and in vivo clustered regulatory interspaced short palindromic repeats (CRISPR)-based therapeutics. These advanced techniques have profound applications in treating highly prevalent neurological diseases and neurodevelopmental disorders including Parkinson's disease, Alzheimer's disease and autism spectrum disorder, as well as rarer diseases such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, lysosomal storage diseases, X-linked adrenoleukodystrophy and oncological diseases. In this article, we present an overview of the latest advances in targeted gene delivery and discuss the challenges and future direction of gene therapy in the treatment of neurological disorders.},
}
@article {pmid31195154,
year = {2019},
author = {Long, J and Xu, Y and Ou, L and Yang, H and Xi, Y and Chen, S and Duan, G},
title = {Polymorphism of Type I-F CRISPR/Cas system in Escherichia coli of phylogenetic group B2 and its application in genotyping.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {74},
number = {},
pages = {103916},
doi = {10.1016/j.meegid.2019.103916},
pmid = {31195154},
issn = {1567-7257},
mesh = {*CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Escherichia coli/*classification/genetics ; Evolution, Molecular ; Genotyping Techniques ; Humans ; Multilocus Sequence Typing/*methods ; Phylogeny ; Plasmids/genetics ; *Polymorphism, Genetic ; },
abstract = {E. coli of phylogenetic group B2 is responsible for many extraintestinal infections, posing a great threat to health. The relatively polymorphic nature of CRISPR in phylogenetically related E. coli strains makes them potential markers for bacterial typing and evolutionary studies. In the current work, we investigated the occurrence and diversity of CRISPR/Cas system and explored its potential for genotyping. Type I-F CRISPR/Cas systems were found in 413 of 1190 strains of E. coli and exhibited the clustering within certain CCs and STs. And CRISPR spacer contents correlated well with MLST types. The divergence analysis of CRISPR showed stronger discriminatory power than MLST, and CRISPR polymorphism was instrumental for differentiating highly closely related strains. The timeline of spacer acquisition and deletion provided important information for inferring the evolution model between distinct serotypes. Identical spacer sequences were shared by strains with the same H-antigen type but not strains with the same O-antigen type. The homology between spacers and antibiotic-resistant plasmids demonstrated the role of Type I-F system in limiting the acquisition of antimicrobial resistance. Collectively, our data presents the dynamic nature of Type I-F CRISPR in E. coli of phylogenetic group B2 and provides new insights into the application of CRISPR-based typing in the species.},
}
@article {pmid31195050,
year = {2019},
author = {Kang, W and Sun, Z and Zhao, X and Wang, X and Tao, Y and Wu, H},
title = {Gene editing based hearing impairment research and therapeutics.},
journal = {Neuroscience letters},
volume = {709},
number = {},
pages = {134326},
doi = {10.1016/j.neulet.2019.134326},
pmid = {31195050},
issn = {1872-7972},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Deafness/*genetics/*therapy ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; Hearing Loss/genetics/therapy ; Humans ; },
abstract = {Hearing impairment affects 1 in 500 newborns worldwide and nearly one out of three people over the age of 65 (WHO, 2019). Hereditary hearing loss is the most common type of congenital deafness; genetic factors also affect deafness susceptibility. Gene therapies may preserve or restore natural sound perception, and have rescued deafness in multiple hereditary murine models. CRISPR-Cas9 and base editors (BEs) are newly developed gene editing technologies that can facilitate gene studies in the inner ear and provide therapeutic approaches for hearing impairment. Here, we present recent applications of gene editing in the inner ear.},
}
@article {pmid31195029,
year = {2019},
author = {O'Meara, D and Nunney, L},
title = {A phylogenetic test of the role of CRISPR-Cas in limiting plasmid acquisition and prophage integration in bacteria.},
journal = {Plasmid},
volume = {104},
number = {},
pages = {102418},
doi = {10.1016/j.plasmid.2019.102418},
pmid = {31195029},
issn = {1095-9890},
mesh = {Bacteria/*virology ; *CRISPR-Cas Systems ; Lysogeny ; *Phylogeny ; Plasmids/*genetics ; Prophages/*physiology ; Viral Plaque Assay ; *Virus Integration ; },
abstract = {CRISPR-Cas is a prokaryotic defense system capable of protecting the cell from damaging foreign genetic elements. However, some genetic elements can be beneficial, which suggests the hypothesis that bacteria with CRISPR-Cas incur a cost of reduced intake of mutualistic plasmids and prophage. Here we present the first robust test of this hypothesis that controls for phylogenic and ecological biases in the distribution of CRISPR-Cas. We filtered the available genomic data (~7000 strains from ~2100 species) by first selecting all pairs of conspecific strains, one with and one without CRISPR-Cas (controlling ecological bias), and second by retaining only one such pair per bacterial family (controlling phylogenetic bias), resulting in pairs representing 38 bacterial families. Analysis of these pairs of bacterial strains showed that on average the CRISPR-Cas strain of each pair contained significantly fewer plasmids than its CRISPR-Cas negative partner (0.86 vs. 1.86). It also showed that the CRISPR-Cas positive strains had 31% fewer intact prophage (1.17 vs. 1.75), but the effect was highly variable and not significant. These results support the hypothesis that CRISPR-Cas reduces the rate of plasmid-mediated HGT and, given the abundant evidence of beneficial genes carried by plasmids, provide a clear example of a cost associated with the CRISPR-Cas system.},
}
@article {pmid31194947,
year = {2019},
author = {Nishitani, N and Ohmura, Y and Nagayasu, K and Shibui, N and Kaneko, S and Ohashi, A and Yoshida, T and Yamanaka, A and Yoshioka, M},
title = {CRISPR/Cas9-mediated in vivo gene editing reveals that neuronal 5-HT1A receptors in the dorsal raphe nucleus contribute to body temperature regulation in mice.},
journal = {Brain research},
volume = {1719},
number = {},
pages = {243-252},
doi = {10.1016/j.brainres.2019.06.009},
pmid = {31194947},
issn = {1872-6240},
mesh = {Animals ; Body Temperature Regulation/*genetics/physiology ; CRISPR-Cas Systems/genetics ; Dorsal Raphe Nucleus/metabolism/*physiology ; Female ; Gene Editing/methods ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neural Inhibition/physiology ; Receptor, Serotonin, 5-HT1A/*genetics/metabolism ; Receptors, Serotonin/metabolism ; Serotonergic Neurons/metabolism ; Serotonin/metabolism ; Serotonin Antagonists/pharmacology ; Serotonin Receptor Agonists/pharmacology ; Serotonin Uptake Inhibitors/pharmacology ; },
abstract = {Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT1A receptor agonist-induced hypothermia was attenuated and antidepressant effect of a selective serotonin reuptake inhibitor (SSRI) was enhanced by microinjection of the viral vector expressing sgHtr1a into the DRN of Cre-dependent Cas9 knock-in mice. These results suggest that this in vivo CRISPR/Cas9-mediated 5-HT receptor gene knockout strategy provides a reliable and low-cost method for elucidating 5-HT receptor functions in specific cell types and brain regions. Further, we demonstrate that the neuronal 5-HT1A receptor in the DRN regulates body temperature and antidepressant effect of SSRI.},
}
@article {pmid31193992,
year = {2019},
author = {Hodges, CA and Conlon, RA},
title = {Delivering on the promise of gene editing for cystic fibrosis.},
journal = {Genes &amp; diseases},
volume = {6},
number = {2},
pages = {97-108},
pmid = {31193992},
issn = {2352-3042},
abstract = {In this review, we describe a path for translation of gene editing into therapy for cystic fibrosis (CF). Cystic fibrosis results from mutations in the CFTR gene, with one allele predominant in patient populations. This simple, genetic etiology makes gene editing appealing for treatment of this disease. There already have been success in applying this approach to cystic fibrosis in cell and animal models, although these advances have been modest in comparison to advances for other disease. Less than six years after its first demonstration in animals, CRISPR/Cas gene editing is in early clinical trials for several disorders. Most clinical trials, thus far, attempt to edit genes in cells of the blood lineages. The advantage of the blood is that the stem cells are known, can be isolated, edited, selected, expanded, and returned to the body. The likely next trials will be in the liver, which is accessible to many delivery methods. For cystic fibrosis, the biggest hurdle is to deliver editors to other, less accessible organs. We outline a path by which delivery can be improved. The translation of new therapies doesn't occur in isolation, and the development of gene editors is occurring as advances in gene therapy and small molecule therapeutics are being made. The advances made in gene therapy may help develop delivery vehicles for gene editing, although major improvements are needed. Conversely, the approval of effective small molecule therapies for many patients with cystic fibrosis will raise the bar for translation of gene editing.},
}
@article {pmid31189706,
year = {2019},
author = {Zhang, Y and Tang, N and Luo, J and Teng, M and Moffat, K and Shen, Z and Watson, M and Nair, V and Yao, Y},
title = {Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines.},
journal = {Journal of virology},
volume = {93},
number = {17},
pages = {},
pmid = {31189706},
issn = {1098-5514},
support = {BBS/E/I/00007032/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Transformed ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Viral/*genetics ; Humans ; Lymphoma/*virology ; Mardivirus/genetics/*pathogenicity ; MicroRNAs/*genetics ; RNA, Viral/genetics ; },
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.},
}
@article {pmid31189656,
year = {2019},
author = {Bando, H and Pradipta, A and Iwanaga, S and Okamoto, T and Okuzaki, D and Tanaka, S and Vega-Rodríguez, J and Lee, Y and Ma, JS and Sakaguchi, N and Soga, A and Fukumoto, S and Sasai, M and Matsuura, Y and Yuda, M and Jacobs-Lorena, M and Yamamoto, M},
title = {CXCR4 regulates Plasmodium development in mouse and human hepatocytes.},
journal = {The Journal of experimental medicine},
volume = {216},
number = {8},
pages = {1733-1748},
pmid = {31189656},
issn = {1540-9538},
mesh = {Animals ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Line, Tumor ; Hepatocytes/*metabolism ; Humans ; Liver/metabolism ; Malaria/*metabolism/parasitology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-kappa B/metabolism ; Plasmodium berghei/*growth &amp; development ; Plasmodium falciparum/*growth &amp; development ; Protein Kinase C/genetics/metabolism ; Proto-Oncogene Proteins c-met/genetics/metabolism ; Receptors, CXCR4/genetics/*metabolism ; Sporozoites/metabolism ; Transfection ; },
abstract = {The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca[2+] levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.},
}
@article {pmid31189559,
year = {2019},
author = {Hakimi, H and Ishizaki, T and Kegawa, Y and Kaneko, O and Kawazu, SI and Asada, M},
title = {Genome Editing of Babesia bovis Using the CRISPR/Cas9 System.},
journal = {mSphere},
volume = {4},
number = {3},
pages = {},
pmid = {31189559},
issn = {2379-5042},
mesh = {Babesia bovis/*genetics ; *CRISPR-Cas Systems ; Gene Deletion ; *Gene Editing ; Green Fluorescent Proteins/genetics ; Humans ; Plasmids/genetics ; Point Mutation ; Tetrahydrofolate Dehydrogenase/genetics ; },
abstract = {Babesia bovis, the most virulent causative agent of bovine babesiosis, is prevalent in tropical and subtropical regions of the world. Although the whole-genome sequence was released more than a decade ago, functional analysis of the genomics of this parasite is hampered by the limited breadth of genetic engineering tools. In this study, we implemented the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system for B. bovis and demonstrated its potential for genome editing. Cas9 and human dihydrofolate reductase (hDHFR) were simultaneously expressed by the B. boviselongation factor-1α bidirectional promoter, and a single guide RNA was expressed via the B. bovisU6 spliceosomal RNA promoter. Using a single plasmid construct, we were able to add an epitope tag to spherical body protein 3 (SBP3), introduce a point mutation into thioredoxin peroxidase 1 (tpx-1) to impair the function of the product, and replace the tpx-1 open reading frame with the other protein. Epitope tagging of SBP3 was efficient using this system, with a negligible number of remaining wild-type parasites and a pure transgenic population produced by allelic replacement of tpx-1 This advancement in genetic engineering tools for B. bovis will aid functional analysis of the genome and underpin characterization of candidate drug and vaccine targets.IMPORTANCEBabesia bovis is the most virulent cause of bovine babesiosis worldwide. The disease consequences are death, abortion, and economical loss due to reduced milk and meat production. Available vaccines are not effective, treatment options are limited, and emergence of drug and acaricide resistance has been reported from different regions. There is an urgent need to identify new drug and vaccine targets. Greater than half of the genes in B. bovis genome, including several expanded gene families which are unique for Babesia spp., have no predicted function. The available genetic engineering tools are based on conventional homologous recombination, which is time-consuming and inefficient. In this study, we adapted the CRISPR/Cas9 system as a robust genetic engineering tool for B. bovis This advancement will aid future functional studies of uncharacterized genes.},
}
@article {pmid31189177,
year = {2019},
author = {Klompe, SE and Vo, PLH and Halpin-Healy, TS and Sternberg, SH},
title = {Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration.},
journal = {Nature},
volume = {571},
number = {7764},
pages = {219-225},
pmid = {31189177},
issn = {1476-4687},
mesh = {Bacterial Proteins/genetics/metabolism ; Base Sequence ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; DNA Transposable Elements/*genetics ; DNA, Bacterial/*genetics/*metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Escherichia coli/genetics ; Gene Editing/*methods ; Genome, Bacterial/genetics ; Integrases/genetics/metabolism ; Mutagenesis, Insertional/*methods ; Mutagenesis, Site-Directed/methods ; RNA, Bacterial/*genetics ; RNA, Guide/genetics ; Substrate Specificity ; Vibrio cholerae/genetics ; },
abstract = {Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.},
}
@article {pmid31188312,
year = {2019},
author = {Rose, BI and Brown, S},
title = {Genetically Modified Babies and a First Application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9).},
journal = {Obstetrics and gynecology},
volume = {134},
number = {1},
pages = {157-162},
doi = {10.1097/AOG.0000000000003327},
pmid = {31188312},
issn = {1873-233X},
mesh = {*CRISPR-Cas Systems ; Female ; Humans ; Infant, Newborn ; *Obstetrics ; Pregnancy ; },
abstract = {The world's first babies with CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-edited genes were born on November 25, 2018. Dr. Jiankui He of Southern University of Science and Technology in Shenzhen performed this gene editing. Dr. He's objectives and an assessment of how well they were achieved are discussed in the context of existing research in this area.},
}
@article {pmid31187895,
year = {2019},
author = {Rao, MC},
title = {Physiology of Electrolyte Transport in the Gut: Implications for Disease.},
journal = {Comprehensive Physiology},
volume = {9},
number = {3},
pages = {947-1023},
doi = {10.1002/cphy.c180011},
pmid = {31187895},
issn = {2040-4603},
mesh = {Animals ; Body Water/metabolism ; Electrolytes/*metabolism ; Gastrointestinal Microbiome/physiology ; Intestinal Absorption/physiology ; Intestinal Diseases/*physiopathology ; Intestinal Mucosa/*metabolism ; Ion Transport/*physiology ; Membrane Transport Proteins/physiology ; Neurotransmitter Agents/physiology ; Sodium-Hydrogen Exchangers/physiology ; Sodium-Potassium-Chloride Symporters/physiology ; },
abstract = {We now have an increased understanding of the genetics, cell biology, and physiology of electrolyte transport processes in the mammalian intestine, due to the availability of sophisticated methodologies ranging from genome wide association studies to CRISPR-CAS technology, stem cell-derived organoids, 3D microscopy, electron cryomicroscopy, single cell RNA sequencing, transgenic methodologies, and tools to manipulate cellular processes at a molecular level. This knowledge has simultaneously underscored the complexity of biological systems and the interdependence of multiple regulatory systems. In addition to the plethora of mammalian neurohumoral factors and their cross talk, advances in pyrosequencing and metagenomic analyses have highlighted the relevance of the microbiome to intestinal regulation. This article provides an overview of our current understanding of electrolyte transport processes in the small and large intestine, their regulation in health and how dysregulation at multiple levels can result in disease. Intestinal electrolyte transport is a balance of ion secretory and ion absorptive processes, all exquisitely dependent on the basolateral Na[+] /K[+] ATPase; when this balance goes awry, it can result in diarrhea or in constipation. The key transporters involved in secretion are the apical membrane Cl[-] channels and the basolateral Na[+] -K[+] -2Cl[-] cotransporter, NKCC1 and K[+] channels. Absorption chiefly involves apical membrane Na[+] /H[+] exchangers and Cl[-] /HCO3 [-] exchangers in the small intestine and proximal colon and Na[+] channels in the distal colon. Key examples of our current understanding of infectious, inflammatory, and genetic diarrheal diseases and of constipation are provided. © 2019 American Physiological Society. Compr Physiol 9:947-1023, 2019.},
}
@article {pmid31186565,
year = {2019},
author = {Cyranoski, D},
title = {Russian biologist plans more CRISPR-edited babies.},
journal = {Nature},
volume = {570},
number = {7760},
pages = {145-146},
doi = {10.1038/d41586-019-01770-x},
pmid = {31186565},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Homozygote ; Humans ; Receptors, CCR5 ; Russia ; },
}
@article {pmid31186504,
year = {2019},
author = {Makokha, GN and Abe-Chayama, H and Chowdhury, S and Hayes, CN and Tsuge, M and Yoshima, T and Ishida, Y and Zhang, Y and Uchida, T and Tateno, C and Akiyama, R and Chayama, K},
title = {Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8462},
pmid = {31186504},
issn = {2045-2322},
mesh = {Base Sequence ; Binding Sites/genetics ; CRISPR-Cas Systems/genetics ; Cell Nucleus/metabolism ; DNA-Binding Proteins/metabolism ; *Gene Expression Regulation, Viral ; Gene Silencing ; Hep G2 Cells ; Hepatitis B/virology ; Hepatitis B virus/*genetics/*physiology ; Humans ; Nucleophosmin ; Promoter Regions, Genetic/genetics ; RNA Recognition Motif/genetics ; RNA Splicing/genetics ; Transcription, Genetic ; Virus Replication/*genetics ; },
abstract = {Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein's broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.},
}
@article {pmid31186424,
year = {2019},
author = {Zhang, B and Ye, Y and Ye, W and Perčulija, V and Jiang, H and Chen, Y and Li, Y and Chen, J and Lin, J and Wang, S and Chen, Q and Han, YS and Ouyang, S},
title = {Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2544},
pmid = {31186424},
issn = {2041-1723},
support = {31770948//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {Amino Acid Sequence ; Bacterial Proteins/chemistry/genetics/*metabolism ; CRISPR-Associated Proteins/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Conformation ; Protein Domains ; RNA Processing, Post-Transcriptional ; RNA, Bacterial/genetics/metabolism ; RNA, Guide/genetics ; Ribonucleases/chemistry/genetics/*metabolism ; Ruminococcus/enzymology/*genetics ; },
abstract = {Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg[2+] ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.},
}
@article {pmid31186306,
year = {2019},
author = {Philip, NS and Escobedo, F and Bahr, LL and Berry, BJ and Wojtovich, AP},
title = {Mos1 Element-Mediated CRISPR Integration of Transgenes in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {8},
pages = {2629-2635},
pmid = {31186306},
issn = {2160-1836},
support = {P40 OD010440/OD/NIH HHS/United States ; R01 NS092558/NS/NINDS NIH HHS/United States ; T32 GM068411/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/*metabolism ; Gene Editing ; Genetic Engineering ; *Transgenes ; Transposases/*metabolism ; },
abstract = {The introduction of exogenous genes in single-copy at precise genomic locations is a powerful tool that has been widely used in the model organism Caenorhabditis elegans Here, we have streamlined the process by creating a rapid, cloning-free method of single-copy transgene insertion we call Mos1 element-mediated CRISPR integration (mmCRISPi). The protocol combines the impact of Mos1 mediated single-copy gene insertion (mosSCI) with the ease of CRISPR/Cas9 mediated gene editing, allowing in vivo construction of transgenes from linear DNA fragments integrated at defined loci in the C. elegans genome. This approach was validated by defining its efficiency at different integration sites in the genome and by testing transgene insert size. The mmCRISPi method benefits from in vivo recombination of overlapping PCR fragments, allowing researchers to mix-and-match between promoters, protein-coding sequences, and 3' untranslated regions, all inserted in a single step at a defined Mos1 loci.},
}
@article {pmid31186067,
year = {2019},
author = {Xiao, Q and Chen, S and Wang, Q and Liu, Z and Liu, S and Deng, H and Hou, W and Wu, D and Xiong, Y and Li, J and Guo, D},
title = {CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4[+] T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4[+] T cell enrichment in humanized mice.},
journal = {Retrovirology},
volume = {16},
number = {1},
pages = {15},
pmid = {31186067},
issn = {1742-4690},
mesh = {Animals ; CD4-Positive T-Lymphocytes/*immunology ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cells, Cultured ; *Gene Editing ; HIV Infections/virology ; Hematopoietic Stem Cells/*cytology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, Transgenic ; RNA, Guide ; Receptors, CCR5/*genetics ; Staphylococcus aureus/enzymology ; },
abstract = {BACKGROUND: The chemokine receptor CCR5, which belongs to the superfamily of G protein-coupled receptors, is the major co-receptor for HIV-1 entry. Individuals with a homozygous CCR5Δ32 mutation have a long lasting and increased resistance to HIV-1 infection. Therefore, CCR5 represents an optimal target for HIV-1/AIDS gene therapy. The CRISPR/Cas9 system has been developed as one of the most efficacious gene editing tools in mammalian cells and the small-sized version from Staphylococcus aureus (SaCas9) has an advantage of easier delivery compared to the most commonly used version from Streptococcus pyogenes Cas9 (SpCas9).<br><br>RESULTS: Here, we demonstrated that CCR5 could be specifically and efficiently edited by CRISPR/SaCas9 together with two sgRNAs, which were identified through a screening of 13 sgRNAs. Disruption of CCR5 expression by lentiviral vector-mediated CRISPR/SaCas9 led to increased resistance against HIV-1 infection in human primary CD4[+] T cells. Moreover, humanized mice engrafted with CCR5-disrupted CD4[+] T cells showed selective survival and enrichment when challenged with CCR5 (R5)-tropic HIV-1 in comparison to mock-treated CD4[+] T cells. We also observed CCR5 could be targeted by CRISPR/SaCas9 in human CD34[+] hematopoietic stem/progenitor cells without obvious differentiation deficiencies.<br><br>CONCLUSIONS: This work provides an alternative approach to disrupt human CCR5 by CRISPR/SaCas9 for a potential gene therapy strategy against HIV-1/AIDS.},
}
@article {pmid31185986,
year = {2019},
author = {Tordjman, J and Majumder, M and Amiri, M and Hasan, A and Hess, D and Lala, PK},
title = {Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells.},
journal = {BMC cancer},
volume = {19},
number = {1},
pages = {561},
pmid = {31185986},
issn = {1471-2407},
mesh = {Animals ; Breast/*cytology ; Breast Neoplasms/*pathology ; CRISPR-Cas Systems ; Cell Movement ; Cell Proliferation ; Cyclooxygenase 2/metabolism ; Epithelial Cells/metabolism ; Epithelial-Mesenchymal Transition ; Female ; Gene Knockout Techniques ; Heterografts ; Humans ; MCF-7 Cells ; Mice ; Mice, Inbred NOD ; Mice, SCID ; MicroRNAs/metabolism ; Protein Isoforms ; RNA, Small Interfering/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Tumor Suppressor Protein p53/metabolism ; },
abstract = {BACKGROUND: Over-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells.<br><br>METHODS: We knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues.<br><br>RESULTS: CPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2R&#x3d2;-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n&#x2009;=&#x2009;105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n&#x2009;=&#x2009;20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2.<br><br>CONCLUSIONS: CPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.},
}
@article {pmid31185896,
year = {2019},
author = {Śmiga, M and Stępień, P and Olczak, M and Olczak, T},
title = {PgFur participates differentially in expression of virulence factors in more virulent A7436 and less virulent ATCC 33277 Porphyromonas gingivalis strains.},
journal = {BMC microbiology},
volume = {19},
number = {1},
pages = {127},
pmid = {31185896},
issn = {1471-2180},
mesh = {Bacterial Proteins/genetics/metabolism ; Bacteroidaceae Infections/microbiology ; Chronic Periodontitis/microbiology ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Bacterial ; Glycosylation ; Humans ; Iron/metabolism ; Metalloproteins/*genetics/metabolism ; Mutation ; Oligonucleotide Array Sequence Analysis ; Oxidative Stress ; Porphyromonas gingivalis/*growth &amp; development/metabolism/pathogenicity ; THP-1 Cells ; Virulence Factors/*genetics/metabolism ; },
abstract = {BACKGROUND: Porphyromonas gingivalis is considered a keystone pathogen responsible for chronic periodontitis. Although several virulence factors produced by this bacterium are quite well characterized, very little is known about regulatory mechanisms that allow different strains of P. gingivalis to efficiently survive in the hostile environment of the oral cavity, a typical habitat characterized by low iron and heme concentrations. The aim of this study was to characterize P. gingivalis Fur homolog (PgFur) in terms of its role in production of virulence factors in more (A7436) and less (ATCC 33277) virulent strains.<br><br>RESULTS: Expression of a pgfur depends on the growth phase and iron/heme concentration. To better understand the role played by the PgFur protein in P. gingivalis virulence under low- and high-iron/heme conditions, a pgfur-deficient ATCC 33277 strain (TO16) was constructed and its phenotype compared with that of a pgfur A7436-derived mutant strain (TO6). In contrast to the TO6 strain, the TO16 strain did not differ in the growth rate and hemolytic activity compared with the ATCC 33277 strain. However, both mutant strains were more sensitive to oxidative stress and they demonstrated changes in the production of lysine- (Kgp) and arginine-specific (Rgp) gingipains. In contrast to the wild-type strains, TO6 and TO16 mutant strains produced larger amounts of HmuY protein under high iron/heme conditions. We also demonstrated differences in production of glycoconjugates between the A7436 and ATCC 33277 strains and we found evidence that PgFur protein might regulate glycosylation process. Moreover, we revealed that PgFur protein plays a role in interactions with other periodontopathogens and is important for P. gingivalis infection of THP-1-derived macrophages and survival inside the cells. Deletion of the pgfur gene influences expression of many transcription factors, including two not yet characterized transcription factors from the Crp/Fnr family. We also observed lower expression of the CRISPR/Cas genes.<br><br>CONCLUSIONS: We show here for the first time that inactivation of the pgfur gene exerts a different influence on the phenotype of the A7436 and ATCC 33277 strains. Our findings further support the hypothesis that PgFur regulates expression of genes encoding surface virulence factors and/or genes involved in their maturation.},
}
@article {pmid31183663,
year = {2019},
author = {Xu, X and Cao, X and Gao, J},
title = {Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system.},
journal = {Transgenic research},
volume = {28},
number = {3-4},
pages = {341-356},
pmid = {31183663},
issn = {1573-9368},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cypriniformes/*genetics ; Fish Proteins/*genetics/metabolism ; *Gene Editing ; *Mutation ; Phenotype ; Sequence Homology ; Skin Diseases/*genetics ; Skin Pigmentation/*genetics ; },
abstract = {CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.},
}
@article {pmid31182835,
year = {2019},
author = {Schober, K and Müller, TR and Gökmen, F and Grassmann, S and Effenberger, M and Poltorak, M and Stemberger, C and Schumann, K and Roth, TL and Marson, A and Busch, DH},
title = {Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function.},
journal = {Nature biomedical engineering},
volume = {3},
number = {12},
pages = {974-984},
doi = {10.1038/s41551-019-0409-0},
pmid = {31182835},
issn = {2157-846X},
mesh = {Antigens, Neoplasm/immunology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Knockout Techniques ; Genes, T-Cell Receptor/genetics ; Genetic Vectors/genetics ; Humans ; Receptors, Antigen, T-Cell, alpha-beta/*chemistry/genetics/*immunology ; Retroviridae/genetics ; T-Lymphocytes/*immunology/*metabolism ; Transduction, Genetic ; Transgenes ; },
abstract = {Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and β-chains combined with orthotopic TCR placement leads to accurate αβ-pairing and results in TCR regulation similar to that of physiological T cells.},
}
@article {pmid31182574,
year = {2019},
author = {Goto, N and Fukuda, A and Yamaga, Y and Yoshikawa, T and Maruno, T and Maekawa, H and Inamoto, S and Kawada, K and Sakai, Y and Miyoshi, H and Taketo, MM and Chiba, T and Seno, H},
title = {Lineage tracing and targeting of IL17RB[+] tuft cell-like human colorectal cancer stem cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {26},
pages = {12996-13005},
pmid = {31182574},
issn = {1091-6490},
mesh = {Animals ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Carcinogenesis ; Cell Differentiation ; Cell Lineage ; Colorectal Neoplasms/*pathology ; Doublecortin-Like Kinases ; Gene Knock-In Techniques ; Humans ; Intestinal Mucosa/cytology/pathology ; Mice ; Mice, Transgenic ; Neoplastic Stem Cells/*pathology ; Octamer Transcription Factors/metabolism ; Primary Cell Culture ; Protein Serine-Threonine Kinases/genetics ; RNA, Small Interfering/metabolism ; Receptors, Interleukin-17/genetics/*metabolism ; Spheroids, Cellular ; Time-Lapse Imaging ; Tumor Cells, Cultured ; Up-Regulation ; Xenograft Model Antitumor Assays ; },
abstract = {Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1[+] tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1[+] mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB[+] CSCs as a cancer therapeutic target.},
}
@article {pmid31182244,
year = {2019},
author = {Bailey, J},
title = {CRISPR-Mediated Gene Editing: Scientific and Ethical Issues.},
journal = {Trends in biotechnology},
volume = {37},
number = {9},
pages = {920-921},
doi = {10.1016/j.tibtech.2019.05.002},
pmid = {31182244},
issn = {1879-3096},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Gene Editing/*ethics/*methods ; Genetic Engineering ; Genome ; },
abstract = {There remains substantial evidence to warrant great concern over the poor efficiency and specificity of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic modification (GM), despite relatively minor improvements compared to other GM methods. These issues cause persistent, adverse, ethical, and scientific consequences for GM animals, which may never be sufficiently resolvable.},
}
@article {pmid31181894,
year = {2019},
author = {Cañadas, IC and Groothuis, D and Zygouropoulou, M and Rodrigues, R and Minton, NP},
title = {RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium.},
journal = {ACS synthetic biology},
volume = {8},
number = {6},
pages = {1379-1390},
doi = {10.1021/acssynbio.9b00075},
pmid = {31181894},
issn = {2161-5063},
support = {G0601176/MRC_/Medical Research Council/United Kingdom ; BB/L013940/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/J014508/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clostridium/*genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Genome, Bacterial/genetics ; Riboswitch/*genetics ; Synthetic Biology ; Theophylline ; },
abstract = {Members of the genus Clostridium represent a diverse assemblage of species exhibiting both medical and industrial importance. Deriving both a greater understanding of their biology, while at the same time enhancing their exploitable properties, requires effective genome editing tools. Here, we demonstrate the first implementation in the genus of theophylline-dependent, synthetic riboswitches exhibiting a full set of dynamic ranges, also suitable for applications where tight control of gene expression is required. Their utility was highlighted by generating a novel riboswitch-based editing tool-RiboCas-that overcomes the main obstacles associated with CRISPR/Cas9 systems, including low transformation efficiencies and excessive Cas9 toxicity. The universal nature of the tool was established by obtaining chromosomal modifications in C. pasteurianum, C. difficile, and C. sporogenes, as well as by carrying out the first reported example of CRISPR-targeted gene disruption in C. botulinum. The high efficiency (100% mutant generation) and ease of application of RiboCas make it suitable for use in a diverse range of microorganisms.},
}
@article {pmid31181342,
year = {2019},
author = {Deyell, M and Ameta, S and Nghe, P},
title = {Large scale control and programming of gene expression using CRISPR.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {124-132},
doi = {10.1016/j.semcdb.2019.05.013},
pmid = {31181342},
issn = {1096-3634},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Expression Regulation/*genetics ; Gene Regulatory Networks/genetics ; Humans ; },
abstract = {The control of gene expression in cells and organisms allows to unveil gene to function relationships and to reprogram biological responses. Several systems, such as Zinc fingers, TALE (Transcription activator-like effectors), and siRNAs (small-interfering RNAs), have been exploited to achieve this. However, recent advances in Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 (CRISPR-Cas9) have overshadowed them due to high specificity, compatibility with many different organisms, and design flexibility. In this review we summarize state-of-the art for CRISPR-Cas9 technology for large scale gene perturbation studies, including single gene and multiple genes knock-out, knock-down, knock-up libraries, and their associated screening assays. We feature in particular the combination of these methods with single-cell transcriptomics approaches. Finally, we highlight the application of CRISPR-Cas9 systems in building synthetic circuits that can be interfaced with gene networks to control cellular states.},
}
@article {pmid31180191,
year = {2019},
author = {},
title = {CRISPR Gene Editing Successfully Treats Lethal Monogenic Lung Disease in Utero.},
journal = {American journal of medical genetics. Part A},
volume = {179},
number = {7},
pages = {1116},
doi = {10.1002/ajmg.a.61213},
pmid = {31180191},
issn = {1552-4833},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Epithelial Cells/metabolism/pathology ; Fetus ; Gene Editing/*methods ; Gene Expression ; Genetic Therapy/*methods ; Humans ; Lung/*metabolism/pathology ; Mice ; Mutation ; Proof of Concept Study ; Pulmonary Surfactant-Associated Protein C/*genetics/metabolism ; Respiratory Insufficiency/*genetics/metabolism/pathology/*therapy ; Respiratory Mucosa/metabolism/pathology ; },
}
@article {pmid31180145,
year = {2019},
author = {Hu, R and Li, H and Lei, Z and Han, Q and Yu, X and Zhou, N and Zhang, X and Mao, Y and Wang, X and Irwin, DM and Niu, G and Tan, H},
title = {Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {10},
pages = {2652-2661},
doi = {10.1002/bit.27084},
pmid = {31180145},
issn = {1097-0290},
support = {//CIHR/Canada ; },
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; *Gene Knock-In Techniques ; HEK293 Cells ; Humans ; Lipopolysaccharide Receptors/biosynthesis/genetics ; Lipopolysaccharides/*analysis ; Lymphocyte Antigen 96/biosynthesis/genetics ; *Models, Biological ; Toll-Like Receptor 4/biosynthesis/genetics ; },
abstract = {A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.},
}
@article {pmid31180051,
year = {2019},
author = {DI Felice, F and Micheli, G and Camilloni, G},
title = {Restriction enzymes and their use in molecular biology: An overview.},
journal = {Journal of biosciences},
volume = {44},
number = {2},
pages = {},
pmid = {31180051},
issn = {0973-7138},
mesh = {CRISPR-Cas Systems ; Chromatin/chemistry/metabolism ; Chromosome Mapping/*history/methods ; Cloning, Molecular/*methods ; DNA/chemistry/genetics/*history/metabolism ; DNA Methylation ; DNA Restriction Enzymes/genetics/*history/metabolism ; History, 20th Century ; History, 21st Century ; Humans ; Molecular Biology/*history/methods ; Nobel Prize ; Nucleotide Mapping/*history/methods ; Transcription Activator-Like Effector Nucleases/genetics/history/metabolism ; },
abstract = {Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the scientists whose pioneering work had explored the activity and sequence specificity of these enzymes, contributing to the definition of their enormous potential as tools for DNA characterization, mapping and manipulation, were awarded the Nobel Prize. In this short review, we celebrate the history of these enzymes in the light of their many different uses, as these proteins have accompanied the history of DNA for over 50 years representing active witnesses of major steps in the field.},
}
@article {pmid31178847,
year = {2019},
author = {Mougari, S and Abrahao, J and Oliveira, GP and Bou Khalil, JY and La Scola, B},
title = {Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {1147},
pmid = {31178847},
issn = {1664-302X},
abstract = {MIMIVIRE is a defense system described in lineage A Mimivirus (Mimiviridae family) that mediates resistance against Zamilon virophage. It is composed of putative helicase and nuclease associated with a gene of unknown function called R349, which contains four 15 bp repeats homologous to the virophage sequence. In a previous study, the silencing of such genes restored virophage susceptibility. Moreover, the CRISPR Cas-4 like activity of the nuclease has recently been characterized. In this study, a recently isolated Mimivirus of lineage A with R349 gene lacking 3 of 4 repeats was demonstrated to be susceptible to Zamilon. To reinforce the importance of the R349 gene in the MIMIVIRE system, we developed and presented, for the first time to our knowledge, a protocol for Mimivirus genomic editing. By knocking out R349 gene in a Mimivirus lineage A, we observed the replication of Zamilon, indicating that this gene is critical in the resistance against this specific group of virophages.},
}
@article {pmid31178391,
year = {2019},
author = {Romero, Z and Lomova, A and Said, S and Miggelbrink, A and Kuo, CY and Campo-Fernandez, B and Hoban, MD and Masiuk, KE and Clark, DN and Long, J and Sanchez, JM and Velez, M and Miyahira, E and Zhang, R and Brown, D and Wang, X and Kurmangaliyev, YZ and Hollis, RP and Kohn, DB},
title = {Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1389-1406},
pmid = {31178391},
issn = {1525-0024},
support = {R25 GM055052/GM/NIGMS NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*genetics/metabolism/therapy ; CRISPR-Cas Systems ; Dependovirus ; Endonucleases/genetics ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Therapy ; Genetic Vectors/genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; *Mutation ; Parvovirinae/genetics ; Tissue Donors ; Transduction, Genetic ; Zinc Finger Nucleases/genetics ; beta-Globins/*genetics ; },
abstract = {Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.},
}
@article {pmid31178131,
year = {2019},
author = {Chen, ZJ and Rong, L and Huang, D and Jiang, Q},
title = {Targeting cullin 3 by miR-601 activates Nrf2 signaling to protect retinal pigment epithelium cells from hydrogen peroxide.},
journal = {Biochemical and biophysical research communications},
volume = {515},
number = {4},
pages = {679-687},
doi = {10.1016/j.bbrc.2019.05.171},
pmid = {31178131},
issn = {1090-2104},
mesh = {Apoptosis ; CRISPR-Cas Systems ; Cell Line ; Cell Survival ; Cullin Proteins/*metabolism ; Humans ; Hydrogen Peroxide/*pharmacology ; Kelch-Like ECH-Associated Protein 1/metabolism ; Lipid Peroxidation ; MicroRNAs/*metabolism ; NF-E2-Related Factor 2/*metabolism ; Oxidative Stress ; Reactive Oxygen Species/metabolism ; Retinal Pigment Epithelium/*metabolism ; Signal Transduction ; Superoxides/metabolism ; },
abstract = {Activation of Nrf2 cascade can protect retinal pigment epithelium (RPE) cells from hydrogen peroxide (H2O2) and other oxidative injury. The current study identified microRNA-601 (miR-601) as a novel cullin 3 (Cul3)-targeting miRNA that activates Nrf2 cascade. In ARPE-19 cells and primary human RPE cells, forced overexpression of miR-601 significantly inhibited Cul3 3'-UTR activity and downregulated Cul3 mRNA/protein expression, leading to Nrf2 protein stabilization and its nuclear translocation as well as expression of anti-oxidant response elements (ARE)-dependent genes (HO1, NQO1 and GCLC). H2O2 treatment increased miR-601 levels in RPE cells. Significantly, ectopic miR-601 overexpression attenuated H2O2-induced oxidative injury and apoptosis in RPE cells. In contrast, miR-601 inhibition promoted Cul3 expression, lowered basal Nrf2 activation, and enhanced H2O2-induced oxidative stress and apoptosis in RPE cells. In ARPE-19 cells, CRISPC/Cas9-mediated knockout (KO) of Cul3 or Keap1 not only mimicked, but also nullified, miR-601-inudced anti-H2O2 actions. Furthermore, Nrf2 silencing by targeted shRNAs abolished miR-601-inudced cytoprotection in H2O2-treated ARPE-19 cells. Taken together, we show that miR-601 activates Nrf2 signaling to protect RPE cells from H2O2 by targeting Cul3.},
}
@article {pmid31178050,
year = {2019},
author = {Huang, L and Dong, H and Zheng, J and Wang, B and Pan, L},
title = {Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion.},
journal = {Microbiological research},
volume = {223-225},
number = {},
pages = {44-50},
doi = {10.1016/j.micres.2019.03.007},
pmid = {31178050},
issn = {1618-0623},
mesh = {Aspergillus niger/enzymology/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; Cytidine Deaminase/*genetics ; Deoxyribonuclease I/genetics ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genes, Fungal/genetics ; Mutagenesis ; },
abstract = {Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.},
}
@article {pmid31177615,
year = {2019},
author = {Williams, MA and O'Grady, J and Ball, B and Carlsson, J and de Eyto, E and McGinnity, P and Jennings, E and Regan, F and Parle-McDermott, A},
title = {The application of CRISPR-Cas for single species identification from environmental DNA.},
journal = {Molecular ecology resources},
volume = {19},
number = {5},
pages = {1106-1114},
doi = {10.1111/1755-0998.13045},
pmid = {31177615},
issn = {1755-0998},
mesh = {Animals ; *Biota ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Environmental/analysis/*genetics ; Gene Editing/*methods ; Nucleic Acid Amplification Techniques/*methods ; Salmo salar/*classification/*genetics ; Sensitivity and Specificity ; Temperature ; },
abstract = {We report the first application of CRISPR-Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR-based techniques to detect a given species from eDNA samples, posing a logistical challenge for on-site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR-Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof-of-concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.},
}
@article {pmid31175856,
year = {2019},
author = {Qu, D and Lu, S and Wang, P and Jiang, M and Yi, S and Han, J},
title = {Analysis of CRISPR/Cas system of Proteus and the factors affected the functional mechanism.},
journal = {Life sciences},
volume = {231},
number = {},
pages = {116531},
doi = {10.1016/j.lfs.2019.06.006},
pmid = {31175856},
issn = {1879-0631},
mesh = {Bacteria/metabolism ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; Databases, Genetic ; Plasmids/genetics ; Proteus/*genetics/*metabolism ; RNA/genetics ; },
abstract = {BACKGROUND: The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion.<br><br>OBJECTIVES: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system.<br><br>METHODS: CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed.<br><br>RESULTS: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids.<br><br>CONCLUSIONS: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.},
}
@article {pmid31175335,
year = {2019},
author = {},
title = {Clever chip designs for diagnostics.},
journal = {Nature biomedical engineering},
volume = {3},
number = {6},
pages = {417-418},
doi = {10.1038/s41551-019-0418-z},
pmid = {31175335},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Graphite ; Macrophages ; Oligonucleotide Array Sequence Analysis ; },
}
@article {pmid31175332,
year = {2019},
author = {Bruch, R and Urban, GA and Dincer, C},
title = {Unamplified gene sensing via Cas9 on graphene.},
journal = {Nature biomedical engineering},
volume = {3},
number = {6},
pages = {419-420},
doi = {10.1038/s41551-019-0413-4},
pmid = {31175332},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Graphite ; },
}
@article {pmid31173713,
year = {2019},
author = {Roper, J and Yilmaz, &#xd6;H},
title = {Breakthrough Moments: Genome Editing and Organoids.},
journal = {Cell stem cell},
volume = {24},
number = {6},
pages = {841-842},
doi = {10.1016/j.stem.2019.05.008},
pmid = {31173713},
issn = {1875-9777},
support = {P30 CA014051/CA/NCI NIH HHS/United States ; R01 CA211184/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Diseases, Inborn/genetics/*pathology ; Genetic Therapy/*methods ; Humans ; Organoids/*pathology/physiology ; },
abstract = {Six years ago, Schwank et al. (2013) adapted CRISPR-Cas9 and organoid technology to repair genetic diseases in patient-derived tissues. We shine a spotlight on how this work has inspired the development of tools to study and correct genetic diseases in experimental systems, with the ultimate goal of treating human disease.},
}
@article {pmid31173076,
year = {2020},
author = {Tsai, SY and Ghazizadeh, Z and Wang, HJ and Amin, S and Ortega, FA and Badieyan, ZS and Hsu, ZT and Gordillo, M and Kumar, R and Christini, DJ and Evans, T and Chen, S},
title = {A human embryonic stem cell reporter line for monitoring chemical-induced cardiotoxicity.},
journal = {Cardiovascular research},
volume = {116},
number = {3},
pages = {658-670},
pmid = {31173076},
issn = {1755-3245},
support = {R35 HL135778/HL/NHLBI NIH HHS/United States ; },
mesh = {Action Potentials/drug effects ; Arrhythmias, Cardiac/*chemically induced/metabolism/physiopathology ; Biomarkers/metabolism ; CRISPR-Cas Systems ; Cardiac Myosins/genetics ; Cardiotoxicity ; *Cell Differentiation ; Cell Line ; Doxorubicin/*toxicity ; Gene Knock-In Techniques ; Genes, Reporter ; Heart Diseases/*chemically induced/genetics/metabolism/pathology ; Human Embryonic Stem Cells/*drug effects/metabolism/pathology ; Humans ; Luminescent Proteins/biosynthesis/genetics ; Myocytes, Cardiac/*drug effects/metabolism/pathology ; Myosin Heavy Chains/genetics ; Oleic Acid/*toxicity ; Time Factors ; },
abstract = {AIMS: Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs.<br><br>METHODS AND RESULTS: We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20&#x2009;days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia.<br><br>CONCLUSION: We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.},
}
@article {pmid31172498,
year = {2019},
author = {Sweeney, CL and Merling, RK and De Ravin, SS and Choi, U and Malech, HL},
title = {Gene Editing in Chronic Granulomatous Disease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1982},
number = {},
pages = {623-665},
doi = {10.1007/978-1-4939-9424-3_36},
pmid = {31172498},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics/immunology ; Cell Line ; Cells, Cultured ; Cloning, Molecular ; *Gene Editing/methods ; Gene Order ; Gene Targeting ; Genetic Vectors ; Granulomatous Disease, Chronic/*genetics/metabolism ; Hematopoietic Stem Cells/cytology/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Neutrophils/cytology/immunology/metabolism ; RNA, Guide ; Reactive Oxygen Species/metabolism ; },
abstract = {Chronic granulomatous disease (CGD) is an immune deficiency characterized by defects in the production of microbicidal reactive oxygen species (ROS) by the phagocytic oxidase (phox) enzyme complex in neutrophils. We have previously described targeted gene editing strategies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases for gene targeting with homology-directed repair in CGD patient stem cells to achieve functional restoration of expression of phox genes and NADPH oxidase activity in differentiated neutrophils. In this chapter, we describe detailed protocols for targeted gene editing in human-induced pluripotent stem cells and hematopoietic stem cells and for subsequent differentiation of these stem cells into mature neutrophils, as well as assays to characterize neutrophil identity and function including flow cytometry analysis of neutrophil surface markers, intracellular staining for phox proteins, and analysis of ROS generation.},
}
@article {pmid31172397,
year = {2019},
author = {Campbell, LA and Richie, CT and Maggirwar, NS and Harvey, BK},
title = {Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {4},
pages = {565-577},
pmid = {31172397},
issn = {1557-1904},
support = {Intramural Research Program/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/administration &amp; dosage/*genetics/metabolism ; CRISPR-Cas Systems/*physiology ; Gene Editing/*methods/trends ; *Gene Transfer Techniques/trends ; Humans ; Inflammation/genetics/metabolism/therapy ; Ribonucleoproteins/administration &amp; dosage/*genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.},
}
@article {pmid31171720,
year = {2019},
author = {Jin, UH and Karki, K and Cheng, Y and Michelhaugh, SK and Mittal, S and Safe, S},
title = {The aryl hydrocarbon receptor is a tumor suppressor-like gene in glioblastoma.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {29},
pages = {11342-11353},
pmid = {31171720},
issn = {1083-351X},
support = {P30 ES023512/ES/NIEHS NIH HHS/United States ; R01 CA202697/CA/NCI NIH HHS/United States ; R01 ES025713/ES/NIEHS NIH HHS/United States ; T32 ES026568/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Brain Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Knockdown Techniques ; *Genes, Tumor Suppressor ; Glioblastoma/*genetics/pathology ; Heterografts ; Humans ; Kynurenine/metabolism ; Mice ; Mice, Nude ; Protein Binding ; Receptors, Aryl Hydrocarbon/*genetics/metabolism ; },
abstract = {The aryl hydrocarbon receptor (AhR) plays an important role in maintaining cellular homeostasis and also in pathophysiology. For example, the interplay between the gut microbiome and microbially derived AhR ligands protects against inflammation along the gut-brain axis. The AhR and its ligands also inhibit colon carcinogenesis, but it has been reported that the AhR and its ligand kynurenine enhance glioblastoma (GBM). In this study, using both established and patient-derived GBM cells, we re-examined the role of kynurenine and the AhR in GBM, observing that kynurenine does not modulate AhR-mediated gene expression and does not affect invasion of GBM cells. Therefore, using an array of approaches, including ChIP, quantitative real-time PCR, and cell migration assays, we primarily focused on investigating the role of the AhR in GBM at the functional molecular and genomic levels. The results of transient and stable CRISPR/Cas9-mediated AhR knockdown in GBM cells indicated that loss of AhR enhances GBM tumor growth in a mouse xenograft model, increases GBM cell invasion, and up-regulates expression of pro-invasion/pro-migration genes, as determined by ingenuity pathway analysis of RNA-Seq data. We conclude that the AhR is a tumor suppressor-like gene in GBM; future studies are required to investigate whether the AhR could be a potential drug target for treating patients with GBM who express this receptor.},
}
@article {pmid31171718,
year = {2019},
author = {Plateau, P and Moch, C and Blanquet, S},
title = {Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {29},
pages = {11311-11322},
pmid = {31171718},
issn = {1083-351X},
mesh = {Binding Sites ; *CRISPR-Cas Systems ; DNA, Bacterial/metabolism ; DNA, Superhelical/metabolism ; Escherichia coli/*drug effects/enzymology ; Escherichia coli Proteins/*metabolism ; Integrases/*metabolism ; Integration Host Factors/metabolism ; Spermidine/*pharmacology ; },
abstract = {Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.},
}
@article {pmid31171706,
year = {2019},
author = {Strecker, J and Ladha, A and Gardner, Z and Schmid-Burgk, JL and Makarova, KS and Koonin, EV and Zhang, F},
title = {RNA-guided DNA insertion with CRISPR-associated transposases.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6448},
pages = {48-53},
pmid = {31171706},
issn = {1095-9203},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Cyanobacteria/*enzymology/genetics ; *DNA Transposable Elements ; Gene Editing/*methods ; *Mutagenesis, Insertional ; *RNA, Guide ; Transposases/*chemistry/genetics/isolation &amp; purification ; },
abstract = {CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.},
}
@article {pmid31169010,
year = {2019},
author = {Aicale, R and Tarantino, D and Maccauro, G and Peretti, GM and Maffulli, N},
title = {Genetics in orthopaedic practice.},
journal = {Journal of biological regulators and homeostatic agents},
volume = {33},
number = {2 Suppl. 1},
pages = {103-117. XIX Congresso Nazionale S.I.C.O.O.P. Societa' Italiana Chirurghi Ortopedici Dell'ospedalita' Privata Accreditata},
pmid = {31169010},
issn = {0393-974X},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Profile ; Humans ; Musculoskeletal Diseases/*genetics ; Musculoskeletal System ; *Orthopedics ; Polymorphism, Single Nucleotide ; Tissue Engineering ; },
abstract = {DNA holds genetic information in the nucleus of eukaryotic cells; and has three different functions: replication, storage of hereditary information, and regulation of cell division. Most studies described the association of single nucleotide polymorphism (SNP) to common orthopaedics diseases and the susceptibility to develop musculoskeletal injuries. Several mutations are associated with osteoporosis, musculoskeletal ailments and other musculoskeletal deformity and conditions. Several strategies, including gene therapy and tissue engineering with mesenchymal stem cells (MSC), have been proposed to enhance healing of musculoskeletal tissues. Furthermore, a recent technique has revolutionized gene editing: clustered regulatory interspaced short palindromic repeat (CRISPR) technology is characterized by simplicity in target design, affordability, versatility, and high efficiency, but needs more studies to become the preferred platform for genome editing. Predictive genomics DNA profiling allows to understand which genetic advantage, if any, may be exploited, and why a given rehabilitation protocol can be more effective in some individual than others. In conclusion, a better understanding of the genetic influence on the function of the musculoskeletal system and healing of its ailments is needed to plan and develop patient specific management strategies.},
}
@article {pmid31168682,
year = {2020},
author = {Herfert, K and Mannheim, JG and Kuebler, L and Marciano, S and Amend, M and Parl, C and Napieczynska, H and Maier, FM and Vega, SC and Pichler, BJ},
title = {Quantitative Rodent Brain Receptor Imaging.},
journal = {Molecular imaging and biology},
volume = {22},
number = {2},
pages = {223-244},
pmid = {31168682},
issn = {1860-2002},
mesh = {Animals ; Biomarkers/metabolism ; Brain/*diagnostic imaging ; Brain Mapping/*methods ; CRISPR-Cas Systems ; Genetic Engineering ; Humans ; Magnetic Resonance Imaging/instrumentation/*methods ; Mice ; Neurotransmitter Agents/metabolism ; Positron-Emission Tomography/instrumentation/*methods ; Rats ; },
abstract = {Positron emission tomography (PET) is a non-invasive imaging technology employed to describe metabolic, physiological, and biochemical processes in vivo. These include receptor availability, metabolic changes, neurotransmitter release, and alterations of gene expression in the brain. Since the introduction of dedicated small-animal PET systems along with the development of many novel PET imaging probes, the number of PET studies using rats and mice in basic biomedical research tremendously increased over the last decade. This article reviews challenges and advances of quantitative rodent brain imaging to make the readers aware of its physical limitations, as well as to inspire them for its potential applications in preclinical research. In the first section, we briefly discuss the limitations of small-animal PET systems in terms of spatial resolution and sensitivity and point to possible improvements in detector development. In addition, different acquisition and post-processing methods used in rodent PET studies are summarized. We further discuss factors influencing the test-retest variability in small-animal PET studies, e.g., different receptor quantification methodologies which have been mainly translated from human to rodent receptor studies to determine the binding potential and changes of receptor availability and radioligand affinity. We further review different kinetic modeling approaches to obtain quantitative binding data in rodents and PET studies focusing on the quantification of endogenous neurotransmitter release using pharmacological interventions. While several studies have focused on the dopamine system due to the availability of several PET tracers which are sensitive to dopamine release, other neurotransmitter systems have become more and more into focus and are described in this review, as well. We further provide an overview of latest genome engineering technologies, including the CRISPR/Cas9 and DREADD systems that may advance our understanding of brain disorders and function and how imaging has been successfully applied to animal models of human brain disorders. Finally, we review the strengths and opportunities of simultaneous PET/magnetic resonance imaging systems to study drug-receptor interactions and challenges for the translation of PET results from bench to bedside.},
}
@article {pmid31167905,
year = {2019},
author = {Wang, C and Jiang, S and Zhang, L and Li, D and Liang, J and Narita, Y and Hou, I and Zhong, Q and Gewurz, BE and Teng, M and Zhao, B},
title = {TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity.},
journal = {Journal of virology},
volume = {93},
number = {16},
pages = {},
pmid = {31167905},
issn = {1098-5514},
support = {R35 CA047006/CA/NCI NIH HHS/United States ; R01 CA047006/CA/NCI NIH HHS/United States ; R01 AI123420/AI/NIAID NIH HHS/United States ; R01 AI137337/AI/NIAID NIH HHS/United States ; P30 CA076292/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/genetics ; Enhancer Elements, Genetic ; Epstein-Barr Virus Infections/*virology ; Gene Editing ; Gene Expression ; *Gene Expression Regulation, Viral ; Gene Knockout Techniques ; Genes, myc ; Herpesvirus 4, Human/*physiology ; Histones/metabolism ; Host-Pathogen Interactions ; Humans ; MEF2 Transcription Factors/genetics/metabolism ; TATA-Binding Protein Associated Factors/*metabolism ; },
abstract = {Super-enhancers (SEs) are clusters of enhancers marked by extraordinarily high and broad chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-κB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESE function, MYC ESEs were cloned upstream of a green fluorescent protein (GFP) reporter. Reporters driven by MYC ESEs 525 kb and 428 kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE-driven luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TBP-associated factor (TAF) family proteins, including TAF8, TAF11, and TAF3, were essential for the activity of the integrated 525ESE-driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout and restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes, including the ETS1 and BCL2 genes. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth.IMPORTANCE SEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not simian virus 40 (SV40) enhancer. These proteins not only were essential for the reporter activity but also were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.},
}
@article {pmid31167792,
year = {2019},
author = {Lessard, CB and Rodriguez, E and Ladd, TB and Minter, LM and Osborne, BA and Miele, L and Golde, TE and Ran, Y},
title = {Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {29},
pages = {11276-11285},
pmid = {31167792},
issn = {1083-351X},
support = {P01 CA166009/CA/NCI NIH HHS/United States ; U54 GM104940/GM/NIGMS NIH HHS/United States ; },
mesh = {Alzheimer Disease/genetics ; Amyloid Precursor Protein Secretases/metabolism ; Animals ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Hydrolysis ; Mice ; Presenilin-1/genetics/*physiology ; Presenilin-2/genetics/*physiology ; Substrate Specificity ; },
abstract = {Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.},
}
@article {pmid31167791,
year = {2019},
author = {McGuire, CM and Collins, MP and Sun-Wada, G and Wada, Y and Forgac, M},
title = {Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {29},
pages = {11248-11258},
pmid = {31167791},
issn = {1083-351X},
support = {R01 GM034478/GM/NIGMS NIH HHS/United States ; },
mesh = {Breast Neoplasms/*enzymology/genetics/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Membrane/enzymology ; Enzyme Inhibitors/pharmacology ; Female ; Humans ; Isoenzymes/antagonists &amp; inhibitors/genetics/*metabolism ; *Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger/genetics ; Vacuolar Proton-Translocating ATPases/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {The vacuolar H[+]-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.},
}
@article {pmid31167143,
year = {2019},
author = {Lopez-Martinez, D and Kupculak, M and Yang, D and Yoshikawa, Y and Liang, CC and Wu, R and Gygi, SP and Cohn, MA},
title = {Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage.},
journal = {Cell reports},
volume = {27},
number = {10},
pages = {2990-3005.e5},
pmid = {31167143},
issn = {2211-1247},
support = {MR/N021002/1/MRC_/Medical Research Council/United Kingdom ; 210640/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; R01 GM067945/GM/NIGMS NIH HHS/United States ; /CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Casein Kinase II/metabolism ; DNA/metabolism ; DNA Damage ; DNA Repair ; Fanconi Anemia/metabolism/pathology ; Fanconi Anemia Complementation Group D2 Protein/chemistry/genetics/*metabolism ; Fanconi Anemia Complementation Group Proteins/*metabolism ; HeLa Cells ; Humans ; Phosphorylation ; Protein Binding ; Protein Structure, Quaternary ; RNA, Guide/metabolism ; Sequence Alignment ; Ubiquitination ; },
abstract = {Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.},
}
@article {pmid31166175,
year = {2019},
author = {Liao, M and Tong, T and Zong, Y and Zhou, X and Cheng, L and Huang, R and Ren, B and Alterovitz, G},
title = {Application of Omics and Bioinformatics Tools in Streptococcus Research.},
journal = {Current issues in molecular biology},
volume = {32},
number = {},
pages = {327-376},
doi = {10.21775/cimb.032.327},
pmid = {31166175},
issn = {1467-3045},
mesh = {Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Chromosome Mapping ; Computational Biology/*methods ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Multiple, Bacterial/*genetics ; *Gene Expression Regulation, Bacterial ; Gene Transfer, Horizontal ; *Genome, Bacterial ; High-Throughput Nucleotide Sequencing ; Host-Pathogen Interactions/genetics ; Humans ; Phylogeny ; Streptococcal Infections/drug therapy/microbiology/pathology ; Streptococcus/classification/drug effects/*genetics/pathogenicity ; Virulence ; },
abstract = {Researchers used to focus on analyzing single gene or protein expression of the microbes. But recently, genome, transcriptome, proteome and metabolome have gained more and more attention. Based on technologies of omics, including genomics, transcriptomics and metabolomics, a large quantity of information about cells, microbes and human, such as the information about phylogeny, virulence, antibiotic resistance and other aspects, has been revealed. Genus Streptococcus is one of the most invasive groups of bacteria that cause both human and animal diseases, threatening public health. In this review, we summarize the application of omics to analyze this genus-Streptococcus.},
}
@article {pmid31294157,
year = {2017},
author = {Abedon, ST},
title = {Phage "delay" towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages.},
journal = {AIMS microbiology},
volume = {3},
number = {2},
pages = {186-226},
pmid = {31294157},
issn = {2471-1888},
abstract = {In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their "vigor" when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such "less-robust" mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically "running away" to more phage-free locations. Delay mechanisms-to the extent that they are less specific in terms of what phages are targeted-collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.},
}
@article {pmid31166168,
year = {2019},
author = {Gong, T and Lu, M and Zhou, X and Zhang, A and Tang, B and Chen, J and Jing, M and Li, Y},
title = {CRISPR-Cas Systems in Streptococci.},
journal = {Current issues in molecular biology},
volume = {32},
number = {},
pages = {1-38},
doi = {10.21775/cimb.032.001},
pmid = {31166168},
issn = {1467-3045},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Conjugation, Genetic ; Gene Editing/methods ; *Gene Expression Regulation, Bacterial ; Gene Transfer, Horizontal ; Genetic Therapy/methods ; Genome, Bacterial ; Humans ; Interspersed Repetitive Sequences ; Isoenzymes/genetics/metabolism ; RNA, Circular/genetics/metabolism ; RNA, Guide/*genetics/metabolism ; Streptococcus/*genetics/immunology/virology ; Streptococcus Phages/*genetics/metabolism ; },
abstract = {Streptococci are one of the most important and common constituents of the host's microbiota and can colonize and live in the upper respiratory and urogenital tract of humans and animals. The CRISPR-Cas systems (i.e., clustered regularly interspaced short palindromic repeat, with CRISPR-associated proteins) found in bacteria and archaea provide sequence-based adaptive immunity against mobile genetic elements, especially in the streptococci. Here, recent research progress on CRISPR-Cas systems in the streptococci is reviewed, including their classification (mainly type I, type II, and type III), physiological function, defense mechanism (CRISPR adaptation, crRNA biogenesis, and target interference) and applications, which are useful for a better understanding of the functions of such systems. Finally, the advances that have been made in streptococci may help in the discovery of further novel CRISPR-Cas systems for use in new technologies and applications in other species.},
}
@article {pmid31166114,
year = {2019},
author = {Garmendia, I and Pajares, MJ and Hermida-Prado, F and Ajona, D and Bértolo, C and Sainz, C and Lavín, A and Remírez, AB and Valencia, K and Moreno, H and Ferrer, I and Behrens, C and Cuadrado, M and Paz-Ares, L and Bustelo, XR and Gil-Bazo, I and Alameda, D and Lecanda, F and Calvo, A and Felip, E and Sánchez-Céspedes, M and Wistuba, II and Granda-Diaz, R and Rodrigo, JP and García-Pedrero, JM and Pio, R and Montuenga, LM and Agorreta, J},
title = {YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.},
journal = {American journal of respiratory and critical care medicine},
volume = {200},
number = {7},
pages = {888-899},
doi = {10.1164/rccm.201807-1292OC},
pmid = {31166114},
issn = {1535-4970},
mesh = {A549 Cells ; Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/drug therapy/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects/*genetics ; Dasatinib/*pharmacology/therapeutic use ; Gene Amplification ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Lung Neoplasms/drug therapy/*genetics ; Mice ; Prognosis ; Proto-Oncogene Mas ; Proto-Oncogene Proteins c-yes/antagonists &amp; inhibitors/*genetics ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.},
}
@article {pmid31165880,
year = {2019},
author = {Hardigan, AA and Roberts, BS and Moore, DE and Ramaker, RC and Jones, AL and Myers, RM},
title = {CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries.},
journal = {Nucleic acids research},
volume = {47},
number = {14},
pages = {e84},
pmid = {31165880},
issn = {1362-4962},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Gene Expression Regulation ; *Gene Library ; High-Throughput Nucleotide Sequencing/methods ; Humans ; MicroRNAs/genetics ; Models, Genetic ; Nucleic Acid Hybridization/*methods ; RNA, Ribosomal/genetics ; RNA, Small Untranslated/*genetics ; Sequence Analysis, RNA/methods ; },
abstract = {In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9-based Depletion of Abundant Species by Hybridization ('DASH') to smRNA-seq, which we have termed miRNA and Adapter Dimer-DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.},
}
@article {pmid31165872,
year = {2019},
author = {Huang, H and Kong, W and Jean, M and Fiches, G and Zhou, D and Hayashi, T and Que, J and Santoso, N and Zhu, J},
title = {A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG).},
journal = {Nucleic acids research},
volume = {47},
number = {14},
pages = {7333-7347},
pmid = {31165872},
issn = {1362-4962},
support = {R01 DE025447/DE/NIDCR NIH HHS/United States ; R01 GM117838/GM/NIGMS NIH HHS/United States ; R33 AI116180/AI/NIAID NIH HHS/United States ; },
mesh = {Aminopyridines/pharmacology ; CD4-Positive T-Lymphocytes/drug effects/metabolism/virology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Demethylation/drug effects ; Dioxygenases/antagonists &amp; inhibitors/*genetics/metabolism ; Gene Expression Regulation, Viral/drug effects ; HEK293 Cells ; HIV Infections/*genetics/metabolism/virology ; HIV-1/drug effects/*genetics/physiology ; Histone Deacetylase Inhibitors/pharmacology ; Histone Demethylases/antagonists &amp; inhibitors/*genetics/metabolism ; Histones/metabolism ; Host-Pathogen Interactions/drug effects/genetics ; Humans ; Hydrazones/pharmacology ; Methylation/drug effects ; Nuclear Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; RNA Interference ; Virus Latency/*genetics ; },
abstract = {Although combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53's function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.},
}
@article {pmid31165867,
year = {2019},
author = {Chen, W and McKenna, A and Schreiber, J and Haeussler, M and Yin, Y and Agarwal, V and Noble, WS and Shendure, J},
title = {Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair.},
journal = {Nucleic acids research},
volume = {47},
number = {15},
pages = {7989-8003},
pmid = {31165867},
issn = {1362-4962},
support = {R00 HG010152/HG/NHGRI NIH HHS/United States ; U01 HG009395/HG/NHGRI NIH HHS/United States ; U41 HG002371/HG/NHGRI NIH HHS/United States ; K99 HG010152/HG/NHGRI NIH HHS/United States ; R01 HG009136/HG/NHGRI NIH HHS/United States ; UM1 HG009408/HG/NHGRI NIH HHS/United States ; },
mesh = {Base Sequence ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; DNA Cleavage ; *DNA End-Joining Repair ; Gene Editing ; Genome, Human/genetics ; Humans ; Models, Genetic ; *Mutation ; Sequence Deletion ; },
abstract = {Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.},
}
@article {pmid31165781,
year = {2019},
author = {Faure, G and Shmakov, SA and Yan, WX and Cheng, DR and Scott, DA and Peters, JE and Makarova, KS and Koonin, EV},
title = {CRISPR-Cas in mobile genetic elements: counter-defence and beyond.},
journal = {Nature reviews. Microbiology},
volume = {17},
number = {8},
pages = {513-525},
pmid = {31165781},
issn = {1740-1534},
mesh = {Archaea/genetics ; Bacteria/genetics ; Bacteriophages/genetics ; *CRISPR-Cas Systems ; DNA Transposable Elements ; *Evolution, Molecular ; *Gene Transfer, Horizontal ; *Interspersed Repetitive Sequences ; Plasmids ; *Recombination, Genetic ; },
abstract = {The principal function of CRISPR-Cas systems in archaea and bacteria is defence against mobile genetic elements (MGEs), including viruses, plasmids and transposons. However, the relationships between CRISPR-Cas and MGEs are far more complex. Several classes of MGE contributed to the origin and evolution of CRISPR-Cas, and, conversely, CRISPR-Cas systems and their components were recruited by various MGEs for functions that remain largely uncharacterized. In this Analysis article, we investigate and substantially expand the range of CRISPR-Cas components carried by MGEs. Three groups of Tn7-like transposable elements encode 'minimal' type I CRISPR-Cas derivatives capable of target recognition but not cleavage, and another group encodes an inactivated type V variant. These partially inactivated CRISPR-Cas variants might mediate guide RNA-dependent integration of the respective transposons. Numerous plasmids and some prophages encode type IV systems, with similar predicted properties, that appear to contribute to competition among plasmids and between plasmids and viruses. Many prokaryotic viruses also carry CRISPR mini-arrays, some of which recognize other viruses and are implicated in inter-virus conflicts, and solitary repeat units, which could inhibit host CRISPR-Cas systems.},
}
@article {pmid31165703,
year = {2019},
author = {Lu, S and Yang, N and He, J and Gong, W and Lai, Z and Xie, L and Tao, L and Xu, C and Wang, H and Zhang, G and Cao, H and Zhou, C and Zhong, L and Zhao, Y},
title = {Generation of Cancer-Specific Cytotoxic PD-1[-] T Cells Using Liposome-Encapsulated CRISPR/Cas System with Dendritic/Tumor Fusion Cells.},
journal = {Journal of biomedical nanotechnology},
volume = {15},
number = {3},
pages = {593-601},
doi = {10.1166/jbn.2019.2712},
pmid = {31165703},
issn = {1550-7033},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Liposomes ; Mice ; *Neoplasms ; Programmed Cell Death 1 Receptor ; T-Lymphocytes ; },
abstract = {T-cell immunotherapy is showing great promise and therefore undergoing intensive developments for cancer treatment. In this study, we applied liposome-encapsulated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) genome editing tool to specifically knock out the programmed death-1 (PD-1) gene from T cells (PD-1[-] T cells). We then activated these cells by dendritic/tumor fusion cells (FCs) and examined their anti-cancer potential. Results showed that, following the antigen presentation and activation by DC/HepG2 FCs, PD-1[-] T cells showed a significantly higher ability than PD-1[+] T cells to proliferate, secrete pro-inflammatory cytokine IFN-γ, and kill HepG2 cells in vitro. Consistently, in vitro activated PD-1[-] T cells inhibited proliferation and induced apoptosis in HepG2 xenografts in vivo, leading to significantly suppressed tumor growth and improved mouse survival. Liposome-encapsulated CRISPR/Cas9 genome editing technology effectively knocked out PD-1 gene in T cells, stimulating T cell activation in response to DC/tumor FCs and affording T cell-mediated cancer immunotherapy. Our study provides evidence to target checkpoint receptors in adoptively transfected T cells, as a novel therapeutic modality for adoptive T cell transfer.},
}
@article {pmid31165372,
year = {2019},
author = {Wang, W and Hou, J and Zheng, N and Wang, X and Zhang, J},
title = {Keeping our eyes on CRISPR: the "Atlas" of gene editing.},
journal = {Cell biology and toxicology},
volume = {35},
number = {4},
pages = {285-288},
doi = {10.1007/s10565-019-09480-w},
pmid = {31165372},
issn = {1573-6822},
mesh = {Acidaminococcus/genetics ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endodeoxyribonucleases/genetics ; Gene Editing/*ethics/methods ; Genome/genetics ; Humans ; Leptotrichia/genetics ; RNA, Guide/genetics ; },
}
@article {pmid31164740,
year = {2019},
author = {Martin-Laffon, J and Kuntz, M and Ricroch, AE},
title = {Worldwide CRISPR patent landscape shows strong geographical biases.},
journal = {Nature biotechnology},
volume = {37},
number = {6},
pages = {613-620},
doi = {10.1038/s41587-019-0138-7},
pmid = {31164740},
issn = {1546-1696},
mesh = {Bias ; CRISPR-Cas Systems/*genetics ; China ; Europe ; Geography/*statistics &amp; numerical data ; Humans ; *Patents as Topic ; United States ; },
}
@article {pmid31164571,
year = {2018},
author = {Nussbaum, L and Telenius, JM and Hill, S and Hirschfeld, PP and Suciu, MC and , and Downes, DJ and Hughes, JR},
title = {High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates.},
journal = {Methods and protocols},
volume = {1},
number = {3},
pages = {},
pmid = {31164571},
issn = {2409-9279},
support = {MC_UU_00016/14/MRC_/Medical Research Council/United Kingdom ; MC_UU_12009/15/MRC_/Medical Research Council/United Kingdom ; 106130/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; 4050189188/MRC_/Medical Research Council/United Kingdom ; },
abstract = {The emergence in recent years of DNA editing technologies-Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors-have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.},
}
@article {pmid31163979,
year = {2019},
author = {Jaffré, F and Miller, CL and Schänzer, A and Evans, T and Roberts, AE and Hahn, A and Kontaridis, MI},
title = {Inducible Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Aberrant Extracellular Regulated Kinase 5 and Mitogen-Activated Protein Kinase Kinase 1/2 Signaling Concomitantly Promote Hypertrophic Cardiomyopathy in RAF1-Associated Noonan Syndrome.},
journal = {Circulation},
volume = {140},
number = {3},
pages = {207-224},
pmid = {31163979},
issn = {1524-4539},
support = {R01 HL114775/HL/NHLBI NIH HHS/United States ; R01 HL102368/HL/NHLBI NIH HHS/United States ; R35 HL135778/HL/NHLBI NIH HHS/United States ; R00 HL125912/HL/NHLBI NIH HHS/United States ; R01 HL122238/HL/NHLBI NIH HHS/United States ; },
mesh = {Adolescent ; CRISPR-Cas Systems/physiology ; Cardiomyopathy, Hypertrophic/*genetics/metabolism ; Cells, Cultured ; Child ; Female ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*physiology ; MAP Kinase Kinase 1/*genetics/metabolism ; MAP Kinase Kinase 2/*genetics/metabolism ; Male ; Mitogen-Activated Protein Kinase 7/*genetics/metabolism ; Myocytes, Cardiac/physiology ; Noonan Syndrome/*genetics/metabolism ; Proto-Oncogene Proteins c-raf/*genetics/metabolism ; },
abstract = {BACKGROUND: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM.<br><br>METHODS: We used patient-derived RAF1[S257L/+] and CRISPR-Cas9-generated isogenic control inducible pluripotent stem cell (iPSC)-derived cardiomyocytes to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease.<br><br>RESULTS: We show that mutant iPSC-derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC-derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC-derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM.<br><br>CONCLUSIONS: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders.},
}
@article {pmid31162951,
year = {2019},
author = {Wang, Y and Kuai, Q and Gao, F and Wang, Y and He, M and Zhou, H and Han, G and Jiang, X and Ren, S and Yu, Q},
title = {Overexpression of TIM-3 in Macrophages Aggravates Pathogenesis of Pulmonary Fibrosis in Mice.},
journal = {American journal of respiratory cell and molecular biology},
volume = {61},
number = {6},
pages = {727-736},
doi = {10.1165/rcmb.2019-0070OC},
pmid = {31162951},
issn = {1535-4989},
mesh = {Adoptive Transfer ; Animals ; Bleomycin/toxicity ; CRISPR-Cas Systems ; Disease Models, Animal ; Hepatitis A Virus Cellular Receptor 2/*blood/deficiency/genetics/*physiology ; Humans ; Idiopathic Pulmonary Fibrosis/chemically induced/genetics/*pathology ; Interleukin-10/biosynthesis ; Lung/pathology ; Macrophages, Alveolar/drug effects/*metabolism/transplantation ; Macrophages, Peritoneal/metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; RAW 264.7 Cells ; RNA, Messenger/biosynthesis ; Transforming Growth Factor beta1/biosynthesis ; },
abstract = {Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder and lacks effective treatments because of unclear mechanisms. Aberrant function of alveolar macrophages is directly linked to pulmonary fibrosis. Here, we show TIM-3 (T-cell immunoglobulin domain and mucin domain-3), a key regulator of macrophage function, aggravates pulmonary fibrosis. TIM-3 mRNA of patients with IPF was analyzed based on the Gene Expression Omnibus and Array Express databases. Lung pathology and profibrotic molecules were assessed in a bleomycin (BLM)-induced pulmonary fibrosis model using wild-type (WT) and TIM-3 transgenic (TIM-3-TG) mice. Macrophage cells, RAW264.7, were then applied to investigate the effect of macrophage TIM-3 under BLM exposure in vitro. Macrophage depletion and adoptive-transfer experiments were finally performed to examine lung morphology and profibrotic molecules. TIM-3 expression was increased both in patients with IPF and in our BLM-induced mouse model. TIM-3-TG mice developed more serious pathological changes in lung tissue and higher expressions of TGF-β1 (transforming growth factor-β1) and IL-10 than WT mice. After BLM treatment, TGF-β1 and IL-10 expression was significantly decreased in RAW264.7 cells after TIM-3 knock-out, whereas it was increased in TIM-3-TG peritoneal macrophages. The scores of pulmonary fibrosis in WT and TIM-3-TG mice were significantly reduced, and there was no difference between them after macrophage depletion. Furthermore, WT mice receiving adoptive macrophages from TIM-3-TG mice also had more serious lung fibrosis and increased expression of TGF-β1 and IL-10 than those receiving macrophages from WT mice. Our findings revealed that overexpressed TIM-3 in alveolar macrophages aggravated pulmonary fibrosis.},
}
@article {pmid31161907,
year = {2019},
author = {Neal, S and de Jong, DM and Seaver, EC},
title = {CRISPR/CAS9 mutagenesis of a single r-opsin gene blocks phototaxis in a marine larva.},
journal = {Proceedings. Biological sciences},
volume = {286},
number = {1904},
pages = {20182491},
pmid = {31161907},
issn = {1471-2954},
mesh = {Animals ; Annelida/*genetics/physiology ; Aquatic Organisms/*genetics/physiology ; *CRISPR-Cas Systems ; Gene Editing ; Larva/genetics/physiology ; Mutagenesis, Site-Directed ; Opsins/*genetics ; Photoreceptor Cells, Invertebrate/metabolism/physiology ; *Phototaxis ; },
abstract = {Many marine animals depend upon a larval phase of their life cycle to locate suitable habitat, and larvae use light detection to influence swimming behaviour and dispersal. Light detection is mediated by the opsin genes, which encode light-sensitive transmembrane proteins. Previous studies suggest that r-opsins in the eyes mediate locomotory behaviour in marine protostomes, but few have provided direct evidence through gene mutagenesis. Larvae of the marine annelid Capitella teleta have simple eyespots and are positively phototactic, although the molecular components that mediate this behaviour are unknown. Here, we characterize the spatio-temporal expression of the rhabdomeric opsin genes in C. teleta and show that a single rhabdomeric opsin gene, Ct-r-opsin1, is expressed in the larval photoreceptor cells. To investigate its function, Ct-r-opsin1 was disrupted using CRISPR/CAS9 mutagenesis. Polymerase chain reaction amplification and DNA sequencing demonstrated efficient editing of the Ct-r-opsin1 locus. In addition, the pattern of Ct-r-opsin1 expression in photoreceptor cells was altered. Notably, there was a significant decrease in larval phototaxis, although the eyespot photoreceptor cell and associated pigment cell formed normally and persisted in Ct-r-opsin1-mutant animals. The loss of phototaxis owing to mutations in Ct-r-opsin1 is similar to that observed when the entire photoreceptor and pigment cell are deleted, demonstrating that a single r-opsin gene is sufficient to mediate phototaxis in C. teleta. These results establish the feasibility of gene editing in animals like C. teleta, and extend previous work on the development, evolution and function of the C. teleta visual system . Our study represents one example of disruption of animal behaviour by gene editing through CRISPR/CAS9 mutagenesis, and has broad implications for performing genome editing studies in a wide variety of other understudied animals.},
}
@article {pmid31161803,
year = {2019},
author = {Garcia-Leon, JA and Vitorica, J and Gutierrez, A},
title = {Use of human pluripotent stem cell-derived cells for neurodegenerative disease modeling and drug screening platform.},
journal = {Future medicinal chemistry},
volume = {11},
number = {11},
pages = {1305-1322},
doi = {10.4155/fmc-2018-0520},
pmid = {31161803},
issn = {1756-8927},
mesh = {Animals ; CRISPR-Cas Systems ; Drug Evaluation, Preclinical/*methods ; Gene Editing/methods ; Humans ; Neurodegenerative Diseases/*drug therapy/genetics/pathology ; Neurogenesis/drug effects ; Neurons/*cytology/drug effects/metabolism/pathology ; Pluripotent Stem Cells/*cytology/metabolism/pathology ; },
abstract = {Most neurodegenerative diseases are characterized by a complex and mostly still unresolved pathology. This fact, together with the lack of reliable disease models, has precluded the development of effective therapies counteracting the disease progression. The advent of human pluripotent stem cells has revolutionized the field allowing the generation of disease-relevant neural cell types that can be used for disease modeling, drug screening and, possibly, cell transplantation purposes. In this Review, we discuss the applications of human pluripotent stem cells, the development of efficient protocols for the derivation of the different neural cells and their applicability for robust in vitro disease modeling and drug screening platforms for most common neurodegenerative conditions.},
}
@article {pmid31161741,
year = {2019},
author = {Lv, J and Wu, S and Wei, R and Li, Y and Jin, J and Mu, Y and Zhang, Y and Kong, Q and Weng, X and Liu, Z},
title = {The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells.},
journal = {Journal of veterinary science},
volume = {20},
number = {3},
pages = {e23},
pmid = {31161741},
issn = {1976-555X},
support = {2016YFA0100200//National Key Research and Development Program of China/China ; 14QC06/NEAU/Northeast Agricultural University/China ; },
mesh = {Animals ; Base Pair Mismatch/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; *Gene Editing/standards ; Genes, erbB-1/genetics ; Nucleic Acid Heteroduplexes/chemistry/*genetics ; RNA, Guide/chemistry/*genetics ; Swine ; },
abstract = {The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.},
}
@article {pmid31161346,
year = {2019},
author = {Balboa, D and Prasad, RB and Groop, L and Otonkoski, T},
title = {Genome editing of human pancreatic beta cell models: problems, possibilities and outlook.},
journal = {Diabetologia},
volume = {62},
number = {8},
pages = {1329-1336},
pmid = {31161346},
issn = {1432-0428},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Diabetes Mellitus/*genetics/*therapy ; *Gene Editing/methods/trends ; Gene Silencing ; Genetic Variation ; *Genome, Human ; Genotype ; Humans ; Insulin-Secreting Cells/*metabolism ; Pluripotent Stem Cells ; Polymorphism, Single Nucleotide ; Risk ; },
abstract = {Understanding the molecular mechanisms behind beta cell dysfunction is essential for the development of effective and specific approaches for diabetes care and prevention. Physiological human beta cell models are needed for this work. We review the possibilities and limitations of currently available human beta cell models and how they can be dramatically enhanced using genome-editing technologies. In addition to the gold standard, primary isolated islets, other models now include immortalised human beta cell lines and pluripotent stem cell-derived islet-like cells. The scarcity of human primary islet samples limits their use, but valuable gene expression and functional data from large collections of human islets have been made available to the scientific community. The possibilities for studying beta cell physiology using immortalised human beta cell lines and stem cell-derived islets are rapidly evolving. However, the functional immaturity of these cells is still a significant limitation. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) has enabled precise engineering of specific genetic variants, targeted transcriptional modulation and genome-wide genetic screening. These approaches can now be exploited to gain understanding of the mechanisms behind coding and non-coding diabetes-associated genetic variants, allowing more precise evaluation of their contribution to diabetes pathogenesis. Despite all the progress, genome editing in primary pancreatic islets remains difficult to achieve, an important limitation requiring further technological development.},
}
@article {pmid31160638,
year = {2019},
author = {Tamura, M and Yonezawa, T and Liu, X and Asada, S and Hayashi, Y and Fukuyama, T and Tanaka, Y and Kitamura, T and Goyama, S},
title = {Opposing effects of acute versus chronic inhibition of p53 on decitabine's efficacy in myeloid neoplasms.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8171},
pmid = {31160638},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Decitabine/pharmacology ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Leukemia, Myeloid, Acute/*drug therapy/genetics/pathology ; Methyltransferases/*antagonists &amp; inhibitors/genetics ; Mice ; Myelodysplastic Syndromes/*drug therapy/genetics/pathology ; Repressor Proteins/genetics ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Decitabine is a DNA methyltransferase inhibitor and is considered a promising drug to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with p53 mutations. However, whether loss of p53 in fact increases the response of MDS/AML cells to decitabine remains unclear. In this study, we assessed the role of p53 in MDS and AML cells treated with decitabine using mouse models for MLL-AF9-driven AML and mutant ASXL1-driven MDS/AML. CRISPR/Cas9-mediated depletion of p53 in MDS/AML cells did not increase, but rather decreased their sensitivity to decitabine. Forced expression of a dominant-negative p53 fragment (p53DD) in these cells also decreased their responses to decitabine, confirming that acute inhibition of p53 conferred resistance to decitabine in AML and MDS/AML cells. In contrast, MLL-AF9-expressing AML cells generated from bone marrow progenitors of Trp53-deficient mice were more sensitive to decitabine in vivo than their wild-type counterparts, suggesting that long-term chronic p53 deficiency increases decitabine sensitivity in AML cells. Taken together, these data revealed a multifaceted role for p53 to regulate responses of myeloid neoplasms to decitabine treatment.},
}
@article {pmid31160565,
year = {2019},
author = {Bandopadhayay, P and Piccioni, F and O'Rourke, R and Ho, P and Gonzalez, EM and Buchan, G and Qian, K and Gionet, G and Girard, E and Coxon, M and Rees, MG and Brenan, L and Dubois, F and Shapira, O and Greenwald, NF and Pages, M and Balboni Iniguez, A and Paolella, BR and Meng, A and Sinai, C and Roti, G and Dharia, NV and Creech, A and Tanenbaum, B and Khadka, P and Tracy, A and Tiv, HL and Hong, AL and Coy, S and Rashid, R and Lin, JR and Cowley, GS and Lam, FC and Goodale, A and Lee, Y and Schoolcraft, K and Vazquez, F and Hahn, WC and Tsherniak, A and Bradner, JE and Yaffe, MB and Milde, T and Pfister, SM and Qi, J and Schenone, M and Carr, SA and Ligon, KL and Kieran, MW and Santagata, S and Olson, JM and Gokhale, PC and Jaffe, JD and Root, DE and Stegmaier, K and Johannessen, CM and Beroukhim, R},
title = {Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2400},
pmid = {31160565},
issn = {2041-1723},
support = {R35 CA210030/CA/NCI NIH HHS/United States ; P30 ES002109/ES/NIEHS NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; R00 CA201592/CA/NCI NIH HHS/United States ; R01 CA219943/CA/NCI NIH HHS/United States ; U54 CA225088/CA/NCI NIH HHS/United States ; R01 CA188228/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Azepines/*pharmacology ; Basic Helix-Loop-Helix Transcription Factors/drug effects/metabolism ; CRISPR-Cas Systems ; Cell Cycle/*drug effects ; Cell Cycle Proteins/drug effects/metabolism ; Cell Line, Tumor ; Cell Lineage ; Cerebellar Neoplasms/*drug therapy/genetics ; Cyclin D2/drug effects/metabolism ; Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors ; Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Humans ; Medulloblastoma/*drug therapy/genetics ; Mice ; Neural Stem Cells/drug effects/metabolism ; Neurogenesis/*drug effects ; Proteins/*antagonists &amp; inhibitors ; Proto-Oncogene Proteins c-myc/genetics ; S Phase/drug effects ; Triazoles/*pharmacology ; },
abstract = {BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.},
}
@article {pmid31160507,
year = {2019},
author = {Chen, X and Tian, X and Xue, L and Zhang, X and Yang, S and Traw, MB and Huang, J},
title = {CRISPR-Based Assessment of Gene Specialization in the Gibberellin Metabolic Pathway in Rice.},
journal = {Plant physiology},
volume = {180},
number = {4},
pages = {2091-2105},
pmid = {31160507},
issn = {1532-2548},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gibberellins/*metabolism ; Mutation/genetics ; Oryza/*genetics/*metabolism ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice (Oryza sativa). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility-suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1, OsKO2, and OsKO5 and neofunctionalization in OsKO3 and OsKO4 Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the "Green Revolution" in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants.},
}
@article {pmid31159834,
year = {2019},
author = {Zhan, H and Zhou, Q and Gao, Q and Li, J and Huang, W and Liu, Y},
title = {Multiplexed promoterless gene expression with CRISPReader.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {113},
pmid = {31159834},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus ; *Gene Expression ; *Genetic Techniques ; Luciferases, Renilla ; Mice ; *Peptide Chain Initiation, Translational ; *Transcriptional Activation ; },
abstract = {BACKGROUND: Genes are comprised of DNA codes and contain promoters and other control elements for reading these codes. The rapid development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has made possible the construction of a novel code-reading system with low dependency on the native control elements.<br><br>RESULTS: We develop CRISPReader, a technology for controlling promoterless gene expression in a robust fashion. We demonstrate that this tool is highly efficient in controlling transcription and translation initiation of a targeted transgene. A notable feature of CRISPReader is the ability to "read" the open reading frames of a cluster of gene without traditional regulatory elements or other cofactors. In particular, we use this strategy to construct an all-in-one AAV-CRISPR-Cas9 system by removing promoter-like elements from the expression cassette to resolve the existing AAV packaging size problem. The compact AAV-CRISPR-Cas9 is also more efficient in transactivation, DNA cleavage, and gene editing than the dual-AAV vector encoding two separate Cas9 elements, shown by targeting both reporter and endogenous genes in vitro and in vivo.<br><br>CONCLUSIONS: CRISPReader represents a novel approach for gene regulation that enables minimal gene constructs to be expressed and can be used in potential biomedical applications.},
}
@article {pmid31159719,
year = {2019},
author = {Li, T and Fang, Z and Peng, H and Zhou, J and Liu, P and Wang, Y and Zhu, W and Li, L and Zhang, Q and Chen, L and Li, L and Liu, Z and Zhang, W and Zhai, W and Lu, L and Gao, L},
title = {Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {444},
pmid = {31159719},
issn = {1471-2164},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Gene Transfer Techniques ; Genetic Engineering ; Genetic Testing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; *Microsatellite Repeats ; Oryza/*genetics ; Plant Proteins/antagonists &amp; inhibitors/*genetics ; Plants, Genetically Modified/*genetics ; Polymorphism, Genetic ; Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/*genetics ; },
abstract = {BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost.<br><br>RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats.<br><br>CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.},
}
@article {pmid31159699,
year = {2018},
author = {Feeney, O and Cockbain, J and Morrison, M and Diependaele, L and Van Assche, K and Sterckx, S},
title = {Patenting Foundational Technologies: Lessons From CRISPR and Other Core Biotechnologies.},
journal = {The American journal of bioethics : AJOB},
volume = {18},
number = {12},
pages = {36-48},
doi = {10.1080/15265161.2018.1531160},
pmid = {31159699},
issn = {1536-0075},
mesh = {Biotechnology/economics/ethics/*legislation &amp; jurisprudence ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Research/legislation &amp; jurisprudence ; Genetics, Medical/economics/ethics/*legislation &amp; jurisprudence ; Health Policy/legislation &amp; jurisprudence ; Humans ; Licensure/legislation &amp; jurisprudence ; Patents as Topic/ethics/*legislation &amp; jurisprudence ; Social Justice ; },
abstract = {In 2012, a new and promising gene manipulation technique, CRISPR-Cas9, was announced that seems likely to be a foundational technique in health care and agriculture. However, patents have been granted. As with other technological developments, there are concerns of social justice regarding inequalities in access. Given the technologies' "foundational" nature and societal impact, it is vital for such concerns to be translated into workable recommendations for policymakers and legislators. Colin Farrelly has proposed a moral justification for the use of patents to speed up the arrival of technology by encouraging innovation and investment. While sympathetic to his argument, this article highlights a number of problems. By examining the role of patents in CRISPR and in two previous foundational technologies, we make some recommendations for realistic and workable guidelines for patenting and licensing.},
}
@article {pmid31158410,
year = {2019},
author = {Josipović, G and Zoldoš, V and Vojta, A},
title = {Active fusions of Cas9 orthologs.},
journal = {Journal of biotechnology},
volume = {301},
number = {},
pages = {18-23},
doi = {10.1016/j.jbiotec.2019.05.306},
pmid = {31158410},
issn = {1873-4863},
mesh = {CRISPR-Cas Systems/*genetics ; DNA, Recombinant/*genetics ; Epigenomics/methods ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Fusion/*genetics ; },
abstract = {Many recent epigenetic studies utilize the advantages of CRISPR/dCas9 based tools in linking certain epigenetic modification with gene expression regulation. Various multifactorial diseases often contain changed epigenetic signatures at many loci, so tools for simultaneously targeting different loci would significantly facilitate the understanding of disease pathogenesis. We tested different dCas9 orthologs (dCjCas9, dNmCas9, dSt1Cas9, dFnCas9, dSaCas9 and dSpCas9) in C-terminal fusion with DNMT3A effector domain to find candidates that potentiate effector domain to perform its function at the target site. We demonstrated that nuclear localization signals (NLS) at both termini of fusion constructs is crucial for both proper nuclear import of such large constructs as well as for maximization of targeted DNA methylation activity. We identified SpCas9, SaCas9 and CjCas9 as potential candidates for the fusion constructs. With further optimization of the SaCas9 ortholog, due to less complex PAM requirements in contrast to CjCas9, we showed that N-terminal fusion with DNMT3A (dSaCas9-DNMT3A) is optimal to exert targeted DNA methylation activity comparable to the dSpCas9-DNMT3A construct. N-terminal fusions showed better results for both Cas9 orthologs, SaCas9 and SpCas9, so it can be used as universal approach for linking different effector domains in order to obtain highly active fusions.},
}
@article {pmid31156675,
year = {2019},
author = {Ren, F and Ren, C and Zhang, Z and Duan, W and Lecourieux, D and Li, S and Liang, Z},
title = {Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {612},
pmid = {31156675},
issn = {1664-462X},
abstract = {Clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system is an efficient targeted genome editing method. Although CRISPR/Cas9-mediated mutagenesis has been applied successfully in grape, few studies have examined the technique's efficiency. To optimize CRISPR/Cas9 editing efficiency in Vitis vinifera, we surveyed three key parameters: GC content of single guide RNA (sgRNA), variety of transformant cells used, and SpCas9 expression levels in transgenic cell mass. Four sgRNAs with differing GC content were designed to target exon sites of the V. vinifera phytoene desaturase gene. Suspension cells of 'Chardonnay' and '41B' varieties were used as the transgenic cell mass. Both T7EI and PCR/RE assays showed that CRISPR/Cas9 editing efficiency increases proportionally with sgRNA GC content with 65% GC content yielding highest editing efficiency in both varieties. Additionally, gene editing was more efficient in '41B' than in 'Chardonnay.' CRISPR/Cas9 systems with different editing efficiency showed different SpCas9 expression level, but compared with GC content of sgRNA, SpCas9 expression level has less influence on editing efficiency. Taken together, these results help optimize of CRISPR/Cas9 performance in grape.},
}
@article {pmid31155345,
year = {2019},
author = {Zhang, H and Li, Z and Daczkowski, CM and Gabel, C and Mesecar, AD and Chang, L},
title = {Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.},
journal = {Cell host &amp; microbe},
volume = {25},
number = {6},
pages = {815-826.e4},
doi = {10.1016/j.chom.2019.05.004},
pmid = {31155345},
issn = {1934-6069},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; Endodeoxyribonucleases/*antagonists &amp; inhibitors/*metabolism/ultrastructure ; Enzyme Inhibitors/*metabolism ; Protein Binding ; Protein Conformation ; RNA, Guide/*metabolism/ultrastructure ; Ribonucleases/*metabolism/ultrastructure ; },
abstract = {CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics.},
}
@article {pmid31155336,
year = {2019},
author = {Wang, P and Zhao, FJ and Kopittke, PM},
title = {Engineering Crops without Genome Integration Using Nanotechnology.},
journal = {Trends in plant science},
volume = {24},
number = {7},
pages = {574-577},
doi = {10.1016/j.tplants.2019.05.004},
pmid = {31155336},
issn = {1878-4372},
mesh = {*Biotechnology ; CRISPR-Cas Systems ; *Crops, Agricultural ; Genetic Engineering ; Genome, Plant ; Nanotechnology ; Plant Cells ; Plants, Genetically Modified ; },
abstract = {Nanomaterial-based delivery systems can deliver functional genes or siRNA into intact plant cells and create transgene-free genetically engineered plants. This system allows highly efficient and organelle-specific delivery that can overcome host-range limitations. This approach will have a diverse range of applications in plant biotechnology and plant biology.},
}
@article {pmid31153632,
year = {2019},
author = {Li, Y and Liu, L and Liu, G},
title = {CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?.},
journal = {Trends in biotechnology},
volume = {37},
number = {8},
pages = {792-795},
doi = {10.1016/j.tibtech.2019.04.012},
pmid = {31153632},
issn = {1879-3096},
mesh = {Biosensing Techniques/*methods/trends ; *CRISPR-Cas Systems ; Diagnostic Tests, Routine/*methods/trends ; Humans ; Molecular Diagnostic Techniques/*methods/trends ; },
abstract = {Performing multiplex detection is still an elusive goal for molecular diagnostics. CRISPR/Cas-based biosensing has demonstrated potential for multiplex detection. Instead of being an insurmountable obstacle, CRISPR/Cas multiplexed biosensing is a realistic challenge with some recent successful applications. Strategic considerations are required to fully explore its potential in multiplex diagnostics.},
}
@article {pmid31152927,
year = {2019},
author = {J, B and Das, A},
title = {An edible fungi Pleurotus ostreatus inhibits adipogenesis via suppressing expression of PPAR γ and C/EBP α in 3T3-L1 cells: In vitro validation of gene knock out of RNAs in PPAR γ using CRISPR spcas9.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {116},
number = {},
pages = {109030},
doi = {10.1016/j.biopha.2019.109030},
pmid = {31152927},
issn = {1950-6007},
mesh = {3T3-L1 Cells ; *Adipogenesis ; Animals ; Base Sequence ; CCAAT-Enhancer-Binding Protein-alpha/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; *Gene Knockout Techniques ; Humans ; Mice ; PPAR gamma/*metabolism ; Pleurotus/*chemistry ; RNA/*metabolism ; },
abstract = {OBJECTIVE: Obesity is now well recognized as a disorder, one that is essentially preventable through changes in lifestyle. Obesity is also a main concern associated with expanded morbidity and mortality from many noncommunicable illnesses (NCDs). The study aimed to determine the antiobesity effect of Pleurotus ostreatus (PO) and its bioactive anthraquinone (AQ). The overall promoter genes CEBPα (CCAAT enhancer binding protein α) and PPARγ (Peroxisome proliferator activated receptor γ) in controlling the homeostasis of glucose was analysed using 3T3-L1 cell line. Finally, an insilico study was carried out using CRISPR software to identify the RNA's involved in adipogenesis especially of the control gene PPARγ.<br><br>MATERIALS AND METHODS: Preliminary screening of the edible fungi and their bio actives led to the marvellous discovery of side effect free agonists for treating obesity (adipogenesis). An edible fungi Pleurotus ostreatus (PO) were analysed in a screening platform with different series of tests for adipocyte differentiation, triglyceride analysis, lipolysis determination, glucose uptake assay, cytotoxicity assay and lipase activity followed by specific gene expression analysis. The gene knockout mechanism was also elucidated by CRISPR spcas 9 tool.<br><br>RESULTS: The antiadipogenic (antiobesity) activity of DMSO extract of PO were found to stimulate the insulin dependent uptake of glucose. The extract also decreased the levels of triglycerides and glycerol accumulation in differentiated adipocyte cells. The binding FABP4 (Fatty acid binding protein) and transport protein FATP1 (Fatty acid transport protein) along with the fat breaking LPL (lipoprotein lipase) was found to be inhibited after the PO treatment at varying concentration (0-300&#x202f;&#x3bc;g/ml). CRISPR spcas9 genome editing software was used as an insilico approach in validating the efficiency of mouse embryonic and human adipogenic cell line (3T3-L1). These tool analysed and found 4 RNAs gene knock out possibilities in PPAR&#x3b3; and their efficiency for further treating obesity.<br><br>CONCLUSION: These novel finding contribute to the confirmation that edible fungi PO and it's bioactive AQ is an adequate supplement for constraining the lipid and triglycerides in differentiated mature adipocytes by reversing the fat deposition. Thereby, forbidding the enzymes linked with fat absorption. Besides, the CRISPR tool identified gene knock out possibilities of control gene PPAR&#x3b3;, will pave a way in further research for treating obesity.},
}
@article {pmid31152780,
year = {2019},
author = {Thomas, M and Parry-Smith, D and Iyer, V},
title = {Best practice for CRISPR design using current tools and resources.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {3-17},
doi = {10.1016/j.ymeth.2019.05.019},
pmid = {31152780},
issn = {1095-9130},
mesh = {Algorithms ; Animals ; CRISPR-Cas Systems/*genetics ; *Databases, Genetic ; Gene Editing/*methods ; *Genomic Library ; Humans ; Mice ; RNA, Guide/genetics ; Software ; },
abstract = {Users facing the task of designing gRNAs for a CRISPR-mutagenesis experiment are typically confronted with a large variety of possible tools and existing libraries. Here we examine the design principles for such resources, and suggest a best practice which allows a user to evaluate and effectively use any of the existing CRISPR design tools or genome-wide libraries.},
}
@article {pmid31150829,
year = {2019},
author = {Harper, JC and Schatten, G},
title = {Are we ready for genome editing in human embryos for clinical purposes?.},
journal = {European journal of medical genetics},
volume = {62},
number = {8},
pages = {103682},
doi = {10.1016/j.ejmg.2019.103682},
pmid = {31150829},
issn = {1878-0849},
mesh = {Female ; Fertilization in Vitro/*trends ; Gene Editing/*trends ; Genetic Testing ; Humans ; Pregnancy ; Preimplantation Diagnosis/*trends ; *Reproductive Techniques, Assisted ; Sperm Injections, Intracytoplasmic ; },
abstract = {Perhaps the two most significant pioneering biomedical discoveries with immediate clinical implications during the past forty years have been the advent of assisted reproductive technologies (ART) and the genetics revolution. ART, including in vitro fertilization (IVF), intracytoplasmic sperm injection and preimplantation genetic testing, has resulted in the birth of more than 8 million children, and the pioneer of IVF, Professor Bob Edwards, was awarded the 2010 Nobel Prize. The genetics revolution has resulted in our genomes being sequenced and many of the molecular mechanisms understood, and technologies for genomic editing have been developed. With the combination of nearly routine ART protocols for healthy conceptions together with almost error-free, inexpensive and simple methods for genetic modification, the question "Are we ready for genome editing in human embryos for clinical purposes?" was debated at the 5th congress on controversies in preconception, preimplantation and Prenatal Genetic Diagnosis, in collaboration with the Ovarian Club Meeting, in November 2018 in Paris. The co-authors each presented scientific, medical and bioethical backgrounds, and the debate was chaired by Professor Alan Handyside. In this paper, we consider whether genome editing is safe and ethical. We conclude that we are currently not ready for genome editing to be used in human embryos for clinical purposes, and we call for a global debate to determine if and when this technology could be used in ART. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬.},
}
@article {pmid31150759,
year = {2020},
author = {Stachler, AE and Schwarz, TS and Schreiber, S and Marchfelder, A},
title = {CRISPRi as an efficient tool for gene repression in archaea.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {76-85},
doi = {10.1016/j.ymeth.2019.05.023},
pmid = {31150759},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/*genetics ; Chromosomes, Archaeal/genetics ; Gene Editing/*methods ; *Gene Expression Regulation, Archaeal ; Gene Knockdown Techniques/methods ; Genes, Archaeal/genetics ; Genes, Essential/genetics ; Haloferax volcanii/*genetics ; Plasmids/genetics ; },
abstract = {In the years following its discovery and characterization, the CRISPR-Cas system has been modified and converted into a multitude of applications for eukaryotes and bacteria, such as genome editing and gene regulation. Since no such method has been available for archaea, we developed a tool for gene repression in the haloarchaeon Haloferax volcanii by repurposing its endogenous type I-B CRISPR-Cas system. Here, we present the two possible approaches for gene repression as well as our workflow to achieve and assess gene knockdown, offer recommendations on protospacer selection and give some examples of genes we have successfully silenced.},
}
@article {pmid31150758,
year = {2019},
author = {Mirza, Z and Karim, S},
title = {Advancements in CRISPR/Cas9 technology-Focusing on cancer therapeutics and beyond.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {13-21},
doi = {10.1016/j.semcdb.2019.05.026},
pmid = {31150758},
issn = {1096-3634},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {"CRISPR" is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, which are a characteristic of the bacterial defense system and Cas9 (or "CRISPR-associated") is a RNA-guided DNA endonuclease or molecular scissor, capable of cutting DNA strands. Both together forms the basis for CRISPR-Cas9 targeted genome editing technology and enables highly specific genomic modifications to an organism's DNA. Recent advent of high-throughput genomics has revolutionizing personalized medicine and enhanced our molecular understanding of human cancers. The development of the CRISPR/Cas9 tool has unveiled advancement of new, simplistic and efficient in vivo model systems in oncology. The usage of CRISPR/Cas9 gene editing systems for curing various cancers promises to be the next great biotechnological breakthrough in medicine. However, urgent attention is needed to assess the functional relevance of novel cancer-associated mutations and translate our molecular knowledge to therapeutics. Herein, we will review the development and applications of the exciting uses of the CRISPR/Cas9 technique for cancer research and therapy with focus on origin, progress, clinical trials, implications, and challenges ahead. Major ethical and safety concerns are perhaps unknown long term consequences of DNA manipulation and irreversibility of this procedure.},
}
@article {pmid31150757,
year = {2019},
author = {Lu, Y and Liang, M and Zhang, Q and Liu, Z and Song, Y and Lai, L and Li, Z},
title = {Mutations of GADD45G in rabbits cause cleft lip by the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT).},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1865},
number = {9},
pages = {2356-2367},
doi = {10.1016/j.bbadis.2019.05.015},
pmid = {31150757},
issn = {1879-260X},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Cleft Lip/genetics/*pathology/veterinary ; Embryo, Mammalian/cytology/metabolism/pathology ; Embryonic Development ; Epithelial-Mesenchymal Transition ; Gene Knockout Techniques/methods ; Homozygote ; Intracellular Signaling Peptides and Proteins/chemistry/*genetics/metabolism ; Lip/pathology ; Mutation ; Protein Structure, Tertiary ; Rabbits ; },
abstract = {The cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects in humans. Genome-wide association studies (GWAS) have been widely used for identifying candidate genes, and different genes or chromosomal regions have shown strong evidence for the presence of causal genes in CL/P. To date, two independent GWAS have identified GADD45G as influencing risk for CL/P. However, there is no animal model evidence about GADD45G related to CL/P. Here, we reported the generation of a novel GADD45G mutated rabbit model by CRISPR/Cas9 and CRISPR-based BE4-Gam systems. The homozygous (GADD45G[-/-]) while not heterozygous (GADD45G[+/-]) pups died after birth due to severe craniofacial defects of unilateral or bilateral cleft lip (CL). Moreover, the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) were also determined in the medial and lateral nasal processes (MNP and LNP) of the embryonic day 13 (E13) GADD45G[-/-] rabbits, which compared with the normal wild type (WT) rabbits. Thus, our study confirmed for the first time that loss of GADD45G lead to CL at the animal level and provided new insights into the crucial role of GADD45G for upper lip formation and fusion.},
}
@article {pmid31149896,
year = {2019},
author = {Torres, SE and Gallagher, CM and Plate, L and Gupta, M and Liem, CR and Guo, X and Tian, R and Stroud, RM and Kampmann, M and Weissman, JS and Walter, P},
title = {Ceapins block the unfolded protein response sensor ATF6α by inducing a neomorphic inter-organelle tether.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31149896},
issn = {2050-084X},
support = {P01 GM111126/GM/NIGMS NIH HHS/United States ; R01 GM024485/GM/NIGMS NIH HHS/United States ; GM111126/NH/NIH HHS/United States ; DP2 OD021007/NH/NIH HHS/United States ; },
mesh = {ATP-Binding Cassette Transporters/metabolism ; Activating Transcription Factor 6/*antagonists &amp; inhibitors/metabolism ; CRISPR-Cas Systems/genetics ; Endoplasmic Reticulum/drug effects/metabolism ; HEK293 Cells ; Hep G2 Cells ; Humans ; Organelles/drug effects/*metabolism ; Peroxisomes/drug effects/metabolism ; Phenotype ; Protein Binding/drug effects ; Small Molecule Libraries/*pharmacology ; *Unfolded Protein Response/drug effects ; },
abstract = {The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.},
}
@article {pmid31149642,
year = {2019},
author = {Aksoy, YA and Nguyen, DT and Chow, S and Chung, RS and Guillemin, GJ and Cole, NJ and Hesselson, D},
title = {Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {198},
pmid = {31149642},
issn = {2399-3642},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *Gene Editing ; Genotype ; Green Fluorescent Proteins/metabolism ; RNA/metabolism ; Recombinational DNA Repair ; Zebrafish/*embryology/*genetics ; },
abstract = {Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.},
}
@article {pmid31148949,
year = {2019},
author = {Wang, J and Fu, D and Senouthai, S and Jiang, Y and Hu, R and You, Y},
title = {Identification of the Transcriptional Networks and the Involvement in Angiotensin II-Induced Injury after CRISPR/Cas9-Mediated Knockdown of Cyr61 in HEK293T Cells.},
journal = {Mediators of inflammation},
volume = {2019},
number = {},
pages = {8697257},
pmid = {31148949},
issn = {1466-1861},
mesh = {Angiotensin II/*pharmacology ; Apoptosis/drug effects ; CRISPR-Cas Systems/*drug effects ; Cell Cycle/drug effects ; Cell Proliferation/drug effects ; Cysteine-Rich Protein 61/genetics/*metabolism ; Gene Ontology ; Gene Regulatory Networks/genetics ; HEK293 Cells ; Humans ; Oligonucleotide Array Sequence Analysis ; RNA, Long Noncoding/metabolism ; RNA, Messenger/metabolism ; },
abstract = {BACKGROUND: The transcriptional networks of Cyr61 and its function in cell injury are poorly understood. The present study depicted the lncRNA and mRNA profiles and the involvement in angiotensin II-induced injury after Cyr61 knockdown mediated by CRISPR/Cas9 in HEK293T cells.<br><br>METHODS: HEK293T cells were cultured, and Cyr61 knockdown was achieved by transfection of the CRISPR/Cas9 KO plasmid. lncRNA and mRNA microarrays were used to identify differentially expressed genes (DEGs). Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine biofunctions and signaling pathways. RT-PCR was used to validate the microarray results. Cells were divided into four groups: control, Cyr61 knockdown, angiotensin II (Ang II) without Cyr61 knockdown, and Ang II with Cyr61 knockdown. CCK8, western blotting, and flow cytometry analysis were carried out to dissect cellular function.<br><br>RESULTS: A total of 23184 lncRNAs and 28264 mRNAs were normalized. 26 lncRNAs and 212 mRNAs were upregulated, and 74 lncRNAs and 233 mRNAs were downregulated after Cyr61 knockdown. Analysis of cellular components, molecular functions, biological processes, and regulatory pathways associated with the differentially expressed mRNAs revealed downstream mechanisms of the Cyr61 gene. The differentially expressed genes were affected for small cell lung cancer, axon guidance, Fc gamma R-mediated phagocytosis, MAPK signaling pathway, focal adhesion, insulin resistance, and metabolic pathways. In addition, Cyr61 expression was increased in accordance with induction of cell cycle arrest and apoptosis and inhibition of cell proliferation induced by Ang II. Knockdown of Cyr61 in HEK293T cells promoted cell cycle procession, decreased apoptosis, and promoted cell proliferation.<br><br>CONCLUSIONS: The Cyr61 gene is involved in Ang II-induced injury in HEK293T cells. Functional mechanisms of the differentially expressed lncRNAs and mRNAs as well as identification of metabolic pathways will provide new therapeutic targets for Cyr61-realated diseases.},
}
@article {pmid31148548,
year = {2019},
author = {Ingle, P and Groothuis, D and Rowe, P and Huang, H and Cockayne, A and Kuehne, SA and Jiang, W and Gu, Y and Humphreys, CM and Minton, NP},
title = {Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8123},
pmid = {31148548},
issn = {2045-2322},
support = {BB/L502030/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/K00283X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L01081X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L013940/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; G0601176/MRC_/Medical Research Council/United Kingdom ; BB/M012336/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Clostridioides difficile/drug effects/*genetics ; Codon, Initiator ; Culture Media ; Erythromycin/*pharmacology ; Escherichia coli ; Frameshift Mutation ; Gene Deletion ; *Gene Editing ; Genetic Vectors ; *Genome, Bacterial ; Methyltransferases/genetics ; Mutagenesis ; Mutation ; Phenotype ; Plasmids/genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; },
abstract = {Understanding the molecular pathogenesis of Clostridioides difficile has relied on the use of ermB-based mutagens in erythromycin-sensitive strains. However, the repeated subcultures required to isolate sensitive variants can lead to the acquisition of ancillary mutations that affect phenotype, including virulence. CRISPR-Cas9 allows the direct selection of mutants, reducing the number of subcultures and thereby minimising the likelihood of acquiring additional mutations. Accordingly, CRISPR-Cas9 was used to sequentially remove from the C. difficile 630 reference strain (NCTC 13307) two ermB genes and pyrE. The genomes of the strains generated (630Δerm* and 630Δerm*ΔpyrE, respectively) contained no ancillary mutations compared to the NCTC 13307 parental strain, making these strains the preferred option where erythromycin-sensitive 630 strains are required. Intriguingly, the cas9 gene of the plasmid used contained a proximal frameshift mutation. Despite this, the frequency of mutant isolation was high (96% and 89% for ermB and pyrE, respectively) indicating that a functional Cas9 is still being produced. Re-initiation of translation from an internal AUG start codon would produce a foreshortened protein lacking a RuvCI nucleolytic domain, effectively a 'nickase'. The mutation allowed cas9 to be cloned downstream of the strong Pthl promoter. It may find application elsewhere where the use of strong, constitutive promoters is preferred.},
}
@article {pmid31148128,
year = {2019},
author = {Kumari, P and Yusuf, F and Gaur, NA},
title = {Novel Microbial Modification Tools to Convert Lipids into Other Value-Added Products.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1995},
number = {},
pages = {161-171},
doi = {10.1007/978-1-4939-9484-7_10},
pmid = {31148128},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular/methods ; Gene Editing/*methods ; Genetic Vectors/genetics ; Lipids/*genetics ; RNA, Guide/genetics ; Transformation, Genetic ; Yeasts/*genetics ; },
abstract = {CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) is a microbial adaptive immune system that has revolutionized the field of molecular biology and genome engineering. The Type II CRISPR system consists of Cas9 nuclease of Streptococcus pyogenes and the RNA complex that guides Cas9 nuclease to a specific sequence of DNA in the genome. The CRISPR-Cas9 technology has reformed our ability to edit DNA and to regulate expression levels of genes of interest to high precision and accuracy. It is a powerful technology, which is used for genome engineering of a wide range of organisms for various applications. Here, we describe a method involving CRISPR-Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms for biotechnological applications in yeast. The complete procedure of genome editing including target sequence selection, cloning gRNA with a target sequence, transformation, and verification of the desired mutation/deletion or insertion can be achieved within 2-3 weeks in yeast.},
}
@article {pmid31147912,
year = {2019},
author = {Kalitsis, P and Zhang, T and Kim, JH and Nielsen, CF and Marshall, KM and Hudson, DF},
title = {Knocking in Multifunctional Gene Tags into SMC Complex Subunits Using Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2004},
number = {},
pages = {91-102},
doi = {10.1007/978-1-4939-9520-2_8},
pmid = {31147912},
issn = {1940-6029},
mesh = {Adenosine Triphosphatases/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Chromosome Mapping/methods ; Chromosomes/*genetics ; DNA-Binding Proteins/*genetics ; Gene Editing/methods ; Genome/genetics ; Multiprotein Complexes/*genetics ; Proteomics/methods ; Vertebrates/genetics ; },
abstract = {Condensin, a highly conserved pentameric chromosome complex, is required for the correct organization and folding of the genome. Here, we highlight how to knock protein tags into endogenous loci to faithfully study the condensin complex in vertebrates and dissect its multiple functions. These include using the streptavidin binding peptide (SBP) to create the first genome-wide map of condensin and perform varied applications in proteomics and enzymology of the complex. The revolution in gene editing using CRISPR/Cas9 has made it possible to insert tags into endogenous loci with relative ease, allowing physiological and fully functional tagged protein to be analyzed biochemically (affinity tags), microscopically (fluorescent tags) or both purified and localized (multifunctional tags). In this chapter, we detail how to engineer vertebrate cells using CRISPR/Cas9 to provide researchers powerful tools to obtain greater precision than ever to understand how the complex interacts and behaves in cells.},
}
@article {pmid31147819,
year = {2019},
author = {Filho, DM and de Carvalho Ribeiro, P and Oliveira, LF and Dos Santos, ALRT and Parreira, RC and Pinto, MCX and Resende, RR},
title = {Enhancing the Therapeutic Potential of Mesenchymal Stem Cells with the CRISPR-Cas System.},
journal = {Stem cell reviews and reports},
volume = {15},
number = {4},
pages = {463-473},
pmid = {31147819},
issn = {2629-3277},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing ; Humans ; *Immunomodulation ; Mesenchymal Stem Cells/*immunology ; },
abstract = {Mesenchymal stem cells (MSCs), also known as multipotent mesenchymal stromal stem cells, are found in the perivascular space of several tissues. These cells have been subject of intense research in the last decade due to their low teratogenicity, as well as their ability to differentiate into mature cells and to secrete immunomodulatory and trophic factors. However, they usually promote only a modest benefit when transplanted in experimental disease models, one of the limitations for their clinical application. The CRISPR-Cas system, in turn, is highlighted as a simple and effective tool for genetic engineering. This system was tested in clinical trials over a relatively short period of time after establishing its applicability to the edition of the mammalian cell genome. Similar to the research evolution in MSCs, the CRISPR-Cas system demonstrated inconsistencies that limited its clinical application. In this review, we outline the evolution of MSC research and its applicability, and the progress of the CRISPR-Cas system from its discovery to the most recent clinical trials. We also propose perspectives on how the CRISPR-Cas system may improve the therapeutic potential of MSCs, making it more beneficial and long lasting.},
}
@article {pmid31147717,
year = {2019},
author = {Park, SH and Lee, CM and Dever, DP and Davis, TH and Camarena, J and Srifa, W and Zhang, Y and Paikari, A and Chang, AK and Porteus, MH and Sheehan, VA and Bao, G},
title = {Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease.},
journal = {Nucleic acids research},
volume = {47},
number = {15},
pages = {7955-7972},
pmid = {31147717},
issn = {1362-4962},
support = {T32 DK060445/DK/NIDDK NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/genetics/*therapy ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Erythrocytes/metabolism ; Gene Editing/*methods ; Genetic Therapy/methods ; Hematopoietic Stem Cell Transplantation/*methods ; Hematopoietic Stem Cells/*metabolism ; Humans ; K562 Cells ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Treatment Outcome ; beta-Globins/*genetics ; },
abstract = {Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.},
}
@article {pmid31147716,
year = {2019},
author = {Su, G and Guo, D and Chen, J and Liu, M and Zheng, J and Wang, W and Zhao, X and Yin, Q and Zhang, L and Zhao, Z and Shi, J and Lu, W},
title = {A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.},
journal = {Nucleic acids research},
volume = {47},
number = {13},
pages = {6737-6752},
pmid = {31147716},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/drug effects ; Cells, Cultured ; Chromatin/genetics/metabolism ; Embryonic Stem Cells/drug effects/*metabolism ; Endoderm/metabolism ; *Enhancer Elements, Genetic/genetics ; Gene Editing ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Gene Ontology ; Homeodomain Proteins/*biosynthesis/genetics ; Intracellular Signaling Peptides and Proteins/biosynthesis/genetics ; Mice ; Promoter Regions, Genetic ; RNA, Small Interfering/genetics ; Recombinant Proteins/metabolism ; Transcription Factors/*biosynthesis/genetics ; Tretinoin/*pharmacology ; },
abstract = {Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.},
}
@article {pmid31147630,
year = {2019},
author = {Roca Paixão, JF and Gillet, FX and Ribeiro, TP and Bournaud, C and Lourenço-Tessutti, IT and Noriega, DD and Melo, BP and de Almeida-Engler, J and Grossi-de-Sa, MF},
title = {Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8080},
pmid = {31147630},
issn = {2045-2322},
mesh = {Acclimatization/*genetics ; Arabidopsis/*physiology ; Arabidopsis Proteins/*genetics/metabolism ; Basic-Leucine Zipper Transcription Factors/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Droughts ; Gene Expression Regulation, Plant/physiology ; Histone Acetyltransferases ; Plants, Genetically Modified/*physiology ; Promoter Regions, Genetic/genetics ; Recombinant Fusion Proteins/genetics ; Transcription Factors/*genetics/metabolism ; },
abstract = {Drought episodes decrease plant growth and productivity, which in turn cause high economic losses. Plants naturally sense and respond to water stress by activating specific signalling pathways leading to physiological and developmental adaptations. Genetically engineering genes that belong to these pathways might improve the drought tolerance of plants. The abscisic acid (ABA)-responsive element binding protein 1/ABRE binding factor (AREB1/ABF2) is a key positive regulator of the drought stress response. We investigated whether the CRISPR activation (CRISPRa) system that targets AREB1 might contribute to improve drought stress tolerance in Arabidopsis. Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Stable transgenic plants expressing chimeric dCas9[HAT] were first generated. Then, we showed that the CRISPRa dCas9[HAT] mechanism increased the promoter activity controlling the β-glucuronidase (GUS) reporter gene. To activate the endogenous promoter of AREB1, the CRISPRa dCas9[HAT] system was set up, and resultant plants showed a dwarf phenotype. Our qRT-PCR experiments indicated that both AREB1 and RD29A, a gene positively regulated by AREB1, exhibited higher gene expression than the control plants. The plants generated here showed higher chlorophyll content and faster stomatal aperture under water deficit, in addition to a better survival rate after drought stress. Altogether, we report that CRISPRa dCas9[HAT] is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.},
}
@article {pmid31147612,
year = {2019},
author = {Pickar-Oliver, A and Gersbach, CA},
title = {The next generation of CRISPR-Cas technologies and applications.},
journal = {Nature reviews. Molecular cell biology},
volume = {20},
number = {8},
pages = {490-507},
pmid = {31147612},
issn = {1471-0080},
support = {UG3 TR002142/TR/NCATS NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; R33 DA041878/DA/NIDA NIH HHS/United States ; R41 AI136755/AI/NIAID NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; U01 HG007900/HG/NHGRI NIH HHS/United States ; R41 GM119914/GM/NIGMS NIH HHS/United States ; P30 AR066527/AR/NIAMS NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Endonucleases/genetics/metabolism ; *Gene Editing ; *Genetic Therapy ; Humans ; *RNA, Guide/genetics/metabolism ; },
abstract = {The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR-Cas-based tools towards clinical use in gene and cell therapies.},
}
@article {pmid31147565,
year = {2019},
author = {Yin, Y and Reed, EF and Zhang, Q},
title = {Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8067},
pmid = {31147565},
issn = {2045-2322},
support = {P01 AI120944/AI/NIAID NIH HHS/United States ; U19 AI128913/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; B-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Exons/genetics ; Feasibility Studies ; Gene Editing/*methods ; Gene Expression Regulation/genetics/immunology ; HLA-B38 Antigen/*genetics/immunology/metabolism ; Herpesvirus 4, Human/genetics ; Heterozygote ; Histocompatibility Testing/*methods ; Homozygote ; Humans ; Polymorphism, Genetic/immunology ; Sequence Deletion ; Transfection ; },
abstract = {The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230-232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.},
}
@article {pmid31147448,
year = {2019},
author = {Hisano, Y and Kono, M and Cartier, A and Engelbrecht, E and Kano, K and Kawakami, K and Xiong, Y and Piao, W and Galvani, S and Yanagida, K and Kuo, A and Ono, Y and Ishida, S and Aoki, J and Proia, RL and Bromberg, JS and Inoue, A and Hla, T},
title = {Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes.},
journal = {The Journal of experimental medicine},
volume = {216},
number = {7},
pages = {1582-1598},
pmid = {31147448},
issn = {1540-9538},
support = {R35 HL135821/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Endothelial Cells/*metabolism ; Fluorescent Antibody Technique ; Gene Editing ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Lymph Nodes/*metabolism ; Lysophospholipids/metabolism ; Mice ; Real-Time Polymerase Chain Reaction ; *Receptor Cross-Talk ; Receptors, G-Protein-Coupled/metabolism ; Receptors, Lysophosphatidic Acid/metabolism ; Sphingosine/analogs &amp; derivatives/metabolism ; Sphingosine-1-Phosphate Receptors/metabolism ; },
abstract = {Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein-coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9-based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/β-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/β-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.},
}
@article {pmid31146387,
year = {2019},
author = {Koeppel, I and Hertig, C and Hoffie, R and Kumlehn, J},
title = {Cas Endonuclease Technology-A Quantum Leap in the Advancement of Barley and Wheat Genetic Engineering.},
journal = {International journal of molecular sciences},
volume = {20},
number = {11},
pages = {},
pmid = {31146387},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Genetic Engineering/*methods ; Hordeum/*genetics ; Plant Breeding/*methods ; Triticum/*genetics ; },
abstract = {Domestication and breeding have created productive crops that are adapted to the climatic conditions of their growing regions. Initially, this process solely relied on the frequent occurrence of spontaneous mutations and the recombination of resultant gene variants. Later, treatments with ionizing radiation or mutagenic chemicals facilitated dramatically increased mutation rates, which remarkably extended the genetic diversity of crop plants. However, a major drawback of conventionally induced mutagenesis is that genetic alterations occur simultaneously across the whole genome and at very high numbers per individual plant. By contrast, the newly emerging Cas endonuclease technology allows for the induction of mutations at user-defined positions in the plant genome. In fundamental and breeding-oriented research, this opens up unprecedented opportunities for the elucidation of gene functions and the targeted improvement of plant performance. This review covers historical aspects of the development of customizable endonucleases, information on the mechanisms of targeted genome modification, as well as hitherto reported applications of Cas endonuclease technology in barley and wheat that are the agronomically most important members of the temperate cereals. Finally, current trends in the further development of this technology and some ensuing future opportunities for research and biotechnological application are presented.},
}
@article {pmid31145755,
year = {2019},
author = {Liu, R and Mendez-Rios, JD and Peng, C and Xiao, W and Weisberg, AS and Wyatt, LS and Moss, B},
title = {SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells.},
journal = {PLoS pathogens},
volume = {15},
number = {5},
pages = {e1007710},
pmid = {31145755},
issn = {1553-7374},
mesh = {A549 Cells ; Genetic Vectors/immunology ; Host Specificity/*immunology ; Humans ; Serine Proteinase Inhibitors/genetics/*immunology ; Vaccinia/immunology/prevention &amp; control/*virology ; Vaccinia virus/*physiology ; Viral Vaccines/*immunology ; *Virus Replication ; },
abstract = {Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.},
}
@article {pmid31144528,
year = {2019},
author = {Liu, L and Cao, J and Chang, Q and Xing, F and Yan, G and Fu, L and Wang, H and Ma, Z and Chen, X and Li, Y and Li, S},
title = {In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System.},
journal = {Human gene therapy},
volume = {30},
number = {9},
pages = {1079-1092},
doi = {10.1089/hum.2019.037},
pmid = {31144528},
issn = {1557-7422},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Copper-Transporting ATPases/*genetics ; Dependovirus/genetics ; *Exons ; *Gene Editing ; Gene Order ; Gene Targeting ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors/administration &amp; dosage/genetics ; Hepatocytes/metabolism ; Lentivirus/genetics ; Mice ; Mice, Transgenic ; Mutation ; RNA, Guide ; Sequence Analysis, DNA ; Transduction, Genetic ; },
abstract = {The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.},
}
@article {pmid31143069,
year = {2019},
author = {Limanskiy, V and Vyas, A and Chaturvedi, LS and Vyas, D},
title = {Harnessing the potential of gene editing technology using CRISPR in inflammatory bowel disease.},
journal = {World journal of gastroenterology},
volume = {25},
number = {18},
pages = {2177-2187},
pmid = {31143069},
issn = {2219-2840},
mesh = {Adaptive Immunity/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; Humans ; Inflammatory Bowel Diseases/genetics/immunology/*therapy ; },
abstract = {The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease (IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease, human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm (Glycine amidinotransferase, mitochondrial), nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore, since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity, protospacer adjacent motifs and crossfire between different target sites. However, these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.},
}
@article {pmid31142834,
year = {2019},
author = {Meeske, AJ and Nakandakari-Higa, S and Marraffini, LA},
title = {Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage.},
journal = {Nature},
volume = {570},
number = {7760},
pages = {241-245},
pmid = {31142834},
issn = {1476-4687},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages/genetics/growth &amp; development/*immunology ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/genetics/*immunology ; DNA Viruses/genetics/growth &amp; development/immunology ; Endodeoxyribonucleases/*metabolism ; Listeria/genetics/growth &amp; development/*immunology/*virology ; RNA, Bacterial/genetics/metabolism ; RNA, Viral/genetics/metabolism ; },
abstract = {Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30-40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers[1-3]. These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids[4,5]. In contrast to most Cas nucleases, which destroy invader DNA[4-7], the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis- and non-specific trans-RNA cleavage[8]. Although it has been hypothesized that Cas13 naturally defends against RNA phages[8], type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages[9,10], suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis- and/or trans-RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans-cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems[11-13], are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage.},
}
@article {pmid31142788,
year = {2019},
author = {Cecconi, D and Brandi, J and Manfredi, M and Serena, M and Dalle Carbonare, L and Deiana, M and Cheri, S and Parolini, F and Gandini, A and Marchetto, G and Innamorati, G and Avanzi, F and Antoniazzi, F and Marengo, E and Tiso, N and Mottes, M and Zipeto, D and Valenti, MT},
title = {Runx2 stimulates neoangiogenesis through the Runt domain in melanoma.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8052},
pmid = {31142788},
issn = {2045-2322},
mesh = {Apoptosis/genetics ; Biomarkers, Tumor/analysis/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Coculture Techniques ; Computational Biology ; Core Binding Factor Alpha 1 Subunit/*metabolism ; Datasets as Topic ; Endoglin/analysis/genetics ; *Gene Expression Regulation, Neoplastic ; Human Umbilical Vein Endothelial Cells ; Humans ; Melanocytes ; Melanoma/blood supply/*genetics/pathology ; Neovascularization, Pathologic/*genetics/pathology ; Platelet Endothelial Cell Adhesion Molecule-1/analysis/genetics ; Primary Cell Culture ; Protein Domains/genetics ; Proteomics ; Skin Neoplasms/blood supply/*genetics/pathology ; Vascular Endothelial Growth Factor A/analysis/genetics ; },
abstract = {Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.},
}
@article {pmid31142767,
year = {2019},
author = {Watanabe, M and Nakano, K and Uchikura, A and Matsunari, H and Yashima, S and Umeyama, K and Takayanagi, S and Sakuma, T and Yamamoto, T and Morita, S and Horii, T and Hatada, I and Nishinakamura, R and Nakauchi, H and Nagashima, H},
title = {Anephrogenic phenotype induced by SALL1 gene knockout in pigs.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {8016},
pmid = {31142767},
issn = {2045-2322},
mesh = {Allografts/supply &amp; distribution ; Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Feasibility Studies ; Female ; Fetal Development/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Humans ; Kidney Transplantation ; Male ; Mutation ; Nephrons/*growth &amp; development ; Pluripotent Stem Cells/physiology ; Regeneration/physiology ; Sus scrofa ; Tissue Engineering/*methods ; Transcription Activator-Like Effector Nucleases/genetics ; Transcription Factors/*genetics ; Zygote/growth &amp; development ; },
abstract = {To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.},
}
@article {pmid31142600,
year = {2019},
author = {Dehler, CE and Lester, K and Della Pelle, G and Jouneau, L and Houel, A and Collins, C and Dovgan, T and Machat, R and Zou, J and Boudinot, P and Martin, SAM and Collet, B},
title = {Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {203},
number = {2},
pages = {465-475},
pmid = {31142600},
issn = {1550-6606},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Cell Line ; Fishes/*metabolism ; Gene Editing/methods ; Interferon Type I/*metabolism ; STAT2 Transcription Factor/*metabolism ; Signal Transduction/*physiology ; Virus Diseases/metabolism ; },
abstract = {IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-γ), but no type III (λ) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in fish.},
}
@article {pmid31142266,
year = {2019},
author = {Léger, S and Costa, MBW and Tulpan, D},
title = {Pairwise visual comparison of small RNA secondary structures with base pair probabilities.},
journal = {BMC bioinformatics},
volume = {20},
number = {1},
pages = {293},
pmid = {31142266},
issn = {1471-2105},
mesh = {Algorithms ; *Base Pairing ; CRISPR-Cas Systems/genetics ; Evolution, Molecular ; Humans ; *Nucleic Acid Conformation ; *Probability ; RNA/*chemistry ; RNA, Guide/genetics ; Virulence/genetics ; Yersinia/pathogenicity ; },
abstract = {BACKGROUND: Predicted RNA secondary structures are typically visualized using dot-plots for base pair binding probabilities and planar graphs for unique structures, such as the minimum free energy structure. These are however difficult to analyze simultaneously.<br><br>RESULTS: This work introduces a compact unified view of the most stable conformation of an RNA secondary structure and its base pair probabilities, which is called the Circular Secondary Structure Base Pairs Probabilities Plot (CS[2]BP[2]-Plot). Along with our design we provide access to a web server implementation of our solution that facilitates pairwise comparison of short RNA (and DNA) sequences up to 200 base pairs. The web server first calculates the minimum free energy secondary structure and the base pair probabilities for up to 10 RNA or DNA sequences using RNAfold and then provides a two panel comparative view that includes CS[2]BP[2]-Plots along with the traditional graph, planar and circular diagrams obtained with VARNA. The CS[2]BP[2]-Plots include highlighting of the nucleotide differences between two selected sequences using ClustalW local alignments. We also provide descriptive statistics, dot-bracket secondary structure representations and ClustalW local alignments for compared sequences.<br><br>CONCLUSIONS: Using circular diagrams and colour and weight-coded arcs, we demonstrate how a single image can replace the state-of-the-art dual representations (dot-plots and minimum free energy structures) for base-pair probabilities of RNA secondary structures while allowing efficient exploration and comparison of different RNA conformations via a web server front end. With that, we provide the community, especially the biologically oriented, with an intuitive tool for ncRNA visualization. Web-server: https://nrcmonsrv01.nrc.ca/cs2bp2plot.},
}
@article {pmid31141512,
year = {2019},
author = {Tu, CF and Chuang, CK and Hsiao, KH and Chen, CH and Chen, CM and Peng, SH and Su, YH and Chiou, MT and Yen, CH and Hung, SW and Yang, TS and Chen, CM},
title = {Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression.},
journal = {PloS one},
volume = {14},
number = {5},
pages = {e0217236},
pmid = {31141512},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems ; Coronavirus Infections/virology ; Cytidine Monophosphate/*analogs &amp; derivatives/genetics ; Diarrhea/virology ; Disease Susceptibility/metabolism ; Enterocytes/pathology ; Female ; Gene Expression Regulation/genetics ; Genetic Predisposition to Disease/*genetics ; Neuraminic Acids ; Porcine epidemic diarrhea virus/*genetics/pathogenicity ; Pregnancy ; Swine ; Swine Diseases/virology ; },
abstract = {The porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene is mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets that were challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs. Four live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I, n = 6, and III, n = 15) and 3-day-old (in exp. II, n = 9) KO and wild-type (WT, same ages in respective exps.) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow's colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals either deceased or euthanized were necropsied and intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.},
}
@article {pmid31140149,
year = {2019},
author = {Patel, VK and Soni, N and Prasad, V and Sapre, A and Dasgupta, S and Bhadra, B},
title = {CRISPR-Cas9 System for Genome Engineering of Photosynthetic Microalgae.},
journal = {Molecular biotechnology},
volume = {61},
number = {8},
pages = {541-561},
pmid = {31140149},
issn = {1559-0305},
mesh = {*Biotechnology ; *CRISPR-Cas Systems ; *Gene Editing ; *Microalgae/genetics/metabolism ; Photobioreactors ; },
abstract = {Targeted genome editing using RNA-guided endonucleases is an emerging tool in algal biotechnology. Recently, CRISPR-Cas systems have been widely used to manipulate the genome of some freshwater and marine microalgae. Among two different classes, and six distinct types of CRISPR systems, Cas9-driven type II system has been widely used in most of the studies for targeted knock-in, knock-out and knock-down of desired genes in algae. CRISPR technology has been demonstrated in microalgae including diatoms to manifest the function of the particular gene (s) and developing industrial traits, such as improving lipid content and biomass productivity. Instead of these, there are a lot of gears to be defined about improving efficiency and specificity of targeted genome engineering of microalgae using CRISPR-Cas system. Optimization of tools and methods of CRISPR technology can undoubtedly transform the research toward the industrial-scale production of commodity chemicals, food and biofuels using photosynthetic cell factories. This review has been focused on the efforts made so far to targeted genome engineering of microalgae, identified scopes about the hurdles related to construction and delivery of CRISPR-Cas components, algae transformation toolbox, and outlined the future prospect toward developing the CRISPR platform for high-throughput genome-editing of microalgae.},
}
@article {pmid31138881,
year = {2019},
author = {Kataoka, Y and Iimori, M and Niimi, S and Tsukihara, H and Wakasa, T and Saeki, H and Oki, E and Maehara, Y and Kitao, H},
title = {Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7964},
pmid = {31138881},
issn = {2045-2322},
mesh = {Arylformamidase/*genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; Cytotoxins/*pharmacology ; Doxycycline/pharmacology ; Founder Effect ; Gene Deletion ; *Gene Expression Regulation, Neoplastic ; HCT116 Cells ; HT29 Cells ; Humans ; Signal Transduction ; Thymidine Kinase/*genetics ; Trifluridine/*pharmacology ; },
abstract = {Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.},
}
@article {pmid31138768,
year = {2019},
author = {Lou, K and Steri, V and Ge, AY and Hwang, YC and Yogodzinski, CH and Shkedi, AR and Choi, ALM and Mitchell, DC and Swaney, DL and Hann, B and Gordan, JD and Shokat, KM and Gilbert, LA},
title = {KRAS[G12C] inhibition produces a driver-limited state revealing collateral dependencies.},
journal = {Science signaling},
volume = {12},
number = {583},
pages = {},
pmid = {31138768},
issn = {1937-9145},
support = {R00 CA204602/CA/NCI NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; F30 CA239476/CA/NCI NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; U54 CA196519/CA/NCI NIH HHS/United States ; R01 CA190408/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cysteine/genetics ; Female ; Genomics ; HEK293 Cells ; Humans ; Lung Neoplasms/genetics/*metabolism ; Mice ; Mice, Nude ; Mutation ; Neoplasm Transplantation ; Oncogenes ; Pancreatic Neoplasms/genetics/*metabolism ; Protein Binding ; Proteomics ; Proto-Oncogene Proteins p21(ras)/*antagonists &amp; inhibitors/genetics ; Sequence Analysis, RNA ; Signal Transduction/drug effects ; },
abstract = {Inhibitors targeting KRAS[G12C], a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)-bound form of KRAS[G12C], sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRAS[G12C] inhibitor in cellular models of KRAS[G12C] mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver-limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRAS[G12C] inhibition. We termed such genes "collateral dependencies" (CDs) and identified two classes of combination therapies targeting these CDs that increased KRAS[G12C] target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.},
}
@article {pmid31138663,
year = {2019},
author = {Marshall, AE and Roes, MV and Passos, DT and DeWeerd, MC and Chaikovsky, AC and Sage, J and Howlett, CJ and Dick, FA},
title = {RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression.},
journal = {Molecular and cellular biology},
volume = {39},
number = {16},
pages = {},
pmid = {31138663},
issn = {1098-5549},
support = {R01 CA228413/CA/NCI NIH HHS/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; MOP 324579//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; *DNA Damage ; Disease Progression ; Humans ; Lung Neoplasms/genetics/metabolism/*pathology ; Mice ; Neoplasm Transplantation ; Reactive Oxygen Species/metabolism ; Retinoblastoma Binding Proteins/*genetics ; *Sequence Deletion ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit.},
}
@article {pmid31138654,
year = {2019},
author = {Lin, DW and Chung, BP and Huang, JW and Wang, X and Huang, L and Kaiser, P},
title = {Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {28},
pages = {10877-10885},
pmid = {31138654},
issn = {1083-351X},
support = {R01 GM066164/GM/NIGMS NIH HHS/United States ; R01 GM074830/GM/NIGMS NIH HHS/United States ; R01 GM128432/GM/NIGMS NIH HHS/United States ; R21 CA242270/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA End-Joining Repair/genetics/physiology ; Fluorescent Antibody Technique/*methods ; Fluorescent Dyes/chemistry ; Gene Editing/methods ; HEK293 Cells ; Humans ; Protein Engineering/*methods ; },
abstract = {Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His6FLAG, His6-Biotin-TEV-RGSHis6, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.},
}
@article {pmid31138644,
year = {2019},
author = {Delfarah, A and Parrish, S and Junge, JA and Yang, J and Seo, F and Li, S and Mac, J and Wang, P and Fraser, SE and Graham, NA},
title = {Inhibition of nucleotide synthesis promotes replicative senescence of human mammary epithelial cells.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {27},
pages = {10564-10578},
pmid = {31138644},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Cellular Senescence ; Epithelial Cells/cytology/metabolism ; Gene Editing ; Glucose/metabolism ; Humans ; Mammary Glands, Human/cytology ; Metabolomics ; Nucleotides/analysis/*metabolism ; Ribonucleoside Diphosphate Reductase/deficiency/genetics/metabolism ; Telomerase/metabolism ; },
abstract = {Cellular senescence is a mechanism by which cells permanently withdraw from the cell cycle in response to stresses including telomere shortening, DNA damage, or oncogenic signaling. Senescent cells contribute to both age-related degeneration and hyperplastic pathologies, including cancer. In culture, normal human epithelial cells enter senescence after a limited number of cell divisions, known as replicative senescence. Here, to investigate how metabolic pathways regulate replicative senescence, we used LC-MS-based metabolomics to analyze senescent primary human mammary epithelial cells (HMECs). We did not observe significant changes in glucose uptake or lactate secretion in senescent HMECs. However, analysis of intracellular metabolite pool sizes indicated that senescent cells exhibit depletion of metabolites from nucleotide synthesis pathways. Furthermore, stable isotope tracing with [13]C-labeled glucose or glutamine revealed a dramatic blockage of flux of these two metabolites into nucleotide synthesis pathways in senescent HMECs. To test whether cellular immortalization would reverse these observations, we expressed telomerase in HMECs. In addition to preventing senescence, telomerase expression maintained metabolic flux from glucose into nucleotide synthesis pathways. Finally, we investigated whether inhibition of nucleotide synthesis in proliferating HMECs is sufficient to induce senescence. In proliferating HMECs, both pharmacological and genetic inhibition of ribonucleotide reductase regulatory subunit M2 (RRM2), a rate-limiting enzyme in dNTP synthesis, induced premature senescence with concomitantly decreased metabolic flux from glucose into nucleotide synthesis. Taken together, our results suggest that nucleotide synthesis inhibition plays a causative role in the establishment of replicative senescence in HMECs.},
}
@article {pmid31138265,
year = {2019},
author = {Luo, Z and Gao, M and Huang, N and Wang, X and Yang, Z and Yang, H and Huang, Z and Feng, W},
title = {Efficient disruption of bcr-abl gene by CRISPR RNA-guided FokI nucleases depresses the oncogenesis of chronic myeloid leukemia cells.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {38},
number = {1},
pages = {224},
pmid = {31138265},
issn = {1756-9966},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Deoxyribonucleases/*metabolism ; Fusion Proteins, bcr-abl/*antagonists &amp; inhibitors ; Gene Editing/*methods ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/*therapy ; Mice ; Signal Transduction ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: The bcr-abl fusion gene encodes BCR-ABL oncoprotein and plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). Current therapeutic methods have limited treatment effect on CML patients with drug resistance or disease relapse. Therefore, novel therapeutic strategy for CML is essential to be explored and the CRISPR RNA-guided FokI nucleases (RFNs) meet the merits of variable target sites and specificity of cleavage enabled its suitability for gene editing of CML. The RFNs provide us a new therapeutic direction to obliterate this disease.<br><br>METHODS: Guide RNA (gRNA) expression plasmids were constructed by molecular cloning technique. The modification rate of RFNs on bcr-abl was detected via NotI restriction enzyme digestion and T7 endonuclease 1 (T7E1) assay. The expression of BCR-ABL and its downstream signaling molecules were determined by western blotting. The effects of RFNs on cell proliferation and apoptosis of CML cell lines and CML stem/progenitor cells were evaluated by CCK-8 assay and flow cytometry. In addition, murine xenograft model was adopted to evaluate the capacity of RFNs in attenuating the tumorigenic ability of bcr-abl.<br><br>RESULTS: The RFNs efficiently disrupted bcr-abl and prematurely terminated its translation. The destruction of bcr-abl gene suppressed cell proliferation and induced cell apoptosis in CML lines and in CML stem/progenitor cells. Moreover, the RFNs significantly impaired the leukemogenic capacity of CML cells in xenograft model.<br><br>CONCLUSION: These results illustrate that the RFNs can target to disrupt bcr-abl gene and may provide a new therapeutic option for CML patients affiliated by drug resistance or disease relapse.},
}
@article {pmid31138220,
year = {2019},
author = {Yin, Y and Hao, H and Xu, X and Shen, L and Wu, W and Zhang, J and Li, Q},
title = {Generation of an MC3R knock-out pig by CRSPR/Cas9 combined with somatic cell nuclear transfer (SCNT) technology.},
journal = {Lipids in health and disease},
volume = {18},
number = {1},
pages = {122},
pmid = {31138220},
issn = {1476-511X},
mesh = {Adiposity ; Animals ; Animals, Genetically Modified ; Base Sequence ; Body Weight ; CRISPR-Cas Systems/*genetics ; Fetus/cytology ; Fibroblasts/metabolism ; *Gene Knockout Techniques ; Gene Targeting ; Genetic Vectors/metabolism ; *Nuclear Transfer Techniques ; RNA, Guide/metabolism ; Receptor, Melanocortin, Type 3/*deficiency/metabolism ; Swine ; },
abstract = {BACKGROUND: Melanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology.<br><br>METHOD: Four CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis.<br><br>RESULTS: Four vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R[-/-] and 10 MC3R[+/-] clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings.<br><br>CONCLUSION: A MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.},
}
@article {pmid31138023,
year = {2019},
author = {Sebastian, J and Hegde, K and Kumar, P and Rouissi, T and Brar, SK},
title = {Bioproduction of fumaric acid: an insight into microbial strain improvement strategies.},
journal = {Critical reviews in biotechnology},
volume = {39},
number = {6},
pages = {817-834},
doi = {10.1080/07388551.2019.1620677},
pmid = {31138023},
issn = {1549-7801},
mesh = {CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; Fumarates/*metabolism ; *Metabolic Engineering ; Rhizopus/genetics/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; },
abstract = {Fumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydrate derived platform chemical. Currently, it is commercially sourced from petrochemicals by chemical conversion. The shift to biochemical synthesis has become essential for sustainable development and for the transition to a biobased economy from a petroleum-based economy. The main limitation is that the concentrations of FA achieved during bioproduction are lower than that from a chemical process. Moreover, the high cost associated with bioproduction necessitates a higher yield to improve the feasibility of the process. To this effect, genetic modification of microorganism can be considered as an important tool to improve FA yield. This review discusses various genetic modifications strategies that have been studied in order to improve FA production. These strategies include the development of recombinant strains of Rhizopus oryzae, Escherichia coli, Saccharomyces cerevisiae, and Torulopsis glabrata as well as their mutants. The transformed strains were able to accumulate fumaric acid at a higher concentration than the corresponding wild strains but the fumaric acid titers obtained were lower than that reported with native fumaric acid producing R. oryzae strains. Moreover, one plausible adoption of gene editing tools, such as Agrobacterium-mediated transformation (AMT), CRISPR CAS-9 and RNA interference (RNAi) mediated knockout and silencing, have been proposed in order to improve fumaric acid yield. Additionally, the introduction of the glyoxylate pathway in R. oryzae to improve fumaric acid yield as well as the biosynthesis of fumarate esters have been proposed to improve the economic feasibility of the bioprocess. The adoption of some of these genetic engineering strategies may be essential to enable the development of a feasible bioproduction process.},
}
@article {pmid31136835,
year = {2019},
author = {Kim, T and Lu, TK},
title = {CRISPR/Cas-based devices for mammalian synthetic biology.},
journal = {Current opinion in chemical biology},
volume = {52},
number = {},
pages = {23-30},
doi = {10.1016/j.cbpa.2019.04.015},
pmid = {31136835},
issn = {1879-0402},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/genetics ; Gene Regulatory Networks ; *Mammals ; RNA Editing ; *Synthetic Biology ; },
abstract = {Since its first demonstration for mammalian gene editing, CRISPR/Cas technology has been widely adopted in research, industry, and medicine. Beyond indel mutations induced by Cas9 activity, recent advances in CRISPR/Cas have enabled DNA or RNA base editing. In addition, multiple orthogonal methods for the spatiotemporal regulation of CRISPR/Cas activity and repurposed Cas proteins for the visualization and relocation of specific genomic loci in living cells have been described. By harnessing the versatility of CRISPR/Cas-based devices and gene circuits, synthetic biologists are developing memory devices for lineage tracing and technologies for unbiased, high-throughput interrogation of combinatorial gene perturbations. We envision that such approaches will enable researchers to gain deeper insights into the translation of genotypes to phenotypes in healthy and diseased states.},
}
@article {pmid31136000,
year = {2019},
author = {Nishiki, I and Yoshida, T and Fujiwara, A},
title = {Complete genome sequence and characterization of virulence genes in Lancefield group C Streptococcus dysgalactiae isolated from farmed amberjack (Seriola dumerili).},
journal = {Microbiology and immunology},
volume = {63},
number = {7},
pages = {243-250},
doi = {10.1111/1348-0421.12716},
pmid = {31136000},
issn = {1348-0421},
mesh = {Animals ; Base Composition ; CRISPR-Cas Systems ; Fishes/*microbiology ; Genome, Bacterial ; Humans ; Phylogeny ; RNA, Ribosomal/genetics ; RNA, Transfer/genetics ; Sequence Analysis, DNA ; Streptococcal Infections/microbiology/*veterinary ; Streptococcus/classification/*genetics/isolation &amp; purification ; Virulence/genetics ; Virulence Factors/*genetics ; *Whole Genome Sequencing ; },
abstract = {Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.},
}
@article {pmid31134585,
year = {2019},
author = {Zhang, J and Wu, Z and Yang, H},
title = {Aminopeptidase N Knockout Pigs Are Not Resistant to Porcine Epidemic Diarrhea Virus Infection.},
journal = {Virologica Sinica},
volume = {34},
number = {5},
pages = {592-595},
pmid = {31134585},
issn = {1995-820X},
mesh = {Animals ; CD13 Antigens/*genetics ; CRISPR-Cas Systems ; Coronavirus Infections/genetics/*veterinary ; Disease Resistance/*genetics ; *Gene Knockout Techniques ; Porcine epidemic diarrhea virus/pathogenicity ; Swine/genetics ; Swine Diseases/enzymology/genetics/*virology ; },
}
@article {pmid31134410,
year = {2019},
author = {Gu, Y and Gao, J and Cao, M and Dong, C and Lian, J and Huang, L and Cai, J and Xu, Z},
title = {Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {35},
number = {6},
pages = {79},
pmid = {31134410},
issn = {1573-0972},
mesh = {*CRISPR-Cas Systems ; DNA Replication ; Escherichia coli/genetics ; Gene Dosage ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Gene Knockout Techniques ; Genetic Engineering/*methods ; Genetic Vectors ; Genomic Instability ; Industrial Microbiology ; Kluyveromyces/genetics ; Pichia/*genetics ; Plasmids/*genetics ; Promoter Regions, Genetic ; RNA, Guide ; Synthetic Biology ; *Transformation, Genetic ; },
abstract = {The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.},
}
@article {pmid31134321,
year = {2019},
author = {Long, J and Xu, Y and Ou, L and Yang, H and Xi, Y and Chen, S and Duan, G},
title = {Diversity of CRISPR/Cas system in Clostridium perfringens.},
journal = {Molecular genetics and genomics : MGG},
volume = {294},
number = {5},
pages = {1263-1275},
pmid = {31134321},
issn = {1617-4623},
mesh = {Bacteriophages/genetics ; CRISPR-Cas Systems/*genetics ; Clostridium perfringens/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computational Biology/methods ; Gene Transfer, Horizontal/genetics ; Genome, Bacterial/genetics ; Phylogeny ; Plasmids/genetics ; Polymorphism, Genetic/genetics ; },
abstract = {Clostridium perfringens is an important pathogen of human and livestock infections, posing a threat to health. The horizontal gene transfer (HGT) of plasmids that carry toxin-related genes is involved in C. perfringens pathogenicity. The CRISPR/Cas system, which has been identified in a wide range of prokaryotes, provides acquired immunity against HGT. However, information about the CRISPR/Cas system in Clostridium perfringens is still limited. In this study, 111 C. perfringens strains with publicly available genomes were used to analyze the occurrence and diversity of CRISPR/Cas system and evaluate the potential of CRISPR-based genotyping in this multi-host pathogen. A total of 59 out of the 111 genomes harbored at least one confirmed CRISPR array. Four CRISPR/Cas system subtypes, including subtypes IB, IIA, IIC, and IIID systems, were identified in 32 strains. Subtype IB system was the most prevalent in this species, which was subdivided into four subgroups displaying subgroup specificity in terms of cas gene content, repeat sequence content, and PAM. We showed that the CRISPR spacer polymorphism can be used for evolutionary studies, and that it can provide discriminatory power for typing strains. Nevertheless, the application of this approach was largely limited to strains that contain the CRISPR/Cas system. Spacer origin analysis revealed that approximately one-fifth of spacers showed significant matches to plasmids and phages, thereby suggesting the implication of CRISPR/Cas systems in controlling HGT. Collectively, our results provide new insights into the diversity and evolution of CRISPR/Cas system in C. perfringens.},
}
@article {pmid31133747,
year = {2019},
author = {Chen, Z and Zhang, Y},
title = {Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development.},
journal = {Nature genetics},
volume = {51},
number = {6},
pages = {947-951},
pmid = {31133747},
issn = {1546-1718},
support = {R01 HD092465/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development/*genetics ; Gene Expression Regulation, Developmental ; Gene Targeting ; *Genetic Association Studies/methods ; *Genome ; Homeodomain Proteins/*genetics ; Mice ; Mice, Knockout ; Zygote/*metabolism ; },
abstract = {How maternal factors in oocytes trigger zygotic genome activation (ZGA) is a long-standing question in developmental biology. Recent studies in 2-cell-like embryonic stem cells (2C-like cells) suggest that transcription factors of the DUX family are key regulators of ZGA in placental mammals[1,2]. To characterize the role of DUX in ZGA, we generated Dux cluster knockout (KO) mouse lines. Unexpectedly, we found that both Dux zygotic KO (Z-KO) and maternal and zygotic KO (MZ-KO) embryos can survive to adulthood despite showing reduced developmental potential. Furthermore, transcriptome profiling of the MZ-KO embryos revealed that loss of DUX has minimal effects on ZGA and most DUX targets in 2C-like cells are normally activated in MZ-KO embryos. Thus, contrary to the key function of DUX in inducing 2C-like cells, our data indicate that DUX has only a minor role in ZGA and that loss of DUX is compatible with mouse development.},
}
@article {pmid31133029,
year = {2019},
author = {Regmi, A and Boyd, EF},
title = {Carbohydrate metabolic systems present on genomic islands are lost and gained in Vibrio parahaemolyticus.},
journal = {BMC microbiology},
volume = {19},
number = {1},
pages = {112},
pmid = {31133029},
issn = {1471-2180},
mesh = {Bacterial Proteins/genetics ; Carbohydrate Metabolism ; Evolution, Molecular ; Gene Transfer, Horizontal ; *Genomic Islands ; Multigene Family ; Phylogeny ; Symporters/*genetics ; Vibrio parahaemolyticus/classification/*genetics ; },
abstract = {BACKGROUND: Utilizing unique carbohydrates or utilizing them more efficiently help bacteria expand and colonize new niches. Horizontal gene transfer (HGT) of catabolic systems is a powerful mechanism by which bacteria can acquire new phenotypic traits that can increase survival and fitness in different niches. In this work, we examined carbon catabolism diversity among Vibrio parahaemolyticus, a marine species that is also an important human and fish pathogen.<br><br>RESULTS: Phenotypic differences in carbon utilization between Vibrio parahaemolyticus strains lead us to examine genotypic differences in this species and the family Vibrionaceae in general. Bioinformatics analysis showed that the ability to utilize D-galactose was present in all V. parahaemolyticus but at least two distinct transporters were present; a major facilitator superfamily (MFS) transporter and a sodium/galactose transporter (SGLT). Growth and genetic analyses demonstrated that SGLT was a more efficient transporter of D-galactose and was the predominant type among strains. Phylogenetic analysis showed that D-galactose gene galM was acquired multiples times within the family Vibrionaceae and was transferred between distantly related species. The ability to utilize D-gluconate was universal within the species. Deletion of eda (VP0065), which encodes aldolase, a key enzyme in the Entner-Doudoroff (ED) pathway, reached a similar biomass to wild type when grown on D-gluconate as a sole carbon source. Two additional eda genes were identified, VPA1708 (eda2) associated with a D-glucuronate cluster and VPA0083 (eda3) that clustered with an oligogalacturonide (OGA) metabolism cluster. EDA2 and EDA3 were variably distributed among the species. A metabolic island was identified that contained citrate fermentation, L-rhamnose and OGA metabolism clusters as well as a CRISPR-Cas system. Phylogenetic analysis showed that CitF and RhaA had a limited distribution among V. parahaemolyticus, and RhaA was acquired at least three times. Within V. parahaemolyticus, two different regions contained the gene for L-arabinose catabolism and most strains had the ability to catabolism this sugar.<br><br>CONCLUSION: Our data suggest that horizontal transfer of metabolic systems among Vibrionaceae is an important source of metabolic diversity. This work identified four EDA homologues suggesting that the ED pathway plays a significant role in metabolism. We describe previously uncharacterized metabolism islands that were hotspots for the gain and loss of functional modules likely mediated by transposons.},
}
@article {pmid31132514,
year = {2019},
author = {Zhang, Y and Feng, J and Wang, P and Xia, J and Li, X and Zou, X},
title = {CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans.},
journal = {Gene},
volume = {709},
number = {},
pages = {8-16},
doi = {10.1016/j.gene.2019.04.079},
pmid = {31132514},
issn = {1879-0038},
mesh = {Ascomycota/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; *Genome, Fungal ; Organisms, Genetically Modified ; Protoplasts/*metabolism ; Saccharomyces cerevisiae/genetics ; *Transformation, Genetic ; },
abstract = {Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.},
}
@article {pmid31131955,
year = {2019},
author = {Tang, J and Lee, T and Sun, T},
title = {Single-nucleotide editing: From principle, optimization to application.},
journal = {Human mutation},
volume = {40},
number = {12},
pages = {2171-2183},
pmid = {31131955},
issn = {1098-1004},
support = {R01 MH083680/MH/NIMH NIH HHS/United States ; R01-MH083680//NIH/NIMH/International ; 81471152 and 31771141//National Natural Science Foundation of China/International ; },
mesh = {Aminohydrolases/*metabolism ; Animals ; CRISPR-Cas Systems ; Cytosine Deaminase/*metabolism ; Gene Editing/*methods ; Gene Silencing ; Humans ; Mice ; Polymorphism, Single Nucleotide ; },
abstract = {Cytosine base editors (CBEs) and adenine base editors (ABEs), which are generally composed of an engineered deaminase and a catalytically impaired CRISPR-Cas9 variant, are new favorite tools for single base substitution in cells and organisms. In this review, we summarize the principle of base-editing systems and elaborate on the evolution of different platforms of CBEs and ABEs, including their deaminase, Cas9 variants, and editing outcomes. Moreover, we highlight their applications in mouse and human cells and discuss the challenges and prospects of base editors. The ABE- and CBE systems have been used in gene silencing, pathogenic gene correction, and functional genetic screening. Single base editing is becoming a new promising genetic tool in biomedical research and gene therapy.},
}
@article {pmid31131829,
year = {2019},
author = {Mangeot, PE},
title = {[Nanoblades: Pseudoviral shuttles for CRISPR-CAS9 delivery].},
journal = {Virologie (Montrouge, France)},
volume = {23},
number = {1},
pages = {3-6},
doi = {10.1684/vir.2019.0759},
pmid = {31131829},
issn = {1267-8694},
mesh = {Biomedical Research/methods/trends ; CRISPR-Cas Systems/*genetics ; Drug Delivery Systems/methods/trends ; *Gene Editing/methods/trends ; *Gene Transfer Techniques ; Genetic Vectors/chemistry/*physiology ; Humans ; Nanotechnology/methods/trends ; Virion/*physiology ; Virus Assembly/physiology ; },
}
@article {pmid31131534,
year = {2019},
author = {Nomura, T and Inoue, K and Uehara-Yamaguchi, Y and Yamada, K and Iwata, O and Suzuki, K and Mochida, K},
title = {Highly efficient transgene-free targeted mutagenesis and single-stranded oligodeoxynucleotide-mediated precise knock-in in the industrial microalga Euglena gracilis using Cas9 ribonucleoproteins.},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2032-2034},
pmid = {31131534},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Euglena gracilis/*growth &amp; development ; *Gene Editing ; Gene Knock-In Techniques ; Microalgae/*genetics ; *Mutagenesis ; Oligodeoxyribonucleotides ; Ribonucleoproteins ; },
}
@article {pmid31131107,
year = {2019},
author = {Sharma, D and Misba, L and Khan, AU},
title = {Antibiotics versus biofilm: an emerging battleground in microbial communities.},
journal = {Antimicrobial resistance and infection control},
volume = {8},
number = {},
pages = {76},
pmid = {31131107},
issn = {2047-2994},
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacterial Infections/drug therapy/prevention &amp; control/radiotherapy ; Biofilms/*drug effects/radiation effects ; *Drug Resistance, Bacterial ; Humans ; Microbiota/*drug effects ; Photochemotherapy ; Quorum Sensing ; },
abstract = {Biofilm is a complex structure of microbiome having different bacterial colonies or single type of cells in a group; adhere to the surface. These cells are embedded in extracellular polymeric substances, a matrix which is generally composed of eDNA, proteins and polysaccharides, showed high resistance to antibiotics. It is one of the major causes of infection persistence especially in nosocomial settings through indwelling devices. Quorum sensing plays an important role in regulating the biofilm formation. There are many approaches being used to control infections by suppressing its formation but CRISPR-CAS (gene editing technique) and photo dynamic therapy (PDT) are proposed to be used as therapeutic approaches to subside bacterial biofim infections, especially caused by deadly drug resistant bad bugs.},
}
@article {pmid31130929,
year = {2019},
author = {Chen, LX and Al-Shayeb, B and Méheust, R and Li, WJ and Doudna, JA and Banfield, JF},
title = {Candidate Phyla Radiation Roizmanbacteria From Hot Springs Have Novel and Unexpectedly Abundant CRISPR-Cas Systems.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {928},
pmid = {31130929},
issn = {1664-302X},
abstract = {The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.},
}
@article {pmid31130364,
year = {2019},
author = {Jia, R and Bonifacino, JS},
title = {Lysosome Positioning Influences mTORC2 and AKT Signaling.},
journal = {Molecular cell},
volume = {75},
number = {1},
pages = {26-38.e3},
pmid = {31130364},
issn = {1097-4164},
support = {Z01 HD001607/ImNIH/Intramural NIH HHS/United States ; ZIA HD001607/ImNIH/Intramural NIH HHS/United States ; },
mesh = {ADP-Ribosylation Factors/deficiency/genetics ; CRISPR-Cas Systems ; Cell Nucleus/*metabolism/ultrastructure ; Culture Media, Serum-Free ; Endosomes/metabolism/ultrastructure ; Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; HeLa Cells ; Humans ; Kinesins/deficiency/genetics ; Lysosomes/*metabolism/ultrastructure ; MEF2 Transcription Factors/deficiency/genetics ; Mechanistic Target of Rapamycin Complex 1/*genetics/metabolism ; Mechanistic Target of Rapamycin Complex 2/*genetics/metabolism ; Proto-Oncogene Proteins c-akt/*genetics/metabolism ; Signal Transduction ; },
abstract = {Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.},
}
@article {pmid31130112,
year = {2019},
author = {Gumer, JM},
title = {The Wisdom of Germline Editing: An Ethical Analysis of the Use of CRISPR-Cas9 to Edit Human Embryos.},
journal = {The New bioethics : a multidisciplinary journal of biotechnology and the body},
volume = {25},
number = {2},
pages = {137-152},
doi = {10.1080/20502877.2019.1606151},
pmid = {31130112},
issn = {2050-2885},
mesh = {*CRISPR-Cas Systems ; Embryo Research/*ethics ; Ethical Analysis ; Gene Editing/*ethics ; Germ-Line Mutation/*ethics ; Humans ; },
abstract = {With recent reports that a Chinese scientist used CRISPR-Cas9 to heritably edit the genomes of human embryos (i.e., germline editing) brought to term, discussions regarding the ethics of the technology are urgently needed. Although certain applications of germline editing have been endorsed by both the National Academy of Sciences (US) and the Nuffield Council (UK), this paper explores the ethical concerns related even to such therapeutic uses of the technology. Additionally, this paper questions whether the technology could ever feasibly be contained to the therapeutic realm. Consequently, this paper necessarily considers the ethical concerns related to enhancement uses of the technology even if only therapeutic applications are initially considered. In light of the concomitant risks, this paper assesses the technology's countervailing benefits to conclude they do not prevail given that similar outcomes can largely be achieved with existing technologies. Consequently, this paper recommends an international ban on germline editing.},
}
@article {pmid31129735,
year = {2019},
author = {Xiong, X and Liu, W and Jiang, J and Xu, L and Huang, L and Cao, J},
title = {Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system.},
journal = {Molecular genetics and genomics : MGG},
volume = {294},
number = {5},
pages = {1251-1261},
pmid = {31129735},
issn = {1617-4623},
mesh = {Agrobacterium/genetics ; Brassica/*genetics ; Breeding/methods ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genes, Plant/genetics ; Genome, Plant/*genetics ; Homozygote ; Mutation/genetics ; Mutation Rate ; Phenotype ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T1 and T2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.},
}
@article {pmid31129119,
year = {2019},
author = {Xu, L and Lau, YS and Gao, Y and Li, H and Han, R},
title = {Life-Long AAV-Mediated CRISPR Genome Editing in Dystrophic Heart Improves Cardiomyopathy without Causing Serious Lesions in mdx Mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1407-1414},
pmid = {31129119},
issn = {1525-0024},
support = {R01 AR064241/AR/NIAMS NIH HHS/United States ; R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cardiomyopathies/*etiology/metabolism/pathology/therapy ; DNA Repair ; Dependovirus/*genetics ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; *Genetic Therapy/methods ; Genetic Vectors/administration &amp; dosage/*genetics ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Duchenne/complications/genetics/therapy ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Previous studies from others and us have demonstrated that CRISPR genome editing could offer a promising therapeutic strategy to restore dystrophin expression and function in the skeletal muscle and heart of Duchenne muscular dystrophy (DMD) mouse models. However, the long-term efficacy and safety of CRISPR genome-editing therapy for DMD has not been well established. We packaged both SaCas9 and guide RNA (gRNA) together into one AAVrh.74 vector, injected two such vectors (targeting intron 20 and intron 23, respectively) into mdx pups at day 3 and evaluated the mice at 19 months. We found that AAVrh.74-mediated life-long CRISPR genome editing in mdx mice restored dystrophin expression and improved cardiac function without inducing serious adverse effects. PCR analysis and targeted deep sequencing showed that the DSBs were mainly repaired by the precise ligation of the two cut sites. Serological and histological examination of major vital organs did not reveal any signs of tumor development or other deleterious defects arising from CRISPR genome editing. These results support that in vivo CRISPR genome editing could be developed as a safe therapeutic treatment for DMD and potentially other diseases.},
}
@article {pmid31128790,
year = {2019},
author = {Zhao, C and Wei, S and Wang, Y},
title = {A guide for drug inducible transcriptional activation with HIT systems.},
journal = {Methods in enzymology},
volume = {621},
number = {},
pages = {69-86},
doi = {10.1016/bs.mie.2019.02.032},
pmid = {31128790},
issn = {1557-7988},
mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; Transcriptional Activation/*drug effects ; },
abstract = {Precise investigation and manipulation of gene function often require modulation in a controlled and dynamic manner. In this chapter, we describe the methods to apply HIT systems for drug inducible transcriptional activation or simultaneous activation and genome editing in human cells. Together with those for editing, which are described in another chapter, HIT systems herein provide a valuable toolbox toward many biological applications, especially when precision and dynamics are required for a functional perturbation.},
}
@article {pmid31128789,
year = {2019},
author = {Zhao, C and Wei, S and Wang, Y},
title = {A guide for drug inducible genome editing with HIT systems.},
journal = {Methods in enzymology},
volume = {621},
number = {},
pages = {53-68},
doi = {10.1016/bs.mie.2019.02.031},
pmid = {31128789},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Humans ; Lentivirus/genetics ; Pharmacology/methods ; Transcription Activator-Like Effector Nucleases/genetics ; Transcription Activator-Like Effectors/genetics ; Transfection/methods ; },
abstract = {Technologies toward precise control of biological events are desired for biomedical research and potential clinical applications. Our recently reported HIT systems based on CRISPR/Cas9 and TAL effectors can achieve temporal and dose dependent drug control for genome editing. Methods are presented for the application of these optimized HIT systems in human cells in this chapter.},
}
@article {pmid31128682,
year = {2019},
author = {Morton, M and AlTamimi, N and Butt, H and Reddy, ASN and Mahfouz, M},
title = {Serine/Arginine-rich protein family of splicing regulators: New approaches to study splice isoform functions.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {283},
number = {},
pages = {127-134},
doi = {10.1016/j.plantsci.2019.02.017},
pmid = {31128682},
issn = {1873-2259},
mesh = {*Alternative Splicing ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Plant Physiological Phenomena ; Protein Isoforms ; Serine-Arginine Splicing Factors/*metabolism ; },
abstract = {Serine/arginine-rich (SR) proteins are conserved RNA-binding proteins that play major roles in RNA metabolism. They function as molecular adaptors, facilitate spliceosome assembly and modulate constitutive and alternative splicing of pre-mRNAs. Pre-mRNAs encoding SR proteins and many other proteins involved in stress responses are extensively alternatively spliced in response to diverse stresses. Hence, it is proposed that stress-induced changes in splice isoforms contribute to the adaptation of plants to stress responses. However, functions of most SR genes and their splice isoforms in stress responses are not known. Lack of easy and robust tools hindered the progress in this area. Emerging technologies such as CRISPR/Cas9 will facilitate studies of SR function by enabling the generation of single and multiple knock-out mutants of SR subfamily members. Moreover, CRISPR/Cas13 allows targeted manipulation of splice isoforms from SR and other genes in a constitutive or tissue-specific manner to evaluate functions of individual splice variants. Identification of the in vivo targets of SR proteins and their splice variants using the recently developed TRIBE (Targets of RNA-binding proteins Identified By Editing) and other methods will help unravel their mode of action and splicing regulatory elements under various conditions. These new approaches are expected to provide significant new insights into the roles of SRs and splice isoforms in plants adaptation to diverse stresses.},
}
@article {pmid31128273,
year = {2019},
author = {Ballard, E and Weber, J and Melchers, WJG and Tammireddy, S and Whitfield, PD and Brakhage, AA and Brown, AJP and Verweij, PE and Warris, A},
title = {Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {130},
number = {},
pages = {98-106},
pmid = {31128273},
issn = {1096-0937},
support = {MR/M026663/1/MRC_/Medical Research Council/United Kingdom ; MR/N006364/1/MRC_/Medical Research Council/United Kingdom ; BB/M010996/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Antifungal Agents/pharmacology ; Aspergillosis/microbiology ; Aspergillus fumigatus/*drug effects/*genetics/growth &amp; development/isolation &amp; purification ; Azoles/*pharmacology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Multiple, Fungal/*genetics ; Ergosterol ; Fungal Proteins/genetics ; Genotype ; Host-Pathogen Interactions ; Humans ; Itraconazole/pharmacology ; Microbial Sensitivity Tests ; Mycelium/drug effects/growth &amp; development ; Phenotype ; *Polymorphism, Single Nucleotide ; },
abstract = {The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213[*] in svf1 and 167[*] in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167[*] in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.},
}
@article {pmid31127573,
year = {2019},
author = {Kan, Y and Hendrickson, EA},
title = {Conversion Tract Analysis of Homology-Directed Genome Editing Using Oligonucleotide Donors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1999},
number = {},
pages = {131-144},
doi = {10.1007/978-1-4939-9500-4_7},
pmid = {31127573},
issn = {1940-6029},
support = {R01 CA190492/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing/*methods ; Genes, Reporter/genetics ; Genetic Vectors/genetics ; Humans ; Lentivirus/genetics ; Luminescent Proteins/chemistry/*genetics ; Oligonucleotides/genetics ; Primary Cell Culture/methods ; *Recombinational DNA Repair ; Transfection/methods ; },
abstract = {Homology-directed genome editing is the intentional alteration of an endogenous genetic locus using information from an exogenous homology donor. A conversion tract is defined as the amount of genetic information that is converted from the homology donor to a given strand of the targeted chromosomal locus. Because of this, conversion tract analysis retrospectively not only elucidates the mechanism of homology-directed genome editing but also provides valuable insights on the conversion efficiency of every nucleotide in the homology donor. Here we describe a blue fluorescent protein-to-green fluorescent protein conversion system that can be conveniently used to measure the efficiency and analyze the lengths of conversion tracts of homology-directed genome editing using oligonucleotide donors in mammalian cells.},
}
@article {pmid31127311,
year = {2019},
author = {Mann, CM and Martínez-Gálvez, G and Welker, JM and Wierson, WA and Ata, H and Almeida, MP and Clark, KJ and Essner, JJ and McGrail, M and Ekker, SC and Dobbs, D},
title = {The Gene Sculpt Suite: a set of tools for genome editing.},
journal = {Nucleic acids research},
volume = {47},
number = {W1},
pages = {W175-W182},
pmid = {31127311},
issn = {1362-4962},
support = {R01 GM055877/GM/NIGMS NIH HHS/United States ; R01 GM063904/GM/NIGMS NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; T32 GM065841/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Databases, Genetic ; *Gene Editing ; Genetic Engineering/*methods ; Humans ; *Software ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org.},
}
@article {pmid31127293,
year = {2019},
author = {Owens, DDG and Caulder, A and Frontera, V and Harman, JR and Allan, AJ and Bucakci, A and Greder, L and Codner, GF and Hublitz, P and McHugh, PJ and Teboul, L and de Bruijn, MFTR},
title = {Microhomologies are prevalent at Cas9-induced larger deletions.},
journal = {Nucleic acids research},
volume = {47},
number = {14},
pages = {7402-7417},
pmid = {31127293},
issn = {1362-4962},
support = {MC_UP_1502/1/MRC_/Medical Research Council/United Kingdom ; MC_UU_00016/2/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Chromosome Breakpoints ; *Chromosome Deletion ; Chromosomes, Mammalian/*genetics/metabolism ; DNA End-Joining Repair ; Deoxyribonuclease I/genetics/metabolism ; Endonucleases/genetics/metabolism ; Gene Editing/*methods ; Mice ; Models, Genetic ; RNA, Guide/genetics/metabolism ; *Sequence Deletion ; },
abstract = {The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.},
}
@article {pmid31127194,
year = {2019},
author = {Sampson, TR and Saroj, SD and Llewellyn, AC and Tzeng, YL and Weiss, DS},
title = {Author Correction: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence.},
journal = {Nature},
volume = {570},
number = {7760},
pages = {E30-E31},
doi = {10.1038/s41586-019-1253-9},
pmid = {31127194},
issn = {1476-4687},
abstract = {Change history: We could not replicate the results in Fig. 2a and g of this Letter, and new information has revealed a flaw in the interpretation of Fig. 2h. As a result, we do not have evidence to support RNA degradation as the mechanism that underlies Cas9-mediated regulation of FTN_1103 mRNA expression; see accompanying Amendment. This has not been corrected online.},
}
@article {pmid31127080,
year = {2019},
author = {Wei, YL and Yang, WX},
title = {Kinesin-14 motor protein KIFC1 participates in DNA synthesis and chromatin maintenance.},
journal = {Cell death &amp; disease},
volume = {10},
number = {6},
pages = {402},
pmid = {31127080},
issn = {2041-4889},
mesh = {Aneuploidy ; CRISPR-Cas Systems/genetics ; Cell Nucleus/metabolism ; Cell Proliferation ; Chromatin/chemistry/*metabolism ; DNA/*biosynthesis ; DNA Replication ; Gene Editing ; HEK293 Cells ; Humans ; Kinesins/deficiency/genetics/*metabolism ; Lamin Type A/metabolism ; Lamin Type B/metabolism ; Mitosis ; S Phase Cell Cycle Checkpoints ; Spindle Apparatus/metabolism ; },
abstract = {The nuclear localization signal (NLS) in kinesin-14 KIFC1 is associated with nuclear importins and Ran gradient, but detailed mechanism remains unknown. In this study, we found that KIFC1 proteins have specific transport characteristics during cell cycle. In the absence of KIFC1, cell cycle kinetics decrease significantly with a prolonged S phase. After KIFC1 overexpression, the duration of S phase becomes shorten. KIFC1 may transport the recombinant/replicate-related proteins into the nucleus, meanwhile avoiding excessive KIFC1 in the cytoplasm, which results in aberrant microtubule bundling. Interestingly, the deletion of kifc1 in human cells results in a higher ratio of aberrant nuclear membrane, and the degradation of lamin B and lamin A/C. We also found that kifc1 deletion leads to defects in metaphase mitotic spindle assembly, and then results in chromosome structural abnormality. The kifc1[-/-] cells finally form micronuclei in daughter cells, and results in aneuploidy and chromosome loss in cell cycle. In this study, we demonstrate that kinesin-14 KIFC1 proteins involve in regulating DNA synthesis in S phase, and chromatin maintenance in mitosis, and maintain cell growth in a nuclear transport-independent way.},
}
@article {pmid31126740,
year = {2019},
author = {Ritchie, C and Cordova, AF and Hess, GT and Bassik, MC and Li, L},
title = {SLC19A1 Is an Importer of the Immunotransmitter cGAMP.},
journal = {Molecular cell},
volume = {75},
number = {2},
pages = {372-381.e5},
pmid = {31126740},
issn = {1097-4164},
support = {R00 CA190896/CA/NCI NIH HHS/United States ; T32 GM007365/GM/NIGMS NIH HHS/United States ; T32 GM007276/GM/NIGMS NIH HHS/United States ; DP2 CA228044/CA/NCI NIH HHS/United States ; DP2 HD084069/HD/NICHD NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Membrane/genetics ; Genome, Human/genetics ; Humans ; Immunity, Innate/*genetics ; Membrane Proteins/genetics ; Neoplasms/*genetics/immunology ; Nucleotides, Cyclic/*genetics/immunology ; Reduced Folate Carrier Protein/*genetics ; Signal Transduction/genetics ; },
abstract = {2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen, we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDA[S]). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.},
}
@article {pmid31126147,
year = {2019},
author = {Diakatou, M and Manes, G and Bocquet, B and Meunier, I and Kalatzis, V},
title = {Genome Editing as a Treatment for the Most Prevalent Causative Genes of Autosomal Dominant Retinitis Pigmentosa.},
journal = {International journal of molecular sciences},
volume = {20},
number = {10},
pages = {},
pmid = {31126147},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Mutation ; Retinitis Pigmentosa/genetics/*therapy ; },
abstract = {: Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of diseases with more than 250 causative genes. The most common form is retinitis pigmentosa. IRDs lead to vision impairment for which there is no universal cure. Encouragingly, a first gene supplementation therapy has been approved for an autosomal recessive IRD. However, for autosomal dominant IRDs, gene supplementation therapy is not always pertinent because haploinsufficiency is not the only cause. Disease-causing mechanisms are often gain-of-function or dominant-negative, which usually require alternative therapeutic approaches. In such cases, genome-editing technology has raised hopes for treatment. Genome editing could be used to i) invalidate both alleles, followed by supplementation of the wild type gene, ii) specifically invalidate the mutant allele, with or without gene supplementation, or iii) to correct the mutant allele. We review here the most prevalent genes causing autosomal dominant retinitis pigmentosa and the most appropriate genome-editing strategy that could be used to target their different causative mutations.},
}
@article {pmid31126133,
year = {2019},
author = {Nadeem, M and Li, J and Yahya, M and Sher, A and Ma, C and Wang, X and Qiu, L},
title = {Research Progress and Perspective on Drought Stress in Legumes: A Review.},
journal = {International journal of molecular sciences},
volume = {20},
number = {10},
pages = {},
pmid = {31126133},
issn = {1422-0067},
mesh = {Acclimatization ; Agriculture ; CRISPR-Cas Systems ; Droughts ; Fabaceae/genetics/*physiology ; Plant Breeding/methods ; Plants, Genetically Modified/genetics/physiology ; Quantitative Trait Loci ; Stress, Physiological ; },
abstract = {Climate change, food shortage, water scarcity, and population growth are some of the threatening challenges being faced in today's world. Drought stress (DS) poses a constant challenge for agricultural crops and has been considered a severe constraint for global agricultural productivity; its intensity and severity are predicted to increase in the near future. Legumes demonstrate high sensitivity to DS, especially at vegetative and reproductive stages. They are mostly grown in the dry areas and are moderately drought tolerant, but severe DS leads to remarkable production losses. The most prominent effects of DS are reduced germination, stunted growth, serious damage to the photosynthetic apparatus, decrease in net photosynthesis, and a reduction in nutrient uptake. To curb the catastrophic effect of DS in legumes, it is imperative to understand its effects, mechanisms, and the agronomic and genetic basis of drought for sustainable management. This review highlights the impact of DS on legumes, mechanisms, and proposes appropriate management approaches to alleviate the severity of water stress. In our discussion, we outline the influence of water stress on physiological aspects (such as germination, photosynthesis, water and nutrient uptake), growth parameters and yield. Additionally, mechanisms, various management strategies, for instance, agronomic practices (planting time and geometry, nutrient management), plant growth-promoting Rhizobacteria and arbuscular mycorrhizal fungal inoculation, quantitative trait loci (QTLs), functional genomics and advanced strategies (CRISPR-Cas9) are also critically discussed. We propose that the integration of several approaches such as agronomic and biotechnological strategies as well as advanced genome editing tools is needed to develop drought-tolerant legume cultivars.},
}
@article {pmid31125685,
year = {2019},
author = {Hameed, A and Shan-E-Ali Zaidi, S and Sattar, MN and Iqbal, Z and Tahir, MN},
title = {CRISPR technology to combat plant RNA viruses: A theoretical model for Potato virus Y (PVY) resistance.},
journal = {Microbial pathogenesis},
volume = {133},
number = {},
pages = {103551},
doi = {10.1016/j.micpath.2019.103551},
pmid = {31125685},
issn = {1096-1208},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural ; Disease Resistance/*genetics ; Gene Editing/methods ; Gene Targeting ; Genes, Plant/genetics ; Genome, Viral ; Models, Theoretical ; Plant Diseases/*genetics/prevention &amp; control/virology ; Plant Viruses/*genetics/immunology/pathogenicity ; Plants/genetics ; Plants, Genetically Modified/immunology/virology ; Potyvirus/*genetics/pathogenicity ; RNA Viruses/*genetics/immunology ; Ribonucleases/genetics ; },
abstract = {RNA viruses are the most diverse phytopathogens which cause severe epidemics in important agricultural crops and threaten the global food security. Being obligatory intracellular pathogens, these viruses have developed fine-tuned evading mechanisms and are non-responsive to most of the prophylactic treatments. Additionally, their sprint ability to overcome host defense demands a broad-spectrum and durable mechanism of resistance. In context of CRISPR-Cas discoveries, some variants of Cas effectors have been characterized as programmable RNA-guided RNases in the microbial genomes and could be reprogramed in mammalian and plant cells with guided RNase activity. Recently, the RNA variants of CRISPR-Cas systems have been successfully employed in plants to engineer resistance against RNA viruses. Some variants of CRISPR-Cas9 have been tamed either for directly targeting plant RNA viruses' genome or through targeting the host genes/factors assisting in viral proliferation. The new frontiers in CRISPR-Cas discoveries, and more importantly shifting towards RNA targeting will pyramid the opportunities in plant virus research. The current review highlights the probable implications of CRISPR-Cas system to confer the pathogen-derived or host-mediated resistance against phytopathogenic RNA viruses. Furthermore, a multiplexed CRISPR-Cas13a methodology is proposed here to combat Potato virus Y (PVY); a globally diverse phytopathogen infecting multiple crops.},
}
@article {pmid31125376,
year = {2019},
author = {Wieghaus, A and Prüfer, D and Schulze Gronover, C},
title = {Loss of function mutation of the Rapid Alkalinization Factor (RALF1)-like peptide in the dandelion Taraxacum koksaghyz entails a high-biomass taproot phenotype.},
journal = {PloS one},
volume = {14},
number = {5},
pages = {e0217454},
pmid = {31125376},
issn = {1932-6203},
mesh = {Biomass ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; *Loss of Function Mutation ; Peptide Hormones/*genetics ; Plant Proteins/*genetics ; Plant Roots/anatomy &amp; histology/*genetics/growth &amp; development ; Taraxacum/anatomy &amp; histology/*genetics/growth &amp; development ; },
abstract = {The Russian dandelion (Taraxacum koksaghyz) is a promising source of inulin and natural rubber because large amounts of both feedstocks can be extracted from its roots. However, the domestication of T. koksaghyz requires the development of stable agronomic traits such as higher yields of inulin and natural rubber, a higher root biomass, and an agronomically preferable root morphology which is more suitable for cultivation and harvesting. Arabidopsis thaliana Rapid Alkalinisation Factor 1 (RALF1) has been shown to suppress root growth. We identified the T. koksaghyz orthologue TkRALF-like 1 and knocked out the corresponding gene (TkRALFL1) using the CRISPR/Cas9 system to determine its impact on root morphology, biomass, and inulin and natural rubber yields. The TkRALFL1 knockout lines more frequently developed a taproot phenotype which is easier to cultivate and harvest, as well as a higher root biomass and greater yields of both inulin and natural rubber. The TkRALFL1 gene could therefore be suitable as a genetic marker to support the breeding of profitable new dandelion varieties with improved agronomic traits. To our knowledge, this is the first study addressing the root system of T. koksaghyz to enhance the agronomic performance.},
}
@article {pmid31125290,
year = {2019},
author = {Demireva, EY and Xie, H and Flood, ED and Thompson, JM and Seitz, BM and Watts, SW},
title = {Creation of the 5-hydroxytryptamine receptor 7 knockout rat as a tool for cardiovascular research.},
journal = {Physiological genomics},
volume = {51},
number = {7},
pages = {290-301},
pmid = {31125290},
issn = {1531-2267},
mesh = {Animals ; *Animals, Genetically Modified ; Blood Pressure/drug effects ; Body Weight/genetics ; CRISPR-Cas Systems ; Cardiovascular Diseases/*genetics ; Cardiovascular System/metabolism ; Exons ; Female ; Frameshift Mutation ; Gene Deletion ; *Gene Knockout Techniques ; Genotype ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin/*genetics ; Serotonin/pharmacology ; },
abstract = {Using CRISPR-Cas9 technology, we created a 5-HT7 receptor global knockout (KO) rat, on a Sprague-Dawley background, for use in cardiovascular physiology studies focused on blood pressure regulation. A stable line carrying indels in exons 1 and 2 of the rat Htr7 locus was established and validated. Surprisingly, 5-HT7 receptor mRNA was still present in the KO rat. However, extensive cDNA and genomic sequencing of KO tissues confirmed an 11 bp deletion in exon 1 and 4 bp deletion in exon 2. The exon 1 deletion resulted in a frameshifted mRNA sequence coding for a nonfunctional protein. While the Htr1B locus was a potential off-target for the guide RNAs designed for exon 2 of Htr7, there were no off-target sequence changes at this locus in the originating founder. When the F2 generation of KO was compared with wild-type (WT) counterparts, neither the male nor female KO rats were different in body size, fat weights, or mass of organs (kidney, heart, and brain) important to blood pressure. Females were smaller in mass than their counterpart males. Clinical measures of plasma from nonfasted rats revealed largely similar values, comparing WT and KO, of glucose, blood urea nitrogen, creatinine, phosphate, calcium, and albumin to name a few. Loss of a functional 5-HT7 receptor was validated by the complete loss of relaxation to the 5-HT1/7 receptor agonist 5-carboxamidotryptamine in the isolated abdominal vena cava. This newly created 5-HT7 receptor KO rat will be of use to investigate the importance of the 5-HT7 receptor in blood pressure regulation.},
}
@article {pmid31124711,
year = {2019},
author = {Straiton, J},
title = {Genetically modified humans: the X-Men of scientific research.},
journal = {BioTechniques},
volume = {66},
number = {6},
pages = {249-252},
doi = {10.2144/btn-2019-0056},
pmid = {31124711},
issn = {1940-9818},
mesh = {*CRISPR-Cas Systems ; DNA/*genetics ; Female ; Gene Editing/*methods ; Genetic Therapy/methods ; Genome, Human ; Human Genetics/methods ; Humans ; Male ; Organisms, Genetically Modified/genetics ; },
}
@article {pmid31124706,
year = {2019},
author = {Fan, J and Xia, Y and Wang, GL},
title = {An improved heteroduplex analysis for rapid genotyping of SNPs and single base pair indels.},
journal = {BioTechniques},
volume = {67},
number = {1},
pages = {6-10},
doi = {10.2144/btn-2019-0012},
pmid = {31124706},
issn = {1940-9818},
mesh = {Base Pairing ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Genotyping Techniques/economics/methods ; Heteroduplex Analysis/economics/*methods ; *INDEL Mutation ; Oryza/genetics ; *Polymorphism, Single Nucleotide ; Time Factors ; },
abstract = {SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always occur in the mutants generated from chemical mutagenesis or CRISPR/Cas9-mediated genome editing. Most of the current SNP and SBP indel genotyping methods are time-consuming and/or require special equipment or reagents. Here, we describe an improved heteroduplex analysis method, named iHDA, that can readily discriminate SNP and SBP indel alleles with specially designed DNA probes that harbor a couple of nucleotides adjacent to the SNP site. By hybridizing with the same probe, SNP and SBP indel alleles form different heteroduplexes, differing in bulge size, which show different mobility on a polyacrylamide gel. Therefore, iHDA is an easy, fast and inexpensive method for SNP and SBP indel genotyping.},
}
@article {pmid31124307,
year = {2019},
author = {Spiegler, S and Rath, M and Much, CD and Sendtner, BS and Felbor, U},
title = {Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson's two-hit hypothesis in vitro.},
journal = {Molecular genetics &amp; genomic medicine},
volume = {7},
number = {7},
pages = {e00755},
pmid = {31124307},
issn = {2324-9269},
mesh = {Adult ; CRISPR-Cas Systems ; Cells, Cultured ; Endothelial Cells/*metabolism ; Gene Editing/*methods ; Genetic Therapy/methods ; Hemangioma, Cavernous, Central Nervous System/*genetics/therapy ; Humans ; KRIT1 Protein/*genetics/metabolism ; Proof of Concept Study ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard-to-transfect cells remains challenging.<br><br>METHODS: In a proof-of-principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high-fidelity Cas9 variant was then transfected into patient-derived BOECs using a ribonucleoprotein complex and a single-strand DNA oligonucleotide. In addition, patient-specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation.<br><br>RESULTS: Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9-induced mutations in predicted off-target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1-deficient BOECs displayed increased resistance to stress-induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1-deficient BOECs largely replaced corrected as well as heterozygous BOECs.<br><br>CONCLUSION: We here demonstrate that a non-viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard-to-transfect endothelial cells (ECs). Comparing patient-derived isogenic CCM1[+/+] , CCM1[+/-] , and CCM1[-/-] ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.},
}
@article {pmid31124015,
year = {2019},
author = {Wang, Z and Yang, L and Qu, S and Zhang, C},
title = {CRISPR-mediated gene editing to rescue haploinsufficient obesity syndrome.},
journal = {Protein &amp; cell},
volume = {10},
number = {10},
pages = {705-708},
pmid = {31124015},
issn = {1674-8018},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Obesity ; },
}
@article {pmid31122933,
year = {2019},
author = {Sahel, DK and Mittal, A and Chitkara, D},
title = {CRISPR/Cas System for Genome Editing: Progress and Prospects as a Therapeutic Tool.},
journal = {The Journal of pharmacology and experimental therapeutics},
volume = {370},
number = {3},
pages = {725-735},
doi = {10.1124/jpet.119.257287},
pmid = {31122933},
issn = {1521-0103},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Transfer Techniques/*trends ; Genetic Therapy/*trends ; Humans ; },
abstract = {CRISPR was first observed in 1987 in bacteria and archaea and was later confirmed as part of bacterial adaptive immunity against the attacking phage. The CRISPR/Cas restriction system involves a restriction endonuclease enzyme guided by a hybrid strand of RNA consisting of CRISPR RNA and trans-activating RNA, which results in gene knockout or knockin followed by nonhomologous end joining and homology-directed repair. Owing to its efficiency, specificity, and reproducibility, the CRISPR/Cas restriction system was said to be a breakthrough in the field of biotechnology. Apart from its application in biotechnology, CRISPR/Cas has been explored for its therapeutic potential in several diseases including cancer, Alzheimer's disease, sickle cell disease, Duchenne muscular dystrophy, neurologic disorders, etc., wherein CRISPR/Cas components such as Cas9/single guide RNA (sgRNA) ribonucleoprotein, sgRNA/mRNA, and plasmid were delivered. However, limitations including immunogenicity, low transfection, limited payload, instability, and off-target binding pose hurdles in its therapeutic use. Nonviral vectors (including cationic polymers, lipids, etc.), classically used as carriers for therapeutic genes, were used to deliver CRISPR/Cas components and showed interesting results. Herein, we discuss the CRISPR/Cas system and its brief history and classification, followed by its therapeutic applications using current nonviral delivery strategies.},
}
@article {pmid31121413,
year = {2019},
author = {Braun, CJ and Hemann, MT},
title = {Functional screens identify coordinators of RNA molecule birth, life, and death as targetable cancer vulnerabilities.},
journal = {Current opinion in genetics &amp; development},
volume = {54},
number = {},
pages = {105-109},
doi = {10.1016/j.gde.2019.04.003},
pmid = {31121413},
issn = {1879-0380},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic/genetics ; Humans ; Mice ; Neoplasms/*genetics ; RNA/genetics ; RNA Stability/*genetics ; RNA, Untranslated/*genetics ; RNA-Binding Proteins/genetics ; },
abstract = {RNA molecules are subject to a complex co-transcriptional and post-transcriptional life cycle, controlled at all stages by RNA binding proteins (RBPs) and non-coding RNAs that influence mRNA stability, splicing, localization, and decay. Together with mechanisms regulating the process of transcription itself, non-coding RNAs and RBPs contribute to a model of para-transcriptional coordination of gene expression, which is utilized during normal tissue physiology and cancer development in order to execute complex gene expression programs. Several key regulators of RNA biology, such as certain splice factors, represent bona fide cancer vulnerabilities, but our understanding of these processes is still far away from being comprehensive. Genetic forward screens utilizing technologies such as transposons, RNAi and CRISPR aid the field in rapidly establishing functional phenotypes and genetic cancer cell addictions. This review focuses on four individual regulatory gene expression processes governed by regulators of the RNA life cycle, the impact of functional genomics on streamlining the discovery process and the role of such mechanisms in tumor biology.},
}
@article {pmid31121300,
year = {2020},
author = {Wandera, KG and Collins, SP and Wimmer, F and Marshall, R and Noireaux, V and Beisel, CL},
title = {An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.},
journal = {Methods (San Diego, Calif.)},
volume = {172},
number = {},
pages = {42-50},
doi = {10.1016/j.ymeth.2019.05.014},
pmid = {31121300},
issn = {1095-9130},
mesh = {Bacteriophages/metabolism ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/metabolism ; CRISPR-Cas Systems/*genetics ; Enzyme Assays/instrumentation/*methods ; Escherichia coli/enzymology/genetics/immunology/virology ; Escherichia coli Proteins/*antagonists &amp; inhibitors/metabolism ; Fluorescence ; Gene Editing/methods ; Genes, Reporter/genetics ; Green Fluorescent Proteins/chemistry/genetics ; Host-Pathogen Interactions/genetics/immunology ; Protein Biosynthesis ; Transcription, Genetic ; Viral Proteins/*metabolism ; },
abstract = {The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.},
}
@article {pmid31119817,
year = {2019},
author = {Sake, HJ and Frenzel, A and Lucas-Hahn, A and Nowak-Imialek, M and Hassel, P and Hadeler, KG and Hermann, D and Becker, R and Eylers, H and Hein, R and Baars, W and Brinkmann, A and Schwinzer, R and Niemann, H and Petersen, B},
title = {Possible detrimental effects of beta-2-microglobulin knockout in pigs.},
journal = {Xenotransplantation},
volume = {26},
number = {6},
pages = {e12525},
doi = {10.1111/xen.12525},
pmid = {31119817},
issn = {1399-3089},
support = {TRR/SFB 127//Deutsche Forschungsgemeinschaft/International ; //DFG via TRR/SFB 127/International ; //cluster of excellence REBIRTH/International ; //Braukmann-Wittenberg-Herz-Stiftung/International ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Female ; Galactosyltransferases/*genetics ; Gene Knockout Techniques/methods ; Histocompatibility Antigens Class I/*genetics ; Nuclear Transfer Techniques ; Pregnancy ; Swine ; *Transplantation, Heterologous/methods ; beta 2-Microglobulin/*genetics ; },
abstract = {BACKGROUND: Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits.<br><br>METHODS: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta-2-microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M-KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules.<br><br>RESULTS: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4&#xa0;weeks due to unexpected disease processes.<br><br>CONCLUSION: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta-2-microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.},
}
@article {pmid31119685,
year = {2019},
author = {Bae, T and Hur, JW and Kim, D and Hur, JK},
title = {Recent trends in CRISPR-Cas system: genome, epigenome, and transcriptome editing and CRISPR delivery systems.},
journal = {Genes &amp; genomics},
volume = {41},
number = {8},
pages = {871-877},
pmid = {31119685},
issn = {2092-9293},
mesh = {Animals ; *CRISPR-Cas Systems ; Epigenomics/*methods ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; Transcriptome ; },
abstract = {BACKGROUND: The CRISPR-Cas systems have emerged as a robust genome editing tool useful in various fields of research. With the discovery and development of the orthologous CRISPR-Cas systems, their genome editing efficiency have improved.<br><br>OBJECTIVE: In this review, we aim to present the recent developments and applications of the CRISPR-Cas systems.<br><br>METHODS: First, we introduce how the advancements of CRISPR technology enabled genome editing to single base precision. Then, we discuss the CRISPR based methods for targeted transcriptional regulation, epigenome editing, and RNA editing. Finally, we review the CRISPR delivery systems highlighting recent attempts to integrate nanotechnology to develop novel CRISPR delivery modalities.<br><br>CONCLUSION: Here, we review the recent trends in CRISPR-based biotechnologies, encompassing genome editing, epigenome regulation and direct RNA targeting and provide an overview of methods employed for CRISPR delivery with an emphasis on the most recent nanotechnology-based delivery strategies. We anticipate that the development of CRISPR based technology will continue to explore novel methods.},
}
@article {pmid31119529,
year = {2019},
author = {Mitsui, R and Yamada, R and Ogino, H},
title = {Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution.},
journal = {Applied biochemistry and biotechnology},
volume = {189},
number = {3},
pages = {810-821},
doi = {10.1007/s12010-019-03040-y},
pmid = {31119529},
issn = {1559-0291},
mesh = {CRISPR-Cas Systems/*genetics ; Directed Molecular Evolution/*methods ; Genome, Fungal/*genetics ; Hydrogen-Ion Concentration ; Mutation ; Random Amplified Polymorphic DNA Technique ; Saccharomyces cerevisiae/cytology/*genetics/*physiology ; Stress, Physiological/*genetics ; },
abstract = {In bioprocesses, a microorganism with high tolerance to various stresses would be advantageous for efficient bio-based chemical production. Yeast Saccharomyces cerevisiae has long been used in the food industry because of its safety and convenience, and genetically engineered S. cerevisiae strains have been constructed and used for the production of various bio-based chemicals. In this study, we developed a novel genome shuffling method for S. cerevisiae using CRISPR-Cas. By using this, the thermotolerant mutant strain T8-292, which can grow well at 39 °C, was successfully created. The strain also showed higher cell viability in low pH and high ethanol concentration. In addition, the differences in genome structure between mutant and parent strains were suggested by random amplified polymorphic DNA PCR method. Our genome shuffling method could be a promising strategy for improvement of various stress tolerance in S. cerevisiae.},
}
@article {pmid31119353,
year = {2019},
author = {Kormanec, J and Rezuchova, B and Homerova, D and Csolleiova, D and Sevcikova, B and Novakova, R and Feckova, L},
title = {Recent achievements in the generation of stable genome alterations/mutations in species of the genus Streptomyces.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {14},
pages = {5463-5482},
doi = {10.1007/s00253-019-09901-0},
pmid = {31119353},
issn = {1432-0614},
mesh = {Bacteriophages/genetics ; Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; Genetic Vectors ; *Genome, Bacterial ; *Microorganisms, Genetically-Modified ; *Mutation ; Plasmids/genetics ; Recombination, Genetic ; Streptomyces/*genetics/virology ; },
abstract = {The bacteria of the genus Streptomyces are the most valuable source of natural products of industrial and medical importance. A recent explosion of Streptomyces genome sequence data has revealed the enormous genetic potential of new biologically active compounds, although many of them are silent under laboratory conditions. Efficient and stable manipulation of the genome is necessary to induce their production. Comprehensive studies in the past have led to a large and versatile collection of molecular biology tools for gene manipulation of Streptomyces, including various replicative plasmids. However, biotechnological applications of these bacteria require stable genome alterations/mutations. To accomplish such stable genome editing, two major strategies for streptomycetes have been developed: (1) integration into the chromosome through Att/Int site-specific integration systems based on Streptomyces actinophages (ΦC31, ΦBT1, VWB, TG1, SV1, R4, ΦJoe, μ1/6) or pSAM2 integrative plasmid; (2) integration by homologous recombination using suicidal non-replicating vectors. The present review is an attempt to provide a comprehensive summary of both approaches for stable genomic engineering and to outline recent advances in these strategies, such as CRISPR/Cas9, which have successfully manipulated Streptomyces strains to improve their biotechnological properties and increase production of natural or new gene-manipulated biologically active compounds.},
}
@article {pmid31119128,
year = {2019},
author = {Eckerstorfer, MF and Dolezel, M and Heissenberger, A and Miklau, M and Reichenbecher, W and Steinbrecher, RA and Waßmann, F},
title = {Corrigendum: An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs).},
journal = {Frontiers in bioengineering and biotechnology},
volume = {7},
number = {},
pages = {90},
doi = {10.3389/fbioe.2019.00090},
pmid = {31119128},
issn = {2296-4185},
abstract = {[This corrects the article DOI: 10.3389/fbioe.2019.00031.].},
}
@article {pmid31116473,
year = {2020},
author = {Qin, L and Li, J and Wang, Q and Xu, Z and Sun, L and Alariqi, M and Manghwar, H and Wang, G and Li, B and Ding, X and Rui, H and Huang, H and Lu, T and Lindsey, K and Daniell, H and Zhang, X and Jin, S},
title = {High-efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {1},
pages = {45-56},
pmid = {31116473},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gossypium/*genetics ; Mutation ; Tetraploidy ; },
abstract = {The base-editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum-Base Editor 3 (GhBE3) base-editing system has been developed to create single-base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32-GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an 'editing window', approximately six-nucleotide windows of -17 to -12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off-target sites predicted by CRISPR-P and Cas-OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole-genome sequencing analyses on two GhCLA-edited and one wild-type plants with about 100× depth showed that no bona fide off-target mutations were detectable from 1500 predicted potential off-target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.},
}
@article {pmid31115652,
year = {2019},
author = {Lee, M and Kim, H},
title = {Therapeutic application of the CRISPR system: current issues and new prospects.},
journal = {Human genetics},
volume = {138},
number = {6},
pages = {563-590},
pmid = {31115652},
issn = {1432-1203},
mesh = {*CRISPR-Cas Systems ; Epigenomics/methods ; Gene Editing/*methods ; Gene Rearrangement ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Humans ; *Mutagenesis ; Reproducibility of Results ; },
abstract = {Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeat (the CRISPR) system has been increasingly applied to therapeutic genome editing. Employment of several viral and non-viral vectors has enabled efficient delivery of the CRISPR system to target cells or tissues. In addition, the CRISPR system is able to modulate the target gene's expression in various ways, such as mutagenesis, gene integration, epigenome regulation, chromosomal rearrangement, base editing and mRNA editing. However, there are still limitations hindering an ideal application of the system: inefficient delivery, dysregulation of the delivered gene, the immune response against the CRISPR system, the off-target effects or the unintended on-target mutations. In addition, there are recent discoveries that have not been yet applied to CRISPR-mediated therapeutic genome editing. Here, we review the overall principles related to the therapeutic application of the CRISPR system, along with new strategies for the further application and prospects to overcome the limitations.},
}
@article {pmid31114911,
year = {2019},
author = {Béguin, P and Chekli, Y and Sezonov, G and Forterre, P and Krupovic, M},
title = {Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei.},
journal = {Nucleic acids research},
volume = {47},
number = {12},
pages = {6386-6395},
pmid = {31114911},
issn = {1362-4962},
mesh = {Archaea/enzymology ; DNA Transposable Elements ; DNA, Archaeal/chemistry ; Euryarchaeota/*enzymology/genetics ; Integrases/*metabolism ; Nucleotide Motifs ; Oligonucleotides ; },
abstract = {Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.},
}
@article {pmid31114866,
year = {2019},
author = {Quan, J and Langelier, C and Kuchta, A and Batson, J and Teyssier, N and Lyden, A and Caldera, S and McGeever, A and Dimitrov, B and King, R and Wilheim, J and Murphy, M and Ares, LP and Travisano, KA and Sit, R and Amato, R and Mumbengegwi, DR and Smith, JL and Bennett, A and Gosling, R and Mourani, PM and Calfee, CS and Neff, NF and Chow, ED and Kim, PS and Greenhouse, B and DeRisi, JL and Crawford, ED},
title = {FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences.},
journal = {Nucleic acids research},
volume = {47},
number = {14},
pages = {e83},
pmid = {31114866},
issn = {1362-4962},
support = {R35 HL140026/HL/NHLBI NIH HHS/United States ; //Wellcome Trust/United Kingdom ; K23 HL138461/HL/NHLBI NIH HHS/United States ; K24 HL133390/HL/NHLBI NIH HHS/United States ; R01 HL110969/HL/NHLBI NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacteria/classification/drug effects/genetics ; Bacterial Infections/diagnosis/genetics/prevention &amp; control ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Drug Resistance, Bacterial/*drug effects/genetics ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Metagenomics/methods ; Reproducibility of Results ; Sensitivity and Specificity ; },
abstract = {The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.},
}
@article {pmid31113993,
year = {2019},
author = {Sarr, A and Bré, J and Um, IH and Chan, TH and Mullen, P and Harrison, DJ and Reynolds, PA},
title = {Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide NUC-1031.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7643},
pmid = {31113993},
issn = {2045-2322},
support = {MR/K001744/1/MRC_/Medical Research Council/United Kingdom ; BB/J004243/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Antineoplastic Agents/therapeutic use/*toxicity ; Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems ; Clinical Trials, Phase I as Topic ; Cytidine Monophosphate/*analogs &amp; derivatives/therapeutic use/toxicity ; Deoxycytidine/analogs &amp; derivatives/therapeutic use/toxicity ; Deoxycytidine Kinase/*genetics/metabolism ; Drug Resistance, Neoplasm/*genetics ; Female ; HEK293 Cells ; Humans ; Ovarian Neoplasms/drug therapy/*genetics ; Pancreatic Neoplasms/drug therapy/*genetics ; },
abstract = {Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.},
}
@article {pmid31113472,
year = {2019},
author = {Bharathy, N and Berlow, NE and Wang, E and Abraham, J and Settelmeyer, TP and Hooper, JE and Svalina, MN and Bajwa, Z and Goros, MW and Hernandez, BS and Wolff, JE and Pal, R and Davies, AM and Ashok, A and Bushby, D and Mancini, M and Noakes, C and Goodwin, NC and Ordentlich, P and Keck, J and Hawkins, DS and Rudzinski, ER and Mansoor, A and Perkins, TJ and Vakoc, CR and Michalek, JE and Keller, C},
title = {Preclinical rationale for entinostat in embryonal rhabdomyosarcoma.},
journal = {Skeletal muscle},
volume = {9},
number = {1},
pages = {12},
pmid = {31113472},
issn = {2044-5040},
support = {P30 CA045508/CA/NCI NIH HHS/United States ; R01 CA143082/CA/NCI NIH HHS/United States ; R01 CA189299/CA/NCI NIH HHS/United States ; },
mesh = {Adolescent ; Animals ; Antineoplastic Agents, Phytogenic/administration &amp; dosage ; Antineoplastic Combined Chemotherapy Protocols/administration &amp; dosage ; Benzamides/administration &amp; dosage/*therapeutic use ; CRISPR-Cas Systems ; Cell Differentiation/drug effects ; Cell Line, Tumor ; Cellular Reprogramming/drug effects/genetics ; Child ; Child, Preschool ; Drug Screening Assays, Antitumor ; Female ; Histone Deacetylase 1/antagonists &amp; inhibitors/genetics ; Histone Deacetylase Inhibitors/administration &amp; dosage/*therapeutic use ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Pyridines/administration &amp; dosage/*therapeutic use ; RNA-Seq ; Rhabdomyosarcoma, Alveolar/drug therapy/enzymology/pathology ; Rhabdomyosarcoma, Embryonal/*drug therapy/enzymology/pathology ; Tumor Burden/drug effects ; Tumor Microenvironment/drug effects/genetics ; Vincristine/administration &amp; dosage ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric cancer population. Survival among metastatic RMS patients has remained dismal yet unimproved for years. We previously identified the class I-specific histone deacetylase inhibitor, entinostat (ENT), as a pharmacological agent that transcriptionally suppresses the PAX3:FOXO1 tumor-initiating fusion gene found in alveolar rhabdomyosarcoma (aRMS), and we further investigated the mechanism by which ENT suppresses PAX3:FOXO1 oncogene and demonstrated the preclinical efficacy of ENT in RMS orthotopic allograft and patient-derived xenograft (PDX) models. In this study, we investigated whether ENT also has antitumor activity in fusion-negative eRMS orthotopic allografts and PDX models either as a single agent or in combination with vincristine (VCR).<br><br>METHODS: We tested the efficacy of ENT and VCR as single agents and in combination in orthotopic allograft and PDX mouse models of eRMS. We then performed CRISPR screening to identify which HDAC among the class I HDACs is responsible for tumor growth inhibition in eRMS. To analyze whether ENT treatment as a single agent or in combination with VCR induces myogenic differentiation, we performed hematoxylin and eosin (H&amp;E) staining in tumors.<br><br>RESULTS: ENT in combination with the chemotherapy VCR has synergistic antitumor activity in a subset of fusion-negative eRMS in orthotopic "allografts," although PDX mouse models were too hypersensitive to the VCR dose used to detect synergy. Mechanistic studies involving CRISPR suggest that HDAC3 inhibition is the primary mechanism of cell-autonomous cytoreduction in eRMS. Following cytoreduction in vivo, residual tumor cells in the allograft models treated with chemotherapy undergo a dramatic, entinostat-induced (70-100%) conversion to non-proliferative rhabdomyoblasts.<br><br>CONCLUSION: Our results suggest that the targeting class I HDACs may provide a therapeutic benefit for selected patients with eRMS. ENT's preclinical in vivo efficacy makes ENT a rational drug candidate in a phase II clinical trial for eRMS.},
}
@article {pmid31112992,
year = {2019},
author = {Zhu, Q and Wang, J and Zhang, L and Bian, W and Lin, M and Xu, X and Zhou, X},
title = {LCK rs10914542-G allele associates with type 1 diabetes in children via T cell hyporesponsiveness.},
journal = {Pediatric research},
volume = {86},
number = {3},
pages = {311-315},
doi = {10.1038/s41390-019-0436-2},
pmid = {31112992},
issn = {1530-0447},
mesh = {Adolescent ; Alleles ; CD3 Complex/metabolism ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Proliferation ; Child ; Child, Preschool ; China ; Diabetes Mellitus, Type 1/*genetics/*immunology ; Female ; Genotype ; Glycated Hemoglobin A/metabolism ; Humans ; Leukocytes, Mononuclear/metabolism ; Lymphocyte Activation ; Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/*genetics ; Male ; Odds Ratio ; *Polymorphism, Single Nucleotide ; T-Lymphocytes/*immunology ; },
abstract = {BACKGROUND: Abnormal lymphocyte-specific protein tyrosine kinase (LCK)-related T cell hyporesponsiveness was discovered in type 1 diabetes (T1D). This study aims to investigate the potential associations between LCK single-nucleotide polymorphisms (SNPs) and the susceptibility of T1D.<br><br>METHODS: DNAs were extracted from blood samples of 589 T1D patients and 596 healthy controls to genotype seven SNPs of the LCK gene using PCR and Sanger sequencing. Associations of these SNPs with the susceptibility of T1D were determined by &#x3c7;[2] test. LCKs were knocked out in peripheral blood mononuclear cells (PBMCs) using CRISPR-Cas9 to investigate the role of LCK SNP in T-lymphocyte activation in T1D.<br><br>RESULTS: SNP rs10914542 but not the other six SNPs of the LCK gene was significantly associated with (C vs. G, odds ratio (OR)&#x2009;=&#x2009;0.581, 95% confidence interval (CI)&#x2009;=&#x2009;0.470-0.718, P value&#x2009;=&#x2009;4.13E&#x2009;-&#x2009;7) the susceptibility of T1D. Peripheral T-lymphocyte activation in response to T cell receptor (TCR)/CD3 stimulation is significantly lower in the rs10914542-G-allele group than in the C-allele group. In vitro experiments revealed that rs10914542 G allele impaired the TCR/CD3-mediated T-cell activation in PBMCs.<br><br>CONCLUSIONS: This study reveals that the G allele of SNP rs10914542 of LCK impairs the TCR/CD3-mediated T-cell activation and increases the risk of T1D.},
}
@article {pmid31112800,
year = {2019},
author = {Singh, DD and Hawkins, RD and Lahesmaa, R and Tripathi, SK},
title = {CRISPR/Cas9 guided genome and epigenome engineering and its therapeutic applications in immune mediated diseases.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {32-43},
doi = {10.1016/j.semcdb.2019.05.007},
pmid = {31112800},
issn = {1096-3634},
support = {R01 AR065952/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/*genetics ; *Genetic Engineering ; Humans ; Immune System Diseases/*drug therapy/*genetics ; },
abstract = {Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.},
}
@article {pmid31112734,
year = {2019},
author = {Gresakova, V and Novosadova, V and Prochazkova, M and Bhargava, S and Jenickova, I and Prochazka, J and Sedlacek, R},
title = {Fam208a orchestrates interaction protein network essential for early embryonic development and cell division.},
journal = {Experimental cell research},
volume = {382},
number = {1},
pages = {111437},
doi = {10.1016/j.yexcr.2019.05.018},
pmid = {31112734},
issn = {1090-2422},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Division/genetics/physiology ; Embryonic Development/genetics/*physiology ; *Gene Expression Regulation, Developmental ; Genes, Lethal ; Genomic Instability ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Multiprotein Complexes ; Nuclear Proteins/*physiology ; Phosphoproteins/metabolism ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Spindle Apparatus/*metabolism ; Zygote/metabolism ; },
abstract = {Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining the genome stability during the cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 leads to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by qRT-PCR profiling and in situ hybridisation [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is multi-interacting protein affecting genome stability on the level of cell division at the earliest stages of development and also by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an additional role in zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp.},
}
@article {pmid31110355,
year = {2019},
author = {Huang, TP and Zhao, KT and Miller, SM and Gaudelli, NM and Oakes, BL and Fellmann, C and Savage, DF and Liu, DR},
title = {Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors.},
journal = {Nature biotechnology},
volume = {37},
number = {6},
pages = {626-631},
pmid = {31110355},
issn = {1546-1696},
support = {DP2 EB018658/EB/NIBIB NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 GM127463/GM/NIGMS NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; T32 GM095450/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; },
mesh = {Adenine/chemistry ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cytosine/chemistry ; Gene Editing/*methods ; Humans ; Nucleotides/chemistry/genetics ; Plasmids/chemistry/genetics ; },
abstract = {Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4-5 nucleotides to up to ~8-9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.},
}
@article {pmid31110297,
year = {2019},
author = {Pei, W and Wang, X and Rössler, J and Feyerabend, TB and Höfer, T and Rodewald, HR},
title = {Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.},
journal = {Nature protocols},
volume = {14},
number = {6},
pages = {1820-1840},
pmid = {31110297},
issn = {1750-2799},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Lineage ; DNA/genetics/metabolism ; DNA Barcoding, Taxonomic/methods ; Female ; *Genes, Reporter ; Genotyping Techniques/methods ; Integrases/genetics ; Male ; Mice ; Mice, Transgenic ; Polymerase Chain Reaction/methods ; Recombination, Genetic ; Sequence Analysis, DNA/*methods ; },
abstract = {Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26[Polylox] reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.},
}
@article {pmid31110156,
year = {2019},
author = {Goswami, MT and VanDenBerg, KR and Han, S and Wang, LL and Singh, B and Weiss, T and Barlow, M and Kamberov, S and Wilder-Romans, K and Rhodes, DR and Feng, FY and Tomlins, SA},
title = {Identification of TP53RK-Binding Protein (TPRKB) Dependency in TP53-Deficient Cancers.},
journal = {Molecular cancer research : MCR},
volume = {17},
number = {8},
pages = {1652-1664},
pmid = {31110156},
issn = {1557-3125},
support = {R01 CA183857/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Apoptosis ; Apoptosis Regulatory Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle ; *Cell Proliferation ; Colonic Neoplasms/genetics/metabolism/*pathology ; Female ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mice ; Mice, Nude ; *Mutation ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53/antagonists &amp; inhibitors/genetics/*metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Tumor protein 53 (TP53; p53) is the most frequently altered gene in human cancer. Identification of vulnerabilities imposed by TP53 alterations may enable effective therapeutic approaches. Through analyzing short hairpin RNA (shRNA) screening data, we identified TP53RK-Binding Protein (TPRKB), a poorly characterized member of the tRNA-modifying EKC/KEOPS complex, as the most significant vulnerability in TP53-mutated cancer cell lines. In vitro and in vivo, across multiple benign-immortalized and cancer cell lines, we confirmed that TPRKB knockdown in TP53-deficient cells significantly inhibited proliferation, with minimal effect in TP53 wild-type cells. TP53 reintroduction into TP53-null cells resulted in loss of TPRKB sensitivity, confirming the importance of TP53 status in this context. In addition, cell lines with mutant TP53 or amplified MDM2 (E3-ubiquitin ligase for TP53) also showed high sensitivity to TPRKB knockdown, consistent with TPRKB dependence in a wide array of TP53-altered cancers. Depletion of other EKC/KEOPS complex members exhibited TP53-independent effects, supporting complex-independent functions of TPRKB. Finally, we found that TP53 indirectly mediates TPRKB degradation, which was rescued by coexpression of PRPK, an interacting member of the EKC/KEOPS complex, or proteasome inhibition. Together, these results identify a unique and specific requirement of TPRKB in a variety of TP53-deficient cancers. IMPLICATIONS: Cancer cells with genomic alterations in TP53 are dependent on TPRKB.},
}
@article {pmid31110048,
year = {2019},
author = {Johnson, K and Learn, BA and Estrella, MA and Bailey, S},
title = {Target sequence requirements of a type III-B CRISPR-Cas immune system.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {26},
pages = {10290-10299},
pmid = {31110048},
issn = {1083-351X},
support = {F31 GM105364/GM/NIGMS NIH HHS/United States ; R01 GM097330/GM/NIGMS NIH HHS/United States ; T32 CA009110/CA/NCI NIH HHS/United States ; },
mesh = {Base Pairing ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; *DNA Cleavage ; *Immune System ; Plasmids/genetics ; RNA/genetics/*metabolism ; Thermotoga maritima/*genetics/*metabolism ; },
abstract = {CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.},
}
@article {pmid31109675,
year = {2019},
author = {Smagghe, G and Zotti, M and Retnakaran, A},
title = {Targeting female reproduction in insects with biorational insecticides for pest management: a critical review with suggestions for future research.},
journal = {Current opinion in insect science},
volume = {31},
number = {},
pages = {65-69},
doi = {10.1016/j.cois.2018.10.009},
pmid = {31109675},
issn = {2214-5753},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Insect Control/*methods ; Insecta/*physiology ; Insecticides ; *Juvenile Hormones ; RNA Interference ; Reproduction/drug effects ; },
abstract = {Of the different approaches to pest control, use of juvenile hormone analogs (e.g. methoprene), molting hormone (20-hydroxyecdysone) analogs (e.g. tebufenozide) and chitin synthesis inhibitors (e.g. diflubenzuron) has dominated this field. Since they adversely interfere with the normal growth and development in one way or another, they have been collectively called as 'insect growth regulators' or IGRs. A lesser known fact is that they all have deleterious effects on reproduction as well as act as ovicides. The raison d'être for this review is to summarize what we have learnt during the last 3-4 decades in the use of these IGRs, how they affect insect reproduction and how we can apply this knowledge to control pest insects. Finally, we present, information on the state of the art use of molecular technologies such as RNAi and CRISPR/Cas9 applications for pest management targeting insect reproduction.},
}
@article {pmid31109137,
year = {2019},
author = {Ji, J and Zhang, C and Sun, Z and Wang, L and Duanmu, D and Fan, Q},
title = {Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9.},
journal = {International journal of molecular sciences},
volume = {20},
number = {10},
pages = {},
pmid = {31109137},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Plant ; Mutagenesis ; Nitrogen Fixation ; RNA, Guide/genetics ; Vigna/*genetics/metabolism ; },
abstract = {Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.},
}
@article {pmid31107563,
year = {2019},
author = {Baffoe-Bonnie, MS},
title = {A justice-based argument for including sickle cell disease in CRISPR/Cas9 clinical research.},
journal = {Bioethics},
volume = {33},
number = {6},
pages = {661-668},
doi = {10.1111/bioe.12589},
pmid = {31107563},
issn = {1467-8519},
mesh = {Anemia, Sickle Cell/*prevention &amp; control/therapy ; Biomedical Research ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Ethnicity ; *Gene Editing ; Genetic Therapy ; Humans ; *Social Justice ; },
abstract = {CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health-care system, and benefit-sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology.},
}
@article {pmid31107171,
year = {2019},
author = {Newby, GA},
title = {Base Editing: Efficient Installation of Point Mutations with Minimal Byproducts.},
journal = {Stem cells and development},
volume = {28},
number = {11},
pages = {712-713},
doi = {10.1089/scd.2019.0080},
pmid = {31107171},
issn = {1557-8534},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematologic Diseases/therapy ; Humans ; Polymorphism, Single Nucleotide/genetics ; },
}
@article {pmid31107154,
year = {2019},
author = {Vakulskas, CA and Behlke, MA},
title = {Evaluation and Reduction of CRISPR Off-Target Cleavage Events.},
journal = {Nucleic acid therapeutics},
volume = {29},
number = {4},
pages = {167-174},
pmid = {31107154},
issn = {2159-3345},
mesh = {CRISPR-Associated Protein 9/genetics/therapeutic use ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genetic Therapy ; Hematopoietic Stem Cells/cytology ; Humans ; Translational Research, Biomedical/trends ; },
abstract = {Introduction of CRISPR/Cas9 methods (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) have led to a huge surge in the use of precision genome editing for research applications. Translational medical efforts are likewise rapidly progressing, and Phase I clinical trials using these techniques have already started. As with any new technology that is applied to medical therapeutics, risks must be carefully defined and steps taken to mitigate side effects wherever possible. Effective methods are now available that permit identification of off-target cleavage events, a major class of potential side effects seen in mammalian genome editing. Off-target prediction algorithms are improving and have utility, but are insufficient to use alone. Empiric methods to define the off-target profile must also be used. Once defined, the frequency of off-target cleavage can be minimized using methods that limit the duration of exposure of the genome to the active genome editing complex, for example, using the ribonucleoprotein (RNP) approach. In addition, Cas9 mutants have been developed that markedly reduce the rate of off-target cleavage compared to the wild-type enzyme. Use of these new tools should become standard practice for medical applications.},
}
@article {pmid31107136,
year = {2019},
author = {Parthiban, K and Perera, RL and Sattar, M and Huang, Y and Mayle, S and Masters, E and Griffiths, D and Surade, S and Leah, R and Dyson, MR and McCafferty, J},
title = {A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing.},
journal = {mAbs},
volume = {11},
number = {5},
pages = {884-898},
pmid = {31107136},
issn = {1942-0870},
mesh = {Animals ; Antibodies, Monoclonal, Humanized/*genetics ; *Antibody Affinity ; Binding Sites, Antibody/*genetics/immunology ; CHO Cells ; CRISPR-Cas Systems ; Complementarity Determining Regions/genetics ; Cricetulus ; Endodeoxyribonucleases ; Flow Cytometry ; Gene Editing ; HEK293 Cells ; Humans ; Immunoglobulin Heavy Chains/genetics ; Mutagenesis, Site-Directed ; Programmed Cell Death 1 Receptor/immunology ; },
abstract = {The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant "solution" involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.},
}
@article {pmid31106778,
year = {2019},
author = {Wang, J and Huang, J and Xu, R},
title = {[Seamless genome editing in Drosophila by combining CRISPR/Cas9 and piggyBac technologies].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {5},
pages = {422-429},
doi = {10.16288/j.yczz.18-345},
pmid = {31106778},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila/*genetics ; *Gene Editing ; *Genome, Insect ; Transposases/*genetics ; },
abstract = {The type2 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.},
}
@article {pmid31106480,
year = {2019},
author = {Bai, X and Zeng, T and Ni, XY and Su, HA and Huang, J and Ye, GY and Lu, YY and Qi, YX},
title = {CRISPR/Cas9-mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis.},
journal = {Insect molecular biology},
volume = {28},
number = {6},
pages = {837-849},
doi = {10.1111/imb.12592},
pmid = {31106480},
issn = {1365-2583},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Compound Eye, Arthropod/*physiology ; Female ; Gene Knockout Techniques ; Insect Proteins/chemistry/*genetics/metabolism ; Male ; Phylogeny ; Pigmentation/*genetics ; Sequence Alignment ; Tephritidae/genetics/*physiology ; },
abstract = {The intensely studied white gene is widely used as a genetic marker in Drosophila melanogaster. Here, we cloned and characterized the white gene in an important pest of the fruit industry, Bactrocera dorsalis, to understand its functional role in pigmentation. We obtained BdWhite knockout strains, based on the wild-type strain, using the CRISPR/Cas9 genome editing system, and found that mutants lost pigmentation in the compound eye and their black head spots. We then examined differences in the expression levels of genes associated with melanin pigmentation between mutants and the wild-type strain using quantitative reverse transcription PCR. We found that transcription levels of the Bd-yellow1 were lower in the head of mutants than in the wild-type strain, and there were no significant differences in expression of the other six genes between mutants and the wild type. Since yellow is critical for melanin biosynthesis (Heinze et al., Scientific Reports. 2017;7:4582), the lower levels of expression of Bd-yellow1 in mutants led to reduced dark pigmentation in head spots. Our results provide the first evidence, to our knowledge, that white may play a functional role in cuticle pigmentation by affecting the expression of yellow.},
}
@article {pmid31106371,
year = {2019},
author = {Labun, K and Montague, TG and Krause, M and Torres Cleuren, YN and Tjeldnes, H and Valen, E},
title = {CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.},
journal = {Nucleic acids research},
volume = {47},
number = {W1},
pages = {W171-W174},
pmid = {31106371},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Databases, Genetic ; Gene Editing/methods ; *Gene Targeting ; Genome/*genetics ; Humans ; RNA, Guide/*genetics ; *Software ; },
abstract = {The CRISPR-Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR-Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR-Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no.},
}
@article {pmid31106355,
year = {2019},
author = {Morio, F and Lombardi, L and Binder, U and Loge, C and Robert, E and Graessle, D and Bodin, M and Lass-Flörl, C and Butler, G and Le Pape, P},
title = {Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {74},
number = {8},
pages = {2230-2238},
doi = {10.1093/jac/dkz204},
pmid = {31106355},
issn = {1460-2091},
mesh = {Amino Acid Substitution ; Antifungal Agents/*pharmacology ; Azoles/*pharmacology ; CRISPR-Cas Systems ; Candida parapsilosis/*drug effects/*genetics ; Candidiasis/microbiology ; Cytochrome P-450 Enzyme System/*genetics ; Drug Resistance, Fungal/genetics ; Gene Editing/*methods ; Humans ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis.<br><br>OBJECTIVES: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis.<br><br>METHODS: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate.<br><br>RESULTS: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole.<br><br>CONCLUSIONS: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.},
}
@article {pmid31105049,
year = {2019},
author = {Park, CY and Sung, JJ and Cho, SR and Kim, J and Kim, DW},
title = {Universal Correction of Blood Coagulation Factor VIII in Patient-Derived Induced Pluripotent Stem Cells Using CRISPR/Cas9.},
journal = {Stem cell reports},
volume = {12},
number = {6},
pages = {1242-1249},
pmid = {31105049},
issn = {2213-6711},
mesh = {*CRISPR-Cas Systems ; *Factor VIII/biosynthesis/genetics ; *Gene Editing ; *Hemophilia A/genetics/metabolism/pathology/therapy ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Mutation ; },
abstract = {Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII) gene. Genome-editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty in preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.},
}
@article {pmid31104397,
year = {2019},
author = {Narimani, M and Sharifi, M and Hakhamaneshi, MS and Roshani, D and Kazemi, M and Hejazi, SH and Jalili, A},
title = {BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression.},
journal = {Iranian biomedical journal},
volume = {23},
number = {6},
pages = {369-378},
pmid = {31104397},
issn = {2008-823X},
mesh = {Apoptosis ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; *Disease Progression ; *Gene Deletion ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute/*genetics/*pathology ; RNA, Guide/metabolism ; Survivin/*genetics ; },
abstract = {BACKGROUND: Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML.<br><br>METHODS: The BIRC5 was silenced permanently in two AML cell lines, HL&#x2011;60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin&#x2011;V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests.<br><br>RESULTS: The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability.<br><br>CONCLUSION: This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.},
}
@article {pmid31104263,
year = {2019},
author = {Wang, L and Li, J},
title = {Expansion of the mutant monkey through cloning.},
journal = {Science China. Life sciences},
volume = {62},
number = {6},
pages = {865-867},
doi = {10.1007/s11427-019-9502-2},
pmid = {31104263},
issn = {1869-1889},
mesh = {ARNTL Transcription Factors/genetics ; Animals ; CRISPR-Cas Systems/genetics ; *Cloning, Molecular ; Gene Editing ; Haplorhini ; *Models, Animal ; Mutation ; Phenotype ; },
}
@article {pmid31102655,
year = {2019},
author = {Rahman, S and Datta, M and Kim, J and Jan, AT},
title = {CRISPR/Cas: An intriguing genomic editing tool with prospects in treating neurodegenerative diseases.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {22-31},
doi = {10.1016/j.semcdb.2019.05.014},
pmid = {31102655},
issn = {1096-3634},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Neurodegenerative Diseases/*genetics/*therapy ; },
abstract = {The CRISPR/Cas genome editing tool has led to a revolution in biological research. Its ability to target multiple genomic loci simultaneously allows its application in gene function and genomic manipulation studies. Its involvement in the sequence specific gene editing in different backgrounds has changed the scenario of treating genetic diseases. By unravelling the mysteries behind complex neuronal circuits, it not only paved way in understanding the pathogenesis of the disease but helped in the development of large animal models of different neuronal diseases; thereby opened the gateways of successfully treating different neuronal diseases. This review explored the possibility of using of CRISPR/Cas in engineering DNA at the embryonic stage, as well as during the functioning of different cell types in the brain, to delineate implications related to the use of this super-specialized genome editing tool to overcome various neurodegenerative diseases that arise as a result of genetic mutations.},
}
@article {pmid31101972,
year = {2019},
author = {Veillet, F and Chauvin, L and Kermarrec, MP and Sevestre, F and Merrer, M and Terret, Z and Szydlowski, N and Devaux, P and Gallois, JL and Chauvin, JE},
title = {The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato.},
journal = {Plant cell reports},
volume = {38},
number = {9},
pages = {1065-1080},
pmid = {31101972},
issn = {1432-203X},
mesh = {Alleles ; CRISPR-Cas Systems ; *Gene Editing ; Solanum tuberosum/*genetics ; Starch Synthase/*genetics ; Tetraploidy ; },
abstract = {The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base-editing strategies, leading to plants with impaired amylose biosynthesis. Genome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant-breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knock out the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss of function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.},
}
@article {pmid31101849,
year = {2019},
author = {Datsomor, AK and Zic, N and Li, K and Olsen, RE and Jin, Y and Vik, JO and Edvardsen, RB and Grammes, F and Wargelius, A and Winge, P},
title = {CRISPR/Cas9-mediated ablation of elovl2 in Atlantic salmon (Salmo salar L.) inhibits elongation of polyunsaturated fatty acids and induces Srebp-1 and target genes.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7533},
pmid = {31101849},
issn = {2045-2322},
mesh = {Animals ; Arachidonic Acid/biosynthesis ; Brain/metabolism ; CRISPR-Cas Systems ; Docosahexaenoic Acids/biosynthesis ; Eicosapentaenoic Acid/biosynthesis ; Fatty Acid Elongases/*genetics/metabolism ; Fatty Acid Synthases/*metabolism ; Fatty Acids, Omega-3/metabolism ; Fatty Acids, Unsaturated/*biosynthesis ; Gene Knockout Techniques ; Lipid Metabolism/genetics ; Muscles/metabolism ; Salmo salar/*genetics ; Sterol Regulatory Element Binding Protein 1/genetics/*metabolism ; },
abstract = {Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.},
}
@article {pmid31101683,
year = {2019},
author = {Schertzer, MD and Thulson, E and Braceros, KCA and Lee, DM and Hinkle, ER and Murphy, RM and Kim, SO and Vitucci, ECM and Calabrese, JM},
title = {A piggyBac-based toolkit for inducible genome editing in mammalian cells.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {1047-1058},
pmid = {31101683},
issn = {1469-9001},
support = {T32 ES007126/ES/NIEHS NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; R25 GM055336/GM/NIGMS NIH HHS/United States ; T32 GM119999/GM/NIGMS NIH HHS/United States ; T32 GM007092/GM/NIGMS NIH HHS/United States ; R01 GM121806/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Engineering ; Humans ; Plasmids/*genetics ; Transposases/genetics/*metabolism ; },
abstract = {We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.},
}
@article {pmid31101673,
year = {2019},
author = {Moffett, HF and Harms, CK and Fitzpatrick, KS and Tooley, MR and Boonyaratanakornkit, J and Taylor, JJ},
title = {B cells engineered to express pathogen-specific antibodies protect against infection.},
journal = {Science immunology},
volume = {4},
number = {35},
pages = {},
pmid = {31101673},
issn = {2470-9468},
support = {T32 AI118690/AI/NIAID NIH HHS/United States ; T32 GM095421/GM/NIGMS NIH HHS/United States ; },
mesh = {3T3 Cells ; Adoptive Transfer/methods ; Animals ; Antibodies, Monoclonal/*immunology ; Antibodies, Neutralizing/*immunology ; Antibodies, Viral/*immunology ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Female ; HEK293 Cells ; Homeodomain Proteins/genetics ; Humans ; Male ; Metabolic Engineering/*methods ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Respiratory Syncytial Virus Infections/*therapy/virology ; Respiratory Syncytial Virus, Human/*immunology ; },
abstract = {Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), HIV, influenza virus, and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine, we developed a genetic engineering strategy in which CRISPR-Cas9 was used to replace endogenously encoded antibodies with antibodies targeting RSV, HIV, influenza virus, or EBV in primary human B cells. The engineered antibodies were expressed efficiently in primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion. Using engineered mouse B cells, we demonstrated that a single transfer of B cells engineered to express an antibody against RSV resulted in potent and durable protection against RSV infection in RAG1-deficient mice. This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.},
}
@article {pmid31100311,
year = {2019},
author = {Li, M and Xie, H and Liu, Y and Xia, C and Cun, X and Long, Y and Chen, X and Deng, M and Guo, R and Zhang, Z and He, Q},
title = {Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {304},
number = {},
pages = {204-215},
doi = {10.1016/j.jconrel.2019.05.019},
pmid = {31100311},
issn = {1873-4995},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; Cell Hypoxia ; Cell Line, Tumor ; Gene Knockdown Techniques ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Lipids/chemistry ; Liposomes ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Metastasis/prevention &amp; control ; Pancreatic Neoplasms/*genetics/pathology ; Survival Rate ; Tumor Microenvironment/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {The hypoxic tumor microenvironment of pancreatic cancer contributes to the progression and metastasis of tumor cells. Downregulation of hypoxia-inducible factor-1α (HIF-1α) with CRISPR/Cas9 is a promising approach to modulate tumor microenvironment and inhibit tumor metastasis. However, the in vivo delivery of CRISPR/Cas9 system remains a challenge. In the present manuscript, a tumor targeted lipid-based CRISPR/Cas9 delivery system was developed to suppress HIF-1α. Plasmids encoding Cas9 and HIF-1α-targeting sgRNA were successfully constructed and coencapsulated in R8-dGR peptide modified cationic liposome with PTX. R8-dGR-Lip exhibited enhanced BxPC-3 cell targeting and deep penetration into tumor spheroids. R8-dGR-Lip/PTX/pHIF-1α successfully downregulated HIF-1α and its downstream molecules VEGF and MMP-9, leading to enhanced antimetastatic effects. Besides, the blockade of HIF-1α also promoted the cytotoxicity of PTX on BxPC-3 cell lines. Compared with pegylated liposomes, R8-dGR-Lip enhanced the distribution in tumor tissues. The targeted delivery of CRISPR/Cas9-HIF-1α system and PTX significantly inhibited tumor growth. More importantly, inhibition of HIF-1α suppressed the metastasis of pancreatic cancer and prolonged survival time. Since CRISPR/Cas 9-HIF-1α hardly affected HIF-1α expression in normal hepatic cells, the designed R8-dGR-Lip/PTX/pHIF-1α did not induce severe toxicity in vivo. This strategy broadened the in vivo application of CRISPR/Cas9 system. Downregulation of HIF-1α may be a feasible approach for antimetastatic therapy.},
}
@article {pmid31099781,
year = {2019},
author = {Kostyusheva, AP and Kostyushev, DS and Brezgin, SA and Zarifyan, DN and Volchkova, EV and Chulanov, VP},
title = {[Small Molecular Inhibitors of DNA Double Strand Break Repair Pathways Increase the ANTI-HBV Activity of CRISPR/Cas9].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {2},
pages = {311-323},
doi = {10.1134/S0026898419010075},
pmid = {31099781},
issn = {0026-8984},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*drug effects/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Breaks, Double-Stranded/*drug effects ; DNA Repair/*drug effects ; DNA, Circular/genetics/metabolism ; DNA, Viral/genetics/*metabolism ; *Hepatitis B virus ; },
abstract = {The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.},
}
@article {pmid31099770,
year = {2019},
author = {Rozov, SM and Deineko, EV},
title = {[Strategies for Optimizing Recombinant Protein Synthesis in Plant Cells: Classical Approaches and New Directions].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {2},
pages = {179-199},
doi = {10.1134/S0026898419020149},
pmid = {31099770},
issn = {0026-8984},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/*trends ; Genes, Essential/genetics ; Plant Cells/*metabolism ; Recombinant Proteins/*biosynthesis/*genetics ; },
abstract = {At present, pharmacologically significant proteins are synthesized in different expression systems, from bacterial to mammalian and insect cell cultures. The plant expression systems (especially suspension cell culture) combine the simplicity and low cost of bacterial systems with the ability to perform eukaryotic-type posttranslational protein modifications. A low (compared with bacterial systems) yield of the target recombinant protein is one of the shortcomings of the plant expression systems. In this review, methods, developed over the past two decades, to increase the level of recombinant gene expression and methods to prevent silencing, caused by a random insertion of the target gene into heterochromatin region, are considered. The emergence of CRISPR/Cas technologies led to the creation of a new approach to increase the gene expression level, directional insertion of "pharmaceutical" protein genes in specific, knowingly transcriptionally active genome regions. The plant cell housekeeping gene loci, actively expressed throughout the interphase, are these regions. The organization of some housekeeping genes, most promising for transferring recombinant protein genes in their loci, is considered in detail.},
}
@article {pmid31099553,
year = {2019},
author = {Wilbie, D and Walther, J and Mastrobattista, E},
title = {Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing.},
journal = {Accounts of chemical research},
volume = {52},
number = {6},
pages = {1555-1564},
pmid = {31099553},
issn = {1520-4898},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*pharmacology ; CRISPR-Cas Systems/*genetics ; Drug Carriers/*chemistry ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; Metal Nanoparticles/*chemistry ; Mice ; RNA, Guide/genetics/pharmacology ; Ribonucleoproteins/genetics/pharmacology ; },
abstract = {The discovery of CRISPR/Cas has revolutionized the field of genome editing. CRIPSR/Cas components are part of the bacterial immune system and are able to induce double-strand DNA breaks in the genome, which are resolved by endogenous DNA repair mechanisms. The most relevant of these are the error-prone nonhomologous end joining and homology directed repair pathways. The former can lead to gene knockout by introduction of insertions and deletions at the cut site, while the latter can be used for gene correction based on a provided repair template. In this Account, we focus on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo. Safe and effective delivery of the CRISPR/Cas components into the nucleus of affected cells is essential for therapeutic gene editing. These components can be delivered in several formats, such as pDNA, viral vectors, or ribonuclear complexes. In the ideal case, the delivery system should address the current limitations of CRISPR gene editing, which are (1) lack of targeting specific tissues or cells, (2) the inability to enter cells, (3) activation of the immune system, and (4) off-target events. To circumvent most of these problems, initial therapeutic applications of CRISPR/Cas were performed on cells ex vivo via classical methods (e.g., microinjection or electroporation) and novel methods (e.g., TRIAMF and iTOP). Ideal candidates for such methods are, for example, hematopoietic cells, but not all tissue types are suited for ex vivo manipulation. For direct in vivo application, however, delivery systems are needed that can target the CRISPR/Cas components to specific tissues or cells in the human body, without causing immune activation or causing high frequencies of off-target effects. Viral systems have been used as a first resort to transduce cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and longevity of Cas expression, which favors off-target events. Viral vectors are as such not the best choice for direct in vivo delivery of CRISPR/Cas. Synthetic vectors can deliver nucleic acids as well, without the innate disadvantages of viral vectors. They can be classed into lipid, polymeric, and inorganic particles, all of which have been reported in the literature. The advantage of synthetic systems is that they can deliver the CRISPR/Cas system also as a preformed ribonucleoprotein complex. The transient nature of this approach favors low frequencies of off-target events and minimizes the window of immune activation. Moreover, from a pharmaceutical perspective, synthetic delivery systems are much easier to scale up for clinical use compared to viral vectors and can be chemically functionalized with ligands to obtain target cell specificity. The first preclinical results with lipid nanoparticles delivering CRISPR/Cas either as mRNA or ribonucleoproteins are very promising. The goal is translating these CRISPR/Cas therapeutics to a clinical setting as well. Taken together, these current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.},
}
@article {pmid31099367,
year = {2019},
author = {Zhu, X and Lv, MM and Liu, JW and Yu, RQ and Jiang, JH},
title = {DNAzyme activated protein-scaffolded CRISPR-Cas9 nanoassembly for genome editing.},
journal = {Chemical communications (Cambridge, England)},
volume = {55},
number = {46},
pages = {6511-6514},
doi = {10.1039/c9cc03172c},
pmid = {31099367},
issn = {1364-548X},
mesh = {Biotin/chemistry ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA, Catalytic/*genetics ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Nanostructures/*chemistry ; Nucleic Acid Hybridization ; RNA, Guide/genetics ; Streptavidin/chemistry ; },
abstract = {A novel self-assembled protein-scaffolded CRISPR-Cas9 nanosystem for facile and efficient gene editing in a DNAzyme-controlled manner has been developed.},
}
@article {pmid31099266,
year = {2019},
author = {Zhou, ZP and Yang, LL and Cao, H and Chen, ZR and Zhang, Y and Wen, XY and Hu, J},
title = {In Vitro Validation of a CRISPR-Mediated CFTR Correction Strategy for Preclinical Translation in Pigs.},
journal = {Human gene therapy},
volume = {30},
number = {9},
pages = {1101-1116},
doi = {10.1089/hum.2019.074},
pmid = {31099266},
issn = {1557-7422},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cystic Fibrosis/*genetics/therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/metabolism ; Disease Models, Animal ; Epithelial Cells/metabolism ; Galactosyltransferases/genetics ; *Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Gene Targeting ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Loci ; Genetic Therapy ; Genetic Vectors/genetics ; Models, Biological ; Mutagenesis, Insertional ; Swine ; Transduction, Genetic ; Transgenes ; },
abstract = {Early efforts in cystic fibrosis (CF) gene therapy faced major challenges in delivery efficiency and sustained therapeutic gene expression. Recent advancements in engineered site-specific endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 make permanent CF transmembrane conductance regulator (CFTR) gene correction possible. However, because of safety concerns of the CRISPR/Cas9 system and challenges in in vivo delivery to inflamed CF airway, CRISPR-based gene correction strategies need to be tested in proper animal models. In this study, we aimed at creating vectors for testing CFTR gene correction in pig models. We constructed helper-dependent adenoviral (HD-Ad) vectors to deliver CRISPR/Cas9 and a donor template (a 6 kb LacZ or 8.7 kb human CFTR expression cassette) into cultured pig cells. We demonstrated precise integration of each donor into the GGTA1 safe harbor through Cas9-induced homology directed repair with 3 kb homology arms. In addition, we showed that both LacZ and hCFTR were persistently expressed in transduced cells. Furthermore, we created a CFTR-deficient cell line for testing CFTR correction. We detected hCFTR mRNA and protein expression in cells transduced with the hCFTR vector. We also demonstrated CFTR function in the CF cells transduced with the HD-Ad delivering the CRISPR-Cas9 system and hCFTR donor at late cellular passages using the membrane potential sensitive dye-based assay (FLIPR[®]). Combined with our previous report on gene delivery to pig airway basal cells, these data provide the feasibility of testing CRISPR/Cas9-mediated permanent human CFTR correction through HD-Ad vector delivery in pigs.},
}
@article {pmid31098740,
year = {2019},
author = {Luz, ACO and da Silva, JMA and Rezende, AM and de Barros, MPS and Leal-Balbino, TC},
title = {Analysis of direct repeats and spacers of CRISPR/Cas systems type I-F in Brazilian clinical strains of Pseudomonas aeruginosa.},
journal = {Molecular genetics and genomics : MGG},
volume = {294},
number = {5},
pages = {1095-1105},
pmid = {31098740},
issn = {1617-4623},
mesh = {Brazil ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genetic Markers/genetics ; Pseudomonas aeruginosa/*genetics ; Transcription, Genetic/genetics ; },
abstract = {CRISPR/Cas is an adaptive immune system found in prokaryotes, with the main function of protecting these cells from invasion and possible death by mobile genetic elements. Pseudomonas aeruginosa is considered a model for type I-F CRISPR/Cas system studies. However, its CRISPR loci characteristics have not yet been thoroughly described, and its function has not yet been fully unraveled. The aims of this study were to find the frequency of the system in Brazilian clinical isolates; to identify the loci sequence, its spacer diversity and its origins; as well as to propose a unified spacer library to aid in future structural studies of the CRISPR loci of P. aeruginosa. We investigated types I-F and I-E gene markers to establish CRISPR/Cas typing, and observed two strains harboring both systems simultaneously, a very rare feature. Through amplification and sequencing of CRISPR loci related to type I-F system, we describe polymorphisms in DRs and 350 spacers, of which 97 are new. The spacers that were identified had their possible organisms or proteins of origin identified. Spacer arrays were grouped in five different CRISPR patterns and the plasticity was inferred by rearrangements in spacer arrays. Here, we perform the first detailed and focused description of CRISPR/Cas elements in Brazilian clinical strains of P. aeruginosa. Our findings reflect active and highly diverse CRISPR loci, and we suggest that CRISPR/Cas may also pose as a transcriptional regulatory mechanism. The structural and diversity features described here can provide insights into the function of CRISPR/Cas in this pathogen and help guide the development of new therapeutic strategies.},
}
@article {pmid31097816,
year = {2019},
author = {Hajian, R and Balderston, S and Tran, T and deBoer, T and Etienne, J and Sandhu, M and Wauford, NA and Chung, JY and Nokes, J and Athaiya, M and Paredes, J and Peytavi, R and Goldsmith, B and Murthy, N and Conboy, IM and Aran, K},
title = {Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor.},
journal = {Nature biomedical engineering},
volume = {3},
number = {6},
pages = {427-437},
pmid = {31097816},
issn = {2157-846X},
support = {R01 EB023776/EB/NIBIB NIH HHS/United States ; R01 HL139605/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Dystrophin/genetics ; Exons/genetics ; Genome ; Graphite/*chemistry ; HEK293 Cells ; Humans ; Immobilized Proteins/*metabolism ; Male ; Muscular Dystrophy, Duchenne/genetics ; Mutation/genetics ; *Nucleic Acid Amplification Techniques ; RNA, Guide/metabolism ; *Transistors, Electronic ; },
abstract = {Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.},
}
@article {pmid31097696,
year = {2019},
author = {Picco, G and Chen, ED and Alonso, LG and Behan, FM and Gonçalves, E and Bignell, G and Matchan, A and Fu, B and Banerjee, R and Anderson, E and Butler, A and Benes, CH and McDermott, U and Dow, D and Iorio, F and Stronach, E and Yang, F and Yusa, K and Saez-Rodriguez, J and Garnett, MJ},
title = {Functional linkage of gene fusions to cancer cell fitness assessed by pharmacological and CRISPR-Cas9 screening.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2198},
pmid = {31097696},
issn = {2041-1723},
mesh = {Antineoplastic Agents/pharmacology ; Biomarkers, Tumor/*genetics ; CRISPR-Cas Systems/*genetics ; Carcinogenesis/genetics ; Cell Line, Tumor ; Datasets as Topic ; Drug Resistance, Neoplasm/genetics ; Early Detection of Cancer/methods ; Gene Expression Profiling/methods ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Fusion/*genetics ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; Humans ; Neoplasms/diagnosis/*genetics ; Sequence Analysis, RNA ; },
abstract = {Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications.},
}
@article {pmid31097693,
year = {2019},
author = {Zhan, T and Ambrosi, G and Wandmacher, AM and Rauscher, B and Betge, J and Rindtorff, N and Häussler, RS and Hinsenkamp, I and Bamberg, L and Hessling, B and Müller-Decker, K and Erdmann, G and Burgermeister, E and Ebert, MP and Boutros, M},
title = {MEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2197},
pmid = {31097693},
issn = {2041-1723},
mesh = {Adenomatous Polyposis Coli Protein/genetics/metabolism ; Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Biopsy ; CRISPR-Cas Systems/genetics ; Carcinogenesis/drug effects ; Cell Line, Tumor ; Cell Plasticity/drug effects ; Colorectal Neoplasms/*drug therapy/pathology ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; HEK293 Cells ; Humans ; Intestines/cytology/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, SCID ; Mitogen-Activated Protein Kinase Kinases/*antagonists &amp; inhibitors/metabolism ; Neoplastic Stem Cells/*drug effects ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Proteomics ; Wnt Signaling Pathway/*drug effects ; Xenograft Model Antitumor Assays ; ras Proteins/metabolism ; },
abstract = {In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/β-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.},
}
@article {pmid31097387,
year = {2020},
author = {Gore-Panter, SR and Van Wagoner, DR},
title = {Editorial commentary: A CRISPR approach to the study and treatment of cardiovascular disease.},
journal = {Trends in cardiovascular medicine},
volume = {30},
number = {2},
pages = {102-103},
pmid = {31097387},
issn = {1873-2615},
support = {R01 HL111314/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Cardiovascular Diseases ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; },
}
@article {pmid31096876,
year = {2019},
author = {Pan, S and Li, Q and Deng, L and Jiang, S and Jin, X and Peng, N and Liang, Y and She, Q and Li, Y},
title = {A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system.},
journal = {RNA biology},
volume = {16},
number = {9},
pages = {1166-1178},
pmid = {31096876},
issn = {1555-8584},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Immunity/*genetics ; Nucleotide Motifs/*genetics ; Nucleotides/genetics ; RNA/*genetics ; RNA Interference ; Sulfolobus/*genetics/*immunology ; },
abstract = {CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.},
}
@article {pmid31095905,
year = {2019},
author = {Javanpour, AA and Liu, CC},
title = {Genetic Compatibility and Extensibility of Orthogonal Replication.},
journal = {ACS synthetic biology},
volume = {8},
number = {6},
pages = {1249-1256},
pmid = {31095905},
issn = {2161-5063},
support = {DP2 GM119163/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA Replication/*genetics ; Gene Editing/*methods ; Genetic Vectors/genetics ; Mutagenesis, Site-Directed/*methods ; Plasmids/genetics ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/*methods ; },
abstract = {We recently developed an orthogonal replication system (OrthoRep) in yeast that allows for the rapid continuous mutagenesis of a special plasmid without mutating the genome. Although OrthoRep has been successfully applied to evolve several proteins and enzymes, the generality of OrthoRep has not yet been systematically studied. Here, we show that OrthoRep is fully compatible with all Saccharomyces cerevisiae strains tested, demonstrate that the orthogonal plasmid can encode genetic material of at least 22 kb, and report a CRISPR/Cas9-based method for expedient genetic manipulations of OrthoRep. It was previously reported that the replication system upon which OrthoRep is based is only stable in respiration-deficient S. cerevisiae strains that have lost their mitochondrial genome (ρ[0] strains). However, here we trace this biological incompatibility to the activity of the dispensable toxin/antitoxin system encoded on the wild-type orthogonal plasmid. Since the toxin/antitoxin system is replaced by genes of interest in any OrthoRep application, OrthoRep is a generally compatible platform for continuous in vivo evolution in S. cerevisiae.},
}
@article {pmid31095680,
year = {2019},
author = {Miao, D and Giassetti, MI and Ciccarelli, M and Lopez-Biladeau, B and Oatley, JM},
title = {Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†.},
journal = {Biology of reproduction},
volume = {101},
number = {1},
pages = {177-187},
pmid = {31095680},
issn = {1529-7268},
support = {R01 HD061665/HD/NICHD NIH HHS/United States ; R21 HD094568/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cattle/embryology ; Cells, Cultured ; Cloning, Organism/*methods/veterinary ; Electroporation/*methods/veterinary ; Embryo Culture Techniques/methods/veterinary ; Embryo Transfer/methods/veterinary ; Embryo, Mammalian/*cytology/metabolism ; Female ; Gene Editing/*methods/veterinary ; Gene Transfer Techniques/veterinary ; Genetic Engineering/*methods/veterinary ; Male ; Mice ; Mice, 129 Strain ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA-Binding Proteins/genetics ; Swine/embryology ; },
abstract = {Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species.},
}
@article {pmid31095294,
year = {2019},
author = {Radovčić, M and Čulo, A and Ivančić-Baće, I},
title = {Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent.},
journal = {FEMS microbiology letters},
volume = {366},
number = {9},
pages = {},
doi = {10.1093/femsle/fnz106},
pmid = {31095294},
issn = {1574-6968},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Helicases/*genetics ; *DNA Replication ; DNA, Bacterial/genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/*genetics ; Plasmids/*genetics ; *Temperature ; },
abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a CRISPR RNA (crRNA)-mediated interference mechanism. In Type I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled ('runaway') replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.},
}
@article {pmid31094569,
year = {2019},
author = {Wang, Y and Li, X and Osmundson, T and Shi, L and Yan, H},
title = {Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate.},
journal = {Foodborne pathogens and disease},
volume = {16},
number = {9},
pages = {604-615},
doi = {10.1089/fpd.2018.2611},
pmid = {31094569},
issn = {1556-7125},
mesh = {Animals ; Anti-Bacterial Agents/pharmacology ; China/epidemiology ; *Drug Resistance, Multiple, Bacterial ; *Food Microbiology ; Foodborne Diseases/*epidemiology/microbiology ; Humans ; Listeria monocytogenes/drug effects/genetics/*isolation &amp; purification ; Whole Genome Sequencing ; },
abstract = {Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA-arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.},
}
@article {pmid31094413,
year = {2019},
author = {Anower-E-Khuda, F and Singh, G and Deng, Y and Gordts, PLSM and Esko, JD},
title = {Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.},
journal = {Glycobiology},
volume = {29},
number = {8},
pages = {582-592},
pmid = {31094413},
issn = {1460-2423},
support = {R01 GM033063/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Hepatocytes/metabolism ; Humans ; Lipoproteins/chemistry/*metabolism ; *Loss of Function Mutation ; N-Acetylglucosaminyltransferases/genetics/*metabolism ; Protein Binding ; Sulfotransferases/genetics/*metabolism ; Syndecan-1/genetics/metabolism ; Triglycerides/chemistry/metabolism ; },
abstract = {Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depend on heparan sulfate and the hepatic proteoglycan, syndecan-1 (SDC1). Alteration of glucosamine N-sulfation by deletion of glucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) and 2-O-sulfation of uronic acids by deletion of uronyl 2-O-sulfotransferase (Hs2st) led to diminished lipoprotein metabolism, whereas inactivation of glucosaminyl 6-O-sulfotransferase 1 (Hs6st1), which encodes one of the three 6-O-sulfotransferases, had little effect on lipoprotein binding. However, other studies have suggested that 6-O-sulfation may be important for TRL binding and uptake. In order to explain these discrepant findings, we used CRISPR/Cas9 gene editing to create a library of mutants in the human hepatoma cell line, Hep3B. Inactivation of EXT1 encoding the heparan sulfate copolymerase, NDST1 and HS2ST dramatically reduced binding of TRLs. Inactivation of HS6ST1 had no effect, but deletion of HS6ST2 reduced TRL binding. Compounding mutations in HS6ST1 and HS6ST2 did not exacerbate this effect indicating that HS6ST2 is the dominant 6-O-sulfotransferase and that binding of TRLs indeed depends on 6-O-sulfation of glucosamine residues. Uptake studies showed that TRL internalization was also affected in 6-O-sulfation deficient cells. Interestingly, genetic deletion of SDC1 only marginally impacted binding of TRLs but reduced TRL uptake to the same extent as treating the cells with heparin lyases. These findings confirm that SDC1 is the dominant endocytic proteoglycan receptor for TRLs in human Hep3B cells and that binding and uptake of TRLs depend on SDC1 and N- and 2-O-sulfation as well as 6-O-sulfation of heparan sulfate chains catalyzed by HS6ST2.},
}
@article {pmid31092866,
year = {2019},
author = {Yamato, T and Handa, A and Arazoe, T and Kuroki, M and Nozaka, A and Kamakura, T and Ohsato, S and Arie, T and Kuwata, S},
title = {Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7427},
pmid = {31092866},
issn = {2045-2322},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Gene Knock-In Techniques/*methods ; Magnaporthe/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100-1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.},
}
@article {pmid31090950,
year = {2019},
author = {Cheong, KH and Koh, JM and Jones, MC},
title = {Black Swans of CRISPR: Stochasticity and Complexity of Genetic Regulation.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {41},
number = {7},
pages = {e1900032},
doi = {10.1002/bies.201900032},
pmid = {31090950},
issn = {1521-1878},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing ; Gene Expression Regulation/*genetics ; Humans ; },
abstract = {Recent waves of controversies surrounding genetic engineering have spilled into popular science in Twitter battles between reputable scientists and their followers. Here, a cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies is presented, focusing in particular on the stochastic nature of cellular control processes.},
}
@article {pmid31090512,
year = {2019},
author = {Bak, RO},
title = {The Potential of CRISPR/Cas9 in Hematotherapy.},
journal = {Stem cells and development},
volume = {28},
number = {11},
pages = {710-711},
doi = {10.1089/scd.2019.0079},
pmid = {31090512},
issn = {1557-8534},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; },
}
@article {pmid31090491,
year = {2019},
author = {Cong, L},
title = {Take Risks and Constantly Challenge the Status Quo.},
journal = {Stem cells and development},
volume = {28},
number = {11},
pages = {709},
doi = {10.1089/scd.2019.0082},
pmid = {31090491},
issn = {1557-8534},
mesh = {CRISPR-Cas Systems/*genetics ; Cell- and Tissue-Based Therapy/*methods ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; },
}
@article {pmid31090220,
year = {2019},
author = {Yeo, WL and Heng, E and Tan, LL and Lim, YW and Lim, YH and Hoon, S and Zhao, H and Zhang, MM and Wong, FT},
title = {Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {9},
pages = {2330-2338},
doi = {10.1002/bit.27021},
pmid = {31090220},
issn = {1097-0290},
mesh = {*CRISPR-Cas Systems ; Francisella/*genetics ; *Gene Editing ; Staphylococcus aureus/*genetics ; Streptococcus thermophilus/*genetics ; },
abstract = {Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high-efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 Cas9 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology-directed repair-mediated knock-in and deletion. In strains where spCas9 was nonfunctional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production, and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.},
}
@article {pmid31089700,
year = {2019},
author = {Makarova, KS and Gao, L and Zhang, F and Koonin, EV},
title = {Unexpected connections between type VI-B CRISPR-Cas systems, bacterial natural competence, ubiquitin signaling network and DNA modification through a distinct family of membrane proteins.},
journal = {FEMS microbiology letters},
volume = {366},
number = {8},
pages = {},
pmid = {31089700},
issn = {1574-6968},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Bacterial/*chemistry ; Escherichia coli/*genetics/metabolism ; Escherichia coli Proteins/*genetics/metabolism ; Genome, Bacterial ; Genomics ; Membrane Proteins/*genetics/metabolism ; *Signal Transduction ; Ubiquitin/*metabolism ; },
abstract = {In addition to core Cas proteins, CRISPR-Cas loci often encode ancillary proteins that modulate the activity of the respective effectors in interference. Subtype VI-B1 CRISPR-Cas systems encode the Csx27 protein that down-regulates the activity of Cas13b when the type VI-B locus is expressed in Escherichia coli. We show that Csx27 belongs to an expansive family of proteins that contain four predicted transmembrane helices and are typically encoded in predicted operons with components of the bacterial natural transformation machinery, multidomain proteins that consist of components of the ubiquitin signaling system and proteins containing the ligand-binding WYL domain and a helix-turn-helix domain. The Csx27 family proteins are predicted to form membrane channels for ssDNA that might comprise the core of a putative novel, Ub-regulated system for DNA uptake and, possibly, degradation. In addition to these associations, a distinct subfamily of the Csx27 family appears to be a part of a novel, membrane-associated system for DNA modification. In Bacteroidetes, subtype VI-B1 systems might degrade nascent transcripts of foreign DNA in conjunction with its uptake by the bacterial cell. These predictions suggest several experimental directions for the study of type VI CRISPR-Cas systems and distinct mechanisms of foreign DNA uptake and degradation in bacteria.},
}
@article {pmid31088931,
year = {2019},
author = {Gruffaz, M and Yuan, H and Meng, W and Liu, H and Bae, S and Kim, JS and Lu, C and Huang, Y and Gao, SJ},
title = {CRISPR-Cas9 Screening of Kaposi's Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells.},
journal = {mBio},
volume = {10},
number = {3},
pages = {},
pmid = {31088931},
issn = {2150-7511},
support = {R01 CA197153/CA/NCI NIH HHS/United States ; R01 CA096512/CA/NCI NIH HHS/United States ; R01 CA124332/CA/NCI NIH HHS/United States ; R01 CA213275/CA/NCI NIH HHS/United States ; R01 DE025465/DE/NIDCR NIH HHS/United States ; R01 CA177377/CA/NCI NIH HHS/United States ; R01 CA132637/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Cycle Checkpoints ; Cell Proliferation/*genetics ; Cell Transformation, Neoplastic/*genetics ; Early Detection of Cancer ; Genes, p53 ; Herpesvirus 8, Human/*pathogenicity ; Humans ; Karyopherins/antagonists &amp; inhibitors/*genetics ; Leukemia, Promyelocytic, Acute ; Liver Neoplasms ; Receptors, Cytoplasmic and Nuclear/antagonists &amp; inhibitors/*genetics ; Sequestosome-1 Protein/genetics ; Stomach Neoplasms ; Tumor Cells, Cultured ; },
abstract = {The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) screening in a unique model of matched primary and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells and identified genes that were growth promoting and growth suppressive for both types of cells, among which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by small interfering RNA (siRNA) knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells and in cell lines of other cancers, including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation, but the mechanisms of p53 activation differed among the different types of cancer cells. p53 activation depended on the formation of promyelocytic leukemia (PML) nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken the data together, we have identified novel growth-promoting and growth-suppressive genes of primary and cancer cells and have demonstrated that XPO1 is a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML- and p62-dependent mechanism of p53 activation in some types of cancer cells.IMPORTANCE Using a model of oncogenic virus KSHV-driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both growth promoting and growth suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated that the exportin XPO1 is a critical factor for the survival of transformed cells. Using a XPO1 inhibitor (KPT-8602) and siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and in growth transformation of KSHV-transformed cells, as well as of gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53, but the mechanisms of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells.},
}
@article {pmid31088860,
year = {2019},
author = {Lorenzini, PA and Chew, RSE and Tan, CW and Yong, JY and Zhang, F and Zheng, J and Roca, X},
title = {Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {905-920},
pmid = {31088860},
issn = {1469-9001},
mesh = {*Alternative Splicing ; CRISPR-Cas Systems ; Carrier Proteins/*genetics ; Cell Hypoxia ; Down-Regulation ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; *Gene Regulatory Networks ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; K562 Cells ; Kruppel-Like Transcription Factors/genetics ; Leukemia, Myeloid, Acute/*genetics ; Mutation ; Myelodysplastic Syndromes/*genetics ; Sequence Analysis, RNA/methods ; },
abstract = {Altered splicing contributes to the pathogenesis of human blood disorders including myelodysplastic syndromes (MDS) and leukemias. Here we characterize the transcriptomic regulation of PRPF40B, which is a splicing factor mutated in a small fraction of MDS patients. We generated a full PRPF40B knockout (KO) in the K562 cell line by CRISPR/Cas9 technology and rescued its levels by transient overexpression of wild-type (WT), P383L or P540S MDS alleles. Using RNA sequencing, we identified hundreds of differentially expressed genes and alternative splicing (AS) events in the KO that are rescued by WT PRPF40B, with a majority also rescued by MDS alleles, pointing to mild effects of these mutations. Among the PRPF40B-regulated AS events, we found a net increase in exon inclusion in the KO, suggesting that this splicing factor primarily acts as a repressor. PRPF40B-regulated splicing events are likely cotranscriptional, affecting exons with A-rich downstream intronic motifs and weak splice sites especially for 5' splice sites, consistent with its PRP40 yeast ortholog being part of the U1 small nuclear ribonucleoprotein. Loss of PRPF40B in K562 induces a KLF1 transcriptional signature, with genes involved in iron metabolism and mainly hypoxia, including related pathways like cholesterol biosynthesis and Akt/MAPK signaling. A cancer database analysis revealed that PRPF40B is lowly expressed in acute myeloid leukemia, whereas its paralog PRPF40A expression is high as opposed to solid tumors. Furthermore, these factors negatively or positively correlated with hypoxia regulator HIF1A, respectively. Our data suggest a PRPF40B role in repressing hypoxia in myeloid cells, and that its low expression might contribute to leukemogenesis.},
}
@article {pmid31087736,
year = {2019},
author = {Maron, L},
title = {Quilting plant chromosomes with CRISPR/Cas9.},
journal = {The Plant journal : for cell and molecular biology},
volume = {98},
number = {4},
pages = {575-576},
doi = {10.1111/tpj.14357},
pmid = {31087736},
issn = {1365-313X},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Plant ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genome, Plant ; },
}
@article {pmid31087560,
year = {2019},
author = {Li, SW and Yu, B and Byrne, G and Wright, M and O'Rourke, S and Mesa, K and Berman, PW},
title = {Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {9},
pages = {2130-2145},
pmid = {31087560},
issn = {1097-0290},
support = {R01 DA036335/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Complement C1s/*genetics/*metabolism ; Cricetulus ; *Gene Knockout Techniques ; HIV Envelope Protein gp120/*biosynthesis/genetics ; *HIV-1 ; *Proteolysis ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s[-/-] MGAT1[-] CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs). The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.},
}
@article {pmid31087001,
year = {2019},
author = {Adiego-Pérez, B and Randazzo, P and Daran, JM and Verwaal, R and Roubos, JA and Daran-Lapujade, P and van der Oost, J},
title = {Multiplex genome editing of microorganisms using CRISPR-Cas.},
journal = {FEMS microbiology letters},
volume = {366},
number = {8},
pages = {},
pmid = {31087001},
issn = {1574-6968},
mesh = {Bacteria/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods/trends ; *Gene Expression Regulation, Bacterial ; Industrial Microbiology/*methods ; },
abstract = {Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.},
}
@article {pmid31086989,
year = {2019},
author = {Sundberg, CD and Hankinson, O},
title = {A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {170},
number = {2},
pages = {310-319},
pmid = {31086989},
issn = {1096-0929},
support = {P30 DK041301/DK/NIDDK NIH HHS/United States ; R21 ES026392/ES/NIEHS NIH HHS/United States ; T32 ES015457/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; Benzo(a)pyrene/toxicity ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cytochrome P-450 CYP1A1/*biosynthesis ; Enzyme Induction ; Heme/biosynthesis ; Mice ; Receptors, Aryl Hydrocarbon/*physiology ; Tumor Cells, Cultured ; },
abstract = {Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.},
}
@article {pmid31086829,
year = {2019},
author = {Burgsdorf, I and Handley, KM and Bar-Shalom, R and Erwin, PM and Steindler, L},
title = {Life at Home and on the Roam: Genomic Adaptions Reflect the Dual Lifestyle of an Intracellular, Facultative Symbiont.},
journal = {mSystems},
volume = {4},
number = {4},
pages = {},
pmid = {31086829},
issn = {2379-5077},
abstract = {"Candidatus Synechococcus feldmannii" is a facultative intracellular symbiont of the Atlanto-Mediterranean sponge Petrosia ficiformis. Genomic information of sponge-associated cyanobacteria derives thus far from the obligate and extracellular symbiont "Candidatus Synechococcus spongiarum." Here we utilized a differential methylation-based approach for bacterial DNA enrichment combined with metagenomics to obtain the first draft genomes of "Ca. Synechococcus feldmannii." By comparative genomics, we revealed that some genomic features (e.g., iron transport mediated by siderophores, eukaryotic-like proteins, and defense mechanisms, like CRISPR-Cas [clustered regularly interspaced short palindromic repeats-associated proteins]) are unique to both symbiont types and absent or rare in the genomes of taxonomically related free-living cyanobacteria. These genomic features likely enable life under the conditions found inside the sponge host. Interestingly, there are many genomic features that are shared by "Ca. Synechococcus feldmannii" and free-living cyanobacteria, while they are absent in the obligate symbiont "Ca. Synechococcus spongiarum." These include genes related to cell surface structures, genetic regulation, and responses to environmental stress, as well as the composition of photosynthetic genes and DNA metabolism. We speculate that the presence of these genes confers on "Ca. Synechococcus feldmannii" its facultative nature (i.e., the ability to respond to a less stable environment when free-living). Our comparative analysis revealed that distinct genomic features depend on the nature of the symbiotic interaction: facultative and intracellular versus obligate and extracellular. IMPORTANCE Given the evolutionary position of sponges as one of the earliest phyla to depart from the metazoan stem lineage, studies on their distinct and exceptionally diverse microbial communities should yield a better understanding of the origin of animal-bacterium interactions. While genomes of several extracellular sponge symbionts have been published, the intracellular symbionts have, so far, been elusive. Here we compare the genomes of two unicellular cyanobacterial sponge symbionts that share an ancestor but followed different evolutionary paths-one became intracellular and the other extracellular. Counterintuitively, the intracellular cyanobacteria are facultative, while the extracellular ones are obligate. By sequencing the genomes of the intracellular cyanobacteria and comparing them to the genomes of the extracellular symbionts and related free-living cyanobacteria, we show how three different cyanobacterial lifestyles are reflected by adaptive genomic features.},
}
@article {pmid31086658,
year = {2019},
author = {Safari, F and Zare, K and Negahdaripour, M and Barekati-Mowahed, M and Ghasemi, Y},
title = {CRISPR Cpf1 proteins: structure, function and implications for genome editing.},
journal = {Cell &amp; bioscience},
volume = {9},
number = {},
pages = {36},
pmid = {31086658},
issn = {2045-3701},
abstract = {CRISPR and CRISPR-associated (Cas) protein, as components of microbial adaptive immune system, allows biologists to edit genomic DNA in a precise and specific way. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which can be programmed with a CRISPR RNA to bind and cleave complementary DNA targets. Cpf1 has recently emerged as an alternative for Cas9, due to its distinct features such as the ability to target T-rich motifs, no need for trans-activating crRNA, inducing a staggered double-strand break and potential for both RNA processing and DNA nuclease activity. In this review, we attempt to discuss the evolutionary origins, basic architectures, and molecular mechanisms of Cpf1 family proteins, as well as crRNA designing and delivery strategies. We will also describe the novel Cpf1 variants, which have broadened the versatility and feasibility of this system in genome editing, transcription regulation, epigenetic modulation, and base editing. Finally, we will be reviewing the recent studies on utilization of Cpf1as a molecular tool for genome editing.},
}
@article {pmid31086349,
year = {2019},
author = {Azvolinsky, A},
title = {Molecular scissors cut in on stem cells.},
journal = {Nature medicine},
volume = {25},
number = {6},
pages = {864-866},
pmid = {31086349},
issn = {1546-170X},
mesh = {Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Clinical Trials as Topic ; Gene Editing/*methods ; Humans ; Nuclear Proteins/genetics ; Repressor Proteins ; Stem Cell Transplantation ; Stem Cells/*metabolism ; beta-Globins/genetics ; beta-Thalassemia/genetics/therapy ; },
}
@article {pmid31086078,
year = {2019},
author = {Ravid, A and Rapaport, N and Issachar, A and Erman, A and Bachmetov, L and Tur-Kaspa, R and Zemel, R},
title = {25-Hydroxyvitamin D Inhibits Hepatitis C Virus Production in Hepatocellular Carcinoma Cell Line by a Vitamin D Receptor-Independent Mechanism.},
journal = {International journal of molecular sciences},
volume = {20},
number = {9},
pages = {},
pmid = {31086078},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics ; Calcifediol/*pharmacology ; Carcinoma, Hepatocellular/*metabolism/*virology ; Cell Line, Tumor ; Cell Survival/drug effects/genetics ; Cholecalciferol/pharmacology ; Hepacivirus/*drug effects/*physiology ; Humans ; Liver Neoplasms/*metabolism/*virology ; Real-Time Polymerase Chain Reaction ; Receptors, Calcitriol/genetics/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; },
abstract = {Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D3 (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D3 exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D3-treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D3 anti-hepatitis C virus effect. We show that vitamin D3 activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D3. This is inferred from the findings that 25(OH)D3 could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D3. Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D3 and 25(OH)D3 was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D3 anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D3 and 25(OH)D3, being 3β-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D3 that is mediated by 25(OH)D3 in a vitamin D receptor-independent mechanism.},
}
@article {pmid31085694,
year = {2019},
author = {McAllister, KN and Sorg, JA},
title = {CRISPR Genome Editing Systems in the Genus Clostridium: a Timely Advancement.},
journal = {Journal of bacteriology},
volume = {201},
number = {16},
pages = {},
pmid = {31085694},
issn = {1098-5530},
support = {R01 AI116895/AI/NIAID NIH HHS/United States ; U01 AI124290/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Clostridium/*genetics/metabolism ; Gene Editing/*methods ; Genome, Bacterial ; },
abstract = {The genus Clostridium is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the health care industry. Historically, even though these bacteria are well known and are commonly studied, the genetic technologies to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic modification systems available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas systems such as using wild-type Cas9, Cas9n, dCas9/CRISPR interference (CRISPRi) and a newly studied Cpf1/Cas12a, are reported. These have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant compounds. Here we discuss the details of the CRISPR-Cas systems as well as the advances and current issues in the developed clostridial systems.},
}
@article {pmid31085639,
year = {2019},
author = {Wang, C and Lu, T and Emanuel, G and Babcock, HP and Zhuang, X},
title = {Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {22},
pages = {10842-10851},
pmid = {31085639},
issn = {1091-6490},
support = {R01 MH113094/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Nucleus/chemistry/metabolism ; Gene Editing ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Image Processing, Computer-Assisted/*methods ; In Situ Hybridization, Fluorescence/*methods ; Molecular Probes/chemistry/genetics/metabolism ; RNA, Guide/chemistry/genetics/metabolism ; *RNA, Long Noncoding/chemistry/genetics/metabolism ; RNA-Binding Proteins/chemistry/metabolism ; Single-Cell Analysis/*methods ; },
abstract = {Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.},
}
@article {pmid31082677,
year = {2019},
author = {Howden, S and Hosseini Far, H and Motazedian, A and Elefanty, AG and Stanley, EG and Lamandé, SR and Bateman, JF},
title = {The use of simultaneous reprogramming and gene correction to generate an osteogenesis imperfecta patient COL1A1 c. 3936 G&gt;T iPSC line and an isogenic control iPSC line.},
journal = {Stem cell research},
volume = {38},
number = {},
pages = {101453},
doi = {10.1016/j.scr.2019.101453},
pmid = {31082677},
issn = {1876-7753},
mesh = {Amino Acid Substitution ; *CRISPR-Cas Systems ; Cell Line ; *Cellular Reprogramming Techniques ; *Collagen Type I/genetics/metabolism ; Collagen Type I, alpha 1 Chain ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Mutation, Missense ; *Osteogenesis Imperfecta/genetics/metabolism/pathology ; },
abstract = {To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we used a simultaneous reprogramming and CRISPR-Cas9 genome editing method to produce an iPSC line with the heterozygous patient mutation (COL1A1 c. 3936 G>T) along with an isogenic gene-corrected control iPSC line. Both IPSC lines had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This osteogenesis imperfecta mutant and isogenic iPSC control line will be of use in exploring disease mechanisms and therapeutic approaches in vitro.},
}
@article {pmid31081744,
year = {2019},
author = {Mussolino, C},
title = {Hematotherapy.},
journal = {Stem cells and development},
volume = {28},
number = {11},
pages = {714},
doi = {10.1089/scd.2019.0078},
pmid = {31081744},
issn = {1557-8534},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunotherapy/*methods ; },
}
@article {pmid31080012,
year = {2019},
author = {Wilkinson, M and Drabavicius, G and Silanskas, A and Gasiunas, G and Siksnys, V and Wigley, DB},
title = {Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.},
journal = {Molecular cell},
volume = {75},
number = {1},
pages = {90-101.e5},
pmid = {31080012},
issn = {1097-4164},
support = {MR/N009258/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; MR/N009258/1/MRC_/Medical Research Council/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Base Sequence ; Binding Sites ; CRISPR-Associated Protein 9/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; Cryoelectron Microscopy ; DNA/*chemistry/genetics/metabolism ; DNA, Intergenic/*chemistry/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; Isoenzymes/chemistry/genetics/metabolism ; Molecular Docking Simulation ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Recombinant Proteins/chemistry/genetics/metabolism ; Streptococcus thermophilus/*genetics/metabolism ; Substrate Specificity ; },
abstract = {CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.},
}
@article {pmid31079595,
year = {2019},
author = {Huang, Y and Ding, Y and Liu, Y and Zhou, S and Ding, Q and Yan, H and Ma, B and Zhao, X and Wang, X and Chen, Y},
title = {Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model.},
journal = {Reproduction, fertility, and development},
volume = {31},
number = {9},
pages = {1533-1537},
doi = {10.1071/RD18485},
pmid = {31079595},
issn = {1031-3613},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Goats ; Microinjections ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL-1 Cas9 mRNA and 25ngμL-1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.},
}
@article {pmid31078796,
year = {2019},
author = {Sayed, S and Paszkowski-Rogacz, M and Schmitt, LT and Buchholz, F},
title = {CRISPR/Cas9 as a tool to dissect cancer mutations.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {36-48},
doi = {10.1016/j.ymeth.2019.05.007},
pmid = {31078796},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cloning, Molecular/methods ; Colorectal Neoplasms/*genetics ; DNA Mutational Analysis/instrumentation/*methods ; Gene Editing/*methods ; Genetic Vectors/genetics ; *Genomic Library ; High-Throughput Nucleotide Sequencing/instrumentation/methods ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics/isolation &amp; purification ; Transfection/instrumentation/methods ; },
abstract = {The CRISPR/Cas9 system is transforming many biomedical disciplines, including cancer research. Through its flexible programmability and efficiency to induce DNA double strand breaks it has become straightforward to introduce cancer mutations into cells in vitro and/or in vivo. However, not all mutations contribute equally to tumorigenesis and distinguishing essential mutations for tumor growth and survival from biologically inert mutations is cumbersome. Here we present a method to screen for the functional relevance of mutations in high throughput in established cancer cell lines. We employ the CRISPR/Cas9 system to probe cancer vulnerabilities in a colorectal carcinoma cell line in an attempt to identify novel cancer driver mutations. We designed 100 high quality sgRNAs that are able to specifically cleave mutations present in the colorectal carcinoma cell line RKO. An all-in-one lentiviral library harboring these sgRNAs was then generated and used in a pooled screen to probe possible growth dependencies on these mutations. Genomic DNA at different time points were collected, the sgRNA cassettes were PCR amplified, purified and sgRNA counts were quantified by means of deep sequencing. The analysis revealed two sgRNAs targeting the same mutation (UTP14A: S99delS) to be depleted over time in RKO cells. Validation and characterization confirmed that the inactivation of this mutation impairs cell growth, nominating UTP14A: S99delS as a putative driver mutation in RKO cells. Overall, our approach demonstrates that the CRISPR/Cas9 system is a powerful tool to functionally dissect cancer mutations at large-scale.},
}
@article {pmid31078694,
year = {2019},
author = {Yan, L and Feng, H and Wang, F and Lu, B and Liu, X and Sun, L and Wang, D},
title = {Establishment of three estrogen receptors (esr1, esr2a, esr2b) knockout lines for functional study in Nile tilapia.},
journal = {The Journal of steroid biochemistry and molecular biology},
volume = {191},
number = {},
pages = {105379},
doi = {10.1016/j.jsbmb.2019.105379},
pmid = {31078694},
issn = {1879-1220},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cichlids/*genetics/physiology ; Estrogen Receptor alpha/*genetics ; Estrogen Receptor beta/*genetics ; Female ; Fish Proteins/*genetics ; Male ; Mutation ; Reproduction ; },
abstract = {Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptors (ESRs) in all vertebrates. To date, distinct roles of estrogen receptors have been characterized only in human and model organisms, including mouse, rat, zebrafish and medaka. Physiological role of estrogen/receptor signaling in reproduction remains poorly defined in non-model organisms. In the present study, we successfully generated esr1, esr2a and esr2b mutant lines in tilapia by CRISPR/Cas9 and examined their phenotypes. Surprisingly, the esr1 mutants showed no phenotypes of reproductive development and function in both females and males. The esr2a mutant females showed significantly delayed ovarian development and follicle growth at 90 and 180 dah, and the development caught up later at 360 dah. The esr2a mutant males showed no phenotypes at 90 dah, and displayed smaller gonads and efferent ducts, less spermatogonia and more abnormal sperms at 180 dah. In contrast, the esr2b mutants displayed abnormal development of ovarian ducts and efferent ducts which failed to connect to the genital orifice, and which in turn, resulted in infertility in female and male, respectively, although they produced gametes in their gonads. Taken together, our study provides evidence for differential functions of esr1, esr2a and esr2b in fish reproduction.},
}
@article {pmid31078316,
year = {2019},
author = {Bruch, R and Urban, GA and Dincer, C},
title = {CRISPR/Cas Powered Multiplexed Biosensing.},
journal = {Trends in biotechnology},
volume = {37},
number = {8},
pages = {791-792},
doi = {10.1016/j.tibtech.2019.04.005},
pmid = {31078316},
issn = {1879-3096},
mesh = {Biosensing Techniques/*methods/trends ; *CRISPR-Cas Systems ; Diagnostic Tests, Routine/*methods/trends ; Humans ; Molecular Diagnostic Techniques/*methods/trends ; },
abstract = {Multiplexed CRISPR/Cas biosensing offers various possibilities for diagnosing different diseases by analyzing many analytes from one single specimen. However, in this relatively new field, nearly no multiplexing approaches exist, as many challenges need to be addressed. We discuss the reasons behind it and possible strategies to push the multiplexing level.},
}
@article {pmid31078270,
year = {2019},
author = {Yang, Q and Xie, H and Zhong, Y and Li, D and Ke, X and Ying, H and Yu, B and Zhang, T},
title = {Severe Fanconi Anemia phenotypes in Fancd2 depletion mice.},
journal = {Biochemical and biophysical research communications},
volume = {514},
number = {3},
pages = {713-719},
doi = {10.1016/j.bbrc.2019.04.201},
pmid = {31078270},
issn = {1090-2104},
mesh = {Anemia/pathology ; Animals ; Base Sequence ; Bone Marrow Cells/drug effects/metabolism/radiation effects ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Fanconi Anemia/*pathology ; Fanconi Anemia Complementation Group D2 Protein/*deficiency/metabolism ; Female ; Hematopoiesis/drug effects/radiation effects ; Mice, Inbred C57BL ; Mice, Knockout ; Mitomycin/pharmacology ; Phenotype ; Radiation, Ionizing ; Stem Cells/drug effects/metabolism/radiation effects ; },
abstract = {Fanconi anemia (FA) is a genetic disorder characterized by congenital malfunction, bone marrow failure and hypersensitivity to DNA damage. FANCD2 protein play the central role in FA pathway. To study the in vivo role of FANCD2, we generated and characterized a new Fancd2 knockout mouse strain with 7bp deletion in Fancd2 gene 5' terminus using Crispr-Cas9 in congenic C57BL/6J background. This Fancd2[-/-] mice displayed similar but overall more severe manifestation than the previous ES cell targeted Fancd2 model. These features include increased embryonic and postnatal lethality rate, higher incidence of microphthalmia, and more severe hypogonadism. The anemia we observed in this Fancd2[-/-] mice has not been described in other FA models. Further study indicated that the hematopoiesis deficiency was associated with increased apoptotic cell death, G2/M phase arrest and hypersensitivity to MMC and IR damage of Fancd2[-/-] bone marrow progenitor cells. Collectively, the resulting Fancd2[-/-] mice with higher resemblance of FA patient symptoms, will be useful in understand the parthenogenesis of pancytopenia and bone marrow failure in FA.},
}
@article {pmid31078083,
year = {2019},
author = {Weber, J and Rad, R},
title = {Engineering CRISPR mouse models of cancer.},
journal = {Current opinion in genetics &amp; development},
volume = {54},
number = {},
pages = {88-96},
doi = {10.1016/j.gde.2019.04.001},
pmid = {31078083},
issn = {1879-0380},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Embryonic Stem Cells/metabolism ; Epigenome/genetics ; Gene Editing ; *Genetic Engineering ; *Genetic Therapy ; Germ Cells/metabolism/transplantation ; Humans ; Mice ; Mutation ; Neoplasms/*genetics/pathology ; },
abstract = {Gene targeting in mammals has revolutionized the study of complex diseases, involving the interaction of multiple genes, cells, and organ systems. In cancer, genetically engineered mouse models deciphered biological principles by integrating molecular mechanisms, cellular processes, and environmental signals. Major advances in manipulative mouse genetics are currently emerging from breakthroughs in gene editing, which open new avenues for rapid model generation. Here, we review recent developments in engineering CRISPR mouse models of cancer. We describe engineering strategies, including germline manipulation of zygotes or embryonic stem cells, direct in vivo somatic gene editing, and ex vivo targeting of cellular transplant models. We also discuss promises and limitations of the expanding spectrum of CRISPR applications, ranging from engineering of simple mutations over complex genomic rearrangements to gene and epigenome regulation. Fast and scalable in vivo CRISPR methodologies pave the way for a new phase of functional cancer genomics.},
}
@article {pmid31077304,
year = {2019},
author = {Trasanidou, D and Gerós, AS and Mohanraju, P and Nieuwenweg, AC and Nobrega, FL and Staals, RHJ},
title = {Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs.},
journal = {FEMS microbiology letters},
volume = {366},
number = {9},
pages = {},
pmid = {31077304},
issn = {1574-6968},
mesh = {Bacteria/*virology ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Evolution, Molecular ; Gene Editing ; Host-Pathogen Interactions/*genetics ; Viral Proteins/*genetics ; },
abstract = {CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.},
}
@article {pmid31077288,
year = {2019},
author = {Wang, L and Li, J},
title = {'Artificial spermatid'-mediated genome editing†.},
journal = {Biology of reproduction},
volume = {101},
number = {3},
pages = {538-548},
doi = {10.1093/biolre/ioz087},
pmid = {31077288},
issn = {1529-7268},
mesh = {Animals ; *Animals, Genetically Modified/embryology/genetics ; CRISPR-Cas Systems/physiology ; Cell Culture Techniques ; Cloning, Organism/methods/veterinary ; Embryo Culture Techniques/veterinary ; Embryonic Stem Cells/cytology ; Gene Editing/*methods/veterinary ; Male ; Spermatids/*cytology/metabolism ; },
abstract = {For years, extensive efforts have been made to use mammalian sperm as the mediator to generate genetically modified animals; however, the strategy of sperm-mediated gene transfer (SMGT) is unable to produce stable and diversified modifications in descendants. Recently, haploid embryonic stem cells (haESCs) have been successfully derived from haploid embryos carrying the genome of highly specialized gametes, and can stably maintain haploidy (through periodic cell sorting based on DNA quantity) and both self-renewal and pluripotency in long-term cell culture. In particular, haESCs derived from androgenetic haploid blastocysts (AG-haESCs), carrying only the sperm genome, can support the generation of live mice (semi-cloned, SC mice) through oocyte injection. Remarkably, after removal of the imprinted control regions H19-DMR (differentially methylated region of DNA) and IG-DMR in AG-haESCs, the double knockout (DKO)-AG-haESCs can stably produce SC animals with high efficiency, and so can serve as a sperm equivalent. Importantly, DKO-AG-haESCs can be used for multiple rounds of gene modifications in vitro, followed by efficient generation of live and fertile mice with the expected genetic traits. Thus, DKO-AG-haESCs (referred to as 'artificial spermatids') combed with CRISPR-Cas technology can be used as the genetically tractable fertilization agent, to efficiently create genetically modified offspring, and is a versatile genetic tool for in vivo analyses of gene function.},
}
@article {pmid31077282,
year = {2019},
author = {Cañez, C and Selle, K and Goh, YJ and Barrangou, R},
title = {Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus.},
journal = {FEMS microbiology letters},
volume = {366},
number = {9},
pages = {},
doi = {10.1093/femsle/fnz105},
pmid = {31077282},
issn = {1574-6968},
mesh = {Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Chromosomes, Bacterial/*genetics ; *Gene Editing ; Genome, Bacterial ; Homologous Recombination ; Plasmids/genetics ; Streptococcus thermophilus/*genetics/*virology ; },
abstract = {CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.},
}
@article {pmid31076628,
year = {2019},
author = {Hocq, R and Bouilloux-Lafont, M and Lopes Ferreira, N and Wasels, F},
title = {σ[54] (σ[L]) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7228},
pmid = {31076628},
issn = {2045-2322},
mesh = {2-Propanol/metabolism ; Bacterial Proteins/genetics/*metabolism ; Butanols/metabolism ; CRISPR-Cas Systems/genetics ; Carbon/*metabolism ; Clostridium beijerinckii/genetics/*metabolism ; Ethanol/metabolism ; Gene Editing/methods ; Glucose/metabolism ; Phenotype ; Point Mutation ; Sigma Factor/deficiency/genetics/*metabolism ; Solvents/metabolism ; },
abstract = {The solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. This strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ[54] sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and RT-qPCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ[54] in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052.},
}
@article {pmid31076459,
year = {2019},
author = {Nasef, M and Muffly, MC and Beckman, AB and Rowe, SJ and Walker, FC and Hatoum-Aslan, A and Dunkle, JA},
title = {Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {948-962},
pmid = {31076459},
issn = {1469-9001},
support = {P30 CA013148/CA/NCI NIH HHS/United States ; R15 GM129671/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine Nucleotides/biosynthesis/*metabolism ; Bacterial Proteins/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/*metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Magnesium/metabolism ; Models, Molecular ; Oligoribonucleotides/biosynthesis/*metabolism ; Polymorphism, Single Nucleotide ; RNA, Bacterial/*metabolism ; Second Messenger Systems ; Staphylococcus epidermidis/*metabolism ; },
abstract = {CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg[2+]-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.},
}
@article {pmid31076406,
year = {2019},
author = {Izaguirre-Carbonell, J and Christiansen, L and Burns, R and Schmitz, J and Li, C and Mokry, RL and Bluemn, T and Zheng, Y and Shen, J and Carlson, KS and Rao, S and Wang, D and Zhu, N},
title = {Critical role of Jumonji domain of JMJD1C in MLL-rearranged leukemia.},
journal = {Blood advances},
volume = {3},
number = {9},
pages = {1499-1511},
pmid = {31076406},
issn = {2473-9537},
support = {R37 CA229751/CA/NCI NIH HHS/United States ; R00 CA168996/CA/NCI NIH HHS/United States ; R01 CA204231/CA/NCI NIH HHS/United States ; R01 AI079087/AI/NIAID NIH HHS/United States ; R01 HL130724/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Editing ; Histone-Lysine N-Methyltransferase/*genetics ; Histones/metabolism ; Humans ; Interleukin-3/metabolism ; Jumonji Domain-Containing Histone Demethylases/chemistry/genetics/*metabolism ; Leukemia, Myeloid, Acute/genetics/*pathology ; Mice ; Mice, Inbred C57BL ; Myeloid-Lymphoid Leukemia Protein/*genetics ; Oxidoreductases, N-Demethylating/chemistry/genetics/*metabolism ; Protein Domains ; RNA, Guide/metabolism ; Signal Transduction ; Transplantation, Heterologous ; Zinc Fingers/genetics ; },
abstract = {JMJD1C, a member of the lysine demethylase 3 family, is aberrantly expressed in mixed lineage leukemia (MLL) gene-rearranged (MLLr) leukemias. We have shown previously that JMJD1C is required for self-renewal of acute myeloid leukemia (AML) leukemia stem cells (LSCs) but not normal hematopoietic stem cells. However, the domains within JMJD1C that promote LSC self-renewal are unknown. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) negative-selection screening and identified a requirement for the catalytic Jumonji (JmjC) domain and zinc finger domain for leukemia cell survival in vitro and in vivo. In addition, we found that histone H3 lysine 36 methylation (H3K36me) is a marker for JMJD1C activity at gene loci. Moreover, we performed single cell transcriptome analysis of mouse leukemia cells harboring a single guide RNA (sgRNA) against the JmjC domain and identified increased activation of RAS/MAPK and the JAK-STAT pathway in cells harboring the JmjC sgRNA. We discovered that upregulation of interleukin 3 (IL-3) receptor genes mediates increased activation of IL-3 signaling upon JMJD1C loss or mutation. Along these lines, we observed resistance to JMJD1C loss in MLLr AML bearing activating RAS mutations, suggesting that RAS pathway activation confers resistance to JMJD1C loss. Overall, we discovered the functional importance of the JMJD1C JmjC domain in AML leukemogenesis and a novel interplay between JMJD1C and the IL-3 signaling pathway as a potential resistance mechanism to targeting JMJD1C catalytic activity.},
}
@article {pmid31075690,
year = {2019},
author = {Hosseini Far, H and Patria, YN and Motazedian, A and Elefanty, AG and Stanley, EG and Lamandé, SR and Bateman, JF},
title = {Generation of a heterozygous COL1A1 (c.3969_3970insT) osteogenesis imperfecta mutation human iPSC line, MCRIi001-A-1, using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {37},
number = {},
pages = {101449},
doi = {10.1016/j.scr.2019.101449},
pmid = {31075690},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Cellular Reprogramming ; Collagen Type I/*genetics ; Collagen Type I, alpha 1 Chain ; *Gene Editing ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Leukocytes, Mononuclear/metabolism/*pathology ; Male ; Middle Aged ; *Mutation ; Osteogenesis Imperfecta/*genetics/pathology ; Phenotype ; },
abstract = {To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we have used gene editing to produce a facsimile of the patient heterozygous COL1A1 mutation in an established control iPSC line. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line and the isogenic parental iPSC line will be of use in exploring osteogenesis imperfecta disease mechanisms and therapeutic approaches in vitro.},
}
@article {pmid31075379,
year = {2019},
author = {Farhat, S and Jain, N and Singh, N and Sreevathsa, R and Dash, PK and Rai, R and Yadav, S and Kumar, P and Sarkar, AK and Jain, A and Singh, NK and Rai, V},
title = {CRISPR-Cas9 directed genome engineering for enhancing salt stress tolerance in rice.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {91-99},
doi = {10.1016/j.semcdb.2019.05.003},
pmid = {31075379},
issn = {1096-3634},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Oryza/*genetics ; Salt Stress/*genetics ; },
abstract = {Crop productivity in rice is harshly limited due to high concentration of salt in the soil. To understand the intricacies of the mechanism it is important to unravel the key pathways operating inside the plant cell. Emerging state-of-the art technologies have provided the tools to discover the key components inside the plant cell for salt tolerance. Among the molecular entities, transcription factors and/or other important components of sensing and signaling cascades have been the attractive targets and the role of NHX and SOS1 transporters amply described. Not only marker assisted programs but also transgenic approaches by using reverse genetic strategies (knockout or knockdown) or overexpression have been extensively used to engineer rice crop. CRISPR/Cas is an attractive paradigm and provides the feasibility for manipulating several genes simultaneously. Here, in this review we highlight some of the molecular entities that could be potentially targeted for generating rice amenable to sustain growth under high salinity conditions by employing CRISPR/Cas. We also try to address key questions for rice salt stress tolerance other than what is already known.},
}
@article {pmid31075206,
year = {2019},
author = {Gou, Y and Liu, W and Wang, JJ and Tan, L and Hong, B and Guo, L and Liu, H and Pan, Y and Zhao, Y},
title = {CRISPR-Cas9 knockout of qseB induced asynchrony between motility and biofilm formation in Escherichia coli.},
journal = {Canadian journal of microbiology},
volume = {65},
number = {9},
pages = {691-702},
doi = {10.1139/cjm-2019-0100},
pmid = {31075206},
issn = {1480-3275},
mesh = {Biofilms/*growth &amp; development ; CRISPR-Cas Systems ; Escherichia coli/cytology/*genetics/metabolism/physiology ; Escherichia coli Proteins/genetics/*metabolism ; Fimbriae, Bacterial/genetics ; Gene Expression Regulation, Bacterial/*genetics ; Microscopy, Electron, Scanning ; Sequence Deletion ; Virulence ; },
abstract = {Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.},
}
@article {pmid31073745,
year = {2019},
author = {Schmöhl, F and Peters, V and Schmitt, CP and Poschet, G and Büttner, M and Li, X and Weigand, T and Poth, T and Volk, N and Morgenstern, J and Fleming, T and Nawroth, PP and Kroll, J},
title = {CNDP1 knockout in zebrafish alters the amino acid metabolism, restrains weight gain, but does not protect from diabetic complications.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {22},
pages = {4551-4568},
pmid = {31073745},
issn = {1420-9071},
mesh = {Amino Acids/*metabolism ; Animals ; Carnosine/metabolism ; Diabetes Complications/*metabolism ; Diabetes Mellitus, Type 2/metabolism ; Dipeptidases/*metabolism ; Gene Knockout Techniques/methods ; Kidney/metabolism ; Weight Gain/*physiology ; Zebrafish ; },
abstract = {The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1[-/-] zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1[-/-] zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala-His and Ser-His. Under diabetic conditions increased carnosine levels in CNDP1[-/-] embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications.},
}
@article {pmid31073223,
year = {2019},
author = {Stanic, K and Reig, G and Figueroa, RJ and Retamal, PA and Wichmann, IA and Opazo, JC and Owen, GI and Corvalán, AH and Concha, ML and Amigo, JD},
title = {The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {7131},
pmid = {31073223},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Embryonic Development ; Hemangioblasts/*metabolism ; Hematopoiesis ; Membrane Proteins/*genetics ; Morpholinos/pharmacology ; Multigene Family ; Zebrafish/blood/*embryology/genetics ; Zebrafish Proteins/genetics ; },
abstract = {The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.},
}
@article {pmid31073172,
year = {2019},
author = {Baumann, V and Wiesbeck, M and Breunig, CT and Braun, JM and Köferle, A and Ninkovic, J and Götz, M and Stricker, SH},
title = {Targeted removal of epigenetic barriers during transcriptional reprogramming.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2119},
pmid = {31073172},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Cell Line ; Cellular Reprogramming/*genetics ; DNA Methylation/genetics ; DNA-Binding Proteins/genetics/metabolism ; *Epigenesis, Genetic ; Gene Editing/methods ; Gene Expression Regulation ; Mice ; Neural Stem Cells/*physiology ; Neuroglia/cytology/physiology ; Promoter Regions, Genetic/genetics ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide/genetics/metabolism ; SOXB1 Transcription Factors/genetics/*metabolism ; Transcription, Genetic/genetics ; },
abstract = {Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers.},
}
@article {pmid31073154,
year = {2019},
author = {Kundert, K and Lucas, JE and Watters, KE and Fellmann, C and Ng, AH and Heineike, BM and Fitzsimmons, CM and Oakes, BL and Qu, J and Prasad, N and Rosenberg, OS and Savage, DF and El-Samad, H and Doudna, JA and Kortemme, T},
title = {Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2127},
pmid = {31073154},
issn = {2041-1723},
support = {R21 EB021453/EB/NIBIB NIH HHS/United States ; T32 GM008284/GM/NIGMS NIH HHS/United States ; R01 GM110089/GM/NIGMS NIH HHS/United States ; R01 GM127463/GM/NIGMS NIH HHS/United States ; R01-GM110089//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; n/a//W. M. Keck Foundation (W.M. Keck Foundation)/International ; R01 AI128214/AI/NIAID NIH HHS/United States ; K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; },
mesh = {Aptamers, Nucleotide/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/*methods ; Ligands ; RNA, Guide/*genetics ; },
abstract = {The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.},
}
@article {pmid31073033,
year = {2019},
author = {Wang, C and Jiang, S and Ke, L and Zhang, L and Li, D and Liang, J and Narita, Y and Hou, I and Chen, CH and Wang, L and Zhong, Q and Ling, Y and Lv, X and Xiang, Y and Guo, X and Teng, M and Tsao, SW and Gewurz, BE and Zeng, MS and Zhao, B},
title = {Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {25},
pages = {9734-9745},
pmid = {31073033},
issn = {1083-351X},
support = {P30 CA076292/CA/NCI NIH HHS/United States ; R01 AI123420/AI/NIAID NIH HHS/United States ; R01 AI137337/AI/NIAID NIH HHS/United States ; R01 CA047006/CA/NCI NIH HHS/United States ; },
mesh = {Biomarkers, Tumor/antagonists &amp; inhibitors/*genetics ; *CRISPR-Cas Systems ; *Cell Proliferation ; Gene Knockout Techniques/*methods ; *Genome, Human ; Humans ; Nasopharyngeal Carcinoma/*genetics/pathology ; Nasopharyngeal Neoplasms/*genetics/pathology ; Proto-Oncogene Mas ; Signal Transduction ; Tumor Cells, Cultured ; },
abstract = {Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.},
}
@article {pmid31072915,
year = {2019},
author = {Zhang, T and Lu, Y and Song, S and Lu, R and Zhou, M and He, Z and Yuan, T and Yan, K and Cheng, Y},
title = {'Double-muscling' and pelvic tilt phenomena in rabbits with the cystine-knot motif deficiency of myostatin on exon 3.},
journal = {Bioscience reports},
volume = {39},
number = {5},
pages = {},
pmid = {31072915},
issn = {1573-4935},
mesh = {Amino Acid Motifs ; Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Female ; *Loss of Function Mutation ; *Muscle, Skeletal/metabolism/pathology ; *Myostatin/genetics/metabolism ; Rabbits ; },
abstract = {Gene mutations at different gene sites will produce totally different phenotypes or biological functions in gene-edited animals. An allelic series of mutations in the myostatin (MSTN) gene can cause the 'double-muscling' phenotype. Although there have been many studies performed on MSTN-mutant animals, there have been few studies that have investigated the cystine-knot motif in exon 3 of MSTN in rabbits. In the current study, CRISPR/Cas9 sgRNA anchored exon 3 of a rabbit's MSTN was used to disrupt the cystine-knot motif to change the MSTN construction and cause a loss of its function. Eleven MSTN-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational MSTN amino acid sequences of the 11 founder rabbits were modeled to the tertiary structure using the SWISS-MODEL, and the results showed that the structure of the cystine-knot motif of each protein in the founder rabbits differed from the wild-type (WT). The MSTN-KO rabbits displayed an obvious 'double-muscling' phenomena, with a 20-30% increase in body weight compared with WT rabbits. In the MSTN-KO rabbits, all of the MSTN[-/-] rabbits showed teeth dislocation and tongue enlargement, and the percentage of rabbits having pelvic tilt was 0% in MSTN[+/+], 0% in MSTN[+/-], 77.78% in female MSTN[-/-] rabbits, and 37.50% in male MSTN[-/-] rabbits. The biomechanical mechanism of pelvic tilt and teeth dislocation in the MSTN-KO rabbits requires further investigation.These newly generated MSTN-KO rabbits will serve as an important animal model, not only for studying skeletal muscle development, but also for biomedical studies in pelvic tilt correction and craniofacial research.},
}
@article {pmid31072871,
year = {2019},
author = {Haroth, S and Feussner, K and Kelly, AA and Zienkiewicz, K and Shaikhqasem, A and Herrfurth, C and Feussner, I},
title = {The glycosyltransferase UGT76E1 significantly contributes to 12-O-glucopyranosyl-jasmonic acid formation in wounded Arabidopsis thaliana leaves.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {25},
pages = {9858-9872},
pmid = {31072871},
issn = {1083-351X},
mesh = {Amino Acid Sequence ; Arabidopsis/genetics/growth &amp; development/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; Cyclopentanes/*chemistry/*metabolism ; Gene Expression Regulation, Plant ; Glycosyltransferases/genetics/*metabolism ; Oxylipins/*chemistry/*metabolism ; Plant Leaves/genetics/growth &amp; development/*metabolism ; Sequence Homology ; Signal Transduction ; },
abstract = {Jasmonoyl-isoleucine (JA-Ile) is a phytohormone that orchestrates plant defenses in response to wounding, feeding insects, or necrotrophic pathogens. JA-Ile metabolism has been studied intensively, but its catabolism as a potentially important mechanism for the regulation of JA-Ile-mediated signaling is not well-understood. Especially the enzyme(s) responsible for specifically glycosylating 12-hydroxy-jasmonic acid (12-OH-JA) and thereby producing 12-O-glucopyranosyl-jasmonic acid (12-O-Glc-JA) is still elusive. Here, we used co-expression analyses of available Arabidopsis thaliana transcriptomic data, identifying four UDP-dependent glycosyltransferase (UGT) genes as wound-induced and 12-OH-JA-related, namely, UGT76E1, UGT76E2, UGT76E11, and UGT76E12 We heterologously expressed and purified the corresponding proteins to determine their individual substrate specificities and kinetic parameters. We then used an ex vivo metabolite-fingerprinting approach to investigate these proteins in conditions as close as possible to their natural environment, with an emphasis on greatly extending the range of potential substrates. As expected, we found that UGT76E1 and UGT76E2 are 12-OH-JA-UGTs, with UGT76E1 contributing a major in vivo UGT activity, as deduced from Arabidopsis mutants with abolished or increased UGT gene expression. In contrast, recombinant UGT76E11 acted on an unidentified compound and also glycosylated two other oxylipins, 11-hydroxy-7,9,13-hexadecatrienoic acid (11-HHT) and 13-hydroxy-9,11,15-octadecatrienoic acid (13-HOT), which were also accepted by recombinant UGT76E1, UGT76E2, and UGT76E12 enzymes. UGT76E12 glycosylated 12-OH-JA only to a low extent, but also accepted an artificial hydroxylated fatty acid and low amounts of kaempferol. In conclusion, our findings have elucidated the missing step in the wound-induced synthesis of 12-O-glucopyranosyl-jasmonic acid in A. thaliana.},
}
@article {pmid31072819,
year = {2019},
author = {Tanaka, H and Arima, Y and Kamimura, D and Tanaka, Y and Takahashi, N and Uehata, T and Maeda, K and Satoh, T and Murakami, M and Akira, S},
title = {Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is critical in IL-17 response.},
journal = {The Journal of experimental medicine},
volume = {216},
number = {6},
pages = {1431-1449},
pmid = {31072819},
issn = {1540-9538},
support = {P01 AI070167/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Cytosol/metabolism ; Encephalomyelitis, Autoimmune, Experimental/pathology ; Endoplasmic Reticulum/drug effects/*metabolism ; I-kappa B Kinase/metabolism ; Inflammation/pathology ; Interleukin-17/*pharmacology ; Interleukin-6/genetics/metabolism ; Mice ; Mutation/genetics ; Phosphorylation ; Protein Serine-Threonine Kinases/metabolism ; Protein Stability/drug effects ; Proteolysis/drug effects ; RNA, Messenger/genetics/metabolism ; Ribonucleases/chemistry/genetics/*metabolism ; Severity of Illness Index ; Signal Transduction/drug effects ; },
abstract = {Regnase-1 (also known as Zc3h12a or MCPIP-1) is an endoribonuclease involved in mRNA degradation of inflammation-associated genes. Regnase-1 is inactivated in response to external stimuli through post-translational modifications including phosphorylation, yet the precise role of phosphorylation remains unknown. Here, we demonstrate that interleukin (IL)-17 induces phosphorylation of Regnase-1 in an Act1-TBK1/IKKi-dependent manner, especially in nonhematopoietic cells. Phosphorylated Regnase-1 is released from the endoplasmic reticulum (ER) into the cytosol, thereby losing its mRNA degradation function, which leads to expression of IL-17 target genes. By using CRISPR/Cas-9 technology, we generated Regnase-1 mutant mice, in which IL-17-induced Regnase-1 phosphorylation is completely blocked. Mutant mice (Regnase-1[AA/AA] and Regnase-1[ΔCTD/ΔCTD]) were resistant to the IL-17-mediated inflammation caused by T helper 17 (Th17) cells in vivo. Thus, Regnase-1 plays a critical role in the development of IL-17-mediated inflammatory diseases via the Act1-TBK1-IKKi axis, and blockade of Regnase-1 phosphorylation sites may be promising for treatment of Th17-associated diseases.},
}
@article {pmid31072420,
year = {2019},
author = {Wang, H and Tang, Z and Li, T and Liu, M and Li, Y and Xing, B},
title = {CRISPR/Cas9-Mediated Gene Knockout of ARID1A Promotes Primary Progesterone Resistance by Downregulating Progesterone Receptor B in Endometrial Cancer Cells.},
journal = {Oncology research},
volume = {27},
number = {9},
pages = {1051-1060},
pmid = {31072420},
issn = {1555-3906},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Down-Regulation ; Endometrial Neoplasms/*genetics/metabolism ; Endometrium/*abnormalities/metabolism ; Female ; Gene Knockout Techniques/*methods ; Humans ; Receptors, Progesterone/*metabolism ; Uterine Diseases/*metabolism ; },
abstract = {Medroxyprogesterone (MPA) is used for the conservative treatment of endometrial cancer. Unfortunately, progesterone resistance seriously affects its therapeutic effect. The purpose of the current study was to investigate the influence of deletion of AT-rich interactive domain 1A (ARID1A) in progesterone resistance in Ishikawa cells. Ablation of ARID1A was conducted through the CRISPR/Cas9 technology. Acquired progesterone-resistant Ishikawa (Ishikawa-PR) cells were generated by chronic exposure of Ishikawa cells to MPA. The sensitivity of the parental Ishikawa, Ishikawa-PR, and ARID1A-deficient cells to MPA and/or LY294002 was determined using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis. In addition, Western blot analysis and reverse transcription-polymerase chain reaction was performed to evaluate the mRNA and protein expression levels of ARID1A, progesterone receptor B (PRB), and P-AKT. Both Ishikawa-PR and ARID1A knockout cells showed insensitivity to MPA, downregulation of PRB, and hyperphosphorylation of AKT compared to the parental Ishikawa cells. Pretreatment with LY294002 significantly enhanced the ability of MPA to suppress proliferation and to induce apoptosis in the parental and Ishikawa-PR cells via the inhibition of AKT activation and upregulation of PRB transcriptional activity. However, the PRB transcriptional activity and insensitivity to MPA were irreversible by LY294002 in ARID1A-deficient cells. Ablation of ARID1A is associated with low PRB expression, which serves an important role in primary progesterone resistance. Akt inhibition cannot rescue PRB or sensitize to MPA in ARID1A knockout cells. These findings suggest that ARID1A may act as a reliable biomarker to predict the response for the combination of AKT inhibitor and MPA treatment.},
}
@article {pmid31071955,
year = {2019},
author = {Chen, SH and Chow, JM and Hsieh, YY and Lin, CY and Hsu, KW and Hsieh, WS and Chi, WM and Shabangu, BM and Lee, CH},
title = {HDAC1,2 Knock-Out and HDACi Induced Cell Apoptosis in Imatinib-Resistant K562 Cells.},
journal = {International journal of molecular sciences},
volume = {20},
number = {9},
pages = {},
pmid = {31071955},
issn = {1422-0067},
mesh = {Acetylation/drug effects ; Apoptosis/drug effects ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/drug effects ; Fusion Proteins, bcr-abl/antagonists &amp; inhibitors/*genetics ; Gene Knockout Techniques ; Histone Deacetylase 1/*genetics ; Histone Deacetylase 2/*genetics ; Histone Deacetylase Inhibitors/pharmacology ; Humans ; Imatinib Mesylate/adverse effects/pharmacology ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/genetics/pathology ; Neoplastic Stem Cells/drug effects ; Panobinostat/pharmacology ; },
abstract = {Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%-20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.},
}
@article {pmid31071912,
year = {2019},
author = {Sheta, R and Bachvarova, M and Macdonald, E and Gobeil, S and Vanderhyden, B and Bachvarov, D},
title = {The polypeptide GALNT6 Displays Redundant Functions upon Suppression of its Closest Homolog GALNT3 in Mediating Aberrant O-Glycosylation, Associated with Ovarian Cancer Progression.},
journal = {International journal of molecular sciences},
volume = {20},
number = {9},
pages = {},
pmid = {31071912},
issn = {1422-0067},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinoma, Ovarian Epithelial/*genetics/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/*genetics ; Female ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; Glycosylation ; Humans ; Mice ; N-Acetylgalactosaminyltransferases/*genetics ; Neoplasm Invasiveness/genetics/pathology ; Ovary/pathology ; RNA, Small Interfering/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.},
}
@article {pmid31071448,
year = {2019},
author = {Tadić, V and Josipović, G and Zoldoš, V and Vojta, A},
title = {CRISPR/Cas9-based epigenome editing: An overview of dCas9-based tools with special emphasis on off-target activity.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {109-119},
doi = {10.1016/j.ymeth.2019.05.003},
pmid = {31071448},
issn = {1095-9130},
mesh = {Animals ; Bacteria/genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Methylation ; Epigenomics/*methods ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Silencing ; Humans ; Optogenetics/methods ; },
abstract = {Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.},
}
@article {pmid31071326,
year = {2019},
author = {Athukoralage, JS and Graham, S and Grüschow, S and Rouillon, C and White, MF},
title = {A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator.},
journal = {Journal of molecular biology},
volume = {431},
number = {15},
pages = {2894-2899},
pmid = {31071326},
issn = {1089-8638},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adenine Nucleotides/*chemistry ; Allosteric Regulation ; CRISPR-Cas Systems ; Catalytic Domain ; Models, Molecular ; Mutagenesis, Site-Directed ; Oligoribonucleotides/*chemistry ; RNA/chemistry/metabolism ; RNA Stability ; Ribonuclease III/chemistry/*genetics/*metabolism ; Second Messenger Systems ; Thermus thermophilus/*enzymology/genetics ; },
abstract = {Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.},
}
@article {pmid31071267,
year = {2019},
author = {Liu, Z and Shan, H and Chen, S and Chen, M and Zhang, Q and Lai, L and Li, Z},
title = {Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {8},
pages = {9210-9219},
doi = {10.1096/fj.201900476RR},
pmid = {31071267},
issn = {1530-6860},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Mutation/genetics ; Polymerase Chain Reaction ; Rabbits ; Rats ; Zygote ; },
abstract = {Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) nickase, enable the efficient conversion of the C·G base pair to T·A in various organisms. However, the currently used rat apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1(rA1)-based BE3 is often inefficient in target Cs that are immediately downstream of a G (GC context). Here, we observed that, with an 11-nt editing window, an optimized activation-induced cytidine deaminase (AID)-Cas9 fusion can efficiently convert C to T in a variety of sequence contexts in rabbits. Strikingly, the enhanced AID-Cas9 fusion (eAID-BE4max) has significant effectiveness of inducing Tyr p.R299H mutation in GC contexts (from 16.67 to 83.33%) in comparison with BE3 in founder rabbits. Furthermore, the engineered AID-Cas9 variants were produced with reduced bystander activity [eAID (N51G)-BE4max] and increased genome-targeting scope (eAID-NG-BE4max). Overall, this work provides a series of improved tools that were generated using optimized AID-Cas9 fusions and associated engineered variants that can be used for efficient and versatile C-to-T base editing, especially in GC contexts.-Liu, Z., Shan, H., Chen, S., Chen, M., Zhang, Q., Lai, L., Li, Z. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.},
}
@article {pmid31071190,
year = {2019},
author = {García-Tuñón, I and Alonso-Pérez, V and Vuelta, E and Pérez-Ramos, S and Herrero, M and Méndez, L and Hernández-Sánchez, JM and Martín-Izquierdo, M and Saldaña, R and Sevilla, J and Sánchez-Guijo, F and Hernández-Rivas, JM and Sánchez-Martín, M},
title = {Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency.},
journal = {PloS one},
volume = {14},
number = {5},
pages = {e0216674},
pmid = {31071190},
issn = {1932-6203},
mesh = {Alleles ; Animals ; Ataxia Telangiectasia Mutated Proteins/genetics ; *CRISPR-Cas Systems ; Cell Line ; Exons ; Gene Editing/methods ; Gene Knockout Techniques/*methods ; Humans ; K562 Cells ; Mice ; Monophenol Monooxygenase/genetics ; Proto-Oncogene Proteins c-abl/genetics ; RNA Splice Sites/*genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.},
}
@article {pmid31070861,
year = {2019},
author = {Ge, Z and Zheng, L and Zhao, Y and Jiang, J and Zhang, EJ and Liu, T and Gu, H and Qu, LJ},
title = {Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants.},
journal = {Plant biotechnology journal},
volume = {17},
number = {10},
pages = {1865-1867},
pmid = {31070861},
issn = {1467-7652},
mesh = {*Arabidopsis ; *CRISPR-Cas Systems ; *Gene Editing ; *Mutagenesis ; Plants, Genetically Modified ; },
}
@article {pmid31069679,
year = {2019},
author = {Warner, BK and Alder, JK and Suli, A},
title = {Genome Editing in Zebrafish Using CRISPR-Cas9: Applications for Developmental Toxicology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1965},
number = {},
pages = {235-250},
doi = {10.1007/978-1-4939-9182-2_16},
pmid = {31069679},
issn = {1940-6029},
support = {R00 HL113105/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development/drug effects ; Gene Editing/*methods ; Gene-Environment Interaction ; Teratogens/toxicity ; Zebrafish/embryology/*genetics ; },
abstract = {Environment-gene interactions have a powerful impact on embryo development. The ability to precisely edit the genome makes it possible to address questions concerning the specific roles that genes or variants play in modulating the response to environmental challenges. In this chapter, we provide a simplified protocol using CRISPR-Cas9 ribonucleoproteins for genome editing in the zebrafish model organism. The genetic manipulation can then be coupled with chemical screens to identify and understand the mechanism behind toxicants or compounds that modulate development.},
}
@article {pmid31069270,
year = {2019},
author = {Qiao, J and Li, W and Lin, S and Sun, W and Ma, L and Liu, Y},
title = {Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {161},
pmid = {31069270},
issn = {2399-3642},
mesh = {Bacterial Proteins/genetics/metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Chromatography, Affinity/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colicins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Escherichia coli/*genetics/metabolism ; Gene Editing/*methods ; Gene Expression ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Ribonucleoproteins/biosynthesis/*genetics/isolation &amp; purification ; },
abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.},
}
@article {pmid31068963,
year = {2019},
author = {Kawall, K},
title = {New Possibilities on the Horizon: Genome Editing Makes the Whole Genome Accessible for Changes.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {525},
pmid = {31068963},
issn = {1664-462X},
abstract = {The emergence of new genome editing techniques, such as the site-directed nucleases, clustered regulatory interspaced short palindromic repeats (CRISPRs)/Cas9, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs), has greatly increased the feasibility of introducing any desired changes into the genome of a target organism. The ability to target a Cas nuclease to DNA sequences with a single-guide RNA (sgRNA) has provided a dynamic tool for genome editing and is naturally derived from an adaptive immune system in bacteria and archaea. CRISPR/Cas systems are being rapidly improved and refined, thereby opening up even more possibilities. Classical plant breeding is based on genetic variations that occur naturally and is used to select plants with improved traits. Induced mutagenesis is used to enhance mutational frequency and accelerate this process. Plants have evolved cellular processes, including certain repair mechanisms that ensure DNA integrity and the maintenance of distinct DNA loci. The focus of this review is on the characterization of new potentials in plant breeding through the use of CRISPR/Cas systems that eliminate natural limitations in order to induce thus far unachievable genomic changes.},
}
@article {pmid31068592,
year = {2019},
author = {Meltzer, H and Marom, E and Alyagor, I and Mayseless, O and Berkun, V and Segal-Gilboa, N and Unger, T and Luginbuhl, D and Schuldiner, O},
title = {Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2113},
pmid = {31068592},
issn = {2041-1723},
support = {615906//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/International ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drosophila/*genetics ; Female ; Gene Editing/*methods ; Male ; Mushroom Bodies/metabolism ; Mutagenesis ; Nervous System/growth &amp; development ; Neuronal Plasticity/genetics ; Neurons/physiology ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.},
}
@article {pmid31068316,
year = {2019},
author = {Zhou, Y and Hui, W and Zhang, H and Zou, L and Zhang, P},
title = {[Establishment of a stable HEK293T cell line with c.392G&gt;T (p.131G&gt;V) mutation site knockout in G6PD gene using CRISPR/Cas9 technique].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {3},
pages = {320-327},
pmid = {31068316},
issn = {1673-4254},
mesh = {*CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Mutation ; Plasmids ; RNA, Guide ; },
abstract = {OBJECTIVE: To establish a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in G6PD gene using CRISPR/Cas9 technique.<br><br>METHODS: We designed 4 pairs of small guide RNA (sgRNA) for G6PD c.392G>T(p.131G>V) mutation site, and constructed exogenous PX458 plasmids expressing Cas9-sgRNA. The plasmids were transfected into HEK293T cells, and the cells expressing GFP fluorescent protein were separated by flow cytometry for further culture. After verification of the knockout efficiency using T7 endonuclease &#x2160;, the monoclonal cells were screened by limiting dilution and DNA sequencing to confirm the knockout. We detected the expressions of G6PD mRNA and protein and examined functional changes of the genetically modified cells.<br><br>RESULTS: We successfully constructed the Cas9-sgRNA exogenous PX458 plasmid based on the c.392G>T(p.131G>V) mutation site of G6PD gene. The editing efficiency of the 4 pairs of sgRNA, as detected by T7E1 enzyme digestion, was 6.74%, 12.36%, 12.54% and 2.94%. Sanger sequencing confirmed that the HEK293T cell line with stable knockout of G6PD c.392G>T(p.131G>V) was successfully constructed. The genetically modified cells expressed lower levels of G6PD mRNA and G6PD protein and showed reduced G6PD enzyme activity and proliferative capacity and increased apoptosis in response to vitamin K3 treatment.<br><br>CONCLUSIONS: We successfully constructed a stable HEK293T cell model with G6PD gene c.392G>T(p.131G>V) mutation site knockout to facilitate future study of gene repair.},
}
@article {pmid31066249,
year = {2019},
author = {Jafarian, A and Shokri, G and Shokrollahi Barough, M and Moin, M and Pourpak, Z and Soleimani, M},
title = {Recent Advances in Gene Therapy and Modeling of Chronic Granulomatous Disease.},
journal = {Iranian journal of allergy, asthma, and immunology},
volume = {18},
number = {2},
pages = {131-142},
pmid = {31066249},
issn = {1735-5249},
mesh = {Animals ; CRISPR-Cas Systems ; Clinical Trials as Topic ; *Genetic Therapy ; Genetic Vectors ; Granulomatous Disease, Chronic/genetics/*therapy ; Hematopoietic Stem Cell Transplantation ; Humans ; Infections/genetics/*therapy ; Mutation/*genetics ; NADPH Oxidases/*genetics ; Phagocytosis/genetics ; },
abstract = {The Chronic granulomatous disease (CGD) is a primary immunodeficiency that characterized by mutations in phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in deficient antimicrobial activity of phagocytic cells and recurrent childhood infections. Hematopoietic stem cell transplantation (HSCT) is a curative option for patients with human leukocyte antigen (HLA) matched donor, when conventional cares and therapies fail. However, in many cases when the patients have not an HLA-matched donor, they need to a method to recapitulate the function of the affected gene within the patient's own cells. Gene therapy is a promising approach for CGD. While, the success of retroviral or lentiviral vectors in gene therapy for CGD has been hampered by random integration and insertional activation of proto-oncogenes. These serious adverse events led to improvement and generations of viral vectors with increased safety characteristics. Gene therapy continues to progress and the advent of new technologies, such as engineered endonucleases that have shown a great promise for the treatment of genetic disease. This review focuses on the application of gene therapy for the CGD, the limitations encountered in current clinical trials, advantages and disadvantages of endonucleases in gene correction and modeling with CRISPR/Cas9 approach.},
}
@article {pmid31066204,
year = {2019},
author = {Jiao, X and Zhang, Y and Liu, X and Zhang, Q and Zhang, S and Zhao, ZK},
title = {Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides.},
journal = {Biotechnology journal},
volume = {14},
number = {7},
pages = {e1900036},
doi = {10.1002/biot.201900036},
pmid = {31066204},
issn = {1860-7314},
mesh = {Basidiomycota/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; },
abstract = {The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon-optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single-guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9-expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome-editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.},
}
@article {pmid31065830,
year = {2019},
author = {Huang, JM and Chang, YT and Shih, MH and Lin, WC and Huang, FC},
title = {Identification and characterization of a secreted M28 aminopeptidase protein in Acanthamoeba.},
journal = {Parasitology research},
volume = {118},
number = {6},
pages = {1865-1874},
pmid = {31065830},
issn = {1432-1955},
mesh = {Acanthamoeba/isolation &amp; purification/*metabolism ; Amebiasis/parasitology ; Aminopeptidases/genetics/*immunology/*metabolism ; CRISPR-Cas Systems ; Complement C3/*metabolism ; Humans ; Lakes/parasitology ; Protozoan Proteins/genetics/*immunology/*metabolism ; Soil/parasitology ; },
abstract = {Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection.},
}
@article {pmid31065786,
year = {2019},
author = {Matsuo, K and Atsumi, G},
title = {CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.},
journal = {Planta},
volume = {250},
number = {2},
pages = {463-473},
pmid = {31065786},
issn = {1432-2048},
mesh = {Arabidopsis/genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Gene Expression ; Green Fluorescent Proteins ; Plant Proteins/genetics/metabolism ; *Plants, Genetically Modified ; RNA Interference ; RNA-Dependent RNA Polymerase/genetics/*metabolism ; Recombinant Proteins ; Tobacco/*genetics/metabolism ; },
abstract = {RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.},
}
@article {pmid31065097,
year = {2019},
author = {Lei, R and Zhai, X and Zhu, W and Qiu, R},
title = {Reboot ethics governance in China.},
journal = {Nature},
volume = {569},
number = {7755},
pages = {184-186},
pmid = {31065097},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; China ; *Ethics, Research/education/history ; Female ; Gene Editing/ethics/legislation &amp; jurisprudence ; History, 20th Century ; History, 21st Century ; Human Experimentation/ethics/history/legislation &amp; jurisprudence ; Humans ; Male ; Research Personnel/education/*ethics/*legislation &amp; jurisprudence ; Science/*ethics/*legislation &amp; jurisprudence ; Translational Research, Biomedical ; },
}
@article {pmid31064825,
year = {2019},
author = {Bertolini, MS and Chiurillo, MA and Lander, N and Vercesi, AE and Docampo, R},
title = {MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca[2+] Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.},
journal = {mBio},
volume = {10},
number = {3},
pages = {},
pmid = {31064825},
issn = {2150-7511},
support = {R01 AI107663/AI/NIAID NIH HHS/United States ; R56 AI107663/AI/NIAID NIH HHS/United States ; },
mesh = {Adaptation, Physiological ; Biological Transport ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium-Binding Proteins/genetics/*metabolism ; Cation Transport Proteins ; Cytosol/chemistry/metabolism ; Gene Knockout Techniques ; Humans ; Mitochondria/*metabolism ; Mitochondrial Membrane Transport Proteins/genetics/*metabolism ; Protozoan Proteins/genetics/*metabolism ; Trypanosoma cruzi/*genetics/pathogenicity ; },
abstract = {The mitochondrial Ca[2+] uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca[2+] uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca[2+] uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca[2+] concentrations ([Ca[2+]]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCETrypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca[2+] sensors and gatekeepers of the MCU, preventing Ca[2+] uptake under resting conditions and favoring it at high cytosolic Ca[2+] concentrations ([Ca[2+]]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca[2+]]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.},
}
@article {pmid31064766,
year = {2019},
author = {Silva-García, CG and Lanjuin, A and Heintz, C and Dutta, S and Clark, NM and Mair, WB},
title = {Single-Copy Knock-In Loci for Defined Gene Expression in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {7},
pages = {2195-2198},
pmid = {31064766},
issn = {2160-1836},
support = {R21 AG056930/AG/NIA NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R01 AG059595/AG/NIA NIH HHS/United States ; R01 AG051954/AG/NIA NIH HHS/United States ; R01 AG044346/AG/NIA NIH HHS/United States ; R01 AG054201/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/*genetics ; *Gene Dosage ; *Gene Expression Regulation ; Gene Knock-In Techniques ; Gene Targeting ; *Genetic Loci ; },
abstract = {We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any DNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any DNA sequence can be introduced as a single copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical, and effective approach for generating single-copy ectopic transgenes in C. elegans.},
}
@article {pmid31064056,
year = {2019},
author = {Soutourina, O},
title = {Type I Toxin-Antitoxin Systems in Clostridia.},
journal = {Toxins},
volume = {11},
number = {5},
pages = {},
pmid = {31064056},
issn = {2072-6651},
mesh = {*Gram-Positive Bacteria ; Humans ; *Toxin-Antitoxin Systems ; },
abstract = {Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.},
}
@article {pmid31063922,
year = {2019},
author = {Basheer, FT and Vassiliou, GS},
title = {Genome-scale drop-out screens to identify cancer cell vulnerabilities in AML.},
journal = {Current opinion in genetics &amp; development},
volume = {54},
number = {},
pages = {83-87},
doi = {10.1016/j.gde.2019.04.004},
pmid = {31063922},
issn = {1879-0380},
support = {MC_PC_12009/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; /DH_/Department of Health/United Kingdom ; C22324/A23015/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; *Drug Screening Assays, Antitumor ; Gene Editing ; Genome, Human/genetics ; Genomics ; Humans ; Leukemia, Myeloid, Acute/*drug therapy/genetics/pathology ; Mutation/genetics ; },
abstract = {Acute myeloid leukemia (AML) is an aggressive cancer that remains lethal to the majority of sufferers. Whilst the mainstay treatments for this condition have remained largely unchanged over the past five decades, progress in deciphering its pathogenesis has accelerated in recent years, propelled in part by advances in cancer genomics and mechanistic studies of leukemogenic mutations. Newer molecular therapies targeting aberrant biological pathways are currently under investigation with a few moving closer to clinical use. However, collectively, these new therapies are not predicted to have a major impact on clinical outcomes and the need for the identification of further therapeutic targets in AML remains critical. Recently the use of CRISPR-Cas9 systems for genome editing and their potential application in genome-wide screening has opened a new frontier for unbiased discovery of therapeutic vulnerabilities in cancer and AML was the first disease in which this technology was systematically applied. In this review we give an overview of recent advances in identifying novel therapeutic vulnerabilities of AML using CRISPR-Cas9 and discuss possible future applications of CRISPR technologies in this field.},
}
@article {pmid31062609,
year = {2019},
author = {Cai, A and Kong, X},
title = {Development of CRISPR-Mediated Systems in the Study of Duchenne Muscular Dystrophy.},
journal = {Human gene therapy methods},
volume = {30},
number = {3},
pages = {71-80},
doi = {10.1089/hgtb.2018.187},
pmid = {31062609},
issn = {1946-6544},
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; Gene Editing ; Humans ; Morpholinos/therapeutic use ; Muscular Dystrophy, Duchenne/genetics/*therapy ; Oxadiazoles/therapeutic use ; },
abstract = {Duchenne muscular dystrophy (DMD) is a severe type of X-linked recessive degenerative muscle disease caused by mutations in the dystrophin (DMD) gene on the X chromosome. The DMD gene is complex, consisting of 79 exons, and mutations cause changes in the DMD mRNA so that the reading frame is altered, and the muscle-specific isoform of the dystrophin protein is either absent or truncated with variable residual function. The emerging CRISPR-Cas9-mediated genome editing technique is being developed as a potential therapeutic approach to treat DMD because it can permanently replace the mutated dystrophin gene with the normal gene. Prenatal DNA testing can inform whether the female fetus is a carrier of DMD, and the male fetus has inherited a mutation from his mother (50% chance of both). This article summarizes the present status of current and future treatments for DMD.},
}
@article {pmid31062017,
year = {2019},
author = {Asoshina, M and Myo, G and Tada, N and Tajino, K and Shimizu, N},
title = {Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells.},
journal = {Nucleic acids research},
volume = {47},
number = {11},
pages = {5998-6006},
pmid = {31062017},
issn = {1362-4962},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems ; Chromosomes, Artificial/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cricetinae ; Cricetulus ; *DNA Replication ; Endonucleases/genetics ; Gene Amplification ; Homologous Recombination ; *Matrix Attachment Regions ; Mice ; Plasmids/*metabolism ; *Replication Origin ; },
abstract = {A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage-fusion-bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.},
}
@article {pmid31061537,
year = {2019},
author = {Soyk, S and Lemmon, ZH and Sedlazeck, FJ and Jiménez-Gómez, JM and Alonge, M and Hutton, SF and Van Eck, J and Schatz, MC and Lippman, ZB},
title = {Duplication of a domestication locus neutralized a cryptic variant that caused a breeding barrier in tomato.},
journal = {Nature plants},
volume = {5},
number = {5},
pages = {471-479},
pmid = {31061537},
issn = {2055-0278},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Domestication ; Epistasis, Genetic/*genetics ; Flowers/growth &amp; development ; Gene Duplication/*genetics/physiology ; Gene Editing/methods ; Genetic Variation/genetics/physiology ; Lycopersicon esculentum/*genetics/growth &amp; development/physiology ; Plant Breeding ; Plants, Genetically Modified ; Quantitative Trait Loci/genetics ; Reproduction/genetics/physiology ; },
abstract = {Genome editing technologies are being widely adopted in plant breeding[1]. However, a looming challenge of engineering desirable genetic variation in diverse genotypes is poor predictability of phenotypic outcomes due to unforeseen interactions with pre-existing cryptic mutations[2-4]. In tomato, breeding with a classical MADS-box gene mutation that improves harvesting by eliminating fruit stem abscission frequently results in excessive inflorescence branching, flowering and reduced fertility due to interaction with a cryptic variant that causes partial mis-splicing in a homologous gene[5-8]. Here, we show that a recently evolved tandem duplication carrying the second-site variant achieves a threshold of functional transcripts to suppress branching, enabling breeders to neutralize negative epistasis on yield. By dissecting the dosage mechanisms by which this structural variant restored normal flowering and fertility, we devised strategies that use CRISPR-Cas9 genome editing to predictably improve harvesting. Our findings highlight the under-appreciated impact of epistasis in targeted trait breeding and underscore the need for a deeper characterization of cryptic variation to enable the full potential of genome editing in agriculture.},
}
@article {pmid31060819,
year = {2019},
author = {Xu, RG and Wang, X and Shen, D and Sun, J and Qiao, HH and Wang, F and Liu, LP and Ni, JQ},
title = {Perspectives on gene expression regulation techniques in Drosophila.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {4},
pages = {213-220},
doi = {10.1016/j.jgg.2019.03.006},
pmid = {31060819},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Drosophila melanogaster/*genetics ; Gene Expression ; Genetic Engineering/*methods ; RNA Interference ; Transcriptional Activation ; },
abstract = {Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.},
}
@article {pmid31060546,
year = {2019},
author = {Carey, K and Ryu, J and Uh, K and Lengi, AJ and Clark-Deener, S and Corl, BA and Lee, K},
title = {Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {25},
pmid = {31060546},
issn = {1472-6750},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Embryo, Mammalian/embryology/*metabolism ; Gene Editing/*methods ; Gene Targeting/*methods ; Mutation ; Sequence Analysis, DNA ; Swine ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined.<br><br>RESULTS: First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5' side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5' side, indicating unpredictability of the events.<br><br>CONCLUSIONS: The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.},
}
@article {pmid31059574,
year = {2019},
author = {Sobh, A and Loguinov, A and Stornetta, A and Balbo, S and Tagmount, A and Zhang, L and Vulpe, CD},
title = {Genome-Wide CRISPR Screening Identifies the Tumor Suppressor Candidate OVCA2 As a Determinant of Tolerance to Acetaldehyde.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {169},
number = {1},
pages = {235-245},
pmid = {31059574},
issn = {1096-0929},
support = {P30 CA077598/CA/NCI NIH HHS/United States ; P42 ES004705/ES/NIEHS NIH HHS/United States ; },
mesh = {Acetaldehyde/*toxicity ; *CRISPR-Cas Systems ; Cell Survival/drug effects ; Cell Transformation, Neoplastic/*chemically induced/*genetics/metabolism/pathology ; DNA Adducts/genetics/metabolism ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study ; HEK293 Cells ; Humans ; K562 Cells ; Neoplasms/*chemically induced/*genetics/metabolism/pathology ; Proteins/*genetics/metabolism ; Risk Assessment ; Tumor Suppressor Proteins/*genetics/metabolism ; },
abstract = {Acetaldehyde, a metabolite of ethanol, is a cellular toxicant and a human carcinogen. A genome-wide CRISPR-based loss-of-function screen in erythroleukemic K562 cells revealed candidate genetic contributors affecting acetaldehyde cytotoxicity. Secondary screening exposing cells to a lower acetaldehyde dose simultaneously validated multiple candidate genes whose loss results in increased sensitivity to acetaldehyde. Disruption of genes encoding components of various DNA repair pathways increased cellular sensitivity to acetaldehyde. Unexpectedly, the tumor suppressor gene OVCA2, whose function is unknown, was identified in our screen as a determinant of acetaldehyde tolerance. Disruption of the OVCA2 gene resulted in increased acetaldehyde sensitivity and higher accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-dG. Together these results are consistent with a role for OVCA2 in adduct removal and/or DNA repair.},
}
@article {pmid31059233,
year = {2019},
author = {Zhang, X and Xia, L and Day, BA and Harris, TI and Oliveira, P and Knittel, C and Licon, AL and Gong, C and Dion, G and Lewis, RV and Jones, JA},
title = {CRISPR/Cas9 Initiated Transgenic Silkworms as a Natural Spinner of Spider Silk.},
journal = {Biomacromolecules},
volume = {20},
number = {6},
pages = {2252-2264},
doi = {10.1021/acs.biomac.9b00193},
pmid = {31059233},
issn = {1526-4602},
mesh = {Animals ; *Animals, Genetically Modified/genetics/metabolism ; *Bombyx/genetics/metabolism ; *CRISPR-Cas Systems ; *Fibroins/biosynthesis/genetics ; Spiders/*genetics ; },
abstract = {Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate spider silk protein genes into the Bombyx mori genome. Native-size spider silk genes (up to 10 kb) were inserted into an intron of the fibroin heavy or light chain (FibH or FibL) ensuring that any sequence changes induced by the CRISPR/Cas9 would not impact protein production. The resulting fibers are as strong as native spider silks (1.2 GPa tensile strength). The transgenic silkworms have been tracked for several generations with normal inheritance of the transgenes. This strategy demonstrates the feasibility of using silkworms as a natural spider silk spinner for industrial production of high-performance fibers.},
}
@article {pmid31056946,
year = {2019},
author = {Stover, JD and Farhang, N and Lawrence, B and Bowles, RD},
title = {Multiplex Epigenome Editing of Dorsal Root Ganglion Neuron Receptors Abolishes Redundant Interleukin 6, Tumor Necrosis Factor Alpha, and Interleukin 1β Signaling by the Degenerative Intervertebral Disc.},
journal = {Human gene therapy},
volume = {30},
number = {9},
pages = {1147-1160},
pmid = {31056946},
issn = {1557-7422},
support = {R01 AR074998/AR/NIAMS NIH HHS/United States ; },
mesh = {Action Potentials ; Biomarkers ; CRISPR-Cas Systems ; Calcium Signaling ; Cytokines/*genetics/metabolism ; *Epigenesis, Genetic ; Female ; Ganglia, Spinal/cytology/*metabolism ; Gene Editing ; Humans ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Intervertebral Disc Degeneration/*genetics/*metabolism/pathology ; Male ; Neurons/*metabolism ; Receptors, Cell Surface/*genetics/metabolism ; *Signal Transduction ; Temperature ; Tumor Necrosis Factor-alpha/genetics/metabolism ; },
abstract = {Back pain is the leading cause of disability worldwide and contributes to significant socioeconomic impacts. It has been hypothesized that the degenerative intervertebral disc (IVD) contributes to back pain by sensitizing nociceptive neurons innervating the IVD to stimuli that would not be painful to healthy patients. However, the inflammatory signaling networks mediating this sensitization remain poorly understood. A better understanding of the underlying mechanisms of degenerative IVD-induced changes in nociception is required to improve the understanding and treatment of back pain. Toward these ends, a novel in vitro model was developed to investigate degenerative IVD-induced changes in dorsal root ganglion (DRG) neuron activation by measuring DRG neuron activity following neuron seeding on human degenerative IVD tissue collected from patients undergoing surgical treatment for back pain. Lentiviral clustered regularly interspaced palindromic repeat (CRISPR) epigenome editing vectors were built to downregulate the inflammatory receptors TNFR1, IL1R1, and IL6st in DRG neurons in single- and multiplex. Multiplex CRISPR epigenome editing of inflammatory receptors demonstrated that degenerative IVD tissue drives thermal sensitization through the simultaneous and redundant signaling of interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), and IL-1β. This work elucidates redundant signaling pathways in neuron interactions with the degenerative IVD and suggests the need for multiplex targeting of IL-6, TNF-α, and IL-1β for pain modulation in the degenerative IVD.},
}
@article {pmid31055869,
year = {2019},
author = {Li, B and Rui, H and Li, Y and Wang, Q and Alariqi, M and Qin, L and Sun, L and Ding, X and Wang, F and Zou, J and Wang, Y and Yuan, D and Zhang, X and Jin, S},
title = {Robust CRISPR/Cpf1 (Cas12a)-mediated genome editing in allotetraploid cotton (Gossypium hirsutum).},
journal = {Plant biotechnology journal},
volume = {17},
number = {10},
pages = {1862-1864},
pmid = {31055869},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gossypium/*genetics ; RNA, Plant ; Sequence Deletion ; Tetraploidy ; },
}
@article {pmid31055665,
year = {2019},
author = {Sukwong, P and Sunwoo, IY and Jeong, DY and Kim, SR and Jeong, GT and Kim, SK},
title = {Enhancement of bioethanol production from Gracilaria verrucosa by Saccharomyces cerevisiae through the overexpression of SNR84 and PGM2.},
journal = {Bioprocess and biosystems engineering},
volume = {42},
number = {9},
pages = {1421-1433},
doi = {10.1007/s00449-019-02139-0},
pmid = {31055665},
issn = {1615-7605},
mesh = {*Biofuels ; CRISPR-Cas Systems ; Ethanol/*metabolism ; Galactose/chemistry/*metabolism ; Gene Expression ; Gracilaria/*chemistry ; Hydrolysis ; *Microorganisms, Genetically-Modified/genetics/growth &amp; development ; *Saccharomyces cerevisiae/genetics/growth &amp; development ; *Saccharomyces cerevisiae Proteins/biosynthesis/genetics ; },
abstract = {A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.},
}
@article {pmid31055134,
year = {2019},
author = {Biswal, AK and Mangrauthia, SK and Reddy, MR and Yugandhar, P},
title = {CRISPR mediated genome engineering to develop climate smart rice: Challenges and opportunities.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {100-106},
doi = {10.1016/j.semcdb.2019.04.005},
pmid = {31055134},
issn = {1096-3634},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Plant/*genetics ; Oryza/*genetics ; },
abstract = {Rice is a staple food crop, which ensures the calorie requirement of half of the world's population. With the continued increase in population, rice will play a key role in achieving the food security. However, in the constantly shrinking scenario of rice fields, the necessity of these extra grains of rice must be met by reducing the yield loss due to various abiotic and biotic stresses. The adverse effects of climate impact both quality and quantity of rice production. One of the most desirable applications of CRISPR/Cas technology would be to develop climate smart rice crop to sustain and enhance its productivity in the changing environment. In this review, we analyze the desirable phenotypes and responsible genetic factors, which can be utilized to develop tolerance against major abiotic stresses imposed by climate change through genome engineering. The possibility of utilizing the information from wild resources to engineer the corresponding alleles of cultivated rice has been presented. We have also shed light on available resources for generating genome edited rice lines. The CRISPR/Cas mediated genome editing strategies for engineering of novel genes were proposed to create a plant phenotype, which can face the adversities of climate change. Further, challenges of off-targets and undesirable phenotype were discussed.},
}
@article {pmid31054950,
year = {2019},
author = {Li, J and Luo, J and Xu, M and Li, S and Zhang, J and Li, H and Yan, L and Zhao, Y and Xia, L},
title = {Plant genome editing using xCas9 with expanded PAM compatibility.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {5},
pages = {277-280},
doi = {10.1016/j.jgg.2019.03.004},
pmid = {31054950},
issn = {1673-8527},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Nucleotide Motifs/*genetics ; },
}
@article {pmid31054854,
year = {2019},
author = {Straathof, AJJ and Wahl, SA and Benjamin, KR and Takors, R and Wierckx, N and Noorman, HJ},
title = {Grand Research Challenges for Sustainable Industrial Biotechnology.},
journal = {Trends in biotechnology},
volume = {37},
number = {10},
pages = {1042-1050},
doi = {10.1016/j.tibtech.2019.04.002},
pmid = {31054854},
issn = {1879-3096},
mesh = {Biofuels ; Biotechnology/*methods ; Genetic Engineering/methods ; Green Chemistry Technology ; Industrial Microbiology/methods ; Metabolic Engineering/methods ; Microbial Consortia/physiology ; Recycling ; Research/*trends ; },
abstract = {Future manufacturing will focus on new, improved products as well as on new and enhanced production methods. Recent biotechnological and scientific advances, such as CRISPR/Cas and various omic technologies, pave the way to exciting novel biotechnological research, development, and commercialization of new sustainable products. Rigorous mathematical descriptions of microbial cells and consortia thereof will enable deeper biological understanding and lead to powerful in silico cellular models. Biological engineering, namely model-based design together with synthetic biology, will accelerate the construction of robust and high-performing microorganisms. Using these organisms, and ambitions towards zero-concepts with respect to emissions and excess resources in bioprocess engineering, industrial biotechnology is expected to become highly integrated into sustainable generations of technology systems.},
}
@article {pmid31054324,
year = {2019},
author = {Kaushik, I and Ramachandran, S and Srivastava, SK},
title = {CRISPR-Cas9: A multifaceted therapeutic strategy for cancer treatment.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {4-12},
pmid = {31054324},
issn = {1096-3634},
support = {R01 CA129038/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Humans ; *Immunotherapy ; Neoplasms/*genetics/immunology/*therapy ; },
abstract = {CRISPR-Cas9 is an RNA guided endonuclease that has revolutionized the ability to edit genome and introduce desired manipulations in the target genomic sequence. It is a flexible methodology and is capable of targeting multiple loci simultaneously. Owing to the fact that cancer is an amalgamation of several genetic mutations, application of CRISPR-Cas9 technology is considered as a novel strategy to combat cancer. Genetic and epigenetic modulations in cancer leads to development of resistance to conventional therapy options. Given the abundance of transcriptomic and genomic alterations in cancer, developing a strategy to decipher these alterations is critical. CRISPR-Cas9 system has proven to be a promising tool in generating cellular and animal models to mimic the mutations and understand their role in tumorigenesis. CRISPR-Cas9 is an upheaval in the field of cancer immunotherapy. Furthermore, CRISPR-Cas9 plays an important role in the development of whole genome libraries for cancer patients. This approach will help understand the diversity in genome variation among the patients and also, will provide multiple variables to scientists to investigate and improvise cancer therapy. This review will focus on the discovery of CRISPR-Cas9 system, mechanisms behind CRISPR technique and its current status as a potential tool for investigating the genomic mutations associated with all cancer types.},
}
@article {pmid31053643,
year = {2019},
author = {Meyer, MB and Benkusky, NA and Kaufmann, M and Lee, SM and Redfield, RR and Jones, G and Pike, JW},
title = {Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {24},
pages = {9518-9535},
pmid = {31053643},
issn = {1083-351X},
support = {R01 DK117475/DK/NIDDK NIH HHS/United States ; },
mesh = {25-Hydroxyvitamin D3 1-alpha-Hydroxylase/*physiology ; Animals ; CRISPR-Cas Systems ; Calcium/*metabolism ; *Enhancer Elements, Genetic ; Female ; Fibroblast Growth Factor-23 ; *Gene Deletion ; *Homeostasis ; Humans ; Kidney/drug effects/*metabolism ; Male ; Methyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Transgenic ; Vitamin D/*analogs &amp; derivatives/pharmacology ; },
abstract = {Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.},
}
@article {pmid31053612,
year = {2019},
author = {Kotov, JA and Kotov, DI and Linehan, JL and Bardwell, VJ and Gearhart, MD and Jenkins, MK},
title = {BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors.},
journal = {The Journal of experimental medicine},
volume = {216},
number = {6},
pages = {1450-1464},
pmid = {31053612},
issn = {1540-9538},
support = {T32 AI083196/AI/NIAID NIH HHS/United States ; T32 AI007313/AI/NIAID NIH HHS/United States ; R01 HD084459/HD/NICHD NIH HHS/United States ; R01 AI103760/AI/NIAID NIH HHS/United States ; R01 AI039614/AI/NIAID NIH HHS/United States ; F31 AI133716/AI/NIAID NIH HHS/United States ; R37 AI027998/AI/NIAID NIH HHS/United States ; R01 AI027998/AI/NIAID NIH HHS/United States ; R01 CA071540/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Cell Lineage ; Co-Repressor Proteins/*metabolism ; Cytokines/metabolism ; F-Box Proteins/metabolism ; Female ; Gene Expression Regulation ; Jumonji Domain-Containing Histone Demethylases/metabolism ; Lymphocyte Subsets/metabolism ; Male ; Mice, Inbred C57BL ; Proto-Oncogene Proteins c-bcl-6/*metabolism ; Receptors, Chemokine/metabolism ; Repressor Proteins/*metabolism ; Signal Transduction ; Streptococcus pyogenes/physiology ; Th17 Cells/*cytology/*metabolism ; },
abstract = {CD4[+] T helper 17 (Th17) cells protect vertebrate hosts from extracellular pathogens at mucosal surfaces. Th17 cells form from naive precursors when signals from the T cell antigen receptor (TCR) and certain cytokine receptors induce the expression of the RORγt transcription factor, which activates a set of Th17-specific genes. Using T cell-specific loss-of-function experiments, we find that two components of the Polycomb repressive complex 1.1 (PRC1.1), BCL6 corepressor (BCOR) and KDM2B, which helps target the complex to unmethylated CpG DNA islands, are required for optimal Th17 cell formation in mice after Streptococcus pyogenes infection. Genome-wide expression and BCOR chromatin immunoprecipitation studies revealed that BCOR directly represses Lef1, Runx2, and Dusp4, whose products inhibit Th17 differentiation. Together, the results suggest that the PRC1.1 components BCOR and KDM2B work together to enhance Th17 cell formation by repressing Th17 fate suppressors.},
}
@article {pmid31053298,
year = {2019},
author = {Maeoka, Y and Okamoto, T and Wu, Y and Saito, A and Asada, R and Matsuhisa, K and Terao, M and Takada, S and Masaki, T and Imaizumi, K and Kaneko, M},
title = {Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5.},
journal = {Biochemical and biophysical research communications},
volume = {514},
number = {2},
pages = {436-442},
doi = {10.1016/j.bbrc.2019.04.168},
pmid = {31053298},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Down-Regulation/drug effects ; Female ; Furosemide/pharmacology ; *Gene Expression Regulation/drug effects ; Gene Knock-In Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Kidney Medulla/*physiology ; Kidney Tubules, Collecting/*metabolism ; Male ; Mice ; Transcription Factors/*metabolism ; Ubiquitin-Protein Ligases/*metabolism ; },
abstract = {Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation.},
}
@article {pmid31053162,
year = {2019},
author = {O'Geen, H and Bates, SL and Carter, SS and Nisson, KA and Halmai, J and Fink, KD and Rhie, SK and Farnham, PJ and Segal, DJ},
title = {Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner.},
journal = {Epigenetics &amp; chromatin},
volume = {12},
number = {1},
pages = {26},
pmid = {31053162},
issn = {1756-8935},
support = {P30 CA093373/CA/NCI NIH HHS/United States ; R01 CA136924/CA/NCI NIH HHS/United States ; R01CA136924/NH/NIH HHS/United States ; FAST//Foundation for Angelman Syndrome Therapeutics/International ; K01CA229995/NH/NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; S10 RR026825/RR/NCRR NIH HHS/United States ; K01 CA229995/CA/NCI NIH HHS/United States ; P30 CA014089/NH/NIH HHS/United States ; R21 HG009742/HG/NHGRI NIH HHS/United States ; R21 CA204563/CA/NCI NIH HHS/United States ; CA204563/NH/NIH HHS/United States ; P30 CA014089/CA/NCI NIH HHS/United States ; HG009742/NH/NIH HHS/United States ; C06 RR012088/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; DNA Methylation ; DNA Methyltransferase 3A ; Enhancer of Zeste Homolog 2 Protein/*genetics/metabolism ; Epigenesis, Genetic ; Gene Editing ; Genetic Engineering/methods ; HCT116 Cells ; Histone Methyltransferases/genetics/metabolism ; Histones/metabolism ; Humans ; Mice ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Receptor, ErbB-2/genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; Transcription Factors/metabolism ; },
abstract = {BACKGROUND: Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.<br><br>RESULTS: Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50&#xa0;days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.<br><br>CONCLUSIONS: These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.},
}
@article {pmid31051255,
year = {2019},
author = {Spiegel, A and Bachmann, M and Jurado Jiménez, G and Sarov, M},
title = {CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {49-58},
doi = {10.1016/j.ymeth.2019.04.016},
pmid = {31051255},
issn = {1095-9130},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques ; DNA End-Joining Repair ; Electroporation/instrumentation/methods ; Gene Knockout Techniques/instrumentation/*methods ; Genetic Loci/genetics ; Genetic Vectors/genetics ; Genotyping Techniques/instrumentation/methods ; HCT116 Cells ; Humans ; Plasmids/genetics ; RNA, Guide/genetics ; *Recombinational DNA Repair ; Reverse Genetics/instrumentation/*methods ; },
abstract = {We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for.},
}
@article {pmid31051141,
year = {2019},
author = {Shariati, SA and Dominguez, A and Xie, S and Wernig, M and Qi, LS and Skotheim, JM},
title = {Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9.},
journal = {Molecular cell},
volume = {74},
number = {3},
pages = {622-633.e4},
pmid = {31051141},
issn = {1097-4164},
support = {F32 GM123576/GM/NIGMS NIH HHS/United States ; K99 GM126027/GM/NIGMS NIH HHS/United States ; R01 GM092925/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Binding Sites/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation/genetics ; Gene Regulatory Networks ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/metabolism ; Nanog Homeobox Protein/*genetics ; Octamer Transcription Factor-3/*genetics ; PAX6 Transcription Factor/*genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Transcription Factors/genetics ; Transcriptional Activation/genetics ; },
abstract = {The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.},
}
@article {pmid31051134,
year = {2019},
author = {Martin, RM and Ikeda, K and Cromer, MK and Uchida, N and Nishimura, T and Romano, R and Tong, AJ and Lemgart, VT and Camarena, J and Pavel-Dinu, M and Sindhu, C and Wiebking, V and Vaidyanathan, S and Dever, DP and Bak, RO and Laustsen, A and Lesch, BJ and Jakobsen, MR and Sebastiano, V and Nakauchi, H and Porteus, MH},
title = {Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.},
journal = {Cell stem cell},
volume = {24},
number = {5},
pages = {821-828.e5},
doi = {10.1016/j.stem.2019.04.001},
pmid = {31051134},
issn = {1875-9777},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Repair ; Dependovirus/*genetics ; Gene Editing/methods ; Gene Frequency ; Genetic Engineering ; Genetic Vectors/genetics ; *Genotype ; Homologous Recombination ; Humans ; Pathology, Molecular ; Pluripotent Stem Cells/*physiology ; Tissue Donors ; },
abstract = {Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.},
}
@article {pmid31051099,
year = {2019},
author = {Maji, B and Gangopadhyay, SA and Lee, M and Shi, M and Wu, P and Heler, R and Mok, B and Lim, D and Siriwardena, SU and Paul, B and Dančík, V and Vetere, A and Mesleh, MF and Marraffini, LA and Liu, DR and Clemons, PA and Wagner, BK and Choudhary, A},
title = {A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.},
journal = {Cell},
volume = {177},
number = {4},
pages = {1067-1079.e19},
pmid = {31051099},
issn = {1097-4172},
support = {R21 AI126239/AI/NIAID NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Protein 9/*antagonists &amp; inhibitors/metabolism ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/physiology ; DNA/metabolism ; Endonucleases/metabolism ; Gene Editing/methods ; Genome ; High-Throughput Screening Assays/*methods ; Small Molecule Libraries ; Streptococcus pyogenes/genetics ; Substrate Specificity ; },
abstract = {The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.},
}
@article {pmid31050154,
year = {2019},
author = {Dong, L and Qi, X and Zhu, J and Liu, C and Zhang, X and Cheng, B and Mao, L and Xie, C},
title = {Supersweet and waxy: meeting the diverse demands for specialty maize by genome editing.},
journal = {Plant biotechnology journal},
volume = {17},
number = {10},
pages = {1853-1855},
pmid = {31050154},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genes, Plant ; Plants, Genetically Modified ; Starch Synthase/*genetics ; Sugars/*analysis ; *Waxes ; Zea mays/*genetics ; },
}
@article {pmid31049151,
year = {2019},
author = {De Giorgi, M and Lagor, WR},
title = {Gene Delivery in Lipid Research and Therapies.},
journal = {Methodist DeBakey cardiovascular journal},
volume = {15},
number = {1},
pages = {62-69},
pmid = {31049151},
issn = {1947-6108},
mesh = {Animals ; CRISPR-Cas Systems ; Cardiovascular Diseases/blood/epidemiology/genetics/*prevention &amp; control ; Dependovirus/genetics ; Dyslipidemias/blood/epidemiology/genetics/*therapy ; Gene Editing/methods ; Genetic Therapy/adverse effects/*methods ; Genetic Vectors ; Humans ; Lipid Metabolism/*genetics ; Lipids/*blood ; Oligonucleotides, Antisense/genetics ; RNA, Small Interfering/genetics ; RNAi Therapeutics/methods ; Treatment Outcome ; },
abstract = {Cardiovascular disease is the leading cause of death worldwide, and elevated lipid levels is a major contributor. Gene delivery, which involves controlled transfer of nucleic acids into cells and tissues, has been widely used in research to study lipid metabolism and physiology. Several technologies have been developed to somatically overexpress, silence, or disrupt genes in animal models and have greatly advanced our knowledge of metabolism. This is particularly true with regard to the liver, which plays a central role in lipoprotein metabolism and is amenable to many delivery approaches. In addition to basic science applications, many of these delivery technologies have potential as gene therapies for both common and rare lipid disorders. This review discusses three major gene delivery technologies used in lipid research-including adeno-associated viral vector overexpression, antisense oligonucleotides and small interfering RNAs, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system-and examines their potential therapeutic applications.},
}
@article {pmid31048719,
year = {2019},
author = {Calatayud, C and Carola, G and Fernández-Carasa, I and Valtorta, M and Jiménez-Delgado, S and Díaz, M and Soriano-Fradera, J and Cappelletti, G and García-Sancho, J and Raya, &#xc1; and Consiglio, A},
title = {CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {6811},
pmid = {31048719},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Calcium/metabolism ; Cell Differentiation ; Cell Tracking ; Cells, Cultured ; Dopaminergic Neurons/cytology/*metabolism ; Flow Cytometry/methods ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; Genes, Reporter ; Humans ; Immunohistochemistry ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Mesencephalon/cytology/physiology ; *Molecular Imaging ; Tyrosine 3-Monooxygenase/*genetics ; },
abstract = {Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases.},
}
@article {pmid31048459,
year = {2019},
author = {Chung, HK and Zou, X and Bajar, BT and Brand, VR and Huo, Y and Alcudia, JF and Ferrell, JE and Lin, MZ},
title = {A compact synthetic pathway rewires cancer signaling to therapeutic effector release.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6439},
pages = {},
pmid = {31048459},
issn = {1095-9203},
support = {P50 GM107615/GM/NIGMS NIH HHS/United States ; R01 GM098734/GM/NIGMS NIH HHS/United States ; R35 GM131792/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenoviridae ; Apoptosis/*genetics ; Bioengineering/*methods ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Endopeptidases/genetics ; Humans ; Models, Theoretical ; Neoplasms/*genetics/pathology/*therapy ; Protein Stability ; Proteolysis ; Receptor, ErbB-2/*antagonists &amp; inhibitors/*genetics/metabolism ; Signal Transduction ; Synthetic Biology ; Transcription, Genetic ; Viral Nonstructural Proteins/genetics ; },
abstract = {An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.},
}
@article {pmid31048007,
year = {2019},
author = {Schuster, M and Kahmann, R},
title = {CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {130},
number = {},
pages = {43-53},
doi = {10.1016/j.fgb.2019.04.016},
pmid = {31048007},
issn = {1096-0937},
mesh = {*CRISPR-Cas Systems ; Fungi/*genetics ; *Gene Editing ; Genome, Fungal ; Mutation ; Oomycetes/*genetics ; },
abstract = {Due to their biotechnological relevance as well as their importance as disease agents, filamentous fungi and oomycetes have been prime candidates for genetic selection and in vitro manipulation for decades. With the advent of new genome editing technologies such manipulations have reached a new level of speed and sophistication. The CRISPR-Cas9 genome editing technology in particular has revolutionized the ways how desired mutations can be introduced. To date, the CRISPR-Cas9 genome editing system has been established in more than 40 different species of filamentous fungi and oomycetes. In this review we describe the various approaches taken to assure expression of the components necessary for editing and describe the varying strategies used to achieve gene disruptions, gene replacements and precise editing. We discuss potential problems faced when establishing the system, propose ways to circumvent them and suggest future approaches not yet realized in filamentous fungi or oomycetes.},
}
@article {pmid31047915,
year = {2019},
author = {Srinivas, S and Hu, Z and Cronan, JE},
title = {Escherichia coli vectors having stringently repressible replication origins allow a streamlining of Crispr/Cas9 gene editing.},
journal = {Plasmid},
volume = {103},
number = {},
pages = {53-62},
pmid = {31047915},
issn = {1095-9890},
support = {R01 AI015650/AI/NIAID NIH HHS/United States ; R37 AI015650/AI/NIAID NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/drug effects/*genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial/*drug effects ; *Genome, Bacterial ; Isopropyl Thiogalactoside/pharmacology ; Mutation ; Plasmids/*chemistry/metabolism ; RNA, Guide/genetics/metabolism ; Replication Origin ; Streptomycin/pharmacology ; Temperature ; },
abstract = {Readily curable plasmids facilitate the construction of plasmid-free bacterial strains after the plasmid encoded genes are no longer needed. The most popular of these plasmids features a temperature-sensitive (Ts) pSC101 origin of replication which can readily revert during usage and cannot be used to construct Ts mutations in essential genes. Plasmid pAM34 which contains an IPTG-dependent origin of replication largely overcomes this issue but is limited by carrying the most commonly utilized antibiotic selection and replication origin. This study describes the construction of an expanded series of plasmid vectors having replication origins of p15a, RSF1030 or RSF1031 that like pAM34 have IPTG-dependent replication. Surprisingly, these plasmids can be cured in fewer generations than pAM34. Derivatives of pAM34 with alternative antibiotic selection markers were also constructed. The utility of these vectors is demonstrated in the construction of a CRISPR-Cas9 system consisting of an IPTG-dependent Cas9 plasmid and a curable guide RNA plasmid having a streptomycin counterselection marker. This system was successfully demonstrated by construction of point mutations, deletions and insertions in the E. coli genome with a very high efficiency and in a shorter timescale than extant methods. The plasmids themselves were readily cured either together or singly from the resultant strains with minimal effort.},
}
@article {pmid31046670,
year = {2019},
author = {Wolter, F and Schindele, P and Puchta, H},
title = {Plant breeding at the speed of light: the power of CRISPR/Cas to generate directed genetic diversity at multiple sites.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {176},
pmid = {31046670},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; Genes, Plant ; *Genetic Variation ; *Plant Breeding ; },
abstract = {Classical plant breeding was extremely successful in generating high yielding crop varieties. Yet, in modern crops, the long domestication process has impoverished the genetic diversity available for breeding. This is limiting further improvements of elite germplasm by classical approaches. The CRISPR/Cas system now enables promising new opportunities to create genetic diversity for breeding in an unprecedented way. Due to its multiplexing ability, multiple targets can be modified simultaneously in an efficient way, enabling immediate pyramiding of multiple beneficial traits into an elite background within one generation. By targeting regulatory elements, a selectable range of transcriptional alleles can be generated, enabling precise fine-tuning of desirable traits. In addition, by targeting homologues of so-called domestication genes within one generation, it is now possible to catapult neglected, semi-domesticated and wild plants quickly into the focus of mainstream agriculture. This further enables the use of the enormous genetic diversity present in wild species or uncultured varieties of crops as a source of allele-mining, widely expanding the crop germplasm pool.},
}
@article {pmid31044159,
year = {2019},
author = {Xu, Q and Li, R and Weng, L and Sun, Y and Li, M and Xiao, H},
title = {Domain-specific expression of meristematic genes is defined by the LITTLE ZIPPER protein DTM in tomato.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {134},
pmid = {31044159},
issn = {2399-3642},
mesh = {Amino Acid Sequence ; Amino Acid Substitution ; Arabidopsis Proteins/chemistry ; CRISPR-Cas Systems ; Crosses, Genetic ; DNA, Plant/genetics ; Feedback, Physiological ; *Gene Expression Regulation, Plant/genetics ; *Genes, Plant ; Homeodomain Proteins/physiology ; Lycopersicon esculentum/*genetics ; Meristem/*metabolism ; *Mutation, Missense ; Plant Proteins/genetics/*physiology ; Plant Shoots/growth &amp; development/metabolism/ultrastructure ; Protein Binding ; Protein Multimerization ; Seedlings/growth &amp; development ; Sequence Alignment ; Sequence Homology, Amino Acid ; Transcription Factors/physiology ; },
abstract = {Shoot meristems, which harbor a small population of stem cells, are responsible for generating new above-ground organs in plants. The proliferation and differentiation of these stem cells is regulated by a genetic pathway involving two key meristematic genes: CLAVATA3 (CLV3) and WUSCHEL (WUS). However, it is not well understood how CLV3 and WUS expression domains in the shoot meristems are specified and maintained during post-embryogenic development. Here, we show that a tomato mutant with fasciated stems, flowers and fruits, due to impaired stem cell activity, is defective in a LITTLE ZIPPER gene denoted as DEFECTIVE TOMATO MERISTEM (DTM). DTM forms a negative feedback loop with class III homeodomain-leucine zipper (HD-ZIP III) transcription factors to confine CLV3 and WUS expression to specific domains of the shoot meristems. Our findings reveal a new layer of complexity in the regulation of plant stem cell homeostasis.},
}
@article {pmid31043511,
year = {2019},
author = {Lavalou, P and Eckert, H and Damy, L and Constanty, F and Majello, S and Bitetti, A and Graindorge, A and Shkumatava, A},
title = {Strategies for genetic inactivation of long noncoding RNAs in zebrafish.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {8},
pages = {897-904},
pmid = {31043511},
issn = {1469-9001},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Expression Regulation ; Gene Knock-In Techniques ; *Gene Silencing ; Organ Specificity ; RNA, Long Noncoding/*genetics ; Sequence Deletion ; Transcription Initiation Site ; Zebrafish/*genetics ; },
abstract = {The number of annotated long noncoding RNAs (lncRNAs) continues to grow; however, their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype with the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcription start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants, due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, highly conserved lncRNA regions can also lead to overexpression of truncated transcripts. In contrast, knock-in of a polyadenylation signal enabled complete inactivation of malat1, the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation, and we propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA-deficient zebrafish.},
}
@article {pmid31043479,
year = {2019},
author = {Walker, MP and Lindner, SE},
title = {Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {24},
pages = {9555-9566},
pmid = {31043479},
issn = {1083-351X},
support = {R01 AI123341/AI/NIAID NIH HHS/United States ; R21 AI130692/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; *Gene Deletion ; Gene Editing/*methods ; Genetic Vectors ; Malaria/parasitology/*veterinary ; Mice ; Plasmids ; Plasmodium yoelii/genetics/isolation &amp; purification ; *RNA, Catalytic ; Rodent Diseases/*parasitology ; },
abstract = {Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causal Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite's growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene of interest to understand its importance and function. However, these approaches can be limited by the availability of selectable markers and the time required to generate transgenic parasites. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a ribozyme-guide-ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free parasites with homology arms as short as 80-100 bp. Additionally, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and desired locus replacement editing events, whereas the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically dead SpCas9 (dSpCas9) to the region upstream of a gene of interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates efficient genetic characterizations of rodent-infectious Plasmodium species.},
}
@article {pmid31041890,
year = {2019},
author = {Qin, K and Liang, X and Sun, G and Shi, X and Wang, M and Liu, H and Chen, Y and Liu, X and He, Z},
title = {Highly efficient correction of structural mutations of 450 kb KIT locus in kidney cells of Yorkshire pig by CRISPR/Cas9.},
journal = {BMC molecular and cell biology},
volume = {20},
number = {1},
pages = {4},
pmid = {31041890},
issn = {2661-8850},
mesh = {Alleles ; Animals ; Breeding/methods ; CRISPR-Cas Systems/*genetics ; Caspase 9/genetics ; Clone Cells ; Gene Editing/*methods ; Genetic Loci/*genetics ; Introns/genetics ; Kidney/*cytology ; *Mutation ; Mutation Rate ; Plasmids/genetics ; Proto-Oncogene Proteins c-kit/*genetics ; Swine/*genetics ; Transfection ; },
abstract = {The white coat colour of Yorkshire and Landrace pig breeds is caused by the dominant white I allele of KIT, associated with 450-kb duplications and a splice mutation (G > A) at the first base in intron 17. To test whether genome editing can be employed to correct this structural mutation, and to investigate the role of KIT in the control of porcine coat colour, we designed sgRNAs targeting either intron 16 or intron 17 of KIT, and transfected Cas9/sgRNA co-expression plasmids into the kidney cells of Yorkshire pigs. The copy number of KIT was reduced by about 13%, suggesting the possibility of obtaining cells with corrected structural mutations of the KIT locus. Using single cell cloning, from 24 successfully expanded single cell clones derived from cells transfected with sgRNA targeting at intron 17, we obtained 3 clones with a single copy of KIT without the splice mutation. Taken together, the 12.5% (3/24) efficiency of correction of structural mutations of 450 kb fragments is highly efficient, providing a solid basis for the generation of genome edited Yorkshire pigs with a normal KIT locus. This provides an insight into the underlying genetic mechanisms of porcine coat colour.},
}
@article {pmid31041571,
year = {2019},
author = {Liang, P and Huang, J},
title = {Off-target challenge for base editor-mediated genome editing.},
journal = {Cell biology and toxicology},
volume = {35},
number = {3},
pages = {185-187},
doi = {10.1007/s10565-019-09474-8},
pmid = {31041571},
issn = {1573-6822},
mesh = {CRISPR-Cas Systems ; Gene Editing/*ethics/*methods ; Humans ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
}
@article {pmid31040331,
year = {2019},
author = {Young, J and Zastrow-Hayes, G and Deschamps, S and Svitashev, S and Zaremba, M and Acharya, A and Paulraj, S and Peterson-Burch, B and Schwartz, C and Djukanovic, V and Lenderts, B and Feigenbutz, L and Wang, L and Alarcon, C and Siksnys, V and May, G and Chilcoat, ND and Kumar, S},
title = {CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {6729},
pmid = {31040331},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Computational Biology/methods ; Gene Editing/*methods ; Genetic Variation ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified ; RNA, Guide ; Reproducibility of Results ; Zea mays/*genetics ; },
abstract = {CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.},
}
@article {pmid31040209,
year = {2019},
author = {Han, X and Wang, M and Duan, S and Franco, PJ and Kenty, JH and Hedrick, P and Xia, Y and Allen, A and Ferreira, LMR and Strominger, JL and Melton, DA and Meissner, TB and Cowan, CA},
title = {Generation of hypoimmunogenic human pluripotent stem cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {21},
pages = {10441-10446},
pmid = {31040209},
issn = {1091-6490},
support = {P30 DK040561/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Knockout Techniques ; Genes, MHC Class I ; Genes, MHC Class II ; Genetic Engineering/*methods ; Humans ; Pluripotent Stem Cells/*immunology ; },
abstract = {Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage "don't-eat me" signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.},
}
@article {pmid31039970,
year = {2018},
author = {Zhou, S and Yu, H and Zhao, X and Cai, B and Ding, Q and Huang, Y and Li, Y and Li, Y and Niu, Y and Lei, A and Kou, Q and Huang, X and Petersen, B and Ma, B and Chen, Y and Wang, X},
title = {Generation of gene-edited sheep with a defined Booroola fecundity gene (FecB[B]) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9.},
journal = {Reproduction, fertility, and development},
volume = {30},
number = {12},
pages = {1616-1621},
doi = {10.1071/RD18086},
pmid = {31039970},
issn = {1031-3613},
mesh = {Animals ; Animals, Genetically Modified ; Bone Morphogenetic Protein Receptors, Type I/*genetics/metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/*methods ; Male ; *Mutation ; Polymorphism, Single Nucleotide ; RNA, Guide ; Sheep ; },
abstract = {Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q>R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.},
}
@article {pmid31039782,
year = {2019},
author = {Lagziel, S and Lee, WD and Shlomi, T},
title = {Inferring cancer dependencies on metabolic genes from large-scale genetic screens.},
journal = {BMC biology},
volume = {17},
number = {1},
pages = {37},
pmid = {31039782},
issn = {1741-7007},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor/*metabolism ; Genes, Essential ; *Genetic Testing ; Humans ; Neoplasms/*genetics ; RNA Interference ; },
abstract = {BACKGROUND: Cancer cells reprogram their metabolism to survive and propagate. Thus, targeting metabolic rewiring in tumors is a promising therapeutic strategy. Genome-wide RNAi and CRISPR screens are powerful tools for identifying genes essential for cancer cell proliferation and survival. Integrating loss-of-function genetic screens with genomics and transcriptomics datasets reveals molecular mechanisms that underlie cancer cell dependence on specific genes; though explaining cell line-specific essentiality of metabolic genes was recently shown to be especially challenging.<br><br>RESULTS: We find that variability in tissue culture medium between cell lines in a genetic screen is a major confounding factor affecting cell line-specific essentiality of metabolic genes-while, quite surprisingly, not being previously accounted for. Additionally, we find that altered expression level of a metabolic gene in a certain cell line is less indicative of its essentiality than for other genes. However, cell line-specific essentiality of metabolic genes is significantly correlated with changes in the expression of neighboring enzymes in the metabolic network. Utilizing a machine learning method that accounts for tissue culture media and functional association between neighboring enzymes, we generated predictive models for cancer cell line-specific dependence on 162 metabolic genes (representing a ~&#x2009;2.2-fold increase compared to previous studies). The generated predictive models reveal numerous novel associations between molecular features and cell line-specific dependency on metabolic genes. Specifically, we demonstrate how cancer cell dependence on one-carbon metabolic enzymes is explained based on cancer lineage, oncogenic mutations, and RNA expression of neighboring enzymes.<br><br>CONCLUSIONS: Considering culture media as well as accounting for molecular features of functionally related metabolic enzymes in a metabolic network significantly improves our understanding of cancer cell line-specific dependence on metabolic genes. We expect our approach and predictive models of metabolic gene essentiality to be a useful tool for investigating metabolic abnormalities in cancer.},
}
@article {pmid31039627,
year = {2019},
author = {Campenhout, CV and Cabochette, P and Veillard, AC and Laczik, M and Zelisko-Schmidt, A and Sabatel, C and Dhainaut, M and Vanhollebeke, B and Gueydan, C and Kruys, V},
title = {Guidelines for optimized gene knockout using CRISPR/Cas9.},
journal = {BioTechniques},
volume = {66},
number = {6},
pages = {295-302},
doi = {10.2144/btn-2018-0187},
pmid = {31039627},
issn = {1940-9818},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genetic Loci ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Zebrafish/genetics ; },
abstract = {CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.},
}
@article {pmid31039344,
year = {2019},
author = {Khosravi, MA and Abbasalipour, M and Concordet, JP and Berg, JV and Zeinali, S and Arashkia, A and Azadmanesh, K and Buch, T and Karimipoor, M},
title = {Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease.},
journal = {European journal of pharmacology},
volume = {854},
number = {},
pages = {398-405},
doi = {10.1016/j.ejphar.2019.04.042},
pmid = {31039344},
issn = {1879-0712},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Carrier Proteins/*genetics ; Fetal Hemoglobin/*metabolism ; *Gene Deletion ; Gene Editing ; Genetic Therapy/*methods ; Humans ; K562 Cells ; Nuclear Proteins/*deficiency/*genetics ; Repressor Proteins ; beta-Thalassemia/genetics/metabolism/*therapy ; gamma-Globins/genetics ; },
abstract = {Hemoglobinopathies, such as β-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of β-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous β-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of β-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating β-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.},
}
@article {pmid31038202,
year = {2019},
author = {Schultz, JC and Cao, M and Zhao, H},
title = {Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {8},
pages = {2103-2109},
doi = {10.1002/bit.27001},
pmid = {31038202},
issn = {1097-0290},
mesh = {Basidiomycota/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Deletion ; Gene Editing/methods ; Genes, Fungal ; Metabolic Engineering/methods ; RNA, Guide/genetics ; },
abstract = {The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA-tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast.},
}
@article {pmid31038108,
year = {2019},
author = {Galzi, JL},
title = {[Gene editing in drug discovery and therapeutic innovation].},
journal = {Medecine sciences : M/S},
volume = {35},
number = {4},
pages = {309-315},
doi = {10.1051/medsci/2019068},
pmid = {31038108},
issn = {1958-5381},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Drug Discovery/history/methods/trends ; *Gene Editing/history/trends ; Genetic Therapy/history/methods/trends ; History, 20th Century ; History, 21st Century ; Humans ; Therapies, Investigational/methods/*trends ; },
abstract = {The idea according to which the most recent therapeutic methods will overcome the more traditional pharmacopoeia is widespread in recent publications. Biomedicine and gene therapies are booming, but we realize, as for other therapeutic approaches, that they suffer intrinsic constraints and limitations and that their most relevant therapeutic fields are complementary to those of traditional drugs. They are now viewed as potentially synergistic with these traditional drugs, rather than competitors. This review puts into perspective the potential of genome editing in the field of drug discovery and therapeutic innovation.},
}
@article {pmid31037730,
year = {2019},
author = {Kina, H and Yoshitani, T and Hanyu-Nakamura, K and Nakamura, A},
title = {Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.},
journal = {Development, growth &amp; differentiation},
volume = {61},
number = {4},
pages = {265-275},
doi = {10.1111/dgd.12607},
pmid = {31037730},
issn = {1440-169X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Drosophila/*genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/*genetics ; Time Factors ; },
abstract = {The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFP[KI]) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFP[KI] strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.},
}
@article {pmid31037510,
year = {2020},
author = {Wang, S and Min, Z and Ji, Q and Geng, L and Su, Y and Liu, Z and Hu, H and Wang, L and Zhang, W and Suzuiki, K and Huang, Y and Zhang, P and Tang, TS and Qu, J and Yu, Y and Liu, GH and Qiao, J},
title = {Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction.},
journal = {Protein &amp; cell},
volume = {11},
number = {1},
pages = {1-22},
pmid = {31037510},
issn = {1674-8018},
mesh = {*Aging, Premature/pathology/therapy ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; *Cockayne Syndrome/pathology/therapy ; DNA Helicases/*genetics ; DNA Repair ; DNA Repair Enzymes/*genetics ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/pathology ; Male ; Mesenchymal Stem Cells/metabolism/pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; *Models, Biological ; Mutation ; Neural Stem Cells/metabolism/pathology ; Poly-ADP-Ribose Binding Proteins/*genetics ; Targeted Gene Repair/*methods ; Transcriptome ; },
abstract = {Cockayne syndrome (CS) is a rare autosomal recessive inherited disorder characterized by a variety of clinical features, including increased sensitivity to sunlight, progressive neurological abnormalities, and the appearance of premature aging. However, the pathogenesis of CS remains unclear due to the limitations of current disease models. Here, we generate integration-free induced pluripotent stem cells (iPSCs) from fibroblasts from a CS patient bearing mutations in CSB/ERCC6 gene and further derive isogenic gene-corrected CS-iPSCs (GC-iPSCs) using the CRISPR/Cas9 system. CS-associated phenotypic defects are recapitulated in CS-iPSC-derived mesenchymal stem cells (MSCs) and neural stem cells (NSCs), both of which display increased susceptibility to DNA damage stress. Premature aging defects in CS-MSCs are rescued by the targeted correction of mutant ERCC6. We next map the transcriptomic landscapes in CS-iPSCs and GC-iPSCs and their somatic stem cell derivatives (MSCs and NSCs) in the absence or presence of ultraviolet (UV) and replicative stresses, revealing that defects in DNA repair account for CS pathologies. Moreover, we generate autologous GC-MSCs free of pathogenic mutation under a cGMP (Current Good Manufacturing Practice)-compliant condition, which hold potential for use as improved biomaterials for future stem cell replacement therapy for CS. Collectively, our models demonstrate novel disease features and molecular mechanisms and lay a foundation for the development of novel therapeutic strategies to treat CS.},
}
@article {pmid31036938,
year = {2019},
author = {Atlasi, Y and Megchelenbrink, W and Peng, T and Habibi, E and Joshi, O and Wang, SY and Wang, C and Logie, C and Poser, I and Marks, H and Stunnenberg, HG},
title = {Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.},
journal = {Nature cell biology},
volume = {21},
number = {5},
pages = {568-578},
pmid = {31036938},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Chromatin/*genetics ; Enhancer Elements, Genetic ; *Epigenesis, Genetic ; Histones/genetics ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; Pluripotent Stem Cells ; Promoter Regions, Genetic ; Receptors, Estrogen/*genetics ; Transcriptome/genetics ; },
abstract = {The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.},
}
@article {pmid31036811,
year = {2019},
author = {Hirano, S and Abudayyeh, OO and Gootenberg, JS and Horii, T and Ishitani, R and Hatada, I and Zhang, F and Nishimasu, H and Nureki, O},
title = {Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1968},
pmid = {31036811},
issn = {2041-1723},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Corynebacterium diphtheriae/enzymology/metabolism ; Crystallography, X-Ray ; DNA Cleavage ; DNA Restriction-Modification Enzymes/*chemistry/*metabolism ; HEK293 Cells ; Humans ; Hydrogen Bonding ; },
abstract = {The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases.},
}
@article {pmid31036344,
year = {2019},
author = {Varble, A and Marraffini, LA},
title = {Three New Cs for CRISPR: Collateral, Communicate, Cooperate.},
journal = {Trends in genetics : TIG},
volume = {35},
number = {6},
pages = {446-456},
pmid = {31036344},
issn = {0168-9525},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation ; RNA Stability ; RNA, Guide/genetics ; Signal Transduction ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated (cas) genes provide protection against invading phages and plasmids in prokaryotes. Typically, short sequences are captured from the genome of the invader, integrated into the CRISPR locus, and transcribed into short RNAs that direct RNA-guided Cas nucleases to the nucleic acids of the invader for their degradation. Recent work in the field has revealed unexpected features of the CRISPR-Cas mechanism: (i) collateral, nonspecific, cleavage of host nucleic acids; (ii) secondary messengers that amplify the immune response; and (iii) immunosuppression of CRISPR targeting by phage-encoded inhibitors. Here, we review these new and exciting findings.},
}
@article {pmid31036069,
year = {2019},
author = {Butt, H and Eid, A and Momin, AA and Bazin, J and Crespi, M and Arold, ST and Mahfouz, MM},
title = {CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {73},
pmid = {31036069},
issn = {1474-760X},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Evolution, Molecular ; Fatty Alcohols ; *Genetic Techniques ; Oryza/*genetics ; Plant Proteins/genetics ; Protein Domains ; Pyrans ; *Spliceosomes ; },
abstract = {Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions.},
}
@article {pmid31036063,
year = {2019},
author = {Zhang, Y and Qi, Y},
title = {CRISPR enables directed evolution in plants.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {83},
pmid = {31036063},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oryza/*genetics ; RNA Splicing ; Spliceosomes ; },
abstract = {A proof-of-concept study has demonstrated the application of CRISPR-Cas9 for directed evolution in rice, engineering crops for desired traits.},
}
@article {pmid31036006,
year = {2019},
author = {Pinto, DO and Scott, TA and DeMarino, C and Pleet, ML and Vo, TT and Saifuddin, M and Kovalskyy, D and Erickson, J and Cowen, M and Barclay, RA and Zeng, C and Weinberg, MS and Kashanchi, F},
title = {Effect of transcription inhibition and generation of suppressive viral non-coding RNAs.},
journal = {Retrovirology},
volume = {16},
number = {1},
pages = {13},
pmid = {31036006},
issn = {1742-4690},
support = {R21 AI074410/AI/NIAID NIH HHS/United States ; AI043894/NH/NIH HHS/United States ; R21 AI078859/AI/NIAID NIH HHS/United States ; R01 AI043894/AI/NIAID NIH HHS/United States ; AI078859/NH/NIH HHS/United States ; AI074410/NH/NIH HHS/United States ; R01MH110262/NH/NIH HHS/United States ; R01 NS099029/NS/NINDS NIH HHS/United States ; },
mesh = {Anti-Retroviral Agents/pharmacology ; Biomimetics ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Gene Expression Regulation, Viral/*drug effects ; Gene Silencing ; HIV-1/drug effects/*genetics ; Humans ; Promoter Regions, Genetic ; RNA, Untranslated/*genetics ; RNA, Viral/genetics ; *Transcription, Genetic ; tat Gene Products, Human Immunodeficiency Virus/chemistry ; },
abstract = {BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo.<br><br>RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1&#xa0;&#xb5;M) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting&#xa0;both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state.<br><br>CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.},
}
@article {pmid31035982,
year = {2019},
author = {Yuan, M and Zhu, J and Gong, L and He, L and Lee, C and Han, S and Chen, C and He, G},
title = {Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {24},
pmid = {31035982},
issn = {1472-6750},
mesh = {Arachis/enzymology/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics/metabolism ; Gene Editing/*methods ; Linoleic Acid/metabolism ; *Mutagenesis ; Oleic Acid/metabolism ; Plant Breeding/methods ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Seeds/enzymology/genetics ; },
abstract = {BACKGROUND: Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models.<br><br>RESULTS: The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content.<br><br>CONCLUSIONS: Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.},
}
@article {pmid31035284,
year = {2019},
author = {Turan, S and Boerstler, T and Kavyanifar, A and Loskarn, S and Reis, A and Winner, B and Lie, DC},
title = {A novel human stem cell model for Coffin-Siris syndrome-like syndrome reveals the importance of SOX11 dosage for neuronal differentiation and survival.},
journal = {Human molecular genetics},
volume = {28},
number = {15},
pages = {2589-2599},
doi = {10.1093/hmg/ddz089},
pmid = {31035284},
issn = {1460-2083},
mesh = {Abnormalities, Multiple/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; *Cell Line ; Cell Proliferation ; Face/abnormalities ; *Gene Dosage ; Gene Editing ; Gene Expression Regulation ; Hand Deformities, Congenital/genetics/metabolism ; Haploinsufficiency ; Human Embryonic Stem Cells/metabolism/*physiology ; Humans ; Intellectual Disability/genetics/metabolism ; Micrognathism/genetics/metabolism ; *Models, Biological ; Neck/abnormalities ; Neural Stem Cells ; Neurodevelopmental Disorders/genetics/*metabolism ; SOXC Transcription Factors/*genetics ; },
abstract = {The SOXC transcription factors Sox4, Sox11 and Sox12, are critical neurodevelopmental regulators that are thought to function in a highly redundant fashion. Surprisingly, heterozygous missense mutations or deletions of SOX11 were recently detected in patients with Coffin-Siris syndrome-like syndrome (CSSLS), a neurodevelopmental disorder associated with intellectual disability, demonstrating that in humans SOX11 haploinsufficiency cannot be compensated and raising the question of the function of SOX11 in human neurodevelopment. Here, we describe the generation of SOX11+/- heterozygous human embryonic stem cell (hESC) lines by CRISPR/Cas9 genome engineering. SOX11 haploinsufficiency impaired the generation of neurons and resulted in a proliferation/differentiation imbalance of neural precursor cells and enhanced neuronal cell death. Using the SOX11+/- hESC model we provide for the first time experimental evidence that SOX11 haploinsufficiency is sufficient to impair key processes of human neurodevelopment, giving a first insight into the pathophysiology of CSSLS and SOX11 function in human neurodevelopment.},
}
@article {pmid31034882,
year = {2019},
author = {Yu, JSL and Yusa, K},
title = {Genome-wide CRISPR-Cas9 screening in mammalian cells.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {29-35},
doi = {10.1016/j.ymeth.2019.04.015},
pmid = {31034882},
issn = {1095-9130},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; Cell Line ; Gene Editing/*methods ; Genetic Testing/methods ; *Genomic Library ; Genomics/*methods ; Humans ; RNA, Guide/genetics ; },
abstract = {Forward genetic screens are a powerful and unbiased approach for uncovering the genetic basis behind a specific phenotype. Genome-wide mutagenesis followed by phenotypic screening represents the ultimate manifestation of this method, directly linking biological phenomena to its corresponding genetic cause. Whilst this has been successful in lower organisms, deployment of genome-wide screens in mammalian systems has been hampered by both limitations of scale and inefficient bi-allelic mutagenesis. CRISPR-Cas9 technology has now largely resolved these issues, whereby delivery of genome-scale gRNA libraries in the presence of gRNA-guided Cas9 endonuclease enables the generation of mutant cell libraries; the perfect platform for performing phenotypic screens. Although the tools are now available for virtually any molecular biology laboratory to conduct such screens, many researchers are daunted by the sheer complexity and scale at which such experiments are performed. This Review will address these concerns, presenting a contextual and practical guide to deploying CRISPR-KO screens in mammalian systems. We will discuss key considerations required in all aspects of screening from initiation to conclusion, which will enable researchers to conduct screens of their own, maximising the potential of this powerful technology.},
}
@article {pmid31034868,
year = {2019},
author = {Darma, R and Lutz, A and Elliott, CE and Idnurm, A},
title = {Identification of a gene cluster for the synthesis of the plant hormone abscisic acid in the plant pathogen Leptosphaeria maculans.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {130},
number = {},
pages = {62-71},
doi = {10.1016/j.fgb.2019.04.015},
pmid = {31034868},
issn = {1096-0937},
mesh = {Abscisic Acid/*metabolism ; Ascomycota/*genetics/*metabolism/pathogenicity ; Brassica napus/microbiology ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; Genes, Fungal/*genetics ; Multigene Family/*genetics ; Plant Diseases/microbiology ; Plant Growth Regulators/*biosynthesis/*genetics ; Polyketide Synthases/genetics ; Secondary Metabolism ; Transcription Factors/genetics ; Up-Regulation ; Virulence/genetics ; },
abstract = {Leptosphaeria maculans is an ascomycetous fungus that causes the disease blackleg on Brassica napus (canola). In spite of the importance of the disease worldwide, the mechanisms of disease development are poorly understood. Secondary metabolites, which are one of the common virulence factors of pathogenic fungi, have not been extensively explored from this fungus. An RNA-seq dataset was examined to find genes responsible for secondary metabolite synthesis by this fungus during infection. One polyketide synthase gene, pks5, was found to be upregulated during the early biotrophic stage of development. In addition to pks5, six other genes adjacent to the pks5 gene, including one encoding a Zn(II)2Cys6 transcription factor abscisic acid-like 7 gene (abl7), were also upregulated during that time. A striking feature of the L. maculans genome is that it contains large AT-rich regions that are gene-poor and large GC-rich regions that are gene rich. This set of seven co-regulated genes is embedded within and separated by two such AT-rich regions. Three of the genes in the cluster have similarities to those known to be involved in the synthesis of abscisic acid (ABA) in other fungi. When L. maculans is grown in axenic culture the genes in this cluster are not expressed and ABA is not produced. Overexpressing abl7, encoding the putative transcription factor, resulted in the transcription of the six adjacent genes in axenic culture and in the production of ABA, as detected by liquid chromatography quadrupole-time-of-flight mass spectrometry analysis. Mutation of two genes of the cluster using CRISPR/Cas9 did not affect pathogenicity on canola cotyledons. The characterization of the ABA gene cluster has led to the discovery of the co-regulation of genes within an AT-rich region by a transcription factor, and the first report of the plant hormone abscisic acid being produced by L. maculans.},
}
@article {pmid31034861,
year = {2020},
author = {Mikuni, T},
title = {Genome editing-based approaches for imaging protein localization and dynamics in the mammalian brain.},
journal = {Neuroscience research},
volume = {150},
number = {},
pages = {2-7},
doi = {10.1016/j.neures.2019.04.007},
pmid = {31034861},
issn = {1872-8111},
mesh = {Animals ; Brain/*metabolism ; Brain Mapping/*methods ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Vectors ; Mammals ; Models, Biological ; Neuroimaging/methods ; Neurons/metabolism ; Single-Cell Analysis ; },
abstract = {A neuron contains thousands of proteins, each of which mediates neuronal processes at distinct subcellular compartments. Thus, precise mapping of each protein with subcellular resolution in the brain is essential to understand neuronal processes at the molecular level. However, no conventional methods have provided rapid, generalizable, and high-throughput readouts for the subcellular localization of endogenous proteins in the mammalian brain. Recently, new methods based on in vivo genome editing have been developed for high-throughput determination of the protein localization with high specificity, resolution and contrast in mammalian brain tissue. In this review, I first describe the merits and demerits of each conventional method for imaging the protein localization in the brain. I then introduce the new genome editing-based methods to discuss their advantages, limitations and future potential in molecular and cellular neuroscience research.},
}
@article {pmid31034471,
year = {2019},
author = {Wang, X and Chen, X and Sun, L and Qian, W},
title = {Canonical cytosolic iron-sulfur cluster assembly and non-canonical functions of DRE2 in Arabidopsis.},
journal = {PLoS genetics},
volume = {15},
number = {4},
pages = {e1008094},
pmid = {31034471},
issn = {1553-7404},
mesh = {Alleles ; Arabidopsis/*genetics/*metabolism ; Arabidopsis Proteins/*genetics/*metabolism ; CRISPR-Cas Systems ; Cytosol/metabolism ; DNA Damage ; DNA Methylation ; Indoleacetic Acids/metabolism ; Iron/*metabolism ; Iron-Sulfur Proteins/*genetics/*metabolism ; Multigene Family ; Mutation ; Protein Interaction Mapping ; Protein Interaction Maps ; Sulfur/*metabolism ; },
abstract = {As a component of the Cytosolic Iron-sulfur cluster Assembly (CIA) pathway, DRE2 is essential in organisms from yeast to mammals. However, the roles of DRE2 remain incompletely understood largely due to the lack of viable dre2 mutants. In this study, we successfully created hypomorphic dre2 mutants using the CRISPR/Cas9 technology. Like other CIA pathway mutants, the dre2 mutants have accumulation of DNA lesions and show constitutive DNA damage response. In addition, the dre2 mutants exhibit DNA hypermethylation at hundreds of loci. The mutant forms of DRE2 in the dre2 mutants, which bear deletions in the linker region of DRE2, lost interaction with GRXS17 but have stronger interaction with NBP35, resulting in the CIA-related defects of dre2. Interestingly, we find that DRE2 is also involved in auxin response that may be independent of its CIA role. DRE2 localizes in both the cytoplasm and the nucleus and nuclear DRE2 associates with euchromatin. Furthermore, DRE2 directly associates with multiple auxin responsive genes and maintains their normal expression. Our study highlights the importance of the linker region of DRE2 in coordinating CIA-related protein interactions and identifies the canonical and non-canonical roles of DRE2 in maintaining genome stability, epigenomic patterns, and auxin response.},
}
@article {pmid31034138,
year = {2019},
author = {Selma, S and Bernabé-Orts, JM and Vazquez-Vilar, M and Diego-Martin, B and Ajenjo, M and Garcia-Carpintero, V and Granell, A and Orzaez, D},
title = {Strong gene activation in plants with genome-wide specificity using a new orthogonal CRISPR/Cas9-based programmable transcriptional activator.},
journal = {Plant biotechnology journal},
volume = {17},
number = {9},
pages = {1703-1705},
pmid = {31034138},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Promoter Regions, Genetic ; Tobacco/*genetics ; Transcription Factors/*genetics ; *Transcriptional Activation ; },
}
@article {pmid31033959,
year = {2019},
author = {Liu, KI and Sutrisnoh, NB and Wang, Y and Tan, MH},
title = {Genome Editing in Mammalian Cell Lines using CRISPR-Cas.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {146},
pages = {},
doi = {10.3791/59086},
pmid = {31033959},
issn = {1940-087X},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Knockout Techniques ; *Genome ; HEK293 Cells ; Humans ; Mammals/*genetics ; Plasmids/genetics ; RNA, Guide/genetics ; Recombinational DNA Repair ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.},
}
@article {pmid31033105,
year = {2019},
author = {Vaughan, A and Yang, IA},
title = {CRISPR-Cas9 technology: A new direction for personalized medicine in respiratory disease?.},
journal = {Respirology (Carlton, Vic.)},
volume = {24},
number = {7},
pages = {614-615},
doi = {10.1111/resp.13570},
pmid = {31033105},
issn = {1440-1843},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Precision Medicine ; *Respiration Disorders ; },
}
@article {pmid31033104,
year = {2019},
author = {Tuncel, A and Corbin, KR and Ahn-Jarvis, J and Harris, S and Hawkins, E and Smedley, MA and Harwood, W and Warren, FJ and Patron, NJ and Smith, AM},
title = {Cas9-mediated mutagenesis of potato starch-branching enzymes generates a range of tuber starch phenotypes.},
journal = {Plant biotechnology journal},
volume = {17},
number = {12},
pages = {2259-2271},
pmid = {31033104},
issn = {1467-7652},
support = {BBS/E/J/00000020/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R012512/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014130/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/J/000PR9799/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/J004561/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {1,4-alpha-Glucan Branching Enzyme/*genetics ; Amylopectin ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Mutagenesis ; Phenotype ; Plant Proteins/*genetics ; Solanum tuberosum/enzymology/*genetics ; Starch ; },
abstract = {We investigated whether Cas9-mediated mutagenesis of starch-branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium-mediated transformation or by PEG-mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low-SBE potato tubers. HPLC-SEC and [1] H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild-type starch. Mutants with strong reductions in SBE2 protein alone had near-normal amylopectin chain-length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9-mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.},
}
@article {pmid31030279,
year = {2019},
author = {Wang, DC and Wang, X},
title = {Off-target genome editing: A new discipline of gene science and a new class of medicine.},
journal = {Cell biology and toxicology},
volume = {35},
number = {3},
pages = {179-183},
doi = {10.1007/s10565-019-09475-7},
pmid = {31030279},
issn = {1573-6822},
mesh = {CRISPR-Cas Systems ; Gene Editing/*ethics/*methods/*trends ; Humans ; },
abstract = {With an increasing growth of genome editing, off-target effects such as non-specific genetic modifications resulting from the designed process of genome editing become a new discipline of gene science and new class medicine. The degree of short-term and long-term side effects and toxicity or dynamics of the primary and secondary off-target genome editing varies with the application of different methodologies of gene editing and measuring, readouts of genetic modifications, or comparison reference. Measurements of dynamic off-target effects caused directly or indirectly by genome editing are critical in clinical application of gene editing. The quality of genome editing methods is one of the decisive factors in the occurrence of off-target effects. Mechanisms by which off-target effects of genome editing occurs are more complex and comprehensive than we expected. The heterogeneity of off-target effects of gene-edited cells at single-cell levels should be defined during the development and formation of cell clusters. In addition to off-target effects on gene-edited cells per se, alterations of gene sequence, structure, dimension, and function of related regulators caused by off-target effects may also influence intercellular communications and interactions between gene-edited cells, between gene-edited cells and non-edited cells, or between non-edited cells. Thus, controlling, measuring, defining, categorizing, and predicting off-target genome editing need to be standardized and prioritized before clinical application of gene editing.},
}
@article {pmid31030226,
year = {2019},
author = {Liu, Z and Chen, M and Shan, H and Chen, S and Xu, Y and Song, Y and Zhang, Q and Yuan, H and Ouyang, H and Li, Z and Lai, L},
title = {Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7).},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {20},
pages = {4155-4164},
pmid = {31030226},
issn = {1420-9071},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Codon ; Dystrophin/genetics/metabolism ; Embryo, Mammalian ; *Founder Effect ; Gene Editing/*methods ; Gene Expression ; Gene Targeting/*methods ; High-Throughput Nucleotide Sequencing ; Microinjections ; Plasmids/chemistry/metabolism ; Poly(A)-Binding Proteins/genetics/metabolism ; Presenilin-1/genetics/metabolism ; RNA, Guide/*genetics/metabolism ; Rabbits ; Trinucleotide Repeats ; Zygote ; },
abstract = {Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.},
}
@article {pmid31029569,
year = {2019},
author = {Ma, K and Han, J and Hao, Y and Yang, Z and Chen, J and Liu, YG and Zhu, Q and Chen, L},
title = {An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {5},
pages = {273-275},
doi = {10.1016/j.jgg.2019.03.005},
pmid = {31029569},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Knockout Techniques ; *Gene Library ; Genes, Plant/*genetics ; *Mutation ; Oryza/*genetics/physiology ; Plant Infertility/*genetics ; },
}
@article {pmid31028650,
year = {2019},
author = {Henriksson, J},
title = {CRISPR Screening in Single Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1979},
number = {},
pages = {395-406},
doi = {10.1007/978-1-4939-9240-9_23},
pmid = {31028650},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/*methods ; Transduction, Genetic/methods ; Transfection/methods ; },
abstract = {The combination of single-cell RNA-seq and CRISPR allows for efficient interrogation of possibly any number of genes, only limited by the sequencing capability. Here we describe the current protocols for CRISPR screening in single cells, from cloning and virus production to generating sequencing data.},
}
@article {pmid31028249,
year = {2019},
author = {Bencheikh, L and Diop, MK and Rivière, J and Imanci, A and Pierron, G and Souquere, S and Naimo, A and Morabito, M and Dussiot, M and De Leeuw, F and Lobry, C and Solary, E and Droin, N},
title = {Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1935},
pmid = {31028249},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Cell Membrane/chemistry/drug effects/metabolism ; Chromatin/chemistry/drug effects/metabolism ; Early Growth Response Protein 1/genetics/metabolism ; Fluorescent Dyes/chemistry ; Gene Editing ; *Gene Expression Regulation ; HEK293 Cells ; Histones/genetics/metabolism ; Humans ; Leukemia, Myelomonocytic, Chronic/*genetics/metabolism/pathology ; Macrophage Colony-Stimulating Factor/metabolism/*pharmacology ; Macrophages/cytology/*drug effects/metabolism ; Maleimides/chemistry ; Primary Cell Culture ; Protein Binding ; RNA, Small Interfering/genetics/metabolism ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists &amp; inhibitors/*genetics/metabolism ; Signal Transduction ; THP-1 Cells ; YY1 Transcription Factor/genetics/metabolism ; ets-Domain Protein Elk-1/genetics/metabolism ; },
abstract = {Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.},
}
@article {pmid31028078,
year = {2019},
author = {Egorova, TV and Zotova, ED and Reshetov, DA and Polikarpova, AV and Vassilieva, SG and Vlodavets, DV and Gavrilov, AA and Ulianov, SV and Buchman, VL and Deykin, AV},
title = {CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene.},
journal = {Disease models &amp; mechanisms},
volume = {12},
number = {4},
pages = {},
pmid = {31028078},
issn = {1754-8411},
mesh = {Animals ; Base Pairing/*genetics ; Biomechanical Phenomena ; CRISPR-Cas Systems/*genetics ; Cell Line ; Child ; Chromatin/metabolism ; Disease Models, Animal ; Dystrophin/*genetics ; Exons/genetics ; Female ; Humans ; Male ; Mice, Inbred C57BL ; Muscles/physiopathology ; Muscular Dystrophy, Duchenne/*genetics/physiopathology ; Phenotype ; RNA, Guide/metabolism ; *Sequence Deletion ; },
abstract = {Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.},
}
@article {pmid31027375,
year = {2019},
author = {Zhang, Y and Luo, J and Tang, N and Teng, M and Reddy, VRAP and Moffat, K and Shen, Z and Nair, V and Yao, Y},
title = {Targeted Editing of the pp38 Gene in Marek's Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System.},
journal = {Viruses},
volume = {11},
number = {5},
pages = {},
pmid = {31027375},
issn = {1999-4915},
support = {BBS/E/I/00007032 and BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Antigens, Viral/*genetics ; *CRISPR-Cas Systems ; Cell Line, Transformed ; DNA End-Joining Repair ; Flow Cytometry ; *Gene Editing ; Gene Targeting ; Genes, Reporter ; Genome, Viral ; Herpesvirus 2, Gallid/*genetics ; Marek Disease/*virology ; Phosphoproteins/*genetics ; RNA, Guide ; },
abstract = {Marek's disease virus (MDV), a lymphotropic α-herpesvirus associated with T-cell lymphomas in chickens, is an excellent model for herpesvirus biology and virus-induced oncogenesis. Marek's disease (MD) is also one of the cancers against which a vaccine was first used. In the lymphomas and lymphoblastoid cell lines (LCLs) derived from them, MDV establishes latent infection with limited gene expression. Although LCLs are valuable for interrogating viral and host gene functions, molecular determinants associated with the maintenance of MDV latency and lytic switch remain largely unknown, mainly due to the lack of tools for in situ manipulation of the genomes in these cell lines. Here we describe the first application of CRISPR/Cas9 editing approach for precise editing of the viral gene phosphoprotein 38 (pp38), a biomarker for latent/lytic switch in MDV-transformed LCLs MDCC-MSB-1 (Marek's disease cell line MSB-1) and MDCC-HP8. Contradictory to the previous reports suggesting that pp38 is involved in the maintenance of transformation of LCL MSB-1 cells, we show that pp38-deleted cells proliferated at a significant higher rate, suggesting that pp38 is dispensable for the transformed state of these cell lines. Application of CRISPR/Cas9-based gene editing of MDV-transformed cell lines in situ opens up further opportunities towards a better understanding of MDV pathogenesis and virus-host interactions.},
}
@article {pmid31026591,
year = {2019},
author = {Yesbolatova, A and Natsume, T and Hayashi, KI and Kanemaki, MT},
title = {Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {73-80},
doi = {10.1016/j.ymeth.2019.04.010},
pmid = {31026591},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; Cell Culture Techniques/methods ; Genetic Vectors/genetics ; HCT116 Cells ; Half-Life ; Humans ; Indoleacetic Acids/*pharmacology ; Luminescent Proteins/genetics/metabolism ; Plant Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Plasmids/genetics ; Proteolysis/*drug effects ; Recombinant Fusion Proteins/genetics/*metabolism ; Transfection/methods ; Ubiquitin-Protein Ligases/antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR-Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.},
}
@article {pmid31026589,
year = {2019},
author = {Kunitake, E and Tanaka, T and Ueda, H and Endo, A and Yarimizu, T and Katoh, E and Kitamoto, H},
title = {CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {130},
number = {},
pages = {82-90},
doi = {10.1016/j.fgb.2019.04.012},
pmid = {31026589},
issn = {1096-0937},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Fungal/genetics ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Genes, Fungal/genetics ; Genetic Loci ; Homologous Recombination ; Ribonucleoproteins/genetics ; Transformation, Genetic ; Ustilaginales/*genetics ; },
abstract = {The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms.},
}
@article {pmid31023625,
year = {2019},
author = {Good, AL and Cannon, CE and Haemmerle, MW and Yang, J and Stanescu, DE and Doliba, NM and Birnbaum, MJ and Stoffers, DA},
title = {JUND regulates pancreatic β cell survival during metabolic stress.},
journal = {Molecular metabolism},
volume = {25},
number = {},
pages = {95-106},
pmid = {31023625},
issn = {2212-8778},
support = {F30 DK105758/DK/NIDDK NIH HHS/United States ; K12 DK094723/DK/NIDDK NIH HHS/United States ; P30 DK019525/DK/NIDDK NIH HHS/United States ; R01 DK122039/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Caspase 3/metabolism ; Caspase 7/metabolism ; Cell Line ; Diabetes Mellitus, Type 2/metabolism ; Fatty Acids ; Gene Expression Regulation ; Glucose/metabolism ; Homeodomain Proteins/genetics ; Humans ; Insulin-Secreting Cells/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Proto-Oncogene Proteins c-jun/*metabolism ; RNA, Messenger/metabolism ; Stress, Physiological/*physiology ; Trans-Activators/genetics ; Transcription Factors ; },
abstract = {OBJECTIVE: In type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in β cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions.<br><br>METHODS: Translating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment.<br><br>RESULTS: TRAP-seq in a PDX1-deficiency model of &#x3b2; cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in &#x3b2; cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of &#x3b2; cell dysfunction, consistent with a maladaptive role for JUND in islets.<br><br>CONCLUSIONS: A translation-centric approach uncovered JUND as a stress-responsive factor in &#x3b2; cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.},
}
@article {pmid31023561,
year = {2019},
author = {Khan, MZ and Haider, S and Mansoor, S and Amin, I},
title = {Targeting Plant ssDNA Viruses with Engineered Miniature CRISPR-Cas14a.},
journal = {Trends in biotechnology},
volume = {37},
number = {8},
pages = {800-804},
doi = {10.1016/j.tibtech.2019.03.015},
pmid = {31023561},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems ; DNA Viruses/*genetics ; Disease Resistance ; Gene Editing/*methods ; Plant Diseases/*prevention &amp; control/*virology ; Plant Viruses/*genetics ; Plants, Genetically Modified/*immunology ; },
abstract = {CRISPR-Cas14a is a highly compact protein with great potential to be used as a guided genome editing tool for single-stranded (ss) DNA cleavage. Recently isolated from noncultured archaea, its unrestricted and sequence-independent cleavage makes it an ideal tool for engineering resistance against economically important plant ssDNA viruses.},
}
@article {pmid31023186,
year = {2019},
author = {Li, Y and Li, S and Li, Y and Xia, H and Mao, Q},
title = {Generation of a novel HEK293 luciferase reporter cell line by CRISPR/Cas9-mediated site-specific integration in the genome to explore the transcriptional regulation of the PGRN gene.},
journal = {Bioengineered},
volume = {10},
number = {1},
pages = {98-107},
pmid = {31023186},
issn = {2165-5987},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Founder Effect ; Gene Editing ; Gene Expression Regulation/drug effects ; Gene Knock-In Techniques/*methods ; Genes, Reporter ; Genome, Human ; HEK293 Cells ; Humans ; Luciferases/*genetics/metabolism ; Progranulins/agonists/*genetics/metabolism ; Promoter Regions, Genetic/drug effects ; Signal Transduction ; Tetradecanoylphorbol Acetate/pharmacology ; Transcription, Genetic/drug effects ; },
abstract = {Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.},
}
@article {pmid31022512,
year = {2019},
author = {Liu, Y and Ma, S and Chang, J and Zhang, T and Chen, X and Liang, Y and Xia, Q},
title = {Programmable targeted epigenetic editing using CRISPR system in Bombyx mori.},
journal = {Insect biochemistry and molecular biology},
volume = {110},
number = {},
pages = {105-111},
doi = {10.1016/j.ibmb.2019.04.013},
pmid = {31022512},
issn = {1879-0240},
mesh = {Animals ; Bombyx/*genetics ; *CRISPR-Cas Systems ; *DNA Methylation ; *Epigenesis, Genetic ; Gene Editing/*methods ; },
abstract = {DNA methylation has been proven to play roles in regulating gene expression, cell fate, disease determination, and chromatin architecture organization in mammals and plants, and is a significant component of epigenetic modification. Compared to mammals or plants, the status and function of DNA methylation are poorly understood in insects, which is partially due to the lack of efficient manipulation tools. In this study, we show that fusion protein of catalytically inactive Cas9 (dCas9) with TET1 can efficiently demethylate genomic DNA of silkworm Bombyx mori, in a programmable target region specific manner. We first developed an all-in-one vector to maximize the targeting efficiency of dCas9-TET1. Then we selected 3 endogenous genes that were previously found to harbor methylated DNA, and designed gRNAs within the methylated region. Co-transfection of dCas9-TET1 and gRNA successfully erased methylation marks near the targeting region, with efficiencies from about 17.50% to 40.00%. Furthermore, targeted demethylation on gene body resulted in increased mRNA transcription level. Unlike the previously widely used decitabine, a methylation inhibitor, dCas9-TET1 is more effective and specific, and has no unwanted impact on whole-genome methylation. DCas9-TET1 provides a powerful tool for investigating the functional significance of DNA methylation in a locus-specific manner, and for exploring the unknown links between methylation and development in insects.},
}
@article {pmid31022459,
year = {2019},
author = {Pandey, PK and Quilichini, TD and Vaid, N and Gao, P and Xiang, D and Datla, R},
title = {Versatile and multifaceted CRISPR/Cas gene editing tool for plant research.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {107-114},
doi = {10.1016/j.semcdb.2019.04.012},
pmid = {31022459},
issn = {1096-3634},
mesh = {Botany/*methods ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Plants/*genetics ; },
abstract = {The ability to create desirable gene variants through targeted changes offers tremendous opportunities for the advancement of basic and applied plant research. Gene editing technologies have opened new avenues to perform such precise gene modifications in diverse biological systems. These technologies use sequence-specific nucleases, such as homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR/Cas) complexes to enable targeted genetic manipulations. Among these, the CRISPR/Cas system has emerged as a broadly applicable and valued gene editing system for its ease of use and versatility. The adaptability of the CRISPR/Cas system has facilitated rapid and continuous innovative developments to the precision and applications of this technology, since its introduction less than a decade ago. Although developed in animal systems, the simple and elegant CRISPR/Cas gene editing technology has quickly been embraced by plant researchers. From early demonstration in model plants, the CRISPR/Cas system has been successfully adapted for various crop species and enabled targeting of agronomically important traits. Although the approach faces several efficiency and delivery related challenges, especially in recalcitrant crop species, continuous advances in the CRISPR/Cas system to address these limitations are being made. In this review, we discuss the CRISPR/Cas technology, its myriad applications and their prospects for crop improvement.},
}
@article {pmid31021314,
year = {2019},
author = {Lee, H and Dhingra, Y and Sashital, DG},
title = {The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31021314},
issn = {2050-084X},
support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; GM115874/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacillus/*enzymology/genetics ; CRISPR-Associated Proteins/*metabolism/ultrastructure ; DNA, Single-Stranded/*metabolism ; Endodeoxyribonucleases/*metabolism/ultrastructure ; Microscopy, Electron ; Protein Conformation ; Protein Interaction Mapping ; Protein Multimerization ; },
abstract = {CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.},
}
@article {pmid31021272,
year = {2018},
author = {Makarova, KS and Wolf, YI and Koonin, EV},
title = {Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?.},
journal = {The CRISPR journal},
volume = {1},
number = {5},
pages = {325-336},
pmid = {31021272},
issn = {2573-1602},
abstract = {As befits an immune mechanism, CRISPR-Cas systems are highly variable with respect to Cas protein sequences, gene composition, and organization of the genomic loci. Optimal classification of CRISPR-Cas systems and rational nomenclature for CRISPR-associated genes are essential for further progress of CRISPR research. These are highly challenging tasks because of the complexity of CRISPR-Cas and their fast evolution, including frequent module shuffling, as well as the lack of universal markers for a consistent evolutionary classification. The complexity and variability of CRISPR-Cas systems necessitate a multipronged approach to classification and nomenclature. We present a brief summary of the current state of the art and discuss further directions in this area.},
}
@article {pmid31021245,
year = {2018},
author = {Lau, CH},
title = {Applications of CRISPR-Cas in Bioengineering, Biotechnology, and Translational Research.},
journal = {The CRISPR journal},
volume = {1},
number = {6},
pages = {379-404},
doi = {10.1089/crispr.2018.0026},
pmid = {31021245},
issn = {2573-1602},
abstract = {CRISPR technology is rapidly evolving, and the scope of CRISPR applications is constantly expanding. CRISPR was originally employed for genome editing. Its application was then extended to epigenome editing, karyotype engineering, chromatin imaging, transcriptome, and metabolic pathway engineering. Now, CRISPR technology is being harnessed for genetic circuits engineering, cell signaling sensing, cellular events recording, lineage information reconstruction, gene drive, DNA genotyping, miRNA quantification, in vivo cloning, site-directed mutagenesis, genomic diversification, and proteomic analysis in situ. It has also been implemented in the translational research of human diseases such as cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnosis, pathogen screening, microbiota remodeling, stem-cell reprogramming, immunogenomic engineering, vaccine development, and antibody production. This review aims to summarize the key concepts of these CRISPR applications in order to capture the current state of play in this fast-moving field. The key mechanisms, strategies, and design principles for each technological advance are also highlighted.},
}
@article {pmid31021242,
year = {2018},
author = {Robertson, L and Pederick, D and Piltz, S and White, M and Nieto, A and Ahladas, M and Adikusuma, F and Thomas, PQ},
title = {Expanding the RNA-Guided Endonuclease Toolkit for Mouse Genome Editing.},
journal = {The CRISPR journal},
volume = {1},
number = {},
pages = {431-439},
doi = {10.1089/crispr.2018.0050},
pmid = {31021242},
issn = {2573-1602},
abstract = {The RNA-guided endonuclease CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is widely used for generating genetically modified mice via zygotic microinjection. Although SpCas9 is a potent mutagen, it requires an NGG proto-spacer adjacent motif (PAM) at the target site, restricting sequence targetability. Here, we show that RNA-guided endonucleases that utilize a range of alternative PAM sequences can edit the mouse genome at the neurog3 (Ngn3) locus: SpCas9 VQR (NGAN PAM), SpCas9 VRER (NGCG), AsCas12a (TTTN), SaCas9 (NNGRRT), and SaCas9 KKH (NNNRRT). Additional experiments targeting tyrosinase and frizzled3 with SaCas9 KKH and its parent protein demonstrated that these endonucleases generated mutations in up to 100% of embryos across three loci. Remarkably, in contrast to wild-type SpCas9, these endonucleases frequently generated mutant embryos that retain unmodified alleles in both template-free and HDR-repair experiments. Our findings broaden PAM recognition options for mouse genome editing and identify SaCas9/SaCas9 KKH as useful alternatives when targeting genes with null lethal phenotypes.},
}
@article {pmid31021236,
year = {2019},
author = {Ding, X and Seebeck, T and Feng, Y and Jiang, Y and Davis, GD and Chen, F},
title = {Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {51-63},
doi = {10.1089/crispr.2018.0036},
pmid = {31021236},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; HMGB1 Protein/genetics/metabolism ; HMGN1 Protein/genetics/metabolism ; Humans ; K562 Cells ; Peptides/genetics ; Recombinant Fusion Proteins/genetics/metabolism ; Streptococcus pyogenes/genetics ; },
abstract = {Bacterial-derived CRISPR-Cas9 nucleases have become a common tool in genome engineering. However, the editing efficiency by even the best-crafted Cas9 nucleases varies considerably with different genomic sites, and efforts to explore the vast natural Cas9 diversity have often met with mixed or little success. Here, we show that modification of the widely used Streptococcus pyogenes Cas9 by fusion with chromatin-modulating peptides (CMPs), derived from high mobility group proteins HMGN1 and HMGB1, histone H1, and chromatin remodeling complexes, improves its activity by up to several fold, particularly on refractory target sites. We further show that this CMP fusion strategy (termed CRISPR-chrom) is also effective in improving the activities of smaller Cas9 nucleases from Streptococcus pasteurianus and Campylobacter jejuni, as well as four newly characterized Cas9 orthologs from Bacillus smithii, Lactobacillus rhamnosus, Mycoplasma canis, and Parasutterella excrementihominis. Our findings suggest that this CRISPR-chrom strategy can be used to improve established Cas9 nucleases and facilitate exploration of novel Cas9 orthologs for genome modification.},
}
@article {pmid31021235,
year = {2019},
author = {Martufi, M and Good, RB and Rapiteanu, R and Schmidt, T and Patili, E and Tvermosegaard, K and New, M and Nanthakumar, CB and Betts, J and Blanchard, AD and Maratou, K},
title = {Single-Step, High-Efficiency CRISPR-Cas9 Genome Editing in Primary Human Disease-Derived Fibroblasts.},
journal = {The CRISPR journal},
volume = {2},
number = {1},
pages = {31-40},
pmid = {31021235},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Cell Culture Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fibroblasts/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genetic Engineering/methods ; Genetic Vectors ; Humans ; Lung/pathology ; Primary Cell Culture/*methods ; Smad Proteins/genetics/metabolism ; },
abstract = {Genome editing is a tool that has many applications, including the validation of potential drug targets. However, performing genome editing in low-passage primary human cells with the greatest physiological relevance is notoriously difficult. High editing efficiency is desired because it enables gene knockouts (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single-step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery in the absence of antibiotic selection or clonal expansion. As proof of concept, we edited two SMAD family members and demonstrated that in response to transforming growth factor beta, SMAD3, but not SMAD2, is critical for deposition of type I collagen in the fibrotic response. The optimization of this workflow can be readily transferred to other primary cell types.},
}
@article {pmid31021234,
year = {2019},
author = {Hwang, S and Maxwell, KL},
title = {Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.},
journal = {The CRISPR journal},
volume = {2},
number = {1},
pages = {23-30},
doi = {10.1089/crispr.2018.0052},
pmid = {31021234},
issn = {2573-1602},
mesh = {Archaea/genetics/immunology/*virology ; Bacteria/genetics/immunology/*virology ; Bacteriophages/*genetics/metabolism ; CRISPR-Associated Protein 9/genetics/immunology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/immunology ; Computational Biology/methods ; DNA Transposable Elements ; Evolution, Molecular ; Gene Editing/methods ; *Genome, Viral ; Plasmids/metabolism ; Prophages/genetics/metabolism ; Pseudomonas Phages/*genetics/metabolism ; Viral Proteins/*genetics/metabolism ; },
abstract = {The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.},
}
@article {pmid31021232,
year = {2019},
author = {Barrangou, R},
title = {CRISPR on the Move in 2019.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {1-2},
doi = {10.1089/crispr.2019.29043.rba},
pmid = {31021232},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; Research/trends ; },
}
@article {pmid31021231,
year = {2019},
author = {Swarts, DC},
title = {Stirring Up the Type V Alphabet Soup.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {14-16},
doi = {10.1089/crispr.2019.29044.dcs},
pmid = {31021231},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Food ; },
}
@article {pmid31021229,
year = {2019},
author = {Davies, K and Charpentier, E},
title = {Finding Her Niche: An Interview with Emmanuelle Charpentier.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {17-22},
doi = {10.1089/crispr.2019.29042.kda},
pmid = {31021229},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Female ; History, 20th Century ; History, 21st Century ; Humans ; },
}
@article {pmid31021228,
year = {2019},
author = {Russell, A},
title = {CRISPR Screens: The Right Tool for the Job.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {9-11},
doi = {10.1089/crispr.2019.29045.adr},
pmid = {31021228},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome ; T-Lymphocytes, Helper-Inducer ; },
}
@article {pmid31021210,
year = {2018},
author = {Dahlberg, L and Groat Carmona, AM},
title = {CRISPR-Cas Technology In and Out of the Classroom.},
journal = {The CRISPR journal},
volume = {1},
number = {2},
pages = {107-114},
pmid = {31021210},
issn = {2573-1602},
abstract = {Student-centered practices, including student-focused research opportunities, enhance biology education and comprehension. One way to support student interest is through research opportunities in faculty laboratories. However, alternatives to traditional research apprenticeships are important for the inclusion of more undergraduate students in CRISPR-Cas-based research. Student interest in CRISPR-Cas technologies serves as a timely focal point for deepening undergraduate student engagement in biology courses. In this article, we describe some of the ongoing efforts to bring CRISPR-Cas technology out of the classroom and into the teaching laboratory.},
}
@article {pmid31021201,
year = {2018},
author = {Crawley, AB and Henriksen, JR and Barrangou, R},
title = {CRISPRdisco: An Automated Pipeline for the Discovery and Analysis of CRISPR-Cas Systems.},
journal = {The CRISPR journal},
volume = {1},
number = {2},
pages = {171-181},
pmid = {31021201},
issn = {2573-1602},
abstract = {CRISPR-Cas adaptive immune systems of bacteria and archaea have catapulted into the scientific spotlight as genome editing tools. To aid researchers in the field, we have developed an automated pipeline, named CRISPRdisco (CRISPR discovery), to identify CRISPR repeats and cas genes in genome assemblies, determine type and subtype, and describe system completeness. All six major types and 23 currently recognized subtypes and novel putative V-U types are detected. Here, we use the pipeline to identify and classify putative CRISPR-Cas systems in 2,777 complete genomes from the NCBI RefSeq database. This allows comparison to previous publications and investigation of the occurrence and size of CRISPR-Cas systems. Software available at http://github.com/crisprlab/CRISPRdisco provides reproducible, standardized, accessible, transparent, and high-throughput analysis methods available to all researchers in and beyond the CRISPR-Cas research community. This tool opens new avenues to enable classification within a complex nomenclature and provides analytical methods in a field that has evolved rapidly.},
}
@article {pmid31021200,
year = {2018},
author = {Klompe, SE and Sternberg, SH},
title = {Harnessing "A Billion Years of Experimentation": The Ongoing Exploration and Exploitation of CRISPR-Cas Immune Systems.},
journal = {The CRISPR journal},
volume = {1},
number = {2},
pages = {141-158},
pmid = {31021200},
issn = {2573-1602},
abstract = {The famed physicist-turned-biologist, Max Delbrück, once remarked that, for physicists, "the field of bacterial viruses is a fine playground for serious children who ask ambitious questions." Early discoveries in that playground helped establish molecular genetics, and half a century later, biologists delving into the same field have ushered in the era of precision genome engineering. The focus has of course shifted-from bacterial viruses and their mechanisms of infection to the bacterial hosts and their mechanisms of immunity-but it is the very same evolutionary arms race that continues to awe and inspire researchers worldwide. In this review, we explore the remarkable diversity of CRISPR-Cas adaptive immune systems, describe the molecular components that mediate nucleic acid targeting, and outline the use of these RNA-guided machines for biotechnology applications. CRISPR-Cas research has yielded far more than just Cas9-based genome-editing tools, and the wide-reaching, innovative impacts of this fascinating biological playground are sure to be felt for years to come.},
}
@article {pmid31021199,
year = {2018},
author = {Canver, MC and Joung, JK and Pinello, L},
title = {Impact of Genetic Variation on CRISPR-Cas Targeting.},
journal = {The CRISPR journal},
volume = {1},
number = {2},
pages = {159-170},
pmid = {31021199},
issn = {2573-1602},
abstract = {The CRISPR-CRISPR-associated (Cas) nuclease system offers the ability to perform unprecedented functional genetic experiments and the promise of therapy for a variety of genetic disorders. The understanding of factors contributing to CRISPR targeting efficacy and specificity continues to evolve. As CRISPR systems rely on Watson-Crick base pairing to ultimately mediate genomic cleavage, it logically follows that genetic variation would affect CRISPR targeting by increasing or decreasing sequence homology at on-target and off-target sites or by altering protospacer adjacent motifs. Numerous efforts have been made to document the extent of human genetic variation, which can serve as resources to understand and mitigate the effect of genetic variation on CRISPR targeting. Here, we review efforts to elucidate the effect of human genetic variation on CRISPR targeting at on-target and off-target sites with considerations for laboratory experiments and clinical translation of CRISPR-based therapies.},
}
@article {pmid31019093,
year = {2019},
author = {Lopez-Gonzalez, R and Yang, D and Pribadi, M and Kim, TS and Krishnan, G and Choi, SY and Lee, S and Coppola, G and Gao, FB},
title = {Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {19},
pages = {9628-9633},
pmid = {31019093},
issn = {1091-6490},
support = {RC1 AG035610/AG/NIA NIH HHS/United States ; RF1 NS101986/NS/NINDS NIH HHS/United States ; P30 NS062691/NS/NINDS NIH HHS/United States ; UL1 TR000124/TR/NCATS NIH HHS/United States ; R01 NS093097/NS/NINDS NIH HHS/United States ; R01 NS101986/NS/NINDS NIH HHS/United States ; R37 NS057553/NS/NINDS NIH HHS/United States ; R01 NS057553/NS/NINDS NIH HHS/United States ; },
mesh = {*Amyotrophic Lateral Sclerosis/genetics/metabolism ; Animals ; Apoptosis Regulatory Proteins/genetics/metabolism ; *C9orf72 Protein/genetics/metabolism ; CRISPR-Cas Systems ; *DNA Repair ; Disease Models, Animal ; Drosophila melanogaster ; *Frontotemporal Dementia/genetics/metabolism ; *Ku Autoantigen/genetics/metabolism ; Repetitive Sequences, Amino Acid ; },
abstract = {GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, we identified a number of genetic modifiers of poly(GR) toxicity. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.},
}
@article {pmid31019072,
year = {2019},
author = {Li, Y and Muffat, J and Omer Javed, A and Keys, HR and Lungjangwa, T and Bosch, I and Khan, M and Virgilio, MC and Gehrke, L and Sabatini, DM and Jaenisch, R},
title = {Genome-wide CRISPR screen for Zika virus resistance in human neural cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {19},
pages = {9527-9532},
pmid = {31019072},
issn = {1091-6490},
support = {T32 GM007287/GM/NIGMS NIH HHS/United States ; R01 MH104610/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R33 AI100190/AI/NIAID NIH HHS/United States ; R01 NS088538/NS/NINDS NIH HHS/United States ; U19 AI131135/AI/NIAID NIH HHS/United States ; },
mesh = {Astrocytes/metabolism/pathology/virology ; *CRISPR-Cas Systems ; Cell Line ; Disease Resistance/*genetics ; Female ; Genome-Wide Association Study ; Humans ; Male ; Neural Stem Cells/metabolism/pathology/*virology ; Virus Replication/*genetics ; Zika Virus/*physiology ; Zika Virus Infection/*genetics/metabolism/pathology ; },
abstract = {Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.},
}
@article {pmid31018865,
year = {2019},
author = {Mehta, D and Stürchler, A and Anjanappa, RB and Zaidi, SS and Hirsch-Hoffmann, M and Gruissem, W and Vanderschuren, H},
title = {Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {80},
pmid = {31018865},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Geminiviridae/*genetics ; Genetic Engineering/*adverse effects/methods ; Host-Pathogen Interactions/*genetics ; Manihot/*genetics/virology ; Plants, Genetically Modified/virology ; },
abstract = {BACKGROUND: Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.<br><br>RESULTS: Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus.<br><br>CONCLUSIONS: Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.},
}
@article {pmid31018108,
year = {2019},
author = {Himeda, CL and Jones, PL},
title = {The Genetics and Epigenetics of Facioscapulohumeral Muscular Dystrophy.},
journal = {Annual review of genomics and human genetics},
volume = {20},
number = {},
pages = {265-291},
doi = {10.1146/annurev-genom-083118-014933},
pmid = {31018108},
issn = {1545-293X},
mesh = {CRISPR-Cas Systems ; Chromatin/chemistry ; Chromosomal Proteins, Non-Histone/*genetics/metabolism ; Chromosomes, Human, Pair 4 ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; Genetic Loci ; Genome, Human ; Homeodomain Proteins/*genetics/metabolism ; Humans ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Facioscapulohumeral/classification/*genetics/metabolism/pathology ; Mutation ; Severity of Illness Index ; },
abstract = {Facioscapulohumeral muscular dystrophy (FSHD), a progressive myopathy that afflicts individuals of all ages, provides a powerful model of the complex interplay between genetic and epigenetic mechanisms of chromatin regulation. FSHD is caused by dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is a pioneer transcription factor that activates a program of gene expression during early human development, after which its expression is silenced in most somatic cells. When misexpressed in FSHD skeletal muscle, the DUX4 program leads to accumulated muscle pathology. Epigenetic regulators of the disease locus represent particularly attractive therapeutic targets for FSHD, as many are not global modifiers of the genome, and altering their expression or activity should allow correction of the underlying defect.},
}
@article {pmid31016787,
year = {2019},
author = {Hu, W and Guo, G and Chi, Y and Li, F},
title = {Construction of Traf3 knockout liver cancer cell line using CRISPR/Cas9 system.},
journal = {Journal of cellular biochemistry},
volume = {120},
number = {9},
pages = {14908-14915},
doi = {10.1002/jcb.28753},
pmid = {31016787},
issn = {1097-4644},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Hep G2 Cells ; Humans ; Liver Neoplasms/*genetics/pathology ; Neoplasm Invasiveness/genetics ; RNA, Guide/genetics ; Sequence Analysis, DNA ; TNF Receptor-Associated Factor 3/*genetics ; },
abstract = {PURPOSE: The gene editing technology in CRISPR/Cas9 system was used to construct the Traf3 knockout HepG2 cell line to explore the role of Traf3 in the development of liver cancer.<br><br>METHODS: Five sgRNA sites were designed for the exons of Traf3. The recombinant plasmid of Lentiviral vector2-Traf3-sgRNA was constructed and transformed into Stbl3 competent cells. The recombinants were screened and sequenced, and the effectiveness of the designed gRNA was verified by sequencing. The constructed vector was transfected into HepG2 cells by lentiviral, and the monoclonal antibody was selected to detect the knockout effect of Traf3 gene in HepG2 cells by Western blot. PCR amplification and gene sequencing were performed to obtain the cell line, which the Traf3 gene was knocked out. MTT and Transwell assays were used to detect the effect of Traf3-knockout on HepG2 cell proliferation and cell invasion, respectively.<br><br>RESULTS: The Lentiviral vector2-sgRNA expression vector was successfully constructed. PCR amplification electrophoresis and gene sequencing showed that the Trep3-knockdown HepG2 cells were successfully constructed. Compared with the wild HepG2 cells group, the proliferation and invasion ability of HepG2 cells were enhanced in the Traf3 knockout group.<br><br>CONCLUSION: Knockout Traf3 gene by CRISPR/Cas9 system enhanced the proliferation, migration, and invasion of HepG2 cells, and provided an effective tool for studying the function and mechanism of Traf3.},
}
@article {pmid31016281,
year = {2019},
author = {Yang, Y and Huang, Y},
title = {The CRIPSR/Cas gene-editing system-an immature but useful toolkit for experimental and clinical medicine.},
journal = {Animal models and experimental medicine},
volume = {2},
number = {1},
pages = {5-8},
pmid = {31016281},
issn = {2576-2095},
abstract = {A Chinese scientist, Jiankui He, and his creation of the world's first genetically altered baby made headlines recently. As a newly developed gene-editing technique, the CRISPR/Cas system should not be applied to human beings for reproductive purposes until it has been extensively tested. However, numerous experimental research studies in human somatic, germline cells, and even in embryos, have been conducted, which have shown CRISPR/Cas to be a useful tool for human genome editing and a potential therapeutic method for future clinical use.},
}
@article {pmid31015728,
year = {2018},
author = {Bae, S and Kim, JS},
title = {Machine learning finds Cas9-edited genotypes.},
journal = {Nature biomedical engineering},
volume = {2},
number = {12},
pages = {892-893},
doi = {10.1038/s41551-018-0327-6},
pmid = {31015728},
issn = {2157-846X},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genotype ; Machine Learning ; },
}
@article {pmid31015699,
year = {2019},
author = {Reardon, S},
title = {CRISPR gene-editing creates wave of exotic model organisms.},
journal = {Nature},
volume = {568},
number = {7753},
pages = {441-442},
pmid = {31015699},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}
@article {pmid31015617,
year = {2018},
author = {Duan, D},
title = {CRISPR alleviates muscular dystrophy in dogs.},
journal = {Nature biomedical engineering},
volume = {2},
number = {11},
pages = {795-796},
pmid = {31015617},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dogs ; *Dystrophin ; Gene Editing ; *Muscular Dystrophy, Duchenne ; },
}
@article {pmid31015438,
year = {2019},
author = {Wang, X and Wang, S and Troisi, EC and Howard, TP and Haswell, JR and Wolf, BK and Hawk, WH and Ramos, P and Oberlick, EM and Tzvetkov, EP and Ross, A and Vazquez, F and Hahn, WC and Park, PJ and Roberts, CWM},
title = {BRD9 defines a SWI/SNF sub-complex and constitutes a specific vulnerability in malignant rhabdoid tumors.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1881},
pmid = {31015438},
issn = {2041-1723},
support = {K99 CA197640/CA/NCI NIH HHS/United States ; R00 CA197640/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; R01 CA113794/CA/NCI NIH HHS/United States ; R01 CA172152/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Chromatin Assembly and Disassembly ; Enhancer Elements, Genetic/genetics ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Mutation ; Promoter Regions, Genetic/genetics ; Protein Domains/drug effects ; RNA, Small Interfering/metabolism ; Rhabdoid Tumor/*genetics/pathology ; SMARCB1 Protein/*genetics/metabolism ; Transcription Factors/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Bromodomain-containing protein 9 (BRD9) is a recently identified subunit of SWI/SNF(BAF) chromatin remodeling complexes, yet its function is poorly understood. Here, using a genome-wide CRISPR-Cas9 screen, we show that BRD9 is a specific vulnerability in pediatric malignant rhabdoid tumors (RTs), which are driven by inactivation of the SMARCB1 subunit of SWI/SNF. We find that BRD9 exists in a unique SWI/SNF sub-complex that lacks SMARCB1, which has been considered a core subunit. While SMARCB1-containing SWI/SNF complexes are bound preferentially at enhancers, we show that BRD9-containing complexes exist at both promoters and enhancers. Mechanistically, we show that SMARCB1 loss causes increased BRD9 incorporation into SWI/SNF thus providing insight into BRD9 vulnerability in RTs. Underlying the dependency, while its bromodomain is dispensable, the DUF3512 domain of BRD9 is essential for SWI/SNF integrity in the absence of SMARCB1. Collectively, our results reveal a BRD9-containing SWI/SNF subcomplex is required for the survival of SMARCB1-mutant RTs.},
}
@article {pmid31015259,
year = {2019},
author = {Jeong, HH and Kim, SY and Rousseaux, MWC and Zoghbi, HY and Liu, Z},
title = {Beta-binomial modeling of CRISPR pooled screen data identifies target genes with greater sensitivity and fewer false negatives.},
journal = {Genome research},
volume = {29},
number = {6},
pages = {999-1008},
pmid = {31015259},
issn = {1549-5469},
support = {R01 GM120033/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Computational Biology/methods/standards ; Gene Editing ; Gene Targeting ; Genetic Association Studies/methods ; *Models, Statistical ; RNA, Guide ; Sensitivity and Specificity ; },
abstract = {The simplicity and cost-effectiveness of CRISPR technology have made high-throughput pooled screening approaches accessible to virtually any laboratory. Analyzing the large sequencing data derived from these studies, however, still demands considerable bioinformatics expertise. Various methods have been developed to lessen this requirement, but there are still three tasks for accurate CRISPR screen analysis that involve bioinformatic know-how, if not prowess: designing a proper statistical hypothesis test for robust target identification, developing an accurate mapping algorithm to quantify sgRNA levels, and minimizing the parameters that need to be fine-tuned. To make CRISPR screen analysis more reliable as well as more readily accessible, we have developed a new algorithm, called CRISPRBetaBinomial or CB[2] Based on the beta-binomial distribution, which is better suited to sgRNA data, CB[2] outperforms the eight most commonly used methods (HiTSelect, MAGeCK, PBNPA, PinAPL-Py, RIGER, RSA, ScreenBEAM, and sgRSEA) in both accurately quantifying sgRNAs and identifying target genes, with greater sensitivity and a much lower false discovery rate. It also accommodates staggered sgRNA sequences. In conjunction with CRISPRcloud, CB[2] brings CRISPR screen analysis within reach for a wider community of researchers.},
}
@article {pmid31015023,
year = {2019},
author = {Le Trionnaire, G and Tanguy, S and Hudaverdian, S and Gleonnec, F and Richard, G and Cayrol, B and Monsion, B and Pichon, E and Deshoux, M and Webster, C and Uzest, M and Herpin, A and Tagu, D},
title = {An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid.},
journal = {Insect biochemistry and molecular biology},
volume = {110},
number = {},
pages = {34-44},
doi = {10.1016/j.ibmb.2019.04.016},
pmid = {31015023},
issn = {1879-0240},
mesh = {Animals ; Aphids/*genetics ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Male ; *Mutagenesis ; },
abstract = {CRISPR-Cas9 technology is a very efficient functional analysis tool and has been developed in several insects to edit their genome through injection of eggs with guide RNAs targeting coding sequences of genes of interest. However, its implementation in aphids is more challenging. Aphids are major pests of crops worldwide that alternate during their life cycle between clonality and sexual reproduction. The production of eggs after mating of sexual individuals is a single yearly event and is necessarily triggered by a photoperiod decrease. Fertilized eggs then experience an obligate 3-month diapause period before hatching as new clonal colonies. Taking into consideration these particularities, we developed in the pea aphid Acyrthosiphon pisum a step-by-step protocol of targeted mutagenesis based on the microinjection within fertilized eggs of CRISPR-Cas9 components designed for the editing of a cuticular protein gene (stylin-01). This protocol includes the following steps: i) the photoperiod-triggered induction of sexual morphs (2 months), ii) the mating and egg collection step (2 weeks), iii) egg microinjection and melanization, iv) the 3-month obligate diapause, v) the hatching of new lineages from injected eggs (2 weeks) and vi) the maintenance of stable lineages (2 weeks). Overall, this 7-month long procedure was applied to three different crosses in order to estimate the impact of the choice of the genetic combination on egg production dynamics by females as well as hatching rates after diapause. Mutation rates within eggs before diapause were estimated at 70-80%. The hatching rate of injected eggs following diapause ranged from 1 to 11% depending on the cross and finally a total of 17 stable lineages were obtained and maintained clonally. Out of these, 6 lineages were mutated at the defined sgRNAs target sites within stylin-01 coding sequence, either at the two alleles (2 lineages) or at one allele (4 lineages). The final germline transmission rate of the mutations was thus around 35%. Our protocol of an efficient targeted mutagenesis opens the avenue for functional studies through genome editing in aphids.},
}
@article {pmid31014355,
year = {2019},
author = {Saeinasab, M and Bahrami, AR and González, J and Marchese, FP and Martinez, D and Mowla, SJ and Matin, MM and Huarte, M},
title = {SNHG15 is a bifunctional MYC-regulated noncoding locus encoding a lncRNA that promotes cell proliferation, invasion and drug resistance in colorectal cancer by interacting with AIF.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {38},
number = {1},
pages = {172},
pmid = {31014355},
issn = {1756-9966},
support = {BFU2014-58027-R//Ministerio de Econom&#xed;a, Industria y Competitividad, Gobierno de Espa&#xf1;a/International ; },
mesh = {Animals ; Apoptosis Inducing Factor/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Colorectal Neoplasms/drug therapy/*genetics/pathology ; Drug Resistance, Neoplasm/genetics ; Female ; Fluorouracil/administration &amp; dosage ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Kaplan-Meier Estimate ; Male ; Mice ; Middle Aged ; Neoplasm Invasiveness/genetics/pathology ; Proto-Oncogene Proteins c-myc/*genetics ; RNA, Long Noncoding/*genetics ; RNA, Small Nucleolar/genetics ; Sequence Analysis, RNA ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Thousands of long noncoding RNAs (lncRNAs) are aberrantly expressed in various types of cancers, however our understanding of their role in the disease is still very limited.<br><br>METHODS: We applied RNAseq analysis from patient-derived data with validation in independent cohort of patients. We followed these studies with gene regulation analysis as well as experimental dissection of the role of the identified lncRNA by multiple in vitro and in vivo methods.<br><br>RESULTS: We analyzed RNA-seq data from tumors of 456 CRC patients compared to normal samples, and identified SNHG15 as a potentially oncogenic lncRNA that encodes a snoRNA in one of its introns. The processed SNHG15 is overexpressed in CRC tumors and its expression is highly correlated with poor survival of patients. Interestingly, SNHG15 is more highly expressed in tumors with high levels of MYC expression, while MYC protein binds to two E-box motifs on SNHG15 sequence, indicating that SNHG15 transcription is directly regulated by the oncogene MYC. The depletion of SNHG15 by siRNA or CRISPR-Cas9 inhibits cell proliferation and invasion, decreases colony formation as well as the tumorigenic capacity of CRC cells, whereas its overexpression leads to opposite effects. Gene expression analysis performed upon SNHG15 inhibition showed changes in multiple relevant genes implicated in cancer progression, including MYC, NRAS, BAG3 or ERBB3. Several of these genes are functionally related to AIF, a protein that we found to specifically interact with SNHG15, suggesting that the SNHG15 acts, at least in part, by regulating the activity of AIF. Interestingly, ROS levels, which are directly regulated by AIF, show a significant reduction in SNHG15-depleted cells. Moreover, knockdown of SNHG15 increases the sensitiveness of the cells to 5-FU, while its overexpression renders them more resistant to the chemotherapeutic drug.<br><br>CONCLUSION: Altogether, these results describe an important role of SNHG15 in promoting colon cancer and mediating drug resistance, suggesting its potential as prognostic marker and target for RNA-based therapies.},
}
@article {pmid31013576,
year = {2019},
author = {Albrechtsen, R and Wewer Albrechtsen, NJ and Gnosa, S and Schwarz, J and Dyrskjøt, L and Kveiborg, M},
title = {Identification of ADAM12 as a Novel Basigin Sheddase.},
journal = {International journal of molecular sciences},
volume = {20},
number = {8},
pages = {},
pmid = {31013576},
issn = {1422-0067},
support = {R146-A9211-16-S2//Danish Cancer Society/International ; },
mesh = {ADAM12 Protein/chemistry/genetics/*metabolism ; Amino Acid Sequence ; Basigin/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Gene Expression ; Gene Knockdown Techniques ; Genes, Reporter ; Humans ; Mutation ; Substrate Specificity ; },
abstract = {The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.},
}
@article {pmid31012023,
year = {2019},
author = {Makhotenko, AV and Khromov, AV and Snigir, EA and Makarova, SS and Makarov, VV and Suprunova, TP and Kalinina, NO and Taliansky, ME},
title = {Functional Analysis of Coilin in Virus Resistance and Stress Tolerance of Potato Solanum tuberosum using CRISPR-Cas9 Editing.},
journal = {Doklady. Biochemistry and biophysics},
volume = {484},
number = {1},
pages = {88-91},
pmid = {31012023},
issn = {1608-3091},
mesh = {CRISPR-Cas Systems ; *Disease Resistance ; *Meristem/genetics/metabolism/virology ; *Nuclear Proteins/genetics/metabolism ; *Osmotic Pressure ; Plant Diseases/*virology ; *Plant Proteins/genetics/metabolism ; Rhabdoviridae/*metabolism ; *Solanum tuberosum/genetics/metabolism/virology ; },
abstract = {The role of the nuclear protein coilin in the mechanisms of resistance of potato Solanum tuberosum cultivar Chicago to biotic and abiotic stresses was studied using the CRISPR-Cas9 technology. For the coilin gene editing, a complex consisting of the Cas9 endonuclease and a short guide RNA was immobilized on gold or chitosan microparticles and delivered into apical meristem cells by bioballistics or vacuum infiltration methods, respectively. Editing at least one allele of the coilin gene considerably increased the resistance of the edited lines to infection with the potato virus Y and their tolerance to salt and osmotic stress.},
}
@article {pmid31011884,
year = {2019},
author = {Raikwar, SP and Kikkeri, NS and Sakuru, R and Saeed, D and Zahoor, H and Premkumar, K and Mentor, S and Thangavel, R and Dubova, I and Ahmed, ME and Selvakumar, GP and Kempuraj, D and Zaheer, S and Iyer, SS and Zaheer, A},
title = {Next Generation Precision Medicine: CRISPR-mediated Genome Editing for the Treatment of Neurodegenerative Disorders.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {4},
pages = {608-641},
pmid = {31011884},
issn = {1557-1904},
support = {I01 BX002477/BX/BLRD VA/United States ; R01 AG048205/AG/NIA NIH HHS/United States ; RO1 AG048205/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods/*trends ; Genetic Therapy/methods/trends ; Humans ; Neurodegenerative Diseases/*genetics/*therapy ; Precision Medicine/methods/*trends ; Treatment Outcome ; },
abstract = {Despite significant advancements in the field of molecular neurobiology especially neuroinflammation and neurodegeneration, the highly complex molecular mechanisms underlying neurodegenerative diseases remain elusive. As a result, the development of the next generation neurotherapeutics has experienced a considerable lag phase. Recent advancements in the field of genome editing offer a new template for dissecting the precise molecular pathways underlying the complex neurodegenerative disorders. We believe that the innovative genome and transcriptome editing strategies offer an excellent opportunity to decipher novel therapeutic targets, develop novel neurodegenerative disease models, develop neuroimaging modalities, develop next-generation diagnostics as well as develop patient-specific precision-targeted personalized therapies to effectively treat neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Frontotemporal dementia etc. Here, we review the latest developments in the field of CRISPR-mediated genome editing and provide unbiased futuristic insights regarding its translational potential to improve the treatment outcomes and minimize financial burden. However, despite significant advancements, we would caution the scientific community that since the CRISPR field is still evolving, currently we do not know the full spectrum of CRISPR-mediated side effects. In the wake of the recent news regarding CRISPR-edited human babies being born in China, we urge the scientific community to maintain high scientific and ethical standards and utilize CRISPR for developing in vitro disease in a dish model, in vivo testing in nonhuman primates and lower vertebrates and for the development of neurotherapeutics for the currently incurable neurodegenerative disorders. Graphical Abstract.},
}
@article {pmid31011186,
year = {2019},
author = {Mimitou, EP and Cheng, A and Montalbano, A and Hao, S and Stoeckius, M and Legut, M and Roush, T and Herrera, A and Papalexi, E and Ouyang, Z and Satija, R and Sanjana, NE and Koralov, SB and Smibert, P},
title = {Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells.},
journal = {Nature methods},
volume = {16},
number = {5},
pages = {409-412},
pmid = {31011186},
issn = {1548-7105},
support = {DP2 HG009623/HG/NHGRI NIH HHS/United States ; DP2 HG010099/HG/NHGRI NIH HHS/United States ; R21 HG009748/HG/NHGRI NIH HHS/United States ; R35 GM124998/GM/NIGMS NIH HHS/United States ; R01 HL125816/HL/NHLBI NIH HHS/United States ; P30 CA034196/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Leukocytes, Mononuclear/metabolism/pathology ; Lymphoma, T-Cell, Cutaneous/genetics/metabolism/pathology ; Mice ; NIH 3T3 Cells ; Proteins/*genetics ; RNA, Guide/genetics ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/*methods ; Skin Neoplasms/genetics/metabolism/pathology ; Transcriptome/*genetics ; Tumor Cells, Cultured ; },
abstract = {Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.},
}
@article {pmid31010827,
year = {2019},
author = {He, X and Wang, Y and Yang, F and Wang, B and Xie, H and Gu, L and Zhao, T and Liu, X and Zhang, D and Ren, Q and Liu, X and Liu, Y and Gao, C and Gu, F},
title = {Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA[Gln]-processing system in human cells.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {23},
pages = {9308-9315},
pmid = {31010827},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Cleavage ; Gene Editing/*methods ; HEK293 Cells ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Transfer, Gln/*metabolism ; },
abstract = {CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNA[Gln]-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNA[Gln]-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.},
}
@article {pmid31010806,
year = {2019},
author = {Kerins, MJ and Liu, P and Tian, W and Mannheim, W and Zhang, DD and Ooi, A},
title = {Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor.},
journal = {Molecular and cellular biology},
volume = {39},
number = {13},
pages = {},
pmid = {31010806},
issn = {1098-5549},
support = {P30 ES006694/ES/NIEHS NIH HHS/United States ; R01 CA226920/CA/NCI NIH HHS/United States ; R01 DK109555/DK/NIDDK NIH HHS/United States ; R21 ES027920/ES/NIEHS NIH HHS/United States ; },
mesh = {Autophagy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation ; *Gene Regulatory Networks/drug effects ; Genome, Human ; Homeostasis ; Humans ; NF-E2-Related Factor 2/*metabolism ; RNA, Guide/*pharmacology ; },
abstract = {The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: ATG12, ATG7, GOSR1, IFT172, NRXN2, RAB6A, VPS37A, and the well-known negative regulator of NRF2, KEAP1 Of these, ATG12, ATG7, KEAP1, and VPS37A are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.},
}
@article {pmid31010727,
year = {2019},
author = {Easmin, F and Hassan, N and Sasano, Y and Ekino, K and Taguchi, H and Harashima, S},
title = {gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing.},
journal = {Journal of bioscience and bioengineering},
volume = {128},
number = {3},
pages = {373-378},
doi = {10.1016/j.jbiosc.2019.02.009},
pmid = {31010727},
issn = {1347-4421},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; *Gene Transfer Techniques ; Genetic Engineering/methods ; Genome, Fungal ; Organisms, Genetically Modified ; Plasmids ; Polymerase Chain Reaction/methods ; Promoter Regions, Genetic ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/genetics/metabolism ; Transformation, Genetic ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.},
}
@article {pmid31009976,
year = {2019},
author = {Assimos, DG},
title = {Re: CRISPR/Cas9-Mediated Glycolate Oxidase Disruption is an Efficacious and Safe Treatment for Primary Hyperoxaluria Type I.},
journal = {The Journal of urology},
volume = {201},
number = {5},
pages = {853-854},
doi = {10.1097/01.JU.0000554109.94248.01},
pmid = {31009976},
issn = {1527-3792},
mesh = {Alcohol Oxidoreductases/genetics ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Hyperoxaluria, Primary/*genetics ; },
}
@article {pmid31009522,
year = {2019},
author = {Yeo, JH and Jung, BK and Lee, H and Baek, IJ and Sung, YH and Shin, HS and Kim, HK and Seo, KY and Lee, JY},
title = {Development of a Pde6b Gene Knockout Rat Model for Studies of Degenerative Retinal Diseases.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {60},
number = {5},
pages = {1519-1526},
doi = {10.1167/iovs.18-25556},
pmid = {31009522},
issn = {1552-5783},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cyclic Nucleotide Phosphodiesterases, Type 6/*genetics ; *Disease Models, Animal ; Electroretinography ; Female ; Gene Knockout Techniques ; In Situ Nick-End Labeling ; Phenotype ; Photography ; Photoreceptor Cells, Vertebrate/*pathology ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Retinal Degeneration/diagnostic imaging/*genetics/*pathology ; Tomography, Optical Coherence ; },
abstract = {PURPOSE: To describe the phenotypes of a newly developed Pde6b-deficient rat model of retinal degeneration.<br><br>METHODS: Pde6b knockout rats were produced by CRISPR-Cpf1 technology. Pde6b knockout rats were evaluated for ocular abnormalities by comparison with wild-type eyes. Eyes were imaged using fundus photography and optical coherence tomography (OCT), stained by hematoxylin and eosin (H&amp;E), and examined by TUNEL assay. Finally, eyes were functionally assessed by electroretinograms (ERGs).<br><br>RESULTS: Pde6b knockout rats exhibited visible photoreceptor degeneration at 3 weeks of postnatal age. The fundus appearance of mutants was notable for pigmentary changes, vascular attenuation with an irregular vascular pattern, and outer retinal thinning, which resembled retinitis pigmentosa (RP) in humans. OCT showed profound retinal thinning in Pde6b knockout rats; the outer nuclear layer (ONL) was significantly thinner in Pde6b knockout rats, with relative preservation of the inner retina at 3 weeks of postnatal age. H&amp;E staining confirmed extensive degeneration of the ONL, beginning at 3 weeks of postnatal age; no ONL remained in the retina by 16 weeks of postnatal age. Retinal sections of Pde6b knockout rats were highly positive for TUNEL, specifically in the ONL. In ERGs, Pde6b knockout rats showed no detectable a- or b-waves at 8 weeks of postnatal age.<br><br>CONCLUSIONS: The Pde6b knockout rat exhibits photoreceptor degeneration. It may provide a better model for experimental therapy for RP because of its slower progression and larger anatomic architecture than the corresponding mouse model. Further studies in this rat model may yield insights into effective therapies for human RP.},
}
@article {pmid31009325,
year = {2019},
author = {Charpentier, E and Elsholz, A and Marchfelder, A},
title = {CRISPR-Cas: more than ten years and still full of mysteries.},
journal = {RNA biology},
volume = {16},
number = {4},
pages = {377-379},
pmid = {31009325},
issn = {1555-8584},
mesh = {Archaea/genetics ; Bacteria/genetics ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; Ecology ; Gene Editing ; Nucleotide Motifs/genetics ; },
}
@article {pmid31009158,
year = {2019},
author = {Rishavy, MA and Hallgren, KW and Zhang, H and Runge, KW and Berkner, KL},
title = {Exon 2 skipping eliminates γ-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency.},
journal = {Journal of thrombosis and haemostasis : JTH},
volume = {17},
number = {7},
pages = {1053-1063},
pmid = {31009158},
issn = {1538-7836},
support = {UL1 RR024989/RR/NCRR NIH HHS/United States ; UL1 TR002548/TR/NCATS NIH HHS/United States ; R01 AG051601/AG/NIA NIH HHS/United States ; R01 HL081093/HL/NHLBI NIH HHS/United States ; R01 HL055666/HL/NHLBI NIH HHS/United States ; },
mesh = {Blood Coagulation/*genetics ; Blood Coagulation Disorders, Inherited/*blood/diagnosis/*genetics ; CRISPR-Cas Systems ; Carbon-Carbon Ligases/*genetics/*metabolism ; *Exons ; Factor IX/metabolism ; Gene Editing ; Genetic Predisposition to Disease ; HEK293 Cells ; Homozygote ; Humans ; *Mutation ; Phenotype ; Protein Processing, Post-Translational ; *RNA Splicing ; },
abstract = {UNLABELLED: Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function.<br><br>BACKGROUND: Mutations in the &#x3b3;-glutamyl carboxylase (GGCX), which is required for vitamin&#xa0;K-dependent (VKD) protein activation, can result in vitamin&#xa0;K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon&#xa0;2 (&#x394;2GGCX). Only &#x394;2GGCX RNA was observed in the patient.<br><br>OBJECTIVES: Loss of exon&#xa0;2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the &#x394;2GGCX mutant is active.<br><br>METHODS: HEK&#xa0;293 cells were edited by the use of CRISPR-Cas9 to eliminate endogenous carboxylase. Recombinant wild-type GGCX and recombinant &#x394;2GGCX were then expressed and tested for carboxylation of the VKD protein factor&#xa0;IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase.<br><br>RESULTS AND CONCLUSIONS: &#x394;2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with &#x394;2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full-length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. &#x394;2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform&#xa0;2; however, &#x394;2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform&#xa0;2 is not physiologically relevant to normal VKD protein carboxylation.},
}
@article {pmid31006810,
year = {2019},
author = {Kounatidou, E and Nakjang, S and McCracken, SRC and Dehm, SM and Robson, CN and Jones, D and Gaughan, L},
title = {A novel CRISPR-engineered prostate cancer cell line defines the AR-V transcriptome and identifies PARP inhibitor sensitivities.},
journal = {Nucleic acids research},
volume = {47},
number = {11},
pages = {5634-5647},
pmid = {31006810},
issn = {1362-4962},
support = {R01 CA174777/CA/NCI NIH HHS/United States ; MR/P009972/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Algorithms ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; DNA Damage ; DNA Repair ; Drug Screening Assays, Antitumor ; Genetic Techniques ; Humans ; Lentivirus ; Male ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Prostatic Neoplasms/*genetics/*metabolism ; Receptors, Androgen/biosynthesis/*genetics ; Sequence Analysis, RNA ; Transcriptome ; },
abstract = {Resistance to androgen receptor (AR)-targeted therapies in prostate cancer (PC) is a major clinical problem. A key mechanism of treatment resistance in advanced PC is the generation of alternatively spliced forms of the AR termed AR variants (AR-Vs) that are refractory to targeted agents and drive tumour progression. Our understanding of how AR-Vs function is limited due to difficulties in distinguishing their discriminate activities from full-length AR (FL-AR). Here we report the development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs. From this, we show that AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitises cells to ionising radiation. Moreover, we demonstrate that AR-Vs interact with PARP1 and PARP2 and are dependent upon their catalytic function for transcriptional activation. Importantly, PARP blockade compromises expression of AR-V-target genes and reduces growth of CRPC cell lines suggesting a synthetic lethality relationship between AR-Vs and PARP, advocating the use of PARP inhibitors in AR-V positive PC.},
}
@article {pmid31006600,
year = {2019},
author = {Bryant, JM and Baumgarten, S and Glover, L and Hutchinson, S and Rachidi, N},
title = {CRISPR in Parasitology: Not Exactly Cut and Dried!.},
journal = {Trends in parasitology},
volume = {35},
number = {6},
pages = {409-422},
doi = {10.1016/j.pt.2019.03.004},
pmid = {31006600},
issn = {1471-5007},
mesh = {Animals ; *CRISPR-Cas Systems ; Congresses as Topic ; Humans ; Parasites/genetics ; Parasitology/*trends ; Research/trends ; },
abstract = {CRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens.},
}
@article {pmid31005787,
year = {2019},
author = {Kobayashi, K and Tsukiyama, T and Nakaya, M and Kageyama, S and Tomita, K and Murai, R and Yoshida, T and Narita, M and Kawauchi, A and Ema, M},
title = {Generation of an OCT3/4 reporter cynomolgus monkey ES cell line using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {37},
number = {},
pages = {101439},
doi = {10.1016/j.scr.2019.101439},
pmid = {31005787},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell Differentiation ; Cell Separation/*methods ; Cells, Cultured ; Embryonic Stem Cells/*cytology/metabolism ; Feeder Cells ; *Gene Editing ; Homologous Recombination ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Macaca fascicularis ; Octamer Transcription Factor-3/antagonists &amp; inhibitors/*genetics ; },
abstract = {Cynomolgus monkey ES (Cyn ES) cells can be generated in a similar manner as human ES cells. However, Cyn ES cells are difficult to maintain in an undifferentiated state by untrained researchers. For easier culture, we generated an OCT3/4-P2A tdTomato IRES Zeocin[R] Cyn ES cell line using CRISPR/Cas9 genome editing technology. The stop codon of the endogenous OCT3/4 locus was replaced with the P2A tdTomato IRES Zeocin[R] pA cassette by homologous recombination. This cell line enables us to isolate pluripotent stem cells and exclude differentiated cells by addition of zeocin, especially for culture without feeder cells.},
}
@article {pmid31005673,
year = {2019},
author = {Pignani, S and Zappaterra, F and Barbon, E and Follenzi, A and Bovolenta, M and Bernardi, F and Branchini, A and Pinotti, M},
title = {Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts.},
journal = {Biochimica et biophysica acta. Gene regulatory mechanisms},
volume = {1862},
number = {6},
pages = {619-624},
doi = {10.1016/j.bbagrm.2019.04.002},
pmid = {31005673},
issn = {1876-4320},
mesh = {Binding Sites ; Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems ; Carcinoma, Hepatocellular ; Endothelial Cells ; Factor VII ; Factor VIII/genetics ; Gene Expression ; Genes, Reporter ; Hep G2 Cells ; Hepatocyte Nuclear Factor 4/genetics ; Humans ; Mutation ; Promoter Regions, Genetic/*genetics ; RNA, Guide ; Receptors, Immunologic/genetics ; Transcription Factors/*genetics ; Transcriptional Activation/*genetics ; },
abstract = {Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.},
}
@article {pmid31005419,
year = {2019},
author = {Seruggia, D and Oti, M and Tripathi, P and Canver, MC and LeBlanc, L and Di Giammartino, DC and Bullen, MJ and Nefzger, CM and Sun, YBY and Farouni, R and Polo, JM and Pinello, L and Apostolou, E and Kim, J and Orkin, SH and Das, PP},
title = {TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network.},
journal = {Molecular cell},
volume = {74},
number = {6},
pages = {1148-1163.e7},
pmid = {31005419},
issn = {1097-4164},
support = {DP2 DA043813/DA/NIDA NIH HHS/United States ; U01 HL100001/HL/NHLBI NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R01 GM112722/GM/NIGMS NIH HHS/United States ; F30 DK103359/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Proliferation ; Cellular Reprogramming ; Embryo, Mammalian ; Embryonic Stem Cells/cytology/*metabolism ; Epigenesis, Genetic ; Fibroblasts/cytology/metabolism ; Gene Editing ; Gene Expression Regulation ; *Gene Regulatory Networks ; HEK293 Cells ; Histones/genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Mice ; Primary Cell Culture ; Protein Isoforms/genetics/metabolism ; Proto-Oncogene Proteins c-myc/*genetics/metabolism ; Signal Transduction ; TATA-Binding Protein Associated Factors/*genetics/metabolism ; },
abstract = {Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.},
}
@article {pmid31004744,
year = {2019},
author = {Couch, T and Murphy, Z and Getman, M and Kurita, R and Nakamura, Y and Steiner, LA},
title = {Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.},
journal = {Experimental hematology},
volume = {74},
number = {},
pages = {19-24.e4},
doi = {10.1016/j.exphem.2019.04.001},
pmid = {31004744},
issn = {1873-2399},
mesh = {CRISPR-Cas Systems ; *Cell Culture Techniques/economics/methods ; *Cell Differentiation ; Cell Line, Transformed ; Erythroblasts/*enzymology ; Gene Editing ; Humans ; K562 Cells ; *Mutation ; *Proto-Oncogene Proteins c-kit/genetics/metabolism ; },
abstract = {A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.},
}
@article {pmid31004551,
year = {2019},
author = {Zhu, C and Zheng, X and Huang, Y and Ye, J and Chen, P and Zhang, C and Zhao, F and Xie, Z and Zhang, S and Wang, N and Li, H and Wang, L and Tang, X and Chai, L and Xu, Q and Deng, X},
title = {Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii).},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2199-2210},
pmid = {31004551},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Citrus/*genetics ; *Gene Editing ; Genome, Plant ; Plants, Genetically Modified ; },
abstract = {Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as 'Mini-Citrus') for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig-N50 2.2 Mb and Scaffold-N50 5.2 Mb) for F. hindsii. In total, 32 257 protein-coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR-modified F. hindsii were predominantly 1-bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.},
}
@article {pmid31004485,
year = {2019},
author = {Jacobsen, T and Liao, C and Beisel, CL},
title = {The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs.},
journal = {FEMS microbiology letters},
volume = {366},
number = {8},
pages = {},
pmid = {31004485},
issn = {1574-6968},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {Acidaminococcus/enzymology/*genetics ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Catalytic Domain ; DNA Cleavage ; Endodeoxyribonucleases/*genetics/metabolism ; Endonucleases/*genetics/metabolism ; Escherichia coli/genetics ; Gene Editing ; HEK293 Cells ; Humans ; *Nucleotide Motifs ; Plasmids/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.},
}
@article {pmid31004423,
year = {2019},
author = {Desgranges, E and Marzi, S and Moreau, K and Romby, P and Caldelari, I},
title = {Noncoding RNA.},
journal = {Microbiology spectrum},
volume = {7},
number = {2},
pages = {},
doi = {10.1128/microbiolspec.GPP3-0038-2018},
pmid = {31004423},
issn = {2165-0497},
mesh = {Animals ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Humans ; RNA, Bacterial/genetics/*metabolism ; RNA, Small Untranslated/*metabolism ; Staphylococcal Infections/microbiology ; Staphylococcus aureus/*genetics/metabolism ; },
abstract = {Regulatory RNAs, present in many bacterial genomes and particularly in pathogenic bacteria such as Staphylococcus aureus, control the expression of genes encoding virulence factors or metabolic proteins. They are extremely diverse and include noncoding RNAs (sRNA), antisense RNAs, and some 5' or 3' untranslated regions of messenger RNAs that act as sensors for metabolites, tRNAs, or environmental conditions (e.g., temperature, pH). In this review we focus on specific examples of sRNAs of S. aureus that illustrate how numerous sRNAs and associated proteins are embedded in complex networks of regulation. In addition, we discuss the CRISPR-Cas systems defined as an RNA-interference-like mechanism, which also exist in staphylococcal strains.},
}
@article {pmid31004275,
year = {2019},
author = {Jiang, M and Hu, H and Kai, J and Traw, MB and Yang, S and Zhang, X},
title = {Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes.},
journal = {Plant molecular biology},
volume = {100},
number = {4-5},
pages = {467-479},
pmid = {31004275},
issn = {1573-5028},
mesh = {CRISPR-Cas Systems ; Gene Knockout Techniques ; Genome, Plant ; Genotype ; Indoleacetic Acids/*metabolism ; Mutation ; Oryza/*genetics/growth &amp; development/metabolism ; Phenotype ; Plant Development/genetics ; Plant Proteins/genetics/metabolism/*physiology ; Plant Roots/genetics/growth &amp; development/metabolism ; Plant Shoots/genetics/growth &amp; development/metabolism ; RNA, Messenger/metabolism ; Signal Transduction ; Transcription Factors/genetics/metabolism/*physiology ; },
abstract = {We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath, mutants with a 13-amino acid deletion showed profoundly greater dwarfing, lateral root developmental disorder, and fertility deficiency, relative to mutants with a single amino acid deletion, demonstrating that those 13 amino acids in Kasalath are essential to gene function. In Wuyunjing24, we predicted that mutants with a single base-pair frameshift insertion would experience premature termination and strong phenotypic defects, but instead these lines exhibited negligible phenotypic difference and normal fertility. Through RNA-seq, we show here that new mosaic transcripts of OsIAA23 were produced de novo, which circumvented the premature termination and thereby preserved the wild-type phenotype. This finding is a notable demonstration in plants that mutants can mask loss of function CRISPR/Cas9 editing of the target gene through de novo changes in alternative splicing.},
}
@article {pmid31004018,
year = {2019},
author = {Patsali, P and Turchiano, G and Papasavva, P and Romito, M and Loucari, CC and Stephanou, C and Christou, S and Sitarou, M and Mussolino, C and Cornu, TI and Antoniou, MN and Lederer, CW and Cathomen, T and Kleanthous, M},
title = {Correction of IVS I-110(G&gt;A) β-thalassemia by CRISPR/Cas-and TALEN-mediated disruption of aberrant regulatory elements in human hematopoietic stem and progenitor cells.},
journal = {Haematologica},
volume = {104},
number = {11},
pages = {e497-e501},
pmid = {31004018},
issn = {1592-8721},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Expression Regulation ; Genetic Therapy ; Hematopoietic Stem Cells/*metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; *Introns ; Mutation ; *Transcription Activator-Like Effector Nucleases ; beta-Globins/*genetics ; beta-Thalassemia/*genetics/therapy ; },
}
@article {pmid31003997,
year = {2019},
author = {Shin, TH and Baek, EJ and Corat, MAF and Chen, S and Metais, JY and AlJanahi, AA and Zhou, Y and Donahue, RE and Yu, KR and Dunbar, CE},
title = {CRISPR/Cas9 PIG -A gene editing in nonhuman primate model demonstrates no intrinsic clonal expansion of PNH HSPCs.},
journal = {Blood},
volume = {133},
number = {23},
pages = {2542-2545},
pmid = {31003997},
issn = {1528-0020},
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Editing/methods ; *Hematopoietic Stem Cells ; Hemoglobinuria, Paroxysmal/*genetics ; Macaca mulatta ; Membrane Proteins/*genetics ; },
}
@article {pmid31003873,
year = {2019},
author = {Barrangou, R and Notebaart, RA},
title = {CRISPR-Directed Microbiome Manipulation across the Food Supply Chain.},
journal = {Trends in microbiology},
volume = {27},
number = {6},
pages = {489-496},
doi = {10.1016/j.tim.2019.03.006},
pmid = {31003873},
issn = {1878-4380},
mesh = {*CRISPR-Cas Systems ; Fermentation ; *Food Chain ; *Food Microbiology ; *Food Supply ; *Gene Editing ; Metabolic Engineering ; *Microbiota ; },
abstract = {The advent of CRISPR-based technologies has revolutionized genetics over the past decade, and genome editing is now widely implemented for diverse medical and agricultural applications, such as correcting genetic disorders and improving crop and livestock breeding. CRISPR-based technologies are also of great potential to alter the genetic content of food bacteria in order to control the composition and activity of microbial populations across the food supply chain, from the farm to consumer products. Advancing the food supply chain is of great societal importance as it involves optimizing fermentation processes to enhance taste and sensory properties of food products, as well as improving food quality and safety by controlling spoilage bacteria and pathogens. Here, we discuss the various CRISPR technologies that can alter bacterial functionalities and modulate the composition of microbial communities in foods. We illustrate how these applications can be harnessed along the food supply chain to manipulate microbiomes that encompass spoilage and pathogenic bacteria as well as desirable starter cultures and health-promoting probiotics.},
}
@article {pmid31002868,
year = {2019},
author = {Satheesh, V and Zhang, H and Wang, X and Lei, M},
title = {Precise editing of plant genomes - Prospects and challenges.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {115-123},
doi = {10.1016/j.semcdb.2019.04.010},
pmid = {31002868},
issn = {1096-3634},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Plant/*genetics ; },
abstract = {The past decade has witnessed unprecedented development in genome engineering, a process that enables targeted modification of genomes. The identification of sequence-specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas system, in particular, has led to precise and efficient introduction of genetic variations into genomes of various organisms. Since the CRISPR/Cas system is highly versatile, cost-effective and much superior to ZFNs and TALENs, its widespread adoption by the research community has been inevitable. In plants, a number of studies have shown that CRISPR/Cas could be a potential tool in basic research where insertion, deletion and/or substitution in the genetic sequence could help answer fundamental questions about plant processes, and in applied research these technologies could help build or reverse-engineer plant systems to make them more useful. In this review article, we summarize technologies for precise editing of genomes with a special focus on the CRISPR/Cas system, highlight the latest developments in the CRISPR/Cas system and discuss the challenges and prospects in using the system for plant biology research.},
}
@article {pmid31002798,
year = {2019},
author = {Lehrbach, NJ and Breen, PC and Ruvkun, G},
title = {Protein Sequence Editing of SKN-1A/Nrf1 by Peptide:N-Glycanase Controls Proteasome Gene Expression.},
journal = {Cell},
volume = {177},
number = {3},
pages = {737-750.e15},
pmid = {31002798},
issn = {1097-4172},
support = {P40 OD010440/OD/NIH HHS/United States ; R01 AG016636/AG/NIA NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Asparagine/metabolism ; Bortezomib/pharmacology ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/chemistry/genetics/*metabolism ; DNA-Binding Proteins/chemistry/genetics/*metabolism ; Endoplasmic Reticulum/metabolism ; Gene Editing ; Gene Expression Regulation/drug effects ; Oxidative Stress ; Proteasome Endopeptidase Complex/genetics/*metabolism ; Protein Subunits/chemistry/genetics/metabolism ; Sequence Alignment ; Transcription Factors/chemistry/genetics/*metabolism ; },
abstract = {The proteasome mediates selective protein degradation and is dynamically regulated in response to proteotoxic challenges. SKN-1A/Nrf1, an endoplasmic reticulum (ER)-associated transcription factor that undergoes N-linked glycosylation, serves as a sensor of proteasome dysfunction and triggers compensatory upregulation of proteasome subunit genes. Here, we show that the PNG-1/NGLY1 peptide:N-glycanase edits the sequence of SKN-1A protein by converting particular N-glycosylated asparagine residues to aspartic acid. Genetically introducing aspartates at these N-glycosylation sites bypasses the requirement for PNG-1/NGLY1, showing that protein sequence editing rather than deglycosylation is key to SKN-1A function. This pathway is required to maintain sufficient proteasome expression and activity, and SKN-1A hyperactivation confers resistance to the proteotoxicity of human amyloid beta peptide. Deglycosylation-dependent protein sequence editing explains how ER-associated and cytosolic isoforms of SKN-1 perform distinct cytoprotective functions corresponding to those of mammalian Nrf1 and Nrf2. Thus, we uncover an unexpected mechanism by which N-linked glycosylation regulates protein function and proteostasis.},
}
@article {pmid31002444,
year = {2019},
author = {Zhu, Y and Lin, Y and Chen, S and Liu, H and Chen, Z and Fan, M and Hu, T and Mei, F and Chen, J and Chen, L and Wang, F},
title = {CRISPR/Cas9-mediated functional recovery of the recessive rc allele to develop red rice.},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2096-2105},
pmid = {31002444},
issn = {1467-7652},
mesh = {*Alleles ; *CRISPR-Cas Systems ; Frameshift Mutation ; Genes, Plant ; Oryza/*genetics ; *Pigmentation ; Sequence Deletion ; },
abstract = {Red rice contains high levels of proanthocyanidins and anthocyanins, which have been recognized as health-promoting nutrients. The red coloration of rice grains is controlled by two complementary genes, Rc and Rd. The RcRd genotype produces red pericarp in wild species Oryza rufipogon, whereas most cultivated rice varieties produce white grains resulted from a 14-bp frame-shift deletion in the seventh exon of the Rc gene. In the present study, we developed a CRISPR/Cas9-mediated method to functionally restore the recessive rc allele through reverting the 14-bp frame-shift deletion to in-frame mutations in which the deletions were in multiples of three bases, and successfully converted three elite white pericarp rice varieties into red ones. Rice seeds from T1 in-frame Rc lines were measured for proanthocyanidins and anthocyanidins, and high accumulation levels of proanthocyanidins and anthocyanidins were observed in red grains from the mutants. Moreover, there was no significant difference between wild-type and in-frame Rc mutants in major agronomic traits, indicating that restoration of Rc function had no negative effect on important agronomic traits in rice. Given that most white pericarp rice varieties are resulted from the 14-bp deletion in Rc, it is conceivable that our method could be applied to most white pericarp rice varieties and would greatly accelerate the breeding of new red rice varieties with elite agronomic traits. In addition, our study demonstrates an effective approach to restore recessive frame-shift alleles for crop improvement.},
}
@article {pmid31001583,
year = {2019},
author = {Cai, Y and Cheng, T and Yao, Y and Li, X and Ma, Y and Li, L and Zhao, H and Bao, J and Zhang, M and Qiu, Z and Xue, T},
title = {In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway.},
journal = {Science advances},
volume = {5},
number = {4},
pages = {eaav3335},
pmid = {31001583},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cyclic Nucleotide Phosphodiesterases, Type 6/genetics ; *DNA Repair ; Electroporation ; Gene Editing/*methods ; Humans ; Mice ; Mice, Inbred C57BL ; RNA, Guide/metabolism ; Rec A Recombinases/*metabolism ; Retinal Degeneration/therapy ; Retinal Rod Photoreceptor Cells/metabolism ; },
abstract = {Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)-based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.},
}
@article {pmid31000663,
year = {2019},
author = {Wienert, B and Wyman, SK and Richardson, CD and Yeh, CD and Akcakaya, P and Porritt, MJ and Morlock, M and Vu, JT and Kazane, KR and Watry, HL and Judge, LM and Conklin, BR and Maresca, M and Corn, JE},
title = {Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6437},
pages = {286-289},
pmid = {31000663},
issn = {1095-9203},
support = {U01 EB029374/EB/NIBIB NIH HHS/United States ; U01 HL145795/HL/NHLBI NIH HHS/United States ; R01 EY028249/EY/NEI NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; DP2 HL141006/HL/NHLBI NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; },
mesh = {Adenoviridae ; Animals ; CRISPR-Associated Protein 9/chemistry/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Chromatin Immunoprecipitation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics ; *DNA Breaks, Double-Stranded ; *DNA Repair ; DNA Repair Enzymes/metabolism ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells ; K562 Cells ; MRE11 Homologue Protein/genetics/*metabolism ; RNA, Guide ; Sequence Analysis, DNA/*methods ; },
abstract = {CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.},
}
@article {pmid31000651,
year = {2019},
author = {Kempton, HR and Qi, LS},
title = {When genome editing goes off-target.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6437},
pages = {234-236},
doi = {10.1126/science.aax1827},
pmid = {31000651},
issn = {1095-9203},
mesh = {Adenine ; CRISPR-Cas Systems ; Cytosine ; *Gene Editing ; Mutation ; *Oryza ; },
}
@article {pmid31000200,
year = {2019},
author = {Xie, J and Li, Q and Zhu, XH and Gao, Y and Zhao, WH},
title = {IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes.},
journal = {Biochemical and biophysical research communications},
volume = {513},
number = {4},
pages = {820-826},
doi = {10.1016/j.bbrc.2019.03.206},
pmid = {31000200},
issn = {1090-2104},
mesh = {Cytokines/*metabolism ; Humans ; Inflammation Mediators/*metabolism ; Lipopolysaccharides/*pharmacology ; Macrophages/drug effects/*metabolism ; Monocytes/drug effects/*metabolism ; NF-kappa B/*metabolism ; RNA, Small Interfering/metabolism ; RNA-Binding Proteins/*metabolism ; THP-1 Cells ; },
abstract = {BACKGROUND: Lipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process.<br><br>METHODS: THP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay.<br><br>RESULTS: In THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-&#x3b1; (TNF-&#x3b1;), interleukin (IL)-1&#x3b2; and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1&#x202f;cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NF&#x3ba;B) complex in nuclei of LPS-treated THP-1&#x202f;cells. Significantly, LPS-induced p65-p52 nuclear translocation and NF&#x3ba;B activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout.<br><br>CONCLUSION: IGF2BP1 promotes LPS-induced NF&#x3ba;B signalling and transcriptional activation in human macrophages and monocytes.},
}
@article {pmid30998900,
year = {2019},
author = {Nussbacher, JK and Tabet, R and Yeo, GW and Lagier-Tourenne, C},
title = {Disruption of RNA Metabolism in Neurological Diseases and Emerging Therapeutic Interventions.},
journal = {Neuron},
volume = {102},
number = {2},
pages = {294-320},
pmid = {30998900},
issn = {1097-4199},
support = {R01 EY029166/EY/NEI NIH HHS/United States ; R01 NS087227/NS/NINDS NIH HHS/United States ; U41 HG009889/HG/NHGRI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; U19 MH107367/MH/NIMH NIH HHS/United States ; T32 CA067754/CA/NCI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Autophagy ; CRISPR-Cas Systems ; Genetic Therapy ; Genetic Vectors ; Homeostasis ; Humans ; Molecular Targeted Therapy ; Nervous System Diseases/genetics/*metabolism/therapy ; Oligoribonucleotides, Antisense/therapeutic use ; Paraneoplastic Syndromes, Nervous System/genetics/metabolism/therapy ; RNA/*metabolism ; RNA Processing, Post-Transcriptional ; RNA Splicing ; RNA Stability ; RNA Transport ; RNA-Binding Proteins/*metabolism ; },
abstract = {RNA binding proteins are critical to the maintenance of the transcriptome via controlled regulation of RNA processing and transport. Alterations of these proteins impact multiple steps of the RNA life cycle resulting in various molecular phenotypes such as aberrant RNA splicing, transport, and stability. Disruption of RNA binding proteins and widespread RNA processing defects are increasingly recognized as critical determinants of neurological diseases. Here, we describe distinct mechanisms by which the homeostasis of RNA binding proteins is compromised in neurological disorders through their reduced expression level, increased propensity to aggregate or sequestration by abnormal RNAs. These mechanisms all converge toward altered neuronal function highlighting the susceptibility of neurons to deleterious changes in RNA expression and the central role of RNA binding proteins in preserving neuronal integrity. Emerging therapeutic approaches to mitigate or reverse alterations of RNA binding proteins in neurological diseases are discussed.},
}
@article {pmid30998719,
year = {2019},
author = {Stevens, RC and Steele, JL and Glover, WR and Sanchez-Garcia, JF and Simpson, SD and O'Rourke, D and Ramsdell, JS and MacManes, MD and Thomas, WK and Shuber, AP},
title = {A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.},
journal = {PloS one},
volume = {14},
number = {4},
pages = {e0215441},
pmid = {30998719},
issn = {1932-6203},
support = {R35 GM128843/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/*genetics ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; *Polymerase Chain Reaction ; RNA, Guide/*genetics ; Sequence Analysis, DNA ; },
abstract = {Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.},
}
@article {pmid30998099,
year = {2019},
author = {Barrangou, R},
title = {Time To Let CRISPR B.E.?.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {67},
doi = {10.1089/crispr.2019.29055.rdb},
pmid = {30998099},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genetic Engineering/*methods ; Humans ; Nucleotides/genetics ; Research/trends ; },
}
@article {pmid30998097,
year = {2019},
author = {Willmann, MR},
title = {Base Editors and Off-Targeting: The Deaminase Matters.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {71-73},
doi = {10.1089/crispr.2019.29054.mrw},
pmid = {30998097},
issn = {2573-1602},
mesh = {Aminohydrolases ; Animals ; CRISPR-Cas Systems ; *Cytosine ; *Gene Editing ; Mice ; Nucleotides ; },
}
@article {pmid30998096,
year = {2019},
author = {Sansbury, BM and Wagner, AM and Tarcic, G and Barth, S and Nitzan, E and Goldfus, R and Vidne, M and Kmiec, EB},
title = {CRISPR-Directed Gene Editing Catalyzes Precise Gene Segment Replacement In Vitro Enabling a Novel Method for Multiplex Site-Directed Mutagenesis.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {121-132},
doi = {10.1089/crispr.2018.0054},
pmid = {30998096},
issn = {2573-1602},
mesh = {Adult ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/methods ; Genetic Therapy/methods ; HEK293 Cells ; Humans ; Mutagenesis/genetics ; Mutagenesis, Site-Directed/*methods ; Mutation/genetics ; Plasmids/genetics ; Polymorphism, Single Nucleotide/genetics ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Much of our understanding of eukaryotic genes function comes from studies of the activity of their mutated forms or allelic variability. Mutations have helped elucidate how members of an intricate pathway function in relation to each other and how they operate in the context of the regulatory circuitry that surrounds them. A PCR-based site-directed mutagenesis technique is often used to engineer these variants. While these tools are efficient, they are not without significant limitations, most notably off-site mutagenesis, limited scalability, and lack of multiplexing capabilities. To overcome many of these limitations, we now describe a novel method for the introduction of both simple and complex gene mutations in plasmid DNA by using in vitro DNA editing. A specifically designed pair of CRISPR-Cas12a ribonucleoprotein complexes are used to execute site-specific double-strand breaks on plasmid DNA, enabling the excision of a defined DNA fragment. Donor DNA replacement is catalyzed by a mammalian cell-free extract through microhomology annealing of short regions of single-stranded DNA complementarity; we term this method CRISPR-directed DNA mutagenesis (CDM). The products of CDM are plasmids bearing precise donor fragments with specific modifications and CDM could be used for mutagenesis in larger constructs such as Bacterial Artificial Chromosome (BACs) or Yeast Artificial Chromosome (YACs). We further show that this reaction can be multiplexed so that product molecules with multiple site-specific mutations and site-specific deletions can be generated in the same in vitro reaction mixture. Importantly, the CDM method produces fewer unintended mutations in the target gene as compared to the standard site-directed mutagenesis assay; CDM produces no unintended mutations throughout the plasmid backbone. Lastly, this system recapitulates the multitude of reactions that take place during CRISPR-directed gene editing in mammalian cells and affords the opportunity to study the mechanism of action of CRISPR-directed gene editing in mammalian cells by visualizing a multitude of genetic products.},
}
@article {pmid30998095,
year = {2019},
author = {Klotz, C},
title = {CRISPR Cinema: Scenes from the Cutting Edge.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {76-78},
doi = {10.1089/crispr.2019.29056.ckl},
pmid = {30998095},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/*ethics ; Humans ; Motion Pictures ; },
}
@article {pmid30998093,
year = {2019},
author = {Abudayyeh, OO and Gootenberg, JS},
title = {Chipping in on Diagnostics.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {69-71},
doi = {10.1089/crispr.2019.29053.oma},
pmid = {30998093},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Dental Porcelain ; Dental Restoration Failure ; *Graphite ; },
}
@article {pmid30998092,
year = {2019},
author = {Yang, B and Yang, L and Chen, J},
title = {Development and Application of Base Editors.},
journal = {The CRISPR journal},
volume = {2},
number = {2},
pages = {91-104},
doi = {10.1089/crispr.2019.0001},
pmid = {30998092},
issn = {2573-1602},
mesh = {APOBEC Deaminases ; Adenine ; Animals ; Bacteria/genetics ; CRISPR-Cas Systems ; Cytosine ; *Gene Editing ; Humans ; Plants/genetics ; },
abstract = {Base editing is emerging as a potent new strategy to achieve precise gene editing. By combining different nucleobase deaminases with Cas9 or Cpf1 proteins, several base editors have recently been developed to achieve targeted base conversions in different genomic contexts. Importantly, base editors have been successfully applied in animals, plants, and bacteria to induce precise substitutions at the single-base level with high efficiency. In this review, we summarize recent progress in the development and application of base editors and discuss some of the future directions of the technology.},
}
@article {pmid30998090,
year = {2019},
author = {Davies, K},
title = {Walk the Line: Debating a Germline Editing Moratorium.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {74-76},
doi = {10.1089/crispr.2019.29052.kda},
pmid = {30998090},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*ethics ; Genetic Engineering/*ethics ; Germ Cells/physiology ; Government Regulation ; Humans ; World Health Organization ; },
}
@article {pmid30998089,
year = {2019},
author = {Trionfini, P and Ciampi, O and Todeschini, M and Ascanelli, C and Longaretti, L and Perico, L and Remuzzi, G and Benigni, A and Tomasoni, S},
title = {CRISPR-Cas9-Mediated Correction of the G189R-PAX2 Mutation in Induced Pluripotent Stem Cells from a Patient with Focal Segmental Glomerulosclerosis.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {108-120},
doi = {10.1089/crispr.2018.0048},
pmid = {30998089},
issn = {2573-1602},
mesh = {Adult ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/methods ; Germ-Line Mutation/genetics ; Glomerulosclerosis, Focal Segmental/*genetics/metabolism/*therapy ; Humans ; Induced Pluripotent Stem Cells/metabolism/physiology ; Kidney Glomerulus/metabolism ; Mutation/genetics ; PAX2 Transcription Factor/analysis/*genetics ; Podocytes/chemistry/metabolism/physiology ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {Focal segmental glomerulosclerosis (FSGS) is defined by focal (involving few glomeruli) and segmental sclerosis of the glomerular tuft that manifests with nephrotic syndrome. Mutations in genes involved in the maintenance of structure and function of podocytes have been found in a minority of these patients. A family with adult-onset autosomal dominant FSGS was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2. Here, we efficiently corrected this point mutation in patient-derived induced pluripotent stem cells (iPSCs) by means of CRISPR-Cas9-based homology-directed repair. The iPSC lines were differentiated into podocytes, which were tested for their motility. Editing the PAX2 p.G189R mutation restored podocyte motility, which was altered in podocytes derived from patient iPSCs.},
}
@article {pmid30998088,
year = {2019},
author = {Torrance, DW},
title = {The Anti-Anti-CRISPR.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {79-80},
doi = {10.1089/crispr.2019.29049.dwt},
pmid = {30998088},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Models, Molecular ; Students ; },
}
@article {pmid30997496,
year = {2019},
author = {Truong, VA and Hsu, MN and Kieu Nguyen, NT and Lin, MW and Shen, CC and Lin, CY and Hu, YC},
title = {CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.},
journal = {Nucleic acids research},
volume = {47},
number = {13},
pages = {e74},
pmid = {30997496},
issn = {1362-4962},
mesh = {Adipogenesis ; Adult Stem Cells/*transplantation ; Animals ; Baculoviridae ; Bone Marrow Transplantation ; Bone Regeneration/*genetics ; CHO Cells ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Chondrogenesis/*genetics ; Cricetulus ; Gene Editing/*methods ; Luminescent Proteins ; *Mesenchymal Stem Cell Transplantation ; PPAR gamma/genetics ; Parietal Bone/injuries/*physiology ; RNA, Guide ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; SOX9 Transcription Factor/genetics ; *Tissue Scaffolds ; *Transcriptional Activation ; Wound Healing/*genetics ; },
abstract = {Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.},
}
@article {pmid30996586,
year = {2019},
author = {Tran, MTN and Khalid, MKNM and Pébay, A and Cook, AL and Liang, HH and Wong, RCB and Craig, JE and Liu, GS and Hung, SS and Hewitt, AW},
title = {Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant.},
journal = {Molecular vision},
volume = {25},
number = {},
pages = {174-182},
pmid = {30996586},
issn = {1090-0535},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Complement Factor H/genetics/metabolism ; Cytosine/metabolism ; Gene Editing/*methods ; Gene Expression ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lac Operon ; Macular Degeneration/genetics/metabolism/pathology ; Mutation ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Thymine/metabolism ; },
abstract = {PURPOSE: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene.<br><br>METHODS: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy.<br><br>RESULTS: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons).<br><br>CONCLUSIONS: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects.},
}
@article {pmid30996265,
year = {2019},
author = {Date, P and Ackermann, P and Furey, C and Fink, IB and Jonas, S and Khokha, MK and Kahle, KT and Deniz, E},
title = {Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {6196},
pmid = {30996265},
issn = {2045-2322},
support = {R01 HD081379/HD/NICHD NIH HHS/United States ; K12 HD001401/HD/NICHD NIH HHS/United States ; R33 HL120783/HL/NHLBI NIH HHS/United States ; UL1 TR001863/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Cerebral Ventricles/pathology ; Cerebrospinal Fluid/*diagnostic imaging ; Cilia/pathology ; Gene Editing/methods ; Humans ; Hydrocephalus/cerebrospinal fluid/diagnostic imaging/*genetics/physiopathology ; *Hydrodynamics ; Membrane Proteins/genetics ; Neural Cell Adhesion Molecule L1/genetics ; Tomography, Optical Coherence/*methods ; Xenopus ; },
abstract = {Cerebrospinal fluid (CSF) flow in the brain ventricles is critical for brain development. Altered CSF flow dynamics have been implicated in congenital hydrocephalus (CH) characterized by the potentially lethal expansion of cerebral ventricles if not treated. CH is the most common neurosurgical indication in children effecting 1 per 1000 infants. Current treatment modalities are limited to antiquated brain surgery techniques, mostly because of our poor understanding of the CH pathophysiology. We lack model systems where the interplay between ependymal cilia, embryonic CSF flow dynamics and brain development can be analyzed in depth. This is in part due to the poor accessibility of the vertebrate ventricular system to in vivo investigation. Here, we show that the genetically tractable frog Xenopus tropicalis, paired with optical coherence tomography imaging, provides new insights into CSF flow dynamics and role of ciliary dysfunction in hydrocephalus pathogenesis. We can visualize CSF flow within the multi-chambered ventricular system and detect multiple distinct polarized CSF flow fields. Using CRISPR/Cas9 gene editing, we modeled human L1CAM and CRB2 mediated aqueductal stenosis. We propose that our high-throughput platform can prove invaluable for testing candidate human CH genes to understand CH pathophysiology.},
}
@article {pmid30996093,
year = {2019},
author = {Luteijn, RD and van Diemen, F and Blomen, VA and Boer, IGJ and Manikam Sadasivam, S and van Kuppevelt, TH and Drexler, I and Brummelkamp, TR and Lebbink, RJ and Wiertz, EJ},
title = {A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.},
journal = {Journal of virology},
volume = {93},
number = {13},
pages = {},
pmid = {30996093},
issn = {1098-5514},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cowpox virus/genetics ; DNA Viruses ; Gene Knockout Techniques ; Genetic Testing ; Golgi Apparatus ; HEK293 Cells ; *Haploidy ; HeLa Cells ; Heparitin Sulfate/*genetics/*isolation &amp; purification/metabolism ; Host Specificity ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; Monkeypox virus/genetics ; N-Acetylglucosaminyltransferases ; Phosphatidylserines/metabolism ; Pinocytosis/*physiology ; Poxviridae/genetics ; Vaccinia/*virology ; Vaccinia virus/*genetics/*metabolism ; Vesicular Transport Proteins/*metabolism ; Virus Attachment ; },
abstract = {Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.},
}
@article {pmid30996089,
year = {2019},
author = {Zhang, W and Yang, F and Zhu, Z and Yang, Y and Wang, Z and Cao, W and Dang, W and Li, L and Mao, R and Liu, Y and Tian, H and Zhang, K and Liu, X and Ma, J and Zheng, H},
title = {Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway.},
journal = {Journal of virology},
volume = {93},
number = {13},
pages = {},
pmid = {30996089},
issn = {1098-5514},
mesh = {Animals ; Antiviral Agents/metabolism/pharmacology ; CRISPR-Cas Systems ; Capsid Proteins/*metabolism ; Cell Line ; Foot-and-Mouth Disease Virus/*drug effects ; Gene Knockout Techniques ; HEK293 Cells ; HSP40 Heat-Shock Proteins/*antagonists &amp; inhibitors/chemistry/genetics/*metabolism ; Host-Pathogen Interactions ; Humans ; Interferon Regulatory Factor-3 ; Interferon-beta/*metabolism ; Lysosomes/*metabolism ; Phosphorylation ; Proteasome Endopeptidase Complex ; Protein Interaction Domains and Motifs ; Signal Transduction/*drug effects ; Viral Proteins/metabolism ; Virus Replication/*drug effects ; },
abstract = {DnaJ heat shock protein family (Hsp40) member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by coimmunoprecipitation and colocalization in FMDV-infected cells. The J domain of DNAJA3 (amino acids 1 to 168) and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss of function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the beta interferon (IFN-β) signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on the IFN-β signaling pathway. Poly(I⋅C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared to that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the host's antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-β signaling pathway.IMPORTANCE This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study is also the first to clarify the mechanism by which VP1 suppressed IFN-β signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-β signaling pathway. These data suggested that DNAJA3 plays an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-β signaling pathway.},
}
@article {pmid30996081,
year = {2019},
author = {Alapati, D and Zacharias, WJ and Hartman, HA and Rossidis, AC and Stratigis, JD and Ahn, NJ and Coons, B and Zhou, S and Li, H and Singh, K and Katzen, J and Tomer, Y and Chadwick, AC and Musunuru, K and Beers, MF and Morrisey, EE and Peranteau, WH},
title = {In utero gene editing for monogenic lung disease.},
journal = {Science translational medicine},
volume = {11},
number = {488},
pages = {},
pmid = {30996081},
issn = {1946-6242},
support = {R01 HL119436/HL/NHLBI NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; T32 HL007586/HL/NHLBI NIH HHS/United States ; U01 HL134745/HL/NHLBI NIH HHS/United States ; R01 HL145408/HL/NHLBI NIH HHS/United States ; K08 HL140178/HL/NHLBI NIH HHS/United States ; I01 BX001176/BX/BLRD VA/United States ; R01 HL132999/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Epithelial Cells/metabolism ; Gene Editing/methods ; Humans ; Lung Diseases/*genetics ; Mice ; Mutation/genetics ; Pulmonary Surfactant-Associated Protein C/genetics ; },
abstract = {Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPC[I73T] mutation. Embryonic expression of Sftpc[I73T] alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant Sftpc[I73T] gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.},
}
@article {pmid30995964,
year = {2019},
author = {Molla, KA and Yang, Y},
title = {CRISPR/Cas-Mediated Base Editing: Technical Considerations and Practical Applications.},
journal = {Trends in biotechnology},
volume = {37},
number = {10},
pages = {1121-1142},
doi = {10.1016/j.tibtech.2019.03.008},
pmid = {30995964},
issn = {1879-3096},
mesh = {Adenine ; Animals ; Base Pairing ; *CRISPR-Cas Systems ; Codon, Terminator ; Cytosine ; Gene Editing/*methods ; Gene Expression ; Genetic Therapy/methods ; Humans ; INDEL Mutation ; Point Mutation ; },
abstract = {Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or RNA without inducing a double-strand DNA break (DSB). A combination of catalytically impaired nuclease variants with different deaminases has yielded diverse base-editing platforms that aim to address the key limitations such as specificity, protospacer adjacent motif (PAM) compatibility, editing window length, bystander editing, and sequence context preference. Because new base editors significantly reduce unintended editing in the genome, they hold great promise for treating genetic diseases and for developing superior agricultural crops. We review here the development of various base editors, assess their technical advantages and limitations, and discuss their broad applications in basic research, medicine, and agriculture.},
}
@article {pmid30995674,
year = {2019},
author = {Grünewald, J and Zhou, R and Garcia, SP and Iyer, S and Lareau, CA and Aryee, MJ and Joung, JK},
title = {Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.},
journal = {Nature},
volume = {569},
number = {7756},
pages = {433-437},
pmid = {30995674},
issn = {1476-4687},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {APOBEC-1 Deaminase/chemistry/genetics/metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cytosine/metabolism ; Deamination ; *Gene Editing ; HEK293 Cells ; Hep G2 Cells ; Humans ; Mutation ; RNA/chemistry/metabolism ; *RNA Editing ; Rats ; Substrate Specificity/*genetics ; Transcriptome/*genetics ; },
abstract = {CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications[1,2]. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex[3,4]. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells[5,6]. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor[7] can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.},
}
@article {pmid30995489,
year = {2019},
author = {MacLeod, G and Bozek, DA and Rajakulendran, N and Monteiro, V and Ahmadi, M and Steinhart, Z and Kushida, MM and Yu, H and Coutinho, FJ and Cavalli, FMG and Restall, I and Hao, X and Hart, T and Luchman, HA and Weiss, S and Dirks, PB and Angers, S},
title = {Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells.},
journal = {Cell reports},
volume = {27},
number = {3},
pages = {971-986.e9},
doi = {10.1016/j.celrep.2019.03.047},
pmid = {30995489},
issn = {2211-1247},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; //CIHR/Canada ; },
mesh = {Animals ; Brain Neoplasms/drug therapy/mortality/*pathology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/genetics ; Endopeptidases/genetics/metabolism ; Endosomal Sorting Complexes Required for Transport/genetics/metabolism ; Female ; Gene Editing/*methods ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Library ; Glioblastoma/drug therapy/mortality/*pathology ; Histone Methyltransferases/metabolism ; Humans ; Mice ; Mice, SCID ; Neoplastic Stem Cells/drug effects/*metabolism ; Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; Survival Analysis ; Temozolomide/*pharmacology/therapeutic use ; Ubiquitin Thiolesterase/genetics/metabolism ; },
abstract = {Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance.},
}
@article {pmid30993331,
year = {2019},
author = {Makarova, KS and Karamycheva, S and Shah, SA and Vestergaard, G and Garrett, RA and Koonin, EV},
title = {Predicted highly derived class 1 CRISPR-Cas system in Haloarchaea containing diverged Cas5 and Cas7 homologs but no CRISPR array.},
journal = {FEMS microbiology letters},
volume = {366},
number = {7},
pages = {},
pmid = {30993331},
issn = {1574-6968},
mesh = {Archaeal Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Archaeal ; Halobacteriaceae/classification/*genetics/metabolism ; Phylogeny ; },
abstract = {Screening of genomic and metagenomic databases for new variants of CRISPR-Cas systems increasingly results in the discovery of derived variants that do not seem to possess the interference capacity and are implicated in functions distinct from adaptive immunity. We describe an extremely derived putative class 1 CRISPR-Cas system that is present in many Halobacteria and consists of distant homologs of the Cas5 and Cas7 protein along with an uncharacterized conserved protein and various nucleases. We hypothesize that, although this system lacks typical CRISPR effectors or a CRISPR array, it functions as a RNA-dependent defense mechanism that, unlike other derived CRISPR-Cas, utilizes alternative nucleases to cleave invader genomes.},
}
@article {pmid30992542,
year = {2019},
author = {Yang, H and Patel, DJ},
title = {CasX: a new and small CRISPR gene-editing protein.},
journal = {Cell research},
volume = {29},
number = {5},
pages = {345-346},
pmid = {30992542},
issn = {1748-7838},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *RNA ; },
}
@article {pmid30991115,
year = {2019},
author = {Nagy, G and Vaz, AG and Szebenyi, C and Takó, M and Tóth, EJ and Csernetics, &#xc1; and Bencsik, O and Szekeres, A and Homa, M and Ayaydin, F and Galgóczy, L and Vágvölgyi, C and Papp, T},
title = {CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {129},
number = {},
pages = {30-39},
doi = {10.1016/j.fgb.2019.04.008},
pmid = {30991115},
issn = {1096-0937},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Endoplasmic Reticulum/*enzymology ; Gene Deletion ; Hydroxymethylglutaryl CoA Reductases/*genetics ; Mevalonic Acid/metabolism ; Microscopy, Fluorescence ; Mucor/*enzymology/*genetics ; Mutation ; },
abstract = {Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the β-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.},
}
@article {pmid30990769,
year = {2019},
author = {Barkau, CL and O'Reilly, D and Rohilla, KJ and Damha, MJ and Gagnon, KT},
title = {Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9.},
journal = {Nucleic acid therapeutics},
volume = {29},
number = {3},
pages = {136-147},
pmid = {30990769},
issn = {2159-3345},
mesh = {CRISPR-Associated Protein 9/antagonists &amp; inhibitors/*genetics/pharmacology ; CRISPR-Cas Systems/drug effects/*genetics ; DNA/drug effects/genetics ; DNA-Binding Proteins/drug effects/*genetics ; *Gene Editing ; Humans ; Nucleotide Motifs/drug effects/genetics ; Oligonucleotides/genetics/pharmacology ; RNA, Guide/adverse effects/genetics/pharmacology ; Streptococcus pyogenes/enzymology ; Tandem Repeat Sequences/drug effects/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.},
}
@article {pmid30988504,
year = {2019},
author = {Kocak, DD and Josephs, EA and Bhandarkar, V and Adkar, SS and Kwon, JB and Gersbach, CA},
title = {Increasing the specificity of CRISPR systems with engineered RNA secondary structures.},
journal = {Nature biotechnology},
volume = {37},
number = {6},
pages = {657-666},
pmid = {30988504},
issn = {1546-1696},
support = {R01 DA036865/DA/NIDA NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; P50 GM098792/GM/NIGMS NIH HHS/United States ; P30 AR066527/AR/NIAMS NIH HHS/United States ; T32 HD040372/HD/NICHD NIH HHS/United States ; DP2 OD008586/OD/NIH HHS/United States ; },
mesh = {Biotechnology/*trends ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Nucleic Acid Conformation ; RNA/chemistry/*genetics ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.},
}
@article {pmid30988386,
year = {2019},
author = {Tsai, JJ and Hsu, WB and Liu, JH and Chang, CW and Tang, TK},
title = {CEP120 interacts with C2CD3 and Talpid3 and is required for centriole appendage assembly and ciliogenesis.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {6037},
pmid = {30988386},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cell Line ; Centrioles/genetics/*metabolism/ultrastructure ; Cilia/genetics/*metabolism/ultrastructure ; Ciliopathies/genetics/metabolism ; Ellis-Van Creveld Syndrome/genetics/metabolism ; Gene Deletion ; HEK293 Cells ; Humans ; Microtubule-Associated Proteins/*metabolism ; Mutation, Missense ; Protein Interaction Maps ; },
abstract = {Centrosomal protein 120 (CEP120) was originally identified as a daughter centriole-enriched protein that participates in centriole elongation. Recent studies showed that CEP120 gene mutations cause complex ciliopathy phenotypes in humans, including Joubert syndrome and Jeune asphyxiating thoracic dystrophy, suggesting that CEP120 plays an additional role in ciliogenesis. To investigate the potential roles of CEP120 in centriole elongation and cilia formation, we knocked out the CEP120 gene in p53-deficient RPE1 cells using the CRISPR/Cas9 editing system, and performed various analyses. We herein report that loss of CEP120 produces short centrioles with no apparent distal and subdistal appendages. CEP120 knockout was also associated with defective centriole elongation, impaired recruitment of C2CD3 and Talpid3 to the distal ends of centrioles, and consequent defects in centriole appendage assembly and cilia formation. Interestingly, wild-type CEP120 interacts with C2CD3 and Talpid3, whereas a disease-associated CEP120 mutant (I975S) has a low affinity for C2CD3 binding and perturbs cilia assembly. Together, our findings reveal a novel role of CEP120 in ciliogenesis by showing that it interacts with C2CD3 and Talpid3 to assemble centriole appendages and by illuminating the molecular mechanism through which the CEP120 (I975S) mutation causes complex ciliopathies.},
}
@article {pmid30988376,
year = {2019},
author = {Pasari, N and Gupta, M and Eqbal, D and Yazdani, SS},
title = {Genome analysis of Paenibacillus polymyxa A18 gives insights into the features associated with its adaptation to the termite gut environment.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {6091},
pmid = {30988376},
issn = {2045-2322},
mesh = {Adaptation, Physiological/*genetics ; Animals ; Bacterial Proteins/genetics/metabolism ; Biofilms ; Cellulase/metabolism ; Enzymes/genetics/metabolism ; Gastrointestinal Microbiome/*physiology ; Genome, Bacterial/*genetics ; Genomics ; Glycoside Hydrolases/metabolism ; Isoptera/*microbiology ; Paenibacillus polymyxa/*physiology ; },
abstract = {Paenibacillus polymyxa A18 was isolated from termite gut and was identified as a potential cellulase and hemicellulase producer in our previous study. Considering that members belonging to genus Paenibacillus are mostly free-living in soil, we investigated here the essential genetic features that helped P. polymyxa A18 to survive in gut environment. Genome sequencing and analysis identified 4608 coding sequences along with several elements of horizontal gene transfer, insertion sequences, transposases and integrated phages, which add to its genetic diversity. Many genes coding for carbohydrate-active enzymes, including the enzymes responsible for woody biomass hydrolysis in termite gut, were identified in P. polymyxa A18 genome. Further, a series of proteins conferring resistance to 11 antibiotics and responsible for production of 4 antibiotics were also found to be encoded, indicating selective advantage for growth and colonization in the gut environment. To further identify genomic regions unique to this strain, a BLAST-based comparative analysis with the sequenced genomes of 47 members belonging to genus Paenibacillus was carried out. Unique regions coding for nucleic acid modifying enzymes like CRISPR/Cas and Type I Restriction-Modification enzymes were identified in P. polymyxa A18 genome suggesting the presence of defense mechanism to combat viral infections in the gut. In addition, genes responsible for the formation of biofilms, such as Type IV pili and adhesins, which might be assisting P. polymyxa A18 in colonizing the gut were also identified in its genome. In situ colonization experiment further confirmed the ability of P. polymyxa A18 to colonize the gut of termite.},
}
@article {pmid30988204,
year = {2019},
author = {Zhang, D and Hurst, T and Duan, D and Chen, SJ},
title = {Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {18},
pages = {8693-8698},
pmid = {30988204},
issn = {1091-6490},
support = {R01 AR069085/AR/NIAMS NIH HHS/United States ; R01 GM063732/GM/NIGMS NIH HHS/United States ; R01 GM117059/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Protein Binding ; RNA, Guide/*chemistry/genetics ; Thermodynamics ; },
abstract = {While CRISPR/Cas9 is a powerful tool in genome engineering, the on-target activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machine-learning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and off-target efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins.},
}
@article {pmid30988165,
year = {2019},
author = {Watanabe, S and Tsuchiya, K and Nishimura, R and Shirasaki, T and Katsukura, N and Hibiya, S and Okamoto, R and Nakamura, T and Watanabe, M},
title = {TP53 Mutation by CRISPR System Enhances the Malignant Potential of Colon Cancer.},
journal = {Molecular cancer research : MCR},
volume = {17},
number = {7},
pages = {1459-1467},
doi = {10.1158/1541-7786.MCR-18-1195},
pmid = {30988165},
issn = {1557-3125},
mesh = {CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Colonic Neoplasms/drug therapy/*genetics/pathology ; Drug Resistance, Neoplasm/genetics ; Exons/genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Lithostathine/*genetics ; Mutation/genetics ; Neoplasm Invasiveness/genetics/pathology ; Neoplastic Stem Cells/metabolism/pathology ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Tumor protein p53 (TP53) mutation is a well-known occurrence at the late phase of carcinogenesis during the adenoma-carcinoma sequence of a sporadic colon cancer. Although numerous reports about clinical information of the patients with colon cancer have suggested that TP53 mutation might be related to various types of malignant potential, the direct effects of this mutation on the malignant potential of colon cancer remain unknown. Notably, no previous report has described a relationship between TP53 mutation and cancer stemness. We therefore aimed to assess the function of a TP53 mutant induced by the CRISPR-Cas9 system in colon cancer cells. In this study, two TP53 mutations, corresponding to exon 3 (TP53E3) and 10 (TP53E10), were generated in LS174T cells derived from a wild-type TP53 human colon cancer via a lentiviral CRISPR-Cas9 system. The loss of function of TP53 resulting from both mutations manifested as resistance to Nutlin3a-induced apoptosis and the downregulation of target genes of TP53. TP53 mutants exhibited an enhanced malignant potential, characterized by accelerated cell growth, invasiveness, chemoresistance, and cancer stemness. Interestingly, TP53E10 but not TP53E3 cells exhibited aberrant transcriptional activity of regenerating family member 1-α (REG1A) and expression of REG1A, resulting in the acquisition of enhanced malignant potential. In conclusion, we demonstrated for the first time that TP53 genomic mutation into human colon cancer cells affects the malignant potential. IMPLICATIONS: These findings suggest that both a loss of function and an aberrant gain of function of TP53 might promote high malignant potentials at the late phase of carcinogenesis in colon cancer.},
}
@article {pmid30988149,
year = {2019},
author = {Sun, Q and Wang, Y and Dong, N and Shen, L and Zhou, H and Hu, Y and Gu, D and Chen, S and Zhang, R and Ji, Q},
title = {Application of CRISPR/Cas9-Based Genome Editing in Studying the Mechanism of Pandrug Resistance in Klebsiella pneumoniae.},
journal = {Antimicrobial agents and chemotherapy},
volume = {63},
number = {7},
pages = {},
pmid = {30988149},
issn = {1098-6596},
mesh = {CRISPR-Cas Systems/genetics ; Colistin/pharmacology ; Gene Editing/*methods ; Klebsiella pneumoniae/*drug effects/genetics ; Tigecycline/pharmacology ; },
abstract = {In this study, a CRISPR/Cas9-mediated genome editing method was used to study the functions of the mgrB, tetA, and ramR genes in mediating colistin and tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). Inactivation of the tetA or ramR gene or the mgrB gene by CRISPR/Cas9 affected bacterial susceptibility to tigecycline or colistin, respectively. This study proved that the CRISPR/Cas9-based genome editing method could be effectively applied to K. pneumoniae and should be further utilized for genetic characterization.},
}
@article {pmid30987660,
year = {2019},
author = {Mou, H and Ozata, DM and Smith, JL and Sheel, A and Kwan, SY and Hough, S and Kucukural, A and Kennedy, Z and Cao, Y and Xue, W},
title = {CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling.},
journal = {Genome medicine},
volume = {11},
number = {1},
pages = {21},
pmid = {30987660},
issn = {1756-994X},
support = {F30 CA239483/CA/NCI NIH HHS/United States ; UL1 TR001453/TR/NCATS NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; S10 OD023540/OD/NIH HHS/United States ; DP2HL137167/HL/NHLBI NIH HHS/United States ; F30 CA232657/CA/NCI NIH HHS/United States ; T32 CA130807/CA/NCI NIH HHS/United States ; 206388/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Actins/genetics ; Animals ; Bile Duct Neoplasms/diagnostic imaging/*genetics/pathology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cholangiocarcinoma/diagnostic imaging/*genetics/pathology ; Gene Knock-In Techniques/*methods ; Genes, Reporter ; Genes, p53 ; *Genes, ras ; Humans ; Mice ; },
abstract = {CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse β-actin, allowing co-expression of reporter genes or oncogenes from the β-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.},
}
@article {pmid30987444,
year = {2019},
author = {Zolotarev, N and Georgiev, P and Maksimenko, O},
title = {Removal of extra sequences with I-SceI in combination with CRISPR/Cas9 technique for precise gene editing in Drosophila.},
journal = {BioTechniques},
volume = {66},
number = {4},
pages = {198-201},
doi = {10.2144/btn-2018-0147},
pmid = {30987444},
issn = {1940-9818},
mesh = {Animals ; *CRISPR-Cas Systems ; Deoxyribonuclease I/genetics ; Drosophila/*genetics ; Drosophila Proteins/genetics ; Female ; Gene Editing/economics/*methods ; Genome, Insect ; Homologous Recombination ; Male ; Recombinases/genetics ; Time Factors ; },
abstract = {The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila. Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recombinase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo. However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I-SceI. The induced homological recombination leads to the removal of the remaining extraneous sequences from the target locus.},
}
@article {pmid30987342,
year = {2019},
author = {Pálinkás, HL and Rácz, GA and Gál, Z and Hoffmann, OI and Tihanyi, G and Róna, G and Gócza, E and Hiripi, L and Vértessy, BG},
title = {CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality.},
journal = {Biomolecules},
volume = {9},
number = {4},
pages = {},
pmid = {30987342},
issn = {2218-273X},
mesh = {Animals ; Blastocyst/metabolism/pathology ; CRISPR-Cas Systems ; Cells, Cultured ; Embryonic Development/*genetics ; *Gene Deletion ; Heterozygote ; Homozygote ; Mice ; Mice, Knockout ; Pyrophosphatases/*genetics/metabolism ; },
abstract = {Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.},
}
@article {pmid30986568,
year = {2019},
author = {Verma, R and Sahu, R and Singh, DD and Egbo, TE},
title = {A CRISPR/Cas9 based polymeric nanoparticles to treat/inhibit microbial infections.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {44-52},
doi = {10.1016/j.semcdb.2019.04.007},
pmid = {30986568},
issn = {1096-3634},
mesh = {Bacterial Infections/*genetics/*therapy ; CRISPR-Cas Systems/*genetics ; *Drug Delivery Systems ; Gene Editing/*methods ; Humans ; Nanomedicine ; Nanoparticles/*administration &amp; dosage ; Polymers/*administration &amp; dosage ; },
abstract = {The latest breakthrough towards the adequate and decisive methods of gene editing tools provided by CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR Associated System), has been repurposed into a tool for genetically engineering eukaryotic cells and now considered as the major innovation in gene-related disorders. Nanotechnology has provided an alternate way to overcome the conventional problems where methods to deliver therapeutic agents have failed. The use of nanotechnology has the potential to safe-side the CRISPR/Cas9 components delivery by using customized polymeric nanoparticles for safety and efficacy. The pairing of two (CRISPR/Cas9 and nanotechnology) has the potential for opening new avenues in therapeutic use. In this review, we will discuss the most recent advances in developing nanoparticle-based CRISPR/Cas9 gene editing cargo delivery with a focus on several polymeric nanoparticles including fabrication proposals to combat microbial infections.},
}
@article {pmid30986258,
year = {2019},
author = {Tormanen, K and Ton, C and Waring, BM and Wang, K and Sütterlin, C},
title = {Function of Golgi-centrosome proximity in RPE-1 cells.},
journal = {PloS one},
volume = {14},
number = {4},
pages = {e0215215},
pmid = {30986258},
issn = {1932-6203},
support = {R01 GM089913/GM/NIGMS NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; },
mesh = {A Kinase Anchor Proteins/genetics/metabolism ; Autoantigens/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Centrosome/*metabolism ; Cytoskeletal Proteins/genetics/metabolism ; Epithelial Cells/cytology/*metabolism ; Gene Deletion ; Golgi Apparatus/genetics/*metabolism ; Humans ; Membrane Proteins/genetics/metabolism ; Retinal Pigment Epithelium/cytology/*metabolism ; },
abstract = {The close physical proximity between the Golgi and the centrosome is a unique feature of mammalian cells that has baffled scientists for years. Several knockdown and overexpression studies have linked the spatial relationship between these two organelles to the control of directional protein transport, directional migration, ciliogenesis and mitotic entry. However, most of these conditions have not only separated these two organelles, but also caused extensive fragmentation of the Golgi, making it difficult to dissect the specific contribution of Golgi-centrosome proximity. In this study, we present our results with stable retinal pigment epithelial (RPE-1) cell lines in which GM130 was knocked out using a CRISPR/Cas9 approach. While Golgi and centrosome organization appeared mostly intact in cells lacking GM130, there was a clear separation of these organelles from each other. We show that GM130 may control Golgi-centrosome proximity by anchoring AKAP450 to the Golgi. We also provide evidence that the physical proximity between these two organelles is dispensable for protein transport, cell migration, and ciliogenesis. These results suggest that Golgi-centrosome proximity per se is not necessary for the normal function of RPE-1 cells.},
}
@article {pmid30983484,
year = {2019},
author = {Cai, J and Huang, S and Yi, Y and Bao, S},
title = {Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells.},
journal = {The Journal of international medical research},
volume = {47},
number = {5},
pages = {2199-2206},
pmid = {30983484},
issn = {1473-2300},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Endometrial Neoplasms/*genetics ; Female ; Gene Expression Regulation, Neoplastic ; *Gene Knockout Techniques ; Humans ; *Microbubbles ; RNA, Guide/metabolism ; RNA, Messenger/genetics/metabolism ; Receptor, ErbB-2/*metabolism ; *Ultrasonics ; },
abstract = {OBJECTIVE: Epidermal growth factor receptor 2 (C-erbB-2) is one of the most frequently mutated oncogenes in human tumors. We aimed to evaluate the knockout efficiency of clustered regularly interspaced short palindromic repeat (CRISPR) technology using ultrasound microbubble transfection to target C-erbB-2 in human endometrial cancer (HEC)-1A cells.<br><br>METHODS: Three single guide RNAs (sgRNAs) targeting C-erbB-2 were designed and used to construct CRISPR/CRISPR-associated (Cas)9-C-erbB-2 plasmids. The constructed plasmids were transfected into HEC-1A cells using ultrasound microbubbles. C-erbB-2 knockout cloned cells were identified by green fluorescence. C-erbB-2 mRNA and protein expression was measured by reverse transcription (RT)-PCR and western blotting, respectively.<br><br>RESULTS: RT-PCR showed that C-erbB-2 mRNA expression was significantly lower in sgRNA1-transfected cells (0.57&#x2009;&#xb1;&#x2009;0.06) than in blank (1.00&#x2009;&#xb1;&#x2009;0.09) and negative-control groups (1.02&#x2009;&#xb1;&#x2009;0.12). Western blotting revealed C-erbB-2 protein expression to be significantly lower in sgRNA1-transfected cells (0.269&#x2009;&#xb1;&#x2009;0.033) than in blank (0.495&#x2009;&#xb1;&#x2009;0.059) and negative-control groups (1.243&#x2009;&#xb1;&#x2009;0.281). However, there was no significant difference in C-erbB-2 protein and mRNA expression in sgRNA2- and sgRNA3-transfected cells compared with controls.<br><br>CONCLUSION: Ultrasound microbubbles can mediate plasmid transfer into HEC-1A cells to interfere with gene expression and knockout C-erbB-2.},
}
@article {pmid30982901,
year = {2019},
author = {Chiang, HC and Zhang, X and Li, J and Zhao, X and Chen, J and Wang, HT and Jatoi, I and Brenner, A and Hu, Y and Li, R},
title = {BRCA1-associated R-loop affects transcription and differentiation in breast luminal epithelial cells.},
journal = {Nucleic acids research},
volume = {47},
number = {10},
pages = {5086-5099},
pmid = {30982901},
issn = {1362-4962},
support = {R01 CA212674/CA/NCI NIH HHS/United States ; R01 CA220578/CA/NCI NIH HHS/United States ; T32 CA148724/CA/NCI NIH HHS/United States ; },
mesh = {BRCA1 Protein/*genetics/metabolism ; Breast/*metabolism ; CRISPR-Cas Systems ; Carcinogenesis ; Cell Differentiation ; *Enhancer Elements, Genetic ; Epithelial Cells/*metabolism ; Estrogen Receptor alpha/genetics ; Female ; Gene Deletion ; *Gene Expression Regulation, Neoplastic ; Genes, BRCA1 ; HEK293 Cells ; Heterozygote ; Humans ; MCF-7 Cells ; Mutation ; Transcription, Genetic ; },
abstract = {BRCA1-associated basal-like breast cancer originates from luminal progenitor cells. Breast epithelial cells from cancer-free BRCA1 mutation carriers are defective in luminal differentiation. However, how BRCA1 deficiency leads to lineage-specific differentiation defect is not clear. BRCA1 is implicated in resolving R-loops, DNA-RNA hybrid structures associated with genome instability and transcriptional regulation. We recently showed that R-loops are preferentially accumulated in breast luminal epithelial cells of BRCA1 mutation carriers. Here, we interrogate the impact of a BRCA1 mutation-associated R-loop located in a putative transcriptional enhancer upstream of the ERα-encoding ESR1 gene. Genetic ablation confirms the relevance of this R-loop-containing region to enhancer-promoter interactions and transcriptional activation of the corresponding neighboring genes, including ESR1, CCDC170 and RMND1. BRCA1 knockdown in ERα+ luminal breast cancer cells increases intensity of this R-loop and reduces transcription of its neighboring genes. The deleterious effect of BRCA1 depletion on transcription is mitigated by ectopic expression of R-loop-removing RNase H1. Furthermore, RNase H1 overexpression in primary breast cells from BRCA1 mutation carriers results in a shift from luminal progenitor cells to mature luminal cells. Our findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation, which could in turn suppress BRCA1-associated tumorigenesis.},
}
@article {pmid30982889,
year = {2019},
author = {Hoffmann, MD and Aschenbrenner, S and Grosse, S and Rapti, K and Domenger, C and Fakhiri, J and Mastel, M and Börner, K and Eils, R and Grimm, D and Niopek, D},
title = {Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.},
journal = {Nucleic acids research},
volume = {47},
number = {13},
pages = {e75},
pmid = {30982889},
issn = {1362-4962},
mesh = {3' Untranslated Regions/genetics ; Binding Sites ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/biosynthesis/*genetics ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Enzyme Activation ; Enzyme Induction ; Gene Editing/*methods ; *Gene Expression Regulation ; Genes, Reporter ; HEK293 Cells ; HeLa Cells ; Hepatocytes/metabolism ; Humans ; Luciferases, Renilla/analysis/genetics ; MicroRNAs ; Myocytes, Cardiac/metabolism ; Organ Specificity ; Protein Isoforms/antagonists &amp; inhibitors ; *Transgenes ; },
abstract = {The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.},
}
@article {pmid30982114,
year = {2019},
author = {Abdelaal, AS and Jawed, K and Yazdani, SS},
title = {CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {46},
number = {7},
pages = {965-975},
pmid = {30982114},
issn = {1476-5535},
mesh = {1-Butanol/*metabolism ; Bioreactors ; CRISPR-Cas Systems ; Escherichia coli/genetics/*metabolism ; Ethanol/metabolism ; Fermentation ; Glucose/metabolism ; Metabolic Engineering ; Operon ; Plasmids ; Xylose/*metabolism ; },
abstract = {Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1-xylose medium.},
}
@article {pmid30981667,
year = {2019},
author = {Zulkefli, KL and Houghton, FJ and Gosavi, P and Gleeson, PA},
title = {A role for Rab11 in the homeostasis of the endosome-lysosomal pathway.},
journal = {Experimental cell research},
volume = {380},
number = {1},
pages = {55-68},
doi = {10.1016/j.yexcr.2019.04.010},
pmid = {30981667},
issn = {1090-2422},
mesh = {CRISPR-Cas Systems/genetics ; Endosomes/*genetics ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Lysosomes/*genetics ; Receptor, IGF Type 2/genetics ; Signal Transduction/genetics ; rab GTP-Binding Proteins/*genetics ; },
abstract = {The small GTPases Rab11a and 11b are key regulators of membrane transport, localised to the recycling endosomes and also early endosomes. The function of Rab11 within the recycling pathway has been well defined, however, the role of Rab11 at the early endosomes remains poorly characterised. Here, we have generated HeLa cell lines devoid of either Rab11a or Rab11b using CRISPR/Cas9 to functionally dissect the roles of these two Rab11 family members in recycling and in the endosomal-lysosomal system. Both Rab11a and Rab11b contribute to the dynamics of tubulation arising from recycling endosomes whereas Rab11a has the major role in recycling of transferrin receptor. Deletion of either Rab11a or Rab11b resulted in the formation of enlarged early endosomes and perturbation of the endosomal-lysosomal pathway. Strikingly, Rab11a knock-out cells showed an increased density of functional late endosomes/lysosomes as well as lysotracker-positive organelles which were primarily concentrated in a perinuclear location, indicating that the homeostasis of the endosome/lysosome pathway had been perturbed. Moreover, in Rab11a knockout cells there was a functional defect in the intracellular recycling of the cation-independent mannose 6-phosphate receptor (CI-M6PR) between the late endosomes and the TGN, a defect associated with enhanced degradation of CI-M6PR. Expression of wild-type Rab11a in Rab11a knockout cells rescued the late endosome/lysosome phenotype. Overall, these results indicate that Rab11a and Rab11b have overlapping and distinct functions and that Rab11a, unexpectedly, plays a central role in the homeostasis of endosomal-lysosomal biogenesis.},
}
@article {pmid30981084,
year = {2019},
author = {Zhang, J and Liu, J and Yang, W and Cui, M and Dai, B and Dong, Y and Yang, J and Zhang, X and Liu, D and Liang, H and Cang, M},
title = {Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats.},
journal = {Theriogenology},
volume = {132},
number = {},
pages = {1-11},
doi = {10.1016/j.theriogenology.2019.03.029},
pmid = {30981084},
issn = {1879-3231},
mesh = {Animals ; *Animals, Genetically Modified ; Base Sequence ; *CRISPR-Cas Systems ; Cloning, Organism ; DNA/genetics ; Embryo, Mammalian ; *Gene Deletion ; Gene Editing ; Genetic Engineering ; Goats/embryology/*genetics ; Myostatin/*genetics ; *Transcription Activator-Like Effector Nucleases ; },
abstract = {The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN[-/-] mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.},
}
@article {pmid30980304,
year = {2019},
author = {Beneke, T and Gluenz, E},
title = {LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1971},
number = {},
pages = {189-210},
doi = {10.1007/978-1-4939-9210-2_9},
pmid = {30980304},
issn = {1940-6029},
support = {15/16_MSD_836338//Medical Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Homologous Recombination ; Leishmania/*genetics ; Plasmids/genetics ; Transfection ; },
abstract = {Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform (LeishGEdit.net) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.},
}
@article {pmid30980303,
year = {2019},
author = {Zirpel, H and Clos, J},
title = {Gene Replacement by Homologous Recombination.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1971},
number = {},
pages = {169-188},
doi = {10.1007/978-1-4939-9210-2_8},
pmid = {30980303},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Targeting ; *Homologous Recombination ; Leishmania/*genetics ; },
abstract = {While homologous recombination-based gene replacement is about to be supplanted by more modern approaches, it is still retaining usefulness for genes that prove to be poor targets for CRISPR/cas-based approaches. Homologous recombination has proven to be relatively robust to minor sequence mismatches between GOI-flanking sequences and the gene replacement constructs, and the faithfulness of recombination events is easily verified by whole-genome sequencing. Moreover, the availability of custom synthetic gene production by numerous service providers should allow for a relatively quick generation of null mutants without the need to introduce additional protein-coding genes beyond the selection markers.},
}
@article {pmid30980103,
year = {2019},
author = {Zhai, Y and Cai, S and Hu, L and Yang, Y and Amoo, O and Fan, C and Zhou, Y},
title = {CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {132},
number = {7},
pages = {2111-2123},
pmid = {30980103},
issn = {1432-2242},
mesh = {Alleles ; Basic Helix-Loop-Helix Transcription Factors/*genetics ; Brassica napus/*genetics ; *CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Genes, Plant ; Phenotype ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Seeds/*physiology ; },
abstract = {The INDEHISCENT (IND) and ALCATRAZ (ALC) gene homologues have been reported to be essential for dehiscence of fruits in Brassica species. But their functions for pod shatter resistance in Brassica napus, an important oil crops, are not well understood. Here, we assessed the functions of these two genes in rapeseed using CRISPR/Cas9 technology. The induced mutations were stably transmitted to successive generations, and a variety of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The results showed that the function of BnIND gene is essential for pod shatter and highly conserved in Brassica species, whereas the BnALC gene appears to have limited potential for rapeseed shatter resistance. The homoeologous copies of the BnIND gene have partially redundant roles in rapeseed pod shatter, with BnA03.IND exhibiting higher contributions than BnC03.IND. Analysis of data obtained from the gene expression and sequence variations of gene copies revealed that cis-regulatory divergences alter gene expression and underlie the functional differentiation of BnIND homologues. Collectively, our results generate valuable resources for rapeseed breeding programs, and more importantly provide a strategy to improve polyploid crops.},
}
@article {pmid30979834,
year = {2019},
author = {Wang, T and Wang, M and Zhang, Q and Cao, S and Li, X and Qi, Z and Tan, Y and You, Y and Bi, Y and Song, Y and Yang, R and Du, Z},
title = {Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {12},
pages = {},
pmid = {30979834},
issn = {1098-5336},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression ; Gene Knockdown Techniques/*methods ; Streptococcus pyogenes ; Yersinia pestis/*genetics ; },
abstract = {Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.},
}
@article {pmid30979813,
year = {2019},
author = {Hunter, FW and Devaux, JBL and Meng, F and Hong, CR and Khan, A and Tsai, P and Ketela, TW and Sharma, I and Kakadia, PM and Marastoni, S and Shalev, Z and Hickey, AJR and Print, CG and Bohlander, SK and Hart, CP and Wouters, BG and Wilson, WR},
title = {Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.},
journal = {Molecular pharmacology},
volume = {95},
number = {6},
pages = {638-651},
doi = {10.1124/mol.118.115196},
pmid = {30979813},
issn = {1521-0111},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Electron Transport/*drug effects ; Gene Expression Regulation/drug effects ; Gene Regulatory Networks/drug effects ; HCT116 Cells ; Humans ; Mitochondria/drug effects/*genetics ; Neoplasms/drug therapy/*genetics ; Nitroimidazoles/*pharmacology ; Phosphoramide Mustards/*pharmacology ; Prodrugs ; RNA, Small Interfering/pharmacology ; Sequence Analysis, RNA/*methods ; },
abstract = {Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ [0] (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ [0] cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.},
}
@article {pmid30978509,
year = {2019},
author = {Chin, JS and Chooi, WH and Wang, H and Ong, W and Leong, KW and Chew, SY},
title = {Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing.},
journal = {Acta biomaterialia},
volume = {90},
number = {},
pages = {60-70},
doi = {10.1016/j.actbio.2019.04.020},
pmid = {30978509},
issn = {1878-7568},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing ; *Gene Transfer Techniques ; Humans ; Indoles/chemistry/pharmacology ; Nanofibers/chemistry ; Polymers/chemistry/pharmacology ; },
abstract = {Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we adsorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration. STATEMENT OF SIGNIFICANCE: Currently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration.},
}
@article {pmid30978482,
year = {2019},
author = {Armario Najera, V and Twyman, RM and Christou, P and Zhu, C},
title = {Applications of multiplex genome editing in higher plants.},
journal = {Current opinion in biotechnology},
volume = {59},
number = {},
pages = {93-102},
doi = {10.1016/j.copbio.2019.02.015},
pmid = {30978482},
issn = {1879-0429},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; Plants ; Plants, Genetically Modified ; RNA, Guide ; },
abstract = {Multiplex genome editing involves the simultaneous targeting of multiple related or unrelated targets. The latter is most straightforward using the CRISPR/Cas9 system because multiple gRNAs can be delivered either as independent expression cassettes with their own promoters or as polycistronic transcripts processed into mature gRNAs by endogenous or introduced nucleases. Multiplex genome editing in plants initially focused on input traits such as herbicide resistance, but has recently expanded to include hormone biosynthesis and perception, metabolic engineering, plant development and molecular farming, with more than 100 simultaneous targeting events reported. Usually the coding region is targeted but recent examples also include promoter modifications to generate mutants with varying levels of gene expression.},
}
@article {pmid30978004,
year = {2019},
author = {Tovell, H and Testa, A and Maniaci, C and Zhou, H and Prescott, AR and Macartney, T and Ciulli, A and Alessi, DR},
title = {Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader.},
journal = {ACS chemical biology},
volume = {14},
number = {5},
pages = {882-892},
pmid = {30978004},
issn = {1554-8937},
support = {MC_U127015387/MRC_/Medical Research Council/United Kingdom ; MC_UU_00018/1/MRC_/Medical Research Council/United Kingdom ; MC_UU_12016/2/MRC_/Medical Research Council/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Class III Phosphatidylinositol 3-Kinases/genetics ; Endosomes/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques ; HEK293 Cells ; Humans ; Protein Serine-Threonine Kinases/genetics ; Proteins/*metabolism ; },
abstract = {Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC50 of 3-10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (Dmax of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.},
}
@article {pmid30976796,
year = {2019},
author = {Zhang, H and Dong, C and Li, L and Wasney, GA and Min, J},
title = {Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {47},
number = {10},
pages = {5420-5428},
pmid = {30976796},
issn = {1362-4962},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; Gene Editing ; Protein Domains ; Protein Structure, Secondary ; RNA/genetics ; RNA, Bacterial/metabolism ; RNA, Guide/metabolism ; Ruminococcus/*metabolism ; },
abstract = {The Type VI-D CRISPR-Cas system employs an RNA-guided RNase Cas13d with minimal targeting constraints to combat viral infections. This CRISPR system contains RspWYL1 as a unique accessory protein that plays a key role in boosting its effector function on target RNAs, but the mechanism behind this RspWYL1-mediated stimulation remains completely unexplored. Through structural and biophysical approaches, we reveal that the full-length RspWYL1 possesses a novel three-domain architecture and preferentially binds ssRNA with high affinity. Specifically, the N-terminus of RspWYL1 harbors a ribbon-helix-helix motif reminiscent of transcriptional regulators; the central WYL domain of RspWYL1 displays a Sm-like β-barrel fold; and the C-terminal domain of RspWYL1 primarily contributes to the dimerization of RspWYL1 and may regulate the RspWYL1 function via a large conformational change. Collectively, this study provides a first glimpse into the complex mechanism behind the RspWYL1-dictated boosting of target ssRNA cleavage in the Type VI-D CRISPR-Cas system.},
}
@article {pmid30976097,
year = {2019},
author = {Marcon, A and Master, Z and Ravitsky, V and Caulfield, T},
title = {CRISPR in the North American popular press.},
journal = {Genetics in medicine : official journal of the American College of Medical Genetics},
volume = {21},
number = {10},
pages = {2184-2189},
pmid = {30976097},
issn = {1530-0366},
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; Gene Editing/*ethics/*trends ; Humans ; North America ; },
abstract = {PURPOSE: CRISPR is often called one of the century's most important discoveries and is commonly discussed in terms of its momentous potential impacts. This study analyzed how CRISPR is discussed in the North American popular press, including how it is defined, and which benefits and risks/concerns are attributed to the technology.<br><br>METHODS: Using the Factiva database, we identified 228 relevant, nonduplicated articles containing either "CRISPR" or "C.R.I.S.P.R.," published in popular US and Canadian news sources between 1 January 2012 and 12 July 2017. Content analysis was performed on the articles.<br><br>RESULTS: CRISPR is most often discussed in the context of human health (83.8%), compared with animals (26.3%) and plants (20.6%). Nearly all articles (96.1%) presented CRISPR's potential benefits; 61.4% of articles presented CRISPR-related risks/concerns, the vast majority of which focused on the uncertainty surrounding CRISPR, specifically with respect to germline modifications.<br><br>CONCLUSIONS: Overall, the discourse suggests a strong promotion of CRISPR, but an element of caution is also evident. Technical as well as ethical, legal, and social risks/concerns play a prominent role. This media portrayal of CRISPR might help facilitate more sophisticated and balanced policy responses, where the scientific potential of the technology is highlighted alongside broader social considerations.},
}
@article {pmid30975893,
year = {2019},
author = {Hartweger, H and McGuire, AT and Horning, M and Taylor, JJ and Dosenovic, P and Yost, D and Gazumyan, A and Seaman, MS and Stamatatos, L and Jankovic, M and Nussenzweig, MC},
title = {HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells.},
journal = {The Journal of experimental medicine},
volume = {216},
number = {6},
pages = {1301-1310},
pmid = {30975893},
issn = {1540-9538},
support = {P01 AI138212/AI/NIAID NIH HHS/United States ; R01 AI129795/AI/NIAID NIH HHS/United States ; UM1 AI100663/AI/NIAID NIH HHS/United States ; },
mesh = {Adoptive Transfer ; Animals ; Antibodies, Neutralizing/immunology ; B-Lymphocytes/*immunology ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; HIV Antibodies/immunology ; HIV Infections/*immunology ; Humans ; INDEL Mutation/genetics ; *Immunity, Humoral ; Mice ; Protein Engineering ; },
abstract = {A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.},
}
@article {pmid30975639,
year = {2019},
author = {Wang, L and Yang, Y and Breton, CA and White, J and Zhang, J and Che, Y and Saveliev, A and McMenamin, D and He, Z and Latshaw, C and Li, M and Wilson, JM},
title = {CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX-knockout mice.},
journal = {Blood},
volume = {133},
number = {26},
pages = {2745-2752},
doi = {10.1182/blood.2019000790},
pmid = {30975639},
issn = {1528-0020},
mesh = {Animals ; Animals, Newborn ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Factor IX/*genetics ; Gene Targeting/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Hemophilia B/*genetics ; Hemostasis/*genetics ; Humans ; Mice ; Mice, Knockout ; },
abstract = {Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.},
}
@article {pmid30975459,
year = {2019},
author = {Dolan, AE and Hou, Z and Xiao, Y and Gramelspacher, MJ and Heo, J and Howden, SE and Freddolino, PL and Ke, A and Zhang, Y},
title = {Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.},
journal = {Molecular cell},
volume = {74},
number = {5},
pages = {936-950.e5},
pmid = {30975459},
issn = {1097-4164},
support = {R00 GM117268/GM/NIGMS NIH HHS/United States ; R01 GM102543/GM/NIGMS NIH HHS/United States ; R35 GM118174/GM/NIGMS NIH HHS/United States ; K99 GM117268/GM/NIGMS NIH HHS/United States ; R35 GM128637/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Endonucleases/chemistry/genetics ; Escherichia coli/genetics ; Gene Editing/methods ; Genome, Human/genetics ; Genomics ; *Human Embryonic Stem Cells ; Humans ; RNA, Guide/*genetics ; Ribonucleoproteins/genetics ; Sequence Deletion/*genetics ; },
abstract = {CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.},
}
@article {pmid30974317,
year = {2019},
author = {Mair, B and Moffat, J and Boone, C and Andrews, BJ},
title = {Genetic interaction networks in cancer cells.},
journal = {Current opinion in genetics &amp; development},
volume = {54},
number = {},
pages = {64-72},
pmid = {30974317},
issn = {1879-0380},
support = {R01 HG005853/HG/NHGRI NIH HHS/United States ; //CIHR/Canada ; },
mesh = {CRISPR-Cas Systems/genetics ; *Epistasis, Genetic ; Gene Editing ; Gene Regulatory Networks/*genetics ; *Genetic Association Studies ; Genotype ; Humans ; Neoplasms/*genetics ; Phenotype ; },
abstract = {The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer.},
}
@article {pmid30973754,
year = {2019},
author = {Chen, G and Ribeiro, CMP and Sun, L and Okuda, K and Kato, T and Gilmore, RC and Martino, MB and Dang, H and Abzhanova, A and Lin, JM and Hull-Ryde, EA and Volmer, AS and Randell, SH and Livraghi-Butrico, A and Deng, Y and Scherer, PE and Stripp, BR and O'Neal, WK and Boucher, RC},
title = {XBP1S Regulates MUC5B in a Promoter Variant-Dependent Pathway in Idiopathic Pulmonary Fibrosis Airway Epithelia.},
journal = {American journal of respiratory and critical care medicine},
volume = {200},
number = {2},
pages = {220-234},
pmid = {30973754},
issn = {1535-4970},
support = {P01 HL108808/HL/NHLBI NIH HHS/United States ; P01 HL110873/HL/NHLBI NIH HHS/United States ; P30 DK065988/DK/NIDDK NIH HHS/United States ; UH3 HL123645/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Endoplasmic Reticulum Stress/genetics ; Endoribonucleases/*genetics/metabolism ; Gene Expression Regulation ; Humans ; Idiopathic Pulmonary Fibrosis/*genetics/metabolism ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Transgenic ; Polymorphism, Genetic ; Primary Cell Culture ; Promoter Regions, Genetic ; Protein Serine-Threonine Kinases/*genetics/metabolism ; Respiratory Mucosa/*metabolism ; X-Box Binding Protein 1/*genetics/metabolism ; },
abstract = {Rationale: The goal was to connect elements of idiopathic pulmonary fibrosis (IPF) pathogenesis, including chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling, distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B (mucin 5B), and associations between IPF risk and polymorphisms in the MUC5B promoter. Objectives: To test whether the endoplasmic reticulum (ER) stress sensor protein ERN2 (ER-to-nucleus signaling 2) and its downstream effector, the spliced form of XBP1S (X-box-binding protein 1), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia. Methods: Primary human airway epithelial (HAE) cells, transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE cells to explore therapeutic potential. Measurements and Main Results: ERN2 regulated MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially upregulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE cells was inhibited by KIRA6 and XBP1 CRISPR-Cas9. Conclusions: A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing an unfolded protein response-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF.},
}
@article {pmid30972865,
year = {2019},
author = {Liu, Y and Wang, Y and Xu, S and Tang, X and Zhao, J and Yu, C and He, G and Xu, H and Wang, S and Tang, Y and Fu, C and Ma, Y and Zhou, G},
title = {Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2143-2152},
pmid = {30972865},
issn = {1467-7652},
mesh = {Agrobacterium ; Araceae/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Transformation, Genetic ; },
abstract = {The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium-mediated transformation to 5-6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9-mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9-mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.},
}
@article {pmid30972412,
year = {2020},
author = {Zeeshan, S and Xiong, R and Liang, BT and Ahmed, Z},
title = {100 Years of evolving gene-disease complexities and scientific debutants.},
journal = {Briefings in bioinformatics},
volume = {21},
number = {3},
pages = {885-905},
doi = {10.1093/bib/bbz038},
pmid = {30972412},
issn = {1477-4054},
mesh = {CRISPR-Cas Systems ; Computational Biology/methods ; Databases, Genetic ; Genetic Diseases, Inborn/*genetics ; *Genetic Predisposition to Disease ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Molecular Sequence Annotation ; },
abstract = {It's been over 100 years since the word `gene' is around and progressively evolving in several scientific directions. Time-to-time technological advancements have heavily revolutionized the field of genomics, especially when it's about, e.g. triple code development, gene number proposition, genetic mapping, data banks, gene-disease maps, catalogs of human genes and genetic disorders, CRISPR/Cas9, big data and next generation sequencing, etc. In this manuscript, we present the progress of genomics from pea plant genetics to the human genome project and highlight the molecular, technical and computational developments. Studying genome and epigenome led to the fundamentals of development and progression of human diseases, which includes chromosomal, monogenic, multifactorial and mitochondrial diseases. World Health Organization has classified, standardized and maintained all human diseases, when many academic and commercial online systems are sharing information about genes and linking to associated diseases. To efficiently fathom the wealth of this biological data, there is a crucial need to generate appropriate gene annotation repositories and resources. Our focus has been how many gene-disease databases are available worldwide and which sources are authentic, timely updated and recommended for research and clinical purposes. In this manuscript, we have discussed and compared 43 such databases and bioinformatics applications, which enable users to connect, explore and, if possible, download gene-disease data.},
}
@article {pmid30971826,
year = {2019},
author = {Behan, FM and Iorio, F and Picco, G and Gonçalves, E and Beaver, CM and Migliardi, G and Santos, R and Rao, Y and Sassi, F and Pinnelli, M and Ansari, R and Harper, S and Jackson, DA and McRae, R and Pooley, R and Wilkinson, P and van der Meer, D and Dow, D and Buser-Doepner, C and Bertotti, A and Trusolino, L and Stronach, EA and Saez-Rodriguez, J and Yusa, K and Garnett, MJ},
title = {Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.},
journal = {Nature},
volume = {568},
number = {7753},
pages = {511-516},
pmid = {30971826},
issn = {1476-4687},
mesh = {Animals ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Drug Discovery/*methods ; Female ; *Gene Editing ; Genome, Human/genetics ; Humans ; Mice ; Microsatellite Instability ; Molecular Targeted Therapy/*methods ; Neoplasm Transplantation ; Neoplasms/classification/*genetics/pathology/*therapy ; Organ Specificity ; Reproducibility of Results ; Synthetic Lethal Mutations/genetics ; Werner Syndrome/genetics ; Werner Syndrome Helicase/genetics ; },
abstract = {Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.},
}
@article {pmid30971823,
year = {2019},
author = {Chan, EM and Shibue, T and McFarland, JM and Gaeta, B and Ghandi, M and Dumont, N and Gonzalez, A and McPartlan, JS and Li, T and Zhang, Y and Bin Liu, J and Lazaro, JB and Gu, P and Piett, CG and Apffel, A and Ali, SO and Deasy, R and Keskula, P and Ng, RWS and Roberts, EA and Reznichenko, E and Leung, L and Alimova, M and Schenone, M and Islam, M and Maruvka, YE and Liu, Y and Roper, J and Raghavan, S and Giannakis, M and Tseng, YY and Nagel, ZD and D'Andrea, A and Root, DE and Boehm, JS and Getz, G and Chang, S and Golub, TR and Tsherniak, A and Vazquez, F and Bass, AJ},
title = {WRN helicase is a synthetic lethal target in microsatellite unstable cancers.},
journal = {Nature},
volume = {568},
number = {7753},
pages = {551-556},
pmid = {30971823},
issn = {1476-4687},
support = {K08 CA198002/CA/NCI NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; T32 CA009172/CA/NCI NIH HHS/United States ; },
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/genetics ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; Humans ; *Microsatellite Instability ; Microsatellite Repeats/*genetics ; Models, Genetic ; Neoplasms/*genetics/pathology ; RNA Interference ; Synthetic Lethal Mutations/*genetics ; Tumor Suppressor Protein p53/metabolism ; Werner Syndrome Helicase/deficiency/*genetics ; },
abstract = {Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics[1]. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins[2]. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach[3]. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.},
}
@article {pmid30971695,
year = {2019},
author = {LaFleur, MW and Nguyen, TH and Coxe, MA and Yates, KB and Trombley, JD and Weiss, SA and Brown, FD and Gillis, JE and Coxe, DJ and Doench, JG and Haining, WN and Sharpe, AH},
title = {A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1668},
pmid = {30971695},
issn = {2041-1723},
support = {U19AI133524/NH/NIH HHS/United States ; T32CA207021/NH/NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; U19 AI133524/AI/NIAID NIH HHS/United States ; T32GM007753/NH/NIH HHS/United States ; T32 CA207021/CA/NCI NIH HHS/United States ; },
mesh = {Adaptive Immunity/*genetics ; Animals ; Bone Marrow Transplantation ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Lineage/genetics/immunology ; Chlorocebus aethiops ; Disease Models, Animal ; Feasibility Studies ; Female ; *Gene Transfer Techniques ; Genetic Testing/*methods ; Genetic Vectors/genetics ; Genomics/methods ; HEK293 Cells ; Humans ; Immunity, Innate/*genetics ; Lymphocytic Choriomeningitis/genetics/immunology/virology ; Lymphocytic choriomeningitis virus/immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics/immunology ; RNA, Guide/genetics ; Transplantation Chimera ; Vero Cells ; },
abstract = {Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8[+] T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.},
}
@article {pmid30970262,
year = {2019},
author = {Ihry, RJ and Salick, MR and Ho, DJ and Sondey, M and Kommineni, S and Paula, S and Raymond, J and Henry, B and Frias, E and Wang, Q and Worringer, KA and Ye, C and Russ, C and Reece-Hoyes, JS and Altshuler, RC and Randhawa, R and Yang, Z and McAllister, G and Hoffman, GR and Dolmetsch, R and Kaykas, A},
title = {Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes.},
journal = {Cell reports},
volume = {27},
number = {2},
pages = {616-630.e6},
doi = {10.1016/j.celrep.2019.03.043},
pmid = {30970262},
issn = {2211-1247},
mesh = {CRISPR-Cas Systems/*genetics ; Genetic Testing/*methods ; Genome/*genetics ; Humans ; Pluripotent Stem Cells/*metabolism ; },
abstract = {Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.},
}
@article {pmid30970016,
year = {2019},
author = {Oppel, F and Tao, T and Shi, H and Ross, KN and Zimmerman, MW and He, S and Tong, G and Aster, JC and Look, AT},
title = {Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.},
journal = {PLoS genetics},
volume = {15},
number = {4},
pages = {e1008039},
pmid = {30970016},
issn = {1553-7404},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Disease Models, Animal ; Erythropoiesis ; Female ; Gene Knockout Techniques ; Globins/genetics ; Humans ; Loss of Function Mutation ; Male ; Neurofibromin 1/deficiency/genetics ; Sarcoma, Experimental/*etiology/genetics/metabolism ; Telomere Homeostasis/genetics ; Tumor Suppressor Protein p53/*deficiency/*genetics ; X-linked Nuclear Protein/*deficiency/*genetics ; Zebrafish/embryology/genetics/metabolism ; Zebrafish Proteins/*deficiency/*genetics ; },
abstract = {The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.},
}
@article {pmid30969416,
year = {2019},
author = {Bourque, K and Devost, D and Inoue, A and Hébert, TE},
title = {Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1947},
number = {},
pages = {169-182},
doi = {10.1007/978-1-4939-9121-1_9},
pmid = {30969416},
issn = {1940-6029},
support = {MOP-130309//CIHR/Canada ; },
mesh = {Bioluminescence Resonance Energy Transfer Techniques/*methods ; Biosensing Techniques/*methods ; *CRISPR-Cas Systems ; Fluorescence ; Gene Editing/*methods ; Humans ; Ligands ; Luciferases, Renilla/*metabolism ; Protein Conformation ; Receptors, G-Protein-Coupled/*chemistry/genetics/*metabolism ; Signal Transduction ; },
abstract = {Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.},
}
@article {pmid30968393,
year = {2019},
author = {Iyer, N and Tcheuyap, VT and Schneider, S and Marshall, V and Jagadeeswaran, P},
title = {Knockout of von Willebrand factor in Zebrafish by CRISPR/Cas9 mutagenesis.},
journal = {British journal of haematology},
volume = {186},
number = {4},
pages = {e76-e80},
pmid = {30968393},
issn = {1365-2141},
support = {R15 DK117384/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; *Gene Editing ; Gene Knockout Techniques ; Gene Targeting ; *Mutagenesis ; RNA, Guide ; Sequence Analysis, DNA ; Zebrafish/*genetics ; von Willebrand Factor/*genetics ; },
}
@article {pmid30967552,
year = {2019},
author = {Pavel-Dinu, M and Wiebking, V and Dejene, BT and Srifa, W and Mantri, S and Nicolas, CE and Lee, C and Bao, G and Kildebeck, EJ and Punjya, N and Sindhu, C and Inlay, MA and Saxena, N and DeRavin, SS and Malech, H and Roncarolo, MG and Weinberg, KI and Porteus, MH},
title = {Gene correction for SCID-X1 in long-term hematopoietic stem cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1634},
pmid = {30967552},
issn = {2041-1723},
mesh = {Animals ; Antigens, CD34/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Codon, Initiator/genetics ; DNA, Complementary/*genetics ; Dependovirus ; Exons/genetics ; Fetal Blood/cytology ; Gene Editing/*methods ; Genetic Vectors/genetics ; Healthy Volunteers ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; Humans ; Interleukin Receptor Common gamma Subunit/*genetics ; Male ; Mice ; Mutation ; Parvovirinae/genetics ; Primary Cell Culture ; Time Factors ; Transduction, Genetic/methods ; Transplantation Chimera/genetics ; Transplantation, Heterologous/methods ; X-Linked Combined Immunodeficiency Diseases/genetics/*therapy ; },
abstract = {Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34[+] HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.},
}
@article {pmid30967548,
year = {2019},
author = {Guichard, A and Haque, T and Bobik, M and Xu, XS and Klanseck, C and Kushwah, RBS and Berni, M and Kaduskar, B and Gantz, VM and Bier, E},
title = {Efficient allelic-drive in Drosophila.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1640},
pmid = {30967548},
issn = {2041-1723},
support = {DP5 OD023098/OD/NIH HHS/United States ; P30 NS047101/NS/NINDS NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; },
mesh = {Agriculture/methods ; *Alleles ; Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/*genetics ; DNA Mutational Analysis ; Drosophila/*genetics ; Female ; Gene Drive Technology/*methods ; Gene Editing/methods ; Inheritance Patterns/genetics ; Male ; Mosaicism ; RNA, Guide/genetics ; },
abstract = {Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.},
}
@article {pmid30967427,
year = {2019},
author = {Yang, R and Li, E and Kwon, YJ and Mani, M and Beitel, GJ},
title = {QuBiT: a quantitative tool for analyzing epithelial tubes reveals unexpected patterns of organization in the Drosophila trachea.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {12},
pages = {},
pmid = {30967427},
issn = {1477-9129},
support = {R01 GM108964/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Triphosphate/chemistry ; Animals ; Body Patterning ; CRISPR-Cas Systems ; Cell Lineage ; Computational Biology/instrumentation ; Crosses, Genetic ; Drosophila/*embryology ; Drosophila Proteins/metabolism ; Epithelium/*embryology ; *Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Membrane Proteins/metabolism ; Models, Biological ; Sodium-Potassium-Exchanging ATPase/metabolism ; Trachea/*embryology ; },
abstract = {Biological tubes are essential for animal survival, and their functions are dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. We therefore developed QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis of the embryonic Drosophila trachea (airway). In the main tube, we find previously unknown anterior-to-posterior (A-P) gradients of cell apical orientation and aspect ratio, and periodicity in the organization of apical cell surfaces. Inferred cell intercalation during development dampens an A-P gradient of the number of cells per cross-section of the tube, but does not change the patterns of cell connectivity. Computationally 'unrolling' the apical surface of wild-type trachea and the hindgut reveals previously unrecognized spatial patterns of the apical marker Uninflatable and a non-redundant role for the Na[+]/K[+] ATPase in apical marker organization. These unexpected findings demonstrate the importance of a computational tool for analyzing small diameter biological tubes.},
}
@article {pmid30967334,
year = {2019},
author = {Zhu, M and Sun, L and Lu, X and Zong, H and Zhuge, B},
title = {Establishment of a transient CRISPR-Cas9 genome editing system in Candida glycerinogenes for co-production of ethanol and xylonic acid.},
journal = {Journal of bioscience and bioengineering},
volume = {128},
number = {3},
pages = {283-289},
doi = {10.1016/j.jbiosc.2019.03.009},
pmid = {30967334},
issn = {1347-4421},
mesh = {Aldehyde Reductase/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Candida/*genetics/metabolism ; Cloning, Molecular/methods ; Ethanol/*metabolism ; Gene Editing/*methods ; Gene Knockout Techniques ; Glucose/metabolism ; Metabolic Engineering/*methods ; Organisms, Genetically Modified ; Xylose/*analogs &amp; derivatives/metabolism ; },
abstract = {Candida glycerinogenes, an industrial yeast with excellent multi-stress tolerance, has been applied to glycerol production for decades. However, its genetic manipulation was limited by the absence of meiosis, the diploid genome, and the lack of molecular tools. We described here the implementation of a transient CRISPR-Cas9 system for efficient genome editing in C. glycerinogenes. By targeting the counterselectable marker genes (TRP1, URA3), single and double gene knock-outs were achieved and the auxotroph obtained can be used as a background for targeting other gene (HOG1) at a mutation efficiency of 80%. Further, a xylonic acid producing C. glycerinogenes strain was constructed by knock-in of the xylose dehydrogenase gene, which produced up to 28 g/L ethanol and 9 g/L xylonic acid simultaneously from simulated lignocellulosic hydrolysate (contained 70 g/L glucose and 24 g/L xylose). These results indicated that the CRSIPR-Cas9 system developed here can facilitate the study of gene functions and metabolic pathways in C. glycerinogenes.},
}
@article {pmid30965672,
year = {2019},
author = {Song, HY and Chien, CS and Yarmishyn, AA and Chou, SJ and Yang, YP and Wang, ML and Wang, CY and Leu, HB and Yu, WC and Chang, YL and Chiou, SH},
title = {Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy.},
journal = {Cells},
volume = {8},
number = {4},
pages = {},
pmid = {30965672},
issn = {2073-4409},
mesh = {Apoptosis ; *Autophagy ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology ; Cell Line ; Exosomes ; Fabry Disease/*complications ; Gene Knockout Techniques ; Human Embryonic Stem Cells/metabolism ; Humans ; *Models, Biological ; Myocytes, Cardiac/metabolism ; Reactive Oxygen Species/metabolism ; Trihexosylceramides/metabolism ; alpha-Galactosidase/*genetics ; },
abstract = {Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.},
}
@article {pmid30964863,
year = {2019},
author = {Ke, H and Ganesan, SM and Dass, S and Morrisey, JM and Pou, S and Nilsen, A and Riscoe, MK and Mather, MW and Vaidya, AB},
title = {Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages.},
journal = {PloS one},
volume = {14},
number = {4},
pages = {e0214023},
pmid = {30964863},
issn = {1932-6203},
support = {K22 AI127702/AI/NIAID NIH HHS/United States ; R01 AI028398/AI/NIAID NIH HHS/United States ; R01 AI100569/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Antimalarials/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cells, Cultured ; Drug Resistance/*genetics ; Electron Transport Complex I/antagonists &amp; inhibitors ; Electron Transport Complex III/antagonists &amp; inhibitors ; Erythrocytes/parasitology ; Gene Knockout Techniques ; Humans ; Malaria, Falciparum/blood/drug therapy/parasitology ; Mitochondria/drug effects/enzymology ; NADH Dehydrogenase/*genetics/metabolism ; Plasmodium falciparum/drug effects/*enzymology/genetics ; Protozoan Proteins/genetics/*metabolism ; Quinolones/pharmacology/therapeutic use ; },
abstract = {The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite's susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.},
}
@article {pmid30963647,
year = {2019},
author = {Liu, Y and Zeng, J and Yuan, C and Guo, Y and Yu, H and Li, Y and Huang, C},
title = {Cas9-PF, an early flowering and visual selection marker system, enhances the frequency of editing event occurrence and expedites the isolation of genome-edited and transgene-free plants.},
journal = {Plant biotechnology journal},
volume = {17},
number = {7},
pages = {1191-1193},
pmid = {30963647},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Plants, Genetically Modified ; RNA, Guide ; Selection, Genetic ; Tobacco/*genetics ; },
}
@article {pmid30962821,
year = {2019},
author = {Naduthodi, MIS and Mohanraju, P and Südfeld, C and D'Adamo, S and Barbosa, MJ and van der Oost, J},
title = {CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1.},
journal = {Biotechnology for biofuels},
volume = {12},
number = {},
pages = {66},
pmid = {30962821},
issn = {1754-6834},
abstract = {BACKGROUND: Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout.<br><br>RESULTS: In this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines.<br><br>CONCLUSION: We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.},
}
@article {pmid30962622,
year = {2019},
author = {Chow, RD and Wang, G and Ye, L and Codina, A and Kim, HR and Shen, L and Dong, MB and Errami, Y and Chen, S},
title = {In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens.},
journal = {Nature methods},
volume = {16},
number = {5},
pages = {405-408},
pmid = {30962622},
issn = {1548-7105},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; T32 HD007149/HD/NICHD NIH HHS/United States ; S10 OD018521/OD/NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Mice ; Neoplasm Metastasis/*genetics ; Sequence Analysis, RNA ; },
abstract = {Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.},
}
@article {pmid30962586,
year = {2019},
author = {Mukherjee-Clavin, B and Mi, R and Kern, B and Choi, IY and Lim, H and Oh, Y and Lannon, B and Kim, KJ and Bell, S and Hur, JK and Hwang, W and Che, YH and Habib, O and Baloh, RH and Eggan, K and Brandacher, G and Hoke, A and Studer, L and Kim, YJ and Lee, G},
title = {Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder.},
journal = {Nature biomedical engineering},
volume = {3},
number = {7},
pages = {571-582},
pmid = {30962586},
issn = {2157-846X},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 AR070751/AR/NIAMS NIH HHS/United States ; R01 NS093213/NS/NINDS NIH HHS/United States ; R01 NS097545/NS/NINDS NIH HHS/United States ; },
mesh = {Adult ; Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; Cell Lineage/genetics ; Cells, Cultured ; Cellular Reprogramming ; Chemokine CCL2/*genetics/metabolism ; Chemokine CXCL1/*genetics/metabolism ; Chemokines ; Embryonic Stem Cells/pathology ; Female ; Gene Editing ; Gene Expression ; Gene Expression Profiling ; *Genetic Diseases, Inborn ; Genetic Predisposition to Disease/genetics ; Human Genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism/pathology ; Male ; Mice ; Mice, Inbred NOD ; Myelin Proteins/genetics/metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Phenotype ; Rats ; SOXE Transcription Factors/genetics/metabolism ; Schwann Cells/*metabolism/pathology ; Transplantation ; },
abstract = {Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.},
}
@article {pmid30962579,
year = {2019},
author = {Song, Y and Park, PMC and Wu, L and Ray, A and Picaud, S and Li, D and Wimalasena, VK and Du, T and Filippakopoulos, P and Anderson, KC and Qi, J and Chauhan, D},
title = {Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma.},
journal = {Leukemia},
volume = {33},
number = {11},
pages = {2685-2694},
pmid = {30962579},
issn = {1476-5551},
support = {P01 CA155258/CA/NCI NIH HHS/United States ; R01 CA050947/CA/NCI NIH HHS/United States ; R01 CA207237/CA/NCI NIH HHS/United States ; P50 CA100707/CA/NCI NIH HHS/United States ; MR/N010051/1/MRC_/Medical Research Council/United Kingdom ; R01 CA222218/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; Bortezomib/pharmacology ; CRISPR-Cas Systems ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Dendritic Cells/cytology ; Drug Evaluation, Preclinical ; HCT116 Cells ; Humans ; Intracellular Signaling Peptides and Proteins/*antagonists &amp; inhibitors/*metabolism ; Lenalidomide/pharmacology ; Mice ; Mice, SCID ; Multiple Myeloma/*metabolism/therapy ; Neoplasm Transplantation ; Proteasome Endopeptidase Complex/metabolism ; Proteasome Inhibitors/*pharmacology ; RNA Interference ; Ubiquitin/chemistry ; },
abstract = {Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.},
}
@article {pmid30962569,
year = {2019},
author = {Lee, HH and Ostrov, N and Wong, BG and Gold, MA and Khalil, AS and Church, GM},
title = {Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.},
journal = {Nature microbiology},
volume = {4},
number = {7},
pages = {1105-1113},
pmid = {30962569},
issn = {2058-5276},
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Culture Media ; Gene Knockdown Techniques ; Gene Library ; Genome, Bacterial/*genetics ; Genomics ; Vibrio/*genetics/growth &amp; development ; },
abstract = {The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time[1,2] and metabolic prowess[3,4]. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.},
}
@article {pmid30962447,
year = {2019},
author = {Taemaitree, L and Shivalingam, A and El-Sagheer, AH and Brown, T},
title = {An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1610},
pmid = {30962447},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics ; Catalysis ; Cell Line, Tumor ; Click Chemistry/economics/*methods ; Copper/chemistry ; Cycloaddition Reaction/methods ; DNA/chemistry/genetics ; DNA Cleavage ; Gene Editing/economics/*methods ; Humans ; Oligonucleotides/chemistry/genetics ; RNA, Guide/*chemistry/genetics ; Triazoles/*chemistry ; },
abstract = {As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.},
}
@article {pmid30962441,
year = {2019},
author = {Gisler, S and Gonçalves, JP and Akhtar, W and de Jong, J and Pindyurin, AV and Wessels, LFA and van Lohuizen, M},
title = {Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1598},
pmid = {30962441},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA/*genetics ; DNA Mutational Analysis ; Gene Editing/*methods ; Genes, Reporter/genetics ; Genetic Loci/genetics ; Genetic Vectors/genetics ; Mice ; Mouse Embryonic Stem Cells ; Mutation Rate ; Plasmids/genetics ; RNA, Guide/*genetics ; },
abstract = {Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.},
}
@article {pmid30962421,
year = {2019},
author = {Zou, Y and Palte, MJ and Deik, AA and Li, H and Eaton, JK and Wang, W and Tseng, YY and Deasy, R and Kost-Alimova, M and Dančík, V and Leshchiner, ES and Viswanathan, VS and Signoretti, S and Choueiri, TK and Boehm, JS and Wagner, BK and Doench, JG and Clish, CB and Clemons, PA and Schreiber, SL},
title = {A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1617},
pmid = {30962421},
issn = {2041-1723},
support = {P30 DK040561/DK/NIDDK NIH HHS/United States ; P50 CA101942/CA/NCI NIH HHS/United States ; T32 HL007627/HL/NHLBI NIH HHS/United States ; U01 CA217848/CA/NCI NIH HHS/United States ; },
mesh = {Aged ; Animals ; Apoptosis/genetics ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Carcinoma, Renal Cell/genetics/*pathology ; Cell Line, Tumor ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Glutathione Peroxidase/genetics/*metabolism ; HEK293 Cells ; Humans ; Iron/metabolism ; Kidney Neoplasms/genetics/*pathology ; Lipid Peroxidation/genetics ; Male ; Mice, Nude ; Middle Aged ; Neoplasm Proteins/genetics/*metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase ; RNA Interference ; Xenograft Model Antitumor Assays ; },
abstract = {Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCCs are distinguished by aberrant lipid and glycogen accumulation and are refractory to a broad range of anti-cancer therapies. Here we identify an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCCs. This vulnerability transcends lineage and genetic landscape, and can be exploited by inhibiting glutathione peroxidase 4 (GPX4) with small-molecules. Using CRISPR screening and lipidomic profiling, we identify the hypoxia-inducible factor (HIF) pathway as a driver of this vulnerability. In renal CCCs, HIF-2α selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Our study suggests targeting GPX4 as a therapeutic opportunity in CCCs, and highlights that therapeutic approaches can be identified on the basis of cell states manifested by morphological and metabolic features in hard-to-treat cancers.},
}
@article {pmid30961525,
year = {2019},
author = {Bao, A and Chen, H and Chen, L and Chen, S and Hao, Q and Guo, W and Qiu, D and Shan, Z and Yang, Z and Yuan, S and Zhang, C and Zhang, X and Liu, B and Kong, F and Li, X and Zhou, X and Tran, LP and Cao, D},
title = {CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {131},
pmid = {30961525},
issn = {1471-2229},
mesh = {Arabidopsis/genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Homozygote ; Mutagenesis, Site-Directed ; Mutation ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic/genetics ; Soybeans/anatomy &amp; histology/*genetics ; Transcription Factors/genetics/*metabolism ; },
abstract = {BACKGROUND: The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants.<br><br>RESULTS: In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean.<br><br>CONCLUSIONS: Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.},
}
@article {pmid30959888,
year = {2019},
author = {Ilyechova, EY and Bonaldi, E and Orlov, IA and Skomorokhova, EA and Puchkova, LV and Broggini, M},
title = {CRISP-R/Cas9 Mediated Deletion of Copper Transport Genes CTR1 and DMT1 in NSCLC Cell Line H1299. Biological and Pharmacological Consequences.},
journal = {Cells},
volume = {8},
number = {4},
pages = {},
pmid = {30959888},
issn = {2073-4409},
mesh = {CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/*genetics ; Cation Transport Proteins/*genetics/metabolism ; Cell Line, Tumor ; Cell Survival/genetics ; Copper/*metabolism ; Copper Transporter 1 ; Gene Expression Regulation, Neoplastic ; Humans ; Ion Transport ; Lung Neoplasms/*genetics ; Transcription Factors/*genetics/metabolism ; },
abstract = {Copper, the highly toxic micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copper is imported by CTR1 (high-affinity copper transporter 1), a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have a cytosol domain which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1 can mediate copper way required for a regulatory copper pool. To verify this hypothesis, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1 KO cells, expression of the DMT1 gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copper concentration dropped, however, in nuclei basal level of copper did not change dramatically. CTR1 KO cells, but not DMT1 KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1 KO, we were able to show that both, CTR1 and DMT1, provided the formation of vital intracellular cuproenzymes (SOD1, COX), but not secretory ceruloplasmin. The loss of CTR1 resulted in a decrease in the level of COMMD1, XIAP, and NF-κB. Differently, the DMT1 deficiency induced increase of the COMMD1, HIF1α, and XIAP levels. The possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.},
}
@article {pmid30959782,
year = {2019},
author = {Lee, C},
title = {CRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge.},
journal = {Molecules (Basel, Switzerland)},
volume = {24},
number = {7},
pages = {},
pmid = {30959782},
issn = {1420-3049},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Viral/genetics ; Humans ; Virus Diseases/*genetics/therapy/virology ; Viruses/*genetics/pathogenicity ; },
abstract = {From its unexpected discovery as a bacterial adaptive immune system to its countless applications as one of the most versatile gene-editing tools, the CRISPR/Cas9 system has revolutionized every field of life science. Virology is no exception to this ever-growing list of CRISPR/Cas9-based applications. Direct manipulation of a virus genome by CRISPR/Cas9 has enabled a systematic study of cis-elements and trans-elements encoded in a virus genome. In addition, this virus genome-specific mutagenesis by CRISPR/Cas9 was further funneled into the development of a novel class of antiviral therapy targeting many incurable chronic viral infections. In this review, a general concept on the CRISPR/Cas9-based antiviral strategy will be described first. To understand the current status of the CRISPR/Cas9-based antiviral approach, a series of recently published antiviral studies involving CRISPR/Cas9-mediated control of several clinically-relevant viruses including human immunodeficiency virus, hepatitis B virus, herpesviruses, human papillomavirus, and other viruses will be presented. Lastly, the potential challenge and future prospect for successful clinical translation of this CRISPR/Cas9-based antiviral method will be discussed.},
}
@article {pmid30959774,
year = {2019},
author = {Bohrer, LR and Wiley, LA and Burnight, ER and Cooke, JA and Giacalone, JC and Anfinson, KR and Andorf, JL and Mullins, RF and Stone, EM and Tucker, BA},
title = {Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.},
journal = {Genes},
volume = {10},
number = {4},
pages = {},
pmid = {30959774},
issn = {2073-4425},
support = {P30 EY025580/EY/NEI NIH HHS/United States ; R01 EY026008/EY/NEI NIH HHS/United States ; T32 GM007337/GM/NIGMS NIH HHS/United States ; RO1-EY026008/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Codon, Nonsense/genetics ; Eye Diseases, Hereditary/*genetics/pathology/*therapy ; Gene Expression Regulation, Developmental/genetics ; Genetic Predisposition to Disease ; *Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells/metabolism/transplantation ; Mutation ; Orphan Nuclear Receptors/*genetics/therapeutic use ; Retinal Degeneration/*genetics/pathology/*therapy ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Vision Disorders/*genetics/pathology/*therapy ; },
abstract = {Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.},
}
@article {pmid30959555,
year = {2019},
author = {Negishi, K and Kaya, H and Abe, K and Hara, N and Saika, H and Toki, S},
title = {An adenine base editor with expanded targeting scope using SpCas9-NGv1 in rice.},
journal = {Plant biotechnology journal},
volume = {17},
number = {8},
pages = {1476-1478},
pmid = {30959555},
issn = {1467-7652},
mesh = {*Adenine ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Oryza/*genetics ; },
}
@article {pmid30959139,
year = {2019},
author = {Sunwoo, IY and Sukwong, P and Jeong, DY and Kim, SR and Jeong, GT and Kim, SK},
title = {Enhancement of galactose consumption rate in Saccharomyces cerevisiae CEN.PK2-1 by CRISPR Cas9 and adaptive evolution for fermentation of Kappaphycus alvarezii hydrolysate.},
journal = {Journal of biotechnology},
volume = {297},
number = {},
pages = {78-84},
doi = {10.1016/j.jbiotec.2019.03.012},
pmid = {30959139},
issn = {1873-4863},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Carbohydrate Metabolism ; *Directed Molecular Evolution ; Ethanol/metabolism ; *Fermentation ; Galactose/*metabolism ; Gene Expression Regulation, Fungal ; Genes, Fungal ; Hydrolysis ; Rhodophyta/*metabolism ; Saccharomyces cerevisiae/genetics/*metabolism ; Temperature ; Transcription, Genetic ; },
abstract = {Ethanol ferrmentation of Kappaphycus alvarezii hydrolysates was performed using wild-type (WT) Saccharomyces cerevisiae CEN.PK2-1, hexokinase 2 deleted (Δhxk2) and adapted strain on high galactose concentrations. The WT and Δhxk2 strains produced 8.9 and 14.67 g/L of ethanol with yield coefficient (YEtOH) of 0.20 and 0.33 (g/g), respectively. However, neither the WT nor Δhxk2strain could utilize all of the galactose, leaving 16.4 and 6.2 g/L of galactose in the fermentation broth, respectively. Therefore, fermentation with S. cerevisiae CEN.PK2-1 adapted to galactose was carried out to increase the ethanol yield coefficient (YEtOH), producing a maximum ethanol concentration of 20.0 g/L with a YEtOH of 0.44 (g/g). Ethanol concentration of adapted strain was 1.36-2.25 times higher than WT and Δhxk2 strains. The adapted yeast exhibited the highest transcript levels of GAL genes. The yeast strain via adaptive yeast strain produced ethanol with a higher titer and yield due to a modular activation of GAL genes than WT or the hxk2 deleted strains.},
}
@article {pmid30958843,
year = {2019},
author = {Wieser, M and Francisci, T and Lackner, D and Buerckstuemmer, T and Wasner, K and Eilenberg, W and Stift, A and Wahrmann, M and Böhmig, GA and Grillari, J and Grillari-Voglauer, R},
title = {CD46 knock-out using CRISPR/Cas9 editing of hTERT immortalized human cells modulates complement activation.},
journal = {PloS one},
volume = {14},
number = {4},
pages = {e0214514},
pmid = {30958843},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *Complement Activation ; Complement C4/chemistry ; Complement C4b/chemistry ; Epithelial Cells/*cytology ; Gene Editing ; *Gene Knockout Techniques ; Humans ; Kidney Tubules/cytology ; Membrane Cofactor Protein/*genetics ; Telomerase/*metabolism ; Telomere/ultrastructure ; gamma-Glutamyltransferase/metabolism ; },
abstract = {The kidney is especially sensitive to diseases associated with overactivation of the complement system. While most of these diseases affect kidney glomeruli and the microvasculature, there is also evidence for tubulointerstitial deposition of complement factors. Complement inactivating factors on cell membranes comprise CD55, CD59 and CD46, which is also termed membrane cofactor protein (MCP). CD46 has been described as localized to glomeruli, but especially also to proximal tubular epithelial cells (RPTECs). However, human cell culture models to assess CD46 function on RPTECs are still missing. Therefore, we here performed gene editing of RPTEC/TERT1 cells generating a monoclonal CD46-/- cell line that did not show changes of the primary cell like characteristics. In addition, factor I and CD46-mediated cleavage of C4b into soluble C4c and membrane deposited C4d was clearly reduced in the knock-out cell line as compared to the maternal cells. Thus, human CD46-/- proximal tubular epithelial cells will be of interest to dissect the roles of the epithelium and the kidney in various complement activation mediated tubulointerstitial pathologies or in studying CD46 mediated uropathogenic internalization of bacteria. In addition, this gives proof-of-principle, that telomerized cells can be used in the generation of knock-out, knock-in or any kind of reporter cell lines without losing the primary cell characteristics of the maternal cells.},
}
@article {pmid30958259,
year = {2019},
author = {Fineran, PC},
title = {Resistance is not futile: bacterial 'innate' and CRISPR-Cas 'adaptive' immune systems.},
journal = {Microbiology (Reading, England)},
volume = {165},
number = {8},
pages = {834-841},
doi = {10.1099/mic.0.000802},
pmid = {30958259},
issn = {1465-2080},
mesh = {Bacteria/*genetics/immunology ; Bacterial Proteins/genetics ; *Bacteriophages/pathogenicity ; *CRISPR-Cas Systems/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Transfer, Horizontal/genetics ; Immunity ; },
abstract = {Bacteria are under a constant pressure from their viruses (phages) and other mobile genetic elements. They protect themselves through a range of defence strategies, which can be broadly classified as 'innate' and 'adaptive'. The bacterial innate immune systems include defences provided by restriction modification and abortive infection, among others. Bacterial adaptive immunity is elicited by a diverse range of CRISPR-Cas systems. Here, I discuss our research on both innate and adaptive phage resistance mechanisms and some of the evasion strategies employed by phages.},
}
@article {pmid30957918,
year = {2019},
author = {Li, L and Yang, Z and Zhu, S and He, L and Fan, W and Tang, W and Zou, J and Shen, Z and Zhang, M and Tang, L and Dai, Y and Niu, G and Hu, S and Chen, X},
title = {A Rationally Designed Semiconducting Polymer Brush for NIR-II Imaging-Guided Light-Triggered Remote Control of CRISPR/Cas9 Genome Editing.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {31},
number = {21},
pages = {e1901187},
doi = {10.1002/adma.201901187},
pmid = {30957918},
issn = {1521-4095},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Genetic Vectors ; HCT116 Cells ; Humans ; Infrared Rays ; Lasers ; Mice, Nude ; Polyethylene Glycols/*chemistry ; Polyethyleneimine/*chemistry ; Semiconductors ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) genome-editing system has shown great potential in biomedical applications. Although physical approaches, viruses, and some nonviral vectors have been employed for CRISPR/Cas9 delivery and induce some promising genome-editing efficacy, precise genome editing remains challenging and has not been reported yet. Herein, second near-infrared window (NIR-II) imaging-guided NIR-light-triggered remote control of the CRISPR/Cas9 genome-editing strategy is reported based on a rationally designed semiconducting polymer brush (SPPF). SPPF can not only be a vector to deliver CRISPR/Cas9 cassettes but also controls the endolysosomal escape and payloads release through photothermal conversion under laser irradiation. Upon laser exposure, the nanocomplex of SPPF and CRISPR/Cas9 cassettes induces effective site-specific precise genome editing both in vitro and in vivo with minimal toxicity. Besides, NIR-II imaging based on SPPF can also be applied to monitor the in vivo distribution of the genome-editing system and guide laser irradiation in real time. Thus, this study offers a typical paradigm for NIR-II imaging-guided NIR-light-triggered remote control of the CRISPR/Cas9 system for precise genome editing. This strategy may open an avenue for CRISPR/Cas9 genome-editing-based precise gene therapy in the near future.},
}
@article {pmid30957847,
year = {2019},
author = {Gong, L and Li, M and Cheng, F and Zhao, D and Chen, Y and Xiang, H},
title = {Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica.},
journal = {Nucleic acids research},
volume = {47},
number = {11},
pages = {5880-5891},
pmid = {30957847},
issn = {1362-4962},
mesh = {Adaptation, Physiological ; CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Primers/genetics ; Escherichia coli/genetics ; Escherichia coli Proteins/metabolism ; Gene Deletion ; Haloarcula/*genetics ; Mutation ; Plasmids/metabolism ; *RNA, Guide ; },
abstract = {Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3' handle, which seriously impaired crRNA stability and interference effects. With 3' handle deletion, further truncation of 5' handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5' handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.},
}
@article {pmid30956982,
year = {2019},
author = {Borisjuk, N and Kishchenko, O and Eliby, S and Schramm, C and Anderson, P and Jatayev, S and Kurishbayev, A and Shavrukov, Y},
title = {Genetic Modification for Wheat Improvement: From Transgenesis to Genome Editing.},
journal = {BioMed research international},
volume = {2019},
number = {},
pages = {6216304},
pmid = {30956982},
issn = {2314-6141},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/growth &amp; development ; Gene Editing/*methods ; *Gene Transfer Techniques ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Triticum/genetics/growth &amp; development ; },
abstract = {To feed the growing human population, global wheat yields should increase to approximately 5 tonnes per ha from the current 3.3 tonnes by 2050. To reach this goal, existing breeding practices must be complemented with new techniques built upon recent gains from wheat genome sequencing, and the accumulated knowledge of genetic determinants underlying the agricultural traits responsible for crop yield and quality. In this review we primarily focus on the tools and techniques available for accessing gene functions which lead to clear phenotypes in wheat. We provide a view of the development of wheat transformation techniques from a historical perspective, and summarize how techniques have been adapted to obtain gain-of-function phenotypes by gene overexpression, loss-of-function phenotypes by expressing antisense RNAs (RNA interference or RNAi), and most recently the manipulation of gene structure and expression using site-specific nucleases, such as CRISPR/Cas9, for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to increase yield, improve nutritional and health-promoting qualities in wheat, and enhance the crop's resistance to various biotic and abiotic stresses.},
}
@article {pmid30956114,
year = {2019},
author = {Roberts, B and Hendershott, MC and Arakaki, J and Gerbin, KA and Malik, H and Nelson, A and Gehring, J and Hookway, C and Ludmann, SA and Yang, R and Haupt, A and Grancharova, T and Valencia, V and Fuqua, MA and Tucker, A and Rafelski, SM and Gunawardane, RN},
title = {Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.},
journal = {Stem cell reports},
volume = {12},
number = {5},
pages = {1145-1158},
pmid = {30956114},
issn = {2213-6711},
mesh = {Actinin/genetics/metabolism ; Amino Acid Sequence ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Myocytes, Cardiac/cytology/*metabolism ; Sarcomeres/*genetics/metabolism ; Sequence Homology, Amino Acid ; Troponin I/genetics/metabolism ; },
abstract = {We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.},
}
@article {pmid30955321,
year = {2019},
author = {Bian, WP and Chen, YL and Luo, JJ and Wang, C and Xie, SL and Pei, DS},
title = {Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA Using Mito-CRISPR/Cas9 System.},
journal = {ACS synthetic biology},
volume = {8},
number = {4},
pages = {621-632},
doi = {10.1021/acssynbio.8b00411},
pmid = {30955321},
issn = {2161-5063},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Repair/genetics ; DNA, Mitochondrial/*genetics ; DNA, Single-Stranded/genetics ; Endonucleases/genetics ; Gene Editing/methods ; Gene Targeting/methods ; HEK293 Cells ; Homologous Recombination/genetics ; Humans ; Mitochondria/genetics ; Mutagenesis/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Zebrafish/*genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {The mitochondria DNA (mtDNA) editing tool, zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system, is a promising approach for the treatment of mtDNA diseases by eliminating mutant mitochondrial genomes. However, there have been no reports of repairing the mutant mtDNA with homologous recombination strategy to date. Here, we show a mito-CRISPR/Cas9 system that mito-Cas9 protein can specifically target mtDNA and reduce mtDNA copy number in both human cells and zebrafish. An exogenous single-stranded DNA with short homologous arm was knocked into the targeting loci accurately, and this mutagenesis could be steadily transmitted to F1 generation of zebrafish. Moreover, we found some major factors involved in nuclear DNA repair were upregulated significantly by the mito-CRISPR/Cas9 system. Taken together, our data suggested that the mito-CRISPR/Cas9 system could be a useful method to edit mtDNA by knock-in strategy, providing a potential therapy for the treatment of inherited mitochondrial diseases.},
}
@article {pmid30954454,
year = {2019},
author = {Gauttam, R and Seibold, GM and Mueller, P and Weil, T and Weiß, T and Handrick, R and Eikmanns, BJ},
title = {A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum.},
journal = {Plasmid},
volume = {103},
number = {},
pages = {25-35},
doi = {10.1016/j.plasmid.2019.04.001},
pmid = {30954454},
issn = {1095-9890},
mesh = {Arginine/biosynthesis ; Argininosuccinate Lyase/genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Citrulline/biosynthesis ; Corynebacterium glutamicum/drug effects/*genetics/metabolism ; Gene Expression Regulation, Bacterial/*drug effects ; Gene Targeting/*methods ; Glucose-6-Phosphate Isomerase/genetics/metabolism ; Isopropyl Thiogalactoside/pharmacology ; Plasmids/*chemistry/metabolism ; RNA, Guide/genetics/metabolism ; Tetracyclines/pharmacology ; },
abstract = {The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.},
}
@article {pmid30954217,
year = {2019},
author = {Eom, SY and Hwang, SH and Yeom, H and Lee, M},
title = {An ATG5 knockout promotes paclitaxel resistance in v-Ha-ras-transformed NIH 3T3 cells.},
journal = {Biochemical and biophysical research communications},
volume = {513},
number = {1},
pages = {234-241},
doi = {10.1016/j.bbrc.2019.03.197},
pmid = {30954217},
issn = {1090-2104},
mesh = {Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/drug effects ; Autophagy/drug effects ; Autophagy-Related Protein 5/*genetics ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; *Drug Resistance, Neoplasm ; Gene Knockout Techniques ; Genes, ras ; Mice ; NIH 3T3 Cells ; Neoplasms/drug therapy/genetics ; Paclitaxel/*pharmacology ; Tubulin Modulators/pharmacology ; },
abstract = {Autophagy plays a contradictory role in cell survival and death. Here, we investigated changes in paclitaxel sensitivity of cells with an ATG5 gene-knockout (KO), incapable of synthesizing an E3 ubiquitin ligase necessary for autophagy. The ATG5 KO in v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3) was established using the CRISPR/Cas9 system. An LC3 immunoblot and a qRT-PCR assay were used to confirm the KO of functional ATG5. We found that the ATG5 KO led to paclitaxel resistance in Ras-NIH 3T3 cells through an ATP-binding cassette (ABC) transporter-independent mechanism. Flow cytometric analyses revealed that paclitaxel induced a remarkable significant G2/M arrest in parental cells, whereas it was relatively less effective in ATG5 KO cells. Additionally, the proportion of early apoptotic cells significantly decreased in ATG5 KO cells treated with paclitaxel than in parental cells. Interestingly, overexpression of ATG5 N-terminal cleavage product in ATG5 KO cells restored their sensitivity to paclitaxel. Taken together, our results suggest that ATG5 KO cells are resistant to paclitaxel due to the inability to produce tATG5.},
}
@article {pmid30953741,
year = {2019},
author = {Koujah, L and Shukla, D and Naqvi, AR},
title = {CRISPR-Cas based targeting of host and viral genes as an antiviral strategy.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {53-64},
pmid = {30953741},
issn = {1096-3634},
support = {R03 DE027147/DE/NIDCR NIH HHS/United States ; R01 DE027980/DE/NIDCR NIH HHS/United States ; R21 DE026259/DE/NIDCR NIH HHS/United States ; R01 EY029426/EY/NEI NIH HHS/United States ; R01 EY024710/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genes, Viral/*genetics ; Host Microbial Interactions/*genetics/immunology/physiology ; Humans ; Virus Diseases/*genetics/immunology/*therapy ; Viruses/*genetics/immunology ; },
abstract = {Viral infections in human are leading cause of mortality and morbidity across the globe. Several viruses (including HIV and Herpesvirus), have evolved ingenious strategies to evade host-immune system and persist life-long. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) is an ancient antiviral system recently discovered in bacteria that has shown tremendous potential as a precise, invariant genome editing tool. Using CRISPR-Cas based system to activate host defenses or genetic modification of viral genome can provide novel, exciting and successful antiviral mechanisms and treatment modalities. In this review, we will provide progress on the CRISPR-Cas based antiviral approaches that facilitate clearance of virus-infected cells and/or prohibit virus infection or replication. We will discuss on the possibilities of CRIPSR-Cas as prophylaxis and therapy in viral infections and review the challenges of this potent gene editing technology.},
}
@article {pmid30953378,
year = {2019},
author = {Kildegaard, KR and Tramontin, LRR and Chekina, K and Li, M and Goedecke, TJ and Kristensen, M and Borodina, I},
title = {CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.},
journal = {Yeast (Chichester, England)},
volume = {36},
number = {5},
pages = {237-247},
pmid = {30953378},
issn = {1097-0061},
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering ; *RNA Interference ; RNA, Small Interfering/genetics ; Recombination, Genetic ; Saccharomyces cerevisiae/*genetics ; Sorbic Acid/analogs &amp; derivatives/metabolism ; },
abstract = {The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.},
}
@article {pmid30952800,
year = {2019},
author = {Valverde, DP and Yu, S and Boggavarapu, V and Kumar, N and Lees, JA and Walz, T and Reinisch, KM and Melia, TJ},
title = {ATG2 transports lipids to promote autophagosome biogenesis.},
journal = {The Journal of cell biology},
volume = {218},
number = {6},
pages = {1787-1798},
pmid = {30952800},
issn = {1540-8140},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; R01 GM114068/GM/NIGMS NIH HHS/United States ; R01 NS063973/NS/NINDS NIH HHS/United States ; R35 GM131715/GM/NIGMS NIH HHS/United States ; R01 GM080616/GM/NIGMS NIH HHS/United States ; },
mesh = {Autophagosomes/*physiology ; *Autophagy ; Autophagy-Related Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Biological Transport ; CRISPR-Cas Systems ; Endoplasmic Reticulum/*metabolism ; HEK293 Cells ; Humans ; Lipids/*physiology ; Vesicular Transport Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.},
}
@article {pmid30952669,
year = {2019},
author = {He, S and Cuentas-Condori, A and Miller, DM},
title = {NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans.},
journal = {Genetics},
volume = {212},
number = {2},
pages = {387-395},
pmid = {30952669},
issn = {1943-2631},
support = {R01 NS081259/NS/NINDS NIH HHS/United States ; R01 NS106951/NS/NINDS NIH HHS/United States ; S10 OD021630/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Caenorhabditis elegans/chemistry/*genetics ; Caenorhabditis elegans Proteins/*analysis/genetics ; Fluorescence ; Gene Editing/*methods ; Green Fluorescent Proteins/*genetics/metabolism ; Membrane Proteins/*analysis/genetics ; Nerve Tissue Proteins/*analysis/genetics ; },
abstract = {GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system.},
}
@article {pmid30952431,
year = {2019},
author = {Ali, M and Mutahir, Z and Riaz, A},
title = {CRISPR/Cas9 engineering of ERK5 identifies its FAK/PYK2 dependent role in adhesion-mediated cell survival.},
journal = {Biochemical and biophysical research communications},
volume = {513},
number = {1},
pages = {179-185},
doi = {10.1016/j.bbrc.2019.03.145},
pmid = {30952431},
issn = {1090-2104},
mesh = {Breast Neoplasms/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Female ; Focal Adhesion Kinase 1/*metabolism ; Focal Adhesion Kinase 2/*metabolism ; Humans ; Mitogen-Activated Protein Kinase 7/*genetics/metabolism ; },
abstract = {Extracellular signal-regulated kinase 5 (ERK5) is now considered a key regulator of breast cancer cell proliferation, migration and invasion. It is also implicated in growth factor induced anti-apoptotic signaling. But its contribution to adhesion-induced survival signaling is not clear. In the present study, using CRISPR/Cas9 editing, we knocked-out ERK5 expression in several cancer cell lines. Then MDA-MB 231 breast cancer cells lacking ERK5 were used to understand its role in adhesion-mediated cell viability. We demonstrated that ERK5 deficient cells exhibited reduced cell attachment to matrix proteins fibronectin and vitronectin. The adhesion ability of these cells was further reduced upon chemical inhibition of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) by PF 431396. FAK/PYK2 inhibited ERK5 knock-out cells also showed markedly reduced cell-viability and increased apoptotic signaling. This was evident from the detection of cleaved PARP and caspase 9 in these cells. Thus, our data suggests a FAK/PYK2 regulated pro-survival role of ERK5 in response to cell adhesion.},
}
@article {pmid30951975,
year = {2019},
author = {Hinterndorfer, M and Zuber, J},
title = {Functional-genetic approaches to understanding drug response and resistance.},
journal = {Current opinion in genetics &amp; development},
volume = {54},
number = {},
pages = {41-47},
doi = {10.1016/j.gde.2019.03.003},
pmid = {30951975},
issn = {1879-0380},
mesh = {Biomarkers, Pharmacological ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/*genetics ; *Drug Screening Assays, Antitumor ; Gene Editing/methods ; Humans ; Neoplasms/*drug therapy/genetics ; RNA Interference ; },
abstract = {Drug development remains a slow and expensive process, while the effective use of established therapeutics is widely hampered by our limited understanding of response and resistance mechanisms. Functional-genetic tools such as CRISPR/Cas9, advanced RNAi methods, and targeted protein degradation, together with other emerging technologies such as time-resolved and single-cell transcriptomics, fundamentally change the way we can search for candidate therapeutic targets and evaluate them before drug development. In addition, for already available therapeutics these tools open vast opportunities for probing response mechanisms and predictive biomarkers, and thereby guide the development of personalized therapies. Here, we review promising applications and remaining limitations of recently established functional-genetic tools for high-throughput screening and the in-depth analysis of candidate targets and established drugs.},
}
@article {pmid30951893,
year = {2019},
author = {Jyoti, A and Kaushik, S and Srivastava, VK and Datta, M and Kumar, S and Yugandhar, P and Kothari, SL and Rai, V and Jain, A},
title = {The potential application of genome editing by using CRISPR/Cas9, and its engineered and ortholog variants for studying the transcription factors involved in the maintenance of phosphate homeostasis in model plants.},
journal = {Seminars in cell &amp; developmental biology},
volume = {96},
number = {},
pages = {77-90},
doi = {10.1016/j.semcdb.2019.03.010},
pmid = {30951893},
issn = {1096-3634},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Variation/genetics ; Homeostasis/*genetics ; *Models, Biological ; Phosphates/*metabolism ; Plants/*genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Transcription Factors/*genetics/*metabolism ; },
abstract = {Phosphorus (P), an essential macronutrient, is pivotal for growth and development of plants. Availability of phosphate (Pi), the only assimilable P, is often suboptimal in rhizospheres. Pi deficiency triggers an array of spatiotemporal adaptive responses including the differential regulation of several transcription factors (TFs). Studies on MYB TF PHR1 in Arabidopsis thaliana (Arabidopsis) and its orthologs OsPHRs in Oryza sativa (rice) have provided empirical evidence of their significant roles in the maintenance of Pi homeostasis. Since the functional characterization of PHR1 in 2001, several other TFs have now been identified in these model plants. This raised a pertinent question whether there are any likely interactions across these TFs. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has provided an attractive paradigm for editing genome in plants. Here, we review the applications and challenges of this technique for genome editing of the TFs for deciphering the function and plausible interactions across them. This technology could thus provide a much-needed fillip towards engineering TFs for generating Pi use efficient plants for sustainable agriculture. Furthermore, we contemplate whether this technology could be a viable alternative to the controversial genetically modified (GM) rice or it may also eventually embroil into a limbo.},
}
@article {pmid30951856,
year = {2019},
author = {Skakic, A and Andjelkovic, M and Tosic, N and Klaassen, K and Djordjevic, M and Pavlovic, S and Stojiljkovic, M},
title = {CRISPR/Cas9 genome editing of SLC37A4 gene elucidates the role of molecular markers of endoplasmic reticulum stress and apoptosis in renal involvement in glycogen storage disease type Ib.},
journal = {Gene},
volume = {703},
number = {},
pages = {17-25},
doi = {10.1016/j.gene.2019.04.002},
pmid = {30951856},
issn = {1879-0038},
mesh = {Antiporters/*genetics ; Apoptosis ; CRISPR-Cas Systems ; Cell Line ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress ; Gene Editing/*methods ; Genetic Markers ; Glycogen Storage Disease Type I/complications/*genetics ; Humans ; Kidney Diseases/*etiology/genetics ; Models, Biological ; Monosaccharide Transport Proteins/*genetics ; Mutation ; *Point Mutation ; Unfolded Protein Response ; },
abstract = {Glycogen storage disease type Ib (GSD Ib) is an autosomal recessive disorder, caused by a deficiency of ubiquitously expressed SLC37A4 protein. Deficiency of SLC37A4 leads to abnormal storage of glycogen in the liver and kidneys, resulting in long-term complications of renal disease and hepatocellular adenomas, whose mechanisms are poorly understood. Molecular markers of the adaptive responses to the metabolic stress caused by a deficiency of SLC37A4, such as markers related to the endoplasmic reticulum (ER) stress and unfolded protein response (UPR), have not been extensively studied. The aim of this study was to investigate the expression of molecular markers of the UPR response and apoptosis related to a deficiency of SLC37A4 in kidney cells. For that purpose, we intended to establish a human kidney cell model system for GSD Ib. The novel variant c.248G>A, found in GSD Ib patients, was introduced into the Flp-In™T-REx™-293 cell line using CRISPR/Cas9-mediated precise gene editing method, resulting in significant decrease of SLC37A4 gene expression. In this model system we used RT-qPCR analysis to investigate the expression of molecular markers of the UPR response (ATF4, DDIT3, HSPA5, and XBP1s) and apoptosis (BCL2, BAX). We demonstrated that under chronic metabolic stress conditions caused by SLC37A4 deficiency, the ER stress-induced UPR was triggered, resulting in suppression of the UPR molecular markers and cell survival promotion (decreased expression levels of ATF4, DDIT3, HSPA5, with the exception of XBP1s). However, persistent metabolic stress overrides an adaptation and induces apoptosis through increased expression of pro-apoptotic markers (decreased ratio of BCL2/BAX genes). We established a cellular model system characterized by a deficiency of SLC37A4, which presents pathological manifestations of GSD Ib in the kidney. Expression analysis in a novel model system supports the hypothesis that renal dysfunction in the GSD Ib is partly due to the ER stress and increased apoptosis.},
}
@article {pmid30951810,
year = {2019},
author = {Li, L and Liu, X and Wei, K and Lu, Y and Jiang, W},
title = {Synthetic biology approaches for chromosomal integration of genes and pathways in industrial microbial systems.},
journal = {Biotechnology advances},
volume = {37},
number = {5},
pages = {730-745},
doi = {10.1016/j.biotechadv.2019.04.002},
pmid = {30951810},
issn = {1873-1899},
mesh = {Actinobacteria/genetics ; Chromosomal Instability ; *Chromosomes ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; Escherichia coli/genetics/metabolism ; Gene Dosage ; Genes ; Genome, Microbial ; Homologous Recombination ; Industrial Microbiology/*methods ; Integrases/genetics/metabolism ; Microorganisms, Genetically-Modified/*genetics ; Multigene Family ; Synthetic Biology/*methods ; Yeasts/genetics/metabolism ; },
abstract = {Industrial biotechnology is reliant on native pathway engineering or foreign pathway introduction for efficient biosynthesis of target products. Chromosomal integration, with intrinsic genetic stability, is an indispensable step for reliable expression of homologous or heterologous genes and pathways in large-scale and long-term fermentation. With advances in synthetic biology and CRISPR-based genome editing approaches, a wide variety of novel enabling technologies have been developed for single-step, markerless, multi-locus genomic integration of large biochemical pathways, which significantly facilitate microbial overproduction of chemicals, pharmaceuticals and other value-added biomolecules. Notably, the newly discovered homology-mediated end joining strategy could be widely applicable for high-efficiency genomic integration in a number of homologous recombination-deficient microbes. In this review, we explore the fundamental principles and characteristics of genomic integration, and highlight the development and applications of targeted integration approaches in the three representative industrial microbial systems, including Escherichia coli, actinomycetes and yeasts.},
}
@article {pmid30951658,
year = {2019},
author = {Trounson, A and Boyd, NR and Boyd, RL},
title = {Toward a Universal Solution: Editing Compatibility into Pluripotent Stem Cells.},
journal = {Cell stem cell},
volume = {24},
number = {4},
pages = {508-510},
doi = {10.1016/j.stem.2019.03.003},
pmid = {30951658},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Induced Pluripotent Stem Cells ; *Pluripotent Stem Cells ; },
abstract = {In this issue of Cell Stem Cell, Xu et al. (2019) demonstrate that editing iPSCs' major histocompatibility antigens may potentially provide a small set of universally compatible stem cell lines for therapies. However, these modifications may result in patient minor histocompatibility responses and deficiencies in their T cell response repertoire to infection and cancer.},
}
@article {pmid30951565,
year = {2019},
author = {Chang, SJ and Jin, SC and Jiao, X and Galán, JE},
title = {Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen.},
journal = {PLoS pathogens},
volume = {15},
number = {4},
pages = {e1007704},
pmid = {30951565},
issn = {1553-7374},
support = {R01 AI114618/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Bacterial Toxins/*metabolism ; Biological Transport ; CRISPR-Cas Systems ; *Endoplasmic Reticulum-Associated Degradation ; HeLa Cells ; Humans ; Intracellular Space/metabolism ; Protein Binding ; Salmonella typhi/genetics/*pathogenicity ; Typhoid Fever/genetics/metabolism/*microbiology ; },
abstract = {Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.},
}
@article {pmid30951393,
year = {2019},
author = {Ruotsalainen, P and Penttinen, R and Mattila, S and Jalasvuori, M},
title = {Midbiotics: conjugative plasmids for genetic engineering of natural gut flora.},
journal = {Gut microbes},
volume = {10},
number = {6},
pages = {643-653},
pmid = {30951393},
issn = {1949-0984},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; *Conjugation, Genetic ; Escherichia coli/drug effects/genetics ; Gastrointestinal Microbiome/*genetics ; Gene Editing ; Genetic Engineering/*methods ; Plasmids/genetics/*physiology ; RNA, Guide/genetics ; beta-Lactam Resistance/genetics ; },
abstract = {The possibility to modify gut bacterial flora has become an important goal, and various approaches are used to achieve desirable communities. However, the genetic engineering of existing microbes in the gut, which are already compatible with the rest of the community and host immune system, has not received much attention. Here, we discuss and experimentally evaluate the possibility to use modified and mobilizable CRISPR-Cas9-endocing plasmid as a tool to induce changes in bacterial communities. This plasmid system (briefly midbiotic) is delivered from bacterial vector into target bacteria via conjugation. Compared to, for example, bacteriophage-based applications, the benefits of conjugative plasmids include their independence of any particular receptor(s) on host bacteria and their relative immunity to bacterial defense mechanisms (such as restriction-modification systems) due to the synthesis of the complementary strand with host-specific epigenetic modifications. We show that conjugative plasmid in association with a mobilizable antibiotic resistance gene targeting CRISPR-plasmid efficiently causes ESBL-positive transconjugants to lose their resistance, and multiple gene types can be targeted simultaneously by introducing several CRISPR RNA encoding segments into the transferred plasmids. In the rare cases where the midbiotic plasmids failed to resensitize bacteria to antibiotics, the CRISPR spacer(s) and their adjacent repeats or larger regions were found to be lost. Results also revealed potential caveats in the design of conjugative engineering systems as well as workarounds to minimize these risks.},
}
@article {pmid30951374,
year = {2019},
author = {Lee, HJ and Yoon, JW and Jung, KM and Kim, YM and Park, JS and Lee, KY and Park, KJ and Hwang, YS and Park, YH and Rengaraj, D and Han, JY},
title = {Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {7},
pages = {8519-8529},
doi = {10.1096/fj.201802671R},
pmid = {30951374},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; Chickens/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genome/genetics ; Germ Cells/*physiology ; Green Fluorescent Proteins/genetics ; Mutagenesis, Insertional/methods ; RNA, Guide/genetics ; Sex Chromosomes/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.},
}
@article {pmid30951293,
year = {2019},
author = {Liu, Y and Ren, CY and Wei, WP and You, D and Yin, BC and Ye, BC},
title = {A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora erythraea Erythromycin Biosynthetic Gene Cluster with Knock-in Bidirectional Promoters.},
journal = {ACS synthetic biology},
volume = {8},
number = {5},
pages = {1134-1143},
doi = {10.1021/acssynbio.9b00024},
pmid = {30951293},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Erythromycin/*biosynthesis ; Flavoproteins/genetics ; Isocitrate Lyase/genetics ; Multigene Family ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Saccharopolyspora/*genetics ; },
abstract = {The regulation of biosynthetic pathways is a universal strategy for industrial strains that overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were tested by reporter mCherry. The SACE_0720 (eryBIV)-SACE_0721 (eryAI) spacer was selected as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs) were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression increased 32 and 79 times, respectively. High performance liquid chromatography results showed that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in bidirectional promoter knock-in recombinant strains. On the basis of the activated strain Ab::Pj23119-PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study showed that bidirectional promoter knock-in and CRISPR interference could regulate gene expression in S. erythraea. This strategy has potential application for biosynthetic gene cluster activation and gene regulation in Actinobacteria.},
}
@article {pmid30950179,
year = {2019},
author = {Bernabé-Orts, JM and Casas-Rodrigo, I and Minguet, EG and Landolfi, V and Garcia-Carpintero, V and Gianoglio, S and Vázquez-Vilar, M and Granell, A and Orzaez, D},
title = {Assessment of Cas12a-mediated gene editing efficiency in plants.},
journal = {Plant biotechnology journal},
volume = {17},
number = {10},
pages = {1971-1984},
pmid = {30950179},
issn = {1467-7652},
mesh = {Arabidopsis/genetics ; *CRISPR-Cas Systems ; Endonucleases ; *Gene Editing ; *Genome, Plant ; Lycopersicon esculentum/genetics ; Mutagenesis ; Sequence Deletion ; Tobacco/genetics ; },
abstract = {The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB-assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target-dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on-target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a-free segregating T2 lines to assess possible off-target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of -10 to -2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off-target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.},
}
@article {pmid30949703,
year = {2019},
author = {Liu, H and Huang, T and Li, M and Li, M and Zhang, C and Jiang, J and Yu, X and Yin, Y and Zhang, F and Lu, G and Luo, MC and Zhang, LR and Li, J and Liu, K and Chen, ZJ},
title = {SCRE serves as a unique synaptonemal complex fastener and is essential for progression of meiosis prophase I in mice.},
journal = {Nucleic acids research},
volume = {47},
number = {11},
pages = {5670-5683},
pmid = {30949703},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/*physiology ; Chromosome Segregation ; DNA-Binding Proteins ; Female ; HEK293 Cells ; Humans ; Male ; Meiosis ; *Meiotic Prophase I ; Mice ; Mice, Knockout ; Nuclear Proteins/genetics/*physiology ; Protein Binding ; Recombination, Genetic ; Spermatocytes/metabolism ; Synaptonemal Complex/*physiology ; Testis/metabolism ; },
abstract = {Meiosis is a specialized cell division for producing haploid gametes from diploid germ cells. During meiosis, synaptonemal complex (SC) mediates the alignment of homologs and plays essential roles in homologous recombination and therefore in promoting accurate chromosome segregation. In this study, we have identified a novel protein SCRE (synaptonemal complex reinforcing element) as a key molecule in maintaining the integrity of SC during meiosis prophase I in mice. Deletion of Scre (synaptonemal complex reinforcing element) caused germ cell death in both male and female mice, resulting in infertility. Our mechanistic studies showed that the synapses and SCs in Scre knockout mice were unstable due to the lack of the SC reinforcing function of SCRE, which is sparsely localized as discrete foci along the central elements in normal synaptic homologous chromosomes. The lack of Scre leads to meiosis collapse at the late zygotene stage. We further showed that SCRE interacts with synaptonemal complex protein 1 (SYCP1) and synaptonemal complex central element 3 (SYCE3). We conclude that the function of SCRE is to reinforce the integrity of the central elements, thereby stabilizing the SC and ensuring meiotic cell cycle progression. Our study identified SCRE as a novel SC fastener protein that is distinct from other known SC proteins.},
}
@article {pmid30949579,
year = {2019},
author = {Pan, Y and Yang, J and Luan, X and Liu, X and Li, X and Yang, J and Huang, T and Sun, L and Wang, Y and Lin, Y and Song, Y},
title = {Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.},
journal = {Science advances},
volume = {5},
number = {4},
pages = {eaav7199},
pmid = {30949579},
issn = {2375-2548},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/*genetics/radiation effects ; Carcinoma, Squamous Cell/genetics/pathology/*therapy ; Cell Proliferation ; Drug Carriers/*chemistry ; Female ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; *Infrared Rays ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mouth Neoplasms/genetics/pathology/*therapy ; Nanoparticles/*administration &amp; dosage/chemistry ; RNA, Guide/genetics/radiation effects ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light-responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as "nanotransducers" that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light-activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.},
}
@article {pmid30948824,
year = {2018},
author = {Lee, B and Lee, K and Panda, S and Gonzales-Rojas, R and Chong, A and Bugay, V and Park, HM and Brenner, R and Murthy, N and Lee, HY},
title = {Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours.},
journal = {Nature biomedical engineering},
volume = {2},
number = {7},
pages = {497-507},
pmid = {30948824},
issn = {2157-846X},
support = {R01 AI117064/AI/NIAID NIH HHS/United States ; R01 EB020008/EB/NIBIB NIH HHS/United States ; R01 EB023776/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal ; Brain/*metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Corpus Striatum/metabolism ; Disease Models, Animal ; Fragile X Mental Retardation Protein/genetics/metabolism ; Fragile X Syndrome/metabolism/*pathology ; Gold/chemistry ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nanoparticles/*chemistry/toxicity ; Neurons/cytology/drug effects/metabolism ; Patch-Clamp Techniques ; Receptor, Metabotropic Glutamate 5/genetics/metabolism ; Thy-1 Antigens/genetics/metabolism ; },
abstract = {Technologies that can safely edit genes in the brains of adult animals may revolutionize the treatment of neurological diseases and the understanding of brain function. Here, we demonstrate that intracranial injection of CRISPR-Gold, a nonviral delivery vehicle for the CRISPR-Cas9 ribonucleoprotein, can edit genes in the brains of adult mice in multiple mouse models. CRISPR-Gold can deliver both Cas9 and Cpf1 ribonucleoproteins, and can edit all of the major cell types in the brain, including neurons, astrocytes and microglia, with undetectable levels of toxicity at the doses used. We also show that CRISPR-Gold designed to target the metabotropic glutamate receptor 5 (mGluR5) gene can efficiently reduce local mGluR5 levels in the striatum after an intracranial injection. The effect can also rescue mice from the exaggerated repetitive behaviours caused by fragile X syndrome, a common single-gene form of autism spectrum disorders. CRISPR-Gold may significantly accelerate the development of brain-targeted therapeutics and enable the rapid development of focal brain-knockout animal models.},
}
@article {pmid30948787,
year = {2019},
author = {Zong, Y and Zhang, CS and Li, M and Wang, W and Wang, Z and Hawley, SA and Ma, T and Feng, JW and Tian, X and Qi, Q and Wu, YQ and Zhang, C and Ye, Z and Lin, SY and Piao, HL and Hardie, DG and Lin, SC},
title = {Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress.},
journal = {Cell research},
volume = {29},
number = {6},
pages = {460-473},
pmid = {30948787},
issn = {1748-7838},
support = {/WT_/Wellcome Trust/United Kingdom ; 204766/WT_/Wellcome Trust/United Kingdom ; 204766/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {AMP-Activated Protein Kinases/biosynthesis/*metabolism ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; *Energy Metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Transgenic ; Microscopy, Fluorescence ; Nutrients/*metabolism ; Phosphorylation ; },
abstract = {AMPK, a master regulator of metabolic homeostasis, is activated by both AMP-dependent and AMP-independent mechanisms. The conditions under which these different mechanisms operate, and their biological implications are unclear. Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.},
}
@article {pmid30948498,
year = {2019},
author = {Zimmer, AM and Pan, YK and Chandrapalan, T and Kwong, RWM and Perry, SF},
title = {Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?.},
journal = {The Journal of experimental biology},
volume = {222},
number = {Pt 7},
pages = {},
doi = {10.1242/jeb.175737},
pmid = {30948498},
issn = {1477-9145},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; Gene Knockout Techniques/methods ; Morpholinos ; Phenotype ; Physiology, Comparative/*methods ; RNA Interference ; Transcription Activator-Like Effector Nucleases ; Zebrafish/*genetics/physiology ; },
abstract = {Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses).},
}
@article {pmid30948423,
year = {2019},
author = {Hu, XF and Zhang, B and Liao, CH and Zeng, ZJ},
title = {High-Efficiency CRISPR/Cas9-Mediated Gene Editing in Honeybee (Apis mellifera) Embryos.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {5},
pages = {1759-1766},
pmid = {30948423},
issn = {2160-1836},
mesh = {Animals ; Base Sequence ; Bees/*genetics ; *CRISPR-Cas Systems ; *Embryo, Nonmammalian ; *Gene Editing ; Gene Knockdown Techniques ; Glycoproteins/chemistry/genetics ; Insect Proteins/chemistry/genetics ; Mutagenesis ; Mutation ; PAX6 Transcription Factor/chemistry/genetics ; RNA, Guide ; },
abstract = {The honeybee (Apis mellifera) is an important insect pollinator of wild flowers and crops, playing critical roles in the global ecosystem. Additionally, the honeybee serves as an ideal social insect model. Therefore, functional studies on honeybee genes are of great interest. However, until now, effective gene manipulation methods have not been available in honeybees. Here, we reported an improved CRISPR/Cas9 gene-editing method by microinjecting sgRNA and Cas9 protein into the region of zygote formation within 2 hr after queen oviposition, which allows one-step generation of biallelic knockout mutants in honeybee with high efficiency. We first targeted the Mrjp1 gene. Two batches of honeybee embryos were collected and injected with Mrjp1 sgRNA and Cas9 protein at the ventral cephalic side and the dorsal posterior side of the embryos, respectively. The gene-editing rate at the ventral cephalic side was 93.3%, which was much higher than that (11.8%) of the dorsal-posterior-side injection. To validate the high efficiency of our honeybee gene-editing system, we targeted another gene, Pax6, and injected Pax6 sgRNA and Cas9 protein at the ventral cephalic side in the third batch. A 100% editing rate was obtained. Sanger sequencing of the TA clones showed that 73.3% (for Mrjp1) and 76.9% (for Pax6) of the edited current-generation embryos were biallelic knockout mutants. These results suggest that the CRISPR/Cas9 method we established permits one-step biallelic knockout of target genes in honeybee embryos, thereby demonstrating an efficient application to functional studies of honeybee genes. It also provides a useful reference to gene editing in other insects with elongated eggs.},
}
@article {pmid30948034,
year = {2019},
author = {Hamdoun, A and Foltz, KR},
title = {Preface.},
journal = {Methods in cell biology},
volume = {151},
number = {},
pages = {xxi-xxii},
doi = {10.1016/S0091-679X(19)30046-9},
pmid = {30948034},
issn = {0091-679X},
support = {R01 ES027921/ES/NIEHS NIH HHS/United States ; R01 ES030318/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Biology/*trends ; Cytological Techniques/*methods ; Genome/genetics ; Genomics/*methods ; Germ Cells/growth &amp; development ; },
}
@article {pmid30948015,
year = {2019},
author = {Lin, CY and Oulhen, N and Wessel, G and Su, YH},
title = {CRISPR/Cas9-mediated genome editing in sea urchins.},
journal = {Methods in cell biology},
volume = {151},
number = {},
pages = {305-321},
pmid = {30948015},
issn = {0091-679X},
support = {R01 GM125071/GM/NIGMS NIH HHS/United States ; R01 GM132222/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cytidine Deaminase/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Gene Targeting/methods ; Genetic Vectors ; Genome/genetics ; Sea Urchins/*genetics/growth &amp; development ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) technology enables rapid, targeted, and efficient changes in the genomes of various model organisms. The short guide RNAs (gRNAs) of the CRISPR/Cas9 system can be designed to recognize target DNA within coding regions for functional gene knockouts. Several studies have demonstrated that the CRISPR/Cas9 system efficiently and specifically targets sea urchin genes and results in expected mutant phenotypes. In addition to disrupting gene functions, modifications and additions to the Cas9 protein enable alternative activities targeted to specific sites within the genome. This includes a fusion of cytidine deaminase to Cas9 (Cas9-DA) for single nucleotide conversion in targeted sites. In this chapter, we describe detailed methods for the CRISPR/Cas9 application in sea urchin embryos, including gRNA design, in vitro synthesis of single guide RNA (sgRNA), and the usages of the CRISPR/Cas9 technology for gene knockout and single nucleotide editing. Methods for genotyping the resultant embryos are also provided for assessing efficiencies of gene editing.},
}
@article {pmid30947515,
year = {2019},
author = {Dunbar, GL and Koneru, S and Kolli, N and Sandstrom, M and Maiti, P and Rossignol, J},
title = {Silencing of the Mutant Huntingtin Gene through CRISPR-Cas9 Improves the Mitochondrial Biomarkers in an In Vitro Model of Huntington's Disease.},
journal = {Cell transplantation},
volume = {28},
number = {4},
pages = {460-463},
pmid = {30947515},
issn = {1555-3892},
mesh = {Biomarkers/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Huntington Disease/*genetics ; Mitochondria/*metabolism ; },
abstract = {During the 25-year history of the American Society for Neural Therapy and Repair (ASNTR) there have been several breakthroughs in the area of neurotherapeutics, which was the case during the 2014-2105 year when one of us (GLD) had the privilege of serving as its president. During that year, the use of a newly developed gene-editing tool, the CRISPR-Cas9 system, started to skyrocket. Although scientists unraveled the use of "clustered regularly interspaced short palindromic repeats" (CRISPR) and its associated genes from the Cas family as an evolved mechanism of some bacterial and archaeal genomes to protect themselves from being hijacked by invasive viral genes, its use as a therapeutic tool was not fully appreciated until further research revealed how this system operated and how it might be developed technologically to manipulate genes of any species. By 2015, this technology had exploded to the point that close to 2,000 papers that used this technology were published during that year alone.},
}
@article {pmid30946744,
year = {2019},
author = {So, CC and Martin, A},
title = {DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells.},
journal = {PLoS genetics},
volume = {15},
number = {4},
pages = {e1008101},
pmid = {30946744},
issn = {1553-7404},
support = {PJT-156330//Canadian Institutes of Health Research/International ; },
mesh = {B-Lymphocytes/*immunology/*metabolism ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/metabolism ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics/immunology ; Humans ; *Immunoglobulin Class Switching ; Immunoglobulin Heavy Chains/genetics ; Models, Genetic ; Proto-Oncogene Proteins c-bcl-6/genetics ; Recombination, Genetic ; *Translocation, Genetic ; },
abstract = {Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5' and 3' overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5' overhangs were removed and 3' overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells.},
}
@article {pmid30945167,
year = {2019},
author = {Patsali, P and Kleanthous, M and Lederer, CW},
title = {Disruptive Technology: CRISPR/Cas-Based Tools and Approaches.},
journal = {Molecular diagnosis &amp; therapy},
volume = {23},
number = {2},
pages = {187-200},
pmid = {30945167},
issn = {1179-2000},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Endonucleases/metabolism ; Gene Editing ; *Genetic Techniques ; Genetic Therapy ; Humans ; },
abstract = {Designer nucleases are versatile tools for genome modification and therapy development and have gained widespread accessibility with the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology. Prokaryotic RNA-guided nucleases of CRISPR/Cas type, since first being adopted as editing tools in eukaryotic cells, have experienced rapid uptake and development. Diverse modes of delivery by viral and non-viral vectors and ongoing discovery and engineering of new CRISPR/Cas-type tools with alternative target site requirements, cleavage patterns and DNA- or RNA-specific action continue to expand the versatility of this family of nucleases. CRISPR/Cas-based molecules may also act without double-strand breaks as DNA base editors or even without single-stranded cleavage, be it as epigenetic regulators, transcription factors or RNA base editors, with further scope for discovery and development. For many potential therapeutic applications of CRISPR/Cas-type molecules and their derivatives, efficiencies still need to be improved and safety issues addressed, including those of preexisting immunity against Cas molecules, off-target activity and recombination and sequence alterations relating to double-strand-break events. This review gives a concise overview of current CRISPR/Cas tools, applications, concerns and trends.},
}
@article {pmid30945166,
year = {2019},
author = {Papasavva, P and Kleanthous, M and Lederer, CW},
title = {Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases.},
journal = {Molecular diagnosis &amp; therapy},
volume = {23},
number = {2},
pages = {201-222},
pmid = {30945166},
issn = {1179-2000},
mesh = {CRISPR-Cas Systems/*genetics ; Genetic Diseases, Inborn/*therapy ; Genetic Therapy ; Humans ; Mutation/genetics ; Rare Diseases/*therapy ; Translational Research, Biomedical ; },
abstract = {Rare diseases pose a global challenge, in that their collective impact on health systems is considerable, whereas their individually rare occurrence impedes research and development of efficient therapies. In consequence, patients and their families are often unable to find an expert for their affliction, let alone a cure. The tide is turning as pharmaceutical companies embrace gene therapy development and as serviceable tools for the repair of primary mutations separate the ability to create cures from underlying disease expertise. Whereas gene therapy by gene addition took decades to reach the clinic by incremental disease-specific refinements of vectors and methods, gene therapy by genome editing in its basic form merely requires certainty about the causative mutation. Suddenly we move from concept to trial in 3 years instead of 30: therapy development in the fast lane, with all the positive and negative implications of the phrase. Since their first application to eukaryotic cells in 2013, the proliferation and refinement in particular of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) prokaryotic RNA-guided nucleases has prompted a landslide of therapy-development studies for rare diseases. An estimated thousands of orphan diseases are up for adoption, and legislative, entrepreneurial, and research initiatives may finally conspire to find many of them a good home. Here we summarize the most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases and take a glimpse at the exciting road ahead.},
}
@article {pmid30944478,
year = {2019},
author = {Pluvinage, JV and Haney, MS and Smith, BAH and Sun, J and Iram, T and Bonanno, L and Li, L and Lee, DP and Morgens, DW and Yang, AC and Shuken, SR and Gate, D and Scott, M and Khatri, P and Luo, J and Bertozzi, CR and Bassik, MC and Wyss-Coray, T},
title = {CD22 blockade restores homeostatic microglial phagocytosis in ageing brains.},
journal = {Nature},
volume = {568},
number = {7751},
pages = {187-192},
pmid = {30944478},
issn = {1476-4687},
support = {R01 GM059907/GM/NIGMS NIH HHS/United States ; T32 GM007365/GM/NIGMS NIH HHS/United States ; R01 AG045034/AG/NIA NIH HHS/United States ; S10 OD020141/OD/NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; F30 AG060638/AG/NIA NIH HHS/United States ; T32 GM120007/GM/NIGMS NIH HHS/United States ; DP1 AG053015/AG/NIA NIH HHS/United States ; R01 CA227942/CA/NCI NIH HHS/United States ; },
mesh = {Aging/drug effects/genetics/*physiology ; Animals ; Brain/*cytology/drug effects/physiology ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cognition/drug effects/physiology ; Female ; Homeostasis/*drug effects/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Microglia/cytology/*drug effects ; N-Acetylneuraminic Acid/chemistry/*pharmacology ; Phagocytosis/*drug effects/genetics ; Sequence Analysis, RNA ; Sialic Acid Binding Ig-like Lectin 2/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-β oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.},
}
@article {pmid30944435,
year = {2019},
author = {Hsu, MN and Hu, YC},
title = {Local magnetic activation of CRISPR.},
journal = {Nature biomedical engineering},
volume = {3},
number = {2},
pages = {83-84},
pmid = {30944435},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Magnetic Phenomena ; },
}
@article {pmid30944431,
year = {2019},
author = {Zhu, H and Zhang, L and Tong, S and Lee, CM and Deshmukh, H and Bao, G},
title = {Spatial control of in vivo CRISPR-Cas9 genome editing via nanomagnets.},
journal = {Nature biomedical engineering},
volume = {3},
number = {2},
pages = {126-136},
pmid = {30944431},
issn = {2157-846X},
support = {PN2 EY018244/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Baculoviridae/genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Endocytosis ; Female ; *Gene Editing ; Genetic Vectors/metabolism ; Humans ; Liver/metabolism ; Luciferases/metabolism ; Magnetic Fields ; *Magnetic Phenomena ; Magnetite Nanoparticles/*chemistry/ultrastructure ; Mice, Nude ; Transgenes ; },
abstract = {The potential of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9)-based therapeutic genome editing is hampered by difficulties in the control of the in vivo activity of CRISPR-Cas9. To minimize any genotoxicity, precise activation of CRISPR-Cas9 in the target tissue is desirable. Here, we show that, by complexing magnetic nanoparticles with recombinant baculoviral vectors (MNP-BVs), CRISPR-Cas9-mediated genome editing can be activated locally in vivo via a magnetic field. The baculoviral vector was chosen for in vivo gene delivery because of its large loading capacity and ability to locally overcome systemic inactivation by the complement system. We demonstrate that a locally applied magnetic field can enhance the cellular entry of MNP-BVs, thereby avoiding baculoviral vector inactivation and causing a transient transgene expression in the target tissue. Because baculoviral vectors are inactivated elsewhere, gene delivery and in vivo genome editing via MNP-BVs are tissue specific.},
}
@article {pmid30944210,
year = {2019},
author = {Xu, J and Pei, D and Nicholson, A and Lan, Y and Xia, Q},
title = {In Silico Identification of Three Types of Integrative and Conjugative Elements in Elizabethkingia anophelis Strains Isolated from around the World.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
pmid = {30944210},
issn = {2379-5042},
support = {SC1 AI112786/AI/NIAID NIH HHS/United States ; },
mesh = {Adaptive Immunity ; Animals ; Bacterial Outer Membrane Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Conjugation, Genetic/*genetics ; Culicidae/microbiology ; DNA, Intergenic/genetics ; Flavobacteriaceae/*genetics/*isolation &amp; purification/pathogenicity ; Flavobacteriaceae Infections/microbiology ; *Genetic Variation ; *Genome, Bacterial ; Genomics ; Global Health ; Humans ; Phylogeny ; Virulence/genetics ; },
abstract = {Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis strains derived from various environmental settings and human infections from different geographic regions around the world from 1950s to the present. Putative integrative and conjugative elements (ICEs) were identified in 31/36 (86.1%) strains in the study. A total of 52 putative ICEs (including eight degenerated elements lacking integrases) were identified and categorized into three types based on the architecture of the conjugation module and the phylogeny of the relaxase, coupling protein, TraG, and TraJ protein sequences. The type II and III ICEs were found to integrate adjacent to tRNA genes, while type I ICEs integrate into intergenic regions or into a gene. The ICEs carry various cargo genes, including transcription regulator genes and genes conferring antibiotic resistance. The adaptive immune CRISPR-Cas system was found in nine strains, including five strains in which CRISPR-Cas machinery and ICEs coexist at different locations on the same chromosome. One ICE-derived spacer was present in the CRISPR locus in one strain. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in architecture and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. The categorization of ICEs will facilitate further investigations of the impact of ICE on virulence, genome epidemiology, and adaptive genomics of E. anophelisIMPORTANCEElizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. Genome comparison identified three types of putative ICEs in 31 of 36 strains. The diversity of ICEs suggests that they had different origins. One of the ICEs was discovered previously from a large E. anophelis outbreak in Wisconsin in the United States; this ICE has integrated into the mutY gene of the outbreak strain, creating a mutator phenotype. Similar to ICEs found in many bacterial species, ICEs in E. anophelis carry various cargo genes that enable recipients to resist antibiotics and adapt to various ecological niches. The adaptive immune CRISPR-Cas system is present in nine of 36 strains. An ICE-derived spacer was found in the CRISPR locus in a strain that has no ICE, suggesting a past encounter and effective defense against ICE.},
}
@article {pmid30942690,
year = {2019},
author = {Chou-Zheng, L and Hatoum-Aslan, A},
title = {A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30942690},
issn = {2050-084X},
support = {K22 AI113106/AI/NIAID NIH HHS/United States ; Career Development Award, 5K22AI113106-02//National Institute of Allergy and Infectious Diseases/International ; CAREER Award, 1749886//National Science Foundation/International ; },
mesh = {*CRISPR-Cas Systems ; DNA, Bacterial/genetics/metabolism ; DNA, Viral/genetics/metabolism ; Endonucleases/*metabolism ; Endoribonucleases/*metabolism ; Interspersed Repetitive Sequences ; Multienzyme Complexes/*metabolism ; Plasmids ; Polyribonucleotide Nucleotidyltransferase/*metabolism ; RNA Helicases/*metabolism ; Staphylococcus Phages/genetics ; Staphylococcus epidermidis/*enzymology/*genetics ; },
abstract = {CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.},
}
@article {pmid30942216,
year = {2019},
author = {Qiao, J and Sun, W and Lin, S and Jin, R and Ma, L and Liu, Y},
title = {Cytosolic delivery of CRISPR/Cas9 ribonucleoproteins for genome editing using chitosan-coated red fluorescent protein.},
journal = {Chemical communications (Cambridge, England)},
volume = {55},
number = {32},
pages = {4707-4710},
doi = {10.1039/c9cc00010k},
pmid = {30942216},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; Chitosan/*chemistry ; DNA, Single-Stranded/metabolism ; Endocytosis ; Escherichia coli/genetics ; Gene Editing/*methods ; Humans ; Luminescent Proteins/*chemistry ; Nanoparticles/chemistry ; Protein Transport ; Ribonucleoproteins/chemistry/*metabolism ; Streptococcus pyogenes/genetics ; },
abstract = {Though plenty of viral and non-viral methods have been rapidly developed for the delivery of the CRISPR/Cas9 system, the direct delivery of Cas9 ribonucleoproteins (RNPs) into the nucleus for genome editing via the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, especially the latter one, remains a challenge. Here we report a delivery vehicle achieved by encapsulating red fluorescent protein (RFP) within chitosan (CS), which can simultaneously deliver engineered Cas9 RNPs with a poly-glutamate peptide tag (E-tag) and DNA donors into a range of cell types with high genome editing efficacy and non-cytotoxicity.},
}
@article {pmid30941642,
year = {2019},
author = {Kumar, R and Kaur, A and Pandey, A and Mamrutha, HM and Singh, GP},
title = {CRISPR-based genome editing in wheat: a comprehensive review and future prospects.},
journal = {Molecular biology reports},
volume = {46},
number = {3},
pages = {3557-3569},
pmid = {30941642},
issn = {1573-4978},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genes, Plant/genetics ; Genetic Engineering/methods ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Poaceae/genetics ; Triticum/*genetics ; },
abstract = {CRISPR technology has vividly increased its applications in last five years for genome editing in a wide range of organisms from bacteria to plants. It is mostly applied in the field of mammalian research. This emerging versatile tool can be utilized in crop improvement by targeting various traits to increase economic value and adaptability of the crop species under changing climate. In plants, Arabidopsis and rice are the most studied plant species in genome editing through CRISPR technology. Wheat is lagging behind in the utilization of CRISPR based genome modifications. The hexaploid, large genome size and the recalcitrant nature in terms of tissue culture are the major obstacles for CRISPR utilization in wheat. Recently, the IWGSC released the high quality of reference genome for wheat which will greatly accelerate the application of CRISPR-based genome engineering in wheat and helps to resolve the global issue of food security in coming decades. The exogenous DNA-free improved mutants with CRISPR technology having desired traits will increase the productivity under biotic and abiotic stress conditions. To address complex traits involving multigene, recently developed multiplex genome editing toolkits can be used. This is a first review of its kind in which the practical utilization and updates on CRISPR validation in wheat along with its future prospects for use of this technology in wheat improvement are comprehensively discussed. Thus, the compiled information will immensely benefit the researchers for utilization of CRISPR system in wheat improvement across the globe.},
}
@article {pmid30941371,
year = {2019},
author = {Sui, C and Jiang, D and Wu, X and Cong, X and Li, F and Shang, Y and Wang, J and Liu, S and Shan, H and Qi, J and Du, Y},
title = {CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication.},
journal = {BioMed research international},
volume = {2019},
number = {},
pages = {7398208},
pmid = {30941371},
issn = {2314-6141},
mesh = {Animals ; Apoptosis/drug effects ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Endoribonucleases/*metabolism ; Gene Editing ; *Gene Knockout Techniques ; Herpesvirus 1, Suid/drug effects/growth &amp; development/*physiology ; Poly I-C/pharmacology ; Pseudorabies/virology ; RNA Stability/genetics ; RNA, Messenger/genetics/metabolism ; RNA, Ribosomal/genetics ; Swine ; Viral Vaccines/immunology ; Virus Replication/drug effects/*physiology ; },
abstract = {Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.},
}
@article {pmid30941126,
year = {2019},
author = {Liu, J and Zhou, G and Zhang, L and Zhao, Q},
title = {Building Potent Chimeric Antigen Receptor T Cells With CRISPR Genome Editing.},
journal = {Frontiers in immunology},
volume = {10},
number = {},
pages = {456},
pmid = {30941126},
issn = {1664-3224},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Graft vs Host Disease/etiology ; Humans ; Immunotherapy, Adoptive/*adverse effects/trends ; Neoplasms/*therapy ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/immunology ; Receptors, Chimeric Antigen/*genetics ; T-Lymphocytes/*immunology ; },
abstract = {Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematological and solid malignancies. However, despite the success of this field, there remain some major challenges, including accelerated T cell exhaustion, potential toxicities, and insertional oncogenesis. To overcome these limitations, recent advances in CRISPR technology have enabled targetable interventions of endogenous genes in human CAR T cells. These CRISPR genome editing approaches have unleashed the therapeutic potential of CAR T cell therapy. Here, we summarize the potential benefits, safety concerns, and difficulties in the generation of gene-edited CAR T cells using CRISPR technology.},
}
@article {pmid30940750,
year = {2019},
author = {Noble, C and Min, J and Olejarz, J and Buchthal, J and Chavez, A and Smidler, AL and DeBenedictis, EA and Church, GM and Nowak, MA and Esvelt, KM},
title = {Daisy-chain gene drives for the alteration of local populations.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {17},
pages = {8275-8282},
pmid = {30940750},
issn = {1091-6490},
support = {DP2 AI136597/AI/NIAID NIH HHS/United States ; R00 DK102669/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Anopheles/genetics ; CRISPR-Cas Systems/*genetics ; Ecology ; Female ; Gene Drive Technology/*methods/*standards ; Genetic Engineering ; HEK293 Cells ; Humans ; Malaria/prevention &amp; control ; Male ; Organisms, Genetically Modified/*genetics ; RNA, Guide/genetics ; },
abstract = {If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. "Daisy-drive" systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.},
}
@article {pmid30940608,
year = {2019},
author = {Alyami, HM and Finoti, LS and Teixeira, HS and Aljefri, A and Kinane, DF and Benakanakere, MR},
title = {Role of NOD1/NOD2 receptors in Fusobacterium nucleatum mediated NETosis.},
journal = {Microbial pathogenesis},
volume = {131},
number = {},
pages = {53-64},
doi = {10.1016/j.micpath.2019.03.036},
pmid = {30940608},
issn = {1096-1208},
mesh = {Biofilms ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Fusobacterium nucleatum/*pathogenicity ; HL-60 Cells ; Histones/metabolism ; Humans ; Leukocyte Elastase/metabolism ; Neutrophils/*immunology/*metabolism ; Nod1 Signaling Adaptor Protein/*metabolism ; Nod2 Signaling Adaptor Protein/*metabolism ; Periodontitis/*metabolism/microbiology ; Peroxidase/metabolism ; Phagocytosis ; Protein-Arginine Deiminase Type 4 ; Protein-Arginine Deiminases/metabolism ; Signal Transduction ; },
abstract = {Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.},
}
@article {pmid30940506,
year = {2020},
author = {Wendisch, VF},
title = {Metabolic engineering advances and prospects for amino acid production.},
journal = {Metabolic engineering},
volume = {58},
number = {},
pages = {17-34},
doi = {10.1016/j.ymben.2019.03.008},
pmid = {30940506},
issn = {1096-7184},
mesh = {*Amino Acids/biosynthesis/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Metabolic Engineering ; *Microorganisms, Genetically-Modified/genetics/metabolism ; },
abstract = {Amino acid fermentation is one of the major pillars of industrial biotechnology. The multi-billion USD amino acid market is rising steadily and is diversifying. Metabolic engineering is no longer focused solely on strain development for the bulk amino acids L-glutamate and L-lysine that are produced at the million-ton scale, but targets specialty amino acids. These demands are met by the development and application of new metabolic engineering tools including CRISPR and biosensor technologies as well as production processes by enabling a flexible feedstock concept, co-production and co-cultivation schemes. Metabolic engineering advances are exemplified for specialty proteinogenic amino acids, cyclic amino acids, omega-amino acids, and amino acids functionalized by hydroxylation, halogenation and N-methylation.},
}
@article {pmid30940138,
year = {2019},
author = {Cai, P and Gao, J and Zhou, Y},
title = {CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {63},
pmid = {30940138},
issn = {1475-2859},
mesh = {Biotechnology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Genome, Fungal ; Saccharomyces cerevisiae/*genetics/metabolism ; },
abstract = {Non-conventional yeasts are playing important roles as cell factories for bioproduction of biofuels, food additives and proteins with outstanding natural characteristics. However, the precise genome editing is challenging in non-conventional yeasts due to lack of efficient genetic tools. In the past few years, CRISPR-based genome editing worked as a revolutionary tool for genetic engineering and showed great advantages in cellular metabolic engineering. Here, we review the current advances and barriers of CRISPR-Cas9 for genome editing in non-conventional yeasts and propose the possible solutions in enhancing its efficiency for precise genetic engineering.},
}
@article {pmid30939239,
year = {2019},
author = {McCarty, NS and Shaw, WM and Ellis, T and Ledesma-Amaro, R},
title = {Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast.},
journal = {ACS synthetic biology},
volume = {8},
number = {4},
pages = {906-910},
doi = {10.1021/acssynbio.9b00041},
pmid = {30939239},
issn = {2161-5063},
support = {BB/R01602X/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/K019791/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Cloning, Molecular/methods ; Gene Editing/methods ; Gene Expression/genetics ; Polymerase Chain Reaction/methods ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/*genetics ; Transcription, Genetic/genetics ; },
abstract = {CRISPR is a versatile technology for genomic editing and regulation, but the expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and expressed from a single transcriptional unit. We demonstrate that these gRNA arrays can stably regulate gene expression in a synergistic manner via dCas9-mediated repression. This approach expands the number of gRNAs that can be expressed in this model organism and may enable the versatile editing or transcriptional regulation of a greater number of genes in vivo.},
}
@article {pmid30937444,
year = {2019},
author = {Almendros, C and Nobrega, FL and McKenzie, RE and Brouns, SJJ},
title = {Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation.},
journal = {Nucleic acids research},
volume = {47},
number = {10},
pages = {5223-5230},
pmid = {30937444},
issn = {1362-4962},
mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Catalytic Domain ; DNA, Bacterial/metabolism ; Escherichia coli/metabolism ; *Gene Expression Regulation, Bacterial ; Gene Fusion ; Genes, Bacterial ; Geobacter/*genetics/metabolism ; Models, Genetic ; *Mutation ; Phylogeny ; Plasmids/genetics ; Sequence Analysis, DNA ; },
abstract = {Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.},
}
@article {pmid30936531,
year = {2019},
author = {Knott, GJ and Thornton, BW and Lobba, MJ and Liu, JJ and Al-Shayeb, B and Watters, KE and Doudna, JA},
title = {Broad-spectrum enzymatic inhibition of CRISPR-Cas12a.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {4},
pages = {315-321},
pmid = {30936531},
issn = {1545-9985},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adaptive Immunity/genetics/physiology ; CRISPR-Cas Systems/genetics/*physiology ; DNA Cleavage ; Endoribonucleases/genetics/metabolism ; Gene Editing/methods ; Protein Multimerization/genetics/physiology ; },
abstract = {Cas12a is a bacterial RNA-guided nuclease used widely for genome editing and, more recently, as a molecular diagnostic. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three such inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity via functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit recognition of double-stranded DNA (dsDNA), with AcrVA4 driving dimerization of Cas12a. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophages with tools to evade CRISPR-Cas12a and support biotechnological applications for which multiple-turnover enzymatic inhibition of Cas12a is desirable.},
}
@article {pmid30936526,
year = {2019},
author = {Dong, L and Guan, X and Li, N and Zhang, F and Zhu, Y and Ren, K and Yu, L and Zhou, F and Han, Z and Gao, N and Huang, Z},
title = {An anti-CRISPR protein disables type V Cas12a by acetylation.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {4},
pages = {308-314},
pmid = {30936526},
issn = {1545-9985},
mesh = {Acetylation ; Acetyltransferases/genetics/metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Moraxella/genetics/*metabolism ; Mutation/genetics ; },
abstract = {Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.},
}
@article {pmid30936525,
year = {2019},
author = {Suresh, SK and Murugan, K and Sashital, DG},
title = {Enzymatic anti-CRISPRs improve the bacteriophage arsenal.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {4},
pages = {250-251},
pmid = {30936525},
issn = {1545-9985},
support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/*physiology ; Immunity/genetics/physiology ; Models, Biological ; },
abstract = {Bacteriophage-encoded anti-CRISPR (Acr) proteins have previously been thought to inhibit CRISPR-mediated immunity by acting as physical barriers against DNA binding or cleavage. Two new studies report that recently discovered type V Acr proteins use enzymatic activities to shut down the Cas12a endonuclease, providing a multi-turnover off-switch for CRISPR-based immunity and technology.},
}
@article {pmid30936372,
year = {2019},
author = {Zhang, Z and Pan, S and Liu, T and Li, Y and Peng, N},
title = {Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers.},
journal = {Journal of bacteriology},
volume = {201},
number = {12},
pages = {},
pmid = {30936372},
issn = {1098-5530},
mesh = {Archaeal Viruses ; CRISPR-Associated Proteins/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial/genetics ; Endonucleases/*genetics/metabolism ; Sulfolobus/*enzymology/*genetics ; Viral Proteins ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity. Recently, we demonstrated that the Csa3a regulator protein triggers CCN protospacer-adjacent motif (PAM)-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (i) in recognizing the 5' PAM and 3' nucleotide motif of protospacers and (ii) in determining both the spacer length and its orientation. Furthermore, we identify amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also that of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency, and the former proteins yielded a high incidence of PAM-dependent atypical spacer integration or of PAM-independent spacer integration. We further demonstrated that in plasmid challenge experiments, overexpressed Cas4-mediated defective spacer acquisition in turn potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition.IMPORTANCE The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The CrenarchaeotaSulfolobus islandicus REY15A carries two cas4 genes (cas4 and csa1) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to those recently reported, were identified to be important for these functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition.},
}
@article {pmid30936113,
year = {2019},
author = {Dandage, R and Després, PC and Yachie, N and Landry, CR},
title = {beditor: A Computational Workflow for Designing Libraries of Guide RNAs for CRISPR-Mediated Base Editing.},
journal = {Genetics},
volume = {212},
number = {2},
pages = {377-385},
pmid = {30936113},
issn = {1943-2631},
support = {299432//CIHR/Canada ; 324265//CIHR/Canada ; 387697//CIHR/Canada ; 364920//CIHR/Canada ; 384483//CIHR/Canada ; },
mesh = {Algorithms ; Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome ; Genomics/*methods ; Humans ; Libraries ; *Mutagenesis ; Polymorphism, Single Nucleotide ; RNA/*genetics ; *Software ; Workflow ; },
abstract = {CRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, Protospacer Adjacent Motif (PAM) recognition sequences, and genomes of many species. Additionally, to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates of editing efficiency, called beditor scores, are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces nonconfounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base editing to model or correct thousands of clinically important human disease mutations.},
}
@article {pmid30935995,
year = {2019},
author = {Aquino-Jarquin, G},
title = {CRISPR-Cas14 is now part of the artillery for gene editing and molecular diagnostic.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {18},
number = {},
pages = {428-431},
doi = {10.1016/j.nano.2019.03.006},
pmid = {30935995},
issn = {1549-9642},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Loci ; *Pathology, Molecular ; },
abstract = {Recently Jennifer Doudna's group discovered the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 14 (Cas14), identified almost exclusively in a superphylum of extremophile archaea. The newly discovered Cas14 possesses a single-stranded (ss)DNA targeting activity - despite being two times smaller than Cas9 - a capability that might confer a defense against viruses with ssDNA genomes. Furthermore, by combining the non-specific ssDNase cleavage activity of Cas14 with isothermal amplification method (DETECTR-Cas14), it can also be promisingly exploited for high-fidelity DNA single-nucleotide polymorphism genotyping, and potentially for detecting ssDNA viruses of undeniable clinical, ecological, and economic importance infecting hosts in all three domains of life. Thus, CRISPR-Cas14 might acquire an exponential expansion in the field of CRISPR diagnostic for infectious and noninfectious diseases.},
}
@article {pmid30935726,
year = {2020},
author = {Rezaei, H and Khadempar, S and Farahani, N and Hosseingholi, EZ and Hayat, SMG and Sathyapalan, T and Sahebkar, AH},
title = {Harnessing CRISPR/Cas9 technology in cardiovascular disease.},
journal = {Trends in cardiovascular medicine},
volume = {30},
number = {2},
pages = {93-101},
doi = {10.1016/j.tcm.2019.03.005},
pmid = {30935726},
issn = {1873-2615},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cardiovascular Diseases/diagnosis/genetics/physiopathology/*therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Therapy/adverse effects/*methods ; Humans ; Stem Cell Transplantation/adverse effects/*methods ; },
abstract = {The CRISPR/Cas9 system is a precisely targeted bacterial defense system, used to control invading viruses. This technology has many potential applications including genetic changes in somatic and germ cells and the creation of knockout animals. Compared to other genome editing techniques such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENS), the CRISPR/Cas9 system is much easier and more efficient. Most importantly, the multifunctional capacity of this technology allows simultaneous editing of several genes. The CRISPR/Cas9 system also potentially has the ability to prevent and treat human diseases. The present article addresses some key points related to the use of the CRISPR/Cas9 system as a powerful tool in cardiovascular research and as a new strategy for the treatment of cardiovascular disease (CVD).},
}
@article {pmid30933966,
year = {2019},
author = {Shoemaker, CJ and Huang, TQ and Weir, NR and Polyakov, NJ and Schultz, SW and Denic, V},
title = {CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor.},
journal = {PLoS biology},
volume = {17},
number = {4},
pages = {e2007044},
pmid = {30933966},
issn = {1545-7885},
support = {K99 GM117218/GM/NIGMS NIH HHS/United States ; R00 GM117218/GM/NIGMS NIH HHS/United States ; R35 GM127136/GM/NIGMS NIH HHS/United States ; P20 GM113132/GM/NIGMS NIH HHS/United States ; },
mesh = {Autophagy/*genetics/*physiology ; CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; Humans ; K562 Cells ; Membrane Proteins/*genetics/metabolism/physiology ; Protein Transport ; },
abstract = {The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.},
}
@article {pmid30932914,
year = {2019},
author = {Neill, US},
title = {A conversation with George Church.},
journal = {The Journal of clinical investigation},
volume = {129},
number = {4},
pages = {1403-1404},
doi = {10.1172/JCI128550},
pmid = {30932914},
issn = {1558-8238},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*history ; History, 20th Century ; History, 21st Century ; Humans ; Sequence Analysis, DNA/*history ; },
}
@article {pmid30932271,
year = {2019},
author = {Yu, JSL and Palano, G and Lim, C and Moggio, A and Drowley, L and Plowright, AT and Bohlooly-Y, M and Rosen, BS and Hansson, EM and Wang, QD and Yusa, K},
title = {CRISPR-Knockout Screen Identifies Dmap1 as a Regulator of Chemically Induced Reprogramming and Differentiation of Cardiac Progenitors.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {7},
pages = {958-972},
pmid = {30932271},
issn = {1549-4918},
support = {//Wellcome Trust/United Kingdom ; WT206194//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Benzamides/*pharmacology ; *CRISPR-Cas Systems ; Cadherins/genetics/metabolism ; Cell Differentiation/drug effects ; Cell Proliferation/drug effects ; Cellular Reprogramming/*drug effects/genetics ; Dioxoles/*pharmacology ; Fibroblasts/cytology/*drug effects/metabolism ; Gene Editing/methods ; Homeobox Protein Nkx-2.5/genetics/metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Muscle, Smooth/cytology/metabolism ; Myocardium/cytology/metabolism ; Myocytes, Cardiac/cytology/drug effects/metabolism ; Primary Cell Culture ; Pyrazoles/*pharmacology ; Pyridines/*pharmacology ; RNA, Guide/genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; Stem Cells/cytology/*drug effects/metabolism ; },
abstract = {Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.},
}
@article {pmid30932232,
year = {2019},
author = {Denner, J and Scobie, L},
title = {Are there better assays to evaluate the risk of transmission of porcine endogenous retroviruses (PERVs) to human cells?.},
journal = {Xenotransplantation},
volume = {26},
number = {4},
pages = {e12510},
doi = {10.1111/xen.12510},
pmid = {30932232},
issn = {1399-3089},
mesh = {Animals ; Biological Assay/economics/*methods ; CRISPR-Cas Systems ; Endogenous Retroviruses/genetics/*physiology ; Genome, Viral ; HEK293 Cells/virology ; Heterografts/*virology ; *Host Specificity ; Humans ; RNA Splicing ; RNA, Messenger/*analysis/biosynthesis/genetics ; RNA, Viral/*analysis/biosynthesis/genetics ; Retroviridae Infections/prevention &amp; control/*transmission ; Retroviridae Proteins/genetics/physiology ; Risk ; Swine/*virology ; *Viral Tropism ; Virus Replication ; },
}
@article {pmid30931913,
year = {2019},
author = {Jordan, B},
title = {[CRISPR babies: technology and transgression].},
journal = {Medecine sciences : M/S},
volume = {35},
number = {3},
pages = {266-270},
doi = {10.1051/medsci/2019034},
pmid = {30931913},
issn = {1958-5381},
mesh = {*Bioethics/trends ; CRISPR-Cas Systems/*genetics ; China ; Cloning, Organism/ethics/methods ; Congresses as Topic ; Disease Resistance/genetics ; *Embryo Research/ethics ; Gene Deletion ; Gene Editing/*ethics ; HIV ; HIV Infections/genetics ; Humans ; Infant, Newborn ; Inventions/ethics ; Mutation ; Receptors, CCR5/genetics ; *Scientific Misconduct/ethics ; },
abstract = {Analysing the data recently presented by Jiankui He and assuming that it is authentic shows that the goal of abolishing the expression of CCR5 may have been reached for one of the resulting twins, although this remains to be proven. However, the canonical delta32 mutation has not been achieved. The various preliminary experiments and controls give some confidence that major off-target modifications have not occurred; again, this is difficult to exclude. Clearly, the requirements of perfect technical mastery of the process have not been met, to say nothing of the requirements for complete transparency and full societal approval.},
}
@article {pmid30931912,
year = {2019},
author = {Chneiweiss, H},
title = {[Back from Hong Kong or ethics at the time of a genetic increase of the human person].},
journal = {Medecine sciences : M/S},
volume = {35},
number = {3},
pages = {263-265},
doi = {10.1051/medsci/2019033},
pmid = {30931912},
issn = {1958-5381},
mesh = {*Bioethics/trends ; CRISPR-Cas Systems/genetics ; Congresses as Topic ; Embryo Research/ethics ; *Gene Editing/ethics/trends ; Genetic Enhancement/*ethics ; Hong Kong ; Humans ; Nontherapeutic Human Experimentation/*ethics ; },
}
@article {pmid30931844,
year = {2019},
author = {Sazonova, MA and Ryzhkova, AI and Sinyov, VV and Sazonova, MD and Khasanova, ZB and Nikitina, NA and Karagodin, VP and Orekhov, AN and Sobenin, IA},
title = {Creation of Cultures Containing Mutations Linked with Cardiovascular Diseases using Transfection and Genome Editing.},
journal = {Current pharmaceutical design},
volume = {25},
number = {6},
pages = {693-699},
doi = {10.2174/1381612825666190329121532},
pmid = {30931844},
issn = {1873-4286},
mesh = {Biolistics ; CRISPR-Cas Systems ; Cardiovascular Diseases/*genetics ; *Gene Editing ; Humans ; Mitochondria/genetics ; *Mutation ; *Transfection ; },
abstract = {OBJECTIVE: In this review article, we analyzed the literature on the creation of cultures containing mutations associated with cardiovascular diseases (CVD) using transfection, transduction and editing of the human genome.<br><br>METHODS: We described different methods of transfection, transduction and editing of the human genome, used in the literature.<br><br>RESULTS: We reviewed the researches in which the creation of &#x441;ell cultures containing mutations was described. According to the literature, system CRISPR/Cas9 proved to be the most preferred method for editing the genome. We found rather promising and interesting a practically undeveloped direction of mitochondria transfection using a gene gun. Such a gun can direct a genetically-engineered construct containing human DNA mutations to the mitochondria using heavy metal particles. However, in human molecular genetics, the transfection method using a gene gun is unfairly forgotten and is almost never used. Ethical problems arising from editing the human genome were also discussed in our review. We came to a conclusion that it is impossible to stop scientific and technical progress. It is important that the editing of the genome takes place under the strict control of society and does not bear dangerous consequences for humanity. To achieve this, the constant interaction of science with society, culture and business is necessary.<br><br>CONCLUSION: The most promising methods for the creation of cell cultures containing mutations linked with cardiovascular diseases, were system CRISPR/Cas9 and the gene gun.},
}
@article {pmid30930513,
year = {2019},
author = {Koonin, EV},
title = {CRISPR: a new principle of genome engineering linked to conceptual shifts in evolutionary biology.},
journal = {Biology &amp; philosophy},
volume = {34},
number = {1},
pages = {9},
pmid = {30930513},
issn = {0169-3867},
abstract = {The CRISPR-Cas systems of bacterial and archaeal adaptive immunity have become a household name among biologists and even the general public thanks to the unprecedented success of the new generation of genome editing tools utilizing Cas proteins. However, the fundamental biological features of CRISPR-Cas are of no lesser interest and have major impacts on our understanding of the evolution of antivirus defense, host-parasite coevolution, self versus non-self discrimination and mechanisms of adaptation. CRISPR-Cas systems present the best known case in point for Lamarckian evolution, i.e. generation of heritable, adaptive genomic changes in response to encounters with external factors, in this case, foreign nucleic acids. CRISPR-Cas systems employ multiple mechanisms of self versus non-self discrimination but, as is the case with immune systems in general, are nevertheless costly because autoimmunity cannot be eliminated completely. In addition to the autoimmunity, the fitness cost of CRISPR-Cas systems appears to be determined by their inhibitory effect on horizontal gene transfer, curtailing evolutionary innovation. Hence the dynamic evolution of CRISPR-Cas loci that are frequently lost and (re)acquired by archaea and bacteria. Another fundamental biological feature of CRISPR-Cas is its intimate connection with programmed cell death and dormancy induction in microbes. In this and, possibly, other immune systems, active immune response appears to be coupled to a different form of defense, namely, "altruistic" shutdown of cellular functions resulting in protection of neighboring cells. Finally, analysis of the evolutionary connections of Cas proteins reveals multiple contributions of mobile genetic elements (MGE) to the origin of various components of CRISPR-Cas systems, furthermore, different biological systems that function by genome manipulation appear to have evolved convergently from unrelated MGE. The shared features of adaptive defense systems and MGE, namely the ability to recognize and cleave unique sites in genomes, make them ideal candidates for genome editing and engineering tools.},
}
@article {pmid30930113,
year = {2019},
author = {Bonafont, J and Mencía, &#xc1; and García, M and Torres, R and Rodríguez, S and Carretero, M and Chacón-Solano, E and Modamio-Høybjør, S and Marinas, L and León, C and Escamez, MJ and Hausser, I and Del Río, M and Murillas, R and Larcher, F},
title = {Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {5},
pages = {986-998},
pmid = {30930113},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Collagen Type VII/*genetics ; Disease Models, Animal ; Epidermolysis Bullosa Dystrophica/genetics/pathology/*therapy ; Exons/genetics ; Frameshift Mutation/genetics ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Humans ; Keratinocytes/metabolism ; Mice ; RNA, Guide/genetics/therapeutic use ; },
abstract = {Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.},
}
@article {pmid30929735,
year = {2019},
author = {Liu, W and He, X and Yang, S and Zouari, R and Wang, J and Wu, H and Kherraf, ZE and Liu, C and Coutton, C and Zhao, R and Tang, D and Tang, S and Lv, M and Fang, Y and Li, W and Li, H and Zhao, J and Wang, X and Zhao, S and Zhang, J and Arnoult, C and Jin, L and Zhang, Z and Ray, PF and Cao, Y and Zhang, F},
title = {Bi-allelic Mutations in TTC21A Induce Asthenoteratospermia in Humans and Mice.},
journal = {American journal of human genetics},
volume = {104},
number = {4},
pages = {738-748},
pmid = {30929735},
issn = {1537-6605},
mesh = {Alleles ; Alternative Splicing ; Animals ; CRISPR-Cas Systems ; China ; Exome ; Flagella/pathology ; Homozygote ; Humans ; Infertility, Male/*genetics ; Male ; Mice ; Microtubule-Associated Proteins/*genetics ; *Mutation ; Phenotype ; Sperm Motility ; Spermatozoa/*abnormalities ; Whole Exome Sequencing ; },
abstract = {Male infertility is a major concern affecting human reproductive health. Asthenoteratospermia can cause male infertility through reduced motility and abnormal morphology of spermatozoa. Several genes, including DNAH1 and some CFAP family members, are involved in multiple morphological abnormalities of the sperm flagella (MMAF). However, these known genes only account for approximately 60% of human MMAF cases. Here, we conducted further genetic analyses by using whole-exome sequencing in a cohort of 65 Han Chinese men with MMAF. Intriguingly, bi-allelic mutations of TTC21A (tetratricopeptide repeat domain 21A) were identified in three (5%) unrelated, MMAF-affected men, including two with homozygous stop-gain mutations and one with compound heterozygous mutations of TTC21A. Notably, these men consistently presented with MMAF and additional abnormalities of sperm head-tail conjunction. Furthermore, a homozygous TTC21A splicing mutation was identified in two Tunisian cases from an independent MMAF cohort. TTC21A is preferentially expressed in the testis and encodes an intraflagellar transport (IFT)-associated protein that possesses several tetratricopeptide repeat domains that perform functions crucial for ciliary function. To further investigate the potential roles of TTC21A in spermatogenesis, we generated Ttc21a mutant mice by using CRISPR-Cas9 technology and revealed sperm structural defects of the flagella and the connecting piece. Our consistent observations across human populations and in the mouse model strongly support the notion that bi-allelic mutations in TTC21A can induce asthenoteratospermia with defects of the sperm flagella and head-tail conjunction.},
}
@article {pmid30929208,
year = {2019},
author = {Kjos, M},
title = {Transcriptional Knockdown in Pneumococci Using CRISPR Interference.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1968},
number = {},
pages = {89-98},
doi = {10.1007/978-1-4939-9199-0_8},
pmid = {30929208},
issn = {1940-6029},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression Regulation, Bacterial/genetics ; Polymerase Chain Reaction ; RNA, Guide/genetics ; Streptococcus pneumoniae/*genetics ; },
abstract = {Sequence-specific knockdown of gene expression using CRISPR interference (CRISPRi) has recently been developed for Streptococcus pneumoniae. By coexpression of a catalytically inactive Cas9-protein (dCas9) and a single guide RNA (sgRNA), CRISPRi can be used to knock down transcription of any gene of interest. Gene specificity is mediated by a 20 bp sequence on the sgRNA, and new genes can be targeted by replacing this 20 bp sequence. Here, a protocol is provided for design of sgRNAs and construction of CRIPSRi strains in S. pneumoniae, based on the vectors published by Liu et al. (Mol Syst Biol 13:931, 2017).},
}
@article {pmid30928637,
year = {2019},
author = {Zhong, Z and Sretenovic, S and Ren, Q and Yang, L and Bao, Y and Qi, C and Yuan, M and He, Y and Liu, S and Liu, X and Wang, J and Huang, L and Wang, Y and Baby, D and Wang, D and Zhang, T and Qi, Y and Zhang, Y},
title = {Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG.},
journal = {Molecular plant},
volume = {12},
number = {7},
pages = {1027-1036},
doi = {10.1016/j.molp.2019.03.011},
pmid = {30928637},
issn = {1752-9867},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Plant ; Oryza/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBEC1 and PmCDA1 cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDA1, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.},
}
@article {pmid30928636,
year = {2019},
author = {Hua, K and Tao, X and Han, P and Wang, R and Zhu, JK},
title = {Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences.},
journal = {Molecular plant},
volume = {12},
number = {7},
pages = {1003-1014},
doi = {10.1016/j.molp.2019.03.009},
pmid = {30928636},
issn = {1752-9867},
mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Gene Editing ; Genome, Plant ; Oryza/*genetics ; },
abstract = {CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.},
}
@article {pmid30928635,
year = {2019},
author = {Ren, B and Liu, L and Li, S and Kuang, Y and Wang, J and Zhang, D and Zhou, X and Lin, H and Zhou, H},
title = {Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice.},
journal = {Molecular plant},
volume = {12},
number = {7},
pages = {1015-1026},
doi = {10.1016/j.molp.2019.03.010},
pmid = {30928635},
issn = {1752-9867},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Plant ; Oryza/*genetics ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM), such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate OsBZR1 gain-of-function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.},
}
@article {pmid30927506,
year = {2019},
author = {Huang, H and Song, X and Yang, S},
title = {Development of a RecE/T-Assisted CRISPR-Cas9 Toolbox for Lactobacillus.},
journal = {Biotechnology journal},
volume = {14},
number = {7},
pages = {e1800690},
doi = {10.1002/biot.201800690},
pmid = {30927506},
issn = {1860-7314},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Bacterial/genetics ; Lactobacillus/*genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Lactobacilli are members of a large family involved in industrial food fermentation, therapeutics, and health promotion. However, the development of genetic manipulation tools for this genus lags behind its relative industrial and medical significance. The development of clustered regularly interspaced short palindromic repeat (CRISPR)-based genome engineering for Lactobacillus is now underway. However, some Lactobacillus species are sensitive to CRISPR-Cas9 induced double strand breaks (DSBs) due to a deficiency in homology-directed repair (HDR), which allows chromosomal genetic editing. Here, phage-derived RecE/T is coupled with CRISPR-Cas9 and the transcriptional activity of broad-spectrum host promoters is assessed to set up a versatile toolbox containing a recombination helper plasmid and a broad host CRISPR-Cas9 editing plasmid, which enables efficient genome editing in Lactobacillus plantarum (L. plantarum) WCFS1 and Lactobacillus brevis (L. brevis) ATCC367. The RecE/T-assisted CRISPR-Cas9 toolbox realizes single gene deletions at an efficiency of 50-100% in seven days. Furthermore, the chromosomal gene replacement of Lp_0537 using a P23 -pyruvate decarboxylase (pdc) expression cassette is accomplished with an efficiency of 35.7%. This study establises a RecE/T-assisted CRISPR genome editing toolbox for L. plantarum WCFS1 and L. brevis ATCC367 and also demonstrate that RecE/T-assisted CRISPR-Cas9 is an effective genome editing system, which can be readily implemented in Lactobacilli.},
}
@article {pmid30927342,
year = {2019},
author = {Noda, T and Sakurai, N and Nozawa, K and Kobayashi, S and Devlin, DJ and Matzuk, MM and Ikawa, M},
title = {Nine genes abundantly expressed in the epididymis are not essential for male fecundity in mice.},
journal = {Andrology},
volume = {7},
number = {5},
pages = {644-653},
pmid = {30927342},
issn = {2047-2927},
support = {P01 HD087157/HD/NICHD NIH HHS/United States ; T32 GM120011/GM/NIGMS NIH HHS/United States ; //Takeda Science Foundation/International ; JP17H01394//Japan Society for the Promotion of Science/International ; R01 HD088412/HD/NICHD NIH HHS/United States ; JP18gm5010001//Japan Agency for Medical Research and Development/International ; JP18K14612//Japan Society for the Promotion of Science/International ; JP25112007//Japan Society for the Promotion of Science/International ; OPP1160866/GATES/Bill &amp; Melinda Gates Foundation/United States ; 20170633//Japan Society for the Promotion of Science/International ; //Ministry of Education, Culture, Sports, Science and Technology/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Epididymis/*metabolism ; Fertility/*genetics ; Gene Knockout Techniques ; Male ; Membrane Proteins/*genetics ; Mice ; Mice, Knockout ; Sperm Maturation/physiology ; Sperm Motility/genetics ; Spermatozoa/*metabolism ; },
abstract = {BACKGROUND: Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation.<br><br>OBJECTIVES: Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis-enriched gene function in&#xa0;vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis.<br><br>MATERIALS AND METHODS: KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild-type females for 3-6&#xa0;months to check fertility.<br><br>RESULTS: We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150&#xa0;kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males.<br><br>DISCUSSION AND CONCLUSION: We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9-mediated KO mice generation accelerates the screening of epididymis-enriched genes for potential functions in reproduction.},
}
@article {pmid30926791,
year = {2019},
author = {Weber, J and de la Rosa, J and Grove, CS and Schick, M and Rad, L and Baranov, O and Strong, A and Pfaus, A and Friedrich, MJ and Engleitner, T and Lersch, R and Öllinger, R and Grau, M and Menendez, IG and Martella, M and Kohlhofer, U and Banerjee, R and Turchaninova, MA and Scherger, A and Hoffman, GJ and Hess, J and Kuhn, LB and Ammon, T and Kim, J and Schneider, G and Unger, K and Zimber-Strobl, U and Heikenwälder, M and Schmidt-Supprian, M and Yang, F and Saur, D and Liu, P and Steiger, K and Chudakov, DM and Lenz, G and Quintanilla-Martinez, L and Keller, U and Vassiliou, GS and Cadiñanos, J and Bradley, A and Rad, R},
title = {PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1415},
pmid = {30926791},
issn = {2041-1723},
support = {MC_PC_12009/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clone Cells ; DNA Transposable Elements/*genetics ; Gene Dosage ; Gene Expression Regulation, Neoplastic ; Genes, Neoplasm ; Genes, Tumor Suppressor ; Genetic Association Studies ; Genetic Testing/*methods ; Humans ; Loss of Heterozygosity ; Lymphoma, B-Cell/*genetics/pathology ; Mice, Inbred C57BL ; Mice, Transgenic ; Receptors, Antigen, B-Cell/metabolism ; Reproducibility of Results ; },
abstract = {B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.},
}
@article {pmid30926729,
year = {2019},
author = {Tapscott, T and Guarnieri, MT and Henard, CA},
title = {Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {11},
pages = {},
pmid = {30926729},
issn = {1098-5336},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Codon, Terminator ; Deoxyribonuclease I/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Methylococcus capsulatus/*genetics ; Oxygenases/genetics ; Plasmids/genetics ; },
abstract = {Methanotrophic bacteria play a crucial role in the Earth's biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9[D10A] nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9[D10A] nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.IMPORTANCE In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.},
}
@article {pmid30926472,
year = {2019},
author = {Wang, F and Wang, L and Zou, X and Duan, S and Li, Z and Deng, Z and Luo, J and Lee, SY and Chen, S},
title = {Advances in CRISPR-Cas systems for RNA targeting, tracking and editing.},
journal = {Biotechnology advances},
volume = {37},
number = {5},
pages = {708-729},
doi = {10.1016/j.biotechadv.2019.03.016},
pmid = {30926472},
issn = {1873-1899},
mesh = {Adenosine Deaminase/genetics ; Aptamers, Nucleotide ; *CRISPR-Cas Systems ; Fluorescent Dyes ; Gene Editing/*methods ; Gene Targeting/*methods ; In Situ Hybridization, Fluorescence ; RNA/*genetics ; RNA Interference ; Sensitivity and Specificity ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.},
}
@article {pmid30926307,
year = {2019},
author = {Zhang, R and Wu, H and Lian, Z},
title = {Bioinformatics analysis of evolutionary characteristics and biochemical structure of FGF5 Gene in sheep.},
journal = {Gene},
volume = {702},
number = {},
pages = {123-132},
doi = {10.1016/j.gene.2019.03.040},
pmid = {30926307},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems ; Computational Biology ; Evolution, Molecular ; Fibroblast Growth Factor 4/classification ; Fibroblast Growth Factor 5/*chemistry/classification/*genetics ; Fibroblast Growth Factors/chemistry/genetics ; Humans ; Mice ; Models, Molecular ; Mutation ; Phylogeny ; Protein Conformation, beta-Strand ; Protein Multimerization ; Receptor, Fibroblast Growth Factor, Type 1/chemistry ; Sequence Alignment ; Sheep/anatomy &amp; histology/*genetics ; Wool/anatomy &amp; histology ; },
abstract = {Fibroblast growth factor (FGF) 5 regulates the development and periodicity of hair follicles, which can affect hair traits. Loss-of-function mutations associated with long-hair phenotypes have been described in several mammalian species. Sheep is an important economic animal, however, the evolution characterizations and biological mechanism of oFGF5 (Ovis aries FGF5) gene are still poorly understood. In this study, oFGF5 gene was obtained by resequencing the whole genome of three Dorper sheep and RACE of two Kazakh sheep FGF5. We proposed FGF5 was phylogenetically related to FGF4 family and oFGF5 clearly orthologed to goat FGF5. Six loci were found from the positive selection results of FGF5 and half of them located on signal peptide. The basically similar rates of function-altering substitutions in sheep and goat lineage and the rest of the mammalian lineage of 365 SNPs indicated that the FGF5 gene was quite conservative during evolution. Homology modeling of the oFGF5 suggested that it has a highly conserved FGF superfamily domain containing 10 β-strands. Furthermore, the protein-protein docking analysis revealed that oFGF5 have the potential to form heterodimers with oFGFR1, the predicted interaction interface of FGF5-FGFR1 heterodimer was formed mainly by residues from FGF superfamily domain. Our observations suggested the evolutionary and structural biology features of oFGF5 might be relevant to its function about hair follicle development and modulating hair growth, and we confirmed our speculation by using the FGF5 gene editing sheep produced by CRISPR/Cas9 technology.},
}
@article {pmid30925937,
year = {2019},
author = {Longmuir, S and Akhtar, N and MacNeill, SA},
title = {Unexpected insertion of carrier DNA sequences into the fission yeast genome during CRISPR-Cas9 mediated gene deletion.},
journal = {BMC research notes},
volume = {12},
number = {1},
pages = {191},
pmid = {30925937},
issn = {1756-0500},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; DNA/*genetics ; *Gene Deletion ; Gene Editing/*methods ; Genes, Fungal/genetics ; Genome, Fungal/*genetics ; Oncorhynchus keta/genetics ; Schizosaccharomyces/*genetics ; Schizosaccharomyces pombe Proteins/genetics/metabolism ; Sequence Analysis, DNA/methods ; Sequence Homology, Nucleic Acid ; Transformation, Genetic ; },
abstract = {OBJECTIVES: The fission yeast Schizosaccharomyces pombe is predicted to encode ~ 200 proteins of < 100 amino acids, including a number of previously uncharacterised proteins that are found conserved in related Schizosaccharomyces species only. To begin an investigation of the function of four of these so-called microproteins (designated Smp1-Smp4), CRISPR-Cas9 genome editing technology was used to delete the corresponding genes in haploid fission yeast cells.<br><br>RESULTS: None of the four microprotein-encoding genes was essential for viability, meiosis or sporulation, and the deletion cells were no more sensitive to a range of cell stressors than wild-type, leaving the function of the proteins unresolved. During CRISPR-Cas9 editing however, a number of strains were isolated in which additional sequences were inserted into the target loci at the Cas9 cut site. Sequencing of the inserts revealed these to be derived from the chum salmon Oncorhynchus keta, the source of the carrier DNA used in the S. pombe transformation.},
}
@article {pmid30925042,
year = {2019},
author = {Stark, JC and Huang, A and Hsu, KJ and Dubner, RS and Forbrook, J and Marshalla, S and Rodriguez, F and Washington, M and Rybnicky, GA and Nguyen, PQ and Hasselbacher, B and Jabri, R and Kamran, R and Koralewski, V and Wightkin, W and Martinez, T and Jewett, MC},
title = {BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts.},
journal = {ACS synthetic biology},
volume = {8},
number = {5},
pages = {1001-1009},
doi = {10.1021/acssynbio.8b00381},
pmid = {30925042},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Cell-Free System ; Drug Resistance, Microbial/genetics ; Gene Editing/methods ; Gene Transfer, Horizontal ; *Genetic Engineering ; Humans ; Optical Imaging ; Synthetic Biology/*education/methods ; },
abstract = {Recent advances in synthetic biology have resulted in biological technologies with the potential to reshape the way we understand and treat human disease. Educating students about the biology and ethics underpinning these technologies is critical to empower them to make informed future policy decisions regarding their use and to inspire the next generation of synthetic biologists. However, hands-on, educational activities that convey emerging synthetic biology topics can be difficult to implement due to the expensive equipment and expertise required to grow living cells. We present BioBits Health, an educational kit containing lab activities and supporting curricula for teaching antibiotic resistance mechanisms and CRISPR-Cas9 gene editing in high school classrooms. This kit links complex biological concepts to visual, fluorescent readouts in user-friendly freeze-dried cell-free reactions. BioBits Health represents a set of educational resources that promises to encourage teaching of cutting-edge, health-related synthetic biology topics in classrooms and other nonlaboratory settings.},
}
@article {pmid30924368,
year = {2019},
author = {Jiang, W and Lian, W and Chen, J and Li, W and Huang, J and Lai, B and Li, L and Huang, Z and Xu, J},
title = {Rapid identification of genome-edited mesenchymal stem cell colonies via Cas9.},
journal = {BioTechniques},
volume = {66},
number = {5},
pages = {231-234},
doi = {10.2144/btn-2018-0183},
pmid = {30924368},
issn = {1940-9818},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Interleukin-10/genetics ; Mesenchymal Stem Cells/*microbiology ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition of specific genes hold a great deal of promise in terms of their therapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previously optimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSC colonies over-expressing IL-10 in the AAVS1 locus.},
}
@article {pmid30923392,
year = {2019},
author = {Rusk, N},
title = {Human immunity to Cas9.},
journal = {Nature methods},
volume = {16},
number = {4},
pages = {286},
doi = {10.1038/s41592-019-0385-z},
pmid = {30923392},
issn = {1548-7105},
mesh = {Adaptive Immunity ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing ; Humans ; },
}
@article {pmid30923209,
year = {2019},
author = {Zaidi, SS and Vanderschuren, H and Qaim, M and Mahfouz, MM and Kohli, A and Mansoor, S and Tester, M},
title = {New plant breeding technologies for food security.},
journal = {Science (New York, N.Y.)},
volume = {363},
number = {6434},
pages = {1390-1391},
doi = {10.1126/science.aav6316},
pmid = {30923209},
issn = {1095-9203},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Food Supply ; *Gene Editing ; Plant Breeding/*methods ; },
}
@article {pmid30923122,
year = {2019},
author = {Kim, H and Bojar, D and Fussenegger, M},
title = {A CRISPR/Cas9-based central processing unit to program complex logic computation in human cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {15},
pages = {7214-7219},
pmid = {30923122},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; *Computers, Molecular ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; },
abstract = {Controlling gene expression with sophisticated logic gates has been and remains one of the central aims of synthetic biology. However, conventional implementations of biocomputers use central processing units (CPUs) assembled from multiple protein-based gene switches, limiting the programming flexibility and complexity that can be achieved within single cells. Here, we introduce a CRISPR/Cas9-based core processor that enables different sets of user-defined guide RNA inputs to program a single transcriptional regulator (dCas9-KRAB) to perform a wide range of bitwise computations, from simple Boolean logic gates to arithmetic operations such as the half adder. Furthermore, we built a dual-core CPU combining two orthogonal core processors in a single cell. In principle, human cells integrating multiple orthogonal CRISPR/Cas9-based core processors could offer enormous computational capacity.},
}
@article {pmid30923099,
year = {2019},
author = {González-Romero, E and Martínez-Valiente, C and García-Ruiz, C and Vázquez-Manrique, RP and Cervera, J and Sanjuan-Pla, A},
title = {CRISPR to fix bad blood: a new tool in basic and clinical hematology.},
journal = {Haematologica},
volume = {104},
number = {5},
pages = {881-893},
pmid = {30923099},
issn = {1592-8721},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering ; *Genetic Therapy ; Hematologic Diseases/genetics/*therapy ; Humans ; Neoplasm Proteins/antagonists &amp; inhibitors/*genetics ; },
abstract = {Advances in genome engineering in the last decade, particularly in the development of programmable nucleases, have made it possible to edit the genomes of most cell types precisely and efficiently. Chief among these advances, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and broad applications in biomedicine. In this review, we focus on the use of CRISPR/Cas9 genome editing in the context of hematologic diseases and appraise the major achievements and challenges in this rapidly moving field to gain a clearer perspective on the potential of this technology to move from the laboratory to the clinic. Accordingly, we discuss data from studies editing hematopoietic cells to understand and model blood diseases, and to develop novel therapies for hematologic malignancies. We provide an overview of the applications of gene editing in experimental, preclinical and clinical hematology including interrogation of gene function, target identification and drug discovery and chimeric antigen receptor T-cell engineering. We also highlight current limitations of CRISPR/Cas9 and the possible strategies to overcome them. Finally, we consider what advances in CRISPR/Cas9 are needed to move the hematology field forward.},
}
@article {pmid30918971,
year = {2019},
author = {Wand, NO and Smith, DA and Wilkinson, AA and Rushton, AE and Busby, SJW and Styles, IB and Neely, RK},
title = {DNA barcodes for rapid, whole genome, single-molecule analyses.},
journal = {Nucleic acids research},
volume = {47},
number = {12},
pages = {e68},
pmid = {30918971},
issn = {1362-4962},
mesh = {Adenoviruses, Human/genetics/isolation &amp; purification ; Bacteriophage lambda/genetics ; Base Sequence ; CRISPR-Cas Systems ; Computer Simulation ; DNA/*chemistry ; DNA, Bacterial/chemistry ; DNA, Viral/chemistry ; Escherichia coli/genetics/isolation &amp; purification ; Fluorescent Dyes ; Genomics/*methods ; Humans ; Klebsiella pneumoniae/genetics ; },
abstract = {We report an approach for visualizing DNA sequence and using these 'DNA barcodes' to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; identifying specific molecules of interest from a dataset containing gigabasepairs of genome; identification of a bacterium from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kb pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest.},
}
@article {pmid30918006,
year = {2019},
author = {Champer, J and Wen, Z and Luthra, A and Reeves, R and Chung, J and Liu, C and Lee, YL and Liu, J and Yang, E and Messer, PW and Clark, AG},
title = {CRISPR Gene Drive Efficiency and Resistance Rate Is Highly Heritable with No Common Genetic Loci of Large Effect.},
journal = {Genetics},
volume = {212},
number = {1},
pages = {333-341},
pmid = {30918006},
issn = {1943-2631},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crosses, Genetic ; Drosophila melanogaster/*genetics ; Female ; Gene Drive Technology/*methods ; *Genetic Loci ; *Genetic Predisposition to Disease ; *Genetic Variation ; Genome-Wide Association Study ; Male ; },
abstract = {Gene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. Clustered regularly interspaced short palindromic repeat (CRISPR) homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population over time. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would eventually halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation postfertilization in the early embryo ranged from 7 to 79% among lines and averaged 42 ± 18%. We performed a genome-wide association study using our results in the DGRP lines, and found that the resistance and conversion rates were not explained by common alleles of large effect, but instead there were several genetic polymorphisms showing weak association. RNA interference knockdown of several genes containing these polymorphisms confirmed their effect, but the small effect sizes imply that their manipulation would likely yield only modest improvements to the efficacy of gene drives.},
}
@article {pmid30917862,
year = {2019},
author = {Li, C and Nguyen, V and Liu, J and Fu, W and Chen, C and Yu, K and Cui, Y},
title = {Mutagenesis of seed storage protein genes in Soybean using CRISPR/Cas9.},
journal = {BMC research notes},
volume = {12},
number = {1},
pages = {176},
pmid = {30917862},
issn = {1756-0500},
mesh = {CRISPR-Cas Systems/*genetics ; Genes, Plant/*genetics ; Mutation/*genetics ; Plant Roots/*genetics ; Plants, Genetically Modified/*genetics ; Seed Storage Proteins/*genetics ; Seeds/*genetics ; Soybeans/*genetics ; },
abstract = {OBJECTIVE: Soybean seeds are an important source of vegetable proteins for both food and industry worldwide. Conglycinins (7S) and glycinins (11S), which are two major families of storage proteins encoded by a small family of genes, account for about 70% of total soy seed protein. Mutant alleles of these genes are often necessary in certain breeding programs, as the relative abundance of these protein subunits affect amino acid composition and soy food properties. In this study, we set out to test the efficiency of the CRISPR/Cas9 system in editing soybean storage protein genes using Agrobacterium rhizogenes-mediated hairy root transformation system.<br><br>RESULTS: We designed and tested sgRNAs to target nine different major storage protein genes and detected DNA mutations in three storage protein genes in soybean hairy roots, at a ratio ranging from 3.8 to 43.7%. Our work provides a useful resource for future soybean breeders to engineer/develop varieties with mutations in seed storage proteins.},
}
@article {pmid30917325,
year = {2019},
author = {Slaymaker, IM and Mesa, P and Kellner, MJ and Kannan, S and Brignole, E and Koob, J and Feliciano, PR and Stella, S and Abudayyeh, OO and Gootenberg, JS and Strecker, J and Montoya, G and Zhang, F},
title = {High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage.},
journal = {Cell reports},
volume = {26},
number = {13},
pages = {3741-3751.e5},
pmid = {30917325},
issn = {2211-1247},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; F30 CA210382/CA/NCI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; Endonucleases/*chemistry/genetics/metabolism ; Enzyme Stability ; Prevotella/*enzymology ; Protein Binding ; Protein Domains ; RNA/chemistry/*metabolism ; Substrate Specificity ; },
abstract = {Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.},
}
@article {pmid30916546,
year = {2019},
author = {Babu, K and Amrani, N and Jiang, W and Yogesha, SD and Nguyen, R and Qin, PZ and Rajan, R},
title = {Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance.},
journal = {Biochemistry},
volume = {58},
number = {14},
pages = {1905-1917},
pmid = {30916546},
issn = {1520-4995},
support = {P20 GM103640/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/genetics/*metabolism ; CRISPR-Associated Protein 9/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/chemistry/genetics/metabolism ; DNA Cleavage ; Gene Editing/*methods ; Models, Molecular ; Mutation, Missense ; Nucleic Acid Conformation ; Proline/chemistry/genetics/*metabolism ; Protein Structure, Secondary ; RNA, Guide/chemistry/genetics/*metabolism ; },
abstract = {CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.},
}
@article {pmid30916397,
year = {2020},
author = {Rutten, MGS and Rots, MG and Oosterveer, MH},
title = {Exploiting epigenetics for the treatment of inborn errors of metabolism.},
journal = {Journal of inherited metabolic disease},
volume = {43},
number = {1},
pages = {63-70},
pmid = {30916397},
issn = {1573-2665},
mesh = {CRISPR-Cas Systems ; Epigenomics/methods ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Metabolism, Inborn Errors/diagnosis/*genetics/*therapy ; },
abstract = {Gene therapy is currently considered as the optimal treatment for inborn errors of metabolism (IEMs), as it aims to permanently compensate for the primary genetic defect. However, emerging gene editing approaches such as CRISPR-Cas9, in which the DNA of the host organism is edited at a precise location, may have outperforming therapeutic potential. Gene editing strategies aim to correct the actual genetic mutation, while circumventing issues associated with conventional compensation gene therapy. Such strategies can also be repurposed to normalize gene expression changes that occur secondary to the genetic defect. Moreover, besides the genetic causes of IEMs, it is increasingly recognized that their clinical phenotypes are associated with epigenetic changes. Because epigenetic alterations are principally reversible, this may offer new opportunities for treatment of IEM patients. Here, we present an overview of the promises of epigenetics in eventually treating IEMs. We discuss the concepts of gene and epigenetic editing, and the advantages and disadvantages of current and upcoming gene-based therapies for treatment of IEMs.},
}
@article {pmid30916347,
year = {2019},
author = {Dreval, K and Lake, RJ and Fan, HY},
title = {HDAC1 negatively regulates selective mitotic chromatin binding of the Notch effector RBPJ in a KDM5A-dependent manner.},
journal = {Nucleic acids research},
volume = {47},
number = {9},
pages = {4521-4538},
pmid = {30916347},
issn = {1362-4962},
support = {P30 CA118100/CA/NCI NIH HHS/United States ; P50 GM085273/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Cell Division/genetics ; Chromatin/*genetics ; Gene Expression Regulation ; Gene Knockout Techniques ; Histone Deacetylase 1/*genetics ; Humans ; Immunoglobulin J Recombination Signal Sequence-Binding Protein/*genetics ; Mice ; Mitosis/genetics ; Promoter Regions, Genetic ; Protein Binding/genetics ; Retinoblastoma-Binding Protein 2/*genetics ; Signal Transduction/genetics ; },
abstract = {Faithful propagation of transcription programs through cell division underlies cell-identity maintenance. Transcriptional regulators selectively bound on mitotic chromatin are emerging critical elements for mitotic transcriptional memory; however, mechanisms governing their site-selective binding remain elusive. By studying how protein-protein interactions impact mitotic chromatin binding of RBPJ, the major downstream effector of the Notch signaling pathway, we found that histone modifying enzymes HDAC1 and KDM5A play critical, regulatory roles in this process. We found that HDAC1 knockdown or inactivation leads to increased RBPJ occupancy on mitotic chromatin in a site-specific manner, with a concomitant increase of KDM5A occupancy at these sites. Strikingly, the presence of KDM5A is essential for increased RBPJ occupancy. Our results uncover a regulatory mechanism in which HDAC1 negatively regulates RBPJ binding on mitotic chromatin in a KDM5A-dependent manner. We propose that relative chromatin affinity of a minimal regulatory complex, reflecting a specific transcription program, renders selective RBPJ binding on mitotic chromatin.},
}
@article {pmid30916346,
year = {2019},
author = {Chen, D and Zhang, Z and Chen, C and Yao, S and Yang, Q and Li, F and He, X and Ai, C and Wang, M and Guan, MX},
title = {Deletion of Gtpbp3 in zebrafish revealed the hypertrophic cardiomyopathy manifested by aberrant mitochondrial tRNA metabolism.},
journal = {Nucleic acids research},
volume = {47},
number = {10},
pages = {5341-5355},
pmid = {30916346},
issn = {1362-4962},
mesh = {Aminoacylation ; Animals ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/genetics/*metabolism ; GTP-Binding Proteins/*genetics/metabolism ; *Gene Deletion ; In Situ Hybridization ; Mitochondria/genetics/metabolism ; Mitochondrial Proteins/metabolism ; Mutation ; Myocytes, Cardiac/metabolism ; Phenotype ; Protein Conformation ; RNA, Transfer/*metabolism ; Transgenes ; Zebrafish/genetics ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {GTPBP3 is a highly conserved tRNA modifying enzyme for the biosynthesis of τm5U at the wobble position of mitochondrial tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). The previous investigations showed that GTPBP3 mutations were associated with hypertrophic cardiomyopathy (HCM). However, the pathophysiology of GTPBP3 deficiency remains elusively. Using the gtpbp3 knockout zebrafish generated by CRISPR/Cas9 system, we demonstrated the aberrant mitochondrial tRNA metabolism in gtpbp3 knock-out zebrafish. The deletion of gtpbp3 may alter functional folding of tRNA, indicated by conformation changes and sensitivity to S1-mediated digestion of tRNAGlu, tRNALys, tRNATrp and tRNALeu(UUR). Strikingly, gtpbp3 knock-out zebrafish displayed the global increases in the aminoacylated efficiencies of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms impaired mitochondrial translation, produced proteostasis stress and altered activities of respiratory chain complexes. These mitochondria dysfunctions caused the alterations in the embryonic heart development and reduced fractional shortening of ventricles in mutant zebrafish. Notably, the gtpbp3 knock-out zebrafish exhibited hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the gtpbp3 knock-out zebrafish recapitulated the clinical phenotypes in HCM patients carrying the GTPBP3 mutation(s). Our findings highlight the fundamental role of defective nucleotide modifications of tRNAs in mitochondrial biogenesis and their pathological consequences in hypertrophic cardiomyopathy.},
}
@article {pmid30914808,
year = {2019},
author = {Reardon, S},
title = {World Health Organization panel weighs in on CRISPR-babies debate.},
journal = {Nature},
volume = {567},
number = {7749},
pages = {444-445},
doi = {10.1038/d41586-019-00942-z},
pmid = {30914808},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Infant ; World Health Organization ; },
}
@article {pmid30914406,
year = {2019},
author = {Mulas, C and Kalkan, T and von Meyenn, F and Leitch, HG and Nichols, J and Smith, A},
title = {Defined conditions for propagation and manipulation of mouse embryonic stem cells.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {6},
pages = {},
pmid = {30914406},
issn = {1477-9129},
support = {MC_UP_1605/2/MRC_/Medical Research Council/United Kingdom ; G1100526/1/MRC_/Medical Research Council/United Kingdom ; BB/G015678/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 091484/Z/10/Z/WT_/Wellcome Trust/United Kingdom ; G1100526/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Cycle ; Cell Differentiation/*genetics ; Coculture Techniques ; Culture Media/chemistry ; Embryo, Mammalian/*cytology ; Embryonic Stem Cells/*cytology ; Humans ; Karyotyping ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/*cytology ; Neurons/cytology ; RNA, Small Interfering/genetics ; Signal Transduction ; },
abstract = {The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.},
}
@article {pmid30914202,
year = {2019},
author = {Kawaguchi, K and Sato, T and Kondo, S and Yamamoto-Hino, M and Goto, S},
title = {Stability of the transamidase complex catalyzing GPI anchoring of proteins.},
journal = {Biochemical and biophysical research communications},
volume = {512},
number = {3},
pages = {584-590},
doi = {10.1016/j.bbrc.2019.03.103},
pmid = {30914202},
issn = {1090-2104},
mesh = {Aminoacyltransferases/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/genetics/*metabolism ; Enzyme Stability ; GPI-Linked Proteins/*metabolism ; Protein Subunits/genetics/metabolism ; },
abstract = {Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). PIG-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex.},
}
@article {pmid30912355,
year = {2019},
author = {Zhang, X and Zhang, Y and Dai, J and Wang, Y and He, W},
title = {[Construction of a new isovalerylspiramycin I producing strain by CRISPR-Cas9 system].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {3},
pages = {472-481},
doi = {10.13345/j.cjb.180282},
pmid = {30912355},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; Homologous Recombination ; },
abstract = {Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.},
}
@article {pmid30912343,
year = {2019},
author = {Fu, J and Yang, F and Xie, H and Gu, F},
title = {[Application and optimization of CRISPR/Cas system in bacteria].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {3},
pages = {341-350},
doi = {10.13345/j.cjb.180429},
pmid = {30912343},
issn = {1872-2075},
mesh = {Animals ; Bacteria ; *CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; Humans ; Mice ; },
abstract = {Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.},
}
@article {pmid30912142,
year = {2019},
author = {Wadhwa, R and Aggarwal, T and Malyla, V and Kumar, N and Gupta, G and Chellappan, DK and Dureja, H and Mehta, M and Satija, S and Gulati, M and Maurya, PK and Collet, T and Hansbro, PM and Dua, K},
title = {Identification of biomarkers and genetic approaches toward chronic obstructive pulmonary disease.},
journal = {Journal of cellular physiology},
volume = {234},
number = {10},
pages = {16703-16723},
doi = {10.1002/jcp.28482},
pmid = {30912142},
issn = {1097-4652},
mesh = {Biomarkers ; Gene Editing ; *Genetic Therapy ; Humans ; Pulmonary Disease, Chronic Obstructive/*metabolism/*therapy ; },
abstract = {Chronic obstructive pulmonary disease accounts as the leading cause of mortality worldwide prominently affected by genetic and environmental factors. The disease is characterized by persistent coughing, breathlessness airways inflammation followed by a decrease in forced expiratory volume1 and exacerbations, which affect the quality of life. Determination of genetic, epigenetic, and oxidant biomarkers to evaluate the progression of disease has proved complicated and challenging. Approaches including exome sequencing, genome-wide association studies, linkage studies, and inheritance and segregation studies played a crucial role in the identification of genes, their pathways and variation in genes. This review highlights multiple approaches for biomarker and gene identification, which can be used for differential diagnosis along with the genome editing tools to study genes associated with the development of disease and models their function. Further, we have discussed the approaches to rectify the abnormal gene functioning of respiratory tissues and various novel gene editing techniques like Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9).},
}
@article {pmid30912094,
year = {2019},
author = {Kieckhaefer, JE and Maina, F and Wells, RG and Wangensteen, KJ},
title = {Liver Cancer Gene Discovery Using Gene Targeting, Sleeping Beauty, and CRISPR/Cas9.},
journal = {Seminars in liver disease},
volume = {39},
number = {2},
pages = {261-274},
pmid = {30912094},
issn = {1098-8971},
support = {K08 DK106478/DK/NIDDK NIH HHS/United States ; U54 CA193417/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/drug therapy/*genetics ; Cell Transformation, Neoplastic/genetics ; Disease Models, Animal ; Gene Targeting/*methods ; Genetic Testing/methods ; Humans ; Liver Neoplasms/drug therapy/*genetics ; Mice ; },
abstract = {Hepatocellular carcinoma (HCC) is a devastating and prevalent cancer with limited treatment options. Technological advances have enabled genetic screens to be employed in HCC model systems to characterize genes regulating tumor initiation and growth. Relative to traditional methods for studying cancer biology, such as candidate gene approaches or expression analysis, genetic screens have several advantages: they are unbiased, with no a priori selection; can directly annotate gene function; and can uncover gene-gene interactions. In HCC, three main types of screens have been conducted and are reviewed here: (1) transposon-based mutagenesis screens, (2) knockdown screens using RNA interference (RNAi) or the CRISPR/Cas9 system, and (3) overexpression screens using CRISPR activation (CRISPRa) or cDNAs. These methods will be valuable in future genetic screens to delineate the mechanisms underlying drug resistance and to identify new treatments for HCC.},
}
@article {pmid30912056,
year = {2019},
author = {Thomsen, EA and Mikkelsen, JG},
title = {CRISPR-Based Lentiviral Knockout Libraries for Functional Genomic Screening and Identification of Phenotype-Related Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {343-357},
doi = {10.1007/978-1-4939-9170-9_21},
pmid = {30912056},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Knockout Techniques ; Genome-Wide Association Study ; Genomics ; Humans ; Phenotype ; RNA Interference ; },
abstract = {Adaptation of the CRISPR system has enabled scientists to probe the genome and interfere with gene function at an unprecedented scale. Adding to the use of CRISPR for generation of individual gene knockout, which is by now conventional, the CRISPR system enables high-throughput functional screening of the genome. By combining the integrative properties of lentiviral vector delivery with the disruptive nature of the CRISPR system, genome-wide CRISPR libraries provide the power to screen among thousands of genes despite the high complexity of the entire genome and identify a list of genes potentially affecting a certain phenotype. Genome-wide CRISPR screening is an advanced technology compiling numerous practical aspects and a series of molecular biology techniques. In this protocol, we describe all steps toward implementing CRISPR knockout screens in your research; we describe the core procedures and key information as well as some tricks and tips needed to successfully perform a CRISPR screen.},
}
@article {pmid30912055,
year = {2019},
author = {Niola, F and Dagnæs-Hansen, F and Frödin, M},
title = {In Vivo Editing of the Adult Mouse Liver Using CRISPR/Cas9 and Hydrodynamic Tail Vein Injection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {329-341},
doi = {10.1007/978-1-4939-9170-9_20},
pmid = {30912055},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Hepatocytes/metabolism ; Liver/metabolism ; Mice ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas9 technology allows facile modification of the genome in virtually any desired way through the use of easily designed plasmid constructs that express a gRNA targeting a genomic site-of-interest and Cas9. Hydrodynamic tail vein injection, on the other hand, is a simple method to deliver "naked" plasmid DNA to 5-40% of the hepatocytes of the liver of adult mice. Here, we describe how these two techniques can be combined to create a workflow for fast, easy, and cost-efficient in vivo genome editing of the adult mouse liver. Using this method, large cohorts of mice with genetically modified livers can be established within 3 weeks to generate models for gene function in normal physiology and diseases of the liver.},
}
@article {pmid30912052,
year = {2019},
author = {Vochozkova, P and Simmet, K and Jemiller, EM and Wünsch, A and Klymiuk, N},
title = {Gene Editing in Primary Cells of Cattle and Pig.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {271-289},
doi = {10.1007/978-1-4939-9170-9_17},
pmid = {30912052},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cattle ; Cells, Cultured ; Gene Editing/*methods ; Livestock/genetics ; RNA, Guide/genetics ; Swine ; },
abstract = {Gene Editing by CRISPR/Cas has revolutionized many aspects of biotechnology within a short period of time. This is also true for the genetic manipulation of livestock species, but their specific challenges such as the lack of stem cells, the limited proliferative capacity of primary cells, and the genetic diversity of the pig and cattle populations need consideration when CRISPR/Cas is applied. Here we present guidelines for CRISPRing primary cells in pig and cattle, with a specific focus on testing gRNA in vitro, on generating single cell clones, and on identifying modifications in single cell clones.},
}
@article {pmid30912051,
year = {2019},
author = {Xiang, X and Li, C and Chen, X and Dou, H and Li, Y and Zhang, X and Luo, Y},
title = {CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {255-269},
doi = {10.1007/978-1-4939-9170-9_16},
pmid = {30912051},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genes, Reporter/genetics ; Swine ; },
abstract = {CRISPR/Cas9 provides a simple and powerful tool for modifying almost any DNA of interest. One promising application of the CRISPR/Cas9 system is for tagging genes with a fluorescence marker or tag peptides. For such a purpose, FLAG, HIS, and HA tags or fluorescence proteins (EGFP, BFP, RFP, etc.) have been broadly used to tag endogenous genes of interest. The advantages of generating fluorescence tagging proteins are to provide easy tracing of the subcellular locations, real-time monitoring the expression and dynamics of the protein in different conditions, which cannot be achieved using traditional immunostaining or biochemistry assays. However, the generation of such a gene-tagged cell line could be technically challenging. In this chapter, we demonstrate the generation of tagging the porcine GAPDH (pGAPDH) gene GFP by CRISPR/Cas9-based homology-directed repair.},
}
@article {pmid30912050,
year = {2019},
author = {Hansen, LA and Füchtbauer, EM},
title = {Genome Editing in Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {249-254},
doi = {10.1007/978-1-4939-9170-9_15},
pmid = {30912050},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Electroporation ; *Gene Editing ; Mice ; RNA, Guide/genetics ; Zygote/metabolism ; },
abstract = {Programmable nucleases like CRISPR/Cas9 enable to edit the mouse genome directly in the zygote. Several methods have been successfully used for this. Here we describe injection into one of the pronuclei of the zygote and electroporation of zygotes. Alternative methods will be mentioned.},
}
@article {pmid30912049,
year = {2019},
author = {Neldeborg, S and Lin, L and Stougaard, M and Luo, Y},
title = {Rapid and Efficient Gene Deletion by CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {233-247},
doi = {10.1007/978-1-4939-9170-9_14},
pmid = {30912049},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Endonucleases/genetics ; Gene Deletion ; *Gene Editing ; Genetic Vectors/genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR/Cas9 is a powerful genetic engineering technology that enables the introduction of genomic changes such as deletions and insertions of specific bits of DNA in cells with high precision. Compared to other programmable DNA nuclease such as ZFNs and TALENs, the specific binding of the Cas9 nuclease is mediated by a small guide RNA (gRNA), which can easily be designed to target any locus in the genome. The ease of generating novel gRNA vectors and its high efficiency has rapidly made CRISPR-Cas9 the dominant tool in gene editing applications, including gene knockout, knockin, tagging, etc. Here we describe our method for rapid and efficient generation of gene knockout or deletion cells using CRISPR/Cas9 within the time span of one month. The design of gRNAs, plasmid cloning, transfection, cell culturing, positive clone selection, and screening can be obtained from this method.},
}
@article {pmid30912048,
year = {2019},
author = {Sergeeva, D and Camacho-Zaragoza, JM and Lee, JS and Kildegaard, HF},
title = {CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {213-232},
doi = {10.1007/978-1-4939-9170-9_13},
pmid = {30912048},
issn = {1940-6029},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/*genetics ; Cricetinae ; Cricetulus ; *Gene Editing ; Genome/*genetics ; },
abstract = {The emergence of CRISPR/Cas9 system as a precise and affordable method for genome editing has prompted its rapid adoption for the targeted integration of transgenes in Chinese hamster ovary (CHO) cells. Targeted gene integration allows the generation of stable cell lines with a controlled and predictable behavior, which is an important feature for the rational design of cell factories aimed at the large-scale production of recombinant proteins. Here we present the protocol for CRISPR/Cas9-mediated integration of a gene expression cassette into a specific genomic locus in CHO cells using homology-directed DNA repair.},
}
@article {pmid30912047,
year = {2019},
author = {Snijders, KE and Cooper, JD and Vallier, L and Bertero, A},
title = {Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {185-209},
doi = {10.1007/978-1-4939-9170-9_12},
pmid = {30912047},
issn = {1940-6029},
support = {FS/11/77/39327/BHF_/British Heart Foundation/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; PSAG028/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; MC_PC_12009/MRC_/Medical Research Council/United Kingdom ; G0701448/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques/*methods ; Humans ; Pluripotent Stem Cells/metabolism ; RNA, Guide/genetics ; },
abstract = {The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease.},
}
@article {pmid30912046,
year = {2019},
author = {Bruntraeger, M and Byrne, M and Long, K and Bassett, AR},
title = {Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {153-183},
doi = {10.1007/978-1-4939-9170-9_11},
pmid = {30912046},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Genome, Human/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism ; RNA, Guide/genetics ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {Genome editing using the CRISPR/Cas9 system has rapidly established itself as an essential tool in the genetic manipulation of many organisms, including human cell lines. Its application to human induced pluripotent stem cells (hiPSCs) allows for the generation of isogenic cell pairs that differ in a single genetic lesion, and therefore the identification and characterization of causal genetic variants. We describe a simple, effective approach to perform delicate manipulations of the genome of hiPSCs through delivery of Cas9 RNPs along with ssDNA oligonucleotide repair templates that can generate mutations in up to 98% of single cell clones and introduce single nucleotide changes at an efficiency of up to 40%. We describe our use of a T7 endonuclease assay to identify active guide RNAs, and a high-throughput sequencing genotyping strategy that allows the identification of correctly edited clones. We also present our experiences of generating single nucleotide changes at 15 sites, which show considerable variability between both guides and target sites in the efficiency at which such changes can be introduced.},
}
@article {pmid30912045,
year = {2019},
author = {Yumlu, S and Bashir, S and Stumm, J and Kühn, R},
title = {Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {137-151},
doi = {10.1007/978-1-4939-9170-9_10},
pmid = {30912045},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics/physiology ; Gene Editing/methods ; Genome, Human/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Plasmids/genetics ; },
abstract = {The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of an utmost importance as these cells carry the potential to be differentiated into any cell lineage. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter covers protocols for the preparation of reagents to target loci of interest, the transfection, and for the genotyping of single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of plasmids enabling multiplex gene targeting.},
}
@article {pmid30912044,
year = {2019},
author = {Laustsen, A and Bak, RO},
title = {Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {127-134},
doi = {10.1007/978-1-4939-9170-9_9},
pmid = {30912044},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Electroporation ; Gene Editing ; Humans ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas9 is an effective and easy-to-use tool for editing the genome of many human cancer cell lines. However, in some hard-to-transfect cell lines and primary cells, gene editing is more challenging. This protocol details an electroporation-based protocol for the delivery of Cas9 protein from Streptococcus pyogenes complexed with chemically modified sgRNAs. We have found this protocol to work very efficiently in numerous cell lines and primary cells that are difficult to transfect by conventional chemical-based transfection methods.},
}
@article {pmid30912042,
year = {2019},
author = {Ryø, LB and Thomsen, EA and Mikkelsen, JG},
title = {Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {93-109},
doi = {10.1007/978-1-4939-9170-9_7},
pmid = {30912042},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genetic Vectors/genetics ; Lentivirus/genetics ; RNA, Guide/*genetics ; },
abstract = {Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.},
}
@article {pmid30912041,
year = {2019},
author = {Yang, S and Wu, Q and Wei, Y and Gong, C},
title = {CRISPR-Cas9 Delivery by Artificial Virus (RRPHC).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {81-91},
doi = {10.1007/978-1-4939-9170-9_6},
pmid = {30912041},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Fluorocarbons ; Gene Editing ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Since its first harnessing in gene editing in 2012 and successful application in mammalian gene editing in 2013, the CRISPR-Cas9 system exerted magnificent power in all gene-editing-related applications, indicating a sharp thrive of this novel technology. However, there are still some critical drawbacks of the CRISPR-Cas9 system that hampered its broad application in gene editing. Efficient delivery of the Cas9 protein and its partner small guide RNA (sgRNA) to the target cells or tissue is one of the technical bottlenecks. CRISPR-Cas9 delivery via DNA plasmids still plays the big role in gene editing methods. With regard to the disadvantages of CRISPR-Cas9 plasmids, the most acute barrier lies in its large size (>10 kb) and the subsequent low transfection efficiency by conventional transfection method. In this chapter, what we present is an easy method by fabricating CRISPR-Cas9 plasmids into nanoparticle system and efficiently delivered into target cells to achieve gene editing.},
}
@article {pmid30912039,
year = {2019},
author = {König, S and Yang, Z and Wandall, HH and Mussolino, C and Bennett, EP},
title = {Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {45-66},
doi = {10.1007/978-1-4939-9170-9_4},
pmid = {30912039},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Editing ; Humans ; INDEL Mutation/genetics ; Leukocytes, Mononuclear/metabolism ; RNA, Guide/genetics ; },
abstract = {Recent developments in gene targeting methodologies such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionized approaches for gene modifications in cells, tissues, and whole animals showing great promise for translational applications. With regard to CRISPR/Cas9, a variety of repurposed systems have been developed to achieve gene knock-out, base editing, targeted knock-in, gene activation/repression, epigenetic modulation, and locus-specific labeling. A functional communality of all CRISPR/Cas9 applications is the gRNA-dependent targeting specificity of the Cas9/gRNA complex that, for gene knock-out (KO) purposes, has been shown to dictate the indel formation potential. Therefore, the objective of a CRISPR/Cas9 KO set up is to identify gRNA designs that enable maximum out-of-frame insertion and/or deletion (indel) formation and thus, gRNA design becomes a proxy for optimal functionality of CRISPR/Cas9 KO and repurposed systems. To this end, validation of gRNA functionality depends on efficient, accurate, and sensitive identification of indels induced by a given gRNA design. For in vitro indel profiling the most commonly used methods are based on amplicon size discrimination or sequencing. Indel detection by amplicon analysis (IDAA™) is an alternative sensitive, fast, and cost-efficient approach ideally suited for profiling of indels induced by Cas9/gRNA with similar sensitivity, specificity, and resolution, down to single base discrimination, as the preferred next-generation sequencing-based indel profiling methodologies. Here we provide a protocol that is based on complexed Cas9/gRNA RNPs delivered to primary peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals followed by quantitative IDAA indel profiling. Importantly, the protocol described benefits from a short "sample-to-data" turnaround time of less than 5 h. Thus, this protocol describes a methodology that provides a suitable and effective solution to validate and quantify the extent of ex vivo CRISPR/Cas9 targeting in primary cells.},
}
@article {pmid30912038,
year = {2019},
author = {Brinkman, EK and van Steensel, B},
title = {Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1961},
number = {},
pages = {29-44},
doi = {10.1007/978-1-4939-9170-9_3},
pmid = {30912038},
issn = {1940-6029},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; INDEL Mutation/genetics ; Mutagenesis ; Mutation/genetics ; Point Mutation/genetics ; },
abstract = {Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.},
}
@article {pmid30912024,
year = {2019},
author = {Klimke, A and Güttler, S and Kuballa, P and Janzen, S and Ortmann, S and Flora, A},
title = {Use of CRISPR/Cas9 for the Modification of the Mouse Genome.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1953},
number = {},
pages = {213-230},
doi = {10.1007/978-1-4939-9145-7_13},
pmid = {30912024},
issn = {1940-6029},
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Genome ; Genotyping Techniques/methods ; Humans ; Male ; Mice/embryology/*genetics ; Mice, Inbred C57BL ; RNA, Guide/genetics ; },
abstract = {The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.},
}
@article {pmid30912013,
year = {2019},
author = {Horn, M and Metge, F and Denzel, MS},
title = {Unbiased Forward Genetic Screening with Chemical Mutagenesis to Uncover Drug-Target Interactions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1953},
number = {},
pages = {23-31},
doi = {10.1007/978-1-4939-9145-7_2},
pmid = {30912013},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Discovery/methods ; Drug Evaluation, Preclinical/*methods ; Drug Resistance ; Embryonic Stem Cells/cytology/drug effects/metabolism ; Gene Editing/*methods ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; *Mutagenesis ; Mutation ; Sequence Analysis, DNA/methods ; },
abstract = {The steadily increasing throughput in next-generation sequencing technologies is revolutionizing a number of fields in biology. One application requiring massive parallel sequencing is forward genetic screening based on chemical mutagenesis. Such screens interrogate the entire genome in an entirely unbiased fashion and can be applied to a number of research questions. CRISPR/Cas9-based screens, which are largely limited to a gene's loss of function, have already been very successful in identifying drug targets and pathways related to the drug's mode of action. By inducing single nucleotide changes using an alkylating reagent, it is possible to generate amino acid changes that perturb the interaction between a drug and its direct target, resulting in drug resistance. This chemogenomic approach combined with latest sequencing technologies allows deconvolution of drug targets and characterization of drug-target binding interfaces at amino acid resolution, therefore nicely complementing existing biochemical approaches. Here we describe a general protocol for a chemical mutagenesis-based forward genetic screen applicable for drug-target deconvolution.},
}
@article {pmid30911135,
year = {2019},
author = {Wu, Y and Zeng, J and Roscoe, BP and Liu, P and Yao, Q and Lazzarotto, CR and Clement, K and Cole, MA and Luk, K and Baricordi, C and Shen, AH and Ren, C and Esrick, EB and Manis, JP and Dorfman, DM and Williams, DA and Biffi, A and Brugnara, C and Biasco, L and Brendel, C and Pinello, L and Tsai, SQ and Wolfe, SA and Bauer, DE},
title = {Highly efficient therapeutic gene editing of human hematopoietic stem cells.},
journal = {Nature medicine},
volume = {25},
number = {5},
pages = {776-783},
pmid = {30911135},
issn = {1546-170X},
support = {K08 DK093705/DK/NIDDK NIH HHS/United States ; 2013137/DDCF/Doris Duke Charitable Foundation/United States ; 2015134/DDCF/Doris Duke Charitable Foundation/United States ; R21 HL145636/HL/NHLBI NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; UL1 TR001102/TR/NCATS NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; R03 DK109232/DK/NIDDK NIH HHS/United States ; DP2 HL137300/HL/NHLBI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; U01 HL117720/HL/NHLBI NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R01 HL137848/HL/NHLBI NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Anemia, Sickle Cell/blood/genetics/therapy ; Base Sequence ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Enhancer Elements, Genetic ; Erythroid Precursor Cells/metabolism ; Fetal Hemoglobin/biosynthesis/genetics ; Gene Editing/*methods ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism ; Humans ; INDEL Mutation ; Nuclear Proteins/genetics ; RNA, Guide/genetics ; Repressor Proteins ; beta-Thalassemia/blood/genetics/therapy ; gamma-Globins/biosynthesis/genetics ; },
abstract = {Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)[1]. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells[2-6]. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.},
}
@article {pmid30911123,
year = {2019},
author = {Toda, E and Koiso, N and Takebayashi, A and Ichikawa, M and Kiba, T and Osakabe, K and Osakabe, Y and Sakakibara, H and Kato, N and Okamoto, T},
title = {An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.},
journal = {Nature plants},
volume = {5},
number = {4},
pages = {363-368},
doi = {10.1038/s41477-019-0386-z},
pmid = {30911123},
issn = {2055-0278},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA, Plant/*genetics ; Gene Editing/*methods ; Genetic Markers/genetics ; Genome, Plant/genetics ; Oryza/*genetics ; Zygote ; },
abstract = {Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants[1,2]. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment[3], CRISPR-Cas9 integration increases the chance of off-target modifications[4], and foreign DNA sequences cause legislative concerns about genetically modified organisms[5]. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca[2+])-mediated transfection in tobacco, Arabidopsis, lettuce, rice[6], Petunia[7], grapevine, apple[8] and potato[9], or into embryo cells by biolistic bombardment in maize[10] and wheat[11]. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes[12] and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.},
}
@article {pmid30910740,
year = {2019},
author = {Hajighasemi, S and Mahdavi Gorabi, A and Bianconi, V and Pirro, M and Banach, M and Ahmadi Tafti, H and Reiner, &#x17d; and Sahebkar, A},
title = {A review of gene- and cell-based therapies for familial hypercholesterolemia.},
journal = {Pharmacological research},
volume = {143},
number = {},
pages = {119-132},
doi = {10.1016/j.phrs.2019.03.016},
pmid = {30910740},
issn = {1096-1186},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell- and Tissue-Based Therapy ; *Genetic Therapy ; Humans ; Hyperlipoproteinemia Type II/genetics/*therapy ; Stem Cells ; },
abstract = {Familial hypercholesterolemia (FH) is a genetic autosomal dominant disorder caused by an impaired receptor-mediated low-density lipoprotein (LDL) removal from the circulation, mainly due to disruptive autosomal co-dominant mutations in the LDL receptor (LDLr) gene, but also less frequently in the apolipoprotein B100 (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. A rare form of autosomal recessive FH has been also described due to LDLr adaptor protein 1 (LDLRAP1) gene mutations. FH is characterized by very high levels of plasma LDL cholesterol associated with the high incidence of premature atherosclerotic cardiovascular disease (CVD). Despite heterozygous FH (HeFH) patients are still poorly recognized and treated, there is today a large availability of drugs (i.e., statins, ezetimibe and PCSK9 inhibitors) allowing theoretically the normalization of plasma LDL cholesterol levels in this population. Homozygous FH patients (HoFH) have a more severe form of FH, characterized by low responsiveness to the conventional lipid-lowering treatment and often associated with unfavorable prognosis in the young age. Inspired by promising outcomes obtained by orthotopic liver transplantation (OLT), scientists are investigating the possibility of correcting the defective LDLr in these patients by using gene therapy approaches to achieve a novel therapeutic solution with high efficiency. In this article, we tried to review the in vitro, ex vivo, and in vivo attempts conducted to correct FH-causing LDLr gene mutations by using different methods of gene delivery, gene editing, and stem cell manipulation. We also discussed some clinical trials performed in this context.},
}
@article {pmid30910031,
year = {2019},
author = {Leto, DE and Kopito, RR},
title = {Methods for genetic analysis of mammalian ER-associated degradation.},
journal = {Methods in enzymology},
volume = {619},
number = {},
pages = {97-120},
doi = {10.1016/bs.mie.2019.01.006},
pmid = {30910031},
issn = {1557-7988},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Endoplasmic Reticulum-Associated Degradation ; Gene Editing/methods ; Humans ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. We also discuss considerations for screen design, execution, and interpretation.},
}
@article {pmid30908954,
year = {2019},
author = {Brandt, K and Barrangou, R},
title = {Applications of CRISPR Technologies Across the Food Supply Chain.},
journal = {Annual review of food science and technology},
volume = {10},
number = {},
pages = {133-150},
doi = {10.1146/annurev-food-032818-121204},
pmid = {30908954},
issn = {1941-1421},
mesh = {Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; *Food Supply ; Gene Editing ; Livestock ; },
abstract = {The food industry faces a 2050 deadline for the advancement and expansion of the food supply chain to support the world's growing population. Improvements are needed across crops, livestock, and microbes to achieve this goal. Since 2005, researchers have been attempting to make the necessary strides to reach this milestone, but attempts have fallen short. With the introduction of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins, the food production field is now able to achieve some of its most exciting advancements since the Green Revolution. This review introduces the concept of applying CRISPR-Cas technology as a genome-editing tool for use in the food supply chain, focusing on its implementation to date in crop, livestock, and microbe production, advancement of products to market, and regulatory and societal hurdles that need to be overcome.},
}
@article {pmid30908848,
year = {2019},
author = {Carnes, RM and Mobley, JA and Crossman, DK and Liu, H and Korf, BR and Kesterson, RA and Wallis, D},
title = {Multi-Omics Profiling for NF1 Target Discovery in Neurofibromin (NF1) Deficient Cells.},
journal = {Proteomics},
volume = {19},
number = {11},
pages = {e1800334},
doi = {10.1002/pmic.201800334},
pmid = {30908848},
issn = {1615-9861},
support = {P30 CA013148/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Neurofibromin 1/*genetics ; Neurogenesis ; Proteomics/*methods ; Signal Transduction ; *Transcriptome ; },
abstract = {Loss of NF1 is an oncogenic driver. In efforts to define pathways responsible for the development of neurofibromas and other cancers, transcriptomic and proteomic changes are evaluated in a non-malignant NF1 null cell line. NF1 null HEK293 cells were created using CRISPR/Cas9 technology and they are compared to parental cells that express neurofibromin. A total of 1222 genes and 132 proteins are found to be differentially expressed. The analysis is integrated to identify eight transcripts/proteins that are differentially regulated in both analyses. Metacore Pathway analysis identifies Neurogenesis NGF/TrkA MAPK-mediated signaling alterations. Next, the data set is compared with other published studies that involve analysis of cells or tumors deficient for NF1 and it is found that 141 genes recur in the sample and others; only thirteen of these genes recur in two or more studies. Genes/proteins of interest are validated via q-RT-PCR or Western blot. It is shown that KRT8 and 14-3-3σ protein levels respond to exogenously introduced mNf1 cDNA. Hence, transcripts/proteins that respond to neurofibromin levels are identified and they can potentially be used as biomarkers.},
}
@article {pmid30908830,
year = {2019},
author = {Jia, H and Orbović, V and Wang, N},
title = {CRISPR-LbCas12a-mediated modification of citrus.},
journal = {Plant biotechnology journal},
volume = {17},
number = {10},
pages = {1928-1937},
pmid = {30908830},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Citrus paradisi/*genetics ; Clostridiales ; *Gene Editing ; Plant Diseases/genetics/microbiology ; Xanthomonas/pathogenicity ; },
abstract = {Recently, CRISPR-Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4 -CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEPthA4 -CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D35 s1 to #D35 s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #Dyao 1 to #Dyao 10. LbCas12a-directed EBEPthA4 -CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35 s1, #D35 s4 and #D35 s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35 s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. Therefore, CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing.},
}
@article {pmid30908774,
year = {2019},
author = {Xiao, G and Yi, Y and Che, R and Zhang, Q and Imran, M and Khan, A and Yan, J and Lin, X},
title = {Characterization of CRISPR-Cas systems in Leptospira reveals potential application of CRISPR in genotyping of Leptospira interrogans.},
journal = {APMIS : acta pathologica, microbiologica, et immunologica Scandinavica},
volume = {127},
number = {4},
pages = {202-216},
doi = {10.1111/apm.12935},
pmid = {30908774},
issn = {1600-0463},
mesh = {*CRISPR-Cas Systems ; *Genetic Variation ; Genome, Bacterial ; *Genotype ; Genotyping Techniques/*methods ; Leptospira interrogans/*classification/enzymology/*genetics ; },
abstract = {Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. However, understanding of the pathogenic mechanism of Leptospira is still elusive due to the limited number of genetic tools available for this microorganism. Currently, the reason for the genetic inaccessibility of Leptospira is still unknown. It is well known that as an acquired immunity of bacteria, Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR-associated gene (CRISPR-Cas) systems can help bacteria against invading mobile genetic elements. In this study, the occurrence and diversity of CRISPR-Cas systems in 41 genomes of Leptospira strains were investigated. Three subtypes (subtype I-B, subtype I-C and subtype I-E) of CRISPR-Cas systems were identified in both pathogenic and intermediate Leptospira species but not in saprophytic species. Noteworthy, the majority of pathogenic species harbor two different types of CRISPR-Cas systems (subtype I-B and subtype I-E). Furthermore, Cas2 protein of subtype I-C in L. interrogans exhibited a metal-dependent DNase activity in a nonspecific manner. CRISPR spacers in subtype I-B are highly conserved within the same serovars and hypervariable across different serovars of L. interrogans. Based on the subtype I-B CRISPR arrays, the serotypes of different L. interrogans strains were easily identified. Investigation of the origin of CRISPR spacers showed that 192 spacers (23.5%) matched to mobile genetic elements, indicating CRISPR-Cas systems may play an important role in the defense of foreign invading DNA.},
}
@article {pmid30906402,
year = {2018},
author = {Kim, JH},
title = {Genetics of Alzheimer's Disease.},
journal = {Dementia and neurocognitive disorders},
volume = {17},
number = {4},
pages = {131-136},
pmid = {30906402},
issn = {2384-0757},
abstract = {Alzheimer's disease (AD) related genes have been elucidated by advanced genetic techniques. Familial autosomal dominant AD genes founded by linkage analyses are APP, PSEN1, PSEN2, ABCA7, and SORL1. Genome-wide association studies have found risk genes such as ABCA7, BIN1, CASS4, CD33, CD2AP, CELF1, CLU, CR1, DSG2, EPHA1, FERMT2, HLA-DRB5-HLA-DRB1, INPP5D, MEF2C, MS4A6A/MS4A4E, NME8, PICALM, PTK2B, SLC24A4, SORL1, and ZCWPW1. ABCA7, SORL1, TREM2, and APOE are proved to have high odds ratio (>2) in risk of AD using next generation sequencing studies. Thanks to the promising genetic techniques such as CRISPR-CAS9 and single-cell RNA sequencing opened a new era in genetics. CRISPR-CAS9 can directly link genetic knowledge to future treatment. Single-cell RNA sequencing are providing useful information on cell biology and pathogenesis of diverse diseases.},
}
@article {pmid30905885,
year = {2019},
author = {Zhang, F},
title = {Exploration of Microbial Diversity to Discover Novel Molecular Technologies.},
journal = {The Keio journal of medicine},
volume = {68},
number = {1},
pages = {26},
doi = {10.2302/kjm.68-002-ABST},
pmid = {30905885},
issn = {1880-1293},
mesh = {Animals ; Archaea/chemistry/classification/genetics/immunology ; Archaeal Proteins/genetics/metabolism ; Bacteria/chemistry/classification/genetics/immunology ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; DNA Transposable Elements ; Eukaryotic Cells/cytology/metabolism ; Gene Editing/*methods ; *Genome ; Humans ; Isoenzymes/genetics/metabolism ; *Molecular Targeted Therapy ; RNA, Guide/*genetics/metabolism ; },
abstract = {Many powerful molecular biology tools have their origin in nature. From restriction enzymes to CRISPR-Cas9, microbes utilize a diverse array of systems to get ahead evolutionarily. We are exploring this natural diversity through bioinformatics, biochemical, and molecular work to better understand the fundamental ways in which microbes and other living organisms sense and respond to their environment and as possible to develop these natural systems as molecular tools and to improve human health. Building on our demonstration that Cas9 can be repurposed for precision genome editing in mammalian cells, we look for novel CRISPR-Cas systems that are different and may have other useful properties. This led to the discovery of several new CRISPR systems, including the CRISPR-Cas13 family that target RNA, rather than DNA. We have developed a toolbox for RNA modulation based on Cas13, including methods for precision base editing, adding to our robust toolbox for DNA based on Cas9 and Cas12. We are expanding our biodiscovery efforts to search for new microbial proteins that may be adapted for applications beyond genome and transcriptome modulation, capitalizing on the growing volume of microbial genomic sequences. We are particularly interested in identifying new therapeutic modalities and vehicles for delivering them into patients. We hope that additional robust tools and delivery options will further accelerate research into human disease and open up new therapeutic possibilities.},
}
@article {pmid30905739,
year = {2019},
author = {Yang, J and Rajan, SS and Friedrich, MJ and Lan, G and Zou, X and Ponstingl, H and Garyfallos, DA and Liu, P and Bradley, A and Metzakopian, E},
title = {Genome-Scale CRISPRa Screen Identifies Novel Factors for Cellular Reprogramming.},
journal = {Stem cell reports},
volume = {12},
number = {4},
pages = {757-771},
pmid = {30905739},
issn = {2213-6711},
support = {/WT_/Wellcome Trust/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; WT098051/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Biomarkers ; CRISPR-Cas Systems ; Cell Line ; Cellular Reprogramming/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Dosage ; *Genome-Wide Association Study ; Germ Layers/cytology/metabolism ; Humans ; Induced Pluripotent Stem Cells ; Mice ; Octamer Transcription Factor-3/genetics/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {Primed epiblast stem cells (EpiSCs) can be reverted to a pluripotent embryonic stem cell (ESC)-like state by expression of single reprogramming factor. We used CRISPR activation to perform a genome-scale, reprogramming screen in EpiSCs and identified 142 candidate genes. Our screen validated a total of 50 genes, previously not known to contribute to reprogramming, of which we chose Sall1 for further investigation. We show that Sall1 augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotent including formation of chimeric mice. We also demonstrate that Sall1 synergizes with Nanog in reprogramming and that overexpression in ESCs delays their conversion back to EpiSCs. Lastly, using RNA sequencing, we identify and validate Klf5 and Fam189a2 as new downstream targets of Sall1 and Nanog. In summary, our work demonstrates the power of using CRISPR technology in understanding molecular mechanisms that mediate complex cellular processes such as reprogramming.},
}
@article {pmid30905411,
year = {2019},
author = {Kasamatsu, A and Uzawa, K and Hayashi, F and Kita, A and Okubo, Y and Saito, T and Kimura, Y and Miyamoto, I and Oka, N and Shiiba, M and Ito, C and Toshimori, K and Miki, T and Yamauchi, M and Tanzawa, H},
title = {Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality.},
journal = {Biochemical and biophysical research communications},
volume = {512},
number = {3},
pages = {486-491},
doi = {10.1016/j.bbrc.2019.03.091},
pmid = {30905411},
issn = {1090-2104},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo Loss/*genetics/pathology ; Embryo, Mammalian/metabolism/*pathology ; *Endoplasmic Reticulum Stress ; Heart/embryology ; Heart Defects, Congenital/*genetics/pathology ; Mice ; Mice, Knockout ; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/*genetics ; },
abstract = {Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2[-/-]) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2[-/-] mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.},
}
@article {pmid30905297,
year = {2019},
author = {de Graeff, N and Jongsma, KR and Johnston, J and Hartley, S and Bredenoord, AL},
title = {The ethics of genome editing in non-human animals: a systematic review of reasons reported in the academic literature.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180106},
pmid = {30905297},
issn = {1471-2970},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/ethics/*veterinary ; },
abstract = {In recent years, new genome editing technologies have emerged that can edit the genome of non-human animals with progressively increasing efficiency. Despite ongoing academic debate about the ethical implications of these technologies, no comprehensive overview of this debate exists. To address this gap in the literature, we conducted a systematic review of the reasons reported in the academic literature for and against the development and use of genome editing technologies in animals. Most included articles were written by academics from the biomedical or animal sciences. The reported reasons related to seven themes: human health, efficiency, risks and uncertainty, animal welfare, animal dignity, environmental considerations and public acceptability. Our findings illuminate several key considerations about the academic debate, including a low disciplinary diversity in the contributing academics, a scarcity of systematic comparisons of potential consequences of using these technologies, an underrepresentation of animal interests, and a disjunction between the public and academic debate on this topic. As such, this article can be considered a call for a broad range of academics to get increasingly involved in the discussion about genome editing, to incorporate animal interests and systematic comparisons, and to further discuss the aims and methods of public involvement. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905296,
year = {2019},
author = {Buchthal, J and Evans, SW and Lunshof, J and Telford, SR and Esvelt, KM},
title = {Mice Against Ticks: an experimental community-guided effort to prevent tick-borne disease by altering the shared environment.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180105},
pmid = {30905296},
issn = {1471-2970},
mesh = {Animals ; Borrelia burgdorferi/*physiology ; CRISPR-Cas Systems/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; Disease Reservoirs/veterinary ; Immunization/methods/*veterinary ; Ixodes/microbiology ; Lyme Disease/prevention &amp; control/*veterinary ; Peromyscus/*immunology ; Rodent Diseases/prevention &amp; control ; },
abstract = {Mice Against Ticks is a community-guided ecological engineering project that aims to prevent tick-borne disease by using CRISPR-based genome editing to heritably immunize the white-footed mice (Peromyscus leucopus) responsible for infecting many ticks in eastern North America. Introducing antibody-encoding resistance alleles into the local mouse population is anticipated to disrupt the disease transmission cycle for decades. Technology development is shaped by engagement with community members and visitors to the islands of Nantucket and Martha's Vineyard, including decisions at project inception about which types of disease resistance to pursue. This engagement process has prompted the researchers to use only white-footed mouse DNA if possible, meaning the current project will not involve gene drive. Instead, engineered mice would be released in the spring when the natural population is low, a plan unlikely to increase total numbers above the normal maximum in autumn. Community members are continually asked to share their suggestions and concerns, a process that has already identified potential ecological consequences unanticipated by the research team that will likely affect implementation. As an early example of CRISPR-based ecological engineering, Mice Against Ticks aims to start small and simple by working with island communities whose mouse populations can be lastingly immunized without gene drive. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905295,
year = {2019},
author = {Ramachandran, G and Bikard, D},
title = {Editing the microbiome the CRISPR way.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180103},
pmid = {30905295},
issn = {1471-2970},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Microbiota ; },
abstract = {Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play a major role in our health. Over the past few years tools derived from the prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify and study organisms with unprecedented ease and efficiency. Here we discuss how various types of CRISPR-Cas systems can be used to modify the genome of gut microorganisms and bacteriophages. CRISPR-Cas systems can also be delivered to bacterial population and programmed to specifically eliminate members of the microbiome. Finally, engineered CRISPR-Cas systems can be used to control gene expression and modulate the production of metabolites and proteins. Together these tools provide exciting opportunities to investigate the complex interplay between members of the microbiome and our bodies, and present new avenues for the development of drugs that target the microbiome. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905294,
year = {2019},
author = {Westra, ER and van Houte, S and Gandon, S and Whitaker, R},
title = {The ecology and evolution of microbial CRISPR-Cas adaptive immune systems.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20190101},
pmid = {30905294},
issn = {1471-2970},
support = {BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity/*genetics ; Archaea/genetics/immunology ; Bacteria/genetics/immunology ; Bacteriophages/genetics/immunology ; CRISPR-Cas Systems/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; },
}
@article {pmid30905293,
year = {2019},
author = {Chevallereau, A and Meaden, S and van Houte, S and Westra, ER and Rollie, C},
title = {The effect of bacterial mutation rate on the evolution of CRISPR-Cas adaptive immunity.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180094},
pmid = {30905293},
issn = {1471-2970},
support = {BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity/*genetics ; Bacteria/*genetics ; CRISPR-Cas Systems/*genetics ; *Evolution, Molecular ; Mutation Rate ; },
abstract = {CRISPR-Cas immune systems are present in around half of bacterial genomes. Given the specificity and adaptability of this immune mechanism, it is perhaps surprising that they are not more widespread. Recent insights into the requirement for specific host factors for the function of some CRISPR-Cas subtypes, as well as the negative epistasis between CRISPR-Cas and other host genes, have shed light on potential reasons for the partial distribution of this immune strategy in bacteria. In this study, we examined how mutations in the bacterial mismatch repair system, which are frequently observed in natural and clinical isolates and cause elevated host mutation rates, influence the evolution of CRISPR-Cas-mediated immunity. We found that hosts with a high mutation rate very rarely evolved CRISPR-based immunity to phage compared to wild-type hosts. We explored the reason for this effect and found that the higher frequency at which surface mutants pre-exist in the mutator host background causes them to rapidly become the dominant phenotype under phage infection. These findings suggest that natural variation in bacterial mutation rates may, therefore, influence the distribution of CRISPR-Cas adaptive immune systems. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905292,
year = {2019},
author = {Pauly, MD and Bautista, MA and Black, JA and Whitaker, RJ},
title = {Diversified local CRISPR-Cas immunity to viruses of Sulfolobus islandicus.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180093},
pmid = {30905292},
issn = {1471-2970},
mesh = {CRISPR-Cas Systems/*immunology ; Hot Springs/microbiology ; Russia ; Sulfolobus/*virology ; Viruses/*immunology ; Wyoming ; },
abstract = {The population diversity and structure of CRISPR-Cas immunity provides key insights into virus-host interactions. Here, we examined two geographically and genetically distinct natural populations of the thermophilic crenarchaeon Sulfolobus islandicus and their interactions with Sulfolobus spindle-shaped viruses (SSVs) and S. islandicus rod-shaped viruses (SIRVs). We found that both virus families can be targeted with high population distributed immunity, whereby most immune strains target a virus using unique unshared CRISPR spacers. In Kamchatka, Russia, we observed high immunity to chronic SSVs that increases over time. In this context, we found that some SSVs had shortened genomes lacking genes that are highly targeted by the S. islandicus population, indicating a potential mechanism of immune evasion. By contrast, in Yellowstone National Park, we found high inter- and intra-strain immune diversity targeting lytic SIRVs and low immunity to chronic SSVs. In this population, we observed evidence of SIRVs evolving immunity through mutations concentrated in the first five bases of protospacers. These results indicate that diversity and structure of antiviral CRISPR-Cas immunity for a single microbial species can differ by both the population and virus type, and suggest that different virus families use different mechanisms to evade CRISPR-Cas immunity. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905291,
year = {2019},
author = {Lopatina, A and Medvedeva, S and Artamonova, D and Kolesnik, M and Sitnik, V and Ispolatov, Y and Severinov, K},
title = {Natural diversity of CRISPR spacers of Thermus: evidence of local spacer acquisition and global spacer exchange.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180092},
pmid = {30905291},
issn = {1471-2970},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Chile ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Italy ; Russia ; Thermus/*genetics/virology ; },
abstract = {We investigated the diversity of CRISPR spacers of Thermus communities from two locations in Italy, two in Chile and one location in Russia. Among the five sampling sites, a total of more than 7200 unique spacers belonging to different CRISPR-Cas systems types and subtypes were identified. Most of these spacers are not found in CRISPR arrays of sequenced Thermus strains. Comparison of spacer sets revealed that samples within the same area (separated by few to hundreds of metres) have similar spacer sets, which appear to be largely stable at least over the course of several years. While at further distances (hundreds of kilometres and more) the similarity of spacer sets is decreased, there are still multiple common spacers in Thermus communities from different continents. The common spacers can be reconstructed in identical or similar CRISPR arrays, excluding their independent appearance and suggesting an extensive migration of thermophilic bacteria over long distances. Several new Thermus phages were isolated in the sampling sites. Mapping of spacers to bacteriophage sequences revealed examples of local acquisition of spacers from some phages and distinct patterns of targeting of phage genomes by different CRISPR-Cas systems. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905290,
year = {2019},
author = {Watson, BNJ and Easingwood, RA and Tong, B and Wolf, M and Salmond, GPC and Staals, RHJ and Bostina, M and Fineran, PC},
title = {Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180090},
pmid = {30905290},
issn = {1471-2970},
support = {BB/H002677/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/G000298/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacteriophages/*genetics ; *CRISPR-Cas Systems ; Pectobacterium/*virology ; Point Mutation ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These 'spacers' provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905289,
year = {2019},
author = {Hoikkala, V and Almeida, GMF and Laanto, E and Sundberg, LR},
title = {Aquaculture as a source of empirical evidence for coevolution between CRISPR-Cas and phage.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180100},
pmid = {30905289},
issn = {1471-2970},
mesh = {*Aquaculture ; Bacteria/*virology ; Bacteriophages/*genetics/immunology ; *Biological Evolution ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution. Existing data from disease surveillance conducted in aquaculture, coupled with growing interest towards phage therapy, may have already resulted in large collections of bacterium and phage isolates. These data, combined with premeditated efforts, can provide empirical evidence on phage-bacterium dynamics such as the bacteriophage adherence to mucus hypothesis, phage life cycles and their relationship with CRISPR-Cas and other immune defences. Typing of CRISPR spacer content in pathogenic bacteria can also provide practical information on diversity and origin of isolates during outbreaks. In addition to providing information of CRISPR functionality and phage-bacterium dynamics, aquaculture systems can significantly impact perspectives on design of phage-based disease treatment at the current era of increasing antibiotic resistance. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905288,
year = {2019},
author = {McKitterick, AC and LeGault, KN and Angermeyer, A and Alam, M and Seed, KD},
title = {Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180089},
pmid = {30905288},
issn = {1471-2970},
support = {R01 AI127652/AI/NIAID NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/*physiology ; CRISPR-Cas Systems/*genetics ; High-Throughput Nucleotide Sequencing ; Interspersed Repetitive Sequences ; Vibrio cholerae/*virology ; },
abstract = {CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905287,
year = {2019},
author = {Bernheim, A and Bikard, D and Touchon, M and Rocha, EPC},
title = {A matter of background: DNA repair pathways as a possible cause for the sparse distribution of CRISPR-Cas systems in bacteria.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180088},
pmid = {30905287},
issn = {1471-2970},
mesh = {Bacteria/*genetics ; CRISPR-Cas Systems/*genetics ; *DNA Repair ; DNA, Bacterial/*genetics ; *Gene Transfer, Horizontal ; },
abstract = {The absence of CRISPR-Cas systems in more than half of the sequenced bacterial genomes is intriguing, because their role in adaptive immunity and their frequent transfer between species should have made them almost ubiquitous, as is the case in Archaea. Here, we investigate the possibility that the success of CRISPR-Cas acquisition by horizontal gene transfer is affected by the interactions of these systems with the host genetic background and especially with components of double-strand break repair systems (DSB-RS). We first described the distribution of systems specialized in the repair of double-strand breaks in Bacteria: homologous recombination and non-homologous end joining. This allowed us to show that such systems are more often positively or negatively correlated with the frequency of CRISPR-Cas systems than random genes of similar frequency. The detailed analysis of these co-occurrence patterns shows that our method identifies previously known cases of mechanistic interactions between these systems. It also reveals other positive and negative patterns of co-occurrence between DSB-RS and CRISPR-Cas systems. Notably, it shows that the patterns of distribution of CRISPR-Cas systems in Proteobacteria are strongly dependent on the epistatic groups including RecBCD and AddAB. Our results suggest that the genetic background plays an important role in the success of adaptive immunity in different bacterial clades and provide insights to guide further experimental research on the interactions between CRISPR-Cas and DSB-RS. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905286,
year = {2019},
author = {Shehreen, S and Chyou, TY and Fineran, PC and Brown, CM},
title = {Genome-wide correlation analysis suggests different roles of CRISPR-Cas systems in the acquisition of antibiotic resistance genes in diverse species.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180384},
pmid = {30905286},
issn = {1471-2970},
mesh = {Anti-Bacterial Agents/pharmacology ; Bacteria/drug effects/*genetics ; Drug Resistance, Microbial/*genetics ; Genome-Wide Association Study ; },
abstract = {CRISPR-Cas systems are widespread in bacterial and archaeal genomes, and in their canonical role in phage defence they confer a fitness advantage. However, CRISPR-Cas may also hinder the uptake of potentially beneficial genes. This is particularly true under antibiotic selection, where preventing the uptake of antibiotic resistance genes could be detrimental. Newly discovered features within these evolutionary dynamics are anti-CRISPR genes, which inhibit specific CRISPR-Cas systems. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally acquired antibiotic resistance genes. To assess that question, we analysed correlations between the CRISPR-Cas, anti-CRISPR and antibiotic resistance gene content of 104 947 reference genomes, including 5677 different species. In most species, the presence of CRISPR-Cas systems did not correlate with the presence of antibiotic resistance genes. However, in some clinically important species, we observed either a positive or negative correlation of CRISPR-Cas with antibiotic resistance genes. Anti-CRISPR genes were common enough in four species to be analysed. In Pseudomonas aeruginosa, the presence of anti-CRISPRs was associated with antibiotic resistance genes. This analysis indicates that the role of CRISPR-Cas and anti-CRISPRs in the spread of antibiotic resistance is likely to be very different in particular pathogenic species and clinical environments. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905285,
year = {2019},
author = {Common, J and Morley, D and Westra, ER and van Houte, S},
title = {CRISPR-Cas immunity leads to a coevolutionary arms race between Streptococcus thermophilus and lytic phage.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180098},
pmid = {30905285},
issn = {1471-2970},
support = {BB/J014400/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M009122/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R010781/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity/*genetics ; Bacteriophages/*immunology/physiology ; CRISPR-Cas Systems/*immunology ; *Evolution, Molecular ; Streptococcus thermophilus/*physiology/virology ; },
abstract = {CRISPR-Cas is an adaptive prokaryotic immune system that prevents phage infection. By incorporating phage-derived 'spacer' sequences into CRISPR loci on the host genome, future infections from the same phage genotype can be recognized and the phage genome cleaved. However, the phage can escape CRISPR degradation by mutating the sequence targeted by the spacer, allowing them to re-infect previously CRISPR-immune hosts, and theoretically leading to coevolution. Previous studies have shown that phage can persist over long periods in populations of Streptococcus thermophilus that can acquire CRISPR-Cas immunity, but it has remained less clear whether this coexistence was owing to coevolution, and if so, what type of coevolutionary dynamics were involved. In this study, we performed highly replicated serial transfer experiments over 30 days with S. thermophilus and a lytic phage. Using a combination of phenotypic and genotypic data, we show that CRISPR-mediated resistance and phage infectivity coevolved over time following an arms race dynamic, and that asymmetry between phage infectivity and host resistance within this system eventually causes phage extinction. This work provides further insight into the way CRISPR-Cas systems shape the population and coevolutionary dynamics of bacteria-phage interactions. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905284,
year = {2019},
author = {Koonin, EV and Makarova, KS},
title = {Origins and evolution of CRISPR-Cas systems.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180087},
pmid = {30905284},
issn = {1471-2970},
mesh = {Archaea/*genetics/immunology ; Bacteria/*genetics/immunology ; CRISPR-Cas Systems/*immunology ; *Evolution, Molecular ; },
abstract = {CRISPR-Cas, the bacterial and archaeal adaptive immunity systems, encompass a complex machinery that integrates fragments of foreign nucleic acids, mostly from mobile genetic elements (MGE), into CRISPR arrays embedded in microbial genomes. Transcripts of the inserted segments (spacers) are employed by CRISPR-Cas systems as guide (g)RNAs for recognition and inactivation of the cognate targets. The CRISPR-Cas systems consist of distinct adaptation and effector modules whose evolutionary trajectories appear to be at least partially independent. Comparative genome analysis reveals the origin of the adaptation module from casposons, a distinct type of transposons, which employ a homologue of Cas1 protein, the integrase responsible for the spacer incorporation into CRISPR arrays, as the transposase. The origin of the effector module(s) is far less clear. The CRISPR-Cas systems are partitioned into two classes, class 1 with multisubunit effectors, and class 2 in which the effector consists of a single, large protein. The class 2 effectors originate from nucleases encoded by different MGE, whereas the origin of the class 1 effector complexes remains murky. However, the recent discovery of a signalling pathway built into the type III systems of class 1 might offer a clue, suggesting that type III effector modules could have evolved from a signal transduction system involved in stress-induced programmed cell death. The subsequent evolution of the class 1 effector complexes through serial gene duplication and displacement, primarily of genes for proteins containing RNA recognition motif domains, can be hypothetically reconstructed. In addition to the multiple contributions of MGE to the evolution of CRISPR-Cas, the reverse flow of information is notable, namely, recruitment of minimalist variants of CRISPR-Cas systems by MGE for functions that remain to be elucidated. Here, we attempt a synthesis of the diverse threads that shed light on CRISPR-Cas origins and evolution. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905283,
year = {2019},
author = {Chabas, H and Nicot, A and Meaden, S and Westra, ER and Tremblay, DM and Pradier, L and Lion, S and Moineau, S and Gandon, S},
title = {Variability in the durability of CRISPR-Cas immunity.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180097},
pmid = {30905283},
issn = {1471-2970},
support = {BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptive Immunity/*genetics ; Bacteriophages/*genetics ; CRISPR-Cas Systems/*immunology ; Streptococcus thermophilus/*immunology/virology ; },
abstract = {The durability of host resistance is challenged by the ability of pathogens to escape the defence of their hosts. Understanding the variability in the durability of host resistance is of paramount importance for designing more effective control strategies against infectious diseases. Here, we study the durability of various clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) alleles of the bacteria Streptococcus thermophilus against lytic phages. We found substantial variability in durability among different resistant bacteria. Since the escape of the phage is driven by a mutation in the phage sequence targeted by CRISPR-Cas, we explored the fitness costs associated with these escape mutations. We found that, on average, escape mutations decrease the fitness of the phage. Yet, the magnitude of this fitness cost does not predict the durability of CRISPR-Cas immunity. We contend that this variability in the durability of resistance may be because of variations in phage mutation rate or in the proportion of lethal mutations across the phage genome. These results have important implications on the coevolutionary dynamics between bacteria and phages and for the optimal deployment of resistance strategies against pathogens and pests. Understanding the durability of CRISPR-Cas immunity may also help develop more effective gene-drive strategies based on CRISPR-Cas9 technology. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905282,
year = {2019},
author = {Gurney, J and Pleška, M and Levin, BR},
title = {Why put up with immunity when there is resistance: an excursion into the population and evolutionary dynamics of restriction-modification and CRISPR-Cas.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180096},
pmid = {30905282},
issn = {1471-2970},
support = {R01 GM091875/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*immunology/virology ; Bacteriophages/*physiology ; CRISPR-Cas Systems/*immunology ; DNA Restriction-Modification Enzymes/*immunology ; *Evolution, Molecular ; Models, Biological ; Population Dynamics ; },
abstract = {Bacteria can readily generate mutations that prevent bacteriophage (phage) adsorption and thus make bacteria resistant to infections with these viruses. Nevertheless, the majority of bacteria carry complex innate and/or adaptive immune systems: restriction-modification (RM) and CRISPR-Cas, respectively. Both RM and CRISPR-Cas are commonly assumed to have evolved and be maintained to protect bacteria from succumbing to infections with lytic phage. Using mathematical models and computer simulations, we explore the conditions under which selection mediated by lytic phage will favour such complex innate and adaptive immune systems, as opposed to simple envelope resistance. The results of our analysis suggest that when populations of bacteria are confronted with lytic phage: (i) In the absence of immunity, resistance to even multiple bacteriophage species with independent receptors can evolve readily. (ii) RM immunity can benefit bacteria by preventing phage from invading established bacterial populations and particularly so when there are multiple bacteriophage species adsorbing to different receptors. (iii) Whether CRISPR-Cas immunity will prevail over envelope resistance depends critically on the number of steps in the coevolutionary arms race between the bacteria-acquiring spacers and the phage-generating CRISPR-escape mutants. We discuss the implications of these results in the context of the evolution and maintenance of RM and CRISPR-Cas and highlight fundamental questions that remain unanswered. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30905281,
year = {2019},
author = {Bradde, S and Mora, T and Walczak, AM},
title = {Cost and benefits of clustered regularly interspaced short palindromic repeats spacer acquisition.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {374},
number = {1772},
pages = {20180095},
pmid = {30905281},
issn = {1471-2970},
mesh = {Bacteria/*genetics ; Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Models, Biological ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated immunity in bacteria allows bacterial populations to protect themselves against pathogens. However, it also exposes them to the dangers of auto-immunity by developing protection that targets its own genome. Using a simple model of the coupled dynamics of phage and bacterial populations, we explore how acquisition rates affect the probability of the bacterial colony going extinct. We find that the optimal strategy depends on the initial population sizes of both viruses and bacteria. Additionally, certain combinations of acquisition and dynamical rates and initial population sizes guarantee protection, owing to a dynamical balance between the evolving population sizes, without relying on acquisition of viral spacers. Outside this regime, the high cost of auto-immunity limits the acquisition rate. We discuss these optimal strategies that minimize the probability of the colony going extinct in terms of recent experiments. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.},
}
@article {pmid30903024,
year = {2019},
author = {Daniel-Moreno, A and Lamsfus-Calle, A and Raju, J and Antony, JS and Handgretinger, R and Mezger, M},
title = {CRISPR/Cas9-modified hematopoietic stem cells-present and future perspectives for stem cell transplantation.},
journal = {Bone marrow transplantation},
volume = {54},
number = {12},
pages = {1940-1950},
doi = {10.1038/s41409-019-0510-8},
pmid = {30903024},
issn = {1476-5365},
mesh = {CRISPR-Cas Systems/*genetics ; Hematopoietic Stem Cell Transplantation/*methods ; Humans ; Stem Cell Transplantation/*methods ; Transplantation Conditioning/*methods ; Transplantation, Homologous/*methods ; },
abstract = {Allogeneic hematopoietic stem cell transplantation (HSCT) is a standard therapeutic intervention for hematological malignancies and several monogenic diseases. However, this approach has limitations related to lack of a suitable donor, graft-versus-host disease and infectious complications due to immune suppression. On the contrary, autologous HSCT diminishes the negative effects of allogeneic HSCT. Despite the good efficacy, earlier gene therapy trials with autologous HSCs and viral vectors have raised serious safety concerns. However, the CRISPR/Cas9-edited autologous HSCs have been proposed to be an alternative option with a high safety profile. In this review, we summarized the possibility of CRISPR/Cas9-mediated autologous HSCT as a potential treatment option for various diseases supported by preclinical gene-editing studies. Furthermore, we discussed future clinical perspectives and possible clinical grade improvements of CRISPR/cas9-mediated autologous HSCT.},
}
@article {pmid30902686,
year = {2019},
author = {Wang, B and Zhu, L and Zhao, B and Zhao, Y and Xie, Y and Zheng, Z and Li, Y and Sun, J and Wang, H},
title = {Development of a Haploid-Inducer Mediated Genome Editing System for Accelerating Maize Breeding.},
journal = {Molecular plant},
volume = {12},
number = {4},
pages = {597-602},
doi = {10.1016/j.molp.2019.03.006},
pmid = {30902686},
issn = {1752-9867},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; *Haploidy ; Plant Breeding/*methods ; Zea mays/*genetics ; },
abstract = {Crop breeding aims to generate pure inbred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize inbred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.},
}
@article {pmid30901150,
year = {2019},
author = {Mehnert, M and Li, W and Wu, C and Salovska, B and Liu, Y},
title = {Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1.},
journal = {Proteomics},
volume = {19},
number = {13},
pages = {e1800438},
doi = {10.1002/pmic.201800438},
pmid = {30901150},
issn = {1615-9861},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/genetics ; Chromatin/physiology ; *Gene Deletion ; Gene Editing/*methods ; HMGN1 Protein/*genetics ; HeLa Cells ; Humans ; Proteomics/*methods ; },
abstract = {CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.},
}
@article {pmid30900787,
year = {2019},
author = {Schmidt, C and Pacher, M and Puchta, H},
title = {Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system.},
journal = {The Plant journal : for cell and molecular biology},
volume = {98},
number = {4},
pages = {577-589},
doi = {10.1111/tpj.14322},
pmid = {30900787},
issn = {1365-313X},
support = {ERC-2016-AdG_741306 CRISBREED/ERC_/European Research Council/International ; },
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/genetics ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA End-Joining Repair ; DNA-Binding Proteins/genetics ; Genetic Engineering/methods ; Genome, Plant/*genetics ; Homologous Recombination ; Sequence Deletion ; },
abstract = {During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end-joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg-cell-specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.},
}
@article {pmid30900179,
year = {2019},
author = {Sauter, EJ and Kutsche, LK and Klapper, SD and Busskamp, V},
title = {Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1942},
number = {},
pages = {101-121},
doi = {10.1007/978-1-4939-9080-1_9},
pmid = {30900179},
issn = {1940-6029},
mesh = {Astrocytes/*cytology ; *CRISPR-Cas Systems ; Cell Differentiation ; Coculture Techniques ; Humans ; Induced Pluripotent Stem Cells/*cytology/transplantation ; Neurodegenerative Diseases/genetics/*therapy ; Neurodevelopmental Disorders/genetics/*therapy ; Neurons/*cytology/transplantation ; Transcription Factors/antagonists &amp; inhibitors/*genetics ; },
abstract = {Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.},
}
@article {pmid30900175,
year = {2019},
author = {Li, M and Hunt, JFVS and Bhattacharyya, A and Zhao, X},
title = {One-Step Generation of Seamless Luciferase Gene Knockin Using CRISPR/Cas9 Genome Editing in Human Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1942},
number = {},
pages = {61-69},
doi = {10.1007/978-1-4939-9080-1_5},
pmid = {30900175},
issn = {1940-6029},
support = {T32 GM008692/GM/NIGMS NIH HHS/United States ; R01 MH118827/MH/NIMH NIH HHS/United States ; R01 MH116582/MH/NIMH NIH HHS/United States ; R21 NS105339/NS/NINDS NIH HHS/United States ; U54 HD090256/HD/NICHD NIH HHS/United States ; R01 MH078972/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Embryonic Stem Cells/cytology/metabolism ; Fragile X Mental Retardation Protein/*genetics ; Fragile X Syndrome/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Luciferases/*metabolism ; },
abstract = {Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.},
}
@article {pmid30899116,
year = {2019},
author = {Soriano, V},
title = {Gene Editing for HIV Cure at the Edge.},
journal = {AIDS reviews},
volume = {21},
number = {1},
pages = {50a-51},
pmid = {30899116},
issn = {1698-6997},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; HIV Infections/*therapy ; HIV-1/*genetics ; Humans ; },
}
@article {pmid30898910,
year = {2019},
author = {Cohen, J},
title = {Tests identify HIV's final redoubt.},
journal = {Science (New York, N.Y.)},
volume = {363},
number = {6433},
pages = {1260-1261},
doi = {10.1126/science.363.6433.1260},
pmid = {30898910},
issn = {1095-9203},
mesh = {Acquired Immunodeficiency Syndrome/*drug therapy/*virology ; Animals ; Anti-Retroviral Agents/*pharmacology ; CRISPR-Cas Systems ; HIV/*drug effects/genetics/*physiology ; Haplorhini ; Humans ; Replicon/drug effects/physiology ; Virus Replication/*drug effects/genetics/*physiology ; },
}
@article {pmid30898877,
year = {2019},
author = {Choquet, K and Forget, D and Meloche, E and Dicaire, MJ and Bernard, G and Vanderver, A and Schiffmann, R and Fabian, MR and Teichmann, M and Coulombe, B and Brais, B and Kleinman, CL},
title = {Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {18},
pages = {7445-7459},
pmid = {30898877},
issn = {1083-351X},
support = {MOP-126141//CIHR/Canada ; MOP-G2-341146-159133-BRIDG//CIHR/Canada ; 201610PJT-377869//CIHR/Canada ; },
mesh = {Down-Regulation/*genetics ; Genes, Recessive ; HeLa Cells ; Hereditary Central Nervous System Demyelinating Diseases/*genetics ; Humans ; *Mutation ; RNA Polymerase III/*genetics ; RNA, Long Noncoding/*genetics ; RNA, Messenger/*genetics ; },
abstract = {RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A→G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.},
}
@article {pmid30898838,
year = {2019},
author = {Price, C and Gill, S and Ho, ZV and Davidson, SM and Merkel, E and McFarland, JM and Leung, L and Tang, A and Kost-Alimova, M and Tsherniak, A and Jonas, O and Vazquez, F and Hahn, WC},
title = {Genome-Wide Interrogation of Human Cancers Identifies EGLN1 Dependency in Clear Cell Ovarian Cancers.},
journal = {Cancer research},
volume = {79},
number = {10},
pages = {2564-2579},
pmid = {30898838},
issn = {1538-7445},
support = {P41 EB015898/EB/NIBIB NIH HHS/United States ; R01 CA223150/CA/NCI NIH HHS/United States ; T32 CA009361/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; *Genome-Wide Association Study ; Humans ; Hypoxia-Inducible Factor-Proline Dioxygenases/*genetics ; Ovarian Neoplasms/*genetics/pathology ; RNA Interference ; },
abstract = {We hypothesized that candidate dependencies for which there are small molecules that are either approved or in advanced development for a nononcology indication may represent potential therapeutic targets. To test this hypothesis, we performed genome-scale loss-of-function screens in hundreds of cancer cell lines. We found that knockout of EGLN1, which encodes prolyl hydroxylase domain-containing protein 2 (PHD2), reduced the proliferation of a subset of clear cell ovarian cancer cell lines in vitro. EGLN1-dependent cells exhibited sensitivity to the pan-EGLN inhibitor FG-4592. The response to FG-4592 was reversed by deletion of HIF1A, demonstrating that EGLN1 dependency was related to negative regulation of HIF1A. We also found that ovarian clear cell tumors susceptible to both genetic and pharmacologic inhibition of EGLN1 required intact HIF1A. Collectively, these observations identify EGLN1 as a cancer target with therapeutic potential. SIGNIFICANCE: These findings reveal a differential dependency of clear cell ovarian cancers on EGLN1, thus identifying EGLN1 as a potential therapeutic target in clear cell ovarian cancer patients.},
}
@article {pmid30898720,
year = {2019},
author = {Herai, RH},
title = {Avoiding the off-target effects of CRISPR/cas9 system is still a challenging accomplishment for genetic transformation.},
journal = {Gene},
volume = {700},
number = {},
pages = {176-178},
doi = {10.1016/j.gene.2019.03.019},
pmid = {30898720},
issn = {1879-0038},
mesh = {CRISPR-Cas Systems ; Gene Editing/ethics/*methods ; Genome, Human ; Humans ; RNA, Guide ; *Transformation, Genetic ; },
abstract = {The recent disclosure of a human embryo subjected to a genetic transformation using the CRISPR/cas9 system give rise to several concerns on ethical questions about its uncontrolled use in humans. Although CRISPR/cas9 has demonstrated its efficiency, this system still lacks the capability to avoid the introduction of undesirable mutations through the target genome. In this Letter, we present several undesirable impacts that CRISPR/cas9 system have in the genetic transformation of the human genome. We briefly discuss, using the very recent literature from distinct high impact journals, the main concerns related to CRISPR/cas9 to deal with off-target effects and how the research community has treated it.},
}
@article {pmid30898719,
year = {2019},
author = {Ryu, N and Kim, MA and Choi, DG and Kim, YR and Sonn, JK and Lee, KY and Kim, UK},
title = {CRISPR/Cas9-mediated genome editing of splicing mutation causing congenital hearing loss.},
journal = {Gene},
volume = {703},
number = {},
pages = {83-90},
doi = {10.1016/j.gene.2019.03.020},
pmid = {30898719},
issn = {1879-0038},
mesh = {Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Exons ; Gene Editing ; Gene Targeting/*methods ; Genetic Therapy ; Hearing Loss/*congenital/genetics ; Humans ; Mice ; Staphylococcus aureus/*metabolism ; Sulfate Transporters/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.},
}
@article {pmid30897320,
year = {2019},
author = {Paul, A and Bharati, J and Punetha, M and Kumar, S and Mallesh, VG and Chouhan, VS and Sonwane, A and Bag, S and Bhure, SK and Maurya, VP and Singh, G and Whitworth, KM and Sarkar, M},
title = {Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {52},
number = {3},
pages = {532-552},
doi = {10.33594/000000038},
pmid = {30897320},
issn = {1421-9778},
mesh = {Animals ; Apoptosis ; Buffaloes/metabolism ; CD36 Antigens/genetics/metabolism ; CD47 Antigen/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Caspase 3/genetics/metabolism ; Cell Survival ; Corpus Luteum/cytology/*metabolism/pathology ; Dinoprost/metabolism ; Down-Regulation ; Female ; Fibroblast Growth Factor 2/genetics/metabolism ; *Gene Editing ; Thrombospondin 1/*genetics/metabolism ; Thrombospondins/genetics/metabolism ; Vascular Endothelial Growth Factor A/genetics/metabolism ; },
abstract = {BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated.<br><br>METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2&#x3b1;, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins.<br><br>RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2&#x3b1; induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells.<br><br>CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2&#x3b1; plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.},
}
@article {pmid30897091,
year = {2019},
author = {Roth, A and Vleurinck, C and Netschitailo, O and Bauer, V and Otte, M and Kaftanoglu, O and Page, RE and Beye, M},
title = {A genetic switch for worker nutrition-mediated traits in honeybees.},
journal = {PLoS biology},
volume = {17},
number = {3},
pages = {e3000171},
pmid = {30897091},
issn = {1545-7885},
mesh = {Animals ; Bees/*enzymology/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Expression Regulation, Developmental/genetics ; Male ; },
abstract = {Highly social insects are characterized by caste dimorphism, with distinct size differences of reproductive organs between fertile queens and the more or less sterile workers. An abundance of nutrition or instruction via diet-specific compounds has been proposed as explanations for the nutrition-driven queen and worker polyphenism. Here, we further explored these models in the honeybee (Apis mellifera) using worker nutrition rearing and a novel mutational screening approach using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. The worker nutrition-driven size reduction of reproductive organs was restricted to the female sex, suggesting input from the sex determination pathway. Genetic screens on the sex determination genes in genetic females for size polyphenism revealed that doublesex (dsx) mutants display size-reduced reproductive organs irrespective of the sexual morphology of the organ tissue. In contrast, feminizer (fem) mutants lost the response to worker nutrition-driven size control. The first morphological worker mutants in honeybees demonstrate that the response to nutrition relies on a genetic program that is switched "ON" by the fem gene. Thus, the genetic instruction provided by the fem gene provides an entry point to genetically dissect the underlying processes that implement the size polyphenism.},
}
@article {pmid30896351,
year = {2019},
author = {Scott, CT and Selin, C},
title = {What to Expect When Expecting CRISPR Baby Number Four.},
journal = {The American journal of bioethics : AJOB},
volume = {19},
number = {3},
pages = {7-9},
doi = {10.1080/15265161.2018.1562793},
pmid = {30896351},
issn = {1536-0075},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; China ; Embryo Research/*ethics/legislation &amp; jurisprudence ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Health Policy/legislation &amp; jurisprudence ; Humans ; Internationality/legislation &amp; jurisprudence ; National Academies of Science, Engineering, and Medicine, U.S., Health and Medicine Division ; United States ; },
}
@article {pmid30895956,
year = {2019},
author = {Karpov, DS and Karpov, VL and Klimova, RR and Demidova, NA and Kushch, AA},
title = {[A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {1},
pages = {91-100},
doi = {10.1134/S0026898419010051},
pmid = {30895956},
issn = {0026-8984},
mesh = {Animals ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; Herpesvirus 1, Human/*physiology ; Plasmids ; Vero Cells ; *Virus Replication ; },
abstract = {Herpesviruses are widespread in the human population. Herpes simplex virus type 1 (HSV1) alone infects more than 3.7 billion people. In most of these, the virus establishes a latent form resistant to the action of all antiviral drugs. Moreover, completely drug-resistant strains of herpesviruses are known, which has prompted the search for alternative approaches to the treatment of herpesviruses, including genome editing with prokaryotic CRISPR/Cas. The CRISPR/Cas9 system of Streptococcus pyogens effectively suppresses HSV1 infection when expressed from genome-integrated lentiviral vectors. However, there are concerns about the safety of this approach. Here we describe the system built upon the plasmid-encoded CRISPR/Cas9 targeted against UL52 and UL29 genes of the HSV1 primase-helicase complex. The construct was transfected into Vero cells with no significant cytotoxic effects detected. Complete suppression of HSV1 infection within two days was observed, raising the possibility that the proposed plasmid-expressed CRISPR/Cas9 system may be used for the screening of genes important for the HSV1 life cycle and for development of novel strategies for targeted therapy of herpesvirus infections.},
}
@article {pmid30895940,
year = {2019},
author = {Bolduc, V and Foley, AR and Solomon-Degefa, H and Sarathy, A and Donkervoort, S and Hu, Y and Chen, GS and Sizov, K and Nalls, M and Zhou, H and Aguti, S and Cummings, BB and Lek, M and Tukiainen, T and Marshall, JL and Regev, O and Marek-Yagel, D and Sarkozy, A and Butterfield, RJ and Jou, C and Jimenez-Mallebrera, C and Li, Y and Gartioux, C and Mamchaoui, K and Allamand, V and Gualandi, F and Ferlini, A and Hanssen, E and , and Wilton, SD and Lamandé, SR and MacArthur, DG and Wagener, R and Muntoni, F and Bönnemann, CG},
title = {A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies.},
journal = {JCI insight},
volume = {4},
number = {6},
pages = {},
pmid = {30895940},
issn = {2379-3708},
support = {K08 NS097631/NS/NINDS NIH HHS/United States ; UM1 HG008900/HG/NHGRI NIH HHS/United States ; ZIA NS003129/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; Collagen Type VI/*genetics ; DNA Mutational Analysis ; Exons/genetics ; Fibroblasts/metabolism/pathology ; Gene Expression ; Genetic Predisposition to Disease/*genetics ; Genetic Therapy/methods ; High-Throughput Nucleotide Sequencing ; Humans ; Introns/genetics ; Muscular Dystrophies/*genetics/*therapy ; Mutation ; RNA Splice Sites ; *RNA Splicing ; RNA, Messenger/metabolism ; Skin/pathology ; },
abstract = {The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI-related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.},
}
@article {pmid30895693,
year = {2019},
author = {Castelli, A and Susani, L and Menale, C and Muggeo, S and Caldana, E and Strina, D and Cassani, B and Recordati, C and Scanziani, E and Ficara, F and Villa, A and Vezzoni, P and Paulis, M},
title = {Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {7},
pages = {876-887},
doi = {10.1002/stem.3006},
pmid = {30895693},
issn = {1549-4918},
mesh = {Aminopterin/metabolism/pharmacology ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Differentiation ; *Chromosomes, Mammalian ; Clone Cells ; Culture Media/chemistry ; Disease Models, Animal ; Gene Editing/methods ; Genetic Therapy/*methods ; Granulocytes/cytology/drug effects/*metabolism ; Granulomatous Disease, Chronic/genetics/metabolism/pathology/*therapy ; Humans ; Hypoxanthine/metabolism/pharmacology ; Hypoxanthine Phosphoribosyltransferase/deficiency/*genetics ; Induced Pluripotent Stem Cells/drug effects/*metabolism/pathology ; Male ; Mice ; NADPH Oxidase 2/deficiency/*genetics ; Proof of Concept Study ; Sequence Deletion ; Thioguanine/metabolism/pharmacology ; Thymidine/metabolism/pharmacology ; X Chromosome/chemistry/metabolism ; },
abstract = {In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.},
}
@article {pmid30895249,
year = {2018},
author = {Hendriks, S and Giesbertz, NAA and Bredenoord, AL and Repping, S},
title = {Reasons for being in favour of or against genome modification: a survey of the Dutch general public.},
journal = {Human reproduction open},
volume = {2018},
number = {3},
pages = {hoy008},
pmid = {30895249},
issn = {2399-3529},
abstract = {STUDY QUESTION: What are the general public's reasons for being in favour of or against the use of genome modification for five potential applications?<br><br>SUMMARY ANSWER: Overall, 43 reasons for being in favour, 45 reasons for being against as well as 26 conditional reasons for the use of genome modification were identified.<br><br>WHAT IS KNOWN ALREADY: Various applications of somatic genome modification are progressing towards clinical introduction and several recent studies have reported on germline genome modification. This has incited a debate on ethical and legal implications and acceptability. There is a growing plea to involve the general public earlier on in the developmental process of science and (bio)technology including genome modification.<br><br>STUDY DESIGN SIZE DURATION: In April 2016, a cross-sectional survey was launched online among the Dutch general public. A documentary on genome modification on public television and calls in social media invited viewers and non-viewers, respectively, to participate.<br><br>The questionnaire introduced five potential future applications of genome modification: modified wheat for individuals with gluten intolerance; somatic modification for individuals with neuromuscular diseases; germline modification to prevent passing on a neuromuscular disease; germline modification to introduce resistance to HIV; and germline modification to increase intelligence. Participants were asked to indicate whether and why they would make use of genome modification in these scenarios. The reasons mentioned were analysed through content analysis by two researchers independently. The proportion of respondents that was willing to modify was described per scenario and associations with respondent characteristics were analysed.<br><br>The survey was completed by 1013 participants. Forty-three reasons for being in favour, 45 reasons for being against as well as 26 conditional reasons for the use of genome modification were identified. These could be categorized into 14 domains: safety of the individuals concerned; effectiveness; quality of life of the individuals concerned; existence of a clinical need or an alternative; biodiversity and ecosystems; animal homo sapiens (i.e. relating to effects on humans as a species); human life and dignity; trust in regulation; justice; costs; slippery slope; argument of nature; parental rights and duties; and (reproductive) autonomy. Participants' willingness to use genome modification was dependent on the application: most participants would eat modified wheat if gluten intolerant (74%), would use genome modification to cure his/her own neuromuscular disease (85%) and would apply germline modification to prevent passing on this neuromuscular disease (66%). A minority would apply germline modification to introduce resistance to HIV (30%) or increase intelligence (16%). Being young (odds ratio (OR) = 0.98 per year increase), being male (OR = 2.38), and having watched the documentary (OR = 1.82) were associated with being willing to apply genome modification in more scenarios.<br><br>Inquiring for reasons through open questions in a survey allowed for a larger sample size and intuitive responses but resulted in less depth than traditional face-to-face interviews. As the survey was disseminated through social media, the sample is not representative of the overall Dutch population, and hence the quantitative results should not be interpreted as such.<br><br>Further public consultation and a more in-depth ethical and societal debate on principles and conditions for responsible use of (germline) genome modification is required prior to future clinical introduction.<br><br>Funded by the University of Amsterdam and University Medical Centre Utrecht. No conflict of interest.<br><br>TRIAL REGISTRATION NUMBER: Not applicable.},
}
@article {pmid30894699,
year = {2019},
author = {Fyfe, I},
title = {Alzheimer disease mice improve with CRISPR-Cas9 gene editing.},
journal = {Nature reviews. Neurology},
volume = {15},
number = {5},
pages = {247},
doi = {10.1038/s41582-019-0172-6},
pmid = {30894699},
issn = {1759-4766},
mesh = {*Alzheimer Disease ; Animals ; CRISPR-Cas Systems ; *Gene Editing ; Mice ; },
}
@article {pmid30894668,
year = {2019},
author = {Sakaguchi, K and Yoneda, M and Sakai, N and Nakashima, K and Kitano, H and Matsuyama, M},
title = {Comprehensive Experimental System for a Promising Model Organism Candidate for Marine Teleosts.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4948},
pmid = {30894668},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Embryo, Nonmammalian ; Fishes/*physiology ; Gene Editing/methods ; Microinjections/methods ; *Models, Animal ; Seawater ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F1 generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.},
}
@article {pmid30894632,
year = {2019},
author = {Jeong, YK and Yu, J and Bae, S},
title = {Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4939},
pmid = {30894632},
issn = {2045-2322},
mesh = {Adenosine/genetics ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular/*methods ; DNA Breaks, Double-Stranded ; DNA Cleavage ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Nucleotide Motifs/*genetics ; Plasmids/genetics ; Proof of Concept Study ; RNA, Guide/genetics ; Substrate Specificity ; },
abstract = {Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.},
}
@article {pmid30894545,
year = {2019},
author = {Sandoz, J and Nagy, Z and Catez, P and Caliskan, G and Geny, S and Renaud, JB and Concordet, JP and Poterszman, A and Tora, L and Egly, JM and Le May, N and Coin, F},
title = {Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1288},
pmid = {30894545},
issn = {2041-1723},
support = {340551/ERC_/European Research Council/International ; },
mesh = {Acetylation ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin/*chemistry/metabolism ; Cockayne Syndrome/*genetics/metabolism/pathology ; Fibroblasts/cytology/metabolism ; Gene Editing ; Gene Expression Regulation ; Histone Acetyltransferases/antagonists &amp; inhibitors/*genetics/metabolism ; Histones/genetics/*metabolism ; Humans ; Models, Biological ; Osteoblasts/cytology/metabolism ; Primary Cell Culture ; Protein Subunits/*genetics/metabolism ; RNA, Guide/genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Signal Transduction ; Transcription Factor TFIIH/*genetics/metabolism ; Transcription Initiation, Genetic ; Xeroderma Pigmentosum/*genetics/metabolism/pathology ; },
abstract = {The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.},
}
@article {pmid30894482,
year = {2019},
author = {Quinney, KB and Frankel, EB and Shankar, R and Kasberg, W and Luong, P and Audhya, A},
title = {Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {14},
pages = {6858-6867},
pmid = {30894482},
issn = {1091-6490},
support = {T32 GM008688/GM/NIGMS NIH HHS/United States ; T32 GM007215/GM/NIGMS NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; R01 GM088151/GM/NIGMS NIH HHS/United States ; R01 GM110567/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Transformed ; Endosomal Sorting Complexes Required for Transport/*genetics/metabolism ; Gene Editing ; Humans ; Intercellular Signaling Peptides and Proteins/genetics/*metabolism ; Multivesicular Bodies/genetics/*metabolism ; Protein Transport/physiology ; },
abstract = {The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.},
}
@article {pmid30894433,
year = {2019},
author = {Otoupal, PB and Ito, M and Arkin, AP and Magnuson, JK and Gladden, JM and Skerker, JM},
title = {Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
pmid = {30894433},
issn = {2379-5042},
mesh = {Basidiomycota/drug effects/*genetics ; *CRISPR-Cas Systems ; Drug Resistance, Fungal ; Fungal Proteins/genetics ; Gene Editing/*methods ; *Genome, Fungal ; Streptothricins/pharmacology ; },
abstract = {Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae Significant advancements in the past few years have bolstered R. toruloides' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.},
}
@article {pmid30894429,
year = {2019},
author = {Myrbråten, IS and Wiull, K and Salehian, Z and Håvarstein, LS and Straume, D and Mathiesen, G and Kjos, M},
title = {CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in Lactobacillus plantarum.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
pmid = {30894429},
issn = {2379-5042},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Cell Wall/enzymology/genetics ; DNA Replication ; Gene Knockdown Techniques/*methods ; *Genes, Essential ; *Genes, cdc ; Lactobacillus plantarum/*genetics ; Phenotype ; Plasmids/genetics ; RNA Interference ; },
abstract = {Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium.IMPORTANCEL. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.},
}
@article {pmid30894372,
year = {2019},
author = {Wang, Z and Feng, X and Molinolo, AA and Martin, D and Vitale-Cross, L and Nohata, N and Ando, M and Wahba, A and Amornphimoltham, P and Wu, X and Gilardi, M and Allevato, M and Wu, V and Steffen, DJ and Tofilon, P and Sonenberg, N and Califano, J and Chen, Q and Lippman, SM and Gutkind, JS},
title = {4E-BP1 Is a Tumor Suppressor Protein Reactivated by mTOR Inhibition in Head and Neck Cancer.},
journal = {Cancer research},
volume = {79},
number = {7},
pages = {1438-1450},
pmid = {30894372},
issn = {1538-7445},
support = {R33 CA225291/CA/NCI NIH HHS/United States ; R01 DE026644/DE/NIDCR NIH HHS/United States ; R01 DE026870/DE/NIDCR NIH HHS/United States ; P30 CA023100/CA/NCI NIH HHS/United States ; T32 GM007752/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*metabolism ; Animals ; Benzoxazoles/pharmacology ; Biomarkers, Tumor/metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/*metabolism ; Cell Line, Tumor ; Cell Proliferation ; Head and Neck Neoplasms/*metabolism/pathology ; Humans ; Mice ; Mice, Knockout ; Phosphorylation ; Prognosis ; Pyrimidines/pharmacology ; Squamous Cell Carcinoma of Head and Neck/*metabolism/pathology ; TOR Serine-Threonine Kinases/*antagonists &amp; inhibitors ; Tumor Suppressor Proteins/*metabolism ; },
abstract = {Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. SIGNIFICANCE: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.},
}
@article {pmid30894153,
year = {2019},
author = {Najah, S and Saulnier, C and Pernodet, JL and Bury-Moné, S},
title = {Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {18},
pmid = {30894153},
issn = {1472-6750},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Gene Editing/*methods ; Homologous Recombination ; RNA, Guide/*genetics ; Streptomyces/genetics ; },
abstract = {BACKGROUND: The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) based technologies have revolutionized genome engineering. While their use for prokaryotic genome editing is expanding, some limitations remain such as possible off-target effects and design constraints. These are compounded when performing systematic genome editing at distinct loci or when targeting repeated sequences (e.g. multicopy genes or mobile genetic elements). To overcome these limitations, we designed an approach using the same sgRNA and CRISPR-Cas9 system to independently perform gene editing at different loci.<br><br>RESULTS: We developed a two-step procedure based on the introduction by homologous recombination of 'bait' DNA at the vicinity of a gene copy of interest before inducing CRISPR-Cas9 activity. The introduction of a genetic tool encoding a CRISPR-Cas9 complex targeting this 'bait' DNA induces a double strand break near the copy of interest. Its repair by homologous recombination can lead either to reversion or gene copy-specific editing. The relative frequencies of these events are linked to the impact of gene editing on cell fitness. In our study, we used this technology to successfully delete the native copies of two xenogeneic silencers lsr2 paralogs in Streptomyces ambofaciens. We observed that one of these paralogs is a candidate-essential gene since its native locus can be deleted only in the presence of an extra copy.<br><br>CONCLUSION: By targeting 'bait' DNA, we designed a 'generic' CRISPR-Cas9 toolkit that can be used to edit different loci. The differential action of this CRISPR-Cas9 system is exclusively based on the specific recombination between regions surrounding the gene copy of interest. This approach is suitable to edit multicopy genes. One such particular example corresponds to the mutagenesis of candidate-essential genes that requires the presence of an extra copy of the gene before gene disruption. This opens new insights to explore gene essentiality in bacteria and to limit off-target effects during systematic CRISPR-Cas9 based approaches.},
}
@article {pmid30894127,
year = {2019},
author = {Barman, HN and Sheng, Z and Fiaz, S and Zhong, M and Wu, Y and Cai, Y and Wang, W and Jiao, G and Tang, S and Wei, X and Hu, P},
title = {Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR/Cas9 system.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {109},
pmid = {30894127},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; Chimera ; Mutagenesis ; Oryza/*genetics/physiology ; Plant Breeding/*methods ; Plant Infertility/*genetics ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Pollen/genetics/growth &amp; development ; Temperature ; },
abstract = {BACKGROUND: Two-line hybrid rice with high yield potential is increasingly popular and the photo- and temperature-sensitive male sterile line is one of the basic components for two-line hybrid rice breeding. The development of male sterile lines through conventional breeding is a lengthy and laborious process, whereas developing thermo-sensitive genic male sterile (TGMS) lines for two-line hybrid breeding by editing a temperature-sensitivity gene by CRISPR/Cas9 is efficient and convenient.<br><br>RESULTS: Here, thermo-sensitive genic male sterility (TGMS) was induced by employing the CRISPR/Cas9 gene editing technology to modify the gene TMS5. Two TGMS mutants, tms5-1 and tms5-2, both lacking any residual T-DNA, were generated in the indica rice cultivar Zhongjiazao17 (cv. YK17) background. When grown at a sub-optimal temperature (22&#x2009;&#xb0;C), both mutants produced viable pollen and successfully produced grain through self-fertilization, but at temperatures 24 and 26&#x2009;&#xb0;C, their pollen was sterile and no grain was set. F1 hybrids derived from the crosses between YK17S (tms5-1) and three different restorer lines outperformed both parental lines with respect to grain yield and related traits.<br><br>CONCLUSION: The YK17S generated by CRISPR/Cas9 system was proved to be a new TGMS line with superior yield potential and can be widely utilized in two-line hybrid breeding of indica rice.},
}
@article {pmid30892626,
year = {2019},
author = {Liu, P and Luk, K and Shin, M and Idrizi, F and Kwok, S and Roscoe, B and Mintzer, E and Suresh, S and Morrison, K and Frazão, JB and Bolukbasi, MF and Ponnienselvan, K and Luban, J and Zhu, LJ and Lawson, ND and Wolfe, SA},
title = {Enhanced Cas12a editing in mammalian cells and zebrafish.},
journal = {Nucleic acids research},
volume = {47},
number = {8},
pages = {4169-4180},
pmid = {30892626},
issn = {1362-4962},
support = {R01 HL093766/HL/NHLBI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; U01 HG007910/HG/NHGRI NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; R35 HL140017/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; Embryo, Nonmammalian ; Endonucleases/*genetics/metabolism ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Homeodomain Proteins/genetics/metabolism ; Humans ; Inverted Repeat Sequences ; Jurkat Cells ; K562 Cells ; Nuclear Localization Signals ; Nucleic Acid Conformation ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Ribonucleoproteins/*genetics/metabolism ; Transcription Factors/genetics/metabolism ; Transfection ; Zebrafish ; Zebrafish Proteins/genetics/metabolism ; },
abstract = {Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.},
}
@article {pmid30891445,
year = {2019},
author = {Eckerstorfer, MF and Dolezel, M and Heissenberger, A and Miklau, M and Reichenbecher, W and Steinbrecher, RA and Waßmann, F},
title = {An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs).},
journal = {Frontiers in bioengineering and biotechnology},
volume = {7},
number = {},
pages = {31},
pmid = {30891445},
issn = {2296-4185},
abstract = {The question whether new genetic modification techniques (nGM) in plant development might result in non-negligible negative effects for the environment and/or health is significant for the discussion concerning their regulation. However, current knowledge to address this issue is limited for most nGMs, particularly for recently developed nGMs, like genome editing, and their newly emerging variations, e.g., base editing. This leads to uncertainties regarding the risk/safety-status of plants which are developed with a broad range of different nGMs, especially genome editing, and other nGMs such as cisgenesis, transgrafting, haploid induction or reverse breeding. A literature survey was conducted to identify plants developed by nGMs which are relevant for future agricultural use. Such nGM plants were analyzed for hazards associated either (i) with their developed traits and their use or (ii) with unintended changes resulting from the nGMs or other methods applied during breeding. Several traits are likely to become particularly relevant in the future for nGM plants, namely herbicide resistance (HR), resistance to different plant pathogens as well as modified composition, morphology, fitness (e.g., increased resistance to cold/frost, drought, or salinity) or modified reproductive characteristics. Some traits such as resistance to certain herbicides are already known from existing GM crops and their previous assessments identified issues of concern and/or risks, such as the development of herbicide resistant weeds. Other traits in nGM plants are novel; meaning they are not present in agricultural plants currently cultivated with a history of safe use, and their underlying physiological mechanisms are not yet sufficiently elucidated. Characteristics of some genome editing applications, e.g., the small extent of genomic sequence change and their higher targeting efficiency, i.e., precision, cannot be considered an indication of safety per se, especially in relation to novel traits created by such modifications. All nGMs considered here can result in unintended changes of different types and frequencies. However, the rapid development of nGM plants can compromise the detection and elimination of unintended effects. Thus, a case-specific premarket risk assessment should be conducted for nGM plants, including an appropriate molecular characterization to identify unintended changes and/or confirm the absence of unwanted transgenic sequences.},
}
@article {pmid30890798,
year = {2019},
author = {Ledford, H},
title = {Bull 'super dads' are being engineered to produce sperm from another father.},
journal = {Nature},
volume = {567},
number = {7748},
pages = {292-293},
doi = {10.1038/d41586-019-00718-5},
pmid = {30890798},
issn = {1476-4687},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; Clustered Regularly Interspaced Short Palindromic Repeats ; Germ Cells ; Male ; RNA-Binding Proteins ; *Spermatozoa ; Swine ; },
}
@article {pmid30890717,
year = {2019},
author = {Harutyunyan, AS and Krug, B and Chen, H and Papillon-Cavanagh, S and Zeinieh, M and De Jay, N and Deshmukh, S and Chen, CCL and Belle, J and Mikael, LG and Marchione, DM and Li, R and Nikbakht, H and Hu, B and Cagnone, G and Cheung, WA and Mohammadnia, A and Bechet, D and Faury, D and McConechy, MK and Pathania, M and Jain, SU and Ellezam, B and Weil, AG and Montpetit, A and Salomoni, P and Pastinen, T and Lu, C and Lewis, PW and Garcia, BA and Kleinman, CL and Jabado, N and Majewski, J},
title = {H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1262},
pmid = {30890717},
issn = {2041-1723},
support = {MC_U132670601/MRC_/Medical Research Council/United Kingdom ; P01 CA196539/CA/NCI NIH HHS/United States ; P41 RR000862/RR/NCRR NIH HHS/United States ; P01-CA196539/NH/NIH HHS/United States ; GM110174/NH/NIH HHS/United States ; TL1TR001880/NH/NIH HHS/United States ; R01 GM110174/GM/NIGMS NIH HHS/United States ; MR/M00094X/1/MRC_/Medical Research Council/United Kingdom ; TL1 TR001880/TR/NCATS NIH HHS/United States ; T32GM008275/NH/NIH HHS/United States ; T32 GM008275/GM/NIGMS NIH HHS/United States ; },
mesh = {Adolescent ; Aged ; Animals ; Brain Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Child ; Chromatin/*metabolism ; CpG Islands/genetics ; DNA Methylation/genetics ; Epigenesis, Genetic ; Female ; Gene Editing/methods ; Gene Expression Regulation, Neoplastic ; Glioblastoma/*genetics/pathology ; HEK293 Cells ; Histone Code/genetics ; Histones/*genetics/metabolism ; Humans ; Lysine/genetics ; Male ; Methionine/genetics ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Mutation ; Neurogenesis/genetics ; Polycomb Repressive Complex 2/*metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.},
}
@article {pmid30890613,
year = {2019},
author = {Gentile, GM and Wetzel, KS and Dedrick, RM and Montgomery, MT and Garlena, RA and Jacobs-Sera, D and Hatfull, GF},
title = {More Evidence of Collusion: a New Prophage-Mediated Viral Defense System Encoded by Mycobacteriophage Sbash.},
journal = {mBio},
volume = {10},
number = {2},
pages = {},
pmid = {30890613},
issn = {2150-7511},
support = {R01 GM116884/GM/NIGMS NIH HHS/United States ; R21 AI140111/AI/NIAID NIH HHS/United States ; R21 AI151264/AI/NIAID NIH HHS/United States ; 54308198/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Host-Parasite Interactions ; Lysogeny ; Mycobacteriophages/*physiology ; Mycobacterium smegmatis/*physiology/*virology ; Prophages/physiology ; },
abstract = {The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 10[23] phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.},
}
@article {pmid30890181,
year = {2019},
author = {Zheng, T and Li, J and Ni, Y and Kang, K and Misiakou, MA and Imamovic, L and Chow, BKC and Rode, AA and Bytzer, P and Sommer, M and Panagiotou, G},
title = {Mining, analyzing, and integrating viral signals from metagenomic data.},
journal = {Microbiome},
volume = {7},
number = {1},
pages = {42},
pmid = {30890181},
issn = {2049-2618},
mesh = {Algorithms ; Anti-Bacterial Agents/*administration &amp; dosage ; Bacteria/*classification/isolation &amp; purification/virology ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Data Mining/*methods ; Feces/microbiology ; Gastrointestinal Microbiome ; Healthy Volunteers ; Humans ; Metagenomics/*methods ; Random Allocation ; },
abstract = {BACKGROUND: Viruses are important components of microbial communities modulating community structure and function; however, only a couple of tools are currently available for phage identification and analysis from metagenomic sequencing data. Here we employed the random forest algorithm to develop VirMiner, a web-based phage contig prediction tool especially sensitive for high-abundances phage contigs, trained and validated by paired metagenomic and phagenomic sequencing data from the human gut flora.<br><br>RESULTS: VirMiner achieved 41.06%&#x2009;&#xb1;&#x2009;17.51% sensitivity and 81.91%&#x2009;&#xb1;&#x2009;4.04% specificity in the prediction of phage contigs. In particular, for the high-abundance phage contigs, VirMiner outperformed other tools (VirFinder and VirSorter) with much higher sensitivity (65.23%&#x2009;&#xb1;&#x2009;16.94%) than VirFinder (34.63%&#x2009;&#xb1;&#x2009;17.96%) and VirSorter (18.75%&#x2009;&#xb1;&#x2009;15.23%) at almost the same specificity. Moreover, VirMiner provides the most comprehensive phage analysis pipeline which is comprised of metagenomic raw reads processing, functional annotation, phage contig identification, and phage-host relationship prediction (CRISPR-spacer recognition) and supports two-group comparison when the input (metagenomic sequence data) includes different conditions (e.g., case and control). Application of VirMiner to an independent cohort of human gut metagenomes obtained from individuals treated with antibiotics revealed that 122 KEGG orthology and 118 Pfam groups had significantly differential abundance in the pre-treatment samples compared to samples at the end of antibiotic administration, including clustered regularly interspaced short palindromic repeats (CRISPR), multidrug resistance, and protein transport. The VirMiner webserver is available at http://sbb.hku.hk/VirMiner/ .<br><br>CONCLUSIONS: We developed a comprehensive tool for phage prediction and analysis for metagenomic samples. Compared to VirSorter and VirFinder-the most widely used tools-VirMiner is able to capture more high-abundance phage contigs which could play key roles in infecting bacteria and modulating microbial community dynamics.<br><br>TRIAL REGISTRATION: The European Union Clinical Trials Register, EudraCT Number: 2013-003378-28 . Registered on 9 April 2014.},
}
@article {pmid30890156,
year = {2019},
author = {Sun, N and Petiwala, S and Wang, R and Lu, C and Hu, M and Ghosh, S and Hao, Y and Miller, CP and Chung, N},
title = {Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {225},
pmid = {30890156},
issn = {1471-2164},
mesh = {Animals ; *CRISPR-Cas Systems ; Carcinoma, Renal Cell/drug therapy/*genetics/pathology ; Cell Proliferation ; Cells, Cultured ; ErbB Receptors/*antagonists &amp; inhibitors/genetics ; Gene Targeting/*methods ; Genetic Testing/*methods ; Genome ; Humans ; Kidney Neoplasms/drug therapy/*genetics/pathology ; Mice ; Protein Kinase Inhibitors/*pharmacology ; },
abstract = {BACKGROUND: Large-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control.<br><br>RESULTS: We designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings.<br><br>CONCLUSIONS: In this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox.},
}
@article {pmid30888429,
year = {2019},
author = {He, W and Chen, J and Gao, S},
title = {Mammalian haploid stem cells: establishment, engineering and applications.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {12},
pages = {2349-2367},
pmid = {30888429},
issn = {1420-9071},
mesh = {Animals ; CRISPR-Cas Systems ; Cloning, Organism/*methods ; DNA Methylation ; Embryonic Stem Cells/*cytology/metabolism ; Female ; Gene Editing/methods ; *Haploidy ; Humans ; Male ; Spermatocytes/cytology/metabolism ; },
abstract = {Haploid embryonic stem cells (haESCs) contain only one set of genomes inherited from the sperm or egg and are termed AG- or PG-haESCs, respectively. Mammalian haESCs show genome-wide hypomethylation and dysregulated imprinting, whereas they can sustain genome integrity during derivation and long-term propagation. In addition, haESCs exhibit similar pluripotency to traditional diploid ESCs but are unique because they function as gametes and have been used to produce semi-cloned animals. More strikingly, unisexual reproduction has been achieved in mice by using haESCs. In combination with a gene editing or screening system, haESCs represent a powerful tool for studies of underlying gene functions and explorations of mechanisms of genetic and epigenetic regulation not only at the cellular level in vitro but also at the animal level in vivo. More importantly, genetically edited AG-haESC lines may further serve as an ideal candidate for the establishment of a sperm bank, which is a highly cost-effective approach, and a wide range of engineered semi-cloned mice have been produced. Here, we review the historical development, characteristics, advantages and disadvantages of haESCs. Additionally, we present an in-depth discussion of the recent advances in haESCs and their potential applications.},
}
@article {pmid30887115,
year = {2019},
author = {Felipe-Medina, N and Gómez-H, L and Condezo, YB and Sanchez-Martín, M and Barbero, JL and Ramos, I and Llano, E and Pendás, AM},
title = {Ubiquitin-specific protease 26 (USP26) is not essential for mouse gametogenesis and fertility.},
journal = {Chromosoma},
volume = {128},
number = {3},
pages = {237-247},
pmid = {30887115},
issn = {1432-0886},
mesh = {Animals ; CRISPR-Cas Systems ; Cysteine Endopeptidases/*genetics ; Female ; Fertility/*genetics ; Gametogenesis/*genetics ; Gene Editing ; Gene Targeting ; Genetic Association Studies ; Genetic Loci ; Germ Cells/metabolism ; Immunohistochemistry ; Male ; Mice ; Mice, Knockout ; Ovary/metabolism ; *Phenotype ; Testis/metabolism ; },
abstract = {Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.},
}
@article {pmid30887099,
year = {2019},
author = {Xiang, X and Luo, L and Nodzyński, M and Li, C and Han, P and Dou, H and Petersen, TS and Liang, X and Pan, X and Qu, K and Yang, L and Dang, Y and Liu, X and Bolund, L and Zhang, X and Tong, G and Xing, Y and Luo, Y and Lin, L},
title = {LION: a simple and rapid method to achieve CRISPR gene editing.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {13},
pages = {2633-2645},
pmid = {30887099},
issn = {1420-9071},
mesh = {Breast/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Female ; Gene Editing/*methods ; Gene Targeting ; Genetic Vectors/*administration &amp; dosage/genetics ; HEK293 Cells ; Humans ; Ovarian Neoplasms/*genetics ; *RNA, Guide ; Uterine Cervical Neoplasms/*genetics ; },
abstract = {The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.},
}
@article {pmid30887096,
year = {2019},
author = {Cui, Y and Zhu, M and Xu, Z and Xu, Q},
title = {Assessment of the effect of ten heading time genes on reproductive transition and yield components in rice using a CRISPR/Cas9 system.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {132},
number = {6},
pages = {1887-1896},
pmid = {30887096},
issn = {1432-2242},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Plant ; *Gene Editing ; Oryza/*genetics ; Photoperiod ; *Plant Breeding ; Plant Proteins/antagonists &amp; inhibitors/*genetics ; *Quantitative Trait Loci ; *Reproduction ; },
abstract = {We demonstrated the effect of heading time genes on reproductive transition and yield components under an identical genetic background using CRISPR/Cas9 gene-editing technology, and we propose that the elite allele will provide a new breeding strategy for rice breeding in high-latitude regions. Heading date is a factor closely associated with grain yield in rice (Oryza sativa L). In recent decades, a number of genes responsible for heading time have been identified, the variation of which contributes to the expansion of the rice cultivation area. However, it is difficult to compare the phenotypic effects of these genes due to the different genetic backgrounds. In this study, we generated 14 heading time mutants using CRISPR/Cas9 gene-editing technology and marker-assisted selection with a japonica Sasanishiki wild-type (WT) genetic background. Photoperiod sensitivity, the relationship between days to heading (DTH), and yield components of mutants were investigated. We found that the yield increases with increases in DTH, but eventually plateaus at maximum and then began to decrease, whereas the biomass continued to increase. The mutants exerted distinctly different effects on DTH and yield components. The convergent double mutants had severe yield reduction compared with single mutants, even with a DTH that was similar to that of single mutants. We also found that an elite mutant of se14 achieved a yield equal to that of the WT, but with heading occurring 10 days earlier. A sequence analysis of 72 cultivars collected from the japonica cultivated zone shows that elite se14 mutants have not been applied to rice breeding. Our study demonstrates the effect of heading time genes on reproductive transition and yield components under an identical genetic background. These results may provide new insights into rice breeding using heading time mutants.},
}
@article {pmid30886437,
year = {2019},
author = {Li, S and Li, J and He, Y and Xu, M and Zhang, J and Du, W and Zhao, Y and Xia, L},
title = {Precise gene replacement in rice by RNA transcript-templated homologous recombination.},
journal = {Nature biotechnology},
volume = {37},
number = {4},
pages = {445-450},
pmid = {30886437},
issn = {1546-1696},
mesh = {Acetolactate Synthase/genetics ; Base Sequence ; Biotechnology ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Gene Targeting/*methods ; *Genes, Plant ; *Homologous Recombination ; Mutation ; Oryza/*genetics ; Plant Proteins/genetics ; Plants, Genetically Modified ; RNA, Plant/genetics ; Recombinant Proteins/genetics ; Recombinational DNA Repair ; Templates, Genetic ; },
abstract = {One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double-stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus. We replaced the rice acetolactate synthase gene (ALS) with a mutated version using a DNA-free ribonucleoprotein complex that contains the recombinant Cpf1, crRNAs, and DRT transcripts. We also produced stable lines with two desired mutations in the ALS gene using TT-HDR.},
}
@article {pmid30886355,
year = {2019},
author = {Varble, A and Meaden, S and Barrangou, R and Westra, ER and Marraffini, LA},
title = {Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci.},
journal = {Nature microbiology},
volume = {4},
number = {6},
pages = {956-963},
pmid = {30886355},
issn = {2058-5276},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/genetics ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/immunology ; Endonucleases ; *Gene Transfer, Horizontal ; Genome, Bacterial ; Genome, Viral ; Plasmids/genetics ; Pseudomonas aeruginosa/genetics ; Sequence Analysis, DNA ; Staphylococcus/*genetics ; Staphylococcus aureus/genetics ; Transduction, Genetic ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral[1] and plasmid[2] invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader's genome into the CRISPR array[1]. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides[3-5] that associate with Cas nucleases to specify a viral target for destruction[6-9]. Although CRISPR-cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer[10-12], the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer.},
}
@article {pmid30886319,
year = {2019},
author = {Saadi, W and Kermezli, Y and Dao, LTM and Mathieu, E and Santiago-Algarra, D and Manosalva, I and Torres, M and Belhocine, M and Pradel, L and Loriod, B and Aribi, M and Puthier, D and Spicuglia, S},
title = {A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4707},
pmid = {30886319},
issn = {2045-2322},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation/drug effects/*genetics ; Cell Line, Tumor ; Gene Knockdown Techniques ; Genetic Loci ; Ionomycin/pharmacology ; Lymphocyte Activation/drug effects/genetics ; Mice ; Proto-Oncogene Proteins c-bcl-2/*genetics ; RNA, Long Noncoding/genetics/*metabolism ; T-Lymphocytes/*physiology ; Tetradecanoylphorbol Acetate/analogs &amp; derivatives/pharmacology ; },
abstract = {Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4[-]CD8[-] double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.},
}
@article {pmid30886178,
year = {2019},
author = {Okamoto, S and Amaishi, Y and Maki, I and Enoki, T and Mineno, J},
title = {Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4811},
pmid = {30886178},
issn = {2045-2322},
mesh = {Base Sequence/genetics ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; DNA, Single-Stranded/genetics ; Feasibility Studies ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Oligonucleotides/genetics ; RNA, Guide/genetics ; Ribonucleoproteins/*genetics ; Transfection/methods ; },
abstract = {Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.},
}
@article {pmid30885348,
year = {2019},
author = {Perumal, E and So Youn, K and Sun, S and Seung-Hyun, J and Suji, M and Jieying, L and Yeun-Jun, C},
title = {PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation of β-catenin and snail/slug in non-small cell lung carcinoma cells.},
journal = {Lung cancer (Amsterdam, Netherlands)},
volume = {130},
number = {},
pages = {25-34},
doi = {10.1016/j.lungcan.2019.01.013},
pmid = {30885348},
issn = {1872-8332},
mesh = {Animals ; Carcinoma, Non-Small-Cell Lung/*genetics/pathology ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epithelial-Mesenchymal Transition ; Female ; Gene Knockdown Techniques ; Humans ; Lung Neoplasms/*genetics/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mutation/*genetics ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Staging ; Neoplasm Transplantation ; PTEN Phosphohydrolase/*genetics ; Signal Transduction ; Snail Family Transcription Factors/genetics/*metabolism ; beta Catenin/genetics/*metabolism ; },
abstract = {OBJECTIVE: Epithelial-mesenchymal transition (EMT) is the key event in distant metastasis of diverse tumors including lung cancer. Recent evidence suggests the involvement of phosphatase and tensin homolog (PTEN) in EMT phenotypes. However, the molecular mechanism of EMT induced by PTEN inactivation is not clear in lung cancer. We aimed to investigate the role of PTEN inactivation in acquisition of EMT in lung cancer cells.<br><br>METHODS: We knocked out the PTEN in PTEN proficient lung cancer cells lines (A549 and NCI-H460) using CRISPR/Cas-9 system and observed the growth, EMT phenotypes, and EMT related molecules. We also explored the in vivo effect of PTEN inactivation on tumor cell growth and distant metastasis using nude mouse injection.<br><br>RESULTS: PTEN knockout (KO) cells showed faster growth, migration and invasion than PTEN wild-type (WT) cells. When we injected the cells into nude mice, PTEN-KO cells showed faster growth and higher metastatic potential. In PTEN-KO cells, the levels of phosphorylated AKT (Ser-473 and Thr-308) were profoundly elevated and the expressions of phosphorylated GSK-3&#x3b2; (Ser9, inactive form) increased, while that of &#x3b2;-catenin decreased. Regarding the EMT markers, the expression of E-cadherin decreased but those of N-cadherin, vimentin and MMP-2 increased in the PTEN-KO cells. Especially, PTEN-KO cells showed the almost complete intra-nuclear shift of &#x3b2;-catenin and no &#x3b2;-catenin signal was observed in the cell membrane. Accordingly, PTEN-KO cells exhibited morphological changes such as loss of cell-to-cell contact, pseudopodia and the round shape, which are the typical phenotypes of EMT. Snail and Slug were also dominantly accumulated in the nucleus after PTEN inactivation.<br><br>CONCLUSION: All these data consistently support that PTEN inactivation contributes to EMT by nuclear translocation of &#x3b2;-catenin and Snail/Slug in lung cancer cells.},
}
@article {pmid30885208,
year = {2019},
author = {Yuan, T and Zhong, Y and Wang, Y and Zhang, T and Lu, R and Zhou, M and Lu, Y and Yan, K and Chen, Y and Hu, Z and Liang, J and Fan, J and Cheng, Y},
title = {Generation of hyperlipidemic rabbit models using multiple sgRNAs targeted CRISPR/Cas9 gene editing system.},
journal = {Lipids in health and disease},
volume = {18},
number = {1},
pages = {69},
pmid = {30885208},
issn = {1476-511X},
mesh = {Animals ; Animals, Genetically Modified ; Apolipoproteins E/genetics ; Atherosclerosis/etiology/genetics ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Gene Editing/*methods ; Gene Knockout Techniques ; Hyperlipidemias/*genetics ; Lipids/blood/genetics ; Lipoproteins/blood/genetics ; Pregnancy ; RNA, Guide ; Rabbits ; Receptors, LDL/genetics ; },
abstract = {OBJECTIVE: To generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis.<br><br>METHODS: CRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis.<br><br>RESULTS: Six knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis.<br><br>CONCLUSION: Large fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.},
}
@article {pmid30884095,
year = {2019},
author = {Xu, CF and Iqbal, S and Shen, S and Luo, YL and Yang, X and Wang, J},
title = {Development of "CLAN" Nanomedicine for Nucleic Acid Therapeutics.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {15},
number = {16},
pages = {e1900055},
doi = {10.1002/smll.201900055},
pmid = {30884095},
issn = {1613-6829},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Heart/physiology ; Humans ; Immune System ; Kupffer Cells/metabolism ; Lactates/*chemistry ; Lipids/chemistry ; Macromolecular Substances ; Nanomedicine/*methods ; Nanoparticles/*chemistry ; Neoplasms/metabolism/*therapy ; Neoplastic Stem Cells/cytology ; Nucleic Acids/chemistry/*therapeutic use ; Polyethylene Glycols/*chemistry ; Zebrafish ; },
abstract = {Nucleic acid-based macromolecules have paved new avenues for the development of therapeutic interventions against a spectrum of diseases; however, their clinical translation is limited by successful delivery to the target site and cells. Therefore, numerous systems have been developed to overcome delivery challenges to nucleic acids. From the viewpoint of clinical translation, it is highly desirable to develop systems with clinically validated materials and controllability in synthesis. With this in mind, a cationic lipid assisted PEG-b-PLA nanoparticle (CLAN) is designed that is capable of protecting nucleic acids via encapsulation inside the aqueous core, and delivers them to target cells, while maintaining or improving nucleic acid function. The system is formulated from clinically validated components (PEG-b-PLA and its derivatives) and can be scaled-up for large scale manufacturing, offering potential for its future use in clinical applications. Here, the development and working mechanisms of CLANs, the ways to improve its delivery efficacy, and its application in various disease treatments are summarized. Finally, a prospective for the further development of CLAN is also discussed.},
}
@article {pmid30883991,
year = {2019},
author = {Moses, C and Kaur, P},
title = {Applications of CRISPR systems in respiratory health: Entering a new 'red pen' era in genome editing.},
journal = {Respirology (Carlton, Vic.)},
volume = {24},
number = {7},
pages = {628-637},
doi = {10.1111/resp.13527},
pmid = {30883991},
issn = {1440-1843},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Humans ; Respiratory Tract Diseases/*genetics ; },
abstract = {Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio-economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.},
}
@article {pmid30883679,
year = {2019},
author = {Xiong, K and Marquart, KF and la Cour Karottki, KJ and Li, S and Shamie, I and Lee, JS and Gerling, S and Yeo, NC and Chavez, A and Lee, GM and Lewis, NE and Kildegaard, HF},
title = {Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {7},
pages = {1813-1819},
pmid = {30883679},
issn = {1097-0290},
support = {R35 GM119850/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Apoptosis/*genetics ; CHO Cells ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; *Caspase 3/genetics/metabolism ; Cricetulus ; *Gene Targeting ; *bcl-2 Homologous Antagonist-Killer Protein/genetics/metabolism ; *bcl-2-Associated X Protein/genetics/metabolism ; },
abstract = {Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB-MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.},
}
@article {pmid30883196,
year = {2019},
author = {Kim, D and Luk, K and Wolfe, SA and Kim, JS},
title = {Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases.},
journal = {Annual review of biochemistry},
volume = {88},
number = {},
pages = {191-220},
doi = {10.1146/annurev-biochem-013118-111730},
pmid = {30883196},
issn = {1545-4509},
support = {R01 AI117839/AI/NIAID NIH HHS/United States ; R01 HL093766/HL/NHLBI NIH HHS/United States ; },
mesh = {APOBEC Deaminases/genetics/metabolism ; Adenosine Deaminase/genetics/metabolism ; Artifacts ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Gene Editing/*methods ; Gene Transfer Techniques ; Genome, Human ; Humans ; Isoenzymes/genetics/metabolism ; Protein Engineering/*methods ; RNA, Guide/*genetics/metabolism ; Software ; },
abstract = {Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.},
}
@article {pmid30882797,
year = {2019},
author = {Lee, J and Jung, MH and Jeong, E and Lee, JK},
title = {Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {144},
pages = {},
doi = {10.3791/59202},
pmid = {30882797},
issn = {1940-087X},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Library ; Humans ; },
abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.},
}
@article {pmid30881132,
year = {2019},
author = {Lu, ZJ and Yu, Q and Zhou, SH and Fan, J and Shen, LF and Bao, YY and Wu, TT and Zhou, ML and Huang, YP},
title = {Construction of a GLUT-1 and HIF-1α gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique.},
journal = {Cancer management and research},
volume = {11},
number = {},
pages = {2087-2096},
pmid = {30881132},
issn = {1179-1322},
abstract = {BACKGROUND: Glucose transporter (GLUT)-mediated glucose uptake is an important process in the development of laryngeal carcinoma, one of the most common malignancies of the head and neck. GLUT-1, together with HIF-1α, is also an indicator of hypoxia. Both proteins play a critical role in glucose uptake and glycolysis in laryngeal carcinoma cells under hypoxic stress. A double gene knockout model in which HIF-1α and GLUT-1 are no longer expressed can provide important information about carcinogenesis in laryngeal carcinoma.<br><br>PURPOSE: In this study we used the CRISPR/Cas 9 system to induce HIF-1&#x3b1; and GLUT-1 double gene knockout in HEp-2 cells and then used the knocked-out cells to study the role of these markers in laryngeal carcinoma, including in chemoradioresistance.<br><br>METHODS: High-grade small-guide RNAs (sgRNAs) of HIF-1&#x3b1; and GLUT-1 were designed using an online tool and inserted into the pUC57-T7-gRNA vector. The recombinant plasmids were transfected into HEp-2 cells and positive cells were screened using the dilution method. Gene mutation and expression were determined by sequence analysis and immunoblotting.<br><br>RESULTS: In HIF-1&#x3b1; and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the HIF-1&#x3b1; genomic sequence was detected, whereas multiple base insertions resulted in frameshift mutations in the GLUT-1 gene. Neither HIF-1&#x3b1; nor GLUT-1 protein was expressed in positive cells. The proliferation, migration, and invasion of HEp-2 cells were significantly decreased afterward. The possible mechanism may be that the inhibition PI3K/AKT/mTOR pathway by HIF-1&#x3b1; and GLUT-1 double gene knockout using CRISPR/Cas9 technique lead to reduction of glucose uptake and lactic acid generation.<br><br>CONCLUSION: Our HIF-1&#x3b1; and GLUT-1 double gene knockout HEp-2 cell model, obtained using a CRISPR/Cas9-based system, may facilitate studies of the pathogenesis of laryngeal carcinoma.},
}
@article {pmid30880567,
year = {2019},
author = {Deryabin, P and Griukova, A and Shatrova, A and Petukhov, A and Nikolsky, N and Borodkina, A},
title = {Optimization of lentiviral transduction parameters and its application for CRISPR-based secretome modification of human endometrial mesenchymal stem cells.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {18},
number = {6-7},
pages = {742-758},
pmid = {30880567},
issn = {1551-4005},
mesh = {CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Cellular Senescence/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endometrium/*physiology ; Female ; Gene Editing/methods ; Genetic Therapy ; Genome/genetics ; HEK293 Cells ; Humans ; Lentivirus/*genetics ; Mesenchymal Stem Cells/*physiology ; Regenerative Medicine/methods ; },
abstract = {Mesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.},
}
@article {pmid30880156,
year = {2019},
author = {Steinberger, J and Robert, F and Hallé, M and Williams, DE and Cencic, R and Sawhney, N and Pelletier, D and Williams, P and Igarashi, Y and Porco, JA and Rodriguez, AD and Kopp, B and Bachmann, B and Andersen, RJ and Pelletier, J},
title = {Tracing MYC Expression for Small Molecule Discovery.},
journal = {Cell chemical biology},
volume = {26},
number = {5},
pages = {699-710.e6},
pmid = {30880156},
issn = {2451-9448},
support = {R35 GM118173/GM/NIGMS NIH HHS/United States ; R01 CA226833/CA/NCI NIH HHS/United States ; SC1 GM086271/GM/NIGMS NIH HHS/United States ; R24 GM111625/GM/NIGMS NIH HHS/United States ; R01 GM092218/GM/NIGMS NIH HHS/United States ; FDN-148366//CIHR/Canada ; },
mesh = {Bufanolides/pharmacology ; CRISPR-Cas Systems/genetics ; Cardiac Glycosides/pharmacology ; Cell Line, Tumor ; Cell Survival/drug effects ; Cytoskeleton/drug effects/metabolism ; Eukaryotic Initiation Factor-2/genetics/metabolism ; Humans ; Proto-Oncogene Proteins c-myc/antagonists &amp; inhibitors/*genetics/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Small Molecule Libraries/*pharmacology ; Transcription, Genetic/*drug effects ; },
abstract = {Our inability to effectively "drug" targets such as MYC for therapeutic purposes requires the development of new approaches. We report on the implementation of a phenotype-based assay for monitoring MYC expression in multiple myeloma cells. The open reading frame (ORF) encoding an unstable variant of GFP was engineered immediately downstream of the MYC ORF using CRISPR/Cas9, resulting in co-expression of both proteins from the endogenous MYC locus. Using fluorescence readout as a surrogate for MYC expression, we implemented a pilot screen in which ∼10,000 compounds were prosecuted. Among known MYC expression inhibitors, we identified cardiac glycosides and cytoskeletal disruptors to be quite potent. We demonstrate the power of CRISPR/Cas9 engineering in establishing phenotype-based assays to identify gene expression modulators.},
}
@article {pmid30880083,
year = {2019},
author = {Palermo, G and Casalino, L and Magistrato, A and Andrew McCammon, J},
title = {Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulations.},
journal = {Journal of structural biology},
volume = {206},
number = {3},
pages = {267-279},
pmid = {30880083},
issn = {1095-8657},
support = {R01 GM031749/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Humans ; Molecular Dynamics Simulation ; *Nucleic Acid Conformation ; Proteins/chemistry/genetics ; RNA Splicing/genetics ; RNA, Untranslated/*genetics/ultrastructure ; },
abstract = {Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Molecular Dynamics (MD) simulations methods to address the mechanistic function of large biomolecular systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomolecular systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystallized so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect atomic-level details of complex biomolecular systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.},
}
@article {pmid30879343,
year = {2019},
author = {Chen, Z and Cai, X and Li, M and Yan, L and Wu, L and Wang, X and Tang, N},
title = {CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors.},
journal = {Journal of enzyme inhibition and medicinal chemistry},
volume = {34},
number = {1},
pages = {799-807},
pmid = {30879343},
issn = {1475-6374},
mesh = {*CRISPR-Cas Systems ; Enzyme Inhibitors/*pharmacology ; Flow Cytometry ; Fluorescence ; Fluorescent Antibody Technique ; HEK293 Cells ; Hep G2 Cells ; High-Throughput Screening Assays ; Humans ; Interleukin-1beta/pharmacology ; Liver/cytology/*drug effects/enzymology ; Prostaglandin-E Synthases/*antagonists &amp; inhibitors/genetics ; RNA Interference ; Real-Time Polymerase Chain Reaction ; Small Molecule Libraries/pharmacology ; },
abstract = {mPGES-1 is a terminal rate-limiting enzyme responsible for inflammation-induced PGE2 production. The inhibition of mPGES-1 has been considered as a safe and effective target for the treatment of inflammation and cancer. However, a specific, efficient, and simple method for high-throughput screening of mPGES-1 inhibitors is still lacking. In this study, we developed a fluorescence imaging strategy to monitor the expression of mPGES-1 via CRISPR/Cas9 knock-in system. Immunofluorescence colocalisation, Sanger sequencing, RNAi, and IL-1β treatment all confirmed the successful construction of mPGES-1 reporter cells. The fluorescence signal intensity of the reporter cells treated with four conventional mPGES-1 inhibitors was considerably attenuated via flow cytometry and fluorescent microplate reader, demonstrating that the reporter cells can be used as an efficient and convenient means for screening and optimising mPGES-1 inhibitors. Moreover, it provides a new technical support for the development of targeted small molecule compounds for anti-inflammatory and tumour therapy.},
}
@article {pmid30879240,
year = {2019},
author = {Luo, J and Padhi, P and Jin, H and Anantharam, V and Zenitsky, G and Wang, Q and Willette, AA and Kanthasamy, A and Kanthasamy, AG},
title = {Utilization of the CRISPR-Cas9 Gene Editing System to Dissect Neuroinflammatory and Neuropharmacological Mechanisms in Parkinson's Disease.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {4},
pages = {595-607},
pmid = {30879240},
issn = {1557-1904},
support = {R01 ES027245/ES/NIEHS NIH HHS/United States ; R00 AG047282/AG/NIA NIH HHS/United States ; R01 NS100090/NS/NINDS NIH HHS/United States ; R01 NS088206/NS/NINDS NIH HHS/United States ; K99 AG047282/AG/NIA NIH HHS/United States ; R01 ES026892/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/immunology ; CRISPR-Cas Systems/*physiology ; Gene Editing/methods/*trends ; Genetic Therapy/methods/*trends ; Humans ; Inflammation/genetics/immunology/therapy ; Neurodegenerative Diseases/genetics/immunology/therapy ; Parkinson Disease/*genetics/immunology/*therapy ; },
abstract = {Chronic and debilitating neurodegenerative diseases, such as Parkinson's disease (PD), impose an immense medical, emotional, and economic burden on patients and society. Due to a complex interaction between genetic and environmental risk factors, the etiology of PD remains elusive. However, the cumulative evidence emerging from clinical and experimental research over the last several decades has identified mitochondrial dysfunction, oxidative stress, neuroinflammation, and dysregulated protein degradation as the main drivers of PD neurodegeneration. The genome-editing system CRISPR (clustered regularly interspaced short palindromic repeats) has recently transformed the field of biotechnology and biomedical discovery and is poised to accelerate neurodegenerative disease research. It has been leveraged to generate PD animal models, such as Parkin, DJ-1, and PINK1 triple knockout miniature pigs. CRISPR has also allowed the deeper understanding of various PD gene interactions, as well as the identification of novel apoptotic pathways associated with neurodegenerative processes in PD. Furthermore, its application has been used to dissect neuroinflammatory pathways involved in PD pathogenesis, such as the PKCδ signaling pathway, as well as the roles of novel compensatory or protective pathways, such as Prokineticin-2 signaling. This review aims to highlight the historical milestones in the evolution of this technology and attempts to illustrate its transformative potential in unraveling disease mechanisms as well as in the development of innovative treatment strategies for PD. Graphical Abstract.},
}
@article {pmid30879090,
year = {2019},
author = {Li, J and Sun, J and Gao, X and Wu, Z and Shang, G},
title = {Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {8},
pages = {3559-3570},
doi = {10.1007/s00253-019-09744-9},
pmid = {30879090},
issn = {1432-0614},
mesh = {CRISPR-Cas Systems ; DNA Primase/*genetics/metabolism ; DNA, Bacterial/genetics ; DNA, Single-Stranded/*genetics ; Escherichia coli/enzymology/*genetics ; Escherichia coli Proteins/*genetics/metabolism ; Gene Editing ; Genetic Engineering/*methods ; Genome, Bacterial/genetics ; Homologous Recombination ; Mutation ; Plasmids/genetics/metabolism ; },
abstract = {Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. Escherichia coli primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.},
}
@article {pmid30878030,
year = {2018},
author = {Makarova, SS and Khromov, AV and Spechenkova, NA and Taliansky, ME and Kalinina, NO},
title = {Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants.},
journal = {Biochemistry. Biokhimiia},
volume = {83},
number = {12},
pages = {1552-1562},
doi = {10.1134/S0006297918120131},
pmid = {30878030},
issn = {1608-3040},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; Plants/*genetics/microbiology/parasitology/virology ; },
abstract = {The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.},
}
@article {pmid30877395,
year = {2019},
author = {Chang, W and Liu, W and Liu, Y and Zhan, F and Chen, H and Lei, H and Liu, Y},
title = {Colorimetric detection of nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification.},
journal = {Mikrochimica acta},
volume = {186},
number = {4},
pages = {243},
pmid = {30877395},
issn = {1436-5073},
support = {21705051//National Natural Science Foundation of China/International ; 21874048//National Natural Science Foundation of China/International ; },
mesh = {Base Sequence ; Biosensing Techniques/methods ; CRISPR-Associated Protein 9/*chemistry ; *CRISPR-Cas Systems ; Colorimetry/*methods ; DNA/*analysis/genetics ; Gold/chemistry ; Limit of Detection ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acid Hybridization ; Oligodeoxyribonucleotides/chemistry/genetics ; Phytophthora infestans/genetics ; },
abstract = {A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA. Graphical abstract Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.},
}
@article {pmid30877356,
year = {2019},
author = {Zhang, YT and Jiang, JY and Shi, TQ and Sun, XM and Zhao, QY and Huang, H and Ren, LJ},
title = {Application of the CRISPR/Cas system for genome editing in microalgae.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {8},
pages = {3239-3248},
doi = {10.1007/s00253-019-09726-x},
pmid = {30877356},
issn = {1432-0614},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Expression Regulation ; Gene Targeting ; Genetic Engineering ; Microalgae/*genetics ; RNA, Guide/genetics ; Transformation, Genetic ; },
abstract = {Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.},
}
@article {pmid30877281,
year = {2019},
author = {Dickenson, D and Darnovsky, M},
title = {Did a permissive scientific culture encourage the 'CRISPR babies' experiment?.},
journal = {Nature biotechnology},
volume = {37},
number = {4},
pages = {355-357},
pmid = {30877281},
issn = {1546-1696},
mesh = {Biotechnology/ethics ; *CRISPR-Cas Systems ; China ; Female ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Humans ; Infant, Newborn ; Pregnancy ; UNESCO ; United Kingdom ; United States ; },
}
@article {pmid30875368,
year = {2019},
author = {Findinier, J and Delevoye, C and Cohen, MM},
title = {The dynamin-like protein Fzl promotes thylakoid fusion and resistance to light stress in Chlamydomonas reinhardtii.},
journal = {PLoS genetics},
volume = {15},
number = {3},
pages = {e1008047},
pmid = {30875368},
issn = {1553-7404},
mesh = {Algal Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Chlamydomonas reinhardtii/genetics/*metabolism/radiation effects ; Chloroplasts/metabolism ; Dynamins/genetics/metabolism ; GTP Phosphohydrolases/genetics/*metabolism ; Gene Knockout Techniques ; Light ; Membrane Fusion ; Microscopy, Electron, Transmission ; Mutation ; Phototrophic Processes ; Recombinant Proteins/genetics/metabolism ; Stress, Physiological ; Thylakoids/*metabolism/radiation effects/ultrastructure ; },
abstract = {Large GTPases of the Dynamin Related Proteins (DRP) family shape lipid bilayers through membrane fission or fusion processes. Despite the highly organized photosynthetic membranes of thylakoids, a single DRP is known to be targeted inside the chloroplast. Fzl from the land plant Arabidopsis thaliana is inserted in the inner envelope and thylakoid membranes to regulate their morphology. Fzl may promote the fusion of thylakoids but this remains to be proven. Moreover, the physiological requirement for fusing thylakoids is currently unknown. Here, we find that the unicellular microalga Chlamydomonas reinhardtii encodes an Fzl ortholog (CrFzl) that is localized in the chloroplast where it is soluble. To explore its function, the CRISPR/Cas9 technology was employed to generate multiple CrFzl knock out strains. Phenotypic analyzes revealed a specific requirement of CrFzl for survival upon light stress. Consistent with this, strong irradiance lead to increased photoinhibition of photosynthesis in mutant cells. Fluorescence and electron microscopy analysis demonstrated that upon exposure to high light, CrFzl mutants show defects in chloroplast morphology but also large cytosolic vacuoles in close contact with the plastid. We further observe that strong irradiance induces an increased recruitment of the DRP to thylakoid membranes. Most importantly, we show that CrFzl is required for the fusion of thylakoids during mating. Together, our results suggest that thylakoids fusion may be necessary for resistance to light stress.},
}
@article {pmid30875366,
year = {2019},
author = {Liaud, N and Horlbeck, MA and Gilbert, LA and Gjoni, K and Weissman, JS and Cate, JHD},
title = {Cellular response to small molecules that selectively stall protein synthesis by the ribosome.},
journal = {PLoS genetics},
volume = {15},
number = {3},
pages = {e1008057},
pmid = {30875366},
issn = {1553-7404},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; S10 RR029668/RR/NCRR NIH HHS/United States ; S10 RR027303/RR/NCRR NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R00 CA204602/CA/NCI NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Cas Systems ; DNA Helicases/antagonists &amp; inhibitors/genetics/metabolism ; Endonucleases/metabolism ; GTP-Binding Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Gene Knockdown Techniques ; Heterocyclic Compounds, 4 or More Rings/pharmacology ; Humans ; K562 Cells ; Models, Biological ; Nuclear Proteins/antagonists &amp; inhibitors/genetics/metabolism ; PCSK9 Inhibitors ; Proprotein Convertase 9/genetics/metabolism ; Protein Biosynthesis/*drug effects ; Ribosomes/drug effects/*metabolism ; Serine Proteinase Inhibitors/pharmacology ; },
abstract = {Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.},
}
@article {pmid30875129,
year = {2019},
author = {Musharova, O and Sitnik, V and Vlot, M and Savitskaya, E and Datsenko, KA and Krivoy, A and Fedorov, I and Semenova, E and Brouns, SJJ and Severinov, K},
title = {Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation.},
journal = {Molecular microbiology},
volume = {111},
number = {6},
pages = {1558-1570},
pmid = {30875129},
issn = {1365-2958},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Intergenic ; Escherichia coli/*genetics ; },
abstract = {CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.},
}
@article {pmid30874768,
year = {2019},
author = {Atmadjaja, AN and Holby, V and Harding, AJ and Krabben, P and Smith, HK and Jenkinson, ER},
title = {CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT).},
journal = {FEMS microbiology letters},
volume = {366},
number = {6},
pages = {},
pmid = {30874768},
issn = {1574-6968},
mesh = {Butanols/metabolism ; Clostridium/*genetics/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ethanol/metabolism ; Fermentation ; Gene Editing/*methods ; Genome, Bacterial ; Polymorphism, Single Nucleotide ; },
abstract = {The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and 'green' chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.},
}
@article {pmid30873832,
year = {2019},
author = {Shan, Y and Zhou, X and Huang, R and Xing, D},
title = {High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity.},
journal = {Analytical chemistry},
volume = {91},
number = {8},
pages = {5278-5285},
doi = {10.1021/acs.analchem.9b00073},
pmid = {30873832},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/*genetics ; Leptotrichia/genetics ; MicroRNAs/*analysis/genetics/metabolism ; },
abstract = {MicroRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression. It has been proved that the aberrant expression of miRNAs is related to disease and miRNAs can serve as potential biomarkers for early tumor diagnosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a is a recently discovered CRISPR-RNA (crRNA) guided RNA manipulation tool. The recognition of target RNA can morphologically activate the robust nonspecific trans ribonuclease activity of Cas13a. This unique property makes Cas13a ideal for nucleic acid detection. Herein, we first exploited CRISPR/LbuCas13a to directly detect miRNAs with high specificity and simplicity. A limit of detection (LOD) as low as 4.5 amol was achieved by this one-step assay within 30 min, and the dynamic range spanned 4 orders of magnitude from 10 amol to 100 fmol. More importantly, single nucleotide variation, even at the end of target miRNA, can be discriminated by rationally programmed crRNA. In addition, the practical application ability of this Cas13a/crRNA-based signal amplification strategy was demonstrated by miRNA quantification in complex biological samples (total small RNA). With excellent reliability, sensitivity, and simple to implement features, this method promises a great potential for early diagnosis of miRNA-related disease. Moreover, the systematic analysis of the crRNA design could provide guidance to further develop Cas13a-based molecular diagnoses.},
}
@article {pmid30873595,
year = {2019},
author = {Porter, SN and Levine, RM and Pruett-Miller, SM},
title = {A Practical Guide to Genome Editing Using Targeted Nuclease Technologies.},
journal = {Comprehensive Physiology},
volume = {9},
number = {2},
pages = {665-714},
doi = {10.1002/cphy.c180022},
pmid = {30873595},
issn = {2040-4603},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *Deoxyribonucleases ; *Gene Editing ; Humans ; },
abstract = {Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, CRISPR-Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR-derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom-edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665-714, 2019.},
}
@article {pmid30872767,
year = {2019},
author = {Wrighton, KH},
title = {Cytosine base editors go off-target.},
journal = {Nature reviews. Genetics},
volume = {20},
number = {5},
pages = {254-255},
pmid = {30872767},
issn = {1471-0064},
mesh = {Animals ; CRISPR-Cas Systems ; *Cytosine ; *Gene Editing ; Mice ; Nucleotides ; },
}
@article {pmid30872621,
year = {2019},
author = {Jin, Y and Lee, A and Oh, JH and Lee, HW and Ha, SJ},
title = {The R229Q mutation of Rag2 does not characterize severe immunodeficiency in mice.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4415},
pmid = {30872621},
issn = {2045-2322},
mesh = {Animals ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/*physiology ; *Disease Models, Animal ; Female ; Gene Editing ; Gene Knock-In Techniques ; Male ; Mice ; Mice, Inbred C57BL ; Mice, SCID ; *Mutation ; Severe Combined Immunodeficiency/*etiology/pathology ; T-Lymphocytes/*immunology ; },
abstract = {RAG1 or RAG2 mutations are associated with defects in V(D)J recombination activity, causing severe immunodeficiency with a wide spectrum of clinical phenotypes. A R229Q mutation of RAG2 was identified in patients with severe combined immunodeficiency (SCID) or Omenn syndrome (OS). Although some factors determining the clinical features between SCID and OS were not clear, the molecular mechanism of OS was studied in a mouse model in which an EGFP tag is fused to Rag2 with the R229Q mutation. To design the human disease model mimicking severe immunodeficiency, we generated Rag2-R229Q knock-in mice without an epitope tag. Mutant mice showed impaired T and B cell differentiation with reduced V(D)J recombination activity; however, the extent to which the R229Q mutation affects severe immunodeficiency was not severe. While Rag2-R229Q mutation under some conditions may cause severe immunological and clinical phenotypes similar to human SCID or OS, R229Q mutation per se did not cause severe immunodeficiency in mice, suggesting that additional factors other than R229Q mutation are required to induce severe immunodeficiency. Thus, our report implies that the effects of genetic background and/or a tagged protein sequence may alter the mouse immune system, revealing the mechanism of phenotypic heterogeneity arising from an identical mutation.},
}
@article {pmid30872606,
year = {2019},
author = {Bhattacharya, D and Van Meir, EG},
title = {A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4437},
pmid = {30872606},
issn = {2045-2322},
support = {P30 CA138292/CA/NCI NIH HHS/United States ; R01 CA235162/CA/NCI NIH HHS/United States ; R01 NS096236/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Electrophoresis, Agar Gel/*methods ; Female ; *Gene Editing ; Genotype ; Genotyping Techniques ; *INDEL Mutation ; Membrane Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; Mice ; Mice, Transgenic ; NAV1.6 Voltage-Gated Sodium Channel/chemistry/*genetics/metabolism ; Nerve Tissue Proteins/antagonists &amp; inhibitors/*genetics/metabolism ; Polymerase Chain Reaction ; },
abstract = {CRISPR gene editing creates indels in targeted genes that are detected by genotyping. Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we developed a simple genotyping method that exploits the differential electrophoretic mobility of homoduplex versus heteroduplex DNA hybrids in high concentration agarose gels. First, the CRISPR target region is PCR amplified and homo- and hetero-duplexed amplicons formed during the last annealing cycle are separated by 4-6% agarose gel electrophoresis. WT/mutant heteroduplexes migrate more slowly and are distinguished from WT or mutant homoduplexes. Heterozygous alleles are immediately identified as they produce two distinct bands, while homozygous wild-type or mutant alleles yield a single band. To discriminate the latter, equal amounts of PCR products of homozygous samples are mixed with wild-type control samples, subjected to one denaturation/renaturation cycle and products are electrophoresed again. Samples from homozygous mutant alleles now produce two bands, while those from wild-type alleles yield single bands. This method is simple, fast and inexpensive and can identify indels >2 bp. in size in founder pups and genotype offspring in established transgenic mice colonies.},
}
@article {pmid30872531,
year = {2019},
author = {Chui, AJ and Okondo, MC and Rao, SD and Gai, K and Griswold, AR and Johnson, DC and Ball, DP and Taabazuing, CY and Orth, EL and Vittimberga, BA and Bachovchin, DA},
title = {N-terminal degradation activates the NLRP1B inflammasome.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6435},
pages = {82-85},
pmid = {30872531},
issn = {1095-9203},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 AI137168/AI/NIAID NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; T32 GM115327/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Antigens, Bacterial/*metabolism ; Apoptosis Regulatory Proteins/genetics/*metabolism ; Bacterial Toxins/*metabolism ; CRISPR-Cas Systems ; Caspase 1/metabolism ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists &amp; inhibitors ; Gene Knockout Techniques ; HEK293 Cells ; Host-Pathogen Interactions/immunology ; Humans ; Immunity, Innate ; Inflammasomes/*metabolism ; Mice ; Proteasome Endopeptidase Complex/metabolism ; *Proteolysis ; Pyroptosis/genetics/*physiology ; RAW 264.7 Cells ; Serine Proteinase Inhibitors/pharmacology ; THP-1 Cells ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule-mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.},
}
@article {pmid30872121,
year = {2019},
author = {Rollins, MF and Chowdhury, S and Carter, J and Golden, SM and Miettinen, HM and Santiago-Frangos, A and Faith, D and Lawrence, CM and Lander, GC and Wiedenheft, B},
title = {Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.},
journal = {Molecular cell},
volume = {74},
number = {1},
pages = {132-142.e5},
pmid = {30872121},
issn = {1097-4164},
support = {R21 AI130670/AI/NIAID NIH HHS/United States ; P30 GM110732/GM/NIGMS NIH HHS/United States ; R01 GM110270/GM/NIGMS NIH HHS/United States ; DP2 EB020402/EB/NIBIB NIH HHS/United States ; S10 OD021634/OD/NIH HHS/United States ; P20 GM103500/GM/NIGMS NIH HHS/United States ; R01 GM108888/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/genetics/immunology/*metabolism ; CRISPR-Associated Proteins/chemistry/genetics/immunology/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy ; DNA, Bacterial/chemistry/genetics/*metabolism ; Models, Molecular ; *Molecular Mimicry ; Nucleic Acid Conformation ; Protein Conformation ; Pseudomonas aeruginosa/*enzymology/genetics/immunology ; RNA, Bacterial/chemistry/genetics/*metabolism ; RNA, Guide/chemistry/genetics/*metabolism ; Structure-Activity Relationship ; Viral Proteins/chemistry/genetics/immunology/*metabolism ; },
abstract = {Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.},
}
@article {pmid30871200,
year = {2019},
author = {Darcis, G and Binda, CS and Klaver, B and Herrera-Carrillo, E and Berkhout, B and Das, AT},
title = {The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape.},
journal = {Viruses},
volume = {11},
number = {3},
pages = {},
pmid = {30871200},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Gene Targeting ; *Genetic Variation ; *Genome, Viral ; HEK293 Cells ; HIV-1/*genetics ; Humans ; RNA, Guide ; Virus Replication/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs.},
}
@article {pmid30871076,
year = {2019},
author = {Guzmán-Zapata, D and Sandoval-Vargas, JM and Macedo-Osorio, KS and Salgado-Manjarrez, E and Castrejón-Flores, JL and Oliver-Salvador, MDC and Durán-Figueroa, NV and Nogué, F and Badillo-Corona, JA},
title = {Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module.},
journal = {International journal of molecular sciences},
volume = {20},
number = {5},
pages = {},
pmid = {30871076},
issn = {1422-0067},
mesh = {Adenine Phosphoribosyltransferase/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Chlamydomonas reinhardtii/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Electroporation/methods ; Gene Editing/methods ; Genes, Reporter/*genetics ; Plasmids/genetics ; RNA, Guide/genetics ; Ribonucleoproteins/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.},
}
@article {pmid30871003,
year = {2019},
author = {Ashley, CL and Abendroth, A and McSharry, BP and Slobedman, B},
title = {Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression.},
journal = {Viruses},
volume = {11},
number = {3},
pages = {},
pmid = {30871003},
issn = {1999-4915},
mesh = {Cells, Cultured ; Cytokines/genetics ; Cytomegalovirus/*immunology ; Fibroblasts/virology ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; *Immunity, Innate ; Interferon Regulatory Factor-3/*genetics/immunology ; Interferon-beta/genetics ; Interferons/*immunology ; Real-Time Polymerase Chain Reaction ; *Signal Transduction ; Ubiquitins/genetics ; Up-Regulation ; },
abstract = {The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.},
}
@article {pmid30870431,
year = {2019},
author = {Thomas, M and Burgio, G and Adams, DJ and Iyer, V},
title = {Collateral damage and CRISPR genome editing.},
journal = {PLoS genetics},
volume = {15},
number = {3},
pages = {e1007994},
pmid = {30870431},
issn = {1553-7404},
support = {14356/CRUK_/Cancer Research UK/United Kingdom ; MR/L007428/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Cycle/genetics ; Cell Division/*genetics ; Gene Editing/trends ; Genetic Therapy/trends ; Genome/*genetics ; *Genomics ; Genotype ; Humans ; Mammals ; Mutation Rate ; Sequence Deletion/genetics ; Zygote/growth &amp; development ; },
abstract = {The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.},
}
@article {pmid30870143,
year = {2019},
author = {Lynch, MR and Tran, MT and Ralto, KM and Zsengeller, ZK and Raman, V and Bhasin, SS and Sun, N and Chen, X and Brown, D and Rovira, II and Taguchi, K and Brooks, CR and Stillman, IE and Bhasin, MK and Finkel, T and Parikh, SM},
title = {TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.},
journal = {JCI insight},
volume = {5},
number = {8},
pages = {},
pmid = {30870143},
issn = {2379-3708},
support = {R01 DK095072/DK/NIDDK NIH HHS/United States ; K01 DK099473/DK/NIDDK NIH HHS/United States ; R35 HL139424/HL/NHLBI NIH HHS/United States ; R01 AG027002/AG/NIA NIH HHS/United States ; P30 DK114809/DK/NIDDK NIH HHS/United States ; },
mesh = {Acute Kidney Injury/chemically induced/*genetics/metabolism ; Animals ; Autophagy/drug effects/genetics ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/*metabolism ; CRISPR-Cas Systems ; Cisplatin/*toxicity ; Gene Transfer Techniques ; Kidney Tubules/cytology/*metabolism ; Lysosomes/*metabolism ; Mice ; Mice, Knockout ; Mice, Transgenic ; Mitochondria/*metabolism ; Mitophagy/genetics ; NAD/metabolism ; *Organelle Biogenesis ; Oxidative Stress/genetics ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/*genetics/metabolism ; Sequence Analysis, RNA ; },
abstract = {Because injured mitochondria can accelerate cell death through the elaboration of oxidative free radicals and other mediators, it is striking that proliferator gamma coactivator 1-alpha (PGC1α), a stimulator of increased mitochondrial abundance, protects stressed renal cells instead of potentiating injury. Here we report that PGC1α's induction of lysosomes via transcription factor EB (TFEB) may be pivotal for kidney protection. CRISPR and stable gene transfer showed that PGC1α knockout tubular cells were sensitized to the genotoxic stressor cisplatin whereas transgenic cells were protected. The biosensor mtKeima unexpectedly revealed that cisplatin blunts mitophagy both in cells and mice. PGC1α not only counteracted this effect but also raised basal mitophagy, as did the downstream mediator nicotinamide adenine dinucleotide (NAD+). PGC1α did not consistently affect known autophagy pathways modulated by cisplatin. Instead RNA sequencing identified coordinated regulation of lysosomal biogenesis via TFEB. This effector pathway was sufficiently important that inhibition of TFEB or lysosomes unveiled a striking harmful effect of excess PGC1α in cells and conditional mice. These results uncover an unexpected effect of cisplatin on mitophagy and PGC1α's exquisite reliance on lysosomes for kidney protection. Finally, the data illuminate TFEB as a novel target for renal tubular stress resistance.},
}
@article {pmid30868974,
year = {2020},
author = {Ri, K and Kim, C and Pak, C and Ri, P and Om, H},
title = {The KLF6 Super Enhancer Modulates Cell Proliferation via MiR-1301 in Human Hepatoma Cells.},
journal = {MicroRNA (Shariqah, United Arab Emirates)},
volume = {9},
number = {1},
pages = {64-69},
doi = {10.2174/2211536608666190314122725},
pmid = {30868974},
issn = {2211-5374},
mesh = {CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cyclin-Dependent Kinase Inhibitor p21/biosynthesis ; Gene Editing/methods ; Gene Expression Regulation, Neoplastic/*genetics ; Hep G2 Cells ; Humans ; Kruppel-Like Factor 6/genetics/*metabolism ; Liver Neoplasms/*genetics/pathology ; MicroRNAs/*genetics ; RNA Interference ; Signal Transduction/genetics ; Tumor Suppressor Protein p53/biosynthesis ; },
abstract = {BACKGROUND: Recent studies have attempted to elucidate the function of super enhancers by means of microRNAs. Although the functional outcomes of miR-1301 have become clearer, the pathways that regulate the expressions of miR-1301 remain unclear.<br><br>OBJECTIVE: The objective of this paper was to consider the pathway regulating expression of miR- 1301 and miR-1301 signaling pathways with the inhibition of cell proliferation.<br><br>METHODS: In this study, we prepared the cell clones that the KLF6 super enhancer was deleted by means of the CRISPR/Cas9 system-mediated genetic engineering. Changes in miR-1301 expression after the deletion of the KLF6 super enhancer were evaluated by RT-PCR analysis, and the signal pathway of miR-1301 with inhibition of the cell proliferation was examined using RNA interference technology.<br><br>RESULTS: The results showed that miR-1301 expression was significantly increased after the deletion of the KLF6 super enhancer. Over-expression of miR-1301 induced by deletion of the KLF6 super enhancer also regulated the expression of p21 and p53 in human hepatoma cells. functional modeling of findings using siRNA specific to miR-1301 showed that expression level changes had direct biological effects on cellular proliferation in Human hepatoma cells. Furthermore, cellular proliferation assay was shown to be directly associated with miR-1301 levels.<br><br>CONCLUSION: As a result, it was demonstrated that the over-expression of miR-1301 induced by the disruption of the KLF6 super enhancer leads to a significant inhibition of proliferation in HepG2 cells. Moreover, it was demonstrated that the KLF6 super enhancer regulates the cell-proliferative effects which are mediated, at least in part, by the induction of p21and p53 in a p53-dependent manner. Our results provide the functional significance of miR-1301 in understanding the transcriptional regulation mechanism of the KLF6 super enhancer.},
}
@article {pmid30868795,
year = {2019},
author = {Shah, SZ and Rehman, A and Nasir, H and Asif, A and Tufail, B and Usama, M and Jabbar, B},
title = {Advances In Research On Genome Editing Crispr-Cas9 Technology.},
journal = {Journal of Ayub Medical College, Abbottabad : JAMC},
volume = {31},
number = {1},
pages = {108-122},
pmid = {30868795},
issn = {1819-2718},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Genomics ; Humans ; },
abstract = {BACKGROUND: The current era of genome engineering has been revolutionized by the evolution of a bacterial adaptive immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) into a radical technology that is making an expeditious progress in its mechanism, function and applicability..<br><br>METHODS: A systematic literature review study was carried out with the help of all available information and online resources..<br><br>RESULTS: In this review, we intend to elucidate different aspects of CRISPR in the light of current advancements. Utilizing a nonspecific Cas9 nuclease and a sequence specific programmable CRISPR RNA (crRNA), this system cleaves the target DNA with high precision. With a vast potential for profound implications, CRISPR has emerged as a mainstream method for plausible genomic manipulations in a range of organisms owing to its simplicity, accuracy and speed. A modified form of CRISPR system, known as CRISPR/Cpf1 that employs a smaller and simpler endonuclease (Cpf1) than Cas9, can be used to overcome certain limitations of CRISPR/Cas9 system. Despite clear-cut innovative biological applications, this technology is challenged by off-target effects and associated risks, thus safe and controlled implementation is needed to enable this emerging technique assist both biological research and translational applications.<br><br>CONCLUSIONS: CRISPR/Cas9 systems will undoubtedly revolutionize the study and treatment of both immunologic and allergic diseases. Concerned authorities should formulate and authorize such laws and regulations that permit the safe and ethical use of this emerging technology for basic research and clinical purposes.},
}
@article {pmid30868519,
year = {2019},
author = {Lander, N and Chiurillo, MA and Docampo, R},
title = {Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1955},
number = {},
pages = {61-76},
doi = {10.1007/978-1-4939-9148-8_5},
pmid = {30868519},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Chagas Disease/parasitology ; DNA Repair ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Humans ; Transfection/methods ; Trypanosoma cruzi/*genetics ; },
abstract = {The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4-5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.},
}
@article {pmid30867524,
year = {2019},
author = {Odamaki, T and Bottacini, F and Mitsuyama, E and Yoshida, K and Kato, K and Xiao, JZ and van Sinderen, D},
title = {Impact of a bathing tradition on shared gut microbe among Japanese families.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4380},
pmid = {30867524},
issn = {2045-2322},
mesh = {Adolescent ; Adult ; *Baths ; Bifidobacterium/genetics/isolation &amp; purification ; CRISPR-Cas Systems/genetics ; Child ; Child, Preschool ; Female ; Gastrointestinal Microbiome/*physiology ; Humans ; Infant ; Japan ; Male ; Middle Aged ; RNA, Ribosomal, 16S/genetics ; Water Microbiology ; Young Adult ; },
abstract = {Sharing of Bifidobacterium longum strains had recently been shown to occur among Japanese family members, a phenomenon that is not confined to mother-infant pairs. In the current study, we investigated if bathtub water is a possible vehicle for the exchange of strains as a consequence of a Japanese custom to share bathtub water by family members during bathing practices. A total of twenty-one subjects from five Japanese families, each consisting of parents with either 2 or 3 children, were enrolled in this study and the fecal microbiota of all participants was determined. Viable bifidobacterial strains were isolated from all bathtub water samples. A subsequent comparative genome analysis using ninety-eight strains indicated that certain strain-sets, which were isolated from feces and bathtub water, share near identical genome sequences, including CRISPR/Cas protospacers. By means of unweighted UniFrac distance analysis based on 16S rRNA gene analysis of 59 subjects from sixteen Japanese families, we showed that the fecal microbiota composition among family members that share bathtub water is significantly closer than that between family members that do not engage in this practice. Our results indicate that bathtub water represents a vehicle for the transmission of gut bacteria, and that the Japanese custom of sharing bathtub water contributes to the exchange of gut microbes, in particular bifidobacteria, among family members.},
}
@article {pmid30867331,
year = {2019},
author = {Dong, OX and Ronald, PC},
title = {Genetic Engineering for Disease Resistance in Plants: Recent Progress and Future Perspectives.},
journal = {Plant physiology},
volume = {180},
number = {1},
pages = {26-38},
pmid = {30867331},
issn = {1532-2548},
support = {R01 GM122968/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Disease Resistance/*genetics ; Genetic Engineering/*trends ; Plant Diseases/virology ; *Plants, Genetically Modified ; RNA Interference ; },
abstract = {A review of the recent progress in plant genetic engineering for disease resistance highlights future challenges and opportunities in the field.},
}
@article {pmid30867327,
year = {2019},
author = {Lombardi, L and Oliveira-Pacheco, J and Butler, G},
title = {Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
pmid = {30867327},
issn = {2379-5042},
mesh = {*CRISPR-Cas Systems ; Candida/*genetics ; Candida tropicalis/genetics ; DNA Barcoding, Taxonomic ; DNA End-Joining Repair ; Gene Editing/methods ; Genetic Vectors ; Genome, Fungal ; Mutation ; Plasmids/*genetics ; Streptothricins/pharmacology ; },
abstract = {Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalisIMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.},
}
@article {pmid30867223,
year = {2019},
author = {Thormann, V and Glaser, LV and Rothkegel, MC and Borschiwer, M and Bothe, M and Fuchs, A and Meijsing, SH},
title = {Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements.},
journal = {Life science alliance},
volume = {2},
number = {2},
pages = {},
pmid = {30867223},
issn = {2575-1077},
mesh = {Animals ; Binding Sites/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromatin/metabolism ; Chromatin Immunoprecipitation ; Gene Editing/*methods ; Gene Expression Regulation ; Humans ; Protein Binding/genetics ; RNA-Seq ; Rats ; Receptors, Glucocorticoid/*genetics ; Regulatory Elements, Transcriptional/genetics ; Response Elements/*genetics ; Transcription Initiation Site ; Transcription, Genetic/genetics ; Transcriptional Activation/genetics ; Transfection ; },
abstract = {The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.},
}
@article {pmid30865899,
year = {2019},
author = {Fang, Z and Weng, C and Li, H and Tao, R and Mai, W and Liu, X and Lu, L and Lai, S and Duan, Q and Alvarez, C and Arvan, P and Wynshaw-Boris, A and Li, Y and Pei, Y and Jin, F and Li, Y},
title = {Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes.},
journal = {Cell reports},
volume = {26},
number = {11},
pages = {3132-3144.e7},
pmid = {30865899},
issn = {2211-1247},
support = {P30 DK020572/DK/NIDDK NIH HHS/United States ; R01 DK113185/DK/NIDDK NIH HHS/United States ; R01 HG009658/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cells, Cultured ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; Diabetes Mellitus, Type 2/*genetics/metabolism ; Gene Expression Profiling ; *Genetic Heterogeneity ; HEK293 Cells ; Histone Acetyltransferases/genetics/metabolism ; Humans ; Insulin/genetics/metabolism ; Insulin-Secreting Cells/*metabolism ; Mice ; Single-Cell Analysis ; *Transcriptome ; },
abstract = {Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct β cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.},
}
@article {pmid30864562,
year = {2019},
author = {Veigl, SJ},
title = {A use/disuse paradigm for CRISPR-Cas systems.},
journal = {Biology &amp; philosophy},
volume = {34},
number = {1},
pages = {13},
pmid = {30864562},
issn = {0169-3867},
}
@article {pmid30864345,
year = {2019},
author = {Berghout, J and Lussier, YA and Vitali, F and Bulyk, ML and Kann, MG and Moore, JH},
title = {Workshop during the Pacific Symposium of Biocomputing, Jan 3-7, 2019: Reading between the genes: interpreting non-coding DNA in high-throughput.},
journal = {Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing},
volume = {24},
number = {},
pages = {444-448},
pmid = {30864345},
issn = {2335-6936},
mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; DNA/*genetics ; Epigenesis, Genetic ; Gene Regulatory Networks ; Genetic Variation ; High-Throughput Nucleotide Sequencing/*statistics &amp; numerical data ; Humans ; Mutation ; RNA, Untranslated/genetics ; Regulatory Elements, Transcriptional ; Sequence Analysis, DNA/*statistics &amp; numerical data ; Systems Biology ; },
abstract = {Identifying functional elements and predicting mechanistic insight from non-coding DNA and noncoding variation remains a challenge. Advances in genome-scale, high-throughput technology, however, have brought these answers closer within reach than ever, though there is still a need for new computational approaches to analysis and integration. This workshop aims to explore these resources and new computational methods applied to regulatory elements, chromatin interactions, non-protein-coding genes, and other non-coding DNA.},
}
@article {pmid30864164,
year = {2019},
author = {Jensen, TI and Axelgaard, E and Bak, RO},
title = {Therapeutic gene editing in haematological disorders with CRISPR/Cas9.},
journal = {British journal of haematology},
volume = {185},
number = {5},
pages = {821-835},
doi = {10.1111/bjh.15851},
pmid = {30864164},
issn = {1365-2141},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Hematologic Diseases/*genetics ; Humans ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9 platform offers an efficient way of making precise genetic changes to the human genome. This can be employed for disruption, addition and correction of genes, thereby enabling a new class of genetic therapies that can be applied to haematological disorders. Here we review recent technological advances in the CRISPR/Cas9 methodology and applications in haematology for curing monogenic genetic disorders and for engineering novel chimeric antigen receptor (CAR) T cells to treat haematological malignancies. Furthermore, we discuss current challenges for full clinical implementation of CRISPR/Cas9, and reflect on future trajectories of the technology.},
}
@article {pmid30863962,
year = {2019},
author = {Wang, X and Lu, J and Lao, K and Wang, S and Mo, X and Xu, X and Chen, X and Mo, B},
title = {Increasing the efficiency of CRISPR/Cas9-based gene editing by suppressing RNAi in plants.},
journal = {Science China. Life sciences},
volume = {62},
number = {7},
pages = {982-984},
doi = {10.1007/s11427-019-9514-9},
pmid = {30863962},
issn = {1869-1889},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Mutation ; Phenotype ; Plant Viruses/genetics ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; Promoter Regions, Genetic ; RNA Interference ; Signal Transduction ; },
}
@article {pmid30863790,
year = {2019},
author = {Savell, KE and Bach, SV and Zipperly, ME and Revanna, JS and Goska, NA and Tuscher, JJ and Duke, CG and Sultan, FA and Burke, JN and Williams, D and Ianov, L and Day, JJ},
title = {A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.},
journal = {eNeuro},
volume = {6},
number = {1},
pages = {},
pmid = {30863790},
issn = {2373-2822},
support = {DP1 DA039650/DA/NIDA NIH HHS/United States ; R00 DA034681/DA/NIDA NIH HHS/United States ; F31 DA042514/DA/NIDA NIH HHS/United States ; R01 MH114990/MH/NIMH NIH HHS/United States ; T32 GM008361/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Brain/cytology/metabolism ; Brain-Derived Neurotrophic Factor/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Adhesion Molecules, Neuronal/metabolism ; Cell Line, Tumor ; Extracellular Matrix Proteins/metabolism ; *Gene Expression Regulation ; *Genetic Techniques ; Male ; Nerve Tissue Proteins/metabolism ; Neurons/cytology/*metabolism ; Primary Cell Culture ; Random Allocation ; Rats, Sprague-Dawley ; Reelin Protein ; Serine Endopeptidases/metabolism ; Transcription, Genetic ; Transcriptome ; },
abstract = {CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.},
}
@article {pmid30862905,
year = {2019},
author = {Connelly, JP and Pruett-Miller, SM},
title = {CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4194},
pmid = {30862905},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome ; *High-Throughput Nucleotide Sequencing ; Rats ; *Sequence Analysis, DNA ; *Software ; },
abstract = {CRISPR-Cas9 technology allows the creation of user-defined genomic modifications in cells and whole organisms. However, quantifying editing rates in pools of cells or identifying correctly edited clones is tedious. Targeted next-generation sequencing provides a high-throughput platform for optimizing editing reagents and identifying correctly modified clones, but the large amount of data produced can be difficult to analyze. Here, we present CRIS.py, a simple and highly versatile python-based program which concurrently analyzes next-generation sequencing data for both knock-out and multiple user-specified knock-in modifications from one or many edited samples. Compared to available NGS analysis programs for CRISPR based-editing, CRIS.py has many advantages: (1) the ability to analyze from one to thousands of samples at once, (2) the capacity to check each sample for multiple sequence modifications, including those induced by base-editors, (3) an output in an easily searchable file format enabling users to quickly sort through and identify correctly targeted clones.},
}
@article {pmid30862357,
year = {2019},
author = {Poondla, N and Chandrasekaran, AP and Heese, K and Kim, KS and Ramakrishna, S},
title = {CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis.},
journal = {Biochemical and biophysical research communications},
volume = {512},
number = {1},
pages = {60-65},
doi = {10.1016/j.bbrc.2019.03.018},
pmid = {30862357},
issn = {1090-2104},
mesh = {Apoptosis/drug effects/genetics/physiology ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics/metabolism ; CRISPR-Cas Systems ; Down-Regulation ; Gene Editing ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Receptors, TNF-Related Apoptosis-Inducing Ligand/*genetics/metabolism ; TNF-Related Apoptosis-Inducing Ligand/*metabolism/pharmacology ; Up-Regulation ; },
abstract = {Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as an anticancer therapy because it mediates apoptosis of several cancer cell types but not normal human cell types. In this study, we implemented genome editing techniques to upregulate DR5 and downregulate cFLIP in HeLa cells to stimulate TRAIL-induced apoptosis. We designed and validated sgRNAs to enrich the endogenous level of DR5 by dead Cas9 (dCas9). Similarly, we designed two sgRNAs to disrupt the cFLIP gene by CRISPR/Cas9. We analyzed the effect of TRAIL on tumor cells by co-transfecting HeLa cells with the best combinations of sgRNAs regulating DR5 and cFLIP genes. TRAIL-induced apoptosis in HeLa cells was evaluated by the γH2AX foci formation assay to check for double-strand break and propidium iodide and Annexin V staining to quantify apoptotic cells. Viable cells were identified by CCK-8 assay, and cleaved-PARP level was evaluated by Western blot. This is the first study to demonstrate that genome editing techniques can be used as an effective combinatorial treatment strategy to induce apoptosis of cancer cells. In particular, enhancement of DR5 expression and inhibition of cFLIP expression by genome editing had a synergistic effect of inhibiting proliferation and inducing apoptosis in TRAIL-resistant HeLa cells. These results suggest that combinatorial treatment strategies mediated by the CRISPR/Cas9 system may be effective for design of other human TRAIL-resistant cell types.},
}
@article {pmid30861315,
year = {2020},
author = {Wirth, NT and Kozaeva, E and Nikel, PI},
title = {Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.},
journal = {Microbial biotechnology},
volume = {13},
number = {1},
pages = {233-249},
pmid = {30861315},
issn = {1751-7915},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Pseudomonas putida/genetics ; Recombination, Genetic ; },
abstract = {Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large-scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user-friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3-5 days. The protocol presented here is based on DNA recombination forced by double-stranded DNA cuts (through the activity of the I-SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR-Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult-to-delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome.},
}
@article {pmid30860482,
year = {2019},
author = {Hong, AL and Tseng, YY and Wala, JA and Kim, WJ and Kynnap, BD and Doshi, MB and Kugener, G and Sandoval, GJ and Howard, TP and Li, J and Yang, X and Tillgren, M and Ghandi, M and Sayeed, A and Deasy, R and Ward, A and McSteen, B and Labella, KM and Keskula, P and Tracy, A and Connor, C and Clinton, CM and Church, AJ and Crompton, BD and Janeway, KA and Van Hare, B and Sandak, D and Gjoerup, O and Bandopadhayay, P and Clemons, PA and Schreiber, SL and Root, DE and Gokhale, PC and Chi, SN and Mullen, EA and Roberts, CW and Kadoch, C and Beroukhim, R and Ligon, KL and Boehm, JS and Hahn, WC},
title = {Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30860482},
issn = {2050-084X},
support = {132943-MRSG-18-202-01-TBG//American Cancer Society/International ; T32 CA136432/CA/NCI NIH HHS/United States ; T32 GM007226/GM/NIGMS NIH HHS/United States ; K12 HD052896/HD/NICHD NIH HHS/United States ; P50 CA101942/CA/NCI NIH HHS/United States ; 328545//CureSearch for Children's Cancer/International ; Young Investigator Award//Alex's Lemonade Stand Foundation for Childhood Cancer/International ; W81XWH-15-1-0659//U.S. Department of Defense/International ; U01 CA176058/CA/NCI NIH HHS/United States ; K08 CA188073/CA/NCI NIH HHS/United States ; Wong Family Award//Dana-Farber Cancer Institute/International ; 14-40-31-HONG//American Association for Cancer Research/International ; OFD BTREC CDA//Boston Children's Hospital/International ; U01 CA217848/CA/NCI NIH HHS/United States ; T32 HG002295/HG/NHGRI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Carcinoma, Medullary/drug therapy/*genetics ; Cell Cycle ; Cell Line, Tumor ; Exome ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Kidney/metabolism ; Kidney Neoplasms/drug therapy/*genetics ; Mice ; Mice, Nude ; Mutation ; Neoplasm Transplantation ; Proteasome Endopeptidase Complex/*genetics ; Proteasome Inhibitors/*pharmacology ; RNA Interference ; SMARCB1 Protein/*genetics ; Sequence Analysis, RNA ; Ubiquitin/chemistry ; Whole Genome Sequencing ; },
abstract = {Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C. Our studies identify a synthetic lethal relationship between SMARCB1-deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.},
}
@article {pmid30860365,
year = {2019},
author = {Qin, P and Park, M and Alfson, KJ and Tamhankar, M and Carrion, R and Patterson, JL and Griffiths, A and He, Q and Yildiz, A and Mathies, R and Du, K},
title = {Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a.},
journal = {ACS sensors},
volume = {4},
number = {4},
pages = {1048-1054},
doi = {10.1021/acssensors.9b00239},
pmid = {30860365},
issn = {2379-3694},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Ebolavirus/genetics/*isolation &amp; purification ; Endoribonucleases/*chemistry ; Fluorometry ; Lab-On-A-Chip Devices ; Leptotrichia/enzymology ; Limit of Detection ; Microfluidic Analytical Techniques/instrumentation/*methods ; Nucleic Acid Hybridization ; Point-of-Care Testing ; RNA, Viral/chemistry/genetics ; },
abstract = {Highly infectious illness caused by pathogens is endemic especially in developing nations where there is limited laboratory infrastructure and trained personnel. Rapid point-of-care (POC) serological assays with minimal sample manipulation and low cost are desired in clinical practice. In this study, we report an automated POC system for Ebola RNA detection with RNA-guided RNA endonuclease Cas13a, utilizing its collateral RNA degradation after its activation. After automated microfluidic mixing and hybridization, nonspecific cleavage products of Cas13a are immediately measured by a custom integrated fluorometer which is small in size and convenient for in-field diagnosis. Within 5 min, a detection limit of 20 pfu/mL (5.45 × 10[7] copies/mL) of purified Ebola RNA is achieved. This isothermal and fully solution-based diagnostic method is rapid, amplification-free, simple, and sensitive, thus establishing a key technology toward a useful POC diagnostic platform.},
}
@article {pmid30859452,
year = {2019},
author = {Zhang, J and Zhu, Z and Yue, W and Li, J and Chen, Q and Yan, Y and Lei, A and Hua, J},
title = {Establishment of CRISPR/Cas9-Mediated Knock-in System for Porcine Cells with High Efficiency.},
journal = {Applied biochemistry and biotechnology},
volume = {189},
number = {1},
pages = {26-36},
doi = {10.1007/s12010-019-02984-5},
pmid = {30859452},
issn = {1559-0291},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Swine ; Transgenes ; },
abstract = {Since the birth of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, the new genome engineering technology has become a hot topic in the scientific community. However, for swine, the system of pig cells' homology directed repair (HDR) is generally unstable and costly. Here, we aim to make knock-in of porcine cells more realizable. The Rosa26 locus was chosen for gene editing. Through the optimization of strategy, an efficient sgRNA was selected by TIDE analysis. Correspondingly, a vector system was constructed for gene insertion in pRosa26 locus by homologous recombination. A large percentage of cells whose gene is edited easily result in apoptosis. To improve the positive rate, culturing systems have been optimized. Sequence alignment and nuclear transfer confirmed that we got two knock-in cell lines and transgene primary porcine fetal fibroblasts (PFFs) successfully. Results showed that the gene editing platform we used can obtain genetically modified pig cells stably and efficiently. This system can contribute to pig gene research and production of transgenic pigs.},
}
@article {pmid30858603,
year = {2019},
author = {Park, H and Oh, J and Shim, G and Cho, B and Chang, Y and Kim, S and Baek, S and Kim, H and Shin, J and Choi, H and Yoo, J and Kim, J and Jun, W and Lee, M and Lengner, CJ and Oh, YK and Kim, J},
title = {In vivo neuronal gene editing via CRISPR-Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {22},
number = {4},
pages = {524-528},
pmid = {30858603},
issn = {1546-1726},
mesh = {Alzheimer Disease/*genetics/*metabolism/therapy ; Amyloid Precursor Protein Secretases/*genetics/metabolism ; Amyloid beta-Peptides/*metabolism ; Animals ; Aspartic Acid Endopeptidases/*genetics/metabolism ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/methods ; Hippocampus/metabolism ; Male ; Mice, Transgenic ; Nanoparticles/administration &amp; dosage ; Neurons/*metabolism ; },
abstract = {In vivo gene editing in post-mitotic neurons of the adult brain may be a useful strategy for treating neurological diseases. Here, we develop CRISPR-Cas9 nanocomplexes and show they were effective in the adult mouse brain, with minimal off-target effects. Using this system to target Bace1 suppressed amyloid beta (Aβ)-associated pathologies and cognitive deficits in two mouse models of Alzheimer's disease. These results broaden the potential application of CRISPR-Cas9 systems to neurodegenerative diseases.},
}
@article {pmid30858339,
year = {2019},
author = {Howden, SE and Vanslambrouck, JM and Wilson, SB and Tan, KS and Little, MH},
title = {Reporter-based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation.},
journal = {EMBO reports},
volume = {20},
number = {4},
pages = {},
pmid = {30858339},
issn = {1469-3178},
support = {UH2 DK107344/DK/NIDDK NIH HHS/United States ; UH3 DK107344/DK/NIDDK NIH HHS/United States ; },
mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Differentiation ; *Chromosome Mapping ; *Gene Expression Profiling ; *Genes, Reporter ; Homeodomain Proteins/genetics ; Humans ; Nephrons/*embryology/*metabolism ; Nerve Tissue Proteins/genetics ; Organogenesis/*genetics ; Organoids ; Pluripotent Stem Cells/cytology/metabolism ; Single-Cell Analysis ; },
abstract = {Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self-renewing Six2-expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell-derived kidney organoids, we performed a similar fate-mapping analysis of the SIX2-expressing lineage in induced pluripotent stem cell (iPSC)-derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro Using CRISPR/Cas9 gene-edited lineage reporter lines, we show that SIX2-expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2[+] cells to contribute to nephron formation over time, but retention of nephron-forming capacity if provided an exogenous WNT signal. Hence, while human iPSC-derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis.},
}
@article {pmid30856357,
year = {2019},
author = {Le Rhun, A and Escalera-Maurer, A and Bratovič, M and Charpentier, E},
title = {CRISPR-Cas in Streptococcus pyogenes.},
journal = {RNA biology},
volume = {16},
number = {4},
pages = {380-389},
pmid = {30856357},
issn = {1555-8584},
mesh = {Adaptation, Physiological/genetics ; CRISPR-Cas Systems/*genetics ; Genetic Loci ; RNA/biosynthesis ; RNA Interference ; Streptococcus pyogenes/*genetics ; },
abstract = {The discovery and characterization of the prokaryotic CRISPR-Cas immune system has led to a revolution in genome editing and engineering technologies. Despite the fact that most applications emerged after the discovery of the type II-A CRISPR-Cas9 system of Streptococcus pyogenes, its biological importance in this organism has received little attention. Here, we provide a comprehensive overview of the current knowledge about CRISPR-Cas systems from S. pyogenes. We discuss how the interplay between CRISPR-mediated immunity and horizontal gene transfer might have modeled the evolution of this pathogen. We review the current literature about the CRISPR-Cas systems present in S. pyogenes (types I-C and II-A), and describe their distinctive biochemical and functional features. Finally, we summarize the main biotechnological applications that have arisen from the discovery of the CRISPR-Cas9 system in S. pyogenes.},
}
@article {pmid30856211,
year = {2019},
author = {Lin, ZQ and Gan, SW and Tung, SY and Ho, CC and Su, LH and Sun, CH},
title = {Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia.},
journal = {PloS one},
volume = {14},
number = {3},
pages = {e0213594},
pmid = {30856211},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Expression Regulation ; Giardia lamblia/cytology/*genetics ; Giardiasis/parasitology ; Humans ; Plasmids/genetics ; Protozoan Proteins/*genetics ; },
abstract = {Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia.},
}
@article {pmid30856164,
year = {2019},
author = {Peterka, M and Kornmann, B},
title = {Miro-dependent mitochondrial pool of CENP-F and its farnesylated C-terminal domain are dispensable for normal development in mice.},
journal = {PLoS genetics},
volume = {15},
number = {3},
pages = {e1008050},
pmid = {30856164},
issn = {1553-7404},
mesh = {Amino Acid Sequence ; Amino Acid Substitution ; Animals ; CRISPR-Cas Systems ; Cell Line ; Chromosomal Proteins, Non-Histone/chemistry/genetics/*metabolism ; Eye Abnormalities/genetics ; Humans ; Intestinal Atresia/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mice, Transgenic ; Microcephaly/genetics ; Microfilament Proteins/chemistry/genetics/*metabolism ; Mitochondria/metabolism ; Mitochondrial Proteins/chemistry/genetics/*metabolism ; Point Mutation ; Prenylation ; Protein Binding ; Protein Interaction Domains and Motifs ; rho GTP-Binding Proteins/chemistry/genetics/*metabolism ; },
abstract = {CENP-F is a large, microtubule-binding protein that regulates multiple cellular processes including chromosome segregation and mitochondrial trafficking at cytokinesis. This multiplicity of functions is mediated through the binding of various partners, like Bub1 at the kinetochore and Miro at mitochondria. Due to the multifunctionality of CENP-F, the cellular phenotypes observed upon its depletion are difficult to interpret and there is a need to genetically separate its different functions by preventing binding to selected partners. Here we engineer a CENP-F point-mutant that is deficient in Miro binding and thus is unable to localize to mitochondria, but retains other localizations. We introduce this mutation in cultured human cells using CRISPR/Cas9 system and show it causes a defect in mitochondrial spreading similar to that observed upon Miro depletion. We further create a mouse model carrying this CENP-F variant, as well as truncated CENP-F mutants lacking the farnesylated C-terminus of the protein. Importantly, one of these truncations leads to ~80% downregulation of CENP-F expression. We observe that, despite the phenotypes apparent in cultured cells, mutant mice develop normally. Taken together, these mice will serve as important models to study CENP-F biology at organismal level. In addition, because truncations of CENP-F in humans cause a lethal disease termed Strømme syndrome, they might also be relevant disease models.},
}
@article {pmid30855744,
year = {2019},
author = {Porteus, MH},
title = {A New Class of Medicines through DNA Editing.},
journal = {The New England journal of medicine},
volume = {380},
number = {10},
pages = {947-959},
doi = {10.1056/NEJMra1800729},
pmid = {30855744},
issn = {1533-4406},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA ; Drug Discovery/*methods ; Gene Editing/history/*methods ; History, 20th Century ; History, 21st Century ; },
}
@article {pmid30854433,
year = {2019},
author = {Min, YL and Li, H and Rodriguez-Caycedo, C and Mireault, AA and Huang, J and Shelton, JM and McAnally, JR and Amoasii, L and Mammen, PPA and Bassel-Duby, R and Olson, EN},
title = {CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells.},
journal = {Science advances},
volume = {5},
number = {3},
pages = {eaav4324},
pmid = {30854433},
issn = {2375-2548},
support = {R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 HL138426/HL/NHLBI NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/*genetics ; *Exons ; Gene Editing ; Gene Expression ; Gene Targeting ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Mice ; Muscular Dystrophy, Duchenne/drug therapy/*genetics ; Myocytes, Cardiac/metabolism ; RNA, Guide ; *Sequence Deletion ; Transduction, Genetic ; },
abstract = {Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.},
}
@article {pmid30853970,
year = {2019},
author = {Parmeciano Di Noto, G and Molina, MC and Quiroga, C},
title = {Insights Into Non-coding RNAs as Novel Antimicrobial Drugs.},
journal = {Frontiers in genetics},
volume = {10},
number = {},
pages = {57},
pmid = {30853970},
issn = {1664-8021},
abstract = {Multidrug resistant bacteria are a serious worldwide problem, especially carbapenem-resistant Enterobacteriaceae (such as Klebsiella pneumoniae and Escherichia coli), Acinetobacter baumannii and Pseudomonas aeruginosa. Since the emergence of extensive and pan-drug resistant bacteria there are few antibiotics left to treat patients, thus novel RNA-based strategies are being considered. Here, we examine the current situation of different non-coding RNAs found in bacteria as well as their function and potential application as antimicrobial agents. Furthermore, we discuss the factors that may contribute in the efficient development of RNA-based drugs, the limitations for their implementation and the use of nanocarriers for delivery.},
}
@article {pmid30853940,
year = {2019},
author = {Jaiswal, S and Singh, DK and Shukla, P},
title = {Gene Editing and Systems Biology Tools for Pesticide Bioremediation: A Review.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {87},
pmid = {30853940},
issn = {1664-302X},
abstract = {Bioremediation is the degradation potential of microorganisms to dissimilate the complex chemical compounds from the surrounding environment. The genetics and biochemistry of biodegradation processes in datasets opened the way of systems biology. Systemic biology aid the study of interacting parts involved in the system. The significant keys of system biology are biodegradation network, computational biology, and omics approaches. Biodegradation network consists of all the databases and datasets which aid in assisting the degradation and deterioration potential of microorganisms for bioremediation processes. This review deciphers the bio-degradation network, i.e., the databases and datasets (UM-BBD, PAN, PTID, etc.) aiding in assisting the degradation and deterioration potential of microorganisms for bioremediation processes, computational biology and multi omics approaches like metagenomics, genomics, transcriptomics, proteomics, and metabolomics for the efficient functional gene mining and their validation for bioremediation experiments. Besides, the present review also describes the gene editing tools like CRISPR Cas, TALEN, and ZFNs which can possibly make design microbe with functional gene of interest for degradation of particular recalcitrant for improved bioremediation.},
}
@article {pmid30853558,
year = {2019},
author = {Xu, H and Wang, B and Ono, M and Kagita, A and Fujii, K and Sasakawa, N and Ueda, T and Gee, P and Nishikawa, M and Nomura, M and Kitaoka, F and Takahashi, T and Okita, K and Yoshida, Y and Kaneko, S and Hotta, A},
title = {Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.},
journal = {Cell stem cell},
volume = {24},
number = {4},
pages = {566-578.e7},
doi = {10.1016/j.stem.2019.02.005},
pmid = {30853558},
issn = {1875-9777},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Female ; *Gene Editing ; HLA Antigens/*genetics/immunology ; Histocompatibility/*immunology ; Humans ; Induced Pluripotent Stem Cells/*immunology/*metabolism ; Male ; Mice ; Mice, Inbred NOD ; },
abstract = {Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.},
}
@article {pmid30852338,
year = {2019},
author = {McDermott, U},
title = {Large-scale compound screens and pharmacogenomic interactions in cancer.},
journal = {Current opinion in genetics &amp; development},
volume = {54},
number = {},
pages = {12-16},
doi = {10.1016/j.gde.2019.02.002},
pmid = {30852338},
issn = {1879-0380},
mesh = {*Biomarkers, Pharmacological ; CRISPR-Cas Systems/genetics ; Computational Biology ; Drug Evaluation, Preclinical/*methods ; Humans ; Neoplasms/*drug therapy/genetics ; *Pharmacogenetics ; },
abstract = {In the last decade, we have witnessed tremendous advances in our understanding of the landscape of the molecular alterations that underpin many of the most prevalent cancers, in the use of automated high throughput platforms for high-throughput drug screens in cancer cells, in the creation of more clinically relevant cancer cell models, in the application of CRISPR genetic screens for novel target identification, and lastly in the development of more useful computational approaches in the pursuit of biomarkers of drug response.},
}
@article {pmid30850620,
year = {2019},
author = {Kaushik, A and Yndart, A and Atluri, V and Tiwari, S and Tomitaka, A and Gupta, P and Jayant, RD and Alvarez-Carbonell, D and Khalili, K and Nair, M},
title = {Magnetically guided non-invasive CRISPR-Cas9/gRNA delivery across blood-brain barrier to eradicate latent HIV-1 infection.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3928},
pmid = {30850620},
issn = {2045-2322},
support = {R01 DA042706/DA/NIDA NIH HHS/United States ; R01 DA037838/DA/NIDA NIH HHS/United States ; R01 DA040537/DA/NIDA NIH HHS/United States ; R21 MH101025/MH/NIMH NIH HHS/United States ; R01 DA027049/DA/NIDA NIH HHS/United States ; R01 DA034547/DA/NIDA NIH HHS/United States ; },
mesh = {Blood-Brain Barrier/*virology ; CRISPR-Cas Systems ; Cells, Cultured ; Drug Delivery Systems ; Gene Editing/methods ; HIV Infections/*therapy/*virology ; *HIV-1/genetics ; Humans ; In Vitro Techniques ; Magnetite Nanoparticles/administration &amp; dosage ; RNA, Guide/*administration &amp; dosage/genetics ; Virus Latency ; },
abstract = {CRISPR-Cas9/gRNA exhibits therapeutic efficacy against latent human immunodeficiency virus (HIV) genome but the delivery of this therapeutic cargo to the brain remains as a challenge. In this research, for the first time, we demonstrated magnetically guided non-invasive delivery of a nano-formulation (NF), composed of Cas9/gRNA bound with magneto-electric nanoparticles (MENPs), across the blood-brain barrier (BBB) to inhibit latent HIV-1 infection in microglial (hμglia)/HIV (HC69) cells. An optimized ac-magnetic field of 60 Oe was applied on NF to release Cas9/gRNA from MENPs surface and to facilitate NF cell uptake resulting in intracellular release and inhibition of HIV. The outcomes suggested that developed NF reduced HIV-LTR expression significantly in comparison to unbound Cas9/gRNA in HIV latent hμglia/HIV (HC69) cells. These findings were also validated qualitatively using fluorescence microscopy to assess NF efficacy against latent HIV in the microglia cells. We believe that CNS delivery of NF (CRISPR/Cas9-gRNA-MENPs) across the BBB certainly will have clinical utility as future personalized nanomedicine to manage neuroHIV/AIDS.},
}
@article {pmid30850590,
year = {2019},
author = {Cullot, G and Boutin, J and Toutain, J and Prat, F and Pennamen, P and Rooryck, C and Teichmann, M and Rousseau, E and Lamrissi-Garcia, I and Guyonnet-Duperat, V and Bibeyran, A and Lalanne, M and Prouzet-Mauléon, V and Turcq, B and Ged, C and Blouin, JM and Richard, E and Dabernat, S and Moreau-Gaudry, F and Bedel, A},
title = {CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1136},
pmid = {30850590},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Chromosome Deletion ; *Chromosomes, Human, Pair 10 ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/metabolism ; *DNA Breaks, Double-Stranded ; Deoxyribonuclease I/*genetics/metabolism ; Fibroblasts/cytology/metabolism ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genome, Human ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; K562 Cells ; Models, Biological ; Porphyria, Erythropoietic/genetics/metabolism/pathology/therapy ; Primary Cell Culture ; RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; Tumor Suppressor Protein p53/genetics/metabolism ; Uroporphyrinogen III Synthetase/*genetics/metabolism ; },
abstract = {CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.},
}
@article {pmid30850537,
year = {2019},
author = {Wong, E and Liao, GP and Chang, JC and Xu, P and Li, YM and Greengard, P},
title = {GSAP modulates γ-secretase specificity by inducing conformational change in PS1.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {13},
pages = {6385-6390},
pmid = {30850537},
issn = {1091-6490},
support = {T32 GM073546/GM/NIGMS NIH HHS/United States ; R01 NS096275/NS/NINDS NIH HHS/United States ; RF1 AG057593/AG/NIA NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 AG047781/AG/NIA NIH HHS/United States ; R01 AG061350/AG/NIA NIH HHS/United States ; },
mesh = {Alzheimer Disease/*metabolism ; Amyloid Precursor Protein Secretases/*metabolism ; CRISPR-Cas Systems ; Catalytic Domain ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Kinetics ; Peptide Fragments/metabolism ; Presenilin-1/*chemistry ; Proteins/genetics/*metabolism ; Receptor, Notch1 ; },
abstract = {The mechanism by which γ-secretase activating protein (GSAP) regulates γ-secretase activity has not yet been elucidated. Here, we show that knockout of GSAP in cultured cells directly reduces γ-secretase activity for Aβ production, but not for Notch1 cleavage, suggesting that GSAP may induce a conformational change contributing to the specificity of γ-secretase. Furthermore, using an active-site-directed photoprobe with double cross-linking moieties, we demonstrate that GSAP modifies the orientation and/or distance of the PS1 N-terminal fragment and the PS1 C-terminal fragment, a region containing the active site of γ-secretase. This work offers insight into how GSAP regulates γ-secretase specificity.},
}
@article {pmid30850374,
year = {2019},
author = {Labun, K and Guo, X and Chavez, A and Church, G and Gagnon, JA and Valen, E},
title = {Accurate analysis of genuine CRISPR editing events with ampliCan.},
journal = {Genome research},
volume = {29},
number = {5},
pages = {843-847},
pmid = {30850374},
issn = {1549-5469},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; *Mutation Rate ; Recombinational DNA Repair/genetics ; Sequence Alignment/methods ; Software ; },
abstract = {We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, and off-target read detection and quantifies insertions, deletions, HDR repair, as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated data sets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.},
}
@article {pmid30850331,
year = {2019},
author = {Zhu, Y and Gao, A and Zhan, Q and Wang, Y and Feng, H and Liu, S and Gao, G and Serganov, A and Gao, P},
title = {Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins.},
journal = {Molecular cell},
volume = {74},
number = {2},
pages = {296-309.e7},
pmid = {30850331},
issn = {1097-4164},
support = {R01 GM112940/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/*genetics ; DNA/chemistry/*genetics ; *Gene Editing ; Humans ; Neisseria meningitidis/enzymology/genetics ; },
abstract = {Anti-CRISPR proteins (Acrs) targeting CRISPR-Cas9 systems represent natural "off switches" for Cas9-based applications. Recently, AcrIIC1, AcrIIC2, and AcrIIC3 proteins were found to inhibit Neisseria meningitidis Cas9 (NmeCas9) activity in bacterial and human cells. Here we report biochemical and structural data that suggest molecular mechanisms of AcrIIC2- and AcrIIC3-mediated Cas9 inhibition. AcrIIC2 dimer interacts with the bridge helix of Cas9, interferes with RNA binding, and prevents DNA loading into Cas9. AcrIIC3 blocks the DNA loading step through binding to a non-conserved surface of the HNH domain of Cas9. AcrIIC3 also forms additional interactions with the REC lobe of Cas9 and induces the dimerization of the AcrIIC3-Cas9 complex. While AcrIIC2 targets Cas9 orthologs from different subtypes, albeit with different efficiency, AcrIIC3 specifically inhibits NmeCas9. Structure-guided changes in NmeCas9 orthologs convert them into anti-CRISPR-sensitive proteins. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms and guidelines for designing regulatory tools in Cas9-based applications.},
}
@article {pmid30849665,
year = {2019},
author = {Swinnen, G and Goossens, A and Colinas, M},
title = {Metabolic editing: small measures, great impact.},
journal = {Current opinion in biotechnology},
volume = {59},
number = {},
pages = {16-23},
doi = {10.1016/j.copbio.2019.02.002},
pmid = {30849665},
issn = {1879-0429},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Metabolic Engineering ; Protein Processing, Post-Translational ; },
abstract = {Metabolic pathways are tightly regulated at the transcriptional and post-translational level, often relying on protein-protein interactions or post-translational protein modifications. Whereas these principles have been established already for a long time, the number of experimentally established cases is expected to rise exponentially in the near future as a result of recent advances in protein-based detection methods. Interactions and modifications are often dependent on only short amino-acid sequences that represent excellent targets for new gene editing technologies by which specific base pairs can be exchanged. Here, we introduce the concept of metabolic editing, which is based on identifying specific amino-acid sequences that are subsequently targeted for gene editing. The proposed workflow will serve for both applied metabolic engineering purposes and proof-of-concept studies in fundamental research.},
}
@article {pmid30849442,
year = {2019},
author = {Liu, B and Saber, A and Haisma, HJ},
title = {CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment.},
journal = {Drug discovery today},
volume = {24},
number = {4},
pages = {955-970},
doi = {10.1016/j.drudis.2019.02.011},
pmid = {30849442},
issn = {1878-5832},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome-editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest here that CRISPR/Cas9 is an effective system to distinguish between mutant and wild-type alleles in cancer. We show that several new potential therapeutic targets, such as CD38, CXCR2, MASTL, and RBX2, as well as several noncoding (nc)RNAs have been identified using CRISPR/Cas9 technology. We also discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic.},
}
@article {pmid30849393,
year = {2019},
author = {Bhate, A and Sun, T and Li, JB},
title = {ADAR1: A New Target for Immuno-oncology Therapy.},
journal = {Molecular cell},
volume = {73},
number = {5},
pages = {866-868},
doi = {10.1016/j.molcel.2019.02.021},
pmid = {30849393},
issn = {1097-4164},
mesh = {Adenosine Deaminase/genetics/immunology/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Survival ; Gene Editing ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Genetic Therapy/*methods ; Humans ; Immunity, Innate ; Immunotherapy/*methods ; Interferon-Induced Helicase, IFIH1/genetics/metabolism ; Neoplasms/enzymology/genetics/immunology/*therapy ; RNA Editing ; RNA, Double-Stranded/genetics/immunology/*metabolism ; RNA-Binding Proteins/genetics/immunology/*metabolism ; },
abstract = {Three recent studies by Ishizuka et al. (2019), Liu et al. (2019), and Gannon et al. (2018) show that deleting RNA editing enzyme ADAR1 could induce higher cell lethality and render tumor cells more vulnerable to immunotherapy, pinpointing ADAR1 as a new immuno-oncology target.},
}
@article {pmid30847946,
year = {2019},
author = {Ishii, T and Schubert, V and Khosravi, S and Dreissig, S and Metje-Sprink, J and Sprink, T and Fuchs, J and Meister, A and Houben, A},
title = {RNA-guided endonuclease - in situ labelling (RGEN-ISL): a fast CRISPR/Cas9-based method to label genomic sequences in various species.},
journal = {The New phytologist},
volume = {222},
number = {3},
pages = {1652-1661},
pmid = {30847946},
issn = {1469-8137},
support = {//CSIRO/International ; Ho1779/28-1//Deutsche Forschungsgemeinschaft/International ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Centromere/metabolism ; Endonucleases/*metabolism ; *Genomics ; RNA, Guide/*metabolism ; Species Specificity ; *Staining and Labeling ; },
abstract = {Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.},
}
@article {pmid30846752,
year = {2019},
author = {Qin, X and Yao, W and Shi, X and Liu, L and Huang, F and Ding, Y and Zhou, Y and Yu, L and Jia, C and Li, S and Rao, C and Wang, J},
title = {Responsive Cells for rhEGF bioassay Obtained through Screening of a CRISPR/Cas9 Library.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3780},
pmid = {30846752},
issn = {2045-2322},
mesh = {Animals ; Biological Assay/*methods ; *CRISPR-Cas Systems ; Epidermal Growth Factor/genetics/*pharmacology ; Gene Knockout Techniques ; Humans ; Loss of Function Mutation ; Mice ; NIH 3T3 Cells ; Reproducibility of Results ; },
abstract = {Bioassay of recombinant protein products is important tests to ensure protein effectiveness. Some recombinant protein products have no cells used in their bioassay but instead use animal models, while others have no suitable method. Here, we developed a method to obtain responsive cells used in bioassay of proteins. After screening of a CRISPR/Cas9 library, we obtained a responsive cell line that grew faster in the presence of rhEGF (recombinant human epidermal growth factor) than that of control cells. We used this cell line for bioassay of rhEGF. This cell line, compared with the control cells, had a 2 day shorter operation time and had lower interference. The responsive cell line is more suitable for use in bioassay of rhEGF.},
}
@article {pmid30846702,
year = {2019},
author = {Park, SJ and Kim, B and Choi, S and Balasubramaniam, S and Lee, SC and Lee, JY and Kim, HS and Kim, JY and Kim, JJ and Lee, YA and Kang, NY and Kim, JS and Chang, YT},
title = {Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1111},
pmid = {30846702},
issn = {2041-1723},
mesh = {Acridines ; Animals ; CRISPR-Cas Systems ; Cation Transport Proteins/*metabolism ; HeLa Cells ; Humans ; Inflammation/*diagnostic imaging/*immunology/metabolism ; *Macrophage Activation ; Mice ; Mice, Knockout, ApoE ; Molecular Probe Techniques ; Molecular Probes ; Plaque, Atherosclerotic/diagnostic imaging/immunology/metabolism ; RAW 264.7 Cells ; },
abstract = {Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.},
}
@article {pmid30846578,
year = {2019},
author = {Normile, D},
title = {China tightens rules on gene editing.},
journal = {Science (New York, N.Y.)},
volume = {363},
number = {6431},
pages = {1023},
doi = {10.1126/science.363.6431.1023-b},
pmid = {30846578},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; China ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Humans ; },
}
@article {pmid30846391,
year = {2019},
author = {Kowalski, PS and Rudra, A and Miao, L and Anderson, DG},
title = {Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {4},
pages = {710-728},
pmid = {30846391},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Cell-Penetrating Peptides/chemistry ; Dendrimers/chemistry ; Drug Delivery Systems/*methods ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; Lipids/chemistry ; Mice ; Nanoparticles/chemistry ; Polymers/chemistry ; RNA, Messenger/genetics/*therapeutic use ; Vaccination/methods ; },
abstract = {mRNA has broad potential as a therapeutic. Current clinical efforts are focused on vaccination, protein replacement therapies, and treatment of genetic diseases. The clinical translation of mRNA therapeutics has been made possible through advances in the design of mRNA manufacturing and intracellular delivery methods. However, broad application of mRNA is still limited by the need for improved delivery systems. In this review, we discuss the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in biomaterials and delivery strategies, and we present an overview of the applications of mRNA-based delivery for protein therapy, gene editing, and vaccination.},
}
@article {pmid30845784,
year = {2019},
author = {Bernard, G and Gagneul, D and Alves Dos Santos, H and Etienne, A and Hilbert, JL and Rambaud, C},
title = {Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory.},
journal = {International journal of molecular sciences},
volume = {20},
number = {5},
pages = {},
pmid = {30845784},
issn = {1422-0067},
mesh = {Agrobacterium/physiology ; CRISPR-Cas Systems ; Chicory/*genetics/microbiology ; Gene Editing/*methods ; Mutation ; Oxidoreductases/*genetics ; Plant Proteins/genetics ; Promoter Regions, Genetic ; },
abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.},
}
@article {pmid30845180,
year = {2019},
author = {de Oliveira, VC and Moreira, GSA and Bressan, FF and Gomes Mariano Junior, C and Roballo, KCS and Charpentier, M and Concordet, JP and Meirelles, FV and Ambrósio, CE},
title = {Edition of TFAM gene by CRISPR/Cas9 technology in bovine model.},
journal = {PloS one},
volume = {14},
number = {3},
pages = {e0213376},
pmid = {30845180},
issn = {1932-6203},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cattle ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Mitochondrial/genetics ; DNA-Binding Proteins/*genetics ; Fibroblasts/physiology ; Gene Dosage/genetics ; Mitochondria/genetics ; Mitochondrial Proteins/*genetics ; Models, Animal ; RNA, Guide/genetics ; Transcription Factors/*genetics ; },
abstract = {The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.},
}
@article {pmid30845151,
year = {2019},
author = {Lischik, CQ and Adelmann, L and Wittbrodt, J},
title = {Enhanced in vivo-imaging in medaka by optimized anaesthesia, fluorescent protein selection and removal of pigmentation.},
journal = {PloS one},
volume = {14},
number = {3},
pages = {e0212956},
pmid = {30845151},
issn = {1932-6203},
mesh = {Anesthesia/methods ; Animals ; Animals, Genetically Modified ; Bungarotoxins/genetics ; CRISPR-Cas Systems/genetics ; Intravital Microscopy/*methods ; Luminescent Proteins/chemistry/genetics ; Microscopy, Fluorescence/methods ; *Models, Animal ; *Oryzias ; Pigmentation/genetics ; Zebrafish ; },
abstract = {Fish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation. We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.},
}
@article {pmid30844400,
year = {2019},
author = {Tabrizi, SJ and Ghosh, R and Leavitt, BR},
title = {Huntingtin Lowering Strategies for Disease Modification in Huntington's Disease.},
journal = {Neuron},
volume = {101},
number = {5},
pages = {801-819},
doi = {10.1016/j.neuron.2019.01.039},
pmid = {30844400},
issn = {1097-4199},
support = {200181/Z/15/Z//Wellcome Trust/United Kingdom ; MR/K023268/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Genetic Therapy/*methods ; Humans ; Huntingtin Protein/*genetics/metabolism ; Huntington Disease/etiology/*therapy ; },
abstract = {Huntington's disease is caused by an abnormally expanded CAG repeat expansion in the HTT gene, which confers a predominant toxic gain of function in the mutant huntingtin (mHTT) protein. There are currently no disease-modifying therapies available, but approaches that target proximally in disease pathogenesis hold great promise. These include DNA-targeting techniques such as zinc-finger proteins, transcription activator-like effector nucleases, and CRISPR/Cas9; post-transcriptional huntingtin-lowering approaches such as RNAi, antisense oligonucleotides, and small-molecule splicing modulators; and novel methods to clear the mHTT protein, such as proteolysis-targeting chimeras. Improvements in the delivery and distribution of such agents as well as the development of objective biomarkers of disease and of HTT lowering pharmacodynamic outcomes have brought these potential therapies to the forefront of Huntington's disease research, with clinical trials in patients already underway.},
}
@article {pmid30844313,
year = {2019},
author = {Shi, Z and Xin, H and Tian, D and Lian, J and Wang, J and Liu, G and Ran, R and Shi, S and Zhang, Z and Shi, Y and Deng, Y and Hou, C and Chen, Y},
title = {Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {6},
pages = {6962-6968},
doi = {10.1096/fj.201802661R},
pmid = {30844313},
issn = {1530-6860},
mesh = {Abnormalities, Multiple/*genetics ; Albinism, Oculocutaneous/*genetics ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Female ; Genotype ; Heart Defects, Congenital/*genetics ; Heart Septal Defects, Atrial/*genetics ; Humans ; Lower Extremity Deformities, Congenital/*genetics ; Male ; *Point Mutation ; Upper Extremity Deformities, Congenital/*genetics ; Xenopus/*embryology ; },
abstract = {Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.},
}
@article {pmid30844312,
year = {2019},
author = {Ko, T and Li, S},
title = {Genome-wide screening identifies novel genes and biological processes implicated in cisplatin resistance.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {6},
pages = {7143-7154},
doi = {10.1096/fj.201801534RR},
pmid = {30844312},
issn = {1530-6860},
mesh = {Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Drug Resistance, Neoplasm ; Eukaryotic Initiation Factor-4E/genetics/metabolism ; Gene Deletion ; Gene Expression Regulation, Neoplastic/*drug effects ; Genome-Wide Association Study ; Humans ; Melanocytes/drug effects ; Melanoma/*drug therapy ; Neurofibromin 2/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; Up-Regulation ; Verteporfin/pharmacology ; beta Catenin/genetics/metabolism ; },
abstract = {Cisplatin-based chemotherapeutic regimens are frequently used for treatments of solid tumors. However, tumor cells may have inherent or acquired cisplatin resistance, and the underlying mechanisms are largely unknown. We performed genome-wide screening of genes implicated in cisplatin resistance in A375 human melanoma cells. A substantial fraction of genes whose disruptions cause cisplatin sensitivity or resistance overlap with those whose disruptions lead to increased or decreased cell growth, respectively. Protein translation, mitochondrial respiratory chain complex assembly, signal recognition particle-dependent cotranslational protein targeting to membrane, and mRNA catabolic processes are the top biologic processes responsible for cisplatin sensitivity. In contrast, proteasome-mediated ubiquitin-dependent protein catabolic process, negative regulations of cellular catabolic process, and regulation of cellular protein localization are the top biologic processes responsible for cisplatin resistance. ZNRF3, a ubiquitin ligase known to be a target and negative feedback regulator of Wnt-β-catenin signaling, enhances cisplatin resistance in normal and melanoma cells independently of β-catenin. Ariadne-1 homolog (ARIH1), another ubiquitin ligase, also enhances cisplatin resistance in normal and melanoma cells. By regulating ARIH1, neurofibromin 2, a tumor suppressor, enhances cisplatin resistance in melanoma but not normal cells. Our results shed new lights on cisplatin resistance mechanisms and may be useful for development of cisplatin-related treatment strategies.-Ko, T., Li, S. Genome-wide screening identifies novel genes and biological processes implicated in cisplatin resistance.},
}
@article {pmid30843540,
year = {2019},
author = {Kim, HY and Kang, SJ and Jeon, Y and An, J and Park, J and Lee, HJ and Jang, JE and Ahn, J and Bang, D and Chung, HS and Jeong, C and Ahn, DR},
title = {Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity.},
journal = {Chemical communications (Cambridge, England)},
volume = {55},
number = {24},
pages = {3552-3555},
doi = {10.1039/c8cc08468h},
pmid = {30843540},
issn = {1364-548X},
mesh = {*Base Composition ; CRISPR-Associated Protein 9/*chemistry ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*chemistry ; DNA Cleavage ; RNA, Guide/*chemistry ; },
abstract = {We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.},
}
@article {pmid30842121,
year = {2019},
author = {Zhao, L and Huang, J and Fan, Y and Li, J and You, T and He, S and Xiao, G and Chen, D},
title = {Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis.},
journal = {Annals of the rheumatic diseases},
volume = {78},
number = {5},
pages = {676-682},
pmid = {30842121},
issn = {1468-2060},
support = {R01 AR054465/AR/NIAMS NIH HHS/United States ; R01 AR055915/AR/NIAMS NIH HHS/United States ; R01 AR062136/AR/NIAMS NIH HHS/United States ; R01 AR070222/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Editing/*methods ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Mice ; Nerve Growth Factor/metabolism ; Osteoarthritis/etiology/*genetics/*therapy ; },
abstract = {OBJECTIVES: Osteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects.<br><br>METHODS: We performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1&#x3b2; and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1&#x3b2;.<br><br>RESULTS: Loss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1&#x3b2; reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1&#x3b2; significantly mitigates the adverse effects of NGF blockade on the joints.<br><br>CONCLUSIONS: CRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1&#x3b2; or IL-1&#x3b2; attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1&#x3b2; provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.},
}
@article {pmid30841961,
year = {2019},
author = {Xu, H and Luo, D and Gao, X and Liu, X and Wang, Q and Sun, A and Shen, J and He, R and Lu, G and Li, K and Zhang, J and Xiao, L},
title = {ZeoR: A Candidate for Assays Testing Enzymatic Activities and Off-Target Effects of Gene-Editing Enzymes.},
journal = {Journal of biomedical nanotechnology},
volume = {15},
number = {4},
pages = {662-673},
doi = {10.1166/jbn.2019.2720},
pmid = {30841961},
issn = {1550-7033},
mesh = {Bleomycin ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering ; },
abstract = {The target lengths of gene-editing enzymes vary from approximately 20 to 80 nucleotides. The ideal reporter genes should be able to tolerate the insertion of target sequences to differentiate the phenotypic changes between in-frame and frame shift mutations for evaluating the enzymatic activities and off-target effects of gene-editing enzymes such as zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats-associated system. This study demonstrated that the zeocin resistance gene (ZeoR) tolerated the insertion of long targets for a variety of tests using a strategy of inserting tandem repeats of NNT. Four genes such as CCR5, AR, HPV 16E7 early gene, and EGFR applications of ZeoR in quantitatively analyzing the off-targets of CRISPR were successful. The data indicated that evaluating the enzymatic activities and off-target effects with assays employing ZeoR had great potential in facilitating the application of gene editing to human gene therapy.},
}
@article {pmid30840895,
year = {2019},
author = {Mogila, I and Kazlauskiene, M and Valinskyte, S and Tamulaitiene, G and Tamulaitis, G and Siksnys, V},
title = {Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits.},
journal = {Cell reports},
volume = {26},
number = {10},
pages = {2753-2765.e4},
doi = {10.1016/j.celrep.2019.02.029},
pmid = {30840895},
issn = {2211-1247},
mesh = {Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Humans ; },
abstract = {The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action of three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). The matured StCsm complex is composed of Cas10:Csm2:Csm3:Csm4:Csm5 subunits and 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit and isolated deletion complexes to reveal the role of individual subunits in complex assembly and function. We show that the Cas10-Csm4 subcomplex binds the 5'-handle of crRNA and triggers Csm3 oligomerization to form a padlock for crRNA binding. We demonstrate that Csm5 plays a key role in target RNA binding while Csm2 ensures RNA cleavage at multiple sites by Csm3. Finally, guided by deletion analysis, we engineered a minimal Csm complex containing only the Csm3, Csm4, and Cas10 subunits and crRNA and demonstrated that it retains all three catalytic activities, thus paving the way for practical applications.},
}
@article {pmid30840660,
year = {2019},
author = {Dupays, L and Towers, N and Wood, S and David, A and Stuckey, DJ and Mohun, T},
title = {Furin, a transcriptional target of NKX2-5, has an essential role in heart development and function.},
journal = {PloS one},
volume = {14},
number = {3},
pages = {e0212992},
pmid = {30840660},
issn = {1932-6203},
support = {MR/R026416/1/MRC_/Medical Research Council/United Kingdom ; FS/15/33/31608/BHF_/British Heart Foundation/United Kingdom ; FC001117/WT_/Wellcome Trust/United Kingdom ; FC001117/CRUK_/Cancer Research UK/United Kingdom ; FC001117/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Bone Morphogenetic Proteins/metabolism ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Proliferation/genetics ; Embryo, Mammalian ; Furin/*genetics/metabolism ; *Gene Expression Regulation, Developmental ; Heart/*embryology ; Homeobox Protein Nkx-2.5/*metabolism ; Mice ; Mice, Transgenic ; Models, Animal ; Mutagenesis ; Mutation ; Myocardium/metabolism ; Myocytes, Cardiac/physiology ; Organogenesis/*genetics ; Stem Cells/physiology ; },
abstract = {The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart.},
}
@article {pmid30840598,
year = {2019},
author = {Schmich, F and Kuipers, J and Merdes, G and Beerenwinkel, N},
title = {netprioR: a probabilistic model for integrative hit prioritisation of genetic screens.},
journal = {Statistical applications in genetics and molecular biology},
volume = {18},
number = {3},
pages = {},
doi = {10.1515/sagmb-2018-0033},
pmid = {30840598},
issn = {1544-6115},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Computational Biology/*statistics &amp; numerical data ; Drosophila melanogaster/genetics ; Genetic Testing/*statistics &amp; numerical data ; *Models, Statistical ; Phenotype ; Receptors, Notch/genetics ; },
abstract = {In the post-genomic era of big data in biology, computational approaches to integrate multiple heterogeneous data sets become increasingly important. Despite the availability of large amounts of omics data, the prioritisation of genes relevant for a specific functional pathway based on genetic screening experiments, remains a challenging task. Here, we introduce netprioR, a probabilistic generative model for semi-supervised integrative prioritisation of hit genes. The model integrates multiple network data sets representing gene-gene similarities and prior knowledge about gene functions from the literature with gene-based covariates, such as phenotypes measured in genetic perturbation screens, for example, by RNA interference or CRISPR/Cas9. We evaluate netprioR on simulated data and show that the model outperforms current state-of-the-art methods in many scenarios and is on par otherwise. In an application to real biological data, we integrate 22 network data sets, 1784 prior knowledge class labels and 3840 RNA interference phenotypes in order to prioritise novel regulators of Notch signalling in Drosophila melanogaster. The biological relevance of our predictions is evaluated using in silico and in vivo experiments. An efficient implementation of netprioR is available as an R package at http://bioconductor.org/packages/netprioR.},
}
@article {pmid30839150,
year = {2019},
author = {Okada, A and Arndell, T and Borisjuk, N and Sharma, N and Watson-Haigh, NS and Tucker, EJ and Baumann, U and Langridge, P and Whitford, R},
title = {CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of male-sterile hexaploid wheat lines for use in hybrid seed production.},
journal = {Plant biotechnology journal},
volume = {17},
number = {10},
pages = {1905-1913},
pmid = {30839150},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Frameshift Mutation ; Gene Knockout Techniques ; Genes, Plant ; *Plant Infertility ; Polyploidy ; *Seeds ; Triticum/*genetics ; },
abstract = {The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male-sterile lines were potentially non-transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.},
}
@article {pmid30838976,
year = {2019},
author = {Wegner, M and Diehl, V and Bittl, V and de Bruyn, R and Wiechmann, S and Matthess, Y and Hebel, M and Hayes, MG and Schaubeck, S and Benner, C and Heinz, S and Bremm, A and Dikic, I and Ernst, A and Kaulich, M},
title = {Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30838976},
issn = {2050-084X},
support = {IIIL5-518/17.004//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst/International ; EXC115/2//Deutsche Forschungsgemeinschaft/International ; IIIL5-519/03/03.001//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst/International ; EXC147/2//Deutsche Forschungsgemeinschaft/International ; },
mesh = {Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; Gene Targeting/*methods ; Humans ; *Mutagenesis ; RNA, Guide/genetics/*metabolism ; },
abstract = {Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.},
}
@article {pmid30838870,
year = {2019},
author = {Wu, M and Feng, Y and Ye, GX and Han, YC and Wang, SS and Ni, HF and Wang, FM and Gao, M and Lv, LL and Liu, BC},
title = {Calcium-sensing receptor activation attenuates collagen expression in renal proximal tubular epithelial cells.},
journal = {American journal of physiology. Renal physiology},
volume = {316},
number = {5},
pages = {F1006-F1015},
doi = {10.1152/ajprenal.00413.2018},
pmid = {30838870},
issn = {1522-1466},
mesh = {Adenine ; Animals ; Benzamides/pharmacology ; CRISPR-Cas Systems ; Calcimimetic Agents/*pharmacology ; Cells, Cultured ; Cinacalcet/*pharmacology ; Collagen/*metabolism ; Cyclohexylamines/pharmacology ; Disease Models, Animal ; Down-Regulation ; Epithelial Cells/*drug effects/metabolism/pathology ; Fibrosis ; Humans ; Kidney Diseases/chemically induced/metabolism/pathology/*prevention &amp; control ; Kidney Tubules, Proximal/*drug effects/metabolism/pathology ; Male ; Phosphorylation ; Rats, Wistar ; Receptors, Calcium-Sensing/*agonists/genetics/metabolism ; Smad2 Protein/metabolism ; Snail Family Transcription Factors/metabolism ; Transforming Growth Factor beta1/pharmacology ; },
abstract = {316: F1006-F1015, 2019. First published March 6, 2019; doi: 10.1152/ajprenal.00413.2018 .-Experimental studies have shown that pharmacological activation of calcium-sensing receptor (CaSR) attenuates renal fibrosis in some animal models beyond modification of bone and mineral homeostasis; however, its underlying mechanisms remain largely unknown. Since excessive collagen deposition is the key feature of fibrosis, the present study aimed to examine whether CaSR was involved in the regulation of collagen expression in rats with adenine diet-induced renal fibrosis and in profibrotic transforming growth factor (TGF)-β1-treated renal proximal tubular epithelial cells (PTECs). The results showed that the CaSR agonist cinacalcet significantly attenuated renal collagen accumulation and tubular injury in adenine diet-fed rats. Additionally, the in vitro experiment showed that profibrotic TGF-β1 significantly increased the expression of collagen and decreased CaSR expression at the mRNA and protein levels in a concentration- and time-dependent manner. Furthermore, the CaSR CRISPR activation plasmid and cinacalcet partially abrogated the upregulation of collagen induced by TGF-β1 treatment. Blockade of CaSR by the CRISPR/Cas9 KO plasmid or the pharmacological antagonist Calhex231 further enhanced TGF-β1-induced collagen expression. Mechanistic experiments found that Smad2 phosphorylation and Snail expression were markedly increased in PTECs treated with TGF-β1, whereas the CaSR CRISPR activation plasmid and cinacalcet substantially suppressed this induction. In summary, this study provides evidence for a direct renal tubular epithelial protective effect of CaSR activation in renal fibrosis, possibly through suppression of collagen expression in PTECs.},
}
@article {pmid30838409,
year = {2019},
author = {Sarno, A and Lundbæk, M and Liabakk, NB and Aas, PA and Mjelle, R and Hagen, L and Sousa, MML and Krokan, HE and Kavli, B},
title = {Uracil-DNA glycosylase UNG1 isoform variant supports class switch recombination and repairs nuclear genomic uracil.},
journal = {Nucleic acids research},
volume = {47},
number = {9},
pages = {4569-4585},
pmid = {30838409},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Nucleus/genetics ; DNA Glycosylases/*genetics ; DNA Repair/*genetics ; DNA Replication/genetics ; DNA, Single-Stranded/genetics ; Gene Knockout Techniques ; Genome/genetics ; Humans ; Immunoglobulin Class Switching/*genetics ; Mice ; Proliferating Cell Nuclear Antigen/genetics ; Protein Isoforms/genetics ; Recombination, Genetic/*genetics ; Uracil/metabolism ; Uracil-DNA Glycosidase/*genetics ; },
abstract = {UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.},
}
@article {pmid30837996,
year = {2019},
author = {Han, P and Dai, Q and Fan, L and Lin, H and Zhang, X and Li, F and Yang, X},
title = {Genome-Wide CRISPR Screening Identifies JAK1 Deficiency as a Mechanism of T-Cell Resistance.},
journal = {Frontiers in immunology},
volume = {10},
number = {},
pages = {251},
pmid = {30837996},
issn = {1664-3224},
mesh = {Adoptive Transfer/methods ; Animals ; Antigen Presentation/immunology ; CRISPR-Cas Systems/*immunology ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; Gene Editing/methods ; Genome/*immunology ; Genome-Wide Association Study/methods ; Janus Kinase 1/*deficiency/immunology ; Melanoma/immunology ; Melanoma, Experimental/immunology ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes/*immunology ; },
abstract = {Somatic gene mutations play a critical role in immune evasion by tumors. However, there is limited information on genes that confer immunotherapy resistance in melanoma. To answer this question, we established a whole-genome knockout B16/ovalbumin cell line by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease technology, and determined by in vivo adoptive OT-I T-cell transfer and an in vitro OT-I T-cell-killing assay that Janus kinase (JAK)1 deficiency mediates T-cell resistance via a two-step mechanism. Loss of JAK1 reduced JAK-Signal transducer and activator of transcription signaling in tumor cells-resulting in tumor resistance to the T-cell effector molecule interferon-and suppressed T-cell activation by impairing antigen presentation. These findings provide a novel method for exploring immunotherapy resistance in cancer and identify JAK1 as potential therapeutic target for melanoma treatment.},
}
@article {pmid30837630,
year = {2019},
author = {Cal, L and Suarez-Bregua, P and Comesaña, P and Owen, J and Braasch, I and Kelsh, R and Cerdá-Reverter, JM and Rotllant, J},
title = {Countershading in zebrafish results from an Asip1 controlled dorsoventral gradient of pigment cell differentiation.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3449},
pmid = {30837630},
issn = {2045-2322},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Agouti Signaling Protein/*genetics ; Amino Acid Sequence ; Animals ; Body Patterning/*genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Targeting ; Genetic Loci ; Loss of Function Mutation ; Phenotype ; Pigmentation/*genetics ; Zebrafish/*genetics ; },
abstract = {Dorso-ventral (DV) countershading is a highly-conserved pigmentary adaptation in vertebrates. In mammals, spatially regulated expression of agouti-signaling protein (ASIP) generates the difference in shading by driving a switch between the production of chemically-distinct melanins in melanocytes in dorsal and ventral regions. In contrast, fish countershading seemed to result from a patterned DV distribution of differently-coloured cell-types (chromatophores). Despite the cellular differences in the basis for counter-shading, previous observations suggested that Agouti signaling likely played a role in this patterning process in fish. To test the hypotheses that Agouti regulated counter-shading in fish, and that this depended upon spatial regulation of the numbers of each chromatophore type, we engineered asip1 homozygous knockout mutant zebrafish. We show that loss-of-function asip1 mutants lose DV countershading, and that this results from changed numbers of multiple pigment cell-types in the skin and on scales. Our findings identify asip1 as key in the establishment of DV countershading in fish, but show that the cellular mechanism for translating a conserved signaling gradient into a conserved pigmentary phenotype has been radically altered in the course of evolution.},
}
@article {pmid30837594,
year = {2019},
author = {Rezza, A and Jacquet, C and Le Pillouer, A and Lafarguette, F and Ruptier, C and Billandon, M and Isnard Petit, P and Trouttet, S and Thiam, K and Fraichard, A and Chérifi, Y},
title = {Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3486},
pmid = {30837594},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Knock-In Techniques/*methods ; Gene Rearrangement/*genetics ; Genetic Loci ; Genotype ; Homologous Recombination ; Male ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/cytology/metabolism ; },
abstract = {The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.},
}
@article {pmid30837529,
year = {2019},
author = {Slesarev, A and Viswanathan, L and Tang, Y and Borgschulte, T and Achtien, K and Razafsky, D and Onions, D and Chang, A and Cote, C},
title = {CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3587},
pmid = {30837529},
issn = {2045-2322},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Chromosome Mapping ; Cricetinae ; Cricetulus ; *Gene Editing ; *Genome ; High-Throughput Nucleotide Sequencing/*methods ; Mammals/*genetics ; },
abstract = {The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.},
}
@article {pmid30837474,
year = {2019},
author = {Zhang, Y and Wang, J and Wang, Z and Zhang, Y and Shi, S and Nielsen, J and Liu, Z},
title = {A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1053},
pmid = {30837474},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; RNA, Guide/*genetics ; RNA, Transfer/*genetics ; Saccharomyces cerevisiae/*genetics ; Sequence Deletion ; Synthetic Biology/methods ; Time Factors ; },
abstract = {With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.},
}
@article {pmid30837341,
year = {2019},
author = {Nasko, DJ and Ferrell, BD and Moore, RM and Bhavsar, JD and Polson, SW and Wommack, KE},
title = {CRISPR Spacers Indicate Preferential Matching of Specific Virioplankton Genes.},
journal = {mBio},
volume = {10},
number = {2},
pages = {},
pmid = {30837341},
issn = {2150-7511},
support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*genetics/*virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial/chemistry/*genetics ; *Genes, Viral ; *Metagenome ; Sequence Homology ; *Water Microbiology ; },
abstract = {Viral infection exerts selection pressure on marine microbes, as virus-induced cell lysis causes 20 to 50% of cell mortality, resulting in fluxes of biomass into oceanic dissolved organic matter. Archaeal and bacterial populations can defend against viral infection using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system, which relies on specific matching between a spacer sequence and a viral gene. If a CRISPR spacer match to any gene within a viral genome is equally effective in preventing lysis, no viral genes should be preferentially matched by CRISPR spacers. However, if there are differences in effectiveness, certain viral genes may demonstrate a greater frequency of CRISPR spacer matches. Indeed, homology search analyses of bacterioplankton CRISPR spacer sequences against virioplankton sequences revealed preferential matching of replication proteins, nucleic acid binding proteins, and viral structural proteins. Positive selection pressure for effective viral defense is one parsimonious explanation for these observations. CRISPR spacers from virioplankton metagenomes preferentially matched methyltransferase and phage integrase genes within virioplankton sequences. These virioplankton CRISPR spacers may assist infected host cells in defending against competing phage. Analyses also revealed that half of the spacer-matched viral genes were unknown, some genes matched several spacers, and some spacers matched multiple genes, a many-to-many relationship. Thus, CRISPR spacer matching may be an evolutionary algorithm, agnostically identifying those genes under stringent selection pressure for sustaining viral infection and lysis. Investigating this subset of viral genes could reveal those genetic mechanisms essential to virus-host interactions and provide new technologies for optimizing CRISPR defense in beneficial microbes.IMPORTANCE The CRISPR-Cas system is one means by which bacterial and archaeal populations defend against viral infection which causes 20 to 50% of cell mortality in the ocean. We tested the hypothesis that certain viral genes are preferentially targeted for the initial attack of the CRISPR-Cas system on a viral genome. Using CASC, a pipeline for CRISPR spacer discovery, and metagenome data from oceanic microbes and viruses, we found a clear subset of viral genes with high match frequencies to CRISPR spacers. Moreover, we observed a many-to-many relationship of spacers and viral genes. These high-match viral genes were involved in nucleotide metabolism, DNA methylation, and viral structure. It is possible that CRISPR spacer matching is an evolutionary algorithm pointing to those viral genes most important to sustaining infection and lysis. Studying these genes may advance the understanding of virus-host interactions in nature and provide new technologies for leveraging CRISPR-Cas systems in beneficial microbes.},
}
@article {pmid30836137,
year = {2019},
author = {Schatoff, EM and Zafra, MP and Dow, LE},
title = {Base editing the mammalian genome.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {100-108},
pmid = {30836137},
issn = {1095-9130},
support = {T32 CA203702/CA/NCI NIH HHS/United States ; K22 CA181280/CA/NCI NIH HHS/United States ; F31 CA224800/CA/NCI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; Cell Line ; DNA/genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; },
abstract = {Base editing is a powerful technology that enables programmable conversion of single nucleotides in the mammalian genome. Base editors consist of a partially active Cas9 nuclease (Cas9[D10A]) tethered to a natural or synthetic DNA modifying enzyme. Though only recently described, BE has already shown enormous potential for basic and translational research, allowing the creation or repair of disease alleles in a variety of cell types and model organisms. In the past 2 years, a vast array of new and modified base editor variants have been described, expanding the flexibility and usefulness of the approach. Though simple in concept, effective implementation of base editing requires an understanding of the advantages and limitations of each of these tools. Here, we provide an overview of the concepts of DNA base editing, and discuss the recent progress toward the development of optimized base editing systems for mammalian cells. In addition, we highlight key technical aspects of designing and executing BE experiments, and provide detailed experimental examples of successful base editing in cell lines and organoids to help guide the effective use of these tools for genome modification.},
}
@article {pmid30835740,
year = {2019},
author = {Hammouda, OT and Böttger, F and Wittbrodt, J and Thumberger, T},
title = {Swift Large-scale Examination of Directed Genome Editing.},
journal = {PloS one},
volume = {14},
number = {3},
pages = {e0213317},
pmid = {30835740},
issn = {1932-6203},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/cytology/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genome ; Oryzias/*genetics ; Zebrafish/*genetics ; },
abstract = {In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.},
}
@article {pmid30835493,
year = {2019},
author = {Chen, K and Wang, Y and Zhang, R and Zhang, H and Gao, C},
title = {CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture.},
journal = {Annual review of plant biology},
volume = {70},
number = {},
pages = {667-697},
doi = {10.1146/annurev-arplant-050718-100049},
pmid = {30835493},
issn = {1545-2123},
mesh = {Breeding ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; Plant Breeding ; },
abstract = {Enhanced agricultural production through innovative breeding technology is urgently needed to increase access to nutritious foods worldwide. Recent advances in CRISPR/Cas genome editing enable efficient targeted modification in most crops, thus promising to accelerate crop improvement. Here, we review advances in CRISPR/Cas9 and its variants and examine their applications in plant genome editing and related manipulations. We highlight base-editing tools that enable targeted nucleotide substitutions and describe the various delivery systems, particularly DNA-free methods, that have linked genome editing with crop breeding. We summarize the applications of genome editing for trait improvement, development of techniques for fine-tuning gene regulation, strategies for breeding virus resistance, and the use of high-throughput mutant libraries. We outline future perspectives for genome editing in plant synthetic biology and domestication, advances in delivery systems, editing specificity, homology-directed repair, and gene drives. Finally, we discuss the challenges and opportunities for precision plant breeding and its bright future in agriculture.},
}
@article {pmid30834930,
year = {2019},
author = {van Sluijs, L and van Houte, S and van der Oost, J and Brouns, SJ and Buckling, A and Westra, ER},
title = {Addiction systems antagonize bacterial adaptive immunity.},
journal = {FEMS microbiology letters},
volume = {366},
number = {5},
pages = {},
pmid = {30834930},
issn = {1574-6968},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics/*physiology ; Drug Resistance, Bacterial/genetics ; Escherichia coli/drug effects/genetics/growth &amp; development/*physiology ; Gene Transfer, Horizontal ; Plasmids/genetics ; Toxin-Antitoxin Systems/genetics/*physiology ; },
abstract = {CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-anti-toxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.},
}
@article {pmid30834641,
year = {2019},
author = {Manna, D and Maji, B and Gangopadhyay, SA and Cox, KJ and Zhou, Q and Law, BK and Mazitschek, R and Choudhary, A},
title = {A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR-Cas9.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {19},
pages = {6285-6289},
pmid = {30834641},
issn = {1521-3773},
support = {R21 AI126239/AI/NIAID NIH HHS/United States ; W911NF1610586//Army Research Office Award/International ; N66001-17-2-4055//Defense Advanced Research Projects Agency/International ; },
mesh = {Binding Sites ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Crystallography, X-Ray ; Gene Editing/*methods ; Humans ; Protein Engineering ; Protein Structure, Tertiary ; Tetrahydrofolate Dehydrogenase/genetics/metabolism ; Transcriptional Activation/drug effects ; Trimethoprim/chemistry/metabolism/pharmacology ; Ultraviolet Rays ; },
abstract = {Several genome engineering applications of CRISPR-Cas9, an RNA-guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small-molecule systems lack wide dynamic range, have background activity in the absence of the small-molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high-intensity light. We previously reported a small-molecule-controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next-generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low-intensity light while rendering the small-molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.},
}
@article {pmid30834637,
year = {2019},
author = {Li, R and Qiu, Z and Wang, X and Gong, P and Xu, Q and Yu, QB and Guan, Y},
title = {Pooled CRISPR/Cas9 reveals redundant roles of plastidial phosphoglycerate kinases in carbon fixation and metabolism.},
journal = {The Plant journal : for cell and molecular biology},
volume = {98},
number = {6},
pages = {1078-1089},
doi = {10.1111/tpj.14303},
pmid = {30834637},
issn = {1365-313X},
mesh = {Arabidopsis/*enzymology/genetics/physiology ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Carbon/*metabolism ; *Carbon Cycle ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosol/metabolism ; Glycolipids/*metabolism ; Glycolysis ; Isoenzymes ; Mutation ; Phenotype ; Phosphoglycerate Kinase/genetics/*metabolism ; Photosynthesis ; Plastids/enzymology ; },
abstract = {Phosphoglycerate kinase (PGK) is a highly conserved reversible enzyme that participates in both glycolysis and photosynthesis. In Arabidopsis thaliana, one cytosolic PGK (PGKc) and two plastidial PGKs (PGKp) are known. It remains debatable whether the two PGKp isozymes are functionally redundant or specialized in plastidial carbon metabolism and fixation. Here, using a pooled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy, we found that plants with single mutations in pgkp1 or pgkp2 were not significantly affected, whereas a pgkp1pgkp2 double mutation was lethal due to retarded carbon fixation, suggesting that PGKp isozymes play redundant functional roles. Metabolomic analysis demonstrated that the sugar-deficient pgkp1pgkp2 double mutation was partially complemented by exogenous sugar, although respiration intermediates were not rescued. Chloroplast development was defective in pgkp1pgkp2, due to a deficiency in glycolysis-dependent galactoglycerolipid biosynthesis. Ectopic expression of a plastid targeting PGKc did not reverse the pgkp1pgkp2 double-mutant phenotypes. Therefore, PGKp1 and PGKp2 play redundant roles in carbon fixation and metabolism, whereas the molecular function of PGKc is more divergent. Our study demonstrated the functional conservation and divergence of glycolytic enzymes.},
}
@article {pmid30833776,
year = {2019},
author = {Kelliher, T and Starr, D and Su, X and Tang, G and Chen, Z and Carter, J and Wittich, PE and Dong, S and Green, J and Burch, E and McCuiston, J and Gu, W and Sun, Y and Strebe, T and Roberts, J and Bate, NJ and Que, Q},
title = {One-step genome editing of elite crop germplasm during haploid induction.},
journal = {Nature biotechnology},
volume = {37},
number = {3},
pages = {287-292},
pmid = {30833776},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; Cytoplasm/genetics ; Gene Editing ; Genome, Plant ; Haploidy ; Plants, Genetically Modified/*genetics/growth &amp; development ; Seeds/*genetics ; Triticum/genetics/growth &amp; development ; Zea mays/*genetics/growth &amp; development ; },
abstract = {Genome editing using CRISPR-Cas9 works efficiently in plant cells[1], but delivery of genome-editing machinery into the vast majority of crop varieties is not possible using established methods[2]. We co-opted the aberrant reproductive process of haploid induction (HI)[3-6] to induce edits in nascent seeds of diverse monocot and dicot species. Our method, named HI-Edit, enables direct genomic modification of commercial crop varieties. HI-Edit was tested in field and sweet corn using a native haploid-inducer line[4] and extended to dicots using an engineered CENH3 HI system[7]. We also recovered edited wheat embryos using Cas9 delivered by maize pollen. Our data indicate that a transient hybrid state precedes uniparental chromosome elimination in maize HI. Edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery. Therefore, edited plants could be used in trait testing and directly integrated into commercial variety development.},
}
@article {pmid30833770,
year = {2019},
author = {Hodgson, J},
title = {CRISPR target prediction remains blunt tool for clinical applications.},
journal = {Nature biotechnology},
volume = {37},
number = {3},
pages = {204-205},
doi = {10.1038/s41587-019-0057-7},
pmid = {30833770},
issn = {1546-1696},
mesh = {Biomedical Research/*trends ; CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Humans ; },
}
@article {pmid30833766,
year = {2019},
author = {},
title = {Vertex ramps up CRISPR repair.},
journal = {Nature biotechnology},
volume = {37},
number = {3},
pages = {205},
doi = {10.1038/s41587-019-0061-y},
pmid = {30833766},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/drug effects ; DNA-Activated Protein Kinase/antagonists &amp; inhibitors/*genetics ; Humans ; Neoplasms/*drug therapy ; Protein Kinase Inhibitors/*therapeutic use ; },
}
@article {pmid30833733,
year = {2019},
author = {Fu, BXH and Smith, JD and Fuchs, RT and Mabuchi, M and Curcuru, J and Robb, GB and Fire, AZ},
title = {Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9.},
journal = {Nature microbiology},
volume = {4},
number = {5},
pages = {888-897},
pmid = {30833733},
issn = {2058-5276},
support = {R01 GM037706/GM/NIGMS NIH HHS/United States ; R35 GM130366/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Deoxyribonuclease I/genetics/*metabolism ; Endonucleases/genetics/*metabolism ; Gene Targeting ; RNA, Guide/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.},
}
@article {pmid30831220,
year = {2019},
author = {Xu, J and Yu, Y and Chen, K and Huang, Y},
title = {Intersex regulates female external genital and imaginal disc development in the silkworm.},
journal = {Insect biochemistry and molecular biology},
volume = {108},
number = {},
pages = {1-8},
doi = {10.1016/j.ibmb.2019.02.003},
pmid = {30831220},
issn = {1879-0240},
mesh = {Animals ; Bombyx/*genetics/*growth &amp; development ; CRISPR-Cas Systems ; Female ; Gene Expression Regulation, Developmental ; Genitalia, Female/growth &amp; development ; Imaginal Discs/*growth &amp; development ; Male ; Mutation ; Signal Transduction ; },
abstract = {As a component of the mediator complex, the intersex (ix) gene product is involved in the sex determination pathway of the Drosophila melanogaster. IX functions together with the female-specific product of doublesex (dsx) at the bottom of the hierarchy to implement female sexual differentiation. Here we analyzed the functions of the ix gene in the model lepidopteran insect Bombyx mori. We found that Bmix is expressed in many tissues and is highly expressed in early pupal stages. We used the transgene-based CRISPR/Cas9 system to generate mutants of the Bmix gene. The Bmix female mutants were sterile and had irregular external genitalia, whereas in the mutant males external genitalia were normal. Mutants of both sexes had normal gonad development and normal splicing of the Bmdsx pre-mRNA, suggesting that Bmix functions independently of Bmdsx. Interestingly, both male and female mutants had defective development of the imaginal disc including wing, antenna, and leg. RNA-seq and gene expression analyses indicated that genes involved in WNT, Hippo, and Hedgehog signaling pathways and wing development genes Bmawd and Bmfng were up-regulated or down-regulated in the Bmix mutants compared with wild-type animals. Our data provide insights into the multiple functions of Bmix in female external genital and imaginal disc development in the silkworm.},
}
@article {pmid30831133,
year = {2019},
author = {Takashima, S and Shinkuma, S and Fujita, Y and Nomura, T and Ujiie, H and Natsuga, K and Iwata, H and Nakamura, H and Vorobyev, A and Abe, R and Shimizu, H},
title = {Efficient Gene Reframing Therapy for Recessive Dystrophic Epidermolysis Bullosa with CRISPR/Cas9.},
journal = {The Journal of investigative dermatology},
volume = {139},
number = {8},
pages = {1711-1721.e4},
doi = {10.1016/j.jid.2019.02.015},
pmid = {30831133},
issn = {1523-1747},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Collagen Type VII/chemistry/*genetics ; *DNA End-Joining Repair ; Epidermolysis Bullosa Dystrophica/genetics/*therapy ; Exons/genetics ; Fibroblasts/transplantation ; Frameshift Mutation ; Gene Editing/methods ; Genes, Recessive/genetics ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Infant ; Mice ; Mutagenesis, Site-Directed ; Mutation ; Primary Cell Culture ; Protein Conformation, alpha-Helical/genetics ; Recombinant Proteins/chemistry/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination or non-homologous end-joining pathways. The non-homologous end-joining pathway, which is activated more frequently than homologous recombination, is prone to introducing small insertions and/or deletions at the double-strand break site, leading to changes in the reading frame. We hypothesized that the non-homologous end-joining pathway is applicable to genetic diseases caused by a frameshift mutation through restoration of the reading frame. Recessive dystrophic epidermolysis bullosa is a hereditary skin disorder caused by mutations in COL7A1. In this study, we applied gene reframing therapy to a recurrent frameshift mutation, c.5819delC, in COL7A1, which results in a premature termination codon. CRISPR/Cas9 targeting this specific mutation site was delivered to recessive dystrophic epidermolysis bullosa patient fibroblasts. After genotyping a large collection of gene-edited fibroblast clones, we identified a significant number (17/50) of clones in which the frameshift in COL7A1 was restored. The reframed COL7 was functional, as shown by triple-helix formation assay in vitro, and was correctly distributed in the basement membrane zone in mice. Our data suggest that mutation site-specific non-homologous end-joining might be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.},
}
@article {pmid30830592,
year = {2020},
author = {Isa, NM and Zulkifli, NA and Man, S},
title = {Islamic Perspectives on CRISPR/Cas9-Mediated Human Germline Gene Editing: A Preliminary Discussion.},
journal = {Science and engineering ethics},
volume = {26},
number = {1},
pages = {309-323},
pmid = {30830592},
issn = {1471-5546},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics ; Genetic Enhancement/ethics ; Germ Cells ; Humans ; *Islam ; Moral Status ; Religion and Science ; Respect ; Value of Life ; },
abstract = {The recent development of CRISPR/Cas9 technology has rekindled the ethical debate concerning human germline modification that has begun decades ago. This inexpensive technology shows tremendous promise in disease prevention strategies, while raising complex ethical concerns about safety and efficacy of the technology, human dignity, tampering with God's creation, and human genetic enhancement. Germline gene editing may result in heritable changes in the human genome, therefore the question of whether it should be allowed requires deep and careful discussion from various perspectives. This paper explores Islamic perspectives on the concerns raised and highlights the ethical principles in Islam that should be taken into consideration when assessing the permissibility of CRISPR/ Cas9-mediated human germline gene editing. As argued in this paper, human germline gene editing would be considered lawful for medical purpose under certain conditions. It should not be applied on humans until the safety and efficacy issues are resolved. Robust ethical guidelines and strict regulations are necessary to preserve human dignity and to prevent premature and misuse of the technology. Maqasid al-shariah's principles of preservation of human life, lineage, and dignity and 'preventing harm takes precedence over securing benefit' are among the guiding principles in assessing the permissibility of CRISPR/Cas9-mediated human germline editing from an Islamic perspective. Further discussions are important to address the controversies as well as to explore the related ethical principles.},
}
@article {pmid30830215,
year = {2019},
author = {Franca, MM and Han, X and Funari, MFA and Lerario, AM and Nishi, MY and Fontenele, EGP and Domenice, S and Jorge, AAL and Garcia-Galiano, D and Elias, CF and Mendonca, BB},
title = {Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.},
journal = {The Journal of clinical endocrinology and metabolism},
volume = {104},
number = {7},
pages = {2827-2841},
pmid = {30830215},
issn = {1945-7197},
support = {R01 HD096324/HD/NICHD NIH HHS/United States ; P30 CA046592/CA/NCI NIH HHS/United States ; P30 DK034933/DK/NIDDK NIH HHS/United States ; R01 HD069702/HD/NICHD NIH HHS/United States ; R21 HD090567/HD/NICHD NIH HHS/United States ; },
mesh = {Adolescent ; Animals ; CRISPR-Cas Systems ; Child ; Female ; Forkhead Box Protein O3/metabolism ; Gene Knockout Techniques ; Heterozygote ; Homozygote ; Humans ; Infertility/genetics ; Litter Size ; Loss of Function Mutation ; Male ; Mice ; Mutation, Missense ; Ovary/metabolism ; Primary Ovarian Insufficiency/*genetics ; RNA Polymerase III/*genetics ; Sexual Maturation/genetics ; Time-to-Pregnancy ; Whole Exome Sequencing ; },
abstract = {CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI.<br><br>OBJECTIVE: To investigate the genetic etiology of idiopathic POI.<br><br>PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology.<br><br>RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles.<br><br>CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.},
}
@article {pmid30828340,
year = {2019},
author = {Muñoz, IV and Sarrocco, S and Malfatti, L and Baroncelli, R and Vannacci, G},
title = {CRISPR-Cas for Fungal Genome Editing: A New Tool for the Management of Plant Diseases.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {135},
pmid = {30828340},
issn = {1664-462X},
}
@article {pmid30828060,
year = {2019},
author = {Çiçek, YA and Luther, DC and Kretzmann, JA and Rotello, VM},
title = {Advances in CRISPR/Cas9 Technology for in Vivo Translation.},
journal = {Biological &amp; pharmaceutical bulletin},
volume = {42},
number = {3},
pages = {304-311},
doi = {10.1248/bpb.b18-00811},
pmid = {30828060},
issn = {1347-5215},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy/*methods ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized therapeutic gene editing by providing researchers with a new method to study and cure diseases previously considered untreatable. While the full range and power of CRISPR technology for therapeutics is being elucidated through in vitro studies, translation to in vivo studies is slow. To date there is no totally effective delivery strategy to carry CRISPR components to the target site in vivo. The complexity of in vivo delivery is furthered by the number of potential delivery methods, the different forms in which CRISPR can be delivered as a therapeutic, and the disease target and tissue type in question. There are major challenges and limitations to delivery strategies, and it is imperative that future directions are guided by well-conducted studies that consider the full effect these variables have on the eventual outcome. In this review we will discuss the advances of the latest in vivo CRISPR/Cas9 delivery strategies and highlight the challenges yet to be overcome.},
}
@article {pmid30827888,
year = {2019},
author = {Esposito, R and Bosch, N and Lanzós, A and Polidori, T and Pulido-Quetglas, C and Johnson, R},
title = {Hacking the Cancer Genome: Profiling Therapeutically Actionable Long Non-coding RNAs Using CRISPR-Cas9 Screening.},
journal = {Cancer cell},
volume = {35},
number = {4},
pages = {545-557},
doi = {10.1016/j.ccell.2019.01.019},
pmid = {30827888},
issn = {1878-3686},
mesh = {Animals ; Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Proliferation/drug effects/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Molecular Targeted Therapy ; Neoplasms/drug therapy/*genetics/metabolism/pathology ; RNA, Long Noncoding/*genetics/metabolism ; },
abstract = {Long non-coding RNAs (lncRNAs) represent a huge reservoir of potential cancer targets. Such "onco-lncRNAs" have resisted traditional RNAi methods, but CRISPR-Cas9 genome editing now promises functional screens at high throughput and low cost. The unique biology of lncRNAs demands screening strategies distinct from protein-coding genes. The first such screens have identified hundreds of onco-lncRNAs promoting cell proliferation and drug resistance. Ongoing developments will further improve screen performance and translational relevance. This Review aims to highlight the potential of CRISPR screening technology for discovering new onco-lncRNAs, and to guide molecular oncologists wishing to apply it to their cancer of interest.},
}
@article {pmid30827034,
year = {2019},
author = {Liu, T and Wang, J and Xie, X and Wang, K and Sui, T and Liu, D and Lai, L and Zhao, H and Li, Z and Feng, JQ},
title = {DMP1 Ablation in the Rabbit Results in Mineralization Defects and Abnormalities in Haversian Canal/Osteon Microarchitecture.},
journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research},
volume = {34},
number = {6},
pages = {1115-1128},
doi = {10.1002/jbmr.3683},
pmid = {30827034},
issn = {1523-4681},
support = {R01DE025014/GF/NIH HHS/United States ; DE025659/GF/NIH HHS/United States ; 2017YFA010501//National Key Research and Development Program of China Stem Cell and Translational Research/International ; 81700980//National Natural Science Foundation of China/International ; 2017M623048//China Postdoctoral Science Foundation/International ; 2018SCU12018//Postdoctoral Research Foundation of Sichuan University/International ; },
mesh = {Animals ; Biomarkers/metabolism ; Bone Matrix/diagnostic imaging/pathology/physiopathology ; CRISPR-Cas Systems/genetics ; *Calcification, Physiologic ; Chondrogenesis ; Extracellular Matrix Proteins/*metabolism ; Familial Hypophosphatemic Rickets/blood/diagnostic imaging/physiopathology ; Femur/diagnostic imaging ; Fibroblast Growth Factor-23 ; Gait ; *Gene Deletion ; Gene Knockout Techniques ; Haversian System/*abnormalities/diagnostic imaging/*physiopathology ; Humans ; Models, Biological ; Osteogenesis ; Rabbits ; Tibia/diagnostic imaging/pathology/physiopathology ; X-Ray Microtomography ; },
abstract = {DMP1 (dentin matrix protein 1) is an extracellular matrix protein highly expressed in bones. Studies of Dmp1 knockout (KO) mice led to the discovery of a rare autosomal recessive form of hypophosphatemic rickets (ARHR) caused by DMP1 mutations. However, there are limitations for using this mouse model to study ARHR, including a lack of Haversian canals and osteons (that occurs only in large mammalian bones), high levels of fibroblast growth factor 23 (FGF23), and PTH, in comparison with a moderate elevation of FGF23 and unchanged PTH in human ARHR patients. To better understand this rare disease, we deleted the DMP1 gene in rabbit using CRISPR/Cas9. This rabbit model recapitulated many features of human ARHR, such as the rachitic rosary (expansion of the anterior rib ends at the costochondral junctions), moderately increased FGF23, and normal PTH levels, as well as severe defects in bone mineralization. Unexpectedly, all DMP1 KO rabbits died by postnatal week 8. They developed a severe bone microarchitecture defect: a major increase in the central canal areas of osteons, concurrent with massive accumulation of osteoid throughout all bone matrix (a defect in mineralization), suggesting a new paradigm, where rickets is caused by a combination of a defect in bone microarchitecture and a failure in mineralization. Furthermore, a study of DMP1 KO bones found accelerated chondrogenesis, whereas ARHR has commonly been thought to be involved in reduced chondrogenesis. Our findings with newly developed DMP1 KO rabbits suggest a revised understanding of the mechanism underlying ARHR. © 2019 American Society for Bone and Mineral Research.},
}
@article {pmid30826063,
year = {2019},
author = {Chang, Y and Geng, F and Hu, Y and Ding, Y and Zhang, R},
title = {Zebrafish cysteine and glycine-rich protein 3 is essential for mechanical stability in skeletal muscles.},
journal = {Biochemical and biophysical research communications},
volume = {511},
number = {3},
pages = {604-611},
doi = {10.1016/j.bbrc.2019.02.115},
pmid = {30826063},
issn = {1090-2104},
mesh = {Animals ; Biomechanical Phenomena ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; LIM Domain Proteins/genetics/*metabolism ; Muscle Proteins/genetics/*metabolism ; Muscle, Skeletal/growth &amp; development/*metabolism/ultrastructure ; Mutation ; Stress, Mechanical ; Zebrafish/genetics/growth &amp; development/*metabolism ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Cysteine and glycine-rich protein 3 (CSRP3) is a striated muscle-specific cytoskeleton protein which participates in cardiac stretch sensing. Mutations in CSRP3 gene cause cardiomyopathies and deregulation of CSRP3 has been found in patients with heart failure and several skeletal muscle diseases. However, the mechanism underneath these disorders still remains poorly understood. Here we generated the first csrp3 knockout zebrafish. csrp3[-/-] embryos showed no gross morphological defects but csrp3 deficient skeletal muscle fibers were prone to lesions upon prolonged stretching force. Further studies revealed csrp3 cooperatively interacted with ilk to maintain skeletal muscle mechanical stability and regulated tcap activation. Thus, our work has established a zebrafish model to investigate the function of csrp3 gene, and provides novel insights towards how csrp3 defects may lead to skeletal myopathies by a mechanistic link between Csrp3 and force stimuli.},
}
@article {pmid30825514,
year = {2019},
author = {Kun, RS and Gomes, ACS and Hildén, KS and Salazar Cerezo, S and Mäkelä, MR and de Vries, RP},
title = {Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation.},
journal = {Biotechnology advances},
volume = {37},
number = {6},
pages = {107361},
doi = {10.1016/j.biotechadv.2019.02.017},
pmid = {30825514},
issn = {1873-1899},
mesh = {*Biomass ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Engineering ; },
abstract = {Fungal strain engineering is commonly used in many areas of biotechnology, including the production of plant biomass degrading enzymes. Its aim varies from the production of specific enzymes to overall increased enzyme production levels and modification of the composition of the enzyme set that is produced by the fungus. Strain engineering involves a diverse range of methodologies, including classical mutagenesis, genetic engineering and genome editing. In this review, the main approaches for strain engineering of filamentous fungi in the field of plant biomass degradation will be discussed, including recent and not yet implemented methods, such as CRISPR/Cas9 genome editing and adaptive evolution.},
}
@article {pmid30824914,
year = {2019},
author = {Smits, AM},
title = {Therapeutic gene editing, making a point.},
journal = {Cardiovascular research},
volume = {115},
number = {4},
pages = {e39-e40},
pmid = {30824914},
issn = {1755-3245},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Therapy ; },
}
@article {pmid30824690,
year = {2019},
author = {Bowler, M and Kong, D and Sun, S and Nanjundappa, R and Evans, L and Farmer, V and Holland, A and Mahjoub, MR and Sui, H and Loncarek, J},
title = {High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {993},
pmid = {30824690},
issn = {2041-1723},
support = {R01 DK108005/DK/NIDDK NIH HHS/United States ; R01 GM101026/GM/NIGMS NIH HHS/United States ; R01 GM114119/GM/NIGMS NIH HHS/United States ; R01 HL128370/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Aurora Kinase A ; CRISPR-Cas Systems ; Cell Cycle Proteins/ultrastructure ; Centrioles/*ultrastructure ; Cilia/*ultrastructure ; DNA-Binding Proteins ; Electron Microscope Tomography/*methods ; HeLa Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Mice ; Mice, Inbred C57BL ; Microscopy, Electron/*methods ; Microtubule Proteins/ultrastructure ; Microtubules/*ultrastructure ; Mitosis ; Protein Serine-Threonine Kinases ; Proto-Oncogene Proteins ; Species Specificity ; Transcription Factors ; },
abstract = {Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole's distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of distal appendage assembly. We further show that distal appendages undergo a dramatic ultra-structural reorganization before mitosis, during which they temporarily lose outer components, while inner components maintain a nine-fold organization. Finally, using electron tomography we reveal that mammalian distal appendages associate with two centriole microtubule triplets via an elaborate filamentous base and that they appear as almost radial finger-like protrusions. Our findings challenge the traditional portrayal of mammalian distal appendage as a pinwheel-like structure that is maintained throughout mitosis.},
}
@article {pmid30823430,
year = {2019},
author = {Loureiro, A and da Silva, GJ},
title = {CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {8},
number = {1},
pages = {},
pmid = {30823430},
issn = {2079-6382},
abstract = {Bacteriophages are pervasive viruses that infect bacteria, relying on their genetic machinery to replicate. In order to protect themselves from this kind of invader, bacteria developed an ingenious adaptive defence system, clustered regularly interspaced short palindromic repeats (CRISPR). Researchers soon realised that a specific type of CRISPR system, CRISPR-Cas9, could be modified into a simple and efficient genetic engineering technology, with several improvements over currently used systems. This discovery set in motion a revolution in genetics, with new and improved CRISPR systems being used in plenty of in vitro and in vivo experiments in recent years. This review illustrates the mechanisms behind CRISPR-Cas systems as a means of bacterial immunity against phage invasion and how these systems were engineered to originate new genetic manipulation tools. Newfound CRISPR-Cas technologies and the up-and-coming applications of these systems on healthcare and other fields of science are also discussed.},
}
@article {pmid30822746,
year = {2019},
author = {Lin, P and Pu, Q and Shen, G and Li, R and Guo, K and Zhou, C and Liang, H and Jiang, J and Wu, M},
title = {CdpR Inhibits CRISPR-Cas Adaptive Immunity to Lower Anti-viral Defense while Avoiding Self-Reactivity.},
journal = {iScience},
volume = {13},
number = {},
pages = {55-68},
pmid = {30822746},
issn = {2589-0042},
support = {R01 AI109317/AI/NIAID NIH HHS/United States ; R01 AI138203/AI/NIAID NIH HHS/United States ; R03 AI097532/AI/NIAID NIH HHS/United States ; R15 AI101973/AI/NIAID NIH HHS/United States ; },
abstract = {CRISPR-Cas systems as adaptive immunity in bacteria and archaea battle against bacteriophages. However, little is known how CRISPR-Cas systems are precisely regulated to effectively eliminate intruders while not inducing self-reactivity. Here, we identify intrinsic negative modulator of CRISPR-Cas that influences interference and adaptation functions. LasI/RhlI-derived autoinducers activate cas operon by enhancing the binding of virulence factor regulator (Vfr) cis-response elements to cas1 promoter, whereas CdpR represses this intracellular signaling and blocks transcription of cas operon. Importantly, inhibition of Vfr reduces cas1 expression and impairs immunization and immune memory mediated by CRISPR-Cas, leading to more severe phage infection but lower self-targeting activities. In addition, CdpR-mediated LasI/RhlI/Vfr intracellular signaling represses cleavage of bacterial endogenous sequences by impeding Cas3 RNA cleavage activity. Thus, CdpR renders important inhibitory effects on CRISPR-Cas systems to avoid possible self-reactivity but potentially heightening infection risk. Our study provides insight into fine regulation of CRISPR-Cas systems for maintaining homeostasis.},
}
@article {pmid30822570,
year = {2019},
author = {Miano, JM and Long, X and Lyu, Q},
title = {CRISPR links to long noncoding RNA function in mice: A practical approach.},
journal = {Vascular pharmacology},
volume = {114},
number = {},
pages = {1-12},
pmid = {30822570},
issn = {1879-3649},
support = {R01 HL132574/HL/NHLBI NIH HHS/United States ; R01 HL136224/HL/NHLBI NIH HHS/United States ; R01 HL138987/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression Regulation ; Humans ; Mice ; Mice, Transgenic ; RNA, Long Noncoding/*genetics/metabolism ; },
abstract = {Next generation sequencing has uncovered a trove of short noncoding RNAs (e.g., microRNAs) and long noncoding RNAs (lncRNAs) that act as molecular rheostats in the control of diverse homeostatic processes. Meanwhile, the tsunamic emergence of clustered regularly interspaced short palindromic repeats (CRISPR) editing has transformed our influence over all DNA-carrying entities, heralding global CRISPRization. This is evident in biomedical research where the ease and low-cost of CRISPR editing has made it the preferred method of manipulating the mouse genome, facilitating rapid discovery of genome function in an in vivo context. Here, CRISPR genome editing components are updated for elucidating lncRNA function in mice. Various strategies are highlighted for understanding the function of lncRNAs residing in intergenic sequence space, as host genes that harbor microRNAs or other genes, and as natural antisense, overlapping or intronic genes. Also discussed is CRISPR editing of mice carrying human lncRNAs as well as the editing of competing endogenous RNAs. The information described herein should assist labs in the rigorous design of experiments that interrogate lncRNA function in mice where complex disease processes can be modeled thus accelerating translational discovery.},
}
@article {pmid30822257,
year = {2019},
author = {Zhou, S and Chen, Y and Gong, X and Jin, J and Li, H},
title = {Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level.},
journal = {Preparative biochemistry &amp; biotechnology},
volume = {49},
number = {4},
pages = {384-390},
doi = {10.1080/10826068.2019.1573196},
pmid = {30822257},
issn = {1532-2297},
mesh = {Animals ; Bevacizumab/genetics ; CHO Cells ; CRISPR-Cas Systems ; Cell Engineering/*methods ; Cricetulus ; Gene Knock-In Techniques/*methods ; Glucagon-Like Peptide 1/analysis/genetics ; Humans ; Immunoglobulin Heavy Chains/*genetics ; Immunoglobulin Light Chains/*genetics ; Recombinant Fusion Proteins/analysis/genetics ; Serum Albumin, Human/analysis/genetics ; },
abstract = {Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006 pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2 pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5 µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17 mg/L over passage 1, 25, and 50.},
}
@article {pmid30820491,
year = {2019},
author = {Lam, TJ and Ye, Y},
title = {CRISPRs for Strain Tracking and Their Application to Microbiota Transplantation Data Analysis.},
journal = {The CRISPR journal},
volume = {2},
number = {1},
pages = {41-50},
pmid = {30820491},
issn = {2573-1599},
support = {R01 AI108888/AI/NIAID NIH HHS/United States ; R01 AI143254/AI/NIAID NIH HHS/United States ; },
mesh = {Bacterial Typing Techniques/*methods ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; DNA, Intergenic/classification/*genetics/metabolism ; Datasets as Topic ; Dysbiosis/microbiology/*therapy ; Fecal Microbiota Transplantation/*methods ; Feces/microbiology ; Gastrointestinal Microbiome/*genetics ; Humans ; Metagenome ; },
abstract = {CRISPR-Cas systems are adaptive immune systems naturally found in bacteria and archaea. Prokaryotes use these immune systems to defend against invaders, which include phages, plasmids, and other mobile genetic elements. Relying on the integration of spacers derived from invader sequences (protospacers) into CRISPR loci (forming spacers flanked by repeats), CRISPR-Cas systems are able to store the memory of past immunological encounters. While CRISPR-Cas systems have evolved in response to invading mobile genetic elements, invaders have also developed mechanisms to avoid detection. As a result of an arms race between CRISPR-Cas systems and their targets, CRISPR arrays typically undergo rapid turnover of spacers through the acquisition and loss events. Additionally, microbiomes of different individuals rarely share spacers. Here, we present a computational pipeline, CRISPRtrack, for strain tracking based on CRISPR spacer content, and we applied it to fecal transplantation microbiome data to study the retention of donor strains in recipients. Our results demonstrate the potential use of CRISPRs as a simple yet effective tool for donor-strain tracking in fecal transplantation and as a general purpose tool for quantifying microbiome similarity.},
}
@article {pmid30820046,
year = {2019},
author = {},
title = {Brave new dialogue.},
journal = {Nature genetics},
volume = {51},
number = {3},
pages = {365},
doi = {10.1038/s41588-019-0374-2},
pmid = {30820046},
issn = {1546-1718},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*legislation &amp; jurisprudence ; Genetic Engineering/*legislation &amp; jurisprudence ; Genome, Human/*genetics ; Humans ; },
}
@article {pmid30819928,
year = {2019},
author = {Zuo, E and Sun, Y and Wei, W and Yuan, T and Ying, W and Sun, H and Yuan, L and Steinmetz, LM and Li, Y and Yang, H},
title = {Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6437},
pages = {289-292},
pmid = {30819928},
issn = {1095-9203},
support = {P01 HG000205/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *Blastomeres ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Cytosine ; DNA Mutational Analysis ; Embryo, Mammalian ; Female ; Gene Editing/*methods ; Genome-Wide Association Study ; Male ; Mice ; Mice, Inbred C57BL ; Mutation ; *Polymorphism, Single Nucleotide ; },
abstract = {Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.},
}
@article {pmid30819898,
year = {2019},
author = {Massey, J and Liu, Y and Alvarenga, O and Saez, T and Schmerer, M and Warmflash, A},
title = {Synergy with TGFβ ligands switches WNT pathway dynamics from transient to sustained during human pluripotent cell differentiation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {11},
pages = {4989-4998},
pmid = {30819898},
issn = {1091-6490},
support = {R01 GM126122/GM/NIGMS NIH HHS/United States ; },
mesh = {Activins/pharmacology ; Adaptation, Biological/drug effects ; Bone Morphogenetic Protein 4/pharmacology ; CRISPR-Cas Systems/genetics ; *Cell Differentiation/drug effects ; Glycogen Synthase Kinase 3 beta/metabolism ; Green Fluorescent Proteins/metabolism ; Human Embryonic Stem Cells/cytology/drug effects/metabolism ; Humans ; Ligands ; Pluripotent Stem Cells/*cytology/drug effects/*metabolism ; Protein Stability/drug effects ; Transforming Growth Factor beta/*metabolism ; Wnt Proteins/metabolism ; *Wnt Signaling Pathway/drug effects ; beta Catenin/metabolism ; },
abstract = {WNT/β-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem cell niches in adults, and is dysregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of β-catenin, the primary transducer of canonical WNT signals, using quantitative live cell imaging. We show that β-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained on differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell lines. Signal attenuation in pluripotent cells is observed even at saturating doses, where ligand stability does not affect the dynamics. TGFβ superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the β-catenin response in a manner independent of their ability to induce new WNT ligand production. Our results reveal how variables external to the pathway, including differentiation status and cross-talk with other pathways, dramatically alter WNT/β-catenin dynamics.},
}
@article {pmid30819013,
year = {2019},
author = {Dong, Y and Peng, T and Wu, W and Tan, D and Liu, X and Xie, D},
title = {Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotides.},
journal = {The Journal of international medical research},
volume = {47},
number = {4},
pages = {1717-1730},
pmid = {30819013},
issn = {1473-2300},
mesh = {Adult ; *CRISPR-Cas Systems ; Child ; Female ; *Gene Editing ; Hearing Loss/*genetics/pathology ; Heterozygote ; *Homozygote ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Male ; Middle Aged ; *Mutation ; Oligonucleotides/administration &amp; dosage ; Pedigree ; Phenotype ; Prognosis ; Receptors, Purinergic P2X2/*genetics ; },
abstract = {BACKGROUND: Heterozygous purinergic receptor p2x gene (P2RX2) c.178G>T (p.V60L) mutations can lead to progressive hearing loss (HL) and increased susceptibility to noise. However, the underlying mechanisms remain unclear. A combination of human induced pluripotent stem cell (hiPSC) technology with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9-mediated gene editing may provide a promising tool to study gene function and treat hereditary deafness in humans.<br><br>METHODS: hiPSC technology and CRISPR/Cas9-mediated gene editing were used to generate heterozygous and homozygous P2RX2 c.178G>T (p.V60L) cell models.<br><br>RESULTS: We generated non-integrative hiPSCs from urine samples derived from three members of a large Chinese family carrying heterozygous P2RX2 c.178G>T mutations (designated P2RX2[+/-]) as a model to study P2RX2-mediated hereditary HL. Furthermore, we used CRISPR/Cas9 and single-stranded donor oligonucleotides to genetically establish homozygous P2RX2 c.178G>T hiPSCs (designated P2RX2[-/-]) from heterozygous patient-specific hiPSCs as a control to further study the pathological gene function.<br><br>CONCLUSIONS: Heterozygous and homozygous P2RX2-mutated hiPSC lines are good models to investigate the pathological mechanisms of P2RX2 mutations in HL pathogenesis. Our findings confirmed our hypothesis that it is feasible and convenient to introduce precise point mutations into genomic loci of interest to generate gene-mutated hiPSC models.},
}
@article {pmid30817647,
year = {2019},
author = {Sabol, RA and Bunnell, BA},
title = {Discussion: CRISPR/Cas9-Mediated BRCA1 Knockdown Adipose Stem Cells Promote Breast Cancer Progression.},
journal = {Plastic and reconstructive surgery},
volume = {143},
number = {3},
pages = {757-758},
doi = {10.1097/PRS.0000000000005391},
pmid = {30817647},
issn = {1529-4242},
mesh = {*Breast Neoplasms ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Stem Cells ; },
}
@article {pmid30817646,
year = {2019},
author = {Zhao, R and Kaakati, R and Liu, X and Xu, L and Lee, AK and Bachelder, R and Li, CY and Hollenbeck, ST},
title = {CRISPR/Cas9-Mediated BRCA1 Knockdown Adipose Stem Cells Promote Breast Cancer Progression.},
journal = {Plastic and reconstructive surgery},
volume = {143},
number = {3},
pages = {747-756},
pmid = {30817646},
issn = {1529-4242},
support = {R01 CA208852/CA/NCI NIH HHS/United States ; R01 CA216876/CA/NCI NIH HHS/United States ; R01 ES024015/ES/NIEHS NIH HHS/United States ; },
mesh = {Adipose Tissue/*cytology ; Adult ; BRCA1 Protein/*genetics ; Breast Neoplasms/genetics/*pathology ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Transformation, Neoplastic/*genetics/pathology ; Culture Media, Conditioned/metabolism ; Disease Progression ; Female ; Gene Knockdown Techniques ; Humans ; Interleukin-8/metabolism ; Middle Aged ; Primary Cell Culture ; Stem Cells/metabolism/*pathology ; Tumor Microenvironment/genetics ; },
abstract = {BACKGROUND: The tumor microenvironment within the breast is rich in adipose elements. The interaction between adipose cells and breast cancer is poorly understood, particularly as it pertains to patients with genetic susceptibility to breast cancer. This study focuses on the phenotype of human adipose-derived stem cells with the BRCA1 mutation and the effect they may have on breast cancer cell behavior.<br><br>METHODS: CRISPR/Cas9 was used to generate de novo BRCA1-knockdown human adipose-derived stem cells. The effect of the BRCA1 knockdown on the adipose-derived stem cell phenotype was compared to wild-type adipose-derived stem cells and patient-derived breast adipose-derived stem cells with known BRCA1 mutations. Interactions between adipose-derived stem cells and the MDA-MB-231 breast cancer cell line were evaluated.<br><br>RESULTS: BRCA1-knockdown adipose-derived stem cells stimulated MDA-MB-231 proliferation (1.4-fold increase on day 4; p = 0.0074) and invasion (2.3-fold increase on day 2; p = 0.0171) compared to wild-type cells. Immunofluorescence staining revealed higher levels of phosphorylated ataxia telangiectasia-mutated activation in BRCA1-knockdown cells (72.9 &#xb1; 5.32 percent versus 42.9 &#xb1; 4.97 percent; p = 0.0147), indicating higher levels of DNA damage. Beta-galactosidase staining demonstrated a significantly higher level of senescence in BRCA1-knockdown cells compared with wild-type cells (7.9 &#xb1; 0.25 percent versus 0.17 &#xb1; 0.17 percent; p < 0.0001). Using quantitative enzyme-linked immunosorbent assay to evaluate conditioned media, the authors found significantly higher levels of interleukin-8 in BRCA1-knockdown cells (2.57 &#xb1; 0.32-fold; p = 0.0049).<br><br>CONCLUSIONS: The authors show for the first time that the BRCA1 mutation affects the adipose-derived stem cell phenotype. Moreover, CRISPR/Cas9-generated BRCA1-knockdown adipose-derived stem cells stimulate a more aggressive behavior in breast cancer cells than wild-type adipose-derived stem cells. This appears to be related to increased inflammatory cytokine production by means of a DNA damage-mediated cell senescence pathway.},
}
@article {pmid30817136,
year = {2019},
author = {Auxillos, JY and Garcia-Ruiz, E and Jones, S and Li, T and Jiang, S and Dai, J and Cai, Y},
title = {Multiplex Genome Engineering for Optimizing Bioproduction in Saccharomyces cerevisiae.},
journal = {Biochemistry},
volume = {58},
number = {11},
pages = {1492-1500},
doi = {10.1021/acs.biochem.8b01086},
pmid = {30817136},
issn = {1520-4995},
support = {BB/M017702/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P02114X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Genetic Engineering/methods ; Metabolic Engineering/methods ; Protein Engineering/*methods ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/*biosynthesis/genetics/metabolism ; Synthetic Biology/*methods ; },
abstract = {The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.},
}
@article {pmid30817134,
year = {2019},
author = {Neukranz, Y and Kotter, A and Beilschmidt, L and Marelja, Z and Helm, M and Gräf, R and Leimkühler, S},
title = {Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.},
journal = {Biochemistry},
volume = {58},
number = {13},
pages = {1786-1798},
doi = {10.1021/acs.biochem.8b01160},
pmid = {30817134},
issn = {1520-4995},
mesh = {Aconitate Hydratase/metabolism ; CRISPR-Cas Systems ; Carbon-Sulfur Lyases/analysis/*metabolism ; Centrosome/*metabolism/ultrastructure ; Coenzymes/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Isocitrate Dehydrogenase/metabolism ; Metalloproteins/metabolism ; Molybdenum Cofactors ; Nucleotidyltransferases/analysis/genetics/*metabolism ; Pteridines/metabolism ; RNA, Transfer/metabolism ; Sulfite Oxidase/metabolism ; Sulfurtransferases/analysis/genetics/*metabolism ; },
abstract = {The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm[5]s[2]U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm[5]s[2]U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.},
}
@article {pmid30816887,
year = {2019},
author = {Graul, AI and Dulsat, C and Pina, P and Cruces, E and Tracy, M},
title = {The year's new drugs and biologics 2018: Part II - News that shaped the industry in 2018.},
journal = {Drugs of today (Barcelona, Spain : 1998)},
volume = {55},
number = {2},
pages = {131-160},
doi = {10.1358/dot.2019.55.2.2974962},
pmid = {30816887},
issn = {1699-3993},
mesh = {*Biological Products ; CRISPR-Cas Systems ; *Drug Approval ; Drug Development ; Drug Industry/*trends ; Hemorrhagic Fever, Ebola ; Orphan Drug Production ; },
abstract = {This eagle's-eye overview of the drug industry in 2018 provides insight into some of last year's top stories, including a large and still growing outbreak of Ebola in the Democratic Republic of the Congo, as well as the potential uses (and abuses) of CRISPR technology. As in previous years, we also review orphan drug development, new regulatory agency-supported programs such as Priority Medicines Scheme (PRIME) and Sakigake, pipeline attrition, and pharma/biotech mergers and acquisitions of note. Finally, we take a glimpse into the crystal ball to anticipate the new drugs that will be approved in 2019.},
}
@article {pmid30816503,
year = {2019},
author = {Rodríguez-Rodríguez, DR and Ramírez-Solís, R and Garza-Elizondo, MA and Garza-Rodríguez, ML and Barrera-Saldaña, HA},
title = {Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review).},
journal = {International journal of molecular medicine},
volume = {43},
number = {4},
pages = {1559-1574},
pmid = {30816503},
issn = {1791-244X},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Models, Genetic ; },
abstract = {Genome editing reemerged in 2012 with the development of CRISPR/Cas9 technology, which is a genetic manipulation tool derived from the defense system of certain bacteria against viruses and plasmids. This method is easy to apply and has been used in a wide variety of experimental models, including cell lines, laboratory animals, plants, and even in human clinical trials. The CRISPR/Cas9 system consists of directing the Cas9 nuclease to create a site‑directed double‑strand DNA break using a small RNA molecule as a guide. A process that allows a permanent modification of the genomic target sequence can repair the damage caused to DNA. In the present study, the basic principles of the CRISPR/Cas9 system are reviewed, as well as the strategies and modifications of the enzyme Cas9 to eliminate the off‑target cuts, and the different applications of CRISPR/Cas9 as a system for visualization and gene expression activation or suppression. In addition, the review emphasizes on the potential application of this system in the treatment of different diseases, such as pulmonary, gastrointestinal, hematologic, immune system, viral, autoimmune and inflammatory diseases, and cancer.},
}
@article {pmid30816314,
year = {2019},
author = {Saito, T and Wada, I and Inoue, N},
title = {Alternative splicing of the Izumo1 gene ensures triggering gamete fusion in mice.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3151},
pmid = {30816314},
issn = {2045-2322},
mesh = {Alternative Splicing/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Exons/genetics ; Female ; Fertilization/*genetics ; Gene Expression Regulation, Developmental/genetics ; Germ Cells/growth &amp; development ; Immunoglobulins/*genetics ; Male ; Membrane Proteins/*genetics ; Mice ; Mice, Knockout ; Oocytes/growth &amp; development/metabolism ; Receptors, Cell Surface/*genetics ; Sperm-Ovum Interactions/genetics ; Spermatozoa/growth &amp; development/metabolism ; },
abstract = {IZUMO1 is a sperm acrosomal membrane protein that is essential for mammalian fertilization through recognition of JUNO on the oocyte surface and accompanying IZUMO1-JUNO complex formation. Here, we report a new Izumo1 gene splicing variant (IZUMO1_v2) with a unique 52-amino-acid-long signal sequence transcribed from Exon 1b. Although the mRNA amount of Izumo1_v2 is 76 times lower than that of the original Izumo1 (IZUMO1_v1) in the testis, the cell-oocyte assay indicates that IZUMO1_v2-expressing COS-7 cells have the ability to attach to the oocyte equivalent of IZUMO1_v1. To clarify the physiological function of IZUMO1_v2, we produced an IZUMO1_v1-specific knockout mouse line with a nine-base deletion adjacent to the initial methionine codon of IZUMO1_v1 by the CRISPR/Cas9 system. The IZUMO1_v1 knockout male mice carry 0.19-fold lower level of IZUMO1 protein in the spermatozoon; however, reduction in fertility was only minimally affected compared to the wild-type mice, suggesting that only a small fraction of IZUMO1 is sufficient for triggering sperm-egg fusion. We propose that the alternative splicing generating IZUMO1_v2 might function as a fail-safe in mouse for when splicing is disturbed.},
}
@article {pmid30816302,
year = {2019},
author = {Zhang, CX and Ye, SB and Ni, JJ and Cai, TT and Liu, YN and Huang, DJ and Mai, HQ and Chen, QY and He, J and Zhang, XS and Zeng, YX and Li, J and Cui, J},
title = {STING signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion.},
journal = {Cell death and differentiation},
volume = {26},
number = {11},
pages = {2314-2328},
pmid = {30816302},
issn = {1476-5403},
mesh = {CRISPR-Cas Systems ; Cell Differentiation/immunology ; Cell Line, Tumor ; Dimerization ; Epstein-Barr Virus Infections/pathology ; Gene Knockout Techniques ; Granulocyte-Macrophage Colony-Stimulating Factor/antagonists &amp; inhibitors ; HEK293 Cells ; Herpesvirus 4, Human/immunology ; Humans ; Interleukin-6/antagonists &amp; inhibitors ; Membrane Proteins/genetics/*metabolism ; Myeloid-Derived Suppressor Cells/*cytology/immunology ; Nasopharyngeal Carcinoma/immunology/*pathology/virology ; Nasopharyngeal Neoplasms/immunology/*pathology/virology ; Phosphorylation/physiology ; Prognosis ; STAT3 Transcription Factor/metabolism ; Suppressor of Cytokine Signaling 1 Protein/metabolism ; Tumor Microenvironment/drug effects/*physiology ; },
abstract = {Stimulator of interferon genes (STING), a major adaptor protein in antiviral innate immune signaling, is considered as one of the most important regulators of antiviral and antitumor immunity. Although STING agonists are now intensively studied in clinical trials as a new class of adjuvants to boost cancer immunotherapy, the tumor-intrinsic role of the STING pathway in shaping the tumor microenvironment remains controversial. Here, we discovered that STING plays a vital role in regulation of myeloid-derived suppressor cell (MDSC) differentiation and antitumor immunity in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). Mechanistic analyses reveal that STING represses NPC-derived MDSC induction by enhancing SOCS1 expression in both tumor cells and MDSCs. SOCS1 physically interacts with STAT3 through its SH2 domain to prevent STAT3 phosphorylation and dimerization, resulting in reduced MDSC induction via inhibition of GM-CSF and IL-6 production. Notably, reduced tumoral STING expression was found to be significantly associated with a poor prognosis for NPC patients. Our findings reveal a novel mechanism linking STING to tumor microenvironmental cytokine production and MDSC induction.},
}
@article {pmid30816277,
year = {2019},
author = {Wan, H and Li, J and Chang, S and Lin, S and Tian, Y and Tian, X and Wang, M and Hu, J},
title = {Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {3188},
pmid = {30816277},
issn = {2045-2322},
mesh = {CRISPR-Associated Proteins/genetics/*ultrastructure ; CRISPR-Cas Systems/*genetics ; Crystallography, X-Ray ; DNA Helicases/genetics/*ultrastructure ; DNA, Intergenic/genetics/ultrastructure ; DNA-Binding Proteins/genetics ; Endodeoxyribonucleases/genetics/*ultrastructure ; Escherichia coli/genetics ; Escherichia coli Proteins/genetics/*ultrastructure ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; },
abstract = {Adaptation in CRISPR-Cas systems enables the generation of an immunological memory to defend against invading viruses. This process is driven by foreign DNA spacer (termed protospacer) selection and integration mediated by Cas1-Cas2 protein. Recently, different states of Cas1-Cas2, in its free form and in complex with protospacer DNAs, were solved by X-ray crystallography. In this paper, molecular dynamics (MD) simulations are employed to study crystal structures of one free and two protospacer-bound Cas1-Cas2 complexes. The simulated results indicate that the protospacer binding markedly increases the system stability, in particular when the protospacer containing the PAM-complementary sequence. The hydrogen bond and binding free energy calculations explain that PAM recognition introduces more specific interactions to increase the cleavage activity of Cas1. By using principal component analysis (PCA) and intramolecular angle calculation, this study observes two dominant slow motions associated with the binding of Ca1-Cas2 to the protospacer and potential target DNAs respectively. The comparison of DNA structural deformation further implies a cooperative conformational change of Cas1-Cas2 and protospacer for the target DNA capture. We propose that this cooperativity is the intrinsic requirement of the CRISPR integration complex formation. This study provides some new insights into the understanding of CRISPR-Cas adaptation.},
}
@article {pmid30815697,
year = {2019},
author = {Sobh, A and Loguinov, A and Yazici, GN and Zeidan, RS and Tagmount, A and Hejazi, NS and Hubbard, AE and Zhang, L and Vulpe, CD},
title = {Functional Profiling Identifies Determinants of Arsenic Trioxide Cellular Toxicity.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {169},
number = {1},
pages = {108-121},
pmid = {30815697},
issn = {1096-0929},
support = {P42 ES004705/ES/NIEHS NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; Arsenic Trioxide/*pharmacology ; CRISPR-Cas Systems ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Gene Editing ; *Gene Expression Profiling ; Gene Expression Regulation, Leukemic ; HEK293 Cells ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/genetics/metabolism/pathology ; Signal Transduction ; Sodium Selenite/pharmacology ; Time Factors ; Transcriptome ; },
abstract = {Arsenic exposure is a worldwide health concern associated with an increased risk of skin, lung, and bladder cancer but arsenic trioxide (AsIII) is also an effective chemotherapeutic agent. The current use of AsIII in chemotherapy is limited to acute promyelocytic leukemia (APL). However, AsIII was suggested as a potential therapy for other cancer types including chronic myeloid leukemia (CML), especially when combined with other drugs. Here, we carried out a genome-wide CRISPR-based approach to identify modulators of AsIII toxicity in K562, a human CML cell line. We found that disruption of KEAP1, the inhibitory partner of the key antioxidant transcription factor Nrf2, or TXNDC17, a thioredoxin-like protein, markedly increased AsIII tolerance. Loss of the water channel AQP3, the zinc transporter ZNT1 and its regulator MTF1 also enhanced tolerance to AsIII whereas loss of the multidrug resistance protein ABCC1 increased sensitivity to AsIII. Remarkably, disruption of any of multiple genes, EEFSEC, SECISBP2, SEPHS2, SEPSECS, and PSTK, encoding proteins involved in selenocysteine metabolism increased resistance to AsIII. Our data suggest a model in which an intracellular interaction between selenium and AsIII may impact intracellular AsIII levels and toxicity. Together this work revealed a suite of cellular components/processes which modulate the toxicity of AsIII in CML cells. Targeting such processes simultaneously with AsIII treatment could potentiate AsIII in CML therapy.},
}
@article {pmid30814697,
year = {2019},
author = {Rusk, N},
title = {Inhibiting CRISPR.},
journal = {Nature methods},
volume = {16},
number = {3},
pages = {218},
doi = {10.1038/s41592-019-0341-y},
pmid = {30814697},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Eukaryotic Cells ; *Gene Editing ; },
}
@article {pmid30814678,
year = {2019},
author = {Guo, M and Zhang, K and Zhu, Y and Pintilie, GD and Guan, X and Li, S and Schmid, MF and Ma, Z and Chiu, W and Huang, Z},
title = {Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry.},
journal = {Cell research},
volume = {29},
number = {4},
pages = {305-312},
pmid = {30814678},
issn = {1748-7838},
support = {P41 GM103832/GM/NIGMS NIH HHS/United States ; R01 GM079429/GM/NIGMS NIH HHS/United States ; U54 GM103297/GM/NIGMS NIH HHS/United States ; S10 OD021600/OD/NIH HHS/United States ; },
mesh = {Bacterial Proteins/*chemistry ; CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Cryoelectron Microscopy/methods ; RNA, Bacterial/*chemistry ; },
abstract = {The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.},
}
@article {pmid30814571,
year = {2019},
author = {Roghanian, M and Semsey, S and Løbner-Olesen, A and Jalalvand, F},
title = {(p)ppGpp-mediated stress response induced by defects in outer membrane biogenesis and ATP production promotes survival in Escherichia coli.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {2934},
pmid = {30814571},
issn = {2045-2322},
mesh = {Adenosine Triphosphate/metabolism ; CRISPR-Cas Systems/genetics ; Cell Membrane/metabolism ; Energy Metabolism/*physiology ; Escherichia coli/genetics/*growth &amp; development/*metabolism ; Escherichia coli Proteins/metabolism ; Gene Expression Regulation, Bacterial/genetics ; Guanosine Pentaphosphate/*metabolism ; Guanosine Tetraphosphate/*metabolism ; Lipopolysaccharides/biosynthesis ; Stress, Physiological/*physiology ; },
abstract = {Cellular growth requires a high level of coordination to ensure that all processes run in concert. The role of the nucleotide alarmone (p)ppGpp has been extensively studied in response to external stresses, such as amino acid starvation, in Escherichia coli, but much less is known about the involvement of (p)ppGpp in response to perturbations in intracellular processes. We therefore employed CRISPRi to transcriptionally repress essential genes involved in 14 vital processes and investigated whether a (p)ppGpp-mediated response would be induced. We show that (p)ppGpp is produced and required for a pertinent stress response during interference with outer membrane biogenesis and ADP synthesis specifically. When these processes were perturbed via the transcriptional repression of essential genes, wild type E. coli MG1655 ceased growing and entered a semi-dormant state, whereas isogenic (p)ppGpp[0] cells continued to grow uncontrollably to the point of lysis. Furthermore, in vivo measurements revealed that the ATP levels were intrinsically offset in (p)ppGpp[0] cells, further indicating a role for the alarmone in cellular energy homeostasis. In summary, our investigation suggests that (p)ppGpp acts as a coordinator of cell growth in response to imbalances in outer membrane biogenesis and adenosine ribonucleotide synthesis, elucidating novel roles for (p)ppGpp in bacterial physiology.},
}
@article {pmid30814462,
year = {2019},
author = {Yoshida, H and Fukushima, Y and Goto, M and Tsuyuki, Y and Takahashi, T},
title = {Analysis of the Type II-A CRISPR-Cas System in Streptococcus canis Isolated from Diseased Companion Animals and One Human Patient in Japan.},
journal = {Japanese journal of infectious diseases},
volume = {72},
number = {4},
pages = {261-265},
doi = {10.7883/yoken.JJID.2018.492},
pmid = {30814462},
issn = {1884-2836},
mesh = {Alleles ; Animals ; Bacteremia/microbiology ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Cattle ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Bacterial/genetics ; Female ; Genome, Bacterial/genetics ; Genotype ; Humans ; Japan/epidemiology ; *Molecular Typing ; Pets/microbiology ; Phylogeny ; Sequence Analysis, DNA ; Streptococcal Infections/epidemiology/*microbiology ; Streptococcus/classification/*genetics/isolation &amp; purification ; },
abstract = {We determined the whole-genome sequence (WGS) of Streptococcus canis strain TA4 harboring the M-like protein gene (scm); the strain was isolated from a human patient presenting with bacteremia. The potential of type II-A clustered regularly interspaced short palindromic repeats (CRISPR) array-based typing was evaluated, and the genetic relation was elucidated between spacer genogroups and scm prevalence and/or polymorphisms among the isolates from 19 diseased companion animals and the human patient. CRISPRFinder and CRISPRCasFinder detected the type II-A locus with the same repeat sequences in strain TA4 and another WGS of S. canis strain, isolated from a cow with mastitis. An optimized PCR-based amplification method was used to sequence the region covering the locus around the leader and terminal repeat sequences. Among the 20 isolates sequenced, 16 strains (including TA4) were identified with the CRISPR array. We conducted comparative analysis of the homologous spacer sequences and performed grouping based on the successive common ancestral spacer types. These 16 isolates were assigned to five genogroups (A to E) with scm being absent in genogroup A. We found a relationship between genogroups C and E and allele type 1 of the deduced M-like protein. These preliminary findings suggest the feasibility of CRISPR array-based typing and a genetic relation between the spacer genogroups and scm prevalence and/or polymorphisms in the isolates.},
}
@article {pmid30813944,
year = {2019},
author = {Yildiz, O and Downes, GB and Sagerström, CG},
title = {Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain.},
journal = {Neural development},
volume = {14},
number = {1},
pages = {5},
pmid = {30813944},
issn = {1749-8104},
support = {R01 NS038183/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Behavior, Animal/physiology ; Body Patterning/genetics/*physiology ; *CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/*physiology ; *Gene Expression Regulation, Developmental ; Germ-Line Mutation ; HEK293 Cells ; Humans ; Locomotion/*physiology ; Nerve Tissue Proteins/*metabolism ; Neural Tube/*embryology ; *Renshaw Cells ; Rhombencephalon/*embryology ; Spinal Cord/*embryology ; Transcription Factors/*metabolism ; *Transcription, Genetic ; Zebrafish ; Zebrafish Proteins/genetics/*metabolism/*physiology ; },
abstract = {BACKGROUND: Functioning of the adult nervous system depends on the establishment of neural circuits during embryogenesis. In vertebrates, neurons that make up motor circuits form in distinct domains along the dorsoventral axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear.<br><br>METHODS: We used CRISPR/Cas9 to generate the first germline mutants for prdm12 and employed this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation.<br><br>RESULTS: We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal touch-evoked escape response. Using luciferase reporter assays, we demonstrate that Prdm12b acts as a transcriptional repressor. We also show that the Bhlhe22 TF binds via the Prdm12b zinc finger domain to form a complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1.<br><br>CONCLUSIONS: We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in formation of the p1 domain - suggesting that prdm12b is not solely required to repress non-p1 fates, but is specifically needed to promote p1 fates.},
}
@article {pmid30813348,
year = {2019},
author = {van Aelst, K and Martínez-Santiago, CJ and Cross, SJ and Szczelkun, MD},
title = {The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a.},
journal = {Genes},
volume = {10},
number = {2},
pages = {},
pmid = {30813348},
issn = {2073-4425},
support = {788405/ERC_/European Research Council/International ; BB/L000873/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; BB/S001239/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Bacterial Proteins/metabolism ; *CRISPR-Cas Systems ; DNA, Superhelical/chemistry/metabolism ; Endodeoxyribonucleases/metabolism ; *R-Loop Structures ; },
abstract = {Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.},
}
@article {pmid30811422,
year = {2019},
author = {Raitskin, O and Schudoma, C and West, A and Patron, NJ},
title = {Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0211598},
pmid = {30811422},
issn = {1932-6203},
support = {BB/L014130/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/T/000PR9816/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/CCG1720/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; Genome, Plant/*genetics ; Plants/*genetics ; },
abstract = {Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.},
}
@article {pmid30811402,
year = {2019},
author = {Zhang, ZJ and Zhang, SS and Niu, BL and Ji, DF and Liu, XJ and Li, MW and Bai, H and Palli, SR and Wang, CZ and Tan, AJ},
title = {A determining factor for insect feeding preference in the silkworm, Bombyx mori.},
journal = {PLoS biology},
volume = {17},
number = {2},
pages = {e3000162},
pmid = {30811402},
issn = {1545-7885},
support = {R01 AG058741/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; Bombyx/*genetics/growth &amp; development/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Chromosomes, Insect/chemistry ; Edible Grain/parasitology ; Feeding Behavior/*physiology ; Fruit/parasitology ; Gene Editing/methods ; Gene Expression ; Genetic Engineering/methods ; Genetic Loci ; HEK293 Cells ; Homozygote ; Humans ; Insect Proteins/*genetics/metabolism ; Larva/genetics/growth &amp; development/metabolism ; Morus/parasitology ; Plant Leaves/parasitology ; RNA, Guide/genetics/metabolism ; Receptors, Cell Surface/*genetics/metabolism ; Taste Perception/*genetics ; },
abstract = {Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.},
}
@article {pmid30810907,
year = {2019},
author = {Selvakumar, GP and Ahmed, ME and Raikwar, SP and Thangavel, R and Kempuraj, D and Dubova, I and Saeed, D and Zahoor, H and Premkumar, K and Zaheer, S and Iyer, S and Zaheer, A},
title = {CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {4},
pages = {537-550},
pmid = {30810907},
issn = {1557-1904},
support = {I01 BX002477/BX/BLRD VA/United States ; R01 AG048205/AG/NIA NIH HHS/United States ; },
mesh = {1-Methyl-4-phenylpyridinium/toxicity ; Animals ; CRISPR-Associated Protein 9/biosynthesis/genetics ; CRISPR-Cas Systems/*physiology ; Cell Line ; Ferritins/biosynthesis/*genetics ; Gene Editing/methods ; Glia Maturation Factor/deficiency/*genetics ; Heme Oxygenase-1/biosynthesis/*genetics ; Membrane Proteins/biosynthesis/*genetics ; Mice ; Mitochondrial Dynamics/*physiology ; NF-E2-Related Factor 2/biosynthesis/*genetics ; Neuroglia/drug effects/*physiology ; Reactive Oxygen Species/metabolism ; },
abstract = {Microglial cells are brain specific professional phagocytic immune cells that play a crucial role in the inflammation- mediated neurodegeneration especially in Parkinson's disease (PD) and Alzheimer's disease. Glia maturation factor (GMF) is a neuroinflammatory protein abundantly expressed in the brain. We have previously shown that GMF expression is significantly upregulated in the substantia nigra (SN) of PD brains. However, its possible role in PD progression is still not fully understood. The Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) protein9 (CRISPR/Cas9) system is a simple, rapid and often extremely efficient gene editing tool at desired loci, enabling complete gene knockout or homology directed repair. In this study, we examined the effect of GMF editing by using the CRISPR/Cas9 technique in BV2 microglial cells (hereafter referred to as BV2-G) on oxidative stress and nuclear factor erythroid 2-related factor 2 (NRF2)/Hemeoxygenase1 (HO-1)-dependent ferritin activation after treatment with (1-methyl-4-phenylpyridinium) MPP[+]. Knockout of GMF in BV2-G cells significantly attenuated oxidative stress via reduced ROS production and calcium flux. Furthermore, deficiency of GMF significantly reduced nuclear translocation of NRF2, which modulates HO-1 and ferritin activation, cyclooxygenase 2 (COX2) and nitric oxide synthase 2 (NOS2) expression in BV2 microglial cells. Lack of GMF significantly improved CD11b and CD68 positive microglial cells as compared with untreated cells. Our results also suggest that pharmacological and genetic intervention targeting GMF may represent a promising and a novel therapeutic strategy in controlling Parkinsonism by regulating microglial functions. Targeted regulation of GMF possibly mediates protein aggregation in microglial homeostasis associated with PD progression through regulation of iron metabolism by modulating NRF2-HO1 and ferritin expression.},
}
@article {pmid30810831,
year = {2019},
author = {Yu, H and Li, H and Li, Q and Xu, R and Yue, C and Du, S},
title = {Targeted Gene Disruption in Pacific Oyster Based on CRISPR/Cas9 Ribonucleoprotein Complexes.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {21},
number = {3},
pages = {301-309},
pmid = {30810831},
issn = {1436-2236},
mesh = {Animals ; Aquaculture/*methods ; *CRISPR-Cas Systems ; Crassostrea/*genetics ; Gene Editing/*methods/standards ; INDEL Mutation/genetics ; },
abstract = {The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few years, the genetic tools for gene functional studies have thus far been limited to RNA interference technology. In this study, we developed a gene editing system for C. gigas based on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 ribonucleoprotein complexes. Two candidate genes, myostatin (MSTN) and Twist, were selected as targets. After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, CRISPR-induced indel mutations were detected in the target genes. The CRISPR/Cas9-induced mutations were predominantly small indel mutations ranging in size from 1 to 24 bp in these two target genes. These results demonstrate that CRISPR/Cas9 can be successfully used as an effective targeted gene editing system in C. gigas. The method reported here provides a powerful tool for gene functional studies in oysters and other marine bivalves, and potentially as a new technology for genetic engineering to improve oyster traits for aquaculture.},
}
@article {pmid30810167,
year = {2019},
author = {Lee, HY and Chen, Z and Zhang, C and Yoon, GM},
title = {Editing of the OsACS locus alters phosphate deficiency-induced adaptive responses in rice seedlings.},
journal = {Journal of experimental botany},
volume = {70},
number = {6},
pages = {1927-1940},
pmid = {30810167},
issn = {1460-2431},
mesh = {Ethylenes/metabolism ; Gene Editing ; Oryza/genetics/growth &amp; development/*physiology ; Phosphates/*deficiency ; Plant Proteins/*genetics/metabolism ; Seedlings/growth &amp; development/physiology ; },
abstract = {Phosphate (Pi) deficiency severely influences the growth and reproduction of plants. To cope with Pi deficiency, plants initiate morphological and biochemical adaptive responses upon sensing low Pi in the soil, and the plant hormone ethylene plays a crucial role during this process. However, how regulation of ethylene biosynthesis influences the Pi-induced adaptive responses remains unclear. Here, we determine the roles of rice 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), the rate-limiting enzymes in ethylene biosynthesis, in response to Pi deficiency. Through analysis of tissue-specific expression of OsACS in response to Pi deficiency and OsACS mutants generated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] genome editing, we found that two members of the OsACS family, i.e. OsACS1 and OsACS2, are involved but differed in their importance in controlling the remodeling of root system architecture, transcriptional regulation of Pi starvation-induced genes, and cellular phosphorus homeostasis. Interestingly, in contrast to the known inhibitory role of ethylene on root elongation, both OsACS mutants, especially OsACS1, almost fail to promote lateral root growth in response to Pi deficiency, demonstrating a stimulatory role for ethylene in lateral root development under Pi-deficient conditions. Together, this study provides new insights into the roles of ethylene in Pi deficiency response in rice seedlings and the isoform-specific function of OsACS genes in this process.},
}
@article {pmid30810036,
year = {2019},
author = {Li, Y and Teng, X and Zhang, K and Deng, R and Li, J},
title = {RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing.},
journal = {Analytical chemistry},
volume = {91},
number = {6},
pages = {3989-3996},
doi = {10.1021/acs.analchem.8b05238},
pmid = {30810036},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Genetic Vectors ; *Genome, Human ; HeLa Cells ; Humans ; MCF-7 Cells ; RNA, Guide/*chemistry/*metabolism ; RNA, Messenger/genetics/*metabolism ; Recombination, Genetic ; },
abstract = {CRISPR/Cas9 has already become a powerful tool for genomic manipulation, and further engineering of the system allows it to be precisely regulated in response to external signals, thus, broadening its application possibilities, such as biosensing or bioimaging. However, most stimuli-responsive CRISPR systems are built based on elaborately designed and engineered inducible Cas9 proteins, and external stimuli are still mostly limited as small molecules and light. To construct more precise and easy-to-build responsive CRISPR systems and broaden their responsive species, we seek to engineer conditional guide RNA, rather than Cas9 protein, to mediate conditional CRISPR corresponding to logic operation. Here, we construct mRNA-sensing CRISPR by gRNA reconfiguration and toehold mediated strand displacement, in which each target site could be independently controlled. We show that switches can be embedded into the gRNA and used as RNA sensors, capable of detecting multiple mRNA inputs orthogonally and providing CRISPR/Cas9 response outputs. NOR and NAND logical gates are also constructed, demonstrating its orthogonality and programmability. This strategy promises potential uses in constructing genetic circuits to detect endogenous mRNAs and initiate cellular responses.},
}
@article {pmid30809786,
year = {2019},
author = {Chen, G and Chu, J},
title = {Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System.},
journal = {Applied biochemistry and biotechnology},
volume = {188},
number = {4},
pages = {1134-1144},
doi = {10.1007/s12010-019-02960-z},
pmid = {30809786},
issn = {1559-0291},
mesh = {Acremonium/*metabolism ; CRISPR-Cas Systems/genetics/physiology ; Gene Editing ; Polyketide Synthases/*metabolism ; RNA, Guide/*economics ; },
abstract = {Acremonium chrysogenum is an important fungal strain used for cephalosporin C production. Many efforts have been made to develop versatile genome-editing tools to better understand the mechanism of A. chrysogenum. Here, we developed a feasible and efficient CRISPR/Cas9 system. Two genes responsible for the synthesis of yellow pigments (sorbicillinoids) were chosen as targets, and plasmids expressing both the Cas9 protein and single-guide RNAs were constructed. After introducing the plasmids into the protoplasts of A. chrysogenum, 83 to 93% albino mutants harboring the expected genomic alteration, on average, were obtained. We have generated two mutant strains that respectively disrupt sorA and sorB by flexible CRISPR/Cas9 system. We further confirmed that the sorbicillinoid biosynthetic gene cluster is regulated by an autoinduction mechanism. This work will lay a solid foundation for gene function research and regulation in the sorbicillinoid biosynthetic pathway.},
}
@article {pmid30809660,
year = {2019},
author = {Killian, T and Buntz, A and Herlet, T and Seul, H and Mundigl, O and Längst, G and Brinkmann, U},
title = {Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids.},
journal = {Nucleic acids research},
volume = {47},
number = {10},
pages = {e55},
pmid = {30809660},
issn = {1362-4962},
mesh = {Antibodies, Bispecific/immunology/*metabolism ; *CRISPR-Cas Systems ; Chromatin/genetics/*metabolism ; DNA/genetics/metabolism ; Gene Editing/*methods ; Humans ; MCF-7 Cells ; Microscopy, Confocal ; Plasmids/*genetics ; Transgenes/genetics ; },
abstract = {We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed 'plasmid-chromatin' deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (∼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.},
}
@article {pmid30809237,
year = {2019},
author = {Sedeek, KEM and Mahas, A and Mahfouz, M},
title = {Plant Genome Engineering for Targeted Improvement of Crop Traits.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {114},
pmid = {30809237},
issn = {1664-462X},
abstract = {To improve food security, plant biology research aims to improve crop yield and tolerance to biotic and abiotic stress, as well as increasing the nutrient contents of food. Conventional breeding systems have allowed breeders to produce improved varieties of many crops; for example, hybrid grain crops show dramatic improvements in yield. However, many challenges remain and emerging technologies have the potential to address many of these challenges. For example, site-specific nucleases such as TALENs and CRISPR/Cas systems, which enable high-efficiency genome engineering across eukaryotic species, have revolutionized biological research and its applications in crop plants. These nucleases have been used in diverse plant species to generate a wide variety of site-specific genome modifications through strategies that include targeted mutagenesis and editing for various agricultural biotechnology applications. Moreover, CRISPR/Cas genome-wide screens make it possible to discover novel traits, expand the range of traits, and accelerate trait development in target crops that are key for food security. Here, we discuss the development and use of various site-specific nuclease systems for different plant genome-engineering applications. We highlight the existing opportunities to harness these technologies for targeted improvement of traits to enhance crop productivity and resilience to climate change. These cutting-edge genome-editing technologies are thus poised to reshape the future of agriculture and food security.},
}
@article {pmid30809070,
year = {2019},
author = {Cyranoski, D},
title = {The CRISPR-baby scandal: what's next for human gene-editing.},
journal = {Nature},
volume = {566},
number = {7745},
pages = {440-442},
pmid = {30809070},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Dissent and Disputes ; Embryo Research/ethics ; Embryonic Development/genetics ; *Ethics, Research ; Female ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Genetic Diseases, Inborn/genetics ; Germ-Line Mutation/*genetics ; HIV Infections/blood/virology ; Humans ; Infant, Newborn ; International Cooperation/legislation &amp; jurisprudence ; Patient Safety ; Receptors, CCR5/deficiency/genetics ; Research Personnel/*ethics/legislation &amp; jurisprudence ; Scientific Misconduct/*legislation &amp; jurisprudence ; Twins/*genetics ; },
}
@article {pmid30809052,
year = {2019},
author = {Maxmen, A},
title = {Faster, better, cheaper: the rise of CRISPR in disease detection.},
journal = {Nature},
volume = {566},
number = {7745},
pages = {437},
pmid = {30809052},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids ; },
}
@article {pmid30809026,
year = {2019},
author = {Clement, K and Rees, H and Canver, MC and Gehrke, JM and Farouni, R and Hsu, JY and Cole, MA and Liu, DR and Joung, JK and Bauer, DE and Pinello, L},
title = {CRISPResso2 provides accurate and rapid genome editing sequence analysis.},
journal = {Nature biotechnology},
volume = {37},
number = {3},
pages = {224-226},
pmid = {30809026},
issn = {1546-1696},
support = {DP2 HL137300/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome, Human/*genetics ; Humans ; Polymorphism, Single Nucleotide/genetics ; RNA Editing/genetics ; Sequence Analysis ; },
}
@article {pmid30808944,
year = {2019},
author = {O'Brien, AR and Wilson, LOW and Burgio, G and Bauer, DC},
title = {Unlocking HDR-mediated nucleotide editing by identifying high-efficiency target sites using machine learning.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {2788},
pmid = {30808944},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *DNA Repair ; *Gene Editing ; *Machine Learning ; Mice ; Mice, Inbred C57BL ; Mutation ; Oligodeoxyribonucleotides/metabolism ; RNA, Guide/metabolism ; },
abstract = {Editing individual nucleotides is a crucial component for validating genomic disease association. It is currently hampered by CRISPR-Cas-mediated "base editing" being limited to certain nucleotide changes, and only achievable within a small window around CRISPR-Cas target sites. The more versatile alternative, HDR (homology directed repair), has a 3-fold lower efficiency with known optimization factors being largely immutable in experiments. Here, we investigated the variable efficiency-governing factors on a novel mouse dataset using machine learning. We found the sequence composition of the single-stranded oligodeoxynucleotide (ssODN), i.e. the repair template, to be a governing factor. Furthermore, different regions of the ssODN have variable influence, which reflects the underlying mechanism of the repair process. Our model improves HDR efficiency by 83% compared to traditionally chosen targets. Using our findings, we developed CUNE (Computational Universal Nucleotide Editor), which enables users to identify and design the optimal targeting strategy using traditional base editing or - for-the-first-time - HDR-mediated nucleotide changes.},
}
@article {pmid30808860,
year = {2019},
author = {Pabon, NA and Zhang, Q and Cruz, JA and Schipper, DL and Camacho, CJ and Lee, REC},
title = {A network-centric approach to drugging TNF-induced NF-κB signaling.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {860},
pmid = {30808860},
issn = {2041-1723},
support = {R01 GM097082/GM/NIGMS NIH HHS/United States ; R35 GM119462/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Drug Development/methods ; Gene Knock-In Techniques ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; I-kappa B Kinase/genetics/metabolism ; Models, Molecular ; NF-kappa B/*metabolism ; Protein Interaction Domains and Motifs/drug effects ; Receptors, Tumor Necrosis Factor, Type I/chemistry/metabolism ; Signal Transduction/*drug effects/physiology ; Systems Biology ; TNF Receptor-Associated Factor 2/chemistry/metabolism ; Transcription Factor RelA/genetics/metabolism ; Tumor Necrosis Factor-alpha/antagonists &amp; inhibitors/*metabolism ; },
abstract = {Target-centric drug development strategies prioritize single-target potency in vitro and do not account for connectivity and multi-target effects within a signal transduction network. Here, we present a systems biology approach that combines transcriptomic and structural analyses with live-cell imaging to predict small molecule inhibitors of TNF-induced NF-κB signaling and elucidate the network response. We identify two first-in-class small molecules that inhibit the NF-κB signaling pathway by preventing the maturation of a rate-limiting multiprotein complex necessary for IKK activation. Our findings suggest that a network-centric drug discovery approach is a promising strategy to evaluate the impact of pharmacologic intervention in signaling.},
}
@article {pmid30808751,
year = {2019},
author = {Litschko, C and Brühmann, S and Csiszár, A and Stephan, T and Dimchev, V and Damiano-Guercio, J and Junemann, A and Körber, S and Winterhoff, M and Nordholz, B and Ramalingam, N and Peckham, M and Rottner, K and Merkel, R and Faix, J},
title = {Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {9},
pages = {3594-3603},
pmid = {30808751},
issn = {1091-6490},
support = {094231/Z/10/Z//Wellcome Trust/United Kingdom ; },
mesh = {Actin Cytoskeleton/chemistry/*genetics ; Actins/genetics ; Animals ; CRISPR-Cas Systems ; Carrier Proteins/*genetics ; Cell Movement/genetics ; Cell Polarity/genetics ; Contractile Proteins/chemistry/*genetics ; Dictyostelium/genetics ; Disease Models, Animal ; Formins ; Humans ; Melanoma, Experimental/*genetics/pathology ; Mice ; Microscopy, Electron ; Muscle Contraction/genetics ; rhoA GTP-Binding Protein/chemistry/genetics ; },
abstract = {The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA[-] /E[-]/H[-] and racE[-] mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.},
}
@article {pmid30808210,
year = {2019},
author = {Li, X and Yu, X and Chen, X and Liu, Z and Wang, G and Li, C and Wong, EYM and Sham, MH and Tang, J and He, J and Xiong, W and Liu, Z and Huang, P},
title = {Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {6},
pages = {6838-6851},
doi = {10.1096/fj.201802155RR},
pmid = {30808210},
issn = {1530-6860},
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; Hair Cells, Auditory/*metabolism ; Hair Cells, Auditory, Inner/*metabolism ; Mechanotransduction, Cellular ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Mice ; Mice, Knockout ; },
abstract = {The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.},
}
@article {pmid30807998,
year = {2019},
author = {Wilson, IBH and Schabussova, I},
title = {Sweet and CRISP(R)y parasite engineering.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {4},
pages = {1126-1127},
pmid = {30807998},
issn = {1083-351X},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Knockout Techniques ; Gene Library ; Glycomics ; Polysaccharides/*genetics/metabolism ; Toxoplasma/*genetics/metabolism ; },
abstract = {Toxoplasma gondii is an intracellular parasite that is highly prevalent within human populations. Its genome encodes a range of enzymes involved in glycan biosynthesis and metabolism. A new study presents a library of CRISPR/Cas9-based glyco-relevant gene knockouts and their examination in glycomic and functional assays. This new resource can pave the way for a better understanding of the role of carbohydrates in infection and immunomodulation by this significant protozoan parasite.},
}
@article {pmid30807691,
year = {2019},
author = {Ji, CH and Kim, H and Kang, HS},
title = {Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces.},
journal = {ACS synthetic biology},
volume = {8},
number = {3},
pages = {577-586},
doi = {10.1021/acssynbio.9b00001},
pmid = {30807691},
issn = {2161-5063},
mesh = {Anthraquinones/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; Gene Expression Regulation, Bacterial ; Genetic Engineering/*methods ; Multigene Family ; Piperidones/metabolism ; Promoter Regions, Genetic ; Saccharomyces cerevisiae/genetics ; Secondary Metabolism/*genetics ; Streptomyces/*genetics/*metabolism ; Synthetic Biology/*methods ; Transcription, Genetic ; },
abstract = {Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as overproduction of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory system that displays a large dynamic range and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: (1) the induction module, (2) the target expression module, and (3) the repressor expression module. Then, these three separate modules were individually optimized in a stepwise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided markerless insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become a useful tool in modulating secondary metabolite production in Streptomyces.},
}
@article {pmid30807689,
year = {2019},
author = {Pristovšek, N and Nallapareddy, S and Grav, LM and Hefzi, H and Lewis, NE and Rugbjerg, P and Hansen, HG and Lee, GM and Andersen, MR and Kildegaard, HF},
title = {Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering.},
journal = {ACS synthetic biology},
volume = {8},
number = {4},
pages = {758-774},
doi = {10.1021/acssynbio.8b00453},
pmid = {30807689},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/genetics ; Cell Engineering/methods ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetulus ; Gene Expression/*genetics ; Mammals/*genetics ; Recombinant Proteins/*genetics ; Recombinases/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Transcription, Genetic/genetics ; },
abstract = {Many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components. Here we show in a systematic manner how multiple components contribute to final net expression of recombinant genes in a characterized integration site. We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5' proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated cassette exchange for fast screening of different expression cassettes. Using the developed toolbox, we show that different 5' proximal regulatory elements generate distinct and robust recombinant gene expression patterns in defined integration sites of CHO cells with a wide range of transcriptional outputs. This approach facilitates the generation of user-defined and product-specific gene expression patterns for programmable mammalian cell engineering.},
}
@article {pmid30806159,
year = {2019},
author = {Tao, Y and Hou, X and Zuo, F and Li, X and Pang, Y and Jiang, G},
title = {Application of nanoparticle-based siRNA and CRISPR/Cas9 delivery systems in gene-targeted therapy.},
journal = {Nanomedicine (London, England)},
volume = {14},
number = {5},
pages = {511-514},
doi = {10.2217/nnm-2018-0522},
pmid = {30806159},
issn = {1748-6963},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Humans ; Nanoparticles/*chemistry ; RNA, Small Interfering/*genetics ; },
}
@article {pmid30806053,
year = {2019},
author = {Guo, M and Yang, L and Du, W and Zhang, T and Lu, H and Wang, L},
title = {[CRISPR/Cas9-mediated foreign gene targeted knock-in into the chicken EAV-HP genome].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {2},
pages = {236-243},
doi = {10.13345/j.cjb.180224},
pmid = {30806053},
issn = {1872-2075},
mesh = {Animals ; *CRISPR-Cas Systems ; Chickens ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knock-In Techniques ; Genome ; HEK293 Cells ; Humans ; },
abstract = {The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein (eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.},
}
@article {pmid30805989,
year = {2019},
author = {Shi, X and Xiao, Z and Zonta, F and Wang, W and Wan, Y and Li, Y and Wang, N and Kuang, Y and Du, M and Dong, J and Wang, J and Yang, G},
title = {Somatic MIWI2 Hinders Direct Lineage Reprogramming From Fibroblast to Hepatocyte.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {6},
pages = {803-812},
pmid = {30805989},
issn = {1549-4918},
mesh = {Albumins/genetics/metabolism ; Animals ; Argonaute Proteins/deficiency/*genetics ; CRISPR-Cas Systems ; Cell Lineage/genetics ; Cell Transdifferentiation/genetics ; Cellular Reprogramming/*genetics ; *Epigenesis, Genetic ; Fibroblasts/cytology/*metabolism ; Gene Regulatory Networks ; Genetic Vectors/chemistry/metabolism ; Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism ; Hepatocyte Nuclear Factor 3-gamma/genetics/metabolism ; Hepatocytes/cytology/*metabolism ; Lentivirus/genetics/metabolism ; Mice ; Mice, Knockout ; RNA, Small Interfering/*genetics/metabolism ; Receptors, Notch/genetics/metabolism ; Signal Transduction ; Transduction, Genetic ; },
abstract = {Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte-like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi-interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. Stem Cells 2019;37:803-812.},
}
@article {pmid30805613,
year = {2019},
author = {Qiu, W and Xu, Z and Zhang, M and Zhang, D and Fan, H and Li, T and Wang, Q and Liu, P and Zhu, Z and Du, D and Tan, M and Wen, B and Liu, Y},
title = {Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system.},
journal = {Nucleic acids research},
volume = {47},
number = {9},
pages = {e52},
pmid = {30805613},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/*genetics ; Chromatin Immunoprecipitation ; DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry/*genetics ; Endonucleases ; Genome/genetics ; Genomics ; HEK293 Cells ; Humans ; Multifunctional Enzymes ; Protein Engineering ; RNA, Guide/chemistry/*genetics ; Repetitive Sequences, Nucleic Acid/*genetics ; Telomere/genetics ; Transcription Factors/genetics ; },
abstract = {The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNAs. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.},
}
@article {pmid30804513,
year = {2019},
author = {Newton, MD and Taylor, BJ and Driessen, RPC and Roos, L and Cvetesic, N and Allyjaun, S and Lenhard, B and Cuomo, ME and Rueda, DS},
title = {DNA stretching induces Cas9 off-target activity.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {3},
pages = {185-192},
pmid = {30804513},
issn = {1545-9985},
support = {206292/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; MC_UP_1102/5/MRC_/Medical Research Council/United Kingdom ; 106954/WT_/Wellcome Trust/United Kingdom ; MC_UP_1102/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 206292/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Bacteriophage lambda/*genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Cleavage ; DNA, Viral/genetics/*metabolism ; Escherichia coli/virology ; Fluorescence Resonance Energy Transfer ; Gene Editing ; Microfluidics ; Microscopy, Confocal ; Optical Tweezers ; RNA, Guide/genetics ; Streptococcus pyogenes/enzymology ; },
abstract = {CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.},
}
@article {pmid30804503,
year = {2019},
author = {Wang, XW and Hu, LF and Hao, J and Liao, LQ and Chiu, YT and Shi, M and Wang, Y},
title = {A microRNA-inducible CRISPR-Cas9 platform serves as a microRNA sensor and cell-type-specific genome regulation tool.},
journal = {Nature cell biology},
volume = {21},
number = {4},
pages = {522-530},
doi = {10.1038/s41556-019-0292-7},
pmid = {30804503},
issn = {1476-4679},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; Embryonic Stem Cells/metabolism ; Genome ; HeLa Cells ; Humans ; Mice ; MicroRNAs ; RNA, Small Interfering/metabolism ; Transcriptional Activation ; Transgenes ; },
abstract = {microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. Measuring miRNA activity in vivo remains a big challenge. Here, using an miRNA-mediated single guide RNA (sgRNA)-releasing strategy and dCas9-VPR to drive a transgene red fluorescent protein, we create an miRNA sensor that can faithfully measure miRNA activity at cellular levels and use it to monitor differentiation status of stem cells. Furthermore, by designing sgRNAs to target endogenous loci, we adapted this system to control the expression of endogenous genes or mutate specific DNA bases upon induction by cell-type-specific miRNAs. Finally, by miRNA sensor library screening, we discover a previously undefined layer of heterogeneity associated with miR-21a activity in mouse embryonic stem cells. Together, these results highlight the utility of an miRNA-induced CRISPR-Cas9 system as miRNA sensors and cell-type-specific genome regulation tools.},
}
@article {pmid30803822,
year = {2019},
author = {Zhang, HX and Zhang, Y and Yin, H},
title = {Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {4},
pages = {735-746},
pmid = {30803822},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Drug Delivery Systems/methods ; Gene Editing/*methods ; Genome ; Humans ; RNA, Messenger/*genetics ; Transcription Activator-Like Effector Nucleases/*genetics ; Transcription, Genetic ; Zinc Finger Nucleases/*genetics ; },
abstract = {Genome-editing technologies based on programmable nucleases have significantly broadened our ability to make precise and direct changes in the genomic DNA of various species, including human cells. Delivery of programmable nucleases into the target tissue or cell is one of the pressing challenges in transforming the technology into medicine. In vitro-transcribed (IVT) mRNA-mediated delivery of nucleases has several advantages, such as transient expression with efficient in vivo and in vitro delivery, no genomic integration, a potentially low off-target rate, and high editing efficiency. This review focuses on key barriers related to IVT mRNA delivery, on developed modes of delivery, and on the application and future prospects of mRNA encoding nuclease-mediated genome editing in research and clinical trials.},
}
@article {pmid30803101,
year = {2019},
author = {Zhan, X and Zhang, F and Zhong, Z and Chen, R and Wang, Y and Chang, L and Bock, R and Nie, B and Zhang, J},
title = {Generation of virus-resistant potato plants by RNA genome targeting.},
journal = {Plant biotechnology journal},
volume = {17},
number = {9},
pages = {1814-1822},
pmid = {30803101},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/*genetics ; Plant Diseases/*genetics/virology ; Potyvirus/*pathogenicity ; RNA, Guide/genetics ; Solanum tuberosum/*genetics/virology ; },
abstract = {CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA-targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.},
}
@article {pmid30802454,
year = {2019},
author = {Hayashida, G and Shioi, S and Hidaka, K and Fujikane, R and Hidaka, M and Tsurimoto, T and Tsuzuki, T and Oda, S and Nakatsu, Y},
title = {Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.},
journal = {Experimental cell research},
volume = {377},
number = {1-2},
pages = {24-35},
doi = {10.1016/j.yexcr.2019.02.020},
pmid = {30802454},
issn = {1090-2422},
mesh = {*CRISPR-Cas Systems ; Colorectal Neoplasms, Hereditary Nonpolyposis/genetics/*pathology ; *Gene Editing ; Genomics/*methods ; HeLa Cells ; Humans ; *Microsatellite Instability ; MutS Homolog 2 Protein/*genetics ; *Mutation ; Phenotype ; },
abstract = {Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI[+] phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.},
}
@article {pmid30802405,
year = {2019},
author = {Yin, J and Hou, S and Wang, Q and Bao, L and Liu, D and Yue, Y and Yao, W and Gao, X},
title = {Microenvironment-Responsive Delivery of the Cas9 RNA-Guided Endonuclease for Efficient Genome Editing.},
journal = {Bioconjugate chemistry},
volume = {30},
number = {3},
pages = {898-906},
doi = {10.1021/acs.bioconjchem.9b00022},
pmid = {30802405},
issn = {1520-4812},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Endonucleases/*metabolism ; Gene Editing/*methods ; Humans ; Matrix Metalloproteinase 2/metabolism ; RNA, Guide/genetics ; *Tumor Microenvironment ; },
abstract = {Successful and efficient delivery of Cas9 protein and gRNA into cells is critical for genome editing and its therapeutic application. In this study, we developed an improved supercharged polypeptide (SCP) mediated delivery system based on dithiocyclopeptide linker to realize the effective genome editing in tumor cells. The fusion protein Cas9-linker-SCP (Cas9-LS) forms positively charged complexes with gRNA in vitro to provide possibilities for gRNA delivery into cells. Under the microenvironment of tumor cells, the dithiocyclopeptide linker, containing matrix metalloproteinase 2 (MMP-2) sensitive sequence and an intramolecular disulfide bond, can be completely disconnected to promote the release of Cas9 protein with the nuclear localization sequence (NLS) in the cytoplasm and transfer to the cell nucleus for highly efficient genome editing, resulting in an obvious increase of indel efficiency in comparison to fusion protein without dithiocyclopeptide linker (Cas9-SCP). Furthermore, Cas9-LS shows no significant cytotoxicity and minimal hemolytic activity. We envision that the microenvironment-responsive Cas9 protein delivery system can facilitate more efficient genome editing in tumor cells.},
}
@article {pmid30802343,
year = {2019},
author = {Raab, N and Mathias, S and Alt, K and Handrick, R and Fischer, S and Schmieder, V and Jadhav, V and Borth, N and Otte, K},
title = {CRISPR/Cas9-Mediated Knockout of MicroRNA-744 Improves Antibody Titer of CHO Production Cell Lines.},
journal = {Biotechnology journal},
volume = {14},
number = {5},
pages = {e1800477},
doi = {10.1002/biot.201800477},
pmid = {30802343},
issn = {1860-7314},
mesh = {Animals ; Antibodies/*metabolism ; CHO Cells/*metabolism ; *CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Engineering/*methods ; Cloning, Molecular ; Cricetulus ; Gene Editing/methods ; Gene Knockdown Techniques ; Genetic Loci ; MAP Kinase Kinase 4 ; MicroRNAs/*genetics/metabolism ; RNA, Guide/genetics ; Staphylococcal Protein A ; Transfection ; },
abstract = {MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L[-1] compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L[-1] , pointing towards the potential of miRNA KO for cell line engineering.},
}
@article {pmid30801858,
year = {2019},
author = {Yu, J and Lu, W and Ge, T and Huang, R and Chen, B and Ye, M and Bai, Y and Shi, G and Songyang, Z and Ma, W and Huang, J},
title = {Interaction Between Sympk and Oct4 Promotes Mouse Embryonic Stem Cell Proliferation.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {6},
pages = {743-753},
doi = {10.1002/stem.2992},
pmid = {30801858},
issn = {1549-4918},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Cytoskeletal Proteins/deficiency/*genetics ; Gene Deletion ; Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Genes, Reporter ; *Genome ; Genomic Instability ; Green Fluorescent Proteins/genetics/metabolism ; Membrane Proteins/deficiency/*genetics ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Nuclear Proteins/deficiency/*genetics ; Octamer Transcription Factor-3/*genetics/metabolism ; Pluripotent Stem Cells/cytology/*metabolism ; Signal Transduction ; Teratoma/genetics/metabolism/pathology ; Tight Junctions/metabolism ; },
abstract = {The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.},
}
@article {pmid30801649,
year = {2019},
author = {Hoshiga, F and Yoshizaki, K and Takao, N and Miyanaga, K and Tanji, Y},
title = {Modification of T2 phage infectivity toward Escherichia coli O157:H7 via using CRISPR/Cas9.},
journal = {FEMS microbiology letters},
volume = {366},
number = {4},
pages = {},
doi = {10.1093/femsle/fnz041},
pmid = {30801649},
issn = {1574-6968},
mesh = {Bacteriophage T4/*genetics/*pathogenicity ; *CRISPR-Cas Systems ; Escherichia coli O157/*virology ; Phage Therapy ; },
abstract = {Phage therapy is getting considerable attention as a method for prophylaxis of food poisoning caused by Escherichia coli O157:H7, an important pathogen causing life-threatening bloody diarrhea. Despite previous studies have shown the feasibility of phage therapy to E. coli O157:H7, promising results have not been obtained in vivo yet. A major drawback of phage therapy is that bacteriophages have high specificity and cannot infect all the sub-strains of a particular pathogenic strain. To overcome this hurdle, we thought to establish a way to artificially expand the host-range of E. coli O157:H7-specific phages. To develop a proof-of-concept for this method, we focused on T2 phage, which cannot infect E. coli O157:H7 strains, and PP01 phage, which displays broad infectivity to them, and attempted to make T2 phage able to infect E. coli O157:H7 as efficiently as PP01. We report the trials of T2 genome editing using the CRISPR/Cas9 system and the modification of both long and short tail fibers of this phage based on comparison with PP01. The resultant recombinant showed the adsorption rate comparable to PP01. Thus, we provided the evidence that the short tail fiber of PP01 plays an important role in adsorption to E. coli O157:H7.},
}
@article {pmid30799057,
year = {2019},
author = {Wang, E and Lu, SX and Pastore, A and Chen, X and Imig, J and Chun-Wei Lee, S and Hockemeyer, K and Ghebrechristos, YE and Yoshimi, A and Inoue, D and Ki, M and Cho, H and Bitner, L and Kloetgen, A and Lin, KT and Uehara, T and Owa, T and Tibes, R and Krainer, AR and Abdel-Wahab, O and Aifantis, I},
title = {Targeting an RNA-Binding Protein Network in Acute Myeloid Leukemia.},
journal = {Cancer cell},
volume = {35},
number = {3},
pages = {369-384.e7},
pmid = {30799057},
issn = {1878-3686},
support = {R01 CA173636/CA/NCI NIH HHS/United States ; P30 CA016087/CA/NCI NIH HHS/United States ; R01 CA194923/CA/NCI NIH HHS/United States ; R01 CA228135/CA/NCI NIH HHS/United States ; R01 HL128239/HL/NHLBI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA216421/CA/NCI NIH HHS/United States ; R01 CA169784/CA/NCI NIH HHS/United States ; P01 CA013106/CA/NCI NIH HHS/United States ; },
mesh = {Alternative Splicing ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; *Gene Regulatory Networks ; Gene Targeting/*methods ; HL-60 Cells ; Homeodomain Proteins/genetics ; Humans ; Jurkat Cells ; Leukemia, Myeloid, Acute/genetics/metabolism/*pathology ; Male ; Mice ; Neoplasm Transplantation ; Prognosis ; Proteomics/*methods ; RNA-Binding Proteins/*genetics/metabolism ; Sequence Analysis, RNA/methods ; Survival Analysis ; *Up-Regulation ; },
abstract = {RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.},
}
@article {pmid30798518,
year = {2019},
author = {Scott, GJ and Gruzdev, A},
title = {Genome Editing in Mouse Embryos with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1960},
number = {},
pages = {23-40},
doi = {10.1007/978-1-4939-9167-9_2},
pmid = {30798518},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics/*physiology ; DNA End-Joining Repair/genetics/physiology ; Embryo, Mammalian/*metabolism ; Gene Editing ; Mice ; Mice, Transgenic ; },
abstract = {Transgenic mouse models can be subdivided into two main categories based on genomic location: (1) targeted genomic manipulation and (2) random integration into the genome. Despite the potential confounding insertional mutagenesis and host locus-dependent expression, random integration transgenics allowed for rapid in vivo assessment of gene/protein function. Since precise genomic manipulation required the time-consuming prerequisite of first generating genetically modified embryonic stem cells, the rapid nature of generating random integration transgenes remained a strong benefit outweighing various disadvantages. The advent of targetable nucleases, such as CRISPR/Cas9, has eliminated the prerequisite of first generating genetically modified embryonic stem cells for some types of targeted genomic mutations. This chapter outlines the generation of mouse models with targeted genomic manipulation using the CRISPR/Cas9 system directly into single cell mouse embryos.},
}
@article {pmid30798517,
year = {2019},
author = {Gruzdev, A and Scott, GJ and Hagler, TB and Ray, MK},
title = {CRISPR/Cas9-Assisted Genome Editing in Murine Embryonic Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1960},
number = {},
pages = {1-21},
doi = {10.1007/978-1-4939-9167-9_1},
pmid = {30798517},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Embryonic Stem Cells/*metabolism ; Gene Editing/*methods ; Mice ; Mice, Transgenic ; Mutation/genetics ; },
abstract = {The study of gene function in normal human physiology and pathophysiology is complicated by countless factors such as genetic diversity (~98 million SNPs identified in the human genome as of June 2015), environmental exposure, epigenetic imprinting, maternal/in utero exposure, diet, exercise, age, sex, socioeconomic factors, and many other variables. Inbred mouse lines have allowed researchers to control for many of the variables that define human diversity but complicate the study of the human genome, gene/protein function, cellular and molecular pathways, and countless other genetic diseases. Furthermore, genetically modified mouse models enable us to generate and study mice whose genomes differ by as little as a single point mutation while controlling for non-genomic variables. This allows researchers to elucidate the quintessential function of a gene, which will further not only our scientific understanding, but provide key insight into human health and disease. Recent advances in CRISPR/Cas9 genome editing have revolutionized scientific protocols for introducing mutations into the mammalian genome. The ensuing chapter provides an overview of CRISPR/Cas9 genome editing in murine embryonic stem cells for the generation of genetically modified mouse models.},
}
@article {pmid30797903,
year = {2019},
author = {Thöne, FMB and Kurrle, NS and von Melchner, H and Schnütgen, F},
title = {CRISPR/Cas9-mediated generic protein tagging in mammalian cells.},
journal = {Methods (San Diego, Calif.)},
volume = {164-165},
number = {},
pages = {59-66},
doi = {10.1016/j.ymeth.2019.02.018},
pmid = {30797903},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/*genetics ; Chromatography, Affinity/instrumentation/methods ; Gene Editing/instrumentation/*methods ; Gene Targeting/instrumentation/*methods ; Genes, Reporter/genetics ; Genetic Vectors/genetics ; Green Fluorescent Proteins/genetics/isolation &amp; purification/metabolism ; HEK293 Cells ; Humans ; Mechanistic Target of Rapamycin Complex 1/genetics/isolation &amp; purification/metabolism ; Microscopy, Confocal/instrumentation/methods ; Microscopy, Fluorescence/instrumentation/methods ; Plasmids/genetics ; Protein Interaction Mapping/instrumentation/*methods ; Proteomics/methods ; Recombinant Fusion Proteins/*genetics/isolation &amp; purification/metabolism ; Signal Transduction/genetics ; Transfection/instrumentation/methods ; },
abstract = {Systematic protein localization and protein-protein interaction studies to characterize specific protein functions are most effectively performed using tag-based assays. Ideally, protein tags are introduced into a gene of interest by homologous recombination to ensure expression from endogenous control elements. However, inefficient homologous recombination makes this approach difficult in mammalian cells. Although gene targeting efficiency by homologous recombination increased dramatically with the development of designer endonuclease systems such as CRISPR/Cas9 capable of inducing DNA double-strand breaks with unprecedented accuracy, the strategies still require synthesis or cloning of homology templates for every single gene. Recent developments have shown that endogenous protein tagging can be achieved efficiently in a homology independent manner. Hence, combinations between CRISPR/Cas9 and generic tag-donor plasmids have been used successfully for targeted gene modifications in mammalian cells. Here, we developed a tool kit comprising a CRISPR/Cas9 expression vector with several EGFP encoding plasmids that should enable tagging of almost every protein expressed in mammalian cells. By performing protein-protein interaction and subcellular localization studies of mTORC1 signal transduction pathway-related proteins expressed in HEK293T cells, we show that tagged proteins faithfully reflect the behavior of their native counterparts under physiological conditions.},
}
@article {pmid30796544,
year = {2020},
author = {Ordon, J and Bressan, M and Kretschmer, C and Dall'Osto, L and Marillonnet, S and Bassi, R and Stuttmann, J},
title = {Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.},
journal = {Functional &amp; integrative genomics},
volume = {20},
number = {1},
pages = {151-162},
pmid = {30796544},
issn = {1438-7948},
mesh = {Alleles ; Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Editing/*methods ; Genome, Plant ; Mutation ; Promoter Regions, Genetic ; Ubiquitin/genetics ; },
abstract = {Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.},
}
@article {pmid30796164,
year = {2019},
author = {O'Connor, CM and Hoffa, MT and Taylor, SE and Avelar, RA and Narla, G},
title = {Protein phosphatase 2A Aα regulates Aβ protein expression and stability.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {15},
pages = {5923-5934},
pmid = {30796164},
issn = {1083-351X},
support = {F30 CA224979/CA/NCI NIH HHS/United States ; TL1 TR002549/TR/NCATS NIH HHS/United States ; T32 CA009676/CA/NCI NIH HHS/United States ; T32 GM008803/GM/NIGMS NIH HHS/United States ; R01 CA240993/CA/NCI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; R01 CA181654/CA/NCI NIH HHS/United States ; },
mesh = {Amyloid beta-Peptides/*biosynthesis/genetics ; Animals ; CRISPR-Cas Systems ; Female ; *Gene Expression Regulation ; HCT116 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Protein Binding ; Protein Phosphatase 2/genetics/*metabolism ; Protein Stability ; },
abstract = {Protein phosphatase 2A (PP2A) represses many oncogenic signaling pathways and is an important tumor suppressor. PP2A comprises three distinct subunits and forms through a highly regulated biogenesis process, with the scaffolding A subunit existing as two highly related isoforms, Aα and Aβ. PP2A's tumor-suppressive functions have been intensely studied, and PP2A inactivation has been shown to be a prerequisite for tumor formation. Interestingly, although partial loss of the Aα isoform is growth promoting, complete Aα loss has no transformative properties. Additionally, in cancer patients, Aα is found to be inactivated in a haploinsufficient manner. Using both cellular and in vivo systems, colorectal and endometrial cancer cell lines, and biochemical and cellular assays, here we examined why the complete loss of Aα does not promote tumorigenesis. CRISPR/Cas9-mediated homozygous Aα deletion resulted in decreased colony formation and tumor growth across multiple cell lines. Protein expression analysis of PP2A family members revealed that the Aα deletion markedly up-regulates Aβ protein expression by increasing Aβ protein stability. Aβ knockdown in control and Aα knockout cell lines indicated that Aβ is necessary for cell survival in the Aα knockout cells. In the setting of Aα deficiency, co-immunoprecipitation analysis revealed increased binding of specific PP2A regulatory subunits to Aβ, and knockdown of these regulatory subunits restored colony-forming ability. Taken together, our results uncover a mechanism by which PP2A Aα regulates Aβ protein stability and activity and suggests why homozygous loss of Aα is rarely seen in cancer patients.},
}
@article {pmid30796102,
year = {2019},
author = {Casden, N and Behar, O},
title = {An approach for accelerated isolation of genetically manipulated cell clones with reduced clonal variability.},
journal = {Journal of cell science},
volume = {132},
number = {6},
pages = {},
doi = {10.1242/jcs.217661},
pmid = {30796102},
issn = {1477-9137},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; Cell Line ; Clone Cells/*physiology ; Colony-Forming Units Assay ; *Culture Media, Conditioned ; Gene Editing ; Humans ; Mutation ; Signal Transduction ; },
abstract = {Genomic editing methods, such as the CRISPR/Cas9 system, are routinely used to study gene function in somatic cells. Owing to the heterogeneity of mutations, it is necessary to purify cell clones grown from high dilution to the point of colony formation, which can be a time-consuming process. Here, we tested a modified approach in which we seeded cells at high dilution, together with non-edited carrier cells. As a comparison, cells were also grown at high dilution with conditioned medium from a high-density culture. When using carrier cells or conditioned medium, the formation of cell colonies is accelerated. Additionally, clones grown with carrier cells are more similar to the parental lines in terms of their tumorigenic properties. Surprisingly, key signaling cascades are highly divergent between clones isolated from low-density cultures, even with conditioned medium, in contrast to clones isolated with carrier cells. Thus, our study uncovers a significant limitation using the common approach of isolating cell clones following genetic modifications and suggests an alternative method that mitigates the problem of heterogeneity of gene expression between clones.This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid30796047,
year = {2019},
author = {Gultekin, Y and Steller, H},
title = {Axin proteolysis by Iduna is required for the regulation of stem cell proliferation and intestinal homeostasis in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {6},
pages = {},
pmid = {30796047},
issn = {1477-9129},
support = {R01 GM060124/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Axin Protein/*metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Crosses, Genetic ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/*metabolism ; Female ; Gene Expression Regulation, Developmental ; Green Fluorescent Proteins/metabolism ; Homeostasis ; Intestines/*physiology ; Male ; Models, Genetic ; Mutation ; Poly-ADP-Ribose Binding Proteins/*metabolism ; RNA Interference ; Recombinant Proteins/metabolism ; Signal Transduction ; Stem Cells/*cytology ; Tankyrases/metabolism ; Ubiquitin-Protein Ligases/*metabolism ; Wnt1 Protein/*metabolism ; },
abstract = {Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-β catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/β-catenin signaling. However, its physiological significance remains to be demonstrated. Here, we generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.},
}
@article {pmid30795863,
year = {2019},
author = {Chen, X and Wei, M and Liu, X and Song, S and Wang, L and Yang, X and Song, Y},
title = {Construction and validation of the CRISPR/dCas9-EZH2 system for targeted H3K27Me3 modification.},
journal = {Biochemical and biophysical research communications},
volume = {511},
number = {2},
pages = {246-252},
doi = {10.1016/j.bbrc.2019.02.011},
pmid = {30795863},
issn = {1090-2104},
mesh = {3T3-L1 Cells ; Adipogenesis ; Animals ; CCAAT-Enhancer-Binding Protein-alpha/*genetics ; *CRISPR-Cas Systems ; Enhancer of Zeste Homolog 2 Protein/*genetics ; Epigenesis, Genetic ; Histone Code ; Histones/*genetics ; Methylation ; Mice ; Promoter Regions, Genetic ; },
abstract = {Cell phenotypes are closely related to the epigenome, which could be precisely regulated by the targeted manipulation of epigenetic marks. Here, we have successfully produced a targeted histone methylation system, which consists of nuclease-null dCas9 protein, the sgRNA fused with PP7 RNA aptamers and the Enhancer of Zeste Homolog 2 (EZH2) fused to PP7 coat protein (PCP). Guided by the dCas9/sgRNA-PP7, the PCP-EZH2 can specifically target gene loci to catalyze 3 methylation of histone H3 lysine 27, resulting in the inhibition of gene expression. This kind of gene inhibition system is supposed to be highly effective, specific and flexible. As a proof-of-concept study, sgRNA targeting C/ebpα promoter region was designed. In the cells co-infected with the dCas9, sgRNA/C/ebpα-PP7 and PCP-EZH2, the expression of C/ebpα gene was significantly reduced via induction of trimethylation to H3K27 on C/ebpα promoter, with the results epigenetically inherited in the daughter cells. In conclusion, our results successfully established a gene modification system consisting of dCas9/sgRNA-PP7 and PCP-EZH2, providing a robust tool for targeted manipulation of gene epigenetic modification and expression.},
}
@article {pmid30795631,
year = {2019},
author = {Bozic, B and Repac, J and Djordjevic, M},
title = {Endogenous Gene Regulation as a Predicted Main Function of Type I-E CRISPR/Cas System in E. coli.},
journal = {Molecules (Basel, Switzerland)},
volume = {24},
number = {4},
pages = {},
pmid = {30795631},
issn = {1420-3049},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coliphages/*genetics ; Computational Biology ; DNA/genetics/metabolism ; DNA, Bacterial/genetics/metabolism ; Escherichia coli/*genetics/metabolism/virology ; *Gene Expression Regulation, Bacterial ; Genetic Loci ; *Genome, Bacterial ; *Genome, Viral ; RNA, Messenger/genetics/metabolism ; Sequence Alignment ; Transcription, Genetic ; },
abstract = {CRISPR/Cas is an adaptive bacterial immune system, whose CRISPR array can actively change in response to viral infections. However, Type I-E CRISPR/Cas in E. coli (an established model system), appears not to exhibit such active adaptation, which suggests that it might have functions other than immune response. Through computational analysis, we address the involvement of the system in non-canonical functions. To assess targets of CRISPR spacers, we align them against both E. coli genome and an exhaustive (~230) set of E. coli viruses. We systematically investigate the obtained alignments, such as hit distribution with respect to genome annotation, propensity to target mRNA, the target functional enrichment, conservation of CRISPR spacers and putative targets in related bacterial genomes. We find that CRISPR spacers have a statistically highly significant tendency to target i) host compared to phage genomes, ii) one of the two DNA strands, iii) genomic dsDNA rather than mRNA, iv) transcriptionally active regions, and v) sequences (cis-regulatory elements) with slower turn-over rate compared to CRISPR spacers (trans-factors). The results suggest that the Type I-E CRISPR/Cas system has a major role in transcription regulation of endogenous genes, with a potential to rapidly rewire these regulatory interactions, with targets being selected through naïve adaptation.},
}
@article {pmid30795542,
year = {2019},
author = {Brane, AC and Tollefsbol, TO},
title = {Targeting Telomeres and Telomerase: Studies in Aging and Disease Utilizing CRISPR/Cas9 Technology.},
journal = {Cells},
volume = {8},
number = {2},
pages = {},
pmid = {30795542},
issn = {2073-4409},
support = {P30 DK079626/DK/NIDDK NIH HHS/United States ; R01 CA178441/CA/NCI NIH HHS/United States ; R01 CA204346/CA/NCI NIH HHS/United States ; },
mesh = {Aging/*genetics ; CRISPR-Cas Systems/*genetics ; Disease/*genetics ; Epigenesis, Genetic ; Humans ; Telomerase/*metabolism ; Telomere/*metabolism ; },
abstract = {Telomeres and telomerase provide a unique and important avenue of study in improving both life expectancy and quality of life due to their close association with aging and disease. While major advances in our understanding of these two biological mediators have characterized the last two decades, previous studies have been limited by the inability to affect change in real time within living cells. The last three years, however, have witnessed a huge step forward to overcome this limitation. The advent of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system has led to a wide array of targeted genetic studies that are already being employed to modify telomeres and telomerase, as well as the genes that affect them. In this review, we analyze studies utilizing the technology to target and modify telomeres, telomerase, and their closely associated genes. We also discuss how these studies can provide insight into the biology and mechanisms that underlie aging, cancer, and other diseases.},
}
@article {pmid30794791,
year = {2019},
author = {Almendros, C and Kieper, SN and Brouns, SJJ},
title = {CRISPR-Cas Systems Reduced to a Minimum.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {641-642},
doi = {10.1016/j.molcel.2019.02.005},
pmid = {30794791},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {In two recent studies in Molecular Cell, Wright et al. (2019) report complete spacer integration by a Cas1 mini-integrase and Edraki et al. (2019) describe accurate genome editing by a small Cas9 ortholog with less stringent PAM requirements.},
}
@article {pmid30794581,
year = {2019},
author = {Babačić, H and Mehta, A and Merkel, O and Schoser, B},
title = {CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-expansion diseases: A systematic review.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0212198},
pmid = {30794581},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Repeat Expansion ; Disease Models, Animal ; Gene Editing/*methods/trends ; Gene Silencing ; *Gene Transfer Techniques ; *Genetic Diseases, Inborn/genetics/pathology/therapy ; Humans ; MEDLINE ; Mice ; *Neuromuscular Diseases/genetics/pathology/therapy ; },
abstract = {INTRODUCTION: The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment.<br><br>METHODS AND DATA ACQUISITION: We searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD.<br><br>RESULTS: We obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review.<br><br>DISCUSSION: Here we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.},
}
@article {pmid30794383,
year = {2019},
author = {Rui, Y and Wilson, DR and Sanders, K and Green, JJ},
title = {Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing.},
journal = {ACS applied materials &amp; interfaces},
volume = {11},
number = {11},
pages = {10472-10480},
pmid = {30794383},
issn = {1944-8252},
support = {R01 CA195503/CA/NCI NIH HHS/United States ; R01 EB022148/EB/NIBIB NIH HHS/United States ; R01 EB016721/EB/NIBIB NIH HHS/United States ; S10 OD016374/OD/NIH HHS/United States ; R01 CA228133/CA/NCI NIH HHS/United States ; },
mesh = {Amines/chemistry ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Survival/drug effects ; DNA/chemistry/*metabolism ; Esters/chemistry ; Gene Editing/*methods ; Humans ; Nanoparticles/chemistry/toxicity ; Plasmids/genetics/metabolism ; Polymers/chemical synthesis/*chemistry ; RNA, Small Interfering/chemistry/*metabolism ; Transfection ; },
abstract = {Functional codelivery of plasmid DNA and RNA oligonucleotides in the same nanoparticle system is challenging due to differences in their physical properties as well as their intracellular locations of function. In this study, we synthesized a series of reducible branched ester-amine quadpolymers (rBEAQs) and investigated their ability to coencapsulate and deliver DNA plasmids and RNA oligos. The rBEAQs are designed to leverage polymer branching, reducibility, and hydrophobicity to successfully cocomplex DNA and RNA in nanoparticles at low polymer to nucleic acid w/w ratios and enable high delivery efficiency. We validate the synthesis of this new class of biodegradable polymers, characterize the self-assembled nanoparticles that these polymers form with diverse nucleic acids, and demonstrate that the nanoparticles enable safe, effective, and efficient DNA-siRNA codelivery as well as nonviral CRISPR-mediated gene editing utilizing Cas9 DNA and sgRNA codelivery.},
}
@article {pmid30793977,
year = {2019},
author = {Pellenz, S and Phelps, M and Tang, W and Hovde, BT and Sinit, RB and Fu, W and Li, H and Chen, E and Monnat, RJ},
title = {New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion.},
journal = {Human gene therapy},
volume = {30},
number = {7},
pages = {814-828},
pmid = {30793977},
issn = {1557-7422},
support = {P01 CA077852/CA/NCI NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Chromosome Breakpoints ; *Chromosomes, Human ; Gene Editing ; Gene Expression ; Gene Knock-In Techniques ; Gene Targeting ; Genetic Loci ; Genome, Human ; Geographic Mapping ; Humans ; *Mutagenesis, Insertional ; *Transgenes ; },
abstract = {This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 in human rhabdomyosarcoma cells. An engineering "toolkit" was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.},
}
@article {pmid30793514,
year = {2019},
author = {Bergeron, F and Boulende Sab, A and Bouchard, MF and Taniguchi, H and Souchkova, O and Brousseau, C and Tremblay, JJ and Pilon, N and Viger, RS},
title = {Phosphorylation of GATA4 serine 105 but not serine 261 is required for testosterone production in the male mouse.},
journal = {Andrology},
volume = {7},
number = {3},
pages = {357-372},
doi = {10.1111/andr.12601},
pmid = {30793514},
issn = {2047-2927},
support = {MOP-14796//Canadian Institutes of Health Research (CIHR)/International ; //Fonds de la Recherche du Qu&#xe9;bec - Sant&#xe9; (FRQS)/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; GATA4 Transcription Factor/*metabolism ; Male ; Mice ; Phosphorylation ; Serine/*metabolism ; Testis/*metabolism ; Testosterone/*biosynthesis ; },
abstract = {BACKGROUND: GATA4 is a transcription factor essential for male sex determination, testicular differentiation during fetal development, and male fertility in the adult. GATA4 exerts part of its function by regulating multiple genes in the steroidogenic enzyme pathway. In spite of these crucial roles, how the activity of this factor is regulated remains unclear.<br><br>OBJECTIVES: Studies in gonadal cell lines have shown that GATA4 is phosphorylated on at least two serine residues-serine 105 (S105) and serine 261 (S261)-and that this phosphorylation is important for GATA4 activity. The objective of the present study is to characterize the endogenous role of GATA4 S105 and S261 phosphorylation in the mouse testis.<br><br>MATERIALS AND METHODS: We examined both previously described GATA4 S105A mice and a novel GATA4 S261A knock-in mouse that we generated by CRISPR/Cas9 gene editing. The male phenotype of the mutants was characterized by assessing androgen-dependent organ weights, hormonal profiles, and expression of multiple testicular target genes using standard biochemical and molecular biology techniques.<br><br>RESULTS: The fecundity of crosses between GATA4 S105A mice was reduced but without a change in sex ratio. The weight of androgen-dependent organs was smaller when compared to wild-type controls. Plasma testosterone levels showed a 70% decrease in adult GATA4 S105A males. This decrease was associated with a reduction in Cyp11a1, Cyp17a1, and Hsd17b3 expression. GATA4 S261A mice were viable and testis morphology appeared normal. Testosterone production and steroidogenic enzyme expression were not altered in GATA4 S261A males.<br><br>DISCUSSION AND CONCLUSION: Our analysis showed that blocking GATA4 S105 phosphorylation is associated with decreased androgen production in males. In contrast, S261 phosphorylation by itself is dispensable for GATA4 function. These results confirm that endogenous GATA4 action is essential for normal steroid production in males and that this activity requires phosphorylation on at least one serine residue.},
}
@article {pmid30793202,
year = {2019},
author = {Jabre, I and Reddy, ASN and Kalyna, M and Chaudhary, S and Khokhar, W and Byrne, LJ and Wilson, CM and Syed, NH},
title = {Does co-transcriptional regulation of alternative splicing mediate plant stress responses?.},
journal = {Nucleic acids research},
volume = {47},
number = {6},
pages = {2716-2726},
pmid = {30793202},
issn = {1362-4962},
support = {P 26333/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {Alternative Splicing/*genetics ; Animals ; Arabidopsis/genetics ; DNA Methylation/genetics ; Epigenesis, Genetic/physiology ; *Gene Expression Regulation, Plant ; Gene Regulatory Networks/genetics ; Humans ; Stress, Physiological/*genetics ; Transcription, Genetic/genetics ; },
abstract = {Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS.},
}
@article {pmid30793048,
year = {2019},
author = {Simeonov, DR and Brandt, AJ and Chan, AY and Cortez, JT and Li, Z and Woo, JM and Lee, Y and Carvalho, CMB and Indart, AC and Roth, TL and Zou, J and May, AP and Lupski, JR and Anderson, MS and Buaas, FW and Rokhsar, DS and Marson, A},
title = {A large CRISPR-induced bystander mutation causes immune dysregulation.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {70},
pmid = {30793048},
issn = {2399-3642},
support = {T32 GM007618/GM/NIGMS NIH HHS/United States ; S10 RR029668/RR/NCRR NIH HHS/United States ; S10 RR027303/RR/NCRR NIH HHS/United States ; T32 GM008568/GM/NIGMS NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; T32 AI007334/AI/NIAID NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cells, Cultured ; DNA Damage ; DNA Repair ; Gene Duplication ; Gene Editing/*methods ; Gene Expression Regulation/immunology ; Interleukin-2 Receptor alpha Subunit/*genetics/immunology ; Mice, Inbred NOD ; *Mutation ; T-Lymphocytes/*immunology/metabolism ; T-Lymphocytes, Regulatory/*immunology/metabolism ; },
abstract = {A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic "bystander" mutations that escape detection by routine targeted genotyping assays.},
}
@article {pmid30792150,
year = {2019},
author = {Bäck, S and Necarsulmer, J and Whitaker, LR and Coke, LM and Koivula, P and Heathward, EJ and Fortuno, LV and Zhang, Y and Yeh, CG and Baldwin, HA and Spencer, MD and Mejias-Aponte, CA and Pickel, J and Hoffman, AF and Spivak, CE and Lupica, CR and Underhill, SM and Amara, SG and Domanskyi, A and Anttila, JE and Airavaara, M and Hope, BT and Hamra, FK and Richie, CT and Harvey, BK},
title = {Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.},
journal = {Neuron},
volume = {102},
number = {1},
pages = {105-119.e8},
doi = {10.1016/j.neuron.2019.01.035},
pmid = {30792150},
issn = {1097-4199},
mesh = {Adult Germline Stem Cells/*metabolism ; Animals ; Brain/*metabolism ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Deoxyribonuclease I/genetics ; Dependovirus ; Disease Models, Animal ; Dopamine Plasma Membrane Transport Proteins/genetics ; Dopaminergic Neurons/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques ; Gene Targeting/*methods ; Genetic Vectors ; Integrases ; Luminescent Proteins/genetics ; Neurons/metabolism ; Promoter Regions, Genetic ; RNA, Guide ; Rats ; Rats, Transgenic ; Tyrosine 3-Monooxygenase/genetics ; },
abstract = {Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.},
}
@article {pmid30791357,
year = {2019},
author = {Fiaz, S and Ahmad, S and Noor, MA and Wang, X and Younas, A and Riaz, A and Riaz, A and Ali, F},
title = {Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities.},
journal = {International journal of molecular sciences},
volume = {20},
number = {4},
pages = {},
pmid = {30791357},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Edible Grain/*genetics/*standards ; Food Quality ; *Gene Editing ; Genome, Plant ; Genomics ; Mutagenesis ; Nutritive Value ; Oryza/*genetics/metabolism ; Quality Improvement ; Starch/metabolism ; },
abstract = {Grain quality improvement is a key target for rice breeders, along with yield. It is a multigenic trait that is simultaneously influenced by many factors. Over the past few decades, breeding for semi-dwarf cultivars and hybrids has significantly contributed to the attainment of high yield demands but reduced grain quality, which thus needs the attention of researchers. The availability of rice genome sequences has facilitated gene discovery, targeted mutagenesis, and revealed functional aspects of rice grain quality attributes. Some success has been achieved through the application of molecular markers to understand the genetic mechanisms for better rice grain quality; however, researchers have opted for novel strategies. Genomic alteration employing genome editing technologies (GETs) like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for reverse genetics has opened new avenues of research in the life sciences, including for rice grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting. Over the past few years many genes that are related to various aspects of rice grain quality have been successfully edited via CRISPR/Cas9 technology. Interestingly, studies on functional genomics at larger scales have become possible because of the availability of GETs. In this review, we discuss the progress made in rice by employing the CRISPR/Cas9 editing system and its eminent applications. We also elaborate possible future avenues of research with this system, and our understanding regarding the biological mechanism of rice grain quality improvement.},
}
@article {pmid30791200,
year = {2020},
author = {Zhou, J and Li, D and Wang, G and Wang, F and Kunjal, M and Joldersma, D and Liu, Z},
title = {Application and future perspective of CRISPR/Cas9 genome editing in fruit crops.},
journal = {Journal of integrative plant biology},
volume = {62},
number = {3},
pages = {269-286},
pmid = {30791200},
issn = {1744-7909},
support = {T32 GM008553/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics/*metabolism ; Fruit/genetics/*metabolism ; Gene Editing/*methods ; Plants, Genetically Modified/genetics/*metabolism ; },
abstract = {Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer-friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.},
}
@article {pmid30790630,
year = {2019},
author = {Cosentino, MS and Oses, C and Vázquez Echegaray, C and Solari, C and Waisman, A and Álvarez, Y and Petrone, MV and Francia, M and Schultz, M and Sevlever, G and Miriuka, S and Levi, V and Guberman, A},
title = {Kat6b Modulates Oct4 and Nanog Binding to Chromatin in Embryonic Stem Cells and Is Required for Efficient Neural Differentiation.},
journal = {Journal of molecular biology},
volume = {431},
number = {6},
pages = {1148-1159},
doi = {10.1016/j.jmb.2019.02.012},
pmid = {30790630},
issn = {1089-8638},
mesh = {Animals ; CRISPR-Cas Systems ; Chromatin/*metabolism ; Embryonic Stem Cells/*cytology/metabolism ; Gene Expression Regulation ; Histone Acetyltransferases/genetics/*metabolism ; Male ; Mice, Nude ; Nanog Homeobox Protein/*metabolism ; *Neurogenesis ; Octamer Transcription Factor-3/*metabolism ; },
abstract = {Chromatin remodeling is fundamental for the dynamical changes in transcriptional programs that occur during development and stem cell differentiation. The histone acetyltransferase Kat6b is relevant for neurogenesis in mouse embryos, and mutations of this gene cause intellectual disability in humans. However, the molecular mechanisms involved in Kat6b mutant phenotype and the role of this chromatin modifier in embryonic stem (ES) cells remain elusive. In this work, we show that Kat6b is expressed in ES cells and is repressed during differentiation. Moreover, we found that this gene is regulated by the pluripotency transcription factors Nanog and Oct4. To study the functional relevance of Kat6b in ES cells, we generated a Kat6b knockout ES cell line (K6b-/-) using CRISPR/Cas9. Fluorescence correlation spectroscopy analyses suggest a more compact chromatin organization in K6b-/- cells and impaired interactions of Oct4 and Nanog with chromatin. Remarkably, K6b-/- cells showed a reduced efficiency to differentiate to neural lineage. These results reveal a role of Kat6b as a modulator of chromatin plasticity, its impact on chromatin-transcription factors interactions and its influence on cell fate decisions during neural development.},
}
@article {pmid30788819,
year = {2019},
author = {Sibbritt, T and Osteil, P and Fan, X and Sun, J and Salehin, N and Knowles, H and Shen, J and Tam, PPL},
title = {Gene Editing of Mouse Embryonic and Epiblast Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1940},
number = {},
pages = {77-95},
doi = {10.1007/978-1-4939-9086-3_6},
pmid = {30788819},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Frameshift Mutation/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Germ Layers/*cytology ; Mice ; Mouse Embryonic Stem Cells/*cytology ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.},
}
@article {pmid30788791,
year = {2019},
author = {Wong, L and Holdridge, B and Engel, J and Xu, P},
title = {Genetic Tools for Streamlined and Accelerated Pathway Engineering in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1927},
number = {},
pages = {155-177},
doi = {10.1007/978-1-4939-9142-6_11},
pmid = {30788791},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cloning, Molecular ; DNA, Fungal ; *Gene Editing/methods ; Gene Expression ; Gene Library ; Genes, Reporter ; *Genetic Engineering ; *Metabolic Engineering/methods ; *Metabolic Networks and Pathways ; Plasmids/genetics ; Promoter Regions, Genetic ; RNA, Catalytic ; RNA, Guide ; Transformation, Genetic ; Yarrowia/*genetics/*metabolism ; },
abstract = {Yarrowia lipolytica is an industrial oleaginous yeast that has many attractive physiological and metabolic characteristics for various biotechnological applications. Although it has a long history of industrial applications, the number of genetic tools available to effectively and efficiently engineer Y. lipolytica still falls behind the vast number of tools available for common organisms such as Escherichia coli and Saccharomyces cerevisiae. In this protocol, we have developed a complete and versatile genetic toolkit tailored for facile genetic manipulation in Y. lipolytica. We created a versatile DNA assembly platform YaliBrick, which can streamline the cloning of large multigene pathways with reused genetic parts. We established a sensitive luciferase reporter assay to characterize a set of 12 native promoters. In addition, we used YaliBrick to generate different gene configurations in multigene constructs. The five-gene biosynthetic pathway of the anticancer, antimicrobial pigment violacein was rapidly assembled in 1 week to demonstrate the simplicity and effectiveness of integrating pathway-balancing strategies with our YaliBrick vectors. In the end, we incorporated CRISPR-Cas9 into our YaliBrick vectors and achieved indel mutation and frameshift gene deletion at the CAN1 (arginine permease) genomic loci of Yarrowia lipolytica. The reported protocol provides a standard procedure to streamline and accelerate metabolic pathway engineering in Yarrowia lipolytica.},
}
@article {pmid30788786,
year = {2019},
author = {Shi, S and Liang, Y and Ang, EL and Zhao, H},
title = {Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1927},
number = {},
pages = {73-91},
doi = {10.1007/978-1-4939-9142-6_6},
pmid = {30788786},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Flow Cytometry ; Gene Order ; *Genetic Engineering ; Genetic Loci ; Genome, Fungal ; Genomics/methods ; Plasmids/genetics ; Real-Time Polymerase Chain Reaction ; Saccharomyces cerevisiae/*genetics ; Transformation, Genetic ; },
abstract = {Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) to enable unprecedented high-efficiency, multicopy, markerless integrations of large biochemical pathways into the S. cerevisiae genome. Detailed protocols are provided on the entire workflow which includes pDi-CRISPR plasmid and donor DNA construction, Di-CRISPR-mediated integration and analysis of integration efficiencies and copy numbers through flow cytometry and quantitative polymerase chain reaction (qPCR).},
}
@article {pmid30787936,
year = {2019},
author = {Charrier, A and Vergne, E and Dousset, N and Richer, A and Petiteau, A and Chevreau, E},
title = {Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {40},
pmid = {30787936},
issn = {1664-462X},
abstract = {Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.},
}
@article {pmid30787439,
year = {2019},
author = {Karakus, U and Thamamongood, T and Ciminski, K and Ran, W and Günther, SC and Pohl, MO and Eletto, D and Jeney, C and Hoffmann, D and Reiche, S and Schinköthe, J and Ulrich, R and Wiener, J and Hayes, MGB and Chang, MW and Hunziker, A and Yángüez, E and Aydillo, T and Krammer, F and Oderbolz, J and Meier, M and Oxenius, A and Halenius, A and Zimmer, G and Benner, C and Hale, BG and García-Sastre, A and Beer, M and Schwemmle, M and Stertz, S},
title = {MHC class II proteins mediate cross-species entry of bat influenza viruses.},
journal = {Nature},
volume = {567},
number = {7746},
pages = {109-112},
pmid = {30787439},
issn = {1476-4687},
support = {HHSN272201400008C/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chickens/genetics/immunology ; Chiroptera/genetics/immunology/metabolism/*virology ; Female ; Gene Expression Profiling ; HLA-DR Antigens/genetics/immunology/metabolism ; Histocompatibility Antigens Class II/genetics/immunology/*metabolism ; *Host Specificity/genetics/immunology ; Humans ; Influenza A virus/*immunology/*physiology ; Male ; Mice ; Mice, Knockout ; Respiratory System/virology ; Swine/genetics/immunology ; Viral Tropism/genetics/immunology ; Virus Replication ; Zoonoses/genetics/*immunology/metabolism/*virology ; },
abstract = {Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats[1,2]. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan[1,3,4], despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.},
}
@article {pmid30787394,
year = {2019},
author = {Lampi, Y and Van Looveren, D and Vranckx, LS and Thiry, I and Bornschein, S and Debyser, Z and Gijsbers, R},
title = {Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {2389},
pmid = {30787394},
issn = {2045-2322},
support = {P30 AI027763/AI/NIAID NIH HHS/United States ; //CIHR/Canada ; },
mesh = {*Adaptor Proteins, Signal Transducing/genetics/physiology ; Cell Line, Tumor ; Gene Knock-In Techniques ; Gene Knockout Techniques ; HEK293 Cells ; HIV Infections/*immunology ; HIV Integrase/metabolism ; HIV-1/*immunology ; Host Microbial Interactions/*immunology ; Humans ; *Transcription Factors/genetics/physiology ; Virus Integration/*immunology ; },
abstract = {To fulfill a productive infection cycle the human immunodeficiency virus (HIV) relies on host-cell factors. Interference with these co-factors holds great promise in protecting cells against HIV infection. LEDGF/p75, encoded by the PSIP1 gene, is used by the integrase (IN) protein in the pre-integration complex of HIV to bind host-cell chromatin facilitating proviral integration. LEDGF/p75 depletion results in defective HIV replication. However, as part of its cellular function LEDGF/p75 tethers cellular proteins to the host-cell genome. We used site-specific editing of the PSIP1 locus using CRISPR/Cas to target the aspartic acid residue in position 366 and mutated it to asparagine (D366N) to disrupt the interaction with HIV IN but retain LEDGF/p75 cellular function. The resulting cell lines demonstrated successful disruption of the LEDGF/p75 HIV-IN interface without affecting interaction with cellular binding partners. In line with LEDGF/p75 depleted cells, D366N cells did not support HIV replication, in part due to decreased integration efficiency. In addition, we confirm the remaining integrated provirus is more silent. Taken together, these results support the potential of site-directed CRISPR/Cas9 mediated knock-in to render cells more resistant to HIV infection and provides an additional strategy to protect patient-derived T-cells against HIV-1 infection as part of cell-based therapy.},
}
@article {pmid30786106,
year = {2019},
author = {Zarei, A and Razban, V and Hosseini, SE and Tabei, SMB},
title = {Creating cell and animal models of human disease by genome editing using CRISPR/Cas9.},
journal = {The journal of gene medicine},
volume = {21},
number = {4},
pages = {e3082},
doi = {10.1002/jgm.3082},
pmid = {30786106},
issn = {1521-2254},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; Models, Animal ; },
abstract = {A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR ("clustered regularly interspaced short palindromic repeats") and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock-in, knock-out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.},
}
@article {pmid30784179,
year = {2019},
author = {Bastet, A and Zafirov, D and Giovinazzo, N and Guyon-Debast, A and Nogué, F and Robaglia, C and Gallois, JL},
title = {Mimicking natural polymorphism in eIF4E by CRISPR-Cas9 base editing is associated with resistance to potyviruses.},
journal = {Plant biotechnology journal},
volume = {17},
number = {9},
pages = {1736-1750},
pmid = {30784179},
issn = {1467-7652},
mesh = {Alleles ; Arabidopsis/genetics/virology ; *CRISPR-Cas Systems ; Disease Resistance/*genetics ; Eukaryotic Initiation Factor-4E/*genetics ; *Gene Editing ; Peas/*genetics/virology ; Plant Diseases/*genetics/virology ; Plant Proteins/genetics ; Plants, Genetically Modified ; Potyvirus/*pathogenicity ; },
abstract = {In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus-resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss-of-function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR-nCas9-cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single-point mutation through C-to-G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome-editing technologies offers a feasible solution for engineering transgene-free genetic resistance in plants, even across species barriers.},
}
@article {pmid30783971,
year = {2019},
author = {Yu, W and Wu, Z},
title = {Use of AAV Vectors for CRISPR-Mediated In Vivo Genome Editing in the Retina.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1950},
number = {},
pages = {123-139},
pmid = {30783971},
issn = {1940-6029},
support = {ZIA EY000443-11//Intramural NIH HHS/United States ; ZIA EY000443-12//Intramural NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics/isolation &amp; purification ; Electroretinography ; *Gene Editing ; Gene Expression ; Gene Transfer Techniques ; Genetic Vectors/*genetics/isolation &amp; purification ; Humans ; Immunohistochemistry ; RNA, Guide ; Retina/*metabolism ; Transduction, Genetic ; Transgenes ; },
abstract = {Degenerative retinal diseases such as retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA) may lead to blindness without effective treatment. With the rapid advancement of the CRISPR/Cas9 genome editing technology, in vivo application of CRISPR/Cas9 holds immense potential for treatment of these diseases. Adeno-associated virus (AAV) vectors are an ideal gene transfer tool for delivery of CRISPR components to the retina. Here, we describe a protocol for utilizing an AAV-based CRISPR/Cas9 system for in vivo genome editing in the retina.},
}
@article {pmid30783736,
year = {2019},
author = {Biswas, S and Li, R and Yuan, Z and Zhang, D and Zhao, X and Shi, J},
title = {Development of methods for effective identification of CRISPR/Cas9-induced indels in rice.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {503-510},
pmid = {30783736},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing ; INDEL Mutation/genetics ; Oryza/*genetics ; },
abstract = {Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.},
}
@article {pmid30783652,
year = {2019},
author = {Lee, HJ and Gopalappa, R and Sunwoo, H and Choi, SW and Ramakrishna, S and Lee, JT and Kim, HH and Nam, JW},
title = {En bloc and segmental deletions of human XIST reveal X chromosome inactivation-involving RNA elements.},
journal = {Nucleic acids research},
volume = {47},
number = {8},
pages = {3875-3887},
pmid = {30783652},
issn = {1362-4962},
support = {T32 HD007396/HD/NICHD NIH HHS/United States ; },
mesh = {Alternative Splicing ; Animals ; *Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Chromosomes, Human, X/*chemistry/metabolism ; Clone Cells ; Exons ; Gene Editing/methods ; Genome, Human ; Humans ; K562 Cells ; Mice ; Promoter Regions, Genetic ; RNA, Guide/genetics/metabolism ; RNA, Long Noncoding/*genetics/metabolism ; *Sequence Deletion ; Species Specificity ; Whole Genome Sequencing ; *X Chromosome Inactivation ; },
abstract = {The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse Xist RNA, how the human XIST RNA controls XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, 11 smaller XIST segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence in situ hybridization experiments. Clones containing en bloc and promoter deletions that consistently displayed no XIST RNAs and a global up-regulation of X-linked genes confirmed that the deletion of XIST reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouse Xist. This Cas9-assisted dissection of XIST allowed us to understand the unique functional domains within the human XIST RNA.},
}
@article {pmid30782847,
year = {2019},
author = {Zhang, Z and Zhan, X and Kim, B and Wu, J},
title = {A proteomic approach identifies SAFB-like transcription modulator (SLTM) as a bidirectional regulator of GLI family zinc finger transcription factors.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {14},
pages = {5549-5561},
pmid = {30782847},
issn = {1083-351X},
support = {R01 NS096068/NS/NINDS NIH HHS/United States ; R21 NS104596/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chromatin ; Gene Editing ; Gene Knock-In Techniques ; Hedgehog Proteins/genetics/*metabolism ; Matrix Attachment Region Binding Proteins/genetics/*metabolism ; Mice ; Mice, Transgenic ; Nerve Tissue Proteins/genetics/*metabolism ; Nuclear Matrix-Associated Proteins/genetics/*metabolism ; Proteomics ; *Signal Transduction ; Zinc Finger Protein Gli3/genetics/*metabolism ; },
abstract = {In Sonic hedgehog (SHH) signaling, GLI family zinc finger (GLI)-mediated diverse gene transcription outcomes are strictly regulated and are important for SHH function in both development and disease. However, how the GLI factors differentially regulate transcription in response to variable SHH activities is incompletely understood. Here, using a newly generated, tagged Gli3 knock-in mouse (Gli3[TAP]), we performed proteomic analyses and identified the chromatin-associated SAFB-like transcription modulator (SLTM) as a GLI-interacting protein that context-dependently regulates GLI activities. Using immunoprecipitation and immunoblotting, RT-quantitative PCR, and ChIP assays, we show that SLTM interacts with all three GLI proteins and that its cellular levels are regulated by SHH. We also found that SLTM enhances GLI3 binding to chromatin and increases GLI3 repressor (GLI3R) form protein levels. In a GLI3-dependent manner, SLTM promoted the formation of a repressive chromatin environment and functioned as a GLI3 co-repressor. In the absence of GLI3 or in the presence of low GLI3 levels, SLTM co-activated GLI activator (GLIA)-mediated target gene activation and cell differentiation. Moreover, in vivo Sltm deletion generated through CRISPR/Cas9-mediated gene editing caused perinatal lethality and SHH-related abnormal ventral neural tube phenotypes. We conclude that SLTM regulates GLI factor binding to chromatin and contributes to the transcriptional outcomes of SHH signaling via a novel molecular mechanism.},
}
@article {pmid30782830,
year = {2019},
author = {Kunova Bosakova, M and Nita, A and Gregor, T and Varecha, M and Gudernova, I and Fafilek, B and Barta, T and Basheer, N and Abraham, SP and Balek, L and Tomanova, M and Fialova Kucerova, J and Bosak, J and Potesil, D and Zieba, J and Song, J and Konik, P and Park, S and Duran, I and Zdrahal, Z and Smajs, D and Jansen, G and Fu, Z and Ko, HW and Hampl, A and Trantirek, L and Krakow, D and Krejci, P},
title = {Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {10},
pages = {4316-4325},
pmid = {30782830},
issn = {1091-6490},
support = {R01 DE019567/DE/NIDCR NIH HHS/United States ; UL1 TR000124/TR/NCATS NIH HHS/United States ; R21 CA195273/CA/NCI NIH HHS/United States ; R01 GM127690/GM/NIGMS NIH HHS/United States ; R01 AR066124/AR/NIAMS NIH HHS/United States ; R01 AR062651/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cilia/*metabolism ; Fibroblast Growth Factors/metabolism ; HEK293 Cells ; Hedgehog Proteins/metabolism ; Humans ; Mice ; Mice, Knockout ; Models, Animal ; Molecular Docking Simulation ; NIH 3T3 Cells ; Phosphorylation ; Protein Interaction Domains and Motifs ; Protein Serine-Threonine Kinases/genetics/*metabolism ; Proteomics ; Receptor, Fibroblast Growth Factor, Type 1/metabolism ; Receptor, Fibroblast Growth Factor, Type 3/genetics/metabolism ; Receptor, Fibroblast Growth Factor, Type 4/metabolism ; Receptors, Fibroblast Growth Factor/genetics/*metabolism ; Signal Transduction ; },
abstract = {Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.},
}
@article {pmid30782165,
year = {2019},
author = {Hannafon, BN and Gin, AL and Xu, YF and Bruns, M and Calloway, CL and Ding, WQ},
title = {Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells.},
journal = {Cell communication and signaling : CCS},
volume = {17},
number = {1},
pages = {13},
pmid = {30782165},
issn = {1478-811X},
support = {U54 GM104938/GM/NIGMS NIH HHS/United States ; P30 CA225520/CA/NCI NIH HHS/United States ; },
mesh = {Biomarkers, Tumor/metabolism ; Breast Neoplasms/*metabolism/*pathology ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Hypoxia/drug effects ; Cell Line, Tumor ; Exosomes/drug effects/*metabolism ; Female ; Gene Ontology ; Green Fluorescent Proteins/metabolism ; Histone Deacetylases/*metabolism ; Humans ; Repressor Proteins/*metabolism ; *Signal Transduction/drug effects ; Tamoxifen/pharmacology ; Trans-Activators ; },
abstract = {BACKGROUND: Exosomes are small membrane-bound vesicles that contribute to tumor progression and metastasis by mediating cell-to-cell communication and modifying the tumor microenvironment at both local and distant sites. However, little is known about the predominant factors in exosomes that contribute to breast cancer (BC) progression. MTA1 is a transcriptional co-regulator that can act as both a co-activator and co-repressor to regulate pathways that contribute to cancer development. MTA1 is also one of the most up-regulated proteins in cancer, whose expression correlates with cancer progression, poor prognosis and increased metastatic potential.<br><br>METHODS: We identified MTA1 in BC exosomes by antibody array and confirmed expression of exosome-MTA1 across five breast cancer cells lines. Ectopic expression of tdTomato-tagged MTA1 and exosome transfer were examined by fluorescent microscopy. CRISPR/Cas9 genetic engineering was implemented to knockout MTA1 in MCF7 and MDA-MB-231 breast cancer cells. Reporter assays were used to monitor hypoxia and estrogen receptor signaling regulation by exosome-MTA1 transfer.<br><br>RESULTS: Ectopic overexpression of tdTomato-MTA1 in BC cell lines demonstrated exosome transfer of MTA1 to BC and vascular endothelial cells. MTA1 knockout in BC cells reduced cell proliferation and attenuated the hypoxic response in these cells, presumably through its co-repressor function, which could be rescued by the addition of exosomes containing MTA1. On the other hand, consistent with its co-activator function, estrogen receptor signaling was enhanced in MTA1 knockout cells and could be reversed by addition of MTA1-exosomes. Importantly, MTA1 knockout sensitized hormone receptor negative cells to 4-hydroxy tamoxifen treatment, which could be reversed by the addition of MTA1-exosomes.<br><br>CONCLUSIONS: This is the first report showing that BC exosomes contain MTA1 and can transfer it to other cells resulting in changes to hypoxia and estrogen receptor signaling in the tumor microenvironment. These results, collectively, provide evidence suggesting that exosome-mediated transfer of MTA1 contributes to BC progression by modifying cellular responses to important signaling pathways and that exosome-MTA1 may be developed as a biomarker and therapeutic target for BC.},
}
@article {pmid30779952,
year = {2019},
author = {Eş, I and Gavahian, M and Marti-Quijal, FJ and Lorenzo, JM and Mousavi Khaneghah, A and Tsatsanis, C and Kampranis, SC and Barba, FJ},
title = {The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated challenges.},
journal = {Biotechnology advances},
volume = {37},
number = {3},
pages = {410-421},
doi = {10.1016/j.biotechadv.2019.02.006},
pmid = {30779952},
issn = {1873-1899},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/genetics ; Food Industry/trends ; Gene Editing/*methods ; Gene Targeting/methods ; Genetic Engineering/*methods ; Humans ; Plants, Genetically Modified/*genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; },
abstract = {The recent progress in genetic engineering has brought multiple benefits to the food and agricultural industry by enhancing the essential characteristics of agronomic traits. Powerful tools in the field of genome editing, such as siRNA-mediated RNA interference for targeted suppression of gene expression and transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) for DNA repair have been widely used for commercial purposes. However, in the last few years, the discovery of the CRISPR-Cas9 system has revolutionized genome editing and has attracted attention as a powerful tool for several industrial applications. Herein, we review current progresses in the utilization of the CRISPR-Cas9 system in the food and agricultural industry, particularly in the development of resistant crops with improved quality and productivity. We compare the CRISPR system with the TALEN and ZFN nucleases-based methods and highlight potential advantages and shortcomings. In addition, we explore the state of the global market and discuss the safety and ethical concerns associated with the application of this technology in the food and agricultural industry.},
}
@article {pmid30779874,
year = {2019},
author = {Rouet, R and Christ, D},
title = {Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins through Receptor Mediated Endocytosis.},
journal = {ACS chemical biology},
volume = {14},
number = {3},
pages = {554-561},
doi = {10.1021/acschembio.9b00116},
pmid = {30779874},
issn = {1554-8937},
mesh = {Asialoglycoprotein Receptor/chemistry/metabolism ; Biological Transport ; CRISPR-Associated Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*physiology ; Cell Line ; Endocytosis/*physiology ; Gene Editing/methods ; Humans ; Oligopeptides/*metabolism ; Ribonucleoproteins/*metabolism ; Signal Transduction ; },
abstract = {We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.},
}
@article {pmid30779043,
year = {2019},
author = {Webster, DE and Roulland, S and Phelan, JD},
title = {Protocols for CRISPR-Cas9 Screening in Lymphoma Cell Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1956},
number = {},
pages = {337-350},
doi = {10.1007/978-1-4939-9151-8_16},
pmid = {30779043},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Genomic Library ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Lentivirus/genetics ; Lymphoma/*genetics ; Plasmids/genetics ; RNA, Guide/genetics ; Transduction, Genetic/methods ; },
abstract = {Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human lymphoma cell lines but are highly amenable for other experimental purposes.},
}
@article {pmid30778240,
year = {2019},
author = {Beyret, E and Liao, HK and Yamamoto, M and Hernandez-Benitez, R and Fu, Y and Erikson, G and Reddy, P and Izpisua Belmonte, JC},
title = {Single-dose CRISPR-Cas9 therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome.},
journal = {Nature medicine},
volume = {25},
number = {3},
pages = {419-422},
pmid = {30778240},
issn = {1546-170X},
support = {P30 CA014195/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Genetic Therapy/*methods ; Lamin Type A/*genetics/metabolism ; *Longevity ; Mice ; Mutation ; Progeria/*genetics ; Protein Isoforms/genetics/metabolism ; },
abstract = {Hutchinson-Gilford progeria syndrome (HGPS) is a rare lethal genetic disorder characterized by symptoms reminiscent of accelerated aging. The major underlying genetic cause is a substitution mutation in the gene coding for lamin A, causing the production of a toxic isoform called progerin. Here we show that reduction of lamin A/progerin by a single-dose systemic administration of adeno-associated virus-delivered CRISPR-Cas9 components suppresses HGPS in a mouse model.},
}
@article {pmid30778239,
year = {2019},
author = {Santiago-Fernández, O and Osorio, FG and Quesada, V and Rodríguez, F and Basso, S and Maeso, D and Rolas, L and Barkaway, A and Nourshargh, S and Folgueras, AR and Freije, JMP and López-Otín, C},
title = {Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome.},
journal = {Nature medicine},
volume = {25},
number = {3},
pages = {423-426},
pmid = {30778239},
issn = {1546-170X},
support = {/WT_/Wellcome Trust/United Kingdom ; 098291/WT_/Wellcome Trust/United Kingdom ; 742067/ERC_/European Research Council/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Lamin Type A/*genetics/metabolism ; Mice ; Point Mutation ; Progeria/genetics/*therapy ; },
abstract = {CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.},
}
@article {pmid30778238,
year = {2019},
author = {Nelson, CE and Wu, Y and Gemberling, MP and Oliver, ML and Waller, MA and Bohning, JD and Robinson-Hamm, JN and Bulaklak, K and Castellanos Rivera, RM and Collier, JH and Asokan, A and Gersbach, CA},
title = {Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy.},
journal = {Nature medicine},
volume = {25},
number = {3},
pages = {427-432},
pmid = {30778238},
issn = {1546-170X},
support = {DP2 OD008586/OD/NIH HHS/United States ; K99 EB023979/EB/NIBIB NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/*genetics/immunology ; Dependovirus ; Disease Models, Animal ; Dystrophin/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Immunity, Cellular/immunology ; Immunity, Humoral/immunology ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne/genetics/*therapy ; },
abstract = {Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy[1-6], however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice[7]; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.},
}
@article {pmid30778200,
year = {2019},
author = {Zhang, Q and Zhang, ZY and Du, H and Li, SZ and Tu, R and Jia, YF and Zheng, Z and Song, XM and Du, RL and Zhang, XD},
title = {DUB3 deubiquitinates and stabilizes NRF2 in chemotherapy resistance of colorectal cancer.},
journal = {Cell death and differentiation},
volume = {26},
number = {11},
pages = {2300-2313},
pmid = {30778200},
issn = {1476-5403},
mesh = {Antineoplastic Agents, Phytogenic/therapeutic use ; Apoptosis/physiology ; CRISPR-Cas Systems/genetics ; Cell Proliferation/physiology ; Colorectal Neoplasms/drug therapy/*pathology ; Deubiquitinating Enzymes/*metabolism ; Drug Resistance, Neoplasm/*physiology ; Endopeptidases/*metabolism ; HCT116 Cells ; HEK293 Cells ; HT29 Cells ; Humans ; Kelch-Like ECH-Associated Protein 1/metabolism ; NF-E2-Related Factor 2/genetics/*metabolism ; Paclitaxel/therapeutic use ; Transcriptional Activation/genetics ; Ubiquitination/physiology ; },
abstract = {The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) is one of the master regulators that control hundreds of genes containing antioxidant response elements (AREs). The NRF2-ARE pathway plays a complex role in colorectal cancer (CRC). NRF2 activity is known to be regulated by KEAP1-CUL3 E3 ligase-mediated ubiquitination, indicating the importance of deubiquitination regulation. However, the deubiquitinase (DUB) of NRF2 remains unknown. Here, by screening a DUB library, we identified DUB3 as a DUB that remarkably stabilized NRF2. Further experiments demonstrated that DUB3 promoted NRF2 stability and transcriptional activity by decreasing the K48-linked ubiquitination of NRF2. Coimmunoprecipitation studies revealed interactions between NRF2 and DUB3, as well as between KEAP1 and DUB3, indicating that NRF2, DUB3, and KEAP1 formed a large functional complex. Importantly, ectopic expression of DUB3 caused NRF2-dependent chemotherapy resistance in colon cancer cell lines. Thus, to the best of our knowledge, our findings are the first to identify DUB3 as a NRF2 DUB and may provide a new strategy against chemotherapy resistance in CRC and other NRF2-related diseases.},
}
@article {pmid30778165,
year = {2019},
author = {Ruf, S and Forner, J and Hasse, C and Kroop, X and Seeger, S and Schollbach, L and Schadach, A and Bock, R},
title = {High-efficiency generation of fertile transplastomic Arabidopsis plants.},
journal = {Nature plants},
volume = {5},
number = {3},
pages = {282-289},
pmid = {30778165},
issn = {2055-0278},
mesh = {Arabidopsis/cytology/genetics/*physiology ; Bacterial Proteins/genetics ; Biolistics/methods ; *CRISPR-Cas Systems ; Cell Culture Techniques ; Chloroplasts/genetics ; Gene Editing ; Gene Knockout Techniques ; Genetic Vectors ; Luminescent Proteins/genetics ; Plant Roots/cytology/genetics ; *Plants, Genetically Modified ; Plastids/*genetics ; Transformation, Genetic ; },
abstract = {The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.},
}
@article {pmid30778069,
year = {2019},
author = {Michaels, YS and Barnkob, MB and Barbosa, H and Baeumler, TA and Thompson, MK and Andre, V and Colin-York, H and Fritzsche, M and Gileadi, U and Sheppard, HM and Knapp, DJHF and Milne, TA and Cerundolo, V and Fulga, TA},
title = {Precise tuning of gene expression levels in mammalian cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {818},
pmid = {30778069},
issn = {2041-1723},
support = {G0902418/MRC_/Medical Research Council/United Kingdom ; MC_UU_00008/1/MRC_/Medical Research Council/United Kingdom ; MC_UU_00016/6/MRC_/Medical Research Council/United Kingdom ; MC_UU_12009/6/MRC_/Medical Research Council/United Kingdom ; },
mesh = {3' Untranslated Regions ; Animals ; B7-H1 Antigen/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation/*genetics ; Genes, BRCA1 ; *Genetic Techniques ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Melanoma, Experimental/genetics/pathology ; Mice, Inbred C57BL ; MicroRNAs/*genetics ; Ovalbumin/genetics ; Recombinant Proteins/genetics/metabolism ; *Response Elements ; Xenograft Model Antitumor Assays ; },
abstract = {Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3'UTR, demonstrating that this versatile tool can be used to tune endogenous genes.},
}
@article {pmid30777520,
year = {2019},
author = {Argani, H},
title = {Genome Engineering for Stem Cell Transplantation.},
journal = {Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation},
volume = {17},
number = {Suppl 1},
pages = {31-37},
doi = {10.6002/ect.MESOT2018.L34},
pmid = {30777520},
issn = {2146-8427},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Diseases, Inborn/diagnosis/genetics/immunology/*therapy ; Genetic Predisposition to Disease ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/immunology/*transplantation ; Phenotype ; Stem Cell Transplantation/adverse effects/*methods ; Treatment Outcome ; },
abstract = {To avoid the ethical issues of embryonic stem cells, genome engineering has focused on inducible pluripotent stem cells, which can develop into all 3 germ layers. The ability to detect methylation patterns in these cells allows research into pluripotency markers. The recently developed CRISPR system has allowed widespread application of genome engineering techniques. The CRISPR-Cas9 system, a potent system for genome editing, can be used for gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. Two types of genome engineering can be initiated: homologous or nonhomologous DNA repair by the Cas9 nuclease. Delivery of the CRISPR-Cas9 and target donor vectors in human pluripotent stem cells can be accomplished via viral and nonviral delivery methods. Nonviral delivery includes lipid-mediated transfection and electroporation. It has become the most common and efficient in vitro delivery method for human pluripotent stem cells. The CRISPR-Cas9 system can be combined with inducible pluripotent stem cells to generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. Knockouts can also be utilized to investigate epigenetic roles and targets, such as investigation of DNA methylation. CRISPR could be combined with human pluripotent stem cells to explore genetic determinants of lineage choice, differentiation, and stem cell fate, allowing investigators to study how various genes or noncoding elements contribute to specific processes and pathways. The CRISPR-Cas9 system can also be used to create null or nucleasedead Cas9, which has no enzymatic activity but has been utilized through fusion with other functional protein domains. In conclusion, RNA-guided genome targeting will have broad implications for synthetic biology, direct perturbation of gene networks, and targeted ex vivo and in vivo gene therapy.},
}
@article {pmid30777488,
year = {2019},
author = {Bassani, F and Zink, IA and Pribasnig, T and Wolfinger, MT and Romagnoli, A and Resch, A and Schleper, C and Bläsi, U and La Teana, A},
title = {Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus.},
journal = {RNA biology},
volume = {16},
number = {5},
pages = {675-685},
pmid = {30777488},
issn = {1555-8584},
mesh = {Archaeal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Peptide Initiation Factors/genetics/*metabolism ; *Protein Biosynthesis ; RNA, Archaeal/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Sulfolobus solfataricus/*growth &amp; development/metabolism ; },
abstract = {Translation factor a/eIF5A is highly conserved in Eukarya and Archaea. The eukaryal eIF5A protein is required for transit of ribosomes across consecutive proline codons, whereas the function of the archaeal orthologue remains unknown. Here, we provide a first hint for an involvement of Sulfolobus solfataricus (Sso) aIF5A in translation. CRISPR-mediated knock down of the aif5A gene resulted in strong growth retardation, underlining a pivotal function. Moreover, in vitro studies revealed that Sso aIF5A is endowed with endoribonucleolytic activity. Thus, aIF5A appears to be a moonlighting protein that might be involved in protein synthesis as well as in RNA metabolism.},
}
@article {pmid30777060,
year = {2019},
author = {Devilder, MC and Moyon, M and Gautreau-Rolland, L and Navet, B and Perroteau, J and Delbos, F and Gesnel, MC and Breathnach, R and Saulquin, X},
title = {Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {14},
pmid = {30777060},
issn = {1472-6750},
mesh = {Antibodies/*genetics/*immunology ; *Antibody Affinity ; B-Lymphocytes/*immunology ; *CRISPR-Cas Systems ; Cytidine Deaminase/*genetics/immunology ; Directed Molecular Evolution ; *Gene Editing ; HEK293 Cells ; HLA Antigens/immunology ; Humans ; Somatic Hypermutation, Immunoglobulin ; },
}
@article {pmid30775458,
year = {2019},
author = {Ono, R and Yasuhiko, Y and Aisaki, KI and Kitajima, S and Kanno, J and Hirabayashi, Y},
title = {Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {57},
pmid = {30775458},
issn = {2399-3642},
mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; DNA/*genetics/metabolism ; DNA Breaks, Double-Stranded ; *DNA Repair ; Embryo, Mammalian ; Escherichia coli/genetics/metabolism ; Exosomes/*genetics/metabolism ; Gene Editing/*ethics ; *Gene Transfer, Horizontal ; *Genome ; Goats ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; NIH 3T3 Cells ; Plasmids/chemistry/metabolism ; Retroelements ; Short Interspersed Nucleotide Elements ; },
abstract = {The CRISPR-Cas9 system has been successfully applied in many organisms as a powerful genome-editing tool. Undoubtedly, it will soon be applied to human genome editing, including gene therapy. We have previously reported that unintentional DNA sequences derived from retrotransposons, genomic DNA, mRNA and vectors are captured at double-strand breaks (DSBs) sites when DSBs are introduced by the CRISPR-Cas9 system. Therefore, it is possible that unintentional insertions associated with DSB repair represent a potential risk for human genome editing gene therapies. To address this possibility, comprehensive sequencing of DSB sites was performed. Here, we report that exosome-mediated horizontal gene transfer occurs in DSB repair during genome editing. Exosomes are present in all fluids from living animals, including seawater and breathing mammals, suggesting that exosome-mediated horizontal gene transfer is the driving force behind mammalian genome evolution. The findings of this study highlight an emerging new risk for this leading-edge technology.},
}
@article {pmid30773816,
year = {2019},
author = {Krupovic, M and Makarova, KS and Wolf, YI and Medvedeva, S and Prangishvili, D and Forterre, P and Koonin, EV},
title = {Integrated mobile genetic elements in Thaumarchaeota.},
journal = {Environmental microbiology},
volume = {21},
number = {6},
pages = {2056-2078},
pmid = {30773816},
issn = {1462-2920},
support = {ANR-17-CE15-0005-01//Agence Nationale de la Recherche/International ; UE 340440//H2020 European Research Council/International ; 685778//Horizon 2020 Framework Programme/International ; intramural program//U.S. National Library of Medicine/International ; RSF 14-14-00988//Campus France/International ; },
mesh = {Archaea/classification/enzymology/*genetics/virology ; Archaeal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *DNA Transposable Elements ; Oxidoreductases/genetics/metabolism ; Phylogeny ; Plasmids/genetics/metabolism ; },
abstract = {To explore the diversity of mobile genetic elements (MGE) associated with archaea of the phylum Thaumarchaeota, we exploited the property of most MGE to integrate into the genomes of their hosts. Integrated MGE (iMGE) were identified in 20 thaumarchaeal genomes amounting to 2 Mbp of mobile thaumarchaeal DNA. These iMGE group into five major classes: (i) proviruses, (ii) casposons, (iii) insertion sequence-like transposons, (iv) integrative-conjugative elements and (v) cryptic integrated elements. The majority of the iMGE belong to the latter category and might represent novel families of viruses or plasmids. The identified proviruses are related to tailed viruses of the order Caudovirales and to tailless icosahedral viruses with the double jelly-roll capsid proteins. The thaumarchaeal iMGE are all connected within a gene sharing network, highlighting pervasive gene exchange between MGE occupying the same ecological niche. The thaumarchaeal mobilome carries multiple auxiliary metabolic genes, including multicopper oxidases and ammonia monooxygenase subunit C (AmoC), and stress response genes, such as those for universal stress response proteins (UspA). Thus, iMGE might make important contributions to the fitness and adaptation of their hosts. We identified several iMGE carrying type I-B CRISPR-Cas systems and spacers matching other thaumarchaeal iMGE, suggesting antagonistic interactions between coexisting MGE and symbiotic relationships with the ir archaeal hosts.},
}
@article {pmid30773774,
year = {2019},
author = {Miyamoto, T and Takada, R and Tobimatsu, Y and Takeda, Y and Suzuki, S and Yamamura, M and Osakabe, K and Osakabe, Y and Sakamoto, M and Umezawa, T},
title = {OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.},
journal = {The Plant journal : for cell and molecular biology},
volume = {98},
number = {6},
pages = {975-987},
doi = {10.1111/tpj.14290},
pmid = {30773774},
issn = {1365-313X},
mesh = {Biomass ; CRISPR-Cas Systems ; Cell Wall/chemistry/metabolism ; Coumaric Acids ; Flavonoids/*metabolism ; Gene Editing ; *Gene Expression Regulation, Plant ; Gene Regulatory Networks ; Lignin/chemistry/*metabolism ; Loss of Function Mutation ; Oryza/chemistry/*genetics/growth &amp; development/metabolism ; Phylogeny ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Propionates/*metabolism ; Transcription Factors/genetics/*metabolism ; Up-Regulation ; },
abstract = {Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.},
}
@article {pmid30773764,
year = {2019},
author = {Wang, T and Liu, Y and Sun, HH and Yin, BC and Ye, BC},
title = {An RNA-Guided Cas9 Nickase-Based Method for Universal Isothermal DNA Amplification.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {16},
pages = {5382-5386},
doi = {10.1002/anie.201901292},
pmid = {30773764},
issn = {1521-3773},
support = {21822402, 21421004//the National Natural Science Foundation of China/International ; 21675052, 21335003//the National Natural Science Foundation of China/International ; 21575089//the National Natural Science Foundation of China/International ; //the Fundamental Research Funds for the Central Universities/International ; B18022, B16017//Programme of Introducing Talents of Discipline to University/International ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; DNA Replication ; DNA, Bacterial/*genetics ; *Nucleic Acid Amplification Techniques ; RNA, Guide/*metabolism ; Salmonella typhimurium/genetics ; *Temperature ; },
abstract = {We have developed an ingenious method, termed Cas9 nickase-based amplification reaction (Cas9nAR), to amplify a target fragment from genomic DNA at a constant temperature of 37 °C. Cas9nAR employs a sgRNA:Cas9n complex with a single-strand nicking property, a strand-displacing DNA polymerase, and two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming, extension, nicking, and displacement reaction steps. Cas9nAR exhibits a zeptomolar limit of detection (2 copies in 20 μL of reaction system) within 60 min and a single-base discrimination capability. More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity, as well as the simple-to-implement, rapid, and isothermal features, Cas9nAR holds great potential to become a routine assay for the quantitative detection of nucleic acids in basic and applied studies.},
}
@article {pmid30773261,
year = {2019},
author = {Liu, Y and Dakou, E and Meng, Y and Leyns, L},
title = {Loss of Emp2 compromises cardiogenic differentiation in mouse embryonic stem cells.},
journal = {Biochemical and biophysical research communications},
volume = {511},
number = {1},
pages = {173-178},
doi = {10.1016/j.bbrc.2019.02.048},
pmid = {30773261},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Differentiation ; Cell Line ; Embryo, Mammalian/cytology/metabolism ; Gastrulation ; *Gene Expression Regulation, Developmental ; Membrane Glycoproteins/*genetics ; Mice ; Mouse Embryonic Stem Cells/*cytology/metabolism ; Myocytes, Cardiac/*cytology/metabolism ; },
abstract = {Isolated mouse embryonic stem cells (mESCs) retain the capacities to self-renew limitlessly and to give rise to all tissues of an adult mouse. A precise understanding of the relationships, mechanisms of action and functions of novel genes involved in mESCs differentiation is crucial to expand our knowledge of vertebrate development. The epithelial membrane protein 2 (EMP2) is a membrane-spanning protein found in epithelial and endothelial cell-cell junctions that has been implicated in the regulation of cell proliferation and migration in normal and tumor tissues. In this study, Emp2 was disrupted in mESCs using the CRISPR/Cas9 technology. We subsequently assessed Emp2 functions by using mouse embryoid bodies (EBs) capable of forming the three germ layers of an embryo in vitro and by further analyzing the emergence of the future cardiac tissue in these EB models. We found that when Emp2 is disrupted, expression of pluripotency markers was up-regulated and/or longer retained in EBs. Additionally, the formation of each germ layer was variously affected during gastrulation and in particular, the formation of mesoderm was delayed. Besides, we discovered that Emp2 was involved in the regulation of the epithelial-mesenchymal transition (EMT) process and in the differentiation of cells into functional cardiomyocytes.},
}
@article {pmid30773093,
year = {2019},
author = {Menon, VR and Ananthapadmanabhan, V and Swanson, S and Saini, S and Sesay, F and Yakovlev, V and Florens, L and DeCaprio, JA and Washburn, MP and Dozmorov, M and Litovchick, L},
title = {DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {18},
number = {5},
pages = {531-551},
pmid = {30773093},
issn = {1551-4005},
support = {P01 CA203655/CA/NCI NIH HHS/United States ; P30 CA016059/CA/NCI NIH HHS/United States ; R01 CA188571/CA/NCI NIH HHS/United States ; R25 GM090084/GM/NIGMS NIH HHS/United States ; R01 CA063113/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/radiation effects ; Cell Line, Tumor ; *DNA Damage/radiation effects ; DNA Repair ; Gamma Rays ; Gene Editing ; Humans ; Metabolic Networks and Pathways ; Mice ; Phosphorylation ; Protein Binding ; Protein Serine-Threonine Kinases/deficiency/genetics/*metabolism ; Protein-Tyrosine Kinases/deficiency/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Tumor Suppressor p53-Binding Protein 1/*metabolism ; Ubiquitin-Protein Ligases/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.},
}
@article {pmid30771412,
year = {2019},
author = {Stephens, CJ and Lauron, EJ and Kashentseva, E and Lu, ZH and Yokoyama, WM and Curiel, DT},
title = {Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {298},
number = {},
pages = {128-141},
pmid = {30771412},
issn = {1873-4995},
support = {F31 DK108624/DK/NIDDK NIH HHS/United States ; R25 GM103757/GM/NIGMS NIH HHS/United States ; R01 CA154697/CA/NCI NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; R41 TR001869/TR/NCATS NIH HHS/United States ; },
mesh = {Adenoviridae/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Female ; Gene Editing/methods ; Gene Knock-In Techniques ; Genetic Therapy/*methods ; Genetic Vectors ; Hemophilia B/genetics/*therapy ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; },
abstract = {Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.},
}
@article {pmid30771271,
year = {2019},
author = {Wang, H and Wang, X and Li, X and Wang, Q and Qing, S and Zhang, Y and Gao, MQ},
title = {A novel long non-coding RNA regulates the immune response in MAC-T cells and contributes to bovine mastitis.},
journal = {The FEBS journal},
volume = {286},
number = {9},
pages = {1780-1795},
doi = {10.1111/febs.14783},
pmid = {30771271},
issn = {1742-4658},
mesh = {Animals ; CRISPR-Cas Systems ; Caseins/metabolism ; Cattle/*genetics/immunology ; Cell Line, Transformed ; Epithelial Cells/*immunology/metabolism ; Escherichia coli/physiology ; Female ; Gene Expression Regulation ; Gene Knockout Techniques ; High-Throughput Nucleotide Sequencing ; Inflammation ; Mammary Glands, Animal/cytology ; Mastitis, Bovine/*genetics ; Organ Specificity ; Quantitative Trait Loci ; RNA, Guide/genetics ; RNA, Long Noncoding/genetics/isolation &amp; purification/*physiology ; Staphylococcus aureus/physiology ; Transforming Growth Factor beta1/metabolism ; Tubulin/genetics ; },
abstract = {The long non-coding RNAs (lncRNAs) are known to transcriptionally regulate a wide spectrum of diseases. Here, we screened for potentially functional lncRNAs in a mammary epithelial cell model of bovine mastitis by RNA-Seq technology and identified a class of previously undetected mastitis-related lncRNAs. A novel lncRNA was widely expressed in a variety of bovine tissues with diverse relative abundance and had a relatively low expression in mammary tissue. Given its predicted target gene is TUBA1C, we name it lncRNA-TUB. We found a higher expression of lncRNA-TUB in mammary epithelial cells that received a proinflammatory stimulus compared to normal cells. Knockout of lncRNA-TUB by the CRISPR/Cas9 system revealed that it plays crucial roles in the morphological shape, proliferation, migration and β-casein secretion of mammary epithelial cells. In addition, lncRNA-TUB mediates Escherichia coli-induced inflammatory factor secretion and Staphylococcus aureus adhesion to epithelial cells. Our results suggest that the lncRNAs identified here function in bovine mastitis, and that lncRNA-TUB affects the basic biological characteristics and functions of bovine mammary epithelial cells in inflammatory conditions, providing valuable insights into the mechanisms of bovine mastitis.},
}
@article {pmid30770867,
year = {2019},
author = {Yang, W and Liu, Y and Tu, Z and Xiao, C and Yan, S and Ma, X and Guo, X and Chen, X and Yin, P and Yang, Z and Yang, S and Jiang, T and Li, S and Qin, C and Li, XJ},
title = {CRISPR/Cas9-mediated PINK1 deletion leads to neurodegeneration in rhesus monkeys.},
journal = {Cell research},
volume = {29},
number = {4},
pages = {334-336},
pmid = {30770867},
issn = {1748-7838},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Macaca mulatta/embryology ; Neurodegenerative Diseases/*metabolism ; Protein Kinases/*genetics/*metabolism ; },
}
@article {pmid30770809,
year = {2019},
author = {Ben Soussia, I and El Mouridi, S and Kang, D and Leclercq-Blondel, A and Khoubza, L and Tardy, P and Zariohi, N and Gendrel, M and Lesage, F and Kim, EJ and Bichet, D and Andrini, O and Boulin, T},
title = {Mutation of a single residue promotes gating of vertebrate and invertebrate two-pore domain potassium channels.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {787},
pmid = {30770809},
issn = {2041-1723},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/genetics/metabolism ; Drosophila ; Evolution, Molecular ; Humans ; Invertebrates ; Membrane Potentials/genetics/*physiology ; Mutation/genetics ; Potassium Channels, Tandem Pore Domain/genetics/*metabolism ; Vertebrates ; },
abstract = {Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.},
}
@article {pmid30770599,
year = {2019},
author = {Liu, CY and Wang, HC},
title = {The fibroblast of radicular cyst facilitate osteoclastogenesis via the autocrine of Fibronectin containing extra domain A.},
journal = {Oral diseases},
volume = {25},
number = {4},
pages = {1136-1146},
doi = {10.1111/odi.13064},
pmid = {30770599},
issn = {1601-0825},
mesh = {Alternative Splicing ; *Fibroblasts ; *Fibronectins ; Humans ; *Osteogenesis ; *Radicular Cyst ; Tumor Microenvironment ; },
abstract = {OBJECTIVE: To investigate the alternative spliced isoforms of Fibronectin (FN) in the stroma of radicular cysts and analyze the associations between these isoforms and the osteoclastogenic effects of fibroblasts.<br><br>METHODS AND MATERIALS: The specimens of radicular cysts were stained with immunohistochemistry, and the associations between each FN isoform and clinical parameters were assessed. The fibroblasts isolated from cysts or jaw bone were cultured to induce the Trap&#xa0;+&#xa0;MNCs. In the conditioned medium, the Fibronectin containing extra domain A (EDA&#xa0;+&#xa0;FN) was neutralized by antibody IST-9, and the EDA exon of fibroblasts was knockout by CRISPR/Cas system, for assessing the osteoclastogenic effects. The mRNA level of FN isoforms and the osteoclastogenesis-related genes were analyzed by quantitive PCR.<br><br>RESULTS: EDA&#xa0;+&#xa0;FN staining was positively associated with the size of the lesions (p&#xa0;<&#xa0;0.05). In contrast with the controls, the ratio of EDA&#xa0;+&#xa0;FN/total FN in the fibroblasts from radicular cysts was significantly higher (p&#xa0;<&#xa0;0.05), and positively associate with Trap&#xa0;+&#xa0;MNCs counting, it was consistent with increased expression of COX-2, IL-6, IL-17, and the RANKL/OPG (p&#xa0;<&#xa0;0.05). The Trap&#xa0;+&#xa0;MNCs counting and osteoclastogenesis-related genes were decreased by IST-9 blocking and EDA exon knockout in fibroblasts, but the blockage of the interaction between EDA&#xa0;+&#xa0;FN and pre-osteoclasts exhibited little effects on Trap&#xa0;+&#xa0;MNCs formation.<br><br>CONCLUSION: The microenvironment of the fibrous capsule of radicular cysts facilitates the splicing of EDA exon, it endues EDA&#xa0;+&#xa0;FN with autocrine effects on fibroblast itself, and it increases the expression of osteoclastogenesis-related genes, by which the osteoclastogenesis in radicular cysts could be initiated.},
}
@article {pmid30770351,
year = {2019},
author = {Sambandam, V and Frederick, MJ and Shen, L and Tong, P and Rao, X and Peng, S and Singh, R and Mazumdar, T and Huang, C and Li, Q and Pickering, CR and Myers, JN and Wang, J and Johnson, FM},
title = {PDK1 Mediates NOTCH1-Mutated Head and Neck Squamous Carcinoma Vulnerability to Therapeutic PI3K/mTOR Inhibition.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {25},
number = {11},
pages = {3329-3340},
pmid = {30770351},
issn = {1557-3265},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 DE024179/DE/NIDCR NIH HHS/United States ; R50 CA221675/CA/NCI NIH HHS/United States ; U01 DE025181/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Humans ; Loss of Function Mutation ; Mice ; Phosphatidylinositol 3-Kinases/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase/*metabolism ; Receptor, Notch1/*genetics/metabolism ; Signal Transduction/drug effects ; Squamous Cell Carcinoma of Head and Neck/drug therapy/*etiology/*metabolism/pathology ; TOR Serine-Threonine Kinases/*metabolism ; },
abstract = {PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression.<br><br>RESULTS: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 [MUT]) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 [MUT] was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 [WT] HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1 [MUT] but not NOTCH1 [WT] HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 [MUT] cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 [WT] HNSCC but had little effect as single agents.<br><br>CONCLUSIONS: Our findings suggest that NOTCH1 [MUT] predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 [WT] HNSCC to PI3K/mTOR pathway inhibitors.},
}
@article {pmid30769780,
year = {2019},
author = {Iwasawa, C and Tamura, R and Sugiura, Y and Suzuki, S and Kuzumaki, N and Narita, M and Suematsu, M and Nakamura, M and Yoshida, K and Toda, M and Okano, H and Miyoshi, H},
title = {Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells.},
journal = {International journal of molecular sciences},
volume = {20},
number = {4},
pages = {},
pmid = {30769780},
issn = {1422-0067},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Ganciclovir/pharmacology ; Gene Editing ; Gene Expression Regulation, Enzymologic/genetics ; Gene Expression Regulation, Viral/genetics ; Genes, Transgenic, Suicide/genetics ; *Genetic Therapy ; Genetic Vectors/therapeutic use ; Humans ; Induced Pluripotent Stem Cells/*enzymology/transplantation ; Lentivirus/genetics ; Nucleotides/biosynthesis/genetics ; Simplexvirus/*enzymology/genetics ; Thymidine Kinase/*genetics ; },
abstract = {Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a "thymidine block", but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.},
}
@article {pmid30769329,
year = {2019},
author = {Frederiksen, HR and Holst, B and Mau-Holzmann, UA and Freude, K and Schmid, B},
title = {Generation of two isogenic iPSC lines with either a heterozygous or a homozygous E280A mutation in the PSEN1 gene.},
journal = {Stem cell research},
volume = {35},
number = {},
pages = {101403},
doi = {10.1016/j.scr.2019.101403},
pmid = {30769329},
issn = {1876-7753},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Amino Acid Substitution ; CRISPR-Cas Systems ; *Cell Line ; *Cellular Reprogramming Techniques ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Mutation, Missense ; *Presenilin-1/genetics/metabolism ; },
abstract = {Alzheimer's disease (AD) is the most common form of dementia. Mutations in the gene PSEN1 encoding Presenilin1 are known to cause familial forms of AD with early age of onset. The most common mutation in the PSEN1 gene is the E280A mutation. iPSCs are an optimal choice for modeling AD, as they can be differentiated in vitro into neural cells. Here, we report the generation of two isogenic iPSC lines with either a homozygous or a heterozygous E280A mutation in the PSEN1 gene. The mutation was introduced into an iPSC line from a healthy individual using the CRISPR-Cas9 technology. Resource table.},
}
@article {pmid30769094,
year = {2019},
author = {Raas, Q and Gondcaille, C and Hamon, Y and Leoni, V and Caccia, C and Ménétrier, F and Lizard, G and Trompier, D and Savary, S},
title = {CRISPR/Cas9-mediated knockout of Abcd1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1864},
number = {5},
pages = {704-714},
doi = {10.1016/j.bbalip.2019.02.006},
pmid = {30769094},
issn = {1879-2618},
mesh = {ATP Binding Cassette Transporter, Subfamily D/*genetics/metabolism ; ATP Binding Cassette Transporter, Subfamily D, Member 1/*genetics/metabolism ; Adrenoleukodystrophy/*genetics/metabolism/pathology ; Animals ; CRISPR-Cas Systems ; Cell Line ; Fatty Acids/metabolism ; Female ; Gene Deletion ; Mice, Inbred C57BL ; Microglia/metabolism/pathology ; },
abstract = {X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the Abcd1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.},
}
@article {pmid30767852,
year = {2019},
author = {Al-Shehabi, H and Fiebig, U and Kutzner, J and Denner, J and Schaller, T and Bannert, N and Hofmann, H},
title = {Human SAMHD1 restricts the xenotransplantation relevant porcine endogenous retrovirus (PERV) in non-dividing cells.},
journal = {The Journal of general virology},
volume = {100},
number = {4},
pages = {656-661},
doi = {10.1099/jgv.0.001232},
pmid = {30767852},
issn = {1465-2099},
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Cell Line ; Endogenous Retroviruses/*physiology ; HEK293 Cells ; Heterografts/*metabolism/*virology ; Humans ; Macrophages/metabolism/virology ; Monocytes/metabolism/virology ; Reverse Transcription/physiology ; SAM Domain and HD Domain-Containing Protein 1/*metabolism ; Swine ; THP-1 Cells ; Transplantation, Heterologous/methods ; Virus Replication/physiology ; },
abstract = {The release of porcine endogenous retrovirus (PERV) particles from pig cells is a potential risk factor during xenotransplantation by way of productively infecting the human transplant recipient. Potential countermeasures against PERV replication are restriction factors that block retroviral replication. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of dNTPs in non-cycling cells starving retroviral reverse transcription. We investigated the antiviral activity of human SAMHD1 against PERV and found that SAMHD1 potently restricts its reverse transcription in human monocytes, monocyte-derived dendritic cells (MDDC), or macrophages (MDM) and in monocytic THP-1 cells. Degradation of SAMHD1 by SIVmac Vpx or CRISPR/Cas9 knock-out of SAMHD1 allowed for PERV reverse transcription. Addition of deoxynucleosides alleviated the SAMHD1-mediated restriction suggesting that SAMHD1-mediated degradation of dNTPs restricts PERV replication in these human immune cells. In conclusion, our findings highlight SAMHD1 as a potential barrier to PERV transmission from pig transplants to human recipients during xenotransplantation.},
}
@article {pmid30767423,
year = {2020},
author = {Wang, J and Zuo, YY and Li, LL and Wang, H and Liu, SY and Yang, YH and Wu, YD},
title = {Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins.},
journal = {Insect science},
volume = {27},
number = {3},
pages = {440-448},
pmid = {30767423},
issn = {1744-7917},
mesh = {Animals ; Bacillus thuringiensis/metabolism ; Bacillus thuringiensis Toxins ; Bacterial Proteins/*pharmacology ; *CD13 Antigens/drug effects/genetics/metabolism ; CRISPR-Cas Systems ; Disease Susceptibility/microbiology ; Endotoxins/*pharmacology ; Gene Knockout Techniques ; Hemolysin Proteins/*pharmacology ; Insect Proteins/drug effects/genetics/metabolism ; Insecticides/pharmacology ; Larva/*drug effects/metabolism/microbiology ; Membrane Proteins/drug effects/genetics/metabolism ; Moths/drug effects/*genetics/metabolism/microbiology ; Pest Control, Biological ; RNA Interference ; },
abstract = {Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence-based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.},
}
@article {pmid30767107,
year = {2019},
author = {Jia, K and Lu, Z and Zhou, F and Xiong, Z and Zhang, R and Liu, Z and Ma, Y and He, L and Li, C and Zhu, Z and Pan, D and Lian, Z},
title = {Multiple sgRNAs facilitate base editing-mediated i-stop to induce complete and precise gene disruption.},
journal = {Protein &amp; cell},
volume = {10},
number = {11},
pages = {832-839},
pmid = {30767107},
issn = {1674-8018},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Mice ; Mice, Knockout ; RNA, Guide/*genetics ; },
}
@article {pmid30767015,
year = {2019},
author = {Lu, Z and Yang, S and Yuan, X and Shi, Y and Ouyang, L and Jiang, S and Yi, L and Zhang, G},
title = {CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis.},
journal = {Nucleic acids research},
volume = {47},
number = {7},
pages = {e40},
pmid = {30767015},
issn = {1362-4962},
mesh = {Bacillus subtilis/*genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Expression Regulation, Bacterial ; Genes, Bacterial/genetics ; Molecular Chaperones/metabolism ; Mutation ; Promoter Regions, Genetic/genetics ; Protein Folding ; RNA, Guide/genetics ; Transcription, Genetic ; Transformation, Genetic ; },
abstract = {Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.},
}
@article {pmid30766726,
year = {2019},
author = {Goeckel, ME and Basgall, EM and Lewis, IC and Goetting, SC and Yan, Y and Halloran, M and Finnigan, GC},
title = {Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae.},
journal = {Fungal biology and biotechnology},
volume = {6},
number = {},
pages = {2},
pmid = {30766726},
issn = {2054-3085},
support = {P20 GM103418/GM/NIGMS NIH HHS/United States ; },
abstract = {BACKGROUND: The bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. Additional research into molecular mechanisms that would allow for control, titration, and inhibition of drive systems is needed.<br><br>RESULTS: In this study, we use artificial gene drives in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences (both NLS and NES) on Cas9 fusion proteins in vivo through fluorescence microscopy and genomic editing. Our results demonstrate that mutational substitutions to nuclear signals and combinatorial fusions can both modulate the level of gene drive activity within a population of cells.<br><br>CONCLUSIONS: These findings have implications for control of traditional nuclease-dependent editing and use of gene drive systems within other organisms. For instance, initiation of a nuclear export mechanism to Cas9 could serve as a molecular safeguard within an active gene drive to reduce or eliminate editing.},
}
@article {pmid30765415,
year = {2019},
author = {Kuil, LE and Oosterhof, N and Geurts, SN and van der Linde, HC and Meijering, E and van Ham, TJ},
title = {Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain.},
journal = {Disease models &amp; mechanisms},
volume = {12},
number = {3},
pages = {},
pmid = {30765415},
issn = {1754-8411},
mesh = {Animals ; Animals, Genetically Modified ; Base Sequence ; Brain/growth &amp; development/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Count ; Cell Proliferation ; *Genetic Testing ; Interleukins/genetics/*metabolism/physiology ; Macrophages/*metabolism ; Microglia/metabolism ; Mutation/genetics ; *Reverse Genetics ; Yolk Sac/*metabolism ; Zebrafish/*metabolism ; Zebrafish Proteins/genetics/*physiology ; },
abstract = {Microglia are brain-resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, by Cas9/gRNA complex injections, followed by Neutral-Red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that, in zebrafish, il34 is required during the earliest seeding of the brain by microglia. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.},
}
@article {pmid30763533,
year = {2019},
author = {Rostøl, JT and Marraffini, L},
title = {(Ph)ighting Phages: How Bacteria Resist Their Parasites.},
journal = {Cell host &amp; microbe},
volume = {25},
number = {2},
pages = {184-194},
pmid = {30763533},
issn = {1934-6069},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*virology ; Bacteriophages/*growth &amp; development ; CRISPR-Cas Systems ; Evolution, Molecular ; *Host-Parasite Interactions ; },
abstract = {Bacteria are under constant attack from bacteriophages (phages), bacterial parasites that are the most abundant biological entity on earth. To resist phage infection, bacteria have evolved an impressive arsenal of anti-phage systems. Recent advances have significantly broadened and deepened our understanding of how bacteria battle phages, spearheaded by new systems like CRISPR-Cas. This review aims to summarize bacterial anti-phage mechanisms, with an emphasis on the most recent developments in the field.},
}
@article {pmid30763088,
year = {2019},
author = {Palermo, G},
title = {Structure and Dynamics of the CRISPR-Cas9 Catalytic Complex.},
journal = {Journal of chemical information and modeling},
volume = {59},
number = {5},
pages = {2394-2406},
doi = {10.1021/acs.jcim.8b00988},
pmid = {30763088},
issn = {1549-960X},
mesh = {Biocatalysis ; *CRISPR-Cas Systems ; Gene Editing ; *Molecular Dynamics Simulation ; Protein Domains ; *Quantum Theory ; },
abstract = {CRISPR-Cas9 is a bacterial immune system with exciting applications for genome editing. In spite of extensive experimental characterization, the active site chemistry of the RuvC domain-which performs DNA cleavages-has remained elusive. Its knowledge is key for structure-based engineering aimed at improving DNA cleavages. Here, we deliver an in-depth characterization by using quantum-classical (QM/MM) molecular dynamics (MD) simulations and a Gaussian accelerated MD method, coupled with bioinformatics analysis. We disclose a two-metal aided architecture in the RuvC active site, which is poised to operate DNA cleavages, in analogy with other DNA/RNA processing enzymes. The conformational dynamics of the RuvC domain further reveals that an "arginine finger" stably contacts the scissile phosphate, with the function of stabilizing the active complex. Remarkably, the formation of a catalytically competent state of the RuvC domain is only observed upon the conformational activation of the other nuclease domain of CRISPR-Cas9-i.e., the HNH domain-such allowing concerted cleavages of double stranded DNA. This structure is in agreement with the available experimental data and remarkably differs from previous models based on classical mechanics, demonstrating also that only quantum mechanical simulations can accurately describe the metal-aided active site in CRISPR-Cas9. This fully catalytic structure-in which both the HNH and RuvC domains are prone to perform DNA cleavages-constitutes a stepping-stone for understanding DNA cleavage and specificity. It calls for novel experimental verifications and offers the structural foundations for engineering efforts aimed at improving the genome editing capability of CRISPR-Cas9.},
}
@article {pmid30762876,
year = {2019},
author = {Scaltriti, E and Carminati, D and Cortimiglia, C and Ramoni, R and Sørensen, KI and Giraffa, G and Zago, M},
title = {Survey on the CRISPR arrays in Lactobacillus helveticus genomes.},
journal = {Letters in applied microbiology},
volume = {68},
number = {5},
pages = {394-402},
doi = {10.1111/lam.13128},
pmid = {30762876},
issn = {1472-765X},
mesh = {Bacteriophages/genetics ; Cheese/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genome, Bacterial/*genetics ; Genotype ; Lactobacillus helveticus/*genetics ; Plasmids/genetics ; },
abstract = {Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacteria that is mainly used in the manufacture of Swiss type and long-ripened Italian hard cheeses. In this study, the presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were analysed in 25 L. helveticus genomes and identified in 23 of these genomes. A total of 40 CRISPR loci were identified and classified into five main families based on CRISPR repeats: Ldbu1, Lsal1, Lhel1, Lhel2 and a new repeat family named Lhel3. Spacers had a size between 30 and 40 bp whereas repeats have an average size of 30 bp, with three longer repeats. The analysis displayed the presence of conserved spacers in 23 of the 40 CRISPR loci. A geographical distribution of L. helveticus isolates with similar CRISPR spacer array profiles were not observed. Based on the presence of the signature protein Cas3, all CRISPR loci belonged to Type I. This analysis demonstrated a great CRISPR array variability within L. helveticus, which could be a useful tool for genotypic strain differentiation. A next step will be to understand the possible role of CRISPR/Cas system for the resistance of L. helveticus to phage infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus, a lactic acid bacteria species widely used as starter culture in the dairy industry has recently also gained importance as health-promoting culture in probiotic and nutraceutical food products. The CRISPR/Cas system, a well-known molecular mechanism that provides adaptive immunity against exogenous genetic elements such as bacteriophages and plasmids in bacteria, was recently found in this species. In this study, we investigated the presence and genetic heterogeneity of CRISPR loci in 25 L. helveticus genomes. The results presented here represent an important step on the way to manage phage resistance, plasmid uptake and genome editing in this species.},
}
@article {pmid30762444,
year = {2019},
author = {Gao, M and Zhang, B and He, Y and Yang, Q and Deng, L and Zhu, Y and Lai, E and Wang, M and Wang, L and Yang, G and Liao, G and Bao, J and Bu, H},
title = {Efficient Generation of an Fah/Rag2 Dual-Gene Knockout Porcine Cell Line Using CRISPR/Cas9 and Adenovirus.},
journal = {DNA and cell biology},
volume = {38},
number = {4},
pages = {314-321},
doi = {10.1089/dna.2018.4493},
pmid = {30762444},
issn = {1557-7430},
mesh = {Adenoviridae/*genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA-Binding Proteins/*deficiency/*genetics ; Gene Knockout Techniques/*methods ; Hydrolases/*deficiency/*genetics ; Mutation ; Swine ; Time Factors ; },
abstract = {The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 × 10[8] human hepatocytes per mouse. We hypothesized that 2-10 × 10[10] human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.},
}
@article {pmid30762338,
year = {2019},
author = {Guo, Y and Bao, C and Ma, D and Cao, Y and Li, Y and Xie, Z and Li, S},
title = {Network-Based Combinatorial CRISPR-Cas9 Screens Identify Synergistic Modules in Human Cells.},
journal = {ACS synthetic biology},
volume = {8},
number = {3},
pages = {482-490},
doi = {10.1021/acssynbio.8b00237},
pmid = {30762338},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Cell Survival/drug effects ; Cell Transformation, Neoplastic/genetics ; Colorectal Neoplasms/etiology/genetics ; Computational Biology/*methods ; Drug Discovery/methods ; Drug Screening Assays, Antitumor/methods ; Gene Editing ; Gene Knockout Techniques ; *Gene Regulatory Networks ; HEK293 Cells ; Humans ; Inflammation/complications/genetics ; Inflammatory Bowel Diseases/complications/genetics ; RNA Interference ; Transcriptome ; Transfection ; Transforming Growth Factor beta1/genetics/metabolism ; },
abstract = {Tumorigenesis is a complex process that is driven by a combination of networks of genes and environmental factors; however, efficient approaches to identifying functional networks that are perturbed by the process of tumorigenesis are lacking. In this study, we provide a comprehensive network-based strategy for the systematic discovery of functional synergistic modules that are causal determinants of inflammation-induced tumorigenesis. Our approach prioritizes candidate genes selected by integrating clinical-based and network-based genome-wide gene prediction methods and identifies functional synergistic modules based on combinatorial CRISPR-Cas9 screening. On the basis of candidate genes inferred de novo from experimental and computational methods to be involved in inflammation and cancer, we used an existing TGFβ1-induced cellular transformation model in colonic epithelial cells and a new combinatorial CRISPR-Cas9 screening strategy to construct an inflammation-induced differential genetic interaction network. The inflammation-induced differential genetic interaction network that we generated yielded functional insights into the genes and functional module combinations, and showed varied responses to the inflammation agents as well as active traditional Chinese medicine compounds. We identified opposing differential genetic interactions of inflammation-induced tumorigenesis: synergistic promotion and suppression. The synergistic promotion state was primarily caused by deletions in the immune and metabolism modules; the synergistic suppression state was primarily induced by deletions in the proliferation and immune modules or in the proliferation and metabolism modules. These results provide insight into possible early combinational targets and biomarkers for inflammation-induced tumorigenesis and highlight the synergistic effects that occur among immune, proliferation, and metabolism modules. In conclusion, this approach deepens the understanding of the underlying mechanisms that cause inflammation to potentially increase the cancer risk of colonic epithelial cells and accelerate the translation into novel functional modules or synergistic module combinations that modulate complex disease phenotypes.},
}
@article {pmid30762073,
year = {2019},
author = {Wu, Z and Chen, Z and Gao, X and Li, J and Shang, G},
title = {Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {6},
pages = {2783-2795},
doi = {10.1007/s00253-019-09654-w},
pmid = {30762073},
issn = {1432-0614},
mesh = {Biotechnology ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; DNA, Single-Stranded/*genetics ; Gene Deletion ; Gene Editing/*methods ; *Genome, Bacterial ; Plasmids/genetics ; Point Mutation ; Pseudomonas putida/*genetics ; },
abstract = {Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.},
}
@article {pmid30761103,
year = {2019},
author = {Broecker, F and Moelling, K},
title = {Evolution of Immune Systems From Viruses and Transposable Elements.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {51},
pmid = {30761103},
issn = {1664-302X},
abstract = {Virus-derived sequences and transposable elements constitute a substantial portion of many cellular genomes. Recent insights reveal the intimate evolutionary relationship between these sequences and various cellular immune pathways. At the most basic level, superinfection exclusion may be considered a prototypical virus-mediated immune system that has been described in both prokaryotes and eukaryotes. More complex immune mechanisms fully or partially derived from mobile genetic elements include CRISPR-Cas of prokaryotes and the RAG1/2 system of vertebrates, which provide immunological memory of foreign genetic elements and generate antibody and T cell receptor diversity, respectively. In this review, we summarize the current knowledge on the contribution of mobile genetic elements to the evolution of cellular immune pathways. A picture is emerging in which the various cellular immune systems originate from and are spread by viruses and transposable elements. Immune systems likely evolved from simple superinfection exclusion to highly complex defense strategies.},
}
@article {pmid30760763,
year = {2019},
author = {Yu, M and Sun, X and Tyler, SR and Liang, B and Swatek, AM and Lynch, TJ and He, N and Yuan, F and Feng, Z and Rotti, PG and Choi, SH and Shahin, W and Liu, X and Yan, Z and Engelhardt, JF},
title = {Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1971},
pmid = {30760763},
issn = {2045-2322},
support = {R24 DK096518/DK/NIDDK NIH HHS/United States ; P01 HL051670/HL/NHLBI NIH HHS/United States ; P30 ES005605/ES/NIEHS NIH HHS/United States ; P30 DK054759/DK/NIDDK NIH HHS/United States ; R01 DK047967/DK/NIDDK NIH HHS/United States ; R24 HL123482/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems/*genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Ferrets ; Gene Knock-In Techniques/*methods ; *Gene Transfer Techniques ; Genes, Reporter/genetics ; Genetic Engineering/*methods ; RNA, Guide/genetics ; Repressor Proteins/genetics ; Viral Proteins/genetics ; },
abstract = {The domestic ferret (Mustela putorius furo) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 "safe harbor" locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26-CAG-[LoxP]tdTomato[StopLoxP]EGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.},
}
@article {pmid30760722,
year = {2019},
author = {Papikian, A and Liu, W and Gallego-Bartolomé, J and Jacobsen, SE},
title = {Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {729},
pmid = {30760722},
issn = {2041-1723},
mesh = {Arabidopsis/*genetics/metabolism ; Arabidopsis Proteins/*genetics/metabolism ; Binding Sites/genetics ; *CRISPR-Cas Systems ; Chromatin/genetics/metabolism ; *DNA Methylation ; Epigenesis, Genetic ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Homeodomain Proteins/genetics/metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic/genetics ; Transcription Factors/genetics/metabolism ; },
abstract = {Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, we present a CRISPR-based methylation targeting system for plants, utilizing a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.},
}
@article {pmid30760609,
year = {2019},
author = {Wensing, L and Sharma, J and Uthayakumar, D and Proteau, Y and Chavez, A and Shapiro, RS},
title = {A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans.},
journal = {mSphere},
volume = {4},
number = {1},
pages = {},
pmid = {30760609},
issn = {2379-5042},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Gene Expression Regulation, Fungal ; Genetic Engineering/*methods ; Promoter Regions, Genetic ; RNA, Guide/genetics ; *Transcription, Genetic ; },
abstract = {Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90 This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen.IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.},
}
@article {pmid30760608,
year = {2019},
author = {Román, E and Coman, I and Prieto, D and Alonso-Monge, R and Pla, J},
title = {Implementation of a CRISPR-Based System for Gene Regulation in Candida albicans.},
journal = {mSphere},
volume = {4},
number = {1},
pages = {},
pmid = {30760608},
issn = {2379-5042},
mesh = {CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Catalase/genetics ; Gene Editing/*methods ; *Gene Expression Regulation, Fungal ; Transcriptional Activation ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast Candida albicans By fusing an allele of Streptococcus pyogenes Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene CAT1, encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of CAT1 controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen.IMPORTANCE CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in Candida albicans using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen.},
}
@article {pmid30760561,
year = {2019},
author = {Enderle, J and Dorn, A and Beying, N and Trapp, O and Puchta, H},
title = {The Protease WSS1A, the Endonuclease MUS81, and the Phosphodiesterase TDP1 Are Involved in Independent Pathways of DNA-protein Crosslink Repair in Plants.},
journal = {The Plant cell},
volume = {31},
number = {4},
pages = {775-790},
pmid = {30760561},
issn = {1532-298X},
mesh = {Arabidopsis/genetics/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; Mutation/genetics ; },
abstract = {DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologs of the universal repair proteases SPARTAN/ weak suppressor of smt3 (Wss1), WSS1A, is essential for DPC repair in Arabidopsis (Arabidopsis thaliana). WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of TYROSYL-DNA PHOSPHODIESTERASE 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.},
}
@article {pmid30759475,
year = {2019},
author = {Schermer, B and Benzing, T},
title = {[Genome Editing with CRISPR/Cas9: First Steps Towards a new Era in Medicine?].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {144},
number = {4},
pages = {276-281},
doi = {10.1055/a-0759-7180},
pmid = {30759475},
issn = {1439-4413},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Humans ; },
abstract = {The emergence of genome editing technologies can be regarded as one of the most groundbreaking revolutions in the history of science. Modern genome editing allows the introduction of precise mutations into the genome of virtually all cells and organisms without leaving any additional trace. Undoubtedly, genome editing with CRISPR/Cas9, often casually referred to as "genetic scissors", will revolutionize medical research and development. However, at the same time it creates a great need for ethical considerations as it might hold risks for both people and the environment that cannot yet be fully assessed. While genome editing is already well established in laboratory research, clinical applications based on genome editing are close. For the first time, targeted corrections of genetic defects in somatic cells, stem cells as well as in the germ line appear technically feasible. This generates possible future scenarios that urgently require broad ethical and social discussions.},
}
@article {pmid30759237,
year = {2019},
author = {Dorsey, BW and Huang, L and Mondragón, A},
title = {Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.},
journal = {Nucleic acids research},
volume = {47},
number = {7},
pages = {3765-3783},
pmid = {30759237},
issn = {1362-4962},
support = {P30 CA060553/CA/NCI NIH HHS/United States ; R35 GM118108/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/chemistry/*ultrastructure ; CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; Crystallography, X-Ray ; *Protein Conformation ; RNA, Bacterial/chemistry ; RNA, Guide/chemistry/genetics ; Staphylococcus epidermidis/*chemistry/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.},
}
@article {pmid30759231,
year = {2019},
author = {Lu, B and Javidi-Parsijani, P and Makani, V and Mehraein-Ghomi, F and Sarhan, WM and Sun, D and Yoo, KW and Atala, ZP and Lyu, P and Atala, A},
title = {Delivering SaCas9 mRNA by lentivirus-like bionanoparticles for transient expression and efficient genome editing.},
journal = {Nucleic acids research},
volume = {47},
number = {8},
pages = {e44},
pmid = {30759231},
issn = {1362-4962},
mesh = {Bacterial Proteins/*genetics/metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Dependovirus/genetics/metabolism ; Gene Editing/*methods ; Genes, Reporter ; Genetic Vectors/chemistry/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lentivirus/genetics/metabolism ; Nanoparticles/chemistry/*metabolism ; Plasmids/chemistry/metabolism ; RNA, Guide/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Sequence Alignment ; Staphylococcus aureus/chemistry/enzymology ; Transfection/*methods ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 (SaCas9) mRNA in each lentivirus-like bionanoparticle (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9. Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.},
}
@article {pmid30755668,
year = {2019},
author = {Kostyushev, D and Kostyusheva, A and Brezgin, S and Zarifyan, D and Utkina, A and Goptar, I and Chulanov, V},
title = {Suppressing the NHEJ pathway by DNA-PKcs inhibitor NU7026 prevents degradation of HBV cccDNA cleaved by CRISPR/Cas9.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1847},
pmid = {30755668},
issn = {2045-2322},
mesh = {Apoptosis ; *CRISPR-Cas Systems ; Catalytic Domain ; Cell Survival ; Chromones/*pharmacology ; DNA End-Joining Repair/*drug effects ; DNA Mutational Analysis ; DNA, Circular/*drug effects ; DNA, Viral/drug effects/*genetics ; Gene Deletion ; Genome, Viral ; Hep G2 Cells ; Hepatitis B virus ; Hepatitis B, Chronic/metabolism/*virology ; High-Throughput Nucleotide Sequencing ; Humans ; Morpholines/*pharmacology ; RNA, Guide/genetics ; },
abstract = {Chronic hepatitis B is a severe liver disease caused by hepatitis B virus (HBV) infection. Covalently closed circular DNA (cccDNA), a super-spiralized, double-stranded form of the HBV genome, is the major determinant of viral persistence. CRISPR/Cas9 nucleases have been recently shown to introduce double-stranded DNA breaks into HBV cccDNA. The inflicted damage results predominantly in erroneous repair of cccDNA by non-homologous end-joining (NHEJ). NHEJ has been suggested to enhance anti-HBV activity of CRISPR/Cas9 and increase cccDNA mutation. In this study, we assessed anti-HBV activity of CRISPR/Cas9 and cccDNA repair outcomes in an altered NHEJ/HR environment. NU7026, a strong inhibitor of NHEJ, prevented CRISPR/Cas9-mediated degradation of cccDNA and resulted in frequent on-target deletions. We conclude that CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonstrate that inhibiting NHEJ impairs cccDNA degradation.},
}
@article {pmid30755626,
year = {2019},
author = {Fernandes, LGV and Guaman, LP and Vasconcellos, SA and Heinemann, MB and Picardeau, M and Nascimento, ALTO},
title = {Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1839},
pmid = {30755626},
issn = {2045-2322},
mesh = {CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Gene Silencing ; Genetic Engineering/*methods ; Leptospira/*physiology ; RNA ; RNA, Guide/*genetics ; Streptococcus pyogenes/*genetics ; },
abstract = {Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.},
}
@article {pmid30755442,
year = {2019},
author = {Howard, TP and Arnoff, TE and Song, MR and Giacomelli, AO and Wang, X and Hong, AL and Dharia, NV and Wang, S and Vazquez, F and Pham, MT and Morgan, AM and Wachter, F and Bird, GH and Kugener, G and Oberlick, EM and Rees, MG and Tiv, HL and Hwang, JH and Walsh, KH and Cook, A and Krill-Burger, JM and Tsherniak, A and Gokhale, PC and Park, PJ and Stegmaier, K and Walensky, LD and Hahn, WC and Roberts, CWM},
title = {MDM2 and MDM4 Are Therapeutic Vulnerabilities in Malignant Rhabdoid Tumors.},
journal = {Cancer research},
volume = {79},
number = {9},
pages = {2404-2414},
pmid = {30755442},
issn = {1538-7445},
support = {T32 CA136432/CA/NCI NIH HHS/United States ; T32 GM007226/GM/NIGMS NIH HHS/United States ; P50 CA101942/CA/NCI NIH HHS/United States ; R00 CA197640/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; R35 CA210030/CA/NCI NIH HHS/United States ; R01 CA113794/CA/NCI NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; F30 CA221087/CA/NCI NIH HHS/United States ; R35 CA197583/CA/NCI NIH HHS/United States ; R50 CA211399/CA/NCI NIH HHS/United States ; R01 CA172152/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; CRISPR-Cas Systems ; Cell Cycle Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; Mice ; Mice, Nude ; Proto-Oncogene Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Proto-Oncogene Proteins c-mdm2/*antagonists &amp; inhibitors/genetics/metabolism ; Rhabdoid Tumor/*drug therapy/genetics/metabolism/pathology ; SMARCB1 Protein/genetics/metabolism ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53/genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.},
}
@article {pmid30755408,
year = {2019},
author = {Hayashi, A and Tanaka, K},
title = {Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {4},
pages = {1153-1163},
pmid = {30755408},
issn = {2160-1836},
mesh = {CRISPR-Cas Systems ; Cloning, Molecular ; Gene Editing/*methods ; Genetic Vectors ; Mutagenesis, Site-Directed ; Point Mutation ; Schizosaccharomyces/*genetics ; Schizosaccharomyces pombe Proteins/chemistry/*genetics ; },
abstract = {The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.},
}
@article {pmid30755401,
year = {2019},
author = {Wu, Y and Liu, SX and Wang, F and Zeng, MS},
title = {Room Temperature Detection of Plasma Epstein-Barr Virus DNA with CRISPR-Cas13.},
journal = {Clinical chemistry},
volume = {65},
number = {4},
pages = {591-592},
doi = {10.1373/clinchem.2018.299347},
pmid = {30755401},
issn = {1530-8561},
mesh = {Base Sequence ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA, Viral/*blood ; Herpesvirus 4, Human/*genetics ; Humans ; Leptotrichia/chemistry ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Temperature ; },
}
@article {pmid30753226,
year = {2019},
author = {Kim, EK and Kim, S and Maeng, YS},
title = {Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0211864},
pmid = {30753226},
issn = {1932-6203},
mesh = {ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics ; CRISPR-Cas Systems/*genetics ; Corneal Dystrophies, Hereditary/*genetics/pathology/therapy ; Epithelial Cells/metabolism/pathology ; Extracellular Matrix Proteins/*genetics ; Extremities/growth &amp; development/pathology ; Gene Editing ; Gene Expression Regulation/genetics ; Gene Knockout Techniques ; *Genetic Therapy ; Humans ; Primary Cell Culture ; RNA, Guide/genetics ; Sequence Deletion/genetics ; Single-Cell Analysis ; Stem Cells/metabolism ; Transforming Growth Factor beta/*genetics ; },
abstract = {Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.},
}
@article {pmid30747044,
year = {2019},
author = {Dyikanov, DT and Vasiluev, PA and Rysenkova, KD and Aleksandrushkina, NA and Tyurin-Kuzmin, PA and Kulebyakin, KY and Rubtsov, YP and Shmakova, AA and Evseeva, MN and Balatskiy, AV and Semina, EV and Rostovtseva, AI and Makarevich, PI and Karagyaur, MN},
title = {Optimization of CRISPR/Cas9 Technology to Knock Out Genes of Interest in Aneuploid Cell Lines.},
journal = {Tissue engineering. Part C, Methods},
volume = {25},
number = {3},
pages = {168-175},
doi = {10.1089/ten.TEC.2018.0365},
pmid = {30747044},
issn = {1937-3392},
mesh = {*Aneuploidy ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques/*standards ; Genes/*genetics ; HeLa Cells ; Hep G2 Cells ; Humans ; Mice ; NIH 3T3 Cells ; },
abstract = {Cell lines represent convenient models to elucidate specific causes of multigenetic and pluricausal diseases, to test breakthrough regenerative technologies. Most commonly used cell lines surpass diploid cells in their accessibility for delivery of large DNA molecules and genome editing, but the main obstacles for obtaining cell models with knockout-targeted protein from aneuploid cells are multiple allele copies and karyotype/phenotype heterogeneity. In the study, we report an original approach to CRISPR-/Cas9-mediated genome modification of aneuploid cell cultures to create functional cell models, achieving highly efficient targeted protein knockout and avoiding "clonal effect" (for the first time to our knowledge).},
}
@article {pmid30745836,
year = {2019},
author = {Chen, J and Wang, H and Bai, J and Liu, W and Liu, X and Yu, D and Feng, T and Sun, Z and Zhang, L and Ma, L and Hu, Y and Zou, Y and Tan, T and Zhong, J and Hu, M and Bai, X and Pan, D and Xing, Y and Zhao, Y and Tian, K and Hu, X and Li, N},
title = {Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163.},
journal = {International journal of biological sciences},
volume = {15},
number = {2},
pages = {481-492},
pmid = {30745836},
issn = {1449-2288},
mesh = {Animals ; Antigens, CD/*genetics ; Antigens, Differentiation, Myelomonocytic/*genetics ; CRISPR-Cas Systems/genetics ; Exons/genetics ; Gene Editing/*methods ; Porcine respiratory and reproductive syndrome virus/*genetics ; Receptors, Cell Surface/*genetics ; Swine ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163[Mut/Mut] pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163[Mut/Mut] pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163[Mut/Mut] pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.},
}
@article {pmid30745494,
year = {2019},
author = {Tobita, T and Kiyozumi, D and Muto, M and Noda, T and Ikawa, M},
title = {Lvrn expression is not critical for mouse placentation.},
journal = {The Journal of reproduction and development},
volume = {65},
number = {3},
pages = {239-244},
pmid = {30745494},
issn = {1348-4400},
mesh = {Animals ; Blood Pressure ; CRISPR-Cas Systems ; Female ; Fetal Growth Retardation/metabolism ; Fetus/metabolism ; Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Lentivirus/metabolism ; Metalloproteases/genetics/*physiology ; Mice ; Mice, Knockout ; Placenta/*physiology ; Placentation/genetics/*physiology ; Pre-Eclampsia ; Pregnancy ; Pregnancy, Animal ; Trophoblasts/metabolism ; },
abstract = {Preeclampsia is a systemic disease caused by abnormal placentation that affects both mother and fetus. It was reported that Laeverin (LVRN, also known as Aminopeptidase Q) was up-regulated in the placenta of preeclamptic patients. However, physiological and pathological functions of LVRN remained to be unknown. Here we characterized Lvrn function during placentation in mice. RT-PCR showed that Lvrn is expressed in both fetus and placenta during embryogenesis, and several adult tissues. When we overexpressed Lvrn in a placenta-specific manner using lentiviral vectors, we did not see any defects in both placentae and fetuses. The mice carrying Lvrn overexpressing placentas did not show any preeclampsia-like symptoms such as maternal high blood pressure and fetal growth restriction. We next ablated Lvrn by CRISPR/Cas9-mediated genome editing to see physiological function. In Lvrn ablated mice, maternal blood pressure during pregnancy was not affected, and both placentas and fetuses grew normally. Collectively, these results suggest that, LVRN is irrelevant to preeclampsia and dispensable for normal placentation and embryonic development in mice.},
}
@article {pmid30745430,
year = {2019},
author = {Kumar, N and Tsai, YH and Chen, L and Zhou, A and Banerjee, KK and Saxena, M and Huang, S and Toke, NH and Xing, J and Shivdasani, RA and Spence, JR and Verzi, MP},
title = {The lineage-specific transcription factor CDX2 navigates dynamic chromatin to control distinct stages of intestine development.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {5},
pages = {},
pmid = {30745430},
issn = {1477-9129},
support = {F32 DK115080/DK/NIDDK NIH HHS/United States ; R01 CA190558/CA/NCI NIH HHS/United States ; R01 DK082889/DK/NIDDK NIH HHS/United States ; U01 DK085532/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CDX2 Transcription Factor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage ; Chromatin/*metabolism ; Female ; *Gene Expression Regulation, Developmental ; Homeodomain Proteins/*metabolism ; Humans ; Intestinal Mucosa/metabolism ; Intestines/*embryology ; Mice ; Mice, Knockout ; Mutation ; Pluripotent Stem Cells/cytology ; Protein Binding ; Protein Domains ; Trans-Activators/metabolism ; },
abstract = {Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic loss of CDX2 triggers homeotic transformation of intestinal fate, whereas adult-onset loss compromises crucial physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program; rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or mature intestinal function. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.},
}
@article {pmid30745359,
year = {2019},
author = {Malfacini, D and Patt, J and Annala, S and Harpsøe, K and Eryilmaz, F and Reher, R and Crüsemann, M and Hanke, W and Zhang, H and Tietze, D and Gloriam, DE and Bräuner-Osborne, H and Strømgaard, K and König, GM and Inoue, A and Gomeza, J and Kostenis, E},
title = {Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {15},
pages = {5747-5758},
pmid = {30745359},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Depsipeptides/*pharmacology ; Enzyme Inhibitors/*pharmacology ; *GTP-Binding Protein alpha Subunits/antagonists &amp; inhibitors/genetics/metabolism ; HEK293 Cells ; Humans ; Hydrophobic and Hydrophilic Interactions ; Mice ; Peptides, Cyclic/*pharmacology ; *Protein Engineering ; },
abstract = {Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.},
}
@article {pmid30745225,
year = {2019},
author = {Soni, S and Kohn, DB},
title = {Chemistry, manufacturing and controls for gene modified hematopoietic stem cells.},
journal = {Cytotherapy},
volume = {21},
number = {3},
pages = {358-366},
doi = {10.1016/j.jcyt.2018.12.001},
pmid = {30745225},
issn = {1477-2566},
mesh = {Blood Component Removal/methods ; CRISPR-Cas Systems/genetics ; Cell Survival ; Endotoxins/analysis ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors ; HEK293 Cells ; *Hematopoietic Stem Cells ; Humans ; Lentivirus/genetics ; Limulus Test ; Mycoplasma ; *Quality Control ; Transduction, Genetic/*methods ; },
abstract = {Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e.g., Zinc finger, TALEN and Crispr/Cas9) are being applied to the treatment of multiple genetic and immunodeficiency disorders. Herein, the current state of the art in manufacturing and critical assays that are required for ex vivo manipulation of stem cells are addressed. Important quality control and safety assays that need to be planned early in the process development phase of these products for regulatory approval are also highlighted.},
}
@article {pmid30744035,
year = {2019},
author = {Chen, YM and Ou, BT and Chen, CY and Chan, HH and Chen, CJ and Wang, RY},
title = {Staufen1 Protein Participates Positively in the Viral RNA Replication of Enterovirus 71.},
journal = {Viruses},
volume = {11},
number = {2},
pages = {},
pmid = {30744035},
issn = {1999-4915},
mesh = {5' Untranslated Regions ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytoskeletal Proteins/genetics/*metabolism ; Enterovirus A, Human/*physiology ; Gene Knockout Techniques ; *Host Microbial Interactions ; Humans ; Protein Biosynthesis ; RNA, Viral/*genetics ; RNA-Binding Proteins/genetics/*metabolism ; *Virus Replication ; },
abstract = {The double-stranded RNA-binding protein Staufen1 (Stau1) has multiple functions during RNA virus infection. In this study, we investigated the role of Stau1 in viral translation by using a combination of enterovirus 71 (EV-A71) infection, RNA reporter transfection, and in vitro functional and biochemical assays. We demonstrated that Stau1 specifically binds to the 5'-untranslated region of EV-A71 viral RNA. The RNA-binding domain 2-3 of Stau1 is responsible for this binding ability. Subsequently, we created a Stau1 knockout cell line using the CRISPR/Cas9 approach to further characterize the functional role of Stau1's interaction with viral RNA in the EV-A71-infected cells. Both the viral RNA accumulation and viral protein expression were downregulated in the Stau1 knockout cells compared with the wild-type naïve cells. Moreover, dysregulation of viral RNA translation was observed in the Stau1 knockout cells using ribosome fractionation assay, and a reduced RNA stability of 5'-UTR of the EV-A71 was also identified using an RNA stability assay, which indicated that Stau1 has a role in facilitating viral translation during EV-A71 infection. In conclusion, we determined the functional relevance of Stau1 in the EV-A71 infection cycle and herein describe the mechanism of Stau1 participation in viral RNA translation through its interaction with viral RNA. Our results suggest that Stau1 is an important host factor involved in viral translation and influential early in the EV-A71 replication cycle.},
}
@article {pmid30742890,
year = {2019},
author = {Wang, Z and Lin, L and Chen, W and Zheng, X and Zhang, Y and Liu, Q and Yang, D},
title = {Neutrophil plays critical role during Edwardsiella piscicida immersion infection in zebrafish larvae.},
journal = {Fish &amp; shellfish immunology},
volume = {87},
number = {},
pages = {565-572},
doi = {10.1016/j.fsi.2019.02.008},
pmid = {30742890},
issn = {1095-9947},
mesh = {Animals ; CRISPR-Cas Systems/immunology ; Chemokines/genetics/immunology ; Cytokines/*genetics/immunology ; Edwardsiella/physiology ; Enterobacteriaceae Infections/immunology/microbiology/*veterinary ; Fish Diseases/*immunology/microbiology ; Gene Knockdown Techniques/veterinary ; Immunity, Innate/*genetics ; Neutrophils/*immunology/metabolism ; Up-Regulation/*genetics ; *Zebrafish ; },
abstract = {Edwardsiella piscicida is a facultative intracellular pathogen that causes hemorrhagic septicemia and haemolytic ascites disease in aquaculture fish. During bacterial infection, macrophages and neutrophils are the first line of host innate immune system. However, the role of neutrophils in response to E. piscicida infection in vivo remains poorly understood. Here, through developing an immersion infection model in the 5 day-post fertilization (dpf) zebrafish larvae, we found that E. piscicida was mainly colonized in intestine, and resulted into significant pathological changes in paraffin sections. Moreover, a dynamic up-regulation of inflammatory cytokines (TNF-α, IL-1β, GCSFb, CXCL8 and MMP9) was detected in zebrafish larvae during E. piscicida infection. Furthermore, a significant recruitment of neutrophils was observed during the E. piscicida infection in Tg(mpx:eGFP) zebrafish larvae. Thus, we utilized the CRISPR/Cas9 system to generate the neutrophil-knockdown (gcsfr[-/-] crispants) larvae, and found a comparative higher mortality and bacterial colonization in gcsfr[-/-] crispants, which reveals the critical role of fish neutrophils in bacterial clearance. Taken together, our results developed an effective E. piscicida immersion challenge model in zebrafish larvae to clarify the dynamic of bacterial infection in vivo, which would provide a better understanding of the action about innate immune cells during infection.},
}
@article {pmid30742127,
year = {2019},
author = {Kleinstiver, BP and Sousa, AA and Walton, RT and Tak, YE and Hsu, JY and Clement, K and Welch, MM and Horng, JE and Malagon-Lopez, J and Scarfò, I and Maus, MV and Pinello, L and Aryee, MJ and Joung, JK},
title = {Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.},
journal = {Nature biotechnology},
volume = {37},
number = {3},
pages = {276-282},
pmid = {30742127},
issn = {1546-1696},
support = {R00 CA218870/CA/NCI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; K99 CA218870/CA/NCI NIH HHS/United States ; },
mesh = {Acidaminococcus/enzymology ; Bacterial Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Endonucleases/*genetics ; Epigenesis, Genetic/genetics ; *Gene Editing ; HEK293 Cells ; Humans ; Mutation ; Ribonucleoproteins/*genetics ; T-Lymphocytes/metabolism ; },
abstract = {Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases[1] has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)[2]. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants[3] to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.},
}
@article {pmid30742049,
year = {2019},
author = {McKenzie, RE and Almendros, C and Vink, JNA and Brouns, SJJ},
title = {Using CAPTURE to detect spacer acquisition in native CRISPR arrays.},
journal = {Nature protocols},
volume = {14},
number = {3},
pages = {976-990},
pmid = {30742049},
issn = {1750-2799},
support = {639707/ERC_/European Research Council/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA Primers/metabolism ; Electrophoresis, Agar Gel ; High-Throughput Nucleotide Sequencing ; Polymerase Chain Reaction/*methods ; },
abstract = {CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 10[5] cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. CAPTURE is a straightforward method that requires only 1 d to enable the detection of spacer acquisition in all native CRISPR systems and facilitate studies aimed both at unraveling the mechanism of spacer integration and more sensitive tracing of integration events in natural ecosystems.},
}
@article {pmid30741685,
year = {2019},
author = {Mohamed, NV and Larroquette, F and Beitel, LK and Fon, EA and Durcan, TM},
title = {One Step Into the Future: New iPSC Tools to Advance Research in Parkinson's Disease and Neurological Disorders.},
journal = {Journal of Parkinson's disease},
volume = {9},
number = {2},
pages = {265-281},
pmid = {30741685},
issn = {1877-718X},
mesh = {Biomedical Research ; CRISPR-Cas Systems ; Coculture Techniques ; Drug Discovery ; Gene Editing ; Humans ; In Vitro Techniques ; *Induced Pluripotent Stem Cells ; Lab-On-A-Chip Devices ; Organoids ; *Parkinson Disease ; },
abstract = {Studying Parkinson's disease (PD) in the laboratory presents many challenges, the main one being the limited availability of human cells and tissue from affected individuals. As PD is characterized by a loss of dopaminergic (DA) neurons in the brain, it is nearly impossible for researchers to access and extract these cells from living patients. Thus, in the past PD research has focused on the use of patients' post-mortem tissues, animal models, or immortalized cell lines to dissect cellular pathways of interest. While these strategies deepened our knowledge of pathological mechanisms in PD, they failed to faithfully capture key mechanisms at play in the human brain. The emergence of induced pluripotent stem cell (iPSC) technology is revolutionizing PD research, as it allows for the differentiation and growth of human DA neurons in vitro, holding immense potential not only for modelling PD, but also for identifying novel therapies. However, to reproduce the complexity of the brain's environment, researchers are recognizing the need to further develop and refine iPSC-based tools. In this review, we provide an overview of different systems now available for the study of PD, with a particular emphasis on the potential and limitations of iPSC as research tools to generate more relevant models of PD pathophysiology and advance the drug discovery process.},
}
@article {pmid30740978,
year = {2019},
author = {Savage, DF},
title = {Cas14: Big Advances from Small CRISPR Proteins.},
journal = {Biochemistry},
volume = {58},
number = {8},
pages = {1024-1025},
pmid = {30740978},
issn = {1520-4995},
support = {DP2 EB018658/EB/NIBIB NIH HHS/United States ; R01 GM127463/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/*metabolism ; *Gene Editing ; Humans ; *RNA Interference ; RNA, Guide/genetics/*metabolism ; },
}
@article {pmid30739529,
year = {2019},
author = {West, KL and Byrum, SD and Mackintosh, SG and Edmondson, RD and Taverna, SD and Tackett, AJ},
title = {Proteomic characterization of the arsenic response locus in S. cerevisiae.},
journal = {Epigenetics},
volume = {14},
number = {2},
pages = {130-145},
pmid = {30739529},
issn = {1559-2308},
support = {UL1 TR000039/TR/NCATS NIH HHS/United States ; R21 ES025268/ES/NIEHS NIH HHS/United States ; R01 GM118760/GM/NIGMS NIH HHS/United States ; P20 GM121293/GM/NIGMS NIH HHS/United States ; S10 OD018445/OD/NIH HHS/United States ; P20 GM103625/GM/NIGMS NIH HHS/United States ; P20 GM103429/GM/NIGMS NIH HHS/United States ; R21 DA041822/DA/NIDA NIH HHS/United States ; },
mesh = {Arsenate Reductases/genetics ; Arsenic/*pharmacology ; Basic-Leucine Zipper Transcription Factors/genetics ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Fungal/drug effects ; Membrane Transport Proteins/genetics ; Promoter Regions, Genetic ; Proteomics/*methods ; Repressor Proteins/genetics ; Saccharomyces cerevisiae/*growth &amp; development/metabolism ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; },
abstract = {Arsenic exposure is a global health problem. Millions of people encounter arsenic through contaminated drinking water, consumption, and inhalation. The arsenic response locus in budding yeast is responsible for the detoxification of arsenic and its removal from the cell. This locus constitutes a conserved pathway ranging from prokaryotes to higher eukaryotes. The goal of this study was to identify how transcription from the arsenic response locus is regulated in an arsenic dependent manner. An affinity enrichment strategy called CRISPR-Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) was used, which provides for the proteomic characterization of a targeted locus. CRISPR-ChAP-MS was applied to the promoter regions of the activated arsenic response locus and uncovered 40 nuclear-annotated proteins showing enrichment. Functional assays identified the histone acetyltransferase SAGA and the chromatin remodelling complex SWI/SNF to be required for activation of the locus. Furthermore, SAGA and SWI/SNF were both found to specifically organize the chromatin structure at the arsenic response locus for activation of gene transcription. This study provides the first proteomic characterization of an arsenic response locus and key insight into the mechanisms of transcriptional activation that are necessary for detoxification of arsenic from the cell.},
}
@article {pmid30739083,
year = {2019},
author = {Komatsubara, AT and Goto, Y and Kondo, Y and Matsuda, M and Aoki, K},
title = {Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {15},
pages = {6062-6072},
pmid = {30739083},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1/genetics/*metabolism ; Ribosomal Protein S6 Kinases, 90-kDa/genetics/*metabolism ; Spectrometry, Fluorescence/methods ; },
abstract = {Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.},
}
@article {pmid30738789,
year = {2019},
author = {Khan, MZ and Zaidi, SS and Amin, I and Mansoor, S},
title = {A CRISPR Way for Fast-Forward Crop Domestication.},
journal = {Trends in plant science},
volume = {24},
number = {4},
pages = {293-296},
doi = {10.1016/j.tplants.2019.01.011},
pmid = {30738789},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural ; *Domestication ; Genome, Plant ; },
abstract = {Precision crop breeding, using genome editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR) systems to improve useful traits in crop plants, holds great potential for the future of agriculture. Using CRISPR-Cas9, recent studies have engineered domestication traits in wild-relative species of tomato crop for higher nutritive value and better adaptation to diverse stresses.},
}
@article {pmid30738580,
year = {2019},
author = {Gong, W and Liu, Y and Qu, H and Liu, A and Sun, P and Wang, X},
title = {The effect of CTCF binding sites destruction by CRISPR/Cas9 on transcription of metallothionein gene family in liver hepatocellular carcinoma.},
journal = {Biochemical and biophysical research communications},
volume = {510},
number = {4},
pages = {530-538},
doi = {10.1016/j.bbrc.2019.01.107},
pmid = {30738580},
issn = {1090-2104},
mesh = {Binding Sites ; CCCTC-Binding Factor/*metabolism ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*genetics ; Cell Line, Tumor ; Chromatin/genetics/metabolism ; Humans ; Liver Neoplasms/*genetics ; Metallothionein/*genetics ; Multigene Family ; *Transcriptional Activation ; },
abstract = {Chromatin spatial organization is essential for transcriptional modulation and stabilization. The pattern of DNA distal interplay form the multiple topological associating domains (TADs), and further assemble the functional compartmentalization with open and expression-active chromatin ("A" compartments) or closed and expression-inactive chromatin ("B" compartments) in genome, whose boundaries were defined by the high enrichment of CCCTC-binding factor (CTCF). Nevertheless, As a potential therapeutic strategy, changing the local chromatin architecture via adding or removing the CTCF binding sites in situ to regulate the transcription activity of genes within one TAD in cancer cells is poorly explored. In present study, we observed that the metallothionein (MT) family were all remarkably decreased in HCC of TCGA database, and MT genes family were located within a TAD of 1.2 Mb at 16q13 in order, and CTCF binding sites were distributed at the both sites of MT gene clusters. Furthermore, CRISPR/Cas9 was employed to destroy the CTCF binding sites at the vicinity of the MT family in human liver hepatocellular carcinoma (HCC) cell lines Huh-7 and HepG2. And the presence of up-regulated transcription of MTs were observed in Huh-7 and HepG2 cells compared to normal liver CRL-12461 cells. Moreover, the presence of the varying DNA interplay as well as H3K4me3 and H3K9me3 modification on different MT genes were observed after CTCF binding domain destruction compared to the control using chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP). Our results determined a potential way to regulate the transcription of a series of genes via changing the local genomic organization for diseases treatment.},
}
@article {pmid30738385,
year = {2019},
author = {Emmert, AS and Vuong, SM and Shula, C and Lindquist, D and Yuan, W and Hu, YC and Mangano, FT and Goto, J},
title = {Characterization of a novel rat model of X-linked hydrocephalus by CRISPR-mediated mutation in L1cam.},
journal = {Journal of neurosurgery},
volume = {132},
number = {3},
pages = {945-958},
doi = {10.3171/2018.10.JNS181015},
pmid = {30738385},
issn = {1933-0693},
abstract = {OBJECTIVE: Emergence of CRISPR/Cas9 genome editing provides a robust method for gene targeting in a variety of cell types, including fertilized rat embryos. The authors used this method to generate a transgenic rat L1cam knockout model of X-linked hydrocephalus (XLH) with human genetic etiology. The object of this study was to use diffusion tensor imaging (DTI) in studying perivascular white matter tract injury in the rat model and to characterize its pathological definition in histology.<br><br>METHODS: Two guide RNAs designed to disrupt exon 4 of the L1cam gene on the X chromosome were injected into Sprague-Dawley rat embryos. Following embryo transfer into pseudopregnant females, rats were born and their DNA was sequenced for evidence of L1cam mutation. The mutant and control wild-type rats were monitored for growth and hydrocephalus phenotypes. Their macro- and microbrain structures were studied with T2-weighted MRI, DTI, immunohistochemistry, and transmission electron microscopy (TEM).<br><br>RESULTS: The authors successfully obtained 2 independent L1cam knockout alleles and 1 missense mutant allele. Hemizygous male mutants from all 3 alleles developed hydrocephalus and delayed development. Significant reductions in fractional anisotropy and axial diffusivity were observed in the corpus callosum, external capsule, and internal capsule at 3 months of age. The mutant rats did not show reactive gliosis by then but exhibited hypomyelination and increased extracellular fluid in the corpus callosum.<br><br>CONCLUSIONS: The CRISPR/Cas9-mediated genome editing system can be harnessed to efficiently disrupt the L1cam gene in rats for creation of a larger XLH animal model than previously available. This study provides evidence that the early pathology of the periventricular white matter tracts in hydrocephalus can be detected in DTI. Furthermore, TEM-based morphometric analysis of the corpus callosum elucidates the underlying cytopathological changes accompanying hydrocephalus-derived variations in DTI. The CRISPR/Cas9 system offers opportunities to explore novel surgical and imaging techniques on larger mammalian models.},
}
@article {pmid30738140,
year = {2019},
author = {Wang, Q and Coleman, JJ},
title = {CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {126},
number = {},
pages = {17-24},
pmid = {30738140},
issn = {1096-0937},
support = {K22 AI100983/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Fungal Proteins/*genetics ; Fusarium/*genetics ; Genes, Fungal ; *Genetic Engineering ; Homologous Recombination ; Hyphae/metabolism ; Qa-SNARE Proteins/genetics ; Spores, Fungal/growth &amp; development ; Virulence ; },
abstract = {Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ∼8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.},
}
@article {pmid30737704,
year = {2019},
author = {Moravec, CE and Pelegri, FJ},
title = {An Accessible Protocol for the Generation of CRISPR-Cas9 Knockouts Using INDELs in Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1920},
number = {},
pages = {377-392},
doi = {10.1007/978-1-4939-9009-2_23},
pmid = {30737704},
issn = {1940-6029},
support = {R01 GM065303/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Computational Biology/methods ; Female ; *Gene Editing ; *Gene Knockout Techniques ; *Gene Targeting/methods ; *INDEL Mutation ; Male ; RNA, Guide/chemical synthesis/genetics ; Zebrafish/*genetics ; },
abstract = {The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.},
}
@article {pmid30737513,
year = {2019},
author = {Lorenzo-Orts, L and Witthoeft, J and Deforges, J and Martinez, J and Loubéry, S and Placzek, A and Poirier, Y and Hothorn, LA and Jaillais, Y and Hothorn, M},
title = {Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants.},
journal = {Nature plants},
volume = {5},
number = {2},
pages = {184-193},
pmid = {30737513},
issn = {2055-0278},
mesh = {5' Untranslated Regions ; Acid Anhydride Hydrolases/*genetics/metabolism ; Anaphase-Promoting Complex-Cyclosome/genetics/metabolism ; Arabidopsis/cytology/*genetics/growth &amp; development ; Arabidopsis Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Cytoplasm/metabolism ; Gene Expression Regulation, Plant ; Mutation ; Open Reading Frames ; Plants, Genetically Modified ; Polyribosomes/genetics/metabolism ; },
abstract = {Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.},
}
@article {pmid30737491,
year = {2019},
author = {Halder, V and Porter, CBM and Chavez, A and Shapiro, RS},
title = {Design, execution, and analysis of CRISPR-Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans.},
journal = {Nature protocols},
volume = {14},
number = {3},
pages = {955-975},
pmid = {30737491},
issn = {1750-2799},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Candida albicans/*genetics ; Diploidy ; *Epistasis, Genetic ; *Gene Deletion ; *Genetic Techniques ; Haploidy ; Homozygote ; Plasmids/genetics ; RNA, Guide/genetics/metabolism ; Transformation, Genetic ; },
abstract = {The study of fungal pathogens is of immediate importance, yet progress is hindered by the technical challenges of genetic manipulation. For Candida species, their inability to maintain plasmids, unusual codon usage, and inefficient homologous recombination are among the obstacles limiting efficient genetic manipulation. New advances in genomic biotechnologies-particularly CRISPR-based tools-have revolutionized genome editing for many fungal species. Here, we present a protocol for CRISPR-Cas9-based manipulation in Candida albicans using a modified gene-drive-based strategy that takes ~1 month to complete. We detail the generation of Candida-optimized Cas9-based plasmids for gene deletion, an efficient transformation protocol using C. albicans haploids, and an optimized mating strategy to generate homozygous single- and double-gene diploid mutants. We further describe protocols for quantifying cell growth and analysis pipelines to calculate fitness and genetic interaction scores for genetic mutants. This protocol overcomes previous limitations associated with genetic manipulation in C. albicans and advances researchers' ability to perform genetic analysis in this pathogen; the protocol also has broad applicability to other mating-competent microorganisms.},
}
@article {pmid30737425,
year = {2019},
author = {Wang, R and Tavano, ECDR and Lammers, M and Martinelli, AP and Angenent, GC and de Maagd, RA},
title = {Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1696},
pmid = {30737425},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Fruit/genetics/physiology ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/genetics/*physiology ; *Mutagenesis ; Phenotype ; Plant Proteins/genetics ; Transcription Factors/*genetics ; },
abstract = {Tomato (Solanum lycopersicum) is a model for climacteric fleshy fruit ripening studies. Tomato ripening is regulated by multiple transcription factors together with the plant hormone ethylene and their downstream effector genes. Transcription Factors APETALA2a (AP2a), NON-RIPENING (NOR) and FRUITFULL (FUL1/TDR4 and FUL2/MBP7) were reported as master regulators controlling tomato fruit ripening. Their proposed functions were derived from studies of the phenotype of spontaneous mutants or RNAi knock-down lines rather than, as it appears now, actual null mutants. To study TF function in tomato fruit ripening in more detail, we used CRISPR/Cas9-mediated mutagenesis to knock out the encoding genes, and phenotypes of these mutants are reported for the first time. While the earlier ripening, orange-ripe phenotype of ap2a mutants was confirmed, the nor null mutant exhibited a much milder phenotype than the spontaneous nor mutant. Additional analyses revealed that the severe phenotype in the spontaneous mutant is caused by a dominant-negative allele. Our approach also provides new insight into the independent and overlapping functions of FUL1 and FUL2. Single and combined null alleles of FUL1 and FUL2 illustrate that these two genes have partially redundant functions in fruit ripening, but also unveil an additional role for FUL2 in early fruit development.},
}
@article {pmid30737174,
year = {2019},
author = {Uribe, RV and van der Helm, E and Misiakou, MA and Lee, SW and Kol, S and Sommer, MOA},
title = {Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.},
journal = {Cell host &amp; microbe},
volume = {25},
number = {2},
pages = {233-241.e5},
doi = {10.1016/j.chom.2019.01.003},
pmid = {30737174},
issn = {1934-6069},
mesh = {CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; Enzyme Inhibitors/*isolation &amp; purification ; Gene Library ; Genetic Testing ; Metagenomics/*methods ; },
abstract = {CRISPR-Cas systems in bacteria and archaea provide immunity against bacteriophages and plasmids. To overcome CRISPR immunity, phages have acquired anti-CRISPR genes that reduce CRISPR-Cas activity. Using a synthetic genetic circuit, we developed a high-throughput approach to discover anti-CRISPR genes from metagenomic libraries based on their functional activity rather than sequence homology or genetic context. We identified 11 DNA fragments from soil, animal, and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain. Further in vivo and in vitro characterization of a subset of these hits validated the activity of four anti-CRISPRs. Notably, homologs of some of these anti-CRISPRs were detected in seven different phyla, namely Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Spirochaetes, and Balneolaeota, and have high sequence identity suggesting recent horizontal gene transfer. Thus, anti-CRISPRs against type II-A CRISPR-Cas systems are widely distributed across bacterial phyla, suggesting a more complex ecological role than previously appreciated.},
}
@article {pmid30735951,
year = {2019},
author = {Lee, HS and Lee, SH and Park, Y},
title = {Enhancement of androgen transcriptional activation assay based on genome edited glucocorticoid knock out human prostate cancer cell line.},
journal = {Environmental research},
volume = {171},
number = {},
pages = {437-443},
doi = {10.1016/j.envres.2019.01.027},
pmid = {30735951},
issn = {1096-0953},
mesh = {Androgen Antagonists ; Androgens/*toxicity ; Cell Line, Tumor ; Glucocorticoids/*metabolism ; Humans ; Male ; Prostatic Neoplasms ; Transcriptional Activation ; },
abstract = {Endocrine-disrupting chemicals (EDCs) interfere with the biological activity of hormones. Among EDC's, (anti-)androgenic compounds potentially cause several androgen-related diseases. To improve the accuracy of an in vitro transactivation assay (TA) for detection of (anti-)androgenic compounds, We established the glucocorticoid receptor (GR) knockout 22Rv1/MMTV cell line by using an RNA-guided engineered nuclease (RGEN)-derived CRISPR/Cas system. The 22Rv1/MMTV GRKO cell line was characterized and validated by androgen receptor (AR)-mediated TA assay compared with the AR-TA assay using 22Rv1/MMTV. In conclusion, the AR-TA assay with the 22Rv1/MMTV GRKO cell line was more accurate, excluding the misleading signals derived from glucocorticoids or equivalent chemicals, and might be an effective method for screening potential (anti-)androgenic compounds.},
}
@article {pmid30735463,
year = {2019},
author = {Salas-Mckee, J and Kong, W and Gladney, WL and Jadlowsky, JK and Plesa, G and Davis, MM and Fraietta, JA},
title = {CRISPR/Cas9-based genome editing in the era of CAR T cell immunotherapy.},
journal = {Human vaccines &amp; immunotherapeutics},
volume = {15},
number = {5},
pages = {1126-1132},
pmid = {30735463},
issn = {2164-554X},
mesh = {Antigens, CD19 ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Immunotherapy, Adoptive ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy ; Receptors, Chimeric Antigen/*therapeutic use ; T-Lymphocytes/immunology ; },
abstract = {The advent of engineered T cells as a form of immunotherapy marks the beginning of a new era in medicine, providing a transformative way to combat complex diseases such as cancer. Following FDA approval of CAR T cells directed against the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma, CAR T cells are poised to enter mainstream oncology. Despite this success, a number of patients are unable to receive this therapy due to inadequate T cell numbers or rapid disease progression. Furthermore, lack of response to CAR T cell treatment is due in some cases to intrinsic autologous T cell defects and/or the inability of these cells to function optimally in a strongly immunosuppressive tumor microenvironment. We describe recent efforts to overcome these limitations using CRISPR/Cas9 technology, with the goal of enhancing potency and increasing the availability of CAR-based therapies. We further discuss issues related to the efficiency/scalability of CRISPR/Cas9-mediated genome editing in CAR T cells and safety considerations. By combining the tools of synthetic biology such as CARs and CRISPR/Cas9, we have an unprecedented opportunity to optimally program T cells and improve adoptive immunotherapy for most, if not all future patients.},
}
@article {pmid30734978,
year = {2019},
author = {Koizumi, Y and Fukushima, J and Kobayashi, Y and Kadowaki, A and Natsui, M and Yamaguchi, T and Imai, Y and Sugiyama, T and Kuba, K},
title = {Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {20},
number = {12},
pages = {1563-1568},
pmid = {30734978},
issn = {1439-7633},
mesh = {Aspergillus niger/metabolism ; CRISPR-Cas Systems ; Cell Survival/drug effects/genetics ; Humans ; Peptides, Cyclic/*pharmacology ; RNA, Guide/genetics ; Squalene Monooxygenase/*genetics/metabolism ; U937 Cells ; },
abstract = {Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1-resistant cells were analyzed by high-throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1-resistant cells, SQLE, which encodes squalene epoxidase, was identified as a candidate gene. SQLE-depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild-type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.},
}
@article {pmid30734775,
year = {2019},
author = {Eoh, J and Gu, L},
title = {Biomaterials as vectors for the delivery of CRISPR-Cas9.},
journal = {Biomaterials science},
volume = {7},
number = {4},
pages = {1240-1261},
doi = {10.1039/c8bm01310a},
pmid = {30734775},
issn = {2047-4849},
mesh = {Biocompatible Materials/*chemistry ; CRISPR-Cas Systems/*genetics ; *Gene Transfer Techniques ; Genetic Vectors/*genetics ; Humans ; },
abstract = {The emergence of the CRISPR-Cas9 gene editing system has brought much hope and excitement to the field of gene therapy and the larger scientific community. However, in order for CRISPR-based therapies to be translated to the clinical setting, there is an urgent need to develop optimized vectors for their delivery. The delivery vector is a crucial determinant of the therapeutic efficacy of gene editing and should be designed to accommodate various factors including the form of the payload, the physiological environment, and the potential immune responses. Recently, biomaterials have become an attractive option for the delivery of Cas9 due to their tunability, biocompatibility and increasing efficacy at drug delivery. Biomaterials offer a unique solution for creating tailored vectors to meet the demands of various applications that cannot be easily met by other delivery methods. In this review, we will discuss the various biomaterial systems that have been used to deliver Cas9 in its plasmid, mRNA and protein forms. In addition, the functions of these materials will be reviewed to understand their roles in Cas9 delivery. Finally, the immune challenges associated with Cas9 and the delivery materials will be discussed as an understanding of the immune responses along with the functions of biomaterials will ultimately guide the field in designing new delivery systems for the clinical applications of CRISPR-Cas9.},
}
@article {pmid30733599,
year = {2019},
author = {Hafford-Tear, NJ and Tsai, YC and Sadan, AN and Sanchez-Pintado, B and Zarouchlioti, C and Maher, GJ and Liskova, P and Tuft, SJ and Hardcastle, AJ and Clark, TA and Davidson, AE},
title = {CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat.},
journal = {Genetics in medicine : official journal of the American College of Medical Genetics},
volume = {21},
number = {9},
pages = {2092-2102},
pmid = {30733599},
issn = {1530-0366},
support = {/DH_/Department of Health/United Kingdom ; },
mesh = {Adult ; Aged ; Aged, 80 and over ; Alleles ; CRISPR-Cas Systems/genetics ; Female ; Fuchs' Endothelial Dystrophy/*genetics/pathology ; *Genetic Predisposition to Disease ; Genotype ; Humans ; Introns/genetics ; Male ; Middle Aged ; Sequence Analysis, DNA ; Single Molecule Imaging ; Transcription Factor 4/*genetics ; Trinucleotide Repeat Expansion/*genetics ; Trinucleotide Repeats/genetics ; },
abstract = {PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1).<br><br>METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n&#x2009;=&#x2009;11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis.<br><br>RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (&#x2265;50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length &#x2265;91 repeats) indicating that expanded alleles behave dynamically.<br><br>CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.},
}
@article {pmid30733567,
year = {2019},
author = {Lee, HK and Willi, M and Smith, HE and Miller, SM and Liu, DR and Liu, C and Hennighausen, L},
title = {Simultaneous targeting of linked loci in mouse embryos using base editing.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1662},
pmid = {30733567},
issn = {2045-2322},
support = {R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cytidine Deaminase/metabolism ; DNA Breaks, Double-Stranded ; Embryo, Mammalian/cytology/*metabolism ; Female ; Gene Editing/*methods ; *Genetic Loci ; Genome ; Male ; Mice ; Mice, Inbred C57BL ; Milk Proteins/*genetics ; Mutation ; Receptor Activity-Modifying Protein 3/*genetics ; Sequence Homology ; Zygote/cytology/*metabolism ; },
abstract = {A particular challenge in genome engineering has been the simultaneous introduction of mutations into linked (located on the same chromosome) loci. Although CRISPR/Cas9 has been widely used to mutate individual sites, its application in simultaneously targeting of linked loci is limited as multiple nearby double-stranded DNA breaks created by Cas9 routinely result in the deletion of sequences between the cleavage sites. Base editing is a newer form of genome editing that directly converts C∙G-to-T∙A, or A∙T-to-G∙C, base pairs without introducing double-stranded breaks, thus opening the possibility to generate linked mutations without disrupting the entire locus. Through the co-injection of two base editors and two sgRNAs into mouse zygotes, we introduced C∙G-to-T∙A transitions into two cytokine-sensing transcription factor binding sites separated by 9 kb. We determined that one enhancer activates the two flanking genes in mammary tissue during pregnancy and lactation. The ability to introduce linked mutations simultaneously in one step into the mammalian germline has implications for a wide range of applications, including the functional analysis of linked cis-elements creating disease models and correcting pathogenic mutations.},
}
@article {pmid30733471,
year = {2019},
author = {Wang, J and Goh, KM and Salem, DR and Sani, RK},
title = {Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1608},
pmid = {30733471},
issn = {2045-2322},
mesh = {Biotechnology ; *Genomics ; Geobacillus/*genetics/*metabolism/physiology ; Polysaccharides, Bacterial/*biosynthesis ; Stress, Physiological ; *Temperature ; },
abstract = {Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.},
}
@article {pmid30733432,
year = {2019},
author = {Mathieu, J and Detraux, D and Kuppers, D and Wang, Y and Cavanaugh, C and Sidhu, S and Levy, S and Robitaille, AM and Ferreccio, A and Bottorff, T and McAlister, A and Somasundaram, L and Artoni, F and Battle, S and Hawkins, RD and Moon, RT and Ware, CB and Paddison, PJ and Ruohola-Baker, H},
title = {Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {632},
pmid = {30733432},
issn = {2041-1723},
support = {P01 GM081619/GM/NIGMS NIH HHS/United States ; U01 HL099997/HL/NHLBI NIH HHS/United States ; R01 GM083867/GM/NIGMS NIH HHS/United States ; U01 HL099993/HL/NHLBI NIH HHS/United States ; R01 GM097372/GM/NIGMS NIH HHS/United States ; P30 DK017047/DK/NIDDK NIH HHS/United States ; },
mesh = {Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems/genetics/physiology ; Cell Line ; Estrone/genetics/metabolism ; Humans ; Immunoprecipitation ; Mechanistic Target of Rapamycin Complex 1/genetics/*metabolism ; Mechanistic Target of Rapamycin Complex 2/genetics/*metabolism ; Proteomics ; Receptors, Estrogen/genetics/metabolism ; Wnt Signaling Pathway/genetics/*physiology ; },
abstract = {To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.},
}
@article {pmid30733242,
year = {2019},
author = {Santiago-Frangos, A and Wiegand, T and Wiedenheft, B},
title = {Cas9 slide-and-seek for phage defense and genome engineering.},
journal = {The EMBO journal},
volume = {38},
number = {4},
pages = {},
pmid = {30733242},
issn = {1460-2075},
mesh = {*Bacteriophages ; *CRISPR-Cas Systems ; Genetic Engineering ; Genome ; },
abstract = {How does the Cas9 nuclease locate a specific 20‐nucleotide target sequence in a crowded intracellular environment packed with mega bases of distracting non‐target DNA? Previously, it was shown that Cas9 finds DNA targets via three‐dimensional diffusion. In this issue of The EMBO Journal, Globyte et al (2019) reveal another dimension of the search process, which involves short‐range one‐dimensional sliding. These results have implications for understanding the natural function of Cas9 and its applications in genome engineering experiments.},
}
@article {pmid30733188,
year = {2019},
author = {Wang, J and Wang, C and Wang, K},
title = {Generation of marker-free transgenic rice using CRISPR/Cas9 system controlled by floral specific promoters.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {46},
number = {1},
pages = {61-64},
doi = {10.1016/j.jgg.2018.11.005},
pmid = {30733188},
issn = {1673-8527},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Flowers/*genetics ; Genetic Engineering/*methods ; Organ Specificity ; Oryza/*genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic/*genetics ; },
}
@article {pmid30731491,
year = {2019},
author = {Kong, X and Ma, L and Chen, E and Shaw, CA and Edelstein, LC},
title = {Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C.},
journal = {Thrombosis and haemostasis},
volume = {119},
number = {5},
pages = {716-725},
pmid = {30731491},
issn = {2567-689X},
support = {R01 HL128234/HL/NHLBI NIH HHS/United States ; },
mesh = {Blood Platelets/*physiology ; Bone Marrow/*physiology ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Chromatin Immunoprecipitation ; Gene Expression Regulation ; Gene Ontology ; Humans ; Induced Pluripotent Stem Cells/*physiology ; MEF2 Transcription Factors/*genetics ; Megakaryocytes/*physiology ; RNA, Small Interfering/genetics ; Regulatory Elements, Transcriptional/*genetics ; Thrombopoiesis/*genetics ; },
abstract = {Megakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.},
}
@article {pmid30731369,
year = {2019},
author = {Tang, B and Gong, T and Zhou, X and Lu, M and Zeng, J and Peng, X and Wang, S and Li, Y},
title = {Deletion of cas3 gene in Streptococcus mutans affects biofilm formation and increases fluoride sensitivity.},
journal = {Archives of oral biology},
volume = {99},
number = {},
pages = {190-197},
doi = {10.1016/j.archoralbio.2019.01.016},
pmid = {30731369},
issn = {1879-1506},
mesh = {Bacterial Proteins/genetics ; Biofilms/*growth &amp; development ; Biomass ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems ; Coculture Techniques ; DNA Helicases ; Dental Caries ; Drug Resistance, Bacterial/genetics ; Drug Tolerance ; Escherichia coli Proteins ; Fluorides/*pharmacology ; Gene Deletion ; Gene Expression Regulation, Bacterial ; Genes, Bacterial/genetics ; In Situ Hybridization, Fluorescence ; Microbial Sensitivity Tests ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Streptococcus mutans/*drug effects/*genetics/*metabolism ; Streptococcus sanguis/physiology ; Transcriptome ; Virulence/genetics ; },
abstract = {OBJECTIVE: The goal of this study was to analyze the impact of cas3 gene on the biofilm formation and virulence gene expression in S. mutans, since our previous studies have found a connection between CRISPR/Cas systems and biofilm formation in S. mutans.<br><br>METHODS: The cas3 gene in-frame deletion strains of S. mutans UA159 was constructed by a two-step transformation procedure and the cas3 mutant strain was complemented in trans. The biofilm biomass was measured by crystal violet staining, and the synthesis of exopolysaccharides (EPS) was measured by the anthrone-sulfuric method. Biofilm analysis and structural imaging was using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) assays. The fluorescence in situ hybridization (FISH) was used to analyze the spatiotemporal interactions between S. mutans and Streptococcus sanguinis. Fluoride sensitivity was determined using fluoride tolerance assays. The expression of biofilm formation related genes was evaluated by qRT-PCR.<br><br>RESULTS: Our results showed that S. mutans cas3 deletion strain formed less biofilm and became less competitive when it was co-cultured with S. sanguinis under fluoride treatment. The expression levels of virulence genes including vicR, gtfC, smu0630 and comDE were significantly downregulated.<br><br>CONCLUSIONS: The cas3 gene in S. mutans could regulate biofilm formation and fluoride resistance, consequently affecting S. mutans competitiveness in a dual-species biofilm model under fluoride treatment. These results also provide a potential strategy for enhancing fluoride specificity, with cas3 gene as a potential genetic target in the modulation of oral microecology and the treatment of dental caries.},
}
@article {pmid30730211,
year = {2019},
author = {Wang, Z and Zhang, Y and Lee, YW and Ivanova, NB},
title = {Combining CRISPR/Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells.},
journal = {BioTechniques},
volume = {66},
number = {1},
pages = {23-27},
pmid = {30730211},
issn = {1940-9818},
support = {R01 GM105772/GM/NIGMS NIH HHS/United States ; R01 GM107092/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Doxycycline/pharmacology ; Gene Expression Regulation ; Gene Knockdown Techniques/*methods ; Genetic Complementation Test ; Human Embryonic Stem Cells/cytology/drug effects/*physiology ; Humans ; Polycomb Repressive Complex 1/genetics ; Proto-Oncogene Proteins/genetics ; RNA, Small Interfering/genetics ; Repressor Proteins/genetics ; SOXF Transcription Factors/genetics ; Transcription Activator-Like Effector Nucleases ; Transgenes ; },
abstract = {Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.},
}
@article {pmid30730208,
year = {2019},
author = {Finkler, M and Ott, A},
title = {Bead-based assay for spatiotemporal gene expression control in cell-free transcription-translation systems.},
journal = {BioTechniques},
volume = {66},
number = {1},
pages = {29-33},
doi = {10.2144/btn-2018-0097},
pmid = {30730208},
issn = {1940-9818},
mesh = {*Cell-Free System ; DNA, Complementary ; Escherichia coli/genetics ; *Gene Expression Regulation ; Genetic Techniques/*instrumentation ; Magnetics ; Nucleic Acid Hybridization ; Protein Biosynthesis ; RNA, Messenger/genetics ; Spatio-Temporal Analysis ; *Transcription, Genetic ; },
abstract = {Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a bead-based method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.},
}
@article {pmid30730047,
year = {2019},
author = {Wang, L and Wang, H and Liu, H and Zhao, Q and Liu, B and Wang, L and Zhang, J and Zhu, J and Bao, R and Luo, Y},
title = {Improved CRISPR-Cas12a-assisted one-pot DNA editing method enables seamless DNA editing.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {6},
pages = {1463-1474},
doi = {10.1002/bit.26938},
pmid = {30730047},
issn = {1097-0290},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; Endodeoxyribonucleases/*genetics ; Escherichia coli/genetics ; Gene Editing/*methods ; Luminescent Proteins/genetics ; Models, Molecular ; Plasmids/genetics ; Protein Engineering ; },
abstract = {As the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (previously known as Cpf1) system cleaves double-stranded DNA and produces a sticky end, it could serve as a useful tool for DNA assembly/editing. To broaden its application, a variety of engineered FnCas12a proteins are generated with expanded protospacer adjacent motif (PAM) requirements. Two variants (FnCas12a-EP15 and EP16) increased the targeting range of FnCas12a by approximately fourfold. They can efficiently recognize a broad range of PAM sequences including YN (Y = C or T), TAC and CAA. Meanwhile, based on our demonstration that FnCas12a is active from 16 to 60°C, we developed an "improved CRISPR-Cas12a-assisted one-pot DNA editing" (iCOPE) method to facilitate DNA editing by combining the crRNA transcription, digestion, and ligation in one pot. By applying iCOPE, the editing efficiency reached 72-100% for two DNA fragment assemblies, and for the 21 kb large DNA construct modification, the editing efficiency can reach 100%. Thanks to the advantages of Cas12a, iCOPE with only one digestion enzyme could replace current a variety of restriction enzymes to perform the cloning in one pot with almost no sequence constraints. Taken together, this study offers an expanded DNA targeting scope of CRISPR systems and could serve as an efficient seamless one-pot DNA editing tool.},
}
@article {pmid30729464,
year = {2019},
author = {Jin, H and Sophocleous, A and Azfer, A and Ralston, SH},
title = {Analysis of Transcriptional Regulation in Bone Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1914},
number = {},
pages = {145-167},
doi = {10.1007/978-1-4939-8997-3_8},
pmid = {30729464},
issn = {1940-6029},
mesh = {Bone and Bones/*cytology ; CRISPR-Cas Systems/genetics ; Cell Culture Techniques/instrumentation/methods ; Cell Line, Tumor ; Cell Separation/instrumentation/methods ; Chromatin Immunoprecipitation/instrumentation/*methods ; Electrophoretic Mobility Shift Assay/instrumentation/*methods ; Flow Cytometry/instrumentation/methods ; Gene Editing/instrumentation/methods ; *Gene Expression Regulation ; Genes, Reporter/genetics ; Humans ; Promoter Regions, Genetic/genetics ; *Transcription, Genetic ; },
abstract = {Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay, five commonly used methods for studying and altering gene transcription.},
}
@article {pmid30729184,
year = {2019},
author = {Tripathi, JN and Ntui, VO and Ron, M and Muiruri, SK and Britt, A and Tripathi, L},
title = {CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {46},
pmid = {30729184},
issn = {2399-3642},
mesh = {Badnavirus/*genetics/pathogenicity ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Chimera/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Genome, Plant ; *Genome, Viral ; Musa/*genetics/virology ; Mutation ; Plant Breeding/*methods ; Plant Diseases/genetics/virology ; Plants, Genetically Modified ; Plasmids/chemistry/metabolism ; RNA, Guide/genetics/metabolism ; Sequence Alignment ; Stress, Physiological ; },
abstract = {Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.},
}
@article {pmid30728517,
year = {2019},
author = {Dolgin, E},
title = {Genomic focus brings tea research to the boil.},
journal = {Nature},
volume = {566},
number = {7742},
pages = {S12-S13},
pmid = {30728517},
issn = {1476-4687},
mesh = {Antioxidants/analysis/metabolism ; CRISPR-Cas Systems ; Caffeine/*analysis/biosynthesis/isolation &amp; purification ; Catechin/analysis/metabolism ; Droughts ; Food, Genetically Modified ; *Genetic Engineering ; Humans ; Internationality ; Methyltransferases/deficiency/genetics ; Plant Breeding/*methods ; Plant Diseases/genetics/microbiology/parasitology/prevention &amp; control ; *Research ; Taste ; Tea/*chemistry/classification/*genetics/standards ; Theobromine/biosynthesis ; Time Factors ; },
}
@article {pmid30728464,
year = {2019},
author = {Ideno, N and Yamaguchi, H and Okumura, T and Huang, J and Brun, MJ and Ho, ML and Suh, J and Gupta, S and Maitra, A and Ghosh, B},
title = {A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9-mediated somatic recombination.},
journal = {Laboratory investigation; a journal of technical methods and pathology},
volume = {99},
number = {8},
pages = {1233-1244},
doi = {10.1038/s41374-018-0171-z},
pmid = {30728464},
issn = {1530-0307},
support = {P01 CA117969/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dependovirus/genetics ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; *Neoplasms, Experimental ; *Pancreatic Neoplasms ; RNA, Guide/genetics ; Recombination, Genetic/*genetics ; },
abstract = {Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single "workhorse" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras [G12D] allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic Kras[G12D] allele at the endogenous locus, and insertion deletion ("indel") and frameshift mutations of targeted tumor suppressor alleles. By using a single "workhorse" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.},
}
@article {pmid30728460,
year = {2019},
author = {Shao, J and Lu, J and Zhu, W and Yu, H and Jing, X and Wang, YL and Wang, X and Wang, XJ},
title = {Derepression of LOXL4 inhibits liver cancer growth by reactivating compromised p53.},
journal = {Cell death and differentiation},
volume = {26},
number = {11},
pages = {2237-2252},
pmid = {30728460},
issn = {1476-5403},
mesh = {A549 Cells ; Animals ; Antimetabolites, Antineoplastic/*pharmacology ; Apoptosis/drug effects ; Azacitidine/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/physiology ; Enzyme Activation/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Hep G2 Cells ; Humans ; Liver Neoplasms/*pathology ; MCF-7 Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Protein Binding/physiology ; Protein-Lysine 6-Oxidase/genetics/*metabolism ; Transplantation, Heterologous ; Tumor Suppressor Protein p53/genetics/*metabolism ; Tumor Suppressor Proteins/genetics/metabolism ; },
abstract = {TP53 is the most frequently mutated gene in human cancer, whereas tumors with wild-type TP53 develop alternative strategies to survive. Identifying new regulators of p53 reactivation would greatly contribute to the development of cancer therapies. After screening the entire genome in liver cancer cells, we identified lysyl oxidase-like 4 (LOXL4) as a novel regulator for p53 activation. We found that 5-azacytidine (5-aza-CR) induces LOXL4 upregulation, with LOXL4 subsequently binding the basic domain of p53 via its low-isoelectric point region. The interaction between LOXL4 and p53 induces the reactivation of compromised p53, resulting in cell death. Furthermore, the nude mouse xenograft model showed that the 5-aza-CR-dependent LOXL4-p53 axis reduces tumor growth. A positive correlation between LOXL4 expression and overall survival in liver cancer patients with wild-type p53 tumors was observed. In conclusion, we found that 5-aza-CR-induced LOXL4 upregulation reactivates wild-type p53 and triggers cell death, which blocks liver cancer development.},
}
@article {pmid30728412,
year = {2019},
author = {Yoshimatsu, S and Okahara, J and Sone, T and Takeda, Y and Nakamura, M and Sasaki, E and Kishi, N and Shiozawa, S and Okano, H},
title = {Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1528},
pmid = {30728412},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Callithrix ; *DNA Breaks, Double-Stranded ; Embryo, Mammalian/*cytology/metabolism ; Embryonic Stem Cells/*cytology/metabolism ; Female ; Forkhead Transcription Factors/antagonists &amp; inhibitors/genetics ; *Gene Editing ; Gene Knock-In Techniques/*methods ; Gene Targeting ; Homologous Recombination ; Humans ; Male ; Models, Animal ; Myelin Proteolipid Protein/antagonists &amp; inhibitors/genetics ; Neural Stem Cells/*cytology/metabolism ; },
abstract = {Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset (Callithrix jacchus), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRISPR-Cas9 system. The use of CRISPR-Cas9, mediated by homologous recombination (HR), enhanced the KI efficiency in marmoset ESCs. Furthermore, we succeeded in performing KI in early-stage marmoset embryos. In the course of the experiments, we found that HR in the marmoset ESCs is innately highly efficient. This suggested that the marmoset possesses a repair mechanism for DNA double-strand breaks. The current study will facilitate the generation of genetically modified marmosets and gene function analysis in the marmoset.},
}
@article {pmid30728393,
year = {2019},
author = {Ramakrishnan, C and Maier, S and Walker, RA and Rehrauer, H and Joekel, DE and Winiger, RR and Basso, WU and Grigg, ME and Hehl, AB and Deplazes, P and Smith, NC},
title = {An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1474},
pmid = {30728393},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cats ; Feces/parasitology ; Female ; Fertilization/drug effects ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Silencing ; Male ; Protozoan Proteins/*genetics ; Protozoan Vaccines/*administration &amp; dosage/pharmacology ; Sequence Analysis, RNA ; Toxoplasma/drug effects/*genetics ; Toxoplasmosis, Animal/*prevention &amp; control/transmission ; Vaccines, Attenuated/*administration &amp; dosage/pharmacology ; },
abstract = {Almost any warm-blooded creature can be an intermediate host for Toxoplasma gondii. However, sexual reproduction of T. gondii occurs only in felids, wherein fertilisation of haploid macrogametes by haploid microgametes, results in diploid zygotes, around which a protective wall develops, forming unsporulated oocysts. Unsporulated oocysts are shed in the faeces of cats and meiosis gives rise to haploid sporozoites within the oocysts. These, now infectious, sporulated oocysts contaminate the environment as a source of infection for people and their livestock. RNA-Seq analysis of cat enteric stages of T. gondii uncovered genes expressed uniquely in microgametes and macrogametes. A CRISPR/Cas9 strategy was used to create a T. gondii strain that exhibits defective fertilisation, decreased fecundity and generates oocysts that fail to produce sporozoites. Inoculation of cats with this engineered parasite strain totally prevented oocyst excretion following infection with wild-type T. gondii, demonstrating that this mutant is an attenuated, live, transmission-blocking vaccine.},
}
@article {pmid30728161,
year = {2019},
author = {Lovell-Badge, R},
title = {CRISPR babies: a view from the centre of the storm.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {3},
pages = {},
doi = {10.1242/dev.175778},
pmid = {30728161},
issn = {1477-9129},
support = {FC001107/CRUK_/Cancer Research UK/United Kingdom ; FC001107/MRC_/Medical Research Council/United Kingdom ; FC001107/WT_/Wellcome Trust/United Kingdom ; MC_U117562207/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*Bioethical Issues ; *Bioethics ; *CRISPR-Cas Systems ; Gene Editing/*ethics ; *Genome, Human ; Humans ; },
abstract = {Towards the end of November 2018, news broke that the Chinese researcher He Jiankui had created the world's first genome-edited babies. This came shortly before the start of the Second International Summit on Human Genome Editing, where researchers, ethicists and others concerned with regulation, social issues and public engagement from around the world gathered to discuss the latest advances in the field. In this Spotlight, I provide my perspective on the events that occurred shortly prior to and at the summit, where He Jiankui gave an account of his activities. I also discuss what was wrong with his approach and how, after more research and with appropriate regulation, clinical applications of germline genome editing in humans may be justifiable.},
}
@article {pmid30727954,
year = {2019},
author = {Boettcher, M and Covarrubias, S and Biton, A and Blau, J and Wang, H and Zaitlen, N and McManus, MT},
title = {Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {107},
pmid = {30727954},
issn = {1471-2164},
support = {K25 HL121295/HL/NHLBI NIH HHS/United States ; 1U01MH105028/NH/NIH HHS/United States ; U01CA168370/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Testing/*methods ; Humans ; Jurkat Cells ; Molecular Typing/*methods ; *RNA, Guide ; },
abstract = {BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells.<br><br>RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA.<br><br>CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.},
}
@article {pmid30726877,
year = {2019},
author = {Kuno, A and Mizuno, S and Takahashi, S},
title = {KOnezumi: a web application for automating gene disruption strategies to generate knockout mice.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {18},
pages = {3479-3481},
pmid = {30726877},
issn = {1367-4811},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Mice ; Mice, Knockout ; RNA, Guide ; *Software ; },
abstract = {SUMMARY: Although gene editing using the CRISPR/Cas9 system enables the rapid generation of knockout mice, constructing an optimal gene disruption strategy is still labourious. Here, we propose KOnezumi, a simple and user-friendly web application, for use in automating the design of knockout strategies for multiple genes. Users only need to input gene symbols, and then KOnezumi returns target exons, gRNA candidates to delete the target exons, genotyping PCR primers, nucleotide sequences of the target exons and coding sequences of expected deletion products. KOnezumi enables users to easily and rapidly apply a rational strategy to accelerate the generation of KO mice using the CRISPR/Cas9 system.<br><br>This web application is freely available at http://www.md.tsukuba.ac.jp/LabAnimalResCNT/KOanimals/konezumi.html.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid30726867,
year = {2019},
author = {Li, Z and Zhou, L and Jiang, T and Fan, L and Liu, X and Qiu, X},
title = {Proteasomal deubiquitinase UCH37 inhibits degradation of β-catenin and promotes cell proliferation and motility.},
journal = {Acta biochimica et biophysica Sinica},
volume = {51},
number = {3},
pages = {277-284},
doi = {10.1093/abbs/gmy176},
pmid = {30726867},
issn = {1745-7270},
mesh = {CRISPR-Cas Systems ; Cell Movement ; Cell Proliferation ; HEK293 Cells ; HeLa Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; Membrane Glycoproteins/physiology ; Proteasome Endopeptidase Complex/metabolism ; Ubiquitin Thiolesterase/*physiology ; Ubiquitination ; beta Catenin/*metabolism ; },
abstract = {The ubiquitin-proteasome system degrades most cellular proteins in eukaryotes. UCH37, also known as UCH-L5, is a deubiquitinase binding to Rpn13, a receptor for ubiquitinated substrates in the 26 S proteasome. But, it remains unclear how UCH37 influences the proteasomal degradation of the ubiquitinated substrates. Because deletion of UCH37 is embryonically lethal in mice, this study aims to investigate the role of UCH37 in proteasomal degradation by constructing the UCH37-deficient cell lines using CRISPR/Cas9 technology. Our results demonstrated that deletion of UCH37 decreased the levels of proteasomal Rpn13, implying that UCH37 might facilitate incorporation of Rpn13 into the proteasome. Meanwhile, deletion of UCH37 decreased the levels of β-catenin and the early endosomal protein Rab8. β-Catenin interacts with TCF/LEF to control transcription, and is involved in development, tissue homeostasis and tumorigenesis. We further found that deletion of UCH37 increased the levels of the ubiquitinated β-catenin and accelerated the hydrogen peroxide-stimulated degradation of β-catenin. Deletion of UCH37 also down-regulated the transcription of c-Myc, a downstream effector of β-catenin, and inhibited cell proliferation and motility. These results raise the possibility that UCH37 maintains the homeostasis of proteasomal degradation reciprocally by assisting the recruitment of the ubiquitin receptor Rpn13 into the proteasome and by reversing ubiquitination of certain critical substrates of the 26 S proteasome.},
}
@article {pmid30726783,
year = {2019},
author = {Tanihara, F and Hirata, M and Nguyen, NT and LE, QA and Hirano, T and Otoi, T},
title = {Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos.},
journal = {The Journal of reproduction and development},
volume = {65},
number = {3},
pages = {209-214},
pmid = {30726783},
issn = {1348-4400},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Blastocyst/cytology ; *CRISPR-Cas Systems ; Cytoplasm/*genetics ; Embryonic Development ; Female ; Fertilization in Vitro ; Gene Editing ; Male ; Microinjections ; *Mosaicism ; Mutation ; Nucleotides/genetics ; Oocytes/cytology ; Swine ; Zygote ; },
abstract = {Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.},
}
@article {pmid30726238,
year = {2019},
author = {Tang, Y and Meister, TR and Walczak, M and Pulkoski-Gross, MJ and Hari, SB and Sauer, RT and Amberg-Johnson, K and Yeh, E},
title = {A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.},
journal = {PLoS biology},
volume = {17},
number = {2},
pages = {e3000136},
pmid = {30726238},
issn = {1545-7885},
support = {R01 AI141366/AI/NIAID NIH HHS/United States ; T32 GM007276/GM/NIGMS NIH HHS/United States ; K08 AI097239/AI/NIAID NIH HHS/United States ; S10 OD018220/OD/NIH HHS/United States ; DP5 OD012119/OD/NIH HHS/United States ; F32 GM116241/GM/NIGMS NIH HHS/United States ; },
mesh = {Apicoplasts/*genetics/metabolism ; CRISPR-Cas Systems ; Erythrocytes/parasitology ; Gene Ontology ; *Genes, Essential ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Luminescent Proteins/genetics/metabolism ; Metalloproteases/genetics/metabolism ; Molecular Sequence Annotation ; Mutagenesis ; *Mutation ; Organelle Biogenesis ; Plasmodium falciparum/*genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; Triose-Phosphate Isomerase/*genetics/metabolism ; Whole Genome Sequencing ; },
abstract = {Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.},
}
@article {pmid30724484,
year = {2019},
author = {Croop, B and Zhang, C and Lim, Y and Gelfand, RM and Han, KY},
title = {Recent advancement of light-based single-molecule approaches for studying biomolecules.},
journal = {Wiley interdisciplinary reviews. Systems biology and medicine},
volume = {11},
number = {4},
pages = {e1445},
doi = {10.1002/wsbm.1445},
pmid = {30724484},
issn = {1939-005X},
mesh = {CRISPR-Cas Systems/genetics ; DNA/*chemistry/metabolism ; Enzyme-Linked Immunosorbent Assay ; Fluorescence Resonance Energy Transfer ; Humans ; *Light ; Microscopy, Fluorescence ; Nanotechnology ; Proteins/analysis/*chemistry/metabolism ; Signal-To-Noise Ratio ; },
abstract = {Recent advances in single-molecule techniques have led to new discoveries in analytical chemistry, biophysics, and medicine. Understanding the structure and behavior of single biomolecules provides a wealth of information compared to studying large ensembles. However, developing single-molecule techniques is challenging and requires advances in optics, engineering, biology, and chemistry. In this paper, we will review the state of the art in single-molecule applications with a focus over the last few years of development. The advancements covered will mainly include light-based in vitro methods, and we will discuss the fundamentals of each with a focus on the platforms themselves. We will also summarize their limitations and current and future applications to the wider biological and chemical fields. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods.},
}
@article {pmid30724156,
year = {2019},
author = {Xue, C and Sashital, DG},
title = {Mechanisms of Type I-E and I-F CRISPR-Cas Systems in Enterobacteriaceae.},
journal = {EcoSal Plus},
volume = {8},
number = {2},
pages = {},
pmid = {30724156},
issn = {2324-6200},
support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaea/genetics ; Bacteriophages/genetics/*physiology ; *CRISPR-Cas Systems ; Enterobacteriaceae/*genetics/immunology/virology ; Escherichia coli/genetics ; Host Microbial Interactions ; Pectobacterium/genetics ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative Enterobacteriaceae family, especially Escherichia coli and Pectobacterium atrosepticum, have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the Enterobacteriaceae. We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.},
}
@article {pmid30723103,
year = {2019},
author = {Hollerer, I and Barker, JC and Jorgensen, V and Tresenrider, A and Dugast-Darzacq, C and Chan, LY and Darzacq, X and Tjian, R and Ünal, E and Brar, GA},
title = {Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {4},
pages = {1045-1053},
pmid = {30723103},
issn = {2160-1836},
support = {DP2 AG055946/AG/NIA NIH HHS/United States ; DP2 GM119138/GM/NIGMS NIH HHS/United States ; U01 DA047729/DA/NIDA NIH HHS/United States ; 003061/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Chromatin Immunoprecipitation ; *Gene Expression Regulation ; Gene Knockdown Techniques ; Histones/metabolism ; Humans ; MCF-7 Cells ; *Models, Genetic ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-mdm2/*genetics ; },
abstract = {We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.},
}
@article {pmid30722791,
year = {2019},
author = {Gonçalves, E and Behan, FM and Louzada, S and Arnol, D and Stronach, EA and Yang, F and Yusa, K and Stegle, O and Iorio, F and Garnett, MJ},
title = {Structural rearrangements generate cell-specific, gene-independent CRISPR-Cas9 loss of fitness effects.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {27},
pmid = {30722791},
issn = {1474-760X},
support = {//Wellcome Trust/United Kingdom ; C44943/A22536//Cancer Research UK/United Kingdom ; 206194//Wellcome Trust/United Kingdom ; 098051//Wellcome Trust/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Genomic Structural Variation ; Genomics/*methods ; Humans ; Neoplasms/genetics ; Ploidies ; Software ; *Whole Genome Sequencing ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed.<br><br>RESULTS: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects.<br><br>CONCLUSION: Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.},
}
@article {pmid30722720,
year = {2019},
author = {Common, J and Westra, ER},
title = {CRISPR evolution and bacteriophage persistence in the context of population bottlenecks.},
journal = {RNA biology},
volume = {16},
number = {4},
pages = {588-594},
pmid = {30722720},
issn = {1555-8584},
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; *Evolution, Molecular ; Host-Pathogen Interactions/genetics ; },
abstract = {Population bottlenecks often cause strong reductions in genetic diversity and alter population structure. In the context of host-parasite interactions, bottlenecks could in theory benefit either the host or the pathogen. We predicted that bottlenecking of bacterial populations that evolve CRISPR immunity against bacteriophages (phage) would benefit the pathogen, because CRISPR spacer diversity can rapidly drive phages extinct. To test this, we bottlenecked populations of bacteria and phage, tracking phage persistence and the evolution of bacterial resistance mechanisms. Contrary to our prediction, bottlenecking worked in the advantage of the host. With some possible exceptions, this effect was not caused by CRISPR immunity. This host benefit is consistent with a dilution effect disproportionately affecting phage. This study provides further insight into how bottlenecking influences bacteria-phage dynamics, the role of dilution in bacteria-phage interactions, and the evolution of host immune systems.},
}
@article {pmid30722048,
year = {2019},
author = {Botella, JR},
title = {Now for the hard ones: is there a limit on CRISPR genome editing in crops?.},
journal = {Journal of experimental botany},
volume = {70},
number = {3},
pages = {734-737},
pmid = {30722048},
issn = {1460-2431},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Fragaria ; *Gene Editing ; Mutagenesis ; },
abstract = {This article comments on the following paper: Martín-Pizarro C, Triviño JC, Posé D. 2019. Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis. Journal of Experimental Botany 70, 885–895.},
}
@article {pmid30718897,
year = {2019},
author = {Shortt, K and Heruth, DP and Zhang, N and Wu, W and Singh, S and Li, DY and Zhang, LQ and Wyckoff, GJ and Qi, LS and Friesen, CA and Ye, SQ},
title = {Identification of Novel Regulatory Genes in APAP Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1396},
pmid = {30718897},
issn = {2045-2322},
mesh = {Acetaminophen/*adverse effects ; Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Databases as Topic ; Gene Expression Regulation ; *Genes, Regulator ; HEK293 Cells ; Hepatocytes/*metabolism/radiation effects ; Humans ; Male ; Mice, Inbred C57BL ; Reproducibility of Results ; Signal Transduction/genetics ; },
abstract = {Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15 mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, and 3) predicted affected biological pathways. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. This study illustrated the power of a genome-wide CRISPR-Cas9 screen to systematically identify novel genes involved in APAP-induced hepatotoxicity and to provide potential targets to develop novel therapeutic modalities.},
}
@article {pmid30718879,
year = {2019},
author = {Waltz, E},
title = {Asexual crops whet industry appetite.},
journal = {Nature biotechnology},
volume = {37},
number = {2},
pages = {109-110},
doi = {10.1038/s41587-019-0031-4},
pmid = {30718879},
issn = {1546-1696},
mesh = {Agriculture/*trends ; Biotechnology/*trends ; CRISPR-Cas Systems ; China ; Crops, Agricultural/*genetics/*physiology ; Genetic Engineering ; *Plant Breeding ; Plants, Genetically Modified/physiology ; *Seeds ; United States ; },
}
@article {pmid30718774,
year = {2019},
author = {Liu, JJ and Orlova, N and Oakes, BL and Ma, E and Spinner, HB and Baney, KLM and Chuck, J and Tan, D and Knott, GJ and Harrington, LB and Al-Shayeb, B and Wagner, A and Brötzmann, J and Staahl, BT and Taylor, KL and Desmarais, J and Nogales, E and Doudna, JA},
title = {CasX enzymes comprise a distinct family of RNA-guided genome editors.},
journal = {Nature},
volume = {566},
number = {7743},
pages = {218-223},
pmid = {30718774},
issn = {1476-4687},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; P01 GM051487/GM/NIGMS NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/chemistry/*classification/metabolism/*ultrastructure ; CRISPR-Cas Systems/*genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism/ultrastructure ; DNA Cleavage ; Escherichia coli/genetics ; Evolution, Molecular ; *Gene Editing ; Gene Silencing ; Genome, Bacterial/genetics ; Genome, Human/genetics ; Humans ; Models, Molecular ; Nucleic Acid Conformation ; Protein Domains ; RNA, Guide/metabolism ; },
abstract = {The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.},
}
@article {pmid30718521,
year = {2019},
author = {Shen, H and Li, L and Teng, Z and Meng, T and Kong, X and Hu, Y and Zhu, Y and Ma, L},
title = {Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1399},
pmid = {30718521},
issn = {2045-2322},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Cycle ; Cell Proliferation ; Gene Editing ; Gene Expression Regulation ; *Gene Knockout Techniques ; HeLa Cells ; Humans ; Luciferases/metabolism ; *Mass Spectrometry ; MicroRNAs/*metabolism ; Proteins/*metabolism ; Proteomics ; Reproducibility of Results ; Signal Transduction ; *Staining and Labeling ; },
abstract = {MicroRNAs (miRNAs) bind to the 3'-untranslated region of target mRNAs in a sequence-specific manner and subsequently repress gene translation. Human miR-26a has been studied extensively, but the target transcripts are far from complete. We first employed the CRISPR-Cas9 system to generate an miR-26a-knockout line in human cervical cancer HeLa cells. The miR26a-knockout line showed increased cell growth and altered proliferation. Proteomics technology of sequential window acquisition of all theoretical mass spectra (SWATH-MS) was utilized to compare the protein abundance between the wild-type and the knockout lines, with an attempt to identify transcripts whose translation was influenced by miR-26a. Functional classification of the proteins with significant changes revealed their function in stress response, proliferation, localization, development, signaling, etc. Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring (PRM). The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification. Among the computationally predicted targets, a subset of the targets was directly regulated by miR-26a, as demonstrated by luciferase assays and Western blotting. This study creates an inventory of miR-26a-targeted transcripts in HeLa cells and provides fundamental knowledge to further explore the functions of miR-26a in human cancer.},
}
@article {pmid30718489,
year = {2019},
author = {Ma, D and Xu, Z and Zhang, Z and Chen, X and Zeng, X and Zhang, Y and Deng, T and Ren, M and Sun, Z and Jiang, R and Xie, Z},
title = {Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {560},
pmid = {30718489},
issn = {2041-1723},
mesh = {Biological Evolution ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Flow Cytometry ; HEK293 Cells ; Humans ; Staphylococcus aureus/genetics/metabolism ; Substrate Specificity ; },
abstract = {Although Cas9 nucleases are remarkably diverse in microorganisms, the range of genomic sequences targetable by a CRISPR/Cas9 system is restricted by the requirement of a short protospacer adjacent motif (PAM) at the target site. Here, we generate a group of chimeric Cas9 (cCas9) variants by replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs. By using a functional assay at target sites with different nucleotide recombinations at PAM position 3-6, we identify several cCas9 variants with expanded recognition capability at NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT PAM sequences. In summary, we provide a panel of cCas9 variants accessible up to 1/4 of all the possible genomic targets in mammalian cells.},
}
@article {pmid30718434,
year = {2019},
author = {Hardy, S and Kostantin, E and Wang, SJ and Hristova, T and Galicia-Vázquez, G and Baranov, PV and Pelletier, J and Tremblay, ML},
title = {Magnesium-sensitive upstream ORF controls PRL phosphatase expression to mediate energy metabolism.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {8},
pages = {2925-2934},
pmid = {30718434},
issn = {1091-6490},
support = {//Wellcome Trust/United Kingdom ; MOP-142497//Canadian Institutes of Health Research/International ; FDN-159923//Canadian Institutes of Health Research/International ; FDN-14366//Canadian Institutes of Health Research/International ; 210692/Z/18/Z//Wellcome Trust/United Kingdom ; },
mesh = {AMP-Activated Protein Kinase Kinases ; CRISPR-Cas Systems ; Cation Transport Proteins ; Cell Cycle Proteins/genetics ; Cellular Reprogramming/*genetics ; Cyclins/genetics ; Energy Metabolism/*genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Regeneration/genetics ; MCF-7 Cells ; Magnesium/metabolism ; Mechanistic Target of Rapamycin Complex 2/genetics ; Membrane Proteins/genetics ; Neoplasm Proteins/genetics ; Neoplasms/*genetics/pathology ; Protein Kinases/genetics ; Protein Tyrosine Phosphatases/*genetics ; },
abstract = {Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5'UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics.},
}
@article {pmid30717767,
year = {2019},
author = {Teng, F and Li, J and Cui, T and Xu, K and Guo, L and Gao, Q and Feng, G and Chen, C and Han, D and Zhou, Q and Li, W},
title = {Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {15},
pmid = {30717767},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Mice ; },
abstract = {CRISPR-Cas12a/Cpf1, a single RNA-guided endonuclease system, provides a promising tool for genome engineering. However, only three Cas12a orthologs have been employed for mammalian genome editing, and the editing efficiency as well as targeting coverage still requires improvements. Here, we harness six novel Cas12a orthologs for genome editing in human and mouse cells, some of which utilize simple protospacer adjacent motifs (PAMs) that remarkably increase the targeting range in the genomes. Moreover, we identify optimized CRISPR RNA (crRNA) scaffolds that can increase the genome editing efficiency of Cas12a.},
}
@article {pmid30717739,
year = {2019},
author = {Tong, Z and Zheng, X and Tong, Y and Shi, YC and Sun, J},
title = {Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {28},
pmid = {30717739},
issn = {1475-2859},
mesh = {Aspergillus niger/*genetics/*metabolism ; CRISPR-Cas Systems ; Citric Acid/*metabolism ; Gene Editing ; *Genome, Fungal ; Genomics ; Industrial Microbiology ; *Metabolic Engineering ; Systems Biology ; },
abstract = {Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.},
}
@article {pmid30717668,
year = {2019},
author = {McDonald, ND and Regmi, A and Morreale, DP and Borowski, JD and Boyd, EF},
title = {CRISPR-Cas systems are present predominantly on mobile genetic elements in Vibrio species.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {105},
pmid = {30717668},
issn = {1471-2164},
support = {T32 GM008550/GM/NIGMS NIH HHS/United States ; 5T32GM008550/NH/NIH HHS/United States ; P20 GM103446/GM/NIGMS NIH HHS/United States ; P20GM103446/NH/NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics ; CRISPR-Associated Proteins/*genetics ; *DNA Transposable Elements ; *Gene Transfer, Horizontal ; *Genomic Islands ; Phylogeny ; *Plasmids ; Sequence Analysis, DNA ; Vibrio/*genetics/metabolism ; },
abstract = {BACKGROUND: Bacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context.<br><br>RESULTS: Herein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer.<br><br>CONCLUSIONS: Our data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.},
}
@article {pmid30717310,
year = {2019},
author = {Lu, C and Pang, D and Li, M and Yuan, H and Yu, T and Huang, P and Li, J and Chen, X and Jiao, H and Xie, Z and Ouyang, H},
title = {CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine miR-17-92 Cluster.},
journal = {Cells},
volume = {8},
number = {2},
pages = {},
pmid = {30717310},
issn = {2073-4409},
mesh = {Animals ; Animals, Genetically Modified ; Base Sequence ; Blastocyst/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Fetus/cytology ; Fibroblasts/metabolism ; Genes, Reporter ; Genome ; Green Fluorescent Proteins/metabolism ; MicroRNAs/*genetics/metabolism ; RNA, Guide/genetics ; RNA, Small Interfering/*metabolism ; Swine ; Transgenes ; },
abstract = {Successful RNAi applications depend on strategies allowing stable and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. In this study, we proposed an endogenous microRNA (miRNA) cluster as a novel integration site for small hairpin RNAs (shRNAs). We successfully integrated exogenous shRNAs at the porcine miRNA-17-92 (pmiR-17-92) cluster via a CRISPR/Cas9-mediated knock-in strategy. The anti-EGFP or anti-CSFV shRNAs could be stably and effectively expressed at the control of the endogenous promoter of the pmiR-17-92 cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous pmiR-17-92 expression in selected transgene (TG) porcine fetal fibroblasts (PFFs) clones. Moreover, these TG PFFs could inhibit the replication of CSFV by half and could be further used for generation of transgenic pigs. Taken together, these results show that our RNA interference (RNAi) expression strategy benefits numerous applications, from miRNA, genome and transgenic research, to gene therapy.},
}
@article {pmid30716580,
year = {2019},
author = {King, MW and Munger, J},
title = {Editing the human cytomegalovirus genome with the CRISPR/Cas9 system.},
journal = {Virology},
volume = {529},
number = {},
pages = {186-194},
pmid = {30716580},
issn = {1096-0341},
support = {R01 AI127370/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cytomegalovirus/*genetics ; Genetic Engineering/*methods ; Genome, Viral/*genetics ; Humans ; },
abstract = {Human Cytomegalovirus (HCMV) is an opportunistic pathogen that causes substantial disease in neonates and immunocompromised individuals. Reverse genetic analysis of the HCMV genome is a powerful tool to dissect the roles that various viral genes play during infection. However, genetic engineering of HCMV is hampered by both the large size of the HCMV genome and HCMV's slow replication cycle. Currently, most laboratories that genetically engineer HCMV employ Bacterial Artificial Chromosome (BAC) mediated recombineering, which is a relatively lengthy process. We explored an alternative method of producing recombinant HCMV using the CRISPR/Cas9 system. We employed both homologous recombination (HR) and Non-homologous end-joining (NHEJ)-based methods, and find that each approach is capable of efficiently mutating the HCMV genome, with optimal efficiencies of 42% and 81% respectively. Our results suggest that CRISPR-mediated genomic engineering of HCMV is competitive with BAC-mediated recombineering and provide a framework for using CRISPR/Cas9 for mutational analysis of the HCMV genome.},
}
@article {pmid30715679,
year = {2019},
author = {Demirci, S and Leonard, A and Haro-Mora, JJ and Uchida, N and Tisdale, JF},
title = {CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges.},
journal = {Advances in experimental medicine and biology},
volume = {1144},
number = {},
pages = {37-52},
doi = {10.1007/5584_2018_331},
pmid = {30715679},
issn = {0065-2598},
mesh = {Anemia, Sickle Cell/*therapy ; *CRISPR-Cas Systems ; Fetal Hemoglobin ; *Gene Editing ; Hematopoietic Stem Cells/cytology ; Humans ; Induced Pluripotent Stem Cells/cytology ; },
abstract = {Sickle cell disease (SCD) is an inherited monogenic disorder resulting in serious mortality and morbidity worldwide. Although the disease was characterized more than a century ago, there are only two FDA approved medications to lessen disease severity, and a definitive cure available to all patients with SCD is lacking. Rapid and substantial progress in genome editing approaches have proven valuable as a curative option given plausibility to either correct the underlying mutation in patient-derived hematopoietic stem/progenitor cells (HSPCs), induce fetal hemoglobin expression to circumvent sickling of red blood cells (RBCs), or create corrected induced pluripotent stem cells (iPSCs) among other approaches. Recent discovery of CRISPR/Cas9 has not only revolutionized genome engineering but has also brought the possibility of translating these concepts into a clinically meaningful reality. Here we summarize genome engineering applications using CRISPR/Cas9, addressing challenges and future perspectives of CRISPR/Cas9 as a curative option for SCD.},
}
@article {pmid30715580,
year = {2019},
author = {Pérez, L and Soto, E and Farré, G and Juanos, J and Villorbina, G and Bassie, L and Medina, V and Serrato, AJ and Sahrawy, M and Rojas, JA and Romagosa, I and Muñoz, P and Zhu, C and Christou, P},
title = {CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.},
journal = {Plant cell reports},
volume = {38},
number = {3},
pages = {417-433},
pmid = {30715580},
issn = {1432-203X},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Endosperm/metabolism ; Mutation/genetics ; Oryza/genetics/*metabolism ; Starch Synthase/genetics/*metabolism ; Waxes/metabolism ; },
abstract = {Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.},
}
@article {pmid30715513,
year = {2019},
author = {Lewis, TW and Barthelemy, JR and Virts, EL and Kennedy, FM and Gadgil, RY and Wiek, C and Linka, RM and Zhang, F and Andreassen, PR and Hanenberg, H and Leffak, M},
title = {Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.},
journal = {Nucleic acids research},
volume = {47},
number = {7},
pages = {3503-3520},
pmid = {30715513},
issn = {1362-4962},
support = {R01 GM122976/GM/NIGMS NIH HHS/United States ; },
mesh = {Alu Elements/*genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA Damage/genetics ; Fanconi Anemia/*genetics/pathology ; Fanconi Anemia Complementation Group D2 Protein/genetics ; Fanconi Anemia Complementation Group Proteins/genetics ; Gene Deletion ; Gene Duplication/genetics ; HeLa Cells ; Hematopoietic Stem Cells/metabolism ; Homologous Recombination/*genetics ; Humans ; Maternal Inheritance/genetics ; Paternal Inheritance/genetics ; Ubiquitin-Conjugating Enzymes/*genetics ; },
abstract = {The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.},
}
@article {pmid30714208,
year = {2019},
author = {Nasseri, S and Nikkho, B and Parsa, S and Ebadifar, A and Soleimani, F and Rahimi, K and Vahabzadeh, Z and Khadem-Erfan, MB and Rostamzadeh, J and Baban, B and Banafshi, O and Assadollahi, V and Mirzaie, S and Fathi, F},
title = {Generation of Fam83h knockout mice by CRISPR/Cas9-mediated gene engineering.},
journal = {Journal of cellular biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/jcb.28381},
pmid = {30714208},
issn = {1097-4644},
abstract = {Family with sequence similarity 83 member H (FAM83H) protein-coding geneplay an essential role in the structural organization, calcification of developing enamel, and keratin cytoskeleton disassembly by recruiting Casein kinase 1 alpha (CSNK1A1) to keratin filaments. In this study, we have applied CRISPR Cas9 nickase (D10A) to knockout (KO) the Fam83h gene in NMRI outbred mice. We generated homozygous Fam83h KO mice (Fam83h [Ko/Ko]) through a premature termination codon, which was validated by Sanger sequencing in F0 generation. Next, we also bred the FAM83H KO for two generations. Reverse-transcription polymerase chain reaction and Western blot analysis approved the Fam83h KO mice. The Fam83h KO mice had evidence of normal morphology at the cervical loops, secretory and maturation stages, and mandibular molars. In comparison with the normal wild-type mice (Fam83h [W/W]), the F2 homozygous KO (Fam83h [Ko/Ko]) had sparse, scruffy coats with small body size and decreased general activity. Also, they had the natural reproductive ability and natural lifespan. In addition, delay in opening the eyes and dry eyes among infant mice were seen. The F1 heterozygous mice looked comparable to the normal wild-type mice (Fam83h [W/W]), which showed autosomal recessive inheritance of these phenotypes. The KO of FAM83H had controversial effects on the development of teeth and the formation of enamel. The phenotype defect in dental development and the enamel formation were seen in three mice among four generations. It can be concluded that null FAM83H in outbred mice not only showed the reported phenotypes in null inbred mouse but also showed normal lifespan and reproductive ability; dental deficiency in three homozygous mice; and the symptoms that were similar to the symptoms of dry eye syndrome and curly coat dog syndrome in all four evaluated KO generations.},
}
@article {pmid30713102,
year = {2019},
author = {Horie, K and Inoue, K and Suzuki, S and Adachi, S and Yada, S and Hirayama, T and Hidema, S and Young, LJ and Nishimori, K},
title = {Oxytocin receptor knockout prairie voles generated by CRISPR/Cas9 editing show reduced preference for social novelty and exaggerated repetitive behaviors.},
journal = {Hormones and behavior},
volume = {111},
number = {},
pages = {60-69},
pmid = {30713102},
issn = {1095-6867},
support = {P51 OD011132/OD/NIH HHS/United States ; P50 MH100023/MH/NIMH NIH HHS/United States ; R01 MH096983/MH/NIMH NIH HHS/United States ; R21 MH114151/MH/NIMH NIH HHS/United States ; R01 MH112788/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Anxiety/genetics/pathology/physiopathology ; Arvicolinae/genetics/*physiology ; Autistic Disorder/*genetics/pathology/physiopathology ; CRISPR-Cas Systems/genetics ; Exploratory Behavior/*physiology ; Female ; Gene Editing/methods ; Gene Knockdown Techniques ; Male ; Mice ; Obsessive Behavior/genetics/pathology ; Oxytocin/metabolism ; Receptors, Oxytocin/*genetics/metabolism ; *Social Behavior ; },
abstract = {Behavioral neuroendocrinology has benefited tremendously from the use of a wide range of model organisms that are ideally suited for particular questions. However, in recent years the ability to manipulate the genomes of laboratory strains of mice has led to rapid advances in our understanding of the role of specific genes, circuits and neural populations in regulating behavior. While genome manipulation in mice has been a boon for behavioral neuroscience, the intensive focus on the mouse restricts the diversity in behavioral questions that can be investigated using state-of-the-art techniques. The CRISPR/Cas9 system has great potential for efficiently generating mutants in non-traditional animal models and consequently to reinvigorate comparative behavioral neuroendocrinology. Here we describe the efficient generation of oxytocin receptor (Oxtr) mutant prairie voles (Microtus ochrogaster) using the CRISPR/Cas9 system, and describe initial behavioral phenotyping focusing on behaviors relevant to autism. Oxtr mutant male voles show no disruption in pup ultrasonic vocalization, anxiety as measured by the open field test, alloparental behavior, or sociability in the three chamber test. Mutants did however show a modest elevation in repetitive behavior in the marble burying test, and an impairment in preference for social novelty. The ability to efficiently generate targeted mutations in the prairie vole genome will greatly expand the utility of this model organism for discovering the genetic and circuit mechanisms underlying complex social behaviors, and serves as a proof of principle for expanding this approach to other non-traditional model organisms.},
}
@article {pmid30712070,
year = {2019},
author = {Wang, X and Liu, X and Dong, R and Liang, C and Reichenberger, EJ and Hu, Y},
title = {Genetic Disruption of Anoctamin 5 in Mice Replicates Human Gnathodiaphyseal Dysplasia (GDD).},
journal = {Calcified tissue international},
volume = {104},
number = {6},
pages = {679-689},
doi = {10.1007/s00223-019-00528-x},
pmid = {30712070},
issn = {1432-0827},
mesh = {Animals ; Animals, Newborn ; Anoctamins/*genetics ; Bone and Bones/pathology ; Cells, Cultured ; *Disease Models, Animal ; Female ; Gene Deletion ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Osteoblasts/pathology/physiology ; Osteogenesis Imperfecta/*genetics/*pathology ; Phenotype ; },
abstract = {Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare skeletal disorder which is mainly characterized by cemento-osseous lesions in mandibles, bone fragility, bowing and diaphyseal sclerosis of tubular bones. GDD is caused by point mutations in Anoctamin-5 (ANO5); however, the disease mechanisms remain unclear. Here we generated Ano5-knockout (KO) mice using a CRISPR/Cas 9 approach to study loss of function aspects of GDD mutations. Homozygous Ano5 knockout mice (Ano5[-/-]) replicate some typical traits of human GDD including massive jawbones, bowing tibia, sclerosis and cortical thickening of femoral and tibial diaphyses. Serum alkaline phosphatase (ALP) levels were elevated in Ano5[-/-] mice as in GDD patients. Calvaria-derived Ano5[-/-] osteoblast cultures show increased osteoblastogenesis, which is consistent with our previous in vitro observations. Bone matrix is hypermineralized, and the expression of bone formation-related factors is enhanced in Ano5[-/-] mice, suggesting that the osteogenic anomaly arises from a genetic disruption of Ano5. We believe this new mouse model will shed more light on the development of skeletal abnormalities in GDD on a cellular and molecular level.},
}
@article {pmid30711520,
year = {2019},
author = {Wang, R and Liu, Y and Liu, L and Chen, M and Wang, X and Yang, J and Gong, Y and Ding, BS and Wei, Y and Wei, X},
title = {Tumor cells induce LAMP2a expression in tumor-associated macrophage for cancer progression.},
journal = {EBioMedicine},
volume = {40},
number = {},
pages = {118-134},
pmid = {30711520},
issn = {2352-3964},
mesh = {Animals ; Biomarkers ; CRISPR-Cas Systems ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Fluorescent Antibody Technique ; *Gene Expression ; Gene Knockdown Techniques ; Gene Targeting ; Humans ; Immunohistochemistry ; Lysosomal-Associated Membrane Protein 2/*genetics/metabolism ; Macrophage Activation/immunology ; Macrophages/immunology/*metabolism/pathology ; Mice ; Neoplasms/*genetics/immunology/mortality/*pathology ; Prognosis ; *Tumor Microenvironment/genetics/immunology ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Tumor cells benefit from tumor-associated macrophages (TAMs) promoting tumor growth and modulating functions of other cells in tumor microenvironment (TME). However, how tumor cells regulate the property of TAMs during tumor invasion remains to be defined.<br><br>METHODS: Mouse tumor models and cancer patients' samples were analyzed to determine LAMP2a expression in TAMs. In vitro mouse primary macrophages were used to assess LAMP2a-modulated macrophage activation, and to verify LAMP2a's target proteins. The effect of LAMP2a-knockdown on tumor progression and TME maintaining was determined by using mouse tumor models.<br><br>FINDINGS: Lysosome associated membrane protein type 2A (LAMP2a) is upregulated in TAMs by tumor cells and important for tumor progression. LAMP2a expression in TAMs, but not in tumor cells, is associated with poor prognosis in breast cancer. LAMP2a inactivation induced by either shRNA or CRISPR/Cas9 prevents TAMs activation and tumor growth. LAMP2a degrades PRDX1 (peroxiredoxin 1) and CRTC1 (CREB-regulated transcription coactivator 1) to promote macrophage pro-tumorigenic activation.<br><br>INTERPRETATION: Our study suggests that tumor cells utilize LAMP2a-PRDX1/CRTC1 axis to modulate TAMs activation and promote tumor growth, reveals the role of LAMP2a in macrophage study and TAM-targeting tumor immunotherapy. FUND: National Natural Science Foundation of China (No. 81602492); National Key Research and Development Program of China (No. 2016YFA0201402).},
}
@article {pmid30710789,
year = {2019},
author = {Wu, X and Mao, S and Ying, Y and Krueger, CJ and Chen, AK},
title = {Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms.},
journal = {Genomics, proteomics &amp; bioinformatics},
volume = {17},
number = {2},
pages = {119-128},
pmid = {30710789},
issn = {2210-3244},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Genetic Loci ; *Genomics ; Molecular Imaging/*methods ; Nanoparticles/chemistry ; },
abstract = {Chromatin conformation, localization, and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states, the requirement for cell fixation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review, we describe currently-available approaches for imaging single genomic loci in cells, with special focus on clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition, we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches, and potential solutions to address these challenges. We anticipate that, with continued refinement of CRISPR-based imaging techniques, significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.},
}
@article {pmid30710623,
year = {2019},
author = {Guo, Z and Sun, D and Kang, S and Zhou, J and Gong, L and Qin, J and Guo, L and Zhu, L and Bai, Y and Luo, L and Zhang, Y},
title = {CRISPR/Cas9-mediated knockout of both the PxABCC2 and PxABCC3 genes confers high-level resistance to Bacillus thuringiensis Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.).},
journal = {Insect biochemistry and molecular biology},
volume = {107},
number = {},
pages = {31-38},
doi = {10.1016/j.ibmb.2019.01.009},
pmid = {30710623},
issn = {1879-0240},
mesh = {Animals ; Bacillus thuringiensis/chemistry ; Bacillus thuringiensis Toxins ; Bacterial Proteins/*pharmacology ; Base Sequence ; CRISPR-Cas Systems ; Endotoxins/*pharmacology ; Gene Knockout Techniques ; Hemolysin Proteins/*pharmacology ; Insect Proteins/*genetics/metabolism ; Insecticide Resistance/*genetics ; Insecticides/*pharmacology ; Larva/drug effects/genetics/growth &amp; development ; Moths/drug effects/*genetics/growth &amp; development ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/*genetics/metabolism ; },
abstract = {Rapid evolution of resistance by insect pests severely jeopardizes the sustainable utilization of biopesticides and transgenic crops that produce insecticidal crystal proteins derived from the entomopathogenic bacterium Bacillus thuringiensis (Bt). Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily C genes ABCC2 and ABCC3 in seven lepidopteran insects, including Plutella xylostella (L.). To further determine the causal relationship between alterations in the PxABCC2 and PxABCC3 genes and Cry1Ac resistance in P. xylostella, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct two knockout strains: the ABCC2KO strain is homozygous for a 4-bp deletion in exon 3 of the PxABCC2 gene, and the ABCC3KO strain is homozygous for a 5-bp deletion in exon 3 of the PxABCC3 gene, both of which can produce only truncated ABCC proteins. Bioassay results indicated that high levels of resistance to the Cry1Ac protoxin were observed in both the ABCC2KO (724-fold) and ABCC3KO (413-fold) strains compared to the original susceptible DBM1Ac-S strain. Subsequently, dominance degree and genetic complementation tests demonstrated that Cry1Ac resistance in both the knockout strains was incompletely recessive, and Cry1Ac resistance alleles were located in the classic BtR-1 resistance locus that harbored the PxABCC2 and PxABCC3 genes, similar to the near-isogenic resistant NIL-R strain. Moreover, qualitative toxin binding assays revealed that the binding of the Cry1Ac toxin to midgut brush border membrane vesicles (BBMVs) in both knockout strains was dramatically reduced compared to that in the susceptible DBM1Ac-S strain. In summary, our CRISPR/Cas9-mediated genome editing study presents, for the first time, in vivo reverse genetics evidence for both the ABCC2 and ABCC3 proteins as midgut functional receptors for Bt Cry1 toxins in insects, which provides new insight into the pivotal roles of both the ABCC2 and ABCC3 proteins in the complex molecular mechanism of insect resistance to Bt Cry1 toxins.},
}
@article {pmid30710419,
year = {2019},
author = {Cunningham-Bryant, D and Sun, J and Fernandez, B and Zalatan, JG},
title = {CRISPR-Cas-Mediated Chemical Control of Transcriptional Dynamics in Yeast.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {20},
number = {12},
pages = {1519-1523},
pmid = {30710419},
issn = {1439-7633},
support = {R35 GM124773/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Expression ; RNA, Guide/*genetics ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Synthetic CRISPR-Cas transcription factors enable the construction of complex gene-expression programs, and chemically inducible systems allow precise control over the expression dynamics. To provide additional modes of regulatory control, we have constructed a chemically inducible CRISPR activation (CRISPRa) system in yeast that is mediated by recruitment to MS2-functionalized guide RNAs. We use reporter gene assays to systematically map the dose dependence, time dependence, and reversibility of the system. Because the recruitment function is encoded at the level of the guide RNA, it is straightforward to target multiple genes and independently regulate expression dynamics at individual targets. This approach provides a new method to engineer sophisticated, multigene programs with precise control over the dynamics of gene expression.},
}
@article {pmid30710307,
year = {2019},
author = {Granata, M and Skarmoutsou, E and Mazzarino, MC and D'Amico, F},
title = {S100A7 in Psoriasis: Immunodetection and Activation by CRISPR technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1929},
number = {},
pages = {729-738},
doi = {10.1007/978-1-4939-9030-6_45},
pmid = {30710307},
issn = {1940-6029},
mesh = {COP9 Signalosome Complex/metabolism ; CRISPR-Cas Systems ; Cell Line ; Fluorescent Antibody Technique ; Humans ; Intracellular Signaling Peptides and Proteins/metabolism ; Keratinocytes/*cytology/metabolism ; Peptide Hydrolases/metabolism ; Psoriasis/genetics/*metabolism ; S100 Calcium Binding Protein A7/*genetics/*metabolism ; Transcriptional Activation ; Up-Regulation ; },
abstract = {Psoriasis, an inflammatory autoimmune skin disease, is the result of a chronic interaction between hyperproliferative keratinocytes and infiltrating activated immune cells. The mechanisms underlying psoriasis pathogenesis remain largely unknown, although a combination of genetic and environmental factors plays an important role in its development. S100A7 is overexpressed in psoriasis, and there is growing evidence that S100A7 may be involved in the pathogenesis of psoriasis. Since the mechanisms underlying S100A7 regulation and function remain elusive, a better understanding of these mechanisms may be useful to uncover additional treatment approaches for psoriasis. Immunohistology provides invaluable tools for a better understanding of the pathogenetic mechanisms of psoriasis. Here, we describe basic methods for immunofluorescence and immunohistochemistry analysis of S100A7 expression in psoriatic patients as well as in S100A7 CRISPR-activated human keratinocyte cell line.},
}
@article {pmid30710124,
year = {2019},
author = {Conklin, BR},
title = {On the road to a gene drive in mammals.},
journal = {Nature},
volume = {566},
number = {7742},
pages = {43-45},
pmid = {30710124},
issn = {1476-4687},
mesh = {Animals ; Automobile Driving ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; *Gene Drive Technology ; Mammals ; Mice ; },
}
@article {pmid30710114,
year = {2019},
author = {Wang, B and Wang, M and Zhang, W and Xiao, T and Chen, CH and Wu, A and Wu, F and Traugh, N and Wang, X and Li, Z and Mei, S and Cui, Y and Shi, S and Lipp, JJ and Hinterndorfer, M and Zuber, J and Brown, M and Li, W and Liu, XS},
title = {Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute.},
journal = {Nature protocols},
volume = {14},
number = {3},
pages = {756-780},
pmid = {30710114},
issn = {1750-2799},
support = {R01 HG008927/HG/NHGRI NIH HHS/United States ; R01 HG008927/NH/NIH HHS/United States ; },
mesh = {*Algorithms ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Gene Dosage ; Genes, Essential ; Genetic Testing/*methods ; Genome ; Glioblastoma/genetics/pathology ; Humans ; Neoplastic Stem Cells/pathology ; },
abstract = {Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.},
}
@article {pmid30710088,
year = {2019},
author = {Hamnett, R and Crosby, P and Chesham, JE and Hastings, MH},
title = {Vasoactive intestinal peptide controls the suprachiasmatic circadian clock network via ERK1/2 and DUSP4 signalling.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {542},
pmid = {30710088},
issn = {2041-1723},
support = {MC_U105170643/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Circadian Clocks/*drug effects/genetics/radiation effects ; Cyclic AMP/metabolism ; Feedback, Physiological/drug effects/radiation effects ; Gene Regulatory Networks/drug effects/radiation effects ; Light ; MAP Kinase Signaling System/*drug effects/radiation effects ; Mice, Knockout ; Protein Biosynthesis/drug effects/radiation effects ; Protein Tyrosine Phosphatases/*metabolism ; Response Elements/genetics ; Suprachiasmatic Nucleus/cytology/drug effects/*physiology/radiation effects ; Transcription, Genetic/drug effects/radiation effects ; Vasoactive Intestinal Peptide/*pharmacology ; },
abstract = {The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.},
}
@article {pmid30709780,
year = {2019},
author = {Heler, R and Wright, AV and Vucelja, M and Doudna, JA and Marraffini, LA},
title = {Spacer Acquisition Rates Determine the Immunological Diversity of the Type II CRISPR-Cas Immune Response.},
journal = {Cell host &amp; microbe},
volume = {25},
number = {2},
pages = {242-249.e3},
pmid = {30709780},
issn = {1934-6069},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Intergenic/*genetics ; DNA, Viral/*genetics ; *Evolution, Molecular ; Staphylococcus Phages/genetics/*growth &amp; development ; Staphylococcus aureus/*enzymology/*genetics ; Streptococcus pyogenes/enzymology/genetics ; },
abstract = {CRISPR-Cas systems provide acquired immunity in prokaryotes. Upon infection, short sequences from the phage genome, known as spacers, are inserted between the CRISPR repeats. Spacers are transcribed into small RNA molecules that guide nucleases to their targets. The forces that shape the distribution of newly acquired spacers, which is observed to be uneven, are poorly understood. We studied the spacer patterns that arise after phage infection of Staphylococcus aureus harboring the Streptococcus pyogenes type II-A CRISPR-Cas system. We observed that spacer patterns are established early during the CRISPR-Cas immune response and correlate with spacer acquisition rates, but not with spacer targeting efficiency. The rate of spacer acquisition depended on sequence elements within the spacer, which in turn determined the abundance of different spacers within the adapted population. Our results reveal how the two main forces of the CRISPR-Cas immune response, acquisition and targeting, affect the generation of immunological diversity.},
}
@article {pmid30709766,
year = {2019},
author = {Kumar, S and Dangi, AK and Shukla, P and Baishya, D and Khare, SK},
title = {Thermozymes: Adaptive strategies and tools for their biotechnological applications.},
journal = {Bioresource technology},
volume = {278},
number = {},
pages = {372-382},
doi = {10.1016/j.biortech.2019.01.088},
pmid = {30709766},
issn = {1873-2976},
mesh = {Biofuels ; *Biotechnology ; Gene Editing ; Hot Temperature ; },
abstract = {In today's scenario of global climate change, there is a colossal demand for sustainable industrial processes and enzymes from thermophiles. Plausibly, thermozymes are an important toolkit, as they are known to be polyextremophilic in nature. Small genome size and diverse molecular conformational modifications have been implicated in devising adaptive strategies. Besides, the utilization of chemical technology and gene editing attributions according to mechanical necessities are the additional key factor for efficacious bioprocess development. Microbial thermozymes have been extensively used in waste management, biofuel, food, paper, detergent, medicinal and pharmaceutical industries. To understand the strength of enzymes at higher temperatures different models utilize X-ray structures of thermostable proteins, machine learning calculations, neural networks, but unified adaptive measures are yet to be totally comprehended. The present review provides a recent updates on thermozymes and various interdisciplinary applications including the aspects of thermophiles bioengineering utilizing synthetic biology and gene editing tools.},
}
@article {pmid30709710,
year = {2019},
author = {Wright, AV and Wang, JY and Burstein, D and Harrington, LB and Paez-Espino, D and Kyrpides, NC and Iavarone, AT and Banfield, JF and Doudna, JA},
title = {A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {727-737.e3},
pmid = {30709710},
issn = {1097-4164},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; S10 OD018174/OD/NIH HHS/United States ; S10 OD020062/OD/NIH HHS/United States ; T32 GM066698/GM/NIGMS NIH HHS/United States ; },
mesh = {Base Pairing ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial/*genetics/metabolism ; Endodeoxyribonucleases/*genetics/metabolism ; Endonucleases/*genetics/metabolism ; Escherichia coli/enzymology/*genetics ; Escherichia coli Proteins/*genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Integrases/*genetics/metabolism ; Nucleotide Motifs ; Substrate Specificity ; },
abstract = {CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.},
}
@article {pmid30707741,
year = {2019},
author = {Ota, H and Ito-Matsuoka, Y and Matsui, Y},
title = {Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions.},
journal = {PloS one},
volume = {14},
number = {2},
pages = {e0211739},
pmid = {30707741},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Proliferation ; Germ Cells ; Male ; Meiosis ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/classification/*genetics ; Spermatids/metabolism ; Spermatocytes/metabolism ; Spermatogenesis/*genetics ; Spermatogonia/metabolism ; Testis/metabolism ; X Chromosome/*genetics ; },
abstract = {MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 3'-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.},
}
@article {pmid30706614,
year = {2019},
author = {Zhang, Z and Hua, L and Gupta, A and Tricoli, D and Edwards, KJ and Yang, B and Li, W},
title = {Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.},
journal = {Plant biotechnology journal},
volume = {17},
number = {8},
pages = {1623-1635},
pmid = {30706614},
issn = {1467-7652},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Agrobacterium ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Plants, Genetically Modified ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Sequence Deletion ; Triticum/*genetics ; },
abstract = {CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium-delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain-regulatory genes, TaCKX2-1, TaGLW7, TaGW2, and TaGW8, in T0 , T1 , and T2 generation plants at an average edit rate of 10% without detecting off-target mutations in the most Cas9-active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160-bp deletion in TaCKX2-D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium-delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.},
}
@article {pmid30706492,
year = {2019},
author = {Khumsupan, P and Donovan, S and McCormick, AJ},
title = {CRISPR/Cas in Arabidopsis: overcoming challenges to accelerate improvements in crop photosynthetic efficiencies.},
journal = {Physiologia plantarum},
volume = {166},
number = {1},
pages = {428-437},
doi = {10.1111/ppl.12937},
pmid = {30706492},
issn = {1399-3054},
mesh = {Arabidopsis/*genetics/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Genome, Plant/genetics ; Photosynthesis/genetics/physiology ; },
abstract = {The rapid and widespread adoption of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technologies has allowed genetic editing in plants to enter a revolutionary new era. In this mini review, we highlight the current CRISPR/Cas tools available in plants and the use of Arabidopsis thaliana as a model to guide future improvements in crop yields, such as enhancing photosynthetic potential. We also outline the current socio-political landscape for CRISPR/Cas research and highlight the growing need for governments to better facilitate research into plant genetic-editing technologies.},
}
@article {pmid30706388,
year = {2019},
author = {Sandoval, IM and Collier, TJ and Manfredsson, FP},
title = {Design and Assembly of CRISPR/Cas9 Lentiviral and rAAV Vectors for Targeted Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1937},
number = {},
pages = {29-45},
doi = {10.1007/978-1-4939-9065-8_2},
pmid = {30706388},
issn = {1940-6029},
mesh = {Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Gene Editing/*methods ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus/*genetics ; RNA, Guide/genetics ; Rats ; Streptococcus pyogenes/enzymology ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system has emerged as an extremely useful tool for biological research and as a potential technology for gene therapy approaches. CRISPR/Cas mediated genome editing can be used to easily and efficiently modify endogenous genes in a large variety of cells and organisms. Furthermore, a modified version of the Cas9 nuclease has been developed that can be used for regulation of endogenous gene expression and labeling of genomic loci, among other applications. This chapter provides an introduction to the basis of the technology and a detail protocol for the most classic application: gene inactivation by CRISPR/Cas9 nuclease system from Streptococcus pyogenes. This workflow can be easily adapted for other CRISPR systems and applications.},
}
@article {pmid30705275,
year = {2019},
author = {Zhang, W and Ding, W and Li, YX and Tam, C and Bougouffa, S and Wang, R and Pei, B and Chiang, H and Leung, P and Lu, Y and Sun, J and Fu, H and Bajic, VB and Liu, H and Webster, NS and Qian, PY},
title = {Marine biofilms constitute a bank of hidden microbial diversity and functional potential.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {517},
pmid = {30705275},
issn = {2041-1723},
mesh = {Biodiversity ; *Biofilms ; CRISPR-Cas Systems/genetics/physiology ; Metagenome/genetics ; Seawater/microbiology ; },
abstract = {Recent big data analyses have illuminated marine microbial diversity from a global perspective, focusing on planktonic microorganisms. Here, we analyze 2.5 terabases of newly sequenced datasets and the Tara Oceans metagenomes to study the diversity of biofilm-forming marine microorganisms. We identify more than 7,300 biofilm-forming 'species' that are undetected in seawater analyses, increasing the known microbial diversity in the oceans by more than 20%, and provide evidence for differentiation across oceanic niches. Generation of a gene distribution profile reveals a functional core across the biofilms, comprised of genes from a variety of microbial phyla that may play roles in stress responses and microbe-microbe interactions. Analysis of 479 genomes reconstructed from the biofilm metagenomes reveals novel biosynthetic gene clusters and CRISPR-Cas systems. Our data highlight the previously underestimated ocean microbial diversity, and allow mining novel microbial lineages and gene resources.},
}
@article {pmid30705093,
year = {2019},
author = {Bourkas, MEC and Arshad, H and Al-Azzawi, ZAM and Halgas, O and Shikiya, RA and Mehrabian, M and Schmitt-Ulms, G and Bartz, JC and Watts, JC},
title = {Engineering a murine cell line for the stable propagation of hamster prions.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {13},
pages = {4911-4923},
pmid = {30705093},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cricetinae ; Gene Editing ; Mice ; Prions/genetics/*metabolism ; },
abstract = {Prions are infectious protein aggregates that cause several fatal neurodegenerative diseases. Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within in vitro amplification systems, they have proved challenging to propagate in cultured cells. Because the murine catecholaminergic cell line CAD5 is susceptible to a diverse range of mouse prion strains, we hypothesized that it might also be capable of propagating nonmouse prions. Here, using CRISPR/Cas9-mediated genome engineering, we demonstrate that CAD5 cells lacking endogenous mouse PrP expression (CAD5-PrP[-/-] cells) can be chronically infected with hamster prions following stable expression of hamster PrP. When exposed to the 263K, HY, or 139H hamster prion strains, these cells stably propagated high levels of protease-resistant PrP. Hamster prion replication required absence of mouse PrP, and hamster PrP inhibited the propagation of mouse prions. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP, suggesting that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of prion strains that can be propagated. We conclude that transfected CAD5-PrP[-/-] cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.},
}
@article {pmid30704988,
year = {2019},
author = {Xu, S and Luk, K and Yao, Q and Shen, AH and Zeng, J and Wu, Y and Luo, HY and Brendel, C and Pinello, L and Chui, DHK and Wolfe, SA and Bauer, DE},
title = {Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia.},
journal = {Blood},
volume = {133},
number = {21},
pages = {2255-2262},
pmid = {30704988},
issn = {1528-0020},
support = {DP2 HL137300/HL/NHLBI NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation ; *Hematopoietic Stem Cells/metabolism/pathology ; Humans ; Point Mutation ; *RNA Splice Sites ; *RNA Splicing ; *beta-Globins/biosynthesis/genetics ; *beta-Thalassemia/genetics/metabolism/therapy ; },
abstract = {The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34[+] hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.},
}
@article {pmid30704485,
year = {2019},
author = {Guo, T and Xin, Y and Zhang, Y and Gu, X and Kong, J},
title = {A rapid and versatile tool for genomic engineering in Lactococcus lactis.},
journal = {Microbial cell factories},
volume = {18},
number = {1},
pages = {22},
pmid = {30704485},
issn = {1475-2859},
mesh = {CRISPR-Cas Systems/genetics ; DNA-Directed RNA Polymerases/genetics ; Enterococcus faecalis/enzymology/genetics ; Gene Editing/*methods ; *Genome, Bacterial ; Genomics ; Lactococcus lactis/*genetics ; Recombinases/genetics ; Recombination, Genetic ; },
abstract = {BACKGROUND: Lactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.<br><br>RESULTS: A high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies&#x2009;>&#x2009;75%. Moreover, seamless genomic DNA deletions (50/100&#xa0;bp) or insertion (a loxP site, 34&#xa0;bp) was efficiently accomplished within 72&#xa0;h.<br><br>CONCLUSIONS: The work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.},
}
@article {pmid30704461,
year = {2019},
author = {Malzahn, AA and Tang, X and Lee, K and Ren, Q and Sretenovic, S and Zhang, Y and Chen, H and Kang, M and Bao, Y and Zheng, X and Deng, K and Zhang, T and Salcedo, V and Wang, K and Zhang, Y and Qi, Y},
title = {Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis.},
journal = {BMC biology},
volume = {17},
number = {1},
pages = {9},
pmid = {30704461},
issn = {1741-7007},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; Temperature ; Zea mays/*genetics ; },
abstract = {BACKGROUND: CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing.<br><br>RESULTS: We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28&#x2009;&#xb0;C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22&#x2009;&#xb0;C, its editing activity was rescued by growing the transgenic plants at 29&#x2009;&#xb0;C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28&#x2009;&#xb0;C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis.<br><br>CONCLUSION: Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.},
}
@article {pmid30704346,
year = {2019},
author = {Acun, A and Zorlutuna, P},
title = {CRISPR/Cas9 Edited Induced Pluripotent Stem Cell-Based Vascular Tissues to Model Aging and Disease-Dependent Impairment.},
journal = {Tissue engineering. Part A},
volume = {25},
number = {9-10},
pages = {759-772},
pmid = {30704346},
issn = {1937-335X},
support = {R01 HL141909/HL/NHLBI NIH HHS/United States ; },
mesh = {Aging/genetics/*metabolism/pathology ; *CRISPR-Cas Systems ; Cell Hypoxia ; Human Umbilical Vein Endothelial Cells/*metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Induced Pluripotent Stem Cells/*metabolism ; *Models, Cardiovascular ; },
abstract = {Modeling human disease as precisely as possible is of upmost importance in understanding the underlying pathology and discovering effective therapies. Therefore, disease models that are highly controlled and composed of human-origin cells that present the disease phenotype are crucial. The human induced pluripotent stem cell (hiPSC)-based tissue model we present in this study is an important example of human-origin tissue model with controlled gene expression. Through CRISPR/Cas9 editing of hypoxia inducible factor 1α in hiPSCs, we developed tissue models that show the age and disease-dependent endothelial deterioration. This model holds promise for various biomedical applications as more realistic disease phenotypes can be created using fully human-origin platforms.},
}
@article {pmid31021187,
year = {2018},
author = {Li, P and Kleinstiver, BP and Leon, MY and Prew, MS and Navarro-Gomez, D and Greenwald, SH and Pierce, EA and Joung, JK and Liu, Q},
title = {Allele-Specific CRISPR-Cas9 Genome Editing of the Single-Base P23H Mutation for Rhodopsin-Associated Dominant Retinitis Pigmentosa.},
journal = {The CRISPR journal},
volume = {1},
number = {1},
pages = {55-64},
pmid = {31021187},
issn = {2573-1602},
abstract = {Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR-Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs that can lead to allele-restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single-guide RNAs to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (Rho)-P23H mice, a model of dominant retinitis pigmentosa. We found that approximately 45% of the mutant P23H allele was edited at the DNA level and that the relative RNA expression of wild-type Rho was about 2.8 times more than that of mutant Rho in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the Rho-P23H retinas at 5 weeks of age. Our proof-of-concept study therefore outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patients. Our study also extends allele-specific editing strategies beyond discrimination within the protospacer adjacent motif sites, with potentially broad applicability to other dominant diseases.},
}
@article {pmid31015611,
year = {2017},
author = {Bashir, S and Kühn, R},
title = {Enhanced precision and efficiency.},
journal = {Nature biomedical engineering},
volume = {1},
number = {11},
pages = {856-857},
doi = {10.1038/s41551-017-0159-9},
pmid = {31015611},
issn = {2157-846X},
mesh = {*CRISPR-Cas Systems ; *DNA Repair ; *Gene Editing ; Oligodeoxyribonucleotides/genetics ; },
}
@article {pmid31015610,
year = {2017},
author = {},
title = {The expanding toolbox for genome engineering.},
journal = {Nature biomedical engineering},
volume = {1},
number = {11},
pages = {853},
doi = {10.1038/s41551-017-0163-0},
pmid = {31015610},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genome/genetics ; Humans ; Translational Research, Biomedical ; },
}
@article {pmid31015609,
year = {2017},
author = {Paulsen, BS and Mandal, PK and Frock, RL and Boyraz, B and Yadav, R and Upadhyayula, S and Gutierrez-Martinez, P and Ebina, W and Fasth, A and Kirchhausen, T and Talkowski, ME and Agarwal, S and Alt, FW and Rossi, DJ},
title = {Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing.},
journal = {Nature biomedical engineering},
volume = {1},
number = {11},
pages = {878-888},
pmid = {31015609},
issn = {2157-846X},
support = {UC4 DK104218/DK/NIDDK NIH HHS/United States ; R01 AI020047/AI/NIAID NIH HHS/United States ; R01 HL107630/HL/NHLBI NIH HHS/United States ; R01 AI077595/AI/NIAID NIH HHS/United States ; R01 DK107716/DK/NIDDK NIH HHS/United States ; U01 HL107440/HL/NHLBI NIH HHS/United States ; U19 HL129903/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; Ectopic Gene Expression ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Rad52 DNA Repair and Recombination Protein/*genetics ; Recombinational DNA Repair ; Tumor Suppressor p53-Binding Protein 1/*genetics ; },
abstract = {Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.},
}
@article {pmid30775868,
year = {2016},
author = {Liang, Z and Zong, Y and Gao, C},
title = {An Efficient Targeted Mutagenesis System Using CRISPR/Cas in Monocotyledons.},
journal = {Current protocols in plant biology},
volume = {1},
number = {2},
pages = {329-344},
doi = {10.1002/cppb.20021},
pmid = {30775868},
issn = {2379-8068},
abstract = {Precise genome modification using artificial nucleases is a powerful tool for in-depth understanding of gene functions and for creating new varieties. The CRISPR/Cas system, derived from an adaptive immunity system in bacteria and archaea, can introduce DNA double-strand breaks (DSBs) into pre-selected genomic loci and lead to loss of gene function due to error-prone non-homologous end joining (NHEJ). RNA-guided nucleases have been widely used in several eukaryotic organisms. In this article, we provide a detailed protocol for designing and constructing gRNA targets, detecting nuclease activity in transient protoplast assays, and identifying mutations in transgenic plants (including rice, wheat and maize). Targeted mutations in T0 plants can be generated in 14 to 18 weeks. © 2016 by John Wiley &amp; Sons, Inc.},
}
@article {pmid30704043,
year = {2019},
author = {Kim, J and Koo, BK and Yoon, KJ},
title = {Modeling Host-Virus Interactions in Viral Infectious Diseases Using Stem-Cell-Derived Systems and CRISPR/Cas9 Technology.},
journal = {Viruses},
volume = {11},
number = {2},
pages = {},
pmid = {30704043},
issn = {1999-4915},
mesh = {Adenoviridae/genetics ; Animals ; *CRISPR-Cas Systems ; Gene Editing ; Host Microbial Interactions/*genetics ; Humans ; Induced Pluripotent Stem Cells/*virology ; Mice ; Norovirus/genetics ; Organoids/*virology ; Virus Diseases/*genetics ; Viruses/genetics/*pathogenicity ; Zika Virus/genetics ; Zika Virus Infection ; },
abstract = {Pathologies induced by viral infections have undergone extensive study, with traditional model systems such as two-dimensional (2D) cell cultures and in vivo mouse models contributing greatly to our understanding of host-virus interactions. However, the technical limitations inherent in these systems have constrained efforts to more fully understand such interactions, leading to a search for alternative in vitro systems that accurately recreate in vivo physiology in order to advance the study of viral pathogenesis. Over the last decade, there have been significant technological advances that have allowed researchers to more accurately model the host environment when modeling viral pathogenesis in vitro, including induced pluripotent stem cells (iPSCs), adult stem-cell-derived organoid culture systems and CRISPR/Cas9-mediated genome editing. Such technological breakthroughs have ushered in a new era in the field of viral pathogenesis, where previously challenging questions have begun to be tackled. These include genome-wide analysis of host-virus crosstalk, identification of host factors critical for viral pathogenesis, and the study of viral pathogens that previously lacked a suitable platform, e.g., noroviruses, rotaviruses, enteroviruses, adenoviruses, and Zika virus. In this review, we will discuss recent advances in the study of viral pathogenesis and host-virus crosstalk arising from the use of iPSC, organoid, and CRISPR/Cas9 technologies.},
}
@article {pmid30703321,
year = {2019},
author = {Geng, P and Leonard, SP and Mishler, DM and Barrick, JE},
title = {Synthetic Genome Defenses against Selfish DNA Elements Stabilize Engineered Bacteria against Evolutionary Failure.},
journal = {ACS synthetic biology},
volume = {8},
number = {3},
pages = {521-531},
doi = {10.1021/acssynbio.8b00426},
pmid = {30703321},
issn = {2161-5063},
mesh = {Acinetobacter/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements/genetics ; Escherichia coli/*genetics ; *Evolution, Molecular ; Genetic Vectors ; Genome, Bacterial ; *Genomic Instability ; Metabolic Engineering/*methods ; Mutation ; Plasmids/genetics ; RNA Interference ; Repetitive Sequences, Nucleic Acid/*genetics ; Synthetic Biology/*methods ; },
abstract = {Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference against mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression. We further show that silencing a transposon in Acinetobacter baylyi ADP1 reduces mutation rates by a factor of 5, nearly as much as deleting all copies of this element from its genome. By deploying CRISPRi-ME on a broad-host-range vector, we have created a generalizable platform for stabilizing the genomes of engineered bacterial cells for applications in metabolic engineering and synthetic biology.},
}
@article {pmid30702927,
year = {2019},
author = {Du, L and Yang, F and Fang, H and Sun, H and Chen, Y and Xu, Y and Li, H and Zheng, L and Zhou, BS},
title = {AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {3},
pages = {4525-4537},
doi = {10.1096/fj.201801559RR},
pmid = {30702927},
issn = {1530-6860},
mesh = {AMP-Activated Protein Kinases/deficiency/genetics/physiology ; Aminoimidazole Carboxamide/*analogs &amp; derivatives/antagonists &amp; inhibitors/pharmacology/toxicity ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/drug effects ; Cell Line, Tumor ; DNA Breaks, Double-Stranded/drug effects ; DNA Replication/drug effects ; Deoxyribonucleotides/metabolism ; Drug Screening Assays, Antitumor ; Gene Knockout Techniques ; Genes, p53 ; Genes, rRNA ; Humans ; Nucleotides/*metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*drug therapy/metabolism/pathology ; RNA, Ribosomal/biosynthesis ; Ribonucleotides/antagonists &amp; inhibitors/metabolism/*pharmacology/toxicity ; Transcription, Genetic/drug effects ; Tumor Suppressor Protein p53/deficiency/physiology ; Uridine/pharmacology ; },
abstract = {It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.},
}
@article {pmid30702425,
year = {2019},
author = {McVeigh, P and Maule, AG},
title = {Can CRISPR help in the fight against parasitic worms?.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30702425},
issn = {2050-084X},
support = {NC/N001486/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Helminths/*genetics ; },
abstract = {The first reports of CRISPR/Cas9 genome editing in flatworms could usher in a new era of research on these dangerous human parasites.},
}
@article {pmid30702189,
year = {2019},
author = {Yahata, T and Mizoguchi, M and Kimura, A and Orimo, T and Toujima, S and Kuninaka, Y and Nosaka, M and Ishida, Y and Sasaki, I and Fukuda-Ohta, Y and Hemmi, H and Iwahashi, N and Noguchi, T and Kaisho, T and Kondo, T and Ino, K},
title = {Programmed cell death ligand 1 disruption by clustered regularly interspaced short palindromic repeats/Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression.},
journal = {Cancer science},
volume = {110},
number = {4},
pages = {1279-1292},
pmid = {30702189},
issn = {1349-7006},
mesh = {Animals ; B7-H1 Antigen/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/genetics ; Cytokines/metabolism ; Disease Progression ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Deletion ; *Gene Editing ; Genetic Loci ; Humans ; *Immunity ; Immunomodulation ; Lymphocytes, Tumor-Infiltrating/immunology/metabolism/pathology ; Macrophages/immunology/metabolism ; Mice ; Neoplasm Metastasis ; Ovarian Neoplasms/*genetics/immunology/*metabolism/pathology ; },
abstract = {Programmed cell death ligand 1 (PD-L1) on tumor cells suppresses anti-tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD-L1 disruption in ovarian cancer. PD-L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. PD-L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD-L1-KO ID8-inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD-L1-KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4[+] T cells, CD8[+] T cells, NK cells and CD11c[+] M1 macrophages were significantly increased, whereas regulatory T cells were significantly decreased in the PD-L1-KO ID8 groups compared with those in their control groups. The intratumoral mRNA expression of interferon-γ, tumor-necrosis factor-α, interleukin (IL)-2, IL-12a, CXCL9 and CXCL10 was significantly stronger, while that of IL-10, vascular endothelial growth factor, CXCL1 and CXCL2 was significantly weaker in the PD-L1-KO ID8 groups. These results indicate that CRISPR/Cas9-mediated PD-L1 disruption on tumor cells promotes anti-tumor immunity by increasing tumor-infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thereby suppressing ovarian cancer progression. These results suggest that PD-L1-targeted therapy by genome editing may be a novel therapeutic strategy for ovarian cancer.},
}
@article {pmid30700901,
year = {2019},
author = {Tang, L},
title = {Optogenetic tools light up phase separation.},
journal = {Nature methods},
volume = {16},
number = {2},
pages = {139},
doi = {10.1038/s41592-019-0310-5},
pmid = {30700901},
issn = {1548-7105},
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans ; Genome ; Green Fluorescent Proteins/metabolism ; Light ; Neurons/*cytology ; Optogenetics/*methods ; Phosphorylation ; Protein Domains ; RNA, Guide/genetics ; },
}
@article {pmid30700876,
year = {2019},
author = {Braun, M and Meacham, D},
title = {UN could lead debate on gene editing.},
journal = {Nature},
volume = {565},
number = {7741},
pages = {567},
doi = {10.1038/d41586-019-00324-5},
pmid = {30700876},
issn = {1476-4687},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Human ; Humans ; United Nations ; },
}
@article {pmid30700796,
year = {2019},
author = {Ojanen, MJT and Uusi-Mäkelä, MIE and Harjula, SE and Saralahti, AK and Oksanen, KE and Kähkönen, N and Määttä, JAE and Hytönen, VP and Pesu, M and Rämet, M},
title = {Intelectin 3 is dispensable for resistance against a mycobacterial infection in zebrafish (Danio rerio).},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {995},
pmid = {30700796},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Codon, Nonsense/genetics ; Cytokines/genetics/*metabolism ; Dexamethasone/pharmacology ; Disease Resistance/immunology ; Embryo, Nonmammalian/drug effects/metabolism/microbiology ; Gene Expression Regulation/drug effects ; Genome ; Lectins/genetics/*metabolism ; Lymphocyte Depletion ; Morpholinos/pharmacology ; Mutation/genetics ; Mycobacterium Infections, Nontuberculous/genetics/immunology/*microbiology ; Mycobacterium marinum/drug effects/*physiology ; Survival Analysis ; Zebrafish/embryology/genetics/*microbiology ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.},
}
@article {pmid30700588,
year = {2019},
author = {Crivello, P and Ahci, M and Maaßen, F and Wossidlo, N and Arrieta-Bolaños, E and Heinold, A and Lange, V and Falkenburg, JHF and Horn, PA and Fleischhauer, K and Heinrichs, S},
title = {Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {202},
number = {6},
pages = {1895-1903},
doi = {10.4049/jimmunol.1800257},
pmid = {30700588},
issn = {1550-6606},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; *HLA-DR beta-Chains/genetics ; Humans ; *RNA, Guide ; },
abstract = {Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing-based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II-expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.},
}
@article {pmid30699972,
year = {2019},
author = {Hadidi, A},
title = {Next-Generation Sequencing and CRISPR/Cas13 Editing in Viroid Research and Molecular Diagnostics.},
journal = {Viruses},
volume = {11},
number = {2},
pages = {},
pmid = {30699972},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering ; Genome, Viral ; *High-Throughput Nucleotide Sequencing ; Humans ; Pathology, Molecular/*trends ; Plants/virology ; Research/trends ; Viroids/*genetics ; },
abstract = {Viroid discovery as well as the economic significance of viroids and biological properties are presented. Next-generation sequencing (NGS) technologies combined with informatics have been applied to viroid research and diagnostics for almost a decade. NGS provides highly efficient, rapid, low-cost high-throughput sequencing of viroid genomes and of the 21[-]24 nt vd-sRNAs generated by the RNA silencing defense of the host. NGS has been utilized in various viroid studies which are presented. The discovery during the last few years that prokaryotes have heritable adaptive immunity mediated through clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated Cas proteins, have led to transformative advances in molecular biology, notably genome engineering and most recently molecular diagnostics. The potential application of the CRISPR-Cas13a system for engineering viroid interference in plants is suggested by targeting specific motifs of three economically important viroids. The CRISPR-Cas13 system has been utilized recently for the accurate detection of human RNA viruses by visual read out in 90 min or less and by paper-based assay. Multitarget RNA tests by this technology have a good potential for application as a rapid and accurate diagnostic assay for known viroids. The CRISPR/Cas system will work only for known viroids in contrast to NGS, but it should be much faster.},
}
@article {pmid30699347,
year = {2019},
author = {Chen, LF and Lin, YT and Gallegos, DA and Hazlett, MF and Gómez-Schiavon, M and Yang, MG and Kalmeta, B and Zhou, AS and Holtzman, L and Gersbach, CA and Grandl, J and Buchler, NE and West, AE},
title = {Enhancer Histone Acetylation Modulates Transcriptional Bursting Dynamics of Neuronal Activity-Inducible Genes.},
journal = {Cell reports},
volume = {26},
number = {5},
pages = {1174-1188.e5},
pmid = {30699347},
issn = {2211-1247},
support = {R33 DA041878/DA/NIDA NIH HHS/United States ; R21 DA041878/DA/NIDA NIH HHS/United States ; DP2 OD008654/OD/NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; },
mesh = {Acetylation ; Action Potentials ; Alleles ; Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Cell Membrane/metabolism ; Enhancer Elements, Genetic/*genetics ; *Gene Expression Regulation ; Histones/*metabolism ; Mice ; Neurons/*metabolism ; Nuclear Proteins/metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-fos/metabolism ; RNA, Messenger/genetics/metabolism ; Transcription Factors/metabolism ; *Transcription, Genetic ; },
abstract = {Neuronal activity-inducible gene transcription correlates with rapid and transient increases in histone acetylation at promoters and enhancers of activity-regulated genes. Exactly how histone acetylation modulates transcription of these genes has remained unknown. We used single-cell in situ transcriptional analysis to show that Fos and Npas4 are transcribed in stochastic bursts in mouse neurons and that membrane depolarization increases mRNA expression by increasing burst frequency. We then expressed dCas9-p300 or dCas9-HDAC8 fusion proteins to mimic or block activity-induced histone acetylation locally at enhancers. Adding histone acetylation increased Fos transcription by prolonging burst duration and resulted in higher Fos protein levels and an elevation of resting membrane potential. Inhibiting histone acetylation reduced Fos transcription by reducing burst frequency and impaired experience-dependent Fos protein induction in the hippocampus in vivo. Thus, activity-inducible histone acetylation tunes the transcriptional dynamics of experience-regulated genes to affect selective changes in neuronal gene expression and cellular function.},
}
@article {pmid30699341,
year = {2019},
author = {Graf, R and Li, X and Chu, VT and Rajewsky, K},
title = {sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing.},
journal = {Cell reports},
volume = {26},
number = {5},
pages = {1098-1103.e3},
pmid = {30699341},
issn = {2211-1247},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *Gene Editing ; Mice, Inbred C57BL ; Nucleotide Motifs/*genetics ; RNA, Guide/*genetics/metabolism ; },
abstract = {Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3' end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.},
}
@article {pmid30699116,
year = {2019},
author = {Leatham-Jensen, M and Uyehara, CM and Strahl, BD and Matera, AG and Duronio, RJ and McKay, DJ},
title = {Lysine 27 of replication-independent histone H3.3 is required for Polycomb target gene silencing but not for gene activation.},
journal = {PLoS genetics},
volume = {15},
number = {1},
pages = {e1007932},
pmid = {30699116},
issn = {1553-7404},
support = {R01 DA036897/DA/NIDA NIH HHS/United States ; R01 GM124201/GM/NIGMS NIH HHS/United States ; R01 GM129132/GM/NIGMS NIH HHS/United States ; R35 GM128851/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Chromatin/genetics ; DNA Replication/genetics ; DNA-Binding Proteins/genetics ; Drosophila melanogaster/genetics ; Epigenesis, Genetic/*genetics ; Gene Silencing ; Histone Code/genetics ; Histones/*genetics ; Humans ; Lysine/*genetics ; Methylation ; Polycomb-Group Proteins/*genetics ; Transcriptional Activation/genetics ; },
abstract = {Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.},
}
@article {pmid30698999,
year = {2019},
author = {Simeonov, DR and Marson, A},
title = {CRISPR-Based Tools in Immunity.},
journal = {Annual review of immunology},
volume = {37},
number = {},
pages = {571-597},
doi = {10.1146/annurev-immunol-042718-041522},
pmid = {30698999},
issn = {1545-3278},
mesh = {Animals ; Autoimmune Diseases/genetics/*immunology ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing ; Genetic Predisposition to Disease ; Genetic Variation ; Humans ; Immunity ; Infections/genetics/*immunology ; Neoplasms/genetics/*immunology ; },
abstract = {CRISPR technology has opened a new era of genome interrogation and genome engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the CRISPR system has been repurposed as an adaptable tool for genome editing and multiple other applications. CRISPR's ease of use, precision, and versatility have led to its widespread adoption, accelerating biomedical research and discovery in human cells and model organisms. Here we review CRISPR-based tools and discuss how they are being applied to decode the genetic circuits that control immune function in health and disease. Genetic variation in immune cells can affect autoimmune disease risk, infectious disease pathogenesis, and cancer immunotherapies. CRISPR provides unprecedented opportunities for functional mechanistic studies of coding and noncoding genome sequence function in immunity. Finally, we discuss the potential of CRISPR technology to engineer synthetic cellular immunotherapies for a wide range of human diseases.},
}
@article {pmid30698726,
year = {2019},
author = {Heitzeneder, S and Sotillo, E and Shern, JF and Sindiri, S and Xu, P and Jones, R and Pollak, M and Noer, PR and Lorette, J and Fazli, L and Alag, A and Meltzer, P and Lau, C and Conover, CA and Oxvig, C and Sorensen, PH and Maris, JM and Khan, J and Mackall, CL},
title = {Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion.},
journal = {Journal of the National Cancer Institute},
volume = {111},
number = {9},
pages = {970-982},
pmid = {30698726},
issn = {1460-2105},
mesh = {Animals ; Cell Line, Tumor ; Cell Membrane/metabolism ; Disease Models, Animal ; Gene Expression ; Gene Expression Profiling ; Humans ; Immunohistochemistry ; Insulin-Like Growth Factor I/metabolism ; Male ; Mice ; Pregnancy-Associated Plasma Protein-A/*metabolism ; Sarcoma, Ewing/*immunology/*metabolism/mortality/pathology ; Signal Transduction ; Transcriptome ; Tumor Burden ; *Tumor Escape ; Tumor Microenvironment/genetics/immunology ; },
abstract = {BACKGROUND: Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.<br><br>METHODS: By comparing RNA expression of cell surface proteins in EWS (n&#x2009;=&#x2009;120) versus normal tissues (n&#x2009;=&#x2009;42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n&#x2009;=&#x2009;5) and controls (n&#x2009;=&#x2009;3). All statistical tests were two-sided.<br><br>RESULTS: EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n&#x2009;=&#x2009;14], mean&#x2009;=&#x2009;397.0, SD&#x2009;=&#x2009;86.1 vs anti-PAPP-A [n&#x2009;=&#x2009;14], mean&#x2009;=&#x2009;311.7, SD&#x2009;=&#x2009;155.0; P =&#x2009;.03; median OS anti-PAPP-A&#x2009;=&#x2009;52.5&#x2009;days, 95% CI&#x2009;=&#x2009;46.0 to 63.0&#x2009;days vs IgG2a&#x2009;=&#x2009;45.0&#x2009;days, 95% CI&#x2009;=&#x2009;42.0 to 52.0&#x2009;days; P&#x2009;=&#x2009;.02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n&#x2009;=&#x2009;15], mean&#x2009;=&#x2009;217.9, SD&#x2009;=&#x2009;148.5 vs IgG2a-CTRL; P&#x2009;<&#x2009;.001; median OS anti-PAPP-A + anti-IGF1R&#x2009;=&#x2009;63.0&#x2009;days, 95% CI&#x2009;=&#x2009;52.0 to 67.0&#x2009;days vs IgG2a-CTRL; P&#x2009;<&#x2009;.001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.<br><br>CONCLUSION: This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.},
}
@article {pmid30698703,
year = {2019},
author = {Egermeier, M and Sauer, M and Marx, H},
title = {Golden Gate-based metabolic engineering strategy for wild-type strains of Yarrowia lipolytica.},
journal = {FEMS microbiology letters},
volume = {366},
number = {4},
pages = {},
doi = {10.1093/femsle/fnz022},
pmid = {30698703},
issn = {1574-6968},
mesh = {CRISPR-Cas Systems ; Glycerol Kinase/genetics ; Industrial Microbiology/*methods ; *Metabolic Engineering ; Yarrowia/enzymology/*genetics ; },
abstract = {The yeast Yarrowia lipolytica represents a future microbial cell factory for numerous applications in a bio-based economy. Outstanding feature of this yeast is the metabolic flexibility in utilising various substrates (sugars, fatty acids, glycerol, etc.). The potential of wild-type isolates of Y. lipolytica to convert glycerol into various value-added compounds is attracting attention of academia and industry. However, the already established tools for efficient engineering of the metabolism of Y. lipolytica are often dependent on genetic features like auxotrophic markers. With the present work we want to introduce a new set of vectors for metabolic engineering strategies, including CRISPR/Cas9 technology. The system is based on GoldenMOCS, a recently established rapid Golden Gate cloning strategy applicable in multiple organisms. We could show that our new GoldenMOCS plasmids are suitable for the extrachromosomal overexpression of the gene glycerol kinase (GUT1) in wild-type isolates of Y. lipolytica resulting in enhanced conversion of glycerol to erythritol and citric acid. Moreover, a GoldenMOCS plasmid for CRISPR/Cas9 mediated genome editing has been designed, which facilitates single gene knock-outs with efficiencies between 6% and 25% in strains with genetic wild-type background.},
}
@article {pmid30697931,
year = {2020},
author = {Gao, YL and Yao, XF and Li, WZ and Song, ZB and Wang, BW and Wu, YP and Shi, JL and Liu, GS and Li, YP and Liu, CM},
title = {An efficient TILLING platform for cultivated tobacco.},
journal = {Journal of integrative plant biology},
volume = {62},
number = {2},
pages = {165-180},
doi = {10.1111/jipb.12784},
pmid = {30697931},
issn = {1744-7909},
mesh = {CRISPR-Cas Systems ; Cadmium/metabolism ; Electrophoresis, Capillary ; Ethyl Methanesulfonate/metabolism ; Mutagenesis/genetics/physiology ; Plant Leaves/genetics/metabolism ; Polymerase Chain Reaction ; Polyploidy ; Tobacco/genetics/*metabolism ; Zinc/metabolism ; },
abstract = {Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.},
}
@article {pmid30697727,
year = {2019},
author = {Karimian, A and Azizian, K and Parsian, H and Rafieian, S and Shafiei-Irannejad, V and Kheyrollah, M and Yousefi, M and Majidinia, M and Yousefi, B},
title = {CRISPR/Cas9 technology as a potent molecular tool for gene therapy.},
journal = {Journal of cellular physiology},
volume = {234},
number = {8},
pages = {12267-12277},
doi = {10.1002/jcp.27972},
pmid = {30697727},
issn = {1097-4652},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Gene Targeting/methods ; Genetic Therapy/*methods ; Humans ; Neoplasms/genetics/*therapy ; RNA Interference ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) is an RNA-guided gene editing tool which offers several advantageous characteristics in comparison with the conventional methods (e.g., zinc finger nucleases and transcription activator-like effector nucleases) such as cost-effectiveness, flexibility, and being easy-to-use. Despite some limitations such as efficient delivery and safety, CRISPR-Cas9 is still the most convenient tool for gene editing purposes. Due to the potential capability of the CRISPR-Cas9 system in genome editing and correction of casual mutations, it can be considered as a possible therapeutic system in the treatment of disorders associated with the genome mutations and in particular cancer treatment. In this review, we will discuss CRISPR-Cas-based gene editing along with its classifications and mechanism of action. Furthermore, the therapeutic application of the CRISPR-Cas9 system in mutational disorders, delivery systems, as well as its advantages and limitations with a special emphasis on cancer treatment will be discussed.},
}
@article {pmid30696716,
year = {2019},
author = {Vicencio, J and Martínez-Fernández, C and Serrat, X and Cerón, J},
title = {Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.},
journal = {Genetics},
volume = {211},
number = {4},
pages = {1143-1154},
pmid = {30696716},
issn = {1943-2631},
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Gene Editing/methods ; *Genes, Reporter ; Green Fluorescent Proteins/*genetics/metabolism ; },
abstract = {CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.},
}
@article {pmid30696702,
year = {2019},
author = {Voutev, R and Mann, RS},
title = {TP901-1 Phage Recombinase Facilitates Genome Engineering in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {4},
pages = {983-986},
pmid = {30696702},
issn = {2160-1836},
support = {R35 GM118336/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Drosophila melanogaster/*genetics ; Genetic Engineering/*methods ; Genetic Vectors ; Genome, Insect ; Recombinases/genetics/*physiology ; Recombination, Genetic ; },
abstract = {Molecular biology techniques have a large impact on biomedical research and the availability of diverse tools to perform genome manipulations advances the ease of executing complicated genetic research. Here, we introduce in the fruit fly another such tool by harnessing the phage recombinase TP901-1 to perform site-directed recombination that leads to recombinase-mediated cassette exchange (RMCE). The TP901-1 system complements already existing recombination systems and enhances genome engineering in the fruit fly and other organisms.},
}
@article {pmid30696428,
year = {2019},
author = {Cho, EY and Ryu, JY and Lee, HAR and Hong, SH and Park, HS and Hong, KS and Park, SG and Kim, HP and Yoon, TJ},
title = {Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes.},
journal = {Journal of nanobiotechnology},
volume = {17},
number = {1},
pages = {19},
pmid = {30696428},
issn = {1477-3155},
mesh = {Animals ; Blood Glucose/drug effects ; *CRISPR-Cas Systems ; Cell Line ; Diabetes Mellitus, Type 2/*therapy ; Dipeptidyl Peptidase 4/*genetics/metabolism ; *Drug Delivery Systems ; Gene Editing ; Gene Targeting ; Genetic Therapy/*methods ; Glucagon-Like Peptide 1/blood ; Humans ; *Lecithins/administration &amp; dosage/chemistry ; *Liposomes/administration &amp; dosage/chemistry ; Mice ; Mice, Knockout ; RNA, Guide/administration &amp; dosage/chemistry/genetics ; },
abstract = {BACKGROUND: Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly.<br><br>RESULTS: In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses.<br><br>CONCLUSIONS: Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.},
}
@article {pmid30693970,
year = {2020},
author = {Phelps, MP and Seeb, LW and Seeb, JE},
title = {Transforming ecology and conservation biology through genome editing.},
journal = {Conservation biology : the journal of the Society for Conservation Biology},
volume = {34},
number = {1},
pages = {54-65},
doi = {10.1111/cobi.13292},
pmid = {30693970},
issn = {1523-1739},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Conservation of Natural Resources ; Ecosystem ; *Gene Editing ; },
abstract = {As the conservation challenges increase, new approaches are needed to help combat losses in biodiversity and slow or reverse the decline of threatened species. Genome-editing technology is changing the face of modern biology, facilitating applications that were unimaginable only a decade ago. The technology has the potential to make significant contributions to the fields of evolutionary biology, ecology, and conservation, yet the fear of unintended consequences from designer ecosystems containing engineered organisms has stifled innovation. To overcome this gap in the understanding of what genome editing is and what its capabilities are, more research is needed to translate genome-editing discoveries into tools for ecological research. Emerging and future genome-editing technologies include new clustered regularly interspaced short palindromic repeats (CRISPR) targeted sequencing and nucleic acid detection approaches as well as species genetic barcoding and somatic genome-editing technologies. These genome-editing tools have the potential to transform the environmental sciences by providing new noninvasive methods for monitoring threatened species or for enhancing critical adaptive traits. A pioneering effort by the conservation community is required to apply these technologies to real-world conservation problems.},
}
@article {pmid30693619,
year = {2019},
author = {Nedorezova, DD and Fakhardo, AF and Nemirich, DV and Bryushkova, EA and Kolpashchikov, DM},
title = {Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {14},
pages = {4654-4658},
doi = {10.1002/anie.201900829},
pmid = {30693619},
issn = {1521-3773},
support = {08-08//Government Council on Grants, Russian Federation/International ; CBET 1706802//National science foundation/International ; CCF 1423219//National Science Foundation/International ; 18-34-00898//Russian Foundation for Basic Research/International ; },
mesh = {Biomarkers, Tumor/chemistry/*metabolism ; DNA, Catalytic/chemistry/metabolism ; DNA, Neoplasm/chemistry/*metabolism ; *Genetic Therapy ; Humans ; Nanostructures/*chemistry ; Nanotechnology ; Neoplasms/metabolism/*therapy ; RNA Folding ; RNA, Messenger/*genetics/metabolism ; },
abstract = {Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme-based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.},
}
@article {pmid30693009,
year = {2018},
author = {Metje-Sprink, J and Menz, J and Modrzejewski, D and Sprink, T},
title = {DNA-Free Genome Editing: Past, Present and Future.},
journal = {Frontiers in plant science},
volume = {9},
number = {},
pages = {1957},
pmid = {30693009},
issn = {1664-462X},
abstract = {Genome Editing using engineered endonuclease (GEEN) systems rapidly took over the field of plant science and plant breeding. So far, Genome Editing techniques have been applied in more than fifty different plants; including model species like Arabidopsis; main crops like rice, maize or wheat as well as economically less important crops like strawberry, peanut and cucumber. These techniques have been used for basic research as proof-of-concept or to investigate gene functions in most of its applications. However, several market-oriented traits have been addressed including enhanced agronomic characteristics, improved food and feed quality, increased tolerance to abiotic and biotic stress and herbicide tolerance. These technologies are evolving at a tearing pace and especially the field of CRISPR based Genome Editing is advancing incredibly fast. CRISPR-Systems derived from a multitude of bacterial species are being used for targeted Gene Editing and many modifications have already been applied to the existing CRISPR-Systems such as (i) alter their protospacer adjacent motif (ii) increase their specificity (iii) alter their ability to cut DNA and (iv) fuse them with additional proteins. Besides, the classical transformation system using Agrobacteria tumefaciens or Rhizobium rhizogenes, other transformation technologies have become available and additional methods are on its way to the plant sector. Some of them are utilizing solely proteins or protein-RNA complexes for transformation, making it possible to alter the genome without the use of recombinant DNA. Due to this, it is impossible that foreign DNA is being incorporated into the host genome. In this review we will present the recent developments and techniques in the field of DNA-free Genome Editing, its advantages and pitfalls and give a perspective on technologies which might be available in the future for targeted Genome Editing in plants. Furthermore, we will discuss these techniques in the light of existing- and potential future regulations.},
}
@article {pmid30692669,
year = {2019},
author = {Rostøl, JT and Marraffini, LA},
title = {Non-specific degradation of transcripts promotes plasmid clearance during type III-A CRISPR-Cas immunity.},
journal = {Nature microbiology},
volume = {4},
number = {4},
pages = {656-662},
pmid = {30692669},
issn = {2058-5276},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/immunology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plasmids/*genetics/metabolism ; Ribonucleases/genetics/immunology ; Staphylococcus epidermidis/genetics/*immunology/metabolism ; },
abstract = {Type III-A CRISPR-Cas systems employ the Cas10-Csm complex to destroy bacteriophages and plasmids, using a guide RNA to locate complementary RNA molecules from the invader and trigger an immune response that eliminates the infecting DNA. In addition, these systems possess the non-specific RNase Csm6, which provides further protection for the host. While the role of Csm6 in immunity during phage infection has been determined, how this RNase is used against plasmids is unclear. Here, we show that Staphylococcus epidermidis Csm6 is required for immunity when transcription across the plasmid target is infrequent, leading to impaired target recognition and inefficient DNA degradation by the Cas10-Csm complex. In these conditions, Csm6 causes growth arrest in the host and prevents further plasmid replication through the indiscriminate degradation of host and plasmid transcripts. In contrast, when plasmid target sequences are efficiently transcribed, Csm6 is dispensable and DNA degradation by Cas10 is sufficient for anti-plasmid immunity. Csm6 therefore provides robustness to the type III-A CRISPR-Cas immune response against difficult targets for the Cas10-Csm complex.},
}
@article {pmid30692104,
year = {2019},
author = {Knoppers, BM and Kleiderman, E},
title = {"CRISPR babies": What does this mean for science and Canada?.},
journal = {CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne},
volume = {191},
number = {4},
pages = {E91-E92},
pmid = {30692104},
issn = {1488-2329},
mesh = {Bioethical Issues ; *CRISPR-Cas Systems ; Canada ; Embryo Research/*ethics ; Female ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Humans ; Infant, Newborn ; Pregnancy ; Reproductive Techniques, Assisted/*ethics/*legislation &amp; jurisprudence ; },
}
@article {pmid30691656,
year = {2019},
author = {Patterson, A and Tokmina-Lukaszewska, M and Bothner, B},
title = {Probing Cascade complex composition and stability using native mass spectrometry techniques.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {87-116},
doi = {10.1016/bs.mie.2018.10.018},
pmid = {30691656},
issn = {1557-7988},
mesh = {Bacteria/*chemistry ; Bacterial Proteins/*chemistry ; CRISPR-Associated Proteins/*chemistry ; CRISPR-Cas Systems ; Mass Spectrometry/methods ; Models, Molecular ; Protein Conformation ; },
abstract = {Adaptive prokaryotic immune systems rely on clustered regularly interspaced short palindromic repeats and their associated genes to provide the components necessary to clear infection by foreign genetic elements. These immune systems are based on highly specific nucleases that bind DNA or RNA and, upon sequence recognition, degrade the bound nucleic acid. Because of their specificity, CRISPR-Cas systems are being co-opted to edit genes in eukaryotic cells. While the general function of these systems is well understood, an understanding of mechanistic details to facilitate engineering and application to this new arena remains a topic of intense study. Here, we present two methods that have been successfully used to study the structure and mechanism of the Type IE CRISPR system, Cascade, from Escherichia coli. We provide the protocol for a typical native mass spectrometry experiment which, because it allows for analysis of a protein complex without disruption of the noncovalent interactions within the complex, can be used to determine complex composition, architecture, and relative affinity between subunits. We, also, provide the protocol for intact protein hydrogen-deuterium exchange mass spectrometry, which provides insight into the overall conformational stability of the complex and changes in complex stability based on conditions such as substrate binding. Investigating the solution-phase structure, stability, and dynamics of these complexes improves the overall understanding of the mechanism facilitating engineered adjustments to function or utility.},
}
@article {pmid30691655,
year = {2019},
author = {Phan, PT and Schelling, M and Xue, C and Sashital, DG},
title = {Fluorescence-based methods for measuring target interference by CRISPR-Cas systems.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {61-85},
pmid = {30691655},
issn = {1557-7988},
support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/*genetics ; Flow Cytometry/methods ; Fluorescence ; Fluorescent Dyes/*analysis/metabolism ; Genes, Reporter ; Green Fluorescent Proteins/*analysis/genetics ; Optical Imaging/methods ; Plasmids/*genetics ; },
abstract = {Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA. Given the importance of Cas effectors in host defense and for biotechnology tools, there has been considerable interest in developing sensitive methods for detecting Cas effector activity through CRISPR interference. In this chapter, we describe an in vivo fluorescence-based method for monitoring plasmid interference in Escherichia coli. This approach uses a green fluorescent protein reporter to monitor varying plasmid levels within bacterial colonies, or to measure the rate of plasmid-loss in bacterial populations over time. We demonstrate the use of this simple plasmid-loss assay for both chromosomally integrated and plasmid-borne CRISPR-Cas systems.},
}
@article {pmid30691654,
year = {2019},
author = {Dillard, KE and Schaub, JM and Brown, MW and Saifuddin, FA and Xiao, Y and Hernandez, E and Dahlhauser, SD and Anslyn, EV and Ke, A and Finkelstein, IJ},
title = {Sortase-mediated fluorescent labeling of CRISPR complexes.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {43-59},
pmid = {30691654},
issn = {1557-7988},
support = {K99 GM097177/GM/NIGMS NIH HHS/United States ; R00 GM097177/GM/NIGMS NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; F31 GM125201/GM/NIGMS NIH HHS/United States ; R01 GM120554/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/*analysis ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cysteine Endopeptidases/*analysis ; Escherichia coli/*chemistry/cytology ; Escherichia coli Proteins/*analysis ; Fluorescent Dyes/*analysis ; Models, Molecular ; Optical Imaging/methods ; Staining and Labeling/methods ; },
abstract = {Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.},
}
@article {pmid30691653,
year = {2019},
author = {Fagerlund, RD and Ferguson, TJ and Maxwell, HWR and Opel-Reading, HK and Krause, KL and Fineran, PC},
title = {Reconstitution of CRISPR adaptation in vitro and its detection by PCR.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {411-433},
doi = {10.1016/bs.mie.2018.10.024},
pmid = {30691653},
issn = {1557-7988},
mesh = {CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; Integration Host Factors/genetics ; Pectobacterium/*genetics ; Polymerase Chain Reaction/methods ; },
abstract = {CRISPR adaptation is the initial step in CRISPR-Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1-Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Structural and biochemical studies from reconstituted complexes have provided mechanistic insight into how CRISPR adaptation occurs; however, these studies have been limited to a narrow subset of CRISPR-Cas types and may not be representative of the other types. Here we describe methods for the purification of the type I-F CRISPR adaptation complex (Cas1:Cas2-3) from Pectobacterium atrosepticum, purification of the DNA architectural protein integration host factor (IHF), and a sensitive PCR-based in vitro integration assay. This assay could easily be used to investigate mechanisms of CRISPR adaptation in other CRISPR-Cas systems, including the roles of accessory proteins.},
}
@article {pmid30691652,
year = {2019},
author = {Nayeemul Bari, SM and Hatoum-Aslan, A},
title = {CRISPR-Cas10 assisted editing of virulent staphylococcal phages.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {385-409},
doi = {10.1016/bs.mie.2018.10.023},
pmid = {30691652},
issn = {1557-7988},
support = {K22 AI113106/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Engineering/methods ; Staphylococcus Phages/*genetics ; Staphylococcus aureus/genetics/*virology ; Staphylococcus epidermidis/genetics/*virology ; },
abstract = {Phages are the most abundant entities in the biosphere and profoundly impact the bacterial populations within and around us. They attach to a specific host, inject their DNA, hijack the host's cellular processes, and replicate exponentially while destroying the host. Historically, phages have been exploited as powerful antimicrobials, and phage-derived proteins have constituted the basis for numerous biotechnological applications. Only in recent years have metagenomic studies revealed that phage genomes harbor a rich reservoir of genetic diversity, which might afford further therapeutic and/or biotechnological value. Nevertheless, functions for the majority of phage genes remain unknown, and due to their swift and destructive replication cycle, many phages are intractable by current genetic engineering techniques. Whether to advance the basic understanding of phage biology or to tap into their potential applications, efficient methods for phage genetic engineering are needed. Recent reports have shown that CRISPR-Cas systems, a class of prokaryotic immune systems that protect against phage infection, can be harnessed to engineer diverse phages. In this chapter, we describe methods to genetically manipulate virulent phages using CRISPR-Cas10, a Type III-A CRISPR-Cas system native to Staphylococcus epidermidis. A method for engineering phages that infect a CRISPR-less Staphylococcus aureus host is also described. Both approaches have proved successful in isolating desired phage mutants with 100% efficiency, demonstrating that CRISPR-Cas10 constitutes a powerful tool for phage genetic engineering. The relatively widespread presence of Type III CRISPR-Cas systems in bacteria and archaea imply that similar strategies may be used to manipulate the genomes of diverse prokaryotic viruses.},
}
@article {pmid30691651,
year = {2019},
author = {Gao, XD and Rodríguez, TC and Sontheimer, EJ},
title = {Adapting dCas9-APEX2 for subnuclear proteomic profiling.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {365-383},
pmid = {30691651},
issn = {1557-7988},
support = {U54 DK107980/DK/NIDDK NIH HHS/United States ; },
mesh = {Ascorbate Peroxidases/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Cloning, Molecular/methods ; HEK293 Cells ; Humans ; Luminescent Proteins/genetics ; Mass Spectrometry/methods ; Nuclear Proteins/*analysis/genetics ; Proteomics/*methods ; Recombinant Fusion Proteins/genetics ; Soybeans/*enzymology/genetics ; },
abstract = {Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR-Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. In this chapter, we describe the dCas9-APEX2 experimental approach for specifically targeting a DNA sequence, enzymatically labeling local proteins with biotin, and quantitatively analyzing the labeled proteome. We also discuss the optimization and extension of this pipeline to facilitate its use in understanding nuclear and chromosome biology.},
}
@article {pmid30691650,
year = {2019},
author = {Mekler, V and Kuznedelov, K and Minakhin, L and Murugan, K and Sashital, DG and Severinov, K},
title = {CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {337-363},
pmid = {30691650},
issn = {1557-7988},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*metabolism ; Bacterial Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; DNA/*metabolism ; DNA Probes/metabolism ; Fluorometry/methods ; RNA, Guide/metabolism ; },
abstract = {CRISPR-Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR-Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR-Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR-Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR-Cas complexes.},
}
@article {pmid30691648,
year = {2019},
author = {Liu, MS and Gong, S and Yu, HH and Taylor, DW and Johnson, KA},
title = {Kinetic characterization of Cas9 enzymes.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {289-311},
pmid = {30691648},
issn = {1557-7988},
support = {R01 AI110577/AI/NIAID NIH HHS/United States ; R01 GM114223/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*enzymology/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Enzyme Assays/instrumentation/methods ; Equipment Design ; Kinetics ; RNA, Guide/metabolism ; },
abstract = {Bacterial adaptive immune systems employ clustered regularly interspaced short palindromic repeats (CRISPR) along with their CRISPR-associated genes (Cas) to form CRISPR RNA (crRNA)-guided surveillance complexes, which target foreign nucleic acids for destruction. Cas9 is unique in that it is composed of a single polypeptide that utilizes both a crRNA and a trans-activating crRNA (tracrRNA) or a single guide RNA to create double-stranded breaks in sequences complementary to the RNA via the HNH and RuvC nuclease domains. Cas9 has become a revolutionary tool for gene-editing applications. Here, we describe methods for studying the cleavage activities of Cas9. We describe protocols for rapid quench-flow and stopped-flow kinetics and interpretation of the results. The protocols detailed here will be paramount for understanding the mechanistic basis for specificity of this enzyme, especially in efforts to improve accuracy for clinical use.},
}
@article {pmid30691647,
year = {2019},
author = {Xiao, Y and Ke, A},
title = {Reconstitution and biochemical characterization of ribonucleoprotein complexes in Type I-E CRISPR-Cas systems.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {27-41},
pmid = {30691647},
issn = {1557-7988},
support = {R35 GM118174/GM/NIGMS NIH HHS/United States ; },
mesh = {Actinomycetales/chemistry/*genetics ; Bacterial Proteins/chemistry/*genetics ; CRISPR-Associated Proteins/chemistry/*genetics ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Conformation ; Protein Conformation ; Ribonucleoproteins/chemistry/*genetics ; },
abstract = {Type I CRISPR-Cas, the most prevalent CRISPR system, features a sequential target searching and degradation process. First, the multisubunit surveillance complex Cascade recognizes the matching dsDNA target flanked by protospacer adjacent motif (PAM), promotes the heteroduplex formation between CRISPR RNA (crRNA) and the target strand (TS) DNA, and displaces the nontarget strand (NTS) DNA, resulting in R-loop formation. The helicase-nuclease fusion enzyme Cas3 is then specifically recruited to Cascade/R-loop, nicks, and processively degrades the DNA target. Here, by using Type I-E CRISPR-Cas system from Thermobifida fusca, we provide protocols for the biochemical reconstitution of the Cascade/R-loop and Cascade/R-loop/Cas3 complexes that allowed for high-resolution structure determination and mechanism investigation. The procedures may be applicable for structural and mechanistic investigations of other Type I CRISPR-Cas systems, and may serve as a reference for the study of other multicomponent protein-nucleic acid complexes.},
}
@article {pmid30691646,
year = {2019},
author = {Hand, TH and Das, A and Li, H},
title = {Directed evolution studies of a thermophilic Type II-C Cas9.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {265-288},
pmid = {30691646},
issn = {1557-7988},
support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; },
mesh = {Actinobacteria/chemistry/*genetics ; Bacterial Proteins/chemistry/*genetics ; CRISPR-Associated Protein 9/chemistry/*genetics ; Directed Molecular Evolution/*methods ; Gene Library ; Models, Molecular ; },
abstract = {Though making up nearly half of the known CRISPR-Cas9 family of enzymes, the Type II-C CRISPR-Cas9 has been underexplored for their molecular mechanisms and potential in safe gene editing applications. In comparison with the more popular Type II-A CRISPR-Cas9, the Type II-C enzymes are generally smaller in size and utilize longer base pairing in identification of their DNA substrates. These characteristics suggest easier portability and potentially less off-targets for Type II-C in gene editing applications. We describe identification and biochemical characterization of a thermophilic Type II-C CRISPR-Cas from Acidothermus cellulolyticus (AceCas9). We describe several library-based methods that enabled us to identify the PAM sequence and elements critical to protospacer mismatch surveillance of AceCas9.},
}
@article {pmid30691645,
year = {2019},
author = {Kissling, L and Monfort, A and Swarts, DC and Wutz, A and Jinek, M},
title = {Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {241-263},
doi = {10.1016/bs.mie.2018.10.028},
pmid = {30691645},
issn = {1557-7988},
mesh = {Animals ; CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Electroporation/methods ; *Gene Deletion ; Gene Editing/*methods ; Genetic Engineering/methods ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas12a is a bacterial RNA-guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.},
}
@article {pmid30691644,
year = {2019},
author = {Karvelis, T and Young, JK and Siksnys, V},
title = {A pipeline for characterization of novel Cas9 orthologs.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {219-240},
doi = {10.1016/bs.mie.2018.10.021},
pmid = {30691644},
issn = {1557-7988},
mesh = {Bacteria/genetics/*metabolism ; Base Pairing ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/genetics/metabolism ; Gene Library ; Phylogeny ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; },
abstract = {In recent years, Cas9 has revolutionized the genome-editing field and enabled a broad range of applications from basic biology to biotechnology and medicine. Cas9 specificity is dictated by base pairing of the guide RNA to the complementary DNA strand, however to initiate hybridization, a short protospacer adjacent motif (PAM) sequence is required in the vicinity of the target sequence. The PAM is recognized by the Cas9 protein and varies between Cas9s. There are thousands of type II CRISPR-Cas9 sequences available in sequence databases. To characterize the PAM recognition diversity provided by Cas9 orthologs, we developed a phylogeny-guided bioinformatics approach and streamlined our experimental procedures for Cas9 expression and RNP complex assembly using cell lysates and in vitro translation mixtures. This approach could be easily adapted for the characterization of other CRISPR-Cas nucleases that require PAM sequences and generate double-strand breaks following target recognition.},
}
@article {pmid30691643,
year = {2019},
author = {Rouillon, C and Athukoralage, JS and Graham, S and Grüschow, S and White, MF},
title = {Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {191-218},
doi = {10.1016/bs.mie.2018.10.020},
pmid = {30691643},
issn = {1557-7988},
support = {//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adenine Nucleotides/genetics/*metabolism ; Archaeal Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Kinetics ; Oligoribonucleotides/genetics/*metabolism ; Second Messenger Systems ; Signal Transduction ; Sulfolobus solfataricus/genetics/*metabolism ; },
abstract = {Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.},
}
@article {pmid30691642,
year = {2019},
author = {Park, KH and An, Y and Woo, EJ},
title = {In vitro assembly of thermostable Csm complex in CRISPR-Cas type III/A system.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {173-189},
doi = {10.1016/bs.mie.2018.10.025},
pmid = {30691642},
issn = {1557-7988},
mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Protein Subunits/genetics ; Recombinant Proteins/genetics ; Temperature ; Thermococcus/*genetics ; },
abstract = {The CRISPR-Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR-Cas system is classified into six types (I-VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2-5) and a crRNA. The Csm complex targets RNA and RNA-dependent single-strand DNA. Here, we present a protocol for in vitro reconstitution of a Csm complex from a hyperthermophilic archaeon Thermococcus onnurineus NA1 (ToCsm complex). The method consists of coexpression and copurification of the subunits, in vitro synthesis of the crRNA and assembly of the ToCsm complex. Purification with heat treatment and affinity and size exclusion chromatography resulted in homogeneous Cas10/Csm4 and Csm2/Csm5 binary complexes, while in vitro transcription with the T7 promoter enabled synthesis of the crRNA. Addition of each component in the presence of the crRNA with a molar ratio of Cas10/Csm4:Csm3:Csm2/Csm5:crRNA=1:3:2:1 yielded an assembled functional Csm complex. This protocol for reconstitution of the Csm complex is presumably applicable to other thermostable effector complexes, which would allow biochemical, structural, or functional studies of the CRISPR-Cas type III/A system in vitro.},
}
@article {pmid30691641,
year = {2019},
author = {Turkowyd, B and Müller-Esparza, H and Climenti, V and Steube, N and Endesfelder, U and Randau, L},
title = {Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {133-171},
doi = {10.1016/bs.mie.2018.11.001},
pmid = {30691641},
issn = {1557-7988},
mesh = {CRISPR-Associated Proteins/genetics ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/genetics ; Microscopy, Fluorescence/methods ; Plasmids/genetics ; RNA, Guide/genetics ; Shewanella putrefaciens/genetics ; Transformation, Genetic ; },
abstract = {Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to scan DNA strands for target regions. Different crRNAs are bound by several CRISPR-associated (Cas) protein subunits that form the stable ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance process requires a sufficient degree of base-complementarity between crRNA and target sequences and relies on the recognition of small DNA motifs, termed protospacer adjacent motifs. Recently, super-resolution microscopy and single-particle tracking methods have been developed to follow individual protein complexes in live cells. Here, we described how this technology can be adapted to visualize the DNA scanning process of Cascade assemblies in Escherichia coli cells. The activity of recombinant Type I-Fv Cascade complexes of Shewanella putrefaciens CN-32 serves as a model system that facilitates comparative studies for many of the diverse CRISPR-Cas systems.},
}
@article {pmid30691640,
year = {2019},
author = {Wade, JT},
title = {High-throughput determination of in vivo DNA sequence preferences for Cas protein binding using Library-ChIP.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {117-132},
doi = {10.1016/bs.mie.2018.10.019},
pmid = {30691640},
issn = {1557-7988},
mesh = {CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics/*metabolism ; Escherichia coli/genetics/*metabolism ; Escherichia coli Proteins/genetics/*metabolism ; Gene Library ; High-Throughput Nucleotide Sequencing/methods ; Protein Binding ; },
abstract = {The specificity of CRISPR-Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR-Cas systems for biotechnology applications. Moreover, the specificity of CRISPR-Cas systems plays an important role in prokaryote evolution due to its role in distinguishing self from nonself. Here, I describe Library-ChIP, a high-throughput method for measuring Cas protein occupancy at many DNA sequence variants in a native prokaryotic host. Library-ChIP can be used to identify the determinants of specificity for Cas protein binding to nucleic acid targets.},
}
@article {pmid30691639,
year = {2019},
author = {Nethery, MA and Barrangou, R},
title = {Predicting and visualizing features of CRISPR-Cas systems.},
journal = {Methods in enzymology},
volume = {616},
number = {},
pages = {1-25},
doi = {10.1016/bs.mie.2018.10.016},
pmid = {30691639},
issn = {1557-7988},
mesh = {Animals ; Bacteria/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computer Simulation ; *Gene Editing/methods ; Genomics/methods ; Humans ; RNA, Guide/genetics ; *Software ; },
abstract = {Pervasive application of CRISPR-Cas systems in genome editing has prompted an increase in both interest and necessity to further elucidate existing systems as well as discover putative novel systems. The ubiquity and power of current computational platforms have made in silico approaches to CRISPR-Cas identification and characterization accessible to a wider audience and increasingly amenable for processing extensive data sets. Here, we describe in silico methods for predicting and visualizing notable features of CRISPR-Cas systems, including Cas domain determination, CRISPR array visualization, and inference of the protospacer-adjacent motif. The efficiency of these tools enables rapid exploration of CRISPR-Cas diversity across prokaryotic genomes and supports scalable analysis of large genomic data sets.},
}
@article {pmid30691438,
year = {2019},
author = {Al Amin, N and Ahmad, N and Wu, N and Pu, X and Ma, T and Du, Y and Bo, X and Wang, N and Sharif, R and Wang, P},
title = {CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max.L).},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {9},
pmid = {30691438},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; *Mutagenesis, Site-Directed ; Mutation ; Plants, Genetically Modified ; Soybeans/*genetics ; },
abstract = {BACKGROUND: Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis.<br><br>RESULTS: Here we demonstrated an efficient sequence specific integration/mutation of FAD2-2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2-2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2-2 loci. The integration of FAD2-2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis.<br><br>CONCLUSION: High frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.},
}
@article {pmid30691408,
year = {2019},
author = {Yamada, S and Shibasaki, M and Murase, K and Watanabe, T and Aikawa, C and Nozawa, T and Nakagawa, I},
title = {Phylogenetic relationship of prophages is affected by CRISPR selection in Group A Streptococcus.},
journal = {BMC microbiology},
volume = {19},
number = {1},
pages = {24},
pmid = {30691408},
issn = {1471-2180},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; Evolution, Molecular ; Genome, Bacterial ; *Phylogeny ; Prophages/*classification ; Streptococcus pyogenes/*genetics/virology ; Virus Integration ; },
abstract = {BACKGROUND: Group A Streptococcus (GAS) is a major human pathogen, which is associated with a wide spectrum of invasive diseases, such as pharyngitis, scarlet fever, rheumatic fever, and streptococcal toxic shock syndrome (STSS). It is hypothesized that differences in GAS pathogenicity are related to the acquisition of diverse bacteriophages (phages). Nevertheless, the GAS genome also harbors clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes, which play an important role in eliminating foreign DNA, including those of phages. However, the structure of prophages in GAS strains is mosaic, and the phylogenetic relationship between prophages and CRISPR is not clear. In this study, we analyzed CRISPR and prophage structure using 118 complete genome sequences of GAS strains to elucidate the relationship between two genomic elements. Additionally, phylogenetic and M-type analyses were performed.<br><br>RESULTS: Of the 118 GAS strains, 80 harbored type I-C and/or II-A CRISPR/cas loci. A total of 553 spacer sequences were identified from CRISPR/cas loci and sorted into 229 patterns. We identified and classified 373 prophages into 14 groups. Some prophage groups shared a common integration site, and were related to M-type. We further investigated the correlation between spacer sequences and prophages. Of the 229 spacer sequence patterns, 203 were similar to that of other GAS prophages. No spacer showed similarity with that of a specific prophage group with mutL integration site. Moreover, the average number of prophages in strains with type II-A CRISPR was significantly less than that in type I-C CRISPR and non-CRISPR strains. However, there was no statistical difference between the average number of prophages in type I-C strains and that in non-CRISPR strains.<br><br>CONCLUSIONS: Our results indicated that type II-A CRISPR may play an important role in eliminating phages and that the prophage integration site may be an important criterion for the acceptance of foreign DNA by GAS. M type, spacer sequence, and prophage group data were correlated with the phylogenetic relationships of GAS. Therefore, we hypothesize that genetic characteristics and/or phylogenetic relationships of GAS may be estimated by analyzing its spacer sequences.},
}
@article {pmid30691366,
year = {2019},
author = {Coller, BS},
title = {Ethics of Human Genome Editing.},
journal = {Annual review of medicine},
volume = {70},
number = {},
pages = {289-305},
doi = {10.1146/annurev-med-112717-094629},
pmid = {30691366},
issn = {1545-326X},
support = {UL1 TR001866/TR/NCATS NIH HHS/United States ; },
mesh = {Advisory Committees ; CRISPR-Cas Systems/*genetics ; Child ; Female ; Gene Editing/*ethics ; Genetic Therapy/*ethics/methods ; Genome, Human/*genetics ; Germ-Line Mutation/genetics ; Humans ; Informed Consent/ethics ; Male ; Risk Assessment ; United States ; },
abstract = {Advances in human genome editing, in particular the development of the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 method, have led to increasing concerns about the ethics of editing the human genome. In response, the US National Academy of Sciences and the National Academy of Medicine constituted a multidisciplinary, international committee to review the current status and make recommendations. I was a member of that committee, and the core of this review reflects the committee's conclusions. The committee's report, issued in February 2017, recommends the application of current ethical and regulatory standards for gene therapy to somatic (nonheritable) human genome editing. It also recommends allowing experimental germline genome editing to proceed if (a) it is restricted to preventing transmission of a serious disease or condition, (b) the edit is a modification to a common DNA sequence known not to be associated with disease, and (c) the research is conducted under a stringent set of ethical and regulatory requirements. Crossing the so-called red line of germline genome editing raises important bioethical issues, most importantly, serious concern about the potential negative impact on individuals with disabilities. This review highlights some of the major ethical considerations in human genome editing in light of the report's recommendations.},
}
@article {pmid30689007,
year = {2019},
author = {Oppel, F and Schürmann, M and Shao, S and Kaltschmidt, B and Kaltschmidt, C and Sudhoff, H},
title = {[Perspectives of genome editing in otorhinolaryngology].},
journal = {HNO},
volume = {67},
number = {3},
pages = {184-189},
pmid = {30689007},
issn = {1433-0458},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Targeting ; Humans ; Mice ; *Otolaryngology ; },
abstract = {BACKGROUND: Recent advances in DNA sequencing technology have enabled researchers to identify the genetic background underlying human illness. In addition, the latest genome editing technology, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9), provides great potential to edit genomic DNA sequences precisely with high efficiency. This technology has been evaluated for treatment of genetic diseases in recently published preclinical studies. Since many such genetic disorders can affect functional structures in the head and neck area, the technology bears high therapeutic potential in otorhinolaryngology.<br><br>OBJECTIVE: In this article, we summarize the concept of CRISPR-Cas9-based therapies, recent achievements in preclinical applications, and future challenges for the implementation of this technology in otolaryngology.<br><br>MATERIALS AND METHODS: Genetic targeting strategies were analyzed or established using genome sequencing data derived from online databases and literature.<br><br>RESULTS: Recent research on animal models has shown that genome editing can be used to treat genetic diseases by specifically targeting mutant genomic loci. For example, one preclinical study in the field of otolaryngology has demonstrated that inherited autosomal dominant deafness in mice can be treated using CRISPR-Cas9. Moreover, the same strategies can be used to establish applications for the treatment of head and neck cancer. The greatest challenge appears to be establishment of a&#xa0;system for the safe and efficient delivery of therapeutic nucleotides in clinics.<br><br>CONCLUSIONS: In theory, genome editing could be used in otolaryngology to target disease-causing genomic loci specifically. However, various challenges have to be overcome until applications can be used clinically.},
}
@article {pmid30688548,
year = {2019},
author = {Arbabi, A and Liu, A and Ameri, H},
title = {Gene Therapy for Inherited Retinal Degeneration.},
journal = {Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics},
volume = {35},
number = {2},
pages = {79-97},
doi = {10.1089/jop.2018.0087},
pmid = {30688548},
issn = {1557-7732},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Genetic Therapy ; Genetic Vectors/chemistry ; Humans ; Photoreceptor Cells, Vertebrate/pathology ; Retinal Degeneration/genetics/pathology/*therapy ; },
abstract = {Inherited retinal degeneration (IRD), a group of rare retinal diseases that primarily lead to the progressive loss of retinal photoreceptor cells, can be inherited in all modes of inheritance: autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), and mitochondrial. Based on the pattern of inheritance of the dystrophy, retinal gene therapy has 2 main strategies. AR, XL, and AD IRDs with haploinsufficiency can be treated by inserting a functional copy of the gene using either viral or nonviral vectors (gene augmentation). Different types of viral vectors and nonviral vectors are used to transfer plasmid DNA both in vitro and in vivo. AD IRDs with gain-of-function mutations or dominant-negative mutations can be treated by disrupting the mutant allele with (and occasionally without) gene augmentation. This review article aims to provide an overview of ocular gene therapy for treating IRDs using gene augmentation with viral or nonviral vectors or gene disruption through different gene-editing tools, especially with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system.},
}
@article {pmid30688002,
year = {2019},
author = {Bi, HL and Xu, J and He, L and Zhang, Y and Li, K and Huang, YP},
title = {CRISPR/Cas9-mediated ebony knockout results in puparium melanism in Spodoptera litura.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1011-1019},
doi = {10.1111/1744-7917.12663},
pmid = {30688002},
issn = {1744-7917},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Female ; Gene Expression ; Genotype ; Melanins/*metabolism ; Phylogeny ; Pigmentation/*genetics ; Pupa/*metabolism ; Spodoptera/*genetics/metabolism ; },
abstract = {Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.},
}
@article {pmid30686786,
year = {2019},
author = {Liu, H and Li, DM and Zhu, LY and Lai, LJ and Yan, WY and Lu, YH and Wei, Y and Huang, YQ and Fang, M and Su, YG and Yang, F and Shu, W},
title = {[Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {1},
pages = {66-75},
doi = {10.16288/j.yczz.18-146},
pmid = {30686786},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; Cell Nucleus ; *Gene Knockout Techniques ; HEK293 Cells ; Hep G2 Cells ; Humans ; Lamin Type A/*genetics ; Mutation ; },
abstract = {The LMNA gene encodes the nuclear Lamin A and Lamin C proteins, and is related to nuclear membrane organization, genome stability and cell differentiation. Abnormal expression of LMNA is ubiquitous in human tumors, and its mutation leads to various forms of laminopathies, including Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), and Hutchinson-Gliford progeria syndrome (HGPS). To further determine the functions of the LMNA gene in cellular physiology, the present study used the CRISPR/Cas9 technique to edit the LMNA gene of 293T and HepG2 cells in vitro, which resulted in two stable LMNA gene knockout (LMNA KO) cell lines. Compared to the respective wild type cells, the LMNA KO cell lines showed decrease in proliferation ability, increase in apoptosis, alteration in cellular morphology and uneven structures in the nucleus membrane. In this study, we report for the first time the results on the construction of LMNA KO immortalized cell lines and characterization of their morphological changes, thereby laying the foundation for the further studies of the LMNA gene functions and pathogenic mutations.},
}
@article {pmid30686760,
year = {2019},
author = {Ronai, I and Griffiths, PE},
title = {The Case for Basic Biological Research.},
journal = {Trends in molecular medicine},
volume = {25},
number = {2},
pages = {65-69},
doi = {10.1016/j.molmed.2018.12.003},
pmid = {30686760},
issn = {1471-499X},
mesh = {Animals ; Biomedical Research ; *CRISPR-Cas Systems ; Gene Editing ; *Genetic Techniques ; Humans ; Polymerase Chain Reaction ; *RNA Interference ; Sequence Analysis, DNA ; },
abstract = {The majority of biomedical and biological research relies on a few molecular biology techniques. Here we show that eight key molecular biology techniques would not exist without basic biological research. We also find that the scientific reward system does not sufficiently value basic biological research into molecular mechanisms.},
}
@article {pmid30686591,
year = {2019},
author = {Mengwasser, KE and Adeyemi, RO and Leng, Y and Choi, MY and Clairmont, C and D'Andrea, AD and Elledge, SJ},
title = {Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets.},
journal = {Molecular cell},
volume = {73},
number = {5},
pages = {885-899.e6},
pmid = {30686591},
issn = {1097-4164},
support = {R01 CA234600/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; BRCA1 Protein/genetics/metabolism ; BRCA2 Protein/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Death ; Cell Line, Tumor ; DNA Damage ; DNA End-Joining Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/*genetics/metabolism ; Endonucleases ; Flap Endonucleases/*genetics/metabolism ; Gene Expression Regulation, Neoplastic ; *Genes, Lethal ; Humans ; Multifunctional Enzymes ; Neoplasms/drug therapy/enzymology/*genetics/pathology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; *RNA Interference ; RNA, Small Interfering/genetics ; *Synthetic Lethal Mutations ; },
abstract = {BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.},
}
@article {pmid30686508,
year = {2019},
author = {Coutton, C and Martinez, G and Kherraf, ZE and Amiri-Yekta, A and Boguenet, M and Saut, A and He, X and Zhang, F and Cristou-Kent, M and Escoffier, J and Bidart, M and Satre, V and Conne, B and Fourati Ben Mustapha, S and Halouani, L and Marrakchi, O and Makni, M and Latrous, H and Kharouf, M and Pernet-Gallay, K and Bonhivers, M and Hennebicq, S and Rives, N and Dulioust, E and Touré, A and Gourabi, H and Cao, Y and Zouari, R and Hosseini, SH and Nef, S and Thierry-Mieg, N and Arnoult, C and Ray, PF},
title = {Bi-allelic Mutations in ARMC2 Lead to Severe Astheno-Teratozoospermia Due to Sperm Flagellum Malformations in Humans and Mice.},
journal = {American journal of human genetics},
volume = {104},
number = {2},
pages = {331-340},
pmid = {30686508},
issn = {1537-6605},
mesh = {*Alleles ; Animals ; Asthenozoospermia/*genetics/*pathology ; CRISPR-Cas Systems ; Cell Cycle Proteins/deficiency ; Cytoskeletal Proteins/*genetics ; Flagella/*genetics ; Humans ; Infertility, Male/genetics/pathology ; Male ; Mice ; Microtubule Proteins/deficiency ; *Mutation ; Proteins ; Spermatozoa/*abnormalities/*pathology ; },
abstract = {Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.},
}
@article {pmid30685869,
year = {2019},
author = {Kc, M and Steer, CJ},
title = {A new era of gene editing for the treatment of human diseases.},
journal = {Swiss medical weekly},
volume = {149},
number = {},
pages = {w20021},
doi = {10.4414/smw.2019.20021},
pmid = {30685869},
issn = {1424-3997},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; },
abstract = {The treatment of human diseases using gene-editing technology has been envisioned for several decades with the realisation that so many were associated with mutations in DNA. The Human Genome Project opened new doors for identifying the genetic bases for human suffering. Research on gene editing has been active since the 1970s, but the technology has seen substantial growth and application just within the past decade. Simply stated, CRISPR technology has become a phenomenon in both biomedical and therapeutics research. Concurrently, cell therapies and pluripotent stem cell research have also been refined and now interfaced with CRISPR technology to enhance and maximise their potential in modelling as well as treatment of human diseases. In this review, we discuss the novel and revolutionary modality of gene editing, as this marks a new era in research and medicine. We also discuss gene-modifying technologies leading to CRISPR, as they are still being used for a wide variety of genomic applications. The modes and challenges for delivery of gene editing components are also discussed. Lastly, we review examples of human diseases that are not only amenable to gene editing techniques, but also show true promise of cure in the early 21st century of genetic correction and gene repair.},
}
@article {pmid30684641,
year = {2019},
author = {Xie, S and Han, S and Qu, Z and Liu, F and Li, J and Yu, S and Reilly, J and Tu, J and Liu, X and Lu, Z and Hu, X and Yimer, TA and Qin, Y and Huang, Y and Lv, Y and Jiang, T and Shu, X and Tang, Z and Jia, H and Wong, F and Liu, M},
title = {Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1865},
number = {6},
pages = {1273-1283},
doi = {10.1016/j.bbadis.2019.01.022},
pmid = {30684641},
issn = {1879-260X},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Differentiation/*genetics ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Microscopy, Electron, Transmission ; *Mutation ; Receptors, Cytoplasmic and Nuclear/*genetics/metabolism ; Retina/embryology/growth &amp; development/ultrastructure ; Retinal Cone Photoreceptor Cells/metabolism/*pathology ; Retinal Degeneration/*genetics/metabolism/pathology ; Retinal Rod Photoreceptor Cells/cytology/*metabolism ; Zebrafish ; Zebrafish Proteins/*genetics/metabolism ; },
abstract = {Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.},
}
@article {pmid30684591,
year = {2019},
author = {Chen, M and Mao, A and Xu, M and Weng, Q and Mao, J and Ji, J},
title = {CRISPR-Cas9 for cancer therapy: Opportunities and challenges.},
journal = {Cancer letters},
volume = {447},
number = {},
pages = {48-55},
doi = {10.1016/j.canlet.2019.01.017},
pmid = {30684591},
issn = {1872-7980},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; Neoplasms/*genetics ; },
abstract = {Cancer is a genetic disease stemming from cumulative genetic/epigenetic aberrations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome editing technology has been extensively applied in various cell types and organisms, both in vitro and in vivo, for efficient gene disruption and gene modification. CRISPR-Cas9 has shown great promise for the treatment of cancer. However, despite its advantages and tremendous potential, numerous challenges, such as fitness of edited cells, editing efficiency, delivery methods and potential off-target effects, remain to be solved for completely clinical application. Here, we present the potential applications and recent advances of CRISPR-Cas9 in cancer therapy, and discuss the challenges that might be encountered in clinical applications.},
}
@article {pmid30684544,
year = {2019},
author = {Rawat, K and Das, S and Vivek Vinod, BS and Vekariya, U and Garg, T and Dasgupta, A and Tripathi, RK},
title = {Targeted depletion of BTF3a in macrophages activates autophagic pathway to eliminate Mycobacterium tuberculosis.},
journal = {Life sciences},
volume = {220},
number = {},
pages = {21-31},
doi = {10.1016/j.lfs.2019.01.035},
pmid = {30684544},
issn = {1879-0631},
mesh = {Autophagosomes/pathology ; Autophagy/drug effects ; Caseins/metabolism ; Humans ; Macrophages/metabolism ; Mycobacterium tuberculosis/*drug effects/metabolism ; Nuclear Proteins/*genetics/*metabolism ; THP-1 Cells/drug effects ; Transcription Factors/*genetics/*metabolism ; },
abstract = {AIMS: β casein fragment peptide (54-59) downregulates Basic Transcription factor 3a (BTF3a) in macrophages and exhibits enhanced clearance of M. bovis BCG and several other intracellular pathogens. However, the direct effect of BTF3a downregulation on Mycobacterium tuberculosis (Mtb) survival and the probable pathways involved have not yet been studied. Therefore, the present study was undertaken to deduce the antimycobacterial significance of BTF3a in human macrophages.<br><br>MAIN METHODS: CRISPR/Cas 9 gRNA was designed to downregulate BTF3a in THP1 derived macrophages. Fold change in BTF3a, p62 and Lamp 1 expression was evaluated through immune blot analysis. CFU assay was done to enumerate the intracellular burden of Mtb H37Rv. LC3B-II turnover and Lamp 1 expression was checked through immunoblotting and also visualized through confocal microscopy. Colocalization of Mtb H37Rv with LC3B, Lysotracker and Rab 7 was visualized through confocal microscopy.<br><br>KEY FINDINGS: The current study identifies BTF3a as a critical host factor assisting intracellular survival of Mtb. In THP1 derived macrophages, infection with Mtb H37Rv resulted in upregulation of BTF3a and targeted depletion of BTF3a resulted in augmented Mtb clearance. Furthermore, BTF3a knockdown demonstrated increased autophagy flux and ameliorated the lysosomal targeting of Mtb containing autophagosomes for lysosomal degradation.<br><br>SIGNIFICANCE: Deep understanding of macrophage-Mtb interactions and their roles in the pathogenesis can offer exciting new therapeutic targets for alternative host-specific adjunct therapies in tuberculosis treatment. The present study highlights a novel and significant role of BTF3a in curbing the intracellular survival of Mtb through modulation of autophagy and lysosome biogenesis.},
}
@article {pmid30684537,
year = {2019},
author = {Lu, F and Gilmour, DS},
title = {Genetic analysis of the RNA polymerase II CTD in Drosophila.},
journal = {Methods (San Diego, Calif.)},
volume = {159-160},
number = {},
pages = {129-137},
pmid = {30684537},
issn = {1095-9130},
support = {R01 GM047477/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Crosses, Genetic ; Drosophila/enzymology/*genetics ; Female ; *Gene Editing ; Male ; *Mutation ; *Protein Domains ; RNA Polymerase II/*genetics/metabolism ; },
abstract = {The Carboxy-terminal Domain (CTD) of RNA polymerase II (Pol II) plays essential roles in regulating gene expression in eukaryotes. Here, we describe multiple genetic approaches for studying the CTD in Drosophila that complement pre-existing molecular analyses of the Pol II CTD in other experimental models. These approaches will allow one to assess the effects of any CTD mutations in a developmentally complex organism. The approaches discussed in this work can in principle, be applied to analyze other transcription components in eukaryotes.},
}
@article {pmid30684509,
year = {2019},
author = {Darwish, M and Nishizono, H and Uosaki, H and Sawada, H and Sadahiro, T and Ieda, M and Takao, K},
title = {Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain.},
journal = {Journal of neuroscience methods},
volume = {317},
number = {},
pages = {149-156},
doi = {10.1016/j.jneumeth.2019.01.010},
pmid = {30684509},
issn = {1872-678X},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems/*genetics ; Cryopreservation/*methods ; Electroporation/*methods ; Embryo, Mammalian/physiology ; Gene Targeting/*methods ; Male ; Mice, Inbred C57BL ; Mutation ; },
abstract = {BACKGROUND: The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging.<br><br>NEW METHOD: We introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1&#x2009;h after thawing.<br><br>RESULTS: Freeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks.<br><br>Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods.<br><br>CONCLUSION: We developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.},
}
@article {pmid30684023,
year = {2019},
author = {Doll, NM and Gilles, LM and Gérentes, MF and Richard, C and Just, J and Fierlej, Y and Borrelli, VMG and Gendrot, G and Ingram, GC and Rogowsky, PM and Widiez, T},
title = {Single and multiple gene knockouts by CRISPR-Cas9 in maize.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {487-501},
pmid = {30684023},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Gene Knockout Techniques/*methods ; Genome, Plant/genetics ; Mutagenesis/genetics ; Zea mays/*genetics ; },
abstract = {The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.},
}
@article {pmid30683899,
year = {2019},
author = {van de Vrugt, HJ and Harmsen, T and Riepsaame, J and Alexantya, G and van Mil, SE and de Vries, Y and Bin Ali, R and Huijbers, IJ and Dorsman, JC and Wolthuis, RMF and Te Riele, H},
title = {Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {768},
pmid = {30683899},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA Repair ; Ear ; Fanconi Anemia/*genetics/*therapy ; Fanconi Anemia Complementation Group F Protein/*genetics ; Fibroblasts ; Gene Editing/*methods ; Genetic Therapy/*methods ; Mice ; Mouse Embryonic Stem Cells ; },
abstract = {Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.},
}
@article {pmid30683865,
year = {2019},
author = {Tan, J and Zhang, F and Karcher, D and Bock, R},
title = {Engineering of high-precision base editors for site-specific single nucleotide replacement.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {439},
pmid = {30683865},
issn = {2041-1723},
mesh = {APOBEC-1 Deaminase/chemistry/*genetics/metabolism ; Amino Acid Transport Systems, Basic/genetics/metabolism ; Base Sequence ; CRISPR-Associated Protein 9/chemistry/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytidine/genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/methods ; Isoenzymes/chemistry/genetics/metabolism ; Mutagenesis, Site-Directed ; RNA, Guide/genetics/metabolism ; Saccharomyces cerevisiae/enzymology/*genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Sensitivity and Specificity ; Thymidine/genetics/metabolism ; },
abstract = {RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.},
}
@article {pmid30683453,
year = {2019},
author = {Ghosh, C and Sarkar, P and Issa, R and Haldar, J},
title = {Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance.},
journal = {Trends in microbiology},
volume = {27},
number = {4},
pages = {323-338},
doi = {10.1016/j.tim.2018.12.010},
pmid = {30683453},
issn = {1878-4380},
mesh = {Animals ; Anti-Bacterial Agents/*therapeutic use ; Antibodies, Monoclonal/therapeutic use ; Antimicrobial Cationic Peptides/therapeutic use ; Bacterial Infections/*therapy ; Bacteriocins/therapeutic use ; Bacteriophages ; Biological Therapy/*methods ; Biotechnology ; CRISPR-Cas Systems ; Disease Models, Animal ; *Drug Resistance, Bacterial/drug effects ; Fecal Microbiota Transplantation ; Genetic Engineering ; Humans ; Microbiota ; Oligonucleotides/therapeutic use ; Phage Therapy/methods ; Probiotics/therapeutic use ; },
abstract = {As more antibiotics are rendered ineffective by drug-resistant bacteria, focus must be shifted towards alternative therapies for treating infections. Although several alternatives already exist in nature, the challenge is to implement them in clinical use. Advancements within biotechnology, genetic engineering, and synthetic chemistry have opened up new avenues towards the search for therapies that can substitute for antibiotics. This review provides an introduction to the various promising approaches that have been adopted in this regard. Whilst the use of bacteriophages and antibodies has been partly implemented, other promising strategies, such as probiotics, lysins, and antimicrobial peptides, are in various stages of development. Propitious concepts such as genetically modified phages, antibacterial oligonucleotides, and CRISPR-Cas9 are also discussed.},
}
@article {pmid30682820,
year = {2019},
author = {Mantravadi, PK and Kalesh, KA and Dobson, RCJ and Hudson, AO and Parthasarathy, A},
title = {The Quest for Novel Antimicrobial Compounds: Emerging Trends in Research, Development, and Technologies.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {8},
number = {1},
pages = {},
pmid = {30682820},
issn = {2079-6382},
support = {R15 GM120653/GM/NIGMS NIH HHS/United States ; R15GM120653/NH/NIH HHS/United States ; },
abstract = {Pathogenic antibiotic resistant bacteria pose one of the most important health challenges of the 21st century. The overuse and abuse of antibiotics coupled with the natural evolutionary processes of bacteria has led to this crisis. Only incremental advances in antibiotic development have occurred over the last 30 years. Novel classes of molecules, such as engineered antibodies, antibiotic enhancers, siderophore conjugates, engineered phages, photo-switchable antibiotics, and genome editing facilitated by the CRISPR/Cas system, are providing new avenues to facilitate the development of antimicrobial therapies. The informatics revolution is transforming research and development efforts to discover novel antibiotics. The explosion of nanotechnology and micro-engineering is driving the invention of antimicrobial materials, enabling the cultivation of "uncultivable" microbes and creating specific and rapid diagnostic technologies. Finally, a revival in the ecological aspects of microbial disease management, the growth of prebiotics, and integrated management based on the "One Health" model, provide additional avenues to manage this health crisis. These, and future scientific and technological developments, must be coupled and aligned with sound policy and public awareness to address the risks posed by rising antibiotic resistance.},
}
@article {pmid30682322,
year = {2019},
author = {Starkuviene, V and Kallenberger, SM and Beil, N and Lisauskas, T and Schumacher, BS and Bulkescher, R and Wajda, P and Gunkel, M and Beneke, J and Erfle, H},
title = {High-Density Cell Arrays for Genome-Scale Phenotypic Screening.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {24},
number = {3},
pages = {274-283},
doi = {10.1177/2472555218818757},
pmid = {30682322},
issn = {2472-5560},
mesh = {CRISPR-Cas Systems ; Cell Line ; Cytological Techniques/*methods ; Endocytosis ; Epidermal Growth Factor/metabolism ; *Genome, Human ; Humans ; Miniaturization ; Phenotype ; RNA Interference ; Robotics ; },
abstract = {Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.},
}
@article {pmid30681833,
year = {2019},
author = {Tian, T and Kang, JW and Kang, A and Lee, TS},
title = {Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {8},
number = {2},
pages = {391-402},
doi = {10.1021/acssynbio.8b00429},
pmid = {30681833},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Metabolic Engineering/methods ; Pentanols/*metabolism ; Promoter Regions, Genetic/genetics ; },
abstract = {CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.},
}
@article {pmid30681273,
year = {2019},
author = {},
title = {Gene Editing Successfully Corrects 2 Amino Acid Disorders: In 2 preclinical studies using CRISPR-mediated gene editing, phenylketonuria and hereditary tyrosinemia type 1 were corrected.},
journal = {American journal of medical genetics. Part A},
volume = {179},
number = {1},
pages = {5-6},
doi = {10.1002/ajmg.a.61026},
pmid = {30681273},
issn = {1552-4833},
mesh = {Amino Acids/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Editing ; Humans ; Hydrolases/*genetics/metabolism ; Mice ; Phenylalanine/genetics/metabolism ; Phenylalanine Hydroxylase/*genetics/metabolism ; Phenylketonurias/genetics/metabolism/*therapy ; Tyrosine/genetics/metabolism ; Tyrosinemias/genetics/*therapy ; },
}
@article {pmid30680434,
year = {2019},
author = {Azam, AH and Tanji, Y},
title = {Bacteriophage-host arm race: an update on the mechanism of phage resistance in bacteria and revenge of the phage with the perspective for phage therapy.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {5},
pages = {2121-2131},
doi = {10.1007/s00253-019-09629-x},
pmid = {30680434},
issn = {1432-0614},
mesh = {Bacteria/genetics/*virology ; Bacterial Proteins/genetics ; Bacteriophages/*growth &amp; development ; CRISPR-Cas Systems/genetics/physiology ; Cell Membrane/physiology ; Phage Therapy/*methods ; RNA Interference/physiology ; Repressor Proteins/genetics ; },
abstract = {Due to a constant attack by phage, bacteria in the environment have evolved diverse mechanisms to defend themselves. Several reviews on phage resistance mechanisms have been published elsewhere. Thanks to the advancement of molecular techniques, several new phage resistance mechanisms were recently identified. For the practical phage therapy, the emergence of phage-resistant bacteria could be an obstacle. However, unlike antibiotic, phages could evolve a mechanism to counter-adapt against phage-resistant bacteria. In this review, we summarized the most recent studies of the phage-bacteria arm race with the perspective of future applications of phages as antimicrobial agents.},
}
@article {pmid30680376,
year = {2019},
author = {Alberti, F and Corre, C},
title = {Editing streptomycete genomes in the CRISPR/Cas9 age.},
journal = {Natural product reports},
volume = {36},
number = {9},
pages = {1237-1248},
doi = {10.1039/c8np00081f},
pmid = {30680376},
issn = {1460-4752},
support = {BB/M017982/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Bacterial/genetics ; Streptomyces/*genetics ; },
abstract = {Covering: up to December 2018 This article aims to highlight advantages, drawbacks and issues that users should consider when implementing the use of CRISPR/Cas9-tools for genome editing in streptomycetes, the most prolific source of antimicrobial natural products to date. Here, we examine four toolkits that have so far been made available for streptomycete in vivo-engineering and one for in vitro-editing, and review how they have been applied over the last three years. Our critical evaluation of these toolkits intends to support potential users in determining what they could achieve, what they should consider and what system they should select/optimise for their application.},
}
@article {pmid30679799,
year = {2019},
author = {Schmidt, L and Heyes, E and Scheiblecker, L and Eder, T and Volpe, G and Frampton, J and Nerlov, C and Valent, P and Grembecka, J and Grebien, F},
title = {CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex.},
journal = {Leukemia},
volume = {33},
number = {7},
pages = {1608-1619},
pmid = {30679799},
issn = {1476-5551},
support = {R01 CA160467/CA/NCI NIH HHS/United States ; F 4704/FWF_/Austrian Science Fund FWF/Austria ; 13008/LLR_/Blood Cancer UK/United Kingdom ; G0701761/MRC_/Medical Research Council/United Kingdom ; 636855/ERC_/European Research Council/International ; MC_UU_12009/7/MRC_/Medical Research Council/United Kingdom ; MC_UU_00016/7/MRC_/Medical Research Council/United Kingdom ; G0900892/MRC_/Medical Research Council/United Kingdom ; },
mesh = {CCAAT-Enhancer-Binding Protein-alpha/*genetics ; *CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; GATA2 Transcription Factor ; Hematopoiesis ; Histone-Lysine N-Methyltransferase/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Leukemia, Myeloid, Acute/drug therapy/genetics/*pathology ; *Mutation ; Myeloid-Lymphoid Leukemia Protein/*antagonists &amp; inhibitors/genetics/metabolism ; Protein Interaction Domains and Motifs/*drug effects ; Proto-Oncogene Proteins/metabolism ; Small Molecule Libraries/*pharmacology ; Tumor Cells, Cultured ; },
abstract = {The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.},
}
@article {pmid30679690,
year = {2019},
author = {Bricogne, C and Fine, M and Pereira, PM and Sung, J and Tijani, M and Wang, Y and Henriques, R and Collins, MK and Hilgemann, DW},
title = {TMEM16F activation by Ca[2+] triggers plasma membrane expansion and directs PD-1 trafficking.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {619},
pmid = {30679690},
issn = {2045-2322},
support = {T32 DK007257/DK/NIDDK NIH HHS/United States ; BB/M022374/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; R01 HL067942/HL/NHLBI NIH HHS/United States ; MR/K015826/1/MRC_/Medical Research Council/United Kingdom ; 1132770/MRC_/Medical Research Council/United Kingdom ; R01 HL119843/HL/NHLBI NIH HHS/United States ; },
mesh = {Anoctamins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Calcium/*metabolism ; Cell Line ; Cell Membrane/*metabolism ; Flow Cytometry ; Humans ; Jurkat Cells ; Lentivirus/genetics ; Microscopy, Confocal ; Phospholipid Transfer Proteins/genetics/*metabolism ; Programmed Cell Death 1 Receptor/genetics/*metabolism ; },
abstract = {TMEM16F is a Ca[2+] -gated ion channel that is required for Ca[2+] -activated phosphatidylserine exposure on the surface of many eukaryotic cells. TMEM16F is widely expressed and has roles in platelet activation during blood clotting, bone formation and T cell activation. By combining microscopy and patch clamp recording we demonstrate that activation of TMEM16F by Ca[2+] ionophores in Jurkat T cells triggers large-scale surface membrane expansion in parallel with phospholipid scrambling. With continued ionophore application,TMEM16F-expressing cells then undergo extensive shedding of ectosomes. The T cell co-receptor PD-1 is selectively incorporated into ectosomes. This selectivity depends on its transmembrane sequence. Surprisingly, cells lacking TMEM16F not only fail to expand surface membrane in response to elevated cytoplasmic Ca[2+], but instead undergo rapid massive endocytosis with PD-1 internalisation. These results establish a new role for TMEM16F as a regulator of Ca[2+] activated membrane trafficking.},
}
@article {pmid30679624,
year = {2019},
author = {Adams, S and Pathak, P and Shao, H and Lok, JB and Pires-daSilva, A},
title = {Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {483},
pmid = {30679624},
issn = {2045-2322},
support = {BB/L019884/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/International ; R21 AI105856/AI/NIAID NIH HHS/United States ; R33 AI105856/AI/NIAID NIH HHS/United States ; OD P40-10939//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; R01 AI050668/AI/NIAID NIH HHS/United States ; AI50688//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; *Liposomes ; *Mutagenesis ; Nematoda/genetics ; *RNA Interference ; RNA, Double-Stranded/genetics ; *Transfection ; },
abstract = {Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.},
}
@article {pmid30679547,
year = {2019},
author = {Shimada, R and Kiso, M and Saga, Y},
title = {ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {515},
pmid = {30679547},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Chimera/genetics/metabolism ; DEAD-box RNA Helicases/analysis/genetics/*metabolism ; Embryonic Stem Cells/cytology/metabolism ; Female ; Gene Deletion ; Gene Expression Regulation, Developmental ; Germ Cells/cytology/*metabolism ; Male ; Mice ; Proto-Oncogene Proteins/analysis/genetics/*metabolism ; RNA-Binding Proteins/analysis/genetics/*metabolism ; Spermatogenesis ; Transcriptome ; },
abstract = {In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen to chimeric embryos containing germ cells derived from recombinant ES cells. DDX6-null germ cells exhibited both similar and distinct defects from those observed in NANOS2-null germ cells. These results demonstrate that NANOS2 function is carried out via both P-body-dependent and -independent mechanisms. RNA-seq analyses further supported the phenotypic differences between DDX6-null and NANOS2-null germ cells, and indicated distinct molecular cascades involved in NANOS2-mediated gene regulation.},
}
@article {pmid30679374,
year = {2019},
author = {Hyun, Y and Vincent, C and Tilmes, V and Bergonzi, S and Kiefer, C and Richter, R and Martinez-Gallegos, R and Severing, E and Coupland, G},
title = {A regulatory circuit conferring varied flowering response to cold in annual and perennial plants.},
journal = {Science (New York, N.Y.)},
volume = {363},
number = {6425},
pages = {409-412},
doi = {10.1126/science.aau8197},
pmid = {30679374},
issn = {1095-9203},
support = {//European Research Council/International ; },
mesh = {Arabidopsis/genetics/physiology ; Arabidopsis Proteins/genetics ; Arabis/genetics/*physiology ; CRISPR-Cas Systems ; *Cold Temperature ; Flowers/*physiology ; Gene Expression Regulation, Plant ; Gene Regulatory Networks ; Meristem/physiology ; MicroRNAs/genetics ; Mutation ; Photoperiod ; Plant Proteins/*genetics ; Signal Transduction ; Transcription Factors/genetics ; },
abstract = {The reproductive strategies of plants are highly variable. Short-lived annuals flower abundantly soon after germination, whereas longer-lived perennials postpone and spatially restrict flowering. We used CRISPR/Cas9 and interspecies gene transfer to understand divergence in reproductive patterns between annual and perennial crucifers. We show that in perennial Arabis alpina, flowering in response to winter cold depends on the floral integrator SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15), whose activity is limited to older shoots and branches during cold exposure. In annuals, this regulatory system is conserved, but cold-induced flowering occurs in young shoots, without requirement for SPL15, through the photoperiodic pathway when plants return to warm. By reconstructing the annual response in perennials, we conclude that characteristic patterns of reproduction in annuals and perennials are conferred through variation in dependency on distinct flowering pathways acting in parallel.},
}
@article {pmid30679258,
year = {2019},
author = {Lemay, ML and Otto, A and Maaß, S and Plate, K and Becher, D and Moineau, S},
title = {Investigating Lactococcus lactis MG1363 Response to Phage p2 Infection at the Proteome Level.},
journal = {Molecular &amp; cellular proteomics : MCP},
volume = {18},
number = {4},
pages = {704-714},
pmid = {30679258},
issn = {1535-9484},
mesh = {Bacterial Proteins/*metabolism ; Bacteriophage P2/*physiology ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genes, Bacterial ; Lactococcus lactis/genetics/growth &amp; development/*virology ; Proteome/*metabolism ; Viral Proteins/metabolism ; },
abstract = {Phages are viruses that specifically infect and eventually kill their bacterial hosts. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated as antibacterial agents for the treatment or prevention of bacterial infections in various sectors. They also play key ecological roles in all ecosystems. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 because of a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (pORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of pORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages' takeover of bacterial cells and provides novel information on phage-host interactions.},
}
@article {pmid30678704,
year = {2019},
author = {Zhu, S and Cao, Z and Liu, Z and He, Y and Wang, Y and Yuan, P and Li, W and Tian, F and Bao, Y and Wei, W},
title = {Guide RNAs with embedded barcodes boost CRISPR-pooled screens.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {20},
pmid = {30678704},
issn = {1474-760X},
mesh = {Bacterial Proteins ; Bacterial Toxins ; *CRISPR-Cas Systems ; Gene Targeting ; Genomics/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; *RNA, Guide ; },
abstract = {We report a new method using re-designed guide RNAs with internal barcodes (iBARs) embedded in their loop regions. Our iBAR approach outperforms the conventional method by producing screening results with much lower false-positive and false-negative rates especially with a high multiplicity of infection (MOI). Importantly, the iBAR approach reduces the starting cells at high MOI significantly with greatly improved efficiency and accuracy compared with the canonical CRISPR screens at a low MOI. This new system is particularly useful when the source of cells is limited or when it is difficult to control viral infection for in vivo screening.},
}
@article {pmid30675057,
year = {2019},
author = {Grunwald, HA and Gantz, VM and Poplawski, G and Xu, XS and Bier, E and Cooper, KL},
title = {Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.},
journal = {Nature},
volume = {566},
number = {7742},
pages = {105-109},
pmid = {30675057},
issn = {1476-4687},
support = {DP5 OD023098/OD/NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; R21 GM129448/GM/NIGMS NIH HHS/United States ; T32 GM007240/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; Breeding ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Chromosomes, Mammalian/genetics ; DNA Breaks, Double-Stranded ; Disease Models, Animal ; Embryo, Mammalian/enzymology/metabolism ; Female ; *Gene Conversion ; Gene Drive Technology/*methods ; Germ-Line Mutation/*genetics ; *Heterozygote ; *Homozygote ; Integrases/genetics/metabolism ; Male ; Mice ; Mice, Transgenic ; Monophenol Monooxygenase/genetics ; RNA, Guide/genetics ; Transgenes/genetics ; },
abstract = {A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity[1-4]. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.},
}
@article {pmid30675053,
year = {2019},
author = {Fessenden, M},
title = {Technologies to watch in 2019.},
journal = {Nature},
volume = {565},
number = {7740},
pages = {521-523},
doi = {10.1038/d41586-019-00218-6},
pmid = {30675053},
issn = {1476-4687},
mesh = {Animals ; Brain Mapping/*trends ; CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy/*trends ; Deep Learning/*trends ; Gene Editing/*trends ; Genomics/*trends ; Humans ; *Neural Pathways ; Single-Cell Analysis/*trends ; Synthetic Biology/*trends ; },
}
@article {pmid30674877,
year = {2019},
author = {Spence, EF and Dube, S and Uezu, A and Locke, M and Soderblom, EJ and Soderling, SH},
title = {In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {386},
pmid = {30674877},
issn = {2041-1723},
support = {MH103374/NH/NIH HHS/United States ; NS059957/NH/NIH HHS/United States ; F31NS092402/NH/NIH HHS/United States ; },
mesh = {Actin Capping Proteins/genetics/metabolism ; Animals ; Biotin ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytoskeletal Proteins/metabolism ; Cytoskeleton/*metabolism ; Dendritic Spines/metabolism ; Excitatory Postsynaptic Potentials/*physiology ; GTPase-Activating Proteins ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Mice, Inbred C57BL ; Microfilament Proteins/genetics/metabolism ; Microtubules/metabolism ; Neurogenesis/genetics/physiology ; Neurons/metabolism ; Proteome/genetics/*metabolism ; *Proteomics ; Synapses/genetics/*metabolism ; },
abstract = {Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynapse. However, the molecular mechanisms that underlie excitatory synaptogenesis are only partially resolved, in part because the internal machinery of developing synapses is largely unknown. To address this, we apply a chemicogenetic approach, in vivo biotin identification (iBioID), to discover aspects of the proteome of nascent synapses. This approach uncovered sixty proteins, including a previously uncharacterized protein, CARMIL3, which interacts in vivo with the synaptic cytoskeletal regulator proteins SrGAP3 (or WRP) and actin capping protein. Using new CRISPR-based approaches, we validate that endogenous CARMIL3 is localized to developing synapses where it facilitates the recruitment of capping protein and is required for spine structural maturation and AMPAR recruitment associated with synapse unsilencing. Together these proteomic and functional studies reveal a previously unknown mechanism important for excitatory synapse development in the developing perinatal brain.},
}
@article {pmid30674557,
year = {2019},
author = {Allen, F and Behan, F and Khodak, A and Iorio, F and Yusa, K and Garnett, M and Parts, L},
title = {JACKS: joint analysis of CRISPR/Cas9 knockout screens.},
journal = {Genome research},
volume = {29},
number = {3},
pages = {464-471},
pmid = {30674557},
issn = {1549-5469},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Bayes Theorem ; *CRISPR-Cas Systems ; Gene Knockout Techniques/*methods ; *Software ; },
abstract = {Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. However, their range of applications remains limited by signal variability from different guide RNAs that target the same gene, which confounds gene effect estimation and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyzes screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification over processing a single screen at a time and outperforms existing methods. This more efficient analysis gives additional hits and allows designing libraries with a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.},
}
@article {pmid30673820,
year = {2019},
author = {Kostyushev, D and Brezgin, S and Kostyusheva, A and Zarifyan, D and Goptar, I and Chulanov, V},
title = {Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {9},
pages = {1779-1794},
pmid = {30673820},
issn = {1420-9071},
mesh = {Antiviral Agents/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Methylation/genetics ; DNA, Circular/*metabolism ; DNA, Viral/*metabolism ; Hep G2 Cells ; Hepatitis B/genetics/*therapy ; Hepatitis B virus/*genetics/*growth &amp; development ; Humans ; RNA, Guide/genetics ; Streptococcus pyogenes/enzymology ; Streptococcus thermophilus/enzymology ; Virus Replication/genetics ; },
abstract = {Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.},
}
@article {pmid30673818,
year = {2019},
author = {Huang, TK and Puchta, H},
title = {CRISPR/Cas-mediated gene targeting in plants: finally a turn for the better for homologous recombination.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {443-453},
pmid = {30673818},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Breaks, Double-Stranded ; Gene Targeting/*methods ; Genome, Plant/genetics ; Homologous Recombination/*genetics ; },
abstract = {We summarize recent progress of CRISPR/Cas9-mediated gene targeting in plants, provide recommendations for designing gene-targeting vectors and highlight the potential of new technologies applicable to plants. Gene targeting (GT) is a tool of urgent need for plant biotechnology and breeding. It is based on homologous recombination that is able to precisely introduce desired modifications within a target locus. However, its low efficiency in higher plants is a major barrier for its application. Using site-specific nucleases, such as the recent CRISPR/Cas system, GT has become applicable in plants, via the induction of double-strand breaks, although still at a too low efficiency for most practical applications in crops. Recently, a variety of promising new improvements regarding the efficiency of GT has been reported by several groups. It turns out that GT can be enhanced by cell-type-specific expression of Cas nucleases, by the use of self-amplified GT-vector DNA or by manipulation of DNA repair pathways. Here, we highlight the most recent progress of GT in plants. Moreover, we provide suggestions on how to use the technology efficiently, based on the mechanisms of DNA repair, and highlight several of the newest GT strategies in yeast or mammals that are potentially applicable to plants. Using the full potential of GT technology will definitely help us pave the way in enhancing crop yields and food safety for an ecologically friendly agriculture.},
}
@article {pmid30673632,
year = {2019},
author = {van Beljouw, SPB and van der Els, S and Martens, KJA and Kleerebezem, M and Bron, PA and Hohlbein, J},
title = {Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis.},
journal = {Physical biology},
volume = {16},
number = {3},
pages = {035001},
doi = {10.1088/1478-3975/ab0162},
pmid = {30673632},
issn = {1478-3975},
mesh = {CRISPR-Cas Systems ; Lactococcus lactis/*isolation &amp; purification ; Luminescent Proteins/*analysis ; Microscopy, Fluorescence ; Photochemical Processes ; },
abstract = {Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.},
}
@article {pmid30672298,
year = {2019},
author = {Qiang, J and Ma, Z and Xie, X and Shi, L and Geng, Y and Hu, J and Liu, R and Liu, N and Zhang, Y},
title = {Multi-omic Analyses Reveal Minimal Impact of the CRISPR-Cas9 Nuclease on Cultured Human Cells.},
journal = {Journal of proteome research},
volume = {18},
number = {3},
pages = {1054-1063},
doi = {10.1021/acs.jproteome.8b00751},
pmid = {30672298},
issn = {1535-3907},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/genetics ; Gene Editing/methods ; Gene Expression Regulation, Enzymologic/genetics ; Histone Code/genetics ; Humans ; Protein Interaction Maps/genetics ; Proteome/*genetics ; Transcriptome/*genetics ; },
abstract = {The CRISPR-Cas9 system is a genomic editing tool widely used in basic research and under investigation for potential applications in gene therapies for human diseases. To accomplish genomic editing, the system requires the expression of a prokaryotic DNA endonuclease enzyme, Cas9, in host cells. Previous studies have mainly focused on the specificity of Cas9 on the host genome, and thus it is unclear whether this bacterium-derived enzyme affects the protein homeostasis of host cells. Here we applied multi-omic analyses, including transcriptome, proteome, phosphoproteome, Cas9-associated protein interactome, protein synthesis, and histone epigenetic modification, to investigate the cellular response of human cells upon the expression of Cas9. We demonstrate that Cas9 has minimal impact on host cells. Our assessment of intracellular effects of Cas9 paves a path for its broad applications in biological studies and potential clinical translations.},
}
@article {pmid30670765,
year = {2019},
author = {Jin, YH and Joo, H and Lee, K and Kim, H and Didier, R and Yang, Y and Shin, H and Lee, C},
title = {Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {277},
pmid = {30670765},
issn = {2045-2322},
support = {R21 NS099813/NS/NINDS NIH HHS/United States ; },
mesh = {Adenoviridae/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Circadian Clocks/genetics ; Gene Editing/methods ; Gene Knockout Techniques/*methods ; Transgenes ; },
abstract = {CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.},
}
@article {pmid30670702,
year = {2019},
author = {Strecker, J and Jones, S and Koopal, B and Schmid-Burgk, J and Zetsche, B and Gao, L and Makarova, KS and Koonin, EV and Zhang, F},
title = {Engineering of CRISPR-Cas12b for human genome editing.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {212},
pmid = {30670702},
issn = {2041-1723},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; },
mesh = {Bacillus/genetics/*metabolism ; Bacterial Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Cell Line ; Deoxyribonucleases/genetics/*metabolism ; *Gene Editing ; *Genome, Human ; Humans ; Models, Molecular ; Mutation ; Protein Conformation ; T-Lymphocytes ; },
abstract = {The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.},
}
@article {pmid30669982,
year = {2019},
author = {Li, R and Liu, C and Zhao, R and Wang, L and Chen, L and Yu, W and Zhang, S and Sheng, J and Shen, L},
title = {CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {38},
pmid = {30669982},
issn = {1471-2229},
mesh = {Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; *Droughts ; *Genes, Plant ; Lycopersicon esculentum/*genetics/physiology ; Mutagenesis ; Stress, Physiological/genetics ; },
abstract = {BACKGROUND: NPR1, nonexpressor of pathogenesis-related gene 1, is a master regulator involved in plant defense response to pathogens, and its regulatory mechanism in the defense pathway has been relatively clear. However, information about the function of NPR1 in plant response to abiotic stress is still limited. Tomato is the fourth most economically crop worldwide and also one of the best-characterized model plants employed in genetic studies. Because of the lack of a stable tomato NPR1 (SlNPR1) mutant, little is known about the function of SlNPR1 in tomato response to biotic and abiotic stresses.<br><br>RESULTS: Here we isolated SlNPR1 from tomato 'Ailsa Craig' and generated slnpr1 mutants using the CRISPR/Cas9 system. Analysis of the cis-acting elements indicated that SlNPR1 might be involved in tomato plant response to drought stress. Expression pattern analysis showed that SlNPR1 was expressed in all plant tissues, and it was strongly induced by drought stress. Thus, we investigated the function of SlNPR1 in tomato-plant drought tolerance. Results showed that slnpr1 mutants exhibited reduced drought tolerance with increased stomatal aperture, higher electrolytic leakage, malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels, and lower activity levels of antioxidant enzymes, compared to wild type (WT) plants. The reduced drought tolerance of slnpr1 mutants was further reflected by the down-regulated expression of drought related key genes, including SlGST, SlDHN, and SlDREB.<br><br>CONCLUSIONS: Collectively, the data suggest that SlNPR1 is involved in regulating tomato plant drought response. These results aid in further understanding the molecular basis underlying SlNPR1 mediation of tomato drought sensitivity.},
}
@article {pmid30669676,
year = {2019},
author = {Lin, CL and Tsai, ML and Lin, CY and Hsu, KW and Hsieh, WS and Chi, WM and Huang, LC and Lee, CH},
title = {HDAC1 and HDAC2 Double Knockout Triggers Cell Apoptosis in Advanced Thyroid Cancer.},
journal = {International journal of molecular sciences},
volume = {20},
number = {2},
pages = {},
pmid = {30669676},
issn = {1422-0067},
mesh = {Acetylation ; Apoptosis/drug effects/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Histone Deacetylase 1/*genetics ; Histone Deacetylase 2/*genetics ; Histone Deacetylase Inhibitors/pharmacology ; Histones/metabolism ; Humans ; Neoplasm Metastasis ; Neoplasm Staging ; Thyroid Neoplasms/drug therapy/*genetics/metabolism/*pathology ; },
abstract = {Anaplastic thyroid carcinoma (ATC) and squamous thyroid carcinoma (STC) are both rare and advanced thyroid malignancies with a very poor prognosis and an average median survival time of 5 months and less than 20% of affected patients are alive 1 year after diagnosis. The clinical management of both ATC and STC is very similar because they are not particularly responsive to radiotherapy and chemotherapy. This inspired us to explore a novel and effective clinically approved therapy for ATC treatment. Histone deacetylase inhibitor (HDACi) drugs are recently FDA-approved drug for malignancies, especially for blood cell cancers. Therefore, we investigated whether an HDACi drug acts as an effective anticancer drug for advanced thyroid cancers. Cell viability analysis of panobinostat treatment demonstrated a significant IC50 of 0.075 µM on SW579 STC cells. In addition, panobinostat exposure activated histone acetylation and triggered cell death mainly through cell cycle arrest and apoptosis-related protein activation. Using CRISPR/Cas9 to knock out HDAC1 and HDAC2 genes in SW579 cells, we observed that the histone acetylation level and cell cycle arrest were enhanced without any impact on cell growth. Furthermore, HDAC1 and HDAC2 double knockout (KO) cells showed dramatic cell apoptosis activation compared to HDAC1 and HDAC2 individual KO cells. This suggests expressional and biofunctional compensation between HDAC1 and HDAC2 on SW579 cells. This study provides strong evidence that panobinostat can potentially be used in the clinic of advanced thyroid cancer patients.},
}
@article {pmid30669572,
year = {2019},
author = {Escobar-Aguirre, S and Arancibia, D and Escorza, A and Bravo, C and Andrés, ME and Zamorano, P and Martínez, V},
title = {Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines.},
journal = {Cells},
volume = {8},
number = {1},
pages = {},
pmid = {30669572},
issn = {2073-4409},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Fishes/*genetics ; Genetic Vectors/*metabolism ; Genome ; HEK293 Cells ; Humans ; Mutation/genetics ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Ribonuclease III/metabolism ; Zebrafish ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function.},
}
@article {pmid30669298,
year = {2019},
author = {Veillet, F and Perrot, L and Chauvin, L and Kermarrec, MP and Guyon-Debast, A and Chauvin, JE and Nogué, F and Mazier, M},
title = {Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor.},
journal = {International journal of molecular sciences},
volume = {20},
number = {2},
pages = {},
pmid = {30669298},
issn = {1422-0067},
mesh = {Agrobacterium/*physiology ; *CRISPR-Cas Systems ; Cytidine/*genetics ; *Gene Editing ; Gene Targeting ; *Gene Transfer Techniques ; Genes, Plant ; Genotyping Techniques ; Lycopersicon esculentum/*genetics ; Sequence Analysis, DNA ; Solanum tuberosum/*genetics ; Transformation, Genetic ; },
abstract = {Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.},
}
@article {pmid30669273,
year = {2019},
author = {Grandvaux, N and McCormick, C},
title = {CSV2018: The 2nd Symposium of the Canadian Society for Virology.},
journal = {Viruses},
volume = {11},
number = {1},
pages = {},
pmid = {30669273},
issn = {1999-4915},
support = {CIHR-MPL-158413//CIHR/Canada ; Conference Grant//Dalhousie Medical Research Foundation/International ; },
mesh = {Awards and Prizes ; Canada ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Congresses as Topic ; Influenza Vaccines ; Respiratory Syncytial Viruses ; *Virology ; },
abstract = {The 2nd Symposium of the Canadian Society for Virology (CSV2018) was held in June 2018 in Halifax, Nova Scotia, Canada, as a featured event marking the 200th anniversary of Dalhousie University. CSV2018 attracted 175 attendees from across Canada and around the world, more than double the number that attended the first CSV symposium two years earlier. CSV2018 provided a forum to discuss a wide range of topics in virology including human, veterinary, plant, and microbial pathogens. Invited keynote speakers included David Kelvin (Dalhousie University and Shantou University Medical College) who provided a historical perspective on influenza on the 100th anniversary of the 1918 pandemic; Sylvain Moineau (Université Laval) who described CRISPR-Cas systems and anti-CRISPR proteins in warfare between bacteriophages and their host microbes; and Kate O'Brien (then from Johns Hopkins University, now relocated to the World Health Organization where she is Director of Immunization, Vaccines and Biologicals), who discussed the underlying viral etiology for pneumonia in the developing world, and the evidence for respiratory syncytial virus (RSV) as a primary cause. Reflecting a strong commitment of Canadian virologists to science communication, CSV2018 featured the launch of Halifax's first annual Soapbox Science event to enable public engagement with female scientists, and the live-taping of the 499th episode of the This Week in Virology (TWIV) podcast, hosted by Vincent Racaniello (Columbia University) and science writer Alan Dove. TWIV featured interviews of CSV co-founders Nathalie Grandvaux (Université de Montréal) and Craig McCormick (Dalhousie University), who discussed the origins and objectives of the new society; Ryan Noyce (University of Alberta), who discussed technical and ethical considerations of synthetic virology; and Kate O'Brien, who discussed vaccines and global health. Finally, because CSV seeks to provide a better future for the next generation of Canadian virologists, the symposium featured a large number of oral and poster presentations from trainees and closed with the awarding of presentation prizes to trainees, followed by a tour of the Halifax Citadel National Historic Site and an evening of entertainment at the historic Alexander Keith's Brewery.},
}
@article {pmid30668910,
year = {2019},
author = {Koopal, B and Kruis, AJ and Claassens, NJ and Nobrega, FL and van der Oost, J},
title = {Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing.},
journal = {ACS synthetic biology},
volume = {8},
number = {2},
pages = {216-222},
doi = {10.1021/acssynbio.8b00347},
pmid = {30668910},
issn = {2161-5063},
mesh = {Amino Acids/*genetics ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Silencing/*physiology ; Promoter Regions, Genetic/genetics ; Transcription, Genetic/genetics ; },
abstract = {The CRISPR-Cas9 nuclease has been repurposed as a tool for gene repression (CRISPRi). This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-mediated repression has been achieved with inducible promoters that regulate the expression of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly leaky dcas9 expression leads to significant background levels of repression of the target gene. In this study, we report on the development of optimized control of dCas9-mediated silencing through additional regulation at the translation level. We have introduced the TAG stop codon in the dcas9 gene in order to insert a synthetic amino acid, l-biphenylalanine (BipA), at a permissive site in the dCas9 protein. In the absence of BipA, a nonfunctional, truncated dCas9 is produced, but when BipA is present, the TAG codon is translated resulting in a functional, full-length dCas9 protein. This synthetic, BipA-containing dCas9 variant (dCas9-BipA) could still fully repress gene transcription. Comparison of silencing mediated by dCas9 to dCas9-BipA revealed a 14-fold reduction in background repression by the latter system. The here developed proof-of-principle system thus reduces unwanted background levels of gene silencing, allowing for tight and timed control of target gene expression.},
}
@article {pmid30668679,
year = {2019},
author = {Stellos, K and Musunuru, K},
title = {Challenges and advances of CRISPR-Cas9 genome editing in therapeutics.},
journal = {Cardiovascular research},
volume = {115},
number = {2},
pages = {e12-e14},
doi = {10.1093/cvr/cvy300},
pmid = {30668679},
issn = {1755-3245},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Mutation ; },
}
@article {pmid30668559,
year = {2019},
author = {Zhu, YN and Wang, LZ and Li, CC and Cui, Y and Wang, M and Lin, YJ and Zhao, RP and Wang, W and Xiang, H},
title = {Artificial selection on storage protein 1 possibly contributes to increase of hatchability during silkworm domestication.},
journal = {PLoS genetics},
volume = {15},
number = {1},
pages = {e1007616},
pmid = {30668559},
issn = {1553-7404},
mesh = {Animals ; Bombyx/genetics/growth &amp; development ; CRISPR-Cas Systems ; Domestication ; *Evolution, Molecular ; Extracellular Matrix/genetics ; Gene Knockout Techniques ; Genome, Insect/genetics ; Insect Proteins/*genetics ; Linkage Disequilibrium ; Metamorphosis, Biological/*genetics ; Phylogeny ; *Selection, Genetic ; Transcriptome/genetics ; },
abstract = {Like other domesticates, the efficient utilization of nitrogen resources is also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources and to advance human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's evolutionary theory on domestication. Storage proteins (SPs), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. In this study, through blast searching on the silkworm genome and further screening of the artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e., the methionine-rich storage protein 1 (SP1), which is clearly divergent from other storage proteins and exhibits increased expression in the ova of domestic silkworms. Knockout of SP1 via the CRISPR/Cas9 technique resulted in a dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the effect in the wild silkworm compared with the domestic type. Larval development and metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated significant higher expression of genes encoding vitellogenin, chorions, and structural components in the extracellular matrix (ECM)-interaction pathway, enzymes in folate biosynthesis, and notably hormone synthesis in the domestic silkworm, compared to both the SP1 mutant and the wild silkworm. Moreover, compared with the wild silkworms, the domestic one also showed generally up-regulated expression of genes enriched in the structural constituent of ribosome and amide, as well as peptide biosynthesis. This study exemplified a novel case in which artificial selection could act directly on nitrogen resource proteins, further affecting egg nutrients and eggshell formation possibly through a hormone signaling mediated regulatory network and the activation of ribosomes, resulting in improved biosynthesis and increased hatchability during domestication. These findings shed new light on both the understanding of artificial selection and silkworm breeding from the perspective of nitrogen and amino acid resources.},
}
@article {pmid30667081,
year = {2019},
author = {Tang, F and Min, L and Seebacher, NA and Li, X and Zhou, Y and Hornicek, FJ and Wei, Y and Tu, C and Duan, Z},
title = {Targeting mutant TP53 as a potential therapeutic strategy for the treatment of osteosarcoma.},
journal = {Journal of orthopaedic research : official publication of the Orthopaedic Research Society},
volume = {37},
number = {3},
pages = {789-798},
doi = {10.1002/jor.24227},
pmid = {30667081},
issn = {1554-527X},
support = {U01 CA151452/CA/NCI NIH HHS/United States ; },
mesh = {Antibiotics, Antineoplastic/therapeutic use ; Bone Neoplasms/*genetics/therapy ; CRISPR-Cas Systems ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Doxorubicin/therapeutic use ; Drug Resistance, Neoplasm ; Drug Synergism ; *Genes, p53 ; *Genetic Therapy ; Humans ; Mutation ; Nitrofurans/pharmacology/*therapeutic use ; Osteosarcoma/*genetics/therapy ; Piperazines/pharmacology/*therapeutic use ; Tumor Suppressor Protein p53/antagonists &amp; inhibitors/genetics ; },
abstract = {Mutant TP53 is a promising therapeutic target in cancers. Considering the current challenges facing the clinical treatment of cancer, as well as the urgent need to identify novel therapeutic targets in osteosarcomas, we aimed to evaluate the clinical significance of mutant TP53 in osteosarcoma patients and to explore the therapeutic effect of targeting mutant TP53 in osteosarcomas. We performed a meta-analysis to investigate the relationship between mutant TP53 and the overall survival of patients with osteosarcoma. A CRISPR-Cas9 system and a TP53 inhibitor, NSC59984, were also used to specifically knock-out and inhibit mutant TP53 in the human osteosarcoma cell lines, KHOS, and KHOSR2. The meta-analysis demonstrated that mutations in the TP53 gene could be used to predict a poor 2-year survival in osteosarcoma patients. We also demonstrated that the expression of mutant TP53 in human osteosarcoma cell lines can be efficiently knocked-out using CRISPR-Cas9, and this decreased the proliferation, migration, and tumor formation activity of these osteosarcoma cells. Moreover, drug sensitivity to doxorubicin was increased in these TP53 knock-out osteosarcoma cells. NSC59984 also showed similar anti-tumor effects as CRISPR-Cas9 targeted TP53 in the osteosarcoma cells in vitro. We have also demonstrated that the knock-out or inhibition of mutant TP53 decreased the expression of the oncogene IGF-1R, anti-apoptotic proteins Bcl-2, and Survivin in osteosarcoma cells. Collectively, these results suggest that mutant TP53 is a promising therapeutic target in osteosarcomas. Therefore, further studies exploring novel strategies to target mutant TP53 may help improve the treatment outcomes of osteosarcoma patients in the clinic. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.},
}
@article {pmid30666960,
year = {2019},
author = {Champer, J and Chung, J and Lee, YL and Liu, C and Yang, E and Wen, Z and Clark, AG and Messer, PW},
title = {Molecular safeguarding of CRISPR gene drive experiments.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30666960},
issn = {2050-084X},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R21AI130635/NH/NIH HHS/United States ; F32AI138476/NH/NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drosophila melanogaster/*genetics ; Gene Drive Technology/*methods ; Gene Editing/methods ; Genetics, Population ; Genotype ; Germ Cells ; Phenotype ; RNA, Guide/*genetics/metabolism ; },
abstract = {CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.},
}
@article {pmid30666557,
year = {2020},
author = {Saber, A and Liu, B and Ebrahimi, P and Haisma, HJ},
title = {CRISPR/Cas9 for overcoming drug resistance in solid tumors.},
journal = {Daru : journal of Faculty of Pharmacy, Tehran University of Medical Sciences},
volume = {28},
number = {1},
pages = {295-304},
pmid = {30666557},
issn = {2008-2231},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Drug Resistance, Neoplasm/*genetics ; Humans ; Neoplasms/drug therapy/*genetics ; },
abstract = {OBJECTIVES: In this review, we focus on the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome editing system, in the identification of resistance mechanisms and in overcoming drug resistance in the most frequent solid tumors.<br><br>DATA ACQUISITION: Data were collected by conducting systematic searching of scientific English literature using specific keywords such as "cancer", "CRISPR" and related combinations.<br><br>RESULTS: The review findings revealed the importance of CRISPR/Cas9 system in understanding drug resistance mechanisms and identification of resistance-related genes such as PBRM1, SLFN11 and ATPE1 in different cancers. We also provided an overview of genes, including RSF1, CDK5, and SGOL1, whose disruption can synergize with the currently available drugs such as paclitaxel and sorafenib.<br><br>CONCLUSION: The data suggest CRISPR/Cas9 system as a useful tool in elucidating the molecular basis of drug resistance and improving clinical outcomes. Graphical abstract The mechanisms of CRISPR/Cas9-mediated genome editing and double-strand breaks (DSBs) repair.},
}
@article {pmid30664777,
year = {2019},
author = {Marx, V},
title = {A rocky road for the maturation of embryo-editing methods.},
journal = {Nature methods},
volume = {16},
number = {2},
pages = {147-150},
doi = {10.1038/s41592-018-0307-5},
pmid = {30664777},
issn = {1548-7105},
mesh = {Animals ; CRISPR-Cas Systems ; Congresses as Topic ; DNA/analysis ; *Embryo, Mammalian ; Gene Editing ; Genetic Diseases, Inborn/therapy ; *Genetic Techniques ; Genome, Human ; Humans ; Mice ; Mosaicism ; Quality Control ; *RNA Editing ; },
}
@article {pmid30664728,
year = {2019},
author = {Guo, M and Ren, K and Zhu, Y and Tang, Z and Wang, Y and Zhang, B and Huang, Z},
title = {Structural insights into a high fidelity variant of SpCas9.},
journal = {Cell research},
volume = {29},
number = {3},
pages = {183-192},
pmid = {30664728},
issn = {1748-7838},
mesh = {CRISPR-Associated Protein 9/*chemistry ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Crystallography, X-Ray ; DNA, Bacterial/*chemistry ; Gene Editing/methods ; Molecular Conformation ; RNA, Guide/*chemistry ; Streptococcus pyogenes/genetics ; },
abstract = {The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9's off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.},
}
@article {pmid30664678,
year = {2019},
author = {Yin, H and Xue, W and Anderson, DG},
title = {CRISPR-Cas: a tool for cancer research and therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {16},
number = {5},
pages = {281-295},
doi = {10.1038/s41571-019-0166-8},
pmid = {30664678},
issn = {1759-4782},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Vectors/administration &amp; dosage ; Humans ; Mice ; Neoplasms/*diagnosis/genetics/*therapy ; Xenograft Model Antitumor Assays ; },
abstract = {In the past decade, the development of a genome-editing technology mediated by CRISPR has made genetic engineering easier than ever, both in vitro and in vivo. CRISPR systems have enabled important advances in cancer research by accelerating the development of study models or as a tool in genetic screening studies, including those aiming to discover and validate therapeutic targets. In this Review, we discuss these applications as well as new potential uses of CRISPR to assist in cancer detection or the development of anticancer therapies.},
}
@article {pmid30664650,
year = {2019},
author = {Campagne, A and Lee, MK and Zielinski, D and Michaud, A and Le Corre, S and Dingli, F and Chen, H and Shahidian, LZ and Vassilev, I and Servant, N and Loew, D and Pasmant, E and Postel-Vinay, S and Wassef, M and Margueron, R},
title = {BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {348},
pmid = {30664650},
issn = {2041-1723},
mesh = {Animals ; B-Lymphocytes/cytology/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics/metabolism ; Cell Line, Tumor ; Drosophila melanogaster ; Gene Editing ; Haploidy ; HeLa Cells ; Histones/genetics/*metabolism ; Humans ; Polycomb Repressive Complex 1/genetics/metabolism ; *Protein Processing, Post-Translational ; Repressor Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Transcription Factors/genetics/metabolism ; Transcriptional Activation ; Tumor Suppressor Proteins/*genetics/metabolism ; Ubiquitin Thiolesterase/*genetics/metabolism ; Ubiquitination ; },
abstract = {In Drosophila, a complex consisting of Calypso and ASX catalyzes H2A deubiquitination and has been reported to act as part of the Polycomb machinery in transcriptional silencing. The mammalian homologs of these proteins (BAP1 and ASXL1/2/3, respectively), are frequently mutated in various cancer types, yet their precise functions remain unclear. Using an integrative approach based on isogenic cell lines generated with CRISPR/Cas9, we uncover an unanticipated role for BAP1 in gene activation. This function requires the assembly of an enzymatically active BAP1-associated core complex (BAP1.com) containing one of the redundant ASXL proteins. We investigate the mechanism underlying BAP1.com-mediated transcriptional regulation and show that it does not participate in Polycomb-mediated silencing. Instead, our results establish that the function of BAP1.com is to safeguard transcriptionally active genes against silencing by the Polycomb Repressive Complex 1.},
}
@article {pmid30664332,
year = {2019},
author = {Pieczynski, JN and Deets, A and McDuffee, A and Lynn Kee, H},
title = {An undergraduate laboratory experience using CRISPR-cas9 technology to deactivate green fluorescent protein expression in Escherichia coli.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {47},
number = {2},
pages = {145-155},
doi = {10.1002/bmb.21206},
pmid = {30664332},
issn = {1539-3429},
mesh = {CRISPR-Cas Systems/*genetics ; Escherichia coli/*genetics ; Gene Expression Regulation, Bacterial/*genetics ; Green Fluorescent Proteins/biosynthesis/*deficiency/*genetics ; *Laboratories ; Software ; Students ; *Universities ; },
abstract = {Undergraduates learn that gene editing in diverse organisms is now possible. How targeted manipulation of genes and genomes is utilized in basic science and biomedicine to address biological questions is challenging for undergraduates to conceptualize. Thus, we developed a lab experience that would allow students to be actively engaged in the full process of design, implementation of a gene editing strategy, and interpretation of results within an 8-week lab period of a Genetics course. The laboratory experience combines two transformative biotechnology tools; the utilization of green fluorescent protein (GFP) as a diagnostic marker of gene expression and the fundamentals and specificity of Clustered Regularly Interspaced Short Palindromic Repeats-cas9 (CRISPR-cas9) gene editing in bacterial cells. The students designed and constructed plasmids that express single guide RNA targeted to GFP, expressed the sgRNA and cas9 in bacteria cells, and successfully deactivated GFP gene expression in the bacterial cells with their designed CRISPR-cas9 tools. Student assessment revealed most students achieved student learning objectives. We conclude this lab experience is an effective and accessible method for engaging students in the scientific practices, knowledge and challenges revolving targeted CRISPR-cas9 gene manipulation. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 145-155, 2019.},
}
@article {pmid30663717,
year = {2019},
author = {Deshpande, A and Chen, BR and Zhao, L and Saddoris, K and Kerr, M and Zhu, N and Mali, P and Deshpande, AJ},
title = {Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {143},
pages = {},
doi = {10.3791/58710},
pmid = {30663717},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Mice ; Mice, Knockout ; },
abstract = {Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.},
}
@article {pmid30663658,
year = {2019},
author = {Tang, N and Zhang, Y and Pedrera, M and Chang, P and Baigent, S and Moffat, K and Shen, Z and Nair, V and Yao, Y},
title = {Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {143},
pages = {},
doi = {10.3791/58193},
pmid = {30663658},
issn = {1940-087X},
support = {BBS/E/I/00007036/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007034/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014262/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Birds/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Vectors/*genetics ; Herpesvirus 1, Meleagrid/*genetics ; },
abstract = {Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.},
}
@article {pmid30663071,
year = {2019},
author = {Jang, YJ and Kim, SA and Seo, SO and Li, L and Han, NS},
title = {Plasmid curing resulted in improved heterologous gene expression in Leuconostoc citreum EFEL2700.},
journal = {Letters in applied microbiology},
volume = {68},
number = {5},
pages = {430-436},
doi = {10.1111/lam.13118},
pmid = {30663071},
issn = {1472-765X},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression/genetics ; Gene Expression Regulation, Bacterial/*genetics ; Leuconostoc/*genetics/*metabolism ; Metabolic Engineering ; Plasmids/*genetics ; beta-Glucosidase/metabolism ; },
abstract = {Leuconostoc citreum EFEL2700 isolated from kimchi was used as a host strain for genetic and metabolic engineering in our previous studies, but the cells of EFEL2700 contained a cryptic plasmid (P-cells). Thus, we created plasmid-free cells (F-cells) using the CRISPR/Cas9 system. In this study, we compared the microbial characteristics of P- and F-cells in terms of growth rate, biochemical properties, transformation efficiency, plasmid copy number and protein expression level. When the growth rate was measured in MRS medium at 30°C, no significant difference (P > 0·01) was observed. Biochemical properties, tested using an API 50CHL kit, showed no differences. Transformation efficiency of F-cells, measured using pCB4270, was higher (1·3 × 10[4] CFU per μg DNA) than that of P-cells (5·0 × 10[3] CFU per μg DNA). Copy number after transformation of pCBBgl was 4-fold higher for F-cells than for P-cells. When β-glucosidase activity was assayed in the above experiment, F-cells showed 3·4-fold higher values than P-cells. In conclusion, this study demonstrates that plasmid curing in L. citreum EFEL2700 improves its characteristics as a gene expression host. SIGNIFICANCE AND IMPACT OF THE STUDY: Leuconostoc citreum EFEL2700 (P-cell) isolated from kimchi is a useful food-grade host for expressing heterologous genes. The presence of a cryptic plasmid is thought to limit efficient gene expression. In this study, we compared the microbial and genetic changes after plasmid curing in this strain. The plasmid-free strain showed improved levels of transformation efficiency, copy number and heterologous gene expression without alterations in phenotypes such as the growth rates and biochemical properties. The resulting strain of L. citreum EFEL2701 (F-cell) can be used as an efficient host for genetic engineering.},
}
@article {pmid30662440,
year = {2018},
author = {Lloyd, KA and Wigerblad, G and Sahlström, P and Garimella, MG and Chemin, K and Steen, J and Titcombe, PJ and Marklein, B and Zhou, D and Stålesen, R and Ossipova, E and Lundqvist, C and Ekwall, O and Rönnelid, J and Mueller, DL and Karlsson, MCI and Kaplan, MJ and Skriner, K and Klareskog, L and Wermeling, F and Malmström, V and Grönwall, C},
title = {Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {3033},
pmid = {30662440},
issn = {1664-3224},
mesh = {Acetylation ; Adult ; Aged ; Animals ; Anti-Citrullinated Protein Antibodies/*immunology/metabolism ; Antigens, Nuclear/*immunology/metabolism ; Apoptosis/immunology ; Arthritis, Rheumatoid/blood/*immunology ; Autoantigens/*immunology/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Cross Reactions ; Extracellular Traps/immunology/metabolism ; Female ; Gene Knockout Techniques ; Histones/immunology/metabolism ; Humans ; Male ; Mice ; Mice, Knockout ; Middle Aged ; Neutrophils/immunology/metabolism ; Primary Cell Culture ; Protein Processing, Post-Translational/immunology ; Protein-Arginine Deiminase Type 4 ; Protein-Arginine Deiminases/genetics/immunology/*metabolism ; },
abstract = {Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.},
}
@article {pmid30662438,
year = {2018},
author = {Cheng, Y and Lun, M and Liu, Y and Wang, H and Yan, Y and Sun, J},
title = {CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {3010},
pmid = {30662438},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Chick Embryo ; Chickens/genetics/*immunology/metabolism ; Fibroblasts ; Gene Knockout Techniques/methods ; *Immunity, Innate ; Interferon-beta/immunology/*metabolism ; Membrane Proteins/immunology/*metabolism ; Primary Cell Culture ; Protein Serine-Threonine Kinases/genetics/*immunology ; RNA, Guide/genetics ; },
abstract = {TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 in chickens remains poorly defined. CRISPR/Cas9 system has made gene-knockout easy to accomplish. Although CRISPR/Cas9 has been used in chicken cells, low mutation efficiency limits its wide application in chickens. In this study, an effective gene-knockout system was developed based on the CRISPR/Cas9 system in chicken embryonic fibroblast DF-1. Two CRISPR/Cas9 plasmids were constructed, TBK1-g1 and TBK1-g2, which express gRNAs targeting different sequences of the chicken TBK1 gene. After transfection and enrichment with puromycin screening, the mutation rates as assessed via T7E1 assay were 88.05 and 89.55%, respectively, and subsequent sequence analysis showed mutation efficiencies of 86.67 and 93.33%. With the limiting-dilution method, a chTBK1 gene-deficiency monoclonal cell line was obtained and was named DF-1-TBK1-C3. The DF-1-TBK1-C3 cells exhibited normal morphology and maintained stable proliferation ability compared to wild-type cells. The gene-overexpression system and luciferase reporter assay showed that IFN-β induction induced by chSTING was almost completely blocked in DF-1-TBK1-C3 cells. With quantitative real-time PCR, we further confirmed the essential role of chTBK1 in the chSTING-mediated IFN-β induction. At last, the study demonstrated that the chTBK1 knockout system is also applicable in primary chick embryo fibroblasts (CEFs). In this study, an effective gene-knockout system was applied in chickens, a TBK1 gene-deleted DF-1 cell line was successfully created using this system, and with the chTBK1 knockout cells, chTBK1 was revealed to be indispensable in STING-mediated IFN-β activation in chicken cells.},
}
@article {pmid30661951,
year = {2019},
author = {Jackson, SA and Birkholz, N and Malone, LM and Fineran, PC},
title = {Imprecise Spacer Acquisition Generates CRISPR-Cas Immune Diversity through Primed Adaptation.},
journal = {Cell host &amp; microbe},
volume = {25},
number = {2},
pages = {250-260.e4},
doi = {10.1016/j.chom.2018.12.014},
pmid = {30661951},
issn = {1934-6069},
mesh = {Bacteriophages/genetics/*growth &amp; development ; *CRISPR-Cas Systems ; DNA, Intergenic/*genetics ; DNA, Viral/*genetics ; *Evolution, Molecular ; Genetic Variation ; Pectobacterium/*genetics/virology ; Serratia/*genetics/virology ; },
abstract = {Many prokaryotes possess CRISPR-Cas adaptive immune systems to defend against viruses and invading mobile genetic elements. CRISPR-Cas immunity relies on genetic memories, termed spacers, for sequence-specific recognition of infections. The diversity of spacers within host populations is important for immune resilience, but we have limited understanding of how CRISPR diversity is generated. Type I CRISPR-Cas systems use existing spacers to enhance the acquisition of new spacers through primed CRISPR adaptation (priming). Here, we present a pathway to priming that is stimulated by imprecisely acquired (slipped) spacers. Slipped spacers are less effective for immunity but increase priming compared with canonical spacers. The benefits of slipping depend on the relative rates of phage mutation and adaptation during defense. We propose that slipped spacers provide a route to increase population-level spacer diversity that pre-empts phage escape mutant proliferation and that the trade-off between adaptation and immunity is important in diverse CRISPR-Cas systems.},
}
@article {pmid30659180,
year = {2019},
author = {Profumo, V and Forte, B and Percio, S and Rotundo, F and Doldi, V and Ferrari, E and Fenderico, N and Dugo, M and Romagnoli, D and Benelli, M and Valdagni, R and Dolfini, D and Zaffaroni, N and Gandellini, P},
title = {LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {307},
pmid = {30659180},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line, Tumor ; Cells, Cultured ; Epithelial Cells/metabolism ; Gene Editing ; Humans ; Male ; MicroRNAs/*genetics ; Promoter Regions, Genetic ; Prostate/*cytology/metabolism ; RNA, Long Noncoding/*genetics ; Ribonuclease III/metabolism ; Transcriptome ; },
abstract = {Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).},
}
@article {pmid30659093,
year = {2019},
author = {Isaji, T and Im, S and Kameyama, A and Wang, Y and Fukuda, T and Gu, J},
title = {A complex between phosphatidylinositol 4-kinase IIα and integrin α3β1 is required for N-glycan sialylation in cancer cells.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {12},
pages = {4425-4436},
pmid = {30659093},
issn = {1083-351X},
mesh = {1-Phosphatidylinositol 4-Kinase/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Gene Knockdown Techniques ; Humans ; Integrin alpha3beta1/genetics/*metabolism ; Membrane Proteins/metabolism ; N-Acetylneuraminic Acid/*metabolism ; Phosphorylation ; Polysaccharides/metabolism ; Protein Binding ; Signal Transduction ; trans-Golgi Network/metabolism ; },
abstract = {Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA-mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3β1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3β1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3β1.},
}
@article {pmid30659022,
year = {2019},
author = {Guo, Q and Li, VZ and Nichol, JN and Huang, F and Yang, W and Preston, SEJ and Talat, Z and Lefrère, H and Yu, H and Zhang, G and Basik, M and Gonçalves, C and Zhan, Y and Plourde, D and Su, J and Torres, J and Marques, M and Habyan, SA and Bijian, K and Amant, F and Witcher, M and Behbod, F and McCaffrey, L and Alaoui-Jamali, M and Giannakopoulos, NV and Brackstone, M and Postovit, LM and Del Rincón, SV and Miller, WH},
title = {MNK1/NODAL Signaling Promotes Invasive Progression of Breast Ductal Carcinoma In Situ.},
journal = {Cancer research},
volume = {79},
number = {7},
pages = {1646-1657},
pmid = {30659022},
issn = {1538-7445},
support = {R01 CA207445/CA/NCI NIH HHS/United States ; R21 CA226567/CA/NCI NIH HHS/United States ; PJT-156269//CIHR/Canada ; MOP-142281//CIHR/Canada ; },
mesh = {Animals ; Breast Carcinoma In Situ/metabolism/*pathology ; Breast Neoplasms/metabolism/*pathology ; CRISPR-Cas Systems ; Carcinoma, Ductal, Breast/metabolism/*pathology ; Cell Line, Tumor ; Cell Proliferation ; Disease Progression ; Female ; Heterografts ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mice ; Mice, Nude ; Nodal Protein/*metabolism ; Protein Serine-Threonine Kinases/genetics/*metabolism ; *Signal Transduction ; },
abstract = {The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro. The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo. In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma.},
}
@article {pmid30659021,
year = {2019},
author = {Sun, T and Patil, R and Galstyan, A and Klymyshyn, D and Ding, H and Chesnokova, A and Cavenee, WK and Furnari, FB and Ljubimov, VA and Shatalova, ES and Wagner, S and Li, D and Mamelak, AN and Bannykh, SI and Patil, CG and Rudnick, JD and Hu, J and Grodzinski, ZB and Rekechenetskiy, A and Falahatian, V and Lyubimov, AV and Chen, YL and Leoh, LS and Daniels-Wells, TR and Penichet, ML and Holler, E and Ljubimov, AV and Black, KL and Ljubimova, JY},
title = {Blockade of a Laminin-411-Notch Axis with CRISPR/Cas9 or a Nanobioconjugate Inhibits Glioblastoma Growth through Tumor-Microenvironment Cross-talk.},
journal = {Cancer research},
volume = {79},
number = {6},
pages = {1239-1251},
pmid = {30659021},
issn = {1538-7445},
support = {R01 CA188743/CA/NCI NIH HHS/United States ; R01 EY013431/EY/NEI NIH HHS/United States ; R01 CA196266/CA/NCI NIH HHS/United States ; R01 CA206220/CA/NCI NIH HHS/United States ; U01 CA151815/CA/NCI NIH HHS/United States ; R01 EY023429/EY/NEI NIH HHS/United States ; R01 CA209921/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Apoptosis ; Biomarkers, Tumor/genetics/metabolism ; Brain/metabolism/pathology ; *CRISPR-Cas Systems ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Glioblastoma/genetics/metabolism/*pathology ; Humans ; Laminin/antagonists &amp; inhibitors/genetics/*metabolism ; Mice ; Mice, Nude ; Nanoparticles/*chemistry ; Neoplastic Stem Cells/metabolism/*pathology ; Prognosis ; Receptors, Notch/genetics/*metabolism ; Signal Transduction ; Survival Rate ; Tumor Cells, Cultured ; *Tumor Microenvironment ; Xenograft Model Antitumor Assays ; },
abstract = {There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4β1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.},
}
@article {pmid30658254,
year = {2019},
author = {Li, Y and Huang, C and Zha, L and Kong, M and Yang, Q and Zhu, Y and Peng, Y and Ouyang, Q and Lu, G and Lin, G and Zhou, D},
title = {Generation of NERCe003-A-3, a p53 compound heterozygous mutation human embryonic stem cell line, by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {34},
number = {},
pages = {101371},
doi = {10.1016/j.scr.2018.101371},
pmid = {30658254},
issn = {1876-7753},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line ; *Gene Editing ; Heterozygote ; Human Embryonic Stem Cells/*cytology ; Humans ; Male ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {p53 is a tumor suppressor gene involved mainly in the regulation of the G1/S cell cycle phase, DNA repair, and senescence. Although p53 is frequently altered in human cancer, the consequences of its depletion in human embryonic stem cells (hESCs) are unknown. We generated NERCe003-A-3, a p53 knockout hESC line, from the normal NERCe003-A hESC line by using CRISPR/Cas9 editing. This cell line maintained a normal 46, XY karyotype. Further analysis suggested that the cells expressed pluripotency-related markers and had the capacity to differentiate in vitro into derivatives of all three germ layers.},
}
@article {pmid30657058,
year = {2019},
author = {Yin, Y and Xu, L and Chang, Y and Zeng, T and Chen, X and Wang, A and Groth, J and Foo, WC and Liang, C and Hu, H and Huang, J},
title = {N-Myc promotes therapeutic resistance development of neuroendocrine prostate cancer by differentially regulating miR-421/ATM pathway.},
journal = {Molecular cancer},
volume = {18},
number = {1},
pages = {11},
pmid = {30657058},
issn = {1476-4598},
support = {R01 CA205001/CA/NCI NIH HHS/United States ; R01 CA172603/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Ataxia Telangiectasia Mutated Proteins/genetics/*metabolism ; Benzamides ; CRISPR-Cas Systems ; Carcinoma, Neuroendocrine/drug therapy/*genetics/pathology ; Cell Line, Tumor ; Cell Movement/drug effects/genetics ; Cell Proliferation/drug effects/genetics ; Cell Survival/drug effects/genetics ; Drug Resistance, Neoplasm ; Humans ; Male ; Mice ; MicroRNAs/genetics/*metabolism ; Morpholines/pharmacology ; N-Myc Proto-Oncogene Protein/*biosynthesis/genetics/metabolism ; Nitriles ; Phenylthiohydantoin/analogs &amp; derivatives/pharmacology ; Prostatic Neoplasms, Castration-Resistant/drug therapy/*genetics/pathology ; Protein Kinase Inhibitors/pharmacology ; Signal Transduction ; Thioxanthenes/pharmacology ; Up-Regulation/drug effects/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: MYCN amplification or N-Myc overexpression is found in approximately 40% NEPC and up to 20% CRPC patients. N-Myc has been demonstrated to drive disease progression and hormonal therapeutic resistance of NEPC/CRPC. Here, we aim to identify the molecular mechanisms underlying the N-Myc-driven therapeutic resistance and provide new therapeutic targets for those N-Myc overexpressed NEPC/CRPC.<br><br>METHODS: N-Myc overexpressing stable cell lines for LNCaP and C4-2 were generated by lentivirus infection. ADT-induced senescence was measured by SA-&#x3b2;-gal staining in LNCaP cells in vitro and in LNCaP xenograft tumors in vivo. Migration, cell proliferation and colony formation assays were used to measure the cellular response after overexpressing N-Myc or perturbing the miR-421/ATM pathway. CRISPR-Cas9 was used to knock out ATM in C4-2 cells and MTS cell viability assay was used to evaluate the drug sensitivity of N-Myc overexpressing C4-2 cells in response to Enzalutamide and ATM inhibitor Ku60019 respectively or in combination.<br><br>RESULTS: N-Myc overexpression suppressed ATM expression through upregulating miR-421 in LNCaP cells. This suppression alleviated the ADT-induced senescence in vitro and in vivo. Surprisingly, N-Myc overexpression upregulated ATM expression in C4-2 cells and this upregulation promoted migration and invasion of prostate cancer cells. Further, the N-Myc-induced ATM upregulation in C4-2 cells rendered the cells resistance to Enzalutamide, and inhibition of ATM by CRISPR-Cas9 knockout or ATM inhibitor Ku60019 re-sensitized them to Enzalutamide.<br><br>CONCLUSIONS: N-Myc differentially regulating miR-421/ATM pathway contributes to ADT resistance and Enzalutamide resistance development respectively. Combination treatment with ATM inhibitor re-sensitizes N-Myc overexpressed CRPC cells to Enzalutamide. Our findings would offer a potential combination therapeutic strategy using ATM kinase inhibitor and Enzalutamide for the treatment of a subset of mCRPC with N-Myc overexpression that accounts for up to 20% CRPC patients.},
}
@article {pmid30656625,
year = {2019},
author = {Hong, H and Daadi, MM},
title = {Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1919},
number = {},
pages = {119-128},
pmid = {30656625},
issn = {1940-6029},
support = {P51 OD011133/OD/NIH HHS/United States ; UL1 TR001120/TR/NCATS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques ; *Cell Differentiation ; Dopaminergic Neurons/*cytology/*metabolism ; Gene Editing ; *Gene Expression ; *Genes, Reporter ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Neural Stem Cells/*cytology/*metabolism ; Parkinson Disease/metabolism ; RNA, Guide ; Tyrosine 3-Monooxygenase/genetics/metabolism ; },
abstract = {Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique.},
}
@article {pmid30656391,
year = {2019},
author = {Plavec, TV and Berlec, A},
title = {Engineering of lactic acid bacteria for delivery of therapeutic proteins and peptides.},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {5},
pages = {2053-2066},
doi = {10.1007/s00253-019-09628-y},
pmid = {30656391},
issn = {1432-0614},
mesh = {Bacterial Proteins/genetics/therapeutic use ; Biological Products/*metabolism/*therapeutic use ; Gene Editing/methods ; Humans ; Lactobacillus/genetics/*metabolism ; Lactococcus lactis/genetics/*metabolism ; Protein Engineering/*methods ; },
abstract = {Lactic acid bacteria (LAB) have a long-term history of use in food industry and are becoming attractive for use in therapy on account of their safety, intrinsic beneficial health effects, and considerable biotechnological potential. The established systems for engineering are combined with novel approaches, such as CRISPR-Cas, to enable the use of LAB as vectors for delivery of various therapeutic molecules. The latter are either secreted or surface displayed and can be used for the treatment or prevention of numerous conditions: inflammatory bowel diseases, infections, autoimmune diseases, and even cancer. This review presents some recent data on engineering of LAB, with the emphasis on the most commonly used genera Lactococcus and Lactobacillus. Their use for the delivery of therapeutic proteins is discussed, while a special focus is given to the delivery of therapeutic peptides. Therapeutically relevant improvements of engineered LAB, such as containment systems, ability to visualize bacteria, or target specific host cells are also addressed. Future engineering of LAB for therapy will adopt the capabilities of synthetic biology, with first examples already emerging.},
}
@article {pmid30655567,
year = {2019},
author = {Supharattanasitthi, W and Carlsson, E and Sharif, U and Paraoan, L},
title = {CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {174},
pmid = {30655567},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Cystatin C/*genetics ; DNA/*genetics ; Epithelial Cells/cytology ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology ; },
abstract = {CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer's disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.},
}
@article {pmid30655519,
year = {2019},
author = {Díez-Villaseñor, C and Rodriguez-Valera, F},
title = {CRISPR analysis suggests that small circular single-stranded DNA smacoviruses infect Archaea instead of humans.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {294},
pmid = {30655519},
issn = {2041-1723},
mesh = {Archaea/*genetics/virology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Viruses/genetics/isolation &amp; purification/*pathogenicity ; DNA, Single-Stranded/*genetics ; Feces/microbiology ; Gastrointestinal Microbiome/*genetics ; Host-Pathogen Interactions/*genetics ; Humans ; Phylogeny ; },
abstract = {Smacoviridae is a family of small (~2.5 Kb) CRESS-DNA (Circular Rep Encoding Single-Stranded (ss) DNA) viruses. These viruses have been found in faeces, were thought to infect eukaryotes and are suspected to cause gastrointestinal disease in humans. CRISPR-Cas systems are adaptive immune systems in prokaryotes, wherein snippets of genomes from invaders are stored as spacers that are interspersed between a repeated CRISPR sequence. Here we report several spacer sequences in the faecal archaeon Candidatus Methanomassiliicoccus intestinalis matching smacoviruses, implicating the archaeon as a firm candidate for a host. This finding may be relevant to understanding the potential origin of smacovirus-associated human diseases. Our results support that CRESS-DNA viruses can infect non-eukaryotes, which would mean that smacoviruses are the viruses with the smallest genomes to infect prokaryotes known to date. A probable target strand bias suggests that, in addition to double-stranded DNA, the CRISPR-Cas system can target ssDNA.},
}
@article {pmid30655324,
year = {2019},
author = {Pegg, HJ and Harrison, H and Rogerson, C and Shore, P},
title = {The RUNX Transcriptional Coregulator, CBFβ, Suppresses Migration of ER[+] Breast Cancer Cells by Repressing ERα-Mediated Expression of the Migratory Factor TFF1.},
journal = {Molecular cancer research : MCR},
volume = {17},
number = {5},
pages = {1015-1023},
doi = {10.1158/1541-7786.MCR-18-1039},
pmid = {30655324},
issn = {1557-3125},
support = {MR/L011840/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Breast Neoplasms/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Core Binding Factor Alpha 2 Subunit/genetics/*metabolism ; Core Binding Factor beta Subunit/*genetics ; Estrogen Receptor alpha/genetics/*metabolism ; Female ; HeLa Cells ; Humans ; MCF-7 Cells ; *Mutation, Missense ; Trefoil Factor-1/genetics/*metabolism ; },
abstract = {Core binding factor β (CBFβ), the essential coregulator of RUNX transcription factors, is one of the most frequently mutated genes in estrogen receptor-positive (ER[+]) breast cancer. Many of these mutations are nonsense mutations and are predicted to result in loss of function, suggesting a tumor suppressor role for CBFβ. However, the impact of missense mutations and the loss of CBFβ in ER[+] breast cancer cells have not been determined. Here we demonstrate that missense mutations in CBFβ accumulate near the Runt domain-binding region. These mutations inhibit the ability of CBFβ to form CBFβ-Runx-DNA complexes. We further show that deletion of CBFβ, using CRISPR-Cas9, in ER[+] MCF7 cells results in an increase in cell migration. This increase in migration is dependent on the presence of ERα. Analysis of the potential mechanism revealed that the increase in migration is driven by the coregulation of Trefoil factor 1 (TFF1) by CBFβ and ERα. RUNX1-CBFβ acts to repress ERα-activated expression of TFF1. TFF1 is a motogen that stimulates migration and we show that knockdown of TFF1 in CBFβ[-/-] cells inhibits the migratory phenotype. Our findings reveal a new mechanism by which RUNX1-CBFβ and ERα combine to regulate gene expression and a new role for RUNX1-CBFβ in the prevention of cell migration by suppressing the expression of the motogen TFF1. IMPLICATIONS: Mutations in CBFβ contribute to the development of breast cancer by inducing a metastatic phenotype that is dependent on ER.},
}
@article {pmid30654914,
year = {2019},
author = {Li, Y and Li, S and Wang, J and Liu, G},
title = {CRISPR/Cas Systems towards Next-Generation Biosensing.},
journal = {Trends in biotechnology},
volume = {37},
number = {7},
pages = {730-743},
doi = {10.1016/j.tibtech.2018.12.005},
pmid = {30654914},
issn = {1879-3096},
mesh = {Biosensing Techniques/*methods/trends ; *CRISPR-Cas Systems ; Gene Editing/*methods/trends ; Humans ; Molecular Diagnostic Techniques/methods/trends ; },
abstract = {Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms.},
}
@article {pmid30654190,
year = {2019},
author = {Moses, C and Nugent, F and Waryah, CB and Garcia-Bloj, B and Harvey, AR and Blancafort, P},
title = {Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System.},
journal = {Molecular therapy. Nucleic acids},
volume = {14},
number = {},
pages = {287-300},
pmid = {30654190},
issn = {2162-2531},
support = {R01 CA170370/CA/NCI NIH HHS/United States ; R01 DA036906/DA/NIDA NIH HHS/United States ; },
abstract = {PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.},
}
@article {pmid30653821,
year = {2019},
author = {Wen, X and Iwata, K and Ikuta, K and Zhang, X and Zhu, K and Ibi, M and Matsumoto, M and Asaoka, N and Liu, J and Katsuyama, M and Yabe-Nishimura, C},
title = {NOX1/NADPH oxidase regulates the expression of multidrug resistance-associated protein 1 and maintains intracellular glutathione levels.},
journal = {The FEBS journal},
volume = {286},
number = {4},
pages = {678-687},
doi = {10.1111/febs.14753},
pmid = {30653821},
issn = {1742-4658},
mesh = {Acrolein/pharmacology ; Animals ; Butanones/pharmacology ; CRISPR-Cas Systems ; Cell Survival ; Cells, Cultured ; Gene Expression Regulation/*drug effects ; Glutathione/*metabolism ; Multidrug Resistance-Associated Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Myocytes, Cardiac/cytology/drug effects/*metabolism ; NADPH Oxidase 1/antagonists &amp; inhibitors/genetics/*metabolism ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Rats ; Reactive Oxygen Species/metabolism ; },
abstract = {The involvement of superoxide-generating NADPH oxidase (NOX) in the cytotoxic effects of cigarette smoke extracts has been documented. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among the different NADPH oxidase isoforms identified so far, NOX1 and NOX4 were found to be expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependent decline in cell viability was observed. Unexpectedly, disruption of Nox1 as well as Nox4 significantly exacerbated cytotoxicity induced by acrolein or MVK. Compared with Nox4-disrupted cells, Nox1-disrupted cells were more vulnerable to acrolein and MVK at lower concentrations. Disruption of Nox1 markedly attenuated the levels of total and reduced glutathione (GSH) in H9c2 clones. Reduction in the cystine level in the culture medium to deplete intracellular GSH significantly exacerbated acrolein or MVK-induced cytotoxicity. Nox1 disruption neither attenuated the level of glutamate-cystine antiporter protein nor the activity of glutamate-cysteine ligase, both rate-limiting factors for GSH synthesis. On the other hand, increased expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was demonstrated in Nox1-disrupted cells. The augmented toxicity of acrolein and MVK in these cells was partially but significantly blunted in the presence of an MRP1 inhibitor, reversan. Taken together, these results show that NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts. A novel crosstalk between NOX1 and MRP1 was demonstrated in this study.},
}
@article {pmid30653574,
year = {2019},
author = {Leynaud-Kieffer, LMC and Curran, SC and Kim, I and Magnuson, JK and Gladden, JM and Baker, SE and Simmons, BA},
title = {A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable.},
journal = {PloS one},
volume = {14},
number = {1},
pages = {e0210243},
pmid = {30653574},
issn = {1932-6203},
mesh = {Aspergillus niger/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Targeting ; Genome, Fungal/*genetics ; Genomics ; },
abstract = {Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.},
}
@article {pmid30652832,
year = {2018},
author = {Węgleński, P},
title = {[Gene editing].},
journal = {Postepy biochemii},
volume = {64},
number = {1},
pages = {9-12},
doi = {10.18388/pb.2018_99},
pmid = {30652832},
issn = {0032-5422},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/*ethics ; Genome/genetics ; Humans ; Metabolic Diseases/genetics ; Mice ; Mutation ; Neoplasms/genetics ; },
abstract = {Development of the gene engineering techniques has raised worries that they will be used for construction of organism endangering humans and environment. In 1975 at the Asilomar conference, geneticists from many countries decided that genetic engineering brings more benefits than threats. In last years a new CRISPR-Cas technique emerged . It allows to make the precise changes in genomes, e.g. to inactivate particular genes or to replace mutated genes by their wild-type alleles. Inactivation in mice of genes corresponding to those whose mutations cause the genetic diseases in man allows to get model organisms for studying the etiology of given disease and for working out the methods of its curing. This technique can be applied for repairing genes whose mutations result in metabolic diseases and cancer. Some voices were raised that the technique can be potentially used for the "improvement" of man, what would create many ethical and social problems. Geneticists, ethicists and lawyers gathered in 2015 at the Washington conference, discussed these problems and proposed rules for their solving.},
}
@article {pmid30652290,
year = {2019},
author = {Liu, G and Li, J and Godwin, ID},
title = {Genome Editing by CRISPR/Cas9 in Sorghum Through Biolistic Bombardment.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1931},
number = {},
pages = {169-183},
doi = {10.1007/978-1-4939-9039-9_12},
pmid = {30652290},
issn = {1940-6029},
mesh = {Base Sequence ; Biolistics/methods ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Edible Grain/*genetics ; Gene Editing/methods ; Genome, Plant/genetics ; Genotype ; Plants, Genetically Modified/genetics ; Sorghum/*genetics ; },
abstract = {The advanced biotechnology CRISPR/Cas9 as a powerful genome editing tool has displayed great potential for improving important agronomic traits such as yield and quality. It has gained momentum worldwide for gene function research of plants in recent years. As for cereals, numerous studies of CRISPR/Cas9 have been reported predominately on rice and quite a few on other cereals including maize, wheat, and barley. In contrast, there are only a couple of reports on sorghum up to date. In this chapter, the CRISPR/Cas9 system has been investigated for sorghum genome editing through biolistic bombardment. Two target genes, cinnamyl alcohol dehydrogenase (CAD) and phytoene desaturase (PDS), have been investigated by CRISPR/Cas9 though bomboarment. Successful genome editing has been achieved within the sorghum genotype Tx430. Furthermore, sequencing PCR product of transgenic plants has confirmed that the CRISPR/Cas9 successfully edited the target gene in sorghum. Both homozygosis and heterozygosis editings of CAD gene have been confirmed in T0 primary transgenic lines through sequencing PCR products. T1 generation of CRISPR plants has been investigated as well. The results illustrated that the edited gene has passed down to next generation. More experiments, such as optimizing promoters for guide RNA (gRNA) and Cas9 in sorghum, are under investigation. Three factors were considered crucial elements to establish an efficient CRISPR/Cas9 system for genome editing in sorghum: (1) an efficient transformation system, (2) the design of targeted gene sequence for gRNA, (3) effective expression of CRISPR components including Cas9 and gRNA.},
}
@article {pmid30652289,
year = {2019},
author = {Sander, JD},
title = {Gene Editing in Sorghum Through Agrobacterium.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1931},
number = {},
pages = {155-168},
doi = {10.1007/978-1-4939-9039-9_11},
pmid = {30652289},
issn = {1940-6029},
mesh = {Agrobacterium/*genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genetic Variation/genetics ; Genome, Plant/*genetics ; Plants, Genetically Modified/genetics ; Sorghum/*genetics/*microbiology ; },
abstract = {The application of CRISPR/Cas to introduce targeted genomic edits is powering research and discovery across the genetic frontier. Applying CRISPR/Cas in sorghum can facilitate the study of gene function and unlock our understanding of this robust crop that serves as a staple for some of the most food insecure regions on the planet. When paired with recent advances in sorghum tissue culture and Agrobacteria technology, CRISPR/Cas can be used to introduce desirable changes and natural genetic variations directly into agriculturally relevant sorghum lines facilitating product development. This chapter describes CRISPR/Cas gene editing and provides high-level strategies and expectations for applying this technology using Agrobacterium in sorghum.},
}
@article {pmid30651362,
year = {2019},
author = {Tso, FY and West, JT and Wood, C},
title = {Reduction of Kaposi's Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene.},
journal = {Journal of virology},
volume = {93},
number = {7},
pages = {},
pmid = {30651362},
issn = {1098-5514},
support = {P30 GM103509/GM/NIGMS NIH HHS/United States ; R01 CA075903/CA/NCI NIH HHS/United States ; U54 CA190155/CA/NCI NIH HHS/United States ; U54 CA221204/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antigens, Viral/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chlorocebus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endothelial Cells/virology ; Epithelial Cells/virology ; HEK293 Cells ; Herpesviridae Infections/virology ; Herpesvirus 8, Human/*genetics ; Humans ; Nuclear Proteins/*genetics ; Sarcoma, Kaposi/*virology ; Vero Cells ; Virus Latency/*genetics ; Virus Replication/genetics ; },
abstract = {Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to the Gammaherpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potential in vivo applications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.},
}
@article {pmid30650001,
year = {2019},
author = {Charo, RA},
title = {Rogues and Regulation of Germline Editing.},
journal = {The New England journal of medicine},
volume = {380},
number = {10},
pages = {976-980},
doi = {10.1056/NEJMms1817528},
pmid = {30650001},
issn = {1533-4406},
mesh = {Bioethical Issues/*legislation &amp; jurisprudence ; CRISPR-Cas Systems ; China ; Embryo Research/ethics/*legislation &amp; jurisprudence ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; *Germ Cells ; *Government Regulation ; Humans ; },
}
@article {pmid30649993,
year = {2019},
author = {Daley, GQ and Lovell-Badge, R and Steffann, J},
title = {After the Storm - A Responsible Path for Genome Editing.},
journal = {The New England journal of medicine},
volume = {380},
number = {10},
pages = {897-899},
doi = {10.1056/NEJMp1900504},
pmid = {30649993},
issn = {1533-4406},
support = {MC_U117562207/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Bioethical Issues/legislation &amp; jurisprudence/*standards ; *CRISPR-Cas Systems ; China ; Congresses as Topic ; Consensus ; Embryo Research/*ethics ; Female ; Fertilization in Vitro ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Genetic Diseases, Inborn/*prevention &amp; control ; *Genetic Therapy ; Germ Cells ; Government Regulation ; Humans ; Preimplantation Diagnosis ; Receptors, CCR5/*genetics ; Twins ; },
}
@article {pmid30649992,
year = {2019},
author = {Rosenbaum, L},
title = {The Future of Gene Editing - Toward Scientific and Social Consensus.},
journal = {The New England journal of medicine},
volume = {380},
number = {10},
pages = {971-975},
doi = {10.1056/NEJMms1817082},
pmid = {30649992},
issn = {1533-4406},
mesh = {Bioethical Issues/legislation &amp; jurisprudence/*standards ; *CRISPR-Cas Systems ; China ; Consensus ; Disease Resistance/genetics ; Embryo Research/*ethics ; Fertilization in Vitro/ethics ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Germ Cells ; *Government Regulation ; Humans ; Preimplantation Diagnosis/ethics ; Receptors, CCR5/*genetics ; Twins ; },
}
@article {pmid30649572,
year = {2019},
author = {Danilo, B and Perrot, L and Mara, K and Botton, E and Nogué, F and Mazier, M},
title = {Efficient and transgene-free gene targeting using Agrobacterium-mediated delivery of the CRISPR/Cas9 system in tomato.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {459-462},
pmid = {30649572},
issn = {1432-203X},
support = {ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/International ; },
mesh = {Agrobacterium/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Targeting/methods ; Lycopersicon esculentum/*genetics ; Transgenes/*genetics ; },
}
@article {pmid30649181,
year = {2019},
author = {Jacquin, ALS and Odom, DT and Lukk, M},
title = {Crisflash: open-source software to generate CRISPR guide RNAs against genomes annotated with individual variation.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {17},
pages = {3146-3147},
pmid = {30649181},
issn = {1367-4811},
support = {202878/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 22398/CRUK_/Cancer Research UK/United Kingdom ; 20412/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genome ; RNA, Guide ; Software ; },
abstract = {SUMMARY: CRISPR/Cas9 system requires short guide RNAs (sgRNAs) to direct genome modification. Most currently available tools for sgRNA design operate only with standard reference genomes, and are best suited for small-scale projects. To address these limitations, we developed Crisflash, a software tool for fast sgRNA design and potential off-target discovery, built for performance and flexibility. Crisflash can rapidly design CRISPR guides against any sequenced genome or genome sequences, and can optimize guide accuracy by incorporating user-supplied variant data. Crisflash is over an order of magnitude faster than comparable tools, even using a single CPU core, and efficiently and robustly scores the potential off-targeting of all possible candidate CRISPR guide oligonucleotides.<br><br>https://github.com/crisflash.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid30648968,
year = {2019},
author = {Voss, JE and Gonzalez-Martin, A and Andrabi, R and Fuller, RP and Murrell, B and McCoy, LE and Porter, K and Huang, D and Li, W and Sok, D and Le, K and Briney, B and Chateau, M and Rogers, G and Hangartner, L and Feeney, AJ and Nemazee, D and Cannon, P and Burton, DR},
title = {Reprogramming the antigen specificity of B cells using genome-editing technologies.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30648968},
issn = {2050-084X},
support = {5R01DE025167-05/NH/NIH HHS/United States ; UM1 AI100663/AI/NIAID NIH HHS/United States ; MR/R008698/1/MRC_/Medical Research Council/United Kingdom ; UL1 TR001114/TR/NCATS NIH HHS/United States ; R01 AI073148/AI/NIAID NIH HHS/United States ; R01 DE025167/DE/NIDCR NIH HHS/United States ; FP7-PEOPLE-2013-IOF//FP7 People: Marie-Curie Actions/International ; Ram&#xf3;n y Cajal Merit Award RYC-2016-21155//Ministerio de Ciencia, Innovacion y Universidades/International ; R37 AI059714/AI/NIAID NIH HHS/United States ; RYC-2016-21155//Ram&#xf3;n y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades/International ; FP7-PEOPLE-2013-IOF//Marie-Curie Fellowship/International ; OPP1183956//Bill and Melinda Gates Foundation/International ; },
mesh = {Antibodies, Neutralizing/immunology ; Antibody Specificity ; Antigen-Antibody Reactions/*genetics ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/metabolism ; Gene Editing/*methods ; HIV Antibodies/immunology ; Humans ; Immunoglobulin Heavy Chains/genetics ; Receptors, Antigen, B-Cell/genetics/immunology ; },
abstract = {We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.},
}
@article {pmid30648435,
year = {2019},
author = {Wasala, NB and Hakim, CH and Chen, SJ and Yang, NN and Duan, D},
title = {Questions Answered and Unanswered by the First CRISPR Editing Study in a Canine Model of Duchenne Muscular Dystrophy.},
journal = {Human gene therapy},
volume = {30},
number = {5},
pages = {535-543},
pmid = {30648435},
issn = {1557-7422},
support = {R01 AR069085/AR/NIAMS NIH HHS/United States ; R01 GM063732/GM/NIGMS NIH HHS/United States ; R01 GM117059/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; Dogs ; *Gene Editing ; Genetic Vectors/genetics ; Humans ; Muscular Dystrophy, Duchenne/*genetics/therapy ; RNA, Guide ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) editing is being considered as a potential gene repair therapy to treat Duchenne muscular dystrophy, a dystrophin-deficient lethal muscle disease affecting all muscles in the body. A recent preliminary study from the Olson laboratory (Amoasii et al. Science 2018;362:89-91) showed robust dystrophin restoration in a canine Duchenne muscular dystrophy model following intramuscular or intravenous delivery of the CRISPR editing machinery by adeno-associated virus serotype 9. Despite the limitation of the small sample size, short study duration, and the lack of muscle function data, the Olson lab findings have provided important proof of principle for scaling up CRISPR therapy from rodents to large mammals. Future large-scale, long-term, and comprehensive studies are warranted to establish the safety and efficacy of CRISPR editing therapy in large mammals.},
}
@article {pmid30648117,
year = {2018},
author = {Wu, J and Min, X and Wang, L and Yang, J and Wang, P and Liu, X and Xia, Y},
title = {Fn14 Deficiency Ameliorates Anti-dsDNA IgG-Induced Glomerular Damage in SCID Mice.},
journal = {Journal of immunology research},
volume = {2018},
number = {},
pages = {1256379},
pmid = {30648117},
issn = {2314-7156},
mesh = {Animals ; Autoantibodies/metabolism ; CRISPR-Cas Systems ; DNA/*immunology ; Disease Models, Animal ; Humans ; Hybridomas/transplantation ; Immunoglobulin G/metabolism ; Kidney Glomerulus/*pathology ; Lupus Erythematosus, Systemic/genetics/*immunology ; Lupus Nephritis/genetics/*immunology ; Mice ; Mice, Inbred MRL lpr ; Mice, Knockout ; Mice, SCID ; Proteinuria ; TWEAK Receptor/genetics/*metabolism ; },
abstract = {Many studies have demonstrated that anti-dsDNA IgG is closely associated with lupus nephritis. Recently, it was found that activation of the fibroblast growth factor-inducible 14 (Fn14) signaling pathway damages glomerular filtration barrier in MRL/lpr lupus-prone mice. However, MRL/lpr mice have high titers of serum autoantibodies other than anti-dsDNA IgG. The aim of this study was to further explore the effect of Fn14 deficiency on anti-dsDNA IgG-induced glomerular damage in severe combined immunodeficiency (SCID) mice that have no endogenous IgG. Fn14 deficiency was generated in SCID mice. The murine hybridoma cells producing control IgG or anti-dsDNA IgG were intraperitoneally injected into mice. In two weeks, the urine, serum, and kidney tissue samples were harvested from mice at sacrifice. It showed that the injection of anti-dsDNA IgG, but not control IgG hybridoma cells, induced proteinuria and glomerular damage in SCID mice. Between the wild-type (WT) and knockout (KO) mice injected with anti-dsDNA IgG hybridoma cells, the latter showed a decrease in both proteinuria and glomerular IgG deposition. The histopathological changes, inflammatory cell infiltration, and proinflammatory cytokine production were also attenuated in the kidneys of the Fn14-KO mice upon anti-dsDNA IgG injection. Therefore, Fn14 deficiency effectively protects SCID mice from anti-dsDNA IgG-induced glomerular damage.},
}
@article {pmid30648020,
year = {2019},
author = {García-Zea, JA and de la Herrán, R and Robles Rodríguez, F and Navajas-Pérez, R and Ruiz Rejón, C},
title = {Detection and variability analyses of CRISPR-like loci in the H. pylori genome.},
journal = {PeerJ},
volume = {7},
number = {},
pages = {e6221},
pmid = {30648020},
issn = {2167-8359},
abstract = {Helicobacter pylori is a human pathogenic bacterium with a high genomic plasticity. Although the functional CRISPR-Cas system has not been found in its genome, CRISPR-like loci have been recently identified. In this work, 53 genomes from different geographical areas are analyzed for the search and analysis of variability of this type of structure. We confirm the presence of a locus that was previously described in the VlpC gene in al lgenomes, and we characterize new CRISPR-like loci in other genomic locations. By studying the variability and gene location of these loci, the evolution and the possible roles of these sequences are discussed. Additionally, the usefulness of this type of sequences as a phylogenetic marker has been demonstrated, associating the different strains by geographical area.},
}
@article {pmid30647913,
year = {2018},
author = {Goodman, SL},
title = {The 3rd Antibody Validation meeting: Bath UK 20-21 [st] September 2018.},
journal = {F1000Research},
volume = {7},
number = {},
pages = {1989},
pmid = {30647913},
issn = {2046-1402},
mesh = {Antibodies/*metabolism/therapeutic use ; CRISPR-Cas Systems/genetics ; Drug Discovery ; Humans ; Reproducibility of Results ; United Kingdom ; },
abstract = {The International Antibody Validation meetings offer a welcome British forum for discussing this important topic, which is existentially crucial for the biological sciences community. Now in its 6th year, the biennial meeting is organized by Andrew Chalmers (University of Bath; CiteAb), this year with Carly Dix (Astra Zeneca). The organizers gathered some 100 members of industry and academia, producers and users, for a day and a half to describe their efforts to ensure that their antibodies have the desired specificity and selectively for well-defined molecular targets. The meeting is largely available as WebCasts (http://www.antibodyvalidation.co.uk/past-events/2018).},
}
@article {pmid30647128,
year = {2019},
author = {Alimov, I and Menon, S and Cochran, N and Maher, R and Wang, Q and Alford, J and Concannon, JB and Yang, Z and Harrington, E and Llamas, L and Lindeman, A and Hoffman, G and Schuhmann, T and Russ, C and Reece-Hoyes, J and Canham, SM and Cai, X},
title = {Bile acid analogues are activators of pyrin inflammasome.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {10},
pages = {3359-3366},
pmid = {30647128},
issn = {1083-351X},
mesh = {Bile Acids and Salts/chemistry/*immunology ; Epithelial Cells/*immunology ; Gastrointestinal Microbiome/*immunology ; Humans ; *Immunity, Mucosal ; Inflammasomes/*immunology ; Intestinal Mucosa/*immunology ; Myeloid Cells/immunology ; Pyrin/*immunology ; THP-1 Cells ; },
abstract = {Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.},
}
@article {pmid30646772,
year = {2019},
author = {Bao, A and Burritt, DJ and Chen, H and Zhou, X and Cao, D and Tran, LP},
title = {The CRISPR/Cas9 system and its applications in crop genome editing.},
journal = {Critical reviews in biotechnology},
volume = {39},
number = {3},
pages = {321-336},
doi = {10.1080/07388551.2018.1554621},
pmid = {30646772},
issn = {1549-7801},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics/growth &amp; development ; Gene Editing/*trends ; Genome, Plant/*genetics ; Plants, Genetically Modified/genetics/growth &amp; development ; },
abstract = {The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) system is an RNA-guided genome editing tool that consists of a Cas9 nuclease and a single-guide RNA (sgRNA). By base-pairing with a DNA target sequence, the sgRNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks (DSBs) that trigger cell repair mechanisms and mutations at or near the DSBs sites. Since its discovery, the CRISPR/Cas9 system has revolutionized genome editing and is now becoming widely utilized to edit the genomes of a diverse range of crop plants. In this review, we present an overview of the CRISPR/Cas9 system itself, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes. We evaluate recent examples of the use of CRISPR/Cas9 for crop plant improvement, and research into the function(s) of genes involved in determining crop yields, quality, environmental stress tolerance/resistance, regulation of gene transcription and translation, and the construction of mutant libraries and production of transgene-free genome-edited crops. In addition, challenges and future opportunities for the use of the CRISPR/Cas9 system in crop breeding are discussed.},
}
@article {pmid30645710,
year = {2019},
author = {Lee, K and Eggenberger, AL and Banakar, R and McCaw, ME and Zhu, H and Main, M and Kang, M and Gelvin, SB and Wang, K},
title = {CRISPR/Cas9-mediated targeted T-DNA integration in rice.},
journal = {Plant molecular biology},
volume = {99},
number = {4-5},
pages = {317-328},
pmid = {30645710},
issn = {1573-5028},
mesh = {Agrobacterium/genetics ; Base Sequence ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; DNA, Bacterial/*genetics ; Exons ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Gene Frequency ; Gene Targeting ; Genes, Plant/genetics ; Genetic Vectors/genetics ; Genome, Plant/*genetics ; INDEL Mutation ; Luminescent Proteins/genetics ; Mutagenesis, Insertional/*methods ; Oryza/*genetics/*metabolism ; Plant Proteins/genetics ; Plants, Genetically Modified/*genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics/metabolism ; Sequence Analysis ; },
abstract = {Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.},
}
@article {pmid30645648,
year = {2019},
author = {Li, Z and Wu, J and Chavez, L and Hoh, R and Deeks, SG and Pillai, SK and Zhou, Q},
title = {Reiterative Enrichment and Authentication of CRISPRi Targets (REACT) identifies the proteasome as a key contributor to HIV-1 latency.},
journal = {PLoS pathogens},
volume = {15},
number = {1},
pages = {e1007498},
pmid = {30645648},
issn = {1553-7374},
support = {R01 GM117901/GM/NIGMS NIH HHS/United States ; S10 OD018174/OD/NIH HHS/United States ; R01 AI150449/AI/NIAID NIH HHS/United States ; P30 AI027763/AI/NIAID NIH HHS/United States ; R01 AI041757/AI/NIAID NIH HHS/United States ; R24 AI067039/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-HIV Agents/therapeutic use ; CD4-Positive T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; HIV Infections/drug therapy/metabolism ; HIV Seropositivity ; HIV-1/*metabolism/pathogenicity ; Humans ; Jurkat Cells ; Proteasome Endopeptidase Complex/*metabolism/physiology ; Proteasome Inhibitors/metabolism/pharmacology ; Transcriptional Elongation Factors ; Virus Activation/drug effects ; Virus Latency/*drug effects/physiology ; },
abstract = {The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.},
}
@article {pmid30645582,
year = {2019},
author = {Chung, JE and Magis, W and Vu, J and Heo, SJ and Wartiovaara, K and Walters, MC and Kurita, R and Nakamura, Y and Boffelli, D and Martin, DIK and Corn, JE and DeWitt, MA},
title = {CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.},
journal = {PloS one},
volume = {14},
number = {1},
pages = {e0208237},
pmid = {30645582},
issn = {1932-6203},
support = {S10 OD018174/OD/NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA, Intergenic/genetics ; Erythroid Cells/*metabolism ; Fetal Hemoglobin/*metabolism ; Gene Editing ; Gene Silencing ; Genotype ; Hematopoietic Stem Cells/metabolism ; Humans ; Phenotype ; Repressor Proteins/*metabolism ; Sequence Deletion/genetics ; Up-Regulation/genetics ; gamma-Globins/genetics ; },
abstract = {Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.},
}
@article {pmid30644359,
year = {2019},
author = {Arunsan, P and Ittiprasert, W and Smout, MJ and Cochran, CJ and Mann, VH and Chaiyadet, S and Karinshak, SE and Sripa, B and Young, ND and Sotillo, J and Loukas, A and Brindley, PJ and Laha, T},
title = {Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30644359},
issn = {2050-084X},
support = {107475/Z/15/Z//Wellcome/International ; R01 CA164719/CA/NCI NIH HHS/United States ; R01CA164719/CA/NCI NIH HHS/United States ; PHD/0111/2557//Thailand Research Fund/International ; APP1117504//National Health and Medical Research Council/International ; APP1085309//National Health and Medical Research Council/International ; PHD/0111/2557//Royal Golden Jubilee PhD Program, Thailand/International ; APP1109829//National Health and Medical Research Council/International ; },
mesh = {Animals ; Bile Ducts, Intrahepatic/*parasitology/*pathology ; CRISPR-Cas Systems/genetics ; Carcinogenesis/pathology ; Cell Line ; Cell Proliferation ; Chronic Disease ; Cricetinae ; Fibrosis ; Gene Editing ; Gene Expression Regulation ; *Gene Knockout Techniques ; Genome ; Granulins/*genetics/metabolism ; Humans ; Hyperplasia ; Mutation/*genetics ; Opisthorchiasis/genetics/parasitology/pathology ; Opisthorchis/*pathogenicity ; Wound Healing ; },
abstract = {Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis.},
}
@article {pmid30644357,
year = {2019},
author = {Ittiprasert, W and Mann, VH and Karinshak, SE and Coghlan, A and Rinaldi, G and Sankaranarayanan, G and Chaidee, A and Tanno, T and Kumkhaek, C and Prangtaworn, P and Mentink-Kane, MM and Cochran, CJ and Driguez, P and Holroyd, N and Tracey, A and Rodpai, R and Everts, B and Hokke, CH and Hoffmann, KF and Berriman, M and Brindley, PJ},
title = {Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30644357},
issn = {2050-084X},
support = {R21AI109532//National Institute of Allergy and Infectious Diseases/International ; PHD/0047/2556//Royal Golden Jubilee Ph.D Program, Thailand/International ; 107475/Z/15/Z//Wellcome/International ; WT 098051//Wellcome/International ; PHD/0011/2555//Thailand Research Fund/International ; HHSN272201000005C/AI/NIAID NIH HHS/United States ; HHSN272201000005I/AI/NIAID NIH HHS/United States ; PHD/0047/2556//Thailand Research Fund/International ; PHD/00531/2556//Thailand Research Fund/International ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromosomes/genetics ; DNA Repair/genetics ; Exons/genetics ; *Gene Editing ; Gene Expression Regulation ; Genetic Loci ; Granuloma/pathology ; Homologous Recombination/genetics ; Humans ; Inflammation/pathology ; Lung/parasitology/pathology ; Mice ; Mutation/genetics ; Ovum/enzymology ; RNA, Messenger/genetics/metabolism ; Ribonucleases/*genetics ; Schistosoma mansoni/*enzymology/*genetics ; Th2 Cells/immunology ; Transgenes ; },
abstract = {CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.},
}
@article {pmid30644027,
year = {2019},
author = {Xu, ZS and Feng, K and Xiong, AS},
title = {CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants.},
journal = {Molecular biotechnology},
volume = {61},
number = {3},
pages = {191-199},
pmid = {30644027},
issn = {1559-0305},
mesh = {Agrobacterium ; CRISPR-Cas Systems ; Citrus sinensis/genetics/*growth &amp; development ; Daucus carota/genetics/*growth &amp; development ; Gene Editing/methods ; Mutagenesis, Site-Directed/*methods ; Mutation Rate ; Plant Proteins/*genetics ; RNA, Guide/genetics ; Sequence Analysis, DNA ; Transformation, Bacterial ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot 'Kurodagosun' resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot 'Deep purple', resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.},
}
@article {pmid30643127,
year = {2019},
author = {Nakamura, M and Srinivasan, P and Chavez, M and Carter, MA and Dominguez, AA and La Russa, M and Lau, MB and Abbott, TR and Xu, X and Zhao, D and Gao, Y and Kipniss, NH and Smolke, CD and Bondy-Denomy, J and Qi, LS},
title = {Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {194},
pmid = {30643127},
issn = {2041-1723},
support = {T32 GM008568/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Eukaryotic Cells ; Gene Editing/*methods ; Gene Regulatory Networks/*genetics ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Intravital Microscopy/methods ; Lentivirus/genetics ; Microscopy, Fluorescence/methods ; Time-Lapse Imaging/methods ; Transduction, Genetic/methods ; Transfection/methods ; },
abstract = {Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.},
}
@article {pmid30642460,
year = {2019},
author = {Javed, MR and Noman, M and Shahid, M and Ahmed, T and Khurshid, M and Rashid, MH and Ismail, M and Sadaf, M and Khan, F},
title = {Current situation of biofuel production and its enhancement by CRISPR/Cas9-mediated genome engineering of microbial cells.},
journal = {Microbiological research},
volume = {219},
number = {},
pages = {1-11},
doi = {10.1016/j.micres.2018.10.010},
pmid = {30642460},
issn = {1618-0623},
mesh = {Bacteria/*genetics/metabolism ; *Biofuels ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fungi/*genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial/genetics ; Genome, Fungal/genetics ; Renewable Energy ; },
abstract = {Geopolitical and economic factors have motivated the scientific community to utilize renewable energy resources. In addition to the modifications in major steps and processes of biofuel production, manipulation of microbial genome engineering tools is essential in order to find sustainable solution of continuous depletion of fossil-fuels. Recently, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9), a prokaryotic molecular immunity system, has emerged as a novel technology for targeted genomic engineering. This genetic machinery seems to be a groundbreaking discovery to engineer the microbial genomes for desired traits such as enhancing the biofuel tolerance, inhibitor tolerance and thermotolerance as well as modifying the cellulases and hemicelluloses enzymes. In this review, a summary of different generations of biofuels, integrated processes of bioconversion of raw materials into biofuels and role of microbes in biofuel production has been presented. However, the ultimate focus of the review is on major discoveries of CRISPR/Cas9-mediated genome editing in microorganisms and exploitation of these discoveries for enhanced biofuel production.},
}
@article {pmid30642400,
year = {2019},
author = {An, H and Skelt, L and Notaro, A and Highley, JR and Fox, AH and La Bella, V and Buchman, VL and Shelkovnikova, TA},
title = {ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles.},
journal = {Acta neuropathologica communications},
volume = {7},
number = {1},
pages = {7},
pmid = {30642400},
issn = {2051-5960},
support = {MRF_MRF-060-0001-RG-SHELK/MRF/MRF/United Kingdom ; SHELKOVNIKOVA/OCT17/968-799/MNDA_/Motor Neurone Disease Association/United Kingdom ; Buchman/Apr13/6096/MNDA_/Motor Neurone Disease Association/United Kingdom ; BUCHMAN/APR13/822-791/MNDA_/Motor Neurone Disease Association/United Kingdom ; Shelkovnikova/Oct17/968-799/MNDA_/Motor Neurone Disease Association/United Kingdom ; },
mesh = {Amyotrophic Lateral Sclerosis/genetics/*metabolism/*pathology ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; Humans ; Intranuclear Inclusion Bodies/*metabolism ; Loss of Function Mutation ; Protein Isoforms/metabolism ; RNA, Long Noncoding/*metabolism ; RNA-Binding Protein FUS/*genetics/*metabolism ; },
abstract = {Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.},
}
@article {pmid30642074,
year = {2019},
author = {Muramoto, T and Iriki, H and Watanabe, J and Kawata, T},
title = {Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Dictyostelium.},
journal = {Cells},
volume = {8},
number = {1},
pages = {},
pmid = {30642074},
issn = {2073-4409},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Dictyostelium/*genetics ; Gene Editing/*trends ; Genetic Vectors/*genetics ; Homologous Recombination ; },
abstract = {In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in Dictyostelium and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms. Using these vectors, precise genome modifications can be achieved within 2[-]3 weeks, beginning with the design of the target sequence. Finally, we discussed future perspectives on the use of CRISPR/Cas9-mediated genome editing in Dictyostelium.},
}
@article {pmid30640520,
year = {2019},
author = {Fan, HH and Yu, IS and Lin, YH and Wang, SY and Liaw, YH and Chen, PL and Yang, TL and Lin, SW and Chen, YT},
title = {P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {4},
pages = {5571-5584},
doi = {10.1096/fj.201802027R},
pmid = {30640520},
issn = {1530-6860},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Targeting/methods ; Germ Cells ; Mice ; Mice, Transgenic/genetics ; Mutagenesis/genetics ; Mutation/*genetics ; Neoplasms/*genetics ; Oncogenes/genetics ; RNA, Guide/genetics ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology facilitates somatic genome editing to reveal cooperative genetic interactions at the cellular level without extensive breeding between different mutant animals. Here we propose a transgenic inducible Cas9 effector-CRISPR mutagen (ICE CRIM) mouse model in which CRISPR/Cas9-mediated somatic mutagenesis events can occur in response to Cre expression. The well-known tumor suppressor gene, Trp53, and 2 important DNA mismatch repair genes, Mlh1 and Msh2, were selected to be our somatic mutagenesis targets. Amplicon-based sequencing was performed to validate the editing efficiency and to identify the mutant allelic series. Crossed with various Cre lines, the Trp53 ICE CRIM alleles were activated to generate targeted cancer gene somatic or germ line mutant variants. We provide experimental evidence to show that an activated ICE CRIM can mutate both targeted alleles within a cell. Simultaneous disruption of multiple genes was also achieved when there were multiple single-guide RNA expression cassettes embedded within an activated ICE CRIM. Our mouse model can be used to generate mutant pools in vivo, which enables a functional screen to be performed in situ. Our results also provide evidence to support a monoclonal origin of hematopoietic neoplasms and to indicate that DNA mismatch repair deficiency accelerates tumorigenesis in Trp53 mutant genetic background.-Fan, H.-H., Yu, I.-S., Lin, Y.-H., Wang, S.-Y., Liaw, Y.-H., Chen, P.-L., Yang, T.-L., Lin, S.-W., Chen, Y.-T. P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.},
}
@article {pmid30640437,
year = {2019},
author = {Gangopadhyay, SA and Cox, KJ and Manna, D and Lim, D and Maji, B and Zhou, Q and Choudhary, A},
title = {Precision Control of CRISPR-Cas9 Using Small Molecules and Light.},
journal = {Biochemistry},
volume = {58},
number = {4},
pages = {234-244},
pmid = {30640437},
issn = {1520-4995},
support = {R21 AI126239/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/antagonists &amp; inhibitors/genetics/metabolism ; *CRISPR-Cas Systems/drug effects ; Gene Expression Regulation ; Genetic Engineering/*methods ; Light ; RNA, Guide ; Recombinant Proteins/genetics/metabolism ; Small Molecule Libraries/chemistry/*pharmacology ; Tacrolimus Binding Proteins/genetics/metabolism ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations, immunogenic response, genotoxicity, and embryonic mosaicism are observed at elevated levels and with prolonged activity of Cas9. Here, we provide a perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas9.},
}
@article {pmid30639735,
year = {2019},
author = {Gürtler, S and Wolke, C and Otto, O and Heise, N and Scholz, F and Laporte, A and Elsner, M and Jörns, A and Weinert, S and Döring, M and Jansing, S and Gardemann, A and Lendeckel, U and Schild, L},
title = {Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1864},
number = {4},
pages = {452-465},
doi = {10.1016/j.bbalip.2019.01.006},
pmid = {30639735},
issn = {1879-2618},
mesh = {Acyltransferases ; Animals ; CRISPR-Cas Systems ; Cardiolipins/*metabolism ; Cell Line, Tumor ; Cell Proliferation ; Energy Metabolism ; Fatty Acids/metabolism ; Gene Knockout Techniques ; Glioma/genetics/*metabolism ; Glycolysis ; Mitochondria/*metabolism ; Oxidative Phosphorylation ; Rats ; Transcription Factors/*genetics/metabolism ; },
abstract = {The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.},
}
@article {pmid30639408,
year = {2019},
author = {Takeshita, D and Sato, M and Inanaga, H and Numata, T},
title = {Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR-Cas Effector Complex.},
journal = {Journal of molecular biology},
volume = {431},
number = {4},
pages = {748-763},
doi = {10.1016/j.jmb.2019.01.009},
pmid = {30639408},
issn = {1089-8638},
mesh = {Amino Acid Sequence ; Bacterial Proteins/*genetics ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; RNA, Bacterial/genetics ; Staphylococcus epidermidis/genetics ; Thermotoga maritima/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR-Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1-Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA. Here we report the crystal structures of the Staphylococcus epidermidis Csm2 (SeCsm2) and Thermoplasma volcanium Csm3 (TvCsm3) at 2.4- and 2.7-Å resolutions, respectively. SeCsm2 adopts a monomeric globular fold by itself, in striking contrast to the previously reported Thermotoga maritima Csm2, which adopted an extended conformation and formed a dimeric structure. We propose that the globular monomeric form is the bona fide structure of Csm2. TvCsm3 forms a filamentous structure in the crystals. The molecular arrangement of TvCsm3 is similar to that of the stacked Cmr4 proteins in the Cmr complex, suggesting the functionally relevant architecture of the present Csm3 structure. We constructed model structures of the Csm complex, which revealed that Csm3 binds the crRNA and periodically deforms the crRNA-target duplex by a similar mechanism to that of Cmr4 in the Cmr complex. The model and mutational analysis suggest that the conserved lysine residue of Csm2 is important for target RNA binding, and Csm2 stabilizes the active structure of the Csm complex to facilitate the reaction.},
}
@article {pmid30639242,
year = {2019},
author = {Kovalski, JR and Bhaduri, A and Zehnder, AM and Neela, PH and Che, Y and Wozniak, GG and Khavari, PA},
title = {The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {830-844.e12},
pmid = {30639242},
issn = {1097-4164},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; R01 AR043799/AR/NIAMS NIH HHS/United States ; R01 CA142635/CA/NCI NIH HHS/United States ; S10 RR027425/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Caco-2 Cells ; Cell Cycle Checkpoints ; Cell Proliferation ; Cell Transformation, Neoplastic/genetics/*metabolism/pathology ; Female ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; Humans ; Mechanistic Target of Rapamycin Complex 2/genetics/*metabolism ; Mice, Hairless ; Mice, SCID ; Mice, Transgenic ; Mutation ; Neoplasms/genetics/*metabolism/pathology ; Protein Binding ; Protein Interaction Domains and Motifs ; *Proteome ; Proteomics/methods ; Tumor Burden ; ras Proteins/genetics/*metabolism ; },
abstract = {Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-, K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycle transcriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia in vivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.},
}
@article {pmid30639240,
year = {2019},
author = {Swarts, DC and Jinek, M},
title = {Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.},
journal = {Molecular cell},
volume = {73},
number = {3},
pages = {589-600.e4},
pmid = {30639240},
issn = {1097-4164},
support = {149393/SNSF_/Swiss National Science Foundation/Switzerland ; 182567/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry/genetics/*metabolism ; Enzyme Activation ; Francisella/*enzymology/genetics ; Gene Editing/*methods ; Models, Molecular ; Nucleic Acid Conformation ; Protein Conformation ; RNA, Guide/chemistry/genetics/*metabolism ; Structure-Activity Relationship ; Substrate Specificity ; },
abstract = {CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.},
}
@article {pmid30639212,
year = {2019},
author = {Kagawa, H and Shimamoto, R and Kim, SI and Oceguera-Yanez, F and Yamamoto, T and Schroeder, T and Woltjen, K},
title = {OVOL1 Influences the Determination and Expansion of iPSC Reprogramming Intermediates.},
journal = {Stem cell reports},
volume = {12},
number = {2},
pages = {319-332},
pmid = {30639212},
issn = {2213-6711},
mesh = {Animals ; Biomarkers/metabolism ; CRISPR-Cas Systems/physiology ; Cell Adhesion Molecules/metabolism ; Cell Proliferation/physiology ; Cellular Reprogramming/*physiology ; DNA-Binding Proteins/*metabolism ; Epithelial Cells/metabolism/physiology ; Epithelial-Mesenchymal Transition/physiology ; Female ; Fibroblasts/metabolism/physiology ; Gene Expression Regulation/physiology ; Induced Pluripotent Stem Cells/*metabolism/*physiology ; Kruppel-Like Factor 4 ; Kruppel-Like Transcription Factors/metabolism ; Lewis X Antigen/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Transcription Factors/*metabolism ; },
abstract = {During somatic cell reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts undergo dynamic molecular changes, including a mesenchymal-to-epithelial transition (MET) and gain of pluripotency; processes that are influenced by Yamanaka factor stoichiometry. For example, in early reprogramming, high KLF4 levels are correlated with the induction of functionally undefined, transiently expressed MET genes. Here, we identified the cell-surface protein TROP2 as a marker for cells with transient MET induction in the high-KLF4 condition. We observed the emergence of cells expressing the pluripotency marker SSEA-1[+] mainly from within the TROP2[+] fraction. Using TROP2 as a marker in CRISPR/Cas9-mediated candidate screening of MET genes, we identified the transcription factor OVOL1 as a potential regulator of an alternative epithelial cell fate characterized by the expression of non-iPSC MET genes and low cell proliferation. Our study sheds light on how reprogramming factor stoichiometry alters the spectrum of intermediate cell fates, ultimately influencing reprogramming outcomes.},
}
@article {pmid30639098,
year = {2019},
author = {Henriksson, J and Chen, X and Gomes, T and Ullah, U and Meyer, KB and Miragaia, R and Duddy, G and Pramanik, J and Yusa, K and Lahesmaa, R and Teichmann, SA},
title = {Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation.},
journal = {Cell},
volume = {176},
number = {4},
pages = {882-896.e18},
pmid = {30639098},
issn = {1097-4172},
support = {/WT_/Wellcome Trust/United Kingdom ; WT206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics/immunology ; Chromatin ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome ; High-Throughput Nucleotide Sequencing ; Humans ; Lymphocyte Activation/genetics/immunology ; Mice ; Mice, Inbred C57BL ; Receptor Cross-Talk/*immunology ; T-Lymphocytes, Helper-Inducer/metabolism ; Th2 Cells/*immunology/*metabolism ; Transcription Factors/metabolism ; },
abstract = {T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.},
}
@article {pmid30638198,
year = {2018},
author = {Breunig, CT and Neuner, AM and Giehrl-Schwab, J and Wurst, W and Götz, M and Stricker, SH},
title = {A Customizable Protocol for String Assembly gRNA Cloning (STAgR).},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {142},
pages = {},
doi = {10.3791/58556},
pmid = {30638198},
issn = {1940-087X},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Genomics/methods ; RNA, Guide/*genetics ; },
abstract = {The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.},
}
@article {pmid30637496,
year = {2019},
author = {Gu, T and Yu, D and Li, Y and Xu, L and Yao, YL and Yao, YG},
title = {Establishment and characterization of an immortalized renal cell line of the Chinese tree shrew (Tupaia belangeri chinesis).},
journal = {Applied microbiology and biotechnology},
volume = {103},
number = {5},
pages = {2171-2180},
doi = {10.1007/s00253-019-09615-3},
pmid = {30637496},
issn = {1432-0614},
mesh = {Animals ; Antigens, Polyomavirus Transforming/genetics ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Line ; Disease Models, Animal ; Epithelial Cells/*metabolism ; Gene Editing/*methods ; HEK293 Cells ; Herpesvirus 1, Human/growth &amp; development ; Humans ; Immunity, Innate/*immunology ; Interferon-beta/biosynthesis ; Keratin-18/biosynthesis ; Kidney/*cytology ; Myxovirus Resistance Proteins/biosynthesis ; Newcastle disease virus/growth &amp; development ; Primary Cell Culture ; Tupaiidae ; Virus Diseases/*immunology ; },
abstract = {The Chinese tree shrew holds a great potential as a viable animal model in biomedical research, especially for infectious diseases and neuropsychiatric disorders. A thorough understanding of the innate immunity, which represents the first line that defends the host against viral infection, of the Chinese tree shrew, is needed. However, the progress is hindered by the lack of a proper cell line for research usage. In this study, we established a cell line that is applicable to the study of tree shrew innate immune responses against viral infections. The Chinese tree shrew primary renal cells (TSPRCs) were immortalized by simian virus 40 large T antigen (SV40LT) transduction, and the immortalized cells were termed TSR6 (tree shrew renal cell #6). TSR6 showed a similar morphology to TSPRCs and expressed the epithelial cell-specific marker cytokeratin 18 (KRT18). In addition, TSR6 could be transfected by transfection reagent and was suitable for CRISPR/Cas9-mediated gene editing. Infection of Newcastle disease virus (NDV) or herpes simplex virus 1 (HSV-1) in TSR6 induced the mRNA expression of tree shrew interferon-β (tIFNB1) and myxovirus resistance protein 1 (tMx1) in a dose- and time-dependent manner. Collectively, we successfully established a tree shrew renal cell line and demonstrated that this cell line was suitable for the study of the innate immune response to viral infections.},
}
@article {pmid30637337,
year = {2018},
author = {Bayer, K and Jahn, MT and Slaby, BM and Moitinho-Silva, L and Hentschel, U},
title = {Marine Sponges as Chloroflexi Hot Spots: Genomic Insights and High-Resolution Visualization of an Abundant and Diverse Symbiotic Clade.},
journal = {mSystems},
volume = {3},
number = {6},
pages = {},
pmid = {30637337},
issn = {2379-5077},
abstract = {Members of the widespread bacterial phylum Chloroflexi can dominate high-microbial-abundance (HMA) sponge microbiomes. In the Sponge Microbiome Project, Chloroflexi sequences amounted to 20 to 30% of the total microbiome of certain HMA sponge genera with the classes/clades SAR202, Caldilineae, and Anaerolineae being the most prominent. We performed metagenomic and single-cell genomic analyses to elucidate the functional gene repertoire of Chloroflexi symbionts of Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism, and respiration. Clade-specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae genomes, but only amino acid utilization by SAR202. While Anaerolineae and Caldilineae import cofactors and vitamins, SAR202 genomes harbor genes encoding components involved in cofactor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPR-Cas systems, eukaryote-like repeat proteins, and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by the fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM) method. Carbohydrate degradation potential was reported previously for "Candidatus Poribacteria" and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus, sponge microbes may not only provide nutrients to the sponge host, but they may also contribute to dissolved organic matter (DOM) recycling and primary productivity in reef ecosystems via a pathway termed the sponge loop. IMPORTANCE Chloroflexi represent a widespread, yet enigmatic bacterial phylum with few cultivated members. We used metagenomic and single-cell genomic approaches to characterize the functional gene repertoire of Chloroflexi symbionts in marine sponges. The results of this study suggest clade-specific metabolic specialization and that Chloroflexi symbionts have the genomic potential for dissolved organic matter (DOM) degradation from seawater. Considering the abundance and dominance of sponges in many benthic environments, we predict that the role of sponge symbionts in biogeochemical cycles is larger than previously thought.},
}
@article {pmid30636321,
year = {2019},
author = {Sung, LY and Wu, MY and Lin, MW and Hsu, MN and Truong, VA and Shen, CC and Tu, Y and Hwang, KY and Tu, AP and Chang, YH and Hu, YC},
title = {Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {5},
pages = {1066-1079},
doi = {10.1002/bit.26915},
pmid = {30636321},
issn = {1097-0290},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics ; Francisella/genetics ; *Gene Knockout Techniques ; *Genetic Engineering ; *Genome, Bacterial ; Staphylococcus aureus/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.},
}
@article {pmid30636320,
year = {2019},
author = {Westbrook, A and Tang, X and Marshall, R and Maxwell, CS and Chappell, J and Agrawal, DK and Dunlop, MJ and Noireaux, V and Beisel, CL and Lucks, J and Franco, E},
title = {Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {5},
pages = {1139-1151},
doi = {10.1002/bit.26918},
pmid = {30636320},
issn = {1097-0290},
mesh = {*CRISPR-Cas Systems ; Cell-Free System/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Models, Chemical ; RNA/*chemistry/metabolism ; *Transcription, Genetic ; *Transcriptional Activation ; },
abstract = {To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.},
}
@article {pmid30636294,
year = {2019},
author = {Mara, K and Charlot, F and Guyon-Debast, A and Schaefer, DG and Collonnier, C and Grelon, M and Nogué, F},
title = {POLQ plays a key role in the repair of CRISPR/Cas9-induced double-stranded breaks in the moss Physcomitrella patens.},
journal = {The New phytologist},
volume = {222},
number = {3},
pages = {1380-1391},
doi = {10.1111/nph.15680},
pmid = {30636294},
issn = {1469-8137},
support = {ANR-10-LABX-0040-SPS//Agence Nationale de la Recherche/International ; ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/International ; },
mesh = {Base Sequence ; Bleomycin/pharmacology ; Bryopsida/drug effects/*genetics/radiation effects ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cisplatin/pharmacology ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; DNA-Directed DNA Polymerase/genetics/*metabolism ; Genomic Instability ; Homologous Recombination/drug effects/radiation effects ; Methyl Methanesulfonate/pharmacology ; Mutation/genetics ; Mutation Rate ; Phenotype ; Ultraviolet Rays ; },
abstract = {Double-stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C-NHEJ) and alternative end joining (Alt-EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt-EJ-mediated DNA repair. Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development. The polq mutant shows the same sensitivity as wild-type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild-type. Furthermore, we show that POLQ is involved in the repair of CRISPR-Cas9-induced double-stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway. This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two-sided HR-mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding.},
}
@article {pmid30635671,
year = {2019},
author = {Schuster, JA and Vogel, RF and Ehrmann, MA},
title = {Characterization and distribution of CRISPR-Cas systems in Lactobacillus sakei.},
journal = {Archives of microbiology},
volume = {201},
number = {3},
pages = {337-347},
doi = {10.1007/s00203-019-01619-x},
pmid = {30635671},
issn = {1432-072X},
mesh = {Bacteriophages/*genetics ; Biological Evolution ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genotype ; Lactobacillus sakei/*genetics ; Polymerase Chain Reaction ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) structures, known as prokaryotes 'immune system', have been successfully applied for genetic engineering and genotyping purposes for a variety of microorganisms. Here we investigated 50 Lactobacillus (L.) sakei genomes and found 13 of them as CRISPR-Cas positive. The majority of positive genomes contain type II-A system, which appears to be widespread across food born lactic acid bacteria. However, a type II-C system with low similarity in Cas protein sequence to related II-C structures is rarely present in the genomes. We depicted a correlation between prophages integrated in the genomes and the presence/absence of CRISPR-Cas systems and identified the novel protospacer adjunction motifs (PAMs) (a/g)AAA for the II-A and (g/a)(c/t)AC for the II-C system including the corresponding tracrRNAs, creating the basis for the development of new Cas-mediated genome editing tools. Moreover, we performed a PCR screening for 81 selected L. sakei isolates and identified 25 (31%) isolates as CRISPR-Cas positive with hypervariable spacer content. Comparative sequence analysis of 33 repeat-spacer arrays resulted in 18 CRISPR genotypes, revealing insights into evolutionary relationships between different strains and illustrating possible applications for the research and development of starter cultures, e.g., the usage for strain differentiation in assertiveness experiments or the development of bacteriophage-resistant strains.},
}
@article {pmid30635443,
year = {2019},
author = {Goto, H and Natsume, T and Kanemaki, MT and Kaito, A and Wang, S and Gabazza, EC and Inagaki, M and Mizoguchi, A},
title = {Chk1-mediated Cdc25A degradation as a critical mechanism for normal cell cycle progression.},
journal = {Journal of cell science},
volume = {132},
number = {2},
pages = {},
doi = {10.1242/jcs.223123},
pmid = {30635443},
issn = {1477-9137},
mesh = {CRISPR-Cas Systems ; Checkpoint Kinase 1/genetics/*metabolism ; *DNA Damage ; *G2 Phase Cell Cycle Checkpoints ; Gene Editing ; HCT116 Cells ; Humans ; *Proteolysis ; *S Phase Cell Cycle Checkpoints ; cdc25 Phosphatases/genetics/*metabolism ; },
abstract = {Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during the DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) through CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell cycle arrest at S or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell cycle progression mainly by inducing Cdc25A degradation.},
}
@article {pmid30635074,
year = {2019},
author = {Taylor, VL and Fitzpatrick, AD and Islam, Z and Maxwell, KL},
title = {The Diverse Impacts of Phage Morons on Bacterial Fitness and Virulence.},
journal = {Advances in virus research},
volume = {103},
number = {},
pages = {1-31},
doi = {10.1016/bs.aivir.2018.08.001},
pmid = {30635074},
issn = {1557-8399},
mesh = {Bacteria/drug effects/genetics/pathogenicity/*virology ; Bacterial Adhesion ; Bacterial Physiological Phenomena ; Bacterial Toxins/genetics ; Bacteriophages/*physiology ; CRISPR-Cas Systems ; Drug Resistance, Bacterial/*genetics ; Host-Pathogen Interactions ; Humans ; Lysogeny ; Phage Therapy/*methods ; Prophages/*genetics ; Quorum Sensing/physiology ; Virulence ; },
abstract = {The viruses that infect bacteria, known as phages, are the most abundant biological entity on earth. They play critical roles in controlling bacterial populations through phage-mediated killing, as well as through formation of bacterial lysogens. In this form, the survival of the phage depends on the survival of the bacterial host in which it resides. Thus, it is advantageous for phages to encode genes that contribute to bacterial fitness and expand the environmental niche. In many cases, these fitness factors also make the bacteria better able to survive in human infections and are thereby considered pathogenesis or virulence factors. The genes that encode these fitness factors, known as "morons," have been shown to increase bacterial fitness through a wide range of mechanisms and play important roles in bacterial diseases. This review outlines the benefits provided by phage morons in various aspects of bacterial life, including phage and antibiotic resistance, motility, adhesion and quorum sensing.},
}
@article {pmid30633974,
year = {2019},
author = {Huang, H and Chai, C and Yang, S and Jiang, W and Gu, Y},
title = {Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii.},
journal = {Metabolic engineering},
volume = {52},
number = {},
pages = {293-302},
doi = {10.1016/j.ymben.2019.01.005},
pmid = {30633974},
issn = {1096-7184},
mesh = {Attachment Sites, Microbiological/genetics ; Bacteriophages/*enzymology/*genetics ; Butyric Acid/metabolism ; CRISPR-Cas Systems ; Clostridium/*genetics/*metabolism ; DNA, Bacterial/genetics ; Fermentation ; Genome, Bacterial/genetics ; Integrases/*genetics/*metabolism ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Plasmids/genetics ; Serine/metabolism ; },
abstract = {The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a "dual integrase cassette exchange" (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.},
}
@article {pmid30633905,
year = {2019},
author = {Oakes, BL and Fellmann, C and Rishi, H and Taylor, KL and Ren, SM and Nadler, DC and Yokoo, R and Arkin, AP and Doudna, JA and Savage, DF},
title = {CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.},
journal = {Cell},
volume = {176},
number = {1-2},
pages = {254-267.e16},
pmid = {30633905},
issn = {1097-4172},
support = {T32 HG000047/HG/NHGRI NIH HHS/United States ; DP2 EB018658/EB/NIBIB NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; S10 RR029668/RR/NCRR NIH HHS/United States ; S10 RR027303/RR/NCRR NIH HHS/United States ; R01 GM127463/GM/NIGMS NIH HHS/United States ; K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Proteins/chemistry ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; DNA/chemistry ; Gene Editing/*methods ; Genome ; Models, Molecular ; RNA/chemistry ; RNA, Guide/genetics ; },
abstract = {The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.},
}
@article {pmid30633431,
year = {2019},
author = {Schiedel, M and Moroglu, M and Ascough, DMH and Chamberlain, AER and Kamps, JJAG and Sekirnik, AR and Conway, SJ},
title = {Chemical Epigenetics: The Impact of Chemical and Chemical Biology Techniques on Bromodomain Target Validation.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {50},
pages = {17930-17952},
doi = {10.1002/anie.201812164},
pmid = {30633431},
issn = {1521-3773},
support = {MR/N009460/1/MRC_/Medical Research Council/United Kingdom ; EP/L015838/1//Engineering and Physical Sciences Research Council/International ; SCHI 1408/1-1//Deutsche Forschungsgemeinschaft/International ; },
mesh = {Acetylation ; Amino Acids/chemistry/metabolism ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Epigenomics/*methods ; Histones/metabolism ; Ligands ; Lysine/metabolism ; Magnetic Resonance Spectroscopy ; Molecular Biology/*methods ; Molecular Probes/chemistry ; Mutagenicity Tests ; Protein Domains/*genetics ; },
abstract = {Epigenetics is currently the focus of intense research interest across a broad range of disciplines due to its importance in a multitude of biological processes and disease states. Epigenetic functions result partly from modification of the nucleobases in DNA and RNA, and/or post-translational modifications of histone proteins. These modifications are dynamic, with cellular machinery identified to modulate and interpret the marks. Our focus is on bromodomains, which bind to acetylated lysine residues. Progress in the study of bromodomains, and the development of bromodomain ligands, has been rapid. These advances have been underpinned by many disciplines, but chemistry and chemical biology have undoubtedly played a significant role. Herein, we review the key chemistry and chemical biology approaches that have furthered our study of bromodomains, enabled the development of bromodomain ligands, and played a critical role in the validation of bromodomains as therapeutic targets.},
}
@article {pmid30633425,
year = {2019},
author = {Kis, A and Hamar, &#xc9; and Tholt, G and Bán, R and Havelda, Z},
title = {Creating highly efficient resistance against wheat dwarf virus in barley by employing CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {17},
number = {6},
pages = {1004-1006},
pmid = {30633425},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/*genetics ; Geminiviridae/*physiology ; Hordeum/*genetics/*virology ; },
}
@article {pmid30632384,
year = {2019},
author = {Gulei, D and Raduly, L and Berindan-Neagoe, I and Calin, GA},
title = {CRISPR-based RNA editing: diagnostic applications and therapeutic options.},
journal = {Expert review of molecular diagnostics},
volume = {19},
number = {2},
pages = {83-88},
doi = {10.1080/14737159.2019.1568242},
pmid = {30632384},
issn = {1744-8352},
support = {UH3 TR000943/TR/NCATS NIH HHS/United States ; R01 CA182905/CA/NCI NIH HHS/United States ; R01 CA222007/CA/NCI NIH HHS/United States ; R01 GM122775/GM/NIGMS NIH HHS/United States ; U54 CA096297/CA/NCI NIH HHS/United States ; U54 CA096300/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA Editing ; },
}
@article {pmid30629682,
year = {2019},
author = {Aslam, MA and Alemdehy, MF and Pritchard, CEJ and Song, JY and Muhaimin, FI and Wijdeven, RH and Huijbers, IJ and Neefjes, J and Jacobs, H},
title = {Towards an understanding of C9orf82 protein/CAAP1 function.},
journal = {PloS one},
volume = {14},
number = {1},
pages = {e0210526},
pmid = {30629682},
issn = {1932-6203},
mesh = {Animals ; Apoptosis ; Apoptosis Regulatory Proteins/genetics/*physiology ; B-Lymphocytes/cytology/metabolism ; CRISPR-Cas Systems ; Caspase 3/metabolism ; Cell Proliferation ; Cells, Cultured ; *DNA Breaks, Double-Stranded ; DNA Damage ; DNA Repair ; DNA Topoisomerases, Type II/*metabolism ; Immunoglobulin Class Switching ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; T-Lymphocytes/cytology/metabolism ; },
abstract = {C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.},
}
@article {pmid30628160,
year = {2019},
author = {Hai, L and Szwarc, MM and Lanza, DG and Heaney, JD and Lydon, JP},
title = {Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {57},
number = {3},
pages = {e23281},
pmid = {30628160},
issn = {1526-968X},
support = {P30 CA125123/CA/NCI NIH HHS/United States ; R01 HD042311/HD/NICHD NIH HHS/United States ; HD042311/HD/NICHD NIH HHS/United States ; U42 HG0063521/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Body Patterning ; Bone and Bones/abnormalities/embryology ; *CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Hindlimb/abnormalities/embryology ; Mice ; Mice, Inbred C57BL ; Promyelocytic Leukemia Zinc Finger Protein/deficiency/*genetics ; },
abstract = {The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (Plzf [KO]) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzf [f/f]). Crossing our Plzf [f/f] mouse with a global cre-driver mouse to generate the Plzf [d/d] bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzf [d/d] bigenic. Similar to the previously reported Plzf [KO] mouse, the Plzf [d/d] mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzf [f/f] mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzf [f/f] mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type.},
}
@article {pmid30628157,
year = {2019},
author = {Ji, X and Du, Y and Li, F and Sun, H and Zhang, J and Li, J and Peng, T and Xin, Z and Zhao, Q},
title = {The basic helix-loop-helix transcription factor, OsPIL15, regulates grain size via directly targeting a purine permease gene OsPUP7 in rice.},
journal = {Plant biotechnology journal},
volume = {17},
number = {8},
pages = {1527-1537},
pmid = {30628157},
issn = {1467-7652},
mesh = {Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; CRISPR-Cas Systems ; Edible Grain/growth &amp; development ; Gene Expression Regulation, Plant ; Nucleobase Transport Proteins/*genetics ; Oryza/enzymology/*genetics ; Plant Proteins/genetics/*metabolism ; Seeds/*growth &amp; development ; },
abstract = {As members of the basic helix-loop-helix transcription factor families, phytochrome-interacting factors regulate an array of developmental responses ranging from seed germination to plant growth. However, little is known about their roles in modulating grain development. Here, we firstly analyzed the expression pattern of rice OsPIL genes in grains and found that OsPIL15 may play an important role in grain development. We then generated knockout (KO) OsPIL15 lines in rice using CRISPR/Cas9 technology, the silencing expression of OsPIL15 led to increased numbers of cells, which thus enhanced grain size and weight. Moreover, overexpression and suppression of OsPIL15 in the rice endosperm resulted in brown rice showing grain sizes and weights that were decreased and increased respectively. Further studies indicated that OsPIL15 binds to N1-box (CACGCG) motifs of the purine permease gene OsPUP7 promoter. Measurement of isopentenyl adenosine, a bioactive form of cytokinin (CTK), revealed increased contents in the OsPIL15-KO spikelets compared with the wild-type. Overall, our results demonstrate a possible pathway whereby OsPIL15 directly targets OsPUP7, affecting CTK transport and thereby influencing cell division and subsequent grain size. These findings provide a valuable insight into the molecular functions of OsPIL15 in rice grains, highlighting a useful genetic improvement leading to increased rice yield.},
}
@article {pmid30626373,
year = {2019},
author = {Hao, Z and Su, X},
title = {Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {2},
pmid = {30626373},
issn = {1472-6750},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cellulose 1,4-beta-Cellobiosidase/genetics ; DNA Fragmentation ; Gene Deletion ; Gene Editing/*methods ; Mutagenesis, Insertional ; RNA, Guide/*genetics ; Trichoderma/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome.<br><br>RESULTS: We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus.<br><br>CONCLUSIONS: Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.},
}
@article {pmid30625150,
year = {2019},
author = {Castel, B and Tomlinson, L and Locci, F and Yang, Y and Jones, JDG},
title = {Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis.},
journal = {PloS one},
volume = {14},
number = {1},
pages = {e0204778},
pmid = {30625150},
issn = {1932-6203},
support = {BB/K003550/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014130/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M003809/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Agrobacterium tumefaciens/genetics ; Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; DNA, Bacterial/*genetics ; Gene Editing/*methods ; Genetic Vectors ; Mutagenesis, Site-Directed/*methods ; Plants, Genetically Modified ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/chemistry/genetics ; Transformation, Bacterial/genetics ; },
abstract = {Bacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.},
}
@article {pmid30623494,
year = {2019},
author = {Wang, J and Liu, M and Zhao, L and Li, Y and Zhang, M and Jin, Y and Xiong, Q and Liu, X and Zhang, L and Jiang, H and Chen, Q and Wang, C and You, Z and Yang, H and Cao, C and Dai, Y and Li, R},
title = {Disabling of nephrogenesis in porcine embryos via CRISPR/Cas9-mediated SIX1 and SIX4 gene targeting.},
journal = {Xenotransplantation},
volume = {26},
number = {3},
pages = {e12484},
doi = {10.1111/xen.12484},
pmid = {30623494},
issn = {1399-3089},
mesh = {Animals ; Blastocyst/metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Targeting ; Genes, Homeobox/*genetics ; Homeodomain Proteins/genetics ; Nuclear Proteins/*metabolism ; Nuclear Transfer Techniques ; Swine ; Trans-Activators/genetics ; Transplantation, Heterologous/methods ; },
abstract = {SIX1 and SIX4 genes play critical roles in kidney development. We evaluated the effect of these genes on pig kidney development by generating SIX1[-/-] and SIX1[-/-] /SIX4[-/-] pig foetuses using CRISPR/Cas9 and somatic cell nuclear transfer. We obtained 3 SIX1[-/-] foetuses and 16 SIX1[-/-] /SIX4[-/-] foetuses at different developmental stages. The SIX1[-/-] foetuses showed a migration block of the left kidney and a smaller size for both kidneys. The ureteric bud failed to form the normal branching and collecting system. Abnormal expressions of kidney development-related genes (downregulation of PAX2, PAX8, and BMP4 and upregulation of EYA1 and SALL1) were also observed in SIX1[-/-] foetal kidneys and confirmed in vitro in porcine kidney epithelial cells (PK15) following SIX1 gene deletion. The SIX1[-/-] /SIX4[-/-] foetuses exhibited more severe phenotypes, with most foetuses showing retarded development at early stages of gestation. The kidney developed only to the initial stage of metanephros formation. These results demonstrated that SIX1 and SIX4 are key genes for porcine metanephros development. The creation of kidney-deficient porcine foetuses provides a platform for generating human kidneys inside pigs using blastocyst complementation.},
}
@article {pmid30622340,
year = {2019},
author = {Domenici, G and Aurrekoetxea-Rodríguez, I and Simões, BM and Rábano, M and Lee, SY and Millán, JS and Comaills, V and Oliemuller, E and López-Ruiz, JA and Zabalza, I and Howard, BA and Kypta, RM and Vivanco, MD},
title = {A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells.},
journal = {Oncogene},
volume = {38},
number = {17},
pages = {3151-3169},
pmid = {30622340},
issn = {1476-5594},
mesh = {Breast/cytology/*metabolism ; Breast Neoplasms/drug therapy/genetics/*metabolism ; Cell Line ; Cell Proliferation ; *Drug Resistance, Neoplasm ; Epithelial Cells/cytology ; Estrogens/pharmacology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; Neoplastic Stem Cells/*metabolism ; SOX9 Transcription Factor/genetics/*metabolism ; SOXB1 Transcription Factors/*metabolism ; Signal Transduction ; Tamoxifen/pharmacology ; Up-Regulation ; },
abstract = {Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.},
}
@article {pmid30622266,
year = {2019},
author = {Kandul, NP and Liu, J and Sanchez C, HM and Wu, SL and Marshall, JM and Akbari, OS},
title = {Transforming insect population control with precision guided sterile males with demonstration in flies.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {84},
pmid = {30622266},
issn = {2041-1723},
support = {K22 AI113060/AI/NIAID NIH HHS/United States ; R21 AI123937/AI/NIAID NIH HHS/United States ; 5K22AI113060/NH/NIH HHS/United States ; 1R21AI123937/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Drosophila/*genetics/physiology ; Female ; Gene Editing/*methods ; Genome, Insect/genetics ; Insect Vectors/*genetics/physiology ; Male ; Models, Biological ; Pest Control, Biological/*methods ; Population Control/methods ; RNA, Guide/genetics ; *Sexual Behavior, Animal ; },
abstract = {The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.},
}
@article {pmid30622252,
year = {2019},
author = {Balmus, G and Pilger, D and Coates, J and Demir, M and Sczaniecka-Clift, M and Barros, AC and Woods, M and Fu, B and Yang, F and Chen, E and Ostermaier, M and Stankovic, T and Ponstingl, H and Herzog, M and Yusa, K and Martinez, FM and Durant, ST and Galanty, Y and Beli, P and Adams, DJ and Bradley, A and Metzakopian, E and Forment, JV and Jackson, SP},
title = {ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {87},
pmid = {30622252},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Ataxia Telangiectasia Mutated Proteins/*genetics/metabolism ; BRCA1 Protein/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; DNA Breaks, Double-Stranded/drug effects ; DNA End-Joining Repair/*genetics ; DNA Ligase ATP/metabolism ; DNA Replication/drug effects/genetics ; Drug Resistance, Neoplasm/*genetics ; Female ; Humans ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mouse Embryonic Stem Cells ; Mutation ; Neoplasms, Experimental/drug therapy/genetics/pathology ; Phthalazines/pharmacology/therapeutic use ; Piperazines/pharmacology/therapeutic use ; Topotecan/pharmacology/therapeutic use ; },
abstract = {Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.},
}
@article {pmid30622144,
year = {2019},
author = {Nasri, M and Mir, P and Dannenmann, B and Amend, D and Skroblyn, T and Xu, Y and Schulze-Osthoff, K and Klimiankou, M and Welte, K and Skokowa, J},
title = {Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response.},
journal = {Blood advances},
volume = {3},
number = {1},
pages = {63-71},
pmid = {30622144},
issn = {2473-9537},
mesh = {Antigens, Differentiation/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; DNA Damage ; Gene Editing/methods ; *Gene Knockout Techniques ; Gene Targeting/methods ; Hematopoietic Stem Cells/*metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Macromolecular Substances/metabolism ; Protein Binding ; RNA, Guide/genetics ; Ribonucleoproteins/*metabolism ; Stress, Physiological/*genetics/radiation effects ; },
abstract = {CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9-gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.},
}
@article {pmid30621855,
year = {2019},
author = {Homanics, GE},
title = {Gene-edited CRISPy Critters for alcohol research.},
journal = {Alcohol (Fayetteville, N.Y.)},
volume = {74},
number = {},
pages = {11-19},
pmid = {30621855},
issn = {1873-6823},
support = {R01 AA010422/AA/NIAAA NIH HHS/United States ; R37 AA010422/AA/NIAAA NIH HHS/United States ; U01 AA020889/AA/NIAAA NIH HHS/United States ; },
mesh = {Alcoholism/*etiology/genetics ; Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Mice ; Rats ; Transcription Activator-Like Effector Nucleases/physiology ; },
abstract = {Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.},
}
@article {pmid30621179,
year = {2019},
author = {Xu, CL and Ruan, MZC and Mahajan, VB and Tsang, SH},
title = {Viral Delivery Systems for CRISPR.},
journal = {Viruses},
volume = {11},
number = {1},
pages = {},
pmid = {30621179},
issn = {1999-4915},
support = {P30EY019007/NH/NIH HHS/United States ; R01EY018213/NH/NIH HHS/United States ; R01EY024698/NH/NIH HHS/United States ; R01EY026682/NH/NIH HHS/United States ; R21AG050437/NH/NIH HHS/United States ; R24EY027285/NH/NIH HHS/United States ; 5P30CA013696/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods ; Genetic Therapy ; *Genetic Vectors ; Genome ; Humans ; Mice ; Models, Animal ; Viruses/*genetics ; },
abstract = {The frontiers of precision medicine have been revolutionized by the development of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 as an editing tool. CRISPR/Cas9 has been used to develop animal models, understand disease mechanisms, and validate treatment targets. In addition, it is regarded as an effective tool for genome surgery when combined with viral delivery vectors. In this article, we will explore the various viral mechanisms for delivering CRISPR/Cas9 into tissues and cells, as well as the benefits and drawbacks of each method. We will also review the history and recent development of CRISPR and viral vectors and discuss their applications as a powerful tool in furthering our exploration of disease mechanisms and therapies.},
}
@article {pmid30619231,
year = {2018},
author = {Berges, M and Michel, AM and Lassek, C and Nuss, AM and Beckstette, M and Dersch, P and Riedel, K and Sievers, S and Becher, D and Otto, A and Maaß, S and Rohde, M and Eckweiler, D and Borrero-de Acuña, JM and Jahn, M and Neumann-Schaal, M and Jahn, D},
title = {Iron Regulation in Clostridioides difficile.},
journal = {Frontiers in microbiology},
volume = {9},
number = {},
pages = {3183},
pmid = {30619231},
issn = {1664-302X},
abstract = {The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 μM) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 μM) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F0F1-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.},
}
@article {pmid30618248,
year = {2019},
author = {Liu, Z and Zhang, Y and Nielsen, J},
title = {Synthetic Biology of Yeast.},
journal = {Biochemistry},
volume = {58},
number = {11},
pages = {1511-1520},
doi = {10.1021/acs.biochem.8b01236},
pmid = {30618248},
issn = {1520-4995},
mesh = {CRISPR-Cas Systems ; Gene Regulatory Networks/genetics ; Genetic Engineering/methods ; Metabolic Engineering/methods ; Promoter Regions, Genetic/genetics ; Protein Engineering/methods/trends ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/*biosynthesis/genetics/metabolism ; Synthetic Biology/*methods/*trends ; },
abstract = {With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.},
}
@article {pmid30618028,
year = {2019},
author = {Mu, W and Tang, N and Cheng, C and Sun, W and Wei, X and Wang, H},
title = {In vitro transcribed sgRNA causes cell death by inducing interferon release.},
journal = {Protein &amp; cell},
volume = {10},
number = {6},
pages = {461-465},
pmid = {30618028},
issn = {1674-8018},
mesh = {*CRISPR-Cas Systems ; Cell Death ; Gene Editing ; HeLa Cells ; Hematopoietic Stem Cells/*cytology ; Humans ; Interferons ; RNA, Guide/*physiology ; T-Lymphocytes/*cytology ; },
}
@article {pmid30617347,
year = {2019},
author = {Peters, JM and Koo, BM and Patino, R and Heussler, GE and Hearne, CC and Qu, J and Inclan, YF and Hawkins, JS and Lu, CHS and Silvis, MR and Harden, MM and Osadnik, H and Peters, JE and Engel, JN and Dutton, RJ and Grossman, AD and Gross, CA and Rosenberg, OS},
title = {Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.},
journal = {Nature microbiology},
volume = {4},
number = {2},
pages = {244-250},
pmid = {30617347},
issn = {2058-5276},
support = {F32 GM108222/GM/NIGMS NIH HHS/United States ; T32 GM007287/GM/NIGMS NIH HHS/United States ; T32 AI060537/AI/NIAID NIH HHS/United States ; R35 GM118061/GM/NIGMS NIH HHS/United States ; R01 AI128214/AI/NIAID NIH HHS/United States ; R01 GM102790/GM/NIGMS NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/pharmacology ; Bacteria/classification/drug effects/*genetics ; Bacterial Proteins/*genetics/metabolism ; Bacteriological Techniques/*methods ; *CRISPR-Cas Systems ; Conjugation, Genetic ; Drug Resistance, Microbial/genetics ; Gene Library ; Gene Regulatory Networks ; Gene Targeting ; Genes, Essential/genetics ; *Genetic Techniques ; Genome, Bacterial/genetics ; },
abstract = {The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans[1-6]. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.},
}
@article {pmid30617277,
year = {2019},
author = {Jang, Y and Choi, J and Park, N and Kang, J and Kim, M and Kim, Y and Ju, JH},
title = {Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering.},
journal = {Experimental &amp; molecular medicine},
volume = {51},
number = {1},
pages = {1-11},
pmid = {30617277},
issn = {2092-6413},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; HLA Antigens/*genetics ; *Histocompatibility ; Humans ; Induced Pluripotent Stem Cells/cytology/*immunology/transplantation ; Male ; Mice ; Mice, Nude ; },
abstract = {Pluripotent stem cell transplantation is a promising regenerative strategy for treating intractable diseases. However, securing human leukocyte antigen (HLA)-matched donor stem cells is extremely difficult. The traditional approach for generating such cells is to establish homozygous pluripotent stem cell lines. Unfortunately, because of HLA diversity, this strategy is too time-consuming to be of practical use. HLA engineering of donor stem cells has been proposed recently as a means to evade graft-versus-host rejection in stem cell allotransplantation. This approach would be advantageous in both time and cost to the traditional method, but its feasibility must be investigated. In this study, we used CRISPR/Cas9 to knockout HLA-B from inducible pluripotent stem cells (iPSCs) with heterogenous HLA-B and showed that the HLA-B knockout iPSCs resulted in less immunogenicity in HLA-B antisera than that in the control. Our results support the feasibility of HLA-engineered iPSCs in stem cell allotransplantation.},
}
@article {pmid30617110,
year = {2019},
author = {Atakpa, P and van Marrewijk, LM and Apta-Smith, M and Chakraborty, S and Taylor, CW},
title = {GPN does not release lysosomal Ca[2+] but evokes Ca[2+] release from the ER by increasing the cytosolic pH independently of cathepsin C.},
journal = {Journal of cell science},
volume = {132},
number = {3},
pages = {},
pmid = {30617110},
issn = {1477-9137},
support = {//Wellcome Trust/United Kingdom ; 101888/WT_/Wellcome Trust/United Kingdom ; BB/P005330/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Biological Transport ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Channels/genetics/metabolism ; Calcium Signaling/*drug effects ; Cathepsin C/genetics/metabolism ; Cell Line, Tumor ; Cytosol/*drug effects/metabolism ; Dipeptides/*pharmacology ; Endoplasmic Reticulum/*drug effects/metabolism ; Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Hydrogen-Ion Concentration/drug effects ; Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Leukocytes/cytology/drug effects/metabolism ; Lysosome-Associated Membrane Glycoproteins/genetics/metabolism ; Lysosomes/drug effects/metabolism ; Ploidies ; Ryanodine Receptor Calcium Release Channel/genetics/metabolism ; },
abstract = {The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca[2+] concentration ([Ca[2+]]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca[2+]]c require Ca[2+] within the endoplasmic reticulum (ER), but they are not mediated by ER Ca[2+] channels amplifying Ca[2+] release from lysosomes. GPN increases [Ca[2+]]c by increasing pHcyt, which then directly stimulates Ca[2+] release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca[2+] release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.},
}
@article {pmid30616826,
year = {2019},
author = {Saunders, CA and Majumdar, R and Molina, Y and Subramanian, BC and Parent, CA},
title = {Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay.},
journal = {Methods in cell biology},
volume = {149},
number = {},
pages = {31-56},
pmid = {30616826},
issn = {0091-679X},
support = {ZIA BC011216/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Biological Assay/*methods ; CRISPR-Cas Systems/genetics ; Cell Differentiation/drug effects ; *Chemotaxis/drug effects ; Dimethyl Sulfoxide/pharmacology ; HEK293 Cells ; Humans ; Sepharose/*chemistry ; },
abstract = {Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible. PLB-985 cells, which are pluripotent hematopoietic cells that can easily be differentiated to neutrophil-like cells, are amenable to genetic manipulations, including the expression of fluorescently tagged proteins-of-interest (POI) and gene editing using the CRISPR/CAS9 system to delete genes-of-interest (GOI). The use of PLB-985 cells can therefore greatly facilitate our understanding of the molecular mechanisms governing neutrophil biology during chemotaxis and serve as a good system to complement results gained from pharmacological inhibition of primary neutrophils. To better study the role and localization of proteins during chemotaxis, the underagarose assay has become a widely used and quantitative assay for measuring several aspects of chemotaxis. The objective of this chapter is to provide protocols for (1) the generation of genetically altered PLB-985 cell lines, (2) the set-up of an underagarose chemotaxis assay, and (3) the analysis of cell movement in chemotactic gradients from an underagarose experiment.},
}
@article {pmid30616753,
year = {2019},
author = {Parameswaran, S and Kundapur, D and Vizeacoumar, FS and Freywald, A and Uppalapati, M and Vizeacoumar, FJ},
title = {A Road Map to Personalizing Targeted Cancer Therapies Using Synthetic Lethality.},
journal = {Trends in cancer},
volume = {5},
number = {1},
pages = {11-29},
doi = {10.1016/j.trecan.2018.11.001},
pmid = {30616753},
issn = {2405-8025},
support = {PJT-156401//CIHR/Canada ; PJT-156309//CIHR/Canada ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Gene Editing ; Genetic Heterogeneity ; Humans ; *Molecular Targeted Therapy/methods ; Neoplasms/*drug therapy/*genetics/metabolism/pathology ; *Precision Medicine/methods ; RNA, Small Interfering/genetics ; Signal Transduction ; *Synthetic Lethal Mutations ; },
abstract = {Targeted therapies rely on the genetic and epigenetic status of the tumor cells and are seen as the most promising approach to treat cancer today. However, current targeted therapies focus on directly inhibiting those molecules that are altered in tumor cells. Unfortunately, targeting these molecules, even with specific inhibitors, is challenging as tumor cells rewire their genetic circuitry to eliminate genetic dependency on these targets. Here, we describe how synthetic lethality approaches can be used to identify genetic dependencies and develop personalized targeted therapies. We also discuss strategies to specifically target these genetic dependencies, using small molecule and biologic drugs.},
}
@article {pmid30616506,
year = {2019},
author = {Dagur, RS and Branch-Woods, A and Mathews, S and Joshi, PS and Quadros, RM and Harms, DW and Cheng, Y and Miles, SM and Pirruccello, SJ and Gurumurthy, CB and Gorantla, S and Poluektova, LY},
title = {Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection.},
journal = {BMC immunology},
volume = {20},
number = {1},
pages = {2},
pmid = {30616506},
issn = {1471-2172},
support = {R24 OD018546/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Genetic Loci ; Glycoproteins/*metabolism ; HIV Infections/genetics/*immunology/*metabolism/*virology ; *HIV-1 ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/immunology/metabolism/virology ; Immune System/cytology/immunology/metabolism ; Immunophenotyping ; Leukocytes, Mononuclear/immunology/metabolism ; Lymphocytes/*immunology/*metabolism/*virology ; Mice ; Mice, Knockout ; Phenotype ; },
abstract = {BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins' chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known.<br><br>RESULTS: We mutated mouse CMAH in the NOD/scid-IL2R&#x3b3;c[-/-] (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8.<br><br>CONCLUSION: NSG-cmah[-/-] mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.},
}
@article {pmid30616338,
year = {2019},
author = {Shi, TQ and Gao, J and Wang, WJ and Wang, KF and Xu, GQ and Huang, H and Ji, XJ},
title = {CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production.},
journal = {ACS synthetic biology},
volume = {8},
number = {2},
pages = {445-454},
doi = {10.1021/acssynbio.8b00478},
pmid = {30616338},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Fungi/*genetics/*metabolism ; Fusarium/*genetics/*metabolism ; Gene Editing/methods ; Genome, Fungal/genetics ; Gibberellins/*metabolism ; },
abstract = {The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research toward making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTBNLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F. fujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What's more, multigene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.},
}
@article {pmid30616324,
year = {2018},
author = {Wang, YD and Liang, QF and Li, ZY and Zhao, CY},
title = {[Application of clustered regularly interspaced short palindromic repeats- associated protein 9 gene editing technology for treatment of HBV infection].},
journal = {Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology},
volume = {26},
number = {11},
pages = {860-864},
doi = {10.3760/cma.j.issn.1007-3418.2018.11.012},
pmid = {30616324},
issn = {1007-3418},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Hepatitis B/*therapy ; *Hepatitis B virus ; Virus Replication ; },
abstract = {A lack of effective drugs and technical means to eradicate hepatitis B virus (HBV) is a bottleneck that limits the ability to fully cure HBV infection. Recently, genome-editing technology based on clustered regularly interspaced short palindromic repeats -associated protein 9 is an emerging technique for editing specific gene loci, which can specifically target HBV covalently closed circular DNA, effectively inhibits HBV DNA replication and regulates HBV functional protein expression, and is expected to become a powerful gene therapy tool for the complete eradication of HBV. Considering this, it has become the focus of attention for scholars at home and abroad that how to use clustered regularly interspaced short palindromic repeats -associated protein 9 to accomplish modification of HBV genomes for complete eradication of HBV. This paper summarizes the latest progress based on the latest research results at home and abroad in the application of clustered regularly interspaced short palindromic repeats -associated protein 9 gene editing technology in anti-HBV infection treatment, and expounds its potential and challenges as a radical cure for HBV infection.},
}
@article {pmid30615645,
year = {2019},
author = {Price, MA and Cruz, R and Baxter, S and Escalettes, F and Rosser, SJ},
title = {CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis.},
journal = {PloS one},
volume = {14},
number = {1},
pages = {e0210121},
pmid = {30615645},
issn = {1932-6203},
mesh = {Bacillus subtilis/*genetics ; Bacterial Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Genetic Engineering ; Industrial Microbiology/methods ; Membrane Transport Proteins/*genetics ; Plasmids ; Subtilisins/*genetics ; Transformation, Bacterial/genetics ; },
abstract = {CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.},
}
@article {pmid30615056,
year = {2019},
author = {Zhu, H and Liang, C},
title = {CRISPR-DT: designing gRNAs for the CRISPR-Cpf1 system with improved target efficiency and specificity.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {16},
pages = {2783-2789},
doi = {10.1093/bioinformatics/bty1061},
pmid = {30615056},
issn = {1367-4811},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Endonucleases ; Gene Editing ; RNA, Guide ; },
abstract = {MOTIVATION: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cpf1 system has been successfully applied in genome editing. However, target efficiency of the CRISPR-Cpf1 system varies among different guide RNA (gRNA) sequences.<br><br>RESULTS: In this study, we reanalyzed the published CRISPR-Cpf1 gRNAs data and found many sequence and structural features related to their target efficiency. With the aid of Random Forest in feature selection, a support vector machine model was created to predict target efficiency for any given gRNAs. We have developed the first CRISPR-Cpf1 web service application, CRISPR-DT (CRISPR DNA Targeting), to help users design optimal gRNAs for the CRISPR-Cpf1 system by considering both target efficiency and specificity. CRISPR-DT will empower researchers in genome editing.<br><br>CRISPR-DT, mainly implemented in Perl, PHP and JavaScript, is freely available at http://bioinfolab.miamioh.edu/CRISPR-DT.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid30614228,
year = {2019},
author = {Li, JR and Walker, S and Nie, JB and Zhang, XQ},
title = {Experiments that led to the first gene-edited babies: the ethical failings and the urgent need for better governance.},
journal = {Journal of Zhejiang University. Science. B},
volume = {20},
number = {1},
pages = {32-38},
pmid = {30614228},
issn = {1862-1783},
mesh = {CRISPR-Cas Systems ; China ; Consent Forms/ethics ; Ethics, Medical ; Female ; Gene Editing/*ethics/legislation &amp; jurisprudence ; Gene Knockout Techniques/ethics ; HIV Infections/genetics/immunology/prevention &amp; control ; Human Experimentation/ethics/legislation &amp; jurisprudence ; Humans ; Infant, Newborn ; Pregnancy ; Professional Misconduct/ethics ; Receptors, CCR5/deficiency/genetics ; },
abstract = {The rapid developments of science and technology in China over recent decades, particularly in biomedical research, have brought forward serious challenges regarding ethical governance. Recently, Jian-kui HE, a Chinese scientist, claimed to have "created" the first gene-edited babies, designed to be naturally immune to the human immunodeficiency virus (HIV). The news immediately triggered widespread criticism, denouncement, and debate over the scientific and ethical legitimacy of HE's genetic experiments. China's guidelines and regulations have banned germline genome editing on human embryos for clinical use because of scientific and ethical concerns, in accordance with the international consensus. HE's human experimentation has not only violated these Chinese regulations, but also breached other ethical and regulatory norms. These include questionable scientific value, unreasonable risk-benefit ratio, illegitimate ethics review, invalid informed consent, and regulatory misconduct. This series of ethical failings of HE and his team reveal the institutional failure of the current ethics governance system which largely depends on scientist's self-regulation. The incident highlights the need for urgent improvement of ethics governance at all levels, the enforcement of technical and ethical guidelines, and the establishment of laws relating to such bioethical issues.},
}
@article {pmid30613384,
year = {2018},
author = {Conboy, I and Murthy, N and Etienne, J and Robinson, Z},
title = {Making gene editing a therapeutic reality.},
journal = {F1000Research},
volume = {7},
number = {},
pages = {},
pmid = {30613384},
issn = {2046-1402},
support = {R01 EB023776/EB/NIBIB NIH HHS/United States ; R01 HL139605/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Muscular Dystrophy, Duchenne/genetics ; Myotonic Dystrophy/genetics ; },
abstract = {This review discusses current bottlenecks in making CRISPR-Cas9-mediated genome editing a therapeutic reality and it outlines recent strategies that aim to overcome these hurdles as well as the scope of current clinical trials that pioneer the medical translation of CRISPR-Cas9. Additionally, this review outlines the specifics of disease-modifying gene editing in recessive versus dominant genetic diseases with the focus on genetic myopathies that are exemplified by Duchenne muscular dystrophy and myotonic dystrophies.},
}
@article {pmid30612741,
year = {2019},
author = {Gasperini, M and Hill, AJ and McFaline-Figueroa, JL and Martin, B and Kim, S and Zhang, MD and Jackson, D and Leith, A and Schreiber, J and Noble, WS and Trapnell, C and Ahituv, N and Shendure, J},
title = {A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.},
journal = {Cell},
volume = {176},
number = {1-2},
pages = {377-390.e19},
pmid = {30612741},
issn = {1097-4172},
support = {UM1 HG009408/HG/NHGRI NIH HHS/United States ; T32 HL007828/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 HL118342/HL/NHLBI NIH HHS/United States ; T32 HG000035/HG/NHGRI NIH HHS/United States ; DP2 HD088158/HD/NICHD NIH HHS/United States ; U24 HG009446/HG/NHGRI NIH HHS/United States ; DP1 HG007811/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromosome Mapping/*methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Enhancer Elements, Genetic/*genetics ; Gene Expression Profiling ; Gene Expression Regulation/*genetics ; Gene Regulatory Networks/genetics ; Genome, Human ; Genome-Wide Association Study ; Genomics ; Humans ; Quantitative Trait Loci ; Transcription Factors/genetics ; },
abstract = {Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.},
}
@article {pmid30612693,
year = {2019},
author = {Platzer, K and Sticht, H and Edwards, SL and Allen, W and Angione, KM and Bonati, MT and Brasington, C and Cho, MT and Demmer, LA and Falik-Zaccai, T and Gamble, CN and Hellenbroich, Y and Iascone, M and Kok, F and Mahida, S and Mandel, H and Marquardt, T and McWalter, K and Panis, B and Pepler, A and Pinz, H and Ramos, L and Shinde, DN and Smith-Hicks, C and Stegmann, APA and Stöbe, P and Stumpel, CTRM and Wilson, C and Lemke, JR and Di Donato, N and Miller, KG and Jamra, R},
title = {De Novo Variants in MAPK8IP3 Cause Intellectual Disability with Variable Brain Anomalies.},
journal = {American journal of human genetics},
volume = {104},
number = {2},
pages = {203-212},
pmid = {30612693},
issn = {1537-6605},
mesh = {Adaptor Proteins, Signal Transducing/chemistry/*genetics/metabolism ; Adolescent ; Animals ; Brain/*abnormalities/diagnostic imaging/*metabolism ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/physiology ; Child ; Child, Preschool ; Computer Simulation ; Female ; Humans ; Intellectual Disability/diagnostic imaging/*genetics ; Locomotion ; Lysosomes/metabolism ; Male ; Models, Molecular ; *Mutation ; Nerve Tissue Proteins/chemistry/*genetics/metabolism ; Whole Exome Sequencing ; Young Adult ; },
abstract = {Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.},
}
@article {pmid30611840,
year = {2019},
author = {Li, J and Handler, AM},
title = {CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa.},
journal = {Gene},
volume = {691},
number = {},
pages = {160-166},
doi = {10.1016/j.gene.2018.12.055},
pmid = {30611840},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; Female ; Gene Editing/*methods ; *Genes, Insect ; Germ-Line Mutation ; Green Fluorescent Proteins/genetics/metabolism ; Male ; Phenotype ; Sequence Deletion ; Tephritidae/genetics/*growth &amp; development ; *Transgenes ; },
abstract = {CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Somatic mutations were identified phenotypically in G0 adult flies at a frequency of 81% based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA do not have a phenotypic effect in XY males. Consistent with this expectation, twelve types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5' sgRNA-1 target site in intersexual adults. However, the 3' sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.},
}
@article {pmid30610648,
year = {2019},
author = {Park, J and Choi, S and Park, S and Yoon, J and Park, AY and Choe, S},
title = {DNA-Free Genome Editing via Ribonucleoprotein (RNP) Delivery of CRISPR/Cas in Lettuce.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {337-354},
doi = {10.1007/978-1-4939-8991-1_25},
pmid = {30610648},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Lettuce/*genetics ; Ribonucleoproteins/*genetics ; },
abstract = {CRISPR/Cas9 nuclease system is getting popular in precise genome editing of both eukaryotic and prokaryotic systems due to its accuracy, programmability, and relative ease of use. CRISPR/Cas systems can be delivered into live cells via plasmid DNA, RNA, and ribonucleoprotein (RNP). Of these, the RNP method is of special interest due to enzymatic action in shorter time and controllability over their activity. In addition, because RNP does not involve DNA, none of unwanted DNA footprints are left in the host genome. Previously, we demonstrated that plant protoplasts can be transfected with functional RNPs and the whole plants can be regenerated from an engineered protoplast. Relative to the published methods, the revised protocols described here should help increase the success rate of whole plant regeneration by reducing damages to the naked protoplast cells.},
}
@article {pmid30610647,
year = {2019},
author = {Liang, Z and Chen, K and Gao, C},
title = {Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {327-335},
doi = {10.1007/978-1-4939-8991-1_24},
pmid = {30610647},
issn = {1940-6029},
mesh = {Biolistics/*methods ; CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Seedlings/genetics ; Triticum/*genetics ; },
abstract = {The great advances in exploiting the CRISPR/Cas9 system are paving the way for targeted genome engineering in plants. Genome editing by direct delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs) into plant cells reduces off-target mutations and avoids the integration of foreign DNA fragments, thus providing an efficient and accurate method for precision crop breeding. Here we describe an RNP-based genome editing protocol for wheat. The protocol covers the in vitro transcription of sgRNA, purification of Cas9 protein, biolistic delivery of CRISPR/Cas9 RNPs, and tissue culture procedures for regenerating testable seedlings.},
}
@article {pmid30610646,
year = {2019},
author = {Mahas, A and Ali, Z and Tashkandi, M and Mahfouz, MM},
title = {Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {311-326},
doi = {10.1007/978-1-4939-8991-1_23},
pmid = {30610646},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Engineering/methods ; Genome, Plant/*genetics ; Plants, Genetically Modified/genetics ; RNA Viruses/genetics ; },
abstract = {Targeted modification of plant genomes is a powerful strategy for investigating and engineering cellular systems, paving the way for the discovery and development of important, novel agricultural traits. Cas9, an RNA-guided DNA endonuclease from the type II adaptive immune CRISPR system of the prokaryote Streptococcus pyogenes, has gained widespread popularity as a genome-editing tool for use in a wide array of cells and organisms, including model and crop plants. Effective genome engineering requires the delivery of the Cas9 protein and guide RNAs into target cells. However, in planta genome modification faces many hurdles, including the difficulty in efficiently delivering genome engineering reagents to the desired tissues. We recently developed a Tobacco rattle virus (TRV)-mediated genome engineering system for Nicotiana benthamiana. Using this platform, genome engineering reagents can be delivered into all plant parts in a simple, efficient manner, facilitating the recovery of progeny plants with the desired genomic modifications, thus bypassing the need for transformation and tissue culture. This platform expands the utility of the CRISPR/Cas9 system for in planta, targeted genome modification. Here, we provide a detailed protocol explaining the methodologies used to develop and implement TRV-mediated genome engineering in N. benthamiana. The protocol described here can be extended to any other plant species susceptible to systemic infection by TRV. However, this approach is not limited to vectors derived from TRV, as other RNA viruses could be used to develop similar delivery platforms.},
}
@article {pmid30610645,
year = {2019},
author = {Shimatani, Z and Ariizumi, T and Fujikura, U and Kondo, A and Ezura, H and Nishida, K},
title = {Targeted Base Editing with CRISPR-Deaminase in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {297-307},
doi = {10.1007/978-1-4939-8991-1_22},
pmid = {30610645},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytidine Deaminase/genetics ; Gene Editing/*methods ; Lycopersicon esculentum/*genetics ; },
abstract = {The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.},
}
@article {pmid30610644,
year = {2019},
author = {Li, J and Meng, X and Li, J and Gao, C},
title = {Gene Replacement by Intron Targeting with CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {285-296},
doi = {10.1007/978-1-4939-8991-1_21},
pmid = {30610644},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Introns/genetics ; Recombinational DNA Repair/*genetics ; },
abstract = {The CRISPR-Cas9 system has become the most widely adopted genome editing platform and is used in an expanding number of organisms, mainly by creating targeted knockouts through non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). It would also be highly desirable to be able to use homology-directed repair (HDR) to perform precise gene editing, for example, by replacing a small section of DNA to substitute one amino acid for another in a given gene product. However, this remains a serious challenge in plants. Here, we describe a recently developed intron-mediated site-specific gene replacement method acting through the NHEJ pathway in which Cas9 simultaneously introduces DSBs in adjacent introns and the donor template. This approach is of general use for replacing targeted gene fragments at specific genomic sites in plants.},
}
@article {pmid30610642,
year = {2019},
author = {Yin, X and Anand, A and Quick, P and Bandyopadhyay, A},
title = {Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {257-268},
doi = {10.1007/978-1-4939-8991-1_19},
pmid = {30610642},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Oryza/*genetics ; },
abstract = {CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improving the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions.},
}
@article {pmid30610641,
year = {2019},
author = {Zhang, Y and Zhang, Y and Qi, Y},
title = {Plant Gene Knockout and Knockdown by CRISPR-Cpf1 (Cas12a) Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {245-256},
doi = {10.1007/978-1-4939-8991-1_18},
pmid = {30610641},
issn = {1940-6029},
mesh = {Arabidopsis/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Gene Knockout Techniques/*methods ; Oryza/genetics ; },
abstract = {CRISPR-Cpf1 (Cas12a) is a class II type V endonuclease, which has been used as a genome editing tool in different biological systems. Here we describe a fast, efficient, and user-friendly system for CRISPR-Cpf1 expression vector assembly. In this system, the Pol II promoter is used to drive the expression of both Cpf1 and its crRNA, with the crRNA flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozyme RNAs for precise crRNA processing. All the components of this system can be modified depending on plant species and experimental goals. Using this system, nearly 100% editing efficiency and 90% gene expression decrease were achieved in rice and Arabidopsis, respectively.},
}
@article {pmid30610640,
year = {2019},
author = {Jia, H and Zou, X and Orbovic, V and Wang, N},
title = {Genome Editing in Citrus Tree with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {235-241},
doi = {10.1007/978-1-4939-8991-1_17},
pmid = {30610640},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; Citrus/*genetics ; Gene Editing/*methods ; Plant Proteins/*genetics ; },
abstract = {CRISPR/Cas9 has been widely employed to edit genome in most of the organisms, including animal, plant, fungus, and microbe. Here we describe the modification of citrus gene CsLOB1 in transgenic citrus by Cas9/sgRNA, a two-component system derived from CRISPR-Cas9. Transgenic citrus plants can be created by Agrobacterium-mediated epicotyl transformation.},
}
@article {pmid30610639,
year = {2019},
author = {Liu, J and Gunapati, S and Mihelich, NT and Stec, AO and Michno, JM and Stupar, RM},
title = {Genome Editing in Soybean with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {217-234},
doi = {10.1007/978-1-4939-8991-1_16},
pmid = {30610639},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Electrophoresis, Polyacrylamide Gel ; Gene Editing/*methods ; Plant Roots/genetics ; Plants, Genetically Modified/*genetics ; Soybeans/*genetics ; Transformation, Genetic/genetics ; },
abstract = {CRISPR/Cas9 mediated genome editing technology has experienced rapid advances in recent years and has been applied to a wide variety of plant species, including soybean. Several platforms have been developed for designing and cloning of single CRISPR targets or multiple targets in a single destination vector. This chapter provides an updated working protocol for applying CRISPR/Cas9 technology to target a single gene or multiple genes simultaneously in soybean. We describe two platforms for cloning single CRISPR targets and multiplexing targets, respectively, and reagent delivery methodologies. The protocols address crucial limiting steps that can limit CRISPR editing in soybean hairy roots, composite plants, and tissue culture-based regenerated whole plants. To date, transgenic soybean plants with mutagenesis in up to three target genes have been obtained with this procedure.},
}
@article {pmid30610638,
year = {2019},
author = {Klimek-Chodacka, M and Oleszkiewicz, T and Baranski, R},
title = {Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {203-215},
doi = {10.1007/978-1-4939-8991-1_15},
pmid = {30610638},
issn = {1940-6029},
mesh = {Anthocyanins/metabolism ; Bony Callus/metabolism ; CRISPR-Cas Systems/*genetics ; Daucus carota/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; },
abstract = {The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.},
}
@article {pmid30610637,
year = {2019},
author = {Nadakuduti, SS and Starker, CG and Voytas, DF and Buell, CR and Douches, DS},
title = {Genome Editing in Potato with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {183-201},
doi = {10.1007/978-1-4939-8991-1_14},
pmid = {30610637},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; Solanum tuberosum/*genetics ; Tissue Culture Techniques ; Transformation, Genetic/genetics ; },
abstract = {Cultivated potato, Solanum tuberosum Group Tuberosum L. (2n = 4x = 48) is a heterozygous tetraploid crop that is clonally propagated, thereby resulting in identical genotypes. Due to the lack of sexual reproduction and its concomitant segregation of alleles, genetic engineering is an efficient way of introducing crop improvement traits in potato. In recent years, genome-editing via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for targeted genome modifications has emerged as the most powerful method due to the ease in designing and construction of gene-specific single guide RNA (sgRNA) vectors. These sgRNA vectors are easily reprogrammable to direct Streptococcus pyogenes Cas9 (SpCas9) to generate double stranded breaks (DSBs) in the target genomes that are then repaired by the cell via the error-prone non-homologous end-joining (NHEJ) pathway or by precise homologous recombination (HR) pathway. CRISPR/Cas9 technology has been successfully implemented in potato for targeted mutagenesis to generate knockout mutations (by means of NHEJ) as well as gene targeting to edit an endogenous gene (by HR). In this chapter, we describe procedures for designing sgRNAs, protocols to clone sgRNAs for CRISPR/Cas9 constructs to generate knockouts, design of donor repair templates and use geminivirus replicons (GVRs) to facilitate gene-editing by HR in potato. We also describe tissue culture procedures in potato for Agrobacterium-mediated transformation to generate gene-edited events along with their molecular characterization.},
}
@article {pmid30610636,
year = {2019},
author = {Reem, NT and Van Eck, J},
title = {Application of CRISPR/Cas9-Mediated Gene Editing in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {171-182},
doi = {10.1007/978-1-4939-8991-1_13},
pmid = {30610636},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/*methods ; Genome, Plant/*genetics ; Lycopersicon esculentum/*genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-/Cas9-mediated gene editing has been demonstrated in a number of food crops including tomato. Tomato (Solanum lycopersicum) is both an important food crop and a model plant species that has been used extensively for studying gene function, especially as it relates to fruit biology. This duality in purpose combined with readily available resources (mutant populations, genome sequences, transformation methodology) makes tomato an ideal candidate for gene editing. The CRISPR/Cas9 system routinely used in our laboratory has been applied to various tomato genotypes and the wild species, Solanum pimpinellifolium. The vector system is based on Golden Gate cloning techniques. Cassettes that contain the neomycin phosphotransferase II (NPTII) selectable marker gene that confers resistance to kanamycin, a human codon-optimized Cas9 driven by the CaMV 35S promoter, and guide RNA (gRNA) under control of the Arabidopsis U6 polymerase promoter are assembled into a T-DNA vector. Generally, we design CRISPR/Cas9 constructs that contain two gRNAs per gene target. However, we have been successful with inclusion of up to eight gRNAs to simultaneously target multiple genes and regions. Introduction of CRISPR-/Cas9-designed constructs into tomato is accomplished by transformation methodology based on Agrobacterium tumefaciens infection of young cotyledon sections and selection on kanamycin-containing medium based on the presence of the NPTII gene. The approaches for the development of CRISPR/Cas9 constructs and genotypic analyses (PCR-based amplicon sequencing and T7 endonuclease) are detailed in this chapter.},
}
@article {pmid30610635,
year = {2019},
author = {Lawrenson, T and Hundleby, P and Harwood, W},
title = {Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {155-170},
doi = {10.1007/978-1-4939-8991-1_12},
pmid = {30610635},
issn = {1940-6029},
support = {BB/N019466/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013511/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Brassica/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Mutagenesis ; Plants, Genetically Modified/*genetics ; },
abstract = {While public and political views on genetic modification (inserting "foreign" genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing offers a faster and simpler approach to gene knockout in both single and multiple genetic locations, within a single or small number of generations, in a way that has not been possible through alternative breeding methods. Here we describe an Agrobacterium-mediated delivery approach to deliver Cas9 and dual sgRNAs into 4-day-old cotyledonary petioles of Brassica oleracea. Mutations are detected in approximately 10% of primary transgenic plants and go on in subsequent T1 and T2 generations to segregate away from the T-DNA. This enables the recovery of non-transgenic, genome-edited plants carrying a variety of mutations at the target locus.},
}
@article {pmid30610634,
year = {2019},
author = {Yu, H and Zhao, Y},
title = {Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {147-154},
doi = {10.1007/978-1-4939-8991-1_11},
pmid = {30610634},
issn = {1940-6029},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Luminescent Proteins/genetics ; Plants, Genetically Modified/genetics ; RNA Editing/genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas9 gene editing technology has successfully introduced modifications at target DNA sequences in many plant species including Arabidopsis. After the target gene is edited, the CRISPR/Cas9 construct needs to be removed to ensure genetic stability and to gain any regulatory approval for commercial applications. However, removal of the transgenes by genetic segregation, backcross, and genotyping is very laborious and time-consuming. The methods we report here allow fast and effective isolation of transgene-free T2 Arabidopsis plants with the desired modifications at the target genes. We express a fluorescence protein mCherry under the control of a seed-specific promoter At2S3 and placed the cassette into the CRISPR/Cas9 vector. Therefore, we can use mCherry as a proxy for the presence of Cas9, and we are able to visually isolate the Cas9-free Arabidopsis plants with heritable mutations at the T2 generation. We targeted two sites in the ABP1 gene to demonstrate the effectiveness of our approach.},
}
@article {pmid30610633,
year = {2019},
author = {Lee, K and Zhu, H and Yang, B and Wang, K},
title = {An Agrobacterium-Mediated CRISPR/Cas9 Platform for Genome Editing in Maize.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {121-143},
doi = {10.1007/978-1-4939-8991-1_10},
pmid = {30610633},
issn = {1940-6029},
mesh = {Agrobacterium/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Mutagenesis/genetics ; Transformation, Genetic/genetics ; Zea mays/*genetics ; },
abstract = {Precise genome engineering can be efficiently made using the revolutionary tool named CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) systems. Adapted from the bacterial immune system, CRISPR/Cas systems can generate highly specific double-strand breaks (DSBs) at the target site, and desired sequence modifications can be introduced during the DSB repair process, such as nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. CRISPR/Cas9 is the most widely used genome editing tool for targeted mutagenesis, precise sequence modification, transcriptional reprogramming, epigenome editing, disease treatment, and many more. The ease of use and high specificity make CRISPR/Cas9 a great tool not only for basic researches but also for crop trait improvements, such as higher grain yield, better tolerance to abiotic stresses, enhanced disease resistance, and better nutritional contents. In this protocol, we present a step-by-step guide to the CRISPR/Cas9-mediated targeted mutagenesis in maize Hi II genotype. Detailed procedures will guide through the essential steps including gRNA design, CRISPR/Cas9 vector construction, Agrobacterium-mediated maize immature embryo transformation, and molecular analysis of the transgenic plants to identify desired mutant lines.},
}
@article {pmid30610632,
year = {2019},
author = {Zhou, J and Zhong, Z and Chen, H and Li, Q and Zheng, X and Qi, Y and Zhang, Y},
title = {Knocking Out MicroRNA Genes in Rice with CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {109-119},
doi = {10.1007/978-1-4939-8991-1_9},
pmid = {30610632},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Genome, Plant/*genetics ; MicroRNAs/*genetics ; Oryza/*genetics ; },
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in plant development and stress responses. Loss-of-function analysis of miRNA genes has been traditionally challenging due to lack of appropriate knockout tools. In this chapter, we describe a method of using CRISPR-Cas9 for knocking out microRNA genes in rice by Agrobacterium-mediated transformation. We also demonstrate single-strand conformation polymorphism (SSCP) as an effective genotyping method for screening CRISPR-Cas9-induced mutations.},
}
@article {pmid30610631,
year = {2019},
author = {Gu, W and Zhang, D and Qi, Y and Yuan, Z},
title = {Generating Photoperiod-Sensitive Genic Male Sterile Rice Lines with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {97-107},
doi = {10.1007/978-1-4939-8991-1_8},
pmid = {30610631},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Oryza/*genetics/*physiology ; *Photoperiod ; Plant Breeding ; Plant Infertility/genetics/*physiology ; },
abstract = {Obtaining photoperiod-sensitive genic male sterility (PGMS) lines is one of the most important steps in two-line hybrid rice breeding. Traditionally, such lines were screened and developed with a classic rice breeding system under both long-day and short-day conditions. The isolation and backcross process used for this could easily last for more than 3 years with a very low success rate. Here, we describe a straightforward method for generating csa-based PGMS lines by using the CRISPR-Cas9 technology in rice.},
}
@article {pmid30610630,
year = {2019},
author = {Malzahn, A and Zhang, Y and Qi, Y},
title = {CRISPR-Act2.0: An Improved Multiplexed System for Plant Transcriptional Activation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {83-93},
doi = {10.1007/978-1-4939-8991-1_7},
pmid = {30610630},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Plant/genetics ; Plant Immunity/genetics/physiology ; Transcriptional Activation/genetics/physiology ; },
abstract = {CRISPR systems have greatly promoted research in genome editing and transcriptional regulation. CRISPR-based transcriptional repression and activation systems will be valuable for applications in engineering plant immunity, boosting metabolic production, and enhancing our knowledge of gene regulatory networks. Multiplexing of CRISPR allows multiple genes to be targeted without significant additional effort. Here, we describe our CRISPR-Act2.0 system which is an improved multiplexing transcriptional activation system in plants.},
}
@article {pmid30610629,
year = {2019},
author = {Tang, X and Zhong, Z and Ren, Q and Liu, B and Zhang, Y},
title = {A Single Transcript CRISPR-Cas9 System for Multiplex Genome Editing in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {75-82},
doi = {10.1007/978-1-4939-8991-1_6},
pmid = {30610629},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plants/*genetics ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/genetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR-Cas9 system has been widely adopted in genome editing. By changing the 20 bp guide sequence, it can easily edit any sequence adjacent to a protospacer adjacent motif (PAM) in a genome. Multiplex genome editing could be accomplished with simultaneous expression of multiple single-guide RNAs (sgRNA). Given that sgRNAs are expressed by Pol III promoters, multiplex genome editing is conventionally done by assembly of multiple complete sgRNA expression cassettes together, which can be a challenge in vector construction. Here, we described a multiplex genome editing system based on a single transcript unit CRISPR-Cas9 (STU CRISPR-Cas9) expression system driven by a single Pol II promoter. It represents a novel approach for multiplex genome editing.},
}
@article {pmid30610628,
year = {2019},
author = {Xie, K and Yang, Y},
title = {A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {63-73},
doi = {10.1007/978-1-4939-8991-1_5},
pmid = {30610628},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; RNA, Transfer/*genetics ; },
abstract = {The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.},
}
@article {pmid30610627,
year = {2019},
author = {Li, R and Char, SN and Yang, B},
title = {Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {47-61},
doi = {10.1007/978-1-4939-8991-1_4},
pmid = {30610627},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Chromosome Deletion ; Gene Editing/methods ; Genome, Plant/*genetics ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; RNA, Guide/genetics ; },
abstract = {Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.},
}
@article {pmid30610626,
year = {2019},
author = {Xie, X and Ma, X and Liu, YG},
title = {Decoding Sanger Sequencing Chromatograms from CRISPR-Induced Mutations.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {33-43},
doi = {10.1007/978-1-4939-8991-1_3},
pmid = {30610626},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Mutation/genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.},
}
@article {pmid30610625,
year = {2019},
author = {Wang, C and Wang, K},
title = {Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {27-32},
doi = {10.1007/978-1-4939-8991-1_2},
pmid = {30610625},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Polymerase Chain Reaction/*methods ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in targeted mutagenesis. However, a significant amount of time and cost is required to screen for the CRISPR/Cas9-induced mutants from a typically large number of initial samples. Here, we describe a cost-effective and sensitive screening technique based on conventional polymerase chain reaction (PCR), termed "annealing at critical temperature PCR" (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants.},
}
@article {pmid30610223,
year = {2019},
author = {Wang, C and Liu, Q and Shen, Y and Hua, Y and Wang, J and Lin, J and Wu, M and Sun, T and Cheng, Z and Mercier, R and Wang, K},
title = {Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes.},
journal = {Nature biotechnology},
volume = {37},
number = {3},
pages = {283-286},
pmid = {30610223},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems/*genetics ; Fertilization/genetics ; Gene Editing ; Genetic Engineering/methods ; Genome, Plant/genetics ; Hybridization, Genetic ; Meiosis/genetics ; Oryza/*genetics/growth &amp; development ; Phenotype ; Plant Proteins/*genetics ; Seeds/*genetics/growth &amp; development ; },
abstract = {Heterosis, or hybrid vigor, is exploited by breeders to produce elite high-yielding crop lines, but beneficial phenotypes are lost in subsequent generations owing to genetic segregation. Clonal propagation through seeds would enable self-propagation of F1 hybrids. Here we report a strategy to enable clonal reproduction of F1 rice hybrids through seeds. We fixed the heterozygosity of F1 hybrid rice by multiplex CRISPR-Cas9 genome editing of the REC8, PAIR1 and OSD1 meiotic genes to produce clonal diploid gametes and tetraploid seeds. Next, we demonstrated that editing the MATRILINEAL (MTL) gene (involved in fertilization) could induce formation of haploid seeds in hybrid rice. Finally, we combined fixation of heterozygosity and haploid induction by simultaneous editing of all four genes (REC8, PAIR1, OSD1 and MTL) in hybrid rice and obtained plants that could propagate clonally through seeds. Application of our method may enable self-propagation of a broad range of elite F1 hybrid crops.},
}
@article {pmid30609814,
year = {2019},
author = {Ortiz-Vitali, JL and Darabi, R},
title = {iPSCs as a Platform for Disease Modeling, Drug Screening, and Personalized Therapy in Muscular Dystrophies.},
journal = {Cells},
volume = {8},
number = {1},
pages = {},
pmid = {30609814},
issn = {2073-4409},
support = {R01 AR068293/AR/NIAMS NIH HHS/United States ; R21 AR071583/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Drug Evaluation, Preclinical ; Gene Targeting ; Humans ; Induced Pluripotent Stem Cells/cytology/*drug effects/*metabolism ; Models, Biological ; Muscular Dystrophy, Duchenne/*drug therapy ; *Precision Medicine ; Transcription Activator-Like Effector Nucleases/metabolism ; Zinc Finger Nucleases/metabolism ; },
abstract = {Induced pluripotent stem cells (iPSCs) are the foundation of modern stem cell-based regenerative medicine, especially in the case of degenerative disorders, such as muscular dystrophies (MDs). Since their introduction in 2006, many studies have used iPSCs for disease modeling and identification of involved mechanisms, drug screening, as well as gene correction studies. In the case of muscular dystrophies, these studies commenced in 2008 and continue to address important issues, such as defining the main pathologic mechanisms in different types of MDs, drug screening to improve skeletal/cardiac muscle cell survival and to slow down disease progression, and evaluation of the efficiency of different gene correction approaches, such as exon skipping, Transcription activator-like effector nucleases (TALENs), Zinc finger nucleases (ZFNs) and RNA-guided endonuclease Cas9 (CRISPR/Cas9). In the current short review, we have summarized chronological progress of these studies and their key findings along with a perspective on the future road to successful iPSC-based cell therapy for MDs and the potential hurdles in this field.},
}
@article {pmid30607331,
year = {2018},
author = {Bayat, H and Naderi, F and Khan, AH and Memarnejadian, A and Rahimpour, A},
title = {The Impact of CRISPR-Cas System on Antiviral Therapy.},
journal = {Advanced pharmaceutical bulletin},
volume = {8},
number = {4},
pages = {591-597},
pmid = {30607331},
issn = {2228-5881},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein nuclease (Cas) is identified as an adaptive immune system in archaea and bacteria. Type II of this system, CRISPR-Cas9, is the most versatile form that has enabled facile and efficient targeted genome editing. Viral infections have serious impacts on global health and conventional antiviral therapies have not yielded a successful solution hitherto. The CRISPR-Cas9 system represents a promising tool for eliminating viral infections. In this review, we highlight 1) the recent progress of CRISPR-Cas technology in decoding and diagnosis of viral outbreaks, 2) its applications to eliminate viral infections in both pre-integration and provirus stages, and 3) various delivery systems that are employed to introduce the platform into target cells.},
}
@article {pmid30606466,
year = {2019},
author = {Liu, L and Yin, M and Wang, M and Wang, Y},
title = {Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race.},
journal = {Molecular cell},
volume = {73},
number = {3},
pages = {611-620.e3},
doi = {10.1016/j.molcel.2018.11.011},
pmid = {30606466},
issn = {1097-4164},
mesh = {Bacteriophages/genetics/*metabolism ; Binding Sites ; Binding, Competitive ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics/metabolism ; Escherichia coli/*enzymology/genetics/virology ; Gene Editing/*methods ; Models, Molecular ; *Molecular Mimicry ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation ; Protein Interaction Domains and Motifs ; RNA, Guide/chemistry/genetics/metabolism ; Structure-Activity Relationship ; Viral Proteins/chemistry/genetics/*metabolism ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems provide prokaryotic cells with adaptive immunity against invading bacteriophages. Bacteriophages counteract bacterial responses by encoding anti-CRISPR inhibitor proteins (Acr). However, the structural basis for their inhibitory actions remains largely unknown. Here, we report the crystal structure of the AcrIIA2-SpyCas9-sgRNA (single-guide RNA) complex at 3.3 Å resolution. We show that AcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region. More specifically, AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection. Thus, phage-encoded AcrIIA2 appears to act as a DNA mimic that blocks subsequent dsDNA binding by virtue of its highly acidic residues, disabling bacterial Cas9 by competing with target dsDNA binding with a binding motif distinct from AcrIIA4. Our study provides a more detailed mechanistic understanding of AcrIIA2-mediated inhibition of SpyCas9, the most widely used genome-editing tool, opening new avenues for improved regulatory precision during genome editing.},
}
@article {pmid30606176,
year = {2019},
author = {Eun, K and Park, MG and Jeong, YW and Jeong, YI and Hyun, SH and Hwang, WS and Kim, SH and Kim, H},
title = {Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {1},
pmid = {30606176},
issn = {1472-6750},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Dogs ; Fibroblasts/physiology ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; *Genes, p53 ; Male ; Neoplasms/*genetics/prevention &amp; control/*veterinary ; },
abstract = {BACKGROUND: Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research.<br><br>RESULTS: We constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing "indel" mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique.<br><br>CONCLUSIONS: Our results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.},
}
@article {pmid30605842,
year = {2019},
author = {Schuster, S and Saravanakumar, S and Schöls, L and Hauser, S},
title = {Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for the STUB1 locus.},
journal = {Stem cell research},
volume = {34},
number = {},
pages = {101378},
doi = {10.1016/j.scr.2018.101378},
pmid = {30605842},
issn = {1876-7753},
mesh = {Adult ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line ; Female ; *Gene Knockout Techniques ; *Genetic Loci ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Ubiquitin-Protein Ligases/*genetics ; },
abstract = {STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Missense and truncating mutations in STUB1 lead to SCAR16. For ideal in vitro disease modelling with isogenic controls, we generated a CHIP knockout cell line from a healthy control with no CHIP functionality, but remaining genomic integrity and verified pluripotency.},
}
@article {pmid30605838,
year = {2019},
author = {Xue, Y and Liao, B and Xie, Y and Li, S and Ma, X and Sun, X},
title = {Establishment of an ectodermal dysplasia related gene EDA Knockout human embryonic stem cell line (WAe001-A-22) by CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {34},
number = {},
pages = {101379},
doi = {10.1016/j.scr.2018.101379},
pmid = {30605838},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/*methods ; Cell Line ; Ectodermal Dysplasia/*genetics/*pathology ; *Gene Knockout Techniques ; Human Embryonic Stem Cells/*cytology ; Humans ; Male ; },
abstract = {EDA is a gene located at Xq13.1. It encodes different isoforms of tumor necrosis factor (TNF) superfamily member ectodysplasin A. Ectodysplasin A is a transmembrane protein which can be cleaved to form a secreted form and interact with EDA receptor to mediate the development of ectoderm. Mutations of the EDA gene are related to ectodermal dysplasia and tooth agenesis. Here, we report the establishment of the EDA gene knockout human embryonic stem (hES) cell line by CRISPR-Cas9 technology. This cell line provides good materials for further studies of the roles ectodysplasin A plays in ectoderm differentiation and tooth development.},
}
@article {pmid30605152,
year = {2019},
author = {},
title = {The blind babymaker.},
journal = {Nature biotechnology},
volume = {37},
number = {1},
pages = {1},
doi = {10.1038/nbt.4341},
pmid = {30605152},
issn = {1546-1696},
mesh = {*CRISPR-Cas Systems ; China ; Congresses as Topic ; *Ethics, Medical ; Female ; Fertilization in Vitro/*ethics/*methods ; Gene Deletion ; Gene Editing ; Genetic Engineering/*ethics/*methods ; Germ-Line Mutation ; Hong Kong ; Humans ; Infant, Newborn ; Preimplantation Diagnosis ; Twins ; },
}
@article {pmid30605150,
year = {2019},
author = {Krimsky, S},
title = {Ten ways in which He Jiankui violated ethics.},
journal = {Nature biotechnology},
volume = {37},
number = {1},
pages = {19-20},
pmid = {30605150},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; Conflict of Interest ; *Ethics, Medical ; Fertilization in Vitro/*ethics ; Gene Editing/*ethics ; Genetics, Medical/*ethics ; Germ Cells ; HIV Infections/prevention &amp; control ; Humans ; Informed Consent ; Mutagenesis ; Parents ; },
}
@article {pmid30604771,
year = {2019},
author = {Sun, J and Carlson-Stevermer, J and Das, U and Shen, M and Delenclos, M and Snead, AM and Koo, SY and Wang, L and Qiao, D and Loi, J and Petersen, AJ and Stockton, M and Bhattacharyya, A and Jones, MV and Zhao, X and McLean, PJ and Sproul, AA and Saha, K and Roy, S},
title = {CRISPR/Cas9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {53},
pmid = {30604771},
issn = {2041-1723},
support = {R01 AG048218/AG/NIA NIH HHS/United States ; P50 AG033514/AG/NIA NIH HHS/United States ; U54 HD090256/HD/NICHD NIH HHS/United States ; R21 AG052404/AG/NIA NIH HHS/United States ; R01 MH078972/MH/NIMH NIH HHS/United States ; },
mesh = {Amyloid Precursor Protein Secretases/*metabolism ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/*genetics/metabolism ; Animals ; Brain/cytology/pathology ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Female ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/administration &amp; dosage/genetics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Injections, Intraventricular ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neurodegenerative Diseases/genetics/pathology/*therapy ; Neurons ; Stereotaxic Techniques ; Transfection ; Treatment Outcome ; },
abstract = {CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-β-cleavage and Aβ production, while up-regulating neuroprotective APP-α-cleavage. APP N-terminus and compensatory APP-homologues remain intact, with no apparent effects on neurophysiology in vitro. Robust APP-editing is seen in human iPSC-derived neurons and mouse brains with no detectable off-target effects. Our strategy likely works by limiting APP and BACE-1 approximation, and we also delineate mechanistic events that abrogates APP/BACE-1 convergence in this setting. Our work offers conceptual proof for a selective APP silencing strategy.},
}
@article {pmid30604745,
year = {2019},
author = {Gdula, MR and Nesterova, TB and Pintacuda, G and Godwin, J and Zhan, Y and Ozadam, H and McClellan, M and Moralli, D and Krueger, F and Green, CM and Reik, W and Kriaucionis, S and Heard, E and Dekker, J and Brockdorff, N},
title = {The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {30},
pmid = {30604745},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 340081/ERC_/European Research Council/International ; BB/M001873/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; R01 HG003143/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Line ; Chromosomal Proteins, Non-Histone/*genetics/metabolism ; CpG Islands ; DNA Methylation/*genetics ; Exons/genetics ; Female ; Fibroblasts ; Gene Knockout Techniques ; Histones/genetics/metabolism ; Male ; Mice ; Point Mutation ; Polycomb-Group Proteins/metabolism ; Transcriptional Activation/*genetics ; *X Chromosome Inactivation ; },
abstract = {The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.},
}
@article {pmid30604066,
year = {2019},
author = {Xu, WH and Liang, DY and Wang, Q and Shen, J and Liu, QH and Peng, YB},
title = {Knockdown of KDM2A inhibits proliferation associated with TGF-β expression in HEK293T cell.},
journal = {Molecular and cellular biochemistry},
volume = {456},
number = {1-2},
pages = {95-104},
pmid = {30604066},
issn = {1573-4919},
mesh = {CRISPR-Cas Systems ; *Cell Proliferation ; F-Box Proteins/genetics/*metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Jumonji Domain-Containing Histone Demethylases/genetics/*metabolism ; Pyrazoles/pharmacology ; Pyrroles/pharmacology ; Transforming Growth Factor beta/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Lysine-specific demethylase 2A (KDM2A, also known as JHDM1A or FBXL11) plays an important role in regulating cell proliferation. However, the mechanisms on KDM2A controlling cell proliferation are varied among cell types, even controversial conclusions have been drawn. In order to elucidate the functions and underlying mechanisms for KDM2A controlling cell proliferation and apoptosis, we screened a KDM2A knockout HEK293T cell lines by CRISPR-Cas9 to illustrate the effects of KDM2A on both biological process. The results indicate that knocking down expression of KDM2A can significantly weaken HEK293T cell proliferation. The cell cycle analysis via flow cytometry demonstrate that knockdown expression of KDM2A will lead more cells arrested at G2/M phase. Through the RNA-seq analysis of the differential expressed genes between KDM2A knockdown HEK293T cells and wild type, we screened out that TGF-β pathway was significantly downregulated in KDM2A knockdown cells, which indicates that TGF-β signaling pathway might be the downstream target of KDM2A to regulate cell proliferation. When the KDM2A knockdown HEK293T cells were transient-transfected with KDM2A overexpression plasmid or treated by TGF-β agonist hydrochloride, the cell proliferation levels can be partial or completely rescued. However, the TGF-β inhibitor LY2109761 can significantly inhibit the KDM2A WT cells proliferation, but not the KDM2A knockdown HEK293T cells. Taken together, these findings suggested that KDM2A might be a key regulator of cell proliferation and cell cycle via impacting TGF-β signaling pathway.},
}
@article {pmid30602779,
year = {2019},
author = {Hurlbut, JB},
title = {Human genome editing: ask whether, not how.},
journal = {Nature},
volume = {565},
number = {7738},
pages = {135},
doi = {10.1038/d41586-018-07881-1},
pmid = {30602779},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Gene Editing/*ethics/*legislation &amp; jurisprudence/trends ; Genetics, Medical/ethics/legislation &amp; jurisprudence/trends ; Genome, Human/*genetics ; Health Policy/legislation &amp; jurisprudence/trends ; Humans ; *Leadership ; Morals ; Public Opinion ; *Social Change ; Stakeholder Participation ; Trust/psychology ; Twins/*genetics ; },
}
@article {pmid30602586,
year = {2019},
author = {Braun, M and Meacham, D},
title = {The trust game: CRISPR for human germline editing unsettles scientists and society.},
journal = {EMBO reports},
volume = {20},
number = {2},
pages = {},
pmid = {30602586},
issn = {1469-3178},
support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Ethics, Research ; *Gene Editing/ethics ; *Germ Cells ; Health Personnel ; Humans ; Scientific Misconduct ; Societies, Scientific ; },
abstract = {The reaction to the alleged birth of two genome‐edited babies in China reveals deeper problems with the oversight of gene editing technologies and public trust in its applications. [Image: see text]},
}
@article {pmid30600536,
year = {2019},
author = {Foss, DV and Hochstrasser, ML and Wilson, RC},
title = {Clinical applications of CRISPR-based genome editing and diagnostics.},
journal = {Transfusion},
volume = {59},
number = {4},
pages = {1389-1399},
doi = {10.1111/trf.15126},
pmid = {30600536},
issn = {1537-2995},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Neoplasms/*diagnosis/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR-based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.},
}
@article {pmid30600447,
year = {2019},
author = {Debbarma, J and Sarki, YN and Saikia, B and Boruah, HPD and Singha, DL and Chikkaputtaiah, C},
title = {Ethylene Response Factor (ERF) Family Proteins in Abiotic Stresses and CRISPR-Cas9 Genome Editing of ERFs for Multiple Abiotic Stress Tolerance in Crop Plants: A Review.},
journal = {Molecular biotechnology},
volume = {61},
number = {2},
pages = {153-172},
pmid = {30600447},
issn = {1559-0305},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/classification/genetics/*physiology ; *Gene Editing ; Gene Expression Regulation, Plant ; Multigene Family/genetics ; Plant Proteins/classification/*genetics/metabolism ; Plants, Genetically Modified ; Signal Transduction ; *Stress, Physiological ; Transcription Factors/classification/*genetics/metabolism ; },
abstract = {Abiotic stresses such as extreme heat, cold, drought, and salt have brought alteration in plant growth and development, threatening crop yield and quality leading to global food insecurity. Many factors plays crucial role in regulating various plant growth and developmental processes during abiotic stresses. Ethylene response factors (ERFs) are AP2/ERF superfamily proteins belonging to the largest family of transcription factors known to participate during multiple abiotic stress tolerance such as salt, drought, heat, and cold with well-conserved DNA-binding domain. Several extensive studies were conducted on many ERF family proteins in plant species through over-expression and transgenics. However, studies on ERF family proteins with negative regulatory functions are very few. In this review article, we have summarized the mechanism and role of recently studied AP2/ERF-type transcription factors in different abiotic stress responses. We have comprehensively discussed the application of advanced ground-breaking genome engineering tool, CRISPR/Cas9, to edit specific ERFs. We have also highlighted our on-going and published R&amp;D efforts on multiplex CRISPR/Cas9 genome editing of negative regulatory genes for multiple abiotic stress responses in plant and crop models. The overall aim of this review is to highlight the importance of CRISPR/Cas9 and ERFs in developing sustainable multiple abiotic stress tolerance in crop plants.},
}
@article {pmid30598579,
year = {2018},
author = {Araújo, T and Khayat, A and Quintana, L and Calcagno, D and Mourão, R and Modesto, A and Paiva, J and Lima, A and Moreira, F and Oliveira, E and Souza, M and Othman, M and Liehr, T and Abdelhay, E and Gomes, R and Santos, S and Assumpção, P},
title = {Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer.},
journal = {World journal of gastroenterology},
volume = {24},
number = {47},
pages = {5338-5350},
pmid = {30598579},
issn = {2219-2840},
mesh = {Argonaute Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Neoplasm Invasiveness/genetics ; Signal Transduction/*genetics ; Stomach Neoplasms/*genetics/pathology ; },
abstract = {AIM: To establish a permanent piwi like RNA-mediated gene silencing 1 (PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.<br><br>METHODS: CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences (Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Wound-healing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit (Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer.<br><br>RESULTS: PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed mRNAs identified a total of 35 genes (5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes (DOCK2, ZNF503, PDE4D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed (being up- or down-regulated after knockout).<br><br>CONCLUSION: Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.},
}
@article {pmid30598555,
year = {2019},
author = {Wilkinson, RA and Martin, C and Nemudryi, AA and Wiedenheft, B},
title = {CRISPR RNA-guided autonomous delivery of Cas9.},
journal = {Nature structural &amp; molecular biology},
volume = {26},
number = {1},
pages = {14-24},
pmid = {30598555},
issn = {1545-9985},
support = {R21 AI130670/AI/NIAID NIH HHS/United States ; P30 GM110732/GM/NIGMS NIH HHS/United States ; R01 GM110270/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; P20 GM103500/GM/NIGMS NIH HHS/United States ; R01 GM108888/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/*physiology ; Gene Editing/methods ; Humans ; RNA, Guide/genetics/*metabolism ; },
abstract = {Cas9 is an endonuclease that can be programed to autonomously deliver diverse effectors to specified genetic addresses. High-resolution structures of this protein and its associated CRISPR RNA guide explain the molecular mechanisms of CRISPR-RNA-guided DNA recognition and provide a molecular blueprint that has facilitated structure-guided functional remodeling. Here we retrace events that led from early efforts to understand the central role of Cas9 in CRISPR-mediated adaptive immunity to contemporary efforts aimed at developing and deploying this enzyme for programmable genetic editing.},
}
@article {pmid30598475,
year = {2019},
author = {Allan, CM and Heizer, PJ and Tu, Y and Sandoval, NP and Jung, RS and Morales, JE and Sajti, E and Troutman, TD and Saunders, TL and Cusanovich, DA and Beigneux, AP and Romanoski, CE and Fong, LG and Young, SG},
title = {An upstream enhancer regulates Gpihbp1 expression in a tissue-specific manner.},
journal = {Journal of lipid research},
volume = {60},
number = {4},
pages = {869-879},
pmid = {30598475},
issn = {1539-7262},
support = {R01 HL087228/HL/NHLBI NIH HHS/United States ; P30 ES006694/ES/NIEHS NIH HHS/United States ; R01 GM065490/GM/NIGMS NIH HHS/United States ; P30 AI036214/AI/NIAID NIH HHS/United States ; K08 HL140198/HL/NHLBI NIH HHS/United States ; R01 HL125335/HL/NHLBI NIH HHS/United States ; T32 CA009523/CA/NCI NIH HHS/United States ; P01 HL090553/HL/NHLBI NIH HHS/United States ; R35 HL139725/HL/NHLBI NIH HHS/United States ; T32 HL007895/HL/NHLBI NIH HHS/United States ; K99 HL123485/HL/NHLBI NIH HHS/United States ; R00 HL123485/HL/NHLBI NIH HHS/United States ; P01 HL146358/HL/NHLBI NIH HHS/United States ; },
mesh = {Adipose Tissue, Brown/*metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Heart ; Humans ; Lipoprotein Lipase/*metabolism ; Mice ; Mice, Inbred Strains ; Receptors, Lipoprotein/analysis/*genetics/metabolism ; Sequence Analysis, DNA ; Triglycerides/blood/metabolism ; },
abstract = {Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1[Enh/Enh]). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia-even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1[Enh/-] mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.},
}
@article {pmid30598393,
year = {2018},
author = {Shan, Q and Baltes, NJ and Atkins, P and Kirkland, ER and Zhang, Y and Baller, JA and Lowder, LG and Malzahn, AA and Haugner, JC and Seelig, B and Voytas, DF and Qi, Y},
title = {ZFN, TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis: A disconnect between somatic and germinal cells.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {45},
number = {12},
pages = {681-684},
pmid = {30598393},
issn = {1673-8527},
support = {T32 GM008347/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/*genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/*metabolism ; Gene Editing ; Gene Targeting/*methods ; Germ Cells, Plant/metabolism ; Homologous Recombination ; Mutagenesis, Insertional/*methods ; Transcription Activator-Like Effector Nucleases/genetics/*metabolism ; },
}
@article {pmid30597534,
year = {2019},
author = {Zhu, T and Tian, D and Zhang, L and Xu, X and Xia, K and Hu, Z and Xiong, Z and Tan, J},
title = {Novel mutations in mevalonate kinase cause disseminated superficial actinic porokeratosis.},
journal = {The British journal of dermatology},
volume = {181},
number = {2},
pages = {304-313},
doi = {10.1111/bjd.17596},
pmid = {30597534},
issn = {1365-2133},
mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cholesterol/metabolism ; DNA Mutational Analysis ; Female ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Keratinocytes/cytology/*pathology ; Male ; Mitochondria/pathology ; Mutation, Missense ; Pedigree ; Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism ; Porokeratosis/*genetics/pathology ; Protein Multimerization/genetics ; Skin/cytology/*pathology ; },
abstract = {BACKGROUND: Disseminated superficial actinic porokeratosis (DSAP) is a rare autosomal dominant disease. In our previous research, we found that a linkage region of DSAP in a large family is located at 12q23·2-q24·1. Subsequently, the mevalonate kinase gene (MVK) was shown to be pathogenic in DSAP.<br><br>OBJECTIVES: To elucidate the mechanism by which MVK mutations lead to keratinocyte apoptosis and DSAP, and to report a new missense mutation, c.566 C>T (p.A189V), in MVK in a Chinese DSAP pedigree.<br><br>METHODS: The half-life of wild-type (WT) MVK protein and mutants was assessed using cycloheximide treatment of cells. Dimerization of MVK was analysed by coimmunoprecipitation and glutathione S transferase pull-down assay. MVK kinase activity, production of cell cholesterol, mitochondrial complex activity and apoptosis were detected, using the corresponding commercial kits, in cells overexpressing MVK WT and mutants.<br><br>RESULTS: Mechanically, we demonstrated that both the pathogenic p.A189V mutant and a sporadic mutation p.H312R (c.935A>G), which we reported previously, have rapid degradation, decreased kinase activity and reduced production of cell cholesterol. Also, we found the p.H312R mutation confers on the MVK protein an inability to dimerize. Further, we demonstrated that the mutants are impaired in mitochondrial function and lead to increased apoptosis.<br><br>CONCLUSIONS: Our results provide an important basis for elucidating the mechanism by which MVK missense mutations contribute to DSAP.},
}
@article {pmid30595438,
year = {2019},
author = {Jiang, F and Liu, JJ and Osuna, BA and Xu, M and Berry, JD and Rauch, BJ and Nogales, E and Bondy-Denomy, J and Doudna, JA},
title = {Temperature-Responsive Competitive Inhibition of CRISPR-Cas9.},
journal = {Molecular cell},
volume = {73},
number = {3},
pages = {601-610.e5},
pmid = {30595438},
issn = {1097-4164},
support = {/HHMI_/Howard Hughes Medical Institute/United States ; DP5 OD021344/OD/NIH HHS/United States ; P01 GM051487/GM/NIGMS NIH HHS/United States ; },
mesh = {Binding, Competitive ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/genetics/*metabolism/ultrastructure ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/genetics/*metabolism/ultrastructure ; Escherichia coli/enzymology/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; Protein Conformation ; Pseudomonas Phages/genetics/*metabolism ; Pseudomonas aeruginosa/*enzymology/genetics/virology ; RNA, Guide/genetics/*metabolism/ultrastructure ; Structure-Activity Relationship ; *Temperature ; Viral Proteins/genetics/*metabolism/ultrastructure ; },
abstract = {CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.},
}
@article {pmid30595385,
year = {2019},
author = {Islam, Z and Inui, T and Ishibashi, O},
title = {Gpr137b is an orphan G-protein-coupled receptor associated with M2 macrophage polarization.},
journal = {Biochemical and biophysical research communications},
volume = {509},
number = {3},
pages = {657-663},
doi = {10.1016/j.bbrc.2018.12.140},
pmid = {30595385},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Polarity ; Gene Expression ; Interleukin-4/*immunology ; Lipopolysaccharides/*immunology ; Macrophages/cytology/*immunology/metabolism ; Mice ; RAW 264.7 Cells ; Receptors, G-Protein-Coupled/genetics/*immunology ; },
abstract = {Macrophages are classified mainly into two subtypes, M1 and M2, which exhibit distinct phenotypes, based on their microenvironment. Although recent studies have suggested that G-protein-coupled receptors (GPCRs) are associated with M1/M2 macrophage polarization, available information on GPCR-mediated macrophage polarization is still limited. In the present study, we identified Gpr137b as an orphan GPCR abundantly expressed in RAW264, a mouse macrophage cell line, and illuminated its role in M2 macrophage polarization. We generated Gpr137b-knockout (Gpr137b-KO) clones of RAW264 cells using the CRISPR/Cas9 genome editing system. Two independent Gpr137b-KO clones were isolated, which were demonstrated to have frameshifting 188-nucleotide deletions at a region containing the ATG start codon of Gpr137b. Consistently, qRT-PCR analysis revealed that the deleted region is not transcribed. We then treated the Gpr137b-KO and wildtype RAW264 cells with interleukin-4 (IL-4) to induce M2 macrophage polarization. Microarray analysis revealed that the IL-4-induced gene expression of representative M2 macrophage markers was significantly reduced in the Gpr137b-KO cells, and this was validated by qRT-PCR analysis. By contrast, M1 macrophage marker gene expression induced by lipopolysaccharide was unaffected by Gpr137b-KO. Collectively, the current study shows that Gpr137b is a possible regulator of M2 macrophage polarization.},
}
@article {pmid30595383,
year = {2019},
author = {Mojumder, S and Sawamura, R and Murayama, Y and Ogura, T and Yamanaka, K},
title = {Functional characterization of UBXN-6, a C-terminal cofactor of CDC-48, in C. elegans.},
journal = {Biochemical and biophysical research communications},
volume = {509},
number = {2},
pages = {462-468},
doi = {10.1016/j.bbrc.2018.12.155},
pmid = {30595383},
issn = {1090-2104},
mesh = {Animal Nutritional Physiological Phenomena ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/*physiology ; Caenorhabditis elegans Proteins/genetics/*metabolism ; Eating ; Gene Deletion ; Hunger ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Lysosomes/genetics/metabolism ; Male ; N-Ethylmaleimide-Sensitive Proteins/*metabolism ; Protein Interaction Maps ; Valosin Containing Protein/*metabolism ; },
abstract = {CDC-48 is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities. Its functional diversity is determined by differential binding of a variety of cofactors. In this study, we analyzed the physiological role of a CDC-48 cofactor UBXN-6 in Caenorhabditis elegans. The amount of UBXN-6 was markedly increased upon starvation, but not with the treatment of tunicamycin and rapamycin. The induction upon starvation is a unique characteristic for UBXN-6 among C-terminal cofactors of CDC-48. During starvation, lysosomal activity is triggered for rapid clearance of cellular materials. We observed the lysosomal activity by monitoring GLO-1::GFP, a marker for lysosome-related organelles. We found that more puncta of GLO-1::GFP were observed in the ubxn-6 deletion mutant after 12 h starvation compared with the wild-type strain. Taken together, we propose that UBXN-6 is involved in clearance of cellular materials upon starvation in C. elegans.},
}
@article {pmid30595124,
year = {2019},
author = {Barton, M and Meyer, MR},
title = {Permissive Role of GPER for Arterial Hypertension.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {73},
number = {2},
pages = {e9-e10},
doi = {10.1161/HYPERTENSIONAHA.118.12159},
pmid = {30595124},
issn = {1524-4563},
mesh = {Animals ; CRISPR-Cas Systems ; *Dysbiosis ; Hypertension/*genetics ; Rats ; Receptors, G-Protein-Coupled ; },
}
@article {pmid30595121,
year = {2019},
author = {Waghulde, H and Galla, S and Chakraborty, S and Joe, B},
title = {Response to Permissive Role of GPER for Arterial Hypertension.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {73},
number = {2},
pages = {e11},
pmid = {30595121},
issn = {1524-4563},
support = {R01 HL143082/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Dysbiosis ; Hypertension/*genetics ; Rats ; Receptors, G-Protein-Coupled ; },
}
@article {pmid30594894,
year = {2019},
author = {Fang, L and Hung, SSC and Yek, J and El Wazan, L and Nguyen, T and Khan, S and Lim, SY and Hewitt, AW and Wong, RCB},
title = {A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9.},
journal = {Molecular therapy. Nucleic acids},
volume = {14},
number = {},
pages = {184-191},
pmid = {30594894},
issn = {2162-2531},
abstract = {Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.},
}
@article {pmid30593779,
year = {2019},
author = {Ghassemi, B and Shamsara, M and Soleimani, M and Kiani, J and Rassoulzadegan, M},
title = {Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing.},
journal = {Analytical biochemistry},
volume = {568},
number = {},
pages = {31-40},
doi = {10.1016/j.ab.2018.12.002},
pmid = {30593779},
issn = {1096-0309},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Embryonic Stem Cells ; Female ; *Gene Editing ; Gene Knockout Techniques/*methods ; Male ; Mice ; Mice, Inbred Strains ; Mice, Knockout ; RNA, Guide/*genetics ; },
abstract = {Animal models possess undeniable utility for progress on biomedical research projects and developmental and disease studies. Transgenic mouse models recreating specific disease phenotypes associated with β-hemoglobinopathies have been developed previously. However, traditional methods for gene targeting in mouse using embryonic stem cells (ESCs) are laborious and time consuming. Recently, CRISPR has been developed to facilitate and improve genomic modifications in mouse or isogenic cell lines. Applying CRISPR to gene modification eliminates the time consuming steps of traditional approach including selection of targeted ESC clones and production of chimeric mouse. This study shows that microinjection of a plasmid DNA encoding Cas9 protein along with dual sgRNAs specific to Hbb-bs gene (hemoglobin, beta adult s chain) enables breaking target sequences at exons 2 and 3 positions. The injections led to a knockout allele with efficiency around 10% for deletion of exons 2 and 3 and 20% for indel mutation.},
}
@article {pmid30593630,
year = {2019},
author = {Qiao, L and Luo, GG},
title = {Functional Characterization of Apolipoproteins in the HCV Life Cycle.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1911},
number = {},
pages = {235-246},
doi = {10.1007/978-1-4939-8976-8_16},
pmid = {30593630},
issn = {1940-6029},
support = {P30 AI027767/AI/NIAID NIH HHS/United States ; },
mesh = {Antibodies, Neutralizing/metabolism ; Apolipoproteins/genetics/*metabolism ; Apolipoproteins E/genetics/metabolism ; Blotting, Western/methods ; CRISPR-Cas Systems ; Cell Line ; Hepacivirus/genetics/growth &amp; development/*physiology ; Hepatitis C/*metabolism ; *Host-Pathogen Interactions ; Humans ; RNA Interference ; RNA, Small Interfering/genetics ; Real-Time Polymerase Chain Reaction/methods ; Transfection/methods ; Viral Envelope Proteins/genetics/*metabolism ; *Virus Assembly ; *Virus Attachment ; },
abstract = {Apolipoprotein E (apoE) plays dual functions in the HCV life cycle by promoting HCV infection and virion assembly and production. ApoE is a structural component on the HCV envelope. It mediates HCV cell attachment through specific interactions with the cell surface receptors such as syndecan-1 (SDC-1) and SDC-2 heparan sulfate proteoglycans (HSPGs). It also interacts with NS5A and E2, resulting in an enhancement of HCV morphogenesis. It can bind HCV extracellularly and promotes HCV infection. It is critical for HCV cell-to-cell transmission and may also play a role in HCV persistence by interfering with the action of HCV-neutralizing antibodies. Other apolipoproteins particularly apoB and apoC1 were also found on the HCV envelope, but their roles in the HCV life cycle remain unclear. In the last decade, a number of genomic, immunological, structural, and cell biology methodologies have been developed and used for determining the importance of apoE in the HCV life cycle. These methods and protocols will continue to be valuable to further understand the importance and the underlying molecular mechanism of various apolipoproteins in HCV infection and pathogenesis.},
}
@article {pmid30593622,
year = {2019},
author = {Wu, X and Dao Thi, VL},
title = {Embryonic or Induced Pluripotent Stem Cell-Derived Hepatocellular Systems for HCV Culture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1911},
number = {},
pages = {121-135},
doi = {10.1007/978-1-4939-8976-8_8},
pmid = {30593622},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; *Cell Differentiation ; Cell Line ; Gene Editing/methods ; Hepacivirus/physiology ; Hepatitis C/genetics ; Hepatocytes/*cytology/metabolism ; Host-Pathogen Interactions ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; },
abstract = {Human-induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) constitute a powerful tool for modeling hepatotropic pathogen infections in cell culture. Meanwhile, CRISPR-Cas9 technology enables precise editing of stem cell genomes to generate patient-specific disease models and thus development of personalized experimental systems. Here we present a detailed stepwise protocol for the differentiation of stem cells to hepatocyte-like cells for HCV studies in cell culture. We also outline the use of an inducible iCRISPR platform for the rapid and efficient modification of host factors of interest to better understand their function during HCV infection.},
}
@article {pmid30593611,
year = {2019},
author = {Liu, Q and Qi, Y and Liang, Q and Song, J and Liu, J and Li, W and Shu, Y and Tao, M and Zhang, C and Qin, Q and Wang, J and Liu, S},
title = {Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny.},
journal = {Science China. Life sciences},
volume = {62},
number = {9},
pages = {1194-1202},
doi = {10.1007/s11427-018-9404-7},
pmid = {30593611},
issn = {1869-1889},
mesh = {Animals ; Breeding/methods ; CRISPR-Cas Systems/genetics ; Female ; Gene Expression Regulation ; Gene Knockout Techniques ; Goldfish ; Hybridization, Genetic ; Male ; Melanins/*metabolism ; Monophenol Monooxygenase/*genetics/*metabolism ; Mutation ; Pigmentation/genetics ; RNA, Messenger ; Sequence Analysis, RNA ; },
abstract = {The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.},
}
@article {pmid30593266,
year = {2018},
author = {Zhang, C and He, X and Kwok, YK and Wang, F and Xue, J and Zhao, H and Suen, KW and Wang, CC and Ren, J and Chen, GG and Lai, PBS and Li, J and Xia, Y and Chan, AM and Chan, WY and Feng, B},
title = {Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells.},
journal = {BMC biology},
volume = {16},
number = {1},
pages = {151},
pmid = {30593266},
issn = {1741-7007},
mesh = {Autophagy-Related Protein-1 Homolog/*genetics ; *CRISPR-Cas Systems ; Carrier Proteins/*genetics ; Cell Line ; *DNA Repair ; Endodeoxyribonucleases ; Gene Knock-In Techniques/*methods ; Intracellular Signaling Peptides and Proteins/*genetics ; Mutagenesis, Insertional ; Nuclear Proteins/*genetics ; Ubiquitins/*genetics ; },
abstract = {BACKGROUND: Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.<br><br>RESULTS: Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1-/- cells and attenuated cytokine-induced cell death in FAT10-/- clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.<br><br>CONCLUSION: Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.},
}
@article {pmid30593263,
year = {2019},
author = {Xie, Z and Tang, Y and Su, X and Cao, J and Zhang, Y and Li, H},
title = {PAX3-FOXO1 escapes miR-495 regulation during muscle differentiation.},
journal = {RNA biology},
volume = {16},
number = {1},
pages = {144-153},
pmid = {30593263},
issn = {1555-8584},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Differentiation/genetics ; Cell Line, Tumor ; Forkhead Box Protein O1/genetics/*metabolism ; Gene Editing ; *Gene Expression Regulation ; Gene Knockdown Techniques ; Genes, Reporter ; Horses ; Humans ; Mice ; MicroRNAs/*genetics ; *Muscle Development/genetics ; PAX3 Transcription Factor/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Sheep ; Swine ; },
abstract = {Pax3 plays an essential role in myogenesis. Previously, we found a tumor-signature chimeric fusion RNA, PAX3-FOXO1 also present during muscle differentiation, raising the possibility of its physiological role. Here we demonstrated that the fusion is needed transiently for muscle lineage commitment. Interestingly, the fusion ortholog was not found in seven mouse muscle differentiation/regeneration systems, nor in other stem cell differentiation systems of another three mammal species. We noticed that Pax3 is expressed at a much lower level in human stem cells, and during muscle differentiation than in other mammals. Given the fact that the fusion and the parental Pax3 share common downstream targets, we reasoned that forming the fusion may be a mechanism for human cells to escape certain microRNA regulation on Pax3. By sequence comparison, we identified 16 candidate microRNAs that may specifically target the human PAX3 3'UTR. We used a luciferase reporter assay, examined the microRNAs expression, and conducted mutagenesis on the reporters, as well as a CRISPR/Cas9 mediated editing on the endogenous allele. Finally, we identified miR-495 as a microRNA that specifically targets human PAX3. Examining several other fusion RNAs revealed that the human-specificity is not limited to PAX3-FOXO1. Based on these observations, we conclude that PAX3-FOXO1 fusion RNA is absent in mouse, or other mammals we tested, the fusion RNA is a mechanism to escape microRNA, miR-495 regulation in humans, and that it is not the only human-specific fusion RNA.},
}
@article {pmid30591527,
year = {2019},
author = {Marconi, C and Di Buduo, CA and LeVine, K and Barozzi, S and Faleschini, M and Bozzi, V and Palombo, F and McKinstry, S and Lassandro, G and Giordano, P and Noris, P and Balduini, CL and Savoia, A and Balduini, A and Pippucci, T and Seri, M and Katsanis, N and Pecci, A},
title = {Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia.},
journal = {Blood},
volume = {133},
number = {12},
pages = {1346-1357},
doi = {10.1182/blood-2018-07-859496},
pmid = {30591527},
issn = {1528-0020},
mesh = {Adolescent ; Adult ; Animals ; Blood Platelets/metabolism/*pathology ; CRISPR-Cas Systems ; Child ; Female ; Follow-Up Studies ; *Genetic Predisposition to Disease ; Hematopoiesis ; Humans ; Male ; Megakaryocytes/metabolism/*pathology ; Middle Aged ; *Mutation ; Pedigree ; Prognosis ; Receptor-Like Protein Tyrosine Phosphatases, Class 3/antagonists &amp; inhibitors/genetics ; Thrombocytopenia/etiology/genetics/*pathology ; Zebrafish ; },
abstract = {Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ . This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41[+] thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.},
}
@article {pmid30590793,
year = {2019},
author = {Laughery, MF and Mayes, HC and Pedroza, IK and Wyrick, JJ},
title = {R-loop formation by dCas9 is mutagenic in Saccharomyces cerevisiae.},
journal = {Nucleic acids research},
volume = {47},
number = {5},
pages = {2389-2401},
pmid = {30590793},
issn = {1362-4962},
support = {R01 ES028698/ES/NIEHS NIH HHS/United States ; R21 ES027937/ES/NIEHS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Cytosine/metabolism ; DNA/*genetics ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; Deamination/genetics ; Mutagenesis/*genetics ; Mutation/*genetics ; Nucleic Acid Conformation ; RNA, Guide/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {Cas9 binds and cleaves specific DNA sequences by inducing the formation of an R-loop between the guide RNA and its genomic target. While targeting of active Cas9 to a genomic locus is highly mutagenic because Cas9 creates DNA double strand breaks, targeting of dead Cas9 (dCas9) is presumed not to be mutagenic, as dCas9 lacks DNA endonuclease activity. Here, we show that dCas9 targeting induces mutations in yeast, particularly when targeted to the non-transcribed strand of a gene. dCas9-induced mutations cluster near the guide RNA target region and are comprised of single nucleotide substitutions, small insertions and deletions, and even complex mutations, depending upon the particular guide RNA target. We show that many of these mutations are a consequence of cytosine deamination events occurring on the non-target strand of the dCas9-induced R-loop, while others are associated with homopolymer instability or translesion DNA synthesis. Targeting of dCas9 by a mismatch-containing guide RNA also increases CAN1 mutation frequency, particularly in an ung1Δ mutant strain, suggesting that dCas9 induces mutations through similar mechanisms at off-target sites. These findings indicate that DNA binding by dCas9 is mutagenic in yeast, likely because dCas9 induces the formation of an R-loop at its target site.},
}
@article {pmid30590691,
year = {2019},
author = {Haldeman, JM and Conway, AE and Arlotto, ME and Slentz, DH and Muoio, DM and Becker, TC and Newgard, CB},
title = {Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing.},
journal = {Nucleic acids research},
volume = {47},
number = {4},
pages = {e23},
pmid = {30590691},
issn = {1362-4962},
support = {R01 DK046492/DK/NIDDK NIH HHS/United States ; },
mesh = {Adenoviridae/genetics ; Animals ; CRISPR-Cas Systems/*genetics ; DNA Transposable Elements/genetics ; Enhancer Elements, Genetic/genetics ; Epigenomics/methods ; Gene Editing/methods ; Gene Expression Regulation/genetics ; Genetic Engineering/*methods ; Genetic Vectors/*genetics ; HEK293 Cells ; Histone Demethylases/genetics ; Homeodomain Proteins/*genetics ; Humans ; Insulinoma/metabolism ; Islets of Langerhans/metabolism ; Lentivirus/genetics ; Mice ; Promoter Regions, Genetic/genetics ; RNA, Guide/genetics ; Rats ; Trans-Activators/*genetics ; Transgenes/*genetics ; },
abstract = {Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.},
}
@article {pmid30590171,
year = {2019},
author = {Ellwanger, JH and Kaminski, VL and Chies, JAB},
title = {CCR5 gene editing - Revisiting pros and cons of CCR5 absence.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {68},
number = {},
pages = {218-220},
doi = {10.1016/j.meegid.2018.12.027},
pmid = {30590171},
issn = {1567-7257},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting ; Humans ; Mutation ; Receptors, CCR5/*genetics ; },
}
@article {pmid30590039,
year = {2018},
author = {Hung, COY and Livesey, FJ},
title = {Altered γ-Secretase Processing of APP Disrupts Lysosome and Autophagosome Function in Monogenic Alzheimer's Disease.},
journal = {Cell reports},
volume = {25},
number = {13},
pages = {3647-3660.e2},
pmid = {30590039},
issn = {2211-1247},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Alzheimer Disease/genetics/*metabolism/pathology ; Amyloid Precursor Protein Secretases/antagonists &amp; inhibitors/*metabolism ; Amyloid beta-Protein Precursor/genetics/*metabolism ; Autophagosomes/*metabolism ; Autophagy ; Axons/metabolism ; CRISPR-Cas Systems/genetics ; Cerebral Cortex/pathology ; Endosomes/metabolism ; Humans ; Lysosomes/*metabolism ; Mutation/genetics ; Presenilin-1/genetics ; *Protein Processing, Post-Translational ; Proteolysis ; },
abstract = {Abnormalities of the endolysosomal and autophagy systems are found in Alzheimer's disease, but it is not clear whether defects in these systems are a cause or consequence of degenerative processes in the disease. In human neuronal models of monogenic Alzheimer's disease, APP and PSEN1 mutations disrupt lysosome function and autophagy, leading to impaired lysosomal proteolysis and defective autophagosome clearance. Processing of APP by γ-secretase is central to the pathogenic changes in the lysosome-autophagy system caused by PSEN1 and APP mutations: reducing production of C-terminal APP by inhibition of BACE1 rescued these phenotypes in both APP and PSEN1 mutant neurons, whereas inhibition of γ-secretase induced lysosomal and autophagic pathology in healthy neurons. Defects in lysosomes and autophagy due to PSEN1 mutations are rescued by CRISPR-knockout of APP. These data demonstrate a key role for proteolysis of the C-terminal of APP by γ-secretase in neuronal dysfunction in monogenic Alzheimer's disease.},
}
@article {pmid30588718,
year = {2019},
author = {Jo, N and Sogabe, Y and Yamada, Y and Ukai, T and Kagawa, H and Mitsunaga, K and Woltjen, K and Yamada, Y},
title = {Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling.},
journal = {Cancer science},
volume = {110},
number = {3},
pages = {926-938},
pmid = {30588718},
issn = {1349-7006},
mesh = {Adenoma/*genetics/pathology ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Gene Editing/methods ; Intestinal Neoplasms/*genetics/pathology ; Kidney Neoplasms/*genetics/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mutation/genetics ; RNA, Guide/*genetics ; Wilms Tumor/*genetics/pathology ; },
abstract = {The emergence of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has dramatically advanced how we manipulate the genome. Regarding in vivo experiments, Cas9-transgenic animals could provide efficient and complex genome editing. However, this potential has not been fully realized partly due to a lack of convenient platforms and limited examples of successful disease modeling. Here, we devised two doxycycline (Dox)-inducible Cas9 platforms that efficiently enable conditional genome editing at multiple loci in vitro and in vivo. In these platforms, we took advantage of a site-specific multi-segment cloning strategy for rapid and easy integration of multiple single guide (sg)RNAs. We found that a platform containing rtTA at the Rosa26 locus and TRE-Cas9 together with multiple sgRNAs at the Col1a1 locus showed higher efficiency of inducible insertions and deletions (indels) with minimal leaky editing. Using this platform, we succeeded to model Wilms' tumor and the progression of intestinal adenomas with multiple mutations including an activating mutation with a large genomic deletion. Collectively, the established platform should make complicated disease modeling in the mouse easily attainable, extending the range of in vivo experiments in various biological fields including cancer research.},
}
@article {pmid30587117,
year = {2018},
author = {Hori, Y and Tanimoto, Y and Takahashi, S and Furukawa, T and Koshiba-Takeuchi, K and Takeuchi, JK},
title = {Important cardiac transcription factor genes are accompanied by bidirectional long non-coding RNAs.},
journal = {BMC genomics},
volume = {19},
number = {1},
pages = {967},
pmid = {30587117},
issn = {1471-2164},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryo, Mammalian/metabolism ; Embryonic Development ; Heart/*growth &amp; development ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/cytology/metabolism ; Myocardium/*metabolism ; Promoter Regions, Genetic ; RNA, Long Noncoding/*genetics/metabolism ; T-Box Domain Proteins/genetics/metabolism ; Transcription Factors/*genetics/metabolism ; },
abstract = {BACKGROUND: Heart development is a relatively fragile process in which many transcription factor genes show dose-sensitive characteristics such as haploinsufficiency and lower penetrance. Despite efforts to unravel the genetic mechanism for overcoming the fragility under normal conditions, our understanding still remains in its infancy. Recent studies on the regulatory mechanisms governing gene expression in mammals have revealed that long non-coding RNAs (lncRNAs) are important modulators at the transcriptional and translational levels. Based on the hypothesis that lncRNAs also play important roles in mouse heart development, we attempted to comprehensively identify lncRNAs by comparing the embryonic and adult mouse heart and brain.<br><br>RESULTS: We have identified spliced lncRNAs that are expressed during development and found that lncRNAs that are expressed in the heart but not in the brain are located close to genes that are important for heart development. Furthermore, we found that many important cardiac transcription factor genes are located in close proximity to lncRNAs. Importantly, many of the lncRNAs are divergently transcribed from the promoter of these genes. Since the lncRNA divergently transcribed from Tbx5 is highly evolutionarily conserved, we focused on and analyzed the transcript. We found that this lncRNA exhibits a different expression pattern than that of Tbx5, and knockdown of this lncRNA leads to embryonic lethality.<br><br>CONCLUSION: These results suggest that spliced lncRNAs, particularly bidirectional lncRNAs, are essential regulators of mouse heart development, potentially through the regulation of neighboring transcription factor genes.},
}
@article {pmid30587106,
year = {2018},
author = {Hwang, GH and Park, J and Lim, K and Kim, S and Yu, J and Yu, E and Kim, ST and Eils, R and Kim, JS and Bae, S},
title = {Web-based design and analysis tools for CRISPR base editing.},
journal = {BMC bioinformatics},
volume = {19},
number = {1},
pages = {542},
pmid = {30587106},
issn = {1471-2105},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Humans ; Internet/*instrumentation ; },
abstract = {BACKGROUND: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed.<br><br>RESULTS: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (<&#x2009;1&#x2009;GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively.<br><br>CONCLUSION: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors.},
}
@article {pmid30586440,
year = {2018},
author = {Cheleuitte-Nieves, C and Gulvik, CA and McQuiston, JR and Humrighouse, BW and Bell, ME and Villarma, A and Fischetti, VA and Westblade, LF and Lipman, NS},
title = {Genotypic differences between strains of the opportunistic pathogen Corynebacterium bovis isolated from humans, cows, and rodents.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0209231},
pmid = {30586440},
issn = {1932-6203},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Cattle ; Corynebacterium/*genetics/*isolation &amp; purification/pathogenicity ; Corynebacterium Infections/microbiology/veterinary ; DNA, Circular ; Female ; Genome, Bacterial ; Genotype ; Humans ; Mice, Nude ; Opportunistic Infections/microbiology/veterinary ; Phylogeny ; RNA, Bacterial ; RNA, Ribosomal, 16S ; Rats ; Virulence Factors/genetics ; },
abstract = {Corynebacterium bovis is an opportunistic bacterial pathogen shown to cause eye and prosthetic joint infections as well as abscesses in humans, mastitis in dairy cattle, and skin disease in laboratory mice and rats. Little is known about the genetic characteristics and genomic diversity of C. bovis because only a single draft genome is available for the species. The overall aim of this study was to sequence and compare the genome of C. bovis isolates obtained from different species, locations, and time points. Whole-genome sequencing was conducted on 20 C. bovis isolates (six human, four bovine, nine mouse and one rat) using the Illumina MiSeq platform and submitted to various comparative analysis tools. Sequencing generated high-quality contigs (over 2.53 Mbp) that were comparable to the only reported assembly using C. bovis DSM 20582T (97.8 ± 0.36% completeness). The number of protein-coding DNA sequences (2,174 ± 12.4) was similar among all isolates. A Corynebacterium genus neighbor-joining tree was created, which revealed Corynebacterium falsenii as the nearest neighbor to C. bovis (95.87% similarity), although the reciprocal comparison shows Corynebacterium jeikeium as closest neighbor to C. falsenii. Interestingly, the average nucleotide identity demonstrated that the C. bovis isolates clustered by host, with human and bovine isolates clustering together, and the mouse and rat isolates forming a separate group. The average number of genomic islands and putative virulence factors were significantly higher (p<0.001) in the mouse and rat isolates as compared to human/bovine isolates. Corynebacterium bovis' pan-genome contained a total of 3,067 genes of which 1,354 represented core genes. The known core genes of all isolates were primarily related to ''metabolism" and ''information storage/processing." However, most genes were classified as ''function unknown" or "unclassified". Surprisingly, no intact prophages were found in any isolate; however, almost all isolates had at least one complete CRISPR-Cas system.},
}
@article {pmid30586281,
year = {2019},
author = {Chang, J and Chen, X and Glass, Z and Gao, F and Mao, L and Wang, M and Xu, Q},
title = {Integrating Combinatorial Lipid Nanoparticle and Chemically Modified Protein for Intracellular Delivery and Genome Editing.},
journal = {Accounts of chemical research},
volume = {52},
number = {3},
pages = {665-675},
pmid = {30586281},
issn = {1520-4898},
support = {R01 EB027170/EB/NIBIB NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Carriers/*chemistry ; Gene Editing/methods ; Gene Transfer Techniques ; Humans ; Lipids/*chemistry ; Mice ; Nanoparticles/*chemistry ; Proteins/*metabolism ; Rats ; },
abstract = {The use of protein to precisely manipulate cell signaling is an effective approach for controlling cell fate and developing precision medicine. More recently, programmable nucleases, such as CRISPR/Cas9, have shown extremely high potency for editing genetic flow of mammalian cells, and for treating genetic disorders. The therapeutic potential of proteins with an intracellular target, however, is mostly challenged by their low cell impermeability. Therefore, a developing delivery system to transport protein to the site of action in a spatiotemporal controlled manner is of great importance to expand the therapeutic index of the protein. In this Account, we first summarize our most recent advances in designing combinatorial lipid nanoparticles with diverse chemical structures for intracellular protein delivery. By designing parallel Michael addition or ring-opening reaction of aliphatic amines, we have generated a combinatorial library of cationic lipids, and identified several leading nanoparticle formulations for intracellular protein delivery both in vitro and in vivo. Moreover, we optimized the chemical structure of lipids to control lipid degradation and protein release inside cells for CRISPR/Cas9 genome-editing protein delivery. In the second part of this Account, we survey our recent endeavor in developing a chemical approach to modify protein, in particular, coupled with the nanoparticle delivery platform, to improve protein delivery for targeted diseases treatment and genome editing. Chemical modification of protein is a useful tool to modulate protein function and to improve the therapeutic index of protein drugs. Herein, we mostly summarize our recent advances on designing chemical approaches to modify protein with following unique findings: (1) chemically modified protein shows selective turn-on activity based on the specific intracellular microenvironment, with which we were able to protein-based targeted cancer therapy; (2) the conjugation of hyaluronic acid (HA) to protein allows cancer cell surface receptor-targeted delivery of protein; (3) the introduction of nonpeptidic boronic acid into protein enabled cell nucleus targeted delivery; this is the first report that a nonpeptidic signal can direct protein to subcellular compartment; and (4) the fusion of protein with negatively supercharged green fluorescent protein (GFP) facilitates the self-assembly of protein with lipid nanoparticle for genome-editing protein delivery. At the end of the Account, we give a perspective of expanding the chemistry that could be integrated to design biocompatible lipid nanocarriers for protein delivery and genome editing in vitro and in vivo, as well as the chemical approaches that we can harness to modulate protein activity in live cells for targeted diseases treatment.},
}
@article {pmid30585915,
year = {2019},
author = {Lin, WV and Stout, JT and Weng, CY},
title = {CRISPR-Cas9 and Its Therapeutic Applications for Retinal Diseases.},
journal = {International ophthalmology clinics},
volume = {59},
number = {1},
pages = {3-13},
doi = {10.1097/IIO.0000000000000252},
pmid = {30585915},
issn = {1536-9617},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Retinal Diseases/genetics/*therapy ; },
}
@article {pmid30584102,
year = {2019},
author = {Xiao, B and Yin, S and Hu, Y and Sun, M and Wei, J and Huang, Z and Wen, Y and Dai, X and Chen, H and Mu, J and Cui, L and Jiang, L},
title = {Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {1},
pages = {255-260},
pmid = {30584102},
issn = {1091-6490},
support = {R01 AI116466/AI/NIAID NIH HHS/United States ; U19 AI089672/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; Erythrocytes/parasitology ; Gene Editing/*methods ; Gene Knockdown Techniques ; Genes, Protozoan/genetics ; Histone Acetyltransferases/genetics ; Histone Deacetylases/genetics ; Humans ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/*genetics/physiology ; Recombinant Proteins ; },
abstract = {Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.},
}
@article {pmid30583862,
year = {2019},
author = {Han, S and Miyoshi, K and Shikada, S and Amano, G and Wang, Y and Yoshimura, T and Katayama, T},
title = {TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia.},
journal = {Biochemical and biophysical research communications},
volume = {509},
number = {1},
pages = {227-234},
doi = {10.1016/j.bbrc.2018.12.109},
pmid = {30583862},
issn = {1090-2104},
mesh = {ADP-Ribosylation Factors/analysis/*metabolism ; CRISPR-Cas Systems ; Carrier Proteins/analysis/metabolism ; Cell Line ; Cilia/genetics/*metabolism/ultrastructure ; Gene Knockout Techniques ; Humans ; Intracellular Signaling Peptides and Proteins ; Phosphoric Monoester Hydrolases/analysis/*metabolism ; Protein Binding ; Proteins/genetics/*metabolism ; },
abstract = {The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.},
}
@article {pmid30583753,
year = {2018},
author = {Caux, F},
title = {[What's new in research?].},
journal = {Annales de dermatologie et de venereologie},
volume = {145 Suppl 7},
number = {},
pages = {VIIS17-VIIS23},
doi = {10.1016/S0151-9638(18)31285-7},
pmid = {30583753},
issn = {0151-9638},
mesh = {Autoimmune Diseases/microbiology ; Carcinoma, Basal Cell/genetics ; Circulating Tumor DNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dermatology/trends ; Epidermolysis Bullosa/immunology ; Gastrointestinal Microbiome/*immunology ; Genetic Therapy ; Humans ; Hypotrichosis/genetics ; Immunotherapy ; *Immunotherapy, Adoptive ; Microfilament Proteins/genetics ; Mutation ; Neoplasm Proteins/blood ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors ; Skin Diseases/genetics/*therapy ; Skin Neoplasms/genetics ; },
abstract = {A traditional lecture given during the annual meeting of the French Society of Dermatology in Paris summarizes the highlights of the scientific literature over the past year. In the current article the selection of the 2017-2018 period retains the following areas of interest: role of microbiome in the response to anti-PD-1 and in autoimmunity, PI3Kδ inhibitors in autoimmune bullous diseases, diagnostic and therapeutic applications of CRISPR/Cas, arrival of CAR-T cells therapy into clinical practice, gene therapy successes, use of targeted therapies in genodermatoses and integration of genetics in primary care. © 2018 Elsevier Masson SAS. All rights reserved.},
}
@article {pmid30583736,
year = {2018},
author = {Häcker, I and Schetelig, MF},
title = {Molecular tools to create new strains for mosquito sexing and vector control.},
journal = {Parasites &amp; vectors},
volume = {11},
number = {Suppl 2},
pages = {645},
pmid = {30583736},
issn = {1756-3305},
mesh = {Aedes/*genetics/physiology ; Animals ; Animals, Genetically Modified ; DNA Transposable Elements/genetics ; Female ; Gene Editing ; Humans ; Infertility, Male ; Male ; Mosquito Control/*methods ; Mosquito Vectors/*genetics/physiology ; Mutagenesis, Site-Directed ; Yellow Fever/*prevention &amp; control/transmission ; },
abstract = {Vector control programs based on population reduction by matings with mass-released sterile insects require the release of only male mosquitoes, as the release of females, even if sterile, would increase the number of biting and potentially disease-transmitting individuals. While small-scale releases demonstrated the applicability of sterile males releases to control the yellow fever mosquito Aedes aegypti, large-scale programs for mosquitoes are currently prevented by the lack of efficient sexing systems in any of the vector species.Different approaches of sexing are pursued, including classical genetic and mechanical methods of sex separation. Another strategy is the development of transgenic sexing systems. Such systems already exist in other insect pests. Genome modification tools could be used to apply similar strategies to mosquitoes. Three major tools to modify mosquito genomes are currently used: transposable elements, site-specific recombination systems, and genome editing via TALEN or CRISPR/Cas. All three can serve the purpose of developing sexing systems and vector control strains in mosquitoes in two ways: first, via their use in basic research. A better understanding of mosquito biology, including the sex-determining pathways and the involved genes can greatly facilitate the development of sexing strains. Moreover, basic research can help to identify other regulatory elements and genes potentially useful for the construction of transgenic sexing systems. Second, these genome modification tools can be used to apply the gained knowledge to build and test mosquito sexing strains for vector control.},
}
@article {pmid30583622,
year = {2018},
author = {Qiu, JR and Su, YK and Song, ZQ and Fang, XS and Li, JY and Zhang, J and Wang, JH},
title = {[Directing construction of CRISPR/Cas9 vector of SmPAL1 in Salvia miltiorrhiza by target efficiency detection in vitro].},
journal = {Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica},
volume = {43},
number = {21},
pages = {4226-4230},
doi = {10.19540/j.cnki.cjcmm.20180726.007},
pmid = {30583622},
issn = {1001-5302},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Metabolic Networks and Pathways/*genetics ; Salvia miltiorrhiza/chemistry/*genetics ; },
abstract = {To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences (SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.},
}
@article {pmid30583530,
year = {2018},
author = {da Silva Duarte, V and Giaretta, S and Campanaro, S and Treu, L and Armani, A and Tarrah, A and Oliveira de Paula, S and Giacomini, A and Corich, V},
title = {A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496.},
journal = {Viruses},
volume = {11},
number = {1},
pages = {},
pmid = {30583530},
issn = {1999-4915},
mesh = {Genome, Bacterial/genetics ; *Genome, Viral ; Lipoproteins/genetics ; Mitomycin/pharmacology ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Prophages/*immunology ; RNA, Untranslated/genetics ; Streptococcus thermophilus/drug effects/*immunology/*virology ; *Virus Integration ; },
abstract = {Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.},
}
@article {pmid30582790,
year = {2019},
author = {Sun, D and Sun, Z and Jiang, H and Vaidya, AM and Xin, R and Ayat, NR and Schilb, AL and Qiao, PL and Han, Z and Naderi, A and Lu, ZR},
title = {Synthesis and Evaluation of pH-Sensitive Multifunctional Lipids for Efficient Delivery of CRISPR/Cas9 in Gene Editing.},
journal = {Bioconjugate chemistry},
volume = {30},
number = {3},
pages = {667-678},
pmid = {30582790},
issn = {1520-4812},
support = {R01 CA194518/CA/NCI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; TL1 TR000441/TR/NCATS NIH HHS/United States ; TL1 TR002549/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/chemistry ; *Gene Editing ; Green Fluorescent Proteins/genetics ; Hemolysis/drug effects ; *Hydrogen-Ion Concentration ; Lipids/chemical synthesis/*chemistry/pharmacology ; Mice ; NIH 3T3 Cells ; Plasmids ; },
abstract = {CRISPR/Cas9 system is a promising approach for gene editing in gene therapy. Effective gene editing requires safe and efficient delivery of CRISPR/Cas9 system in target cells. Several new multifunctional pH-sensitive amino lipids were designed and synthesized with modification of the amino head groups for intracellular delivery of CRISPR/Cas9 system. These multifunctional pH-sensitive amino lipids exhibited structurally dependent formulation of stable nanoparticles with the DNA plasmids of CRISPR/Cas9 system with the sizes ranging from 100 to 200 nm. The amino lipid plasmid DNA nanoparticles showed pH-sensitive hemolysis with minimal hemolytic activity at pH 7.4 and increased hemolysis at acidic pH (pH = 5.5, 6.5). The nanoparticles exhibited low cytotoxicity at an N/P ratio of 10. Expression of both Cas9 and sgRNA of the CRISPR/Cas9 system was in the range from 4.4% to 33%, dependent on the lipid structure in NIH3T3-GFP cells. The amino lipids that formed stable nanoparticles with high expression of both Cas9 and sgRNA mediated high gene editing efficiency. ECO and iECO mediated more efficient gene editing than other tested lipids. ECO mediated up to 50% GFP suppression based on observations with confocal microscopy and nearly 80% reduction of GFP mRNA based on RT-PCR measurement in NIH3T3-GFP cells. The multifunctional pH-sensitive amino lipids have the potential for efficient intracellular delivery of CRISPR/Cas9 for effective gene editing.},
}
@article {pmid30582653,
year = {2019},
author = {Tang, X and Ren, Q and Yang, L and Bao, Y and Zhong, Z and He, Y and Liu, S and Qi, C and Liu, B and Wang, Y and Sretenovic, S and Zhang, Y and Zheng, X and Zhang, T and Qi, Y and Zhang, Y},
title = {Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing.},
journal = {Plant biotechnology journal},
volume = {17},
number = {7},
pages = {1431-1445},
pmid = {30582653},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genome, Plant ; Oryza/*genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species-specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first-generation single transcript unit (STU)-Cas9 system, STU-Cas9-RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU-Cas9 systems and one STU-Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU-Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU-Cas9-Csy4 and STU-Cas9-tRNA systems showed more robust genome editing efficiencies than our first-generation STU-Cas9-RZ system and the conventional mixed dual promoter system. We further applied the STU-Cas9-tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU-based PmCDA1 base editor system is highly efficient in rice. The STU-Cas12a system, based on Cas12a' self-processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.},
}
@article {pmid30582453,
year = {2019},
author = {Guo, F and Sun, Y and Wang, X and Wang, H and Wang, J and Gong, T and Chen, X and Zhang, P and Su, L and Fu, G and Su, J and Yang, S and Lai, R and Jiang, C and Liang, P},
title = {Patient-Specific and Gene-Corrected Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Short QT Syndrome.},
journal = {Circulation research},
volume = {124},
number = {1},
pages = {66-78},
doi = {10.1161/CIRCRESAHA.118.313518},
pmid = {30582453},
issn = {1524-4571},
mesh = {Action Potentials/*genetics ; Adult ; Arrhythmias, Cardiac/*genetics/metabolism/physiopathology ; CRISPR-Cas Systems ; Case-Control Studies ; Cell Line ; Cell Lineage ; ERG1 Potassium Channel/*genetics/metabolism ; Female ; *Gain of Function Mutation ; Gene Editing/*methods ; Genetic Predisposition to Disease ; Heart Rate/*genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Middle Aged ; *Mutation, Missense ; Myocytes, Cardiac/*metabolism ; Phenotype ; Single-Cell Analysis/*methods ; Time Factors ; },
abstract = {RATIONALE: Short QT syndrome (SQT) is a rare but arrhythmogenic disorder featured by shortened ventricular repolarization and a propensity toward life-threatening ventricular arrhythmias and sudden cardiac death.<br><br>OBJECTIVE: This study aimed to investigate the single-cell mechanism of SQT using patient-specific and gene-corrected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).<br><br>METHODS AND RESULTS: One SQT patient carrying missense mutation T618I in potassium voltage-gated channel subfamily H member 2 (KCNH2) was recruited as well as 2 healthy control subjects in this study. Control and SQT patient-specific iPSCs were generated from skin fibroblasts using nonintegrated Sendai virus. The KCNH2 T618I mutation was corrected by genome editing in SQT iPSC lines to generate isogenic controls. All iPSCs were differentiated into iPSC-CMs using monolayer-based differentiation protocol. SQT iPSC-CMs exhibited abnormal action potential phenotype featured by shortened action potential duration and increased beat-beat interval variability, when compared with control and gene-corrected iPSC-CMs. Furthermore, SQT iPSC-CMs showed KCNH2 gain-of-function with increased rapid delayed rectifying potassium current (IKr) density and enhanced membrane expression. Gene expression profiling of iPSC-CMs exhibited a differential cardiac ion-channel gene expression profile of SQT. Moreover, QTc of SQT patient and action potential durations of SQT iPSC-CMs were both normalized by quinidine, indicating that quinidine is beneficial to KCNH2 T618I of SQT. Importantly, shortened action potential duration phenotype observed in SQT iPSC-CMs was effectively rescued by a short-peptide scorpion toxin BmKKx2 with a mechanism of targeting KCNH2.<br><br>CONCLUSIONS: We demonstrate that patient-specific and gene-corrected iPSC-CMs are able to recapitulate single-cell phenotype of SQT, which is caused by the gain-of-function mutation KCNH2 T618I. These findings will help elucidate the mechanisms underlying SQT and discover therapeutic drugs for treating the disease by using peptide toxins as lead compounds.},
}
@article {pmid30582405,
year = {2019},
author = {Ng, LY and Ma, HT and Liu, JCY and Huang, X and Lee, N and Poon, RYC},
title = {Conditional gene inactivation by combining tetracycline-mediated transcriptional repression and auxin-inducible degron-mediated degradation.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {18},
number = {2},
pages = {238-248},
pmid = {30582405},
issn = {1551-4005},
mesh = {ATPases Associated with Diverse Cellular Activities/genetics ; CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/genetics ; Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cyclin A/genetics ; Cyclin-Dependent Kinase 2/genetics ; Gene Expression ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Indoleacetic Acids/*metabolism ; *Proteolysis ; Response Elements/genetics ; Retroviridae/genetics ; Tetracyclines/*metabolism ; Transcriptional Activation/*genetics ; Transfection ; },
abstract = {Characterizing the functions of essential cell cycle control genes requires tight and rapid inducible gene inactivation. Drawbacks of current conditional depletion approaches include slow responses and incomplete depletion. We demonstrated that by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system together, AID-tagged proteins can be downregulated more efficiently than the individual technology alone. When used in conjunction with CRISPR-Cas9-mediated disruption of the endogenous locus, this system facilitates the analysis of essential genes by allowing rapid and tight conditional depletion, as we have demonstrated using several cell cycle-regulatory genes including cyclin A, CDK2, and TRIP13. The vectors constructed in this study allow expression of AID-fusion proteins under the control of tetracycline-controlled promoters and should be useful in studies requiring rapid and tight suppression of gene expression in mammalian cells.},
}
@article {pmid30581191,
year = {2019},
author = {Persaud, A and Desine, S and Blizinsky, K and Bonham, VL},
title = {A CRISPR focus on attitudes and beliefs toward somatic genome editing from stakeholders within the sickle cell disease community.},
journal = {Genetics in medicine : official journal of the American College of Medical Genetics},
volume = {21},
number = {8},
pages = {1726-1734},
pmid = {30581191},
issn = {1530-0366},
support = {Z99 HG999999/ImNIH/Intramural NIH HHS/United States ; ZIA HG200394/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*epidemiology/genetics ; Attitude ; CRISPR-Cas Systems/*genetics ; *Decision Making ; Focus Groups/methods ; *Gene Editing ; Humans ; Parents ; Stakeholder Participation ; },
abstract = {PURPOSE: Genome editing holds both tremendous therapeutic promise and significant potential risk. Sickle cell disease (SCD), the most commonly inherited blood disorder, is a frontline candidate for the clinical applications of this tool. However, there is limited knowledge of patient community values and concerns regarding this new technology. This study aims to investigate the perspectives of three key decision-makers (patients, parents, and physicians) toward participation in future CRISPR-mediated somatic genome editing clinical trials.<br><br>METHODS: We utilized a mixed-methods approach, involving an educational video tool, two-part survey, and 15 moderated, audio-recorded focus groups, which were conducted in seven U.S. cities.<br><br>RESULTS: Study participants expressed hope that genome editing technology would rechart the course for SCD, but concerns related to involvement burden, uncertainty of clinical outcomes, and equity in access were identified. Major themes emerged from the focus groups: facilitators of, and barriers to, participation in future somatic genome editing clinical trials; information pertinent to the decision-making process; persons from whom participants would seek counsel before making a decision; and recommendations for the research community on meaningful engagement as clinical trials are designed and approved.<br><br>CONCLUSION: The advent of genome editing has renewed hope for the SCD community, but caution tempers this optimism.},
}
@article {pmid30581144,
year = {2019},
author = {Edraki, A and Mir, A and Ibraheim, R and Gainetdinov, I and Yoon, Y and Song, CQ and Cao, Y and Gallant, J and Xue, W and Rivera-Pérez, JA and Sontheimer, EJ},
title = {A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {714-726.e4},
pmid = {30581144},
issn = {1097-4164},
support = {UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; R21 HD089566/HD/NICHD NIH HHS/United States ; R01 GM125797/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics/metabolism ; Dependovirus/genetics ; Embryo Transfer ; Female ; Gene Editing/*methods ; Genetic Vectors ; HEK293 Cells ; Humans ; K562 Cells ; Liver/*enzymology ; Mice, Inbred C57BL ; Neisseria meningitidis/*enzymology ; Nucleotide Motifs ; Proprotein Convertase 9/*genetics/metabolism ; RNA, Guide/genetics/metabolism ; Substrate Specificity ; Zygote/metabolism ; },
abstract = {CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.},
}
@article {pmid30581143,
year = {2019},
author = {Leto, DE and Morgens, DW and Zhang, L and Walczak, CP and Elias, JE and Bassik, MC and Kopito, RR},
title = {Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation.},
journal = {Molecular cell},
volume = {73},
number = {2},
pages = {377-389.e11},
pmid = {30581143},
issn = {1097-4164},
support = {F32 GM113370/GM/NIGMS NIH HHS/United States ; F32 GM113378/GM/NIGMS NIH HHS/United States ; S10 RR027431/RR/NCRR NIH HHS/United States ; R01 GM074874/GM/NIGMS NIH HHS/United States ; S10 RR025518/RR/NCRR NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Endoplasmic Reticulum-Associated Degradation/drug effects/genetics ; Genome-Wide Association Study/*methods ; HEK293 Cells ; Humans ; K562 Cells ; Kinetics ; Proteasome Endopeptidase Complex/genetics/*metabolism ; Protein Folding ; Proteolysis ; *Proteostasis/drug effects/genetics ; Ricin/pharmacology ; Substrate Specificity ; Ubiquitin-Protein Ligases/genetics/metabolism ; Ubiquitination ; },
abstract = {The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.},
}
@article {pmid30578845,
year = {2019},
author = {Carpenter, MA and Law, EK and Serebrenik, A and Brown, WL and Harris, RS},
title = {A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.},
journal = {Methods (San Diego, Calif.)},
volume = {156},
number = {},
pages = {79-84},
pmid = {30578845},
issn = {1095-9130},
support = {/HHMI/Howard Hughes Medical Institute/United States ; P01 CA234228/CA/NCI NIH HHS/United States ; R37 AI064046/AI/NIAID NIH HHS/United States ; },
mesh = {Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cytidine Deaminase/deficiency/genetics ; Exons ; Gene Deletion ; Gene Editing/*methods ; Genetic Vectors/chemistry/metabolism ; HEK293 Cells ; Humans ; Integrases/*genetics/metabolism ; Introns ; Lentivirus/*genetics/metabolism ; MCF-7 Cells ; Minor Histocompatibility Antigens/genetics ; MutS Homolog 2 Protein/deficiency/genetics ; RNA, Guide/*genetics/metabolism ; *Recombination, Genetic ; SAM Domain and HD Domain-Containing Protein 1/deficiency/genetics ; },
abstract = {A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.},
}
@article {pmid30578646,
year = {2019},
author = {Cornelissen, LAM and Blanas, A and van der Horst, JC and Kruijssen, L and Zaal, A and O'Toole, T and Wiercx, L and van Kooyk, Y and van Vliet, SJ},
title = {Disruption of sialic acid metabolism drives tumor growth by augmenting CD8[+] T cell apoptosis.},
journal = {International journal of cancer},
volume = {144},
number = {9},
pages = {2290-2302},
pmid = {30578646},
issn = {1097-0215},
mesh = {Animals ; Apoptosis/*immunology ; CD8-Positive T-Lymphocytes/*immunology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Colorectal Neoplasms/genetics/*pathology ; Disease-Free Survival ; Glycosylation ; Humans ; Lymphocyte Count ; Mice ; Mice, Inbred C57BL ; N-Acylneuraminate Cytidylyltransferase/*genetics ; Sialic Acids/*metabolism ; Tumor Escape/*immunology ; Tumor Microenvironment/genetics/immunology ; },
abstract = {Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti-tumor immunity remain unstudied. Here, we report that CRISPR/Cas9-mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38-Sia[null]) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38-MOCK cells. This enhanced tumor growth of MC38-Sia[null] cells could be attributed to decreased CD8[+] T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor-draining lymph nodes remained unaffected. In addition, MC38-Sia[null] cells were able to induce CD8[+] T cell apoptosis in an antigen-independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence-free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.},
}
@article {pmid30576956,
year = {2019},
author = {Carlin, AF and Shresta, S},
title = {Genome-wide approaches to unravelling host-virus interactions in Dengue and Zika infections.},
journal = {Current opinion in virology},
volume = {34},
number = {},
pages = {29-38},
pmid = {30576956},
issn = {1879-6265},
support = {KL2 TR001444/TR/NCATS NIH HHS/United States ; R01 AI116813/AI/NIAID NIH HHS/United States ; R21 AI127988/AI/NIAID NIH HHS/United States ; R21 NS100477/NS/NINDS NIH HHS/United States ; R21 AI140063/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Dengue/*genetics/virology ; Dengue Virus/*genetics/pathogenicity ; Genomics ; *Host-Pathogen Interactions ; Humans ; Zika Virus/*genetics/pathogenicity ; Zika Virus Infection/*genetics/virology ; },
abstract = {Genomics approaches are increasingly utilized to probe host-viral interactions and identify mechanisms of viral pathogenesis and host-subversion. Here we review recent studies that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 screens, transcriptomics and epigenomics to gain insight into Dengue and Zika virus infections in humans. We discuss the benefits and limitations of recently utilized techniques that separate virally infected cells from neighboring uninfected cells to identify the mechanisms by which these viruses regulate host responses. We conclude by discussing how these approaches can best advance our understanding of Dengue and Zika virus pathogenesis in humans.},
}
@article {pmid30576937,
year = {2019},
author = {Zhang, YQ and Pei, JH and Shi, SS and Guo, XS and Cui, GY and Li, YF and Zhang, HP and Hu, WQ},
title = {CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {111},
number = {},
pages = {76-85},
doi = {10.1016/j.biopha.2018.12.048},
pmid = {30576937},
issn = {1950-6007},
mesh = {Base Sequence ; CRISPR-Cas Systems/*physiology ; Carcinogenesis/genetics/metabolism ; Cell Line, Tumor ; Cell Movement/*physiology ; Gastric Mucosa/metabolism/pathology ; Gene Knockdown Techniques/*methods ; Humans ; Neoplasm Invasiveness/*genetics/pathology ; Proto-Oncogene Proteins c-ets/deficiency/*genetics ; Random Allocation ; Stomach Neoplasms/*genetics/metabolism/pathology ; },
abstract = {Gastric cancer is one of the most common malignant tumors worldwide and has the second highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Here, we evaluated PDEF expression in 30 cases of human gastric carcinoma and the corresponding peritumoral tissues, using immunohistochemistry and immunofluorescence. Significantly higher levels of PDEF were detected in tumors compared to peritumoral tissues. We then investigated PDEF expression in the gastric cancer cell lines SGC and AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. Cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells were evaluated using CCK-8, flow cytometry, scratch wound, and transwell assays, respectively. The results illustrated that PDEF-knockout inhibited AGS cell proliferation, migration, and invasion. Taken together, the results imply that PDEF plays important roles in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer.},
}
@article {pmid30576352,
year = {2018},
author = {Itakura, Y and Inagaki, S and Wada, H and Hayashi, S},
title = {Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0209058},
pmid = {30576352},
issn = {1932-6203},
mesh = {Animals ; Behavior, Animal/physiology ; CRISPR-Cas Systems/genetics ; Drosophila/growth &amp; development/*metabolism ; Drosophila Proteins/genetics/*metabolism ; Embryo, Nonmammalian/metabolism ; Epidermis/metabolism/pathology ; Extracellular Matrix/*metabolism ; Gene Knockout Techniques ; Larva/growth &amp; development/metabolism ; Osmolar Concentration ; Trachea/metabolism ; },
abstract = {The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small-molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.},
}
@article {pmid30575820,
year = {2019},
author = {Saenz, DT and Fiskus, W and Manshouri, T and Mill, CP and Qian, Y and Raina, K and Rajapakshe, K and Coarfa, C and Soldi, R and Bose, P and Borthakur, G and Kadia, TM and Khoury, JD and Masarova, L and Nowak, AJ and Sun, B and Saenz, DN and Kornblau, SM and Horrigan, S and Sharma, S and Qiu, P and Crews, CM and Verstovsek, S and Bhalla, KN},
title = {Targeting nuclear β-catenin as therapy for post-myeloproliferative neoplasm secondary AML.},
journal = {Leukemia},
volume = {33},
number = {6},
pages = {1373-1386},
pmid = {30575820},
issn = {1476-5551},
support = {R01 CA173877/CA/NCI NIH HHS/United States ; R35 CA197589/CA/NCI NIH HHS/United States ; },
mesh = {Acetanilides/pharmacology ; Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Nucleus/*drug effects/metabolism/pathology ; *Drug Synergism ; Heterocyclic Compounds, 3-Ring/pharmacology ; Humans ; Leukemia, Myeloid, Acute/complications/*drug therapy/metabolism/pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Myeloproliferative Disorders/complications/*drug therapy/metabolism/pathology ; Nitriles ; Protein Kinase Inhibitors/*pharmacology ; Pyrazoles/pharmacology ; Pyrimidines ; Signal Transduction ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; beta Catenin/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear β-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of β-catenin or treatment with BC2059 that disrupts binding of β-catenin to TBL1X (TBL1) depleted nuclear β-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of β-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of β-catenin and BETP antagonists against post-MPN sAML BPCs.},
}
@article {pmid30575812,
year = {2019},
author = {Burgess, DJ},
title = {Genome editing for disease locus dissection.},
journal = {Nature reviews. Genetics},
volume = {20},
number = {2},
pages = {67},
doi = {10.1038/s41576-018-0091-1},
pmid = {30575812},
issn = {1471-0064},
mesh = {CRISPR-Cas Systems ; *Cardiovascular Diseases ; *Gene Editing ; Haplotypes ; Humans ; Risk Factors ; },
}
@article {pmid30575746,
year = {2018},
author = {Sanson, KR and Hanna, RE and Hegde, M and Donovan, KF and Strand, C and Sullender, ME and Vaimberg, EW and Goodale, A and Root, DE and Piccioni, F and Doench, JG},
title = {Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {5416},
pmid = {30575746},
issn = {2041-1723},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Genomic Library ; Streptococcus pyogenes ; },
abstract = {The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.},
}
@article {pmid30575740,
year = {2018},
author = {Zabaleta, N and Barberia, M and Martin-Higueras, C and Zapata-Linares, N and Betancor, I and Rodriguez, S and Martinez-Turrillas, R and Torella, L and Vales, A and Olagüe, C and Vilas-Zornoza, A and Castro-Labrador, L and Lara-Astiaso, D and Prosper, F and Salido, E and Gonzalez-Aseguinolaza, G and Rodriguez-Madoz, JR},
title = {CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {5454},
pmid = {30575740},
issn = {2041-1723},
mesh = {Alcohol Oxidoreductases/*antagonists &amp; inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Hyperoxaluria, Primary/*therapy ; Male ; Mice ; Nephrocalcinosis/prevention &amp; control ; Oxalates/*urine ; },
abstract = {CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1[-/-] mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.},
}
@article {pmid30575381,
year = {2019},
author = {Kim, SH and Lu, W and Ahmadi, MK and Montiel, D and Ternei, MA and Brady, SF},
title = {Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster Refactoring Approach.},
journal = {ACS synthetic biology},
volume = {8},
number = {1},
pages = {109-118},
pmid = {30575381},
issn = {2161-5063},
support = {R35 GM122559/GM/NIGMS NIH HHS/United States ; U01 GM110714/GM/NIGMS NIH HHS/United States ; },
mesh = {Biological Products/metabolism ; CRISPR-Cas Systems/genetics/physiology ; Data Mining ; Metagenomics/methods ; Promoter Regions, Genetic/genetics ; Sesterterpenes/*metabolism ; },
abstract = {Most natural product biosynthetic gene clusters identified in bacterial genomic and metagenomic sequencing efforts are silent under laboratory growth conditions. Here, we describe a scalable biosynthetic gene cluster activation method wherein the gene clusters are disassembled at interoperonic regions in vitro using CRISPR/Cas9 and then reassembled with PCR-amplified, short DNAs, carrying synthetic promoters, using transformation assisted recombination (TAR) in yeast. This simple, cost-effective, and scalable method allows for the simultaneous generation of combinatorial libraries of refactored gene clusters, eliminating the need to understand the transcriptional hierarchy of the silent genes. In two test cases, this in vitro disassembly-TAR reassembly method was used to create collections of promoter-replaced gene clusters that were tested in parallel to identify versions that enabled secondary metabolite production. Activation of the atolypene (ato) gene cluster led to the characterization of two unprecedented bacterial cyclic sesterterpenes, atolypene A (1) and B (2), which are moderately cytotoxic to human cancer cell lines. This streamlined in vitro disassembly- in vivo reassembly method offers a simplified approach for silent gene cluster refactoring that should facilitate the discovery of natural products from silent gene clusters cloned from either metagenomes or cultured bacteria.},
}
@article {pmid30575220,
year = {2019},
author = {Matsumura, R and Akashi, M},
title = {Role of the clock gene Period3 in the human cell-autonomous circadian clock.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {24},
number = {2},
pages = {162-171},
doi = {10.1111/gtc.12664},
pmid = {30575220},
issn = {1365-2443},
mesh = {Base Sequence ; Bone Neoplasms/genetics/*metabolism/pathology ; *CRISPR-Cas Systems ; *Circadian Clocks ; Circadian Rhythm ; Humans ; Osteosarcoma/genetics/*metabolism/pathology ; Period Circadian Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Sequence Homology ; Tumor Cells, Cultured ; },
abstract = {Previous studies have shown that mouse Period3 (mPer3) is dispensable for the generation of autonomous oscillations in the circadian clock. However, human studies have suggested that human Period3 (hPer3) may have more important roles in the core clock machinery than mPer3. To investigate the role of hPer3 protein in the cell-autonomous circadian oscillator, we conducted gene knockout of the hPer3 gene in human bone osteosarcoma epithelial cells using genome-editing technology. We examined the circadian transcription of endogenous clock genes in hPer3-deficient cell clones and found that hPer3-deficient cells showed a phase advance in circadian transcription compared to wild-type cells. We subsequently transfected wild-type and mutant cells with an adenovirus carrying a luciferase gene whose expression was driven by a clock gene promotor, and monitored bioluminescence in real time. Cosinor analysis showed that the circadian period length in all hPer3-deficient cells was significantly shorter than that in wild-type cells, demonstrating that the phase advance in endogenous clock gene expression in hPer3-deficient cell clones was attributable to a shortened circadian period length rather than a phase shift. Together these findings are consistent with previous studies in mice lacking functional mPer3, indicating that the Per3 protein functions similarly in both mice and humans.},
}
@article {pmid30574724,
year = {2018},
author = {Taylor-Sands, M and Gyngell, C},
title = {Legality of Embryonic Gene Editing in Australia.},
journal = {Journal of law and medicine},
volume = {26},
number = {2},
pages = {356-373},
pmid = {30574724},
issn = {1320-159X},
mesh = {Animals ; Australia ; *CRISPR-Cas Systems ; Embryo, Mammalian ; Gene Editing/ethics/*legislation &amp; jurisprudence ; Humans ; },
abstract = {The CRISPR-cas9 genome editing system (CRISPR) has been used to make precise and heritable changes to a diverse range of animals. The use of CRISPR to edit embryonic cells initially raised widespread criticism and calls for an international ban. However, the rapid development of genome editing has prompted governments around the world to review the regulatory frameworks that oversee genetic technologies. In Australia, the Prohibition of Human Cloning for Reproduction Act 2002 (Cth) and the Research Involving Human Embryos Act 2002 (Cth) expressly regulate the use of genome editing in early human embryos. This article analyses how these two Acts regulate research involving CRISPR and the implications of this for research practices in Australia. We argue that, given the current regulatory uncertainty around the legality of genome editing research in Australia, legislative reform is needed and propose reforms to provide greater clarity in this area.},
}
@article {pmid30573836,
year = {2019},
author = {Strack, R},
title = {Precision genome editing.},
journal = {Nature methods},
volume = {16},
number = {1},
pages = {21},
pmid = {30573836},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; Fibroblasts/metabolism ; *Gene Editing ; Genome, Human ; Genotype ; Humans ; Machine Learning ; Mutation ; },
}
@article {pmid30573681,
year = {2019},
author = {Ogawa, S and Matsuoka, Y and Takada, M and Matsui, K and Yamane, F and Kubota, E and Yasuhara, S and Hieda, K and Kanayama, N and Hatano, N and Tokumitsu, H and Magari, M},
title = {Interleukin 34 (IL-34) cell-surface localization regulated by the molecular chaperone 78-kDa glucose-regulated protein facilitates the differentiation of monocytic cells.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {7},
pages = {2386-2396},
pmid = {30573681},
issn = {1083-351X},
mesh = {Animals ; Cell Differentiation/*physiology ; Cell Line ; Cell Membrane/genetics/*metabolism ; Dendritic Cells, Follicular/cytology/*metabolism ; Endoplasmic Reticulum Chaperone BiP ; Gene Expression Regulation/*physiology ; Heat-Shock Proteins/genetics/*metabolism ; Interleukins/*biosynthesis/genetics ; Male ; Mice ; Mice, Inbred BALB C ; Monocytes/cytology/*metabolism ; },
abstract = {Interleukin 34 (IL-34) constitutes a cytokine that shares a common receptor, colony-stimulating factor-1 receptor (CSF-1R), with CSF-1. We recently identified a novel type of monocytic cell termed follicular dendritic cell-induced monocytic cells (FDMCs), whose differentiation depended on CSF-1R signaling through the IL-34 produced from a follicular dendritic cell line, FL-Y. Here, we report the functional mechanisms of the IL-34-mediated CSF-1R signaling underlying FDMC differentiation. CRIPSR/Cas9-mediated knockout of the Il34 gene confirmed that the ability of FL-Y cells to induce FDMCs completely depends on the IL-34 expressed by FL-Y cells. Transwell culture experiments revealed that FDMC differentiation requires a signal from a membrane-anchored form of IL-34 on the FL-Y cell surface, but not from a secreted form, in a direct interaction between FDMC precursor cells and FL-Y cells. Furthermore, flow cytometric analysis using an anti-IL-34 antibody indicated that IL-34 was also expressed on the FL-Y cell surface. Thus, we explored proteins interacting with IL-34 in FL-Y cells. Mass spectrometry analysis and pulldown assay identified that IL-34 was associated with the molecular chaperone 78-kDa glucose-regulated protein (GRP78) in the plasma membrane fraction of FL-Y cells. Consistent with this finding, GRP78-heterozygous FL-Y cells expressed a lower level of IL-34 protein on their cell surface and exhibited a reduced competency to induce FDMC differentiation compared with the original FL-Y cells. These results indicated a novel GRP78-dependent localization and specific function of IL-34 in FL-Y cells related to monocytic cell differentiation.},
}
@article {pmid30573670,
year = {2019},
author = {Globyte, V and Lee, SH and Bae, T and Kim, JS and Joo, C},
title = {CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion.},
journal = {The EMBO journal},
volume = {38},
number = {4},
pages = {},
pmid = {30573670},
issn = {1460-2075},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; DNA/*genetics/metabolism ; *Gene Editing ; Genetic Engineering/*methods ; *Nucleotide Motifs ; Sequence Homology ; Streptococcus pyogenes/*genetics/metabolism ; Substrate Specificity ; },
abstract = {The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.},
}
@article {pmid30573669,
year = {2019},
author = {Al-Raawi, D and Jones, R and Wijesinghe, S and Halsall, J and Petric, M and Roberts, S and Hotchin, NA and Kanhere, A},
title = {A novel form of JARID2 is required for differentiation in lineage-committed cells.},
journal = {The EMBO journal},
volume = {38},
number = {3},
pages = {},
pmid = {30573669},
issn = {1460-2075},
mesh = {CRISPR-Cas Systems ; *Cell Differentiation ; *Cell Lineage ; Embryonic Stem Cells/*cytology/metabolism ; HEK293 Cells ; Humans ; Keratinocytes/*cytology/metabolism ; Polycomb Repressive Complex 2/antagonists &amp; inhibitors/genetics/*metabolism ; Protein Binding ; Protein Isoforms ; },
abstract = {Polycomb repressive complex-2 (PRC2) is a group of proteins that play an important role during development and in cell differentiation. PRC2 is a histone-modifying complex that catalyses methylation of lysine 27 of histone H3 (H3K27me3) at differentiation genes leading to their transcriptional repression. JARID2 is a co-factor of PRC2 and is important for targeting PRC2 to chromatin. Here, we show that, unlike in embryonic stem cells, in lineage-committed human cells, including human epidermal keratinocytes, JARID2 predominantly exists as a novel low molecular weight form, which lacks the N-terminal PRC2-interacting domain (ΔN-JARID2). We show that ΔN-JARID2 is a cleaved product of full-length JARID2 spanning the C-terminal conserved jumonji domains. JARID2 knockout in keratinocytes results in up-regulation of cell cycle genes and repression of many epidermal differentiation genes. Surprisingly, repression of epidermal differentiation genes in JARID2-null keratinocytes can be rescued by expression of ΔN-JARID2 suggesting that, in contrast to PRC2, ΔN-JARID2 promotes activation of differentiation genes. We propose that a switch from expression of full-length JARID2 to ΔN-JARID2 is important for the up-regulation differentiation genes.},
}
@article {pmid30573379,
year = {2018},
author = {Xing, S and Jia, M and Wei, L and Mao, W and Abbasi, UA and Zhao, Y and Chen, Y and Cao, M and Zhang, K and Dai, Z and Dou, Z and Jia, W and Li, B},
title = {CRISPR/Cas9-introduced single and multiple mutagenesis in strawberry.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {45},
number = {12},
pages = {685-687},
doi = {10.1016/j.jgg.2018.04.006},
pmid = {30573379},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems ; Fragaria/*genetics/growth &amp; development/metabolism ; Gene Editing ; Genome, Plant ; *Mutagenesis ; Plant Proteins/genetics/metabolism ; },
}
@article {pmid30572690,
year = {2018},
author = {Aman, R and Mahas, A and Butt, H and Aljedaani, F and Mahfouz, M},
title = {Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis.},
journal = {Viruses},
volume = {10},
number = {12},
pages = {},
pmid = {30572690},
issn = {1999-4915},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/*methods ; Genome, Viral ; Potyvirus/*genetics ; RNA/genetics ; *RNA Interference ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA.},
}
@article {pmid30572641,
year = {2018},
author = {Foltz, LP and Howden, SE and Thomson, JA and Clegg, DO},
title = {Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {19},
number = {12},
pages = {},
pmid = {30572641},
issn = {1422-0067},
mesh = {Atrophy ; CRISPR-Cas Systems/*genetics ; Eye Proteins/metabolism ; *Gene Editing ; Humans ; Nerve Growth Factors/metabolism ; Phagocytosis ; Pigmentation ; Retinal Photoreceptor Cell Outer Segment/metabolism/pathology ; Retinal Pigment Epithelium/metabolism/*pathology ; Serpins/metabolism ; },
abstract = {Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.},
}
@article {pmid30572386,
year = {2018},
author = {Liang, WJ and Cui, CC and Duan, GC and Liu, HY and Xu, YK and Xi, YL and Yang, HY and Chen, SY},
title = {[Identification and evaluation on methods with upstream flank sequences of CRISPR1, regarding Escherichia coli and Shigella].},
journal = {Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi},
volume = {39},
number = {12},
pages = {1607-1610},
doi = {10.3760/cma.j.issn.0254-6450.2018.12.013},
pmid = {30572386},
issn = {0254-6450},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics/isolation &amp; purification ; Genotype ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA ; Shigella/classification/*genetics/isolation &amp; purification ; },
abstract = {Objective: To analyze the effect of the identification and evaluation of Escherichia (E.) coli and Shigella, based on the upstream flanking sequences of CRISPR1. Methods: Both CRISPR and cas sequences were obtained through the BLAST with repeating sequences against the publicly complete genome in GenBank that related to E. coli and Shigella. Clustal X was used to perform multi-sequences alignment of the flanking sequences. PCR method was used to amplify the upstream flanking sequences of CRISPR1 in order to appraise the effect of identification and evaluation of upstream flanking sequences on E. coli and Shigella, which were based on the upstream flanking sequences of CRISPR1. Results: The results showed that 73.4% of the strains containing the I-E CRISPR/Cas that belonged to the phylogroups A, B1, D while 8.4% strains carried the I-F CRISPR/Cas. Another 17.2% of the strains owned CRISPR3-4 (non-CRISPR/Cas) only belonged to the phylogroups B2. All the Shigella strains carried I-E CRISPR/Cas. More than 99% of similarity the CRISPR1 upstream-flanking sequences was seen in E. coli (except B2) and Shigella and E. coli (B2). Both sensitivity and specificity were greater than 91% after PCR amplification in the region to identify the E.coli and Shigella. Conclusion: The upstream of CRISPR1 could achieve a preliminary identification effect on E.coli and Shigella.},
}
@article {pmid30571788,
year = {2018},
author = {Chakraborty, S and von Mentzer, A and Begum, YA and Manzur, M and Hasan, M and Ghosh, AN and Hossain, MA and Camilli, A and Qadri, F},
title = {Phenotypic and genomic analyses of bacteriophages targeting environmental and clinical CS3-expressing enterotoxigenic Escherichia coli (ETEC) strains.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0209357},
pmid = {30571788},
issn = {1932-6203},
support = {R01 AI055058/AI/NIAID NIH HHS/United States ; R37 AI055058/AI/NIAID NIH HHS/United States ; },
mesh = {Adult ; Bacteriophages/genetics/*pathogenicity ; Bangladesh ; CRISPR-Cas Systems/immunology ; Child, Preschool ; Diarrhea/microbiology/*prevention &amp; control ; Enterotoxigenic Escherichia coli/immunology/isolation &amp; purification/metabolism/*virology ; Environmental Pollution/*prevention &amp; control ; Escherichia coli Infections/microbiology/*prevention &amp; control ; Escherichia coli Proteins/metabolism ; Feces/microbiology ; Fimbriae Proteins/metabolism ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Water Microbiology ; },
abstract = {Diarrhea due to infection of enterotoxigenic Escherichia coli (ETEC) is of great concern in several low and middle-income countries. ETEC infection is considered to be the most common cause of diarrhea in Bangladesh and is mainly spread through contaminated water and food. ETEC pathogenesis is mediated by the expression of enterotoxins and colonization factors (CFs) that target the intestinal mucosa. ETEC can survive for extended time periods in water, where they are likely to be attacked by bacteriophages. Antibiotic resistance is common amongst enteric pathogens and therefore is the use of bacteriophages (phage) as a therapeutic tool an interesting approach. This study was designed to identify novel phages that specifically target ETEC virulence factors. In total, 48 phages and 195 ETEC isolates were collected from water sources and stool samples. Amongst the identified ETEC specific phages, an enterobacteria phage T7, designated as IMM-002, showed a significant specificity towards colonization factor CS3-expressing ETEC isolates. Antibody-blocking and phage-neutralization assays revealed that CS3 is used as a host receptor for the IMM-002 phage. The bacterial CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) defence mechanism can invoke immunity against phages. Genomic analyses coupled with plaque assay experiments indicate that the ETEC CRISPR-Cas system is involved in the resistance against the CS3-specific phage (IMM-002) and the previously identified CS7-specific phage (IMM-001). As environmental water serves as a reservoir for ETEC, it is important to search for new antimicrobial agents such as phages in environmental water as well as the human gut. A better understanding of how the interplay between ETEC-specific phages and ETEC isolates affects the ETEC diversity, both in environmental ecosystems and within the host, is important for the development of new treatments for ETEC infections.},
}
@article {pmid30570178,
year = {2019},
author = {Wan, Y and Guo, R and Deng, M and Liu, Z and Pang, J and Zhang, G and Wang, Z and Wang, F},
title = {Efficient generation of CLPG1-edited rabbits using the CRISPR/Cas9 system.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {54},
number = {3},
pages = {538-544},
doi = {10.1111/rda.13394},
pmid = {30570178},
issn = {1439-0531},
mesh = {Animals ; Animals, Newborn ; Base Sequence ; *CRISPR-Cas Systems ; Hypertrophy/*genetics ; *Models, Animal ; Muscle, Skeletal/*growth &amp; development ; *Mutation ; Phenotype ; Rabbits ; },
abstract = {The sheep callipyge (CLPG) phenotype, a well-known muscular hypertrophy syndrome, is caused by an A-to-G transition in the CLPG1 locus. The mechanisms of CLPG phenotype are very complicated and remain to be further studied. Lacking suitable animal models containing CLPG mutations may partially contribute to these unanswered mechanisms. In this study, we confirmed that the CLPG1 locus, especially the 12-bp CLPG1 motif, is conserved in mammalian animals including rabbit. Then, we generated seven CLPG1-edited rabbits with 100% efficiency using CRISPR/Cas9 system combined with cytoplasm injection technology. All the newborn rabbits were mosaicism with numerous kinds of mutations around the target sites. Among the nine screened potential off-target sites (POTs) for the two sgRNAs used in this study, none off-target effect was detected. This indicated that we efficiently and precisely generated CLPG1-edited rabbits, and we believe that these newly generated rabbits will do help to unravel the mechanisms of the CLPG phenotype in the future.},
}
@article {pmid30569609,
year = {2019},
author = {Parry, G and Harrison, CJ},
title = {GARNet gene editing workshop.},
journal = {The New phytologist},
volume = {221},
number = {2},
pages = {640-643},
doi = {10.1111/nph.15573},
pmid = {30569609},
issn = {1469-8137},
support = {//New Phytologist Trust/International ; //Bristol Centre for Agricultural Innovation/International ; BB/M004376/1//UKRI BBSRC- funded GARNet2020/International ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Endonucleases ; *Gene Editing/legislation &amp; jurisprudence ; Legislation as Topic ; Plants/*genetics ; Plants, Genetically Modified ; },
}
@article {pmid30569414,
year = {2019},
author = {Dadheech, N and James Shapiro, AM},
title = {Human Induced Pluripotent Stem Cells in the Curative Treatment of Diabetes and Potential Impediments Ahead.},
journal = {Advances in experimental medicine and biology},
volume = {1144},
number = {},
pages = {25-35},
doi = {10.1007/5584_2018_305},
pmid = {30569414},
issn = {0065-2598},
mesh = {CRISPR-Cas Systems ; Diabetes Mellitus, Type 1/*therapy ; Diabetes Mellitus, Type 2/*therapy ; Humans ; Induced Pluripotent Stem Cells/*cytology ; *Islets of Langerhans Transplantation ; *Stem Cell Transplantation ; Transplantation, Autologous ; },
abstract = {The successful landmark discovery of mouse and human inducible pluripotential stem cells (iPSC's) by Takahashi and Yamanaka in 2006 and 2007 has triggered a revolution in the potential generation of self-compatible cells for regenerative medicine, and further opened up a new avenue for "disease in dish" drug screening of self-target cells (Neofytou et al. 2015). The introduction of four 'Yamanaka' transcription factors through viral or other transfection of mature cells can induce pluripotency and acquired plasticity. These factors include transduction with octamer-binding transcription factor-4 (Oct-4), nanog homeobox (Nanog), sex-determining region Y-box-2 (Sox-2) and MYC protooncogene (cMyc). Such cells become iPSC's (Takahashi and Yamanaka 2006). These reprogrammed cells exhibit increased telomerase activity and have a hypomethylated gene promotor region similar to embryonic stem cells (ESC's). These milestone discoveries have generated immense hope that diseases such as diabetes could be treated and effectively cured by transplantation of self-compatible, personalized autologous stem cell transplantation of β-cells that release physiological insulin under glycemic control (Maehr et al. 2009; Park et al. 2008) (Fig. 1). Diabetes is a profligate disease of disordered glucose metabolism resulting from an absolute or relative deficiency of insulin, the consequences of which lead to immense socio-economic societal burden. While there are many different types of diabetes, the two major types (type 1 diabetes (T1DM) and type 2 diabetes (T2DM) are caused respectively by immune-mediated destruction (T1DM) or malfunctioning (T2DM) insulin-producing β-cells within the endocrine pancreas, the islets of Langerhans (Atkinson et al. 2011; Holman et al. 2015; You and Henneberg 2016). Almost 425 million people are affected by the global burden of diabetes, and this is predicted to increase by 48% (629 million) by 2045 (International Diabetes Federation Atlas 8th Ed 2018). Whole pancreas or islet cell transplantation offer an effective alternative to injected insulin, but both require lifelong potent immunosuppression to control both allo-and autoimmunity. Whole pancreas transplantation involves invasive complex surgery and is associated with greater morbidity and occasional mortality, while islet transplantation involves a minimally invasive intraportal hepatic infusion. Generally, whole pancreas transplantation provides greater metabolic reserve, but this may be matched by cumulative multiple islet infusions to achieve insulin independence. An additional challenge of islet transplantation is progressive loss of complete insulin independence over time, which may be multifactorial, the dominant factor however being ineffective control of autoimmunity. Both whole pancreas and islet transplantation are restricted to patients at risk of severe hypoglycemia that cannot be stabilized by alternate means, or in recipients that are already immunosuppressed in order to sustain a kidney or other solid organ transplant. The risks of chronic immunosuppression and the scarcity of human organ donors mean that both of these transplantation therapies cannot presently be extended to the broader diabetic population (Shapiro 2011; Shapiro et al. 2006). Recent progress in xenotransplantation of multiple knock-out 'humanized' pig islets could offer one potential solution, perhaps aided by clustered regularly interspaced short palindromic repeats/CRISPR associated-9 (CRISPR/Cas-9) gene editing approaches, but this remains to be proven in practice. Human stem cell derived new β-cell products could effectively address the global supply challenge for broad application across all forms of diabetes, but recurrent autoimmunity may still remain an insurmountable challenge. Considerable progress in the generation of human stem cell derived SC-β cells from ESC, iPS and other adult cell sources such as mesenchymal stem cells (MSCs) offer huge hope that a personalized, 'syngeneic' cell could be transplanted without risk of alloimmunity, thereby securing sufficient supply to meet future global demand (Cito et al. 2018).},
}
@article {pmid30568241,
year = {2019},
author = {Cornel, MC and Howard, HC and Lim, D and Bonham, VL and Wartiovaara, K},
title = {Moving towards a cure in genetics: what is needed to bring somatic gene therapy to the clinic?.},
journal = {European journal of human genetics : EJHG},
volume = {27},
number = {3},
pages = {484-487},
pmid = {30568241},
issn = {1476-5438},
mesh = {CRISPR-Cas Systems ; Clinical Trials as Topic ; *Congresses as Topic ; Gene Editing/methods ; Genetic Therapy/*methods ; Genetics, Medical/*methods ; Humans ; },
abstract = {Clinical trials using somatic gene editing (e.g., CRISPR-Cas9) have started in Europe and the United States and may provide safe and effective treatment and cure, not only for cancers but also for some monogenic conditions. In a workshop at the 2018 European Human Genetics Conference, the challenges of bringing somatic gene editing therapies to the clinic were discussed. The regulatory process needs to be considered early in the clinical development pathway to produce the data necessary to support the approval by the European Medicines Agency. The roles and responsibilities for geneticists may include counselling to explain the treatment possibilities and safety interpretation.},
}
@article {pmid30568213,
year = {2018},
author = {Knoepfler, P},
title = {Gene editing: sloppy definitions mislead.},
journal = {Nature},
volume = {564},
number = {7736},
pages = {345},
doi = {10.1038/d41586-018-07802-2},
pmid = {30568213},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genome ; },
}
@article {pmid30566855,
year = {2018},
author = {Li, B and Zeng, C and Li, W and Zhang, X and Luo, X and Zhao, W and Zhang, C and Dong, Y},
title = {Synthetic Oligonucleotides Inhibit CRISPR-Cpf1-Mediated Genome Editing.},
journal = {Cell reports},
volume = {25},
number = {12},
pages = {3262-3272.e3},
pmid = {30566855},
issn = {2211-1247},
support = {R01 HL136652/HL/NHLBI NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Proteins/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/metabolism ; *Gene Editing ; Genetic Loci ; *Genome ; HEK293 Cells ; Humans ; Oligonucleotides/*metabolism ; Phosphorothioate Oligonucleotides/metabolism ; RNA/metabolism ; Ribonucleoproteins/metabolism ; },
abstract = {Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1. Time-dependent inhibitory effects were validated in multiple loci of different human cells. Further studies demonstrated that PS-modified DNA oligonucleotides were able to serve as Cpf1 inhibitors in a sequence-independent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder target DNA binding and recognition by Cpf1. Consequently, these synthetic DNA molecules expand the sources of CRISPR inhibitors, providing a platform to inactivate Cpf1-mediated genome editing.},
}
@article {pmid30566249,
year = {2019},
author = {Moore, A},
title = {"Memetic Engineering", Please! Thought, Values and Behaviour Are as Important as Technology!.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {41},
number = {1},
pages = {e1800242},
doi = {10.1002/bies.201800242},
pmid = {30566249},
issn = {1521-1878},
mesh = {*CRISPR-Cas Systems ; *Evolution, Molecular ; *Gene Editing ; Humans ; },
}
@article {pmid30565988,
year = {2018},
author = {Lv, W and Qiao, L and Petrenko, N and Li, W and Owens, AT and McDermott-Roe, C and Musunuru, K},
title = {Functional Annotation of TNNT2 Variants of Uncertain Significance With Genome-Edited Cardiomyocytes.},
journal = {Circulation},
volume = {138},
number = {24},
pages = {2852-2854},
pmid = {30565988},
issn = {1524-4539},
support = {R01 GM104464/GM/NIGMS NIH HHS/United States ; R01 HL118744/HL/NHLBI NIH HHS/United States ; },
mesh = {Action Potentials/drug effects ; CRISPR-Cas Systems/genetics ; Cardiomyopathy, Dilated/genetics/pathology ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology ; Isoproterenol/pharmacology ; Myocytes, Cardiac/cytology/drug effects/metabolism ; Polymorphism, Single Nucleotide ; Troponin T/*genetics ; },
}
@article {pmid30565897,
year = {2019},
author = {Li, Y and Li, AC and Xu, Q},
title = {Intracellular Delivery of His-Tagged Genome-Editing Proteins Enabled by Nitrilotriacetic Acid-Containing Lipidoid Nanoparticles.},
journal = {Advanced healthcare materials},
volume = {8},
number = {6},
pages = {e1800996},
pmid = {30565897},
issn = {2192-2659},
support = {R21 EB024041/EB/NIBIB NIH HHS/United States ; R01 EB027170/EB/NIBIB NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; DMR1452122//National Science Foundation/International ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Cell Survival/drug effects ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Histidine/genetics/metabolism ; Humans ; Lipids/*chemistry ; Nanoparticles/*chemistry/toxicity ; Nitrilotriacetic Acid/*chemistry ; Oligopeptides/genetics/metabolism ; Transfection ; },
abstract = {Protein- and peptide-based therapeutics with high tolerance and specificity along with low off-target effects and genetic risks have attracted tremendous attention over the last three decades. Herein, a new type of noncationic lipidoid nanoparticle (LNP) is reported for His-tagged protein delivery. Active lipidoids are synthesized by conjugating a nitrilotriacetic acid group with hydrophobic tails (EC16, O16B, and O17O) and nanoparticles are formulated in the presence of nickel ions and helper lipids (cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). It is demonstrated that the newly developed LNPs are capable of delivering various His-tagged proteins including green fluorescent protein (GFP), (-30)GFP-Cre recombinase, and CRISPR/Cas9 ribonucleoprotein into mammalian cells.},
}
@article {pmid30565410,
year = {2019},
author = {Zhao, S and Xing, Z and Liu, Z and Liu, Y and Liu, X and Chen, Z and Li, J and Yan, R},
title = {Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system.},
journal = {Pest management science},
volume = {75},
number = {7},
pages = {1921-1932},
doi = {10.1002/ps.5305},
pmid = {30565410},
issn = {1526-4998},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Eye Color/genetics ; Gene Editing/methods ; Germ Cells ; INDEL Mutation ; Mutagenesis ; RNA Editing ; RNA, Messenger ; Sex Determination Processes ; Tephritidae/embryology/*genetics ; },
abstract = {BACKGROUND: Bactrocera dorsalis (Hendel), a very destructive insect pest of many fruits and vegetables, is widespread in many Asian countries. To facilitate control of this pest, it is essential to investigate its genetics and gene function using targeted gene disruption.<br><br>RESULTS: Here, we describe successful targeted mutagenesis of the white and transformer genes in B. dorsalis through use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. Co-injection of the white sgRNA and Cas9 mRNA into B. dorsalis embryos caused eye color change, and the white mutations in the germline were heritable. CRISPR-mediated knockout of the sex determination gene transformer (tra) in B. dorsalis resulted in a male-biased sex ratio and adult flies with abnormal outer and interior reproductive organs. Small indels and substitutions were induced by CRIRPR for both genes.<br><br>CONCLUSION: Our data demonstrate that somatic and germline genome engineering of the pest B. dorsalis can be performed efficiently using the CRISPR/Cas9 system, opening the door to the use of the CRISPR-mediated method for functional annotations of genes in B. dorsalis and for its population control using, for example, such as gene drive. &#xa9; 2018 Society of Chemical Industry.},
}
@article {pmid30563863,
year = {2018},
author = {Felce, JH and Sezgin, E and Wane, M and Brouwer, H and Dustin, ML and Eggeling, C and Davis, SJ},
title = {CD45 exclusion- and cross-linking-based receptor signaling together broaden FcεRI reactivity.},
journal = {Science signaling},
volume = {11},
number = {561},
pages = {},
pmid = {30563863},
issn = {1937-9145},
support = {MC_UU_12010/4/MRC_/Medical Research Council/United Kingdom ; 098274/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; MR/K01577X/1/MRC_/Medical Research Council/United Kingdom ; 104924/14/Z/14/WT_/Wellcome Trust/United Kingdom ; 091911/WT_/Wellcome Trust/United Kingdom ; 100262/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; MC_UU_12010/9/MRC_/Medical Research Council/United Kingdom ; 207547/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 107375/Z/15/Z/WT_/Wellcome Trust/United Kingdom ; 207547/WT_/Wellcome Trust/United Kingdom ; MC_UU_12025/MRC_/Medical Research Council/United Kingdom ; MC_UU_00008/4/MRC_/Medical Research Council/United Kingdom ; MC_UU_00008/9/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Degranulation ; Cross-Linking Reagents/chemistry ; Immunoglobulin E/*metabolism ; Integrins/metabolism ; Leukemia, Basophilic, Acute/*immunology/metabolism/pathology ; Leukocyte Common Antigens/genetics/*metabolism ; Mast Cells/*immunology/metabolism ; Rats ; Receptors, IgE/antagonists &amp; inhibitors/genetics/*metabolism ; Tumor Cells, Cultured ; },
abstract = {For many years, the high-affinity receptor for immunoglobulin E (IgE) FcεRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FcεRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FcεRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FcεRI-ligand engagement. Similarly to the T cell receptor, FcεRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion-based receptor triggering could function together with cross-linking-based FcεRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion-based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.},
}
@article {pmid30563861,
year = {2018},
author = {Lock, JT and Alzayady, KJ and Yule, DI and Parker, I},
title = {All three IP3 receptor isoforms generate Ca[2+] puffs that display similar characteristics.},
journal = {Science signaling},
volume = {11},
number = {561},
pages = {},
pmid = {30563861},
issn = {1937-9145},
support = {R01 DE014756/DE/NIDCR NIH HHS/United States ; R01 DE019245/DE/NIDCR NIH HHS/United States ; R37 GM048071/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Calcium/*metabolism ; *Calcium Signaling ; HEK293 Cells ; Humans ; Inositol 1,4,5-Trisphosphate/*metabolism ; Inositol 1,4,5-Trisphosphate Receptors/antagonists &amp; inhibitors/genetics/*metabolism ; Kinetics ; Protein Isoforms ; },
abstract = {Inositol 1,4,5-trisphosphate (IP3) evokes Ca[2+] release through IP3 receptors (IP3Rs) to generate both local Ca[2+] puffs arising from concerted openings of clustered IP3Rs and cell-wide Ca[2+] waves. Imaging Ca[2+] puffs with single-channel resolution yields information on the localization and properties of native IP3Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP3Rs. Here, we used TIRF and light-sheet microscopy to image Ca[2+] puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP3R type 1, 2, or 3. Photorelease of the IP3 analog i-IP3 in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca[2+] flux was similar among isoforms, indicating that clusters of different IP3R isoforms contain comparable numbers of active channels. Our results show that all three IP3R isoforms cluster to generate local Ca[2+] puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells.},
}
@article {pmid30562583,
year = {2019},
author = {Bruni, GO and Zhong, K and Lee, SC and Wang, P},
title = {CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {124},
number = {},
pages = {1-7},
pmid = {30562583},
issn = {1096-0937},
support = {R21 AI121451/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Genes, Fungal ; Genetic Vectors ; Orotate Phosphoribosyltransferase/genetics ; Orotic Acid/analogs &amp; derivatives/pharmacology ; *Point Mutation ; Rhizopus/drug effects/enzymology/*genetics ; Uracil ; },
abstract = {Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.},
}
@article {pmid30562388,
year = {2018},
author = {Xiong, T and Rohm, D and Workman, RE and Roundtree, L and Novina, CD and Timp, W and Ostermeier, M},
title = {Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0209408},
pmid = {30562388},
issn = {1932-6203},
support = {DP1 DK105602/DK/NIDDK NIH HHS/United States ; },
mesh = {Binding Sites/genetics ; CRISPR-Cas Systems/*genetics ; CpG Islands/genetics ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; DNA Methylation/genetics ; Escherichia coli ; Gene Editing/*methods ; Mutagenesis/genetics ; Protein Engineering/*methods ; Protein Interaction Domains and Motifs/genetics ; RNA, Guide/genetics ; Recombinant Fusion Proteins/*genetics/metabolism ; },
abstract = {Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo gene regulation could have therapeutic and research applications. We have previously developed a dCas9-MC/MN protein for targeting CpG methylation. dCas9-MC/MN is composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused to a nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed dCas9-MC/MN to methylate target sites in E. coli and human cells but also caused some low-level off-target methylation. Here, in E. coli, we show that shortening the dCas9-MC linker increases methylation of CpG sites located at select distances from the dCas9 binding site. Although a shortened linker decreased methylation of other CpGs proximal to the target site, it did not reduce off-target methylation of more distant CpG sites. Instead, targeted mutagenesis of the methyltransferase's DNA binding domain, designed to reduce DNA affinity, significantly and preferentially reduced methylation of such sites.},
}
@article {pmid30561900,
year = {2019},
author = {Luhur, A and Klueg, KM and Zelhof, AC},
title = {Generating and working with Drosophila cell cultures: Current challenges and opportunities.},
journal = {Wiley interdisciplinary reviews. Developmental biology},
volume = {8},
number = {3},
pages = {e339},
pmid = {30561900},
issn = {1759-7692},
support = {P40 OD010949/OD/NIH HHS/United States ; P40OD010949/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Drosophila/*cytology/*genetics/metabolism ; Drosophila Proteins/*genetics/metabolism ; *Gene Editing ; *Gene Expression Regulation ; Phenotype ; },
abstract = {The use of Drosophila cell cultures has positively impacted both fundamental and biomedical research. The most widely used cell lines: Schneider, Kc, the CNS and imaginal disc lines continue to be the choice for many applications. Drosophila cell lines provide a homogenous source of cells suitable for biochemical experimentations, transcriptomics, functional genomics, and biomedical applications. They are amenable to RNA interference and serve as a platform for high-throughput screens to identify relevant candidate genes or drugs for any biological process. Currently, CRISPR-based functional genomics are also being developed for Drosophila cell lines. Even though many uniquely derived cell lines exist, cell genetic techniques such the transgenic UAS-GAL4-based Ras[V12] oncogene expression, CRISPR-Cas9 editing and recombination mediated cassette exchange are likely to drive the establishment of many more lines from specific tissues, cells, or genotypes. However, the pace of creating new lines is hindered by several factors inherent to working with Drosophila cell cultures: single cell cloning, optimal media formulations and culture conditions capable of supporting lines from novel tissue sources or genotypes. Moreover, even though many Drosophila cell lines are morphologically and transcriptionally distinct it may be necessary to implement a standard for Drosophila cell line authentication, ensuring the identity and purity of each cell line. Altogether, recent advances and a standardized authentication effort should improve the utility of Drosophila cell cultures as a relevant model for fundamental and biomedical research. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes.},
}
@article {pmid30560399,
year = {2019},
author = {Hryhorowicz, M and Grześkowiak, B and Mazurkiewicz, N and Śledziński, P and Lipiński, D and Słomski, R},
title = {Improved Delivery of CRISPR/Cas9 System Using Magnetic Nanoparticles into Porcine Fibroblast.},
journal = {Molecular biotechnology},
volume = {61},
number = {3},
pages = {173-180},
pmid = {30560399},
issn = {1559-0305},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Fibroblasts/*cytology ; Gene Editing/*methods ; Heat-Shock Proteins/*genetics ; Magnetic Fields ; Magnetite Nanoparticles/administration &amp; dosage/chemistry ; Plasmids/*administration &amp; dosage/genetics ; Polyethyleneimine/chemistry ; Swine ; Transfection ; },
abstract = {Genetically modified pigs play an important role in agriculture and biomedical research; hence, new efficient methods are needed to obtain genetically engineered cells and animals. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system represents an effective genome editing tool. It consists of two key molecules: single guide RNA (sgRNA) and the Cas9 endonuclease that can be introduced into the cells as one plasmid. Typical delivery methods for CRISPR/Cas9 components are limited by low transfection efficiency or toxic effects on cells. Here, we describe the use of magnetic nanoparticles and gradient magnetic field to improve delivery of CRISPR/Cas9 constructs into porcine fetal fibroblasts. Polyethylenimine-coated nanoparticles with magnetic iron oxide core were used to form magnetic plasmid DNA lipoplexes. CRISPR/Cas9 construct was prepared to induce site-specific cutting at the porcine H11 locus. Quantitative assessment of genomic cleavage by sequence trace decomposition demonstrated that the magnetofection efficiency was more than 3.5 times higher compared to the classic lipofection method. The Tracking of Indels by Decomposition web tool precisely determined the spectrum of indels that occurred. Simultaneously, no additional cytotoxicity associated with the utilization of magnetic nanoparticles was observed. Our results indicate that magnetofection enables effective delivery of the CRISPR/Cas9 construct into porcine fetal fibroblasts with low cell toxicity.},
}
@article {pmid30559443,
year = {2018},
author = {Becker, JR and Cuella-Martin, R and Barazas, M and Liu, R and Oliveira, C and Oliver, AW and Bilham, K and Holt, AB and Blackford, AN and Heierhorst, J and Jonkers, J and Rottenberg, S and Chapman, JR},
title = {The ASCIZ-DYNLL1 axis promotes 53BP1-dependent non-homologous end joining and PARP inhibitor sensitivity.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {5406},
pmid = {30559443},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/R017549/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; BRCA1 Protein/*genetics ; Breast Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cytoplasmic Dyneins/*metabolism ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; Female ; Genomic Instability/genetics ; HEK293 Cells ; Humans ; MCF-7 Cells ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Transcription Factors/*metabolism ; Tumor Suppressor p53-Binding Protein 1/*genetics ; },
abstract = {53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub-DYNLL1-as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1's recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers.},
}
@article {pmid30559437,
year = {2019},
author = {Shao, Y and Lu, N and Cai, C and Zhou, F and Wang, S and Zhao, Z and Zhao, G and Zhou, JQ and Xue, X and Qin, Z},
title = {A single circular chromosome yeast.},
journal = {Cell research},
volume = {29},
number = {1},
pages = {87-89},
pmid = {30559437},
issn = {1748-7838},
mesh = {CRISPR-Cas Systems ; Chromosomes, Artificial, Yeast/*genetics ; DNA, Circular/*genetics ; Microorganisms, Genetically-Modified/*genetics ; Saccharomyces cerevisiae/*genetics ; },
}
@article {pmid30559432,
year = {2019},
author = {Breinig, M and Schweitzer, AY and Herianto, AM and Revia, S and Schaefer, L and Wendler, L and Cobos Galvez, A and Tschaharganeh, DF},
title = {Multiplexed orthogonal genome editing and transcriptional activation by Cas12a.},
journal = {Nature methods},
volume = {16},
number = {1},
pages = {51-54},
pmid = {30559432},
issn = {1548-7105},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; *Transcriptional Activation ; },
abstract = {CRISPR-Cas9-based combinatorial perturbation approaches for orthogonal knockout and gene activation have been impeded by complex vector designs and co-delivery of multiple constructs. Here, we demonstrate that catalytically active CRISPR-Cas12a fused to a transcriptional-activator domain enables flexible switching between genome editing and transcriptional activation by altering guide length. By leveraging Cas12a-mediated CRISPR-RNA array processing, we illustrate that Cas12a-VPR enables simplified multiplexed knockout and transcriptional activation in vitro and in vivo.},
}
@article {pmid30559380,
year = {2019},
author = {Ishizuka, JJ and Manguso, RT and Cheruiyot, CK and Bi, K and Panda, A and Iracheta-Vellve, A and Miller, BC and Du, PP and Yates, KB and Dubrot, J and Buchumenski, I and Comstock, DE and Brown, FD and Ayer, A and Kohnle, IC and Pope, HW and Zimmer, MD and Sen, DR and Lane-Reticker, SK and Robitschek, EJ and Griffin, GK and Collins, NB and Long, AH and Doench, JG and Kozono, D and Levanon, EY and Haining, WN},
title = {Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade.},
journal = {Nature},
volume = {565},
number = {7737},
pages = {43-48},
pmid = {30559380},
issn = {1476-4687},
support = {T32 CA009172/CA/NCI NIH HHS/United States ; T32 HG002295/HG/NHGRI NIH HHS/United States ; },
mesh = {Adenosine Deaminase/*deficiency/genetics/*metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/*drug effects ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*drug effects/genetics ; Female ; Histocompatibility Antigens Class I/immunology ; Immunotherapy ; Inflammation/genetics/immunology ; Interferon-Induced Helicase, IFIH1/metabolism ; Interferons/immunology ; Melanoma, Experimental/*drug therapy/*genetics/immunology/radiotherapy ; Mice ; Mice, Inbred C57BL ; Phenotype ; Programmed Cell Death 1 Receptor/*antagonists &amp; inhibitors ; RNA Editing ; RNA, Double-Stranded/genetics ; RNA-Binding Proteins/genetics/*metabolism ; Receptors, G-Protein-Coupled/metabolism ; },
abstract = {Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8[+] T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.},
}
@article {pmid30559373,
year = {2019},
author = {Hultquist, JF and Hiatt, J and Schumann, K and McGregor, MJ and Roth, TL and Haas, P and Doudna, JA and Marson, A and Krogan, NJ},
title = {CRISPR-Cas9 genome engineering of primary CD4[+] T cells for the interrogation of HIV-host factor interactions.},
journal = {Nature protocols},
volume = {14},
number = {1},
pages = {1-27},
pmid = {30559373},
issn = {1750-2799},
support = {F30 DK120213/DK/NIDDK NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; R01 AI120694/AI/NIAID NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; K22 AI136691/AI/NIAID NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; U19 AI118610/AI/NIAID NIH HHS/United States ; P30 AI027763/AI/NIAID NIH HHS/United States ; U19 AI106754/AI/NIAID NIH HHS/United States ; P01 AI063302/AI/NIAID NIH HHS/United States ; DP2 DA042423/DA/NIDA NIH HHS/United States ; P01 AI090935/AI/NIAID NIH HHS/United States ; },
mesh = {Antibodies/pharmacology ; Antigens, CD/genetics/immunology ; CD4-Positive T-Lymphocytes/drug effects/immunology/*metabolism/virology ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Nucleus/drug effects/immunology/metabolism/virology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation/methods ; Gene Editing/*methods ; Genome, Human ; HIV-1/genetics/*immunology ; *High-Throughput Screening Assays ; Host-Pathogen Interactions/genetics/*immunology ; Humans ; Lymphocyte Activation ; Primary Cell Culture ; RNA, Guide/genetics/metabolism ; Ribonucleoproteins/genetics/immunology ; },
abstract = {CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4[+] T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4[+] T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4[+] T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.},
}
@article {pmid30559276,
year = {2019},
author = {Hudson, AM and Mannix, KM and Gerdes, JA and Kottemann, MC and Cooley, L},
title = {Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {1},
pages = {},
pmid = {30559276},
issn = {1477-9129},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; R01 GM043301/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; P40 OD010949/OD/NIH HHS/United States ; T32 GM007499/GM/NIGMS NIH HHS/United States ; RC1 GM091791/GM/NIGMS NIH HHS/United States ; },
mesh = {Actin Cytoskeleton/genetics/*metabolism ; Actins/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster ; Microfilament Proteins/genetics/*metabolism ; Mutagenesis ; Oocytes/cytology/*metabolism ; Proteasome Endopeptidase Complex/genetics/*metabolism ; *Proteolysis ; *Ubiquitination ; },
abstract = {During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3[Kelch]) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3[Kelch] ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3[Kelch] substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.},
}
@article {pmid30559100,
year = {2018},
author = {Xin, GW and Hu, XX and Wang, KJ and Wang, XC},
title = {[Cas9 protein variant VQR recognizes NGAC protospacer adjacent motif in rice].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {12},
pages = {1112-1119},
doi = {10.16288/j.yczz.18-126},
pmid = {30559100},
issn = {0253-9772},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Oryza/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is the third-generation genome editing tools that was developed and widely used in recent years. However, Streptococcus pyogenes Cas9 (SpCas9) in this system could only recognize NGG PAM (protospacer adjacent motif), which largely restricts the range of genome editing. The VQR (D1135V/R1335Q/T1337R) variant of SpCas9 could recognize NGAA, NGAG and NGAT PAMs in rice. However, whether VQR variant could recognize NGAC PAM remains unclear. In this study, three low editing efficiency sites of the VQR variant, NAL1-Q1, NAL1-Q2 and LPA1-Q, were selected for genome editing using the improved CRISPR/VQR system. The improved CRISPR/VQR system effectively edited these target sites, and the gene editing efficiency was 9.75%, 43.90% and 29.26% respectively. To ensure the recognition of NGAC PAM by the improved CRISPR/VQR system, two NGAC PAM containing sites (NAL-C and GL1-C) in the NARROW LEAF 1 (NAL1) for leaf length and GLOSSY1 (GL1) genes for wax biosynthesis were selected for genome editing in rice in this study, and 57 transgenic plants were obtained. The PCR amplification and sequencing results showed that 27 plants (47.36%) had mutation in the NAL1-C site, 44 plants (77.19%) had mutation in the GL1 gene, and 26 plants (45.61%) had mutation in the NAL-C and GL1-C sites. Further analysis revealed that there were four types of mutations caused by the CRISPR/VQR system, respectively for the hybrid mutation, biallelic mutation, chimeric mutation and homozygous mutations. Among them, heterozygous mutation and biallelic mutation were dominant changes. These results indicated that the improved CRISPR/VQR system could efficiently edit the NGAC PAM sites of the rice and produce abundant mutant types. This study provides a theoretical basis for NGAC PAM editing in rice and other related plants.},
}
@article {pmid30559097,
year = {2018},
author = {Liu, CX and Geng, LZ and Xu, JP},
title = {[Detection methods of genome editing in plants].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {12},
pages = {1075-1091},
doi = {10.16288/j.yczz.18-079},
pmid = {30559097},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Engineering ; Genome, Plant ; Plants/*genetics ; },
abstract = {The life science has entered a new chapter with the revolutionary implementation of the CRISPR/Cas9 genome editing technology in various living organisms. With the unique flexibility, feasibility and extendibility, the CRISPR/Cas9 technology greatly accelerates genetic engineering research, as well as plant molecular breeding. However, it has become a challenge to screen for and identify genome-edited plants at early stages in a rapid and high-throughput fashion, due to the massive number of plants produced from transformation process. In this review, we summarize the molecular methods developed in recent years to identify genome-edited plants. We compare their advantages and disadvantages, and the scope of application. In addition, we provide insights of the development trend of detection methods for plant genome editing. This review will serve as a reference for future genome editing research in plants.},
}
@article {pmid30559070,
year = {2019},
author = {Recchia, A},
title = {AAV-CRISPR Persistence in the Eye of the Beholder.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {1},
pages = {12-14},
pmid = {30559070},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Campylobacter jejuni/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics ; Gene Editing ; Mice ; Retina ; },
}
@article {pmid30558714,
year = {2018},
author = {Faure, JD and Napier, JA},
title = {Europe's first and last field trial of gene-edited plants?.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30558714},
issn = {2050-084X},
support = {BBS/E/C/000I0420//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Brassicaceae/*genetics/*growth &amp; development ; Crops, Agricultural/genetics/*growth &amp; development ; European Union ; Gene Editing/*legislation &amp; jurisprudence ; Plants, Genetically Modified/*genetics/*growth &amp; development ; United Kingdom ; },
abstract = {On 5 June this year the first field trial of a CRISPR-Cas-9 gene-edited crop began at Rothamsted Research in the UK, having been approved by the UK Department for Environment, Food &amp; Rural Affairs. However, in late July 2018, after the trial had started, the European Court of Justice ruled that techniques such as gene editing fall within the European Union's 2001 GMO directive, meaning that our gene-edited Camelina plants should be considered as genetically modified (GM). Here we describe our experience of running this trial and the legal transformation of our plants. We also consider the future of European plant research using gene-editing techniques, which now fall under the burden of GM regulation, and how this will likely impede translation of publicly funded basic research.},
}
@article {pmid30558146,
year = {2018},
author = {Ding, F and Hu, Q and Wang, M and Zhang, S},
title = {Knockout of SlSBPASE Suppresses Carbon Assimilation and Alters Nitrogen Metabolism in Tomato Plants.},
journal = {International journal of molecular sciences},
volume = {19},
number = {12},
pages = {},
pmid = {30558146},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems ; Carbon/*metabolism ; Gene Editing ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Lycopersicon esculentum/genetics/*growth &amp; development/metabolism ; Nitrogen/*metabolism ; Phosphoric Monoester Hydrolases/*genetics ; Photosynthesis ; Plant Proteins/genetics/metabolism ; Ribulosephosphates/metabolism ; },
abstract = {Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme in the Calvin[-]Benson cycle and has been documented to be important in carbon assimilation, growth and stress tolerance in plants. However, information on the impact of SBPase on carbon assimilation and nitrogen metabolism in tomato plants (Solanum lycopersicum) is rather limited. In the present study, we investigated the role of SBPase in carbon assimilation and nitrogen metabolism in tomato plants by knocking out SBPase gene SlSBPASE using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology. Compared with wild-type plants, slsbpase mutant plants displayed severe growth retardation. Further analyses showed that knockout of SlSBPASE led to a substantial reduction in SBPase activity and as a consequence, ribulose-1,5-bisphosphate (RuBP) regeneration and carbon assimilation rate were dramatically inhibited in slsbpase mutant plants. It was further observed that much lower levels of sucrose and starch were accumulated in slsbpase mutant plants than their wild-type counterparts during the photoperiod. Intriguingly, mutation in SlSBPASE altered nitrogen metabolism as demonstrated by changes in levels of protein and amino acids and activities of nitrogen metabolic enzymes. Collectively, our data suggest that SlSBPASE is required for optimal growth, carbon assimilation and nitrogen metabolism in tomato plants.},
}
@article {pmid30556811,
year = {2018},
author = {Lu, A and Wawro, P and Morgens, DW and Portela, F and Bassik, MC and Pfeffer, SR},
title = {Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30556811},
issn = {2050-084X},
support = {R56 DK037332/DK/NIDDK NIH HHS/United States ; DP2 HD084069/HD/NICHD NIH HHS/United States ; R37 DK037332/DK/NIDDK NIH HHS/United States ; DP2HD084069/NH/NIH HHS/United States ; T32 HG000044/NH/NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; NIDDK 37332/NH/NIH HHS/United States ; R01 DK037332/DK/NIDDK NIH HHS/United States ; },
mesh = {A549 Cells ; Base Sequence ; CRISPR-Cas Systems ; Endosomes/metabolism ; Exocytosis ; Exosomes/genetics ; Extracellular Vesicles/*genetics ; Gene Regulatory Networks ; *Genome, Human ; HEK293 Cells ; HeLa Cells ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; MicroRNAs/*genetics ; Multivesicular Bodies/metabolism ; },
abstract = {Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.},
}
@article {pmid30555184,
year = {2018},
author = {Palermo, G and Chen, JS and Ricci, CG and Rivalta, I and Jinek, M and Batista, VS and Doudna, JA and McCammon, JA},
title = {Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.},
journal = {Quarterly reviews of biophysics},
volume = {51},
number = {},
pages = {},
pmid = {30555184},
issn = {1469-8994},
support = {R01 GM031749/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry ; *CRISPR-Cas Systems ; Catalytic Domain ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; *Gene Editing ; Humans ; *Molecular Dynamics Simulation ; Principal Component Analysis ; },
abstract = {Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1-3 regions 'sense' nucleic acids, 'regulate' the HNH conformational transition, and ultimately 'lock' the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1-3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.},
}
@article {pmid30555080,
year = {2018},
author = {Turner, AN and Andersen, RS and Bookout, IE and Brashear, LN and Davis, JC and Gahan, DM and Davis, JC and Gotham, JP and Hijaz, BA and Kaushik, AS and Mcgill, JB and Miller, VL and Moseley, ZP and Nowell, CL and Patel, RK and Rodgers, MC and Patel, RK and Shihab, YA and Walker, AP and Glover, SR and Foster, SD and Challa, AK},
title = {Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene generated using CRISPR-Cas9 RNPs.},
journal = {Journal of genetics},
volume = {97},
number = {5},
pages = {1315-1325},
pmid = {30555080},
issn = {0973-7731},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites/genetics ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/embryology/metabolism ; Holoprosencephaly/genetics ; Intracellular Signaling Peptides and Proteins/chemistry/*genetics ; Models, Molecular ; *Mutation ; Phenotype ; Protein Domains ; Ribonucleoproteins/*genetics/metabolism ; Zebrafish/embryology/genetics/metabolism ; Zebrafish Proteins/chemistry/*genetics ; },
abstract = {Nodal-related protein (ndr2) is amember of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.},
}
@article {pmid30555042,
year = {2019},
author = {Li, B and Cao, Y and Meng, G and Qian, L and Xu, T and Yan, C and Luo, O and Wang, S and Wei, J and Ding, Y and Yu, D},
title = {Targeting glutaminase 1 attenuates stemness properties in hepatocellular carcinoma by increasing reactive oxygen species and suppressing Wnt/beta-catenin pathway.},
journal = {EBioMedicine},
volume = {39},
number = {},
pages = {239-254},
pmid = {30555042},
issn = {2352-3964},
mesh = {Adult ; Aged ; Animals ; Big Data ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/drug therapy/genetics/metabolism/*pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Glutaminase/antagonists &amp; inhibitors/*genetics/*metabolism ; Hep G2 Cells ; Humans ; Liver Neoplasms/drug therapy/genetics/metabolism/*pathology ; Male ; Mice ; Middle Aged ; Neoplasm Transplantation ; Neoplastic Stem Cells/drug effects/*metabolism ; Prognosis ; Reactive Oxygen Species/*metabolism ; Up-Regulation ; Wnt Signaling Pathway ; },
abstract = {BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignant disease with poor prognosis. Recent advances suggest the existence of cancer stem cells (CSCs) within liver cancer, which are considered to be responsible for tumor relapse, metastasis, and chemoresistance. However, novel therapeutic approaches for eradicating CSCs are yet to be established. Here, we aimed to identify the role of glutaminase 1 (GLS1) in stemness, and the feasibility that GLS1 serves as a therapeutic target for elimination CSCs as well as the possible mechanism.<br><br>METHODS: Publicly-available data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was mined to unearth the association between GLS1 and stemness phenotype. Using big data, human tissues and multiple cell lines, we gained a general picture of GLS1 expression in HCC progression. We generated stable cell lines by lentiviral-mediated overexpression or CRISPR/Cas9-based knockout. Sphere formation assays and colony formation assays were employed to analyze the relationship between GLS1 and stemness. A series of bioinformatics analyses and molecular experiments including qRT-PCR, immunoblotting, flow cytometry, and immunofluorescence were employed to investigate the role of GLS1 in regulating stemness in vitro and in vivo.<br><br>FINDINGS: We observed GLS1 (both KGA and GAC isoform) is highly expressed in HCC, and that high expression of GAC predicts a poor prognosis. GLS1 is exclusively expressed in the mitochondrial matrix. Upregulation of GLS1 is positively associated with advanced clinicopathological features and stemness phenotype. Targeting GLS1 reduced the expression of stemness-related genes and suppressed CSC properties in vitro. We further found GLS1 regulates stemness properties via ROS/Wnt/&#x3b2;-catenin signaling and that GLS1 knockout inhibits tumorigenicity in vivo.<br><br>INTERPRETATION: Targeting GLS1 attenuates stemness properties in HCC by increasing ROS accumulation and suppressing Wnt/&#x3b2;-catenin pathway, which implied that GLS1 could serve as a therapeutic target for elimination of CSCs.},
}
@article {pmid30554945,
year = {2019},
author = {Chakrabarti, AM and Henser-Brownhill, T and Monserrat, J and Poetsch, AR and Luscombe, NM and Scaffidi, P},
title = {Target-Specific Precision of CRISPR-Mediated Genome Editing.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {699-713.e6},
pmid = {30554945},
issn = {1097-4164},
support = {FC001152/WT_/Wellcome Trust/United Kingdom ; FC001110/CRUK_/Cancer Research UK/United Kingdom ; FC001110/WT_/Wellcome Trust/United Kingdom ; FC001152/CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Proliferation ; Chromatin/genetics/metabolism ; Chromatin Assembly and Disassembly ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics/metabolism ; *Gene Deletion ; Gene Editing/*methods ; HEK293 Cells ; Hep G2 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; *Mutagenesis, Insertional ; Nucleotide Motifs ; RNA, Guide/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.},
}
@article {pmid30552382,
year = {2020},
author = {Zeng, X and Liu, G and Peng, W and He, J and Cai, C and Xiong, W and Chen, S and Yang, M and Dong, Z},
title = {Combined deficiency of SLAMF8 and SLAMF9 prevents endotoxin-induced liver inflammation by downregulating TLR4 expression on macrophages.},
journal = {Cellular &amp; molecular immunology},
volume = {17},
number = {2},
pages = {153-162},
pmid = {30552382},
issn = {2042-0226},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cytokines/metabolism ; Down-Regulation/*genetics ; Hepatitis/*etiology/*immunology/metabolism/mortality ; Lipopolysaccharides/*adverse effects ; MAP Kinase Signaling System/*genetics ; Macrophage Activation/genetics ; Macrophages/*immunology/metabolism ; Membrane Proteins/*deficiency/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; RAW 264.7 Cells ; Signaling Lymphocytic Activation Molecule Family/*deficiency/genetics ; Survival Rate ; Toll-Like Receptor 4/*metabolism ; Transfection ; },
abstract = {Classical signaling lymphocyte activating molecule (SLAM) family receptors are abundant within many types of immune cells, whereas the nonclassical SLAM family receptors SLAMF8 and SLAMF9, which uniquely lack cytoplasmic signaling motifs, are highly expressed by myeloid cells. Due to the potential redundancy, whether these two receptors regulate macrophage function remains largely unknown. Here, we show that SLAMF8 and SLAMF9 co-regulate macrophage-mediated liver inflammation. To overcome the redundancy, we generated mice that simultaneously lacked SLAMF8 and SLAMF9 using CRISPR-Cas9 technology. Although macrophage differentiation was not altered by the combined deficiency of SLAMF8 and SLAMF9, the loss of these two receptors significantly protected against lipopolysaccharide (LPS)-induced liver injury. SLAMF8 and SLAMF9 double-deficient mice had a prolonged survival rate and less infiltration of inflammatory cells. The depletion of macrophages using clodronate liposomes abolished the effects of SLAMF8 and SLAMF9 deficiencies on LPS-induced liver injury, which demonstrates that these receptors are required for macrophage activation following LPS challenge. Moreover, the deficiency of SLAMF8 and SLAMF9 suppressed the secretion of inflammatory cytokines by downregulating the expression of Toll-like receptor-4 (TLR4), a receptor that specifically binds LPS, which led to decreased mitogen-activated protein kinases (MAPK) signaling activation. Notably, combined injections of truncated extracellular SLAMF8 and SLAMF9 proteins significantly alleviated LPS-induced liver injury. Thus, our findings provide insights into the role of SLAMF8 and SLAMF9 in endotoxin-induced liver injury and suggest that SLAMF8 and SLAMF9 are potential therapeutic targets for acute hepatic injury.},
}
@article {pmid30551175,
year = {2018},
author = {Møller, HD and Lin, L and Xiang, X and Petersen, TS and Huang, J and Yang, L and Kjeldsen, E and Jensen, UB and Zhang, X and Liu, X and Xu, X and Wang, J and Yang, H and Church, GM and Bolund, L and Regenberg, B and Luo, Y},
title = {CRISPR-C: circularization of genes and chromosome by CRISPR in human cells.},
journal = {Nucleic acids research},
volume = {46},
number = {22},
pages = {e131},
pmid = {30551175},
issn = {1362-4962},
mesh = {Base Sequence ; Biosensing Techniques ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Chromosomes, Human, Pair 18/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; DNA, Circular/*genetics/metabolism ; Fibroblasts ; Fluorescent Dyes/chemistry/metabolism ; Gene Editing/*methods ; Genes, Reporter ; Genetic Loci ; Genetic Vectors/chemistry/metabolism ; Genome, Human ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Luminescent Proteins/genetics/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.},
}
@article {pmid30549429,
year = {2019},
author = {Xu, J and Chen, RM and Chen, SQ and Chen, K and Tang, LM and Yang, DH and Yang, X and Zhang, Y and Song, HS and Huang, YP},
title = {Identification of a germline-expression promoter for genome editing in Bombyx mori.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {991-999},
doi = {10.1111/1744-7917.12657},
pmid = {30549429},
issn = {1744-7917},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*genetics ; CRISPR-Cas Systems ; Cell Line ; Embryo, Nonmammalian ; *Gene Editing ; Genes, Reporter ; Green Fluorescent Proteins ; *Promoter Regions, Genetic ; Transformation, Genetic ; },
abstract = {Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmβ4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.},
}
@article {pmid30549238,
year = {2019},
author = {Wang, J and Meng, X and Hu, X and Sun, T and Li, J and Wang, K and Yu, H},
title = {xCas9 expands the scope of genome editing with reduced efficiency in rice.},
journal = {Plant biotechnology journal},
volume = {17},
number = {4},
pages = {709-711},
pmid = {30549238},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant/*genetics ; Oryza/*genetics ; },
}
@article {pmid30549232,
year = {2019},
author = {Schwefel, K and Spiegler, S and Ameling, S and Much, CD and Pilz, RA and Otto, O and Völker, U and Felbor, U and Rath, M},
title = {Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening.},
journal = {Journal of cellular and molecular medicine},
volume = {23},
number = {3},
pages = {1771-1783},
pmid = {30549232},
issn = {1582-4934},
mesh = {Alleles ; Apoptosis ; Apoptosis Regulatory Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; CRISPR-Cas Systems ; *Clonal Evolution ; Endothelium, Vascular/metabolism/*pathology ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing ; Human Umbilical Vein Endothelial Cells ; Humans ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; MicroRNAs/genetics ; *Mutation ; Neovascularization, Pathologic/*etiology/metabolism/pathology ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {CCM3, originally described as PDCD10, regulates blood-brain barrier integrity and vascular maturation in vivo. CCM3 loss-of-function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral- and plasmid-free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss-of-function CCM3 variants into human ECs and studied the molecular and functional effects of long-term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real-time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3-deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long-term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3-deficient dysfunctional ECs contributes to CCM formation.},
}
@article {pmid30548045,
year = {2019},
author = {Tong, Y and Weber, T and Lee, SY},
title = {CRISPR/Cas-based genome engineering in natural product discovery.},
journal = {Natural product reports},
volume = {36},
number = {9},
pages = {1262-1280},
doi = {10.1039/c8np00089a},
pmid = {30548045},
issn = {1460-4752},
mesh = {Biological Products/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Bacterial/genetics ; Genome, Fungal/genetics ; Metabolic Engineering/methods ; },
abstract = {Covering: up to February, 2018 This review briefly introduces and summarizes current knowledge about the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) - CRISPR/Cas system and how it was engineered to become one of the most important and versatile genome editing techniques that are currently revolutionizing the whole field of molecular biology. It aims to highlight and discuss the applications and remaining challenges of CRISPR/Cas (mainly focusing on CRISPR/SpCas9)-based genome editing in natural product discovery. The organisms covered include bacteria such as Streptomyces, Corynebacteria, and Myxobacteria; filamentous fungi such as Aspergillus, Beauveria, and Ganoderma; microalgae; and some plants. As closing remarks, the prospects of using CRISPR/Cas in natural product discovery will be discussed.},
}
@article {pmid30547885,
year = {2018},
author = {Bezares-Calderón, LA and Berger, J and Jasek, S and Verasztó, C and Mendes, S and Gühmann, M and Almeda, R and Shahidi, R and Jékely, G},
title = {Neural circuitry of a polycystin-mediated hydrodynamic startle response for predator avoidance.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30547885},
issn = {2050-084X},
mesh = {Animals ; Annelida/*genetics/physiology ; Behavior, Animal/*physiology ; CRISPR-Cas Systems ; Cilia/genetics/physiology ; Larva/genetics/physiology ; Locomotion/genetics/physiology ; Muscles/physiology ; Mutation ; Neurons/*physiology ; TRPP Cation Channels/*genetics ; },
abstract = {Startle responses triggered by aversive stimuli including predators are widespread across animals. These coordinated whole-body actions require the rapid and simultaneous activation of a large number of muscles. Here we study a startle response in a planktonic larva to understand the whole-body circuit implementation of the behaviour. Upon encountering water vibrations, larvae of the annelid Platynereis close their locomotor cilia and simultaneously raise the parapodia. The response is mediated by collar receptor neurons expressing the polycystins PKD1-1 and PKD2-1. CRISPR-generated PKD1-1 and PKD2-1 mutant larvae do not startle and fall prey to a copepod predator at a higher rate. Reconstruction of the whole-body connectome of the collar-receptor-cell circuitry revealed converging feedforward circuits to the ciliary bands and muscles. The wiring diagram suggests circuit mechanisms for the intersegmental and left-right coordination of the response. Our results reveal how polycystin-mediated mechanosensation can trigger a coordinated whole-body effector response involved in predator avoidance.},
}
@article {pmid30547274,
year = {2019},
author = {Sun, D and Wang, L and Mao, X and Fei, M and Chen, Y and Shen, M and Qiu, J},
title = {Chemical transformation mediated CRISPR/Cas9 genome editing in Escherichia coli.},
journal = {Biotechnology letters},
volume = {41},
number = {2},
pages = {293-303},
doi = {10.1007/s10529-018-02639-1},
pmid = {30547274},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems ; Chemical Phenomena ; Electroporation ; Escherichia coli/*genetics ; Gene Editing/*economics/*methods ; Genome, Bacterial ; Transformation, Bacterial ; },
abstract = {OBJECTIVES: To develop a convenient chemical transformation mediated CRISPR/Cas9 (CT-CRISPR/Cas9) system for genome editing in Escherichia coli.<br><br>RESULTS: Here, we have constructed a CT-CRISPR/Cas9 system, which can precisely edit bacterial genome (replacing, deleting, inserting or point mutating a target gene) through chemical transformation. Compared with the traditional electroporation mediated CRISPR/Cas9 (ET-CRISPR/Cas9) system, genome editing with the CT-CRISPR/Cas9 system is much cheaper and simpler. In the CT-CRISPR/Cas9 system, we observed efficient genome editing on LB-agar plates. The CT-CRISPR/Cas9 system has successfully modified the target gene with the editing template flanked by short homologous DNA fragments (~&#x2009;50&#xa0;bp) which were designed in primers. We used the lab-made CaCl2 solution to perform the CT-CRISPR/Cas9 experiment and successfully edited the genome of E. coli. Potential application of the CT-CRISPR/Cas9 system in high-throughput genome editing was evaluated in two E. coli strains by using a multiwell plate.<br><br>CONCLUSIONS: Our work provides a simple and cheap genome-editing method, that is expected to be widely applied as a routine genetic engineering method.},
}
@article {pmid30546095,
year = {2019},
author = {Fineran, PC},
title = {CRISPR-Cas impedes archaeal mating.},
journal = {Nature microbiology},
volume = {4},
number = {1},
pages = {2-3},
doi = {10.1038/s41564-018-0326-0},
pmid = {30546095},
issn = {2058-5276},
mesh = {Archaea/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Archaeal ; },
}
@article {pmid30546061,
year = {2018},
author = {Noguchi, T and Suzuki, M and Mutoh, N and Hirata, Y and Tsuchida, M and Miyagawa, S and Hwang, GW and Aoki, J and Matsuzawa, A},
title = {Nuclear-accumulated SQSTM1/p62-based ALIS act as microdomains sensing cellular stresses and triggering oxidative stress-induced parthanatos.},
journal = {Cell death &amp; disease},
volume = {9},
number = {12},
pages = {1193},
pmid = {30546061},
issn = {2041-4889},
mesh = {Apoptosis/drug effects/*genetics ; Autophagy/drug effects ; CRISPR-Cas Systems ; Cell Death/drug effects/*genetics ; Cell Line, Tumor ; Cephalosporins/pharmacology ; Gene Knockout Techniques ; Humans ; Macrophages/drug effects ; Oxidative Stress/*genetics ; Sequestosome-1 Protein/antagonists &amp; inhibitors/*genetics ; Ubiquitination/genetics ; },
abstract = {Aggresome-like induced structures (ALIS) have been described as ubiquitinated protein-containing aggresomes transiently formed in response to various stresses. In this study, we provide evidence that ALIS composed of SQSTM1/p62 act as a key determinant of oxidative stress-induced parthanatos, which is newly discovered and distinct from regular programmed cell death. Interestingly, we first found that chemical stresses induced by particular chemical drugs, such as several cephalosporin antibiotics, cause oxidative stress-mediated parthanatos, accompanied by the ALIS formation. Blocking the ALIS formation potently suppressed the parthanatos, and p62 knockout cells exhibited the attenuated ALIS formation and high resistance to parthanatos. Moreover, we also found that the redox-sensing activity of p62 is required for nuclear accumulation of the p62-based ALIS, resulting in the induction of parthanatos. Together, our results demonstrate unexpected functions of p62 and ALIS as cell death mediators sensing oxidative stress, and thus uncover a novel mechanism whereby p62 mediates parthanatos.},
}
@article {pmid30546007,
year = {2018},
author = {Jiang, S and Wen, N and Li, Z and Dube, U and Del Aguila, J and Budde, J and Martinez, R and Hsu, S and Fernandez, MV and Cairns, NJ and , and , and Harari, O and Cruchaga, C and Karch, CM},
title = {Integrative system biology analyses of CRISPR-edited iPSC-derived neurons and human brains reveal deficiencies of presynaptic signaling in FTLD and PSP.},
journal = {Translational psychiatry},
volume = {8},
number = {1},
pages = {265},
pmid = {30546007},
issn = {2158-3188},
support = {U01 AG052411/AG/NIA NIH HHS/United States ; R01 AG057777/AG/NIA NIH HHS/United States ; RF1 AG058501/AG/NIA NIH HHS/United States ; U01 AG058922/AG/NIA NIH HHS/United States ; P01 AG003991/AG/NIA NIH HHS/United States ; K01 AG046374/AG/NIA NIH HHS/United States ; R01 AG044546/AG/NIA NIH HHS/United States ; P50 AG005681/AG/NIA NIH HHS/United States ; UF1 AG032438/AG/NIA NIH HHS/United States ; R01 AG056293/AG/NIA NIH HHS/United States ; RF1 AG053303/AG/NIA NIH HHS/United States ; RF1 AG044546/AG/NIA NIH HHS/United States ; P01 AG026276/AG/NIA NIH HHS/United States ; },
mesh = {Aged ; Aged, 80 and over ; Animals ; Brain/*metabolism ; CRISPR-Cas Systems ; Female ; Frontotemporal Lobar Degeneration/genetics/*metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Mice, Inbred C57BL ; Neurons/*metabolism ; Receptors, GABA/metabolism ; Signal Transduction ; Supranuclear Palsy, Progressive/genetics/*metabolism ; Transcriptome ; tau Proteins/genetics/*metabolism ; },
abstract = {Mutations in the microtubule-associated protein tau (MAPT) gene cause autosomal dominant frontotemporal lobar degeneration with tau inclusions (FTLD-tau). MAPT p.R406W carriers present clinically with progressive memory loss and neuropathologically with neuronal and glial tauopathy. However, the pathogenic events triggered by the expression of the mutant tau protein remain poorly understood. To identify the genes and pathways that are dysregulated in FTLD-tau, we performed transcriptomic analyses in induced pluripotent stem cell (iPSC)-derived neurons carrying MAPT p.R406W and CRISPR/Cas9-corrected isogenic controls. We found that the expression of the MAPT p.R406W mutation was sufficient to create a significantly different transcriptomic profile compared with that of the isogeneic controls and to cause the differential expression of 328 genes. Sixty-one of these genes were also differentially expressed in the same direction between MAPT p.R406W carriers and pathology-free human control brains. We found that genes differentially expressed in the stem cell models and human brains were enriched for pathways involving gamma-aminobutyric acid (GABA) receptors and pre-synaptic function. The expression of GABA receptor genes, including GABRB2 and GABRG2, were consistently reduced in iPSC-derived neurons and brains from MAPT p.R406W carriers. Interestingly, we found that GABA receptor genes, including GABRB2 and GABRG2, are significantly lower in symptomatic mouse models of tauopathy, as well as in brains with progressive supranuclear palsy. Genome wide association analyses reveal that common variants within GABRB2 are associated with increased risk for frontotemporal dementia (P < 1 × 10[-3]). Thus, our systems biology approach, which leverages molecular data from stem cells, animal models, and human brain tissue can reveal novel disease mechanisms. Here, we demonstrate that MAPT p.R406W is sufficient to induce changes in GABA-mediated signaling and synaptic function, which may contribute to the pathogenesis of FTLD-tau and other primary tauopathies.},
}
@article {pmid30545937,
year = {2019},
author = {Sharpe, LJ and Howe, V and Scott, NA and Luu, W and Phan, L and Berk, JM and Hochstrasser, M and Brown, AJ},
title = {Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {7},
pages = {2436-2448},
pmid = {30545937},
issn = {1083-351X},
support = {F32 GM113456/GM/NIGMS NIH HHS/United States ; R01 GM046904/GM/NIGMS NIH HHS/United States ; R37 GM046904/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cholesterol/genetics/*metabolism ; Enzyme Activation/genetics ; HEK293 Cells ; Humans ; Membrane Proteins/genetics/*metabolism ; Proteasome Endopeptidase Complex/genetics/metabolism ; Protein Domains ; *Proteolysis ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Valosin Containing Protein/genetics/metabolism ; },
abstract = {The E3 ligase membrane-associated ring-CH-type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum. It controls levels of several known protein substrates, including a key enzyme in cholesterol synthesis, squalene monooxygenase. However, beyond its own autodegradation, little is known about how MARCH6 itself is regulated. Using CRISPR/Cas9 gene-editing, MARCH6 overexpression, and immunoblotting, we found here that cholesterol stabilizes MARCH6 protein endogenously and in HEK293 cells that stably express MARCH6. Conversely, MARCH6-deficient HEK293 and HeLa cells lost their ability to degrade squalene monooxygenase in a cholesterol-dependent manner. The ability of cholesterol to boost MARCH6 did not seem to involve a putative sterol-sensing domain in this E3 ligase, but was abolished when either membrane extraction by valosin-containing protein (VCP/p97) or proteasomal degradation was inhibited. Furthermore, cholesterol-mediated stabilization was absent in two MARCH6 mutants that are unable to degrade themselves, indicating that cholesterol stabilizes MARCH6 protein by preventing its autodegradation. Experiments with chemical chaperones suggested that this likely occurs through a conformational change in MARCH6 upon cholesterol addition. Moreover, cholesterol reduced the levels of at least three known MARCH6 substrates, indicating that cholesterol-mediated MARCH6 stabilization increases its activity. Our findings highlight an important new role for cholesterol in controlling levels of proteins, extending the known repertoire of cholesterol homeostasis players.},
}
@article {pmid30545858,
year = {2018},
author = {Dzau, VJ and McNutt, M and Bai, C},
title = {Wake-up call from Hong Kong.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6420},
pages = {1215},
doi = {10.1126/science.aaw3127},
pmid = {30545858},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics/*standards ; *Genome, Human ; Hong Kong ; Humans ; Stakeholder Participation ; },
}
@article {pmid30544778,
year = {2018},
author = {Mayo-Muñoz, D and He, F and Jørgensen, JB and Madsen, PK and Bhoobalan-Chitty, Y and Peng, X},
title = {Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2.},
journal = {Viruses},
volume = {10},
number = {12},
pages = {},
pmid = {30544778},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Viral/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome, Viral ; Mutation ; Polymerase Chain Reaction ; Rudiviridae/*genetics ; Sulfolobus/*virology ; Viral Proteins/genetics/metabolism ; },
abstract = {Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (Δarrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses.},
}
@article {pmid30544514,
year = {2018},
author = {Feng, Z and Zhang, Z and Hua, K and Gao, X and Mao, Y and Botella, JR and Zhu, JK},
title = {A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis.},
journal = {International journal of molecular sciences},
volume = {19},
number = {12},
pages = {},
pmid = {30544514},
issn = {1422-0067},
mesh = {Arabidopsis/genetics/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics/physiology ; Cell Division/genetics/physiology ; Gene Editing ; Genome, Plant/genetics ; Mutation/genetics ; Plants, Genetically Modified/genetics/*metabolism ; Promoter Regions, Genetic/genetics ; },
abstract = {The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4[-]10%) and T2 (32.5[-]46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications.},
}
@article {pmid30543773,
year = {2018},
author = {Billker, O},
title = {CRISPRing the Elephant in the Room.},
journal = {Cell host &amp; microbe},
volume = {24},
number = {6},
pages = {754-755},
doi = {10.1016/j.chom.2018.11.015},
pmid = {30543773},
issn = {1934-6069},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Plasmodium/*genetics ; Toxoplasma/*genetics ; },
abstract = {The importance of guanylyl-cyclases (GCs) in apicomplexa has remained elusive due to the large size of the genes. Two recent studies, including Brown and Sibley, 2018 in this issue of Cell Host &amp; Microbe, make elegant use of genome editing with CRISPR-Cas9 to characterize roles of GCs in Toxoplasma and Plasmodium.},
}
@article {pmid30543180,
year = {2018},
author = {Menzies, SA and Volkmar, N and van den Boomen, DJ and Timms, RT and Dickson, AS and Nathan, JA and Lehner, PJ},
title = {The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30543180},
issn = {2050-084X},
support = {/WT_/Wellcome Trust/United Kingdom ; 102770/Z/13/Z/WT_/Wellcome Trust/United Kingdom ; 210688/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; 100140/WT_/Wellcome Trust/United Kingdom ; PhD studentship/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cholesterol/metabolism ; Humans ; Hydroxymethylglutaryl CoA Reductases/*genetics ; Membrane Proteins/*genetics/metabolism ; Mice ; Proteolysis ; Receptors, Autocrine Motility Factor/*genetics ; Ubiquitin-Conjugating Enzymes/genetics ; Ubiquitin-Protein Ligases/*genetics ; Ubiquitination ; },
abstract = {UNLABELLED: Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.<br><br>EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).},
}
@article {pmid30543064,
year = {2019},
author = {Li, C and Zhang, B},
title = {Genome Editing in Cotton Using CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1902},
number = {},
pages = {95-104},
doi = {10.1007/978-1-4939-8952-2_8},
pmid = {30543064},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting ; Gossypium/*genetics ; Mutagenesis ; Plants, Genetically Modified ; RNA, Guide/genetics ; Reproducibility of Results ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as effective strategy for generating site-specific mutations. Recently, CRISPR/Cas9-mediated genome editing system have been rapidly optimized and applied in crop genetic improvement. In this chapter, we present a simple and high-efficiency method for crating targeted gene mutation in allotetraploid cotton genome using CRISPR/Cas9 system. This chapter will employ GhMYB25-like A and GhMYB25-like D that derived from upland cotton (Gossypium hirsutum) A subgenome and the D subgenome, respectively, as an example to introduce the procedure of how to generate effective mutations in cotton genome using CRISPR/Cas9-based biotechnology. Based on our previous results, this CRISPR/Cas9 system can induce a proportion of 14.2-21.4% fragment truncation events in GhMYB25-like A and GhMYB25-like D genome sites. In addition, PCR product sequencing results suggest that the mutation frequencies that occurred in GhMYB25-like A and GhMYB25-like D DNA sites are 100% and 98.8%, respectively. More important, the off-target-caused mutation events have not been detected in our transgenic plants, even one of the putative off-target site only have one nucleotide mismatch with the designed GhMYB25 sgRNA. Thus, this CRISPR/Cas9 method might be an effective approach for targeted mutagenesis in cotton genome.},
}
@article {pmid30542997,
year = {2019},
author = {Slipek, NJ and Varshney, J and Largaespada, DA},
title = {CRISPR/Cas9-Based Positive Screens for Cancer-Related Traits.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1907},
number = {},
pages = {137-144},
doi = {10.1007/978-1-4939-8967-6_11},
pmid = {30542997},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Genome, Human ; Genomics/*methods ; Humans ; Neoplasm Proteins/antagonists &amp; inhibitors/*genetics ; Neoplasms/*genetics/pathology ; *Quantitative Trait Loci ; },
abstract = {Since the advent of large-scale, detailed descriptive cancer genomics studies at the beginning of the century, such as The Cancer Genome Atlas (TCGA), labs around the world have been working to make this data useful. Data like these can be made more useful by comparison with complementary functional genomic data. One new example is the application of CRISPR/Cas9-based library screening for cancer-related traits in cell lines. Such screens can reveal genome-wide suppressors of tumorigenesis and metastasis. Here we describe the use of widely available lentiviral libraries for such screens in cultured cell lines.},
}
@article {pmid30542996,
year = {2019},
author = {Adelmann, CH and Wang, T and Sabatini, DM and Lander, ES},
title = {Genome-Wide CRISPR/Cas9 Screening for Identification of Cancer Genes in Cell Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1907},
number = {},
pages = {125-136},
doi = {10.1007/978-1-4939-8967-6_10},
pmid = {30542996},
issn = {1940-6029},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques ; *Genome, Human ; Genome-Wide Association Study/*methods ; Genomics/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Neoplasm Proteins/antagonists &amp; inhibitors/*genetics ; Neoplasms/diagnosis/*genetics ; Phenotype ; Tumor Cells, Cultured ; },
abstract = {In this protocol, pooled sgRNA libraries targeting thousands of genes are computationally designed, generated using microarray-based synthesis techniques, and packaged into lentiviral particles. Target cells of interest are transduced with the lentiviral sgRNA pools to generate a collection of knockout mutants-via Cas9-mediated genomic cleavage-and screened for a phenotype of interest. The relative abundance of each mutant in the population can be monitored over time through high-throughput sequencing of the integrated sgRNA expression cassettes. Using this technique, we outline strategies for the identification of cancer driver genes and genes mediating drug response.},
}
@article {pmid30542190,
year = {2018},
author = {Mallapaty, S},
title = {Engineering a biomedical revolution.},
journal = {Nature},
volume = {564},
number = {7735},
pages = {S66-S68},
doi = {10.1038/d41586-018-07692-4},
pmid = {30542190},
issn = {1476-4687},
mesh = {Animals ; Animals, Genetically Modified ; Animals, Laboratory/genetics ; Authorship ; Bibliometrics ; Biomedical Engineering/statistics &amp; numerical data ; Biomedical Research/economics/ethics/legislation &amp; jurisprudence/*statistics &amp; numerical data ; CRISPR-Cas Systems/genetics ; Child ; China ; Disease Models, Animal ; Embryo Research/ethics ; Embryonic Stem Cells/transplantation ; Foreign Professional Personnel/supply &amp; distribution ; Gene Editing/ethics ; Haplorhini/genetics ; Humans ; Male ; Rabbits ; },
}
@article {pmid30523370,
year = {2019},
author = {Hu, W and Zi, Z and Jin, Y and Li, G and Shao, K and Cai, Q and Ma, X and Wei, F},
title = {CRISPR/Cas9-mediated PD-1 disruption enhances human mesothelin-targeted CAR T cell effector functions.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {68},
number = {3},
pages = {365-377},
doi = {10.1007/s00262-018-2281-2},
pmid = {30523370},
issn = {1432-0851},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; GPI-Linked Proteins/*genetics ; Humans ; Immunotherapy, Adoptive ; Lymphocyte Activation ; Mesothelin ; Mice ; Neoplasms, Experimental/therapy ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/*genetics ; Receptors, Antigen, T-Cell/*genetics/immunology ; },
abstract = {The interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligands on a target tumour may limit the capacity of chimeric antigen receptor (CAR) T cells to eradicate solid tumours. PD-1 blockade could potentially enhance CAR T cell function. Here, we show that mesothelin is overexpressed in human triple-negative breast cancer cells and can be targeted by CAR T cells. To overcome the suppressive effect of PD-1 on CAR T cells, we utilized CRISPR/Cas9 ribonucleoprotein-mediated editing to disrupt the programmed cell death-1 (PD-1) gene locus in human primary T cells, resulting in a significantly reduced PD-1[hi] population. This reduction had little effect on CAR T cell proliferation but strongly augmented CAR T cell cytokine production and cytotoxicity towards PD-L1-expressing cancer cells in vitro. CAR T cells with PD-1 disruption show enhanced tumour control and relapse prevention in vivo when compared with CAR T cells with or without αPD-1 antibody blockade. Our study demonstrates a potential advantage of integrated immune checkpoint blockade with CAR T cells in controlling solid tumours and provides an alternative CAR T cell strategy for adoptive transfer therapy.},
}
@article {pmid30523326,
year = {2018},
author = {Chakradhar, S},
title = {Treatments that made headlines in 2018.},
journal = {Nature medicine},
volume = {24},
number = {12},
pages = {1785-1787},
doi = {10.1038/s41591-018-0292-3},
pmid = {30523326},
issn = {1546-170X},
mesh = {Alzheimer Disease/genetics/therapy ; Amyloidosis/genetics/therapy ; CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Humans ; beta-Thalassemia/genetics/therapy ; },
}
@article {pmid30523088,
year = {2018},
author = {Normile, D},
title = {For China, a CRISPR first goes too far.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6419},
pages = {1091},
doi = {10.1126/science.362.6419.1091},
pmid = {30523088},
issn = {1095-9203},
mesh = {*Bioethical Issues ; *CRISPR-Cas Systems ; China ; Disease Resistance/*genetics ; Gene Editing/*ethics ; HIV Infections/genetics ; Humans ; *Public Policy ; },
}
@article {pmid30523087,
year = {2018},
author = {Cohen, J},
title = {What now for human genome editing?.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6419},
pages = {1090-1092},
doi = {10.1126/science.362.6419.1090},
pmid = {30523087},
issn = {1095-9203},
mesh = {*Bioethical Issues ; *CRISPR-Cas Systems ; China ; Disease Resistance ; Gene Editing/*ethics ; *Genome, Human ; HIV Infections/genetics ; Humans ; *Social Control, Formal ; },
}
@article {pmid30523077,
year = {2019},
author = {Yan, WX and Hunnewell, P and Alfonse, LE and Carte, JM and Keston-Smith, E and Sothiselvam, S and Garrity, AJ and Chong, S and Makarova, KS and Koonin, EV and Cheng, DR and Scott, DA},
title = {Functionally diverse type V CRISPR-Cas systems.},
journal = {Science (New York, N.Y.)},
volume = {363},
number = {6422},
pages = {88-91},
doi = {10.1126/science.aav7271},
pmid = {30523077},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; DNA/*chemistry ; Databases, Protein ; Deoxyribonucleases/chemistry ; Escherichia coli ; Gene Library ; Nucleic Acid Conformation ; RNA, Guide/*chemistry ; Ribonucleases/*chemistry ; },
abstract = {Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.},
}
@article {pmid30540754,
year = {2018},
author = {Ramanagoudr-Bhojappa, R and Carrington, B and Ramaswami, M and Bishop, K and Robbins, GM and Jones, M and Harper, U and Frederickson, SC and Kimble, DC and Sood, R and Chandrasekharappa, SC},
title = {Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.},
journal = {PLoS genetics},
volume = {14},
number = {12},
pages = {e1007821},
pmid = {30540754},
issn = {1553-7404},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Damage ; Fanconi Anemia/*genetics/physiopathology ; Female ; Fertility/genetics/physiology ; Frameshift Mutation ; Gene Knockout Techniques ; Humans ; Male ; Phenotype ; RNA Splicing/genetics ; Sex Determination Processes/genetics/physiology ; Sexual Development/genetics/physiology ; Zebrafish/*genetics/growth &amp; development/physiology ; Zebrafish Proteins/genetics/physiology ; },
abstract = {Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.},
}
@article {pmid30540740,
year = {2018},
author = {Kallimasioti-Pazi, EM and Thelakkad Chathoth, K and Taylor, GC and Meynert, A and Ballinger, T and Kelder, MJE and Lalevée, S and Sanli, I and Feil, R and Wood, AJ},
title = {Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair.},
journal = {PLoS biology},
volume = {16},
number = {12},
pages = {e2005595},
pmid = {30540740},
issn = {1545-7885},
support = {102560/Z/13/Z//Wellcome Trust/United Kingdom ; //Medical Research Council/United Kingdom ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics/physiology ; DNA Breaks, Double-Stranded ; DNA Repair/genetics/*physiology ; Endonucleases/metabolism ; Gene Editing/methods ; Genome ; Heterochromatin/*genetics/*physiology ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/physiology ; Mutagenesis, Insertional ; Mutagens ; Mutation/genetics ; Recombinational DNA Repair/physiology ; Sequence Deletion ; },
abstract = {Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.},
}
@article {pmid30540494,
year = {2019},
author = {Violante, S and Achetib, N and van Roermund, CWT and Hagen, J and Dodatko, T and Vaz, FM and Waterham, HR and Chen, H and Baes, M and Yu, C and Argmann, CA and Houten, SM},
title = {Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {3},
pages = {4355-4364},
pmid = {30540494},
issn = {1530-6860},
support = {R01 DK113172/DK/NIDDK NIH HHS/United States ; },
mesh = {ATP-Binding Cassette Transporters/deficiency/genetics/*physiology ; Animals ; CRISPR-Cas Systems ; Carnitine/analogs &amp; derivatives/metabolism ; Carnitine O-Palmitoyltransferase/antagonists &amp; inhibitors/deficiency/physiology ; Fatty Acids/*metabolism ; HEK293 Cells ; Humans ; Lauric Acids/metabolism ; Membrane Proteins/metabolism ; Mice ; Mice, Knockout ; Mitochondria/enzymology ; Oxidation-Reduction ; Palmitic Acid/metabolism ; Peroxisomal Bifunctional Enzyme/deficiency ; Peroxisomal Multifunctional Protein-2/deficiency/genetics/*physiology ; Peroxisomes/*enzymology ; Recombinant Proteins/metabolism ; },
abstract = {Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid β-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.},
}
@article {pmid30539697,
year = {2018},
author = {Zheng, R and Fang, X and He, L and Shao, Y and Guo, N and Wang, L and Liu, M and Li, D and Geng, H},
title = {Generation of a Primary Hyperoxaluria Type 1 Disease Model Via CRISPR/Cas9 System in Rats.},
journal = {Current molecular medicine},
volume = {18},
number = {7},
pages = {436-447},
doi = {10.2174/1566524019666181212092440},
pmid = {30539697},
issn = {1875-5666},
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Glyoxylates/metabolism ; *Hyperoxaluria, Primary/genetics/pathology/urine ; Mitochondria/genetics/metabolism ; *Nephrocalcinosis/genetics/pathology/urine ; Oxalates/urine ; Rats ; Rats, Transgenic ; Transaminases/*deficiency ; },
abstract = {BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an inherited disease caused by mutations in alanine-glyoxylate aminotransferase (AGXT). It is characterized by abnormal metabolism of glyoxylic acid in the liver leading to endogenous oxalate overproduction and deposition of oxalate in multiple organs, mainly the kidney. Patients of PH1 often suffer from recurrent urinary tract stones, and finally renal failure. There is no effective treatment other than combined liver-kidney transplantation.<br><br>METHODS: Microinjection was administered to PH1 rats. Urine samples were collected for urine analysis. Kidney tissues were for Western blotting, quantitative PCR, AGT assays and histological evaluation.<br><br>RESULTS: In this study, we generated a novel PH1 disease model through CRISPR/Cas9 mediated disruption of mitochondrial localized Agxt gene isoform in rats. Agxt-deficient rats excreted more oxalate in the urine than WT animals. Meanwhile, mutant rats exhibited crystalluria and showed a slight dilatation of renal tubules with mild fibrosis in the kidney. When supplied with 0.4% ethylene glycol (EG) in drinking water, mutant rats excreted greater abundance of oxalate and developed severe nephrocalcinosis in contrast to WT animals. Significantly elevated expression of inflammation- and fibrosisrelated genes was also detected in mutants.<br><br>CONCLUSION: These data suggest that Agxt-deficiency in mitochondria impairs glyoxylic acid metabolism and leads to PH1 in rats. This rat strain would not only be a useful model for the study of the pathogenesis and pathology of PH1 but also a valuable tool for the development and evaluation of innovative drugs and therapeutics.},
}
@article {pmid30539545,
year = {2019},
author = {Waśko, U and Zheng, Z and Bhatnagar, S},
title = {Visualization of Xist Long Noncoding RNA with a Fluorescent CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1870},
number = {},
pages = {41-50},
doi = {10.1007/978-1-4939-8808-2_3},
pmid = {30539545},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cloning, Molecular ; Female ; Fluorescent Antibody Technique ; Gene Editing ; Genes, Reporter ; Histones/metabolism ; Mice ; *Molecular Imaging/methods ; RNA, Guide ; RNA, Long Noncoding/*genetics/metabolism ; Transfection ; },
abstract = {X-inactive specific transcript (Xist) is a long noncoding RNA that is essential for initiating and maintaining epigenetic silencing of one copy of the X chromosome in mammalian females. But the mechanism by which Xist localizes and spreads on the X chromosome and facilitates transcriptional silencing remains largely unknown. This limited understanding, at least in part, is due to the technical difficulties in the visualization and functional characterization of Xist. Development of a successful method for Xist tracking is a key to better understanding of the X chromosome silencing, as well as to gain insight into the regulatory role of other long noncoding RNAs. Here, we describe an alternative method for visualization of Xist lncRNA in cells using a CRISPR/Cas9-based approach. This strategy is relatively simple approach to track Xist at different stages of cell differentiation, providing mechanistic insights into the initiation, maintenance, and establishment of X inactivation.},
}
@article {pmid30538335,
year = {2019},
author = {Hatzi, K and Geng, H and Doane, AS and Meydan, C and LaRiviere, R and Cardenas, M and Duy, C and Shen, H and Vidal, MNC and Baslan, T and Mohammad, HP and Kruger, RG and Shaknovich, R and Haberman, AM and Inghirami, G and Lowe, SW and Melnick, AM},
title = {Histone demethylase LSD1 is required for germinal center formation and BCL6-driven lymphomagenesis.},
journal = {Nature immunology},
volume = {20},
number = {1},
pages = {86-96},
pmid = {30538335},
issn = {1529-2916},
support = {K99 CA212276/CA/NCI NIH HHS/United States ; P01 CA013106/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R35 CA220499/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/*physiology ; CRISPR-Cas Systems ; Carcinogenesis ; DNA, Intergenic/genetics ; Germinal Center/immunology/*pathology ; Histone Demethylases/genetics/*metabolism ; Hyperplasia ; Immunological Synapses/genetics ; Introns/genetics ; Lymphoma/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Proto-Oncogene Proteins c-bcl-6/genetics/*metabolism ; },
abstract = {Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.},
}
@article {pmid30537986,
year = {2018},
author = {Chen, N and Zhao, G and Yan, X and Lv, Z and Yin, H and Zhang, S and Song, W and Li, X and Li, L and Du, Z and Jia, L and Zhou, L and Li, W and Hoffman, AR and Hu, JF and Cui, J},
title = {A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {218},
pmid = {30537986},
issn = {1474-760X},
support = {I01 BX002905/BX/BLRD VA/United States ; BX002905/VA/VA/United States ; },
mesh = {Breast Neoplasms/*metabolism ; CRISPR-Cas Systems ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism ; DNA Methylation ; *Gene Expression Regulation, Neoplastic ; Humans ; Mixed Function Oxygenases/metabolism ; Neoplasm Metastasis ; Proto-Oncogene Mas ; Proto-Oncogene Protein c-fli-1/*metabolism ; Proto-Oncogene Proteins/metabolism ; },
abstract = {BACKGROUND: Friend leukemia virus integration 1 (FLI1), an ETS transcription factor family member, acts as an oncogenic driver in hematological malignancies and promotes tumor growth in solid tumors. However, little is known about the mechanisms underlying the activation of this proto-oncogene in tumors.<br><br>RESULTS: Immunohistochemical staining showed that FLI1 is aberrantly overexpressed in advanced stage and metastatic breast cancers. Using a CRISPR Cas9-guided immunoprecipitation assay, we identify a circular RNA in the FLI1 promoter chromatin complex, consisting of FLI1 exons 4-2-3, referred to as FECR1.Overexpression of FECR1 enhances invasiveness of MDA-MB231 breast cancer cells. Notably, FECR1 utilizes a positive feedback mechanism to activate FLI1 by inducing DNA hypomethylation in CpG islands of the promoter. FECR1 binds to the FLI1 promoter in cis and recruits TET1, a demethylase that is actively involved in DNA demethylation. FECR1 also binds to and downregulates in trans DNMT1, a methyltransferase that is essential for the maintenance of DNA methylation.<br><br>CONCLUSIONS: These data suggest that FECR1 circular RNA acts as an upstream regulator to control breast cancer tumor growth by coordinating the regulation of DNA methylating and demethylating enzymes. Thus, FLI1 drives tumor metastasis not only through the canonical oncoprotein pathway, but also by using epigenetic mechanisms mediated by its exonic circular RNA.},
}
@article {pmid30523254,
year = {2018},
author = {Yamaguchi, H and Hopf, FW and Li, SB and de Lecea, L},
title = {In vivo cell type-specific CRISPR knockdown of dopamine beta hydroxylase reduces locus coeruleus evoked wakefulness.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {5211},
pmid = {30523254},
issn = {2041-1723},
support = {R01 AG047671/AG/NIA NIH HHS/United States ; R01 MH087592/MH/NIMH NIH HHS/United States ; R01 MH102638/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Dopamine beta-Hydroxylase/*genetics/metabolism ; Electric Stimulation ; Female ; Locus Coeruleus/cytology/*metabolism/physiology ; Male ; Mice ; Mice, Knockout ; Mice, Transgenic ; NIH 3T3 Cells ; Neurons/metabolism/physiology ; Norepinephrine/metabolism ; Sleep/genetics/physiology ; Wakefulness/*genetics/physiology ; },
abstract = {Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal. Optogenetic stimulation of LC-NE neurons induces immediate sleep-to-wake transitions. However, LC neurons also secrete other neurotransmitters in addition to NE. To interrogate the role of NE derived from the LC in regulating wakefulness, we applied in vivo cell type-specific CRISPR/Cas9 technology to disrupt the dopamine beta hydroxylase (dbh) gene selectively in adult LC-NE neurons. Unilateral dbh gene disruption abolished immediate arousal following optogenetic stimulation of LC. Bilateral LC-specific dbh disruption significantly reduced NE concentration in LC projection areas and reduced wake length even in the presence of salient stimuli. These results suggest that NE may be crucial for the awakening effect of LC stimulation and serve as proof-of-principle that CRISPR gene editing in adult neurons can be used to interrogate gene function within genetically-defined neuronal circuitry associated with complex behaviors.},
}
@article {pmid30537191,
year = {2019},
author = {Qin, R and Li, J and Li, H and Zhang, Y and Liu, X and Miao, Y and Zhang, X and Wei, P},
title = {Developing a highly efficient and wildly adaptive CRISPR-SaCas9 toolset for plant genome editing.},
journal = {Plant biotechnology journal},
volume = {17},
number = {4},
pages = {706-708},
pmid = {30537191},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant/*genetics ; Plants/*genetics ; Streptococcus pyogenes/*enzymology/genetics ; },
}
@article {pmid30537089,
year = {2019},
author = {Zhao, P and You, Q and Lei, M},
title = {A CRISPR/Cas9 deletion into the phosphate transporter SlPHO1;1 reveals its role in phosphate nutrition of tomato seedlings.},
journal = {Physiologia plantarum},
volume = {167},
number = {4},
pages = {556-563},
doi = {10.1111/ppl.12897},
pmid = {30537089},
issn = {1399-3054},
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/*genetics/physiology ; Phosphate Transport Proteins/*genetics ; Phosphates/*physiology ; Phylogeny ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Seedlings/genetics/physiology ; },
abstract = {In vascular (Arabidopsis thaliana) and non-vascular (Physcomitrella patens) plants, PHOSPHATE 1 (PHO1) homologs play important roles in the acquisition and transfer of phosphate. The tomato genome contains six genes (SlPHO1;1-SlPHO1;6) homologous to AtPHO1. The six proteins have typical characteristics of the plant PHO1 family, such as the three Syg1/Pho81/XPRI (SPX) subdomains in the N-terminal portion and one ERD1/XPR1/SYG1 (EXS) domain in the C-terminal portion. Phylogenetic analysis revealed that the SlPHO1 family is subdivided into three clusters. A pairwise comparison indicated that SlPHO1;1 showed the highest level of sequence identity/similarity (67.39/76.21%) to AtPHO1. SlPHO1;1 deletion mutants induced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 displayed typical phenotypes of Pi starvation, such as decreased shoot fresh weight and increased root fresh weight, therefore having a greater root-to-shoot ratio. Mutants also accumulated more anthocyanin and had more soluble Pi content in the root and less in the shoot. These results indicate that SlPHO1;1 plays an important role in Pi transport in the tomato at seedling stage.},
}
@article {pmid30535997,
year = {2019},
author = {Lopez, J and Tait, SWG},
title = {Application of Mito-Priming to Generate BCL-2 Addicted Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1877},
number = {},
pages = {45-60},
doi = {10.1007/978-1-4939-8861-7_3},
pmid = {30535997},
issn = {1940-6029},
support = {C40872/A20145//Cancer Research UK/United Kingdom ; },
mesh = {Apoptosis/physiology ; CRISPR-Cas Systems/physiology ; Cell Line ; HEK293 Cells ; Humans ; Mitochondria/metabolism ; Mitochondrial Membranes/metabolism ; Proto-Oncogene Proteins c-bcl-2/*metabolism ; bcl-X Protein/metabolism ; },
abstract = {The majority of apoptotic stimuli trigger cell death through the mitochondrial pathway of apoptosis. Invariably, mitochondrial apoptosis requires engagement of mitochondrial outer membrane permeabilization or MOMP to initiate cell death. We have developed a new method, called mito-priming, that allows for rapid and synchronous induction of mitochondrial apoptosis in an on-target manner. Mito-priming uses coexpression of pro- and antiapoptotic Bcl-2 proteins to render cells sensitive to the addition of Bcl-2 targeting BH3-mimetic drugs. This chapter describes how to design mito-priming constructs and apply them to generate mito-primed lines. Second, we describe how to validate cell death sensitivity of mito-primed lines using different methods. Finally, we describe how to generate MOMP-resistant cell lines, using CRISPR-Cas9 mediated deletion of BAX and BAK. Facilitating the investigation of mitochondrial apoptosis, mito-priming provides a clean, robust way to induce mitochondrial apoptosis both in vitro and in vivo.},
}
@article {pmid30535704,
year = {2019},
author = {Rattanapornsompong, K and Ngamkham, J and Chavalit, T and Jitrapakdee, S},
title = {Generation of Human Pyruvate Carboxylase Knockout Cell Lines Using Retrovirus Expressing Short Hairpin RNA and CRISPR-Cas9 as Models to Study Its Metabolic Role in Cancer Research.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1916},
number = {},
pages = {273-288},
doi = {10.1007/978-1-4939-8994-2_26},
pmid = {30535704},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Genetic Vectors ; Humans ; Neoplasms/genetics/pathology ; Promoter Regions, Genetic ; Pyruvate Carboxylase/*genetics ; RNA, Guide/genetics ; RNA, Small Interfering/*genetics ; Retroviridae/genetics ; },
abstract = {We report two protocols to generate human pyruvate carboxylase knockdown and knockout cell lines using short hairpin RNA (shRNA) and CRISPR-Cas9 technologies. The first protocol involved cloning of a shRNA cassette targeted to human pyruvate carboxylase (PC) under the control of a U6 promoter in a retrovirus-based vector. The stable knockdown cells were achieved following infection of retroviruses expressing shRNA in target cells followed by selecting these in medium containing puromycin. The second protocol describes a CRISPR Cas9-knockout cell constructed by cloning of single guide RNA (gRNA) targeted to the human pyruvate carboxylase gene placed adjacent to Cas 9 in the pSpCas9(BB)-2A-GFP vector. The knockout cells can be selected by sorting the cells expressing GFP. We also describe protocols for detecting the level of PC mRNA and protein in the knockdown or knockout cells using qPCR and Western blot analyses, respectively. The above protocols allow investigators to create PC deficient cell lines as a tool to study role of this enzyme in cancer research.},
}
@article {pmid30535125,
year = {2019},
author = {Chae, YC and Kim, JY and Park, JW and Kim, KB and Oh, H and Lee, KH and Seo, SB},
title = {FOXO1 degradation via G9a-mediated methylation promotes cell proliferation in colon cancer.},
journal = {Nucleic acids research},
volume = {47},
number = {4},
pages = {1692-1705},
pmid = {30535125},
issn = {1362-4962},
mesh = {Apoptosis/genetics ; Azepines/pharmacology ; CRISPR-Cas Systems/genetics ; Cell Proliferation/drug effects ; Colonic Neoplasms/*genetics/pathology ; DNA Methylation/genetics ; Female ; Flow Cytometry ; Forkhead Box Protein O1/*genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; HCT116 Cells ; Histocompatibility Antigens/*genetics ; Histone-Lysine N-Methyltransferase/antagonists &amp; inhibitors/*genetics ; Humans ; Male ; Quinazolines/pharmacology ; S-Phase Kinase-Associated Proteins/*genetics ; Tissue Array Analysis ; Ubiquitination/genetics ; },
abstract = {Posttranslational modifications of the Forkhead family transcription factor, FOXO1, have been known to have important regulatory implications in its diverse activities. Several types of modifications of FOXO1, including acetylation, phosphorylation, and ubiquitination, have been reported. However, lysine methylation of FOXO1 has not yet been identified. Here, we reported that FOXO1 is methylated by G9a at K273 residue in vitro and in vivo. Methylation of FOXO1 by G9a increased interaction between FOXO1 and a specific E3 ligase, SKP2, and decreased FOXO1 protein stability. In addition, G9a expression was increased by insulin and resulted in insulin-mediated FOXO1 degradation by K273 methylation. Tissue array analysis indicated that G9a was overexpressed and FOXO1 levels decreased in human colon cancer. Cell proliferation assays revealed that G9a-mediated FOXO1 methylation increased colon cancer cell proliferation. Fluorescence-activated cell sorting (FACS) analysis indicated that apoptosis rates were higher in the presence of FOXO1 than in FOXO1 knock-out cells. Furthermore, we found that G9a protein levels were elevated and FOXO1 protein levels were decreased in human colon cancer patients tissue samples. Here, we report that G9a specific inhibitor, BIX-01294, can regulate cell proliferation and apoptosis by inhibiting G9a-mediated FOXO1 methylation.},
}
@article {pmid30532129,
year = {2018},
author = {Mádrová, L and Krijt, M and Barešová, V and Václavík, J and Friedecký, D and Dobešová, D and Součková, O and Škopová, V and Adam, T and Zikánová, M},
title = {Mass spectrometric analysis of purine de novo biosynthesis intermediates.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0208947},
pmid = {30532129},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Chromatography, Liquid ; DNA/*biosynthesis/chemistry ; Gene Editing ; HeLa Cells ; Humans ; Purines/*biosynthesis/chemistry ; RNA/*biosynthesis/chemistry ; *Tandem Mass Spectrometry ; },
abstract = {Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.},
}
@article {pmid30531984,
year = {2019},
author = {Siu, KH and Chen, W},
title = {Riboregulated toehold-gated gRNA for programmable CRISPR-Cas9 function.},
journal = {Nature chemical biology},
volume = {15},
number = {3},
pages = {217-220},
doi = {10.1038/s41589-018-0186-1},
pmid = {30531984},
issn = {1552-4469},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli ; Gene Editing/*methods ; Gene Expression Regulation/*physiology ; RNA, Guide/genetics/physiology ; Riboswitch/*genetics/physiology ; },
abstract = {Predictable control over gene expression is essential to elicit desired synthetic cellular phenotypes. Although CRISPR-Cas9 offers a simple RNA-guided method for targeted transcriptional control, it lacks the ability to integrate endogenous cellular information for efficient signal processing. Here, we present a new class of riboregulators termed toehold-gated gRNA (thgRNA) by integrating toehold riboswitches into sgRNA scaffolds, and demonstrate their programmability for multiplexed regulation in Escherichia coli with minimal cross-talks.},
}
@article {pmid30531939,
year = {2019},
author = {Endo, M and Mikami, M and Endo, A and Kaya, H and Itoh, T and Nishimasu, H and Nureki, O and Toki, S},
title = {Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM.},
journal = {Nature plants},
volume = {5},
number = {1},
pages = {14-17},
pmid = {30531939},
issn = {2055-0278},
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Plant ; Nucleotide Motifs ; Oryza/*genetics ; Plants, Genetically Modified/genetics ; RNA, Guide ; Transformation, Genetic ; },
abstract = {Streptococcus pyogenes Cas9 (SpCas9) is widely used for genome editing and requires NGG as a protospacer adjacent motif (PAM). Here, we show that the engineered SpCas9 (SpCas9-NGv1) can efficiently mutagenize endogenous target sites with NG PAMs in the rice and Arabidopsis genomes. Furthermore, we demonstrate that the SpCas9-NGv1 nickase fused to cytidine deaminase mediates C-to-T substitutions near the 5' end of the target sequence.},
}
@article {pmid30530241,
year = {2019},
author = {Goh, YJ and Barrangou, R},
title = {Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli.},
journal = {Current opinion in biotechnology},
volume = {56},
number = {},
pages = {163-171},
doi = {10.1016/j.copbio.2018.11.009},
pmid = {30530241},
issn = {1879-0429},
mesh = {CRISPR-Cas Systems/*genetics ; Drug Delivery Systems ; Gene Editing ; Genetic Engineering/*methods ; Lactobacillus/*genetics ; Probiotics/*metabolism ; },
abstract = {Our evolving understanding on the mechanisms underlying the health-promoting attributes of probiotic lactobacilli, together with an expanding genome editing toolbox have made this genus an ideal chassis for the development of living therapeutics. The rising adoption of CRISPR-based technologies for prokaryotic engineering has demonstrated precise, efficient and scalable genome editing and tunable transcriptional regulation that can be translated into next-generation development of probiotic lactobacilli with enhanced robustness and designer functionalities. Here, we discuss how these tools in conjunction with the naturally abundant and diverse native CRISPR-Cas systems can be harnessed for Lactobacillus cell surface engineering and the delivery of biotherapeutics.},
}
@article {pmid30530202,
year = {2019},
author = {Cheng, R and Gong, L and Li, Z and Liang, YK},
title = {Rice BIG gene is required for seedling viability.},
journal = {Journal of plant physiology},
volume = {232},
number = {},
pages = {39-50},
doi = {10.1016/j.jplph.2018.11.006},
pmid = {30530202},
issn = {1618-1328},
mesh = {Blotting, Western ; CRISPR-Cas Systems ; Cell Death/physiology ; Chlorophyll/metabolism ; Gene Knockdown Techniques ; Genes, Plant/genetics/*physiology ; Lipid Peroxidation ; Oryza/*genetics/metabolism/physiology ; Phylogeny ; Plant Proteins/*genetics/physiology ; Seedlings/*genetics/metabolism/physiology ; Sequence Analysis, DNA ; },
abstract = {Arabidopsis BIG (AtBIG) gene encodes an enormous protein that is required for auxin transport. Loss of AtBIG function not only profoundly changes plant architecture but also alters plant adaptability to environmental stimuli. A putative homolog of AtBIG exists in the rice genome, but no function has been ascribed to it. In this study, we focus on the characterization of the gene structure and function of OsBIG. Sequence and phylogenetic analysis shows that the homologs of OsBIG have high amino acid conservation in several domains across species. Transgenic rice plants in which the expression of OsBIG was disrupted through the CRISPR/Cas9 system-mediated genome editing were used for phenotypic analysis. The Osbig/- plants show high levels of cell death, enhanced electrolyte leakage and membrane lipid peroxidation, and reduced chlorophyll content, which likely accounted for the seedling lethality. Moreover, gene expression between Osbig/- and wild-type plants analyzed by RNA-seq indicates that a number of metabolic and hormonal pathways including ribosome, DNA replication, photosynthesis, and chlorophyll metabolism were significantly perturbed by OsBIG deficiency. In summary, OsBIG gene is integral to the normal growth and development in rice.},
}
@article {pmid30529535,
year = {2019},
author = {Liu, P and Wang, W and Zhao, J and Wei, D},
title = {Screening novel β-galactosidases from a sequence-based metagenome and characterization of an alkaline β-galactosidase for the enzymatic synthesis of galactooligosaccharides.},
journal = {Protein expression and purification},
volume = {155},
number = {},
pages = {104-111},
doi = {10.1016/j.pep.2018.12.001},
pmid = {30529535},
issn = {1096-0279},
mesh = {Bacteria/*enzymology/*genetics/metabolism ; CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; Galactose/metabolism ; Galactosides/metabolism ; Lac Operon ; Metagenome ; Models, Molecular ; Oligosaccharides/metabolism ; Protein Multimerization ; Substrate Specificity ; beta-Galactosidase/*genetics/metabolism ; },
abstract = {βgalactosidases have wide industrial applications in lactose hydrolysis and transglycosylation reactions. Therefore, there is a need to mine novel and high-quality β-galactosidases with good tolerance and novel features from harsh environments and genomic databases. In this study, an Escherichia coli β-galactosidase-deficient host, ΔlacZ(DE3)pRARE, was constructed by the CRISPR-Cas9 system for screening active β-galactosidases. Of thirty selected β-galactosidases, twelve novel enzymes showed β-galactosidase activity, four of which were purified for further study. BGal_375 exhibited maximal activity at pH 8 and 50 °C. The concentrations of two types of galactooligosaccharides, tri- and tetra-saccharides, produced by BGal_375, reached 64.53 g/l and 8.32 g/l, respectively. BGal_375 displayed a Km value of 1.65 mM and kcat value of 53 s[-1] for p-nitrophenyl-β-d-galactopyranoside (pNPG). BGal_137, BGal_144-3, and BGal_145-2 showed promising hydrolytic activity for pNPG. BGal_137 is a homodimer while BGal_144-3, BGal_145-2, and BGal_375 were all monomeric. This study provided an efficient solution for the identification of new β-galactosidases from metagenomic data, and an alkaline β-galactosidase efficient for the synthesis of galactooligosaccharides was obtained, which is important for potential industrial applications.},
}
@article {pmid30529475,
year = {2019},
author = {O'Halloran, DM},
title = {Genome aware CRISPR gRNA target prediction for parasitic nematodes.},
journal = {Molecular and biochemical parasitology},
volume = {227},
number = {},
pages = {25-28},
doi = {10.1016/j.molbiopara.2018.12.001},
pmid = {30529475},
issn = {1872-9428},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genome, Helminth ; Mutation ; Nematoda/*genetics/metabolism ; RNA, Guide/*genetics/metabolism ; RNA, Helminth/*genetics/metabolism ; },
abstract = {The pace of research towards a genetic model to understand the unique molecular biology of parasitic nematodes has increased recently. This research has developed a diverse suite of genetic tools for a variety of parasitic nematodes. CRISPR/Cas9 technology in particular offers much promise as a game changing tool for researchers studying parasitic nematodes. Unlike RNAi, which depends on diverse nematode effectors to silence gene expression, the effectors for CRISPR/Cas9 mutations are typically supplied by the experimenter, making gene editing via CRISPR/Cas9 ideal for testing on genetically intractable nematode systems. To facilitate the development of CRISPR/Cas9 technology for parasitic nematodes, I here describe a tool for identifying gRNA targets and diagnostic primers to a user supplied sequence. The software attempts to minimize non-specific targets by interrogating the genomes of parasitic nematodes. This software is freely available online and features an intuitive interface to help researchers design effective CRISPR experiments for parasitic nematodes.},
}
@article {pmid30529264,
year = {2019},
author = {Shi, X and He, W and Guo, S and Zhang, B and Ren, S and Liu, K and Sun, T and Cui, J},
title = {RNA-seq Analysis of the SCN1A-KO Model based on CRISPR/Cas9 Genome Editing Technology.},
journal = {Neuroscience},
volume = {398},
number = {},
pages = {1-11},
doi = {10.1016/j.neuroscience.2018.11.052},
pmid = {30529264},
issn = {1873-7544},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Epilepsies, Myoclonic/genetics/metabolism ; *Gene Editing ; Gene Expression ; *Gene Knockout Techniques/methods ; Mice ; NAV1.1 Voltage-Gated Sodium Channel/*deficiency/genetics ; RNA, Messenger/metabolism ; Sequence Analysis, RNA ; Signal Transduction ; },
abstract = {Dravet syndrome (DS) is a disease that is primarily caused by the inactivation of the SCN1A-encoded voltage-gated sodium channel alpha subunit (Nav1.1). In this study, we constructed an SCN1A gene knockout model using CRISPR/Cas9 genome editing technology to deprive the Nav1.1 function in vitro. With mRNA-seq analysis we found abundant gene changes after SCN1A knockout, which associated with various signaling pathways, such as cancer pathways, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and pathways involved in HTLV-I infection. We also noticed changes in the spliceosome, decreased glycolytic capacity, disturbances in calcium signaling pathways, and changes in the potassium, sodium, chloride, and calcium plasma channels after SCN1A knockout. In this study, we have been the first time to discover these changes and summarize them here and hope it would provide some clue for the study of Nav1.1 in the nervous system.},
}
@article {pmid30528226,
year = {2019},
author = {Przybilla, MJ and Ou, L and Tăbăran, AF and Jiang, X and Sidhu, R and Kell, PJ and Ory, DS and O'Sullivan, MG and Whitley, CB},
title = {Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B.},
journal = {Molecular genetics and metabolism},
volume = {126},
number = {2},
pages = {139-150},
doi = {10.1016/j.ymgme.2018.11.002},
pmid = {30528226},
issn = {1096-7206},
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Fluorometry ; Gangliosidosis, GM1/genetics/*pathology ; Gene Editing ; Mental Status and Dementia Tests ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mucopolysaccharidosis IV/genetics/*pathology ; Mutation ; Mutation, Missense ; Phenotype ; beta-Galactosidase/genetics/*metabolism ; },
abstract = {Deficiencies in the lysosomal hydrolase β-galactosidase (β-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene, Glb1, was targeted to generate both models of β-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1[W274L]), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of β-gal (β-gal[-/-]). Glb1[W274L] mice showed a significant reduction in β-gal enzyme activity (8.4-13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, β-gal[-/-] mice were devoid of β-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal[-/-] mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of β-galactosidase deficiency with residual enzyme activity has been developed.},
}
@article {pmid30528088,
year = {2019},
author = {Song, X and Xu, P and Meng, C and Song, C and Blackwell, TS and Li, R and Li, H and Zhang, J and Lv, C},
title = {lncITPF Promotes Pulmonary Fibrosis by Targeting hnRNP-L Depending on Its Host Gene ITGBL1.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {2},
pages = {380-393},
pmid = {30528088},
issn = {1525-0024},
support = {P01 HL092870/HL/NHLBI NIH HHS/United States ; },
mesh = {Aged ; Animals ; Blotting, Northern ; Blotting, Western ; CRISPR-Cas Systems/genetics/*physiology ; Cell Line ; Cell Movement/genetics/physiology ; Chromatin Immunoprecipitation ; Chromatography, Liquid ; Female ; Fluorescent Antibody Technique ; Heterogeneous-Nuclear Ribonucleoprotein L/genetics/*metabolism ; Humans ; Idiopathic Pulmonary Fibrosis/genetics/*metabolism/*pathology ; Immunoprecipitation ; In Situ Hybridization ; Male ; Mice ; Middle Aged ; Models, Biological ; Myofibroblasts/cytology/metabolism ; RNA, Long Noncoding/genetics/*metabolism ; Rats ; Real-Time Polymerase Chain Reaction ; },
abstract = {The role of long non-coding RNA (lncRNA) in idiopathic pulmonary fibrosis (IPF) is poorly understood. We found a novel lncRNA-ITPF that was upregulated in IPF. Bioinformatics and in vitro translation verified that lncITPF is an actual lncRNA, and its conservation is in evolution. Northern blot and rapid amplification of complementary DNA ends were used to analyze the full-length sequence of lncITPF. RNA fluorescence in situ hybridization and nucleocytoplasmic separation demonstrated that lncITPF was mainly located in the nucleus. RNA sequencing, chromatin immunoprecipitation (ChIP)-qPCR, CRISPR-Cas9 technology, and promoter activity analysis showed that the fibrotic function of lncITPF depends on its host gene integrin β-like 1 (ITGBL1), but they did not share the same promoter and were not co-transcribed. Luciferase activity, pathway inhibitors, and ChIP-qPCR showed that smad2/3 binds to the lncITPF promoter, and TGF-β1-smad2/3 was the upstream inducer of the fibrotic pathway. Furthermore, RNA-protein pull-down, liquid chromatography-mass spectrometry (LC-MS), and protein-RNA immunoprecipitation showed that lncITPF regulated H3 and H4 histone acetylation in the ITGBL1 promoter by targeting heterogeneous nuclear ribonucleoprotein L. Finally, sh-lncITPF was used to evaluate the therapeutic effect of lncITPF. Clinical analysis showed that lncITPF is associated with the clinicopathological features of IPF patients. Our findings provide a therapeutic target or diagnostic biomarker for IPF.},
}
@article {pmid30527810,
year = {2019},
author = {Rago, F and DiMare, MT and Elliott, G and Ruddy, DA and Sovath, S and Kerr, G and Bhang, HC and Jagani, Z},
title = {Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.},
journal = {Biochemical and biophysical research communications},
volume = {508},
number = {1},
pages = {109-116},
doi = {10.1016/j.bbrc.2018.09.009},
pmid = {30527810},
issn = {1090-2104},
mesh = {Animals ; Antineoplastic Agents/administration &amp; dosage ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*drug therapy/*genetics/metabolism ; Cell Line, Tumor ; DNA Helicases/*genetics/metabolism ; Female ; Gene Knockout Techniques ; Humans ; Isoquinolines/administration &amp; dosage ; Lung Neoplasms/*drug therapy/*genetics/metabolism ; Mice ; Mice, Nude ; Mutation ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists &amp; inhibitors/genetics ; Nuclear Proteins/*genetics/metabolism ; Oxidative Phosphorylation/drug effects ; Proteolysis ; Sulfonamides/administration &amp; dosage ; Transcription Factors/*antagonists &amp; inhibitors/*genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses.},
}
@article {pmid30527703,
year = {2019},
author = {Larouche, J and Aguilar, CA},
title = {New Technologies To Enhance In Vivo Reprogramming for Regenerative Medicine.},
journal = {Trends in biotechnology},
volume = {37},
number = {6},
pages = {604-617},
doi = {10.1016/j.tibtech.2018.11.003},
pmid = {30527703},
issn = {1879-3096},
mesh = {Animals ; CRISPR-Cas Systems ; *Cellular Reprogramming ; Gene Editing ; Humans ; Mice ; Nanomedicine ; *Regenerative Medicine ; Synthetic Biology ; },
abstract = {Cellular identity and state are determined by a collection of molecular components that are specified during development and stabilized thereafter to maintain and protect tissue functions. Alteration of the molecular elements (gene expression program and chromatin state) as a result of disease or age can induce somatic cells to assume different identities or modulate functions. Therapeutic use of this technique, called 'cellular reprogramming', is very promising for regenerative medicine, but implementation of reprogramming-based strategies in vivo has been precluded by technological and safety limitations. Recent advances in transcriptional control and improved transmembrane delivery strategies now offer exciting potential to more efficiently reprogram cell fates as well as to control the reprogramming timeline and scale of delivery to improve safety.},
}
@article {pmid30526668,
year = {2018},
author = {Wang, M and Wang, H and Wen, Y and Chen, X and Liu, X and Gao, J and Su, P and Xu, Y and Zhou, W and Shi, L and Zhou, J},
title = {MEIS2 regulates endothelial to hematopoietic transition of human embryonic stem cells by targeting TAL1.},
journal = {Stem cell research &amp; therapy},
volume = {9},
number = {1},
pages = {340},
pmid = {30526668},
issn = {1757-6512},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Endothelial Cells/*cytology/metabolism ; Gene Deletion ; Hematopoiesis ; Hematopoietic Stem Cells/*cytology/metabolism ; Homeodomain Proteins/*metabolism ; Human Embryonic Stem Cells/*cytology/*metabolism ; Humans ; Megakaryocytes/cytology ; T-Cell Acute Lymphocytic Leukemia Protein 1/*metabolism ; Transcription Factors/*metabolism ; },
abstract = {BACKGROUND: Despite considerable progress in the development of methods for hematopoietic differentiation, efficient generation of transplantable hematopoietic stem cells (HSCs) and other genuine functional blood cells from human embryonic stem cells (hESCs) is still unsuccessful. Therefore, a better understanding of the molecular mechanism underlying hematopoietic differentiation of hESCs is highly demanded.<br><br>METHODS: In this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. We deleted MEIS2 gene in hESCs using the CRISPR/CAS9 technology and induced them to hematopoietic differentiation, megakaryocytic differentiation.<br><br>RESULTS: In this study, we found that MEIS2 deletion impairs early hematopoietic differentiation from hESCs. Furthermore, MEIS2 deletion suppresses hemogenic endothelial specification and endothelial to hematopoietic transition (EHT), leading to the impairment of hematopoietic differentiation. Mechanistically, TAL1 acts as a downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 deletion exerts minimal effects on megakaryocytic differentiation and platelet generation from hESCs.<br><br>CONCLUSIONS: Our findings advance the understanding of human hematopoietic development and may provide new insights for large-scale generation of functional blood cells for clinical applications.},
}
@article {pmid30526490,
year = {2018},
author = {Li, X and Ma, Y and Liang, S and Tian, Y and Yin, S and Xie, S and Xie, H},
title = {Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle.},
journal = {BMC genomics},
volume = {19},
number = {1},
pages = {889},
pmid = {30526490},
issn = {1471-2164},
mesh = {Bacterial Secretion Systems/genetics ; CRISPR-Cas Systems/genetics ; Conserved Sequence/genetics ; Genes, Bacterial ; Genetic Variation ; *Genome, Bacterial ; *Genomics ; Multigene Family ; Operon/genetics ; Pectobacterium/*genetics/isolation &amp; purification/*pathogenicity ; Phenotype ; Species Specificity ; },
abstract = {BACKGROUND: Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).<br><br>RESULTS: Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.<br><br>CONCLUSIONS: This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.},
}
@article {pmid30526248,
year = {2018},
author = {Shahid, M and Lee, MY and Piplani, H and Andres, AM and Zhou, B and Yeon, A and Kim, M and Kim, HL and Kim, J},
title = {Centromere protein F (CENPF), a microtubule binding protein, modulates cancer metabolism by regulating pyruvate kinase M2 phosphorylation signaling.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {17},
number = {24},
pages = {2802-2818},
pmid = {30526248},
issn = {1551-4005},
support = {UL1 TR000124/TR/NCATS NIH HHS/United States ; U24 DK097154/DK/NIDDK NIH HHS/United States ; U01 DK103260/DK/NIDDK NIH HHS/United States ; U01 DP006079/DP/NCCDPHP CDC HHS/United States ; R01 DK100974/DK/NIDDK NIH HHS/United States ; },
mesh = {Anoikis ; Apoptosis ; CRISPR-Cas Systems/genetics ; Carrier Proteins/*metabolism ; Cell Line, Tumor ; Chromosomal Proteins, Non-Histone/genetics/*metabolism ; Epithelial-Mesenchymal Transition ; Gene Editing ; Glucose/metabolism ; Humans ; Male ; Membrane Proteins/*metabolism ; Microfilament Proteins/genetics/*metabolism ; Phosphorylation ; Prostatic Neoplasms/metabolism/pathology ; Proteome/metabolism ; Signal Transduction ; Thyroid Hormones/*metabolism ; Tight Junctions/metabolism ; },
abstract = {Prostate cancer (PC) is the most commonly diagnosed cancer in men and is the second leading cause of male cancer-related death in North America. Metabolic adaptations in malignant PC cells play a key role in fueling the growth and progression of the disease. Unfortunately, little is known regarding these changes in cellular metabolism. Here, we demonstrate that centromere protein F (CENPF), a protein associated with the centromere-kinetochore complex and chromosomal segregation during mitosis, is mechanically linked to altered metabolism and progression in PC. Using the CRISPR-Cas9 system, we silenced the gene for CENPF in human PC3 cells. These cells were found to have reduced levels of epithelial-mesenchymal transition markers and inhibited cell proliferation, migration, and invasion. Silencing of CENPF also simultaneously improved sensitivity to anoikis-induced apoptosis. Mass spectrometry analysis of tyrosine phosphorylated proteins from CENPF knockout (CENPF[KO]) and control cells revealed that CENPF silencing increased inactive forms of pyruvate kinase M2, a rate limiting enzyme needed for an irreversible reaction in glycolysis. Furthermore, CENPF[KO] cells had reduced global bio-energetic capacity, acetyl-CoA production, histone acetylation, and lipid metabolism, suggesting that CENPF is a critical regulator of cancer metabolism, potentially through its effects on mitochondrial functioning. Additional quantitative immunohistochemistry and imaging analyzes on a series of PC tumor microarrays demonstrated that CENPF expression is significantly increased in higher-risk PC patients. Based on these findings, we suggest the CENPF may be an important regulator of PC metabolism through its role in the mitochondria.},
}
@article {pmid30526040,
year = {2018},
author = {Shabbir, MA and Wu, Q and Shabbir, MZ and Sajid, A and Ahmed, S and Sattar, A and Tang, Y and Li, J and Maan, MK and Hao, H and Yuan, Z},
title = {The CRISPR-cas system promotes antimicrobial resistance in Campylobacter jejuni.},
journal = {Future microbiology},
volume = {13},
number = {},
pages = {1757-1774},
doi = {10.2217/fmb-2018-0234},
pmid = {30526040},
issn = {1746-0921},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Campylobacter jejuni/drug effects/*genetics/growth &amp; development/isolation &amp; purification/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/*physiology ; Drug Resistance, Bacterial/*physiology ; Gene Expression Profiling ; *Gene Expression Regulation, Bacterial ; Microbial Sensitivity Tests ; Sequence Deletion ; },
abstract = {AIM: The purpose of current study is to find out relationship between cas9 gene and antimicrobial resistance in Campylobacter jejuni NCTC11168.<br><br>MATERIALS &amp; METHODS: The involvement of the cas9 gene in antimicrobial resistance of C. jejuni was determined by assessment of minimum inhibitory concentration, clustered regularly interspaced short palindromic repeats (CRISPR)-cas gene expression in standard strains, in vitro resistance development and transcriptome analysis of a cas9 deletion mutant and wild strains.<br><br>RESULTS: Increased expression of CRISPR-related genes was observed in standard strains. We also observed that &#x394;cas9 mutant strain is more sensitive to antibiotics than its wild strain. Transcriptome analysis revealed that cas9 gene regulate several genes to promote antimicrobial resistance in C. jejuni.<br><br>CONCLUSION: CRISPR-cas system plays role in the enhancement of antimicrobial resistance in C. jejuni.},
}
@article {pmid30525482,
year = {2019},
author = {Boyle, WS and Twaroski, K and Woska, EC and Tolar, J and Reineke, TM},
title = {Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells.},
journal = {Bioconjugate chemistry},
volume = {30},
number = {2},
pages = {418-431},
doi = {10.1021/acs.bioconjchem.8b00760},
pmid = {30525482},
issn = {1520-4812},
support = {DP2 OD006669/OD/NIH HHS/United States ; R01 AR059947/AR/NIAMS NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; R01 HL108627/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Fibroblasts/metabolism ; Heparin/*analogs &amp; derivatives ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Plasmids/*administration &amp; dosage/genetics ; Transcriptional Activation ; Transfection/*methods ; Trehalose/*analogs &amp; derivatives ; },
abstract = {Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5-13 kbp) in comparison to common reporter plasmids (∼5-8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.},
}
@article {pmid30525474,
year = {2019},
author = {Niu, TC and Lin, GM and Xie, LR and Wang, ZQ and Xing, WY and Zhang, JY and Zhang, CC},
title = {Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120.},
journal = {ACS synthetic biology},
volume = {8},
number = {1},
pages = {170-180},
doi = {10.1021/acssynbio.8b00437},
pmid = {30525474},
issn = {2161-5063},
mesh = {Anabaena/*genetics ; Bacterial Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; DNA Polymerase I/genetics ; Gene Editing ; Genome, Bacterial/*genetics ; },
abstract = {CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a "two-spacers" strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.},
}
@article {pmid30521608,
year = {2018},
author = {Wu, K and Shirk, PD and Taylor, CE and Furlong, RB and Shirk, BD and Pinheiro, DH and Siegfried, BD},
title = {CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in fall armyworm moth (Spodoptera frugiperda).},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0208647},
pmid = {30521608},
issn = {1932-6203},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Female ; *Gene Knockdown Techniques ; Insect Proteins/*genetics/metabolism ; Male ; Mutagenesis, Site-Directed ; Pest Control, Biological ; Phenotype ; Phylogeny ; Spodoptera/anatomy &amp; histology/*genetics/metabolism ; },
abstract = {The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is an important pest of maize in the Americas and has recently been introduced into Africa. Fall armyworm populations have developed resistance to control strategies that depend on insecticides and transgenic plants expressing Bacillus thuringiensis toxins. The study of various resistance mechanisms at the molecular level and the development novel control strategies have been hampered by a lack of functional genomic tools such as gene editing in this pest. In the current study, we explored the possibility of using the CRISPR/Cas9 system to modify the genome of FAW. We first identified and characterized the abdominal-A (Sfabd-A) gene of FAW. Sfabd-A single guide RNA (sgRNA) and Cas9 protein were then injected into 244 embryos of FAW. Sixty-two embryos injected with Sfabd-A sgRNA hatched. Of these hatched embryos, twelve developed into larvae that displayed typical aba-A mutant phenotypes such as fused segments. Of the twelve mutant larvae, three and five eventually developed into female and male moths, respectively. Most mutant moths were sterile, and one female produced a few unviable eggs when it was outcrossed to a wild-type male. Genotyping of 20 unhatched Sfabd-A sgRNA-injected embryos and 42 moths that developed from Sfabd-A sgRNA-injected embryos showed that 100% of the unhatched embryos and 50% of the moths contained indel mutations at the Sfabd-A genomic locus near the guide RNA target site. These results suggest that the CRISPR/Cas9 system is highly efficient in editing FAW genome. Importantly, this gene editing technology can be used to validate gene function to facilitate an understanding of the resistance mechanism and lead to the development of novel pest management approaches.},
}
@article {pmid30520985,
year = {2019},
author = {Toymentseva, AA and Altenbuchner, J},
title = {New CRISPR-Cas9 vectors for genetic modifications of Bacillus species.},
journal = {FEMS microbiology letters},
volume = {366},
number = {1},
pages = {},
doi = {10.1093/femsle/fny284},
pmid = {30520985},
issn = {1574-6968},
mesh = {Bacillus/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods/trends ; Genetic Vectors/*genetics ; },
abstract = {Genetic manipulation is a fundamental procedure for the study of gene and operon functions and new characteristics acquisition. Modern CRISPR-Cas technology allows genome editing more precisely and increases the efficiency of transferring mutations in a variety of hard to manipulate organisms. Here, we describe new CRISPR-Cas vectors for genetic modifications in bacillary species. Our plasmids are single CRISPR-Cas plasmids comprising all components for genome editing and should be functional in a broad host range. They are highly efficient (up to 97%) and precise. The employment and delivery of these plasmids to bacillary strains can be easily achieved by conjugation from Escherichia coli. During our research we also demonstrated the absence of compatibility between CRISPR-Cas system and non-homologous end joining in Bacillus subtilis.},
}
@article {pmid30520725,
year = {2018},
author = {OhAinle, M and Helms, L and Vermeire, J and Roesch, F and Humes, D and Basom, R and Delrow, JJ and Overbaugh, J and Emerman, M},
title = {A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30520725},
issn = {2050-084X},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; CFAR New Investigator Award, P30 AI027757/AI/NIAID NIH HHS/United States ; DP1 DA039543/DA/NIDA NIH HHS/United States ; P30 CA015704-43/CA/NCI NIH HHS/United States ; P30 DK056465/DK/NIDDK NIH HHS/United States ; R01 AI030927/AI/NIAID NIH HHS/United States ; CCEH Pilot Grant, DK56465/DK/NIDDK NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI30927/AI/NIAID NIH HHS/United States ; R37 AI030927/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD/genetics/immunology ; Antigens, Differentiation/genetics/immunology ; Antiviral Restriction Factors ; CRISPR-Cas Systems ; Carrier Proteins/genetics/immunology ; Cell Line, Tumor ; Epithelial Cells/drug effects/*immunology/virology ; GPI-Linked Proteins/genetics/immunology ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Vectors/chemistry/immunology ; HEK293 Cells ; HIV-1/drug effects/*genetics/growth &amp; development/immunology ; *Host-Pathogen Interactions ; Humans ; Interferon-alpha/pharmacology ; Lentivirus/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/immunology ; Nuclear Proteins/deficiency/*genetics/immunology ; Phosphotransferases (Alcohol Group Acceptor)/deficiency/*genetics/immunology ; RNA-Binding Proteins ; Receptors, CCR5/genetics/immunology ; Receptors, CXCR4/genetics/immunology ; Repressor Proteins ; Signal Transduction ; THP-1 Cells ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; Viral Tropism/genetics ; Virus Assembly/drug effects ; Virus Replication/drug effects ; },
abstract = {UNLABELLED: Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized.<br><br>EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).},
}
@article {pmid30520487,
year = {2018},
author = {Han, S and Wei, S and Wang, X and Han, X and Zhang, M and Su, M and Li, Y and Guo, J and Zeng, W and Liu, J and Gao, Y and Shen, L},
title = {Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.},
journal = {Integrative biology : quantitative biosciences from nano to macro},
volume = {10},
number = {12},
pages = {780-790},
doi = {10.1039/c8ib00109j},
pmid = {30520487},
issn = {1757-9708},
mesh = {Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytochrome P-450 CYP3A/*metabolism ; Gene Editing ; Gene Expression Profiling ; Hep G2 Cells ; Hepatocytes ; Humans ; Light ; Liver/*metabolism ; Liver, Artificial ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; *Transcriptional Activation ; },
abstract = {Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.},
}
@article {pmid30520167,
year = {2019},
author = {Zhou, Q and Zhan, H and Liao, X and Fang, L and Liu, Y and Xie, H and Yang, K and Gao, Q and Ding, M and Cai, Z and Huang, W and Liu, Y},
title = {A revolutionary tool: CRISPR technology plays an important role in construction of intelligentized gene circuits.},
journal = {Cell proliferation},
volume = {52},
number = {2},
pages = {e12552},
pmid = {30520167},
issn = {1365-2184},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Gene Regulatory Networks ; *Genes, Synthetic ; Genetic Therapy/methods ; Humans ; Neoplasms/genetics/therapy ; },
abstract = {With the development of synthetic biology, synthetic gene circuits have shown great applied potential in medicine, biology, and as commodity chemicals. An ultimate challenge in the construction of gene circuits is the lack of effective, programmable, secure and sequence-specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system, a CRISPR-associated RNA-guided endonuclease Cas9 (CRISPR-associated protein 9)-targeted genome editing tool, has recently been applied in engineering gene circuits for its unique properties-operability, high efficiency and programmability. The traditional single-targeted therapy cannot effectively distinguish tumour cells from normal cells, and gene therapy for single targets has poor anti-tumour effects, which severely limits the application of gene therapy. Currently, the design of gene circuits using tumour-specific targets based on CRISPR/Cas systems provides a new way for precision cancer therapy. Hence, the application of intelligentized gene circuits based on CRISPR technology effectively guarantees the safety, efficiency and specificity of cancer therapy. Here, we assessed the use of synthetic gene circuits and if the CRISPR system could be used, especially artificial switch-inducible Cas9, to more effectively target and treat tumour cells. Moreover, we also discussed recent advances, prospectives and underlying challenges in CRISPR-based gene circuit development.},
}
@article {pmid30519946,
year = {2019},
author = {Hoepfner, D and McAllister, G and Hoffman, GR},
title = {CRISPR/Cas9-Based Chemogenomic Profiling in Mammalian Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1888},
number = {},
pages = {153-174},
doi = {10.1007/978-1-4939-8891-4_9},
pmid = {30519946},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Cell Survival/drug effects/genetics ; Dose-Response Relationship, Drug ; Gene Editing ; *Gene Expression Profiling/methods ; Gene Expression Regulation/*drug effects ; Genetic Vectors/genetics ; *Genomics/methods ; Haploinsufficiency ; Humans ; Lentivirus/genetics ; RNA, Guide ; },
abstract = {Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. It is based on identifying cellular hypersensitivity and resistance caused by individual gene modulations with genome-wide coverage. Due to the requirement of bar-coded, genome-wide deletion collections, high-resolution experiments of this nature have historically been limited to fungal systems. Pooled RNAi reagents have enabled similar attempts in mammalian cells but efforts have been hampered by significant off-target effects and experimental noise. The CRISPR/Cas9 system for the first time enables precise DNA editing at defined loci in a genome-wide fashion. Here we present the detailed protocol that leverages the CRISPR/Cas9 system for chemogenomic profiling and target identification of diverse chemical probes.},
}
@article {pmid30518723,
year = {2019},
author = {Ayabe, S and Nakashima, K and Yoshiki, A},
title = {Off- and on-target effects of genome editing in mouse embryos.},
journal = {The Journal of reproduction and development},
volume = {65},
number = {1},
pages = {1-5},
pmid = {30518723},
issn = {1348-4400},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Embryo, Mammalian ; Gene Editing/*methods ; Mice ; Mice, Inbred C57BL ; *Mutagenesis ; Reproducibility of Results ; Whole Genome Sequencing ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based genome editing technology has enabled manipulation of the embryonic genome. Unbiased whole genome sequencing comparing parents to progeny has revealed that the rate of Cas9-induced mutagenesis in mouse embryos is indistinguishable from the background rate of de novo mutation. However, establishing the best practice to confirm on-target alleles of interest remains a challenge. We believe that improvement in editing strategies and screening methods for founder mice will contribute to the generation of quality-controlled animals, thereby ensuring reproducibility of results in animal studies and advancing the 3Rs (replacement, reduction, and refinement).},
}
@article {pmid30518653,
year = {2019},
author = {Isken, O and Postel, A and Bruhn, B and Lattwein, E and Becher, P and Tautz, N},
title = {CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.},
journal = {Journal of virology},
volume = {93},
number = {5},
pages = {},
pmid = {30518653},
issn = {1098-5514},
mesh = {Animals ; Bovine Virus Diarrhea-Mucosal Disease/virology ; CRISPR-Cas Systems/*genetics ; Cattle ; Cattle Diseases/virology ; Cell Line ; Diarrhea Virus 1, Bovine Viral/*genetics ; Fetal Proteins/*genetics ; Gene Knockout Techniques ; Genome, Viral/genetics ; HEK293 Cells ; Humans ; Molecular Chaperones/*genetics/metabolism ; RNA Helicases/genetics/metabolism ; RNA, Viral/*biosynthesis/genetics ; Serine Endopeptidases/genetics/metabolism ; Swine ; Viral Nonstructural Proteins/*genetics/metabolism ; Virus Replication/genetics ; },
abstract = {Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.},
}
@article {pmid30518324,
year = {2018},
author = {Nishihara, M and Higuchi, A and Watanabe, A and Tasaki, K},
title = {Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri.},
journal = {BMC plant biology},
volume = {18},
number = {1},
pages = {331},
pmid = {30518324},
issn = {1471-2229},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Color ; Flowers/anatomy &amp; histology/*genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; Lamiales/anatomy &amp; histology/*genetics ; Plants, Genetically Modified ; Sequence Analysis, DNA ; },
abstract = {BACKGROUND: CRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.).<br><br>RESULTS: We attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene.<br><br>CONCLUSIONS: These results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.},
}
@article {pmid30507925,
year = {2018},
author = {Evans, BA and Pickerill, ES and Vyas, VK and Bernstein, DA},
title = {CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {141},
pages = {},
pmid = {30507925},
issn = {1940-087X},
support = {R15 AI130950/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Gene Editing/*methods ; Humans ; Plasmids ; RNA, Guide/genetics ; },
abstract = {This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.},
}
@article {pmid30507915,
year = {2018},
author = {Bialek, JK and Walther, T and Hauber, J and Lange, UC},
title = {CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {141},
pages = {},
doi = {10.3791/58572},
pmid = {30507915},
issn = {1940-087X},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome ; HIV Infections/*genetics ; HIV-1/*genetics ; Humans ; Jurkat Cells ; RNA, Guide/genetics ; *Virus Integration ; },
abstract = {Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites. In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed. By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.},
}
@article {pmid30506264,
year = {2019},
author = {Brune, T and Kunze-Schumacher, H and Kölling, R},
title = {Interactions in the ESCRT-III network of the yeast Saccharomyces cerevisiae.},
journal = {Current genetics},
volume = {65},
number = {2},
pages = {607-619},
pmid = {30506264},
issn = {1432-0983},
mesh = {CRISPR-Cas Systems ; Endosomal Sorting Complexes Required for Transport/*metabolism ; Endosomes/metabolism ; Epistasis, Genetic ; Gene Deletion ; Mutation ; Phenotype ; Protein Binding ; Protein Transport ; Saccharomyces cerevisiae/drug effects/*genetics/*metabolism ; },
abstract = {Here, we examine the genetic interactions between ESCRT-III mutations in the yeast Saccharomyces cerevisiae. From the obtained interaction network, we make predictions about alternative ESCRT-III complexes. By the successful generation of an octuple deletion strain using the CRISPR/Cas9 technique, we demonstrate for the first time that ESCRT-III activity as a whole is not essential for the life of a yeast cell. Endosomal sorting complex required for transport (ESCRT)-III proteins are membrane remodeling factors involved in a multitude of cellular processes. There are eight proteins in yeast with an ESCRT-III domain. It is not clear whether the diverse ESCRT-III functions are fulfilled by a single ESCRT-III complex or by different complexes with distinct composition. Genetic interaction studies may provide a hint on the existence of alternative complexes. We performed a genetic mini-array screen by analyzing the growth phenotypes of all pairwise combinations of ESCRT-III deletion mutations under different stress conditions. Our analysis is in line with previous data pointing to a complex containing Did2/CHMP1 and Ist1/IST1. In addition, we provide evidence for the existence of a novel complex consisting of Did2/CHMP1 and Vps2/CHMP2. Some of the interactions on Congo red plates could be explained by effects of ESCRT-III mutations on Rim101 signaling.},
}
@article {pmid30504911,
year = {2018},
author = {Ogawa, Y and Terao, M and Hara, S and Tamano, M and Okayasu, H and Kato, T and Takada, S},
title = {Mapping of a responsible region for sex reversal upstream of Sox9 by production of mice with serial deletion in a genomic locus.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {17514},
pmid = {30504911},
issn = {2045-2322},
support = {25132713//Japan Society for the Promotion of Science (JSPS)/International ; 24-3//National Center for Child Health and Development (NCCHD)/International ; 29-11//National Center for Child Health and Development (NCCHD)/International ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Conserved Sequence ; Disease Models, Animal ; Female ; *Gene Deletion ; Gonadal Dysgenesis, 46,XY/*genetics ; Humans ; Male ; Mice ; Mice, Mutant Strains ; SOX9 Transcription Factor/*genetics ; },
abstract = {Sox9 plays critical roles in testis formation. By mapping four familial cases of disorders of sexual development, a 32.5 kb sequence located far upstream of SOX9 was previously identified as being a commonly deleted region and named the XY sex reversal region (XYSR). To narrow down a responsible sequence in XYSR, we generated mutant mice with a series of deletions in XYSR by application of the CRISPR/Cas9 system, using a mixture of sgRNAs targeting several kilobase (kb) intervals in the region. When the whole XYSR corresponding sequence in mice was deleted in XY karyotype individuals, the mutation resulted in female offspring, suggesting that an expression mechanism of SOX9/Sox9 through XYSR is conserved in human and mouse. Male-to-female sex reversal was found in mice with a 4.8 kb deletion. We identified a sequence conserved among humans, mice, and opossum, the deletion of which (783 bp) in mice resulted in male-to-female sex reversal. The sequence includes a recently reported critical gonad enhancer for Sox9. Although it cannot be concluded that the human sequence is responsible for XYSR, it is likely. This method is applicable for fine mapping of responsible sequences for disease-causing deletions especially with regard to rare diseases.},
}
@article {pmid30504875,
year = {2018},
author = {Hsu, JY and Fulco, CP and Cole, MA and Canver, MC and Pellin, D and Sher, F and Farouni, R and Clement, K and Guo, JA and Biasco, L and Orkin, SH and Engreitz, JM and Lander, ES and Joung, JK and Bauer, DE and Pinello, L},
title = {CRISPR-SURF: discovering regulatory elements by deconvolution of CRISPR tiling screen data.},
journal = {Nature methods},
volume = {15},
number = {12},
pages = {992-993},
pmid = {30504875},
issn = {1548-7105},
support = {K08 DK093705/DK/NIDDK NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; K99 HG009917/HG/NHGRI NIH HHS/United States ; F30 DK103359/DK/NIDDK NIH HHS/United States ; R03 DK109232/DK/NIDDK NIH HHS/United States ; DP2 HL137300/HL/NHLBI NIH HHS/United States ; R35 HG011324/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; Genetic Engineering/*methods ; Genome, Human ; Genomics/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Further information on research design is available in the Nature Research Reporting Summary linked to this article.},
}
@article {pmid30504872,
year = {2018},
author = {Ikeda, K and Uchida, N and Nishimura, T and White, J and Martin, RM and Nakauchi, H and Sebastiano, V and Weinberg, KI and Porteus, MH},
title = {Efficient scarless genome editing in human pluripotent stem cells.},
journal = {Nature methods},
volume = {15},
number = {12},
pages = {1045-1047},
doi = {10.1038/s41592-018-0212-y},
pmid = {30504872},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Embryonic Stem Cells/cytology/*metabolism ; *Gene Editing ; Gene Expression Regulation ; *Genome, Human ; *Homologous Recombination ; Humans ; Pluripotent Stem Cells/cytology/*metabolism ; RNA, Small Interfering/*genetics ; },
abstract = {Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.},
}
@article {pmid30504868,
year = {2018},
author = {Rusk, N},
title = {Recording transcriptional activity.},
journal = {Nature methods},
volume = {15},
number = {12},
pages = {999},
doi = {10.1038/s41592-018-0242-5},
pmid = {30504868},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; RNA/*analysis/*genetics ; *Transcription, Genetic ; },
}
@article {pmid30504842,
year = {2018},
author = {Rice, CM and Davies, LC and Subleski, JJ and Maio, N and Gonzalez-Cotto, M and Andrews, C and Patel, NL and Palmieri, EM and Weiss, JM and Lee, JM and Annunziata, CM and Rouault, TA and Durum, SK and McVicar, DW},
title = {Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {5099},
pmid = {30504842},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Flow Cytometry ; Immunoblotting ; Immunohistochemistry ; Mice ; Mice, Knockout ; Microscopy, Confocal ; Mitochondria/*metabolism ; Neutrophils/metabolism/*physiology ; Oxidation-Reduction ; Oxidative Phosphorylation ; Oxidative Stress ; Reactive Oxygen Species/metabolism ; Signal Transduction/physiology ; },
abstract = {Neutrophils are a vital component of immune protection, yet in cancer they may promote tumour progression, partly by generating reactive oxygen species (ROS) that disrupts lymphocyte functions. Metabolically, neutrophils are often discounted as purely glycolytic. Here we show that immature, c-Kit[+] neutrophils subsets can engage in oxidative mitochondrial metabolism. With limited glucose supply, oxidative neutrophils use mitochondrial fatty acid oxidation to support NADPH oxidase-dependent ROS production. In 4T1 tumour-bearing mice, mitochondrial fitness is enhanced in splenic neutrophils and is driven by c-Kit signalling. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and T cell suppression when glucose utilisation is restricted. Consistent with these findings, peripheral blood neutrophils from patients with cancer also display increased immaturity, mitochondrial content and oxidative phosphorylation. Together, our data suggest that the glucose-restricted tumour microenvironment induces metabolically adapted, oxidative neutrophils to maintain local immune suppression.},
}
@article {pmid30504793,
year = {2018},
author = {Zuckermann, M and Hlevnjak, M and Yazdanparast, H and Zapatka, M and Jones, DTW and Lichter, P and Gronych, J},
title = {A novel cloning strategy for one-step assembly of multiplex CRISPR vectors.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {17499},
pmid = {30504793},
issn = {2045-2322},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Cloning, Molecular/methods ; *Gene Editing ; Gene Order ; Genes, Reporter ; Genetic Vectors/*genetics ; Humans ; RNA, Guide/genetics ; Sequence Analysis, DNA ; Workflow ; },
abstract = {One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.},
}
@article {pmid30504723,
year = {2018},
author = {Xiao, Z and Wan, J and Nur, AA and Dou, P and Mankin, H and Liu, T and Ouyang, Z},
title = {Targeting CD44 by CRISPR-Cas9 in Multi-Drug Resistant Osteosarcoma Cells.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {51},
number = {4},
pages = {1879-1893},
doi = {10.1159/000495714},
pmid = {30504723},
issn = {1421-9778},
mesh = {Adolescent ; Adult ; Antibiotics, Antineoplastic/pharmacology/therapeutic use ; Bone Neoplasms/drug therapy/*genetics/pathology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Child ; Child, Preschool ; Doxorubicin/pharmacology/therapeutic use ; *Drug Resistance, Neoplasm ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Hyaluronan Receptors/*genetics ; Infant ; Male ; Neoplasm Invasiveness/genetics/pathology/prevention &amp; control ; Osteosarcoma/drug therapy/*genetics/pathology ; Young Adult ; },
abstract = {BACKGROUND/AIMS: Drug resistance is the main difficulty for the current treatment for osteosarcoma. Cluster of differentiation 44 (CD44) is a receptor for hyaluronic acid (HA) and HA-binding has been proven to participate in various biological tumor activities, including tumor progression, metastasis and drug resistance. In this study, we aimed to determine the effects of CD44 on migration, invasion, proliferation, and the drug-sensitivity of osteosarcoma.<br><br>METHODS: 96 human osteosarcoma tissues from 56 patients were collected to evaluate the expression of CD44 in osteosarcoma tissue by immunohistochemistry. CRISPR-Cas9 system was used to specifically silence CD44 in drug-resistant cell lines (KHOSR2 and U-2OSR2). The migration and invasion activities of cells was demonstrated by wound healing and transwell invasion assay. The proliferation speed of the cells was detected under 3D cell culture condition. Drug resistance of cells was detected by MTT and drug uptake assay.<br><br>RESULTS: The immunohistochemistry results demonstrated that a high level of CD44 may predict poor survival and higher potential of metastasis, recurrence and drug resistance in patients with osteosarcoma. After knocking-out of CD44 by the CRISPR-Cas9 system, not only the migration and invasion activities of osteosarcoma cells were significantly inhibited, but the drug sensitivity was also enhanced.<br><br>CONCLUSION: CD44 silencing could inhibit the development of osteosarcoma migration, invasion, proliferation and ameliorate drug resistance to current treatment in osteosarcoma. This study applies new strategy to target CD44, which may improve the prognosis of osteosarcoma.},
}
@article {pmid30504366,
year = {2019},
author = {Poe, AR and Wang, B and Sapar, ML and Ji, H and Li, K and Onabajo, T and Fazliyeva, R and Gibbs, M and Qiu, Y and Hu, Y and Han, C},
title = {Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance in Drosophila.},
journal = {Genetics},
volume = {211},
number = {2},
pages = {459-472},
pmid = {30504366},
issn = {1943-2631},
support = {R01 NS099125/NS/NINDS NIH HHS/United States ; R21 OD023824/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila/genetics ; Epidermis/metabolism ; *Gene Dosage ; *Genetic Techniques ; Loss of Function Mutation ; *Mutagenesis ; Neurons/metabolism ; Organ Specificity ; RNA, Guide/genetics ; Transgenes ; },
abstract = {Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes.},
}
@article {pmid30504364,
year = {2019},
author = {Farboud, B and Severson, AF and Meyer, BJ},
title = {Strategies for Efficient Genome Editing Using CRISPR-Cas9.},
journal = {Genetics},
volume = {211},
number = {2},
pages = {431-457},
pmid = {30504364},
issn = {1943-2631},
support = {/HHMI/Howard Hughes Medical Institute/United States ; P40 OD010440/OD/NIH HHS/United States ; R01 GM030702/GM/NIGMS NIH HHS/United States ; R15 GM117548/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Gene Editing/*methods ; },
abstract = {The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and nonmodel organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-strand breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at a considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. We found a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9-targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.},
}
@article {pmid30504134,
year = {2019},
author = {Ge, XA and Hunter, CP},
title = {Efficient Homologous Recombination in Mice Using Long Single Stranded DNA and CRISPR Cas9 Nickase.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {1},
pages = {281-286},
pmid = {30504134},
issn = {2160-1836},
support = {U19 CA179513/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; DNA End-Joining Repair/genetics ; DNA Repair/genetics ; DNA, Single-Stranded/genetics ; Deoxyribonuclease I/*genetics ; Heterozygote ; Homologous Recombination/*genetics ; Mice ; Mutation/genetics ; RNA, Guide/*genetics ; },
abstract = {The CRISPR/Cas9 nickase mutant is less prone to off-target double-strand (ds)DNA breaks than wild-type Cas9 because to produce dsDNA cleavage it requires two guide RNAs to target the nickase to nearby opposing strands. Like wild-type Cas9 lesions, these staggered lesions are repaired by either non-homologous end joining or, if a repair template is provided, by homologous recombination (HR). Here, we report very efficient (up to 100%) recovery of heterozygous insertions in Mus musculus produced by long (>300 nt), single-stranded DNA donor template-guided repair of paired-nickase lesions.},
}
@article {pmid30503774,
year = {2019},
author = {Wang, L and Mo, CY and Wasserman, MR and Rostøl, JT and Marraffini, LA and Liu, S},
title = {Dynamics of Cas10 Govern Discrimination between Self and Non-self in Type III CRISPR-Cas Immunity.},
journal = {Molecular cell},
volume = {73},
number = {2},
pages = {278-290.e4},
pmid = {30503774},
issn = {1097-4164},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; F32 GM128271/GM/NIGMS NIH HHS/United States ; R00 GM107365/GM/NIGMS NIH HHS/United States ; },
mesh = {*Autoimmunity ; Bacterial Proteins/genetics/*immunology/metabolism ; CRISPR-Associated Proteins/genetics/*immunology/metabolism ; CRISPR-Cas Systems/genetics/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/*immunology ; Escherichia coli/enzymology/genetics/immunology ; Kinetics ; Microscopy, Fluorescence ; Mutation ; Nucleic Acid Conformation ; Protein Conformation ; RNA, Bacterial/chemistry/genetics/*immunology/metabolism ; *Self Tolerance ; Signal Transduction ; Single Molecule Imaging/methods ; Staphylococcus aureus/enzymology/genetics/immunology ; Staphylococcus epidermidis/enzymology/genetics/immunology ; Structure-Activity Relationship ; },
abstract = {Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.},
}
@article {pmid30503773,
year = {2019},
author = {Jia, N and Mo, CY and Wang, C and Eng, ET and Marraffini, LA and Patel, DJ},
title = {Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity.},
journal = {Molecular cell},
volume = {73},
number = {2},
pages = {264-277.e5},
pmid = {30503773},
issn = {1097-4164},
support = {S10 RR029205/RR/NCRR NIH HHS/United States ; P41 GM103310/GM/NIGMS NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; F32 GM128271/GM/NIGMS NIH HHS/United States ; R01 GM104962/GM/NIGMS NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; DP1 GM128184/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; U19 CA179564/CA/NCI NIH HHS/United States ; },
mesh = {*Autoimmunity ; Bacterial Proteins/genetics/immunology/*metabolism/ultrastructure ; CRISPR-Associated Proteins/genetics/immunology/*metabolism/ultrastructure ; *CRISPR-Cas Systems/genetics/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics/immunology ; Cryoelectron Microscopy ; Deoxyribonucleases/genetics/immunology/*metabolism/ultrastructure ; Escherichia coli/enzymology/genetics/immunology ; Gene Expression Regulation, Bacterial ; Models, Molecular ; Multiprotein Complexes ; Mutation ; Nucleic Acid Conformation ; Protein Conformation ; *RNA Stability ; RNA, Bacterial/genetics/immunology/*metabolism/ultrastructure ; RNA-Binding Proteins/genetics/immunology/*metabolism/ultrastructure ; Structure-Activity Relationship ; Thermococcus/enzymology/genetics/immunology ; },
abstract = {Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus Csm[crRNA] binary, Csm[crRNA]-target RNA and Csm[crRNA]-target RNA[anti-tag] ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of Csm[crRNA] adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into Csm[crRNA] complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.},
}
@article {pmid30503261,
year = {2018},
author = {Ma, S and Viola, R and Sui, L and Cherubini, V and Barbetti, F and Egli, D},
title = {β Cell Replacement after Gene Editing of a Neonatal Diabetes-Causing Mutation at the Insulin Locus.},
journal = {Stem cell reports},
volume = {11},
number = {6},
pages = {1407-1415},
pmid = {30503261},
issn = {2213-6711},
mesh = {Base Sequence ; Blood Glucose/metabolism ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Codon, Initiator/genetics ; Diabetes Mellitus/*genetics ; *Gene Editing ; *Genetic Loci ; Homeostasis ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Infant, Newborn ; Insulin/*genetics ; Insulin-Secreting Cells/*metabolism ; Mutation/*genetics ; RNA Stability/genetics ; },
abstract = {Permanent neonatal diabetes mellitus (PNDM) can be caused by insulin mutations. We generated induced pluripotent stem cells from fibroblasts of a patient with PNDM and undetectable insulin at birth due to a homozygous mutation in the translation start site of the insulin gene. Differentiation of mutant cells resulted in insulin-negative endocrine stem cells expressing MAFA, NKX6.1, and chromogranin A. Correction of the mutation in stem cells and differentiation to pancreatic endocrine cells restored insulin production and insulin secretion to levels comparable to those of wild-type cells. Grafting of corrected cells into mice, followed by ablating mouse β cells using streptozotocin, resulted in normal glucose homeostasis, including at night, and the stem cell-derived grafts adapted insulin secretion to metabolic changes. Our study provides proof of principle for the generation of genetically corrected cells autologous to a patient with non-autoimmune insulin-dependent diabetes. These cases should be readily amenable to autologous cell therapy.},
}
@article {pmid30503210,
year = {2019},
author = {You, L and Ma, J and Wang, J and Artamonova, D and Wang, M and Liu, L and Xiang, H and Severinov, K and Zhang, X and Wang, Y},
title = {Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.},
journal = {Cell},
volume = {176},
number = {1-2},
pages = {239-253.e16},
pmid = {30503210},
issn = {1097-4172},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins ; CRISPR-Associated Proteins/chemistry/*ultrastructure ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; DNA/chemistry ; Deoxyribonucleases/metabolism ; Endoribonucleases/metabolism ; Models, Molecular ; RNA/chemistry ; RNA, Bacterial/chemistry ; RNA, Guide/chemistry ; Ribonucleases/metabolism ; },
abstract = {Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.},
}
@article {pmid30503205,
year = {2018},
author = {Stella, S and Mesa, P and Thomsen, J and Paul, B and Alcón, P and Jensen, SB and Saligram, B and Moses, ME and Hatzakis, NS and Montoya, G},
title = {Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.},
journal = {Cell},
volume = {175},
number = {7},
pages = {1856-1871.e21},
doi = {10.1016/j.cell.2018.10.045},
pmid = {30503205},
issn = {1097-4172},
mesh = {Bacterial Proteins/*chemistry/genetics ; *CRISPR-Cas Systems ; Catalysis ; *DNA Cleavage ; DNA, Single-Stranded/*chemistry/genetics ; Francisella/*chemistry/genetics ; Gene Editing ; RNA, Guide/*chemistry/genetics ; },
abstract = {Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.},
}
@article {pmid30502398,
year = {2019},
author = {Montiel-Gonzalez, MF and Diaz Quiroz, JF and Rosenthal, JJC},
title = {Current strategies for Site-Directed RNA Editing using ADARs.},
journal = {Methods (San Diego, Calif.)},
volume = {156},
number = {},
pages = {16-24},
pmid = {30502398},
issn = {1095-9130},
support = {R01 NS087726/NS/NINDS NIH HHS/United States ; },
mesh = {Adenosine/metabolism ; Adenosine Deaminase/*genetics/metabolism ; Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Genome, Human ; Humans ; Inosine/metabolism ; Mutagenesis, Site-Directed/*methods ; Oligoribonucleotides, Antisense/genetics/metabolism ; Protein Domains ; *RNA Editing ; RNA, Guide/genetics/metabolism ; RNA, Messenger/*genetics/metabolism ; RNA-Binding Proteins/*genetics/metabolism ; },
abstract = {Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I's base pair like guanosines (G's). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA. The main challenge in doing so is to re-direct ADAR's catalytic activity towards A's that are not naturally edited, a process termed Site-Directed RNA Editing (SDRE). Recently, a handful of labs have taken up this challenge and two basic strategies have emerged. The first involves redirecting endogenous ADAR to new sites by making editable structures using antisense RNA oligonucleotides. The second also utilizes antisense RNA oligonucleotides, but it uses them as guides to deliver the catalytic domain of engineered ADARs to new sites, much as CRISPR guides deliver Cas nucleases. In fact, despite the intense current focus on CRISPR-Cas9 genome editing, SDRE offers a number of distinct advantages. In the present review we will discuss these strategies in greater detail, focusing on the concepts on which they are based, how they were developed and tested, and their respective advantages and disadvantages. Though the precise and efficient re-direction of ADAR activity still remains a challenge, the systems that are being developed lay the foundation for SDRE as a powerful tool for transient genome editing.},
}
@article {pmid30502320,
year = {2019},
author = {Wang, M and Sintim, HO},
title = {Discriminating cyclic from linear nucleotides - CRISPR/Cas-related cyclic hexaadenosine monophosphate as a case study.},
journal = {Analytical biochemistry},
volume = {567},
number = {},
pages = {21-26},
doi = {10.1016/j.ab.2018.11.022},
pmid = {30502320},
issn = {1096-0309},
mesh = {CRISPR-Cas Systems/*genetics ; Circular Dichroism ; Fluorescent Dyes/chemistry ; Gene Editing/methods ; Hydrogen-Ion Concentration ; Nucleic Acid Conformation ; Oligonucleotides/chemistry/*metabolism ; },
abstract = {Nucleic acids exist in biological systems as linear and cyclic forms and in most cases the biology of the cyclic form is different from the linear form of exactly the same sequence. Case examples are cyclic nucleotides, second messengers in both prokaryotes and eukaryotes whereby the cyclic forms account for their interesting biological profiles and the actions of the cyclic nucleotides are terminated upon phosphodiesterase hydrolysis into linear forms. For mono and dinucleotides, it has been shown that vast conformational changes that accompany the hydrolysis of the cyclized form allow for discrimination between the cyclized and linear forms. As the ring size increases, it becomes difficult to use conformational or structural differences alone to discriminate between cyclic and linear nucleotides. Here we reveal that for the recently discovered CRISPR/Cas-related cyclic hexaadenosine monophosphate, it is possible to discriminate between the cyclized and linear forms. The structures of c-HexaAMP and linear form are different in acidic media and this structural difference facilitated a simple and practical detection platform for this interesting and new bacterial immunity-related molecule, and we also demonstrate that it is possible to distinguish between linear and cyclized polynucleotides using simple spectroscopic techniques, such as CD and fluorescence-based methods.},
}
@article {pmid30501735,
year = {2018},
author = {Li, WY and Gao, QP and Liu, H},
title = {[CRISPR/Cas9 System and Its Advances in Gene Therapy of Hematologic Diseases--Review].},
journal = {Zhongguo shi yan xue ye xue za zhi},
volume = {26},
number = {6},
pages = {1863-1867},
doi = {10.7534/j.issn.1009-2137.2018.06.048},
pmid = {30501735},
issn = {1009-2137},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Hematologic Diseases/*therapy ; Humans ; Molecular Biology ; },
abstract = {In recent years, with the development of gene editing technology, the site-specific genome can be modified. The curability of genetic disease may be achieved by the use of gene editing techniques. As the simplicity, high specificity and economical efficiency, much attention has been paid to the CRISPR/Cas9 system, which was been widely used in research of molecular biology and other fields of life science. In this review, the mechanism for CR1SPR/Cas9 system and the progress of gene therapy, such as for hemophilia, beta(?)thalassaemia and chronic myeloid leukemia were summarized briefly.},
}
@article {pmid30501614,
year = {2018},
author = {Zhang, Y and Massel, K and Godwin, ID and Gao, C},
title = {Applications and potential of genome editing in crop improvement.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {210},
pmid = {30501614},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Gene Editing ; *Plant Breeding ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; },
abstract = {Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism's genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.},
}
@article {pmid30501172,
year = {2019},
author = {Marreddy, RKR and Wu, X and Sapkota, M and Prior, AM and Jones, JA and Sun, D and Hevener, KE and Hurdle, JG},
title = {The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection.},
journal = {ACS infectious diseases},
volume = {5},
number = {2},
pages = {208-217},
pmid = {30501172},
issn = {2373-8227},
support = {P20 GM103466/GM/NIGMS NIH HHS/United States ; R21 AI126755/AI/NIAID NIH HHS/United States ; },
mesh = {Anti-Bacterial Agents/*pharmacology ; Biosynthetic Pathways ; CRISPR-Cas Systems ; Clostridioides difficile/*drug effects/enzymology/genetics ; Crystallography, X-Ray ; DNA, Antisense ; Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/*antagonists &amp; inhibitors/genetics ; Fatty Acids/biosynthesis ; Gene Silencing ; },
abstract = {Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein (ACP) reductase II (FabK), which catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed that C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using ClosTron mutagenesis and by growth inhibition upon gene silencing with CRISPR interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazole-derived inhibitor (1). The inability of fatty acids to bypass FASII inhibition is likely due to the function of the transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti- C. difficile drugs that treat CDI but avoid collateral damage to the gut microbiota.},
}
@article {pmid30517747,
year = {2019},
author = {Gorter de Vries, AR and Couwenberg, LGF and van den Broek, M and de la Torre Cortés, P and Ter Horst, J and Pronk, JT and Daran, JG},
title = {Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.},
journal = {Nucleic acids research},
volume = {47},
number = {3},
pages = {1362-1372},
pmid = {30517747},
issn = {1362-4962},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Cell Proliferation/genetics ; DNA Breaks, Double-Stranded ; *Diploidy ; *Gene Editing ; Heterozygote ; Homozygote ; Humans ; Loss of Heterozygosity/*genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.},
}
@article {pmid30517736,
year = {2019},
author = {O'Reilly, D and Kartje, ZJ and Ageely, EA and Malek-Adamian, E and Habibian, M and Schofield, A and Barkau, CL and Rohilla, KJ and DeRossett, LB and Weigle, AT and Damha, MJ and Gagnon, KT},
title = {Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity.},
journal = {Nucleic acids research},
volume = {47},
number = {2},
pages = {546-558},
pmid = {30517736},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; DNA Cleavage ; DNA, A-Form/chemistry ; RNA, Bacterial/*chemistry/metabolism ; Ribonucleoproteins/metabolism ; Streptococcus pyogenes/enzymology/genetics ; Structure-Activity Relationship ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.},
}
@article {pmid30517145,
year = {2018},
author = {Park, S and Gilmour, SJ and Grumet, R and Thomashow, MF},
title = {CBF-dependent and CBF-independent regulatory pathways contribute to the differences in freezing tolerance and cold-regulated gene expression of two Arabidopsis ecotypes locally adapted to sites in Sweden and Italy.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0207723},
pmid = {30517145},
issn = {1932-6203},
mesh = {Acclimatization/genetics/physiology ; Alleles ; Arabidopsis/*genetics/growth &amp; development/*physiology ; Arabidopsis Proteins/*genetics/*physiology ; CRISPR-Cas Systems ; Ecotype ; Flowers/genetics/growth &amp; development ; Freezing ; Gene Expression Regulation, Plant ; Gene Regulatory Networks ; Genes, Plant ; Italy ; Mutagenesis ; Plants, Genetically Modified ; Regulon ; Sweden ; Trans-Activators/*genetics/*physiology ; Transcription Factors/genetics/physiology ; },
abstract = {Arabidopsis thaliana (Arabidopsis) increases in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. The CBF regulatory pathway, which contributes to cold acclimation, includes three genes-CBF1, CBF2 and CBF3-encoding closely-related transcription factors that regulate the expression of more than 100 genes-the CBF regulon-that impart freezing tolerance. Here we compare the CBF pathways of two Arabidopsis ecotypes collected from sites in Sweden (SW) and Italy (IT). Previous studies showed that the SW ecotype was more freezing tolerant than the IT ecotype and that the IT ecotype had a nonfunctional CBF2 gene. Here we present results establishing that the difference in CBF2 alleles contributes to the difference in freezing tolerance between the two ecotypes. However, other differences in the CBF pathway as well as CBF-independent pathways contribute the large majority of the difference in freezing tolerance between the two ecotypes. The results also provided evidence that most cold-induced CBF regulon genes in both the SW and IT ecotypes are coregulated by CBF-independent pathways. Additional analysis comparing our results with those published by others examining the Col-0 accession resulted in the identification of 44 CBF regulon genes that were conserved among the three accessions suggesting that they likely have important functions in life at low temperature. The comparison further supported the conclusion that the CBF pathway can account for a large portion of the increase in freezing tolerance that occurs with cold acclimation in a given accession, but that CBF-independent pathways can also make a major contribution.},
}
@article {pmid30517136,
year = {2018},
author = {Boonyalai, N and Collins, CR and Hackett, F and Withers-Martinez, C and Blackman, MJ},
title = {Essentiality of Plasmodium falciparum plasmepsin V.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0207621},
pmid = {30517136},
issn = {1932-6203},
support = {FC001043/MRC_/Medical Research Council/United Kingdom ; FC001043/CRUK_/Cancer Research UK/United Kingdom ; FC001043/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Aspartic Acid Endopeptidases/*genetics/metabolism/*physiology ; CRISPR-Cas Systems ; Erythrocytes/metabolism/parasitology ; Gene Expression Regulation ; Humans ; Mutation/genetics ; Plasmodium falciparum/genetics/*metabolism ; Protease Inhibitors ; Protein Processing, Post-Translational ; Protozoan Proteins/metabolism ; },
abstract = {The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested early in the ring-to-trophozoite transition in the erythrocytic cycle following gene excision. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway.},
}
@article {pmid30517011,
year = {2019},
author = {Li, Z and Zhou, X and Wei, M and Gao, X and Zhao, L and Shi, R and Sun, W and Duan, Y and Yang, G and Yuan, L},
title = {In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9.},
journal = {Nano letters},
volume = {19},
number = {1},
pages = {19-28},
doi = {10.1021/acs.nanolett.8b02689},
pmid = {30517011},
issn = {1530-6992},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; ELAV-Like Protein 1/*chemistry/genetics ; Exosomes/*chemistry/genetics ; Gene Transfer Techniques ; Humans ; Mice ; MicroRNAs/*chemistry/genetics ; RNA, Small Interfering/chemistry/genetics ; RNA-Binding Proteins/chemistry/genetics ; Tetraspanin 29/*chemistry/genetics ; },
abstract = {In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as a kind of rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNA of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacting with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriched miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognized the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for enhanced RNA cargo encapsulation into engineered exosomes, which in turn functions in the recipient cells.},
}
@article {pmid30515147,
year = {2018},
author = {Lima, ARJ and Siqueira, AS and de Vasconcelos, JM and Pereira, JS and de Azevedo, JSN and Moraes, PHG and Aguiar, DCF and de Lima, CPS and Vianez-Júnior, JLSG and Nunes, MRT and Xavier, LP and Dall'Agnol, LT and Goncalves, EC},
title = {Insights Into Limnothrix sp. Metabolism Based on Comparative Genomics.},
journal = {Frontiers in microbiology},
volume = {9},
number = {},
pages = {2811},
pmid = {30515147},
issn = {1664-302X},
abstract = {Currently only four genome sequences for Limnothrix spp. are publicly available, and information on the genetic properties of cyanobacteria belonging to this genus is limited. In this study, we report the draft genome of Limnothrix sp. CACIAM 69d, isolated from the reservoir of a hydroelectric dam located in the Amazon ecosystem, from where cyanobacterial genomic data are still scarce. Comparative genomic analysis of Limnothrix revealed the presence of key enzymes in the cyanobacterial central carbon metabolism and how it is well equipped for environmental sulfur and nitrogen acquisition. Additionally, this work covered the analysis of Limnothrix CRISPR-Cas systems, pathways related to biosynthesis of secondary metabolites and assembly of extracellular polymeric substances and their exportation. A trans-AT PKS gene cluster was identified in two strains, possibly related to the novel toxin Limnothrixin biosynthesis. Overall, the draft genome of Limnothrix sp. CACIAM 69d adds new data to the small Limnothrix genome library and contributes to a growing representativeness of cyanobacterial genomes from the Amazon region. The comparative genomic analysis of Limnothrix made it possible to highlight unique genes for each strain and understand the overall features of their metabolism.},
}
@article {pmid30514786,
year = {2018},
author = {Lee, J and Mir, A and Edraki, A and Garcia, B and Amrani, N and Lou, HE and Gainetdinov, I and Pawluk, A and Ibraheim, R and Gao, XD and Liu, P and Davidson, AR and Maxwell, KL and Sontheimer, EJ},
title = {Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.},
journal = {mBio},
volume = {9},
number = {6},
pages = {},
pmid = {30514786},
issn = {2150-7511},
support = {R01 GM125797/GM/NIGMS NIH HHS/United States ; FDN-15427//CIHR/Canada ; PJT-152918//CIHR/Canada ; },
mesh = {Bacteriophages/*chemistry ; CRISPR-Associated Protein 9/*antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; HEK293 Cells ; Haemophilus parainfluenzae/virology ; Humans ; Neisseriaceae/virology ; Viral Proteins/genetics/*metabolism ; },
abstract = {In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.},
}
@article {pmid30514784,
year = {2018},
author = {Musharova, O and Vyhovskyi, D and Medvedeva, S and Guzina, J and Zhitnyuk, Y and Djordjevic, M and Severinov, K and Savitskaya, E},
title = {Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation.},
journal = {mBio},
volume = {9},
number = {6},
pages = {},
pmid = {30514784},
issn = {2150-7511},
support = {R01 GM104071/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Bacterial/genetics ; DNA, Intergenic/*genetics ; Escherichia coli/*genetics ; },
abstract = {CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a "directionality" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.},
}
@article {pmid30513774,
year = {2018},
author = {Cai, Y and Chen, L and Sun, S and Wu, C and Yao, W and Jiang, B and Han, T and Hou, W},
title = {CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean.},
journal = {International journal of molecular sciences},
volume = {19},
number = {12},
pages = {},
pmid = {30513774},
issn = {1422-0067},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Genes, Plant ; *Genome, Plant ; Homozygote ; Inheritance Patterns/genetics ; Mutagenesis, Site-Directed ; Mutation/genetics ; Mutation Rate ; Phenotype ; Sequence Deletion ; Soybeans/*genetics ; },
abstract = {At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 'transgene-free' homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.},
}
@article {pmid30513694,
year = {2018},
author = {Bailey, CG and Metierre, C and Feng, Y and Baidya, K and Filippova, GN and Loukinov, DI and Lobanenkov, VV and Semaan, C and Rasko, JE},
title = {CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer.},
journal = {International journal of molecular sciences},
volume = {19},
number = {12},
pages = {},
pmid = {30513694},
issn = {1422-0067},
mesh = {Animals ; CCCTC-Binding Factor/*genetics/*metabolism ; CRISPR-Cas Systems ; Cell Proliferation/genetics/physiology ; Cell Survival/genetics/*physiology ; Endometrial Neoplasms/*genetics ; Female ; *Gene Editing ; Haploinsufficiency/genetics/physiology ; Humans ; K562 Cells ; Mice ; RNA, Small Interfering/genetics ; },
abstract = {CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf[+/-]) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf[+/-] MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf[+/-] MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF's role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.},
}
@article {pmid30513349,
year = {2019},
author = {Amann, T and Hansen, AH and Kol, S and Hansen, HG and Arnsdorf, J and Nallapareddy, S and Voldborg, B and Lee, GM and Andersen, MR and Kildegaard, HF},
title = {Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.},
journal = {Metabolic engineering},
volume = {52},
number = {},
pages = {143-152},
doi = {10.1016/j.ymben.2018.11.014},
pmid = {30513349},
issn = {1096-7184},
mesh = {Animals ; Antigens, CD/biosynthesis/genetics ; CHO Cells/*metabolism ; CRISPR-Cas Systems ; Complement C1 Inhibitor Protein/*biosynthesis ; Cricetinae ; Cricetulus ; Glycosylation ; Humans ; Metabolic Engineering/*methods ; Recombinant Proteins/biosynthesis ; Sialyltransferases/biosynthesis/genetics ; alpha 1-Antitrypsin/*biosynthesis ; },
abstract = {Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin[®]-C and Cinryze[®], respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.},
}
@article {pmid30513193,
year = {2018},
author = {Lainšček, D and Kadunc, L and Keber, MM and Bratkovič, IH and Romih, R and Jerala, R},
title = {Delivery of an Artificial Transcription Regulator dCas9-VPR by Extracellular Vesicles for Therapeutic Gene Activation.},
journal = {ACS synthetic biology},
volume = {7},
number = {12},
pages = {2715-2725},
doi = {10.1021/acssynbio.8b00192},
pmid = {30513193},
issn = {2161-5063},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chemical and Drug Induced Liver Injury/pathology/therapy ; Disease Models, Animal ; Extracellular Vesicles/genetics/*metabolism/transplantation ; Gene Editing/*methods ; Gene Expression Regulation ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Hepatocyte Growth Factor/genetics ; Humans ; Liver/metabolism/pathology ; Mice ; Mice, Inbred BALB C ; Plasmids/genetics/metabolism ; },
abstract = {The CRISPR/Cas system has been developed as a potent tool for genome engineering and transcription regulation. However, the efficiency of the delivery of the system into cells, particularly for therapeutic in vivo applications, remains a major bottleneck. Extracellular vesicles (EVs), released by eukaryotic cells, can mediate the transfer of various molecules, including nucleic acids and proteins. We show the packaging and delivery of the CRISPR/Cas system via EVs to the target cells, combining the advantages of both technological platforms. A genome editing with designed extracellular vesicles (GEDEX) system generated by the producer cells can transfer the designed transcriptional regulator dCas9-VPR complexed with appropriate targeting gRNAs enabling activation of gene transcription. We show functional delivery in mammalian cells as well in the animals. The therapeutic efficiency of in vivo delivery of dCas9-VPR/sgRNA GEDEX is demonstrated in a mouse model of liver damage counteracted by upregulation of the endogenous hepatocyte growth factor, demonstrating the potential for therapeutic applications.},
}
@article {pmid30511631,
year = {2018},
author = {Farooq, R and Hussain, K and Nazir, S and Javed, MR and Masood, N},
title = {CRISPR/Cas9; A robust technology for producing genetically engineered plants.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {64},
number = {14},
pages = {31-38},
pmid = {30511631},
issn = {1165-158X},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Genetic Engineering/*methods ; Mutation/genetics ; Plants, Genetically Modified/*genetics ; Transgenes ; },
abstract = {CRISPR/Cas9 is a technology evolved from modified type II immune system of bacteria and archaea. Exploitation of this bacterial immune system in all eukaryotes including plants may lead to site-specific targeted genome engineering. Genome engineering is objectively utilized to express/silence a trait harbouring gene in the plant genome. In this review, different genetic engineering techniques including classical breeding, RNAi and genetic transformation and synthetic sequence-specific nucleases (zinc finger nucleases; ZFNs and transcription activator-like effector nuclease; TALENs) techniques have been described and compared with advanced genome editing technique CRISPR/Cas9, on the basis of their merits and drawbacks. This revolutionary genome engineering technology has edge over all other approaches because of its simplicity, stability, specificity of the target and multiple genes can be engineered at a time. CRISPR/Cas9 requires only Cas9 endonuclease and single guide RNA, which are directly delivered into plant cells via either vector-mediated stable transformation or transient delivery of ribonucleoproteins (RNPs) and generate double-strand breaks (DSBs) at target site. These DSBs are further repaired by cell endogenous repairing pathways via HDR or NHEJ. The major advantage of CRISPR/Cas9 system is that engineered plants are considered Non-GM; can be achieved using in vitro expressed RNPs transient delivery. Different variants of Cas9 genes cloned in different plasmid vectors can be used to achieve different objectives of genome editing including double-stranded DNA break, single-stranded break, activate/repress the gene expression. Fusion of Cas9 with fluorescent protein can lead to visualize the expression of the CRISPR/Cas9 system. The applications of this technology in plant genome editing to improve different plant traits are comprehensively described.},
}
@article {pmid30511203,
year = {2019},
author = {Zhu, W and Saw, D and Weiss, M and Sun, Z and Wei, M and Shaligram, S and Wang, S and Su, H},
title = {Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice.},
journal = {Translational stroke research},
volume = {10},
number = {5},
pages = {557-565},
pmid = {30511203},
issn = {1868-601X},
support = {R01 NS027713/NS/NINDS NIH HHS/United States ; R01 HL122774/HL/NHLBI NIH HHS/United States ; },
mesh = {Activin Receptors, Type II/*genetics ; Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Endothelial Cells/metabolism ; Female ; Gene Expression ; Genetic Vectors ; Intracranial Arteriovenous Malformations/*genetics ; Male ; Mice, Inbred C57BL ; Mutation ; },
abstract = {Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.},
}
@article {pmid30510770,
year = {2018},
author = {Teng, F and Cui, T and Feng, G and Guo, L and Xu, K and Gao, Q and Li, T and Li, J and Zhou, Q and Li, W},
title = {Repurposing CRISPR-Cas12b for mammalian genome engineering.},
journal = {Cell discovery},
volume = {4},
number = {},
pages = {63},
pmid = {30510770},
issn = {2056-5968},
abstract = {The prokaryotic CRISPR-Cas adaptive immune systems provide valuable resources to develop genome editing tools, such as CRISPR-Cas9 and CRISPR-Cas12a/Cpf1. Recently, CRISPR-Cas12b/C2c1, a distinct type V-B system, has been characterized as a dual-RNA-guided DNA endonuclease system. Though being active in vitro, its cleavage activity at endogenous genome remains to be explored. Furthermore, the optimal cleavage temperature of the reported Cas12b orthologs is higher than 40 °C, which is unsuitable for mammalian applications. Here, we report the identification of a Cas12b system from the Alicyclobacillus acidiphilus (AaCas12b), which maintains optimal nuclease activity over a wide temperature range (31 °C-59 °C). AaCas12b can be repurposed to engineer mammalian genomes for versatile applications, including single and multiplex genome editing, gene activation, and generation of gene mutant mouse models. Moreover, whole-genome sequencing reveals high specificity and minimal off-target effects of AaCas12b-meditated genome editing. Our findings establish CRISPR-Cas12b as a versatile tool for mammalian genome engineering.},
}
@article {pmid30510202,
year = {2018},
author = {Møller-Olsen, C and Ho, SFS and Shukla, RD and Feher, T and Sagona, AP},
title = {Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {17559},
pmid = {30510202},
issn = {2045-2322},
support = {BB/N011872/1//Biotechnology and Biological Sciences Research Council (BBSRC)/International ; },
mesh = {Cell Line ; Epithelial Cells/*microbiology/pathology ; Escherichia coli/*virology ; Escherichia coli Infections/*therapy ; Humans ; Phage Therapy/*methods ; Podoviridae/genetics/*growth &amp; development ; *Urinary Tract Infections/microbiology/therapy ; Urothelium/*microbiology ; },
abstract = {Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host and can be genetically modified for different applications. However, whether and how bacteriophages can kill intracellular bacteria in human cells remains elusive. Here, using CRISPR/Cas selection, we have engineered a fluorescent bacteriophage specific for E. coli K1, a nosocomial pathogen responsible for urinary tract infections, neonatal meningitis and sepsis. By confocal and live microscopy, we show that engineered bacteriophages K1F-GFP and E. coli EV36-RFP bacteria displaying the K1 capsule, enter human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP efficiently kills intracellular E. coli EV36-RFP in T24 human urinary bladder epithelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy.},
}
@article {pmid30510171,
year = {2019},
author = {Daly, RA and Roux, S and Borton, MA and Morgan, DM and Johnston, MD and Booker, AE and Hoyt, DW and Meulia, T and Wolfe, RA and Hanson, AJ and Mouser, PJ and Moore, JD and Wunch, K and Sullivan, MB and Wrighton, KC and Wilkins, MJ},
title = {Viruses control dominant bacteria colonizing the terrestrial deep biosphere after hydraulic fracturing.},
journal = {Nature microbiology},
volume = {4},
number = {2},
pages = {352-361},
doi = {10.1038/s41564-018-0312-6},
pmid = {30510171},
issn = {2058-5276},
mesh = {Bacteriophages/classification/genetics/*physiology ; Biodiversity ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Firmicutes/classification/genetics/*growth &amp; development/*virology ; Hydraulic Fracking ; Metagenome ; *Microbial Consortia/genetics ; Oil and Gas Fields/*microbiology/*virology ; Virus Activation ; },
abstract = {The deep terrestrial biosphere harbours a substantial fraction of Earth's biomass and remains understudied compared with other ecosystems. Deep biosphere life primarily consists of bacteria and archaea, yet knowledge of their co-occurring viruses is poor. Here, we temporally catalogued viral diversity from five deep terrestrial subsurface locations (hydraulically fractured wells), examined virus-host interaction dynamics and experimentally assessed metabolites from cell lysis to better understand viral roles in this ecosystem. We uncovered high viral diversity, rivalling that of peatland soil ecosystems, despite low host diversity. Many viral operational taxonomic units were predicted to infect Halanaerobium, the dominant microorganism in these ecosystems. Examination of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) spacers elucidated lineage-specific virus-host dynamics suggesting active in situ viral predation of Halanaerobium. These dynamics indicate repeated viral encounters and changing viral host range across temporally and geographically distinct shale formations. Laboratory experiments showed that prophage-induced Halanaerobium lysis releases intracellular metabolites that can sustain key fermentative metabolisms, supporting the persistence of microorganisms in this ecosystem. Together, these findings suggest that diverse and active viral populations play critical roles in driving strain-level microbial community development and resource turnover within this deep terrestrial subsurface ecosystem.},
}
@article {pmid30500879,
year = {2019},
author = {Sun, J and Liu, H and Liu, J and Cheng, S and Peng, Y and Zhang, Q and Yan, J and Liu, HJ and Chen, LL},
title = {CRISPR-Local: a local single-guide RNA (sgRNA) design tool for non-reference plant genomes.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {14},
pages = {2501-2503},
doi = {10.1093/bioinformatics/bty970},
pmid = {30500879},
issn = {1367-4811},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Plant ; RNA, Guide ; },
abstract = {SUMMARY: CRISPR-Local is a high-throughput local tool for designing single-guide RNAs (sgRNAs) in plants and other organisms that factors in genetic variation and is optimized to generate genome-wide sgRNAs. CRISPR-Local outperforms other sgRNA design tools in the following respects: (i) designing sgRNAs suitable for non-reference varieties; (ii) screening for sgRNAs that are capable of simultaneously targeting multiple genes; (iii) saving computational resources by avoiding repeated calculations from multiple submissions and (iv) running offline, with both command-line and graphical user interface modes and the ability to export multiple formats for further batch analysis or visualization. We have applied CRISPR-Local to 71 public plant genomes, using both CRISPR/Cas9 and CRISPR/cpf1 systems.<br><br>CRISPR-Local can be freely downloaded from http://crispr.hzau.edu.cn/CRISPR-Local/.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid30498995,
year = {2019},
author = {Malik, S and Oyaghire, S and Bahal, R},
title = {Applications of PNA-laden nanoparticles for hematological disorders.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {6},
pages = {1057-1065},
pmid = {30498995},
issn = {1420-9071},
mesh = {Animals ; CRISPR-Cas Systems ; DNA/*chemistry/genetics ; Gene Editing/methods ; Hematologic Diseases/genetics/*therapy ; Humans ; Molecular Structure ; Nanoparticles/*chemistry ; Nucleic Acid Conformation ; Peptide Nucleic Acids/*chemistry/genetics ; },
abstract = {Safe and efficient genome editing has been an unmitigated goal for biomedical researchers since its inception. The most prevalent strategy for gene editing is the use of engineered nucleases that induce DNA damage and take advantage of cellular DNA repair machinery. This includes meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems. However, the clinical viability of these nucleases is marred by their off-target cleavage activity (≥ 50% in RNA-guided endonucleases). In addition, in vivo applications of CRISPR require systemic administration of Cas9 protein, mRNA, or DNA, which presents a significant delivery challenge. The development of nucleic acid probes that can recognize specific double-stranded DNA (dsDNA) regions and activate endogenous DNA repair machinery holds great promise for gene editing applications. Triplex-forming oligonucleotides (TFOs), which were introduced more than 25 years ago, are among the most extensively studied oligomeric dsDNA-targeting agents. TFOs bind duplex DNA to create a distorted helical structure, which can stimulate DNA repair and the exchange of a nearby mutated region-otherwise leading to an undesired phenotype-for a short single-stranded donor DNA that contains the corrective nucleotide sequence. Recombination can be induced within several hundred base-pairs of the TFO binding site and has been shown to depend on triplex-induced initiation of the nucleotide excision repair pathway and engagement of the homology-dependent repair pathway. Since TFOs do not possess any direct nuclease activity, their off-target effects are minimal when compared to engineered nucleases. This review comprehensively covers the advances made in peptide nucleic acid-based TFOs for site-specific gene editing and their therapeutic applications.},
}
@article {pmid30498103,
year = {2018},
author = {Normile, D},
title = {Shock greets claim of CRISPR-edited babies.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6418},
pages = {978-979},
doi = {10.1126/science.362.6418.978},
pmid = {30498103},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; China ; Embryo, Mammalian ; Female ; Gene Editing/*ethics ; HIV Infections/genetics/*prevention &amp; control/transmission ; Humans ; Infectious Disease Transmission, Vertical/*prevention &amp; control ; Paternal Inheritance ; Receptors, CCR5/*genetics ; Twins ; },
}
@article {pmid30488760,
year = {2018},
author = {Mattapally, S and Pawlik, KM and Fast, VG and Zumaquero, E and Lund, FE and Randall, TD and Townes, TM and Zhang, J},
title = {Human Leukocyte Antigen Class I and II Knockout Human Induced Pluripotent Stem Cell-Derived Cells: Universal Donor for Cell Therapy.},
journal = {Journal of the American Heart Association},
volume = {7},
number = {23},
pages = {e010239},
pmid = {30488760},
issn = {2047-9980},
support = {R01 HL114120/HL/NHLBI NIH HHS/United States ; U01 HL134764/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Genes, MHC Class I/*genetics ; Genes, MHC Class II/*genetics ; Humans ; Induced Pluripotent Stem Cells/*transplantation ; Male ; Myocytes, Cardiac/transplantation ; Stem Cell Transplantation/*methods ; Transplantation, Homologous/methods ; },
abstract = {Background We aim to generate a line of "universal donor" human induced pluripotent stem cells (hi PSC s) that are nonimmunogenic and, therefore, can be used to derive cell products suitable for allogeneic transplantation. Methods and Results hi PSC s carrying knockout mutations for 2 key components (β2 microglobulin and class II major histocompatibility class transactivator) of major histocompatibility complexes I and II (ie, human leukocyte antigen [HLA] I/ II knockout hi PSC s) were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene-editing system and differentiated into cardiomyocytes. Pluripotency-gene expression and telomerase activity in wild-type (WT) and HLAI / II knockout hi PSC s, cardiomyocyte marker expression in WT and HLAI / II knockout hi PSC -derived cardiomyocytes, and assessments of electrophysiological properties (eg, conduction velocity, action-potential and calcium transient half-decay times, and calcium transient increase times) in spheroid-fusions composed of WT and HLAI / II knockout cardiomyocytes, were similar. However, the rates of T-cell activation before (≈21%) and after (≈24%) exposure to HLAI / II knockout hi PSC -derived cardiomyocytes were nearly indistinguishable and dramatically lower than after exposure to WT hi PSC -derived cardiomyocytes (≈75%), and when WT and HLAI / II knockout hi PSC -derived cardiomyocyte spheroids were cultured with human peripheral blood mononuclear cells, the WT hi PSC -derived cardiomyocyte spheroids were smaller and displayed contractile irregularities. Finally, expression of HLA -E and HLA -F was inhibited in HLAI / II knockout cardiomyocyte spheroids after coculture with human peripheral blood mononuclear cells, although HLA -G was not inhibited; these results are consistent with the essential role of class II major histocompatibility class transactivator in transcriptional activation of the HLA -E and HLA-F genes, but not the HLA -G gene. Expression of HLA -G is known to inhibit natural killer cell recognition and killing of cells that lack other HLAs. Conclusions HLAI / II knockout hi PSC s can be differentiated into cardiomyocytes that induce little or no activity in human immune cells and, consequently, are suitable for allogeneic transplantation.},
}
@article {pmid30488155,
year = {2019},
author = {Liu, X and Liu, H and Wang, M and Li, R and Zeng, J and Mo, D and Cong, P and Liu, X and Chen, Y and He, Z},
title = {Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs.},
journal = {Transgenic research},
volume = {28},
number = {1},
pages = {141-150},
pmid = {30488155},
issn = {1573-9368},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Binding Sites ; Breeding ; CRISPR-Cas Systems/*genetics ; Fibroblasts/metabolism ; Gene Expression Regulation, Developmental ; Insulin-Like Growth Factor II/*genetics ; Introns/genetics ; Meat ; Muscle Development/*genetics ; Repressor Proteins/*genetics ; Swine ; *Zinc Fingers ; },
abstract = {Insulin-like growth factor 2 (IGF2) plays an important role in the development of the foetus and in post-natal growth and development. A SNP within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 in skeletal muscle and major effects on muscle growth, heart size, and fat deposition. This favourable mutation is common in Western commercial pig populations, but is not present in most indigenous Chinese pig breeds. Here, we described the efficient disruption of the ZBED6 binding site motif in intron 3 of IGF2 by CRISPR/Cas9 in porcine embryonic fibroblasts (PEFs) from the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Disruption of the binding motif led to a drastic up-regulation of IGF2 expression in PEFs and enhanced myogenic potential and cell proliferation of PEFs. IGF2-edited pigs were then generated using somatic cell nuclear transfer. Enhanced muscle development was evident in one pig with biallelic deletion of the ZBED6 binding site motif, implying that the release of ZBED6 repression has a major effect on porcine muscle development. Our study confirmed the important effect of a mutation in the ZBED6 binding site motif on IGF2 expression and myogenesis, thus providing the basis for breeding a new line of Liang Guang Small Spotted pigs with improved lean meat percentage, a trait of great commercial value to pig producers.},
}
@article {pmid30488027,
year = {2018},
author = {Aikawa, C and Nakajima, S and Karimine, M and Nozawa, T and Minowa-Nozawa, A and Toh, H and Yamada, S and Nakagawa, I},
title = {NLRX1 Negatively Regulates Group A Streptococcus Invasion and Autophagy Induction by Interacting With the Beclin 1-UVRAG Complex.},
journal = {Frontiers in cellular and infection microbiology},
volume = {8},
number = {},
pages = {403},
pmid = {30488027},
issn = {2235-2988},
mesh = {Autophagosomes/metabolism ; Autophagy/*physiology ; Beclin-1/genetics/*metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; HeLa Cells ; Host-Pathogen Interactions/physiology ; Humans ; Lysosomes/metabolism ; Mitochondrial Proteins/genetics/*metabolism ; Streptococcal Infections/*metabolism ; Streptococcus pyogenes/*metabolism/pathogenicity ; Tumor Suppressor Proteins/genetics/*metabolism ; },
abstract = {Group A Streptococcus (GAS) can invade epithelial cells; however, these bacteria are targeted and eventually destroyed by autophagy. Members of the Nod-like receptor (NLR) family are thought to be critical for the autophagic response to invasive bacteria. However, the intracellular sensors within host cells that are responsible for bacterial invasion and the induction of autophagy are largely unknown. Thus, our aim was to examine the role of one such NLR, namely NLRX1, in invasion and autophagy during GAS infection. We found that GAS invasion was markedly increased in NLRX1 knockout cells. This led to the potentiation of autophagic processes such as autophagosome and autolysosome formation. NLRX1 was found to interact with Beclin 1 and UVRAG, members of Beclin1 complex, and knockout of these proteins inhibited invasion and autophagy upon GAS infection. Especially, NLRX1 interacted with Beclin 1 via its NACHT domain and this interaction was responsible for the NLRX1-mediated inhibition of invasion and autophagic processes including autophagosome and autolysosome formation during GAS infection. These findings demonstrate that NLRX1 functions as a negative regulator to inactivate the Beclin 1-UVRAG complex, which regulates invasion and autophagy during GAS infection. Thus, our study expands our knowledge of the role of NLRX1 during bacterial invasion and autophagy and could lead to further investigations to understand pathogen-host cell interactions, facilitating novel targeted therapeutics.},
}
@article {pmid30487289,
year = {2019},
author = {Miao, K and Zhang, X and Su, SM and Zeng, J and Huang, Z and Chan, UI and Xu, X and Deng, CX},
title = {Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {4},
pages = {1142-1151},
pmid = {30487289},
issn = {1083-351X},
mesh = {Achondroplasia/genetics/*therapy ; Animals ; *CRISPR-Cas Systems ; Female ; Gene Editing ; *Gene Targeting ; Male ; Mice ; Mice, Knockout ; *Mutation ; Receptor, Fibroblast Growth Factor, Type 3/genetics/*physiology ; },
abstract = {CRISPR/Cas9 is a powerful technology widely used for genome editing, with the potential to be used for correcting a wide variety of deleterious disease-causing mutations. However, the technique tends to generate more indels (insertions and deletions) than precise modifications at the target sites, which might not resolve the mutation and could instead exacerbate the initial genetic disruption. We sought to develop an improved protocol for CRISPR/Cas9 that would correct mutations without unintended consequences. As a case study, we focused on achondroplasia, a common genetic form of dwarfism defined by missense mutation in the Fgfr3 gene that results in glycine to arginine substitution at position 374 in mice in fibroblast growth factor receptor 3 (Fgfr3-G374R), which corresponds to G380R in humans. First, we designed a GFP reporter system that can evaluate the cutting efficiency and specificity of single guide RNAs (sgRNAs). Using the sgRNA selected based on our GFP reporter system, we conducted targeted therapy of achondroplasia in mice. We found that we achieved higher frequency of precise correction of the Fgfr3-G374R mutation using Cas9 protein rather than Cas9 mRNA. We further demonstrated that targeting oligos of 100 and 200 nucleotides precisely corrected the mutation at equal efficiency. We showed that our strategy completely suppressed phenotypes of achondroplasia and whole genome sequencing detected no off-target effects. These data indicate that improved protocols can enable the precise CRISPR/Cas9-mediated correction of individual mutations with high fidelity.},
}
@article {pmid30487141,
year = {2019},
author = {Zeng, W and Dai, X and Sun, J and Hou, Y and Ma, X and Cao, X and Zhao, Y and Cheng, Y},
title = {Modulation of Auxin Signaling and Development by Polyadenylation Machinery.},
journal = {Plant physiology},
volume = {179},
number = {2},
pages = {686-699},
pmid = {30487141},
issn = {1532-2548},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/drug effects/*physiology ; Arabidopsis Proteins/genetics/*metabolism ; Benzamides/pharmacology ; CRISPR-Cas Systems ; Carrier Proteins/genetics/metabolism ; Cleavage Stimulation Factor/genetics/*metabolism ; Gene Expression Regulation, Plant ; Indoleacetic Acids/*metabolism ; Mutation ; Naphthols/pharmacology ; Plants, Genetically Modified ; Polyadenylation/*physiology ; Signal Transduction ; Transcription Factors/genetics/metabolism ; },
abstract = {Polyadenylation influences gene expression by affecting mRNA stability, transport, and translatability. Here, we report that Cleavage stimulation Factor 77 (AtCstF77), a component of the pre-mRNA 3'-end polyadenylation machinery, affects polyadenylation site (PAS) selection in transcripts of some auxin signaling genes in Arabidopsis (Arabidopsis thaliana). Disruption of AtCstF77 reduced auxin sensitivity and decreased the expression of the auxin reporter DR5-GFP Null mutations of cstf77 caused severe developmental defects, but were not lethal as previously reported. cstf77-2 genetically interacted with transport inhibitor response 1 auxin signaling f-box 2 auxin receptor double mutants, further supporting that polyadenylation affects auxin signaling. AtCstF77 was ubiquitously expressed in embryos, seedlings, and adult plants. The AtCstF77 protein was localized in the nucleus, which is consistent with its function in pre-mRNA processing. We observed that PASs in transcripts from approximately 2,400 genes were shifted in the cstf77-2 mutant. Moreover, most of the PAS shifts were from proximal to distal sites. Auxin treatment also caused PAS shifts in transcripts from a small number of genes. Several auxin signaling or homeostasis genes had different PASs in their transcripts in the cstf77-2 mutant. The expression levels of AUXIN RESISTANT 2/INDOLE-3-ACETIC ACID 7 were significantly increased in the cstf77-2 mutant, which can partially account for the auxin resistance phenotype of this mutant. Our results demonstrate that AtCstF77 plays pleiotropic and critical roles in Arabidopsis development. Moreover, disruption of AtCstF64, another component of the polyadenylation machinery, led to developmental defects and reduced auxin response, similar to those of the cstf77-2 mutant. We conclude that AtCstF77 affects auxin responses, likely by controlling PAS selection of transcripts of some auxin signaling components.},
}
@article {pmid30486613,
year = {2018},
author = {Lee, SH and Kim, S and Hur, JK},
title = {CRISPR and Target-Specific DNA Endonucleases for Efficient DNA Knock-in in Eukaryotic Genomes.},
journal = {Molecules and cells},
volume = {41},
number = {11},
pages = {943-952},
pmid = {30486613},
issn = {0219-1032},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; DNA Repair ; Deoxyribonuclease I/*metabolism ; Eukaryota/genetics ; Gene Editing ; Gene Knock-In Techniques/*methods ; Genetic Engineering ; Genetic Therapy ; Genome/*genetics ; Humans ; },
abstract = {The discovery and mechanistic understanding of target-specific genome engineering technologies has led to extremely effective and specific genome editing in higher organisms. Target-specific genetic modification technology is expected to have a leading position in future gene therapy development, and has a ripple effect on various basic and applied studies. However, several problems remain and hinder efficient and specific editing of target genomic loci. The issues are particularly critical in precise targeted insertion of external DNA sequences into genomes. Here, we discuss some recent efforts to overcome such problems and present a perspective of future genome editing technologies.},
}
@article {pmid30485810,
year = {2018},
author = {Lee, S and Cieply, B and Yang, Y and Peart, N and Glaser, C and Chan, P and Carstens, RP},
title = {Esrp1-Regulated Splicing of Arhgef11 Isoforms Is Required for Epithelial Tight Junction Integrity.},
journal = {Cell reports},
volume = {25},
number = {9},
pages = {2417-2430.e5},
pmid = {30485810},
issn = {2211-1247},
support = {P30 AR057217/AR/NIAMS NIH HHS/United States ; R01 AR066741/AR/NIAMS NIH HHS/United States ; R56 AR066741/AR/NIAMS NIH HHS/United States ; },
mesh = {Alopecia/pathology ; Alternative Splicing/*genetics ; Animals ; Animals, Newborn ; CRISPR-Cas Systems/genetics ; Cell Membrane Permeability ; Epithelial Cells/*metabolism ; Exons/genetics ; Inflammation/pathology ; Keratinocytes/metabolism ; Mesoderm/metabolism ; Mice, Knockout ; Myosin Light Chains/metabolism ; Phosphorylation ; Protein Isoforms/genetics/metabolism ; RNA-Binding Proteins/*metabolism ; Rho Guanine Nucleotide Exchange Factors/*genetics/metabolism ; Tight Junctions/*metabolism ; p21-Activated Kinases/metabolism ; rhoA GTP-Binding Protein/metabolism ; },
abstract = {The epithelial-specific splicing regulators Esrp1 and Esrp2 are required for mammalian development, including establishment of epidermal barrier functions. However, the mechanisms by which Esrp ablation causes defects in epithelial barriers remain undefined. We determined that the ablation of Esrp1 and Esrp2 impairs epithelial tight junction (TJ) integrity through loss of the epithelial isoform of Rho GTP exchange factor Arhgef11. Arhgef11 is required for the maintenance of TJs via RhoA activation and myosin light chain (MLC) phosphorylation. Ablation or depletion of Esrp1/2 or Arhgef11 inhibits MLC phosphorylation and only the epithelial Arhgef11 isoform rescues MLC phosphorylation in Arhgef11 KO epithelial cells. Mesenchymal Arhgef11 transcripts contain a C-terminal exon that binds to PAK4 and inhibits RhoA activation byArhgef11. Deletion of the mesenchymal-specific Arhgef11 exon in Esrp1/2 KO epithelial cells using CRISPR/Cas9 restored TJ function, illustrating how splicing alterations can be mechanistically linked to disease phenotypes that result from impaired functions of splicing regulators.},
}
@article {pmid30485515,
year = {2018},
author = {Zhang, Y and Wang, Y and Wang, X and Ji, Y and Cheng, S and Wang, M and Zhang, C and Yu, X and Zhao, R and Zhang, W and Jin, J and Li, T and Zuo, Q and Li, B},
title = {Acetyl-coenzyme A acyltransferase 2 promote the differentiation of sheep precursor adipocytes into adipocytes.},
journal = {Journal of cellular biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/jcb.28080},
pmid = {30485515},
issn = {1097-4644},
abstract = {The acetyl CoA acyltransferase 2 (ACAA2) is a key enzyme of the fatty acid oxidation pathway, catalyzing the last step of the mitochondrial beta oxidation, thus playing an important role in the fatty acid metabolism. The purpose of this study was to investigate the effect of knocking out ACAA2 on the expression of genes lipoprteinlipase (LPL), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase, fat mass and obesity-associated gene, adipocyte fatty acid-binding protein (AP2) in precursor adipocytes and their differentiation into adipocytes. The knockout vector was constructed using CRISPR-Cas RNA-guided nuclease technology with an efficiency of 23.80%, and the vector was transfected into precursor adipocyte cells, while an overexpression vector of the ACAA2 gene was also transfected in another group of preadipocytes. Quantitative polymerase chain reaction showed that the expression of the PPAR-γ, LPL, and AP2 was significantly lower in the knockout compared with the overexpression group, while there was no difference in cell growth. After induction of adipocyte precursor cells into adipocytes using dexamethasone, insulin, and IBMX, oil red staining showed a significantly different number of lipid droplets in the knockout group. These results provide a preliminary indication for a possible involvement of the ACAA2 gene in adipocyte differentiation in vitro.},
}
@article {pmid30485088,
year = {2019},
author = {Xue, L and Tang, B and Chen, W and Luo, J},
title = {Prediction of CRISPR sgRNA Activity Using a Deep Convolutional Neural Network.},
journal = {Journal of chemical information and modeling},
volume = {59},
number = {1},
pages = {615-624},
doi = {10.1021/acs.jcim.8b00368},
pmid = {30485088},
issn = {1549-960X},
mesh = {CRISPR-Cas Systems/*genetics ; Computational Biology/*methods ; *Deep Learning ; Gene Editing ; Models, Molecular ; Nucleic Acid Conformation ; RNA, Guide/chemistry/*genetics ; },
abstract = {The CRISPR-Cas9 system derived from adaptive immunity in bacteria and archaea has been developed into a powerful tool for genome engineering with wide-ranging applications. Optimizing single-guide RNA (sgRNA) design to improve efficiency of target cleavage is a key step for successful gene editing using the CRISPR-Cas9 system. Because not all sgRNAs that cognate to a given target gene are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective sgRNAs. Despite considerable efforts to date, it still remains a big challenge to accurately predict functional sgRNAs directly from large-scale sequence data. We propose DeepCas9, a deep-learning framework based on the convolutional neural network (CNN), to automatically learn the sequence determinants and further enable the identification of functional sgRNAs for the CRISPR-Cas9 system. We show that the CNN method outperforms previous methods in both (i) the ability to correctly identify highly active sgRNAs in experiments not used in the training and (ii) the ability to accurately predict the target efficacies of sgRNAs in different organisms. Besides, we further visualize the convolutional kernels and show the match of identified sequence signatures and known nucleotide preferences. We finally demonstrate the application of our method to the design of next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. We expect that DeepCas9 will assist in reducing the numbers of sgRNAs that must be experimentally validated to enable more effective and efficient genetic screens and genome engineering. DeepCas9 can be freely accessed via the Internet at https://github.com/lje00006/DeepCas9 .},
}
@article {pmid30484125,
year = {2019},
author = {Cameron Coates, R and Blaskowski, S and Szyjka, S and van Rossum, HM and Vallandingham, J and Patel, K and Serber, Z and Dean, J},
title = {Systematic investigation of CRISPR-Cas9 configurations for flexible and efficient genome editing in Corynebacterium glutamicum NRRL-B11474.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {46},
number = {2},
pages = {187-201},
pmid = {30484125},
issn = {1476-5535},
mesh = {*CRISPR-Cas Systems ; Corynebacterium glutamicum/*genetics ; Gene Deletion ; *Gene Editing ; Plasmids/genetics ; Polymorphism, Single Nucleotide ; RNA, Guide/genetics/isolation &amp; purification ; },
abstract = {This study details a reliable and efficient method for CRISPR-Cas9 genome engineering in the high amino acid-producing strain of Corynebacterium glutamicum, NRRL-B11474. Our investigation demonstrates that a plasmid-encoded single-guide RNA paired with different edit-encoding fragments is sufficient to generate edits without the addition of an exogenous recombinase. This approach leverages a genome-integrated copy of the cas9 gene for reduced toxicity, in combination with a single plasmid carrying the targeting guide RNA and matching edit fragment. Our study systematically investigated the impact of homology arm length on editing efficiency and demonstrates genome editing with homology arm lengths as small as 25 bp for single-nucleotide polymorphisms and 75 bp for 100 bp sequence swaps. These homology arm lengths are smaller than previously reported for other strains of C. glutamicum. Our study finds that C. glutamicum NRRL-B11474 is not amenable to efficient transformation with plasmids containing the BL1, NG2, or CC1 origins of replication. This finding differs from all previously reported approaches to plasmid-based CRISPR-Cas9 or Cpf1 editing in other strains of C. glutamicum. Two alternative origins of replication (CG1 and CASE1) can be used to successfully introduce genome edits; furthermore, our data demonstrate improved editing efficiency when guide RNAs and edit fragments are encoded on plasmids carrying the CASE1 origin of replication (compared to plasmids carrying CG1). In addition, this study demonstrates that efficient editing can be done using an integrated Cas9 without the need for a recombinase. We demonstrate that the specifics of CRISPR-Cas9 editing configurations may need to be tailored to enable different edit types in a particular strain background. Refining configuration parameters such as edit type, homology arm length, and plasmid origin of replication enables robust, flexible, and efficient CRISPR-Cas9 editing in differing genetic strain contexts.},
}
@article {pmid30483283,
year = {2018},
author = {Nadakuduti, SS and Buell, CR and Voytas, DF and Starker, CG and Douches, DS},
title = {Genome Editing for Crop Improvement - Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.).},
journal = {Frontiers in plant science},
volume = {9},
number = {},
pages = {1607},
pmid = {30483283},
issn = {1664-462X},
abstract = {Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop.},
}
@article {pmid30483260,
year = {2018},
author = {Bray, C and Wright, D and Haupt, S and Thomas, S and Stauss, H and Zamoyska, R},
title = {Crispr/Cas Mediated Deletion of PTPN22 in Jurkat T Cells Enhances TCR Signaling and Production of IL-2.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {2595},
pmid = {30483260},
issn = {1664-3224},
support = {WT096669AIA//Wellcome Trust/International ; WT205014/Z/16/Z//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Autoimmunity ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Humans ; Immunization ; Interleukin-2/*metabolism ; Jurkat Cells ; Lymphocyte Activation ; Mice ; Mutation/*genetics ; Protein Tyrosine Phosphatase, Non-Receptor Type 22/*genetics/metabolism ; Receptors, Antigen, T-Cell/*metabolism ; Signal Transduction ; T-Lymphocytes/*physiology ; },
abstract = {A single nucleotide polymorphism, C1858T, in the gene encoding the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) results in one of the strongest genetic traits associated with autoimmune disease outside of the Major Histocompatibility Complex (MHC) genes. However, the consequences of this polymorphism, which introduces an arginine to tryptophan substitution at amino acid 620, for the function of PTPN22 protein is unclear and conflicting results have been obtained in human compared to mouse cells expressing this variant phosphatase. In mouse the variant appears to be a loss-of-function allele resembling a milder form of the null allele, while studies in human cells have reported it to be a gain-of-function mutation. To address whether the phosphatase has distinct functions in mouse vs. human T cells, we used CRISPR gene-editing to generate the first example of human PTPN22-KnockOut (KO) T cells. By comparing isogenic human T cells which express or lack PTPN22, we showed that PTPN22 KO T cells displayed enhanced expression of IL-2 and CD69 upon stimulation with cognate antigen. PTPN22 KO cells also showed increased Erk phosphorylation upon stimulation with weak antigen, but the difference was diminished in response to strong antigen, indicating that PTPN22 plays a more critical role in regulating weak-antigen responses. These data are in keeping with a role for PTPN22 in determining the threshold of stimulation required to activate T cells, a critical function of autoimmune pathogenesis. Our data indicate that PTPN22 has comparable functions in mouse and human T cells, and that the conflicting results in the literature regarding the impact of the point mutation are not due to differences in the activity of PTPN22 itself, but may be related to interactions with other proteins or splice variation.},
}
@article {pmid30482929,
year = {2018},
author = {Cyranoski, D and Ledford, H},
title = {Genome-edited baby claim provokes international outcry.},
journal = {Nature},
volume = {563},
number = {7733},
pages = {607-608},
doi = {10.1038/d41586-018-07545-0},
pmid = {30482929},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; China ; Embryo Research/*ethics/*legislation &amp; jurisprudence ; Female ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; HIV/physiology ; HIV Infections/genetics/prevention &amp; control ; Humans ; Infant, Newborn ; *Internationality ; Male ; Practice Guidelines as Topic ; Punishment ; Receptors, CCR5/deficiency/genetics/metabolism ; Receptors, CXCR4/genetics/metabolism ; *Scientific Misconduct ; Twins/*genetics ; Virus Internalization ; },
}
@article {pmid30482801,
year = {2019},
author = {Li, M and Liu, X and Dai, S and Xiao, H and Wang, D},
title = {High Efficiency Targeting of Non-coding Sequences Using CRISPR/Cas9 System in Tilapia.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {1},
pages = {287-295},
pmid = {30482801},
issn = {2160-1836},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genome/genetics ; Germ Cells ; Sequence Deletion/genetics ; Tilapia/*genetics ; },
abstract = {The CRISPR/Cas9 has been successfully applied for disruption of protein coding sequences in a variety of organisms. The majority of the animal genome is actually non-coding sequences, which are key regulators associated with various biological processes. In this study, to understand the biological significance of these sequences, we used one or dual gRNA guided Cas9 nuclease to achieve specific deletion of non-coding sequences including microRNA and 3' untranslated region (UTR) in tilapia, which is an important fish for studying sex determination and evolution. Co-injection of fertilized eggs with single gRNA targeting seed region of miRNA and Cas9 mRNA resulted in indel mutations. Further, co-injection of fertilized eggs with dual gRNAs and Cas9 mRNA led to the removal of the fragment between the two target loci, yielding maximum efficiency of 11%. This highest genomic deletion efficiency was further improved up to 19% using short ssDNA as a donor. The deletions can be transmitted through the germline to the next generation at average efficiency of 8.7%. Cas9-vasa 3'-UTR was used to increase the efficiency of germline transmission of non-coding sequence deletion up to 14.9%. In addition, the 3'-UTR of the vasa gene was successfully deleted by dual gRNAs. Deletion of vasa 3'-UTR resulted in low expression level of vasa mRNA in the gonad when compared with the control. To summarize, the improved CRISPR/Cas9 system provided a powerful platform that can assist to easily generate desirable non-coding sequences mutants in non-model fish tilapia to discovery their functions.},
}
@article {pmid30482756,
year = {2019},
author = {Dunn, SJ and Li, MA and Carbognin, E and Smith, A and Martello, G},
title = {A common molecular logic determines embryonic stem cell self-renewal and reprogramming.},
journal = {The EMBO journal},
volume = {38},
number = {1},
pages = {},
pmid = {30482756},
issn = {1460-2075},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Self Renewal/*genetics ; Cells, Cultured ; Cellular Reprogramming/*genetics ; Computational Biology ; Embryonic Stem Cells/*physiology ; Epigenesis, Genetic/*physiology ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Regulatory Networks/*physiology ; Mice ; Mouse Embryonic Stem Cells/physiology ; Pluripotent Stem Cells/physiology ; },
abstract = {During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.},
}
@article {pmid30482592,
year = {2018},
author = {Gravelle, R},
title = {In search of: Suggesting a course of action for the scientific community to research potential impacts of heritable gene editing on the maternal carrier.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {522-524},
doi = {10.1053/j.semperi.2018.09.014},
pmid = {30482592},
issn = {1558-075X},
mesh = {CRISPR-Cas Systems/*genetics ; Embryonic Development/*genetics ; Female ; Gene Editing/*methods ; Genetic Predisposition to Disease/*genetics ; *Genetic Therapy/methods ; Humans ; Mothers ; Pregnancy ; *Reproductive Health ; },
abstract = {Prospective parents with a high risk of transmission of a disease-causing mutation may want to have an unaffected genetically related child. With advances in scientific technologies, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), they may be able to do so through heritable gene editing of the human germline at the pre-implantation stage. CRISPR technology in the reproduction and fertility context could potentially correct mutations in the germline, allow for the production of embryos that are free from a mutation and terminate the transmission of a disease-causing mutation from parent to child. As reproductive technologies evolve, a gap in the available literature exists that fails to address the potential impacts of edited fetal DNA on the maternal carrier. Both critical technical issues related to employing CRISPR, and germline editing based technologies in human reproduction and long-term impacts need to be studied and clarified to ensure positive application and outcomes for both offspring and mother.},
}
@article {pmid30482591,
year = {2018},
author = {Vigliotti, VS and Martinez, I},
title = {Public health applications of CRISPR: How children's health can benefit.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {531-536},
pmid = {30482591},
issn = {1558-075X},
support = {P30 MH062294/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy/*trends ; Child ; Culicidae/genetics ; Disease Models, Animal ; *Gene Editing ; Humans ; Influenza, Human ; Insect Vectors/*genetics ; Lyme Disease ; Malaria ; Pediatrics/*methods/trends ; Proviruses/genetics ; Public Health/*methods/trends ; Swine/genetics ; Ticks/genetics ; Transplants ; },
abstract = {Children under the age of five years old face significant mortality risks around the world. Public health innovations, particularly gene-editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) could help to reduce the risk of death in children under the age of five years old. For example, CRISPR-based strategies could reduce infectious disease morbidity by gene editing mosquitoes to prevent transmission of malaria. CRISPR gene editing technology could also help to screen for influenza virus and prevent it from replicating; influenza is a particularly difficult to treat and severe virus causing many deaths in children. The lack of liver, kidney, and heart donations for children on the organ donation waiting list could also benefit from CRISPR. Gene editing of pigs to reduce rejection rates and associated risks of porcine endogenous retroviruses could allow for the utilization of pig organs for transplant. Here we review proposed applications of gene-editing technology in public health and discuss its potential to reduce child mortality and morbidity globally.},
}
@article {pmid30482590,
year = {2018},
author = {Memi, F and Ntokou, A and Papangeli, I},
title = {CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {487-500},
doi = {10.1053/j.semperi.2018.09.003},
pmid = {30482590},
issn = {1558-075X},
mesh = {Bioethical Issues ; *CRISPR-Cas Systems ; Evidence-Based Medicine ; *Gene Editing/ethics/trends ; *Genetic Research/ethics ; Genetic Therapy/*ethics/*methods/trends ; Humans ; Morals ; },
abstract = {Gene therapy carries the potential to treat more than 10,000 human monogenic diseases and benefit an even greater number of complex polygenic conditions. The repurposing of CRISPR/Cas9, an ancient bacterial immune defense system, into a gene-editing technology has armed researchers with a revolutionary tool for gene therapy. However, as the breadth of research and clinical applications of this technology continues to expand, outstanding technical challenges and ethical considerations will need to be addressed before clinical applications become commonplace. Here, we review CRISPR/Cas9 technology and discuss its benefits and limitations in research and the clinical context, as well as ethical considerations surrounding the use of CRISPR gene editing.},
}
@article {pmid30481169,
year = {2018},
author = {Tian, S and Muneeruddin, K and Choi, MY and Tao, L and Bhuiyan, RH and Ohmi, Y and Furukawa, K and Furukawa, K and Boland, S and Shaffer, SA and Adam, RM and Dong, M},
title = {Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.},
journal = {PLoS biology},
volume = {16},
number = {11},
pages = {e2006951},
pmid = {30481169},
issn = {1545-7885},
support = {R01 NS080833/NS/NINDS NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R21 NS106159/NS/NINDS NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; },
mesh = {Bacterial Toxins/metabolism ; CRISPR-Cas Systems ; Endosomes/metabolism ; Genome-Wide Association Study/methods ; Glycolipids/metabolism ; Glycosphingolipids ; Glycosylation ; Golgi Apparatus/metabolism/physiology ; HEK293 Cells ; HeLa Cells ; Humans ; Loss of Function Mutation/genetics ; Membrane Proteins/metabolism ; Membrane Transport Proteins/metabolism/physiology ; Oncogene Proteins/metabolism ; Protein Transport ; Ricin/*genetics/metabolism ; Shiga Toxins/*genetics/metabolism ; Trihexosylceramides/metabolism/physiology ; },
abstract = {Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.},
}
@article {pmid30480643,
year = {2019},
author = {Cowan, PJ and Hawthorne, WJ and Nottle, MB},
title = {Xenogeneic transplantation and tolerance in the era of CRISPR-Cas9.},
journal = {Current opinion in organ transplantation},
volume = {24},
number = {1},
pages = {5-11},
doi = {10.1097/MOT.0000000000000589},
pmid = {30480643},
issn = {1531-7013},
mesh = {Animals ; Animals, Genetically Modified ; Antibodies, Heterophile/*genetics ; CRISPR-Cas Systems/*immunology ; Humans ; Swine ; Transplantation, Heterologous/*methods ; },
abstract = {PURPOSE OF REVIEW: The use of genetically modified donor pigs has been integral to recent major advances in xenograft survival in preclinical nonhuman primate models. CRISPR-Cas9 gene editing technology has dramatically accelerated the development of multimodified pigs. This review examines the current and projected impact of CRISPR-Cas9-mediated donor modification on preventing rejection and potentially promoting tolerance of porcine xenografts.<br><br>RECENT FINDINGS: CRISPR-Cas9 has been used to engineer several genetic modifications relevant to xenotransplantation into pigs, including glycosyltransferase knockouts (GGTA1, CMAH, &#x3b2;4GALNT2, A3GALT2 and combinations thereof), other knockouts (SLA-I, ULBP1, PERV and GHR), and one knock-in (anti-CD2 monoclonal antibody transgene knocked into GGTA1). Although the use of these pigs as donors in preclinical nonhuman primate models has been limited to a single study to date, in-vitro analysis of their cells has provided invaluable information. For example, deletion of three of the glycosyltransferases progressively decreased the binding and cytotoxicity of preexisting immunoglobulin G and immunoglobulin M in human sera, suggesting that this 'triple-KO' pig could be a platform for clinical xenotransplantation.<br><br>SUMMARY: CRISPR-Cas9 enables the rapid generation of gene-edited pigs containing multiple tailored genetic modifications that are anticipated to have a positive impact on the efficacy and safety of pig-to-human xenotransplantation.},
}
@article {pmid30480076,
year = {2019},
author = {Franke, FC and Müller, J and Abal, M and Medina, ED and Nitsche, U and Weidmann, H and Chardonnet, S and Ninio, E and Janssen, KP},
title = {The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.},
journal = {Cellular and molecular gastroenterology and hepatology},
volume = {7},
number = {1},
pages = {33-53},
pmid = {30480076},
issn = {2352-345X},
mesh = {Adaptor Proteins, Signal Transducing/chemistry/*metabolism ; Amino Acid Motifs ; CRISPR-Cas Systems/genetics ; Colorectal Neoplasms/*metabolism/*pathology ; *Epithelial-Mesenchymal Transition ; HCT116 Cells ; HEK293 Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Nuclear Proteins/chemistry/*metabolism ; Phenotype ; Protein Binding ; Signal Transduction ; Tumor Suppressor Proteins/chemistry/deficiency/*metabolism ; src Homology Domains ; },
abstract = {BACKGROUND &amp; AIMS: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation.<br><br>METHODS: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by&#xa0;the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set.<br><br>RESULTS: SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in&#xa0;vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy.<br><br>CONCLUSIONS: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.},
}
@article {pmid30479331,
year = {2018},
author = {Zhou, W and Hu, L and Ying, L and Zhao, Z and Chu, PK and Yu, XF},
title = {A CRISPR-Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {5012},
pmid = {30479331},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/*analysis ; Fluorescence ; HEK293 Cells ; Humans ; Nucleic Acid Amplification Techniques/*methods ; Nucleotides/genetics ; },
abstract = {Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR-Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.},
}
@article {pmid30478778,
year = {2019},
author = {Ye, C and Chen, J and Cheng, X and Zhou, S and Jiang, S and Xu, J and Zheng, H and Tong, W and Li, G and Tong, G},
title = {Functional analysis of the UL24 protein of suid herpesvirus 1.},
journal = {Virus genes},
volume = {55},
number = {1},
pages = {76-86},
pmid = {30478778},
issn = {1572-994X},
mesh = {Animals ; CRISPR-Cas Systems ; Chlorocebus aethiops ; DNA, Viral ; Female ; HeLa Cells ; Herpesvirus 1, Suid/*physiology ; Homologous Recombination ; Humans ; Mice ; Mutation ; Protein Transport ; Pseudorabies/metabolism/virology ; RNA, Guide/genetics ; Thymidine Kinase/genetics/metabolism ; Trigeminal Nucleus, Spinal/virology ; Vero Cells ; Viral Load ; Viral Proteins/genetics/*metabolism ; Virulence ; },
abstract = {The UL24 homologous genes are conserved in alphaherpesviruses. However, the proximity of the UL24 gene and the UL23 gene encoding for thymidine kinase (TK) in the genome of suid herpesvirus 1 (SuHV-1) makes it difficult to mutate UL24 without affecting the expression of the TK gene, and thus functional studies of the UL24 gene have lagged behind. In this study, CRISPR/Cas9 and homologous recombination were adopted to generate UL24 and TK mutant viruses. Deletion of either the UL24 or the TK gene resulted in significantly reduced SuHV-1 replication and spread capacity in Vero cells. However, UL24-deleted virus still maintained a certain degree of lethality in mice, while TK-deleted viruses completely lost their lethality in mice. Similarly, neurovirulence of UL24-deleted virus in mice was not significantly affected compared to parental virus. In comparison, infection with the TK-deleted viruses resulted in significantly reduced neurovirulence and complete loss of lethality. In addition, and for the first time, viral UL24 protein was found to be expressed late during SuHV-1 infection; enhanced green fluorescence protein (eGFP) labeled UL24 protein was shown to be localized in the nucleus via heterologous expression. In conclusion, the UL24 gene of SuHV-1 encodes a nuclear-localized viral protein and acts as a minor virulence-associated factor compared to the TK gene.},
}
@article {pmid30478371,
year = {2018},
author = {Datta, MS and Kishony, R},
title = {A spotlight on bacterial mutations for 75 years.},
journal = {Nature},
volume = {563},
number = {7733},
pages = {633-644},
doi = {10.1038/d41586-018-07521-8},
pmid = {30478371},
issn = {1476-4687},
mesh = {Animals ; Bacteria/cytology/*genetics/virology ; Bacteriophages/pathogenicity ; CRISPR-Cas Systems/genetics ; Escherichia coli/cytology/genetics/virology ; *Evolution, Molecular ; History, 20th Century ; History, 21st Century ; Microbiology/*history ; Models, Genetic ; Molecular Biology/*history ; Mutagenesis ; *Mutation ; Poisson Distribution ; Selection, Genetic/*genetics ; Streptococcus thermophilus/cytology/genetics/virology ; },
}
@article {pmid30478310,
year = {2018},
author = {Chin, HS and Li, MX and Tan, IKL and Ninnis, RL and Reljic, B and Scicluna, K and Dagley, LF and Sandow, JJ and Kelly, GL and Samson, AL and Chappaz, S and Khaw, SL and Chang, C and Morokoff, A and Brinkmann, K and Webb, A and Hockings, C and Hall, CM and Kueh, AJ and Ryan, MT and Kluck, RM and Bouillet, P and Herold, MJ and Gray, DHD and Huang, DCS and van Delft, MF and Dewson, G},
title = {VDAC2 enables BAX to mediate apoptosis and limit tumor development.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4976},
pmid = {30478310},
issn = {2041-1723},
mesh = {Animals ; *Apoptosis ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*metabolism/*pathology ; Embryonic Development ; HCT116 Cells ; HeLa Cells ; Humans ; Mice, Inbred C57BL ; Mitochondria/metabolism ; Promoter Regions, Genetic/genetics ; Voltage-Dependent Anion Channel 2/*metabolism ; bcl-2 Homologous Antagonist-Killer Protein/metabolism ; bcl-2-Associated X Protein/*metabolism ; },
abstract = {Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.},
}
@article {pmid30478289,
year = {2019},
author = {Turgeman-Grott, I and Joseph, S and Marton, S and Eizenshtein, K and Naor, A and Soucy, SM and Stachler, AE and Shalev, Y and Zarkor, M and Reshef, L and Altman-Price, N and Marchfelder, A and Gophna, U},
title = {Pervasive acquisition of CRISPR memory driven by inter-species mating of archaea can limit gene transfer and influence speciation.},
journal = {Nature microbiology},
volume = {4},
number = {1},
pages = {177-186},
pmid = {30478289},
issn = {2058-5276},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA, Intergenic/*genetics ; Gene Transfer, Horizontal/*genetics ; Genetic Speciation ; Haloferax mediterranei/*genetics/growth &amp; development ; Haloferax volcanii/*genetics/growth &amp; development ; },
abstract = {CRISPR-Cas systems provide prokaryotes with sequence-specific immunity against viruses and plasmids based on DNA acquired from these invaders, known as spacers. Surprisingly, many archaea possess spacers that match chromosomal genes of related species, including those encoding core housekeeping genes. By sequencing genomes of environmental archaea isolated from a single site, we demonstrate that inter-species spacers are common. We show experimentally, by mating Haloferax volcanii and Haloferax mediterranei, that spacers are indeed acquired chromosome-wide, although a preference for integrated mobile elements and nearby regions of the chromosome exists. Inter-species mating induces increased spacer acquisition and may result in interactions between the acquisition machinery of the two species. Surprisingly, many of the spacers acquired following inter-species mating target self-replicons along with those originating from the mating partner, indicating that the acquisition machinery cannot distinguish self from non-self under these conditions. Engineering the chromosome of one species to be targeted by the other's CRISPR-Cas reduces gene exchange between them substantially. Thus, spacers acquired during inter-species mating could limit future gene transfer, resulting in a role for CRISPR-Cas systems in microbial speciation.},
}
@article {pmid30478237,
year = {2018},
author = {Bassalo, MC and Garst, AD and Choudhury, A and Grau, WC and Oh, EJ and Spindler, E and Lipscomb, T and Gill, RT},
title = {Deep scanning lysine metabolism in Escherichia coli.},
journal = {Molecular systems biology},
volume = {14},
number = {11},
pages = {e8371},
pmid = {30478237},
issn = {1744-4292},
mesh = {CRISPR-Cas Systems ; Escherichia coli/genetics/*metabolism ; Escherichia coli Proteins/genetics/metabolism ; Gene Library ; Lysine/*metabolism ; *Metabolic Networks and Pathways ; *Mutation ; Phenotype ; },
abstract = {Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a single-protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks. These sophisticated networks hinder our understanding of the phenotype of interest and limit our capabilities to rewire cellular functions. Here, we leveraged CRISPR-EnAbled Trackable genome Engineering to attempt a parallel and high-resolution interrogation of complex networks, deep scanning multiple proteins associated with lysine metabolism in Escherichia coli We designed over 16,000 mutations to perturb this pathway and mapped their contribution toward resistance to an amino acid analog. By doing so, we identified different routes that can alter pathway function and flux, uncovering mechanisms that would be difficult to rationally design. This approach sets a framework for forward investigation of complex multigenic phenotypes.},
}
@article {pmid30478227,
year = {2019},
author = {Katayama, T and Nakamura, H and Zhang, Y and Pascal, A and Fujii, W and Maruyama, JI},
title = {Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {3},
pages = {},
pmid = {30478227},
issn = {1098-5336},
mesh = {Aspergillus oryzae/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Editing ; Genetic Engineering ; Genome, Fungal ; Industrial Microbiology ; Mutagenesis ; Plasmids/genetics/metabolism ; },
abstract = {Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability.IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.},
}
@article {pmid30478157,
year = {2019},
author = {Liang, C and Zhao, J and Lu, J and Zhang, Y and Ma, X and Shang, X and Li, Y and Ma, X and Liu, M and Wang, X},
title = {Development and Characterization of MDR1 (Mdr1a/b) CRISPR/Cas9 Knockout Rat Model.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {47},
number = {2},
pages = {71-79},
doi = {10.1124/dmd.118.084277},
pmid = {30478157},
issn = {1521-009X},
mesh = {ATP Binding Cassette Transporter, Subfamily B/*genetics/metabolism ; Administration, Oral ; Animals ; Brain/metabolism ; CRISPR-Cas Systems/genetics ; Cytochrome P-450 CYP3A/analysis/genetics/metabolism ; Digoxin/administration &amp; dosage/*pharmacokinetics ; Female ; Gene Knockout Techniques/methods ; Intestine, Small/metabolism ; Kidney/metabolism ; Liver/metabolism ; Male ; Models, Animal ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Sprague-Dawley ; Rats, Transgenic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 (Mdr1a/b) double-knockout (KO) rat model was generated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO rats. Further pharmacokinetic studies of digoxin, a typical substrate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of Mdr1a/b (-/-) rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type rats. In general, a new Mdr1a/b (-/-) rat model has been successfully generated and characterized. This rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.},
}
@article {pmid30477585,
year = {2018},
author = {Chong, ZS and Ohnishi, S and Yusa, K and Wright, GJ},
title = {Pooled extracellular receptor-ligand interaction screening using CRISPR activation.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {205},
pmid = {30477585},
issn = {1474-760X},
support = {/WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Genomics/*methods ; Humans ; Ligands ; Receptors, Cell Surface/*analysis ; },
abstract = {Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale.},
}
@article {pmid30477421,
year = {2018},
author = {Zhang, S and Zhang, R and Song, G and Gao, J and Li, W and Han, X and Chen, M and Li, Y and Li, G},
title = {Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat.},
journal = {BMC plant biology},
volume = {18},
number = {1},
pages = {302},
pmid = {30477421},
issn = {1471-2229},
mesh = {Agrobacterium tumefaciens/*genetics ; *CRISPR-Cas Systems ; *Gene Targeting ; Genes, Plant ; *Mutagenesis ; Triticum/*genetics ; },
abstract = {BACKGROUND: Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation.<br><br>RESULTS: In the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences.<br><br>CONCLUSIONS: Our results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.},
}
@article {pmid30476497,
year = {2019},
author = {Fujii, M and Clevers, H and Sato, T},
title = {Modeling Human Digestive Diseases With CRISPR-Cas9-Modified Organoids.},
journal = {Gastroenterology},
volume = {156},
number = {3},
pages = {562-576},
doi = {10.1053/j.gastro.2018.11.048},
pmid = {30476497},
issn = {1528-0012},
mesh = {CRISPR-Cas Systems/*genetics ; Colorectal Neoplasms/genetics/pathology ; Esophageal Neoplasms/genetics/pathology ; Female ; Gastrointestinal Diseases/genetics/pathology ; Gastrointestinal Neoplasms/*genetics/*pathology ; Gastrointestinal Tract/pathology ; *Genetic Predisposition to Disease ; Humans ; Male ; *Organoids ; Pancreatic Neoplasms/genetics/pathology ; Sensitivity and Specificity ; },
abstract = {Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients' tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system.},
}
@article {pmid30476227,
year = {2019},
author = {Oughtred, R and Stark, C and Breitkreutz, BJ and Rust, J and Boucher, L and Chang, C and Kolas, N and O'Donnell, L and Leung, G and McAdam, R and Zhang, F and Dolma, S and Willems, A and Coulombe-Huntington, J and Chatr-Aryamontri, A and Dolinski, K and Tyers, M},
title = {The BioGRID interaction database: 2019 update.},
journal = {Nucleic acids research},
volume = {47},
number = {D1},
pages = {D529-D541},
pmid = {30476227},
issn = {1362-4962},
support = {BB/F010486/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; R01 OD010929/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Data Curation ; *Databases, Factual ; Drug Discovery ; Genes ; Humans ; Mice ; Protein Interaction Mapping ; },
abstract = {The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the curation and archival storage of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2018 (build 3.4.164), BioGRID contains records for 1 598 688 biological interactions manually annotated from 55 809 publications for 71 species, as classified by an updated set of controlled vocabularies for experimental detection methods. BioGRID also houses records for >700 000 post-translational modification sites. BioGRID now captures chemical interaction data, including chemical-protein interactions for human drug targets drawn from the DrugBank database and manually curated bioactive compounds reported in the literature. A new dedicated aspect of BioGRID annotates genome-wide CRISPR/Cas9-based screens that report gene-phenotype and gene-gene relationships. An extension of the BioGRID resource called the Open Repository for CRISPR Screens (ORCS) database (https://orcs.thebiogrid.org) currently contains over 500 genome-wide screens carried out in human or mouse cell lines. All data in BioGRID is made freely available without restriction, is directly downloadable in standard formats and can be readily incorporated into existing applications via our web service platforms. BioGRID data are also freely distributed through partner model organism databases and meta-databases.},
}
@article {pmid30476163,
year = {2019},
author = {Hofmann, A and Falk, J and Prangemeier, T and Happel, D and Köber, A and Christmann, A and Koeppl, H and Kolmar, H},
title = {A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast.},
journal = {Nucleic acids research},
volume = {47},
number = {1},
pages = {509-520},
pmid = {30476163},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression Regulation/genetics ; Genes, Reporter/genetics ; Models, Theoretical ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/*genetics ; Single-Cell Analysis ; Trans-Activators/*genetics ; *Transcription, Genetic ; Transcriptional Activation/genetics ; },
abstract = {The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis.},
}
@article {pmid30476078,
year = {2019},
author = {Blanas, A and Cornelissen, LAM and Kotsias, M and van der Horst, JC and van de Vrugt, HJ and Kalay, H and Spencer, DIR and Kozak, RP and van Vliet, SJ},
title = {Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells.},
journal = {Glycobiology},
volume = {29},
number = {2},
pages = {137-150},
pmid = {30476078},
issn = {1460-2423},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Colorectal Neoplasms/*genetics/metabolism/pathology ; Fucosyltransferases/*genetics/metabolism ; Mice ; Polysaccharides/*genetics/metabolism ; *Transcriptional Activation ; Tumor Cells, Cultured ; },
abstract = {Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically, in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase 9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the well-known murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9 enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4 and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression stability of the induced enzymes. Moreover, Lewisx expression was found to influence core-fucosylation, sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is a promising method of transcriptional gene activation with broad application possibilities in glycobiology and oncology research.},
}
@article {pmid30473704,
year = {2018},
author = {Teague, WJ and Jones, MLM and Hawkey, L and Smyth, IM and Catubig, A and King, SK and Sarila, G and Li, R and Hutson, JM},
title = {FGF10 and the Mystery of Duodenal Atresia in Humans.},
journal = {Frontiers in genetics},
volume = {9},
number = {},
pages = {530},
pmid = {30473704},
issn = {1664-8021},
abstract = {Background: Duodenal atresia (DA) is a congenital obstruction of the duodenum, which affects 1 in 7000 pregnancies and requires major surgery in the 1st days of life. Three morphological DA types are described. In humans, the association between DA and Down syndrome suggests an underlying, albeit elusive, genetic etiology. In mice, interruption of fibroblast growth factor 10 (Fgf10) gene signaling results in DA in 30-50% of embryos, supporting a genetic etiology. This study aims to validate the spectrum of DA in two novel strains of Fgf10 knock-out mice, in preparation for future and translational research. Methods: Two novel CRISPR Fgf10 knock-out mouse strains were derived and embryos generated by heterozygous plug-mating. E15.5-E19.5 embryos were genotyped with respect to Fgf10 and micro-dissected to determine the presence and type of DA. Results: One twenty seven embryos (32 wild-type, 34 heterozygous, 61 null) were analyzed. No wild-type or heterozygous embryos had DA. However, 74% of Fgf10 null embryos had DA (49% type 1, 18% type 2, and 33% type 3). Conclusion: Our CRISPR-derived strains showed higher penetrance of DA due to single-gene deletion of Fgf10 in mice than previously reported. Further, the DA type distribution in these mice more closely reiterated that observed in humans. Future experiments will document RNA and protein expression of FGF10 and its key downstream signaling targets in normal and atretic duodenum. This includes exploitation of modern, high-fidelity developmental tools, e.g., Fgf10 [flox/+]-tomato[flox/flox] mice.},
}
@article {pmid30472287,
year = {2019},
author = {Durand, GA and Raoult, D and Dubourg, G},
title = {Antibiotic discovery: history, methods and perspectives.},
journal = {International journal of antimicrobial agents},
volume = {53},
number = {4},
pages = {371-382},
doi = {10.1016/j.ijantimicag.2018.11.010},
pmid = {30472287},
issn = {1872-7913},
mesh = {Actinobacteria/metabolism ; Anti-Bacterial Agents/*isolation &amp; purification ; CRISPR-Cas Systems ; Chromosome Mapping ; Drug Discovery/*methods ; Drug Resistance, Multiple, Bacterial/genetics ; Fungi/metabolism ; Gastrointestinal Microbiome/*drug effects ; Humans ; },
abstract = {Antimicrobial resistance is considered a major public-health issue. Policies recommended by the World Health Organization (WHO) include research on new antibiotics. No new class has been discovered since daptomycin and linezolid in the 1980s, and only optimisation or combination of already known compounds has been recently commercialised. Antibiotics are natural products of soil-living organisms. Actinobacteria and fungi are the source of approximately two-thirds of the antimicrobial agents currently used in human medicine; they were mainly discovered during the golden age of antibiotic discovery. This era declined after the 1970s owing to the difficulty of cultivating fastidious bacterial species under laboratory conditions. Various strategies, such as rational drug design, to date have not led to the discovery of new antimicrobial agents. However, new promising approaches, e.g. genome mining or CRISPR-Cas9, are now being developed. The recent rebirth of culture methods from complex samples has, as a matter of fact, permitted the discovery of teixobactin from a new species isolated from soil. Recently, many biosynthetic gene clusters were identified from human-associated microbiota, especially from the gut and oral cavity. For example, the antimicrobial lugdunin was recently discovered in the oral cavity. The repertoire of human gut microbiota has recently substantially increased, with the discovery of hundreds of new species. Exploration of the repertoire of prokaryotes associated with humans using genome mining or newer culture approaches could be promising strategies for discovering new classes of antibiotics.},
}
@article {pmid30471466,
year = {2019},
author = {White, CW and Johnstone, EKM and See, HB and Pfleger, KDG},
title = {NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing.},
journal = {Cellular signalling},
volume = {54},
number = {},
pages = {27-34},
doi = {10.1016/j.cellsig.2018.11.018},
pmid = {30471466},
issn = {1873-3913},
mesh = {Bioluminescence Resonance Energy Transfer Techniques/*methods ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Luciferases/chemistry ; Receptor, Adenosine A2B/*analysis ; },
abstract = {Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A2B. However, in cells expressing gene-edited Nluc/A2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (Kd = 21.4 nM). Additionally, at gene-edited Nluc/A2B receptors we derived pharmacological parameters of ligand binding; Kd as well as Kon and Koff for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A2B were used to determine the pKi of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.},
}
@article {pmid30470963,
year = {2019},
author = {Li, Y and Yan, F and Wu, H and Li, G and Han, Y and Ma, Q and Fan, X and Zhang, C and Xu, Q and Xie, X and Chen, N},
title = {Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {46},
number = {1},
pages = {81-90},
pmid = {30470963},
issn = {1476-5535},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Chromosomes, Bacterial/*genetics ; Cloning, Molecular ; *DNA Fragmentation ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics ; Gene Deletion ; Gene Editing ; Genes, Bacterial ; Plasmids/genetics ; RNA, Guide/metabolism ; Streptococcus pyogenes/genetics ; },
abstract = {Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3-4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.},
}
@article {pmid30470841,
year = {2018},
author = {Otsuka, K and Tomita, M},
title = {Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {17309},
pmid = {30470841},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell Cycle ; Cell Cycle Proteins ; *DNA Breaks, Double-Stranded ; *DNA Repair ; Fluorescent Antibody Technique ; Genetic Vectors/*administration &amp; dosage ; Image Processing, Computer-Assisted/*methods ; Kinetics ; Mice ; NIH 3T3 Cells ; Radiation, Ionizing ; Tumor Suppressor p53-Binding Protein 1/antagonists &amp; inhibitors/genetics/*metabolism ; },
abstract = {Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated "Focicles" harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn). We used CRISPR/Cas9-mediated genome editing to obtain Focicle knock-in cell lines in NIH3T3 cells, which were subject to X-ray irradiation that induced comparable numbers of Focicle and endogenous-53BP1 foci. In addition, the Focicle probes enabled the kinetic analysis of both DSB repair and cell-cycle arrest/progression after irradiation, demonstrating that the Focicle knock-in cells progressed to cell division after DNA damage elimination. These newly developed probes can help to gain a better understanding of the dynamics of DSB repair and cell-cycle control to in turn guide cancer treatment development and cancer-risk assessments.},
}
@article {pmid30470168,
year = {2018},
author = {Gao, Z and Herrera-Carrillo, E and Berkhout, B},
title = {Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA.},
journal = {RNA biology},
volume = {15},
number = {12},
pages = {1458-1467},
pmid = {30470168},
issn = {1555-8584},
mesh = {Bacterial Proteins/*metabolism ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Chromosomes/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*metabolism ; *Gene Editing ; Gene Expression Regulation ; Genes, Reporter ; Humans ; RNA, Catalytic/chemistry/*genetics ; },
abstract = {The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3'-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1.1 to 5.2 fold. We also demonstrate that this design can enhance CRISPR-based gene activation. We thus generated an improved CRISPR-Cpf1 system for more efficient gene editing and gene regulation.},
}
@article {pmid30468812,
year = {2019},
author = {Vimalraj, S and Saravanan, S and Anuradha, D and Chatterjee, S},
title = {Models to investigate intussusceptive angiogenesis: A special note on CRISPR/Cas9 based system in zebrafish.},
journal = {International journal of biological macromolecules},
volume = {123},
number = {},
pages = {1229-1240},
doi = {10.1016/j.ijbiomac.2018.11.164},
pmid = {30468812},
issn = {1879-0003},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Models, Biological ; *Neovascularization, Physiologic ; Zebrafish/*metabolism ; },
abstract = {Angiogenesis is a distinct process which follows sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA) forming the basis for various physiological and pathological scenarios. Angiogenesis is a double edged sword exerting both desirable and discernible effects owing to the referred microenvironment. Therapeutic interventions to promote angiogenesis in regenerative medicine is essential to achieve functional syncytium of tissue constructs while, angiogenic inhibition is a key therapeutic target to suppress tumor growth. In the recent years, clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) based gene editing approaches have been gaining considerable attention in the field of biomedical research owing to its ease in tailoring targeted genome in living organisms. The Zebrafish model, with adequately high-throughput fitness, is a likely option for genome editing and angiogenesis research. In this review, we focus on the implication of Zebrafish as a model to study IA and furthermore enumerate CRISPR/Cas9 based genome editing in Zebrafish as a candidate for modeling different types of angiogenesis and support its candidature as a model organism.},
}
@article {pmid30467400,
year = {2018},
author = {Yan, Y and Finnigan, GC},
title = {Development of a multi-locus CRISPR gene drive system in budding yeast.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {17277},
pmid = {30467400},
issn = {2045-2322},
support = {P20 GM103418/GM/NIGMS NIH HHS/United States ; Hatch Project 1013520//USDA | National Institute of Food and Agriculture (NIFA)/International ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Dosage ; Gene Editing/methods ; Genetic Loci ; RNA, Guide/*genetics ; Saccharomycetales/genetics/*growth &amp; development ; Streptococcus pyogenes/enzymology/genetics ; },
abstract = {The discovery of CRISPR/Cas gene editing has allowed for major advances in many biomedical disciplines and basic research. One arrangement of this biotechnology, a nuclease-based gene drive, can rapidly deliver a genetic element through a given population and studies in fungi and metazoans have demonstrated the success of such a system. This methodology has the potential to control biological populations and contribute to eradication of insect-borne diseases, agricultural pests, and invasive species. However, there remain challenges in the design, optimization, and implementation of gene drives including concerns regarding biosafety, containment, and control/inhibition. Given the numerous gene drive arrangements possible, there is a growing need for more advanced designs. In this study, we use budding yeast to develop an artificial multi-locus gene drive system. Our minimal setup requires only a single copy of S. pyogenes Cas9 and three guide RNAs to propagate three gene drives. We demonstrate how this system could be used for targeted allele replacement of native genes and to suppress NHEJ repair systems by modifying DNA Ligase IV. A multi-locus gene drive configuration provides an expanded suite of options for complex attributes including pathway redundancy, combatting evolved resistance, and safeguards for control, inhibition, or reversal of drive action.},
}
@article {pmid30467164,
year = {2018},
author = {Wang, T},
title = {Paring down to the essentials.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6417},
pages = {904},
doi = {10.1126/science.aav6872},
pmid = {30467164},
issn = {1095-9203},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Knockout Techniques ; *Gene Library ; Genes, Essential ; Genetic Testing/*methods ; Genome-Wide Association Study ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Loss of Function Mutation ; },
}
@article {pmid30466900,
year = {2019},
author = {Monteiro, R and Pires, DP and Costa, AR and Azeredo, J},
title = {Phage Therapy: Going Temperate?.},
journal = {Trends in microbiology},
volume = {27},
number = {4},
pages = {368-378},
doi = {10.1016/j.tim.2018.10.008},
pmid = {30466900},
issn = {1878-4380},
mesh = {Animals ; Anti-Bacterial Agents ; Bacteria/genetics/virology ; Bacterial Infections/*therapy ; Bacteriophages/classification/*genetics/physiology ; CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial ; Endotoxins ; Genetic Engineering ; Humans ; Phage Therapy/*methods ; Transduction, Genetic ; Virulence ; },
abstract = {Strictly lytic phages have been consensually preferred for phage therapy purposes. In contrast, temperate phages have been avoided due to an inherent capacity to mediate transfer of genes between bacteria by specialized transduction - an event that may increase bacterial virulence, for example, by promoting antibiotic resistance. Now, advances in sequencing technologies and synthetic biology are providing new opportunities to explore the use of temperate phages for therapy against bacterial infections. By doing so we can considerably expand our armamentarium against the escalating threat of antibiotic-resistant bacteria.},
}
@article {pmid30466730,
year = {2019},
author = {Duester, G},
title = {Knocking Out Enhancers to Enhance Epigenetic Research.},
journal = {Trends in genetics : TIG},
volume = {35},
number = {2},
pages = {89},
pmid = {30466730},
issn = {0168-9525},
support = {R01 AR067731/AR/NIAMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation/*genetics ; DNA Methylation/genetics ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; Gene Knockout Techniques/methods ; Histones/genetics ; Humans ; Promoter Regions, Genetic ; },
}
@article {pmid30466578,
year = {2018},
author = {Pandiarajan, R and Grover, A},
title = {In vivo promoter engineering in plants: Are we ready?.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {277},
number = {},
pages = {132-138},
doi = {10.1016/j.plantsci.2018.10.011},
pmid = {30466578},
issn = {1873-2259},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Promoter Regions, Genetic/*genetics ; },
abstract = {Engineering plant promoter sequence for optimal expression of a gene has been a long standing goal for plant scientists. In recent times, Sequence Specific Nucleases (SSNs) like CRISPR/Cas9 are enabling researchers to achieve this goal, in vivo in the genome. It is well known that SSNs have met with unprecedented success in rapid transgene free crop improvement largely by targeting the coding sequence. Here, we discuss the strategies being employed by plant scientists in targeting SSNs to non-coding promoter regions/Cis Regulatory Elements (CRE). We collectively refer all such endeavors as in vivo promoter engineering (IPE). We further classify the IPE efforts into CRE addition, CRE deletion/disruption, promoter swap/insertion and targeted promoter polymorphism. Till date, IPE has proven useful in altering plant architecture in tomato, developing resistance against Xanthomonas sp in rice and citrus, and engineering drought tolerance in maize. However it is quite challenging to achieve predictable changes in gene expression using IPE at this point. In future years, data generated from high throughput techniques to investigate non coding genome may immensely augment the efforts in this direction. As IPE does not involve addition of the transgene for modifying crop traits, it will be relatively more conducive to public acceptance in crop improvement programs.},
}
@article {pmid30466537,
year = {2018},
author = {Burian, M and Yazdi, AS},
title = {NLRP1 Is the Key Inflammasome in Primary Human Keratinocytes.},
journal = {The Journal of investigative dermatology},
volume = {138},
number = {12},
pages = {2507-2510},
doi = {10.1016/j.jid.2018.08.004},
pmid = {30466537},
issn = {1523-1747},
mesh = {Adaptor Proteins, Signal Transducing ; Apoptosis Regulatory Proteins ; CRISPR-Cas Systems ; Gene Editing ; Humans ; *Inflammasomes ; Interleukin-1beta ; *Keratinocytes ; NLR Family, Pyrin Domain-Containing 3 Protein ; NLR Proteins ; },
abstract = {The epidermis is the primary area of contact between the body and the environment, and it distinguishes between harmful exposures and those that should be tolerated. Discrimination between insults, and in particular the recognition of danger signals such as UVB, is mediated by innate immune receptors. Inflammasomes are one major innate mechanism that activate inflammatory caspases. In human keratinocytes, the importance of inflammasomes and the sensing of UVB and other danger signals are a matter of debate. Fenini et al. now provide evidence that the NLRP1 (rather than the NLRP3) inflammasome plays a key role in UVB sensing and subsequent IL-1β and -18 secretion by human keratinocytes.},
}
@article {pmid30466456,
year = {2018},
author = {Wong, NK and Huang, CL and Islam, R and Yip, SP},
title = {Long non-coding RNAs in hematological malignancies: translating basic techniques into diagnostic and therapeutic strategies.},
journal = {Journal of hematology &amp; oncology},
volume = {11},
number = {1},
pages = {131},
pmid = {30466456},
issn = {1756-8722},
mesh = {Gene Expression Regulation, Neoplastic/*genetics ; Hematologic Neoplasms/*genetics ; Humans ; RNA, Long Noncoding/*genetics ; },
abstract = {Recent studies have revealed that non-coding regions comprise the vast majority of the human genome and long non-coding RNAs (lncRNAs) are a diverse class of non-coding RNAs that has been implicated in a variety of biological processes. Abnormal expression of lncRNAs has also been linked to different human diseases including cancers, yet the regulatory mechanisms and functional effects of lncRNAs are still ambiguous, and the molecular details also need to be confirmed. Unlike protein-coding gene, it is much more challenging to unravel the roles of lncRNAs owing to their unique and complex features such as functional diversity and low conservation among species, which greatly hamper their experimental characterization. In this review, we summarize and discuss both conventional and advanced approaches for the identification and functional characterization of lncRNAs related to hematological malignancies. In particular, the utility and advancement of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system as gene-editing tools are envisioned to facilitate the molecular dissection of lncRNAs via different knock-in/out strategies. Besides experimental considerations specific to lncRNAs, the roles of lncRNAs in the pathogenesis and progression of leukemia are also highlighted in the review. We expect that these insights may ultimately lead to clinical applications including development of biomarkers and novel therapeutic approaches targeting lncRNAs.},
}
@article {pmid30465094,
year = {2019},
author = {Saika, H and Mori, A and Endo, M and Toki, S},
title = {Targeted deletion of rice retrotransposon Tos17 via CRISPR/Cas9.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {455-458},
pmid = {30465094},
issn = {1432-203X},
mesh = {CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Gene Editing/methods ; Genome, Plant/genetics ; Oryza/*genetics ; Retroelements/*genetics ; Terminal Repeat Sequences/genetics ; },
abstract = {A successful example of transposon deletion via CRISPR/Cas9-mediated genome editing suggests a novel alternative approach to plant breeding. Transposition of transposable elements (TEs) can affect adjacent genes, leading to changes in genetic traits. Expression levels and patterns, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus can be affected. Artificial modification of loci adjacent to TEs, or TEs themselves, by genome editing could mimic the translocation of TEs that occurs in nature, suggesting that it might be possible to produce novel plants by modification of TEs via genome editing. To our knowledge, there are no reports thus far of modification of TEs by genome editing in plants. In this study, we performed targeted deletion of the Tos17 retrotransposon, which is flanked at both ends by long terminal repeat (LTR) sequences, via genome editing in rice. We succeeded in targeted mutagenesis of the LTR, and targeted deletion between LTRs, in calli transformed with CRISPR/Cas9 vectors for the Tos17 LTR. Moreover, we also successfully obtained regenerated plants derived from transformed calli and plants homozygous for lacking Tos17 in the next generation. Taken together, our results demonstrate successful deletion of the Tos17 retrotransposon from the rice genome by targeted mutagenesis using CRISPR/Cas9. We believe that this strategy could be applied to other TEs in many plant species, providing a rapid breeding technology as an alternative means to re-activate expression of agronomically important genes that have been inactivated by TE insertion, especially in plants such as fruit trees, in which it is difficult to maintain parental agronomical traits by cross-breeding due to high heterozygosity.},
}
@article {pmid30464460,
year = {2018},
author = {Cheng, WJ and Chen, LC and Ho, HO and Lin, HL and Sheu, MT},
title = {Stearyl polyethylenimine complexed with plasmids as the core of human serum albumin nanoparticles noncovalently bound to CRISPR/Cas9 plasmids or siRNA for disrupting or silencing PD-L1 expression for immunotherapy.},
journal = {International journal of nanomedicine},
volume = {13},
number = {},
pages = {7079-7094},
pmid = {30464460},
issn = {1178-2013},
mesh = {Animals ; B7-H1 Antigen/*metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Death ; Cell Line, Tumor ; Cell Survival ; Endocytosis ; Gene Silencing ; Humans ; *Immunotherapy ; Mice ; Nanoparticles/*chemistry/ultrastructure ; Particle Size ; Plasmids/metabolism ; Polyethyleneimine/*chemistry ; Proton Magnetic Resonance Spectroscopy ; RNA, Small Interfering/genetics/*metabolism ; Serum Albumin, Human/*chemistry ; Static Electricity ; Stearic Acids/*chemistry ; Transfection ; },
abstract = {PURPOSE: In this study, a double emulsion method for complexing plasmids with stearyl poly-ethylenimine (stPEI) as the core to form human serum albumin (HSA) (plasmid/stPEI/HSA) nanoparticles (NPs) was developed for gene delivery by non-covalently binding onto plasmid/stPEI/HSA nanoparticles with CRISPR/Cas9 or siRNA, which disrupts or silences the expression of programmed cell death ligand-1 (PD-L1) for immunotherapy.<br><br>MATERIALS AND METHODS: Chemically synthesized stearyl-polyethyenimine (stPEI)/plasmids/HSA nanoparticles were maded by double emulsion method. They were characterized by dynamic light scattering (DLS), transmission electron microscope and also evaluated by in vitro study on CT 26 cells.<br><br>RESULTS: stPEI was synthesized by an N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction, and we found that the degree of substitution was ~1.0 when the ratio of PEI to stearic acid was 1:7 in the reaction. Then, two sgRNA sequences were selected and evaluated for their ability to knock out PD-L1 by decreasing its expression by about 20%. Based on the trend of particle size/zeta potential values as a function of ratio, F25P1 containing 25 &#x3bc;g of plasmid/stPEI/HSA NPs noncovalently bound to 1 &#x3bc;g plasmids via charge-charge interactions was found to be optimal. Its particle size was about 202.7&#xb1;4.5 nm, and zeta potential was 12.60&#xb1;0.15 mV. In an in vitro study, these NPs showed little cytotoxicity but high cellular uptake. Moreover, they revealed the potential for transfection and PD-L1 knockout in an in vitro cell model. Furthermore, F25P1S0.5 containing 25 &#x3bc;g of plasmid/stPEI/HSA NPs noncovalently bound to 1 &#x3bc;g of plasmids and 0.5 &#x3bc;g siRNA was prepared to simultaneously deliver plasmids and siRNA. An in vitro study demonstrated that the siRNA did not interfere with the transfection of plasmids and showed a high-transfection efficiency with a synergistic effect on inhibition of PD-L1 expression by 21.95%.<br><br>CONCLUSION: The plasmids/stPEI/HSA NPs could be a promising tool for gene delivery and improved immunotherapy.},
}
@article {pmid30464262,
year = {2018},
author = {He, YJ and Meghani, K and Caron, MC and Yang, C and Ronato, DA and Bian, J and Sharma, A and Moore, J and Niraj, J and Detappe, A and Doench, JG and Legube, G and Root, DE and D'Andrea, AD and Drané, P and De, S and Konstantinopoulos, PA and Masson, JY and Chowdhury, D},
title = {DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.},
journal = {Nature},
volume = {563},
number = {7732},
pages = {522-526},
pmid = {30464262},
issn = {1476-4687},
support = {R01 CA142698/CA/NCI NIH HHS/United States ; R01 CA208244/CA/NCI NIH HHS/United States ; },
mesh = {BRCA1 Protein/*deficiency/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromosome Aberrations ; Cytoplasmic Dyneins/*metabolism ; DNA/*metabolism ; DNA Damage/drug effects ; Drug Resistance, Neoplasm/drug effects ; Female ; Gene Editing ; *Genes, BRCA1 ; Genomic Instability/drug effects ; Homologous Recombination/drug effects ; Humans ; MRE11 Homologue Protein/*metabolism ; Mutation ; Ovarian Neoplasms/genetics/pathology ; Platinum/pharmacology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Protein Binding ; *Recombinational DNA Repair/drug effects ; Transcription Factors/metabolism ; },
abstract = {Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.},
}
@article {pmid30463938,
year = {2019},
author = {Gas-Pascual, E and Ichikawa, HT and Sheikh, MO and Serji, MI and Deng, B and Mandalasi, M and Bandini, G and Samuelson, J and Wells, L and West, CM},
title = {CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology.},
journal = {The Journal of biological chemistry},
volume = {294},
number = {4},
pages = {1104-1125},
pmid = {30463938},
issn = {1083-351X},
support = {R01 GM037539/GM/NIGMS NIH HHS/United States ; R21 AI123161/AI/NIAID NIH HHS/United States ; P41 GM103390/GM/NIGMS NIH HHS/United States ; R01 GM084383/GM/NIGMS NIH HHS/United States ; T32 AI060546/AI/NIAID NIH HHS/United States ; R01 AI110638/AI/NIAID NIH HHS/United States ; P41 RR018502/RR/NCRR NIH HHS/United States ; P41 GM103490/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Knockout Techniques ; Gene Library ; *Glycomics ; Polysaccharides/*genetics/*metabolism ; Toxoplasma/*genetics/*metabolism ; },
abstract = {Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but like nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes, but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, a novel HexNAc-GalNAc-mucin-type O-glycan, and Tn-antigen; identified the glycosyltransferases for assembling novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans; and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes.},
}
@article {pmid30463021,
year = {2018},
author = {Kalinava, N and Ni, JZ and Gajic, Z and Kim, M and Ushakov, H and Gu, SG},
title = {C. elegans Heterochromatin Factor SET-32 Plays an Essential Role in Transgenerational Establishment of Nuclear RNAi-Mediated Epigenetic Silencing.},
journal = {Cell reports},
volume = {25},
number = {8},
pages = {2273-2284.e3},
pmid = {30463021},
issn = {2211-1247},
support = {P40 OD010440/OD/NIH HHS/United States ; R01 GM111752/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/genetics/*physiology ; *Epigenesis, Genetic ; Germ Cells/metabolism ; Heterochromatin/*metabolism ; Histone Methyltransferases/genetics/*physiology ; Histones/metabolism ; Lysine/metabolism ; Mutation/genetics ; *RNA Interference ; RNA, Double-Stranded/metabolism ; Transcription, Genetic ; },
abstract = {The dynamic process by which nuclear RNAi engages a transcriptionally active target, before the repressive state is stably established, remains largely a mystery. Here, we found that the onset of exogenous dsRNA-induced nuclear RNAi in C. elegans is a transgenerational process, and it requires a putative histone methyltransferase (HMT), SET-32. By developing a CRISPR-based genetic approach, we found that silencing establishment at the endogenous targets of germline nuclear RNAi also requires SET-32. Although SET-32 and two H3K9 HMTs, MET-2 and SET-25, are dispensable for the maintenance of silencing, they do contribute to transcriptional repression in mutants that lack the germline nuclear Argonaute protein HRDE-1, suggesting a conditional role of heterochromatin in the maintenance phase. Our study indicates that (1) establishment and maintenance of siRNA-guided transcriptional repression are two distinct processes with different genetic requirements and (2) the rate-limiting step of the establishment phase is a transgenerational, chromatin-based process.},
}
@article {pmid30462638,
year = {2018},
author = {LaRoche-Johnston, F and Monat, C and Coulombe, S and Cousineau, B},
title = {Bacterial group II introns generate genetic diversity by circularization and trans-splicing from a population of intron-invaded mRNAs.},
journal = {PLoS genetics},
volume = {14},
number = {11},
pages = {e1007792},
pmid = {30462638},
issn = {1553-7404},
mesh = {Bacteria/*genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Base Sequence ; DNA Transposable Elements/genetics ; Evolution, Molecular ; Genetic Variation ; Humans ; *Introns ; Lactococcus lactis/genetics/metabolism ; Models, Genetic ; Nucleic Acid Conformation ; RNA/chemistry/genetics/metabolism ; RNA, Bacterial/chemistry/*genetics/metabolism ; RNA, Circular ; RNA, Messenger/chemistry/genetics/metabolism ; Retroelements ; Spliceosomes/genetics ; Trans-Splicing ; },
abstract = {Group II introns are ancient retroelements that significantly shaped the origin and evolution of contemporary eukaryotic genomes. These self-splicing ribozymes share a common ancestor with the telomerase enzyme, the spliceosome machinery as well as the highly abundant spliceosomal introns and non-LTR retroelements. More than half of the human genome thus consists of various elements that evolved from ancient group II introns, which altogether significantly contribute to key functions and genetic diversity in eukaryotes. Similarly, group II intron-related elements in bacteria such as abortive phage infection (Abi) retroelements, diversity generating retroelements (DGRs) and some CRISPR-Cas systems have evolved to confer important functions to their hosts. In sharp contrast, since bacterial group II introns are scarce, irregularly distributed and frequently spread by lateral transfer, they have mainly been considered as selfish retromobile elements with no beneficial function to their host. Here we unveil a new group II intron function that generates genetic diversity at the RNA level in bacterial cells. We demonstrate that Ll.LtrB, the model group II intron from Lactococcus lactis, recognizes specific sequence motifs within cellular mRNAs by base pairing, and invades them by reverse splicing. Subsequent splicing of ectopically inserted Ll.LtrB, through circularization, induces a novel trans-splicing pathway that generates exon 1-mRNA and mRNA-mRNA intergenic chimeras. Our data also show that recognition of upstream alternative circularization sites on intron-interrupted mRNAs release Ll.LtrB circles harboring mRNA fragments of various lengths at their splice junction. Intergenic trans-splicing and alternative circularization both produce novel group II intron splicing products with potential new functions. Overall, this work describes new splicing pathways in bacteria that generate, similarly to the spliceosome in eukaryotes, genetic diversity at the RNA level while providing additional functional and evolutionary links between group II introns, spliceosomal introns and the spliceosome.},
}
@article {pmid30462300,
year = {2019},
author = {Shen, CC and Hsu, MN and Chang, CW and Lin, MW and Hwu, JR and Tu, Y and Hu, YC},
title = {Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation.},
journal = {Nucleic acids research},
volume = {47},
number = {3},
pages = {e13},
pmid = {30462300},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/biosynthesis/*genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; *Gene Expression Regulation ; Humans ; Kinetics ; Mutagenesis ; Mutation ; Protein Biosynthesis ; Transcription, Genetic ; },
abstract = {CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.},
}
@article {pmid30461368,
year = {2019},
author = {Bose, P and Armstrong, GAB and Drapeau, P},
title = {Neuromuscular junction abnormalities in a zebrafish loss-of-function model of TDP-43.},
journal = {Journal of neurophysiology},
volume = {121},
number = {1},
pages = {285-297},
doi = {10.1152/jn.00265.2018},
pmid = {30461368},
issn = {1522-1598},
support = {//CIHR/Canada ; },
mesh = {3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology ; Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Calcium Channel Agonists/pharmacology ; Calcium Channels, L-Type/metabolism ; DNA-Binding Proteins/*genetics/*metabolism ; Disease Models, Animal ; Genotype ; *Loss of Function Mutation ; Motor Activity/drug effects/physiology ; Neuromuscular Junction/drug effects/growth &amp; development/*metabolism/*pathology ; Synaptic Transmission/drug effects/physiology ; TDP-43 Proteinopathies/drug therapy/metabolism/pathology ; Zebrafish ; Zebrafish Proteins/*genetics/*metabolism ; },
abstract = {Almost 90% of amyotrophic lateral sclerosis (ALS) cases are characterized by the presence of aggregates of insoluble, misfolded cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43). Distal axonopathy with impaired neuromuscular junctions (NMJs) before motor neuron degeneration or clinical onset of symptoms has been hypothesized as an early pathology in ALS. However, synaptic defects at the NMJ caused by TDP-43 mutations have not been characterized. In this study, we examined a previously reported zebrafish line expressing the tardbp[Y220X/Y220X] variant, which results in an unstable and degraded protein. These tardbp[-/-] larvae, however, mature normally due to the upregulated expression of an alternative splice variant of the tardbp paralog tardbp-like, or tardbpl. We generated a mutant line with a CRISPR/Cas9-mediated 5-base pair deletion encompassing the ATG start codon of tardbpl and in-crossed these with tardbp[-/-] mutants to obtain tardbp[-/-] and tardbpl[-/-] double mutants, herein referred to as hom/hom. We subsequently characterized morphological, coiling, locomotor, synaptic, and NMJ structural abnormalities in the hom/hom mutants and in their genotypic controls. We observed that hom/hom mutants displayed gross morphological defects, early lethality, reduced locomotor function, aberrant quantal transmission, and perturbed synapse architecture at the NMJ. We further employed pharmacological manipulations in an effort to rescue phenotypic defects and observed that tardbp[+/-]; tardbpl[-/-] (herein referred to as het/hom) mutants, but not hom/hom mutants, were sensitive to chronic treatments of BAY K 8644, an L-type calcium channel agonist. This result highlights the importance of partial vs. complete loss of allelic functions of TDP-43. NEW &amp; NOTEWORTHY This study highlights the importance of partial vs. complete loss of allelic functions of TDP-43 in a zebrafish loss of function model, thus making it an attractive tool for drug screening approaches.},
}
@article {pmid30461029,
year = {2018},
author = {Jansson, S},
title = {Gene-edited plants: What is happening now?.},
journal = {Physiologia plantarum},
volume = {164},
number = {4},
pages = {370-371},
doi = {10.1111/ppl.12853},
pmid = {30461029},
issn = {1399-3054},
mesh = {Agriculture/legislation &amp; jurisprudence/methods/trends ; *CRISPR-Cas Systems ; Gene Editing/legislation &amp; jurisprudence/*methods/trends ; Plants/*genetics ; Plants, Genetically Modified ; },
}
@article {pmid30460568,
year = {2019},
author = {Lawrenson, T and Harwood, WA},
title = {Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1900},
number = {},
pages = {217-232},
doi = {10.1007/978-1-4939-8944-7_14},
pmid = {30460568},
issn = {1940-6029},
support = {BB/N019466/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013511/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA, Plant/genetics/isolation &amp; purification ; Gene Knockout Techniques/*methods ; Genetic Loci ; Genome, Plant ; Hordeum/*genetics ; Mutation/genetics ; Polymerase Chain Reaction ; RNA, Guide/genetics ; Sequence Analysis, DNA ; Transgenes ; },
abstract = {Knockout mutants are an invaluable reverse genetics tool which have not been well developed in crop species compared to models like Arabidopsis. However, the emergence of CRISPR/Cas9 has changed this situation making the generation of such mutants accessible to many crops including barley. A single T-DNA construct can be transformed into barley immature embryos and stable transgenic lines regenerated through tissue culture which contain targeted mutations. Mutations are detected in T0 plants and go on in subsequent T1 and T2 generations to segregate from T-DNA, leaving lines which are non-transgenic and carrying a variety of mutations at the target locus. These mutations can be targeted to a particular gene of interest in order to bring about a loss of function creating a knockout mutant.},
}
@article {pmid30459943,
year = {2018},
author = {Tang, Y and Fu, Y},
title = {Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing.},
journal = {Cell &amp; bioscience},
volume = {8},
number = {},
pages = {59},
pmid = {30459943},
issn = {2045-3701},
abstract = {Artificial nuclease-dependent DNA cleavage systems (zinc-finger nuclease, ZFN; transcription activator like effectors, TALENs) and exogenous nucleic acid defense systems (CRISPR/Cas) have been used in the new era for genome modification. The most widely used toolbox for genome editing, modulation and detection contains Types II, V and VI of CRISPR/Cas Class 2 systems, categorized and characterized by Cas9, Cas12a and Cas13 respectively. In this review, we (1) elaborate on the definition, classification, structures of CRISPR/Cas Class 2 systems; (2) advance our understanding of new molecular mechanisms and recent progress in their applications, especially beyond genome-editing applications; (3) provide the insights on the specificity, efficiency and versatility of each tool; (4) elaborate the enhancement on specificity and efficiency of the CRISPR/Cas toolbox. The expanding and concerted usage of the CRISPR/Cas tools is making them more powerful in genome editing and other biotechnology applications.},
}
@article {pmid30459428,
year = {2018},
author = {Huo, Y and Li, T and Wang, N and Dong, Q and Wang, X and Jiang, T},
title = {Cryo-EM structure of Type III-A CRISPR effector complex.},
journal = {Cell research},
volume = {28},
number = {12},
pages = {1195-1197},
pmid = {30459428},
issn = {1748-7838},
mesh = {Bacterial Proteins/*chemistry ; CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Cryoelectron Microscopy/methods ; Models, Molecular ; Multiprotein Complexes/chemistry ; Protein Conformation ; RNA, Bacterial/chemistry ; Thermococcus/*metabolism ; },
}
@article {pmid30459378,
year = {2018},
author = {Callaway, E},
title = {Ban on 'gene drives' is back on the UN's agenda - worrying scientists.},
journal = {Nature},
volume = {563},
number = {7732},
pages = {454-455},
doi = {10.1038/d41586-018-07436-4},
pmid = {30459378},
issn = {1476-4687},
mesh = {Animals ; Anopheles/genetics ; CRISPR-Cas Systems/genetics ; Consensus ; Disease Eradication ; Disease Vectors ; *Dissent and Disputes ; Ecosystem ; Gene Drive Technology/*legislation &amp; jurisprudence ; Gene Editing ; Humans ; International Cooperation/legislation &amp; jurisprudence ; Research Personnel/*psychology ; Safety Management/*legislation &amp; jurisprudence ; *Uncertainty ; United Nations/*legislation &amp; jurisprudence ; },
}
@article {pmid30459263,
year = {2019},
author = {Wang, D and Samsulrizal, NH and Yan, C and Allcock, NS and Craigon, J and Blanco-Ulate, B and Ortega-Salazar, I and Marcus, SE and Bagheri, HM and Perez Fons, L and Fraser, PD and Foster, T and Fray, R and Knox, JP and Seymour, GB},
title = {Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.},
journal = {Plant physiology},
volume = {179},
number = {2},
pages = {544-557},
pmid = {30459263},
issn = {1532-2548},
support = {BB/M025918/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Wall/chemistry/metabolism ; Enzymes/*genetics/metabolism ; Esterification ; Fruit/*physiology ; Galactans/genetics/metabolism ; Gene Expression Regulation, Plant ; Gene Silencing ; Lycopersicon esculentum/genetics/*physiology ; Mutation ; Pectins/genetics/immunology/*metabolism ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.},
}
@article {pmid30458018,
year = {2018},
author = {Bazan, B and Wiktor, M and Maszczak-Seneczko, D and Olczak, T and Kaczmarek, B and Olczak, M},
title = {Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4.},
journal = {PloS one},
volume = {13},
number = {11},
pages = {e0207521},
pmid = {30458018},
issn = {1932-6203},
mesh = {Amino Acid Motifs/*genetics ; Amino Acid Sequence/genetics ; CRISPR-Cas Systems/genetics ; Dipeptides/chemistry/genetics ; Endoplasmic Reticulum/*chemistry/genetics ; Glucosamine/analogs &amp; derivatives/chemistry ; Glycosylation ; Golgi Apparatus/*chemistry/genetics ; Hep G2 Cells ; Humans ; Lysine/chemistry/genetics ; Nucleotide Transport Proteins/antagonists &amp; inhibitors/*chemistry/genetics ; Signal Transduction ; Uridine Diphosphate Sugars/chemistry ; },
abstract = {SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.},
}
@article {pmid30457360,
year = {2018},
author = {Zhen, S and Lu, J and Chen, W and Zhao, L and Li, X},
title = {Synergistic Antitumor Effect on Bladder Cancer by Rational Combination of Programmed Cell Death 1 Blockade and CRISPR-Cas9-Mediated Long Non-Coding RNA Urothelial Carcinoma Associated 1 Knockout.},
journal = {Human gene therapy},
volume = {29},
number = {12},
pages = {1352-1363},
doi = {10.1089/hum.2018.048},
pmid = {30457360},
issn = {1557-7422},
mesh = {Animals ; Antineoplastic Agents/*pharmacology ; B7-H1 Antigen/*antagonists &amp; inhibitors/metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cytokines/metabolism ; Dendritic Cells/drug effects/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; *Gene Knockout Techniques ; Gene Targeting ; Humans ; Mice, SCID ; RNA, Guide/metabolism ; RNA, Long Noncoding/*genetics/metabolism ; Tumor Microenvironment/drug effects ; Urinary Bladder Neoplasms/genetics/*pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Targeted therapy produces objective responses in bladder cancer patients, although the responses can be short. Meanwhile, response rates to immune therapy are lower, but the effects are more durable. Based on these findings, it was hypothesized that urothelial carcinoma associated 1 (UCA1)-targeted therapy could synergize with programmed cell death 1 (PD-1) blockade to enhance antitumor activity. To test this hypothesis, the effects of CRISPR-Cas9 targeting of UCA1 and PD-1 were assessed in vitro and in vivo. It was found that gRNA/cas9-targeted UCA1 induced apoptosis of 5637 bladder cancer cells, whereas PD-1 gene knockout could be achieved by electroporation of gRNA/cas9 targeting PD-1, as detected by polymerase chain reaction. In 5637 cell-xenografted humanized SCID mice, stimulation with CRISPR-Cas9 systems, immune phenotypes, and cytokine expression of human dendritic cells (DCs) was detected by flow cytometry, and polymerase chain reaction, respectively. The results of these assays suggested that the gRNA/cas9 treatment upregulated expression of CD80, CD83, and CD86 and significantly increased interleukin (IL)-6, IL-12, and IL-23 and tumor necrosis factor alpha mRNA levels. Co-administration of anti-PD-1 and anti-UCA1 treatment suppressed tumor growth and markedly improved survival of 5637 xenografted mice. Additionally, the combination treatment increased interferon gamma production by T cells that subsequently enhanced the expression of Th1-associated immune-stimulating genes to reduce transcription of regulatory/suppressive immune genes and reshape the tumor microenvironment from an immunosuppressive to a stimulatory state. Finally, anti-UCA1 treatment was shown to induce interferon gamma-dependent programmed cell death ligand 1 expression within 5637 xenograft tumors in vivo. Together, these results demonstrate potent synergistic effects of a combination therapy using LncRNA UCA1-targeted therapy and immune checkpoint blockade of PD-1, thus supporting the translational potential of this combination strategy for clinical treatment of bladder cancer.},
}
@article {pmid30455686,
year = {2018},
author = {Machado-Pineda, Y and Cardeñes, B and Reyes, R and López-Martín, S and Toribio, V and Sánchez-Organero, P and Suarez, H and Grötzinger, J and Lorenzen, I and Yáñez-Mó, M and Cabañas, C},
title = {CD9 Controls Integrin α5β1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {2474},
pmid = {30455686},
issn = {1664-3224},
mesh = {ADAM17 Protein/genetics/immunology/*metabolism ; Antibodies, Monoclonal/metabolism ; CRISPR-Cas Systems ; Cell Adhesion ; Fibronectins/metabolism ; Gene Knockdown Techniques ; Humans ; Integrin alpha5beta1/metabolism ; K562 Cells ; Leukocytes/*immunology ; Neoplastic Cells, Circulating/*immunology ; Protein Binding ; Tetraspanin 29/*metabolism ; },
abstract = {Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17.},
}
@article {pmid30454954,
year = {2018},
author = {Ylä-Herttuala, S},
title = {CRISPR/Cas9 and p53: An Odd Couple Requiring Relationship Management.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {12},
pages = {2711},
pmid = {30454954},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; DNA Repair ; Gene Editing ; Genomics/methods ; Humans ; Tumor Suppressor Protein p53/*genetics ; },
}
@article {pmid30452298,
year = {2018},
author = {Rich, K and Terry, SF},
title = {CRISPR-Cas9: New Heights, New Hesitations.},
journal = {Genetic testing and molecular biomarkers},
volume = {22},
number = {11},
pages = {635-636},
doi = {10.1089/gtmb.2018.0267},
pmid = {30452298},
issn = {1945-0257},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*ethics/methods ; Humans ; },
}
@article {pmid30451898,
year = {2018},
author = {Santofimia-Castaño, P and Lan, W and Bintz, J and Gayet, O and Carrier, A and Lomberk, G and Neira, JL and González, A and Urrutia, R and Soubeyran, P and Iovanna, J},
title = {Inactivation of NUPR1 promotes cell death by coupling ER-stress responses with necrosis.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16999},
pmid = {30451898},
issn = {2045-2322},
support = {R01 CA178627/CA/NCI NIH HHS/United States ; R01 DK052913/DK/NIDDK NIH HHS/United States ; },
mesh = {Acinar Cells ; Adenosine Triphosphate/metabolism ; Animals ; Apoptosis ; Basic Helix-Loop-Helix Transcription Factors/antagonists &amp; inhibitors/genetics/metabolism ; CRISPR-Cas Systems ; *Cell Death ; Cells, Cultured ; DNA-Binding Proteins/*physiology ; *Endoplasmic Reticulum Stress ; Humans ; Mice ; Mice, Knockout ; Mitochondria/metabolism/*pathology ; Mitophagy ; *Necrosis ; Neoplasm Proteins/antagonists &amp; inhibitors/genetics/metabolism/*physiology ; Oxidative Phosphorylation ; Pancreas/metabolism/pathology ; Pancreatic Neoplasms/metabolism/*pathology ; Pancreatitis/metabolism/*pathology ; },
abstract = {It was already described that genetic inhibition of NUPR1 induces tumor growth arrest. In this paper we studied the metabolism changes after NUPR1 downregulation in pancreatic cancer cells, which results in a significant decrease of OXPHOS activity with a concomitant lower ATP production which precedes the necrotic cell death. We demonstrated that NUPR1 downregulation induces a mitochondrial failure with a loss of the mitochondrial membrane potential, a strong increase in ROS production and a concomitant relocalization of mitochondria to the vicinity of the endoplasmic reticulum (ER). In addition, the transcriptomic analysis of NUPR1-deficient cells shows a decrease in the expression of some ER stress response-associated genes. Indeed, in ER stressors-treated cells with thapsigargin, brefeldin A or tunicamycin, a greater increase in necrosis and decrease of ATP content was observed in NUPR1-defficent cells. Finally, in vivo experiments, using acute pancreatitis which induces ER stress as well as NUPR1 activation, we observed that NUPR1 expression protects acinar cells from necrosis in mice. Importantly, we also report that the cell death observed after knocking-down NUPR1 expression is completely reversed by incubation with Necrostatin-1, but not by inhibiting caspase activity with Z-VAD-FMK. Altogether, these data enable us to describe a model in which inactivation of NUPR1 in pancreatic cancer cells results in an ER stress that induces a mitochondrial malfunction, a deficient ATP production and, as consequence, the cell death mediated by a programmed necrosis.},
}
@article {pmid30451839,
year = {2018},
author = {Bolukbasi, MF and Liu, P and Luk, K and Kwok, SF and Gupta, A and Amrani, N and Sontheimer, EJ and Zhu, LJ and Wolfe, SA},
title = {Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4856},
pmid = {30451839},
issn = {2041-1723},
support = {R01 AI117839/AI/NIAID NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; R01 HL093766/HL/NHLBI NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics/metabolism ; *Base Sequence ; *CRISPR-Cas Systems ; Carrier Proteins/genetics/metabolism ; Endonucleases/*genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering ; Genome, Human ; HEK293 Cells ; Humans ; Jurkat Cells ; K562 Cells ; Mutant Chimeric Proteins/*genetics/metabolism ; Neisseria meningitidis/enzymology/genetics ; Nuclear Proteins/genetics/metabolism ; Repressor Proteins ; *Sequence Deletion ; Staphylococcus aureus/enzymology/genetics ; },
abstract = {The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.},
}
@article {pmid30451675,
year = {2018},
author = {Croteau, FR and Rousseau, GM and Moineau, S},
title = {[The CRISPR-Cas system: beyond genome editing].},
journal = {Medecine sciences : M/S},
volume = {34},
number = {10},
pages = {813-819},
doi = {10.1051/medsci/2018215},
pmid = {30451675},
issn = {1958-5381},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Gene Editing/*methods/trends ; Genetic Engineering/methods/*trends ; Genetic Therapy/methods/trends ; *Genome ; Humans ; },
abstract = {CRISPR-Cas is an adaptive immune system used by many microbes to defend against nucleic acids invasion such as viral genomes. The microbial system uses its CRISPR locus to store genetic information that will generate short CRISPR RNAs. The latter with endonucleases (Cas) prevent future viral infections. Parts of this system were exploited to develop a powerful genome editing tool that was adapted for a variety of organisms. The ability of the CRISPR-Cas9 technology to effectively and precisely cut a targeted genomic DNA region has the potential to may be one day cure genetic diseases. The malleability of this editing tool also offers a wide range of possibilities from modulations of gene expression to epigenetic modifications. The natural CRISPR loci found in bacteria can be used to differentiate microbial strains or to study the interactions between bacteria and its habitat. Addressing CRISPR-Cas fundamentals in microbes and its popular use in eukaryotes, this review presents an update on a system that has revolutionized biological sciences.},
}
@article {pmid30449619,
year = {2018},
author = {Shifrut, E and Carnevale, J and Tobin, V and Roth, TL and Woo, JM and Bui, CT and Li, PJ and Diolaiti, ME and Ashworth, A and Marson, A},
title = {Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function.},
journal = {Cell},
volume = {175},
number = {7},
pages = {1958-1971.e15},
pmid = {30449619},
issn = {1097-4172},
support = {F30 DK120213/DK/NIDDK NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics/immunology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; *Genome, Human ; Genome-Wide Association Study ; Humans ; T-Lymphocytes/cytology/*immunology ; },
abstract = {Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.},
}
@article {pmid30447330,
year = {2019},
author = {Kim, CL and Lee, GM},
title = {Improving recombinant bone morphogenetic protein-4 (BMP-4) production by autoregulatory feedback loop removal using BMP receptor-knockout CHO cell lines.},
journal = {Metabolic engineering},
volume = {52},
number = {},
pages = {57-67},
doi = {10.1016/j.ymben.2018.11.003},
pmid = {30447330},
issn = {1096-7184},
mesh = {Animals ; Antimetabolites/pharmacology ; Bone Morphogenetic Protein 4/*biosynthesis/*genetics ; Bone Morphogenetic Protein Receptors/antagonists &amp; inhibitors/*genetics ; CHO Cells ; CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Feedback, Physiological ; Gene Expression Profiling ; Gene Knockout Techniques ; Homeostasis ; Methotrexate/pharmacology ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Recombinant Proteins ; },
abstract = {A Chinese hamster ovary (CHO) cell line producing recombinant human bone morphogenetic protein-4 (rhBMP-4) (CHO-BMP-4), which expresses essential components of BMP signal transduction, underwent autocrine BMP-4 signaling. RNA seq analysis on CHO host cells (DG44) treated with rhBMP-4 (20 µg/mL) suggested that rhBMP-4 induced signaling in CHO cells could be a critical factor in limiting rhBMP-4 production and should be removed to improve rhBMP-4 production in recombinant CHO (rCHO) cells. The inhibition of autocrine BMP signaling in CHO-BMP-4 cells by the addition of LDN-193189, a chemical inhibitor of BMP receptor type I, significantly increased the mRNA expression levels of rhBMP-4. To establish BMP signaling-free host cells, a BMP receptor, the BMPRIA or BMPRII gene in DG44 cells, was knocked out using CRISPR/Cas9 gene-editing technology. Using three different knockout (KO) host cell lines as well as a DG44 wild-type (wt) cell line, rCHO cell clones producing rhBMP-4 were generated by a stepwise selection with increasing methotrexate concentrations. KO-derived clones showed a significantly higher maximum rhBMP-4 concentration than wt-derived clones in both batch and fed-batch cultures. Unlike wt-derived clones, KO-derived cell clones were able to produce higher amounts of hBMP-4 transcripts and proteins in the stationary phase of growth and did not experience growth inhibition induced by rhBMP-4. The mean maximum rhBMP-4 concentration of KO host-derived clones was approximately 2.4-fold higher than that of wt-derived clones (P < 0.05). Taken together, the disruption of BMP signaling in CHO cells by knocking out the BMP receptor significantly improved rhBMP-4 production.},
}
@article {pmid30447221,
year = {2019},
author = {Adli, M},
title = {The Biology and Application Areas of CRISPR Technologies.},
journal = {Journal of molecular biology},
volume = {431},
number = {1},
pages = {1-2},
doi = {10.1016/j.jmb.2018.11.012},
pmid = {30447221},
issn = {1089-8638},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; },
}
@article {pmid30446870,
year = {2019},
author = {Liu, Q and Wang, C and Jiao, X and Zhang, H and Song, L and Li, Y and Gao, C and Wang, K},
title = {Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems.},
journal = {Science China. Life sciences},
volume = {62},
number = {1},
pages = {1-7},
doi = {10.1007/s11427-018-9402-9},
pmid = {30446870},
issn = {1869-1889},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Gene Editing/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; *Mutation ; Reproducibility of Results ; },
abstract = {The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM (available at https://doi.org/www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing (NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.},
}
@article {pmid30446729,
year = {2019},
author = {Cloney, R},
title = {The oracle of inDelphi predicts Cas9 repair outcomes.},
journal = {Nature reviews. Genetics},
volume = {20},
number = {1},
pages = {4-5},
pmid = {30446729},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide ; },
}
@article {pmid30446639,
year = {2018},
author = {Wang, H and Shi, Y and Wang, L and Liu, S and Wu, S and Yang, Y and Feyereisen, R and Wu, Y},
title = {CYP6AE gene cluster knockout in Helicoverpa armigera reveals role in detoxification of phytochemicals and insecticides.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4820},
pmid = {30446639},
issn = {2041-1723},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/deficiency/*genetics ; Embryo, Nonmammalian ; Female ; Gene Editing/methods ; Gene Expression ; *Genome, Insect ; Inactivation, Metabolic/*genetics ; Insect Proteins/deficiency/*genetics ; Insecticide Resistance/*genetics ; Insecticides/*metabolism/pharmacology ; Larva/drug effects/enzymology/genetics/growth &amp; development ; Lethal Dose 50 ; Male ; Moths/drug effects/enzymology/*genetics/growth &amp; development ; Multigene Family ; Mutation ; RNA, Guide/genetics/metabolism ; Reverse Genetics ; },
abstract = {The cotton bollworm Helicoverpa armigera, is one of the world's major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.},
}
@article {pmid30446374,
year = {2019},
author = {Gajula, KS},
title = {Designing an Elusive C•G→G•C CRISPR Base Editor.},
journal = {Trends in biochemical sciences},
volume = {44},
number = {2},
pages = {91-94},
doi = {10.1016/j.tibs.2018.10.004},
pmid = {30446374},
issn = {0968-0004},
mesh = {CRISPR-Cas Systems/*genetics ; Cytidine Deaminase/genetics/metabolism ; DNA/*genetics ; DNA Glycosylases/genetics/metabolism ; Gene Editing/*methods ; Humans ; },
abstract = {Protein engineering advances, including DNA repair manipulation of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) machinery, have paved the way for the first set of DNA precision base editors (C•G→T•A and A•T→G•C), with wide-ranging implications for treating many human genetic diseases. By utilizing the latest protein evolution advances, a hypothetical model for the first transversion (C•G→G•C) base editor can now be proposed.},
}
@article {pmid30445568,
year = {2019},
author = {Schaefer, M and Clevert, DA and Weiss, B and Steffen, A},
title = {PAVOOC: designing CRISPR sgRNAs using 3D protein structures and functional domain annotations.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {13},
pages = {2309-2310},
pmid = {30445568},
issn = {1367-4811},
mesh = {Algorithms ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide ; Software ; },
abstract = {SUMMARY: Single-guide RNAs (sgRNAs) targeting the same gene can significantly vary in terms of efficacy and specificity. PAVOOC (Prediction And Visualization of On- and Off-targets for CRISPR) is a web-based CRISPR sgRNA design tool that employs state of the art machine learning models to prioritize most effective candidate sgRNAs. In contrast to other tools, it maps sgRNAs to functional domains and protein structures and visualizes cut sites on corresponding protein crystal structures. Furthermore, PAVOOC supports homology-directed repair template generation for genome editing experiments and the visualization of the mutated amino acids in 3D.<br><br>PAVOOC is available under https://pavooc.me and accessible using modern browsers (Chrome/Chromium recommended). The source code is hosted at github.com/moritzschaefer/pavooc under the MIT License. The backend, including data processing steps, and the frontend are implemented in Python 3 and ReactJS, respectively. All components run in a simple Docker environment.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid30444997,
year = {2018},
author = {Mo, CY and Marraffini, LA},
title = {If You'd Like to Stop a Type III CRISPR Ribonuclease, Then You Should Put a Ring (Nuclease) on It.},
journal = {Molecular cell},
volume = {72},
number = {4},
pages = {608-609},
doi = {10.1016/j.molcel.2018.10.048},
pmid = {30444997},
issn = {1097-4164},
support = {/HHMI/Howard Hughes Medical Institute/United States ; F32 GM128271/GM/NIGMS NIH HHS/United States ; DP1 GM128184/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine Nucleotides ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oligoribonucleotides ; *Ribonucleases ; },
abstract = {Athukoralage et al. (2018) identify a new class of nuclease that degrades cyclic oligoadenylate (cOA), a second messenger that activates non-specific RNA degradation by the type III CRISPR-Cas accessory RNase Csm6/Csx1. This discovery provides a mechanism for regulating the degradation of foreign transcripts during the type III CRISPR immune response.},
}
@article {pmid30443021,
year = {2018},
author = {Riad, A and Zeng, C and Weng, CC and Winters, H and Xu, K and Makvandi, M and Metz, T and Carlin, S and Mach, RH},
title = {Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16845},
pmid = {30443021},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; *Endocytosis ; Gene Editing ; HeLa Cells ; Humans ; Insulin/metabolism ; Ligands ; Membrane Proteins/*metabolism ; Protein Binding ; Receptors, LDL/*metabolism ; Receptors, Progesterone/*metabolism ; Receptors, sigma/*metabolism ; Somatostatin/metabolism ; },
abstract = {CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (σ2R) radioligands, [[125]I]RHM-4 or [[3]H]DTG, but a reduction in the receptor affinity of both radioligands was observed. TMEM97 KO resulted in a complete loss of binding of [[125]I]RHM-4 and a significant reduction in binding of [[3]H]DTG. TMEM97 KO and PGRMC1 KO resulted in an equal reduction in the rate of uptake of fluorescently-tagged or [3]H-labeled LDL, and knocking out both proteins did not result in a further rate of reduction of LDL uptake. Confocal microscopy and Proximity Ligation Assay studies indicated a clear co-localization of LDLR, PGRMC1 and TMEM97. These data indicate that the formation of a ternary complex of LDLR-PGRMC1-TMEM97 is necessary for the rapid internalization of LDL by LDLR.},
}
@article {pmid30442934,
year = {2018},
author = {Lee, HK and Willi, M and Miller, SM and Kim, S and Liu, C and Liu, DR and Hennighausen, L},
title = {Targeting fidelity of adenine and cytosine base editors in mouse embryos.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4804},
pmid = {30442934},
issn = {2041-1723},
support = {R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; },
mesh = {Adenine/*metabolism ; Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cytosine/*metabolism ; Embryo, Mammalian ; Gene Editing/*methods ; Genetic Loci ; Mice ; Microinjections ; *Point Mutation ; RNA, Guide/genetics/metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Zygote ; },
abstract = {Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyze on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more than 430 alleles generated from mouse zygotic injections using newly generated and published sequencing data. ABE predominantly generates anticipated A•T-to-G•C edits. Among CBEs, SaBE3 and BE4, result in the highest frequencies of anticipated C•G-to-T•A products relative to editing byproducts. Together, these findings highlight the remarkable fidelity of ABE in mouse embryos and identify preferred CBE variants when fidelity in vivo is critical.},
}
@article {pmid30442929,
year = {2018},
author = {Koh, B and Abdul Qayum, A and Srivastava, R and Fu, Y and Ulrich, BJ and Janga, SC and Kaplan, MH},
title = {A conserved enhancer regulates Il9 expression in multiple lineages.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4803},
pmid = {30442929},
issn = {2041-1723},
support = {R01 GM123314/GM/NIGMS NIH HHS/United States ; R01 AI129241/AI/NIAID NIH HHS/United States ; T32 AI060519/AI/NIAID NIH HHS/United States ; P30 CA082709/CA/NCI NIH HHS/United States ; U54 DK106846/DK/NIDDK NIH HHS/United States ; T32 DK007519/DK/NIDDK NIH HHS/United States ; R01 AI057459/AI/NIAID NIH HHS/United States ; R03 AI135356/AI/NIAID NIH HHS/United States ; T32 GM077229/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage/genetics/*immunology ; Conserved Sequence ; *Enhancer Elements, Genetic ; Female ; Gene Editing ; Gene Expression Regulation ; Humans ; Interleukin-9/*genetics/immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Primary Cell Culture ; Protein Binding ; T-Lymphocytes, Helper-Inducer/cytology/*immunology ; Transcription Factors/*genetics/immunology ; },
abstract = {Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.},
}
@article {pmid30442778,
year = {2018},
author = {Kory, N and Wyant, GA and Prakash, G and Uit de Bos, J and Bottanelli, F and Pacold, ME and Chan, SH and Lewis, CA and Wang, T and Keys, HR and Guo, YE and Sabatini, DM},
title = {SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6416},
pages = {},
pmid = {30442778},
issn = {1095-9203},
support = {R01 CA103866/CA/NCI NIH HHS/United States ; S10 OD020142/OD/NIH HHS/United States ; T32 GM007287/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R37 AI047389/AI/NIAID NIH HHS/United States ; R01 CA129105/CA/NCI NIH HHS/United States ; },
mesh = {Biological Transport ; CRISPR-Cas Systems ; Carbon/metabolism ; Genetic Testing ; Humans ; Jurkat Cells ; K562 Cells ; Mitochondria/*metabolism ; Serine/*metabolism ; Sodium-Glucose Transporter 1/genetics/*metabolism ; },
abstract = {One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.},
}
@article {pmid30442540,
year = {2019},
author = {Claes, C and Van Den Daele, J and Boon, R and Schouteden, S and Colombo, A and Monasor, LS and Fiers, M and Ordovás, L and Nami, F and Bohrmann, B and Tahirovic, S and De Strooper, B and Verfaillie, CM},
title = {Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2.},
journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association},
volume = {15},
number = {3},
pages = {453-464},
doi = {10.1016/j.jalz.2018.09.006},
pmid = {30442540},
issn = {1552-5279},
mesh = {Amyloid beta-Protein Precursor/genetics/metabolism ; Animals ; Brain ; CRISPR-Cas Systems ; Cells, Cultured ; Escherichia coli ; Membrane Glycoproteins/*deficiency/genetics ; Mice, Transgenic ; Microglia/*metabolism ; Monocytes/*metabolism ; Phagocytosis ; Plaque, Amyloid/*metabolism ; Pluripotent Stem Cells ; Presenilin-1/genetics/metabolism ; Receptors, Immunologic/*deficiency/genetics ; },
abstract = {INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2[R47H] mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2[+/R47H] human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2[+/R47H] mutation and loss of TREM2 on phagocytosis.<br><br>METHODS: We generated isogenic TREM2[+/R47H], TREM2[+/-], and TREM2[-/-] hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1[+/-] mouse brains.<br><br>RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2[+/-] and TREM2[-/-] hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2[+/R47H] progeny.<br><br>DISCUSSION: In&#xa0;vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2[+/R47H] mutation but was significantly impaired in TREM2[+/-] and TREM2[-/-] progeny.},
}
@article {pmid30442181,
year = {2018},
author = {Ji, X and Si, X and Zhang, Y and Zhang, H and Zhang, F and Gao, C},
title = {Conferring DNA virus resistance with high specificity in plants using virus-inducible genome-editing system.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {197},
pmid = {30442181},
issn = {1474-760X},
mesh = {Arabidopsis ; *CRISPR-Cas Systems ; *Disease Resistance ; *Geminiviridae ; Gene Editing/*methods ; Tobacco ; },
abstract = {The CRISPR/Cas9 system has recently been engineered to confer resistance to geminiviruses in plants. However, we show here that the usefulness of this antiviral strategy is undermined by off-target effects identified by deep sequencing in Arabidopsis. We construct two virus-inducible CRISPR/Cas9 vectors that efficiently inhibit beet severe curly top virus (BSCTV) accumulation in both transient assays (Nicotiana benthamiana) and transgenic lines (Arabidopsis). Deep sequencing detects no off-target effect in candidate sites of the transgenic Arabidopsis. This kind of virus-inducible genome-editing system should be widely applicable for generating virus-resistant plants without off-target costs.},
}
@article {pmid30439390,
year = {2019},
author = {Yamaguchi, H and de Lecea, L},
title = {In vivo cell type-specific CRISPR gene editing for sleep research.},
journal = {Journal of neuroscience methods},
volume = {316},
number = {},
pages = {99-102},
pmid = {30439390},
issn = {1872-678X},
support = {R01 AG047671/AG/NIA NIH HHS/United States ; R01 MH087592/MH/NIMH NIH HHS/United States ; R01 MH102638/MH/NIMH NIH HHS/United States ; R01 MH116470/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Brain/metabolism/*physiology ; *CRISPR-Cas Systems ; *Gene Editing ; Sleep/*genetics ; },
abstract = {Sleep is an innate behavior conserved in all animals and, in vertebrates, is regulated by neuronal circuits in the brain. The conventional techniques of forward and reverse genetics have enabled researchers to investigate the molecular mechanisms that regulate sleep and arousal. However, functional interrogation of genes in specific cell subtypes in the brain remains a challenge. Here, we review the background of newly developed gene-editing technologies using engineered CRISPR/Cas9 system and describe the application to interrogate gene functions within genetically-defined brain cell populations in sleep research.},
}
@article {pmid30429597,
year = {2018},
author = {Fei, JF and Lou, WP and Knapp, D and Murawala, P and Gerber, T and Taniguchi, Y and Nowoshilow, S and Khattak, S and Tanaka, EM},
title = {Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).},
journal = {Nature protocols},
volume = {13},
number = {12},
pages = {2908-2943},
doi = {10.1038/s41596-018-0071-0},
pmid = {30429597},
issn = {1750-2799},
mesh = {Ambystoma mexicanum/*genetics ; Animals ; Animals, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Frameshift Mutation ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Phenotype ; RNA, Guide/genetics ; },
abstract = {Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.},
}
@article {pmid30429497,
year = {2018},
author = {Sun, B and Zheng, A and Jiang, M and Xue, S and Yuan, Q and Jiang, L and Chen, Q and Li, M and Wang, Y and Zhang, Y and Luo, Y and Wang, X and Zhang, F and Tang, H},
title = {CRISPR/Cas9-mediated mutagenesis of homologous genes in Chinese kale.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16786},
pmid = {30429497},
issn = {2045-2322},
mesh = {Brassica/*genetics ; CRISPR-Cas Systems/*genetics ; China ; Genes, Plant/*genetics ; Genetic Association Studies ; *Mutagenesis, Site-Directed ; Mutation ; Plant Proteins/genetics ; Plants, Genetically Modified ; *Sequence Homology, Nucleic Acid ; },
abstract = {The clustered regulatory interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has developed into a powerful gene-editing tool that has been successfully applied to various plant species. However, studies on the application of the CRISPR/Cas9 system to cultivated Brassica vegetables are limited. Here, we reported CRISPR/Cas9-mediated genome editing in Chinese kale (Brassica oleracea var. alboglabra) for the first time. A stretch of homologous genes, namely BaPDS1 and BaPDS2, was selected as the target site. Several stable transgenic lines with different types of mutations were generated via Agrobacterium-mediated transformation, including BaPDS1 and BaPDS2 double mutations and BaPDS1 or BaPDS2 single mutations. The overall mutation rate reached 76.47%, and these mutations involved nucleotide changes of fewer than 10 bp. The clear albino phenotype was observed in all of the mutants, including one that harbored a mutation within an intron region, thereby indicating the importance of the intron. Cleavage in Chinese kale using CRISPR/Cas9 was biased towards AT-rich sequences. Furthermore, no off-target events were observed. Functional differences between BaPDS1 and BaPDS2 were also assessed in terms of the phenotypes of the respective mutants. In combination, these findings showed that CRISPR/Cas9-mediated targeted mutagenesis can simultaneously and efficiently modify homologous gene copies of Chinese kale and provide a convenient approach for studying gene function and improving the yield and quality of cultivated Brassica vegetables.},
}
@article {pmid30429256,
year = {2019},
author = {Strich, JR and Chertow, DS},
title = {CRISPR-Cas Biology and Its Application to Infectious Diseases.},
journal = {Journal of clinical microbiology},
volume = {57},
number = {4},
pages = {},
pmid = {30429256},
issn = {1098-660X},
mesh = {Animals ; Archaea/genetics ; Bacteria/genetics ; *CRISPR-Cas Systems ; Communicable Disease Control ; Communicable Diseases/*diagnosis/microbiology ; Gene Editing ; Host-Pathogen Interactions/*genetics ; Humans ; Mice ; Pathology, Molecular ; Point-of-Care Systems ; },
abstract = {Infectious diseases remain a global threat contributing to excess morbidity and death annually, with the persistent potential for destabilizing pandemics. Improved understanding of the pathogenesis of bacteria, viruses, fungi, and parasites, along with rapid diagnosis and treatment of human infections, is essential for improving infectious disease outcomes worldwide. Genomic loci in bacteria and archaea, termed clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, function as an adaptive immune system for prokaryotes, protecting them against foreign invaders. CRISPR-Cas9 technology is now routinely applied for efficient gene editing, contributing to advances in biomedical science. In the past decade, improved understanding of other diverse CRISPR-Cas systems has expanded CRISPR applications, including in the field of infectious diseases. In this review, we summarize the biology of CRISPR-Cas systems and discuss existing and emerging applications to evaluate mechanisms of host-pathogen interactions, to develop accurate and portable diagnostic tests, and to advance the prevention and treatment of infectious diseases.},
}
@article {pmid30429198,
year = {2019},
author = {Li, L and Li, W and Chen, N and Zhao, H and Xu, G and Zhao, Y and Pan, X and Zhang, X and Zhou, L and Yu, D and Li, A and Hu, JF and Cui, J},
title = {FLI1 Exonic Circular RNAs as a Novel Oncogenic Driver to Promote Tumor Metastasis in Small Cell Lung Cancer.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {25},
number = {4},
pages = {1302-1317},
doi = {10.1158/1078-0432.CCR-18-1447},
pmid = {30429198},
issn = {1557-3265},
mesh = {A549 Cells ; Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/blood/genetics/pathology ; Cell Cycle/genetics ; Cell Movement/genetics ; Cell Proliferation/genetics ; Exons/genetics ; Exosomes/genetics ; Heterografts ; Humans ; Mice ; MicroRNAs/*genetics ; Neoplasm Metastasis ; Proto-Oncogene Protein c-fli-1/antagonists &amp; inhibitors/*genetics ; RNA, Circular/*genetics/isolation &amp; purification ; RNA, Small Interfering/genetics ; Small Cell Lung Carcinoma/blood/*genetics/pathology ; rho-Associated Kinases/*genetics ; },
abstract = {PURPOSE: The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3' region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC.<br><br>EXPERIMENTAL DESIGN: The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non-small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver.<br><br>RESULTS: Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1).<br><br>CONCLUSIONS: This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584-ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.},
}
@article {pmid30428163,
year = {2019},
author = {Lin, CY and Nozawa, T and Minowa-Nozawa, A and Toh, H and Aikawa, C and Nakagawa, I},
title = {LAMTOR2/LAMTOR1 complex is required for TAX1BP1-mediated xenophagy.},
journal = {Cellular microbiology},
volume = {21},
number = {4},
pages = {e12981},
doi = {10.1111/cmi.12981},
pmid = {30428163},
issn = {1462-5822},
mesh = {Blotting, Western ; CRISPR-Cas Systems/genetics ; Cell Line ; HeLa Cells ; Humans ; Immunoprecipitation ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Macroautophagy/genetics/*physiology ; Microscopy, Fluorescence ; Neoplasm Proteins/genetics/*metabolism ; Salmonella/*pathogenicity ; },
abstract = {Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium-containing endosomes, and LAMTOR2 was recruited to bacterium-containing damaged endosomes in a LAMTOR1-dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen-containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.},
}
@article {pmid30428077,
year = {2019},
author = {Martín-Pizarro, C and Triviño, JC and Posé, D},
title = {Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis.},
journal = {Journal of experimental botany},
volume = {70},
number = {3},
pages = {885-895},
pmid = {30428077},
issn = {1460-2431},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Flowers/*genetics/growth &amp; development/metabolism ; Fragaria/*genetics/metabolism ; High-Throughput Nucleotide Sequencing ; MADS Domain Proteins/*genetics/metabolism ; Mutagenesis ; Phylogeny ; Plant Proteins/*genetics/metabolism ; Pollen/*genetics/growth &amp; development/metabolism ; Polyploidy ; Sequence Alignment ; },
abstract = {The B-class of MADS-box transcription factors has been studied in many plant species, but remains functionally uncharacterized in Rosaceae. APETALA3 (AP3), a member of this class, controls petal and stamen identities in Arabidopsis. In this study, we identified two members of the AP3 lineage in cultivated strawberry, Fragaria × ananassa, namely FaAP3 and FaTM6. FaTM6, and not FaAP3, showed an expression pattern equivalent to that of AP3 in Arabidopsis. We used the CRISPR/Cas9 genome editing system for the first time in an octoploid species to characterize the function of TM6 in strawberry flower development. An analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed highly efficient genome editing already present in the T0 generation. Phenotypic characterization of the mutant lines indicated that FaTM6 plays a key role in anther development in strawberry. Our results validate the use of the CRISPR/Cas9 system for gene functional analysis in F. × ananassa as an octoploid species, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.},
}
@article {pmid30427863,
year = {2018},
author = {Ismail, NIB and Kato, Y and Matsuura, T and Watanabe, H},
title = {Generation of white-eyed Daphnia magna mutants lacking scarlet function.},
journal = {PloS one},
volume = {13},
number = {11},
pages = {e0205609},
pmid = {30427863},
issn = {1932-6203},
mesh = {ATP-Binding Cassette Transporters/genetics ; Animals ; CRISPR-Cas Systems ; Daphnia/*genetics/physiology ; Drosophila Proteins/genetics ; Drosophila melanogaster/genetics ; Eye/growth &amp; development ; Eye Color/*genetics ; Eye Proteins/genetics ; *Genomics ; Pigmentation/*genetics ; RNA Interference ; },
abstract = {The crustacean Daphnia magna is an important model in multi-disciplinary scientific fields such as genetics, evolutionary developmental biology, toxicology, and ecology. Recently, the draft genome sequence and transcriptome data became publicly available for this species. Genetic transformation has also been achieved via the introduction of plasmid DNA into the genome. The identification of a screenable marker gene and generation of mutant strains are essential to further advance D. magna functional genomics. Because crustaceans are closely related to insects, we hypothesized that, similar to Drosophila genetic studies, eye color-related genes can function as marker genes in Daphnia. We searched orthologs of Drosophila eye pigment transporters White, Scarlet, and Brown in the genome of D. magna. Amino acid sequence alignment and phylogenetic analysis suggested that D. magna has six white and one scarlet orthologs, but lacks the brown ortholog. Due to the multiplicity of white orthologs, we analyzed the function of the scarlet ortholog, DapmaSt, using RNA interference. DapmaSt RNAi embryos showed disappearance of black pigments both in the compound eye and in the ocellus, suggesting that DapmaSt is necessary for black pigmentation in Daphnia eyes. To disrupt DapmaSt using the Crispr/Cas9 system, we co-injected DapmaSt-targeting gRNAs with Cas9 mRNAs into eggs and established white-eyed DapmaSt mutant lines that lack eye pigments throughout their lifespan. Our results suggest that DapmaSt can be used as a transformation marker in D. magna and the DapmaSt mutants would be an important resource for genetic transformation of this species in the future.},
}
@article {pmid30427207,
year = {2019},
author = {Kang, J and Kang, Y and Kim, YW and You, J and Kang, J and Kim, A},
title = {LRF acts as an activator and repressor of the human β-like globin gene transcription in a developmental stage dependent manner.},
journal = {Biochemistry and cell biology = Biochimie et biologie cellulaire},
volume = {97},
number = {4},
pages = {380-386},
doi = {10.1139/bcb-2018-0303},
pmid = {30427207},
issn = {1208-6002},
mesh = {CRISPR-Cas Systems/genetics ; Cells, Cultured ; DNA-Binding Proteins/*metabolism ; *Gene Expression Regulation, Developmental ; Humans ; Repressor Proteins/*metabolism ; Transcription Factors/*metabolism ; *Transcription, Genetic ; *Transcriptional Activation ; beta-Globins/*genetics ; },
abstract = {Leukemia/lymphoma-related factor (LRF; a hematopoietic transcription factor) has been suggested to repress fetal γ-globin genes in the human adult stage β-globin locus. Here, to study the role of LRF in the fetal stage β-globin locus, we knocked out its expression in erythroid K562 cells, in which the γ-globin genes are mainly transcribed. The γ-globin transcription was reduced in LRF knock-out cells, and transcription factor binding to the β-globin locus control region hypersensitive sites (LCR HSs) and active histone organization in the LCR HSs were disrupted by the depletion of LRF. In contrast, LRF loss in the adult stage β-globin locus did not affect active chromatin structure in the LCR HSs and induced the fetal γ-globin transcription. These results indicate that LRF may act as an activator and repressor of the human β-like globin gene transcription in a manner dependent on developmental stage.},
}
@article {pmid30426459,
year = {2019},
author = {Wu, W and Chen, T},
title = {Ribonucleoproteins Mediated Efficient In Vivo Gene Editing in Skin Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1879},
number = {},
pages = {75-86},
doi = {10.1007/7651_2018_115},
pmid = {30426459},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Cloning, Molecular/methods ; Gene Editing/methods ; Genetic Therapy/methods ; Mice ; RNA, Guide/metabolism ; Ribonucleoproteins/*metabolism ; Skin/*metabolism ; Stem Cells/*metabolism ; },
abstract = {The clustered regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system functions like an adaptive immune system in a variety of microbes and has recently been engineered as a powerful tool for manipulating genomic sequences in a huge variety of cell types. In mammals, CRISPR/Cas9 has the potential to bring curative therapies to patients with genetic diseases, although it remained unknown whether suitable in vivo methods for its use are feasible. It is now appreciated that the efficient delivery of these genome-editing tools into most tissue types, including skin, remains a major challenge. Here, we describe a detailed protocol for performing in vivo gene editing of genomic sequences in mouse skin stem cells using Cas9/sgRNAs ribonucleoproteins in combination with electrotransfer technology. We here present all of the required methods needed for the protocol, including molecular cloning, in vitro sgRNA expression and sgRNA purification, Cas9 protein purification, and in vivo delivery of cas9 ribonucleoproteins. This protocol provides a novel in vivo gene editing strategy using ribonucleoproteins for skin stem cells and can potentially be used as curative treatment for genetic diseases in skin and other somatic tissues.},
}
@article {pmid30426198,
year = {2019},
author = {Hahn, F and Nekrasov, V},
title = {CRISPR/Cas precision: do we need to worry about off-targeting in plants?.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {437-441},
pmid = {30426198},
issn = {1432-203X},
support = {BB/P016855/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Mutation/genetics ; Plants/genetics ; Software ; },
abstract = {The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.},
}
@article {pmid30425321,
year = {2018},
author = {Zhang, B and Ye, W and Ye, Y and Zhou, H and Saeed, AFUH and Chen, J and Lin, J and Perčulija, V and Chen, Q and Chen, CJ and Chang, MX and Choudhary, MI and Ouyang, S},
title = {Structural insights into Cas13b-guided CRISPR RNA maturation and recognition.},
journal = {Cell research},
volume = {28},
number = {12},
pages = {1198-1201},
pmid = {30425321},
issn = {1748-7838},
mesh = {Bacterial Proteins/*chemistry ; CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; Flavobacteriaceae/*metabolism ; Models, Molecular ; Multiprotein Complexes/*chemistry ; Protein Conformation ; RNA, Bacterial/*chemistry ; },
}
@article {pmid30425154,
year = {2018},
author = {Høyland-Kroghsbo, NM and Muñoz, KA and Bassler, BL},
title = {Temperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity in Pseudomonas aeruginosa.},
journal = {mBio},
volume = {9},
number = {6},
pages = {},
pmid = {30425154},
issn = {2150-7511},
support = {R01 GM065859/GM/NIGMS NIH HHS/United States ; R37 GM065859/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacteriophages ; Biofilms ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Pseudomonas aeruginosa/*genetics/*growth &amp; development ; Quorum Sensing/genetics ; *Temperature ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. In Pseudomonas aeruginosa, quorum sensing (QS) induces the CRISPR-Cas defense system at high cell density when the risk of bacteriophage infection is high. Here, we show that another cue, temperature, modulates P. aeruginosa CRISPR-Cas. Increased CRISPR adaptation occurs at environmental (i.e., low) temperatures compared to that at body (i.e., high) temperature. This increase is a consequence of the accumulation of CRISPR-Cas complexes, coupled with reduced P. aeruginosa growth rate at the lower temperature, the latter of which provides additional time prior to cell division for CRISPR-Cas to patrol the cell and successfully eliminate and/or acquire immunity to foreign DNA. Analyses of a QS mutant and synthetic QS compounds show that the QS and temperature cues act synergistically. The diversity and level of phage encountered by P. aeruginosa in the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures.IMPORTANCEP. aeruginosa is a soil dwelling bacterium and a plant pathogen, and it also causes life-threatening infections in humans. Thus, P. aeruginosa thrives in diverse environments and over a broad range of temperatures. Some P. aeruginosa strains rely on the CRISPR-Cas adaptive immune system as a phage defense mechanism. Our discovery that low temperatures increase CRISPR adaptation suggests that the rarely occurring but crucial naive adaptation events may take place predominantly under conditions of slow growth, e.g., during the bacterium's soil dwelling existence and during slow growth in biofilms.},
}
@article {pmid30425074,
year = {2019},
author = {Doumpas, N and Lampart, F and Robinson, MD and Lentini, A and Nestor, CE and Cantù, C and Basler, K},
title = {TCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes.},
journal = {The EMBO journal},
volume = {38},
number = {2},
pages = {},
pmid = {30425074},
issn = {1460-2075},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Gene Expression Profiling/*methods ; Gene Expression Regulation ; HEK293 Cells ; Humans ; TCF Transcription Factors/*genetics/metabolism ; *Transcription, Genetic ; Whole Exome Sequencing/methods ; Wnt Signaling Pathway ; beta Catenin/*metabolism ; },
abstract = {During canonical Wnt signalling, the activity of nuclear β-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of β-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that β-catenin occupied specific genomic regions in the absence of TCF/LEF Finally, we revealed the existence of a transcriptional activity of β-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as β-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of β-catenin that bypasses the TCF/LEF transcription factors.},
}
@article {pmid30424953,
year = {2019},
author = {Lattanzi, A and Meneghini, V and Pavani, G and Amor, F and Ramadier, S and Felix, T and Antoniani, C and Masson, C and Alibeu, O and Lee, C and Porteus, MH and Bao, G and Amendola, M and Mavilio, F and Miccio, A},
title = {Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {1},
pages = {137-150},
pmid = {30424953},
issn = {1525-0024},
mesh = {Anemia, Sickle Cell/genetics/metabolism/therapy ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Genetic Therapy/*methods ; Hematopoietic Stem Cells/cytology/*metabolism ; Hemoglobinopathies/genetics/metabolism/therapy ; Heterogeneous-Nuclear Ribonucleoproteins/genetics/metabolism ; Humans ; Plasmids/genetics ; RNA, Guide/genetics/metabolism ; beta-Thalassemia/genetics/metabolism/therapy ; },
abstract = {Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9[+] HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.},
}
@article {pmid30423295,
year = {2018},
author = {Celik, H and Koh, WK and Kramer, AC and Ostrander, EL and Mallaney, C and Fisher, DAC and Xiang, J and Wilson, WC and Martens, A and Kothari, A and Fishberger, G and Tycksen, E and Karpova, D and Duncavage, EJ and Lee, Y and Oh, ST and Challen, GA},
title = {JARID2 Functions as a Tumor Suppressor in Myeloid Neoplasms by Repressing Self-Renewal in Hematopoietic Progenitor Cells.},
journal = {Cancer cell},
volume = {34},
number = {5},
pages = {741-756.e8},
pmid = {30423295},
issn = {1878-3686},
support = {T32 CA113275/CA/NCI NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; UL1 TR000448/TR/NCATS NIH HHS/United States ; T32 HL007088/HL/NHLBI NIH HHS/United States ; F31 DK111058/DK/NIDDK NIH HHS/United States ; F31 DK114951/DK/NIDDK NIH HHS/United States ; UL1 TR002345/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Self Renewal/*genetics/physiology ; Cell Transformation, Neoplastic/*genetics/pathology ; Female ; Gene Deletion ; Gene Knockdown Techniques ; Genes, Tumor Suppressor ; Humans ; Leukemia, Myeloid, Acute/*genetics/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myelodysplastic Syndromes/*genetics/pathology ; Myeloproliferative Disorders/*genetics/pathology ; N-Myc Proto-Oncogene Protein/metabolism ; Polycomb Repressive Complex 2/*genetics/metabolism ; RUNX1 Translocation Partner 1 Protein/metabolism ; Transplantation, Heterologous ; },
abstract = {How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDSs) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPNs or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.},
}
@article {pmid30423072,
year = {2018},
author = {Lin, J and Wong, KC},
title = {Off-target predictions in CRISPR-Cas9 gene editing using deep learning.},
journal = {Bioinformatics (Oxford, England)},
volume = {34},
number = {17},
pages = {i656-i663},
pmid = {30423072},
issn = {1367-4811},
mesh = {Algorithms ; Area Under Curve ; CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing ; Logistic Models ; },
abstract = {MOTIVATION: The prediction of off-target mutations in CRISPR-Cas9 is a hot topic due to its relevance to gene editing research. Existing prediction methods have been developed; however, most of them just calculated scores based on mismatches to the guide sequence in CRISPR-Cas9. Therefore, the existing prediction methods are unable to scale and improve their performance with the rapid expansion of experimental data in CRISPR-Cas9. Moreover, the existing methods still cannot satisfy enough precision in off-target predictions for gene editing at the clinical level.<br><br>RESULTS: To address it, we design and implement two algorithms using deep neural networks to predict off-target mutations in CRISPR-Cas9 gene editing (i.e. deep convolutional neural network and deep feedforward neural network). The models were trained and tested on the recently released off-target dataset, CRISPOR dataset, for performance benchmark. Another off-target dataset identified by GUIDE-seq was adopted for additional evaluation. We demonstrate that convolutional neural network achieves the best performance on CRISPOR dataset, yielding an average classification area under the ROC curve (AUC) of 97.2% under stratified 5-fold cross-validation. Interestingly, the deep feedforward neural network can also be competitive at the average AUC of 97.0% under the same setting. We compare the two deep neural network models with the state-of-the-art off-target prediction methods (i.e. CFD, MIT, CROP-IT, and CCTop) and three traditional machine learning models (i.e. random forest, gradient boosting trees, and logistic regression) on both datasets in terms of AUC values, demonstrating the competitive edges of the proposed algorithms. Additional analyses are conducted to investigate the underlying reasons from different perspectives.<br><br>The example code are available at https://github.com/MichaelLinn/off_target_prediction. The related datasets are available at https://github.com/MichaelLinn/off_target_prediction/tree/master/data.},
}
@article {pmid30423065,
year = {2018},
author = {Peng, H and Zheng, Y and Zhao, Z and Liu, T and Li, J},
title = {Recognition of CRISPR/Cas9 off-target sites through ensemble learning of uneven mismatch distributions.},
journal = {Bioinformatics (Oxford, England)},
volume = {34},
number = {17},
pages = {i757-i765},
doi = {10.1093/bioinformatics/bty558},
pmid = {30423065},
issn = {1367-4811},
mesh = {Base Composition ; CRISPR-Cas Systems ; Genome ; High-Throughput Nucleotide Sequencing/*methods ; Machine Learning ; RNA, Guide/genetics ; Software ; },
abstract = {MOTIVATION: CRISPR/Cas9 is driving a broad range of innovative applications from basic biology to biotechnology and medicine. One of its current issues is the effect of off-target editing that should be critically resolved and should be completely avoided in the ideal use of this system.<br><br>RESULTS: We developed an ensemble learning method to detect the off-target sites of a single guide RNA (sgRNA) from its thousands of genome-wide candidates. Nucleotide mismatches between on-target and off-target sites have been studied recently. We confirm that there exists strong mismatch enrichment and preferences at the 5'-end close regions of the off-target sequences. Comparing with the on-target sites, sequences of no-editing sites can be also characterized by GC composition changes and position-specific mismatch binary features. Under this novel space of features, an ensemble strategy was applied to train a prediction model. The model achieved a mean score 0.99 of Aera Under Receiver Operating Characteristic curve and a mean score 0.45 of Aera Under Precision-Recall curve in cross-validations on big datasets, outperforming state-of-the-art methods in various test scenarios. Our predicted off-target sites also correspond very well to those detected by high-throughput sequencing techniques. Especially, two case studies for selecting sgRNAs to cure hearing loss and retinal degeneration partly prove the effectiveness of our method.<br><br>The python and matlab version of source codes for detecting off-target sites of a given sgRNA and the supplementary files are freely available on the web at https://github.com/penn-hui/OfftargetPredict.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid30422347,
year = {2019},
author = {Sim, SB and Kauwe, AN and Ruano, REY and Rendon, P and Geib, SM},
title = {The ABCs of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera and Ceratitis.},
journal = {Insect molecular biology},
volume = {28},
number = {2},
pages = {277-289},
doi = {10.1111/imb.12550},
pmid = {30422347},
issn = {1365-2583},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Knockout Techniques ; Mutation ; Tephritidae/*genetics ; },
abstract = {Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by nonhomologous end join repair of clustered regularly interspaced short palindromic repeats and the CRISPR associated protein 9 (Cas9) (commonly known as CRISPR/Cas9) has been demonstrated to be an efficient method for creating knock-out mutants and can be utilized to create germline mutations in Tephritidae. In this paper, we describe detailed methods to knockout the white gene in three tephritid species in the genera Anastrepha, Bactrocera and Ceratitis, including the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the variables in tephritid systems that directed method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.},
}
@article {pmid30420783,
year = {2019},
author = {Cheng, AZ and Yockteng-Melgar, J and Jarvis, MC and Malik-Soni, N and Borozan, I and Carpenter, MA and McCann, JL and Ebrahimi, D and Shaban, NM and Marcon, E and Greenblatt, J and Brown, WL and Frappier, L and Harris, RS},
title = {Epstein-Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity.},
journal = {Nature microbiology},
volume = {4},
number = {1},
pages = {78-88},
pmid = {30420783},
issn = {2058-5276},
support = {F30 CA200432/CA/NCI NIH HHS/United States ; R21 CA206309/CA/NCI NIH HHS/United States ; T32 CA009138/CA/NCI NIH HHS/United States ; U54 GM103368/GM/NIGMS NIH HHS/United States ; R21 AI138793/AI/NIAID NIH HHS/United States ; T32 GM008244/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Catalytic Domain/genetics ; Cell Line ; Cytidine Deaminase/*antagonists &amp; inhibitors ; Genome, Viral/genetics ; HEK293 Cells ; Herpesvirus 4, Human/growth &amp; development/*metabolism ; Herpesvirus 8, Human/*metabolism ; Humans ; Minor Histocompatibility Antigens ; RNA Interference ; RNA, Small Interfering/genetics ; Ribonucleotide Reductases/genetics/*metabolism ; Viral Proteins/genetics/*metabolism ; },
abstract = {The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication[1-4]. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism[1-4]. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers[5], utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF2[6,7], binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi's sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.},
}
@article {pmid30420468,
year = {2019},
author = {Au, V and Li-Leger, E and Raymant, G and Flibotte, S and Chen, G and Martin, K and Fernando, L and Doell, C and Rosell, FI and Wang, S and Edgley, ML and Rougvie, AE and Hutter, H and Moerman, DG},
title = {CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {1},
pages = {135-144},
pmid = {30420468},
issn = {2160-1836},
support = {P40 OD010440/OD/NIH HHS/United States ; R24 OD023041/OD/NIH HHS/United States ; PJT-148549//CIHR/Canada ; MOP 93719//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Caenorhabditis elegans/*genetics ; Gene Deletion ; *Gene Editing ; Gene Knockout Techniques ; Gene Targeting ; Homologous Recombination/*genetics ; Mutagenesis/genetics ; },
abstract = {The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events.},
}
@article {pmid30420296,
year = {2018},
author = {Kofler, N and Kraschel, KL},
title = {Treatment of heritable diseases using CRISPR: Hopes, fears, and reality.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {515-521},
doi = {10.1053/j.semperi.2018.09.012},
pmid = {30420296},
issn = {1558-075X},
mesh = {*CRISPR-Cas Systems ; Decision Making ; Evidence-Based Medicine ; *Fear ; Gene Editing/*methods/trends ; Genetic Predisposition to Disease/*genetics ; Genetic Therapy/*trends ; *Hope ; Humans ; *Reproductive Health ; },
abstract = {CRISPR gene editing is poised to transform the therapeutic landscape for diseases of genetic origin. The ease and agility by which CRISPR can make specific changes to DNA holds great promise not only for the treatment of heritable diseases, but also their prevention through germline editing. CRISPR-based therapeutic strategies are currently under development for numerous monogenic diseases. These strategies range from proof of concept studies demonstrating pre-fertilization gamete editing to recently initiated clinical trials for postnatal ex vivo therapies. The promise of CRISPR's human genome editing potential has captivated the public's attention. It is of paramount importance that medical professionals who work with patients who may have or carry a monogenic heritable disease understand CRISPR technology in order to have informed and compassionate discussions with their patients. Understanding CRISPR means understanding its evolving therapeutic applications' nuances, limitations, and barriers to access as well as the regulatory landscape they inhabit. In this piece we provide a review of the promises and pitfalls of CRISPR germline gene editing and their implications for patient decision-making throughout various stages of the reproductive process.},
}
@article {pmid30420295,
year = {2018},
author = {Kraschel, KL and Kofler, N},
title = {Introduction.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {485-486},
doi = {10.1053/j.semperi.2018.09.018},
pmid = {30420295},
issn = {1558-075X},
mesh = {*CRISPR-Cas Systems ; Evidence-Based Medicine ; Gene Editing/*trends ; Humans ; },
}
@article {pmid30418623,
year = {2019},
author = {Eisemann, T and Costa, B and Harter, PN and Wick, W and Mittelbronn, M and Angel, P and Peterziel, H},
title = {Podoplanin expression is a prognostic biomarker but may be dispensable for the malignancy of glioblastoma.},
journal = {Neuro-oncology},
volume = {21},
number = {3},
pages = {326-336},
pmid = {30418623},
issn = {1523-5866},
mesh = {Animals ; Antineoplastic Agents, Alkylating/pharmacology ; Apoptosis/*genetics ; Brain ; Brain Neoplasms/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/*genetics ; Cell Survival/drug effects ; Cells, Cultured ; Disease Progression ; Flow Cytometry ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Glioblastoma/*genetics ; Humans ; Membrane Glycoproteins/*genetics ; Mice ; Neoplasm Invasiveness/genetics ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Prognosis ; Temozolomide/pharmacology ; Transcriptome ; Tumor Cells, Cultured ; },
abstract = {BACKGROUND: Treatment options of glioblastoma, the most aggressive primary brain tumor with frequent relapses and high mortality, are still very limited, urgently calling for novel therapeutic targets. Expression of the glycoprotein podoplanin correlates with poor prognosis in various cancer entities, including glioblastoma. Furthermore, podoplanin has been associated with tumor cell migration and proliferation in vitro; however, experimental data on its function in gliomagenesis in vivo are still missing. Hence, we have functionally investigated the impact of podoplanin on glioblastoma in a preclinical mouse model to evaluate its potential as a therapeutic target.<br><br>METHODS: Fluorescence activated cell sorting, genome-wide expression analysis, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated deletion of podoplanin in patient-derived human glioblastoma cells were combined with organotypic brain slice cultures and intracranial injections into mice.<br><br>RESULTS: We defined a malignant gene signature in tumor cells with high podoplanin expression. The increase and/or maintenance of high podoplanin expression in serial transplantations and in podoplaninlow-sorted glioblastoma cells during outgrowth indicated the association of high podoplanin expression and poor outcome. Unexpectedly, similar rates of proliferation, apoptosis, angiogenesis, and invasion were observed in control and podoplanin-deleted tumors. Accordingly, neither tumor growth nor survival was affected upon podoplanin loss.<br><br>CONCLUSION: We report that tumor progression occurs independently of podoplanin. Thus, in contrast to previous suggestions, blocking of podoplanin does not represent a promising therapeutic approach. However, as podoplanin is associated with tumor aggressiveness and progression, we propose the cell surface protein as a biomarker for poor prognosis.},
}
@article {pmid30418590,
year = {2019},
author = {Gordeeva, J and Morozova, N and Sierro, N and Isaev, A and Sinkunas, T and Tsvetkova, K and Matlashov, M and Truncaite, L and Morgan, RD and Ivanov, NV and Siksnys, V and Zeng, L and Severinov, K},
title = {BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.},
journal = {Nucleic acids research},
volume = {47},
number = {1},
pages = {253-265},
pmid = {30418590},
issn = {1362-4962},
support = {R01 GM107597/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenine/metabolism ; Bacillus cereus/genetics ; Bacteriophage lambda/*genetics ; CRISPR-Cas Systems/genetics ; DNA Methylation/*genetics ; Escherichia coli/*genetics ; Methyltransferases/genetics ; Nucleotide Motifs/*genetics ; Phosphoric Monoester Hydrolases/genetics ; },
abstract = {Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.},
}
@article {pmid30418108,
year = {2019},
author = {Aung, MS and San, T and San, N and Oo, WM and Ko, PM and Thet, KT and Urushibara, N and Kawaguchiya, M and Sumi, A and Kobayashi, N},
title = {Molecular characterization of Staphylococcus argenteus in Myanmar: identification of novel genotypes/clusters in staphylocoagulase, protein A, alpha-haemolysin and other virulence factors.},
journal = {Journal of medical microbiology},
volume = {68},
number = {1},
pages = {95-104},
doi = {10.1099/jmm.0.000869},
pmid = {30418108},
issn = {1473-5644},
mesh = {Adult ; Aged ; Aged, 80 and over ; Bacterial Proteins/*genetics ; Bacterial Typing Techniques ; Coagulase/genetics ; Female ; Genotype ; Hemolysin Proteins/genetics ; Humans ; Male ; Multilocus Sequence Typing ; Myanmar ; Phylogeny ; Staphylococcal Infections/*microbiology ; Staphylococcal Protein A/genetics ; Staphylococcus/enzymology/*genetics/isolation &amp; purification ; Virulence Factors/*genetics ; Young Adult ; },
abstract = {PURPOSE: Staphylococcus argenteus is a novel emerging species of coagulase-positive staphylococcus that is genetically closely related to Staphylococcus aureus. To elucidate the molecular differences in the virulence factors (staphylocoagulase, protein A, alpha-haemolysin, enterotoxin-like toxin and staphylokinase) between these staphylococcal species, S. argenteus that had recently been isolated in Myanmar (five nasal isolates and four clinical isolates) were analysed.<br><br>METHODOLOGY: The nucleotide sequences of the virulence factors were determined by PCR and direct sequencing, followed by phylogenetic analysis by mega6 and multiple alignment by clustalw using the published sequence data for S. aureus and S. argenteus.<br><br>RESULTS: Six S. argenteus isolates belonged to MLST sequence type (ST) 2250, while others belonged to ST4625, ST2198 and ST2854. The novel staphylocoagulase (coa) genotype XIV and the novel coa-XI subtype (XId) were identified in an ST2198 isolate and all other isolates, respectively. Among the S. argenteus isolates, the protein A and alpha-haemolysin genes showed high sequence identity (96-98&#x200a;%&#x2009;and >99&#x200a;%, respectively), while lower identity was observed between S. argenteus and S. aureus (88-91&#x200a;%&#x2009;and 86&#x200a;%, respectively), with both species showing phylogenetically distinct clusters. Similar findings were found for the staphylococcal enterotoxin (SE)-like toxin genes selw, selx and sely. In contrast, the staphylokinase genes were almost identical between these two species. All of the coa-XId isolates had a CRISPR/Cas locus at the site of orfX without having SCCmec, whereas an ST2198 isolate lacked this locus.<br><br>CONCLUSION: The primary virulence factors (staphylocoagulase, protein A andalpha-haemolysin) as well as the SE-like toxins of S. argenteus were genetically discriminated from those of S. aureus, revealing the presence of the novel coa-type/subtype (coa-IXd, XIV) in S. argenteus.},
}
@article {pmid30417500,
year = {2019},
author = {Wahiduzzaman, M and Karnan, S and Ota, A and Hanamura, I and Murakami, H and Inoko, A and Rahman, ML and Hyodo, T and Konishi, H and Tsuzuki, S and Hosokawa, Y},
title = {Establishment and characterization of CRISPR/Cas9-mediated NF2[-/-] human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma.},
journal = {Cancer science},
volume = {110},
number = {1},
pages = {180-193},
pmid = {30417500},
issn = {1349-7006},
mesh = {Adolescent ; Adult ; Aged ; Aged, 80 and over ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Child, Preschool ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/*genetics/metabolism/pathology ; Male ; Mesothelioma/*genetics/metabolism/pathology ; Mesothelioma, Malignant ; Middle Aged ; Neurofibromin 2/*genetics/metabolism ; Pleural Neoplasms/*genetics/metabolism/pathology ; Receptor, Fibroblast Growth Factor, Type 2/*genetics ; Sequence Homology, Nucleic Acid ; Young Adult ; },
abstract = {Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.},
}
@article {pmid30417194,
year = {2018},
author = {Liu, BY and He, XY and Zhuo, RX and Cheng, SX},
title = {Reversal of tumor malignization and modulation of cell behaviors through genome editing mediated by a multi-functional nanovector.},
journal = {Nanoscale},
volume = {10},
number = {45},
pages = {21209-21218},
doi = {10.1039/c8nr07321j},
pmid = {30417194},
issn = {2040-3372},
mesh = {Aptamers, Nucleotide/*chemistry/metabolism ; CRISPR-Cas Systems/genetics ; Cell Movement ; Cell Survival ; Drug Carriers/chemistry ; Focal Adhesion Protein-Tyrosine Kinases/deficiency/genetics ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Microscopy, Confocal ; Mucin-1/genetics/metabolism ; Nanoparticles/*chemistry ; Oligodeoxyribonucleotides/chemistry/metabolism ; Plasmids/chemistry/metabolism ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {To effectively reverse tumor malignization by genome editing, a multi-functional self-assembled nanovector for the delivery of a genome editing plasmid specifically to tumor cells was developed. The nanovector core consisting of protamine and calcium carbonate entrapping the CRISPR-Cas9 plasmid is decorated by aptamer incorporated heparin. Owing to a high affinity between a MUC1 specific aptamer and mucin 1 (MUC1) overexpressed in tumor cells as well as the interaction between AS1411 and nucleolin on the tumor cell surface and cell nuclei, the nanovector can target the nuclei of tumorous cells for the knockout of focal adhesion kinase (FAK). Notably, the genome editing mediated by our delivery systems can effectively modulate cell behaviors and thus reverse tumor malignization. Up-regulated p53, p16, p21, E-cadherin, CD80, MICA, MICB and Fas, together with down-regulated MMP-9, vimentin, VEGF, TGF-β, CD47 and CD133 in genome edited cells indicate that the genome editing system can inhibit cancerous cell growth, prevent tumor invasion and metastasis, reverse tumor-induced immune suppression, and inhibit cancer stemness. More importantly, the edited cells can maintain the modulated cellular function after succeeding subcultures.},
}
@article {pmid30417100,
year = {2018},
author = {Wang, L and Ozark, PA and Smith, ER and Zhao, Z and Marshall, SA and Rendleman, EJ and Piunti, A and Ryan, C and Whelan, AL and Helmin, KA and Morgan, MA and Zou, L and Singer, BD and Shilatifard, A},
title = {TET2 coactivates gene expression through demethylation of enhancers.},
journal = {Science advances},
volume = {4},
number = {11},
pages = {eaau6986},
pmid = {30417100},
issn = {2375-2548},
support = {K08 HL128867/HL/NHLBI NIH HHS/United States ; R35 CA197569/CA/NCI NIH HHS/United States ; R50 CA211428/CA/NCI NIH HHS/United States ; T32 CA070085/CA/NCI NIH HHS/United States ; },
mesh = {Breast Neoplasms/genetics/*metabolism/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; Cohort Studies ; DNA Methylation ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; *Demethylation ; Dioxygenases ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; Estrogen Receptor alpha/antagonists &amp; inhibitors/genetics/*metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Survival Rate ; Tumor Cells, Cultured ; },
abstract = {The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.},
}
@article {pmid30415764,
year = {2018},
author = {Pan, A and Kraschel, KL},
title = {CRISPR diagnostics: Underappreciated uses in perinatology.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {525-530},
doi = {10.1053/j.semperi.2018.09.016},
pmid = {30415764},
issn = {1558-075X},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods/trends ; Gene Targeting/*methods/trends ; Humans ; Molecular Diagnostic Techniques/*methods/trends ; Perinatology/*methods/trends ; Point-of-Care Systems ; Secondary Prevention/*methods ; },
abstract = {CRISPR-based therapeutics have the potential to revolutionize the treatment of hereditary diseases, but current efforts to translate research to the bedside face significant technical, regulatory, and ethical hurdles. In this article, we discuss an underappreciated application of CRISPR: diagnostic testing, and argue that: (1) CRISPR diagnostics are poised to disrupt diagnostic practices including perinatal screening and (2) since CRISPR diagnostics pose minimal technical, regulatory and ethical hurdles (unlike CRISPR therapeutic uses) they are likely to be clinically relevant before CRISPR-based therapies, and thus warrant medical community's attention.},
}
@article {pmid30415343,
year = {2019},
author = {Char, SN and Li, R and Yang, B},
title = {CRISPR/Cas9 for Mutagenesis in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1864},
number = {},
pages = {279-293},
doi = {10.1007/978-1-4939-8778-8_19},
pmid = {30415343},
issn = {1940-6029},
mesh = {Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/instrumentation/*methods ; Genetic Vectors/genetics ; Genome, Plant/genetics ; *Mutagenesis ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; RNA, Guide/genetics ; Tissue Culture Techniques/instrumentation/methods ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.},
}
@article {pmid30415341,
year = {2019},
author = {Schmidt, C and Pacher, M and Puchta, H},
title = {DNA Break Repair in Plants and Its Application for Genome Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1864},
number = {},
pages = {237-266},
doi = {10.1007/978-1-4939-8778-8_17},
pmid = {30415341},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/drug effects/radiation effects ; DNA Repair/*genetics ; DNA, Plant/*genetics ; Gene Editing/instrumentation/methods ; Genetic Engineering/instrumentation/*methods ; Genome, Plant/*genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {Genome engineering is a biotechnological approach to precisely modify the genetic code of a given organism in order to change the context of an existing sequence or to create new genetic resources, e.g., for obtaining improved traits or performance. Efficient targeted genome alterations are mainly based on the induction of DNA double-strand breaks (DSBs) or adjacent single-strand breaks (SSBs). Naturally, all organisms continuously have to deal with DNA-damaging factors challenging the genetic integrity, and therefore a wide range of DNA repair mechanisms have evolved. A profound understanding of the different repair pathways is a prerequisite to control and enhance targeted gene modifications. DSB repair can take place by nonhomologous end joining (NHEJ) or homology-dependent repair (HDR). As the main outcome of NHEJ-mediated repair is accompanied by small insertions and deletions, it is applicable to specifically knock out genes or to rearrange linkage groups or whole chromosomes. The basic requirement for HDR is the presence of a homologous template; thus this process can be exploited for targeted integration of ectopic sequences into the plant genome. The development of different types of artificial site-specific nucleases allows for targeted DSB induction in the plant genome. Such synthetic nucleases have been used for both qualitatively studying DSB repair in vivo with respect to mechanistic differences and quantitatively in order to determine the role of key factors for NHEJ and HR, respectively. The conclusions drawn from these studies allow for a better understanding of genome evolution and help identifying synergistic or antagonistic genetic interactions while supporting biotechnological applications for transiently modifying the plant DNA repair machinery in favor of targeted genome engineering.},
}
@article {pmid30414698,
year = {2019},
author = {Goodspeed, A and Jean, A and Costello, JC},
title = {A Whole-genome CRISPR Screen Identifies a Role of MSH2 in Cisplatin-mediated Cell Death in Muscle-invasive Bladder Cancer.},
journal = {European urology},
volume = {75},
number = {2},
pages = {242-250},
pmid = {30414698},
issn = {1873-7560},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; P30 CA046934/CA/NCI NIH HHS/United States ; R50 CA221675/CA/NCI NIH HHS/United States ; T32 GM007635/GM/NIGMS NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; Biomarkers, Tumor/*genetics ; *CRISPR-Cas Systems ; Cell Death/drug effects ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Databases, Genetic ; Drug Resistance, Neoplasm/genetics ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; MutS Homolog 2 Protein/*genetics ; Neoplasm Invasiveness ; Phenotype ; Urinary Bladder Neoplasms/*drug therapy/genetics/metabolism/pathology ; Whole Genome Sequencing/*methods ; },
abstract = {BACKGROUND: The response to first-line, platinum-based treatment of muscle-invasive bladder cancer has not improved in 3 decades.<br><br>OBJECTIVE: To identify genes that influence cisplatin resistance in bladder cancer.<br><br>We performed a whole-genome CRISPR screen in a bladder cancer cell line to identify genes that mediate resistance to cisplatin.<br><br>Targeted validation was performed in two bladder cancer cell lines. The top gene candidate was validated in a publicly available bladder cancer dataset.<br><br>RESULTS AND LIMITATIONS: From the CRISPR screen, we identified MSH2 as the most significantly enriched gene and mismatch repair as the most significantly enriched pathway that promoted resistance to cisplatin. Bladder cancer cells with knockdown of MSH2 showed a reduction in cisplatin-mediated apoptosis. MSH2 loss did not impact the sensitivity to other chemotherapies, including the cisplatin analog oxaliplatin. Bladder tumors with low MSH2 protein levels, quantified using reverse-phase protein array, showed poorer survival when treated with cisplatin- or carboplatin-based therapy; these results require future validation using immunohistochemistry. Additionally, results are retrospective from patients with primarily high-grade tumors; thus, validation in a controlled clinical trial is needed.<br><br>CONCLUSIONS: We generated in vitro evidence that bladder cancer cell lines depleted of MSH2 are more resistant to cisplatin. We additionally found an association between low MSH2 in bladder tumors and poorer patient survival when treated with platinum-based chemotherapy. If successfully validated prospectively, MSH2 protein level could assist in the selection of patients for chemotherapy.<br><br>PATIENT SUMMARY: We report the first evidence that MSH2 protein level may contribute to chemotherapy resistance observed in muscle-invasive bladder cancer. MSH2 has potential as a biomarker predictive of response to platinum-based therapy.},
}
@article {pmid30414124,
year = {2019},
author = {Hutchinson, LD and Bozatzi, P and Macartney, T and Sapkota, GP},
title = {Generation of Endogenous BMP Transcriptional Reporter Cells Through CRISPR/Cas9 Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1891},
number = {},
pages = {29-35},
doi = {10.1007/978-1-4939-8904-1_4},
pmid = {30414124},
issn = {1940-6029},
support = {MC_UU_00018/6/MRC_/Medical Research Council/United Kingdom ; MC_UU_12016/3/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Bone Morphogenetic Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; *Gene Editing ; *Genes, Reporter ; Humans ; Promoter Regions, Genetic ; RNA, Guide ; Recombinant Proteins ; Signal Transduction ; *Transcription, Genetic ; },
abstract = {Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.},
}
@article {pmid30413738,
year = {2018},
author = {Tan, Z and Shan, J and Rak-Raszewska, A and Vainio, SJ},
title = {Embryonic Stem Cells Derived Kidney Organoids as Faithful Models to Target Programmed Nephrogenesis.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16618},
pmid = {30413738},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian/*cytology/metabolism ; Embryonic Stem Cells/*cytology/physiology ; Gene Expression Regulation, Developmental ; Mesoderm/*cytology/metabolism ; Mice ; Mice, Knockout ; Nephrons/*cytology/metabolism ; *Organogenesis ; Organoids/*cytology/metabolism ; Signal Transduction ; Wnt4 Protein/antagonists &amp; inhibitors/*physiology ; },
abstract = {The kidney is a complex organ that is comprised of thousands of nephrons developing through reciprocal inductive interactions between metanephric mesenchyme (MM) and ureteric bud (UB). The MM undergoes mesenchymal to epithelial transition (MET) in response to the signaling from the UB. The secreted protein Wnt4, one of the Wnt family members, is critical for nephrogenesis as mouse Wnt4[-/-] mutants fail to form pretubular aggregates (PTA) and therefore lack functional nephrons. Here, we generated mouse embryonic stem cell (mESC) line lacking Wnt4 by applying the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9). We describe here, differentiation of the wild type and Wnt4 knockout mESCs into kidney progenitors, and such cells induced to undergo nephrogenesis by the mouse E11.5 UB mediated induction. The wild type three-dimensional (3D) self-organized organoids depict appropriately segmented nephron structures, while the Wnt4-deficient organoids fail to undergo the MET, as is the case in the phenotype of the Wnt4 knockout mouse model in vivo. In summary, we have established a platform that combine CRISPR/Cas9 and kidney organoid technologies to model kidney development in vitro and confirmed that mutant organoids are able to present similar actions as in the in vivo studies.},
}
@article {pmid30413649,
year = {2019},
author = {Li, CJ and Jiang, C and Liu, Y and Bell, T and Ma, W and Ye, Y and Huang, S and Guo, H and Zhang, H and Wang, L and Wang, J and Nomie, K and Zhang, L and Wang, M},
title = {Pleiotropic Action of Novel Bruton's Tyrosine Kinase Inhibitor BGB-3111 in Mantle Cell Lymphoma.},
journal = {Molecular cancer therapeutics},
volume = {18},
number = {2},
pages = {267-277},
pmid = {30413649},
issn = {1538-8514},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R21 CA202104/CA/NCI NIH HHS/United States ; R50 CA221675/CA/NCI NIH HHS/United States ; },
mesh = {Agammaglobulinaemia Tyrosine Kinase/antagonists &amp; inhibitors/*genetics ; Animals ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Drug Resistance, Neoplasm/drug effects ; Female ; Gene Editing ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Lymphoma, Mantle-Cell/*drug therapy/genetics ; Mice ; Piperidines/*administration &amp; dosage/pharmacology ; Protein Kinase Inhibitors/*administration &amp; dosage/pharmacology ; Pyrazoles/*administration &amp; dosage/pharmacology ; Pyrimidines/*administration &amp; dosage/pharmacology ; Signal Transduction/drug effects ; Stress, Physiological/drug effects ; Xenograft Model Antitumor Assays ; },
abstract = {Bruton's tyrosine kinase (BTK) is a key mediator of BCR-dependent cell growth signaling and a clinically effective therapeutic target in mantle cell lymphoma (MCL). The molecular impact of BTK inhibition remains unclear particularly in hematopoietic malignancies. We analyzed the molecular mechanisms of BTK inhibition with the novel inhibitor BGB-3111 (zanubrutinib) in MCL models. The efficacy of BGB-3111 was investigated using growth proliferation/cell viability and apoptosis assays in MCL cell lines and patient-derived xenograft (PDX) MCL cells. The activity and mechanisms of BGB-3111 were further confirmed using a cell line xenograft model, an MCL PDX mouse model, and a human phosphokinase profiler array and reverse phase protein array. Finally, the mechanisms related to resistance to BTK inhibition were analyzed by creating cell lines with low levels of BTK using CRISPR/Cas 9 genome editing. We found that inhibition of BTK leads to suppression of tumor growth, which was mediated via potent suppression of AKT/mTOR, apoptosis, and metabolic stress. Moreover, targeted disruption of the BTK gene in MCL cells resulted in resistance to BTK inhibition and the emergence of novel survival mechanisms. Our studies suggest a general efficacy of BTK inhibition in MCL and potential drug resistance mechanism via alternative signaling pathways.},
}
@article {pmid30413470,
year = {2018},
author = {Kim, D and Kim, JS},
title = {DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA.},
journal = {Genome research},
volume = {28},
number = {12},
pages = {1894-1900},
pmid = {30413470},
issn = {1549-5469},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Chromatin/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *High-Throughput Nucleotide Sequencing ; Humans ; Reproducibility of Results ; },
abstract = {To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.},
}
@article {pmid30412052,
year = {2018},
author = {Balboa, D and Saarimäki-Vire, J and Borshagovski, D and Survila, M and Lindholm, P and Galli, E and Eurola, S and Ustinov, J and Grym, H and Huopio, H and Partanen, J and Wartiovaara, K and Otonkoski, T},
title = {Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30412052},
issn = {2050-084X},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Proliferation/genetics ; Diabetes Mellitus/*genetics/pathology ; Endoplasmic Reticulum/genetics ; Endoplasmic Reticulum Stress/*genetics ; Female ; Humans ; Induced Pluripotent Stem Cells/*chemistry/metabolism ; Infant, Newborn ; Insulin-Secreting Cells/chemistry/metabolism ; Male ; Mice ; Mutation ; Proinsulin/chemistry/*genetics ; Protein Folding ; Sequence Analysis, RNA ; Signal Transduction ; Single-Cell Analysis ; },
abstract = {Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.},
}
@article {pmid30411440,
year = {2019},
author = {Dong, Z and Wu, S and Zhu, C and Wang, X and Li, Y and Chen, X and Liu, D and Qiang, L and Baas, PW and Liu, M},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish.},
journal = {Traffic (Copenhagen, Denmark)},
volume = {20},
number = {1},
pages = {71-81},
pmid = {30411440},
issn = {1600-0854},
support = {R01 NS028785/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Kinesins/*genetics ; Motor Neurons/cytology/metabolism/physiology ; *Mutation ; *Nerve Regeneration ; *Neuronal Outgrowth ; Zebrafish ; Zebrafish Proteins/*genetics ; },
abstract = {KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.},
}
@article {pmid30409884,
year = {2018},
author = {Theisen, DJ and Davidson, JT and Briseño, CG and Gargaro, M and Lauron, EJ and Wang, Q and Desai, P and Durai, V and Bagadia, P and Brickner, JR and Beatty, WL and Virgin, HW and Gillanders, WE and Mosammaparast, N and Diamond, MS and Sibley, LD and Yokoyama, W and Schreiber, RD and Murphy, TL and Murphy, KM},
title = {WDFY4 is required for cross-presentation in response to viral and tumor antigens.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6415},
pages = {694-699},
pmid = {30409884},
issn = {1095-9203},
support = {T32 CA009621/CA/NCI NIH HHS/United States ; P30 AR073752/AR/NIAMS NIH HHS/United States ; F30 DK108498/DK/NIDDK NIH HHS/United States ; R01 CA193318/CA/NCI NIH HHS/United States ; T32 AI007163/AI/NIAID NIH HHS/United States ; U19 AI109948/AI/NIAID NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; P30 DK020579/DK/NIDDK NIH HHS/United States ; U19 AI109725/AI/NIAID NIH HHS/United States ; R01 CA190700/CA/NCI NIH HHS/United States ; U42 OD011185/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Antigens, Neoplasm/*immunology ; Antigens, Viral/*immunology ; Basic-Leucine Zipper Transcription Factors/genetics/physiology ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Cross-Priming/*genetics ; Genetic Testing ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Repressor Proteins/genetics/physiology ; Toxoplasma/immunology ; Toxoplasmosis/immunology ; },
abstract = {During the process of cross-presentation, viral or tumor-derived antigens are presented to CD8[+] T cells by Batf3-dependent CD8α[+]/XCR1[+] classical dendritic cells (cDC1s). We designed a functional CRISPR screen for previously unknown regulators of cross-presentation, and identified the BEACH domain-containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1s in mice. However, WDFY4 was not required for major histocompatibility complex class II presentation, nor for cross-presentation by monocyte-derived dendritic cells. In contrast to Batf3 [-/-] mice, Wdfy4 [-/-] mice displayed normal lymphoid and nonlymphoid cDC1 populations that produce interleukin-12 and protect against Toxoplasma gondii infection. However, similar to Batf3 [-/-] mice, Wdfy4 [-/-] mice failed to prime virus-specific CD8[+] T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in antiviral and antitumor immunity.},
}
@article {pmid30409154,
year = {2018},
author = {Duan, J and Lu, G and Hong, Y and Hu, Q and Mai, X and Guo, J and Si, X and Wang, F and Zhang, Y},
title = {Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {192},
pmid = {30409154},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; *Genome ; Green Fluorescent Proteins/genetics/*metabolism ; HEK293 Cells ; Hep G2 Cells ; Hepatocytes/cytology/*metabolism ; Humans ; Image Processing, Computer-Assisted ; Mice ; Mice, Inbred C57BL ; Molecular Imaging/*methods ; Telomere/genetics/*metabolism ; },
abstract = {CRISPR/dCas9 is a versatile tool that can be used to recruit various effectors and fluorescent molecules to defined genome regions where it can modulate genetic and epigenetic markers, or track the chromatin dynamics in live cells. In vivo applications of CRISPR/dCas9 in animals have been challenged by delivery issues. We generate and characterize a mouse strain with dCas9-EGFP ubiquitously expressed in various tissues. Studying telomere dynamics in these animals reveals surprising results different from those observed in cultured cell lines. The CRISPR/dCas9 knock-in mice provide an important and versatile tool to mechanistically study genome functions in live animals.},
}
@article {pmid30406958,
year = {2019},
author = {Ortega-Escalante, JA and Jasper, R and Miller, SM},
title = {CRISPR/Cas9 mutagenesis in Volvox carteri.},
journal = {The Plant journal : for cell and molecular biology},
volume = {97},
number = {4},
pages = {661-672},
doi = {10.1111/tpj.14149},
pmid = {30406958},
issn = {1365-313X},
support = {/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Mutagenesis/genetics/physiology ; Volvox/*genetics ; },
abstract = {Volvox carteri and other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis in V. carteri using CRISPR/Cas9 components expressed from transgenes. We used V. carteri nitrate reductase gene (nitA) regulatory sequences to conditionally express Streptococcus pyogenes Cas9, and V. carteri U6 RNA gene regulatory sequences to constitutively express single-guide RNA (sgRNA) transcripts. Volvox carteri was bombarded with both Cas9 vector and one of several sgRNA vectors programmed to target different test genes (glsA, regA and invA), and transformants were selected for expression of a hygromycin-resistance marker present on the sgRNA vector. Hygromycin-resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA) were tested for Cas9 and sgRNA expression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously described glsA mutants, and sequencing of the glsA gene in independent mutants revealed short deletions within the targeted region of glsA, indicative of Cas9-directed non-homologous end joining. Similarly, bombardment of a morphologically wild-type strain with the Cas9 plasmid and sgRNA plasmids targeting regA or invA yielded regA and invA mutant transformants/progeny, respectively (at rates of 0.1-100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development in V. carteri, and of developmental novelty in the volvocine algae.},
}
@article {pmid30405244,
year = {2018},
author = {Shen, MW and Arbab, M and Hsu, JY and Worstell, D and Culbertson, SJ and Krabbe, O and Cassa, CA and Liu, DR and Gifford, DK and Sherwood, RI},
title = {Predictable and precise template-free CRISPR editing of pathogenic variants.},
journal = {Nature},
volume = {563},
number = {7733},
pages = {646-651},
pmid = {30405244},
issn = {1476-4687},
support = {K01 DK101684/DK/NIDDK NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HG008363/HG/NHGRI NIH HHS/United States ; T32 GM087237/GM/NIGMS NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 HG008754/HG/NHGRI NIH HHS/United States ; 1R01HG010372/NH/NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R01 HG010372/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; DNA Repair/genetics ; Fibroblasts/metabolism/pathology ; Gene Editing/*methods/*standards ; HCT116 Cells ; HEK293 Cells ; Hermanski-Pudlak Syndrome/*genetics/pathology ; Humans ; K562 Cells ; *Machine Learning ; Menkes Kinky Hair Syndrome/*genetics/pathology ; Reproducibility of Results ; Substrate Specificity ; *Templates, Genetic ; },
abstract = {Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5-11% of Cas9 guide RNAs targeting the human genome are 'precise-50', yielding a single genotype comprising greater than or equal to 50% of all major editing products. We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky-Pudlak syndrome and Menkes disease. This study establishes an approach for precise, template-free genome editing.},
}
@article {pmid30405210,
year = {2018},
author = {Elaswad, A and Khalil, K and Ye, Z and Liu, Z and Liu, S and Peatman, E and Odin, R and Vo, K and Drescher, D and Gosh, K and Qin, G and Bugg, W and Backenstose, N and Dunham, R},
title = {Effects of CRISPR/Cas9 dosage on TICAM1 and RBL gene mutation rate, embryonic development, hatchability and fry survival in channel catfish.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16499},
pmid = {30405210},
issn = {2045-2322},
mesh = {Adaptor Proteins, Vesicular Transport/chemistry/*genetics ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Chromosomal Proteins, Non-Histone/chemistry/*genetics ; Congenital Abnormalities/diagnosis/genetics ; Embryonic Development/*genetics ; Genetic Association Studies ; Genetic Predisposition to Disease ; Ictaluridae/*physiology ; Mortality ; *Mutation ; *Mutation Rate ; Open Reading Frames ; Phenotype ; Reproduction/genetics ; },
abstract = {The current study was conducted to assess the effects of microinjection of different dosages of guide RNA (gRNA)/Cas9 protein on the mutation rate, embryo survival, embryonic development, hatchability and early fry survival in channel catfish, Ictalurus punctatus. Guide RNAs targeting two of the channel catfish immune-related genes, toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) and rhamnose binding lectin (RBL) genes, were designed and prepared. Three dosages of gRNA/Cas9 protein (low, 2.5 ng gRNA/7.5 ng Cas9, medium, 5 ng gRNA/15 ng Cas9 and high, 7.5 ng gRNA/22.5 ng Cas9) were microinjected into the yolk of one-cell embryos. Mutation rate increased with higher dosages (p < 0.05). Higher dosages increased the mutation frequency in individual embryos where biallelic mutations were detected. For both genes, microinjection procedures increased the embryo mortality (p < 0.05). Increasing the dosage of gRNA/Cas9 protein increased the embryo mortality and reduced the hatching percent (p < 0.05). Embryonic development was delayed when gRNAs targeting RBL gene were injected. Means of fry survival time were similar for different dosages (p > 0.05). The current results lay the foundations for designing gene editing experiments in channel catfish and can be used as a guide for other fish species.},
}
@article {pmid30403660,
year = {2018},
author = {Rousset, F and Cui, L and Siouve, E and Becavin, C and Depardieu, F and Bikard, D},
title = {Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors.},
journal = {PLoS genetics},
volume = {14},
number = {11},
pages = {e1007749},
pmid = {30403660},
issn = {1553-7404},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics/virology ; *Genes, Essential ; *Genetic Association Studies ; *Genome, Bacterial ; *Genome-Wide Association Study ; Host-Pathogen Interactions ; },
abstract = {High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr. We then apply the screen to discover E. coli genes required by phages λ, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional λ capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.},
}
@article {pmid30401651,
year = {2019},
author = {Zhou, C and Liu, H and Yuan, F and Chai, H and Wang, H and Liu, F and Li, Y and Zhang, H and Lu, F},
title = {Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing.},
journal = {International journal of biological macromolecules},
volume = {122},
number = {},
pages = {329-337},
doi = {10.1016/j.ijbiomac.2018.10.170},
pmid = {30401651},
issn = {1879-0003},
mesh = {Bacillus licheniformis/*genetics ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {A highly efficient genome editing system for Bacillus licheniformis was developed based on single-plasmid CRISPR/Cas9. For highly efficient genome editing the shuttle vector pWH1520 was selected to construct the knockout plasmids. A construct harboring a pS promoter driving cas9 endonuclease expression, a strong pLY-2 promoter driving the transcription of a single guide RNA was demonstrated as being the most effective. To verify the feasibility of the method the uprT gene coding uracil phosphoribosyltransferase was selected as the reporter gene. The efficiency of introducing nucleotide point mutations and single gene deletion reached an editing efficiency of up to 99.2% and 97.3%, respectively. After a upp-deficient strain was engineered, the system and strain were applied to introduce genomic deletions of another two genes, amyL and chiA (encoding amylase and chitinase, respectively) with about 90% deletion efficiency. As two native extracellular proteins with relatively high secretion in the host, amylase and chitinase can hamper the secretion and expression of alkaline protease. It was demonstrated that the mutant with deletions of the two genes effectively improved the alkaline protease yield by 24.8%. The results illustrated that the establishment of a CRISPR/Cas9 system for Bacillus licheniformis is of significance, and confirmed the system's high efficiency. The system provides support for effective molecular modification and metabolic regulation of Bacillus licheniformis, and offers promise for applications in genetic modification of other industrially relevant Bacillus species.},
}
@article {pmid30400943,
year = {2018},
author = {Guo, X and Dean, A},
title = {CRISPR/Cas9 offers a new tool for studying the role of chromatin architecture in disease pathogenesis.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {185},
pmid = {30400943},
issn = {1474-760X},
support = {DK075033/DK/NIDDK NIH HHS/United States ; },
mesh = {CCCTC-Binding Factor/genetics/*metabolism ; *CRISPR-Cas Systems ; *Chromatin ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; Prostatic Neoplasms/genetics/*pathology ; Protein Binding ; *Regulatory Elements, Transcriptional ; },
abstract = {A recent study used CRISPR/Cas9 to reveal long-range looping between disease-related genes and their regulatory elements that is mediated by the CCCTC-binding factor (CTCF) in prostate cancer.},
}
@article {pmid30400928,
year = {2018},
author = {Yao, Y},
title = {Genome editing: from tools to biological insights.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {186},
pmid = {30400928},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genetic Engineering ; *Genome ; },
}
@article {pmid30400804,
year = {2018},
author = {Smith, JL and Mou, H and Xue, W},
title = {Understanding and repurposing CRISPR-mediated alternative splicing.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {184},
pmid = {30400804},
issn = {1474-760X},
support = {DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; },
mesh = {*Alternative Splicing ; *CRISPR-Cas Systems ; *Exons ; Gene Editing/*methods ; Humans ; RNA, Guide/*genetics ; },
abstract = {Two new studies refine our understanding of CRISPR-associated exon skipping and redefine its utility in engineering alternative splicing.},
}
@article {pmid30400152,
year = {2018},
author = {Koslová, A and Kučerová, D and Reinišová, M and Geryk, J and Trefil, P and Hejnar, J},
title = {Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.},
journal = {Viruses},
volume = {10},
number = {11},
pages = {},
pmid = {30400152},
issn = {1999-4915},
mesh = {Animals ; Avian Leukosis/*genetics/*virology ; Avian Leukosis Virus/*physiology ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line ; Chickens ; Disease Resistance/*genetics ; *Gene Editing ; Genes, Viral ; Genetic Techniques ; Genetic Vectors/genetics ; RNA, Guide ; Receptors, Virus/*genetics/metabolism ; },
abstract = {Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.},
}
@article {pmid30399648,
year = {2019},
author = {Wang, M and Qiao, J and Yu, C and Chen, H and Sun, C and Huang, L and Li, C and Geisler, M and Qian, Q and Jiang, A and Qi, Y},
title = {The auxin influx carrier, OsAUX3, regulates rice root development and responses to aluminium stress.},
journal = {Plant, cell &amp; environment},
volume = {42},
number = {4},
pages = {1125-1138},
doi = {10.1111/pce.13478},
pmid = {30399648},
issn = {1365-3040},
mesh = {Aluminum/*toxicity ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Indoleacetic Acids/metabolism ; Oryza/drug effects/*growth &amp; development/metabolism ; Plant Growth Regulators/metabolism ; Plant Proteins/metabolism/*physiology ; Plant Roots/drug effects/*growth &amp; development/metabolism ; Plants, Genetically Modified ; Polymerase Chain Reaction ; },
abstract = {In rice, there are five members of the auxin carrier AUXIN1/LIKE AUX1 family; however, the biological functions of the other four members besides OsAUX1 remain unknown. Here, by using CRISPR/Cas9, we constructed two independent OsAUX3 knock-down lines, osaux3-1 and osaux3-2, in wild-type rice, Hwayoung (WT/HY) and Dongjin (WT/DJ). osaux3-1 and osaux3-2 have shorter primary roots (PRs), decreased lateral root (LR) density, and longer root hairs (RHs) compared with their WT. OsAUX3 expression in PRs, LRs, and RHs further supports that OsAUX3 plays a critical role in the regulation of root development. OsAUX3 locates at the plasma membrane and functions as an auxin influx carrier affecting acropetal auxin transport. OsAUX3 is up-regulated in the root apex under aluminium (Al) stress, and osaux3-2 is insensitive to Al treatments. Furthermore, 1-naphthylacetic acid accented the sensitivity of WT/DJ and osaux3-2 to respond to Al stress. Auxin concentrations, Al contents, and Al-induced reactive oxygen species-mediated damage in osaux3-2 under Al stress are lower than in WT, indicating that OsAUX3 is involved in Al-induced inhibition of root growth. This study uncovers a novel pathway alleviating Al-induced oxidative damage by inhibition of acropetal auxin transport and provides a new option for engineering Al-tolerant rice species.},
}
@article {pmid30399413,
year = {2019},
author = {Li, T and Zhu, L and Xiao, B and Gong, Z and Liao, Q and Guo, J},
title = {CRISPR-Cpf1-mediated genome editing and gene regulation in human cells.},
journal = {Biotechnology advances},
volume = {37},
number = {1},
pages = {21-27},
doi = {10.1016/j.biotechadv.2018.10.013},
pmid = {30399413},
issn = {1873-1899},
mesh = {Aptamers, Nucleotide/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Endonucleases/*genetics ; Gene Editing/*methods ; Gene Expression Regulation/genetics ; Humans ; Ribonucleases/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) system is being championed as a robust and flexible tool for genome editing. Compared with CRISPR associated protein 9 (Cas9), the CRISPR from Prevotella and Francisella 1 (Cpf1) protein has some distinct characteristics, including RNase activity, T-rich protospacer adjacent motif (PAM) preference and generation of sticky cutting ends. The extremely low propensity of off-target effects and relatively high editing efficiency represent prominent advantages of Cpf1 over Cas9. CRISPR-Cpf1, alone or fused with function domains, has broadly expanded the applications such as multiplex gene knockout, transcriptional repression or activation and epigenome editing in a drug controlled way. Meanwhile, the modification of CRISPR RNAs (crRNAs) with aptamer RNA achieves great promotion on genome editing. Moreover, disease-associated gene manipulation in mice, tumor mutation detection in patients with cancers, and more yet to come, represent growing demands of CRISPR-Cpf1 in clinical genome therapy. In this review, we summarized the unique properties of Cpf1 and the molecular mechanisms underlying CRISPR-Cpf1 on gene editing and regulation in human cells.},
}
@article {pmid30399165,
year = {2018},
author = {Wu, C and Jiao, Y and Shen, M and Pan, C and Cheng, G and Jia, D and Zhu, J and Zhang, L and Zheng, M and Jia, J},
title = {Clustering-local-unique-enriched-signals (CLUES) promotes identification of novel regulators of ES cell self-renewal and pluripotency.},
journal = {PloS one},
volume = {13},
number = {11},
pages = {e0206844},
pmid = {30399165},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Self Renewal/*genetics ; Chromatin Immunoprecipitation ; *Embryonic Stem Cells ; *Epigenesis, Genetic ; Histone Code/genetics ; Histone-Lysine N-Methyltransferase/*genetics ; Mice ; Promoter Regions, Genetic ; Protein Processing, Post-Translational ; Regulatory Sequences, Nucleic Acid ; },
abstract = {BACKGROUND: Key regulators of developmental processes can be prioritized through integrated analysis of ChIP-Seq data of master transcriptional factors (TFs) such as Nanog and Oct4, active histone modifications (HMs) such as H3K4me3 and H3K27ac, and repressive HMs such as H3K27me3. Recent studies show that broad enrichment signals such as super-enhancers and broad H3K4me3 enrichment signals play more dominant roles than short enrichment signals of the master TFs and H3K4me3 in epigenetic regulatory mechanism. Besides the broad enrichment signals, up to ten thousands of short enrichment signals of these TFs and HMs exist in genome. Prioritization of these broad enrichment signals from ChIP-Seq data is a prerequisite for such integrated analysis.<br><br>RESULTS: Here, we present a method named Clustering-Local-Unique-Enriched-Signals (CLUES), which uses an adaptive-size-windows strategy to identify enriched regions (ERs) and cluster them into broad enrichment signals. Tested on 62 ENCODE ChIP-Seq datasets of Ctcf and Nrsf, CLUES performs equally well as MACS2 regarding prioritization of ERs with the TF's motif. Tested on 165 ENCODE ChIP-Seq datasets of H3K4me3, H3K27me3, and H3K36me3, CLUES performs better than existing algorithms on prioritizing broad enrichment signals implicating cell functions influenced by epigenetic regulatory mechanism in cells. Most importantly, CLUES helps to confirm several novel regulators of mouse ES cell self-renewal and pluripotency through integrated analysis of prioritized broad enrichment signals of H3K4me3, H3K27me3, Nanog and Oct4 with the support of a CRISPR/Cas9 negative selection genetic screen.<br><br>CONCLUSIONS: CLUES holds promise for prioritizing broad enrichment signals from ChIP-Seq data. The download site for CLUES is https://github.com/Wuchao1984/CLUESv1.},
}
@article {pmid30398641,
year = {2018},
author = {Chi, B and O'Connell, JD and Iocolano, AD and Coady, JA and Yu, Y and Gangopadhyay, J and Gygi, SP and Reed, R},
title = {The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex.},
journal = {Nucleic acids research},
volume = {46},
number = {22},
pages = {11939-11951},
pmid = {30398641},
issn = {1362-4962},
support = {R35 GM122524/GM/NIGMS NIH HHS/United States ; R01 GM043375/GM/NIGMS NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics/metabolism/pathology ; CRISPR-Cas Systems ; Carrier Proteins/genetics/metabolism ; DNA Helicases/genetics/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Humans ; Muscular Atrophy, Spinal/*genetics/metabolism/pathology ; Nuclear Matrix-Associated Proteins/deficiency/*genetics ; Nuclear Proteins/genetics/metabolism ; Positive Transcriptional Elongation Factor B/genetics/metabolism ; RNA Polymerase II/genetics/metabolism ; RNA, Transfer/genetics/metabolism ; RNA-Binding Protein EWS/deficiency/*genetics ; RNA-Binding Protein FUS/deficiency/*genetics ; RNA-Binding Proteins/*genetics ; Ribonucleoprotein, U1 Small Nuclear/genetics/metabolism ; Spliceosomes/chemistry/metabolism ; TATA-Binding Protein Associated Factors/deficiency/*genetics ; Transcription Factors, TFII/genetics/metabolism ; },
abstract = {Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.},
}
@article {pmid30397814,
year = {2019},
author = {Bandyopadhyay, A and Yin, X and Biswal, A and Coe, R and Quick, WP},
title = {CRISPR-Cas9-Mediated Genome Editing of Rice Towards Better Grain Quality.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1892},
number = {},
pages = {311-336},
doi = {10.1007/978-1-4939-8914-0_18},
pmid = {30397814},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; Edible Grain/*genetics/*standards ; Food Quality ; *Gene Editing ; Oryza/*genetics ; RNA, Guide ; Transformation, Genetic ; },
abstract = {With continued economic development in Asia the demand for high yielding varieties with premium grain quality traits is set to increase. This presents a significant challenge to plant breeders because varieties must be tailored to meet regional preferences. It is already apparent that traditional breeding techniques cannot meet this challenge and so emerging genomics technologies will have to be utilized. Genome editing tools afford the ability to efficiently and precisely manipulate the genome. Among these, the bacterial clustered, regularly interspaced, short palindromic repeat (CRISPR) associated protein 9 (Cas9) or CRISPR-Cas9 has emerged as the easiest, most economic, and efficient technology to undertake genome editing in rice. This technique allows precise site-specific gene modification or integration. In this chapter we present a method for utilizing CRISPR-Cas9 for improving grain quality traits in rice; this should enable molecular breeders to quickly and efficiently produce high yielding rice varieties tailored to meet specific cultural and regional requirements for grain quality.},
}
@article {pmid30397647,
year = {2018},
author = {Chatterjee, P and Jakimo, N and Jacobson, JM},
title = {Minimal PAM specificity of a highly similar SpCas9 ortholog.},
journal = {Science advances},
volume = {4},
number = {10},
pages = {eaau0766},
pmid = {30397647},
issn = {2375-2548},
mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Computational Biology/*methods ; *Gene Editing ; Genetic Engineering ; Genome, Human ; HEK293 Cells ; Humans ; Sequence Homology ; Streptococcus/*enzymology ; Substrate Specificity ; },
abstract = {RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5'-NNG-3' PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.},
}
@article {pmid30397343,
year = {2019},
author = {Özcan, A and Pausch, P and Linden, A and Wulf, A and Schühle, K and Heider, J and Urlaub, H and Heimerl, T and Bange, G and Randau, L},
title = {Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum.},
journal = {Nature microbiology},
volume = {4},
number = {1},
pages = {89-96},
doi = {10.1038/s41564-018-0274-8},
pmid = {30397343},
issn = {2058-5276},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Mass Spectrometry ; Microscopy, Electron ; Plasmids/genetics ; RNA, Bacterial/*genetics ; RNA, Guide/*genetics ; Rhodocyclaceae/*genetics ; Sequence Analysis, RNA ; },
abstract = {Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.},
}
@article {pmid30395968,
year = {2019},
author = {Pirouz, M and Ebrahimi, AG and Gregory, RI},
title = {Unraveling 3'-end RNA uridylation at nucleotide resolution.},
journal = {Methods (San Diego, Calif.)},
volume = {155},
number = {},
pages = {10-19},
pmid = {30395968},
issn = {1095-9130},
support = {R01 CA211328/CA/NCI NIH HHS/United States ; R01 GM086386/GM/NIGMS NIH HHS/United States ; },
mesh = {3' Flanking Region ; Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Computational Biology/*methods ; DNA-Binding Proteins/genetics/metabolism ; Exoribonucleases/deficiency/genetics ; Gene Editing/methods ; Mice ; Mouse Embryonic Stem Cells ; Nucleotidyltransferases/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA Stability ; RNA, Guide/genetics/metabolism ; RNA, Messenger/*genetics/metabolism ; RNA, Small Nuclear/*genetics/metabolism ; Spliceosomes/genetics/metabolism ; Uridine/*metabolism ; },
abstract = {Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.},
}
@article {pmid30395160,
year = {2019},
author = {Canver, MC and Bauer, DE and Maeda, T and Pinello, L},
title = {DrugThatGene: integrative analysis to streamline the identification of druggable genes, pathways and protein complexes from CRISPR screens.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {11},
pages = {1981-1984},
pmid = {30395160},
issn = {1367-4811},
support = {K08 DK093705/DK/NIDDK NIH HHS/United States ; R03 DK109232/DK/NIDDK NIH HHS/United States ; DP2 HL137300/HL/NHLBI NIH HHS/United States ; R01 DK111455/DK/NIDDK NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; R56 DK105001/DK/NIDDK NIH HHS/United States ; F30 DK103359/DK/NIDDK NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; Humans ; Software ; },
abstract = {MOTIVATION: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease system has allowed for high-throughput, large scale pooled screens for functional genomic studies. To aid in the translation of functional genomics to therapeutics, we developed DrugThatGene (DTG) as a web-based application that streamlines analysis of potential therapeutic targets identified from functional genetic screens.<br><br>RESULTS: Starting from a gene list as input, DTG offers automated identification of small molecules along with supporting information from human genetic and other relevant databases. Furthermore, DTG aids in the identification of common biological pathways and protein complexes in conjunction with associated small molecule inhibitors. Taken together, DTG aims to expedite the identification of small molecules from the abundance of functional genetic data generated from CRISPR screens.<br><br>DTG is an open-source and free software available as a website at http://drugthatgene.pinellolab.org. Source code is available at: https://github.com/pinellolab/DrugThatGene, which can be downloaded in order to run DTG locally.},
}
@article {pmid30394685,
year = {2019},
author = {Li, P and Fu, X and Zhang, L and Li, S},
title = {CRISPR/Cas-based screening of a gene activation library in Saccharomyces cerevisiae identifies a crucial role of OLE1 in thermotolerance.},
journal = {Microbial biotechnology},
volume = {12},
number = {6},
pages = {1154-1163},
pmid = {30394685},
issn = {1751-7915},
mesh = {CRISPR-Associated Protein 9 ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fatty Acids, Unsaturated/metabolism ; Gene Editing/*methods ; Genetic Testing/methods ; Genetics, Microbial/*methods ; *Heat-Shock Response ; Hot Temperature ; Lipid Peroxidation ; Saccharomyces cerevisiae/genetics/*radiation effects ; Stearoyl-CoA Desaturase/genetics/*metabolism ; *Thermotolerance ; *Transcriptional Activation ; },
abstract = {CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) screening has been proved to be an efficient method to study functional genomics from yeast to human. In this study, we report the development of a focused CRISPR/Cas-based gene activation library in Saccharomyces cerevisiae and its application in gene identification based on functional screening towards improved thermotolerance. The gene activation library was subjected to screening at 42°C, and the same library cultured at 30°C was set as a control group. After five successive subcultures, five clones were randomly picked from the libraries cultured at 30 and 42°C, respectively. The five clones selected at 30°C contain the specificity sequences of five different single guide RNAs, whereas all the five clones selected at 42°C contain the specificity sequence of one sgRNA that targets the promoter region of OLE1. A crucial role of OLE1 in thermotolerance was identified: the overexpression of OLE1 increased fatty acid unsaturation, and thereby helped counter lipid peroxidation caused by heat stress, rendering the yeast thermotolerant. This study described the application of CRISPR/Cas-based gene activation screening with an example of thermotolerant yeast screening, demonstrating that this method can be used to identify functional genes in yeast.},
}
@article {pmid30394652,
year = {2018},
author = {Borton, MA and Daly, RA and O'Banion, B and Hoyt, DW and Marcus, DN and Welch, S and Hastings, SS and Meulia, T and Wolfe, RA and Booker, AE and Sharma, S and Cole, DR and Wunch, K and Moore, JD and Darrah, TH and Wilkins, MJ and Wrighton, KC},
title = {Comparative genomics and physiology of the genus Methanohalophilus, a prevalent methanogen in hydraulically fractured shale.},
journal = {Environmental microbiology},
volume = {20},
number = {12},
pages = {4596-4611},
doi = {10.1111/1462-2920.14467},
pmid = {30394652},
issn = {1462-2920},
support = {1342701//National Science Foundation Dimensions of Biodiversity/International ; FE0024297//Department of Energy's National Energy Technology Laboratory/International ; //U.S. Department of Energy Joint Genome Institute/International ; DE-AC02-05CH11231//Office of Science of the U.S. Department of Energy/International ; },
mesh = {Ecosystem ; Genome, Bacterial ; *Hydraulic Fracking ; Metagenome ; Methanosarcinaceae/genetics/*physiology ; Natural Gas ; Oil and Gas Fields ; },
abstract = {About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes (MAGs). Utilizing six other previously sequenced isolate genomes and MAGs, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs.},
}
@article {pmid30394131,
year = {2018},
author = {Daigneault, BW and Vilarino, M and Rajput, SK and Frum, T and Smith, GW and Ross, PJ},
title = {CRISPR editing validation, immunostaining and DNA sequencing of individual fixed bovine embryos.},
journal = {BioTechniques},
volume = {65},
number = {5},
pages = {281-283},
doi = {10.2144/btn-2018-0051},
pmid = {30394131},
issn = {1940-9818},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cattle/*embryology/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/analysis/genetics ; Embryo, Mammalian/chemistry/metabolism/ultrastructure ; Gene Editing/*methods ; Immunohistochemistry/methods ; Optical Imaging/methods ; Sequence Analysis, DNA/methods ; Tissue Fixation/methods ; },
abstract = {CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. The techniques described facilitate investigation of functional genomics in bovine embryos compatible with gene editing in livestock after zygotic CRISPR microinjection. These methods avoid traditional avenues that necessitate the use of gene-edited cell lines followed by nuclear transfer that hinder efficiency, limit physiological relevance and contribute to technical challenges.},
}
@article {pmid30394044,
year = {2018},
author = {Ali, S and Park, SK and Kim, WC},
title = {The Pragmatic Introduction and Expression of Microbial Transgenes in Plants.},
journal = {Journal of microbiology and biotechnology},
volume = {28},
number = {12},
pages = {1955-1970},
doi = {10.4014/jmb.1808.08029},
pmid = {30394044},
issn = {1738-8872},
mesh = {Antibodies/metabolism ; Bioreactors ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Disease Resistance/genetics ; Enzymes/biosynthesis ; Food Quality ; Gene Editing/methods ; *Gene Transfer Techniques ; Genes, Plant ; Genetic Engineering/*methods ; Plants/*genetics/metabolism/*microbiology ; Plants, Genetically Modified ; Secondary Metabolism ; Stress, Physiological ; *Transgenes ; Vaccines/biosynthesis ; },
abstract = {Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of plants as "bioreactors" for the production of recombinant proteins, industrially important enzymes, vaccines, antimicrobial compounds, and other valuable secondary metabolites. We discuss the applicable and cost-effective approaches of transgenesis in different plants, as well as the limitations thereof. We subsequently present the contemporary developments in targeted genome editing systems that have facilitated the process of genetic modification and manifested stable and consumer-friendly genetically modified plants and their products. Finally, this article presents the different approaches and demonstrates the introduction and expression of microbial transgenes for the improvement of plant resistance to pathogens and abiotic stress conditions and the production of valuable compounds, together with the promising research progress in targeted genome editing technology. We include a special discussion on the highly efficient CRISPR-Cas system helpful in microbial transgene editing in plants.},
}
@article {pmid30394035,
year = {2018},
author = {Chen, F and Yang, P and Zhu, J},
title = {[Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {34},
number = {10},
pages = {1679-1692},
doi = {10.13345/j.cjb.180033},
pmid = {30394035},
issn = {1872-2075},
mesh = {Blotting, Western ; *CRISPR-Cas Systems ; ErbB Receptors/genetics ; Gene Expression ; Gene Expression Regulation ; *Gene Knockout Techniques ; Gene Products, rev/*genetics ; HEK293 Cells ; Humans ; Lentivirus ; Progranulins/*genetics ; Promoter Regions, Genetic ; RNA, Messenger ; },
abstract = {In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.},
}
@article {pmid30393919,
year = {2019},
author = {Wang, L and Zheng, W and Liu, S and Li, B and Jiang, X},
title = {Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {20},
number = {5},
pages = {634-643},
doi = {10.1002/cbic.201800629},
pmid = {30393919},
issn = {1439-7633},
mesh = {Acquired Immunodeficiency Syndrome/therapy ; Animals ; CRISPR-Cas Systems/*genetics/immunology ; Cell Line ; Gene Editing/methods ; Genetic Therapy/*methods ; Heredodegenerative Disorders, Nervous System/therapy ; Humans ; Molecular Targeted Therapy ; Nanoparticles/therapeutic use ; Neoplasms/therapy ; },
abstract = {Precise editing of the genome of a living body is a goal pursued by scientists in many fields. In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome-editing systems have become a revolutionary toolbox for gene editing across various species. However, the low transfection efficiency of the CRISPR/Cas9 system to mammalian cells in vitro and in vivo is a big obstacle hindering wide and deep application. In this review, recently developed delivery strategies for various CRISPR/Cas9 formulations and their applications in treating gene-related diseases are briefly summarized. This review should inspire others to explore more efficient strategies for CRISPR system delivery and gene therapy.},
}
@article {pmid30393777,
year = {2018},
author = {Zhang, F and Zhao, S and Ren, C and Zhu, Y and Zhou, H and Lai, Y and Zhou, F and Jia, Y and Zheng, K and Huang, Z},
title = {CRISPRminer is a knowledge base for exploring CRISPR-Cas systems in microbe and phage interactions.},
journal = {Communications biology},
volume = {1},
number = {},
pages = {180},
pmid = {30393777},
issn = {2399-3642},
abstract = {CRISPR-Cas systems not only play key roles in prokaryotic acquired immunity, but can also be adapted as powerful genome editing tools. Understanding the native role of CRISPR-Cas systems in providing adaptive immunity can lead to new CRISPR-based technologies. Here, we develop CRISPRminer, a knowledge base and web server to comprehensively collect and investigate the knowledge of CRISPR-Cas systems and generate instructive annotations, including CRISPR arrays and Cas protein annotation, CRISPR-Cas system classification, self-targeting events detection, microbe-phage interaction inference, and anti-CRISPR annotation. CRISPRminer is user-friendly and freely available at http://www.microbiome-bigdata.com/CRISPRminer.},
}
@article {pmid30393194,
year = {2019},
author = {Hussain, W and Mahmood, T and Hussain, J and Ali, N and Shah, T and Qayyum, S and Khan, I},
title = {CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases.},
journal = {Gene},
volume = {685},
number = {},
pages = {70-75},
doi = {10.1016/j.gene.2018.10.072},
pmid = {30393194},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gene Expression Regulation ; Genetic Therapy ; *Genomics/methods ; Homologous Recombination ; Humans ; RNA, Guide ; },
abstract = {Genes, are the functional units of heredity that used as an instructors to make proteins either to become the functional or structural part of the cell. Hence, the proteins get more attention because most of the life functions depends on it. Any mutation or alteration in genome sequences results in complete loss of function or formation of abnormal protein which leads to hereditary disorder. Gene therapy on the other hand, used as a remedy, a process that make correction in the gene which is responsible for genomic disorders. The treatment of disease state depends on the understanding of their genetic basis. While, numerous molecular genome editing tools have been developed and are being utilized to translate the abstract of gene therapy into reality, but the problem is still a mystery. The genome editing molecular scissors can be applied to dictate the selected genetic products that can have the therapeutic power. Thus, editing the specific sequences depends on the type of strategies being used by a molecule such is HDR or NHEJ. CRISPR/Cas9 editing technology can use in disease model to study the genitival disorders. One side the CRISPR technology seemed to be extremely accurate but on the other side it has some harmful effects i.e. Cas9 proteins sometimes cuts the similar sequences other than the specific targeted and Off-targeting Sequences etc. Urgent attention and improvement are needed for various implication of CRISPR/Cas9 technology, including the delivery, precision and control over the mention system. This review presents the current scenario of genome editing in vivo and its implications for the future of human genetic disease treatment as well as genome throughput potency.},
}
@article {pmid30393033,
year = {2018},
author = {Schaks, M and Singh, SP and Kage, F and Thomason, P and Klünemann, T and Steffen, A and Blankenfeldt, W and Stradal, TE and Insall, RH and Rottner, K},
title = {Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.},
journal = {Current biology : CB},
volume = {28},
number = {22},
pages = {3674-3684.e6},
pmid = {30393033},
issn = {1879-0445},
mesh = {Actins/metabolism ; Adaptor Proteins, Signal Transducing/chemistry/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Dictyostelium/cytology/genetics/*metabolism ; Mice ; Multiprotein Complexes/*metabolism ; Nerve Tissue Proteins/antagonists &amp; inhibitors/physiology ; Neuropeptides/antagonists &amp; inhibitors/metabolism ; Protein Conformation ; Pseudopodia/*physiology ; Tumor Cells, Cultured ; Wiskott-Aldrich Syndrome Protein Family/chemistry/genetics/metabolism ; rac GTP-Binding Proteins/antagonists &amp; inhibitors/metabolism ; rac1 GTP-Binding Protein/antagonists &amp; inhibitors/*metabolism ; },
abstract = {Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.},
}
@article {pmid30392975,
year = {2018},
author = {Smith, JGW and Owen, T and Bhagwan, JR and Mosqueira, D and Scott, E and Mannhardt, I and Patel, A and Barriales-Villa, R and Monserrat, L and Hansen, A and Eschenhagen, T and Harding, SE and Marston, S and Denning, C},
title = {Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits.},
journal = {Stem cell reports},
volume = {11},
number = {5},
pages = {1226-1243},
pmid = {30392975},
issn = {2213-6711},
support = {SP/15/9/31605/BHF_/British Heart Foundation/United Kingdom ; RG/15/6/31436/BHF_/British Heart Foundation/United Kingdom ; RG/11/20/29266/BHF_/British Heart Foundation/United Kingdom ; RM/13/30157/BHF_/British Heart Foundation/United Kingdom ; MR/M017354/1/MRC_/Medical Research Council/United Kingdom ; NC/C013105/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; PG/14/59/31000/BHF_/British Heart Foundation/United Kingdom ; FS/16/17/31663/BHF_/British Heart Foundation/United Kingdom ; NC/C013202/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; RG/14/1/30588/BHF_/British Heart Foundation/United Kingdom ; P47352/CRM/BHF_/British Heart Foundation/United Kingdom ; MR/L012618/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Actins/*genetics ; Arrhythmias, Cardiac/pathology/physiopathology ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Calcium Signaling ; Cardiomyopathy, Hypertrophic/*pathology/physiopathology ; Gene Editing ; Heart Defects, Congenital/pathology/physiopathology ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Mutation/*genetics ; Myocardial Contraction ; Myocytes, Cardiac/metabolism/*pathology ; Tissue Engineering ; },
abstract = {Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca[2+] handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations.},
}
@article {pmid30391781,
year = {2019},
author = {Zhang, P and Kang, B and Xie, G and Li, S and Gu, Y and Shen, Y and Zhao, X and Ma, Y and Li, F and Si, J and Wang, J and Chen, J and Yang, H and Xu, X and Yang, Y},
title = {Genomic sequencing and editing revealed the GRM8 signaling pathway as potential therapeutic targets of squamous cell lung cancer.},
journal = {Cancer letters},
volume = {442},
number = {},
pages = {53-67},
doi = {10.1016/j.canlet.2018.10.035},
pmid = {30391781},
issn = {1872-7980},
mesh = {Animals ; Antineoplastic Agents/therapeutic use ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Carcinoma, Squamous Cell/drug therapy/*genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cyclic AMP/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/drug therapy/*genetics/metabolism/pathology ; Mice, Inbred NOD ; Mice, SCID ; Mitogen-Activated Protein Kinases/metabolism ; Molecular Targeted Therapy ; Mutation ; Receptors, Metabotropic Glutamate/*genetics/metabolism ; Signal Transduction ; Transcriptional Activation ; Tumor Burden ; Tumor Cells, Cultured ; *Whole Exome Sequencing ; },
abstract = {The study sought to explore novel genetic aberration driving squamous cell lung carcinoma (LUSC). The whole exome (WES), whole genome (WGS) and target region (TS) sequencings and CRISPR-Cas9 genome editing techniques were integrated to explore and validate novel targeting candidates from LUSC primary tumors and corresponding patient-derived xenografts (PDXs). Seven genes (FGFR2, GRM1,PIK3CG, PIK3CA,ZFHX4, CSMD3, GRM8) with high frequencies of both single nucleotide variants (SNVs) and copy number variants (CNVs), and two genes (CLDN1 and RIT1) only with CNVs were identified by bioinformatics analysis. The functions of these candidates were validated through CRISPR-Cas9 system in primary PDX cells. Furthermore, we focused on the genetic and functional analysis of Metabotropic glutamate receptor 8 (GRM8), whose transcriptional activation was elucidated to promote the survival of LUSC tumor cell through inhibiting cAMP pathway and activating MAPK pathway. The SNV identified in GRM8, A112G, activated downstream signaling pathway and induced cell proliferation, which could be reversed by cAMP stimulator and MEK inhibitor. In conclusion, the components of GRM8 signaling pathway could serve as potential targets of squamous cell lung cancer carrying GRM8 activating variants.},
}
@article {pmid30391674,
year = {2019},
author = {Lee, M and Wang, C and Jin, SW and Labrecque, MP and Beischlag, TV and Brockman, MA and Choy, JC},
title = {Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1.},
journal = {Free radical biology &amp; medicine},
volume = {130},
number = {},
pages = {278-287},
doi = {10.1016/j.freeradbiomed.2018.10.441},
pmid = {30391674},
issn = {1873-4596},
mesh = {A549 Cells ; Astrocytes/metabolism ; CRISPR-Cas Systems/genetics ; Cell Hypoxia/*genetics ; Cytokines/metabolism/*pharmacology ; Gene Expression Regulation/drug effects ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Interferon-gamma/drug effects/genetics/pharmacology ; Interleukin-1/pharmacology ; Nitric Oxide/biosynthesis/metabolism ; Nitric Oxide Synthase Type II/*genetics ; Promoter Regions, Genetic/genetics ; Response Elements/genetics ; Tumor Necrosis Factor-alpha/pharmacology ; },
abstract = {The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at -4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.},
}
@article {pmid30391410,
year = {2018},
author = {Zhou, X and Jiang, G and Yang, L and Qiu, L and He, P and Nong, C and Wang, Y and He, Y and Xing, Y},
title = {Gene diagnosis and targeted breeding for blast-resistant Kongyu 131 without changing regional adaptability.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {45},
number = {10},
pages = {539-547},
doi = {10.1016/j.jgg.2018.08.003},
pmid = {30391410},
issn = {1673-8527},
mesh = {Alleles ; Base Sequence ; CRISPR-Cas Systems ; China ; Disease Resistance ; Magnaporthe/*physiology ; Oryza/*genetics/immunology/microbiology ; Plant Breeding ; Plant Diseases/genetics/*immunology/microbiology ; Plant Proteins/genetics/immunology ; Recombination, Genetic ; },
abstract = {The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131), a leading japonica variety in Northeast China. In this study, two rice lines, KP1 and KP2-Hd1, were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131, respectively. However, both lines headed later than KY131. RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1, and the linkage drag of Hd1 was broken by recombination. On the other hand, no known flowering genes were introgressed into KP1. Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles. Due to its suppression role in heading under long-day conditions, Ghd8 was chosen as the target for gene editing to disrupt its function. Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations, which were easy to detect via agarose gel electrophoresis. All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1, even than KY131, under natural long-day conditions, which ensures its growth in Northeast China. This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.},
}
@article {pmid30391232,
year = {2018},
author = {Chen, H and Li, Y and Du, C and Li, Y and Zhao, J and Zheng, X and Mao, Q and Xia, H},
title = {Aptazyme-mediated direct modulation of post-transcriptional sgRNA level for conditional genome editing and gene expression.},
journal = {Journal of biotechnology},
volume = {288},
number = {},
pages = {23-29},
doi = {10.1016/j.jbiotec.2018.10.011},
pmid = {30391232},
issn = {1873-4863},
mesh = {Aptamers, Nucleotide ; CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Expression ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Theophylline ; },
abstract = {RNA-guided endonuclease Cas9 derived from microbial CRISPR-Cas adaptive immune systems is a powerful tool for genome editing, which has been widely used in eukaryotic systems, prokaryotic systems, and plants. However, the off-target effects caused by Cas9/sgRNA remain a major concern. Currently, the efforts to reduce the off-target effects mainly focus on improving the targeting specificity of sgRNA/Cas9, regulating the activity of the Cas9 protein or the sgRNA, and controlling the time window of their expression. In this study, a novel system was established to regulate the post-transcriptional sgRNA level by small molecule-controlled aptazyme. This system was shown to reduce the off-target effects caused by Cas9/sgRNA, while enabling precise temporal control over gene editing and regulatory activity. This new system could provide a potentially safer and more powerful tool for genome editing and therapeutic application.},
}
@article {pmid30390160,
year = {2019},
author = {Smirnikhina, SA and Anuchina, AA and Lavrov, AV},
title = {Ways of improving precise knock-in by genome-editing technologies.},
journal = {Human genetics},
volume = {138},
number = {1},
pages = {1-19},
pmid = {30390160},
issn = {1432-1203},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Genetic Engineering/*methods ; Humans ; },
abstract = {Despite the recent discover of genome-editing methods, today we can say these approaches have firmly entered our life. Two approaches-knocking out malfunctioning gene allele or correcting the mutation with precise knock-in-can be used in hereditary monogenic diseases treatment. The latter approach is relatively ineffective. Modern data about the ways of repair of double-strand DNA breaks formed by nucleases are presented in this review. The main part of the review is devoted to the ways of increasing precise and effective knock-in: inhibition of non-homologous end joining and stimulation of homology-directed repair key factors, use of small molecules with unknown mechanism of action, cell-cycle synchronization and cell-cycle-dependent activity of Cas9, donor molecule design, selection, alternative methods for insertion and other approaches.},
}
@article {pmid30390050,
year = {2018},
author = {Osakabe, Y and Liang, Z and Ren, C and Nishitani, C and Osakabe, K and Wada, M and Komori, S and Malnoy, M and Velasco, R and Poli, M and Jung, MH and Koo, OJ and Viola, R and Nagamangala Kanchiswamy, C},
title = {CRISPR-Cas9-mediated genome editing in apple and grapevine.},
journal = {Nature protocols},
volume = {13},
number = {12},
pages = {2844-2863},
doi = {10.1038/s41596-018-0067-9},
pmid = {30390050},
issn = {1750-2799},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genome, Plant ; Malus/*genetics ; *Mutagenesis ; Mutation ; Plants, Genetically Modified/genetics ; Plasmids/genetics ; Vitis/*genetics ; },
abstract = {The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR-Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR-Cas9 RNPs takes as little as 2-3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.},
}
@article {pmid30389991,
year = {2018},
author = {Yen, J and Fiorino, M and Liu, Y and Paula, S and Clarkson, S and Quinn, L and Tschantz, WR and Klock, H and Guo, N and Russ, C and Yu, VWC and Mickanin, C and Stevenson, SC and Lee, C and Yang, Y},
title = {TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16304},
pmid = {30389991},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Electroporation/methods ; Female ; Fetal Hemoglobin/*genetics/metabolism ; Filtration/economics/instrumentation/*methods ; Gene Editing/*methods ; Genetic Therapy/economics/instrumentation/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; Hemoglobinopathies/genetics/therapy ; Humans ; Membranes, Artificial ; Mice ; Models, Animal ; RNA, Guide/genetics ; Ribonucleoproteins/*genetics ; Transplantation, Autologous ; },
abstract = {CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.},
}
@article {pmid30389791,
year = {2018},
author = {Guan, X and Luo, Z and Sun, W},
title = {A peptide delivery system sneaks CRISPR into cells.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {44},
pages = {17306-17307},
pmid = {30389791},
issn = {1083-351X},
mesh = {Adipocytes/*metabolism ; Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Gene Transfer Techniques ; Mice ; Nuclear Receptor Interacting Protein 1/genetics/metabolism ; Peptides/*genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has developed into a powerful platform for genome editing in various types of cells and tissues with single-nucleotide precision, but limited delivery options hamper its application in real-world settings. A new study by Shen et al. describes the use of an amphipathic peptide to deliver Cas9/sgRNA ribonucleoprotein complexes, leading to the disruption of GFP genes in cells and mice. Disruption of the Nrip1 gene in isolated pre-adipocytes led to a "browning" phenotype, pointing to new options in the fight against diabetes and obesity.},
}
@article {pmid30389787,
year = {2018},
author = {Lynch, JM and Li, B and Katoli, P and Xiang, C and Leehy, B and Rangaswamy, N and Saenz-Vash, V and Wang, YK and Lei, H and Nicholson, TB and Meredith, E and Rice, DS and Prasanna, G and Chen, A},
title = {Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {52},
pages = {20137-20156},
pmid = {30389787},
issn = {1083-351X},
mesh = {Amino Acid Substitution ; Animals ; Crystallins/genetics/metabolism ; Cytoskeletal Proteins/genetics/*metabolism ; Disease Models, Animal ; Eye Proteins/genetics/*metabolism ; Female ; Glaucoma/genetics/*metabolism/pathology ; Glycoproteins/genetics/*metabolism ; Humans ; Male ; Mice, Mutant Strains ; Microtubule-Associated Proteins/genetics/metabolism ; *Mutation, Missense ; Protein Binding ; Rats, Sprague-Dawley ; Trabecular Meshwork/*metabolism/pathology ; alpha-Crystallin B Chain/genetics/*metabolism ; },
abstract = {Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.},
}
@article {pmid30389355,
year = {2019},
author = {Campbell, LA and Coke, LM and Richie, CT and Fortuno, LV and Park, AY and Harvey, BK},
title = {Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {1},
pages = {151-163},
pmid = {30389355},
issn = {1525-0024},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/drug effects/genetics/*physiology ; Gene Editing/*methods ; HEK293 Cells ; HIV Long Terminal Repeat/genetics/physiology ; Heterogeneous-Nuclear Ribonucleoproteins/genetics/*metabolism ; Humans ; Lipopolysaccharides/pharmacology ; Mutation/genetics ; Proviruses/genetics/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Vesiculovirus/genetics/metabolism ; },
abstract = {Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.},
}
@article {pmid30389228,
year = {2018},
author = {Neuhaus, CP and Zacharias, RL},
title = {Compassionate use of gene therapies in pediatrics: An ethical analysis.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {508-514},
doi = {10.1053/j.semperi.2018.09.010},
pmid = {30389228},
issn = {1558-075X},
mesh = {*CRISPR-Cas Systems ; Compassionate Use Trials/*ethics ; Decision Making ; *Ethical Analysis ; Genetic Therapy/*ethics ; Humans ; Infant ; Leukemia/genetics/immunology/*therapy ; Pediatrics ; Remission Induction ; Treatment Outcome ; },
abstract = {In this commentary, we raise concerns about the compassionate use of CRISPR-mediated gene therapies in pediatric and perinatal patients. There is already a precedent for obtaining gene therapies for pediatric patients through compassionate use programs, and the recent passage of a federal Right to Try law may contribute to an increase in the number of patients who seek access to investigational products outside of a clinical trial. Clinicians, nurses, drug companies, and parents need support as they grapple with whether compassionate use of CRISPR-mediated gene therapies is the right thing to pursue for a child. We raise three issues to consider in that decision: (1) the effects of compassionate use on scientific research; (2) hype and harms of gene therapies; and (3) the limits and scope of parental authority.},
}
@article {pmid30388811,
year = {2018},
author = {Dion, MB and Labrie, SJ and Shah, SA and Moineau, S},
title = {CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualization.},
journal = {Viruses},
volume = {10},
number = {11},
pages = {},
pmid = {30388811},
issn = {1999-4915},
support = {143924//CIHR/Canada ; },
mesh = {Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; Data Mining ; Databases, Nucleic Acid ; Gene Editing ; Sequence Analysis, DNA ; *Software ; User-Computer Interface ; },
abstract = {The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.},
}
@article {pmid30388412,
year = {2018},
author = {Gonatopoulos-Pournatzis, T and Wu, M and Braunschweig, U and Roth, J and Han, H and Best, AJ and Raj, B and Aregger, M and O'Hanlon, D and Ellis, JD and Calarco, JA and Moffat, J and Gingras, AC and Blencowe, BJ},
title = {Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.},
journal = {Molecular cell},
volume = {72},
number = {3},
pages = {510-524.e12},
doi = {10.1016/j.molcel.2018.10.008},
pmid = {30388412},
issn = {1097-4164},
support = {//CIHR/Canada ; },
mesh = {Alternative Splicing/*physiology ; Animals ; Autistic Disorder/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Exons/physiology ; Genetic Engineering/*methods ; Humans ; Mice ; Nerve Tissue Proteins ; Neurogenesis ; Neurons ; RNA Precursors/physiology ; RNA Splice Sites/*physiology ; RNA Splicing/physiology ; RNA-Binding Proteins ; Ribonucleoproteins ; Serine-Arginine Splicing Factors ; Spliceosomes ; },
abstract = {Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.},
}
@article {pmid30388409,
year = {2018},
author = {Terns, MP},
title = {CRISPR-Based Technologies: Impact of RNA-Targeting Systems.},
journal = {Molecular cell},
volume = {72},
number = {3},
pages = {404-412},
pmid = {30388409},
issn = {1097-4164},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA ; Gene Editing/*methods ; Genetic Engineering/*methods ; Humans ; RNA/genetics ; RNA, Guide/genetics ; },
abstract = {DNA-targeting CRISPR-Cas systems, such as those employing the RNA-guided Cas9 or Cas12 endonucleases, have revolutionized our ability to predictably edit genomes and control gene expression. Here, I summarize information on RNA-targeting CRISPR-Cas systems and describe recent advances in converting them into powerful and programmable RNA-binding and cleavage tools with a wide range of novel and important biotechnological and biomedical applications.},
}
@article {pmid30388408,
year = {2018},
author = {Abbott, TR and Qi, LS},
title = {Evolution at the Cutting Edge: CRISPR-Mediated Directed Evolution.},
journal = {Molecular cell},
volume = {72},
number = {3},
pages = {402-403},
doi = {10.1016/j.molcel.2018.10.027},
pmid = {30388408},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Directed DNA Polymerase ; Genomics ; Nucleotides ; },
abstract = {In a recent issue of Nature, Halperin et al. (2018) develop a new technology to continuously diversify specific genomic loci by combining CRISPR-Cas9 with error-prone DNA polymerases.},
}
@article {pmid30387552,
year = {2018},
author = {Kumlehn, J and Pietralla, J and Hensel, G and Pacher, M and Puchta, H},
title = {The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology.},
journal = {Journal of integrative plant biology},
volume = {60},
number = {12},
pages = {1127-1153},
doi = {10.1111/jipb.12734},
pmid = {30387552},
issn = {1744-7909},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics/metabolism ; Gene Editing/methods ; Plant Breeding ; Plants/*genetics ; Synthetic Biology/*methods ; },
abstract = {Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)-associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction, resulting in mutations by non-homologous recombination. Strategies for performing such experiments - from the design of guide RNA to the use of different transformation technologies - are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNA-binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.},
}
@article {pmid30386377,
year = {2018},
author = {Guzina, J and Chen, WH and Stankovic, T and Djordjevic, M and Zdobnov, E and Djordjevic, M},
title = {In silico Analysis Suggests Common Appearance of scaRNAs in Type II Systems and Their Association With Bacterial Virulence.},
journal = {Frontiers in genetics},
volume = {9},
number = {},
pages = {474},
pmid = {30386377},
issn = {1664-8021},
abstract = {In addition to its well-established defense function, CRISPR/Cas can also exhibit crucial non-canonical activity through endogenous gene expression regulation, which was found to mainly affect bacterial virulence. These non-canonical functions depend on scaRNA, which is a small RNA encoded outside of CRISPR array, that is typically flanked by a transcription start site (TSS) and a terminator, and is in part complementary to another small CRISPR/Cas-associated RNA (tracrRNAs). Identification of scaRNAs is however largely complicated by the scarcity of RNA-Seq data across different bacteria, so that they were identified only in a relatively rare CRISPR/Cas subtype (IIB), and the possibility of finding them in other Type II systems is currently unclear. This study presents the first effort toward systematic detection of small CRISPR/Cas-associated regulatory RNAs, where obtained predictions can guide future experiments. The core of our approach is ab initio detection of small RNAs from bacterial genome, which is based on jointly predicting transcription signals - TSS and terminators - and homology to CRISPR array repeat. Particularly, we employ our improved approach for detecting bacterial TSS, since accurate TSS detection is the main limiting factor for accurate small RNA prediction. We also explore how our predictions match to available RNA-Seq data and analyze their conservation across related bacterial species. In Type IIB systems, our predictions are consistent with experimental data, and we systematically identify scaRNAs throughout this subtype. Furthermore, we identify scaRNA:tracrRNA pairs in a number of IIA/IIC systems, where the appearance of scaRNAs co-occurs with the strains being pathogenic. RNA-Seq and conservation analysis show that our method is well suited for predicting CRISPR/Cas-associated small RNAs. We also find possible existence of a modified mechanism of CRISPR-associated small RNA action, which, interestingly, closely resembles the setup employed in biotechnological applications. Overall, our findings indicate that scaRNA:tracrRNA pairs are present in all subtypes of Type II systems, and point to an underlying connection with bacterial virulence. In addition to formulating these hypotheses, careful manual curation that we performed, makes an important first step toward fully automated predictor of CRISPR/Cas-associated small RNAs, which will allow their large scale analysis across diverse bacterial genomes.},
}
@article {pmid30386178,
year = {2018},
author = {Shankar, S and Sreekumar, A and Prasad, D and Das, AV and Pillai, MR},
title = {Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design.},
journal = {Cancer cell international},
volume = {18},
number = {},
pages = {169},
pmid = {30386178},
issn = {1475-2867},
abstract = {BACKGROUND: Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome.<br><br>METHODS: HPV 16[+ve] cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole.<br><br>RESULTS: Although ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity.<br><br>CONCLUSIONS: Our study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.},
}
@article {pmid30385564,
year = {2018},
author = {Kofler, N and Collins, JP and Kuzma, J and Marris, E and Esvelt, K and Nelson, MP and Newhouse, A and Rothschild, LJ and Vigliotti, VS and Semenov, M and Jacobsen, R and Dahlman, JE and Prince, S and Caccone, A and Brown, T and Schmitz, OJ},
title = {Editing nature: Local roots of global governance.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6414},
pages = {527-529},
doi = {10.1126/science.aat4612},
pmid = {30385564},
issn = {1095-9203},
mesh = {Animals ; Anthozoa/genetics ; *Bioethical Issues ; *CRISPR-Cas Systems ; Conservation of Natural Resources/*methods ; Coral Reefs ; Gene Editing/*ethics/*methods ; *Gene-Environment Interaction ; },
}
@article {pmid30383747,
year = {2018},
author = {Combs, PA and Fraser, HB},
title = {Spatially varying cis-regulatory divergence in Drosophila embryos elucidates cis-regulatory logic.},
journal = {PLoS genetics},
volume = {14},
number = {11},
pages = {e1007631},
pmid = {30383747},
issn = {1553-7404},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Drosophila/*genetics ; Drosophila Proteins/genetics ; *Embryo, Nonmammalian ; Gene Editing ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Targeting ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Spatial patterning of gene expression is a key process in development, yet how it evolves is still poorly understood. Both cis- and trans-acting changes could participate in complex interactions, so to isolate the cis-regulatory component of patterning evolution, we measured allele-specific spatial gene expression patterns in D. melanogaster × simulans hybrid embryos. RNA-seq of cryo-sectioned slices revealed 66 genes with strong spatially varying allele-specific expression. We found that hunchback, a major regulator of developmental patterning, had reduced expression of the D. simulans allele specifically in the anterior tip of hybrid embryos. Mathematical modeling of hunchback cis-regulation suggested a candidate transcription factor binding site variant, which we verified as causal using CRISPR-Cas9 genome editing. In sum, even comparing morphologically near-identical species we identified surprisingly extensive spatial variation in gene expression, suggesting not only that development is robust to many such changes, but also that natural selection may have ample raw material for evolving new body plans via changes in spatial patterning.},
}
@article {pmid30382614,
year = {2019},
author = {Cook, PJ and Ventura, A},
title = {Cancer diagnosis and immunotherapy in the age of CRISPR.},
journal = {Genes, chromosomes &amp; cancer},
volume = {58},
number = {4},
pages = {233-243},
pmid = {30382614},
issn = {1098-2264},
support = {P30-CA008748/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Therapy/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; Neoplasms/diagnosis/immunology/*therapy ; },
abstract = {The explosion in genome editing technologies that has occurred in the past decade has revolutionized cancer research and promises to improve cancer diagnosis and therapy. Ongoing efforts include engineering of chimeric antigen receptor-T cells using clustered regularly interspaced short palindromic repeats (CRISPR) to generate a safer, more effective therapy with improved performance in immunologically "cold" tumors, as well as clever adaptations of CRISPR enzymes to allow fast, simple, and sensitive detection of specific nucleotide sequences. While still in their infancy, CRISPR-based cancer therapeutics and diagnostics are developing at an impressive speed and it is likely they will soon impact clinical practice. Here, we summarize their history and the most recent developments.},
}
@article {pmid30382180,
year = {2019},
author = {Wang, HC and Wang, P and Chen, YW and Zhang, Y},
title = {Bevacizumab or fibronectin gene editing inhibits the osteoclastogenic effects of fibroblasts derived from human radicular cysts.},
journal = {Acta pharmacologica Sinica},
volume = {40},
number = {7},
pages = {949-956},
pmid = {30382180},
issn = {1745-7254},
mesh = {Bevacizumab/*pharmacology ; Exons ; Fibroblasts/*drug effects/metabolism ; Fibronectins/*genetics ; Gene Editing ; Humans ; Osteogenesis/*drug effects ; Protein Domains/genetics ; Radicular Cyst/genetics/*metabolism/pathology ; Vascular Endothelial Growth Factor A/metabolism ; },
abstract = {Fibronectin (FN) is a main component of extracellular matrix (ECM) in most adult tissues. Under pathological conditions, particularly inflammation, wound healing and tumors, an alternatively spliced exon extra domain A (EDA) is included in the FN protein (EDA[+]FN), which facilitates cellular proliferation, motility, and aggressiveness in different lesions. In this study we investigated the effects of EDA[+]FN on bone destruction in human radicular cysts and explored the possibility of editing FN gene or blocking the related paracrine signaling pathway to inhibit the osteoclastogenesis. The specimens of radicular cysts were obtained from 20 patients. We showed that the vessel density was positively associated with both the lesion size (R = 0.49, P = 0.001) and EDA[+]FN staining (R = 0.26, P = 0.022) in the specimens. We isolated fibroblasts from surgical specimens, and used the CRISPR/Cas system to knockout the EDA exon, or used IST-9 antibody and bevacizumab to block EDA[+]FN and VEGF, respectively. Compared to control fibroblasts, the fibroblasts from radicular cysts exhibited significantly more Trap[+]MNCs, the relative expression level of VEGF was positively associated with both the ratio of EDA[+]FN/total FN (R = 0.271, P = 0.019) and with the number of Trap[+]MNCs (R = 0.331, P = 0.008). The knockout of the EDA exon significantly decreased VEGF expression in the fibroblasts derived from radicular cysts, leading to significantly decreased osteoclastogenesis; similar results were observed using bevacizumab to block VEGF, but block of EDA[+]FN with IST-9 antibody had no effect. Furthermore, the inhibitory effects of gene editing on Trap[+]MNC development were restored by exogenous VEGF. These results suggest that EDA[+]FN facilitates osteoclastogenesis in the fibrous capsule of radicular cysts, through a mechanism mediated by VEGF via an autocrine effect on the fibroblasts. Bevacizumab inhibits osteoclastogenesis in radicular cysts as effectively as the exclusion of the EDA exon by gene editing.},
}
@article {pmid30381865,
year = {2019},
author = {Wang, Y and Song, H and Wang, W and Zhang, Z},
title = {Generation and characterization of Megf6 null and Cre knock-in alleles.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {57},
number = {2},
pages = {e23262},
doi = {10.1002/dvg.23262},
pmid = {30381865},
issn = {1526-968X},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Lineage ; Gene Knock-In Techniques/*methods ; Intercellular Signaling Peptides and Proteins/*genetics/metabolism ; Mesoderm/cytology/metabolism ; Mice ; },
abstract = {Megf6, a member of MEGF (multiple EGF-like domains) protein family, is a conserved high molecular weight protein with 30 EGF-like domains. Although many members of the MEGF protein family are essential for embryonic development and homeostasis, the role of Megf6 in development and physiology is still unknown. Here, we generated Megf6-deficient mice using CRISPR-Cas9 technique and showed that Megf6 is dispensable for embryonic development. We also constructed the Megf6[Cre] allele to study Megf6-expressing cell lineages. Our results showed that Megf6-expressing cells contribute to the periotic mesenchyme and its derivatives, skin epidermis, certain cells in brain and ribs. Therefore, the Megf6[Cre] allele can be a useful tool for conditional deletion in these tissues, in particular for periotic mesenchyme deletion.},
}
@article {pmid30379982,
year = {2018},
author = {Liu, W and Zhang, Y and Li, S and Li, Y},
title = {Rapid and efficient CRISPR-mediated genome editing with cloning-free method.},
journal = {Acta biochimica et biophysica Sinica},
volume = {50},
number = {11},
pages = {1173-1175},
doi = {10.1093/abbs/gmy107},
pmid = {30379982},
issn = {1745-7270},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; },
}
@article {pmid30379827,
year = {2018},
author = {Sugano, SS and Nishihama, R and Shirakawa, M and Takagi, J and Matsuda, Y and Ishida, S and Shimada, T and Hara-Nishimura, I and Osakabe, K and Kohchi, T},
title = {Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0205117},
pmid = {30379827},
issn = {1932-6203},
mesh = {Arabidopsis/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genetic Vectors ; *Genome, Plant ; Immunoblotting ; Marchantia/*genetics/metabolism ; Mutation ; Organisms, Genetically Modified ; Phenotype ; },
abstract = {Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.},
}
@article {pmid30379614,
year = {2019},
author = {McInally, SG and Hagen, KD and Nosala, C and Williams, J and Nguyen, K and Booker, J and Jones, K and Dawson, SC},
title = {Robust and stable transcriptional repression in Giardia using CRISPRi.},
journal = {Molecular biology of the cell},
volume = {30},
number = {1},
pages = {119-130},
pmid = {30379614},
issn = {1939-4586},
support = {R01 AI077571/AI/NIAID NIH HHS/United States ; R21 AI119791/AI/NIAID NIH HHS/United States ; T32 GM007377/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Nucleus/metabolism ; Flagella/metabolism ; Gene Knockdown Techniques ; Genes, Reporter ; Giardia/*genetics ; Kinesins/metabolism ; Nuclear Localization Signals ; Phenotype ; Protozoan Proteins/metabolism ; *Transcription, Genetic ; Trophozoites/metabolism ; },
abstract = {Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.},
}
@article {pmid30379597,
year = {2019},
author = {Min, YL and Bassel-Duby, R and Olson, EN},
title = {CRISPR Correction of Duchenne Muscular Dystrophy.},
journal = {Annual review of medicine},
volume = {70},
number = {},
pages = {239-255},
pmid = {30379597},
issn = {1545-326X},
support = {R01 DK099653/DK/NIDDK NIH HHS/United States ; R01 HL138426/HL/NHLBI NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Dystrophin/*genetics ; Gene Editing ; Genetic Predisposition to Disease/epidemiology ; Genetic Therapy/*methods ; Humans ; Mice ; Muscular Dystrophy, Duchenne/epidemiology/*genetics/physiopathology/*therapy ; Mutation ; Prevalence ; Prognosis ; Risk Assessment ; Severity of Illness Index ; Treatment Outcome ; },
abstract = {The ability to efficiently modify the genome using CRISPR technology has rapidly revolutionized biology and genetics and will soon transform medicine. Duchenne muscular dystrophy (DMD) represents one of the first monogenic disorders that has been investigated with respect to CRISPR-mediated correction of causal genetic mutations. DMD results from mutations in the gene encoding dystrophin, a scaffolding protein that maintains the integrity of striated muscles. Thousands of different dystrophin mutations have been identified in DMD patients, who suffer from a loss of ambulation followed by respiratory insufficiency, heart failure, and death by the third decade of life. Using CRISPR to bypass DMD mutations, dystrophin expression has been efficiently restored in human cells and mouse models of DMD. Here, we review recent progress toward the development of possible CRISPR therapies for DMD and highlight opportunities and potential obstacles in attaining this goal.},
}
@article {pmid30378731,
year = {2019},
author = {Adkar, SS and Wu, CL and Willard, VP and Dicks, A and Ettyreddy, A and Steward, N and Bhutani, N and Gersbach, CA and Guilak, F},
title = {Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing.},
journal = {Stem cells (Dayton, Ohio)},
volume = {37},
number = {1},
pages = {65-76},
pmid = {30378731},
issn = {1549-4918},
support = {R43 AR072166/AR/NIAMS NIH HHS/United States ; R21 DA041878/DA/NIDA NIH HHS/United States ; R01 AG046927/AG/NIA NIH HHS/United States ; R01 AR048182/AR/NIAMS NIH HHS/United States ; R01 AG015768/AG/NIA NIH HHS/United States ; U01 HG007900/HG/NHGRI NIH HHS/United States ; R21 AR067467/AR/NIAMS NIH HHS/United States ; R41 GM119914/GM/NIGMS NIH HHS/United States ; R03 AR061042/AR/NIAMS NIH HHS/United States ; R21 AR065956/AR/NIAMS NIH HHS/United States ; DP2 OD008586/OD/NIH HHS/United States ; R21 AR072870/AR/NIAMS NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; S10 OD010707/OD/NIH HHS/United States ; R01 AR048852/AR/NIAMS NIH HHS/United States ; T32 DK108742/DK/NIDDK NIH HHS/United States ; R01 AR070864/AR/NIAMS NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; P01 AR050245/AR/NIAMS NIH HHS/United States ; T32 GM007171/GM/NIGMS NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; T32 EB018266/EB/NIBIB NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Chondrogenesis/*genetics ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; },
abstract = {The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2019;37:65-76.},
}
@article {pmid30377374,
year = {2018},
author = {Ma, H and Tu, LC and Naseri, A and Chung, YC and Grunwald, D and Zhang, S and Pederson, T},
title = {CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging.},
journal = {Nature methods},
volume = {15},
number = {11},
pages = {928-931},
pmid = {30377374},
issn = {1548-7105},
support = {K99 GM126810/GM/NIGMS NIH HHS/United States ; R01 GM102515/GM/NIGMS NIH HHS/United States ; U01 DA047733/DA/NIDA NIH HHS/United States ; U01 EB021238/EB/NIBIB NIH HHS/United States ; U01 DA040588/DA/NIDA NIH HHS/United States ; R01 GM123541/GM/NIGMS NIH HHS/United States ; },
mesh = {A549 Cells ; Binding Sites ; *CRISPR-Cas Systems ; Fluorescent Dyes/*chemistry ; Genes, Reporter ; *Genome, Human ; Genomics/*methods ; HEK293 Cells ; Humans ; Microscopy, Fluorescence ; Molecular Imaging/*methods ; RNA, Guide/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA scaffolds have been adapted to carry multiple binding sites for fluorescent proteins to enhance brightness for live cell imaging of genomic loci. However, many of these modifications result in guide RNA instability and thus produce lower genome-labeling efficiency than anticipated. Here we introduce CRISPR-Sirius, based on octet arrays of aptamers conferring both enhanced guide RNA stability and brightness, and provide initial biological applications of this platform.},
}
@article {pmid30377362,
year = {2018},
author = {Bubeck, F and Hoffmann, MD and Harteveld, Z and Aschenbrenner, S and Bietz, A and Waldhauer, MC and Börner, K and Fakhiri, J and Schmelas, C and Dietz, L and Grimm, D and Correia, BE and Eils, R and Niopek, D},
title = {Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.},
journal = {Nature methods},
volume = {15},
number = {11},
pages = {924-927},
doi = {10.1038/s41592-018-0178-9},
pmid = {30377362},
issn = {1548-7105},
mesh = {*Biosensing Techniques ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Epigenomics ; *Gene Editing ; Genome ; HEK293 Cells ; Humans ; Light ; *Optogenetics ; Phototropins/*chemistry ; *Protein Engineering ; Streptococcus pyogenes/enzymology ; },
abstract = {Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.},
}
@article {pmid30377348,
year = {2018},
author = {Rusk, N},
title = {Keeping CRISPR in check.},
journal = {Nature methods},
volume = {15},
number = {11},
pages = {857},
doi = {10.1038/s41592-018-0207-8},
pmid = {30377348},
issn = {1548-7105},
mesh = {Bacteriophages/*genetics ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting ; Humans ; },
}
@article {pmid30377319,
year = {2018},
author = {Wight, AJ},
title = {Strict EU ruling on gene-edited crops squeezes science.},
journal = {Nature},
volume = {563},
number = {7729},
pages = {15-16},
doi = {10.1038/d41586-018-07166-7},
pmid = {30377319},
issn = {1476-4687},
mesh = {Agriculture/economics/*legislation &amp; jurisprudence ; Animals ; Belgium ; Brazil ; Breeding/legislation &amp; jurisprudence ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/economics/*genetics ; *European Union ; Evaluation Studies as Topic ; France ; Gene Editing/*legislation &amp; jurisprudence ; Humans ; Plants, Genetically Modified/*genetics ; },
}
@article {pmid30377126,
year = {2018},
author = {Zhang, H and Chen, M and Fang, T and Zhang, T and Ni, W},
title = {[Establishment and verification of a mouse model of Gata4 gene H435Y mutation].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {38},
number = {10},
pages = {1245-1249},
pmid = {30377126},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems ; GATA4 Transcription Factor/*genetics ; Gene Targeting/*methods ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Point Mutation/*genetics ; Polymerase Chain Reaction ; },
abstract = {OBJECTIVE: To establish a mouse model of H435Y mutation of Gata4 gene using CRISPR/Cas9- mediated gene targeting.<br><br>METHODS: The single-stranded guide RNA (sgRNA) specific to the H435Y loci of Gata4 gene was designed based on the sequence of Gata4 gene. After activity assessment, the active sgRNA and Cas9 were in vitro transcribed into RNA and microinjected along with the donor DNA fragment with point mutations into fertilized mouse eggs. The microinjected eggs were transferred into pseudopregnant mice to obtain the F0 generation mice with the target Gata4 gene mutation confirmed by PCR and gene sequencing. Gata4 gene mutations in the offsprings of the F0 generation mice were analyzed.<br><br>RESULTS: Gene sequencing confirmed the successful establishment of mouse models carrying H435Y mutation of Gata4 gene in 4 of the F0 generation mice. The positive F0 generation mice were crossed with wild-type C57BL/6J mice to obtain the F1 generation mice, and PCR confirmed the presence of H435Y mutations of Gata4 gene in 6 of the F1 mice. Then F2 generation mice were obtained by F1 generation matting with each other. PCR showed that H435Y mutation of Gata4 gene in F2 mice was found, indicating the mousemodel of Gata4 gene mutation in H435Y was established and propagated successfully.<br><br>CONCLUSIONS: We successfully established Gata4 gene H435Y mutant mouse models using CRISPR/Cas9 technique.},
}
@article {pmid30376985,
year = {2018},
author = {Hong, A},
title = {CRISPR in personalized medicine: Industry perspectives in gene editing.},
journal = {Seminars in perinatology},
volume = {42},
number = {8},
pages = {501-507},
doi = {10.1053/j.semperi.2018.09.008},
pmid = {30376985},
issn = {1558-075X},
mesh = {Anemia, Sickle Cell/*genetics/therapy ; *CRISPR-Cas Systems ; Evidence-Based Medicine ; *Gene Editing/trends ; Genetic Therapy/*trends ; Humans ; *Precision Medicine/trends ; Risk Assessment ; beta-Thalassemia/*genetics/therapy ; },
abstract = {While research in gene editing technology has been active since the 1990s, recent advances in CRISPR ease of use have sparked significant enthusiasm in the biomedical community. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing has the potential to shape the therapeutic landscape of genetically-defined diseases, with key players such as CRISPR Therapeutics, Intellia, and Editas leading the way in β-thalassemia and sickle cell disease. In coming years, clinical development of CRISPR technology promises to change how we think about monogenic and polygenic diseases, though data is still in its infancy. While concerns regarding safety and administration of CRISPR persist and researchers are still seeking to better understand its effect on the body's ability to recognize and repair damaged DNA, the applications for personalized medicine are exciting and wide-ranging. This review examines potential applications of CRISPR for human therapeutics, with a private sector focus detailing anticipated clinical advances and evolving challenges along the way.},
}
@article {pmid30375858,
year = {2019},
author = {White, J and Dhingra, SK and Deng, X and El Mazouni, F and Lee, MCS and Afanador, GA and Lawong, A and Tomchick, DR and Ng, CL and Bath, J and Rathod, PK and Fidock, DA and Phillips, MA},
title = {Identification and Mechanistic Understanding of Dihydroorotate Dehydrogenase Point Mutations in Plasmodium falciparum that Confer in Vitro Resistance to the Clinical Candidate DSM265.},
journal = {ACS infectious diseases},
volume = {5},
number = {1},
pages = {90-101},
pmid = {30375858},
issn = {2373-8227},
support = {R01 AI090141/AI/NIAID NIH HHS/United States ; R01 AI103947/AI/NIAID NIH HHS/United States ; R01 AI093380/AI/NIAID NIH HHS/United States ; R01 AI103058/AI/NIAID NIH HHS/United States ; U19 AI089688/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Antimalarials/*pharmacology ; Binding Sites ; CRISPR-Cas Systems ; Crystallography, X-Ray ; Dihydroorotate Dehydrogenase ; Drug Resistance/*genetics ; Gene Editing ; Humans ; Malaria, Falciparum ; Oxidoreductases Acting on CH-CH Group Donors/antagonists &amp; inhibitors/*genetics ; Plasmodium falciparum/*drug effects/enzymology/*genetics ; *Point Mutation ; Pyrimidines/*pharmacology ; Triazoles/*pharmacology ; },
abstract = {Malaria is one of the most challenging human infectious diseases, and both prevention and control have been hindered by the development of Plasmodium falciparum resistance to existing therapies. Several new compounds with novel mechanisms are in clinical development for the treatment of malaria, including DSM265, an inhibitor of Plasmodium dihydroorotate dehydrogenase. To explore the mechanisms by which resistance might develop to DSM265 in the field, we selected for DSM265-resistant P. falciparum parasites in vitro. Any of five different amino acid changes led to reduced efficacy on the parasite and to decreased DSM265 binding to P. falciparum DHODH. The DSM265-resistant parasites retained full sensitivity to atovaquone. All but one of the observed mutations were in the DSM265 binding site, and the remaining C276F was in the adjacent flavin cofactor site. The C276F mutation was previously identified in a recrudescent parasite during a Phase IIa clinical study. We confirmed that this mutation (and the related C276Y) accounted for the full level of observed DSM265 resistance by regenerating the mutation using CRISPR/Cas9 genome editing. X-ray structure analysis of the C276F mutant enzyme showed that conformational changes of nearby residues were required to accommodate the larger F276 residue, which in turn led to a restriction in the size of the DSM265 binding pocket. These findings underscore the importance of developing DSM265 as part of a combination therapy with other agents for successful use against malaria.},
}
@article {pmid30375719,
year = {2018},
author = {Pickett, CJ and Zeller, RW},
title = {Efficient genome editing using CRISPR-Cas-mediated homology directed repair in the ascidian Ciona robusta.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {56},
number = {11-12},
pages = {e23260},
pmid = {30375719},
issn = {1526-968X},
support = {R21 DC013180/DC/NIDCD NIH HHS/United States ; 1R21DC01318001A1/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Ciona/*genetics ; Fetal Proteins/genetics/metabolism ; Gene Editing/*methods ; POU Domain Factors/genetics/metabolism ; T-Box Domain Proteins/genetics/metabolism ; },
abstract = {Eliminating or silencing a gene's level of activity is one of the classic approaches developmental biologists employ to determine a gene's function. A recently developed method of gene perturbation called CRISPR-Cas, which was derived from a prokaryotic adaptive immune system, has been adapted for use in eukaryotic cells. This technology has been established in several model organisms as a powerful and efficient tool for knocking out or knocking down the function of a gene of interest. It has been recently shown that CRISPR-Cas functions with fidelity and efficiency in Ciona robusta. Here, we show that in C. robusta CRISPR-Cas mediated genomic knock-ins can be efficiently generated. Electroporating a tissue-specific transgene driving Cas9 and a U6-driven gRNA transgene together with a fluorescent protein-containing homology directed repair (FP-HDR) template results in gene-specific patterns of fluorescence consistent with a targeted genomic insertion. Using the Tyrosinase locus to optimize reagents, we first characterize a new Pol III promoter for expressing gRNAs from the Ciona savignyi H1 gene, and then adapt technology that flanks gRNAs by ribozymes allowing cell-specific expression from Pol II promoters. Next, we examine homology arm-length efficiencies of FP-HDR templates. Reagents were then developed for targeting Brachyury and Pou4 that resulted in expected patterns of fluorescence, and sequenced PCR amplicons derived from single embryos validated predicted genomic insertions. Finally, using two differentially colored FP-HDR templates, we show that biallelic FP-HDR template insertion can be detected in live embryos of the F0 generation.},
}
@article {pmid30375512,
year = {2019},
author = {Rakovic, A and Ziegler, J and Mårtensson, CU and Prasuhn, J and Shurkewitsch, K and König, P and Paulson, HL and Klein, C},
title = {PINK1-dependent mitophagy is driven by the UPS and can occur independently of LC3 conversion.},
journal = {Cell death and differentiation},
volume = {26},
number = {8},
pages = {1428-1441},
pmid = {30375512},
issn = {1476-5403},
mesh = {CRISPR-Cas Systems ; Humans ; Microtubule-Associated Proteins/*metabolism ; Proteasome Endopeptidase Complex/*metabolism ; Protein Kinases/*metabolism ; Tumor Cells, Cultured ; Ubiquitin/*metabolism ; },
abstract = {The Parkinson's disease (PD)-related ubiquitin ligase Parkin and mitochondrial kinase PINK1 function together in the clearance of damaged mitochondria. Upon mitochondrial depolarization, Parkin translocates to mitochondria in a PINK1-dependent manner to ubiquitinate outer mitochondrial membrane proteins. According to the current model, the ubiquitin- and LC3-binding adaptor protein SQSTM1 is recruited to mitochondria, followed by their selective degradation through autophagy (mitophagy). However, the role of the ubiquitin proteasome system (UPS), although essential for this process, still remains largely elusive. Here, we investigated the role of the UPS and autophagy by applying the potassium ionophore Valinomycin in PINK1-deficient human fibroblasts and isogenic neuroblastoma cell lines generated by CRISPR/Cas9. Although identical to the commonly used CCCP/FCCP in terms of dissipating the mitochondrial membrane potential and triggering complete removal of mitochondria, Valinomycin did not induce conversion of LC3 to its autophagy-related form. Moreover, FCCP-induced conversion of LC3 occurred even in mitophagy-incompetent, PINK1-deficient cell lines. While both stressors required a functional UPS, the removal of depolarized mitochondria persisted in cells depleted of LC3A and LC3B. Our study highlights the importance of the UPS in PINK1-/Parkin-mediated mitochondrial quality control. In contrast, activation of autophagy, monitored through conversion of LC3, is likely induced by depolarizing-agent-induced toxicity in a PINK1-/Parkin-independent manner.},
}
@article {pmid30375401,
year = {2018},
author = {Nakamura, T and Nakajima, K and Ohnishi, T and Yoshikawa, T and Nakanishi, M and Takumi, T and Tsuboi, T and Kato, T},
title = {Quantitative evaluation of incomplete preweaning lethality in mice by using the CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16025},
pmid = {30375401},
issn = {2045-2322},
support = {16815678//Japan Agency for Medical Research and Development (AMED)/International ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genes, Lethal/genetics ; Genome/genetics ; Genotype ; Mice ; Mice, Knockout ; Mutation ; Phenotype ; Vesicular Transport Proteins/*genetics ; *Weaning ; },
abstract = {Various molecular biology techniques implementing genome editing have made it possible to generate mouse mutants for nearly all known genes; as a result, the International Mouse Phenotyping Consortium (IMPC) database listing the phenotypes of genetically modified mice has been established. Among mouse phenotypes, lethality is crucial to evaluate the importance of genes in mouse survival. Although many genes are reported to show "preweaning lethality, incomplete penetrance" in the IMPC database, the survival rates of homozygous knockout mice are highly variable. Here, we propose the lethal allele index (LAI), the ratio of the observed number of mice with homozygous knockout (KO) to the theoretically predicted number of homozygous KO mice, as a simple quantitative indicator of preweaning lethality. Among the mice mutants registered as incompletely lethal in IMPC, the LAI calculated from the genotypes of F1 mice tended to be lower in disease-related genes, and correlated with the frequency of loss-of-function (LOF) alleles in humans. In genome-edited mice using CRISPR/Cas9, the number of mice with homozygous frameshift alleles seemed to be associated with lethality. We edited the Ehd1 gene in cell lines as well as mice using CRISPR/Cas9, and found that the genotype distribution was significantly different. The LAI calculated from these data was similar to the value calculated from the IMPC data. These findings support the potential usefulness of the LAI as an index of preweaning lethality in genome-edited mice.},
}
@article {pmid30375064,
year = {2019},
author = {Takeda, Y and Suzuki, S and Tobimatsu, Y and Osakabe, K and Osakabe, Y and Ragamustari, SK and Sakamoto, M and Umezawa, T},
title = {Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.},
journal = {The Plant journal : for cell and molecular biology},
volume = {97},
number = {3},
pages = {543-554},
doi = {10.1111/tpj.14141},
pmid = {30375064},
issn = {1365-313X},
mesh = {Acrolein/*analogs &amp; derivatives/metabolism ; Biomass ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Coumaric Acids ; Lignin/*metabolism ; Loss of Function Mutation ; Mixed Function Oxygenases/genetics/*metabolism ; Oryza/enzymology/*genetics ; Plant Proteins/genetics/metabolism ; Propionates/metabolism ; },
abstract = {The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.},
}
@article {pmid30374457,
year = {2018},
author = {England, WE and Kim, T and Whitaker, RJ},
title = {Metapopulation Structure of CRISPR-Cas Immunity in Pseudomonas aeruginosa and Its Viruses.},
journal = {mSystems},
volume = {3},
number = {5},
pages = {},
pmid = {30374457},
issn = {2379-5077},
support = {T32 AI078876/AI/NIAID NIH HHS/United States ; },
abstract = {Viruses that infect the widespread opportunistic pathogen Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57-64, 2015, https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly available P. aeruginosa sequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts that P. aeruginosa viruses face can be applied to study of P. aeruginosa gene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control of P. aeruginosa infection. IMPORTANCE Pseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human "islands." These data set the stage for exploring metapopulation dynamics occurring within and between isolated "island" populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains.},
}
@article {pmid30373918,
year = {2019},
author = {Oser, MG and Fonseca, R and Chakraborty, AA and Brough, R and Spektor, A and Jennings, RB and Flaifel, A and Novak, JS and Gulati, A and Buss, E and Younger, ST and McBrayer, SK and Cowley, GS and Bonal, DM and Nguyen, QD and Brulle-Soumare, L and Taylor, P and Cairo, S and Ryan, CJ and Pease, EJ and Maratea, K and Travers, J and Root, DE and Signoretti, S and Pellman, D and Ashton, S and Lord, CJ and Barry, ST and Kaelin, WG},
title = {Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.},
journal = {Cancer discovery},
volume = {9},
number = {2},
pages = {230-247},
pmid = {30373918},
issn = {2159-8290},
support = {P50 CA101942/CA/NCI NIH HHS/United States ; R35 CA210068/CA/NCI NIH HHS/United States ; T32 CA009172/CA/NCI NIH HHS/United States ; K08 CA222657/CA/NCI NIH HHS/United States ; R01 CA213404/CA/NCI NIH HHS/United States ; //Wellcome Trust/United Kingdom ; K08 CA208008/CA/NCI NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; P50 CA165962/CA/NCI NIH HHS/United States ; /CRUK_/Cancer Research UK/United Kingdom ; R01 CA076120/CA/NCI NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; Aurora Kinase B/genetics/*metabolism ; CRISPR-Cas Systems ; *Cell Proliferation ; Chromosome Segregation ; Drug Resistance, Neoplasm ; Female ; *Genes, Tumor Suppressor ; Humans ; Lung Neoplasms/drug therapy/metabolism/*pathology ; Mice ; Mice, Nude ; *Mutation ; Retinoblastoma Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Signal Transduction ; Small Cell Lung Carcinoma/drug therapy/metabolism/*pathology ; Tumor Cells, Cultured ; Ubiquitin-Protein Ligases/antagonists &amp; inhibitors/genetics/*metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 [-/-] SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 [-/-] SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 [-/-] cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 [-/-] SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 [-/-] SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.},
}
@article {pmid30372609,
year = {2018},
author = {Bourgeois, L and Pyne, ME and Martin, VJJ},
title = {A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast.},
journal = {ACS synthetic biology},
volume = {7},
number = {11},
pages = {2675-2685},
doi = {10.1021/acssynbio.8b00339},
pmid = {30372609},
issn = {2161-5063},
mesh = {Alkaloids/biosynthesis ; CRISPR-Cas Systems/genetics ; Carbon-Nitrogen Ligases/genetics ; DNA Copy Number Variations ; Gene Editing/methods ; *Genome, Fungal ; Metabolic Engineering/*methods ; Saccharomyces cerevisiae/*genetics ; },
abstract = {A fundamental undertaking of metabolic engineering involves identifying and troubleshooting metabolic bottlenecks that arise from imbalances in pathway flux. To expedite the systematic screening of enzyme orthologs in conjunction with DNA copy number tuning, here we develop a simple and highly characterized CRISPR-Cas9 integration system in Saccharomyces cerevisiae. Our engineering strategy introduces a series of synthetic DNA landing pads (LP) into the S. cerevisiae genome to act as sites for high-level gene integration. LPs facilitate multicopy gene integration of one, two, three, or four DNA copies in a single transformation, thus providing precise control of DNA copy number. We applied our LP system to norcoclaurine synthase (NCS), an enzyme with poor kinetic properties involved in the first committed step of the production of high-value benzylisoquinoline alkaloids. The platform enabled rapid construction of a 40-strain NCS library by integrating ten NCS orthologs in four gene copies each. Six active NCS variants were identified, whereby production of (S)-norcoclaurine could be further enhanced by increasing NCS copy number. We anticipate the LP system will aid in metabolic engineering efforts by providing strict control of gene copy number and expediting strain and pathway engineering campaigns.},
}
@article {pmid30371662,
year = {2018},
author = {Nguyen, BH and Ishii, M and Maxson, RE and Wang, J},
title = {Culturing and Manipulation of O9-1 Neural Crest Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {140},
pages = {},
pmid = {30371662},
issn = {1940-087X},
support = {K01 DE026561/DE/NIDCR NIH HHS/United States ; R01 DE016320/DE/NIDCR NIH HHS/United States ; R01 DE019650/DE/NIDCR NIH HHS/United States ; R03 DE025873/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Culture Techniques ; Cell Differentiation ; Cell Line ; Mice ; Multipotent Stem Cells/*cytology ; Myocytes, Smooth Muscle ; Neural Crest/*cytology ; Osteoblasts ; RNA, Small Interfering/genetics ; },
abstract = {Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.},
}
@article {pmid30370841,
year = {2019},
author = {Sinha, R and Shukla, P},
title = {Antimicrobial Peptides: Recent Insights on Biotechnological Interventions and Future Perspectives.},
journal = {Protein and peptide letters},
volume = {26},
number = {2},
pages = {79-87},
pmid = {30370841},
issn = {1875-5305},
mesh = {Animals ; Anti-Infective Agents/*chemical synthesis/*pharmacology ; Antimicrobial Cationic Peptides/*chemical synthesis/*pharmacology ; Biotechnology ; CRISPR-Cas Systems ; Drug Resistance, Microbial ; Gene Editing/methods ; Gene Expression Regulation ; Humans ; Plants, Genetically Modified ; },
abstract = {With the unprecedented rise of drug-resistant pathogens, particularly antibiotic-resistant bacteria, and no new antibiotics in the pipeline over the last three decades, the issue of antimicrobial resistance has emerged as a critical public health threat. Antimicrobial Peptides (AMP) have garnered interest as a viable solution to this grave issue and are being explored for their potential antimicrobial applications. Given their low bioavailability in nature, tailoring new AMPs or strategizing approaches for increasing the yield of AMPs, therefore, becomes pertinent. The present review focuses on biotechnological interventions directed towards enhanced AMP synthesis and revisits existing genetic engineering and synthetic biology strategies for production of AMPs. This review further underscores the importance and potential applications of advanced gene editing technologies for the synthesis of novel AMPs in future.},
}
@article {pmid30369475,
year = {2018},
author = {Zhang, GS and Yang, Y and Zhang, LM and Dai, XH},
title = {[Application of machine learning in the CRISPR/Cas9 system].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {9},
pages = {704-723},
doi = {10.16288/j.yczz.18-135},
pmid = {30369475},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Expression Regulation ; Genetic Engineering/instrumentation/*methods ; Humans ; *Machine Learning ; RNA Editing ; },
abstract = {The third generation of the CRISPR/Cas9-mediated genome fixed-point editing technology has been widely used in the field of gene editing and gene expression regulation. How to improve the on-target efficiency and specificity of this system, as well as reduce its off-target effects are always the bottleneck in its development. Machine learning provides novel methods to the problems of the CRISPR/Cas9 system, and CRISPR/Cas9-based machine learning has recently become a very hot research topic. In this review, we firstly outline the mechanism of the CRISPR/Cas9 system. Subsequently, we elaborate the current issues of CRISPR/Cas9, including low efficiency and potential off-target effects, and sequence-recognizing limitation from protospacer adjacent motif (PAM). Finally, we summarize the applications of methods within the machine learning framework for optimizing the CRISPR/Cas9 system, such as optimized single-guide RNA (sgRNA) design, CRISPR/Cas9 cleavage efficiency prediction, off-target effects evaluation, gene knock-out as well as high-throughput functional genetic screening and prospects for development.},
}
@article {pmid30369472,
year = {2018},
author = {Chen, YO and Bao, Y and Ma, HZ and Yi, ZY and Zhou, Z and Wei, WS},
title = {[Gene editing technology and its research progress in China].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {10},
pages = {900-915},
doi = {10.16288/j.yczz.18-195},
pmid = {30369472},
issn = {0253-9772},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems ; China ; *Gene Editing ; Genetic Engineering ; Plants, Genetically Modified/*genetics ; Research/trends ; },
abstract = {Gene editing is a technology for precision modification of genome sequences or expressing transcripts. Early versions of such techniques include homing endonuclease, zinc finger endonuclease and transcription-like activator effectors. The CRISPR/Cas9 system has recently emerged as a powerful and versatile gene editing tool for a broad spectrum of applications. In this review, we discuss the recent development and applications of gene editing technology, and particularly focus on the progress in China, with the emphasis on mechanistic studies, development of new and extended technologies, the applications in animals, plants and the therapeutics.},
}
@article {pmid30368976,
year = {2019},
author = {Tanihara, F and Hirata, M and Nguyen, NT and Le, QA and Hirano, T and Takemoto, T and Nakai, M and Fuchimoto, DI and Otoi, T},
title = {Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation.},
journal = {Animal science journal = Nihon chikusan Gakkaiho},
volume = {90},
number = {1},
pages = {55-61},
doi = {10.1111/asj.13129},
pmid = {30368976},
issn = {1740-0929},
mesh = {Animals ; *Blastocyst ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Electroporation ; Female ; Fertilization in Vitro ; Gene Editing/*methods ; Homeodomain Proteins/*genetics ; *Mutation ; RNA, Guide/genetics ; Swine/*genetics ; Trans-Activators/*genetics ; *Zygote ; },
abstract = {Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.},
}
@article {pmid30368519,
year = {2018},
author = {Aryal, NK and Wasylishen, AR and Lozano, G},
title = {CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo.},
journal = {Cell death &amp; disease},
volume = {9},
number = {11},
pages = {1099},
pmid = {30368519},
issn = {2041-4889},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *Base Pair Mismatch ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; DEAD-box RNA Helicases/genetics/metabolism ; DNA End-Joining Repair ; Embryo, Mammalian ; Gene Editing/*methods/standards ; Mice ; Mice, Transgenic ; *Mutation ; RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; Ribonuclease III/genetics/metabolism ; },
}
@article {pmid30367669,
year = {2018},
author = {Alkan, F and Wenzel, A and Anthon, C and Havgaard, JH and Gorodkin, J},
title = {CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {177},
pmid = {30367669},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Endonucleases/metabolism ; *Gene Editing ; Humans ; Nucleic Acids/*chemistry ; RNA, Guide/*genetics ; Substrate Specificity ; },
abstract = {BACKGROUND: Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment.<br><br>RESULTS: We present an approximate binding energy model for the Cas9-gRNA-DNA complex, which systematically combines the energy parameters obtained for RNA-RNA, DNA-DNA, and RNA-DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components.<br><br>CONCLUSIONS: The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/ .},
}
@article {pmid30367555,
year = {2019},
author = {Xu, R and Qin, R and Li, H and Li, J and Yang, J and Wei, P},
title = {Enhanced genome editing in rice using single transcript unit CRISPR-LbCpf1 systems.},
journal = {Plant biotechnology journal},
volume = {17},
number = {3},
pages = {553-555},
pmid = {30367555},
issn = {1467-7652},
support = {2016ZX08010-002-008//Genetically Modified Breeding Major Projects/International ; 31501239//National Natural Science Foundation of China/International ; 31701405//National Natural Science Foundation of China/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; Clostridiales/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Oryza/*genetics ; },
}
@article {pmid30367407,
year = {2019},
author = {Chen, J and Wang, C and Kuang, S},
title = {Transdifferentiation of Muscle Satellite Cells to Adipose Cells Using CRISPR/Cas9-Mediated Targeting of MyoD.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1889},
number = {},
pages = {25-41},
pmid = {30367407},
issn = {1940-6029},
support = {R01 AR071649/AR/NIAMS NIH HHS/United States ; },
mesh = {Adipocytes/cytology/metabolism ; Adipogenesis/genetics ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Cell Transdifferentiation/*genetics ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; *Gene Targeting ; Humans ; Immunohistochemistry ; Mice, Knockout ; MyoD Protein/*genetics/metabolism ; RNA, Guide ; Satellite Cells, Skeletal Muscle/*cytology/*metabolism ; },
abstract = {Brown adipocytes dissipate energy through non-shivering thermogenesis mediated by UCP1 protein, hence representing a powerful target to overcome obesity due to energy surplus. However, brown adipocytes are scarce in adult humans, especially in obese subjects, urging the development of novel strategies to boost the number of these thermogenic adipocytes from a therapeutical perspective. In this regard, transdifferentiation of myoblasts into brown adipocytes represents a promising approach. Here, we describe a method that we have recently developed to transdifferentiate myoblasts into brown adipocytes through CRISPR/Cas9-medidated targeting of MyoD, the master myogenic regulatory factor.},
}
@article {pmid30367000,
year = {2019},
author = {Šulčius, S and Šimoliūnas, E and Alzbutas, G and Gasiūnas, G and Jauniškis, V and Kuznecova, J and Miettinen, S and Nilsson, E and Meškys, R and Roine, E and Paškauskas, R and Holmfeldt, K},
title = {Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {1},
pages = {},
pmid = {30367000},
issn = {1098-5336},
mesh = {Aphanizomenon/*virology ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Viral/analysis ; Genome, Viral/*genetics ; Lithuania ; Phylogeny ; Siphoviridae/classification/*genetics ; },
abstract = {While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria.IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.},
}
@article {pmid30366936,
year = {2018},
author = {Prykhozhij, SV and Berman, JN},
title = {Zebrafish knock-ins swim into the mainstream.},
journal = {Disease models &amp; mechanisms},
volume = {11},
number = {10},
pages = {},
pmid = {30366936},
issn = {1754-8411},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; *Gene Knock-In Techniques ; Point Mutation/genetics ; Zebrafish/*genetics ; },
abstract = {The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study.},
}
@article {pmid30366497,
year = {2018},
author = {Nomura, W and Matsumoto, D and Sugii, T and Kobayakawa, T and Tamamura, H},
title = {Efficient and Orthogonal Transcription Regulation by Chemically Inducible Artificial Transcription Factors.},
journal = {Biochemistry},
volume = {57},
number = {45},
pages = {6452-6459},
doi = {10.1021/acs.biochem.8b00741},
pmid = {30366497},
issn = {1520-4995},
mesh = {Antibiotics, Antineoplastic/pharmacology ; *CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation/*drug effects ; Gibberellins/*pharmacology ; Humans ; Luciferases/*metabolism ; Plant Growth Regulators/pharmacology ; Promoter Regions, Genetic ; Sirolimus/*pharmacology ; Tacrolimus Binding Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Transcription Factors/antagonists &amp; inhibitors/genetics/*metabolism ; *Transcriptional Activation ; },
abstract = {The DNA-binding specificity of genome editing tools can be applied to gene regulation. Recently, multiple artificial transcription factors (ATFs) were shown to synergistically and efficiently regulate gene expression. Chemically triggered protein associations are useful for functional regulation at specific timings. A combination of several inducible protein association systems could enable the regulation of multiple genes at different loci with independent timing. We applied the FKBP-rapamycin-FRB and GAI-gibberellin-GID systems for gene regulation using multiple TALEs and dCas9. By the combined use of currently available systems, reporter gene assays were performed; the results indicated that gene expression was regulated by rapamycin or gibberellin in the presence of the FRB or GAI effector domains, respectively. Furthermore, the activation of endogenous genes was differentially regulated by the system. This success suggests the usability of the chemically inducible multiple ATFs for the time-dependent regulation of multiple genes, such as the case for cellular phenomena that are dependent on the programmable timing of expression and the differential expression of multiple genes.},
}
@article {pmid30365569,
year = {2018},
author = {Zhao, Q and Busch, B and Jiménez-Soto, LF and Ishikawa-Ankerhold, H and Massberg, S and Terradot, L and Fischer, W and Haas, R},
title = {Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation.},
journal = {PLoS pathogens},
volume = {14},
number = {10},
pages = {e1007359},
pmid = {30365569},
issn = {1553-7374},
mesh = {Antigens, Bacterial/genetics/*metabolism ; Antigens, CD/genetics/*metabolism ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Adhesion Molecules/antagonists &amp; inhibitors/genetics/*metabolism ; GPI-Linked Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Integrins/antagonists &amp; inhibitors/genetics/*metabolism ; Protein Transport ; Stomach Neoplasms/genetics/*metabolism/pathology ; Tumor Cells, Cultured ; },
abstract = {Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like molecular apparatus extended by an external pilus-like rod structure that binds host cell surface integrin heterodimers. It is still largely unclear how the interaction of the cag-T4SS finally mediates translocation of the CagA protein into the cell cytoplasm. Recently certain carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), acting as receptor for the Hp outer membrane adhesin HopQ, have been identified to be involved in the process of CagA host cell injection. Here, we applied the CRISPR/Cas9-knockout technology to generate defined human gastric AGS and KatoIII integrin knockout cell lines. Although confocal laser scanning microscopy revealed a co-localization of Hp and β1 integrin heterodimers on gastric epithelial cells, Hp infection studies using the quantitative and highly sensitive Hp β-lactamase reporter system clearly show that neither β1 integrin heterodimers (α1β1, α2β1 or α5β1), nor any other αβ integrin heterodimers on the cell surface are essential for CagA translocation. In contrast, deletion of the HopQ adhesin in Hp, or the simultaneous knockout of the receptors CEACAM1, CEACAM5 and CEACAM6 in KatoIII cells abolished CagA injection nearly completely, although bacterial binding was only reduced to 50%. These data provide genetic evidence that the cag-T4SS-mediated interaction of Hp with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of Hp with CEACAM receptors is facilitating CagA translocation by the cag-T4SS of this important microbe.},
}
@article {pmid30363939,
year = {2018},
author = {Donà, V and Perreten, V},
title = {Comparative Genomics of the First and Complete Genome of "Actinobacillus porcitonsillarum" Supports the Novel Species Hypothesis.},
journal = {International journal of genomics},
volume = {2018},
number = {},
pages = {5261719},
pmid = {30363939},
issn = {2314-436X},
abstract = {"Actinobacillus porcitonsillarum" is considered a nonpathogenic member of the Pasteurellaceae family, which phenotypically resembles the pathogen Actinobacillus pleuropneumoniae. Previous studies suggested that "A. porcitonsillarum" may represent a new species closely related to Actinobacillus minor, yet no full genome has been sequenced so far. We implemented the Oxford Nanopore and Illumina sequencing technologies to obtain the highly accurate and complete genome sequence of the "A. porcitonsillarum" strain 9953L55. After validating our de novo assembly strategy by comparing the A. pleuropneumoniae S4074[T] genome sequence obtained by Oxford Nanopore Technology combined with Illumina reads with a PacBio-sequenced S4074[T] genome from the NCBI database, we performed comparative analyses of the 9953L55 genome with the A. minor type strain NM305[T], A. minor strain 202, and A. pleuropneumoniae S4074[T]. The 2,263,191 bp circular genome of 9953L55 consisted of 2168 and 2033 predicted genes and proteins, respectively. The lipopolysaccharide cluster resembled the genetic organization of A. pleuropneumoniae serotypes 1, 9, and 11, possibly explaining the positive reactions observed previously in serotyping tests. In contrast to NM305[T], we confirmed the presence of a complete apxIICABD operon in 9953L55 and 202 accounting for their hemolytic phenotype and Christie-Atkins-Munch-Petersen (CAMP) reaction positivity. Orthologous gene cluster analysis provided insight into the differential ability of strains of the A. minor/"porcitonsillarum" complex and A. pleuropneumoniae to ferment lactose, raffinose, trehalose, and mannitol. The four strains showed distinct and shared transposable elements, CRISPR/Cas systems, and integrated prophages. Genome comparisons based on average nucleotide identity and in silico DNA-DNA hybridization confirmed the close relationship among strains belonging to the A. minor/"porcitonsillarum" complex compared to other Actinobacillus spp., but also suggested that 9953L55 and 202 belong to the same novel species closely related to A. minor, namely, "A. porcitonsillarum." Recognition of the taxon as a separate species would improve diagnostics and control strategies of pig pleuropneumonia.},
}
@article {pmid30362590,
year = {2019},
author = {Khambhati, K and Bhattacharjee, G and Singh, V},
title = {Current progress in CRISPR-based diagnostic platforms.},
journal = {Journal of cellular biochemistry},
volume = {120},
number = {3},
pages = {2721-2725},
pmid = {30362590},
issn = {1097-4644},
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/analysis ; Humans ; *Molecular Diagnostic Techniques ; Polymerase Chain Reaction ; RNA/analysis ; Signal Processing, Computer-Assisted ; },
abstract = {The CRISPR-Cas system is a key technology for genome editing and regulation in a wide range of organisms and cell types. Recently, CRISPR-Cas-based diagnostic platform has shown idealistic properties for pathogen detection. Integrating the CRISPR-Cas platform along with lateral flow system allows rapid, sensitive, specific, cheap, and reliable diagnostic. It has the potential to be in frontline for not only pathogen detection during the epidemic outbreak, but also cancer, and genetic diseases.},
}
@article {pmid30362544,
year = {2019},
author = {Khadempar, S and Familghadakchi, S and Motlagh, RA and Farahani, N and Dashtiahangar, M and Rezaei, H and Gheibi Hayat, SM},
title = {CRISPR-Cas9 in genome editing: Its function and medical applications.},
journal = {Journal of cellular physiology},
volume = {234},
number = {5},
pages = {5751-5761},
doi = {10.1002/jcp.27476},
pmid = {30362544},
issn = {1097-4652},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Targeting/*methods ; Genetic Therapy/*methods ; Humans ; RNA, Guide/genetics/metabolism ; },
abstract = {The targeted genome modification using RNA-guided nucleases is associated with several advantages such as a rapid, easy, and efficient method that not only provides the manipulation and alteration of genes and functional studies for researchers, but also increases their awareness of the molecular basis of the disease and development of new and targeted therapeutic approaches. Different techniques have been emerged so far as the molecular scissors mediating targeted genome editing including zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). CRISPR-Cas9 is a bacterial immune system against viruses in which the single-strand RNA-guided Cas9 nuclease is linked to the targeted complementary sequences to apply changes. The advances made in the transfer, modification, and emergence of specific solutions have led to the creation of different classes of CRISPR-Cas9. Since this robust tool is capable of direct correction of disease-causing mutations, its ability to treat genetic disorders has attracted the tremendous attention of researchers. Considering the reported cases of nonspecific targeting of Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard, significant advances have been made in choosing guide RNA, new enzymes and methods for identifying misplaced targeting. Here, we highlighted the history and various direct aspects of CRISPR-Cas9, such as precision in genomic targeting, system transfer and its control over correction events with its applications in future biological studies, and modern treatment of diseases.},
}
@article {pmid30361373,
year = {2018},
author = {Kratochwil, CF and Liang, Y and Gerwin, J and Woltering, JM and Urban, S and Henning, F and Machado-Schiaffino, G and Hulsey, CD and Meyer, A},
title = {Agouti-related peptide 2 facilitates convergent evolution of stripe patterns across cichlid fish radiations.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6413},
pages = {457-460},
doi = {10.1126/science.aao6809},
pmid = {30361373},
issn = {1095-9203},
mesh = {Agouti-Related Protein/genetics/*physiology ; Animals ; *Biological Evolution ; CRISPR-Cas Systems ; Chromosome Mapping ; Cichlids/*anatomy &amp; histology/genetics/*physiology ; Gene Knockout Techniques ; Genetic Loci ; Mutation ; *Skin Pigmentation/genetics ; },
abstract = {The color patterns of African cichlid fishes provide notable examples of phenotypic convergence. Across the more than 1200 East African rift lake species, melanic horizontal stripes have evolved numerous times. We discovered that regulatory changes of the gene agouti-related peptide 2 (agrp2) act as molecular switches controlling this evolutionarily labile phenotype. Reduced agrp2 expression is convergently associated with the presence of stripe patterns across species flocks. However, cis-regulatory mutations are not predictive of stripes across radiations, suggesting independent regulatory mechanisms. Genetic mapping confirms the link between the agrp2 locus and stripe patterns. The crucial role of agrp2 is further supported by a CRISPR-Cas9 knockout that reconstitutes stripes in a nonstriped cichlid. Thus, we unveil how a single gene affects the convergent evolution of a complex color pattern.},
}
@article {pmid30361258,
year = {2018},
author = {McDiarmid, TA and Au, V and Loewen, AD and Liang, J and Mizumoto, K and Moerman, DG and Rankin, CH},
title = {CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance.},
journal = {Disease models &amp; mechanisms},
volume = {11},
number = {12},
pages = {},
pmid = {30361258},
issn = {1754-8411},
support = {MOP 130287//CIHR/Canada ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/chemistry/genetics ; Chemotaxis/drug effects ; Choice Behavior/drug effects ; *Conserved Sequence ; Gene Deletion ; Gene Dosage ; Gene Editing ; *Gene Targeting ; Genes, Helminth ; *Genetic Variation ; Humans ; Neurons/drug effects/metabolism ; PTEN Phosphohydrolase/chemistry/genetics ; Phenotype ; Sodium Chloride/pharmacology ; },
abstract = {Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole-exome and whole-genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization, based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans, that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene phosphatase and tensin homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosensory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E), and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss of function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines the identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.},
}
@article {pmid30359560,
year = {2019},
author = {Mehravar, M and Shirazi, A and Nazari, M and Banan, M},
title = {Mosaicism in CRISPR/Cas9-mediated genome editing.},
journal = {Developmental biology},
volume = {445},
number = {2},
pages = {156-162},
doi = {10.1016/j.ydbio.2018.10.008},
pmid = {30359560},
issn = {1095-564X},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Genetic Therapy/adverse effects/methods ; Humans ; Male ; Models, Genetic ; *Mosaicism ; Mutation ; Pregnancy ; },
abstract = {The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.},
}
@article {pmid30358434,
year = {2019},
author = {McCullough, KT and Boye, SL and Fajardo, D and Calabro, K and Peterson, JJ and Strang, CE and Chakraborty, D and Gloskowski, S and Haskett, S and Samuelsson, S and Jiang, H and Witherspoon, CD and Gamlin, PD and Maeder, ML and Boye, SE},
title = {Somatic Gene Editing of GUCY2D by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque.},
journal = {Human gene therapy},
volume = {30},
number = {5},
pages = {571-589},
pmid = {30358434},
issn = {1557-7422},
support = {P30 EY003039/EY/NEI NIH HHS/United States ; R01 EY024280/EY/NEI NIH HHS/United States ; R01 EY025555/EY/NEI NIH HHS/United States ; T32 EY007132/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Dependovirus/*genetics ; Electroretinography ; *Gene Editing ; Genes, Reporter ; Genetic Vectors/genetics ; Guanylate Cyclase/*genetics/metabolism ; Macaca ; Mice ; Mice, Knockout ; Molecular Imaging/methods ; Promoter Regions, Genetic ; RNA, Guide/chemistry/genetics ; Receptors, Cell Surface/*genetics/metabolism ; Retina/*metabolism/pathology ; },
abstract = {Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.},
}
@article {pmid30358158,
year = {2019},
author = {Maguire, JA and Cardenas-Diaz, FL and Gadue, P and French, DL},
title = {Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.},
journal = {Current protocols in stem cell biology},
volume = {48},
number = {1},
pages = {e64},
pmid = {30358158},
issn = {1938-8969},
support = {U01 HL099656/HL/NHLBI NIH HHS/United States ; U01 HL134696/HL/NHLBI NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting/*methods ; Genome, Human ; Humans ; Mutation ; *Pluripotent Stem Cells ; },
abstract = {Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley &amp; Sons, Inc.},
}
@article {pmid30357987,
year = {2019},
author = {Binder, WH},
title = {The Past 40 Years of Macromolecular Sciences: Reflections on Challenges in Synthetic Polymer and Material Science.},
journal = {Macromolecular rapid communications},
volume = {40},
number = {1},
pages = {e1800610},
doi = {10.1002/marc.201800610},
pmid = {30357987},
issn = {1521-3927},
mesh = {Macromolecular Substances/chemical synthesis/chemistry ; Materials Science ; Polymers/chemical synthesis/*chemistry ; },
abstract = {Technology and science are often successful in discontinuities ("disruptive innovations" or "leapfrogging"), in turn allowing true, big societal development by entire changes in technology rather than by minuscule stepwise improvements. Examples are the emergence of modern computer science by inventing the field-effect transistor rather than further fine-tuning the "Röhrentransistor"; the development of (organic) light-emitting diodes in advance of the "Gasglühstrumpf"; CRISPR/Cas exceeding any previous genetic method or Ziegler-Natta polymerization enabling stereoregular polypropylene (PP) and high-density polyethylene (HDPE) in advance of free-radical polymerization. Where may the frogs in polymer science in the future "jump" to? Contemplating past achievements in (synthetic) polymer science, such as living polymerization, "click" chemistry, supramolecular chemistry, the potentially "leaping" areas of self-healing and (bio)degradable materials, amyloids, and biomaterials are reflected upon.},
}
@article {pmid30357811,
year = {2019},
author = {Jiao, J and Jin, Y and Zheng, M and Zhang, H and Yuan, M and Lv, Z and Odhiambo, W and Yu, X and Zhang, P and Li, C and Ma, Y and Ji, Y},
title = {AID and TET2 co-operation modulates FANCA expression by active demethylation in diffuse large B cell lymphoma.},
journal = {Clinical and experimental immunology},
volume = {195},
number = {2},
pages = {190-201},
pmid = {30357811},
issn = {1365-2249},
mesh = {Animals ; Antineoplastic Agents/pharmacology ; B-Lymphocytes/metabolism ; Bortezomib/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Transformation, Neoplastic/metabolism ; Cytidine Deaminase/genetics/*metabolism ; *DNA Demethylation ; DNA-Binding Proteins/*metabolism ; Dioxygenases ; Disease Models, Animal ; Fanconi Anemia Complementation Group A Protein/*genetics/metabolism ; Gene Expression Regulation/genetics ; Gene Knockout Techniques ; Humans ; Immunoglobulin Class Switching/genetics ; Lymphoma, Large B-Cell, Diffuse/*drug therapy/immunology/*pathology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Proto-Oncogene Mas ; Proto-Oncogene Proteins/*metabolism ; Somatic Hypermutation, Immunoglobulin/genetics ; },
abstract = {Diffuse large B cell lymphoma (DLBCL) is traced to a mature B malignance carrying abnormal activation-induced cytidine deaminase (AID) expression. AID activity initially focuses on deamination of cytidine to uracil to generate somatic hypermutation and class-switch recombination of the immunoglobulin (Ig), but recently it has been implicated in DNA demethylation of genes required for B cell development and proliferation in the germinal centre (GC). However, whether AID activity on mutation or demethylation of genes involves oncogenesis of DLBCL has not been well characterized. Our data demonstrate that the proto-oncogene Fanconi anaemia complementation group A (FANCA) is highly expressed in DLBCL patients and cell lines, respectively. AID recruits demethylation enzyme ten eleven translocation family member (TET2) to bind the FANCA promoter. As a result, FANCA is demethylated and its expression increases in DLBCL. On the basis of our findings, we have developed a new therapeutic strategy to significantly inhibit DLBCL cell growth by combination of the proteasome inhibitor bortezomib with AID and TET2 depletion. These findings support a novel mechanism that AID has a crucial role in active demethylation for oncogene activation in DLBCL.},
}
@article {pmid30357503,
year = {2019},
author = {Peng, F and Liu, X and Wang, X and Chen, J and Liu, M and Yang, Y and Bai, Z},
title = {Triple deletion of clpC, porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {46},
number = {1},
pages = {67-79},
pmid = {30357503},
issn = {1476-5535},
mesh = {Bacterial Proteins/*genetics/metabolism ; Batch Cell Culture Techniques ; Bioreactors ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Corynebacterium glutamicum/*genetics ; *Gene Deletion ; Gene Editing ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; Green Fluorescent Proteins/genetics/metabolism ; Multigene Family ; Natriuretic Peptide, Brain/genetics/metabolism ; Peptide Fragments/genetics/metabolism ; Plasmids/genetics ; Reproducibility of Results ; Ubiquitin/genetics/metabolism ; },
abstract = {In our previous work, a two-plasmid CRISPR/Cas9 system was constructed for genome editing in Corynebacterium glutamicum. To increase the transformation efficiency and simplify the plasmid curing steps, an all-in-one CRISPR/Cas9 system was constructed for efficient genome editing. In addition, to research proteolysis during the production of recombinant proteins and generate a host for enhanced expression of recombinant proteins, the system was used to delete three genes, clpC, porB, and mepA in C. glutamicum CGMCC1.15647, which encoded the Clp protease subunit ClpC, anion selective channel protein B, and metallopeptidase A, respectively. After the evaluation of different plasmids and hosts, small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide (SUMO-NT-proBNP), an important protein used for the diagnosis of mild heart failure was successfully expressed in the triple mutant ΔclpCΔporBΔmepA, which exhibit threefold higher levels of protein expression compared with the wild-type. In conclusion, we created a simplified CRISPR tool for genome editing in C. glutamicum, provided a method to generate a host for enhanced expression of recombinant proteins and successfully expressed SUMO-NT-proBNP in C. glutamicum. This tool and method will greatly facilitate genetic engineering and metabolic optimization of this important platform organism.},
}
@article {pmid30357478,
year = {2019},
author = {Baliga, P and Shekar, M and Venugopal, MN},
title = {Investigation of direct repeats, spacers and proteins associated with clustered regularly interspaced short palindromic repeat (CRISPR) system of Vibrio parahaemolyticus.},
journal = {Molecular genetics and genomics : MGG},
volume = {294},
number = {1},
pages = {253-262},
pmid = {30357478},
issn = {1617-4623},
mesh = {Adaptive Immunity ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; DNA, Intergenic ; Genome, Bacterial ; Vibrio parahaemolyticus/*genetics/immunology ; },
abstract = {Vibrio parahaemolyticus, a ubiquitous bacterium of the marine environment is an important food-borne pathogen responsible for gastroenteritis worldwide. In this study, we aimed to investigate the occurrence and diversity of the CRISPR-Cas system in V. parahaemolyticus genomes using a bioinformatics approach. The CRISPR-Cas system functions as an adaptive immune system in prokaryotes that provides immunity against foreign genetic elements. In total, 570 genomes V. parahaemolyticus genomes were analyzed of which 200 confirmed for the presence of CRISPR-Cas system. The CRISPR-Cas loci were further analyzed for their repeats, spacers and associated Cas proteins. Among the 200 V. parahaemolyticus strains analyzed, 16 (8%) strains possessed the CRISPR-Cas system of complete subtype I-F, while the remaining 184 (92%) harbored the minimalistic type, a subtype I-F variant. Orphan CRISPR repeats and Cas genes were found in one strain each. The CRISPR-associated direct repeat had an unit length of 28 bases. The number of repeat units in each array ranged from 3 to 5 or 5-41 depending on whether they belonged to the minimalistic or complete subtype-IF CRISPR-Cas system, respectively. Of the 768 spacers analyzed in this study, 295 were found to be unique to V. parahaemolyticus. Homology analysis of the conserved spacers revealed matches to plasmids, phages and gut viruses and self chromosomes. Among the CRISPR-associated proteins, Cas5 and Cas7 proteins were found to be conserved. However, variations were seen in the Cas6 protein, which could be grouped into four different types based on their protein length as well as amino acid composition. We present here the diversity and main features of the CRISPR-Cas system in V. parahaemolyticus, which could provide valuable insights in elucidating the role and mechanism of CRISPR/Cas elements in this pathogen.},
}
@article {pmid30357163,
year = {2018},
author = {Zheng, L and Shi, J and Mu, Y},
title = {Dynamics changes of CRISPR-Cas9 systems induced by high fidelity mutations.},
journal = {Physical chemistry chemical physics : PCCP},
volume = {20},
number = {43},
pages = {27439-27448},
doi = {10.1039/c8cp04226h},
pmid = {30357163},
issn = {1463-9084},
mesh = {Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Endonucleases/genetics/*metabolism ; *Mutation ; Protein Binding/genetics ; Protein Domains ; Static Electricity ; },
abstract = {CRISPR-Cas9, a powerful genome editing tool, has widely been applied in biological fields. Since the discovery of CRISPR-Cas9 as an adaptive immune system, it has been gradually modified to perform precise genome editing in eukaryotic cells by creating double-strand breaks. Although it is robust and efficient, the current CRISPR-Cas9 system faces a major flaw: off-target effects, which are not well understood. Several Cas9 mutants show significant improvement, with very low off-target effects; however, they also show relatively lower cleavage efficiency for on-target sequences. In this study, the dynamics of wild-type Cas9 from Streptococcus pyogenes and a high fidelity Cas9 mutant have been explored using molecular dynamics simulations. It was found that the mutations cause decreased electrostatic interactions between Cas9 and the R-loop. Consequently, the flexibility of the tDNA/sgRNA heteroduplex is decreased, which may explain the lower tolerance of mismatches in the heteroduplex region. The mutations also affect the protein dynamics and the correlation networks among Cas9 domains. In mutant Cas9, weakened communications between two catalytic domains as well as a slight opening of the conformation induced by the mutations account for the lower on-target cleavage efficiency and probably the lower off-target efficiency as well. These findings will facilitate more precise Cas9 engineering in future.},
}
@article {pmid30356064,
year = {2018},
author = {Wang, X and Li, X and Wang, T and Wu, SP and Jeong, JW and Kim, TH and Young, SL and Lessey, BA and Lanz, RB and Lydon, JP and DeMayo, FJ},
title = {SOX17 regulates uterine epithelial-stromal cross-talk acting via a distal enhancer upstream of Ihh.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4421},
pmid = {30356064},
issn = {2041-1723},
support = {R01 HD042311/HD/NICHD NIH HHS/United States ; R01 HD067721/HD/NICHD NIH HHS/United States ; HD-042311//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/International ; Z1AES103311-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/International ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Endometrium/metabolism ; Female ; GATA2 Transcription Factor/genetics/metabolism ; HMGB Proteins/genetics/*metabolism ; Hedgehog Proteins/genetics/metabolism ; Hepatocyte Nuclear Factor 3-beta/genetics/metabolism ; Leukemia Inhibitory Factor/genetics/metabolism ; Mice ; SOXF Transcription Factors/genetics/*metabolism ; Transcriptome/genetics ; Uterus/*metabolism ; },
abstract = {Mammalian pregnancy depends on the ability of the uterus to support embryo implantation. Previous studies reveal the Sox17 gene as a downstream target of the Pgr-Gata2-dependent transcription network that directs genomic actions in the uterine endometrium receptive for embryo implantation. Here, we report that ablating Sox17 in the uterine epithelium impairs leukemia inhibitory factor (LIF) and Indian hedgehog homolog (IHH) signaling, leading to failure of embryo implantation. In vivo deletion of the SOX17-binding region 19 kb upstream of the Ihh locus by CRISPR-Cas technology reduces Ihh expression specifically in the uterus and alters proper endometrial epithelial-stromal interactions, thereby impairing pregnancy. This SOX17-binding interval is also bound by GATA2, FOXA2, and PGR. This cluster of transcription factor binding is common in 737 uterine genes and may represent a key regulatory element essential for uterine epithelial gene expression.},
}
@article {pmid30355950,
year = {2018},
author = {Xu, J and Barone, S and Zahedi, K and Brooks, M and Soleimani, M},
title = {Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {50},
number = {4},
pages = {1361-1375},
doi = {10.1159/000494596},
pmid = {30355950},
issn = {1421-9778},
support = {I01 BX001000/BX/BLRD VA/United States ; },
mesh = {Animals ; Bicarbonates/metabolism ; CRISPR-Cas Systems/genetics ; Diuresis/drug effects ; Dogs ; Furosemide/pharmacology ; In Situ Hybridization ; Kidney/*metabolism ; Kidney Tubules, Collecting/metabolism ; Madin Darby Canine Kidney Cells ; Mice ; Mice, Knockout ; Oligoribonucleotides, Antisense/metabolism ; Plasmids/genetics/metabolism ; Sodium/urine ; Sodium Chloride/metabolism ; Sodium-Bicarbonate Symporters/deficiency/genetics/*metabolism ; },
abstract = {BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney.<br><br>METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice.<br><br>RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis.<br><br>CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.},
}
@article {pmid30355756,
year = {2018},
author = {Tessadori, F and Roessler, HI and Savelberg, SMC and Chocron, S and Kamel, SM and Duran, KJ and van Haelst, MM and van Haaften, G and Bakkers, J},
title = {Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders.},
journal = {Disease models &amp; mechanisms},
volume = {11},
number = {10},
pages = {},
pmid = {30355756},
issn = {1754-8411},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cardiovascular Diseases/*genetics ; Disease Models, Animal ; *Gene Editing ; Gene Knock-In Techniques ; Genetic Testing ; Heterozygote ; Humans ; Mutation/genetics ; Nucleotides/*genetics ; Zebrafish/*genetics ; },
abstract = {The zebrafish (Danio rerio) has become a popular vertebrate model organism to study organ formation and function due to its optical clarity and rapid embryonic development. The use of genetically modified zebrafish has also allowed identification of new putative therapeutic drugs. So far, most studies have relied on broad overexpression of transgenes harboring patient-derived mutations or loss-of-function mutants, which incompletely model the human disease allele in terms of expression levels or cell-type specificity of the endogenous gene of interest. Most human genetically inherited conditions are caused by alleles carrying single nucleotide changes resulting in altered gene function. Introduction of such point mutations in the zebrafish genome would be a prerequisite to recapitulate human disease but remains challenging to this day. We present an effective approach to introduce small nucleotide changes in the zebrafish genome. We generated four different knock-in lines carrying distinct human cardiovascular-disorder-causing missense mutations in their zebrafish orthologous genes by combining CRISPR/Cas9 with a short template oligonucleotide. Three of these lines carry gain-of-function mutations in genes encoding the pore-forming (Kir6.1, KCNJ8) and regulatory (SUR2, ABCC9) subunits of an ATP-sensitive potassium channel (KATP) linked to Cantú syndrome (CS). Our heterozygous zebrafish knock-in lines display significantly enlarged ventricles with enhanced cardiac output and contractile function, and distinct cerebral vasodilation, demonstrating the causality of the introduced mutations for CS. These results demonstrate that introducing patient alleles in their zebrafish orthologs promises a broad application for modeling human genetic diseases, paving the way for new therapeutic strategies using this model organism.},
}
@article {pmid30355591,
year = {2018},
author = {Boel, A and De Saffel, H and Steyaert, W and Callewaert, B and De Paepe, A and Coucke, PJ and Willaert, A},
title = {CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.},
journal = {Disease models &amp; mechanisms},
volume = {11},
number = {10},
pages = {},
pmid = {30355591},
issn = {1754-8411},
mesh = {Animals ; Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Gene Knock-In Techniques ; *Genome ; Germ Cells/metabolism ; Injections ; Mutation/*genetics ; Oligodeoxyribonucleotides/*metabolism ; Recombinational DNA Repair/*genetics ; *Templates, Genetic ; Zebrafish/*genetics ; },
abstract = {Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.},
}
@article {pmid30355043,
year = {2018},
author = {VandenDriessche, T and Chuah, MK},
title = {Getting Into the Rhythm With CRISPR.},
journal = {Circulation research},
volume = {123},
number = {8},
pages = {928-930},
doi = {10.1161/CIRCRESAHA.118.313876},
pmid = {30355043},
issn = {1524-4571},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Ryanodine Receptor Calcium Release Channel ; *Tachycardia, Ventricular ; },
}
@article {pmid30355031,
year = {2018},
author = {Pan, X and Philippen, L and Lahiri, SK and Lee, C and Park, SH and Word, TA and Li, N and Jarrett, KE and Gupta, R and Reynolds, JO and Lin, J and Bao, G and Lagor, WR and Wehrens, XHT},
title = {In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia.},
journal = {Circulation research},
volume = {123},
number = {8},
pages = {953-963},
pmid = {30355031},
issn = {1524-4571},
support = {T32 HL139430/HL/NHLBI NIH HHS/United States ; R56 HL131649/HL/NHLBI NIH HHS/United States ; R01 HL136389/HL/NHLBI NIH HHS/United States ; R01 HL091947/HL/NHLBI NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; R01 HL089598/HL/NHLBI NIH HHS/United States ; R01 HL117641/HL/NHLBI NIH HHS/United States ; },
mesh = {Action Potentials/genetics ; Animals ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Calcium Signaling/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Predisposition to Disease ; Genetic Therapy/*methods ; Genetic Vectors ; Heart Rate/genetics ; Mice, Inbred C57BL ; Mice, Transgenic ; *Mutation ; Phenotype ; RNA, Guide/genetics ; Ryanodine Receptor Calcium Release Channel/*genetics/metabolism ; Tachycardia, Ventricular/genetics/metabolism/physiopathology/*therapy ; },
abstract = {RATIONALE: Autosomal-dominant mutations in ryanodine receptor type 2 (RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.<br><br>OBJECTIVE: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).<br><br>METHODS AND RESULTS: Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 (Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca[2+] spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca[2+] transients.<br><br>CONCLUSIONS: AAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.},
}
@article {pmid30354961,
year = {2018},
author = {Pasricha, SR and Drakesmith, H},
title = {Hemoglobinopathies in the Fetal Position.},
journal = {The New England journal of medicine},
volume = {379},
number = {17},
pages = {1675-1677},
doi = {10.1056/NEJMcibr1809628},
pmid = {30354961},
issn = {1533-4406},
support = {G0901149/MRC_/Medical Research Council/United Kingdom ; MC_UU_00008/10/MRC_/Medical Research Council/United Kingdom ; MC_UU_12010/10/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Anemia, Sickle Cell/*drug therapy/genetics ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Erythroid Cells/metabolism ; Fetal Hemoglobin/*genetics ; *Gene Expression Regulation ; Humans ; Molecular Targeted Therapy ; eIF-2 Kinase/*antagonists &amp; inhibitors/genetics/metabolism ; },
}
@article {pmid30354846,
year = {2019},
author = {Yu, N and Yang, J and Mishina, Y and Giannobile, WV},
title = {Genome Editing: A New Horizon for Oral and Craniofacial Research.},
journal = {Journal of dental research},
volume = {98},
number = {1},
pages = {36-45},
pmid = {30354846},
issn = {1544-0591},
support = {R01 DE020843/DE/NIDCR NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Repair ; Endonucleases ; *Gene Editing ; *Genetic Therapy ; Humans ; },
abstract = {Precise and efficient genetic manipulations have enabled researchers to understand gene functions in disease and development, providing a platform to search for molecular cures. Over the past decade, the unprecedented advancement of genome editing techniques has revolutionized the biological research fields. Early genome editing strategies involved many naturally occurring nucleases, including meganucleases, zinc finger nucleases, and transcription activator-like effector-based nucleases. More recently, the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nucleases (CRISPR/Cas) system has greatly enriched genetic manipulation methods in conducting research. Those nucleases generate double-strand breaks in the target gene sequences and then utilize DNA repair mechanisms to permit precise yet versatile genetic manipulations. The oral and craniofacial field harbors a plethora of diseases and developmental defects that require genetic models that can exploit these genome editing techniques. This review provides an overview of the genome editing techniques, particularly the CRISPR/Cas9 technique, for the oral and craniofacial research community. We also discuss the details about the emerging applications of genome editing in oral and craniofacial biology.},
}
@article {pmid30354811,
year = {2018},
author = {Waghulde, H and Cheng, X and Galla, S and Mell, B and Cai, J and Pruett-Miller, SM and Vazquez, G and Patterson, A and Vijay Kumar, M and Joe, B},
title = {Attenuation of Microbiotal Dysbiosis and Hypertension in a CRISPR/Cas9 Gene Ablation Rat Model of GPER1.},
journal = {Hypertension (Dallas, Tex. : 1979)},
volume = {72},
number = {5},
pages = {1125-1132},
pmid = {30354811},
issn = {1524-4563},
support = {R01 HL020176/HL/NHLBI NIH HHS/United States ; R01 HL112641/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dysbiosis/*physiopathology ; Gastrointestinal Microbiome/*physiology ; Hypertension/*physiopathology ; RNA, Guide ; Rats ; Rats, Inbred Dahl ; Receptors, G-Protein-Coupled/*genetics ; },
abstract = {G-protein-coupled estrogen receptor, Gper1, has been implicated in cardiovascular disease, but its mechanistic role in blood pressure control is poorly understood. Here, we demonstrate that genetically salt-sensitive hypertensive rats with complete genomic excision of Gper1 by a multiplexed guide RNA CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated proteins) approach present with lower blood pressure, which was accompanied by altered microbiota, different levels of circulating short chain fatty acids, and improved vascular relaxation. Microbiotal transplantation from hypertensive Gper1[+/+] rats reversed the cardiovascular protective effect exerted by the genomic deletion of Gper1. Thus, this study reveals a role for Gper1 in promoting microbiotal alterations that contribute to cardiovascular pathology. However, the exact mechanism by which Gper1 regulates blood pressure is still unknown. Our results indicate that the function of Gper1 is contextually dependent on the microbiome, whereby, contemplation of using Gper1 as a target for therapy of cardiovascular disease requires caution.},
}
@article {pmid30354074,
year = {2018},
author = {Löbs, AK and Schwartz, C and Thorwall, S and Wheeldon, I},
title = {Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {7},
number = {11},
pages = {2647-2655},
doi = {10.1021/acssynbio.8b00331},
pmid = {30354074},
issn = {2161-5063},
mesh = {Acetates/*metabolism ; CRISPR-Cas Systems/*genetics ; Citric Acid Cycle/genetics ; Electron Transport Chain Complex Proteins/genetics ; Gene Editing ; Kluyveromyces/genetics/*metabolism ; Metabolic Engineering/methods ; Mitochondria/*metabolism ; },
abstract = {The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.},
}
@article {pmid30353530,
year = {2019},
author = {Pei, W and Burgess, SM},
title = {Microinjection in Zebrafish for Genome Editing and Functional Studies.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {459-474},
doi = {10.1007/978-1-4939-8831-0_26},
pmid = {30353530},
issn = {1940-6029},
support = {ZIA HG200386/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/growth &amp; development ; CRISPR-Cas Systems ; Chaperonin 60/genetics ; Gene Editing/*methods ; Microinjections/*methods ; Zebrafish/*embryology/genetics ; },
abstract = {A major strength of zebrafish as a model organism is their rapid, in vitro development. The easy access to embryos compared to mammals, allows larval molecular and cellular composition to be manipulated by microinjection, providing a powerful avenue for biological and translational studies. Here, we describe the essential steps and different applications of microinjection in zebrafish for genome editing and functional studies, along with some experimental tips that are critical for microinjection success.},
}
@article {pmid30353529,
year = {2019},
author = {Iyer, J and DeVaul, N and Hansen, T and Nebenfuehr, B},
title = {Using Microinjection to Generate Genetically Modified Caenorhabditis elegans by CRISPR/Cas9 Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {431-457},
doi = {10.1007/978-1-4939-8831-0_25},
pmid = {30353529},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Gene Editing/methods ; Genetic Engineering/methods ; Microinjections/*methods ; },
abstract = {In this chapter, we describe the procedure for generating genetically modified Caenorhabditis elegans using microinjection via the Cas9-mediated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing technique. Specifically, we describe the detailed method of performing CRISPR editing by microinjection using the Cloning-free Co-CRISPR method described by the Seydoux lab. This microinjection protocol can also be used for CRISPR editing with protocols from other labs as well as for a variety of other editing techniques including Mos1-mediated single-copy transgene insertions (MosSCI), transcriptional activator-like nucleases (TALENs), and zinc-finger nucleases (ZFNs). Further, this microinjection protocol can also be used for injecting plasmid DNA to generate heritable extrachromosomal arrays for gene expression and mosaic analysis, performing RNAi by injection and delivering RNA, dyes or other molecules into the C. elegans germline.},
}
@article {pmid30353526,
year = {2019},
author = {Fan, Z and Yang, M and Regouski, M and Polejaeva, IA},
title = {Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {373-390},
doi = {10.1007/978-1-4939-8831-0_22},
pmid = {30353526},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cells, Cultured ; Cloning, Organism/*methods ; Embryo Culture Techniques ; Female ; Fibroblasts/*cytology/metabolism ; Gene Knockout Techniques/*methods ; Goats ; Nuclear Transfer Techniques ; RNA, Guide/genetics ; },
abstract = {The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of pooled CRISPR/Cas9 targeting vectors.},
}
@article {pmid30353524,
year = {2019},
author = {Li, R and Miao, J and Wang, Z},
title = {Production of Genetically Engineered Porcine Embryos by Handmade Cloning.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {347-360},
doi = {10.1007/978-1-4939-8831-0_20},
pmid = {30353524},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cells, Cultured ; Cloning, Organism/*methods ; Embryo Culture Techniques/*methods ; Endonucleases/metabolism ; Genetic Engineering/*methods ; RNA, Guide/genetics ; Single-Cell Analysis ; Swine ; },
abstract = {Genetic engineering is essential to realize the full potentials of pigs both as livestock and as animal models of human disease. With the development of new genetic engineering technologies, such as the clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system, the porcine genome can be engineered with high efficiency. In this chapter, we describe a protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells, the procedures to establish single-cell-derived porcine fibroblast cell colonies carrying the desired genetic modifications, and the handmade cloning (HMC) technique to generate cloned embryos ready for embryo transfer.},
}
@article {pmid30353523,
year = {2019},
author = {Yang, D and Xu, J and Chen, YE},
title = {Generation of Rabbit Models by Gene Editing Nucleases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {327-345},
doi = {10.1007/978-1-4939-8831-0_19},
pmid = {30353523},
issn = {1940-6029},
support = {R01 HL117491/HL/NHLBI NIH HHS/United States ; R01 HL129778/HL/NHLBI NIH HHS/United States ; R01 HL147527/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/*growth &amp; development ; CRISPR-Cas Systems ; Endonucleases/*metabolism ; Gene Editing/*methods ; Microinjections ; Rabbits ; },
abstract = {Due to the lack of germline transmitting pluripotent stem cells (PSCs) cell lines and the extreme difficulty of somatic cell nuclear transfer (SCNT) in rabbit, the gene targeting technology in rabbit was lagging far behind those in rodents and in farm animals. As a result, the development and application of genetically engineered rabbit model are much limited. With the advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas9, it is now possible to produce gene targeting (i.e., knockout, knockin) rabbits with high success rates. In this chapter, we describe a comprehensive, step-by-step protocol for rabbit genome editing based on gene editing nucleases with specific emphasis of CRISPR/Cas9.},
}
@article {pmid30353520,
year = {2019},
author = {Alcantar, TM and Rairdan, XY},
title = {Microinjection and Oviduct Transfer Procedures for Rat Model Generation with CRISPR-Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {273-294},
doi = {10.1007/978-1-4939-8831-0_16},
pmid = {30353520},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Nucleus/genetics ; Cytoplasm/genetics ; Embryo Transfer ; Female ; Gene Editing ; Gene Knockout Techniques ; Microinjections/*methods ; *Models, Animal ; Oviducts/*metabolism ; Rats ; Rats, Sprague-Dawley ; Species Specificity ; Superovulation ; },
abstract = {Since the first knockout rat model was generated with zinc-finger nucleases (ZFNs) by Geurt's group in 2009, the demand for making targeted rat models has increased tremendously. The advent of the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system provides researchers with a more efficient method for producing modified animals, which has since then been developed and applied in rat. Since we established a rat model production system at our facility in 2014, we have consistently generated rat models. Due to differences in physiology and embryology between mouse and rat, species-specific protocols for superovulation conditions, microinjection, and embryo transfer (among others) are required. There are over 100 rat strains, and Sprague Dawley is one of the commonly used outbred strains in biomedical research. In this chapter, we describe in detail a range of topics including donor and recipient preparation, microinjection setup, CRISPR reagent preparation, and oviduct transfer procedures for making rat models in the Sprague Dawley background.},
}
@article {pmid30353519,
year = {2019},
author = {Sakurai, T and Kamiyoshi, A and Ohtsuka, M and Gurumurthy, CB and Sato, M and Shindo, T},
title = {Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {257-271},
doi = {10.1007/978-1-4939-8831-0_15},
pmid = {30353519},
issn = {1940-6029},
mesh = {Albumins/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Gene Editing/*methods ; Hepatocytes/*metabolism ; Hydrodynamics ; Mice ; Mice, Transgenic ; RNA, Guide/genetics ; Single-Cell Analysis/*methods ; },
abstract = {The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called "sCAT" mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.},
}
@article {pmid30353515,
year = {2019},
author = {Liu, Y and Du, Y and Xie, W and Zhang, F and Forrest, D and Liu, C},
title = {Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {191-210},
pmid = {30353515},
issn = {1940-6029},
support = {Z99 HL999999/ImNIH/Intramural NIH HHS/United States ; ZIC HL005907/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; Gene Knockout Techniques/*methods ; Gene Targeting ; Homeodomain Proteins/*genetics ; Mice ; Mice, Knockout ; Microinjections ; Oligonucleotides/*genetics ; RNA, Guide/genetics ; Trans-Activators/*genetics ; Zygote/metabolism ; },
abstract = {Conditional knockout (cKO) mice are extremely valuable for biomedical research because they enable detailed analyses of gene functions in a tissue- or temporally-specific fashion. The conventional method for generating cKO mice is time consuming and labor intensive, which involves making a large gene-targeting construct, transfecting and screening many embryonic stem (ES) cell clones, injecting positive ES clones into blastocysts to produce chimeric mice, and breeding the chimeras to transmit the targeted gene through the germline. Recently developed CRISPR technology has revolutionized the way genetically engineered organisms are created. Knockout and knockin mice can now be made by directly injecting zygotes with Cas9, sgRNA, and donor DNA. In theory, cKO mice can be generated by simultaneously inserting two loxP sites using two sgRNAs and two oligonucleotides as donors, but in practice the probability of obtaining cKO mice in one step is still very low, partly because the efficiency of oligo-mediated knockin is much lower than non-homologous end joining (NHEJ) and partly because co-cutting juxtaposed sites in one allele at the same time often leads to the deletion of the entire fragment between the two cutting sites. Therefore, many laboratories prefer to insert the two loxP sites sequentially, i.e., generating mice with one loxP first and then use embryos collected from these mice to insert the second loxP site. In this chapter, we describe our procedures and timeline using this sequential method to make a Six6 cKO mouse line as a demonstration of its feasibility.},
}
@article {pmid30353514,
year = {2019},
author = {Qin, W and Wang, H},
title = {Delivery of CRISPR-Cas9 into Mouse Zygotes by Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {179-190},
doi = {10.1007/978-1-4939-8831-0_10},
pmid = {30353514},
issn = {1940-6029},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Electroporation/*methods ; Female ; Gene Editing/methods ; Mice ; Mutation ; Zygote/growth &amp; development/*metabolism ; },
abstract = {The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including the mice. In this scheme, components of the CRISPR-Cas9 system are delivered into the mouse zygote and mutant mice carrying genetic modifications derived. Although microinjection has been the technology of choice, electroporation has also emerged and been proven to be effective delivering CRISPR-Cas9 reagents into the mouse zygote. Here, we describe the experimental protocol employing electroporation to deliver CRISPR-Cas9 reagents into mouse embryos and derive gene-edited mutant mice.},
}
@article {pmid30353513,
year = {2019},
author = {Scott, MA and Hu, YC},
title = {Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {169-178},
doi = {10.1007/978-1-4939-8831-0_9},
pmid = {30353513},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Cytochalasin B/pharmacology ; Female ; Gene Editing/*methods ; Male ; Mice ; Microinjections/*methods ; Nuclear Transfer Techniques ; Rats ; Zygote/*growth &amp; development/metabolism ; },
abstract = {Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rate. Because this method uses a much wider injection needle than that in pronuclear injection, it allows a larger volume of the editing materials to be transported into the zygotes, leading to an increase in the targeting efficiency. This also eliminates the clogging issues seen regularly in pronuclear injection. Moreover, Cytochalasin B that is used to soften zygotes during piezo-driven microinjection has been suggested a role in improving the knockin efficiency, which provides an additional benefit to use this injection method.},
}
@article {pmid30353512,
year = {2019},
author = {Du, Y and Xie, W and Zhang, F and Choi, U and Liu, C and Sweeney, CL},
title = {Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {139-168},
pmid = {30353512},
issn = {1940-6029},
support = {Z99 HL999999/ImNIH/Intramural NIH HHS/United States ; ZIC HL005907/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo Transfer/*methods ; Exons ; Immunocompromised Host ; Mice ; Mice, Knockout ; Microinjections ; NADPH Oxidase 2/*genetics ; RNA, Guide/genetics ; },
abstract = {NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl/]SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use the knockout of the X-linked Cybb gene as an example to describe our procedures for genetically modifying NSG mice using the CRISPR/Cas9 method. Briefly, two sgRNAs were designed and made to target exon 1 and exon 3 of the Cybb gene, and either sgRNA was then microinjected together with Cas9 mRNA into fertilized eggs collected from NSG mice. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. Offspring born to the foster mothers were genotyped by PCR and DNA sequencing. In this chapter, we describe our experiment procedures in detail and report our genotyping results for demonstrating that NSG mice can be genetically modified using the CRISPR/Cas9 technology in a highly efficient manner.},
}
@article {pmid30353511,
year = {2019},
author = {Liu, P and Li, Y and Lei, J and Dong, L},
title = {Creating Knockin Alleles in Mouse Embryonic Stem Cells by CRISPR/Cas9-Mediated Homologous Recombination Without Drug Selection.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {115-137},
doi = {10.1007/978-1-4939-8831-0_7},
pmid = {30353511},
issn = {1940-6029},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cells, Cultured ; Gene Knock-In Techniques/*methods ; Germ Cells/*cytology ; Homologous Recombination ; Mice ; Mouse Embryonic Stem Cells/*cytology ; },
abstract = {The rapidly evolving CRISPR/Cas9-mediated genome editing provides the convenience of genome manipulation directly in mouse zygotes for a number of genomic manipulations; but knockins of large insertions prove to be relatively inefficient at least with double-strand DNA as targeting constructs. Here, we describe an alternative approach to the direct genome editing in mouse zygotes by generating knockin alleles in mouse embryonic stem cells first with CRIPSR-mediated homologous recombination. Our results show this approach is efficient and requires no drug selection in mouse embryonic stem cells as in classic gene targeting. As the result, knockin alleles across many target loci are created in mouse embryonic stem cells and readily transmitted through germline. The knockin alleles created in ES cells can also serve as valuable tools for in vitro stem cell differentiation.},
}
@article {pmid30353505,
year = {2019},
author = {Xu, W},
title = {Microinjection and Micromanipulation: A Historical Perspective.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1874},
number = {},
pages = {1-16},
doi = {10.1007/978-1-4939-8831-0_1},
pmid = {30353505},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Gene Targeting ; Genetic Engineering ; History, 20th Century ; History, 21st Century ; Humans ; Microinjections/*history/trends ; Micromanipulation/*history/trends ; Nanotechnology ; },
abstract = {Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its preciseness in mechanical retrieval, or delivery of biological materials, which in some cases is simply necessary or more effective than other retrieval/delivery means. Microinjection is favored for its straightforwardness in transferring contents from micromolecules to macromolecules and from organelles to cells. Microinjection/micromanipulation has been practiced over the century like an art form. Variations in handlings and instruments can be tolerated to a surprising degree with satisfactory outcomes. Throughout the century, microinjection developed as an indispensable tool along with the evolution of biomedical fields: from transgenics to gene targeting, from animal cloning to human infertility treatment, from nuclease-guided genetic engineering to RNA-guided genome editing (Fig. 1). The birth of the CRISPRology rejuvenated microinjection. For microinjection/micromanipulation, the second century has already begun with the early arrival of computerized instrumentation and lately of the high-throughput nanomanipulators potentially operable by artificial intelligence. As we yin-yang both systemic and precision approaches in research and medicine, microinjection will no doubt continue to find its unique place in the future.},
}
@article {pmid30353175,
year = {2018},
author = {Raj, B and Gagnon, JA and Schier, AF},
title = {Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR-Cas9 barcodes by scGESTALT.},
journal = {Nature protocols},
volume = {13},
number = {11},
pages = {2685-2713},
pmid = {30353175},
issn = {1750-2799},
support = {DP1 HD094764/HD/NICHD NIH HHS/United States ; R01 HD085905/HD/NICHD NIH HHS/United States ; U01 MH105960/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Brain/growth &amp; development/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Lineage/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Nonmammalian ; Gene Editing/*methods ; Gene Library ; Organ Specificity ; RNA, Guide/genetics/metabolism ; Single-Cell Analysis/methods ; *Transcriptome ; Zebrafish/*genetics/growth &amp; development/metabolism ; },
abstract = {Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR-Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods. The recorded lineages are captured, along with thousands of cellular transcriptomes, to build lineage trees with hundreds of branches representing relationships among profiled cell types. Here, we provide details for (i) generating transgenic zebrafish; (ii) performing multi-timepoint barcode editing; (iii) building scRNA-seq libraries from brain tissue; and (iv) concurrently amplifying lineage barcodes from captured single cells. Generating transgenic lines takes 6 months, and performing barcode editing and generating single-cell libraries involve 7 d of hands-on time. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration, or disease.},
}
@article {pmid30353159,
year = {2018},
author = {Parthasarathy, S},
title = {Use the patent system to regulate gene editing.},
journal = {Nature},
volume = {562},
number = {7728},
pages = {486-488},
doi = {10.1038/d41586-018-07108-3},
pmid = {30353159},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clinical Trials as Topic/ethics/legislation &amp; jurisprudence ; Congresses as Topic ; European Union ; Gene Editing/*ethics/*legislation &amp; jurisprudence/statistics &amp; numerical data ; *Government Regulation ; History, 19th Century ; History, 20th Century ; History, 21st Century ; Human Embryonic Stem Cells ; Humans ; Licensure ; Patents as Topic/history/*legislation &amp; jurisprudence/statistics &amp; numerical data ; Swine/genetics ; United States ; },
}
@article {pmid30352733,
year = {2018},
author = {Huo, X and Zhang, X and Liu, Y and Sun, Y and Ren, Y and Li, C and Du, X and Chen, Z},
title = {Instability of microsatellites linked to targeted genes in CRISPR/Cas9-edited and traditional gene knockout mouse strains.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {45},
number = {10},
pages = {553-556},
doi = {10.1016/j.jgg.2018.07.010},
pmid = {30352733},
issn = {1673-8527},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques/*adverse effects/methods ; Genomic Instability ; Mice/*genetics ; Mice, Knockout ; *Microsatellite Repeats ; },
}
@article {pmid30352624,
year = {2018},
author = {Majeed, M and Soliman, H and Kumar, G and El-Matbouli, M and Saleh, M},
title = {Editing the genome of Aphanomyces invadans using CRISPR/Cas9.},
journal = {Parasites &amp; vectors},
volume = {11},
number = {1},
pages = {554},
pmid = {30352624},
issn = {1756-3305},
mesh = {Animals ; Aphanomyces/enzymology/*genetics/pathogenicity ; CRISPR-Cas Systems ; Fish Diseases/parasitology ; Gene Targeting ; Genome ; Perciformes/parasitology ; Ribonucleoproteins/genetics ; Serine Proteases/genetics ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is increasingly being used for genome editing experiments. It is a system to add, delete and/or replace parts of a gene in situ in a time- and cost-efficient manner. The genome of many organisms has been edited using this system. We tested the CRISPR/Cas9 system in Aphanomyces invadans, an oomycete, which is the causative agent of epizootic ulcerative syndrome (EUS) in many fish species. Extracellular proteases produced by this oomycete are believed to play a role in EUS virulence.<br><br>METHODS: We designed three single guide-RNAs (gRNA) to target A. invadans serine protease gene. These gRNAs were individually combined with the Cas9 to form ribo-nucleo-protein (RNP) complex. A. invadans protoplasts were then transfected with RNP complexes. After the transfection, the target gene was amplified and subjected to sequencing. Zoospores of A. invadans were also transfected with the RNP complex. Three groups of dwarf gourami (Trichogaster lalius) were then experimentally inoculated with (i) non-treated A. invadans zoospores; (ii) RNP-treated A. invadans zoospores; and (iii) autoclaved pond water as negative control, to investigate the effect of edited serine protease gene on the virulence of A. invadans in vivo.<br><br>RESULTS: Fluorescence microscopy showed sub-cellular localization of RNP complex in A. invadans protoplasts and zoospores. Sequencing results from the protoplast DNA revealed a point mutation in the target gene. A matching mutation was also detected in zoospores after similar treatment with the same RNP complex. In vivo results showed that the CRISPR/Cas9-treated A. invadans zoospores did not produce EUS clinical signs in the fish. These results were then confirmed by histopathological staining of the muscle sections using Gomori's methenamine silver nitrate and hematoxylin and eosin stains.<br><br>CONCLUSIONS: Results obtained in this study indicate that the RNP complex caused effective mutation in the target gene. This hindered the production of serine protease, which ultimately impeded the manifestation of EUS in the fish. Our methods thus establish a promising approach for functional genomics studies in A. invadans and provide novel avenues to develop effective strategies to control this pathogen.},
}
@article {pmid30352492,
year = {2018},
author = {Lee, HW},
title = {Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript.},
journal = {Molecules and cells},
volume = {41},
number = {10},
pages = {917-922},
pmid = {30352492},
issn = {0219-1032},
mesh = {4-1BB Ligand/*genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genomics/*methods ; Humans ; },
abstract = {The CRISPR-Cas system is a well-established RNA-guided DNA editing technique widely used to modify genomic DNA sequences. I used the CRISPR-Cas9 system to change the second and third nucleotides of the triplet TCT of human TNSFSF9 in HepG2 cells to TAG to create an amber stop codon. The TCT triplet is the codon for Ser at the 172[nd] position of TNSFSF9. The two substituted nucleotides, AG, were confirmed by DNA sequencing of the PCR product followed by PCR amplification of the genomic TNFSF9 gene. Interestingly, sequencing of the cDNA of transcripts of the edited TNFSF9 gene revealed that the TAG had been re-edited to the wild type triplet TCT, and 1 or 2 bases just before the triplet had been deleted. These observations indicate that CRISPR-Cas9-mediated editing of bases in target genomic DNA can be followed by spontaneous re-editing (correcting) of the bases during transcription.},
}
@article {pmid30352154,
year = {2018},
author = {Shao, Y and Lu, N and Qin, Z and Xue, X},
title = {CRISPR-Cas9 Facilitated Multiple-Chromosome Fusion in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {7},
number = {11},
pages = {2706-2708},
doi = {10.1021/acssynbio.8b00397},
pmid = {30352154},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Chromosomes, Fungal/genetics/*metabolism ; Gene Editing/*methods ; Gene Fusion/*genetics ; Homologous Recombination ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Eukaryotic cells usually contain multiple linear chromosomes. Recently, we artificially created a functional single-chromosome yeast via sequential two-chromosome fusion utilizing the high performance of the CRISPR-Cas9 system and homologous recombination in Saccharomyces cerevisiae. In this paper, we adapted this method for the simultaneous fusion of multiple chromosomes. We demonstrated the fusion of two, two-chromosome sets with a 75% positive rate and three-chromosome fusions with a 50% positive rate. We also found that by using an additional selection marker, the positive rate of two-chromosome fusions reached 100%. Due to the simplicity, efficiency, and portability of this method, we expect that it can be easily adapted for multiple-chromosome fusions in other organisms.},
}
@article {pmid30352075,
year = {2018},
author = {Tanihara, F and Hirata, M and Nguyen, NT and Le, QA and Hirano, T and Takemoto, T and Nakai, M and Fuchimoto, DI and Otoi, T},
title = {Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0206360},
pmid = {30352075},
issn = {1932-6203},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Electroporation ; Gene Deletion ; Gene Editing/*methods ; Genotype ; Histiocytoma, Benign Fibrous/etiology/genetics ; Neoplasms/*etiology/genetics ; Phenotype ; Swine ; Tumor Suppressor Protein p53/*genetics ; Zygote/cytology/metabolism ; },
abstract = {TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs.},
}
@article {pmid30350208,
year = {2019},
author = {Hu, N and Malek, SN},
title = {Gene Disruption Using CRISPR-Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1881},
number = {},
pages = {201-209},
doi = {10.1007/978-1-4939-8876-1_16},
pmid = {30350208},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair/genetics ; Gene Editing/instrumentation/*methods ; Gene Targeting/instrumentation/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics ; Transduction, Genetic/instrumentation/methods ; },
abstract = {The emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology provides tools for researchers to modify genomes in a specific and efficient manner. The Type II CRISPR-Cas9 system enables gene editing by directed DNA cleavage followed by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Here, we described the use of the Type II CRISPR-Cas9 system in detail from designing the guides to analyzing the desired gene disruption events.},
}
@article {pmid30350207,
year = {2019},
author = {Wu, K and Malek, SN},
title = {CRISPR/Cas9-Based Gene Dropout Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1881},
number = {},
pages = {185-200},
doi = {10.1007/978-1-4939-8876-1_15},
pmid = {30350207},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/instrumentation/*methods ; Gene Library ; Gene Targeting/instrumentation/*methods ; Genomics/instrumentation/*methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/instrumentation/methods ; Humans ; RNA, Guide/genetics ; Real-Time Polymerase Chain Reaction/instrumentation/methods ; Sequence Analysis, DNA/instrumentation/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based technology enables efficient and precise perturbations of target genomic sites. Combining the endonuclease Cas9 and a pooled guide RNA library allows for systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Here, we describe a complete protocol for scalable CRISPR/Cas9-based dropout screening for essential genes from focused genomic regions to whole genomes.},
}
@article {pmid30350199,
year = {2019},
author = {Murthy, T and Paul, KV and Minella, AC and Pillai, MM},
title = {The Development and Use of Scalable Systems for Studying Aberrant Splicing in SF3B1-Mutant CLL.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1881},
number = {},
pages = {83-99},
doi = {10.1007/978-1-4939-8876-1_7},
pmid = {30350199},
issn = {1940-6029},
support = {R01 HL098608/HL/NHLBI NIH HHS/United States ; R01 HL104070/HL/NHLBI NIH HHS/United States ; R01 HL133406/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Culture Techniques/instrumentation/methods ; Cell Line ; Computational Biology/instrumentation/*methods ; Datasets as Topic ; Gene Editing/instrumentation/methods ; High-Throughput Nucleotide Sequencing/instrumentation/methods ; Humans ; Induced Pluripotent Stem Cells ; Leukemia, Lymphocytic, Chronic, B-Cell/blood/*genetics/pathology ; Mice ; Mouse Embryonic Stem Cells ; Mutation ; Phosphoproteins/*genetics ; Protein Domains/genetics ; RNA Splice Sites/genetics ; RNA Splicing/genetics ; RNA Splicing Factors/*genetics ; Sequence Analysis, RNA/instrumentation/*methods ; Software ; },
abstract = {Mutational landscape of CLL is now known to include recurrent non-synonymous mutations in SF3B1, a core splicing factor. About 5-10% of newly diagnosed CLL harbor these mutations which are typically limited to HEAT domains in the carboxyl-terminus of the protein. Importantly, the mutations are not specific to CLL but also present in several unrelated clonal disorders. Analysis of patient samples and cell lines has shown the primary splicing aberration in SF3B1-mutant cells to the use of novel or "cryptic" 3' splice sites (3SS). Advances in genome-editing and next-generation sequencing (NGS) have allowed development of isogenic models and detailed analysis of changes to the transcriptome with relative ease. In this manuscript, we focus on two relevant methods to study splicing factor mutations in CLL: development of isogenic scalable cell lines and informatics analysis of RNA-Seq datasets.},
}
@article {pmid30349006,
year = {2018},
author = {Daou, S and Barbour, H and Ahmed, O and Masclef, L and Baril, C and Sen Nkwe, N and Tchelougou, D and Uriarte, M and Bonneil, E and Ceccarelli, D and Mashtalir, N and Tanji, M and Masson, JY and Thibault, P and Sicheri, F and Yang, H and Carbone, M and Therrien, M and Affar, EB},
title = {Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4385},
pmid = {30349006},
issn = {2041-1723},
support = {R01 CA198138/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics/physiology ; Cell Line ; Cell Line, Tumor ; Cells, Cultured ; Drosophila ; Drosophila Proteins/genetics/*metabolism ; Fluorescent Antibody Technique ; Humans ; Immunoblotting ; Immunoprecipitation ; RNA, Small Interfering/genetics ; Repressor Proteins/genetics/metabolism ; Tumor Suppressor Proteins/genetics/*metabolism ; Ubiquitin Thiolesterase/genetics/*metabolism ; Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Ubiquitination/genetics/physiology ; },
abstract = {The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1.},
}
@article {pmid30348755,
year = {2019},
author = {Yourik, P and Fuchs, RT and Mabuchi, M and Curcuru, JL and Robb, GB},
title = {Staphylococcus aureus Cas9 is a multiple-turnover enzyme.},
journal = {RNA (New York, N.Y.)},
volume = {25},
number = {1},
pages = {35-44},
pmid = {30348755},
issn = {1469-9001},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; DNA/genetics/metabolism ; DNA Breaks, Double-Stranded ; Gene Editing ; Kinetics ; RNA, Guide/genetics/metabolism ; Species Specificity ; Staphylococcus aureus/*enzymology/genetics ; Streptococcus pyogenes/enzymology/genetics ; Substrate Specificity ; },
abstract = {Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics, we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.},
}
@article {pmid30348199,
year = {2018},
author = {Ravindranathan, S and Nguyen, KG and Kurtz, SL and Frazier, HN and Smith, SG and Koppolu, BP and Rajaram, N and Zaharoff, DA},
title = {Tumor-derived granulocyte colony-stimulating factor diminishes efficacy of breast tumor cell vaccines.},
journal = {Breast cancer research : BCR},
volume = {20},
number = {1},
pages = {126},
pmid = {30348199},
issn = {1465-542X},
support = {R01 CA172631/CA/NCI NIH HHS/United States ; R15 CA176648/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast/pathology ; Breast Neoplasms/immunology/pathology/*therapy ; CRISPR-Cas Systems ; Cancer Vaccines/administration &amp; dosage/*immunology ; Cell Line, Tumor/immunology/radiation effects/transplantation ; Disease Models, Animal ; Female ; Gene Deletion ; Granulocyte Colony-Stimulating Factor/genetics/*immunology ; Humans ; *Immunogenicity, Vaccine ; Mice ; Mice, Inbred BALB C ; Neoplasm Recurrence, Local/immunology/*prevention &amp; control ; Treatment Outcome ; },
abstract = {BACKGROUND: Although metastasis is ultimately responsible for about 90% of breast cancer mortality, the vast majority of breast-cancer-related deaths are due to progressive recurrences from non-metastatic disease. Current adjuvant therapies are unable to prevent progressive recurrences for a significant fraction of patients with breast cancer. Autologous tumor cell vaccines (ATCVs) are a safe and potentially useful strategy to prevent breast cancer recurrence, in a personalized and patient-specific manner, following standard-of-care tumor resection. Given the high intra-patient and inter-patient heterogeneity in breast cancer, it is important to understand which factors influence the immunogenicity of breast tumor cells in order to maximize ATCV effectiveness.<br><br>METHODS: The relative immunogenicity of two murine breast carcinomas, 4T1 and EMT6, were compared in a prophylactic vaccination-tumor challenge model. Differences in cell surface expression of antigen-presentation-related and costimulatory molecules were compared along with immunosuppressive cytokine production. CRISPR/Cas9 technology was used to modulate tumor-derived cytokine secretion. The impacts of cytokine deletion on splenomegaly, myeloid-derived suppressor cell (MDSC) accumulation and ATCV immunogenicity were assessed.<br><br>RESULTS: Mice vaccinated with an EMT6 vaccine exhibited significantly greater protective immunity than mice vaccinated with a 4T1 vaccine. Hybrid vaccination studies revealed that the 4T1 vaccination induced both local and systemic immune impairments. Although there were significant differences between EMT6 and 4T1 in the expression of costimulatory molecules, major disparities in the secretion of immunosuppressive cytokines likely accounts for differences in immunogenicity between the cell lines. Ablation of one cytokine in particular, granulocyte-colony stimulating factor (G-CSF), reversed MDSC accumulation and splenomegaly in the 4T1 model. Furthermore, G-CSF inhibition enhanced the immunogenicity of a 4T1-based vaccine to the extent that all vaccinated mice developed complete protective immunity.<br><br>CONCLUSIONS: Breast cancer cells that express high levels of G-CSF have the potential to diminish or abrogate the efficacy of breast cancer ATCVs. Fortunately, this study demonstrates that genetic ablation of immunosuppressive cytokines, such as G-CSF, can enhance the immunogenicity of breast cancer cell-based vaccines. Strategies that combine inhibition of immunosuppressive factors with immune stimulatory co-formulations already under development may help ATCVs reach their full potential.},
}
@article {pmid30346516,
year = {2018},
author = {Markusková, B and Lichvariková, A and Szemes, T and Korenová, J and Kuchta, T and Drahovská, H},
title = {Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese.},
journal = {FEMS microbiology letters},
volume = {365},
number = {23},
pages = {},
doi = {10.1093/femsle/fny257},
pmid = {30346516},
issn = {1574-6968},
mesh = {Bacteriocins/genetics ; CRISPR-Cas Systems/genetics ; Cheese/*microbiology ; Computational Biology ; DNA Restriction-Modification Enzymes/genetics ; Drug Resistance, Bacterial ; Genes, Bacterial ; *Genome, Bacterial ; High-Throughput Nucleotide Sequencing ; Hydrolases/genetics ; Lactobacillales/classification/*genetics/isolation &amp; purification ; Metabolic Networks and Pathways/genetics ; Molecular Sequence Annotation ; Ornithine Decarboxylase/genetics ; Prophages/genetics ; Slovakia ; },
abstract = {Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction-modification system, all strains carried 1-6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0-24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction-modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.},
}
@article {pmid30345399,
year = {2018},
author = {Yao, R and Liu, D and Jia, X and Zheng, Y and Liu, W and Xiao, Y},
title = {CRISPR-Cas9/Cas12a biotechnology and application in bacteria.},
journal = {Synthetic and systems biotechnology},
volume = {3},
number = {3},
pages = {135-149},
pmid = {30345399},
issn = {2405-805X},
abstract = {CRISPR-Cas technologies have greatly reshaped the biology field. In this review, we discuss the CRISPR-Cas with a particular focus on the associated technologies and applications of CRISPR-Cas9 and CRISPR-Cas12a, which have been most widely studied and used. We discuss the biological mechanisms of CRISPR-Cas as immune defense systems, recently-discovered anti-CRISPR-Cas systems, and the emerging Cas variants (such as xCas9 and Cas13) with unique characteristics. Then, we highlight various CRISPR-Cas biotechnologies, including nuclease-dependent genome editing, CRISPR gene regulation (including CRISPR interference/activation), DNA/RNA base editing, and nucleic acid detection. Last, we summarize up-to-date applications of the biotechnologies for synthetic biology and metabolic engineering in various bacterial species.},
}
@article {pmid30344094,
year = {2018},
author = {Mohr, G and Silas, S and Stamos, JL and Makarova, KS and Markham, LM and Yao, J and Lucas-Elío, P and Sanchez-Amat, A and Fire, AZ and Koonin, EV and Lambowitz, AM},
title = {A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.},
journal = {Molecular cell},
volume = {72},
number = {4},
pages = {700-714.e8},
pmid = {30344094},
issn = {1097-4164},
support = {R01 GM037706/GM/NIGMS NIH HHS/United States ; R01 GM037949/GM/NIGMS NIH HHS/United States ; ZIA LM000073-22//Intramural NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Base Sequence/genetics ; CRISPR-Associated Proteins/*physiology ; CRISPR-Cas Systems/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/*physiology ; DNA ; Endonucleases ; Marinomonas/genetics/metabolism ; Phylogeny ; RNA/biosynthesis ; RNA-Directed DNA Polymerase/*physiology ; Substrate Specificity ; },
abstract = {Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.},
}
@article {pmid30343903,
year = {2018},
author = {Dillard, KE and Brown, MW and Johnson, NV and Xiao, Y and Dolan, A and Hernandez, E and Dahlhauser, SD and Kim, Y and Myler, LR and Anslyn, EV and Ke, A and Finkelstein, IJ},
title = {Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex.},
journal = {Cell},
volume = {175},
number = {4},
pages = {934-946.e15},
pmid = {30343903},
issn = {1097-4172},
support = {F31 GM125201/GM/NIGMS NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Actinomycetales/*enzymology ; Bacterial Proteins/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/*metabolism ; *CRISPR-Cas Systems ; DNA, Viral/metabolism ; Protein Multimerization ; Single Molecule Imaging ; },
abstract = {CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.},
}
@article {pmid30343902,
year = {2018},
author = {Wroblewska, A and Dhainaut, M and Ben-Zvi, B and Rose, SA and Park, ES and Amir, ED and Bektesevic, A and Baccarini, A and Merad, M and Rahman, AH and Brown, BD},
title = {Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.},
journal = {Cell},
volume = {175},
number = {4},
pages = {1141-1155.e16},
pmid = {30343902},
issn = {1097-4172},
support = {U19 AI128949/AI/NIAID NIH HHS/United States ; R01 AI113221/AI/NIAID NIH HHS/United States ; R01 CA190400/CA/NCI NIH HHS/United States ; R33 CA182377/CA/NCI NIH HHS/United States ; U24 AI118644/AI/NIAID NIH HHS/United States ; S10 OD023547/OD/NIH HHS/United States ; R01 CA154947/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Epitopes/chemistry/classification/genetics ; Flow Cytometry/*methods ; Genomics/*methods ; HEK293 Cells ; Humans ; Immunophenotyping/methods ; Jurkat Cells ; Mass Spectrometry/*methods ; Mice, Inbred BALB C ; Proteome/chemistry/classification/genetics ; Single-Cell Analysis/*methods ; THP-1 Cells ; },
abstract = {CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.},
}
@article {pmid30342154,
year = {2019},
author = {Batool, S and Argyropoulos, KV and Azad, R and Okeoma, P and Zumrut, H and Bhandari, S and Dekhang, R and Mallikaratchy, PR},
title = {Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1863},
number = {1},
pages = {232-240},
pmid = {30342154},
issn = {1872-8006},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; SC1 GM122648/GM/NIGMS NIH HHS/United States ; },
mesh = {Aptamers, Nucleotide/*chemistry ; B-Lymphocytes/*metabolism ; Burkitt Lymphoma/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Dimerization ; Epitopes/*chemistry ; HEK293 Cells ; Humans ; Immunoglobulin M/chemistry ; Lentivirus/genetics ; Leukocytes, Mononuclear/cytology ; Ligands ; Lymphoma, B-Cell/metabolism ; Plasmids/metabolism ; Protein Binding ; Protein Engineering ; Puromycin/chemistry ; SELEX Aptamer Technique ; Temperature ; Waldenstrom Macroglobulinemia/metabolism ; },
abstract = {Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.},
}
@article {pmid30341435,
year = {2018},
author = {Lazzarotto, CR and Nguyen, NT and Tang, X and Malagon-Lopez, J and Guo, JA and Aryee, MJ and Joung, JK and Tsai, SQ},
title = {Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq.},
journal = {Nature protocols},
volume = {13},
number = {11},
pages = {2615-2642},
pmid = {30341435},
issn = {1750-2799},
support = {DP1 GM105378/GM/NIGMS NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; R35GM118158/NH/NIH HHS/United States ; U01HL145793/NH/NIH HHS/United States ; R01GM107427/NH/NIH HHS/United States ; DP1GM105378/NH/NIH HHS/United States ; 2017093/DDCF/Doris Duke Charitable Foundation/United States ; R01 GM107427/GM/NIGMS NIH HHS/United States ; U01 HL145793/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Circular/*genetics/metabolism ; Gene Editing/*methods ; Gene Library ; Genome, Human ; High-Throughput Nucleotide Sequencing/*statistics &amp; numerical data ; Humans ; Lymphocytes/cytology/metabolism ; Osteoblasts/cytology/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and off-target) of CRISPR-Cas9 nucleases by selective sequencing of nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are treated with CRISPR-Cas9 ribonucleoprotein complexes, and nuclease-linearized DNA fragments are then ligated to adapters for high-throughput sequencing. The primary advantages of CIRCLE-seq as compared with other in vitro methods for defining genome-wide genome editing activity are (i) high enrichment for sequencing nuclease-cleaved gDNA/low background, enabling sensitive detection with low sequencing depth requirements; and (ii) the fact that paired-end reads can contain complete information on individual nuclease cleavage sites, enabling use of CIRCLE-seq in species without high-quality reference genomes. The entire protocol can be completed in 2 weeks, including time for gRNA cloning, sequence verification, in vitro transcription, library preparation, and sequencing.},
}
@article {pmid30340831,
year = {2018},
author = {Li, XF and Li, SY and Dai, CM and Li, JC and Huang, DR and Wang, JY},
title = {PP2A inhibition by LB-100 protects retinal pigment epithelium cells from UV radiation via activation of AMPK signaling.},
journal = {Biochemical and biophysical research communications},
volume = {506},
number = {1},
pages = {73-80},
doi = {10.1016/j.bbrc.2018.10.077},
pmid = {30340831},
issn = {1090-2104},
mesh = {AMP-Activated Protein Kinases/*genetics/metabolism ; Animals ; Bridged Bicyclo Compounds, Heterocyclic/*pharmacology ; CRISPR-Cas Systems ; Cell Line ; Cell Survival/drug effects/radiation effects ; Enzyme Activation ; Enzyme Inhibitors/*pharmacology ; Epithelial Cells/cytology/*drug effects/metabolism/radiation effects ; Gene Editing ; Gene Expression Regulation ; Humans ; Mice ; NADP/metabolism ; Piperazines/*pharmacology ; Primary Cell Culture ; Protein Phosphatase 2/antagonists &amp; inhibitors/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Retinal Pigment Epithelium/cytology/drug effects/metabolism/radiation effects ; Signal Transduction ; Sunscreening Agents/*pharmacology ; Ultraviolet Rays/adverse effects ; },
abstract = {AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19 cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.},
}
@article {pmid30340791,
year = {2018},
author = {Teplow, DB},
title = {Preface.},
journal = {Progress in molecular biology and translational science},
volume = {159},
number = {},
pages = {xi-xii},
doi = {10.1016/S1877-1173(18)30129-7},
pmid = {30340791},
issn = {1878-0814},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Gene Editing ; Half-Life ; RNA, Catalytic/metabolism ; Receptors, G-Protein-Coupled/*metabolism ; },
}
@article {pmid30340786,
year = {2018},
author = {Chandrasekaran, AP and Song, M and Kim, KS and Ramakrishna, S},
title = {Different Methods of Delivering CRISPR/Cas9 Into Cells.},
journal = {Progress in molecular biology and translational science},
volume = {159},
number = {},
pages = {157-176},
doi = {10.1016/bs.pmbts.2018.05.001},
pmid = {30340786},
issn = {1878-0814},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; *Gene Transfer Techniques ; Genetic Vectors/metabolism ; Humans ; Stem Cells/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is comprised of repetitive bases followed by short fragments of DNA from a previously invading organism that provide immunity to the most prokaryotic organisms. An RNA-dependent spacer is required for CRISPR/Cas9 to recognize the target DNA. Delivery of the CRISPR/Cas9-guide RNA (gRNA) complex to any cell results in modification of the target sequence. The CRISPR/Cas9-mediated genome editing technique is currently in the spotlight and has several research interests, including molecular medicine and agriculture. There are several factors that hinder the delivery of this complex, such as the large size of the plasmid or high dosage of the chemical agent. There are several methods available to deliver CRISPR/Cas9 and its components to the target cells. It includes viral, non-viral and physical methods to deliver plasmid or ribonucleoprotein (RNP) of CRISPR components. But in vivo CRISPR/Cas9 delivery remains challenging to the researchers due to insertional mutagenesis, targeted delivery, immunogenicity, and off-targets. However, studies suggesting that the CRISPR/Cas9-RNP delivery can overcome these hurdles. Here, we review the various methods for delivery of CRISPR/Cas9 and gRNA to several cell lines, highlighting the limitations of each approach, and suggest possible alternative methods.},
}
@article {pmid30340581,
year = {2018},
author = {Liu, X and Wang, M and Qin, Y and Shi, X and Cong, P and Chen, Y and He, Z},
title = {Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {66},
pmid = {30340581},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Crossing Over, Genetic ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Endodeoxyribonucleases/genetics/metabolism ; Gene Targeting/*methods ; Genome ; Humans ; Plasmids/genetics ; Receptors, CCR5/genetics ; },
abstract = {BACKGROUND: Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.<br><br>RESULTS: We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17-0.45%) that was used to knock in an approximately 5-kb long DNA fragment.<br><br>CONCLUSION: We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.},
}
@article {pmid30340517,
year = {2018},
author = {Guo, T and Feng, YL and Xiao, JJ and Liu, Q and Sun, XN and Xiang, JF and Kong, N and Liu, SC and Chen, GQ and Wang, Y and Dong, MM and Cai, Z and Lin, H and Cai, XJ and Xie, AY},
title = {Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {170},
pmid = {30340517},
issn = {1474-760X},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; DNA-Binding Proteins/metabolism ; *Gene Editing ; Gene Knockout Techniques ; *Genome ; HEK293 Cells ; Humans ; Liver/metabolism ; Mice ; Mutagenesis, Insertional/genetics ; Reproducibility of Results ; *Sequence Deletion ; },
abstract = {BACKGROUND: Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.<br><br>RESULTS: In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent +&#x2009;1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n&#x2009;+&#x2009;1"-, or "3n&#x2009;+&#x2009;2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n&#x2009;+&#x2009;1"-bp, "3n&#x2009;+&#x2009;2"-bp can overcome +&#x2009;1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.<br><br>CONCLUSIONS: NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.},
}
@article {pmid30340514,
year = {2018},
author = {Schoonenberg, VAC and Cole, MA and Yao, Q and Macias-Treviño, C and Sher, F and Schupp, PG and Canver, MC and Maeda, T and Pinello, L and Bauer, DE},
title = {CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {169},
pmid = {30340514},
issn = {1474-760X},
support = {R03DK109232/DK/NIDDK NIH HHS/United States ; K08 DK093705/DK/NIDDK NIH HHS/United States ; DP2OD022716/HL/NHLBI NIH HHS/United States ; R03 DK109232/DK/NIDDK NIH HHS/United States ; DP2 HL137300/HL/NHLBI NIH HHS/United States ; R01 DK111455/DK/NIDDK NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; P01HL032262/HL/NHLBI NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; K08DK093705/DK/NIDDK NIH HHS/United States ; R00HG008399/HG/NHGRI NIH HHS/United States ; R56 DK105001/DK/NIDDK NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; *Gene Editing ; *Genome ; Humans ; Molecular Sequence Annotation ; Mutagenesis/*genetics ; Open Reading Frames/*genetics ; Protein Structure, Secondary ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; },
abstract = {CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro .},
}
@article {pmid30340417,
year = {2018},
author = {Van Houdt, R and Provoost, A and Van Assche, A and Leys, N and Lievens, B and Mijnendonckx, K and Monsieurs, P},
title = {Cupriavidus metallidurans Strains with Different Mobilomes and from Distinct Environments Have Comparable Phenomes.},
journal = {Genes},
volume = {9},
number = {10},
pages = {},
pmid = {30340417},
issn = {2073-4425},
abstract = {Cupriavidus metallidurans has been mostly studied because of its resistance to numerous heavy metals and is increasingly being recovered from other environments not typified by metal contamination. They host a large and diverse mobile gene pool, next to their native megaplasmids. Here, we used comparative genomics and global metabolic comparison to assess the impact of the mobilome on growth capabilities, nutrient utilization, and sensitivity to chemicals of type strain CH34 and three isolates (NA1, NA4 and H1130). The latter were isolated from water sources aboard the International Space Station (NA1 and NA4) and from an invasive human infection (H1130). The mobilome was expanded as prophages were predicted in NA4 and H1130, and a genomic island putatively involved in abietane diterpenoids metabolism was identified in H1130. An active CRISPR-Cas system was identified in strain NA4, providing immunity to a plasmid that integrated in CH34 and NA1. No correlation between the mobilome and isolation environment was found. In addition, our comparison indicated that the metal resistance determinants and properties are conserved among these strains and thus maintained in these environments. Furthermore, all strains were highly resistant to a wide variety of chemicals, much broader than metals. Only minor differences were observed in the phenomes (measured by phenotype microarrays), despite the large difference in mobilomes and the variable (shared by two or three strains) and strain-specific genomes.},
}
@article {pmid30340091,
year = {2018},
author = {Anderson, RH and Kerkvliet, JG and Otta, JJ and Ross, AD and Leiferman, PC and Hoppe, AD and Francis, KR},
title = {Generation of a CLTA reporter human induced pluripotent stem cell line, CRMi001-A-1, using the CRISPR/Cas9 system to monitor endogenous clathrin trafficking.},
journal = {Stem cell research},
volume = {33},
number = {},
pages = {95-99},
pmid = {30340091},
issn = {1876-7753},
support = {F30 NS106788/NS/NINDS NIH HHS/United States ; P20 GM103548/GM/NIGMS NIH HHS/United States ; P20 GM103620/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Clathrin/*metabolism ; Genes, Reporter ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; },
abstract = {The most highly studied endocytic pathway, clathrin-dependent endocytosis, mediates a wide range of fundamental processes including nutrient internalization, receptor recycling, and signal transduction. In order to model tissue specific and developmental aspects of this process, CRISPR/Cas9 genomic editing was utilized to fluorescently label the C-terminus of clathrin light chain A (CLTA) within the phenotypically normal, parental CRMi001-A human induced pluripotent stem cell line. Successfully edited cells were isolated by fluorescently activated cell sorting, remained karyotypically normal, and maintained their differentiation potential. This cell line facilitates imaging of endogenous clathrin trafficking within varied cell types.},
}
@article {pmid30339905,
year = {2018},
author = {Liu, BY and He, XY and Zhuo, RX and Cheng, SX},
title = {Tumor targeted genome editing mediated by a multi-functional gene vector for regulating cell behaviors.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {291},
number = {},
pages = {90-98},
doi = {10.1016/j.jconrel.2018.10.018},
pmid = {30339905},
issn = {1873-4995},
mesh = {*CRISPR-Cas Systems ; Cyclin-Dependent Kinases/*genetics ; Gene Editing/*methods ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Genetic Vectors/*genetics/therapeutic use ; Humans ; MCF-7 Cells ; Neoplasms/genetics/*therapy ; Plasmids/genetics/therapeutic use ; },
abstract = {For effective regulation of cell behaviors and prevention of tumor development by genome editing, we constructed multi-functional self-assembled nanoparticles based on natural polymers to deliver CRISPR-Cas9 plasmid to tumorous cells. The CRISPR based gene editing plasmid to knockout CDK11 gene was complexed with protamine sulfate, and then the complex was decorated by a multi-functional outer layer composed of an endosomolytic peptide (KALA) and aptamer AS1411 incorporated carboxymethyl chitosan. The resultant multi-functional nanoparticles, which exhibit significantly enhanced delivery efficiency, can specifically deliver the plasmid into tumor cell nuclei owing to the favorable effects of KALA in cellular uptake and endosomal escape, together with the cancer cell and cell nucleus targeting capability of AS1411 ligands. The genome editing mediated by the nanoparticles leads to a dramatic decrease (>75%) in CDK11 expression, which results in further modulation of cancer cells with significant down-regulation of the proteins (MMP-9 and VEGF) involved in tumor development and metastasis as well as up-regulation of the tumor suppressor protein p53. More importantly, the detection of immune-related proteins after genome editing shows that the significantly enhanced Fas, CD80, MICA, MICB, and HLA-1 expression and decreased CD47 and MUC1 expression, indicating the genome editing is favorable for reversal of tumor-induced immunosuppression and prevention of tumor development.},
}
@article {pmid30338908,
year = {2018},
author = {Zhou, L and Peng, R and Zhang, R and Li, J},
title = {The applications of CRISPR/Cas system in molecular detection.},
journal = {Journal of cellular and molecular medicine},
volume = {22},
number = {12},
pages = {5807-5815},
pmid = {30338908},
issn = {1582-4934},
mesh = {Bacteria/isolation &amp; purification ; Base Sequence ; CRISPR-Cas Systems/*genetics ; *Molecular Diagnostic Techniques ; Mutation/genetics ; Polymorphism, Single Nucleotide/genetics ; Viruses/isolation &amp; purification ; },
abstract = {The Streptococcus pyogenes CRISPR/Cas system has found widespread applications as a gene-editing and regulatory tool for the simultaneous delivery of the Cas9 protein and guide RNAs into the cell, thus making the recognition of specific DNA sequences possible. The recent study that shows that Cas9 can also bind to and cleave RNA in an RNA-programmable manner is suggestive of potential utility of this system as a universal nucleic-acid recognition tool. To increase the signal intensity of the CRISPR/Cas system, a signal amplification technique has to be exploited appropriately; this requirement is also a challenge for the detection of DNA or RNA. Furthermore, the CRISPR/Cas system may be used to detect point mutations or single-nucleotide variants because of the specificity of the recognition between the target sequence and the CRISPR/Cas system. These lines of evidence make this technique capable of detecting pathogens during infection via analysis of their DNA or RNA. Thus, here we summarize applications of the CRISPR/Cas system to the recognition and detection of DNA and RNA molecules as well as the signal amplification. We also describe its potential ability to detect mutations and single-nucleotide variants. Finally, we sum up its applications to testing for pathogens and potential barriers for its implementation.},
}
@article {pmid30338631,
year = {2019},
author = {Zhu, Z and Kang, X and Lor, VS and Weiss, D and Olszewski, N},
title = {Characterization of a semidominant dwarfing PROCERA allele identified in a screen for CRISPR/Cas9-induced suppressors of loss-of-function alleles.},
journal = {Plant biotechnology journal},
volume = {17},
number = {2},
pages = {319-321},
pmid = {30338631},
issn = {1467-7652},
support = {US-4813-15C//US Israel Binational Agriculture Research and Development/International ; 201606050088//State Scholarship Fund from China Scholarship Council/International ; },
mesh = {Alleles ; CRISPR-Cas Systems ; Genes, Dominant ; Germination ; Gibberellins/*metabolism ; Loss of Function Mutation ; Lycopersicon esculentum/*genetics/growth &amp; development/physiology ; Models, Molecular ; Mutagenesis ; Plant Growth Regulators/*metabolism ; Plant Proteins/genetics/*metabolism ; Seeds/genetics/growth &amp; development/physiology ; Sequence Alignment ; },
}
@article {pmid30338024,
year = {2018},
author = {Parise, D and Parise, MTD and Viana, MVC and Muñoz-Bucio, AV and Cortés-Pérez, YA and Arellano-Reynoso, B and Díaz-Aparicio, E and Dorella, FA and Pereira, FL and Carvalho, AF and Figueiredo, HCP and Ghosh, P and Barh, D and Gomide, ACP and Azevedo, VAC},
title = {First genome sequencing and comparative analyses of Corynebacterium pseudotuberculosis strains from Mexico.},
journal = {Standards in genomic sciences},
volume = {13},
number = {},
pages = {21},
pmid = {30338024},
issn = {1944-3277},
abstract = {Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster.},
}
@article {pmid30337455,
year = {2018},
author = {Harrington, LB and Burstein, D and Chen, JS and Paez-Espino, D and Ma, E and Witte, IP and Cofsky, JC and Kyrpides, NC and Banfield, JF and Doudna, JA},
title = {Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6416},
pages = {839-842},
pmid = {30337455},
issn = {1095-9203},
support = {/HHMI/Howard Hughes Medical Institute/United States ; T32 GM008295/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaeal Proteins/*chemistry/*classification/genetics ; CRISPR-Associated Proteins/*chemistry/*classification/genetics ; *DNA Cleavage ; DNA, Single-Stranded/*chemistry ; Datasets as Topic ; Endodeoxyribonucleases/*chemistry/*classification/genetics ; Evolution, Molecular ; Metagenomics ; Phylogeny ; },
abstract = {CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.},
}
@article {pmid30336982,
year = {2018},
author = {Bardet, M and Seeholzer, T and Unterreiner, A and Woods, S and Krappmann, D and Bornancin, F},
title = {MALT1 activation by TRAF6 needs neither BCL10 nor CARD11.},
journal = {Biochemical and biophysical research communications},
volume = {506},
number = {1},
pages = {48-52},
doi = {10.1016/j.bbrc.2018.10.029},
pmid = {30336982},
issn = {1090-2104},
mesh = {B-Cell CLL-Lymphoma 10 Protein/deficiency/*genetics ; CARD Signaling Adaptor Proteins/deficiency/*genetics ; CRISPR-Cas Systems ; Enzyme Activation/drug effects ; Gene Editing/methods ; Gene Expression Profiling ; Gene Expression Regulation ; Guanylate Cyclase/deficiency/*genetics ; HEK293 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; Jurkat Cells ; Lymphocyte Activation/drug effects ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/*genetics/metabolism ; Signal Transduction ; TNF Receptor-Associated Factor 6/*genetics/metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; },
abstract = {The MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) paracaspase couples antigen receptors on lymphocytes to downstream signaling events. Activation of MALT1 is known to involve stimulus-dependent CBM complex formation, that is, the recruitment of BCL10-bound MALT1 to a CARD-Coiled Coil protein. Beyond this canonical, CBM-dependent mechanism of MALT1 activation, recent studies suggest that MALT1 protease activity may be triggered by alternative mechanisms. For instance, the E3-ligase TRAF6 can activate MALT1 proteolytic function and induce MALT1 auto-cleavage. However, the interplay between CBM and TRAF6 with regard to MALT1 activation has remained incompletely elucidated. Here, by generating CRISPR/Cas9-derived knock-out Jurkat T-cells, we show that TRAF6 was dispensable for CARD11/BCL10-dependent MALT1 activation upon T-cell stimulation. However, ectopically-expressed TRAF6 could induce MALT1 activity in Jurkat T-cells devoid of either CARD11 or BCL10. These data provide unequivocal evidence that TRAF6-mediated MALT1 activation does not require the upstream scaffold CARD11 or the interaction between MALT1 and BCL10. Thus, TRAF6 may be part of a previously unidentified non-canonical pathway that triggers MALT1 protease activity independently of canonical CBM signalosomes.},
}
@article {pmid30335949,
year = {2018},
author = {Schneider, N and Gäbelein, C and Wiener, J and Georgiev, T and Gobet, N and Weber, W and Meier, M},
title = {Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins.},
journal = {ACS chemical biology},
volume = {13},
number = {11},
pages = {3049-3053},
doi = {10.1021/acschembio.8b00594},
pmid = {30335949},
issn = {1554-8937},
mesh = {Actins/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; *Codon, Terminator ; Genetic Engineering/methods ; HEK293 Cells ; Hemagglutinin Glycoproteins, Influenza Virus/genetics ; Humans ; Luminescent Proteins/genetics ; Lysine/analogs &amp; derivatives/genetics ; Lysine-tRNA Ligase/genetics/*metabolism ; Methanosarcina barkeri/enzymology ; Microscopy, Fluorescence ; Peptide Fragments/genetics ; Protein Engineering/*methods ; RNA, Transfer/genetics ; Tumor Suppressor Protein p53/genetics/*metabolism ; },
abstract = {We here present a method that combines genetic code expansion with CRISPR/Cas9 genome engineering to label endogenously expressed proteins with high spatiotemporal resolution. The method exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with noncanonical amino acids to create stop codon read through events. To demonstrate the functionality of the method, we pulse labeled endogenous β-actin and tumor protein p53 with a minimally invasive HA tag at their C-termini. Targeting the protein label with a proximity ligation assay plus real time imaging facilitates seamless quantification of the protein synthesis rate and spatial localization at the single cell level. The presented approach does not interfere with any physiological control of cellular expression, nor did we observe any perturbation of endogenous protein functions.},
}
@article {pmid30335163,
year = {2018},
author = {Park, J and Lee, H and Han, N and Kwak, S and Lee, HT and Kim, JH and Kang, K and Youn, BH and Yang, JH and Jeong, HJ and Kang, JS and Kim, SY and Han, JW and Youn, HD and Cho, EJ},
title = {Long non-coding RNA ChRO1 facilitates ATRX/DAXX-dependent H3.3 deposition for transcription-associated heterochromatin reorganization.},
journal = {Nucleic acids research},
volume = {46},
number = {22},
pages = {11759-11775},
pmid = {30335163},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems ; Carrier Proteins/*genetics/metabolism ; Cell Cycle Proteins/genetics/metabolism ; Cell Differentiation ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; Co-Repressor Proteins ; Female ; Gene Editing ; Gene Expression Regulation, Developmental ; HEK293 Cells ; Heterochromatin/*chemistry/metabolism ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; Histones/*genetics/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/*genetics/metabolism ; Male ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Molecular Chaperones ; Muscle Development/*genetics ; Muscle, Skeletal/cytology/growth &amp; development/metabolism ; NIH 3T3 Cells ; Nuclear Proteins/*genetics/metabolism ; RNA, Long Noncoding/antagonists &amp; inhibitors/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Transcription, Genetic ; X-linked Nuclear Protein/*genetics/metabolism ; },
abstract = {Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.},
}
@article {pmid30334617,
year = {2019},
author = {Oh, SA and Seki, A and Rutz, S},
title = {Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells.},
journal = {Current protocols in immunology},
volume = {124},
number = {1},
pages = {e69},
doi = {10.1002/cpim.69},
pmid = {30334617},
issn = {1934-368X},
mesh = {Animals ; CD4-Positive T-Lymphocytes/cytology/*metabolism ; CD8-Positive T-Lymphocytes/cytology/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Humans ; Mice ; Mice, Knockout ; Ribonucleoproteins/*genetics/metabolism ; Transfection ; },
abstract = {CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level. This approach enables rapid target discovery and validation in both mouse and human primary T cells. © 2018 by John Wiley &amp; Sons, Inc.},
}
@article {pmid30333047,
year = {2018},
author = {Balabaskaran-Nina, P and Desai, SA},
title = {Diverse target gene modifications in Plasmodium falciparum using Bxb1 integrase and an intronic attB.},
journal = {Parasites &amp; vectors},
volume = {11},
number = {1},
pages = {548},
pmid = {30333047},
issn = {1756-3305},
mesh = {Animals ; CRISPR-Cas Systems ; Genetic Engineering/*methods ; Genetic Vectors ; Humans ; Integrases/*genetics ; *Introns ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/enzymology/*genetics ; Transfection ; },
abstract = {Genetic manipulation of the human malaria parasite Plasmodium falciparum is needed to explore pathogen biology and evaluate antimalarial targets. It is, however, aggravated by a low transfection efficiency, a paucity of selectable markers and a biased A/T-rich genome. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. We recently devised a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. Although this attB is silent and without effect on intron splicing or protein translation and function, it allows efficient gene modification with minimal risk of unwanted changes at other genomic sites. We describe the range of applications for this new method as well as specific cases where it is preferred over CRISPR-Cas9 and other technologies. The advantages and limitations of various strategies for endogenous gene editing are also discussed.},
}
@article {pmid30332653,
year = {2018},
author = {Simões-Sousa, S and Littler, S and Thompson, SL and Minshall, P and Whalley, H and Bakker, B and Belkot, K and Moralli, D and Bronder, D and Tighe, A and Spierings, DCJ and Bah, N and Graham, J and Nelson, L and Green, CM and Foijer, F and Townsend, PA and Taylor, SS},
title = {The p38α Stress Kinase Suppresses Aneuploidy Tolerance by Inhibiting Hif-1α.},
journal = {Cell reports},
volume = {25},
number = {3},
pages = {749-760.e6},
pmid = {30332653},
issn = {2211-1247},
support = {/WT_/Wellcome Trust/United Kingdom ; 11913/CRUK_/Cancer Research UK/United Kingdom ; 203141/WT_/Wellcome Trust/United Kingdom ; MR/L006839/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {*Aneuploidy ; *Apoptosis ; CRISPR-Cas Systems ; Chromosomes, Human/*genetics ; Colonic Neoplasms ; Glycolysis ; Humans ; *Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Mitogen-Activated Protein Kinase 14/antagonists &amp; inhibitors/genetics/*metabolism ; Signal Transduction ; Tumor Cells, Cultured ; },
abstract = {Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1α and that inhibiting Hif-1α restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1α to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.},
}
@article {pmid30332651,
year = {2018},
author = {De Maio, A and Yalamanchili, HK and Adamski, CJ and Gennarino, VA and Liu, Z and Qin, J and Jung, SY and Richman, R and Orr, H and Zoghbi, HY},
title = {RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins.},
journal = {Cell reports},
volume = {25},
number = {3},
pages = {726-736.e7},
pmid = {30332651},
issn = {2211-1247},
support = {C06 RR029965/RR/NCRR NIH HHS/United States ; U54 HD083092/HD/NICHD NIH HHS/United States ; P30 HD024064/HD/NICHD NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; R37 NS027699/NS/NINDS NIH HHS/United States ; P01 HD040301/HD/NICHD NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; DNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Female ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Male ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nerve Tissue Proteins/*physiology ; *RNA Splicing ; RNA Splicing Factors/antagonists &amp; inhibitors/genetics/*metabolism/physiology ; RNA-Binding Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Spliceosomes ; },
abstract = {RNA splicing entails the coordinated interaction of more than 150 proteins in the spliceosome, one of the most complex of the cell's molecular machines. We previously discovered that the RNA-binding motif protein 17 (RBM17), a component of the spliceosome, is essential for survival and cell maintenance. Here, we find that it interacts with the spliceosomal factors U2SURP and CHERP and that they reciprocally regulate each other's stability, both in mouse and in human cells. Individual knockdown of each of the three proteins induces overlapping changes in splicing and gene expression of transcripts enriched for RNA-processing factors. Our results elucidate the function of RBM17, U2SURP, and CHERP and link the activity of the spliceosome to the regulation of downstream RNA-binding proteins. These data support the hypothesis that, beyond driving constitutive splicing, spliceosomal factors can regulate alternative splicing of specific targets.},
}
@article {pmid30332437,
year = {2018},
author = {Morozova, KN and Suldina, LA and Malankhanova, TB and Grigor'eva, EV and Zakian, SM and Kiseleva, E and Malakhova, AA},
title = {Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0204735},
pmid = {30332437},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Clone Cells/metabolism/ultrastructure ; Endoplasmic Reticulum/ultrastructure ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Huntingtin Protein/antagonists &amp; inhibitors/*genetics ; Huntington Disease/*genetics/*pathology ; Lysosomes/ultrastructure ; Microscopy, Electron, Transmission ; Mitochondria/ultrastructure ; Mutant Proteins/*genetics ; Mutation ; *Trinucleotide Repeat Expansion ; },
abstract = {Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.},
}
@article {pmid30329221,
year = {2018},
author = {Gong, T and Tang, B and Zhou, X and Zeng, J and Lu, M and Guo, X and Peng, X and Lei, L and Gong, B and Li, Y},
title = {Genome editing in Streptococcus mutans through self-targeting CRISPR arrays.},
journal = {Molecular oral microbiology},
volume = {33},
number = {6},
pages = {440-449},
doi = {10.1111/omi.12247},
pmid = {30329221},
issn = {2041-1014},
mesh = {Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; Dental Caries/microbiology ; Dental Plaque/microbiology ; Gene Editing/*methods ; Glucosyltransferases/genetics ; Humans ; Microscopy, Electron, Scanning ; Streptococcus mutans/*genetics/pathogenicity ; Virulence Factors/genetics ; },
abstract = {Streptococcus mutans is the primary etiological agent of human dental caries. Its major virulence factors, glucosyltransferases (Gtfs), utilize sucrose to synthesize extracellular polysaccharides (EPS), leading to the formation of dental plaque biofilm. The current study was designed to develop a novel self-targeting gene editing technology that targeted gtfs to inhibit biofilms formation. The CRISPR-Cas system (ie, clustered regularly interspaced short palindromic repeat, with CRISPR-associated proteins) provides sequence-specific protection against foreign genetic materials in archaea and bacteria, and has been widely developed for genomic engineering. The first aim of this study was to test whether components of the CRISPR-Cas9 system from S mutans UA159 is necessary to defend against foreign DNA. The data showed that a suitable PAM site, tracrRNA, Cas9, and RNase III are indispensable elements to perform normal function of S mutans CRISPR-Cas9 system. Based on these results, we designed self-targeting CRISPR arrays (containing spacer sequences identifying with gtfB) and cloned them onto plasmids. Afterward, we transformed the plasmids and editing templates into UA159 (self-targeting) to acquire desired mutants. Our data showed that this technology performed well and was able to successfully edit gtfB or gtfBgtfC genes. This resulted in high reduction in EPS synthesis and was able to breakdown biofilm formation, which is also a promising tool for dental clinics in order to prevent the formation of S mutans biofilms in the future.},
}
@article {pmid30329048,
year = {2019},
author = {Tu, Z and Zhao, H and Li, B and Yan, S and Wang, L and Tang, Y and Li, Z and Bai, D and Li, C and Lin, Y and Li, Y and Liu, J and Xu, H and Guo, X and Jiang, YH and Zhang, YQ and Li, XJ},
title = {CRISPR/Cas9-mediated disruption of SHANK3 in monkey leads to drug-treatable autism-like symptoms.},
journal = {Human molecular genetics},
volume = {28},
number = {4},
pages = {561-571},
pmid = {30329048},
issn = {1460-2083},
support = {R01 NS036232/NS/NINDS NIH HHS/United States ; R01 NS101701/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Autism Spectrum Disorder/diagnostic imaging/*drug therapy/genetics/pathology ; Behavior, Animal/drug effects ; Brain/diagnostic imaging/*drug effects/pathology ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Exons ; Fluoxetine/*administration &amp; dosage ; Humans ; Interpersonal Relations ; Macaca fascicularis/genetics ; Mice ; Mutation ; Nerve Tissue Proteins/*genetics ; },
abstract = {Monogenic mutations in the SHANK3 gene, which encodes a postsynaptic scaffold protein, play a causative role in autism spectrum disorder (ASD). Although a number of mouse models with Shank3 mutations have been valuable for investigating the pathogenesis of ASD, species-dependent differences in behaviors and brain structures post considerable challenges to use small animals to model ASD and to translate experimental therapeutics to the clinic. We have used clustered regularly interspersed short palindromic repeat/CRISPR-associated nuclease 9 to generate a cynomolgus monkey model by disrupting SHANK3 at exons 6 and 12. Analysis of the live mutant monkey revealed the core behavioral abnormalities of ASD, including impaired social interaction and repetitive behaviors, and reduced brain network activities detected by positron-emission computed tomography (PET). Importantly, these abnormal behaviors and brain activities were alleviated by the antidepressant fluoxetine treatment. Our findings provide the first demonstration that the genetically modified non-human primate can be used for translational research of therapeutics for ASD.},
}
@article {pmid30328670,
year = {2018},
author = {Yang, Y and Wang, YH and Chen, XE and Tian, D and Xu, X and Li, K and Huang, YP and He, L},
title = {CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon.},
journal = {Insect science},
volume = {25},
number = {6},
pages = {1017-1024},
doi = {10.1111/1744-7917.12647},
pmid = {30328670},
issn = {1744-7917},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation, Developmental ; Larva/*growth &amp; development ; Lepidoptera/enzymology/*genetics/*growth &amp; development ; Mutation ; Pest Control, Biological ; Phenotype ; Tyrosine 3-Monooxygenase/*deficiency/*genetics ; },
abstract = {Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.},
}
@article {pmid30328555,
year = {2019},
author = {Shin, HY and Hennighausen, L and Yoo, KH},
title = {STAT5-Driven Enhancers Tightly Control Temporal Expression of Mammary-Specific Genes.},
journal = {Journal of mammary gland biology and neoplasia},
volume = {24},
number = {1},
pages = {61-71},
pmid = {30328555},
issn = {1573-7039},
mesh = {Animals ; CCCTC-Binding Factor/metabolism ; CRISPR-Cas Systems/genetics ; Cytokines/metabolism ; *Enhancer Elements, Genetic ; Female ; Gene Editing/methods ; *Gene Expression Regulation, Developmental ; Gene Regulatory Networks ; Genetic Loci ; Lactation/*genetics ; Mammary Glands, Animal/*growth &amp; development/metabolism ; Milk Proteins/metabolism ; Pregnancy ; Prolactin/metabolism ; RNA-Seq ; STAT5 Transcription Factor/genetics/*metabolism ; },
abstract = {The de novo formation of milk-secreting mammary epithelium during pregnancy is regulated by prolactin through activation of the transcription factor STAT5, which stimulates the expression of several hundred mammary-specific genes. In addition to its key role in activating gene expression in mammary tissue, STAT5, which is ubiquitously expressed in most cell types, implements T cell-specific programs controlled by interleukins. However, the mechanisms by which STAT5 controls cell-specific genetic programs activated by distinct cytokines remain relatively unknown. Integration of data from genome-wide surveys of chromatin markers and transcription factor binding at regulatory elements may shed light on the mechanisms that drive cell-specific programs. Here, we have illustrated how STAT5 controls cell-specific gene expression through its concentration and an auto-regulatory enhancer supporting its high levels in mammary tissue. The unique genomic features of STAT5-driven enhancers or super-enhancers that regulate mammary-specific genes and their dynamic remodeling in response to pregnancy hormone levels are described. We have further provided biological evidence supporting the in vivo function of a STAT5-driven super-enhancer with the aid of CRISPR/Cas9 genome editing. Finally, we discuss how the functions of mammary-specific super-enhancers are confined by the zinc finger protein, CTCF, to allow exclusive activation of mammary-specific genes without affecting common neighboring genes. This review comprehensively summarizes the molecular pathways underlying differential control of cell-specific gene sets by STAT5 and provides novel insights into STAT5-dependent mammary physiology.},
}
@article {pmid30327559,
year = {2018},
author = {Wallace, M and Green, CR and Roberts, LS and Lee, YM and McCarville, JL and Sanchez-Gurmaches, J and Meurs, N and Gengatharan, JM and Hover, JD and Phillips, SA and Ciaraldi, TP and Guertin, DA and Cabrales, P and Ayres, JS and Nomura, DK and Loomba, R and Metallo, CM},
title = {Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues.},
journal = {Nature chemical biology},
volume = {14},
number = {11},
pages = {1021-1031},
pmid = {30327559},
issn = {1552-4469},
support = {R01 AI114929/AI/NIAID NIH HHS/United States ; R01 CA172667/CA/NCI NIH HHS/United States ; P01 HL110900/HL/NHLBI NIH HHS/United States ; R01 CA188652/CA/NCI NIH HHS/United States ; R01 HL126945/HL/NHLBI NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; },
mesh = {3T3 Cells ; Adipocytes/cytology ; Adipose Tissue/*enzymology ; Amino Acids, Branched-Chain/*metabolism ; Animals ; CRISPR-Cas Systems ; Carnitine O-Acetyltransferase/metabolism ; Cytosol/metabolism ; Fatty Acid Synthases/*metabolism ; Fatty Acids/*biosynthesis ; Female ; Hypoxia ; Lentivirus/genetics ; Lipogenesis ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Obese ; Obesity/*enzymology ; RNA, Small Interfering/metabolism ; },
abstract = {Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.},
}
@article {pmid30326628,
year = {2018},
author = {Chen, G and Xiong, L and Wang, Y and He, L and Huang, R and Liao, L and Zhu, Z and Wang, Y},
title = {ITGB1b-Deficient Rare Minnows Delay Grass Carp Reovirus (GCRV) Entry and Attenuate GCRV-Triggered Apoptosis.},
journal = {International journal of molecular sciences},
volume = {19},
number = {10},
pages = {},
pmid = {30326628},
issn = {1422-0067},
mesh = {Animals ; *Apoptosis ; CRISPR-Cas Systems ; Carps/*genetics/*virology ; Clathrin/metabolism ; Endocytosis ; Fish Diseases/*genetics/*virology ; Gene Knockout Techniques ; Integrin beta1/*genetics ; Phenotype ; Phylogeny ; Reoviridae/classification/*physiology ; *Virus Internalization ; },
abstract = {Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b[-/-]) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b[-/-] rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b[-/-] rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.},
}
@article {pmid30324523,
year = {2019},
author = {Aregger, M and Chandrashekhar, M and Tong, AHY and Chan, K and Moffat, J},
title = {Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1869},
number = {},
pages = {169-188},
doi = {10.1007/978-1-4939-8805-1_15},
pmid = {30324523},
issn = {1940-6029},
support = {342551//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Data Analysis ; Gene Knockout Techniques ; Gene Library ; *Genetic Testing ; Genomics/*methods ; High-Throughput Nucleotide Sequencing ; Lentivirus/*genetics ; Mammals/*genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.},
}
@article {pmid30324446,
year = {2019},
author = {Feehan, JM and Stanar, P and Tam, BM and Chiu, C and Moritz, OL},
title = {Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1834},
number = {},
pages = {193-207},
doi = {10.1007/978-1-4939-8669-9_14},
pmid = {30324446},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Fluorescent Antibody Technique ; Gene Editing ; Gene Expression ; Gene Knockout Techniques ; Genes, Reporter ; Humans ; Mice ; Phenotype ; RNA, Guide ; Retinal Degeneration/*genetics/*metabolism/pathology ; Xenopus laevis ; },
abstract = {Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout. Our techniques can produce high-frequency gene editing in X. laevis, permitting analysis in the F0 generation.},
}
@article {pmid30324436,
year = {2019},
author = {Benati, D and Marigo, V and Recchia, A},
title = {CRISPR/Cas9 Gene Editing In Vitro and in Retinal Cells In Vivo.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1834},
number = {},
pages = {59-74},
doi = {10.1007/978-1-4939-8669-9_4},
pmid = {30324436},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Cloning, Molecular ; Computational Biology/methods ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Gene Targeting ; HeLa Cells ; Humans ; Mice ; Polymerase Chain Reaction ; RNA, Guide ; Retina/*cytology/*metabolism ; Rhodopsin/genetics ; },
abstract = {CRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knockdown of human rhodopsin (RHO) gene carrying the P23H mutation in vitro, in engineered HeLa cells, and in vivo, in P23H RHO transgenic mice. To this aim, we report two molecular assays: site-specific PCR on P23H RHO cells treated with CRISPR/Cas9 and Western blotting analysis on retinal cells prepared from P23H RHO transgenic mice electroporated with CRISPR/Cas9 and GFP plasmids.},
}
@article {pmid30323812,
year = {2018},
author = {Krischuns, T and Günl, F and Henschel, L and Binder, M and Willemsen, J and Schloer, S and Rescher, U and Gerlt, V and Zimmer, G and Nordhoff, C and Ludwig, S and Brunotte, L},
title = {Phosphorylation of TRIM28 Enhances the Expression of IFN-β and Proinflammatory Cytokines During HPAIV Infection of Human Lung Epithelial Cells.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {2229},
pmid = {30323812},
issn = {1664-3224},
mesh = {A549 Cells ; Analysis of Variance ; Animals ; CRISPR-Cas Systems/genetics ; Chlorocebus aethiops ; DEAD Box Protein 58/metabolism ; Epithelial Cells/*virology ; Gene Knockdown Techniques ; Gene Transfer Techniques ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Influenza A virus/*metabolism ; Influenza, Human/*metabolism ; Interferon-beta/*metabolism ; Lung/*virology ; Phosphorylation ; Receptors, Immunologic ; Ribosomal Protein S6 Kinases, 90-kDa/metabolism ; Tripartite Motif-Containing Protein 28/*genetics/*metabolism ; p38 Mitogen-Activated Protein Kinases/metabolism ; },
abstract = {Human infection with highly pathogenic avian influenza viruses (HPAIV) is often associated with severe tissue damage due to hyperinduction of interferons and proinflammatory cytokines. The reasons for this excessive cytokine expression are still incompletely understood, which has hampered the development of efficient immunomodulatory treatment options. The host protein TRIM28 associates to the promoter regions of over 13,000 genes and is recognized as a genomic corepressor and negative immune regulator. TRIM28 corepressor activity is regulated by post-translational modifications, specifically phosphorylation of S473, which modulates binding of TRIM28 to the heterochromatin-binding protein HP1. Here, we identified TRIM28 as a key immune regulator leading to increased IFN-β and proinflammatory cytokine levels during infection with HPAIV. Using influenza A virus strains of the subtype H1N1 as well as HPAIV of subtypes H7N7, H7N9, and H5N1, we could demonstrate that strain-specific phosphorylation of TRIM28 S473 is induced by a signaling cascade constituted of PKR, p38 MAPK, and MSK1 in response to RIG-I independent sensing of viral RNA. Furthermore, using chemical inhibitors as well as knockout cell lines, our results suggest that phosphorylation of S473 facilitates a functional switch leading to increased levels of IFN-β, IL-6, and IL-8. In summary, we have identified TRIM28 as a critical factor controlling excessive expression of type I IFNs as well as proinflammatory cytokines during infection with H5N1, H7N7, and H7N9 HPAIV. In addition, our data indicate a novel mechanism of PKR-mediated IFN-β expression, which could lay the ground for novel treatment options aiming at rebalancing dysregulated immune responses during severe HPAIV infection.},
}
@article {pmid30323337,
year = {2018},
author = {Ding, Y and Gong, C and Huang, D and Chen, R and Sui, P and Lin, KH and Liang, G and Yuan, L and Xiang, H and Chen, J and Yin, T and Alexander, PB and Wang, QF and Song, EW and Li, QJ and Wood, KC and Wang, XF},
title = {Synthetic lethality between HER2 and transaldolase in intrinsically resistant HER2-positive breast cancers.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4274},
pmid = {30323337},
issn = {2041-1723},
support = {P50 CA190991/CA/NCI NIH HHS/United States ; F30 CA206348/CA/NCI NIH HHS/United States ; W81XWH-16-1-0618//U.S. Department of Defense (DOD)/International ; T32 GM007171/GM/NIGMS NIH HHS/United States ; K12 HD043446/HD/NICHD NIH HHS/United States ; W81XWH-16-1-0703//U.S. Department of Defense (DOD)/International ; },
mesh = {Breast Neoplasms/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Death/drug effects ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Female ; Genetic Testing ; HEK293 Cells ; Humans ; Lapatinib/pharmacology ; Metabolic Flux Analysis ; NADP/metabolism ; Pentose Phosphate Pathway ; Receptor, ErbB-2/*genetics ; Synthetic Lethal Mutations/*genetics ; Transaldolase/*genetics ; },
abstract = {Intrinsic resistance to anti-HER2 therapy in breast cancer remains an obstacle in the clinic, limiting its efficacy. However, the biological basis for intrinsic resistance is poorly understood. Here we performed a CRISPR/Cas9-mediated loss-of-function genetic profiling and identified TALDO1, which encodes the rate-limiting transaldolase (TA) enzyme in the non-oxidative pentose phosphate pathway, as essential for cellular survival following pharmacological HER2 blockade. Suppression of TA increases cell susceptibility to HER2 inhibition in two intrinsically resistant breast cancer cell lines with HER2 amplification. Mechanistically, TA depletion combined with HER2 inhibition significantly reduces cellular NADPH levels, resulting in excessive ROS production and deficient lipid and nucleotide synthesis. Importantly, higher TA expression correlates with poor response to HER2 inhibition in a breast cancer patient cohort. Together, these results pinpoint TA as a novel metabolic enzyme possessing synthetic lethality with HER2 inhibition that can potentially be exploited as a biomarker or target for combination therapy.},
}
@article {pmid30323312,
year = {2018},
author = {Rees, HA and Liu, DR},
title = {Base editing: precision chemistry on the genome and transcriptome of living cells.},
journal = {Nature reviews. Genetics},
volume = {19},
number = {12},
pages = {770-788},
pmid = {30323312},
issn = {1471-0064},
support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 GM065400/GM/NIGMS NIH HHS/United States ; R01 GM065865/GM/NIGMS NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; /HHMI_/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *Endonucleases ; Gene Editing/*methods ; Humans ; *Transcriptome ; },
abstract = {RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products. In this Review, we summarize base-editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision and in vivo delivery of base editors and discuss limitations and future directions of base editing for research and therapeutic applications.},
}
@article {pmid30323295,
year = {2018},
author = {Dong, C and Fontana, J and Patel, A and Carothers, JM and Zalatan, JG},
title = {Author Correction: Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4318},
doi = {10.1038/s41467-018-06909-4},
pmid = {30323295},
issn = {2041-1723},
abstract = {In the original version of the Supplementary Information file associated with this Article, the sequence '1x MS2 scRNA.b2' was incorrectly given as 'GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCGCACATGAGGATCACCCATGTGCTTTTTT' and should have read 'GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACATGAGGATCACCCATGTGCTTTTTTT'. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.},
}
@article {pmid30323226,
year = {2018},
author = {Beisel, CL},
title = {CRISPR tool puts RNA on the record.},
journal = {Nature},
volume = {562},
number = {7727},
pages = {347-349},
doi = {10.1038/d41586-018-06869-1},
pmid = {30323226},
issn = {1476-4687},
support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *RNA ; },
}
@article {pmid30323083,
year = {2018},
author = {Hatoum-Aslan, A},
title = {CRISPR Methods for Nucleic Acid Detection Herald the Future of Molecular Diagnostics.},
journal = {Clinical chemistry},
volume = {64},
number = {12},
pages = {1681-1683},
doi = {10.1373/clinchem.2018.295485},
pmid = {30323083},
issn = {1530-8561},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids ; Pathology, Molecular ; },
}
@article {pmid30323019,
year = {2019},
author = {Sega, AG and Mis, EK and Lindstrom, K and Mercimek-Andrews, S and Ji, W and Cho, MT and Juusola, J and Konstantino, M and Jeffries, L and Khokha, MK and Lakhani, SA},
title = {De novo pathogenic variants in neuronal differentiation factor 2 (NEUROD2) cause a form of early infantile epileptic encephalopathy.},
journal = {Journal of medical genetics},
volume = {56},
number = {2},
pages = {113-122},
doi = {10.1136/jmedgenet-2018-105322},
pmid = {30323019},
issn = {1468-6244},
support = {R01 HD081379/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; Basic Helix-Loop-Helix Transcription Factors/*genetics ; CRISPR-Cas Systems ; Child, Preschool ; Female ; Gene Expression ; Gene Knockdown Techniques ; Humans ; Larva/genetics ; Magnetic Resonance Imaging ; Male ; Mutation, Missense ; Neuropeptides/*genetics ; Spasms, Infantile/diagnostic imaging/etiology/*genetics ; Whole Exome Sequencing ; Xenopus laevis/embryology/genetics ; },
abstract = {BACKGROUND: Early infantile epileptic encephalopathies are severe disorders consisting of early-onset refractory seizures accompanied often by significant developmental delay. The increasing availability of next-generation sequencing has facilitated the recognition of single gene mutations as an underlying aetiology of some forms of early infantile epileptic encephalopathies.<br><br>OBJECTIVES: This study was designed to identify candidate genes as a potential cause of early infantile epileptic encephalopathy, and then to provide genetic and functional evidence supporting patient variants as causative.<br><br>METHODS: We used whole exome sequencing to identify candidate genes. To model the disease and assess the functional effects of patient variants on candidate protein function, we used in vivo CRISPR/Cas9-mediated genome editing and protein overexpression in frog tadpoles.<br><br>RESULTS: We identified novel de novo variants in neuronal differentiation factor 2 (NEUROD2) in two unrelated children with early infantile epileptic encephalopathy. Depleting neurod2 with CRISPR/Cas9-mediated genome editing induced spontaneous seizures in tadpoles, mimicking the patients' condition. Overexpression of wild-type NEUROD2 induced ectopic neurons in tadpoles; however, patient variants were markedly less effective, suggesting that both variants are dysfunctional and likely pathogenic.<br><br>CONCLUSION: This study provides clinical and functional support for NEUROD2 variants as a cause of early infantile epileptic encephalopathy, the first evidence of human disease caused by NEUROD2 variants.},
}
@article {pmid30322741,
year = {2019},
author = {Kilcher, S and Loessner, MJ},
title = {Engineering Bacteriophages as Versatile Biologics.},
journal = {Trends in microbiology},
volume = {27},
number = {4},
pages = {355-367},
doi = {10.1016/j.tim.2018.09.006},
pmid = {30322741},
issn = {1878-4380},
mesh = {Bacteria/virology ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Drug Resistance, Multiple, Bacterial ; Gene Editing ; *Genetic Engineering ; Genome, Viral ; Homologous Recombination ; Humans ; *Phage Therapy ; Virus Assembly ; },
abstract = {Viruses of bacteria (bacteriophages or phages) are highly evolved nanomachines that recognize bacterial cell walls, deliver genetic information, and kill or transform their targets with unparalleled specificity. For a long time, the use of genetically modified phages was limited to phage display approaches and fundamental research. This is mostly because phage engineering has been a complex and time-consuming task, applicable for only a few well characterized model phages. Recent advances in sequencing technology and molecular biology gave rise to rapid and precise tools that enable modification of less-well-characterized phages. These methods will pave the way for the development of modular designer-phages as versatile biologics that efficiently control multidrug-resistant bacteria and provide novel tools for pathogen detection, drug development, and beyond.},
}
@article {pmid30321477,
year = {2018},
author = {Chen, W and Werdann, M and Zhang, Y},
title = {The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster.},
journal = {The FEBS journal},
volume = {285},
number = {23},
pages = {4378-4393},
pmid = {30321477},
issn = {1742-4658},
support = {P20 GM103650/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Drosophila melanogaster ; Female ; *Gene Editing ; Indoleacetic Acids/*pharmacology ; Locomotion ; Male ; Nervous System/drug effects/*metabolism ; Period Circadian Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Plant Growth Regulators/*pharmacology ; Proteolysis/*drug effects ; },
abstract = {Tools that allow inducible and reversible depletion of target proteins are critical for biological studies. The plant-derived auxin-inducible degradation system (AID) enables the degradation of target proteins tagged with the AID motif. This system has been recently employed in mammalian cells as well as in Caenorhabditis elegans and Drosophila. To test the utility of the AID approach in the nervous system, we used circadian locomotor rhythms as a model and applied the AID method to temporally and spatially degrade PERIOD (PER), a critical pacemaker protein in Drosophila. We found that the period locus can be efficiently tagged with the AID motif by CRISPR/Cas9-based genome editing without disrupting PER function. Moreover, we demonstrated that the AID system could be used to induce rapid and efficient protein degradation in the nervous system as shown by effects on circadian and sleep behaviors. Furthermore, the protein degradation by AID was rapidly reversible after auxin removal. Together, our results show that the AID system provides a powerful tool for behavior studies in Drosophila.},
}
@article {pmid30320547,
year = {2018},
author = {Vuolo, L and Stevenson, NL and Heesom, KJ and Stephens, DJ},
title = {Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30320547},
issn = {2050-084X},
support = {MR/K018019/1/MRC_/Medical Research Council/United Kingdom ; MR/P000177/1/MRC_/Medical Research Council/United Kingdom ; BB/L014181/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/N000420/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptor Proteins, Signal Transducing/chemistry/*metabolism ; Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Carrier Proteins/chemistry/*metabolism ; Cell Line ; Cell Membrane/metabolism ; Cilia/*metabolism/ultrastructure ; Dyneins/*metabolism ; Gene Knockout Techniques ; Humans ; Mice ; Mutation/genetics ; Phenotype ; Tumor Suppressor Proteins/metabolism ; },
abstract = {The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.},
}
@article {pmid30319822,
year = {2018},
author = {Lone, BA and Karna, SKL and Ahmad, F and Shahi, N and Pokharel, YR},
title = {CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering.},
journal = {Genetics research international},
volume = {2018},
number = {},
pages = {3797214},
pmid = {30319822},
issn = {2090-3154},
abstract = {Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.},
}
@article {pmid30319599,
year = {2018},
author = {Brajic, A and Franckaert, D and Burton, O and Bornschein, S and Calvanese, AL and Demeyer, S and Cools, J and Dooley, J and Schlenner, S and Liston, A},
title = {The Long Non-coding RNA Flatr Anticipates Foxp3 Expression in Regulatory T Cells.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {1989},
pmid = {30319599},
issn = {1664-3224},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Forkhead Transcription Factors/*metabolism ; Gene Expression Profiling ; Gene Expression Regulation ; Lymphocyte Activation/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; RNA, Long Noncoding/genetics/*metabolism ; Self Tolerance/*genetics ; Signal Transduction/genetics/immunology ; T-Lymphocytes, Regulatory/*immunology/metabolism ; },
abstract = {Mammalian genomes encode a plethora of long non-coding RNA (lncRNA). These transcripts are thought to regulate gene expression, influencing biological processes from development to pathology. Results from the few lncRNA that have been studied in the context of the immune system have highlighted potentially critical functions as network regulators. Here we explored the nature of the lncRNA transcriptome in regulatory T cells (Tregs), a subset of CD4[+] T cells required to establish and maintain immunological self-tolerance. The identified Treg lncRNA transcriptome showed distinct differences from that of non-regulatory CD4[+] T cells, with evidence of direct shaping of the lncRNA transcriptome by Foxp3, the master transcription factor driving the distinct mRNA profile of Tregs. Treg lncRNA changes were disproportionally reversed in the absence of Foxp3, with an enrichment for colocalisation with Foxp3 DNA binding sites, indicating a direct coordination of transcription by Foxp3 independent of the mRNA coordination function. We further identified a novel lncRNA Flatr, as a member of the core Treg lncRNA transcriptome. Flatr expression anticipates Foxp3 expression during in vitro Treg conversion, and Flatr-deficient mice show a mild delay in in vitro and peripheral Treg induction. These results implicate Flatr as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.},
}
@article {pmid30318302,
year = {2018},
author = {Liu, Y and Yu, C and Daley, TP and Wang, F and Cao, WS and Bhate, S and Lin, X and Still, C and Liu, H and Zhao, D and Wang, H and Xie, XS and Ding, S and Wong, WH and Wernig, M and Qi, LS},
title = {CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming.},
journal = {Cell stem cell},
volume = {23},
number = {5},
pages = {758-771.e8},
pmid = {30318302},
issn = {1875-9777},
support = {DP5 OD017887/OD/NIH HHS/United States ; U01 DA047732/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Cellular Reprogramming/*genetics ; Female ; *Gene Editing ; Male ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed/*methods ; Neurons/*cytology/*metabolism ; Rats ; Rats, Sprague-Dawley ; Transcription Factors/*genetics/metabolism ; },
abstract = {Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.},
}
@article {pmid30318144,
year = {2018},
author = {Wang, H and Xu, X and Nguyen, CM and Liu, Y and Gao, Y and Lin, X and Daley, T and Kipniss, NH and La Russa, M and Qi, LS},
title = {CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.},
journal = {Cell},
volume = {175},
number = {5},
pages = {1405-1417.e14},
pmid = {30318144},
issn = {1097-4172},
support = {DP5 OD017887/OD/NIH HHS/United States ; T32 GM008568/GM/NIGMS NIH HHS/United States ; U01 DA047732/DA/NIDA NIH HHS/United States ; U01 EB021240/EB/NIBIB NIH HHS/United States ; },
mesh = {Abscisic Acid/pharmacology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Chromatin/genetics/metabolism ; Coiled Bodies/genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Loci ; *Genome ; Humans ; In Situ Hybridization, Fluorescence ; S Phase Cell Cycle Checkpoints/drug effects ; },
abstract = {Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.},
}
@article {pmid30317650,
year = {2019},
author = {Liang, W and van Wersch, S and Tong, M and Li, X},
title = {TIR-NB-LRR immune receptor SOC3 pairs with truncated TIR-NB protein CHS1 or TN2 to monitor the homeostasis of E3 ligase SAUL1.},
journal = {The New phytologist},
volume = {221},
number = {4},
pages = {2054-2066},
doi = {10.1111/nph.15534},
pmid = {30317650},
issn = {1469-8137},
support = {9975971//NSF Plant Genome Program Award/International ; //China Scholarship Council (CSC)/International ; //NSERC-Discovery program/International ; //CFI/International ; },
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/genetics/*metabolism ; Autoimmunity ; Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Deletion ; Gene Expression Regulation, Plant ; *Homeostasis ; NLR Proteins/genetics/metabolism ; Protein Binding ; Receptors, Cell Surface/*metabolism ; Receptors, Immunologic/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {Intracellular nucleotide binding (NB) and leucine-rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings have revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition. Here, by using the CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR-NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1. Overaccumulation of SAUL1 is monitored by the SOC3-TN2 pair, while SAUL1's disappearance is guarded by the SOC3-CHS1 pair. SOC3 forms a head-to-head genomic arrangement with CHS1 and TN2, indicative of transcriptional co-regulation. Such intricate cooperative interactions can probably enlarge the recognition spectrum and increase the functional flexibility of NLRs, which can partly explain the overwhelming occurrence of NLR gene clustering in higher plants.},
}
@article {pmid30317623,
year = {2019},
author = {Cai, M and Li, S and Shuai, Y and Li, J and Tan, J and Zeng, Q},
title = {Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells.},
journal = {Journal of cellular physiology},
volume = {234},
number = {6},
pages = {9184-9193},
doi = {10.1002/jcp.27595},
pmid = {30317623},
issn = {1097-4652},
mesh = {Apoptosis/*drug effects ; Apoptosis Regulatory Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Human Umbilical Vein Endothelial Cells/*drug effects/metabolism/pathology ; Humans ; Mitochondria/drug effects/metabolism/pathology ; Mitochondrial Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Receptors, Tumor Necrosis Factor, Type I/genetics/metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha/*pharmacology ; bcl-Associated Death Protein/genetics/metabolism ; },
abstract = {Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.},
}
@article {pmid30317409,
year = {2019},
author = {Peterson, TA and MacLean, AG},
title = {Current and Future Therapeutic Strategies for Lentiviral Eradication from Macrophage Reservoirs.},
journal = {Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology},
volume = {14},
number = {1},
pages = {68-93},
pmid = {30317409},
issn = {1557-1904},
support = {P51 OD011104/OD/NIH HHS/United States ; R01 NS104016/NS/NINDS NIH HHS/United States ; R21 MH113517/MH/NIMH NIH HHS/United States ; T32 OD011124/OD/NIH HHS/United States ; },
mesh = {Animals ; Anti-HIV Agents/*therapeutic use ; CRISPR-Cas Systems ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; HIV Infections/*therapy ; HIV-1/drug effects/physiology ; Humans ; Lentivirus ; Macrophages/*virology ; Virus Latency/drug effects/physiology ; },
abstract = {Macrophages, one of the most abundant populations of leukocytes in the body, function as the first line of defense against pathogen invaders. Human Immunodeficiency virus 1 (HIV-1) remains to date one of the most extensively studied viral infections. Naturally occurring lentiviruses in domestic and primate species serve as valuable models to investigate lentiviral pathogenesis and novel therapeutics. Better understanding of the role macrophages play in HIV pathogenesis will aid in the advancement towards a cure. Even with current efficacy of first- and second-line Antiretroviral Therapy (ART) guidelines and future efficacy of Long Acting Slow Effective Release-ART (LASER-ART); ART alone does not lead to a cure. The major challenge of HIV eradication is viral latency. Latency Reversal Agents (LRAs) show promise as a possible means to eradicate HIV-1 from the body. It has become evident that complete eradication will need to include combinations of various effective therapeutic strategies such as LASER-ART, LRAs, and gene editing. Review of the current literature indicates the most promising HIV eradication strategy appears to be LASER-ART in conjunction with viral and receptor gene modifications via the CRISPR/Cas9 system. Graphical abstract A multimodal approach to HIV treatment including gene editing, LASER-ART, and latency reversal agents may provide a means to achieve HIV eradication.},
}
@article {pmid30317145,
year = {2019},
author = {Bron, PA and Marcelli, B and Mulder, J and van der Els, S and Morawska, LP and Kuipers, OP and Kok, J and Kleerebezem, M},
title = {Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria.},
journal = {Current opinion in biotechnology},
volume = {56},
number = {},
pages = {61-68},
doi = {10.1016/j.copbio.2018.09.004},
pmid = {30317145},
issn = {1879-0429},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Food Microbiology ; Gene Editing/*methods ; Gene Transfer, Horizontal ; Lactobacillales/*genetics ; Transduction, Genetic ; },
abstract = {The ever-expanding genomic insight in natural diversity of lactic acid bacteria (LAB) has revived the industrial interest in traditional and natural genetic mobilization methodologies. Here, we review recent advances in horizontal gene transfer processes in LAB, including natural competence, conjugation, and phage transduction. In addition, we envision the possibilities for industrial strain improvement arising from the recent discoveries of molecular exchanges between bacteria through nanotubes and extracellular vesicles, as well as the constantly expanding genome editing possibilities using the CRISPR-Cas technology.},
}
@article {pmid30315870,
year = {2018},
author = {Larroude, M and Rossignol, T and Nicaud, JM and Ledesma-Amaro, R},
title = {Synthetic biology tools for engineering Yarrowia lipolytica.},
journal = {Biotechnology advances},
volume = {36},
number = {8},
pages = {2150-2164},
pmid = {30315870},
issn = {1873-1899},
support = {BB/R01602X/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Metabolic Engineering ; *Synthetic Biology ; *Yarrowia/genetics/metabolism ; },
abstract = {The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.},
}
@article {pmid30315482,
year = {2019},
author = {Whitworth, KM and Rowland, RRR and Petrovan, V and Sheahan, M and Cino-Ozuna, AG and Fang, Y and Hesse, R and Mileham, A and Samuel, MS and Wells, KD and Prather, RS},
title = {Resistance to coronavirus infection in amino peptidase N-deficient pigs.},
journal = {Transgenic research},
volume = {28},
number = {1},
pages = {21-32},
pmid = {30315482},
issn = {1573-9368},
mesh = {Aminopeptidases/deficiency/*genetics ; Animals ; Animals, Genetically Modified/*genetics/virology ; CRISPR-Cas Systems ; Coronavirus/genetics/*pathogenicity ; Coronavirus Infections/*genetics/virology ; Enterocytes/enzymology/virology ; Porcine epidemic diarrhea virus/pathogenicity ; Swine ; Transmissible gastroenteritis virus/pathogenicity ; },
abstract = {The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.},
}
@article {pmid30312667,
year = {2019},
author = {Raas, Q and Saih, FE and Gondcaille, C and Trompier, D and Hamon, Y and Leoni, V and Caccia, C and Nasser, B and Jadot, M and Ménétrier, F and Lizard, G and Cherkaoui-Malki, M and Andreoletti, P and Savary, S},
title = {A microglial cell model for acyl-CoA oxidase 1 deficiency.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1864},
number = {4},
pages = {567-576},
doi = {10.1016/j.bbalip.2018.10.005},
pmid = {30312667},
issn = {1879-2618},
mesh = {Acyl-CoA Oxidase/*deficiency/genetics ; Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; Gene Editing ; Hydrogen Peroxide/metabolism ; Mice ; Microglia/*cytology/metabolism ; *Models, Biological ; *Mutation ; Neurodegenerative Diseases/*genetics ; Oxidative Stress ; },
abstract = {Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β-oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1β and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal β-oxidation defect on oxidative stress, inflammation and cellular functions.},
}
@article {pmid30312651,
year = {2019},
author = {Graham, GV and Conlon, JM and Abdel-Wahab, YH and Flatt, PR},
title = {Glucagon-like peptides-1 from phylogenetically ancient fish show potent anti-diabetic activities by acting as dual GLP1R and GCGR agonists.},
journal = {Molecular and cellular endocrinology},
volume = {480},
number = {},
pages = {54-64},
doi = {10.1016/j.mce.2018.10.011},
pmid = {30312651},
issn = {1872-8057},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cricetinae ; Cricetulus ; Cyclic AMP/metabolism ; Fishes/*metabolism ; Glucagon-Like Peptide 1/administration &amp; dosage/chemistry/*pharmacology ; Glucagon-Like Peptide-1 Receptor/*antagonists &amp; inhibitors/metabolism ; HEK293 Cells ; Humans ; Hypoglycemic Agents/*pharmacology ; Insulin Secretion/drug effects ; Mice ; *Phylogeny ; Rats ; Receptors, Glucagon/*agonists/metabolism ; },
abstract = {Glucagon-like peptides-1 (GLP-1)from phylogenetically ancient fish (lamprey, dogfish, ratfish, paddlefish and bowfin) and from a teleost, the rainbow trout produced concentration-dependent stimulations of insulin release from clonal β-cells and isolated mouse islets. Lamprey and paddlefish GLP-1 were the most potent and effective. Incubation of BRIN-BD11 cells with GLP-1 receptor (GLP1R) antagonist, exendin-4 (9-39) attenuated insulinotropic activity of all peptides whereas glucagon receptor (GCGR) antagonist [des-His[1],Pro[4],Glu[9]] glucagon amide significantly decreased the activities of lamprey and paddlefish GLP-1 only. The GIP receptor antagonist GIP (6-30) Cex-K[40] [Pal] attenuated the activity of bowfin GLP-1. All peptides (1 μM) produced significant increases in cAMP concentration in CHL cells transfected with GLP1R but only lamprey and paddlefish GLP-1 stimulated cAMP production in HEK293 cells transfected with GCGR. Intraperitoneal administration of lamprey and paddlefish GLP-1 (25 nmol/kg body weight) in mice produced significant decreases in blood glucose and increased insulin concentrations comparable to the effects of human GLP-1. Lamprey and paddlefish GLP-1 display potent insulinotropic activity in vitro and glucose-lowering activity in vivo that is mediated through GLP1R and GCGR so that these peptides may constitute templates for design of new antidiabetic drugs.},
}
@article {pmid30312021,
year = {2019},
author = {Huang, B and Johansen, KH and Schwartzberg, PL},
title = {Efficient CRISPR/Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes.},
journal = {Current protocols in immunology},
volume = {124},
number = {1},
pages = {e62},
pmid = {30312021},
issn = {1934-368X},
support = {//Wellcome Trust/United Kingdom ; ZIA HG000123-18//Intramural NIH HHS/United States ; ZIA HG000123-19//Intramural NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Mice ; Mice, Transgenic ; *Mutagenesis ; T-Lymphocytes/cytology/immunology/*metabolism ; },
abstract = {The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling, and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene-silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9-based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation of many cell types with greater precision and ease. This article outlines a protocol for CRISPR/Cas9-mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs. © 2018 by John Wiley &amp; Sons, Inc.},
}
@article {pmid30311353,
year = {2019},
author = {Liu, T and Yang, WQ and Xie, YG and Liu, PW and Xie, LH and Lin, F and Li, CY and Gu, JB and Wu, K and Yan, GY and Chen, XG},
title = {Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1045-1054},
doi = {10.1111/1744-7917.12645},
pmid = {30311353},
issn = {1744-7917},
mesh = {Aedes/*genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Insect Vectors/*genetics ; Kynurenine 3-Monooxygenase/genetics ; Male ; Mutation ; },
abstract = {Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.},
}
@article {pmid30310080,
year = {2018},
author = {Idoko-Akoh, A and Taylor, L and Sang, HM and McGrew, MJ},
title = {High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {15126},
pmid = {30310080},
issn = {2045-2322},
support = {BBS/E/D/20320000/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013759/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013732/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Alleles ; Animals ; Base Sequence ; Binding Sites ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; Gene Order ; Genetic Vectors/genetics ; Germ Cells/cytology/*metabolism ; Heterozygote ; INDEL Mutation ; Mutation ; Protein Binding ; Sequence Analysis, DNA ; },
abstract = {Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.},
}
@article {pmid30309916,
year = {2018},
author = {Eldred, KC and Hadyniak, SE and Hussey, KA and Brenerman, B and Zhang, PW and Chamling, X and Sluch, VM and Welsbie, DS and Hattar, S and Taylor, J and Wahlin, K and Zack, DJ and Johnston, RJ},
title = {Thyroid hormone signaling specifies cone subtypes in human retinal organoids.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6411},
pages = {},
pmid = {30309916},
issn = {1095-9203},
support = {T32 GM007231/GM/NIGMS NIH HHS/United States ; T32 GM007445/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 EY025598/EY/NEI NIH HHS/United States ; R00 EY024648/EY/NEI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Embryonic Stem Cells/metabolism ; *Gene Expression Regulation, Developmental ; Humans ; Mutation ; Organoids/*growth &amp; development/metabolism ; Proteolysis ; Retina/cytology/*growth &amp; development ; Retinal Cone Photoreceptor Cells/*classification ; Thyroid Hormones/*metabolism ; },
abstract = {The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone-degrading and -activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.},
}
@article {pmid30309895,
year = {2018},
author = {Liu, Y and Wei, Z and Zhang, Y and Ma, X and Chen, Y and Yu, M and Ma, C and Li, X and Cao, Y and Liu, J and Han, J and Yang, X and Duan, Y},
title = {Activation of liver X receptor plays a central role in antiviral actions of 25-hydroxycholesterol.},
journal = {Journal of lipid research},
volume = {59},
number = {12},
pages = {2287-2296},
pmid = {30309895},
issn = {1539-7262},
mesh = {Animals ; Blotting, Western ; CRISPR-Cas Systems/genetics ; Hep G2 Cells ; Herpesvirus 1, Human/metabolism ; Humans ; Hydroxycholesterols/*metabolism ; Interferon-gamma/metabolism ; Liver X Receptors/*metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; RAW 264.7 Cells ; Real-Time Polymerase Chain Reaction ; Sulfotransferases/metabolism ; },
abstract = {Production of 25-hydroxycholesterol (25HC), a potent inhibitor of viral infection, is catalyzed by cholesterol 25-hydroxylase (CH25H). We previously reported that 25HC induced CH25H expression in a liver X receptor (LXR)-dependent manner, implying that LXR can play an important role in antiviral infection. In this study, we determined that activation of LXR by 25HC or synthetic ligands [T0901317 (T317) or GW3965] inhibited infection of herpes simplex virus type 1 (HSV-1) or MLV-(VSV)-GFP in HepG2 cells or RAW 264.7 macrophages. Genetic deletion of LXRα, LXRβ, or CH25H expression in HepG2 cells by CRISPR/Cas9 method increased cell susceptibility to HSV-1 infection and attenuated the inhibition of LXR on viral infection. Lack of interferon (IFN)-γ expression also increased cell susceptibility to viral infection. However, it attenuated, but did not block, the inhibition of LXR on HSV-1 infection. In addition, expression of CH25H, but not IFN-γ, was inversely correlated to cell susceptibility to viral infection and the antiviral actions of LXR. Metabolism of 25HC into 25HC-3-sulfate (25HC3S) by cholesterol sulfotransferase-2B1b moderately reduced the antiviral actions of 25HC because 25HC3S is a weaker inhibitor of HSV-1 infection than 25HC. Furthermore, administration of T317 to BALB/c mice reduced HSV-1 growth in mouse tissues. Taken together, we demonstrate an antiviral system of 25HC with involvement of LXR activation, interaction between CH25H and IFN-γ, and 25HC metabolism.},
}
@article {pmid30307989,
year = {2018},
author = {Ng, KP and Manjeri, A and Lee, LM and Chan, ZE and Tan, CY and Tan, QD and Majeed, A and Lee, KL and Chuah, C and Suda, T and Ong, ST},
title = {The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0205254},
pmid = {30307989},
issn = {1932-6203},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/*drug effects ; Arginase/*antagonists &amp; inhibitors/genetics/metabolism ; Arginine/*analogs &amp; derivatives/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Drug Screening Assays, Antitumor ; Enzyme Assays ; Feasibility Studies ; Gene Knockout Techniques ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/pathology ; Primary Cell Culture ; RNA, Small Interfering/metabolism ; },
abstract = {Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1-α and HIF2-α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor-NOHA overcame hypoxia-mediated resistance towards BCR-ABL1 kinase inhibitors. While nor-NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor-NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor-NOHA on cellular respiration. In conclusion, we show that nor-NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor-NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor-NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.},
}
@article {pmid30307929,
year = {2018},
author = {Sheridan, C},
title = {Sangamo's landmark genome editing trial gets mixed reception.},
journal = {Nature biotechnology},
volume = {36},
number = {10},
pages = {907-908},
pmid = {30307929},
issn = {1546-1696},
mesh = {Biotechnology ; CRISPR-Cas Systems ; Clinical Trials as Topic ; *Gene Editing ; Humans ; Mucopolysaccharidosis II/enzymology/*genetics/*therapy ; Zinc Finger Nucleases/genetics ; },
}
@article {pmid30307765,
year = {2019},
author = {Zhang, G and Isaji, T and Xu, Z and Lu, X and Fukuda, T and Gu, J},
title = {N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {2},
pages = {2823-2835},
doi = {10.1096/fj.201801478R},
pmid = {30307765},
issn = {1530-6860},
mesh = {CRISPR-Cas Systems ; *Cell Adhesion ; *Cell Movement ; *Cell Proliferation ; *Epithelial-Mesenchymal Transition ; Glycosylation ; HeLa Cells ; Humans ; N-Acetylglucosaminyltransferases/antagonists &amp; inhibitors/genetics/*metabolism ; Phosphorylation ; Signal Transduction ; },
abstract = {N-Glycans are involved in numerous biologic processes, such as cell adhesion, migration, and invasion. To distinguish the functions of complex high-mannose types of N-glycans, we used the clustered, regularly interspaced, short palindromic repeats/Cas9 system to establish N-acetylglucosaminyltransferase (GnT)-I-knockout (KO) cells. Loss of GnT-I greatly induced cell-cell adhesion and decreased cell migration. In addition, the expression levels of epithelial-mesenchymal transition (EMT) markers such as α-SMA, vimentin, and N-cadherin were suppressed, whereas the expression of claudin-1 was promoted, suggesting a mesenchymal-epithelial transition-like phenotype, an opposite process to the EMT, was occurred in the KO cells. The phosphorylation levels of Smad-2, epidermal growth factor receptor, and integrin-mediated focal adhesion kinase (FAK) were consistently suppressed. Furthermore, the restoration of GnT-I in the KO cells suppressed the cell-cell adhesion and augmented the expression of EMT markers as well as that of FAK activation. The expression levels of integrins were upregulated in the KO cells, although their functions were decreased, whereas their expression levels were downregulated in the rescued cells, which suggests a negative feedback loop between function and expression. Finally, we also found that the expression of GnT-I was important for cell survival, resistance to cancer drugs, and increased colony formation. The results of the present study demonstrate that GnT-I works as a switch to turn on/off EMT, which further supports the notion that on most surface receptors, the N-glycans differentially play essential roles in biologic functions.-Zhang, G., Isaji, T., Xu, Z., Lu, X., Fukuda, T., Gu, J. N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.},
}
@article {pmid30307510,
year = {2019},
author = {Yang, N and Wu, N and Zhang, L and Zhao, Y and Liu, J and Liang, X and Ren, X and Li, W and Chen, W and Dong, S and Zhao, S and Lin, J and Xiang, H and Xue, H and Chen, L and Sun, H and Zhang, J and Shi, J and Zhang, S and Lu, D and Wu, X and Jin, L and Ding, J and Qiu, G and Wu, Z and Lupski, JR and Zhang, F},
title = {TBX6 compound inheritance leads to congenital vertebral malformations in humans and mice.},
journal = {Human molecular genetics},
volume = {28},
number = {4},
pages = {539-547},
pmid = {30307510},
issn = {1460-2083},
support = {UM1 HG006542/HG/NHGRI NIH HHS/United States ; },
mesh = {Adolescent ; Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Child ; Child, Preschool ; Congenital Abnormalities/diagnostic imaging/*genetics/physiopathology ; Disease Models, Animal ; Female ; Haploinsufficiency ; Humans ; Infant ; Male ; Mice ; Mutation ; Phenotype ; Scoliosis/diagnostic imaging/*genetics/physiopathology ; Spine/*abnormalities/diagnostic imaging/physiopathology ; T-Box Domain Proteins/*genetics ; },
abstract = {Congenital vertebral malformations (CVMs) are associated with human TBX6 compound inheritance that combines a rare null allele and a common hypomorphic allele at the TBX6 locus. Our previous in vitro evidence suggested that this compound inheritance resulted in a TBX6 gene dosage of less than haploinsufficiency (i.e. <50%) as a potential mechanism of TBX6-associated CVMs. To further investigate this pathogenetic model, we ascertained and collected 108 Chinese CVM cases and found that 10 (9.3%) of them carried TBX6 null mutations in combination with common hypomorphic variants at the second TBX6 allele. For in vivo functional verification and genetic analysis of TBX6 compound inheritance, we generated both null and hypomorphic mutations in mouse Tbx6 using the CRISPR-Cas9 method. These Tbx6 mutants are not identical to the patient variants at the DNA sequence level, but instead functionally mimic disease-associated TBX6 variants. Intriguingly, as anticipated by the compound inheritance model, a high penetrance of CVM phenotype was only observed in the mice with combined null and hypomorphic alleles of Tbx6. These findings are consistent with our experimental observations in humans and supported the dosage effect of TBX6 in CVM etiology. In conclusion, our findings in the newly collected human CVM subjects and Tbx6 mouse models consistently support the contention that TBX6 compound inheritance causes CVMs, potentially via a gene dosage-dependent mechanism. Furthermore, mouse Tbx6 mutants mimicking human CVM-associated variants will be useful models for further mechanistic investigations of CVM pathogenesis in the cases associated with TBX6.},
}
@article {pmid30306365,
year = {2018},
author = {Givens, BE and Naguib, YW and Geary, SM and Devor, EJ and Salem, AK},
title = {Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Therapeutics.},
journal = {The AAPS journal},
volume = {20},
number = {6},
pages = {108},
pmid = {30306365},
issn = {1550-7416},
support = {P30 CA086862/CA/NCI NIH HHS/United States ; P30 ES005605/ES/NIEHS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/*methods ; *Gene Transfer Techniques ; Genetic Therapy/*methods ; Humans ; Nanoparticles/*chemistry ; },
abstract = {The recent progress in harnessing the efficient and precise method of DNA editing provided by CRISPR/Cas9 is one of the most promising major advances in the field of gene therapy. However, the development of safe and optimally efficient delivery systems for CRISPR/Cas9 elements capable of achieving specific targeting of gene therapy to the location of interest without off-target effects is a primary challenge for clinical therapeutics. Nanoparticles (NPs) provide a promising means to meet such challenges. In this review, we present the most recent advances in developing innovative NP-based delivery systems that efficiently deliver CRISPR/Cas9 constructs and maximize their effectiveness.},
}
@article {pmid30305306,
year = {2018},
author = {Wang, X and Huang, R and Zhang, L and Li, S and Luo, J and Gu, Y and Chen, Z and Zheng, Q and Chao, T and Zheng, W and Qi, X and Wang, L and Wen, Y and Liang, Y and Lu, L},
title = {A severe atherosclerosis mouse model on the resistant NOD background.},
journal = {Disease models &amp; mechanisms},
volume = {11},
number = {10},
pages = {},
pmid = {30305306},
issn = {1754-8411},
mesh = {Animals ; Apolipoproteins E/genetics ; Atherosclerosis/*pathology ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Female ; Gene Deletion ; Hyperlipidemias/pathology ; Male ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Knockout ; Receptors, LDL/genetics ; },
abstract = {Atherosclerosis is a complex disease affecting arterial blood vessels and blood flow that could result in a variety of life-threatening consequences. Disease models with diverged genomes are necessary for understanding the genetic architecture of this complex disease. Non-obese diabetic (NOD) mice are highly polymorphic and widely used for studies of type 1 diabetes and autoimmunity. Understanding atherosclerosis development in the NOD strain is of particular interest as human atherosclerosis on the diabetic and autoimmune background has not been successfully modeled. In this study, we used CRISPR/Cas9 genome editing to genetically disrupt apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) expression on the pure NOD background, and compared phenotype between single-gene-deleted mice and double-knockout mutants with reference to ApoE-deficient C57BL/6 mice. We found that genetic ablation of Ldlr or Apoe in NOD mice was not sufficient to establish an atherosclerosis model, in contrast to ApoE-deficient C57BL/6 mice fed a high-fat diet (HFD) for over 12 weeks. We further obtained NOD mice deficient in both LDLR and ApoE, and assessed the severity of atherosclerosis and immune response to hyperlipidemia in comparison to ApoE-deficient C57BL/6 mice. Strikingly, the double-knockout NOD mice treated with a HFD developed severe atherosclerosis with aorta narrowed by over 60% by plaques, accompanied by destruction of pancreatic islets and an inflammatory response to hyperlipidemia. Therefore, we succeeded in obtaining a genetic model with severe atherosclerosis on the NOD background, which is highly resistant to the disease. This model is useful for the study of atherosclerosis in the setting of autoimmunity.},
}
@article {pmid30305304,
year = {2018},
author = {Fang, B and Ren, X and Wang, Y and Li, Z and Zhao, L and Zhang, M and Li, C and Zhang, Z and Chen, L and Li, X and Liu, J and Xiong, Q and Zhang, L and Jin, Y and Liu, X and Li, L and Wei, H and Yang, H and Li, R and Dai, Y},
title = {Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs.},
journal = {Disease models &amp; mechanisms},
volume = {11},
number = {10},
pages = {},
pmid = {30305304},
issn = {1754-8411},
mesh = {Animals ; Animals, Genetically Modified ; Apolipoproteins E/*deficiency/genetics ; Atherosclerosis/blood/*pathology ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cholesterol/blood ; Diet, High-Fat ; Feeding Behavior ; Fetus/cytology ; Fibroblasts/metabolism ; Hyperlipoproteinemia Type II/blood/pathology ; INDEL Mutation/genetics ; Nuclear Transfer Techniques ; Phenotype ; RNA, Guide/metabolism ; Swine ; Swine, Miniature ; Triglycerides/blood ; },
abstract = {Miniature pigs have advantages over rodents in modeling atherosclerosis because their cardiovascular system and physiology are similar to that of humans. Apolipoprotein E (ApoE) deficiency has long been implicated in cardiovascular disease in humans. To establish an improved large animal model of familial hypercholesterolemia and atherosclerosis, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 system (CRISPR/Cas9) was used to disrupt the ApoE gene in Bama miniature pigs. Biallelic-modified ApoE pigs with in-frame mutations (ApoE[m/m]) and frameshift mutations (ApoE[-/-]) were simultaneously produced. ApoE[-/-] pigs exhibited moderately increased plasma cholesterol levels when fed with a regular chow diet, but displayed severe hypercholesterolemia and spontaneously developed human-like atherosclerotic lesions in the aorta and coronary arteries after feeding on a high-fat and high-cholesterol (HFHC) diet for 6 months. Thus, these ApoE[-/-] pigs could be valuable large animal models for providing further insight into translational studies of atherosclerosis.},
}
@article {pmid30302894,
year = {2019},
author = {Varkonyi-Gasic, E and Wang, T and Voogd, C and Jeon, S and Drummond, RSM and Gleave, AP and Allan, AC},
title = {Mutagenesis of kiwifruit CENTRORADIALIS-like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering.},
journal = {Plant biotechnology journal},
volume = {17},
number = {5},
pages = {869-880},
pmid = {30302894},
issn = {1467-7652},
mesh = {Actinidia/anatomy &amp; histology/*genetics/growth &amp; development ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Ectopic Gene Expression/genetics ; Flowers/anatomy &amp; histology/*genetics/growth &amp; development ; Gene Editing ; Genes, Plant/genetics/physiology ; Plant Proteins/genetics/physiology ; },
abstract = {Annualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. Previously, multiple kiwifruit CENTRORADIALIS (CEN)-like genes have been identified as potential repressors of flowering. In this study, CRISPR/Cas9- mediated manipulation enabled functional analysis of kiwifruit CEN-like genes AcCEN4 and AcCEN. Mutation of these genes transformed a climbing woody perennial, which develops axillary inflorescences after many years of juvenility, into a compact plant with rapid terminal flower and fruit development. The number of affected genes and alleles and severity of detected mutations correlated with the precocity and change in plant stature, suggesting that a bi-allelic mutation of either AcCEN4 or AcCEN may be sufficient for early flowering, whereas mutations affecting both genes further contributed to precocity and enhanced the compact growth habit. CRISPR/Cas9-mediated mutagenesis of AcCEN4 and AcCEN may be a valuable means to engineer Actinidia amenable for accelerated breeding, indoor farming and cultivation as an annual crop.},
}
@article {pmid30301924,
year = {2018},
author = {Leidy-Davis, T and Cheng, K and Goodwin, LO and Morgan, JL and Juan, WC and Roca, X and Ong, ST and Bergstrom, DE},
title = {Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {15028},
pmid = {30301924},
issn = {2045-2322},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Bcl-2-Like Protein 11/*genetics ; Blastocyst/cytology/*metabolism ; CRISPR-Cas Systems ; Embryonic Stem Cells/cytology/*metabolism ; Gene Editing ; Humans ; Introns/genetics ; Mice ; Microinjections ; RNA, Guide/genetics ; Zygote/growth &amp; development/*metabolism ; },
abstract = {Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3' of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment.},
}
@article {pmid30301855,
year = {2018},
author = {Bando, H and Lee, Y and Sakaguchi, N and Pradipta, A and Ma, JS and Tanaka, S and Cai, Y and Liu, J and Shen, J and Nishikawa, Y and Sasai, M and Yamamoto, M},
title = {Inducible Nitric Oxide Synthase Is a Key Host Factor for Toxoplasma GRA15-Dependent Disruption of the Gamma Interferon-Induced Antiparasitic Human Response.},
journal = {mBio},
volume = {9},
number = {5},
pages = {},
pmid = {30301855},
issn = {2150-7511},
mesh = {Animals ; Antigens, Protozoan/immunology ; CRISPR-Cas Systems ; Cell Line ; Coculture Techniques ; Hepatocytes/drug effects/immunology/parasitology ; Host-Parasite Interactions/immunology ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism ; Interferon-gamma/*pharmacology ; Interleukin-1beta/metabolism ; Mice ; Mice, Inbred C57BL ; Monocytes/immunology/parasitology ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Nitric Oxide Synthase Type II/*immunology ; Protozoan Proteins/*immunology ; Toxoplasma/*immunology/pathogenicity ; Virulence Factors/*immunology ; },
abstract = {Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host's NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor.IMPORTANCEToxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.},
}
@article {pmid30301773,
year = {2018},
author = {Ma, F and Qi, H and Hu, YF and Jiang, QR and Zhang, LG and Xue, P and Yang, FQ and Wang, R and Ju, Y and Uchida, H and Zhang, Q and Sodmergen, },
title = {The Mitochondrial Endonuclease M20 Participates in the Down-Regulation of Mitochondrial DNA in Pollen Cells.},
journal = {Plant physiology},
volume = {178},
number = {4},
pages = {1537-1550},
pmid = {30301773},
issn = {1532-2548},
mesh = {Amino Acid Motifs ; Arabidopsis/genetics/growth &amp; development ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; DNA, Mitochondrial/*metabolism ; Deoxyribonucleases/genetics/isolation &amp; purification/*metabolism ; Down-Regulation ; Endonucleases/genetics/*metabolism ; Gene Expression Regulation, Plant ; Mitochondrial Proteins/genetics/isolation &amp; purification/metabolism ; Plants, Genetically Modified ; Pollen/cytology/*genetics/metabolism ; Sequence Homology, Amino Acid ; Zea mays/*genetics/metabolism ; },
abstract = {Maintaining the appropriate number of mitochondrial DNA (mtDNA) molecules is crucial for supporting mitochondrial metabolism and function in both plant and animal cells. For example, a substantial decrease in mtDNA levels occurs as a key part of pollen development. The molecular mechanisms regulating mtDNA copy number are largely unclear, particularly with regard to those that reduce mtDNA levels. Here, we identified and purified a 20-kD endonuclease, M20, from maize (Zea mays) pollen mitochondria. We found M20 to be an His-Asn-His/Asn (H-N-H/N) nuclease that degrades linear and circular DNA in the presence of Mg[2+] or Mn[2+] Arabidopsis (Arabidopsis thaliana) AtM20, which shared high sequence similarity with maize M20, localized to the mitochondria, had a similar H-N-H/N structure, and degraded both linear and circular DNA. AtM20 transcript levels increased during pollen development, in parallel with a rapid reduction in mtDNA. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing techniques were used to generate knockout lines of AtM20 (atm20), which exhibited a significant delay in the reduction in mtDNA levels in pollen vegetative cells but normal mtDNA levels in somatic cells. The delayed reduction in pollen mtDNA levels was rescued by the transgenic expression of AtM20 in atm20 plants. This study thus uncovers an endonucleolytic DNase in plant mitochondria and its crucial role in reducing mtDNA levels, pointing to the complex mechanism regulating mtDNA levels in plants.},
}
@article {pmid30298816,
year = {2018},
author = {Libby, AR and Joy, DA and So, PL and Mandegar, MA and Muncie, JM and Mendoza-Camacho, FN and Weaver, VM and Conklin, BR and McDevitt, TC},
title = {Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30298816},
issn = {2050-084X},
support = {LAI-C1408015//California Institute for Regenerative Medicine/International ; P01HL089707/NH/NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; Instrumentation Grant, NIH S10 RR028962/NH/NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; U01HL100406/NH/NIH HHS/United States ; T32 HD007470/HD/NICHD NIH HHS/United States ; R01HL130533/NH/NIH HHS/United States ; F31 HL140907/HL/NHLBI NIH HHS/United States ; P01 HL089707/HL/NHLBI NIH HHS/United States ; Center Core Grant, NIH P30 AI027763/NH/NIH HHS/United States ; U01 HL100406/HL/NHLBI NIH HHS/United States ; NSF-CBET 0939511//Emergent Behaviors of Integrated Cellular Systems/International ; R01 EY028249/EY/NEI NIH HHS/United States ; S10 RR028962/RR/NCRR NIH HHS/United States ; F31HL140907/HL/NHLBI NIH HHS/United States ; P30 AI027763/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD/*genetics ; CRISPR-Cas Systems/genetics ; Cadherins/*genetics ; Cell Communication/genetics ; Cell Differentiation/*genetics ; Cell Lineage/genetics ; Gene Knockdown Techniques ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Morphogenesis/genetics ; rho-Associated Kinases/*genetics ; },
abstract = {Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.},
}
@article {pmid30297964,
year = {2018},
author = {Ting, PY and Parker, AE and Lee, JS and Trussell, C and Sharif, O and Luna, F and Federe, G and Barnes, SW and Walker, JR and Vance, J and Gao, MY and Klock, HE and Clarkson, S and Russ, C and Miraglia, LJ and Cooke, MP and Boitano, AE and McNamara, P and Lamb, J and Schmedt, C and Snead, JL},
title = {Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.},
journal = {Nature methods},
volume = {15},
number = {11},
pages = {941-946},
doi = {10.1038/s41592-018-0149-1},
pmid = {30297964},
issn = {1548-7105},
mesh = {CD8-Positive T-Lymphocytes/cytology/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Gene Editing ; *Genome, Human ; HEK293 Cells ; Hematopoietic Stem Cells/cytology/*metabolism ; Humans ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4[+] T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34[+] hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.},
}
@article {pmid30297904,
year = {2018},
author = {Villiger, L and Grisch-Chan, HM and Lindsay, H and Ringnalda, F and Pogliano, CB and Allegri, G and Fingerhut, R and Häberle, J and Matos, J and Robinson, MD and Thöny, B and Schwank, G},
title = {Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.},
journal = {Nature medicine},
volume = {24},
number = {10},
pages = {1519-1525},
pmid = {30297904},
issn = {1546-170X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA/genetics/therapeutic use ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing ; Genetic Therapy/methods ; Humans ; Liver/metabolism/pathology ; Liver Diseases/genetics/metabolism/pathology/*therapy ; Mice ; Phenylalanine/blood ; Phenylalanine Hydroxylase/*genetics/therapeutic use ; Phenylketonurias/genetics/metabolism/pathology/*therapy ; Recombinational DNA Repair/genetics ; },
abstract = {CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)[enu2] mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)[1], using recently developed CRISPR-Cas-associated base editors[2-4]. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pah[enu2] gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pah[enu2] mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.},
}
@article {pmid30297903,
year = {2018},
author = {Rossidis, AC and Stratigis, JD and Chadwick, AC and Hartman, HA and Ahn, NJ and Li, H and Singh, K and Coons, BE and Li, L and Lv, W and Zoltick, PW and Alapati, D and Zacharias, W and Jain, R and Morrisey, EE and Musunuru, K and Peranteau, WH},
title = {In utero CRISPR-mediated therapeutic editing of metabolic genes.},
journal = {Nature medicine},
volume = {24},
number = {10},
pages = {1513-1518},
pmid = {30297903},
issn = {1546-170X},
support = {R01 HL118744/HL/NHLBI NIH HHS/United States ; R01 HL126875/HL/NHLBI NIH HHS/United States ; T32 HL007843/HL/NHLBI NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Editing ; *Genetic Therapy ; Genetic Vectors/genetics/therapeutic use ; Humans ; Oxidoreductases/*genetics/therapeutic use ; Proprotein Convertase 9/*genetics/therapeutic use ; Tyrosinemias/genetics/pathology/*therapy ; },
abstract = {In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.},
}
@article {pmid30297902,
year = {2018},
author = {Seo, H and Kim, JS},
title = {Towards therapeutic base editing.},
journal = {Nature medicine},
volume = {24},
number = {10},
pages = {1493-1495},
pmid = {30297902},
issn = {1546-170X},
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; Liver Diseases/*genetics ; Mice ; },
}
@article {pmid30297850,
year = {2018},
author = {Serebrenik, YV and Shalem, O},
title = {CRISPR mutagenesis screening of mice.},
journal = {Nature cell biology},
volume = {20},
number = {11},
pages = {1235-1237},
doi = {10.1038/s41556-018-0224-y},
pmid = {30297850},
issn = {1476-4679},
mesh = {Amino Acids ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Germ Cells ; Mice ; Mutagenesis ; Neoplasm Proteins ; },
}
@article {pmid30297533,
year = {2018},
author = {Ali, R and Al-Kawaz, A and Toss, MS and Green, AR and Miligy, IM and Mesquita, KA and Seedhouse, C and Mirza, S and Band, V and Rakha, EA and Madhusudan, S},
title = {Targeting PARP1 in XRCC1-Deficient Sporadic Invasive Breast Cancer or Preinvasive Ductal Carcinoma In Situ Induces Synthetic Lethality and Chemoprevention.},
journal = {Cancer research},
volume = {78},
number = {24},
pages = {6818-6827},
doi = {10.1158/0008-5472.CAN-18-0633},
pmid = {30297533},
issn = {1538-7445},
mesh = {Apoptosis ; Breast Neoplasms/*metabolism ; CRISPR-Cas Systems ; Carcinoma, Intraductal, Noninfiltrating/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Chemoprevention ; DNA Breaks, Double-Stranded ; DNA Repair/drug effects ; Female ; Germ-Line Mutation ; HeLa Cells ; Humans ; Indazoles/pharmacology ; Neoplasm Invasiveness ; Neoplasm Recurrence, Local ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Piperidines/pharmacology ; Poly (ADP-Ribose) Polymerase-1/*metabolism ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Spheroids, Cellular ; Synthetic Lethal Mutations ; X-ray Repair Cross Complementing Protein 1/*metabolism ; },
abstract = {: Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS. SIGNIFICANCE: These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells.},
}
@article {pmid30296255,
year = {2018},
author = {Baskoylu, SN and Yersak, J and O'Hern, P and Grosser, S and Simon, J and Kim, S and Schuch, K and Dimitriadi, M and Yanagi, KS and Lins, J and Hart, AC},
title = {Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration.},
journal = {PLoS genetics},
volume = {14},
number = {10},
pages = {e1007682},
pmid = {30296255},
issn = {1553-7404},
support = {T32 MH020068/MH/NIMH NIH HHS/United States ; T32 NS062443/NS/NINDS NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Amyotrophic Lateral Sclerosis/*genetics/pathology ; Animals ; Animals, Genetically Modified ; Base Sequence ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/*genetics ; Cholinergic Neurons/metabolism/*pathology ; Disease Models, Animal ; Gain of Function Mutation ; Gene Frequency ; Gene Knock-In Techniques ; Glutamic Acid/metabolism ; Humans ; Loss of Function Mutation ; Motor Neurons/metabolism/*pathology ; Superoxide Dismutase/*genetics ; Superoxide Dismutase-1/*genetics ; },
abstract = {Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.},
}
@article {pmid30296034,
year = {2018},
author = {Tomlinson, T},
title = {A Crispr Future for Gene-Editing Regulation: a Proposal for an Updated Biotechnology Regulatory System in an Era of Human Genomic Editing.},
journal = {Fordham law review},
volume = {87},
number = {1},
pages = {437-483},
pmid = {30296034},
issn = {0015-704X},
mesh = {Biotechnology/*ethics/legislation &amp; jurisprudence/*standards ; *CRISPR-Cas Systems ; Embryo Research/*ethics/history/legislation &amp; jurisprudence ; Gene Editing/*ethics/legislation &amp; jurisprudence/*standards ; *Government Regulation/history ; History, 20th Century ; Humans ; Stem Cell Research/ethics/history/legislation &amp; jurisprudence ; United States ; },
abstract = {Recent developments in gene-editing technology have enabled scientists to manipulate the human genome in unprecedented ways. One technology in particular, Clustered Regularly Interspaced Short Pallindromic Repeat (CRISPR), has made gene editing more precise and cost-effective than ever before. Indeed, scientists have already shown that CRISPR can eliminate genes linked to life-threatening diseases from an individual's genetic makeup and, when used on human embryos, CRISPR has the potential to permanently eliminate hereditary diseases from the human genome in its entirety. These developments have brought great hope to individuals and their families, who suffer from genetically linked diseases. But there is a dark side: in the wrong hands, CRISPR could negatively impact the course of human evolution or be used to create biological weaponry. Despite these possible consequences, CRISPR remains largely unregulated due to the United States's outdated regulatory scheme for biotechnology. Moreover, human embryo research, which is likely critical to maximizing the therapeutic applications of CRISPR, is not easily undertaken by scientists due to a number of federal and state restrictions aimed at preventing such research. This Note examines the possible benefits and consequences of CRISPR and discusses the current regulations in both the fields of biotechnology and human embryo research that hamper the government's ability to effectively regulate this technology. Ultimately, this Note proposes a new regulatory scheme for biotechnology that focuses on the processes used to create products using CRISPR, rather than the products themselves, with a focus on enabling ethical research using human embryos to maximize the potential benefits of CRISPR.},
}
@article {pmid30295654,
year = {2018},
author = {Du, L and Zhou, A and Sohr, A and Roy, S},
title = {An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {139},
pages = {},
pmid = {30295654},
issn = {1940-087X},
support = {R00 HL114867/HL/NHLBI NIH HHS/United States ; R35 GM124878/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Drosophila/*genetics/metabolism ; Gene Editing/*methods ; Gene Expression Regulation ; RNA, Guide/genetics ; },
abstract = {Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a "Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated" (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.},
}
@article {pmid30295650,
year = {2018},
author = {Kudyba, HM and Cobb, DW and Florentin, A and Krakowiak, M and Muralidharan, V},
title = {CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {139},
pages = {},
pmid = {30295650},
issn = {1940-087X},
support = {R00 AI099156/AI/NIAID NIH HHS/United States ; R01 AI130139/AI/NIAID NIH HHS/United States ; T32 AI060546/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Malaria, Falciparum/*genetics ; Parasites/*genetics ; },
abstract = {Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.},
}
@article {pmid30295162,
year = {2019},
author = {Ali, Q},
title = {Non-conventional therapeutic technique to replace CRISPR bacteria from biofilm by inducible lysogen.},
journal = {Journal of biological dynamics},
volume = {13},
number = {sup1},
pages = {151-178},
doi = {10.1080/17513758.2018.1527958},
pmid = {30295162},
issn = {1751-3766},
mesh = {Algorithms ; Bacteria/*genetics ; *Biofilms/growth &amp; development ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Lysogeny/*genetics ; Models, Biological ; },
abstract = {Bacteriophage can be an effective means of regulating bacterial populations when conditions allow phage invasion of bacterial colonies. Phage can either infect and lyse a host cell, or insert their DNA into the host cell genome; the latter process is called lysogeny. The clustered regularly interspaced short palindromic repeat (CRISPR) system, linked with CRISPR-associated (Cas) genes, is a regulatory system present in a variety of bacteria which confers immunity against bacteriophage. Studies of the group behaviour of bacteria with CRISPR/Cas systems have provided evidence that CRISPR in lysogenized bacteria can cause an inability to form biofilm. This allows CRISPR-immune bacteria in biofilms to effectively resist phage therapy. Our recent work has described a potential therapeutic technique to eradicate CRISPR-immune bacteria from a biofilm by a continuous influx of lysogens carrying an identical phage sequence. However, this model predicted that the CRISPR-immune population could persist for long times before eradication. Our current focus is on the use of diverse lysogens against CRISPR-capable bacterial populations. The goal of this work is to find a suitable strategy which can eradicate bacteria with a CRISPR system through the influx of finite amounts of distinct lysogens over fixed intervals.},
}
@article {pmid30293118,
year = {2018},
author = {Yang, X and Huang, B and Deng, L and Hu, Z},
title = {Progress in gene therapy using oncolytic vaccinia virus as vectors.},
journal = {Journal of cancer research and clinical oncology},
volume = {144},
number = {12},
pages = {2433-2440},
pmid = {30293118},
issn = {1432-1335},
mesh = {Animals ; CRISPR-Cas Systems ; Cancer Vaccines/genetics/immunology ; Gene Targeting ; *Genetic Therapy/methods ; Genetic Vectors/*genetics ; Humans ; Immunotherapy ; Neoplasms/genetics/immunology/therapy ; *Oncolytic Virotherapy/methods ; Oncolytic Viruses/*genetics ; Vaccinia virus/*genetics ; },
abstract = {BACKGROUND: Vaccinia virus was widely used in the World Health Organization's smallpox eradication campaign and is currently a promising vector for gene therapy owing to its unique characteristics. Vaccinia virus can selectively replicate and propagate productively in tumor cells, resulting in oncolysis. In addition, rapid viral particle production, wide host range, large genome size (approximately 200 kb), and safe handling render vaccinia virus a suitable vector for gene therapy.<br><br>MATERIALS AND METHODS: Cancer vaccines and gene therapy are being studied in clinical trials and experiment researches. However, we put forward unique challenges of optimal selection of foreign genes, administration and modification of VACV, personalized medicine, and other existing problems, based on current researches and our own experiments.<br><br>CONCLUSION: This review presents an overview of the vaccinia virus from its mechanisms to medical researches and clinical trials. We believe that the solution to these problems will contribute to understanding mechanisms of VACV and provide a theoretical basis for clinical treatment.},
}
@article {pmid30292351,
year = {2018},
author = {Ryan, CJ and Bajrami, I and Lord, CJ},
title = {Synthetic Lethality and Cancer - Penetrance as the Major Barrier.},
journal = {Trends in cancer},
volume = {4},
number = {10},
pages = {671-683},
doi = {10.1016/j.trecan.2018.08.003},
pmid = {30292351},
issn = {2405-8025},
support = {//Cancer Research UK/United Kingdom ; 103049/Z/13/Z//Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Computational Biology ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; Molecular Targeted Therapy/methods ; Neoplasms/genetics/*therapy ; Oncogenes/*genetics ; *Penetrance ; RNA Interference ; Synthetic Lethal Mutations/*genetics ; },
abstract = {Synthetic lethality has long been proposed as an approach for targeting genetic defects in tumours. Despite a decade of screening efforts, relatively few robust synthetic lethal targets have been identified. Improved genetic perturbation techniques, including CRISPR/Cas9 gene editing, have resulted in renewed enthusiasm for searching for synthetic lethal effects in cancer. An implicit assumption behind this enthusiasm is that the lack of reproducibly identified targets can be attributed to limitations of RNAi technologies. We argue here that a bigger hurdle is that most synthetic lethal interactions (SLIs) are not highly penetrant, in other words they are not robust to the extensive molecular heterogeneity seen in tumours. We outline strategies for identifying and prioritising SLIs that are most likely to be highly penetrant.},
}
@article {pmid30291759,
year = {2019},
author = {Li, J and Manghwar, H and Sun, L and Wang, P and Wang, G and Sheng, H and Zhang, J and Liu, H and Qin, L and Rui, H and Li, B and Lindsey, K and Daniell, H and Jin, S and Zhang, X},
title = {Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants.},
journal = {Plant biotechnology journal},
volume = {17},
number = {5},
pages = {858-868},
pmid = {30291759},
issn = {1467-7652},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Genetic Variation/genetics ; Genome, Plant/genetics ; Genome-Wide Association Study ; Gossypium/*genetics ; Mutation/genetics ; Plants, Genetically Modified/genetics ; *Whole Genome Sequencing ; },
abstract = {The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9-edited plants. To identify on- and off-target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9-edited cotton plants targeted to three genes, and three negative (Ne) control and three wild-type (WT) plants. In total, 4188-6404 unique single-nucleotide polymorphisms (SNPs) and 312-745 insertions/deletions (indels) were detected in 14 Cas9-edited plants compared to WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer-adjacent motif (PAM), we demonstrated that the most variations following Cas9-edited are due either to somaclonal variation or/and pre-existing/inherent variation from maternal plants, but not off-target effects. Of a total of 4413 potential off-target sites (allowing ≤5 mismatches within the 20-bp sgRNA and 3-bp PAM sequences), the WGS data revealed that only four are bona fide off-target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off-target sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off-target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.},
}
@article {pmid30291498,
year = {2019},
author = {Yoo, KH and Hennighausen, L and Shin, HY},
title = {Dissecting Tissue-Specific Super-Enhancers by Integrating Genome-Wide Analyses and CRISPR/Cas9 Genome Editing.},
journal = {Journal of mammary gland biology and neoplasia},
volume = {24},
number = {1},
pages = {47-59},
pmid = {30291498},
issn = {1573-7039},
mesh = {Animals ; Breast Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics/pathology ; *Enhancer Elements, Genetic ; Female ; Gene Editing/*methods/trends ; *Gene Expression Regulation, Developmental ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Humans ; Mammary Glands, Animal/*growth &amp; development/pathology ; Mammary Glands, Human/*growth &amp; development/pathology ; Mice ; Mutagenesis ; RNA, Guide/genetics ; Whole Genome Sequencing/*methods/trends ; },
abstract = {Recent advances in genome-wide sequencing technologies have provided researchers with unprecedented opportunities to discover the genomic structures of gene regulatory units in living organisms. In particular, the integration of ChIP-seq, RNA-seq, and DNase-seq techniques has facilitated the mapping of a new class of regulatory elements. These elements, called super-enhancers, can regulate cell-type-specific gene sets and even fine-tune gene expression regulation in response to external stimuli, and have become a hot topic in genome biology. However, there is scant genetic evidence demonstrating their unique biological relevance and the mechanisms underlying these biological functions. In this review, we describe a robust genome-wide strategy for mapping cell-type-specific enhancers or super-enhancers in the mammary genome. In this strategy, genome-wide screening of active enhancer clusters that are co-occupied by mammary-enriched transcription factors, co-factors, and active enhancer marks is used to identify bona fide mammary tissue-specific super-enhancers. The in vivo function of these super-enhancers and their associated regulatory elements may then be investigated in various ways using the advanced CRISPR/Cas9 genome-editing technology. Based on our experience targeting various mammary genomic sites using CRISPR/Cas9 in mice, we comprehensively discuss the molecular consequences of the different targeting methods, such as the number of gRNAs and the dependence on their simultaneous or sequential injections. We also mention the considerations that are essential for obtaining accurate results and shed light on recent progress that has been made in developing modified CRISPR/Cas9 genome-editing techniques. In the future, the coupling of advanced genome-wide sequencing and genome-editing technologies could provide new insights into the complex genetic regulatory networks involved in mammary-gland development.},
}
@article {pmid30291334,
year = {2019},
author = {Humbert, O and Laszlo, GS and Sichel, S and Ironside, C and Haworth, KG and Bates, OM and Beddoe, ME and Carrillo, RR and Kiem, HP and Walter, RB},
title = {Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2.},
journal = {Leukemia},
volume = {33},
number = {3},
pages = {762-808},
pmid = {30291334},
issn = {1476-5551},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Exons/*genetics ; Hematopoiesis/*genetics ; Humans ; Immunotherapy/methods ; Leukemia, Myeloid, Acute/*genetics/immunology/therapy ; Sequence Deletion/*genetics ; Sialic Acid Binding Ig-like Lectin 3/*genetics ; },
}
@article {pmid30291308,
year = {2018},
author = {Chanock, SJ},
title = {Gene editing reveals the effect of thousands of variants in a key cancer gene.},
journal = {Nature},
volume = {562},
number = {7726},
pages = {201-202},
doi = {10.1038/d41586-018-06022-y},
pmid = {30291308},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Oncogenes ; },
}
@article {pmid30291237,
year = {2018},
author = {Xu, C and Lu, Z and Luo, Y and Liu, Y and Cao, Z and Shen, S and Li, H and Liu, J and Chen, K and Chen, Z and Yang, X and Gu, Z and Wang, J},
title = {Targeting of NLRP3 inflammasome with gene editing for the amelioration of inflammatory diseases.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4092},
pmid = {30291237},
issn = {2041-1723},
mesh = {Amino Acid Sequence ; Animals ; Apoptosis Regulatory Proteins/metabolism ; CRISPR-Cas Systems ; Calcium-Binding Proteins/metabolism ; Caspase Activation and Recruitment Domain ; Female ; *Gene Editing ; *Genetic Therapy ; Inflammasomes/*genetics ; Inflammation/*therapy ; Male ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/*genetics/metabolism ; Peritonitis/immunology ; Protein Conformation ; },
abstract = {The NLRP3 inflammasome is a well-studied target for the treatment of multiple inflammatory diseases, but how to promote the current therapeutics remains a large challenge. CRISPR/Cas9, as a gene editing tool, allows for direct ablation of NLRP3 at the genomic level. In this study, we screen an optimized cationic lipid-assisted nanoparticle (CLAN) to deliver Cas9 mRNA (mCas9) and guide RNA (gRNA) into macrophages. By using CLAN encapsulating mCas9 and gRNA-targeting NLRP3 (gNLRP3) (CLANmCas9/gNLRP3), we disrupt NLRP3 of macrophages, inhibiting the activation of the NLRP3 inflammasome in response to diverse stimuli. After intravenous injection, CLANmCas9/gNLRP3 mitigates acute inflammation of LPS-induced septic shock and monosodium urate crystal (MSU)-induced peritonitis. In addition, CLANmCas9/gNLRP3 treatment improves insulin sensitivity and reduces adipose inflammation of high-fat-diet (HFD)-induced type 2 diabetes (T2D). Thus, our study provides a promising strategy for treating NLRP3-dependent inflammatory diseases and provides a carrier for delivering CRISPR/Cas9 into macrophages.},
}
@article {pmid30290202,
year = {2019},
author = {Chen, S and Liu, H and Liang, W and Hong, L and Zhang, B and Huang, L and Guo, X and Duan, G},
title = {Insertion sequences in the CRISPR-Cas system regulate horizontal antimicrobial resistance gene transfer in Shigella strains.},
journal = {International journal of antimicrobial agents},
volume = {53},
number = {2},
pages = {109-115},
doi = {10.1016/j.ijantimicag.2018.09.020},
pmid = {30290202},
issn = {1872-7913},
mesh = {Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/*genetics ; Chloramphenicol/pharmacology ; Chloramphenicol Resistance/genetics ; DNA Transposable Elements/genetics ; DNA, Intergenic/*genetics ; Drug Resistance, Multiple, Bacterial/*genetics ; Gene Transfer, Horizontal/*genetics ; Humans ; Microbial Sensitivity Tests ; Shigella/drug effects/*genetics/isolation &amp; purification ; },
abstract = {Multidrug-resistant (MDR) Shigella strains are an enormous threat to public health. Antimicrobial resistance genes are frequently located on plasmids, phages and integrons, which enter bacterial cells by horizontal gene transfer (HGT). CRISPR-Cas systems are adaptive prokaryotic immune systems in bacteria that confer resistance to foreign genetic material such as phages and other mobile genetic elements. However, this may come at a cost of inhibiting the acquisition of other beneficial genes through HGT. This study investigated how Shigella strains regulate the activity of the CRISPR-Cas system spontaneously when they require an exogenous gene necessary for survival. Insertion sequence (IS) elements were identified in cas genes, such as IS600 in cse2, ISSfl2 in cas6e and IS629 in cse1-cas3. The number of spacers in CRISPR-Cas arrays in strains containing an IS was less than that for strains with no IS. Interestingly, fewer spacers were also found in MDR Shigella isolates. Furthermore, an antimicrobial-resistant strain was constructed by electrotransformation of a resistance plasmid in order to detect changes in the CRISPR-Cas system. It was found that the cse2 gene had a new IS (IS600) in the antimicrobial-resistant strain. Bioinformatics analyses showed that the IS600 insertion hotspot was TGC-GGC in the cse2 gene, and the tertiary structure of the Cse2 protein was different with IS600. IS600 caused a five-order of magnitude decrease in relative expression of the cse2 gene. This study sheds mechanistic light on CRISPR-Cas-mediated HGT of antimicrobial resistance genes in Shigella spp. isolates.},
}
@article {pmid30289463,
year = {2018},
author = {Zhang, S and Voigt, CA},
title = {Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design.},
journal = {Nucleic acids research},
volume = {46},
number = {20},
pages = {11115-11125},
pmid = {30289463},
issn = {1362-4962},
mesh = {Amino Acid Motifs ; Binding Sites ; *CRISPR-Cas Systems ; Escherichia coli/*genetics ; *Gene Expression Regulation, Bacterial ; Gene Regulatory Networks ; Metabolic Engineering ; Mutation ; Promoter Regions, Genetic ; RNA, Guide/*metabolism ; Synthetic Biology ; },
abstract = {Large synthetic genetic circuits require the simultaneous expression of many regulators. Deactivated Cas9 (dCas9) can serve as a repressor by having a small guide RNA (sgRNA) direct it to bind a promoter. The programmability and specificity of RNA:DNA basepairing simplifies the generation of many orthogonal sgRNAs that, in theory, could serve as a large set of regulators in a circuit. However, dCas9 is toxic in many bacteria, thus limiting how high it can be expressed, and low concentrations are quickly sequestered by multiple sgRNAs. Here, we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF). Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter. The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9. Further, PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF). A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates; however, the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold. This work introduces a non-toxic variant of dCas9, critical for its use in applications in metabolic engineering and synthetic biology, and exposes a limitation in the number of regulators that can be used in one cell when they rely on a shared resource.},
}
@article {pmid30289378,
year = {2018},
author = {Tashkandi, M and Ali, Z and Aljedaani, F and Shami, A and Mahfouz, MM},
title = {Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.},
journal = {Plant signaling &amp; behavior},
volume = {13},
number = {10},
pages = {e1525996},
pmid = {30289378},
issn = {1559-2324},
mesh = {Begomovirus/*pathogenicity ; CRISPR-Cas Systems/genetics/*physiology ; Lycopersicon esculentum/*virology ; Plant Diseases/genetics/*prevention &amp; control/virology ; },
abstract = {CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.},
}
@article {pmid30287957,
year = {2018},
author = {Lemmon, ZH and Reem, NT and Dalrymple, J and Soyk, S and Swartwood, KE and Rodriguez-Leal, D and Van Eck, J and Lippman, ZB},
title = {Rapid improvement of domestication traits in an orphan crop by genome editing.},
journal = {Nature plants},
volume = {4},
number = {10},
pages = {766-770},
doi = {10.1038/s41477-018-0259-x},
pmid = {30287957},
issn = {2055-0278},
mesh = {Arabidopsis/genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Crop Production/*methods ; *Domestication ; Gene Editing/*methods ; Lycopersicon esculentum/genetics ; Physalis/*genetics/growth &amp; development ; Plants, Genetically Modified ; },
abstract = {Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing breeding in orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. We developed genomic resources and efficient transformation in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) and used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size, thereby improving these major productivity traits. Thus, translating knowledge from model crops enables rapid creation of targeted allelic diversity and novel breeding germplasm in distantly related orphan crops.},
}
@article {pmid30287919,
year = {2018},
author = {Bajor, M and Zych, AO and Graczyk-Jarzynka, A and Muchowicz, A and Firczuk, M and Trzeciak, L and Gaj, P and Domagala, A and Siernicka, M and Zagozdzon, A and Siedlecki, P and Kniotek, M and O'Leary, PC and Golab, J and Zagozdzon, R},
title = {Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents.},
journal = {British journal of cancer},
volume = {119},
number = {7},
pages = {873-884},
pmid = {30287919},
issn = {1532-1827},
mesh = {Animals ; Antioxidants/*administration &amp; dosage/pharmacology ; Ascorbic Acid/administration &amp; dosage/pharmacology ; Breast Neoplasms/genetics/*therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Diterpenes, Kaurane/*administration &amp; dosage/pharmacology ; Female ; Gene Knockdown Techniques/*methods ; Glucose Oxidase/administration &amp; dosage/pharmacology ; Humans ; MCF-7 Cells ; Mice ; Oxidative Stress/drug effects ; Peroxiredoxins/*genetics ; RNA Interference ; Up-Regulation/drug effects ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells.<br><br>METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples.<br><br>RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate.<br><br>CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.},
}
@article {pmid30287096,
year = {2018},
author = {Wienert, B and Martyn, GE and Funnell, APW and Quinlan, KGR and Crossley, M},
title = {Wake-up Sleepy Gene: Reactivating Fetal Globin for β-Hemoglobinopathies.},
journal = {Trends in genetics : TIG},
volume = {34},
number = {12},
pages = {927-940},
doi = {10.1016/j.tig.2018.09.004},
pmid = {30287096},
issn = {0168-9525},
mesh = {Adult ; CRISPR-Cas Systems/genetics ; Gene Editing/trends ; Genetic Therapy/*trends ; Hemoglobinopathies/blood/*genetics/pathology ; Humans ; Mutation ; beta-Globins/*genetics/therapeutic use ; gamma-Globins/*genetics/therapeutic use ; },
abstract = {Disorders in hemoglobin (hemoglobinopathies) were the first monogenic diseases to be characterized and remain among the most common and best understood genetic conditions. Moreover, the study of the β-globin locus provides a textbook example of developmental gene regulation. The fetal γ-globin genes (HBG1/HBG2) are ordinarily silenced around birth, whereupon their expression is replaced by the adult β-globin genes (HBB primarily and HBD). Over 50 years ago it was recognized that mutations that cause lifelong persistence of fetal γ-globin expression ameliorate the debilitating effects of mutations in β-globin. Since then, research has focused on therapeutically reactivating the fetal γ-globin genes. Here, we summarize recent discoveries, focusing on the influence of genome editing technologies, including CRISPR-Cas9, and emerging gene therapy approaches.},
}
@article {pmid30286211,
year = {2018},
author = {Ye, W and Chew, M and Hou, J and Lai, F and Leopold, SJ and Loo, HL and Ghose, A and Dutta, AK and Chen, Q and Ooi, EE and White, NJ and Dondorp, AM and Preiser, P and Chen, J},
title = {Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway.},
journal = {PLoS pathogens},
volume = {14},
number = {10},
pages = {e1007298},
pmid = {30286211},
issn = {1553-7374},
support = {R01 NS099064/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell-Derived Microparticles/*immunology ; Cells, Cultured ; Cytoplasm/metabolism ; Erythrocytes/*immunology/metabolism/parasitology ; Humans ; Interferon-Induced Helicase, IFIH1/antagonists &amp; inhibitors/genetics/*metabolism ; Killer Cells, Natural/*immunology/metabolism/parasitology ; Lymphocyte Activation ; Malaria, Falciparum/*immunology/metabolism/parasitology ; Plasmodium falciparum/*immunology/isolation &amp; purification ; },
abstract = {Natural killer (NK) cells provide the first line of defense against malaria parasite infection. However, the molecular mechanisms through which NK cells are activated by parasites are largely unknown, so is the molecular basis underlying the variation in NK cell responses to malaria infection in the human population. Here, we compared transcriptional profiles of responding and non-responding NK cells following exposure to Plasmodium-infected red blood cells (iRBCs) and identified MDA5, a RIG-I-like receptor involved in sensing cytosolic RNAs, to be differentially expressed. Knockout of MDA5 in responding human NK cells by CRISPR/cas9 abolished NK cell activation, IFN-γ secretion, lysis of iRBCs. Similarly, inhibition of TBK1/IKKε, an effector molecule downstream of MDA5, also inhibited activation of responding NK cells. Conversely, activation of MDA5 by liposome-packaged poly I:C restored non-responding NK cells to lyse iRBCs. We further show that microvesicles containing large parasite RNAs from iRBCs activated NK cells by fusing with NK cells. These findings suggest that NK cells are activated through the MDA5 pathway by parasite RNAs that are delivered to the cytoplasm of NK cells by microvesicles from iRBCs. The difference in MDA5 expression between responding and non-responding NK cells following exposure to iRBCs likely contributes to the variation in NK cell responses to malaria infection in the human population.},
}
@article {pmid30285700,
year = {2018},
author = {Tang, JX and Chen, D and Deng, SL and Li, J and Li, Y and Fu, Z and Wang, XX and Zhang, Y and Chen, SR and Liu, YX},
title = {CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {61},
pmid = {30285700},
issn = {1472-6750},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exons ; *Gene Editing ; Gene Knockdown Techniques/*methods ; INDEL Mutation ; Introns ; Mice/*genetics ; Mice, Inbred C57BL ; RNA Precursors/*genetics ; *RNA Splicing ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing.<br><br>RESULTS: In the present study, we showed that gene knockdown model also could be generated using CRISPR-mediated gene editing by disrupting the boundary of exon and intron in mice (C57BL/6&#xa0;J). CRISPR induced indel at the boundary of exon and intron (5' splice site) caused alternative splicing and produced multiple different mRNAs, most of these mRNAs introduced premature termination codon causing down expression of the gene.<br><br>CONCLUSIONS: These results showed that alternative splicing mutants were able to generate through CRISPR-mediated genome editing by deleting the boundary of exon and intron causing disruption of 5' splice site. Although alternative splicing was an unexpected outcome, this finding could be developed as a technology to generate gene knockdown models or to investigate pre-mRNA splicing.},
}
@article {pmid30285624,
year = {2018},
author = {Howells, RM and Craze, M and Bowden, S and Wallington, EJ},
title = {Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9.},
journal = {BMC plant biology},
volume = {18},
number = {1},
pages = {215},
pmid = {30285624},
issn = {1471-2229},
support = {BB/J019356/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Agrobacterium/genetics ; *CRISPR-Cas Systems ; DNA, Bacterial ; Gene Editing/*methods ; Genome, Plant ; Hordeum/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Transformation, Genetic ; Triticum/*genetics ; },
abstract = {BACKGROUND: The use of CRISPR/Cas9 systems could prove to be a valuable tool in crop research, providing the ability to fully knockout gene function in complex genomes or to precisely adjust gene function by knockout of individual alleles.<br><br>RESULTS: We compare gene editing in hexaploid wheat (Triticum aestivum) with diploid barley (Hordeum vulgare), using a combination of single genome and tri-genome targeting. High efficiency gene editing, 11-17% for single genome targeted guides and 5% for tri-genome targeted guides, was achieved in wheat using stable Agrobacterium-mediated transformation. Gene editing in wheat was shown to be predominantly heterozygous, edits were inherited in a Mendelian fashion over multiple generations and no off-target effects were observed. Comparison of editing between the two species demonstrated that more stable, heritable edits were produced in wheat, whilst barley exhibited continued and somatic editing.<br><br>CONCLUSION: Our work shows the potential to obtain stable edited transgene-free wheat lines in 36&#xa0;weeks through only two generations and that targeted mutagenesis of individual homeologues within the wheat genome is achievable with a modest amount of effort, and without off-target mutations or the need for lengthy crossing strategies.},
}
@article {pmid30285540,
year = {2019},
author = {Smith, CIE and Zain, R},
title = {Therapeutic Oligonucleotides: State of the Art.},
journal = {Annual review of pharmacology and toxicology},
volume = {59},
number = {},
pages = {605-630},
doi = {10.1146/annurev-pharmtox-010818-021050},
pmid = {30285540},
issn = {1545-4304},
mesh = {Clinical Trials as Topic ; Humans ; Morpholinos/therapeutic use ; Nucleic Acids/metabolism ; Oligonucleotides/*therapeutic use ; Oligonucleotides, Antisense/therapeutic use ; },
abstract = {Oligonucleotides (ONs) can interfere with biomolecules representing the entire extended central dogma. Antisense gapmer, steric block, splice-switching ONs, and short interfering RNA drugs have been successfully developed. Moreover, antagomirs (antimicroRNAs), microRNA mimics, aptamers, DNA decoys, DNAzymes, synthetic guide strands for CRISPR/Cas, and innate immunity-stimulating ONs are all in clinical trials. DNA-targeting, triplex-forming ONs and strand-invading ONs have made their mark on drug development research, but not yet as medicines. Both design and synthetic nucleic acid chemistry are crucial for achieving biologically active ONs. The dominating modifications are phosphorothioate linkages, base methylation, and numerous 2'-substitutions in the furanose ring, such as 2'-fluoro, O-methyl, or methoxyethyl. Locked nucleic acid and constrained ethyl, a related variant, are bridged forms where the 2'-oxygen connects to the 4'-carbon in the sugar. Phosphorodiamidate morpholino oligomers, carrying a modified heterocyclic backbone ring, have also been commercialized. Delivery remains a major obstacle, but systemic administration and intrathecal infusion are used for treatment of the liver and brain, respectively.},
}
@article {pmid30285246,
year = {2019},
author = {Chen, W and Zhang, G and Li, J and Zhang, X and Huang, S and Xiang, S and Hu, X and Liu, C},
title = {CRISPRlnc: a manually curated database of validated sgRNAs for lncRNAs.},
journal = {Nucleic acids research},
volume = {47},
number = {D1},
pages = {D63-D68},
pmid = {30285246},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/*methods ; *Databases, Genetic ; *Gene Editing ; RNA, Guide/*genetics ; RNA, Long Noncoding/*genetics ; Reproducibility of Results ; Software ; User-Computer Interface ; },
abstract = {The CRISPR/Cas9 system, as a revolutionary genome editing tool for all areas of molecular biology, provides new opportunities for research on lncRNA's function. However, designing a CRISPR/Cas9 single guide RNA (sgRNA) for lncRNA is not easy with an unwarrantable effectiveness. Thus, it is worthy of collecting validated sgRNAs, to assist in efficiently choosing sgRNA with an expected activity. CRISPRlnc (http://www.crisprlnc.org or http://crisprlnc.xtbg.ac.cn) is a manually curated database of validated CRISPR/Cas9 sgRNAs for lncRNAs from all species. After manually reviewing more than 200 published literature, the current version of CRISPRlnc contains 305 lncRNAs and 2102 validated sgRNAs across eight species, including mammalian, insect and plant. We handled the ID, position in the genome, sequence and functional description of these lncRNAs, as well as the sequence, protoacceptor-motif (PAM), CRISPR type and validity of their paired sgRNAs. In CRISPRlnc, we provided the tools for browsing, searching and downloading data, as well as online BLAST service and genome browse server. As the first database against the validated sgRNAs of lncRNAs, CRISPRlnc will provide a new and powerful platform to promote CRISPR/Cas9 applications for future functional studies of lncRNAs.},
}
@article {pmid30284145,
year = {2018},
author = {Lau, CH and Suh, Y},
title = {In vivo epigenome editing and transcriptional modulation using CRISPR technology.},
journal = {Transgenic research},
volume = {27},
number = {6},
pages = {489-509},
pmid = {30284145},
issn = {1573-9368},
support = {R01 CA180126/CA/NCI NIH HHS/United States ; R01 GM104459/GM/NIGMS NIH HHS/United States ; U19 AG056278/AG/NIA NIH HHS/United States ; P01 AG017242/AG/NIA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; *Epigenomics ; *Gene Editing ; Humans ; Promoter Regions, Genetic ; *Transcription, Genetic ; },
abstract = {The rapid advancement of CRISPR technology has enabled targeted epigenome editing and transcriptional modulation in the native chromatin context. However, only a few studies have reported the successful editing of the epigenome in adult animals in contrast to the rapidly growing number of in vivo genome editing over the past few years. In this review, we discuss the challenges facing in vivo epigenome editing and new strategies to overcome the huddles. The biggest challenge has been the difficulty in packaging dCas9 fusion proteins required for manipulation of epigenome into the adeno-associated virus (AAV) delivery vehicle. We review the strategies to address the AAV packaging issue, including small dCas9 orthologues, truncated dCas9 mutants, a split-dCas9 system, and potent truncated effector domains. We discuss the dCas9 conjugation strategies to recruit endogenous chromatin modifiers and remodelers to specific genomic loci, and recently developed methods to recruit multiple copies of the dCas9 fusion protein, or to simultaneous express multiple gRNAs for robust epigenome editing or synergistic transcriptional modulation. The use of Cre-inducible dCas9-expressing mice or a genetic cross between dCas9- and sgRNA-expressing flies has also helped overcome the transgene delivery issue. We provide perspective on how a combination use of these strategies can facilitate in vivo epigenome editing and transcriptional modulation.},
}
@article {pmid30284144,
year = {2018},
author = {Vilarino, M and Suchy, FP and Rashid, ST and Lindsay, H and Reyes, J and McNabb, BR and van der Meulen, T and Huising, MO and Nakauchi, H and Ross, PJ},
title = {Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.},
journal = {Transgenic research},
volume = {27},
number = {6},
pages = {525-537},
pmid = {30284144},
issn = {1573-9368},
support = {MR/L006537/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *Animals, Genetically Modified ; Biopsy ; *Blastocyst ; *CRISPR-Cas Systems ; Embryo Transfer ; *Embryo, Mammalian ; Embryonic Development ; Female ; Gene Editing/methods/*veterinary ; Homeodomain Proteins/*genetics ; Male ; Mosaicism ; *Mutation ; Sheep ; Trans-Activators/*genetics ; },
abstract = {The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.},
}
@article {pmid30284045,
year = {2019},
author = {Majumdar, S and Terns, MP},
title = {CRISPR RNA-guided DNA cleavage by reconstituted Type I-A immune effector complexes.},
journal = {Extremophiles : life under extreme conditions},
volume = {23},
number = {1},
pages = {19-33},
pmid = {30284045},
issn = {1433-4909},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; },
mesh = {Archaeal Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Pyrococcus furiosus/enzymology/*genetics ; },
abstract = {Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA. Here, we reconstituted functional Type I-A effector crRNPs in vitro with recombinant Csa proteins and synthetic crRNA and characterized properties of crRNP assembly, target DNA recognition and cleavage. Six proteins (Csa 4-1, Cas3″, Cas3', Cas5a, Csa2, Csa5) are essential for selective target DNA binding and cleavage. Native gel shift analysis and UV-induced RNA-protein crosslinking demonstrate that Cas5a and Csa2 directly interact with crRNA 5' tag and guide sequences, respectively. Mutational analysis revealed that Cas3″ is the effector nuclease of the complex. Together, our results indicate that DNA cleavage by Type I-A crRNPs requires crRNA-guided and protospacer adjacent motif-dependent target DNA binding to unwind double-stranded DNA and expose single strands for progressive ATP-dependent 3'-5' cleavage catalyzed by integral Cas3' helicase and Cas3″ nuclease crRNP components.},
}
@article {pmid30283411,
year = {2018},
author = {Terceti, MS and Vences, A and Matanza, XM and Dalsgaard, I and Pedersen, K and Osorio, CR},
title = {Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity.},
journal = {Frontiers in microbiology},
volume = {9},
number = {},
pages = {2155},
pmid = {30283411},
issn = {1664-302X},
abstract = {The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the toxR gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPR-Cas systems and putative new mobile genetic elements. This study provides sound evidence that P. damselae subsp. damselae outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen.},
}
@article {pmid30283135,
year = {2018},
author = {Schmidt, F and Cherepkova, MY and Platt, RJ},
title = {Transcriptional recording by CRISPR spacer acquisition from RNA.},
journal = {Nature},
volume = {562},
number = {7727},
pages = {380-385},
doi = {10.1038/s41586-018-0569-1},
pmid = {30283135},
issn = {1476-4687},
mesh = {CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Clostridiales/enzymology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/analysis/genetics ; Escherichia coli/cytology/drug effects/genetics ; Genes, Bacterial/genetics ; Herbicides/pharmacology ; *High-Throughput Nucleotide Sequencing ; Oxidative Stress/genetics ; Paraquat/pharmacology ; RNA/*analysis/*genetics ; Transcription, Genetic/*genetics ; },
abstract = {The ability to record transcriptional events within a cell over time would help to elucidate how molecular events give rise to complex cellular behaviours and states. However, current molecular recording technologies capture only a small set of defined stimuli. Here we use CRISPR spacer acquisition to capture and convert intracellular RNAs into DNA, enabling DNA-based storage of transcriptional information. In Escherichia coli, we show that defined stimuli, such as an RNA virus or arbitrary sequences, as well as complex stimuli, such as oxidative stress, result in quantifiable transcriptional records that are stored within a population of cells. We demonstrate that the transcriptional records enable us to classify and describe complex cellular behaviours and to identify the precise genes that orchestrate differential cellular responses. In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record-seq) could be used to reconstruct transcriptional histories that describe complex cell behaviours or pathological states.},
}
@article {pmid30282831,
year = {2018},
author = {Fan, Z and Perisse, IV and Cotton, CU and Regouski, M and Meng, Q and Domb, C and Van Wettere, AJ and Wang, Z and Harris, A and White, KL and Polejaeva, IA},
title = {A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene.},
journal = {JCI insight},
volume = {3},
number = {19},
pages = {},
pmid = {30282831},
issn = {2379-3708},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Cystic Fibrosis/*genetics/pathology ; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics ; *Disease Models, Animal ; Female ; Fibrosis ; Gallbladder/pathology ; Gene Knockout Techniques ; Humans ; Liver/pathology ; Lung/pathology ; Male ; Nuclear Transfer Techniques ; Pancreas/pathology ; Phenotype ; *Sheep ; Species Specificity ; },
abstract = {Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The major cause of limited life span in CF patients is progressive lung disease. CF models have been generated in 4 species (mice, rats, ferrets, and pigs) to enhance our understanding of the CF pathogenesis. Sheep may be a particularly relevant animal to model CF in humans due to the similarities in lung anatomy and development in the two species. Here, we describe the generation of a sheep model for CF using CRISPR/Cas9 genome editing and somatic cell nuclear transfer (SCNT) techniques. We generated cells with CFTR gene disruption and used them for production of CFTR-/- and CFTR+/- lambs. The newborn CFTR-/- sheep developed severe disease consistent with CF pathology in humans. Of particular relevance were pancreatic fibrosis, intestinal obstruction, and absence of the vas deferens. Also, substantial liver and gallbladder disease may reflect CF liver disease that is evident in humans. The phenotype of CFTR-/- sheep suggests this large animal model will be a useful resource to advance the development of new CF therapeutics. Moreover, the generation of specific human CF disease-associated mutations in sheep may advance personalized medicine for this common genetic disorder.},
}
@article {pmid30282704,
year = {2018},
author = {Hasan, MH and Davis, LE and Bollavarapu, RK and Mitra, D and Parmar, R and Tandon, R},
title = {Dynamin Is Required for Efficient Cytomegalovirus Maturation and Envelopment.},
journal = {Journal of virology},
volume = {92},
number = {24},
pages = {},
pmid = {30282704},
issn = {1098-5514},
mesh = {Animals ; Carrier Proteins/metabolism ; Cell Survival ; Cells, Cultured ; Cytomegalovirus/*physiology ; Dynamins/*genetics/metabolism ; Fibroblasts/cytology/metabolism/*virology ; Gene Expression Regulation, Viral ; Gene Knockout Techniques ; Glycoproteins/metabolism ; Immediate-Early Proteins/metabolism ; Mice ; Viral Envelope Proteins/metabolism ; Viral Load ; Viral Proteins/*metabolism ; Virus Internalization ; },
abstract = {Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate-early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells, as well as in mock-induced parental MEF, at early times postinfection, although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 h postinfection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids), indicating successful viral DNA replication, capsid assembly, and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore)-treated MEF showed a phenotype similar to TKO cells upon mouse cytomegalovirus infection, confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.IMPORTANCE Viruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV)-infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment [vAC]) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. We show here that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules, as well as by newer generation nucleotide-based therapeutics (e.g., small interfering RNA, CRISPR/CAS gRNA, etc.).},
}
@article {pmid30282659,
year = {2018},
author = {George, L and Indig, FE and Abdelmohsen, K and Gorospe, M},
title = {Intracellular RNA-tracking methods.},
journal = {Open biology},
volume = {8},
number = {10},
pages = {},
pmid = {30282659},
issn = {2046-2441},
mesh = {Aptamers, Nucleotide/chemistry/genetics/metabolism ; Bacteriophages/chemistry/genetics/metabolism ; CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Humans ; *In Situ Hybridization, Fluorescence ; RNA, Messenger/chemistry/genetics/*metabolism ; RNA, Untranslated/chemistry/genetics/*metabolism ; Sequence Analysis, RNA ; *Single-Cell Analysis ; },
abstract = {RNA tracking allows researchers to visualize RNA molecules in cells and tissues, providing important spatio-temporal information regarding RNA dynamics and function. Methods such as fluorescent in situ hybridization (FISH) and molecular beacons rely on complementary oligonucleotides to label and view endogenous transcripts. Other methods create artificial chimeric transcripts coupled with bacteriophage-derived coat proteins (e.g. MS2, λN) to tag molecules in live cells. In other approaches, endogenous RNAs are recognized by complementary RNAs complexed with noncatalytic Cas proteins. Each technique has its own set of strengths and limitations that must be considered when planning an experiment. Here, we discuss the mechanisms, advantages, and weaknesses of in situ hybridization, molecular beacons, MS2 tagging and Cas-derived systems, as well as how RNA tracking can be employed to study various aspects of molecular biology.},
}
@article {pmid30280895,
year = {2018},
author = {Ke, M and Liu, J and Chen, W and Chen, L and Gao, W and Qin, Y and He, A and Chu, B and Tang, J and Xu, R and Deng, Y and Tian, R},
title = {Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.},
journal = {Analytical chemistry},
volume = {90},
number = {21},
pages = {12574-12583},
doi = {10.1021/acs.analchem.8b02667},
pmid = {30280895},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems ; Chromatography, Affinity/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; Mass Spectrometry/*methods ; Multiprotein Complexes/*analysis/metabolism ; Protein Serine-Threonine Kinases/genetics/metabolism ; Proteome/*analysis ; Proteomics/*methods ; },
abstract = {Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.},
}
@article {pmid30279181,
year = {2018},
author = {Jin, JJ and Lv, W and Xia, P and Xu, ZY and Zheng, AD and Wang, XJ and Wang, SS and Zeng, R and Luo, HM and Li, GL and Zuo, B},
title = {Long noncoding RNA SYISL regulates myogenesis by interacting with polycomb repressive complex 2.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {42},
pages = {E9802-E9811},
pmid = {30279181},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; *Epigenesis, Genetic ; Gene Silencing ; Mice ; Mice, Knockout ; Muscle Development/*physiology ; Polycomb Repressive Complex 2/genetics/*metabolism ; Promoter Regions, Genetic ; RNA, Long Noncoding/genetics/*metabolism ; },
abstract = {Although many long noncoding RNAs (lncRNAs) have been identified in muscle, their physiological function and regulatory mechanisms remain largely unexplored. In this study, we systematically characterized the expression profiles of lncRNAs during C2C12 myoblast differentiation and identified an intronic lncRNA, SYISL (SYNPO2 intron sense-overlapping lncRNA), that is highly expressed in muscle. Functionally, SYISL promotes myoblast proliferation and fusion but inhibits myogenic differentiation. SYISL knockout in mice results in significantly increased muscle fiber density and muscle mass. Mechanistically, SYISL recruits the enhancer of zeste homolog 2 (EZH2) protein, the core component of polycomb repressive complex 2 (PRC2), to the promoters of the cell-cycle inhibitor gene p21 and muscle-specific genes such as myogenin (MyoG), muscle creatine kinase (MCK), and myosin heavy chain 4 (Myh4), leading to H3K27 trimethylation and epigenetic silencing of target genes. Taken together, our results reveal that SYISL is a repressor of muscle development and plays a vital role in PRC2-mediated myogenesis.},
}
@article {pmid30278987,
year = {2019},
author = {Rui, Y and Wilson, DR and Green, JJ},
title = {Non-Viral Delivery To Enable Genome Editing.},
journal = {Trends in biotechnology},
volume = {37},
number = {3},
pages = {281-293},
pmid = {30278987},
issn = {1879-3096},
support = {R01 CA228133/CA/NCI NIH HHS/United States ; R01 EB022148/EB/NIBIB NIH HHS/United States ; },
mesh = {Drug Delivery Systems/*methods/trends ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genome, Human ; Humans ; },
abstract = {Genome-editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biological research. Each biotechnology consists of a DNA-binding protein that can be programmed to recognize and initiate double-strand breaks (DSBs) for site-specific gene modification. These technologies have the potential to be harnessed to cure diseases caused by aberrant gene expression. To be successful therapeutically, their functionality depends on their safe and efficient delivery into the cell nucleus. This review discusses the challenges in the delivery of genome-editing tools, and highlights recent innovations in non-viral delivery that have potential to overcome these limitations and advance the translation of genome editing towards patient care.},
}
@article {pmid30278376,
year = {2018},
author = {Wei, R and Yuan, F and Wu, Y and Liu, Y and You, K and Yang, Z and Chen, Y and Getachew, A and Wang, N and Xu, Y and Zhuang, Y and Yang, F and Li, YX},
title = {Construction of a GLI3 compound heterozygous knockout human embryonic stem cell line WAe001-A-20 by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {32},
number = {},
pages = {139-144},
doi = {10.1016/j.scr.2018.09.010},
pmid = {30278376},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics/*physiology ; Cell Line ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Stem Cells/cytology/metabolism ; Human Embryonic Stem Cells/*cytology/*metabolism ; Humans ; Male ; Middle Aged ; Nerve Tissue Proteins/*genetics ; Zinc Finger Protein Gli3/*genetics ; },
abstract = {The human GLI3 protein has a dual function as a transcriptional activator or repressor of hedgehog signaling, depending on the proteolytic processing forms of GLI3. In this study, we established a compound heterozygous GLI3 mutant human embryonic stem cell line (WAe001-A-20) through CRISPR/Cas9 editing. The WAe001-A-20 cells carried two deletions on two different alleles of exon 2 of GLI3, respectively, which resulted in a frame shift and early termination in the translation of GLI3. Moreover, WAe001-A-20 maintains a normal karyotype, parental cell morphology, pluripotent phenotype and the ability to differentiate into three germ layers. Resource table.},
}
@article {pmid30278126,
year = {2018},
author = {Wu, Z and Zhang, L and Qiao, D and Xue, H and Zhao, X},
title = {Functional Analyses of Cassette Chromosome Recombinase C2 (CcrC2) and Its Use in Eliminating Methicillin Resistance by Combining CRISPR-Cas9.},
journal = {ACS synthetic biology},
volume = {7},
number = {11},
pages = {2590-2599},
doi = {10.1021/acssynbio.8b00261},
pmid = {30278126},
issn = {2161-5063},
mesh = {Bacterial Proteins/*genetics/metabolism ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Chromosomes, Bacterial/*genetics/metabolism ; Methicillin Resistance/genetics ; Methicillin-Resistant Staphylococcus aureus/*genetics ; Penicillin-Binding Proteins/genetics ; Recombinases/*genetics/metabolism ; Sequence Alignment ; Staphylococcus aureus/genetics ; },
abstract = {Worldwide occurrence of methicillin-resistant Staphylococcus aureus (MRSA) poses enormous challenges for both communities and health care settings. Cassette chromosome recombinases (Ccr) specifically perform excision and acquisition of a staphylococcal cassette chromosome mec (SCC mec) in staphylococci and are responsible for the spread of methicillin resistance. This study explored the roles of CcrC2, a recently discovered Ccr, in the horizontal transfer of SCC mec and developed a potential means to control the spread of methicillin resistance. Knockout of CcrC2 completely aborted the excision of SCC mec, while overexpression of CcrC2 partially removed the SCC mec from the genome and transformed methicillin-resistant Staphylococcus aureus (MRSA) into methicillin-susceptible Staphylococcus aureus (MSSA). Moreover, two nucleotide residues (G5C6) in the direct repeat sequence within an att site were found to be critical for excision and acquisition efficiencies. To block the horizontal transfer of methicillin resistance, a SCC mec killer system was developed by combining the CcrC2-mediated SCC mec excision and the mecA-targeting CRISPR-Cas9 machinery. The SCC mec killer transformed MRSA to MSSA and disrupted the mecA-carrying SCC mec intermediate, thereby eliminating methicillin resistance determinant mecA gene inside a MRSA cell and blocking the horizontal transfer of SCC mec. The SCC mec killer was versatile for efficiently removing multiple types of SCC mec elements. It is envisioned that this approach could offer a new means to control the spread of methicillin resistance.},
}
@article {pmid30276556,
year = {2018},
author = {Xue, T and Liu, K and Chen, D and Yuan, X and Fang, J and Yan, H and Huang, L and Chen, Y and He, W},
title = {Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {34},
number = {10},
pages = {154},
pmid = {30276556},
issn = {1573-0972},
mesh = {Alcohol Dehydrogenase/*genetics/*metabolism ; Analysis of Variance ; Base Sequence ; *CRISPR-Cas Systems/genetics ; Cloning, Molecular ; DNA, Fungal/analysis ; Escherichia coli/genetics ; Ethanol/*metabolism ; Fermentation ; Gene Deletion ; Gene Editing ; Gene Expression Regulation, Fungal ; Gene Knockout Techniques/methods ; Genetic Engineering/*methods ; Genome, Fungal/genetics ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Saccharomyces cerevisiae/*genetics/*metabolism ; Saccharomyces cerevisiae Proteins/*genetics/*metabolism ; Sequence Alignment ; Transformation, Genetic ; },
abstract = {Bioethanol, as a form of renewable and clean energy, has become increasingly important to the energy supply. One major obstacle in ethanol production is developing a high-capacity system. Existing approaches for regulating the ethanol production pathway are relatively insufficient, with nonspecific genetic manipulation. Here, we used CRISPR/Cas9 technology to disrupt the alcohol dehydrogenase (ADH) 2 gene via complete deletion of the gene and introduction of a frameshift mutation in the ADH2 locus. Sequencing demonstrated the accurate knockout of the target gene with 91.4% and near 100% targeting efficiency. We also utilized genome resequencing to validate the mutations in the ADH2 mutants targeted by various single-guide RNAs. This extensive analysis indicated the mutations in the CRISPR/Cas9-engineered strains were homozygous. We applied the engineered Saccharomyces cerevisiae strains for bioethanol production. Results showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain. This work illustrates the applicability of this highly efficient and specific genome engineering approach to promote the improvement of bioethanol production in S. cerevisiae via metabolic engineering. Importantly, this study is the first report of the disruption of a target gene, ADH2, in S. cerevisiae using CRISPR/Cas9 technology to improve bioethanol yield.},
}
@article {pmid30275595,
year = {2018},
author = {Tang, L},
title = {Does the number of chromosomes matter?.},
journal = {Nature methods},
volume = {15},
number = {10},
pages = {761},
doi = {10.1038/s41592-018-0164-2},
pmid = {30275595},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; *Chromosomes, Fungal ; Saccharomyces cerevisiae/*genetics/growth &amp; development ; Saccharomyces cerevisiae Proteins/*genetics ; },
}
@article {pmid30275594,
year = {2018},
author = {Willi, M and Smith, HE and Wang, C and Liu, C and Hennighausen, L},
title = {Mutation frequency is not increased in CRISPR-Cas9-edited mice.},
journal = {Nature methods},
volume = {15},
number = {10},
pages = {756-758},
doi = {10.1038/s41592-018-0148-2},
pmid = {30275594},
issn = {1548-7105},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mice ; *Mutation ; *Mutation Rate ; },
}
@article {pmid30275584,
year = {2018},
author = {Marx, V},
title = {Base editing a CRISPR way.},
journal = {Nature methods},
volume = {15},
number = {10},
pages = {767-770},
doi = {10.1038/s41592-018-0146-4},
pmid = {30275584},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Human ; Humans ; *Mutation ; },
}
@article {pmid30275576,
year = {2018},
author = {Rusk, N},
title = {Better base editors.},
journal = {Nature methods},
volume = {15},
number = {10},
pages = {763},
doi = {10.1038/s41592-018-0154-4},
pmid = {30275576},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Cytidine Deaminase ; *Gene Editing ; Proteins ; },
}
@article {pmid30275529,
year = {2018},
author = {Li, Q and Li, Y and Yang, S and Huang, S and Yan, M and Ding, Y and Tang, W and Lou, X and Yin, Q and Sun, Z and Lu, L and Shi, H and Wang, H and Chen, Y and Li, J},
title = {CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development.},
journal = {Nature cell biology},
volume = {20},
number = {11},
pages = {1315-1325},
doi = {10.1038/s41556-018-0202-4},
pmid = {30275529},
issn = {1476-4679},
mesh = {Amino Acids/*genetics/metabolism ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Embryo, Mammalian/cytology/embryology/metabolism ; Female ; Gene Editing/*methods ; Germ Cells/cytology/growth &amp; development/*metabolism ; HEK293 Cells ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mice, Inbred ICR ; Neoplasm Proteins/*genetics/metabolism ; },
abstract = {CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.},
}
@article {pmid30275376,
year = {2018},
author = {Zhu, S and Yu, X and Li, Y and Sun, Y and Zhu, Q and Sun, J},
title = {Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {19},
number = {10},
pages = {},
pmid = {30275376},
issn = {1422-0067},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genes, Plant ; Genetic Vectors/metabolism ; Gossypium/*genetics ; Mutagenesis/genetics ; Mutation/genetics ; Plants, Genetically Modified ; RNA, Guide/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4[-]100% and 92.9[-]100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.},
}
@article {pmid30275337,
year = {2018},
author = {Ling, L and Raikhel, AS},
title = {Serotonin signaling regulates insulin-like peptides for growth, reproduction, and metabolism in the disease vector Aedes aegypti.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {42},
pages = {E9822-E9831},
pmid = {30275337},
issn = {1091-6490},
support = {R01 AI036959/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/*physiology ; Animals ; CRISPR-Cas Systems ; Female ; Insect Proteins/*metabolism ; Insulin/*metabolism ; Mosquito Vectors/*physiology ; Peptide Fragments/*metabolism ; Receptors, Serotonin/chemistry/genetics/*metabolism ; *Reproduction ; Serotonin/*metabolism ; Signal Transduction ; },
abstract = {Disease-transmitting female mosquitoes require a vertebrate blood meal to produce their eggs. An obligatory hematophagous lifestyle, rapid reproduction, and existence of a large number of transmittable diseases make mosquitoes the world's deadliest animals. Attaining optimal body size and nutritional status is critical for mosquitoes to become reproductively competent and effective disease vectors. We report that blood feeding boosts serotonin concentration and elevates the serotonin receptor Aa5HT2B (Aedes aegypti 5-hydroxytryptamine receptor, type 2B) transcript level in the fat-body, an insect analog of the vertebrate liver and adipose tissue. Aa5HT2B gene disruption using the CRISPR-Cas9 gene-editing approach led to a decreased body size, postponed development, shortened lifespan, retarded ovarian growth, and dramatically diminished lipid accumulation. Expression of the insulin-like peptide (ILP) genes ilp2 and ilp6 was down-regulated while that of ilp5 and ilp4 was up-regulated in response to Aa5HT2B disruption. CRISPR-Cas9 disruption of ilp2 or ilp6 resulted in adverse phenotypes similar to those of Aa5HT2B disruption, while ilp5 CRISPR-Cas9 disruption had exactly the opposite effect on growth and metabolism, with significantly increased body size and elevated lipid stores. Simultaneous CRISPR-Cas9 disruption of Aa5HT2B and ilp5 rescued these phenotypic manifestations. Aa5HT2B RNAi silencing rendered ilp6 insensitive to serotonin treatment in the cultured fat-body, suggesting a regulatory link between Aa5HT2B and ILP6. Moreover, CRISPR-Cas9 ilp6 disruption affects expression of ilp-2, -5, and -4, pointing out on a possible role of ILP6 as a mediator of the Aa5HT2B action.},
}
@article {pmid30275336,
year = {2018},
author = {Sago, CD and Lokugamage, MP and Paunovska, K and Vanover, DA and Monaco, CM and Shah, NN and Gamboa Castro, M and Anderson, SE and Rudoltz, TG and Lando, GN and Munnilal Tiwari, P and Kirschman, JL and Willett, N and Jang, YC and Santangelo, PJ and Bryksin, AV and Dahlman, JE},
title = {High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {42},
pages = {E9944-E9952},
pmid = {30275336},
issn = {1091-6490},
support = {T32 EB006343/EB/NIBIB NIH HHS/United States ; T32 EB021962/EB/NIBIB NIH HHS/United States ; T32 GM008433/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Endothelial Cells/cytology/*metabolism ; *Gene Editing ; *Gene Transfer Techniques ; HEK293 Cells ; Hepatocytes/cytology/metabolism ; High-Throughput Screening Assays ; Humans ; Lipids/*chemistry ; Mice ; Mice, Inbred C57BL ; Nanoparticles/*administration &amp; dosage/chemistry ; RNA, Guide/chemistry/*genetics ; RNA, Messenger/chemistry/*genetics ; },
abstract = {Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.},
}
@article {pmid30275281,
year = {2018},
author = {Zhang, H and Pan, H and Zhou, C and Wei, Y and Ying, W and Li, S and Wang, G and Li, C and Ren, Y and Li, G and Ding, X and Sun, Y and Li, GL and Song, L and Li, Y and Yang, H and Liu, Z},
title = {Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {20},
pages = {},
doi = {10.1242/dev.168906},
pmid = {30275281},
issn = {1477-9129},
mesh = {Amino Acid Transport Systems, Acidic/metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cochlea/metabolism ; Deafness/genetics/physiopathology ; Disease Models, Animal ; Electrophysiological Phenomena ; Gene Editing/*methods ; Membrane Proteins/metabolism ; Mice ; Molecular Motor Proteins/metabolism ; Mutation/genetics ; Zygote/*metabolism ; },
abstract = {In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.},
}
@article {pmid30274819,
year = {2018},
author = {Song, Y and Zhang, Y and Chen, M and Deng, J and Sui, T and Lai, L and Li, Z},
title = {Functional validation of the albinism-associated tyrosinase T373K SNP by CRISPR/Cas9-mediated homology-directed repair (HDR) in rabbits.},
journal = {EBioMedicine},
volume = {36},
number = {},
pages = {517-525},
pmid = {30274819},
issn = {2352-3964},
mesh = {Albinism, Oculocutaneous/*genetics/metabolism ; *Amino Acid Substitution ; Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Enzyme Activation ; Female ; Gene Editing ; Genetic Association Studies ; Genetic Loci ; Genetic Predisposition to Disease ; Melanins ; Monophenol Monooxygenase/*genetics/metabolism ; Mutation ; *Polymorphism, Single Nucleotide ; Rabbits ; *Recombinational DNA Repair ; },
abstract = {BACKGROUND: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association.<br><br>METHODS: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin.<br><br>FINDINGS: We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level.<br><br>INTERPRETATION: Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. FUND: The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team.},
}
@article {pmid30274789,
year = {2018},
author = {Lin, L and He, X and Zhao, T and Gu, L and Liu, Y and Liu, X and Liu, H and Yang, F and Tu, M and Tang, L and Ge, X and Liu, C and Zhao, J and Song, Z and Qu, J and Gu, F},
title = {Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {11},
pages = {2650-2657},
pmid = {30274789},
issn = {1525-0024},
mesh = {Bacterial Proteins/chemistry/genetics/therapeutic use ; CRISPR-Cas Systems/*genetics ; Endonucleases/chemistry/*genetics/therapeutic use ; Francisella/*enzymology ; Gene Editing/*methods ; Genome, Human/genetics ; HEK293 Cells ; Humans ; },
abstract = {FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.},
}
@article {pmid30274788,
year = {2018},
author = {Wang, Y and Hao, L and Wang, H and Santostefano, K and Thapa, A and Cleary, J and Li, H and Guo, X and Terada, N and Ashizawa, T and Xia, G},
title = {Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {11},
pages = {2617-2630},
pmid = {30274788},
issn = {1525-0024},
support = {K08 AR064836/AR/NIAMS NIH HHS/United States ; },
mesh = {3' Untranslated Regions ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; HEK293 Cells ; Humans ; Muscle, Skeletal/growth &amp; development ; Myocytes, Cardiac/physiology ; Myotonic Dystrophy/*genetics/pathology/therapy ; Myotonin-Protein Kinase/*genetics ; Neural Stem Cells/*physiology ; Neurons/physiology ; RNA 3' Polyadenylation Signals/genetics ; RNA, Guide ; Transfection ; Trinucleotide Repeat Expansion/*genetics ; },
abstract = {Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3' UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3' UTR is a viable approach to develop therapeutic genome editing for DM1.},
}
@article {pmid30274509,
year = {2018},
author = {Huang, JF and Shen, ZY and Mao, QL and Zhang, XM and Zhang, B and Wu, JS and Liu, ZQ and Zheng, YG},
title = {Systematic Analysis of Bottlenecks in a Multibranched and Multilevel Regulated Pathway: The Molecular Fundamentals of l-Methionine Biosynthesis in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {7},
number = {11},
pages = {2577-2589},
doi = {10.1021/acssynbio.8b00249},
pmid = {30274509},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Carbon/metabolism ; Chromatography, High Pressure Liquid ; Escherichia coli/genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Bacterial ; Mass Spectrometry ; Metabolic Engineering/methods ; Methionine/analysis/*biosynthesis ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; },
abstract = {To produce chemicals and fuels from renewable resources, various strategies and genetic tools have been developed to redesign pathways and optimize the metabolic flux in microorganisms. However, in most successful cases, the target chemicals are synthesized through a linear pathway, and regular methodologies for the identification of bottlenecks and metabolic flux optimization in multibranched and multilevel regulated pathways, such as the l-methionine biosynthetic pathway, have rarely been reported. In the present study, a systematic analysis strategy was employed to gradually reveal and remove the potential bottlenecks limiting the l-methionine biosynthesis in E. coli. 80 genes in central metabolism and selected amino acids biosynthetic pathways were first repressed or upregulated to probe their effects on l-methionine accumulation. The l-methionine biosynthetic pathway was then modularized and iteratively genetic modifications were performed to uncover the multiple layers of limitations and stepwise improve the l-methionine titer. The metabolomics data further revealed a more evenly distributed metabolic flux in l-methionine biosynthesis pathway of the optimal strain and provided valuable suggestions for further optimization. The optimal strain produced 16.86 g/L of l-methionine in 48 h by fed-batch fermentation. This work is the first to our knowledge to systematically elucidate the molecular fundamentals of multilevel regulation of l-methionine biosynthesis. It also demonstrated that the systematic analysis strategy can boost our ability to identify the potential bottlenecks and optimize the metabolic flux in multibranched and multilevel regulated pathways for the production of corresponding chemicals.},
}
@article {pmid30269252,
year = {2019},
author = {An, X and Li, L and Wu, S},
title = {In vivo tunable CRISPR mediates efficient somatic mutagenesis to generate tumor models.},
journal = {Protein &amp; cell},
volume = {10},
number = {6},
pages = {450-454},
pmid = {30269252},
issn = {1674-8018},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing ; *Genetic Therapy ; HeLa Cells ; Humans ; Mice ; Mutagenesis ; *Neoplasms/genetics/therapy ; },
}
@article {pmid30269229,
year = {2018},
author = {Hong, KQ and Liu, DY and Chen, T and Wang, ZW},
title = {Recent advances in CRISPR/Cas9 mediated genome editing in Bacillus subtilis.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {34},
number = {10},
pages = {153},
pmid = {30269229},
issn = {1573-0972},
mesh = {Bacillus subtilis/*genetics ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; Gene Editing/*methods ; Genes, Bacterial/genetics ; Molecular Biology/methods ; },
abstract = {Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.},
}
@article {pmid30267758,
year = {2019},
author = {Dong, H and Xiang, H and Mu, D and Wang, D and Wang, T},
title = {Exploiting a conjugative CRISPR/Cas9 system to eliminate plasmid harbouring the mcr-1 gene from Escherichia coli.},
journal = {International journal of antimicrobial agents},
volume = {53},
number = {1},
pages = {1-8},
doi = {10.1016/j.ijantimicag.2018.09.017},
pmid = {30267758},
issn = {1872-7913},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/*genetics ; Escherichia coli/classification/drug effects/*genetics ; Escherichia coli Proteins/*genetics ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; Plasmids/*genetics ; RNA, Bacterial/genetics/metabolism ; },
abstract = {The transfer of multi-drug-resistance plasmids by bacterial conjugation is largely responsible for the development of drug resistance in bacteria, and causes serious problems in the treatment of infectious diseases. Since the first discovery of plasmid-borne colistin resistance gene mcr-1 was reported in late 2016, this gene has been found in a great number of Escherichia coli and other Gram-negative pathogens separated from different types of sources worldwide. The elimination of plasmids carrying mcr-1 and restoration of polymyxin sensitivity has very important clinical significance because polymyxins are frequently used as last-resort antibiotics to treat extensively drug-resistant Gram-negative bacterial infections. A host-independent conjugative plasmid was constructed in this study, and an engineered CRISPR/Cas9 system was used to remove plasmid harbouring mcr-1 from bacteria. This study found that this conjugative plasmid can not only be used as a new tool to remove resistance plasmids and sensitize the recipient bacteria to antibiotics, but can also make the recipient cell acquire immunity against mcr-1. This strategy provides a novel method to counteract the ever-worsening spread of mcr-1 among bacterial pathogens.},
}
@article {pmid30267031,
year = {2018},
author = {Winters, IP and Murray, CW and Winslow, MM},
title = {Towards quantitative and multiplexed in vivo functional cancer genomics.},
journal = {Nature reviews. Genetics},
volume = {19},
number = {12},
pages = {741-755},
doi = {10.1038/s41576-018-0053-7},
pmid = {30267031},
issn = {1471-0064},
mesh = {Animals ; *CRISPR-Cas Systems ; *Genes, Neoplasm ; Genomics/*methods ; Humans ; Mice ; Neoplasms/*genetics/metabolism/pathology ; },
abstract = {Large-scale sequencing of human tumours has uncovered a vast array of genomic alterations. Genetically engineered mouse models recapitulate many features of human cancer and have been instrumental in assigning biological meaning to specific cancer-associated alterations. However, their time, cost and labour-intensive nature limits their broad utility; thus, the functional importance of the majority of genomic aberrations in cancer remains unknown. Recent advances have accelerated the functional interrogation of cancer-associated alterations within in vivo models. Specifically, the past few years have seen the emergence of CRISPR-Cas9-based strategies to rapidly generate increasingly complex somatic alterations and the development of multiplexed and quantitative approaches to ascertain gene function in vivo.},
}
@article {pmid30266827,
year = {2018},
author = {Vaid, S and Camp, JG and Hersemann, L and Eugster Oegema, C and Heninger, AK and Winkler, S and Brandl, H and Sarov, M and Treutlein, B and Huttner, WB and Namba, T},
title = {A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {20},
pages = {},
doi = {10.1242/dev.169276},
pmid = {30266827},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Embryo, Mammalian/metabolism ; Ependymoglial Cells/*metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/*metabolism ; Humans ; Lateral Ventricles/embryology ; Mice, Inbred C57BL ; Neocortex/*cytology/*embryology/metabolism ; PAX6 Transcription Factor/metabolism ; Stem Cells/cytology ; },
abstract = {A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.},
}
@article {pmid30266728,
year = {2018},
author = {Morovic, W and Roos, P and Zabel, B and Hidalgo-Cantabrana, C and Kiefer, A and Barrangou, R},
title = {Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline.},
journal = {Applied and environmental microbiology},
volume = {84},
number = {23},
pages = {},
pmid = {30266728},
issn = {1098-5336},
mesh = {Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics/metabolism ; Bifidobacterium animalis/*drug effects/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/drug effects ; Gene Expression Regulation, Bacterial/drug effects ; Genome, Bacterial/drug effects ; Tetracycline/*pharmacology ; Tetracycline Resistance ; Transcription, Genetic/*drug effects ; },
abstract = {Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.},
}
@article {pmid30266652,
year = {2018},
author = {Kantor, B and Tagliafierro, L and Gu, J and Zamora, ME and Ilich, E and Grenier, C and Huang, ZY and Murphy, S and Chiba-Falek, O},
title = {Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {11},
pages = {2638-2649},
pmid = {30266652},
issn = {1525-0024},
support = {R01 NS085011/NS/NINDS NIH HHS/United States ; },
mesh = {Brain/metabolism/pathology ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques ; Cell Differentiation/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; DNA Methylation/*genetics ; DNA Methyltransferase 3A ; Dopaminergic Neurons/metabolism/pathology ; Gene Editing ; Gene Expression Regulation/genetics ; *Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells/metabolism/transplantation ; Introns/genetics ; Parkinson Disease/*genetics/pathology/therapy ; RNA, Guide/genetics ; alpha-Synuclein/*genetics ; },
abstract = {Elevated levels of SNCA have been implicated in the pathogenesis of Parkinson's disease (PD), while normal physiological levels of SNCA are needed to maintain neuronal function. We ought to develop new therapeutic strategies targeting the regulation of SNCA expression. DNA methylation at SNCA intron 1 regulates SNCA transcription, and PD brains showed differential methylation levels compared to controls. Thus, DNA methylation at SNCA intron 1 is an attractive target for fine-tuned downregulation of SNCA levels. Here we developed a system, comprising an all-in-one lentiviral vector, for targeted DNA methylation editing within intron 1. The system is based on CRISPR-deactivated Cas9 (dCas9) fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A). Applying the system to human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons from a PD patient with the SNCA triplication resulted in fine downregulation of SNCA mRNA and protein mediated by targeted DNA methylation at intron 1. Furthermore, the reduction in SNCA levels by the guide RNA (gRNA)-dCas9-DMNT3A system rescued disease-related cellular phenotype characteristics of the SNCA triplication hiPSC-derived dopaminergic neurons, e.g., mitochondrial ROS production and cellular viability. We established that DNA hypermethylation at SNCA intron 1 allows an effective and sufficient tight downregulation of SNCA expression levels, suggesting the potential of this target sequence combined with the CRISPR-dCas9 technology as a novel epigenetic-based therapeutic approach for PD.},
}
@article {pmid30266091,
year = {2018},
author = {Mao, Y and Yang, X and Zhou, Y and Zhang, Z and Botella, JR and Zhu, JK},
title = {Manipulating plant RNA-silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {149},
pmid = {30266091},
issn = {1474-760X},
support = {NA//Chinese Academy of Sciences/International ; NA//Youth Innovation Promotion Association, CAS/International ; },
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Mutation ; *RNA Interference ; RNA, Plant/*metabolism ; Viral Proteins/metabolism ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level.<br><br>RESULTS: Here, we evaluate the expression levels of the two CRISPR/Cas9 components and the gene editing efficiency in a set of Arabidopsis mutants involved in RNA silencing. We find that mutants defective in the post-transcriptional gene-silencing pathway display significantly higher Cas9 and sgRNA transcript levels, resulting in higher mutagenesis frequencies than wild-type controls. Accordingly, silencing of AGO1 by introduction of an AGO1-RNAi cassette into the CRISPR/Cas9 vector provides an increase in gene editing efficiency. Co-expression of the viral suppressor p19 from the tomato bushy stunt virus to suppress the plant RNA-silencing pathway shows a strong correlation between the severity of the phenotypic effects caused by p19 and the gene editing efficiency of the CRISPR/Cas9 system for two different target genes, AP1 and TT4.<br><br>CONCLUSIONS: This system has useful practical applications in facilitating the detection of CRISPR/Cas9-induced mutations in T1 plants as well as the identification of transgene-free T2 plants by simple visual observation of the symptom severity caused by p19. Our study shows that CRISPR/Cas9 gene editing efficiency can be improved by reducing RNA silencing in plants.},
}
@article {pmid30265349,
year = {2018},
author = {Muhr, M and Paulat, M and Awwanah, M and Brinkkötter, M and Teichmann, T},
title = {CRISPR/Cas9-mediated knockout of Populus BRANCHED1 and BRANCHED2 orthologs reveals a major function in bud outgrowth control.},
journal = {Tree physiology},
volume = {38},
number = {10},
pages = {1588-1597},
doi = {10.1093/treephys/tpy088},
pmid = {30265349},
issn = {1758-4469},
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques ; Phenotype ; Plant Leaves/genetics/growth &amp; development ; Plant Proteins/*genetics/metabolism ; Plant Stems/genetics/*growth &amp; development ; Populus/*genetics/*growth &amp; development/metabolism ; Transcription Factors/*genetics/metabolism ; },
abstract = {The TCP-type transcription factors BRANCHED1 and BRANCHED2 shape plant architecture by suppressing bud outgrowth, with BRANCHED2 only playing a minor role in Arabidopsis. Here, we investigated the function of orthologs of these genes in the model tree Populus. We used CRISPR/Cas9 to generate loss-of-function mutants of previously identified Populus BRANCHED1-1 and BRANCHED2-1 candidate genes. BRANCHED1-1 mutants exhibited strongly enhanced bud outgrowth. BRANCHED2-1 mutants had an extreme bud outgrowth phenotype and possessed two ectopic leaves at each node. While BRANCHED1 function is conserved in poplar, BRANCHED2, in contrast to its Arabidopsis counterpart, plays an even more critical role in bud outgrowth regulation. In addition, we identified a new, not yet reported association of this gene to leaf development.},
}
@article {pmid30262816,
year = {2018},
author = {Kragesteen, BK and Spielmann, M and Paliou, C and Heinrich, V and Schöpflin, R and Esposito, A and Annunziatella, C and Bianco, S and Chiariello, AM and Jerković, I and Harabula, I and Guckelberger, P and Pechstein, M and Wittler, L and Chan, WL and Franke, M and Lupiáñez, DG and Kraft, K and Timmermann, B and Vingron, M and Visel, A and Nicodemi, M and Mundlos, S and Andrey, G},
title = {Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis.},
journal = {Nature genetics},
volume = {50},
number = {10},
pages = {1463-1473},
doi = {10.1038/s41588-018-0221-x},
pmid = {30262816},
issn = {1546-1718},
mesh = {Animals ; CRISPR-Cas Systems ; Chromatin/*chemistry/genetics/metabolism ; Chromatin Assembly and Disassembly/genetics ; DNA/chemistry/metabolism ; Embryo, Mammalian ; Enhancer Elements, Genetic/*physiology ; Forelimb/embryology/metabolism ; Gene Expression Regulation, Developmental/genetics ; Hindlimb/*embryology/metabolism ; Mice ; Mice, Transgenic ; *Molecular Conformation ; Morphogenesis/*genetics ; Nucleic Acid Conformation ; Paired Box Transcription Factors/genetics/*physiology ; },
abstract = {The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity and specificity in vivo and its disturbance can result in gene misexpression and disease.},
}
@article {pmid30262503,
year = {2018},
author = {He, S and Del Viso, F and Chen, CY and Ikmi, A and Kroesen, AE and Gibson, MC},
title = {An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis.},
journal = {Science (New York, N.Y.)},
volume = {361},
number = {6409},
pages = {1377-1380},
doi = {10.1126/science.aar8384},
pmid = {30262503},
issn = {1095-9203},
mesh = {Animals ; Bacterial Proteins ; Body Patterning/*genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Endoderm/cytology/growth &amp; development ; Endonucleases ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques/methods ; Genes, Homeobox/genetics/*physiology ; Larva/cytology/genetics/growth &amp; development ; Mutagenesis ; RNA, Small Interfering/genetics ; Sea Anemones/cytology/genetics/*growth &amp; development ; Transcription Factors/genetics/*physiology ; },
abstract = {Hox genes encode conserved developmental transcription factors that govern anterior-posterior (A-P) pattering in diverse bilaterian animals, which display bilateral symmetry. Although Hox genes are also present within Cnidaria, these simple animals lack a definitive A-P axis, leaving it unclear how and when a functionally integrated Hox code arose during evolution. We used short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9 mutagenesis to demonstrate that a Hox-Gbx network controls radial segmentation of the larval endoderm during development of the sea anemone Nematostella vectensis. Loss of Hox-Gbx activity also elicits marked defects in tentacle patterning along the directive (orthogonal) axis of primary polyps. On the basis of our results, we propose that an axial Hox code may have controlled body patterning and tissue segmentation before the evolution of the bilaterian A-P axis.},
}
@article {pmid30261908,
year = {2018},
author = {Dank, A and Smid, EJ and Notebaart, RA},
title = {CRISPR-Cas genome engineering of esterase activity in Saccharomyces cerevisiae steers aroma formation.},
journal = {BMC research notes},
volume = {11},
number = {1},
pages = {682},
pmid = {30261908},
issn = {1756-0500},
mesh = {*CRISPR-Cas Systems ; Carboxylic Ester Hydrolases ; Clustered Regularly Interspaced Short Palindromic Repeats ; Esterases/*genetics/metabolism ; Fermentation ; *Gene Editing ; Mutation ; *Odorants ; Saccharomyces cerevisiae/*enzymology ; Saccharomyces cerevisiae Proteins ; },
abstract = {OBJECTIVE: Saccharomyces cerevisiae is used worldwide for the production of ale-type beers. This yeast is responsible for the production of the characteristic fruity aroma compounds. Esters constitute an important group of aroma active secondary metabolites produced by S. cerevisiae. Previous work suggests that esterase activity, which results in ester degradation, may be the key factor determining the abundance of fruity aroma compounds. Here, we test this hypothesis by deletion of two S. cerevisiae esterases, IAH1 and TIP1, using CRISPR-Cas9 genome editing and by studying the effect of these deletions on esterase activity and extracellular ester pools.<br><br>RESULTS: Saccharomyces cerevisiae mutants were constructed lacking esterase IAH1 and/or TIP1 using CRISPR-Cas9 genome editing. Esterase activity using 5-(6)-carboxyfluorescein diacetate (cFDA) as substrate was found to be significantly lower for &#x394;IAH1 and &#x394;IAH1&#x394;TIP1 mutants compared to wild type (WT) activity (P&#x2009;<&#x2009;0.05 and P&#x2009;<&#x2009;0.001, respectively). As expected, we observed an increase in relative abundance of acetate and ethyl esters and an increase in ethyl esters in &#x394;IAH1 and &#x394;TIP1, respectively. Interestingly, the double gene disruption mutant &#x394;IAH1&#x394;TIP1 showed an aroma profile comparable to WT levels, suggesting the existence and activation of a complex regulatory mechanism to compensate multiple genomic alterations in aroma metabolism.},
}
@article {pmid30261221,
year = {2018},
author = {Mollanoori, H and Shahraki, H and Rahmati, Y and Teimourian, S},
title = {CRISPR/Cas9 and CAR-T cell, collaboration of two revolutionary technologies in cancer immunotherapy, an instruction for successful cancer treatment.},
journal = {Human immunology},
volume = {79},
number = {12},
pages = {876-882},
doi = {10.1016/j.humimm.2018.09.007},
pmid = {30261221},
issn = {1879-1166},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; Neoplasms/genetics/immunology/*therapy ; Receptors, Chimeric Antigen/genetics/*immunology/metabolism ; Reproducibility of Results ; T-Lymphocytes/*immunology/metabolism ; Treatment Outcome ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) technology, an acquired immune system in bacteria and archaea, has provided a new tool for accurately genome editing. Using only a single nuclease protein in complex with 2 short RNA as a site-specific endonuclease made it a simple and flexible genome editing tool to target nearly any genomic locus. Due to recent developments in therapeutic engineered T cell and effective responses of CD19-directed chimeric antigen receptor T cells (CART19) in patients with B-cell leukemia and lymphoma, adoptive T cell immunotherapy, particularly CAR-T cell therapy became a rapidly growing field in cancer therapy and recently Kymriah and Yescarta (CD19-directed CAR-T cells) were approved by FDA. Therefore, the combination of CRISPR/Cas9 technology as a genome engineering tool and CAR-T cell therapy (engineered T cells that express chimeric antigen receptors) may lead to further improvement in efficiency and safety of CAR-T cells. This article reviews mechanism and therapeutic application of CRISPR/Cas9 technology, accuracy of this technology, cancer immunotherapy by CAR T cells, the application of CRISPR technology for the production of universal CAR T cells, improving their antitumor efficacy, and biotech companies that invested in CRISPR technology for CAR-T cell therapy.},
}
@article {pmid30261168,
year = {2019},
author = {Hidalgo-Cantabrana, C and Goh, YJ and Barrangou, R},
title = {Characterization and Repurposing of Type I and Type II CRISPR-Cas Systems in Bacteria.},
journal = {Journal of molecular biology},
volume = {431},
number = {1},
pages = {21-33},
doi = {10.1016/j.jmb.2018.09.013},
pmid = {30261168},
issn = {1089-8638},
mesh = {Archaea/*genetics ; Bacteria/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Plasmids/genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR-Cas systems constitute the adaptive immune system of bacteria and archaea, as a sequence-specific nucleic acid targeting defense mechanism. The sequence-specific recognition and cleavage of Cas effector complexes has been harnessed to developed CRISPR-based technologies and drive the genome editing revolution underway, due to their efficacy, efficiency, and ease of implementation in a broad range of organisms. CRISPR-based technologies offer a wide variety of opportunities in genome remodeling and transcriptional regulation, opening new avenues for therapeutic and biotechnological applications. To repurpose CRISPR-Cas systems for these applications, the various elements of the system need to be first identified and functionally characterized in their native host. Bioinformatic tools are first used to identify putative CRISPR arrays and their associated genes, followed by a comprehensive characterization of the CRISPR-Cas system, encompassing predictions for guide and target sequences. Subsequently, interference assays and transcriptomic analyses should be performed to probe the functionality of the CRISPR-Cas system. Once an endogenous CRISPR-Cas system is characterized as functional, they can be readily repurposed by delivering an engineered synthetic CRISPR array or a small RNA guide for targeted gene manipulation. Alternatively, developing a plasmid-based system for heterologous expression of the necessary CRISPR components can enable exploitation in other organisms. Altogether, there is a wide diversity of native CRISPR-Cas systems in many bacteria and most archaea that await functional characterization and repurposing for genome editing applications in prokaryotes.},
}
@article {pmid30260998,
year = {2018},
author = {Veach, RA and Wilson, MH},
title = {CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line.},
journal = {PloS one},
volume = {13},
number = {9},
pages = {e0204487},
pmid = {30260998},
issn = {1932-6203},
support = {I01 BX002190/BX/BLRD VA/United States ; I01 BX004258/BX/BLRD VA/United States ; P30 DK114809/DK/NIDDK NIH HHS/United States ; R01 DK093660/DK/NIDDK NIH HHS/United States ; },
mesh = {Acute Kidney Injury/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cisplatin/pharmacology ; Gene Knock-In Techniques ; Gene Targeting ; Genes, Reporter ; Genetic Engineering ; Glucose/pharmacology ; Green Fluorescent Proteins/genetics ; Hepatitis A Virus Cellular Receptor 1/*genetics/*metabolism ; Homologous Recombination ; Humans ; Kidney Tubules, Proximal/cytology/drug effects/*metabolism ; Luciferases/genetics ; Up-Regulation/drug effects ; },
abstract = {We used the CRISPR/Cas9 system to knock-in reporter transgenes at the kidney injury molecule-1 (KIM-1) locus and isolated human proximal tubule cell (HK-2) clones. PCR verified targeted knock-in of the luciferase and eGFP reporter at the KIM-1 locus. HK-2-KIM-1 reporter cells responded to various stimuli including hypoxia, cisplatin, and high glucose, indicative of upregulation of KIM-1 expression. We attempted using CRISPR/Cas9 to also engineer the KIM-1 reporter in telomerase-immortalized human RPTEC cells. However, these cells demonstrated an inability to undergo homologous recombination at the target locus. KIM-1-reporter human proximal tubular cells could be valuable tools in drug discovery for molecules inhibiting kidney injury. Additionally, our gene targeting strategy could be used in other cell lines to evaluate the biology of KIM-1 in vitro or in vivo.},
}
@article {pmid30260951,
year = {2018},
author = {Hara, T and Maejima, I and Akuzawa, T and Hirai, R and Kobayashi, H and Tsukamoto, S and Tsunoda, M and Ono, A and Yamakoshi, S and Oikawa, S and Sato, K},
title = {Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007647},
pmid = {30260951},
issn = {1553-7404},
mesh = {Adaptor Proteins, Vesicular Transport ; Amyloid Precursor Protein Secretases/*metabolism ; Animals ; Behavior, Animal ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cerebral Cortex/*growth &amp; development/metabolism ; Chromosome Deletion ; Chromosome Disorders/genetics ; Chromosomes, Human, Pair 1/genetics ; Disease Models, Animal ; Female ; *Gene Expression Regulation, Developmental ; Humans ; Lysosomes/metabolism ; Male ; Membrane Glycoproteins/genetics/*metabolism ; Mice ; Mice, Knockout ; Neural Stem Cells ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Receptors, Notch/metabolism ; },
abstract = {Rer1 is a retrieval receptor for endoplasmic reticulum (ER) retention of various ER membrane proteins and unassembled or immature components of membrane protein complexes. However, its physiological functions during mammalian development remain unclear. This study aimed to investigate the role of Rer1-mediated quality control system in mammalian development. We show that Rer1 is required for the sufficient cell surface expression and activity of γ-secretase complex, which modulates Notch signaling during mouse cerebral cortex development. When Rer1 was depleted in the mouse cerebral cortex, the number of neural stem cells decreased significantly, and malformation of the cerebral cortex was observed. Rer1 loss reduced γ-secretase activity and downregulated Notch signaling in the developing cerebral cortex. In Rer1-deficient cells, a subpopulation of γ-secretase complexes and components was transported to and degraded in lysosomes, thereby significantly reducing the amount of γ-secretase complex on the cell surface. These results suggest that Rer1 maintains Notch signaling by maintaining sufficient expression of the γ-secretase complex on the cell surface and regulating neural stem cell maintenance during cerebral cortex development.},
}
@article {pmid30260068,
year = {2019},
author = {Ebrahimi, S and Teimoori, A and Khanbabaei, H and Tabasi, M},
title = {Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome.},
journal = {Reviews in medical virology},
volume = {29},
number = {1},
pages = {e2009},
doi = {10.1002/rmv.2009},
pmid = {30260068},
issn = {1099-1654},
mesh = {CRISPR-Associated Protein 9/*metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Viruses/*genetics ; Gene Editing/*methods ; Recombination, Genetic ; },
abstract = {The recent development of the Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a genome editing system, has many potential applications in virology. The possibility of introducing site specific breaks has provided new possibilities to precisely manipulate viral genomics. Here, we provide diagrams to summarize the steps involved in the process. We also systematically review recent applications of the CRISPR/Cas9 system for manipulation of DNA virus genomics and discuss the therapeutic potential of the system to treat viral diseases.},
}
@article {pmid30260055,
year = {2019},
author = {Zheng, W and Li, Q and Sun, H and Ali, MW and Zhang, H},
title = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9-mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae).},
journal = {Insect molecular biology},
volume = {28},
number = {2},
pages = {222-234},
doi = {10.1111/imb.12540},
pmid = {30260055},
issn = {1365-2583},
mesh = {Animals ; *CRISPR-Cas Systems ; Flight, Animal/physiology ; Gene Editing/*methods ; Insect Proteins/*physiology ; Muscles/physiology ; Tephritidae/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a versatile, efficient and heritable gene editing tool that can be useful for genome engineering. Bactrocera dorsalis (Hendel) is a major pest of agriculture that causes great economic losses. We used the B. dorsalis multiple edematous wings (Bdmew) gene as the target gene to explore the effectiveness of CRISPR/Cas9 for B. dorsalis genome manipulation. We studied the physiological functions of the Bdmew gene, particularly those related to muscle development. Site-specific genome editing was feasible using direct microinjection of specific guide RNA and the Cas9-plasmid into B. dorsalis embryos. Mutation frequencies ranged from 12.1 to 30.2% in the injected generation. Mosaic G0, with the mew mutation, was heritable to the next generation. The G1 displayed a series of defective phenotypes including muscle weakness, flightlessness, failure to eclose, wing folds and unbalanced movement. These results demonstrated that CRISPR/Cas9 can act as a highly specific, efficient, heritable tool for genome manipulation in B. dorsalis and this has significance for gene function research and genetic control of pests. The Bdmew gene possesses key functions in muscle development of B. dorsalis. Bdmew mutations cause a series of serious defects by interfering with muscle development and may provide a means for controlling B. dorsalis via a gene-based method such as gene drive.},
}
@article {pmid30259067,
year = {2018},
author = {Xiang, G and Ren, J and Hai, T and Fu, R and Yu, D and Wang, J and Li, W and Wang, H and Zhou, Q},
title = {Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {75},
number = {24},
pages = {4619-4628},
pmid = {30259067},
issn = {1420-9071},
mesh = {Alleles ; Animal Husbandry/methods ; Animals ; Breeding/methods ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Genotype ; Insulin-Like Growth Factor II/*genetics ; *Introns ; Male ; Meat/analysis ; Mutation ; Phenotype ; *Regulatory Sequences, Nucleic Acid ; Swine/*genetics ; },
abstract = {Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.},
}
@article {pmid30258941,
year = {2018},
author = {Karlapudi, AP and T C, V and Tammineedi, J and Srirama, K and Kanumuri, L and Prabhakar Kodali, V},
title = {In silico sgRNA tool design for CRISPR control of quorum sensing in Acinetobacter species.},
journal = {Genes &amp; diseases},
volume = {5},
number = {2},
pages = {123-129},
pmid = {30258941},
issn = {2352-3042},
abstract = {CRISPR genome editing utilizes Cas9 nuclease and single guide RNA (sgRNA), which directs the nuclease to a specific site in the genome and makes a double-stranded break (DSB). Design of sgRNA for CRISPR-Cas targeting, and to promote CRISPR adaptation, uses a regulatory mechanism that ensures maximum CRISPR-Cas9 system functions when a bacterial population is at highest risk of phage infection. Acinetobacter baumannii is the most regularly identified gram-negative bacterium infecting patients. Recent reports have demonstrated that the extent of diseases caused by A. baumannii is expanding and, in a few cases, now surpasses the quantity of infections caused by P. aeruginosa. Most Acinetobacter strains possess biofilm-forming ability, which plays a major role in virulence and drug resistance. Biofilm bacteria use quorum sensing, a cell-to-cell communication process, to activate gene expression. Many genes are involved in biofilm formation and the mechanism to disrupt the biofilm network is still not clearly understood. In this study, we performed in silico gene editing to exploit the AbaI gene, responsible for biofilm formation. The study explored different tools available for genome editing to create gene knockouts, selecting the A. baumannii AbaI gene as a target.},
}
@article {pmid30258161,
year = {2018},
author = {DeWeerdt, S},
title = {The genomics of brain cancer.},
journal = {Nature},
volume = {561},
number = {7724},
pages = {S54-S55},
pmid = {30258161},
issn = {1476-4687},
mesh = {Adult ; Animals ; Brain Neoplasms/diagnosis/*genetics/*therapy ; CRISPR-Cas Systems/genetics ; Chromosome Deletion ; Chromosomes, Human, Pair 19/genetics ; Gene Editing ; *Genetic Predisposition to Disease ; Genome, Human/*genetics ; *Genomics ; Glioblastoma/diagnosis/genetics/therapy ; Glioma/diagnosis/genetics/therapy ; Humans ; Isocitrate Dehydrogenase/genetics ; Mice ; Mutation ; Oligodendroglioma/diagnosis/pathology/therapy ; *Pharmacogenomic Testing ; Precision Medicine ; Prognosis ; Survival Analysis ; Telomere Homeostasis/genetics ; Treatment Outcome ; World Health Organization ; },
}
@article {pmid30258105,
year = {2018},
author = {Hamada, H and Liu, Y and Nagira, Y and Miki, R and Taoka, N and Imai, R},
title = {Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14422},
pmid = {30258105},
issn = {2045-2322},
mesh = {Biolistics/*methods ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Meristem/genetics ; Mutagenesis ; Mutation ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Triticum/*genetics ; },
abstract = {The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T1 plants identified plants homozygous for the three homeologous genes, which were all derived from one T0 plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve in planta genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.},
}
@article {pmid30258061,
year = {2018},
author = {Sharma, AK and Nymark, M and Sparstad, T and Bones, AM and Winge, P},
title = {Transgene-free genome editing in marine algae by bacterial conjugation - comparison with biolistic CRISPR/Cas9 transformation.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14401},
pmid = {30258061},
issn = {2045-2322},
support = {GBMF4966//Gordon and Betty Moore Foundation/International ; GBMF4966//Gordon and Betty Moore Foundation/International ; GBMF4966//Gordon and Betty Moore Foundation/International ; },
mesh = {*Biolistics ; *CRISPR-Cas Systems ; *Conjugation, Genetic ; Diatoms/*genetics ; Escherichia coli/*genetics ; Genetic Vectors/*genetics ; Plasmids/*genetics ; *Transformation, Genetic ; },
abstract = {The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.},
}
@article {pmid30255296,
year = {2019},
author = {Adames, NR and Gallegos, JE and Peccoud, J},
title = {Yeast genetic interaction screens in the age of CRISPR/Cas.},
journal = {Current genetics},
volume = {65},
number = {2},
pages = {307-327},
pmid = {30255296},
issn = {1432-0983},
support = {R01 GM078989/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Diploidy ; Gene Editing ; Gene Library ; Genetic Engineering/methods ; Genetic Testing/methods ; *Genome, Fungal ; Genomics/*methods ; Mutation ; Saccharomyces cerevisiae/genetics ; Synthetic Lethal Mutations ; Yeasts/*genetics ; },
abstract = {The ease of performing both forward and reverse genetics in Saccharomyces cerevisiae, along with its stable haploid state and short generation times, has made this budding yeast the consummate model eukaryote for genetics. The major advantage of using budding yeast for reverse genetics is this organism's highly efficient homology-directed repair, allowing for precise genome editing simply by introducing DNA with homology to the chromosomal target. Although plasmid- and PCR-based genome editing tools are quite efficient, they depend on rare spontaneous DNA breaks near the target sequence. Consequently, they can generate only one genomic edit at a time, and the edit must be associated with a selectable marker. However, CRISPR/Cas technology is efficient enough to permit markerless and multiplexed edits in a single step. These features have made CRISPR/Cas popular for yeast strain engineering in synthetic biology and metabolic engineering applications, but it has not been widely employed for genetic screens. In this review, we critically examine different methods to generate multi-mutant strains in systematic genetic interaction screens and discuss the potential of CRISPR/Cas to supplement or improve on these methods.},
}
@article {pmid30254645,
year = {2018},
author = {Li, H and Sheng, C and Liu, H and Wang, S and Zhao, J and Yang, L and Jia, L and Li, P and Wang, L and Xie, J and Xu, D and Sun, Y and Qiu, S and Song, H},
title = {Inhibition of HBV Expression in HBV Transgenic Mice Using AAV-Delivered CRISPR-SaCas9.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {2080},
pmid = {30254645},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; DNA, Viral/genetics/immunology ; *Dependovirus ; *Genetic Therapy ; *Hepatitis B virus/genetics/immunology ; *Hepatitis B, Chronic/genetics/immunology/pathology/therapy ; Mice ; Mice, Transgenic ; *Transduction, Genetic ; },
abstract = {The chronic production of hepatitis B viral (HBV) antigens could cause inflammation and necrosis, leading to elevation of liver enzymes from necrotic hepatocytes, hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. However, no current treatment is capable of significantly reducing HBsAg expression in patients. Our previous studies had confirmed the ability of CRISPR-Cas9 in disrupting HBV cccDNA. Here, to inhibit HBV expression efficiently in the mouse model of chronic HBV infection, the miniaturized CRISPR-SaCas9 system compatible with a HBV core region derived guide-RNA had been packaged in recombinant adeno-associated virus (AAV) type 8, which lowered the levels of serum HBsAg, HBeAg, and HBV DNA efficiently in HBV transgenic mice during 58 days continuous observation after vein injection. It further confirms the potential of the CRISPR-Cas9 technique for use in hepatitis B gene therapy.},
}
@article {pmid30254261,
year = {2018},
author = {Serif, M and Dubois, G and Finoux, AL and Teste, MA and Jallet, D and Daboussi, F},
title = {One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3924},
pmid = {30254261},
issn = {2041-1723},
mesh = {Adenine Phosphoribosyltransferase/genetics/metabolism ; Algal Proteins/genetics/metabolism ; Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems ; Diatoms/*genetics/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Microalgae/genetics/metabolism ; Multienzyme Complexes/genetics/metabolism ; Orotate Phosphoribosyltransferase/genetics/metabolism ; Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism ; Reproducibility of Results ; Ribonucleoproteins/genetics/metabolism ; Sequence Homology, Amino Acid ; Transfection/*methods ; },
abstract = {Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.},
}
@article {pmid30254203,
year = {2018},
author = {Foster, AJ and Martin-Urdiroz, M and Yan, X and Wright, HS and Soanes, DM and Talbot, NJ},
title = {CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14355},
pmid = {30254203},
issn = {2045-2322},
support = {294702/ERC_/European Research Council/International ; BB/ BB/N009959/1//Biotechnology and Biological Sciences Research Council (BBSRC)/International ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Magnaporthe/*genetics/metabolism/*physiology ; Melanins/biosynthesis ; Mutation ; Oryza/*microbiology ; Plant Diseases/*microbiology ; Polymorphism, Single Nucleotide ; Ribonucleoproteins/*metabolism ; },
abstract = {The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.},
}
@article {pmid30254120,
year = {2018},
author = {Cernak, P and Estrela, R and Poddar, S and Skerker, JM and Cheng, YF and Carlson, AK and Chen, B and Glynn, VM and Furlan, M and Ryan, OW and Donnelly, MK and Arkin, AP and Taylor, JW and Cate, JHD},
title = {Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.},
journal = {mBio},
volume = {9},
number = {5},
pages = {},
pmid = {30254120},
issn = {2150-7511},
mesh = {Biotechnology ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genes, Mating Type, Fungal/genetics ; *Genetic Engineering ; Kluyveromyces/*genetics/metabolism ; Lipid Metabolism ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/*methods ; Temperature ; Thermotolerance ; },
abstract = {Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.},
}
@article {pmid30254056,
year = {2018},
author = {Luttrell, LM and Wang, J and Plouffe, B and Smith, JS and Yamani, L and Kaur, S and Jean-Charles, PY and Gauthier, C and Lee, MH and Pani, B and Kim, J and Ahn, S and Rajagopal, S and Reiter, E and Bouvier, M and Shenoy, SK and Laporte, SA and Rockman, HA and Lefkowitz, RJ},
title = {Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.},
journal = {Science signaling},
volume = {11},
number = {549},
pages = {},
pmid = {30254056},
issn = {1937-9145},
support = {R01 HL118369/HL/NHLBI NIH HHS/United States ; I01 BX003188/BX/BLRD VA/United States ; R01 DK055524/DK/NIDDK NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 HL016037/HL/NHLBI NIH HHS/United States ; R35 GM126955/GM/NIGMS NIH HHS/United States ; R01 HL056687/HL/NHLBI NIH HHS/United States ; R01 GM122798/GM/NIGMS NIH HHS/United States ; T32 GM007171/GM/NIGMS NIH HHS/United States ; 14843//CIHR/Canada ; MOP-74603//CIHR/Canada ; K08 HL114643/HL/NHLBI NIH HHS/United States ; P01 HL075443/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Membrane/metabolism ; Enzyme Activation ; Gene Deletion ; Gene Editing ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; MAP Kinase Signaling System ; Phosphorylation ; RNA, Small Interfering/*metabolism ; Receptors, Adrenergic, beta-2/metabolism ; Receptors, G-Protein-Coupled/*metabolism ; *Signal Transduction ; beta-Arrestins/*metabolism ; },
abstract = {G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.},
}
@article {pmid30254054,
year = {2018},
author = {Gurevich, VV and Gurevich, EV},
title = {Arrestins and G proteins in cellular signaling: The coin has two sides.},
journal = {Science signaling},
volume = {11},
number = {549},
pages = {},
pmid = {30254054},
issn = {1937-9145},
support = {R01 EY011500/EY/NEI NIH HHS/United States ; R01 NS065868/NS/NINDS NIH HHS/United States ; R21 DA030103/DA/NIDA NIH HHS/United States ; R35 GM122491/GM/NIGMS NIH HHS/United States ; },
mesh = {*Arrestins ; *CRISPR-Cas Systems ; Phosphorylation ; RNA, Small Interfering ; beta-Arrestins ; },
abstract = {Several studies have suggested that arrestin-mediated signaling by GPCRs requires G protein activation; however, in this issue of Science Signaling, Luttrell et al. documented arrestin-dependent activation of ERK1/2 by a number of GPCRs. These studies do not contradict each other, but illustrate the complexity of cellular signaling that cannot and should not be reduced to simplistic models.},
}
@article {pmid30253789,
year = {2018},
author = {Gaj, T and Perez-Pinera, P},
title = {The continuously evolving CRISPR barcoding toolbox.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {143},
pmid = {30253789},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Two articles recently described the development of CRISPR technologies that have the potential to fundamentally transform the barcoding and tracing of mammalian cells.},
}
@article {pmid30253761,
year = {2018},
author = {Chen, D and Tang, JX and Li, B and Hou, L and Wang, X and Kang, L},
title = {CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {60},
pmid = {30253761},
issn = {1472-6750},
mesh = {Animal Migration ; Animals ; *CRISPR-Cas Systems ; Exons ; Frameshift Mutation ; Gene Editing/*methods ; Grasshoppers/*genetics/physiology ; INDEL Mutation ; Insect Proteins/genetics/metabolism ; *RNA Splicing ; Receptors, Odorant/genetics/metabolism ; Sequence Deletion ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame exon skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo.<br><br>RESULTS: We showed that frameshift indels also induced complete or stochastic exon skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55&#xa0;bp at the boundary of intron 3 and exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete exon 4 skipping, whereas the lacking 22&#xa0;bp in exon 4 of LmigOr35 only resulted in stochastic exon 4 skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and exon to disrupt the 3' splicing site causing completely exon skipping, or alternatively induce small insertions or deletions in the exon to stochastic alter splicing causing the stochastic exon skipping.<br><br>CONCLUSIONS: These results indicated that complete or stochastic exon skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although exon skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.},
}
@article {pmid30252954,
year = {2018},
author = {Trothe, J and Ritzmann, D and Lang, V and Scholz, P and Pul, &#xdc; and Kaufmann, R and Buerger, C and Ertongur-Fauth, T},
title = {Hypotonic stress response of human keratinocytes involves LRRC8A as component of volume-regulated anion channels.},
journal = {Experimental dermatology},
volume = {27},
number = {12},
pages = {1352-1360},
doi = {10.1111/exd.13789},
pmid = {30252954},
issn = {1600-0625},
mesh = {Anions/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Epidermis/*metabolism ; Fluoresceins/chemistry ; Gene Expression Profiling ; HEK293 Cells ; Humans ; Keratinocytes/*cytology/metabolism ; Membrane Proteins/*metabolism ; Osmoregulation ; Osmosis ; Osmotic Pressure ; Protein Multimerization ; Sequence Analysis, RNA ; },
abstract = {The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume-regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT- and NHEK-LRRC8A knockout cells by using CRISPR-Cas9. I[-] influx assays using halide-sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein-AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.},
}
@article {pmid30252203,
year = {2018},
author = {Ding, M and Liu, Y and Li, J and Yao, L and Liao, X and Xie, H and Yang, K and Zhou, Q and Liu, Y and Huang, W and Cai, Z},
title = {Oestrogen promotes tumorigenesis of bladder cancer by inducing the enhancer RNA-eGREB1.},
journal = {Journal of cellular and molecular medicine},
volume = {22},
number = {12},
pages = {5919-5927},
pmid = {30252203},
issn = {1582-4934},
mesh = {Aged ; Apoptosis/drug effects ; CRISPR-Cas Systems/genetics ; Carcinogenesis/drug effects/genetics/*pathology ; Cell Line, Tumor ; Cell Movement/drug effects/genetics ; Cell Proliferation/drug effects/genetics ; Down-Regulation/drug effects ; Enhancer Elements, Genetic/*genetics ; Estrogens/*adverse effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; RNA/*genetics ; RNA, Small Interfering/metabolism ; Up-Regulation/drug effects/genetics ; Urinary Bladder Neoplasms/genetics/*pathology ; },
abstract = {In recent years, studies have shown that enhancer RNAs (eRNAs) can be transcribed from enhancers. Increasing evidence has revealed that eRNAs play critical roles in the development of various cancers. Oestrogen-associated eRNAs are closely related to breast cancer. In view of the gender differences in bladder cancer (BCa), we suppose that oestrogen-associated eRNAs are also involved in tumorigenesis of BCa. In our study, we first demonstrated that eGREB1 derived from the enhancer of an oestrogen-responsive gene-GREB1 was up-regulated in BCa tissues, and the expression level of eGREB1 is positively associated with the histological grade and TNM stage of BCa. Knockdown of eGREB1 by CRISPR-Cas13a could inhibit cell proliferation, migration and invasion and induce apoptosis in BCa cells T24 and 5637. Besides, we exhibited the promoting effect of oestrogen on BCa cells. What's more, down-regulation of eGREB1 could improve the malignant biological characteristics of BCa cells induced by oestrogen. In conclusion, our data indicated that eGREB1 plays oncogenic role and oestrogen may promote the occurrence and progression of BCa by inducing eGREB1 production. Our findings provide new insights into the prevention of BCa and develop a novel therapeutic target for the treatment of BCa.},
}
@article {pmid30251692,
year = {2018},
author = {Luanpitpong, S and Poohadsuan, J and Samart, P and Kiratipaiboon, C and Rojanasakul, Y and Issaragrisil, S},
title = {Reactive oxygen species mediate cancer stem-like cells and determine bortezomib sensitivity via Mcl-1 and Zeb-1 in mantle cell lymphoma.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1864},
number = {11},
pages = {3739-3753},
doi = {10.1016/j.bbadis.2018.09.010},
pmid = {30251692},
issn = {1879-260X},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/drug effects ; Biopsy ; Bortezomib/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Free Radical Scavengers/pharmacology ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Lymph Nodes/pathology ; Lymphoma, Mantle-Cell/*drug therapy/pathology ; Middle Aged ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists &amp; inhibitors/genetics/*metabolism ; Neoplasm Recurrence, Local/pathology/prevention &amp; control ; Neoplastic Stem Cells/drug effects/metabolism ; Primary Cell Culture ; Reactive Oxygen Species/*metabolism ; Zinc Finger E-box-Binding Homeobox 1/*metabolism ; },
abstract = {Mantle cell lymphoma (MCL) is an aggressive, incurable non-Hodgkin B-cell lymphoma with good initial response to therapy then subsequently relapse. Cancer stem cells (CSCs) are considered to be an underlying cause of these inevitable drug resistance and tumor regrowth, but how CSCs are regulated is largely unknown. We demonstrate here for the first time the existence of CSC-like subpopulations that are modulated by reactive oxygen species (ROS) in MCL cell lines and patient-derived primary cells in an inverse correlation with bortezomib (BTZ) sensitivity. Using various known donors and inhibitors of cellular superoxide (O2[-]), hydrogen peroxide (H2O2) and hydroxyl radical (OH), we unveil their distinct roles in the regulation of CSC-like subpopulations and thus MCL response to BTZ. O2[-] inhibits CSC-like cells and sensitizes BTZ-induced apoptosis, whereas H2O2 conversely enriches CSC-like cells and protects against apoptosis and OH has minimal effects. We further observed that an anti-apoptotic Mcl-1 and a transcription factor Zeb-1 are favorable targets of O2[-] and H2O2, respectively. Using small molecule inhibition, ectopic expression and CRISPR/Cas9-mediated gene manipulation, we verified the roles of Mcl-1 and Zeb-1 in CSC and apoptosis regulation by O2[-] and H2O2. Our findings provide a novel mechanistic insight into the significance of redox status of MCL cells in determining their drug response via CSC-like subpopulations, which are imperative to a better understanding of therapeutic resistance and relapse.},
}
@article {pmid30251682,
year = {2018},
author = {Montioli, R and Desbats, MA and Grottelli, S and Doimo, M and Bellezza, I and Borri Voltattorni, C and Salviati, L and Cellini, B},
title = {Molecular and cellular basis of ornithine δ-aminotransferase deficiency caused by the V332M mutation associated with gyrate atrophy of the choroid and retina.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1864},
number = {11},
pages = {3629-3638},
doi = {10.1016/j.bbadis.2018.08.032},
pmid = {30251682},
issn = {1879-260X},
mesh = {CRISPR-Cas Systems/genetics ; Coenzymes/metabolism ; Enzyme Assays ; Gene Knockout Techniques ; Gyrate Atrophy/drug therapy/*genetics/pathology ; HEK293 Cells ; Holoenzymes/genetics/metabolism ; Humans ; Mutagenesis, Site-Directed ; Ornithine-Oxo-Acid Transaminase/*genetics/metabolism ; Point Mutation ; Protein Aggregation, Pathological/drug therapy/*genetics/pathology ; Pyridoxal Phosphate/*metabolism ; Pyridoxine/pharmacology/therapeutic use ; Recombinant Proteins/genetics/metabolism ; Treatment Outcome ; Vitamin B Complex/*pharmacology/therapeutic use ; },
abstract = {Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5'-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic-γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs.},
}
@article {pmid30251625,
year = {2018},
author = {Emmer, BT and Hesketh, GG and Kotnik, E and Tang, VT and Lascuna, PJ and Xiang, J and Gingras, AC and Chen, XW and Ginsburg, D},
title = {The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30251625},
issn = {2050-084X},
support = {R35 HL135793/HL/NHLBI NIH HHS/United States ; T32 HL007853/HL/NHLBI NIH HHS/United States ; R35-HL135793T/HL/NHLBI NIH HHS/United States ; T32-HL007853/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Biotinylation ; CRISPR-Cas Systems/genetics ; Endoplasmic Reticulum/metabolism ; Gene Deletion ; HEK293 Cells ; Humans ; Membrane Proteins/*metabolism ; Mutagenesis/genetics ; Proprotein Convertase 9/*metabolism ; Protein Binding ; Recombinant Fusion Proteins/metabolism ; Secretory Pathway ; },
abstract = {PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.},
}
@article {pmid30251378,
year = {2019},
author = {da Silva Xavier, A and de Almeida, JCF and de Melo, AG and Rousseau, GM and Tremblay, DM and de Rezende, RR and Moineau, S and Alfenas-Zerbini, P},
title = {Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex.},
journal = {Molecular plant pathology},
volume = {20},
number = {2},
pages = {223-239},
pmid = {30251378},
issn = {1364-3703},
mesh = {Adaptive Immunity/physiology ; Bacteriophages/genetics/metabolism/pathogenicity ; CRISPR-Cas Systems/*genetics ; Ralstonia solanacearum/*genetics/*virology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are flanked by associated (Cas) genes. CRISPR-Cas systems are found in the genomes of several microbes and can act as an adaptive immune mechanism against invading foreign nucleic acids, such as phage genomes. Here, we studied the CRISPR-Cas systems in plant-pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR-Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR-Cas types I-E and II-C were found, with I-E being the most common. The presence of the same CRISPR-Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. In addition, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstoniasolanacearum strains CFBP2957 and K60[T] were challenged with a virulent phage, and the CRISPR arrays of bacteriophage-insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-protospacer adjacent motif (PAM) delivery system, and no resistance was observed against phage phiAP1. Our results show that the CRISPR-Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy.},
}
@article {pmid30251319,
year = {2019},
author = {Martin, RM and Moniruzzaman, M and Mucci, NC and Willis, A and Woodhouse, JN and Xian, Y and Xiao, C and Brussaard, CPD and Wilhelm, SW},
title = {Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance.},
journal = {Environmental microbiology},
volume = {21},
number = {6},
pages = {1942-1956},
doi = {10.1111/1462-2920.14425},
pmid = {30251319},
issn = {1462-2920},
support = {//Kenneth &amp; Blaire Mossman Endowment/International ; //NIOZ/International ; LP130100311//ARC-Linkage/International ; //University of Tennessee/International ; DEB 1240870//National Science Foundation/International ; IOS 141528//National Science Foundation/International ; },
mesh = {Australia ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cyanobacteria/physiology/*virology ; Ecosystem ; Fresh Water/microbiology/virology ; Genome, Viral ; Genomics ; *Host Specificity ; Nitrogen ; Virus Physiological Phenomena ; Viruses/genetics/*isolation &amp; purification ; },
abstract = {Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen-fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus-host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV-01T, a previously isolated cyanosiphovirus, and its host, C. raciborskii strain Cr2010. Analyses suggest that CrV-01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV-01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR-Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV-01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV-01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus-host interactions in an ecologically important bloom-forming cyanobacterium.},
}
@article {pmid30250197,
year = {2018},
author = {Buchrieser, J and Oliva-Martin, MJ and Moore, MD and Long, JCD and Cowley, SA and Perez-Simón, JA and James, W and Venero, JL},
title = {RIPK1 is a critical modulator of both tonic and TLR-responsive inflammatory and cell death pathways in human macrophage differentiation.},
journal = {Cell death &amp; disease},
volume = {9},
number = {10},
pages = {973},
pmid = {30250197},
issn = {2041-4889},
support = {WTISSF121302/WT_/Wellcome Trust/United Kingdom ; 090532/Z/09/Z/WT_/Wellcome Trust/United Kingdom ; G0900747 91070/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Adaptor Proteins, Vesicular Transport/metabolism ; Apoptosis/*physiology ; CRISPR-Cas Systems/genetics ; Caspase 8/metabolism ; Cell Differentiation/*physiology ; Gene Editing ; Gene Knockout Techniques ; Hematopoiesis/physiology ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Inflammation/*metabolism ; Macrophages/*metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/genetics/*metabolism ; Toll-Like Receptor 3/*metabolism ; Toll-Like Receptor 4/*metabolism ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {In this study, we took advantage of human-induced pluripotent stem cells (hiPSC) and CRISPR/Cas9 technology to investigate the potential roles of RIPK1 in regulating hematopoiesis and macrophage differentiation, proinflammatory activation, and cell death pathways. Knock-out of RIPK1 in hiPSCs demonstrated that this protein is not required for erythro-myeloid differentiation. Using a well-established macrophage differentiation protocol, knock-out of RIPK1 did not block the differentiation of iPSC-derived macrophages, which displayed a similar phenotype to WT hiPSC-derived macrophages. However, knock-out of RIPK1 leads to a TNFα-dependent apoptotic death of differentiated hiPSC-derived macrophages (iPS-MΦ) and progressive loss of iPS-MΦ production irrespective of external pro-inflammatory stimuli. Live video analysis demonstrated that TLR3/4 activation of RIPK1 KO hiPSC-derived macrophages triggered TRIF and RIPK3-dependent necroptosis irrespective of caspase-8 activation. In contrast, TLR3/4 activation of WT macrophages-induced necroptosis only when caspases were inhibited, confirming the modulating effect of RIPK1 on RIPK3-mediated necroptosis through the FADD, Caspase-8 pathway. Activation of these inflammatory pathways required RIPK3 kinase activity while RIPK1 was dispensable. However, loss of RIPK1 sensitizes macrophages to activate RIPK3 in response to inflammatory stimuli, thereby exacerbating a potentially pathological inflammatory response. Taken together, these results reveal that RIPK1 has an important role in regulating the potent inflammatory pathways in authentic human macrophages that are poised to respond to external stimuli. Consequently, RIPK1 activity might be a valid target in the development of novel therapies for chronic inflammatory diseases.},
}
@article {pmid30248362,
year = {2018},
author = {Hübner, A and Keil, GM and Kabuuka, T and Mettenleiter, TC and Fuchs, W},
title = {Efficient transgene insertion in a pseudorabies virus vector by CRISPR/Cas9 and marker rescue-enforced recombination.},
journal = {Journal of virological methods},
volume = {262},
number = {},
pages = {38-47},
doi = {10.1016/j.jviromet.2018.09.009},
pmid = {30248362},
issn = {1879-0984},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial/*genetics ; Genetic Vectors ; Genome, Viral/genetics ; Herpesvirus 1, Suid/*genetics ; Mutagenesis, Insertional/*methods ; Open Reading Frames ; Rabbits ; *Recombination, Genetic ; Swine ; *Transgenes ; },
abstract = {For development of vectored vaccines against porcine pathogens the genome of the pseudorabies virus vaccine strain Bartha (PrV-Ba) was previously cloned as an infectious bacterial artificial chromosome (BAC), containing the bacterial replicon and a reporter gene cassette encoding enhanced green fluorescent protein (EGFP) at the nonessential glycoprotein G locus. To facilitate substitution of this insertion, this BAC was now modified by deletion of the adjacent promoter and initiation codon of the essential glycoprotein D (gD) gene of PrV-Ba. Furthermore, rabbit kidney (RK13) cells stably expressing Cas9 nuclease and an EGFP gene-specific guide RNA were prepared to induce site specific cleavage of the BAC DNA. After co-transfection of these cells with the modified BAC and recombination plasmids containing expression cassettes for new transgenes flanked by PrV DNA sequences including the intact 5'-end of the gD gene, >95% of the recombinants exhibited the desired gene substitutions, while no EGFP-expressing progeny virus was detectable. This approach was used for insertion and expression of the open reading frames E199L, CP204L (p30) and KP177R (p22) of African swine fever virus. The studies revealed that codon adaptation significantly enhanced expression of E199L, and that the chimeric CAG promoter increased transgene expression compared to cytomegalovirus immediate-early promoters.},
}
@article {pmid30248107,
year = {2018},
author = {Wu, S and Zhang, M and Yang, X and Peng, F and Zhang, J and Tan, J and Yang, Y and Wang, L and Hu, Y and Peng, Q and Li, J and Liu, Y and Guan, Y and Chen, C and Hamer, MA and Nijsten, T and Zeng, C and Adhikari, K and Gallo, C and Poletti, G and Schuler-Faccini, L and Bortolini, MC and Canizales-Quinteros, S and Rothhammer, F and Bedoya, G and González-José, R and Li, H and Krutmann, J and Liu, F and Kayser, M and Ruiz-Linares, A and Tang, K and Xu, S and Zhang, L and Jin, L and Wang, S},
title = {Genome-wide association studies and CRISPR/Cas9-mediated gene editing identify regulatory variants influencing eyebrow thickness in humans.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007640},
pmid = {30248107},
issn = {1553-7404},
support = {BB/I021213/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromosomes, Human/genetics ; Eyebrows/*growth &amp; development ; Forkhead Transcription Factors/genetics ; Gene Editing ; Genetic Loci/*genetics ; Genome-Wide Association Study ; Genotype ; Humans ; *Phenotype ; Polymorphism, Single Nucleotide ; SOXB1 Transcription Factors/genetics ; Selection, Genetic ; },
abstract = {Hair plays an important role in primates and is clearly subject to adaptive selection. While humans have lost most facial hair, eyebrows are a notable exception. Eyebrow thickness is heritable and widely believed to be subject to sexual selection. Nevertheless, few genomic studies have explored its genetic basis. Here, we performed a genome-wide scan for eyebrow thickness in 2961 Han Chinese. We identified two new loci of genome-wide significance, at 3q26.33 near SOX2 (rs1345417: P = 6.51×10(-10)) and at 5q13.2 near FOXD1 (rs12651896: P = 1.73×10(-8)). We further replicated our findings in the Uyghurs, a population from China characterized by East Asian-European admixture (N = 721), the CANDELA cohort from five Latin American countries (N = 2301), and the Rotterdam Study cohort of Dutch Europeans (N = 4411). A meta-analysis combining the full GWAS results from the three cohorts of full or partial Asian descent (Han Chinese, Uyghur and Latin Americans, N = 5983) highlighted a third signal of genome-wide significance at 2q12.3 (rs1866188: P = 5.81×10(-11)) near EDAR. We performed fine-mapping and prioritized four variants for further experimental verification. CRISPR/Cas9-mediated gene editing provided evidence that rs1345417 and rs12651896 affect the transcriptional activity of the nearby SOX2 and FOXD1 genes, which are both involved in hair development. Finally, suitable statistical analyses revealed that none of the associated variants showed clear signals of selection in any of the populations tested. Contrary to popular speculation, we found no evidence that eyebrow thickness is subject to strong selective pressure.},
}
@article {pmid30248089,
year = {2018},
author = {Chabas, H and Lion, S and Nicot, A and Meaden, S and van Houte, S and Moineau, S and Wahl, LM and Westra, ER and Gandon, S},
title = {Evolutionary emergence of infectious diseases in heterogeneous host populations.},
journal = {PLoS biology},
volume = {16},
number = {9},
pages = {e2006738},
pmid = {30248089},
issn = {1545-7885},
support = {BB/N017412/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; 109776/Z/15/Z//Wellcome Trust/United Kingdom ; BB/R010781/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; Bacteriophages/physiology ; Biodiversity ; *Biological Evolution ; Communicable Diseases/*microbiology/parasitology/*virology ; Disease Resistance ; *Host-Pathogen Interactions ; Humans ; Models, Biological ; Probability ; },
abstract = {The emergence and re-emergence of pathogens remains a major public health concern. Unfortunately, when and where pathogens will (re-)emerge is notoriously difficult to predict, as the erratic nature of those events is reinforced by the stochastic nature of pathogen evolution during the early phase of an epidemic. For instance, mutations allowing pathogens to escape host resistance may boost pathogen spread and promote emergence. Yet, the ecological factors that govern such evolutionary emergence remain elusive because of the lack of ecological realism of current theoretical frameworks and the difficulty of experimentally testing their predictions. Here, we develop a theoretical model to explore the effects of the heterogeneity of the host population on the probability of pathogen emergence, with or without pathogen evolution. We show that evolutionary emergence and the spread of escape mutations in the pathogen population is more likely to occur when the host population contains an intermediate proportion of resistant hosts. We also show that the probability of pathogen emergence rapidly declines with the diversity of resistance in the host population. Experimental tests using lytic bacteriophages infecting their bacterial hosts containing Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR-Cas) immune defenses confirm these theoretical predictions. These results suggest effective strategies for cross-species spillover and for the management of emerging infectious diseases.},
}
@article {pmid30247490,
year = {2018},
author = {Kyrou, K and Hammond, AM and Galizi, R and Kranjc, N and Burt, A and Beaghton, AK and Nolan, T and Crisanti, A},
title = {A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.},
journal = {Nature biotechnology},
volume = {36},
number = {11},
pages = {1062-1066},
pmid = {30247490},
issn = {1546-1696},
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Cas Systems/*genetics ; DNA-Binding Proteins ; Exons/genetics ; Female ; *Gene Drive Technology ; Gene Targeting ; Insect Proteins/genetics/*metabolism ; Introns/genetics ; Male ; Mosquito Vectors/*genetics ; Sex Determination Processes/genetics ; },
abstract = {In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR-Cas9-targeted disruption of the intron 4-exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR-Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7-11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.},
}
@article {pmid30245733,
year = {2018},
author = {Hsu, DS and Kornepati, AV and Glover, W and Kennedy, EM and Cullen, BR},
title = {Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth in vivo.},
journal = {Future virology},
volume = {13},
number = {7},
pages = {475-482},
pmid = {30245733},
issn = {1746-0794},
support = {R21 CA198050/CA/NCI NIH HHS/United States ; },
abstract = {AIM: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor in vivo.<br><br>MATERIALS &amp; METHODS: We grew HPV16[+], patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs.<br><br>RESULTS &amp; CONCLUSION: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9. These studies provide proof of principle for the hypothesis that CRISPR/Cas has the potential to be used to selectively treat HPV-induced tumors in humans.},
}
@article {pmid30245013,
year = {2018},
author = {Sharon, E and Chen, SA and Khosla, NM and Smith, JD and Pritchard, JK and Fraser, HB},
title = {Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing.},
journal = {Cell},
volume = {175},
number = {2},
pages = {544-557.e16},
pmid = {30245013},
issn = {1097-4172},
support = {R01 GM097171/GM/NIGMS NIH HHS/United States ; T32 GM007276/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Chromosome Mapping ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genetic Variation/genetics ; Genetic Vectors ; Genome ; High-Throughput Nucleotide Sequencing/*methods ; Saccharomyces cerevisiae/*genetics ; Yeasts/genetics ; },
abstract = {A major challenge in genetics is to identify genetic variants driving natural phenotypic variation. However, current methods of genetic mapping have limited resolution. To address this challenge, we developed a CRISPR-Cas9-based high-throughput genome editing approach that can introduce thousands of specific genetic variants in a single experiment. This enabled us to study the fitness consequences of 16,006 natural genetic variants in yeast. We identified 572 variants with significant fitness differences in glucose media; these are highly enriched in promoters, particularly in transcription factor binding sites, while only 19.2% affect amino acid sequences. Strikingly, nearby variants nearly always favor the same parent's alleles, suggesting that lineage-specific selection is often driven by multiple clustered variants. In sum, our genome editing approach reveals the genetic architecture of fitness variation at single-base resolution and could be adapted to measure the effects of genome-wide genetic variation in any screen for cell survival or cell-sortable markers.},
}
@article {pmid30244670,
year = {2018},
author = {Koonin, EV},
title = {Open questions: CRISPR biology.},
journal = {BMC biology},
volume = {16},
number = {1},
pages = {95},
pmid = {30244670},
issn = {1741-7007},
mesh = {Archaea/genetics/immunology/*physiology ; Bacterial Physiological Phenomena/*genetics/immunology ; CRISPR-Cas Systems/immunology/*physiology ; },
abstract = {CRISPR-Cas systems, the purveyors of adaptive immunity in archaea and bacteria and sources of the new generation of genome engineering tools, have been studied in exquisite molecular detail. However, when it comes to biological functions, ecology, and evolution of CRISPR-Cas, many more intriguing questions remain than there are answers.},
}
@article {pmid30244462,
year = {2018},
author = {Liu, S},
title = {Mesenchymal Stem Cell Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1868},
number = {},
pages = {145-150},
doi = {10.1007/978-1-4939-8802-0_15},
pmid = {30244462},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics ; Cell Separation ; Chromosomes, Human/metabolism ; Gene Editing ; Gene Knockout Techniques ; Humans ; Mesenchymal Stem Cells/*cytology ; Synovial Membrane/cytology ; Tissue Engineering/*methods ; },
abstract = {Mesenchymal stem cells (MSC) are multipotent stem cells that display the capacity to generate the tissue in which they reside. MSC have been used as progenitor cells to engineer cartilage implants that can be used to repair chondral and osteochondral lesions, or as trophic producers of bioactive factors to initiate endogenous regenerative activities in the arthritic joint. Targeted gene therapy might further enhance the capacity of MSC for chondrogenesis. By using a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein genomic manipulation technique, target-gene-modified MSC would be a promising therapeutic option for regeneration of diseased joints in the treatment of RA.},
}
@article {pmid30244429,
year = {2018},
author = {Yang, Z and Chen, S and Xue, S and Li, X and Sun, Z and Yang, Y and Hu, X and Geng, T and Cui, H},
title = {Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model.},
journal = {Biotechnology letters},
volume = {40},
number = {11-12},
pages = {1507-1518},
pmid = {30244429},
issn = {1573-6776},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; CRISPR-Cas Systems/*genetics ; Endogenous Retroviruses/*genetics ; Female ; Gene Editing/*methods ; Gene Knockout Techniques ; Male ; Promoter Regions, Genetic/genetics ; Zebrafish/*genetics ; },
abstract = {OBJECTIVES: To investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.<br><br>RESULTS: Here we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.<br><br>CONCLUSIONS: The transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.},
}
@article {pmid30244086,
year = {2018},
author = {Kim, MS and Shin, MJ and Kim, KH},
title = {Increase of viral hemorrhagic septicemia virus growth by knockout of IRF9 gene in Epithelioma papulosum cyprini cells.},
journal = {Fish &amp; shellfish immunology},
volume = {83},
number = {},
pages = {443-448},
doi = {10.1016/j.fsi.2018.09.025},
pmid = {30244086},
issn = {1095-9947},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Fishes ; Gene Editing ; Gene Knockout Techniques ; Interferon Type I/immunology ; Interferon-Stimulated Gene Factor 3, gamma Subunit/*genetics ; Novirhabdovirus/*physiology ; Poly I-C/pharmacology ; Virus Replication ; },
abstract = {Viral hemorrhagic septicemia virus (VHSV) has been a notorious pathogen in freshwater and marine fish. Due to the lack of effective treatment measures against VHSV disease, the development of prophylactic vaccines has been required, and methods that can produce high-titered viruses would be advantageous in producing cost-effective vaccines. Type I interferon (IFN) responses are the key elements of vertebrates' antiviral activities, and IFN-stimulated gene factor 3 (ISGF3) complex formed through type I IFNs up-regulates the expression of IFN-stimulated genes (ISGs). IFN regulatory factor 9 (IRF9) is a key component of ISGF3, so the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity. In this study, to increase the replication of VHSV, we generated IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contains an EPC cell's U6 promoter-driven guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette. In the clones of IRF9 knockout EPC cells, there were no increase in ISG15 gene by poly I:C, and in Mx1 gene by both poly I:C and VHSV. Interestingly, although the increased folds were conspicuously lower than control EPC cells, the expression of ISG 15 gene in all the IRF9 knockout clones was significantly increased by VHSV infection. Control EPC cells pre-treated with poly I:C did not show any CPE when infected with VHSV, however, IRF9 knockout EPC cells showed CPE by VHSV infection in spite of being pretreated with poly I:C. The replication of VHSV in IRF9 knockout EPC cells was significantly faster and higher than that in control EPC cells indicating that the IRF9 knockout-mediated decrease of type I IFN responses allowed VHSV to replicate efficiently. Considering an economical aspect for the production of fish vaccines, the present IRF9 knockout EPC cells can be used to get higher-titered VHSV.},
}
@article {pmid30243976,
year = {2018},
author = {Zhang, B and Xia, Q and Wang, Q and Xia, X and Wang, J},
title = {Detecting and typing target DNA with a novel CRISPR-typing PCR (ctPCR) technique.},
journal = {Analytical biochemistry},
volume = {561-562},
number = {},
pages = {37-46},
doi = {10.1016/j.ab.2018.09.012},
pmid = {30243976},
issn = {1096-0309},
mesh = {CRISPR-Cas Systems/*genetics ; DNA, Viral/*analysis/*genetics ; Human Papillomavirus DNA Tests/*methods ; Humans ; Papillomaviridae/*genetics/*isolation &amp; purification ; Polymerase Chain Reaction/*methods ; },
abstract = {This study develops a new method for detecting and typing the interested DNAs based on CRISPR, which was named as ctPCR3.0, representing CRISPR- or Cas9/sgRNA-typing PCR, version 3.0. This technique detects target DNA in just one homogeneous step: quantitative PCR (qPCR) amplifying the Cas9/sgRNA-cleaved DNA samples. By directly adding Cas9 and sgRNA into the qPCR reaction and giving an additional isothermal incubation before qPCR program, the target DNA can be homogeneously detected in as few as 2 h. Without opening the detecting tube in the whole detection process, ctPCR3.0 can be used to detect target DNA as the traditional qPCR detection. The technique was fully verified by detecting the cloned HPV L1 genes of 10 high-risk human papillomavirus (HPV) subtypes. The technique also successfully detected the L1 and E6-E7 genes of two highest-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, the ctPCR3.0 method was validated by successfully detecting HPVs in many clinical samples. By performing these detections, this study thus provides a new CRISPR-based DNA detection and typing platform and a ready-to-use HPV clinical detection technique. The platform has wide application in clinical diagnosis.},
}
@article {pmid30243490,
year = {2018},
author = {Lu, R and Yuan, T and Wang, Y and Zhang, T and Yuan, Y and Wu, D and Zhou, M and He, Z and Lu, Y and Chen, Y and Fan, J and Liang, J and Cheng, Y},
title = {Spontaneous severe hypercholesterolemia and atherosclerosis lesions in rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7.},
journal = {EBioMedicine},
volume = {36},
number = {},
pages = {29-38},
pmid = {30243490},
issn = {2352-3964},
mesh = {Animals ; Animals, Genetically Modified ; Atherosclerosis/*genetics/metabolism/*pathology ; Biomarkers ; CRISPR-Cas Systems ; Coronary Artery Disease/genetics/metabolism/pathology ; Disease Models, Animal ; *Exons ; Female ; Gene Targeting ; Genotype ; Hypercholesterolemia/diagnosis/*genetics/metabolism ; Inflammation Mediators/blood ; Leukocyte Count ; Lipids/blood ; Male ; Plaque, Atherosclerotic/pathology ; Rabbits ; Receptors, LDL/*deficiency ; Sequence Deletion ; },
abstract = {Rabbits (Oryctolagus cuniculus) have been the very frequently used as animal models in the study of human lipid metabolism and atherosclerosis, because they have similar lipoprotein metabolism to humans. Most of hyperlipidemia and atherosclerosis rabbit models are produced by feeding rabbits a high-cholesterol diet. Gene editing or knockout (KO) offered another means of producing rabbit models for study of the metabolism of lipids and lipoproteins. Even so, apolipoprotein (Apo)E KO rabbits must be fed a high-cholesterol diet to induce hyperlipidemia. In this study, we used the CRISPR/Cas9 system anchored exon 7 of low-density lipoprotein receptor (LDLR) in an attempt to generate KO rabbits. We designed two sgRNA sequences located in E7:g.7055-7074 and E7:g.7102-7124 of rabbit LDLR gene, respectively. Seven LDLR-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational LDLR amino acid sequences of the 7 founder rabbits were subjected to tertiary structure modeling with SWISS-MODEL, and results showed that the structure of EGF-A domain of each protein differs from the wild-type. All the founder rabbits spontaneously developed hypercholesterolemia and atherosclerosis on a normal chow (NC) diet. Analysis of their plasma lipids and lipoproteins at the age of 12 weeks revealed that all these KO rabbits exhibited markedly increased levels of plasma TC (the highest of which was 1013.15 mg/dl, 20-fold higher than wild-type rabbits), LDL-C (the highest of which was 730.00 mg/dl, 35-fold higher than wild-type rabbits) and TG accompanied by reduced HDL-C levels. Pathological examinations of a founder rabbit showed prominent aortic atherosclerosis lesions and coronary artery atherosclerosis.In conclusion, we have reported the generation LDLR-KO rabbit model for the study of spontaneous hypercholesterolemia and atherosclerosis on a NC diet. The LDLR-KO rabbits should be a useful rabbit model of human familial hypercholesterolemia (FH) for the simulations of human primary hypercholesterolemia and such models would allow more exact research into cardio-cerebrovascular disease.},
}
@article {pmid30242813,
year = {2018},
author = {Hildebrandt, CC and Marron, JM},
title = {Justice in CRISPR/Cas9 Research and Clinical Applications.},
journal = {AMA journal of ethics},
volume = {20},
number = {9},
pages = {E826-833},
doi = {10.1001/amajethics.2018.826},
pmid = {30242813},
issn = {2376-6980},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Databases, Genetic/ethics ; Ethics, Clinical ; Ethics, Research ; Ethnicity ; Gene Editing/*ethics ; Genetic Therapy/*ethics ; *Health Equity ; *Healthcare Disparities ; Humans ; Minority Groups ; *Social Justice ; },
abstract = {CRISPR/Cas9 is a rapidly developing gene editing technology that will soon have many clinical applications. As with many other new technologies, somatic gene editing with CRISPR/Cas9 raises concerns about equitable access to therapies by historically disenfranchised racial and ethnic minorities. We describe justice concerns related to CRISPR/Cas9, including its potential impact on historically mistreated populations through underrepresentation of minorities in genomic databases and the potential for disparate access to somatic gene therapies when they become clinically available. We then describe ongoing work that aims to address these justice concerns. We conclude by highlighting important considerations to ensure equitable access to therapies going forward, including enhancing diversity in genomic sequencing efforts, improving education and transparency, and building partnerships with underserved and socially disenfranchised communities.},
}
@article {pmid30242691,
year = {2018},
author = {Wang, Q and Chung, CY and Rosenberg, JN and Yu, G and Betenbaugh, MJ},
title = {Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1850},
number = {},
pages = {237-257},
doi = {10.1007/978-1-4939-8730-6_16},
pmid = {30242691},
issn = {1940-6029},
support = {S10 OD016230/OD/NIH HHS/United States ; },
mesh = {Animals ; Antibodies/genetics/*metabolism ; CHO Cells ; CRISPR-Cas Systems/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology ; Cricetulus ; Gene Editing/methods ; Glycosylation ; Recombinant Proteins/genetics/metabolism ; },
abstract = {Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of fucose significantly enhances antibody dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) to disrupt the α-1,6-fucosyltranferase (FUT8) gene and subsequently inhibit α-1,6-fucosylation on antibodies expressed in CHO cells.},
}
@article {pmid30242690,
year = {2018},
author = {Kellner, K and Solanki, A and Amann, T and Lao, N and Barron, N},
title = {Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1850},
number = {},
pages = {221-235},
doi = {10.1007/978-1-4939-8730-6_15},
pmid = {30242690},
issn = {1940-6029},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/genetics/physiology ; Cell Engineering ; Cricetinae ; Cricetulus ; MicroRNAs/genetics/*metabolism ; Recombinant Proteins/genetics/*metabolism ; },
abstract = {MicroRNAs with their unique ability to target hundreds of genes have been highlighted as powerful tools to improve bioprocess behavior of cells. The common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, which requires the introduction and expression of extra genetic material. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate Chinese hamster ovary (CHO) cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in controlling CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We showed that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures making it a potentially interesting target to improve bioprocess performance of CHO cells.},
}
@article {pmid30242682,
year = {2018},
author = {Parola, C and Mason, DM and Zingg, A and Neumeier, D and Reddy, ST},
title = {Genome Engineering of Hybridomas to Generate Stable Cell Lines for Antibody Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1850},
number = {},
pages = {79-111},
doi = {10.1007/978-1-4939-8730-6_7},
pmid = {30242682},
issn = {1940-6029},
mesh = {Animals ; Antibodies, Monoclonal ; CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing/*methods ; Humans ; Hybridomas/*metabolism ; },
abstract = {From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines, we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing. Initially, the antibody variable light (VL) chain is deleted by multiplexed Cas9 cleavage; subsequently, the variable heavy (VH) chain is replaced by a fluorescent reporter gene (mRuby) by Cas9-assisted homology-directed repair (HDR). This cell line is customized by replacing mRuby with a synthetic antibody (consisting of a VL, light constant region and VH) by once again using Cas9-assisted HDR. Due to hybridomas' inherent ability to surface display and secrete antibodies, they provide a suitable host for both the selection and the production process, substantially streamlining the process for stable cell line generation, and thus we refer to this platform as plug-and-(dis)play (PnP) hybridomas.},
}
@article {pmid30242271,
year = {2018},
author = {Guo, Y and Jardin, BD and Zhou, P and Sethi, I and Akerberg, BN and Toepfer, CN and Ai, Y and Li, Y and Ma, Q and Guatimosim, S and Hu, Y and Varuzhanyan, G and VanDusen, NJ and Zhang, D and Chan, DC and Yuan, GC and Seidman, CE and Seidman, JG and Pu, WT},
title = {Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3837},
pmid = {30242271},
issn = {2041-1723},
support = {17IRG33410894//American Heart Association (American Heart Association, Inc.)/International ; U01 HL131003/HL/NHLBI NIH HHS/United States ; T32 HL007572/HL/NHLBI NIH HHS/United States ; UM1 HL098166/HL/NHLBI NIH HHS/United States ; 2UM1 HL098166//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/International ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; Chromatin/metabolism ; Female ; Gene Expression Regulation ; Male ; Mice ; Mice, Knockout ; Mutagenesis ; Myocytes, Cardiac/*physiology ; Serum Response Factor/*metabolism ; Transcriptome ; },
abstract = {After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal's lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.},
}
@article {pmid30242113,
year = {2018},
author = {Penfold, L and Woods, A and Muckett, P and Nikitin, AY and Kent, TR and Zhang, S and Graham, R and Pollard, A and Carling, D},
title = {CAMKK2 Promotes Prostate Cancer Independently of AMPK via Increased Lipogenesis.},
journal = {Cancer research},
volume = {78},
number = {24},
pages = {6747-6761},
pmid = {30242113},
issn = {1538-7445},
support = {MC_U120027537/MRC_/Medical Research Council/United Kingdom ; MRF_MRF-023-0001-S-PENFO/MRF/MRF/United Kingdom ; MRF_MRF-023-0003-RG-PENFO/MRF/MRF/United Kingdom ; R01 CA197160/CA/NCI NIH HHS/United States ; },
mesh = {AMP-Activated Protein Kinases/*genetics ; Adenocarcinoma/genetics ; Animals ; Benzimidazoles/chemistry ; CRISPR-Cas Systems ; Calcium-Calmodulin-Dependent Protein Kinase Kinase/*genetics ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cell Survival ; Female ; Gene Deletion ; Humans ; *Lipogenesis ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Naphthalimides/chemistry ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms/genetics/*pathology ; Receptors, Androgen/genetics ; Signal Transduction ; },
abstract = {: New targets are required for treating prostate cancer, particularly castrate-resistant disease. Previous studies reported that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) expression is increased in human prostate cancer. Here, we show that Camkk2 deletion or pharmacologic inhibition protects against prostate cancer development in a preclinical mouse model that lacks expression of prostate-specific Pten. In contrast, deletion of AMP-activated protein kinase (Ampk) β1 resulted in earlier onset of adenocarcinoma development. These findings suggest for the first time that Camkk2 and Ampk have opposing effects in prostate cancer progression. Loss of CAMKK2 in vivo or in human prostate cancer cells reduced the expression of two key lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase. This reduction was mediated via a posttranscriptional mechanism, potentially involving a decrease in protein translation. Moreover, either deletion of CAMKK2 or activation of AMPK reduced cell growth in human prostate cancer cells by inhibiting de novo lipogenesis. Activation of AMPK in a panel of human prostate cancer cells inhibited cell proliferation, migration, and invasion as well as androgen-receptor signaling. These findings demonstrate that CAMKK2 and AMPK have opposing effects on lipogenesis, providing a potential mechanism for their contrasting effects on prostate cancer progression in vivo. They also suggest that inhibition of CAMKK2 combined with activation of AMPK would offer an efficacious therapeutic strategy in treatment of prostate cancer. SIGNIFICANCE: These findings show that CAMKK2 and its downstream target AMPK have opposing effects on prostate cancer development and raise the possibility of a new combined therapeutic approach that inhibits CAMKK2 and activates AMPK.},
}
@article {pmid30242021,
year = {2018},
author = {Lizotte, PH and Hong, RL and Luster, TA and Cavanaugh, ME and Taus, LJ and Wang, S and Dhaneshwar, A and Mayman, N and Yang, A and Kulkarni, M and Badalucco, L and Fitzpatrick, E and Kao, HF and Kuraguchi, M and Bittinger, M and Kirschmeier, PT and Gray, NS and Barbie, DA and Jänne, PA},
title = {A High-Throughput Immune-Oncology Screen Identifies EGFR Inhibitors as Potent Enhancers of Antigen-Specific Cytotoxic T-lymphocyte Tumor Cell Killing.},
journal = {Cancer immunology research},
volume = {6},
number = {12},
pages = {1511-1523},
pmid = {30242021},
issn = {2326-6074},
support = {R01 CA135257/CA/NCI NIH HHS/United States ; U54 HL127365/HL/NHLBI NIH HHS/United States ; },
mesh = {Afatinib/administration &amp; dosage ; Animals ; Antibodies, Monoclonal, Humanized/administration &amp; dosage ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cell Line, Tumor ; Coculture Techniques ; Drug Screening Assays, Antitumor/*methods ; ErbB Receptors/*antagonists &amp; inhibitors ; Head and Neck Neoplasms/drug therapy ; High-Throughput Screening Assays/*methods ; Humans ; Luciferases, Firefly/genetics ; Mice, Inbred C57BL ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors ; Protein Kinase Inhibitors/*pharmacology ; Squamous Cell Carcinoma of Head and Neck/drug therapy ; T-Lymphocytes, Cytotoxic/*drug effects/immunology ; },
abstract = {We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8[+] T cells specifically recognizing the model antigen in an H-2b-restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8[+] cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.},
}
@article {pmid30241607,
year = {2018},
author = {Zhang, C and Konermann, S and Brideau, NJ and Lotfy, P and Wu, X and Novick, SJ and Strutzenberg, T and Griffin, PR and Hsu, PD and Lyumkis, D},
title = {Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.},
journal = {Cell},
volume = {175},
number = {1},
pages = {212-223.e17},
pmid = {30241607},
issn = {1097-4172},
support = {DP5 OD021396/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U54 GM103368/GM/NIGMS NIH HHS/United States ; DP5 OD021369/OD/NIH HHS/United States ; P30 CA014195/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryoelectron Microscopy/methods ; Endonucleases/metabolism ; HEK293 Cells ; Humans ; Molecular Conformation ; RNA/genetics ; RNA, Guide/genetics/*physiology/ultrastructure ; Ribonucleases/metabolism/*physiology/ultrastructure ; },
abstract = {CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.},
}
@article {pmid30241032,
year = {2018},
author = {Han, YQ and Ming, SL and Wu, HT and Zeng, L and Ba, G and Li, J and Lu, WF and Han, J and Du, QJ and Sun, MM and Yang, GY and Wang, J and Chu, BB},
title = {Myostatin knockout induces apoptosis in human cervical cancer cells via elevated reactive oxygen species generation.},
journal = {Redox biology},
volume = {19},
number = {},
pages = {412-428},
pmid = {30241032},
issn = {2213-2317},
mesh = {A549 Cells ; Animals ; Antioxidants/pharmacology ; Apoptosis/*genetics ; CRISPR-Cas Systems/genetics ; Cachexia/*pathology ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytochromes c/metabolism ; Epoxy Compounds/pharmacology ; Fatty Acids/metabolism ; Female ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Lipid Metabolism/physiology ; Membrane Potential, Mitochondrial/genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mitochondria/genetics/metabolism ; Myostatin/*genetics ; Oxidation-Reduction ; Reactive Oxygen Species/*metabolism ; Uterine Cervical Neoplasms/genetics/*pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Myostatin (Mstn) is postulated to be a key determinant of muscle loss and cachexia in cancer. However, no experimental evidence supports a role for Mstn in cancer, particularly in regulating the survival and growth of cancer cells. In this study, we showed that the expression of Mstn was significantly increased in different tumor tissues and human cancer cells. Mstn knockdown inhibited the proliferation of cancer cells. A knockout (KO) of Mstn created by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) induced mitochondria-dependent apoptosis in HeLa cells. Furthermore, KO of Mstn reduced the lipid content. Molecular analyses demonstrated that the expression levels of fatty acid oxidation-related genes were upregulated and then increased rate of fatty acid oxidation. Mstn deficiency-induced apoptosis took place along with generation of reactive oxygen species (ROS) and elevated fatty acid oxidation, which may play a role in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, apoptosis induced by Mstn KO was partially rescued by antioxidants and etomoxir, thereby suggesting that the increased level of ROS was functionally involved in mediating apoptosis. Overall, our findings demonstrate a novel function of Mstn in regulating mitochondrial metabolism and apoptosis within cancer cells. Hence, inhibiting the production and function of Mstn may be an effective therapeutic intervention during cancer progression and muscle loss in cachexia.},
}
@article {pmid30239926,
year = {2018},
author = {Li, XL and Li, GH and Fu, J and Fu, YW and Zhang, L and Chen, W and Arakaki, C and Zhang, JP and Wen, W and Zhao, M and Chen, WV and Botimer, GD and Baylink, D and Aranda, L and Choi, H and Bechar, R and Talbot, P and Sun, CK and Cheng, T and Zhang, XB},
title = {Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.},
journal = {Nucleic acids research},
volume = {46},
number = {19},
pages = {10195-10215},
pmid = {30239926},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Cells, Cultured ; Gene Editing/*methods ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Jurkat Cells ; K562 Cells ; Mice ; Transfection ; Up-Regulation/genetics ; bcl-X Protein/*genetics ; },
abstract = {Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.},
}
@article {pmid30239882,
year = {2018},
author = {Tóth, E and Czene, BC and Kulcsár, PI and Krausz, SL and Tálas, A and Nyeste, A and Varga, &#xc9; and Huszár, K and Weinhardt, N and Ligeti, Z and Borsy, A&#xc9; and Fodor, E and Welker, E},
title = {Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.},
journal = {Nucleic acids research},
volume = {46},
number = {19},
pages = {10272-10285},
pmid = {30239882},
issn = {1362-4962},
mesh = {Animals ; Base Sequence ; Binding Sites/genetics ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/metabolism/*physiology ; Francisella/*enzymology ; HEK293 Cells ; Humans ; Mammals ; Mice ; Moraxella/*enzymology ; Protein Binding ; Substrate Specificity ; Tumor Cells, Cultured ; },
abstract = {Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.},
}
@article {pmid30239876,
year = {2018},
author = {Han, W and Stella, S and Zhang, Y and Guo, T and Sulek, K and Peng-Lundgren, L and Montoya, G and She, Q},
title = {A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding.},
journal = {Nucleic acids research},
volume = {46},
number = {19},
pages = {10319-10330},
pmid = {30239876},
issn = {1362-4962},
mesh = {Adenine Nucleotides/biosynthesis/*metabolism ; Adenosine Triphosphate/metabolism ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Catalysis ; Membrane Proteins/chemistry/*metabolism ; Oligoribonucleotides/biosynthesis/*metabolism ; Protein Binding ; Ribonucleases/chemistry/metabolism ; Ribonucleoproteins/metabolism ; *Second Messenger Systems ; Signal Transduction ; Substrate Specificity ; Sulfolobus ; },
abstract = {Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α-RNP) was purified from the native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α-RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme-substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.},
}
@article {pmid30239713,
year = {2018},
author = {Haverkamp, THA and Geslin, C and Lossouarn, J and Podosokorskaya, OA and Kublanov, I and Nesbø, CL},
title = {Thermosipho spp. Immune System Differences Affect Variation in Genome Size and Geographical Distributions.},
journal = {Genome biology and evolution},
volume = {10},
number = {11},
pages = {2853-2866},
pmid = {30239713},
issn = {1759-6653},
mesh = {Bacteria/*genetics/immunology ; Gene Transfer, Horizontal ; *Genome, Bacterial ; Hydrothermal Vents/*microbiology ; Oil and Gas Fields/*microbiology ; *Phylogeny ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Thermosipho species inhabit thermal environments such as marine hydrothermal vents, petroleum reservoirs, and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents, and hotsprings. Comparative genomics of 15 Thermosipho genomes separated them into three distinct species with different habitat distributions: The widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. Moreover, the species can be differentiated on the basis of genome size (GS), genome content, and immune system composition. For instance, the T. africanus genomes are largest and contained the most carbohydrate metabolism genes, which could explain why these isolates were obtained from ecologically more divergent habitats. Nonetheless, all the Thermosipho genomes, like other Thermotogae genomes, show evidence of genome streamlining. GS differences between the species could further be correlated to differences in defense capacities against foreign DNA, which influence recombination via HGT. The smallest genomes are found in T. affectus that contain both CRISPR-cas Type I and III systems, but no RM system genes. We suggest that this has caused these genomes to be almost devoid of mobile elements, contrasting the two other species genomes that contain a higher abundance of mobile elements combined with different immune system configurations. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA.},
}
@article {pmid30238143,
year = {2018},
author = {Shi, TQ and Huang, H and Kerkhoven, EJ and Ji, XJ},
title = {Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system.},
journal = {Applied microbiology and biotechnology},
volume = {102},
number = {22},
pages = {9541-9548},
pmid = {30238143},
issn = {1432-0614},
mesh = {Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Metabolic Engineering/*methods/trends ; Yarrowia/*genetics/*metabolism ; },
abstract = {The oleaginous yeast Yarrowia lipolytica is widely used for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products. Consequently, it is becoming increasingly popular for metabolic engineering applications. Multiple gene manipulation tools including URA blast, Cre/LoxP, and transcription activator-like effector nucleases (TALENs) have been developed for metabolic engineering in Y. lipolytica. However, the low efficiency and time-consuming procedures involved in these methods hamper further research. The emergence of the CRISPR/Cas system offers a potential solution for these problems due to its high efficiency, ease of operation, and time savings, which can significantly accelerate the genomic engineering of Y. lipolytica. In this review, we summarize the research progress on the development of CRISPR/Cas systems for Y. lipolytica, including Cas9 proteins and sgRNA expression strategies, as well as gene knock-out/knock-in and repression/activation applications. Finally, the most promising and tantalizing future prospects in this area are highlighted.},
}
@article {pmid30237553,
year = {2018},
author = {Ridler, C},
title = {CRISPR therapy shows promise in Duchenne muscular dystrophy.},
journal = {Nature reviews. Neurology},
volume = {14},
number = {11},
pages = {632-633},
doi = {10.1038/s41582-018-0078-8},
pmid = {30237553},
issn = {1759-4766},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dogs ; *Dystrophin ; Gene Editing ; *Muscular Dystrophy, Duchenne ; },
}
@article {pmid30237509,
year = {2019},
author = {Fauster, A and Rebsamen, M and Willmann, KL and César-Razquin, A and Girardi, E and Bigenzahn, JW and Schischlik, F and Scorzoni, S and Bruckner, M and Konecka, J and Hörmann, K and Heinz, LX and Boztug, K and Superti-Furga, G},
title = {Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking.},
journal = {Cell death and differentiation},
volume = {26},
number = {6},
pages = {1138-1155},
pmid = {30237509},
issn = {1476-5403},
support = {P 29250/FWF_/Austrian Science Fund FWF/Austria ; },
mesh = {CRISPR-Cas Systems/genetics ; Cation Transport Proteins/deficiency/*genetics/metabolism ; Cell Death ; Cell Line ; Cell Survival ; *Chromosome Mapping ; HEK293 Cells ; Humans ; Necroptosis/*genetics ; Receptors, Tumor Necrosis Factor, Type I/genetics/*metabolism ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.},
}
@article {pmid30237447,
year = {2018},
author = {Sheth, RU and Wang, HH},
title = {DNA-based memory devices for recording cellular events.},
journal = {Nature reviews. Genetics},
volume = {19},
number = {11},
pages = {718-732},
pmid = {30237447},
issn = {1471-0064},
support = {R01 AI132403/AI/NIAID NIH HHS/United States ; T32 GM008224/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Computers, Molecular ; },
abstract = {Measuring biological data across time and space is critical for understanding complex biological processes and for various biosurveillance applications. However, such data are often inaccessible or difficult to directly obtain. Less invasive, more robust and higher-throughput biological recording tools are needed to profile cells and their environments. DNA-based cellular recording is an emerging and powerful framework for tracking intracellular and extracellular biological events over time across living cells and populations. Here, we review and assess DNA recorders that utilize CRISPR nucleases, integrases and base-editing strategies, as well as recombinase and polymerase-based methods. Quantitative characterization, modelling and evaluation of these DNA-recording modalities can guide their design and implementation for specific application areas.},
}
@article {pmid30237405,
year = {2018},
author = {Wang, Q and Zhang, B and Xu, X and Long, F and Wang, J},
title = {CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14126},
pmid = {30237405},
issn = {2045-2322},
support = {61571119//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Viral/genetics ; Female ; Human papillomavirus 16/genetics ; Humans ; Papillomaviridae/genetics ; Polymerase Chain Reaction/*methods ; },
abstract = {This study develops a new method for detecting and typing target DNA based on Cas9 nuclease, which was named as ctPCR, representing Cas9-sgRNA- or CRISPR-typing PCR. The technique can detect and type target DNA easily, rapidly, specifically, and sensitively. This technique detects target DNA in three steps: (1) amplifying target DNA with PCR by using a pair of universal primers (PCR1); (2) treating PCR1 products with a process referred to as CAT, representing Cas9 cutting, A tailing and T adaptor ligation; (3) amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers (gs-primers) (PCR2). This method was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. This method was also verified by detecting the L1 and E6-E7 genes of two high-risk HPVs (HPV16 and 18) in cervical carcinoma cells and many clinical samples. In this method, PCR1 was performed to determine if the detected DNA sample contained the target DNA (such as virus infection), while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample (such as virus subtypes). Based on these proof-of-concept experiments, this study provides a new CRISPR/Cas9-based DNA detection and typing method.},
}
@article {pmid30237171,
year = {2018},
author = {Benoit, CR and Stanton, AE and Tartanian, AC and Motzer, AR and McGaughey, DM and Bond, SR and Brody, LC},
title = {Functional and phylogenetic characterization of noncanonical vitamin B12-binding proteins in zebrafish suggests involvement in cobalamin transport.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {45},
pages = {17606-17621},
pmid = {30237171},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Protein Domains ; *Transcobalamins/chemistry/genetics/metabolism ; Vitamin B 12/chemistry/genetics/metabolism ; *Zebrafish/genetics/metabolism ; Zebrafish Proteins/chemistry/genetics/metabolism ; },
abstract = {In humans, transport of food-derived cobalamin (vitamin B12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an α-domain and a β-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the α-domain. These β-domain-only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2[-/-] zebrafish.},
}
@article {pmid30237156,
year = {2018},
author = {de Miguel Beriain, I},
title = {Human dignity and gene editing: Using human dignity as an argument against modifying the human genome and germline is a logical fallacy.},
journal = {EMBO reports},
volume = {19},
number = {10},
pages = {},
pmid = {30237156},
issn = {1469-3178},
mesh = {Adult Germline Stem Cells ; CRISPR-Cas Systems/genetics ; Dissent and Disputes ; Gene Editing/*ethics ; Genome, Human/*genetics ; Genomics/*ethics ; Germ Cells/growth &amp; development ; Humans ; *Personhood ; },
abstract = {A common argument against gene editing the human germline is the notion of dignity as linked to the human genome. But this based on a logical fallacy and should rather prompt us to consider gene editing for therapeutic purposes in order to preserve human dignity. [Image: see text]},
}
@article {pmid30233563,
year = {2018},
author = {Naeimi Kararoudi, M and Hejazi, SS and Elmas, E and Hellström, M and Naeimi Kararoudi, M and Padma, AM and Lee, D and Dolatshad, H},
title = {Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {1711},
pmid = {30233563},
issn = {1664-3224},
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Markers ; *Heterografts ; Humans ; *Inverted Repeat Sequences ; },
abstract = {Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.},
}
@article {pmid30232659,
year = {2018},
author = {Li, Z and Zhao, J and Muhammad, N and Wang, D and Mao, Q and Xia, H},
title = {Establishment of a HEK293 cell line by CRISPR/Cas9-mediated luciferase knock-in to study transcriptional regulation of the human SREBP1 gene.},
journal = {Biotechnology letters},
volume = {40},
number = {11-12},
pages = {1495-1506},
doi = {10.1007/s10529-018-2608-2},
pmid = {30232659},
issn = {1573-6776},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Gene Expression Regulation/*genetics ; Gene Knock-In Techniques/*methods ; HEK293 Cells ; Humans ; Luciferases/*genetics ; Sterol Regulatory Element Binding Protein 1/*genetics/metabolism ; },
abstract = {OBJECTIVES: To establish a HEK293 cell line with a luciferase knock-in reporter controlled by the endogenous SREBP1 promoter for investigating transcriptional regulation of the SREBP1 gene.<br><br>RESULTS: PCR confirmed the site-specific integration of a single copy of the exogenous luciferase gene into one allele of the genome and a 14&#xa0;bp deletion of the targeted sequence in the other. Luciferase activity was directly correlated with the promoter activity of the endogenous SREBP1 gene in the HEK293-SREBP1-T2A-luciferase-KI cell line cell line.<br><br>CONCLUSIONS: We successfully generated a&#xa0;novel luciferase knock-in reporter system, which will be very useful for studying transcriptional regulation of the SREBP1 gene and for screening drugs or chemical molecules that regulate SREBP1 gene expression.},
}
@article {pmid30232454,
year = {2018},
author = {Athukoralage, JS and Rouillon, C and Graham, S and Grüschow, S and White, MF},
title = {Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate.},
journal = {Nature},
volume = {562},
number = {7726},
pages = {277-280},
pmid = {30232454},
issn = {1476-4687},
support = {BB/G011400/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M000400/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M021017/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adenine Nucleotides/*metabolism ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/*classification/metabolism ; CRISPR-Cas Systems/*genetics ; Endoribonucleases/*chemistry/genetics/isolation &amp; purification/*metabolism ; Kinetics ; Models, Molecular ; Oligoribonucleotides/*metabolism ; RNA, Small Interfering/genetics/metabolism ; Second Messenger Systems ; Sulfolobus solfataricus/*enzymology/genetics ; },
abstract = {The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids[1-3]. Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA[4,5]. Cyclic oligoadenylate, in turn, binds to and activates ribonucleases and other factors-via a CRISPR-associated Rossman-fold domain-and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the 'off-switch' that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This 'ring nuclease' is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight to the CRISPR system.},
}
@article {pmid30232365,
year = {2018},
author = {Hikita, T and Miyata, M and Watanabe, R and Oneyama, C},
title = {Sensitive and rapid quantification of exosomes by fusing luciferase to exosome marker proteins.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {14035},
pmid = {30232365},
issn = {2045-2322},
support = {JP1005457//Japan Science and Technology Agency (JST)/International ; },
mesh = {A549 Cells ; Animals ; CRISPR-Cas Systems ; Cell Line ; Exosomes/chemistry/*genetics ; HCT116 Cells ; HT29 Cells ; Humans ; Mice ; Nanoparticles ; Nanotechnology/*methods ; Tetraspanin 30/*genetics ; },
abstract = {Exosomes have emerged as important mediators of intercellular communication. Although their modes of action have been elucidated, the molecular mechanisms underlying their secretion, sorting of molecules, uptake into recipient cells, and biological distribution in vivo remain elusive. Here, we present a novel system for quantifying secreted exosomes by introducing ectopic or CRISPR/Cas9-mediated knock-in of luciferase-fusion exosome markers such as CD63. This luciferase-based method makes it possible to measure exosomes secreted into the culture medium with high linearity and wide dynamic range in a high-throughput manner. We demonstrate that data obtained by luminescent quantification are well correlated with data obtained by conventional nanoparticle tracking analysis under multiple conditions. In addition, our system is capable of evaluating the recipient cells or tissues that take up exosomes, as well as visualizing exosomes in vivo. The proposed system represents a powerful tool for understanding the molecular mechanisms underlying exosome production, uptake, and long-term distribution.},
}
@article {pmid30232196,
year = {2018},
author = {Nachmanson, D and Lian, S and Schmidt, EK and Hipp, MJ and Baker, KT and Zhang, Y and Tretiakova, M and Loubet-Senear, K and Kohrn, BF and Salk, JJ and Kennedy, SR and Risques, RA},
title = {Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).},
journal = {Genome research},
volume = {28},
number = {10},
pages = {1589-1599},
pmid = {30232196},
issn = {1549-5469},
support = {R01 CA160674/CA/NCI NIH HHS/United States ; R01 CA181308/CA/NCI NIH HHS/United States ; T32 CA009515/CA/NCI NIH HHS/United States ; R44 CA221426/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; DNA Fragmentation ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Ovarian Neoplasms/*genetics ; Sequence Analysis, DNA/*methods ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.},
}
@article {pmid30231914,
year = {2018},
author = {Ibraheim, R and Song, CQ and Mir, A and Amrani, N and Xue, W and Sontheimer, EJ},
title = {All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {137},
pmid = {30231914},
issn = {1474-760X},
support = {129056-RSG-16-093//American Cancer Society/International ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; HL137167/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; GM1115911/GM/NIGMS NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; HL131471/HL/NHLBI NIH HHS/United States ; },
mesh = {4-Hydroxyphenylpyruvate Dioxygenase/genetics ; Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Dependovirus/*genetics ; *Gene Editing ; Genetic Vectors/administration &amp; dosage ; Mice ; Mice, Inbred C57BL ; Neisseria meningitidis/enzymology ; Oxidoreductases/genetics ; Plasmids/administration &amp; dosage ; Proprotein Convertase 9/genetics ; Tyrosinemias/therapy ; },
abstract = {BACKGROUND: Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.<br><br>RESULTS: Here, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail-vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >&#x2009;35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo.<br><br>CONCLUSIONS: Our findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.},
}
@article {pmid30231020,
year = {2018},
author = {Magee, CL and Kleyn, PW and Monks, BM and Betz, U and Basnet, S},
title = {Pre-existing technological core and roots for the CRISPR breakthrough.},
journal = {PloS one},
volume = {13},
number = {9},
pages = {e0198541},
pmid = {30231020},
issn = {1932-6203},
mesh = {Animals ; Bacteria/enzymology/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics/metabolism ; Gene Editing/*methods ; Genome ; Humans ; *Patents as Topic ; },
abstract = {This paper applies objective methods to explore the technological origins of the widely acclaimed CRISPR breakthrough in the technological domain of genome engineering. Previously developed patent search techniques are first used to recover a set of patents that well-represent the genome editing domain before CRISPR. Main paths are then determined from the citation network associated with this patent set allowing identification of the three major knowledge trajectories. The most significant of these trajectories for CRISPR involves the core of genome editing with less significant trajectories involving cloning and endonuclease specific developments. The major patents on the core trajectory are consistent with qualitative expert knowledge of the topical area. A second set of patents that we call the CRISPR roots are obtained by finding the patents directly cited by the recent CRISPR patents along with patents cited by that set of patents. We find that the CRISPR roots contain 8 key patents from the genome engineering main path associated with restriction endonucleases and the expected strong connection of CRISPR to prior genome editing technology such as Zn finger nucleases. Nonetheless, analysis of the full CRISPR roots shows that a very wide array of technological knowledge beyond genome engineering has contributed to achieving the CRISPR breakthrough. Such breadth in origins is not surprising since "spillover" is generally perceived as important and previous qualitative studies of CRISPR have shown not only technological breadth in origins but scientific breadth as well. In addition, we find that the estimated rate of functional performance improvement of the CRISPR roots set is about 9% per year compared to the genome engineering set (~4% per year). These estimates indicate below average rates of improvement and may indicate that CRISPR (and perhaps yet undiscovered) genome engineering developments could evolve in effectiveness over an upcoming long rather than short time period.},
}
@article {pmid30231016,
year = {2018},
author = {Dunn-Fletcher, CE and Muglia, LM and Pavlicev, M and Wolf, G and Sun, MA and Hu, YC and Huffman, E and Tumukuntala, S and Thiele, K and Mukherjee, A and Zoubovsky, S and Zhang, X and Swaggart, KA and Lamm, KYB and Jones, H and Macfarlan, TS and Muglia, LJ},
title = {Anthropoid primate-specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length.},
journal = {PLoS biology},
volume = {16},
number = {9},
pages = {e2006337},
pmid = {30231016},
issn = {1545-7885},
support = {R01 HD091527/HD/NICHD NIH HHS/United States ; R21 HD090196/HD/NICHD NIH HHS/United States ; T32 GM063483/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Chromosomes, Artificial, Bacterial/genetics ; Corticotropin-Releasing Hormone/*genetics/metabolism ; Female ; Gene Regulatory Networks ; Homeodomain Proteins/metabolism ; Humans ; Male ; Mice, Transgenic ; Mutagenesis, Insertional/genetics ; Parturition ; Placenta/*metabolism ; Pregnancy ; Primates/*genetics ; Protein Binding ; Retroelements/*genetics ; Sequence Deletion ; Species Specificity ; Terminal Repeat Sequences/genetics ; Transcription Factors/metabolism ; Trophoblasts/metabolism ; },
abstract = {Pregnancy and parturition are intricately regulated to ensure successful reproductive outcomes. However, the factors that control gestational length in humans and other anthropoid primates remain poorly defined. Here, we show the endogenous retroviral long terminal repeat transposon-like human element 1B (THE1B) selectively controls placental expression of corticotropin-releasing hormone (CRH) that, in turn, influences gestational length and birth timing. Placental expression of CRH and subsequently prolonged gestational length were found in two independent strains of transgenic mice carrying a 180-kb human bacterial artificial chromosome (BAC) DNA that contained the full length of CRH and extended flanking regions, including THE1B. Restricted deletion of THE1B silenced placental CRH expression and normalized birth timing in these transgenic lines. Furthermore, we revealed an interaction at the 5' insertion site of THE1B with distal-less homeobox 3 (DLX3), a transcription factor expressed in placenta. Together, these findings suggest that retroviral insertion of THE1B into the anthropoid primate genome may have initiated expression of CRH in placental syncytiotrophoblasts via DLX3 and that this placental CRH is sufficient to alter the timing of birth.},
}
@article {pmid30229432,
year = {2019},
author = {Shan, L and Wang, D and Mao, Q and Xia, H},
title = {Establishment of a DGKθ Endogenous Promoter Luciferase Reporter HepG2 Cell Line for Studying the Transcriptional Regulation of DGKθ Gene.},
journal = {Applied biochemistry and biotechnology},
volume = {187},
number = {4},
pages = {1344-1355},
doi = {10.1007/s12010-018-2890-4},
pmid = {30229432},
issn = {1559-0291},
mesh = {CRISPR-Cas Systems/*genetics ; Diacylglycerol Kinase/*genetics ; *Gene Expression Regulation, Enzymologic ; Gene Knock-In Techniques ; Genes, Reporter/*genetics ; Hep G2 Cells ; Humans ; Luciferases/*genetics ; Promoter Regions, Genetic/*genetics ; Transcription Factors/metabolism ; *Transcription, Genetic ; },
abstract = {DGKθ protein expression levels are closely related to the development of diseases including diabetes, cancer, and neuronal disease. To investigate the transcriptional regulation of the DGKθ gene, we used CRISPR/Cas9 to generate a DGKθ endogenous promoter luciferase reporter HepG2 cell line, in which the endogenous DGKθ promoter controls the expression of the luciferase reporter gene. To test the cell line, FXR, the transcription factor for upregulating the expression of DGKθ gene, was used to validate the cell line. Furthermore, the reported agonists for the expression of DGKθ, cAMP and GW4064, the known inhibitor for DGKθ enzyme activity, R59949, and a potential regulator for DGKθ enzyme expression, EGCG (the major catechin in green tea), were applied to the reporter cell line. The results indicated that these reagents could significantly regulate the expression of reporter luciferase. Finally, four transcription factors (E2F1, c-Myc, USF1, and Bmal1) potentially binding to the DGKθ gene's upstream promoter region were tested. DGKθ expression was upregulated by c-Myc and downregulated by E2F1, which was also confirmed in wild-type HepG2 cells. We found that the cell line's luciferase activity was directly correlated with DGKθ endogenous promoter activity, suggesting that it is liable and sensitive for studying DGKθ transcriptional regulation. The study provides a useful tool for high-throughput drug screening for the treatment of DGKθ-involved diseases.},
}
@article {pmid30228869,
year = {2018},
author = {Reimer, KA and Neugebauer, KM},
title = {Blood Relatives: Splicing Mechanisms underlying Erythropoiesis in Health and Disease.},
journal = {F1000Research},
volume = {7},
number = {},
pages = {},
pmid = {30228869},
issn = {2046-1402},
support = {U54 DK106857/DK/NIDDK NIH HHS/United States ; R01 HL133406/HL/NHLBI NIH HHS/United States ; },
mesh = {Cell Differentiation ; *Erythropoiesis ; Hematologic Diseases/*genetics/*physiopathology ; Humans ; *RNA Splicing ; RNA, Messenger/*genetics ; Transcriptome ; },
abstract = {During erythropoiesis, hematopoietic stem and progenitor cells transition to erythroblasts en route to terminal differentiation into enucleated red blood cells. Transcriptome-wide changes underlie distinct morphological and functional characteristics at each cell division during this process. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA-the most highly expressed transcript in erythroblasts-was the focus of many seminal studies on the mechanisms of pre-mRNA splicing. We now understand that pre-mRNA splicing plays an important role in shaping the transcriptome of developing erythroblasts. Recent advances have provided insight into the role of alternative splicing and intron retention as important regulatory mechanisms of erythropoiesis. However, dysregulation of splicing during erythropoiesis is also a cause of several hematological diseases, including β-thalassemia and myelodysplastic syndromes. With a growing understanding of the role that splicing plays in these diseases, we are well poised to develop gene-editing treatments. In this review, we focus on changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing.},
}
@article {pmid30228232,
year = {2018},
author = {Fedele, PL and Willis, SN and Liao, Y and Low, MS and Rautela, J and Segal, DH and Gong, JN and Huntington, ND and Shi, W and Huang, DCS and Grigoriadis, G and Tellier, J and Nutt, SL},
title = {IMiDs prime myeloma cells for daratumumab-mediated cytotoxicity through loss of Ikaros and Aiolos.},
journal = {Blood},
volume = {132},
number = {20},
pages = {2166-2178},
doi = {10.1182/blood-2018-05-850727},
pmid = {30228232},
issn = {1528-0020},
mesh = {Antibodies, Monoclonal/*pharmacology ; Antibody-Dependent Cell Cytotoxicity/drug effects ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Ikaros Transcription Factor/*genetics ; Multiple Myeloma/*drug therapy/*genetics ; },
abstract = {Recent studies have demonstrated that the immunomodulatory drugs (IMiDs) lead to the degradation of the transcription factors Ikaros and Aiolos. However, why their loss subsequently leads to multiple myeloma (MM) cell death remains unclear. Using CRISPR-Cas9 genome editing, we have deleted IKZF1/Ikaros and IKZF3/Aiolos in human MM cell lines to gain further insight into their downstream gene regulatory networks. Inactivation of either factor alone recapitulates the cell intrinsic action of the IMiDs, resulting in cell cycle arrest and induction of apoptosis. Furthermore, evaluation of the transcriptional changes resulting from their loss demonstrates striking overlap with lenalidomide treatment. This was not dependent on reduction of the IRF4-MYC "axis," as neither protein was consistently downregulated, despite cell death occurring, and overexpression of either factor failed to rescue for Ikaros loss. Importantly, Ikaros and Aiolos repress the expression of interferon-stimulated genes (ISGs), including CD38, and their loss led to the activation of an interferon-like response, contributing to MM cell death. Ikaros/Aiolos repressed CD38 expression through interaction with the nucleosome remodeling and deacetylase complex in MM. IMiD-induced loss of Ikaros or treatment with interferon resulted in an upregulation of CD38 surface expression on MM cells, priming for daratumumab-induced NK cell-mediated antibody-dependent cellular cytotoxicity. These results give further insight into the mechanism of action of the IMiDs and provide mechanistic rationale for combination with anti-CD38 monoclonal antibodies.},
}
@article {pmid30227135,
year = {2018},
author = {Borsenberger, V and Onésime, D and Lestrade, D and Rigouin, C and Neuvéglise, C and Daboussi, F and Bordes, F},
title = {Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica.},
journal = {Journal of molecular biology},
volume = {430},
number = {21},
pages = {4293-4306},
doi = {10.1016/j.jmb.2018.08.024},
pmid = {30227135},
issn = {1089-8638},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Fungal ; Promoter Regions, Genetic ; Yarrowia/*genetics ; },
abstract = {Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide.},
}
@article {pmid30226608,
year = {2018},
author = {Takao, A and Yoshikawa, K and Karnan, S and Ota, A and Uemura, H and De Velasco, MA and Kura, Y and Suzuki, S and Ueda, R and Nishino, T and Hosokawa, Y},
title = {Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling.},
journal = {Oncology reports},
volume = {40},
number = {5},
pages = {2455-2466},
doi = {10.3892/or.2018.6683},
pmid = {30226608},
issn = {1791-2431},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; *Genetic Therapy ; Humans ; Immunity, Cellular/immunology ; Male ; Mice ; MicroRNAs/genetics ; Microarray Analysis ; PTEN Phosphohydrolase/*genetics ; Prostate/immunology/pathology ; Prostatic Neoplasms/*genetics/immunology/pathology ; Proto-Oncogene Proteins c-akt/genetics ; },
abstract = {Phosphatase and tensin homolog (PTEN) deficiency is associated with development, progression, and metastasis of various cancers. However, changes in gene expression associated with PTEN deficiency have not been fully characterized. To explore genes with altered expression in PTEN‑deficient cells, the present study generated a PTEN‑knockout cell line (ΔPTEN) from a mouse prostate cancer‑derived cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 (CRISPR/Cas9) gene editing system. Following transfection of the CRISPR/Cas9 construct, DNA sequencing was performed to identify deletion of the Pten locus and PTEN inactivation was verified by western blotting. The ΔPTEN cell line exhibited enhanced RAC‑alpha serine/threonine‑protein kinase phosphorylation and cyclin D1 expression. In addition, an increase in cell proliferation and colony formation was observed in the ΔPTEN cell line. Gene expression profiling experiments were analyzed with microarray and microRNA (miRNA) arrays. In the microarray analysis, 111 genes exhibited ≥10‑fold increased expression compared with the parent strain and mock cell line and 23 genes were downregulated. The only miRNA with increased expression of 10‑fold or more was mmu‑miR‑210‑3p. Genes with enhanced expression included genes involved in the development, progression, and metastasis of cancer such as Tet methylcytosine dioxygenase 1, twist family BHLH transcription factor 2, C‑fos‑induced growth factor and Wingless‑Type MMTV Integration Site Family, Member 3, and genes involved in immunosuppression such as Arginase 1. The results of the present study suggest that PTEN deficiency mobilizes a variety of genes critical for cancer cell survival and host immune evasion.},
}
@article {pmid30226584,
year = {2018},
author = {Liu, Q and Fan, D and Adah, D and Wu, Z and Liu, R and Yan, QT and Zhang, Y and Du, ZY and Wang, D and Li, Y and Bao, SY and Liu, LP},
title = {CRISPR/Cas9‑mediated hypoxia inducible factor‑1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma.},
journal = {Oncology reports},
volume = {40},
number = {5},
pages = {2547-2557},
pmid = {30226584},
issn = {1791-2431},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/*genetics/pathology/surgery ; Cell Line, Tumor ; *Chemoembolization, Therapeutic ; Gene Knockout Techniques ; Hepatic Artery/surgery ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Liver/pathology/surgery ; Liver Neoplasms/*genetics/pathology/surgery ; Mice ; Tumor Hypoxia/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Transarterial embolization (TAE) is a palliative option commonly used for the treatment of advanced, unresectable hepatocellular carcinoma (HCC). However, patient prognosis in regards to overall survival has not improved with this method, mainly due to hypoxia‑inducible factor‑1α (HIF‑1α)‑induced angiogenesis and invasiveness. Thus, it is hypothesized that HIF‑1α may be an ideal knockout target for the treatment of HCC in combination with TAE. Thus, in the present study, HIF‑1α knockout was conducted in human liver cancer SMMC‑7721 cells and a xenograft HCC model was established using a lentivirus‑mediated CRISPR/Cas system (LV‑Cas) with small guide RNA‑721 (LV‑H721). Furthermore, hepatic artery ligation (HAL) was used to mimic human transarterial chemoembolization in mice. The results revealed that HIF‑1α was highly expressed in both HCC patient tissues and SMMC‑7721‑induced tumor tissues. The HIF‑1α knockout in SMMC‑7721 cells significantly suppressed cell invasiveness and migration, and induced cell apoptosis under CoCl2‑mimicking hypoxic conditions. Compared with the control groups, HAL + LV‑H721 inhibited SMMC‑7721 tumor growth in orthotopic HCC and markedly prolonged the survival of HCC‑bearing mice, which was accompanied by a lower CD31 expression (tumor angiogenesis) and increased apoptosis in the tumor cells. These findings demonstrated a valuable antitumor synergism in combining CRISPR/Cas9‑mediated HIF‑1α knockout with TAE in mice and highlighted the possibility that HIF‑1α may be an effective therapeutic knockout target in combination with TAE for HCC treatment.},
}
@article {pmid30224644,
year = {2018},
author = {Giacomelli, AO and Yang, X and Lintner, RE and McFarland, JM and Duby, M and Kim, J and Howard, TP and Takeda, DY and Ly, SH and Kim, E and Gannon, HS and Hurhula, B and Sharpe, T and Goodale, A and Fritchman, B and Steelman, S and Vazquez, F and Tsherniak, A and Aguirre, AJ and Doench, JG and Piccioni, F and Roberts, CWM and Meyerson, M and Getz, G and Johannessen, CM and Root, DE and Hahn, WC},
title = {Mutational processes shape the landscape of TP53 mutations in human cancer.},
journal = {Nature genetics},
volume = {50},
number = {10},
pages = {1381-1387},
pmid = {30224644},
issn = {1546-1718},
support = {T32 GM007226/GM/NIGMS NIH HHS/United States ; U01 CA199253/CA/NCI NIH HHS/United States ; K08 CA218420/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; U01 CA176058/CA/NCI NIH HHS/United States ; UL1 TR001102/TR/NCATS NIH HHS/United States ; },
mesh = {A549 Cells ; Alleles ; CRISPR-Cas Systems ; Cells, Cultured ; DNA Mutational Analysis ; Databases, Genetic ; High-Throughput Nucleotide Sequencing ; Humans ; Mutagenesis/*physiology ; *Mutation ; Neoplasms/*genetics/pathology ; Sequence Analysis, DNA ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations[1,2]. Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models[3-8]. To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database[9,10], we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations.},
}
@article {pmid30224454,
year = {2018},
author = {Oberhofer, G and Ivy, T and Hay, BA},
title = {Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {40},
pages = {E9343-E9352},
pmid = {30224454},
issn = {1091-6490},
support = {T32 GM007616/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila melanogaster ; Female ; Fertility/genetics ; *Gene Targeting ; },
abstract = {A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work in Drosophila and mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs in Drosophila melanogaster targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.},
}
@article {pmid30224411,
year = {2018},
author = {Gavilan, MP and Gandolfo, P and Balestra, FR and Arias, F and Bornens, M and Rios, RM},
title = {The dual role of the centrosome in organizing the microtubule network in interphase.},
journal = {EMBO reports},
volume = {19},
number = {11},
pages = {},
pmid = {30224411},
issn = {1469-3178},
mesh = {A Kinase Anchor Proteins/genetics ; Antigens/genetics ; CRISPR-Cas Systems ; Cell Cycle Proteins ; Cell Line ; Centrosome/*metabolism ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; Cytoskeletal Proteins/genetics ; Gene Knockout Techniques ; Golgi Apparatus/metabolism ; Humans ; Interphase/*physiology ; Intracellular Signaling Peptides and Proteins/genetics ; Microtubules/genetics/*metabolism ; Nerve Tissue Proteins/genetics ; Tubulin/metabolism ; },
abstract = {Here, we address the regulation of microtubule nucleation during interphase by genetically ablating one, or two, of three major mammalian γ-TuRC-binding factors namely pericentrin, CDK5Rap2, and AKAP450. Unexpectedly, we find that while all of them participate in microtubule nucleation at the Golgi apparatus, they only modestly contribute at the centrosome where CEP192 has a more predominant function. We also show that inhibiting microtubule nucleation at the Golgi does not affect centrosomal activity, whereas manipulating the number of centrosomes with centrinone modifies microtubule nucleation activity of the Golgi apparatus. In centrosome-free cells, inhibition of Golgi-based microtubule nucleation triggers pericentrin-dependent formation of cytoplasmic-nucleating structures. Further depletion of pericentrin under these conditions leads to the generation of individual microtubules in a γ-tubulin-dependent manner. In all cases, a conspicuous MT network forms. Strikingly, centrosome loss increases microtubule number independently of where they were growing from. Our results lead to an unexpected view of the interphase centrosome that would control microtubule network organization not only by nucleating microtubules, but also by modulating the activity of alternative microtubule-organizing centers.},
}
@article {pmid30224336,
year = {2018},
author = {Wang, H and Park, H and Liu, J and Sternberg, PW},
title = {An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {11},
pages = {3607-3616},
pmid = {30224336},
issn = {2160-1836},
support = {K99 GM126137/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R24 OD023041/OD/NIH HHS/United States ; T32 GM007616/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/genetics ; *Gene Editing ; Mutation ; },
abstract = {Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2 We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.},
}
@article {pmid30224156,
year = {2018},
author = {Marzec, M and Hensel, G},
title = {Targeted Base Editing Systems Are Available for Plants.},
journal = {Trends in plant science},
volume = {23},
number = {11},
pages = {955-957},
doi = {10.1016/j.tplants.2018.08.011},
pmid = {30224156},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genetic Engineering/methods ; Plants/*genetics ; },
abstract = {Use of RNA-guided endonucleases for targeted genome editing is one of the most important breakthrough discoveries of the 21st century. Recent studies have described modifications of this precise base editing technique that open up a new dimension to plant genome editing.},
}
@article {pmid30223022,
year = {2019},
author = {Seidl, CI and Fulga, TA and Murphy, CL},
title = {CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation.},
journal = {Osteoarthritis and cartilage},
volume = {27},
number = {1},
pages = {140-147},
doi = {10.1016/j.joca.2018.09.001},
pmid = {30223022},
issn = {1522-9653},
support = {G0902418/MRC_/Medical Research Council/United Kingdom ; 20902/ARC_/Arthritis Research UK/United Kingdom ; },
mesh = {Adult ; Aged ; Aged, 80 and over ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems/*genetics ; Cartilage, Articular/*cytology/metabolism ; Cell Survival/genetics ; Chondrocytes/*metabolism ; Collagen Type II/*metabolism ; Female ; Gene Editing/*methods ; Humans ; Male ; Matrix Metalloproteinase 13/*metabolism ; Middle Aged ; Spheroids, Cellular/metabolism ; Transfection ; Young Adult ; },
abstract = {OBJECTIVE: To investigate the efficacy of CRISPR-Cas9 mediated editing in human chondrocytes, and to develop a genome editing approach relevant to cell-based repair.<br><br>METHODS: Transfection of human articular chondrocytes (both healthy and osteoarthritic) with ribonucleoprotein complexes (RNP) containing Cas9 and a crisprRNA targeting exon2 of MMP13 was performed to assess editing efficiency and effects on MMP13 protein levels and enzymatic activity. Using spheroid cultures, protein levels of a major target of MMP13, type II collagen, were assessed by western blot and immunofluorescence.<br><br>RESULTS: With an editing efficiency of 63-74%, secreted MMP13 protein levels and activity were significantly reduced (percentage decrease 34.14% without and 67.97% with IL-1&#x3b2; based on median values of MMP13 enzymatic activity, N&#xa0;=&#xa0;7) comparing non-edited with edited cell populations using an exon-targeting gRNA resulting in premature stop codons through non-homologous end joining (NHEJ). Accumulation of cartilage matrix protein type II collagen was enhanced in edited cells in spheroid culture, compared to non-edited controls.<br><br>CONCLUSION: CRISPR-Cas9 mediated genome editing can be used to efficiently and reproducibly establish populations of human chondrocytes with stably reduced expression of key genes of interest without the need for clonal selection. Such an editing approach has the potential to greatly enhance current cell-based therapies for cartilage repair.},
}
@article {pmid30222933,
year = {2018},
author = {Zhang, H and Bahamondez-Canas, TF and Zhang, Y and Leal, J and Smyth, HDC},
title = {PEGylated Chitosan for Nonviral Aerosol and Mucosal Delivery of the CRISPR/Cas9 System in Vitro.},
journal = {Molecular pharmaceutics},
volume = {15},
number = {11},
pages = {4814-4826},
pmid = {30222933},
issn = {1543-8392},
support = {R01 HL138251/HL/NHLBI NIH HHS/United States ; },
mesh = {Administration, Inhalation ; CRISPR-Cas Systems/*genetics ; Chitosan/chemistry ; DNA/*administration &amp; dosage ; Feasibility Studies ; Gene Editing/instrumentation/*methods ; HEK293 Cells ; Humans ; Nanostructures/*chemistry ; Nebulizers and Vaporizers ; Polyethylene Glycols/chemistry ; Transfection/instrumentation/*methods ; },
abstract = {Chitosan has been widely employed to deliver nucleic acids such as siRNA and plasmids. However, chitosan-mediated delivery of a gene-editing system has not been reported yet. In this study, poly(ethylene glycol) monomethyl ether (mPEG) was conjugated to chitosan with different molecular weights (low molecular weight and medium molecular weight chitosan) achieving a high degree of substitution as identified by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance ([1]H NMR) spectra. PEGylated chitosan/pSpCas9-2A-GFP nanocomplexes were formed at different N/ P (amine group to phosphate group) ratios and characterized in terms of size and zeta potential. The nanocomplexes developed showed the capability to protect loaded nucleic acids from DNase I digestion and from the stresses of nebulization. In addition, we demonstrated that the PEG conjugation of chitosan improved the mucus-penetration capability of the formed nanocomplexes at N/ P ratios of 5, 10, 20, and 30. Finally, PEGylated low molecular weight chitosan nanocomplexes showed optimal transfection efficiency at an N/ P ratio of 20, while PEGylated medium molecular weight chitosan nanocomplexes showed an optimal transfection efficiency at an N/ P ratio of 5 at pH 6.5 and 6.8. This study established the basis for the delivery of a gene-editing system by PEGylated chitosan nanocomplexes.},
}
@article {pmid30222779,
year = {2018},
author = {Shi, CH and Rubel, C and Soss, SE and Sanchez-Hodge, R and Zhang, S and Madrigal, SC and Ravi, S and McDonough, H and Page, RC and Chazin, WJ and Patterson, C and Mao, CY and Willis, MS and Luo, HY and Li, YS and Stevens, DA and Tang, MB and Du, P and Wang, YH and Hu, ZW and Xu, YM and Schisler, JC},
title = {Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007664},
pmid = {30222779},
issn = {1553-7404},
support = {T32 GM008320/GM/NIGMS NIH HHS/United States ; R01 GM061728/GM/NIGMS NIH HHS/United States ; R37 HL065619/HL/NHLBI NIH HHS/United States ; R35 GM118089/GM/NIGMS NIH HHS/United States ; R25 GM089569/GM/NIGMS NIH HHS/United States ; R01 GM075156/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems/genetics ; *Cognition ; Disease Models, Animal ; Female ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Models, Molecular ; Motor Activity/*genetics ; Mutagenesis, Site-Directed ; Phenotype ; Point Mutation ; Protein Domains/*genetics ; Protein Multimerization/genetics ; Rats ; Rats, Sprague-Dawley ; Spinocerebellar Ataxias/congenital/*genetics ; Ubiquitin-Protein Ligases/*genetics/metabolism ; },
abstract = {CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.},
}
@article {pmid30222773,
year = {2018},
author = {Kurata, M and Wolf, NK and Lahr, WS and Weg, MT and Kluesner, MG and Lee, S and Hui, K and Shiraiwa, M and Webber, BR and Moriarity, BS},
title = {Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays.},
journal = {PloS one},
volume = {13},
number = {9},
pages = {e0198714},
pmid = {30222773},
issn = {1932-6203},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Microarray Analysis ; Plasmids/genetics ; Promoter Regions, Genetic ; RNA, Guide/*genetics ; },
abstract = {The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.},
}
@article {pmid30222730,
year = {2018},
author = {Dorn, A and Röhrig, S and Papp, K and Schröpfer, S and Hartung, F and Knoll, A and Puchta, H},
title = {The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007674},
pmid = {30222730},
issn = {1553-7404},
mesh = {Arabidopsis/*physiology ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Catalytic Domain/genetics ; DNA Repair/*physiology ; DNA Topoisomerases, Type I/genetics/*metabolism ; DNA, Cruciform/genetics ; Endonucleases/genetics/*metabolism ; Escherichia coli Proteins/genetics/metabolism ; Gene Knockout Techniques ; Holliday Junction Resolvases/genetics/metabolism ; Mutagenesis ; Phenotype ; Plant Roots/growth &amp; development ; Plants, Genetically Modified ; Zinc Fingers/*genetics ; },
abstract = {Topoisomerase 3α, a class I topoisomerase, consists of a TOPRIM domain, an active centre and a variable number of zinc-finger domains (ZFDs) at the C-terminus, in multicellular organisms. Whereas the functions of the TOPRIM domain and the active centre are known, the specific role of the ZFDs is still obscure. In contrast to mammals where a knockout of TOP3α leads to lethality, we found that CRISPR/Cas induced mutants in Arabidopsis are viable but show growth retardation and meiotic defects, which can be reversed by the expression of the complete protein. However, complementation with AtTOP3α missing either the TOPRIM-domain or carrying a mutation of the catalytic tyrosine of the active centre leads to embryo lethality. Surprisingly, this phenotype can be overcome by the simultaneous removal of the ZFDs from the protein. In combination with a mutation of the nuclease AtMUS81, the TOP3α knockout proved to be also embryo lethal. Here, expression of TOP3α without ZFDs, and in particular without the conserved ZFD T1, leads to only a partly complementation in root growth-in contrast to the complete protein, that restores root length to mus81-1 mutant level. Expressing the E. coli resolvase RusA in this background, which is able to process Holliday junction (HJ)-like recombination intermediates, we could rescue this root growth defect. Considering all these results, we conclude that the ZFD T1 is specifically required for targeting the topoisomerase activity to HJ like recombination intermediates to enable their processing. In the case of an inactivated enzyme, this leads to cell death due to the masking of these intermediates, hindering their resolution by MUS81.},
}
@article {pmid30222362,
year = {2018},
author = {Chen, EC and Broccatelli, F and Plise, E and Chen, B and Liu, L and Cheong, J and Zhang, S and Jorski, J and Gaffney, K and Umemoto, KK and Salphati, L},
title = {Evaluating the Utility of Canine Mdr1 Knockout Madin-Darby Canine Kidney I Cells in Permeability Screening and Efflux Substrate Determination.},
journal = {Molecular pharmaceutics},
volume = {15},
number = {11},
pages = {5103-5113},
doi = {10.1021/acs.molpharmaceut.8b00688},
pmid = {30222362},
issn = {1543-8392},
mesh = {ATP Binding Cassette Transporter, Subfamily B, Member 1/*genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Dogs ; Drug Evaluation, Preclinical/methods ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Madin Darby Canine Kidney Cells ; Permeability ; },
abstract = {Permeability assays are commonly conducted with Madin-Darby canine kidney (MDCK) cells to predict the intestinal absorption of small-molecule drug candidates. In addition, MDCK cells transfected to overexpress efflux transporters are often used to identify substrates. However, MDCK cells exhibit endogenous efflux activity for a significant proportion of experimental compounds, potentially leading to the underestimation of permeability and confounded findings in transport studies. The goal of this study was to evaluate canine Mdr1 knockout MDCK (gMDCKI) cells in permeability screening and human MDR1 substrate determination in a drug discovery setting. The gMDCKI cells were established by CRISPR-Cas9-mediated knockout of the canine Mdr1 gene in MDCKI wildtype (wt) cells. A comparison of efflux ratios (ER) between MDCKI wt and gMDCKI showed that out of 135 compounds tested, 38% showed efflux activity in MDCKI wt, while no significant efflux was observed in gMDCKI cells. Apparent permeability (Papp) from apical-to-basolateral (A-to-B) and basolateral-to-apical were near unity in gMDCKI cells, which approximated passive permeability, and 17% of compounds demonstrated increases in their Papp A-to-B values. Overexpression of human MDR1 in gMDCKI (gMDCKI-MDR1) cells enabled substrate determination without the contribution of endogenous efflux, and the assay was able to deconvolute ambiguous results from MDCKI-MDR1 and identify species differences in substrate specificity. An analysis of 395 and 474 compounds in gMDCKI and gMDCKI-MDR1, respectively, suggested physicochemical properties that were associated with low permeability correlated with MDR1 recognition. Poorly permeable compounds and MDR1 substrates were more likely to be large, flexible, and more capable of forming external hydrogen bonds. On the basis of our evaluation, we concluded that gMDCKI is a better cell line for permeability screening and efflux substrate determination than the MDCK wt cell line.},
}
@article {pmid30222305,
year = {2018},
author = {Wang, Y and Ma, B and Abdeen, AA and Chen, G and Xie, R and Saha, K and Gong, S},
title = {Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery.},
journal = {ACS applied materials &amp; interfaces},
volume = {10},
number = {38},
pages = {31915-31927},
pmid = {30222305},
issn = {1944-8252},
support = {K25 CA166178/CA/NCI NIH HHS/United States ; R01 HL129785/HL/NHLBI NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Oxidation-Reduction ; Plasmids/*administration &amp; dosage ; Polymers ; RNA, Messenger/*administration &amp; dosage ; Transfection ; },
abstract = {Gene therapy holds great promise for the treatment of many diseases, but clinical translation of gene therapies has been slowed down by the lack of safe and efficient gene delivery systems. Here, we report two versatile redox-responsive polyplexes (i.e., cross-linked and non-crosslinked) capable of efficiently delivering a variety of negatively charged payloads including plasmid DNA (DNA), messenger RNA, Cas9/sgRNA ribonucleoprotein (RNP), and RNP-donor DNA complexes (S1mplex) without any detectable cytotoxicity. The key component of both types of polyplexes is a cationic poly(N, N'-bis(acryloyl)cystamine- co-triethylenetetramine) polymer [a type of poly(N, N'-bis(acryloyl)cystamine-poly(aminoalkyl)) (PBAP) polymer] containing disulfide bonds in the backbone and bearing imidazole groups. This composition enables efficient encapsulation, cellular uptake, controlled endo/lysosomal escape, and cytosolic unpacking of negatively charged payloads. To further enhance the stability of non-crosslinked PBAP polyplexes, adamantane (AD) and β-cyclodextrin (β-CD) were conjugated to the PBAP-based polymers. The cross-linked PBAP polyplexes formed by host-guest interaction between β-CD and AD were more stable than non-crosslinked PBAP polyplexes in the presence of polyanionic polymers such as serum albumin, suggesting enhanced stability in physiological conditions. Both cross-linked and non-crosslinked polyplexes demonstrated either similar or better transfection and genome-editing efficiencies, and significantly better biocompatibility than Lipofectamine 2000, a commercially available state-of-the-art transfection agent that exhibits cytotoxicity.},
}
@article {pmid30222157,
year = {2018},
author = {Sorlien, EL and Witucki, MA and Ogas, J},
title = {Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {138},
pages = {},
pmid = {30222157},
issn = {1940-087X},
support = {P30 CA023168/CA/NCI NIH HHS/United States ; R21 CA182197/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques/*methods ; Zebrafish/*genetics ; },
abstract = {Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.},
}
@article {pmid30220031,
year = {2018},
author = {Kim, MS and Kim, KH},
title = {CRISPR/Cas9-mediated knockout of HIF-1α gene in epithelioma papulosum cyprini (EPC) cells inhibited apoptosis and viral hemorrhagic septicemia virus (VHSV) growth.},
journal = {Archives of virology},
volume = {163},
number = {12},
pages = {3395-3402},
doi = {10.1007/s00705-018-4018-0},
pmid = {30220031},
issn = {1432-8798},
mesh = {Animals ; *Apoptosis ; CRISPR-Cas Systems ; Cell Line ; Cyprinidae/virology ; Fish Diseases/*genetics/metabolism/physiopathology/virology ; Gene Knockout Techniques ; Hemorrhagic Septicemia, Viral/*genetics/metabolism/physiopathology/virology ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism ; Novirhabdovirus/genetics/*growth &amp; development/physiology ; },
abstract = {Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1β, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection.},
}
@article {pmid30219987,
year = {2018},
author = {Hu, L and Zhang, H and Yang, Q and Meng, Q and Han, S and Nwafor, CC and Khan, MHU and Fan, C and Zhou, Y},
title = {Promoter variations in a homeobox gene, BnA10.LMI1, determine lobed leaves in rapeseed (Brassica napus L.).},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {131},
number = {12},
pages = {2699-2708},
pmid = {30219987},
issn = {1432-2242},
mesh = {Alleles ; Amino Acid Sequence ; Brassica napus/anatomy &amp; histology/*genetics ; CRISPR-Cas Systems ; Chromosome Mapping ; *Genes, Homeobox ; *Genes, Plant ; Genetic Linkage ; Plant Leaves/*anatomy &amp; histology ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; *Promoter Regions, Genetic ; Transcription Factors/genetics ; },
abstract = {BnA10.LMI1 positively regulates the development of leaf lobes in Brassica napus, and cis-regulatory divergences cause the different allele effects. Leaf shape is an important agronomic trait, and large variations in this trait exist within the Brassica germplasm. The lobed leaf is a unique morphological characteristic for Brassica improvement. Nevertheless, the molecular basis of leaf lobing in Brassica is poorly understood. Here, we show that an incompletely dominant locus, BnLLA10, is responsible for the lobed-leaf shape in rapeseed. A LATE MERISTEM IDENTITY1 (LMI1)-like gene (BnA10.LMI1) encoding an HD-Zip I transcription factor is the causal gene underlying the BnLLA10 locus. Sequence analysis of parental alleles revealed no sequence variations in the coding sequences, whereas abundant variations were identified in the regulatory region. Consistent with this finding, the expression levels of BnLMI1 were substantially elevated in the lobed-leaf parent compared with its near-isogenic line. The knockout mutations of BnA10.LMI1 gene were induced using the CRISPR/Cas9 system in both HY (the lobed-leaf parent) and J9707 (serrated leaf) genetic backgrounds. BnA10.LMI1 null mutations in the HY background were sufficient to produce unlobed leaves, whereas null mutations in the J9707 background showed no obvious changes in leaf shape compared with the control. Collectively, our results indicate that BnA10.LMI1 positively regulates the development of leaf lobes in B. napus, with cis-regulatory divergences causing the different allelic effects, providing new insights into the molecular mechanism of leaf lobe formation in Brassica crops.},
}
@article {pmid30217854,
year = {2018},
author = {Wang, Y and Wang, S and Chen, W and Song, L and Zhang, Y and Shen, Z and Yu, F and Li, M and Ji, Q},
title = {CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae.},
journal = {Applied and environmental microbiology},
volume = {84},
number = {23},
pages = {},
pmid = {30217854},
issn = {1098-5336},
mesh = {Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytidine Deaminase/*metabolism ; Gene Editing/*methods ; Genetic Engineering/*methods ; *Genome, Bacterial ; Klebsiella pneumoniae/*genetics ; },
abstract = {Klebsiella pneumoniae is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant K. pneumoniae Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation in K. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in K. pneumoniae By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in K. pneumoniae using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the blaKPC-2 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of the two editing tools will significantly facilitate the genetic engineering of K. pneumoniaeIMPORTANCE Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the blaKPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.},
}
@article {pmid30217531,
year = {2019},
author = {Spille, JH and Hecht, M and Grube, V and Cho, WK and Lee, C and Cissé, II},
title = {A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {153},
number = {},
pages = {35-45},
doi = {10.1016/j.ymeth.2018.09.004},
pmid = {30217531},
issn = {1095-9130},
support = {DP2 CA195769/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing/*methods ; Mice ; Mouse Embryonic Stem Cells/*metabolism ; RNA, Messenger/*analysis/chemistry/metabolism ; Single Molecule Imaging/*methods ; },
abstract = {The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.},
}
@article {pmid30217040,
year = {2018},
author = {Pan, Q and Wang, J and Gao, Y and Cui, H and Liu, C and Qi, X and Zhang, Y and Wang, Y and Wang, X},
title = {The Natural Large Genomic Deletion Is Unrelated to the Increased Virulence of the Novel Genotype Fowl Adenovirus 4 Recently Emerged in China.},
journal = {Viruses},
volume = {10},
number = {9},
pages = {},
pmid = {30217040},
issn = {1999-4915},
mesh = {Adenoviridae/classification/*genetics ; Adenoviridae Infections/*veterinary ; Animals ; CRISPR-Cas Systems ; Chickens ; China/epidemiology ; Ducks ; Gene Editing ; *Genome, Viral ; Genotype ; Phylogeny ; Poultry Diseases/diagnosis/epidemiology/*virology ; *Sequence Deletion ; Virulence/genetics ; },
abstract = {Since 2015, severe hydropericardium-hepatitis syndrome (HHS), caused by a highly pathogenic fowl adenovirus 4 (FAdV-4), emerged in China. In our previous study, the FAdV-4 has been identified as a novel genotype with a unique 1966-bp nucleotide deletion (1966Del) between open reading frame 42 and 43. In this study, the natural 1966Del was frequently identified among 17 clinical isolates and other reported Chinese clinical strains. To investigate the relationship between 1966Del and the increased virulence of the novel FAdV-4, a CRISPR/Cas9 operating platform for FAdV-4 was developed for the first time in this study. Based on this platform, a Re1966 strain was rescued, inserted the relative 1966Del sequence of a nonpathogenic strain KR5. In the pathogenicity study, the Re1966 strain retained high virulence for specific-pathogen-free chickens, similar to the parental wild-type HLJFAd15, although the survival time of chickens infected with Re1966 was much longer. Therefore, the natural 1966Del was identified as a non-essential site for the increased virulence of the emerged novel FAdV-4. Although further research on the virulence-determining region or point within the genome of the novel FAdV-4 is needed, the CRISPR/Cas9 operating platform for the novel FAdV-4 was developed and successfully applied to edit the genomic DNA for the first time, and it provides a novel powerful tool for both basic virology studies and vaccine vector development of FAdVs.},
}
@article {pmid30215766,
year = {2019},
author = {Lee, J and Lim, H and Jang, H and Hwang, B and Lee, JH and Cho, J and Lee, JH and Bang, D},
title = {CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system.},
journal = {Nucleic acids research},
volume = {47},
number = {1},
pages = {e1},
pmid = {30215766},
issn = {1362-4962},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; DNA/*genetics ; Escherichia coli/genetics ; Genome, Bacterial/*genetics ; Genome, Human/*genetics ; Humans ; Sequence Analysis, DNA ; },
abstract = {Existing methods to enrich target regions of genomic DNA based on PCR, hybridization capture, or molecular inversion probes have various drawbacks, including long experiment times and low throughput and/or enrichment quality. We developed CRISPR-Cap, a simple and scalable CRISPR-based method to enrich target regions of dsDNA, requiring only two short experimental procedures that can be completed within two hours. We used CRISPR-Cap to enrich 10 target genes 355.7-fold on average from Escherichia coli genomic DNA with a maximum on-target ratio of 81% and high enrichment uniformity. We also used CRISPR-Cap to measure gene copy numbers and detect rare alleles with frequencies as low as 1%. Finally, we enriched coding sequence regions of 20 genes from the human genome. We envision that CRISPR-Cap can be used as an alternative to other widely used target-enrichment methods, which will broaden the scope of CRISPR applications to the field of target enrichment field.},
}
@article {pmid30214055,
year = {2018},
author = {Kusano, H and Ohnuma, M and Mutsuro-Aoki, H and Asahi, T and Ichinosawa, D and Onodera, H and Asano, K and Noda, T and Horie, T and Fukumoto, K and Kihira, M and Teramura, H and Yazaki, K and Umemoto, N and Muranaka, T and Shimada, H},
title = {Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {13753},
pmid = {30214055},
issn = {2045-2322},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Vectors/genetics ; Mutagenesis/genetics ; Plants, Genetically Modified/*genetics/growth &amp; development ; RNA, Guide/*genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Solanum tuberosum/*genetics/growth &amp; development ; Starch Synthase/*genetics ; },
abstract = {CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5'-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.},
}
@article {pmid30213958,
year = {2018},
author = {Lager, AM and Corradin, OG and Cregg, JM and Elitt, MS and Shick, HE and Clayton, BLL and Allan, KC and Olsen, HE and Madhavan, M and Tesar, PJ},
title = {Rapid functional genetics of the oligodendrocyte lineage using pluripotent stem cells.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3708},
pmid = {30213958},
issn = {2041-1723},
support = {F31 NS083354/NS/NINDS NIH HHS/United States ; S10 OD016164/OD/NIH HHS/United States ; P30 CA043703/CA/NCI NIH HHS/United States ; R01 NS093357/NS/NINDS NIH HHS/United States ; R01 NS095280/NS/NINDS NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Cell Lineage ; DNA Mutational Analysis ; Genetic Association Studies ; Genetic Engineering ; Genotype ; Induced Pluripotent Stem Cells ; Lentivirus ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons/metabolism ; Oligodendroglia/*cytology ; Pluripotent Stem Cells/*cytology ; },
abstract = {Oligodendrocyte dysfunction underlies many neurological disorders, but rapid assessment of mutation-specific effects in these cells has been impractical. To enable functional genetics in oligodendrocytes, here we report a highly efficient method for generating oligodendrocytes and their progenitors from mouse embryonic and induced pluripotent stem cells, independent of mouse strain or mutational status. We demonstrate that this approach, when combined with genome engineering, provides a powerful platform for the expeditious study of genotype-phenotype relationships in oligodendrocytes.},
}
@article {pmid30213867,
year = {2018},
author = {Huynh, N and Zeng, J and Liu, W and King-Jones, K},
title = {A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {11},
pages = {3593-3605},
pmid = {30213867},
issn = {2160-1836},
mesh = {Animals ; CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/genetics ; Drosophila/*genetics ; Drosophila Proteins/genetics ; Exocrine Glands/*metabolism ; Gene Expression Regulation ; Mixed Function Oxygenases/genetics ; },
abstract = {Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.},
}
@article {pmid30213538,
year = {2018},
author = {Suzuki, M and Hayashi, T and Inoue, T and Agata, K and Hirayama, M and Suzuki, M and Shigenobu, S and Takeuchi, T and Yamamoto, T and Suzuki, KT},
title = {Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration.},
journal = {Developmental biology},
volume = {443},
number = {2},
pages = {127-136},
doi = {10.1016/j.ydbio.2018.09.008},
pmid = {30213538},
issn = {1095-564X},
mesh = {Animals ; Animals, Genetically Modified ; Breeding/methods ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Developmental Biology/methods ; Gene Knockout Techniques ; Phenotype ; Pleurodeles/*genetics/metabolism ; Regeneration/*genetics ; Ribonucleoproteins/genetics/metabolism ; Sequence Analysis, DNA/methods ; },
abstract = {Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase, pax6, tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.},
}
@article {pmid30213032,
year = {2018},
author = {Ho, BX and Loh, SJH and Chan, WK and Soh, BS},
title = {In Vivo Genome Editing as a Therapeutic Approach.},
journal = {International journal of molecular sciences},
volume = {19},
number = {9},
pages = {},
pmid = {30213032},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Gene Editing/*methods ; Hemophilia A/genetics/metabolism ; Humans ; Mucopolysaccharidosis II/genetics/metabolism ; Mutation/genetics ; Transcription Activator-Like Effector Nucleases/*genetics ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.},
}
@article {pmid30212742,
year = {2018},
author = {Du, J and Yin, N and Xie, T and Zheng, Y and Xia, N and Shang, J and Chen, F and Zhang, H and Yu, J and Liu, F},
title = {Quantitative assessment of HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide-mediated DSB repair.},
journal = {DNA repair},
volume = {70},
number = {},
pages = {67-71},
doi = {10.1016/j.dnarep.2018.09.002},
pmid = {30212742},
issn = {1568-7856},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; Homologous Recombination/*genetics ; Humans ; Oligodeoxyribonucleotides/*metabolism ; },
abstract = {Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two major mechanisms for the repair of DNA double-strand breaks (DSBs) in eukaryotic cells. Previously, we designed an assay for detecting NHEJ activity by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, however, this approach cannot be used to predict the activity of HR repair. Hence, we developed a novel method that is capable of quantitatively measuring both HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide (ODN)-mediated DSB repair. In the present experimental procedures, the CRISPR/Cas9 plasmid was cotransfected with single-stranded ODN (ssODN) or blunt-ended double-stranded ODN (dsODN), both of which harbored a unique marker sequence. After the induction of site-specific DSBs by CRISPR/Cas9 system, the ssODN, functioned as the donor template for HR repair, could insert the marker sequence into the DSB sites, while the dsODN was embedded in the DSB sites through NHEJ pathway. Next, by means of PCR analysis using a specific primer for the marker sequence and the primers that flank the DSB sites, the relative amount of integrated marker sequence in the genomic DNA could be quantitatively determined. The correlation between the marker sequence abundance and the HR and NHEJ activities was confirmed by using the selective HR and NHEJ inhibitors. This accessible and rapid quantitative assay for HR and NHEJ activities might be useful for the future research of the DSB repair mechanisms.},
}
@article {pmid30210501,
year = {2018},
author = {Hammerschmidt, SI and Werth, K and Rothe, M and Galla, M and Permanyer, M and Patzer, GE and Bubke, A and Frenk, DN and Selich, A and Lange, L and Schambach, A and Bošnjak, B and Förster, R},
title = {CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {1949},
pmid = {30210501},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Transformed ; *Cell Movement/genetics/immunology ; Dendritic Cells/cytology/*immunology ; *Homeodomain Proteins/genetics/immunology ; Mice ; Mice, Transgenic ; *Receptors, CCR7/genetics/immunology ; *Signal Transduction/genetics/immunology ; Stem Cells/cytology/*immunology ; },
abstract = {To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca[2+] sensor GCaMP6S to visualize in real-time chemokine-induced Ca[2+] signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo.},
}
@article {pmid30210230,
year = {2018},
author = {Zelinka, CP and Sotolongo-Lopez, M and Fadool, JM},
title = {Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration.},
journal = {Molecular vision},
volume = {24},
number = {},
pages = {587-602},
pmid = {30210230},
issn = {1090-0535},
support = {R21 EY025410/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Codon, Terminator/genetics ; *Disease Models, Animal ; Frameshift Mutation/genetics ; Gene Targeting ; Immunoblotting ; Polymorphism, Restriction Fragment Length ; RNA, Messenger/genetics ; Retinal Degeneration/*genetics/pathology ; Retinal Rod Photoreceptor Cells/*pathology ; Rhodopsin/*genetics ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {PURPOSE: Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1-1.<br><br>METHODS: One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes.<br><br>RESULTS: Using gRNAs that targeted highly conserved regions of rh1-1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5'-coding sequence of rh1-1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3'-coding region of rh1-1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane.<br><br>CONCLUSIONS: The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.},
}
@article {pmid30209659,
year = {2018},
author = {Bressan, RB and Pollard, SM},
title = {Genome Editing in Human Neural Stem and Progenitor Cells.},
journal = {Results and problems in cell differentiation},
volume = {66},
number = {},
pages = {163-182},
doi = {10.1007/978-3-319-93485-3_7},
pmid = {30209659},
issn = {0080-1844},
mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Neural Stem Cells/cytology/*metabolism ; Pluripotent Stem Cells/cytology/metabolism ; },
abstract = {Experimental tools for precise manipulation of mammalian genomes enable reverse genetic approaches to explore biology and disease. Powerful genome editing technologies built upon designer nucleases, such as CRISPR/Cas9, have recently emerged. Parallel progress has been made in methodologies for the expansion and differentiation of human pluripotent and tissue stem cells. Together these innovations provide a remarkable new toolbox for human cellular genetics and are opening up vast opportunities for discoveries and applications across the breadth of life sciences research. In this chapter, we review the emergence of genome editing technologies and how these are being deployed in studies of human neurobiology, neurological disease, and neuro-oncology. We focus our discussion on CRISPR/Cas9 and its application in studies of human neural stem and progenitor cells.},
}
@article {pmid30209390,
year = {2018},
author = {Akcakaya, P and Bobbin, ML and Guo, JA and Malagon-Lopez, J and Clement, K and Garcia, SP and Fellows, MD and Porritt, MJ and Firth, MA and Carreras, A and Baccega, T and Seeliger, F and Bjursell, M and Tsai, SQ and Nguyen, NT and Nitsch, R and Mayr, LM and Pinello, L and Bohlooly-Y, M and Aryee, MJ and Maresca, M and Joung, JK},
title = {In vivo CRISPR editing with no detectable genome-wide off-target mutations.},
journal = {Nature},
volume = {561},
number = {7723},
pages = {416-419},
pmid = {30209390},
issn = {1476-4687},
support = {P30 DK043351/DK/NIDDK NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*methods/*standards ; Genome/*genetics ; Humans ; INDEL Mutation ; Male ; Mice ; Mice, Inbred C57BL ; *Mutation ; Proprotein Convertase 9/genetics ; Substrate Specificity/*genetics ; Transgenes/genetics ; },
abstract = {CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications[1-6] but identifying unwanted off-target mutations is important for clinical translation[7]. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.},
}
@article {pmid30209359,
year = {2018},
author = {Allison, SJ},
title = {Targeting methylation.},
journal = {Nature reviews. Nephrology},
volume = {14},
number = {11},
pages = {658},
doi = {10.1038/s41581-018-0062-y},
pmid = {30209359},
issn = {1759-507X},
mesh = {*CRISPR-Cas Systems ; *DNA Methylation ; Fibrosis ; Gene Expression ; Humans ; Protein Processing, Post-Translational ; },
}
@article {pmid30209206,
year = {2018},
author = {Cubbon, A and Ivancic-Bace, I and Bolt, EL},
title = {CRISPR-Cas immunity, DNA repair and genome stability.},
journal = {Bioscience reports},
volume = {38},
number = {5},
pages = {},
pmid = {30209206},
issn = {1573-4935},
support = {BB/M020541-1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; DNA Repair/*genetics ; Gene Editing ; Genome, Human/genetics ; Genomic Instability/*genetics ; Humans ; },
abstract = {Co-opting of CRISPR-Cas 'Interference' reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data are emerging about the identities of DNA repair enzymes that support genome editing in human cells. At the same time, it is becoming apparent that CRISPR-Cas systems functioning in their native environment, bacteria or archaea, also need DNA repair enzymes. In this short review, we survey how DNA repair and CRISPR-Cas systems are intertwined. We consider how understanding DNA repair and CRISPR-Cas interference reactions in nature might help improve the efficacy of genome editing procedures that utilise homologous or analogous systems in human and other cells.},
}
@article {pmid30209174,
year = {2018},
author = {Sivan, G and Glushakow-Smith, SG and Katsafanas, GC and Americo, JL and Moss, B},
title = {Human Host Range Restriction of the Vaccinia Virus C7/K1 Double Deletion Mutant Is Mediated by an Atypical Mode of Translation Inhibition.},
journal = {Journal of virology},
volume = {92},
number = {23},
pages = {},
pmid = {30209174},
issn = {1098-5514},
mesh = {Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems ; HeLa Cells ; Host Specificity ; Host-Pathogen Interactions ; Humans ; Intracellular Signaling Peptides and Proteins ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Methyltransferases/antagonists &amp; inhibitors/genetics/*metabolism ; Nuclear Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; *Protein Biosynthesis ; Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; RNA, Viral ; *Sequence Deletion ; Vaccinia/*genetics/metabolism/virology ; Vaccinia virus/genetics/isolation &amp; purification/physiology ; *Virus Replication ; },
abstract = {Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide short interfering RNA (siRNA) screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host range restriction factor along with additional candidates, including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knockout (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80S and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained nonpermissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L, or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other's vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA, and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L, or G3 binding proteins, suggesting a novel mechanism of host restriction.},
}
@article {pmid30208656,
year = {2018},
author = {Sun, Q and Lin, L and Liu, D and Wu, D and Fang, Y and Wu, J and Wang, Y},
title = {CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.},
journal = {International journal of molecular sciences},
volume = {19},
number = {9},
pages = {},
pmid = {30208656},
issn = {1422-0067},
mesh = {Ascomycota/physiology ; Brassica napus/*genetics ; *CRISPR-Cas Systems ; Disease Resistance ; *Gene Editing ; Gene Expression Regulation, Plant ; Genome, Plant ; Mutagenesis ; Mutation ; Plant Diseases/*genetics/microbiology ; Plant Proteins/*genetics ; Plants, Genetically Modified/*genetics ; Transcription Factors/*genetics ; },
abstract = {Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T0) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T1 generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T1 plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.},
}
@article {pmid30208287,
year = {2018},
author = {Stanley, SY and Maxwell, KL},
title = {Phage-Encoded Anti-CRISPR Defenses.},
journal = {Annual review of genetics},
volume = {52},
number = {},
pages = {445-464},
doi = {10.1146/annurev-genet-120417-031321},
pmid = {30208287},
issn = {1545-2948},
mesh = {Bacteria/*genetics/virology ; Bacteriophages/*genetics/pathogenicity ; CRISPR-Cas Systems/*genetics ; *Evolution, Molecular ; Gene Editing ; Viral Proteins/genetics ; },
abstract = {The battle for survival between bacteria and bacteriophages (phages) is an arms race where bacteria develop defenses to protect themselves from phages and phages evolve counterstrategies to bypass these defenses. CRISPR-Cas adaptive immune systems represent a widespread mechanism by which bacteria protect themselves from phage infection. In response to CRISPR-Cas, phages have evolved protein inhibitors known as anti-CRISPRs. Here, we describe the discovery and mechanisms of action of anti-CRISPR proteins. We discuss the potential impact of anti-CRISPRs on bacterial evolution, speculate on their evolutionary origins, and contemplate the possible next steps in the CRISPR-Cas evolutionary arms race. We also touch on the impact of anti-CRISPRs on the development of CRISPR-Cas-based biotechnological tools.},
}
@article {pmid30206235,
year = {2018},
author = {Wang, H and Guo, W and Mitra, J and Hegde, PM and Vandoorne, T and Eckelmann, BJ and Mitra, S and Tomkinson, AE and Van Den Bosch, L and Hegde, ML},
title = {Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in Amyotrophic Lateral Sclerosis.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3683},
pmid = {30206235},
issn = {2041-1723},
support = {I01 BX002466/BX/BLRD VA/United States ; P01 CA092584/CA/NCI NIH HHS/United States ; R01 ES012512/ES/NIEHS NIH HHS/United States ; R01 NS088645/NS/NINDS NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA/*metabolism ; DNA Damage/*genetics ; DNA Ligases/metabolism ; DNA Repair/*genetics ; Gene Knockout Techniques ; Genes, Dominant ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Models, Biological ; Mutation/*genetics ; *Oxidative Stress ; Poly(ADP-ribose) Polymerases/metabolism ; RNA-Binding Protein FUS/*genetics ; Reactive Oxygen Species/metabolism ; X-ray Repair Cross Complementing Protein 1/metabolism ; },
abstract = {Genome damage and defective repair are etiologically linked to neurodegeneration. However, the specific mechanisms involved remain enigmatic. Here, we identify defects in DNA nick ligation and oxidative damage repair in a subset of amyotrophic lateral sclerosis (ALS) patients. These defects are caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction. We discover that loss of nuclear FUS caused DNA nick ligation defects in motor neurons due to reduced recruitment of XRCC1/LigIII to DNA strand breaks. Moreover, DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in motor neurons generated therefrom are rescued by CRISPR/Cas9-mediated correction of mutation. Our findings uncovered a pathway of defective DNA ligation in FUS-linked ALS and suggest that LigIII-targeted therapies may prevent or slow down disease progression.},
}
@article {pmid30206203,
year = {2018},
author = {Wang, S and Liu, Z and Ye, Y and Li, B and Liu, T and Zhang, W and Liu, GH and Zhang, YA and Qu, J and Xu, D and Chen, Z},
title = {Ectopic hTERT expression facilitates reprograming of fibroblasts derived from patients with Werner syndrome as a WS cellular model.},
journal = {Cell death &amp; disease},
volume = {9},
number = {9},
pages = {923},
pmid = {30206203},
issn = {2041-4889},
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Cellular Reprogramming/*physiology ; Cellular Reprogramming Techniques/*methods ; Fibroblasts/*cytology ; Gene Knockout Techniques ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Telomerase/metabolism ; Topoisomerase Inhibitors/pharmacology ; Werner Syndrome/*pathology ; Werner Syndrome Helicase/*genetics ; },
abstract = {The induced pluripotent stem cell (iPSC) technology has provided a unique opportunity to develop disease-specific models and personalized treatment for genetic disorders, and is well suitable for the study of Werner syndrome (WS), an autosomal recessive disease with adult onset of premature aging caused by mutations in the RecQ like helicase (WRN) gene. WS-derived fibroblasts were previously shown to be able to generate iPSCs; however, it remains elusive how WS-derived iPSCs behave and whether they are able to mimic the disease-specific phenotype. The present study was designed to address these issues. Unexpectedly, we found that a specific WS fibroblast line of homozygous truncation mutation was difficult to be reprogrammed by using the Yamanaka factors even under hypoxic conditions due to their defect in induction of hTERT, the catalytic unit of telomerase. Ectopic expression of hTERT restores the ability of this WS fibroblast line to form iPSCs, although with a low efficiency. To examine the phenotype of WRN-deficient pluripotent stem cells, we also generated WRN knockout human embryonic stem (ES) cells by using the CRISPR/Cas9 method. The iPSCs derived from WS-hTERT cells and WRN-/- ESCs are fully pluripotent, express pluripotent markers and can differentiate into three germ layer cells; however, WS-iPSCs and WRN-/- ESCs show S phase defect in cell cycle progression. Moreover, WS-iPSCs and WRN-/- ESCs, like WS patient-derived fibroblasts, remain hypersensitive to topoisomerase inhibitors. Collectively, WS-derived iPSCs and WRN-/- ESCs mimic the intrinsic disease phenotype, which may serve as a suitable disease model, whereas not be good for a therapeutic purpose without gene correction.},
}
@article {pmid30206189,
year = {2018},
author = {Palazón-Riquelme, P and Worboys, JD and Green, J and Valera, A and Martín-Sánchez, F and Pellegrini, C and Brough, D and López-Castejón, G},
title = {USP7 and USP47 deubiquitinases regulate NLRP3 inflammasome activation.},
journal = {EMBO reports},
volume = {19},
number = {10},
pages = {},
pmid = {30206189},
issn = {1469-3178},
support = {/WT_/Wellcome Trust/United Kingdom ; MR/N029992/1/MRC_/Medical Research Council/United Kingdom ; 104192/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Deubiquitinating Enzymes/chemistry/genetics ; Gene Knockdown Techniques ; Humans ; Inflammasomes/genetics/metabolism ; Inflammation/*genetics/pathology ; Interleukin-18/genetics ; Interleukin-1beta/genetics ; Macrophages/metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/*genetics ; Signal Transduction/genetics ; Ubiquitin Thiolesterase/*genetics ; Ubiquitin-Specific Peptidase 7/*genetics ; Ubiquitin-Specific Proteases ; Ubiquitination/genetics ; },
abstract = {The assembly and activation of the inflammasomes are tightly regulated by post-translational modifications, including ubiquitin. Deubiquitinases (DUBs) counteract the addition of ubiquitin and are essential regulators of immune signalling pathways, including those acting on the inflammasome. How DUBs control the assembly and activation of inflammasomes is unclear. Here, we show that the DUBs USP7 and USP47 regulate inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation, independently of transcription, by preventing ASC oligomerisation and speck formation. We also provide evidence that the ubiquitination status of NLRP3 itself is altered by inhibition of USP7 and USP47. Interestingly, we found that the activity of USP7 and USP47 increased in response to inflammasome activators. Using CRISPR/Cas9 in the macrophage cell line THP-1, we show that inflammasome activation is reduced when both USP7 and USP47 are knocked down. Altogether, these data reveal a new post-transcriptional role for USP47 and USP7 in inflammation by regulating inflammasome activation and the release of the pro-inflammatory cytokines IL-1β and IL-18, and implicate dual USP7 and USP47 inhibitors as potential therapeutic agents for inflammatory disease.},
}
@article {pmid30205881,
year = {2018},
author = {Cao, J and Xiao, Q and Yan, Q},
title = {The multiplexed CRISPR targeting platforms.},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {53-61},
pmid = {30205881},
issn = {1740-6749},
support = {R21 CA187862/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting/*methods ; Genetic Therapy/*methods ; Humans ; },
abstract = {The discovery and engineering of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the past several years have revolutionized biomedical research. The CRISPR technology showed great potential to advance detection, prevention, and treatment of human diseases in the near future. Compared to previous developed genome editing approaches, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR-based systems have numerous advantages. One example is that the CRISPR systems can be easily adopted to efficiently target multiple genes simultaneously. Several strategies and toolboxes have been developed to achieve multiplexed targeting using the CRISPR systems. In this short review, we will discuss the principle, approach, and application of these strategies.},
}
@article {pmid30205880,
year = {2018},
author = {Naert, T and Vleminckx, K},
title = {CRISPR/Cas9 disease models in zebrafish and Xenopus: The genetic renaissance of fish and frogs.},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {41-52},
doi = {10.1016/j.ddtec.2018.07.001},
pmid = {30205880},
issn = {1740-6749},
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; *Gene Editing ; Gene Targeting ; Genetic Therapy/methods ; Genomics ; Mutation ; Xenopus ; Zebrafish ; },
abstract = {The speed by which clinical genomics is currently identifying novel potentially pathogenic variants is outperforming the speed by which these can be functionally (genotype-phenotype) annotated in animal disease models. However, over the past few years the emergence of CRISPR/Cas9 as a straight-forward genome editing technology has revolutionized disease modeling in vertebrate non-mammalian model organisms such as zebrafish, medaka and Xenopus. It is now finally possible, by CRISPR/Cas9, to rapidly establish clinically relevant disease models in these organisms. Interestingly, these can provide both cost-effective genotype-phenotype correlations for gene-(variants) and genomic rearrangements obtained from clinical practice, as well as be exploited to perform translational research to improve prospects of disease afflicted patients. In this review, we show an extensive overview of these new CRISPR/Cas9-mediated disease models and provide future prospects that will allow increasingly accurate modeling of human disease in zebrafish, medaka and Xenopus.},
}
@article {pmid30205879,
year = {2018},
author = {Sürün, D and von Melchner, H and Schnütgen, F},
title = {CRISPR/Cas9 genome engineering in hematopoietic cells.},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {33-39},
doi = {10.1016/j.ddtec.2018.08.001},
pmid = {30205879},
issn = {1740-6749},
mesh = {Acquired Immunodeficiency Syndrome/genetics/therapy ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Targeting/methods ; Genetic Therapy/*methods ; Hematologic Diseases/genetics/therapy ; Humans ; Neoplasms/genetics/therapy ; },
abstract = {The development of genome editing tools capable of modifying specific genomic sequences with unprecedented accuracy has opened up a wide range of new possibilities in targeted gene manipulation. In particular, the CRISPR/Cas9 system, a repurposed prokaryotic adaptive immune system, has been widely adopted because of its unmatched simplicity and flexibility. In this review we discuss achievements and current limitations of CRISPR/Cas9 genome editing in hematopoietic cells with special emphasis on its potential use in ex vivo gene therapy of monogenic blood disorders, HIV and cancer.},
}
@article {pmid30205877,
year = {2018},
author = {Xu, CL and Park, KS and Tsang, SH},
title = {CRISPR/Cas9 genome surgery for retinal diseases.},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {23-32},
pmid = {30205877},
issn = {1740-6749},
support = {R21 AG050437/AG/NIA NIH HHS/United States ; R01 EY018213/EY/NEI NIH HHS/United States ; R01 EY026682/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; P30 EY019007/EY/NEI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy/methods ; Genome, Human ; Humans ; Retinal Diseases/*genetics/*therapy ; },
abstract = {Retinal diseases that impair vision can impose heavy physical and emotional burdens on patients' lives. Currently, clustered regularly interspaced short palindromic repeats (CRISPR) is a prevalent gene-editing tool that can be harnessed to generate disease model organisms for specific retinal diseases, which are useful for elucidating pathophysiology and revealing important links between genetic mutations and phenotypic defects. These retinal disease models are fundamental for testing various therapies and are indispensible for potential future clinical trials. CRISPR-mediated procedures involving CRISPR-associated protein 9 (Cas9) may also be used to edit genome sequences and correct mutations. Thus, if used for future therapies, CRISPR/Cas9 genome surgery could eliminate the need for patients with retinal diseases to undergo repetitive procedures such as drug injections. In this review, we will provide an overview of CRISPR/Cas9, discuss the different types of Cas9, and compare Cas9 to other endonucleases. Furthermore, we will explore the many ways in which researchers are currently utilizing this versatile tool, as CRISPR/Cas9 may have far-reaching effects in the treatment of retinal diseases.},
}
@article {pmid30205876,
year = {2018},
author = {Christidi, E and Huang, HM and Brunham, LR},
title = {CRISPR/Cas9-mediated genome editing in human stem cell-derived cardiomyocytes: Applications for cardiovascular disease modelling and cardiotoxicity screening.},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {13-21},
doi = {10.1016/j.ddtec.2018.06.002},
pmid = {30205876},
issn = {1740-6749},
mesh = {Animals ; *CRISPR-Cas Systems ; Cardiotoxicity/*genetics/therapy ; Cardiovascular Diseases/*genetics/therapy ; Cell Differentiation/genetics ; *Gene Editing ; Genome, Human ; Humans ; Myocytes, Cardiac/cytology/physiology ; Stem Cells/cytology/*physiology ; },
abstract = {Cardiovascular diseases (CVDs) are leading causes of death worldwide, and drug-induced cardiotoxicity is among the most common cause of drug withdrawal from the market. Improved models of cardiac tissue are needed to study the mechanisms of CVDs and drug-induced cardiotoxicity. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) have provided a major advance to our ability to study these conditions. Combined with efficient genome editing technologies, such as CRISPR/Cas9, we now have the ability to study with greater resolution the genetic causes and underlying mechanisms of inherited and drug-induced cardiotoxicity, and to investigate new treatments. Here, we review recent advances in the use of hPSC-CMs and CRISPR/Cas9-mediated genome editing to study cardiotoxicity and model CVD.},
}
@article {pmid30205875,
year = {2018},
author = {Bellin, M},
title = {Crispr/Cas9 homologous recombination (HR).},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {1-2},
doi = {10.1016/j.ddtec.2018.08.006},
pmid = {30205875},
issn = {1740-6749},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Homologous Recombination ; Humans ; },
}
@article {pmid30205462,
year = {2018},
author = {Hidalgo-Cantabrana, C and Sanozky-Dawes, R and Barrangou, R},
title = {Insights into the Human Virome Using CRISPR Spacers from Microbiomes.},
journal = {Viruses},
volume = {10},
number = {9},
pages = {},
pmid = {30205462},
issn = {1999-4915},
support = {support//North Carolina Agricultural Foundation/International ; internal support//North Carolina State University/International ; },
mesh = {*Biodiversity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Metagenomics ; *Microbiota ; Viruses/*classification/genetics/*isolation &amp; purification ; },
abstract = {Due to recent advances in next-generation sequencing over the past decade, our understanding of the human microbiome and its relationship to health and disease has increased dramatically. Yet, our insights into the human virome, and its interplay with important microbes that impact human health, is relatively limited. Prokaryotic and eukaryotic viruses are present throughout the human body, comprising a large and diverse population which influences several niches and impacts our health at various body sites. The presence of prokaryotic viruses like phages, has been documented at many different body sites, with the human gut being the richest ecological niche. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins constitute the adaptive immune system of bacteria, which prevents attack by invasive nucleic acid. CRISPR-Cas systems function by uptake and integration of foreign genetic element sequences into the CRISPR array, which constitutes a genomic archive of iterative vaccination events. Consequently, CRISPR spacers can be investigated to reconstruct interplay between viruses and bacteria, and metagenomic sequencing data can be exploited to provide insights into host-phage interactions within a niche. Here, we show how the CRISPR spacer content of commensal and pathogenic bacteria can be used to determine the evidence of their phage exposure. This framework opens new opportunities for investigating host-virus dynamics in metagenomic data, and highlights the need to dedicate more efforts for virome sampling and sequencing.},
}
@article {pmid30205198,
year = {2018},
author = {Zoppo, M and Lombardi, L and Rizzato, C and Lupetti, A and Bottai, D and Papp, C and Gácser, A and Tavanti, A},
title = {CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {120},
number = {},
pages = {19-29},
doi = {10.1016/j.fgb.2018.09.001},
pmid = {30205198},
issn = {1096-0937},
mesh = {Animals ; CRISPR-Cas Systems ; Candida parapsilosis/*genetics/growth &amp; development/pathogenicity ; Candidiasis/microbiology ; Cell Adhesion ; Female ; *Genes, Fungal ; Humans ; Mice ; Mice, Inbred BALB C ; Mouth Mucosa/*microbiology ; Mutagenesis ; Virulence/genetics ; },
abstract = {Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis.},
}
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